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Synthesis of Curcumin Analogues Biconjugates as

Potential Antitumor Agents in Isolated Human Cells





Reem Ibraheem Al-Wabli
"B. Pharm. Sci."



1426 A.H.
2006 A.D.




Submitted In Partial Fulfillment of the Requirement for the
Master's Degree in the Department of
Pharmaceutical Chemistry
At the College of Pharmacy,
King Saud University















,---- ,-~

----- ;--- ,-,---- 4--- ----- ,----- ,-~- -'=- ---
----- =--- _-- ;-,- ,--~='-- ~=-- - .

i
Contents
Page

Acknowledgement, ix

1. Summary, x

2. Introduction,. 1
2.1 Overview,. 1

2.2 Biological Properties of Curcumin and its Derivatives, 2
2.2.1 Antioxidant Properties of Curcumin,.......2
2.2.1.1 Mechanism of Antioxidant Action of Curcumin and
its Derivatives,.2
2.2.1.2 Physiological Antioxidant Mechanism of Curcumin
and Synergism with Water-Soluble Antioxidants,..5
2.2.2 Anti-inflammatory Properties of Curcumin,.......6
2.2.2.1 Mechanism of Anti-inflammatory Action,.7
2.2.3 Anticancer Properties of Curcumin,...8
2.2.3.1 Mechanism of Anti-carcinogenic Activity of
Curcumin,........9
2.2.4 Chemopreventive Properties of Curcumin,11
2.2.5 Antibacterial, Antifungal and Antiparasitic Properties of Curcumin,12
2.2.6 Antiviral Properties of Curcumin,..13
2.2.7 Antihistaminic Properties of Curcumin,.....13
2.2.8 Curcumin for Treatment of Skin Diseases,....13
2.3 Classification of Curcumin Analogs,.14
2.3.1 Naturally Occuring Diarylheptanoids,...14
2.3.2 Modified Diarylheptanoids,...19
2.3.2.1 Curcuminoids,....19
2.3.2.2 Cyclic Diarylheptanoids,....27
2.3.2.3 Non-heptanoid Derivatives,....32

3. Research Objectives,.....37

4. Results and Discussion,.42
4.1. Chemistry42
4.1.1 1,7-Bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (1)
and 1,7-bis(4-hydroxy-3-ethoxyphenyl)-1,6-heptadiene-3,5-
dione,......42

4.1.2 Di-O-Chloroacetylcurcumin (3a) and di-O-chloropropionyl-
curcumin (3b),... 54
ii
4.1.2.1 Method A....54
4.1.2.2 Method B56

4.1.3 2-Chloroethylamine monohydrochloride and bis(2-chloroethyl)-
amine hydrochloride,...63

4.1.4 1,7-Bis(4-Alkyl(cycloalkyl or heteroaryl)aminoacyloxy)-
3-(methoxyphenyl)-1,6-heptadiene-3,5-dione (5a-n) and
1,7-bis(4-Alkyl(cycloalkyl or heteroaryl)aminoacyloxy)-
3-(ethoxyphenyl)-1,6-heptadiene-3,5-dione (6a-n),..64

4.1.5 1,7-Bis(4-(4-substituted sulfanilamido)acyloxy)-3-(methoxy-
phenyl)-1,6-heptadiene-3,5-dione (7a,b) and 1,7-bis(4-(4-
substituted sulfanilamido)acyloxy)-3-(ethoxyphenyl)-1,6-
heptadiene-3,5-dione (7c-f),..74

4.1.6 Di-O-Adamantoylcurcumin (8a) and di-O-adamantoylethyl
curcumin (8b),...75

4.1.7 Di-O-Heptanoylcurcumin (8c) and di-O-heptanoylethyl
curcumin (8d),...78

4.1.8 Di-O-(2-Thienoyl)curcumin (8e) and di-O-thienoylethyl
curcumin (8f),...83

4.1.9 Attempt Reacting Curcumin with Chlorosulfonyl isocyanate,.............87

4.2 Anticancer Screening,...88

4.2.1 Introduction,..88
4.2.2 Discussion of the Results,.....92
4.2.3 Conclusion,...96

5. Experimental Part,.100
5.1 General,....100

5.2 Chemical Procedures,...............101

5.2.1 1,7-Bis(4-hydroxy-3-methoxyphenyl)-1,6-hepta diene-
3,5-dione (1) and 1,7-bis(4-hydroxy-3-ethoxyphenyl)-
1,6 heptadiene (2),..101

5.2.2 Di-O-Chloroacetylcurcumin (3a) and di-O-
chloropropionylcurcumin (3b), 103
5.2.2.1 Method A,.103
5.2.2.2 Method B,.105

5.2.3 2-Chloroethylamine monohydrochloride and
bis(2-chloroethyl)amine hydrochloride,109
iii

5.2.4 1,7-Bis(4-Alkyl(cycloalkyl or heteroaryl)amino-
acyloxy)-3-(methoxyphenyl)-1,6-heptadiene-
3,5-dione (5a-n) and 1,7-Bis(4-Alkyl(cycloalkyl-
or heteroaryl)aminoacyloxy)-3-(methoxyphenyl)-
1,6-heptadiene-3,5-dione (6a-n),....110

5.2.5 1,7-Bis(4-(4-substituted sulfanilamido)acyloxy)-3-
(methoxyphenyl)-1,6-heptadiene-3,5-dione (7a,b)
and 1,7-bis(4-(4-substituted sulfanilamido)acyloxy)-
3-(ethoxyphenyl)-1,6-heptadiene-3,5-dione (7c-f ),...117

5.2.6 Di-O-Adamantoylcurcumin (8a) and di-O-
adamantoylethyl curcumin (8b), 119

5.2.7 Di-O-Heptanoylcurcumin (8c) and di-O-
heptanoylethyl curcumin (8d),121

5.2.8 Di-O-(2-Thienoyl)curcumin (8e) and di-O-
(2-thienoyl)ethyl curcumin (8f),...123

5.3 Anticancer Screening,..125
5.3.1 Materials and Methods,. ...125
5.3.1.1 Cytotoxicity to Mammalian Cells,125
5.3.1.2 Interaction with Topoisomerases, .....................132


8. References,..135


Arabic Summary,

iv
List of Figures
Page
Figure 1:
1
H NMR spectrum of curcumin (1),......49

Figure 2:
1
H NMR spectrum of ethylcurcumin (2),..50

Figure 3:
13
C NMR spectrum of ethylcurcumin (2),...51

Figure 4: Mass spectrum of ethylcurcumin (2),.........52

Figure 5:
1
H NMR spectrum of di-O-chloroacetylcurcumin (3a),.55

Figure 6:
1
H NMR spectrum of 3-methoxy-4-chloroacetyloxy-
benzaldehyde (4a),..........57

