aharmac. Ther. Vol. 52, pp.

1-34, 1991 0163-7258/91

hinted in Great Britain. All rights reserved © 1992 Pergam¢

ipecialist Subject Editor: E. HAMEL

THE CHEMISTRY OF TAXOL

DAVID G. I. KINGSTON
Department of Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0212,
U.S.A.

Abstract--The chemistry of the ] accr diterpenoid taxol is reviewed, with an emphasis on

isolation and analysis, structural modifications, partial synthesis, and structure-activity relationshil~~S.

CONTENTS
1. Introduction and Scope 1
2. Isolation and Structure of Taxol 2
2.1. Historical background 2
2.2. Structure elucidation of t~ 2
2.3. Structures of related taxa 4
2.4. Sources of taxol and rela 4
2.5. NMR spectra of taxol 7
2.6. Mass
vlass spectrometry of taxol 9
2.7. X-ra
(-ray crystallography of taxol 9
2.8. Analysis of taxol 9
3. Chemistry of Taxol 10
3.1. H~Iydrolysis of taxol 10
Epimerization of taxol at C-7 11
kcylation of taxol 13
)xidation 14
3.5. Reduction 15
3.6. Reaction with electrophiles 15
3.7. Preparation of labeled taxols 17
4. Synthesis of Taxol 18
4.1. Syntheses from baccatin III 18
4.2. Approaches to total synthesis 21
5. Biosynthesis and Biotransformation of Taxol 24
5.1. Biosynthesis of taxol and related taxanes 24
5.2. Biotransformation of taxol 25
6. Structure-Activity Relationships of Taxol Derivatives 26
3accatin III analogs
Side chain modified analogs
~ng modified analogs
~nimal test data
ledgements
2es

1. I J . N I IR
~LO/ LD/ U
U LC~T
I IIUO
I .N A N D SCOPE
./'klNl./ ~L,L/I"I:~, This
I n I 8 revi
review IS pan oz a scnes mat ¢

The discovery of the novel diterpenoid* anticancer inhibitors of microtubule function, and t~
agent taxol in 1971 by M o n r o e Wall and his collab- eluded because its primary mechanism of a
orators (Wani et al., 1971) ranks in retrospect as one the microtubule level. Because of the impc
of the most significant discoveries ever made in taxol and because of the rapidly developing
the field of naturally occurring anticancer agents. this area of study, the discussion of taxol k,
extracts have been used as anticancer two parts. 1
uries, only a handful of plant-derived including it~
ts has been found to show clinically cations, pal
and taxol is clearly a member of this relationship
of the inter
tubules will
! D . G . I . KINGSTON
A©O OA¢ Taxinino (O-¢innamoyltaxicin-II triacetate)
O-cinnamoyltaxicin-I tdacotato
%**0
0
OAc
FIG. 1. Structures of taxinine and O-cinnamoyltaxicin-I triacetate.
relationships. Similarly, the many ap-
proaehes to the taxane ring system a are
not discussed in detail, in part because the total
synthesis of taxol has not yet been achieved. Various
partial syntheses of taxol and its analogs are, how-
ever, included. A full discussion of the taxane diter-
penoids will be published elsewhere (Kingston et al.,
1992b), and a discussion of synthetic approaches has
recently appeared (Swindell, 1991).
N A N D S T R U C T U R E O F TAXOL
2. ISOLATION
2.1. HISTORICAL BACKGROUND
nmemorial it has been known that the
From time immemorial
few, such as the English yew, Taxus
~tain toxic constituents. In the middle
oi me nmeteenm th centur'
century a mixture ofoI toxic
toxin alkaloids
alKaloms
was isolated from T. baccata and given the name
taxine (Lucas, 1856), but the solution of the structural
problems posed by these complex compounds proved
too challenging for the techniques of that day. It was
early recognized, however, that the taxines had an
unusual skeletal structure, which is now known as the
taxane skeleton. Other compounds containing this
skeleton were isolated from both T. baccata and the
" cuspidata, and the structures of some
ounds were elucidated in the 1960s.
acture of the diterpenoid taxinine
composition product of taxine, was
63 (Kurono et al., 1963), and Lythgoe
kers published a series of ten papers
l structural assignments of several
RiO 0 P~
PhCOO
R=H
R=OH
Ph
taxane diterpenoids as summarized m a review artk
on Taxus alkaloids (Lythgoe, 1968). The derivati
O-cinnamoyltaxicin-I triacetate (Fig. 1), obtain
from the alkaloid taxine-I in T. baecata by Hofmal
elimination and acetylation, is a typical example
e
Harrison a]
the structures elucidated by Lythgoe (Hal
Lythgoe, 1966; Harrison et al., 1966).
The development of nuclear magnetic resonan
spectroscopy as a powerful structural tool in t
1960s, together with the advent of X-ray crystallogr
phy on a routine basis, enabled work on taxa
ids to advance much more rapidly. In pe
diterpenoid
ticular, Hadsall and his coworkers isolated nineto
different taxane
ta derivatives and reported them in
series of seven brief reports over the peri(
1966-1975. Among these derivatives was the dit(
penoid baccatin-III, which although originally
signed a different structure was finally assigned t
structure shown in Fig. 2 (Della Casa de Marcano
and Haisall, 1975). This and other work on the taxane
diterpenoids until 1979 has been well reviewed by
Miller (1980). The work on taxol itself until 1985 is
summarized in an excellent review by Suffness and
Cordell (1985), and a review on Taxus alkaloids has
recently appeared (Bleehert and Gu6nard, 1990).
2.2.
As an on
of novelan
collaborati(
Wall and 17
of the wes
et al., 1971
PhCONH
: 0
Ph~OCHs
 The chemistry of taxol
o

\
°W °"

FIG. 3. Structure and numbering of taxol.

scheme they succeeded in isolating tk Live indicated an unusual lack of reactivity of the C-1
cytotoxic and antileukemic
nmeugeimc compound compounu taxo~ ~rom me the hydroxyl
nyuroxy~ gr group in baecatin III, and proved to be
bark in 0.02% yield. The structure of taxol was harbinger of o problems to come in the semisynthes
recognized to be ~e that of a taxane diterpenoid based of taxol.
on its 1H-NMRLspectrum, but the full solution of the Structura
Structurally, taxol is differentiated from most oth¢
structural problem lena re,
required the use of X-ray crystal- taxane diterpenoids by its ester side chain at C- 13 an
niques. Mild methanolysis of taxol by its oxetane ring D; it can be viewed as tk
yl ester and a tetraol. The ester was N-benzoyl-t//-phenylisoserine ester of baccatin II
~,lac.~t~ta~,~.~ o fy X-ray
f ~ .~-xu.v crystallography
~ tjoLgaxvba~lp.Aa J tas
* o the
L a I ~ yp-bro-
-oa~- The
x a a ~ conventional
conven
~ alv~axLI~aI~a planar
IJ1~ax~L representation
t~VL~o~xa*~*avax of
~ 1 Fig.
a A ~ . . 3 is
o ao i
IaA

mobenzoate derivative, and the tetraol was character- fact rather misleading, since taxol actually has a
ized as the bisiodoacetate (Fig. 2). These data, structure best described as an inverted cup shape, in
together with an analysis of the IH-NMR spectrum which the ester side chain lies across the opening of
of taxol, enabled Wall to assign the structure of taxol the cup. The representation of taxol shown in Fig. 4
as shown in Fig. 3. The numbering system shown for was obtained by using the Chem-3D Plus program,
taxol is that recommended by IUPAC (IUPAC, and clearly illustrates its three-dimensional shape.
Commission on the Nomenclature of Organic Chem- Because of the difficulty of representing taxol and
istry, 1978). It is interesting to note that Wall's other taxanes in two dimensions, two different rep-
only a bisiodoacetate from the tetraol resentations
used in this
o, , ,,~ . proposed b
showing the
groups accu
used by Ly'
.o~ \ / ""-d.~ ~ ° " popularadvanta
of
advantage, as pointed out by Lythgoe (1961
orientation of the 16- and 17-methyl gro
reverse of that expected. The 16-methyl grc
is fl with respect to the 19-methyl group as a
must be shown with a hatched bond, whi
/o~.-"'- methyl group is • but must be shown wit]
t3~
o o~ bond. Thes~
// . ~ , , ~ . designation
.-\-- Chemical
I N ~ system for
taxol is nar~
specifically
 D. G. I. KINGSTON

AcO O OH AcO O OH

H O ' " " ~

HOo0~ 0 (.x*) H~." ~ , , ~ / ~I ff'~ .i.'0
. (,nd*) 14" HO" "if: dAcV
°
.
(.xo) ~1 PhCO0
(*ndo)
FIG. 5. Structure of baccatin III showin~ the 16Bifl and 17~ methyl ~rouos in both conventional
yl groups
epresentations.

2aR-[2act,4fl,4afl,6fl,9ct(ctR*,flS*),l~,12ct,12a~t,12bct]]. such as cephalotaxine. The alternative na name of ta

Neither the systematic name nor its associated num- B was thus proposed by Potier and his collaborat(co
bering system will be used further in this review, but who isolated cephalomannine from the trunk tn bar~
a knowledge of the system is helpful in conducting Taxus baccata (Chauvi6re et al., 1981; S6nilh S et
literature searches in Chemical Abstracts. 1984a). In this system taxol becomes taxol ta A, ,'
other compounds differing in the nature of the N-~
2.3. STRUCTURESOF RELATED group can be named as taxols C, D, and so on. B
taxol B and eephalomannine are included in Chem
Over one hundred
aunorea compounos , u n d s wire me taxane xane Abstracts;tracts as alternative names, but the original na
skeleton have been isolated from various Taxus of cephal( ~halomannine has gained the widest curre
species. While it is not the purpose of this review to and will thus be used in this review.
list all these taxanes, it is necessary to note the 10
The 10-deacetyl derivatives of both taxol
structures of s;ome closely related compounds, since cephalom~ halomannine have been isolated from T. wallit
., .,
important information on struc- ana (McLaughlin et al., 1981), and T. bacc
:elationships in this area. This section (Chauvi6n
Chauvi6re et al., 1981; S6nilh et al., 1984a) (Fig.
Lttun /o~uaCa uon
tl taxanes
taAatt~a that
unaL contain
~ ,uitLalil aa side
a l u m , chain
~,ltaiii lTl ' lh
e forme
tformer
ormer aauth~
uthors aalso
lso iisolated
solated 19-nyOroxylaaccatln
19-hydrox ,bacce
similar to that of taxol and those that are closely III (Fig. 10), while the latter authors isolated several
related to baccatin III. new taxol analogs. These included six with a xylosyl
The diterpenoid cephalomannine (Fig. 7) was iso- group at C-7, two of which had an N-hexanoyl group
lated from a plant originally identified as Cephalo- replacing the N-benzoyl group of taxol and were thus
taxus manii (Powell et al., 1979), but later shown to named as derivatives of taxoi C. Two other deriva-
be Taxus wallichiana (Miller et al., 1981). The struc- tives had fl-hydroxybutyryl groups in place of the
ture of this compound was confirmed by mild 10-acetyl groups of taxol and cephalomannine
methanolysis to a_ mixtu_re of five products,, two. of. (Fig. 9). These authors also obtained baccatin III,
entitled as baccatin III and the methyl 10-deacety
Fig. 7; the structure of the methyl ester T. baccata
by X-ray crystallography. The remain- 10-Deac
tucts were identified as 10-deacetylbac- a 'post-tax
~i-baccatin III, and lO-deacetyl-7-epi- et al., 198~
axol and lfl-hydroxybaccatin I (Fig. 8) taxol and
ated from this plant, tion that h
ification of plant
The unfortunate misidentification ~lant material 'pretaxol' fraction of T. brevifolia bark yi,
led to a name for the new diterpenoid which obscures taxol and 10-deacetyl-10-oxo-7-epi-taxo
its close relationship to taxol and erroneously implies (Huang et al., 1986). Potier and his cc
a relationship to authentic Cephalotaxus alkaloids have isolated over thirty new taxanes fr,
taxus spicata (Ettouati et al., 1988, 198.(
oA© 0o ,~u
014 these compounds has a taxol-like side ch;
~© but several
are relateq
compound
scheme fox
Section 5
.,,o ~ ~, j M .."Ik "n
 The chemistry of taxol

,P o.
L = 'o¢~
OH

.
r
OH = OAc
=. =
OH PhCOO
FIG. 7. Structure ~nnine (taxol B) and its ester side chain.

