Analytical Profiles

of

Drug Substances
Volume 16
Edited by

Klaus Florey
The Squibb Institute for Medical Research
New Brunswick, New Jersey

Contributing Editors

Abdullah A. Al-Badr

Gerald S. Brenner

1987

ACADEMIC PRESS, INC.
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EDITORIAL BOARD

Abdullah A. Al-Badr
Steven A. Benezra
Gerald S. Brenner
Glenn A. Brewer, Jr.
Nicholas DeAngelis

Lee T. Grady
Eugene Inman
Joseph Mollica
Milton D. Yudis
John Zarembo

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Library of Congress Cataloging in Publication Data
(Revised for volume 16)
Analytical profiles o f drug substances.
Compiled under the auspices o f the Pharmaceutical
Analysis and Control Section, Academy of Pharmaceutical
Sciences.
Includes bibliographical references and indexes.
1. Drugs-Analysis-Collected works. 2. Chemistry,
Pharmaceutical-Collected works. I. Florey, Klaus,
11. Brewer, Glenn A. 111. Academy of Pharmaceutical
Sciences. Pharmaceutical Analysis Control Section.
[DNLM: 1. Drugs-Analysis-Yearbooks.
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RS 189.A58
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ISBN 0-12-260816-X
(v. 16 : alk. paper)
PRINTED IN THE UNITED STATES OF AMERICA
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AFFILIATIONS OF
EDITORS AND CONTRIBUTORS

Mohammad A . Abounassif, College of Pharmacy, King Saud University,
Riyadh, Saudi Arabia
Abdul Fattah A . Afify, College of Pharmacy, King Saud University,
Riyadh, Saudi Arabia
Abdullah A . Al-Budr, King Saud University, Riyadh, Saudi Arabia
Mohammed A . Al-Yahya, College of Pharmacy, King Saud University,
Riyadh, Saudi Arabia
Salim A . Babhair, College of Pharmacy, King Saud University, Riyadh,
Saudi Arabia
Jos H . Beijnen, Slotervaart Hospital, Amsterdam, The Netherlands
Steven A . Benezra, Wellcome Research Laboratories, Research Triangle
Park, North Carolina
Gerald S. Brenner, Merck Sharp & Dohme Research Laboratories, West
Point, Pennsylvania
Glenn A . Brewer, Jr., The Squibb Institute for Medical Research, New
Brunswick, New Jersey
Auke Bult, Faculty of Pharmacy, State University of Utrecht, Utrecht,
The Netherlands
Nicholas J. DeAngelis, Wyeth Laboratories, Philadelphia, Pennsylvania
Humeida A. El-Obeid, King Saud University, Riyadh, Saudi Arabia
Klaus Florey, The Squibb Institute for Medical Research, New Brunswick, New Jersey
Dennis K . G . Gorecki, College of Pharmacy, University of Saskatchewan,
Saskatoon, Saskatchewan, Canada
Lee T . Grady, The United States Pharmacopeia, Rockville, Maryland
Mahmoud M . A . Hassan, King Saud University, Riyadh, Saudi Arabia
Eugene L. Inman, Lilly Research Laboratories, Indianapolis, Indiana
vii

...

Vlll

AFFILIATIONS

Dominic P . I p , Merck Sharp & Dohme Research Laboratories, West
Point, Pennsylvania
Casimir A . Janicki, McNeil Pharmaceutical, Spring House, Pennsylvania
Vijay K . Kapoor, Department of Pharmaceutical Sciences, Panjab University, Chandigarh, India
Alice E . Loper, Merck Sharp & Dohme Research Laboratories, West
Point, Pennsylvania
David J . Mazzo, Travenol Laboratories, Morton Grove, Illinois
A . G. Mekkawi, College of Pharmacy, King Saud University, Riyadh,
Saudi Arabia
Joseph A . Mollica, Pharmaceuticals, Du Pont, Wilmington, Delaware
Jaber S. Mossa, College of Pharmacy, King Saud University, Riyadh,
Saudi Arabia
Farid J . Muhtadi, King Saud University, Riyadh, Saudi Arabia
Davide Pitrc?, Faculty of Pharmacy, University of Milan, Milan, Italy
James T. Stewart, College of Pharmacy, University of Georgia, Athens,
Georgia
Riccardo Stradi, Faculty of Pharmacy, University of Milan, Milan, Italy
Abdul Hameed U . Kader Taragan, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
Mohammad Tariq, College of Pharmacy, King Saud University, Riyadh,
Saudi Arabia
WillyJ . M . Underberg, Faculty of Pharmacy, State University of Utrecht,
Utrecht, The Netherlands
Roger K . Verbeeck, College of Pharmacy, University of Saskatchewan,
Saskatoon, Saskatchewan, Canada
Milton D . Yudis, Schering-Plough, Kenilworth, New Jersey
John E . Zarembo, W. H. Rorer, Inc., Fort Washington, Pennsylvania

PREFACE

Although the official compendia define a drug substance as to identity,
purity, strength, and quality, they normally do not provide other physical
or chemical data, nor do they list methods of synthesis or pathways of
physical or biological degradation and metabolism. Such information is
scattered through the scientific literature and the files of pharmaceutical
laboratories.
I perceived a need to supplement the official compendial standards of
drug substances with a comprehensive review of such infoimation, and
sixteen years ago the first volume of Anulyticul Profiles of Drug Substances was published under the auspices of the Pharmaceutical Analysis and Control Section of the APhA Academy of Phaimaceutical Sciences. That we were able to publish one volume per year is a tribute to
the diligence of the editors to solicit articles and even more so to the
enthusiastic response of our authors, an international group associated
with pharmaceutical firms, academic institutions, and compendial authorities. I would like to express my sincere gratitude to them for making
this venture possible.
Over the years, we have had queries concerning our publication policy. Our goal is to cover all divg substances of medical value and, therefore, we have welcomed any articles of interest to an individual contributor. We also have endeavored to solicit profiles of the most useful and
used medicines, but many in this category still need to be profiled.
Klaus Florey

ix

BROMAZEPAM

Mahmoud M.A. H a h a n , PhO6eAhOh 06 PhahmaceuLLcd C h m h L t y ,
Depah;tment od PhahmaceuLLcd C h m h L t y , CoMege 06
Phanmacy, King Saud U n L v m L t q , Riyadh, Saudi Axabia.
Mohammad A. A b u u m n i d , AnbhXant Phodeddon 04 PhamaceuLic d C h m h i x y , Heud 04 Xhe DepdmenX 06 P h a m a c e d i c d
C h m h L t y , College 06 Phahmacy, King Saud U n i v m i , t y ,
Riyadh, Saudi Arrabia.

1. H i s t o r y and T h e r a p e u t i c Category
2.

Description
2.1

Nomenclature

2.2

Formulae

2.3

Molecular Weight

ANALYTICAL PROFILES OF DRUG SUBSTANCES
VOLUME 16

1

Copyright 0 1987 by Academic Press, Inc.
All rights of reproduction in any form reserved.

MAHMOUD M. A. HASSAN AND MOHAMMAD A. ABOUNASSIF

2

.3.

4,

Physical Properties

3.1

Appearance, Color, Odor and Taste

3.2

Melting Range

3.3

D i s s o c i a t i o n Constants

3.4

Crystal Structure

Spectral Properties

4.1

U l t r a v i o l e t Spectrum

4.2

I n f r a r e d Spectrum

4.3

Nuclear Magnetic Resonance S p e c t r a

4.4

Mass Spectrum

5.

Synthesis

6.

Metabolism

7.

Pharmacokinetic S t u d i e s

8.

Methods of Analysis

8.1

Elemental Analysis

8.2

I d e n t i f i c a t i o n Tests

8.3

Titrimetric

8.4 Spectrophotometric
8.5 Polarographic
8.6 Atomic Absorption

9.

8.7

Radioimunoassays

8.8

Chromatographic

References

BR 0MAZEPAM

3

A n a l y t i c a l P r o f i l e of Bromazepam
1. H i s t o r y and Therapeutic Category
The l a s t q u a r t e r c e n t u r y , and i n p a r t i c u l a r t h e decade
1952-1962, has witnessed a break-through i n t h e t r e a t m e n t
and outlook of t h e mentally ill. Two groups of drugs
were developed namely psychotic and psychotropic a g e n t s .
Among t h e l a s t named psychotropic a g e n t s , t h e benzodiazep i n e s . These drugs such as diazepam, chlordiazepoxide
and oxazepam have been shown t o e x e r t a n x i o l y t i c , s l e e p
inducing, muscle r e l a x a n t and a n t i c o n v u l s a n t e f f e c t s i n
g r e a t e r or l e s s e r degree. Search continued f o r making
more d e r i v a t i v e s t o produce s u i t a b l e drug f o r s p e c i f i c
cases or symptoms. I n t h i s way nitrazepam and more
r e c e n t l y flurazepam have been found t o be s p e c i a l l y e f f e c t i v e s l e e p inducers and clonazepam e x e r t s even more
p o t e n t l y t h e a n t i c o n w l s a n t a c t i v i t y known a l r e a d y f o r
diazepam.
Search has continued and i n 1974 bromazepam w a s marketed
as a new psychotropic drug of t h e benzodiazepine s e r i e s
but belonging t o a new c l a s s of pyridyl-benzodiazepines.
It possesses c e r t a i n biochemical f e a t u r e s which d i s t i n guish it from o t h e r benzodiazepines. It has been shown
t o e x e r t a more profound a n x i o l y t i c e f f e c t t h a n o t h e r
similar substances.
2.

Description
2.1

Nomenclature
2.1.1

Chemical Names

7-Brom0-1,3-dihydro-5-(~2-pyridyl)-2H-l,~benzodiazepine-2-one.
2.1.2

Generic Name
Bromazepam.

2.1.3

Trade Names
Lectopam; Lexomil; Lexotan; Lexotanil.

4

MAHMOUD M. A. HASSAN AND MOHAMMAD A. ABOUNASSIF

2.2

Formulae
2.2.1

Ehpirical
C14H10BrN

2.2.2

0

3

Structural

H

2.2.3

Research Number (1)
Ro 5-3350.

2.2.4

Chemical A b s t r a c t s R e g i s t r y Number

[ 1812-30-21
2.3

Molecular Weight
316.16

3.

Physical Properties
3.1

Appearance, Color, Odor and T a s t e
A p a l e yellow o d o r l e s s c r y s t a l l i n e s o l i d ( 2 ) .

3.2

Melting Range

BROMAZEPAM

3.3

5

D i s s o c i a t i o n Constants
Bromazepam h a s t h r e e pKa v a l u e s o f 2.5, 5.2 and
11.8 corresponding t o p r o t o n a t i o n at t h e azomethine
and p y r i d i n e n i t r o g e n atoms, and d e p r o t o n a t i o n a t
t h e n i t r o g e n atom i n p o s i t i o n 1, r e s p e c t i v e l y .
These pKa v a l u e s were determined by s p e c t r a l and
polarographic a n a l y s i s ( 3 ) .

3.4

Crystal Structure

(4)

The c r y s t a l s t r u c t u r e of bromazepam w a s determined
u s i n g c r y s t a l s from amyl a c e t a t e - e t h a n o l o b t a i n e d
by slow e v a p o r a t i o n . The i n t e n s i t y d a t a w e r e
c o l l e c t e d on a n Enraf-Nonius CAD-4 d i f f r a c t o m e t e r ,
c r y s t a l s i z e 0 . 3 x 0.25 x 0.2 mm, c e l l dimentions
from s e t t i n g a n g l e s o f 25 r e f l e c t i o n s and g r a p h i t e
monochromated MoKa r a d i a t i o n . Bromazepam i s monoc l i n i c w i t h space groups P 2 l / c (non-centrosymetric)
The c e l l dimentions are a = 10.304 ( 4 ) , b = 15.897
( 5 ) , c = 8.122 ( 3 ) A', 6 = 106.8 (3)', U = 1273.6 AO,
Z = 4 , Dx = 1.649 Mg m-3, p(MoKa, A = 0.71069 A')
= 3.13 mm-1, F(000) = 6 3 2 , room t e m p e r a t u r e ,
R = 0.040 f o r 1470 observed r e f l e c t i o n s .

.

I n bromazepam t h e 7-membered r i n g i s i n a b o a t
conformat i o n . The a n g l e between t h e benzo-moeity
o f t h e 1,4-benzodiazepine system and t h e h e t e r o c y c l i c r i n g system i n t h e ? - p o s i t i o n i s 60.3 ( 6 ) ' .
F i n a l atomic parameters f o r bromazepam are l i s t e d i n
Table 1; bond l e n g t h s , bond a n g l e s and s e l e c t e d
t o r s i o n a n g l e s are l i s t e d i n Table 2. The atomic
numbering scheme i s i l l u s t r a t e d i n F i g . 1. Bond
l e n g t h s and a n g l e s g e n e r a l l y a g r e e w e l l w i t h v a l u e s
found i n analogous molecules ( 5 - 7 ) . The N ( l ) - C ( 2 )
formal s i n g l e bond i s s h o r t e n e d t o 1.351 ( 5 ) A' i n
bromazepam and t h e d i s p o s i t i o n o f bonds at N ( 1 ) i s
n e a r p l a n a r s o t h a t t h e geometry of t h e bond
resembles t h a t of a normal double bond ( c f . t o r s i o n
a n g l e s i n Table 2 ) ( 6 ) . The N ( 4 ) - C ( 5 ) l e n g t h i n
bromazepam corresponds t o t h a t o f a C = N bond and
t h e C ( 5 ) - C ( l ' ) and C ( 5 ) - C ( l l ) l e n g t h s are w i t h i n t h e
a c c e p t e d range (1.48-1.50 A') for a C(sp2)-C(sp2)
s i n g l e bond. There i s , t h e r e f o r e , no evidence f o r
any e l e c t r o n d e l o c a l i z a t i o n between t h e 5-pyridyl
r i n g i n bromazepam and t h e 1,4-benzodiazepine
system.

a,

c
0

w

c,

F

(ud

BROMAZEPAM

7

The seven-membered r i n g i s i n a c y c l o h e p t a t r i e n e l i k e b o a t conformation, C ( 1 0 ) and C ( 1 1 ) forming t h e
' s t e r n ' and C(3) t h e 'bow'.
The bow a n g l e s , 60.3
( 8O) i n bromazepam and t h e s t e r n a n g l e 30.8 (8' )

.

Table 1.

4

F r a c t i o n a l atomic c o o r d i n a t e s ( x 1 0 ) w i t h e . s . d . ' s
i n p a r e n t h e s e s and e q u i v a l e n t i s o t r o p i c t e m p e r a t u r e
f a c t o r s ( ~ x2 1 0 3 )

X

-

Y

-

617 (1)
-1404 ( 3 )
-528 ( 2 )
-2169 ( 2 )
-1209 ( 2 )
-1298 ( 3 )
-2004 ( 3 )
-1626 ( 5 )
-520 ( 3 )
237 ( 3 )
726 ( 3 )
449 ( 3 )
-324 ( 3 )
-821 ( 2 )
-1841 ( 3 )
-1400 ( 3 )
-2190 ( 3 )
-2820 ( 4 )
-2650 ( 3 )

Z

-

U
eq

184
177
94

102

95

110

163
86

101
104

108
119
78
75
79
110

126
125
99

The major conformational d i f f e r e n c e between t h i s
compound and o t h e r known 5-phenyl-1,4-benzodiazep i n e s i s i n t h e o r i e n t a t i o n of t h e 5-aryl r i n g . The
a n g l e between t h e mean p l a n e o f t h e 5-aryl r i n g and
t h e 'benzo' p l a n e i s 60.3 (6') i n bromazepam and
75.5 (9') i n o t h e r benzodiazepines ( 8 ) .

8

MAHMOUD M. A. HASSAN AND MOHAMMAD A. ABOUNASSIF

Table 2. Molecular dimensions
(b) Bond angles
(")

1.351

1.214

1.453
1.278
1.483
1.394
1 371

119
113
127.1
122.1
114.6
123.3
111.3

-

1.376

1.362
1 397
1* 395

(31

(3)

(4)
(5)
(4)
(4)

(4)

117.4 ( 4 )
126.1 ( 4 )

116.3 (4)

1.402

117.6 (3)

1.892

-

121.3
120.3
119.8
119.8

-

-

(4)

(4)
(4)
(3)

L

1.378 ( 6)

-

C ( 11) -C( 10)-N( 1)
C( 10)-c( 11)-c( 6)
c( 1O)-C( ll)-C( 5)
C( 6 ) -c( 11) -c( 5 )
C( 5 ) -C( 1' ) -N( 2 ' )
c(5 -C(l')-C(2')

c(5 -c(if)-c(6I)

119.5
121.2
119.5
116.9
123.5
118.1
112.8
119.0
117.9

-

(4)

(4)
(4)

(4)

(4)
(4)

(4)
(4)
(4)

120.7 ( 4 )

c(2 )-c( 1'1-c( 6')
N ( 2 )-c(it)-c(6') 1 2 2 . 3 (4)
C( 2 )-C( 2')-C( 3') 116.8 ( 4 )
C( 1 ) -C( 2')-C( 3')
C( 1 ) - C ( 2')-F
C( 3 ' ) -C( 2 ' ) -F
c( 2 ' )-C(3')-C(4'
N( 2')-C( 3' )-C( 4 ' ) 124.4 (5)
c( 3' )-c( 4 ' )-c( 5 ' ) 117.8 (5)
C( 4 ' )-C( 5')-~(6') 119.2 ( 5 )
C( 5' 1-C( 6 ' 1-C( 1') 1 1 9 . 4 ( 5 )

BROMAZEPAM

(c)

9

Selected torsion angels

C( l O ) - N ( 1 ) - C ( 2)-C( 3)
N ( l ) - C ( p ) - C ( 3)-N(4)
c(2)-C(3)-N(b)-C( 5 )
C ( 3 ) - N ( 4 ) -C( 5 ) -C( 11)
N ( 4 ) - C ( 5 ) -C( ll)-C( 10)
C ( 5 ) - C ( 11)-C( 10)-N( 1)

4.

(O);

-1.1

-70.3

72.3

-2.7
-37.6
-1.2

e.s.d.'s

ca

0.6'

C(ll)-C(lO)-N(l)-C(2)
39.8
4 ) - C ( 5 ) -C( 1' )-N( 2 ' ) 141.6
4 ) -C( 5 ) -C( 1' ) -C( 2 ' )
C( 11)-C( 5 ) - C ( 1' ) -N( 2 ' ) -39.8
C( I l ) - C ( 5)-C( 1' )-C( 2 ' )
-

N(
Nf

Spectral Properties

4.1

U l t r a v i o l e t Spectrum
The u l t r a v i o l e t spectrum o f bromazepam i n methanol
i s shown i n F i g . 2. The s p e c t r a of bromazepam i n
1 N NaOH and 1N H C 1 are p r e s e n t e d i n F i g . 3 and Fig.
4 , r e s p e c t i v e l y . The
and maximum wavelengths
cm
are given i n Table 3 ( 9 ) . These v a l u e s a g r e e s w i t h
t h e p u b l i s h e d d a t a (10-12).
Levillain (13), has
s t u d i e d t h e r e l a t i o n s h i p of s t r u c t u r e and t h e W
a b s o r p t i o n c h a r a c t e r i s t i c s of a s e r i e s of 1,4-benzod i a z e p i n e s , i n c l u d i n g bromazepam, c o n s i d e r i n g t h e
electronic distribution of t h e various substituent s
and t h e s t e r e o c h e m i s t r y . The spectrum of bromazepam
i s c o n s i s t e n t w i t h t h a t o f o t h e r benzodiazepines w i t h
s i m i l a r s t r u c t u r e . Als o bromazepam was i d e n t i f i e d
i n s o l i d dosage forms by W a b s o r p t i o n spectrometry

q%

(14).

Table 3.
Solvent
Methanol

1 N NaOH

1N HC1

UV S p e c t r a l c h a r a c t e r i s t i c s

max ( n m )
320

258

1%

cm

54.54

233

398.08
1004.78

350
268
238

493.77
794.25

348

270
238

59.33

39 * 23
386.60
552.15

The above v a l u e s a g r e e s w i t h t h a t of bromazepam.

90 80 70 60 M 40 3a 2G 10 .

r( i* .-C N n h P 1cI 0 E e a V CI 5 0. 3 c w m M .11 z z 4 3 .

. 250 The U V s p e c t r u m of bromazeparn in IN HCI. 4..91 81 7( 6t 5( 4 f 200 Fi:.

16. . 1200. 890.3150 .2 I n f r a r e d Spectrum An i n f r a r e d a b s o r p t i o n spectrum o f potassium bromide d i s p e r s i o n o f bromazepam (Roche .1590. 2 .1570.Reference Standard Lot 0401055) i s shown i n F i g . 1600. 1230. 5. 1340. Table 4. 1040.17) l i s t e d i n Table 4 .h-Trisubst i t u t e d aromatic and o . Bromazepam was i d e n t i f i e d i n s o l i d dosage forms by I R a b s o r p t i o n spectrometry (14).4-tris u b s t i t u t e d aromatic Out o f p l a n e CH deformat i o n of o r t h o . 1385. 1090. 1000. A l s o 2-substituted pyridine. 990.d i s u b s t i t u t e d aromatic.2940. 1320. The s p e c t r a l band assignments (9.2.14~0 Aromatic C = C s t r e t c h 815 Out o f p l a n e CH-deformat i o n of 1. Other c h a r a c t e r i s t i c bands a r e 1440. I n f r a r e d h a s been used f o r i d e n t i f i c a t i o n o f bromazepam w i t h o t h e r psychotropic drugs ( 1 8 ) . 3080 N-H stretch 3000. I n f r a r e d s p e c t r a l assignments o f bromazepam Wave number (em-') Assignment (Vibration m o M 3220.d i s u b s t i t u t e d aromatic 1 . and 790 em-1.A 4.2860 C-H stretch 1695 C = 0 stretch 1615 C = N s t r e t c h of both benzodiazepine and pyridine.

.

The sample w a s d i s s o l v e d i n DMSO d6 and t h e spectrum was recorded on a 400 MHz NMR spectrometer.22 7.3.23 7. using TMS as an i n t e r n a l r e f e r e n c e standard.41 7. The proton assignments are l i s t e d i n Table 5.1 'H-NMR Spectrum A t y p i c a l 'H-NMR spectrum o f bromazepam i s shown i n Fig.49 7.15 BROMAZEPAM 4.irig 4. H Table 5. NMR spectrum recorded on a Jeol-FX-100 90 MHz s p e c t r o x e t e r i s a l s o shown i n Fig 6a.86 8.57 (s) (d) (s) (m) (m) (m) (d) (d) 10. Proton p o s i t ion c-3 c-9 C-6 c-5' C-8 c-4' PMR c h a r a c t e r i s t i c s of bromazepam Group -CH2 -9H -6H -5'H( -8H -4'H( -3'H( -6'H( (Benzenoid) ( " " ) F'yridine ) (Benzenoid) Fyridine) c-3' Fyridine) c-6' Pyridine) N-1 .95 8.l H (Diazepine) (disappeared a f t e r D20 exchange ) s = singlet d = doublet Chemical s h i f t ppm and split?. The spectrum a f t e r a d d i t i o n of D20 i s shown i n F i g . 6.44 7. 7.3 Nuclear Magnetic Resonance S p e c t r a 4.66 (s) rn = m u l t i p l e t .

I Lr .

7 . 'H-NYR Fig. 6s. 17 spectrum of bromazepam i n DMSO d6 and TMS. 'H-NMR spectrum of bromazepam i n DMSO d6 and TYS after DzO t=xchsngr. .BROMAZEPAM F i g .

3. 127. on J e o l FX-100 90 MHz i n s t r u m e n t . ABOUNASSIF 18 4.24 ( d ) 122. Carbon No.63 ( s ) c-9 c-6' c-5: c-4 c-3' s = singlet d = doublet Chemical s h i f t 6 (ppm) 123.72 ( s ) c-5 56.81 ( s ) t = triplet .50 ( d ) C-8 133. 100 mg/ 1 m l ) .94 ( s f c-3 C-5a Carbon No. 0 9 Table 6.49 ( s ) 155. r e s p e c t i v e l y . and F i g . Sample t u b e 1 0 mm and t e t r a m e t h y l s i l a n e as a n i n t e r n a l r e f e r e n c e s t a n d a r d a t 2OoC were used. 9 .28 ( s ) 133.34 ( d ) 148. Both were recorded over 5000 Hz range i n d e u t e r a t e d dimethyl s u l f o x i d e (DMSO d 6 ) . 8.96 ( d ) 124.55 ( s ) C-6 c-7 113. HASSAN AND MOHAMMAD A. ( c a n e . The carbon chemical s h i f t s a r e a s s i g n e d on t h e b a s i s o f t h e chemical s h i f t t h e o r y and t h e off-resonance s p l i t t i n g p a t t e r n ( T a b l e 6 ) . A.89 ( t ) 167. c-2 5' Carbon chemical s h i f t s of bromazepam Chemical s h i f t 6 (ppm) 169.MAHMOUD M.2 13C-NMR Spectra 13C-NMR noise-decoupled and off-resonance s p e c t r a of bromazepam are shown i n F i g .79 ( d ) C-la c-2' 138.87 ( a ) 136.

19 BROMAZEPAM Fig. 9 . Pig. 13C-NMR off-resonance spectrum of bromazepam in DUSO and TUS. . . “C-NMR noise-decoupled Spectrum o f bmmazepam i n OMS0 and TUS. 8 .

The i o n i z i n g e l e c t r o n beam energy w a s at 70 eV.33 289 33. It shows a molecular i o n !d+ a t m/e 316 ( r e l a t i v e i n t e n s i t y 34.09 287 35.88 236 316 34.MAHMOUD M.87) and M+ + 1 a t m / e 317 ( r e l a t i v e i n t e n s i t y 63.70 207 30 37 314 26. 11. HASSAN AND MOHAMMAD A.00 ( b a s e Peak) 315 60.87 208 100. A.95 90 35. The most prominent fragments of bromaz epam m/e Relative i n t e n s i t y m/e Relative i n t e n s i t y 317 63.89 288 59.54 286 52.28 78 31 90 259 19.84 79 19.4 Mass Spectrum The E l mass spectrum o f bromazepam (Roche Reference Standard.84 206 23. 10.67 43. ABOUNASSIF 20 4. Lot 0401055) was o b t a i n e d on a Finigan 1020 quadrupole mass spectrometer and shown i n Fig. I t s GC t r a c e i s shown i n Fig.08 51 25.69 261 19.56 - - .95 104 21.88).36 77 24. Some of t h e most prominent i o n s a r e given i n Table 7 (9). Table 7.24 179 30.

F i g . 10. EI-mass spectrum of bromazeparn. .

ABOUNASSIF 22 5.MAHMOUD M. A. HASSAN AND MOHAMMAD A.HC1 2 2 p y r i d i n e /ref lux I 0 . Synthesis D i f f e r e n t r o u t e s f o r t h e s y n t h e s i s o f bromazepam were r e p o r t e d (19-21). Route I Br [11 H CH NH COOC2H5.

BROMAZEPAM 23 2-(2-Amino-5-bromobenzoylpyridine [ 41 was prepared from 2-phenacylpyridine p-bromophenyl hydrazone [ 11 through cyclization with concentrated hydrochloric acid to give 5-bromo-2-phenyl-3-( 2-pyridy1)indole [ 21. . A mixture of [4].4-benzodiazepines. This compound was oxidized with chromium trioxide to give 2-(2-benzamido-5-bromobenzoy1)pyridine [3] ( 2 9 . .24). Br2 H I [5 1 in glacial acetic acid I \ / V 0 [41 Alternatively. glycine ethyl ester hydrochloride and pyridine was refluxed to give bromazepam [ 5 ] . the key intermediate compound [4] was prepared by bromination of 2-(2-aminobenzoyl)pyridine [6] with bromine and glacial acetic acid (23. 2 2 ) which on hydrolysis with concentrated hydrochloric acid afforded the desired compound 2( 2-amino-5-bromobenzoy1)pyridine [4] This compound is considered to be the starting material for the synthesis of 1.

ABOUNASSIF 24 Route 3 H O BrCOCH2Br H Br Heat -6 [ 51 Bromazepam I n t h i s r o u t e 2-(2-aminobenzoyl)pyridine [ 41 w a s t r e a t e d with bromoacetyl bromide in g l a c i a l a c e t i c a c i d .MAHMOUD M. A. The crude bromoacetyl d e r i v a t i v e [ 71 w a s converted d i r e c t l y t o bromazepam [ 5 ] by r e a c t i o n with ammonia through t h e intermediate aminoacetamidobenzoyl p y r i d i n e (20). HASSAN AND MOHAMMAD A. [a] .

T h i s compound i s prepared a c c o r d i n g t o t h e method of Sheehan and Hess d e v i s e d f o r t h e s y n t h e s i s o f p e p t i d e s ( 2 5 . The methylene c h l o r i d e s o l u t i o n a f t e r removal of N. 2-( 2-aminoacetamido-5-bromobenzoy1)pyridine.N'-dicyclohexylurea w a s c o n c e n t r a t e d i n vacuo a t room t e m p e r a t u r e . A m i x t u r e of t h e 2-(2-amino-5-bromobenzoyl)pyrid i n e and carbobenzoxyglycine i n methylene c h l o r i d e w a s added. .a L I CH3COOH [ 51 An a l t e r n a t i v e method (20)f o r t h e s y n t h e s i s of bromazepam proceeded v i a t h e carbobenzoxyglyclyl d e r i v a t i v e [ 9 ] . Cleavage o f t h e carbobenzoxy group w i t h hydrogen bromide i n g l a c i a l a c e t i c a c i d (27) gave bromazepam [ 5 ] d i r e c t l y without i s o l a t i o n of t h e i n t e r m e d i a t e . 2 6 ) .BROMAZEPAM Route 25 4 CH2 0- \ -/ [41 H-N H c o \ - QI IC Br Br ' - 4 . The r e s i d u e w a s d i s s o l v e d i n benzene and chromatographed t o g i v e [ g ] .

A.MAHMOUD M. HASSAN AND MOHAMMAD A. ABOUNASSIF 26 Route 5 Clarke e t a1 (1980) (28) have described i n d e t a i l s t h e p r e p a r a t i o n of bromazepam [ 5 ] from t h e corresponding benzophenone by t h e use of hexamine and hydrochloric a c i d as o u t l i n e d below: H Br / t - + 3MeC6H12N4C1 t NH4C1 t 3HC02Me .

27 .BROMAZEPAM Three main by-product were i d e n t i f i e d : two i m i d a z o l i d i nones [lo]. [ll] and a n aminobenzodiazepine ( 1 2 ) .

but i t s e x c r e t i o n by r a t s was v e r y l i m i t e d . T h i s i s t h e f i r s t r e p o r t e d i n s t a n c e o f N4-oxidation o f a 1. The i s o l a t e d u r i n a r y m e t a b o l i t e was assumed t o be a 3-hydroxylated d e r i v a t i v e o f 2-amino-5-bromobenzoylpyridine [ 1 5 ] . OH The b i o t r a n s f o r m a t i o n of bromazepam.MAHMOUD M. r a t and mouse ( 3 1 ) . m e t a b o l i t e [ 1 5 ] and 2-( 2amino-5-bromobenzoy1)pyridine [ 4 ] were d e t e c t e d i n t h e e x c r e t a o f t h e human.1 4 C were t h e conjugated forms of 3hydroxy bromazepam [13] accounting f o r 13-30% of t h e dose. Four m e t a b o l i t e s w e r e i d e n t i f i e d and t h e i r e x c r e t i o n w a s q u a n t i t a t i v e l y determined. was s t u d i e d i n f o u r s p e c i e s namely human. dogs but not i n r a t s f i t s t h e same p a t t e r n of o t h e r lY4-benzodiazepine-2-one i n a f f o r d i n g conjugated 3-hydroxylated d e r i v a t i v e (32). The i d e n t i f i c a t i o n and q u a n t i t a t i o n of t h e d i f f e r e n t m e t a b o l i t e s were c a r r i e d o u t by t h i n l a y e r chromatography u s i n g s e v e r a l s o l v e n t systems. The s t r u c t u r a l e l u c i d a t i o n w a s based on e l e m e n t a l a n a l y s i s . t h e Nq-oxide [14] was a minor m e t a b o l i t e i n dog u r i n e . Conjugated [131 w a s a l s o t h e major m e t a b o l i t e i n dogs and mice a d m i n i s t e r e d 14C-bromazepam. Metabolism The f i r s t r e p o r t e d m e t a b o l i c work on bromazepam w a s t h a t published by Sawada i n 1972 ( 2 9 . T h i s s t u d y w a s c a r r i e d o u t b o t h i n dogs. mass spectrometry and n u c l e a r magnetic resonance s t u d i e s . r a b b i t s and r a t s . . 3 0 ) . This s t u d y has shown t h a t t h e metabolism o f bromazepm i n human. A. ABOUNASSIF 28 6. Together w i t h [13]. dog.4-benzodiazepine-2-one. r a t and mouse. dog. Although t h e p y r i d y l N-oxide [I81 d e r i v a t i v e o f bromazepam w a s not d e t e c t e d as a m e t a b o l i t e i n any s p e i c e s . The major u r i n a r y m e t a b o l i t e s e x c r e t e d by t h r e e humans given s i n g l e 1 2 mg o r a l doses of bromazepam-5. and 2-( 2-amino-5-bromo-3-hydroxybenzoyl)pyridine [15] accounting f o r 4-25% of t h e dose. HASSAN AND MOHAMMAD A.

( 6' -N-ac etylL-cystein-S-y1)-pyridyl] -2H1.3-dihydro-5-(2pyridyl)-2H-1.12 190-196O 16 Bromazepam mercapturic acid methylester( 7-bromo-1. 293. Table 8 shows the chemical names and physical properties of bromazepam and its major metabolites.3-dihydro-5-[2'-( 6I-methylthio)pyridyl]-2H-1. Isolation of the mer- .h-benzodiazepin-2-one 4-oxide. Chemical names and physical properties of bromazepam and metabolites No.2-one (methylthiobromazepam) [17]. 5 Chemical name 7-Bromo-1.12 97* 5-99O 15 2-Amino-5-brom0-3-hydroxybenzoylpyridine.4-benzodiazepin .4-benzodiazepin-2-one (bromazepam) Molecular weight Melting point 316. 277.2 263O dec.16 237438.3-dihydro-5-(2pyridyl)-2H-1.50 dec - 13 . 228 - 17 18 Met hylthiobromazepam Pyridyl N-oxide of bromazepam - - Taleishi and Shimizu 1976 (34) has recently reported the isolation of a methylthio-containing metabolite which was identified as 7-bromo-l. Also they have shown that the methionine donor theory (34) is very unlikely in the case of the formation of methylthiobromazepam. 4 2-Amino-5-bromobenzoylpyridine.4-benzodiazepin-2-one.BROMAZEPAM 29 de Silva et al. Table 8.3dihydro-5. 1974 (33) have reported a sensitive and specific electron-capture GLC assay for bromazepm and its major metabolites (see under methods of analysis).[ 2 ' . 332 2 198-2ooo 14 7-Bromo-1. 332.

I 0 01 e Y Y M E .

4-benzodiazepin-2-one (bromazepam rnercapturate methylester) was synthesised from bromazepam "-oxide and methyl N-acetyl-L-cysteinate in acetic anhydride according to the pyridine-N-oxide .3-dihydro-5-[2'(1' -ox0 ) -pyridyl 3 -2H-1.N-acetylL-cysteinate-5-yl)-pyridyl]-2H-1. its identification and subsequent conversion of the mercapturic acid to methylthiobromazepam in rat liver preparation was reported (35). 7-Bromo-l.BROMAZEPAM 31 capturic acid conjugate of bromazepam from bile of rat given the drug. [5. H H I O II CH3 [51 ' COOH I H H Bromazepam mercapturic acid I [MeY 14 C -SAM] Br [I71 : S .C] bromazepam and bromazepam-N'-oxide ('j'-bromo-1.( 6'-methyl . Scheme 1 shows a proposed pathway for the biotransformation of bromazepam to methylthiobromazepam in the rat.2-one were employed. 14 In this study non-radioactive bromazepam.3-dihydro-5-[ 2 I .h-benzodiazepin . A proposed pathway f o r the biotransformation of bromazepam to methylthiobromazepam in the rat.@ 14CH3 Scheme I.

ABOUNASSIF 32 n u c l e o p h i l i c s u b s t i t u t i o n d e s c r i b e d by Bauer and Dickerhofe ( 3 6 ) . The r e s u l t s i n d i c a t e d t h a t 6'-methylthiobromazepam i s o l a t e d p r e v i o u s l y i n t h e r a t u r i n e i s formed a t l e a s t i n p a r t v i a t h e m e r c a p t u r i c a c i d and t h a t rat l i v e r c o n t a i n s enzyme(s) capable of c a t a l y s i n g t h e conversion from t h e mercapturic a c i d t o methylthiobromazepam. A. Chemical r e a c t i o n s o f bromazepam and i t s known m e t a b o l i t e s are shown i n Scheme 2. HASSAN AND MOHAMMAD A. . Chemical names and p h y s i c a l p r o p e r t i e s of bromazepam and i t s m e t a b o l i t e s are l i s t e d i n Table 8.MAHMOUD M. H OH I I1 I ""3 COOH Bromazepam mercapturic acid 0 [I71 Scheme 2. Chemical r e a c t i o n s of bromazepam and i t s known m e t a bolites.

peak plasma l e v e l s b e i n g reached about an hour a f t e r o r a l a d m i n i s t r a t i o n . 13 f o r i n t r a v e n o u s and o r a l a d m i n i s t r a t i o n a r e p r a c t i c a l l y i d e n t i c a l . Bromazepam i s mainly e l i m i n a t e d i n t h e u r i n e . The important pharmacokinetic parameters have been c a l c u l a t e d from t h e plasma c o n c e n t r a t i o n . ~ 1 ( B ) = 1 9 . F i g .l i f e = 1 8 . The a s s a y can b e used a l s o for r o u t i n e plasma l e v e l monitoring. compared w i t h 22. u n l i k e most o t h e r benzodiazepines. T h i s i s c o n s i d e r a b l y l e s s t h a n f o r diazepam and most o t h e r benzodiazepines.gl/kgm following s i n g l e o r a l However. Absorpt i o n and d i f f u s i o n o f t h e s u b s t a n c e t a k e s p l a c e r a p i d l y . 5 hours 3 mg/day.BROMAZEPAM 33 Pharmacokinetic S t u d i e s D i s t r i b u t i o n and e l i m i n a t i o n of bromazepam h a s been s t u d i e d i n 4 human s u b j e c t s f o l l o w i n g s i n g l e o r a l and i n t r a v e n o u s doses o f 6 mg 14C-bromazepam ( 3 7 ) . C1 = t o t a l T 2 (B) body plasma c l e a r a n c e = 347 m l / m i n and VdR = a p p a r e n t volume of d i s t r i b u t i o n = O. r e c t i l i n e a r p a t t e r n which i s p r a c t i c a l l y completed a f t e r 120 hours. u s i n g m u l t i p l e dosing w i t h dose o f 6 mg/day. E x c r e t i o n o f t h e m e t a b o l i t e s c l o s e l y p a r a l l e l s t h a t of t h e unchanged s u b s t a n c e .l i f e f o r t h e e l i m i n a t i o n of bromazepam from t h e plasma is 2 0 . 5 hours. . and i s a t t r i b u t e d t o t h e i n c r e a s e d p o l a r i t y of t h e molecule due t o t h e p r e s e n c e o f t h e pyridyl radical (38). excret i o n f o l l o w s a r e g u l a r . 1976 (40) have r e p o r t e d plasma c o n c e n t r a t i o n s and t i m e p r o f i l e s which i n agreement w i t h t h e above mentioned work. 1 hours following i n t r a v e n o u s a d m i n i s t r a t i o n . which showed t h a t about 70% of t h e substance p r e s e n t i s bound. it w a s found t h a t T 2 = 243 ml/min and Vdg = 071/kg. Binding o f bromazepam by plasma p r o t e i n s w a s s t u d i e d by e q u i l i b r a t i o n d i a l y s i s . H a l f . and. 1 - = e l i m i n a t i o n h a l f . Another work d e s c r i b e d t h e a p p l i c a t i o n of gas l i q u i d chromatography f o r pharmacokinetic s t u d i e s i n man ( 3 9 ) . Kaplan e t a l .5 h o u r s f o r t o t a l r a t i o a c t i v i t y (unchanged bromazepam p l u s m e t a b o l i t e s ) . 1 2 and F i g .

MAHMOUD M. 13 Bromazepam + metabolites Fig. 13. Fig. injection o f 6. 12.0 mg..... ABOUNASSIF 34 Fig. HASSAN AND MOHAMMAD A. . 12 -Bromazepam .V. A. 14C-Bromazepam in plasma (pg/ml) after oral administration of 6.0 mg. Fig. 14C-Bromazepam and total radioactivity (ug/ml) in plasma after I..

e. : Ethyl a c e t a t e .1 Elemental h a l y s i s The elemental composition of bromazepam i s Element % Theoretical C 53.BROMAZEPAM 8.06 I d e n t i f i c a t i o n Tests 8.1 Color Reactions Reported c o l o r r e a c t i o n s for bromazepam a r e as follows ( 4 1 ) : Diazocoupling React i o n with 1.3dinitrobenzene In In 1 M N a O H 2 M HC1 I n DMSO (CH3)bN OH Bromaze- Pam Orange red Red v i o l e t Yellow Yellow Orange brown i 8.conc. MERCK pre- coated p l a t e s 0.25 mm. Methods of Analysis 8.18 H 8.2 Thin-Layer Chromatogram (42) Layer : S i l i c a g e l F254. ammonia (100 + 1) ( p r e p a r e f r e s h l y ) without s a t u r a t i o n o f t h e chamber atm0sphel.2.2.2 3-19 Br 25.28 N 13 * 29 0 5. Mobile phase .

A. Rf-value : Bromazepam approx. Spray t h e d r i e d p l a t e w i t h a f r e s h l y prepared s o l u t i o n of f e r r o u s s u l f a t e . d r y f o r a s h o r t moment and spray t h e n w i t h an ammonia (1%): Bromazepam appears as a v i o l e t spot. Observe under short-wave W-light: decrease o f t h e fluorescence through bromazepam. 3 min and f i l t e r . Front d i s t a n c e : 1 2 cm Detect i o n : 1. c r y s t a l s i n t h e form of s m a l l t a b l e t s were obtained a f t e r one hour ( F i g . The drop being s t i r r e d w i t h a s l i g h t s c r a t c h i n g of t h e g l a s s t o promote t h e formation of c r y s t a l s . HASSAN AND MOHAMMADA. ABOUNASSIF 36 Application : 1 0 p l of each s o l u t i o n (sample and comparison) Sample s o l u t i o n : Shake 400 mg of t a b l e t powder w i t h 5 ml of chloroform for approx.2. Bromazepam comparison solut i o n : Dissolve 60 mg of bromazepam i n 25 m l of chloroform. 7H20 solution i n 1 0 0 m l of d i s t . 0. 14a). 8. 2.MABMOUD M. .6 Ferrous s u l f a t e : Dissolve 1 g of FeS04.3 Microcrystal T e s t s Bromazepam w a s i d e n t i f i e d i n s o l u t i o n by m i c r o c r y s t a l t e s t s i n microgram q u a n t i t i e s (43) * A micro drop of t h e t e s t s o l u t i o n ( i n 50% e t h a n o l ) w a s placed on t h e g l a s s cover s l i p and a micro drop of t h e reagent was added. With potassium cadmium i o d i d e . The appearance of t h e c r y s t a l s w a s recorded as follows: 1. water.

1 Non-aqueous T i t r a t i o n Among s e v e r a l 1. compounds having a b a s i c d i p y r i d y l t y p e bond s t r u c t u r e which has e x c e l l e n t metal complexing p r o p e r t i e s .4 Spectrophotometric Unlike o t h e r 1. bromazepam w a s t i t r a t e d with p e r c h l o r i c a c i d i n dioxane or tetrabutylammonium hydroxide i n benzene-methanol ( 4 3 ) . white on black back ground were formed a f t e r one hour ( F i g . one of i t s p r i n c i p a l m e t a b o l i t e s . With gold bromide hydrochloride. 2 . -N \c - HN- C The method has been a p p l i e d ( 4 6 ) t o t h e determination o f bromazepam i n b i o l o g i c a l m a t e r i a l s by adding methanol s o l u t i o n of f e r r o u s c h l o r i d e t o t h e bromazepam obtained a f t e r e x t r a c t i o n from blood o r u r i n e and measuring t h e absorbance of t h e s o l u t i o n a t 580 nm. Sabatino -e t a 1 ( 4 5 ) reported t h a t f e r r o u s i o n s form purple complexes with p y r i d y l benzodiazepin-2-ones.h-benzodiazepine d e r i v a t i v e s .37 BROMAZEPAM 2.b-benzodiazepines.3 Titrimetric 8. a ' . The procedure w a s a l s o a p p l i e d t o t h e determination o f bromazepam i n r a t blood ( 4 7 ) .d i p y r i d y l t y p e moiety enables bromazepam t o form complexes w i t h d i v a l e n t metal i o n s .3. 1 4 b ) . . c r y s t a l s i n t h e form of round t r i f e r c a t e b l a c k i s h s t r u c t u r e s . t h e e x i s t e n c e of t h e a. 14c ) . c r y s t a l s i n t h e form of flowers of d e n d r i t e s were obtained i n s t a n t l y ( F i g . The s e n s i t i v i t i e s o f t h e t e s t s were 0 . 8. 0. The method proved t o be s p e c i f i c for bromazepam w i t h no i n t e r f e r e n c e from 2-amino-5-bromobenzoylpyridine. With potassium mercuric i o d i d e . 1 microgram r e s p e c t i v e l y .5 and 0 . 8. 3.

14c. Microcrystals of bromazepam with potassium mercuric iodide. Microcrystals of bromazepam with gold bromide hydrochloride. . Microcrystals of bromazepam with potassium cadmium iodide. 14c Fig. 14b. 14a Fig. Fig. 14b Fig. Fig.Fig. 14a.

1 N methanolic s u l f u r i c a c i d (= test solution). as w e l l as some o t h e r benzodiazepines.A E = e x t i n c t i o n o f t h e sample s o l u t i o n A = average weight (mg) B = weight of sample (mg) 300 = E ( 1 % . pKa v a l u e s and mechanism o f h y d r o l y s i s were a l s o r e p o r t e d u s i n g W spectrophotometry ( 3 ) .BROMAZEPAM 39 Smyth e t a1 (48) s t u d i e d t h e c h e l a t i o n o f bromazepam w i t h F e ( I I ) . Calculation: mg of bromazepam t a b l e t . D i l u t e t o volume w i t h 0 . C o l o r i m e t r i c d e t e r m i n a t i o n o f bromazepam. Hoffnann . Add about 200 m l of 0.0 m l o f t h e c l e a r f i l t r a t e t o 100.v i s . Another W-spectrophotometric method used by F. Method. 1 N methanolic s u l f u r i c a c i d . spectrophotometry. 1 cm)-value of bromazepam a t 285 nm (maximum) i n 0. and Co(I1) u s i n g s p e c t r o s c o p i c t e c h n i q u e s such as W .1 N methanolic s u l f u r i c a c i d . 1 N methanolic s u l f u r i c a c i d as a b l a n k u s i n g 1 em-quartz c e l l s . o f Analysis: With a spectrophotometer measure t h e e x t i n c t i o n o f t h e t e s t s o l u t i o n a t 285 nm (max. = E.0 g o f 98% s u l f u r i c a c i d t o 1000 m l w i t h methanol.) a g a i n s t 0 .B .L a Roche & Co Limited i s as f o l l o w s : ( 5 0 ) Assay: The d e t e r m i n a t i o n should b e c a r r i e d o u t w i t h i n one hour under subdued d a y . Methanolic s u l f u r i c a c i d : D i l u t e 5.l i g h t . w a s a l s o performed a f t e r r e a c t i o n w i t h a modified Marquis's r e a g e n t ( 4 9 ) . F i l t e r a part of t h i s solution (discard t h e first 1 5 m l ) and d i l u t e 10. 25000 300 .0 m l w i t h 0 . P r e p a r e t h i s s o l u t i o n a t l e a s t 24 hours b e f o r e u s e . P u l v e r i z e 20 t a b l e t s and a c c u r e t e l y weigh approximat e l y 1000 mg of t a b l e t powder i n t o a 250 ml-volumetric f l a s k . .1 N methanolic s u l f u r i c a c i d and shake f o r 1 5 min o r t r e a t w i t h u l t r a s o n i c s . C u ( I I ) .

whereas t h e P e z due due t o t h e r e d u c t i o n o f t h e c a r b o n y l group o f t h e m e t a b o l i t e s 2-amino-5-bromobenzoylpyridine and 2-amino-3-hydroxy-5-bromobenzoylpyridine occured a t -0. Trace l e v e l s of bromazepam i n blood w a s determined by d i f f e r e n t i a l p u l s e polarography ( 5 1 ) . 7 D e S i l v a et a1 ( 3 3 ) d e s c r i b e d a d i f f e r e n t i a l p u l s e polarographic method f o r t h e d e t e r m i n a t i o n o f bromazepam and i t s m e t a b o l i t e s . The method has a recovery o f 62. Polarography w a s c a r r i e d o u t u s i n g phosphate b u f f e r as t h e s u p p o r t i n g e l e c t r o l y t e w i t h pH o f 5. 2-amino-3-hydroxy-5-bromobenzoylpyridine and 2-amino5-bromobenzoylpyridine i n u r i n e . due t o t h e r e d u c t i o n o f t h e azomethine group o f bromazepam and i t s 3-hydroxym e t a b o l i t e occured a t -0.8 i n t h e r a n g e of 10-1000 ng bromazepam/l ml o f blood.630 and -0.535 and -0. and t h e ( > C = 0 ) carbonyl group o f i t s benzophenone m e t a b o l i t e s .1% 7.635 V v e r s u s calomel e l e c t rode. The peak p o t e n t i a l . HASSAN AND MOHAMMAD A.555 V v e r s calomel e l e c t r o d e r e s p e c t i v e l y .MAHMOUD M.5-azomet h i n e bond o f bromazepam w a s measured a t -0.5 Polarographic The ease of r e d u c t i o n of t h e azomethine ( > C = N4) group o f bromazepam and i t s 3-hydroxy metabo i t e . 15). and f o r t h e i d e n t i f i c a t i o n o f any one o r more of several 1 . 4 benzodiazepines i n c l u d i n g bromazepam ( 5 2 ) .5. r e s p e c t i v e l y ( F i g .6 Atomic Absorption Gonzales-Perez e t a1 ( 5 3 ) d e s c r i b e d a method f o r t h e d e t e r m i n a t i o n o f bromazepam by atomic a b s o r p t i o n spectrophotometry. A. * Polarography w a s a l s o used t o s t u d y acid-base and complexing behaviour o f bromazepam ( 3 ) .p a i r formed by t h e bromazepamn i c k e l (11) c a t i o n i c c h e l a t e and p e r c h l o r a t e from aqueous t o m e t h y l i s o b u t y l k e t o n e (MIBK) s o l u t i o n s . The method i s based on t h e e x t r a c t i o n o f t h e i o n . S e n s i t i v i t y l i m i t s ranged between 50 and 1 0 0 ng/5 ml u r i n e . Ep. 8. promotes t h e i r q u a n t i t a t i o n i n t h e subnanogram range by d i f f e r e n t i a l p u l s e polarography. . The c u r r e n t r e s u l t i n g from r e d u c t i o n o f t h e 4.610 V v e r s u s s i l v e r c h l o r i d e e l e c t r o d e . 3-hydroxy-bromazepam. ABOUNASSIF 40 8.

5 phosphate buffer as the supporting electrolyte. authentic standard mixture.750 POTENrlAL VOLTS VERSUSSATURATED CAZOMELELECTROOE Cb) Fig. I.0. Differential pulse polarograms of: (a) I and I 1 (b) I 1 1 and IV in 1. control urine blank. 111. Bromazepam 11. IV. 2-Amino-5-bromobenzoylpyridine 3-Hydroxymetabolite of bromazepm 2-Amino-3-hydroxy-5-brombenzoylpyridine . authentic recovered from urine. B.BROMAZEPAM 41 I . Key: A. and C.350 I I -0550 -0. 15.0 M pH 5.

G.MAHMOUD M. The method i s based upon competit i o n between [ 3H]flunitrzepam and b i o l o g i c a l l y a c t i v e benzodiazepines i n b i o l o g i c a l f l u i d s f o r brain-specific receptors.1 Paper Chromatography Bromazepam w a s s e p a r a t e d from o t h e r benzodiazepines by u s i n g a t h i n l a y e r system: Merck aluminium o x i d e F254 and CHC13 . A r a d i o r e c e p t o r a s s a y f o r benzodiazepines i n c l u d i n g bromazepam i n human blood. The method t h a t showed a d e t e c t i o n l i m i t o f 2 X 10-6M.PhMe . 8. The s p o t s were l o c a t e d by s h o r t W wavelength and a c i d i f i e d potassium i o d o p l a t i n a t e ( 57 ) . s a l i v a and u r i n e w a s developed ( 5 5 ) . HASSAN AND MOHAMMAD A. Other method.7 Radioimunoassay Robinson e t a1 (54 ) d e s c r i b e d f o u r radioimmunoassays f o r t h e d e t e r m i n a t i o n of bromazepam and o t h e r benzodiazepines.8. 81 s i l i c a g e l loaded paper system developed i n CHCl3-EtOH (49:l). was a p p l i e d t o t h e d e t e r m i n a t i o n of bromazepam i n drugs. 8. ABOUNASSIF 42 The absorbance of n i c k e l i n t h e o r g a n i c phase w a s determined a t t h e n i c k e l resonance a 232. dry form and easy t o handle.8 Chromatographic 8. and r e q u i r e o n l y 75 m l o f blood. t h e antiserum from t h e Finit-tox serum benzodiazepine a a s s a y k i t w a s used w i t h [-H]flunitrazepam and proved t o be s u i t a b l e f o r detecting sub-therapeutic l e v e l s o f a l l benzodiazepines t e s t e d except one. The method i s u s e f u l t o c l i n i c a l t o x i c o l o g y p r a c t i c e . prepared i n a s t a b l e . The a s s a y i s p a r t i c u l a r l y a p p l i c a b l e t o blood samples of f o r e n s i c i n t e r e s t which may b e hemolyzed or decomposing. A.0 nm l i n e . d e s c r i b e d t h e d e t e r m i n a t i o n o f benzodiazepines i n serum u s i n g t h e enzyme m u l t i p l i e d immuno assay-single t e s t (EMIT-ot) drug d e t e c t i o n system ( 5 6 ) .E t O H (40:60:2) complemented by a Whatman S. I n t h e most s e n s i t i v e method. plasma. The method i s s p e c i f i c f o r b i o l o g i c a l l y a c t i v e benzodiazepines. .

c o n c e n t r a t e d ammonia ( 50 : 50 :3) and : Heptane/ chloroform/ ethanol/conc e n t r a t e d ammonia (50:50:20:1) S i l i c a g e l Chloroform/acetone (gO:lO). d i a z o t i z a t i o n and c o u p l i n g w i t h N-( 1-naphthy1)ethylenediamine t o form an azo-dye. Solvent systems and d e t e c t i o n methods f o r br oma z epam Detection Support Solvent system S i l i c a AR 7GF-5 (twc diment ional TLC ) Hept a n e / e t hyl a c e t a t e/ethanol. Thin-layer chromatographic systems and d e t e c t i o n methods d e s c r i b e d f o r t h e i d e n t i f i c a t i o n and d e t e r m i n a t i o n of bromazepam a r e summarized i n Table 9 . S i l i c a g e l p l a t e s were used w i t h methylacetate-ammonia 33% (1OO:l) as a s o l v e n t system. Table 9 .6-0. 59 60 . The q u a n t i t a t i o n o f t h e s p o t s w a s performed e i t h e r by measuring t h e W r e f l e c t a n c e of t h e p l a t e o r by t h e hydrolys i s o f bromazepam t o 2-amino-5-bromobenzoylp y r i d i n e on t h e p l a t e .8. .43 BROMAZEPAM 8.l i g h t (254 nm: 50 10% w/v ~ $ 0 4 h e a t a t 120' for 1 0 minutes chamber w i t h n i t r o u s fumes Spray w i t h anapht h y l e t hylmediamine R. Bromazepam h a s an Rf v a l u e o f 0. which was t h e n e v a l u a t e d d e n s i t o m e t r i c a l l y . d r a g e n d o r f f and iodoplati n a te reagents Ref. W .65. The recovery from plasma was found t o be over 90%. G benzene/isopropanol/ammonia ( 85 :15 :10) S i l i c a g e l Diisopropylether-toluenee t h y l a c e t a t e methanol di6o *254 ethylamine (70:15:10:5:2) - W-light Bratton-Marshall.2 Thin-Layer Chromatography Haefelfinger (58) has reported a specific and s e n s i t i v e method for t h e d e t e r m i n a t i o n o f bromazepam i n plasma by q u a n t i t a t i v e t h i n l a y e r chromatography.

The experimental c o n d i t i o n s used f o r t h e a n a l y s i s o f t h e drug i n b i o l o g i c a l f l u i d s a r e summarized i n Table ( 1 0 ) . ABOUNASSIF 44 Thin l a y e r chromatographic d e t e c t i o n and i d e n t i f i c a t i o n of bromazepam among o t h e r lY4-benzodiazepines h a s a l s o been r e p o r t e d (61-64). The presence o f e l e c t r o n e g a t i v e f u n c t i o n a l groups ( t h e halogen i n t h e 7 .l i q u i d chromatography (EC-GLC).8. A. . t h e r e f o r e a l l ( e x c e p t one) t h e above mentioned methods a p p l i e d (EC-GLC) t o t h e d e t e r m i n a t i o n o f t h e drug.b-benzodiazepine-2-0ne o r as t h e hydrolyzed 2-amino-5-bromobenzoylp y r i d i n e (ABBP) (33. Most of t h e s e methods d e s c r i b e d t h e d e t e r m i n a t i o n o f t h e compound e i t h e r as t h e i n t a c t 1.c a p t u r e g a s . Recently.MAHMOUD M. it h a s been coupled w i t h mass spectrometry (71). t h e s e methods are c a p a b l e of d e t e c t i n g nanogram amounts o f bromazepam i n b i o l o g i c a l f l u i d s where t h e y compete w i t h radioimmunoassays. 8.p o s i t i o n and t h e 2-carbonyl) r e n d e r s bromazepam amenable t o s e n s i t i v e d e t e r m i n a t i o n by e l e c t r o n . Bromazepam h a s a l s o been assayed as t h e "-methyl derivative (70). I n p r i n c i p l e . HASSAN AND MOHAMMAD A. 65-69).3 Gas Chromatography Numerous methods f o r t h e g a s chromatographic a n a l y s i s o f bromazepam i n b i o l o g i c a l f l u i d s have been r e p o r t e d .

6 EC 67 265" 4 FI 68 270° 3-4 EC 70 3-4 EC 69 OV-17 220 N2 on Gas Chrom Q (60/80mesh) Ar-CH4 ( 90-10 1 214O Bromazepam 10% UCC-W-98 on Chromosorb W AW DMC: (80/100mesh) N2 N'-Methylbromazepam 0. Ar-cH4 silicone) on Gas Chrom Q ( 6 0 / 8 0 Rest (9O:lO) Bromazepam Bromazepam Column temp * )etector .Table 10. Gas liquid chromatographic conditions f o r the determination of bromazepam and its metabolites Drug o r metabolite Column packing Carrier 2-Amino-5-bromo-benzoylpyridine (ABBP) 2% Carbowax 20M-TPA on silanized Gas ChromP (100-120 mesh) 2-Amino-5-bromo-benzoylpyridine (ABBP) 3% OV 225 (methyl-phenylcyanopropyl.5% OV-17 Chromosorb G HP (100/200 mesh) N2 4% SE-52 silica capillary column He Programmed 130-260' 20% UCC-W on Chromosorb W AW DMCS (80/100mesh) He ProgramMass 71 med spectro100-310° .5 EC 66 3% OV-17 on Gas Chrom Q (60/80mesh) Ar-CH4 245 O (90:lO) 6 EC 33 3% 240° 9.meter - Ar = Air . RT ns - 20 mi 5 2e: - 6-7 EC 65 12.

The column used w a s a 15 cm S u p e l c o s i l LC18 and t h e mobile phase c o n s i s t e d o f a m i x t u r e o f methanol/phosphate b u f f e r .4 High P r e s s u r e Liquid Chromatography A high p r e s s u r e l i q u i d chromatographic procedure f o r t h e d e t e r m i n a t i o n o f bromazepam i n human plasma has been d e s c r i b e d ( 7 2 ) .v / v ) b e f o r e i n j e c t i o n i n t o t h e chromatograph. ABOUNASSIF 46 8. and T O O ml of s o l u t i o n c o n t a i n i n g 20 ml 10% t e t r a b u t y l ammonium hydroxide i n 1 L water. 30 m l MeCN. 5 . A. I n o r d e r t o determine t h e lower t h e r a p e u t i c range of drug l e v e l s i n serum (73 ) . A f t e r s o l v e n t e x t r a c t i o n a t pH 9 .6% KH PO (1:l) a d j u s t e d t o pH 3. t h e u r i n e samples were incubated t o e f f e c t enzymatic deconjugat i o n b e f o r e e x t r a c t i o n w i t h dichloromethane. t h e a n a l y s i s w a s performed by HPLC on a reverse-phase column w i t h MeCN 0. For t h e d e t e r m i n a t i o n of t h e m e t a b o l i t e s . 2 4 Heizmann et a1 ( 7 5 ) r e p o r t e d a HPLC method f o r t h e d e t e r m i n a t i o n o f bromazepam i n plasma and of i t s main m e t a b o l i t e s i n u r i n e .8. The i n t e r n a l s t a n d a r d w a s carbamazepine and d e t e c t i o n w a s by W spectroscopy. w a s chromatographed on an ~ ~ Micropack 1 8 MCH 1 0 column u s i n g UV d e t e c t o r and perchlorate/MeCN s o l u t i o n as eluent. The unchanged drug was e x t r a c t e d from plasma w i t h dichloromethane. A f t e r e x t r a c t i o n from plasma. t h e e x t r a c t w a s evaporate& and t h e r e s i d u e w a s suspended i n acetonitrile/tetrabutylammonium hydroxide (60:40. pH 7 . A n a l y s i s w a s performed on a 1-1 Bondpack c18 column w i t h mobile phase made from 20 m l MeOH. and t h e r e s i d u e was subseq u e n t l y analysed by r e v e r s e d phase HPLC w i t h W d e t e c t i o n (230 n m ) .MAHMOUD M. The r e t e n t i o n times r a n g i n g from 6 t o 20 minutes. Hochmuth et a1 (74) d e s c r i b e d a simple and r a p i d method f o r t h e t o x i c o l o g i c a l a n a l y s i s of some benzodiazepines and a n t i d e p r e s s a n t s i n c l u d i n g bromazepam. HASSAN AND MOHAMMAD A. . bromazepam. a t a flow r a t e of 1 ml/min. among o t h e r b a s i c drug.

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BUSULPHAN Muhammad T d q " and AbduReah. Inc. A . Saudi h u b & . Riyadh. . ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 16 Copyright 0 1987 by Academic Press. CoReegc 06 P h m a c y . Al-Ba&** *U~pumincnX06 P h a c o L o g y and "Vepahtment 06 PhmacclLticd CCZmhfiy. King Saud UniuemLty. 53 A11 rights of reproduction in any form reserved.

2 2.5 3.4 Identification Titrimetric Methods Chromatographic Methods Nuclear Magnetic Resonance Methods References .MOHAMMADTARIQ AND ABDULLAH A. Nomenclature Formulae Molecular Weight Elemental Composition Appearance.3 7.2 3.4 Melting Point Solubility Storage Spectral Properties 4. Toxicology 7. Methods of Analysis 7.1 7.3 2.4 2. Pharmacokinetics 6.2 7. Description 2. Color and Odor Physical Properties 3.3 3.1 2. Foreword 2.1 3. AL-BADR 54 1. Synthesis 5.

possessing significant cytotoxic activity and is the drug of preference in the treatment of chronic myelocytic or granulocytic leukaemia. myasthenia gravis (20) and chronic granulomatous disease in children (21). Description 2. .2 Generic Name Busulphan.1.4-Butanediylbismethane sulphonate. which has been widely used as antineoplastic drug since its discovery in 1953 (1).4-di(methylsulfonoxy)butane. Sulfabutin. It has recently been proposed as a drug of choice in polycythaemia and thrombocythemia (2-13). 55 Foreword Busulphanis a chemotherapeutic agent. NSC 750. 1. Tetramethylene bis (methanesulfonate) (27-29) 2.4-Bis (methanesulf0noxy)butane. The drug is also used as reproductive sterilant for insect (22-23) and as a general immunosuppressant for organ transplant in the treatment of autoimmune disease (24-26). This alkylating agent belongs to the series of alkanesulfonic acid esters. Busulphanhas also been used for the treatment of bronchial carcinoma (18-19) . Mielucin (27). Methanesulfonic acid tetramethylene ester. Myeleukon. 1. CB 2041.3 Trade Names Myleran. 2. 1. Myeloleukon. Misulban. 2.1. 1. WR 19508 Busulfanum (29). Mitosan.1 Chemical Names 1.1.4-Butanediol dimethanesulfonate. GT 41.BUSULPHAN 1.4-di(methanesulfonyloxy)butane. Myelosan.1 Nomenclature 2. .

S. 3. protect from light (30).03% (27).5 0 . 26. AL-BADR 56 2. Appearance. 32). 5.2 119O.3 Storage Busulphan should be kept in a well-closed container. Color and Odor A white almost odorless crystalline powder (29. 32). at 20°.31 2. 38.3 Molecular Weight 246.2.2. very slightly soluble in alcohol (27. (27).26%. in 750 parts of water (in which it is slowly hydrolyzed) and in 25 parts of acetone.3 CAS No. Physical Properties 3. 3. H.4 (31) Elemental Composition C.1 Melting Point 114-118O. [55-98-17 (27) 2.2 Structural 0 0 0 0 I1 II H C-S-O-CH2-CH2-CH2-CH2-O-S-CH3 3 It II 2. 2. 29.98%.73%.MOHAMMAD TARIQ AND ABDULLAH A. . 3. Solubility Soluble. 116'.

The assignments of the characteristic bands in the infrared spectrum is shown below: -1 3.BUSULPHAN 3. ethanol and in water was scanned from 200 to 400 nm using Varian DMS 90 Spectrophotometer.4. The drug is dissolved and its spectrum determined on a T60 A NMR spectrometer using TMS internal standard.) 1000-770 S-0-C stretch 1 Proton Nuclear Magnetic Resonance ( H NMR) Spectrum The 1H NMR spectrum of Busulphan Figure 2.4 Spectral Properties 3.1 Ultraviolet Spectrum The ultraviolet spectrum of busulphan in methanol. 3.4. It was recorded on a Pye-Unicam SP 1025 infrared spectrophotometer.4.) 1180 S ( = 0)2 stretch (Symm.3 Frequency cm Assignments 2960-3060 CH stretch 1430-1415 -CH2-0 vibration 1352 S ( = 0 > 2 stretch (Assymm. is shown in in DMSO-db Varian as the Assignment of the chemical shifts to the different protons is shown below: .2 Infrared Spectrum The infrared spectrum of busulphan as KBr disc is shown in Figure 1. No absorbance has been noticed .

Wavelenqth urn 3500 3ooo 2500 zoo0 1800 1600 1400 1200 Wavenumber Figure 1 : Infrared spectrum of Busulphan. KBr disc. loo0 800 Jo 625 .

0 6. a . TM: - a . . .CH3 i. . ch W 1 - . .O Figure 2 : Proton nuclear magnetic Resonance spectrum of Busulphan DMSO -dg using TMS as reference standard in . .-0 0 ' .0 7. 8. . .I I " ' ' 500 I 400 ' * I ' I 300 6 ! 5 4 3 2 CH3-~-O-CH2-CHj-CH~CH. " " " " ' I 0 1 -S. . . " 9 1 .

68-1.52 Quartet 1 and 6 69.CH3 carbon chemical shifts are assigned on basis of the chemical shift theory and off-resonance splitting pattern and shown below: Chemical shift (6) Multiplicity Carbon assignments 24. Figure 3 and 4 represent the proton-decoupled and off-resonance spectra respectively.MOHAMMAD TARIQ AND ABDULLAH A.40 Triplet 2 and 5 .70 Triplet 3 and 4 36. 3.90 Multiplet 3 and 4 (Cg2) 3.1-4.10 Sing1et 1 and 6 (Cg3) 4.4.40 Mu1tiplet 2 and 5 (Cl12) *Please refer to Figure 2. AL-BADR 60 Chemical shift (6) Multiplicity Proton assignment* 1.4 Carbon-13 Nuclear Magnetic Resonance Spectra (C-13 NMR) The carbon-13 NMR spectra of busulphan in DMSO-d6 using TMS as an internal reference are obtained using a Jeol FX 100 MHz spectrometer at an ambient temperature. 0 0 0 0 6 11 5 4 3 2 II CH3-S-0-CH CH CH CH -0-S iI 2 2 2 2 11 The the the are 1 .

decoupled carbon . .I3 nuclear magnetic resonance spectrum of Busulphan in DMSO.Figure 3 : Proton .dgusing TMS as reference standard.

Ill Figure 4 I : Off- I resonance carbon-13 nuclear magnetic resonance spectrum of Busulphan in DMSO-dG using TMS as reference standard. .

4-butanediol [HOCH CH CH CH OH] 2 2 2 2 with methanesulfonyl chloride [CH SO2C1] in the 3 presence of pyridine ( 3 1 . Other major fragment are at m/e 175. 111. 97. 55 and 42.5 Mass Spectrum The electron impact (EI) mass spectrum of busulphan at 70 eV recorded on Varian Mat 311 mass spectrometer and the direct chemical ionization (DCI) mass spectrum obtained with Finnigan 4000 mass spectrometer are shown in figures 5 and 6 respectively. the molecularion peak was not detected. 4. The CI spectrum (Figure 6 ) is simple and shows a base peak at m/e 151 and at molecular ion peak at 247 corresponding to (M + 1) and minor peak at m/e 125. Synthesis By esterifying 1. The (EI) spectrum (Figure 5) shows a base peak at m/e 7 1 . A proposed mechanism of fragmentation of the EI fragments is shown in scheme 1.4. 79. 122.BUSULPHAN 63 3. 33) as shown below: 0 II CH3-S-C1 II + OH-CH2CH2CH2CH2-OH 0 0 II 0 II CH3-S-O-CH2CH2CH2CH2-O-E-CH3 11 0 0 Pyridine c r . 109.

AL-BADR 64 -29568 175 150 200 2 50 300 Electron impact (El) mass spectrum of Busulphan. . 100 Figure 5 : 100 150 200 250 300 350 Figure 6:Ckmical imitation(CI1 mass spectrum of Busulphan.MOHAMMAD TARIQ AND ABDULLAH A.

.rl .m-0 +o.X +OX m 5 I =--a 0 I 'c: 0 m I 8 t - m X V . U 0 w .I c' 0 Y o= 1 I 0 I m=o Xm U 1 m X U O I +o=(.= P 0 -51 0 0 co m I I +o= m= c i 0 \ 0 re: .

/ ‘ 0 + OH iI (4*7”J.+ . m/e 71 .CH3 0 .-m3 0 m/e 246 + OH + 0 OH 0 0 m / e 175 Scheme 1: Continued.

m P +O 0 . 0 0 I m +O=T=O E 67 m T.1.. I II --m= 51 0 -m=o P 0 Im T. 4 .

05 hr-1 (SD). I t i s l a r g e l y e x c r e t e d i n t h e u r i n e as s u l p h u r . f o r t h e i n i t i a l p h a s e of r a d i o l a b e l e d d r u g i n plasma. AL-BADR 68 5. Metabolism and E x c r e t i o n Busulphan i s r e a d i l y absorbed from t h e g a s t r o i n t e s t i n a l t r a c t and r a p i d l y d i s a p p e a r s from t h e blood. A f t e r o r a l d o s i n g an i n i t i a l l o g phase of about 0. The major p o r t i o n of busulphan i s probably e l i m i n a t e d by enzymatic a c t i v i t y r a t h e r t h a n pure chemical d e g r a d a t i o n (36). and between 20% and 95% was removed w i t h i n 3 t o 5 minutes. Vodopick et a l .5 t o 2 h o u r s was observed b e f o r e measureable blood l e v e l s were achieved. Pharmacokinetics Absorption. A f t e r o r a l 3H-labeled busulphan. t h e compound might be bound b o t h r e v e r s i b l y and i r r e v e r s i b l y ( c o v a l e n t l y ) t o blood component (34). Only about 1% of a dose of busulphan i s e x c r e t e d as unchanged drug w i t h i n 2 4 h o u r s . (36) found a prompt r i s e i n t h e plasma l e v e l of r a d i o a c t i v i t y .c o n t a i n i n g m e t a b o l i t e ( 2 9 ) . The e l i m i n a t i o n rate c o n s t a n t averaged 0. followed by a r a p i d f a l l and t h e n a g r a d u a l r i s e i n r a d i o a c t i v i t y . The plasma AUC w a s l i n e a r l y r e l a t e d t o t h e d o s e . t h e t i m e absorpt i o n ends.27 ? 0. The l o g t i m e f o r t h e s t a r t of a b s o r p t i o n . The c l i n i c a l r e s p o n s e u s u a l l y b e g i n s w i t h i n 1 t o 2 weeks a f t e r i n i t i a t i o n of t h e r a p y .4 t o 6 mg. Due t o i t s a l k y l a t i n g a c t i v i t y .5 h o u r . a f t e r i n t r a v e n o u s d o s i n g . t h e r a d i o a c t i v i t y i n t h e blood r a p i d l y d e c r e a s e d . Busulphan i s e l i m i n a t e d w i t h t$ of about 2. The plasma c o n c e n t r a t i o n t i m e d a t a c o u l d be f i t t e d t o a zero-order a b s o r p t i o n one-component open model. which i s peaked w i t h i n t h e f i r s t h o u r . The metabolism t a k e s p l a c e mainly i n t h e l i v e r . It a p p e a r s t o be p r a c t i c a l l y c o m p l e t e l y e x c r e t e d i n t h e u r i n e as methane-sulphonic a c i d (35) Nadkarni -e t a1 ( 4 ) u s i n g 14C and 35S-labeled busulphan found t h a t . and t h e a b s o r p t i o n rate c o n s t a n t showed .MOHAMMAD TARIQ AND ABDULLAH A. Ehrsson e t a1 (37) have s t u d i e d t h e busulphan k i n e t i c s and r e p o r t e d t h a t k i n e t i c s were followed i n p a t i e n t w i t h c h r o n i c m y e l o c y t i c leukemia a f t e r o r a l dose of 2.

34. 2 9 ) . about 1.and Ik-labelled busulphan. About 1% of the drug is excreted unchanged in urine over 24 hours. The fate of the drug in man has been studied after the administration of radiolabelled drug followed by measurement of total radioactivity in plasma and urine (4. 6.3-butanediol.BUSULPHAN 69 some inter-individual variation. and methanessulphonic acid were identified as principal metabolites. Wiygul et a1 (39) have studied metabolism of busulphan in the boll weevil. Jones and Campbell (40) have studied the metabolism. most of the metabolites have not been identified (38).4-Butanediol. aminoacids. malonic acid.53. succinic acid.09% as busulphan. 42.34% of total radioactivity was recovered from the feces. When one-day male boll weevils were force-fed 3H. The most important side effect of busulphan in high doses are thrombocytopenia and damage to haemotological tissues (45. The drug is rapidly eleminated from the plasma and is reported to be extensively metabolized. are also reported (41. The results obtained suggest that cycloalkylation of thiol groups by busulphan may represent a normal detoxification route rather than a mechanism of action. reactions and biological activities of some cyclic dimethanesulphonates. sulfolanes. Toxicity Recently Bishop and Wassom (44) have published a detailed review article on the toxicity of busulphan incorporating around 290 references. Relevance to the mechanism of action of busulphan (Myleran). at 72 hours after injection. Other studies on the metabolism of busulphan in b o l l weevils. 1. Most metabolism of busulphan took place within 24 hours postingestion. 2. . 43). 36. including methane-sulphonic acid and 3-hydroxytetrahydrothiophene-1. twelve metabolites have been isolated. 37).l-dioxide. malic acid.

an odor c h a r a c t e r i s t i c of methanesulfonic acid i s p e r c e p t i b l e . - Methods of A n a l y s i s 7. a c i d i f y w i t h d i l u t e d h y d r o c h l o r i c a c i d . A) Fuse about 100 mg w i t h about 100 mg of p o t a s s i u m n i t r a t e and a p e l l e t of potassium hydroxide weighing approximately 250 mg. known as "Busulphan lung". B) To 100 mg add 10 m l of water and 5 m l of 1 N sodium hydroxide. A w a s t i n g o r Addison . c a t a r a c t s ( 6 8 ) ) c e l l u l a r d y s p l a s i a of pancreas ( 6 9 ) . and add 1 drop . A c i d i f y t h e second p o r t i o n of t h e s o l u t i o n w i t h d i l u t e d s u l f u r i c a c i d . AL-BADR 70 I n t e r s t e t i a l pulmonary f i b r o s i s . t h e n t o b l u e . 8 2 .1 Identification The USP XX (1980) ( 3 2 ) d e s c r i b e s t h e f o l l o w i n g i d e n t i f i c a t i o n t e s t s f o r busulphan. To one p o r t i o n add 1 d r o p of potassium permanganate TS.1 0 0 ) . d i s s o l v e t h e r e s i d u e i n water. a w h i t e p r e c i p i t a t e i s formed. and f i n a l l y t o emeraldgreen.MOHAMMAD TARIQ AND ABDULLAH A. and add a few d r o n s of barium c h l o r i d e TS. Busulphan i s p o t e n t i a l l y mutagenic (104 114) and c a r c i n o g e n i c i n man (115 123). a d m i n i s t e r e d d u r i n g t h e t h i r d trimester of pregnancy. 81.l i k e syndrome has o c c u r r e d i n some p a t i e n t s r e c e i v i n g l o n g term t h e r a p y w i t h Busulphan (59. Cool. 6 6 ) . 86) and r e p r o d u c t i v e t o x i c i t y of busulphan (87 . T e r a t o g e n i c e f f e c t of t h i s drug i n animals h a s been r e p o r t e d by s e v e r a l workers (102 . Heat u n t i l a c l e a r s o l u t i o n i s o b t a i n e d . C) Cool t h e s o l u t i o n o b t a i n e d i n I d e n t i f i c a t i o n Test B . t h e p u r p l e c o l o r changes t o v i o l e t .) 85. Other a d v e r s e e f f e c t s include endocardia1 f i b r o s i s (67). can a d v e r s e l y a f f e c t f e t a l growth (101). i s as w e l l r e c o p i s e d s i d e e f f e c t busulphan t h e r a p y (54-64). - 7.1 0 6 ) . 65. Busulphan. . and d i v i d e i t i n t o two e q u a l p o r t i o n s . a d r e n a l i n s u f f i c i e n c y (70) and bone marrow damage (71-83) Laboratory s t u d i e s i n animals have shown s i g n i f i c a n t immunosuppressive ( 4 8 .

Add 0. Wash t h e condenser w i t h a small q u a n t i t y of water. E x t r a c t i n t h e same way w i t h two f u r t h e r 10-ml q u a n t i t i e s of a c e t o n e .1 Aqueous B r i t i s h Pharmacopoeia (1980) (30) d e s c r i b e d an aqueous t i t r a t i o n procedure f o r t h e a s s a y of busulphan a s f o l l o w s : To 0.2 T i t r i m e t r i c Methods 7. s e a l t h e t u b e and h e a t a t 300° f o r two hours.BUSULPHAN 71 of potassium permanganate TS: t h e c o l o r of t h e permanganate i s n o t d i s c h a r g e d . 7.2.1 M sodium hydroxide VS i s e q u i v a l e n t t o 0. decant t h e l i q u i d through a f i l t e r i n t o a C a r i u s t u b e and e v a p o r a t e t o s m a l l volume under a j e t of warm a i r . It i s n e c e s s a r y t o e x t r a c t t h e busulphan w i t h a c e t o n e and decompose i t i n a s e a l e d t u b e w i t h n i t r i c acid a s follows: T r i t u r a t e an a c c u r a t e l y weighed q u a n t i t y of powdered t a b l e t s e q u i v a l e n t t o about 2 mg of busulphan w i t h 10 m l of h o t a c e t o n e . c o o l and t i t r a t e w i t h 0. 7. Each m l of 0.1 M sodium hydroxide VS.2. hence t h e f l a s k combustion method i s n o t a p p l i c a b l e becuase of t h e l a r g e amount of o r g a n i c matter. A f t e r opening t h e . u s i n g p h e n o l p h t h a l e i n s o l u t i o n as i n d i c a t o r .01232 g of C6H1406S2.25 g of t h e drug add 20 m l of w a t e r and b o i l g e n t l y under a r e f l u x condenser f o r t h i r t y minutes. d e c a n t i n g and e v a p o r a t i n g a f t e r each e x t r a c t i o n and e v a p o r a t i n g t o d r y n e s s a f t e r t h e t h i r d e x t r a c t i o n . B r i t i s h Pharmacopoeia (1980) (30) h a s a l s o r e p o r t e d similar procedures f o r t h e i d e n t i f i c a t i o n of busulphan.2 Gravimetric Busulphan t a b l e t s are s u g a r c o a t e d and c o n t a i n o n l y m i l l i g r a m q u a n t i t i e s .5 m l of fuming n i t r i c a c i d .

0 ml) was mixed with 0.d. dissolve the residue in water and acidify with concentrated hydrochloric acid. After addition of 0. 1-2 pl of solution was injected into Varian 3700 GC equipped with a constant current 63Ni lectroncapture detector. cool and weigh. ignite at 800° for one hour. An OV-I fused silica cappillary column (25 m x 0. 7. detector and injector port temperatures at 145OC.3.MOHAMMAD TARIQ AND ABDULLAH A.) with a film thickness of 0. The method comprise a new derivatization technique for extraction of busulphan from plasma in a single step combined with high separation efficiency and sensitivity by using capillary column in combination with electron capture detection. heat on a water-bath for three hours. The derivatization of busulphan was done directly in plasma by adding sodium iodide in acetone.4 ml n-heptane the reaction was carried out at 7OoC for 70 minutes under stirring.1 Gas Chromatographic Method Hassan and Ehrrson (125) developed a gas chromatographic method for the determination of busulphan in plasma with electron capture detection. The helium was used as carrier gaswith a flow rate of 2 ml/min. AL-BADR 12 Carius tube transfer its contents to a small dish with 15 ml of water. evaporate to dryness. The organic phase was separated for analysis on gas chromatography. Each mg of residue = 0. filter through a platinum filtering crucible.1 ml of 1.5-bis(methanesulfoxy)pentane in acetone and sodium iodide in water (1 ml acetone and 8 M Na' iodide). Add an excess of 10 per cent barium chloride solution. Plasma sample (1.the inlet pressure was .3 mm i.52 um (HewlettPackard) was used. The instrument was operated isothermally with the oven. 25OoC and 2OO0C respectively.5276 mg of busulphan (124).0 ug/ml of 1.3 Chromatographic Methods 7.

) w a s packed w i t h 10% SP-2401 on 100-120 Supelcoport and w a s o p e r a t e d a t 140°using a helium flow r a t e of 20 ml/min. The mininam! d e t e c t a b l e c o n c e n t r a t i o n was 5.0 pg/ml) i n acetone and e x t r a c t e d w i t h 4 m l of methylene c h l o r i d e u s i n g a mechanical shaker (100 s t r o k e s / m i n ) The o r g a n i c phase w a s s e p a r a t e d and evaporated t o d r y n e s s . The s t a n d a r d curve u s i n g plasma w a s l i n e a r w i t h i n t h e range of 5-300 ng/ml.v. A s p l i t r a t i o of 1 : 10 w a s used. d .05 m l of n-hexane and 0. 1. 1 m l of 1 M sodium i o d i d e i n a c e t o n e w i t h a r e a c t i o n t i m e of 20 minutes a t 7OoC. . Nitrogen was used a s make up g a s and added through t h e hydrogen i n l e t a t a rate of 30 ml/min i n t h e d e t e c t o r base.BUSULPHAN 73 0.0 M 1 of plasma was mixed w i t h 0.4-diiodobutane.4 b a r .D. .6% a t 100 ng/ml and ? 4. The a l i q u o t (1-2 pg of n-hexane) w a s used f o r t h e a n a l y s i s by GC-MS.5 m x 2 mm i . The p r e c i s i o n of t h i s method t 3. The s t a n d a r d curve w a s l i n e a r over a range of 10-400 ng/ml.7 x 10-16 mole/sec.9% (c.2 Gas Chromatography - Mass Spectrometry (GC-MS) Ehrsson and Hassan (126) developed a chromatographic method f o r t h e determinat i o n of busulphan i n plasma by GC-MS w i t h s e l e c t e d i o n monitoring.3% a t 10 ng/ml (n = 5 ) . After a d d i t i o n 0. d e r i v a t i z a t i o n was done by adding 0 . A h i g h s e p a r a t i o n e f f i c i e n c y and s e n s i t i v i t y w a s o b t a i n e d u s i n g t h i s method.v. Busulphan e x t r a c t e d from plasma with methylene c h l o r i d e and converted t o 1. t h e r e t e n t i o n t i m e w a s 8 minutes.LKB 2091 w i t h an i o n i z i n g energy of 70 e V was used. 3 % (c.3.1 m l of i n t e r n a l s t a n d a r d (1. The i n j e c t o r and t h e i o n s o u r c e temperature w e r e 230° and 27OoC. respectively. The r e l a t i v e S.) a t t h e 100 ng/ml (n = 5 ) . was 2.5-pentanediol d i m e t h a n e s u l f o n a t e 1.) a t t h e 10 ng/ml l e v e l (n = 5) and ? 2 . The column(1.1 m l of water t h e m i x t u r e was v i g o r o u s l y shaken. 7.

AL-BADR 74 7. P u l s e width Pulse r e p e t i t i o n t i m e FTD d a t a p o i n t s Observation t i m e = = 3 = 9. F o u r i e r t r a n s f o r m s p e c t r o m e t e r equipped w i t h 5 mm p r o t o n probe and computer w i t h 24 K memory. The s p e c t r o m e t r y c o n d i t i o n s were: ambient t e m p e r a t u r e = 30OC. frequency 1 h = 200. . The i n s o l u b l e matter i s allowed t o s e t t l e and 0. 8 2.5 t o 1 m l of upper l a y e r i s t r a n s f e r r e d t o NMR t u b e . I n t e g r a t e t h e broad t r i p l e t a t about 4. The c o n t e n t s were shaked v i g o r o u s l y .06 MHz. 61. t h e v i a l was c l o s e d w i t h a septum and crimper s e a l e d w i t h an aluminium seal. caped and shaken.MOHAMMAD TARIQ AND ABDULLAH A. The NMR s p e c t r a were r e c o r d e d w i t h a 200 MHz. an a c c u r a t e l y weighed q u a n t i t y of powder e q u i v a l e n t t o 6 mg of busulphan i s t r a n s f e r r e d t o a 15 m l v i a l .3 ppm due t o t h e 4-methylene p r o t o n s (-CH2-O-S02-) of busulphan and t h e s i n g l e t s and about 4. I n t e g r a l v a l u e of signal r e p r e s e n t methanamine. 11. Formula weight of busulphan 14. 2 M I of chloroform w a s added u s i n g a s y r i n g e by i n t r o d u c i n g t h e n e e d l e t h r o u g h septum. where I n t e g r a l v a l u e of signal r e p r e s e n t i n g bulsulphan. 20 G r a m of busulphan t a b l e t s are f i n e l y powdered. Wise b o r e .7 ppm due t o t h e 12 methylene p r o t o n s of t h e i n t e r n a l s t a n d a r d .2 m i n u t e s .58. Weight i n mg of m e t h e n m i n e t a k e n f o r t h e analysis. 1 Drop of r e f e r e n c e s t a n d a r d (50 mg t e t r a m e t h y l s i l a n e (TMS) i n 10 m l chloroform) was added.68 mg of methenamine i n 10 m l of chloroform) was added.4 NMR S p e c t r o m e t r i c Methods a) Truczan and Lau-Cm(l27) d e s c r i b e d a s i m p l e NMR s p e c t r o s c o p i c method f o r t h e a s s a y of busulphan i n t a b l e t s . Observation frequency range = 1600 H2. To t h i s 1 m l of i n t e r n a l s t a n d a r d s o l u t i o n (11./ES) x Au = As = Ev Es = = C = c.68.13 sec. 14 sec. The q u a n t i t y of busulphan i n mg i s o b t a i n e d from:(AU/AS) x (EV. Formula wieght of methenamine/l2.

Syed Rafatullah. . Tanvir A. for his technical assistance and to Mr. was synthesized and proved to undergo cyclization to THF by intramolecular alkylation. From the data obtained. it is concluded that 4-methansulphonyloxybutanol is unlikely to be responsible for the biological action of busulphan. The half-life of this first-order reaction in aqueous solution at 3 7 O was determined to be approximately 12 minutes at pH 3 as well as at pH 7.BUSULPHAN b) 15 Feit and Rastrup-Andersen (128) have investigated the hydrolysis of busulphan by means of NMR spectroscopy. King Saud Univeristy. 4-methanesulphonybutanol. The rather unstable intermediate. Butt. College of Pharmacy. Assistant Researcher. The final product was found to be tetrahydrofuran (THF). secretary to the Pharmaceutical Chemistry Dept. Acknowledgements The authors would like to thank Mr.4. for his secretarial services.

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E8tOn and W. Immunology 9.W. Econ. W . 968-970 Glover. 14. K. 66(1).P. 194. Enatomol.F.N.S. M. Mutation 411-420 (1971). Huff and S. Earle. Moore and H. Iyengar and V. W. Generoso. Kar. H. Jpn. 94. (1962). 447-465 (1961). Flint. H. Fertil. 84.M. 93-117 (1974). N. Taft. Agarwal. 453-459 (1975). Reprod. 90. Craig. 319- Res. H. V. 47-53 (1973). (1968). 87.W. 156. J. . 16. J. 181. 95. 322 (1972). Wschr. A. Pharmacol. 99.. 13-22 (1963). 92. Repr. J. J. Monoyer. Craig and T. Bollag. Biol. 165-170 P. A. 36. 11. C. Jones and H. 2. Jackson.W. Experentia.D.. 1716-1731 (1975). 85. Baisakh. Nature.H. J.W. 95. Kamboj and H.W. S. Tange. B. 96. ----Indian J. Fox. SOC.M. W. R. 268 (1953). Bhatia.BUSULPHAN 81 83. 98. M. Fertil. 355-365 (1979). K.B. 88.W. Klassen. Reprod.K. D. 63(12). 393-395 (1954).R. Berenbaum. Ikegani and Takeuchi . J. Jackson. Pathol.M. 66(1) 9698 (1975). 24(1). (1959). (Paris).5. 2. Jackson. H. 149-157 Craig. 93.C. 354 (1958).W. 84. H. Nature. Jackson. W. Fertil.M. Pharmacol. Med. Fertil. Fox and H. Jpn. Res. SOC. Br. 1184-1185 (1962). B. T.I. 97. B.. B. Fox and A. J. Reprod. A.. Acta. J. Schweiz 89. J. 86. Econ. Partington and B. Stout.F. W. Carig. Fox and A . Mamura. Jackson. Med. Jackson. Bollag. J. Entomol. B. Chandra.353- Fox. 91.W.W.

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O.fhy.CHLORAMBUCIL Mohammad T L q * and AbduReah A . ke-Bad&** *UepcuLtment 06 P h m a c o R o g y . Box 2 4 5 7 . Riyadh-17451. ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 16 85 Copyright 01987 by Academic Press.t 06 Phacu~laceuLicaI Chern&in. P. College 06 P h m a c y . . All rights of reproduction in any form resewed. S a u d i RtLabia. **UepahGncn. Inc. K i m j Saud LlniLImiXy.

7 Melting Point Extraction Solubility Moisture Content and Hygroscopicity Dissociation Constant Storage Spectral Properties 4.2 2.5 2.3 8. Description 2.5 3.4 3. Toxicity 8.1 3.4 2. Therapeutic Uses 7. AL-BADR 86 1.2 3. Synthesis 5.6 3. Nomenclature Formulae Molecular Weight Elemental Composition Appearance. Color and Odor Crystal Properties Physical Properties 3.2 8.4 References Identification Titrimetric Methods Spectrophotometric Methods Chromatographic Methods .MOHAMMAD TARIQ AND ABDULLAH A.3 2.6 3. History 2. Methods of Analysis 8. Pharmacokinetics 6.3 3.1 2.1 8.

B. C. p-Di(2-chloroethyl)aminophenyl butyric acid (9 -12).1. 4-[ 4-Di-( 2-chloroethyl ) aminophenyl] butyric acid. Hodgkin's and non-Hodgkin's lymphoma ( 6 ) . 1348. and breast carcinoma (7). History This aromatic derivative of mechlorethamine was synthesized first at the Chester Beatty Research Institute in England. . Chloroambucil. These include chronic lymphocytic leukemia (4. choriocarcinoma. 4-[4-(Bis(2-chloroethyl)amino)phenyl]butanoic acid. 2. 2.1 Chemical Names 4-[Bis( 2-chloroethyl)amino]benzenebutanoic acid.1. It is most often employed in the long-term maintenance management of chronic lymphoet a1 (8) found that a response cytic leukemia.2). It was synthesized among other aromatic derivatives of the chloroethylmines by the British chemists. Moore rate of 19% was achieved in 52 advanced breast cancer patients. Linfolysin (10-12) - . 2.3 Trade Names Chlorambucil . Everett.2 Generic Names Chlorambucil. 4-[ p. ~-[p-Di(2-chloroethyl)aminophenyl]butyric acid.5). Chloraminophene.87 CHLORAMBUCIL 1. (11) Amboclorin.1. ovarian carcinoma.[ Bis ( 2-chloroethyl) amino]phenyl ] butyric acid.1 Nomenclature 2.N-di-2-chloroethyl-y-p-aminophenylbutyric acid. Description 2. N. Roberts and Ross Chlorambucil has shown activity in a variety (1. of human malignancies (3). NSC 3088 Chlorbuti- num (10-12). Leukeran.

27%. Color and Odor A w h i t e or off-white c r y s t a l l i n e or g r a n u l a r powder with a s l i g h t odor (11).5 C1 23. H 6.2.4 CAS R e g i s t r y No.30%.1. 0 10. .3 Molecular Weight 304.20 2. 2. 2. 3.52% ( 1 0 ) .2 . N 4.6 Crystal Properties F l a t t e n e d n e e d l e s from petroleum e t h e r ( 1 0 ) .MOHAMMAD TARIQ AND ABDULLAH A.1. AL-BADR 88 2.2 Formulae 2. Wiswesser Line N o t a t i o n 2.60%.1 Empirical (14J 5 ) C14H19C12N02 2.31%. (10.4 . Appearance.1 Properties Melting P o i n t Melts between 64' and 69' (12).2.5 (13) QV3R DN2G2G 2. ( 305-03-3) . 3. Physical. Structural C 1CH 2CH2 \ / N CH CH CH COOH C1CH2CH2 2.15) Elemental Composition C 55.

7. S o l u b l e a t 2OoC i n 1 . and minima a t 225 nm and 281 nm ( 1 2 ) . and i n 2 p a r t s of a c e t o n e . 1 cm 8 5 ) . determined by F i s c h e r t i t r a t i o n (14).3 Solubility P r a c t i c a l l y i n s o l u b l e i n water (14). 3. .6 (16).5 p a r t s o f chloroform.8 3. i n 2. s o l u b l e i n e t h e r ( 1 0 ) and i n d i l u t e s o l u t i o n of a l k a l i hydroxides (11). 5 % . 1 cm 650) and 302 nm ( E 1%. respectively. 5 p a r t s of a l c o h o l .1 U l t r a v i o l e t Spectrum The u l t r a v i o l e t spectrum of chlorambucil i n methanol e x h i b i t s maxima a t 258 nm ( E 1%.4 Moisture Content and Hygroscopicity Not more t h a n O .89 CHLORAMBUCIL 3. Storage It should be s t o r e d i n a c o o l p l a c e i n a i r t i g h t . 3. The W spectrum o f chlorambucil i n w a t e r and i n methanol which was scanned from 200 t o 400 nm u s i n g DMS 90 Varian spectrophotometer. 3. The W s p e c t r a i n w a t e r and i n methanol a r e shown i n Figs [l] and [ 2 ] .5 D i s s o c i a t i o n Constant pKa v a l u e 5.2 Extract ion Chlorambucil i s e x t r a c t e d by o r g a n i c s o l v e n t s from aqueous a c i d s o l u t i o n s ( 1 2 ) . 3. It e x h i b i t s a b s o r p t i o n maxima a t 257 nm and a t 302 nm and minima a t 224 and 280 nm.7 Spectral Properties 3. l i g h t r e s i s t a n t c o n t a i n e r s (11).

lo . AL-BADR 2 so. . 70 .MOHAMMAD TARIQ AND ABDULLAH A. .LO 40. 80 90 - ' 80 .20 10. 50 . .10 A 2 00 250 Wavelenplh 2 00 IS0 Wardepth 300 300 150 I50 F i g u r e 2 : U l t r a v i o l e t Spectrum o f Chlorambucil i n Methanol.so .

3. The s p e c t r a l assignments are l i s t e d below:- 10 9 H C ~H~ c iC &@ / C1CH2CH2 10' 9' 7' 6' 4 3 2 1 CH2-CH2-CH2-COOH .1 3 NMR) The C-13 NMR n o i s e decoupled and o f f resonance s p e c t r a a r e shown i n Figs.91 CHLORAMBUCIL 3. The spectrum w a s o b t a i n e d a s K B r d i s c and i s recorded i n Pye-Unicam SP 1025 i n f r a r e d spectrophotometer. 830 cm-1. [ 4 ] and 151. [ 3 ] .7.2 I n f r a r e d Spectrum The i n f r a r e d spectrum of chlorambucil i s shown i n F i g .3 Carbon-13 Nuclear Magnetic Resonance S p e c t r a ( C . 1465 and Clarke ( 1 2 ) r e p o r t e d t h e p r i n c i p a l peak a r e 1695. r e s p e c t i v e l y . Both were recorded i n d e u t e r a t e d chloroform on a Jeol-XL-100 MHz NMR s p e c t r o m e t e r u s i n g t e t r a m e t h y l s i l a n e as a r e f e r e n c e s t a n d a r d . 1520 and 1229 cm-l.7. The f r e q u e n c i e s and t h e i r s t r u c t u r a l assignments are as follows:Frequency (em-') Assignment 1720 O H s t r e t c h and CH stretch C = 0 acid 1622 C = C aromatic 1450 CH deformation 3100-29'70 730 C-C1 stretch Other f i n g e r p r i n t bands a r e 1530.

.L 92 Wavenumber Figure 3 : Infrared Spectrum of Chlorambucil as KBr Disc.

93 . YO . d Figure 4 : "C-NMR .r Y E c .r Spectrum o f Chlorambucil in CDC13 (Noise Decoupled).. 0 W L v) .

.\ a I a a Figure 5 : 13C-NMR Spectrum o f Chlorambucil i n CDC13 (Off-Resonance).

4 t 6 s = singlet 3. Fig. [ 61.5 O H ( a c i d i c ) exchangea b l e w i t h D20.95 CHLORAMBUCIL Chemical s h i f t ( PPm 1 Carbon No.A NMR spectrometer u s i n g t e t r a m e t h y l s i l a n e (TMS) as t h e i n t e r n a l standard . The spectrum w a s recorded on Varian T ~ O .6-7.08 Aromatic p r o t o n s S i n g l e t a t 11.5 t 10 and 10' 40.1 d 8 144.2 S and 6' 129.4 Mult i p l ic ity d = doublet t = triplet I Proton Magnetic Resonance S p e c t r a ( H-NMR) 1 The 60 MHz H-NME3 spectrum of chlorambucil i n d e u t e r a t e d chloroform i s shown i n F i g . [ T I . The f o l l o w i n g are t h e s t r u c t u r a l assignments: Chemical s h i f t (ppm) Assignments M u l t i p l e t a t 1.3 1 S 2 33.8 -(CH ) -CO p r o t o n s S i n g l e t a t 3.60 23 -N-CH2CH2 p r o t o n s M u l t i p l e t a t 6.7. .4" t 4 26.8-2.6 d 7 and 7' 112.4 t 5 130.8" t 3 33.3 S 9 and 9' 53. 180.

..1 6-0 I ... v 0 100 200 300 400 --... .. ... 6 I I I 1 4-0 3-0 2. .. .. ... . .- TY I [ ...".. . .. . . ... .0 14 Figure 6 : 'H-NMR Spectrum o f Chlorambucil i n Deuterated Chloroform... l .. . I Qo 7-0 .. .4 500 ..'.... "...... .. 0 . . I 5-0 ..

200 100 L' Figure 7 : 'H-NMR Spectrum o f Chlorambucil in Deuterated Chloroform + D20. 0 .

The spectrum shows a pronounced peak r e s u l t i n g from t h e loss of HC1 (& . i o n source p r e s s u r e o f 0. The spectrum i s shown i n F i g . A proposed mechanism of f r a g m e n t a t i o n and t h e m / e o f t h e major fragments i s given i n Scheme 1.7. The chemical i o n i s a t i o n ( C I ) spectrum ( F i g . The mass s p e c t r a l assignments o f t h e prominant i o n s under C I conditions a r e given i n t h e following m/e Fragment 306 [ M + 2H]+ 305 MH+ 304 M+ - 303 [MH 286 [M - 268 [M . AL-BADR 98 3.36) c o n s t i t u t e t h e base peak a t m / e 268 and a molecular i o n peak at m / e 304. i o n s o u r c e t e m p e r a t u r e o f 1 5 0 O c and emission c u r r e n t o f 300 PA. ion s o u r c e temperature of 1 8 0 O c and a n emission c u r r e n t of 300 uA. A proposed f r a g m e n t a t i o n pathway for t h e fragment i n t h e E I spectrum i s shown i n Schemes l a and lb.13 Torr. The spectrum is dominated by m / e 254 i o n ( b a s e peak) r e s u l t i n g from t h e l o s s of CH2C1 and H .2 H C 1 ] + H20]+ - - H20] + (H2 + C H 2 C l ] + . [ 9 ] ) i s o b t a i n e d on a Finnigan 4000 mass s p e c t r o m e t e r w i t h i o n e l e c t r o n energy of 1 0 0 e V .HCl]' 270 [M + 2 H 254 [MH 232 [M . r e c o r d e d on Varian Mat 311 mass s p e c t r o m e t e r u s i n g i o n source p r e s s u r e of 10-6 T o r r . [ a ] .MOHAMMAD TARIQ AND ABDULLAH A.5 Mass Spectrum The e l e c t r o n impact ( E I ) mass spectrum o f chlorambucil a t 70 e V .

c 50.0 WIE 50 100 1so ZOO 250 Figure 8 : Mass Spectrum o f Chlorambucil ( E I ) . 300 .1oo.

I 1oo. I0 310 100 .c I 3 5O.C 'r MI€ 200 220 240 260 'i 2 a 300 320 Figure 9 : Mass Spectrum of Chlorambucil 340 (CI).

W 4 a. 4 4 0 m 0 w E rd +J .d s8 m a a. 4 E \ r . 0 PI 0 k a . M E \ 4 00 a.z 0 N 8 6N L W -N M 0 a.rl rd t.. k k 0 w w a . 4 E \ +z I' Y z-z u +z Ill db.

.0 m/e 254 H Scheme l b : Proposed mechanism of fragmentation of chlorambucil.

103 CHLORAMBUCIL 4.CH. Synthesis E v e r e t t et g (1) have s y n t h e s i z e d chlorambucil and some o t h e r n i t r o g e n mustard. The s y n t h e s i s have been r e p o r t e d (17.CH. C~CH~CH~ 2J Esterification Reduction Pd/CaCO. L 3 Ethylene oxide CH3COOH POC 1 CH2CH2CH2COOCH3 HOCH. a HzN L - I Hydrolysis P .18) as f o l l o w s : 0 N i tr a t ion CH2CH2CH2COOH a 02N -(Ot 02N CH2CH2CH2COOH CH2CH2CH2COOCH3 CH2CH2CH2COOCH3 0 HOCH2CH2 4 / a- / C1CH2CH2 ClCH CH \IN L CH2CH2CH2COOCH3 CH2CH2CH2COOH ClCH .

has been i d e n t i f i e d . S i x t y percent of drug r a d i o a c t i v i t y w a s excreted i n t o t h e u r i n e by 24 h r .l i f e of about 2. S i g n i f i c a n t drug binding t o gamma g l o b u l i n p r o t e i n s has been observed. The u l t i m a t e m e t a b o l i t e ( s ) of chlorambucil i n man has y e t t o be defined and f u r t h e r work i s r e q u i r e d t o confirm t h e s e i n i t i a l pharmacokinetic observations (21). with t h e m a j o r i t y of t h e remainder e x i s t i n g as t i s s u e as tissue-bound drug ( 2 2 ) .5 h r i n man. 2[ k-N. absorpt i o n from t h e g a s t r o . Although t h e i n t r a venous r o u t e produced higher c o n c e n t r a t i o n s . Pharmacokinetics 5. Metabolism and Excretion Oral absorption of chlorambucil i s adequate and r e l i a b l e . A n hour a f t e r a d m i n i s t r a t i o n by e i t h e r r o u t e t h e r a t e of metabolism w a s s i m i l a r and s u f f i c i e n t t o make a c o n t r i b u t i o n t o t h e a c t i v i t y of t h e drug ( 2 0 ) . D i s t r i b u t i o n . f a i r l y homogeneous t i s s u e d i s t r i b u t i o n w a s noted.MOHAMMAD TARIQ AND ABDULLAH A. a l s o c a l l e d phenylacetic mustard.1 Absorption. and a t one h r t h e h i g h e s t t i s s u e c o n c e n t r a t i o n i s found i n t h e l i v e r . This m e t a b o l i t e i s known t o have an a l k y l a t i n g a c t i o n i n animals. The drug h a s a l s o been shown t o d i s t r i b u t e w e l l i n t o a s c i t i c f l u i d a f t e r subcutaneous administ-ation. I n r a t s . Because a p o r t i o n of t h e drug molecule has l i p o p h i l i c . AL-BADR 104 5. McLean e t a1 ( 2 0 ) reported two m e t a b o l i t e s one w a s i d e n t i f i e d as t h e @-oxidation yroduct of chloramb u c i l . peakplasma concentrations were achieved i n 40 t o 7 0 minutes.i n t e s t i n a l t r a c t w a s consist e n t l y r a p i d and appeared t o be complete. However. Apparently no drug i s excreted i n t h e f e c e s . an aminophenyla c e t i c a c i d d e r i v a t i v e which has a h a l f . There appears t o be extensive metabolic degradation of t h e drug and a major m e t a b o l i t e .N-bis( 2-chloroethyl)aminophenyl] a c e t i c a c i d .l i f e i n plasma of approximately 90 minutes. and it i s almost completely metabolized ( 1 9 ) . r a d i o a c t i v e drug appear t o be c l e a r e d from t h e blood r a p i d l y . The drug has a h a l f . Dorr and W i l l i a m ( 2 1 ) e x t e n s i v e l y reviewed t h e d i s p o s i t i o n of chlorambucil i n human being and experimental animals.

i . The t o t a l dose on average i s about 400 mg per course but i n i n d i v i d u a l p a t i e n t s it v a r i e s g r e a t l y with t h e form of t h e d i s e a s e . i n p a r t i c u l a r . During t h e subsequent 5 y e a r s t h e substance w a s s t u d i e d i n q u i t e some d e t a i l . s p l e e n and l i v e r . remission can again be obtained i n some p a t i e n t s by t h e same drug ( 2 3 ) . Both regimens commonly include prednisone. A f t e r t h e onset of a r e l a p s e . The t h e r a p e u t i c uses of chlorambucil have been discussed by Larionov ( 2 3 ) . The t h e r a p e u t i c e f f e c t does not come a t once. e .CHLORAMBUCIL 105 p r o p e r t i e s . 0 mg/kg were w e l l t o l e r a t e d and d i d not produce undue marrow t o x i c i t y .2 mg/kg. i n p a r t i c u l a r . The drug may a l s o be administered on an i n t e r m i t t e n t b a s i s ( 0 . 6. The maintenance dose of t h e compound i s from 0. 5 2 . previous t r e a t m e n t . t h u s n e c e s s i t a t i n g withdrawal of t h e p r e p a r a t i o n .03 t o 0 . neutropenia and thrombocytopenia may appear. i n t h a t v a r i a n t known as f o l l i c u l a r lymphoma Galton and T i l l ( 2 4 ) Altman e t a1 ( 2 5 ) . On t h e i n t e r m i t t e n t schedule. 4 mg/kg every four weeks ( 2 8 ) . 1 mg/kg. The drug i s given o r a l l y i n t a b l e t form i n doses from 0 . Moore et a1 ( 8 ) used a continuous d a i l y dose of 0 . 1 0. 6 t o 25 mg d a i l y . Chlorambucil g i v e s t h e b e s t e f f e c t i n chronic lymphoid leumaemia both i n t h e leukaemic and aleukaemic form and. 1 t o 0. f a l l i n lymphocytosis and sometimes i n c r e a s e i n n e u t r o p h i l s i n t h e blood. Towards t h e end of t h e course . The remission l a s t s from 3 months t o 1-3 y e a r s . Therapeutic Uses C l i n i c a l t r i a l s of c h l o r m b u c i l were i n s t i t u t e d i n 1954. The e f f e c t c o n s i s t s remission expressed.4 mg/kg. i n r e d u c t i o n of t h e lymph nodes. i n c r e a s e d haemoglobin. I n chronic lymphoid leukaemias. e . t h e s t a g e . but u s u a l l y i n t h e 2nd t o 3rd week a f t e r s t a r t ing t h e drug. 6-12 mg d a i l y f o r 4-8 weeks. 1 0. t h e condition of t h e organism. i . Israels _ e t -a1 (261 Doan e t a1 ( 2 7 ) . The course of treatment u s u a l l y l a s t s 3-9 weeks.2 mg/kg. chlorambucil i s given i n doses of 0 . e t c . A n e f f e c t i v e biweekly dosing schedule . A t t h e s e doses s i d e e f f e c t s i n t h e g a s t r o i n t e s t i n a l t r a c t a r e r a r e l y observed. 2-6 mg d a i l y . i . some f a t ( d e p o t ) drug s t o r a g e may occur. monthly pulse doses as high as 1 . e . . This could e x p l a i n t h e prolonged c l i n i c a l e f f e c t of chlorambucil o c c a s i o n a l l y observed i n man.

AL-BADR 106 w i t h l e s s e n e d hematologic t o x i c i t y i s a l s o r e p o r t e d : 0. and lymphopenia. chlorambucil can a l s o cause a l v e o l a r d y s p l a s i a and pulmonary f i b r o s i s w i t h long-term u s e ( 3 3 ) . Thus myelosuppression i s t h e common d o s e . C e n t r a l nervous system s t i m u l a t i o n h a s occurred w i t h chlorambucil but i s uncommon u n l e s s l a r g e doses are used. Spermatogenesis i s a l s o depressed w h i l e t h e p a t i e n t i s on chlorambucil t h e r a p y ( 3 4 ) . o c c u r s w i t h prolonged u s e . may o c c u r . I r r e v e r s i b l e bone marrow damage.MOHAMMAD TARIQ AND ABDULLAH A. A good c l i n i c a l response t o drug d i s c o n t i n u a n c e and c o r t i c o s t e r o i d s w a s n o t e d i n t h i s c a s e . 7. 1 mg/kg increments t o t o x i c i t y o r remission ( 5 ) .4 mg/kg every 2 weeks. 3 0 ) . Lerner ( 3 2 ) has f u r t h e r a s s o c i a t e d c h r o n i c chlorambucil t h e r a p y w i t h a high i n c i d e n c e o f secondary a c u t e myelogenous leukemia. - S i m i l a r t o some o t h e r a l k y l a t i n g a g e n t s . Methods o f A n a l y s i s 8. i r r i t a b i l i t y . LD 0 i n rats i n a s i n g l e a d m i n i s t r a t i o n i s 21-23 mg/kg ( 3 5 ) . Bone marrow d e p r e s s i o n . shaking o c c a s i o n a l l y . however. 4 g of chlorambucil w i t h 1 0 m l o f 2 M hydrochloric a c i d and a l l o w t o s t a n d f o r t h i r t y minutes. i n c l u d i n g n e u t r o p e n i a .5 5 mg/kg have occurred without f a t a l i t i e s b u t w i t h moderate t o x i c i t y c h a r a c t e r i z e d by vomiting.1 Identification B r i t i s h Pharmacopoeia 1980 ( 9 ) d e s c r i b e s t h e following i d e n t i f i c a t i o n t e s t s : 1) Mix 0 . I n a d v e r t e n t t o x i c i n g e s t i o n s i n c h i l d r e n o f 1. Chromosomal damage i s w e l l documented f o r t h i s agent although t h e e x a c t mechanism i s not w e l l known ( 3 1 ) . thrombocytop e n i a . and h y p e r a c t i v i t y followed by l e t h a r g y and e v e n t u a l m i l d pancytopenia ( 2 9 . F i l t e r . 8. i n c r e a s i n g by 0 . wash t h e . a g i t a t i o n .l i m i t i n g t o x i c i t y with chlorambucil. G a s t r o i n t e s t i n a l d i s t r e s s w i t h l a r g e r doses o c c u r s but i s u s u a l l y not s e r i o u s l Rare h e p a t i t i s and d i s t u r b a n c e s o f l i v e r f u n c t i o n have a l s o been r e p o r t e d . Toxicity According t o Dorr and W i l l i a m ( 2 1 ) chlorambucil i s one of t h e b e s t t o l e r a t e d o r a l a l k y l a t i n g a g e n t s .

3 ) Mix 0. each o f 1 m l . . t h e n add 4 drops of s i l v e r n i t r a t e ( t e s t s o l u t i o n ) : no opalescence i s observed immediately (absence of c h l o r i d e i o n ) . Warm t h e s o l u t i o n on a steam b a t h : opalescence develops ( presecne o f i o n i s a b l e c h l o r i n e ) . C r y s t a l s of phenylhydrazinium c h l o r i d e are formed. a f t e r washing with two q u a n t i t i e s . e x h i b i t s maxima o n l y a t t h e same wavelength as t h a t o f a s i m i l a r s o l u t i o n o f USP Chlorambucil Reference Standard. To a f u r t h e r 1 0 m l add 0. a b u f f c o l o r e d p r e c i p i t a t e i s produced.6 g w i t h 0.CHLORAMBUCIL 107 r e s i d u e with two q u a n t i t i e s . 2 ) D i s s o l v e 50 mg i n 5 ml o f a c e t o n e . f i l t e r immediately. a c c u r a t e l y weighed. add 1 0 m l o f water.7 KPa (about 5 t o r r ) f o r t h r e e h o u r s . 5 m l of potassium m e r c u r i i o d i d e s o l u t i o n . Pharmacopeia XX ( 1 4 ) d e s c r i b e t h e f o l l o w i n g t e s t s f o r t h e i d e n t i f i c a t i o n o f chlorambucil: 1) The i n f r a r e d a b s o r p t i o n spectrum of a 1 i n 125 s o l u t i o n i n carbon d i s u l f i d e . a f t e r d r y i n g over phosphorous pentoxide a t a p r e s s u r e not exceedi n g 0. o f a b s o l u t e e t h a n o l and d r y i n g a t 105O. h e a t f o r twenty seconds. and a l l o w t o stand f o r t h i r t y minutes.2 T i t r i m e t r i c Methods U . i n 1 0 ml o f a c e t o n e . which. Add 1 drop of 2 N s u l f u r i c a c i d . S . add 0 .S. 2) To 1 0 ml of t h e mixed f i l t r a t e and washings r e s e r v e d i n t e s t f o r i d e n t i f i c a t i o n . and d i l u t e w i t h water t o 1 0 m l . s t i r r i n g o c c a s i o n a l l y . o f w a t e r . Melting p o i n t o f t h e r e s i d u e . about 1 4 6 O .5 m l of potassium permanganate solution t h e purple color i s discharged. Pharmacopeia XX ( 1 4 ) d e s c r i b e s t h e following method: Dissolve about 200 mg o f c h l o r a m b u c i l . each o f 1 0 m l . and r e s e r v e t h e mixed f i l t r a t e s and washi n g s for t e s t f o r i d e n t i f i c a t i o n 2. 8.2 m l o f phenylhydrazine. U. i n a 1 mm c e l l . w i t h decomposition. add 2 m l of a b s o l u t e e t h a n o l . h e a t i n a water b a t h f o r t e n minutes. have a m e l t i n g p o i n t of about 245' .

0 ) (1m l ) . Carry o u t a b l a n k determination. t r a n s f e r t h e m i x t u r e t o a 100-ml graduated f l a s k w i t h t h e a i d of w a t e r . add 20 m l of M sodium hydroxide and b o i l under a r e f l u x condenser f o r one hour. u s i n g 3 m l o f ammonium i r o n (111) s u l f a t e s o l u t i o n as i n d i c a t o r .3.3 Spectrophotometric Methods 8. d i s s o l v e d i n a 5% s o l u t i o n of dimethylacetamide i n e i t h e r e t h a n o l o r 0. F i l t e r and t i t r a t e t h e excess of s i l v e r n i t r a t e i n 50 m l o f t h e f i l t r a t e w i t h 0 .2 I n f r a r e d S p e c t r o m e t r i c Method Kozlov and Sbezhneva (37) r e p o r t e d an i n f r a r e d s p e c t r o s c o p i c a n a l y s i s of chloramb u c i l i n pharmaceutical p r e p a r a t i o n s . Mix t h e drug (10 t o 70 ug) . mix. 8. 1 N sodium hydroxide VS. B r i t i s h Pharmacopoeia f o l l o w i n g method: 1980 ( 9 ) d e s c r l b e s t h e To 0. add N-KOH i n 90% e t h a n o l ( 0 . Cool. 5% b ( p . 1 N sodium hydroxide i s e q u i v a l e n t t o 30. and add s u f f i c i e n t water t o produce 100 m l . 1 nil).1 M s i l v e r n i t r a t e VS i s e q u i v a l e n t t o 0. 1 M ammonium t h i o c y a n a t e VS. with phthalate buffer s o lu tio n (pH 4 . S p e c i a l l y t o determine t h e amount of a c t i v e . d i l u t e w i t h e t h a n o l a t 600 mp w i t h i n 2 o r 3 min.3. AL-BADR 108 and t i t r a t e w i t h 0 .n i t r o b e n z y 1 ) p y r i d i n e s o l u t i o n i n acetone (1 m l ) and 0.01521 g of C14H19C12N02* 8.9% N a C l s o l u t i o n . Dopan. and d i l u t e w i t h e t h a n o l t o 4 m l . 1 M s i l v e r n i t r a t e VS and 5 m l o f n i t r i c a c i d . c o o l i n i c e . U r a c i l Mustard and Mustargen i n plasma. Each m l o f 0.5 g of chlorambucil. add 50 m l o f 0 .MOHAMMAD TARIQ AND ABDULLAH A. Each m l of 0 .1 C o l o r i m e t r i c Method P e t e r i n g and Van Giessen ( 3 6 ) d e s c r i b e d a c o l o r i m e t r i c procedure f o r d e t e r m i n a t i o n o f n i t r o g e n mustards i n c l u d i n g Chlorambucil . u s i n g p h e n o l p h t h a l e i n TS as t h e i n d i c a t o r .9% N a C l s o l u t i o n (1m l ) .42 mg o f C14HlgC12N02. Heat a t 80' f o r 20 min. .