Figure 7:
1
H NMR spectrum of 3-ethoxy-4-chloroacetyloxy-
benzaldehyde (4c),..58

Figure 8:
1
H NMR spectrum of di-O-chloroacetylethyl curcumin (3c),...60

Figure 9: Mass spectrum of di-O-chloroacetylethyl curcumin (3c),....................61

Figure 10:
1
H NMR spectrum of di-O-chloropropionylethyl curcumin (3d),62

Figure 11:
1
HNMR spectrum of 1,7-bis(4-adamantylaminoacetyloxy)-
3-(methoxyphenyl)-1,6-heptadiene-3,5-dione (5a),65

Figure 12:
1
H NMR spectrum of 1,7-bis(4-bis(2-chloroethyl)amino-
acetyloxy)-3- (methoxyphenyl)-1,6-heptadiene-3,5- dione (5e),...68

Figure 13:
1
H NMR spectrum of 1,7-bis(4-adamantylaminopropionyl-
oxy)-3-(methoxyphenyl)-1,6-heptadiene-3,5-dione (5h),..........................69

Figure 14: Mass spectrum of 1,7-bis(4-adamantylaminopropionyl-
oxy)-3-(methoxyphenyl)- 1,6-heptadiene-3,5-dione (5h),70

Figure 15: Mass spectrum of 1,7-bis(4-(methylthiadiazolyl)amino-
propionyloxy)-3- (methoxyphenyl)-1,6-heptadiene-3,5- dione (5j),.........72

Figure 16: Mass spectrum of di-O-Adamantoylcurcumin (8a),...............................76

Figure 17:
1
H NMR spectrum of di-O-heptanoylethyl curcumin (8d),..80

Figure 18:
13
C NMR spectrum of di-O-heptanoylethyl curcumin (8d),.81

Figure 19: 2D
1
H NMR (COSY) spectrum of di-O-heptanoylethyl
curcumin (8d),..81

v
Figure 20:
1
H,
13
C NMR (C,H correlation) spectrum of di-O-heptanoyl-
ethyl curcumin (8d),... .82

Figure 21:
1
H NMR spectrum of di-O-(2-Thienoyl)curcumin (8e),...84

Figure 22: Mass spectrum of di-O-(2-thienoyl)ethyl curcumin (8f),..85




vi
List of Charts
Page
Chart 1: Possible Fragmentation Pattern for compound 2,53
Chart 2: Possible Fragmentation Pattern for compound 3c,...61
Chart 3: Possible Fragmentation Pattern for compound 5a,..66
Chart 4: Possible Fragmentation Pattern for compound 5h,..71
Chart 5: Possible Fragmentation Pattern for compound 5j,...73
Chart 6: Possible Fragmentation Pattern for compound 8a,..77
Chart 7: Possible Fragmentation Pattern for compound 8f,..86
Chart 8: Cytotoxic Effect of the Synthesized Compounds to
SK-Mel Cells,128

Chart 9: Cytotoxic Effect of the Synthesized Compounds to KB Cells,.129

Chart 10: Cytotoxic Effect of the Synthesized Compounds to BT-549 Cells,130

Chart 11: Cytotoxic Effect of the Synthesized Compounds to SK-OV-3 Cells,.....131


vii
List of Tables
Page
Table 1: Physicochemical Data of di-O-chloroacetylcurcumin (3a)
and di-O-chloropropionylcurcumin (3b),104

Table 2: Physicochemical Data of the Synthesized Compounds 4a-d,....106

Table 3: Physicochemical Data of the Synthesized Compounds 3a-d,..108

Table 4: Physicochemical Data of the Synthesized Compounds 5a-n and 6a-n,114

Table 5: Physicochemical Data of the Synthesized Compounds 7a-f,...118

Table 6: Physicochemical Data of the Synthesized Compounds 8a-f, 124

Table 7: Cytotoxicity of the Synthesized Compounds to Mammalian Cells, 126

Table 8: Anticancer Activity of the Synthesized Compounds
(Inhibition of Topoisomerase Activity),.. 134
















viii
List of Schemes
Page

Scheme 1: Synthetic Routes for the Preparation of Curcumin (1) and Ethyl
curcumin (2) and Synthetic Routes for the Preparation of di-O-
chloroacylcurcumin (3a,b) and di-O-chloroacylethyl curcumin (3c,d),39


Scheme 2: Synthetic Routes for the Preparation of 1,7-Bis(4-Alkyl (cycloalkyl
or heteroaryl)aminoacyloxy)-3-(methoxyphenyl)-1,6-heptadiene-
3,5-dione (5a-n) and 1,7-bis(4-alkyl (cycloalkyl or heteroaryl)-
aminoacyloxy)-3-(ethoxyphenyl)-1,6-heptadiene-3,5-dione (6a-n)
Synthetic Routes for the Preparation of 1,7-Bis(4-(4-substituted
sulfanilamido)acyloxy)-3-(methoxyphenyl)-1,6-heptadiene-
3,5-dione (7a,b) and 1,7-bis(4-(4-substituted sulfanilamido)
acyloxy)-3-(ethoxyphenyl)-1,6-heptadiene-3,5-dione (7c-f),...40


Scheme 3: Synthetic Routes for the Preparation of Di-O-Adamantoyl-
curcumin (8a), di-O-adamantoylethyl curcumin (8b),
di-O-Heptanoylcurcumin (8c), di-O-heptanoylethyl curcumin (8d),
di-O-(2-Thienoyl)curcumin (8e) and di-O-(2-thienoyl)ethyl
curcumin (8f),..41














ix
Acknowledgement

All my thanks to Allah, the Almighty (Praise Be To Him) who enabled me to
achieve all goals.

I would like to express my deepest and special thanks to my main supervisor,
Professor Dr. Khairia M. Youssef, Pharmaceutical Chemistry, College of Pharmacy,
King Saud University, for the initiation and supervision of the project.

I feel indebted to my co-supervisor, Professor Dr. Omaima M.A. Alam El-Din,
Pharmaceutical Chemistry Department, College of Pharmacy, KSU, for her constant
guidance, assistance and full support throughout the realization of the work.

I extend my thanks to all my educators and colleagues in the Pharmaceutical
Chemistry Department, College of Pharmacy, KSU, for their advice and
encouragement.

I appreciate very much the help of Dr. Abeer Al-Alfy, Pharmacology Department,
KSU and Dr. Shabana Khan, National Center for Natural Products Research,
University of Mississippi, USA, for performing the Cytotoxicity Tests.

I also extend my thanks to Hessa Al-Telassy and Maher Al-Jabal, Central
Laboratory, KSU, for performing the different Spectra.