AcO OAc
1~Hydmxybaccatin I
RI=OAc R2=H

1~-Hydroxy-7a-deacetylbaccatin I
""o/~ RI=H R2=OH
AcO¢'I°~~ (
HO AcO
FIG. 8. Structures of baccatin I derivatives from T. ba
baccata and T. wallichiana.

~ho

RaCONH
,,," - H
H

HO PhCOO
Taxol (Taxol A) R~fH R2=Ac RpCsHs
Cephalomannine (Taxol B) R~=H R2=Ac Ra=CHsCH=C(CH~)
7-Xylosyl-10-deacetyltaxol R~=~-xylose R~H Rs=CHeHs
7-Xylosyl-
,ylosy 10-deacetyltaxol
y B R~=l~-xylose R2=H Rs=CHsCH=C(CHa)
Xylosyl-10-deacetyltaxol C R~=l~-xylose Rz=k
Xylosyltaxol R~=~-xylose R==,~
Xylosyltaxol B R~=13-xylose R2=,~
Xylosyltaxol C R~=13-xylose R==A
)-Deacetyltaxol R~=R==H
)-Deacetyltaxol B Rm=R2=H
)-(l~-Hydroxybutyryl)- R~=I-
10-deacetyltaxol
1U-(~-t-tyoroxyouIyryl)-
)-(13-Hydmxybutyryl)- r'hfn, n2=~,ns~ntun/~.,n2~,u
R,=I-
10-deacetyltaxol B Rs=CHsCH-C(CHs)
FIG. 9. Structures of taxol analogs.

of this compound, or for baccatin III and ]0-deacetyi lower yields. Thus in one such isolation 27
baccatin III, both of which can be converted into bark was expected to yield a total of about
~.l). Much of this work has not been taxol, or ab,
?roprietary reasons, but some trends of taxol fro
From the published work. is severely
1as initially obtained from T. brevifolia above requi
02% yield (Wani et al., 1971), and roughly thn
ale isolations have given somewhat T. brevifolia
 D. G. I. KINGSTON

R=O /3 ~Rs 19-HydroxybaccatinIII RI=Rs=H R2=Ac R3=O R4=OI-

CH=R,L BaccatinIII RI=R4=Rs=H R2=Ac R3_-O
./ ~ " ~ BaccatinVl RI=R2=Rs=Ac Ra=c(OAc,~H R4=H
RI=R2=R4=Rs=H R3=O

""" "

PhCOO
FIG. 10. Structures of baccatin III derivatives from T. baccata and T. wallichiana.

that T. brevifolia bark could never b( ma- it is known that other surveys have been carried o
nent solution to the supply problem
~roblem ot
ol taxol, since the but have not been publzshed
~ublished for
tor proprietary
proprletat reasoz
tree is slow growing and takes up to 200 years to thus Croomt reported on ornamental Taxus Tax= cultiv~
reach maturity; it has been estimated that there are as sustainable abundant plant materials ffor clinic
enough trees available to maintain a supply of taxol taxol studies, but provided no analytic~ ,tical data
only for the next 5-10 years.* support this claim. Of the three published studies,
s t
Taxol has also been detected or isolated in various first (Witherup et al., 1990) reported on the taxol a
other Taxus species. Wall's original 7ted 10-deacetylbaccatin III content of six Taxus speci~
that taxol had been isolated from 7" and The taxol content of both needles and stems w
T. baccata (Wani et al., 1971), but vere determined by HPLC, and the highest amount
given. Both tax<:ol and cephalomannine were isolated 0.01%) was found in T. X m e d i a cv. Hick
weight (0.0
~iana; a mixture of roots, stems, and
from T. wallichiana: needles, with
wi! comparable amounts in T. cuspidata (
;ed, and taxol was obtained with a
leaves were used. Capitata, T. canadensis, and T. brevifolia needh
0.001% yield (Miller
Miller et al., 1981). The trunk bark of Taxol content
cont~ of the stems was typically one half,
duced taxol with a 0.016% yield and
T. baccata produced less, that of
o the needles. The 10-deacetylbaccatin ]
0.0064% yield (S6nilh et al., 1984a). content of the stems and needles of the same plat
baccatin III and 10-deacetyl baccatin was also determined;
d in this case the highest yie
III can both be converted into taxol in high yield (0.02%) was found in T. baccata cv. Repandens
(Section 4.1), and thus the availability of these corn- needles, with other species yielding one half this
pounds is almost as important as that of taxol. amount or less.
Indeed, in view of the finding that the semisynthetic The second study (Vidensek et al., 1990) reported
taxol analog taxotere ~ (Fig. 12) is reported to have only on taxol content, but analyzed many more plant
an improved activity as compared with taxol (Man- samples, particularly of T. brevifolia. This study
gatal et al., 1989), baccatin III or its 10-deacetyl demonstrated that taxol content is highly variable,
derivative may ultimately prove to be more valuable even within the same species. Thus the 15 bark
than taxol, L[ of T. brevifolia ~ave a range of taxol content
samples
was originally isolated from heart- of 0.0001-4
ccata with an unreported yield (Chan 0.00003-0.(
td its correct structure was reported in the first grc
Lsa de Marcano and Halsall, 1975). It lia needles,
ated from the trunk bark of T. baccata the present
yield (S6nilh et al., 1984a), and from highest av,
and leaves of T. wallichiana in an seedlings a~
unspecified yield (Miller et al., 1981). Its 10-deacetyl amounts, although the latter datum is bas
derivative was obtained from T. baccata trunk bark two samples of undefined taxonomy.
with 0.02% yield (Chauvi6re et al., 1981), and also
from T. brevifolia bark in an unspecified yield n o
(Kingston et al., 1982).
The results of survevs for the occurrence of
~f three surveys / ~| J
,'acetylbaccatin III have appeared, and
PhCONH.
_ =
989) Presentation to the Division of Cancer
onal Cancer Institute, Board of Scientific Pa
zesda, MD, October 12. oH
 The chemistry of taxol

14o~,9 mOH Under these circumstances the semisynthetic

/ ~ " ' ~ x m~ ' ~ ! becomes the best route, since it opens u p ttu~ lJu~
bgs such
taxotere.
O~J"NH 0 The third option for taxol production is i that (
p n / - t , , . . . . . ~ o . . . . . . ~ o plant tissue culture. Very little has been pui )ublished i
" T~ ~ ... this area, but anecdotal evidence suggests sts that
th~ sever
se,
OH Pheoo groups have tried this approach with some some sue SUCCeS
FIG. 12. Structure of taxotere. The only reports of success thus far have been 1~ tho~ t
of Christen e t aL (1989) and Ellis e t a l . , t but the,, 1
T h ~ t h ; , A ~+,,,~. . . . o~d ~ ,~,,,,h, ~ , , ~ , ~ , ~ , ~ r~T ~SAA
The third study used a newly de~ preliminary communications have not yet been fo
technique to carry out semiquanti mi- lowed with full accounts of the work, and it is thl
nations of oI taxol-like
taxol-llKe materials in " various i a x u s ano lad difficult
. . . . . . . . . .to
. . .assess
. . . . . . . .the
. . significance
,,. . . . . . . . . . . of
. . . .these
. . . . . results, l
related genera. Both leaves and stem-bark of T. view of the importance of taxol, however, howe it
b a c c a t a contained taxol-like materials in yields of up expected that work in this area will continu will continue and wi
to 0.05% for leaves and 0.08% for stem-bark (Jaziri ultimately be successful.
e t al., 1991).
The final approach to taxol production is that(
The major conclusions from these three studies are total synthesis. Although no total synthesi ~nthesis of taxq
,L . . . . . . , . . . . . . . :.
that taxol content is very variable, t. . . . . . . . . . :~t.,~ t.. . . . ~,. . . . . . . :_
:ain has yet appeared, a synthesis
s) of the simpl pler taxa)
species, and/or cultivars, appear to Lble taxusin has been published (Holton et aI., 1988) an
amounts of taxoi and, in some cases, tyl- will be described briefly in Section 4.2. The synthes
baccatin III. The ['he prospects are thus good that a of u~ taxol
mxul is in aan extremely challenging task, and to da
careful survey of )f all the available species and cultivars there is no evidence that a total synthesis will t
of T a x u s will ,ield one or more plants that produce competitive with the other routes, but the ingenui(
taxol or 10-deacet cetylbaccatin III in adequate amounts and skill of synthetic organic chemists in addrcssir
to justify commercial ~ercial production and harvesting. It is this problem should not be underestimated.
that appropriate plant breeding tech- Based on on the current state of the art, itis likelyth:
e applied to enhance the yield of the T. b r e v i f rolia
o l i d bark will remain the preferred source (
deslrect taxanes.* ;.* It isIS particularly encouraging ' that
tlaat taxol for
. . . . . . .for the
. . . . next
. . . .th . . . . . . 2-3 jyears.
. . . . . . .After
. . . . . . .this
. . . . it
. . is eprobab
.......
the taxol precursor 10-deacetylbaccatin III is avail- that one or more cultivars of T a x u s will become the
able from yew leaves (needles), since this compound best source of taxol, while the semisynthetic route
could thus be obtained from yew plantation clippings from 10-deacetylbaccatin III could also become a
on a sustained yield basis, viable alternative in the same or a slightly longer time
The details of the partial synthesis of taxol are frame. If success at plant tissue culture or total
given in Section 4.1, but it is appropriate here to synthesis is achieved, one of the approaches could
compare this approach to that of direct isolation of become important some time after the year 2000.
taxol. The available data (Chauvidre e t al., 1981; Finally, studies currently in progress in our group
!., 1990) suggest that 10-deacetylbac- and in othe
9e slightly more available than taxol, the taxol bil
would not commend the semisynthetic ongoing str
since the required chemical synthesis the develop~
~ut two additional factors come into ble structur~
that the yield of the key intermediate, time frame.
ight be increased significantly by some
form of chemical manipulations designed to convert 2.5. N M R SPECTRAOF TAXOL
taxol and 10-deacetylbaccatin III into this com-
pound, and thus make it easier to isolate from the The 400 MHz ~H-NMR spectrum of taxc
crude plant extract. A second and even more corn- in Fig. 13. It is a very nice spectrum to
pelling consideration is that ultimately taxol probably since the signals for most of the protons arc
will riot be the drug of choice; taxotere ® (Fig. 12) or over•~.a .wide~ range, of chemical . . . . shifts, . . and
alog will become the preferred drug. relatively fe'
range. Assil
have been i
(1990) Approaches to improvement of
~axus. In: Workshop on Taxol and Taxus: 1980; Mille
re Perspectives, National Cancer Institute, The most r
une 26, 1990. Chmurny, I
 D. G. I. KINGSTON

.+, . . . . . . . . r . . . . . . . . . i . . . . . . . ~ . . . . . . . . . i . . . . . . . . . ,

9 8 7 6 5 4 3 2 1 0

FIG. 13. 4 4MR spectrum of taxol in CDCI~.