T r i m e t h y l s i l y l a t e d chlorambucil i s . 0 .( 2 chloroethyl)ar. T h i s i s submitted t o GLC column ( 5 0 cm X 2 mm) of 3% of OV-17 on gas Chrom Q ( 1 0 0 t o 1 2 0 mesh) a f t e r 40 seconds a t 190°C t h e column t e m p e r a t u r e i s r a p i d l y r a i s e d t o 31OoC. The c o n c e n t r a t i o n o f t h e drug i s c a l c u l a t e d u s i n g a c a l i b r a t i o n curve. The f i l t e r e d e x t r a c t w a s d i l u t e d t o 50 m l w i t h e t h a n o l . w i t h p u r i f i e d N2 a t room t e m p e r a t u r e and s e v e r a l drops o f methylene c h l o r i d e were added t o d r y r e s i d u e t o form t r i m e t h y l s i l y l d e r i v a t i v e .3.4 Mass S p e c t r o m e t r i c Methods e t -a1 (39) developed a s e n s i t i v e and Chang _ s p e c i f i c method f o r t h e d e t e r m i n a t i o n of chlorambucil and i t s m e t a b o l i t e s i n b i o l o g i c a l samples. 7 0 eV-m. 1 g of sample of t h e p r e p a r a t i o n i s e x t r a c t e d i n 100 m l o f acetone and t h e absorbance o f a l i q u o t i s measured a t 770 cm-1. The method i s based on s e l e c t e d i o n monitoring d e t e c t i o n f o l l o w i n g simple e x t r a c t i o n of t h e p a r e n t compound. i t s m e t a b o l i t e and i n t e r n a l s t a n d a r d (Chlorambucil-d8) from plasma and u r i n e samples.3.ino]phenyl a c e t i c a c i d i n 0. To determine chlorambucil and b [ b i s .5 m l plasma or u r i n e add 0.s detection i s u s e d . 8.109 CHLORAMBUCIL 11 8. A p o r t i o n of t h i s s o l u t i o n w a s f u r t h e r d i l u t e d as r e q u i r e d w i t h e t h a n o l and t h e absorbance measured a t 250 nm v s e t h a n o l .5 m l of 4% HCl04 ( p e r c h l o r i c a c i d ) and [2H8] chlorambucil as i n t e r n a l s t a n d a r d . The e r r o r does not exceed f 1%.3 ingredients i n carcinostatic preparations c o n t a i n i n g chlorambucil. U l t r a v i o l e t Spectrometric )Jethod El-Tarras e t a 1 (38) developed a simple method f o r t h e d e t e r m i n a t i o n of chlorambucil i n pharmaceutical p r e p a r a t i o n s . The powdered t a b l e t s are e x t r a c t e d i n t o e t h a n o l (4 X 1 0 ml). The mixture i s e x t r a c t e d w i t h e t h y l a c e t a t e and The o r g a n i c l a y e r w a s d r i e d h e x m e (1:l).

1 M b o r a t e b u f f e r (pH 9 . It i s t h e n . AL-BADR 110 monitored a t m / e 3?6 and 328. A Varian MAT mass spectrometer i n combination w i t h Varian Aerograph 1400 g a s chromatograph with a column o f 0.3%. A f t e r developing and drying t h e chromatogram. The d e t e c t i o n l i m i t i s about 8 ng/ml f o r b o t h d e t e r m i n a t i o n s and t h e r e l a t i v e s t a n d a r d d e v i a t i o n 3% f o r chlorambucil and 5% prednimustine a t 30-60 ng/rnl l e v e l . The compounds are determined u s i n g fragmentography.4. are used. and t h a t of t h e i n t e r n a l s t a n d a r d a t m / e 383 and 385.4 Chromatographic Methods 8. The mean recovery and s t a n d a r d d e v i a t i o n were 94. it i s sprayed w i t h 3% s o l u t i o n of r e a g e n t i n acetophenone dimethylformamide ( 7 : 3 ) c o n t a i n i n g 0 . t h e d e r i v a t i v e of t h e m e t a b o l i t e of chlorambucil a t m / e 298. hexane ( 3 : 7 ) and s e p a r a t i o n i s done by p a r t i t i o n a f t e r adding 0 . prepared by i n t r o d u c i n g e i g h t 2H atoms i n t o t h e b i s . Analogues. 0 ) . * et a1 ( 4 0 ) d e s c r i b e d an a n a l y t i c a l Jakhammer procedure f o r simultaneous d e t e r m i n a t i o n of chlorambucil and prednimustine i n plasma. The s p o t i s a p p l i e d on a s h e e t o r precoated p l a t e s . The drug samples are d i s s o l v e d i n methanol.3 1.5% o f Carbowax 20 M on Chromosorb W-HP.1 Thin Layer Chromatograph (TLC) Norpoth e t a1 ( 4 1 ) d e s c r i b e d a TLC method for t h e d e t e r m i n a t i o n o f a l k y l a t i n g cytos t a t i c a g e n t s on a t h i n l a y e r p l a t e w i t h i s o n i c o t i n a l d e h y d e benzothiazol-2-ylhydrazone. Chlorambucil i n t h e aqueous phase add predenimustine i n o r g a n i c phase are each t r a n s e s t e r i f i e d w i t h methanol-3F3 d i e t h y l e t h e r a t e ( 4 : l ) .( 2-chloroethy1)amino group of b o t h compounds are used as i n t e r n a l s t a n d a r d s and c a r r i e r s . 8. 1 M phosphate 0 . The method i n v o l v e s e x t r a c t i o n and s e p a r a t i o n followed by d e r i v a t i z a t i o n t o chloramb u c i l methyl e s t e r and mass fragmentography. The drugs are e x t r a c t e d i n t o chloroform. 0 1 m l of c o n c e n t r a t i o n H C 1 p e r 100 m l .MOHAMMAD TARIQ AND ABDULLAH A.

The organic phase w a s s e p a r a t e d and e x t r a c t e d with 0 .91m l ) was added. 5 m l of 0 . Chlorambucil produces r e d s p o t . The aqueous phase w a s s e p a r a t e d and h e a t e d f o r 1 5 minutes a t 80°C t o c o n v e r t chlorambucil i n t o t e t r a h y d r o t h i a z i n e d e r i v a t i v e .05 m l a l l y l b r o m i d e were added and t h e mixture w a s shaken f o r 30 min a t 2 5 O C . s u l p h i d e f o r 5 minutes.5 M t e t r a b u t y l ammonium. 1 M. To t h i s O . d e t e c t i o n . 0 5 m l o f 12 M NaOH. 2 m l of 0. 2 m l ) .2 Gas Liquid Chromatography ( G L C ) Ehrsson _ et _ a1 (42) d e s c r i b e d a GLC t e c h n i q u e f o r d e t e r m i n a t i o n of chlorambucil i n plasma by g a s . An a l i q u o t o f o r g a n i c phase was s e p a r a t e d and evaporated t o dryness with n i t r o g e n g a s .l i q u i d chromatography w i t h s e l e c t e d ion-monitoring. For q u a n t i t a t i v e d e t e r m i n a t i o n t h e c o l o r i n t e n s i t y o f t h e spot i s measured u s i n g chromatogram spectrophotometer . The o r g a n i c phase (1-2 mcg) w a s i n j e c t e d i n GLC ( a t Z50°C) on a column of 5% of OV-17 on gas-Chrome-Q o p e r a t e d with 70 eV. The r e s i d u e w a s d i s s o l v e d i n methanol-water 0 . The i n t e r n a l s t a n d a r d used w a s [%8] chlorambucil.111 CHLORAMBUCIL p l a c e d over a b a t h o f acetophenone and b a t h and p l a t e are h e a t e d a t 2OO0C for 20 minutes.2 ml methylene c h l o r i d e and 0. 0. phosphoric a c i d . then c o n c e n t r a t e d phosphoric a c i d (0. 1 M sod. a c e t i c a c i d (75:15:10) and a n a l y s e d on g a s chromatography. 8.~g o f chlorambucil p e r m l w a s mixed w i t h h y d r o c h l o r i c a c i d . or phosphate b u f f e r ( 0 . 2 m l o f plasma sample c o n t a i n i n g 8 l.4. The mixture w a s e x t r a c t e d with methylene c h l o r i d e ( 2 m l ) f o r 30 minutes. 0 . t h e cooled p l a t e i s t h e n sprayed w i t h t r i e t h y l a m i n e . s .m. The peaks were monitored a t m / e 305 and 313 f o r chlorambucil and . A l t e r n a t i v e l y t h e plasma samples w e r e a d j u s t e d t o pH 3 w i t h 1 M phosphoric a c i d and e x t r a c t e d w i t h e t h y l e n e d i c h l o r i d e f o r 30 minutes. and H2S removed w i t h N2 f o r 5 minutes.

The a p p a r a t u s c o n s i s t e d o f a Waters A s s o c i a t e s ~ 6 0 0 0pump. The c o e f f i c i e n t of v a r i a t i o n f o r 10ng/ml o f chlorambucil w a s 5%.MOHAMMAD TARIQ AND ABDULLAH A.6 8 O C . Leff and Bardsley ( 4 4 ) have r e p o r t e d pharmacokinetic of chlorambucil i n o v a r i a n carcinoma u s i n g a new HPLC assay. a Waters A s s o c i a t e s Model U6K i n j e c t o r . The absorbance o f e l u t e w a s monitored a t 254. a 10 x 0.4. phenyla c e t i c mustard and predenimustine i n human plasma. The t u b e s w e r e c e n t r i f u g e d a t 600 pg f o r 10 minutes a t 4 O C u n t i l t h e thawed aqueous phase c o u l d be s e p a r a t e d . L i n e a r g r a d i e n t o r e l u t i o n w a s done at 2 m l p e r minute w i t h aqueous 0. 2 m l of d r i e d e t h y l a c e t a t e e x t r a c t was evaporated t o dryness i n a stream o f n i t r o g e n at 4 5 O C . a me-Unicam LC3 v a r i a b l e .1 Bondapak c18 column.a c e t i c a c i d (from 40 t o 0 % ) i n methanol d u r i n g 1 0 minutes followed by i s o c r a t i c e l u t i o n w i t h methanol. One m l plasma w a s e x t r a c t e d w i t h 2 ml of e t h y l a c e t a t e . T h i s method gave a good s e p a r a t i o n of chloramb u c i l i n l e s s t h a n 1 2 m i n u t e s . t h e pooled e t h y l a c e t a t e e x t r a c t s were d r i e d over anhydrous sodium s u l p h a t e f o r 1 hour a t room t e m p e r a t u r e .175 M . 8. t h e emulsion formed by vigorous a g i t a t i o n on a Whirlimixer was f r o z e n by immersing t h e t u b e s i n a methanol carbondioxide b a t h a t . D e t e c t o r response w a s r e c t i l i n e a r over a range o f 5 t o 1000 ng o f t h e compound.11of t h e e x t r a c t w a s i n j e c t e d on a 1. The r e s i d u e w a s d i s s o l v e d i n 100 m l o f e t h y l a c e t a t e f o r chromatographic a n a l y s i s .46 cm s t e e l column packed w i t h S 5 ODs.05 t o 10m.3 High-pressure Liquid Chromatography (HPLC) Newel1 et a1 ( 4 3 ) developed a HPLC method f o r e s t i m a t i o n o f c h l o r a m b u c i l . The recovery o f chlorambucil w a s c o n s t a n t over t h e range o f 0. AL-BADR 112 2 [ H8] chlorambucil r e s p e c t i v e l y . 50 1. The procedure w a s r e p e a t e d with f u r t h e r 2 m l of e th y la c e ta te . and 280 nm simultaneously.

A p a r t i s i l PXS-10/25 ODS column ( 2 5 cm X 4. 0 1 M a c e t a t e b u f f e r . The mobile phase was water: methanol 20:80 ( v / v ) w i t h 0. f o r f a s t e r e l u t i o n .4-( 4-( 2- chloroethyl-2-hydroxyethylamino)-phenyl) b u t y r i c acid.1% ammonium a c e t a t e ( w / v ) The flow r a t e used w a s 1 ml min-1 ( = 500 p s i ) .5 ( 6 5 : 5 : 3 0 ) .32 a u f s (254 nm d e t e c t o r ) . . Zakaria and Brown ( 4 5 ) r e p o r t e d a r a p i d a s s a y f o r plasma chlorambucil and i t s m e t a b o l i t e ( p h e n y l a c e t i c mustard) u s i n g reversed-phase l i q u i d chromatography. C a l i b r a t i o n graphs are r e c t i l i n e a r over t h e range o f i n t e r e s t . Ekrsson e t a1 ( 4 7 ) d e s c r i b e d a reverse phase HPLC method t o s t u d y d e g r a d a t i o n o f chlorambucil i n aqueous s o l u t i o n . d .113 CHLORAMBUCIL wavelength W d e t e c t o r o p e r a t i n g at 254 nm.6 mm) i s used for t h e a n a l y s i s .0 . S e p a r a t i o n was performed on a reversed-phase column (Zorbax C-8. Plasma ( 2 0 o r 30 ~ 1 i)s i n j e c t e d d i r e c t l y i n t h e chromatographic system. D e l . 0 m l min-l. a chromegabond MC-18 column ( 1 5 cm X 4. ) and m e t h a n o l . Dupont I n s t r u m e n t s . 6 ml/min was used as t h e mobile phase. The mobile phase being methanol .a c e t o n i t r i l e . and t h e r e c o r d e r w a s set a t 0. are determined i n aqueous methanol. Chloramb u c i l and i t s d e g r a d a t i o n products. The chlorambucil s o l u t i o n i s . 5 o r 1 . W i l l i n g t o n .0. Simultaneous d e t e c t i o n a t 280 and 254 nm a l l o w s picomole amounts t o be determined. pH 4.6 mm i . r e s p e c t i v e l y . a t a flow rate of 1 . A 1 0 p1 full l o o p volume w a s q u a n t i t a t i v e l y i n j e c t e d . The highp r e s s u r e l i q u i d chromatograph w a s equipped w i t h a fixed-volume l o o p i n j e c t o r and a f i x e d wavelength d e t e c t o r .6 mm) o r . r e s p e c t i v e l y ) a t 1 . 6 pm average p a r t i c l e s i z e . which i n c o r p o r a t e s a 5 cm Co: P e l 1 ODS pre-column f o r sample c l e a n up. A 250 X 4. reversed-phase column w a s used. Chatterji _ et _ a1 ( 4 6 ) have s t u d i e d k i n e t i c s of chlorambucil h y d r o l y s i s u s i n g highp r e s s u r e l i q u i d chromatography.02 M KH2P04 (1:l o r 11:9.

Reversed-phase HPLC a n a l y s i s i s done on a column (15 cm X 4 mm) o f Li-Chrosorb RP-18 ( 5 urn). Absorbance was monitored a t 263 nm and recorded on a Data Module. Tanvir A . Methanollwater c o n t a i n i n g 0. A t d i f f e r e n t t i m e i n t e r v a l s e x t r a c t i o n i s done with e t h y l a c e t a t e .7 nm w a s used f o r d e t e r m i n a t i o n o f t h e concentration. Syed A l i Jawad f o r t h e t e c h n i c a l a s s i s t a n c e and Mr. .01 M phosphoric a c i d i s used as mobile phase. The t u b e s w e r e c e n t r i fuged f o r 2 minutes and t h e s u p e r n a t e n t w a s r a p i d l y f r o z e n i n s o l i d carbondioxide-acetone t o complete d e p r o t e i n i s a t i o n .2% a c e t i c a c i d (13:7) as mobile phase. 2 m l under a n i t r o g e n atmosphere. The a l i q u o t s o o b t a i n e d w a s analysed on Water's Radial-PAK c18 ( 1 0 u m ) c a r t r i d g e . Ahmed _ et _ a1 (48) d e s c r i b e d a q u a n t i t a t i v e method for d e t e r m i n a t i o n of chlorambucil i n plasma by r e v e r sed-phas e h i gh-pe r f ormanc e l i q u i d chromatography. t h e solvent i s evaporated t o 0 . Acknowledgement The a u t h o r s would l i k e t o t h a n k Mr.114 MOHAMMAD TARlQ AND ABDULLAH A. The plasma samples a r e d e p r o t e i n i s e d by adding 4 volumes o f a c e t o n i t r i l e a t pH 3 . A W d e t e c t o r a d j u s t e d a t 253. AL-BADR prepared by adding 1 0 mg o f compound i n 0. The t u b e s a r e c e n t r i f u g e d for 2 m i n u t e s .5 m l methanol and t h e volume i s made upto 100 m l with d i s t i l l e d water. w i t h a c e t o n i t r i l e 0. A s t a n d a r d curve was developed u s i n g a c e t o n i t r i l e s o l u t i o n o f chlorambucil s t a n d a r d . B u t t f o r t h e s e c r e t a r i a l a s s i s t a n c e i n t y p i n g t h e manuscript.

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Analytical Control McNeil Pharmaceutical Spring House.CHLORZOXAZONE James T. . Stability 7. Taste 3.4 Mass Spectra 4. Janicki Director.7 Melting Range 4.1 Infrared Spectrum 4.7 High Performance Liquid Chromatography 5. Molecular Weight 2. Odor. Formula.5 X-Ray Diffraction Pattern 4.6 Thermal Analysis 4. Georgia Casimir A.2 Ultraviolet Absorption Spectrum 4. Synthesis 4 . All rights of reproduction in any form reserved. Stewart Department of Medicinal Chemistry and Pharmacognosy College of Pharmacy University of Georgia Athens.1 Names.4 Gas Chromatography 5. Pharmacokinetics. Inc.9 Colorimetry 5.11 Polarizing Microscopy 5. Pennsylvania Foreword.10 Color Test 5.8 Solubility 4.3 Nuclear Magnetic Resonance Spectra 4. 2.8 Non-Aqueous Titrimetry 5.1 Fluorescence Spectrophotometry 5.3 Iodometric Titrimetry 5. Therapeutic Category Description 2.9 pKa 5 .6 Paper Chromatography 5. Physical Properties 4.2 Appearance. History. Drug Metabolic Products.12 Potentiometry 6 . ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 16 119 Copyright 0 1987 by Academic Press.2 Ultraviolet Spectrophotometry 5. and Bioavailabi1ity 1. Methods of Analysis 5. Color.5 Thin-Layer Chromatography 5.

5 High Performance Liquid Chromatography 10. Identification and Determination in Body Fluids and Tissues 8.11 Urine Metabolism 7.3 High Performance Liquid Chromatography 8.1 Drug Metabolic Products 7.4 Potentiometry 9. JANICKI 120 7.2 Pharmacokinetics and Bioavailability 8.4 Gas Chromatography 9.2 Ultraviolet Spectrophotometry 9. STEWART AND CASIMIR A. Identification and Determination in Pharmaceuticals 9.1 Colorimetry 9.1 Ultraviolet Spectrophotometry 8.JAMES T.3 Fluorescence Spectrophotometry 9.2 Fluorescence Spectrophotometry 8.12 In-vitro Metabolism 7. References .

CHLORZOXAZOIW 1. myositis. and muscle strains. Chlorzoxazone was ranked among the top 100 most widely prescribed drugs for many years ( 3 ) . molecular weight Generic name . Description 2. 2-Hydroxy-5-Chlorobenzoxazole. Chemical Abstracts Registry No. The drug was reported to be useful in the treatment of painful spasm of skeletal motor muscles by acting at spinal levels of the central nervous system (2). Taste . .Odorless colorless crystals or a white to creamy white crystalline powder with a bitter taste. The skeletal muscle relaxant properties were first discovered by Marsh at McNeil Laboratories In the late 1950s (1). The drug exhibits minimal adverse effects and almost no gastrointestinal irritation. Therapeutic Category Chlorzoxazone is a centrally active muscle relaxant for the treatment of painful muscle spasms associated with muscloskeletal disorders.2 Appearance. such as fibrositis. 169. Onset of therapeutic activity is observed within one hour and the duration usually lasts up to 6 hours. Chemical names . History. 5-Chloro-2hydroxybenzoxazole.58 2.1 Names. under the trade name. 5-Chlorobenzoxazolidone.5 Chloro-2-(3H)-benzoxazolone. formula. 5-Chloro-2-benzoxazolol. 121 Foreword. Color.Chlorzoxazone Trade name Marketed by McNeil Pharmaceutical -__. Wt. Odor. sprains. spondylitis. 2. Paraflex. 95-25-0 - C7H4C1N02 Mol. 5-Chlorbenzoxazolin-2-one. bursitis.

1 Infrared I SDectrum .8 5.5 34.JAMES T.4 33.8 16. STEWART AND CASIMIR A.1 23.1 67.6 36.7 19.2 79. It can also be prepared from the reaction of sodium ethoxide with 2-ethoxycarbonylamino4-chlorophenol in tetralin (6). ClCOCl Physical Properties 4.7 .0 6.8 49.4 69.7 37. Synthesis Chlorzoxazone is synthesized by either the acid hydrolysis of 2-amino-5-chlorobenzoxazole (4) or by treating 2-amino-4-chlorophenol with phosgene in ethyl acetate (5).8 1330 1300 1262 1153 1101 1064 964 923 867 845 805 53.4 70. -1 cm 3470 3155 3117 3087 3057 2978 2282 1924 1885 1772 1621 1482 1463 %T 72. t 4.3 26.8 41.1 66.7 54.6 70.1 58.0 67.1 46.6 27.2 30.An FTIR sDectrum of chlorzoxazone is shown in Figure 1.8 713 600 591 552 417 394 382 352 234 216 210 32.2 45.5 13.3 39.2 46.2 3. JANICKI 122 3.3 -1 cm % T 1367 43.3 35.2 21.2 -1 % T cm 732 52.1 8.

.. I I i i I i I i Fig.... 1 FTIR spectrum of chlorzoxazone .11i - I rI r I rI I I I I I r ... .

JAMES T.7 f 131.2 C NH d Proton NMR Proton fi (DDm) r a 2.A solution of the drug in absolute methanol shows a maximum at 282 ? 2 nm and a m i n i m u m a i 248 _t 2 nm.36 HDO C 7.1 h 154.7 g 142.4 DMSO-dc J b 109.3. -- 4. There is an inflection at about 228 GII! ( s e e Figure 2). JANICKI 124 4.8 C 110.8 ~~ ~ Y .2 Ultraviolet Absorption Spectrum .13 e 7.4 e 127. Nuclear lvlagnetic hpectra .10 d 7. b 3.r --I f .Resenance nuclear magnetic resonance spectra are shown in Figures 3 and 4.28 f 11.8 d 121.49 DMSO-d. b C 13C NMR Carbon 6(ppm) a 39.13C and proton . STEWART AND CASIMIR A.

2 W spectrum of chlorzoxazone in methanol .CHLORZOXAZONE 350 220 WAVELENGTH ( n m ) Fig.

3 13C-NMR spectrum of chlorzoxazone in deuterated dimethylsulfoxide .I Fig.

1 3....r I 8.1 2. 4 .1 PPM 5.1 6..-.1 1.--I---* .1 Fig....1 .I 7.0 'H-NMR spectrum of chlorzoxazone in deuterated dimethylsulfoxide .7 7 1d.127 "-2.0 4.

JANICKI 128 Spectra .7 Melting Range .4-dihydroxybenzaldehyde. Fluorescamine gave the highest intensity product and was used to analyze chlorzoxazone in the 0.3 mg/ml). potassium bromide and potassium bromate) with 0. 4. Methods of Analysis 5.4 pg/ml range. soluble in aqueous solutions of ammonia and alkali hydroxides. 5.3.4 Gas Chromatography - Kaempe has chromatographed chlorzoxazone on a packed 15% Dexsil 300 on HP .Conney et a1 utilized a back-extraction approach to quantitate chlorzoxazone at 289 nm (9). fluorescamine.Chlorzoxazone is very slightly soluble in water (0. 4. sparingly soluble in ethanol and methanol. 4.1 N sodium thiosulfate after potassium iodide was added to a cooled solution (11). respectively (8).4 EM ionization (CI) mass spectra are shown in Figures 5 and 6. STEWART AND CASIMIR A.0.3. 5. and salicylaldehyde to form fluorophores (7). The same authors also reported that chlorzoxazone exhibits native fluorescence in chloroform using excitation and emission wavelengths of 286 and 310 nm.The melting point range is 189-194°C.27 . 4.2 .2 Ultraviolet Spectrophotometry .A DSC thermogram of chlorzoxazone is shown in Figure 8.6 Thermal Analysis .3 * - Iodometric Titrimetr Beral et a1 titrated c h l o r z o x a z o d t r e a t m e n t with boiling 10% sulfuric acid.9 pKa - The pKa of chlorzoxazone in 8. 2. The drug can also be directly determined at 282 nm in absolute methanol (10) 5. soluble in chloroform (1 g in It is freely 250 ml) and ether (1 g in 60 ml). 5.Electron impact (EI) and chemical 4.JAMES T.5 X-Ray Diffraction . 4.Stewart and Chan have studied the reaction of chlorzoxazone with dansyl chloride.1 Fluorescence Spectrophotometry .8 Solubility .Figure 7 shows an X-ray diffraction pattern for chlorzoxazone powder.

. I.[ASS 130 200 40 60 Fig.. I 140 .. I. .. I . . . .11 9 90 80 - 70 - I 60 50 - 113 30 - 20 51 63 d I d.. 5 Electron impact (EI) mass spectrum of chlorzoxazone 30 100 120 160 .

MH+ '0°1 80 - 60 - %I a !! 50 Fig. 6 90 I30 m/e I 70 210 250 Chemical ionization (CI) mass spectrum of chlorzoxazone .

90 - 80 - 70 - 60 - 50 - e W e 1 Fig. 7 X-Ray diffraction pattern of chlorzoxazone .

MI TEMPERATURE (K) Fig.5. SCAN RATE: 0.M 42O.00 deg/rnxn 380.00 ' L O T 2933 1.00 mg WT: *. MI 5.00 5th 01 DSC .00 7 CHLORZOXAZONE.M 4M. 8 DSC thermogram of chlorzoxazone 460.M 4 h no bu).

Parker et a1 attempted to chromatograph the drug on SE-30 (0.reported that chlorzoxazone can be chromatographed on a 2.d.4 m i.d. Retention time was 0.CHLORZOXAZONE 133 Chromosorb W 801100 mesh glass column (6 ft x 6.5% SE-30 and 3% OV-17 at 200 and 220°C column temperatures. respectively (17).69 relative to diphenhydramine.5% SE-30 on 80/100 mesh Chromosorb W acid-washed HMDS treated 5 ft x 4 mm i. Nitrogen was used as carrier gas at 50 ml/min a. Beckett et a1 . glass column packed with 3% OV-1 on 60/80 mesh Gas Chrom Q (14).00 1. Desiraju et a1 chromatographed chlorzoxazone on a 6 ft x 4 mm i. 5. They used the retention data to perform qualitative analysis of the drug based on Kovat's Retention Indices. glass column containing 2.62 relative to caffeine using a column temperature of 220°C (12).5% SE-30 on 80/100 mesh Chromosorb G acid-washed HMOs treated will separate chlorzoxazone using a column temperature of 173°C (16). glass column at a column temperature of 225°C (15). Retention time is given relative to n-alkanes.nd FID detection was utilized.05% on glass microbeads 60/80 mesh) at 150 and 165°C column temperatures with no success (13).5 Thin-Layer Chromatography Stationary Phase Mobile Phase Silica Gel HF254 Chloroform-methanol Chloroform-methanol Chloroform-methanol Chloroform-methanol Chloroform-methanol Kieselgel 60F254 19:l 9:l 4:l 7:3 3:2 Toluene ethylacetate 85% formic acid (50:45:5) 18 0. The retention time was 0.69 19 .d.25 minutes using a column flow rate of 30 ml/minute. Pedroso and Moraes have chromatographed chlorzoxazone on 2. Ardrey and Moffat also reported that a 2 m x 4 mm i.87 1.58 0.).00 0.d.32 0. Column temperature was 130°C and retention time was 3.

95 Bonded C18F Methanol 0.35 0.55 - 19 Toluene-dioxane methanol ammonium hydroxide (20:50:20:10) Toluene Acetone 2N Acetic Acid (30:65:5) - - - 0. JANICKI 0.6819 - Toluene Isopropanol concd.8320 20 Silica Gel 60F254 Toluene Isopropanol Ethyl Acetate 2N Acetic Acid (10:35:35:20) 0.32 20 0.134 Kieselgel 60F254 Kieselgel 60F254 Silica Gel 60F254 JAMES T.5% Phosphoric Acid 3% Sodium Chloride ( 4 0 : 10:50) Bonded C18F Isopropanol Methanol 0.5% Phosphoric Acid 3% Sodium Chloride (23:23:10:44) Bonded C18F Bonded C18F - - - Tetrahydrofuran Methanol 0.33 20 20 0. STEWART AND CASIMIR A.5% Phosphoric Acid 3% Sodium Chloride (28:28:10:34) Methanol 2 N Ammonia 3% Sodium Chloride (60:10:30) - 20 0.3220 Bonded C18F Isopropanol 0.60 . ammonium hydroxide (70:29 :1) 0.5% Phosphoric Acid 3% Sodium Chloride (60:10:30) 0.

4720 Isopropanol Methanol 2N Ammonia 3% Sodium Chloride (23:23:10:44) - 0.Clarke reported that chlorzoxazone gave an R of 0.6 Isopropanol 2N Ammonia 3% Sodium Chloride (40:10:50) 0.93 using a mobile phase of 87 :13 n-butanol-waEer containing 0.35 Methanol Isopropanol Tetrahydrofuran 3 % Sodium Chloride (17:17:17:49) 0.32 Ace tone 3% Sodium Chloride (50:50) 0.4320 Tetrahydrofuran Methanol 2N Ammonia 3% Sodium Chloride (28:28:10:34) 0.4620 Methanol 3 % Sodium Chloride (60 :40) 0.5520 - Acetonitrile 3% Sodium Chloride (50:50) 0.36 20 20 20 20 Paper Chromatography .18 Isopropanol 3% Sodium Chloride (40:60) 0.25 Tetrahydrofuran 3% Sodium Chloride (43:57) 20 0.48% citric acid (21) * .135 CHLORZOXAZONE Bonded C18F Bonded C18F Bonded C18F Bonded C18F Bonded C18F Bonded C18F Bonded C18F Bonded C18F Bonded C18F 5.

A line r calibrati n plot was obtained within the 3 x 10-8 to 5 x 10-3 M.8 5. The drug can also be titrated in dimethylsulfoxide using O.JAMES T. 5.Chlorzoxazone is titrated with 0.10 Color Test .The Koppanyi-Zwikker -violet color (27). Chlorzoxazone was effectively separated from acetaminophen in a mixture with 50:50 methanol-water at a 2. Non-Aqueous Titrimetry .12 test gives a - Potentiometry A potentiometric method for the determination of chlorzoxazone. Storage at temperatures for . and a 1:l acetonitrile-water mixture (10).Sanghoi reported a colorimetric procedure for chlorzoxazone based on base hydrolysis of the drug followed by diazotization of the product with nitrous acid formed -in situ (26).11 Polarizing Microscopy .Stewart a1 have chromatographed chlorzoxazone on an octadecylsilane column using varying proportions of absolute methanol-distilled water (22).7 5.range of chlorzoxazone. 5.Watanabe et al.0 ml/min flow rate using refractive index detection. 6 Stability Chlorzoxazone is stable in the solid state for up to 5 years at room temperature. Colorimetry . The drug has also been chromatographed on an octadecylsilane column using 60:40 watermethanol (23).9 High Performance Liquid Chromatography . JANICKI 136 5. 5. Upon refluxing the drug with 3N sodium hydroxide. STEWART AND CASIMIR A.1 N sodium methoxide in dimethvlformamide using thymol blue as indicator ( 2 4 ) . These methods utilized ultraviolet detection at 280 nm. Acidification of the reaction mixture yields carbon dioxide which is sensed by the gas-permeable membrane electrode.1N propanolic potassium hydroxide as titrant and metanil yellow as visual indicator (25). is described by Tagami and Muramoto (29). based on the use of a carbon dioxide gas-sensing electrode. The color measured at 405 nm is stable for 60 minutes and obeys Beer's Law in the 4-32 pg/ml range. reported that polarizing microscopy can be used as a qualitative identification tool for chlorzoxazone (28).. aminophenol and sodium carbonate are formed.

6-Hydroxychlorzoxazone had little or no muscle relaxant activity when tested in mice and rats. humidity conditions of 40"C/80% relative humidity and artificial sunlight (1000 foot candles) €or up to 4 weeks have not caused degradation of the product. The reaction was stopped by addition of 3N hydrochloric acid. and Bioavailabi1ity 7.12 In-vitro metabolism . 7. Studies showed that the metabolite is eliminated as the glucuronide conjugate to the extent of 74% of the dose. Pharmacokinetics. produced by substitution of hydrogen for chlorine and hydroxylation of a neighbor ring-carbon atom (30). a 5-chloro-2. Drug Metabolic Products.1 Drug Metabolic Products 7. It has been established that the liver is the principal site of metabolism. 7. . In 1982. Temperatures for up to 60"C.Chlorzoxazone was incubated with fortified homogenates of mouse and rat liver. Ether extraction of urine yielded a residue which was identified as 6-hydroxychlorzoxazone (9). the solution is generally not stable. Chlorzoxazone yields 2-amino-4-chlorophenol as its major degradation product.The metabolic product of chlorzoxazone was isolated from urine of human subjects administered an oral dose of the drug. An aqueous suspension of the drug in distilled water or acidic media is generally stable but in solutions with a pH greater than 7. Tablets of chlorzoxazone have also been found to be stable for up to 5 years at room temperature. and 6-hydroxybenzoxazolone. Spectrophotometric assay of a sample preparation identified 6-hydroxy-chlorzoxazone as the major metabolite (31).4-dihydroxyacetanilid produced from 6-hydroxychlorzoxazone by ring cleavage and acetylation of the amino moiety.11 Urine metabolism . Twele and Spiteller identified two additional chlorzoxazone metabolites from human urine.CHLORZOXAZONE 137 up to 80°C and in artificial sunlight conditions (1000 foot candles) for up to 4 weeks did not cause significant degradation.

and the drug extracted into chloroform for the measurement step. Identification and Determination in Body Fluids and Tissues 8 . respectively.0 f 39. Less than 1% of the drug is excreted unchanged in urine.12 f 0.29 2. while liver.JAMES T. The organic phase was separated and the drug was back extracted into 0.Stewart and Chan reported the quantitation of chlorzoxazone from plasma and/or -urine samples ( 8 ) . Chlorzoxazone was extracted from the biological sample at pH 2. 8 with hydrochloric acid.73 2 0.2 Fluorescence Spectrophotometry .5% isoamyl alcohol was shaken with the urine sample for 60 minutes. 1 Ultraviolet Spectrophotometry . Excitation and emission wavelengths were 286 and 310 nm. Chlorzoxazone is widely distributed in plasma and tissues. JANICKI 138 7. 8. The organic phase was re-extracted with strong base. 8. STEWART AND CASIMIR A.3 minutes 4084 f 284 pg min/ml 13. The apparent volume of distribution of approximately 14 liters suggests that the drug is not widely distributed and that it would be confined to the circulatory system and possibly the extracellular fluid . muscle.2 Pharmacokinetics and Bioavailability . Petroleum ether containing 1.28 2 2.Conney et a1 reported the extraction and estimation of chlorzoxazone in urine ( 9 ) .70 f 5.07 liters 0.9 hrI: hr hr ml/min There is rapid absorption and elimination of chlorzoxazone. . the pH adjusted to pH 6 .3 pg/ml 38 f 3.5% isoamyl alcohol.48 148.5 N sodium hydroxide.5 with petroleum ether containing 1. Absorbance read at 289 nm followed Beers Law. Recovery of drug from urine was 93 f 2%. brain and kidney concentrations are one-half or less that found in plasma.08 1.3 f 2.The following pharmacokinetic data were obtained following a 750 mg oral dose of chlorzoxazone to human subjects ( 1 4 ) : Peak plasma concentration Peak time AUC (0-10 hr) Apparent Volume of Distribution (V/F) Elimination Rate Constant (K) Absorption Rate Constant (ka) Elimination Half Life (t ) Apparent Clearance (KV/F)' 36. The drug concentration in fat is twice the plasma concentration.

0 ml/min flow rate.3. Another HPLC assay for chlorzoxazone in plasma reported by Ng at McNeil Pharmaceutical (32) used at 1:l acetonitrile-distilled water mobile phase. Stewart and Chan also reported a procedure for the analysis of chlorzoxazone based upon solvent extraction of the drug from human plasma or urine followed by chemical derivatization with fluorescamine (7).).6 mm.06 .42%. Stewart and Carter reported modifications in the above mentioned HPLC assay by Honigberg.13 . Retention times for the hydroxy compound and drug were approximately 4 and 8 minutes. The limit of detection for each compound was calculated to be 8 0 ng at signallnoise = 2. The separation was achieved on an octadecylsilane column (30 cm x 3.3. an octadecylsilane column (25 cm x 4. i.0 vg/ml for plasma and urine. The ethyl acetate was evaporated to dryness and the residue dissolved in methanol prior to injection into the HPLC .Honigberg.CHLORZOXAZONE 139 Drug concentration arid fluorescence were linear from 0.5 minutes). Percent recoveries of chlorzoxazone from plasma and urine were 86.63 2 1.2 pg/ml and 0. A plasma sample was made acidic with hydrochloric acid and extracted once with ethyl acetate.9 mm. respectively.) at a flow rate of 2 ml/min with UV detection at 280 nm and a mobile phase of 40:60 absolute methanoldistilled water. Stewart and Coldren in which an octadecylsilane solid-phase extraction column was used t o separate chlorzoxazone and 6-hydroxychlorzoxazone from human serum (33).24 2 4.37 2 2. respectively.d. Stewart. respectively. Ethyl 5-chloro-2-hydroxycarbanilate was used as internal standard (retention time of 4. respectively. i.d. Percent recoveries were 90.9 minutes. and Coldren reported an HPLC assay for chlorzoxazone and 6-hydroxy-chlorzoxazone in plasma based on ether extraction of the compounds from acidic plasma (22). respectively. Linear detection range was 2-10 pg/ml using excitation and emission wavelengths of 370 and 505 nm.28 and .3 High Performance Liquid Chromatography . Limits of detection were 6 0 and 130 ng/ml for plasna and urine..66 and 95. a 2. and UV detection at 280 nm. Retention time for the drug is 2. 8.

An aliquot of the powder equivalent to 40-60 mg of chlorzoxazone was hydrolyzed with 20% sodium hydroxide solution. and the reaction allowed to proceed at 42°C for 20 minutes.d. The two compounds were detected at the glassy carbon electrode using a cell potential of + 1300 mV.Twenty tablets were weighed and powdered in a dry mortar and pestle. 5 minutes. carrier gas (nitrogen at 30 ml/min). 3 .4 Gas Chromatography - Desiraju et a1 reported a packed column method for the quantitation of chlorzoxazone in plasma samples ( 1 4 ) .99. cooled. 30 times the previous limit of detection using 280 nm. The GC parameters were flame ionization detection (30 and 300 ml/min flow rates for hydrogen and air. 8.5 ng. The ethyl acetate is separated andevaporated to dryness. Extraction efficiency for chlorzoxazone was 88 It: 8% (n = 6 ) . The calibration curve was linear in the 0.9% range utilizing the assay procedure. 6 ft x 4 mm i. 9. The retention times of chlorzoxazone and internal standard were 3.5 .JAMEST.25 and 4 . The limit of detection was 0 .25 ug/ml range. 5 with phenacetin as internal standard. respectively. 0 5 M sodium dihydrogen phosphate. glass column packed with 3% OV-1 on 6 0 / 8 0 mesh Gas Chrom Q.JANICKI 140 86. The separation was achieved on an octadecylsilane column using a mobile phase of 5 0 : 5 0 acetonitrile-aqueous 0 . respectively. An aliquot of the mixture is injected into the gas chromatograph. n-hexadecane.06 It: 3. Identification and Determination in Pharmaceuticals Colorimetry . 9. and column temperature of 130°C. respectively). the tube is capped. and reacted with nitrous acid to yield the diazonium salt. whose absorbance measured at 405 nm obeyed Beer's Law in the 4-32 ug/ml range ( 2 6 ) .58% for drug and metabolite.STEWART AND CASIMIRA. pH 4 . Recovery of chlorzoxazone in bulk powder and tablet samples was in the 9 8 . The drug is extracted from acidified plasma with ethyl acetate containing the internal standard. Acetaminophen and indomethacin were shown to interfere with the method. Pyridine and acetic anhydride are added to the residue. 5 ug/ml using 1 ml of plasma. These modifications halved the original HPLC analysis time and increased detection for each compound to 2.1 .

discarding the first 20 mL of the filtrate. Dilute with methanol to volume. with a suitable spectrophotometer. Filter.Kirschner of McNeil Pharmaceutical reported an assay for the drug in tablets as follows (10): Standard Preparation: Dissolve a suitable quantity of USP Chlorzoxazone KS.4 Fluorescence Spectrophotometry .2 141 Ultraviolet Spectrophotometry . using methanol as the blank. and mix. and pipet 2 ml of the filtrate into a 100-ml volumetric flask. accurately weighed. Procedure: Concomitantly determine the absorbances of the Assay and Standard Preparations at 282 nm. Potentiometry . These same authors also demonstrated that chlorzoxazone could be successfully analyzed in a tablet dosage form chemical derivatization with fluorescamine to form a fluorophore (7). of USP chlorzoxazone RS in the Standard Preparation and Au and As are the absorbances of the Assay and Standard Preparations. After stirring and centrifugation. Transfer an accurately weighed portion of the powder. the supernatant acetone solution was removed and . respectively. Twenty tablets were powdered and a portion equivalent to about 424 mg of drug was weighed and extracted with 20 ml of acetone. in methanol. in which C is the concentration. equivalent to about 100 mg of chlorzoxazone. Dilute with methanol to volume. 9. and shake by mechanical means for about 15 minutes. and mix. and dilute quantitatively and stepwise with methanol t o obtain a solution having a known concentration of about 20 pg/mL. Recovery of drug was in the 98-101% range. in pg per ml. in mg. Assay Preparation: Weigh and finely powder not less than 20 Chlorzoxazone tablets.Tagami and Muramoto described a method based on the use of a carbon dioxide gas-sensing electrode ( 2 9 ) .CHLORZOXAZONE 9.Stewart and Chan used the intrinsic fluorescence of chlorzoxazone to assay for the drug in commercial tablets containing acetaminophen ( 8 ) . to a 100-ml volumetric flask. Add about 80 ml of warm methanol. of chlorzoxazone in the portion of tablets taken by the formula 5C (Au/As).3 9. Calculate the quantity.

Therap. thru Chem. minutes Component 2. m-chloroaniline. U S Patent 2. Sci. In the method. J.5 High Performance Liquid Chromatography . References 1.. p-aminophenol (a degradation prozuct of acetaminophen).W.The USP XXI method for Chlorzoxazone with Acetaminophen tablets is a gradient HPLC method (34). KD. A. D. Sci. Pharmacy Times.24. Pharmacol. Stewart and C. Fort Washington. 9. Exptl. McNeil Laboratories.1 m-Chloroaniline 7.W. 5. 577 (1961). Hung.. Chan. F.P. 895. J.. Current Therapeutic Research (1975) 17. The residue was refluxed for 2 hr with 50 ml of 3N sodium hydroxide and after acidification. the carbon dioxide was determined using a gas permeable electrode. This extraction procedure was performed four times.. Leszkovszky. J. A.. 2. 68 (1979) 910-912. Inc. Literature Services Section. 7. Anal. J. Conney. Patent 150. 6.JAMES T. JANICKI 142 diluted with additional acetone. Burns. N. 53. PA. 3. 128 (1960) 333. Marsh. 877 (1959). 5507b (1964). Sam and J .3 Acetaminophen 6. 48 (1982) 23-32. Harsanyi.6% with a standard deviation of 0. Letters. Pharmacol. 9.T. Chan. Stewart and C.5 Internal Standard (phenacetin) 8. 129 (1960) p 75. An example of the separation is as follows: Retention time.1 Chlorzoxazone 10. Therap. 8. 4. .. Korbonitis and G. J. Trousof and J. K.T. The collected acetone fractions were evaporated to dryness. 60. Plampin. Pharm.2 p-Chlorophenol 10. Mean recovery of chlorzoxazone was 99. Abstr. N .H. acetaminophen and the internal standard phenacetin. (1978) 667-680. Exptl. (1964) 538-544.0 2-Amino-4-chlorophenol p-Aminophenol 7.Pharm. Roszkowski. p-chlorophenol and 2-amino-4-chlorophenol are separated from chlorzoxazone. J. Bl1 (9). J.p 123. Toffler.3 9.J. STEWART AND CASIMIR A.

K.G. Spring House. Ullah. Bull. Pharm. 31. Ng. I. London. N. Bucur. 18 (1982) 183-195. J. Joshi. 46 (1970) 211-216.C. Egli and S. Rioqulm. Stewart. (1974). Anal. and I. Yokoyama and T. 1986. Pharmacol. Twele and G. 28. 124 (1984) 1167-1170. m.. T.L. London. HonigberE J. E. and K. Cadwallader. K. p 34. Pharm. R. R. 32. Burns. Desiraju. Umeda. Rev. 249-256. Parker. A. Paulo. Clarke. Egli andS. Moffat. Pedroso and E.L.C. 1 7 . Beckett. (1979) 253-255. Anal.D. J. PA (1986). Stewart and H.G. Tagami and Y. Fontan and P." 2nd Edition.C. Stewart. 32 (1984) 1018-1024.A. Moraes. 22 * J. R. 24. V. 21. N. Arch. 68. (Bucharest) 16 (1965) 322. The Pharmaceutical Press.R. Chem. 68.. Spiteller. Conney and J.T. p 465. Rev.H. Nayak and K. Coldren. Univ. 759-763. Ed. Forsch. J. B.H. Sci. and J. Chim. 27. N.L.K.. The Pharmaceutical Press. 14. g . A . Tanner. 1986. 12.29. 145. Dubal. Reral. 18. Yamaoka. Chem. Kaempe. 72 (1983) 991-994. 13. Japan Pharmacopeia. Carter. Gamentzy and I. Spring House. 34 (1962) 757. Sanghoi. J.. Chromatogr." Volume 1.L. 16. 19 (1967) 273. p 157. Pharm. Biomed. A.M.K. Exptl..C. (1979) 32-36. Kirschner.. Pharmac.A. Moffat.R. Kirk. Personal communication. Japan.W.E. E.C. Keller.G.J.J. 26. S. "Isolation and Identification of Drugs. 32 (1982) 30. Honigberg. Society of Japanese Pharmacopeia. 128 (1960) 340..." 2nd Edition. Ther. A. Pharm. Appl. Ap0th. Arzneim.F.L. Ed. Fresenius 2.. D. Y. 15. Sci. ..T. 25. J. Renzi. 33. V.CHLORZOXAZONE 143 10. Chromatogr. Chemi. McNeil Pharmaceutical. Farm... 2 (1984) 20. "Clarke's Isolation and Identification of Drugs. 9th Ed. McNeil Pharmaceutical. Yakugaku Zasshi.T. R. H. Moffat. I. K. Indian Drugs. A.C. and J. 380 (1986) 177-183. Sci. PA. Coldren. J.W.. London (1969). Dtsch. J. 23. R. Personal Communication. C.-Ztg.S. J. Chromatogr. 1. MuramotFChem.. 295 (1979) 398-401.D.. Pharm. Watanabe. J. Kuroda. 18 (1981) 299-302. p 193. Honigberg. Ng. Pharm. Ed. The Pharmaceutical Press. Quade-Henkel. Tlarke's Isolation and Identification of Drugs. V. (1986). S. 11. Tucker and A.. I. Schnekenburger and M.L. 19.-l05 (1985) -481-490.T.

1986. (1985). STEWART AND CASIMIR A. MD. L i t e r a t u r e surveyed t h r o u g h December. 2 1st E d i t i o n . B e t h e s d a . p . U n i t e d S t a t e s Pharmacopeial Convention. JANICKI Second Supplement. JAMES T. . U n i t e d S t a t e s P h a r n a c o p e i a . 1 8 2 7 .144 34.

CYCLOSPORINE *Mahmaud M.mhin. Saudi Atrabiu. AL-Yahya 06 P h c u u n a c W c d Chmbin. *Pho6ehboh PhOdeAhotr 06 Phahmacognob y . King Saud U n i w m L t y . King Saud U n i w m L t y .the MediciMae Akomatic and Pohonoun Plan& Runahch Cenhn. Inc. Riyadh. CoUege 04 P h m a c y . VhecZoh 06 . **kshoch~& ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 16 145 Copyright 0 1987 by Academic Press.thy. Saudi k a b i a . CoLtege 06 Phahmacy.A.thy. All rights of reproduction in any form reserved. H c u a n and **Mohammed A . Ve-ed 06 Phahmacognon y. Vep&evLt 06 P h m a c e d c d Che. . Riyadh.

Acknowledgement . Metabo1ism 8.2 Formulae 2.2 11. References 13.2 Cyclosporine Analogs 6.5 Crystal Properties Melting Range Solubility Optical Rotation Spectral Properties 4. HASSAN AND MOHAMMED A.4 3.4 Conformation 2.2 3.1 Cyclosporine 6. Description 2.4 Appearance.2. Physical Properties 3.3 3.1 3.3 Molecular Weight 2. S y nthesis 6.4 Elemental Composition Introduction to Analysis Radioimmunoassay High Performance Liquid Chromatography 12. Odor 3.3 Degradation and Structural Elucidation 2. Taste.3 11.3 Structure-Activity Relationship 7. AL-YAHYA 146 1. Pharmacological Activity and Clinical Applications 10.2. Pharmacokinetics 9.1 Nomenclature 2. Iso lation 5. Biosynthesis 6. A. Color. Pharmaceutical Forms 11.1 11.MAHMOUD M. History and Therapeutic Category 2. Methods of Analysis 11.

9 ) .1 Nomenclature 2.2 Generic Names Cyclosporine (USAN) and i s adopted f o r t h e b a s i c s t r u c t u r e . ) R i f a i or Cylindrocarpon luciderm Booth ( 3 ) . The main component i n normal f e r m e n t a t i o n b r o t h s w a s named c y c l o s p o r i n A ( A ) . A s r e p o r t e d by Bore1 e t a1 ( 5 . c i c l o s p o r i n (INN name. H i s t o r y and T h e r a p e u t i c Category It w a s from Hardanger Vidda. meanwhile t h e f o l l o w i n g names h a s been a c c e p t e d i n o t h e r c o u n t r i e s . England.1 Chemical Names Cyclosporin A. Today c y c l o s p o r i n e can be regarded as t h e m o s t important r e c e n t i n n o v a t i o n i n t h e f i e l d of organ t r a n s plantation. c y c l o s p o r i n (BAN name). Sandimmun . c i c y l o s p o r i n e . a b l e a k t r e e l e s s p l a t e a u i n t h e South o f Norway. now known as c y c l o s p o r i n e . 2.1. Cyclosporine. WHO).1. F r a n c e .147 CYCLOSPORINE Cyclosporine 1. D e s c r i p t i o n 2. formerly ( 2 ) d e s i g n a t e d as Trichoderma p o l y s p o r m (Link ex P e r s .3 Trade Names Sandimmune . 2. S e v e r a l m e t a b o l i t e s o f t h i s fungus have been i s o l a t e d of which c y c l o s p o r i n s A . 2. c y c l o s p o r i n e i s a s e l e c t i v e immunosuppressive drug w i t h a n t i f u n g a l and antiinflammatory p r o p e r t i e s .1. C and G t o have so f a r been shown t o p o s s e s s s t r o n g immunosuppressive a c t i v i t y . The fungus i s o l a t e d from t h i s sample w a s c a l l e d Tolypocladium i n f l a t m Gams (1). S w i t z e r l a n d and o t h e r s . t h a t a s o i l sample w a s o b t a i n e d i n t h e e a r l y s e v e n t i e s .

Hydrolytic cleavage of c y c l o s p o r i n e f u r n i s h e d 11 amino a c i d s as fragments. which has t h e Rc o n f i g u r a t i o n . N . It has t h e molecular formula C62HlllN11012 as deduced by NMR and mass s p e c t r a . V a l i n p o s i t i o n 5. AL-YAHYA 148 2. Ala i n p o s i t i o n 7 . e l u c i d a t e d as t h e n e u t r a l c y c l i c o l i g o p e p t i d e It i s composed of e l e v e n amino a c i d r e s i d u e s . N-MeLeu i n p o s i t i o n 4. D-Ala i n p o s i t i o n 8 . 1 0 and 11. Also with an x-ray c r y s t a l o g r a p h i c a n a l y s i s of t h e iodo d e r i v a t i v e [ l a ] . Ten are known a c i d s .Oa c y l m i g r a t i o n .2.6. A. a l l having t h e S-configurat i o n o f t h e n a t u r a l L-amino a c i d s .2 Structural 2.9 and 1 0 . 1) was determined by chemical d e g r a d a t i o n (4) and x-ray c r y s t a l l o g r a p h i c a n a l y s i s (10. 9 . except f o r t h e D-alanine i n p o s i t i o n 8 and t h e non-chiral s a r c o s i n e (N-methylglycine) i n p o s i t i o n 3. hydrophobic c y c l i c p e p t i d e composed o f 11 amino a c i d r e s i d u e s .M e V a l i n p o s i t i o n 11 and MeBmt i n p o s i t i o n 1.2. among them an a r t i f a c t o f a new C9-amino a c i d . a b a s i c rearrangement product formed from c y c l o s p o r i n e by N.1 Ehpiricd N O C 6 2 H l l l 11 1 2 2.MAHMOUD M.2 Formulae 2.2. s p e c t r o s c o p i c and c r y s t a l l o g r a p h i c e v i d e n c e . 1) i s a n e u t r a l . Ten o f t h e - .3 Cyclosporine ( F i g . t h e s t r u c t u r e of c y c l o s p o r i n e w a s . On t h e b a s i s of t h e chemical. HASSAN AND MOHAMMED A.are N-methylated. 6 . Sar-in p o s i t i o n 3. 3. Seven amino a c i d s i n p o s i t i o n s 1. 4. a l l having t h e L-configuration of t h e n a t u r a l amino a c i d s . t h e y are a-Abu i n p o s i t i o n 2 . and S a r i n p o s i t i o n 3. Degradation and S t r u c t u r a l E l u c i d a t i o n The s t r u c t u r e o f c y c l o s p o r i n e [l] ( F i g . Seven a c i d s a r e N-methylated. except f o r t h e D-Ala i n p o s i t i o n 8.ll). The amino a c i d sequence w a s determined by Edman d e g r a d a t i o n o f i s o c y c l o s p o r i n .

N-dimcthyl-~-threoninc (see text). Struciure of cyclosporine 1 (schematic). I . McBmt nyl]-4.u A Fig. - (4R)-4-[(€)-2-bute- .

AL-YAHYA 150 J cyclorporine not obtoincd Ib cyclic derivotivool the Nmdhyl-C9-amino ocid 1 cyclosporlnc 1 IS OCYCLOSWRINE onhydromrthylthio Ic hydontoin de rivot ive Edman d e g r a d a t i o n with CHI-NCS under c a r e l u l l y chosen mild conditions p e p t i d e hydrolysis w i t h c h r o m a tog raphy.MAHMOUD M. HASSAN AND MOHAMMED A.. A.I TlOAcl It -+ t--Zn I HOAc cy cloiporlnc 1 n . 6 N HCI followed by ion erchange n 'F.

9 and 10. polar features of an N-methyl-L-threonine which is substituted at the end of the carbon chain by butenyl and methyl groups. Hydrolysis of cyclosporine followed by ion exchange chromatography afforded a cyclic derivative [lb]of the MeBmt minoacid. Alanine (Ala) in position 7.4 Conformation of Cyclosporine The conformation of cyclosporine in the crystal and in solution was reported (11). and in accord with amino acid nomenclature. Sarcosine (S. This amino acid was hitherto not known in the free form. 4R. O-acylmigration of the methylvalyl moiety and furnishes isocyclosporine [lc]. 2. is now designated as (4R)-4-[(E )-2-butenyl]4-N-dimethyl-L-threonine and abbreviated Thus the novel amino acid has the MeBmt . The novel amino acid was found to have the composition ( 2S. Valine (Val) in position 5. A modified Edman degradation of isocyclosporine [1] using methyl isothiocyanate produced an anhydrothiohydantoin-derivative [l] and thus established MeBmt as the first amino acid in the peptide sequence. N-methylleucine (MeLeu) in positions 4. The .CYCLOSPORINE 151 eleven ring members are derivatives of known aliphatic amino acids: a-aminobutyric acid (Abu) in position 2. D-Alanine (D-Ala) in position 8. 6. and N-methylvaline (Me Val) in position 11. 3R.2.ar) in position 3. since only antifacts and derivatives were obtained from degradation experiments on cyclosporine(4). 6E)-3-hydroxy-bmethyl-2-( methylamino)-6-octenoic acid. The complete sequence was established by repetitive Edman degradation. Acidic treatment of cyclosporin [l] in the absence of water effects an N. The amino acids were readily characterised following acid hydrolysis of cyclosporine.

and can b e brought about by r e l a t i v e l y s m a l l r o t a t i o n i n t h e backbone t o r s i o n a n g l e s i n t h e r e g i o n MeLeu-6 t o MeLeu-9. The side-chain conformations o f M e V a l and V a l and t h r e e o f t h e f o u r MeLeu-residues (MeLeu -4. T h i s r o t a t i o n which i s e n e r g e t i c a l l y allowed. The side-chain conformation o f MeBmt i n s o l u t i o n can be d e r i v e d from t h e X-ray conformation simply by means o f 120' r o t a t i o n about t h e cl. t o t h e carbonyl 0-atom of D-&a-8 The conformation change needed t o c o n v e r t t h e X-ray backbone i n t o t h e NMR backbone i s small. 2 ) .B-bond. whereas i n s o l u t i o n a b i f u r c a t e d H-bond t o b o t h t h e c a r b o n y l 0-atom o f MeLeu-6 and itself. 3 ) . HASSAN AND MOHAMMED A. could be t h e r e s u l t from t h e formation of an i n t r a m o l e c u l a r H-bond between t h e B-OH and t h e carbonyl 0-atom of MeBmt r e p l a c i n g t h e i n t e r m o l e c u l a r Hbond found i n t h e c r y s t a l . and t h e d e t a i l e d backbone conformation i n t h e r e g i o n o f D. Cyclosporine molecule i s highly lipophilic. p r i n c i p a l l y around t h e 7. Here t h e X-ray s t r u c t u r e c l e a r l y shows one H-bond from D-Ala-8 ( N H ) t o MeLeu-6 ( C O ) . b o t h i n t h e c r y s t a l l i n e s t a t e and i n s o l u t i o n ( F i g .8 ( F i g . and p o i n t i n g o u t i n t o s o l v e n t i n t h e NMR s t u d y ) .and 8residues. A l a r g e p o r t i o n o f t h e backbone ( r e s i d u e s 1-6) a d o p t s an a n t i p a r a l l e l & p l e a t e d s h e e t conformation which c o n t a i n s t h r e e t r a n s a n n u l a r H-bonds . -6 and -9) are i d e n t i c a l i n c r y s t a l and i n s o l u t i o n . Only t h a t o f MeLeu-10 i s r o t a t e d by 120°. therefore t h e s o l i d s t a t e conformation and t h a t deduced from NMR s t u d i e s i n a p o l a r s o l v e n t s a r e c l o s e l y s i m i l a r .152 MAHMOUD M. It assumes a r a t h e r r i g i d conformation. AL-YAHYA conformational a n a l y s i s was based on X-ray c r y s t a l l o g r a p h y i n t h e c r y s t a l and on NMR spectroscopy i n s o l u t i o n u s i n g d i f f e r e n t l i p o p h i l i c s o l v e n t s . t h e side-chain conformation o f MeLeu-10.A l a . The o n l y d i f f e r e n c e s a r e t h e side-chain conformation o f MeBmt ( a major change between t h e c h a i n f o l d e d i n upon t h e molecule i n t h e solid s t a t e . A.

. b) Computer-generated conformation in apolar solvents (in solution the distal atoms of Abu-2 and YeBmt-1 have a high flexibility). 153 Stereoview of cyclosporine. a) Solid-state conformation.CYCLOSPORINE Fig. 2.

Fig. Superposition of the solid-state conformation (thin line. 3. .) and the computer-generated conformation of cyclosporine in apolar solvent (thick lines).

in which the MeE3mt side-chain. known to be important for immunosuppressive activity.amide linkgage in the molecule between the two adjacent N-methylleucine residues 9 and 10. As a consequence of this rather rigid conformation of the cyclosporine skeleton. This means that. in MeBmt-1 and ~ e ~ e u -they 6 are pointing downwards. The side-chains of the remaining aminoacids lie more or less in the plane of the peptide ring.155 CY CLOSPORINE and is markedly twisted. in Abu-2.pro -S proton of the methylene group of Sar 3 is axial. the CC group of Abu-2 and the N-CH? group of MeLeu-4 are up and the CC gEoup of Sar-3 and NH group of Val-5 are down . and the side-chain of MeLeu-4 is requatorial. in a manner suggestive of special function (Fig. 4). in the orientations chosen in Figures l and 2. stands out. This loop contains the only cis. six amino acids have their side-chains directed quasi-perpendicular to the plane of the peptide ring. probiscis . Val-5 and MeLeu-6 they are pointing upwards. 2.2. The resulting overall molecular shape in apolar solution is that of the previously postulated "butterfly" (10). The remaining H-bond of a y-type and serves to hold the backbone in a folded L-shaped.like.2.6 Clinical Code CyAIOL 27-400 . The sarcosine in position 3 and N-methylleucine in position 4 participate in a type 11B-turn (12-14).5 CAS Registery Number Cyclosporine [79217-60-0] Cyclosporine A [ 59865-13-31 2. The remaining residues 7-11 form an open loop.

A. MAHMOUD M. The backbone conformation corresponds to that of iodocyclosporine. HASSAN AND MOHAMMED A. . 4. AL-YAHYA Two roughly orthogonal views of a model of native cyclosporine in the conformation suggested as probable.156 Fig.

4 Appearance. 5 1 ( 36508 r e f l e x i o n r ) Cell dimen-. cm-3 4272(I 2.157 CY CLOSPORINE 2.1 X-ray D i f f r a c t i o n C r y s t a l Data X-ray c r y s t a l s t r u c t u r e s o f c y c l o s p o r i n e and i t s i o d o d e r i v a t i v e [ I a ] were d e t e r m i n e d (10. Taste.1 Crystal Properties 3.3 Molecular Weight 1202.ll).5 Diffractometer CAD4 ( E n r a f Nonius) Crystallisation from a c e t o n e Radiation C a a ( Graphite monochromat o r ) C r y s t a l form colourless. 0 4 2 g.64 2. C r y s t a l and D i f f r a c t i o n Data Molecular f o r mula N O C 6 2 H l l l 11 12 Molecules per c e l l z = 4 Molecular weight 1202.50(1).) c = 41.3 tan 0 Crystal s i z e ca. Odor White p r i s m a t i c c r y s t a l s . sions I n t e n s i t ie: measured Intensities significant 5057 ( u n i q u e ) C r y s t a l dens i t y (calc.0°+0. T a b l e 1.1. P h y s i c a l P r o p e r t i e s 3. 0.2 x 0. Ow = 1. .242(33 A. C o l o r . 3.3 mm ( tmax = 120s) Space group Sphere of ref lexion sine/X 4 0 . p r is m a ti c Intwsity scans w/2e = 1. V = 7896A dc = 1 .02 x 0. A summary o f t h e c r y s t a l d a t a and r e f r a c t o m e t r y for c y c l o s p o r i n e i s g i v e n i n Table 1 (11).0.2 a ( T ) / I = 0.

5b) w i t h 50% p r o b a b i l i t y v i b r a t i o n a l e l l i p s o i d s of t h e atomic thermal motion (16) which d i s p l a y t h e r e l a t i v e l y s m a l l .35 (2'1. a = ( 1 0 . ( c. The conformation of c y c l o s p o r i n e observed i n t h e c r y s t a l ( F i g . A = 1. M = 1327.i n d i c a t i n g conformat i o n a l r i g i d i t y . b = 1 9 . A c r y s t a l o f approximate dimensions 0 . Fig. 4 7 5 ( 5 ) . 4). The a b s o l u t e c o n f i g u r a t i o n w a s n o t determined as p a r t of the structure analysis. I)-. isotopic v ib r a tio n s of t h e backbone atoms .i n c o n t r a s t t o t h e more f l e x i b l e side-chain atoms whose v i b r a t i o n a l amplitudes i n c r e a s e w i t h t h e d i s t a n c e from (a). . z = 2. It became apparent d u r i n g t h e a n a l y s i s . 3 x 0. 6 0 ( 1 ) . t h a t A l a .03. p a r t i c u l a r l y t h o s e of MeLeu-9 are a p p a r e n t . c = 21.8 has t h e D-configuration (15). 5b. U = 4264 A o 3 .158 MAHMOUD M. The iodocyclosporine showed t h e a n t i p a r e l l e l & p l e a t e d s h e e t conformation of r e s i d u e s 1-6. 6a shows t h e backbone of t h e c y c l i c p e p t i d e w i t h t h e 4-. space group ~2~ . 5 : l monoclinic. A. c o l o r l e s s '62'110 11 1 2 prisms from n-heptene.. t h e open l o o p of r e s i d u e s 7-11. g r a p h i t e monochromator.s t r a n d s adding t o t h e s t a b i l i t y of @-fragment. f3 = 99. Three H-bonds b r i d g e t h e two s h o r t @. and t h a t of c y c l o s p o r i n e i n F i g . N O . AL-YAHYA The c r y s t a l d a t a for iodocyclosporine (10). however . Dc = 1. N 0 + .05(1)A0. A s t e r e o v i e w o f iodocyclosporine molecule i s shown i n F i g . 2 . CUKci r a d i a t i o n . since sufficient of t h e h y d r o l y s i s p r o d u c t s could r e l i a b l y be i d e n t i f i e d as L-amino-acids. 5a.a n g l e s and some s t r u c t u r a l f e a t u r e s . and w.7 x 0.54187 A o . and t h e r a t h e r l a r g e t h e r m a l v i b r a t i o n s of some of t h e side-chain atoms . HASSAN AND MOHAMMED A.4 mm w a s used on an Enraf-Nonius CAD4-F automatic d i f f r a c t o meter.

A stereoscopic view of the iodocyclosporine molecule showing 50% probability ellipsoids of thermal vibration./! Fig. The Cg amino acid is at centre left on the forward side of the molecule and the conventional sequence then extends upwards at the front through a-aminobutyric acid. . glycine etc. One of the hydrogen bonds of the 8-pleated sheet is indicated (dotted) in the centre and the interesting hydrogen bond Ala-8 4 Meleu-6 appears at centre right. 5a.

5b.Fig.probability ellipsoids. ORTEP drawing of the crystal structure of cyclosporine. Anisotropic atomic vibrations are represented by 50% . The numbers shown are residue numbers. .

12 Ao). \ bet a Backbone conformation of crystalline cyclosporine with indication of the loop and 6-fragment. $I. .26 AO. The dashed lines indicate B-bonds. C 7:3. N 9 : 3 . 5 4 AO. C 8:3. N 10:3. 6 a .04 Ao. The torsion angles 6. 0 7:3. the dotted lines close contacts from CH3N of MeVal to different atoars (0 11:3.36 Ao.loop Fig.41 A O . and w and in brackets the computermodeled values for the NMR conformation are given.

6b.F i g . Stereoview of a space-filling model of cyclosporine showing the side chain of between MeLeu-6 and-Meleu-4 into the ply of the &fragment .

i n d i c a t e d t h e NH groups . e t h a n o l . b u t v e r y s o l u b l e i n a l l o t h e r o r g a n i c s o l v e n t s .IR spectrophotometer i s shown i n Fig. and MeLeu r e s i d u e s are i n t h e s t a g g e r e d conformation. i n s o l u b l e i n water and n-hexane.3 Solubility The compound i s n e u t r a l . The s y n t h e t i c c y c l o s p o r i n e m.2 I n f r a r e d Spectrum The i n f r a r e d c h a r a c t e r i s t i c s i n methylenec h l o r i d e ( C H 2 C 1 2 ) and i n c a r b o n t e t r a c h l o r i d e ( C C l 4 ) were r e p o r t e d ( 4 . Spectral Properties 3. e t h e r and chloroform.89' ( C = 0.p. l l ) . 3.2 Melting Range White p r i s m a t i c n e e d l e s from acetone a t . 149-150° (18). 6 b ) . 3. 7.5 i n methanol). - 1. a c e t o n e .5. 3. t h e MeLeu s i d e c h a i n s being extended. V a l .4 Optical Rotation [a]:' 3. m. Rather unexpected i s t h e conformation o f t h e Cg-alkylene s i d e chain of MeBmt-1. It i s v e r y s o l u b l e i n methanol. 148-151' (17).5 (17) - 244' ( C = 0.5. r i c h i n hydrophobic amino a c i d s .15'. The I R spectrum i n methylene c h l o r i d e recorded on a P e r k i n E l m e r 21.163 CYCLOSPORINE The s i d e c h a i n s of t h e Abu.p.1 U l t r a v i o l e t Spectrum The W spectrum o f c y c l o s p o r i n e i n methanol w a s recorded on a Beck~nann-DK2 spectrophotometer shows only an end a b s o r p t i o n a t 200 nm ( 4 ) 3.6 i n c h l o r o f o r m ) . It s h o u l d be n o t e d t h a t s o l u t i o n s t u d i e s . it i s n e a t l y f o l d e d i n t o t h e p l y of t h e 6-pleated s h e e t which a l l o w s t h e molecule t o adopt a g l o b u l a r compact shape ( F i g .

IR-spectrum of cyclosporine in CH2C12.Fig. . 7.

Roughly . i d e n t i f i c a t i o n o f s p i n systems were o b t a i n e d .3 Nuclear Magnetic Resonance S p e c t r a 1 'H-NMR. 5 = 16 Hz) ( 4 ) . It i s e v i d e n t from t h e s p e c t r a t h a t one conformation s t r o n g l y dominates.5. 3. The spectrum shows a l s o an i n t e n s i v e amidecarbonyl bond a t 1630-1670 cm-1.6 9 ) w a s recorded on Bruker HX-360. 90 MHz instrument ( 4 ) .3.165 CYCLOSPORINE are involved i n H-bonding. A completely d i f f e r e n t s i t u a t i o n i s observed i n DMSO d6 s o l u t i o n .1 'H-NMR Spectra 1 The H-NMR spectrum of c y c l o s p o r i n e ( F i g . A p a r t o f 1~-NMR spectrum o f c y c l o s p o r i n e i n ~ 6 ( F~i g . where a m i x t u r e o f a t l e a s t seven conformations l e a d s t o a spectrum of h i g h complexity.5.1Oq4 M i n C C 1 4 s o l u t i o n .10-2 t o 4.5-3. However. 13C-NMR and 15N-NMR s p e c t r a i n deuterochloroform and deuterobenzene by a combination of homonuclear and h e t e r o nuclear two d i m e n t i o n a l t e c h n i q u e s were d e s c r i b e d by Kessler e t a1 ( 1 9 ) . Their absorption bonds ( 3330. Some minor peaks s p e c i a l l y i n t h e N-methyl r e g i o n (2. 13C-NMR and H-NMR-NOE-difference s p e c t r a of cyclosporine i n deuterochloroform and deuterobenzene were r e p o r t e d ( 4 ~ 8 . 3. 11).5 ppm) are v i s i b l e i n d i c a t i n g t h e presence o f a n o t h e r conformation i n s l o w exchange. t h e complete assignment of lH-NMR. This h a s proved t h e E-configurat i o n o f t h e double bond i n c y c l o s p o r i n e by u s i n g t h e double resonance t e c h n i q u e (JE. From t h e s e s p e c t r a . 360 MHz i n s t r u m e n t . 3290 cm-l i n C C l 4 s o l u t i o n and 3400-3330 cm-1 i n CH2C12 s o l u t i o n ) are independent o f t h e c o n c e n t r a t i o n i n t h e range o f 4. 8 ) i n CDC13 and TMS w a s recorded on Bruker HX-gO-E.

8 . TMS -- 0 ppm. 0 T 'H-NMR-spectrum 6 5 I 3 2 * 0 ??a I of cyclosporine by 90 !Mz in CDcl3.8 0 Fig. .

6 A l i p h a t i c f3-.8.5 N-methyl groups 2.3 -6 P P ~ a-protons as w e l l as t h e v i n y l i c protons 4. h C H-c c h b 0 t F i g .1 .6 C-methyl groups 0.2.167 CYCLOSPORINE speaking. yand 6 protons 1.3.8 ppm The proton chemical shifts for cyclosporine i n CDC13 and C6D6 are l i s t e d i n Table 2 . b) Decoupled s n e c t r m keeping lock on H-C(n) of Cg As c ) Decoupled spectrum keeping lock on H-C(6) of Q As . f i v e more o r l e s s separated s p e c t r a l regions are distinguishable: NH protons 7 . 1 Part of the H-NMR-spectrum of cyclosporlne by 360 llHz in CsDs. 9.8 ppm .1.7 PPRI pprn . a) Undecoupled spectrum.

36 C6D6 3.and chemical shifts of cyclosporine [I] in C D C l 13C-NMR Amino-acid residue Group MeBmt: CH3N ~~ co H-C(a ) H-C(B) OH 3-52 5.33 U’O * 73 59.07 2.13 0.97 169.30 36.76 17.7 4.62 2 and C G D ~ ~ ) l3C-NMR lH-NMR CDC13 3 1.51 Abu NH co 7.65 58.35 1.20 3.5 2.96 74.75 74. Residue 1 h.68 d 126.04 s 174.69 2.87 1.30 126.32 d 18.71 5.47 5.96 - - q 131.65 33.99 d 35.15 5.64 5-52 1-75 7.72 5.21 5.45 3.73 - - - 5.04 16.63 t 36.38 8.72 4.96 18.93 - C6D6 CDC13 3.74 - 9 s d d 34.63 2.95 - 35.82 3.41 1-73 0.29 .76 q 129.26 - - - 173.Table 2.

50 s - 169.74 4.87 0.40 q 10.10 co - 3.76 4.60 2.20 35.49 q 21.6-1.35 s 55.42 0.46 C6D6 48.11 2.06 t m3N co 5 CDCl C6D6 2K 2K H-C( a) 3 13C-NMR H-NMR residue 170.79 H-C( Y) 0.28 22.18 q 25.32 q CH N 3 H-C( ~ 1 ) 5. TOb) .12 Val 32K H-C( 6) 1.27 1.39 170. 3.40 - H-C( a ) 4.98 0.89 3 49.58 1.91 7.54 26.11 9. Residue No.08 - 5.21 56.74 1.51 a 31.00 - 3.93 9 39. (Continued) Amino-acid Group CDCl 4 Sar MeLeu 32K 5-03 5.12 5.58 171.73 39.59 31.Table 2.99 t 37 * 01 24.90 d 23.86 d 25.01 50 37 t.80 50.79 24.34 H1-C( B) 2.

3 r W I A ( u r l 0 170 ? M m (u 0‘ m Ln d M 0 cu m L n cu M ul I a d d I co t- ? .o r i A O O W \ 0 0 wlnmrJ&w d ri I W - * .W RW V M d V e 4 0 d c . . ? 1 . .

co 171 L n d L n L n ? ? ? ? ti m t i c o o c u r . .J J L P m UJ cu t- co I L n M cu ? 0 I L n cs cu r- M m co . gt- cucu 5 9 L n L n .

06 1.70 co CH3( 32K 0.17 23.96 174.98 C6D6 CDC13 32K 0.93 d 58.85 q 29 .86 9 24. Residue No.84 2.84 2.47 170.98 57.85 q 24.49 0.81 29. ( Continued) Amino-acid residue CDCl MeLeu - H-C(CX) 5.36 42.17 - 29.35 2.83 q 169.10 6) H-C(y) CH3(61) MeVal 13C-NMR C6D6 2K 6 m3N - 21.24 1.81 172.73 t 58.29 2.79 24.89 CH 3N H1-C( 11 3 2K CH3(62) 10 lH-NMR Group 2.13 1.55 d 25.05 t 2.99 .42 s 30.Table 2 .27 29.38 q 22.14 H-C( 6 ) 2.34 1.98 co - - - H-C(CX) 5-15 5.98 0.62 1.85 5.41 s 30.42 1.61 5-27 57.71 2.16 23. 6bb) 2.54 d 40.

75 q 0 .38 0. a m r i m oco d o 173 e 4 W 0. The H-NMR data were obtained from a conventional w lk k n ) Gik Ld a 13c-NMR CDC1. . The signals of MeLeu-4(6 ) and MeLe~-9(6~) as well as those of MeLeu-6(6 ) and MeLeu-lO(6 ) 2 1 2 in C D may be exchanged).26 q 20.$-COSY with 2K data points in f2 at 300 MHz b.66 20.59 0 18. 66 .96 1 At 296 K in pprn from internal TMS. Group 0 1H-NMR Pi 3 Amino-acid residue k 0 Residue ( Continued) WDV) 19. . 32K spectrum or from the cross-sections of a IH.Table 2. .

whereas 49 C-signals a r e i n t h e a l i p h a t i c r e g i o n .2 - 3 in C6D6 259.2 13C-NMR Spectra I n t h e broadband-decoupled 13C-NMR s p e c t r a i n CDC13 or C6D6 were r e p o r t e d ( 1 9 ) .5 Abu - 256. 15N-NMR chemical s h i f t s o f 1. A l l carbonyl C-atoms r e s o n a t e i n t h e range between 169 and 175 ppm.261. AL-YAHYA 174 3.2 - 260.261. 6 spectrum (22-26) w a s a l s o performed i n CDC13 The chemical s h i f t f o r a l l carbons and m u t l i p l i c i t i e s i n C X 1 3 and i n C6D6 are given i n Table 2 .5.6 Val - 255.3 15N-NMR Spectra AH-decoupled 15N-NMR spectrum shows eleven N-signals ( T a b l e 3 ) . 2 1 ) .255.1 Abu - 256.258. but these s p l i t when t h e s o l v e n t i s changed. The two o l e f i n i c C-atoms of MeBmt are a l s o i n a t y p i c a l range 126-132 ppm.1 263. 360 MHz instrument i s shown i n Fig. 6 ppm from N H 4 l 5 N O 3 ~~ i n C6D6 i n CDC13 - 248. HASSAN AND MOHAMMED A. Table 3.5.2 . A.0 258. Almost a l l 62 Cs i g n a l s a r e r e s o l v e d and only few signals overlap.8 A7 . i n an e x t e r n a l c a p i l l a r y .3. P a r t o f t h e l 3 C . The number of a t t a c h e d p r o t o n s were o b t a i n e d v i a t h e u s u a l DEPT t e c h n i q u e ( 2 0 .4 V a l 264.2 - 265.9 ~8 .m spectrum ( t h e carbonyl r e g i o n ) of c y c l o s p o r i n e i n CDC13 measured on a Bruker-WH-360.3 A8 258.1 .1 A7 256.9 .1 263.8 260.8 248. 1 0 (11).3 - i n CDCl 259.MAHMOUD M.3. 3. A h e t e r o n u c l e a r 2D-J.265.7 .9 257.

''C-NNFI . 6 .1 1 m I73 -6 F i g . 10. The CO sign81 of residue 8 (shaded) lowest from those attached to NE groups (residues 1 . and 7). Those CO groups whose signals shifted downfield are marked In black. [PpnJ 1 m 170 spectrum of cyclosporlne: CO region. 4 .

AL-YAHYA 176 3.10-6 T o m . t h e a c c e l e r a t i o n rate 4. 4. The e l e c t r o n energy w a s 7 0 e V .831 i 0.6. t h e temperature of t h e i o n s o u r c e goo and t h e power f a c t o r f o r m u l t i p l i e r 106. R i f a i ( 2 ) . The peak a t m/e 1183 i s formed by eliminat i o n o f a molecule o f water.6. A s t h e M+ v a n i s h e s a peak a t m / e 1183 i n c r e a s e s i n i n t e n s i t y and t h e spectrum remains s t a b l e . T h i s fungus w a s p r e v i o u s l y known as Trichoderma polysporum (Link ex P r e s . NH' a t m / e 1203 and ( M + N a + ) a t m / e 1225. T h i s w a s i n agreement w i t h t h a t deduced from NMR and o t h e r f i n d i n g s . The a c c e l e r a t i o n energy r a t e 6 KV.841).6-8 KV. A molec u l a r i o n peak M+ of m / e 1201.6 f o r t h e d i h y d r o c y c l o s p o r i n e . Other mass s p e c t r a l d a t a w i l l be p r e s e n t e d under metabolism.1 EI Spectrum The e l e c t r o n impact spectrum o f c y c l o s p o r i n e w a s measured on a CEC-mass spectrometer 21-llOB ( 4 ) . I s o l a t i o n o f Cyclosporine Dreyfuss e t a 1 ( 2 ) and o t h e r s ( 4 . 3.003 and 1201. A. A molecular i o n peak Mc a t m / e 1202. Another FD spectrum w a s r e p o r t e d (18). The molecules w a s u n s t a b l e g i v i n g a molecular i o n peak a t m/e 1201 which v a n i s h e s q u i c k l y . t h e temperat u r e of t h e source 280-300°C and a p r e s s u r e of 10-5 .6 Mass Spectrum 3.8 w a s o b t a i n e d and m / e 1203.2 FD Spectrum The f i e l d d e s o r p t i o n mass spectrum w a s measured on a MAT 711 mass spectrometer ( 4 ) . HASSAN AND MOHAMMED A. .MAHMOUD M. The e x a c t mass measurement were made at 1183. 27-30) have r e p o r t e d t h e i s o l a t i o n of c y c l o s p o r i n e from o t h e r f u n g a l m e t a b o l i t e s of t h e f u n g i Tolypocladium i n f l a t u m Gams. The molecular f o r m u l a o b t a i n e d by t h i s measurement w a s C62HlllN11012 ( p h y s i c a l molecular mass 1201.003.842 f 0.

The former w a s homogen i z e d t h r e e t i m e s w i t h methanol-water (9:l) and t h e combined f i l t r a t e s were c o n c e n t r a t e d under vacuum t o remove t h e methanol. t h e cyclosporines were e l u t e d i n t h e following o r d e r : D . The f r a c t i o n s o b t a i n e d by chloroform-methanol ( 98 :5 :1 .2 mm) w i t h e t h y l a c e t a t e s a t u r a t e d w i t h water. The crude r e s i d u e o b t a i n e d w a s d e f a t t e d with 90% methanol and petroleum e t h e r . c y c l o s p o r i n e s A . B and C . The p o l a r i t y of t h e e l u t i n g s o l v e n t w a s g r a d u a l l y i n c r e a s e d by adding methanol t o chloroform. The f i l t r a t e was evaporated t o g i v e c y c l o s p o r i n A as an amorphous powder.d i c h l o r o e t h a n e r e s u l t e d i n an a d d i t i o n a l p o r t i o n of crude material. The . N RRL 8044. Those f r a c t i o n s which c o n t a i n t h e c y c l o s p o r i n e ( a s a m i x t u r e ) were pooled and t h e n s e p a r a t e d i n t o t h e components by s i l i c a g e l column chromatography (Merck . A . F r a c t i o n s c o n t a i n e d c y c l o s p o r i n -C w a s o b t a i n e d w i t h chloroform-methanol (97:3).e t h y l a c e t a t e as t h e e l u e n t . Working-up of t h e c u l t u r e f i l t r a t e by e x t r a c t i o n w i t h 1 . The t o p f r a c t i o n s w e r e d i s s o l v e d i n t o l u e n e and a p p l i e d t o column chromatography o f aluminium oxide u s i n g t o l u e n e . 5 ) c o n t a i n e d c y c l o s p o r i n A . The water s o l u t i o n w a s e x t r a c t e d t h r e e times w i t h l . Then a f t e r t h e s e p a r a t i o n of t h e o r g a n i c l a y e r it w a s c o n c e n t r a t e d under vacuum.d i c h l o r o e t h a n e and t h e o r g a n i c l a y e r s were evaporated t o d r y n e s s . I n accordance t o t h e i r p o l a r i t y . Another r e p o r t e d procedure ( 2 7 ) w a s as f o l l o w s : The f e r m e n t a t i o n b r o t h w a s s e p a r a t e d i n t o t h e m y c e l i a l cake and t h e c u l t u r e f i l t r a t e . T h i s was grown i n an a e r o b i c submerged culture. The combined e x t r a c t e d were submitted t o g e l f i l t r a t i o n on Sephadex LH-20 w i t h methanol. The product o b t a i n e d was d i s s o l v e d i n chloroform and a p p l i e d t o a column chromatography of s i l i c a g e l . 0. The f r a c t i o n s c o n t a i n i n g c y c l o s p o r i n A were s u b j e c t e d t o g e l f i l t r a t i o n with Sephadex LH-20 w i t h methanol.CYCLOSPORINE 177 The s t r a i n used i s Tolypocladium i n f l a t u m d e s i g n a t e d No. A r e p o r t e d method of i s o l a t i o n of c y c l o s p o r i n e from f e r m e n t a t i o n media w a s as f o l l o w s ( 4 ) : The f e r m e n t a t i o n medium w a s e x t r a c t e d w i t h n-butylacetate. The crude c y c l o s p o r i n e s were f u r t h e r p u r i f i e d by c r y s t a l l i z a t i o n ( c y c l o s p o r i n e G from e t h e r / p e t r o l e u m e t h e r a t room t e m p e r a t u r e . C and D f r o m a c e t o n e a t . The f r a c t i o n s o b t a i n e d were evaporated and d i s s o l v e d i n a l c o h o l . 2 . t h e n t r e a t e d w i t h c h a r c o a l . 2 . G.15OC).063-0.

HASSAN AND MOHAMMED A. t h u s i n d i c a t i n g t h a t Nm e t h y l a t i o n of t h e amino a c i d s o c c u r r e d s i m u l t a n e o u s l y . It w a s demonstrated t h a t t h e N-methyl groups of t h e molec u l e and t h e methyl group i n t h e y . I n e n n i a t i n .p o s i t i o n o f t h e u n s a t u r a t e d amino a c i d MeBmt a r e i n t r o d u c e d as i n t a c t methyl groups from methionine and t h a t t h e remaining carbons of t h e MeBmt moiety a r e d e r i v e d from t h e headt o . 'H. Ruegger _ et _ a1 ( 4 ) and Traber e t a1 ( 2 8 . I R . 5.33) have r e p o r t e d t h e b i o s y n t h e s i s of c y c l o s p o r i n e and o t h e r f u n g a l metab o l i t e s . 3H.32.and 13C-labeled p r e c u r s o r s w e r e f e d t o t h e c u l t u r e and t h e p o s i t i o n o f i n c o r p o r a t i o n o f t h e l a b e l i n c y c l o s p o r i n e w a s determined by NMR s p e c t r o s c o p y . They were found t o be i d e n t i c a l i n a l l r e s p e c t s w i t h a u t h e n t i c samples i s o l a t e d from f e r m e n t a t i o n i n complex n u t r i e n t media as p u b l i s h e d by Dreyfuss e t a 1 ( 2 ) . a n analogous example of non-ribosomal c y c l i c p e p t i d e s y n t h e s i s by f u n g i .178 MAHMOUD M. t h e results o b t a i n e d by t h e Zocher group ( 3 3 ) w i t h short-term f e e d i n g experiments u s i n g l a b e l e d a c e t a t e and methionine. 5 and 7 o f t h e MeBmt u n i t and no '3C i n c o r p o r a t i o n i n t o any o t h e r amino a c i d . i n which probably o n l y i n c o r p o r a t i o n of . C. m e l t i n g p o i n t . D and G w e r e c h a r a c t e r i z e d by a n a l y t i c a l methods ( t h i n l a y e r chromatography. Zocher and Kleinkauf ( 32) demonstrated t h a t a m u l t i f u n c t i o n a l enzyme c a r r i e s o u t t h e N-methylation o f c o n s t i t u e n t amino a c i d s following an a c t i v a t i o n s t e p when t h e a c i d s are bound t o t h e enzyme as t h i o e s t e r s . From t h e r e s u l t s o f Kobel e t a1 ( 3 1 ) t h e s i t e of b i o s y n t h e s i s of t h e Bmt u n i t remains u n c l e a r . 3. o p t i c a l r o t a t i o n ) and s p e c t r a l d a t a ( U . The l3C-NMR spectrum o f t h e e n r i c h e d c y c l o s p o r i n e d e r i v e d from [ l-13C]acetate showed f o u r enhanced s i g n a l s corresponding t o t h e carbon atoms 1.and l3C-NMR. B. Supporting evidence f o r a similar mechanism i n c y c l o s p o r i n e b i o s y n t h e s i s w a s provided by t h e o b s e r v a t i o n o f Zocher e t a1 ( 3 3 ) t h a t [14C] s a r c o s i n e w a s n o t ' n c o r p o r a t e d and t h a t t h e r a d i o a c t i v i t y from [Me-16C]methionine i n c o r p o r a t e d i n t o each N-methylated amino a c i d w a s d i r e c t l y proport i o n a l t o t h e number o f t h e corresponding amin a c i d r e s i d u e s i n c y c l o s p o r i n e . AL-YAHYA r e s u l t i n g pure c y c l o s p o r i n e s A. B i o s y n t h e s i s of Cyclosporine Kobel _ e t -a1 ( 3 1 ) and o t h e r s (27. mass s p e c t r u m ) . A. I n t h e i n i t i a l b i o s y n t h e t i c s t u d i e s o f Kobel _ et _ a1 (31) . 3 0 ) .t a i l condensation of f o u r a c e t a t e u n i t s . However.

Synthesis of the heptapeptide H-Me-C9-AbuSar-MeLeu-Val-MeLeu-Ala-benzylester (18. TV. Building of the tetrapeptide BOC-MeLeu-ValMeLeu-Ala-benzylester (18). The strategy of the total synthesis of cyclosporine has involved the following steps:- I. suggest that the amino acid Bmt is not biosynthesized on the enzyme matrix but at some other site before incorporation into cyclosporine. The synthesis of the hexapeptide BOC-AbuSar-MeLeu-Val-MeLeu-Ala-benzylester (18). Synthesis 6. 11. Cyclization of the undecapeptide with a free amino group and free carboxyl group to produce cyclosporine (48.179 CYCLOSPORINE label in the N-methyl group occurred.47) .46). 111. VI .48).51). VIII. Formation of the undecapeptide BOC-Ala-MeLeuMeLeu-MeVal-Me-Cg-Abu-Sar-MeLeu-Val-MeLeuAla-benzylester (18. VII.37. 34-38) has reported the total synthesis of cyclosporine and analogues. Preparation of the dipeptide BOC-Abu-Sarbenzylester (18.50.1 Cyclosporine Winger (18. The synthesis of the N-methyl-C-9-amino acid (Scheme 1) (36. V. Building of the tetrapeptide BOC-D-Ala-MeLeuMeLeu-MeVal-benzylester (37). 6. . 39-44).

HASSAN AND MOHAMMED A. 5E) -2-hydroxy-3-methyl5-heptenal (2S.3R. 3R.180 tio- MAHMOUD M. 7 Steps b COOEI (ZR.2. 6E) -3-hydroxy-4methyl-2-(methylamino)-6-octenoic acid (MeBmt) Scheme 1.SE)-3-rnethyl-S-heptene1. 3R.2-diol "II_ c y k c y (2R. A.4. AL-YAHYA F:.3R)-3-methyl-l. Synthesis of MeBmt . 4R.-butanetrio1 6 Steps (2R.

The carboxy groups were g e n e r a l l y a c t i v a t e d using a v a r i a t i o n of t h e mixed p i v a l i c anhydride method r e p o r t e d by Zaoral ( 4 6 ) and adapted f o r t h e N-methylamino a c i d d e r i v a t i v e s ( 3 7 ) . 2 . The intramolecular hydrogen bonds between t h e amide groups of t h i s l i n e a r p e p t i d e could o p e r a t e so as t o s t a b i l i z e t h e open chain i n folded conformations approximating t h e c y c l i c s t r u c t u r e of cyclosporine and t h u s assist c y c l i z a t i o n . a fragment-condensation technique i n t r o d u c i n g t h e amino a c i d MeBmt a t t h e end of t h e s y n t h e s i s w a s used.CYCLOSPORINE VIII. There were two main reasons f o r choosing t h i s s t r a t e g y : 1. For t h e s y n t h e s i s of t h e undecapeptide. 1) t h e p e p t i d e bond between t h e L-alanine i n p o s i t i o n 7 and t h e D-alanine i n p o s i t i o n 8 w as chosen f o r t h e c y c l i z a t i o n s t e p . I n t h i s way.2OoC with p i v a l o y l c h l o r i d e i n t h e presence of 2 e q u i v a l e n t s of a t e r t i a r y base such as N-methylmorpholine before adding t h e amino a c i d o r p e p t i d e e s t e r s t o be coupled i n t h e form o f t h e i r f r e e bases. 35.2OoC and subsequent . 34. t h e number o f s t e p s a f t e r t h e i n t r o d u c t i o n of t h i s amino a c i d w a s minimized. t h e r e f o r e . bond formation between t h e only consecutive p a i r of non-methylated amino a c i d s i n cyclosporine appeared t h e l o g i c a l choice f o r t h e c y c l i z a t i o n s t e p .45) . 37 1 S t r a t e g y Used f o r t h e Synthesis o f Cyclosporine For t h e s y n t h e s i s of cyclosporine [l]( F i g . T h i s s t r a t e g y allowed slow anhydride formation i n chloroform a t . The bond formation between N-methylated amino a c i d s i s more d i f f i c u l t t h a n between non-methylated amino a c i d s (37. 181 T o t a l Synthesis of Cyclosporine (18. The f r e e bases were obtained by removal of t h e Boc-protecting groups with t r i f l u o r o a c e t i c a c i d a t . Scheme 5 shows t h e s t e p sequence used f o r j o i n i n g t h e ( p r o t e c t e d ) p e p t i d e fragments.

t h e f i n a l amide l i n k a g e 1 0 w a s made by coupling Boc-D-Ala-MeLeu-MeLeu-MeVal-OH with t h e heptapeptide i n presence of N-methylmorphol i n e i n CH2C12 at room temperature using t h e reagent (1H-1. This isopropylidene-protecting group was r e a d i l y introduced by r e f l u x i n g t h e amino a c i d i n acetone and has t h e advantage of avoiding epimerization of t h e amino a c i d MeBmt during peptide-bond formation. A f t e r h y d r o l y s i s of t h e .3-benzotriazol-l-yloxy)tris (dimethylamino)-phosphonium hexafluorophosphate ( B t O P ( NMe2)'3 PFZ) developed by Castro et a1 ( 4 8 ) . 6 and 7 i n t h a t o r d e r . A f t e r removal of t h e isopropylidene-protecting group (1 equiv. t h e p r o t e c t e d MeBmt amino a c i d w a s s t a b i l i z e d by a d d i t i o n of N-methylmorpholine (1 e q u i v . The t e t rapept i d e Boc-D-Ala-MeLeu-MeLeuMeVal-OBzl w a s synthesized from t h e l e f t t o t h e r i g h t by forming bonds 1. Subsequent coupling (bond 8 ) w i t h t h e p r e v i o u s l y synthesized d i p e p t i d e t h e n furnished t h e hexapeptide Boc-Abu-SarMeLeu-Val-MeLeu-Ala-OBzl.2.182 MAHMOUD M. HASSAN AND MOHAMMED A. To prepare t h e p r o t e c t e d heptapeptide from t h e p r o t e c t e d MeBmt amino a c i d and t h e hexapeptide. A. Bond 4 was formed on synt h e s i s of t h e d i p e p t i d e Boc-Abu-Sar-OBzl. On p r e p a r a t i o n . of 1 N HCl/MeOH). 2 and 3 (Scheme 2 ) . t h e hydroxyand N-methylamino f u n c t i o n s were p r o t e c t e d i n t h e form of a dimethyloxazolidine derivat i v e . This means t h a t t h e oxazolidine r i n g r e t a i n s t h e thermodynamically more s t a b l e t r a n s c o n f i g u r a t i o n of s u b s t i t u e n t s during carboxy a c t i v a t i o n and peptide formation. t h e dicyclohexylcarbodiimide ( D C C I ) coupling method w a s used i n presence of N-hydroxybenzotriazole ( 4 7 ) . t h e benzyloxyp r o t e c t i n g groups of t h e p e p t i d e i n t e r mediates were removed h y d r o g e n o l y t i c a l l y with palladium/carbon i n ethanol. AL-YAHYA n e u t r a l i z a t i o n with NaHC03. The t e t r a p e p t i d e Boc-MeLeu-Val-MeLeu-AlaOBzl w a s synthesized from t h e r i g h t t o t h e l e f t by forming bonds 5 . ) . but before a c t i v a t i o n . During incorporat i o n of t h e amino a c i d MeBmt.

Bold arrows: slralrgy lor the synthesis o l the pcptidcs. >-OH isopropylidcne. .proleacd MeBml. Boc-rrrrbuloxycarbonyl. For delailr see teat.A lo --A lo t. BzI -bcnzyl.183 CYCLOSPORINE step sequence for the peptide bond formation 1 - 4 - iiiTiiiiii 1 0-Alo 8 HeLeu HeLeu 9 10 HeVol 11 H e 6 m t Abu Sor MeLeu Vol HeLeu Alo 1 HeLcu-cHcVol~HeB~t-cAbu~Sar t HeLeu t 0 .Syniherir olcyclosporinc 1 .H rLeu-Vol --HeLeu - Scheme 2.

By using t h e mixed phosphonic anhydride method described by Wissmann and K l e i n e r ( 4 9 ) o r t h e pentafluorophenol-DCCI complex of Kovacs ( 5 0 ) t o e f f e c t c y c l i z a t i o n of t h e unprotected undecapeptide . Metabolism of Cyclosporine Maurer e t a1 ( 5 2 ) have r e p o r t e d t h e d i s p o s i t i o n of cyclosporine i n man and s e v e r a l animal s p e c i e s and a l s o s t r u c t u r a l e l u c i d a t i o n of i t s m e t a b o l i t e s .5% y i e l d with r e s p e c t t o t h e amino a c i d MeBmt. 3H-labeled cyclosporine with s p e c i f i c a c t i v i t i e s ranging from 25 t o 80 uCi/mg w a s prepared b i o s y n t h e t i c a l l y by feeding submerged c u l t u r e s of a s e l e c t e d s t r a i n of t h e fungus inflatum G a m s with [methyl-H3] methionine as l a b e l e d precursor ( 3 1 ) . The metabol i t e s were i s o l a t e d from dog and human u r i n e and from rat b i l e and f a e c e s . 3H-NMR a n a l y s i s revealed t h e incorporated tritium t o be l o c a t e d i n t h e seven Nmethyl groups and i n t h e methyl group a t p o s i t i o n 4 (C. t h e y i e l d o f cyclosporine could be increased t o 65%. BtOP(NMe).2OoC) . 11).) of amino a c i d ( F i g .e x t r a c t a b l e s of cyclosporine were i s o l a t e d from u r i n e of dog and man and from r a t b i l e and f a e c e s ( F i g . 1 . r.184 MAHMOUD M. N-methylmorpholine). . Using t h e fragment-condensation technique described h e r e it i s now p o s s i b l e t o synthesize cyclosporine very e f f i c i e n t l y i n 27. 6 g of cyclosporine can be produced s t a r t i n g from 1 g of t h e amino a c i d MeBmt . AL-YAHYA e s t e r group (NaOH a t O°C) and removal of t h e Boc group (CF3COOH a t . 12). Thus. PFZ ( 4 8 ) . t h e unprotected undecapeptide w a s c y c l i z e d a t room temperature t o cyclosporine 1 (0. 7. The radiochemical p u r i t y of t h e l a b e l e d substance w a s b e t t e r t h a n 95%. The s e p a r a t i o n of m e t a b o l i t e s was performed by HPLC. Nine e t h e r . HASSAN AND MOHAMMED A. The y i e l d of c r y s t a l l i n e cyclosporine w a s 62%.0002 M CH2C12. A. 1 equiv. The s t r u c t u r e s of t h e i d e n t i f i e d m e t a b o l i t e s were used t o e s t a b l i s h t h e biotransformation processes.

11. c H 0 r) .. 1) .Site and type of biotransformation in cyclosporine F i g .

F i g . HASSAN AND MOHAMMED A.I &A8 0 b A I I I R ---Ctiz-:ti*... C 4 ti cti. h H I 2I 0 .. O C .... ti 1220..61 .. 6 ~ On OH n 081 n CH./: 'cn..C!l Cn.-N f cl'I ... 011 Rl Cyclosporinc n 011 1! 9 ho. $ . C H -NI ...64 A ~ I 1218.64 1218.. ....i - .. .. 12.N I CH. CHI Cli.MAHMOUD M.C. 1...ite on R4 n n It CH] OH n n CH) n on n -I n H of( Cn..64 /9 C H ch-cnZ z c r .N ....62 1234. I CI(.C O . I . n oti ciil n n ?! H II n II II Other modif i r r t ion 1214.iII. AL-YAHYA 186 Structure of cyclosDorine and i t s m e t a b o l i t e s .* CkI.64 1214... n or.. .CH 1 I b I 0..CH . CII.64 1188.. I CH...I AI CO CH.. - -10 16 I1 - Ho I ccul a r ueiqht Rl R -I - R2 Vet~bo.C . 6 co h-R2 . CH+ *3 ----- tj CH. n M 1202 .CH-NI CHj I 1. CI. A.u 0 .

1 0 . The c y c l i c o l i g o p e p t i d e s t r u c t u r e of c y c l o s p o r i n e i s p r e s e r v e d i n a l l i d e n t i f i e d metab o l i t e s . The i d e n t i f i c a t i o n o f a d i h y d r o x y l a t e d and N-demethylated d e r i v a t i v e of c y c l o s p o r i n e ( m e t a b o l i t e 9 ) i n d i c a t e s f u r t h e r o x i d a t i o n t o occur on e i t h e r t h e d i h y d r o x y l a t e d m e t a b o l i t e 1 6 by N-demethylation o f t h e N-methylleucine 4 o r t h e primary N-demethylcyclosporine 2 1 by hydroxyl a t i o n o f b o t h N-methylleucines 6 and 9. and o f m e t a b o l i t e 17 on t h e N-methylleucine 9 g e n e r a t e s d i h y d r o x y l a t e d d e r i v a t i v e s of c y c l o s p o r i n e ( m e t a b o l i t e s 8 . F u r t h e r o x i d a t i o n o f metab o l i t e l on e i t h e r one o f t h e N-methylleucines 4 and 6 o r on t h e Cg-amino a c i d 1. Oxidative N-demethylation. namely f o u r of 11 amino a c i d s .p o s i t i o n o f t h e N-methylleucines 4. seems t o occur o n l y on t h e N-methylleucine 4. undergo r e g i o s p e c i f i c o x i d a t i v e modif i c a t i o n s . S i t e and t y p e of b i o t r a n s f o r m a t i o n i n c y c l o s p o r i n e are summarized i n F i g . . and 1 6 ) .p o s i t i o n or t h e amino a c i d 1 a t t h e q . 6 . M e t a b o l i t e s 1 and 17 r e s u l t from h y d r o x y l a t i o n o f e i t h e r t h e N-methylleucine 9 a t t h e y . t h e r e a c t i o n s involved i n t h e b i o d e g r a d a t i o n o f t h e molecule are l i m i t e d and produce o n l y a r e l a t i v e l y s m a l l number o f m e t a b o l i t e s .CYCLOSPORINE 187 Despite t h e complex s t r u c t u r e o f c y c l o s p o r i n e . 13) are based on t h e s t r u c t u r e o f t h e i s o l a t e d m e t a b o l i t e s . and 9. T y p i c a l phase I1 r e a c t i o n s l i k e c o n j u g a t i o n w i t h s u l f u r i c a c i d or g l u c u r o n i c a c i d could n o t be d e t e c t e d . Metabolite 21 r e p r e s e n t s the product o f N-demethylation on t h e N-methylleucine 4. The r e g i o i s o m e r i c monohydroxylated c y c l o s p o r i n e s 1 and 17 and t h e N-demethyl a t e d c y c l o s p o r i n e 2 1 are primary m e t a b o l i t e s . The r e s u l t i n g p r o d u c t s a r e l i p o p h i l i c as i n d i c a t e d by t h e i r occurrence i n t h e e t h e r phase of the liquid-liquid extraction. so f a r . Hydroxylation r e a c t i o n s appear t o be r e s t r i c t e d t o t h e rl-position o f amino a c i d 1 (Cg-amino a c i d ) and t h e y . 11. Metabolism mainly c o n s i s t s o f o x i d a t i v e p r o c e s s e s ( p h a s e I r e a c t i o n s ) . The proposed pathways f o r t h e b i o t r a n s f o r m a t i o n o f c y c l o s p o r i n e ( F i g .p o s i t i o n . The N-methylleuc i n e s 6 and 9 a r e s u b j e c t o f a s i n g l e r e a c t i o n ( h y d r o x y l a t i o n ) whereas amino a c i d 1 and t h e N-methyll e u c i n e 4 are s u b s t r a t e s f o r two t y p e s of transformat i o n : h y d r o x y l a t i o n and i n t r a m o l e c u l a r e t h e r formation o r h y d r o x y l a t i o n and N-dernethylation. Only a l i m i t e d number o f t h e c y c l o s p o r i n e s u b u n i t s .

Proposed pathways for the biotransformation of cyclosporine. .8 t t 21 'En. I 10 Fig. 13.

The spectroscopic data of two of them were compatible with hydroxylated and N-demethylated derivatives of cyclosporine. It should be noted that metabolites 10 and found in man (53). . whereas those of the third indicated a dihydroxylated derivative. may be derived from 17 (monohydroxycyclosporine) by intramolecular addition of the 6-hydroxyl group to the double bond of the C9-amino acid 1.CYCLOSPORINE 189 Metabolite 18. containing a cyclic ether moiety (tetrahydrofuran). An artifact with a similar cyclic ether structure has previsouly been observed among the acid hydrolysis products of cyclosporine. 99% Number of components 10-12(9 of which have been characterised as metabolites). 16 were not 8. Three other metabolites possessed the basic cyclic structure of the parent drug. metabolism and elimination of cyclosporine have been investigated by many authors - ( 52-57) The pharmacokinetic data on cyclosporine in man as follows : Absorption Ab solute or a1 bioavailabi1ity 20-50% (mean 34%) Plasma peak concentration time 1-6 hours Di stribut ion Binding to plasma proteins go% (20OC) Apparent volume of distribution 3. This cyclization is suggested to proceed by a nonenzymatic mechanism. distribution. Pharmacokinetics of cyclosporine The absorption. Their structures remain incompletely determined.5 1/Kg Metabolism Extent of metabolism Ca.

10-12 metab o l i t e s have been d e t e c t e d i n human plasma.51/Kgm. S t r u c t u r a l m o d i f i c a t i o n s c o n s i s t e d of monoand di-hydroxylation and N-demethylation a t v a r i o u s p o s i t i o n s on t h e c y c l o s p o r i n e . kidney. Nine metab o l i t e s have been i s o l a t e d and i d e n t i f i e d . A. O f t h e t o t a l amount of drug i n t h e plasma. p a n c r e a s . I n whole blood. t h y r o i d and r e n a l f a t .b a s e d s o l u t i o n . t h e h i g h e s t t i s s u e c o n c e n t r a t i o n s of r a d i o a c t i v i t y were found i n l i v e r . about 50% o f c y c l o s p o r i n e i s t a k e n up by e r y t h r o c y t e s and 10-20% by l e u c o c y t e s . t h e o r a l dose form of c y c l o s p o r i n e i n an o l i v e . a d r e n a l s . AL-YAHYA Elimination Terminal e l i m i n a t i o n h a l f . . I n a d d i t i o n t o t h e p a r e n t d r u g .o i l . approximately 90% i s protein-bound.l i v e s from blood o r plasma o f 14-26 hours have been r e p o r t e d . thymus. I n man t h e t o t a l u r i n a r y e x c r e t i o n o f r a d i o a c t i v i t y i n t h e p e r i o d up t o 96 hours f o l l o w i n g a s i n g l e dose o f 3H-cyclosporine w a s 6% of t h e dose. The t o t a l a p p a r e n t volume o f d i s t r i b u t i o n was 3. Cyclosporine i s slowly b u t e x t e n s i v e l y metabolised by l i v e r . t h e drug was found t o be d i s t r i b u t e d according t o a three-compartment d i s t r i b u t i o n model. From blood concentration-time d a t a f o l l o w i n g i n t r a v e n o u s a d m i n i s t r a t i o n i n man. The drug and i t s m e t a b o l i t e s are e x c r e t e d mainly v i a t h e l i v e r and o n l y t o a s l i g h t e x t e n t v i a t h e k i d n e y s . HASSAN AND MOHAMMED A.47 l/h/Kg because of i t s v e r y low w a t e r . The a b s o l u t e o r a l b i o a v a i l a b i l i t y from t h i s s o l u t i o n ( i n s t e a d y s t a t e ) i s 20-50% (mean 34%).s o l u b i l i t y .1% o f t h e o r & dose. Terminal e l i m i n a t i o n h a l f .MAHMOUD M.l i f e 14-26 hours T o t a l body c l e a r a n c e 0. unchanged cyclos p o r i n e accounted f o r o n l y 0.27-0. mostly t o l i p o p r o t e i n s . I n animal experiments using3H-cyclosporine. a l l c o n t a i n e d t h e i n t a c t c y c l i c o l i g o p e p t i d e s t r u c t u r e of t h e p a r e n t molecule.

The hemopoietic tissues in immunosuppressed animals are not affected. such as in the treatment of autoirnune and other diseases. are currently under investigations. 10. Today cyclosporine is used successfully to prevent graft rejection following bone marrow and organ transplantations. The first transplantation in humans with the aid of cyclosporine were reported in 1978 by Calne et a1 (60) f o r kidney and by F’owles et a1 (61) f o r bone marrow. and specific for T-lymphocytes. cyclosporine is a selective immunosuppressive drug with antifungal and antiinflammatory properties.00 .80 16.90 H 9-30 N 0 12. Bueding _ et _ a1 (58) demonstrated an antishistosomal activity and Thommen-Scott ( 59) an antimalarial effect of cyclosporine. 11. Other possible applications. both antiparasitic activities being independent of immunosuppression.1 Elemental Composition Cyclosporine (C H N 0 ): 62 111 11 12 C 61. Pharmaceutical Forms Cyclosporine ( Sandimmun) is available as : a) Drinking Solution: 100 mg/ml cyclosporine dissolved in olive oil and labrafil. It has been shown to strongly suppress the production of haemagglutinating antibodies against sheep erythrocytes in mice and to be effective in inhibiting both humoral and cellmediated immune responses. Pharmacological Activity and Clinical Application As reported by Bore1 et a1 (5-9).Methods of Analysis 11. b) Intravenous Preparation: Ampoules of 5 m l (250 mg cyclosporine) and 1 ml (50 mg cyclosporine).191 CYCLOSPORINE 9 . Its effect is reversible.

The technical complexity of this method encouraged several investigators.5 to 3 h after collection. The first method reported was the radioimmunoassay (RIA) of Donatsch (62)which is now available as a kit and has been used extensively during the clinical development for cyclosporine. Other RIA procedures were reported ( 54. Niederberger et a1 (68) developed a high pressure liquid chromatogri:phy ( HPLC) method based on gradient elution. 11. Non-temperature-standardized protocol: About 0. - 63-67) One method described recently by Randall et a1 (64) is as follows: Sample Preparation: Venous blood was collected in heparinized tubes from 38 post-transplant patients receiving cyclosporine. The main criticism of the RIA method has been the cross-reactivity of the antisera with some of the circulatinc metabolites of cyclosporine.55. is also applicable to blood samples. AL-YAHYA 192 11. (69-75) to reinvestigate the method. . The plasma and whole-blood specimens were both then stored at . just before their next dose and 12 h after their last. 63-67). The method was initially developed for plasma. and as shown later. an aliquot of each sample f o r whole-blood analysis was set aside and centrifuged the remaining specimen at room temperature to remove cells.55. The results of these activities are shown in Table 4.3 Radioimmunoassay ( RIA) Several radioimmunoassay procedures have been developed for both the analysis and immunopharmacological monitoring of cyclosporine ( 54. A.20%. Subsequent to the introduction of the RIA method.2 Introduction to Analysis Several methods have been developed for the analysis of cyclosporine in plasma and or blood.MAHMOUD M. HASSAN AND MOHAMMED A.

. and 180 min. I n a d d i t i o n . 6.L o f T r i s b u f f e r ( 0 . 5 ) . samples s t o r e d a t room temperature or 4°C were c e n t r i f u g e d a t room t e m p e r a t u r e . B a s l e . followed by c e n t r i f u g a t i o n a t 37OC and prompt removal of t h e plasma. 1 5 . A t t h e s e s p e c i f i e d t i m e s .CYCLOSPORINE 193 Temperature-standardized p r o t o c o l : After c o l l e c t i o n . and 37OC. Measurement of c y c l o s p o r i n e : Cyclosporine w a s measured by radioimmunoassay. samples were r e f r i g e r a t e d for no l o n g e r t h a n 4 h . S w i t z e r l a n d .e q u i l i b r a t e d t o 37OC by i n c u b a t i o n i n a water b a t h f o r 30 min. t h e n e q u i l i b r a t e d i n a 37OC w a t e r b a t h for 0.5. F r e e and bound f r a c t i o n s were s e p a r a t e d by e x t r a c t i o n w i t h c h a r c o a l for min a t 4 O C . room t e m p e r a t u r e ) The r e s u l t i n g plasma w a s s t o r e d f r o z e n a t -2OOC u n t i l analysis. 2. The r a d i o a c t i v i t y was measured i n each specimen w i t h a l i q u i d s c i n t i l l a t i o n . 4 . TOO 1J. room t e m p e r a t u r e ( a b o u t 22OC). p H 8 . 0. t h e n c e n t r i f u g e d without d e l a y (1100 X g . 60. t h e t r e a t e d a l i q u o t s of samples s t o r e d a t room t e m p e r a t u r e and 4 O C f o r t h e above t i m e s b y r e . 5 mol/L. t h e n i n c u b a t i n g for 2 h a t room t e m p e r a t u r e . u s i n g t r i t i a t e d c y c l o s p o r i n e as t r a c e r and an a n t i b o d y i n a k i t s u p p l i e d by Sandoz L t d . t h o s e s t o r e d a t 3 7 O C were c e n t r i f u g e d a t 37OC and t h e plasma was decanted and f r o z e n . 1. D u p l i c a t e 5 pL p a t i e n t ' s samples were mixed with 1 0 0 pL of t r a c e r . 30. 1 0 min. 10.25. 1 2 0 .e q u i l i b r a t i n g t o 3 7 O C by i n c u b a t i n g for 1 5 min i n a 37OC w a t e r b a t h . and t h e plasma was immediately decanted and stored a t -2OOC u n t i l a n a l y s i s . All specimens were c e n t r i f u g e d a t 3 7 O C . r e . Time and t e m p e r a t u r e experiments: A l i q u o t s of whole blood from p a t i e n t s r e c e i v i n g c y c l o s p o r i n e were i n c u b a t e d for 0. E q u i l i b r a t i o n time experiment: A l i q u o t s o f whole blood from p a t i e n t s r e c e i v i n g c y c l o s p o r i n e w e r e maintained a t 4 O C f o r 4 h a f t e r c o l l e c t i o n . and 100 UL o f sheep a n t i s e r u m t o c y c l o s p o r i n e . and 24 h a t 4'12.

) I n s t r u m e n t a t i o n and chromatograghic c o n d i t i o n s : A Waters Instrument ( M i l f o r d . AL-YAHYA c o u n t e r . A r e c e n t HPLC method w a s r e p o r t e d by Kates and L a t i n i ( 7 5 ) and i s as follows : Chemicals and r e a g e n t s : Cyclosporine and t h e i n t e r n a l s t a n d a r d .S. Model 7lOB sample i n j e c t o r and Model 730 d a t a module a r e employed. U. The a n a l y t i c a l recovery o f c y c l o s p o r i n e i n plasma and whole blood supplemented w i t h t h e drug w a s about 75%. c y c l o s p o r i n D ( F i g . This i s because.) RP-8 MLPC a n a l y t i c a l c a r t r i d g e . 69-75). t h e a n t i b o d i e s c r o s s r e a c t w i t h t h e m e t a b o l i t e s .A. u s i n g a quench-corrected c o u n t i n g program. a c e t o n i t r i l e . F L ) . Model 480 v a r i a b l e wavelength UV d e t e c t o r . ( S a n t a Clara.A.A. purhcased from A l d r i c h (Milwaukee. Glass-distilled diethyl ether.194 MAHMOUD M. The column used i s a Brown-Lee Labs. 5 4 . q-hexane. The plasma c y c l o s p o r i n e r e s u l t s were c o r r e c t e d f o r hematocrit by m u l t i p l y i n g t h e u n c o r r e c t e d plasma c y c l o s p o r i n e r e s u l t s by t h e p a t i e n t ' s hematocrit / O . U. Hialeah. W I .) Model 6 0 0 0 ~HPLC pump.). A. HASSAN AND MOHAMMED A. CA. U. and methanol are o b t a i n e d from Burdick and Jackson Labs. MA. The HPLC c o n d i t i o n s used f o r t h e a n a l y s i s o f c y c l o s p o r i n e a r e l i s t e d i n Table 5. M I . C o r r e c t i o n f o r h e m a t o c r i t : Hematocrits of samples f o r c y c l o s p o r i n e a n a l y s i s were measured as p a r t o f t h e r o u t i n e b l o o d count on a Coulter S Plus (Coulter Instruments.A. S w i t z e r l a n d ) . 4 High Performance Liquid Chromatography S e v e r a l HPLC procedures were r e p o r t e d for t h e e s t i m a t i o n o f c y c l o s p o r i n e i n blood and o r plasma a s w e l l as immunopharmacological monit o r i n g o f c y c l o s p o r i n e ( 5 3 .S. are o b t a i n e d from Sandoz (Basle. These methods were developed t o overcome t h e drawbacks o f t h e R I A methods p a r t i c u l a r l y t h e overestimation of cyclosporine i n b i o l o g i c a l f l u i d s . Tris(hydroxymethy1)ainomethane i s U. 14). t h e n o m a l v a l u e f o r t h e hematocrit.S.39. (Muskegon. 1 1 . 67.S.

S . They a r e rocked f o r 20 min on a labquake r o c k e r (Lab. The mobile phase i s c o n t i n u o u s l y r e c y c l s e d and r e p l a c e d about every two weeks.A. ) The e f f l u e n t from t h e column i s monitored a t a wavelength o f 215 nm. The . ) column h e a t e r . CAY U . These columns a r e p r e p a r e d by washing w i t h 6 ml of methanol and t h e n 6 ml o f water under vacuum. 3 ml o f 0 . The flow-rate o f t h e mobile phase i s 0.S. CA. Berkeley. The columns used are t h e 3 ml cyano d i s p o s a b l e e x t r a c t i o n columns. Baker. p a r t i c l e s i z e 1 0 m). The d i e t h y l e t h e r i s p i p e t t e d i n t o a c l e a n t u b e and t h e blood or serum residue i s d i s c a r d e d .) and c e n t r i fuged for 20 min t o s e p a r a t e t h e e t h e r and aqueous l a y e r s . Mobile phase: The mobile phase c o n s i s t s o f a simple mixture of a c e t o n i t r i l e . P h i l l i p s b u r g ..D.w a t e r ( 72:28).6 mm I. They a r e l e f t approximately one h a l f t o t h r e e q u a r t e r s f u l l of w a t e r u n t i l t h e sample i s added.195 CYCLOSPORINE ( 1 0 ern X 4. s i. This m i x t u r e i s f i l t e r e d and degassed by vacuum and s o n i c a t i o n . 8 ) and 1 0 m l o f d i e t h y l e t h e r . To t h e d i e t h y l e t h e r i s added 200 p1 o f 75% methanol i n w a t e r .). U. screw-capped t u b e s . N J . 1 M T r i s b u f f e r (pH 9 . Only t h e d i e t h y l e t h e r i s e v a p o r a t e d . l e a v i n g 150200 p 1 o f t h e methanol-water remaining.A. The d i e t h y l e t h e r i s t h e n evaporated a t room temperat u r e w i t h a g e n t l e stream of n i t r o g e n .6 ml/min which produces a precolumn p r e s s u r e o f about 34 bars ( 500 p. The h e a t e r i s l e f t on a t a l l t i m e s and a c o n s t a n t flow i s m a i n t a i n e d through t h e column. . T . P r e p a r a t i o n o f e x t r a c t i o n columns: The e x t r a c t i o n o f c y c l o s p o r i n e i n v o l v e s t h e use o f a Baker-10 SPE e x t r a c t i o n system ( J . U. The column i s m a i n t a i n e d a t 7 O o C with an Eldex (Menlo Park. E x t r a c t i o n procedure: One ml o f whole blood or serum i s added t o PTFE-lined.S. To t h i s are added a n o t h e r 100 u1 of methanol. To t h e blood or serum i s added 450 ng o f i n t e r n a l s t a n d a r d . I n d u s t r i e s . A . A 3-em RP-8 guard c a r t r i d g e i s p o s i t i o n e d a t t h e head o f t h e column.