Finally, I am grateful to my parents, husband and all my family for their
encouragement and support.






x
Abstract

It is well-documented that curcumin, the major constituent of turmeric powder
extracted from the rhizome of curcuma longa, possesses a broad spectrum of
biological activity. The most pronounced are: antioxidant, anti-inflammatory,
anticancer, chemopreventive, antibacterial, antifungal, antiparasitic, antiviral and
antihistaminic activities. Other closely related natural congeners, modified
diarylheptanoids as curcuminoids, cyclic diarylheptanoids and non-heptanoid
derivatives were either isolated from plants or synthesized and screened for various
biological effects. Guided by the above-mentioned information, it was designed to
synthesize novel drug candidates containing different alkylamino-, cycloalkylamino-
and aminoheterocyclic moieties attached through an acyl bridge to the phenolic
groups of the curcumin nucleus. In addition, some curcumin esters were also
designed.
A discussion of the theoretical basic concepts for some already acceptable
methods for the synthesis of the designed compounds is included. Referring to the
available knowledge in the literature for the preparation of structurally related
compounds, well-documented methods of synthesis such as acylation, amination and
esterification were adopted. In addition the elegant one step condensation of an
appropriate aromatic aldehyde with acetylacetone-boric oxide complex method was
also used for the preparation of the intermediate compounds.
Actually, forty new final compounds have been synthesized along with curcumin
and ethyl curcumin and eight key intermediates. These are:
- The starting materials: curcumin (1) and ethyl curcumin (2).
- The key intermediates:
* di-O-chloroacetylcurcumin (3a), di-O-chloropropionylcurcumin (3b), di-O-
xi
chloroacetylethyl curcumin (3c) and di-O-chloropropionyl ethyl curcumin
(3d)
from various starting materials using direct and indirect methods.
*3-Methoxy-4-chloroacetyloxybenzaldehyde (4a), 3-methoxy-4-chloro-
propionyloxybenzaldehyde (4b), 3-ethoxy-4-chloroacetyloxybenzaldehyde
(4c) and 3-ethoxy-4-chloropropionyloxybenzaldehyde (4c).
- The target products:
*1,7-Bis(4-Alkyl(cycloalkyl or heteroaryl)aminoacetyloxy)-3-
(methoxyphenyl)-
1,6-heptadiene-3,5-dione (5a-g).
*1,7-Bis(4-Alkyl(cycloalkyl or heteroaryl)aminopropionyloxy)-3-(methoxy-
phenyl)-1,6-heptadiene-3,5-dione (5h-n).
*1,7-Bis(4-Alkyl(cycloalkyl or heteroaryl)aminoacetyloxy)-3-(ethoxyphenyl)-
1,6-heptadiene-3,5-dione (6a-g).
*1,7-Bis(4-Alkyl(cycloalkyl or heteroaryl)aminopropionyloxy)-3-
(ethoxyphenyl)
1,6-heptadiene-3,5-dione (6h-n).
*1,7-Bis(4-(4-substituted sulfanilamido)acyloxy)-3-(methoxyphenyl)-1,6-
heptadiene-3,5-dione (7a,b).
*1,7-bis(4- (4-substituted sulfanilamido)acyloxy)-3-(ethoxyphenyl)-1,6-
heptadiene-3,5-dione (7c-f).
*Di-O-Adamantoylcurcumin (8a) and di-O-adamantoylethyl curcumin (8b).
*Di-O-Heptanoylcurcumin (8c) and di-O-heptanoylethyl curcumin (8d).
*Di-O-(2-Thienoyl)curcumin (8e) and di-O-(2-thienoylethyl curcumin (8f).
The structures of the synthesized compounds were confirmed by using various
spectroscopic tools including IR,
1
H-NMR,
13
C-NMR and mass spectroscopy.
xii
Some selected members of the newly synthesized compounds were screened for
their anticancer activity by determination of their cytotoxic activity which was
compared with that of curcumin, ethyl curcumin and doxorubicin.
In this context, the in vitro cytotoxic activity was assessed against several cell
lines including human cancer cell line SK-MEL (malignant, melanoma), KB
(epidermal carcinoma, oral), BT-459 (ductal carcinoma, breast), and SK-OV-3 (ovary
carcinoma). Vero cells, derived from monkey kidney fibroblasts, and LLC-PK1, from
pig kidney epithelial tissue, were used representing noncancerous cells.
Some of the synthesized compounds, namely 1,7-bis(4-(5-methyl- thiadiazol-2-
yl)- aminoacetyloxy)-3-(methoxyphenyl)-1,6-heptadiene-3,5-dione (5c), 1,7-bis(4-
bis(2-chloroethyl)aminoacetyloxy)-3-(methoxyphenyl)-1,6-heptadiene-3,5-dione (5e),
1,7-bis (4-(5-methylthiadiazol-2-yl)aminopropionyloxy)-3-(methoxyphenyl)-1,6-
heptadiene-3,5-dione (5j) and 1,7-bis(4-(6-methoxybenzothiazol-2-
yl)aminoacetyloxy)-3-(ethoxy- phenyl)-1,6-heptadiene-3,5-dione (6b) showed
cytotoxic activity against various cancerous cell lines with no or little effect on the
noncancerous cells.
The mechanism of cytotoxicity was also investigated by evaluating the inhibition
of the topoisomerases I and II.
In addition, none of the tested compounds showed any inhibition of the
topoisomerase I and exhibited very low inhibition of the catalytic activity of
topoisomerase II which does not correlate with the cytotoxic effect indicating that the
cytotoxicity of these compounds should be explained through a mechanism of action
different from inhibition of topoisomerases.
1
2. Introduction
2.1. Overview

Curcumin (1), [diferuloylmethane, 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-
hepta- diene-3,5-dione], is a well known acyclic diarylheptanoid which has been
identified as the major constituent of turmeric powder extracted from the rhizome of
the plant curcuma longa
[1,2]
.


HO OH
O O
H
3
CO OCH
3

HO OH
OH O
H
3
CO OCH
3

(a) (b)
(1)

Curcuma longa is a plant belonging to the ginger family found in south and
southeast tropical Asia
[3]
. The rhizome has been used for centuries as a spice and as a
coloring agent in many foods. It is the common oriental spice that gives the curry
powder its yellowish color and is frequently used in Indian and Thai cooking. In the
same way, the traditional uses of turmeric in folk medicine are multiple and many of
its therapeutic effects have been confirmed by contemporary scientific research.
Recently, numerous studies have demonstrated the remarkable antioxidant and free
radical scavenging activities of curcumin
[4-7]
. Also, it has long been used as a natural
occurring medicine for the treatment of inflammatory diseases
[8-12]
. In recent years,
several studies have shown that curcumin possesses antiproliferative activities against
tumor cells in vitro
[13]
. Curcumin is a potent inhibitor of tumor intiation in vivo
[14,15]

and is also a potent chemopreventive agent inhibiting tumor promotion in skin, oral,
intestinal and colon carcinogenesis
[16,17]
. Besides, it possesses several other biological
2
activities including antibacterial
[18]
, antiviral
[19]
, antihepatotoxic
[20]
, hypotensive
[21]

and anticholesterolemic
[22]
activities.