that of taxol: thus acylation at the 2

. . . . . . j . .
~os-
. . . . . . . . . . . . . . . . . . . r . . . . .
assigned by conventional 1D methods. A rece
. . m-

ition causes a downfield shift of about 0.8 ppm manuscript (J. A. Beutler, G. N. Chmurny, B.
(Mellado et al. , 1984), and removal of the C-13 ester Hilton, S. Brobst,
] S. A. Look and K. M. Witheru
side chain causes ses the C-13 proton to move upfield submitted for
I publication) reports the assignment
almost 1.4 ppm; a; this latter assignment was used to ]3C-NMR spectrum of taxol by 2D methods; t
the ]3C-N/V
,'~l~'r';#"v (I,"~'rYIP, ,'~nn'~11(:
onfusion in the literature about the IaC-NMR~assignments of taxol are shown in Fig. ]
tion position of baccatin III (Magri et A recent manuscript reports the spectrum of tax
• . is worth notingv that the ~H-NMR obtained by a solid/liquid intermolecular transJ
spectrum of taxol is quite solvent dependent. Thus dynamic nuclear polarization technique (H. C. Dorn,
the C-20 protons appear as an AB quartet with some C. Tsiao, K. H. Tsai, G. Samaranayake and D. G. I.
additional coupling in CDC13, but as a broad singlet Kingston, submitted for publication). In this method
in CD3OD. dynamic nuclear polarization enhancement was gen-
The ~3C-NMR spectra of taxol (Magri, 1985) and erated at low field using nitroxides immobilized on
of baccatin III (Rojas et al., 1983) were originally silica gel in contact with the dissolved sample. The

7.49(m)
) ~ i 7"40(m)
2.23(S) 6.27(s)
7.74(dd,1.2,8.3) *,u e,~,~ 14 2

X 1.79(.,1.5) / %~

"r.01(d,U) % "r"f I . , .... "-' I

z.s,e.9) " H I ..~*" X

Hf "°O.3.61,(bs) .~ = -.1 7 H 8 + ~ ~ + I'""H 4"19'dd,I"0,8"5)

.~ ' ~ .~,
.,'~K 4.78id,2.7)
4.78(d,2.7) "~ H ~. 1.98 == I~
I1 H H 4.30(ddd,1.1,0.8,8.4)
42(m) ~ / ,JF 0.,. .0 ~ ,
\ 2.35(dd,9.0,15.4) / ~ \
7.48(m) 2.28(ddd,O.6,9.0,15.3) /
/
"-- 7.35(tt,1.6,7.3) A

[I "]++"
 The chemistry of taxol

O
71.2

OH

129,0 C ,~,~3..0-..H ~/ I 21..

]1 ~ IOI 142"01 ~" ,~o~3.2 74"9t
12..s ~,, . . v i... II ..... '2.3 9.0
= 0

6~ / C H 3 22.6
~27.0
128.7
OH
07.0%~0 "~?0.'
16 0
ri2S.1

C
130.2

128.7

133.7

FIG. 15. "C-NMR

MR assignments of taxol.

polarized sample le was then monitored with improved
tion at high field. Because of the
spectral resolution
experimental conditions
~nditions used, ]H-NMR signals with
relaxation times, such as the aro-
;ns, showed dipolar dominated or
ncements, while signals with short
relaxation times showed only small enhancements,
Interestingly, the C-10 acetate and the C-16, 17, and
19 methyl groups showed larger enhancements than
the C-4 acetate and the C-18 methyl group,
suggesting that the convex face of the taxol molecule
is the one that interacts with the silica surface.
2.6. MASS SPECTROMETRYOF TAXOL
discoverers of taxol reported a mol-
umably obtained under electron ioniz-
ks) at m / z 853, but later workers only
it ion peaks under these conditions
~81). The FAB mass spectrum oftaxol
olecular ions at M H +, M N a +, and
,,ment ions are observed corresponding
..... ' ....... "............................. " ....... "
to loss of the protonated C-13 side chain and to the
loss of acetic acid (Magri, 1985).
Recently, a detailed analysis of the FAB mass
spectrum of taxol has been carried out, using colli-
sional activation to produce daughter ion spectra, but
the= r~ltlt~ h~ve; ~n
so f:~r
far nnlv ]'~en
only been nublished
published in
in abstract
abstract
bisiodoacetate and p-bromobenzoate derivative
(Fig. 2) determined during the original structm
elucidation of taxol have never been published. Hov
ever, X-ray structures of baccatin V (Castellano an
Hodder, 1973)
19 and of taxotere (Fig. 16) (Gu~rittq
Voegelein et al., 1990) have been published. T~
structure of taxotere (Fig. 16) clearly shows the
cup-like shape of the taxane skeleton of taxol and its
relationship to the C-13 side chain.
2.8. ANALYSISOF TAXOL
The need to carry out extensive surveys of T a x u s
species for their taxol content and to monitor taxol
in clinical sl
analytical rr
graphic me
groups are
initial resull
et al., 1991~
Most o f '
HPLC method was developed to separate t
cephalomannine, 10-deacetylcephalomanr
catin III, and 10-deacetylbaccatin I I I ,
et al., 1989). Both isocratic and gradient rev
methods were tested, using cyano and phen
silica gel columns.
arations, bu Both columns gave adeq

solvent and
and decrea
AY CRYSTALLOGRAPHYOF TAXOL
phase for t
no X-ray crystal structure of taxol has starting wit
and even the X-ray structures of the to MeOH:
10 D . G . I . KINGSTON

Ic

ay structure of taxotere.

discussed (Witherup
nerup et at.,
al., 1990).
~ v ) . Th
~ne analysis otf ~.
T. aoequateJy
adequately, and also separated several other co
brevifolia for taxol content previously noted was pounds from a T. brevifolia extract.
carried out usin sing Cl8 columns and M e O H : H : O ,
68 : 32 as the solvent,
lvent (Vidensek et al., 1990). A method
for analysis of taxol in T. chinensis has also been 3. CHEMISTRY O F TAXOL
i .1 i r~r
and Liu, 1989). Very recently another
3.1. HYDROLYSISOF TAXOL
separation of taxol and cephaloman-
published (Cardellina. 1991). This The first reactions carried out on taxol were hydt
method uses a cyanopropyl column with hexane: lytic in nature, as an aid to structure elucidation. As
iPrOH (2 : 1), and may be more amenable to prepara- previously noted, mild methanolysis of taxoi gave the
tive work than the reverse phase systems previously methyl ester of the side chain and a tetraol, which was
described, identified as 10-deacetylbaccatin III (Fig. 2) (Wani et
Analysis for taxol in biological fluids has also used al., 1971). Mild methanolysis of cephalomannine,
HPLC. After evaluating C8, cyanopropyl, and phenyl however, gave a mixture of six products, identified as
bonded phases, Riley and his coworkers (Rizzo et al., baccatin III (19%), 10-deacetylbaccatin III (19%),
1990) selected a Cs bonded phase column with a 10-deacetylcephalomannine (5%), baccatin V (17%),
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ff MeOH :NaOAc buffer (0.02 M, pH 10-deacetyl

as their system for analysis of taxol in ester was a
and urine. A reverse phase HPLC vigorous 1
used for analysis of taxol in human yielded the
ine during the Phase I trials of taxol product: tt
! al., 1987); in this case a Cls column complex an
a gradient of H20:MeCN, 65:35 to been carrk
0:100, as the mobile phase. Similarly, a reverse phase products by HPLC over a time period
method was used for the analysis of taxol and its (Ringel and Horwitz, 1987); under the col
decomposition products in cell culture media (Ringel this study (pH 9, MeOH) taxol was conver!
and Horwitz, 1987) and in rat bile (Monsarrat et al., ily to baccatin III, with much smaller a
1990). 10-deacetyltaxol, 7-epitaxol, 10-deacetyl-
Two TLC methods have also been developed for baccatin V, 10-deacetylbaccatin V, and 1~
txol in plant extracts (Stasko et al., baccatin II
ystem a cyano modified silica gel plate A selecti
two dimensional development using ester side c
base (CH2C12: hexane: AcOH, 9 : l0: l) developed |
phase (H20 : MeCN: MeOH :THF, et aL, 1986)
,elopment. The other system used a or cephalot
 The chemistry of taxol
RiO O R= O

I ,

"- OH
PhCO0

baccatin III RI=AC, R2---OH, R3:H

10-doacotylbaccatin III RI=R3:H R~--OH
baccatin V R1--Ac, R2=H, R3---OH
I0-d¢ :in V RI=R2=H R3---OH
Fm.
l'lCJ. I /. r r o o u c t s of
Ol methanolysis of taxol B.

the borohydride, and does not proceed if this yield some additional products in low yie deld, whic
group is acylated or converted to a silyl ether, were characterized as the 2,10-dideacyl derivatix
The reaction thus offers a pathway for the con- and the 2,4,10-trideacyl-13-de(triethylsilyl) derivatix
version of cephalomannine into taxol by conversion (Fig. 20). An alternate interpretation of these
the resul
baceatin III
to baccatin 1II and reacylation
reacvlation a,, as described in is that the neighboring
nei~hborin~ group
~rnun effect is not relevan
Section 4.1. but that access to the C-4 acetate is blocked by t~
Studies have also been carried out J ton bulky C-13 triethylsilyl group.
group on the selecuve selective deac,oeacylauon
dation ot ( t~accatm III, Ill,
with the conceept that reacylation might produce
3.2. EPIMERIZATIONOF TAXOLAT C-7
novel derivatives es with improved properties (Sama-
ranayake, 1990). ). Hydrogenation of baccatin III over The C-7 hydroxyl group of taxol is part of
Pt yields a bexah ahydro derivative by reduction of the fl-hydroxycycarbonyl system encompassing C-7, C-
the A 11 double bond is completely and C-9, anda as such is subject to epimerizatiol
uction under these conditions. Hydro- presumably via a retro-aldol/aldol mechanism (Fi
t y u t ; SLUO.leS w ~ere
r e tcarried
;ariletl oout
ut oon
n the
t i l e 7-triethylsilyl
/-trletnyl~llyl 21). Thus
LI). thUS !t r e a t m e n t
treatment oof cephalomannine
l c e p n a l o m a n n l n e vwith
~m ba~
oase

ether of this bexahydrobaccatin III, to avoid prob- yielded derivatives both of baccatin III and of 7-epi-
lems with epimerization at C-7 (see below, Section baccatin III, or baccatin V (Fig. 17) (Miller et al.,
3.2). Mild hydrolysis of 7-(triethylsilyl)-hexahydro- 1981), and 10-deacetyltaxol and 10-deacetylcephalo-
baccatin III with methanolic sodium methoxide at mannine were both partially converted to their
25°C yielded the 10-deacetyl derivative and the 4,10- 7-epi isomers on standing in aqueous methanol
dideacetyl derivative to about equal extents; there (McLaughlin et al., 1981). The driving force for the
was no indication at all of the formation of any epimerization may be in part due to the formation of
2-deacyl or 2,10-dideacyl product. Further hydrolysis a hydrogen bond between the 7-epi-hydroxyl group
of partially deacylated derivatives and the C-4
to 2-debenzoyl-4,10-dideacetyl-7-tri- gen bond is
in III (Fig. 19). (Castellano
ydrolysis of the tertiary C-4 acetoxy be an overx
of the secondary C-2 acyloxy group epimerizatic
ae due to a 'neighboring group' effect 1981), and il
droxyl group. This group is actually at equilibril
located relatively close to the C-4 acetate (Fig. 16), deacetyl derivatives appear to epimerize rr
and can thus act as a nucleophile to attack the readily than the corresponding acetylated d
C-4 acetoxy group intramolecularly. In support of The reason for this is not fully clear, but is
this hypothesis, treatment of 7,13-di(triethylsilyl)- associated with hydrogen bonding from
hexahydrobaccatin III under the same conditions as hydroxyl group to the C-9 carbonyl grot
used earlier gave only the 10-deacetyl derivative, associated activation of the C-9 carbonyl
l base for an extended time period did retro-aldol

RCONH
Bu4NBH4
o
12 D . G . I . KINGSTON

AcO. 0 OSIEI~ HO 10 o0 OSiEts

NaOMa
H MaOH H
o.~ po

J
J

.o "d
HO

FIG. 19. Hydrolysis of 7-(triethylsilyl) hexahydrobaccatin

hexah3 III.