. The column i s t h e n washed w i t h 3 ml of 25% a c e t o n i t r i l e i n w a t e r and 6 m l of n-hexane. The r e s i d u e i s e l u t e d onto t h e column w i t h water. This procedure has a lower l i m i t of s e n s i t i v i t y below 50 ng/ml. 1 4 ) . A. P r e p a r a t i o n of c a l i b r a t i o n s t a n d a r d s : S t o c k s o l u t i o n s of c y c l o s p o r i n e and c y c l o s p o r i n D are prepared i n methanol and s t o r e d i n amber b o t t l e s a t room t e m p e r a t u r e . Cyclosporine and c y c l o s p o r i n D are t h e n d l u t e d off t h e column w i t h t h r e e washings o f 200 p l of methanol. T h i s s o l u t i o n i s used t o p r e p a r e s t a n d a r d s for t h e c a l i b r a t i o n curve.196 MAHMOUD M. The columns a r e t h e n d r i e d by drawing through a i r for 4 min. The s t o c k s o l u t i o n of c y c l o s p o r i n e i s prepared i n a c o n c e n t r a t i o n of 10 n g / p l . HASSAN AND MOHAMMED A. The methanol i s c o l l e c t e d i n t o d i s p o s a b l e t u b e s and evaporated t o dryness w i t h a stream of n i t r o g e n a t 5OoC. These are t h e n t r e a t e d as d e s c r i b e d i n t h e e x t r a c t i o n procedure. The r e s u l t i n g r e s i d u e i s d i s s o l v e d i n 200 p 1 of t h e mobile phase. The c a l i b r a t i o n c u r v e samples are prepared by adding amounts of cyclos p o r i n e (50-800 ng) t o whole blood or serum from a normal v o l u n t e e r . A s t o c k s o l u t i o n of c y c l o s p o r i n D i s prepared i n a c o n c e n t r a t i o n of 30 n g / u l . AL-YAHYA methanol-water i s t h e n t r a n s f e r r e d t o a 3 m l Baker e x t r a c t i o n column. An a l i q u o t o f 1 0 0 p 1 i s t h e n i n j e c t e d o n t o t h e column ( F i g .

. I1 = cyclosporin D. Chromatograms of extracted whole blood samples. not taking cyclosporine. (B) blood to which has been added 200 ng/rnl of cyclosporine and the internal standard. ( A ) Blood from normal healthy volunteer. 14.C d I n 0 71 I 11 \ L F i g . (C) blood from a patient who was taking cyclosporine ( t h e concentration in this patient sample is 304 nglml). internal standard. Peaks: I = cyclosporine.

19 75 p r o t e i n p r e c i p i t a t i o n and i n j e c t i o n . I n t . = I s o c r a t i c . Radioimmunoassay. .External. 54955. * 1-20 . Ex:. 30 100 - Ext Very l o n g 25 High Int Short 20 High Blood/plasma HPLC-Grad. Int . Plasma Long 31 High Int . Plasma Long Serum Long .Table 4. = Internal. 30 73 74 15 67 . I s o c r . Ref. = G r a d i e n t . ihort s emiaut oma- 8 High Ext 50 High Int HPLC-Isocr. . - 20 High Int . Blood/plasma Long 100 High Int HPLC-Grad. Col-Swit = Column s w i t c h i n g . a d d i t i o n a l sample m a n i p u l a t i o n . =. Int . 63-67 68 5 10 69 70 30 71 20 72 63 45 15 . Blood/plasma HPLC-Col Swit Blood/plasma HPLC-Isocr .Swit 3loodlplasma HPLC-Isocr "Short: **RIA: ted) . Time( min) Metabolite x-react i v i t y ~ RIA** HPLC-Isocr Chromat o- Very Long: Grad. HPLC-Isocr. Plasma Long 100 High . . Comparison o f p u b l i s h e d methods o f a n a l y s i s o f c y c l o s p o r i n e analysis Sample t y p e Sample* preparation Detect i o n limit pg/litre) Specificity S t an dardization Ext . Long . Serum Long 30-50 High Int HPLC-Isocr. % . Long: e x t r a c t i o n & e v a p o r a t i o n . HPLC-Col .

6 Acetonitrilel w a t e r 72:28 cal cartridge. 40 X 4. Column I. . A l t e x p e r i s o r b RP-8 . 5- 6. A l t e r n a t i v e column. Acetonitrile1 water 55:45 v/v 3. D . A c e t o n i t r i l e l water 72:28 vlv 4. 10 cm x 4.2 mm I . A c e t o n i t r i l e 1 methanollwat e r 35 :20 :45 v/v/v 2. A l t e x pumps AX-110.0 UV a t 202 nm 1-7 W at 210 nm I C o n s i s t s of 6 solvent s.6 mm I . 5 urn. c ~ 1 0. HPLC c o n d i t i o n s used f o r t h e a n a l y s i s of c y c l o s p o r i n e Flow Sample Instrument rate Mobile phase Column m l /min I Blood/snrum Waters Model RP-8 YLPC a n a l y t i 6000~ . a t 75Oc. 1. D . a t 70Oc. .2 UV a t 215 nm (mins) 3. a t 7OoC. PA. 5 urn. 3-em RP-8 guard c a r t r i d g e . UV d e t e c t o r LCD 725 Uvikon Recorder Tarkan 600 W+W A l t e x m i croproc essor 420. A l t e x p e r i s o r b RP-2 40 X 3. blood/ plasma .6 mm I . s u p e l c o . D ..Table 5.6 mm I . Beckman U l t r a s p h e r e O D s . . D . Column 11. D . 30-40 urn.Blood/ 1 Technicon Acet o n i t r i l e / LC-8 S u p e l c o s i l . I Tetentior time Detect i o n 9. 150 X 4. Guard column. .6 mm I . Tetrahydrofuran Acetonitrile/ water 90110 v/v Methanol . I n c . 150 X 4. . . water 55:45 v/v B e l l e f o n t e . 30-40 urn. Lichrosorb RP-18.

46 cm. Sample Serum Serum ( Continued) 1 Instrument Varian Vist a HPLC system. . I Waters syst e m cont r o l l e d with M-660 s o l vent programmer . 25 cm X 4. Flow Retention time Detect ion 3ef rate (mins ) (ml/min 1 14. I Mobile phase Beckman u l t r a s p h e r e ODs.Table 5. 35:65 and increasing proport ions 4 X 0. Beckman u l t r a s p h e r e .4 cm. Precolumn Vydac C18.46 and UV a t 210 nm 74 .6 methanol / w a t er mm.1 IUV a t 73 1-5 20. 47 :20 :33 by Vol 5 pm. u n t i l 5:95 a t Both columns a t 1 5 mins.A c e t o n i t r i l e / o c t y l . 25 X 0. S t a r t i n g with t r ifluoroacet i c acidlacetonitrile 5 um. Maintained a t 72Oc. 7OoC.

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C l i n k . Kovacs "Rcemization and Coupling N . 2. 459 (1981). Chem. i n t h e ' P e p t i d e s ' eds. . Kahan. F. A . A. 2 . G. X V . J . D. J . Bueding. Keller. Academic P r e s s . (1983) p. S i n c l a i r . Vozeh. T. P r o t e i n Res.. Wissmann. 1980. 1327 (1978). Thommen-Scott. J . D. J .R. J . Vol. Suppl. Res.120 (1984). 60.D. Wall. AL-YAHYA 204 49. 380 (1981). Angew. McKenzie and W. HASSAN AND MOHAMMED A. Ther. B. W. Koegler. P e p t . White. M. D i s p o s i t . Michot. Cha. S c h r e i e r and B. . McMaster. p . Lancet 2 . Lancet i i . H . Wang. 57.S. Vol.E. W .B. Pharmacol. S t i l l e r . 1983. McMichael. 1 2 9 ( 1 9 8 0 ) . Agents A c t i o n s .R. Vol. 50 - H. New York-London. 4. N. C l i n . Calne.E. G. 2. K l e i n e r . Evans. D . Wenk. T . e t a l . 4 .M. XV .. T h i r u . X V . M e r r i f i e l d . Pentlow. Miederberger. E . K . Keown. 77 (1981). S. 53. 48s. 1323 (1978). 2413. p. No. 2409. 638 (1983). 59.B. J . M. F o l l a t h . H . I n t . e t a l .p r o t e c t e d Amino Acid and P e p t i d e Active E s t e r s " . F. 30. 51. Gratwohl. No. 55 * J. A . S . 92. 12. 61. 2438. B.S. C. P. Dunn. E n g l . Wang and R. . 54. and T. P. T r a n s p l a n t a t i o n Proceedings. . a J . D. 9.C.MAHMOUD M. Vol. N. Kay. 18. Craddock. T r a n s p l a n t a t i o n Proceedings. McElwain. Maurer. H. Beveridge . E. R . Howson.A. 52 * G. . - 56. Cum. T r a n s p l a n t a t i o n Proceedings .B. Suppl. Stawecki. L o o s l i . S . 1. Agents A c t i o n s . R o l l e s . Angew. Hawkins and Y. Ther. 11. No. Chem. K.H. Gross and J Meinenhofer. 58. A. p . C a r r u t h e r s . H.N. Barrett. Maurer. 1 (1983). 5 (1981).L. G . J . P . I n t . R. Newburger and B. Sloane. J . Drug Metab. Ed. J .N. Beveridge. J . 4. Tam. Wood. Supp-1. J . 133 ( 1 9 8 0 ) . Powles. J .R.

A. J . 68. J. G. J . 27. S t i l l e r . The Lancet. Agostino. 32 70. Sawchuk. Niederberger. Ulan.F. Buren. R . Robinson. Chromatogr. 1 5 ( 4 Suppl. . R .C. . W. Keown. 309. 182. Comun. E . Kntes. B a r r y . Comun. Pharm. W. R . J . 30( 11). C a r t i e r . Res. Appl. Chromatogr. W. R a n d a l l . 75. 64. R .S.J.. l'oges..T. Lawrence. 66. 72. B o i l e a u . Homergen. lOOP ( 1 9 8 0 ) . Lin. G. Beveridge. 19-32 (1981)..205 CYCLOSPORINE 62.Y.N. Niederberger. Wall. E . A . Ono. G. H. Keown. 36 ( 1 9 8 2 ) . P a t h o l Pharmacol. . R .G. N . 1).P. LeGrue. S . 37. S i n c l a i r .D. 71* K. S t i l l e r .P. March 28 (1981). R. W. Chem.T. 1368 . C . R . Kreis. T r a b e r . R. Leyland-Jones.... Gmur. J . S. P. R. W . W. 74. P r o c e e d . D. 73. 2269 ( 1 9 8 2 ) .N. 2. J . 9. 28. 2(1).. L. Allwood. Schaub. P. R . Transplantation. D . M. J . Yee.R.A. M. 441 (1984). Chem. Nussbaumer. Appl. C l i n . Chem. H . Chem. M. Kerman. 180 (1983). Transp. J . Biomed. B. 15(1). M. Abisch. B. 446 ( 1 9 8 3 ) . L a t i n i . Pharmacol. 67. 1812 (1984).D. Howson. Keller. J . P. Koegler. Immunoassay. .D. S. J . Ried. Chem. C l i n . W . Schran.. P. 3.A. P r o c e e d . High Resolut Chromatogr Chromatogr. 431 ( 1 9 8 2 ) .L. Trapp. Lanpacis.454 ( 1 9 8 0 ) - 69. 424 ( 1 9 8 2 ) . Payne. C l i n . Y . J . Kennedy. B . Freeman. Kahan. 65. H. M. C l i n . (1981). ( S u p p l ) . Transp. J . R .C. M. Howson. 63. W. C a r r a t h e r s . Kahan. J . A. D . C l a r k . Rush. 2403 ( 1 9 8 3 ) . Biomed. C a r r u t h e r s . Donatsch. C. W. C. C . Newburger. J e f f e r y . E.

A. King Saud University. Saudi Arabia for his continuous and indispensable assistance throughout the preparation of this profile. AL-YAHYA 13.206 MAHMOUD M. Abdalla I. Acknowledgement The authors would like to express their sincere gratitude to Mr. . HASSAN AND MOHAMMED A. College of Pharmacy. Riyadh. Babikir of the Medicinal Aromatic and Poisonous Plants Research Center.

Physical Properties 4. Odor 3.8 Solubility 4. Ip and Gerald S . 1 1 Partition Behavior 4. History and Therapeutic Properties 2. 7 Thermal Behavior 4.9 Crystal Properties 4.Nuclear Magnetic Resonance Spectrum 4 .1 Nomenclature 2. Inc.Nuclear Magnetic Resonance Spectrum 1% .6 Optical Rotation 4 .4 Trade Names 2.2 Generic Name 2. ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 16 207 Copyright 0 1987 by Academic Press.3 Laboratory Codes 2.12 Confocmational Isomers ::. .1. Description 2 .1.1.3 Appearance.10 Dissociation Constants 4 .1.1.1 infrared Spectrum . Brenner 1.2 Formula and Molecular Weight 2. All rights of reproduction in any form reserved.1 Chemical Name 2. Synthesis 4.4 Ultraviolet Spectrum 4. Color.5 Mass Spectrum 4.5 CAS Registry Number 2.ENALAPRIL MALEATE Dominic P.

2 Solution Stability 7. Determination in Biological Fluids 9.1 5. References .2 5.1 Solid State Stability 6.4 5.DOMINIC P.3. IP AND GERALD S.2 High Performance Liquid Chromatography Identification Tests Titration 6. Stability and Degradation 6.5 Elemental Analysis Colorimetric/Spectrophotometric Chromatographic 5.1 Thin-Layer Chromatography 5. Methods of Analysis 5. Pharmacokinetics and Metabolism 8. BRENNER 208 5.3 5.3.

Several review articles give a detailed account of the history. it is the compound that is used in o r a l dosage forms. ixltenedioate(1:l)salt (2)-2- . chernistry and pharmacology of the drug.1 Chemical Name ( S) -1-[N-[ 1-( ethoxycarbonyl) -3phenylpropyll-Galany11 ->proline.209 ENALAPRIL MALEATE 1. Enalapril maleate. History and Therapeutic Properties Enalapril is the ethyl ester of a long-acting angiotensin converting enzyme inhibitor.1 Nomenclature 2. 2. CH3 I e C H 2 C H 2 y H N H C H -CO -N COOH COOCHPCH3 Enalapril I CH3 I CHZCHzCHNHCH -CO -N COOH COOH Enalaprilat Since the oral absorption of enalapril is superior to that of enalaprilat. Enalapril is a pro-drug. Enalaprilat. the deesterified parent diacid of enalapril is the form of the drug that is administered intravenously. (2-6). enalaprilat. following oral administration. discovered and developed by the Merck Sharp and Dohme Research Laboratories (l). Description 2. is indicated f o r the treatment of essential and renovascular hypertension. design. it is bioactivated by hydrolysis of the ethyl ester to enalaprilat.1. which is the active angiotensin converting enzyme inhibitor.

1.1. Color. Synthesis Enalapril maleate has been prepared by the scheme outlined in Figure 1 ( 7 ) .5 CAS Registry Number 76095-16-4 2.2 Formula and Molecular Weight mol w t 492.1. odorless powder 3.3 Appearance.53 2.3 Laboratory Codes G154. Xanef 2. IP AND GERALD S. Renitec. MK-0421 2.DOMINIC P. Tnnovace.4 Trade Names Vasotec. crystalline.739-001D.2 Generic Name Enalapril Maleate 2. Pres. BRENNER 210 2. The more biologically active diastereomer (SSS) is isolated . as a mixture of syn and anti isomers. Reduction of the imine bond in 3 results in formation of the diastereomeric mixture 4 (SSS and RSS).1. Ethyl 2-0x0-4-phenylbutanoate (1) is condensed with L-alanyl-L-proline (2) to give Schiff base 3. Odor Enalapril maleate is a white to off-white.

a T 0" 5 l - A" a f 3' 5 0" aJ m c1 aJ c m rl I4 . I I 0 .o= t z C U I z + 0.A I4 m e w 0 w tn aJ c C h U v) .

structured) 1750 1727 1645 1570 1500-1390 1375 1355 1187 750 695 4. Assignments for the characteristic bands in the spectrum are listed in Table I.1 Infrared Spectrum The infrared spectrum is shown in Figure 2 (10). out-of-plane hydrogen bending (5adjacent aromatic hydrogens) Phenyl out-of-plane ring bending ’H-Nuclear Magnetic Resonance Spectrum The proton magnetic resonance spectrum is shown in Figure 3 (11).2 Assignment Aromatic C-H Stretch Methyl and methylene C-H asymmetric stretch / \NH$ Stretch Acid carbonyl stretch Ester carbonyl stretch Amide carbonyl stretch Monohydrogen maleate carboxyl stretch Mainly methylene bending Methyl symnetric bending Methylene bending Ester C-0 stretch In-phase. Table I Wavenumber. The spectrum was obtained as a potassium bromide pellet using a Perkin-Elmer Model 281B spectrophotometer. The spectrum was obtained using a Varian Associates Model SC300 spectrometer at a concentration of 0.34% overall yield. In addition to this synthetic route. 4. 8.DOMINIC P. Physical Properties 4. Active protons exchange with D20 in .55% w/v in deuterium oxide (D20). BRENNER 212 by fractional crystallization of the maleate salts to yield enalapril maleate (5) of greater than 99% purity in 32 . an-’ 3100-3010 2970 and 2920 2800-2200 (Broad. others have also been described in the literature (1. IP AND GERALD S. 9).

10 N E Bb 0 8 0 L2 0 0 s 0 Li 0 0 9 0 0 ? 0 0 F 0 0 E 0 5: N 0 0 0 rr) 0 5: lo .

The P r o t o n Magnetic Resonance Spectrum of E n a l a p r i l Maleate .-m -m Figure 3 .

02 m 2.9 ppm.60 3.30 t 1. H\ m C II /COOH H/'\COOH o Table I1 hemical Shift (ppm) Multiplicity '€I6. For several protons in the molecule. The notations 'major' and 'minor' denote the relative abundance of the two rotamers relative to each other in the spectrum.02 m 2.75 m 2. A tabulation of the chemical shifts and assignments for the numbered structure below is shown in Table 11. The phenomena has been attributed to the existence of rotational conformers as in the case of captopril in aqueous solution ( 1 2 ) .97 4.8-4. 1.27 4.54 d 1.95 3.80 3. slow rotation about the proline amide C-N bond gives rise to paired signals.09 Assignment CE3CH 0- Alanyl C% (minor) Alanyl C% (major1 Proline C-4 proton (minor) Proline C-4 proton (major) Proline C-3 protons (minor) Proline C-3 protons (major) C-3 ' protons C-4' protons Proline C-5 protons C-2' proton (minor) C-2' proton (major) CH3CZ20a ' proton (minor) .33 2. Figure 4 shows an expanded scale spectrum which m r e clearly illustrates the signals pattern.59 d 1.33 m 2.ENALAPRIL MALEATE 215 solution and appear in the HOD signal at approximately 4.

2 The Proton Magnetic Resonance Expanded Spectrum of Enalapril Maleate .0 PPM 2.0 Figure 4 . 3.--csr I 6.0 4.0 1.

The internal standard was tetramethylsilane.9 s.41 m m Assignment a' proton (major) -H @ L'C02H Aryl protons Aryl protons -m -o&p 13C Nuclear Magnetic Resonance Spectrum The l3C magnetic resonance spectrum shown in Figure 5 was run using a Varian Associate Model XL200 spectrometer at a concentration of 8 . acquisition time was 0. .ENALAPRIL MALEATE 217 hemical Shift 'H. 4 % w/v in deuterated methanol (13). 6 (ppm) Multiplicity 4.3 4. the two rotamers are designated 'major' and 'minor' in order of their relative abundances. As discussed above. The pulse width was 4 ps.33 9 6.37 7.33 S 7. The assignments for the numbered structure below are shown in Table 111.

v 160 Figure 5. PPM 50 The Carbon-13 Magnetic Resonance Spectrum of Enalapril Maleate .

9-65.5 176.1 24 -9 27. An exact assignment of the signals in this region was not made. c-5" 138. Major differences between spectra of the two compounds occur between 31. between 54.9 Naleate -HcSHPhenyl C-1" 170. C-2' and -gH2CH3 including signaIs attributable to rotamers.8 . An exact assignmnt of the signals in this region was not made.8 62.5 171. C-4' and C-4.219 ENALAPRIL MALEATE Table 111 Chemical 13c.7 35.4 65.9-35.7 arise from alanyl.5-176.6 65. C-3 proline including signals corresponding to rotamers.3 62.4 176.2 171.8 Proline C5 57.7 61.2 p p m arise from the four carbons C-3'. However.7 The signals at 170.6 172. 6 (ppm) 16. 129.0 35.2 Assignment iE:$2H3 Alanyl ?[I3 (major) (minor) The signals at 24.4 61. Alanyl C-a.3 Phenyl C-4" 131.7 The signals at 57. ester and maleate carbonyl carbons.8 33.8 and 34. definitive assignments were not made for the carbonyl carbons. proline.4 18. Carbon-13 NMR may also distinguish enalapril mleate and 'ts RSS diastereomer.2 17.9 58.0 ppm.0 142.7 ppm arise from the four carbons Proline C-2'.0 172.4 Phenyl C-2".7 and 60.7 31.2 131. 49. and phenyl c-3". C-6".

about 8. 16OOC) of the diagnostic fragment ions at m/e 358. does not make it useful for characterization or quantitation of the compound.0991 mg/ml. good molecular ion and pseudomolecular ion [M+H]+ formation (376. However. . Molecular ion information for enalapril base is also absent in the spectrum obtained by direct probe EI at 70 eV and 20 eV. 254. typical of an unconjugated phenyl moiety. 70 eV. The low intensity and lack of well-defined maxima in the ultraviolet absorption spectrum of enalapril maleate. this technique is not preferred for accurate measurement of small quantities of either compound in the other. 4.7 ppm.5 Mass Spectrum The direct probe electron impact (EI) mass spectrum of enalapril maleate as shown in Figure 7 was obtained on the VG MM7035'mass spectrometer employing 70 eV electron energy and a probe temperature of 16OoC (14). Figure 6 shows the ultraviolet absorption spectra of enalapril maleate in N/10 methanolic hydrochloric acid at concentrations of 0. 234.4 Ultraviolet Spectrum Enalapril maleate is characterized by inflections or "shoulders" at about 251 and 263 y%and barely discernible maxima at abut 21J nm (A1 cm. 208.DOMINIC P. respectively. Such information is only possible when a gross sample overload is employed. BRENNER 220 ppm and between 168. 257.6 and 170.991 mg/ml and 0.6). 285. Accurate mass measurements (high resolution. Under field desorption (FD)/MS however. 4. about 15) and abut 267 nm (A1 cm. 313. [MH20] atm/e 358 and other major fragment ions. Under the condition of analysis the maleic acid moiety is pumped off before the peptide derivative is vaporized. The low resolution EI 70 eV spectrum (Figure 7) shows a base peak at m/e at 91 (benzylic ion). IP AND GERALD S. 377) is observed (Figure 8 ) .

c 2 1 al 0 0 c *u1 a 1 C \ 220 240 260 280 300 320 340 220 240 260 280 300 320 340 Wavelength ( n m I F i g u r e 6.991 mg/ml and (b) N i l 0 Methanolie H y d r o c h l o r i c Acid. C o n c e n t r a t i o n : 0. C o n c e n t r a t i o n : 0 . The U l t r a v i o l e t Absorption Spectrum of E n a l a p r i l Maleate i n ( a ) N / 1 0 Methanolic H y d r o c h l o r i c Acid. ~ 9 9 1 mg/ml .

The Low R e s o l u t i o n (EI) Mass Spectrum of Enalapril Maleate .u) )r c 0) U 60- x 0 0) a 50 k 'T' 150 250 350 Mass Figure 7.80 c .

The F i e l d D e s o r p t i o n (FD) Mass Spectrum of E n a l a p r i l Maleate .223 Figure 8.

BRENNER 224 169. indicating accompanying decompostion during melting of the conpound (. of enalapril maleate (three asymnetric centers) in methanol is. 160.6 < 0.) A DTA curve for enalapril maleate obtained on a DuPont 1090 equipped with a DTA head is given in Figure 10. -42.1 . 4.143- By capillary melting point determination (USP Class Ia method) enalapril maleate melts at 144OC (15).3' (c=l%). and 149OC at 2OoC/ min. Table IV Solvent Water Methanol Ethanol Isopropanol Acetone Acetonitrile Dimethylformamide Chloroform Diethyl ether Hexane Polyethylene glycol 300 Solubility (mg/ml) 25 200 80 1.1 3. 4. 9 1 and 70 facilitated a proposed fragmention pattern as shown in Figure 9.14OOC at 2OC/min.4 9.3 >400 0.6 Optical Rotation The optical rotation.1 < 0.DOMINIC P. The peak temperature of the endotherm varies with heating rate.1 < 0. 4.7 Thermal Behavior Enalapril maleate exhibits a single melting endotherm by differential thermal analysis (DTA) under a stream of nitrogen.8 Solubility The following approximate solubility data (Table IV) have been obtained at ambient temperature. 126. 168. IP AND GERALD S. - .

t 3 ###BOT_TEXT###quot; I +5 +* r 4 Figure 9. A Proposed Fragmentation Pattern to Explain the Mass Spectrum of Enalapril Maleate .

-( ar 3 k FL( M 'rl .r r .- O r N r o d . (D 0 d- N (0 0 " " N 0 N N 0 0 E 0 2 0 5 0 2 0 ? 0 (D 0 t ar m ar U 2 4 4 a k 'rl m m 4 wG 0 u-i al > 3 k u fi m 4 k al c b -4 n b 0 .

9 Crystal Properties Enalapril maleate is a white to off-white crystalline corrpound.35 at 25OC for enalapril (18). 4. 4. referred to as Form I and Form I1 are very similar in energy. differing only by 0. The compound is polymorphic. these plymrphs. The two rotamers are distinguishable on the NMR .5 buffer 4.5 buffer mineral oil/pH 5. A pH versus solubility profile constructed to compare theoretical points to actual data is shown in Figure 11 (16). % 4. TWO nonsolvated polylmrphs have been detected and characterized by high resolution spec oscopic NMR) techniques ( F T I R .11 Partition Behavior At room temperature. However.221 ENALAPRIL MALEATE The solubility of enalapril maleate increases with pH.5 buffer mineral oil/pH 4. solid state and solution calorimetry ( 1 7 ) . resulting in a slow equilibrium between two rotational conformers (cis and trans).12 Conformational Isomers Proton NMR and carbon-13 NMR spectra of enalapril maleate are characterized by several paired signals attributed to slow rotation about the proline amide bond. respectively. Raman.97 and 5. The X-ray powder diffraction spectra for Form I and Form I1 of enalapril maleate are shown in Figures 12 and 13.6 Kcal/ml. The spectra were obtained using a Philips Electronics X-ray diffractometer using CuKa irradiation. Solvent Pairs n-Octanol/pH 7 buffer mineral oil/pH 3. enalapril maleate does not partition out of the the aqueous media listed below into mineral oil or n-octanol (19).10 Dissociation Constants Aqueous acidicfiasic potentiometric titration yielded pKa values of 2.

pH Calculated Pts Experimental Pts .Solubility Profile of Enalapril Maleate .DOMINIC P. BRENNER 228 1000000 100000 100 10 PH -D- o Figure 1 1 . IP AND GERALD S.

F i g u r e 12. Powder X-Ray D i f f r a c t i o n P a t t e r n of E n a l a p r i l Maleate. Form I .

In P 9 R In (u m In 0 O* f E H H N 0 F a d a N .d fd fd rl wc 0 w E al PI d a:: N .

23 1 ENALAPRIL MALEATE time scale. The procedure is stability indicating: the degradation products of enalapril and excipients in the dosage forms do not interfere. HPLC has provided firm evidence for their existence by demonstrating the chromatographic behavior of enalapril maleate at various temperatures (20). hydrogen and nitrogen was reported as follows: Element C H N % Calculated % 58. This method is suitable for content uniformity and dissolution assays of enalapril maleate capsules and tablets. 5.53 6.2 Colorimetric/Spectrophotometric Due to the lack of strong W absorption maxima.75 5. L-154.55 5. One approach has been an autoanalyzer colorimetric assay procedure (21). in addition to the early eluting maleic acid peak ( 1. the rotamer peaks have coalesced to yield a single symmetrical peak. pH 2/CHC13 mixture and is then assayed spectrophotometrically at 415 nm after extraction with chloroform.739-001D22 for carbon. Figure 14 shows chromtograms obtained by reverse-phase HPLC at column oven temperatures of 10°C and 80°C.53 6. In addition. In this approach enalapril maleate forms a yellow dye complex with bromothymol blue in acidic aqueous. At 10°C. m r e sensitive ways for the analysis of enalapril maleate are needed. the enalapril base peak is resolved into two distinct rotamer peaks. Analysis of enalapril in pharmaceutical dosage forms via flow injection analysis has recently been reported (22). At 8OoC.2 minute).82 5. Methods of Analysis 5.1 Elemental Analysis Analysis of Merck Sharp & Dohme reference l o t . .68 Found 58. Enalapril is determined by extraction as an ion-pair with bromthyml blue in an unsegmented flow system.

IP AND GERALD S. : N Time (Minutes HPLC Chromatograms of Enalapril Maleate at 10" and 80°C . BRENNER 232 80"C 10°C a0 c r Time (Minutes 1 Figure 14.DOMINIC P.

6 (enalapril base ) acetic acid 90:10 :1) 0.1) Solvent System Rf (Approximate) A (chloroform/methanol/ 0.3 Chromatographic 5.55 (enalapril base ) 0.7 (enalapril base and maleic acid) 0. Detection of components is accomplished by visualization of dried plates under short wavelength (254 nm) ultraviolet light.35 (enalaprilat) Rf (Approximate) 0.45 (maleic acid and enalaprilat) These solvent systems use a silica gel adsorbent containing an ultraviolet-fluorescence quench indicator (Analtech GF or Whatman K1F or E Merck Silica Gel G60 type plates). the low sensitivity of the method (detection limit 1 to 2 % ) renders it unsuitable for the .3.see Section 6.1 Thin-Layer Chromatography Three solvent systems (23) for the thinlayer chromatography of enalapril maleate are given below.233 ENALAPRIL MALEATE 5. The systems separate enalapril base from its salt forming acid (maleic acid) and its hydrolysis product (enalaprilat .2 (maleic acid) 0 (enalaprilat) B (n-Butanol/water/ acetic acid 3:l:l) Solvent System C (n-Butanol/toluene/ water/acetone/acetic acid 1:l:l:l:l) 0. Iodine vapor yields a mre intense spot for the enalapril base but does not visualize the maleic acid. In the systems reported above.

1). (c) content uniformity assays of enalapril maleate in tablets (26) and (d) dissolution of enalapril maleate in tablets ( 2 7 ) .3. 5. Detection is by uv at 210 or 215 nm. The significance of specifying a high column temperature has been discussed in section 4. With respect to the sodium .5 Titration Enalapril maleate can be determined by potentiometric titration with aqueous sodium hydroxide and perchloric acid. (b) analysis of enalapril mleate and degradates in tablets (2 5 ). 7 ) and complexation with bromothyml blue (section 5.4 Identification Tests Identification of enalapril maleate can be carried out by infrared absorption (section 4.2). Reverse-phase HPLC conditions are employed.3. Supportive evidence for identification can be obtained by differential thermal analysis (section 4 . HPLC (section 5.2) and thin-layer chromatography (section 5.DOMINIC P.12. A mobile phase of aqueous phosphate buffer and organic modifier (acetonitrile or a mixture of acetonitrile and methanol) and an oven temperature of 7OoC or 80° provide the selectivity and sensitivity required for satisfactory chromatography.2 High Performance Liquid Chromatography (HpLC) Several HPLC methods have been developed which are suitable for: (a) quantification of enalapril maleate and process impurities in the bulk substance ( 2 4 ) . 5.3.0 ml/min.8 or 2. A summary of these HPLC systems is given in Table V. IP AND GERALD S. 5. Flow rate is normally 1. BRENNER 234 detection and quantification of low levels of impurities in enalapril maleate bulk substance.1).

& 0 ru '9 v X . 0.d X 0- 0 w 0 In 0 u 07 0 v . mRJ 4J . obl o c a. x 9 0 a3 Y N a z a x W X W w 0 OD 9 cv X F! 2 4:. .. obl d . cv-l JJ c u 0 % X w W In 0 V m v a .

The mass balance between loss OF enalapril maleate and formation of I1 and I11 has been demonstrated.1N perchloric acid in glacial acetic acid as the titrant and an anhydrous electrode system. Degradation can be induced.g. 6. however.1N sodium hydroxide is the titrant and a combination pH electrode is employed as the electrode system. Enalapril maleate tablets are stable when protected from elevated temperatures and high humidity. BREWER 236 hydroxide titration. The compound stored at room temperature (amber glass) for four years shows no evidence of degradation by HPLC analysis. Crystalline enalapril maleate is a very stable solid. 0. if tablets are stored at 4OoC and 75% RH in open containers. e. Stability and Degradation 6. The perchloric acid titration employs 0. The structure of these compounds are shown in Figure 15. Maximum solution stability occurred around pH 3 . IP AND GERALD S.1 Solid State Stability There are two major potential modes of degradation for enalapril maleate: (a) hydrolysis of the ethyl ester to 11. tgO . enalaprilat. 6. Metrohm #EA 441/5 silversilver chloride electrode filled with 0.2 Solution Stability The stability of enalapril maleate in aqueous buffer solutions has been studied as a function of pH in the range of 2-7 (28). The major degradation product is 11. resulting in enalapril maleate loss of about 10% or greater for three months.DOMINIC P. Less than 2% of degradation can be induced by storage at 8OoC for 3 weeks (in screw-cap vials) and < 1% of decomposition at 4OoC and 75% RH (open vial) for 16 weeks. the free acid and (b) cyclization to a diketopiperazine derivative 111. The rate of enalapril loss and the mode of degradation are dependent upon the solution pH. versus a glass electrode is used. At room temperature.1N lithium perchlorate in glacial acetic acid (or acetic anhydride).

IN I I V In I. 0 m v) h v) 3 C U 0 a u m U aJ crr: rl m U C ri Ll .d m a C 'd w 0 W .

The primary route of excretion of the drug is renal. Following oral administration of enalapril maleate to man ( 3 3 ) peak serum concentrations of enalapril and enalaprilat occur within 0. The area under the serum concentration-time curves for enalaprilat and urinary recoveries for enalaprilat and the total drug did not differ significantly between the fed and fasted conditions. Pharmacokinetics and Metabolism The pharmacokinetics and metabolism of enalapril maleate follming oral administration have been the subject of a number of investigations and reviews (4. I11 was the major degradate but the fraction of I1 was increased. Based on urinary recovery of the total drug (enalapril plus its active metabolite. The in vitro conversion of enalapril to enalaprilat in human autopsy tissues has been examined (34). At pH 5 and ahve. I11 was the major degradation product while I1 represented only a minor fraction of the loss. Human cadaver liver was the only tissue in which significant conversion was demonstrated. 7.238 DOMINIC P. Enalapril absorption is not influenced by the presence of food in the gastrointestinal tract (35).5 W/ml at pH 2 and 5. A t pH 3 . Approximtely 94% of the dose is recovered in the urine and feces as enalaprilat or enalapril. enalaprilat) absorption is at least 61%. Enalapril. Bioactivation appears to be largely post-absorptive and there is no evidence for metabolism of enalapril beyond bioactivation to enalaprilat in man. For up to 20% loss at pH 2. BRENNER (time required for 10% loss of starting material) values are 262 days and 114 days €or 0. The pharmacokinetics of enalapril maleate was determined in normal volunteers after repeated single daily . respectively. I1 accounted for the bulk of enalapril loss with I11 present only in trace quantities. The drug was administered to normal volunteers both in the fasting state and with a normal meal. is rapidly absorbed and bioactivated extensively to enalaprilat. when administered orally.5 and 3 to 4 hours respectively. 29-32). IP AND GERALD S.5 to 1.

The enalaprilat component of the mixture is determined by the enzyme assay which uses as a substrate. An enzyme inhibition assay for enalaprilat has also been developed .4 columns. Urinary radioactivity may be separated into enalapril and enalaprilat by thin-layer chromatography * Enalaprilat can also be determined by its in vitro inhibition of angiotensin converting enzyme (37). The product. Serum profiles show little accumulation of enalprilat over the course of the study. feces or plasm samples may be counted by liquid scintillation techniques for total radioactivity. urine and homogenates of feces are placed on xAD. An average effective half-life for accumulation of approximately 11 hours was calculated from urine data. Those of primary utility include radiometric. 8. Following administration of the radioactive drug. A The physiological disposition and metabolism of enalapril maleate in laboratory animals has been studied with both radiolabeled drug and an enzyme-inhibition assay (37). the tripeptide carbobenzoxycarbonyl-phenylalanyl-histidyl leucine and converting e n z w isolated from porcine plasm. Plasm. An average steady state recovery of enalaprilat is achieved by the third or fourth dose. urine. Determination in Biological Fluids number of procedures have been developed for the analysis of enalapril and enalaprilat in biological Eluids. apparently representing a small fraction of the administered dose that has been bound to angiotensin converting enzyme. histidyl-leucine is then quantified by fluorescence after reaction with o-phthalaldehydc ( 3 8 ) . washed and then eluted with methanol to yield enalapril and enalaprilat. enzyrw inhibition and radioimuno-methods. For the determination of the total drug (enalapril plus enalaprilat) samples are hydrolyzed at high pH and then reassayed for enalaprilat. Enalapril levels may then be calculated as the difference between the total drug and enalaprilat prior to hydrolysis. The serum concentration profile of enalaprilat is characterized by a prolonged terminal phase.ENALAPRIL MALEATE 239 doses for eight days (36). The amount bound does not increase with dose indicating a saturable site of binding.

The total drug is measured as enalaprilat after sample hydrolysis by rat liver homogenate. The radiolabeled substrate employed is [flHI hippuryl-kglycylL-glycine. Acknowledgements The authors wish to thank Mrs.thereby eliminating derivatization with o-phthalaldehyde and fluorescence detection of the enzyme roducts. Enalaprilat has also been determined in serum and urine samples by radioimmunassay procedures. BRENNER 240 which uses radiolabeled substrate ( 3 9 ) . Betty Moyer €or typing the manuscript and Ms. Enalapril can then be calculated as the difference between enalaprilat before and after hydrolysis. [ 3HI hippuric acid is extracted into a water-immiscible scintillation cocktail and then quantified by counting in a liquid scintillation spectrometer. (40. The product. IP AND GERALD S.41). The radiolabeled component is obtained by iodination of the CH2CH2-c A -N COOH Lisinopril .DOMINIC P.c -c -N I I 51 *’ COOH NH2 amidine derived from the reaction of lisinopril and methyl p-hydroxybenzimidate. Florence Berg for performing the literature search. In one such procedure ( 4 0 ) antibodies for the radioimunoassay are raised in rabbits using an immunogen prepared by coupling the lysine analog of enalaprilat (lisinopril) to albumin with difluoro-dinitrobenzene. .

A. Taub. G. Patchett. E. W. C. 4 2 . W. A. Patchett. A. Inc. J. L. Lohr.) US 4. Sweet. Harris. temroeke. Br. Doyle and A. L. Thorsett. Lohr. Inc. Patchett. 2. Org.829. Tristram. 2. E. D. unpublished data. Gross. D.. J. Ruyle. E. T. E. 2816 (1984). Ther 216. E. L. Ikeler. R. Raven Press. H. Ferguson. D. 18. M. E. (1982). Conner and R. Ikeler. H. Pharmac. J. Wyvratt. S. Aster. S. Maycock. C. Hirschmann. D. Backlund. Proc. P. J. T. Sweet. Joshua.. S. 12. E. V. G. Urn. Weitz. E. H. Tristram (Merck and Co. W. A. - 5. Anal. A. Gross. 10. Wyvratt. A. A. Bearn Eds. C. Larijani. 7. N. N. D. J. H. Pat. H. Tristram. J. New York. Morris. A. J. Chem. T.Y. A. and J. Appl. Harris. R. F. L. E. A. D. 526 . N. M. 1984. R. Fed. M.. 201s (1984). 552 (1981). A. Springer. 4. Ulm. E. P. A. Arison and A. B. 49.401. References 1. J. Pharrn. Wyvratt (Merck and Co. J. Vlasses. W. Greenlee. A. W. Chem. L. A. Patchett in "Hypertension and the Angiotensin System: Therapeutic Approaches". D. D. Wyvratt and E.) Eur. 8. Hoffsomer. T. Isab. W. 4 . E. Exp. unpublished data. S. Robinson. S. Patchett. P. Merck Sharp and Dohme Research Laboratories.. E. 27 (1985).374.. Joshua. Vassal and C. 6. Wu. 167 (1983). C. T. Harris. D. Stone. Sweet. J. A. Clin. G. Bohn. E. A. M. Rothrock. Ondeyka. H. 3. M. Payne. 11.ENALAPRIL MALEATE 24 I 9. Pharmacol. Merck Sharp and Dohme Research Laboratories. D. 280 (1980). Nature 288. S. M. P. A. 9. Peterson. J. Patchett. A. M. C. 12. Clin. W. M. Rabenstein and A. E. J. E. C. Wenger. K. D. Vassil and C. Stone. Tristram. H.

J. unpublished data. formerly of Merck Sharp and Dohme Research Laboratories. 14. D. P. McCauley. P. 18. T. Merck Sharp and Dohme Research Laboratories. Mazzo. 15. R. S. 16. 5. Lindenbaum. unpublished data. personal comunication. J. DeMarcO. Merck Sharp and Dohme Research Laboratories. Bergstcorn. D. 23. Merck Sharp and Dohme Research Laboratories. G. Bell. 26. Nerck Sharp and Dohme Research Laboratories. 175. 19. 27. Brenner. unpublished data.. . J. R. personal comunication. Roman. personal comunication. A. McCauley. Draper. Konieczny. 28. IP AND GERALD S. unpublished data. personal comunication. G. unpublished data. J. J. of Pharm. A. Acta. Merck Sharp and D o h Research Laboratories. Int. Douglas. J. 22. formerly of Merck Sharp and Dohme Research Laboratories and C. D. Nerck Sharp and Dohme Research Laboratories. M. Kato. formerly of Merck Sharp and Dohme Research Laboratories. 20. Ramjit. Stevenson? S. J. Merck Sharp and Dohm Research Laboratories. personal comnunication. 17. personal comunication. unpublished data.242 DOMINIC P.339 (1985). R. M. BRENNER 13. Merck Sharp and Dohrne Research Laboratories. Anal Chim. 24. 25. personal comnunication. A. Merck Sharp and Dohme Research Laboratories. H. Klein and J. Roman. Merck Sharp and Dohme Research Laboratories. 21. W. Ip. Merck Sharp and Dohme Research Laboratories. G. 183 (1986).

K. B r o o k s . 357 (1982). 29. E. T. Lant. J . J. Hirhens. A. F. Drug. P. personal communication. Lant. Ligand Q. P. 41. A. B. J. . McNabb. 32.F. Gomez. 31. Swanson. Pharmacol. E. Metab. I. 31 (1985). Noormhamed and A. W. Swanson. F. 30. Merck Sharp and Dohme Research Laboratories. I . K. M. J. J. B. J a r r o t t . A c t u a l . L. T i l l . Perry. 75. McNabb and F. H. Gomez. Roth. Pharm. C. I r v i n and K. Vlasses. C. Mulcahy. 512 (1986). 401 (1985). R. J. P. M. E. Hichens. Ferguson. W.. 38. Larmour. Drug Metab. B r .. Clin. U l m . Reinharz and M. E. Biopharm. P. 33.701 (1985). 36. 206. S. Hand and W. E. Clin. L.ENALAPRIL MALEATE 243 28. 198 (1986). Y. D. 148. B. Biochem. The l i t e r a t u r e w a s reviewed t o J u l y . A. H. L.. Todd and R. . H. Ng. A. 21. A. D. 34. Biollaz. 1 5 (1982). 40. H. Stauber. 14. M. A. R. L. 35. T i l l . Hand. 37. Duncan. Anal. 4. Vassil and E. R. H. 19. Harris. A. J . V a s s i l . T i l l . Cubelal and C. Bolognese. B r .. Drugs 31. Drug Dispos. 39. Heel. W. Noormohamed. J.. 136 (1970). C. Alpaugh and S. U l m . H. A. L. Pharm Sci. Acta. Sci. Hypertens. J. 10. F. Biochem. H. E. J. Pharmacol. B e r g q u i s t . A. Worland and B. A. Johnston. Rev. 2. Biophys. 73. Dispos. J. 1655 (1984). 14 . 2. H. 99 (1983). E. A. W. H. A. N. R. M e d . 1986. 43 (1981). 273 (1984). R. B. J. Piquillaud. 37 (1984). Weinstein. C. deLuna. R. Schnelling. Jackson. U l m . T. TOCCO. Brunner and J. E. N. Hichens.

King Saud U n i v m L t y . ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 16 245 Copylight 0 1987 by Academic Press. Uepan. . Muktadi M. A d i h y CoUege F.J. Riyadh. AmmaZic a n d P o h a n o u n P&m% R e n u c h Cevtta. and AbduR FaMah A .A. M e d i c i d . All rights of reproduction in any form reserved. H u b a n . Muktadi.HOMATROPINE HYDROBROMIDE F a i d J.A. Saudi A h a b i a . Mahmoud M.lmeMA: od Phatunacognony. Hanun A.A.M. Qep'ahtment 06 Phatunacagnony. Inc. A d i d y : : : 06 Phanmacy.

5. Odor and Taste 2. Methods of Analysis .2 Total Synthesis of Mandelic Acid 4.1 Total Synthesis o f Tropine 4.FARID J.3 Crystal Structure 2.4 X-Ray Powder Diffraction 2.1 W Spectrum 2.2 Formulae 1. 1.4 Elemental Composition 1.5 Appearance.2 IR Spectrum 2. Description 1.3 Nuclear Magnetic Resonance Spectra 2. 3.3 Molecular Weight 1.5.2 Solubility 2.6 Dissociation Constant 1.5.7 pH Range Physical Properties 2.4 Mass Spectrum Preparation of Homatropine Hydrobromide 4.3 Total Synthesis of Homatropine 5. Therapeutic Uses and Pharmacology 6. Color. Drug Stability 7. Total Synthesis 4. MUHTADI ETAL.5. 246 1.1 Melting Point 2.1 Nomenclature 1.5 Spectral Properties 2.

5 Polarographic Methods 7.7 Chromatographic Methods Acknowledgement References 247 .HOMATROPINE HYDROBROMIDE 7.6 Spectrophotometric Methods 7.4 Complexornetry 7.1 Tests of Identification 7.3 Titrimetric Methods 7.2 Microcrystal Formation 7.

1. D e s c r i p t i o n 1. 5aH-tropane-3a-01 mandelate ( e s t e r ) hydrobromide.2.2 a. 248 1. 2 . 8-methyl-8a z a b i c y c l o [ 3. Generic Names Homatropine Hydrobromide.2.1 Empirical CIGH2103N.2 Formulae 1. Benzeneacetic a c i d .2..3 y l e s t e r hydrobromide . Mandelyltropine Hydrobromide.FARID J. 1 ] oct-3-yl e s t e r .1.1. Tropine mandelate Hydrobromide .17 o c t . 1.2 HBr Structural 7 HO OH II I 0. DL-Hornatropine Hydrobromide. The hydrobromide of t r o p i n e mandelate . Bufopto. l a H .C-CH .3 Trade Names Homatrisol. a-hydroxy-.Hydroxybenzene a c e t i c a c i d 8-methyl-8a z a b i c y c l o [ 3 .1 Nomenclature 1.1. 1. endo-( 2 ) . MUHTADI ETAL. Homatrocel. hydrobromide.1 Chemical Names 1.

and t h e boat form may w i l l be favored because o f t h e in c re a s e d i n t e r a c t i o n s i n v o l v i n g t h e dimethylene b rid g e i n t h e c h a i r conformation ( 7 ). Hornatropine would be t h e same as a t r o p i n e .2. 2.4 H Br Wiswesser Line Notation T56 A ANTJ A -GOVYGR 6 EH 1. Study of t h e dipole-moment and Kerr-constant measurements o f a number of tro p a n e d e r i v a t i v es h as shown t h a t t h e p i p e r i d i n e r i n g i s i n t h e c h a i r form w i t h t h e N-methyl e q u a t o r i a l ( 4 ) . . t h e a-3 -s u b s titu e n t i s of greet e r bulk th an t h e hydroxyl.3 CAS Reg istry No. t h e predominant conformation i s t h e p i p e r i d i n e r i n g i n a deformed c h a i r form t o gether w i t h a minor amount i n t h e boat form (6 I* HO Tropine I n a t r o p i n e .2. 5 (1) Stereo ch em istry B a m i n a t i o n o f t h e M s p e c t r a of some tro p a n e d e u t e r o h a l i d e s has shown t h a t t h e N-substitue n t i n tro p an es i s predominantly e q u a t o r i a l ( 2 1. X-ray a n a l y s i s o f t r o p i n e hydrobromide has shown t h e presence of c h a i r conformation ( 3 ) . 1. I n t r o p i n e . Another study of t h e dipole-moments and NMR s p e c t r a o f some tr o p a n e d e r i v a t i v e s have confirmed t h a t t h e p i p e r i d i n e r i n g i s i n t h e chair conformation w i t h t h e N-methyl group predominantly e q u a t o r i a l ( 5 ) .249 HOMATROPINE HYDROBROMIDE 1. however. [Sl-56-91 C16H2103N.

T h i s s t u d y showed s t r o n g c r o s s . MUHTADI ETAL. T h i s broadening a r i s e s a s a consequence of e c l i p s i n g o f t h e s e p r o t o n s i n t h e boat conformer ( 9 ) . 250 II N -CH3 e / 011 0-c-CH ‘‘6HS A d e t a i l e d review i s a v a i l a b l e f o r t h e boat o r c h a i r conformation i n t r o p i n e s ( 8 ) . Carbon-13 magnetic resonance s t u d y has a l s o suggest e d a n o n . I t i s s u g g e s t e d t h a t t h e p i p e r i d i n e r i n g of t r o p a n e s is somewhat d i f f e r e n t i n conformation from t h e normal c h a i r form ( 1 0 ) .r i n g i n t r a m o l e c u l a r i n t e r a c t i o n s of t h e t y p e N---C---O and N---H-0were i n d i c a t e d by t h e broadening of t h e p r o t o n s i g n a l due t o t h e c o u p l i n g between 1(5)-H and 2(4)-H p r o t o n s i n t h e boat conformer compared with t h e c h a i r .33 356.c h a i r conformations i n t r o p a n e d e r i. 1 A p p l i c a t i o n of H-NMR c o n t a c t s h i f t s u s i n g n i c k e l and c o b a l t d i a c e t y l a c e t o n a t e s . Me 1.FARID J.3 Molecular Weight 275. t o t r o p i n e s h a s demonstrated t h a t t h e six-membered r i n g of t r o p i n e s is i n a n e a r l y s e m i p l a n e r form. Other PMR s t u d y suggested a p r e f e r e n c e f o r t h e b o a t conformation i n s e v e r a l t r o p a n e d e r i v a t i v e s .vat i v e s (11).3 (Homatropine) (Homat r o p i n e HBr) .

3 Crystal Structure The c r y s t a l s t r u c t u r e o f b o t h homatropine and homat r o p i n e hydrobromide were r e p o r t e d (16). C o l o r . 1 2 m l a l c o h o l a t 60°.47% (Homatropine HBr). 2 2 . 17.5 Appearance. 3.43% Il-lomatropine) .9(2O0) (14) pH Range -A 2% s o l u t i o n i n water has a pH 5 .6 D i s s o c i a t i o n Constant pKa 1. 5 . o d o r l e s s .4 Elemental Cormosition C .217O (with decomposition) O t h e r m e l t i n g p o i n t s were a l s o r e p o r t e d : 215 (with decomposition) f 15) 217-218 (with decomposition) (1) 2.69%. 2 2 % . Orthorhombic. C r y s t a l s o f homatropine were o b t a i n e d by r e c r y s t a l l i z a t i o n from e t h a n o l . The s o l u b i l i t y i n c r e a s e s r a p i d l y as t h e t e E p e r a t u r e rises (12 ) . 69. 1. 1.79%.94%. bipyramidal p r i s m s (from w a t e r ) . 0 9 % . H R r ) were o b t a i n e d by r e c r y s t a l - . 4 4 % . a 5. N . with a b i t t e r t a s t e ( 1 2 ) .25 1 HOMATROPINE HYDROBROMIDE 1. H . pH (1%aqueous s o l u t i o n ) : 5.1 Melting P o i n t (12) 214 . Odor and T a s t e C o l o r l e s s c r y s t a l s o r a w h i t e c r y s t a l l i n e powder. 7. 53. H. 6 .7 9. 13. odorless (13).67% s o l u t i o n i n water i s i s o . 0 .2 Solubility One gram d i s s o l v e s i n 6 ml water.93%. 420 m l chlornform. 2. 5 t o 7 (12 ) .o s m o t i c w i t h serum.4 (13). N . 0 . i'hysical P r o p e r t i. 2.es 2. I n s o l u b l e i n e t h e r . C . B r . 40 m l a l c o h o l . whereas t h e c r y s t a l s of homatropine hydrobromide (C16H21N03.

4 19. I n t e r p l a n n a r d i s t a n c e and r e l a t i v e i n t e n s i t y are t a b u l a t e d i n t a b l e 2 .3 16. . The scanning speed o f t h e goniometer used was 0. a(A") b(A") c(A") Homatropine 14.02 2 e / s e c .21 Homatropine HBr 10. T a b l e 1. The monochromator was a s i n g l e curved c r y s t a l ( y = l . MUHTADIETAL. .25 1. The C r y s t a l S t r u c t u r e o f Homatrop i n e and of Homatropine H B r .000 c o u n t s a t r e c o r d e r speed o f 4 mm/28.46 8 Pcab X-ray Powder D i f f r a c t i o n The X-ray d i f f r a c t i o n p a t t e r n o f homatropine hydrobromide was determined with a P h i l i p s X-ray d i f f r a c t i o n s p e c t r o g o n i o m e t e r equipped w i t h PW 1730 generator. The u n i t was a l i g n e d and examined. t h e u n i t c e l l s must be assumed t o c o n t a i n e q u a l numbers of L . r e c o r d e r f u l l s c a l e was 10.1 6. The lower level and t h e upper l e v e l of t h e s i g n a l c o n t r o l waz 35 E 75 r e s pectively.5918A0) The u n i t was equipped with P h i l i p s PM 821.a d D-molecules ( 1 6 ) .0 p r i n t i n g recorded d i g i t a l p r i n t e r . In b o t h homatropine and homatropine hydrobromide t h e n i t r o g e n atom may be o p t i c a l l y a c t i v e .22 4 P211/c 1. 252 l i z a t i o n from water. u s i n g a S o l i c o n s t a n d a r d . Divergance s l i t and t h e r e c e i v i n g s l i t were 1".4 Density (g. The X-ray p a t t e r n of homatropine hydrobromide i s p r e s e n t e d i n F i g . 1. t o a t t a i n maximum o p e r a t i o n c o n d i t i o n s .FARID J. s i n c e t h e s p a c e groups a r e centrosymmetric. 5 15. O s c i l l a t i o n and Weissenberg photographs were used t o determine t h e space group (Table 1 ) . High i n t e n s i t y X-ray t u b e o p e r a t e d a t 40 KV and 20 mA.cmR3) observed c a l c u l a t e d Z Space group 1.97 1. X-ray r a d i a t i o n was provided by a copper t a r g e t (Cuanode 2000 W ) .48 Compound 2.

15 5.80 1.5% 10.5% 21.15 2.07 2.98 6.4% 10.7% 46.9% 12.7% 24.43 3.2% 11.0% 85.19 3.35 2.72 2.4% 39. I/Io = relat i v e i n t e n s i t y (based on t h e h i g h e s t i n t e n s i t y of 100).52 2.3% 48.6% 14.29 2.2% 22.97 ~ 17.61 2.35 4.0% 36.0% 22.3% 22.7% 14.2% 38.00 2.47 2.96 3.9% 13.17 3.12 3.57 3.30 3.8% 17.58 2.1% 11.0% 11.28 4.00 5.5% ~~ d = i n t e r p l a n a r distance.9% 100.1% 15.7% 13.7% 21.9% 11.7% 20.7% 20.3% 20.0% 13.8% 17. .87 7.87 1.5% 12.64 2.98 1.1% 19.41 6.50 3.03 1.7% 13.23 3.0% 13.2% 20.48 4.07 3.50 2. J 1 lY 1 X-RAY DIFFRACTION PATTEW OF HCMATROPINE HYDROIIRO\IIDL.84 4.93 1.32 2.1% 22.8% 2.1% 73.77 11.3% 16.04 4.26 2.5% 25.64 4.253 HOMATROPINE HYDROBROMIDE Table 2 : X-Ray Powder D i f f r a c t i o n P a t t e r n of Homatropine Hydrobromide - ~ ~~ 6.1% 11.83 2.

FARID J. MUHTADI ETAL.

254

2.5

Spectral Properties
2.5.1

U l t r a v i o l e t Spectrum
The UV spectrum of homatropine hydrobromide
i n methanol ( F i g . 2 ) was scanned from 200
t o 350 nm u s i n g DMS 90 Varian S p e c t r o p h o t o meter. I t e x h i b i t s t h e f o l l o w i n g UV d a t a
(Table 3 ) .
Table 3 . UV C h a r a c t e r i s t i c s o f Homatropine

Xmax. a t nm
252
258
264.5
273

(A 1%,lcm)

E

224.28
26 7
228.5
78.32

6.3
7.5
6.42
2.2

Other r e p o r t e d UV s p e c t r a l d a t a € o r homatropine i n 0.1 N s u l f u r i c acid ( 1 7 ) .
Xmax. a t 252 mu ( E 1%, 1 cm 4 . 8 ) , 258 mu
( E 1%, 1 cm 6.1) and 264 mu ( E 1%, 1 cm 5 ) .

Amax. i n 0 . 1 N h y d r o c h l o r i c a c i d a t 254 nm
( E 1%, 1 cm = 3 ) ; a t 259 nm ( E 1% , 1 cm = 5 . 2 )
and 266 nm. ( 1 8 ) .
2.5.2

I n f r a r e d Spectrum
The I R spectrum of homatropine hydrobromide
as KBr-disc was r e c o r d e d on a Perkin E l m e r
580 B I n f r a r e d S p e c t r o m e t e r t o which i n f r a r e d
d a t a s t a t i o n i s attached (Fig. 3 ) .
The s t r u c t u r a l assignment have been c o r r e l a t e d w i t h t h e f o l l o w i n g f r e q u e n c i e s (Table 4 ) .

Table 4. I R C h a r a c t e r i s t i c of Homatropine

Ass i gnment

Frequency cm-’
Base
3350
2940,3050

1735

HBr
3270
2965
2 80 0 - 2.585
1755

OH ( s t r e t c h )
CH ( s t r e t c h )

+

NH

R

-0-C- ( e s t e r )

255

HOMATROPINE HYDROBROMIDE

200

250

300

350

Fig. 2 : ‘THE W SPECTRUM OF HOMATROPINE IIYDROl3KOMIDE IN PE’MANOL

1

I

3500

3000

I

I

I

I

2500

2000

I

I

I

19

1

I

1

1

1

1600

1400

1200

1000

1800

Fig. 3 : ‘IHE IR SPECTRLM OF HOMATRDPINE HYDROBRDMIDE AS KBr DISC.

1

800

1

600

257

HOMATROPINE HY DROBROMIDE

Frequency cm -1
Ease

Assignment

HBr

1600
145 0,1500
1100,1065
755,700

1600
1475
1170,1070
735,700

C=C (aromatic)
C=C (aromatic)
C-0-C (ether)
5 H (monosubstituted
aromatic)

Other characteristic bands are 2750, 2710, 1450,
1420, 1395, 1378, 1328, 1288, 1265, 1195, 1105,
1025, 960, 885, 820 and 615 cm-l.
Other IR data for homatropine and the hydrobromide
salt have been reported (1,14,17).
2.5.3

Nuclear Magnetic Resonance
2.5.3.1

'H-NMR Spectra
The 'H-NMR spectra o f homatropine
hydrobromide in DMSO-D6 and in D20
were recorded on a Varian T-60A, 60
MHz NMR spectrometer iising TMS (tet-

8

A H 3

ramethylsilane) as an internal reference. These are shown in F i g . 4
and 5 respectively. The following
structural assignment have been made
(Table 5).

76&
>
H
$0

'

" OH

Oi4
12

13

0- C-CH
9

10

1
'6
Table 5. The H-NMR Characterjstics of Homatropine
Group
5 aromatic protons
(12,13,14,15,16 H)
10 H (OH)
10 H

Chemical Shift (pmm)
DMSO-d6
D2°
7.33 (s)

7,4 (s)

6.03 (d)
5.13 (d)

5.33 ( s )

J
L

.
.

1

In

. . . .

I

In

. I. . .
,

.

,

,

1 ,
l

6 0

so

.

,

.

.
.
.
PPWIII

,
l

,
.

an

,
.

I
.

.

.

I

,
.
1 0

,
.

.

,
.

I

.
.

.

1
*

.

,
I

Y O

Fig. 4 : THE 'H-NMR SPECTRUM HOMATROPINE HYDROBROMIDE IN D I W - L ) ~

.
.

L
10

Fig. 5 : THE 'H-NMR SPECTRUM OF HOMATROPINE HYDROBROMIDE IN D20.

FARID J. MUWTADIETAL.

260

Group

Chemical S h i f t (pmm)
DMSO- d6
D2°

3H

4.70 ( t )

5.03 ( t )

1 H and 5 H

3.71 ( b s )

3.66 ( b s )

8-N-Me

2.63 (s)

2.67 (s)

2,4,6,7 H

2.4-1.27

(m)

2.4-1.8

(m)

s = s i n g l e t , d = d o u b l e t , t = t r i p l e t , bs=broad s i n g l e t
1
Other H-NMR d a t a f o r homatropine hydrobromide was
r e p o r t e d ( 1 ) . Ohashi e t a 1 (10 ) have r e p o r t e d
'H n u c l e a r magnetic resonance c o n t a c t s h i f t s o f some
t r o p a n e s i n c l u d i n g homatropine i n CDC13 a t 220 MHz.
The c o n t a c t s h i f t s used were n i c k e l and c o b a l t d i a cetylacetonates.
2.5.3.2

13C-NMR

The I3C -NMR complete l y decoupled
and o f f resonance s p e c t r a of homat r o p i n e hydrobromide a r e p r e s e n t e d
i n Fig.6 and Fig. 7 r e s p e c t i v e l y .
Both were r e c o r d e d over 5000 Hz
range i n a mixture o f DMSO-D6 and
CDC13 (conc. 100 mg/ml), on a J o e l
FX-100-90 MHz s p e c t r o m e t e r . A samp l e t u b e ' l 0 mm and TMS a s i n t e r n a l
s t a n d a r d a t 20' were used. The
carbon chemical s h i f t s are a s s i g n e d
on t h e b a s i s o f t h e chemical s h i f t
t h e o r y and t h e o f f - r e s o n a n c e s p l i t t i n g p a t t e r n (Table 6 ) .

8

7

12
II

I

0- C-CH
9 10

13

Fig. 6 : 'IHE l 3 C - I W W L E T E L Y DECOUPLED SPECTRUM OF HOMATROPINE HYDROBNNIDE.

Fig.

:

THE

'3C-W

OFF-RESONANCE S P E m M OF Wl'p.OPINE HWWBROFlIDE.

263

HOMATROPINE HYDROBROMIDE

Table 6 .
Carbon
No.
‘1

Carbon Chemical S h i f t s of Homatropine

Chemical S h i f t
[PPml
59.18 (d)
32.11 ( t )

c2

70.74

c3

(d)

32.11 ( t )

‘4

59.18 (d)

c5

21.19 ( t )

‘6

21.19 ( t )

c7

40.21 ( 9 )

‘8

Carbon
No.
‘9

5 0
cll
5 2

‘13
14
5 5
‘16

Chemical S h i f t
[PPml
169.38 (s)
62.76 (d)
137.38 (s)
126.34 (d)
124.76 (d)
125.99 (d)
124.76 (d)
126.34 (d)

s = singlet, d = doublet, t = t r i p l e t , q = quartet.

2.5.4

Mass Spectrum
The mass s p e c t r u m o f homatropine o b t a i n e d by
e l e c t r o n impact i o n i z a t i o n a t 70 eV, was
r e c o r d e d on a Finigan-Mat 5100 Mass S p e c t r o meter. The s p e c t r u m ( F i g . 8) shows a molec u l a r i o n peak M+ a t m/e 275. The b a s e peak
i s a t m/e 124.
The most prominent f r a g m e n t s , t h e i r r e l a t i v e
i n t e n s i t i e s and some proposed i o n f r a g m e n t s
are shown i n t a b l e 7.

Table 7 .

m/e

Mass Fragements o f Homatropine
Relative Intensity %

275

4.4

234

1.2

193

1.2

165

1.3

142

6.4

140

4.6

125

10.5

Ions
-

M+

-

+

[IT)]’

I-

Fig. 8 : 'THE MASS SF'ECIRLN OF ~ l R O P I t E

HOMATROPINE HYDROBROMIDE

m/e
-

Relative Intensity %

265

Ions
125-H

124

100

122

1.6

107

7.5

106

3.8

105

6.1

96

14

95

9.3

96 -H

94

21.5

95-H

83

20.3

82

29.2

79

14.3

77

19.2

68

5.9

67

19.1

68 -H

55

6.7

[CH2 =N=CH-CH2]

O t h e r mass s p e c t r a l
r e p o r t e d (19,20).

-

+

data

of homatropine have been

+

FARID J. MUHTADI ETAL.

266

3.

P r e p a r a t i o n of Homatropine Hydrobromide
Atropine i s hydrolyzed w i t h barium hydroxide s o l u t i o n t o
g i v e t r o p i n e and t r o p i c a c i d .
The t r o p i n e i s h e a t e d w i t h mandelic a c i d i n t h e p r e s e n c e
o f hydrogen c h l o r i d e . Ammonia i s added, and t h e l i b e r a t e d homatropine i s e x t r a c t e d with chloroform. The c h l o roform s o l u t i o n i s e v a p o r a t e d t o d r y n e s s . The r e s i d u e
i s t r e a t e d with hydrobromic a c i d and t h e homatropine
hydrobromide o b t a i n e d is p u r i f i e d by r e c r y s t a l l i z a t i o n
(21, 2 2 ) . The p r e p a r a t i o n i s o u t l i n e d i n Scheme I .
Scheme I .

H

f) / CH20H
0-C-CH
\C6H
Hydrolysis

eoi
6
+
HOOC -CH-CHZ OH

HOOC\

Ho’cH@

1

0

0-E-cii’

OH

‘6HS

Homatropine

HOMATROPINE HYDROBROMIDE

267

An alternative method of hydrolyzing atropine was achieved

microbically by the action of Arthrobacter a t r o p i n i (23).

4. Total Synthesis
Since Homatropine is the tropine ester of mandelic acid,
schemes for the total synthesis of tropine and of mandelic
acid were reported.
4.1

Total Synthesis of Tropine
Four schemes for the total synthesis of tropine are
known. Scheme I1 was also modified to give a much
better yield.
Scheme 11: Willstatter's total synthesis of tropine
(24) *
Suberone (cycloheptanone) [l] is reduced to suberol
which is treated with hydrogen iodide to give suberyl
iodide [2]. This is treated with potassium hydroxide
in ethanol to give cycloheptene [ 3 ] . Cycloheptene
is brominated to give 1,Z-dibromocycloheptane [4]
which is treated with dimethylamine to yield dimethylaminocyclohept-2-ene [5]. The latter is converted
to cyclohepta-1,3-diene [6] by exhaustive methylation.
[6] is brnminated at 1,4-positions to give 1,4-dibromocyclohept-2-ene [7]. Elimination of two moles of
the hydrogen bromide of [7] is effected by quinaline
to give cycloheptatriene [ 8 ] .
Substance [8] is treated with hydrogen bromide to
give bromocyclohepta-3,5-diene [ 9 ] which is reacted
with dimethylamine to give dimethyl aminocyclohepta2,4-diene [ l o ] . The latter is treated with sodium in
ethanol followed by bromination to give 1,2-dibromo5-dimethylamino-cycloheptane [ll].
This is warmed in
ether when intramolecular alkylation occurs to give
2-bromotropane methobromide [12]. Hydrogen bromide
is eliminated from [12] by the action of alkali to
yield tropidine methobromide [ 1 3 ] . This is transformed to tropidine methochloride [ 1 4 ] by the action
of potassium iodide followed by the action of silver
chloride. Sub-rtance [14] is pyrolized to give tropidine [15].
Hydrogen bromide is added to an acetic acid solution
of tropidine [15] to yield 3-bromotropane [16! which
is hydrolysed with 10% sulfuric acid at 2@0-21@0 to
give pseudotropine [ 171. JI-tropine [ 171 is oxidized
with chromium trioxide to give tropinone [18]. This
ketone is reduced with zinc and hydriodic acid to
tropine [ 191.

FARID J. MUHTADI ETAL.

268

Scheme 11 : W i l l s t a t t e r ' s t o t a l s y n t h e s i s o f a t r o p i n e

QBr

exhaust

L
methyln

[4

Br

q u i n o l i ne
1500~

*

warm
i n Et20
[I21

f

Br

Br

[111

I

Br

G N -CH 3 CH3 \ /"=" CH3 CH-OH I31 [GI + [41 I51 .269 HOMATROPINE HY DROBROMIDE Scheme T I l : Robinson's t o t t i l s y n t h e s i s of atropine CH-OH CH3NH2 121 cond.

270 Scheme 1 1 1 : Robinson's s y n t h e s i s (25) Succindialdehyde [ 11 i s condensed with methylamine [ 2 ] t o g i v e t h e condensate b i s c a r b i n olamine [3]. The y i e l d can be improved by s u b s t i t u t i o n of t h e more r e a c t i v e acetone d i c a r b o x y l a t e o r i t s e s t e r f o r acetone. Scheme IV-: Willstatter I s second s y n t h e s i s ( 2 6 ) S u c c i n y l d i a c e t i c e s t e r [ 11 i s condensed with methylamine [ 2 ] t o g i v e diethyl-N-methylpyrr o l e d i a c e t a t e [ 31. Hydrolysis of [5] with 10% s u l f u r i c a c i d g i v e s e t h y l t r o p i none-2-carboxylic a c i d [ 61. Succindialdehyde [l] i s condensed w i t h m t h y l a mine [21 t o g i v e b i s c a r b i n o l m i n e [31. Tropinone [61 i s reduced w i t h z i n c and hydriodic a c i d t o t r o p i n e [71. The c i s form of [41 is c y c l i z e d i n t h e presence of Na and p-cymene t o g i v e e t h y l tropinone-2-carboxylate [ 51. This i s warmed with hydrochloric a c i d t o g i v e t r o pinone [61. This i n t u r n condensed w i t h acet o n e [ 4 ] t o give t r o p i n o n e [ 5 ] ( T h i s mixture i s allowed t o s t a n d i n water a t o r d i n a r y t e m p e r a t u r e f o r h a l f an h o u r ) . MUHTADI ETAL. The l a t t e r i s heated t o y i e l d tropinone [TI which i s reduced w i t h z i n c and hydriodic a c i d t o t r o p i n e [a].FARID J. I31 i s condensed w i t h calcium acetonedicarboxyl a t e [4] t o af1'31-d t h e condensate [51. Tropinone [ 5 ] i s reduced with z i n c and hydriodic acid t o tropine [6].5-dimethoxy t e t r a hydrofuran as follows : H+ CH3NH2HC. Scheme V: Tropinone can a l s o be s y n t h e s i z e d ( 2 7 ) u s i n g methylamine hydrochloride a c e t o n d i c a r b o x y l i c a c i d and g e n e r a t i n g succindialdehyde i n s i t u by t h e a c t i o n of a c i d on 2.l CO( CH2COOH)2 "0 CHO . This i s reduced (H2+Pt) t o a f f o r d diethyl-N-methylpyrrolidinediacet a t e [41.

..-COOC I HI CH2-COOC 1 C- I H CH2.(-------------- CH= C- CH2-COOC H \ ' A CH -C1 2 I III CR -C- I 2 1 H 2 5 CH 1 I .'.HOMATROPINE HYDROBROMIDE .C- [4 CH= 111 I CH2COOCa W i l l s t a t t e r ' s second s y n t h e s i s + H2NCH3 CH= 27 1 * II 2 5 H 2 5 . Scheme 111: Robinson's :li s y n t h e s i s ( y i e l d improvement) CH-OH f NHeCH3 121 I11 cond CH-OIf [31 Scheme I& Cli- CH2-COOC CH2COOCa \ c=o + / I H3CT 1 c=o I CH -C-CH2-COOC 2 (51 1 H COOCa H 2 5 [21 H 2 5 H .-.c- i 2 i II CN 31 CH2- cI H - I I3] CH2-COOC Cli3 CH2-COOC CH-COOH I c=o I &I2 (61 2 5 2 5 [41 H H CH.

FARID J. 212 CH2 . Scheme I1 Acetophenone [ l ] i s c o n v e r t e d i n t o d i c h l o r o a c e t o phenone [ 2 ] by t h e a c t i o n of c h l o r i n e i n a c e t i c a c i d .CH2 Zn HI L [71 4. 4. [ 2 ] i s hydrolyzed w i t h c o l d c o n c e n t r a t e d hydroc h l o r i c a c i d t o y i e l d mandelic a c i d [3] (28). Scheme I Benzaldehyde [ l ] i s t r e a t e d w i t h a m i x t u r e of a s a t u r a t e d s o l u t i o n of sodium hydrogen s u l f i t e and aqueous sodium cyanide t o g i v e m a n d e l o n i t r i l e [ 2 ] .2 CH2 . [ 2 ] i s t r e a t e d with sodium hydroxide t o a f f o r d t h e sodium s a l t o f mandelic a c i d .CH I \ / -CH2 I I C=O . .C H I Me-N CH2 . MUHTADI ETAL. Scheme I11 Amygdalin [ 13 i s t r e a t e d w i t h fuming h y d r o c h l o r i c a c i d t o produce m a n d e l o n i t r i l e [ 2 ] which i s t h e n hydrolyzed w i t h h y d r o c h l o r i c a c i d t o g i v e mandelic a c i d [ 3 ] (30). which i s t r e a t e d w i t h h y d r o c h l o r i c a c i d t o g i v e mandelic a c i d [ 3 ] ( 2 9 ) .C H 2 [81 T o t a l S y n t h e s i s of Mandelic Acid S e v e r a l schemes f o r t h e t o t a l s y n t h e s i s a r e known (28-30).CH .3 T o t a l S y n t h e s i s of Homatropine Tropine i s t h e n e s t e r i f i e d w i t h (+) -mandelic a c i d i n t h e presence of hydrogen c h l o r i d e ( F i s c h e r S p e i e r e s t e r f i c a t i o n ) t o g i v e ( 2 ) -homatropine.

HOMATROPINE HYDROBROMIDE 213 Total Synthesis of Mandelic Acid Scheme I NaCN NaHSO Scheme I1 @Lo c12 CH3COOH Scheme I11 HO HO b / / CHC 1 2 [21 i) NaOH ii) HCI .

Homatropine i s an a n t i c h o l i n e r g i c d r u g w i t h e f f e c t s s i m i l a r t o h u t much weaker (about o n e . Therapel. b u t more o f t e n i n form of 2 t o 5% hornatropine hydrobromide ophthalmic s o l u t i o n s (12. b u t it produces a l e s s s a t i s f a c t o r y m y d r i a s i s i n c h i l d r e n (18).31). . t h e r a t e of hydrol y s i s i s a minimum a t pH 3 .o c u l a r p r e s s u r e (18). Some of t h e s e compounds ( a t r o p i n e and homatropine) when a p p l i e d l o c a l l y t o t h e eye can cause mydriasis and c y c l o p l e g i a (31).34). showed no decomposition a f t e r f i v e y e a r s when examined according t o t h e B.P. 5 t o 4.t e n t h ) t h a n t h o s e o f atropine (12. MUHTADI ETAL. Homatropine i s used a s a 2% s o l u t i o n i n c a s t o r o i l .FARID J. less prolonged and i s z o r e r e a d i l y c o n t r o l l e d by p h y s o s t i g mine ( 1 2 ) .31l.itic Uses and Pharmacological E f f e c t s Homatropine i s used a s a m y d r i a t i c and c y c l o p l e g i c d r u g i n ophthalmology. I t i s p r e f e r r e d t o a t r o p i n e f o r d i a g n o s t i c purposes because i t s a c t i o n i s more r a p i d . 5 5 % . I t i s seldom used i n t e r n a l l y (12). A t 25". Homatropine has a l s o l e s s tendency t h a n a t r o p i n e t o i n c r e a s e t h e i n t r a . The e f f e c t of homatropine i s e x e r t e d i n 15 t o 30 minutes and p a s s e s o f f i n 1 2 t o 24 h o u r s . In aqueous s o l u t i o n . 7 . generally t h e belladonna a l k a l o i d s a r e absorbed r a p i d l y from t h e g a s t r o i n t e s t i n a l t r a c t . I t j s sornetimes used with c o c a i n e which enhances i t s m y d r i a t i c action (12). ( 1 5 ) . when it i s a d m i n i s t e r e d subcutaneously 6.8 (33. i n a i r t i g h t c o n t a i n e r a t room temperature and p r o t e c t e d from l i g h t . 214 5. The pH of t h i s s o l u t i o n f e l l from 4 . Hydrolysis i s c a t a l y z e d by hydrogen i o n s and hydroxide i o n s (18). l o s t 2% of i t s homatropine c o n t e n t a f t e r a u t o c l a v i n g f o r 20 minutes a t 120" (12). A s o l u t i o n c o n t a i n i n g homatropine hydrobromide 1%and b o r i c a c i d 1 . 5 t o 3. Drug S t a b i l i t y Homatropine hydrobromide i n t h e d r y s t a t e . They a l s o e n t e r t h e c i r c u l a t i o n when a p p l i e d l o c a l l y t o t h e mucosal s u r f a c e s o f t h e body (31). I t has been r e p o r t e d (32) t h a t hornatropine i n doses o f 0 . induces b r a d y c a r d i a i n man.0 mg. homatropine h y d r o l y s e s t o t r o p i n e andmandelic a c i d .

A 2 p e r c e n t w/v s o l u t i o n y i e l d s t h e r e a c t i o n c h a r a c t e r i s t i c o f a l k a l o i d s and t h e r e a c t i o n s c h a r a c t e r i s t i c o f bromides.Another c o l o r t e s t was r e p o r t e d a s f o l l o w s ( 3 7 ) : Homatropine i s e v a p o r a t e d t o d r y n e s s w i t h a mixture of fuming n i t r i c a c i d .1 I d e n t i f i c a t i o n Tests The f o l l o w i n g i d e n t i f i c a t i o n t e s t s a r e mentioned i n t h e B. . ( 3 5 ) . - A s i m p l e .a c e t i c anhydride ( 4 : 3 ) and 5% met hano 1i c t e t rame t h y 1ammonium hydroxide so l u t ion i s added t o a s o l u t i o n of t h e r e s i d u e i n a c e t o n e . e x h i b i t s maxima only a t t h e same wavelengths a s t h a t of a s i m i l a r p r e p a r a t i o n of USP Homatropine Hydrobromide Reference Standard . a yellow c o l o r develops which t u r n t o r e d on warming. add a s l i g h t e x c e s s o f 10M ammonia and shake w i t h 5 m l of chloroform. t h e c o l o r d e v e l o p s and remains c o n s t a n t f o r 6 t o 8 minutes. .1.HOMATROPINE HYDROBROMIDE 7. t h e c r y s t a l s were m i c r o s c o p i c a l l y examined (39) . 215 Methods o f A n a l y s i s 7. a methanolic bromophenol b l u e i s added t o g i v e b l u e s t a b l e c o l o r .2 M i c r o c r v s t a l Tests Homatropine hydrobromide was d i s s o l v e d i n water (10 mg i n 10 m l ) .P.Other i d e n t i f i c a t i o n t e s t s have been a l s o mentioned (38) * 7. - Dissolve 10 mg o f homatropine hydrobromide i n 1 m l of water. . .S. ( 1 5 ) . 5 m l of a 2 p e r c e n t w/v s o l u t i o n of mercury (11) c h l o r i d e i n e t h a n o l ( 6 0 % ) . - The f o l l o w i n g t e s t i s mentioned i n t h e U. Evaporate t h e c h l o r o form l a y e r t o d r y n e s s on a water b a t h and add 1 . I t i s p o s s i b l e t o determine t h e a l k a l o i d q u a n t i t a t i v e l y by measuring t h e c o l o r a t 570 ni. The i n f r a r e d a b s o r p t i o n spectrum of a potassium bromide d i s p e r s i o n of i t . After s p e c i f i c time. 1 t o 2 d r o p s of t h i s s o l u t i o n was t r e a t e d w i t h t h e r e a g e n t on a m i c r o s c o p i c a l g l a s s s l i d e .P. r a p i d and s e n s i t i v e t e s t €or t h e d e t e c t i o n of homatropine and o t h e r a l k a l o i d s i n p h y s i o l o g i c a l f l u i d s such a s s a l i v a and u r i n e i s r e p o r t e d (36) To t h e s o l u t i o n o f a l k a l o i d s .

9 ) .3 minutes ( F i g .1 Wagner's r e a g e n t was added t o t h e t e s t s o l u t i o n . and c o o l i n an i c e b a t h . and h e a t t h e s o l u t i o n j u s t t o b o i l i n g . 1 0 ) . .2.40. minute i r r e g u l a r orange c r y s t a l s were formed a f t e r 1 . Add 10 m l of d i l u t e n i t r i c a c i d ( 1 i n 1 5 ) . 7.2. - A p o t e n t r i o m e t r i c t i t r a t i o n of homatropine hydrobromide i n g a l e n i c a l p r e p a r a t i o n s w i t h sodium t e t r a p h e n y l b o r o n was d e s c r i b e d (43) .P (35) .S. i n water t o make 50 m l and mix.d i c h l o r o e t h a n e (42).2.OSN c e r i c ammonium n i t r a t e u n t i l t h e pink c o l o r i s discharged.D i s s o l v e about 400 of Homatropine Hydrohro- mide. add 5 m l of sodium hydroxide TS. 7.FARID J.3 D r a g e n d r o f f ' s r e a g e n t was added t o t h e t e s t s o l u t i o n . 0 1 M .2 .3 T i t r i m e t r i r Determinations 7. MUHTADI ETAL.HBr. T r a n s f e r 10 m l of t h i s s o l u t i o n t o a b e a k e r .1 Aqueous The f o l l o w i n g a s s a y i s mentioned i n U. a c c u r a t e l y weighed. Concomitantly add 5 m l o f d i l u t e n i t r i c a c i d (1 i n 15) t o a second 10 m l p o r t i o n o f t h e s o l u t i o n of Homat r o p i n e Hydrobromide. 276 7.t e t r a p h e n y l b o r a t e . Other m i c r o c r y s t a l t e s t s have a l s o been r e p o r t e d (17.3.907 mg of CI6HZ1NO3. Each m l o f t h e d i f f e r e n c e i n volumes of 0. r a d i a t i n g r o d s and i r r e g u l a r b l a d e s were formed a f t e r 10 minutes ( F i g . minute irregular brown b l a d e s were formed a f t e r 2-3 minutes ( F i g . add w a t e r t o make 50 m l . add water t o make 50 m l . - Homatropine was determined by t i t r a t i o n w i t h aqueous 0 . 7. and c o o l i n an i c e b a t h . w i t h t e t r a bromophenolphthalein e t h y l e s t e r as an i n d i c a t o r i n 1. t i t r a t e w i t h O.2 P i c r i c a c i d was added t o t h e t e s t s o l u t i o n .41). 1 1 ) .05N c e r i c ammonium n i t r a t e r e q u i r e d i s e q u i v a l e n t t o 8. Add 1 d r o p o f n i t r o p h e n a n t h r o l i n e TS t o each s o l u t i o n and while keeping t h e s o l u t i o n s c o l d .

Hornatropine HBr with picric acid reagent a- Fig. 11 Hornatropine HBr with Dragendorff's reagent . 9.HOMATROPINE HY DROBROMIDE 277 Fig. 10. Hornatropine HBr with Wagner's reagent ~~ Fig.

The combined e x t r a c t s a r e evaporated and water and 0. 1 mmol (44) could n o t be determined.Homatropine hydrobromide was t i t r a t e d i n dimethyl sulphoxide medium w i t h 0. Each m l of 0 . 1 N i o d i n e i n 0.15 M potassium i o d i d e s o l u t i o n .P. - 7. (15) * Dissolve 0 . 7.2 Non-aqueous . . 1 M p e r c h l o r i c a c i d VS i s e q u i v a l e n t t o 0. and t h e r e s u l t s were s a t i s f a c t o r y by a l l t h r e e t e c h n i q u e s . 3 gm i n 20 m l of anhydrous g l a c i a l a c e t i c a c i d .4 - An a l t e r n a t i v e method i n v o l v e s d i r e c t t i t r a - t i o n of homatropine hydrobromide w i t h p e r c h l o r i c a c i d i n a c e t i c anhydride medium u s i n g naphthalbenzein o r Sudan r e d B a s i n d i c a t o r (45) Other non-aqueous t i t r a t i o n has been a l s o r e p o r t e d (46).05M EDTA t o a yellow end p o i n t .1Ms i l v e r n i t r a t e a r e added.03563 gm of C16H21N03. The mercury i n t h e s o l u t i o n i s t h e n t i t r a t e d w i t h 0. add 10 m l o f mercury (IT) a c e t a t e s o l u t i o n and c a r r y o u t Method I f o r non-aqueous t i t r a t i o n . 1 N t h a l l i u m s u l f a t e and 3 p a r t s 0 . The use of t h i s p r e c i p i t a t i o n r e a g e n t f o r an i n d i r e c t d e t e r m i n a t i o n of s m a l l amounts of homatrop i n e i s based on t h e d e t e r m i n a t i o n of t h a l l i u m i n the p r e c i p i t a t e (47). End-point was determined by conductometric.3. Appendix V I I I A. The p r e c i p i t a t i o n i n a n e u t r a l medium i s most s e n s i t i v e . p o t e n t i o m e t r i c and p o l a r i m e t r i c t e c h n i q u e s . MUHTADI ETAL.FARID J. 3 sodium hydroxide s o l u t i o n was r e p o r t e d ( 4 8 ) . Amounts of 0 .1 M AgN03.Another method u t i l i z i n g merceuric-potassium i o d i d e r e a g e n t i n 0 . - Complexometry P r e c i p i t a t i o n of homatropine a s a t h a l i u m complex u s i n g one p a r t 0 . 218 .HBr. The d e t e r m i n a t i o n i s based on t h e e x t r a c t i o n of t h e p r e c i p i t a t e formed with chloroform.The f o l l o w i n g a s s a y i s mentioned i n t h e B. Methylthymolblue i s used a s i n d i c a t o r and s u f f i c i e n t 20% h e x a m e t h y l e n e t e t r a mine s o l u t i o n i s added t o g i v e a b l u e c o l o r . . d e t e r m i n i n g t h e end-point p o t e n t i o m e t r i c a l l y .

HOMATROPINE HYDROBROMIDE 7. mix and s e t a s i d e f o r 1 h r . 7.Absorpt i o m e t r i c d e t e r m i n a t i o n o f homatropine hydrobromide i n eye d r o p s ( 5 3 ) . . 2 5 t o 2 mg of . a l l o w t h e e v o l u t i o n of gas t o s u b s i d e and measure t h e e x t i n c t i o n o f t h e s o l u t i o n a t 540 nm. add s a t u r a t e d aqueous hydroxylammonium c h l o r i d e s o l u t i o n ( 1 ml) and 1 0 . 5 M potasium hydroxide (1 ml) . 5 m l of sample t o 2 5 m l and t o 1 m l of t h e r e s u l t i n g s o l u t i o n [ 0 .i n on t h e c u r v e i s p o s s i b l e i n a l k a l i n e s o l u t i o n s . S e m i q u a n t i t a t i v e determinat i o n from t h e depth o f t h e c h a r a c t e r i s t i c C u t s . Remove t h e f l a s k from t h e b a t h .6. 2 t o 1 .1 M hydrochloric acid (1 ml) and mix a g a i n . Add 4 M h y d r o c h l o r i c a c i d ( 2 ml) t o g i v e pH 1 . 3 7 M F e r r i c chloride solution i n 0.1 Colorimetry .6 Spectrophotometric methods 7. . add 0 . - A s t a b i l i t y i n d i c a t i n g a s s a y f o r t h e determinat i o n of homatropine hydrobromide and methobromide and t h e i r d e g r a d a t i o n p r o d u c t s i n g a l e n i c a l p r e p a r a t i o n s was r e p o r t e d ( 5 2 ) . 4 .5 - 279 Polarographic The o s c i l l o p o l a r o g r a p h i c behaviour of homatropine and o t h e r a l k a l o i d s was r e p o r t e d ( 4 9 ) .A c o l o r i m e t r i c method f o r t h e d e t e r m i n a t i o n of homatropine and o t h e r r e l a t e d a l k a l o i d s was r e p o r t e d ( 5 1 ) . The product i s made a l k a l i n e with h o t 20% sodium hydroxide and t h e c o l o r which develops i s measured. The d e p o l a r i z i n g p o t e n t i a l s were r e f e r r e d t o t h e p o t e n t i a l s of T1 i o n s . D i l u t e 2 . and a g r a p h i t e one a s a r e f e r e n c e . m i x . The method i s based on t h e n i t r a t i o n o f t h e drug w i t h a s o l u t i o n of 20% potassium n i t r a t e i n c o n c e n t r a t e d s u l f u r i c a c i d a t 50-60' f o r 30 minutes. A dropping mercury e l e c t r o d e s e r v e d a s a p o l a r i z a b l e e l e c t r o d e .Another o s c i l l o p o l a r o g r a p h i c method f o r i d e n t i f i c a t i o n o f homatropine hydrobromide i n a l k a l o i d a l m i x t u r e s was a l s o d e s c r i b e d (50). The method i s as follows:To 5 ml o f prepared s o l u t i o n ( z = 3 mg o f a l k a l o i d ) i n a 5Q m l f l a s k p l a c e d i n an i c e w a t e r b a t h .

05 gm) was d i s s o l v e d i n 50 m l t o l u e n e and from t h i s s o l u t i o n .Homatropine was determined u s i n g t e t r a - bromophenolphthalein e t h y l e s t e r a t pH 8-10. t h e s t a n d a r d s a t 20.a c o n i t i c a n h y d r i d e i n 100 m l r e d i s t i l l e d a c e t i c anhydride.25 mg i n t h e f i n a l s o l u t i o n t h e mean e r r o r i s + 3 % . The method i s as f o l l o w s : D i s s o l v e 1 mg of sample i n 100 m l of water and t o 1 m l of t h e s o l u t i o n add 3 m l o f mM-bromocresol p u r p l e and 2 m l o f b u f f e r s o l u t i o n (pH 4 ) . - C o l o r i m e t r i c d e t e r m i n a t i o n of t h e t h a l i u m complex of homatropine hydrobromide u s i n g c r y s t a l v i o l e t was a l s o r e p o r t e d ( 5 8 ) . MUHTADI ETAL. . E x t r a c t t h e c o l o r e d 1:l p r o d u c t i n t o c h l o r o f o r m (2x5 ml) . I t i s as f o l l o w s : The sample (0.t o l u e n e m i x t u r e and absorbancy measured a t 535 nm. 280 homatropine hydrobromide] add 2. The s o l u t i o n s were h e a t e d f o r 45 minutes on a steam b a t h and c o o l e d f o r 15 minutes i n t h e d a r k .50 and 100 y / m l were p r e p a r e d . .5 and t h e a d d i t i o n compound formed was e x t r a c t e d w i t h 1 . d i l u t e t h e combined e x t r a c t s t o 10 m l w i t h c h l o r o form and measure t h e absorbance a t 405 t o 410 nm. The s e n s i t i v i t y of t h e method i s 0. 5 m l of F o l i n r e a g e n t . A f t e r 10 m i n u t e s .56). measure t h e e x t i n c t i o n of t h e b l u e s o l u t i o n w i t h u s e of a r e d f i l t e r . 3 .5 m l o f water. Each s t a n d a r d ( 2 m l ) was d i l u t e d w i t h 2 m l t o l u e n e and 1 m l r e a g e n t p r e p a r e d by d i s s o l v i n g 0. Aqueous s o l u t i o n s o f t h e s a l t was b a s i f i e d w i t h ammonia and e x t r a c t e d w i t h t o l u e n e . 3 m l of 5 % sodium hydroxide s o l u t i o n and a f t e r 5 minutes. w i t h 20: 80 a c e t i c a n h y d r i d e .FARID J.Another c o l o r i m e t r i c d e t e r m i n a t i o n o f homatropine hydrobromide has been a l s o r e p o r t e d (54). Then were d i l u t e d t o 10 m l . - Other c o l o r i m e t r i c d e t e r m i n a t i o n of homat r o p i n e by c i s . Hydrolysis p r o d u c t s of t h e drug does n o t i n t e r f e r e u n l e s s p r e s e n t i n t h r e e f o l d amounts.25 gm c i s . 2 .a c o n i t i c anhydride h a s been d e s c r i b e d (55. .d i c h l o r o e t h a n e and e x t i n c t i o n i s measured a t 560 nm (57). The r e l a t i v e e r r o r of t h e method does n o t exceed 2 1%.

1 o r No. 8 gm of c i t r i c a c i d Paper Whatman No. Table 8 i n c l u d e s paper Table 8 . Drying t h e paper i n a i r f o r 5 t o 10 minutes.7.4 .V. Solvent system R -€ value - Reference (59) .BuOH. chromatographic c o n d i t i o n s used €or homatropine.H2 0 (10: 1 : 10) Descending t e c h n i q u e 0. o r Iodoplatinate 0.1 Paper chromatography Homatropine can be d e t e c t e d by paper chromatography.2% I o d i n e s o l u t i o n spray BuOH-C2H5C 02H-H20 (10: 1: 10) Other paper chromatography h a s a l s o been r e p o r t e d (61). V .1 dipped U.3 impregnated by d i p p i n g i n 10% s o l u t i o n of T r i b u t y r i n i n a c e t o n e and d r y i n g i n a i r . (60) . 1 paper impreg.acetate buffer Location under U . Descending t e c h n i que. Light (254 mu) o r Iodoplatinate spray 0.3 Clark (17) . Paper Chromatography of Hornatropine Conditions Detecting reagent Whatman No. .7.AC OH.7 A 7.But an o 1 (pH 4.HCO 2H.96 Clark (17) i n a mixture of 130 water and 870 of n.H 2O (40 : 10 :50) BUOH .58) i n 5% s o l u t i o n of sodium dihydrogen c i t r a t e and d r i e d Whatman No.5% aqueous ammonia s a t u r a t e d w i t h octanol n a t e d w i t h 7 % m e t h a n o l i c o c t a n o l .

wat e r . Table 9 t h i n l a y e r chromatographic c o n d i t i o n s used f o r homatropine.L.5 : 100) s i l i c a gel G acidified iodoplatinate o r under U .15 Clark ( 17) 0. (254 nm) Ethanol-pyridine-water : 6 : 4) (1 alumina iodoplatinate spray Chloroform-acetone-diethylamine ( 5 : 4 : 1) Acetone .7. V .7.25 % ammonia solution : 6) (90 : 4 K i e s e l g e l GF 254 Kieselgel G Dragendorf f s p r a y The upper phase of But ano 1.C. includes Ce 1l u l ose R f- value Reference 0. .87 (65) Dragendorff e t h y l a c e t a t e reagent - (64) Dragendorff s p r a y - (65) f o r homatropine h a s been a l s o r e p o r t e d ( 6 6 ) . S o l v e n t system Thin l a y e r chromatography of Eomatropine Conditions Detecting reagent S t r o n g ammonia s o l u t i o n : methanol (1.a c e t i c ac id-wat e r ( 10 : 1 : 5) Other T. Table 9.2 Thin l a y e r chromatography (TLC) Homatropine can b e d e t e c t e d by t h i n l a y e r chromatography.

2 . . 7.4 Column chromatography Chromatographic a s s a y of homatropine hydrobromide and o t h e r r e l a t e d a l k a l o i d s a r e quant i t a t i v e l y e l u t e d w i t h 95% a c e t o n e from a column packed w i t h b a s i c alumina (70).3 283 Electrophoresis . 7.16 r e s p e c t i v e l y ( 6 7 . 10. After e l u t i o n . 104.7. 6 8 ) . . t h e e l u a t e was d i l u t e d w i t h w a t e r and homatropine hydrobromide i s t i t r a t e d w i t h 0 .5 Gas chromatography (GC) The gas chromatography f o r homatropine a r e l i s t e d i n t a b l e (lo).100. The p u r i t y of homatropine hydrobromide can be determined on alumina column as f o l l o w s (71) ' Homatropine hydrobromide (0.105. 4 . 3 .7.7. 5 4 . . i n t h e p r e s e n c e of u n i v e r s a l buffer The r e l a t i v e d i s p l a c e m e n t s a t pH 2 ./cm f o r 3 h r s .HOMATROPINE HYDROBROMIDE 7. 1 N h y d r o c h l o r i c a c i d u s i n g bromophenol b l u e as an i n d i c a t o r .1 gm) was e l u t e d o v e r a column packed w i t h b a s i c alumina (5 gm) by u s i n g aqueous a c e t o n e as s o l v e n t .4 were 6 3 .5 and 11.E l e c t r o p h o r e s i s of homatropine was performed on Whatman No.Other e l e c t r o p h o r e s i s f o r s e v e r a l a l k a l o i d s have been r e p o r t e d ( 6 9 ) . 4 . 3 . 6 . 8 .1 p a p e r (18x46 cm) a t 8 v.

hydrogen (22 ml/min. - C a r r i e r gas 2.39 r e l a t i v e (50 ml/min. Column t e n p e r a t u r e 225".) t o codeine a i r (300 ml/min. Gas Chrom(R)Q.I.D.7 ml/min. 2 mm) column t e m p e r a t u r e 160". I . (1. The GC of Homatropine Ref.I.) F. 250-270° 15 % QF-1 on 60-80 mesh.I.) F . D .) Clark (17) 5% SE-30 on 60-80 mesh Chromosorb W AW (5 f t x 1/8 d i a m e t e r ) Column temperature 230'.) F . .230. Nitrogen (50 ml/min. Detector Retention _ t i_ me Column c o n d i t i o n Nitrogen (50 ml/min) F. I . Nitrogen (50 ml/min.8m x 2mm) Column t e m p e r a t u r e 250".5% SE-30 on 80-100 mesh chromosorb W (5 F t x 4mm i n t e r n a l d i a m e t e r ) .) t o codeine a i r (300 ml/min. Nitrogen (SO ml/min.Table 1 0 . 2 (min) (73) . 5% SE-30 on 60-80 mesh a c i d washed Chromosorb W a t 190. D .) Clark (17) 3% XE-60 s i l i c o n e n i t r i l e polymer on 100-120 mesh Chromosorb W.D. Column t e m p e r a t u r e 225'. hydrogen 0. Other gas chromatographic a n a l y s i s have been r e p o r t e d (75-78) - - 4 .210. Nitrogen (30.) 0.5m x 3 .36 r e l a t i v e (50 ml/min.D. hydrogen 0. F.44 r e l a t i v e t o codeine Clark (17) 3% J X R on s i l a n i s e d G Chrom P on 100-120 mesh (1.) F.D.I.

A d i f f e r e n t i a l r e f r a c t i v e index d e t e c t o r and a UV d e t e c t o r o p e r a t i n g a t 254 nm were used t o monitor t h e eluate. .6 285 High P r e s s u r e Liquid Chromatography (HPLC) Fell _ et _ a l . 6 mm) packed with sil-X a b s o r b e n t w i t h 28% aqueous ammonia t e t r a h y d r o f u r a n (1: 100) as t h e s o l v e n t and w i t h a column i n l e t p r e s s u r e o f 500 l b p e r s q . Sharma of Department of Pharmdcognosy.t o l u i c a c i d . Uday C.s t e e l column (1 meter x 4 . (73) have r e p o r t e d an a n a l y s i s of homatropine hydrobromide by r e v e r s e d .HOMATROPINE HY DROBROMIDE 7. The compound was s e p a r a t e d on a s t a i n l e s s . i n c h . 5 ) .p r e s s u r e l i q u i d chromatography.p h a s e h i g h . Homatrop i n e hydrobromide was determined on a column of H y p e r s i l ODS ( 5 um) with 5 0 mM.7. King Saud U n i v e r s i t y €or h i s s e c r e t a r i a l assistance i n t h e reproduction of t h e manuscript.a c e t o n i t r i l e ( 3 : l ) as t h e mobile phase and d e t e c t i o n a t 254 nm. S t u t z and Sass (80) have d e s c r i b e d a high-speed. c o l l e g e o f Pharmacy. R e c t i l i n e a r c a l i b r a t i o n graph was o b t a i n e d f o r homatropine hydrobromide ( i n eye d r o p s ) i n t h e range 0 t o 4 mg ml-1. high p r e s s u r e l i q u i d chromatography of t r o p a n e a l k a l o i d s i n c l u d i n g homatropine. ACKNOWLEDGEMENT The a u t h o r s would l i k e t o thank Mr. Sodium a c e t a t e i n 10 mM-tetrabutylammonium s u l f a t e (pH 5 . The i n t e r n a l s t a n d a r d was p .

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153 (1965). Tokar. ( I t a l . 931 (1971). J . Weisel. ( I s t a n b u l ) . 8 3 ( 1 9 7 7 ) . Farmaco (Pavia) Ed. Marini . J . (Ned. 1646 ( 1 9 7 0 ) . I s t v a n . 52. B . D . Simonyi and G . H i n c a l . J. 54. B . O. A . 26 ( 3 ) . 348 ( 1 9 5 6 ) . Brantner? Gyogysze22 ( 1 2 ) . M. 63. Rend. N. 1315 ( 1 9 7 1 ) . and P . 62. P r a t . Marini . 206 (1977). H . 35 ( 9 3 . r e s z e t (Budapest) . Palumbo and S . and B. K. Pharm. L. Uno.N.. Eczac.A. C e s k o s l . Coch Frugoni. Pharm.B e t t o l o and J . (Budapest) 21 ( 6 ) . Vamos. 56. J . Gore and J . Tsubouchi. M . M. M i k u l l a . 26 ( l ) . 803 ( 1 9 5 2 ) . 65 ( 1 9 6 2 ) . - 64. J . G . ) 8 6 . Magyar Kem. 58. G. Gyogyszereszet. Pharmazie 11. (Tokyo). J . Kakac. S c i . Farmatsiya.HOMATROPINE HYDROBROMIDE 289 I . Bubon. 533 (1956). 104 (421. L . Ebel. . B u l l . Pharm. Szasz and A.G. 65. 57. C . Macek. D i j k h u i s Pharm. Chem. 467 (1978). and S. and Pharmacol. W . S a n i t a (Uruguay) 21. 394 ( 1 9 8 3 ) . Chem.T. 60. Feldman and B . B r a n t n e r . Agric. Robb. 17. Coch Frugoni. 1324 (1956). D. 62 51. Guven and A. 68. Zyka. L.B e t t o l o and J u a n A. . 319 (1958). 55. 24 Adshead. (1969) 67. S. . (lo). Horak and J .). Farni. 34 ( 1 9 7 7 ) . A . Akopyan. 1st. 12 ( 8 ) . 59. Apoth. Ambrus. 289-93. 2237 ( 1 9 7 6 ) . Yamamoto. Gazz. I b i d . ( 9 ) . and T. I . 53. and K . J . 1317 . 1 ( 2 ) . 4. Fak. F o l y . Senov. . 59 ( 1 1 ) . Hacaperkova. S . Weekbl. D t . Farmatsiya. M . Ambrus.C. s u p e r . S a c c a . and A. 61. P.. 111 ( 2 5 ) . K. - 66. Vamos. Z t g . Chim. Mecm. Hara. B i o l . G .

. and W.G. V . 35. Shami. Chang. 447 (1961). Berg. E.K. 71. H. Quercia. Brochmann . 122 (1970). 75. Cardini and B. 76. H. V. Bohme. Tingstand. 19 (2) . J . . Brochmann-Hanssen and A. 70.FARID J. Softly. J. 31 (Suppl. Berg. Dudzinski. H. Arch. St. 107 (12). B. Kirk. Gorbowski. and J . 290 69. 77. Haney. and E. 51. 'Tauber. 1095 (1962).Hanssen and C. - 78. and G. (Ital. - 79. M. farm. 25 ( 2 ) . Johnstone. J.. 737 (1968). Sci. Sci. (1973).D. Boll. E. Parker. A. Tauber. Pharm.L.J. K. J . Quercia. Ed. 806 (1973). E. Pharm. Teodosiu. Farmacia (Bucharest) 10..R. J. Fontan. 72. Stamm and E. 296 (1965)... 293. G .utz.. Pharmacol. prat.. Pharm. B.. Stamm. B. 62 (81. Pharm. S. 73. 13.. Apreotesei and M. Anal. Arch. 294. Smith. C. MUHTADI ETAL. 315 (1961). C. 74. 294 ( 7 ) . and S. J.R. C. 356 (1963). Fontan. Pharm. 1283 (1973). Chim. 45 (12). 342 (1960). Tucci. Pharm.. 321 (1962). 62.F. G. 2134.. Chem.).B. 80. J.J. Farmaco. Sci. Chem.) 20 (1979). Svendsen. and P.H. Analyt. Fell. Lachman. Bohme. Chromatog.F. Sass.L.

ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 16 29 1 Copyright 0 1987 by Academic Press.MEBENDAZOLE A . AL-Budrz* und M . A . T d / t i q * * *Vepu. All rights of reproduction in any form reserved.ahtmeMA: 06 PhatunuzceuLlcal C h m i ~ f i yand VcpahXment** 0 6 Phahmacology. . King Saud UnivehhLty. Coflecje ozj Phahmucy. P. Riyudh-11451. Box 2457. Saudi Ahabia. Inc.O.

2 2.2 Identification Titrimetric Methods 8 . Forward.5 3.A. Pharmacokinetics 7. Methods of Analysis 8. AL-BADR AND M. TARIQ 292 1. Toxicity 8. A. Spectral Properties 5.4 Chromatographic Methods 8. Synthesis 6.3 2.5 Polarographic Method 8. Description 2.1 8. Color and Taste Physical properties 4. 3 Spectrophotometric Methods 8.1 2. Nomenclature Formulae Molecular Weight Elemental Composition Appearance.4 2.6 Radioimmunoassay Method References . History 2.

Oxiben. Parelmin.3 (7). Vermirax. T r o t i l . H i s t o r y Mebendazole i s a broad spectrum a n t h e l m i n t i c a g e n t s y n t h e s i z e d and developed by J a n s s e n Pharmaceutica.5-benzoyl-1H-benzimidzol-2-yl. Vermox. and Whipworms ( T r i c h u r i s ) . R 17. Mebendacin. ) . Lomper. Methyl-5-benzoylbenzimidazol-2-ylcarbamate.1.1 Nomenclature 2.1. Mebendozol M K . Mebenix. Nemasole. Pantelmin.635 2.1. (6) CAS R e g i s t r y Number [31431-29-71 2. . Mebendazole. Methyl 5-benzoyl-2-benzimidozolecarbamate. Description 2. 5-Benzoyl-2-benzimidazolecarbamic a c i d methyl ester. methyl e s t e r . 2. t h e drug became a v a i l a b l e i n numerous c o u n t r i e s around t h e world.6. Methyl-(5-benzoyl-1H-benzimidazol-2-yl) carbamat e. Beerse.MEBENDAZOLE 293 Foreword. and Zakor (1.2 (6) Generic N a m e Mebendazole. Equivuarm p l u s Forverm. I t produces high c u r e rates i n i n f e s t a t i o n s by A s c a r i s . hookworms (Ancylostoma and Necator S p p . Granverm. Besantin. S i r b e n . E s a d i r a s e . Gendal. Research Laboratody. Telmin.1 Chemical Names (5-Benzoyl-1H-benzimidazol-2-yl)-carbamic a c i d methyl ester. I t i s a l s o a c t i v e i n some (about 40%) Dwarf tapeworm (Hymenolepis) i n f e s t a t i o n s (2-5). Belgium. Mebutar. A f t e r i t s i n t r o d u c t i o n t h e r e i n 1972. threadworms ( E n t e r o b i u s ) . i n c l u d i n g t h e United S t a t e s and Canada (1). 8-11). Carbamic acid. Trade Names P r o p r e t a r y Names Bendrax.

4 Elemental Composition C .2.3 0 Molecular Weight 295.2.294 A. readily soluble in formic acid (9 E 11). decomposition (6-8 6 l o ) . 288.2 Structural H I 2. C o l o r and Taste Off-white to slightly yellow amorphous powder and not unpleasant to taste.25% 2.1 Melting Point Melts at about 290°.1 Empirical 2. TARIQ 2.5'. 3. 2.2 Formulae 2.23%.3 Hygroscopicity Mebendazole is not hygroscopic and it is stable in air.30 and 295 ( 7 E 9 ) . ether. 65. Physical Properties 3. 4. ethanol. (10-12) 3.2 above 280' with Solubility Almost insoluble in water. 0. and dilute mineral acids. chloroform. 14. H .5 Appearance. 16.44%.08%. A. AL-BADR AND M. N . 3. .

MEBENDAZOLE

3.4

295

Extraction
Mebendazole i s e x t r a c t e d by chloroform from aqueous
a l k a l i n e solutions (11).

4.

Spectral Properties
4.1

U l t r a v i o l e t Spectrum
The u l t r a v i o l e t a b s o r p t i o n spectrum o f mebendazole
o b t a i n e d from a s o l u t i o n i n n e u t r a l methanol i n t h e
r e g i o n o f 200 t o 400 nm u s i n g DMS 90 s p e c t r o p h o t o meter i s shown i n F i g u r e 1. Three a b s o r p t i o n
maxima a t about 210, 247 and 310 nm and two minima
a t 228 and 273 nm were observed.
C l a r k e (11) r e p o r t e d t h e f o l l o w i n g :
Mebendazole i n 0.05% f o r m i c a c i d i n isopropanol;
maxima a t 248 nm ( E l % , 1 cm 1005) and 313 nm
( E l % , 1 cm 5 1 8 ) , minima a t 230 nm and 275 nm.
Mebendazole i n 0 . 0 1 N sodium hydroxide i n i s o p r o p a n o l , maxima a t 270 nm ( E l % , 1 cm 802) and 355 nm
( E l % , 1 cm 653), minima a t 245 nm and 302 nm.
Mebendazole i n 0 . 1 N h y d r o c h l o r i c a c i d i n i s o p r o p a n o l , maxima a t 234 nm ( E l % , 1 cm 1000) and
288 nm ( E l % , 1 cm 5 2 4 ) , minima a t 215 nm and 266 nm.

4.2

I n f r a r e d Spectrum
The i n f r a r e d spectrum o f mebendazole i s p r e s e n t e d ;
i n F i g u r e 2. The spectrum was o b t a i n e d from K B r
d i s c u s i n g a Pye Unicam SP 1025 i n f r a r e d
s p e c t r o p h o t o m e t e r . The s p e c t r a l a s s i g n m e n t s are
presented i n t h e following t a b l e .

A. A. AL-BADR AND M.TARIQ

2%

200

250

300Wavelength 350

400

(nm)
FIGURE I : ULTRAVIOLET SPECTRUM OF MEEENDAZOLE IN METHANOL

-E 20.

*20

;"

0

C

0

e

.-

m
C

0;

FIGURE 2 . INFRARED S P E C T R U M

OF M E B E N D A Z O L E

( KBr d i s c )

298

A. A. AL-BADR AND M. TARIQ

Frequency cm

-1

Assignment

34 15

NH s t r e t c h

2500-2950

CH s t r e t c h

1720

C = 0 (amide) s t r e t c h

1650

C = 0 stretch

1530
1600

I

141 0-1460
700- 765
878

1

900)

C = C stretch

CH3-0 (C-0) s t r e t c h
Monosubstituted benzene

1 , 2 , 4 T r i s u b s t i t u t e d benzene

C l a r k e (11) r e p o r t e d t h e p r i n c i p a l peaks (KBr d i s c )
a r e 705, 1260, 1590, 1635 and 1730 cm-1.
4.3

1
'H Nuclear Magnetic Resonance ( H NMR) Spectrum
1
The H NMR spectrum of mebendazole i s shown i n
F i g u r e 3. The drug i s d i s s o l v e d i n T r i f l u o r o a c e t i c a c i d (TFA) and i t s spectrum determined on a
Varian T60 A NMR s p e c t r o m e t e r u s i n g TMS ( t e t r a m e t h y l s i l a n e ) as t h e i n t e r n a l s t a n d a r d . Assignment
of t h e chemical s h i f t s t o t h e d i f f e r e n t p r o t o n s is
shown be low:

Chemical S h i f t
PPM (6)
7.5 - 8.3
4.08

Multiplicity

P r o t o n Assignment

mu 1t ip 1et

Aromatic

singlet

methyl

FIGURE 3: PROTON MAGNETIC RESONANCE SPECTRUM OF MEBENDAZOLE IN

TRIFLOUROACE T I C
ACID(TFAI

300

A. A. AL-BADR AND M. TARIQ

4.4

I 3 C Nuclear Magnetic Resonance (13C NMR) Spectrum

The I3C NMR s p e c t r a of mebendazole i n formic a c i d
u s i n g TMS ( t e t r a m e t h y l s i l a n e ) as an i n t e r n a l
s t a n d a r d a r e o b t a i n e d u s i n g a J o e l FX 100 MHz
s p e c t r o m e t e r a t an ambient t e m p e r a t u r e . F i g u r e s 4
and 5 r e p r e s e n t t h e 1H-decoupled and o f f - r e s o n a n c e
spectra respectively.
/
0

.,

The carbon assignments a r e based on chemical s h i f t
v a l u e s and by comparison with model compounds and
a r e shown below:
Chemical S h i f t
PPM ( 6 )

Multiplicity

C a r bon Ass ignm en t

57.5

quartet

155.8

singlet

148.4

singlet

139.2

s i n g 1e t

134.4

singlet

118.2

doublet

130.5

singlet

130.9

doublet

115.8

doublet

200.5

singlet

136.7

singlet

132.9

doublet

131.3

doublet

‘13 and ‘15

136.3

doublet

‘14

c1

c2
c3

c4
c5

‘6
c7

c9

clo
5 1
‘12

and ‘16

MEBENDAZOLE

301

FIGURE L : PROTON OECOUPLEO CARBON-13 NMR SPECTRUM OF MEBENDAZOLE IN FORMIC ACID

FIGURE 5 : OF-RESONANCE CARB3N-I3 NMR SPECTRUM

OF

MEBENDAZOLE IN FORMIC

ACID

A. A. AL-BADR AND M. TARIQ

302

4.5

Mass Spectrum and Fragmentometry
F i g u r e 6 shows t h e 70 eV e l e c t r o n impact ( E I ) mass
spectrum o b t a i n e d on Varian MAT 311 mass s p e c t r o meter usi ng ion s o u r c e p r e s s u r e of 10-6 T o r r , i o n
s o u r ce t e mpe ra ture o f 18OoC and an emission c u r r e n t
of 300 PA. The spectrum is dominated by m/e 186
i o n ( ba se peak) r e s u l t i n g from t h e l o s s of CH30H
and C6H5 fragments. A proposed mechanism of
f r ag m enta ti on and t h e mass/charge r a t i o s of t h e
major fragments i s given i n Scheme 1 (a ar,d b ) .
Chemical i o n i s a t i o n (CI) spectrum i s p r e s e n t e d i n
F i g u r e 7 and i s obt a i ne d on a Finnigan 4000 mass
s p e ct r ome t e r wit h ion e l e c t r o n energy of 100 eV,
ion s ourc e p r e s s u r e o f 0 . 3 T o r r , ion s o u r c e
t e m p e r a ture of 15OoC and emission c u r r e n t of 300 PA.
The mass s p e c t r a l assignment of t h e prominent i o n s
under C I c o n d i t i o n s i s gi ve n below:

m/e

Species

296

M+ + 1

2 95

M+

218

M+

-

C6H5

MEBENDAZOLE

303

2

lBO(

1

50.C

324.2
J

2180
208 \

I86

,330

50.0

336.2

235.8 251 6 2Ul

32832.

I05

263
218

2 95

M /E
FIGURE 7

MASS SPECTRUM OF MEBENDAZOLE ( c l )

b

.+

a

&@+.

H

G

[@!$==
7

m/e 295

N?-NH-c-o

-cH3

'\
0-C'
O+

- co

____)

E

m/e 77

-+0

H

NH

- \C - OCH3

-CH30H

[-

m/e 105

Ill
O 1

IA
-co
*

C-HNy@
-, i ,

m/e 158

L

m/186
m/e 218

Scheme l a :

Proposed mechanism of fragmentation of mebendazole.

/4

m/e 130

m/e 295

m/e 295

N7rNH-C
0 '&
+

OI

s
W

-CH30H

t

Figure lb:

m/e 263

= O

-co

4 m/e

Proposed mechanism of f r a g m e n t a t i o n o f mebendazole.

235

A. A. AL-BADR AND M.TARIQ

5.

Synthesis
1- Mebendazole was prepared (13) from 3,4(NH2)2C,H,CoC6H5
and NHz(CH3S)C = N - COOCH3
according to the following scheme.

0

II

NH3

C

0

HN

v

' 3
c

0

II

C-NH-C-0-CH3

.CH3

11

H2N,
,C = N-C-0-CH3

s,

0

CH3
NH -c-0II
0

U

11

0

CH3

-

MEBENDAZOLE

2-

307

Mebendazole was also prepared in 52% yield by
decomposition of methyl 7-benzoyl-lH-2,1,4benzothiadiazine-3-y1 carbamate in methanol with
2 N HC1. The latter compound is prepared in two
stages from 4 - b e n z o y l - 2 - n i t r o a n i l i n e (14)

.

Two steps

0
II

*

-

CH30H

CH3 2N HC1
H

6.