2.2. Biological Properties of Curcumin and its Derivatives
2.2.1. Antioxidant Properties of Curcumin

Curcumin (1) has a potent antioxidant activity
[23-25]
and has received attention as a
promising nutraceutical or as a component of designer foods for its cancer preventive
ability
[26]
. Curcumin, having a unique conjugated structure including two
methoxylated phenols (1a) and an enol form of -diketone (1b), its structure shows a
typical radical trapping ability as a chain breaking antioxidant. The antioxidant
mechanism of curcumin and curcumin related phenols, also found in edible or
medicinal plants, has attracted much attention
[7,27-29]
, however, it is still not well
understood.

2.2.1.1. Mechanism of Antioxidant Action of Curcumin and its Derivatives

2.2.1.1.a. It is usually assumed that the phenol moiety is responsible for the
antioxidant activities of the plant phenolic compounds. Consequently, the free radical
chemistry of curcumin and other o-methoxyphenols, has focused on its phenolic
rings
[30-33]
. Generally, the non enzymatic antioxidant process of the phenolic materials
is thought to be divided into the following two stages (Chart i)
[34]
:




3

Stage I :
S-OO

+ AH SOOH + A


Stage II :
A

+ X

non radical materials

Where: S: is the oxidized substance.
AH: is the phenolic antioxidant.
A

: is the antioxidant radical.


X

:

is another radical species or the same as A

.

Chart i

Although the first stage is a reversible process, the second stage is irreversible and
must produce stable radical terminated compounds. During the course of the
antioxidant mechanism study of curcumin, it was shown that dimerization was a main
termination process of the radical reaction of curcumin itself
[35]
. On the other hand in
the food system, the antioxidant coexists with a large amount of oxidizable
biomolecules such as poly- unsaturated lipids. These biomolecules produce reactive
peroxyl radicals during their oxidation, which may act as X

and couple with the


antioxidant radical A

in the second step of the antioxidation process


[23]
.

2.2.1.1.b. Other studies have pointed to the possible involvement of the -diketone
moiety in the antioxidant mechanism of curcumin and its derivatives
[35,36]
. A
hydrogen atom donation from the -diketone moiety to a lipid alkyl or a lipid peroxyl
radical was described as a potentially more important antioxidant action of
curcumin
[29]
. In the case of hydrogen atom donation to a bis-allylic radical (e.g.
linoleic acid radical), the following reaction occurs (Chart ii):
4



Chart ii

It is possible that the resonance stabilized -oxoalkylcurcumin radical
[29]
, with
unpaired electron density distributed between three carbons and two oxygens, adds
oxygen at the central carbon atom to become a peroxyl radical. This occurs in some
curcumin derivatives as in trimethylcurcumin which generates potentially damaging
peroxyl radicals because of its inability of molecular reorganization. This would be an
undesirable reaction because peroxyl radicals propagate lipid peroxidation which is
highly damaging to the cell membranes
[29]
. In comparison, when the addition of
oxygen is inefficient, the curcumin radicals may react with each other or with other
free radicals to yield stable products as curcumin dimers
[35]
.

2.2.1.1.c. Recently, it was proposed that the -diketone moiety alone does not have
antioxidant properties. Apparently, the presence of both -diketone and phenol is
necessary for optimal antioxidant function of curcumin. Therefore, one of the
5
curcumin oxyl radicals generated by its antioxidant action undergoes "molecular
reorganization", i.e., the initially generated curcumin -oxoalkyl radical undergoes
rapid intramolecular shift and is transformed into the phenoxyl radical as a result of
higher resonance stabilization afforded in this phenoxyl radical

(Chart iii)
[7]
:

Chart iii

2.2.1.2. Physiological Antioxidant Mechanism of Curcumin and
Synergism with Water-soluble Antioxidants

Pure curcumin is an extremely potent lipid soluble antioxidant. It positions itself
within the cell membrane where it intercepts lipid (peroxyl) radicals and becomes a
phenoxyl radical. Being more polar than curcumin, the phenoxyl radical may travel to
the surface of the membrane, where it may be repaired by any water-soluble
antioxidant as follows (Chart iv)
[7]
:





6


Chart iv


2.2.2. Anti-inflammatory Properties of Curcumin

Curcumin exhibits remarkable anti-inflammatory properties and has been used as
a naturally occurring medicine for the treatment of inflammatory diseases
[8-12]
.
7
Reports have shown that curcumin has an inhibitory effect on arachidonic acid-
induced inflammation and on arachidonic acid metabolism through the inhibition of
both cyclooxygenase and lipoxygenase pathways. It inhibits 5-lipoxygenase activity
in human neutrophils and cyclooxygenase in bovine seminal vesicles
[37]
. In the same
way, curcumin is a potent inhibitor of both epidermal cyclooyxgenase and
lipoxygenase activity
[38]
, prostaglandins and leukotrienes biosynthesis
[39,40]
.

2.2.2.1. Mechanism of Anti-inflammatory Action

2.2.2.1.a. Although the mechanism of anti-inflammatory action of curcumin remains
unclear, it has been shown to inhibit the activation of nuclear kappa B (NF-
K
B) and
AP-1 transcription factors required for induction of many pro-inflammatory
mediators. Curcumin also blocks the isopentenyl pyrophosphate-induced release of
chemokines macrophage inflammatory protein-1 alpha and -1 beta
[41]
.

2.2.2.1.b. Curcumin decreases the activity of serum aspartate transaminase (AST) and
alkaline phosphatase (ALP). It reduces the inflammatory response by decreasing the
concentration of prostaglandins and fatty acids
[42]
.

2.2.2.1.c. Curcumin lowers the release of lysosomal enzymes and eicosanoids and
decreases the incorporation of [
3
H]arachidonic acid into macrophage lipids by
increasing the secretion of 6-keto-PGF
1
[43]
.

2.2.2.1.d. Curcumin may represent a novel therapeutic agent targeting the vasculature
for inflammatory conditions, such as inflammatory bowel disease, by inhibiting the
expression of vascular cell adhesion (VCAM-1), intercellular adhesion and E-selectin
8
(ICAM-1) as well as monocyte adhesion in tumor necrosis factor-
/lipopolysaccharide (TNF/LPS)-activated HIMECs through blockade of NF-B and
C-Jun N-terminal kinase activity
[44]
.

2.2.2.1.e. Curcumin strongly reduces the protein and mRNA levels of the isoformic
nitric oxide synthase (iNOS) in lipopolysaccharide activated macrophage and blocks
LPS-induced binding of nuclear factor-kB (NF-
K
B) transcription factor necessary for
the iNOS induction to its double stranded oligonucleotide probe
[45]
.