n of taxol to 7-epi-taxol was observed
when taxol was heated with the free radical initiator
azobis(isobutyronitrile) (AIBN)(Huang et al., 1986).
The reaction proceeded cleanly and in high yield with
the first batch of AIBN used, but later attempts to
repeat this action have given poor or negative results,
The reason for this discrepancy is not known, but
presumably associated with a trace impurity in one of
the batches of AIBN. 7-Epi-taxol has also been
isolated from T. brevifolia (Huang et al., 1986), and
epimerization of taxol to 7-epi-taxol has been ob-
served in cell culture medium; baceatin III is also

AcO ,O OSIEI3
o~OO OSIE
13 NIOMa
aSI MaOH EtsSl

~/~ ~ Major
produ(:t
 The chemistry of taxol

AoO. _O OH AoO O O AoOl /O OH

o-x -o ;;o'\
PhOOO
PhCOO PhCOO
FlG. 21. Mechanism of epimerization of baccatin III in hydroxylic solvents (SOH).

formed in small quantities under these conditions group with Zn/AcOH yielded an unprotected OX;
/11-----:
_ A t - - - - - _ J TT:-----~--_ srhoo\ "rl- . . . . . . . ~.
(Ringel and Horwitz, 1987). zolone (Magri and Kingston, 1988). The unprotecte
oxazolone was also prepared from taxol by treatmel
3.3. ACYLATIONOF TAXOL with carbonyldiimidazole (Deutsch et al., 1989).
In addition to acetylation of taxol, vanous
vari oth(
Taxol has three free hydroxyl groups, but the one 2'-acyltaxols have been prepared in attem Ipts to d
at C-1 is tertiary and thus inert to normal acylation velop water-soluble prodrugs of taxol. The T~ 2'-po
conditions. The 7-hydroxyl group is relatively ition is the preferred position for this appro )roach, sin(
crowded, so mild acetylation of taxol yields the acyl derivatives at this position are readily hhydrolyze
2'-acetate
9 preferentially. Acylation
' _ ~ r ~ t ~ t a nr@t'ar@nti*llv un
Arvl~tinn u nclar
m n r F .vionr . . . . . . . .
for- and could thus readily be converted to taxol in viv
. . . . . .

ous conditions leads to the 2',7-diac ,ery The fact that these derivatives are the easiest !
mild hydrolysis of this converts it t, :ate prepare is of course an added bonus.
(Mellado et el., 1984) (Fig. 22). The first derivatives of this type were prepared lc
A better way of preparing 7-acyltaxols is through Magri and Kingston (1988), who prepared 2'-su~
the use of more ~re selective protecting groups. The cinyltaxol aand 2'-(fl-alanyl)taxol formate (Fig. 24
chloroacetyl group oup has worked well in our hands for Very simila
similar types of derivatives were prepared t
this purpose, since ince it is readily removable from the Zalkow and and his collaborators (Deutsch et el., 1989
treatment with thioethanolamine or These ~se inves
investigators prepared 2'-succinyltaxol and 2
oist silica gel (Samaranayake, 1990). glutaryltaxc
dtaxol and their sodium, triethanolamm(
lloroethyloxycarbonyl derivative
i ne z,z,z-mcnioroemyioxycaroonyt aenvauve has
nas nium, and N-methylglucammonium salts. They also
also been used: it is selectively removable by treat- prepared the amide of 2'-glutaryltaxol and 3-(di-
ment with zinc and acetic acid (Chenu et el., 1987). methylamino)-l-propylamine, and attempted without
The 2,2,2-trichloroethyloxycarbonyl (troc)group success to prepare 2'-glycyltaxol and 2'-(phenyl-
also can function as an electrophilic center for intra- alanyl)taxol (Fig. 24). Various dimethylaminoacyl
molecular displacement. Thus treatment of 2',7- derivatives of taxol in both the 2'- and 7-positions
di(troc)taxol with the base 1,8-diazabicyclo[5.4.0]- have also been prepared by Stella and Mathew
undec-7-ene (DBU) yielded an oxazolone as the (1990).
single major product (Fig. 23). Treatment of this In
In recent
recent work Kingston and Zhao have nrenarad
dilute HCI during work-up also gave three new i
,1 oxazolone, while removal of the troc solubilizing

AaO. o OH
b
O ~ " ~ l Ao2OtPY PhCONH

OH PhCOO AcO PhCOO

I A©20/DCCIDI
12h r.t.

o "Y~/L, I I ,..co. phCO,,

II I @'~ ~ A --CH.OH/H.O
14 D.G.I. KINGSTON

AcO O OCOOCH=CCI
3 AcO O OCOOCH=(

PhCONH O ~ ~ - " DBU~ Ph O ~ l ~ ~~ ~

Ph PhCON
CI3CCH2OCOO PhCOO ~O PhCOO

O I HCI
Zn/AcON J
J AcO O OCOOCH=CCI
3
AcO

Ph o

HN/ - vn = . . . .
'~O PhCOO
PhCOO
O
O
FIG. 23. af oxazolone derivatives of taxol.

was prepared by the Michael addition of sodium In additi

metabisulfite to 2'-acryloyltaxol, and the two sulfo-
amidosuccinyl derivatives were prepared by a new
method involvin ing the use of tetrabutylammonium
aaqueous solvent to avoid aqueous
he accompanying degradation of the
tcyltaxol (Zhao and Kingston, 1991)
(Fig. 24).
All of the water-soluble analogs described showed
improved solubility as compared with taxol. The three
last compounds described, for example, are between
100-200 times more soluble than taxoi. The 2'-(fl-
alanyl)taxol prepared by Magri and Kingston (1988)
was rather unstable, reverting spontaneously to taxol
in less than 20 hr at 37°C and pH 6.6, and reacting
methanol solution. The 2'-glycyl and
d)taxols that Zalkow attempted to pre-
asly even less stable, and could not be
ioactivity of these water-soluble taxol
be discussed in Section 6.1.
iition to the 2'-acyl derivatives, 2'-silyl eth(
have been prepared by treatment of taxol with t
ppropnate silyl chloride. Both 2'-(triethylsilyl) ta~
appropriate
It-butyldimethylsilyl)taxol have been prepar
and 2'-(t-bl
(Fig. 25)(?(Magri and Kingston, 1988; Magri, 198
lation studies on baccatin III will be describ
Acylatio]
in Section 4.1.
3.4. OXIDATION
Oxidation of taxol could in principle occur either
at the 2'- or the 7-position. Here, however, the
reactivity order is reversed as compared to acylation,
and the 7-position is the most reactive. Thus oxi-
dation of taxol with Jones' reagent under mild con-
ditions yields 7-oxotaxol, while the use of excess
reagent anc
form 2',7-d
ing with the
added metl~
and Kingst

AoO O

' i
OR
OH
PhCOO
iAC(

2'-succinyltaxol R=COCH
2'-(13-alanyl)taxolformate R=COCH:
2'-glutaryltaxol R-COCH
2'-glutarylamino amide derivative R=CO(CI-
 The chemistry of taxol

~o
---I I/
o OH
V "
osmium tetroxide under catalytic condition,,
_] L o ~,- - - ,- ~ , . ~ in a very clean conversion to a diol (Fig. ~2!) . • ~LAI
was unaffected by these conditions, and could
cou] readil
be separated from the more polar diol byy simple
sin ttas
PhCONH O column chromatography. This process thus provid(
Ph" Y. ^"" 0 a simple and efficient method to separate ttaxol an
cephalomannine (Kingston et al., 1992a).
OR PhCOO
3.5. REDUCTION
2'-(triothylsilyl)taxol RfSiEt3 Taxol is very resistant to reduction reactions.~.Th~ ?
2'-(t-butyldimet hylsilyl)taxol R, hydrogenation of taxol under mild conditions leave
Fl(:;. 25. Silyl taxol derivati~ the compound unchanged, while more vigorous col
ditions
OltlOnS lead leao to the me reduction reOucuon of ot the
me phenyl
pne rin!
Treatment of 7-oxotaxol with 1,8-diazabicycloun- (Magri, 1985). The C-9 carbonyl group is is also
a resis
decene (DBU) in dichloromethane at 25°C or even by ant to reduction under various conditions, Slpecifical
including hydride reagents, such as sodium sodiun boroh2
chromatography on silica gel gave a ring-D seCO dride. Inspection of the structure of taxo taxol (Fig.,
product in quantitative yield. This ring-opened
product could be reduced to its dihydro derivative, shows that this group is in a very crowded
crowded envirot
. . . . . ~ _ _ J ~,~ ~'c___ _". . . . . . . VEt_ ~_ _J ~_~'__ _
~ne ment, and is thus inaccessible to reducing agents. 32
but this proved to be unstable and yi
red only reduction reaction that taxol undergoes readi
by the process of retro-Claisen conden
is the reductive cleavage of the C-13 ester side chai
by lactone formation (Fig. 27) (Magri
on, discussed in Section 3.1.
1986).
~halomannine differs from taxol in having a
Oxidation of taxol with activated manganese diox- Cephalon
~osed double bond, and this can readily be reduce
ide under mild basic conditions yielded 13-oxo- exposeddot
on catalytic ytic hydrogenation (D. G. I. Kingston an
resumably by hydrolysis of the C-13
baccatin III, presumably
C. A. Ivey, unpublished work). In all other respec
and oxidation of the liberated allylic
~halomannine is as unreactive towards reducir
) (Wani et al., 1971). In support of this cephalomar
agents as taxol.
cetylbaccatin III can be oxidized with
chromium trioxide in pyridine to its 13-oxo deriva-
tive; this reaction is reversible in that the 13-oxo 3.6. REACTION WITH ELECTROPHILF.~
derivative can be reduced stereoselectively to yield As already noted, taxol is very labile to basic
10-deacetylbaccatin III (Fig. 28) (S6nilh et al., 1984b). reagents, but is reasonably stable to acids, surviving
Oxidation ofcephalomannine could also be carried treatment with cold dilute sulfuric acid unchanged.
out with osmium tetroxide, since this compound has However, it does react with various Lewis acids under
an accessible double bond in the side chain. The more vigorous conditions.
separation of taxol and cephalomannine is a difficult Reaction of taxol with ZnBr 2 in MeOH at room
mg very careful chromatography, and temperature
ation is usually not possible in a single of 10-deace
, oxidation of cephalomannine with 30); the 7-et

CrO=/H*

,,oo,.._=
H
o
X ~
i i
PhCONH
...... itl V.r,

OH PhCOO OH PhCOO

CrOI/H*

AoO O O

o ,,.o. PhOONH
16 D . G . I . KINGSTON

AcO .0 0 DBU ~ AcO 0 0

,OH
PhCO0
OH PhCO0 OH

AcO
I Hz/Pt

O O

.h°O,, O
PhCONH 0

:
OH PhCO0 ~O'"r~O
OH PhCOO
FIG. 1 and reactions o f D-SCCOtaxo].

HO O OH HO O OH

CrO~'py ~
N
N"BH4

O
O
.