0

Pharmacokinetics
6.1 Absorption, Distribution, Metbolism and Extraction

Mebendazole is poorly absorbed by gastrointestinal
tract after oral administration (10). Only
5-10% of the orally administered frug is found
in the blood (1). The poor intestinal absorption
of mebendazole is of therapeutic advantage in the
treatment of intestinal helminthis. However,
for tissue dwelling organism high oral doses are
required ( 1 5 ) .
The absorption of drug in man is markedly enhanced
when the drug is used together with the meals, as
concomitent fat ingestion ehances its intestinal
absorption. Following oral administration peak
plasma level reach in 2-4 hours (16-18).
Munst et a1 (19) reported a significant variation
in plasma mebendazole level in human subjects
following oral ingestion. According to this study
the plasma half-life was ranging between 1 . 5 and

308

A. A. AL-BADR AND M. TARIQ

5 . 5 h o u r s . The peak c o n c e n t r a t i o n following o r a l
a d m i n i s t r a t i o n were d e t e c t e d a t 30 minutes t o 2
hours of t h e dose. Mebendazole is metabolized
p r i m a r i l y i n l i v e r by d e c a r b o x y l a t i o n (20) , t h e
major m e t a b o l i t e was 2 -amino-5 (6-benzimidazolyl
phenylketone. B r a i t h w a i t e et a1 (21) r e p o r t e d t h a t
mebendazole is more l i p o p h i l i c t h a n any of i t s
m e t a b o l i t e s . The d i s t r i b u t i o n s t u d y by same a u t h o r
showed t h a t 63.3% of mebendazole was d i s t r i b u t e d
i n plasma and 36.7% i n c e l l u l a r f r a c t i o n . Most
of t h e drug i n plasma (95%) was p r e s e n t i n t h e
p r o t e i n bound form. The c o n c e n t r a t i o n of mebendazole
i n l i v e r was c o n s i d e r a b l y h i g h e r as compared t o t h e
f a t t y t i s s u e . G i l b a l d i and Perrier (22) r e p o r t e d
t h a t s t e a d y s t a t e AUC of mebendazole i s independent
of a b s o r p t i o n r a t e i n human s u b j e c t s . Approximately
90% o f t h e o r a l l y a d m i n i s t e r e d dose o f mebendazole
i s e l i m i n a t e d p r i m a r i l y a s unchanged drug i n
faeces. About 5-10% of t h e a d m i n i s t e r e d drug may
be recovered i n t h e form o f i t s c o n j u g a t e s , and
metabolites i n t h e urine (11).
I n r a t s about 85% o f an I . V . dose was e l i m i n a t e d
with t h e b i l e and remainder w i t h t h e u r i n e . The
m a j o r i t y o f t h e dose was recovered a s conjugated
m e t a b o l i t e s (23-24). I n a n o t h e r s t u d y (25), t h r e e
m e t a b o l i t e s of mebendazole were i s o l a t e d from t h e
b i l e o f r a t s and i d e n t i f i e d as : methyl-5(a-hydroxybenzyl)-2-benzimidazole carbamate; 2amino-5- (a-hydroxybenzyl) benzimidazole and 2-amino5-benzoylbenzimidazole.
7.

Toxicity
G a s t r o i n t e s t i n a l symptoms i n c l u d i n g c o n s t i p a t i o n have
been r e p o r t e d i n f r e q u e n t l y f o l l o w i n g use o f mebendazole
(26). High doses may on o c c a s i o l s b e a s s o c i a t e d w i t h
mild
colic
p a i n o r d i a r r h o e a as r e p o r t e d by
C h a v a r r i a ( 2 7 ) u s i n g doses of u p t o 300 mg B I D i n t h e
t r e a t m e n t of t a e n i a s i s

.

Acute and c h r o n i c t o x i c i t y
s t u d i e s i n animals
i n d i c a t e a wide range between t h e r a p e u t i c and t o x i c
d o s e s . The LD50 (mg/kg o r a l l y ) was more t h a n 640 mg/kg in
r a b b i t s and dogs and more t h a n 1280 mg/kg i n mice and rats
(11). S i g n i f i c a n t haematologic, biochemical, o r pathol o g i c a b n o r m a l i t i e s were n o t found i n animals given
40 mg/kg d a i l y f o r 13 weeks b u t were noted i n r a t s

MEBENDAZOLE

309

given a d a i l y dosage o f 130 mg/kg. Mebendazole i s
c o n t r a i n d i c a t e d i n p r e g n a n t women because i t
h a s shown embryotoxic and t e r a t o g e n i c a c t i v i t y i n
s i n g l e p e r o r a l dose a s low as 10 mg/kg (18, 2 8 , 2 9 ) .
Rare cases o f leukopenia i n human s u b j e c t s f o l l o w i n g
t h e a d m i n i s t r a t i o n o f mebendazole was a l s o reporqed ( 3 0 ) .
I n high d o s e s 40-50 mg/kg/day, t h e drug has been
a s s o c i a t e d w i t h s e v e r e i r r e v e r s i b l e n e u t r o p e n i a . No
s i g n i f i c a n t CNS e f f e c t s i n t h e p a t i e n t s t a k i n g mebendazole
t h e r a p y has been r e p o r t e d e x c e p t s l i g h t headache and
dizziness (31).
8.

Methods o f A n a l y s i s
8.1

Identification
I n f r a r e d Spectrum
The i n f r a r e d a b s o r p t i o n spectrum of a
potassium bromide d i s p e r s i o n o f i t , p r e v i o u s l y
d r i e d , e x h i b i t maxima o n l y a t t h e same
wavelength as t h a t o f a s i m i l a r p r e p a r a t i o n of
USP mebendazole RS ( 3 2 ) .
X-ray D i f f r a c t i o n
The X-ray powder d i f f r a c t i o n d a t a f o r i d e n t i f y i n g mebendazole and o t h e r a n t h e l m i n t i c s have
been o b t a i n e d by d i f f r a c t o m e t e r and Debye S c h e r r e r camera t e c h n i q u e s ( 3 3 ) . The d a t a were
t a b u l a t e d i n terms of t h e l a t t i c e s p a c i n g s and
the r e l a t i v e i n t e n s i t i e s of t h e lines.
P a t t e r n s u s i n g t h r e e d i f f e r e n t X-ray wavelengths
with t h e camera method a r e compared w i t h each
o t h e r and w i t h t h e d i f f r a c t o m e t e r p a t t e r n .

8.2

T i t r i m e t r i c Method
USP XX (1980) (32) d e s c r i b e d t h e f o l l o w i n g

t i t r i m e t r i c method f o r t h e a s s a y o f mebendazole:

Dissolve about 225 mg of mebendazole, a c c u r a t e l y
weighed, i n 30 m l o f g l a c i a l a c e t i c a c i d . T i t r a t e
w i t h 0 . 1 N p e r c h l o r i c a c i d VS, d e t e r m i n i n g t h e
end-point p o t e n t i o m e t r i c a l l y , u s i n g a calomelg l a s s e l e c t r o d e system. Perform a blank determinat i o n , and make any n e c e s s a r y c o r r e c t i o n . Each ml

Mebendazole ( i n amounts u p t o 250 mg) i n anhydrous a c e t i c a c i d medium can be t i t r a t e d w i t h 0 . AL-BADR AND M. (34) developed t h e two t i t r i m e t r i c methods t o determine t h e mebendazole i n pharmac e u t i c a l p r e p a r a t i o n s . t h e powder was d i s s o l v e d i n 20 m l o f 1 M p e r c h l o r i c a c i d i n g l a c i a l acetic a c i d . development o f a p e r s i s t e n t green c o l o r was d e s i g n a t e d a s end p o i n t .53 mg Of C16H13N303' Wahbi and Onsy.TARIQ 3 10 o f 0 . Blank d e t e r m i n a t i o n was performed and t h e n e c e s s a r y c o r r e c t i o n s were made.1 N perchloric acid i n g l a c i a l a c e t i c acid u s i n g c r y s t a l v i o l e t a s i n d i c a t o r . D i r e c t t i t r a t i o n and back t i t r a t i o n methods are mentioned a s f o l l o w s : a) D i r e c t T i t r a t i o n Method 200 Mg o f mebendazole was t a k e n i n d r y c o n i c a l f l a s k and d i s s o l v e d i n 2 m l o f 99% formic a c i d followed by t h e a d d i t i o n o f 60 m l o f g l a c i a l a c e t i c a c i d . These methods a r e reproducible with standard deviation of 0.T i t r a t i o n Method 200 Mg o f mebendazole was t a k e n i n d r y c o n i c a l f l a s k . T h i s s o l u t i o n was t i t r a t e d with 0 . Results a g r e e with t h o s e o b t a i n e d by spectrophotometry a t 313 nm ( 3 5 ) . 8. 1 N sodium acetate i n glacial acetic acid. development o f b l u i s h green a s i n d i c a t o r . T h i s s o l u t i o n was t i t r a t e d w i t h 0.3 Spectrophotometric Methods 8. A.5% c r y s t a l v i o l e t solution a s indicator.A.1 U l t r a v i o l e t S p e c t r o m e t r i c Methods -- P a t e 1 e t a1 (36) r e p o r t e d two s p e c t r o p h o t o metric methods f o r t h e e s t i m a t i o n o f . b) B a c k . 1 N p e r c h l o r i c a c i d i s e q u i v a l e n t t o 29. crystal violet was used a s i n d i c a t o r .3%. The c a l c u l a t i o n was done by u s i n g t h e e q u i v a l e n c e f a c t o r which is 1 m l o f 0 . 1 N p e r c h l o r i c a c i d i s e q u a l t o 29.3. 1 M p e r c h l o r i c a c i d ( a l s o i n a c e t i c a c i d ) u s i n g 0.53 mg o f mebendazole.

After 30 min. developed f o r 15 cm and d r i e d f o r 30 minutes a t 115O. The absorbance of t h i s s o l u t i o n was measured i n a 1 cm c e l l a t 288 nm wavelength u s i n g a spectrophotometer.3.5:0. In t a b l e t s . Thin-layer chromatography was run on s i l i c a g e l i n chloroform : e t h y l a c e t a t e . t h e absorbance was measured a t 420 nm. 1 M 1 of f i l t r a t e taken t o a 100 m l f l a s k and d i l u t e d t o volume w i t h d i s t i l l e d w a t e r . a s o l u t i o n o f t h e drug i n formic a c i d i s d i l u t e d w i t h i s o p r o p a n o l and t r e a t e d w i t h MeOH and 30% potassium hydroxide.MEBENDAZOLE 311 mebendazole i n raw m a t e r i a l . Wahbi and Osny (34) r e p o r t e d a s p e c t r o p h o t o metric method o f a n a l y s i s of mebendazole i n t a b l e t s with a s t a n d a r d d e v i a t i o n o f 1 . 1 cm) v a l u e a t 288 nm o r by u s i n g a s u i t a b l e c a l i b r a t i o n curve. a c e t o n e : formic a c i d (70:80:9. t h i s method gave a r e c o v e r y o f 97. 0 . The drug i s t h e n e x t r a c t e d i n t o chloroform and measured a t 313 nm. The mixture was shaken f o r 10 minutes and f i l t e r e d through a d r y f i l t e r paper.2 Phosphorescence Method Mebendazole h a s been r e p o r t e d t o show good phosphorescence a t room temperature when determined on Whatman 42 f i l t e r paper u s i n g Pb ( I V ) and T 1 ( I ) a s phosphorescence enhancing a g e n t s .6% o f mebendazole (37) * . In a n o t h e r methods. 4 % . Powdered t a b l e t s e q u i v a l e n t t o 50 mg of mebendazole were t a k e n i n t o 50 m l s t a n d a r d f l a s k followed by 10 m l o f 70% p e r c h l o r i c a c i d . 2 M 1 o f 70% p e r c h l o r i c a c i d d i l u t e d w i t h 100 m l of d i s t i l l e d w a t e r was used a s b l a n k . C a l c u l a t i o n o f c o n c e n t r a t i o n was done by t a k i n g 566 a s t h e (E 1%. 8.5). A s o l u t i o n of t h e sample i n formic a c i d was d i l u t e d w i t h a mixture o f chloroform : methanol (17:3).

The c a l i b r a t i o n graph was l i n e a r f o r 0 .3. t h e s o l u t i o n was s p o t t e d on t o Whatman 42 f i l t e r p a p e r and d r i e d a t 65 f o r 5 t o 10 m i n u t e s . I n t h i s method 2 t o 5 mg o f mebendazole i s d i s s o l v e d i n 10 m l of 1 M sodium h y d r o x i d e and h e a t e d f o r 1 h r on a b o i l i n g water b a t h .3 F l u o r i m e t r i c Method Abdel F a t t a h et a1 (38) have r e p o r t e d a f l u o r i m e t r i c d e t e r m i n a t i o n method o f mebendazole i n p h a r m a c e u t i c a l dosage forms a f t e r a l k a l i n e h y d r o l y s i s . t h e c o e f f i c i e n t o f v a r i a t i o n was 9. S o l v e n t system: Chloroform : methanol : formic a c i d (90:5:5). 8.4 C o l o r i m e t r i c Method Rana et& (39) have d e s c r i b e d a method f o r c o l o r i m e t r i c d e t e r m i n a t i o n of mebendazole i n t a b l e t s and s u s p e n s i o n b y adding hydroxyl amine h y d r o c h l o r i d e . The l i m i t of d e t e c t i o n was 0 .97%.56%. .27 t o 2.04 t o 100.5% (n = 1 9 ) . 1 f o r mebendazole w i t h r e c t i l i n e a r c a l i b r a t i o n graphs u p t o 10 and 50 ppm ( i n t h e f i n a l s o l u t i o n ) . 0 pg/ml of mebendazole . 8. For 8 ng o f mebendazole p e r s p o t . Absorbent: S i l i c a g e l HF 254 (Merck) . 1 M sodium h y d r o x i d e ( 4 9 : 1 ) . AL-BADR AND M.4.A. w i t h c o e f f i c i e n t o f v a r i a t i o n of 2. For p h a r m a c e u t i c a l f o r m u l a t i o n s . A. 4 t o 2 .3. dicyclohexylcarbodiimide and f e r r i c c h l o r i d e t o a s o l u t i o n o f t a b l e t s (or a d i l u t i o n o f a s u s p e n s i o n ) i n isopropanol and formic a c i d and measuring t h e absorbance a t 520 nm.4 Chromatographic Methods 8.1 Thin-Layer Chromatography (TLC) C l a r k e (11) h a s d e s c r i b e d t h e f o l l o w i n g TLC system f o r t h e i d e n t i f i c a t i o n o f mebendazole. r e c o v e r y o f mebendazole ranged from 99. After d i l u t i o n w i t h methanol. The f l u o r e s c e n c e was measured a t 460 nm ( e x c i t a t i o n a t 365 nm). TARIQ 312 8.

and develop t h e chromatogram i n a s o l v e n t system c o n s i s t i n g o f chloroform.p r e s s u r e Liquid Chromatography (HPLC) A s e n s i t i v e method f o r s e p a r a t i o n and s i m u l t a n e o u s d e t e r m i n a t i o n o f mebendazole and hydroxymebendazole i n human plasma o r c y s t l i q u i d sample u s i n g f l u b e n d a z o l e as . 0 m l o f 98 p e r c e n t f o r m i c a c i d i n a 10-ml v o l u m e t r i c f l a s k . add chloroform t o volume. 8. i n t h e chromatogram o f t h e t e s t s o l u t i o n i s l a r g e r o r more i n t e n s e t h a n t h e main s p o t o b t a i n e d from t h e d i l u t e d S t a n d a r d s o l u t i o n . add a m i x t u r e o f chloroform and 98 p e r c e n t formic a c i d ( 9 : l ) t o volume. and no s p o t . The method i s d e s c r i b e d as follows: D i s s o l v e 50 mg i n 1 .4. Rf : 0. o t h e r t h a n t h e main s p o t . T r a n s f e r 1 .l a y e r chromatographic p l a t e . S i m i l a r l y p r e p a r e a s o l u t i o n of USP Mebendazole RS i n t h e same medium having a c o n c e n t r a t i o n o f 5 mg p e r m l . mark t h e s o l v e n t f r o n t .25-mm l a y e r o f chromatog r a p h i c s i l i c a g e l m i x t u r e . c o a t e d w i t h a 0.2 H i g h . t h e S t a n d a r d s o l u t i o n . a l l o w t h e s o l v e n t t o e v a p o r a t e . Remove t h e p l a t e from t h e d e v e l o p i n g chamber. and mix. The R f v a l u e of t h e main s p o t o b t a i n e d from t h e t e s t s o l u t i o n c o r r e s p o n d s t o t h a t o b t a i n e d from t h e S t a n d a r d s o l u t i o n . and mix ( d i l u t e d S t a n d a r d s o l u t i o n ) . methanol. and examine t h e p l a t e under s h o r t .53.f o u r t h s o f t h e l e n g t h o f t h e p l a t e . United S t a t e s Pharmacopeia "USPXX 1980 (32) have used TLC t o determine t h e p u r i t y o f mebendazole. and 98 p e r c e n t formic a c i d (90:5:5) u n t i l t h e s o l v e n t f r o n t h a s moved about t h r e e .w a v e l e n g t h u l t r a v i o l e t l i g h t . 0 m l o f t h i s S t a n d a r d s o l u t i o n t o a 200-ml v o l u m e t r i c f l a s k . s p o t 10-1-11 p o r t i o n s of t h e t e s t s o l u t i o n . On a s u i t a b l e t h i n . d r a g e n d o r f f s p r a y (Location under u l t r a v i o l e t l i g h t ) .MEBENDAZOLE 313 V i s u a l i z i n g a g e n t : I o d i n e vapour. and t h e d i l u t e d S t a n d a r d s o l u t i o n . Allow t h e s p o t s t o d r y .

p r e s s u r e l i q u i d chromatography pump. A f t e r t h e spikedplasma was p a sse d through t h e c a r t r i d g e . Queensferry Clwyd. 6 mm X 5 cm) was packed with S p h er i s o r b 5 ). The e l ec t r o ch e m i c a l d e t e c t o r equipped with a r o t a t i n g d i s c working e l e c t r o d e was u se d . 6 m l of methanol e l u t e d t h e f i v e compounds from c a r t r i d g e . 0 phosphate b u f f e r .314 A. A.).K. 5 m l o f 40% methanol i n water and 0. a t a flow r a t e of 2. The methods elimin a t e t h e need f o r s o l v e n t e x t r a c t i o n a s such. 0 . The mobile phase c o n s i s t e d of 20% 2-propanol i n a pH 7 . AL-BADR AND M. 6 m l o f methanol was ev a p o r a t ed t o d r y n e ss i n .TARIQ i n t e r n a l s t a n d a r d by high-performance l i q u i d chromatography w ith e le c tr o c h e m ic a l detector i s described (40).5 ml/min. methyl 5fa-hydroxybenzyl) -2-benzimidazole carbamate.rm ODS (Phase Sep.(a-hydroxybenzyl) -benzimidazole and w i t h 5 . it was washed with 20 m l o f d i s t i l l e d w a t e r . g r a d i e n t e l u t i o n . - Allan e t a 1 (41) r e p o r t e d two h ig h performance l i q u i d chromatographic methods f o r d et er m i n a t i o n o f mebendazole and i t s m e t a b o l i t e s i n human plasma u sin g a r a p i d Sep Pak C18 e x t r a c t i o n . The l i m i t of d e t e c t i o n f o r mebendazole and hydroxymebendazole was 5 and 2.30 pg. The chromatographic c o n d i t i o n s : The column ( 4 . Samples were a d j u s t e d t o pH 6 with d i l u t e h y d r o c h l o r i c a c i d o r sodium c a r b o n a te s o l u t i o n and e x t r a c t e d by p a s s i n g through a Sep Pak C18 c a r t r i d g e f i t t e d t o a l u e r lock g l a s s s y r i n g e . One method i n c l u d e s i s o c r a t i c e l u t i o n and o t h e r . t h i s 1 . 2-amino-5-benzoyl-benzimidazole and 2amino-5. 0 pg o f ethyl-5-benzoylbenzimid a z o l e carbamate as i n t e r n a l s t a n d a r d i n t h e range o f 10 ng . The n e x t 1 . The e l u e n t was d e l i v e r e d by Kipp 9208 h i g h . U. The g r a d i e n t e l u t i o n system p r o v i d es s u p e r i o r r e s o l u t i o n s Plasma samples ( 5 ml) was s p i k e w i t h major m e t a b o l i t e s o f mebendazole.5 ng/ml r e s p e c t i v e 1y _- .4 m l of methanol.

6 mm I.D.D. 150 1-11 were i n j e c t e d . . The c o n t e n t s were c e n t r i f u g e d and aqueous phase was a s p i r a t e d and t h e chloroform l a y e r was evaporated under reduced p r e s s u r e . 2 M 1 plasma o f t h e p a t i e n t s was a l k a l i n i z e d w i t h 8 ml of sodium c a r b o n a t e b u f f e r (0.05 M. Flow r a t e Pressure : 1 . 1200-1300 p . USA) equipped w i t h f i x e d wavelength d e t e c t o r (254 nm.) LiChrosorb. 8-ul flow c e l l ) . USA) Model 3380 A i n t e g r a t o r r e c o r d e r . 5 (55:45 pump B) . a Rheodyne (Berkeley. RP-8 10-ym reversed-phase packing. s . The i n s t r u m e n t used was A l t e x Model 322 MP HPLC ( A l t e x S c i e n t i f i c . CA. 2 mm I.05 M pH 5 . 7 ml/min. A n a l y s i s u s i n g Waters Model M6000A High-pressure Liquid Chromatograph equipped w i t h Wisp Model 710 i n j e c t o r and a Perkin-Elmer W d e t e c t o r Model LC55.3) and was e x t r a c t e d with 10 m l o f chloroform by shaking i n a g l a s s t u b e f o r 15 m i n u t e s . Mobile phase : Methanol-water (55:45 pump A) and methanol-aqueous ammonium phosphate 0. pH 11. USA) Model 7120 i n j e c t o r and Hewlett-Packard (Avondale. i . For mebendazole t h e d e t e r m i n a t i o n limits a r e 20 ng/ml ( i s o c r a t i c system) and 10 ng/ml (gradient system). PA. A l i q u o t e s of 20 1~1were i n j e c t e d i n t o HPLC system. A precolumn (35 x 3 .MEBENDAZOLE 315 p o i n t e d and t h e r e s i d u e was d i s s o l v e d i n 100 1-11 DMSO. CA.) dry packed with C o r a s i l C18 was a l s o used. -- K a r l a g a n i s e t a 1 (42) developed an HPLC method f o r t h e d e t e r m i n a t i o n o f mebendazole i n b i o l o g i c a l samples. Chromatographic C o n d i t i o n s : Column (250 x 4 . Berkeley. The r e s i d u e was d i s s o l v e d i n 200 1~1mobile phase.

pH 6 .05 M potassium hydrogen phosphate b u f f e r (pH 7. 80. d .e x t r a c t e d w i t h 6 m l o f petroleum e t h e r and c e n t r i f u g e d . t h e r e s i d u e was d i s s o l v e d i n 0. by volume) . A. d e t e c t i o n r a t e i n t h e range o f 6. The s o l v e n t was d r i e d and d i s s o l v e d i n 60 ~1 of e t h y l acetate and 20 p1 was i n j e c t e d i n chromatograph (Model ALC 202/204 Waters Associates) f o r analysis. s u p e r n a t e n t was d i s c a r d e d t h e aqueous f r a c t i o n was a l k a l i n i z e d w i t h 2 m l 0 .5/0. 200 and 400 ng of mebendazole.A. TARIQ 316 Chromatographic Condi tions: Column : 25 cm x 3 mm ( i . AL-BADR AND M. 5 . r a p i d and s p e c i f i c HPLC method €or q u a n t i t a t i v e d e t e r m i n a t i o n of mebendazole i n plasma.0) and e x t r a c t e d w i t h 7 m l of e t h y l a c e t a t e . C a l i b r a t i o n curve was pr epar ed u s i n g 4 0 . The o r g a n i c l a y e r was removed and d r i e d . 1 Mg o f c ic lobe nda z ole was used as a i n t e r n a l s t a n d a r d . .001 N HC1 and r e . ) LiChrosorb S1 60 5 m . Alton -e t a1 (43) developed a simple. 2 M 1 o f plasma was d i l u t e d w i t h 2 n i l o f 0.02 a b s o r p t i o n u n i t s full scale). 8 ml/min Pressure : 400 p s i Column t e mpe ra ture : Ambient De t e c tor wavelength : 307 nm Recorder : 5 mV (0.1. Flow r a t e : 0 . Mobile phase : Acetonitrile/water-saturated ch 1o r o f orm/2 5% (weight / volume) ammonia i n water (75/92. 0 1 N NaOH and e x t r a c t e d t w i c e with e t h y l acetate.117 ng/ml.

The benzimidazoles were d i s s o l v e d i n p u r e formic a c i d (5 ml) t h e n i n c o n c e n t r a t e d h y d r o c h l o r i c a c i d (5 ml) and i n 50% (%) aqueous e t h a n o l (90 m l ) .05 M KH2P04-NaOH b u f f e r (pH 6 . Flubendazole was used as i n t e r n a l s t a n d a r d .15 ug/ml f o r e a c h sample. ii. and C were used.d . Flow r a t e : 2 . 5 ml/min Pressure : 2500 p s i Column temperature : Ambient Detector wavelength : UV (313 nm). B . Mixture C was e l u t e d . ) reversed-phase .Normal p a r t i t i o n chromatography w i t h isocratic elution. Mixtures A and C c o n t a i n mebendazole. Mobile phase : 0.a c e t o n i t r i l e ( 7 3 : 2 7 v/v). Three d i f f e r e n t m i x t u r e s A .p h a s e g r a d i e n t e l u t i o n with t h e solvent containing aceton i t r i l e .MEBENDAZOLE 317 Chromatographic C o n d i t i o n s : Column : u Bondapak C-18 300 x 3 .aqueous 1%HzS04 w i t h t h e programme: 10% a c e t o n i t r i l e f o r 2 minutes t h e n from 10% a c e t o n i t r i l e t o 30% a t a r a t e of 5% p e r minute. The s o l u t i o n s A and B were s e p a r a t e d by r e v e r s e d . i n t h e q u a n t i t y o f 0. The procedure i n v o l v e s two d i f f e r e n t chromatography systems: i- Reversed-phase g r a d i e n t . A simple and r a p i d method t o d e t e c t t h e presence of d i f f e r e n t benzimidazole compounds i n drug p r e p a r a t i o n s have been r e p o r t e d ( 4 4 ) . 0 mm ( i . T h i s method can be used t o measure plasma mebendazole l e v e l as low as 10 ng/ml. 0 ) .

Flow r a t e : 80 m l h .d .l De te c t or sensitivity : 0 . 7 mm i . 5 AUFS Column press ure De te c t or wavelength - 500 p s i : (UV) 254 nm This method would be u s e f u l i n t h e q u a l i t y c o n t r o l of raw m a t e r i a l s i n t h e q u a n t i f i c a t i o n o f benzimidazoles i n f o o d s t u f f s of animal o r i g i n . 5 mg/ml) was added t o each .7 mm i .20 mg of mebendazole was a c c u r a t e l y measured o u t €or each d e t e r m i n a t i o n . AL-BADR AND M. I n i t i a l l y f o r d e t e r m i n a t i o n of r e t e n t i o n time each of t h e benzimidazoleswas s e p a r a t e l y chromatographed.A. A. Column LiChrosorb RP 8 . e q u i v a l e n t t o about 10 . ) Mobile phase : Methylene c h l o r i d e + i s o propyl a l c o h o l (90 + 10) a c e t o n i t r i l e (5O:SO). A simpl e . d . and i n t h e d e t e c t i o n of residues.microp a r t i c u l a t e (15 cm 4 . 20 T a b l e t s were t a k e n and ground i n f i n e powder.a c e t o n i t r i l e ( 5 0 : 5 0 ) . TARIQ 318 i s o c r a t i c a l l y on a amino phase chemically bonded t o s i l i c a g e l (Type NH2) w i t h t h e s o l v e n t : methylene c h l o r i d e + i s o p r o p y l a l c o h o l (90 + 10) . a c c u r a t e and r e p r o d u c i b l e HPLC method h a s been d e s c r i b e d (45) f o r deter minat i o n of mebendazole i n t a b l e t s . The powder was e x t r a c t e d wi th 5 m l o f formic a c i d . ) LiChrosorb NH2 (Merck) (15 cm x 4. Chromatographic Condit ions: Chromatograph : Varian LC 8500 HPLC w i t h UV d e t e c t o r . 5 M 1 o f s a l i c y l a mide s o l u t i o n ( 8 .

5 % formic acid (30:60). 50 o r 100 mg/kg. A t 1 2 . Behm et a1 (47) have measured t h e c o n c e n t r a t i o n o f mebendazole and i t s major m e t a b o l i t e s by HPLC i n s h e e p plasma a t 12. Flow r a t e : 1 . Chromatographic C o n d i t i o n s : Chromatograph: Waters A s s o c i a t e s Model 440 Column u : Bondapak C-18 Mobile phase : T e t r a h y d r o f u r a n . 25. Two major m e t a b o l i t e s were d e t e c t e d . After o r a l a d m i n i s t r a t i o n o f t h e p r o d r u g . 5 mg/kg t h e peak plasma c o n c e n t r a t i o n s occured between n i n e and 24 h o u r s f o r a l l dose r a t e s and d e c l i n e r a p i d l y . Pressure : 1500 p s i . was more t h a n twice t h a t o b t a i n e d a f t e r g i v i n g rats an equimolar amount o f mebendazole.15%.5. t h e prodrug was r a p i d l y c o n v e r t e d t o mebendazole and t h e area under t h e blood level vs time c u r v e o f mebendazole. and t h e i r known o r expected m e t a b o l i t e s and d e g r a d a t i o n p r o d u c t s i n aqueous media and r a t blood was developed ( 4 6 ) . mebendazole and known m e t a b o l i t e s o f mebendazole were d e t e c t e d i n r a t s g i v e n t h e prodrug. Temperature : 25-28OC.0 . i t s prodrug. t h e i r . Only t h e p r o d r u g .58 .100.MEBENDAZOLE 319 e x t r a c t as i n t e r n a l s t a n d a r d . i n rats dosed w i t h p r o d r u g . 9 ml/min. 4-arnin0-3-(3~-methoxycarbonyl 2 -thioureido)benzophenone . An HPLC method f o r t h e e s t i m a t i o n mebendazole. Detect o r wavelength : (UV) 254 nm The r e c o v e r y was between 99. The m i x t u r e was c e n t r i f u g e d and t h e s u p e r n a t e n t was used f o r HPLC d e t e r m i n a t i o n .

A 20 m l of t h i s s o l u t i o n (corresponding t o 0 . TARIQ 320 c o n c e n t r a t i o n s exceeded t h a t o f mebendazole a t a l l dose r a t e s . 0 .5 P o l a r o g r ap h i c Method P i n z a u t i -e t a1 (48) d e s c r i b e d a d i r e c t . A. 4 mg o f mebendazole) was t r a n s f e r r e d i n t o p o l a r o g r a p h i c c e l l (Metrohm E506 r e c o r d i n g polarograph equipped w i t h polarography s t a n d ) . Mebendazole was converted t o methyl [5-[4-(2-aminoethyl) ben zoy 11-1H-ben zimidazol . 8. A q u a n t i t y o f t h e powder e q u i v a l e n t t o 50 mg o f mebendazole was t r a n s f e r r e d t o 100 m l v o l u m e t r i c f l a s k .A. and e s t a b l i s h e d t h e optimum c o n d i t i o n s f o r t h e d ete r m in a tio n o f t h i s drug i n dosage form. 3 m l o f 1 M p e r c h l o r i c a c i d was added and t h e s o l u t i o n d i l u t e d t o volume w ith t h e McIlvaine b u f f e r pH 2 . AL-BADR AND M.2 M sodium phosphate with 17. S i x t a b l e t s o f mebendazole (about 0 . 8.18 v o l . Deaeration and polarography were performed on u s i n g Metrohm EA 290 hanging mercury drop system. 3 g) were ground. 6 (2.c u r r e n t p o l a r o g r ap h i c r ed u c t i o n method o f mebendazole a t t h e dropping-mercury e l e c t r o d e . 0. A c a l i b r a t i o n curve was p r e p a r e d u s i n g 5 t o 50 pg/ml mebendazole s o l u t i o n p r e p a r ed i n McIlvaine b u f f e r .6 Radioimmunoassay Method M i c h i e l s -e t a 1 (49) developed an e x tr e m e ly h i g h l y s p e c i f i c radioimmunoassay procedure f o r t h e d e t e r m i n at i o n o f mebendazole i n plasma.2.6 m l o f 70% p e r c h l o r i c a c i d was added and t h e m ix tu r e s t i r r e d f o r 10 minutes and d i l u t e d t o volume w i t h water. . The suspension was f i l t e r e d under s u c t i o n through a f i n e p o r s i t y g l a s s c r u c i b l e .82 v o l .y l ] carbamate T h is hapten was coupled t o bovine serum albumin u s i n g a water s o l u b l e carbodiimide as f o llo w s: . The number of e l e c t r o n s involved i n t h e r e d u c t i o n was c a l c u l a t e d a t a mebendazole c o n c e n t r a t i o n o f 1 x 10-4 M u s i n g a s o l i d s t a t e c o n t r o l p o t e n t i a l c o u l o m e t r i c a p p a r a t u s . To t h i s powder 8.b e performed with a l i m i t of d e t e c t i o n 100 ng/ml ( 4 8 ) . u s i n g t h i s method mebendazole a n a l y s i s c o u l d . 1 M c i t r i c a c i d ) . 2 M1 p o r t i o n of t h e f i l t r a t e was t r a n s f e r r e d t o a 50 m l volumetric f l a s k .

. 0 II BSA-C-HN-CH2-CH2 Synthesis of the hapten and the mebendazole-protein conjugate used f o r the immunization of the rabbits (49). Bovine Serum Albumin (BSA). H 0 I. 1 N a!a7 H H 2 NCH2CH2 0 It NH-C-0-CH3 1.MEBEND AZOLE 321 ~ T NHH - 0 0 ICI -0- C H ~ II CH3-C-NH-CH2CH2 N HC1. DMF/MeOH carbodi imide /HC1 2 .

f o r h i s t e c h n i c a l a s s i s t a n c e and Mr. A. t h e r a b b i t serum bound s p e c i f i c a l l y n e a r l y 50% o f added 0 .b o d i e s .322 A. T a n v i r A . Unmetabolized mebendazole was measured by d i r e c t radioimmunoassay u s in g a n t i . TARIQ The r e s u l t i n g co n j cg a t e was used t o immunize r a b b i t s according t o co n v e n t i o n a l p r o c e d u r e s. . AL-BADR AND M. Syed R a f a t u l l a h . Acknowledeements The a u t h o r s would l i k e t o Thank M r . Blood was c o l l e c t e d on EDTA and plasma was o b ta in e d a f t e r c e n t r i f u g a t i o n of t h e blood a t 2200 g f o r 15 minutes. Butt f o r h i s s e c r e t a r i a l s e r v i c e s . Under t h e s t a n d a r d i z e d a s s a y c o n d i t i o n . M et ab o l i t es d i d n o t i n t e r f e r e with t h e binding of parent drug t o antibodies. Un sp e c if ic a d s o r p t i o n t o plasma c o n s t i t u e n t s d i d n o t exceed 2 % . The method is q u i t e s e n s i t i v e t o a s s a y mebendazole i n plasma a t c o n c e n t r a t i o n as low a s 100 picogram/ml. 2 ng H3-Mebendazole a t 1/100 serum d i l u t i o n . A s s i s t a n t Researcher. The a n t i serum e l i c i t e d i n t h i s way was t e s t e d f o r i t s a b i l i t y t o b i n d s p e c i f i c a l l y mebendazole.

K . Goodman and G i l l m a n ' s . N e w York. Ann. J . page 98 (1982).M. 8.G. "Annual Drug Data Report". "Textbook o f Pharmacology".. The E x t r a Pharmacopoeia. . page 1018 (1975).A. B . . page 137 (1979). J . Med. page 1182 (1980). 2. C h a v a r r i a . Med. " D i c t i o n e r y o f Organic Compoundsrr. Wolfe and J . - 4.. 91. I n c . 1408 (1974). 1412 (1974). Wagner and A. Wershing. N . E a s t o n . page 747 (1976). Krupp. 6 t h Ed. M . 970 (1973). 2. 15. J . " I s o l a t i o n and I d e n t i f i c a t i o n o f Drugs". 6. De N o l l i n and H . . London. 59. J .637 (1971) through CA 74: 1000475 (1971). 14. Merck 6 Co.D. 2nd Ed. page 1056 (1975). page 3644 (1982). U. Inc. The 10. 9 t h Ed.. M i l l e r . P e n n s y l v a l n i a . O f f e n . A . L i t t l e and C . . Vanden.MEBENDAZOLE 323 References 1. "Remington's Pharmaceutical Sciences". . J . 7. M. . 151 (1974). 9.277 (1974) through CA 81: 25669K (1974). Volume I V Y Champan and Hall Ltd. J . P a r a s i t o l . Rahway. 230. Barcelona. J . Osol .D. 1 5 t h Ed. . . M a r t i n d a l e . JAMA.350. Murdoch. JAMA. . Vol. Keystone.029. 3. London. 5 t h Ed.S. M . Oxford. 11. "The Merck Indextr. 13. and J . M. page 374 (1980). Bowman and M . Mac M i l l a n P u b l i s h i n g Co.. 1. C l a r k e . S a n t o s . I n t e r n . . 23. S . E. I. Trop. R .230. Ger. London. P u b l i s h e r s . 11. Brit. Pharmaceutical Press. Blackwell S c i e n t i f i c P u b l i c a t i o n s . Rand. The Pharmacological B a s i s o f T h e r a p e u t i c s " .S. 582 (1979). Am.P.C.C. 28th Ed. 12. Mack P u b l i s h i n g Co. The Pharmaceutical Press. . - 5. S . USA. Vol. 11* E. Spain. . W. .

600 (1975). J.R. 23. Watson. J . TARIQ 16.G. 14. J . Levin. Pharmacokinet. R a r i t a n New J e r s e y .A. Med. Munst. C l i n . AL-BADR AND M. Allan and T. Allan and T. 25.. 1356 (1980). "Basic and C l i n i c a l Pharmacology". 118 (1977). G . Kale. 8.H. J . J . Eur. J . Hyg. D. J . P . Schuermans and H . Pharmacol. Watson. Pharmacokinet. 26. Tro. Allan and T. Tro.. Pharmac. Pharmacol.. Miskovitz and N . 0.. Am.R. Pharmacol. 26. Hz. L . 27. B r a i t h w a i t e . 20. Morris and S. - 21. J . O f f i c i a l Product Monograph.A.R. . 28. P. C a l i f o r n i a . M. Drug Metab. 31. Rawlins.. I. Am. V . 18. 8.249. C . J . Palmer. R . C l i n . J . J . R . "Pharmacokinetics" Marcel Dekker. Watson. L. Karlaganis and J . Toxicol. P . C l i n . J . W. Middle E a s t E d i t i o n . 175 (1980). P e r r i e r . JAMA.. Pharmac. Med. 175 J a v i t t . . L. R . 2 2 . V a n p a r i j s . 2929 (1983). 19. Brugmans. Drug Me'tab. B i r c h e r . Eur. 2 1 7 .E.. 17. B r . M. Otho Pharmac. 608 (1982). 30. Am. 24. 8 (1975) . G . B r . 11 J u n e . B.S.0. C o l i s t e .F. l J 375 . . 313 (1971). 453 (1982). Dawson.. E u r . C h a v a r r i a . J . G i b a l d i and D. V . (1982).. J . (1980).324 A. J . Roberts. J . Watson. 161 (1982). L . J . O. . Eur.F. 91 (1975). Med. 7.V. 29. Corp.. 29. M. R i t s c h e l and G . A. JAMA.D. Med. P. R . D . New York (1975). Gould. Allan and T. 131 (1982). C l i n . M. 285. Eur. Vermox T a b l e t s .P. M. 373 (1983). Theinpont. R . 18. Tro. 24. Lange Medical P u b l i c a t i o n s . Katzung. B e r t i l s s o n and L . I n t . 22.R. Hammer. Wijngaarden. C l i n . A. Lauwers.

Da-Peng Wang and Hsin-Shang. 43. C. Drug M o n i t o r .P. - 44. Chim. Pharm. J . . R. 154. A. J .R. Acta. 66. A . [ I n t Symp. F. C . 36. A l t o n . De Moerloose. Hashim and F. E . Res.. G . J o u r n a l o f Chromatography. Anal.A. 333 (1981). Patel. 8 . 2 . Drug Metab. Gandhi. Pharm. P a t e l . T a l a n t a . S c i . Rana. 20 (1978). Assoc. P . United S t a t e Pharmacopeia USP XX. P . Page 335 (1981). 34. P a t r i c k and J . Anal. De Moerloose “Biolumin Chemilumin . Patel. J . 37 (1983). . V e t . O o s t e r h u i s . P. . . - Patel.325 MEBENDAZOLE 32.T. K a r l a g a n i s .V. Appl . and Chromatogr. 161 (1983). Chem. I n d i a n J . A b d e l . Wetsteyn and C .A.. Chemilumin] . I n d i a n Drugs. 25. 716 (1978). B i r c h e r . High Resolu. E u r . 40. C . 35. T. M . 42. Chromatogr.P. 141 (1979).A. 40. J . O f f .M. Therap. J . A n a l y s t . 41. P a t e l . P a t e l and V . 39.R. McGuire. Lo. Owen. B. Watson. Baeyens. W. J . 37. S c i . L . Mourot. 880 (1979). 18 D . F. A l l a n . 311 (1980). R . Acta. N . J . B. G . B . Bryant. I n d i a n J .R. W . 183. 47. 38. Gayot. Abdel F a t t a h . J. H. 46. page 465 (1980). G . J . Behm. J . A . T.M. Onsy. B o i s s e a u and G . 329 (1983). Watson. 33. Pharmacokinet. Underwood. V a n B o x t e l . 109.A. J . R. 215 (1984).R. C . 76 (1978). Dave and M. . Gandhi.68.M. Goodman and T. (1984). R . 6 . F. Wahbi and S . Commun. Munst and J . P a t e l and V .T. Biolumin. A . Anal.F a t t a h . 351 (1983). Anal. K . 99. T. . 371 (1978).R. Dawson. A . S c i . 34. 669 45. Chim. C o r n i s h and C . 40. Pharm. Baeyens and P .

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U n i v e r s i t y o f Milan 1.5 Formula and Molecular Weight 1.METOCLOPRAMIDE HYDROCHLORIDE Davide P i t r g and R i c c a r d o S t r a d i F a c u l t y o f Pharmacy.2 Chemical names 1.1 I n f r a r e d Spectrum 2.6 Appearance.1 Nomenclature 1.4 Trade names 1. Physical Properties 2.2 Nuclear Magnetic Resonance S p e c t r a ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 16 321 Copyright 01987 by Academic Press. . Odor and Taste 2. Color.3 G e n e r i c names 1. Description 1. All rights of reproduction in any form reserved. Inc.

5 Ultraviolet Spectrophotometric Analysis 6.DAVIDE PITRe AND RICCARDO STRADI 328 2.8 2.2 Nonaqueous Titration 6.4 Colorimetric Analysis 6.12 3.8 Thin-Layer Chromatography 6. Metabolism and Pharmacokinetics 5.2 2.3 Complexometric Analysis 6. Stability 5.11 2.6 2.2.4 2.9 2.3 2.6 Fluorimetric Analysis 6. Methods of Analysis Elementa1 6.9 High Performance Thin-Layer Chromatography 6.3 Acute Toxicity 6.2 Pharmacokinetics 5. References .7 2. Powder Diffraction Crystal Structure Melting Range Solubility PKa PH Partition Coefficient Manufacturing Procedures Synthesis 3.12 7.10 Gas Liquid Chromatography 6.5 2. Determination in Body Fluids and Tissues 8.7 Paper Chromatography 6. 'H-NMR 13 C-NMR Ultraviolet Spectrum Mass Spectrum Fluorescence Spectrum X-Ray.2.11 High Performance Liquid Chromatography 6.1 Identification Tests 6.1 Metabolism 5.1 4.1 2.10 2.

Gifuken-Japan) Primperan (Delagrange-Paris) Reglan (Robins.U.4 Trade Names Cerucal (Arzneimittelwerk-Dresden) Elieten (Nippon Kayaku.IX Metoclopramidhydrochloride DAC 1979 Metoclopramidum hydrochloricum 2.2 Chemical Names 329 Benzamide. rnonohydrate CAS 54143-57-6 anhydrous CAS 7831-21-5 4-Amino-5-chloro-N-[2(diethylamino)ethyllo-anisasamide 1. USA) .3 Generic Names Metoclopramide Hydrochloride USAN-BP 1980-F. Description 1.1 Nomenclature 1. Richmond.AB-DDR 1.METOCLOPRAMIDE HYDROCHLORIDE 1. Tokyo-Japan) Emperal (Neofarma-Helsinki) Maxeran ( Delagrange-Paris) Plasil ( Lepetit-Milan) Peraprin (Taiyo. 4-amino-5-chloro-N-[2-(diethylamino) ethyl 3-2-methoxy. monohydrochloride.

CH-2 CH2-N OCH3 . H C l .Wt. = 354. odorless (1) . H 0 1 4 22 3 2 2 Mol. Odor.HCI * H20 \ c2 CI NHp C 1.DAVIDE PITRh AND RICCARDO STRADI 330 1. C o l o r .6 H C1N 0 .3 Appearance. Taste A w h i t e o r a l m o s t w h i t e c r y s t a l l i n e powder.5 Formula and Molecular Weight C0 NH I .

3400. 2500. -1 The f o l l o w i n g bands (cm ) were a s s i g n e d and a r e r e p o r t e d i n Table 1. 3300.1 I n f r a r e d Spectrum The i n f r a r e d s p e c t r u m of rnetocloprarnide hydrochlor i d e .33 1 METOCLOPRAMIDE HYDROCHLORIDE 2. 3460 VNH 2860. 2980 VC H SP3 3030 VC H SP2 2100. 2660. The spectrum was r e c o r d e d w i t h a s o l i d sample d i s c composed of 1 m g o f compound/200 m g KBr on a P e r k i n E l m e r Model 1310. Table 1 Wave Number Assignments . Physical Properties 2. i s p r e s e n t e d i n F i g . 2710 v N+H 1600 v c=o 1540 6NH 1270 v C-0-C 700 v c-c1 VOH (Amide) (Asymmetric) . 3200. 3340. 2950. 1.

Fig. 1 - I n f r a r e d Spectrum of Metocloprarnide Hydrochloride .

0-3. 2 . The chemical s h i f t s a r e l i s t e d i n t h e Table 2. CONH 10.exch.72 9 2 NH-CH -2 3.exch.exch.33 t 6 N-(CH2-CH -3 ) 3.2 Nuclear Magnetic Resonance S p e c t r a 2.03 broad s 2 .95 S 3 OCH 6.44 t 1.333 METOCLOPRAMIDE HYDROCHLORIDE 2.2.72 S 1 H (arorn.2 m 6 CH -N-(CH -2 -2 -CH 3. Table 2 Moltepli city Number of protons Assignments ~ 1.) 8.) 7. shown i n F i g .60 s 1 H~ (arom.85 broad s 1. was obtained i n DMSO-d with a Varian spec6 trometer EM-390 o p e r a t i n g a t 90 MHz. NH 6. hH 3 2 6 2 ) 3 2 .1 1 H-NMR 1 H-NMR spectrum o f metoclopramide h y d r o c h l o r i d e .

Fig. 2 - 1 H-NMR (90 MHz) Spectrum of Metocloprarnide h y d r o c h l o r i d e i n DMSO-d 6 .

18 S C 8 110.46 S 2. 4 .07 S 12 167.89 q OCH 6 98.CH 5 55.763 10 m/l) was o b t a i n e d (60) with a Beckman Mod.p.391 MHz.27 t N(CH -CH ) -2 3 2 4 51.15 t 3 48.3 CH -N(C2H512 -2 -2 1 5 3 (aromatic) or C or C 5 1 ‘6 C 4 c2 c=o U l t r a v i o l e t Spectrum The u l t r a v i o l e t spectrum of metoclopramide hydro-5 c h l o r i d e i n w a t e r ( c = 5. 24 s p e c t r o p h o t o m e t e r and i t is shown i n Fig.m) Mu1 t i p 1i c i t y Assignments N.38 q 2 35. 3.40 t NH.335 METOCLOPRAMIDE HYDROCHLORIDE 2.2 13 C-NMR 13 The C-NMR spectrum o f metoclopramide hydrochlor i d e i s shown i n F i g .00 d c3 7 109.61 S C 9 131.43 d 10 148. N(CH2.CH ) -3 2 1 8.2.57 S 11 158. Table 3 Line (p. The chemical s h i f t s a r e l i s t e d i n Table 3. The spectrum was r e c o r d e d i n D 0 2 w i t h Varian XL.200 s p e c t r o m e t e r o p e r a t i n g a t 50.

i' . 3 jl I : i i I h 13 H-NMR (90 MHz) Spectrum of Metoclopramide hydrochloride in DMSO-d 6 .i I 8 - - . . ' ! 1 Fig.

4 ! - . Ultraviolet Spectrum of Metoclopramide hydrochloride in Water 331 .METOCLOPRAMIDE HYDROCHLORIDE i Fig.

4 The mass spectrum of metoclopramide h y d r o c h l o r i d e was recorded ( F i g .23 227 0.66 141 1.96 86 100 + c1QCH3NH2 4fCH3 J$ro c1 0 II NH2 c1 -0"' NH2 c1 CH +/CZHS -N 2.43 169 0.\C2H5 . 5 ) on Finnigan 1020 s p e c t r o m e t e r by c o w e n t i o n a l e l e c t r o n impact i o n i s a t i o n a t 70 eV.78 99 27.DAVIDE PITRG AND RICCARDO STRADI 338 Mass Spectrum 2. Prominent fragments and t h e i r r e l a t i v e i n t e n s i t y a r e shown i n Table 4.60 181 6.90 Attribution ~ + 201 7. Table 4 m/e Intensity 229 0.

cL.tI3.oo t M n : SIFWI6 172 2128 B P X M'E: P6 PIC: 167616. 50. 1W.w.'86 9zs7.H22.cL. 5 281 - 2Qe""'"- 285 ~ " 26 " 29s " ' Electron impart Mass Spectrum of Monocloprarnide hydrochloride m .02. 96 367616.Q 10. WREi Cll.1 99 WE mss S P E C T M Wrn.02.METOCLOPRAMIDE HYDROCHLORIDE 339 Ell% N.1 se.E: DulR: STRW16 (72 '108 t 2128 RlCi n. I 270 i' 272 I M Fig. 173712. 188.

6. The emission spectrum of the compound consists of a single peak at 355 nm. The spectra obtained in water (5 mcg/ml). The X-ray diffraction data are given in Table 5. it shows a sharp peak at 353 nm and a second peak at about 273-276 nm. When excited at 353 nm.6 The X-ray powder diffraction data of metoclopramide hydrochloride was determined (2) by a Philips Powder Diffractometer PW 1710 with nichel-filtered copper radiation (1. 40 KV.are given in Fig. SLITS: lo-0. TIME CONSTANT: 1".54051 A) with the following instrumental conditions: TUBE: BF type. 40 mA.5 A solution of metoclopramide hydrochloride in water exhibits fluorescence when excited with ultraviolet light.008°xl'1. X-Ray Powder Diffraction 2. DETE TOR:PW1711 proportional counter + discriminator. 2 . SCANNING SPEED: 0. SPECIMEN HOLDER: Niskanen. on a spectrophotometer Perkin-Elmer MPE 44A.1 mm-lo.DAVIDE PITRB AND RICCARDO STRADI 340 Fluorescence Spectrum 2. CU/Ni. SCALE: 2x10 cps. PAPER SPEED 1 cmxlo.

METOCLOPRAMIDE HYDROCHLORIDE Fig. 6 - F l u o r e s c e n c e Spectrum of Metoclopramide hydrochloride i n water 341 .

33 2.46 7.54051/2sin diam.01 1.31 2.16 2. d(A)* 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 9.26 2.99 2.47 3.28 6.26 5.48 4.03 3.86 2.18 4.02 5.64 6.04 2.81 1.23 4.29 2.71 1.74 2.78 2.41 3.87 4.82 3.71 4.36 3.95 2.51 2.71 362 3.15 3. ** Relative intensities are based on highest intensity of 100 .05 2.63 12 8 6 11 12 10 20 13 8 21 11 10 9 13 18 10 8 10 11 11 10 8 9 8 8 7 9 PLUS OTHER LINES c5 Interplanar distances = 1.82 2.95 3.53 2.69 2.92 2.43 2.07 3.DAVIDE PITRB AND RICCARDO STRADI 342 Table 5 N.23 8. 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 50 51 52 3.32 I&** 5 25 20 22 13 65 8 20 10 38 45 30 31 10 20 20 6 47 35 17 10 58 30 75 100 11 I&*+ N.78 3.50 3.81 1.27 4.59 2.64 6.14 2.60 4.98 6.

c=7. whereas it is practically insoluble in ether (10). The dihydrochloride monohydrate melts in the range of 148O with decomposition.477 A .60 Dihydrochloride 48 9 6 0. b = 1 8. Table Solubility of metocloprarnide g/100 m l at 25O ~~ Solvent Base Water Ethanol 95 Ethanol Benzene Chloroform 0. Z = 2.9 At 25O metoclopramide monohydrochloride is soluble in 0. (11).METOCLOPRAMIDE HYDROCHLORIDE 343 Crystal Structure 2.10 6.7 The metoclopramide hydrochloride monohydrate is monoclinic.p.02 2. Melting Range 2.13 and Y = 101.8 Employing the USP method the metoclopramide monohydrochloride monohydrate melts at 182-185O(5) and with terma1 microscopy the melting begins at 181O and completes at 183O(6).90 1.055 (3).7 g of water. space group P2 /n with a = 12. The metoclopramide free base ( C A S 364-62-5) is triclinic. p = 81. The melting points f o r metoclopramide free base are about 148O(7) or m. c = 17.553(2). The structure was solved by direct methods and refined to R = 0. For dihydrochloride is also reported an eutectic melting point of 160° with phenacetin and of 117O with benzanilide(7). a = 114.120(4) A and p= 98. 146-148O(8).10 0.90 0.728.138(3). A study of structure-activity relations shows an analogy between metoclopramide and apomorphine(4) . Solubility 2. 3 g of ethanol (96 per cent) and 55 g of chloroform.23O.20. space group P1 with a = 13.06(7) Z = 4 . Solubility of the base and of dihydrochloride are reported in Table (6).10 .310 b = 8.

The determination was 1 carried out spectrometricalfy in aqueous solution and the values are a mean of 16 determinations with a standard deviation of 0.6 and 6.42. 2.03 and 0.71 and pK = 0.10 €5 Metoclopramide hydrochloride shows two ionisation costants.DAVIDE PITRfi AND RICCARDO STRADI 344 2.667 Calculated from R. 2.12 Partition Coefficient The partition coefficient in octanol/water has been determined(l3) by reversed-phase HPLC at 20°.5 according to BP 1980. The hydrochloride gives Experimental LogP = 2.A.11 pH Range The pH of a 10% water solution must be between 4. Deckker (14) LogP = 2.76 .02 (12). pic = 9.

as starting product. In Scheme A. In scheme B the first step in the synthesis is the etherester (VI). a compound whose properties are economically excellent. was found at pH = 7.2 Synthesis 345 Among the synthetic methods leading to metoclopramide (I) it seems suitable to report only the ones of greatest interest that are those using p-aminosalicilic acid (II). investigated at different pH values. Manufacturing Procedures 3. The two main processes are listed in Fig 7. The protection of the amino aromatic group was not considered necessary (16).6 while the maximum degradation was found at pH = 2.METOCLOPRAMIDE HYDROCHLORIDE 3.N-diethylethylendiamine(l7). the acid (1I)is acetylated to (111) and then treated with methyl sulfate to give the ether-ester (IV). . This compound is reacted with (C H ) N-CH CH -NH to the corresponding amide. referring to the first invention(l5). One of the degradation products was identified as N. Stabi1ity Aqueous solution The maximum stability of metoclopramide. 4. The direct chlorination (18) of (VI) with sodium ipochloride gives (VII) which is transformed in excellent yields into (I). The 25 2 2 2 subsequent Zesacetylation leads to metoclopramide ( 1).

Scheme A FOOH FOOCH3 COOCH3 COOCH3 FOOCH3 COOH I 60 b Fig. 7 - Synthesis of Metoclopramide .

R R 1 2 -NHCH CH N(C H ) 2 2 252 -CH -NHCH CH NHC H 2 2 2 5 -C H -NHCH CH NH 2 2 2 -C Hg -OH -CH3 V -NHCH CH N(C H ) 2 2 2 5 2 -H VI -NHCH CH N(C2H512 2 2 -H -NH 2 -CH VIII -NHCH COOH 2 -C H IX -NHCH = CH -CH -NHCH CH NHCOCH 2 2 3 -CH I I1 I11 IV VII X 3 3 3 2 3 3 3 REFERENCES .1 Metabolism The metabolites identified in the in vitro and in vivo studies are listed in Table 7. Table 7 Non conjugated metabolites found CI @ O R 2 NH2 N. Metabolism and Pharmacokinetics 5. in which metoclopramide is indicated as (I).METOCLOPRAMIDE HYDROCHLORIDE 341 5.

7 4.0 1.0) is the main metabolite in human urine.7 65.as N -glucoronide and N sulfonate were identified. (20). dogs.8 6.0. were found metabolites (11) (IV) (V) (VII) (VIII). In particular in rat urine.o 18. % of the administered .0 5. Metabolic products were quantified in rabbit urines (21) (Ii) 4 and (IV) but also unchanged (I). Pharmacokinetic 5. but the main metabolite was found to be unchanged metoclopramide originated from the sulfate and glucuronide. Another product of N-oxidation of 4 (I) was also found. Table 8 14 Excretion of C-metoclopramide in rats.7 0.8 r 0-24 24-48 48-72 10 20 100 urinary excr.348 DAVIDE PITRe AND RICCARDO STRADI By studies of the in vitro metabolism of (I) using hepatic fractions from several animal species (1) (2) five metabolites (11) (111) (IV) (V) (VI) were detected. dose F = F U F 20 1. and humans Dosage p. while in dog urine were present metabolites (V) (VI) (VIII) (IX) (X). After enzimatic hydrolisis was identified metabolite (VIII)(CAS 52702.04. Identification studies of the metabolites in urinary excretes of rat and dog (20) after enzimatic hydrolysis evidenced some compounds already known from the in vitro studies and some new ones corresponding to (VII) (VIII) IX) (X).9 1.3 ---- 77.9 8. Metoclopramide N -sulfonate (CAS 2726042. dogs and humans show that the drug is distributed within a few minutes after oral administration and it is eliminated mainly in the urine of the first 24 hrs.2).6 fecal excr.8 8.2 14 The studies of pharmacokinetics of C-metoclopramide in rats. In human urine the excretion was found to be 60% in the first 24 hours.3 0.5 1.4 1. m g k hours Rats Dogs U = humans U F U 71.

9 . f o r s o l u t i o n s 49.6 p = 165.7-76. Methods of A n a l y s i s 6. 50.v.67 f o r t h e base. 0 2 . 5 . The f o l l o w i n g r e s u l t s were o b t a i n e d : f o r t a b l e t s 57.I/? plasma c l e a r a n c e was 10. The shape o f t h e plasma l e v e l c u r v e i n t h e s e s u b j e c t s f i t s a two-compartment model w i t h a d i s t r i b u t i o n o f T 1 a = 4.0. C 1 . 5.08. Acute T o x i c i t y 5. 7. 7.01. N. 8 3 . 21.2 a) I n f r a r e d Spectroscopic Test BP 1980 (10) c i t e s t h e u s e of t h e i n f r a r e d abs o r p t i o n spectrum o f a potassium c h l o r i d e d i s p e r s i o n o f t h e rnetoclopramide h y d r o c h l o r i d e i n a c c o r dance w i t h t h e r e f e r e n c e s p e c t r u m r e p r o d u c e d i n t h e appendix. v .3 (LD ) of t h e metoclopraThe a c u t e t o x i c i t y i .12.7% and f o r c a p s u l e s 54. 10. 47. H . 0.9 ml/min g ( 2 2 ) ( 2 3 ) . I d e n t i f i c a t i o n Tests 6. N . . 9. 50 mide d i h y d r o c h l o r i d e is 85.8%. C 1 . 20.3%.7 mg/kg f o r t h e r a t and 71 mg/kg f o r t h e mice ( 2 5 ) . 0.1 E l e m e n t a l Composition H . B e r n e r and co-workers ( 2 4 ) r e p o r t e d t h e s t u d y o f b i o a v a i l a b i l i t y of commercial f o r m u l a t i o n s o f metoclopramide i n male h e a l t h y v o l u n t e e r s .40. 1 4 .7 min. N . and a n e l i m i n a t i o n o f T .9.1 f o r t h e 2 monohydrate C .METOCLOPRAMIDE HYDROCHLORIDE 349 The pharriiacokinetic of metoclopramide H C 1 was s t u d i e d i n normal male v o l u n t e e r s a f t e r i.6-80. 1 1 . C 1 . 6. dosage o f 10 mg. H 0 .4. C./2The body min. G. 11. 5 6 . 6 . 1 2 . 8 9 . H .50 f o r t h e a n h y d r o u s form C.

in the range 230 to 350 nm of a 2-cm layer of a 0. is titrated with EDTA using Eriochromo Black T as indicator (26). This is another identification test recommended by B. 6. / d) Crystal Tests - Gold Cyanide solution needles.1980 (10). Lead iodide solution .DAVIDE PITRk AND RICCARDO STRADI 350 b) Ultraviolet Spectroscopic Test The light absorption. 6. / Vitali's test: pale-yellow/light brown (sensitivity : 1.0 ug) (7). C) Color Tests With a solution of 4-dimethylaminobenzaldehyde yellow-orange(1). (7). .001 per cent w/v solution in 0.feathery rosettes (sensitivity: 1 in 1000) (7). absorbance at 273 nm about 0.P. ammonium vanadate test .69.01M hydrochloric acid exhibits two maxima.light brown (sensitivity : 1. at 273 nm and 303 nm.3 Nonaqueous Titration The metocloropramide hydrochloride may be titrated (10) potentiometrically in glacial acetic acid containing mercuric acetate with perchloric acid in glacial acetic acid as titrant.0 ug). amberlite IR 120 in the Zn corresponding to the absorbed metoclopramide. The released quantity.79 and at 309 nm about 0. sometimes in rosettes (sensitivity: 1 in 1000).4 Complessometric Analysis The method is ba ed on the use of a column of 5+ form.

1-11 32 + 500 1-14 33 475 20-120 34 + 495 1-11 35 NH Sulfamate 4 Diazotization + 440 0.4-dihydroxybenzoic acid . They are listed in the following table 9.METOCLOPRAMIDE HYDROCHLORIDE 6. max Bear's law Ref. P-dinitrostilbene 390 -- 29 4 DiazotizationtN(1-naphthyl) 525 2 0. .5 35 1 Colorimetric Analyses The colorimetric assays have been the most widely used especially to determine metoclopramide in pharmaceutical preparations.ug/ml -- 1 Citric acid in Ac 0 600 2-14 27 2 NH4SCN t Co (N03)2 620 -- 28 3 Z a . Table 9 Colorimetric assays of Metoclopramide N.4-4 30-11 ethylendiamine 5 HNO 6 Diazotization to form the nitrous 2 derivative 375 10-60 31 + a-Naphthyl- 510 0.8 36 amine 7 Diazotization 8 Dragendorff's 9 Diazotization 10 R-Salt 2.

8 Paper Chromatography Several chromatographic assays are summarized in the following table 10.DAVIDE PITRB AND RICCARDO STRADI 352 6.ug ml . E(1%. Iodoplatinate spray RfxlOO 45 II 77 II 39 1% of chloranyl in Bz - Ref. The me-1 thod. After the extraction of the base in chloroform. -8 Chromatography 6. . with detection limit of 3x10 .58 Phosphate buffer pH = 7.V.6 Ultraviolet Spectrophotometric Analysis B.7 Fluorimetric Analysis Fluorimetric measurements are carried out in pH=2 buffer solution on the basis of intense emission at 360 nm that occurs when the sample is exci ed at 310 nm. the metoclopramide can be determined by measurement of absorbance at 305 nm.8% and a coefficient of variation of 0.4 Toluene: Methanol : conc.9% (37).6 g of citric acid in 130 ml H 0 and 2 870 ml BuOH Acetate buffer pH = 4.9-1. Table 10 Solvent system 4.8-101. was successfully applied to analyses of pharmaceutical formulations with a recovery of 100. lcm) = 265. NH40H Paper Detection U.P 1980 (10) has described a spectrophotometric assay for the quantitative determination of metoclopramide tablets. 6.

Table 11 Solvent system I I1 III IV V VI VII VIII IX X XI XI1 Plate A.METOCLOPRAMIDE HYDROCHLORIDE 353 Paper: A Whatman N 1. b l o t t i n g and drying a t 25O f o r 1 hour. B Whatman N 1 N 3. C Whatman N 1 - - - 6. s h e e t 11 x 6 cm buffered by dipping i n a 5% s o l u t i o n o f sodium c i t r a t e .B C C C C C C F C C F F R f x 100 40 10 46 16 54 29 55 - 98 68 47 13 Reference .9 Thin-Layer Chromatographx The methods f o r s e p a r a t i o n and d e t e c t i o n o f metoclopramide a r e summirized i n t a b l e 11. s h e e t 17x 19 cm impregnated by dipping i n a 10% s o l u t i o n of t r i b u t y r i n i n acetone and drying i n air.

5:100) I1 Methanol-chloroform (1:4) I11 1. .1N KOH and dried . Analtech (6) Kodak Silice Fluorescente K301 Silica dipped in 0. D.Spray solution: 1% solution sodium nitrite in HC1 N and then with 0. 0. . .88) (70: 15:2) IV n-Butanol-acetic acid-water (4:l:l) V Isopropanol-ammonia solution (sp.88) ( 80:4:5) VI VII VIII IX X Butanol-acetic acid-water (4:l:l) Isopropanol-NH OH-water (80:4:5) 4 Dioxan-NH OH-benzene (8:l:l) 4 Chloroform-methanol-28% ammonia (10:4:1) Chloroform-methanol-dioxane-28% ammonia (90:14:10:3) XI Methanol-ammonia (100:1. F.Spray solution: 2 g of iodic acid dissolved in 10 m l H20 and made up to volume of 100 m l with 90% (w/v) sulfuric acid (5).DAVIDE PITRfi AND RICCARDO STRADI 354 Solvent Sytem I Strong ammonia solution: methanol (1.8 E. C. Plate A.2-Dichloroethane-ethanol-ammonia solution (sp.Spray solution: 0.2% solution of chloranil in acetonitrile followed by heating the plate at 100-105° for 2 min. 20 x 20 cm coated with silica gel Pre-coated silica gel G (Merck) Silica1 gel 60 F (Merck) 254 GF Plate. B.gr.gr.5) XI1 Acetone Detection - Acidified iodoplatinate spray .0.Ultraviolet light (250 nm).4% of N-(1-naphthy1)-ethylaminediammonium dichloride as coupling agent in methanol. Glassplate.

Detector and injection temperature were 350° and 250OC. Retention time: 1. oven 250 and detection 350.K. . 6.87. .10 355 High Performance Thin-Layer Chromatography HPTLC was performed on metoclopramide base using NH OH HPTLC Fertiplatten Kiegelgel 6OF(Merck)and Me0H:CHCl 3: 4 25% (80:85:0. The compound may be quantified by means of excitation fluorimetry at 312 nm and emission at 365 nm integrating the peak area with a videointegrator. Working conditions: glass column (1.2) as solvent. E .1 min for diazepam.METOCLOPRAMIDEHYDROCHLORIDE 6. The procedure involved the extraction of the drug from an alkalinized aqueous solution in benzene and derivatization with heptafluorobutirric anhydride. coated with 80-160 mesh. chromosorb W. The determination of the derivative was done using diazepam as internal standard. with N as carrier gas (50 ml min ) 2 and a flame ionization detector.76 min for the metoclopramide and 9. Tamond.) packed with 3% OV-1 on Chromosorb W-HP (100 to 120 mesh) and operated at -1 15Oo-25O0 (lO°C/min).2 m x 2 inc. Daldrup and co-workers (44) described a GLC assay method on a glass-column (6 ft x 2 inc. Axelson (49)have been determined the mctoclopramide in picogram quantities in plasma 63 with a Ni-electron capture detector.11 Gas Liquid Chromatography T. A mixture of argon-methane (95:5) was employed with a flow -1 rate of 40 ml/min The retention times were 3. J . GLC has been used as the method for the determination of the drug and its 15 metabolites in biological fluids (45) (46) (47) (48)and Y. The results are linear from 160 ug to 20 ug with a standard / / deviation of 2-5% (43). packed 3% OV-17. Injection 2 5 8 O . Internal standard: 2-amino-5-chlorobenzophenone.

Determination of metoclopramide in Body Fluids and Tissues The determination of metoclopramide in biological fluids of man and of various animal species is the object of many communications as reported below: Blood (Plasma.. Block et a1 ( 5 0 ) determined metoclopramide using reverse-phase HPLC with an RP-18 column under isocratic conditi n -4. flow rate 2.2 ml/min 3 2 4 Detection at 308 nm. Flow rate 1.( propylamino ethyl]-Z-me thoxybenz m i de ) W. Elution after 5 min. 7.(l) used a column 15 cm x . .5 min for the internal standard (4-amino-5-chloro-ND. Detect4 ion 280 nm. (30:70:0.5) at room temperature.75:24.5 mm packed with silica gel M 171.12 High-pressure Liquid Chromatography -? Teng et al.9:0.1). (CH 0H:H 0:NH OH conc. Serum) Colorimetry: (11) : ( 3 ) (41) (40) (52) HPLC: (24) TLC GC : (45) (46) (47) (53) (48) HPLC : (54) (55) (20) (50) (53) (56) (57) GC-Mass Spectrometry: (15) Urine Colorimetry : (21) (52) TLC : (21) (40) (39) HPCL : (20) (56) GC : (54) (19) GLC-Mass Spectrometry: (58) Saliva HPLC : ( 59) .0 ml/min and CH OH3 -CHC1 -NH OH conc. The retention times of metoclopramide is 2.8 min and 4.356 DAVIDE PITRB AND RICCARDO STRADI 6.

METOCLOPRAMIDE HYDROCHLORIDE Bile Colorimetry : (21) Feces Radioactivity with 14C-metoclopramide : (20) Microsomal fraction of liver homogenate HPLC : (39) 357 .

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S. Swajber.De Moerloox Analist 103. Sane. Taka. Clin. K. J. 1356 (1978) A. 36. 214 . Sawant Indian Drugs 19.H. 157 (1979) - - - .Emschermann. Abd El-Kader J. P.A. Clin. Z.405 (1979) G. M. Vie1 J. 6508 (1977) D.V. 177. C. Jorndas Argent Toxicol. Bhatkar. Haase. J. C. Kahn. Levillan. 32.R.Pharmaco1. Kahn. E. Balyens. 387 (1983) CA 99-181559q W. 239 (1982) CA 96-223389s D. F. Lacroix. J. Pradego Indian Drugs 20. K. Chromatogr.M.N.Pharmaco1. Davies. Agralo. Beckett. 26. Emmanuel.N. Chudasama Indian Drugs 3. Jarzebinski. Dubois. 227 (1979) O. 73 (1982) CA 98-78232v R. D. B.Pharmaco1. Schuppan. Kamalapurkar. U. J. P. Actas 1st. Calleja Rev. R. I.M. Araldi. G. Shingbal. Heller Arzneim.J. Segura J. 24.CA 79-73800 J. 133 (1981) CA 95-1758 8 9 k J.Pharm. Naik.Kamalapurttar. Mashiter.Y. 12 (1975) CA 80-100249k P. 205 (1983) CA 99-164094a O. 237 (1978) J. Skingbal. A. Emmanuel. 194 (Pub. D. Anal. Naik Indian Durgs 18. Priolkar Indian Drugs 20. H.G. D. 172. Vogtle-Junkert.METOCLOPRAMIDE HYDROCHLORIDE 359 D. (Buenos Aires). Klos Pharm.Forsch. W. Huinzing. 4. Pandit. R.T. C. S. Bateman. 115. Forsch. 5 .Pharm.V. 108 (1982) CA 98-221907b R. 9 . V. A.Farm. Dobrecky. CA 95-121218~ D. 298 (1983) CA 99-10952~ J. J. H.B. Leuschner. 28.S.S.V.J. Nagvenkar East Pharm. 203 (19811. 32 (1976) D. Bateman. 441 (1981) CA 96-24856s O.S. Beiner. Davies Br. 34. Chromatog.D. Okamantar. J. Yogyanaranan East Pharm. 1972) 1.S.D. 401 (1978) G. K. K. Bulgach. Pol. Glaottke. Ambardaker Indian Drugs 20. S. T. Wagener Arzneim.M. 169 (1982) C. N. Belg. A. M. Schmidt.M. Maskiter Br. Segura J. Bakke.

Res.H. 5 . (1984) Y.Sci. Hooper. 3. 109 (1980) G. P. 413 (1981) 45) L. S. Verbic Comput. M. Sci. D. M. Axelson J. Eddie.Pharm.Chromatogr.E. Skelson. Bugatti.Forsch. Chem.K. Faculty of Pharmacy. J. . J. K.P. Rurak. 1675 (1970) J. Stanley. Wazer Neuropsychobiology lo. Hasman. Barier.M.B. R. Sckikawa Chem. Wright. E.Pharm.Pharm. T. CA 90-66423~ W. 595 (1970) T. Gibbs. Arita.P.D. Pitel. 223 (1986) T. A . 183. M. 1073 (1978) W. J.McErlane. Axelson. Bylsma-Havell. Popovic Ther. 215 (1983) C. Choen. 1254 (1979) M. 1106 (1982) 43 M. 67. 308. 18. - . J. M. Vergin J. Pingoud Fresenius Z. 2. A. J. D. Anal. 276. Bishop-Frendling. 319 (1983) Y.W.E. 315 ( 19781. Gill. R. D. Lab. Hori. 1073 (1978) R. Axelson J . 28. G.Tam. Daldrup. Michalke 441 Fresenius 2. Leppard. 2.Sci.M. Tyrer J. J. Axelson. 46 B. J. (Tokyo).E.P.C. Stram Pharm.Pharm.H.Ito. 453 (1983) J. suppl. 1. Kapil. J. Pingoud. W.I. 67. 301. Th.E. Tam.Chem.W. Pharm. Susanto. Riggs. Price J. M. Block. Luce Ann.Moffat Analyst 107.D. F. Price J. 108 (1984) Y.E.Ana1. F. McDermed. Tam. G.E.Dugley. Ongley J. 1041 (1981) G. 68.B.E. R. P. Reid Methodol Surv Biochem.DAVIDE PITRE AND RICCARDO STRADI 360 A. Prosek. 273. 77 (1984) J.Drug Monit. Chromatogr. J.L. R. Stead. Ongley. I.Fr.J. 173 (1980) K. Hisayasu. Personal Communication 42 - . MacMorland.K.Chromatogr. Khan. H.Watre.K.M. Ross-Lee. J. Kjeldrap Arzneim. University of Milan.Sci.Pharm.D. R. Bull. B. E. Block.

8 Differential Scanning Calorimetry 4 . The Netherlands 1. Beijnen Slotervaart HospitalfNetherlands Cancer Institute Amsterdam.2 Stability in Pharmaceutical Formulations 6 .4 Mass Spectrum 4. 4 Gel Filtration Chromatography 5. The Netherlands Auke Bult and Willy J.11 Dissociation Constants 4.12 Electrochemistry Methods of Analysis 5 .M. 3 Cljnical Toxicity 7 . 1 Ultraviolet-Visible Spectrum 4 .MITOMYCIN C J o s H. 1 Elemental Analysis 5.7 Melting Point 4. . Colour. Historv Description 2. 5. Molecular Weight 2.6 Identification and Purity Tests in Official Compendia Stability. 3 Thin Layer and Paper Chromatography 5 . 2 Clinical Antiturnour Activity 7 . 9 Solubility 4. 2. Chemical Synthesis Physical Properties 4 .6 Optical Rotation 4.10 Partition Coefficient 4. 7. All rights of reproduction in any form reserved. Department of Pharmaceutical Analysis State University of Utrecht Utrecht.2 Appearance. 4 Pharmacokinetics Determination in Body Fluids References ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 16 361 Copyright 0 1987 by Academic Press. 2 Infrared Spectrum 4. Underberg Faculty of Pharmacy. 8.1 Name.5 High Performance Liquid Chromatography 5. 6. 3 Stability in Biological Media Pharmacology 7 .1 Stability in Aqueous Solutions 6.5 Circular Dichroism (CD) Spectrum 4. Degradation 6. Odour Biosynthesis. 3.2 Ultraviolet Spectrophotometry 5 . 1 Mechanism of Action 7 .Inc.3 Nuclear Magnetic Resonance (NMR) Spectrum 4. Formula. 4.

Formula. Extensive chemical degradation studies.JOSH. .1 Name. cumulative bone marrow suppression. DESCRIPTION 2. clinical experience with mitomycin C has shown that it causes severe toxicity in particular a delayed. from the same actinomyces strain (2). Unfortunately.3 1 ) . led by Wakaki. This administration mode has been shown to be very effective and devoid of serious haematological toxicities. . intravesically administered. Several mitomycin derivatives have since been isolated from Streptomyces species (3-8). 362 1. In 1958 another Japanese group. X-ray diffraction crystallography. The molecular weight is 334. Mitomycin C is rarely used as a single agent except for the treatment of superficial cancer of the urinary bladder. Mitomycin C was selected for further development after it had been established that mitomycin C possessed profound antineoplastic activity and that the therapeutic index was more favourable than that of related mitomycin congeners. These pigmented compounds were obtained by extraction and chromatographic purification of the fermentation broth filtrate of Streptomyces caespitosus. BEIJNEN ETAL. The drug has activity against an array of solid tumours such as adenocarcinomas of the gastrointestinal tract. succeeded in the isolation of a mitomycin derivative. Molecular Weight The generic name is mitomycin C (50-07-7). chromatographic and spectroscopic analysis and biosynthesis studies have contributed to the elucidation of the structure and absolute stereochemistry of mitomycin C and related mitomycins (9-23a). The drug is marketed under the trade names of MutamycinB. HISTORY In 1956 Hata and coworkers (1) first reported about the isolation of a new group of antitumour antibiotics which they named mitomycins. called mitomycin C.13. breast and stomach cancer the reader is referred to review articles and books ( 2 4 . For more specific information about the clinical usefulness of mitomycin C combinations in lung. 2. Mitomycin-C The molecular formula for mitomyKyowa@ and Ametycine@ cin C is CI5Hl8N4O5.

1 Ultraviolet-Visible Spectrum The ultraviolet spectrum of mitomycin C ( ~ x ~ O .6-dimethoxytoluene as starting material by Kishi and collaborators (36). CHEMICAL SYNTHESIS By using 13C. 21. In the visible region a broad band of low intensity is present. Colour. investigators have studied the biosynthetic pathways of mitomycins (20.3 23. The total chemical synthesis of mitomycin C has been accomplished from 2.000 209 . 3. BIOSYNTHESIS. 32-35). L-methionine. L-arginine.olet crystalline powder which is o d o u r l e s s . 3H and 15N-labeled precursors in feeding experiments. 14C. Molar absorptivities (E) and A1 cm values are reported in Table I (9). The spectrum was recorded by using a Shimadzu UV-200 double beam spectrophotometer in 1 cm quartz cell. At pH values over 10 the absorption maximum at 360 nm shifts to 295 nm due to keto-enol tautomerization and deprotonation of the 7-amino quinone part of the mitomyzin C molecule (Figure 2)(37).2 Appearance.~ Min) water is depicted in Figure 1. The synthesis route is lengthy (47 steps) and complex. Table I 360 555 UV-VIS Spectral Data for Mitomycin C in Methanol(9) 689 6. Feeding studies have revealed that D-glucosamine. pyruvate and D-erythrose are candidates as biosynthetic precursors of the mitomycines (21). PHYSICAL PROPERTIES 4. 4. Odour Blue-vi.MITOMYCIN C 363 2. Although considerable progress has been made in elucidating the biosynthesis routes the exact pathway remains to be established. L-citrulline.

I 400 MMC 01 absorbance 300 250 wavelength I nrn) 350 200 MMC- Figure 2.364 JOS H. Keto-enol tautomerization and deprotonation of mitomycin C (MMC) .BEIJNEN ETAL.

. 1 1 . Table I1 Infrared Assignments for Mitomycin C -1 wavenumber (cm ) assignment 346OS. . '"1 0 . 1558' V(C=O) of quinone . 3260' V(NH) and 1735' carbarnate and 7C-NH2 V(C=O) of carbamate function 16OOs. . . 331OS. .2 Infrared Spectrum The infrared spectrum of mitomycin C in a potassium bromide pellet is presented in Figure 3 . 1 . . . Assignments o f a number of prominent bands is listed in Table 11. . . . V (NH2) of aziridine. The spectrum was recorded with a Jouan-Jasco IRA-grating infrared spectrophotometer.MITOMYCIN C 365 4. . . . . . Infrared spectrum of mitomycin C . . . Figure 3 .

57 doublet 1 H-3 ' 4. BEIJNEN ETAL. H-9.20 to lower ppm values (spectrum F i g . 366 4.43 quartet 1 H-10' 7. Proton exchange with deuterium oxide results in disappearance of NH.19 singlet 3 OCH3 ( C . The spectral assignments are presented in Table 111 (in pyridine-d ) and are derived from L o w and Begleiter ( 3 8 ) .02 quartet 1 H-9 4.10 triplet 1 NH (Cl-C2) 2.13 3. Irrigularitges in intensity comparison of the signals make the assignments tentative.72 quartet 1 H-2 1 H-1 3. while the H-3. Table I11 'H NMR Assignments for Mitomycin C in pyridine-d5 ~ ~~~~ Chemical shift 6 (ppm) Multiplicity Number of atoms Assignment 2.9 ) 3.13 MHz (37a).3 5. . The spectrum (detail) is presented in Figure 4 and refers to the situation after proton exchange.53 doublet 1 H. NH and H-1 signals.46 MHz (37a).JOSH. 4 ) .6 broad 4 NH2(C-7) and CONH2 The natural abundance carbon 13 NMR spectrum was recorded under the same experimental conditions except that the frequency was 75.10 to 0.00 singlet 3 CH3 (C-6) 2. The assignments of Keller and Hornemann are followed ( 3 9 ) . 2 H-10 and H-10' signals shift 0.11 triplet 1 H-10 5. The proton-noise decoupled spectrum is presented in Figure 5 and the spectral assignments are summarized in Table IV.3 Nuclear Magnetic Resonance fNMR) Spectrum The proton NMR spectrum was recorded in pyridine-d containing tetramethylsilane as internal reference and wi ?h the use of a Bruker WM-300 spectrometer a t frequency 300.

. . . 5. . .O chemical shift (ppm) Figure 4 . . . . .O . 1 . . . . . 1 . . 367 . I . . 1 4 . .0 . . . Proton NMR spectrum of mitomycin C (detail) 2. . . . .MITOMYCIN C 1 . 3.0 . .1 . . .

180 160 140 120 100 80 chemlcal shift (ppm) Figure 5.BEIJNEN ETAL.368 JOSH. I3c NMR spectrum of mitomycin c 60 40 20 0 .

1 C-lOa 176. chemical ionization (CI) . Direct insertion probe CI mass spectra were obtained by using a Finnigan 3200 quadrupole mass spectrometer combined with a Finnigan 6000 data system. EI mass spectrometry was performed by direct probe analysis on a Kratos MS-80 mass spectrometer.5 c-10 104.7 c-1 44.3 ~~~ a ~~ ~ obscured by solvent 4.8 C-9a 110. Source conditions were: 200 OC source temperature. 70 eV electron energy and 100 PA ionising current.4 Mass Spectrum Electron impact (EI) mass spectroscopy of mitomycins. 42). Methane was used as rhe ionising gas. His observations were confirmed in later studies (41.8 32. field desorption (FD) and fast atom bombardment (FAB) in the positive and negative ion mode. For this analytical profile electron impact (EI).3 C-6 106. 10 p m tungsten wire FD emitters containing carbon microneedles . spectra of mitomycin C (lot nr. FD mass spectra were obtained with a Varian MAT 711 double focussing mass spectrometer equipped with a MAT 100 data acquisition unit. 010783) were recorded. has been investigated in detail by Van Lear (40).6 c-3 62.20 mA and the multiplier voltage 1800 V.7 C-8a 149.2 c-9 49.6 c-2 36.5 OCH3 (C-9a) 50. The emission current was 0.6a c-7 156.MITOMYCIN C 369 13C NMR Assignments for Mitomycin C in pyridine-d 5 Table IV Chemical shift 6(ppm) ~ Ass-ignment ~~ CH3 (C-6) 8. including mitomycin C.1 C-5a 158.7 C-8 c-5 178.

370 Figure 6.360 242 292 I 3ab Figure 7. BEIJNEN ETAL. EI mass spectrum of mitomycin C .JOSH. CI mass spectrum of mitomycin C x 4 .

1280 with composition C15H18N405 (calculated mass 334. micromass ZAB-2F mass spectrometer. 8.G. 7. 9 and 10 and the major ion assignments are given in Table V. An emission current of 12 mA was used to desorb the sample. an instrument with reverse geometry and fitted with a high field magnet and coupled to a V. FAB mass spectrometry was carried out using a V.MITOMYCIN C 371 with an average length of 30 p m were used. 11-250 data system. The ion source temperature was 70 'C. The sample was loaded in thioglycerol solution onto a stainless steel probe and bombarded with Xenon atoms having a 8 keV energy. 4 Figure 8. FD mass spectrum of mitomycin C . The recorded spectra are depicted in Figures 6.G.1277). The mitomycin C sample was dissolved in methanol and then loaded onto the emitters by the dipping technique. A high resolution mass spectrum exhibited the molecular ion at m/z 334.

21s I88 288 388 480 Figure 9/10. FAB(+) mass spectrum (upper spectrum) and FAB(-) mass spectrum (lower spectrum) of mitomycin C . 38. 18. 68. 88 18. 98. 2r 58.3 72 JOSH. BEIJNEN ETAL. 37 28 18 e 388 L 488.

91 and 215 signals come from the thioglycerol matrix FAB (-) 334 333 M-' 29 1 (M-CONH) - (M-H)- m/z 107.MITOMYCIN C Table V 373 Mass Spectral Data for Mitomycin C m/z assignment FD 334 335 M+* (M+H)+ FAB (+) - 358 (M+H+Na) 357 (M+Na ) 336 (M+2 H)+ 335 274 (M+H) ( (M+H)-02CNH3) 242 ( ( (M+H) -H~COH) -O~CNH~) + + + + + m/z 57. 215 and 321 signals come from the thioglycerol matrix m/z 139 and 265 signals cannot be assigned with certainty CI - 363 335 302 274 EI - (M-H3COH)+ + 242 (M-02CNH2) ( (M-H3COH) -02CNH2)+ 334 M+ 302 (M-H~COH)+ 291 (M-OCWH) 273 259 (M-0 242 ( (M-H3COH) -02CNH2) + + CNH3) ( (M-02CNH2)-CH3) + + .

CD spectrum of mitomycin C .5 Circular Dichroism (CD) Spectrum The chiral centers at C1. 374 4. C2.JOSH. is shown in Figure 11. +A c F i g u r e 11. BEIJNEN ETAL.OOl%). The CD spectrum of mitomycin C in a methanolic solution (c=O. determined by using a Jobin Yvon Dichrograph 111. C9a and C9 in the mitomycin C molecule are responsible for the CD Cotton effects at 355 nm and 395 nm.

the heating rate was 5 K/min and the sample size was 3 mg ( 4 4 ) .73 temperature ("C) ~ 0.9 Solubility Mitomycin C is readily soluble in methanol.0180 0. The optical rotatory dispersion (Om)spectrum of mitomycin C has been reported by Hornemann et al. 4. The following solubility data have been reported ( 4 5 ) . determined by suspending an excess amount of mitomycin C in a solvent.7 Melting Point In the literature no melting point for mitomycin C is mentioned except for the statement that it should be over 300 " C (11). ( 4 3 ) .8 Differential Scanning Calorimetry In nitrogen atmosphere no transitions were recorded up to 330 OC in a closed (air tied) sample holder.MITOMYCIN C 375 4 .0234~10-~ 25 25 37 25 . 6 Optical Rotation The optical rotation of mitomycin C in methanol (c-0. This analysis was performed with a Thorn NPL Automatic Polarimeter Type 243 at 589 nm. Table VI Solubilities of Mitomycin C solvent solubility (mM) water sesame oil isopropylmyristate hexane 2.1%) was determined to be + 2 3 2 " . 4. 4. acetonitrile normale saline and water. In an open sample holder an undefined large exothermic decomposition takes place from 215 "C.0193 0. The applied apparatus was a Feteram 111. but is nearly insoluble in hydrocarbon solutions. followed by filtration and HPLC analysis.

BEIJNEN ETAL.42 (90+10) (50+50) (90+10) (80+20) dichloromethane/2-propanol (50+50) a reference 0.51 2. 0.07M phosphate buffer pH = 7.27. 0. 0.10 Partition Coefficient The partition coefficient for mitomycin C has been measured in various organic phaseldistilled water systems.85 1. The results have been combined in Table VII. the N-4 nitrogen and the aziridine nitrogen.26.32 0.s in degradation studies (49-51) although the titrimetrical determination also has been influenced by degradation.11 Dissociation Constants The mitomycin C molecule contains several prototropic functions. Therefore the acid dissociation constants for these €unctions were derived from titrations with stable analogs (37).41.ationship.44 at room temperature.2) and ethyl acetate (7. Basic groups are the 7-amino group. .52 1. Table V I I Partition Coefficients for Mitomycin C solvent chloroform n-octanol ethyl acetate 1-pent an1 chloroform/2-propano1 chloroform/2-propanol chloroform/l-pentanol chloroform/l -pentan01 partition coefficient 0. The pKa of the aziridine nitrogen has been determined titrimetrically (11) and kinetically from the pH-rate re1.JOS H. 4. 48) have been used with success €or the isolation of mitomycins from the aqueous fermentation broths of Streptomyces species. Mitomycin C also has acidic properties with an (apparent) pKa 12.4 The organic solvents chloroform (1. 376 4.12 1.50 1.04 the aqueous phase was 0. Due to rapid degradation the pKa values of the conjugated acid concerning the 7-amino group and N-4 nitrogen can not be deduced from titration experiments with intact mitomycin C. These acidic properties emerge after keto-enol tautomerization of the 7-amino quinoid moiety which precedes deprotonation in alkaline medium (Figure 2)(37). Results are listed in Table VIIT.

the extremely high reactivity of the hydroquinone form of mitomycin C. Direct current polarographic curves for mitompcin C are shown in Figure 1 2 .MITOMYCIN C Table V I I I 377 Prototropic Functions of Mitomycin C pKa(25"C) function determination method N-4 7-amino aziridine -1.44 7-amino reference 4 .74 via pH-rate profile 2.3 potentiometric titration 3. 5 2 . 52-54) and aprotic solvents ( 5 5 ) . This conversion triggers a complicated pattern of chemical consecutive reactions. This is due to: rapid degradation of mitomycin C at p H < 3 and p H > 1 2 . Electrochemistry of mitomycin C has been studied in aqueous solutions (50. 52-54) and cyclic voltammetry ( 5 0 .8 2 via pH-rate profile via pH-rate profile 2.50(49 spectrophotometry 12. Rapid degradation of mltomycin C within the time scale of recording a direct current polarographic curve at p H < 3 and pH > 1 2 strongly influences the shape and heights of these curves ( 5 0 ) .2 spectrophotometry spectrophotometry -1. 5 3 . the fact that chemical degradation products as well as electrochemically generated degradation products of mitomycin C can be reduced within the region of reduction potentials of mitomycin C itself ( 5 0 ) . 1 2 Electrochemistry Due to the presence of the quinone ring of the mitomycin C molecule the compound can be reduced and transformed into its hydroquinone form. 5 5 ) . by using polarography ( 5 0 . Although much progress has been made in the elucidation of the processes occurring during and following electrochemical reduction of mitomycin C the exact complicated mechanisms have not yet been unravelled (50). .2 via pH-rate profile 2.

378 JOS H.BEIJNENETAL. -4 Direct current polarographic curves of mitomycin C (10 M) . I I I 5 PH = S O I 0 I -500 I -1000 I -1500 potential (mV) Figure 12.

86 24.02 0.55 5.43 16. Some representative systems have been summarized in Table X.80 (12) (12) (12) (57) (57) 0. This assay is linear in the range 2x10. 379 METHODS OF ANALYSIS 5. N and 0 was reported to be as follows: Table IX El-emental Analysis of Mltomycin C (11) element % theory % found C H 53. Table X Thin Layer and Paper Chromatography of Mitomycin C plate solvent (v/v) Rf silicagel butanol (sat.2 Ultraviolet Spectrophotometry Mitomycin C has been determined by using UV spectrophotometry at 360 nm &n methan-qjl solution. with water) ethyl acetate (sat. The mitomycin C spot can be located by: irradiation with UV light of 254 nm.56 .1 Elemental Analysis The elemental analysis of a purified mitomycin C sample for C. H.57 16. with water) reference 0.MITOMYCIN C 5.98 53.56 (58) (59) (57) (60) (60) (60) 0.83 5.88 0.12 0. 5.57 0.76 23.5M NH40H (10:6) paper butanol-water (84:16) methanol-benzene-water (1:1:2) ethyl acetate (sat.75 0.13 N 0 5.11 0. followed by heating the plate at 110°C for 15 minutes by which mitomycin C appears as a pink spot (56). examination under daylight (mitomycin C has a blue-violet colour) .02 0.57 ~ ~_________ ~ 1-octanol-acetone-ligroin (9O-115°C) (2:5:5) chloroform-methanol (9: 1) cellulose isobutyric acid-0.3 Thin Layer and Paper Chromatography A diverse array of thin layer chromatographic systems have been described in the literature for analysing mitomycin C and derivatives (55). with water) methanol-benzene-water (10:5:5) isopropylalcohol-1ZNH OH (2:l) 4 methano 1 0.to 4x10 M y with a precision of ? 1%. spraying with a 1 in 100 solution of ninhydrin in alcohol.

BEIJNENETAL. 0 2 M NH HCO solution as eluent. but detection at 254 nm. 7 2 . 75. 91. By using a 1 . 5 . 6 9 . such as removal of oxvgen from the mobile phase and damping of pulsating flow of the solvent delivery system ( 9 3 ) . Detection by W absorbance measurements at 365 nm is usually preferred ( 5 5 . 6 1 . 7 5 .380 JOS H. 5 ~ 4 3 4 1 . 87-90). 92). is also reported ( 6 6 . The polarographic electrochemical detection mode is an elegant alternative for the detection of mitomycin C ( 5 0 . 5 cm Sephadex G-25 (fine) column the elution volume of mitomycin C is 96 m l ( 5 7 ) .5 High Performance Liquid Chromatography HPLC procedures reported in the literature have been developed mainly for the analysis of mitomycin C and derivatives in decompositinn mixtures ( 5 1 . which is less sensitive. tissues) ( 4 6 . 4 Cel Filtration Chromatography For the separation of mitomycin C from its degradation products and alkylation adducts Tomasz et a1 (57. 93). but demands extensive experimental precautions to be taken.7 1 ) in biological matrices (plasma. 6 5 . 5. . 7 6 ) because of the high specificity and sensitivity.8 6 ) and other systems such a s (enzyme containing) culture media ( 6 2 . bile. urine. Important HPLC systems have been enumerated in Table XI. 5 9 .6 4 ) utilized pel filtration chromatography on Sephadex G-25 columns and 0 .

5% (v/w) phosphate buffer pH 7.3)~ methanol-water (30:70. w/w) (59) acetonitrile-water (25:75) + 0.w/w) + 0.03M potassium phosphate buffer (12.0125M sodium acetate (66) methanol-1 mM octanesulfonicacid sodium salt solution (30:7O.01 M phosphate buffer (pH 6.5:87.01M phosphate buffer pH 7.w/w) + 0.025M (66) sodium acetate acetonitrile-water (20:80) + 0.0)(30:7O.v/v) (pH* 4.2% cetrimide + 0.0 (20:70:10) acetonitrile-0.2% sodium laurylsulphate + 0.pm) p-Bondapak C18 (10 pm) Lichrosorb RP18 (10 pm) Lichrosorh RP8 (5 p'm) Ultrasphere ODX Hypersil-MOS (5 pm) Hewlett-Packard C8 (10 pm) Radial Pak C18 (10 pm) Reversed Phase HPLC methanol-water (35: 65) methanol-0.0 methanol-water-phosphate buffer pH 7.5% (v/w) tetrabutylammoniumbromide solution (20%.w/w) + 0.w/w) methanol-water (30:70. gradient from 0% to 50% methanol Lichrosorb RP18 (10 pm) Lichrosorb RP18 ( l o p ) Hypersil ODS (5 pm) Hypersil ODS (5 pm) Resolve C18 (5 pm) Porasil P (10 pm) Porasil (10 pm) Silica Si-60 a ~~ reference (77-80) (46) (67) (82) (91) (75) (94) (42) Ion Pair HPLC (65) methanol-water (40:63.5) acetonitrile-0.05M phosphate buffer pH 7 (10:90) acetonitrile-water.Table XI High Performance Liquid Chromatography for Mitomycin C ~~ ~~ column mobile phase p-Bondapak C 1 8 (10 .5)a (85) Normal Phase HPLC tetrahydrofuran-methanol-isooctane-dichloro~t~ane (3:7:15:75) chloroform-methanol (9:1) ethyl acetate-methanol (95:l) ethyl acetate-methanol-water-dichloromethane (97:2:1:1) pH values of organic modifier-water mixtures . gradient from 5% to 41% acetonitrile methanol-0.0 + 0.5X (v/w) glacial acetic acid (~H*=4.5% (v/w) phosphate buffer pH 7.

A protonated 2-amino function may direct incoming water molecules to a cis configuration. In the cascade of reactions an intermediate is proposed.6 Identification and Purity Tests in Official Compendia In the United States Pharmacopeia XXI (56) the monographs "Mitomycin" and "Mitomycin for Injection" have been included. 107-110).BEIJNEN ETAL. alkali.0. Whenever the amino function in the intermediate is not protonated the directing electrostatic power is absent and a reacting water molecule may approach the C1 carbonium ion from both .2-cis-l-hgdroxy-2.1 Stability in Aqueous Solutions Mitomycin C and related mitomycins are not stable when stored as aqueous solutions (11-13. 82. 108). The overall degradation scheme is given in Figure 13. having a positive charge at C1 (49. In the mitosene compounds the C9a methoxy group has been cleaved. 1 3 ) . 55.2-trans.7-diaminomitosenes. Under drastic acidic conditions progressive degradation reactions lead to hydrolysis of the 7-amino group and the C10 carbamate side chain (9-11. Degradation is accelerated by acid. Aziridine ring opening occurs with configurational retention at C2 and water attachment at C1 with inversion as well. 5. (buffer) ions and high temperatures (108). as retention of the C1 stereochemistry yielding 1. a double bond between C 9 and C9a has been introduced and the aziridine moiety has been opened.7-diaminomitosenes. At p H < 7 mitomycin and at C is converted into l-hydroxy-2. Furthermore Mltomycin and Mitomycin for Injection (a dry mixture of mitomycin C and mannitol) must meet the requirements of specific tests described in the USP XXI such as: Safety Tests. STABILITY. This electrophilic center serves as target for attacking (nucleophilic) water molecules resulting in the formation o f l-hydroxv-2.and 1. 51. 96-108). Pyrogen Tests and Sterility Tests (56). The degree of protonation of the 2-amino function in the proposed intermediate species (pKa:2.0 and 8. The water content of the raw material may not exceed 5%. In these monographs the identification of mitomycin C is based on IR and UV spectroscopy and chromatography by which the sample should exhibit the same spectroscopic and chromatographic properties as a similar preparation of a USP Mitamycin Reference Standard. The pH of a solution containing 5 mg/ml should be between 6.382 JOS H. DEGRADATION 6. 57. Depressor Substances Tests.8(65)) determines the C1 stereochemistry of the resulting mitosenes. 59.7-diaminomitosenes pH > 7 it is hydrolysed into 7-hydroxymitosane. respectively. The mechanism of the initial degradation step of mitomycin C in acid is shown in Figure 14 (107. 65-68. 6.

51.MITOMYCIN C 383 sides with roughly equal chances. At pH=l the cisltrans ratio is about 4 while at pH=6 the ratio approaches 1 (65). 103. These are important observations which lend credibility to the hypothesis that acid activation may play a role in the in vivo mitomycin C DNA alkylation. 107.i \ cis. For further detailed kinetic information is referred to the literature (49. 108).7-diaminomitosenes are generated (57. Overall degradation scheme of mitomvcin C trans -mitosene NH. Some kinetic data are summarized in Table X I I . + i.hydroxymitosane Figure 13. 100-102). 111-114). When mitomycin C degrades in acidic medium in the presence o f nucleophiles such as acetate. 67. . Under prolonged severe alkaline treatment the quinone chromophore is destroyed ( 5 9 ) . 55. In alkaline solution (pH 7 to 13) the 7-amino group of the mitomycin C molecule is substituted by a hydroxy function (59. phosphate or ever! DNA parts. l-nucleophile-2.mitosene mitomycin C \ OH- 7 . In huffered media the degradation kinetics o f mitomycin C can be described adequately by (pseudo) first order kinetics. This explains the remarkable influence of pH on the C 1 stereochemistry of the resulting mitosenes.

" L 0 1 0 II c N-H Figure 14.OH . Acid degradation mechanism of rnitomycin C + CH. ..384 JOSH. BEIJNEN ETAL.

71.OM phosphate buffer. 116-120).1~1O-~ (105) 1. perchloric acid solution.4.4~10-~ (107) 4. 0. 25'C/ HPLC pH 4.87.Ox (65) 1. An overview of stability data of mitomycin C in infusion fluids is given in Table XIII. 37'C/ HPLC pH 9. depending strongly on the pH of the final solution (68). More specific and extended data are described in the references concerned (68. 2OoC/ HPLC pH 2.2~10-~ (114) 1. 0.3~ (67) 6. 0.l ) 1. 0.1M phosphate buffer.0. 20°C/ HPLC 1. .0.M O-~ reference (65) 4. pH 3.001M acetate buffer. 40°C/ spectrophotometry pH 7. 70.MITOMYCIN C Table XI1 385 Degradation Kinetics for Mitomycin C conditions/method pH 0.1. In this state the drug is stable for at least two years if kept dry and in well closed containers at room temperature in the dark (115).86.1M phosphate buffer. Ametycine lo@ : 10 mg mitomycin C + 240 mg sodium chloride) as excipients. After reconstitution the drug has only limited stability.64.8~10-~ (104) 5.1M phosphate buffer. 25OC/ HPLC kobs ( s . 37"C/ spectrophotometry pH 11.2 Stability in Pharmaceutical Formulations Mitomycin C is commercially available in a lyophilized form containing mannitol (Mutamycin@ : 5 mg mitomycin C + 10 mg Mannitol) or sodium chloride (Mitomycin C-Kyowam :10 mg mitomycin C + 240 mg sodium chloride. 0.1M phosphate buffer.

Table XI11 Stability of Mitomycin C in Infusion Fluids at Room Temperature and .6 mg/ml 99% remained after 4 weeks storage at -30°C 90 =24h .4 mg/ml ~~ 5% dextrose = 78 h stable for 7 days = 12 tgO = tgO = h 15 days 1 h 0.3 O O C infusion fluid concentration stability reference 5% dextrose 50 pglml tgO = 3 . t50 0.9% sodium chloride 0.9% sodium chloride pg/ml mg/ml 93% remained after 5 days sterile water 50 2 5% dextrose 50 pg/ml 26% remained after 12 hours 0.75 50 pg/ml 0. 4 mg/ml t 0.9% sodium chloride 50 pglml t50 phosphate buffer pH 7. 0 h.9% sodium chloride 0 .

In Mark's M-20 culture medium with fetal calf serum. (86) published an extehsive study concerning the handling of mitomycin C biological samples. adjusted to neutral values. den Hartigh et al. Proctor and Gaulden (90) observed that the amount of mitomycin C was reduced by 2 9 % after 30 minutes incubation. the pH of urine samples should always be checked and. . Ignorance of the chemical stability of the drug during sampling and sample pre-treatment procedures may lead the researcher to misinterpretations. however. supplemented with serum (69).extracts of biological samples may be kept for at least 24 h. The in vitro half life of mitomycin C was determined to be > 14 days under conditions of the clonogenic assay ( 21).mitomycin C samples should be protected from long-term exposure to daylight in order to prevent analyte degradation. provided analysis is carried out within a period of 2 h. . Fischer's or MEM culture media.9% sodium chloride solution (68. . analysis should take place within 3 weeks as longer storage at this temperature may induce considerable reduction of the mitomycin C concentration. 70. separation of plasma and red blood cells should take place within half an hour after collection of the samples. 119. RPMI. Mitomycin C is fairly stable (70-802 of the drug remained) when incubated for 5 days at 37°C in Medium E. Recently. either dry or dissolved in methanol at 4 ° C or 20°C prior to analysis. 116. in the dark.MITOMYCIN C 387 Further research is required to resolve the problem of conflicting stability data on mitomycin C in 0. . The following recommendations and guidelines were given by these authors (86): .whole blood samples should be placed in ice immediately after sampling.3 Chemical instability of mitomycin C in biological fluids and tissues may be a complicating factor in the bioanalysis of the drug.repeated freezing and thawing of plasma and urine samples does not influence the analytical results of the mitomycin C assay.plasma and urine samples may be stored in a freezer at -2OOC. 120). if necessary. as a low pH will induce mitomycin C decomposition and the formation of precipitates upon which mitomycin C may be adsorbed: . Stability in Biological Media 6.plasma and urine samples need not necessarily be placed in the refrigerator or freezer immediately after preparation. .

126). BEIJNENETAL. 144). Under aerobic conditions. is not cytotoxic although some interaction with nucleic acids may exist (122). by which a monofu ctional alkylation adduct is formed. Reduction can be mediated by enzyme systems (87-89. JOS H. 111-113. . The semiquinone radical is also believed to be capable of producing interstrand cross-links between complimentary strands of DNA (87. More studies are necessary in order to elucidate which of the proposed mechanisms or combination is actually responsible for mitomycin C induced cytotoxicity in vivo. but then. 103. Reduction of mitomycin C may follow either one or two electron reduction steps. in its oxidized form. 133-136). leading to cell damage and can contribute in this manner to mitomycin C induced cytotoxicity. PHARMACOLOGY 7. activation by acid is a requisite (57. Acid catalyzed degradation of mitomycin C (Figure 14) occurs through reaction intermediates possessing an electrophilic C1 center which is the target for nucleophilic nucleic acid attack.388 7. The alkylating capacity is unmasked by reductive or acidic activation. nucleotides and nucleosides have been reported (62-64. The drug as such. Chemical structures of monofunctionally linked mitomycin C adducts with DNA. In the absence of any reducing agent or enzymes mitomycin C is also capable of coupling covalently with DNA or nucleic acid parts. 129-132) or by chemical reducing agents (58. An intramolecular 9 SN -displacement of the carbamate moiety leads to the bifunctional cross-linked adduct F (Figure 15) (128). Mitomycin C must be activated before it is capable of alkylating DNA (123-128).1 Mechanism of Action Mitomycin C induced cytotoxicity is believed to be mediated by DNA binding. the semiquinone radical formed after a one electron reduction can transfer its electron to oxygen to generate superoxide anion under regeneration of the oxidized quinone (138-142). The interaction of mitomycin C with DNA is more than 9OX of the monofunctional type. A subsequent rearrangement in species C leads to the formation of the quinone methide D which acts as target for nucleophilic nucleic acid attack. 137). An outline of the reductive activation pathway is given in Figure 15. Reactive oxygen species can cause DNA strand scission and membrane lipid peroxidation. The conversion of mitomycin C into its reduced form destabilizes the compound drastically and almost instantaneously the C9a methoxy group is lost (Figure 15). 143.

Reductive activation mechanism of mitomycin C .MITOMYCIN C 389 OH OH Figure 15.

Metabolites have never been identified. 7. Mitomycin C is usually used in combination with other cytostatics (28-30). Extravasation of mitomycin C causes severe cellulitis accompanied with chronic pain and ulceration (151). 7. Pharmacokinetic data after intra-arterial administration are available (73). stomatits. 155).4 Pharmacokinetics Following intravenous bolus injection of mitomycin C the plasma concentration-time curves show a bi-phasic.390 JOSH. 28.2 Clinical Antitumour Activity Single agent activity of mitomycin C has been demonstrated against various solid neoplasms in humans such as breast cancer (29. stomach cancer (146). . After intravesical instillation. . diarrhea. exponential decline (72. Following topical intravesical mitomycin C therapy for bladder cancer dermatological toxicity has been encoutered in some cases (154). Mitomycin C pharmacokinetics (at clinically relevant doses) is not dose dependent (72. non-small cell lung cancer (148) . 145) . Others include nausea. vomiting. hemolytic-uremic syndrome and renal failure have occasionally also been documented as due to the drug. BEIJNEN ETAL. pulmonary fibrosis.3 Clinical Toxicity Delayed cumulative myelosuppression is the most important toxicity. limited absorption of mitomycin C into the systemic circulation occurs (30. anorexia. Absorption after oral administration is very irregular(l56). Encouraging results have been obtained on the treatment of superficial transitional cell carcinoma of the bladder when mitomycin C is administered intravesically (28-30. skin rashes and alopecia (24. Interstitial pneumonitis. squamous cell carcinoma of the uterine cervix (28) and head and neck cancer (28). 155). 150). 157). pancreatic cancer (147).l49). 7. colorectal cancer (29). Maj o r routes of elimination are liver metabolism and urinary excretion.

Aliquots of 10-20 PI are injected into the chromatograph. of chloroform-2-propanol ( l + l .7 4 ) . HPLC: column: p Bondapak C18 ( 3 0 cm x 3 . 1 5 8 . . 1 6 4 ) . 1 6 6 ) .1 6 1 .8 6 . 391 DETERMINATION IN BODY FLUIDS The analysis of mitomycin C in biological samples is hampered by its instability. 9 mm i. The following procedure is followed for plasma samples: 0 . high-performance differential pulse polarography ( 5 4 ) and HPLC assays ( 4 6 . 9 5 .01 M phosphate buffer pH 6. 0 or 10. The following techniques have been used: microbiological assays ( 7 4 . The residue is dissolved in 50-100 p l of methanol with the aid of a vortextype mixer. immunological assay ( 1 6 3 . flow rate 1. detection: 365 nm. 0 . low concentrations and the polar nature of the mitomycin C molecule (55.0-methanol (70+30. 1 0 4 .5 or 1.0 ml samples is mixed with the internal standard porfiromycin (about 500 ng) and with 2 .. 7 2 . ( 4 6 . 93-96. particle size 10 pm). The HPLC assay designed by Den Hartigh et al.0 ml/min. 0.MITOMYCIN C 8. w/w).d. the clear supernatant is transferred into a conical tube and evaporated to dryness at 30-40°C under nitrogen. respectively. An overview of published determination methods of mitomycin C in body fluids is given in Table X I V .0 ml. w/w). bioassay by using a repair deficient mutant of Chinese hamster ovary cells ( 1 6 2 ) . 7 2 ) has shown to be very suitable for pharmacokinetic studies ( 7 2 . After shaking for 1 minute and centrifugating for 5 minutes ( 2 5 0 0 g). 2 . 7 6 ) . mobile phase: 0. 5 . 1 5 5 ) .

coli B B. tissues. urine lI0 1. urine serum. bile plasma. urine no 2 no no no 10 500 60 E.Table X I V Determination Methods for Mitomycin C in Body Fluids matrix sample pretreatment a b determination limit(ng/ml) .s 200 25 (54) (54) . urine no 4 (163) polarography plasma. urine plasma.test organism reference microbiological assays ~~~ W tJ blood.subtilis E. urine.coli R E. coli bioassay with hamster ovary cells plasma 1 no (162) immunological assays serum. urine plasma.

.1.S. urine plasma. 77.5 ml ( 9 4 ) samples C the internal standard is porfiromycin d RP: reversed phase chromatography.S. Yes Yes plasma. no no no 1. yes no plasma. 7 5 .S. no no no w plasma.0 ml ( 4 6 .1.0 ml (54.: sample pre-treatment by liquid-liquid extraction. ascit e s serum.s.Table XIV (continued) matrix high performance liquid chromatography sample preinternal chrom to ra hic determination a C g g p treatment standard mode limit(ng/rnl) - serum.s. urine Yes Yes 1 RP RP RP 1 5 RP RP 40 (serum) 1 5 0 (urine) 4 (plasma) 2 (plasma) 500 (urine) 1 (plasma) 200 (urine) 1 (plasma) reference RP (gradient) RP NP 10 10 RP RP RP 25 10 RP RP a 1. 1. ascites plasma plasma plasma.1.1. 162). 1.1. Yes plasma. 1. deproteination with methanol 1. urine plasma. 1 6 3 ) . 7 8 . NP: normal phase chromatography. 2.1. b determination limits for 25 p1 ( 8 0 ) . urine 1. 1. urine. 72-74. urine serum serum 1. 9 5 ) and 2.: sample pre-treatment by liquid-solid extraction.1. urine 1. bile.2 ml (160).s. plasma. 1. 0. 100 p l ( 8 4 ) . urine.1. (plasma) 1. urine 1. 50 p l ( 8 5 . 82. 1. 1.

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&Lty. ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 16 403 06 Copyright 01987 by Academic Press. King Saud UVLive. All rights of reproduction in any form reserved. Riyadh-77451.thy and **Vep&men. A . lahiq" *DepahAnent 06 Phanmacaficak? Chemhin. Al-Ba&* and Ad. Inc. P.O. Box 2457. (Saudi A m b h l . .NEOSTIGMINE A . College 06 P h m a c y .t Phatrmacotogy.

3 Molecular Weight 2.7 Polarographic Method References .2 Formulae 2.5 Chromatographic Methods 8.4 Elemental Composition 2. Toxicity 8. A.1 Identification 8. AL-BADR AND M. Pharmacokinetics 6.A.5 Appearance.TARIQ 404 1.3 Biological Method 8. Methods o f Analysis 8.6 Ion-Selective Electrodes Method 8.1 Nomenclature 2. History 2. Physical Properties 4. Therapeutic and Other Uses 7. Color. Synthesis 5. Odor and Taste 3.2 Titrimetric Methods 8.4 Spectrophotometric Methods 8. Description 2.

N . Neostigmine was a l s o found u s e f u l i n p a r a l y t i c i l l e u s .N-trimethyl bromide.t r i m e t h y l a n i l i n i u m bromide. (m-Hydroxyphenyl) t r i m e t h y l ammonium methyl s u l p h a t e dimethylcarbamate.1 Chemical Names 3. t h e r e s p o n s e t o t i t a n i c s t i m u l a t i o n i s improved a l o n g w i t h symptomatic improvement i n muscle s t r e n g t h ( 7 ) .NEOSTIGMINE 1. N . (m-Hydroxypheny1)timethylammonium bromide dimethyl carbamate.[ [ (dimethylamino)carbonyl] oxy] -N . 3. was i n t r o d u c e d i n t o t h e r a p e u t i c s i n 1931 f o r i t s s t i m u l a n t a c t i o n on t h e i n t e s t i n a l t r a c t .N. I t was r e p o r t e d i n d e p e n d e n t l y by Remen (5) and Walker (6) t o be e f f e c t i v e i n t h e symptomatic t h e r a p y of m y a s t h e n i a g r a v i s which is due t o d e f e c t i n s y n a p t i c t r a n s m i s s i o n a t neuromuscular j u n c t i o n .[ [ (Dimethylamino)carbonyl]oxy] . Neostigmine ( P r o s t i g m i n ) . 2. 3 ) i n e l u c i d a t i n g t h e chemical b a s i s o f t h e a c t i v i t y of physostigmine. N .Ntrimethylbenzaminium methyl s u l p h a t e . a t o n y o f u r i n a r y b l a d d e r and i n glaucoma ( 8 . N . 3-(Dimethylcarbamoyloxyphenyl)trimethyl- ammonium bromide.N . 3.1. (3-Dimethylcarbamoxyphenyl)trimethylamonium methyl s u l p h a t e (10-13). 405 Historv As a r e s u l t o f t h e b a s i c r e s e a r c h o f Stedman and a s s o c i a t e s ( 1 . Aeschlimann and R e i n e r t (4) s y s t e m a t i c a l l y i n v e s t i g a t e d a s e r i e s o f s u b s t i t u t e d phenyl esters o f a l l y l c a r b a m i c a c i d s . .1 Nomenclature 2. a most promising member o f t h i s series. 9 ) . Benzenaminium. trimethylbenzenaminium bromide 3.N . When t h e patient i s given an a p p r o p r i a t e dose o f n e o s t igmine. Description 2. 2 .(Dimethylcarbamoyloxy) -N .[ [ (Dimethylamino) carbonyl loxy] -N .

J u v a s t i g m i n e .1.1. P h i 10st igmine . Neostigmine methyl s u l p h a t e I n t r a s t i g m i n a . Neostigmine DCF. Leostigmin ( 1 3 . N e o s t i g m i n i i bromidum. Neos t igmin i bromidum.14).2 Generic Names Neostigmine bromide. S t i g l y n .A.3 Trade Names Neost i pmine bromide P r o s t i g m i n .1. P r o s t i g m i n .2. Konstigmin. Hodostin. N e o e s s e r i n . Mestastigmin.5 Neostigmine Neostigmine bromide Neostigmine methyl s u l p h a t e (14) Wiswesser Line N o t a t i o n 1 N 1 & VOR CK-& E (Neostigmine bromide) (15) 2. Stigmosan (13.1. J u v a s t i g m i n .1 Empirical C H BrN202 1 2 19 C13H22N206S Neostigmine bromide Neostigmine methyl s u l p h a t e .14). 2.4 CAS R e g i s t r y Number 59-99-4 114-80-7 51-60-5 2. Neoeserine. NFN. Normastigmin. 2. Eus t igmine . 1 4 ) . A.2 Formulae 2. Metastigmin. Normastigmin. Neostigmium bromatum (13. Synstigmine. P r o s t i g m i n . AL-BADR AND M.TARIQ 2. P r o s e r i n e .

5 47. Physical Properties 3. N 9. N 8 .2 Structural CH ‘I x0 ’- X = Br- II c -N .401 NEOSTIGMINE 2.4 Elemental Composition C 0 C 0 2. 46.53%. 28. Color. s l i g h t l y hygroscopic powder w i t h a b i t t e r t a s t e (14) * 3.36%.1 Melting P o i n t Neost igmine bromide C r y s t a l s from a l c o h o l and e t h e r melt a t 167’ w i t h decomposition ( 1 3 ) . Odor and Taste Odorless.CH3 ‘CH3 Neostigmine bromide X = CH3SOi Neostigmine methyl s u l p h a t e 2. S 9. B r 26. H 6 . Neostigmine methyl s u l p h a t e C r y s t a l s from a l c o h o l melt between 142OC and 145OC (13) * . Appearance. 6 3 % .24%. 10.69%.59%. H 6.2.3 Molecular Weipht Neostigmine bromide 303. c o l o r l e s s c r y s t a l o r a white c r y s t a l l i n e .32%.2 Neostigmine methyl s u l p h a t e 334.4 2. 3 8 % .71% (methyl s u l p h a t e ) .55% (bromide).

7 Spectral Properties 3. 3.1 U l t r a v i o l e t SDectrum The UV spectrum o f neostigmine bromide i n e t h a n o l i s given i n Figure 1.1 cm 1 8 ) . The aqueous s o l u t i o n i s a c i d i f i e d w i t h d i l u t e a c e t i c a c i d .5 Moisture Content and H y g r o s c o p i c i t y Not more than I%. The methanol e x t r a c t w i l l c o n t a i n most o f t h e q u a t e r n a r y ammonium compound and may b e p u r i f i e d by paper chromatography ( 1 6 ) . Clarke (11) r e p o r t e d t h e following: Neostigmine methyl s u l f a t e i n 1 N H2SO4. 3. Moderately s o l u b l e i n chloroform and e t h a n o l .6 - Storage I t should b e s t o r e d i n a i r t i g h t c o n t a i n e r s p r o t e c t e d from l i g h t ( 1 0 ) .2 Extraction Neostigmine s a l t s a r e q u a t e r n a r y ammonium compounds and a r e s o l u b l e i n water.f r e e ) n o t more than 0 . evaporated t o dryness and e x t r a c t e d w i t h methanol. determined by d r y i n g a t 1 0 5 O C (10) 3.f r e e w a t e r and t i t r a t e a t pH 7 . TARIQ 408 3. The spectrum was recorded on Varian spectrophotometer model DMS 90. 3.7. 2 m l i s r e q u i r e d (17). maxima a t 260 nm (E 1 % . A. I n s o l u b l e i n e t h e r (11). . AL-BADR AND M. I t shows two maxima a t 260 nm and 266 nm.4 Acidity Dissolve 0 . 0 w i t h 0 . 0 2 N sodium hydroxide ( c a r b o n a t e .1 cm 20) and 266 nm (E 1%. 3. 2 0 g i n 20 m l carbon d i o x i d e .3 Solubility Neostigmine s a l t s a r e f r e e l y s o l u b l e i n w a t e r .A.