2.2.3. Anticancer Properties of Curcumin

Cancer is the most progressive and devastating disease posing a threat of mortality
to the entire world despite significant advances in medical technology for its diagnosis
and treatment. Recently, considerable attention has been focused on identifying
naturally occurring substances capable of inhibiting, retarding or reversing the process
of multistage carcinogenesis. Wide arrays of phenolic substances, particularly those
present in dietary and medicinal plants, have been reported to possess substantial anti-
carcinogenic and anti-mutagenic effects
[46]
. Curcumin is capturing the attention of
cancer investigators worldwide because of its potent inhibitory properties against
human malignancies
[47]
. Several studies in recent years have shown that curcumin
possesses anti-tumor promoting and anti-proliferating activity against tumor cells in
vitro
[15,48-50]
. Curcumin exhibits remarkable cytotoxic effect on various cancer cells
[51-
53]
. It induces apoptotic cell death in human promyelocytic leukemia HL-60 cells and
human oral squamous carcinoma HSC-4 cells
[54]
. Curcumin also shows a potent anti-
carcinogenic activity against a broad range of tumors, including skin, forestomach,
duodenal and colon carcinogenesis
[16,55-57]
. Curcumin was found to be a potent
9
cytotoxic agent against bladder tumor (MBT and UMUC) cell lines
[58]
and human
prostate (LNCaP) and (DU145) cancer cells
[59,60]
. In addition, curcumin decreases the
Ehrlich's ascites carcinoma (EAC) cell number by the induction of apoptosis in the
tumor cells
[61]
. Curcumin and some of its derivatives are also known as potent
inhibitors of angiogenesis
[62]
whose inhibition is responsible for the broad spectrum of
anti-carcinogenic activity of these compounds. Some curcumin metabolites are
reported to exhibit significant inhibition of corneal neovasculari- zation
[63,64]
. In
addition, some curcumin derivatives are described as potent inhibitors of several cells
line including HOS (bone cancer)
[65]
, 1A9 (breast cancer)
[65]
and MCF-7 (breast
cancer) cell lines
[66]
.

2.2.3.1. Mechanism of Anti-carcinogenic Activity of Curcumin

The anti-carcinogenic properties of curcumin in animals have been demonstrated
by its inhibition of tumor initiation induced by various carcinogens
[13,14,67,68]
.
However, the cellular and molecular mechanisms underlying curcumin induced
apoptosis are not clear. Some of these mechanisms may include:
2.2.3.1.a. Curcumin may cause tumor cells death by upregulation of the proto-
oncoprotein Bax, release of cytochrome c from the mitochondria and activation of
caspase-3
[61,69]
.

2.2.3.1.b. Another mechanism suggests that compounds like curcumin possessing
antioxidant or anti-inflammatory activities may inhibit the tumor promotion and cell
proliferation. The anti-proliferating activity of curcumin can be explained by the
inhibitory effect of arachidonic acid metabolism via the cyclooxygenase and
10
lipoxygenase pathways. The products of arachidonic acid metabolism can activate the
protein kinase C and so may play an important role in the tumor and in the
proliferation process
[70]
. Lipoxygenase-dependent growth has been reported for
various malignant cell lines, such as, mouse melanoma
[71]
, neuroblastoma
[72]
and
MCF-7 human breast cancer cells
[73]
. Curcumin is also a potent inhibitor of other
oxygen generating enzymes such as xanthine dehydrogenase-oxidase. It is also a
potent inhibitor of protein kinase C, EGF-receptor tyrosine kinase and 1-kB kinase
thereby suppressing the tumor promotion by blocking the signal transduction pathway
in the target cells
[74]
.

2.2.3.1.c. Curcumin may cause the intracellular Ca
+2
uptake into mitochondria via a
uniporter pathway involving the execution of apoptosis
[69]
.

2.2.3.1.d. Inhibition of tumor growth and metastasis have been postulated to be due to
the inhibition of angiogenesis. Angiogenesis is the process of new blood vessels
formation by endothelial cells. The process is complex and involves several steps
such as membrane degradation by proteolytic enzymes secreted by endothelial cells,
chemotactic migration toward the stimulus, proliferation of these cells and formation
of vascular loops. Each step of the process is tightly controlled by regulatory factors
that stimulate or inhibit angiogenesis. However, these controlled mechanisms are
often disordered in several pathological diseases including cancer. Angiogenesis is a
crucial process for the outgrowth of cancer cells and the spreading into other
tissues
[75]
.

2.2.3.1.e. Curcumin induces the glutathione-S-transferase (GST) enzyme and partly
prevents the decrease in cellular glutathione (GSH). Studies showed that the cystinyl
11
thiol group of GSH is an important site of thiocarbamoylation by the cytotoxic agent
phenethyl isothiocyanate during induction of apoptosis leading to depletion of cellular
GSH by efflux of the GSH conjugate
[76,77]
.

2.2.3.1.f. One aspect of the antitumor activity of curcumin during the promotion stage
of mammary gland tumorigenesis may be linked to the reduction of free radicals.
Nitric oxide (NO) has been found to inflict damage on important biomolecules, and
the overproduction of NO in diseases may be implicated in carcinogenesis and tumor
progression. It is reported that the presence of three isoforms of nitric oxide synthases
(iNOS) and nitric oxide (NO) generation in the mammary gland correlate with the
mammary gland development and mammary carcinogenesis. Curcumin was found to
be effective and useful in the pathophysiology of the mammary gland. It has the
ability to inhibit the induceable nitric oxide synthase (iNOS) induction by
lipopolysaccharide (LPS) in the mammary gland and scavenges NO radicals
[78]
.

2.2.3.1.g. Other mechanisms for the cytotoxic action of curcumin against cancer cell
involve modulation of lymphocytes mediated in immune function by increased
mucosal CD4
+
T cells and B cells in animals treated with curcumin
[79]
and
mobilization of endogenous copper
[80]
.

2.2.4. Chemopreventive Properties of Curcumin

Curcumin is a potent chemopreventive agent that has been entered into phase I
clinical trials for chemoprevention by National Cancer Institute (NCI)
[26]
. The
chemoprevention effects of curcumin have been attributed to various properties
including its anti-angiogenesis action
[62,64]
which limits the blood supply to rapidly
12
growing malignant cells, its stimulation of phase I and phase II detox systems e.g.
inhibition of COX-1 and COX-2 enzymes, and stimulation of glutathione-S-
transferase
[81,82]
, its interference with cell growth by inhibition of protein kinases, and
especially its neutralization of carcinogenic free radicals
[30-33]
.

Curcumin significantly
inhibits the activity of the isoenzymes of cytochrome P-450 involved in the
metabolism of some carcinogens
[83]
.

It is possible that any one, more than one, or all
of these biological, biochemical and chemical mechanisms are responsible for the
anticarcinogenic potential of curcumin. Curcumin possesses a potent preventive
action on radiation-induced initiation of mammary tumorigenesis in rats
[84]
. It exerts
skin cancer chemopreventive effects
[85]
, cytoprotective activity
[86]
and a protective
effect on aflatoxin-induced mutagenicity and hepatocarcinogenicity
[87]
. Later,
curcumin was further demonstrated to have an antimetastatic effect in mice. It inhibits
SK-Hep-1 hepatocellular carcinoma cell invasion in vitro and suppresses matrix
metalloproteinase-9 secretion
[20]
.