PhCOO PhCOO

FIG. 28. Oxidation of 10-deacetylbaecatin III.

that this isomer is the more stable form in the
10-deacetyl series (Jitrangsri, 1986; Samaranayake
et al., 1991).
The reaction of taxol with zinc chloride has also
been carried out by Potier and his collaborators
(Gu6ritte-Voegelein et al., 1987), but this reaction
ve been done under more vzgorous
led to a ring-opened product similar
ed below,
found change accompanied the reac-
ith triethyloxonium tetrafluoroborate
agent). Reaction with this powerful
der mild conditions, followed by an
aqueous worK-up,
u yzelded
'ielded a mixture ot
of products
)roducts with
one predominating. This product was shown to be the
20, O-secotaxol derivative 20-acetoxyl-4-deacetyl-5-
epi-20, O-secotaxoi (Fig. 31) by a combination of
spectroscopic techniques and chemical conversion to
its isopropylidene derivative (Samaranayake et al.,
Treatment of either taxol or the Meerwein prod-
uct with acetyl chloride yielded a second novel
product, which was shown to have the structure
indicated in Fig. 31. This product is presumably
formed either by a cationic mechanism or poss-
ibly by a concerted rearrangement in which hydro-
gen abstr~
is concerte
acetate fro~
1991).
The rec(
chloride pr
a taxol der
contracted
contracted,A-ring (:Samaranayake et al., 19
ment of 2',7-di(triethylsilyl) taxol with tr
fonyl chloride and triethylamine, followed
tection, yielded the 11(15---1)abeotaxol de
Fig. 32. This compound possesses all the
groups of taxol except for the C-I hydro
but the skel

OAc 0 OH

OsO4
"NH O
 The chemistry of taxol

HO O OH HO oO OH

PhCONH 0 ~ PhCONH 0

OH PhCOO OH PhCOO

FIG. 30. Products of reaction of taxol with ZnBr2.

AcO /O OH AoO O OAc

hO0,, 0 / / PhCONH

Ph
OH
0""~ O'

PhCO0 OAc
OH Ph

Ok=
& °°~

PhCO0 OAc
OA©

20-Acetoxyl-4-deacetyl-5-epl-20,O.! Acetylohlorldeproduct
FIG. 31. Structures of l reagent and acetyl chloride reaction products.

bioactivity of this
Lhis important derivative will be dis- affinity labeled
labe derivative and a fluorescent labele
cussed in Section 6.3. derivative. The
' photoaffinity labeled derivative w~
prepared from 4-(l-azi-2,2,2-trifluoroethyl)benzo
EPARAT1ON qOF LABELED TAXOLS
3.7. PREPARATION Nassal, 1983) and taxol by protection of tax(
acid (Nassa
at the 2'-pc)osition with chloroacetyl chloride, acyh
l in Section 6.2 below, acylation of tion with ttthe azibenzoic acid at the 7-position, an
position does not change its tubulin- deprotectiol
protection (Samaranavake, 1990). The resultiv
assembly activity significantly. It has thus been poss- azibenzoyltaxol (Fig. 33) had an ICs0 comparable
ible to prepare various 7-acyl taxols with specific with that of taxol, indicating that it was an excellent
labels for biological studies on taxol, promoter of microtubule assembly and valuable for
The initial studies of taxol binding to microtubules photoaflinity labeling of tubulin; studies in this area
were carried out using a tritiated taxol prepared by a are in progress.
specially developed tritium exchange procedure The fluorescent labeled taxol was prepared by
with 3H20 o v e r rhodium on alumina (Parness and acylation of a 2'-protected taxol at the 7-position with
Horwitz, 1981). This procedure gave a mixture of five N-(Cbz)-/~-alanine, deprotection, and reaction of the
J I- . . . . . . . . . J . . . . . ".__.z . . . . z'..1 1-
;ver, and required careful chromatog- //-alanyl ar
ate the labeled taxol from the other resulting dt
)ducts. A simpler method of preparing same ICs0
taxol derivative was thus developed and also sh,
:etic anhydride to prepare 7-[3H- is thus a po
lenu et al., 1987). The labeled 7-acetyl- the biologic
C50 for stabilization of microtubules techniques q
a b o u t [Wlce m a tt oz
of [axol,
taxol aand is [thus
n o 15 useful as a pprobe
n u s useiul robe
for biological studies.
Since taxol binds reversibly to microtubules, there
4. SYNTHESIS OF TAXOL
has been interest in the preparation of labeled taxols
that could assist biological studies of this binding. At this time the total synthesis of taxol 11
Two such derivatives have been prepared: a photo- been achieved, but three different partial

1. Et~lSIClllm/dllzole Ph(
2. MsCI/NEt;
18 D.G.I. KINGSTON

AcO o oa tive TLC to give both isomers as pure comt

similar sequence of reactions, but using the
PhCONH 0
hydroxyamination methodology in place of o osmiu
tetroxide alone, yielded a mixture of the two t, ami
Ph
~ " .-- A
° " " ~ " v " ~ r 6H V " 'c' ~d '° alcohol derivatives shown in Fig. 34, tog(ether wi
their respective diastereomers (Srnilh et al., ai 19841
OH PhCO0 An improved version of the hydroxy;aminati amin
approach led to the synthesis of taxol aas w ,well
azlbenzoyl taxol several additional side chain analogs (Mang atal :atal et
R. co 1989). Thus 7-(2,2,2-trichloroethyloxycarbonyi)~yloxycarbonyi) b~
CFa
catin III was converted to its 13-cinnamate ester est
reaction with cinnamic acid, dicyclohexylcarbo~
danllyl taxol R m COCH2CH2NHSO2~" imide (DCC), and DMAP. This ester was then su
m jected to hydroxyamination with N-chloro-N-sodi N-chlorc
N(eH~h tert-butylcarbamate, silver nitrate, and osmiu
tetroxide in acetonitrile. The reaction gave negligit
stereochemical induction under these conditions, cond b
FIG. 33. Labeled taxol derivatives, of tt
some stereocontrol (up to 80% the desir
stereoisomers)~) was obtained with chiral amines
from baccatin III have been publishe, :tial inducers. However, both regioisomers were obtaine
syntheses will thus be discussed first, Lion and thus the yield of the desired stereo and regiois
will conclude with a discussion of o: I to mer was low. In the 10-deacetyl series the yield w
the taxane skeleton. 12-25%, depending
d on conditions, and yields we
not cited forfe the 10-acetyl series. Deprotection of t
t-BOC group,
grol benzoylation, and removal of the tr
4. I. SYNTHESES
YNTHESES FROM BACCATIN III
group then completed the synthesis of taxol (Fig. 3
the synthesis of taxol from baccatin The yield of o these last three steps was 60%, giving
:eptively simple, since the key step is overall yielield of taxol from 7-troc-baccatin III
LIII~ I~IL~IIII~GtlklII n UofI the
tlI~ kC-13
a-l.J hydroxyl
IlffUlk/Affl group
~lt/U 1 ) with
VVII.II a
(l about
O .UI3UL 14°A
l14%.
"r /ll, /A
a- similar ~process
L.~IIIIIIKI/. /l~/t.4~OO was
VI(J,O 6also
1OI) used
UOFU I.k/

suitably protected derivative of the side chain acid. prepare 10-deacetyltaxol from 10-deacetylbaccatin
However, the C-13 hydroxyl group of baccatin III is III.
very hindered, and may also be hydrogen bonded to One interesting and important finding from this
the C-4 acetoxy carbonyl group. Acylation at this approach is that N-t-butoxycarbonyl-10-deacetyl-
position is thus very difficult. As an example of this taxol, which was prepared from 10-deacetylbaccatin
hindrance, baccatin III is preferentially acetylated at III by a simple modification of the route described,
C-7 rather than C-13, and preparation of 13-acetyl- showed excellent in vivo anticancer activity (Colin
baccatin III required protection of the 7-position, et al., 1988). This compound has been named tax-
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
th excess acetic anhydride in the otere®, an,
lridine and 4-dimethylaminopyridine The seco
deprotection (Magri et al., 1986). developed t
on study of 10-deacetylbaccatin III tion with C
ut by Potier and his collaborators essentially
elein et al., 1986). Under mild con- baccatin II
20°C) the 7-acetate and 7,10-diacetate forcing co]
were formed in about equal amounts. Under more synthesized
,nthesized by Greene and his collaborat
vigorous conditions (48 hr, 60°C) the 7,10-diacetate et al., 1986) in a pathway built around
and 7,10,13-triacetate were formed in equal amounts, epoxidation reaction (Fig. 36). Epoxidati
while under the most vigorous conditions (24 hr, cinnamyl alcohol with t-butyl hydropero:
80°C) only the triacetate was formed. These results presence of titanium isopropoxide and (
suggest that the order of reactivity of the hydroxyl tartrate yielded the epoxide in 61% yield
eacetylbaccatin III is 7 > 10>>13. enantiomer
Lese results, Potier then developed a yielded the
is of taxol and various analogs from went regio
nd 10-deacetyl baccatin III. Initial trimethylsil
d conversion of 10-deacetylbaccatin was benzo,.
)c) derivative, acylation at C-13 with duction of
 The chemistry of taxol

HO O OH 00~OCOOOH:OO~

.o~o HO"~ ~.o"°'

~ ~V ~
I "\
PhCOO
~ ~
PhC06 O

AgCN,toluene
110 Q

.o cc~,c.~ocoo._~ ocooc.,cc~,
. ,,v -- un 1 . 0804 --':-,,z'--~~,.~'~r~
OH 0 3. Separation ~ I "~,

HO PhCOO PhCOO
1. CINaNR/AgNO:/OsO4
2. 7.n/AoOH
3. SelmrMIonof isomers

HO O OH

,2%
Ao o
NHR PhCOO OH PhCOO

R ,, SO2CsH4CHs or COOCaHs
FIG. 34. Synthesisof taxol side chain analogs.

et al., 1990). In this approach (Fig. 37) the readily pholine N-oxide (NMMO) and dihydroquinidine 4-
Lyl cinnamate was hydroxylated by chlorobenz(
)rocedure, using catalytic amounts of recrystallize
ide in the presence of N-methylmor- material in

~o o ocooc.,cc,~
"~*'~,1 hydr°xyemlnetl°n
O ~ ~ ButOOONH

,h .o PhCO0
,h OH
o. PhCO0
o
+ otherisomers [ (CHs)sSII
I
AoO O OH

% J ~ " ~ ~1. PhCOCl

~ ~ ~ 2"Zn/A°OH NH'
20 D . G . I . KINGSTON

• RuCI~,Halo4 0
ph~"~CH2OH ~ 2. CH2N2 ~--
(+)-DET p 1.12OH P OOCH3

1. MesSIN3
2. PhCOCI

PhCONH
: o N, o
II H2/Pd ph~ ~ L ~ O C I . I ~
ph" ~ ~ O C H s
i
OF
S
OCOPh
FIG. 36. Synthesis of the taxol side chain acid.

OsO4, NMMO, OH / - O\
1. TsCUEt3N
phff~,,/COOCH~ DQCB ~- COOCH:
2. K~COs =-
Ph P~ COOCH3
r
OH
F~C te route to the key epoxide.

gave only the C-2

:-2 tosylate, and treatment of this with In his studies
stud on the synthesis of some side ch~
base yielded the ae same epoxide as that prepared in modified taxol
t~ analogs, he coupled 7-(troc)baccal
their earlier synthesis.
tthesis. The remaining steps of the syn- III with va
various protected acids (Fig. 40). He fou
thesis were modified dified slightly, and the final ester was that the coupling
cc of fl(benzamido) acids proceed
L~--:--- J :- AILIn~
/0 overall yield from methyl cinnamate, with unexlpectedly higher rates than coupling
:oach to the taxol side chain acid has tier aci~
simpler acids: thus the top reaction shown in Fig.
shed; this pathway involves a fl-lactam proceeded much more slowly than the bottom re.~
as the key intermediate (Palomo et al., 1990) (Fig. tion, even though the degree of steric hindrance is
38). Reduction of the indicated azetidine-2,3-dione comparable for both substrates. This result was ex-
with sodium borohydride gave an ~-hydroxy ~- plained by postulating the intermediacy ofa 5,&dihy-
lactam as a single diastereomer. Protection of the dro-6-ketooxazine derivative (Fig. 40). 5,6-Dihydro-
hydroxyl group and N-dearylation gave the protected 6-ketooxazines have been shown to be acylation
~-lactam which was opened with methanolic tri- reagents,and would be less sterically demanding than
methylchlorosilane and benzoylated to yield the de- their acyclic counterparts; it is thus very probable
sired taxol side chain, that the synthesis of taxol by Greene described above
of the taxol side chain and baccatin proceeds t
:hieved by the pathway shown in Fig. (Swindell e
,1988). The starting material used was The fin~
=atin III, available from T. baccata reported b)
s of about 0.1% (Chauvi6re et al., is the coup
; was converted to its 7-(triethylsilyl) baccatin II]
then acetylated to give 7-(triethylsilyl) an excellen
baccatin Ill. Treatment
['reatment of this protected
arotected baccatin
baccatm have yet to be pubhshed. An enantloselq
derivative with the side chain acid previously pre- thesis of the key fl-lactam has recently beer
pared (protected as its 1-ethoxyethyl ether) in the (Ojima et aL, 1991), which increases the v~
presence of di-2-pyridyl carbonate (DPC) and approach significantly.
D M A P gave an 80% yield of coupled product at 50% In evaluating the three synthetic app~
conversion, or a 60% yield at 85% conversion, appears that the second Potier-Greene ar
Le protecting groups with 0.5% HCl the Holton
8% overall yield from 10-deacetylbac- strategies fi
ning no recycling. III. The yic
sm of the key coupling reaction of this superior in
been clarified by a recent paper by but the Pot
is collaborators (Swindell et al., 1991). and enanti(
 The chemistry of taxol
o. ....ph ,o.. /h ClC,.COO. ..i'h
* NISH4 °°*"J--"'J "" CICHaCOCI, CsHsN
c..c,..,o-- °///
"**"
,i
""
,,'.