NEOSTIGMINE lot 2 00 2SOWavelength 300 (nm) 350 400 Figure I: Ultraviolet spectrum of neostigmine bromide in methanol. .

TARIQ 410 3.T60 A NMR s p e c t r o m e t e r u s i n g sodium 2.2-dimethyl 2 .7. AL-BADR AND M.s i l a p e n t ane. and was recorded on Pye-Unicam I R s p e c t r o p h o t o meter model SP 1025. potassium bromide d i s c t h e p r i n c i p a l peaks a r e 1711. The drug was dissolved in deuterium o x i d e (DzO) and i t s spectrum was determined on a Varian . .7.N vibration (aliphatic) CH (aromatic) bending vibrations Clarke (11) r e p o r t e d t h e f o l l o w i n g : Neostigmine bromide.3 1 Proton Nuclear Magnetic Resonance ( H NMR) Spectrum 1 The H NMR spectrum o f neostigmine methyl s u l p h a t e i s shown i n F i g u r e 3. 1215 and 1154 cm-1. The f r e q u e n c i e s and t h e s t r u c t u r a l assignments a s shown below: Frequency cm-l 3020- 1 2900 .s u l p h o n a t e (DSS) a s t h e i n t e r n a l standard. A.) 1730 1610 1 1595) 1490-1450 1030 1 1070) 780 and 895 As s ignment CH s t r e t c h C = 0 (amide) s t r e t c h C = C s t r e t c h (aromatic) CH bending v i b r a t i o n s C .5 .A.2 I n f r a r e d Spectrum .(IR) The IR spectrum of n e o s t i g m i n e bromide i n K B r d i s c i s p r e s e n t e d i n F i g u r e 2 . 3.

i .

.o I .o 0 NMR spectrum of neostigmine methyl sulphate in D20 with TMS as internal reference.0 Figure 3: Proton 6.0 S.0 7.I -- f I 8.OPpm(6k.O 3 -0 2 .

F i g u r e s 4 and 5 r e p r e s e n t t h e proton-decoupled and o f f resonance s p e c t r a r e s p e c t i v e l y .4 Proton assignment 1 m u l t i p l e t s Aromatic p r o t o n s Carbon-13 Nuclear Magnetic Resonance (C-13 NMR) Spectrum The C-13 NMR s p e c t r a o f neostigmine methyl s u l p h a t e i n deuterium oxide (D2O) u s i n g DSS (sodium 2.15-7.S O i -C 5 3 0 " -N 0 1 CH ..66-8.12 1 7.CH3 2 The carbon chemical s h i f t s were a s s i g n e d on t h e b a s i s o f t h e chemical s h i f t t h e o r y and t h e o f f .s i l a p e n t a n e 5-sulphonate) as an i n t e r n a l r e f e r e n c e were o b t a i n e d u s i n g a J e o l FX 100 MHz s p e c t r o m e t e r a t an ambient t e m p e r a t u r e .2-dimethyl 2 .04) II singlets -C-N(CH -3 ) 2 3.NEOSTIGMINE 413 Assignment o f t h e chemical s h i f t s t o t h e d i f f e r e n t p r o t o n s i s shown below: Chemical shift (ppm) Multiplicity 0 3.r e s o n a n c e s p l i t t i n g p a t t e r n and a r e shown below: .80 s i n g 1e t CH. 72 singlet 4.30 3.7.( g 3 ) 3.SO4 3.60 7. 12 CH3 I+ 13 CH3 .

A.414 A. . AL-BADR AND M.TARIQ Figure 4 : Proton -decoupled carbon-I3 NMR spectrum Of n-stigmine methyl wlphate in D20 with DSS as internal reference. Figure 5 : Off -Resonance carbon -13 NMR spectrum of neostigmine methyl sulphate in 9 0 with DSS a s internal reference.

8 quartet 2 158.NEOSTIGMINE 415 Chemical shift (ppm) 3.9 quartet 1 38.7. 11 and 12 57.9 quartet 13 Mass Spectrum The e l e c t r o n impact ( E I ) mass spectrum a t 70 e V was r e c o r d e d on Varian MAT 311 mass s p e c t r o m e t e r i s shown i n F i g u r e 6 .8 s i n gl e t 4 126. In t h e spectrum an ion a t m/e 428 was observed which most p r o b a b l y a r i s e s from a recombination o f fragments.5 singlet 8 117. The spectrum shows a b a s e peak a t m/e 72.6 quartet 10.3 singlet 3 149.8 doublet 7 154. The most prominent i o n s a r e shown below: .1 doublet 9 59.5 Multiplicity Carbon assignment 38.0 doub 1e t 6 119.9 doublet 5 134.

Figure 6: Electron impld (El)mass spectrum Of neostigmine methyl sulphate. .

I+ 1' L-.CON (CH. 42 8 OCON ( CH3) 1 CH2 The chemical i o n i s a t i o n ( C I ) mass spectrum was o b t a i n e d on Finnigan 4000 mass s p e c t r o m e t e r and is shown i n F i g u r e 7 .).NEOSTIGMINE 417 Fragment 72 208 356 CON ( c H ~+ ) ~ @- 0-CON (cH~$+ 428 . The spectrum shows i o n s at m/e 1 2 7 ( t h e b a s e peak) and a t m/e 303 and both a r e probably a r i s e d from a recombination o f fragments. The most prominent i o n s a r e shown below: .

.Figure 7 : Chemical ionization ( C I mass Spectrum Of neostigmine methyl sulphate.

by combination w i t h methyl bromide.NEOSTIGMINE 419 m/e 127 Fragment 0 II CH30-S-O-CH3 II 0 . . e .n i t r o a n i l i n e by o r d i n a r y s y n t h e t i c methods.COC1 (made from dimethylamine and carbonyl c h l o r i d e ) . The 3-hydroxydimethyla n i l i n e i s d i s s o l v e d i n an a l c o h o l i c s o l u t i o n o f t h e c a l c u l a t e d amount of potassium hydroxide. t h e dimethylcarbamic e s t e r o f 3-hydroxy-NNN-trimethylanil i n i u m bromide: (18. y i e l d s t h e required quaternary s a l t . Me2N. which i s made from 3 . and t h e s o l u t i o n o f t h e potassium d e r i v a t i v e so formed is t r e a t e d w i t h dimethylcarbamoyl c h l o r i d e .t l+ H 209 303 4. 1 9 ) .i . T h i s g i v e s a t e r t i a r y b a s e which. and t h e s o l u t i o n o f t h e potassium hydroxide. l+ Synthesis Neostigmine bromide can b e p r e p a r e d from 3-hydroxydim e t h y l a n i l i n e .

OK OCON (CH3) CH3S0i 0-C-N. A. ”0 CH3 . except t h a t i n t h e l a s t s t a g e t h e secondary amine i s combined with methyl sulphate t o form t h e s a l t [ (R-AMe3)MeSOi] (18).TARIQ 420 OCON (CH3) OCON (CH3) Neostigmine methylsulphate can be made i n the same way.A. AL-BADR AND M.

CH3S0i OCOCl + OCON (CH3) .42 1 NEOSTIGMINE A modification of t h i s route is t o convert t h e hydroxydimethylani l i n e i n t o t h e q u a r t e r n a r y s a l t (by combination w i t h methyl bromide o r methyl s u l p h a t e ) . and t h e n t o t r e a t t h e s a l t with dimethylcarbamoyl c h l o r i d e (18). 9 - + N(CH3)2 OK ClCON (CH3) P Neostigmine can be s y n t h e s i z e d e i t h e r d i r e c t l y from 3-hydroxydimethylaniline with phosgene t o g i v e t h e carbonyl c h l o r i d e and t h e n with dimethylamine t o g i v e 3-dimethylaminophenyldimethylurethane o r t h e l a t t e r may be prepared from t h e sodium s a l t of t h e s t a r t i n g m a t e r i a l and dimethylcarbamic c h l o r i d e . Y OCON (CH31 ON a COC 1 2 N OH 2- . In e i t h e r c a s e t h e product i s converted t o t h e m e t h y l s u l p h a t e by t r e a t m e n t with dimethyl s u l p h a t e (20).

t h e e q u i v a l e n t o r a l dose may be 30 mg o r more. 98 p e r c e n t is metabolized i n 10 minutes. . t h e y may n o t p r e s e n t a b a r r i e r t o t h e d i f f u s i o n o f q u a t e r n a r y amines such as n e o s t i g mine. ( 2 2 ) I t s t r a n s f e r from plasma t o l i v e r c e l l s and then t o b i l e is probably p a s s i v e i n c h a r a c t e r . When neostigmine was given by mouth t o 2 p a t i e n t s with myasthenia g r a v i s less t h a n 5 % was found unchanged i n t h e u r i n e ( 1 4 ) . E l i m i n a t i o n h a l f .0 mg. less t h a n 5% o f t h e o r a l dose i s e x c r e t e d i n t h e u r i n e unchanged. O f neostigmine t h a t reaches t h e l i v e r .l i f e o f less than one minute. Large o r a l doses may prove t o x i c i f i n t e s t i n a l a b s o r p t i o n i s enhanced f o r any reason and t h e q u a t e r n a r y a l c o h o l and p a r e n t compound are e x c r e t e d i n t h e u r i n e . 20% of an o r a l dose is e x c r e t e d i n t h e u r i n e and 50% i n t h e faeces. such t h a t much l a r g e r doses a r e needed t h a n by t h e p a r e n t e r a l r o u t e . Pharmacokinetics Neostigmine is absorbed p o o r l y a f t e r o r a l a d m i n i s t r a t i o n . 5 t o 2. Since c e l l u l a r membranes permit t h e passage o f plasma p r o t e i n s s y n t h e s i z e d i n l i v e r i n t o t h e blood stream through c a p i l l a r y walls o r lymphatic v e s s e l s .A. P y r i d o s t i g mine and i t s q u a t e r n a r y a l c o h o l are a l s o t h e predominant e n t i t i e s found i n u r i n e a f t e r a d m i n i s t r a t i o n o f t h i s drug t o man (21). P o s s i b l y t h e r a p i d h e p a t i c metabolism o f neostigmine p r o v i d e s a downhill g r a d i e n t f o r t h e c o n t i n u a l d i f f u s i o n of t h i s compound ( 2 3 ) . TARIQ 422 5. Neostigmine was r a p i d l y e l i m i n a t e d from t h e plasma o f 5 p a t i e n t s t o whom 5 mg o f t h e methylsulphate had been given t o a n t a g o n i s e r e s i d u a l neuromuscular b l o c k . The plasma c o n c e n t r a t i o n o f neostigmine d e c l i n e d t o about 8% o f i t s i n i t i a l v a l u e a f t e r 5 minutes with a d i s t r i b u t i o n h a l f . AL-BADR AND M. A. A c e r t a i n amount may be hydrolyzed slowly by plasma c h o l i n e s t e r a s e .l i f e ranged from about 15 t o 30 minutes. Trace amounts o f neostigmine could be d e t e c t e d i n t h e plasma a f t e r one hour ( 1 4 ) . Whereas t h e e f f e c t i v e p a r e n t e r a l dose o f neostigmine i n man i s 0 . Following i n t r a m u s c u l a r a d m i n i s t r a t i o n about 65% o f a dose i.s e x c r e t e d i n t h e u r i n e unchanged ( 1 0 ) .

The a n t i c h o l i n e s t e r a s e a c t i o n s o f ne ost igmi ne are r e v e r s i b l e .b l a d d e r . For e x p e l l i n g i n t e s t i n a l f l a t u s p r i o r t o r a di ogra phy o f t h e g a l l . and less f r e q u e n t l y t o i n c r e a s e a c t i v i t y o f smooth muscle ( 1 4 ) . I t is used mainly f o r i t s a c t i o n on s k e l e t a l muscle. I t is a l s o used i n t h e t r e a t m e n t o f p a r a l y t i c i l e u s postoperative urinary retention. 5 mg d a i l y given i n d i v i d e d dose s by subcutaneous. o r u r e t e r s . t h e h e a r t . Neostigmine can b e employed as a d i a g n o s t i c t e s t . i n which c o n d i t i o n ne ostigmine a g g r a v a t e s t h e symptoms. u r i n a r y b l a d d e r . e s p e c i a l l y a f t e r symptoms have been pur posely a c c e n t u a t e d by t h e a d m i n i s t r a t i o n o f q u i n i n e . a s i n g l e dose o f 500 ug h a s sometimes been used ( 1 4 ) . b u t f o r t u n a t e l y i t s e f f e c t s a r e more prominent on c e r t a i n s t r u c t u r e s t h a n on o t h e r s . The drug i n h i b i t s c h o l i n e s t e r a s e a c t i v i t y and p r o l o n g s and i n t e n s i f i e s t h e m u s c a r i n i c and n i c o t i n i c e f f e c t s o f a c e t y l c h o l i n e . kidneys . t h e p u p i l . 5 t o 1 mg. i n d o s es o f 0 . b e i n g p a r t i c u l a r l y e f f e c t i v e on t h e bowel. Neostigmine i s used i n c o n d i t i o n s o f u r i n a r y b l a d d e r a t o n y due t o p o s t a n e s t h e t i c d e p r e s s i o n o r t o n e u r o l o g i c a l d i s o r d e r s (19). Neostigmine h a s widespread a c t i o n s . blood p r e s s u r e . Neostigmine is used as d i a g n o s t i c t e s t a gent i n myotonia c o n g e n i t a . Neostigmine me t hylsul pha t e i s used i n t h e t r e a t m e n t o f myasthenia g r a v i s i n u s u a l d o s e s o f 1 t o 2 .NEOSTIGMINE 6. Neostigmine i s used t o p i c a l l y t o t r e a t primary open-angle glaucoma and i n . and s e c r e t i o n s b e i n g a f f e c t e d t o a much lesser e x t e n t i n doses t h a t are o r d i n a r i l y e f f e c t i v e on r e l a t e d s t r u c t u r e s (19) . I t proba bly a l s o has d i r e c t e f f e c t s on s k e l e t a l muscle f i b r e s . and s k e l e t a l muscle. i n t r a m u s c u l a r . I t a c t s a t t h e e s t e r a t i c s i t e o f t h e enzyme t o form t h e i n a c t i v e dimethylcarbamoyl enzyme. 423 T h e r a n e u t i c and Ot he r Uses Neostigmine is a q u a t e r n a r y ammonium a n t i c h o l i n e s t e r a s e . o r i n t r a v e n o u s i n j e c t i o n a c c ordi ng t o t h e s e v e r i t y o f t h e c o n d i t i o n (14). I t may be used as a d i a g n o s t i c agent f o r e a r l y pregnancy o r t o t r e a t delayed menstr uation: given i n t r a m u s c u l a r l y on t h r e e s u c c e s s i v e days i t w i l l i n d u c e m e nst rua ti on w i t h i n 72 hours a f t e r t h e l a s t dose u n l e s s t h e p a t i e n t i s pre gna nt .

c o n j u n c t i v a l co n g e stio n . i n c r e a s e d b r o c h i a l s e c r e t i o n combined wi t h b r o n c h o c o n s t r i c t i o n . a n o r e x i a. abdominal cramps and d i a r r h o e a . Symptoms of overdosage i n c l u d e sweating. muscle cramps. f e a r .TARlQ t h e emergency t r ea t m e n t o f primary a c u te . r e s t l e s s n e s s . and add 1 m l o f diazoaminobenzene- .1 Identification a) To 0 .a n g le glaucoma. The major symptom o f overdosage i n myasthena g r a v i s i s i n c r e a s e d muscular weakness ( 1 4 ) . Heat q u i c k l y on an o i l . Cool. b r a d y c a r d ia and hypotension. i n v o l u n t a r y d e f a e c a t i o n and u r i n a t i o n f l u s h i n g . Methods o f Analysis 8. add 0. watery n a s a l d i s c h a r g e . nausea and vomiting . e r u c t a t i o n .5 m l of sodium hydroxide s o l u t i o n and e v a p o r a te t o d r y n e s s on a water-bath. c i l i a spasm. Miosis lasts 1 2 t o 36 h o u r s . e x c e s s i v e dreaming. m i o s i s . lachrymation. A. AL-BADR AND M. a g i t i o n . 1 m l o f a 1 p e r c e n t w/v s o l u t i o n . 8. brow ache. Neostigmine methyl s u l p h a t e . T o x i c i t y and S i d e E f f e c t s Adverse and s i d e e f f e c t s o f neostigmine i n c l u d e s a l i v a t i o n . I t i s c o n t r a i n d i c a t e d i n a s t h m a t i c p a t i e n t s .b a t h t o about 250' and m a in ta in a t t h i s temperature f o r about t h i r t y seconds. Quinidine i n t e r f e r e s w i t h t h e a c t i o n o f neostigmine ( 1 9 ) . c o n v u lsio n s. c o o l i n i c e water. I t h as a l s o been s t a t e d t h a t p a r a d o x ic a l e f f e c t could o c c u r due t o i n t e r a c t i o n between n i c o t i n i c and muscarinic a c t i o n s . and coma.424 A. d i s s o l v e t h e r e s i d u e i n 1 m l o f water. Longer-acting a n t i c h o l i n e s t e r a s e s a r e p r e f e r r e d f o r t h e t r e a t m e n t o f accommod a t i v e e s o t r o p i a (19). a consequence of t h i s could b e some evidence o f an a c c e l e r a t i o n p u l s e . by i n c r e a s i n g i n t e s t i n a l m o t i l i t y .r a t e and e l e v a t i o n o f blood p r e s s u r e . s c a t t e r e d f a s i c u l a t i o n s and e v e n t u a l s e v e r e weakness and p a r a l y s i s . 7. Death may f o llo w due t o cardiac a r r e s t o r central respiratory paralysis and pulmonary oedema. may cause d i s r u p t i o n o f i n t e s t i n a l s u t u r e l i n e s (10). I t should n o t be employed al o n g with c h o l i n e esters e x c e p t f o r ophthalmologic u s e. nystagmus.

8. p r e v i o u s l y d r i e d a t 1 0 5 O f o r 3 h o u r s . Repeat t h e o p e r a t i o n w i t h o u t t h e s u b s t a n c e b e i n g examined. The i n f r a r e d a b s o r p t i o n spectrum o f a potassium bromide d i s p e r s i o n o f i t .15 g i n 20 m l of w a t e r . c o l l e c t t h e d i s t i l l a t e i n 50 m l o f 0 . 1 N s u l p h u r i c a c i d u n t i l t h e t o t a l volume r e a c h e s about 200 m l .425 NEOSTIGMINE sulphonic acid. Huang -e t a1 (24) d e s c r i b e d a s i m p l e .2. Pass a c u r r e n t o f steam through t h e m i x t u r e .c u r r e n t o s c i l l o p o l a r o g r a p h i c t i t r a t i o n i n pharmaceutical a n a l y s i s .006688 g of Cl3HZ2N2O6S.02 N NaOH u s i n g methyl r e d s o l u t i o n as i n d i c a t o r . Each m l o f 0. p l a t i n i c i o d i d e s o l u t i o n . r a p i d and accurate method u s i n g t h e a p p l i c a t i o n of a l t e r n a t i n g . e x h i b i t s maxima o n l y a t t h e same wavelengths as t h a t of a s i m i l a r p r e p a r a t i o n o f USP Neostigmine Bromide Reference Standard (12).1 Aqueous T i t r a t i o n B r i t i s h Pharmacopoeia (1973) (17) r e p o r t e d t h e f o l l o w i n g procedure f o r t h e a s s a y of neostigmine methyl s u l p h a t e : D i s s o l v e 0. t r a n s f e r t o a semimicro ammonia d i s t i l l a t i o n a p p a r a t u s . In . t h e d i f f e r e n c e between t h e t i t r a t i o n s represents t h e amount o f a c i d r e q u i r e d t o n e u t r a l i s e t h e dimethylamine formed from t h e n e o s t i g m i n e .02 N s u l p h u r i c a c i d i s e q u i v a l e n t t o 0.2 T i t r i m e t r i c Methods 8. and t i t r a t e t h e e x c e s s o f a c i d w i t h 0.bunches o f curved ( s e n s i t i v i t y : 1 i n 400) (11). and add 20 m l of a 50 p e r c e n t w/v s o l u t i o n o f sodium hydroxide. C r y s t a l t e s t s . A s o l u t i o n (1 i n 50) responds t o t h e t e s t o f bromide (neostigmine bromide) (12). Micro: l e a d i o d i d e s o l u t i o n b r a n c h i n g n e e d l e s ( s e n s i t i v i t y : 1 i n 400) . a cherry-red colour i s produced ( 1 5 ) .

a c e t a t e . t h e d i s appearance o f t h e i n c i s i o n o f sodium t e t r a p h e n y l b o r a t e on t h e curve of d/E/dt vs E b e i n g used t o i n d i c a t e t h e end p o i n t . . t h e c o n t e n t s were f i l t e r e d .100. The end-point was d e t e c t e d by means o f c o l o r change o f t h e i n d i c a t o r i n t h e o r g a n i c phase without t r a n s f e r of i n d i c a t o r between phases. dodecyle s u l p h a t e and mercury caused interference. 4 2 % .01 N TI2S04. Tsubouchi -e t a 1 (25) r e p o r t e d a method o f a n a l y s i s o f neostigmine u s i n g t h e a p p l i c a t i o n o f one-phase end-point change system i n two phase t i t r a t i o n t o amine drug a n a l y s i s . A.426 A. .2% and t h e c o e f f i c i e n t o f v a r i a t i o n was 0 . To t h e i n j e c t i o n s o l u t i o n ( 2 0 ml) c o n t a i n i n g 10 mg o f neostigmine methyl s u l p h a t e were added 10 m l of 0 . 0 1 N sodium t e t r a p h e n y l b o r a t e and 10 m l o f a c e t a t e b u f f e r s o l u t i o n (pH 5 . The s o l u t i o n was d i l u t e d t o 50 m l w i t h w a t e r . 2 1 mM) o f neostigmine bromide and 5 m l o f t h e a p p r o p r i a t e b u f f e r s o l u t i o n was added. Neostigmine i n aqueous s o l u t i o n ( 5 o r 10 mM) was determined by t i t r a t i o n w i t h aqueous 0 . 5 ) . 3 . 0 1 M t e t r a p h e n y l b o r a t e with tetrabromo p h e n o l p h t h a l i e n e t h y l ester a s an i n d i c a t o r i n t h e o r g a n i c phase ( 1 . a f t e r 5 minutes t h e p r e c i p i t a t e was formed. 5 . The r e c o v e r y range from 9 9 . 3 ) . papaverine . A 25 m l p o r t i o n o f f i l t r a t e was t r e a t e d w i t h 8 drops o f 40% sodium hydroxide s o l u t i o n and t i t r a t e d w i t h 0. i n b o r a t e phosphate b u f f e r medium (pH 5 . 25 M 1 o f aqueous s o l u t i o n ( 0 .d i c h l o r o e t h a n e ) .TARIQ t h i s method neostigmine methyl s u l p h a t e i n i n j e c t i o n s was t i t r a t e d w i t h sodium t e t r a p h e n y l b o r a t e . 2 . Diamandis and C h r i s t o p o u l o s ( 2 6 ) developed a p o t e n t i o m e t r i c t i t r a t i o n o f neostigmine bromide i n pharmaceutical compounds by t i t r a t i n g them w i t h sodium t e t r a p h e n y l b o r a t e . Various common i o n s ( i n c l u d i n g c a r b o n a t e . The r e s u l t s were c o r r e c t e d f o r a blank v a l u e . and t a n n a t e ) did not i n t e r f e r e but thiamine . AL-BADR AND M. c i t r a t e .7 .

T h i s method was adopted f o r d e t e r m i n a t i o n o f neostigmine i n powders. and add 40 m l of sodium hydroxide s o l u t i o n (1 i n 1 0 ) . . T r a n s f e r a q u a n t i t y o f t h e powder.15 o f neostigmine bromide. . 0 2 N s u l p h u r i c a c i d i s e q u i v a l e n t t o 6. Each m l of 0 . I ! . Each m l o f 0. add 20 m l o f a 50% w/v s o l u t i o n o f sodium hydroxide and 0 . i n j e c t i o n s . i n a 500 m l Kjeldahl f l a s k .02 N s u l p h u r i c a c i d i s e q u i v a l e n t t o 0. . e q u i v a l e n t t o 0. and make t h e n e c e s s a r y c o r r e c t i o n .36 m l p e r minute with 0.NEOSTIGMINE 421 The s o l u t i o n was t i t r a t e d p o t e n t i o m e t r i c a l l y a t 0. t o a semi-micro ammonia d i s t i l l a t i o n a p p a r a t u s . Perform a blank d e t e r m i n a t i o n .688 mg o f C13H22N206S' B r i t i s h Pharmacopoeia (1973) (17) r e p o r t e d t h e f o l l o w i n g procedure f o r t h e a s s a y of n e o s t igmine t a b l e t s : Weigh and powder 2 t a b l e t s . a c c u r a t e l y weighed.02 N s u l p h u r i c a c i d . United S t a t e s Pharmacopeia X I X (1980) (12) d e s c r i b e d t h e f o l l o w i n g procedure f o r t h e a s s a y o f neostigmine methyl s u l p h a t e : Place about 100 mg o f neostigmine methyl s u l p h a t e . 5 m l o f a 2% s o l u t i o n o f octan-2-01 i n l i q u i d p a r a f f i n and complete t h e a s s a y d e s c r i b e d under neostigmine methyl s u l p h a t e . add methyl p u r p l e TS t o t h e s o l u t i o n i n t h e r e c e i v e r . . d i s t i l about 150 m l o f t h e c o n t e n t s of t h e f l a s k . d i s s o l v e i n 150 m l o f w a t e r . and t i t r a t e with 0. drops and s y r u p s . (above) b e g i n i n g a t t h e words "Pass a c u r r e n t o f steam . Connect t h e f l a s k by means o f a d i s t i l l a t i o n t r a p t o a well-cooled condenser t h a t d i p s i n t o 25 m l o f b o r i c a c i d s o l u t i o n ( 1 i n 2 5 ) . The end-point was d e t e c t e d by a t e t r a p h e n y l b o r a t e s e l e c t i v e e l e c t r o d e .006064 g o f C12H19BrN202.01 M sodium t e t r a p h e n y l b o r a t e a t 2 2 O 5 20 w i t h continuous s t i r r i n g .

p o i n t . A.2. and t i t r a t e with 0 . add 5% HgII a c e t a t e s o l u t i o n . and t i t r a t e with 0.p o i n t . i n a m i x t u r e o f 70 m l of g l a c i a l a c e t i c a c i d and 20 m l o f m e r c u r i c a c e t a t e TS. Surmann et a 1 (28) r e p o r t e d a t i t r a t i o n method f o r t h e pharmaceutical h y d r o c h l o r i d e s and hydrobromides i n non-aqueous media. and make any n e c e s s a r y c o r r e c t i o n .1% g e n t i a n v i o l e t i n a c e t i c a c i d c o n t a i n i n g 3% m e r c u r i c a c e t a t e as indicator. add 4 d r o p s o f c r y s t a l v i o l e t TS. Kracmarova and Kracmar (29) r e p o r t e d t h e f o l l o w i n g non-aqueous tit r a t i o n : Evaporate t h e sample o f t h e eye d r o p s (5 ml) on a w a t e r b a t h t o d r y n e s s . Each m l of 0 .1 N perchloric acid t o a b l u e e n d . Bayer and Posgay (27) s u g g e s t e d a p e r c h l o r i c a c i d t i t r a t i o n method f o r t h e d e t e r m i n a t i o n o f neostigmine i n pharmaceutical p r e p a r a t i o n s . 1 N p e r c k l o r i c a c i d t o a b l u e e n d .A.2 T i t r i m e t r i c Methods Non-aqueous T i t r a t i o n United S t a t e Pharmacopeia X I X (1980) (12) d e s c r i b e d t h e f o l l o w i n g procedure f o r t h e a s s a y of neostigmine bromide: Dissolve about 750 mg of neostigmine bromide a c c u r a t e l y weighed. The neostigmine s o l u t i o n was t i t r a t e d with 0 .2% i n anhydrous a c e t i c a c i d ) (2 d r o p s ) a s i n d i c a t o r . AL-BADR AND M.32 mg o f C12H19BrN202 (12). 1 N p e r c h l o r i c a c i d i s e q u i v a l e n t t o 30. add c r y s t a l v i o l e t (0. d i s s o l v e t h e r e s i d u e i n anhydrous a c e t i c a c i d (2 x 20 m l ) . Perform a blank determinat i o n . T h i s method i n v o l v e d i r e c t t i t r a t i o n o f drug w i t h p e r c h l o r i c a c i d i n a c e t i c anhydride medium. w i t h n a p h t h o l benzein o r Sudan Red B an i n d i c a t o r f o r hydroc h l o r i d e s f o r hydrobromides r e s p e c t i v e l y .TARIQ 8. 1 N p e r c h l o r i c a c i d which h a s been s t a n d a r d i z e d with anhydrous potassium c a r b o n a t e w i t h 0.

The drug sample i s d i s s o l v e d i n water and t h e solution is t r e a t e d with 2-3 m l of d i l u t e s u l p h u r i c a c i d . 1 N sodium t h i o s u l p h a t e . d i l u t e d t o 25 m l with w a t e r and f i l t e r e d . 5 M 1 o f sample c o n t a i n i n g 5 ug of neostigmine i s mixed w i t h 1 m l o f h o r s e serum. When b o t h a r e p r e s e n t t h e sum o f t h e two i s determined by above method and only p i l o c a r p i n e i s determined a l k a l i m e t r i c a l l y . 2-3 m l o f 10% potassium i o d i d e s o l u t i o n and 5 m l of 0 .2% cresol r e d s o l u t i o n and 37 m l o f w a t e r . a r g e n t i m e t r i c a l l y o r mercurim e t r i c a l l y . 8. After 15 minutes add 5 m l o f 3% a c e t y l c h o l i n e p e r c h l o r a t e s o l u t i o n and r e a d j u s t . The r e l a t i v e e r r o r i n determining t h e two d r u g s i n eye-drops is 3. 10 m l o f f i l t r a t e i s t i t r a t e d with 0 .2. Neostigmine methyl s u l p h a t e is determined by t h e d i f f e r e n c e . Neostigmine methyl s u l p h a t e and p i l o c a r p i n e HC1 a r e determined by r e a c t i o n with i o d i n e s o l u t i o n i n d a r k . t h e d i f f e r e n c e between t h e t i t r e s corresponds t o neostigmine bromide.3 I o d i m e t r i c Methods Koka (30) developed an i o d i m e t r i c determinat i o n o f p r o s e r i n e (neostigmine methyl s u l p h a t e ) i n drug f o r m u l a t i o n s .56 and 2. 1 m l o f 0.3 B i o l o g i c a l Method Buckles and Bullock (32) r e p o r t e d t h e a p p l i c a t i o n o f enzyme i n h i b i t i o n t o t h e e s t i m a t i o n o f s m a l l q u a n t i t i e s o f drugs p o s s e s s i n g a n t i c h o l i n e s t r a s e a c t i v i t y .85 r e s p e c t i v e l y .NEOSTIGMINE 429 (5 ml). Mitchenko and Kirichenko (31) r e p o r t e d t h e u s e of i o d i m e t r i c method f o r t h e determinat i o n o f neostigmine methyl s u l p h a t e and p i l o c a r p i n e HC1 i n eye-drops. 9 . 8. f i l t e r i n g t h e m i x t u r e and t i t r a t i n g u n r e a c t e d i o d i n e w i t h sodium t h i o s u l p h a t e s o l u t i o n . 1 N i o d i n e . The m i x t u r e i s h e a t e d t o 4OoC and pH a d j u s t e d t o 7 . mix and t i t r a t e a g a i n t o a b l u e endp o i n t . The method is based on t h e i n h i b i t i o n of t h e pseudocholinesterase a c t i v i t y of horse serum.

i t s absorbance b e i n g measured a t 615 nm as a r e a g e n t b l a n k . 5 m l o f 0. The p e r c e n t a g e i n h i b i t i o n i s a l i n e a r f u n c t i o n o f t h e l o g c o n c e n t r a t i o n of n e o s t i g m i n e methyl sulphate.8 ) and 2 m l o f a 0. 0 . Belikov -e t a1 (34) have used sulphonepht h a l e i n dyes as e x t r a c t i o n r e a g e n t s f o r t h e e x t r a c t i o n o f q u a t e r n a r y ammonium compounds. 5 % . 5 m l of 0. C o r r e c t f o r nonenzymic h y d r o l y s i s by c o n d u c t i n g an experiment w i t h 1 m l b u f f e r i n s t e a d o f h o r s e serum and c a r r y o u t a b l a n k w i t h 5 m l w a t e r i n s t e a d o f sample. (33) d e s c r i b e d a s p e c t r o photometric determination o f neostigmine i n t a b l e t s and ampoules. A l t e r n a t i v e l y 1 0 m l of chloroform e x t r a c t was t r e a t e d w i t h 10 ml of 0 . 0 .1% dye . d i s t r i b u t i o n c o e f f i c i e n t and s e n s i t i v i t y of reactions f o r t h e ion a s s o c i a t i o n complexes o f n e o s t i g m i n e methyl s u l p h a t e . 8 and 8 . Xmax. T a b l e t p r e p a r a t i o n s c o n t a i n i n g n e o s t i g m i n e methyl s u l p h a t e were powdered and d i s s o l v e d i n 50 m l o f w a t e r and 1 m l of s o l u t i o n was mixed w i t h 3 m l o f c i t r a t e .1% s o l u t i o n o f bromothymol b l u e i n 0 .1 C o l o r i m e t r i c Methods Belal et a l . 0 1 N sodium hydroxide. They r e p o r t e d t h e optimum pH v a l u e . The m i x t u r e was e x t r a c t e d w i t h chloroform b e f o r e measurement o f i t s absorbance a t 416 nm vs a r e a g e n t b l a n k . 0 d u r i n g 15 minutes by dropwise a d d i t i o n o f 0. 8. 0 1 N sodium hydroxide and t h e aqueous l a y e r was s e p a r a t e d and made up t o 25 m l with water.p h o s p h a t e b u f f e r s o l u t i o n (pH 7 . 9 . TARIQ 430 t h e pH t o 7 . I n a g e n e r a l procedure f o r d e t e r m i n i n g t h e q u a t e r n a r y ammonium compounds. The c a l i b r a t i o n graph were r e c t i l i n e a r f o r 0 .025 N sodium h y d r o x i d e and r e c o r d t h e volume of a l k a l i used.1 .01% s o l u t i o n of compound is added t o 5 m l o f a b u f f e r s o l u t i o n o f a p p r o p r i a t e pH.A. 6 mg. The r e c o v e r y by t h i s method was 1 0 0 .4. 2 . A. AL-BADR AND M.4 S p e c t r o p h o t o m e t r i c Methods 8. Maintain t h e pH between 7 .

An a l i q u o t of i n j e c t i o n preparation equivalent t o 5 mg o f n e o s t i g m i n e methyl s u l p h a t e was a c i d i f i e d and any phenol o r p-hydroxy b e n z o a t e p r e s e r v a t i v e p r e s e n t was e x t r a c t e d w i t h chloroform. Kracmarova and Kracmar (29) d e s c r i b e d a s p e c t r o p h o t o m e t r i c method f o r e v a l u a t i o n of n e o s t i g m i n e bromide and n e o s t i g m i n e methyl sulphate with regards t o t h e degradation p r o d u c t s . Tsubouchi (35) r e p o r t e d a s p e c t r o p h o t o m e t r i c d e t e r m i n a t i o n o f o r g a n i c c a t i o n s by s o l v e n t e x t r a c t i o n w i t h tetrabromophenolp h t h a l i e n e t h y l e s t e r .01 WM of n e o s t i g m i n e was e x t r a c t e d w i t h 2 m l o f 0. The m i x t u r e was d i l u t e d t o 10 m l w i t h water. The r e s i d u a l aqueous s o l u t i o n was made a l k a l i n e and h e a t e d on steam b a t h f o r 30 m i n u t e s and t h e 3.2. 8. d i l u t e t o 25 m l w i t h water and shake f o r 2 minutes w i t h 1 0 m l of 1.(dimethylamino) phenol formed is determined by UV-spectrophotometry.(dimethy1amino)phenol.11%. To 5 m l o f i n j e c t i o n s o l u t i o n . Relative e r r o r i s less t h a n 2 % .2 U l t r a v i o l e t SDectronhotometric Method Rita (36) developed a u l t r a v i o l e t spectrophotometric assay o f neostigmine methyl s u l p h a t e as t h e a l k a l i n e h y d r o l y s i s p r o d u c t . 2 m l o f 0 . The absorbance was r e c o r d e d a t 261 nm f o r n e o s t i g m i n e bromide o r a t 260 nm f o r n e o s t i g m i n e methyl s u l p h a t e o r a t 266 nm f o r b o t h o f t h e compounds.07% e t h a n o l i c tetrabromophenolphthalien e t h y l e s t e r (potassium s a l t ) and 5 m l o f b o r a t e phosphate b u f f e r (pH l o ) .4. 5 1. . 3.11 of t h e sample c o n t a i n i n g less t h a n 0. S e p a r a t e and f i l t e r t h e o r g a n i c phase and measure i t s absorbance a t 615 nm a g a i n s t r e a g e n t b l a n k . t h e complex is e x t r a c t e d i n t o 5 m l o f chloroform and absorbance o f o r g a n i c phase i s measured a t 400-434 nm.43 I NEOSTIGMINE s o l u t i o n i s added.dichloroethane. The c o e f f i c i e n t o f v a r i a t i o n was 1. 0 5 N H2SO4 was added.

bromoc r e s o l p u r p l e s o l u t i o n (2 g i n 15 m l o f water and 3 . r e p e a t t h e e x t r a c t i o n w i t h 2 m l of chloroform and d i l u t e t h e combined e x t r a c t s w i t h chloroform t o 50 m l .4. C h a r a c t e r i s t i c band f o r neostigmine bromide and n e o s t i g m i n e methyl s u l p h a t e a r e observed. TARIQ 432 Mytchenko and Kyrychenko (37) r e p o r t e d a spectrophotometric determination o f p r o s e r i n e ( n e o s t igmine methyl s u l p h a t e ) i n m e d i c i n a l f o r m u l a t i o n s . Kracmarova and Kracmar (29) r e p o r t e d s p e c t r o p h o t o m e t r i c a n a l y s i s of n e o s t i g m i n e i n i n f r a r e d r e g i o n . . Shake f o r 1 minute i n a s e p a r a t i n g f u n n e l .I R Mynka et a1 (38) r e p o r t e d t h e i n f r a r e d s p e c t r o s c o p y o f drugs o f t h e amide class.3 I n f r a r e d S p e c t r o p h o t o m e t r i c Methods . 6 ) (5 m l ) .4. 6 6 % . A.5% s o l u t i o n i s f i l t e r e d and t h e absorbance o f f i l t r a t e i s d i r e c t l y r e c o r d e d a t 260 nm. A n a l y s i s can be done w i t h a maximum e r r o r o f 1 . The t a b l e t s are g r i n d e d i n w a t e r and shaked. AL-BADR AND M. The I R spectrum of n e o s t i g m i n e methyl s u l p h a t e was determined and i n t e r p r e t e d by measurement a t 1732 cm-l. and f i l t e r t h e chloroform l a y e r . 6 6 % . 5 ml) add b u f f e r s o l u t i o n (pH 4 . 8. T h i s method can a l s o be used f o r a n a l y s i s o f n e o s t i g m i n e i n eye drops ( c o n t a i n i n g a l s o p i l o c a r p i n e HC1) a f t e r d i l u t i o n . 8. 2 m l o f 0 .potassium hydroxide d i l u t e d t o 250 m l w i t h w a t e r ) (S ml) and chloroform (25 m l ) .4 Photometric Method Kracmarova and Kracmar (29) r e p o r t e d a p h o t o m e t r i c method €or t h e e v a l u a t i o n o f n e o s t i g m i n e bromide and n e o s t i g m i n e methyl s u l p h a t e as f o l l o w s : To t h e i n j e c t i o n s o l u t i o n ( 1 . 1 N . The accuracy o f method i s w i t h i n ? 0 .A. Grind t h e sample w i t h l i q u i d p a r a f f i n (12 d r o p s ) and r e c o r d t h e spectrum i n a sodium c h l o r i d e c e l l i n t h e range 3-15 1-1. 0.

N e o s t i g m i n e an d m -h y d r oxyphen y 1t r i m e t h y 1ammon iurn bromide are p r e c i p i t a t e d from u r i n e w i t h aqueous bromine. 1 p a p e r .1 Paper Chromatographic Method Giebelmann (39) developed a p a p e r chromatog r a p h i c d e t e c t i o n f o r phenoxy compounds having a phenyl e s t e r f u n c t i o n a l group using t h e following s o l v e n t s . butanol-conc-aqueous ammonia-water (3: 1:4) as s o l v e n t . . 8. and Dragendorff r e a g e n t as t h e d e t e c t i n g a g e n t . methanol a c e t o n e t r i e t h a n o l a m i n e (100: 100: 3) and b u t a n o l water ( 6 : l ) . The p r e c i p i t a t e i s e x t r a c t e d with 50% methanol. 3 dimethylaminophenol and (3-hydroxyphenyl) trimethylammonium s a l t s . shake f o r 30 seconds. d i l u t e t o 100 m l w i t h w a t e r and measure t h e e x t i n c t i o n a t 570 nm. 3 .5-10 1-16 of n e o s t i g m i n e can be d e t e c t e d w i t h M i l l o n ' s reagent.86. The Rf v a l u e o f n e o s t i g m i n e was between 0 .5. (40) r e p o r t e d an i n v e s t i g a t i o n on t h e metabolism o f n e o s t i g m i n e i n p a t i e n t s w i t h myasthenia g r a v i s . Kracmarova and Kracmar ( 2 9 ) d e s c r i b e d procedures f o r t h e determination o f n e o s t i g m i n e bromide and n e o s t igmine methyl sulphate i n various pharmaceutical preparat i o n s .5 Chromatographic Methods 8. As l i t t l e as 2. 1 N potassium hydroxide (20 m l ) . 2 N h y d r o c h l o r i c a c i d and s u b m i t t e d t o chromatography on Whatman NO 541 p a p e r w i t h butanol-ethanol-water-acetic a c i d (32: 8 : 1 2 : 1) as s o l v e n t . Scott et a l . a s w e l l as f o r t h e chromatographic control of t h e degradation products. T h i s e x t r a c t is a p p l i e d t o Amberlite CG-50 r e s i n a t pH 6. 5 . w i t h t h e u s e o f Whatman No.butanol-20% formic a c i d ( 4 : 1 ) . Ten b a s e s a r e e l u t e d w i t h 0 . f i l t e r t h e aqueous l a y e r .0 .NEOSTIGMINE 433 To a 25 m l p o r t i o n add 0 .

a b s o r p t i o n . i n t a n k 8 x 11 x 15% i n . t h e paper i s f o l d e d i n t o a c y l i n d e r and c l i p p e d . Location under u l t r a v i o l e t l i g h t . 1 o r No. 15 t o 20 minutes. S o l v e n t : 4 .844. 5 8 ) .A. Time of run. This may be used f o r s e v e r a l weeks i f water i s added from time t o time t o keep t h e s p e c i f i c g r a v i t y a t 0. 5 1-11 of a 1%s o l u t i o n . I t can be s t o r e d immediately. s h e e t 14 x 6 i n . Sample: 2 . Development: Ascending. weak r e a c t i o n . A. b l o t t i n g . s h e e t 17 x 19 cm. b u f f e r e d by d i p p i n g i n a 5% s o l u t i o n o f sodium hydrogen c i t r a t e .b u t a n o l . 8 g of c i t r i c a c i d i n a m i x t u r e of 130 m l o f water and 870 m l o f n . i n 2 N a c e t i c a c i d i f possible. and d r y i n g a t 250 f o r 1 hour. otherwise i n 2 N h y d r o c h l o r i c a c i d . A p l a t e .843 t o 0. 4 s h e e t s b e i n g run a t a time. weak r e a c t i o n . Sample: 5 p1 o f 1 t o 5% s o l u t i o n s i n e t h a n o l o r chloroform. Development: Ascending. Rf 0. 5 h o u r s . TARIQ 434 Clarke (11) d e s c r i b e d t h e f o l l o w i n g : (1) Paper: Whatman No. . Solvent: A c e t a t e b u f f e r (pH 4 . t h e c y l i n d e r is i n s e r t e d i n t h e b e a k e r c o n t a i n i n g t h e s o l v e n t which i s n o t removed from t h e oven. E q u i l i b r a t i o n : The b e a k e r c o n t a i n i n g t h e solvent is equilibrated i n a thermostatically c o n t r o l l e d o v e r a t 95O f o r about 1 5 minutes. 3. 1.g l a s s d i s k t h i c k l y smeared with s i l i c o n e g r e a s e may s e r v e as a cover. locat ion reagents : Iodoplati na te spray . ( 2 ) Paper: Whatman No. 2 N sodium hydroxide. bromocresol green s p r a y . implegnated by d i p p i n g i n a 10% s o l u t i o n o f t r i b u t y r i n i n acetone and d r y i n g i n a i r .20. AL-BADR AND M. o r ethanol. Time of run.

2 Thin-Layer Chromatography (TLC) Anand (41) r e p o r t e d a t h i n . The samples o f neostigmine were a p p l i e d on TLC p l a t e s c o a t e d w i t h alumina-D and were developed f o r 30 minutes w i t h chloroform-benzenemethanol (5:4:1) t o g i v e a good s e p a r a t i o n . Neostigmine was d e t e c t e d w i t h i o d i n e vapours o r w i t h D r a g e n d o r f f ' s r e a g e n t . . (3) Paper and sample a s i n 2 above. 8. The drug i s d e t e c t e d and determined by TLC on c e l l u l o s e and u l t r a v i o l e t o r v i s i b l e s p e c t r o p h o t o m e t r y is done a f t e r f o r m a t i o n o f c o l o r e d compound w i t h 3.00. P o r s t and Kny (42) d e s c r i b e d a n a l y s i s o f neostigmine eye drops u s i n g TLC system and a b s o r p t i o n s p e c t r o s c o p y . a b s o r p t i o n . c o a t e d w i t h a s l u r r y c o n s i s t i n g o f 30 g o f s i l i c a g e l G i n 60 m l o f w a t e r t o g i v e . Development: as i n 2 above. Neostigmine h a s a maxima a t 261 nm. E q u i l i b r a t i o n : The peak c o n t a i n i n g t h e solvent is equilibrated i n a thermostatic a l l y c o n t r o l l e d oven a t 86' f o r about 15 minutes. Rf 1.66.NEOSTIGMINE 435 Location: Under u l t r a v i o l e t l i g h t .l a y e r chromatog r a p h i c and s p e c t r o p h o t o m e t r i c i n v e s t i g a t i o n on t h e i d e n t i f i c a t i o n o f a p h e n o l i c i m p u r i t y i n n e o s t i g m i n e .5. a b s o r p t i o n . 4 ) .a l a y e r 0. Clarke (11) d e s c r i b e d t h e f o l l o w i n g : P l a t e : G l a s s p l a t e . Location: Under u l t r a v i o l e t l i g h t . The u l t r a v i o l e t spectrophotometric technique is a l s o used f o r q u a n t i t a t i v e e s t i m a t i o n . S o l v e n t : Phosphate b u f f e r (pH 7 .25 mm l i k e and d r i e d a t 110 f o r 1 hour. Rf 0. 20 x 20 cm.S-dichloro-pbenzoquinonechlorimine o r sodium n i t r i t e o r 4-dimethylaminobenzaldehyde.

2 5 i n c h e s O. Ward et a 1 (44) d e s c r i b e d a simple GLC a s s a y of neostigmine u s i n g d i e t h y l analog o f neostigmine a s i n t e r n a l s t a n d a r d . 2 1. AL-BADR AND M. The t e m p e r a t u r e was m a i n t a i n e d a t 205OC and t h e flow r a t e o f n i t o g e n was 30 m l p e r minute.11 o f chloroform. i n a t a n k .) packed w i t h Diatomite CQ c o a t e d w i t h 3% o f OV-1. The e x t r a c t i s e v a p o r a t e d and t h e r e s i d u e is d i s s o l v e d i n methanol.5 : 100). Pyridostigmine bromide was used a s i n t e r n a l s t a n d a r d . I t should be changed a f t e r two r u n s . 2 .02. 3% o f OV-17 o r 3 .5. TARIQ 436 Sample: acid. A. 1 u1 o f a 1%s o l u t i o n i n 2 N a c e t i c S o l v e n t : S t r o n g ammonia s o l u t i o n : methanol (1. cm. R f 0.A. 30 minutes. a n i t r o g e n s e l e c t i v e d e t e c t o r was used. The d e t e c t i o n l i m i t was 5 ng/ml and t h e r e was no i n t e r f e r e n c e with o t h e r b a s i c d r u g s . Plasma c o n t a i n i n g neostigmine i s washed w i t h e t h y l e t h e r and b u f f e r e d w i t h potassium iodide-glycine s o l u t i o n .3 Gas Liquid Chromatographic Methods e t a1 (43) developed a s e n s i t i v e and Chan -s e l e c t i v e a n a l y t i c a l method f o r t h e measurement o f neostigmine i n human plasma u s i n g gas chromatography. Development: 2 1 x 21 x 1 0 covered w i t h evaporation. then iodideg l y c i n e . Location r e a g e n t : A c i d i f i e d i o d o p l a t i n a t e s p r a y . 8. Ascending.D.5 1-11 o f m e t h a n o l i c s o l u t i o n was i n j e c t e d i n t o gas chromatographic g l a s s column (2 m x 0 . Neostigmine and i t s analog were d i s s o l v e d i n 25 1. 8 % of SE-30 and s i l a n i s e d . p o s i t i v e r e a c t i o n .r1o f chloroform s o l u t i o n was i n j e c t e d i n t o a . t h e end o f t h e t a n k b e i n g f i l t e r paper t o assist Time o f r u n . E q u i l i b r a t i o n : The s o l v e n t i s allowed t o stand i n t h e tank f o r 1 hour.d r u g complexes are e x t r a c t e d i n t o dichloromethane.

The l i m i t of d e t e c t i o n was 5 ng p e r m l . sodium hydrogen phosphate (0. 2 m x 2 mm I . De Ruyter et a1 (45) developed a r e v e r s e d phase. The method i s based on e x t r a c t i o n of neostigmine i n t o w a t e r . D .s a t u r a t e d dichloromethane. For neostigmine e l u t i o n was c a r r i e d o u t w i t h a s o l u t i o n c o n t a i n i n g sodium h e p t a n e s u l p h o n a t e (0. The c o e f f i c i e n t o f v a r i a t i o n was 1 .5 mM) i n aqueous 20% a c e t o n i t r i l e . 3 5 . The e x t r a c t s a r e s u b m i t t e d t o chromatography on a v a r i e t y o f r e v e r s e d phased columns f i t t e d with 214 nm d e t e c t o r s . For d e t e r m i n a t i o n o f neostigmine i n b i o l o g i c a l f l u i d s .1 7 on Gas-Chrom Q i s used a t 210' with helium (40 ml/minute) a s a carrier gas and flame i o n i z a t i o n d e t e c t o r .01 M) and t e t r a m e t h y l ammonium c h l o r i d e (2. The mobile phase (2 m l p e r minute) was a d j u s t e d t o pH 3 with s u l p h u r i c a c i d . The r e l a t i v e r e t e n t i o n time of t h e drug and t h e s t a n d a r d is 1 t o 1 .p a i r l i q u i d chromatography o f q u a r t e r n a r y ammonium compounds €or t h e determination of pyridostigmine. and edrophonium i n b i o l o g i c a l f l u i d s .01 M ) . i o n . 6 mm) o f U l t r a s p h e r e o c t y l ( 5 pm) provided with an RP-2 pre-column was used.437 NEOSTIGMINE gas chromatograph equiped w i t h a flame i o n i s a t i o n d e t e c t o r and 1 . g l a s s column packed with 5 % O V . n e o s t i g m i n e . Davison e t a 1 (46) d e s c r i b e d a method f o r simultaneous monitoring of plasma l e v e l of neostigmine and p y r i d o s t i g m i n e i n man. For b i o l o g i c a l e x t r a c t s a column (15 cm x 4 . t h e drug i s e x t r a c t e d i n t o chloroform w i t h added 3-diethylcarbamoyloxy-"N-trimethylanil inium i o d i d e as t h e i n t e r n a l s t a n d a r d . 5 % .e x t r a c t i o n is done w i t h t e t r a b u t y l ammonium hydrogen s u l p h a t e t o enhance t h e r e c o v e r y t o more t h a n 86%. edrophonium was used a s an i n t e r n a l s t a n d a r d . The c a l i b r a t i o n graphs were r e c t i l i n e a r f o r u p t o 400 ng per m l . t h e n b a c k . The a s s a y i n v o l v e p r e l i m i n a r y ion p a i r .

p a i r e x t r a c t i o n o f t h e drugs and t h e i r m e t a b o l i t e s i n t o dichloromethane and i n (dichloromethane-acetone) . and t h e methyl i o d i d e r e l e a s e d i s measured w i t h a 63Ni e l e c t r o n . . The method i n v o l v e s a p r e l i m i n a r y . Chan and Dehghan (48) d e s c r i b e d a GLC method f o r i s o l a t i o n and d e t e r m i n a t i o n o f neostigmine. r e s p e c t i v e l y . s e l e c t i v e i o n . On t h e column t h e q u a r t e r n a r y ammonium i o d i d e s a r e t h e r m a l l y decomposed. o p e r a t e d a t 14SoC-17O0C with helium o r n i t r o g e n a s c a r r i e r gas (20 m l p e r ml). 8 m x 2 mm) o f Porapak Q (800-100 mesh). The q u a r t e r n a r y s a l t s i n t h e serum o r u r i n e a r e first converted w i t h potassium t r i i o d i d e (K13) i n t o t h e i r i o d i e s which a r e e x t r a c t e d i n t o chloroform. The e x t r a c t i s a n a l y s e d by GLC (10% o f OV-17 on Chromosorb W-AW (100-120 mesh) with a n i t r o g e n . Pohlmann and Cohan (47) developed a s i m p l i f i e d d e t e c t i o n o f q u a r t e r n a r y ammonium compounds by gas chromatography.s e n s i t i v e d e t e c t o r . The e x t r a c t i s evaporated i n vacuum and t h e r e s i d u e is d i s s o l v e d i n water o r hexane. AL-BADR AND M. The a n a l y s i s o f e x t r a c t was done by GLC on a column o f 3% (drug) o r 10% ( m e t a b o l i t e ) o f OV-17 on Chromosorb W AW. Extraction y i e l d s a r e a t l e a s t 95% and down t o 1 0 f-mol of q u a r t e r n a r y ammonium compound can be d e t e c t e d w i t h good r e p r o d u c i b i l i t y . A. Chromosorb 101 (80-100 mesh) o r Chromosorb 105 (80-100 mesh). The s o l u t i o n is i n j e c t e d on t o a column ( 1 . p y r i d o s t i g m i n e and t h e i r m e t a b o l i t e s i n human b i o l o g i c a l f l u i d s .c a p t u r e detector. TARIQ e x t r a c t i o n o f t h e drugs and i n t e r n a l s t a n d a r d s (3-diproypl carbamoyloxy) 1-methyl pyridinium bromide) w i t h t h e u s e o f potassium i o d i d e and o f g l y c i n e b u f f e r .438 A. The method h a s been a p p l i e d t o p y r i d o s t i g m i n e . neostigmine and a c e t y l c h o l i n e . The calib r a t i o n graphs a r e r e c t i l i n e a r and reproduced o v e r t h e range 5-100 ng p e r m l o f e i t h e r drugs i n 3 m l samples.

8. l . T h i s method i s based on t h e h y d r o l y s i s of neostigmine and n i t r a t i o n o f r e s u l t i n g phenol. sodium t e t r a p e n t y l b o r a t e .s e n s i t i v e d e t e c t o r . As l i t t l e as 3 ng/ml o f neostigmine and upto 50 ng/ml o f i t s m e t a b o l i t e can be determined i n b i o l o g i c a l samples. (See The end p o i n t was d e t e c t e d by a Section 8.lO-phenanthroline . The s o l v e n t s used f o r t h e s e compounds were 1. a major m e t a b o l i t e o f neostigmine.1.439 NEOSTIGMINE with a n i t r o g e n . The s e l e c t i v i t i e s r e l a t i v e t o u n i v a l e n t c a t i o n s were mostly b e t t e r than 10-4. followed by polarography o f . 5 mg/ml o f t h e drug.6 I o n .7diphenyl. 1 M w i t h a response o f 59 mV p e r decade change i n t h e c o n c e n t r a t i o n o f t h e drug.Z-dichloroethane and n i t r o b e n z e n e . For t h e d e t e r m i n a t i o n o f 3-hydroxytrimethyl a n i l i n i u m i o n . 3-hydroxy-N-methylpyridinium ion was used a s a i n t e r n a l s t a n d a r d .).S e l e c t i v e E l e c t r o d e s Method Kina et a1 (49) d e s c r i b e d a method of neostigmine a n a l y s i s based on i o n .2. 8.1.7 P o l a r o g r a p h i c Method Novotny (50) devised a p o l a r o g r a p h i c method f o r t h e d e t e r m i n a t i o n o f neostigmine i n pharmaceutical p r e p a r a t i o n s having a c o n c e n t r a t i o n o f 0 . The exchange components used were c r y s t a l v i o l e t . The c h o i c e of s o l v e n t s a f f e c t e d with s e l e c t i v i t i e s o f t h e membranes b u t t h e c h o i c e o f ion-exchange components d i d n o t . tetraphenylborate-selective e l e c t r o d e . The e l e c t r o d e s could be used i n a n a l y s i s of mixed pharmaceutical preparations.s e l e c t i v e e l e c t r o d e s s e n s i t i v e t o some o r g a n i c compounds used a s drugs. The u s e f u l c o n c e n t r a t i o n range o f e l e c t r o d e s was 10 VM t o 0 .p h e n a n t h r o l i n e . 4. l O . d i p i c r y l a m i n e .a s s o c i a t i o n e x t r a c t i o n method. Liquid membrane e l e c t r o d e s s e n s i t i v e t o n e o s t i g mine have been made by t h e i o n . Diamandis and C h r i s t o p o u l o s (26) r e p o r t e d a p o t e n t i o m e t r i c t i t r a t i o n o f neostigmine and o t h e r pharmaceutical compound i n pharmaceutical formul a t i o n with sodium t e t r a h y d r o b o r a t e .

King Saud U n i v e r s i t y f o r h i s t e c h n i c a l assist a n c e and Mr. 10 M 1 of KOH s o l u t i o n (20%) is added and t h e volume made upto 1 4 . 5 m l w i t h water.A. C o l l e g e of Pharmacy. The e s t i m a t i o n i s c a r r i e d out by pol a rogra phy. The neostigmine s o l u t i o n ( 0 . TARIQ r e s u l t i n g d e r i v a t i v e . . Butt €or h i s s e c r e t a r i a l a s s i s t a n c e i n t y p i n g t h e manuscript. 6 m l of 0 . 5 M 1 of wa te r and 2 m l o f conc. Syed A l i Jawad. both with and wit hout a known amount of neostigmine. 1 bll o f KOH s o l u t i o n (20%) i s added and warmed on t h e b a t h f o r 20 minutes and a ga i n e va por ated t o dryness. w i t h a galvanometer s e n s i t i v e t o 10-9 amp. s o l u t i o n o f t h e unknown c o n c e n t r a t i o n o f neostigmine i s t r e a t e d as d e s c r i b e d above. AL-BADR AND M. Acknowledgements The a u t h o r s would l i k e t o thank Mr. To determine t h e c o n c e n t r a t i o n o f neostigmine i n an unknown s o l u t i o n . A. HN03 (65%) i s added and warmed f o r 20 minutes and cooled. 1 % ) i s e vapor ated t o dr yness i n a 50 m l be a ke r on a water b a t h . Ta nvir A. 0 . 2 t o 0 .

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&**. Rubha. . M-Badrr* ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 16 445 Copyright 0 1987 by Academic Press. S a L h A .PIRENZEPINE DIHYDROCHLORIDE ffumeiida A . and A b d u U a h A . Inc. All rights of reproduction in any form reserved. ER-Ob&id*.

2 7. 3 Radioimmunoassays 6. Pharmacology Antisecretory Effects Efficacy Adverse React ions Tissue Selectivity 7 .4 Acknowledgement Re ferences .3 2.1 7. Melting Range Solubility Distribution Ratio Spectral Properties Crystal Structure Synthesis 4.4 2.3 7. Description 1.2 Distribution 6 . Pharmacokinetics 6.5 Appearance 2.HUMEIDA A.1 2. 3 Metabolism 6 . 4 Excretion 7.1 Nomenclature 1. 446 Contents 1. 4 Elemental Composition 1. Physico-chemical Properties 2.2 Chromatographic Methods 5 . Methods of Analysis 5.1 Spectrophotometric Methods 5.2 Formulae 1 .5 3. EL-OBEID ETAL. Stability 5. 3 Molecular Weight 1 .1 Absorption 6. 5 Mechanism of Action 7.2 2.

2 (base) Structural I CH2- . G a s t e r i l .p y r i d o [ 2 . 1. 11. G a s t r o z e p i n .3-b] [ 1 . Ulcosan.1. 3-b] [ 1 . Tabe. 447 Descrivt ion 1.11-Dihydro-ll-[(4-methyl-l-piperazinyl) a c e t y l ] -6H-pyrido[ 2.1 Nomenclature 1.3 Trade Names B i s v a n i l .[ (4-Methyl-1-piperazinlyl) a c e t y l ] .PIRENZEPINE DIHYDROCHLORIDE 1. P i r e n z e p i n e 1.1.2 Formulae 1.1 Empirical (dihydrochl o r i d e ) C 9H2 3C 12N502 C19H21N502 1. 1. Leblon.6 (5 H) -one d i h y d r o c h l o r i d e monohydrate. H 2 0 NwN CH3 . 4 ] b e n z o d i a z e p i n 6-one d i h y d r o c h l o r i d e monohydrate.2.1 Chemical Names 5.2 G e n e r i c Names L-S 519.2. 4 ] b e n z o d i a z e p i n .1.2HC1.

1.4 Elemental Composition Dihydrochloride salt: C 53.11% (2).46%. [ 29868-97-11 as dihydrochloride salt. 2. N 19. 1.3 Molecular Weight 424. Free base: C 64. N 16.3 CAS No. 2.52%. H 6.HUMEIDA A. Physico-chemical Properties 2.94%.42 as free base. C1 16. [28797-61-71 as free base. H 5. 448 1. Pirenzepine is unusual because of its pronounced hydrophilic properties. 1.1 Melting Range Pirenzepine dihydrochloride melts in the range 250-252OC (corrected) with decomposition (I). but its physioco-chemical properties differ considerably from those of other tricyclic compounds which are used as centrally-acting drugs.78%.33 as dihydrochloride salt 351.3 Distribution Ratio Pirenzepine is a tricyclic compound. slightly soluble in methanol and practically insoluble in ether (1).02%.71% (1). 0 9.5 Appearance Pirenzepine dihydrochloride is a white crystalline substance (2). Comparison of the distribution coefficient of pirenzepine with those of other . 2. 0 7.2 Solubility Pirenzepine dihydrochloride is readily soluble in water.93%. EL-OBEID ETAL.50%.2.

PIRENZEPINE DIHYDROCHLORIDE 449 t r i c y c l i c drugs a t pH 7. Each o f t h e f i v e elements can be a l l o c a t e d a hydrophobic s u b s t i t u e n t consant (TX) and compared with t h o s e assigned f o r corresponding s t r u c t u r a l elements i n imipramine molecule (Table 2 ) .4 $Ppe---Chlorpromazine 3160 CNS e f f e c t s + 1600 + 250 + Dibenzepine 51 + Pirenzepine 0. . A p o s i t i v e T X value i n d i c a t e s contribution t o t h e l i p o p h i l i c c h a r a c t e r of t h e molecule and a negative T X value i n d i c a t e s contribution t o h y d r o p h i l i c i t y of t h e molecules. Drug - H 7. Table 1: D i s t r i b u t i o n c o e f f i c i e n t of pirenzepine and o t h e r t r i c y c l i c drugs.23 - Cyprohept adine Imipramin Eberlein -e t a1 (3) analysed t h e pirenzepine molecule i n t o f i v e s t r u c t u r a l elements. The r e s u l t s depicted i n Table 1 show t h a t pirenzepine i s 200-13000 l e s s l i p i d s o l u b l e . has been c a r r i e d out (3).4.

50 \ / N-COCH2- nx . Fragment structure TX -CH2-CH2- +10.80 LogBase (experiment a l pH 7 . 4 ) -0.HUMEIDA A. H CH2CH2CH2 ' CH. CH. 65 1. EL-OBEID E T A L .4 9 + 1. .1 .9 7 n ~ ~ LogBase ( c a l c u l a t e d ) + 5.96 + l . fN I 1 4 - n 5 - I m ip r am i n e Pirenzepine ~~~~ Fragment No.96 + \ / N-CH2CH2CH2- Fragment structure + 0.0 .96 + O .64 .02 . 010 LogBase(experimental) + 4. 450 Table 2 : Contribution o f t h e d i f f e r e n t fragments of p i r e n z e p i n e and imipramine m o l e c u l e s t o t h e i r distribution coefficients.12 (calculated) LogBase +O.

PIRENZEPINE DIHYDROCHLORIDE 45 1 I t can b e shown t h a t t h e s t r u c t u r a l elements 1 and 4. A t p h y s i o l o g i c a l pH. 2. I t is a l s o reported t h a t pirenzepine i n 0 . which i n d i c a t e s t h a t p i r e n z e p i n e a s t h e uncharged free b a s e w i l l be d i s t r i b u t e d uniformly between t h e o c t a n o l and wa te r . Fragments 1 and 4 i n p i r e n z e p i n e c o n s i s t o f amide groupings each w i t h two d i p o l e s which can i n t e r a c t s t r o n g l y with water molecules.4.2 I n f r a r e d Spectrum The i n f r a r e d a b s o r p t i o n spectrum o f p i r e n z e p i n e o b t a i n ed from a potassium bromide d i s p e r s i o n was r e c o r d e d on a Pye . however. As w i l l b e shown l a t e r . c o n t r i b u t e l a r g e l y t o t h e h y d r o p h i l i c i t y of p i r e n z e p i n e . t h e pronounced h y d r o p h i l i c p r o p e r t i e s o f p i r e n z e p i n e e x p l a i n many o f t h e s p e c i f i c pharmac o k i n e t i c c h a r a c t e r i s t i c s of t h e drug.4. t h e r a t i o s h i f t s even more towards w at er s o l u b i l i t y due i o n i z a t i o n o f t h e molecule. The r e s u l t s a g r e e w i t h t h o s e r e p o r t e d i n t h e l i t e r a t u r e (4.0. Adding up t h e i n d i v i d u a l hydrophobic c o n s t a n t s f o r p i r e n z e p i n e m o l e cu l ar fragments a l o g P of 0. shown i n F i gu r e 1. The spectrum. 1 N sodium hydroxide s o l u t i o n shows a b s o r p t i o n maximum a t 295 nm ( 4 ) . i n d i c a t i n g t h a t about 5 times more o f t h e compound i s d i s t r i b u t e d i n t h e aqueous phase t h an i n o c t a n o l . 5 ) .01 i s o b t a i n e d f o r t h e whole molecule. i s c h a r a c t e r i z e d by a maximum a t 283 nm and a minimum a t 264 nm.4) t h e ex p e r i m e n t al l y determined l o g P is .64 (Table 2 ) . 2. Under p h y s i o l o g i c a l c o n d i t i o n s (pH 7. Under t h e same c o n d i t i o n s imipramine accumulates much more s t r o n g l y i n t h e o r g a n i c phase.4 Spectral Properties 2. i n p a r t i c u l a r .1 U l t r a v i o l e t Suectrum The u l t r a v i o l e t a b s o r p t i o n spectrum o f p i r en z ep i n e i n met h an o 1/HC 1 was ob t a i n ed on a Cary 219 s p e c t r o ph o to m e te r . The p y r i d y l group of fragment 2 i n p i r e n z e p i n e i s a l s o expected t o c o n t r i b u t e t o t h e h y d r o p h i l i c i t y of t h e molecule.

. EL-OBEIDETAL. Figure 1 : ULTRAVIOLET SPECTRUM OF PIRENZEPINE DItiYDRDQiLORIDE IN METHANOLIC HCl.452 HUMEIDA A.

H outof-plane bending. 'H Nuclear Magnetic Resonance ('H NMR) SDectrum Pirenzepine s o l u t i o n i n D 2 0 was used t o obtain t h e proton magnetic spectrum on a Varian-T60 NMR spectrometer with TMS a s i n t e r n a l standard.3 t -Amine s a l t N-H 785. 2. and assignment of t h e chemical s h i f t s t o t h e d i f f e r e n t protons is summarized below: . 3463 Amide N-H s t r e t c h 3240 Water molecule Aromatic C-H s t r e t c h 3020. C N stretch.) 1479.C . The spectrum i s shown i n Figure 3.PIRENZEPINE DIHYDROCHLORIDE 453 Unicam SP 1025 spectrometer and i s shown i n Figure 2 . The c h a r a c t e r i s t i c bands of t h e spectrum with t h e assignments a r e l i s t e d below: Frequency (cm-l) Assignment 3520. 1502. 2990 2700 . 1678 Amide C = 0 1605. 1370 Aryl C 800.4. 760 -* - N stretch Aromatic C . 1585. 1432 1 Aromatic C .2400 stretch 1 7 1 7 .

600 400 200 .Microns P VI P 4000 Wavenumber 3000 2500 2000 1800 1600 1400 1000 800 Figure 2 : INFRARED SPECTRUM OF PIRENZEPINE DIHYDROCHLORIDE. KBr DISC.

. 7. . . .0 0 8. . . . . l . . . .I I 400 3 200 100 0 I7 16 Rl u I l . 1 . . l . . 1 . . . . l . l . . . .0 Figure 3 : H' SIR SPECIRUM CF PIRENZEPINE DIHYDROCMRIDE IN DZO WITH ?Hs As IkTERUL REFERENCE. . 1 . . . .O ( 6 ) 4-0 3-0 2-0 1.0 60(ppm)Ei. . .

HUMEIDA A. The carbon chemical s h i f t s .4. assigned on b a s i s of t h e off-resonance s p l i t t i n g p a t t e r n and t h e t h e o r i e s of chemical s h i f t s .7 Mu 1t i p l e t Aromat ic-H 6 8.4 13C Nuclear Magnetic Resonance ( 13C NMR) Spectrum The 13C NMR s p e c t r a of pirenzepine i n a mixture of D20 and formic acid using dioxane a s i n t e r n a l reference a r e obtained using a J e o l FX 100 90 MHz spectrometer a t ambient temperature.3 . r e s p e c t i v e l y .4 Mu 1t i p l e t Aromatic-H 1 2.05 Sing 1e t -N-CH -3 3 3. EL-OBEIDETAL.55 Mu I t i p 1et -CH -(piperazine) -2 8 0 It 4.C - 2 7. of protons 3.15 Mult i p l e t . Figure 4 and 5 show t h e IH-decoupled and off-resonance s p e c t r a . are presented below: . 456 0 I 14 = c 1 2 n N -CH3 1 13 Multiplicity 18 N CH~- Chemical s h i f t (6) 15 I U 6 Proton Assignment No.

4 Figure 4 : 'ti DEaWPLED 13C tW SPEclRuM OF PIRENZEPINE DIHYDROMLORIDE I N DzO/ RIRMIC ACID FlIXlURE W I ' M DIOXANE AS INNWU. a' Figure 5 : .PIRENZEPINE DIHYDROCHLORIDE 451 Dioxan 67. REFERNECE.

83 Quartet 50.15 Doublet 132.31 Sing 1et Carbon Assignment ‘18 ‘15’ ‘16 ‘14’ ‘17 ‘13 clo ‘8 c7 ‘3 ‘6’ c9 c4 c7’ c2 c31 c21 ‘12 ‘6 .41 Doublet 128.43 Doublet 143.57 Singlet 132. 458 IY 10 121 0-c I CH2l3 Chemical S h i f t (61 18 NWN CH3 17 16 Multiplicity 44.88 Doub 1e t 130.87 Triplet 57.72 Triplet 51.77 Triplet 122.22 Doub 1e t 128.24 Singlet 139.57 Doublet 131.74 Doub 1e t 135.61 Singlet 146. EL-OBEIDETAL.21 Singlet 165.77 Singlet 165.HUMEIDA A.

4. P2 a = 13. presented i n Figure 6 . 3 Torr. was obtained on Varian MAT 311 mass spectrometer using ion source pressure of Torr.160 [7]. The mass s p e c t r a l assignments of t h e prominent ions under DCI conditions a r e shown i n Table 3.5 459 Mass SDectra The 70 eV e l e c t r o n impact mass spectrum of pirenzepine.__ 2. ion source temperature of 18OoC and an emission current o f 300 PA. Table 3. m/e Species 392 [M + C ~ H ~ I + 380 [M . Pirenzepine monohydrate monoclinic.PIRENZEPINE DIHYDROCHLORIDE 2. ion source pressure o f 0 . The spectrum is dominated by a quasi-molecular ion (M + 1 ) . A proposed fragmentation p a t t e r n and t h e mass/charge r a t i o s o f t h e major fragments i s shown i n Scheme 1.5 Mass s p e c t r a l assignments o f pirenzepine using DCI with methane a s reagnet gas. ion source temperature of 150°C and emission current o f 300 PA. b 1/c monohydrate and Kojecwas t o be = 12. The d i r e c t chemical i o n i z a t i o n (DCI) spectrum (Figure 7) was obtained on a Finnigan 4000 mass spectrometer using methane gas a reagent with ion e l e c t r o n energy of 100 eV. Two peaks appearing a t m/e 380 and m/e 392 a r e a t t r i b u t e d t o t h e t r a n s f e r o f carbocations from t h e c a r r i e r gas.766 [Sly . + 352 MH+ 35 1 M+ CZH51+ Crystal S t r u c t u r e The c r y s t a l s t r u c t u r e o f pirenzepine f r e e base was s t u d i e d by Ruzic-Toros Prodic (6). The molecular ion i s d e t e c t a b l e a t m/e 351 and t h e base peak a t m/e 113.

Figure 7 : DIRECT WICAL IONIZATION W S SPECIIW OF PIRENZEPINE. . EL-OBEID ETAL. Figure 6 : ELECTRON IMPACT W S SPECTHW! OF PSWZEPINE.460 HUMEIDA A.

m/e 56 . -cH3- Proposed f r a p e n t a t i o n of pirenzepine.CHJ 0 = C-CH-N CH3 .140 H m/e 211 Scheme 1.H @% N 0 -238 =c I %lac.C L u dh 0 =c I ____t +p 'd -CH -43 3-N = CH2 N H2C = m/e 113 5 m/e 351 - -42 =N=CHC 2 2 m/e 113 + w =CH2 m / e 70 @ N+3 ~cH 1 m/e 71 - tH 1 ~ A ~ 1N . -N H. DN -CH.

. . a r e a f f e c t e d by c o n j u g a t i o n .2 [4]O. The seven-membered d i a z e p i n e r i n g is f o l d e d a t “51 . 3.H[5] . -1 = 0.9[41° was o b t a i n e d f o r t h e d i h e d r a l a n g l e between t h e benzene and t h e p y r i d i n e r i n g s . F i n a l R = 0. 462 c = 12.470[3]Ao. The w a t e r molecule is involved i n hydrogen bonds with t h e amide group o f t h e d i a z e p i n e r i n g . . Deviation from t h e v a l u e s expected €or t h e given atom type and h y b r i d i z a t i o n could n o t be e x p l a i n e d by t h e thermal v i b r a t i o n s of t h e atoms (Table 5 ) . EL-OBEID ETAL.372[4]Ao.H(W)1 N[4’](2. 2 [ 41°.C [ 8 ] . Bond d i s t a n c e s and a n g l e s (Table 4) i n t h e f u s e d r i n g s .907[ 31 Ao) . i n v o l v i n g t h e s e atoms. “51 .352[3]. The p y r i d i n e and benzene r i n g s a r e p l a n a r while t h e p i p e r a z i n e r i n g i n a c h a i r conformation.. The r e l a t i v e o r i e n t a t i o n o f t h e p y r i d i n e and p i p e r a z i n e r i n g .. -- Trummlitz e t a l . N [ 4 ’ ] o f t h e p i p e r a z i n e r i n g is hydrogen-bonded t o t h e water molecule by O(W).HUMEIDA A.045 f o r 2681 observed r e f l e x i o n s . Molecular packing i s a l s o i n f l u e n c e d by van d e r Wall i n t e r a c t i o n s . The s h o r t e n i n g o f C[7] .439 171 A’. 1.C[6] and “111 t o form a b o a t conformations. . . w i t h a d i h e d r a l a n g l e o f 2 2 . d i a z e p i n e and p y r i d i n e .128 mm = . p i r e n z e p i n e monohydrochloride (Figure 10) Molecular mechanics (MMPI) and semiempirical quantum chemical (MNDO) c a l c u l a t i o n s showed t h a t t h e c a l c u l a t e d minimum energy conformations o f t h e t r i c y c l e and of t h e e x o c y c l i c amide group are .. Dc = 1 .800[3])and O(W)-H(W)2 O[ 61 c2.8 Ao3. A v a l u e o f 60. B = 113. with N[1][3. u s i n g X-ray a n a l y s i s . U Z = 4.087[2]Ao. “1’1 o f 3. 2 7 7 Mgm-3. O[W]I2.. i n t h e benzene r i n g could be due t o i n t e r m o l e c u l a r c o n t a c t s . The confonnat i o n o f t h e molecule is shown i n F i g u r e 9 and Table 6. e n a b l e s a s h o r t i n t r a m o l e c u l a r c o n t a c t “11 . determined t h e c r y s t a l s t r u c t u r e s o f p i r e n z e p i n e d i h y d r o c h l o r i d e and i t s monoprotonated form.821[3]A0).. benzene. The s t r u c t u r a l formula and atomic numbering o f p i r e n z e p i n e i s given i n F i g u r e 8. p(Mo Ka) 1920. ( 7 ) .

315 (3) 1.454 (3) 1 . 5 (2) 121 (2) 1 .cj( 1 3 j -c (12) C (6) -C (14) -C (7) C(6) -C (14) -C (15) c (7) -c (14) -c (15) C ( 10) -C (15) -N ( 1 1) C(10) -c (15) -c (14) N (11) -C (15) -C (14) N (11) -C (16) -0 (16) N (11) -C (16) -C (17) 0 (16) -C (16) -C (17) C (16) -C (17) -N ( 1 ' ) 1 2 4 . 7 (2) 123 (1) 118 (1) 129.9 (2) 115 (1) 1 2 1 (1) 1 1 9 . 3 8 3 (4) 0.6 1 1 7 . 0 5 (3) 0 .362 (3) 1 . 4 3 8 (3) 1 .9 3 (3) 0 .6 1 2 0 . 0 0 (3) 1 .0 119.387 (3) C(Z)-N(l)-C(12) c (1) -c (2) -c (3) N (1) -C ( 2 ) -H (2) C(3)-C(Z)-H(2) c (2) -c (3) -c (4) C (2) -C (3) -H( 3) C (4) -C (3) -H (3) c (3) -c (4) -c (13) C(3)-C (4)-H(4) C ( 1 3) -C (4) -H (4) C(6)-N(S)-C(13) C(6)-N(S)-H(5) C (13) -N (5) -H (5) N (5) -C (6) -0 (6) N (5) -C (6) -C (14) O(6) -C (6) -C (14) C(8) -C (7) -C (14) C (8) -C (7) -H (7) C (14) -C (15) C(16)-O(16) C (16) -C ( 1 7) C (17) -N (1' ) C(17)-H(17)1 C ( 1 7) -H ( 1 7) 2 N(l')-C(Z') N ( 1 ' ) -C ( 6 ' ) C (2 ' ) -C ( 3 ' ) C (2 ' ) -H(2 ' ) 1 C (2 ' ) -H (2 ' ) 2 C ( 3 ' ) -N (4 ' ) C(3')-H(3')1 C ( 3 ' ) -H ( 3 ' ) 2 N ( 4 ' ) -C ( 5 ' ) N ( 4 ' ) -C ( 7 ' ) C (5 ' ) -C ( 6 ' ) C ( 5 ' ) -1-1 ( 5 ' ) 1 C ( 5 ' ) -H ( 5 ' ) 2 C ( 6 ' ) -H(6 ' ) 1 C (6 ' ) -H ( 6 ' ) 2 C ( 7 ' ) -H ( 7 ' ) C ( 7 ' ) -H( 7 ' ) 2 C (7 I ) -H ( 7 ' ) 3 O(W)-H(W)1 O(W)-H(W)2 116.PIRENZEPINE DIHYDROCHLORIDE T a b l e 4.1 123.5 1 2 0 .4 6 0 (3) 1.0 1 (2) 1 .94 (2) 1 .502 (4) 1.0 1 (3) 1. 6 (2) 120 ( 1 ) 120 (1) 1 1 8 .452 (3) 1 .97 (4) 1 .03 (2) 1 .3 8 8 (3) 1.8 117. 3 (2) 121.0 4 (2) 1 .362 (2) 1. 9 7 (2) 1. 4 0 3 (3) 0. 0 1 (2) 1 .0 4 (2) 1 .0 125.9 1 1 8 .8 1 2 3 .468 (5) 1.2 1 1 8 .364 (4) 0 .3 113. 3 4 8 (3) 1. 3 7 5 (3) 0. 4 3 3 (3) 1.0 4 (2) 1 .8 116.86 (2) 1 .0 118.4 6 5 (3) 1. 3 9 7 (4) 0. 463 Bond d i s t a n c e s (A) and a n g l e s (O) C(lO)-C(15) C(lO)-H(lO) N(l1)-C(12) N(l1)-C(15) N(l1)-C(16) C(12)-C(13) 1 .462 (4) 1.2 .5 120. 0 3 (3) 1. 9 9 (2) 1 .7 (2) 122. 2 3 1 (3) 1 . 7 8 (5) C (12) -N ( 1 1 ) -C (16) C (15) -N (11) -C(16) N ( 1 ) -C ( 12 ) -N ( 1 1 ) N ( l ) -C (12) -C (13) N (11) -C (12) -C(13) C(4) -C (13) -N (5) C (4) -C (13) -C (12) ~ ( s.372 (4) 1.97 (2) 1.8 (2) 114 ( 1 ) 115 (1) 119.229 (3) 1 . 0 5 (2) 1 .9 1 2 1 .2 (2) 1 2 0 . 3 9 5 (3) 0 .3 (2) 1 1 9 .09 (3) 0.05 (2) 1 .377 ( 4 ) 1 .1 1 1 8 .5 0 5 (4) 1 .92 ( 2 ) 1 .97 (2) 1 .9 120. 3 8 1 (3) 0. 4 8 4 (4) 1 .5 1 3 (4) 1.

7 (2) 1 1 0 . N and 0..a* a * .6 4.7 4.1 4.4 (2) 123 (1) 117 (1) 1 1 9 ..5 3.2 4.0 (2) 111.3 4.4 4.9 (2) 1 1 0 .2 6.9 (2) 110. f o r H) and i s o t r o p i c thermal parameters (x l o 2 ) U eq U eq i s derived from t h e a n i s o t r o p i c thermal parameters by 1 = . C . 1 (2) 1 1 0 .5 3.HUMEIDA A.8 (2) (2) (2) (2) (2) (2) (2) (3) (3) (3) (2) (2) (2) (2) (2) (2) (2) (2) (3) (2) .7 4.2 (3) 120 (2) 120 (2) 120.8 5. 6 (2) 109. 4 (2) 1 2 1 (1) 119 (1) 1 1 4 . 6 (2) 110 (4) f o r C .3 7. 3 (1) C (1 7) -N ( 1 ' ) -C (2 ' ) C(17)-N(lf)-C(6') C (2 ' ) -N ( 1 ' ) -C (6 ' ) N ( 1 ' ) -C (2 ' ) -C ( 3 ' ) C(2')-C(3')-N(4') C(3' ) -N ( 4 ' ) -C(S') C (3 ' ) -N ( 4 ' ) -C ( 7 ' ) C(5 ) -N(4' ) -C ( 7 I ) N (4 ') -C ( 5 ') -C (6' ) N ( 1 ' ) -C(6') -C(S') H(W) l-O(W) -H(W) 2 Final atomic coordinates (x 5 x 10 lo4 111. 3 (2) 1 1 0 . 9 (2) 110.C .8 (2) 1 0 9 . 118 (2) 120. . a * . U.6 3..4 3.1 5.. 464 Table 4.9 3. Contd .7 3. a .. EL-OBEIDETAL. a 3 1 J 1J J 3 1 j or U 6263 6199 6380 6615 7023 6726 7131 5060 4228 4186 4978 6635 6537 6695 5866 5818 7373 7400 8146 8712 z Y X (1) (1) (1) (1) (1) (1) (1) (1) (2) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) 1423 1306 372 -503 -1266 -1540 -2330 -1465 -932 143 698 716 588 -404 -922 162 1361 1426 1997 1375 (1) (1) (2) (1) (1) (1) (1) (1) (2) (2) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) 6087 4985 4557 5255 7162 8057 8634 8510 8677 8615 8379 7947 6761 6403 8266 8216 8752 9729 8376 7805 ueq('42) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (I) (1) (2) (1) 4.2 6.2 4. C (14) -C (7) -H (7) C (7) -C (8) -C (9) C (7) -C (8) -H (8) C (9) -C (8) -H (8) C (8) -C (9) -C (10) C (8) -C (9) -H(9) C(l0)-C(9) -H(9) C (9)-C (10) -C (15) C(9)-C(lO)-H(lO) C(lS)-C(lO)-H(lO) C( 12) -N(11) -C(15) Table 5.

4 (3) 4 .9 ( 6 ) 4 .9 (2) 6 .. 465 .0 (7) 6 .5 (8) 11 (1) 9 (1) 1 0 (1) 1 0 (1) 1 4 (2) . 7 (7) 7. 6 (8) 6 .2 ( 6 ) 6 .4 ( 4 ) 7. 1 (7) 5.2 (6) 4.. 3 (9) 6 .7 (7) 6 .1 (6) 7. 7 (2) 8 . 6 (9) 8 . 6 (7) 7 .3 (6) 5 .2 ( 6 ) 3.0 (8) 5 .1 (8) 5. 0 (2) 5. U Y X 9214 9766 10604 10082 9536 11162 2165 603 632 676 737 509 366 362 496 868 768 857 978 918 1017 951 1068 1012 917 1175 1152 1057 162 223 (1) (2) (1) (2) (2) (3) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (3) (3) (3) (3) (4) 2042 1377 696 28 688 63 2153 195 32 -118 -175 -222 -133 53 143 237 259 248 255 91 184 -46 -44 112 21 -45 55 -38 167 217 (1) (2) (1) (1) (2) (3) (1) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (3) (3) (3) (3) (4) 7195 6583 7417 8008 8627 6827 8845 451 375 499 696 854 886 871 834 909 780 661 777 594 621 741 857 929 904 747 648 615 843 950 or Ue9(A2) Z (2) (2) (1) (2) (2) (3) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (3) (3) (3) (3) (4) 4 . 9 (3) 5 .PIRENZEPINE DIHYDROCHLORIDE Table 5. Contd. 8 (3) 5 . 3 (7) 5.

HUMEIDA A. EL-OBEID ETAL.

466

Figure

8 : STRUCTURAL FORMULA OF PIRENZEPINE WITH ATOMIC NUMBERING (6).

Figure 9 :

A VIEW OF THE CRYSTAL STRUCTURE SHOWING
THE PACKING, HYDROGEN BONDS AND CONFOR-MATION OF THE BENZODIAZEPINE AND
PIPERAZINE R I N G S ( 6 ) .

Table 6.

Torsion a n g l e s

C (12) -N (1) -C (2) -C (3)
N ( l ) -C(2) -C(3) -C (4)
C (2) -C (3) -C (4) -C (13)
C(3) -C (4) -C(13)-C (12)
C (3) -C (4) -C (13) -N (5)
C (4)-C (13) -C(12) -N (1)
C(4) -C(13)-C (12) -N(11)
C(13)-C (12) -N(1) -C(2)
C (7) -C (8) -C (9) -C (10)
C(8)-C(S)-C(lO)-C(15)
C (9) -C (10) -C (15) -C (14)
C (9)-C(lO)-C(15) -N(11)
C (10) -C (15) -C (14) -C (7)
C(10) -C(15) -C(14) -C(6)
C(15) -C (14) -C (7) -C (8)
C (14) -C (7) -C(8) -C (9)
N(S)-C(6)-C(14)-C(15)
C(6) -C (14) -C (15)-N (11)

(O)

1.4
1.5
- 2 .0
- 0 .4
-174.7
3.6
-178.9
-4.1
0.3
- 0 .5
1.1
1 7 8 .7
- 1 .4
1 7 7 .8
1.1
-0.6
-42.4
0 .2

(3)
(4)
(3)
(3)
(2)
(3)
(2)
(3)
(3)
(3)
(3)
(2)
(3)
(2)
(3)
(3)
(3)
(3)

C (14) -C (15) -N (11) -C (12)
N (11 ) -C (12) -C (13) -N (5)
C ( 12) -C ( 13) -N (5) -C (6)
C ( 1 3 ) -N (5) -C (6) -C (14)
C (15) -N (11) -C (12) -C (13)
C (13) -N (5) -C (6) - 0 ( 6 )
C(12) -N(11) -C(16) -0(16)
C(12) -N(11) -C(16) -C(17)
N (11) -C (16) -C (17) -N ( 1 ' )
C (16) -C (17) -N (1' ) -C (2 I )
C (17) -N (I ) -C (2 ' ) -C ( 3 ' )
N ( 1 ' ) -C (2 ' ) -C ( 3 ' ) -N ( 4 ' )
C (2 ) -C ( 3 ) -N (4 ' ) -C ( 5 )
C (2 I ) -C ( 3 ) -N (4 ' ) -C ( 7 ' )
C ( 3 ' ) -N (4 I ) -C ( 5 I ) -C ( 6 ' )
N (4 ' ) -C ( 5 I ) -C (6' ) -N (1I )
C(S')-C(6') -N(1 I ) - C ( 2 ' )

6 9 .5
-4.7
3 8 .3
5.3
-64.5
-178.8
179.8
0.2
-50.0
165.2
-178.4
-57.9
58 .7
-178.7
-58.6
5 8 .6
-57.7

( 2)
(3)
( 3)
(3)
(2)
(2)
(2)
(3)
(2)
(1)
(1)
(2)
(3)
(2)
(2)
(2)
(2)

HUMEIDA A. EL-OBEIDETAL.

468

Figure 10: STEREO-PAIRS OF DRAWINGS FOR THE MOLECULAR
STRUCTURES OF PIRENZEPINE DIHYDROCHLORIDE(a)AND
PI RENfEPlNE MONOHYDROCHLORIDE(b) OBTAINED
FROM X-RAY ANALYSIS ( 7 ) .

a-

1.491 (3)
1.503(6)

Figure l l

:

ATOM NUMBERING SCHEME AND CRYSTALLOGRAPHIC BOND
ANGLES

(A,

STANDARD DEVIATONS IN PARENTHESES)

FOR PIRENZEFINE DIWDROCHLORIDE ( FIRST VALUE) AND
PIRENZEPINE MONOHYDROCHLORIDE (SECOND VALUE I( 7).

PIRENZEPINE DIHYDROCHLORIDE

469

i n agreement w i t h t h e c r y s t a l s t r u c t u r e s . The
t r i c y c l i c r i n g system c o n s i s t s of t h e p l a n a r
r i n g s A and C and nonplanar r i n g B (Figure 1 1 ) .
For b o t h compounds t h e seven-membered r i n g B
h a s a d i s t a r t e d boat shape. This r i n g i s f o l d e d
a l o n g a f o l d i n g l i n e p a s s i n g through N 1 and t h e
middle o f t h e bond N 8 - C9. T h i s s i m i l a r i t y i n
t h e geometry o f t h e t r i c y c l i c systems o f b o t h
compounds does n o t h o l d f o r t h e s i d e c h a i n s
which have t o t a l l y d i f f e r e n t arrangements with
r e s p e c t t o t h e t r i c y c l i c system i n both compounds
( F i g u r e s 10 and 1 2 ) . The s t e r i c s i t u a t i o n is
q u i t e s i m i l a r along t h e bond N 1 - C16, i n t h a t
t h e carbonyl groups are almost i n e c l i p s e d
p o s i t i o n s t o N 1 - C15. S i g n i f i c a n t d i f f e r e n c e s
are observed, however, a l o n g t h e C16 - C 1 7 bond.
For t h e d i h y r o c h l o r i d e N18 i s trans t o
N 1 (T N18-Cl7-Cl6-Nl = 171.3') whereas i n t h e
monohydrochloride N18 and N 1 a r e i n gauche
p o s i t i o n s (T N18-Cl7-Cl6-Nl = 64.1').
In t h i s
arrangement t h e p i p e r a z i n e r i n g w i l l be s i t u a t e d
below t h e t r i c y c l i c r i n g system i n t h e c r y s t a l
s t r u c t u r e o f p i r e n z e p i n e monohydrochloride.
The c r y s t a l s t r u c t u r e s o f b o t h s a l t s showed
s t r o n g N-H .
C 1 - hydrogen bonds f o r t h e p r o t o nated nitrogens of t h e piperazine rings. A
r e l a t i v e l y weak 0
C 1 - hydrogen bonds a l s o
e x i s t f o r t h e d i s t o r t e d w a t e r molecule i n t h e
c r y s t a l l a t t i c e i n t h e dihydrochloride salts.
The amide n i t r o g e n N8 o f t h e monohydrochloride
s a l t shows a f u r t h e r N-H ... C 1 - hydrogen bond
which i s n o t observed i n t h e d i h y d r o c h l o r i d e
structure.

..

...

3.

Synthesis
P i r e n z e p i n e , t o g e t h e r with o t h e r 1 1 - s u b s t i t u t e d 11Hpyrido[ 2 ,3-b] [ 1 , 5 ] benzodiazepin-5 (6H) -ones , had
been prepared by D r . Karl Thomae ( 8 ) . The compounds
are p r e p a r e d by t r e a t m e n t of an 11H-pyrido[ 2 ,3-b] [ 1,51
benzodiazepin-5 (6H) -one with a dihalogen compound,
XCH2COX' i n which X and X I may be a l i k e o r d i f f e r e n t
and d e s i g n a t e an atom o f C 1 , B r and I . The 11h a l o a c e t y l i n t e r m e d i a t e is t h e n t r e a t e d with t h e
a p p r o p r i a t e amine. Thus, a b o i l i n g s o l u t i o n o f 2 1 g
11H-pyrido[ 2 ,3-b] [ 1,51 benzodiazepin-5 (6H) -one) i n 300 m l

HUMEIDA A. EL-OBEIDETAL.

470

C16 -NI

C 17- C I6
HI71

N 18-C I7
HI8

HI72

Figure 12 : NEWMAN PROJECTIONS DOWN THE BONDS ClbNl, c r C 1 6 &

v8-Ci7

FOR PIRENZEPINE DIHYDROCHLORIDE ( ABOVE ) AND THE
MONOHYDROCHLORIDE (BELOW) ( 7 1.

PIRENZEPINE DIHYDROCHLORIDE

47 1

a b s o l u t e dioxane t r e a t e d dropwise sim u lta n e o u sly i n
30 min w i t h 15.8 g ClCH2COC1 i n 40 m l a b s o l u t e dioxane
and 1 4 . 4 g E t 3 N i n 30 m l a b s o l u t e dioxane, t h e m ix tu r e
r e f l u x e d f o r 8 h r and t h e h o t f i l t r a t e e v a p o r a te d i n
vacuo gave ll-chloroacetyl-11H-pyrido[2,3-b][1,5]
benzodiazepin-5 (6H) -one (Scheme 2 ) . The c h l o r o a c e t y l
d e r i v a t i v e and N-methylpiperazine i n a b s o l u t e a l c o h o l
r e f l u x e d f o r 18 h r f i l t e r e d and t h e hot f i l t r a t e
e v a p o r a t e d i n vacuo t o g i v e p i r e n z e p i n e .

C 1CH, COC 1
%

N
H

0

I
=c
I

CH2C1

H

H2C -N

0

wNCH3

Scheme 2 .

Synthesis of pirenzepine

HUMEIDA A. EL-OBEID ETAL.

412

4.

Stability
S t a b i l i t y of p i r e n z e p i n e depends on b o t h m o istu r e
c o n t e n t s and pH and t h e e f f e c t o f t h e m o istu r e c o n t e n t s
could be minimized by a d j u s t i n g t o optimum pH ( 9 ) .
P i r e n z e p i n e d i h y d r o c h l o r i d e i n t a b l e t was shown
s t a b l e t o exposure t o l i g h t a t 50 - 5 5 O . No change
i n c o l o r , smell, t a s t e o r g e n e r a l appearance o r
presence o f d eg r a d a t i o n p r o d u ct s was observed (10).

5.

Methods of A n a l y s i s
5.1

S p e ct r o p h o t o m et r i c Methods
The d e t er m i n a t i o n of p i r e n z e p i n e d i h y d r o c h l o r i d e
s p e c t r o p h o t o m e t r i c a l l y u s i n g AA method have
r e c e n t l y been r e p o r t e d ( 4 ) . The mean p e r c e n ta g e
r e c o v er y f o r t a b l e t s , each l a b e l l e d t o c o n t a i n
25 mg,was 100.2 -+ 1 . 1 5 . The drug shows maximum
a b s o r p t i o n a t 280 nm i n h y d r o c h l o r i c a c i d and a t
295 nm i n 0 . 1 N sodium hydroxide due t o k e to - e n o l
tautomerism. AA a t 300 nm was found t o be
l i n e a r l y r e l a t e d t o t h e drug c o n c e n t r a t i o n o v e r
a range of 10-80 Ug/ml with a r e l a t i v e s t a n d a r d
d e v i a t i o n of 0 . 4 % .
A r a p i d and d i r e c t method f o r t h e d e te r m in a tio n

o f p i r e n z e p i n e d i h y d r o c h l o r i d e i n t h e p r e se n c e
o f d eg r a d a t i o n p r o d u ct s u s i n g f i r s t d e r i v a t i v e
spectrophotometry h as a l s o been r e p o r t e d ( 5 ) .
The method was a p p l i e d u s i n g s y n t h e t i c m ix tu r e s
o f t h e i n t a c t drug and d e g r a d a tio n p r o d u c ts. The
procedure i s s u i t a b l e f o r m o n ito r in g t h e drug
s t a b i 1i t y

.

5.2

Chromatographic Eilethods
5.2.1

Thin-Layer Chromatographic (TLC) Methods
Thin l a y e r chromatography have been used
f o r t h e analysis o f pirenzepine (10).
P r e c o a t ed TLC p l a t e s with 0 . 2 mm s i l i c a
g e l FZs4 were used.

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413

High-pressure Liquid Chromatography (HPLC)
Daldrup -e t a1 (11) have used reversed-phase
l i q u i d chromatography a s a t e s t f o r
s c r e e n i n g pharmaceuticals , drugs and
insecticides.
High performance r e v e r s e - p h a s e l i q u i d
chromatography r e t e n t i o n d a t a f o r
p i r e n z e p i n e and o t h e r compounds (560
compounds) a r e given. The r e l a t i v e r e t e n t i o n
times were c a l c u l a t e d a s t h e r a t i o s of
r e t n t i o n times of compound and r e f e r e n c e
compound, 5- (p-methylphenyl) -5-phenyl
hydantoin. The UV d e t e c t o r wavelength was
220 nm, where most of t h e compounds gave
response. Two s o l v e n t programs and a
prepacked column C - 1 8 SIL-X-10 were used
f o r the analysis.
Babhair (12) r e p o r t e d a s i m p l e and s e n s i t i v e
method for t h e d e t e r m i n a t i o n o f p i r e n z e p i n e ,
i n dosage forms and i n b i o l o g i c a l f l u i d s ,
u s i n g h i g h - p e r formance 1i q u i d chromatography w i t h U . V . d e t e c t o r . A t a b l e t o f
25 mg drug was ground, suspended i n 1 0 m l
o f w a t e r , shaken and t h e n f i l t e r e d . A
known volume o f t h e f i l t r a t e i s a d j u s t e d
t o a p p r o p r i a t e c o n c e n t r a t i o n . Twenty 1.11
o f t h i s s o l u t i o n were i n j e c t e d . Plasma o r
u r i n e samples were made a l k a l i n e w i t h
ammonia b e f o r e e x t r a c t ion with chloroform
which was evaporated and t h e r e s i d u e was
d i s s o l v e d i n t h e mobile phase, 20 1-1 o f t h i s
s o l u t i o n were i n j e c t e d . The d e t e r m i n a t i o n
l i m i t f o r q u a n t i t a t i o n was about 1 pg/ml o f
p i r e n z e p i n e . Complete s e p a r a t i o n of t h e
drug was achieved i n about 5 . 4 minutes
under t h e p r e s e n t chromatographic c o n d i t i o n s .
A 30 X 3.9 cm i . d. commercially a v a i l a b l e
s t a i n l e s s s t e e l C-18 column was used.
Mobile phase c o n s i s t e d o f a c e t o n i t r i l e ,
methanol and 5% a c e t i c a c i d (70:40:15).
High-performance l i q u i d chromatographic
determination of pirenzepine dihydrochloride
i n i t s pharmaceutical f o r m u l a t i o n have a l s o

HUMEIDA A. EL-OBEIDETAL.

474

been r e p o r t e d (13). Normal phase l i q u i d
chromatography has been performed on a
Micropack Si-10 column u s i n g ammonium hydrox i d e (28-30% NH3) i n methanol (0.75 : 99.25%
v/v) mobile phase and a flow r a t e o f 2 ml/min.
Clobazam h as been used a s i n t e r n a l s t a n d a r d
with r e t e n t i o n times o f 1 . 9 and 2.8 minutes
f o r clobazam and p i r e n z e p i n e d i h y d r o c h l o r i d e ,
r e s p e c t i v e l y a t 254 nm. T a b l e t s each l a b e l l e d
t o c o n t ai n 25 mg p i r e n z e p i n e d i h y d r o c h l o r i d e
gave mean p er c e n t ag e result o f 99.98 2 0 . 4 .
The method have a l s o been r e p o r t e d t o be a
s t a b i l i t y i n d i c a t i n g method.
5.3

Radioimmunoassay
A radioimmunoassay system developed f o r p i r e n z e p i n e

was used t o s p e c i f y t h e r e q u i re m e n ts f o r u s i n g such
system i n pharmacokinetics and t o i l l u s t r a t e t h e
general strategy i n establishing s p e c i f i c i t y , sensit i v i t y and r e l i a b i l i t y ( 1 4 ) . The t y p e o f e r r o r i n
t h e f i n a l d a t a was c o n s i d er e d . I t is a widespread
assumption t h a t weak c r o s s - r e a c t i o n s i n m e t a b o l i t e s
r e s u l t i n a r e l a t i v e e r r o r and, t h e r e f o r e , have no
r e l e v a n c e t o t h e d e t e c t i o n o f t h e p a r e n t drug. In
c o n t r a s t t o t h i s a s s u p t i o n , it was shown t h a t such
weak c r o s s - r e a c t i v i t y i n b i o l o g i c a l samples g i v e s
r i s e t o an a b s o l u t e e r r o r which i s independent o f t h e
c o n c e n t r a t i o n and analogous t o t h a t caused by a b la n k
v a l u e i n chemical a n a l y s i s .
Tanswell and Zahn (15) a p p l i e d monoclonal a s s a y s f o r
s t u d y i n g t h e pharmacokinetics o f p i r e n z e p i n e .

6.

Pharmacokinetics
The marked physicochemical p r o p e r t i e s o f p i r e n z e p i n e
r e s u l t i n t h e drug b ei n g h i g h l y h y d r o p h i l i c a t p h y s i o l o g i c a l pH. T h i s p r o p e r t y determines t h e f a t e o f p i r e n z e p i n e
i n t h e organism due t o t h e l i m i t e d a b i l i t y of t h e drug t o
p e n e t r a t e l i p i d membranes. T h i s l i m i t e d p e n e t r a t i o n i s
r e f l e c t e d i n i t s a b s o r p t i o n , d i s t r i b u t i o n , b io tr a n sf o r m a t i o n and e x c r e t i o n .
6.1

Absorption
O r a l l y a d m i n i s t e r e d p i r e n z e p i n e was almost
completely absorbed by r a b b i t s and dogs b u t o n l y
about 11%absorbed by r a t s and t h e blood l e v e l
maxima reached a f t e r 3 h r (16).

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475

After o r a l a d m i n i s t r a t i o n o f aqueous s o l u t i o n s o r
t a b l e t s 20-30% o f t h e dose is absorbed ( 1 7 ) . In
an i n t e r n a t i o n a l pharmacokinetic s t u d i e s 20% o f t h e
o r a l dose a d m i n i s t e r e d t o t h e panel o f 87
v o l u n t e e r s was absorbed (18). Uniform plasma
l e v e l s were observed i n t h i s l a r g e panel from
10 d i f f e r e n t c o u n t r i e s a f t e r a s i n g l e o r a l dose
o f 50 mg, with minimal i n t e r p a t i e n t v a r i a t i o n .
Peak serum c o n c e n t r a t i o n were approximately 50 ng/ml
o c c u r r i n g w i t h i n 2 h r s . After o r a l dose o f 20 mg/kg,
t h e drug appeared i n t h e blood a t a c o n c e n t r a t i o n
o f 0 . 1 7 ug/ml o n l y 15 min a f t e r a d m i n i s t r a t i o n .
Afterwards, however, i n c r e a s e i n t h e c o n c e n t r a t i o n
was shown r e a c h i n g t h e maximum of 0 . 4 2 ug/ml
approximately 3 h r a f t e r a d m i n i s t r a t i o n ( 1 9 ) . The
r e s u l t s a r e comparable t o t h o s e o f Hammer e t_a1
(16) who r e p o r t e d t h a t t h e maximum blood
c o n c e n t r a t i o n was a t t a i n e d 3 h r a f t e r a d m i n i s t r a t i o n
a t t h e l e v e l of 0.55 ug/ml.
After o r a l a d m i n i s t r a t i o n o f 25 mg o f t h e drug, t h e
maximum blood l e v e l occurred i n 2 - 3 h r and was
about 24 ng/ml. Computer s i m u l a t i o n was used t o
d e r i v e a s i n g l e dosage scheme f o r p i r e n z e p i n e
c o n s i s t i n g o f an i n i t i a l dose o f 50 mg and
maintenance dose o f 25 mg a t 12 h r - i n t e r v a l s .
T h i s schedule maintained a maximum plasma l e v e l
o f -> 24.1 ng/ml i n normal s u b j e c t s ( 1 7 ) .

The maximum plasma l e v e l appeared 2 h r a f t e r t h e
a d m i n i s t r a t i o n o f 1 2 . 5 - 150 mg p i r e n z e p i n e o r a l l y
t o volunteers (20). After multiple administration
(25 - 50 mg a day) t h e plasma l e v e l was i n c r e a s e d
f o r t h e f i r s t 3 days o f a d m i n i s t r a t i o n and remained
constant t h e r e a f t e r .
Simulated plasma l e v e l s of p i r e n z e p i n e a f t e r an o r a l
t h e r a p e u t i c dosage regimen (50 mg, twice d a i l y ) on
t h e b a s i s o f s i n g l e a d m i n i s t r a t i o n d a t a , showed
s t e a d y s t a t e w i t h i n 2 days o f t r e a t m e n t (18).
P i r e n z e p i n e a d m i n i s t e r e d i . m . was completely
absorbed i n r a b b i t s and dogs. Rats t h a t p o o r l y
absorb o r a l p i r e n z e p i n e showed complete a b s o r p t i o n
o f i . m . dose o f t h e drug.
Using monoclonal a s s a y s (15), plasma p i r e n z e p i n e
l e v e l s and u r i n a r y e x c r e t i o n were measured over time

HUMEIDA A. EL-OBEID ETAL.

476

i n 10 h e a l t h y male s u b j e c t s a f t e r 10 mg i . v . or
i . m . dose, i n randomized c r o s s o v e r s t u d y . The
d a t a were f i t t e d t o a 3-compartment open model.
Absorption a f t e r i . m . i n j e c t i o n was r a p i d and
complete (mean b i o a v a i l a b i l i t y 1 0 0 . 3 % ) .
After i . v . b o l u s i n j e c t i o n h i g h plasma l e v e l peaks
are reached which last only f o r a s h o r t time
p e r i o d s i n c e due t o t h e d i s t r i b u t i o n o f p i r e n z e p i n e
i n t o body t i s s u e s t h e plasma l e v e l d e c l i n e s r a p i d l y
with an i n i t i a l h a l f - l i f e o f about 5 min. (17, 2 1 ) .
The peak c o n c e n t r a t i o n a f t e r i . v . a d m i n i s t r a t i o n
(22) was much h i g h e r than t h e t h e r a p e u t i c l e v e l
a f t e r o r a l a d m i n i s t r a t i o n . In t h e f i r s t minutes
a f t e r i . v . i n j e c t i o n o f 10 mg p i r e n z e p i n e ( 0 . 1 5
mg/kg) , t h e plasma c o n c e n t r a t i o n s were almost
1 0 - f o l d h i g h e r than t h e s t e a d y s t a t e l e v e l reached
a f t e r d a i l y doses o f o r a l 100 mg. I n less t h a n
one hour, however, t h e plasma l e v e l o f t h e i . v .
dose i s comparable t o t h e s t e a d y s t a t e c o n c e n t r a t i o n s after o r a l a d m i n i s t r a t i o n (18).
The a b s o r p t i o n o f S . C . dose o f p i r e n z e p i n e i s very
swift and comparable t o t h a t o f i . v . (4) and
u n l i k e t h a t o f i . m . r e p o r t e d by Hammer -e t a 1 (16).
6.2

Distribution
Informations provided by whole-animal a u t o r a d i o g r a phy of r a t 10 minutes a f t e r i n t r a v e n o u s i n j e c t i o n
o f 2 mg/kg o f r a d i o a c t i v e p i r e n z e p i n e (15, 2 1 ) ,
showed t h a t t h e drug is widely d i s t r i b u t e d i n t h e
body. The r a d i o a c t i v e drug i s found i n a l l
i n t e r n a l organs and s k e l e t a l muscles. No r a d i o a c t i v i t y was d e t e c t e d i n t h e b r a i n o f t h e r a t o r
t h e f e t u s e s o f g e s t a t i n g mouse s u g g e s t i n g t h a t t h e
blood-brain b a r r i e r r e p r e s e n t s a genuine b a r r i e r
t o p i r e n z e p i n e and t h a t t h e drug does n o t c r o s s
t h e p l a c e n t a l b a r r i e r . Continuous i n t r a v e n o u s
i n f u s i o n o f p i r e n z e p i n e over a p e r i o d o f 2 days a t
a r a t e o f 2 mg/hr/8-9 kg baboon (10 times t h e
human i n f u s i o n d o s e ) , showed t h a t t h e h i g h e s t
l e v e l s a r e i n t h e organs r e s p o n s i b l e f o r t h e
e l i m i n a t i o n of t h e drug i . e . l i v e r and kidneys.
The o t h e r i n t e r n a l organs, t h e s k i n and s k e l e t a l
muscles, however, showed a h i g h e r p i r e n z e p i n e
c o n c e n t r a t i o n p e r g of t i s s u e t h a n t h e plasma.

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477

Extremely low drug c o n c e n t r a t i o n was found i n
t h e brain.
The d i s t r i b u t i o n o f p i r e n z e p i n e i n t h e t i s s u e s
a f t e r one-time o r a l a d m i n i s t r a t i o n was i n v e s t i g a t e d
by Kobayashi -e t a 1 (19) a f t e r i s o l a t i o n o f t i s s u e s
and whole-body a u t o r a d i o g r a p h y t e c h n i q u e . After
3 h r , t h e time o f maximum blood c o n c e n t r a t i o n ,
h i g h c o n c e n t r a t i o n of p i r e n z e p i n e was observed i n
t i s s u e s l i k e l i v e r , kidney, p a n c r e a s , lung,
p i t u i t a r y g l a n d , s a l i v a r y g l a n d , a d r e n a l gland e t c .
These r e s u l t s , t h e r e f o r e , agree w i t h t h o s e o f
Hammer and Koss (21) i n t h a t r a d i o a c t i v e p i r e n z e p i n e
was found d i s t r i b u t e d i n a l l t i s s u e s e x c e p t t h e
c e n t r a l nervous system. The r a d i o a c t i v i t y
d i s t r i b u t e d i n t h i s way d e c r e a s e d as time p a s s e d .
Twenty-four hours a f t e r a d m i n i s t r a t i o n , t h e
c o n c e n t r a t i o n was s l i g h t l y h g i h o n l y i n t h e
g a s t r o i n t e s t i n a l t r a c t and l i v e r . R a d i o a c t i v i t y
i n t h e t e s t i s showed a tendency t o d e c r e a s e
g r a d u a l l y d u r i n g 72 h r . Kaubisch et a1 (23)
observed s t r o n g r a d i o a c t i v i t y d i s t r i b u t e d i n t h e
g a s t r o i n t e s t i n a l t r a c t , kidney and s a l i v a r y g l a n d s
i n a whole-body a u t o r a d i o g r a p h y performed a f t e r
i . v. a d m i n i s t r a t i o n of 14C-pirenzepine i n a dose
o f 2 mg/kg. The p r e s e n c e o f r a d i o a c t i v i t y i n t h e
g a s t r o i n t e s t i n a l t r a c t was c o n s i d e r e d t o r e s u l t
from t h e e x c r e t i o n o f t h e r a d i o a c t i v i t y i n t o t h e
i n s i d e o f t h e i n t e s t i n a l t r a c t v i a t h e blood
v e s s e l s and i n t e s t i n a l w a l l . T h i s i s s u p p o r t e d
by t h e f i n d i n g o f Kobayashi e t a 1 (19) who
observed c l e a n p i c t u r e s o f r a d i o a c t i v i t y
d i s t r i b u t i o n a t t h e wall o r mucus o f t h e stomach
and i n t e s t i n e s i n a u t o r a d i o g r a p h y 3 h r and 9 h r
a f t e r a d m i n i s t r a t i o n and c o n s i d e r e d t h a t t h e
r a d i o a c t i v i t y was s e c r e t e d from t h e g a s t r o i n t e s t i n a l
wall o r was d e p o s i t e d t h e r e .
L -

--

After m u l t i p l e o r a l d o s e , Kobayashi e t a 1 ( 1 9 ) ,
observed a s l i g h t tendency towards accumulation
i n t h e l i v e r , kidney and t e s t i s whereas r a d i o a c t i v i t y d e c r e a s e d s l o w l y i n one-day a d m i n i s t r a t i o n .
However, t h e r a d i o a c t i v e c o n c e n t r a t i o n i n each
t i s s u e did not increase i n proportion t o t h e
a d m i n i s t r a t i o n times b u t d e c r e a s e d as time p a s s e d
and l e f t o n l y t r a c e s one week a f t e r t h e t e r m i n a t i o n
o f a d m i n i s t r a t i o n . In m u l t i p l e a d m i n i s t r a t i o n

HUMEIDA A. EL-OBEIDETAL.

almost a l l of t h e r a d i o a c t i v i t y was found
excreted i n t o u r i n e and feces 24 h r a f t e r each
administration. I t i s , t h e r e f o r e , considered
t h a t t h e accumulation of t h e drug i n t h e body
is small. Jaup and Blomstrand (24) measured
serum and cerebrospinal concentrations of
pirenzepine i n healthy volunteers a f t e r t h e r a p e u t i c
dosage over 2-5 days. The cerebrospinal f l u i d to-serum r a t i o was 0.095 t o 1, i n d i c a t i n g t h a t
pirenzepine passed t h e blood-brain b a r r i e r , but
only t o a small e x t e n t . Pirenzepine concentrations
i n t h e cerebrospinal f l u i d was about 10% those
of serum.
That t h e penetration of pirenzepine through t h e
blood-brain b a r r i e r i s very limited was a l s o
demonstrated by o t h e r s t u d i e s (18, 25-27).
6.3

Metabolism
Pirenzepine undergoes only s l i g h t metabolism
i r r e s p e c t i v e of the route of administration and
t h e r e i s no s i g n i f i c a n t f i r s t - p a s s e f f e c t . Only
small amounts of metabolites have been detected
(16-18, 21), of which t h e desmethyl d e r i v a t i v e
accounted f o r s i g n i f i c a n t l y less t h a n 10% of t h e
dose. The desmethyl compound has no pharmacological
e f f e c t . The t r i c y c l i c moiety, despiperazinly
pirenzepine was a l s o detected a s a metabolite i n
n e g l i g i b l e amounts ( 1 7 ) . Unlike cimetidine,
pirenzepine does not i n h i b i t t h e h e p a t i c mixed
function oxidase system (28).

6.4

Excretion
In man as well as i n o t h e r animals, pirenzepine i s
excreted unchanged i n u r i n e and feces ( 1 7 , 18, 21).
Due t o t h e s p e c i a l physicochemical p r o p e r t i e s of
pirenzepine only about 10% of t h e dose i s bound
t o plasma p r o t e i n s (17, 20) and hence it i s almost
completely f i l t e r e d i n t o t h e kidneys. The volume
of d i s t r i b u t i o n of pirenzepine i s i d e n t i c a l t o
t h e e x t r a c e l l u l a r space which i n d i c a t e s t h a t
pirenzepine r a p i d l y e q u i l i b r a t e s between plasma
and t h e e x t r a c e l l u l a r f l u i d s of t h e peripheral
t i s s u e s . The t o t a l plasma clearance of
pirenzepine i s 255 ml/min. Because of i t s limited

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479

l i p i d s o l u b i l i t y , t h e drug cannot be reabsorbed
from t h e renal tubules s o t h a t t h e renal clearance
of 129 ml/min corresponds t o the glumerular
f i l t r a t i o n r a t e . Hammer -e t a1 (16) reported t h a t
approximately equal amounts of pirenzepine were
excreted v i a t h e b i l e and u r i n e . The h e p a t i c
clearance, mostly b i l i a r y excretion i s about
125 ml/min (17).
Following o r a l administration o f a t h e r a p e u t i c
dose of labeled drug, 82% of t h e substance
excreted i n u r i n e i s unchanged, a s is 92% of t h a t
i n t h e feces. The drug i s eliminated from the
organism f u l l y and almost equally i n u r i n e and
f e c e s , elimination being almost complete a f t e r
4 days (17). Kobayashi -e t a1 (19) reported t h a t
i n s i n g l e a s well as i n m u l t i p l e administration,
almost a l l of t h e dose was excreted i n u r i n e and
feces. In i . v . administration, 44% and 50% of t h e
administered dose were excreted i n t o u r i n e and
f e c e s , r e s p e c t i v e l y 24 h r a f t e r administration and
46% and 53% r e s p e c t i v e l y , 4 days a f t e r administr a t i o n . Similar r e s u l t s were obtained with
subcutaneous administration. Hammer -e t a1 (16)
reported t h a t 36% and 56% of t h e administered
dose were excreted i n u r i n e and f e c e s , r e s p e c t i v e l y ,
following i . v . administration. 42% of a 1 0 mg
dose was a l s o reported (15) eliminated r e n a l l y
with t o t a l clearance o f 253 ml/min and renal
clearance of 107 ml/min. The mean t r a n s i t time
was 9 . 3 h r . A urinary excretion r a t e of 7-12%
was reported by Ohashi -e t a1 (20) within 4 days
a f t e r multiple administration of pirenzepine
(25-50 mg, 3 times a day f o r 4 days). Hammer e t
a1 (16) reported approximately 4% o f the admingt e r e d dose was excreted i n u r i n e a f t e r o r a l
administration. According t o Kobayashi -e t a1 (19)
about 8% was excreted i n u r i n e a f t e r s i n g l e o r a l
administration and about 4.5% i n multiple
administration. The d i f f e r e n c e i n t h e value was
a t t r i b u t e d t o t h e d i f f e r e n c e i n absorption r a t e s
between f a s t e d and nonfasted animals.
Elimination h a l f - l i f e of 10 h r was reported (21)
following i . v . dose o f 8 mg pirenzepine t o humans.
Oral administration o f 25 mg t a b l e t s t o
volunteers r e s u l t e d i n elimination h a l f - l i f e of

480

HUMEIDA A. EL-OBEID ETAL.