2.2.5. Antibacterial, Antifungal and Antiparasitic Properties
of Curcumin

The antibacterial activity of curcumin bioconjugates has been tested particularly
for -lactamase producing microorganisms
[18]
. Turmeric oil was also tested for
antibacterial activity against Bacillus cereus, Bacillus coagulans, Bacillus subtilis,
Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa
[88]
. Turmeric
oil was tested for its antifungal activity against Aspergillus flavus, Aspergillus
parasiticus, Fusarium moniliforme and Penicillium digitatum
[89]
. When combined
with amphotericin B or fluconazole, curcumin provided a greater fungicidal effect in
the treatment of systemic fungal infections such as candidiasis and candidemia
[90]
.
13
Curcumin was found to be an antiparasitic agent of natural sources being cytotoxic
against African trypanosoma in vitro
[91]
. It was also observed that curcumin possesses
nematocidal activity against visceral Larva migrans
[92]
and against the second-stage
larvae of dog roundworm, Toxocara canis
[93]
.

2.2.6. Antiviral Properties of Curcumin

Curcumin is currently undergoing clinical trials for AIDS patients and its effect
has been determined on purified human immunodeficiency virus type 1 (HIV-1)
integrase. The anti-integrase activity of curcumin could be due to an intramolecular
stacking of the two phenyl rings that bring the hydroxyl groups into close proximity.
This HIV-1 integrase inhibition may contribute to the antiviral activity of curcumin.
These observations suggest new strategies for antiviral drug development based upon
curcumin as a lead compound for the development of inhibitors of HIV-1 integrase
[19]
.

2.2.7. Antihistaminic Properties of Curcumin

Curcuminoids were also found to inhibit the histamine release from rat peritoneal
mast cells
[94]
. These compounds potentially suppress the histamine release probably
through blockade of the degranulation of the mast cells following a rise in the
intracellular Ca
+2
levels induced by some types of histamine releasers
[95]
.

2.2.8. Curcumin for Treatment of Skin Diseases

Curcumin and curcumin analogs, being angiogenesis inhibitors, can also be used
to treat skin disorders. Curcumin inhibits angiogenesis, in part, by inhibition of the
14
basic fibroblast growth factor (bFGF). Representative skin disorders to be treated by
curcumin and its analogs include malignant diseases such as angiosarcoma, basal cell
carcinoma, malignant melanoma and Kaposi's sarcoma. Non malignant diseases
include psoriasis, acne, rosacea, eczema, seborrheic keratosis and actinic keratosis
[96]
.
Curcumin derivatives were also used as antioxidants in cosmetics for the protection of
keratinous tissue against environmental aggressors such as smoke and UV
radiation
[97]
.

2.3. Classification of Curcumin Analogs
2.3.1. Naturally Occuring Diarylheptanoids

Besides curcumin, there are some closely related natural congeners
such as desmethoxycurcumin (2) and bis-desmethoxycurcumin (3)
[98]
.
R
1
HO
O O
R
2
OH


(1) R
1
= R
2
= OCH
3
(curcumin)
(2) R
1
= H, R
2
= OCH
3
(desmethoxycurcumin)
(3) R
1
= R
2
= H (bis-desmethoxycurcumin)

Desmethoxycurcumin (2) was recently found to be selectively active against KB
cells (nasopharynx epidermoid carcinoma) with an ED
50
value of 1g/ml
[65]
and a
potent inhibitor of angiogenesis
[75]
. It was the most potent among the three curcumin
analogs as an anti-inflammatory agent
[99]
.
Curcumin has attracted few model studies although it has been isolated as early as
1815
[98]
.

It was crystallized by Daube
[100]
and its structure was elucidated in 1910 by
15
Lampe and co-workers
[101]
, who later completed its synthesis
[102]
. Renewed interest
has been evoked by the recent discovery of relatives sharing the 1,7-diarylheptanoid
skeleton.
Phenolic diarylheptanoids, including 4-hydroxycinnamoyl-(4-hydroxy-3-
methoxy- cinnamoyl)methane (2) and bis-(4-hydroxycinnamoyl)methane (4), have
been both isolated from Curcuma species
[103]
.
O O
R
OH
HO

(4) R = H

The male flowers (catkins) of certain Alnus species contain 1,7-diphenylhepta-4,6-
dien-3-one (5), 1,7-diphenyl-5-hydroxyhept-1-en-3-one (6), 1,7-diphenyl-5-
hydroxyheptan-3-one (7) and 1,7-diphenylheptane-3,5-diol (8)
[104-106]
while

Centrolobium species contain 1,7-bis(3-hydroxyphenyl)-3-heptanol ((-)centrolobol)
(9)
[107]
.
O

(5)


OH O

(6)

OH
R R
X Y

16

(7) R = H, X,Y = O
(8) R = X = H, Y = OH
(9) R = OH, X =Y = H
Numerous chemical investigations of Ginger
[108]
, the rhizome of Zingiber
officinale Roscoe (Zingiberaceae), have led to the isolation and identification of some
diarylheptanoids from the ethanol extract of the rhizomes of Z. officinale
[109]
.

These
new compounds are: (3S,5S)-3,5-diacetoxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-
heptane (10) and its derivatives, (5S)-5-acetoxy-1,7-bis(4-hydroxy-3-
methoxyphenyl)- heptan-3-one (11) and its derivatives, and the diarylheptenone 1,7-
bis(4-hydroxy-3-methoxyphenyl)-hept-4-en-3-one (12).
OCH
3
OH
HO
H
3
CO
OCOCH
3
H
3
COCO

(10)

OCH
3
OH
HO
H
3
CO
O OCOCH
3

(11)

H
3
CO
HO
O
OCH
3
OH

(12)

The Aceraeae plant Acer nikoense MAXIM is indigenous to Japan and its stem
bark has been used as a folk medicine for the treatment of hepatic disorders and eye
diseases
[110]
. Among the chemical constituents of this folk medicine, the
17
diarylheptanoid (13) and the diarylheptanoid glycoside aceroside VIII (14) were
characterized
[110-114]
.