~OCHs i CISIMlla,CHaOH

PhCONH N.I'Ii O
: O PhCOCI, NEts

ph " = " ~ ~ O C H i
ph. , ~ CHa i
:
6H OH

FIG. 38. Synthesis of the taxol side chain by Palomo et al. (1990).

taxol models by partial synthesis. The approach of 4.2. APPROACHESTO TOTAL SYNTHESIS
Swindell has already been alluded to: this approach
can be characterized as the synthesis, ha At this time no total synthesis of taxol has bee
modified side chain. reported, and only one total synthesis of a natural
Two groups have prepared analog,, 2tct occurring taxane has been accomplished. A detaile
side chain but a highly modified 'taxane' nucleus, treatment of
ol all the varied synthetic approaches to tt
)eAmicis (DeAmicis, 1988) prepared
Pirrung and DeAmicis taxane skeleton
skel that have been used by differet
racemic taxol side
ide chain from commercially available workers is beyond
1 the scope of this review; a fulh
racemic threo- rphenylserine and optically active side discussion of these approaches is contained in
chain by a moclification of the original Greene route, recent review
revie (Blechert and Gu6nard, 1990) and v~
ctive side chain was then coupled with also be forthcoming
for in a future review (Kingstc
diiodoalkanes in order to prepare et al., 1992).
199, In this section we will thus simp
esters (Fii
symmetrical diesters (Fig. 42). H<
However none ofo1- the describe the one synthesis
describ 'nthesis of
o1 the enantiomer ofor a
three diesters prepared showed any tubulin-assembly natural taxane that has been published to date, as an
activity, indication of the complexity of the task of the
Fillion (1990) has prepared a naphthalene carbo- synthesis of taxol.
lactone ester of the taxol sidechain (Fig. 43). Regret- The one taxane that has been synthesized is taxusin
tably no biological data was presented on this (Holton et al., 1988), or rather enantiotaxusin, since
ester, the natural product used as a starting material had

HO r~ _.. AcOI oO OSIEt~

1. EI3SlCI, CsHsN \
2. CH3COCI, CsHsN ~--

PhCOO
PhCONH O
DPC, DMAP
Ph OH CsHsCHs
OCHCHs
I
OCHiCHs

AcO .o oN
)-q)..
T 1 -"°'"'°.oo..
..,,t.
~.2 D . G . I . KINGSTON

AcO. .O OCOOCH.aCCI
3 AcO O OH

I I ~. 6c.,oc.,cc~ \ xl" T "

I" ..~ L J, Dec, o ~ e o Y/ I.,, I
.,L " " X . . _ _ / a . . " x / \ ,.,n,,oo. - II I ",,
HO~'~ O t~
: - :
OCOPh OH OCOPh
PhCONH
~,,,COOH DPC. DMAP
CI3CCH2OCH2()

Ph
.I)-.o 1.7-(troc)baccatln III
2. ZnlAoOH o
:.~o,,"~ :d%,:o
C13CCI'12OCH20 OH OCOPh
FIG. 40. Synthesis of taxol analol [1 et al. (1991) showing the postulated 5,6-dihydro-6-keto-
oxazine intermediate.

the incorrect absolute

bsolute configuration to give natural
taxusin. The atpproach involves the conversion of
patchouli alcohol
lol via previously developed chemistry
: the complete synthetic pathway is
44. Clearly this pathway represents a
highly sophisticated and yet efficient approach to
taxusin syn,nthesis: when the reactions are carried o
in the most
mosl efficient way the overall yield is greal
than 20%.
The task of modifying the taxusin synthesis
allow the synthesis
s'. of baccatin III (and hence tax(
is clearly a formidable one, and it is not clear at this

Ph
Aco..~ A~o.,,.,, .,"'Ph Aco,,.,,, ,,"'Ph
COCI
J iPr2NEt O~.~ CAN ~
I CH2C12 .N NH

,
OCHs

~0,%,o ,,",,,Ph ElO~ , . , , O,,,,,o ,,",,,PI

,j j, -oo°, ;, n
N Nit
NeOPh
I 7-(trlethylsllyl)ba¢callnIII
DMAP,pyrldlne,25°

O ~ O H÷ ~--- PhCONHi

,~ ph ~
 The chemistry of taxol
PhCONH
= O
)hCONH O O NHCOPh

Ph HO
OH OH

x. \(c.d" . o,

FIG. 42. Dimeric taxol side chains prepared by DeAmicis

and Pirrung (DeAmicis, 19
_ _ J ~ . . . . . . /T'%_&__:_.'_ 1Noo\
F]G. 43. Naphthalene carbolaetone ester of taxol side cha
lr'~.-- A~ XT----L~L--I . . . . . . L--I----~ . . . . . *__ _L'. .... 1-~'J-

tBuLI -~ ~ ~ , ~ t-BuOOH
TllOIPr)4

O
O ~ BF'J'EtzO

1. LDA/TMSCI
~2. PhSeCI ~PDC
-3. H202 ~OMF
4. MeOH/K2CO2
HO
I 1. BrCH2CHBrOCHs
2. BusSnH, AIBN/PhNMe2

2 c~cooo.- o,,,~o~7~./,~ e.'coo / .0 "

1. CH3COOOH
1. OMe 1.(CHs)aC(OCHs)2TsOH I~ 2. TI(OIPr)4
M"O × 2. But~aSlTf, pyrldlne
"L K.CO._ MmOH
O" 2. H aO O O 3. K2

~ Sml2, THF
1. L
X 1" FeCh' Ac20 X 2. mCPBA
2. NaOCH:, CHsOH 3. Bu4NF

~
O O ~,, O O .~ Ace OAc
~. I 3. T~40, pyrldlne 4. HCI ~ ~
~ 4. t-BueNa, THF 5. Ac20, pyrldlne
elm

Ac,O~
'O'rBS O
).4 D.G.I. KINGSTON

;tage whether the synthesis could be modified in an epoxide rings. These experiments do not
appropriate way. Thus, although it is likely that the hypothesis that the taxane skeleton l ; b I U I ] I I
9accatin III will ultimately yield to total synthesis, it from verticillene-type intermediates, since sinc( cycli2
i ss also quite unlikely that a commercially feasible tions of this type can be quite sensitive to the actt
sy;ynthetic route to taxol will be developed before the conditions. They do suggest, however, that tha the bi
end of this century. genetic sequence is much more subtle than l9reviou, r ....

supposed, perhaps involving different geo eometric

eometl
positional isomers of geranylgeranyl pyrophosr pyrop )ha
5. BIOSYNTHESIS A N D or possibly involving alternative reactive interme~ inten
i
B I O T R A N S F O R M A T I O N OF TAXOL ates
a t ~ e such
ennh a ase radicals.
r~rl;pale T h ~ f'aPt
The fact tthat
h a t the
t h ~ ttaxane
av~n ske
skelet
~ 1 n . . . . . . . . . . . . . T . . . . . . . . ~ . . . . . T ....... is a rare one also suggests that the biosyn ~nthe
5.1. BIOSYNTHESISOF TAXOL AND REI ~ES
pathway to it is unusual.
NO studies have yet been publishec ;yn- The formation of the oxetane ring of taxol has be
thesis of taxol. However, some biosynthetic work on investigated in some model studies (Swi Swindell a
compounds related to the side chain has appeared, Britcher, 1986; Francl et al., 1990). It was propos
and various speculations and model reactions for the that the characteristic 3-oxetanol ring syste 'stem of tax
biosynthesis of the taxane ring system have also been could be formed from the fl epoxide of a taxane t~ su
published. as baccatin I, but solvolytic studies on a fl epoxi
The most commonlyy accepted hy hypothesis
t for the (Fig. 47) failed to yield any oxetane and gave ga inste
formation of the taxanes is that they a :om a ketoalcohol product.
geranylgeranyl pyrophosphate by e cy- An alternative proposal for the biosynthesis of t
clization, possibly
Oly via cembrene
V1:4 U UIIII.)IUII~ orl vq
O VUII.Ii.;IIII~III; I I I Lter-
UI.- oxetane
U AULd.IIU 1ring
111 has been made by Potier and his colla
mediates (Fig. 45) (Harrison and Lythgoe, 1966; orators (G~ Gurritte-Voegelein et al., 1987). He not
Erdtman et aL, 1964). It has also been suggested that the existence
existent of three major groups of taxanes: grol
the taxanes are related to quassin (Ueda et al., 1963), A with an exocyclic
q methylene at C-4, group B wi
but this seems less likely. However, model studies on )oxide (oxirane) at C-4, and group C with
a fl-epoxid
~clization of cembrenes (Dauben et al., oxetane ring. Many of the group A compoun
icillene and epoxyverticillenes (Begley contain ester groups at C-5 that are structura
tiled to yield products with a taxane related to the t taxol C-13 ester side chain and Pot
skeleton. Thus treatment of verticillene, verticillene thus suggested that the C-13 ester side chain of taxol
7,8-epoxide, verticillol 7,8-epoxide, or anhydroverti- is derived by intramolecular transfer from C-5.
cillol 7,8-epoxide with Lewis acids (Fig. 46) yielded Although C-5 and C-13 appear to be far apart in the
only products resulting from rearrangements of the conventional representation of the taxane skeleton,

S /

I
 The chemistry of taxol

H H

vertlclllene vortiolllene 7,8-epoxkle

HO " '"

vertlcinol 7,8-epoxlde Inhyclrovertlolllol 7,8-epoxlde

FIG. 46. Comp ~il to cyclize to the taxane ring system.

they are fairly close c~ose m ' a 3-dimens

~-mmenslona~ structure ure stereospecifically,
s[ereospec~n with loss of the 3-pro-R proton an
(Fig. 4) and Potier's )tier's suggestion is thus a reasonable retention of the 3-pro-S proton (Fig. 49). It is proi
one. Further elaboration aboration of the resulting allylic alco- the biosynthesis of the taxol side chai
able that t]
hol is proposedi to lead to the oxetane ring through occurs by a similar pathway, but no experiment~
an epoxide which ich undergoes ring-opening to a ter- results on this conversion have been reported.
d cyclization to an oxetane (Fig. 48).
side chain is the only part of the
5.2. BIOTRANSFORMATIONOF TAXOL
LO.AO, I I g ; 3 U I A W l i /rich
bli aactual
bLUg/ biosynthetic
UIU~lltllget|b studies
O I.UUI~O have
llGVlv

been carded out. Acid hydrolysis of taxine
yields (3R)-dimethylamino-3-phenylpropionic acid
(Winterstein's acid) (Baxter et al., 1962), and biosyn-
thetic studies on this acid, which is related to the taxol
side chain, have been carried out by Leete and Bodem
(1966) and Haslam (Plattet aL, 1984). In T. baccata
phenylalanine serves as the best precursor of
Winterstein's acid, and the bioconversion occurs
Studies of the biotransformation of taxol are only
just beginning to appear. Analysis of taxol in human
plasma obtained as part of clinical studies has been
carded out in three cases. In the first two cases
(Longnecker et al., 1987; Wiernik et al., 1987) no
metabolites were detected. In the third case (Rizzo
et al., 1990) a new peak eluting before taxol was
detected in the plasma of a cancer patient by reverse