--

11 h r (21). Ohashi e t a1 (20) reported an
elimination h a l f - l i f e of 13 h r . The long eliminat i o n h a l f - l i f e was a t t r i b u t e d t o slow r e d i s t r i b u t i o n
of pirenzepine from t h e t i s s u e s r a t h e r than t o
slow excretion (21).

Intravenous administration o f 14C-pirenzepine t o
dams i n a dose of 2 mg/kg caused t h e appearance
o f r a d i o a c t i v i t y i n m i l k almost equivalent t o
t h a t i n blood (19).
Pretreatment of healthy volunteers with t h e r a p e u t i c
doses of pirenzepine over 5 days showed no
s i g n i f i c a n t e f f e c t on t h e absorption, d i s t r i b u t i o n
o r elimination of a n t i p y r i n e (28).
7.

Pharmaco 1ogy
7.1

Antisecretory E f f e c t s
Secretory s t u d i e s with pirenzepine c l e a r l y
demonstrate t h a t pirenzepine suppresses both basal
and stimulated acid and pepsin s e c r e t i o n (30-52).
Intravenous pirenzepine reduces b a s a l acid secret i o n by 90% and pentagastrin-stimulated
s e c r e t i o n by approximately 50% ( 3 0 ) . F r i t s c h
-e t a1 (31) reported t h a t o r a l 50 mg of pirenzepine,
i n p a t i e n t s with duodenal u l c e r , i n h i b i t e d t h e
basal acid s e c r e t i o n by 39.9% and peptone-induced
acid output by 21.3%. The percentage i n h i b i t i o n
n e a r l y doubled i f t h e same dose is given a f t e r
a treatment with 50 mg pirenzepine twice d a i l y
f o r 3 t o 7 days. No acute e f f e c t on serum g a s t r i n
l e v e l s can be demonstrated by a s i n g l e dose of
pirenzepine. After pretreatment only small
increase of basal g a s t r i n l e v e l s i s observed,
serum g a s t r i n does not change during stimulation.
Oral pirenzepine (25 mg) reduces acid output by
50% and pentagastrin-stimulated s e c r e t i o n by
30-40%. Doubling t h e dose increases t h e degree
of i n h i b i t i o n of stimulated s e c r e t i o n by
10%
(30). Jaup -e t a1 (32) reported t h a t pirenzepine
50 mg PO b . i . d . reduced b a s a l g a s t r i c a c i d
s e c r e t i o n by 54% a t two hours following t h e l a s t
dose. Pentagastrin-stimulated s e c r e t i o n o f
hydrochloric acid (1 mcg/kg/hr) by continuous i . v .
infusion was reduced by 31%. Secretory s t u d i e s by

Stockbrugger et a1 (37) s t u d i e d t h e e f f e c t o f t h r e e d i f f e r e n t doses o f p i r e n z e p i n e on a c i d s e c r e t i o n s t i m u l a t e d by modified shamfeeding (MSF) on h e a l t h y v o l u n t e e r s . i n s t i m u l a t e d a c i d o u t p u t (0 120 min) by 45%. p e n t a g a s t r i n . reduced b a s a l o u t p u t (0-30 min) by 48%. The drug i n h i b i t e d t h e v a g a l l y t r a n s m i t t e d a c i d s e c r e t i o n by about 45% even when t h e v a g a l l y s t i m u l a t e d a c i d s e c r e t i o n amounts t o more t h a n 50% o f t h e p e n t a g a s t r i n . Mignon e t a1 (38) r e p o r t e d a d o s e . . t o dogs 10 min b e f o r e i n f u s i o n o f bombesin. P i r e n z e p i n e 10 mg given t o duodenal u l c e r p a t i e n t s 45 min and a g a i n 10 min b e f o r e t h e s t a r t of MSF blocked 48% o f t h e response (39). by 12. randomized s t u d i e s on p a t i e n t s w i t h dyspepsia.s t i m u l a t e d s e c r e t i o n . m .PIRENZEPINE DIHYDROCHLORIDE 48 1 J a u p -e t a1 (33) c l e a r l y demonstrated t h a t p i r e n z e p i n e s u p p r e s s e s b a s a l . d . 5 mg/kg o f p i r e n z e p i n e showed 53% r e d u c t i o n i n a c i d secret i o n . In a d o u b l e . Oral p i r e n z e p i n e 25 mg b . When t h e a c i d s e c r e t i o n was c a l c u l a t e d a s volume c o r r e c t e d f o r d u o d e n a l . Bianchi Porro et a1 (34) r e p o r t e d a 48% d e c r e a s e o f g a s t r i c s e c r e t i o n i n t h e first hour and 30% i n t h e second hour a f t e r 50 mg o r a l dose o f p i r e n z e p i n e . 50 mg b .r e l a t e d r e d u c t i o n o f meal-stimulated a c i d response w i t h i n c r e a s i n g doses o f i . I n Heidenhain pouch b o t h doses f u l l y prevented a c i d s e c r e t i o n . i . These e f f e c t s of pirenzepine a r e simila r t o those of a tro p i n e b u t d i f f e r e n t from t h e e f f e c t s o f c i m e t i d i n e . Pirenzepine i n j e c t e d during t h e secretory plateau . 3 % and 42.7%. by 24%.5% and w i t h 50 mg t . m . s i g n i f i c a n t l y i n h i b i t e d t h e h y p e r s e c r e t i o n induced by t h e p e p t i d e i n t h e main stomach and i n a dose o f 100 ug/kg p i r e n z e p i n e v e r t u a l l y a b b o l i s h e d t h e a c i d response t o bombesin ( 4 0 ) . placebo c o n t r o l l e d .b l i n d . Eugenides -e t a1 (36) r e p o r t e d t h a t histamines t i m u l a t e d a c i d o u t p u t p e r two hours i n h e a l t h y v o l u n t e e r s was reduced with 25 mg p i r e n z e p i n e b .g a s t r i c r e f l u x and p y r o l i c l o s s e s . d . p i r e n z e p i n e . o r 50 mg t . 59% and 66% r e s p e c t i v e l y . t h e corresponding f i g u r e s were 33.s t i m u l a t e d and insulin-stimulated acid secretion in a dose-related manner. d . P i r e n z e p i n e caused a r e d u c t i o n t o 20% o f t h e concentr a t i o n o f t h e a c i d produced by vagal s t i m u l a t i o n (35). -- P i r e n z e p i n e (25 pg/kg) a d m i n i s t e r e d i .6%. i . R e s u l t s with 0 . i . 3 6 . s . d . d . 58% and 48% r e s p e c t i v e l y .

The published world l i t e r a t u r e (from 1977 t o 1981) on t h e e f f i c a c y of pirenzepine had been t h e s u b j e c t o f a review and commentary by Bianchi Porro and P e t r i l l o (53). 53-130). but only 55% i f t h e dosage of t h e drug was 75 mg o r less. v . Acid and pepsinogen s e c r e t i o n induced by bethanechol i n i s o l a t e d perfused mouse stomach a r e i n h i b i t e d by pirenzepine (41).2 Efficacy Pirenzepine i s e f f e c t i v e i n t h e treatment of g a s t r i c and duodenal u l c e r s . 10 min before a meal t e s t ) f u l l y prevented a c i d s e c r e t i o n f o r Hiedenhain pouch. I t i s a l s o e f f e c t i v e i n nonu l c e r dyspepsia and i n preventing u l c e r recurrence.5% and 80% with doses of 25 and 100 g/kg respectively. induced by bombesin. . In 12 prospective randomized double-blind placebo-controlled s t u d i e s pirenzepine was administered t o 475 duodenal u l c e r p a t i e n t s with incidence of endoscopically proven healing of 74% when the p a t i e n t s received d a i l y dosage o f 100 o r 150 mg pirenzepine.or meal-stimulated g a s t r i n r e l e a s e . Pirenzepine (25 and 100 ug/kg i . Similar healing rates were observed (72% when d a i l y dose of pirenzepine was 100 o r 150 mg. produced a mean peak i n h i b i t i o n of acid output from t h e main stomach of 76. The review examined t h e d a t a published i n Europe which deal with pirenzepine i n t h e treatment of g a s t r i c and duodenal u l c e r s . Pirenzepine i n combination therapy with cimetidine can be used s u c c e s s f u l l y f o r t h e treatment o f c i m e t i d i n e .482 HUMEIDA A. The l i t e r a t u r e r e p o r t s a number of s t u d i e s on t h e e f f i c a c y of pirenzepine i n t h e treatment of g a s t r i c and duodenal u l c e r s (43. The reviewers subjected t h e . Texter and R e i l l y (83) evaluated the c l i n i c a l evidence concerning t h e healing e f f e c t of pirenzepine i n g a s t r i c and duodenal u l c e r s . The drug showed no e f f e c t on bombesin . 7. taking i n t o account only those r e s u l t s obtained from c o n t r o l l e d endoscopic t r i a l s . EL-OBEID ETAL. 55% when t h e dose was 75 mg o r l e s s ) i n 4 randomized doubleb l i n d placebo -controlled s t u d i e s i n which 84 p a t i e n t s with g a s t r i c u l c e r were admitted.r e s i s t a n t conditions such as duodenal u l c e r and Zollinger-Ellison syndrome. In another review.

i n t h e treatment of duodenal u l c e r s i n a placebo-controlled study. t h e r a p e u t i c s t u d i e s on u l c e r healing t o mathematical treatments. The doseresponse curve f o r duodenal u l c e r incorporating 16 d a t a p o i n t s from 8 t r i a l s i n 530 p a t i e n t s . The a n a l y s i s a l s o shows t h a t endoscopic healing from g a s t r i c and duodenal u l c e r s i s l i n e a r with respect t o time upto 6 weeks ( r = 0. The r e l a p s e r a t e a f t e r treatment with pirenzepine was lower than a f t e r treatment with cimetidine. 100 mg daily.75.7%. which increased t o 72% with 150 mg dose f o r 8 weeks. The e f f e c t of pirenzepine and cimetidine on healing. Brunner (94) reported t h a t pirenzepine is equally o r a t least n o t e s s e n t i a l l y l e s s e f f e c t i v e than cimetidine i n t h e treatment o f duodenal u l c e r . Although t h e a n a l s i s is not s t r i c t l y mathematically c o r r e c t . r e s u l t e d i n a c o r r e l a t i o n c o e f f i c i e n t r = 0. showed endoscopic healing r a t e o f 66.001) and suggests t h a t healing r a t e s improve with time on therapy. This observation on time course is strengthened by t h e r e s u l t s of a l a r g e Japanese m u l t i c e n t r e t r i a l on pirenzepine. The pred i c t e d healing r a t e a t 150 mg was 79%. P < 0. The t h e r a p e u t i c s t u d i e s reviewed included a l a r g e number o f p a t i e n t s i n Europe and Japan. Pirenzepine produced healing i n 14 out o f 18 p a t i e n t s (78%) following 6 weeks o f treatment as compared t o 7 of 20 p a t i e n t s (35%) receiving placebo. Therapeutic s t u d i e s on g a s t r i c u l c e r . symptoms and r e l a p s e r a t e of duodenal u l c e r was s t u d i e d by Eichenberger -e t a1 (95). it suggests t h a t pirenzepine h e a l s duodenal u l c e r compared t o placebo with a p o s i t i v e r e l a t i o n s h i p between dose and e f f e c t . Do -e t a1 (96) reported t h e e f f i c a c y of pirenzepine. Relief o f daytime and nighttime pain was g r e a t e r i n pirenzepine-treated p a t i e n t s .005.887 with P < 0.PIRENZEPINE DIHYDROCHLORIDE 483 results of t h e previous double-blind. a s well as lower consumption of antacid. . using 75 mg/day pirenzepine f o r 8 weeks. 343 of whom received pirenzepine. No s i g n i f i c a n t d i f f e r e n c e s i n u l c e r healing between t h e e f f i c a c y o f t h e drugs when 1 gm/day o f cimetid i n e and 75 mg/day pirenzepine were used.

57. 107). EL-OBEID ETAL. 115).484 HUMEIDA A. 60. whereas lower doses of 75 mg d a i l y o r l e s s have produced lower response r a t e s ( z 55%) (53. -- Pirenzepine was shown by Dal Monte e t a1 (98) t o be e f f e c t i v e i n t h e maintenance treatment of duodenal u l c e r s t o prevent u l c e r recurrence when given i n o r a l doses o f 100 mg d a i l y f o r one year.7%. were given pirenzepine 50 mg d a i l y f o r 6 months. who i n t e r r u p t e d treatment completely without pirenzepine. 106). i n t h e following 6 months. 114. followed by 50 mg b . Hoffenberg (103) reported pirenzepine i n o r a l dose of 50 mg b . . These results a r e supported by more recent c l i n i c a l s t u d i e s with pirenzepine i n o r a l d a i l y doses of 100 150 mg and t h i s appears t o be t h e optimal dose (107 .150 mg has been e f f e c t i v e i n producing healing of 74% of p a t i e n t s with duodenal u l c e r s i n c o n t r o l l e d c l i n i c a l t r i a l s (range 52 90%) (53. d . d . 104.7%. However.113). i n i t i a l l y . These d a t a suggest t h a t longer courses of therapy (6-8 weeks) may be indicated. was no more e f f e c t i v e than placebo i n t h e treatment of duodenal u l c e r (48% versus 40% complete healing). 89. Pirenzepine i n o r a l d a i l y doses of 100 . 54. doses of 75 mg d a i l y o r l e s s produce lower healing rates (53. Sixteen p a t i e n t s completing treatment. - Available c l i n i c a l t r i a l s on g a s t r i c u l c e r suggest t h a t o r a l doses of 100-150 mg d a i l y can produce healing in approximately 72% of p a t i e n t s with gast r i c u l c e r . f o r 4 weeks. In 16 o t h e r p a t i e n t s . d . and placebo i n t h e prevention o f duodenal u l c e r r e l a p s e i n a one-year study (102). 55. 76. 104. 74. Pirenzepine has a l s o been e f f e c t i v e i n doses of 50 mg d a i l y f o r one year (100. i . 105). f o r 4 weeks. i . s i m i l a r t o duodenal u l c e r . with endoscopic recurrence i n 62%. c l i n i c a l recurrence of duodenal u l c e r occured i n 68. Cheli -e t a1 (97) reported t h a t pirenzepine i n 100 mg o r a l l y d a i l y f o r 28 days r e s u l t e d i n c l i n i c a l cure i n 34 of 44 duodenal u l c e r p a t i e n t s . o t h e r i n v e s t i g a t o r s have reported no s i g n i f i c a n t d i f f e r e n c e between pirenzepine 25 mg o r a l l y b . i . r e s u l t e d i n u l c e r r e l a p s e i n 18.

121). P a l p i t a t i o n s occurred i n s e v e r a l p a t i e n t s i n t h i s study with doses of 50 mg.b l i n d multicenter study Brunner et a1 (108) evaluated t h e e f f i c a c y of pirenzepine 100 mg d a i l y . Short-term pirenzepine therapy (100 mg d a i l y f o r 4 weeks) was reported e f f e c t i v e i n improving t h e endoscopic appearance of acute c o n j e s t i v e and e r o s i v e g a s t r i t i s and non-ulcer-associated severe duodenitis (118).PIRENZEPINE DIHYDROCHLORIDE 485 Oral 50 mg d a i l y dose of pirenzepine was reported e f f e c t i v e i n t h e treatment o f non-ulcer dyspepsia i n a double-blind comparative t r i a l i n 59 p a t i e n t s f o r a period of 4 weeks (116). Both drugs have been equally e f f e c t i v e i n preventing duodenal u l c e r recurrence following healing with conventional treatment with e i t h e r drug (99. 81. t h i s cimetidine dose. The drug was s i m i l a r l y e f f e c t i v e a s cimetidine 1 gm d a i l y f o r 4 weeks. 101. 108). Porro e t a1 (109) have a l s o reported t h a t pirenzepine 150 mg PO d a i l y was comparable t o cimetidine 1 gm d a i l y i n t h e treatment o f a c t i v e duodenal u l c e r i n 90 p a t i e n t s t r e a t e d f o r a period of 4 weeks (72% versus 75% r e s p e c t i v e l y ) . t h e e f f i c a c y of both drugs was approximately 70%. 107. The authors i n d i c a t e t h a t high doses of pirenzepine should be avoided due t o antimuscarinic e f f e c t s such a s dry mouth and p a l p i t a t i o n s and t h a t cimetidine i s i n d i c a t e d as agent of choice f o r reduction of g a s t r i c a c i d s e c r e t i o n . Combination therapy with cimetidine has been e f f e c t i v e i n Zollinger-Ellison syndrome r e s i s t a n t t o cimetidine alone (121). In a s i n g l e . however. 120. had a g r e a t e r i n h i b i t o r y e f f e c t on g a s t r i c basal a c i d s e c r e t i o n (122). 68. A d a i l y dose o f 100 mg was a l s o shown t o be e f f e c t i v e (117). i n h i b i t i o n of p e n t a g a s t r i n stimulated acid s e c r e t i o n was similar t o t h a t o f an o r a l 200 mg cimetidine. Comparative e f f i c a c y of pirenzepine 100 mg PO d a i l y and cimetidine 1 gm PO d a i l y i n 6-week treatment o f duodenal u l c e r i n o u t p a t i e n t s has been indicated by s e v e r a l c o n t r o l led c l i n i c a l t r i a l s (67. -- A s i n g l e 400 mg o r a l dose of cimetidine demonstrated approximately twice t h e a n t i s e c r e t o r y a c t i v i t y of 50 mg pirenzepine s i n g l e o r a l dose i n both basal and stimulated g a s t r i c a c i d s e c r e t i o n following pentagastrin stimulation. In most c l i n i c a l s t u d i e s . With pirenzepine i n doses o f 50 mg.

except f o r more frequently reported antimuscarinic e f f e c t s i n pirenzepine-treated p a t i e n t s . pirenzepine had no e f f e c t s on CNS. Londong -e t a1 (125) reported t h a t concomitant pirenzepine and cimetidine therapy r e s u l t i n s i g n i f i c a n t l y g r e a t e r suppression o f stimulated acid s e c r e t i o n than e i t h e r drug alone. (50 mg before b r e a k f a s t and dinner) compared with t h a t of cimetidine 1 gm d a i l y (200 mg t . urinary bladder c o n t r a c t i o n o r motor function of g a s t r o e n t e r i c t r a c t (124). I n t r a g a s t r i c microbial concentrations were increased s i g n i f i c a n t l y with both drugs but remained within normal limits. EL-OBEIDETAL. o r a l dose (low-dose therapy) i n t h e treatment of duodenal u l c e r s i n multicenter t r i a l (125).486 HUMEIDA A. . Oral 300 mg d a i l y dose r a n i t i d i n e was reported (104) equally e f f e c t i v e as o r a l 100 mg d a i l y dose of pirenzepine i n t h e treatment of duodenal u l c e r i n one c o n t r o l l e d study (response rates of 87% i n each group). i . Pirenzepine was equally e f f e c t i v e as a t r o p i n e as g a s t r i c a n t i s e c r e t o r y agent. i . r e s p e c t i v e l y . d . but unlike a t r o p i n e . s . Pain r e l i e f was achieved r a p i d l y with both drugs and t h e incidence of s i d e e f f e c t s was s i m i l a r . Following 4 weeks of therapy. t h e endoscopist was blinded a s t o treatment regimens. Pirenzepine i n an o r a l dose of 50 mg b . ) i n t h e treatment of endoscopically-proven duodenal u l c e r i n 254 p a t i e n t s . I t i s suggested t h a t combination therapy may e x h i b i t s y n e r g i s t i c e f f e c t s on p a r i e t a l c e l l function and could be advantageous i n p a t i e n t s w i t h gastrinoma. d . mydriasis. d . i n c r e a s e i n h e a r t r a t e . which was not s t a t i s t i c a l l y s i g n i f i c a n t . i . Endoscopy was repeated a f t e r 4 weeks of treatment with both regimens. was reported s i m i l a r l y e f f e c t i v e as cimetidine 400 mg b . with meals and 400 mg h . No adverse e f f e c t s on i n t r a g a s t r i c milieu were reported. n i t r i l e concentrat i o n p r i o r t o o r following treatment did not exceed t h e normal range. Side e f f e c t s were s i m i l a r i n each group. healing occured i n 27 of 37 pirenzepine-treated p a t i e n t s (73%) and 29 of 38 cimetidine-treated p a t i e n t s ( 7 6 % ) . Healing r a t e s of 64% and 73% occurred with pirenzepine and cimetidine.

The combination o f p i r e n z e p i n e 50 mg h .L Unlike c i m e t i d i n e . Addition o f p i r e n z e p i n e t o a n t a c i d t h e r a p y r e s u l t e d i n a more s u s t a i n e d pH e l e v a t i o n t h a n t h a t observed with t w i c e t h e amount o f a n t a c i d a d m i n i s t e r e d a l o n e . S t u d i e s p r e s e n t e d so f a r do n o t show t h a t t h e drug i s more e f f e c t i v e t h a n c i m e t i d i n e o r r a n i t i d i n e and no s i g n i f i c a n t d i f f e r e n c e s i n t o x i c i t y have been observed. given f o r a p e r i o d o f 6-12 weeks was e f f e c t i v e i n a l l of 7 p a t i e n t s with a c t i v e recurrent duodenal u l c e r . These r e s u l t s s u g g e s t t h e e f f i c a c y of combined H2-antagonist and p i r e n z e p i n e t h e r a p y i n h i g h l y r e c u r r e n t o r r e s i s t a n t duodenal u l c e r s (127).i l l p a t i e n t s . . i .E l l i s o n syndrome.PIRENZEPINE DIHYDROCHLORIDE 487 u l c e r s and h y p e r s e c r e t i o n r e s i s t a n t t o s i n g l e drug t h e r a p y . . s . s u g g e s t t h a t p i r e n z e p i n e may be very u s e f u l i n t h e t r e a t m e n t o f r e s i s t a n t duoden a l u l c e r and Z o l l i n g e r . and r a n i t i d i n e 150 mg b .a g e n t ( c i m e t i d i n e o r r a n i t i d i n e ) t h e r a p y . Combination t h e r a p y w i t h H2-receptor a n t a g o n i s t s h a s proven more e f f e c t i v e t h a n H2-antagonist t h e r a p y a l o n e (118. inhibitory effects o x i d a s e system and concomitantly w i t h t h i s system (128). d . however. i n t h e p r e v i o u s y e a r . and i n p r o p h y l a x i s o f stress u l c e r bleeding i n c r i t i c a l l y . p i r e n z e p i n e appears t o l a c k on t h e h e p a t i c mixed f u n c t i o n it should be safe t o a d m i n i s t e r drugs t h a t a r e metabolized by The a v a i l a b l e i n f o r m a t i o n s s u g g e s t t h a t p i r e n z e p i n e p o s s e s s e s s e l e c t i v e a n t i m u s c a r i n i c e f f e c t s and minimal CNS effects. Weberg et a1 (126) h a s demonstrated t h a t p i r e n z e p i n e enhances and prolongs t h e e f f e c t s o f a n t a c i d s on i n t r a g a s t r i c a c i d i t y . 1 2 7 ) . P i r e n z e p i n e i n combination t h e r a p y w i t h H2-receptor a n t a g o n i s t s . Continuous maintenance t h e r a p y with r a n i t i d i n e 150 mg d a i l y and p i r e n z e p i n e 50 mg d a i l y r e s u l t e d i n prevention of u l c e r relapse i n 5 p a t i e n t s who. c o n t i n u a l l y r e l a p s e d while r e c e i v i n g s i n g l e . Mignon e t a1 (121) r e p o r t e d t h e e f f e c t i v e n e s s o f combination t h e r a p y o f p i r e n z e p i n e and c i m e t i d i n e i n 3 of 4 p a t i e n t s w i t h Z o l l i n g e r E l l i s o n syndrome who were unresponsive t o cimet i d i n e a l o n e .

i . which was approximately equipotent i n suppression of g a s t r i c a c i d . o r R-hyoscyamine 0. Diarrhea has a l s o occurred with pirenzepine therapy. EL-OBEIDETAL. 106. i . d . ( o r a l l y ) i n comparison with a t r o p i n e 0. d . reported pirenzepine (50 mg o r a l l y ) showed no e f f e c t s on g a s t r i c emptying. however. i . its delaying e f f e c t on g a s t r i c emptying was i n s i g n i f i c a n t . 134).5 mg i . Dry mouth is t h e most common s i d e e f f e c t of pirenzepine. Bianchi Porro e t a1 (34). t h e incidence appears t o be l e s s than t h a t of constipation (23.3 Adverse Reactions The major s i d e e f f e c t s of pirenzepine a r e due t o i t s a n t i c h o l i n e r g i c a c t i v i t y which i n d i c a t e s t h a t t h e s e l e c t i v i t y of t h e drug is n o t absolute and t h e a n t i c h o l i n e r g i c e f f e c t s can occur which appear t o be dose-dependents.6% of p a t i e n t s receiving doses of 150 mg o r a l d a i l y dose (54).488 HUMEIDA A. Jaup (135) measured esophageal p e r i s t a l t i c a c t i v i t y a f t e r pirenzepine. v . i n h i b i t e d s a l i v a t i o n compared t o placebo (131) but not a s much as R-hyoscyamine i n a dose o f 0. Despite t h e considerable e f f e c t s of pirenzepine on a n t r a l m o t i l i t y . 10 mg i . Constipation has occurred with pirenzepine i n approximately 2. 7. S a l i v a r y s e c r e t i o n e f f e c t s have been noted i n many t h e r a p e u t i c t r i a l s . Dry mouth does not appear t o be severe enough t o warrant withdrawal of treatment i n most cases (106).5% with doses of 75 mg d a i l y o r l e s s (54).6 mg b .5% of p a t i e n t s receiving pirenzepine i n doses o f 150 mg d a i l y . Similar r e s u l t s were reported by Stacher -e t a1 (133) when healthy men received e i t h e r 50 mg pirenzepine twice d a i l y o r placebo. The l a t t e r two agents reduced esophageal p e r i s t a l t i c pressure compared t o placebo while pirenzepine showed I . Dry mouth i s reported t o occur i n 13. however. d . Ten of 18 p a t i e n t s i n t h e t r i a l s reported dry mouth with pirenzepine and 17 of 18 on R-hyoscyamine. The incidence is reduced t o 4% with doses of 100 mg d a i l y and t o 2. v . d . pirenzepine i n a dose of 50 mg b . o r 50 mg b . In t h e pharmacological s t u d i e s i n man. Pirenzepine i n high doses i n h i b i t s t h e c o n t r a c t i l e (132) a c t i v i t y of t h e stomach and colon (132).6 mg b . i .

145) observed decreases i n c o n t r a s t t o a t r o p i n e and another t r i a l (144) showed no change. 137) have shown decreased amplitude of esophageal contractions as well as i n h i b i t i o n of lower esophageal s p i n c t e r pressure with p a r e n t e r a l administration of pirenzepine. suggesting a reduction i n ACTH s e c r e t i o n . chymotrypsin. pepsin and g a s t r i c a c i d s e c r e t i o n s a r e decreased. Oral pirenzepine s l i g h t l y decreased (.PIRENZEPINE DIHYDROCHLORIDE 489 marked e f f e c t s only s h o r t l y a f t e r i . d . p a n c r e a t i c polypeptide. vasoactive i n t e s t i n a l polypeptide. a reduction i n basal c o r t i s o l l e v e l s occurred with pirenzepine. pyruvate. Masala e t a1 (147) reported t h a t pirenzepine i n s i n g l e doses o f 75 mg reduced p r o l a c t i n l e v e l s i n females but produced only minimal e f f e c t s on p r o l a c t i n i n males. The responsiveness o f t h e adrenal glands t o exogenous ACTH was unchanged. l a c t a t e . dosing. One study (146) noted a s l i g h t prolongation o f g a s t r i n postp r a n d i a l l y . m . while o t h e r s (143. Other s t u d i e s (136. Pancreatic s e c r e t i o n volume. The drug has not produced adrenal i n s u f f i c i e n c y . when plasma l e v e l s were high. (138) and g a s t r i c smooth muscle e f f e c t s a r e s l i g h t a t 0 . i n s u l i n . Despite pirenzepine e f f e c t s i n a l t e r i n g esophageal and colonic m o t i l i t y . amylase. a c e t o a c e t a t e . The e f f e c t s of intravenous and o r a l pirenzepine on various g a s t r o i n t e s t i n a l hormones and metab o l i c parameters a r e s i m i l a r t o those observed f o r antimuscarinic agents except f o r t h e lack of depression of pancreatic bicarbonate s e c r e t i o n (141. (131). 142) and p o s s i b l y lack of g a s t r i n (143-145). 3 mg/kg i . Pirenzepine produced no changes i n g a s t r i c i n h i b i t o r y polypeptide. S i n g l e doses o f 75 mg pirenzepine were reported by Alagna -e t a1 (148) t o produce no d i r e c t e f f e c t upon t h e adrenal glands. l i p a s e . glucagon. (139) o r 50 mg b . 2 mg/kg i . However. -- . t h e a v a i l a b l e d a t a does not suggest t h e drug w i l l increase t h e r i s k of p e p t i c g a s t r o esophageal r e f l u x (140). m . p r o l a c t i n and growth hormone. Colonic m o t i l i t y is reduced by 0 . s e c r e t i n . neurotensin. enteroglucagon. v .15%) p e r i s t a l t i c amplitude compared t o placebo while R-hyoscyamine decreased t h i s parameter over 50%. i . f r e e f a t t y a c i d s . somatostatin. t r y p s i n .. f3-hydroxybutyrate.

m. 132). 7. however. Kuhn -e t a1 (150) reported t h a t pirenzepine (8 mg i. Pharmacological and c l i n i c a l s t u d i e s have shown t h i s a c t i o n of pirenzepine t o be s e l e c t i v e .490 HUMEIDA A.3% of p a t i e n t s receiving pirenzepine i n doses o f 100 mg d a i l y ( 8 3 ) . 83). Urinary r e t e n t i o n has been reported i n 0.3% of p a t i e n t s receiving o r a l d a i l y doses of 150 mg (75.4 Tissue S e l e c t i v i t y Pirenzepine blocks t h e acetylcholine receptors of t h e p a r i e t a l c e l l s o f the stomach and i s thereby a marked i n h i b i t o r of g a s t r i c acid s e c r e t i o n both i n animals and men. reported t h a t only small number of p a t i e n t s t r e a t e d with pirenzepine complained about impairment o f v i s i o n . 33. no e f f e c t s on h e a r t r a t e were observed (83). mental confusion. In most s t u d i e s . 45) and much higher doses a r e needed t o i n h i b i t s a l i v a t i o n (132) and contraction of stomach and colon (138) o r t o induce tachycardia (52. Blurred vision and double vision have occurred during p i renz ep i n e t r e a t men t i n approximate 1y 6. a s t h e n i a and headache have been reported i n a few p a t i e n t s (152. 151). Studies i n healthy volanteers showed t h a t no psychotropic e f f e c t s occurred t h a t a r e commonly encountered with t r i c y c l i c a n t i d e p r e s s a n t s . Brunner (94) however. s i n c e i n doses which s i g n i f i c a n t l y i n h i b i t g a s t r i c secretion. i n d i c a t i n g t h a t pirenzepine lack appreciable CNS t o x i c i t y (154). Hoffenberg (103) described amblyopia i n 24% of p a t i e n t s receiving dose of 100 mg d a i l y . s i d e e f f e c t s typical of a n t i c h o l i n e r g i c agents do not occur o r minimal (30. Dizziness. 153). . Pirenzepine i n dose of 50 mg d a i l y caused p a l p i t a t i o n s (122). Visual disturbances have been reported i n 42% o f s u b j e c t s (149). Fink and Erwin (149) a l s o reported lack of CNS e f f e c t s by pirenzepine with doses upto 150 mg. Tachycardia has been observed during pirenzepine treatment (103).) decreased t h e pulse r a t e and s y s t o l i c and d i a s t o l i c blood pressure. This can be explained on t h e bases o f pirenzepine s e l e c t i v i t y and a l s o t o hydrophilic c h a r a c t e r i s t i c s . EL-OBEIDETAL. CNS t o x i c i t y has been minimal i n most s t u d i e s with pirenzepine (83.

Atropine caused a dose-dependent i n c r e a s e i n h e a r t r a t e which. Pirenzepine was 16. t h e s e l e c t i v e antagonism o f c a r b a c h o l . could only be o b t a i n e d w i t h p i r e n z e p i n e i n doses 10 t o 100 times g r e a t e r t h a n with a t r o p i n e . 156) compared t h e effects o f p i r e n z e p i n e and R-hyoscyamine when t a k e n i n e q u i p o t e n t doses i n i n h i b i t i n g g a s t r i c s e c r e t i o n . Jaup et a1 (131. Both drugs a f f e c t e d t h e s a l i v a r y s e c r e t i o n b u t t h e i n h i b i t i o n by p i r e n z e p i n e was s i g n i f i c a n t l y less t h a n t h a t by R-hyoscyamine. Atropine caused a dose-dependent inhibition of t h e gastrointes tin a l tr a n s p o r t w h i l e p i r e n z e p i n e .PIRENZEPINE DIHYDROCHLORIDE 49 1 Engelhorn (155) has e v a l u a t e d t h e s e l e c t i v i t y o f p i r e n z e p i n e and a t r o p i n e and h i s experiments i n d i c a t e d s u b s t a n t i a l d i f f e r e n c e s between t h e i n f l u e n c e o f e i t h e r a n t i m u s c a r i n i c agent on g a s t r i c u l c e r and g a s t r i c s e c r e t i o n on one hand and on smooth muscle organs on t h e o t h e r . is t h e c o m p e t i t i v e i n h i b i t i o n by t h e drug of t h e carbachol e f f e c t on t h e spontaneously b e a t i n g guinea p i g atrium ( 1 5 7 ) .5 times weaker than a t r o p i n e i n reducing s a l i v a t i o n i n r a b b i t and is 100 times less e f f e c t i v e than a t r o p i n e i n doubling p a p i l l a r y diameter. The minimal e f f e c t s o f p i r e n z e p i n e on t h e CNS have been a t t r i b u t e d t o t h e drug s e l e c t i v i t y and h y d r o p h i l i city. 7. however. s i g n i f i c a n t d i f f e r e n c e s a r e demonstrated i n i s o l a t e d muscle preparations a f t e r acetylcholine stimulation as well a s after transmural stimulation. Gastric emptying was s i g n i f i c a n t l y delayed by R-hyoscyamine compared t o p i r e n z e p i n e . P u p i l s i z e and n e a r p o i n t d i s t a n c e were s i g n i f i c a n t l y a f f e c t e d by R-hyoscyamine b u t n o t by p i r e n z e p i n e . d i d n o t cause a d e c r e a s e b u t r a t h e r an i n c r e a s e . Swallowing induced eosphageal p e r i s t a l s i s was s i g n i f i c a n t l y i n h i b i t e d i n 51%. even a t h i g h e r d o s e s .5 Mechanism o f Action I t i s g e n e r a l l y accepted t h a t p i r e n z e p i n e i s an a n t i m u s c a r i n i c drug.s t i m u l a t e d a c i d s e c r e t i o n i n t h e p e r f u s e d r a t stomach w h i l e no such antagonism was n o t i c e d when h i s t a m i n e o r .u l c e r and a n t i s e c r e t o r y e f f e c t s o f p i r e n z e p i n e approach t h o s e o f a t r o p i n e . Although t h e a n t i . Among o t h e r examples demonstrating t h i s .

1 0 0 .f o l d weaker a t t h e A-type.15 mg/kg.6 (10-fold weaker) and 4 . m . ev i d en c e s have been p r o v id e d (164-166) o f t h e e x i s t e n c e o f t h r e e d i s t i n c t i v e t y p e s of muscarinic receptors c l a s s i f i e d a s types A. In t h e same system t h e l o g a f f i n i t y f o r a t r o p i n e i s about 8. i n d i c a t i n g t h a t t h e pharmacological r e sp o n se o f p i r e n z e p i n e as compared t o t h a t o f a t r o p i n e w i l l b e about 1 0 . pharmacological i n v e s t i g a t i o n on i s o l a t e d t i s s u e s and s t u d i e s i n man have shown t h a t p i r e n z e p i n e d i s t i n g u i s h e s between d i f f e r e n t s u b c l a s s e s o f m u sc a r in ic r e c e p t o r s (83. t h e conduction t i s s u e s i n t h e h e a r t c o n t a i n B-type r e c e p t o r s w h i le t h e ileum seems t o have b o t h t y p e B and C r e c e p t o r s and t h e CNS b o t h t y p e A and B r e c e p t o r s . 155). by p i r e n z e p i n e . blocked o n l y t h e m u s c a r in ic e f f e c t s o f a c e t y l c h o l i n e without a l t e r i n g t h e n i c o t i n i c component (151). i .f o ld weaker a t t h e B-type and 400-fold weaker a t t h e C-type. o f t h e m u s ca r i n i c response t o bethanechol on conscious c a t s with g a s t r i c f i s t u l a (157) and t h e f i n d i n g t h a t p i r e n z e p i n e . The myocardium have predominantly C . Using p i r e n z e p i n e a s novel t o o l t h a t r e v e a l s h e t e r o g e n e i t y o f m u sc a r in ic r e c e p t o r s .492 HUMEIDA A. In most of t h e cases t h e r e i s good q u a n t i t a t i v e agreement between t h e b i n d i n g a n a l y s i s and t h e pharmacological r e s u l t s (132. EL-OBEID ETAL. 150.r e c e p t o r s . S i m i l a r t o o t h e r a n t i m u s c a r i n i c a g e n t s .5 t o 9 . B a ld i (158) have demonstrated t h a t p i r e n z e p i n e (0. The pharmacological d a t a a v a i l a b l e on t h e potency of p i r e n z e p i n e as well a s d a t a from b in d in g s t u d i e s s u g g e s t t h a t i n t h e calf sy m p a th e tic g a n g l i a t h e r e seems t o be predominantly Ar e c e p t o r s and i n t h e r a t s y m p a th e tic g a n g l i a more o f B. Evidence f o r t h e h e t e r o g e n e i t y o f m u s c ar i n i c r e c e p t o r s i s a l s o p r e s e n t e d (171-173) using t h e s e l e c t i v e ganglionic receptor agonist . as well a s t h e s e l e c t i v e antagonism. p i r en z e p i n e a f f e c t s o n ly t h e volume and et a1 a c i d o u t p u t . f o r t h e Bt y p e about 6. With p i r e n z e p i n e t h e l o g a f f i n i t y c o n s t a n t f o r t h e A-type is about 7. B and C. i n g r e a t e r amount t h a t a t r o p i n e . S t u d i e s on r e c e p t o r b i n d i n g . and n o t g a s t r i c pH (83).f o l d f u r t h e r weaker f o r t h e C-type. 159-170). p e n t a g a s t r i n was used as a s t i m u l u s (48). ) i n h i b i t s t h e motor response i n t h e human colon t o a c h o l i n e r g i c s t i m u l u s w i t h p r o stig m in e .7.r e c e p t o r s .

175). Tanvir A.PIRENZEPINE DIHYDROCHLORIDE 493 McN-A-343 [ 4. I t appears t h a t MI and M2 receptors correspond t o t h e A-type and C-type receptors r e s p e c t i v e l y . B u t t f o r typing t h i s manuscript. Other mechanism f o r pirenzepine ulcer-heating e f f e c t s were a l s o reported (176). Further i n v e s t i gations a r e needed on pirenzepine and McN-A-343 t o determine which nomenclature t o follow. Pirenzepine may a l s o have a cytoprotective e f f e c t i n addition t o i t s a n t i s e c r e t o r y e f f e c t s (174. Acknowledgement The authors would l i k e t o thank M r . Those receptors stimulated s e l e c t i v e l y by McN-A-343 have been named as M1-receptors and those stimulated by conventional muscarinic drugs are c a l l e d M2-receptors. . The compound s e l e c t i v e l y stimulates t h e muscarinic receptors on ganglionic receptors with minimal influence on t h e muscarinic receptors i n t h e h e a r t and smooth muscle.(m-chlorophenylcarbamoyloxy) -2butenyltrimethylammonium c h l o r i d e ] .

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STREPTOMYCIN

J a b e r S . Mossa, Abdul Hameed U . Kader Taragan,
and Mahmoud M.A. Hassan,

J a b e r S . Mossa, A s s o c i a t e P r o f e s s o r o f Pharmacognosy,
C o l l e g e of Pharmacy, King Saud U n i v e r s i t y , Riyadh,
Saudi A r a b i a .
Abdul Hameed U . Kader Taragan, L e c t u r e r o f Pharmacognosy,
Department o f Pharmacognosy, C o l l e g e o f Pharmacy,
King Saud U n i v e r s i t y , Riyadh, S a u d i A r a b i a .

Mahmoud M.A. Hassan, P r o f e s s o r of Medicinal Chemistry,
Department of Pharm. Chem., C o l l e g e o f Pharmacy,
King Saud U N i v e r s i t y , Riyadh, Saudi Arabia.

ANALYTICAL PROFILES OF DRUG SUBSTANCES

VOLUME 16

507

Copyright 0 1987 by Academic Press, Inc.
All rights of reproductionin any form reserved.

JABER S . MOSSA ETAL.

508

1. History
2. Description
2.1
2.2
2.3
2.4
2.5

Nomenclature
Formulae
Molecular Weight
Appearance, Color, Odor and Taste
pH Range.

3. Physical Properties
3.1 Melting Range
3.2 Solubility
3.3 Loss on hying
3.4 Optical Rotation
3.5 Crystal Structure
3.6 Spectral Properties

4. Production of Streptomycin
5. Synthesis of Streptomycin

6. Biosynthesis of Streptomycin
7. Pharmacology and Therapeutic Category
8. Pharmacokinetics

8.1 Absorption
8.2 Distribution
8.3 Excretion
8.4 Half -life
9. Toxicity

10. Dosage
11. Pharmaceutical Forms
12. Mechanism of Action
13. Methods of Analysis

STREPTOMYCIN

13.1
13.2
13.3
13.4
13.5
13.6
13.7
13.8
13.9

Elemental Composition
Identification
Titrimetric Methods
Gasometric
Electrometric
Spectroscopic Methods
Chromatographic Methods
Bio-Assay Methods.
Biochemical Methods.

Acknowledgement
References

509

JABER S . MOSSA ETAL.

510

1. HISTORY

Streptomycin is a water soluble organic base biosynthesised
by appropriate strain of the actinomycetes,SXkepXornqcU
g h i n m . Prior to the development of Penicillin Waksman and
his collaborators began to study the production of antibiotic substances in the department of microbiology of New
Jersey Agricultural Experiment Station, Rutgers University
in 1939 (1). In the succeeding years they examined some
thousands of actinomycetes, hundreds of fungi and many bacteria and extracted from their culture media a number of
antibacterial substances. Particular attention was devoted to obtaining substances which acted prominently on gram
negative bacteria. As a result of this work, the organism
producing streptomycin was isolated from heavily manured
soil and from a chicken's throat (2,3). The first publication on streptomycin was made by Schatz, Bugie and Waksman
(3) in 1944. A complete bibliography of the work on streptomycin from 1944-1952 was recorded by Waksman in "The Literature on Streptomycin" (4). The cultural descriptions and
the nutritional requirement of ShepXornqcu g h i n w are also
discussed by Waksman ( 5 , 6 ) . The early promising clinical
reports of 1944 based on crude Streptomycin prepared in the
Merck Research Laboratories showed the importance of the
crude drug and started an intensive development program for
its large-scale production. The general line of development of the work on streptomycin have closely followed
those of the work on penicillin. Rapid progress in the
study of streptomycin was greatly helped by the experience
gained with penicillin and by the fact that there already
existed organizations for large-scale manufacture and
exploitation of the latter.
2. DESCRIPTION
2.1 Nomenclature
2 . 1 1 Chemical Names

a) O-2-Deoxy-2-(methylamino)-a-L-glycopyranosyl-

(1 + 2)-0-5-deoxy-3-C-formyl-a-L-lyxofuranosyl-

(1 + 4)-N,N' -bis (aminoiminomethyl)-D-streptamine ( 7 ) .

b) 4-0-(2-0-(2-Deoxy-2-methylamino- a -L-glycopyranosyl)-5-deoxy-3-C-formyl-a-L-lyxofuranosyl)-N

N' -diamidino-D -streptamhe (8).

STREPTOMYCIN

511

c) 2,4-Diguanidino-3,5,6-trihydroxycyclohexyl5-deoxy-2-0-(2-deoxy-2-methylamino-a -L-glucopyranosyl ) -3-formy1-a - L 1yxopentanofuranoside (9).
d) N-Methyl-L-glucosamidinostreptosidostreptidine (10).
e) 2- (N-methyl-a-L-glucopyranosaminido)- 3-Cforinyl-5-desoxy-L-aldopentofuranose (11) .
2.12 Generic Name
Streptomycin A.

(12)

2.13 Trade Names
a)
b)
c)
d)
e)
f)
g)
h)
i)
j)

Estreptomicina
Streptomycinum
Streptomyseen
Streptomycini
Streptovex
Stryzolin
Crystamycin
Dimycin
Oroject NS
Streptopen

2.2 Formulae
2.21 Empirical

a) Streptomycin = C21H39N7012
b) Streptomycin sulphate = (C21H39N,012)23H2S04
c) Streptomycin Hydrochloride =
C21H39N7012.3HC1.
d) Streptomycin calcium chloride =
(C21H39N7012. 3HC1) CaC12

JABER S. MOSSA ETAL.

512

2 . 2 2 Structural

MH

NH- C-NH2

0

H3C NH

HO

Dl

Streptomycin i s an o p t i c a l l y a c t i v e , t r i a c i d i c
base, C21H39N7012; possessing an aldehydic
carbonyl group. The high proportion of oxygen
molecule i s c h a r a c t e r i s t i c of a carbohydrate.
Streptomycin [ l ] i s made up o f t h r e e components :
S t r e p t i d i n e [ Z ] , S t r e p t o s e [ 3 1 , and N-methylL-glucosamine [ 4 ] , linked together by g l y c o s i d i c
bonds (16).

STREPTOMYCIN

513

NH

I

Streptomycin

PI

5 14

JABER S. MOSSA ETAL.

2.23 Structural elucidation
The difficult task of elucidating the structure
of streptomycin was carried out principally by
four groups of workers led by K. Folkers,
0. Wintersteiner, H.E. Carter and M.L. Wolfrom.
Their work has excellently reviewed f o r several
times (11, 17-21). The structural elucidation of
streptomycin is based on studies o f its various
degradation products.
1) On acid hydrolysis, the weaker glycosidic bond
in streptomycin between streptidine and streptose
splits to give streptidine [21 and streptobiosamine 151 (16,22-26).
2) Mild methanolysis o f streptomycin [ 11 with
methanolic hydrogen chloride gives streptidine
[ 21 and methyl streptobiosaminide dimethyl acetal
hydrochloride [61. Treating [6] with ethyl mercaptan and hydrogen chloride gives ethyl thiostreptobiosaminide diethyl mercaptal hydrochloride [ 7 1 which is also obtained by the action of
ethyl mercaptan and hydrogen chloride on streptomycin. By Raney-nickel and subsequent acid hydrolysis o f [71 , it gives N-methyl-L-glucosamine [4]
and bisdeoxy-streptose 181 (27-28).
3) Hydrolysis of streptomycin xith N1 sodium hydroxideofor three minutes at 100 or eighteen hours
at 40 yields a weakly acid substance, which has
been characterized as maltol [9! (29).

qCH3
OH

0

The degradation reactions of streptomycin is well
summarized in Scheme I.

f

t

JABER S . MOSSA ETAL.

516

Streptidine
is a meso Form of 1,3Streptidine 121, ~8~18~604,
diguanido-2,4,5,6-tetrahydrocyclohexane (24,25,
30, 31). The structure of streptidine [21 was
confirmed by synthesis from D-glucosamine (32,33).
Streptobioasamine
The structure of Nitrogen containing disaccharide,
streptobiosamine [51 was determined by a study of
hydrolysis products of the methyl ether and the
ethyl thio-ether formed by the action of hydrogen chloride in methanol and ethyl mercaptan respectively (27, 29, 34, 35). The hydrolysis of
methyl-streptobiosaminide dimethyl acetal gave
N-methylglucosamine 141 (27,28). The structure of
N-methyl-L-glucosamine has been established by
synthesis from L-arabinose (36). Because of its
instability, it is very difficult to isolate streptose [ 31 , the second component of streptobiosamine
[51. The molecular formula CgH1005 was calculated
from the known formulae of streptomycin 111 ,
Streptidine [ 2 ] and N-methyl-L-glucosamine [41
and its structure was deduced by identifying oxidation and reduction products. It was finally
shown to be 3-C-formyl-5-deoxy-L-lyxose, a pentose
containing the reactive aldehyde group associated
with streptomycin molecule (29,37-40). The
structure of streptomycin [I] was finally confirmed by total synthesis (Scheme 11).
2.24 CAS Registry Number
1) Streptomycin
2) Streptomycin sulphate
3) Streptomycin Hydrochloride

(57-92-1)
(3810- 74-0)
(6160-32-3)

2.25 Wiswesser Line Notation
Streptomycin sulphate :
T60TJ BlQ CQ -DQ EM1 FO- DT50TJ B CV H CQ EO
AL6T J BQ CQ DMYZ UM EQ EMYZU M WSQQ 3.

(41)

STREPTOMYCIN

517

2.26 Stereochemistry and Absolute Configuration
The chemistry of the component fragments of streptomycin was thoroughly studied and their structures were established (23-40). The absolute configuration of streptomycin has been elucidated by
chemical (applying Reeve's Copper complexing
method to various degradation products of streptomycin, dihydrostreptomycin and streptobiosamine)
(42,43) and optical correlations (44). The absolute stermch emistry of streptose was established
to be (R) at C-4 and (S) at C-2 (37,40,45). The
streptidine was shown to possess all mrn configuration (46). Although streptidine is a meso
form, the asymmetric attachment of streptobiosamine to it causes each of the ring carbon of
streptidine in streptomycin to be asymmetric. The
asymmetry of streptidine ring in streptomycin is
1(R), 2(R), 3(S), 4(R), 5(R), 61s). The streptobiosamine moiety of streptomycin has been shown to
be attached to position 4 of streptidine in either
R or S absolute configuration (46-48). The three
units of the streptomycin are attached together
by glycosidal bonds which were shown to be formed
by condensations between :
(i) the C-2 hydroxyl o f streptose and C-1 hydroxyl
of N-methyl-L-glucosamine (28,38,39,49-51).
(ii) C-1 hydroxyl of streptose and a hydroxl
group of streptidine that is adjacent to only
one guanidino group (i.e., position 4)
(22,38,39,52-54).
All glycosidic linkages have an a-S configuration
which was confirmed by NMR and crystallographic
studies (44, 55-57). By using the configurational
assignment, the structure of streptomycin in
complete stereochemical details may be written
as indicated below :

JABER S. MOSSAETAL.

518

YH2
C=NH
I
N-H

HOCH2

OH

-I

\,

- 11-

YH2

L-I

II

H NH

HO

2 . 3 Molecular Weight

a)
b)
c)
d)

Streptomycin
Streptomycin
Streptomycin
Streptomycin

=

sulphate =
hydrochloride =
calcium chloride =

581.6
1457.4
691.0
1492.9 (8)

2 . 4 Appearance, Color, Odor and Taste

Streptomycin occurs as white to slightly pink o r pale
brownish powder or granules; odor: odorless o r with
a slight c d o r ; taste : slightly bitter. Streptomycin
is hygroscopic and may deliquensce on exposure to air
but not affected by air or light ( 1 3 ) .
2 . 5 PH Range

per cent w/v solution of streptomycin has a pH
between 5 and 7.
A 25

-84: 2 .12) : 25 [a1D 1. Model 241 MC and found t o be [a125 . chloroform.0) The s p e c i f i c r o t a t i o n €or s t r e p t o m y c i n s u l p h a t e was determined i n our l a b o r a t o r y on a P e r k i n E l m e r Polar i m e t e r . 2 So 1u b i 1it y 1) Streptomycin : I t i s very s o l u b l e i n w a t e r .75' i n D water (C = 1. p r a c t i c a l l y i n s o l u b l e i n a l c o h o l a c e t o n e . Streptomycin h y d r o c h l o r i d e . c h l o r o form and e t h e r ( 8 ) . 4) Streptomycin calcium c h l o r i d e : I t is v e r y s o l u b l e i n water. e t h e r and petroleum e t h e r ( 1 3 ) . PHYSICAL PROPERTIES 3 . p r a c t i c a l l y i n s o l u b l e i n a l c o h o l . O ) . 3 . o f merc u r y . c h l o r o form and e t h e r ( 8 ) . O ) Water (C -1. . n o t more t h a n 5 p e r c e n t (13). e t h e r and petroleum e t h e r ( 8 ) . 3 . 3) Streptomycin h y d r o c h l o r i d e : I t i s v e r y s o l u b l e i n water. The f o l l o w i n g o p t i c a l r o t a t i o n s were r e p o r t e d f o r t h e various s a l t s of streptomycin (7. 3. 1 Melting Range I n d e f i n i t e (12). f o r 3 hours.519 STREPTOMYCIN 3 .4 O m i c a l R o t a t i o n Streptomycin i s o p t i c a l l y a c t i v e w i t h l e v 0 r o t a t i o n . almost i n s o l u b l e i n a l c o h o l ( 9 5 % ) . 3 Loss on Drying Strephomycin l o s e s when d r i e d o v e r phosphorus p e n t o x i d e a t 60 C and a t a p r e s s u r e n o t exceeding 5 mm. 2) Streptomycin s u l p h a t e : I t i s v e r y s o l u b l e i n water.7 6 Solvent Water (C = l . p r a c t i c a l l y i n s o l u b l e i n a l c o h o l . chloroform. . Streptomycin calcium c h l o r i d e .

C21H40Ng012 [ 1%H2Se04]. MOSSA ETAL. 1670. 1110 and 1040 cm- . 5 5 g . 4H20 c r y s t a l l i z e s from aqueous methanol as monoclinic n e e d l e s . Other r e p o r t e d I R d a t a 41) a r e 3330.2N su. 2 ) . 1470. ~ m .JABER S. 3. s a c e groupoC2. 5 4 g . a = 17. D = 1 . e t a1 (57) have r e p o r t e d t h e X-ray c r y s t a l l o g r a p h i c a n a l y s i s of s t r e p t o m y c i n oxime s e l e n a t e .~Dc. (vb) * C-0-C-stretch (vb) * Y I vb = Very broad. 3.6 Spectral Properties 3 . cm-3 f o r f o u r formula u n f t s p e r c e l l .phuric a c i d . S t r e p mycin oxime selenate'.5 Crystal Structure Neidle. 5. 520 3. Streptomyc i n i n 0. Three maxima were observed a t 241.62 I n f r a r e d spectrum The i n f r a r e d spectrum of s t r e p t o m y c i n s u l p h a t e a s K B r d i s c recorded on a Pye Unicam i n f r a r e d spectrophotometer i s shown i n F i g . Frequency cm -1 Assignment 3100-3500 NH and OH s t r e t c h (vb). c = 16.2 ( 9 ) . showed a maxima a t 280 nm w i t h E l 4 = 0.6.1710 1480 1100-1200 -C=O and -C=NH s t r e t c h ( v b ) . 6 1 U l t r a v i o l e t spectrum The UV spectrum o f s t r e p t o m y c i n s u l p h a t e i n water was scanned from 200 t o 400 nm on DMS 100 Varian AG Spectrophotometer (Fig.13 B = 108 .2 nm. The [OOl] p r o j e c t i o n o f one streptomycin oxime i o n i n selenate c r y s t a l i s shown i n F i g .36. b = 14. 1cm 3. 270 and 328. 1390. The s t r u c t u r a l assignments have been c o r r e l a t e d with t h e f o l l o w i n g f r e q u e n c i e s (Table 1 ) : Table 1 : I R C h a r a c t e r i s t i c s of Streptomycin Sulphate. 1. = 1 .10. B. 1630.

2 : UV Spectrum of Streptomycin Sulphate in Water. 1 : [OOlI P r o j e c t i o n o f one Streptomycin oxime i o n i n t h e Selenate C r y s t a l .STREPTOMYCIN F i g . 521 . Fig.

1 1200 1 1000 800 I .80 t 1 2000 1800 1600 I 1400 Fig. 3 : IR S p e c m ~ aof Streptomycin Sdphrte as K k diw.

23 .53 H-1 Streptose 3 -ti Formyl s t r e p t o s e 5.631 1 H-NMR Spectra 1 The H-NMR s p e c t r a of streptomycin i n D 0 ( F i g . NH 8 I 1 OH CH 7* 3 Table 2 : PblR C h a r a c t e r i s t i c s o f Stremomycin Sulphate Chemical Group. 2.30 5.523 STREPTOMYCIN 3 .83 3.56 2 .N Me of N-methyl glucosamine.06 3.86 1. shift. 4 . D20 H-1 N-methylglucosamjne 5. The proton chemical s h i f t s a r e shown i n Table 2 . 6 3 Nuclear Magnetic Resonance Spectra 3. 4 ) were recorded on a T60A MHz N M i spectrometer with TMS a s i n t e r n a l reference.Sec-Me o f S t r e p t o s e .

. . . . . . I . . 1 .0 3. . 4 : H-NMW Spectrum of Streptomycin Sulphatr iii I)$) and TMS.0 m (4) 4. 1 I . 1 . 8.1 1 . . . . . I . 1 .0 5. . I ' . . . r.0 I Fig. .0 I I I I . I . I I 1 . . 1 . . 1. 6.0 Z.0 . I .e 0 .

82 108.81 186.11 63.20 Multiplicity d d d d d d S S d d S d 4 d d d d d d t 4 .50 73.61 o r 97.80 108. Table 3 : Carbon Chemical S h i f t s o f Streptomycin Sulphate.31 108.61 o r 97.33 160.11 64.632 "C-NMR Spectra The n a t u r a l abundance C-13 NMR n o i s e .21 80.90-MHz i n s t r u m e n t . Carbon No. 1 2 3 4 5 6 7 8 1' 2' 3' 4' 5' 6' 1" 2'7 3" 4" 5" 6" 7'' Chemical S h i f t 6 ppm 57.14 75.11 61. The carbon chemical s h i f t s were a s s i g n e d on t h e b a s i s o f t h e t h e o r y of chemical s h i f t and SFORD s p l i t t i n g p a t t e r n (Table 3 ) .33 74.89 80.STREPTOMYCIN 525 3.d e coupled and s i n g l e frequency o f f .61 o r 97.11 71.52 160.67 34.r e s o n a n c e decoupled (SFORD) s p e c t r a ( F i g .92 84.13 60. 5 and F i g . 6 ) i n deuterium o x i d e were o b t a i n e d on a J o e l FX 90 .97 15.06 87.

5 : 13C-NMR Noisc-decoupkd Spectrum of Streptomycin SuIphate in D20 and TMS. .Fig.

6 : 13C-NMR Off-Resonance Spectrum of Streptomycin Sulphate in D 2 0 and TMS.1 hli Fig. .

3 3 (s) 57.33 (d) 64.8 (5) H2N 60. 82(d) - C 0 . i 1(d) 108.61 o r 97.05 (d) .06 ( d ) 87.13(d) 160.JABER S.11 (d) 9 7 . MOSSA ETAL.1 1 (d) 80.14 (d) 0 74. 528 1 6 0 .HN H 0 63.21 (d) 73.

PRODUCTION OF STREPTOMYCIN Streptomycin i s produced by t h e micro-organism b. SXtepXomycu g & e u can b i o s y n t h e s i z e s t r e p t o m y c i n i n a medium c o n t a i n i n g g l u c o s e . A b r i e f o u t l i n e of t h e p r o d u c t i o n o f s t r e p t o m y c i n i s well summarised below (104): a ) P r o d u c t i o n o f Spore Suspension The s p o r e s o f h%%eptumqcu g h i n ~ .s c a l e p r o d u c t i o n h a s been accomplished by e i t h e r s u r f a c e c u l t u r e o r deep c u l t u r e p r o c e s s ( 6 4 . b) P r e p a r a t i o n o f Medium A l l t h e s o l i d c o n s t i t u e n t s o f t h e c u l t u r e medium a r e p l a c e d i n t h e ferinenter through t h e c h a r g e h o l e and w a t e r i s added t o make up t h e volume. However. provided with s t i r r e r and inoculum measuring t a n k ) i s .STREPTOMYCIN 529 4 . 6 5 ) .b ( 5 8 ) . 1 0 5 ) . sodium c i t r a t e and i n o r g a n i c s a l t s . A f t e r s h u t t i n g o f f t h e steam. f o r l a r g e s c a l e p r o d u c t i o n . S t e r i l i z a t i o n i s c a r r i e d o u t a t 1 5 l b p r e s s u r e f o r 2 5 minutes. c ) P r e p a r a t i o n o f t h e inoculum The s t e r i l i z e d medium i n t h e s e e d t a n k (115 L .trreptomyca g h h w . c o r n s t e e p l i q u o r o r f e r m e n t a t i o n s o l u b l e s (61-88). I t i s a l s o produced by o t h e r s t r a i n s of organisms l i k e Acfinomyca cj1obhppohu. The medium i s s t e r i l i z e d by t h e d i r e c t i n t r o d u c t i o n of steam. y e a s t a u t o l y s a t e . One o r more of t h e s e a r e used f o r i n d u s t r i a l b i o s y n t h e s i s .s c a l e p r o d u c t i o n o f s t r e p t o m y c i n . The s p o r e s are e i t h e r suspended i n s t e r i l e water o r i n skimmed m i l k which i s i n o c u l a t e d w i t h t h e medium i n t h e shake f l a s k s . bAh. 89-103) . The s i t r r e r is working d u r i n g s t e r i l i z a t i o n .epXumycu g d b u (60). water i s passed through t h e j a c k e t and an a i r p r e s s u r e o f 10 l b i s maintained i n t h e f e r m e n t e r . pink v a r i a n t o f bRtreptumqca g h i n e u n (591. c a p a c i t y . but much h i g h e r y i e l d s are o b t a i n e d by u s i n g t h e media c o n t a i n i n g a l s o an o r g a n i c s o u r c e o f n i t r o g e n such a s meat e x t r a c t . c o o l i n g and ferment a t i o n . Soybean f l o u r . Tremendous amount of work h a s been done on t h e p r o d u c t i o n of s t r e p t o m y c i n (64 . a p p r o p r i a t e s t r a i n o f baheptomyca g&mb i s used.r e~q ~u i r e d f o r i n o c u l a t i o n i s o b t a i n e d from r a p i d l y growing c u l t u r e on s o l i d medium (64. The l a r g e . The deep c u l t u r e p r o c e s s i s more o f t e n used f o r l a r g e .

Flow Sheet 1 : The production of Streptomycin.530 JABER S . . MOSSA ETAL.

a i r s p a r g e r and a i r o u t l e t . After t h e i n o c u l a t i o n . The p r o d u c t i o n of s t r e p t o m y c i n is given i n flow sheet I . The t o t a l s y n t h e s i s of s t r e p t o m y c i n was achieved by Sumio Umezawa e t a l .000 u n i t s p e r l i t r e ) w i l l be reached i n 72 h o u r s . S o l v e n t e x t r a c t i o n techniques (180-188). t h e niycelium i s grown f o r 36 hours w i t h an a e r a t i o n r a t e of 100 L p e r minute. a heavy growth of mycelium i s o b t a i n e d . SYNTHESIS OF STREPTOMYCIN Although s t r e p t o m y c i n was t h e f i r s t aminoglycoside a n t i b i o t i c t o be d i s c o v e r e d . The r e a c t i o n s involved a r e i n shown i n Scheme I 1 €j summarized below: Condensation o f blocked d e r i v a t i v e o f d i h y d r o . d) Fermentation The f e r m e n t e r s a r e made up o f s t a i n l e s s s t e e l . followed by hydrol y s i s o f t h e S c h i f f b a s e w i t h 50% a c e t i c a c i d and . The f i n a l c o n c e n t r a t i o n o f s t r e p tomycin (50. a n t i foam n o z z l e . F u r t h e r p u r i f i c a t i o n i s c a r r i e d o u t by chromatography on alumina (118-122) o r i o n exchange resins (123-164) and by p r e c i p i t a t i o n w i t h v a r i o u s b a s e p r e c i p i t a n t s (165-179). About twenty l i t e r s o f t h i s mycelium suspension i s blown i n t o t h e measuring t a n k and f i n a l l y t o t h e f e r m e n t e r s through p i p e .192). e) I s o l a t i o n and P u r i f i c a t i o n The s t r e p t o m y c i n from t h e c u l t u r e f l u i d i s s e p a r a t e d by a d s o r p t i o n on t h e c h a r c o a l and by subsequent e l u t i o n w i t h methanolic hydrochloric acid o r other s u i t a b l e solvents (106-117). After t h e inoculum from t h e s e e d tank h a s been added t o t h e ferment e r .STREPTOMYCIN 53 1 i n o c u l a t e d w i t h t h e c o n t e n t o f shake f l a s k . having t h e c a p a c i t y of 15000 g a l l o n s and provided w i t h s i t r r e r . i n 1973 (191. After 36 h o u r s . e l e c t r o d i a l y s i s (189) and e l e c t r o o s m o t i c (190) procedures a r e a l s o employed. 5 . Then t h e f e r m e n t a t i o n 0 0 i s c a r r i e d o u t a t an optimum t e m p e r a t u r e of 25 -30 C . a e r a t i o n i s s t a r t e d a t once.l i n e . Foaming d u r i n g f e r m e n t a t i o n i s c o n t r o l l e d by t h e a d d i t i o n o f a n t i f o a m i n g a g e n t s . i t was n o t s y n t h e s i s e d u n t i l some 30 y e a r s l a t e r .s t r e p t o s e (benzyl a-L-dihydro-streptose)[lO] with t h e glucosamine d e r i v a t i v e (L-glucopyranosyl b r o m i d e ) [ l l ] i n benzene i n t h e p r e s e n c e of m e r c u r i c c y a n i d e .

JABER S . 532 SCHEME I1 . MOSSA ETAL.

STREPTOMYCIN 533 1111 phcg-J PhCOO 0 0 1191 PhCOO .

JABER S. MOSSA ETAL. 534 phcol&j PhCOO 0 NH I1 NHCNH2 1221 tlH NHCNHz I OH .

Streptomycin is formed from glucose as a primary precursor (193). . streptose and N-methyl-glucosamine are well summarised by Horner (193) E Snell (194).n i t r o p h en o x y c a r b o n y lc h lo r id e (NaOH. aq. acetone) gave the protected streptobiosaminide[ 121. 6. benzoyl and acetyl groups. Finally condensation of [211 with di-N-acety1-di-N- b e n z y l o x y c a r b o n y l .0 . Biosynthesis of the components of streptomycin. BIOSYNTHESIS OF STREPTOMYCIN Biosynthesis of streptomycin has been quite extensively studied. carbonate.c y c l o h e x y l i d e n e s t r e p t i d i n e gave the 4-0-glycoside [22].05 N Ba(OH2) and dioxane successively removed the carbamate. Removal of the remaining protecting groups with 50% acetic acid followed by hydrogenolysis with Palladium black gave dihydrostreptomycin [23].STREPTOMYCIN 535 reaction with methyl chloroformate (Na2Cog. Aqueous MeOH) gave [ 141 which was identical with benzyl a-S-dihydrostreptobioasminide. Partial hydrolysis of [181(75% Acetic acid) removed the isopropylidene group giving a diol which was transformed (p-N02C6H ococ 1. Direct treatment of [ 151 with p-nitrophenoxycarbonylchloride o r phosgene followed by benzolyation also gave [191. This was converted into the dibenzoyl derivative [18]. NMethvlation of [121 gave [131 Deacetylation followed by deisopropylidenation (IN HC1.2-dimethoxypropane (p-toluenesulfonic acid) gave a diisopropylidene derivative and further blocking by use of p . aqueous acetone) gave [ 161. The reaction of [201 with thionyl chloride at room temperature offered the a-glycosyl chloride [211. Partial hydrolysis of [161 (25% AcOH in MeOH) gave the diol 1171. Hydrolysis of 1141 with 10% Ba(OH)2 affored benzyl a-L-dihydro-Streptobiosaminide [ 151 . Subsequent mild oxidation of [23] afforded streptomycin[l] . streptidine. pyridine) into the carbonate [19]: Hydrogenofysis of the glycoside linkages (Palladium Black-dioxane) gave free sugar 1201. But the yield is very poor. General indication of the metabolic relationships of glucose L241 to the various components of streptomycin is shown in Scheme 111. Treatment of [ 151 with 2. Hydrolysis of 1221 with 0.

on HO Mvoinotitol - H.JABER S. MOSSA ETAL.yo3n on OH . 536 SCHEME I11 Tranunnular O<QH Icarranttemcnt.

u n i t s i s l a b e l e d a t t h e c o r r e s p o n d i n g carbon (Scheme IV). i n t h e s t r e p t o m y c i n b i o s y n t h e t i c pathway (204). S h i g e l L a . i n combination w i t h . Some o f t h e i n t e r m e d i a t e s i n t h e b i o s y n t h e t i c pathway of s t r e p t o m y c i n have been i s o l a t e d . T h i s was e s t a b l i s h e d by s e v e r a l groups (195-203).254). g h i n w was c a t a l i z e d and t r a n s f e r r e d t o d i h y d r o s t r e p t o s y l s t r e p t i d i n e 6-phosphate [ 2 5 ] which i n t u r n s was c o n v e r t e d t o dihydrostreptomycin 6 phosphate [ 2 6 ] .6-dideoxyglucose-dTDP which i n t u r n was c o n v e r t e d t o dihydrostreptose-dTDP. i n P o t l . They concluded t h a t t h e nucleotide-bound N-methyl4-glucosamine moiety i n S. I t i s a v a l u a b l e t h e r a p e u t i c a g e n t f o r a l l forms of t u b e r c u l o s i s (210-252). d e c a l h . The r e a c t i o n i s summarized i n Scheme V . I t i s h i g h l y s p e c i f i c and one o f t h e most e f f e c t i v e a g e n t f o r t h e t r e a t m e n t o f a l l forms o f p l a u g e (255). E. A . I t i s a l s o e f f e c t i v e a g a i n s t h&phyLococcud uLbw (257).&jCOC&. b a c t e r o i d e s .Lo carnnin and i n combination w i t h t e t r a c y c l i n e .STREPTOMYCIN 537 Experiments w i t h l a b e l e d compounds demonstrated t h a t t h e s p e c i f i c a l l y labeled glucose [24] i s converted i n t o s t r e p tomycin [ l ] i n which each of t h e s u b . t e ~ i n f e c t i o n s and i n b r u c e l l o s i s (256. Streptomycin i s a d r u g o f second c h o i c e i n i n f e c t i o n s due t o s u s c e p t i b l e s t r a i n s o f a n a e r o b i c bfiCLp. Recent s t u d i e s e s t a b l i s h e d t h e p r e s e n c e o f s t r e p t i d i n e . i n d h e n z a e . S t r e p t o mycin i s e f f e c t i v e a g a i n s t Tularemia (253. F u r t h e r o x i d a t i o n of [ 2 6 ] gave s t r e p t o mycin-6-phosphate[27] (207) hid h y d r o l y s i s y i e l d s t r e p t o mycin [ l ] (208). AmobacXm a e h o g e n a . 7 .6 phosphate and dihydro-streptose-dTDP. it i s c o n s i d e r e d t o be t h e d r u g o f c h o i c e i n i n f e c t i o n s w i t h SfiepXococcU v h i d a v m and EvLtmococcu ( 2 5 6 ) . I n combinat i o n w i t h P e n i c i l l i n G . Since s t r e p t o m y c i n i s e f f e c t i v e a g a i n s t H. both Gram p o s i t i v e and Gram n e g a t i v e (209).258-264). b u t some o f t h e key i n t e r m e d i a t e s a r e n o t y e t d e t e c t e d . PHARMACOLOGY AND THERAPEUTIC CATEGORY Streptomycin i s an aminoglycoside a n t i b i o t i c which i s a c t i v e a g a i n s t a wide range of b a c t e r i a . BhuceRea and Vibh. K&bbLelLa pneurnanhe and S&andl?a. Recently Kniep and Grisebach (206) e s t a b l i s h e d t h e r o l e o f d i h y d r o s t r e p t o s y l s t r e p t i d i n e 6-phosphate [ 2 5 ] i n t h e b i o s y n t h e s i s o f s t r e p tomycin. Nothing i s known y e t r e g a r d i n g t h e i n t e r m e d i a t e between g l u c o s e and N-methylglucosamine. CULL. Some i n t e r m e d i a t e s between g l u c o s e and d i h y d r o s t r e p t o s e a r e known as a r e s u l t o f t h e work o f Grisebach (205) who demonstrated t h a t [14C-] glucose-dTDP was c o n v e r t e d t o 4-Keto4.

0 OH ~241 #q* I * 0 H3C OH STREPTOSE HQ N-METHYL- CH20H # CH3NH OH * [11 L-GLUCOSAMINE STREPTOBIOSAMINE .NH SCHEME I V II NH NHCNH II STREPTIDINE i-koH HO -.

STREPTOMYCIN 539 SCHEME V nvo 1251 H$NM OM OH Ill 1261 .

The e f f e c t i v e minimum serum c o n c e n t r a t i o n i s about 7 mg/ml ( 1 5 ) . Streptomycin i s r a p i d l y absorbed when a d m i n i s t e r e d by subcutaneous.287). d i p h t h e r i a (279) and i n murine l e p r o s y (280-283). Very l i t t l e enters i n t o cerebrospinal f l u i d except i n meningitis (256.3 E x c r e t i o n About 50-80% o f s t r e p t o m y c i n i n j e c t e d i s e x c r e t e d i n u r i n e unchanged i n 24 hours (15. 1 5 ) . About 0.288. 8. 1 Absorption Streptomycin is absorbed p o o r l y and i r r e g u l a r l y from t h e g a s t r o i n t e s t i n a l t r a c t (255. Streptomycin can a l s o be absorbed through rectum when a d m i n i s t e r e d as cocoa b u t t e r o r carbowax m i x t u r e (286. However.285. p e r i c a r d i a l .256. Streptomycin i s a l s o e f f e c t i v e f o r typhoid f e v e r (274-277) . 8 .5% is e x c r e t e d i n t h e b r e a s t m i l k i n 24 hours and 1%i s e x c r e t e d i n b i l e (15.MOSSA ETAL.285.2 Distribution No m e t a b o l i t e of streptomycin has so f a r been d e t e c t e d . 8. i n t r a m u s c u l a r o r i n t r a v e n o u s r o u t e s (255. 540 o t h e r a n t i b i o t i c s it i s useful i n t h e treatment of r e s p i r a t o r y t r a c t i n f e c t i o n s . Most of t h e o r a l dose i s e l i m i n a t e d i n t h e f a e c e s (15.289) .285). T h e r e f o r e . whooping cough (278) . The drug p e n e t r a t e s r a p i d l y i n t o t h e p e r i t o n e a l .256).291).286). f a l l i n g t o 5 t o 10 mg/ml a t 12 hours (9.JABER S. P a r e n t r a l a d m i n i s t r a t i o n must be employed t o e n s u r e t h e r a p e u t i c a l l y a d e q u a t e plasma and t i s s u e l e v e l s of s t r e p t o m y c i n .265-273). p l e u r a l and s y n o v i a l f l u i d s .290. About one t h i r d t h e c i r c u l a t i n g st-reptomycin i s bound t o serum p r o t e i n . . b a c t e r i a l m e n i n g i t i s and u r i n a r y t r a c t i n f e c t i o n s (256.52 h o u r s . t h e o r a l r o u t e h a s been recommended i n t h e t r e a t m e n t o f i n t e s t i n a l d i s o r d e r s due t o s t r e p t o m y c i n s e n s i t i v e s t r a i n s o f b a c t e r i a o r t o reduce t h e b a c t e r i a l f l o r a of t h e i n t e s t i n e p r i o r t o o p e r a t i o n on i n t e s t i n a l t r a c t . t h e o r a l route i s not s u i t a b l e f o r administration of s t r e p tomycin f o r s y s t e m i c a c t i o n . PHARMACOKINETICS 8 .284). After an i n t r a m u s c u l a r dose of 1 g o f streptomycinipeak serum c o n c e n t r a t i o n s o f 25 t o 50 mg/ml a r e a t t a i n e d i n 0. Streptomycin i s widely d i s t r i b u t e d t o a l l organs e x c e p t b r a i n .

500 m i l l i g r a m s by mouth every e i g h t hours (304). In r e n a l f u n c t i o n impairment. 292-303). bone marrow and l i v e r r a r e l y o c c u r s a t t h e usual t h e r a p e u t i c t i s s u e l e v e l . c a u s i n g d e a f n e s s which i s permanent. 1 0 . t h e r e b y d i s t o r t i n g t h e t r a n s l a t i o n of t h e g e n e t i c code.l i f e o f s t r e p t o m y c i n i s about 2 . The d r u g a l s o c a u s e s headache. T h i s w i l l be i n c r e a s e d i n t h e new born and i n a d u l t s above 40 y e a r s .P. A s an i n t e r n a l a n t i s e p t i c . MECHANISM OF ACTION The a n t i b a c t e r i a l a c t i o n of s t r e p t o m y c i n i s a consequence o f i t s combination w i t h t h e 30s ribosomes o f s u s c e p t i b l e organisms.l i f e may be i n c r e a s e d t o 2-5 days (15) * 9 . 11. 307).STREPTOMYCIN 541 8. v e r t i g o . . Damage t o r e n a l t u b u l e s .P. t h e p r o t e i n s y n t h e s i s i s d i s r u p t e d and t h e c e l l d i e s (256. (305). nausea. hydrochloride B. TOXICITY A l l e r g i c r e a c t i o n s a r e r e l a t i v e l y v e r y common w i t h t h e a d m i n i s t r a t i o n o f s t r e p t o m y c i n .286.288. sulphate i n j e c t i o n B.P. Application of s t r e p t o mycin d i r e c t l y t o t h e peritoneum o r i t s i n t r a v e n o u s i n j e c t i o n i n l a r g e doses may cause d e a t h by p a r a l y s i s o f t h e r e s p i r a t i o n (285. 5 hours i n young a d u l t s . r e n a l i r r i t a t i o n and n e u r o t o x i c i t y . DOSAGE About 0 . f e v e r and s k i n r a s h e s .S. a r t h r a l g i a . PHARMACEUTICAL FORMS 1) 2) 3) 4) Streptomycin Streptomycin Streptomycin Streptomycin s u l p h a t e U.P. (8) * (8) - 1 2 . The u s u a l a l l e r g i c symptoms a r e e o s i n o p h i l i a . As a r e s u l t o f t h i s c o n d i t i o n t h e wrong amino a c i d s a r e i n c o r p o r a t e d i n t o t h e n a s c e n t p o r t i o n o f t h e p r o t e i n molecule. 5 t o 1 gram by i n t r a m u s c u l a r i n j e c t i o n d a i l y o r a t long i n t e r v a l s . The most s e r i o u s t o x i c e f f e c t i s damage t o t h e e i g h t h c r a n i a l n e r v e . 306. b l u r r i n g o f v i s i o n .4 Half-life Serum h a l f . t h e h a l f . (304). calcium c h l o r i d e B.

2 g o f s t r e p t o m y c i n i n a m i x t u r e o f 2 m l o f methyl a l c o h o l and 0 . 1 m l o f s u l p h u r i c a c i d .01% (7). b) Boil a small q u a n t i t y of s t r e p t o m y c i n w i t h 1 N sodium hydroxide f o r a few minutes and add a s l i g h t excess o f h y d r o c h l o r i c a c i d and a few d r o p s o f f e r r i c c h l o r i d e t e s t s o l u t i o n . a b r i l l i a n t v i o l e t c o l o r i s produced. The s o l u t i o n i s a g a i n h e a t e d f o r 5 minutes and cooled.2 I d e n t i f i c a t i o n 13.76% N = 16.7% H = 6.JABER S. f i l t e r i f n e c e s s a r y and allow t o s t a n d a t about 250. a ) D i s s o l v e about 0 . METHODS OF ANALYSIS 1 3 . 13. 13.22 Color T e s t s The f o l l o w i n g c o l o r t e s t s have been d e s c r i b e d f o r t h e i d e n t i f i c a t i o n of streptomycin : a ) Glucosamine Test i ) A 10 mg of s t r e p t o m y c i n i s d i s s o l v e d i n 1 m l o f water. About 1 m l o f e t h a n o l and 25 m l o f c o n c e n t r a t e d h y d r o c h l o r i c a c i d . 1 Elemental Composition c = 43. Then 2 m l of 2 N sodium hydroxide and 1 m l o f 20% aqueous a c e t y l a c e t o n e a r e added.86% 0 = 33.21 Pharmacopeial T e s t s The f o l l o w i n g t e s t s have been d e s c r i b e d i n B r i t i s h Pharmacopoeia f o r t h e i d e n t i f i c a t i o n of s t r e p t o m y c i n (304). 2 m l o f 1 N h y d r o c h l o r i c a c i d is added and t h e s o l u t i o n is h e a t e d on a steam b a t h f o r 10 minutes. MOSSA ETAL.1 g o f t r i n i t r o p h e n o l and c o o l . The m e l t i n g p o i n t o f t h e p r e c i p i t a t e a f t e r r e c r y s t a l l i s a t i o n from hot water i s about 283%. 542 13. c r y s t a l s o f s t r e p t i d i n e sulphate separate i n t h e course of 2 o r 3 d a y s . D i s s o l v e t h e c r y s t a l s i n 10 ml of h o t water c o n t a i n i n g 0.

STREPTOMYCIN 543 a r e t h e n added. a f t e r t h e c o l o r becomes b r i g h t green-yellow. i v ) When s t r e p t o m y c i n i s t r e a t e d w i t h sodium hypobromite i n s t r o n g a l k a l i n e s o l u t i o n . 10% potassium f e r r i c y a n i d e and 10% sodium hydroxide. c ) Other c o l o r T e s t s i ) An a l c o h o l i c s o l u t i o n o f s t r e p t o m y c i n gives a pink color with sulphuric acid (315). A cherry red color i s i i ) To about 2 m l o f an aqueous s o l u t i o n o f s t r e p t o m y c i n . A pink c o l o r i s developed on a d d i t i o n o f 2 m l o f a s o l u t i o n o f E h r l i c h ' s aldehyde (309).313). i i ) Aqueous s o l u t i o n o f s t r e p t o m y c i n when t r e a t e d with d i a c e t y l i n t h e presence o f oxygen g i v e s a pink c o l o r which i s i n t e n s i f i e d by t h e a d d i t i o n o f l-napht h o l (311). a v o l a t i l e bromo amine i s produced which turns acidified starch iodide solutions t o dark b l u e c o l o r (314). a r e d c o l o r i s produced (312. . 1 m l i s d i l u t e d t o 100 m l w i t h w a t e r ) . produced (308). The m i x t u r e i s h e a t e d f o r 10 minutes i n a b o i l i n g water b a t h and t h e n cooled. b) Guanido Test i ) A 1 0 mg o f s t r e p t o m y c i n i s d i s s o l v e d i n 1 m l o f water and 1 m l of o x i d i z e d n i t r o p n r s s i d e r e a g e n t i s added ( t h e r e a g e n t i s p r e p a r e d by mixing equal volumes of 10% sodium n i t r o p r u s s i d e . a r e d c o l o r devel o p s (310). 1 m l of a 2% s o l u t i o n o f a c e t y l a c e t o n e i n w a t e r and 1 m l o f 1 N sodium hydroxide a r e added. i n t h a t o r d e r . i i i ) When s t r e p t o m y c i n i s t r e a t e d with l-napht h o l and sodium hypobromite.

neutralize with 2N hydrochloric acid and add 1 ml in excess. c) Water-blue Test A dilute solution of streptomycin prevents the formation of the blue color when treated with a culture of KLeebtA.JABER S.eRea pfiewnoniae containing water blue dye (319). iii) With Sakaguchi solution (100 mg o f boric acid in 10 ml of concentrated sulphuric acid) streptomycin gives pale-green after 3-5 minutes (315).3 Titrimetric Methods 13.f!ucLh. prevents the usual color change from leucoform to red (317. prevents the coagulation of the milk in the usual way (316) . add 15 ml of Nessler's reagent and 1 ml of 10% potassium iodide.05N sodium thiosulphate solution.7 to 1 g of Streptomycin in 15 ml of water. 13. Shake and titrate the excess of iodine with 0. 544 ii) An alcoholic solution of streptomycin produces a yellowish-red color with phosphoric acid (315). .1N iodine solution. Then add 20 ml of water.23 Microbiological Tests a) Test with Streptococcus lactis Streptomycin when treated with milk inoculated with SRhepAococcud .318). The method is as follows : Dissolve about 0. 13. Add 15 ml of 0.31 Aqueous Delaby and Stephan (320) have reported an iodimetric method for the determination o f streptomycin. b) Triphenyltetrazolium Chloride (TTC) Test When streptomycin is treated with a culture of SR/LepAocaccw t h m o p k i e e u d on a sterile milk medium containing triphenyltetrazolium chloride.MOSSA ETAL.

13 32 Non-aaueous Penau e t a1 (325) have d e s c r i b e d a nonaquous method of d e t e r m i n a t i o n o f a n t i b i o t i c s i n c l u d i n g . it i s a c i d i f i e d with 1 N sulphuric a c i d .2% p u r e s t r e p t o m y c i n s u l p h a t e showed v a l u e s o f 89.7 and 8 8 . T h i s method can a l s o b e employed f o r t h e d e t e r m i n a t i o n of s t r e p t o m y c i n i n c u l t u r e media (323). 2 m l o f 1 N .2.1N s u l p h u r i c a c i d and 0. T h i s modified method g i v e s r e s u l t s w i t h s h a r p e r end p o i n t . A f t e r c o o l i n g .STREPTOMYCIN 545 An i o d o m e t r i c method i n v o l v i n g t h e r e a c t i o n o f s t r e p t o m y c i n w i t h sodium hyphobromite has been a l s o r e p o r t e d (321). 4 % . Huttenrauch and Keiner (322) have d e s c r i b e d a t i t r i m e t r i c method f o r t h e d e t e r m i n a t i o n o f s t r e p t o m y c i n s u l p h a t e .2 g potassium i o d i d e . was r e p o r t e d by Chandra e t a 1 (324). 88. then d i l u t e d t o 100 m l w i t h water and about 0 . The method i s a s follows : The sample c o n t a i n i n g s t r e p t o m y c i n s u l p h a t e i s added t o 5-10 m l b a s i c sodium hyphobrom i t e s o l u t i o n and a i r i s swept s u c c e s s i v e l y through t h e sample and 2 U-tubes each cont a i n i n g 5 m l o f 0. M o d i f i c a t i o n of Huttenrauch and Keiner method by r e p l a c i n g c e r i c s u l p h a t e s o l u t i o n w i t h p o t assium permanganate s o l u t i o n . The s o l u t i o n i s set a s i d e f o r 7 minutes and t h e n t i t r a t e d w i t h 0. A n a l y s i s o f 4-8 mg p o r t i o n s o f 90. 1 m l o f 2% f e r r i c c h l o r i d e s o l u t i o n is added. A f t e r sweeping a i r (10-15 b u b b l e s / s e c ) through t h e system €or about 15 m i n u t e s . t h e l i b e r a t e d i o d i n e i s t i t r a t e d w i t h 0. The method i s a s follows : The sample c o n t a i n i n g about 50 t o 150 mg o f streptomycin sulphate is dissolved i n 25 m l o f w a t e r .1-0.005N sodium t h i o s u l p h a t e .01N c e r i c s u l p h a t e u n t i l t h e c h a r a c t e r i s t i c red colour i s discharged.sodium hydroxide i s added and t h e m i x t u r e i s h e a t e d a t looo f o r 5 minutes.

0 mg/ml. are determined by measuring t h e volume of n i t r o g e n evolved by s t r e p t i d i n e f r a c t i o n c.5 Electrometric 13. The c o n c e n t r a t i o n d i f f u s i o n current curve i s l i n e a r f o r concent r a t i o n s from 0 . add s u f f i c i e n t b e n z i d i n e ( a s 0.05M b e n z i d i n e s o l u t i o n i n a c e t i c a c i d i s added. The s o l u t i o n o f s t r e p t o m y c i n i n 3% t e t r a m e t h y l ammonium . l m l o f 0. The s p e c i f i e d amount o f S t r e p tomycin s u l p h a t e is d i s s o l v e d i n p e r c h l o r i c a c i d and 2 m l of e t h y l e n e g l y c o l .6/30 mg o f s t r e p t o m y c i n base.05M solution i n g l a c i a l acetic acid) t o p r e c i p i t a t e 95 % of t h e s u l p h a t e . A polarographic a n a l y s i s has been developed ( 3 2 9 ) . Pure s t r e p t o m y c i n i n s o l u t i o n o r i n complex p r e p a r a t i o n s o r i n a s s o c i a t i o n w i t h p e n i c i l l i n s . The procedure i s a s f o l l o w s : To a 0. and o t h e r s by d i r e c t t i t r a t i o n o f p e r c h l o r i c a c i d i n g l a c i a l a c e t i c a c i d . 546 s t r e p t o m y c i n .1N s o l u t i o n of t h e sample i n g l a c i a l a c e t i c a c i d . G a u t i e r and P e l l e r i n (326) have r e p o r t e d a nonaqueous method o f e s t i m a t i o n o f s u l p h a t e o f t h e o r g a n i c b a s e s l i k e s t r e p t o m y c i n . Then about 0. 1 t o 1 . 5 % t h i a z o l e yellow i n e t h a n o l ) is added and t h e e x c e s s o f p e r c h l o r i c a c i d i s t i t r a t e d with a c i d potassium p h t h a l a t e s o l u t i o n . 13. a l l o w t h e p r e c i p i t a t e t o s e t t l e and t i t r a t e t h e s u p e r n a t a n t l i q u i d with 0. 13.1N H C 1 0 4 a c i d i s e q u i v a l e n t t o 581. 2 d r o p s of i n d i c a t o r (1% thymolphthalein i n e t h a n o l o r 0 .51 P o l a r o g r a p h i c Streptomycin i s r e d u c i b l e a t t h e dropping mercury electrode.4 Gasometric Gasometric d e t e r m i n a t i o n o f s t r e p t o m y c i n was r e p o r t e d by Yamagishi and Yokoo ( 3 2 7 ) and V i a l a (328).f tlie molecule under t h e a c t i o n o f sodium h y p o c h l o r i t e o r sodium hypobromite and compari n g it with t h a t f r e e d by r e f e r e n c e s o l u t i o n s o f guanidine hydrochloride o r guanidine a c e t a t e . After 10 minutes. MOSSA ETAL. dihydros t r e p t o m y c i n .JABER S.1 N p e r c h l o r i c acid i n g l a c i a l a c e t i c a c i d .

2 8 1 . The pH of the solution was adjusted to 1 2 . 2 9 volts.Solutions containing 100-400 y /ml were used for the determination. Plotting the diffusion current against concentration resulted in a straight 1ine. The rate of decomposition o f streptomycin in alkaline solution was polarographically studied by Bricker and Vail ( 3 3 0 ) .45 volts vs a mercury electrode. 3 3 6 ) . 5 8 . with mercury capillaries of drop times between 2 and 3 seconds at 50 cm height. 4 by adding 2 ml of 1 N sodium hydroxide. the Eh is 1. 5 2 Amperometric Streptomycin forms water insoluble salt with various anionic dyes. For all concentrations the half wave potential (E%) was 1 . A Tinsley Mark 19 pen recording polarograph is used. Doan and Riedel (332) have reported a polarographic method for the estimation of streptomycin sulphate. Goodey et a1 (331) has described a polarographic method for the assay of streptomycin in fermenter broth and associated recovery stages. 1 3 . Caplis et a1 (334) have reported a method f o r determination o f antibiotics including streptomycin by alternating-current polarography. Lithium hydroxide is used as base electrolyte. The limit o f identification of streptomycin sulphate was 0 .STREPTOMYCIN 541 hydroxide is polarographed at 13. . This can be used €or amperometric estimation of streptomycin ( 3 3 7 ) . 8 7 y/ml.7 2 . A survey of the applications of polarographic methods in the production control of antibiotics including streptomycin was described by Weissbuch and Unterman ( 3 3 5 .6O. Cunha (333) has published a similar polarographic method f o r determination of streptomycin sulphate. A mercury drop rate of 4 . The dissolved oxygen was removed by bubbling nitrogen through the sample f o r 5 minutes after that the surface was flushed with nitrogen.5 seconds was maintained.

adjust to pH 9 . Sample solutions containing 2-4 pg ml-1 of streptomycin . pH 2. The potential is maintained at -0. separate and dilute the aqueous phase to 50 ml.8 by addition of 2N hydrochloric acid and titrate the streptomycin potentiometrically with 0. MOSSA ETAL.08 volts. centrifuge.53 Potentiometric Machek (339) has carried out a potentiometric titration for the determination of streptomycin in mixed preparations.8 and is titrated with standard solution of the dye.6. 548 The method is based on the precipitation of streptomycin with a trivalent dye which is synthesised by coupling diazotized para rosaniline with 1-naphthol-4-sulphonic acid. 13. Extract a 1 ml aliquot of the filtered upper layer with 10 ml of phosphate buffer solution (pH 7. 5 by addition of 2N sodium hydroxide. Mix the filtered lower layer with 20 ml of butyl acetate. add Barium chloride (2ml) and shake for 1 minute.JABER S. adjust the pH of a 20 ml aliquot to 5. The method for the determination of tetracycline hydrochloride with suspension of E b C h d C k i d cofi and a potentiometric C02 sensor (HNC Model 10-22-00 or an Orion Model 95-02) has been extended to the determination of streptomycin.1N NaOH to pH 9.2) and determine the penicillin in the extract iodimetrically or microbiologically. Konopik (338) has described the application of metal salt solutions to amperometric determination of organic compounds including streptomycin. The method is as follows : Mix the sample with 15 ml o f water and 20 ml of butyl acetate and adjust to pH 2. A potentiometric microbiological assay for the antibiotic substances including streptomycin was proposed (340). About 1-3 mg of streptomycin is dissolved in 5 to 10 ml of 0.1 by addition of 2N hydrochloric acid and shake for five minutes.02M triethylamine citrate buffer.

STREPTOMYCIN 549 was used. To 5 . Maltol r e a c t s w i t h f e r r i c i o n s t o g i v e a p u r p l e c o l o r (304) and w i t h t h e F o l i n . The e x t i n c t i o n i s n o t less t h a n 90. o m i t t i n g t h e s u b s t a n c e t o be examined. 0 m l o f t h i s s o l u t i o n add 5 . f o r t h e estimation of s t r e p tomycin i s d e s c r i b e d i n t h e European Pharmacopoeia (342). The c a l i b r a t i o n graph was r e c t i l i n e a r w i t h range 0.5N s u l p h u r i c a c i d and s u f f i c i e n t water t o produce 25 m l and mix. using s t r e p t o m y c i n s u l p h a t e CRS i n s t e a d o f t h e s u b s t a n c e t o be examined.C i o c a l t e u phenol r e a g e n t i t produces a b l u e c o l o r (341). add 3 m l of a 1 . C a l c u l a t e t h e percentage with reference t o t h e subst a n c e t o be examined and t h e streptomyc i n s u l p h a t e CRS.2N sodium hydroxide and h e a t f o r e x a c t l y 10 minutes i n a water b a t h . 6 1 C o l o r i m e t r i c Methods 13. 0 m l o f 0. The procedure i s as f o l l o w s : Dissolve 0 .0 p e r c e n t o f t h a t o b t a i n e d by c a r r y i n g o u t t h e operations simultaneously. A colorimetric assay involving t h e maltol formation. measure t h e e x t i n c t i o n o f a 2 cm l a y e r a t about 525 nm. l g o f s t r e p t o m y c i n i n water and d i l u t e t o 100.33 t o 16.6 S p e c t r o s c o p i c Methods 1 3 . This f a c t may be used f o r t h e a s s a y o f streptomycin. 13.1 . E x a c t l y 20 minutes a f t e r t h e a d d i t i o n of t h e f e r r i c ammonium s u l p h a t e .0 m l with t h e same s o l v e n t . b o t h d r i e d f o r 4 hours .67 pg m l .611 The m a l t o l Method When s t r e p t o m y c i n is h e a t e d i n d i l u t e a l k a l i n e s o l u t i o n i t forms m a l t o l (29). u s i n g a s t h e compensation l i q u i d a s o l u t i o n p r e p a r e d i n t h e same manner. Cool i n i c e f o r e x a c t l y 5 minutes. 5 p e r c e n t w/v s o l u t i o n o f f e r r i c ammonium s u l p h a t e i n 0.

This method is recommended f o r r o u t i n e u s e . The method i s based on t h e formation o f m a l t o l from s t r e p t o mycin. a t 60' o v er phosphorus p e n to x id e a t a p r e s s u r e n o t exceeding 5 T o r r . The sta n d a r d c u r v e s p r e p a r e d from p u r e str e p to m y c in are linear. provided t h a t dosage was moder a t e . Pharmacopoeia of United S t a t e s . t h e phenol r e a g e n t i s used and t h e c o l o r i s read a t 775 nm. Eisenman and B r i c k e r (345) have d e s c r i bed a c o l o r i m e t r i c method €or t h e e s t i mation o f s t r ep t om y c in i n which t h e m a l t o l i s s e p a r a t e d from t h e r e a c t i o n m i x t u r e by steam d i s t i l l a t i o n . I f less t h ea n 500 u n i t s a r e p r e s e n t .550 JABER S. Bagdasarian and Michalska (346) have r e p o r t e d the m a lto l method f o r d e te r m in a t i o n of s t r e p t om y c in i n u r i n e without e x t r a c t i n g t h e s tr e p to m y c in p r i o r t o t h e a s s a y . I f more th a n 500 u n i t s o f streptomycin a r e p r e s e n t t h e colorimet r i c r e a c t i o n w i t h f e r r i c i o n is employed and t h e c o l o r i s read a t 550 nm. 1955 (343) d e s c r i b e d a similar m a l t o l c o l o r i metric a s s a y f o r d e t e r m i n a t i o n o f s t r e p tomycin i n s t r e p t o d u o c i n €or I n j e c t i o n .S. c o n c e n t r a t e s and c l i n i c a l samples. MOSSA ETAL. b u t cannot be a p p l i e d t o determining s t r e p to m y c in i n c o n c e n tr a t i o n below 100 y/ml. U. u r i n e and b r o t h . The method i s s u c c e s s f u l l y a p p l i e d f o r t h e d et er m i n a t i o n of str e p to m y c in i n ferment a t i o n b r o t h s . . s e p a r a t i o n of t h e m a lto l from t h e i n t e r f e r i n g s u b s t a n c e s by ext r a c t i o n with chloroform and subsequent d e t er m i n a t i o n by e i t h e r phenol r e a g e n t o r a c i d f e r r i c ammonium s u l p h a t e . Boxer e t a1 (344) have r e p o r t e d a s i m p l e and r a p i d c o l o r i m e t r i c a s s a y f o r streptomycin i n c l i n i c a l p r e p a r a t i o n s .P.

Bruns e t a 1 ( 3 4 9 ) . A blank was run c o n c u r r e n t l y . c o n s i s t i n g o f t h e same sample without h e a t . 3 . have r e p o r t e d a c o lo rimetric method f o r t h e d e t e r m i n a t i o n o f s t r ep t o m y c i n from t h e b r o t h .8 . The c o n t e n t i s determined by m a l t o l method. The absorbance was r e a d a f t e r a d d i t i o n of f e r r i c ammonium s u l p h a t e . After t h e a d d i t i o n o f 2 m l of 2N sodium hydroxide t o 5 m l o f t h e b r o t h . 5 on s i l i c a g e l and extract e d i n s u l p h u r i c a c i d . The a n t i b i o t i c s are c o l l e c t e d from t h e s o l u t i o n a t pH 8 . The m a l t o l t h u s s e p a r a t e d was a u t o m a t i c a l l y t r e a t e d w i t h stream o f f e r r i c c h l o r i d e i n 2. F e r r a r i e t a1 (351) have adapted t h e m a l t o l method f o r automated a n a l y s i s o f s t r ep t o m y c i n i n f e r m e n ta tio n media u s i n g d i a l y s i s t o s e p a r a t e m a l t o l from t h e i n t e r f e r i n g s u b s t a n c e s . F u r t h e r development o f t h e a u t o . They claimed t h a t h ig h p r e c i s i o n could .a n a l y z e r method f o r e v a l u a t i o n of str e p to m y c in were d e s c r i b e d by Shaw and Fortune ( 3 5 2 ) . Desai e t a1 (350) have d e s c r i b e d a d i r e c t method f o r e s t i m a t i o n o f s t r e p tomycin i n f e r m en te r b r o t h sample. Final d e t e r m i n a t i o n of str e p to m y c in was done by Maltol method. The b r o t h sample was d e p r o t e i n i z e d by t h e a d d i t i o n o f 4 m l of t r i c h l o r o a c e t i c a c i d . f i l t e r e d c e n t r i f u g e d and d i l u t e d .STREPTOMYCIN 551 Doery and Mason (347) and Ka r tse v a e t a 1 (348) s e p a r a t e d str e p to m y c in from o t h e r f e r m e n t a t i o n p r o d u c t s by i o n exchange chromatography on c a t i o n exchange r e s i n before convertion i n t o maltol.2N h y d r o c h l o r i c a c i d and t h e e x t i n c t i o n o f t h e r e a c t i o n product a t 530 nm was a u to m a t i c a l l y r e c o r d e d . E r r o r s were claimed t o be less t h a n with c o n v e n tio n a l manual methods. t h e m i x t u r e was heated i n a b o i l i n g water b a t h f o r t h r e e minutes and c o o le d .

552 JABER S.5 by adding 0. Krystyna (354) h a s r e p o r t e d t h e m a l t o l c o l o r i m e t r i c method €or t h e determinat i o n of streptomycin residues i n milk. a f t e r 10 minutes t h e absorbance was measured a t 660 nm and r e f e r r e d t o a c a l i b r a t i o n graph. Iodimetric-absorptiometric method was adopted by Sauciuc and Ionescu (353) f o r micro-determination o f s t r e p t o m y c i n i n f e r m e n t a t i o n l i q u i d .e x t r a c t e d i n t o w a t e r a t a l k a l i n e pH.02 g N-naphthyl-ethylenediamine d i h y d r o c h l o r i d e and 5g hydroxylammonium c h l o r i d e ) .055N phosphoric a c i d t h e n t r e a t e d w i t h f r e s h l y d i l u t e d 0. 200 m l acetic a c i d .1N sodium hydroxide f o r 4 minutes.f o r t i f i e d milk samples were cooled. d e p r o t e i n i z e d with z i n c s u l p h a t e i n t h e p r e s e n c e of barium hydroxide. The s o l u t i o n i s t h e n a d j u s t e d t o pH 7. . concent r a t e d and a g a i n d e f a t t e d w i t h l i g h t petroleum. The aqueous e x t r a c t was mixed w i t h F o l i n C i o c a l t e u phenol r e a g e n t .72g Sulphanilamide 0. A n t i b i o t i c . MOSSA ETAL. 6 g potassium bromide and l m l h y d r o c h l o r i c a c i d ) is added followed by a s t r o n g j e t o f 1 m l of r e a g e n t B (100 m l water. be maintained even w i t h c o n s i d e r a b l e s i m p l i f i c a t i o n t o a l l o w f o r h i g h throughp u t o f samples. Then t h e sample was t r e a t e d w i t h sodium hydroxide and h e a t e d on t h e water b a t h . The method i s a s follows : The sample c o n t a i n i n g 25-150 Ug o f s t r e p t o m y c i n is h e a t e d w i t h 0. After 10 minutes t h e e x t i n c t i o n of t h e s o l u t i o n is measured a g a i n s t t h e blank s o l u t i o n which is s i m i l a r l y t r e a t e d .001N i o d i n e i n potassium i o d i d e s o l u t i o n . 0. d e f a t t e d by c e n t r i f u g a t i o n . After 5 t o 10 minutes 5 m l of r e a g e n t A (300 m l water. The m a l t o l formed was e x t r a c t e d i n t o chloroform from t h e a c i d i c medium and b a c k .

Minor modifications have been suggested by Borowiecka (359). feeds (357) and urine (358).5 ml. 13. The procedure is as follows : 10 ml o f oxidized nitroprusside reagent is added to 10 ml of aqueous sample solution containing approximately 0.362).1 mg of streptomycin o r dihydrostreptomycin.water is added to bring to 2. The urine sample is treated with acetone and centrifuged after 30 minutes. To an aliquot of the clear solution. in which a pink color is generated by the reaction of the guanidine moiety with 3iacetyl and . Based on this principle Valedinskaya (363) has described a colorimetric method for estimation o f streptomycin. Then the precipitate is stirred with water and filtered o r centrifuged. then treated with nitroprusside reagent an3 the absorbance is measured. Kartseva and Bruns (360) and Wahbi et a1 (361). A working curve is prepared with the appropriate compound following the same procedure.STREPTOMYCIN 553 The maltol method has been extensively used in studies of stability (355) and in the analysis of streptomycin in pharmaceutical preparations (356). The color is stable for about 4 hour. Halliday (311) described the application of the modified Voges-Proskauer reaction to streptomycin. Vieira and Pereira (364) have adapted the nitroprusside method for the determination of streptomycin in urine.6 12 Guanido reaction Method Both streptomycin and dihydrostreptomycin form orange-red color with oxidized nitroprusside reagent (310. After 3 minutes the absorbance is determined at 490 nm against a reagent blank prepared by substituting 10 ml of water for the sample aliquot.

Forty minutes later determine the extinction at 525 MI in a 1 cm cell against water. reagents. and so on.313) has been adapted by Buck et a1 (369) to the analysis of streptomycin and dihydrostreptomycin.4 per cent diacetyl solution. 1 ml of 20% potassium hydroxide solution and 1 ml of 10% solution of 1-naphthol in anhydrous ethanol. Ashton et a1 (366) and Banerjee (367). ethanolic sodium hydroxide is added and the mixture is set aside at room temperature for 45 minutes. using spectrophotometric determination of the red color formed by the reaction of 1-naphthol and sodium hypobromite with the antibiotics. A standard graph should be prepared for each determination to allow for differences in room temperature. Schulz and Posada (368) have developed a colorimetric method involving guanidino group reaction for determination of streptomycin. add 15 ml of water and then 1 ml of 0. using the same procedure for color development as described for the sample. The Sakaguchi reaction (312.554 JABER S.18 of streptomycin sulphate per ml. The procedure i s as follows : . Mix the contents of the tube by inversion after each addition. The extinction is then measured at 555 nm and the streptomycin content is calculated with reference to a standard solution containing 50 1. Prepare a standard graph from suitable dilutions of a standard streptomycin solution. based on the Sakaguchi reaction. Natarajan and Tayal (370) have reported a rapid method of colorimetric estimation of streptomycin. 1-napthol. in that order.7-diol. Determine the potency of the diluted sample solution from the standard graph. The aqueous sample solution is treated with an ethanolic reagent containing diacetyl and naphthalene-2. MOSSA ETAL. Quantitative analytical procedure was developed by Szafir and Bennett (365). The method is as follows : Transfer 2 ml of the sample solution to a test tube.

M o d i f i c a t i o n of t h e above method by u s i n g 8hydroxyquinoline was d e s c r i b e d by T i b a l d i (372). t h e mixture i s shaken v i g o r o u s l y and 1 m l o f 40% u r e a s o l u t i o n i s added i n 1 5 second. can be used q u a n t i t a t i v e l y (309). and add 5 m l o f a b s o l u t e e t h a n o l .555 STREPTOMYCIN Mix a n aqueous s o l u t i o n o f t h e sample w i t h 1 m l o f 10% sodium hydroxide. add 1 m l of sodium hypobramite r e a g e n t and 10 m l o f c a r b o n t e t r a c h l o r i d e . The method i s as f o l l o w s : 5 m l O f t h e s o l u t i o n o f t h e sample i s t r e a t e d w i t h 2 m l o f 1-naphthol r e a g e n t .5 m l o f N-bromos u c c i n i m i d e s o l u t i o n . After b e i n g shaken and set a s i d e f o r 3 minutes. The r e s u l t s were comparable w i t h t h o s e from m i c r o b i o l o g i c a l method. r e s u l t s being r e f e r r e d t o standard curves.613 Glucosamine r e a c t i o n Method The glucosamine t e s t f o r t h e i d e n t i f i c a t i o n of s t r e p t o m y c i n u s i n g a c e t y l a c e t o n e and E h r l i c h ' s r e a g e n t . The a p p l i c a t i o n of t h e Sakaguchi r e a c t i o n f o r t h e c o l o r i m e t r i c d e t e r m i n a t i o n o f streptomyc i n was a l s o r e p o r t e d by Fontes ( 3 7 1 ) . w i t h a b l u e f i l t e r .02% e t h a n o l i c 1-naphthol.p o s i t i v e a n t i b i o t i c s . Shake well. The p r o c e d u r e of t h e method i s as follows : . A v i v i d r e d c o l o u r devel o p s . shake a g a i n and measure t h e e x t i n c t i o n o f t h e aqueous p h a s e a t 530 nm i n a 1 cm c u v e t t e with a r e a g e n t b l a n k . The r e l a t i v e e r r o r o f t h e method i s f 4 % . Kamata e t a1 (374) have d e s c r i b e d a c o l o r i m e t r i c a s s a y f o r s t r e p tomycin. t h e mixt u r e i s r a p i d l y poured i n t o 0. t h e e x t i n c t i o n o f which is r e a d i n 5 minutes a g a i n s t b l a n k s . S z i l a g y i and Szabo (373) have r e p o r t e d a simil a r c o l o r i m e t r i c method f o r t h e a n a l y s i s o f S a k a g u c h i . u s i n g N-bromosuccinimide. After 15 m i n u t e s . and 2 m l o f 0. 13. Based on t h i s p r i n c i p l e .

30 m l o f concent r a t e d h y d r o ch l o r i c a c i d and a b s o l u t e e t h a n o l ) .225 m l a c e t y l a c e t o n e . c o o l .614 Other Methods The aldehyde group o f str e p to m y c in was expl o i t e d by Marshall e t a1 (358) who developed a q u a n t i t a t i v e p r o ce d u r e based on t h e format i o n o f a co l o r ed semicarbazone with 4-[4(p-chlorophenazo) -1 -naphthyl ] semicarbazide.5 m l o f r e a g e n t A (2. 1 m l 1N h y d r o c h l o r i c a c i d and water) . n e u t r a l i z e and d i l u t e t o 10 m l .JABER S . s t o p p e r . c o o l . Mattick (377) h a s r e p o r t e d a c o l o r i m e t r i c method f o r t h e d e t e r m i n a t i o n o f a n t i b i o t i c s . 113. t h e e x c e s s o f l e a d is removed by sodium s u l p h a t e and f i l t e r e d . To about 2 m l o f t h i s s o l u t i o n add 5 .d i n i t r o p h e n y l h y d r a z i n e which reacts w i t h s t r e p tomycin t o form a yellow hydrazone. Boil f o r 1 hour. add 10 m l . o f e t h a n o l . S a v i t s k a y a and Kartseva (376) used 2 . 4 . Measure t h e i n d e n t s i t y o f t h e c o l o r a t 532 nm. 2 m l o f th e E h r lic h ' s aldehyde r e a g e n t is t h e n added and t h e red c o l o r developed i s compared w i t h a sta n d a r d s o l u t i o n o f s t r ep t o m y c i n and w i t h a blank of u r i n e sample. The s o l u t i o n i s h ea t ed i n a w a te r h a t h f o r 10 minu t e s and co o l ed . The u r i n e sample c o n t a i n i n g s t r e p to m y c in i s t r e a t e d w i t h l ea d a c e t a t e . b o i l f o r 20 minutes. The a c e t y l acetone-Ehrlich's aldehyde method h a s been adapted by Masquelier (375) f o r t h e d e t e r m i n a t i o n o f s t r e p t o m y c in i n u r i n e . The method i s r e l i a b l e o n l y f o r samples o f high p u r i t y . The c o l o r developed i s measured a t 580 nm.8 o f Ehrlich'saldehyde.5N s u l p h u r i c a c i d . f i n a l l y add r e a g e n t B (0. The e x c e s s r e a g e n t is e x t r a c t e d w i t h b u t y l acetate and t h e c o n c e n t r a t i o n o f t h e str e p to m y c in i s determined from c a l i b r a t i o n curve p r e p a r e d with pure streptomycin. The ex c e s s of t h e r e a g e n t i s e x t r a c t e d w i t h chloroform and h y d r o c h l o r i c a c i d i s added. MOSSA ETAL. add 1 m l o f 2. To about 1 m l o f t h e f i l t r a t e 1 m l o f a c e t y l a c e to n e r e a g e n t and 2 d r o p s o f sodium hydroxide a r e added.75 m l 2 N sodium c a r b o n a t e 3 4 . 556 To 4 m l of s t r ep t o m y c i n s o l u t i o n . 13.

b o r a t e b u f f e r and 2 m l of o x a l i c a c i d d i h y d r a z i d e r e a g e n t . washed w i t h et h an o l and d i s s o l v e d i n ac e t o n e water m i x t u r e . The p r e c i p i t a t e t h u s formed i s separ a t e d . 5 m l of t h i s solut i o n i s t r e a t e d with 5 m l of f e r r i c n i t r a t e and t h e c o n c e n t r a t i o n o f str e p to m y c in i s d e t e r mined c o l o r i m e t r i c a l l y . About 1 m l of t h e sample i s t r e a t e d with 2 m l o f p h o s p h a t e . S t r e p t o mycin i s h ea t ed w i t h w ate r a t 40° and e q u a l volume o f ammonium r e i n e c k a t e s o l u t i o n i s added. d i l u t e d w i t h water and neut r a l i z e d with n i t r i c acid.c i t r a t e .STREPTOMYCIN 557 i n c l u d i n g s t r ep t o m y c i n i n m i l k which is based on t h e i n h i b i t i o n o f t h e r e d u c t i o n of n i t r a t e t o n i t r o u s o x i d e by Miwiacoccus pqkogcnu. The s o l u t i o n i s th e n t r e a t e d w ith sodium hydroxide. The n i t r o u s o x i d e produced i s determined color i m e t r i c a l l y a f t e r d i a z o t i s a t i o n w i t h su lp h a n i l i c a c i d and c o u p l i n g with l-naphthylamine. Using p i c r i c a c i d r e a g e n t . B a r i l a r i and K a t z (378) have r e p o r t e d a method u s i n g copper t a r t r a t e and phosphomolybdic a c i d reagents f o r colorimetric determination o f s t r ep t o m y c i n . After c o o l i n g t h e phosphomolybdic a c i d i s added and t h e b l u e c o l o r developed i s r e a d p h o t o c o l o r i m e t r i c a l l y . The test sample and . The b l u e o r v i o l e t c o l o r r e s u l t i n g i s read colorimetrically. Agrawal and D u tta (382) have developed a r a p i d c o l o r i m e t r i c a s s a y method f o r s t r ep t om y c in i n m ix tu r e w i t h d i h y d r o s t r e p t o m y c i n . A p h o t o c o l o r i m e t r i c method f o r t h e determina- t i o n o f s t r e p t o m y c i n w i t h r e i n e c k a t e s a l t was d e s c r i b e d by B a r i l a r i e t a1 ( 3 7 9 ) . The f r e s h l y p r e p a r e d s o l u t i o n o f streptomycin s u l p h a t e is heated f o r 8 minu t e s w i t h 2 m l of w a t er and 2 m l of copper t a r t r a t e s o l u t i o n . C o l o r i m e t r i c d e t e r m i n a t i o n o f aldehyde and k e t o n es w i t h o x a l i c a c i d d ih y d r a z id e was adap t e d by Bartos and B u r t i n (381) f o r d e te r m in a t i o n o f s t r ep t o m y c i n . Then a c e to n e i s evap o r a t e d . The a p p l i c a t i o n o f t h i s method i n t h e pharmaceutical a n a l y s i s was r e p o r t e d by Basu and D utta (380). The m ix tu r e i s allowed t o s t a n d f o r 4 h o u r s .

by salt formation with bromothymol blue was reported by Nour El-Deen et a1 (384). 558 the blank are treated with 3 ml of 0. A spectrophotometric method for the estimation of streptomycin by salt formation with tropaeolin 000. Dihydrostreptomycin does not give this color reaction. decant and wash the precipitate with 10 n i l of cold dilute sulphuric acid. but can be determined after being converted into streptomycin. cooled diluted to 10 ml and the extinction is measured at 500 nm. Dissolve the residue in 2 ml of sodium hydroxide. The method is .1% picric acid. cooled and the absorbance is read at 490 nm. After the disappearance of the yellow color. Barilari et a1 (385) have reported a new photocolorimetric method for the determination o f streptomycin. Yavors'kli (383) has reported the application of dinitrobenzene and their derivatives as reagent in colorimetric analysis of organic medicinal substances including streptomycin.JABER S. A new sensitive colorimetric determination of streptomycin by thiobarbituric acid method was proposed by Erno (387). centrifuge. adjust to 100 ml with water. MOSSA ETAL.2 ml of 5% sodium hydroxide. dilute sulphuric acid. boiled. Mix and cool in ice for one hour. 0 . and 3 ml of cold aqueous solution Tropaeolin 000. A new spectrophotometric method for the assay of streptomycin. Dilute a mixture of 5 ml of the solution and 1 ml of dilute sulphuric acid to 50 ml and measure the extinction at 485 nm. The method is as follows : To about 3 ml of sample in water add about 1 ml o f cold. heated for 3 minutes. Streptomycin solution is treated with potassium iodate solution and sodium arsenite solution. 0 3 ml of phenol and 4 ml of sulphuric acid are added. was reported by Nour El-Deen et a1 (384) and Beltagy et a1 (386).