HO
OR
OH

(13) R = H
(14) R = Glc
6_1
Api

Diarylheptanoids (15,16) have also been isolated from the roots of Juglans
mandshurica MAXIMOWICZ (Juglandaceae) which have been used as a folk
medicine for treatment of cancer in Korea
[115-120]
. Compound (16) showed weak
cytotoxicities against the HT-29 (human colon carcinoma) and MCF-7 (human breast
carcinoma) cell lines with IC
50
of 41.3g/ml and >50 g/ml, respectively
[115]
.
HO
OCH
3
OH
O
HO
H

(15)

HO
OCH
3
OH
O OCH
3

(16)

As regards the chemical components of the rhizomes of Temu Lawak, Curcuma
xanthorrhiza (Zingiberaceae), several diarylheptanoids have been isolated
[121,122]
,
including

(3S,5S)-1,7-bis(4-hydroxy-3-methoxyphenyl)heptane-3,5-diol (17) and (1)-
1-hydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-6-heptene-3,5-dione (18), in addition
to dihydrocurcumin (19), hexahydrocurcumin (20) and curcumin (1).
18

H
3
CO
HO
OCH
3
OH
OH
OH

(17)

H
3
CO
HO
O O
OCH
3
OH
OH

(18)
H
3
CO
HO
O O
OCH
3
OH

(19)

HO
OCH
3
OH
O
OH
H
3
CO

(20)

Another new diarylheptanoid
[121]
, (3R,5R)-1-(4-hydroxyphenyl)-7-phenyl-
heptane-3,5-diol (21), was also isolated by the same team-workers from the rhizomes
of Alpinia officinarum (Zingiberaceae).
OH
OH
OH

(21)

The naturally occurring diarylheptanoid, yakuchinone B (22), displayed a dual
inhibition of 5-lipoxygenase and cyclooxygenase enzymes in vitro
[123]
. However, this
19
compound (22) did not exhibit reproducible in vivo inhibition of LTB
4
biosynthesis or
anti-inflammatory activity.
O
H
3
CO

(22)

2.3.2. Modified Diarylheptanoids
2.3.2.1. Curcuminoids

In an effort to impart more potent in vitro inhibition and confer in vivo activity in
this chemical class of compounds, the structure-activity relationships (SAR) of
various curcumin analogs was explored. This led to the development of novel series
of synthetic curcuminoids that were investigated for various purposes.
Previously, methylation with methyl iodide was reported to provide di-O-
methylcurcumin (23)
[124,125]
.
H
3
CO OCH
3
O O
H
H
3
CO OCH
3

(23)

Later, it was found that treatment of curcumin (1) with methyl iodide in refluxing
acetone gave mainly the tetramethyl derivative (24), although some trimethyl
derivative (25) was also obtained
[107]
.
H
3
CO OCH
3
O O
OCH
3
H
3
CO

(24)
20

H
3
CO OCH
3
O O
OCH
3
H
3
CO
CH
3
H

(25)

Trimethylcurcumin (25) was found to be active against a narrow spectrum of cell
lines including 1A9 (ovarian cancer) and HOS (bone cancer)
[65]
. Similarly,
diacetylcurcumin (26) was readily prepared by treating curcumin with acetic
anhydride
[124]
.
H
3
CO
H
3
COCO
O
OCH
3
OCOCH
3
O
H

(26)

Hydrogenation of curcumin (1) was also investigated under a variety of conditions
and catalysts; however, mixtures of tetrahydrocurcumin (27), hexahydrocurcumin
(20) and octahydrocurcumin (28) were always obtained
[107,121,123,126]
. Saturation of the
olefinic bond of curcumin gave compounds (20,28) with abolished cytotoxic activity.
Therefore, the presence of the conjugated -diketone in a cyclic carbon chain appears
to play an important role for cytotoxicity in this class of compounds
[65]
.
OCH
3
OH
HO
H
3
CO
O O
H

(27)



21


H
3
CO
HO
OH OH
OCH
3
OH

(28)

In another report, the methylthiomethyl derivative (29) of curcumin was prepared
from curcumin for the improvement of its antioxidant activity and the effectiveness of
the new compound (29) was confirmed
[127]
.
O O
S
H
3
C
S
CH
3
OH
OCH
3
HO
OCH
3

(29)

A series of hydrazinocurcumins
[65,75,123]
(30,31) and hydrazinotetrahydro-
curcumins
[123]
(32) was synthesized to enhance some biological activity of curcumin.
These derivatives were found to be inhibitors of 5-lipoxygenase and cylooxygenase
activities in rat basophilic leukemia cells. Their angiogenic activity was also
evaluated. Compounds from these series were found to inhibit the proliferation of
bovine aortic endothelial cells (BAECs) at a nanomolar concentration of IC
50
= 520
nM without discernable cytotoxicity
[75]
. These compounds were prepared by treating
curcumin and dihydrocurcumin (19) with hydrazine hydrate to give the corresponding
pyrazole derivatives
[65,75,123]
.



22
N NH
R
2
OH
R
1
HO

(30)
(a) R
1
= R
2
= OCH
3

(b) R
1
= OCH
3
, R
2
= H
(c) R
1
= R
2
= H

N N
R
2
OH
R
1
HO
O
OH

(31)
(a) R
1
= R
2
= OCH
3

(b) R
1
= OCH
3
, R
2
= H
(c) R
1
= R
2
= H

N NH
OCH
3
OH
H
3
CO
HO

(32)

Conversion of curcumin to the pyrazole analog (30a) resulted in a more potent 5-
lipoxygenase inhibitor while the reduced analog (32) was 53-fold less active than
compound (30a)
[123]
. Thus, it would appear that at least one of the olefinic bonds of
curcumin (1) is necessary for potent 5-lipoxygenase inhibition and that pyrazoles
might retain or enhance the 5-lipoxygenase inhibitory properties in this class of
compounds
[123]
. Hydrazinocurcumin (30a) also showed a broad cytotoxicity spectrum
against a wide range of human tumor cell lines including HOS (bone cancer), CAKI-1
23
(renal cancer), MCF-7 (breast cancer), 1A9 (ovarian cancer) and HepG2 (liver
cancer)
[65]
. In another series of curcumin derivatives with altered potencies against
HIV-integrase as probes for biochemical mechanisms of drug action, three curcumin
analogs, namely dicaffeoylmethane (33), caffeoylferuloylmethane (34) and rosmarinic
acid (35), were found to be very potent as inhibitors of HIV-1 integrase activity
showing potencies comparable to that of curcumin. These three analogs had at least
one catechol structure suggesting that the methoxy groups do not play a key role in
potency. Two of these potent analogs (33) and (35) were examined for their effects on
the strand transfer reaction using a precleaved substrate corresponding to the product
of the 3'-processing reaction. Both analogs were able to inhibit the strand transfer
activity of HIV-1 integrase in this assay
[126]
.
HO
HO
O O
OH
OH

(33)


HO
HO
O O
OCH
3
OH


(34)


HO
HO
O
O COOH
OH
OH

(35)

In order to study the anti-inflammatory activity of some curcumin analogs, a
series of curcumin derivatives (36a-f)
[99]
was prepared and the inhibition of the
24
carrageenan-induced edema by these compounds was established. It was deduced that
the para hydroxy groups in curcumin were important for anti-inflammatory activity
and that this activity was enhanced when, in combination with the para hydroxy
groups, the meta positions were occupied with alkyl groups. Since the methyl
derivatives were more active than the corresponding ethyl and tertiary butyl
derivatives, it was suggested that steric hindrance was involved
[8,99]
. Probably an
electron donating substituent in the para position was also favorable for activity.
R
3
O
R
2
O
H
R
4
R
1
R
2
R
3
R
1
R4