OH""

%o.
~.6 D . G . I . KINGSTON
. A
t, c .--
OR OR
AcO OR OR

2--5& O NMe2 MII2N 0

'%OH
""O Ph Ph 0 "~"
H~ ~ OH : II OH HO OCOR
OCOR

AcO OR OR
AcO OI

Me2NH O
o
i il I "\
h ~ O , ,:, ' I %"~
' = or"
'/OH HO OCOR
=
HO - - OCOR N'~OH %ell 3

AoO O OH

PhCONH O

°
Ph : OH =
HO OCOPh

FIG. 48. Proposed biogenesis of taxol from a type A precursor.

phase HPLC. This peak was not due to 7-epitaxol, in Fig. 50; other metabolites appeared also to be
since this elutes after taxol, and it was thus assumed hydroxylated taxols, but their precise structures were
to be due to a new and as yet unidentified metabolite not elucidated.
o f taxol. Treatment of taxol with cell culture media
in the presence or absence of cells does however
convert it in part to 7-epitaxol (Ringel and Horwitz, 6. S T R U C T U R E - A C T I V I T Y RELATIONSHIPS
1987). Thus taxol was approximately 50% converted c ~ T Ax,c~T r ~ i v AT T V ~
the cell culture medium of the J774-2
z2 hr, but the conversion is reversible Discussi(
was partially converted to taxol under taxol deriw
tions, many differ
of taxol in rat bile has been described the various
~t al. (1990). These workers found that that the nt
e injected taxol was recovered in urine, assays, evel
and no urinary metabolites were detected. On the depend on the exact conditions of the as
other hand, over 40% of the injected taxol was found major classes of assay have been used.
in rat bile, either unchanged or as one of the nine (1) Microtubule assembly assays. Th
metabolites detectable by HPLC. Baccatin III was the mechanism of action of taxol is as an
only metabolite that had lost the ester side chain; the agent. Unlike other plant-derived antirr
other metabolites all appeared to have intact side agents such as colchicine, podophyllotoxi
f these metabolites were identified as vinca alkal,
ed taxol derivatives V and VI shown taxol actua
 The chemistry of taxol

P388 cells (a mouse lymphocytic leukemia

'-
~ ~,,,'o
o"
R
R1 ~ (3) Assays in the mouse. Animal tests haveh
Metabolltl V RlmOH
Metabollt4)VI RImH R2mmOH
FIG. 50. Metabolites of taxol in rat bile.
stabilizes the microtubules so formed against depoly-
merization by calcium ions and by low temperature
(Schiff et al., 1979; Parness and I- gl).
Assays for microtubule-assembly prc be
carried out either by measuring the of
microtubule assembi
sembly (Swindell et al., 1991) or by
measuring the initial
nitial rate of microtubule disassembly
(Lataste et al., 1984
1984). Other investigators have studied
the displacement nt of labeled taxol from microtubules
bv an excess of unlabeled compound (Parness et al.,
of the different methods used, the
in assembly assays will be presented as
data relative to taxol, thus minimizing differences due
to the different methodologies used.
(2) Mammalian cell culture toxicity. Cytotoxicity of
taxol derivatives has usually been determined with
KB cells (human carcinoma of the nasopharynx),
J774-2 cells (a mouse macrophage-like cell line), or
Results in these three cell lines appear to b~ UIu;'mu
comparable to each other and to result from
frorr tubuliJ
assembly assays, although there are some differeno
d
in situations where biotransformation of a tax
Ac derivative can occur under the conditions of c the cx
culture assay.
bee
carried out in P388 leukemia, B16 melanoma,
melan( an
Jse ofhu
other mouse-based assays, including the use of huma
R, tumors as xenografts in athymic mice. The data dat; f{
these assays are much more limited than for the fir
H2lm I1
two assays,
~ •
and will thus be considered- separatel
In the discussion that follows, taxol analogs
ana wil
variations in the C-13 side chain will be consider(
c
first, followed by ring-modified taxol analog
Because it is chemically simpler to modif)~¢ the sic
chain, more work has been done in this area.
6.1. BACCATINIII ANALOGS
The most significant change possible in the sic
chain is its deletion, which then gives baccatin I
(Fig. 2). It was
v recognized early on that the side cha
is necessaryy for the full activity of taxol, since baq
catin III is about 1700-fold less cytotoxic to KB (:el
than taxol. The bioactivities of several baccatin I
derivatives have been determined, and are shown
Table 1. These
Th data indicate that baccatin III deriv
tives are all much less effective than taxol as inhibitors
of disassembly of mammalian tubulin, ranging from
38-fold to 680-fold less active. Interestingly, however,
there is much less difference between the activity of
taxol and baccatin III derivatives towards tubulin
from the amoeba Physarum polycephala (Lataste

TABLE 1. Tubulin Assembly Activity and Cytotoxicity of Baccatin III Derivatives

OCOPh M

Compound R~ R2 R3 R,
1 Baccatin III OH OAc = O OH 52 1.3 1.7 x I,
2 10-Deacetylbaccatin III OH OH =O OH 46 1.0 4 x I(
3 7-Acetyl-10-deacetylbaccatin III OH OH =O OAc 82 0.9
4 7-Acetylbaccatin III OH OAc =O OAc 384
5 10-Deacetylbaccatin V OH OH =O ~t-OH 250 3.3
6 I 0-Deacetyl- 13-oxobaccatin III =O OH =O OH 350 5.3
7 10-Deacetvl-
yl- 1l l,
l. 12-dihvdro- 11-
12-dihydro- 11-
/-13-oxobaccatin III =O OH =O OH
~tylbaccatinIII OAc OAc =O OAc
VI OAc OAc fl-Ac OAc
yl-10-oxobaccatin Ill OH = 0 = 0 OH
xybaccatin I
~ybaccatin III
g". . . . :- ~....:- bTT.:---- *..'h~..l'.-- C.--~ ID[. . . . . . . . . . . . i ..... L--t
 The chemistry of taxol

TABLE 3. Tubulin Assembly Activity of lO-Deacetyltaxol Derivatives with Modified Side Chains"
HO .0 OH

Bioactivity
".-,t'l Tubulin-
disassembly Cytotoxicity
a R4 PhCO0
inhibition P388
No. Rl R2 R3 R4 iC5o~, b EC~o~l
1 PhCONH H OH H 1.3
2 H PhCONH H OH 4
3 OH H CONH H l0
4 H OH H PhCONH 170
5e PhCONH H H OH 1.3
6d H PhCONH OH H 1.3
7 H H H H 17
8 H H OH H 4.5
9 H H H OH 3.5
I0 d H ButOCONH H H 2.3
11d ButOCONH H H H 4.1 12
12 TsNH H OH H 5
13 OH H "sNH H 15
14 ButOCONH H OH H 0.5 0.13
15 H ButOCC H OH 30 > 10
16 OH H IJU'UIJUN
)CONH 1"1 H 10 4
17 H OH H ButOCONH 160
18d ButOCONH H H OH 1.8
19d H ButOCONH OH H 4.3
20d NH 2 H H OH 30
21 d H NH2 OH H 30
(CH2)3CONH H OH H 1
CbH4CONH H OH H 5.5 >4
H R3 R2 H 23
25c OH H OH H 3
aData from Gurritte-Voegelein et al. (1991). blCs0 = concentration of drug leading to a 50*/, inhibition of the rate of
mammalian microtubule disassembly. IC50~ = ICso/IC~0(taxol). IC50 for taxol = 0.5/aM. Numbers larger than unity thus
correspond to less active analogs, cEC~0= concentration of drug leading to 50*/, inhibition in the amount of cell growth.
ECs0~'l= ECs0/ECs0(taxol). ECs0 for taxol = 0.27/~g/mL. dThe structures of each pair of erythro compounds may be
reversed. CThreo compounds: 2'R YS + 2'S,3'R.

et al., 1984), with at least one baccatin III derivative units o f / z g / m L , while the J774-2 d a t a are expressed
y 3) being m o r e active t h a n taxol in u n i t s o f p
.urn tubulin. The reason for this m a j o r can be sign
t k n o w n , b u t it is apparently not due and withou
actors such as the different conditions T h e tub~
ably o f the two tubulins. It is thus derived p r
the binding site o n Physarum micro- Gu~ritte-Vc
,~ t o l e r a n t t h a n the c o r r e s p o n d i n g site Voegelein
o n m a m m a l i a n tubulin. However However, baccatin
baccatln III 111 a n d its (Swmdell et al., 1991). The m a j o r conclu
(Swindell
derivatives were m u c h less cytotoxic t h a n taxol can be derived from a study o f these data,
towards Physarum or Plasmodium a m o e b a l cultures, n a t i o n with the scantier cytotoxicity d a t a
so either the baccatin derivatives are n o t available to eated below.
tubulin u n d e r these in vivo conditions or o t h e r factors (1) Some activity is retained even with s
are involved, t h a t are very different from t h a t o f taxol.
t r u n c a t e d si
activities wi
DE CHAIN MODIFIED ANALOGS
tubulin ass
tata o n taxols modified in the C-13 cytotoxicity
I is summarized in Tables 2-3. Table 322-fold.
Ltion o n t u b u l i n assembly or disassem- (2) The
30 D . G . I . KINGSTON