The method i n v o l v e s f o r m a t i o n o f a t e r n a r y complex i n a medium c o n t a i n i n g 40% of methanol and 1%of p o l y v i n y l p y r r o l i d i n o n e . When t h e brown c o l o r i s d i s a p p e a r e d .5M s u l p h u r i c a c i d . After 20 minutes t h e mixture i s c e n t r i f u g e d and t h e absorbance o f t h e s u p e r n a t a n t l i q u i d is measured a t 560 nm.05 m l o f 0. 1 m l o f 1 mM PrC13 and 6 ml o f e t h a n o l added. . The blood sample is t r e a t e d with 1 m l o f water and 0 . A new and s e l e c t i v e s p e c t r o p h o t o m e t r i c d e t e r mination o f s t r e p t o m y c i n u s i n g O-hydroxyhydroq u i n o n e p h t h a l e i n [2'.7'-tetrahydroxyspiro (isobenzofuran. was r e p o r t e d (391).STREPTOMYCIN 559 s p e c i f i c and s e n s i t i v e . A p h o t o m e t r i c method f o r t h e d e t e r m i n a t i o n of s t r e p t o m y c i n and o t h e r aminoglycoside a n t i b i o t i c s was proposed by Alykov (392). Alykov (390) has d e s c r i b e d an a b s o r p t i o m e t r i c method f o r t h e d e t e r m i n a t i o n o f amino g l y c o s i d e a n t i b i o t i c s i n b i o l o g i c a l f l u i d s . 9 ' .6'. 1 Acid Black 24 w i t h t h e a n t i b i o t i c s . 5 m l o f t r i c h l o r o a c e t i c a c i d s o l u t i o n and c e n t r i f u g e d . The method i n v o l v e s t h e measurement of t h e d e c r e a s e i n t h e absorbance a t 575 nm. due t o Acid b l a c k S caused by p r e c i p i t a t i o n o f a complex o f C . 1( 3 H ) . The m i x t u r e i s h e a t e d f o r 10 minutes and t h e n t h e e x t i n c t i o n of t h e s o l u t i o n i s measured a t 415 nm a g a i n s t a b l a n k .3'. The m o d i f i c a t i o n o f t h i o b a r b i t u r i c a c i d method was r e p o r t e d by Erno e t a l . a f t e r 15 minutes.(9H) xanthen) -3-one) ] and manganese. follows : The procedure i s a s The s o l u t i o n c o n t a i n i n g s t r e p t o m y c i n i s mixed w i t h 0. a 2% s o l u t i o n o f sodium a r s e n i t e i n 5 m l h y d r o c h l o r i c a c i d i s added. (388) and Fukumoto Hisako (389). To t h e s u p e r n a t a n t l i q u i d 1 m l of s t i l b a z o c h r o m e P r e a g e n t i n c i t r a t e b u f f e r s o l u t i o n . 3 . 1 m l o f t h i o b a r b i t u r a t e r e a g e n t is added. Streptomycin and o t h e r amino g l y c o s i d e s form a complex w i t h stilbazochrome P i n a c e t a t e o r c i t r a t e b u f f e r medium. Measurements a r e made a t 570 nm a t pH 10 t o 1 0 .1M potassium i o d a t e i n 0.

boiled for 15 minutes. After drying the colored bands were transferred to test tubes and shaken with 5 ml of propan-2-01 containing 5 drops of ammonium hydroxide and the tubes were centrifuged. About 0 .3M.0 ml of the solution was treated with 3 ml of SmM-brucine. 5 to 3. which exhibits an absorption maximum at 322 nm in alkaline solution. The residue was dissolved in 20 ml of warm water and diluted to 50 ml. the chromatogram was developed in a solution of l g of ninhydrin in 50 ml of 95% ethanol-anhydrous acetic acid (5:l).JABER S. About 10 l. Therefore the streptomycin can be assayed by measuring the absorbance at 322 nm before and after hydrolysis in sodium hydroxide solution (396). cooled and the absorbance was measured at 430-450 nm. 1. The absorbance spectrum o f supernatant solution was recorded in the range 400 to 800 nm against water. Divakar et a1 (395) have reported a spectrophotometric method for the determination of streptomycin. 13 62 Ultraviolet Methods When streptomycin is heated with 0. Katz (397) has reported an ultraviolet spectroscopic method for the determination of .sodium metaperiodate and 2 ml of 2. it produce maltol (L9). The solution is then diluted. MOSSA ETAL. About 10 ml of streptomycin solution was boiled under reflux f o r 45 minutes with 10 ml o f 2M hydrochloric acid and the excess of the acid was removed in vacuo.1N sodium hydroxide. Ninhydrin spectrophotometric method has been adapted f o r the determination of aminoglycoside antibiotics in pharmaceutical preparations (394). tetracycline and chloramphenicol using brucine and sodium periodate.M.-sulphuric acid.5 ml of 5m. 560 Eriochrome Slack T was used as a precipitant in the photometric determination of streptomycin and other antibiotics in biological fluids (393).11portions of the solutions of streptomycin in water were applied to precoated Silica gel plate.

Faure and Blanquet (401) have developed an a n a l y t i c a l procedure f o r t h e d e t e r m i n a t i o n of s t r e p t o m y c i n . 6 3 F l u o r i m e t r i c Methods Boxer and J e l i n e k (398) have r e p o r t e d a f l u o r i metric method f o r t h e d e t e r m i n a t i o n o f s t r e p tomycin i n blood and s p i n a l f l u i d . 1 3 . s e p a r a t e d . n e u t r a l and b a s i c compounds are s e p a r a t e d from t h e s t r o n g l y b a s i c s t r e p t o m y c i n hydrazone by e x t r a c t i o n from t h e s o l u t i o n with benzyl a l c o h o l .STREPTOMYCIN 561 s t r e p t o m y c i n i n f e e d s .2-naphthaquinone-4-suIphonate (400). The s t r e p t o m y c i n i n f e e d s was e x t r a c t e d w i t h d i l u t e s u l p h u r i c a c i d . f u r t h e r s e p a r a t e d . A continuous-flow Auto-Analyzer f l u o r i m e t r i c procedure f o r t h e d e t e r m i n a t i o n of l a c t i c a c i d i n m i l k was a p p l i e d t o t h e d e t e c t i o n o f a n t i b i o t i c s ( 4 0 7 ) . The d e t a i l e d s t u d i e s o f f l u o r e s c e n c e s p e c t r a were made i n subsequent p u b l i c a t i o n s (402. The procedure was based on t h e . n e u t r a l i z e d . conc e n t r a t e d and p u r i f i e d by i o n exchange.2-naphthaquinone-4-sulphonate s o l u t i o n . produce a s t r o n g f l u o r e s c e n c e w i t h sodium 1. The method h a s been extended t o determine s t r e p t o m y c i n i n u r i n e and t i s s u e s (399). Aqueous s o l u t i o n o f s t r e p t o m y c i n was t r e a t e d w i t h sodium hydroxide and sodium 1. The aqueous base c o n t a i n i n g s t r e p t o m y c i n hydrazone i s c e n t r i f u g e d and t h e intens i t y of t h e f l u o r e s c e n c e o f t h e clear s o l u t i o n i s measured.403). Based on t h i s p r i n c i p l e . The s t r e p t o s e moiety was c o n v e r t e d i n t o m a l t o l and q u a n t i t a t i v e l y determined by measuring i t s u l t r a v i o l e t absorbance. Streptomycin and dihydrostreptomycin i n a l k a l i n e s o l u t i o n . A p p l i c a t i o n o f t h e method f o r t h e d e t e r m i n a t i o n o f s t r e p t o m y c i n i n blood serum o r plasma was a l s o e x p l o r e d (404-406). The b a s i s o f t h i s method i s t h e formation o f hydrazone o f s t r e p t o m y c i n w i t h f l u o r e s c e n t 9-hydrazinoa c r i d i n e h y d r o c h l o r i d e . The e x c e s s of t h e r e a g e n t t o g e t h e r w i t h hydrazones of a c i d i c . Then t h e s o l u t i o n was set a s i d e i n t h e d a r k €or 1 hour a t 20° and t h e n t h e f l u o r e s c e n c e was measured.

blood and u r i n e . 13. MOSSA ETAL.8 t o 6 . The method i n v o l v e s e x t r a c t i o n of f l u o r e s c e i n complexan-Pr3+ aminoglycoside s p e c i e s from an aqueous medium o f pH 5. Applicat i o n o f t h e above method f o r d e te r m in a tio n of .1M sodium f l u o r i d e and F l u o r 2 me tr ic d e t e r m i n a t i o n o f f l u o r e s c e i n complexan i n t h i s aqueous s o l u t i o n a t 533 nm. John e t a1 (410) used column o f c a t i o n exchange r e s i n . R es o lu tio n s such a s s t r e p tomycin and kanamycin from oleandomycin were d e s c r i b e d . The method i s s u i t a b l e f o r t h e d e t e r m i n a t i o n o f s t r e p t om y c in and t h e r e l a t e d a n t i b i o t i c s i n blood. Amberlite IRC-50 t o s e p a r a t e streptomycin and s t r ep t om y c in B from fermentat i o n b r o t h s . a n t i b i o t i c being determined c o l o r i m e t r i c a l l y [maltol method). 562 i n h i b i t i o n by a n t i b i o t i c s o f t h e a b i l i t y of s t a r t e r c u l t u r e s t o produce l a c t i c a c i d . Alykov (408) has d e s c r i b e d a new f l u o r i m e t r i c method f o r t h e d et er m i n a tio n o f aminoglycoside a n t i b i o t i c s .71 Column Chromatography Column chromatographic (CC) method h a s been used e x t e n s i v e l y f o r t h e s e p a r a t i o n and p u r i f i c a t i o n of s t r e p t o m y c i n. The method a p p e a r s t o have a p p l i c a b i l i t y i n t h e i s o l a t i o n o f t h e a n t i b i o t i c s from b i o l o g i c a l f l u i d s such a s c u l t u r e media. S t . Prot e i n s a r e removed from t h e blood samples by p r e c i p i t a t i o n with t r i c h l o r o a c e t i c acid. w ith good a g r e e ment w i t h m i c r o b i o l o g i c a l procedure. S e v e r a l r e p o r t s have been p u b l i s h e d r e g a r d i n g t h e a p p l i c a t i o n o f c h a r c o a l (lQ6-117). a c i d washed alumina (118-122) and i o n exchange r e s i n s (123-164) f o r t h e s e p a r a t i o n and p u r i f i c a t i o n o f s t r e p t o mycin.JABER S. 7 Chromatographic Methods 13. 2 i n t o isoamyl alcohol-benzene (1 :1) r e e x t r a c t i o n o f f l u o r e s c e i n complexan a s s o c i a t e d w i t h t h e aminoglycoside i n t o aqueous 0.s i l i c a t e f o r p u r i f i c a t i o n o f b a s i c a n t i b i o t i c s . milk and u r i n e . Okumura e t a1 (409) have used columns o f a magnesium a l u m i n o .

S t o d o l l a e t a1 (423) Kowezyk (424) have a p p l i e d similar method f o r t h e s e p a r a t i o n o f s t r ep t om y c in . P e t e r s o n and Reineke (425) d e s c r i b e d a s i m i l a r method f o r t h e d e t e r m i n a t i o n o f s t r ep to m y c in i n s a l t . The p o s i t i o n o f t h e a n t i b i o t i c s were r e v e a l e d by bioautography on a g a r . Wj-nsten and Eigen (421) were s u c c e s s f u l i n r e s o l v i n g m i x t u r es o f s tr e p to m y c in and c l o s e l y r e l a t e d s u b s t a n c e s . t h e r e s o l u t i o n o f components of str e p to m y c in complex (416). . E f f e c t s o f s a l t s on t h e movement and zone shape of str e p to m y c in i n p ap e r chromatography was d i s c u s s e d by So k o lsk i and Lummis (426). t h e s e p a r a t i o n o f str e p to m y c in from f e r m e n t a t i o n b r o t h s u s i n g continuous p r o c e s s (417) and e v a l u a t i o n o f v a r i o u s exchangers f o r s e p a r a t i n g c o l o r e d i m p u r i t i e s (418). h b e r l i t e CG 120 i o n exchange r e s i n was u t i l i z e d by Conca and Pazdera (412) f o r s e p a r a t i o n of s t r e p t i d i n e i n s t r ep t o m y c i n .p ip e r id in e t o l u e n e s u l p h o n i c a c i d m ix tu r e was used as a d e v e l o p i n g a g e n t .STREPTOMYCIN 563 s t r e p t o m y c i n i n animal f e e d (397) and i n formul a t i o n s (411) were r e p o r t e d . 3% ammonium c h l o r i d e was found t o be n e c e s s a r y f o r good s e p a r a t i o n . Comparative s t u d y o f c a r b o x y l i c c a t i o n exchangersin r e s p e c t s o f t h e s o r p t i o n and d e s o r p t i o n o f s t r ep t o m y c i n was r e p o r t e d (413).f r e e p r e p a r a t i o n s and a p p l i c a t i o n s o f t h e method t o s t u d i e s o f b i o s y n t h e s i s were p u b lish e d by Hunter e t a1 (199). The p o s i t i o n o f t h e a n t i b i o t i c were d e t e c t e d by means o f t h e Sakaguchi r e a g e n t . 13. Solomons and Regna (422).72 Paper Chromatography Horne and P o l l a r d ( 4 2 0 ) have d e s c r i b e d a method f o r i d e n t i f i c a t i o n o f s tr e p to m y c in by u s i n g p a p e r . Other r e p o r t s have d e s c r i b e d i n d e t a i l s t h e u s e o f p e r m u t i t (414). B u ta n o l. phenoxy-acetic a c i d c a t i o n exchanger and p h e n o l i c r e s i n s (415).s t r i p chromatogram. The chromatographic behaviour of str e p to m y c in on Sephadex G-10 h a s been d e s c r i b e d by S t o r l (419).

The d e v e lo p e r used was p r ep a r e d by mixing e q u a l volume o f amyl a l c o h o l c o n t a i n i n g 1%o f b i s . Albu-Budai and Unterman (432) r e p o r t e d a quant i t a t i v e procedure based on paper chromatograp h i c r e s o l u t i o n . l a c t i c a c i d .( 2 . Paper p r e v i o u s l y impregnated w i t h phosphate b u f f e r a t pH 7 and sodium hydroxide-pentachlorophenol-butanol as developing s o l v e n t were U s e d .a c e to n e t o . p r e v i o u s l y impregnated with phosphate b u f f e r o f pH 7 were used.17 and 0 . The Rf v a l u e s of s t r ep t o m y c i n i n t h e above s o l v e n t systems were 0.5% sodium c h l o r i d e s o l u t i o n i n b o r a t e b u f f e r .564 JABER S .d i a c e t y l o r by bioautogrsphy on a n u t r i e n t a g a r p l a t e seeded w i t h s p o r e s o f B a W bub-. MOSSA ETAL. To d e t e c t t h e compounds. T h i s i n c l u d e s potassium permanganate (433). e l u t i o n and sp e c tr o p h o to m e tr ic d e t e r m i n a t i o n after c o l o r development w i t h MN 261 paper s t r i p s 1-naphthol-diacetyl. i o d i n e (434). d i p t h e chromatogram i n p e r i o d a t e ac e t o n e and t h en i n b e n z i d i n e . Couling and Goodey (437) have d e s c r i b e d a new method o f s t r ep t o m y c i n chromatography and i t s u s e i n t h e examination of t h e r e a c t i o n between s t r e p t o m y c i n and ammonia.e t h y l h e x y l ) phosphate and 0. The former used b u t a n o l . Other r e p o r t s o f r e s o l u t i o n have been p u b l i shed by Makisumi (429). A d e t a i l e d s t u d y by Heding (427. The developing s o l v e n t were pentachlorophenol-butanol-sodium hydroxide-water. r e a g e n t (9). sodium p e r i o d a t e w i t h p i c r i c a c i d (435). The s p o t s were d e t e c t e d e i t h e r by u s i n g a s p r a y o f 1 . water (4: 1:1:2) and b u t a n o l : 6N h y d r o c h l o r i c a c i d ( 7 : 3 ) as developing r e a g e n t and Sakaguchi's r e a g e n t and J a f f e ' s r e a g e n t f o r d e t e c t i o n . p i c r i c a c i d m i x t u r e (436) and bromine-starchpotassium i o d i d e .428) produced a system c a p a b l e of c l e a r l y r e s o l v i n g s t r e p t o mycin. t h e a n i l i n e . p y r i d i n e .n a p h t h o l . Numerous s p r a y r e a g e n t s have been used i n t h e d e t e c t i o n o f s t r ep t o m y c in based l a r g e l y on c o lo r i m e t r y . a c e t i c a c i d . and i t s r e l a t e d compounds w i t h a development time o f 8 hours. Kondo e t a1 (430) and Pant and Agrawal (431). 0 8 r e s p e c t i v e l y .

The s p o t s o b t a i n e d from streptomycin by TLC procedure have been e l u t e d w i t h phosphate b u f f e r and examined m i c r o b i o l o g i c a l l y w i t h B a W bubLLLi. A p p l i c a t i o n 4-Dimethylaminobenzaldehyde-antimony t r i c h l o r i d e a s a d e t e c t i n g agent f o r a n t i b i o t i c i n TLC was r e p o r t e d by Mathis and . The former used t h e p l a t e s c o a t e d with sephadex and t h e l a t t e r used t h e c e l l u l o s e l a y e r . S a t 0 and Ikeda (445) were a b l e t o r e s o l v e streptomycin and i t s dihydro-and dihydrodeoxy d e r i v a t i v e s by ascending TLC procedure. Simil a r claims were made by Katayama and Ikeda (446). The s o l v e n t system and t h e Rf v a l u e s are r e p o r t e d i n t a b l e .b i o a u t o g r a p h i c method h a s been employed f o r t h e s e p a r a t i o n and i d e n t i f i c a t i o n o f streptomycin (447. 4.6 s p o r e s A s i m i l a r procedure h a s been adapted by Zuidweg e t a1 (449) and Hamann e t a 1 (450) t o r e s o l v e streptomycin and o t h e r a n t i b i o t i c s .448) . 1 3 . 7 3 Thin-Layer Chromatography Thin l a y e r chromatographic t e c h n i q u e was ext e n s i v e l y used € o r t h e s e p a r a t i o n and i d e n t i f i c a t i o n o f s t r e p t o m y c i n and i t s r e l a t e d compounds by Kondo (430) and Borowiecka (438). VonSchantz e t a1 (439) s t u d i e d t h e a p p l i c a t i o n of metal p l a t e s f o r t h i n l a y e r d e t e c t i o n o f pharmaceutical compounds. Voigt and Bared (443) and Katayama and Ibeda (444) have r e p o r t e d two-dimensional TLC method f o r s e p a r a t i o n and i d e n t i f i c a t i o n of s t r e p t o mycin and i t s r e l a t e d compounds.STREPTOMYCIN 565 g i v e yellow o r white s p o t i n a b l u e background. S e v e r a l r e p o r t s have appeared on t h e a p p l i c a t i o n o f t h i n l a y e r chromatography i n i d e n t i f y i n g aminoglycoside a n t i b i o t i c s . o r d i p i n a mixture o f d i a c e t y l and 1-naphthol t o g i v e r e d s p o t on a white background. Thin l a y e r . The former used p l a t e s c o a t e d w i t h a c t i v a t e d carbon whereas t h e latter used p l a t e s c o a t e d e i t h e r w i t h s i l i c a g e l G o r Kieselguhr G.

62-07 440 0.4 441 0. PropanolEthyl acetate-waterAmmonium hydroxide (50:10:30:10).32 0. Spray Reagent 1. 2.32 442 . potassium permanganate and sodium hydroxide mixture. Silica gel G Butanol-watermethanol-tolune -p-sulphonic acid (40:20:10:1) Sodium Nitroprusside. > J Rf value Reference 0. Silica gel G / Aluminium oxide. Ninhydrin. 3 . 10% aqueous solution Copper sulphate with 2% ammonia. Silica gel G 1) Acetic Acid : butanol (7 :2) 2) Benzene-acetone acetic acid 2:2:1.Table 4 : TLC of Streptomycin Adsorbent Solvent System.

The chromatographic conditions are : Mobile phase : 0. antibiotics like streptomycin were determined by HPLC on a column packed with Spherisorb SS-ODs. The column was fitted with a reversed-phase guard column and elution was carried out at 1 mljmin. The elutes were monitored by spectrophotometric detection. at 25O. Whall (456) has reported a HPLC method for the determination of streptomycin and dihydrostreptomycin sulphate. Forty seven solvent systems were investigated in deciding on optimum conditions for resolution of streptomycin and related compounds. Solutes being detected at 195 nm. 3 ml of each solution is then diluted to 50 ml with mobile phase containing 0.025 M sodium phosphate in aqueous acetonitrile. Visualization was made by a spray reagent of sodium nitroprussidepotassium permanganate.0.01M ammonium dihydrogen phosphate . 1 3 . The method is as follows: Samples were dissolved in water and diluted to 50 ml. was used as an eluent. HPLC assay of pyrazinoic acid in human plasma in the presence o f antituberculosis drugs using an automatic sampler was proposed by Ratti et a1 ( 4 5 7 ) . This method could also be used to determine streptomycin. 7 4 High Performance Liquid Chromatography Bagon (455) has developed an assay of antibiotics in pharmaceutical preparations using reversed-phase high-pressure liquid chromatography.acetonitrile ( 3 :7 ) . More extensive examination of streptomycin group was reported by Heding (453) and More recently Borowiecka ( 4 5 4 ) . Aqueous methanol at a flow rates of upto 1 ml/min. A 25 pl portion was subjected to HPLC on a column packed with LI Bondapak C18.STREPTOMYCIN 567 Schmitt (451). Adapting this method.02 M sodium hexanesulphonate . Paunez (452) described a TLC method utalizing the layer o f ion-exchange resin. adjusted to pH 6 .. .

6 mm) packed with ultrasphere c18 (5 pm) with methanol . The aminoglycoside antibiotics were separated at ambient temperature on a column (25 cm x 4.50 mM-pentafluoropropionic acid (21:29). The electropherogram was run €or 3 hours at 500 V and the spots were developed with Monastero's alkaline nitroprusside-ferric cyanide reagent. methanol . the potency was determined microbiologically by laid down the papers on agar plates seeded with suitable organisms.2 M trifluoroacetic acid as mobile phase and refractive index detection.75 Electrophoresis Foster and Ashton (459) described a method for separation of streptomycin and related compounds by paper electrophoresis. Inchauspe and Samain (458) have described a ion-pair reversed-phase high performance liquid chromatographic method for separating aminoglycoside antibiotics using perfluorinated carboxylic acids.50 mM . MOSSA ETAL. The elutes are detected at 254 nm. methanol .JABER S.50 mM-heptafluorobutyric acid (3:2). The test solutions were applied to the paper which is then wetted with a buffer solution of pH 5. 568 Column condition : Column of 25 cm length 4. 13. Takahashi and Amano (460) employed the paper electrophoresis method for the identification o f various antibiotics. After passage o f current for 16 hours the paper was removed and dried. Philippe et a1 (461) have reported a similar method of electrophoresis f o r the separation of streptomycin and dihydrostreptomycin.camphorsulphonic acid (29:21) o r 0. 2) naphtharesorcinol spray. The spots were revealed by one of following reagents : 1) diacetyle. 3) Elson Morgans reagent. Whatman 3MM paper in borate buffer at pH 9 were used. Alternatively. Flow rate at 2 rnl/min.6 mm width packed with Lichrosorb-NH2. Other reports o f resolution of streptomycin and other antibiotics have been published by Paris .

They also described a two dimensional technique using a combination of electrophoresis and chromatography on silica gel (466). Katayama and Ikeda (465) achieved a similar resolution using electrophoresis layers of silica gel.569 STREPTOMYCIN and Theallet (462) Lightbrown and de Rossi (463).tieid. The antibiotics could be separated on microscopic slides coated with agarose gel. The former obtained a better resolution by using the solvent systems of an aqueous phase containing 0. Gorge and Monteoliva (467) used two dimensional techniques on paper (chromatography in one direction and electrophoresis in the second) €or identification of many guanidino compounds including streptomycin. After electrophoresis at 50 V per cm. the antibiotics were located by Bioautography on agar seeded with BaCieeUn c5ub. for 5 to 30 minutes. Application of electrphoresis to the identification of several aminoglycoside antibiotics has been reported by Garber and Dobrecky (468) and Ochab (469). Craig countercurrent distribution technique has been extensively employed for the separation of streptomycin from mannosidostreptomycin by Plaut and McCormack (472) and O'Keeffe et a1 (473). 13. Langner et a1 (470) have described a method of determination of antibiotics including streptomycin in mixtures at ultra-micro levels by rapid low-voltage electrophoresis.5% sodium bicarbonate and 1% sodium chloride and solvent phase .76 Counter-Current Distribution Titus and Fried (471) have reported the u s e of counter-current distribution technique f o r the analysis of streptomycin preparation. The application of electrophoresis for separation and identification o f the antibiotics in Pharmaceutical mixtures was reported by Apreotesei and Teodosiu (464). The Craig technique of counter-current distribution was employed to show the presence of a related substance in crude streptomycin by distribution between butanol and water.

d i f f u s i o n methods w i t h t h e s u i t a b l e inoculum o f t h e fol lowing t e s t organisms have been r e p o r t e d : Enchenicki cakX (475-477). Resazurin d i s c ha s been employed by Shahani and Badami (511).8 Bio-Assay Methods The p r i n c i p l e o f t h e b i o l o g i c a l a s s a y i s based on t h e comparison o f t h e i n h i b i t i o n of t h e growth o f b a c t e r i a produced by known c o n c e n t r a t i o n o f t h e s t a n d a r d p r e p a r a t i o n wi th t h a t produced by measured c o n c e n t r a t i o n o f t h e sample be ing t e s t e d . S. The method h a s been e x t e n s i v e l y used i n determin a t i o n o f stre pt omyc i n i n b i o l o g i c a l body f l u i d (495-499).c u r r e n t d i s t r i b u t i o n t e c hnique s employed f o r t h e a n a l y s i s of v a r i o u s compounds i n c l u d i n g s t r e p t o m y c i n . u r i n e (500). Meyer (474) h a s d i s c u s s e d i n d e t a i l s t h e d i f f e r e n t methods o f c o u n t e r . 570 (Pentasol-amyl a l c o h o l ) c o n t a i n i n g 5% s t e a r i c a c i d and t h e latter employed a system composed o f l a u r i c a c i d i n amyl a l c o h o l and aqueous phospha t e -bora te b u f f e r . food and animal f e e d (501-505) and pha rmaceutical p r e p a r a t i o n s (506). M o d i f i c a t i o n s of a g a r d i f f u s i o n method have been r e p o r t e d . B a W cetrew v a r . mgcoidu (486-487).thepRococcub h c . B a W cd~cf.81 Agar-Diffusion Method on S o l i d Media Many a g a r . Koelzer and Giesen ( 5 0 8 ) . t i n (491) K L e b n i d h pneumonkw (492) L a c t o b a c i U u b&atLicw (493) a c i d r e s i s t a n t microbacterium V-5(494). 13. Waksman and R e i l l y (512) a p p l i e d Agar-str eak method €or a s s a y i n g st reptomycin and o t h e r a n t i The potency o f t h e a n t i b i o t i c substances: b i o t i c s p r e p a r a t i o n s can be determined by . R e i l l y and Sobe rs (509). Paper d i s k method h a s been adapted f o r t h e d e t e r m i n a t i o n o f str eptomycin by Loo e t a1 (507).JABER S. MOSSA ETAL. BacAYw n u b U (478-483). B a r n ficheni~onmn (485).&ns (484). and Kanazawa & Kuramata (510). 1 3 . SXaphyLocaccw awrew (488490).

STREPTOMYCIN 571 i n c o r p o r a t i n g graded d i l u t i o n s i n s t a n d a r d n u t r i e n t a g a r media i n p e t r i p l a t e s and s t r e a k i n g t h e agar surface with selected t e s t b a c t e r i a . McGuire e t a 1 (513) h as suggested a new l i n e a r d i f f u s i o n method f o r m i c r o b i o l o g i c a l a ssa y o f s t r e p t o m y c i n . p a p e r . Dewart e t a1 (517) and Light Brown e t a 1 (518) have d e s c r i b e d a d i f f u s i o n a s s a y f o r a n t i b i o t i c s by an automated procedure. t h e a r e a o f zones of i n h i b i t i o n a r e measured.c e l l method (514) and modified d r o p .d i s h e s .d isk . p u lp d i s k and s u p e r p o s i t i o n a s s a y mthods was r e p o r t e d by I s h i d a e t a1 ( 5 1 6 ) . g . . 50 o r 70 1. a f t e r incubation. Improvements o f Agar d i f f u s i o n method f o r a s s a y i n g s t r e p t o m y c i n were a l s o r e p o r t e d (520. . B r i t i s h Pharmacopoeia 1958 (304) and Egyptian Pharmacopoeia 1953 (13) d e s c r i b e a m ic r o b io lo g i c a l Assay method f o r d e t e r m i n a t i o n o f s t r ep t o m y c i n .521). S l i d e . Assay Procedure : F i l l i n uniform t h i c k n e s s p e t r i . t h e n t o a d i s p e n s i n g u n i t t h a t d i s p e n s e s measured volumes ( e . A comparative s t u d y o f cup.p l a t e method were a l s o r e p o r t e d . The method i s as follows : Disposable p l a s t i c P e t r i d i s h e s loaded with a g a r p r e p a r e d f o r a s s a y are c a r r i e d a u t o m a t i c a l l y t o a d e v i c e t h a t punches h o l e s i n t h e a g a r . t h e potency is r e a d and expressed as t h e r e c i p r o c a l o f t h e h i g h e s t d i l u t i o n i n h i b i t i n g t h e d i f f e r e n t t e s t organisms.11) o f t e s t o r s t a n d a r d solution i n t o t h e holes. o r r e c t a n g u l a r t r a y s . After o v e r n i g h t i n c u b a t i o n . with a g a r c u l t u r e medium which h a s been p r e v i o u s l y inoculated with a s u i t a b l e quantity of a susp en s i o n o f s p o r e s o f a s u i t a b l e s t r a i n o f BacLUuh h u b U . t o a d e p th o f 3 t o 4 mm. Continuous au t o m a t i c m i c r o b i o l o g i c a l a s s a y o f a n t i b i o t i c s was r e p o r t e d by Shaw and Duncombe (519). The method was based on t h e measurement of r e s p i r a t o r y carbon d i o x i d e .

F i l l i n t o t h e cylinders o r holes. and from t h e r e s u l t s t h e potency of t h e l a t t e r is c a l c u l a t e d . and then incubate a t about 30° t o 37OC f o r about 16 hours. r e s p e c t i v e l y . may be placed on each. o r any o t h e r numbers which can be conveniently adjusted t o t h e s i z e of t h e agar p l a t e . and 92. Place on the s u r f a c e of the inoculated medium. t h e s o l u t i o n s of t h e s t a n dard preparation and of t h e sample t o be t e s t e d i n a l t e r n a t e order by means of a p i p e t t e which d e l i v e r s a standard amount o f s o l u t i o n s u f f i c i e n t almost t o f i l l t h e holes when they a r e used. L i m i t of Error: The limits of e r r o r (P=O. . pH 8 . u n t i l used. .5 t o 2 u n i t s per m l and s o l u t i o n s of t h e sample t o be t e s t e d presumed t o be o f t h e same order of concentration. by means of a s t e r i l e borer. Place t h e p l a t e s i n a r e f r i g e r a t o r f o r about 2 hours. 5 t o 8 mm i n diameter. e i g h t c y l i n d e r s . 89. 85Oand l l S O . s i x .50 where 80 cylinders o r holes a r e used f o r t h e standard preparation of streptomycin and f o r t h e sample t o be t e s t e d .. where 10 cylinders o r holes a r e used. MOSSAETAL.. Bore i n t h e medium. porcelain o r aluminium.572 JABER S. holes. small s t e r i l e cylinders of uniform s i z e approximately 10 mm high o r having an i n t e r n a l diameter of approximately 5 mm. i n place of c y l i n d e r s . Prepare i n s t e r i l e phosphate buffer s o l u t i o n . Measure.99) t h a t can be obtained with t h i s method are 79 and 1 2 1 per c e n t . with t h e g r e a t e s t possible accuracy.50 where 40 c y l i n d e r s o r holes are used. Five. 0 . t h e diameters of t h e i n h i b i t i o n zones produced by t h e varied concentrations of t h e standard preparation of Streptomycin and of t h e sample t o be t e s t e d . made of g l a s s . Allow t h e inoculated p l a t e s t o dry a t room temperature and keep a t 5OC.50 and 107.50 and 110. s o l u t i o n s of t h e standard preparation o f streptomycin. where 20 cylinders o r holes a r e used. of known concentrations. and keep a t about 150OC. as f o r example 0.

i n c u b a t e d a t 37O f o r 4 h o u r s . The p e r c e n t a g e o f transmission read i n a photoelectric c o l o r i m e t e r . A microbiological assay of a n t i b i o t i c s based on i n h i b i t i o n of ammonia p r o d u c t i o n was r e p o r t e d by Kobos (527).821 T u r b i d i m e t r i c Method Oswald and Knudsen (522) have r e p o r t e d a t u r b i d i m e t r i c method f o r a s s a y o f s t r e p tomycin.STREPTOMYCIN 513 13. serum and c e r e b r o s p i n a l f l u i d were r e p o r t e d by Gibb (523) and Whitlock e t a l ( 5 2 4 ) . 13. one w i t h graded amounts o f s t a n d a r d s t r e p t o m y c i n and o t h e r w i t h t h e unknown are i n o c u l a t e d w i t h t h e same amount o f a s t a n d a r d s u s pension o f t h e t e s t organism.822 Other Methods Bonifas and Chesni (525) have developed t h e d i l u t i o n method f o r t h e d e t e r m i n a t i o n o f s t r e p t o m y c i n : K ~ e b p bn m~o n ~ iae was allowed t o grow a t pH 9-9. t h e pH o f t h e m i x t u r e was .. The r e a c t i o n s h i f t e d towards t h e a c i d s i d e upon s p l i t t i n g t h e s u g a r . Decreasing amounts o f s t r e p t o m y c i n were added t o t h e medium. cofi i n 6% peptone medium. Dilution-method was extended t o determine s t r e p t o m y c i n i n u r i n e and body f l u i d s (526). The t e s t s o l u t i o n was i n c u b a t e d f o r 2 hours a t 37O w i t h a s u s p e n s i o n o f E .5 i n t h e s y n t h e t i c medium of Monod t o which s u g a r and i n d i c a t o r c r e s o l r e d were added. A p p l i c a t i o n s of t h e t u r b i d i m e t r i c method € o r t h e a n a l y s i s of s t r e p t o m y c i n i n u r i n e . The i n h i b i t i n g amount o f s t r e p tomycin was determined by n o t i n g t h e d i l u t i o n i n t h e t u b e immediately p r e c e d i n g t h a t i n which t h e s e was t h e f i r s t change of color of t h e indicator.82 Liquid C u l t u r e Method 13. Potency of t h e sample under t e s t can be r e a d d i r e c t l y from t h e s t a n dard curve. Two series of t u b e s .

9 Biochemical Methods A radioimmuno a s s a y (528. A l t a f Hussain Naqvi and M r . Acknwo 1edgement The a u t h o r s s i n c e r e l y thank Mr. On mixing o f t r a c e r .529) was developed f o r d e t e r mination o f s t r e p t o m y c i n i n plasma and u r i n e . Schwenzer and Anhalt (530) have r e p o r t e d an automated f l u o r e s c e n c e p o l a r i z a t i o n immunoassay f o r str e p to m y c in i n blood serum. Uday C. 13. . The method i n v o l v es enzyme-catalysed i n t r o d u c t i o n o f a l a b e l l e d group i n t o t h e molecule and s e p a r a t i o n o f t h e l a b e l l e d product by paper chromatography b e f o r e counting . 514 a d j u s t e d w i t h sodium hydroxide and t h e c o n c e n t r a t i o n of ammonia was determined w i t h u s e of an Orion 95-10 ammonia s e n s o r . Sharm €or t h e i r s e c r e t a r i a l h e l p . I n t h e a s s a y d e s c r i b e d f l u o r e s c e i n l a b e l l e d streptomycin was used a s t h e tracer and a n t i serum a g a i n s t streptomycin was r a i s e d i n r a b b i t s .JABER S. sample and a n tib o d y . MOSSA ETAL. t h e p o l a r i z a t i o n o f t r a c e r f l u o r e s c e n c e was measured i n an Abbott TDX f l u o r i m e t e r .

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Scand.. _ 49. T h e a l l e t . Y. 5 7 . Acta. B e r e t t a and A. Bagon..C. Ann. Ann.W. 1. C . 29. F o s t e r and G . cJ. - 303. i 3 B O r O W i e C i c a . P h i l i p p e . F r a n c e . 463. Assoc. 1 0 . 604 453. 6E34 (1985).A. 3D90 (1983). 300 (1967). A p r e o t e s e i and M. MOSSA ETAL.. Katayama and H. I b i d . 81 53. Teodosiu. . 99 (1973). Whall. 26. Anal. 465. 42. A . C . 2. Abst. R a t t i . France. 15. C. 180 (1963). 3086 (1970). 561 (1954). 1E20 (1980). Farmacia. Ed. 24. J u l i o Lopez Gorge and Miguel Monteoliva. M. 6 7 . S c i . 33. Ashton. Chromatogr. . B i n j i Takahashi and Y a s u j i Amano. 456.. 27711984). . Rev. 454. Heding. 90. 458. G. 10511973). 35816j (1970). 89 (1981). I b i d . 37. 2854 (1974). 219.. C . J. Abst. J . C. 2855 (1974). Analyst. 7 2 . (1954). Anal. Phys. d e Rossi. 459. 211 (197Y). 436 (1962). Chromatogr. 47. S Ochab P o l . J o l c h i n e and 0. T o s e l l i . 468.. 172. Prat. 96642t (1967). 3123 (1959). J . Anal. Chem. E. Anal. N a t u r e .546 (1957). Paris and J . Chem. Pharm. Phamac. Pap. I n s t . 9335 (1959).. 25. 26. . Abst. 457. 958 (1953) 460. 4375 (1965). Abst. T. A l c a r a z . A . T . J . 130 (1968). . l k e d a . J . 466. 469. Inchauspe. 226 (1982). Res. . Chromatogr. Pharm. Pharm. 6E28 (1982).. C. Commun. . C . Abst. Abst. Lightbrown and P.. 461. P . 44. Anal. A b s t . Chromatogr. Anal. 89(1965). Chromatogr. Argent. . 6 . Kay R. A n t i b i o t i c s 7. R . K . A . Bernareggi. C . Anal. A . 321(1962) 464. Samain.. D. J. 38.JABER S . 462. H. A. A b s t . 3. Idem. 455. 6 2 . Dobrecky. Garber and J . . E. 16746 (1962). (1969).. High Kesolut. Pharmac. Anal. 467. J. Bioquim. Farmaco. 63. B . . 25. J . J.

J. S a b a t h . 2.T.209 (1964). C . 4193 94. S c i e n c e . 33. C l i n . 18639 (1965). 474. Jr. Meyer.C.605 STREPTOMYCIN 470. 15884 ( 1 9 6 3 ) . d 9 ( 1 9 6 8 ) . Phytopathology. 2615 ( 1 9 5 5 ) . SOC. S o i l . Anal.. Fcrnandez. 485. and F i n l a n d . Med. L. C . (1946). J . 7 . B e r l i n . 70. 473. S e i x a s . S t a m p f . 103. S. J i r o Tsukada and S o s h i c h i Kamegaya. . M. Hunt. . Zasey. 60. V .W. 478. 7 . 471. 4894 ( 1 9 6 1 ) .. A n t i b i o t i k i . E. Waksman. D. 57. S e i d l e r . Antao... 168. R e g i n a l d H. S t i l l e r .nb.. C . 40 ( 1 9 4 5 ) . 75. C . J. O'Keeffe. 28072 (1970). 56 4 0 .H. 7192 ( 1 9 4 9 ) . C. 480. F. A . . Expan. 476. M. 2908 (19641. C. M. Chem. Meiji S h i k a Kenkyu Nempo.D. 484. . A . P o r t . Ohem. J. H... Y. L . 2 .A. N i c l s e n and H . 1 0 2 . P . . A. H a l l and Donald P. 2264 11949). C a r r e c o . and S c h r o t h . 4 2 . Young. A . A . J ... S u v o r k i n a . 55. 43. 5 9 . 17. . 8 4 6 . 7 1 1 0 3 s ( 1 9 6 9 ) . Siegert . A . C . Andrew D.. 567 (1970). K . MeCorrnack. . Fro:cmhold a n d 5 3 . 472.A. Welch. P r i c e . 18. A . A .. Chem. 2186 ( 1 9 4 6 ) . 6 1 . Lab.A. 4 ( 1 9 6 0 ) . C . 482. 281 (1955). David Prarner and Robert L. A . I : l e i s c h w i r t s c h a f t . M. M i c r o b i o l . C . A . M. Elwood T i t u s and J o s e f F r i e d .W. Am. C . Chem.A. 483. J . 8 3 (1970) . D r u z h i n i n a and D . Rev. C. D o l l i v e r and E.Teuf'e1. 73.. 479. 392 (1947). C . 71.N. and Mary B . A . 39. 107921b ( 1 9 7 1 ) . 48 ( 1 9 6 2 ) . A . Abst. U. 2530 ( 1 9 6 2 ) . 825 481. S t a r k e y . Brit. Sands. E . I . 557 ( 1 9 7 3 ) . - 475. . Ag. 41.N. B . .Langner. P l a u t and R . C .Ruch. Chemolter. B i o l . . 7361 (1948). 886 ( 1 9 4 8 ) . Am. Farm. F e l l .. ii. C. 2452 ( 1 9 4 9 ) . (1945). Antirnici. Tech. . C .. S c i e n c e . Y. G.E. 6 3 .A. L . Sci. SOC. 477. 168 (1960). Simoes. . (1Y42). J . 4190 ( 1 9 4 7 ) .

Georges Sanchez and Andre Lamensans. 13. E p t l . . 372 (1949 45. 488. R. 39. 1158t (1971). Hector Enrique Cammarota. C . U. B i o l . Am. 39. 3.. f a l s . Ann. J . Veisfeiler and T. Microbiol. Nauk. 492. A . . Mitchison and C . Gosta Wallmark. S t e b b i n s and H . C . A n t i b i o t i k i . . A .A. C. C . fac. . quim. - 494. Med. Proc. Ya Kivman and I . L. 50. C . 3120 (1952). 46. E . L . 1010 (1968). . J .Werber and Leo Loewe. 3550 (1950). 31701d (1969). . 9594 (1951). J . Proc. 496. 17. J . . C . 493.ty McCray. C l i n Path. A . C . 4640 (1945).. cienc. 397 (1951).. A . Med. 75. 43. 490. Trudy Akad. 7076 (1949).R. (1962). Erna A l t u r e . C. 47. 145 (1945). J a c q u e t and L. Acta Path. A . A .A. A . 20. . Scand. . 29 . Yu. MOSSA ETAL. 3169 (1947). r e n d . 58. 489. C . Baudolino Mussa. S. 59. A n t i b i o t i c Med. O l i t z k i . SOC. 88 (1952). 1189 (1947). 44. 498. Vakulenko.. Compt. 63. 277(1946). 184 (1949). Osgood and S. - 500. . 50. 41. N . E x p t l . A . A n t i b i o t i k i i i k h Primenenie. A .M.S. 2 . 16. .N. D. E .A. C . S t a f f Meetings Mayo Clinic. K. Proc. D. SOC.S.. C . 4341 (1945). A .H. (1971). 5 (1953). . 1268 (1947). . Minerva p e d i a t . Rubksova.S.K. A . . Med. S p i c e r . 93 11947).702 495.9484 (1956). 497. 606 486.. A . 41. 37116 (1956). 46. M i c r o b i o l . 2 2 . 255 (1945). 331 Antibiotiki. 1. f r a u d e s .S.. 50. Gen. 14854 (1956). B i o l . 499. Ya Geitman. 3. . 5996 (1953). 491. O l i t z k i and Z . C . e t .JABER S. 5 3 (1952).A.B.R. Robinson. G. Rec. S t e e g . 317 (1956). 70. C. 487. C . Heilman and Bet. A . C . Zykova. 5979 (1963). 224. 27.. Graham.

S. H. Anal. B. Abst. 504. . C h r i s t i n e K e i l l y . A n t i b i o t i k i .. and T. . C . Davis . Masako. A . 40496w 507. J.) (1971). (1969).71. C . A .A. 8 . 10027 (1950).H. Anal. Suvorkina. 840 ( 1 9 4 9 ) . C . D a i l y . . 3973 (1961).A. 39. A . 514. Ed. 19. P a r k e and W. 52. 44. Bact. Skell.Chizuru 86 and Haruta. Eng. 502.41. A b s t . O f f i c . (1971). Anal. I n v e s t . J. Y. Badami. C h r i s t i n e R e i l l y and Hazel overby S o b e r s . Shokuhin E i s e i g a k u Z a s s h i . I b i d . A . 2550 (1962). Thornbery. A . G. 7160(1953).A. 742 (1961). 1130 ( 1 9 4 7 ) .. J. 75. 3196 (1959). 3064 (1950). .Mayernik. 73. H. 679 59682t (1969). 117193e (1971). 9. 44. 556 (1945). - - 51u.J.M. Chem. Chem. A. Abst. and Chemotherapy. Savage and J . Agric. b i o l . 17. J .A.N. . E. . Hajime. Ind. 508. Druzhinina.M. 115799h [ 1971). Waksman and H. T. . Giesen. J. Anal. Anal. Y.. . Kanzaki.. 8. M . O f f .. . 469 (1952). C . 12. 6 .. Epidemiol . 2877 (1963). A n t i b i o t i c 2 .A. 75. K l i n . 1 9 . . 44. C. 48 (1971). 44. 4908 (1945). W. 159 (1971). 40. W. C . 33(1961). MikroC .V. Anal. Ass. 4 7 . J . P a s t e u r . Abst. J . Anal. . (1970). 5U3.H. Koelzer and J . 7Cl (1945). I n s t . 10029(1950). J. 44. Med. .Ksnazawa ~ 511.E. John E h r l i c h . E. Tebyakina and E. 28. 513. 4 4 . Ann. S y l v e s t e r . Misao. B o l e s t i . 1442 (1949). . C. C . 1 0 .. Murakami. Katz and J . Yves Chabberc and Bernard Sureau. C. Infek. K. C l i n . . Druzhinina and D. Spock.607 STREPTOMYCIN 5U1. Shahani and M. 509. 50. Chem. 75. 2495 (1946).A. Fujimoto.V. S. Diary S c i . C. (1958).C. A b s t . A . J. 505.M. 2678 506. J . McGuire. Ass.Kurarnata Nippon Kagaku Ryohogakukai Z a s s h i . Loo.. P. Mayernik.S. 1510 512.N. McGuire. 825 (1962). 7.

15595 (1963). 1D49 (1978). Med.D. 4 . E x p t l .. . Acta.. 524. .JABER S. Z e n t r . Tohoku. D. Med. 46. Methodol. O ’ N e i l and Ralph E . 4200 (1950). 521. 155 (1951). A . O r i g .O. R. J r . Bonifas and Y. C .. C. J a c k P r e s b e r g . 37. . 4 5 . Bryan. Belge. F. 54. I e r u s a l i m s k i i . . Anal. and S. Carl Hornberger. W. Neronova. J. Anal..A. 47. R. 45. Kobos. Ind. ASSOC. Hunt. 355(1948). 1729 (1961). Ken K a t a g i r i and Reiko Chida. 608 515. W. 3 2 . J. Andrew D.. J. 273 c (1984). Georg Hennberg and Wolfgang Waterstraat. Abst.9610 (1951). 103 (1970). 526. C . C l i n .G. J0nes. N. 525. Whitlock. Duncombe.A. 8. 258(1950). Abst. 5755(1951). 694(1963). .E. 45. . 528. 104. J r . El-Nakeep and R. 9E25 (1985). 829Y7d (1970).. Edward Roche. P a r a s i t e n k I Abt. 433 (1959). Oswald and L i l a F. Naudts and W.K. Am. A . V. Abst. 60865 [1969). Michelle A. Lhoest. Mary L o u i s e G a u t s c h i and Helene Drusch. Sharpe and A. 28.. Mikrobiologiya. S t a n f o r d Med. I s a a c s o n . 5 3 7 2 2 2 5 2 (1959).. 522. 517. Lab.M. Gibb.. R. -.. 5.A. Louis Jensen. 3596 (1952).T. Arden W. C. Am. 34.D.Analys. Bakt. George V. C .t. M. . J . Abst.H. Anal. C. Simpson. Chim. 188(1951).. 523. 17. . A. V . C. 8. Pharm. 61 (1950). 20. Knudsen. J . 14 (1951).M. Arzneim-Forsch. Biochem. 3035 (1951). 45.Cutlex.. 1825 (1949). . Blair. C. Dev.A. A . A . 519. A . 3448 [ 1951).. C . 59. A n a l y s t 8 .. 39. 235 (1976). . Forrey. C. L. Nakao I s h i d a . 4E34 (1979). Light-brown. B u l l . 70. A . C . Konova and N.A. I . Broadbridge. E x p e r i e n t i a .. E l i z a b e t h J. 37. Yousef. 272 (1967). Fred A. 520. I 518. MOSSA ETAL. 527. 201 [1979).A. J. Technol. Chesni. 4 3 . Anal. 516. 72. 164. Tashman. E .. Shaw and K. . T a p l i n . Chin!. C . Med. 97(1950). Dewart. Mary C. Anal. 44.A. P.

530. 6 D 68 (1980). . P . 683 (1983) . 5D54 (1984). 38. Anal. Abst. Abst. 24. 23. Schwenzer and J .STREPTOMYCIN 609 529. T. 46.G. Anhalt.. Koroleva. K. Antimicrob Agents Chemother. Anal. Ya Goncharskaya and V. 859 (1979). A n t i b i o t i k i .S.

2 Ultraviolet Spectrum 2.3 Optical Rotation 2.6 Colorimetric Analysis 6. Color. Formula and Molecular Weight 1.4 Bioassay 6. Odor 1.71 Ultraviolet 6. References ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 16 61 1 Copyright 0 1987 by Academic Press.5 Solubility 2.9 Chromatographic Analysis 6.1 Infrared Spectrum 2. Metabolism and Phannacokinetics 6. Description 1.94 High Performance Liquid Chromatography 6.6 Crystal Properties 3.3 Titrimetric Analysis 6.91 Paper Chromatography 6.10 Miscellaneous 7. Physical Properties 2. Kapoor 1.5 Polarographic Analysis 6.72 Infrared 6. Limits of Tolerance 8.1 Elemental Analysis 6.93 Gas Chromatography 6.2 Appearance. Stability and Degradation 5.4 Melting Range 2.92 Thin-Layer Chromatography 6.7 Spectrophotometric Analysis 6.8 Spectrofluorometric Analysis 6. Inc. Synthesis 4.2 Identification Color Test 6. AU riehts of reproductionin any form reserved. Methods of Analysis 6.THIABENDAZOLE Vijay X.1 Name. .3 Salts 2.

98. 1.5 2. Formula and Molecular Weight Thiabendazole. Physical Properties 2. 2. was developed by scientists of Merck laboratories1 in 1961. about 0. 906 and 74OCm-1. in the range 230 to 350 tun. Odor White to cream-colored. The CAS Registry number is 148-79-8. Thiabendazole is 2-(4-thiazolyl)-1H_-benzimidazole. fumarate and salicylate which had limited solubility in water.VIJAY K. The hydrochloride (sublimed at 265O) and sulfate (melted at 262-6O) have been prepared.1 Infrared Spectrum The infrared spectrum4 (KBr disc) of thiabendazole is presented in Figure 1. Color.KAPOOR 612 1 Description 1.47 and at 302 nm.2 Appearance. Molecular Weight: 201-25 10H7N'3 1.2 Ultraviolet Spectrum The light absorption. at 243 ?h~ and at 302 nm. about 0. The principal peaks are at 1408.1N hydrochloric acid exhibits two maxima.0004 per cent w/v solution in 0. an anthelmintic and antifungal agent.2 Watersoluble lactate of thiabendazole has also been prepared along with the citrate. odorless or almost odorless powder. . of a 2-cm layer of a 0. absorbance at 243 nm.1 Name.3 Salts Thiabendazole is a weak base. The most common proprietory name is Mintezol .

66 3.33 0. 2. 2. Infrared Spectrum of Thiabendazole 2.5 slightly soluble soluble . mg/ml practically insoluble 6.3 Optical Rotation Thiabendazole exhibits no optical activity.4 Melting Range The melting range6 of thiabendazole is between 296O and 303O.5 Solubility The solubility7 of thiabendazole at ordinary room temperature is as follows: Solvent Water Alcohol Chlorofonn Ether Acetone Dilute mineral acids Solubility.THIABENDAZOLE 613 Wavelength microns 6 7 8 9 10 11 12 131415 90 80 70 60 50 40 30 20 l10 o I 2000 I 1800 I I I 1600 1400 1200 I 1000 I I 800 650 Wavenum ber Figure 1.

but are twisted by loo with respect to each other.030?8) A. and are tabulated in terms of the lattice spacings and the relative intensities of the line. long. O-Nitroaniline was condensed with lactic acid or-the acid halide to give g-lactoyl2-nitroacetanilide which on oxidation followed by bromination gave ~-(bromopyruvoyl)-2-nitroanilide.9 The data were obtained by diffractometer and Debye-Scherrer camera techniques.421 g cm Intensity data were collected on an automatic diffractometert the structural parameters were refined by full-matrix least-squares to an index of 0. Molecules are linked together by N ( 1 ) H(l)---N(14) hydrogen bonds to form chains parallel to the axis.052(7).998(4) . X-ray powder diffraction data for thiabendazole is given in Table 1. Alternate methods of synthesis havs also been described.414 and 1. . There are erght formula units per cell.* Crystals of thiabendazole are orthorhombic. and = 10.442(10) %. The C-C bond connecting the two ring systems is 1. In a patent18 the synthesis of thiabendazole is reported through another route (Scheme 11).13 - . b = 10.066 f o r 1805 reflections.6 Crystal Properties Crystal structure of thiabendazole is described. Synthesis Brown et a1. The latter on refluxing overnight with thiofomamide hydrate followed by zinc dust reduction gave thiabendazole. Patterns using three different X-ray wavelengths w i t h the camera method are given. with a = 17.l from Merck laboratories first reported the synthesis (Scheme I) of thiabendazole by the reaction of 4-thiazolecarboxamide with O-phenylenediamine in polyphos horic acid at 250° for three hours. space group p s.or 3 S-labeled thiabendazole i s also described.VIJAY K. The two ring systems are approximately planar.11812 Synthesis of 14C. observed -3 and calculated densities are 1. 3.KAPOOR 614 2.

Table 1 X-Ray Diffraction Data f o r Thiabendazole Diffractometer CuKa d (ft) 1/11 6.515 20418 2.64 5056 8 4 7 43* 5 007" - 4r23* 3.525 2 327 - - -14 loo* 43* 2 -10 -33 -- 2 Camera corn CuKa d (1) - 6.316 2.055 20022 2.81 5. d <A) 1/11 d (A) 1/11 5.21* 4001 3071 3 38* 3.02 3.321 2.14.76 5057 5oO3* 4021~ 4.808 12 17 21 5 4 2.419 2.72 3 39* 3027 2.519 7 7 7 5 - 20311 63* - loo* 20 43* 7 I -.26 3010 20806 2 0 685 20630 2.76 5 04" 4.70 3 0 38* 3025 I -- 21 2.71 3 0 38* 3026 3011 2 0803 2.056 2 0 000 14 75* loof 5 27 5 1* 7 4 5057 5oO3* 4021* (c.132 2. 3.57 -17 6076 - -1113 6.252 2.517 2.253 20137 2.814 20695 2.062 2 1 20025 *Most intense lines 1/11 17 I 18 68* loo* 3 26 51* 5 3 19 6 3 12 1 9 4 5 7 4 CrKa. 8 I 7 .681 20629 2.

Alternate N-H I c=o I I c=o CHI 'NH2 aNo2 iiya 1 synthetic pathway t o thiabendazole . Synthesis of thiabcndazole + UNo2 CHaCHOHCOR N-H R =OH. 250°/ 3 hr Scheme I.KAPOOR 616 $7 PolYphosphoric acid NHpCO N .VIJAY K. C I I I H COH I c=o CH3 aNo2 N -H I c=o I c =o I 0r2/0O0 CHI Br HC II Zn/HCl NH-C Scheme N n.

carboxamide Figure 2. thiazol-4ylamidine and methyl thiazol-4-carboxylate.16 A recent study involving photooxidation of thiabendazole in methanol in presence and absence of a photosensitizer. methyleneblue also identified dimethyl oxalate. has been reported14-17 to be slightly uv (sunlight) sensitive. benzimidazole-2-carboxamide and benzimidazole as the main products of photolysis. however. triazole-4-carboxamide. Benzi midosole Benzimidozole-2-corboxomide CH3OOCNHCO Methyl benrim~dozole. 87% in the urine and 5% in . methyl benzimidazole-2-carboxylate. benzimidazole-2carboxamide.2 -corboxylote 1hiozote -4 c.6 Thiabendazole.f N-car bomethoxy) .19 Figure 2 gives the structures of the main degradative products. Photolytic degradation products of thiabendazole have been identified as benzimidazole.14. Moin products of photolysis of thiabendazole A recent review discusses the different breakdown products due to environmental factors of various fungicides including thiabendazole 2o 5. . thiazole-4-(pcarbomethoxy)carboxamlde. Stability and Degradation In general thiabendazole is a stable compound. I 3 Thiabendazole is rapidly absorbed from the gastrointestinal tract and peak plasma levels of 13 to 18 pg/ml are found within an hour of ingestion.sa .THIABENDAZOLE 617 4. The drug and its metabolites are excreted rapidly. The USP XXI NF XVI directs that it should be packed and stored in well-closed containers. Metabolism and Pharmacokinetics Thiabendazole is metabolized chiefly to 5hydroxythiabendazole which is excreted in the urine conjugated either B S glucuronide or as t h e sulfate (Figure 3) .

22 Wilson & a1. 21 small amounts of both unchanged thiabendazole and free 5-hydroxythiabendazole may be found in both the plasma and urine.24 A single o r a l dose of desmethylimipramine (80 mg/kg) administered to rats inhibited the hydroxylation of thiabendazole by 45% in vitro at 5 hr after dosage but did not decrease cytochrome P-450. Gastrointestinal absorption and secretion of thiabendazole has been studied. Metabolism o f thiabendazole the faeces within 48 hours.23 have reported that 5-hydroxylation of thiabendazole in rat liver microsomal preparation is dependent on cytochrome P-450 and has a Km of 3.484pole of 5hydroxythiabendazole produced/hr/mg of protein. inhibition was less at 5 hr.92 x 10-431 and Vmax of 0.VIJAY K.KAPOOR 618 Thiabendazole 5 -Hydroxythiabendazole Hp mN Sulfate conjugate N Glucuronide conjugate Figure 3 . A single oral dose of ethoxyquin (200 mg/kg) to rats inhibited the hydroxylation by 65% in vitro at 1 hr after dosage. Bauer - . Oral ethoxyquin (400 mg/kg) or desmethylimipramine (80 rng/kg) administered 30 minutes before oral thiabendazole (100 and 200 mg/kg. respectively) delayed absorption of thiabendazole and resulted in its decreased plasma concentrations. Desmethylimipramine and ethoxyquin are reported to inhibit the hepatic microsomal hydroxylation of thiabendazole. 5-Hydroxythiabendazole gave a type If binding spectrum w i t h cytochrome P-450.

586 About 5 mg of the sample are dissolved in 5 ml of 0.76 l/kg.2 ml/rnin/kg.51 3. .17 hr.28 and avian29 species. and 27. When diluted with water to 100 ml a blue or blue-violet color is developed6.12 hydrochloric acid and 3 mg of E-phenylenedimine dihydrochloride are added f01 lowed by shaking to dissolve the contents.62 N 20. The half-life. The findings are consistent with the literature data that metabolism and excretion of thiabendazole are more extensive in cattle than in sheep. respectively. the glucuronide and sulfate conjugates accumulated extensively despite hemodialysis and hemoperfusion.1 g of zinc dust is then mixed and the mixture allowed to stand f o r 2 minutes. % Found C 59. volume of distribution. 5 ml of a solution prepared by dissolving 20 g of ferric ammonium sulphate in 7 5 rnl of water.68 59074 H 3.2 Identification Color Test The following color reaction can be used to identify a sample of thiabendazole. 2.88 20. Methods of Analysis 6. About 0.3 Titrimetric Analysis The titration with perchloric acid is the method of choice to assay thiabendazole.57 S 15.268 27 sheep.99 6.25 have studied the phamacokinetics of thiabendazole and its metabolites in an anephric patient undergoing hernodialysis and hemoperfusion. Pharmacokinetics of thiabendazole has been studied in cattle.1 Elemental Analysis The elemental analysis of thiabendazole is as follows: 11 E 1ement % Calcd.586 6. and 10 ml of 1E sulphuric acid are then added. and clearance of thiabendazole were 1. While thiabendazole and the 5-hydro= metabolite did not accumulate during multiple dosing.THIABENDAZOLE 619 et a1.93 15.

30 6. from citrus fruit and bananas were applied as spots to silica gel in P e t r i dishes which were then uniformly inoculated with Penicillium cyclopium spores. Thiabendazole showed one wave peak in the polarogram when a short drop time (0. Fungicide concentration was detennined from comparison of inhibition zones with those of a standard 48-hr incubation.32 Graphium ulmf3 and Sporobolomyces roseus. Y . the size of the inhibition zone is measured (Figure 4 ) . After speciEic quantities of acetic snhydride. 31-38 These methods have been employed f o r determining the residue of the fungicide on plant materials or in soil.1E perchloric acid to a hlue-green end point is carried out. A titration method utilizing the complex formed by thiabendazole with silver Ions is also described.1N perchloric acid is equivalent to 20. mercuric aceta%e and two drops of c r y s t a l violet soluticn are added.37 After cold pre-incubation followed by incubatlon at 2 7 O .13 mg of CIOH7N3S. polarography and differential pulse polarography. Detection threshold was 0. Detection was most sensitive at pH 8 . Each r n l of 0.KAPOOR The sample is dissolved i n glsclal acetic acid. 39 The electrochemical behavior of thfabendazole was investigated by sampled doc. A blank determination is performed and any necessary correction is made. The lowest de%ectable concentration of thiabendazole in a sand soil was In another method 5 extracts found to be 1.5 Polaroqraphic Analysis A polarographic method for determining thiabendazole is described. Other microorganisms employed in the bioassa studies are Penicillium exansum.620 VIJAY K.0 ) i g / g .4 Bioassay Several microbiological assay methods using sensitive fungi for the quantitative determination of thiabendazole have been reported. A simple and rapid bioassay €or the direct determination of thiabendazole residue in soil involves placing pellets composed of mixtures of soil (200-500 my> and agar on an agar medium preinoculated with the test organism Penicillium digitatum.4 s ) was used. the titration with 0.38 6.5 p g thiabendazole.

(c) further purif ication by reextraction into dil. Differential pulse polarography has been used for quantitative determination of thiabendazole In citrus fruit peels.40-43 The method involves: (a) complete extraction of thiabendazole from Eeed w i ’ 3 1 C.THIABENDAZOLE c “E 250 c Y . The detection limit was 0.2N H C 1 in SO% methanol.5 ppm.6 Colarimetric Analysis Thiabendazole can be analyzed colorimetrically in feeds.-C 2 4 6 8 10 12 Thiabendazole concentration in soil ( p g / g ) Figure 4. ( d ) reduction with .200 621 t C 0 c .raction with chlorof o m from a l k a l i n e solution. 6.-2 . (h) separation from other suEstences by ext. Effect of thiabendazole concentration in-soil on growth inhibition of Penicfllium di-itatum using the soil-agar pellet technique.vte H C l .. inhibition is expressed a s the square Grow + The current measured was proportional to the concentration.

and the recovery was 95-100% from feed containing 1% thiabendazole.hite. The relative standard deviation was 1.3% zinc dust in glycerol.oroform with alkaline buffered bromocresol green is also 6escribed.1-2.2%. purification by acid-base procedure.1g H2SO4. (el subsequent oxidation with a ferric solution to form a thiazine dye. A modified method was used to determine the residues of thiabendazole in citrus fruits-45 A method for estimating thiabendazole in the milligram and submilligram ranges (0. A simple colorimetric method involving the reaction of thiabendazole in ch1.47 The analysis is completed by spectrophotometric measurement. and measuring the absorbance of the acid solution at 302-3 nm. In another purification procedure the .46 A modified method in which absorbance of the complex was measured at 311 nm permitted estimation of 2 pg/ml thiabendazole.~~ In the automated system the solution was reduced with 0.1-1.0 mg/ml can be estimated by this method.7 Spectrophotometric Analysis 6.0 mg/ml) has been descrlbed which involves measuring of absorbance of a copper-thiabendazole-tetramethylguanidine complex at 350-400 nm. KAPOOR zinc dust in the presence of pphenylenediamine to form a complex. by !~.17% pphenylenediamine in 2N H2SO4 and 15% Fe NH4 (SOg)2 in 0. Thiabendazole in concentration range of 0. and (f) extraction of the dye into butanol and measurement at 605 nm against reference standard thiabendazole.48 6. An improved procedure involves shaking an alkaline aqueous suspension containing thiabendazole with solution of cupric acetate and 1-dimethylamino-2-propanol in chloroform to produce a green color in the chloroform phase. The glue complex was measured at 610 nm.49-60 The method in general involves extraction with ethyl acetate-ammonium hydroxide.71 Ultraviolet Thiabendazole is estimated in various fruits as a residual fungicide by ultraviolet spectrophotometry. The method gives most accurate and reproducible he method described above was automated results. and the products were coupled with 0.622 VIJAY K.

~-dimethylformamide and adding 1 g of anhydrous sodium s u l p h a t e .63-%8 can be detected i n c r o p s by acetone e x t r a c t i o n followed by p a r t i t i o n i n g i n acetone-water and e t h y l a c e t a t e and clean-up on magnesium oxidec e l i t e .9 described.05 pg/ml. The cornpound i s t h e n q u a n t i t a t i v e l y measured by d i r e c t fluorometry.THIABENDAZOLE 623 f r u i t p e e l s o r p u l p s are extracted with chloroEorm. Chromatographic A n a l y s i s 6.5 and 16.a l u n f n i u m oxide column.12 g of t h i a b e n d a z o l e sample i n 5 m l of l?. The e x c i t a t i o n and emission wavelengths are 305 and 345 nm. 63 An automated f l u o r o m e t r i c method f o r t h e d e t e r m i n a t i o n of t h i a b e n d a z o l e i s a l s o d e s c r i b e d which gives t h e minirnun l e v e l of d e t e c t i o n of t h e f u n g i c i d e asw0. and t h i a b e n d a z o l e e x t r a c t e d a t pH 9. 11. 6.02 ppm thl sbendazole. Absorbance a t scanning between 2.72 Infrared Thiabendazole can a l s o be analyzed by i n f r a r e d spectrophotometry. r e s p e c t i v e l y f o r thiabendazole.09 )xn is used f o r q u a n t i t a t i o n . 68 6. T h e method i s r e p o r t e d t o be s e n s i t i v e t o 0. o r 0. t h e e x t r a c t i s a c i d i f i e d and c h l o r o f o m evaporated.5. Aspect of p r o t e i n i n t e r f e r e n c e i n t h i a b e n d a z o l e d e t e r m i n a t i o n i s discussed.03 ppm i n whole c i t r u s f r u i t .54.8 Spectrofluorometric A n a l y s i s Thiabendazole is a n a l zed by spectroThe f u n g i c i d e f l u o r o m e t r i c method also. Using t h i s method t h i a b e n d a z o l e w a s determined spectrophotom e t r i c a l l y a t 302-3 nm a t c o n c e n t r a t i o n s of 0.91 Paper Chromatoqraphy The f o l l o w i n g system h a s been 4 .62 The method i n v o l v e s d i s s o l v i n g 0.7 )un. The a c i d i c c o n c e n t r a t e i s cleaned up with chloroform.6* A s e n s i t i v e f l u o r o m e t r i c method f o r d e t e r m i n a t i o n of t h i a z o l e c o n t a i n i n g compound i s r e c e n t l y described. The s t a t i s t i c a l a n a l y s i s of m u l t i p l e d e t e r m i n a t i o n by t h i s method gave a r e l a t i v e e r r o r o f \ ( 2 % and a c o e f f i c i e n t of v a r i a t i o n 4 1%.61 6.1 pprn i n 100 g peel or pulp. A p o r t i o n of t h e s u p e r n a t a n t i s used for i n f r a r e d spectrophotometry.

sheet 1 4 x 6 i n .4) 71. Examination under ultraviolet light b. buffered by dipping i n a 5% s o l u t i o n of sodium d ihydr ogen c i tret e . 4. These are t a b u l a t e d below: S o l v e n t System Reference Benzene-acetic acid-acetone-water 70 (10:4: 1:0. b l o t t i n g .KAPOOR 624 Paper: Solvent: Equilibration: Development: Location: Rft (l:6) Khatrnan Noel.69 allowing t h e paper t o react with i o d i n e vapor for 1 minute.l a y e r chromatog r a p h i c s y s t e m s u s i n g s i l i c a g e l or si"l1ca g e l GF254 p l a t e s f o r i d e n t i f i c a t i o n and e v a l u a t i o n of thiabenda z o l e have been r e p o r t e d .92 Thin-Layer Chromatography A v a r i e t y of t h i n .06 .72 Benzene-acetic acid-acetone-water (70:20: 10: 2) Ethyl a c e t a t e .8 CJ of c i t r i c a c i d in a mixture of 330 m l of water and 870 ml of r p b u t a n o l . and drying a t 2 5 O for 1 hour. A n a l t e r n a t e system employs acetone-water i n ascending mode which g i v e s Rf v a l u e of Location is done by t h i a b e n d a z o l e as 0. Time of run. 5 hours a.e t h y l methyl ketone65.67 formic acid-water (50: 30: 10: 10) Ethyl acetate-benzene (50:50) D i e t h y l e t h e r . Fotassium permanganate spray 0. 6. None Ascending.g l a c i a l acetic acidmethanol (100:5:2) 74 Acetone 74 73 .VIJAY K. Bromocresol green spray C.49.

A n enzyme i n h i b i t i o n . G u a n t i t a t i v e relations were e s t a b l i s h e d b e t w e e n spot s u r f a c e a r e a and t h e amount of f u n g i c i d e .THIABENDAZOLE 625 Solvent. System Reference L i g h t petroleum (60-80°)-acetone (30:lO) Chloroform-acetone (80: 20) 74 75 Using aluminium o x i d e F254 n e u t r a l p l a t e 74 d i e t h y l ether-methanol i s t h e s y s tern employed.e t h a n o l arnmonia (60:10:10:2) 60 - 41 31 21 43 58 52 38 D e t e c t i o n of s p o t of t h i a b e n d a z o l e on t h e p l a t e h a s been c a r r i e d o u t by d i f f e r e n t methods such as v i s u a l i z a t i o n under u l t r a v i o l e t l i g h t . 7 4 8 7 6 # 7 7 by s p r a y i n g with potassium iodobismuthate solution followed by exposure t o bromine vaposs74 o r by exposure t o i o d i n e vapors.et h a n o 1 ammonia (70: 15: 15:2) Toluene-ethyl a c e t a t e .e t k i y 1 ace t a t e. Cole e t h a s g i v e n t h e Rf v a l u e s of t h i a b e n d a z x e i n d i f f e r e n t s o l v e n t systems. These a r e g i v e n below: R f Value x 100 S o l v e n t System E t h y l acetate 55 E t h y l a c e t a t e s a t u r a t e d wit. 76 A b i o l o g i c a l method f o r d e t e c t i o n and e v a l u a t i o n of t h i a b e n d a z o l e h a s a l s o been d e s c r i b e d O 7 3 I t i s done by s p r a y i n g t h e p l a t e s with spores of any of t h e t h i r t y t w o fungal. species f o l l o w e d b y i n c u b a t i o n .h ammonia E t h y l acetate-toluene-anunonia (60:40:2) E t h y l acetate-toluene-ammonia (40:60: 2) E t h y l acetate-toluene-anunonia (20:80:2) Toluene-ethanol-ammonia (80:20:2) Toluene-ethyl a c e t a t e .e t h a n o l ammonia ( 60 :10 :30 :2 1 To 1uene.

It p e r m i t s d e t e m l n a t i o n i n c o n c e n t r a t i o n s down t o 0. column temperature 230O.l ppm.VIJAY K.8~.a b s o r p t i o n photometry with measurement at 302 nrn o r f l u o r o m e t r i c a l l y a t 355 nm.1aye r chsornatoy r aphy want i t a t i v e determination of thiabendazole has been c a r r i e d oiit s p e c t r o p h o t o m e t r i c a l l 70.67.1g HC1. direct e v a l u a t i o n on t h e chromatogram h a s t h e advantage t h a t it saves t i m e and r e q u i r e s less substance p e r spot.67. Nitrogen was used as a c a r r i e r g a s a t a flow-rate of . Hey used SE-30 a s s t a t i o n a r y phase with a n i t r o g e n . P r i n c i p a l o p e r a t i n g c o n d i t i o n s were: 1.KAPOOR 626 75 technique i s a l s o reported. g l a s s column packed with 5% OV-101 on Gas Chrom G (HP) (80-100 mesh). The concentrated e x t r a c t was chromatographed on a lo”/.50 Samples were made a l k a l i n e with ammonia s o l u t i o n and macerated w i t h e t h y l a c e t a t e and filtered. using t h i n .o1e050.72. Otteneder and FIezel65 have r e p o r t e d a method by which thiabenda z o l e can be Getermined d i r e c t l y on t h e chromatogram by r e f l e c t a n c e . The l i m i t o f s 3 d e t e c t i o n was of t h e o r d e r of 0. The m i n i m u m amount of thiabendazole detectable by t h i s method was 0.D.82-94 An e a r l i e r method t o determine t h e f u n g i c i d e i n whole c i t r u s f r u i t and i n c i t r u s and bansna pulp is d e s c r i b e d by Mestres e t al.93 G a s Chroniatoqraphy Gas chromatographic methods have been used t o determine r e s i d u e of thiabenda. 6.5 m x 3 mm I.53. Dc-200 on Gas-Chrom Q a t 240O. Thiabendazole w a s determined by Nose e t al.71. . 8 4 a s g-pentafluorobenzoyl d e r i v a t i v e by e l e c t r o n c a p t u r e g a s chromatography following r e a c t i o n of thiabendazole with pentafluorobenzoyl c h l o r i d e .s e n s i t i v e Tanaka and Fujimoto83 determined detection thiabendazole a s i t s methyl d e r i v a t i v e w i t h flame i o n i z a t i o n d e t e c t i o n and a column of lo”/.1 pj. DC-200 s t a t i o n a r y phase using FPD set i n t h e mode f o r determining s u l f u r . with e x c i t a t i o n a t 313 nm.79 or fluorometrically65.01 ppm. and making t h e aqueous phase a l k a l i n e and r e e x t r a c t i n g back i n t o e t h y l a c e t a t e . Compared with t h e e l u t i o n and subsequent photometry. T h e f i l t r a t e was cleaned up by e x t r a c t i o n i n t o 0.78.~1. i n j e c t i o n p o r t and d e t e c t o r temperature 270°.

g-dimethyl derivative of fj-hydroxy= thiabendazole. 5 On-column methylation converts these compounds to their Emethyl and E. . separator temperature 260°.g-dimethyl derivatives. Tjan and Jansen66 have used pentafluorobenzoyl bromide f o r derivatization of thiabendazole. argon-methane carrier gas (95 + 5) at a flow-rate of 60 ml/min.THIABENDAZOLE 627 40 ml/min. Thiabendazole is extracted with methanol and the extract is washed with phexane saturated with methanol. respectively. A combined gas Chromatography mass spectrometry confirniatory assay for thiabendazole and 5-hydroxythiabendazole at 0. Collinge and Nairfalisegg have determined thiabendazole in curds (artificial marmalades) and orange and lemon marmalades - B -- . 2-methylindole is added as an internal standard.8"A ammonia wzter (60:40) as mobile phase.83 m x 4 mm I . Isshiki et a l o g 6 have described a simple HPLC method for eetermining thiabendazole in fruits. The HPLC determination is carried out u s i n g LiChrosorb RP-8 as stationary phase and methanol-2. Bardalaye and Wheeler85 employed this technique to determine thiabendazole in yams with operating conditions as: 1.1 pg thiabendazole/ Recoveries are 92%. D . Identification and quantitation are achjeved by selective ion monitoring of M-1.94 High Performance Liquid Chromatoqraphy In recent years high performance liquid chromatography (HPLC) has become one of the major analytical tools to determine the residual thiabendazole alone96-100 or simultaneously with other addit. Temperatures of oven. Recently. 6. respectively. detector and injectors used were 240 300 and 200°. The carrier gas was helium at a flow-rate of 30 ml/min. Combined gas chromatography-mass spectrometry was operated with column temperature 2 3 0 ° . M and Etl ions from E-methylthiabendazole and the I4 and M-15 i o n s from IJ.iveslOl-lll in fruits. The detection is carried out fluorometrically and the limit of detection is 0.1 p m in animal tissue isolates has been developed. glass column packed with 5% OV-17 on 80-100 mesh Gas-Chrom 0. ion source temperature 290° and flash heater temperature 270'.

1°6 A rapid. A simultaneous HPLC determination of thiabendazole. respectively.628 VIJAY K.0 ml/min. The method should be of particular use for monitoring thiabendazole therapy in .PA of ammonium nitrate and 0. range 0. the column temperature was 22-26O and the detection wavelength was 305 nm. quantitation of the separate metabolites was possible.1 pg/ml serum for thiabendazole and 0. A special adaptation of the chromatographic procedure was utilized for the determination of 5-hydroxythiabendazole metabolites in the sera of uremic patients. An ionggair HPLC has been described by Belinky.4 pg/ml serum for 5-hydroxythiabendazole. 2-methylindole.lo0 have described a detailed procedure for the separation of thiabendazole in fruits in which the uv-absorbing Contaminants are removed by fractional separation with an aqueous-ethyl acetate system. The flow-rate in column was 1. detector uv (298 nm).112 for the simultaneous determination of thiabendazole and unconjugated 5-hydroxythiabendazole in human serum. v/v % ) I flow rate.08 ppm.16 M KH2PO4 water ( 5 0 : 5 0 . 1.0 ml/min. and buffered solution-methanol ( 3 :2) for marmalades. 0-phenylphenol and diphenyl residues in cityus fruits without prior clean up has been described by Kitada et a1.6 mm x 300 mmt eluant. which can contain large mounts of interfering fluorescent substances.KAPOOR by using aqueous solution containing 0. Thus. and sample size 5 pl. Detection limits were found to be 0. Sample pretreatment consists only of protein precipitation with acetonitrile containing the internal standard. sensitive and precise HPLC method using fluorescence detection has been developed by Watts et a1. Unisil C-18 10 p 4. Yamada et &. pressure.01 AUF. 60 kg/cm2. A microenzymatic method for the conversion of the glucuronide and sulfate esters of 5-hydroxythiabendazole was also developed using P-glucuronidase and sulfatase.7% of ammonia (buffered solution)-methanol (1:1) as mobile phase for curds. and it was shown that 1 ppm of thiabendazole in the fruits could be estimated within 10%. The detection limit for this method was found to be 0. The HPLC conditions for thiabendazole determination reported aret column. methanol0.

A . \ British Pharmacopoeia 1980. Limits of Tolerance Tolerances in ppm for the residues of the fungicide thiabendazole in or on various raw agricultural commodities established under the Federal Food. J. 15517f (1962) 3. C. R. S . R. L. References H. p.l20 eggs. Brown. London. J. M. 1969.4 for banana pulp119 and rnilki123 1 for hubbard squashr124 3 for bananas119 and rough rice1125 5 f o r strawberriesol26 6 €or su ar beetsol21 8 for rice hullst127 10 for a les.480t 69. S . rice straw. D. HPIC methods for determining thiabendazole in waste waters1 138114 and in textile productsll5-117 are also described. Peterson.. L. 4500 . 1764 (1961) 2.A. Ilves.$25 sugar beet tops135 and wheat grains. Bezou. L o H.491. 131 15 for cantaloupes. I.10 Miscellaneous A radioactive indicator method for the determination of thiabendazole residues has been described. Matzuk. Sarett. French Patent 1. E .THIABENDAZOLE 629 patients to eliminate the potentially toxic metabolites. 1. 151 and soyabeanso122 0. 83. L o H. R . "Isolation and Identification 5. M. A . H. 59241g (1968).llg 0.1 for dried beans. C . Chem. p. Clarke. Her Lajesty's Stationary Office.118 7. Pharmaceutical Society of Great Britain. of Drugs". London. J. W. Patent 3. U . Drug. Egerton. C. I. G .l29 citrus fruits 130 grapes. J.415. A. C.121 meat.P93 pears 128 potatoesl31.017. Campbell and A. Brown. 0 56.8 4. and Cosmetic Act fixed by the United States Environmental Protection Agency are: 0. Harris. Yakstis.13~ 8 8. 568.02 for sweet otatoes. SOC. 6.l 8 carrots. D. 1980. A. C. Cuckler.126 and 40 for rnushro0m. Vol. Am. Sarett and H.

Inc. Lyr and C. Jones. H. A. W.347. Akad. G . R . 259 (1965) 11.A. C. V.981). 7. R . Matzuk. H. O r s . P o 1045. D. I n t .KAPOOR 630 U n i t e d S t a t e s Pharmacopeia XXI National Formulary XVI. C. 16. II. D.8 327 (1974)/(Pub 19751. F. Underwood. E. symp.. 8 6 8 4 77954a (1968). J. 6 6 8 161 (1983) 10 . ) .A. J.VIJAY K. J.lertel. 147233d (1978) ... H. 14 T. F. . Bombay. E. Twentye i g h t h Edn. Prasad (Eds.). 12 Profiles i n Drug Syntheses. P f i s t e r and J. Hashim and F. . K o l l o n i t s c h . A u s t . VandenHeuvel. J. 1982. (1. Buchenauer. The Pharmaceutical P r e s s . - 77 (1976). S l e t z i n g e r . M a r t i n d a l e The E x t r a Pharmacopoeia. M d . R..-Verlag. 88. R. J. 8. Harmsn and N. 145869f (1973). 2298 (1973). 17 G . C. C h a m . Sect. C. K. J. O f f . Chem.. 0 775 (1982) 35. 2. J. Bokin Bobai. R o c k v i l l e . A. Brown. 1 5 9 5 4 ~(1982) 18 G.. L. Anal. B. Gogte ( E d . Buhs. 28 399 (1964). E. J. A c t a C r y s t a l l o g r . 30. 1985. .908. N. R. 9.A. B e r l i n . Vol. E.0 79. Fuse and H. R. Owen. Systemfungiz. C h a . Agric. R. Gokul P u h l i s h e r s . M. J. Raynolds and A.S. 107.). p. J. . E..8 96. B I 29. Chemosphere. . R. Marsh. Med. P. TOCCO. Jacob. D. P a t e n t 3. Grenda. A. C a r l i n . Cheni.A. 13. G a l and M. 2 3 8 704 (1975) 15 H. Tana. J. U n i t e d S t a t e s Pharmacopeial Convention. M. U. 1982. Trenner. A. J. 28 273 Crank and A. Walker. A s s O C . T r u s and R. V. B. B. Food Chem. London. 8 1985. P o l t e r (Eds. I.. Wolf and F. A . h a t k i n s . Mursyidi.

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60. 1219251. Prov. Pharm. 188293s (1978). Trav. V . C. 0 Pavoni. Tanabe.A. C. Romminger and H. . 56123~(1975).20 601 (1979).A. Csonka. 59 . Nauka. Hopper Nahrung. C.A. Lozar. Shokuhin Eiseigaku Zasshi. 131 (1974). z 0 193 (1970). 3587lp (1974) Rajzman. C.-Unters. Chim. J. 17925~(1983) C. J. Campo and J. Pharm. C. Chem. .8 58 153 (1980). Montpellier. Logan. Trav. A n a l . Off. Lebensm.A.8 145043a (1980) 9 2 0 Pavoni. Unione I t a l . CXIIPO. Tourte and M. M.. R e c . Chim. T. Ann. 52. Mestres.8 P a r t e ~ c i . Expert. Mestres.0 2.0 85. SOC. Pharm. J.A. R. 50. Copeland and R. C. Mestres. 2. Tourte. R.0 800 . 2 0 120 (1974). C. A n a l y s t (London). C. Lab. Mestres. F. R. 50. .A. Kondo and H. 54.A.. Campo and J. Prov. Karageorgiev.A. 971 (1975).O'Neill. A. J. 33 (1977). 2 0 407 (1976). K. 18664~(1971). B..0 8l. M i l l e r . M o n t p e l l i e r .THIABENDAZOLE 633 47. C. 14. 58. B o l l . Gould and E. Chim.# 82. 4 0 1490 53. A s s o c . 3 91 (1974). Tourte. 55. 49 . 2964g (1981). R e s . 2 0 179 (1973). C. Unione I t a l . &f 160 (1970) 51. G.. 167887d (1974). SOC. 79 (1972). M. Kesavan.A. L.8 88. Forsch. R. Trav..8 94. Z. CXIIPO. P a r t e Scf. 9 9 . R. Tourte and M. Gnaegi. Agric. M o n t p e l l i e r . 56. Fals. 26. . 57 D. Lab. Bras. 83 (1978). .# 75.1 (1976). 417 (1982). 48 C. M. F i t o p a t o l . Hey. Mihara.0 9 0 8 163162b (1979) R. Boll. Gradinar. G. H.A. SOC.

Assoc. Sao Paulo. K. J.. 104225j (1986). Fink.... T. 403 (1975). N. 69. Farrow and R. 196470~(1980) 73.A.. Ott. 2. 109. 93. J. S. 85 (1969). 67 (1980): C. 63. 75. Lyon. J. Baker. Chem. 1330 (1973) 64 D. J.-Rundsch. Pharm. T. 56. Pierson and P. 67 276 (1984). 56. Tab. Joshi. 19523f (1981). Off. Lab. . K. 70 E. Shim. K. M. Chert. 693 (1979). SOC. Unione Ital. 4 4 0 4 8 ~(1980). C. 108592g (1971)F - 62.. Inst. T. 0 J. C. Yajima and J. A.. H. Sect. Chromatogr. C. 1003 (1971). 223 (1973). Ben-Adz. Hezel. Tancogne and R. 5 1 8 7 (1984). J. E... B. 110. Jeffus and C.. Arq. M. Chem.. Kenner. 275 (1980). Otteneder and U. Becker. 68 3 . 74 . Sinha. W. Hamadi. Assoc... P. R. Aharonson and A. Assoc. 22. J. W.2. 75. T j a n and L. KAPOOR 634 61. 588 - 0 11. T r a v . 9 G. Biol.. Lord. Chromatogr. J. 16. Anal. Anal. Anal. off. Piedade. PTOV. 104. O f f . 8 1 8 174 (197310 G.VIJAY K. Off. Chambon. CoAo. 65 H.. Kroeller. Hoodless. Chim. Pharm. F. 66 . J. - F. Modi.. Chem. Deut. Chiti and G.. Fresenius'z.A. 318. 95. Delon. Vyas and L. 160 (1975). R. 3 (1978). (1980) G. Anal. Parte Sci. W. Sci. 658 710 2.A*.A. Ann. J. Anal. Cayley and A. Chem. 54. Piedade. J. S. Burgers. Chromatogr. Sci. Assoc. E. Boll. K. 92. Pestic. Lebensm. C . Bull. Tolan and D. 181 (1975). M. J. 69 0.

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Meriwether. 26 May 1972. 8 Sept 1982.0 10 A p r 7985. Regist.A. 46067.A. 2 0 2 7 2 9 ~ (1985) Fed. 38229.A. 122. 30 Oct 1974.. 4922h (1984). 8 74.8 92. 45 (1331.. 47 (1’741. C . 29789t (1975). C . Regist. 119. M. Fed. 9 July 1980. 37 (19618 212787 7 8 0 2 8 7 4 ~(1973) C. 116494q (1977). 126. T . Regist.KAPOOR 638 117.A*. 123 (1985)..A. Regist. . Fed. 50 (691.8 19 Mar 1986. 127 1280 129 130.. C.Am. Watanabe. 36. 3 6 7 0 4 4 ~(19$6) Fed. 10670. Regist. 3 A p r 1985. 1 3 0 7 5 2 ~(1980) C. S . & I 1 3 5 3 3 5 ~( 1 9 8 n . 46073. 121. 39488.A. 100. c. 118.. 4S (1331. 104.. C. R e g i s t .VIJAY K. Radioanal... C. J. 2 8 NoV 19808 45 (2311.. Regist. C.980) 3907. C. 131. 7 O c t 1972. C. 48 (223).8 2 0 46820k (1972) 125 Fed. 130751b (1980). 32782.. 2 8 Jun 19778 42 (1241. Fed. . Mori.osenblum anc? H. C. C. Fed.. .A. Amemiya.A. R e g i s t . Chern. Siizuki and Y. A . 51 (531... 37 (103). 63918s (1981). 9448. 161179h (1982).A. . R e g i s t .A. 124. Fed. S a k a i .8 Fed..A. Fed. 93. 1230 - 17 Nav 1983. 97. C. .. 87. K.. P. 9 J u l 1980. 2 2 Jan 1980. Kenkyu NenpoTokyo-toritsu Eisel Kenkyusha. 58 379 (1970). 50 (641. Ikeda. 39 (2lO)# 82.A. Regist.. 45 (141.A.. Regist. Fed. 94. Regist. 93. 79068. C. 98714m (1971). K. 13195r 1 0 2 8 183870a (1985). 102. T.. 52304. 14106. Fed. Fed. C. 120. 145132d (1. Regist.

38 (121). ..A.. C.A.THIABENDAZOLE 132. 64669~(1073) C. 16644. 50 ( 6 9 ) . 2 0 2 7 2 7 ~ (1985). R e g i s t . R e g i s t . 14104. . Fed. 133. 102. 2. 25 Jun 1973. 10 hpr 1385. 639 Fed. .

MAZZO* AND ALICE E. WEST POINT. ILLINO1 S #RESEARCH FELLOW. MERCK SHARP AND DOHME RESEARCH LABORATORIES. . TRAVENOL LABORATORIES. PENNSYLVANIA ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 16 641 Copyright 0 1987 by Academic Press. PARENTERALS RESEARCH AND DEVELOPMENT. MORTON GROVE. AU rights of reproduction in any form reserved. Inc. PHARMACEUTICAL RESEARCH AND DEVELOPMENT. LOPER# * MANAGER.TIMOLOL MALEATE DAVID J.

Introduction 1.me.1 Chemical . Synthesis 4.1 Proton NMR 4.7.2 Carbon-13 NMR 4.. MAZZO AND ALICE E.6 Electroanalytical Behavior 4.1 Infrared Spectrum 4. Physical Properties 4.2 Definition 2. Odor 3. LOPER 642 1. Formu a.2 History Description 2.7.4 Mass Spectrum 4. Color.2 Differential Thermal Analysis Behavior .3 Ultraviolet Spectrum 4.2 Nuclear Magnetic Resonance Spectrum 4.3 Appearance.DAVID J. 2.2. Molecular Weight 2.1 Therapeutic Category 1.7 Thermoanalytical Behavior 4.2.1 Melting Point 4.5 Optical Rotation 4.

1 5.3 4.3 Elemental Analysis 5.2 Solution Stability .2 Titrimetric Analysis 5. Identification Tests 5.2 Ultraviolet Spectrophotometry via Flow Injection Analysis 5.1.8 Solubilities 4.7.2 High Performance Liquid Chromatography Stability - Degradation 6.1.4 6.1.1 Ultraviolet Spectrophotometry 5.3 Spectrophotometric Analysis 5.4.1 Solid State Stability 6.3.3.9 Crystal Properties 4.4.TIMOLOL MALEATE 643 Thermogravimetric Analysis Behavior 4.3.2 Infrared Spectroscopy 5. Methods of Analysis 5.11 Dissociation Constant 5.1 Thin-layer Chromatography 5.10 Hygroscopicity 4.1 Direct Ultraviolet Spectrophotometry 5.3 Specific Rotation Chromatographic Analysis 5.

Determination in Biological Matrices 9. 9.1 Absorption and Bioavailability 7.2.1 Assay 9. LOPER 644 7.DAVID J.3 Stability Testing References . Biopharmaceutics and Metabolism 7.2 Dosage Uniformity 9. Determination in Pharmaceuticals 10.2 Metabolism 7.2 Potency 9.3 Pharmacokinetics 8.1 Dissolution Testing 9.2.2. MAZZO AND ALICE E.

A number of adverse reactions considered to be causally related to timolol maleate have been reported. However. Timolol maleate. a number of adverse effects have been reported with other betaadrenergic blocking agents and should be considered potential adverse effects of timolol maleate.1 Therapeutic Category (1) Timolol maleate is a beta adrenergic blocker which is non-selective between beta and beta adrenergic receptors.TIMOLOL MALEATE 1. have been reported (1) . available for oral dosing as tablets and for injection and ophthalmic dosing as distinct sterile aqueous solutions. Timolol maleate is effective in lowering intraocular pressure and is widely used in patients with openangle glaucoma and aphakic glaucoma. severe respiratory reactions and cardiac reactions. direct myocardial depressant or local anesthetic (membrane-stabilizing)activity. Timolol maleate is also indicated both for the treatment of hypertension (alone or in combination with other anti-hypertensive agents. For example. It dhes not ha6e significant intrinsic sympathomimetic. is usually well tolerated with most adverse effects being mild and transient. including death due to bronchospasm in patients with asthma and rarely death in association with cardiac failure. especially thiazide-type diuretics) and to reduce cardiovascular mortality and the risk of reinfarction in patients who have survived the acute phase of myocardial infarction and who are clinically stable. 645 Introduction 1.

A search of Chemical Abstracts (1966 . Timolol maleate.5thiadiazol-3-yl)oxy]-2-propanol. was first synthesized in the Merck-Frosst Laboratories in Montreal.2. Formula.1986) produced over 210 unique bibliographic citations for works dealing with timolol maleate.2. belonging to the thiadiazole class of compounds. since its introduction in pharmaceutical formulations.DAVID J. Canada.1986) and Pharmaceutical Abstracts (1974 . The first non-patent literature reference to timolol maleate appeared in 1972 (2). (2)-butenedioate (1:l) salt. Description 2. Molecular Weight The accepted chemical name for timolol maleate (MK-950) is: (S)-l-[(l.l- dimethylethyl)-aminol-3-[[4-(4morpholinyl)-1. The CAS registry number is 26921-17-5.5-thiadiazol-3- ylloxyl-2-propanol.5- thiazide.2. Other names which have been used for timolol maleate include the maleate salts of (s)-(-)-3-(3-tert-butyl-amino2-hydroxypropoxy)-4-morpholino-l.5- thiadiazole and (-)-1-(tert- butylamino)-3-[(4-morpholino-1.2. MAZZO AND ALICE E. (-)-3-morpholino-4-(3-tert- butylamino-2-hydroxypropoxy)-1. has gained a wide acceptance as an anti-hypertensive and anti-glaucoma agent.2 History Timolol maleate. . LOPER 646 1. 2.1 Chemical Name.

3. Color. Timolol. The synthetic route is presented in Figure 1. odorless. This substituted mannitol (I11 is then oxidized in tetrahydrofuran with lead tetraacetate to the substituted glyceraldehyde (1111. crystalline powder. The starting material. It has tradenames of BLOCADRENs and TIMOPTICs. In a separate reaction sequence.3 Appearance. hydrogenated with t-butylamine ( I V ) and treated with benzaldehyde to form a substituted phenyloxazolidine (V). Odor Timolol maleate is a white. 2. is reacted with acetone and the product (11) is washed with benzene and crystallized from hexane.2 647 Definition Timolol maleate possesses an asymmetric carbon atom in its chemical structure and is provided as the levoisomer. Synthesis Timolol maleate has been prepared through a series of synthetic steps beginning with D-mannitol and acetone (3).TIMOLOL MALEATE 2. when referred to. indicates the free base (CAS Registry Number = 26839-75-8). C13H24N403S-C4H404 Molecular Weight 432. I.49 g/mole .

c/0-?H2 HO -CHe I HO-C-H I HO-C-H I H-C-OH (CH3)zCO I H3C' '0-C-H I Pb(0Ach HO-C-H I H-C-OH I H-C-0.H3C. o&N\C(CH3)3 /CH3 'CH3 II I . Synthetic route to timolol maleate. H-C-OH I CHDH I HeC-O/ C [""' /o-i:6] H. X .C/~'O-CH (CH3)aCNH2 HOCHzCHCHzNHC(CH3)3 I OH + 6/ m rn Y m S \ Y Ip H N. CHO ..N Ix Figure 1.

Both of these spectra are consistent with the structure of timolol maleate. 4.649 TIMOLOL MALEATE aminoacetonitrile hydrochloride (VI) is reacted in the presence of chlorine with sulfur monochloride to form a chlorinated thiadiazole (VII). Frequency assignments for some of the characteristic bands are listed in Table I. Table I Infrared Spectral Assignments for Timolol Maleate Frequency ( cm’l) 3250 3000 1700 1200 1100 Assiqnment 0-H N-H C=N c-o(c-OH) c-0-c stretch stretch stretch stretch stretch The infrared spectrum of timolol maleate taken in a mineral oil mull is shown in Figure 3 (4). A Digilab Model FTS-15C Fourier transform infrared spectrophotometer was used to acquire the spectrum. Physical Properties 4. is reacted with morpholine to form VIII. Compounds VI and VIII are reacted to timolol free base (1x1 which is then converted to timolol maleate by refluxing with maleic acid in acetone ( X ) . in turn. . which.1 Infrared Spectrum The infrared spectrum of timolol maleate taken in a KBr pellet is shown in Figure 2 (4).

1 1 1 4000 3800 1 1 1 1 1 1 3600 3400 1 1 1 1 3200 1 1 1 1 3000 1 1 1 1 1 1 2800 2600 1 1 1 1 2400 1 1 1 1 2200 1 1 1 1 1 1 1 l 2000 1800 1 l 1 l 1600 l V l l l 1400 l l l l 1200 l l l l l l I l i I I I I I I Wavenumbers Figure 2. . I 1000 900800 700 600500 Infrared spectrum of timolol maleate taken in a KBr pellet.

Mineral oil contributes to the spectrum at ca. . 1350-1450.1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ”. Infrared spectrum of timolol maleate taken in a mineral oil mull. 15001750 and 2875-3000 wavenumbers. 1 1 3700 1 1 3500 1 3300 1 1 3100 2900 2700 2500 2300 2100 1900 1700 1500 1300 1100 900 800700600 Wavenumbers Figure 3.

2 Nuclear Magnetic Resonance Spectrum 4.02 M solution using d DMSO as the solvent. The spectrum was acquired from an approximately 0. - . s ) . LOPER 652 4.1 Proton NMR Spectrum The proton magnetic resonance spectrum of timolol maleate was obtained using a Nicolet NT 360 spectrometer.2. The spgctrum is shown in Figure 4 and the spectral assignments are listed in Table I1 ( 4 .DAVID J. MAZZO AND ALICE E.

A 20 1 r I ill LL / 10 " ' I " 7 ' 8 Figure 4. . b 4 Proton NMR spectrum of timolol maleate.

LOPER 654 Table I1 Re lat ive No.Cg2 -N 2 2*> 3.CHf 3.03 2 HC=Cg 8.CHpE& .CH.35 - 3. Protons Assignment d .97 1 -0-g 6.1 2 I -C=C-COOH I - c02 .DMSO sglvent effect .38 2 I I H - I + -N-C(CH3)3 I H 20.Cgf ‘CH 0 .2 1 residual H20/HOD .47 4 CH .CHI 0 4.DAVID J.72 4 -N 4.C H h / -2 0 -N ‘CH2 . MAZZO AND ALICE E.4 2 0-C€12- 5. 3.

0 -CHOH-(C@) - 56.9 72.5 -NH-C- 43.5 M timolol maleate fglution in d . Table I11 CI3 NMR Spectral Assignments for Timolol Maleate 6 (ppm) Assignment 167. The C spectral assignments are listed in Table 111.9 C(CH313 .7 -NHCH2-(Cy) 24.DMSO (5).4 C02H/C02- 153.6 O-(CH2-l2 ( a ) 65.0 -0cH2-(Ca) 65.2.3 c3 149.8 c4 I I HC=CH 135.TIMOLOL MALEATE 4.2 655 CI3 NMR Spectrum The CI3 NMR spectrum shown in Figure 5 was obtained on a Varian CFT-20 spectrometer using a 0.

.I I I I I I I I I I 1 180 170 160 150 145 130 120 110 100 90 I I I I I I I I 1 80 70 60 50 40 30 20 10 0 (PPm) Figure 5. CI3 NMR spectrum of timolol maleate.

Principle fragmentations of the morpholine ring are seen . b) The C and C carbons are equivalent. theregore. A strong M-minus-methyl peak is observed at m/e = 301. on?y a single line is observed for these two carbons. 4. This spectrum was obtained using an LKB model 9000 mass spectrometer operated at 70 eV in the direct probe-electron impact mode.3 Ultraviolet Spectrum The ultraviolet absorption spectrum of timolol maleate in 0. The absortivity in absorbance units drug solution in a 1 cm is 200 at 294 nm.1 N aqueous hydrochloric acid is characterized by maxima at approximately 210 nm and 294 nm. Loss of C H produces a fragment at m/e = 259 w h e h lacks the intensity to be seen in a high resolution spectrum. there$ore. 4. The W in Figure 6 was obtained using a Hewlett-Packard Model HP8451A diode array spectrophotometer. The fragmentation pattern observed is consistent with the chemical structure of timolol.TIMOLOL MALEATE 657 1 t 1 I a) The C and C carbons are equivalent.4 Mass Spectrum A mass spectrum of timolol is shown in Figure 7. The molecular ion is noted at m/e = 316. The loss of C H N(H)-CH gives rise to m/e = 230 with4t8e peak it m/e = 86 representing this lost group. onfy a single line is observed for these two carbons. The loss of CIHION leads to m/e = 244. Both the proton and CI3 NMR spectra are consistent with the timolol maleate structure.

f 0 658 8 0 w .

I 100 80 60 Relative Intensity 40 130 57 20 144 154 0 3 50 100 - 150 187 188 I 200 229 230 l A 250 A 286 316 l 300 (mle) Figure 7. Low resolution mass spectrum of timolol maleate taken in the electron impact mode. .

The peak at m/e = 243 is due to the loss of C H 0 from the molecular ion. having one chiral carbon. The levorotatory enantiomer is the biologically active form. Fragmentation of the heterocyclic nucleus produces ions at m/e = 144 and possible M-144 at m/e = 172. The origin of this peak is apparent from the metastable peak at m/e = 196 which corresponds to the transition from m/e = 301 to m/e = 243.2 " ) (6). The optical rotation of a 5% (w/v) solution of timolol maleate in i50 N aqueous HCt5at 25 "C and 405 nm is -12.DAVID J. . is optically active. MAZZO AND ALICE E. A MacClafferty rearrangement leads to m/e = 187 and two ions at m/e = 130 (see Figure 8). LOPER at m/e = 286 and m/e = 272.5 Optical Rotation Timolol maleate. 4.5 (due to the same decomposition). This fraAe8t also appears as a characteristic doubly charged ion at m/e = 121.2" ( A D = -4.

0415) (1 30.1232) I (major) / -(CH. CH.r f7 U N 0 L mle 187. mle112 Schematic representation of the mass spectral fragmentation of timolol maleate.0075) (minor) Figure 8.0057 (130.O @ CH3 I = NKC-CH. = N @ H r N\s.) 154.0625) CH.0616 (154. f/ 0 NH mle 130.0407 (187.=CH-CH -H.-0-CH-CH. . I CH.

An additional endotherm with a peak temperature of ca.DAVID J.7. 8 "C. 4. This curve was obtained using a Perkin-Elmer Series 7 DSC scanning from 40 "C to 2 5 0 "C at 1 0 "C/minute. 4.6 Electroanalytical Behavior Timolol maleate is electrochemically active and it undergoes a single irreversible electro-oxidation.1 Melting Point The melting point of timolol maleate varies between 201.7 Thermoanalytical Behavior 4. 5 'C as determined by the USP class Ia capillary method (6). A primary endotherm corresponding to melting is observed with an actual peak temperature of 2 0 5 . a silver/silver chloride reference electrode and a platinum auxillary electrode. 2 1 5 'C is noted corresponding to compound decomposition. LOPER 662 4. MAZZO AND ALICE E.01 M aqueous KC1 is +730 mV (vs Ag/AgCl). .2 Differential Thermal Analysis Behavior The differential thermal analysis (DSC) behavior of timolol maleate under nitrogen is shown in Figure 10. The E of an approximately 40 vg/mL skliition of timolol maleate in 0. Figure 9 shows the voltammogram of this solution obtained using a Princeton Applied Research Model 174A Polarographic Analyzer with a glassy carbon working electrode.7.5 'C and 2 0 2 .

6 0.3 0.1 M K C 1 (aq).0 1.5 0.2 0.0 Figure 9.8 0.4 0. AgIAgCI) Scanning voltammogram of timolol maleate in 0.1 0.7 0.2 Potential (Volts vs.9 1.1 1. I I I I I I 1 I I I I 0. .

0 I 125.0 0.0 - 20.0 225. . Differential scanning calorimetry behavior of timolol maleate.0 I 200.0 80.0 75.100.0 - - n =3 U 70.0 90.0 I 100.0 10.0 - c m 40.0 -p ' I I 50.0 30.0 - 50.0 60.0 Temperature ("C) Figure 10.0 1 150.0 I 175.

P. From 2 0 5 .TIMOLOL MALEATE 665 Thermogravimetric Analysis Behavior 4. Table IV Solubility of Timolol Maleate at Room Temperature Solvent Water Methanol Ethanol Chloroform Propylene Glycol Ether Cyclohexane Isooctane Solubility Soluble Soluble Soluble Sparingly Soluble Sparingly Soluble Practically Insoluble Practically Insoluble Practically Insoluble . 4.8 Solubilities The solubilities of timolol maleate in a variety of solvents at room temperature ( ~ 2 5"C) is presented in Table IV (6). Note that these solubilities are stated in terms of the current U.523 mg of timolol maleate was used. A sample of 3.S. A typical TGA curve for timolol maleate is shown in Figure 11.8 "C. definitions ( 7 ) . 3 5 0 " C there is an approximately 80% weight loss due to decomposition/vaporization. 8 " C to ca.3 Thermogravimetric analysis of timolol maleate indicates no weight loss until 205.7. The curve was obtained using a PerkinElmer Series 7 TGA scanning from 30 "C to 4 0 0 "C at 1 0 "C/minute.

0 8 cv 2 8 8 ? 9 m 0 .I I I I I I I 0 8m 8 s: cu 8rr.

4 23.68 5.0 38.44 4.8 26. No polymorphs of timolol maleate have been reported.4 22.33 4.95 7.4 14.49 8.2 14.47 4.1 29.63 2.56 4.96 5. Figure 1 2 shows the x-ray powder diffraction pattern of timolol maleate obtained with a Phillips Electronics model APD3720 powder diffractometer using CuKa radiation.36 3.8 24.82 2.32 2.24 4.76 6.0 26.97 3.74 3.6 16.0 19.8 15.5 20.1 9.03 2. Table V X-Ray Powder Diffraction of Timolol Maleate Peak Angle ( 2 0 " ) 7.67 4.16 3.5 19.6 34.3 29.31 4.90 4.9 11.6 20.8 20.1 19.9 667 crystal Properties ( 8 ) Timolol maleate exists as a crystalline powder.3 18.5 31.07 3.86 3.3 D Spacinq 12.29 3.42 3.9 21.5 27.23 5.14 3.29 (I/Imax)(%) 13 13 6 100 27 12 30 43 62 24 18 52 54 67 75 35 9 50 45 7 16 60 32 13 5 26 12 19 14 .0 23.1 28.8 39.TIMOLOL MALEATE 4.58 3. Table V lists the interplanar distances and the relative intensities of the major lines in the x-ray powder diffraction pattern of timolol maleate.

.

pH of a saturated aqueous solution of timolol maleate is 2 4. No hydrates of timolol maleate have been reported. equimolar sample and standard solutions will exhibit absorbances at 2 294 nm (Xmax) whi Q differ by no more than 3% (AX&= 2 0 0 ) . Quantitatively. exists both as a stable crystalline hemihydrate and anhydrous compound.2. A solution in 0.1 Identification Tests 5.10 Hygroscopicity (9) The maleate salt of timolol is a stable anhydrous compound. 5.2 Infrared Spectroscopy Infrared spectroscopy may also . 4.TIMOLOL MALEATE 669 4. however.1.1 N HC1 (aq) scanned from 200 nm to 400 nm qualitatively exhibits the same absorbance characteristics at identical wavelengths as does a similarly prepared and concomitantly measured solution of a timolol maleate standard.11 Dissociation Constant ( 9 ) The pKa of timolol base obtained by potentiometric titration in water at The native 25 " C is approximately 9.1 Ultraviolet Spectrophotometry (10) Ultraviolet spectrophotometry is used to identify timolol maleate. The free base of timolol. 5. Anhydrous timolol maleate is nonhygroscopic when stored under ambient temperature conditions (-25 " C ) and relative humidity conditions ranging from 3 3 % to 76%.1. Methods of Analysis 5.

A platinum rfng reference electrode is to be used.15%. An accurately weighed sample ( = 800 mg) is dissolved in = 90 mL glacial acetic acid and titrated with 0. 7.71%. 5. 10.1 N perchloric acid in dioxane to a green endpoint.35%. For potentiometric titrations.DAVID J. 5. sulfur. The infrared spectrum prepared as a mineral oil dispersion compares qualitatively (with maxima only at the same frequencies) to the spectrum of a similarly prepared timolol maleate standard. 17. Alternatively. the endpoint may be determined potentiometrically as that point at which the greatest change in potential per unit volume change of titrant is noted.1 Direct Ultraviolet Spectrophotometry . MAZZO AND ALICE E.3 Spectrophotometric Analysis 5.3 Elemental Analysis Timolol maleate may be identified through the use of elemental analysis.1. LOPER 670 be used to identify timolol maleate.2 Titrimetric Analysis (10) Timolol maleate may be determined by perchloric acid titration to a visual or potentiometric endpoint. The results of an elemental weight percent determination of carbon. hydrogen and oxygen are compared to the respective theoretical values of 49. drying it and filling with 0.17%.65% and 15. the electrode system should consist of an indicating electrode prepared by emptying a sleeve type calomel electrode.1 N LiClO in acetic anhydride. nitrogen.3. 5.

where : = Concentration of the sample solution = Concentration of the standard ‘std solution = Absorbance at 2 9 4 nm of the Asam sample solution = Absbrbance at 2 9 4 nm of the standard solution = Absorbance at 2 9 4 nm of the Ab drug free solution matrix 5. In the case of the flow injection technique assay.3.1 N HC1 (as) with the net absorbance of a standard in 0. The net absorbance is calculated by subtracting the drug free matrix contribution to absorbance at the wavelength of determination from the absorbance of the drug solution at the same wavelength.1 N HC1 (as) of known concentration.TIMOLOL MALEATE 67 1 Timolol maleate exhibits an ultraviolet absorption band near 2 9 4 nm attributed to the thiadiazole core structure. This absorption is the basis for the quantitative determination of the drug.2 Ultraviolet Spectrophotometry via Flow Injection Analysis Ultraviolet absorbance is also used as the detection mode for timolol maleate determinations by flow injection analysis (11). Assay of the compound is performed by comparing the net absorbance at 2 9 4 nm of a sample dissolved in 0. timolol maleate sample and .

Specific rotation. The resulting changes in UV absorbance at 294 nm of the stream are measured relative to the analyte-free stream.4 Chromatographic Analysis 5.7" and -12. LOPER 672 standard solutions are periodically injected into a flowing stream. calculated on the dried basis. Using a solution of = 50 mg/mL.3.1 Thin Layer Chromatography (10) Normal-phase thin layer chromatography on silica gel has been employed for timolol maleate.5'.4. A reference spot for impurity purposes applied as 10 pL of = 20 pg/mL solution of timolol maleate in methanol is also employed. R for timolol maleate in this &stem is = 0. Calculation of the concentration of timolol is made in an identical fashion as direct UV spectrophotometry. 5. is between -11.3 Specific Rotation The specific rotation of timolol maleate is determined in 0. in a 10 cm cell is determined with a suitable photoelectric polarimeter.7. 5. Detection is made under visible light after the developed and air-dried plate has been exposed .DAVID J. 25 "C. A developing solvent system consisting of chloroform/methanol/(28% ammonia water) [80/20/1 (v/v)l is used to develop a spot resulting from 10 pL of an approximately 5 mg/mL solution of the drug in methanol. MAZZO AND ALICE E. the angle of rotation at 405 nm.1 N HC1 (aq).

20 pL injections of a timolol maleate solution are made.6 mm i. Under these conditions.2 High Performance Liquid Chromatography (12) High performance liquid chromatography may be employed to analyze timolol maleate. 5. 6. Sensitivity is estimated to be within an order of magnitude of the amount of timolol maleate in the reference spot solution ( = 200 ng drug).Degradation Solid State Stability ( 1 3 ) Timolol maleate is an extremely stable compound at room temperature in the solid state. timolol is separated from its known potential process impurities.1 . For example.00 mL/min are used. A chromatographic system consisting of a column (300 mm x 4. In fact.d. prolonged exposure to extremes of temperature and humidity caused minimal degradation of the solid compound. Stability 6. timolol maleate stored for = 2 months at 105 O C in an air atmosphere (ambient relative humidity) showed no difference in potency by TLC chromatographic .613 TIMOLOL MALEATE to iodine vapor for 2 hours.005 M aqueous hexane sulfonic acid (pH adjusted to 1 with acetic acid).4. Detection is by UV absorbance at 280 nm. Typically.) packing with p-phenyl stationary phase (10 pm particle size) has been used with a mobile phase consisting of 1 part methanol and 2 parts 0. A column temperature of 30 'C and a flow rate of 2.

Solutions of timolol maleate prepared in each of 0. Timolol maleate exhibited surface discoloration when exposed in an open dish to intense UV light ( z 20 times normal sunlight). show any loss of potency by UV assay nor were any decomposition products detected by TLC.) Timolol maleate. Timolol maleate can be degraded in solution if exposed to elevated temperatures or intense ultraviolet radiation. MAZZO AND ALICE E. LOPER 614 profile when compared to a sample of unstressed solid. This sample did not. also exhibited a loss in potency of 25% (by W assay).1 N HC1 (aq) (pH = 1) and phosphate buffer (pH = 7 . 6. A solution prepared in distilled water and stored identically .2 Solution Stability ( 1 3 ) Timolol maleate is extremely stable in aqueous solutions (protected from light) with no decomposition being detected in injectable formulations stored at room temperature for = 5 years.DAVID J. however. Two degradates were isolated from this sample and were identified as the cis/trans isomer pair of timolol 0-fumarate ester and timolol 0-maleate ester (Figure 13). 2 ) and then stored in sealed glass vials for = 2 months at 105 OC yielded considerable amounts of degradation products. Only heating timolol maleate to above its melting point and keeping it molten for 10 minutes produced discoloration and an approximate 5% loss in potency. when heated at 95 O C for = 3 weeks in an air atmosphere (100% relative humidity). (TLC of this sample did detect small quantities of several unknown decomposition products. The solution decomposition of the drug is pH dependent with maximum stability occurring near pH = 4.

.CHCH.NHb-CH. 0 N \ S / N I 0 I c=o 1 CH3 I HC = CHCOOH cis and trans Figure 13.n OCH. Potential solid state degradates of timolol maleate exposed to 105 " C / l O O % relative humidity.

Aqueous solutions of timolol maleate (pH 5 ) have also been shown to be susceptible to degradation when exposed to intense ultraviolet radiation.2.DAVID J. Figure 14 depicts the proposed degradation mechanism and indicates the three modes of thermally induced solution degradation.5 ) under autoclave conditions indicated that timolol maleate degrades at the rate of approximately l%/hour at 120 OC.5-thiadiazole. Three decomposition products were isolated from solutions which were autoclave degraded. 7. Maximum blood plasma concentrations ranging from 10 ng/mL to 100 ng/mL are attained within 1 to 2. Biopharmaceutics and Metabolism 7.4 hours after either acute or .1 Absorption and Bioavailability Timolol maleate is rapidly and completely absorbed after oral administration (14).2. (1) rearrangement to isotimolol. LOPER 676 was found to be virtually the same as unstressed control solution. Evidence of the three thermal degradation products mentioned above as well as several unidentified degradation products was found in this solution. it is recommended that aqueous timolol maleate solutions be stored protected from light. and (3) oxidation followed by ether cleavage to form 4-hydroxy-3-morpholino-l. MAZZO AND ALICE E. Because of the potential for light-induced degradation. Studies of timolol aqueous solutions (pH .5thiadiazole 1-oxide. Exposure for 2 hours to U V light equivalent to = 20 times normal sunlight produced approximately 2% loss in potency. (2) ether cleavage to form 4-hydroxy-3morpholino-1.

Proposed aqueous solution degradation mechanism for timolol maleate in solutions exposed to autoclave conditions. n IIOCH. I I N\ / CH.NHC-CH.OH I o=N'rrr(opH Y SY lsotimolol FH3 CH~NHC-CH. I "\_/". N S Timolol n CH.2.5-thiadiazole Figure 14. OH CH.CHCH. .. I 'OH 0 4-hydroxy-3-morpholino1.2.5-thiadiazole l-oxide 4-hydroxy-3-morpholino1. I n oWNllfoH N N I ' 'S CH.

The effect of food on the rate and extent of oral absorption of timolol maleate is not significant (19) Timolol maleate administered directly to the eye as an ophthalmic drop appears to be rapidly absorbed. Only 20% of the timolol . dose of 72% of the I4C label is excreted within 84 hours with 66% in the urine and 6% in the feces.221. 7.5 mg to 20 mg of timolol maleate twice daily (15-16). producing a decrease in intraocular pressure within three hours (20). The bioavailability of oral timolol is reported to be 61% to 75% of a reference intravenous dose (17-18).timolol maleate to man. producing peak blood plasma levels of no more than 2 to 5 ng/mL at 0.DAVID J. Supporting studies in rabbits indicate that timolol reaches peak levels in most tissues of the eye at ten minutes after ocular administration with blood serum concentrations peaking at 30 to 60 minutes (23). Bioavailability of less than 100% is attributed to first-pass metabolic extraction by the liver after oral administration rather than to incomplete gastrointestinal absorption (14).5 hours after instillation of 2 drops of 0.2 Metabolism After orf4 administration of a 4 mg C. MAZZO AND ALICE E. LOPER 678 chronic administration of 2.5% timolol maleate ophthalmic solution in each eye (21).5 hours to 1. Timolol free base is rapidly absorbed through intact skin (24). Some small but statistically significant cardiovascular effects have been reported after ophthalmic administration (20. Systemic absorption of timolol also occurs after ocular administration.

Over 80% of the blood plasma radioactivity represents timolol metabolites (14). Oxidation of the basic oxypropanolamine side chain of orally administered timolol maleate results in two other minor urinary metabolites in man.l-dimethylethyl)aminol-2-hydroxypropoxy}-1.C H ~ .thiadiazol-3-yl}-N-(2-hydroxyethyl) glycine.C ( C H ~ ) ~ N N bH ’S‘ Metabolite I1 l-(l. respectively.N H .2.timolol maleate produces two major urinary metabolites in man (25).5-thiadiazol-3-ylloxy}-2-propanol.5.N H . Metabolites I11 and IV represent 6% and 3 % . Oxidation and hydrolytic cleavage of !be morpholine ring of an oral dose of C.~ O--C HC -HC H ~ . HO-CH2.l-dimethylethylamino)-3-{[4-(2-hydroxyethylamino)-1.TIMOLOL MALEATE 679 dose is recovered unchanged from the urine.251. H O .CH HOOC-CH< \ S/ * 0-CH -CH-CH2-NH-C(CH3)3 bH Metabolite I N-{{4-[3-(l.C H ~ .2. . of administered radioactivity (26). Metabolite I represents approximately 30% of the total urinary radioactivity while Metabolite I1 accounts for 10% (14.

Other metabolites in rodents result from hydroxylation and oxidation of either the morpholino ring or the oxypropanolamine side chain (25). I1 and I11 shows only Metabolite I1 to have significant 6-adrenergic blocking activity. activity was only one-seventh that of timolol maleate in anesthetized dogs (26). These four metabolites are also produced by other species.5-thiadiazol-3-yl}oxy]propanoic acid H-CH2NH-C-CH2-OH O w N - I \ / CH3 S Metabolite IV l-~[(1.2. LOPER 680 .3 Pharmacokinetics Oral administration of timolol maleate in doses ranging from 5 mg to 20 mg .1-dimethyl-2-hydroxy)-ethyllamino}-3-{[4(4-morpholinyl)-1. N\s/ N OH Metabolite I11 2-hydroxy-3-[{4-(4-morpholinyl)-l. Metabolite I11 represents the major metabolic pathway for timolol maleate in the dog. However.5-thiadiazol-3-yl]oxy}-2propanol. 7. MAZZO AND ALICE E. Biological testing of synthetic samples of Metabolites I.DAVID J.2..