2-4-fold less active in the tubulin-assembly assay and from the 2'-hydroxyl group (compound 8
12-fold less active in the cytotoxicity assay. The 3'-butoxycarbamate group (compound 10 u1 tm).
2'-(t-butyldimethylsilyl) taxol derivative (Table 2, A comparison of cytotoxicity data with wit tubu
~ntry 4) is very much less cytotoxic than taxol, but'
entr assembly or disassembly activity is instructiveinstructi (Tab
this may be due to the steric bulk of the silyl group
this 2 and 3). The major conclusions to emerlge from
rather than the loss of the free hydroxyl group,
rather study of these data are that the activities of ( most
(3) The 3'-phenyl group appears to contribute to the analogs broadly parallel their tubulin-astubulin-asseml
~mbly
the overall activity of taxol. Although no close -disassembly activities. As an example, th the six s~
analogs were prepared, the lactic acid derivatives thetic analogs prepared by Swindell et al. ((195
(Table 2 compounds 10 and 11) are about one half (Table 2, entries 10-15) show the same trends in i t
as active as the corresponding phenyllactates (com- J774.2 cell culture assay as they do in the tubuli tul
pounds 12 and 13). The p-hydroxyp] tive assembly assay, although the magnitude of t
24 (Table 2) is slightly more activ ulin changes is larger in the cell culture assay. SimilaJ
disassembly inhibitor than taxol, but it is less cyto- four 10-deacetyltaxol analogs (Table 3, entries ent 14-1
toxic than taxol. 23) have cytotoxicities which broadly pallrallel th
(4) The 3'-N-benzoyl group can be replaced with tubulin-disassembly activities. The major excepti
other N-acyl groups with little loss of activity to this rule occurs in the case of 2'-acyl dderivativ
(cephalomannine, Table 2, entry 3) or with an in- which are hydrolyzed in vivo to the correspondi corr
crease in activity y ((Table 2, entry "y 19)
19). T-hydroxyl~ydroxyl comrcompound. Such derivatives are al not ve
(5) Removal of the 3'-N-benzoyl 1 s in potent tubulin disassembly inhibitors, but they
loss of activity (Table 2, entry 23). It n at quite cytotoxic (Table 2, entry 2). This cytotoxicity
this time whether a ccarboxamide
[ l e t :a ~Eoox~tmlue glI U L I p Lt:~ ~ L I ~ IhI l~i
~ is greatly
~ reduced if the T-hydroxyl group is convert
[ I ; ~ t l y 1~(.11
tivity,
essential for activit ' but the relatively good activity to a hydro ydrolytically stable group such as a t-but
of the desamino no derivatives 12 and 13 (Table 2) dimethylsil'. Lylsilyl ether (Table 2, entry 4), although
her groups could provide activity,
suggest that other noted earlier earlic it is possible that the steric bulk of tl
(6) The naturere of the polar groups at C-2' and C-3' group also has a negative effect on activity.
to a surprising extent without major These resultsre have been discussed in terms
Thus the 2',3'-diol (Table 3, entry 25) molecular modeling i of the taxol side chain (Swind
less active than taxol in the tubulin- et al. 1991 ). The major conclusion reached by the
disassembly assay, and compounds with the 2'- and authors is that the conformation of the side chain is
3'-groups interchanged can still show some (albeit not strongly influenced by the taxane skeleton, and
reduced) activity; this loss appears however to be they propose that the taxol recognition site on micro-
greater in cytotoxicity than in tubulin-disassembly tubules possesses a hydrophobic cleft designed to
activity (Table 2, entries 17 and 21). accept a side chain with its functionality preorganized
(7) The stereochemistry at C-2' makes an import- by stereochemistry and hydrogen bonding to re-
ant contribution to activity. In a model series this semble that of taxotere 8.
effect is more pronounced when there is an amide The fact that baccatin III analogs have the same
3' (Table 2, entries 14 and 15) than affinity as
ot (Table 2, entries 12 and 13). In the also been d
however, the effect is rather variable. These auth
-deacetyl series (Table 3) inversion at tubulin inv
o difference to tubulin disassembly followed b)
there is an N-benzoyl group present by specific
nd 6) but reduces activity by either
4-fold or 8-IOld when there is
d ' an N-t-BOC
/V-t-l~OCS group, 6.3. RING MODIFIEDANALOGS
depending on which of the structures shown as 18 and
19 has which activity. Data on taxol analogs modified on the !
(8) A major stereochemical effect is observed when system are shown in Table 4. The major c
the 2'- and 3'-groups are interchanged. The com- to emerge from these data are as follows
pound with the natural configuration (Table 2, entry (1) Acylation at C-7 (entries 8, 12)
aore active in the tubulin disassembly significantl,
one with the unnatural configuration of taxol.
(2) Atta(
ge in activity for certain modifications slightly inc
additive, at least in some cases. Thus activity (en
vity in going from compound 7 (Table (3) Rem
 The chemistry of taxol

TAeLE4. Activity o f Ring-Modified Taxols

Bioactivity

Cytotoxicity
Tubulin
disassembly J774.2 KB
No. Compound iCs0~l a ECsor~lb ECs0~I b Ref
1 Taxol 1.0 1.0 1.0 c,d,e,f
2 Cephalomannine 1.5 1.4 3.2 c,g,f
3. 10-Deacetyltaxol 1.3, 3.5 2.8 22 c,g,h,i
4 4-Deacetylcephalomannine 4.2 26 g,h
5 lO-Deacetyl-7-epitaxol 3.5 28 h,j
6 lO-Deacetyl-7-epicephalomanni 38 h
7 7-Epitaxol 3.0 1.5 3.0 j,k
8 7-Acetyltaxol 2.0 1.3 400 d,e,l,n
9 7-Xylosyltaxol 0.4 d
10 10-Deacetyl-7-xylosyltaxol 0.6 d
11 7-Xylosylcephalomannine 0.5 d
12 7-Glutaryltaxol 1 i
13 7,10-Diglutaryltaxotere 2 i
14 7,10-Diglycyltaxotere 1.2 i
15 7-(Phenylalanyl)taxotere 1 i
16 2',7-Diacetyltaxol 11.4 g
17 2',7-Diacetyl- 10-deacetyltaxol 2.8 g
18 2-Debenzoyl-2-(3-hydroxybenzc
. . . . . j - - ~- -.j . . . . . .,, . . . . . . j - / . . . . . . 1 41 n o
19 7-Oxotaxol > 103 m
20 2'-Acetyl-7-oxotaxol > 105 m
21 2',7-Dioxotaxol >106 m
22 10-Oxo- 10-deacetyltaxol > 103 k
23 Lactone (Fig. 27) > 105 m
24 20.O-secotaxol
~ecotaxol (Fig. 31) >21 > 105 m
chloride product (Fig. 31) > 105 m
-*l)abeotaxol (Fig. 32) 3 > 105 m
alCs0 = concentration of drug leading to a 50% inhibition of the rate of mammalian microtubule disassembly.
ICs0'°~= ICs0/ICso(taxol). IC50 for taxol = 0.5#M. Numbers larger than unity thus correspond to less active analogs.
bECso = concentration of drug leading to 50% inhibition in the amount of cell growth. ECs0~l= ECs0/ECso(taxol) in the
indicated cell line. ECs0 data for taxol: KB 1 x 10-5 ltg/mL; J774.2 9 x 10-2/~mol/L. ¢Chauvi6re et al. (1981). dLataste et
al. (1984). eMellado et al. (1984). fMiller et al. (1981). gParness et al. (1982). hMcLaughlin et al. (1981). iGu6ritte-Voegelein
et al. (1991). JRingel and Horwitz (1987). kHuang et aL (1986). IChenu et al. (1987). "Kingston et al. (1990). nln LI210
lymphocytic leukemia. °Monsarrat et al. (1990).

disassembly activity and cytotoxicity, so clearly there (8) Contraction of ring A does not reduce the
. . . . . . . . . . . . . . . . . . . . . . . . . .

between side chain and ring effects, tubulin-disa

ent of polar groups at both C-7 and spite of the
e activity slightly reduced (entries 13, this convers
however m
ation at C-7 slightly reduces activity finding is i
dichotomy
n at C-7 reduces activity significantly cytotoxicity
(entry 19). This result should be viewed with caution, the instability of A-nortaxol in cell culture n
however, since 7-oxotaxol is somewhat unstable and its failure to enter the cell. In this case the ¢
undergoes oxetane ring-opening in base (Magri and between cytotoxicity and tubulin-assemb]
Kingston, 1986). It is thus possible that this process could readily be understood, and A-nort~
could have occurred under cell culture conditions, become the basis of a new series of compo
(7) Opening of the oxetane ring drastically reduces taxol-like activity in which the stability r
ld tubulin disassembly inhibition ac- addressed i
~). Although 1H-NMR evidence indi- correlation
~nformational changes accompanying yet underst
ing (Samaranayake et al., 1991), an (9) A ch
olecular modeling study suggests that hydroxyben
lot a large one (J. A. Beutler, personal sembly inhi
32 D . G . I . KINGSTON

T.riLE 5. Animal Test Data on Taxol Analogs

In vivo bioactivity

No.
F~iO. Compound Tumor* T/C (dose) b Re
1 Taxol P388 157(20) 149 (5) c
BI6 283(5) 254(25) c
MX-I CRa(15)-84(10) CR(6.7) e
LI210 131 (20) c
2 Cephalomannine P388 152-180(1-3.3) f
3 10-Deacetyltaxol P388 135 (16) g
4 10-Deacetyleephalomannine P388 130 (16) g
5 Taxotere ® P388 > 175 h
LI210 > 150 h
B16 > 150 h
6 2'-Acetyltaxol P388 140(20) 130(I0) i
7 7-Acetyltaxol P388 130(26) 127(13) i
8 2',7-Diacetyltaxol P388 104 (40) 100 (20) i
2'-Substituted taxols: 2'-substituent
9 CO(CH2)NH 3+ H C O 0 - P388 153(40) 140(40), 138(20) j
10 CO(CH2)2COOH P388 129 (44) 122 (22) j
11 CO(CH2)2COOH BI6 185(40) 154(20) 154(5) j
12 CO(CH2)COONa BI6 218(40) 201 (20) 154(2.5) j
13 CO(CH2)2COO - HN + (CH2CH2OF BI6 314 (27) 230 (7) j
14 CO(CH2)2COO-N-methylglucamm BI6 241 (50) 176 (25) 134 (12.5) j
15 CO(CHz)3COONa nl6 339(49) 192(12) 159(6) j
16 CO(CH2)3COO
~tSLR)- HN
HI~ +
~ (CH2CH2OI-
[L;H2t;I-12UH)3 Bi6 300(53) 291 (13) 207(7) j
17 CONH(CH2)3N(CH3)2HCI
CO(CH2)3CONH(CH2)3N(CH3)21 BI6 352(20) 352(10) 188(5)
18 COCH2N(CH3) 2 MX-I CR (20) CR (13)-53 (8.9)
19 COCH2CH2N(CH2CH3)2HC1 MX-I CR(18) CR(9) CR(4.5)
2',7-Disubstituted
)stituted taxols: substituent
20 CO(CH2)2COOH BI6 114(44) 105(22) 124(11) j
COONa BI6 166(44) 109(22) 149(11) j
,ted taxols: substituent
(CH3)2 MX-I CR (33) - 9 3 (16.5) 23 8.2) e
aTumor system used: P388 lymphocytic leukemia, LI210 lymphoid leukemia, BI6 melanoma, or MX-I mammary
xenograft, as indicated, bT/C values for P388, B16, and LI210 are expressed as median lifetime of test animals divided by
the median lifetime of control animals, times 100. T/C values for MX-1 are expressed as (change in treated tumor
weight/initial tumor weight) times 100 for tumors that regress. The dose (in mg/kg) is given in brackets. °Suffness and Cordell
(1985). dCR = complete remission. 'Stella and Mathew (1990). fMiller et al. (1981). mMcLaughlin et al. (1981). hLavelle et
al. (1989). iMagri and Kingston (1988). JDeutsch et al. (1989).

understand more fully those features o f the taxol and the functional groups of O-cinnamoyltaxicin-I.
molecule which are necessary for activity. J. chem. Soc. 2964-2971.
BEGL~, M.
Total syn!
L4. ANIMAL TEST DATA to the ta
4907-4924
e animal test data on taxol and its BLECXF.aT,S
nmarized in Table 5. The major point The Alkal
Lthese data is that some T-substituted pp. 195-23
CARDELLINA,
~etter activity in the B16 m e l a n o m a cephaloma
system than taxol. This is particularly true o f the CAS~LL^NO,E. E. and HODDER, O. J. R. (1973)
glutaryl amide derivative prepared by Deutsch et al. and molecular structure of the diterpenoid b~
(1989) (entry 17). Diacylation at C-2' and C-7 did not naturally occurring oxetan with a taxane sk~
give useful activity, however (entries 20, 21). Crystallogr. Sect. B 29:. 2566-2570.
C H A N , W . R . , H A L S A L L , T. G., H O R N B Y , G. IV
A . W . , SABEL, W . , BJAMER, K . , F E R G U S O N , G . a
Acknowledgements--I thank my coworkers who have SON, J. M. (1966) Taxa-4(16),ll-diene-5,,,
-L--~J __.:.t --__ . L . . . . .
-". . . . . . . .
the excitement ofr our
..... J~ . . . . .
studies tr .
on this . . .
tetraol, .
a . . . . . . .

:ule: their names are included in the refer- yew (T. ba

rom my group. I also thank Drs Potier, derivative.
for sending me manuscripts or preprints of CIO~,
k, Dr M. Suffness for careful reading of the and Porto
Dr J. Merola for preparing Fig. 16 from biochimiql
L. (Taxac
 The chemistry of taxol

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