6 mL/minute ( 2 7 ) . Half-life is independent of the administered dose. 3. 1 hours. The mean apparent half-life of elimination of unmetabolized drug from plasma is approximately 2 . Timolol maleate appears to follow simple linear kinetics over the dosage range studied. Determination in Biological Matrices Timolol maleate may be determined in blood plasma and urine by isolation and . Reported half-lives for timolol maleate elimination from plasma. area under the plasma concentration versus time curve is proportional to the administered dose ( 1 5 ) . although plasma half-lives as long as five hours have been reported in normal volunteers ( 2 7 ) . 1 7 1 .161. 8. plasma levels decrease biexponentially with time ( 2 8 ) . Following intravenous administration.3 to 4 . are comparable to the oral half-life. Fifty percent of maximum inhibition of exercise-induced tachycardia is produced by timolol maleate plasma concentrations of 3 to 4 ng/mL. 5 hours ( 1 5 .TIMOLOL MALEATE 681 results in peak plasma concentrations of timolol maleate within one to two hours. The mean renal clearance of unchanged timolol maleate in normal volunteers was reported as 6 9 . followed by an exponential decline of plasma concentrations with time ( 1 5 ) . Maximal suppression of exercise-induced tachycardia. a measure of @-blockade. occurs when timolol maleate plasma levels reach 27 to 30 ng/mL. Pharmacokinetic parameters do not change upon one week chronic administration of oral timolol maleate ( 1 5 ) . The pharmacodynamics of oral timolol maleate have been well characterized (15.

The Ni electron-capture detector was operated in the pulse mode of voltage with a pulse interval of 5 0 microseconds. respectively.64 cm column packed with 1% OV-17 on 80-100 mesh Gas Chrom Q. The diheptafluorobutyryl derivatives are recovered in n-heptane. the carrier gas. 185 "C and 300 "C.63re maintained at 75 mL per minute. oven and detector temperatures of 250 OC. LOPER 682 derivatization followed by gas-liquid chromatography with electron capture detection (29). which is evaporated at 60 "C under a stream of nitrogen to concentrate the sample. After evaporation of the methylene chloride. the acidic phase alkalinized. respectively. Timolol and the internal standard. The chromatograph is operated isothermally with injection port. A capillary column gas-liquid chromatographic-mass spectrometric assay monitoring for timolol with maleate and C-timolol maleate in blood plasma is reported to have a sensitivity of 0.DAVID J.6 and 6. This method provides the capability to examine administration of drug and its stable isotope-labeled counterpart simultaneously for comparison of two different routes of administration.1 mL of alkalinized urine into 4% isoamyl alcohol in heptane. Limits of detection are 2 ng/mL in plasma and 20 ng/mL in urine. desmethyltimolol. The compounds are then back-extracted into 0. The selectefiion . are extracted from either 1.1 mL of heptaflurobutyrylimidazole-ethyl acetate reagent and immersion in a boiling water bath for one hour.83 m X 0. 1 to 5 p 1 of sample is chromatographed on a 1. and 10% methane in argon.5 ng/mL (30).1 N hydrochloric acid. Helium. and the compounds extracted into methylene chloride.0 mL of alkalinized plasma or 0. timolol maleate and the internal standard are derivatized by addition of 0. MAZZO AND ALICE E. Retention times of derivatized timolol maleate and internal standard are 7. the purge gas.0 minutes. Typically.

25 mm capillary column coated with SE-30. One to two ~ J Lare injected onto a 16 m X 0. containing H-timolol maleate as an internal standard.5 to 1. through a cartridge containing an octadecyl-silica bonded reversed-phase packing. Back extraction from cyclohexane-toluene into an aqueous phase followed by extraction into methylene chloride precedes evaporation and derivatization with bis(trimethylsily1)-trifluoroacetamidepyridine.TIMOLOL MALEATE 683 isolation procedure prior to assay consists of passing 9.5 mL of plasma. 89 and 95. Selected ion monitoring at m/e 86. base peaks corresponqing to the side chains f f timolol maleate.6 m i. ~ Other gas-liquid chromatographic methods employing mass fragmentographic detection (31) or nitrogen-selective flame ionization detection (18) have been reported. The splitless injection port temperature is 260 "C and oven temperature is 225 " C . Carrier gas (helium) velocity is 30 cm/second. The cartridge is flushed and the compounds are then eluted with acetonitrile0.05 N hydrochloric acid.8 mamp and electron multiplier setting of 1900 V. A 25 cm X 4. High performance liquid chromatography with electrochemical detection may also be employed for analysis of timolol maleate in human blood plasma and breast milk (32). The mass spectrometer is operated in the electron-impact ionization mode at an ionizing potential of 70 eV. C-timolol maleate and H-timolol maleate.2 M sodium dihydrogen phosphate :88% orthophosphoric acid :water (500:200:3:297) pumped at a flow .d. allows construction of a standard curve from the ratios Intensity86/Intensity95 and Intensitygg/ Intensityg5. column packed with PXS 5/25 Partisil ODS 3 (5 pm) is used with a mobile phase of methanol :0. respectively. a filament emission current of 0. which is subsequently alkalinized.

Timolol maleate and the internal standard. For plasma or serum. are extracted from biological matrices with diethyl ether.2 V for maximum sensitivity.1 Dissolution Testing The dissolution of timolol maleate tablets is evaluated based on methodology and apparatus described in USP XXI(33). 9. propranolol for plasma or serum. a hexane wash of the organic phase is sufficient to prepare the acidic aqueous phase for injection of 100 pL into the chromatographic column. pindolol for breast milk. LOPER 684 rate of 1. Calibration curves are linear from the 2 ng/mL limit of detection up to 100 ng/mL. After introduction of the tablet.5 "C and basket speed is 100 rpm. Timolol maleate and internal standard are once again extracted into dilute orthophosphoric acid and the hexane wash and assay are performed as for plasma. filtered aliquots of the dissolution medium are withdrawn for assay for timolol maleate content against appropriate standards. The wavelength for detection is 295 nm. Determination in Pharmaceuticals 9. The acidic dissolution medium ( 5 0 0 mL of 0.1 N hydrochloric acid) is maintained at 37 5 0. the acidic aqueous phase must be alkalinized and the compounds back-extracted into ether. MAZZO AND ALICE E. A glassy carbon working electrode is employed with a silver/silver chloride reference electrode and a platinum auxillary electrode. The applied potential for oxidative electrochemical detection is +1. A mobile phase consisting of pH 2.8 phosphate . Compounds are extracted from the organic phase into dilute orthophosphoric acid. For breast milk.DAVID J.0 mL per minute. The official assay is a high performance liquid chromatographic method developed for a system with an ultraviolet detector.

Tablets are prepared for assay by weighing and finely powdering not less than twenty tablets. About 15 p 1 of dissolution sample is injected onto the column. Section 9.TIMOLOL MALEATE 685 buffer: methanol (3:2) is pumped at a rate of 1. An accurately weighed portion of powder is dispersed in 0. Water is then added and the dispersion is shaken for ten minutes. ultraviolet spectrophotometry via flow injection analysis and high performance liquid chromatography). After dilution to volume with water.9 mm column packed with octadecylbonded silica.8 mL/minute through a 30 cm X 3.2 Potency Testing 9.1 Assay (33) Official compendial methods are available for both timolol maleate tablets and ophthalmic solution. The supernatant liquid may be assayed with appropriate standards with the chromatographic system outlined above (Dissolution. as outlined in the previous sections (direct ultraviolet spectrophotometry. Alternative assay methods.2. Acetonitrile is added followed by an additional five minutes of sonication. 9.1) or by an alternative assay if an official cornpendial method is .05 M monobasic sodium phosphate and sonicated for five minutes. can be used if an official cornpendial method is not required. Not less than 80% of the labeled amount of timolol maleate is dissolved in 20 minutes. the sample may be centrifuged to remove dispersed solids from the timolol maleate solution.

The aqueous phase in each separator is then transferred to a second set of separators for an additional toluene extraction.686 DAVID J. The sulfuric acid extracts are diluted to volume with additional acid solution. The extracts are assayed against a 0. after extraction. Additional pH 9. Timolol in the combined toluene solutions is extracted into four portions of 0.7 buffer is added to the toluene remaining in the first set of separators and. MAZZO AND ALICE E. The official compendia1 assay for timolol maleate opthalmic solution requires that an aliquot of the solution be diluted to volume with water to produce a 0. The timolol maleate ophthalmic solution must . the aqueous phase is transferred to the second set of separators.1 N sulfuric acid blank at 2 9 4 nm with an ultraviolet spectrophotometer. the aqueous phase is discarded and the toluene from both sets of separators is combined.7 carbonate buffer. LOPER not required. Timolol is extracted into toluene from the alkaline aqueous phase. After shaking.5 mg per mL timolol maleate solution.0 percent and not more than 110. Tablets must contain not less than 90. Five mL each of this solution and reference standard solutions are transferred to separators and made basic with pH 9. The aqueous phase in the second set of separators is then discarded.0 percent of the labeled amount of timolol maleate.1 N sulfuric acid.

assayed individually must exhibit 85. Typically sample preparation involves the disintegration and dissolution of single tablets in solvents identical to those used for assay. A set of extractions and other sample preparation steps identical to those described in the potency assay ( 9 .0 percent of label claim range ( 3 4 ) . not more than one may be outside the 85. all as previously described. ultraviolet spectrophotometry via flow injection analysis and reversedphase high performance liquid chromatography with ultraviolet detection.0 percent range. 9.0 percent. Ten tablets.0 to 115. an additional twenty tablets must be tested. If one unit is outside the label claim range or the relative standard deviation is greater than 6.0 percent of the label claim and have a relative standard deviation less than or equal to 6. 2 . Of the thirty tested.2. are acceptable techniques for uniformity determinations. or both.2 Dosage Uniformity ( 3 3 ) Tablets must also meet official compendia1 standards for uniformity of dosage units.0 to 115. 1 ) are then employed.0 percent nor more than 110.0 percent. . Direct ultraviolet spectrophotometry. 0 to 125.0 percent of label claim. and that must be within the 7 5 .TIMOLOL MALEATE 687 contain not less than 90.

timolol maleate tablets carry a five year expiration dating.3 Stability Testing ( 3 3 ) Timolol maleate is tested for stability using the high performance liquid chromatographic method described in the USP XXI.2.DAVID J.LOPER 688 9. MAZZO AND ALICE E. . Based upon data generated in accelerated and room temperature stability studies.

S.A. R. 4) J. Williams.. 10th edition. Inc. personal communication. PA. Medical Economics Company. Med. 1983. U. Inc. Department of Analytical Research. Publisher. 9) E.TIMOLOL MALEATE 689 References Physicians Desk Reference. 1972 and 1972. A. Department of Pharmaceutical Research and Development. U.S.S. 1987. 3655663 and 3657237 (to Merck and Co.A.A. personal communication. United States of America Patents 3619370. H. West Point. Department of Pharmaceutical Research and Development. U. personal communication.. U.. U. Bondi. 41st edition.A. Barnhart.. B. W. monograph no. Merck and Company. respectively.A.A.. Oberholtzer. Merck Sharp and Dohme Research Laboratories. L. PA.. 1971. M. 6) The Merck Index. Yates.S. editor. 9284. Merck Sharp and Dohme Research Laboratories. personal communication. Edward R. McCauley. Pitzenberger and J. Chem 15(6). C. Department of Medicinal Chemistry.A. The United States Pharmacopeia.S. 21st revision.. Rahway. U. pp. and Joseph V. 1985. S. NJ. Gibson. R. Murphy. Weinstock. 1249-1251. PA. NJ. Merck Sharp and Dohme Research Laboratories. 1353... J.S. Ryan. Martha Windholtz. Rahway. S.S. pg. K. Oradell. NJ. Wasson. NJ. pg. 651-655 (1972). West Point. Stuart. W. J. Rahway.. Mack Publishing Company. PA. S.). Easton. U. West Point. 7. K. Merck Sharp and Dohme Research Laboratories.S.A. 1340-1342. U. M. . H.

16 1 R. F. Huber and R. R. MAZZO AND ALICE E. I. Pharmacol. S. Pharmacol. Vandenheuvel. Koponen-and U. W. West Point. K. Roman. Tsianco. Lowenthal. Eidelson. 24. 19 1 R. 227 (1983). H. P. A. Clin. Ferguson. G. 14. Demetriades and W. Clin. 3. West Point. Br. Tobert. D. W. 676 (1982). 1063-1064. personal communication. 21st revision.. G. TOCCO. J. P. 32. G. U. Else. 12 1 P.A. B. A. Nykanen. F. Disp. T. P. 1985. P. Huber. Brenner. Holmes. 20 1 M. I. U. Acta 181. Hagerman.A. Mannisto. Pharmacol. Hucker. Bigley.B. Pharmacol. EuE. Eur. Korner. A. I. Gruber and W. Mack Publishing Company. Merck Sharp and Dohme Research Laboratories. B. Cook and G. PA. Tway. Department of Pharmaceutical Research and Development. Pharmacol. 13 R. H. Anal. P. L. J. Ashley and P. Druq Metab. Jennings. J. P.A. R. J. 18 0. 16. Clin. E. Ther 27. DeLuna. 241-244 (1986). Edwards. H. 361 ( 1975 1. J. J. Merck Sharp and Dohme Research Laboratories. . 719 (1982). Johnson. Wilson. Abrams. Department of Analytical Research.. Davies.S. 14 I D. personal communication. V. T. 1 51 A.DAVID J. A. 111 F. J. 14. B. Vlasses. Firor. B. Mantyla. Affrime. 431 (1978). Lamminsivu. M C. (19801. J. PA.'Pharmacol. U. Bobik. B. Easton. Chim. 243 (1979). Koplin.. W. J. Holmes. G. pp.S. L. Clin. Onesti.A. R. Sorensen and I. Clin. Shirk. S. PA. 0. Duncan. 471 Eur. E. Clin. Grob. 17 1 T. W. Ther. J.S. F. and G. LOPER 690 10 1 The United States Pharmacopeia.

D. Share. V. K. 6 4 . 23 C. TOCCO. Duncan. Med. D. 5 4 7 ( 1 9 8 0 ) . 24 1 P. K. Smith. Pitone. 2 3 6 (1980). E. S. 1 8 7 9 ( 1 9 7 5 ) . Kim. J. M. Clementi. Vedin. LeDouarec. W. N. T. Clin. Widmark. K. L. Vandenheuvel. 5. Shirk. H. Ferguson. F. Lotti and J. Loper. Pharmacokin. Vandenheuvel. J. J. Affrime. E. A. Weber. R. Wasson. J. TOCCO. Lefebvre. Pharm. A. 9 5 ( 1 9 8 0 ) . H. J. 43 ( 1 9 8 2 1.TIMOLOL MALEATE 2 11 G. Clin. M. E. L. G. J. A. K. Schmitt. TOCCO. 30 J. 27 1 D. 3 5 . Disp. Ann. Ther.'J. 25 1 D. Arison. J. Busby. Pharm. H. 28 1 J. 1 9 7 ( 1 9 8 6 ) . C. C. E. M. L. 22 C. Sci. Seideman. Walker. B. J. B. R. B. 6 0 6 ( 1 9 7 8 ) . J. A.A. Ellsworth and D. Med. 26 1 B. W. 0. Ophthalmol. A. Pharmacol. A. Ther. Int. 2 3 . Vandenheuvel. Hall. W. 8. V. J. A. 5 7 3 (1981). Scheigetz. W. J. 0. J. Walker and W. M. Ferguson and W. T. K. Fourtillan. 1111 ( 1 9 8 0 ) . Pharmacol. H. Drug Metab. T. Sci. N. R. Girault and P. Pharmacol. Vlasses. Leier. DeLuna. J. DeLuna and A. J. Courtois. Hichens. A. 2 7 9 (1984). 1 1 7 8 (1980). D. Hensens. V. 2 3 . B. E. Ribeiro. Rotmensch. Arch. 2 3 . B. J. 29 1 D. Kristianson and C. N. Calissendorff. Davies. R. Bondi. Sci. R. Alvan. Wilhelmsson. Swartz and G. Pharm. Onesti. 6 9 . Chem. C. W. A. 69 1 . J. J. Clin. J. W. P. A. J. Widmark and G. J. F. R. Duncan. Nancarrow. R. Clin. R. 104. A. P. Lowenthal. Baker and P. 9 8 . Carlin. Rooney. 70. Abrams and R. 31 J.

U. 21st revision. pp. Jack. 1063-1064. 34) Ibid. J . Gregg and D.A. Mack Publishing Company.DAVID J. 1277-1278. 244 (1984). 305.S. Chromatog. B. MAZZO AND ALICE E. 33) The United States Pharmacopeia. R. . 1985. pp. Easton. PA.LOPER 692 32) M..

3.2. Registry Numbers Molecular weight Elemental Composition Appearance.1.2. Verbeeck College of Pharmacy University of Saskatchewan Saskatoon. .3. 3.4. Melting Range Solubility Stability and Storage Dissociation Constant Crystal structure Differential Scanning Calorimetry Spectral Properties 3.3.7.7.4. Fluorescence Spectrophotometry 3.1. 2.2.3.1.2.7. 3. Introduction 1. 2. Empirical 2. Nomenclature 2.5. Therapeutic Category 2. Description 2. Color and Odor Patent Information Phvsico-Chemical ProDerties 3.1.6. 2.5.3.3.2. Ultraviolet Spectrum 3. Structural 2.J.1.1. Infrared Spectrum 3.7.2. 3. All rights of reproductionin any form reserved. 3.1. Proprietary Names Formulae 2. 2.TRAZODONE HYDROCHLORIDE DeMiS K. Nuclear Magnetic Resonance (NMR) Spectra ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 16 693 Copyright 0 1987 by Academic Press.2. 2. Chemical Name 2. Saskatchewan.2. 3.2. Generic Name 2. History 1. 3. Canada 1.6.7. Inc. Gorecki and Roger K.1.1.4.

GORECKI AND ROGER K. Chemical Ionization (CI) 4.2.2.2. High Performance Liquid Chromatography (HPLC) 7. Proton Magnetic Resonance (PMR) Spectra 3.2.3. Carbon-13 Nuclear Magnetic Resonance Spectrum (~%-NMEX) 3.1. Elimination 6. Pharmacokinetics 5. References .2. Thin Layer Chromatography (TLC) 6. Synthesis 5. G.1. Methods of Analysis 6. Chromatographic Analysis 6. VERBEECK 694 3.2. Absorption 5.1.7.5. Distribution 5.DENNIS K. Identification 6.4.5. Electron Impact (EI) 3.2.4. Gas-Liquid Chromatography (GLC) 6.7.5.3.1. Mass Spectrometry 3.1.7.2.7.7.

therefore. Description 2. 1.1.3). The molecule consists of a triazolopyridine ring and a phenyl piperazinylalkyl moiety. considered particularly useful in the geriatric population (5).TRAZODONE HYDROCHLORIDE 695 1.2.4-triazolo-[4. The history. and therefore. Introduction 1. Chemical Name 2-{3-[4-(3-chlorophenyl)-l-piperazinyll propyl)-1. In addition. Therapeutic Category Trazodone hydrochloride is an antidepressant drug indicated in the symptomatic treatment of moderate to severe depression.1.3-a]pyridin-3(2H)-one monohydrochloride .2. History Trazodone hydrochloride was originally synthesized in 1966 by Palazzo and Silvestrini (1) in the chemical laboratories of the firm Francesco Angelini.1. pharmacology and clinical data on trazbdone hydrochloride is well documented in a number of review articles and monographs ( 3 .1. It is the first triazolopyri -dine derivative to be used clinically. Its development was a result of an organized approach rather than fortuitous discovery and was based on the hypothesis that the mechanisms responsible for physical and mental pain are the same (2. Its major advantages include a low incidence of anticholinergic and cardiovascular side effects along with minimal stimulatory effects upon dopamine and norepinephrine receptors and is. an analytical profile has been described (9). Nomenclature 2. 2.8 ) . represents a new chemical class of antidepressant drugs.

Proprietary Names Desyrel. 17. N. odorless crystals (plates)with a bitter taste. Formulae 2. Thombran.1. Pat. 2.2. C1 17. Molecular Weight 408.696 DENNIS K. Color and W o r White. 3.1.33 2. Patent Information U.68%.92% 2. G.S. Pragmazone. Appearance.2. 55. Registry Numbers Chemical abstracts. 1. Elemental Composition C.068 (11).5.6. Structural 2.89%. VERBEECK 2. GORECKI AND ROGER K. Molipaxin. Empirical C19H22C1N50 HC1 2.2.117.2. .3. Trazodone:19794-93-5 Trazodone hydrochloride:25332-39-2 2. Trittico 2.4.381.3.1. Pat. 0. HI 5.15%.36%. Brit. Generic Name Trazodone hydrochloride 2.3. 3. Manegan.2.009 (1968 to Angelini Francesco)(l).2.

Melting Range The melting point for trazodone free base is 96OC (9).1O.345 mg m. Under vacuum decomposihion does not occur and a melting range of 231-232.14 (9).4. 3. structure of trazodone hydrochloride (58). is 6.. 691 Physico-Chemical Properties 3.866[31. Bond lengths and angles are tabulated in Tables 1 and 2.5 C is reported (9).230[21. light resistant gontainers and protected from temperatures in excess of 40 C (13). 3. b. X-ray diffraction data were obtained using an Oak Ridge diffractometer employing molybdenum Kolradiation with a niobium filter. The megsured and calculated densities are 1. Trazodone hydrochloride. sparingly soluble in water. Solubility Trazodone hydrochloride is soluble in chloroform.respectively for Z=4.crystals are monoclinic with space group182?3c a8d with cell dimension8 a=16.3. 3. .5. ethanol and methanol (10) and insoluble in common organic solvents (9). =93. C H C1N 0-HC1. Stability and Storage Trazodone hydrochloride tablets should be stored at room temperature in tight. A.66[2lr c=11. Dissociation Constant The reported pKa for trazodone. 3.1. Crystal Structure The crystal and molecular structure for trazodone hydrochloride has been determined (14).04(1) and Z=4 with the final R factor of 0.344 and 1. in 50% ethanol. Figure 1 depicts the 3-dimensional.2. This value was obtained potentiometrically using a glass-calomel electrode.TRAZODONE HYDROCHLORIDE 3.042 for 3017 reflections. X-ray derived. The hydrochboride salt melts with decomposition in the range 222-228 C (grlOr1l).

X-Ray Derived. Structure of Trazodone Hydrochloride. Three-Dimensional. .Figure 1.

451 1.463 Distances C(17)-C(18) C(18)-N(13) N(16)-C(19) C(19)-C(20) C(20)-C(21) C(21)-C(22) C(22)-C(23) C(23)-C(24) C(24)-C(19) C(22)-C1(2) 0 A - 1.419 1.491 1.363 1.337 1.378 1.308 1.394 1.391 1.383 1.386 1.222 1.344 1.180 1.506 1. 0 Intramolecular Bond Lengths ( A ) of Trazodone Hydrochloride 0 0 A - D i s tances A - 1.Table 1.495 1.512 1.518 1.052 2.421 1.497 1.407 1.872 3.455 1.384 1.349 1.394 N(2)-C(10) C( lO)-C(ll) C(ll)-C(12) C( 12)-N( 13) N( 13)-H(13) N( 13)-C1(1) H( 13)-C1(1) N( 13)-C( 14) C (14)-C ( 15) C (15)-N( 16) N(16)-C(17) 1.747 .498 0.384 1.380 1.

Table 2. Bond Angles ( " ) of Trazodone Hydrochloride Angles 0 (1) -C (1)-N(l) 0 (1)-C( 1)-N(8) C(l)-N(2)-C(lO) C (l)-N( 2)-N( 3) C ( 1)-N (8) -C ( 9) N(2)-C(l)-N(8) C( 10)-N( 2)-N(3) N( 2)-N( 3)-C(9) N(3)-C(9)-C(4) N (3) -C (9)-N (8) C(9) -C(4) -c(5) C(4)-C(5)-C(6) C (5)-C( 6)-C ( 7 ) C(6)-C(7)-N(8) ("> 130 128 125 114 108 103 121 104 130 112 119 122 121 118 Angles C(7)-N(8)-C(9) N(8)-C(g)-C(4) C(l)-N(8)-C(7) N(2)-C(lO)-C(11) C( lO)-C(ll)-C( 12) C(ll)-C(l2)-N(13) C(12)-N(13)-C(14) C(12)-N(13)-H(13) N (13) -H (13)-C1(1) N (13)-C (14)-C( 15) C ( 14)-C ( 15)-N (16) C(15)-N(16)-C(17) N(16)-C(17)-C(18) C(17)-C(18)-N(13) (") 123 118 129 113 112 113 109 114 172 112 111 111 111 111 Angles C( 18)-N( 13) -C ( 1 4 ) C(17)-N(16)-C(19) C(15)-N(16)-C(19) N( 16)-C (19)-C(20) N(16)-C(19)-C(24) C(19)-C(20)-C(21) c(20)-c (211-c(221 C(21)-C(22)-C(23) C(22)-C(23)-C(24) C( 23)-c(24)-c( 19) c (24) -c (19) -c (20) Cl(2) -C( 23)-C(22) C1(2)-C(23)-C(24) 108 118 117 121 121 120 122 117 118 120 118 119 118 .

100 11. 278 and 312 nm while that in water displays maxima at 211. The latter are in agreement in previously reported data (9.TRAZODONE HYDROCHLORIDE 70 1 This study revealed a number of interesting features about trazodone which may be useful in proposing structural requirements for neuroleptic activity.7. Data obtained using a Perkin Elmer DSC 1B instrument was used as a purity index for trazodone. Spectral Properties 3. the triazolopyridine nucleus (including C(10)) and the chlorophenyl moiety are planar groups . 1 cm) and molar absorptivity values for trazodone hydrochloride in water are s m r i z e d in Table 3 (9).6. The piperazine ring is in the normal chair conformation with “13) extending upward and N(16) down. Ultraviolet Characteristics of Trazodone Hydrochloride max (m) 211 246 274 312 A( 106. 256.101.730 3.840 . Table 3. In addition. Ultraviolet Spectrum The ultraviolet spectrum of trazodone hydrochloride was scanned from 200 to 360 nm with a Gilford Response spectrophotometer at a concentration of 30 and 75!~moles/Lin methanol and 30 Umoles/L water (Figure 2).1.lcm) 1225 287 94 94 E 50.040 3. Torsiogal angles about N(2)-C(10)(93. 3. 3. 246. The bond lengths and angles about N(13) resemble that of a protonated2 mine and those associated with N(16) demonstrate sp character due to the attached chlorophEny1 ring. The reported A (l%. 275 and 312 nm. The spectra in methanol show maxima at 213. Differential Scanning Calorimetry A differential scanning calorimetry (DSC) curve for trazodone hydrochloride has been reported (9).2 ) suggest that the propylene side chain is fully extended.2 1 C(ll)-C(12) (178. Each spectrum is characterized by two well defined and two less prominent peaks.7.

---. Ultraviolet Spectra of Trazodone Hydrochloride (-.A 10 Figure 2 .. . methanol. water).

4-triazolo-[4.7. Other characteristic bands appe r at 1540. 3.13 MHz. 3. COSY and heteronuclear correlation experiments.7. S=Strong.3-al pyridin-3 ( 2H)-ones . 703 Fluorescence Spectrophotometry Trazodone hydrochloride.2. The spectrum was obtained with a Beckman AccuLab 4 infrared spectrophotometer from a compressed KBr disc.7. Nuclear Magnetic Resonance (NMR) Spectra 3. 1447. Spectral assignments were confirmed by homodecoupling. like most 2-alkyl-substituted-l. 1350.4.TRAZODONE HYDROCHLORIDE 3.2. W=Weak . 1491. Structural assignments of some of the characteristic absorption bands are presented in Table 4. .2460 1705 1640 1595 W M S M S Aliphatic CH +N-H stretching C=O stretching triazoline C=N stretch aromatic C=C stretch M=Medium.3.7. shows an intense fluorescence-when irradiated with ultraviolet light. 943 and 745 cm-'i Principle peaks. Table 4.1. Infrared Characteristics of Trazodone Hydrochloride Frequency (cm-' ) Intensity Assignment 2975-2840 2535. wavenumbers and structural assignments are in agreement with previously published results (9.4. 1273.lO). Proton Magnetic Resonance (PMR) Spectra The PMR spectra of trazodone base and trazodone hydrochloride were recorded on a Bruker AM 300 NMR spectrometer employing a frequency of 300. The excitation and emission wavelengths for trazodone are 317 and 455 nm respectively (9). Infrared Spectrum The infrared spectrum of trazodone hydrochloride is shown in Figure 3.

I 8 9 I 1 1200 10 1 1000 11 I 12 1 800 14 16 10 600 .1 okooo 3 4 I I 3000 I MICRONS 5 I I I 2000 I I 1600 I800 WAVENUMBER Figure 3. 6 7 I 1400 CM Infrared Spectrum of Trazodone Hydrochloride-KBr Disc.

8 ppm is illustrated in Figure 5. Chemical shifts and assignments are given in Table 6.76 6.75 7.11 6. These data are consistent with the results reported using a 60 MHz instrument ( 9 ) . Scale expansion $n the region 6.46 4.TRAZODONE HYDROCHLORIDE 705 The spectrum of trazodone base obtained in CDCl is presented in Figure 4. .13 2. Scale expansion in the region 6.49 2.5 to 8. The chemical shifts.09 3.56 2.4 to 7.05 d m m m m t t t t m 1 3 1 2 1 2 4 4 2 1 1 314. multiplicities and assignments are compiled in Table 5.0 ppm is depicted in Figure 7. m=multiplet The PMR spectrum of trazodone hydrochloride in DMSO-d is shown in Figure 6. Proton Magnetic Resonance Assignments for Trazodone Base 3 i /y\ /-7 9 *\ 1 12 10 13 5 6 11 U Chemical Number Shift of 6 (ppm) Multiplicities Protons Assignment 7. Table 5.6 8 517 2 9 13 12 11 10 d=doublet. t=triplet.84 6. It can be seen that protonation of the piperzine ring nitrogen nearest the propyl chain affects the chemical shift and splitting pattern of adjacent protons in the piperazine ring.

0 5.5 3.0 Figure 4 .0 .0 6.5 4.5 5. LL 2.0 3.5 7.0 P r o t o n Magnetic Resonance Spectrum of Trazodone Base i n CDC13.5 6.0 PPM 4. 7.5 2.UCJ I " " I " " I ~ " ' " ' 1 " ' " " ' ' " I ' " ' I " " I ' " ' I ' ' ~ ' I " " I ' ' " 8.

6 I 6.6 I 6.8 Figure 5.0 PPM I 6.r 1 - 7.4-7.4 Proton Magnetic Resonance Spectrum o f Trazodone Base in CDCl with Scale 3 Expansion (6. ' I 7.6 I I 7.4 I 7. .8 ppm).2 I 7.

0 1 I I 1 6.I " " " " ' I 8.5 I 1 1 1 1 ' 7.5 1 1 " [ ' I 6.0 PPM 1 1 1 4.0 1 1 1 ~ I 3. 7.0 Figure 6.0 1 l 1 1 ~ 2.5 I I I ~I 3. ] 1 1 1 .0 1 1 I l 5.0 Proton Magnetic Resonance Spectrum of Trazodone Hydrochloride in DMSO-d6.5 " l 1 ' I 5.5 ' 1 I I 1 4.5 1 1 1 1 2.

6 Proton Magnetic Resonance Spectrum of Trazodone Hydrochloride in DMSO-d 6 with Scale Expansion (6.5 I 7.6 I 7.7 I 7.6 I 6.9 Figure 7.1 I 7.3 I PPM 7.9 I 6.5-8.2 I 7.r I 7.7 I -6. I 7.0 ppm).8 1 7.0 I 6. .4 I 7.

Spectral assignments were confirmed by C-13m-1 heteronuclear correlation experiments and published chemical shift data.7. Proton Magnetic Resonance Assignments for Trazodone Hydrochloride Chemical Number Shift of 6 (ppm) Multiplicities Protons Assignment 7.2.13 12. t-triplet m=multiplet 3.87 7.25 7. dd=doublet.64 4. and assignments are outlined in Table 7.95 6.47 MHz with broad band proton decoupling. .14.4. Carbon-13 Nuclear Magnetic Resonance Spectrum (IjC-NMR) The 13C-NMR spectrum of trazodone hydrochloride base obtained in DMSO-d6 is given in Figure 8.25 m m m dd dd m t d d m m 1 3 1 1 1 1 2 2 2 6 2 1 31416 8 5 7 2 9 1 2 .710 DENNIS K.15 2.doublet.13 11. VERBEECK Table 6. G. The spectrum was obtained at ambient temperature on a Bruker AM 300 NMR spectrometer at 75.86 3. GORECKI AND ROGER K.04 6.53 3.15 10 d=doublet.01 3.86 6. Chemical shifts.

' l ' ~ 140 ' l ' 120 . ' l ' 20 Carbon-13 Nuclear Magnetic Resonance Spectrum of Trazodone Hydrochloride in DMSO-d 6' . ' " . ' 60 . ' . ' l ' 100 ~ ' .d 1 ' ~ 160 Figure 8. PPM ' 80 . ' l 40 ' .

51 130. .2% and a base peak at 205.5.84 130.5.06 133.23 44.88 141.12 3.47 123.s or 9 8 3 13 17 16 15 14 Mass Spectrometry 3. . Electron Impact (EI) An electron impact mass spectrum of trazodone hydrochloride is presented in Figure 9.69 147.78 Assignment 4. The fragmentation pattern leading to the various ions is outlined below.712 DENNIS K.92 113. This was obtained by direct solid insertion probe at an electron eneljgy of 70 eV and a source temperature of 180 C using a VG Analytical MM 16F single focussing mass spectrometer linked to a VG 2035 data system+ The spectrum shows a molecular ion peak M at a mass/charge (m/z) ratio of 371 with a relative intensity of 13. VERBEECK Table 7. s or 9 4. Prominent diagnostic ions are observed at m/z 70.5 or 9 2 10 12 Chemical Shift 6 (ppm) 114.82 119. 176.1. Carbon-13 Nuclear Mametic _.54 22.7.99 110.66 42.79 50. 231 and 278.76 52. 166. G. Assignment 6 1 7 11 4 .~~ Resonance Assignments forTrazodone Hydrochloride Chemical Shift 6 (ppm) 150.03 115. 136. GORECKI AND ROGER K.7. 209.

16.2.18). This spectrum was also determined on a VG Analytical MM 16F mass spectrometer. however.TRAZODONE HYDROCHLORIDE 713 20:91J 231 Mass spectra of trazodone and other related compounds have been reported elsewhere (9. Alternatively. Subsequent treatment with N-(3-chlorophenyl)piperazine gives trazodone. The original synthesis reported by Palazzo and Silvestrini (1) consisted of the condensation of l-(3-chloropheny1)-4-(3-~hloropropyl)-piperazine with the sodium salt of I.2.17. 3.15. The more important methods are outlined in Scheme 1. 4. The spectrum shows a pseudo molecular ion at a mass to charge ratio of 372. One method involves the condensation of the usual triazopyridine (I) with bromochloropropane to yield the 2-(3-~hloropropyl)-derivative (11). More recently. This product is then chlorinated with thionyl chloride and subsequently reacted with 3-chloroaniline to yield trazodone. .5. All of the reported routes employ 1.4-triazo-[4.20).19. Chemical Ionization (CI) The chemical ionization spectrum of trazodone hydrochloride is shown in Figure 10. Other related patented methods were simultaneously developed in the same laboratories (11). using ammonia as a reagent gas.7. Synthesis A number of synthetic pathways have been described for the synthesis of trazodone hydrochloride (1.11. modications of existing pathways were reported (19). I is alkylated with bromochloropropane and treated with diethanolamine to form the di-alcohol (111).3-a]-pyridin3(2H)-one (I) or 2-substituted derivatives as stzrting matFria1.

1s I 231 I 1 1 Figure 10. Mass Spectrum of Trazodone HydrochlorideChemical Ionization.GORECKI AND ROGER K.714 DENNIS K. 375 . Mass Spectrum of Trazodone Hydrochloride Electron Impact. G. VERBEECK m 100- 80 > c - H cn -6 c 60 - z - W > 0 100 50 1-50 250 200 300 350 MASSKHARGE RAT I0 Figure 9.

HC1 TRAZODONE c1 HYDROCHLORIDE Synthesis of trazodone hydrochloride 0 Scheme 1.Cl-(CH ) -N 2 3 w CH2 CH20H c1 I CH2CH 20H HC1 I1 . .

Excretion of trazodone in human breast milk has been studied and found to be very small ( 3 2 ) .Olvg/mL respectively ( 2 9 ) . exposure to babies via breast milk would be minimal. Distribution Trazodone is a weak base with a pKa of 6. Following oral ingestion of a single 100 mg trazodone hydrochloride dose average m a x i m plasma concentrations of 1. Little information is available in man concerning its distribution in organs and body fluids.27).61 Pg/mt and 1.5 to 1. G .25.14. GORECKI AND ROGER K.0.311. trazodone is rapidly absorbed. exists mostly in the unionized form at physiological pH.0 pg/mL (21. therefore. Pharmacokinetics 5.14 (91. The average milk/plasma ratio was 0. Absorption Following oral administration to man. 5. and therefore. Peak plasma concentrations following oral administration of a single 50 mg dose range from 0.66 ug/mL were reported for a capsule and liquid formulation respectively ( 2 2 ) . Ingestion of food delays the absorption of trazodone from the gastrointestinal tract but does not seem to affect its bioavailability (23.0 hours in fasting subjects (21-26).28).24.5 to 4. 5. 0. The time to reach m a x i m plasma concentrations typically varies from 0. In vitro protein binding studies with human plasma have shown that trazodone is 96.716 DENNIS K. Trazodone is highly bound to plasma proteins. 98 and 97% bound at concentrations of 1.2. VERBEECK The synthesis of 14-C-labelled trazodone hydrochloride has also been published ( 2 0 ) . Oral and intramuscular bioavailability of trazodone is almost complete as shown by the virtually identical plasma concentration-time profiles obtained following oral intramuscular and intravenous administration to healthy volunteers ( 2 3 ) .24-26). Studies in laboratory animals show that trazodone readily crosses the blood brain barrier but is minimally transferred across the placenta (30. The drug distributes in a relatively large volume: Vd values reported in the literature or calculated from published data range approximately from 1-2 L/kg for healthy volunteers (21. .1 and O.22.1. This procedure also employs the classical condensation reaction described in the original patent. Studies in rats and rabbits have shown that food delays the oral absorption by decreasing the transfer rate of drug from the stomach to the duodenum ( 2 3 ) .

Whether these small concentrations contribute to the overall effects of trazodone remains unclear. 11) while N-oxidation occurs on a piperazine nitrogen (N-oxide.25.28. 111).271.32).24.22. involving biliary excretion. When treated with Liebermanns Test reagent at 100 C (sodium nitrite-sulphuric acid) trazodone gives a transient violet color whereas with Mandelin's reagent (ammonium vanadate-sulphuric acid) a grey color develops which later changes to violet (10). Methods of Analysis 6.3-a]-pyridin-2-yl propionic acid ( IV) and 1-m-chlorophenylpiperazine ( V ).34).32).1. most as metabolites. approximately 1% of 'frazodone plasma concentrations. I) and in the triazolopyridine ring (dihydrodiol. Approximately 70-75% of an oral dose of the drug is excreted in urine within 72 hours of administration. A possible diurnal variation in clearance may be responsible.3. N-oxidation and hydrolysis (Scheme 2) (34).2. to 400 mL/min. Total body clearance (assuming an oral bioavailability of 100%) ranges from 150 mL/min. Results from animal studies indicate that trazodone does not induce its own metabolites (38). Small concentrations of 1-m-chlorophenylpiperazine. plasma concentrations of trazodone in man decline in a biphasic manner with a mean half-life of approximately 4 hours during the period 3-10 hours following dosing and a terminal elimination half-life of 6-12 hours (21. have been reported in man (37). (21. Hydroxylation occurs in the benzene ring (4-hydroxyphenyl metabolite. Patients treated with trazodone on a twice daily dosage schedule show significantly higher plasma levels in the morning as compared to the levels at night (33).TRAZODONE HYDROCHLORIDE 717 5. Only less than 1% of the dose is recovered in urine as unchanged drug (26.361. Trazodone is extensively metabolized via hydroxylation. Identification Trazodone hydrochloride reacts with certain reagents to produce characteristic colors which may be useful for its identificahion. . a pharmacologTcally active metabolite ( 35. mainly as trazodone metabolites (23. A hydrolytic reaction results in the formation of 3-0~0-1. 6.4-triazo10[4. The remainder of an oral dose is excreted in feces. Elimination In general.

Biotransformation pathways of trazodone in man ./ QOH OH Cl 0 TRAZODONE \ GLUCURONIDE IV 0 I GLUCURONIDE Scheme 2.

System I1 (10) Absorbent: Silica gel G. Thin-Layer Chromatography ( T L C ) A number of thin-layer chromatographic systems have been developed for the determination of trazodone. sulphuric acid with glacial acetic acid. rhodanine.TRAZODONE HYDROCHLORIDE 719 An infrared spectroscopic method is described which is useful for the rapid emergency identification of certain psychtropic drugs including trazodone (39). . Chromatographic Analysis 6. ammonium sulphocyanide and cobaltous chloride or potassium dichromate.25 mm treated with potassium hydroxide in methanol. A variety of detection methods have been used and most systems use silica gel plates as absorbent. A summary of some of the reported TLC methods is given below.41.5) ii) cyclohexane.42)and quantitation (43. ninhydrin.40. These systems have been employed for the identification (9. System I ( 9 ) Absorbent: Merck Kieselgel-0. 6. ethano1:NH (35:5) Detection: ultra-viol& light (366 MI). 0. Infrared spectra are determined as compressed potassium bromide discs from dried chloroform extracts.25 mm Mobile Phase: i) cyclohexane-benzene-diethylamine (5:4:1) ii) chloroform-methanol-isopropanol-ether-NH (60:25:25:2) i ii ) n-butanol :wate? :NH ( 20:10:2 1 iv) abs.2.1.toluene:diethyl3 amine (75:15:10) Detection: ultra-violet light.2.10. Dragendorff's reagent or acidified iodoplatinate solution.45) of the drug both as pure drug substance as well as in biological fluids and tissues. The method employs chloroform extraction of patient's urine at different pH's and utilizes the infrared finger print region and spectra of authentic standards to confirm identity. Mobile Phase: i) methano1:NH (100:1.44.

47). G . biopharmaceutics and pharmacokinetic studies (16. 0. 0. iodoplatinate solution followed by Dragendorff's reagent 6.DENNIS K. Detectors employed include flame ionization (FID).5) ii) cyclohexane. Gas-Liquid Chromatography (GLC) A number of GLC procedures have been published for the determination of trazodone.S) ii) ethy1acetate:methanol:amonia ( 85:10:5) Detection: 5% sulphuric acid.46) Chromatography of trazodone is readily accomplished without derivatization using a variety of stationary phases in packed or capillary columns. 0. as well as in therapeutic and overdose stiuations.25 nun Mobile Phase: i) methano1:ammonia (100:l.25 mm Mobile Phase: i) methano1:NH (100:1. The conditions for the reported GLC methods are as follows: .28.to1uene:diethyl3 amine (75:15:10) iii) ch1oroform:methanol (9:l) iv) acetone Detection: Dragendorff's reagent System IV ( 4 2 ) Absorbent: Silica gel 60. nitrogen-phosphorous (NPD) and mass spectrometry (MS).2. F254. GORECKI AND ROGER K.25 nun Mobile Phase: i) ethylacetate:methanol:30% NH3 (85:10:5) ii) cyc1ohexane:toluene:diethylamine (65:25:10) iii) ethy1acetate:chloroform (50:50) iv) acetone Detection: 10% sulphuric acid followed by Dragendorff's reagent and acidified iodoplatinate solution System V ( 4 5 ) Absorbent: Silica gel. VERBEECK 720 System 111 (40) Absorbent: Silica gel.10. F254.2. The technique has been used for routine detection of the drug (9.17).(45.19.

240 C. Retention time: 3. D .5% SE-30 on chromosorb G 80-100 Carrier gas: nitrogen. Detector: F I D (or NPD) In addition: i) Column: 1. hold 5 min. presumably F I D System I1 (10) Type of sample: general Column: 2 m x 4 mm I . retention index . injector 32OoC Retention time: approximately 3. &3 lb/inch 2 Temperature: column 268 C. DB-1 fused-silica capillary (0. hold 7 min.8 m column with 3% SP 2250 on Supegcoport 100-120 Temperape: 10 C/min. injector.5 min. Temperature: column.10 min. 33 mL/min. glass packed with 2.8 m column with 3% SP 2100 on Supegcoport 100-120 Temperape: 13 C/min from 160-320 C. 270 C Retention time: 2 min. from 140-300 C. Detector: not stated.25wm film) Carrier gas: hydrogen. Detector: F I D and MS System Iv ( 4 5 ) Type of sample: biological fluids and tissue Column: 4 m x 0. 265 C Retention time: 2.5% OV-17 on Chromosorb W HP 80-100 Carrier gas: Nitrogen. Retention time: 3. D . Temperature: column. Tevrature: column.89 min. Detector: NPD (MS) .10 Retention index: 3250 Detector: F I D or NPD System 111 (16) Type of sample: plasma ( rat) Column: 80 cm x 4 mm I . D .87 min.D. 48 cwsec. glass column packed with 1% OV-1 on Chromosorb Q 100-120 Carrier gas: nitrogen. glass column packed with 1.25 mm I. Detector: NJ?D (MS) ii) Column: 1.TRAZODONE HYDROCHLORIDE 72 1 System I (9) Type of sample: drug substance Column: 6 ft x 1/4" I . 45 mt/min.

GORECKI AND ROGER K.D.2 min. to 255 CA injector. Temperature: 270 C Retention time: 4.. 30 ml/min. Others include .43. most of which have been employed in analysis of biological fluids and tissues for unchanged drug and metabolites. DB-1 fused-silica capillary (0. 260 C. Detector: NPD System VI (47) Type of sample: plasma.3. separator oven 255 C Retention time: 3 min.1 min. G . glass column packed with 3% OV-101 on Chromosorb W HP 80-100 Carrier gas: helium. 310 C. nickel capillary column packed with 20% UCC-W (Hewlett Packard) on Chromosorb W AW DMCS 80-100 Carrier gas: helium. Temperahure: column.D.25 mm I. 30 p/min Tevrature: column.54). 250 C.50-52. Detector: MS High Performance Liquid Chromatography 3HPLC ) Several HPLC methods have been developed for the determination of trazodone. 20 $pin. VERBEECK 722 System V (28) of sample: plasma Column: 1.25 pm film) Carrier gas: helium.23 m x 2 mm I.32.48. injector. 275OC Retention time: 5. The majority of the procedures utilize reverse-phase chromatography with mobile phases comprised of an aqueous buffer with organic solvent modifiers (21. detector. brain tissue (rat) Column: 1 m x 3 mm I. 270 C Detector: MS System VIII (18) 'rype of sample: plasma 6. Detector: NPD (MS) System V I I (17) Type of sample: urine (rat) Column: 60 cm x 1 mm I. injector. Teperature: c o l p . from 100-300 C. 7 p/min. 170 C for 1 gin.D. 15 C/min.2.DENNISK.D. "ype Column: 4 m x 0. glass column packed with 3% OV-1 on Gas Chrom Q 80-100 Carrier gas: nithogen. 2000cm/sec.

5): acetonitrile (39:47:14) Temperature: ambient Flow rate: 1.04 M triethylamine Temperature: ambient Flow rate: 1.6 mm trimethyl silyl (5urn) Mobile phase: acetonitri1e:phosphate buffer (pH 3.8 min.6): methanol: acetonitrile (47:37:16) Temperature: ambient Flow rate: 2.2 min. Retention time: 3. fluorescence and electrochemical oxidation.01M (pH 4.56. Detection: W at 211 mm System I11 (49) Type of sample: plasma and tissue Column: 250 x 4. Detection: UV at 206 nm System I1 (32) Type of sample: breast milk Column: 150 x 3.5 mL.1 mL/min. Retention time: 8 min. System I (48) Type of sample: serum or plasma Coluum: 150 x 4.5 mL/min.6 mm Bondapak C18 (10 V M ) Mobile phase: phosphate buffer 0.7): acetonitrhle (72:8) Temperature: 60 C Flow rate: 2. Detection: W at 214 nm .57) and normal-phase systems (53. Detection: W at 214 nm System Iv (50) Type of sample: plasma Column: 300 x 4. A summary of the reported HPLC systems is presented below.082 M (PH 6./min.551. Detector systems include ultra-violet ( w ) at various wavelengths.02 M heptane sulfonic acid and 0.0) (27:73) with 0.5 mL/min.6 mm Supelcosil LC-8 DB Mobile phase: phosphate buffer 0.9 mm Nova-Pak C18 (4p m ) Mobile phase: methano1:phosphate buffer 0.TRAZODONE HYDROCHLORIDE 723 reversed-phase with ion-pairing agents (49.05 M (pH 4. Retention time: 12. Retention time: 5 min.

0 (1:1) Temperature: ambient Flow rate: 2 mL/min. 6 mm LiChrosorb l o w 8 Mobile phase: acetonitrile: phosphate buffer..0 ml/min.5): acetonitrile (60:38:2) Detection: W at 250 nm System VIII (21) of sample: plasma Column: 250 x 4. retention time ( 1 0 : 3 . 0. 2 v . -I. 0. Detection: W at 242 nm System VII ( 4 3 ) Type of sample: serum and urine Column: ODs (C18) Mobile phase: methanol: 0. pH 6 . GORECKI AND ROGER K.25 min. pH 3.1 M (pH 7. 8 ) . 10 mM.0): acetgnitrile (10:3. 60 C. electrochemical. 8 min. 1.1 M (pH 3. Retention time: 3 .6%.5 mL/min. Detection: Uv at 254 nm System IX (53) Type of sample: drug substance Column: 125 mm Spherisorb S5W Silica "ype Mobile phase: methanolic ammonium perchlorate.0): acgtonitrile (10:3).8).05 M sulphuric acid ( 1 8 : l ) Temperature: ambient Flow rate: 2 mL/min. retention time. 6 mm Ultrasphere ODs (octadecylsilyl) 5 p m) Mobile phase: i) phosphate buffer.6 rmn Spherisorb S5 ODs Mobile phase: acetonitrile: 0. Relative retention time: 0 . ii) phosphate buffer 0. 3 1 Detection: W at 254 nm. 8 min. VERBEECK System V (51) Type of sample: serum Column: 250 x 4 . 1. 3.1 .011 M phosphate buffer (pH 7. 7 Temperature: ambient Flow rate: 2 mL/min. 65 C. Detection: W at 240 nm System VI ( 5 2 ) Type of sample: biological fluids Column: 250 x 4 . Retention tinre: 6 . 8 min.724 DENNIS K. 0 .

5 !nL/min. Retention time: 13 min.6 mm Ultrasphere octyl (5urn) Mobile phase: acetonitrile: water (50:50) with 0. Retention time: 7.7 min.TRAZODONE HYDROCHLORIDE 125 System X ( 5 4 ) Type of sample: plasma Columns: 250 x 4.O/min. Xex=320 nm.7): tetrahydrofuran: acetonitrile (90:5:5) Temperature: 45'~ Flow rate: 1.005 M of each of sodium heptane sulfonate and n-nonylamine Temperature: ambient Flow rate: 2. Detection: Fluorescence.5 min.05 M I pH 3.6 mm Bondapak C18 (10um) Mobile phase: phosphate buffer.15 V .2 mL/min.6 mm LC-1 (trimethysilyl) ( 5 urn) Mobile phase: phosphate buffer (0. Detection: W at 254 nm System XI (55) Type of sample: biological fluids Column: 125 x 5 mm Spherisorb S5W Silica ( 5 pm) Mobile phase: methanol with 0. 0. Detection: Fluorescence. 300 x 4. Aem=440 nm System XI11 (57) Type of sample: plasma Column: 250 x 4.0): acetonitrile (9O:lO) with 0.A ex=200nm System XI1 (56) Type of sample: plasma Column: 150 x 4.05% of each of tetramethylammmonium hydroxide and perchloric acid (70%) Temperature: ambient Flow rate: l.6 mm MC-18 with 20% carbon load (5 pm).2 M (pH 4.0 mL/min. Detection: electrochemical. +1. Retention time: 39 min.02% perchloric acid Temperature: ambient Flow rate: 2. Retention time: 3.

Ed. 2.M. Rahway.W. Silvestrini. London. Edizioni Luigi Pozzi. De Greqorio. A new approach to the therapy of depression. pp. L. 5. Rickels and 8. 10.N. B35.S. Amsterdam: Excerpta Medica. 11. Am. B. The Merck Index.52146W (1968). Karger. References 1. Gershon. in Pharmacological and Biochemical Properties of Drug Substances. Saarma and B. Ed.. 400 (1973). Evansville. .P. 13. S. Clarke's Isolation and Identification of Drugs. Ban and B. M. Brit. Life Sciences. Avery. GORECKI AND ROGER K. Patent No. S. MOrOZOV. Association.126 DENNIS K. Al-Yassiri. pp. Silvestrini. J. Eds. 9. Silvestrini.E. B.C.S. Silvestrini. Int.. 1-10. Lisciani and M. 14. Vol. 2449 (1981). Ed. 401 (1981). U. 1981. V. R.. Silvestrini. A.. J. A. 1986. G. 1980. B. 3. 1983 p. DC. M. Cioli. A. Brogden. Chiari. 94-119. 8. R. 10th edition. Speight and G. 498 (1979). 1. Trazodone in Modern Problems of Pharmacopsychiatry. N. Silvestrini. Trazodone: A New Broad Spectrum Antidepressant.A.A. Windholz. CA:69. Mead Johnson and Co. Drugs. Washington. Desyrel (Trazodone hydrochloride) Product Monograph.C. Eds. Silvestrini. Baiocchi. Heel..S. pp. Frigerio and P. 4. Trazodone. 1978. Merck and Co.117.67429 q (1968). Acta Cryst. U.. 587 (1968). Arzneim-Forsch.M. Roma. Indiana. . 3. 2. Goldberg.Vol 9. CA:69. G . 28. 12. S. 1370. Hawkinson. J. Palazzo and B. 23. Burberi and B. 1035-1036.381 ... 6. Fillers and S.. T.I. Eds. Ankier and R. 3. In Proceedings of "Depression and the Role of Trazodone in Antidepressant Therapy". 009. VERBEECK 7. Pharm.. Bridges. The Pharmaceutical Press.K. . R. G. M. Neuropharmacol.068.. Patent No. S. K. 1974. 7. 1. Ridolfi.J. Inc. Moffat. T. Catanese..

. 2084 (1976). G. E.E. 305. Farm. Koss and U. Pharmacol. Yamato and T. Gershon. GiaMangeli and L.I. 18. Sugiyama. F. 1984. J. pp. 27. 505 (1981). 22. J. 11-20... Clin. K. Bullen. 16. 20. R. Mackenthun and J. W.I. 11. D. Pharmacology. Pharmacol. .. 1980. Bayer. 9. 631 (1975). 8. R. B. 125 (1983). 26. R. Pantarotto.E. D. Koss and U. Boll. Russell. Z. A. M.I.I. 6.W. Boll. A. Arzneim-Forsch. Eds. 1978. A. 26. C. R.J. Xenobiotica.. Zopitar. A. 28.TRAZODONE HYDROCHLORIDE 727 15. C.. 112.. Frigerio. J. Compd. 152 (1981). Gammans. R. Yamato. as.. Ankier. 16. Morozov. 114. Bosch: Trazodone as an example-drug levels in blood and brain.W. 24. Chromatogr. Russell. Chromatogr. J.R. 30. Ankier and G. S. Saarma and 8.V. Shader. P.H. Br. J. Pathy and S.. K. J. Greenblatt and R. Kono. Bosch. Ward. F. S. Ankier. Clin. Javch. Excerpta Medica. J. G. 25.S. Chim. Br. T. 29. 17. 18. Covington 339. S. Roma.. 309 ( 1984 1. M. Belvedere. Amsterdam. Pharm. In "Trazodone--A New Borad-spectrum Antidepressant". Gammans. Clin. Gammans.W. Chromatogr.K. In Proceedings of "Depression and the Role of Trazodone in Antidepressant Therapy". L.E. Br. J.S.W.J. Edizioni Luigi Pozzi. Baiocchi and M.K. A.. and J.W. Hosp. Kimber. Silvestrini. Frigerio and C. 23. Secchi. Pharmacol. Baiocchi. Prox and A. Fujita. L.W. A. 206 (1975). Carpenter and C. Kerns. Takahashi and T. 28. 303 (1985). 295 (1976). Farm. - 19. Zimmer.. J. 371 (1983). Graham.J. Clin. Fujita. Harcus. A. Pfleger. Rickels and B. 113. Lab. S. 431 (1984). Personal communication. Abernethy.E. Maurer and K. 723 (1973). Chim.. Giannangeli. H. M. Silvestrini. 42 (1984).R.R. Rogers. Pharmacokinetics and metabolism of trazodone in different species. 21. Martin.

6. G. Psychopharmacol. 9". 43. Pharmacol. 367 (1986).A. 413 (1981). J. J. 538 (1984). Pharmacol. Eds. M. Clementi and S. Pala. CA:96. Psychopharmacol. Sci. P. Fong. 9. Musumarra. 12. Palazzo. F. Pharm. Giannangeli and G. J. Silvestrini. McKenna. M. 24. T. Zanini. 39. G . G. J. . Clementi and G . 22.. 8. Basel. 41 (1984). J. Smith. - 34. Frigerio.. Suzuki: Teratogenicity and placental transfer of trazodone. 308. Anderson and M. Pharm. S. Anal. 33. Pecknold. Verbeeck. 151 (1985). Musumarra. Romano.-Forsch. Putzolu. G. Anal. Ballabio. F. G. Caccia. Wilkins and G. Bull.C. A. Giovanni. 605 (1982). 38. M. 4. Romano.. Chromatogr. J. Boll. 126695k (1986).. Sansone. G. GORECKI AND ROGER K. 504 (1985). 33. Daldrup. Fujita. 35. Toxicol. In "Modern Problems of Pharmacopsychiatry.. Xenobiotica. Y. Ross and E. 45. DeCosmo. L. P. Giulietti. 40 (1976). S. SOC. Melzacka and J.H.R. Archuleta. Nat.N. 1974. Napoli. Scarlata. Pharmacol. S. A. J. . 42. S. Arzneim. 350.G. 217 (1984). W. Clin. Chem. Br. S. 93. 223 (1986). J.G. 44. Cirma. Resuscitation. T. Zanini and S. 125 (1981). Baxter. Ban and B. J. T. Pharmacol.H. C. R. Karger. Baiocchi. S. Samanin. M.. CA: 608. Vetulani. Fresenius Z. Garattini. 477 (1981). VERBEECK 31. G . Caccia. Vol. Bondoli. Clin.B.M. Schiano di Zenise and P.F. 37. S. Chromatoyr. 34. Scotto di Tella. J. 1699 (1974). 32. 41. Sabato and A.. L. Palazzo. Adamus. M. G... 36.29498m (1982). DiNunzio. Ricci... Susanto and P. A. Yamato.K. A. Pharm. C. Scarlata. Takahashi and T.G. Wold. L. G. 22. Michalke. 40. 37. S. Baiocchi. R.728 DENNIS K. M. Garattini and M. 313 (1974).

Y. J. Wennig Simultaneous determination of benzodiazepines and antidepressants in emergency toxicology by HPLC. Caldwell and R. Marzouk.H. Suckow. Chem. Pappalardo. Toxicol. B. 187 (1985). H.N. Res. 1983. 210. Br. 30. 19 (1975). J. M. 1984. J. Chromatogr.W. I. Flanagan. Lew. 442 (1985).A. 143-149. Psychiat. P. 374. J.G. Chan. 47. Amsterdam. R. 8. 342. . Chromatogr. In "Topics in Forensic and Analytical Toxicology". 55. 48. Chromatogr..J. Liquid Chromatogr. Mitchard and T. McKiMon and R. Schreck and R. Stewart and S. 20. 58. Betts.- 51.L. J. 56..M.A. R. Ed.A. S. Wong. R. 8. 6.. Devane. College Park. J.TRAZODONE HYDROCHLORIDE 729 46. 91 (1984).. .L. Liquid Chromatogr. Bell and K. J. Fanelli. 230 (1984)... Ohlson. 191 (1985). pp. Elsevir. A.M. Zanini. Anal.B. Caccia. 2195 (1983). 388 ( 1986 1. - 49. M. J. S. G. J.. Gerson. Flanagan. B.H.. Ballabio. Chromatogr. 57. S. Gupta and M. S. Computer Science Centre. S. 2. Miller and C. Clin. M. In "The XTAL system of crystallographic programs.. M. Guiso and M. R. Munday. J. Pharmacol.J. 1379 (1985).L. F..D. Hochmuth. Robert. R. Mais. 69 (1986). Clin. Chromatogr. Root and G. Kendal. R. Jain. 54. 323. 50. M.F. .J. J. I. 53. 311 (1981). Wong and N. 52. Hall.R.Y. Jane. 345.. University of Maryland. J. Waugh. Draz and N.

Delbaere. McKay for determining the mass spectra and to Dr. Peter Pavlakidis for performing the literature search and providing some spectral data. .DENNIS K. Acknowledgements The authors would like to express sincere appreciation to Mr. G. VERBEECK 730 8. W. Dr. S . Leek for determining and Dr. M. Sardessai. Special thanks are extended to Mrs. Dimmock for assistance in analyzing the nuclear magnetic resonance spectra. Quail and Mr.R. L. Loewen for interpretation of X-ray diffraction data and providing the three-dimensional structure of trazodone hydrochloride. Dr. Shuttleworth for typing this manuscript. to Dr. G . C. D. Reid and Dr. GORECKI AND ROGER K. to Mr. J. G.

G .ticcd ~ h m h h y C o l l e g e 0 6 P h m a c y . A .U. Mehhawi* and A .YOHIMBINE A .i. King Saud UVLivmLty.ul ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 16 73 1 Copyright 0 1987 by Academic Press.t 06 Humnaceu. P. Box 2 4 5 7 . All rights of reproduction in any form IeSeNed. . Al-Ua&** *vep&eitt 06 Phahmacagnany **'Depcmhen. Riyadh-] 1451 (Saudi Ahab. Inc.

2.4 8. Total Synthesis of Yohimbine 7.2.1 8.2 1. Natural Sources and Isolation 5.2 8. A. Pharmacology and Medicinal Uses 8. Description 1.2.2.2. Spectral Properties 4.4 Nomenclature Formulae Molecular Weight Elemental Composition 2.3 8.5 Acknowledgements References Gravimetric Methods Titrimetric Methods Spectrophotometric Methods Spectrofluorimetric Methods Chromatographic Methods . MEKKAWI AND A.3 1.2 Qualitative Analysis Quantitative Analysis 8. G . Physical Properties 3. Methods of Analysis 8.1 8.A. Biosynthesis of Yohimbine 6. AL-BADR 732 1.1 1.

( 4 ) ...2 . I t was known s i n c e a n c i e n t t i m e s . Aphrodine ( 3 .1.1.YOHIMBINE 133 From t h e h i s t o r i c a l p o i n t of view yohimbine i s t h e most i m p o r t a n t of t h e complex i n d o l e a l k a l o i d t y p e s . as an a p h r o d i s i a c by t h e West A f r i c a n n a t i v e s . 4 .1. -. 1.1 UVOl Empirical ‘2 l H 26°3N2 . 5 . (4). .. Formulae 1. It o c c u r s i n v a r i o u s t r o p i c a l trees such P a u s i n y s t a l i a yohimbe P i e r r e and r e l a t e d s p e i c e s . 4 ) . 6 ) .1 Nomenclature 1....: MT F6 ON&TTTTJ T Q UVOl &GH .1 Chemical Name 17a-Hydroxyyohimban-16a-carboxylic methyl e s t e r ( 3 . 1. 1. Corynine..3 Wiswesser Line N o t a t i o n a) Yohimbine b a s e : T F6 D5 C666 E M ONbTTTTJ T Q b) 1.. i n t h e c u r d e drug b a r k o r t h e impure form.2 acid G e n e r i c Names Quebrachine. Yohimbine D5 C666 E --h y d r o c h l o r i d e . Aspidosperma quebrachoblanco S c h l e c h t and as a minor a l k a l o i d i n some Rawaolfia The f i r s t i s o l a t i o n of t h e drug w a s r e p o r t e d by S p i e g e l i n 1896 (1) and Witkop (2) i n 1943 s u g g e s t e d its c o r r e c t constitution.2. Description 1.

3 d I OH Molecular Weight 354.39%. 6 . Physical Properties 2. A. Odour and Taste 2. chloroform and sparingly soluble in diethyl ether (4.90%.45 1.3 - 241'.16%. . freely soluble in ethanol.2 Colorless needles when crystallized from ethanol.A. Melting Point 240' 2. H 7.2 Structural 10 11 0 1. N 7. colorless or white in bulk.54%.4 Elemental Composition C 71. Taste is bitter. odourless ( 4 .01 mm (7). G .6). Sublimation Under vacuum the base sublimes at 159°/0.4 Solubility Almost insoluble in water. 7 ) . 2.2. MEKKAWI AND A. 0 13. 2.1 Color. AL-BADR 134 1.

p.p.1 N H2SO4 were scanned from 200 to 400 nm using Varian DMS 90 spectrophotometer and the following maxima and (E 1%.234OC) and the colorless methiodide plates from acetone have the m. 282 and 225 nm were recorded. [ a ] i O+ EtOH ( 9 ) .3 (H20) (7).N-diacetyl derivative melting point is 1 8 3 O C ( 7 .250OC.p. 8 ) .1 an) at 280 nm was 225.YOHIMBINE 2.302OC . 249 .6 Specific Rotations of Base and Salts Various specific rotations have been recorded for the base. followed by solidification and remelting at 278OC. the tarkarate hexahydrate m. . the hydrochloride 103. + 62. 1 cm) values were found: In methanol: 3 maxima at 289. 1 cdfound were : 210 at 289 nm. at 272 nm was 233 and at 221 nm was 1110. spectra of yohimbine in methanol and in 0. + 56 in in EtOH ( 7 ) .1 N H2S04 (6) were as follows: (E 1%. at 272 nm 208 and 220 nm 1064. the nitrate m. In the acidic solution a shift to lower wavelengths with some hyperchromic effect was observed (E 1%.p. 213OC.2' 107. 1 an) at 278 nm 204. 247 at 282 nm and 1079 at 225 nm.9' in pyridine. The melting point for the 0-acetate is 1 3 3 O C and the 0. 2. 233' . Previous reports in 0.p. 269-270°C. 3oOo . Figure 1 illustrates the UV spectrum behaviour for both the methanolic and the acidic solutions of yohimbine.1 Ultraviolet Spectrum The U. The (E 1%. + 3. Spectral Properties 3. The thiocyanate is of rectangular crystals (m.V.5 735 Salts and Their Melting Point The salts are well crystalline and the hydrochloride is colorless plates m.

736 A. AL-BADR . MEKKAWI AND A. A. G .

741. 1025. 1160 o r 1197 o r 1436 cm-1. 3.3. 5 . 6 ) . 750 and 702 cm-’.stretch CH - 2900) 2960) 1730 stretch C = 0 . 1000.2 737 I n f r a r e d Spectrum The i n f r a r e d spectrum of yohimbine h y d r o c h l o r i d e i s p r e s e n t e d i n F i g u r e 2.3 Nuclear Mangetic Reonance S p e c t r a (NMR) 3. I R d a t a of yohimbine h y d r o c h l o r i d e have a l s o been r e p o r t e d (3. I t shows t h e following : -1 Frequency (cm ) Assignments 3540 OH . The spectrum i s o b t a i n e d from K B r d i s c on a Perkin-Elmer I n f r a r e d Spectrophotometer model 580 B. 1150. 970.aromatic 1460) 1472)) 1448) Other c h a r a c t e r i s t i c a b s o r p t i o n bands are : 1172.1 P r o t o n Nuclear Magnetic Resonance (PMR) Spectra The PMR spectrum of yohimbine h y d r o c h l o r i d e i n DMSO-db and i n DMSO-d6 p l u s D20 were shown i n F i g u r e s 3a and 3b r e s p e c t i v e l y .stretch (free) 3190 NH . The s p e c t r a were r e c o r d e d on a Varian T60 A NMR s p e c t r o m e t e r u s i n g t e t r a m e t h y l s i l a n e . C l a r k e ( 6 ) r e p o r t e d t h e p r i n c i p l a l peaks a t 1705.stretch C = C .YOHIMBINE 3.

3 4 7 6 5 8 9 10 11 12 13 1461qm 90 * 80- * 90 * 80 * 20 70. r-’ 0. 38003500 3ooo 2500 2000 Wavenumber -10 1800 1600 1400 1200 Figure 2 : Infrared spectrum of yohimbine. Fac 10.Wavelength urn 100. 1000 800 0 625 . HBr disc.

139 YOHIMBINE . O 8. .d ~using TMS as reference standard. I .0 6. .. . . - I - - 1 300 80 7. 200 . .0 7. .0 0 Figure 3a: Proton magnetic resonance spectrum of yohimbine hydrochloride in D M s 0 .O 30 2.0 60 WPPM(~)L. . '7 I . ' . . 1500 - I 400 ' I .O 30 2.0 S.0 I0 0 Pigurc 3b: Proton magnetic resonance spectrum of yohimbinc hydrochloride in D M s 0 .0 1. . .OPpM(r)L. . . . I . . .d ~ PIUS @ o using TMS a5 reference standard. . . .

The carbon assignments are based on chemical shift values and off-resonance spectrum and are listed below: 0 I OH . The structrual assignments as follows: Chemical shift (PPMJ Assignments Multiplets at 0.4 Carbon-13 Nuclear Magnetic Resonance Spectrum (C-13 NMR) The proton-decoupled and off-resonance carbon-13 NMR spectrum of yohimbine have been determined on Jeol FX 100 MHz spectrometer at an ambient temperature and are presented in Figures 4 and 5 respectively.A. 3. Singlet at 10.4 Aromatic protons Singlet at 4.05 NH (exchangable with D20).7-7.60 CH2 and CH protons Singlet at 3. AL-BADR 740 (TMS) as reference standard.60 CH3 protons Multiplet at 6. The sample was dissolved in deuterated chloroform and DMSO-dC. A.65 OH (exchangable with D 2 0 ) . G . Mills et a1 (3) have reported the proton NMR spectrum of yohimbine. MEKKAWI AND A. using TMS as reference standard.7-4.

TMS 0 Figure 5 : Off.decoupled carbon -13 NMR spectrum of yohimbine in CDCI3 and D M s 0 .resonance carbon-13NMR spectrum of yohimbin inCDCi3 and DMSO-ds using TMS a reference standard. .d ~using TMS as reference standard.(OHIMBINE 741 sil TMS u Figure4: Proton.

3 Triplet c2 1 174.0 Doublet c12 136.5 Doublet c11 111.8 Doublet 2‘ 0 61.MEKKAWI AND A.0 Singlet ‘8 117.A.4 Doublet ‘16 66. 0.2 Doublet c3 52.7 Triplet ‘14 36.2 Triplet ‘19 39.6 Quartet 2 ‘ 3 .8 S i n g 1e t c22 51.9 S i n gl e t c7 127. AL-BADR 142 Chemical s h i f t ( 6 ) Multiplicity Assignment 135.1 S i n g1e t ‘13 34.9 Triplet ‘18 23.5 Doublet clo 120.8 Doublet ‘17 31.5 Doublet ‘15 52.6 Triplet ‘5 21. A.1 Singlet c2 70.7 Triplet 6 ‘ 106.6 Doublet ‘9 118.

N a t u r a l Sources and I s o l a t i o n Yohimbine i s t h e major a l k a l o i d of t h e bark of P a u s i n y s t a l i a yohimba (Syn.YOHIMBINE 3. working on Rauwolfia s p e c i e s . benzene Le H i r -e t a1 (8) a l s o used o r chloroform (14. v o m i t o r i a ( l 3 ) . l O ) .15). The C I spectrum ( F i g . Other m a s s spectrum d a t a h a s a l s o been r e p o r t e d (3) * 4. Although p r e p a r a t i v e HPLC w a s n o t used i n t h e r o u t i n e i s o l a t i o n of such compounds b u t t h e t e c h n i q u e might be of r o u t i n e u s e i n t h e f u t u r e . 6 ) shows a b a s e peak a t m / e 353 and a n o t h e r peak a t m/e 354 (&) of almost e q u a l abundance ( 9 8 % ) . Corynanthe yohimbe) and a l s o p r e s e n t i n t h e r e l a t e d s p e c i e s p. t h e yohimbine t a r t a r a t e c r y s t a l l i z e d immediately while a-yohimbine t a r t a r a t e remained i n s o l u t i o n and c r y s t a l l i z e d l a t e r .t a r t a r i c a c i d . where yohimbine i n a m i x t u r e w i t h t h e o t h e r isomers (a-yohimbine) w a s b o i l e d w i t h d .used p r e p a r a t i v e t h i n .l a y e r chromatography as a r o u t i n e t e c h n i q u e f o r t h e i s o l a t i o n and c h a r a c t e r i s a t i o n of i n d o l e a l k a l o i d s . 7) shows a b a s e peak a t m / e 355 (M+ + 1 ) . s e r p e n t i n a ) ( 4 . m a c r o c e r a s . . Other fragments are a t m / e 337 and a t m / e 323. R. Court and h i s s t u d e n t s (16-ZO). The i s o l a t i o n of yohimbine from t h e b a r k of s p e c i e s c o n t a i n i n g i t as t h e major a l k a l o i d can be done by t h e method of L e H i r -e t a1 ( 8 ) . ( f . g . The drug powder i s t r e a t e d w i t h Na2C03. 4 . d r i e d and e x t r a c t e d w i t h a s u i t a b l e o r g a n i c s o l v e n t e. Rubiaceae) ( 7 . &.g. m / e 169 and a t m / e 156. The E I spectrum (Fig. P.5 143 Mass S p e c t r a The e l e c t r o n impact ( E I ) m a s s spectrum a t 70 e V r e c o r d e d on Varian Mat 311 mass s p e c t r o m e t e r and t h e chemical i o n i s a t i o n (CI) mass spectrum o b t a i n e d w i t h F i n n i g a n 4000 mass s p e c t r o m e t e r are shown i n F i g u r e s 6 and 7 r e s p e c t i v e l y . t r i l l e s i i ( f . p a n i c u l a t a and 2. c a f f r a ( l l ) . o t h e r major fragments a r e a t m / e 184. The e x t r a c t i o n of t h e a l k a l o i d s from t h e i r n a t u r a l s o u r c e s i s g e n e r a l l y c a r r i e d o u t by t h e c o n v e n t i o n a l method. &. Apocynaceae). t e t r a p h y l l a (12) and &. column chromatographic s e p a r a t i o n . 8 ) . S e v e r a l Rauwolfia s p e c i e s w e r e r e p o r t e d t o c o n t a i n yohimbine ( e .

MEKKAWI AND A.0- 50.A. 100.0- 50. A.0169 467' 100.0- 18912. 504 383 337 323 M E 300 320 I 340 360 380 400 420 440 Figure7 : Chemical ionization (CI )mass spectrum of yohimbine . G. 100. AL-BADR 744 -9904.

4. The tryptamide [lo] is then converted to the cis-diol [ll] using solution of osmium tetraoxide in tetrahydrofuran added to a solution of amide [lo] in pyridineTHF and cooled at dry-ice-acetone temperature.8. This aldehyde can then isomerize to [8] which cyclizes to yohimbine. and results in the formation of strictosidine [3]. Hydrogenation of the keto-diol [ll] over Adams' catalyst in ethanol gave a good yield of the desired triol [12]. subsequent hydrolysis to the glycidic acid [5] and then final decarboxylation to the ketoaldehyde [ 6 ] .4-dione [2]. p-Benzophenone [l] was condensed with lY4-butadiene to give the adduct 1. Hydrolysis of [16] by means of aqueous hydrochloric . Intermediate [12] was converted to the dialdehyde [13] by cleavage of the triol amide [12] in aqueous acetone and then was cyclized -in situ using phosphoric acid and heating at 70°.5.9.1O-cis-hexahydronaphthalene1. Scheme 1 illustrates how yohimbine is biosynthesized from tryptamine and secologanin. The ketoaldehyde [6] was oxidized to the corresponding ketoacid [7] using silver oxide in the presence of alkali.21-dehydrocorynantheine aldehyde [7] by D-ring closure.YOHIMBINE 5. The keto acid [7] was then converted to its acid chloride [8] and the latter was reacted with tryptamine [9] in the presence of pyridine yielding tryptamide [lo]. The lactol [14] was then transformed into the lactollactam methyl ether [15] by means of p-toluenesulfonic acid catalyzed methanolysis. 6. Lithium aluminium hydride reduction of [15] in THF afforded the lactol ether base [16]. A second glycosidase enzyme converts strictosidine via a series of reactive intermediates [4]-[6] to 4. 745 Biosynthesis of Yohimbine The major features of the probable biosynthetic pathway to yohimbine were summarized by Atta-ur-Rahman and Basha (21). where intermediate [14] was obtained. Selective reduction by zinc metal and acetic acid gives the octahydronaphthalenedione [3] which was converted to glycidic acid ester [4]. The condensation of tryptamine [l] with secologanin [2] is catalysed by the enzyme strictosidine synthetase which controls the C-ring closure mechanism. Total Synthesis of Yohimbine The following total synthesis of yohimbine was described by Van Tamelen et a1 (22).

O c-0-cs3 11 0 + H20 . [21 r 1 I + + HH 22 00 ' 1 I L -Glucose CB oc CH3-0-C 3 \ U II OH CH30-C II Scheme 1: - Biosynthesis of Yohimbine. A.A. AL-BADR 746 . *v .MEKKAWI AND A. G.glucose Glu. I OH .

160' Silver oxide/alkali H O H O [61 [51 Oxalyl c h l o r i d e 0 HZ . ) i n pyridine 0 Scheme 2: [91 T o t a l S y n t h e s i s of Yohimbine.Condn. ~ .YOHIMBINE -@ 141 Zn-AcOH Diels-Alder t --. -- 0 0 111 [21 COOEt Aqueous NaOH ( h o t ) under nitrogen Ethyl chloroacetate K t-butoxide/benzene H O [41 [31 COOH (1) Cu powder i n d i e t h y l g l y c o l a t e 155 .

AL-BADR 748 I H HL=O OH H OH H LAH i n THF OCH3 OH [I61 Scheme 2: [I71 Continued. G . A. . MEKKAWI AND A.A.

[211 * .295' OCOCH3 [ 191 Metaperiodate Cleavage o = c 'OCHO H Chromic anhydride at Oo/H2 SO4 Scheme 2 : Continued.YOHIMBINE 749 - 285 [ 181 .

MEKKAWI AND A.Chromatography ii. G . AL-BADR 750 i.L-camphorsulphonic acid CH3-O-C 'OH ' H \ 0 Scheme 2.A. A. 1 - . [ 23 Continued.

L e c r u b i e r e t a1 (29) i n v e s t i g a t e d t h e p o s s i b l e u s e of yohimbine t o improve t h e c o n d i t i o n of o r t h o s t a t i c hypotension induced by clomipramine. t o improve p a r a t h e s i a of t h e lower limbs i n 4 p a t i e n t s o u t of 6 d i a b e t i c p a t i e n t s .5 e q u i v . The r e s o l u t i o n of t h e s y n t h e t i c b a s e was accomplished by means of l-camphor s u l p h o n i c a c i d where pseudoyohimbine e p i m i e r i z e s t o yohimbine[23].0-3.3 3 ) . G e n e r a l CNS e x c i t a t i o n i n c l u d i n g e l e v a t e d blood p r e s s u r e .p e r i o d a t e . Pharmacology and Medicinal Uses Yohimbine produces a c o m p e t i t i v e a . S t e e r s e t a1 (31) . i n c r e a s e d motor a c t i v i t y .YOHIMBINE 75 1 a c i d gave t h e l a c t o l b a s e 1171. These i n c l u d e v a s o p r e s s i n release and hence m a n i f e s t s an a n t i d i u r e t i c e f f e c t . Morales e t a1 (30) r e p o r t e d t h e a b i l i t y of 6 mg of yohimbine. The' o l i f i n i c bond i n t h e c y c l i c e n o l e t h e r system w a s o x i d i z e d u s i n g osmium t e t r a o x i d e . The c r u d e p e r i o d a t e was d i s s o l v e d i n methanola c e t o n e and t r e a t e d a t Oo w i t h chromic a n h y d r i d e (2. g i v e n 3 times a day. where t h e e n o l e t h e r [19] have been formed. 7. A t high doses i t a l s o blocks postsynaptic a-adrenergic receptors.p y r i d i n e . The a c e t a t e s a l t [18] w a s subjected t o pyrolysis c o n d i t i o n s . The ozmylation w a s c a r r i e d i n t h e e n o l e t h e r a t . The l a t t e r w a s t h e n c o n v e r t e d t o t h e l a c t o l acetate [ 1 8 ] ( a s acetate s a l t ) by t r e a t m e n t w i t h a c e t i c a n h y d r i d e . a s t h e a c e t a t e s a l t . It a l s o b l o c k s 5-HT r e c e p t o r s . where t h e d i o l [ 2 0 ] was o b t a i n e d . It h a s a l i t t l e e f f e c t on smooth muscle and r e a d i l y p e n e t r a t e s t h e c e n t r a l n e r v o u s system and produces a complex p a t t e r n of r e s p o n s e s i n d o s e s lower t h a n t h e r e q u i r e d t o produce p e r i p h e r a l aa d r e n e r g i c blockade. pseudoyohimbine [ 2 2 ] was o b t a i n e d (low y i e l d ) u s i n g alumina chromatography. P a r e n t r a l a d m i n i s t r a t i o n may i n d u c e s w e a t i n g . ) i n s u l f u r i c a c i d f o r 30 m i n u t e s . A f t e r removal of t h e forrnyl group. i r r i t a b i l i t y and t r e m o r s a l s o occured. i n c r e a s e d h e a r t r a t e . M e t a p e r i o d a t e c l e a v a g e of 1.2g l y c o l [ 2 0 ] gave t h e 0-formate of pseudoyohimbaldehyde [ 2 1 ] . where a l i m i t e d amount of a c e t a t e s a l t under 0 . 0 1 mm p r e s s u r e i n a s u b l i m e r was exposed t o a metal b a t h p r e v i o u s l y h e a t e d t o 285-295'. Scheme 2 i l l u s t r a t e s t h e sequence of s y n t h e s i s d e s c r i b e d above. n a u s e a and vomiting (23 .a d r e n e r g i c blockade of l i m i t e d d u r a t i o n and i t i s r e l a t i v e l y s e l e c t i v e f o r t h e presynaptic a2-receptor.7 8 O i n THF-pyridine. The c o n t r o v e r s i a l a p h r o d i s i a c p r o p e r t y of yohimbine was r e c e n t l y i n v e s t i g a t e d by many a u t h o r s .

With V i t a l i ' s t e s t t h e c o l o u r sequence i s y e l l o w / y e l l o w / v i o l e t ( 6 ) . 3 3 ) found t h a t i n p a t i e n t s w i t h c l e a r o r g a n i c impotence. However.1 pg). MEKKAWI AND A. h a s now r e c e i v e d s u p p o r t from a s t u d y c a r r i e d o u t b y Clark e t a1 ( 3 4 ) .1 Color Tests Yohimbine when r e a c t e d upon by ammonium molybdate g i v e s a b l u e c o l o r t h a t changes slowly t o green ( s e n s i t i v i t y 0.2 M i c r o c r y s t a l Tests Wachsmuth (35) determined t h e p r e c i p i t a t i n g p r o p e r t i e s of f l a v i n i c a c i d ( i n e t h a n o l d i e t h y l e t h e r s o l u t i o n ) on s e v e r a l a l k a l o i d a l and o t h e r n i t r o g e n c o n t a i n i n g s u b s t a n c e s and r e p o r t e d t h a t abundant p r e c i p i t a t e s were o b t a i n e d w i t h yohimbine ( s e n s i t i v i t y 1:lOO). a t l e a s t i n r a t s .1. Condra e t a 1 (32) s t u d i e d t h e e f f e c t i v e n e s s of yohimbine i n t h e t r e a t m e n t of impotence and Nakatau e t a1 ( 3 3 ) have s t u d i e d t h e pharmacokinetics of yohimbine i n man. Methods of A n a l y s i s 8.025 pg). G . The teams of Condra and Nakatau ( 3 2 . t r e a t m e n t w i t h yohimbine h y d r o c h l o r i d e e x h i b i t e d a p o s i t i v e r e s p o n s e r a t e of approximately 30% and p o s t u l a t e d t h a t t h e f a i l u r e t o improve t h e c o n d i t i o n s of t h e o t h e r two t h i r d s of p a t i e n t s may be due t o i n t e r i n d i v i d u a l d i f f e r e n c e s i n t h e pharmacokinetics of t h e drug which a p p e a r s t o be c l e a r e d i n i n d i v i d u a l s by metabolism.1. i n 8.v i t r o p r e p a r a t i o n s of human and r a b b i t i-n v i v o on r a b b i t s and concluded t h a t . w i t h ammonium vanadate t e s t i t g i v e s a b l u e changing t o preen and f a d i n g away ( s e n s i t i v i t y i s 0. Habib and Aziz ( 3 6 ) found t h a t yohimbine g i v e s i r r e g u l a r l a r g e r o s e t t e s of curved h a i r s w i t h 0. yohimbine e x h i b i t s a . t h e p o s s i b l e e f f e c t i v e n e s s of yohimbine as an a p h r o d i s i a c . -i n .s p e c i f i c t e s t s a r e t h e x a n t h y d r o l r e a g e n t and 4-dimethylamino benzaldehyde i n m e t h a n o l i c HC1.5% aqueous ammonium r e i n e c k a t e .A.1 Q u a l i t a t i v e Analysis 8. Other n o n . AL-BADR 152 s t u d i e d t h e pharmacological e f f e c t s of yohimbine i n p e n i s and the penis.a d r e n e r g i c b l o c k i n g p r o p e r t i e s and does n o t a f f e c t catecholamine l e v e l i n t h i s t i s s u e . 8. A.

.1 G r a v i m e t r i c Methods Raymond (11) reviewed methods used a t h i s t i m e i n gravimetric analysis. C h a r a c t e r i s t i c c r y s t a l s with d i f f e r e n t reagents obtained i n o u r l a b o r a t o r y .2.o r . The a l k a l o i d s were p r e c i p i t a t e d w i t h an a l c o h o l i c s o l u t i o n of H C 1 i n a b e a k e r and t h e aqueous a l c o h o l i c p a r t was washed twice w i t h e t h e r and methyl a c e t a t e . t h e c r y s t a l l i n e yohimbineH C l was f i l t e r e d on a weighed f i l t e r p a p e r . are i l l u s t r a t e d i n F i g u r e s 8-17. The o r g a n i c s o l v e n t was e v a p o r a t e d .t r i c h l o r o e t h y l e n e mixed i n t h e r e c e i v e r w i t h aqueous o x a l i c a c i d s o l u t i o n . 8.2 Q u a n t i t a t i v e Analysis 8. 8.YOHIMBINE 153 which can be i d e n t i f i e d m i c r o s c o p i c a l l y . The secondary a l k a l o i d s remained i n s o l u t i o n a f t e r c r y s t a l l i s a t i o n of yohimbine HC1. t h e aqueous a c i d s o l u t i o n w a s t r e a t e d w i t h NH40H s o l u t i o n t o l i b e r a t e t h e a l k a l o i d s which were t h e n e x t r a c t e d w i t h e t h e r . With potassium c y a n i d e s o l u t i o n . y o h i m b i n e g i v e s r o s e t t e s of r o d s and w i t h sodium c a r b o n a t e s o l u t i o n i t g i v e s r o s e t t e s of r o d s o r dense r o s e t t e s ( s e n s i t i v i t y i n b o t h r e a g e n t s 1: 1500) ( 4 ) . A f t e r b e i n g l e f t 24 h o u r s i n a r e f r i g e r a t o r .2. He extracted powdered bark of yohimbehe w i t h e t h e r : chloroform ( 4 : l ) m i x t u r e u s i n g 10 m l of s o l v e n t f o r each gram of b a r k m a t e r i a l Feldhoff (15) m o d i f i e d t h e t e c h n i q u e s u s e d b e f o r e him by l i b e r a t i n g t h e b a s e s i n t h e w i t h Na2C03 t r e a t m e n t bark of Yohimbe and e x t r a c t i n g t h e d r u g i n a s o x h l e t w i t h d i . washed and d r i e d a t 102% t o c o n s t a n t w e i g h t .2 T i t r i m e t r i c Methods Precipitation Titration V y t r a s and Riha (37) c a r r i e d o u t p r e c i p i t a t i o n t i t r i m e t r y for 9 alkaloids including yohimbine u s i n g sodium t e t r a p h e n y l b o r a t e r e a g e n t and a C r y t u r valinomycin i o n selective electrode.

MEKKAWI AND A. Figure91 Radiating pointing plates of phimbine with mercuric chloride reagent ( a f t e r 14minutes). . Figure 10 : Orange coloured radiating relatively wide crystal plates of yohimbine with potassium chromate reagent (after 9 minutes ).A. A. G . AL-BADR 754 Figure8: Rosettes of circular plates of yohimbine with picric acide reagent ( a f t e r 3 minutes 1.

. Figure12.YOHIMBINE 155 Figurell : Tagged plates of crystals of yohirnbine with Dragendorf f's reagent ( after 5 minutes ). White crystalline minute rods of yohimbine after reaction w i t h lead iodide reagent (immediately produced ) Figure 13: Crystal of tetragonal or hexagonal structures a f t e r addition of Marme's reagent (after 5 minutes).

A. Figure 16:immediate sandy crystals w i t h Wagner's reagent. AL-BADR 156 Figure 14 Radiating rod clusters of yohlrnblne and Naf03 solution (after 10 minutes) Figure IS : Crystal plates. MEKKAWI AND A. Fine clusters of rods with KCN solution (after 15minutes) . A. characteristic. to reaction of yohimbine with NH4S CN aqueous solution ( a f t e r 5 . G .7 minutes). Figure 17.

I n p r e s e n c e of o t h e r i n d o l e c o n t a i n i n g compounds.3 mp. and t h e c o l o r w a s allowed t o develop. p a p a v e r i n e . To t h i s . d i s t i l l e d water was added t o make 10 m l of m i x t u r e and d e n s i t y was measured p h o t o m e t r i c a l l y u s i n g a f i l t e r S 39 o r S 49.3 S p e c t r o p h o t o m e t r i c Methods a) Colorimetry C o l o r i m e t r i c a s s a y s of t h e weakly b a s i c t e r t i a r y a l k a l o i d w e r e f i r s t done on c o l o r r e a c t i o n s c h a r a c t e r i s t i c of t h e i n d o l e group ( 8 . quinone . I n a 10 m l v o l u m e t r i c f l a s k t o 1 m l of t h e s o l u t i o n c o n t a i n i n g 0.05 . tungstophosphoric o r molybdophosphoric a c i d a t pH 1 o r 7.2. Bobtelsky and B r a z i l y (38) s t u d i e d t h e b e h a v i o u r of micro amounts of yohimbine t o g e t h e r w i t h n i t r o n . such as t h o s e n a t u r a l l y present i n t h e n a t u r a l source.such a method i s of l i t t l e v a l u e . of t h e allcaloid s a l t . . 8. The mechanism of t h i s r e a c t i o n w a s s t u d i e d by P i n d u r (40). a t r o p i n e . shaken. They found t h a t t h e i n f l u e n c e of t h e a c i d i t y of t h e s o l u t i o n was d i f f e r e n t i f t h e o r g a n i c molecule c o n t a i n e d 1 o r 2 h e t e r o c y c l i c N atoms. Kolsck (39) used t h e c o l o u r r e a c t i o n of yohimbine i n d o l e n u c l e u s w i t h p-dimethylaminobenzaldehyde and measured t h e c o l o u r produced p h o t o m e t r i c a l l y w i t h f i l t e r S 39 o r S 49.05 m l of a 5% H202 ( o r 1%NaN02) s o l u t i o n w a s added.c o n t a i n i n g compounds. However. i f d e s i r e d w i t h a d d i t i o n of l i t t l e e t h a n o l . The method i s summarized a s f o l l o w s : Yohimbine-HC1 w a s d i s s o l v e d i n water.YOHIMBINE b) M i cr 0. n i c o t i n e . 2 m l of p-dimethylylaminobenzaldehyde w e r e added and t h e m i x t u r e w a s l e f t t o c o o l f o r 10 minutes and 0. 29). s t r y c h n i n e . morphine o r phenanthrol i n e when t i t r a t e d h e t e r o m e t r i c a l l y w i t h t u n g s t o s i l i c i c .he t e r ome t r i c T i t r a t i o n I n t h e i r t r i a l t o analyze l a r g e organic n i t r o g e n .

reported that yohimbine at pH 7 had its maximum activation at 270 mu and its maximum emission of fluorescence at 360 mu. The color is stable for 5 hr.4 Spectrofluorimetric Methods In their spectrophotofluorimetric study of a vast number of organic compounds of pharmaceutical interest. 0 ) and 1 ml of 0. cooled and the extinction wasmeasured at 515 mu.758 A. 8.5%. The yohimbine-methyl orange complex was extracted successively with chloroform. Underfried et a1 ( 4 5 ) using a Xe arc . The yohimbine-methyl orange complex color as a quantitative method was utilized by Mohamed and Elsayed ( 4 4 ) for assaying the base in tablet formulations. MEKKAWI AND A. G . injections or mixtures after extracting the alkaloid by a suitable method The method was also used by Budavaris a1 ( 4 3 ) . The indole nucleus of the two compounds forms a colored compound with xanthydrol and the extinction is measured at 515 mu. For 5 determinations the recovery was 97.source emitting a continuum from 200-800 mu in their spectrofluorometer. In this method a 50-150 p g of yohimbine were dissolved in 5 ml of a freshly prepared solution o f 0 .1% methyl orange solution in 20% ethanol. diluted to 25 ml with chloroform and the extinction was measured at 421 nm. The procedure can be used for tablets. .102.2. 4 % xanthydrol in glacial acetic acid Containing 1% v/v HC1 heated on a boiling water bath for 15 minutes. A solution of the powdered tablets ( e 5 0 mg of yohimbine hydrochloride) in 50 ml of H20 was prepared A measured volume containing about 300 mg of tablet base was diluted to 5 0 ml. A.6 . 4 2 ) photometrically with xanthydrol reagent in presence of other nitrogen containing compounds. AL-BADR Jung assayed reserpine and yohimbine ( 4 1 . A 2 m portion of this solution was transferred to a separating-funnel containing 2 ml of buffer (pH 4 .

A similar solution of standard yohimbine was prepared. They extracted 1 g of the bark with ethyl ether after alkalinisation with 10% aqueous ammonia. The ethereal extract was re-extracted with 0. The chloroformic extract was chromatographed on a MN 300 Polygram cellulose plate impregnated with formamide : dimethylformamide : acetone (4:3:12) and developed with heptane : ethylmethyl ketone : 0.YOHIMBINE 759 Chiang and Chen (46) assayed yohimbine after increasing its fluorescence in solutions by subjecting its heated solution in presence of H202 to ultraviolet light. Auterhoff and Schroeder ( 4 7 ) carried a similar assay and evaluated yohimbine in Yohimbe bark. 1 ml of 3% H202 and 4 ml of 30% acetic acid were added. was extracted with CHC13 : CH3OH (100 ml).1 N H2SO4 solution and the acid solution was basified with NH40H. extracted with chloroform.49) . the solvent removed under reduced pressure at 35OC and the residue dissolved in 10 ml of 30% acetic acid.25% aqueous ammonia (4:2:1) for 15 cm and the zone of Rf 0. To 1 ml of this solution. After excitation at 365 nm the fluorescence was measured at 496 nm. Both solutions were heated for 2 hr at 80°C. ajmaline and yohimbine may be separated by thin-layer chromatography and determined by spectrophotometry or spectrofluorometry as described previously for indole alkaloids by some authors (48. Clarke ( 6 ) reported the quantitative estimation of yohimbine as follows: The drug when in a mixture consisting of reserpine. concentrated and diluted to 10 ml with chloroform. The extract was filtered.2. cooled and the volume was adjusted to 10 ml with 30% acetic acid. correspondin? to yohimbine.

2.2.2.843 t o 0. . A.A. Development: Ascending.L. ( I t can and d r y i n g a t 25' f o r 1 hour. b) Paper: Whatman No.844.5. 1 o r No. i n 2 N a c e t i c acid i f possible. G . week r e a c t i o n . 4 s h e e t s b e i n g run a t a t i m e . weak r e a c t i o n . Location: Under u l t r a v i o l e t l i g h t .5 Chromatographic Methods 8.8 of c i t r i c a c i d i n a m i x t u r e of 130 m l of w a t e r and 870 m l of n-butanol. Sample: 2 . AL-BADR 760 8. 5 ~1 of a 1X s o l u t i o n . 3 . Time o f r u n . s h e e t 16 x 4 i n c h .C. MEKKAWI AND A. 2 N sodium h y d r o x i d e .) Z a f f a r o n i ' s system (50) w a s t r i e d by Machovicova ( 51 ) on p a p e r chromatography and was found t o be s u i t a b l e f o r t h e s e p a r a t i o n of r e s e r p i n e . s h e e t 1 7 x 19 cm.1 Paper Chromatography (P. by Auterhoff and Schroeder (47) i s d e s c r i b e d i n s e c t i o n 8. S o l v e n t : 4. b u f f e r e d by d r i p p i n g i n a 5% s o l u t i o n of sodium dihydrogen c i t r a t e . 1 . or ethanol. impregnated by d i p p i n g i n a 10% s o l u t i o n of t r i b u t y r i n i n a c e t o n e and drying i n a i r . p a l e blue fluorescence. b l o t t i n g . otherwise i n 2 N h y d r o c h l o r i c . 5 hour. be s t o r e d i n d e f i n i t e l y ) .L.C. bromocresol green s p r a y . r e s e r p i c a c i d and yohimbine. location reagent : i o d o p l a t i n a t e s p r a y . T h i s may be used f o r s e v e r a l weeks i f w a t e r added from t i m e t o time t o keep t h e s p e c i f i c g r a v i t y a t 0.4 above C l a r k e (6) r e p o r t e d t h e f o l l o w i n g paper chromatography systems f o r t h e s e p a r a t i o n of yohimbine: a) Paper: Whatman No. Q u a n t i t a t i o n of yohimbine u s i n g P. i n a t a n k 8 x 11 x 15% i n c h .

) S i l i c a g e l G.L. L o c a t i o n : Under u l t r a v i o l e t l i g h t . Rf v a l u e Chromatography (T. 6 4 .04.g l a s s d i s k t h i c k l y smeared w i t h s i l i c o n e g r e a s e may s e r v e a s a cover. 15 t o 20 m i n u t e s . However. blue fluorescence. Development: Location: 0. Development: Ascending. t h e c y l i n d e r i s i n s e r t e d i n t h e beaker c o n t a i n i n g t h e s o l v e n t which i s n o t removed from t h e oven. The p a p e r i s f o l d e d i n t o a c y l i n d e r and c l i p p e d . 1 M NaOH o r 0. Phosphate b u f f e r (pH 7 . Solvent: Acetate b u f f e r (pH 4.1 M KHSO4 developed by 5 s o l v e n t systems were used by Fike (52) i n t h e s t u d y of t h e b e h a v i o u r of 140 b a s i c d r u g s i n c l u d i n g yohimbine and t h e Rf v a l u e s of t h o s e d r u g s w e r e r e c o r d e d and s u c c e s s f u l s e p a r a t i o n w a s achieved b u t none of t h e i n d o l e a l k a l o i d s r e l a t e d t o yohimbine w e r e i n c l u d e d i n t h e s t u d y . Rf v a l u e 0 .58). 4 ) . Thin-Layer . p l a t e s p r e a p r e d w i t h 0 . Court and .2. T i m e of r u n .C. A p l a t e .2 A s above i n (b) A s above i n ( b ) . E q u i l i b r a t i o n : The beaker c o n t a i n i n g the solvent i s equilibrated i n a t h e r m o s t a t i c a l l y c o n t r o l l e d oven a t 86' f o r about 15 m i n u t e s . E q u i l i b r a t i o n : The beaker c o n t a i n i n g the solvent i s equilibrated i n a t h e r m o s t a t i c a l l y c o n t r o l l e d oven a t 95' f o r about 15 m i n u t e s . 8.5.YOHIMBINE 76 1 Sample: 5 1-11of 1 t o 5% s o l u t i o n i s e t h a n o l o r chloroform. c) Paper and Sample: A s above i n ( b ) Solvent: .

AL-BADR and Habib (16) and Court and Timmins ( 1 7 ) . Q u a n t i t a t i o n p h o t o d e n s i t o m e t r i c a l l y on s i l i c a g e l l a y e r s of a l k a l o i d s of p l a n t s o u r c e s w a s c a r r i e d o u t by Massa e t a1 (53) who found t h a t f l u o r s c e n c e r e f l e c t a n c e r e a d i n g s of unsprayed s p o t s of f l u o r e s c e n t a l k a l o i d s were f a v o u r a b l e i n a c t i v a t e d s i l i c a g e l p l a t e s .MEKKAWI AND A.C. Nine s o l v e n t s systems were used and s u i t a b l e c o n c e n t r a t i o n s of s t a n d a r d s were r u n and t h e s t u d y w a s done on 25 a l k a l o i d s of which 1 4 a l k a l o i d s sprayed w i t h Dragendorff-Schuette r e a g e n t . 0. Court and Habib (16) q u a n t i t a t e d 10 Rauwolfia a l k a l o i d s by combined p r e p a r a t i v e T. A. However. and e x c i t e d a t 358 nm.L. c o a t e d w i t h a s l u r r y c o n s i s t i n g of 30 g of s i l i c a p.25 mm t h i c k and d r i e d a t l l O o f o r 1 h o u r .L. . s e r p e n t i n e .el G i n 60 m l of w a t e r t o g i v e a l a y e r 0. i n t h e i r s t u d i e s on Rauwolfia a l k a l o i d s and t h e i r behaviour on T.C. a-yohimbine. They used a double-beam p h o t o d e n s i t o m e t e r w i t h r e c o r d e r and i n t e g r a t o r f u n c t i o n i n g i n t h e v i s i b l e and u l t r a v i o l e t l i g h t s . I o d i n e o r d i c h l o r o a c e t i c a c i d and h e a t . have piven v a l u a b l e i n f o r m a t i o n s on t h e c o l o u r r e a c t i o n s and R f v a l u e s of such r e l a t e d a l k a l o i d s on s i l i c a g e l l a y e r s employing 10 s o l v e n t systems and emphasis can b e c o n c e n t r a t e d on t h e r e l a t e d a l k a l o i d s : Yohimbine.162 A. and c o l o r i m e t r y and found t h a t t h e i r method w a s b e s t s u i t a b l e f o r B-carboline a l k a l o i d s . s a r p a g i n e and a j m a l i c i n e which come i n t h e s t r o n g l y b a s i c f r a c t i o n of Rauwolfia r o o t e x t r a c t . were d e t e c t e d a t 400 nm and 11 o t h e r s were determined by i n h i b i t i o n of f l u o r e s c e n c e a t 528 nm and 13 a l k a l o i d s e s t i m a t e d by t h e i r n a t u r a l f l u o r e s c e n c e o r induced by a s u i t a b l e r e a g e n t such as KMnO4. Clarke ( 6 ) d e s c r i b e d t h e f o l l o w i n g TLC system f o r t h e i s o l a t i o n of yohimbine: P l a t e : Glass p l a t e s 20 x 20 c m .

The i n s t r u m e n t s e t t i n g s were a s f o l l o w s : I n j e c t i o n p o r t t e m p e r a t u r e . Using c h o l e s t e r o l a s a s t a n d a r d . Location reagent: Acidified i d o p l a t i n a t e Rf v a l u e 0.) Dusci and Hackett (54) d e s c r i b e d a d i r e c t p r o c e d u r e f o r a n a l y s i s of yohimbine and o t h e r n e u t r a l drugs i n t i s s u e s . i n a t a n k 2 1 x 2 1 x 10 cm. C l a r k e ( 6 ) r e p o r t e d t h e f o l l o w i n g GC system f o r t h e s e p a r a t i o n of yohimbine. two r u n s . Column: W AW. d . 5% SE-30 on 60-80 mesh Chromosorb 5 f t x 118 i n c h i n t e r n a l d i a m e t e r s t a i n l e s s s t e e l column. 8. Solvent: 1 p1 of a 1%s o l u t i o n i n 2 N a c e t i c S t r o n g ammonia s o l u t i o n : methanol I t should be changed a f t e r (1. E q u i l i b r a t i o n : The s o l v e n t i s allowed t o s t a n d i n t h e t a n k f o r 1 hour. Time of run: 30 m i n u t e s .2. spray. p o s i t i v e reaction. t h e r e l a t i v e r e t e n t i o n t i m e (RRT) of yohimbine was 0. . i n c r e a s e d a t 8OC/min t o 29OoC and t h e f i n a l t e m p e r a t u r e was h e l d f o r 4 m i n u t e s . 30OoC.5.3 (G. n i t r o g e n c a r r i e r g a s flow r a t e . d e t e c t o r t e m p e r a t u r e . 80-100 mesh. Nitrogen.89. They used Hewlett-Packard gas chromatograph model 5700A equipped w i t h a f l a m e i o n i z a t i o n d e t e c t o r . t h e end of t h e t a n k b e i n g covered w i t h f i l t e r paper t o a s s i s t e v a p o r a t i o n . Oven t e m p e r a t u r e programmed as 15OOC h e l d f o r 2 m i n u t e s .YOHIMBINE 163 Sample: acid. 60 ml/min.L.5 : 1 0 0 ) . 25OoC. g l a s s column pakced w i t h 3% OV-17 on Gas Chrom Q.C. Development: Ascending. Column t e m p e r a t u r e : Carrier Gas: 23OoC.55. The column w a s a 4 f t x 4 mm i .

. A. hydrogen 22 m l p e r minute.2.5. f o r performing t h e m i c r o c r y s t a l t e s t s B u t t .d. Rodgers ( 5 6 ) w a s a b l e t o a n a l y s e s e v e r a l classes of t r a n q u i l i z e r drugs i n c l u d i n g Rauwolfia a l k a l o i d s by t h i s t e c h n i q u e u s i n g a d s o r p t i o n columns of s i l i c a g e l and c a t i o n exchangers. f o r t y p i n g l i k e t o thank M r .4 0.1 cm]of yohimbine i n U. G .V. wavelength can be v a r i e d t o t h e optimum.7 m l p e r minute. HPLC on a l k a l o i d s u s i n g v a r i o u s t y p e s of chromatographic s e p a r a t i o n modes w a s c a r r i e d o u t by v a r i o u s a u t h o r s .A. Pharm. Yohimbine s e p a r a t i o n on a d o s r p t i o n HPLC w a s s u c c e s s f u l and V e r p o o r t e and Svendsen ( 5 5 ) were a b l e t o s e p a r a t e i t from r e s e r p i n e on Mercksorb S 1 70 ( 5 pm) column u s i n g 6 systems of e l u e n t and a Packard Model 8200 l i q u i d chromatograph equipped w i t h a 254 nm and 280 nm UV d e t e c t o r w a s used.) Dept. Retention t i m e : 8. S e c r e t a r y t o P h a r m a c e u t i c a l Chemistry t h e manuscript. MEKKAWI AND A.38 r e l a t i v e t o c o d e i n e .V. D e t e c t o r : Flame i o n i z a t i o n d e t e c t o r . However. The column t e m p e r a t u r e w a s maintained a t 2OoC and p r e s s u r e s of 50-250 kg/cm2 were employed t o c o n t r o l flow rates.). Acknowledgements The a u t h o r would of Pharmacognosy and t o T a n v i r A. High-speed L i q u i d Chromatography(HPLC) The advent of HPLC i n t h e f i e l d o f s e p a r a t i o n and q u a n t i t a t i o n of almost a l l t y p e s of chemical m i x t u r e s made t h e t a s k s of t h e a n a l y t i c a l chemists f a r e a s i e r t h a n two decades ago. t h e r e l a t i v e l y high[E 1%. AL-BADR 764 Gas Flow: 30. Dept. The column w a s a s t a i n l e s s s t e e l t u b e (30 cm x 2 mm i. Amir S h e h a t a (B. l i g h t a t 226 nm makes t h e d e t e c t i o n of minute q u a n t i t i e s p o s s i b l e i f t h e i n s t r u m e n t U.

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12. 16. 53 Caffeine. 4. 10. 207 Dihydroergotoxine methanesulfonate. 573 Allopurinol. 2. 4. I Amitriptyline hydrochloride. 15 Chlordiazepoxide hydrochloride. 518 Ascorbic acid. 139 Cefoxitin. 5. 7% Clidinium bromide.79: 4. 113 Cocaine hydrochloride. 19 Amphotericin B. 1. 1 Amikacin sulfate. 13. 10. 1 Baclofen. 127 Amoxicillin.61 Chlorpheniramine maleate. 39. 502 Ampicillin. 1 Cefotaxime. I Aminosalicylic acid. 7. 14. 95 Chloroquine phosphate. 32 Azathioprine. 10. 15. 15. 10. 6. 37 Amiloride hydrochloride. 2. 15. I Chlorzoxazone. 149 Digoxin. 16. I Atenolol. 91 Clotrimazole. 3. 1. see Vitamin D3 Cimetidine. 155 Dapsone. 491 Digitoxin. 83. 197 Clonazepam. 35 Aminophylline. 11. 8. 10. 14. 2. 66 Cypropheptadine. 9. 87 Dexamethasone. 6. 4. 5. 518 Chloroquine. 101 Chlorthalidone. 9. 14. 12.53 Cyclosporine. 105 Diflunisal. 8. 1. 519 Diatrizoic acid. 85 Chlorambucil. 1. 245 Benzocaine. 71 Bromazepam. 10. 9. 119 Cholecalciferol. 4. 14. 12. 14. 3. 225 Cloxacillin sodium. 169 Cephalexin. 6. 13. 107 Cefamandole nafate. 701 Chlordiazepoxide. 15. 1. 4. 9. I Bromocriptine methanesulfonate. 5. 16. 3. 11.CUMULATIVE INDEX Bold numerals refer to volume numbers Acetaminophen. 8. 21 Cephalothin sodium. 43 Chlorprothixene. 10. 145 Clindamycin hydrochloride. 21 Chloral hydrate. 12. 9. 15 I Codeine phosphate. 527 Bendroflumethiazide. 4. 9. 11. 11. 27 Captopril. 556 Diazepam. I . 11. 3.61 Clorazepate dipotassiurn. 145 Cyclothiazide. 43 Bretylium tosylate. 518 Dibenzepin hydrochloride. 1 1 1 Amantadine. 183 Cyclizine. 2. 551 Acetohexamide. 79 Carbamazepine. 45 Aspirin. I . 4. 73 Benzyl benzoate. 71 Calcitriol. 5.47. I. 10. 1 Atropine. 13. I Arninoglutethimide. 502 Cycloserine. 139 Cyanocobalamin.93 Colchicine. 729 Cefazolin. 75 Clofibrate. sodium. 77 : 15. 55 Betamethasone dipropionate. 7. 2. 11. 4. 137. 11. 6. 63 Chlortetracycline hydrochloride. 14. 597 Benperidol 14. 6. 29 Bacitracin. 1: 7. 163. 9. 16. 87 Cefaclor. I .1. 127 Cisplatin. 16. 9. 319 Cephradine. 518. I Alpha-tocopheryl acetate. 7. 8. 7. 125. 5. 85 Chloramphenicol. 181 Dibucaine and dibucaine hydrochloride. 47 Busulphan. 1. 2. 13. 4. 81 769 . 15. 83 Camphor. 7.

520. 10. 8. 10. 2. 168 Dobutamine hydrochloride. 16. 231 Ethynodiol diacetate. 277 Homatropine hydrobromide. 307 Gentamicin sulfate. 1. 307 Methylphenidate hydrochloride. 487 . 9. 199: 12. 245 Hydralazine hydrochloride. 233 Epinephrine. 499 Nadolol. 49. 12. 325 Metronidazole. 2. 11. 8. 313 Fluorouracil. 9. 327 Metoprolol tartrate. 8. 524 Fluphenazine hydrochloride. 265 Meperidine hydrochloride. 4. 183 Disulfiram. 391 lsosorbide dinitrate. 4. 10. 183 Isopropamide. 455. 16. 275. 13. 1. 10. 10. 9. 453 Natamycin. 713 Diperodon. 721 Isoproterenol. 323 Mitomycin C. 5. 209 CUMULATIVE INDEX Hydroxyzine dihydrochloride. 7. 365. 13. 101. 15. 219. 337 Gluthethimide. 327 Minocycline. 173 Diphenoxylate hydrochloride. 3. 207. 367 Lorazepam. 7. 333 lopanoic acid. 761 Lithium carbonate. 259 Ketamine. 16. 207 Ephedrine hydrochloride. 16. 251 Haloperidol. 4. 357 Hexestrol. 9 9 Diphenhydramine hydrochloride. 6. 9. 14. 8. 21 1 loddmide. 732 Nalidixic acid. 179 Griseofulvin. 10. 161 Flucytosine. 171 Echothiophate iodide. 315 Levallorphan tartrate. 363 Metocloprarnide hydrochloride. 583 Guanabenz acetate. 245. 2. 319 Halcinonide. 339 Levarterenol bitartrate. 573. 4. 14. 731 Glibenclamide. 225 Lidocaine base and hydrochloride. 297 Ketoprofen. 175 Meprobamate. 427 Methyclothiazide. 5. 2. 12. 3. 305 Nabilone. 573 Estradiol. 5 . 189 Levothyroxine sodium. 6. 1. 15. 257 Doxorubicine. 319 Imipramine hydrochloride. 295. 4. 2. 12. 601 Methaqualone. 37 Indomethacin.473 Methyprylon. 2. 253 Etomidate. 6. 9. 5. 295 Isoniazide. 149 Disopyramide phosphate. 2. 11. 12. 297 Hydroxyprogesterone caproate. 443 Ketotifen. 7. 221 Fluoxymesterone. 7. 291 Mefloquine hydrochloride. 15. 273 Ergotamine tartrate. 245 Droperidol. 5. 351 Methotrexate. 8. 5 . 587 6-Mercaptopurine. 277 Hydroflumethiazide. 9. 7. 215 Heroin. 3. 555 Levodopa. 9. 14. 191 Fenoprofen calcium 6. 7. 14. 397 Maprotiline hydrochloride. 556 Kanamycin sulfate. 11. 283 Estradiol valerate. 2. 573. 233 Emetine hydrochloride. 3. 520. 10. 15. 393 Mebendazole. 12. 403 Nitrazepam. 519 Flurazepam hydrochloride. 7. 4. 193 Ergonovine maleate. 135 Ethambutol hydrochloride. 10. 4. 11. 361 Moxalactam disodium. 347 Hexetidine. 513 Neornycin. 11. 1. 9. 245. 225. 283 Methoxsalen. 9. 14. 181 Isocarboxazid. 730 Fluphenazine enanthate. 10. 520 Methimazole. 283 Hydrochlorothiazide. 11. 2. 157 Melphalan. 139 Gramicidin. 8. 7. 315. 13. 119. 3. 209. 405 Hydrocortisone. 15. 16. 597 Heparin sodium. 2. 4. I13 Erythromycin. 341 Halothane. 6. 281 Flufenamic acid. 6 . 5 . 159 Erythromycin estolate. 337 lodipamide. 13. 25 I Fluphenazine decanoate. 375 Methadone hydrochloride. 263. 139 Dopamine hydrochloride. 15. 6. 13. 239 Khellin. 3.770 Dioctyl sodium sulfosuccinate. 115 Fludrocortisone acetate. 9. 14. 8. 14. 371 Naloxone hydrochloride. 1. 2. 371 Leucovorin calcium. 8. 12. 8. 11. 10. 289 Enalapril maleate. 2. 4. 9. 15. 399 Neostigrnine. 10. 16. 343 Mestranol. 7. 5. 8. 192 Estrone.

423 Timolol maleate. 4. 16. 13. 4. 13. 521 77 1 Secobarbital sodium. 647 Vinblastine sulfate. 4. 13. 509 Tropicamide. 13. 5 . 457 Pseudoephedrine hydrochloride. 273 Testolactone. 601 Penicillin-G benzothine. 14. 11. 383 Phenformin hydrochloride. 61 I Thiosrrepton. 507 Strychnine. 341 Oxazepam. 135 Yohimbine. I . 4. 13. 384. 15. 6. 10. 5 . 571. 5. 1. 11. 563 Penicillamine. 1. 452 Tetracycline hydrochloride.483 Phenylpropanolamine hydrochloride. 12. 13. 467 Rutin. 501. 593 Triamcinolone acetonide. 8. 515 Terpin hydrate. 2. 10. 12. 16. 639 Procainamide hydrochloride. 579 Triclobisonium chloride. 11. 7. 401 Sulfamethoxazole. 1. 8. 16. 3. 521 Sulfasalazine. 9. 11. 3. 4. 5. 691 Sulfadiazine. 1.483 Phenylephrine hydrochloride. 427 Penicillin-V. 4. 521. 5. 463 Quinidine sulfate. 12. 523.441 Oxyphenbutazone. 2 . 15. 487. 375 Pirenzepine dih ydrochloride. 521. 651 Triamcinolone hexacetonide. 641 Tolbutamide. 385 Piperazine estrone sulfate. 343 Silver sulfadiazine. 319. 529 Vidarabine. 737 Rifampin. 2. 12. 477 Tybamate.431 Streptomycin. 10. 367. 557 Trimethaphan camsylate. 7. 665 Salicylamide. 533 Testosterone enanthate. 719 Trazodone hydrochloride. 571. 2. 519 Norethindrone.CUMULATIVE INDEX Nitrofurantoin. 403 Promethazine hydrochloride. IS. 6. 4. 673 . 357. 249 Pentazocine.409 Probenecid. 10. 3. 543 Triflupromazine hydrochloride. 107 Triprolidine hydrochloride. 4. 301. 14. 11. 545 Trimethobenzamide hydrochloride. 333 Oxytocin. 557. 345 Nitroglycerin. 423: 11. 7. 13. 6. 565 Tubocurarine chloride. 7. 466 Thiabendazole. 563 Succinycholine chloride. 15. 3. 731 Zomepirac sodium. 14. 615 Triamcinolone diacetate. 417 Pilocarpine. 573 Sulphamerazine. 521. 13. 429 Proparacaine hydrochloride. 6. 3. I . 1. 5 . 10. 551 Trimethoprim. 4.447 Pyrimethamine. IS. 513. 361 Phenazopyridine hydrochloride. 2. 6 . 16. 5. 547 Ranitidine. 573 Noscapine. 416. 487 Salbutamol. 6. 12. 445 Trimipramine maleate. 10. 507 Trifluoperazine hydrochloride. 12. 520. 4. 12. 407 Nystatin. 4. 5. SO9 Primidone. 705 Tripelennamine hydrochloride. 423 Propiomazine hydrochloride. 693 Triamcinolone. 7. 14. 5. 249 Phenylhutazone. 15. 445 Piroxicam. 5. 13. 463 Vitamin Dlr 13. 655 Warfarin. 443 Vincristine sulfate.489 Pyrazinamide. 1. 243 Xylometazoline hydrochloride. 4. 2. 483 Quinine hydrochloride. 233. 2. 429 Phenobarbital.465 Phenelzine sulfate. 1. 2. 4. 2. 2. 16. 515 Sulfisoxazole. 463 Penicillin C. 4. potassium. 439 Propoxyphene hydrochloride. 5 . 2. 553 Sodium nitroprusside. 359 Phenoxymethyl penicillin potassium. I. 487 Sulindac. 524: 11. 433 Pyridoxine hydrochloride. 397. 13. 13. 16.494 Valproate sodium and valproic acid. 523 Sulfamethazine. 623 Saccharin. 294 Nonriptyline hydrochloride. 15. 781 Spironolactone. 771 Phenytoin. 3. 598 Propylthiouracil. 333 Procarbazine hydrochloride. 268 Norgestrel. 533 Reserpine. 13. 467. 12. 683 Trioxsalen. 7. 8. 9. 6. 557. 597 Theophylline.