(36)

R
1
R
2
R
3
R
4


(a) H CH
3
OCH
3
CH
3

(b) H C
2
H
5
OH C
2
H
5

(c) H i-C
3
H
7
OH i-C
3
H
7

(d) OCH
3
H H H
(e) H H Cl H
(f) H t-C
4
H
9
OH t-C
4
H
9


Furthermore, some curcumin bioconjugates, namely di-O-glycinoyl curcumin
(37), di-O-glycinoyl-C
4
-glycylcurcumin (38), 5-deoxy-5-curcuminylthymidine (39)
and 2-deoxy-2-curcuminyluridine (40), have been synthesized. The antibacterial
activity of these bioconjugates has been tested particularly for -lactamase-producing
microorganisms
[18]
. The

results showed that the amino acid bioconjugates were
approximately two times more active than the nucleoside bioconjugates. The
25
hydrophilic nature of these amino acid bioconjugates may help in active transport
across cellular membrane.
H
3
CO
H
2
N-H
2
C-OC-O
OCH
3
O-CO-CH
2
-NH
2
O O

(37)
H
3
CO
H
2
N-H
2
C-OC-O
OCH
3
O-CO-CH
2
-NH
2
O O
CO-CH
2
-NH
2


(38)

H
3
CO OH
H
3
CO OH
O
O
N
HN
O
O
O
H
OH
CH
3

OCH
3
OH
OCH
3
HO
O
O
N
HN
O
O
O
H
HO
OH


(39) (40)

Fluorinated diarylheptanoids (41a-g) were also prepared and evaluated for their
cytotoxic effect against a panel of human tumor cell lines. The ortho fluorinated
compound (41a) having a fluorine atom on each benzene ring had a broad
cytotoxicity spectrum while the remaining fluorinated diarylheptanoids were inactive
(41 b-g)
[65]
.
26
R
4
R
4
R
3
R
2
R
1
R
1
R
2
R
3
O OH


(41)
R
1
R
2
R
3
R
4

(a) F H H H
(b) H F OCH
3
H
(c) H CF
3
F H
(d) H H OCF
3
H
(e) F H F H
(f) F H OCH
3
H
(g) F H H OCH
3


Glycosidation
[128]
of curcumin has recently been reported by acetobromoglucose
in the presence of triethylbenzylammonium bromide (Et
3
BnN
+
Br
-
) to give the
monoglucoside (42) and the diglucoside (43) in 8 and 3% yields respectively
[129]
.
Later, symmetrical and unsymmetrical glycosylcurcuminoids (44, 45) were obtained
in a one-step condensation of the appropriately glycosylated aromatic aldehyde with
acetylacetone-boric oxide complex
[129]
.
O
H
3
CO OCH
3
OH
O OH
O
HO
HO
OH
HOH
2
C

(42)


O
H
3
CO OCH
3
O
O OH
O
HO
HO
OH
HOH
2
C
O
HO
OH
OH
CH
2
OH

(43)


27
OH O
R
1
OR
2
R
1
HO


(44)

(a) R
1
= OCH
3
, R
2
= Glc-Ac
(b) R
1
= H, R
2
= Glc-Ac
(c) R
1
= OCH
3
, R
2
= Gal-Ac


O OH
OCH
3
OGlc
H
3
CO
GlcO

(45)


2.3.2.2. Cyclic Diarylheptanoids

The m,m-bridged biphenyls myricanol (46)
[107]
, myricanone (47)
[130]
(Myrica
nagi), 13-oxomyricanol (48)
[65]
, asadanin (49) and its relatives
[107,131]
(Ostrya
japonica) are closely related in structure to curcumin. Of these compounds,
myricanone (47) and 13-oxomyricanol (48) exhibited potent antitumor promoting
effects on 12-O-tetradecanoyl- phorbol-13-acetate (TPA)-induced mouse skin
carcinogenesis
[65]
. Furthermore, myricanone (47) inhibited papilloma formation
initiated by peroxynitrite
[65]
.
OH
OCH
3
OH H
3
CO
X


(46) X = H, OH
(47) X = O
28

OH
H
3
CO
HO
H
3
CO
OH
O

(48)

OH HO
O
OH
OH HO

(49)

Two other cyclic diarylheptanoids (50, 51) were isolated from the roots of Juglans
mandshurica MAXIMOWTEZ (Juglandaceae); however, they were inactive when
assayed by the tetrazolium-based colorimetric assay (MTT cytotoxicity assay) against
the human colon carcinoma (HT-29) and the human breast carcinoma (MCF-7) cells
[115]
.
O
O
HO
H
3
CO
H
OH

(50)

29
O
HO
H
3
CO
H OH

(51)

During the course of some studies on bio-active constituents of natural medicines
and medicinal foodstuffs, cyclic diarylheptanoids, namely acerosides B
1
(52), B
2
(53)
and aceroketoside (54), were isolated from the stem bark of A. nikoense and their
inhibitory effects on nitric oxide (NO) production in lipopolysaccharide (LPS)
activated macrophages were determined
[110]
.
O
O
OH
O
H
OH
OH
OH
HO

(52)

O
O
O
H
OH
OH
OH
HO
OH

(53)

30
O
HO
O
O
O
O
OH
OH
O
OH OH
HO


(54)

Two other cyclic diarylheptanoids, namely acerogenin B (55) and acerogenin K
(56) were isolated from the same plant and showed inhibitory activity of the release of
-hexosaminidase induced by dinitrophenylated bovine serum albumin (DNP-BSA)
from RBL-2H3 cells sensitized with anti-DNP Ig E. The activities of these two
compounds (55) and (56) were stronger than those of the two antiallergic compounds,
tranilast and ketotifen fumarate
[110,132]
.
O
HO
OH


(55)

HO
HO
OH


(56)
31
Among the same series, 2 novel cyclic diarylheptanoid glucosides; namely;
myricatomentoside I (59) and myricatomentoside II (60) were isolated from the
branches of Myrica gale var. tomentosa
[133]
and a diphenyl ether-type diarylheptanoid
(61) was isolated from the fruits of Rhoiptelea chiliantha DIEL et HAND.-MAZZ
(Rhoipteleaceae)
[134]
. The chemical structures of the isolated cyclic diarylheptanoids
were elucidated; however, there were no report about their pharmacological activities.
H
3
CO
Glu-O
O
HO
O

O-Glu
H
3
CO
H
3
CO
HO
OH
O

(57) (58)

H
3
CO
H
3
CO OH
H
OH
O

(59)

In another report, the synthesis and complexation properties of two new bis-
curcuminoids bearing a diphenylmethane bridge (60) and a crown ether chain (61)
were studied
[135]
.
H
3
CO
HO
HO
H
3
CO
OCH
3
OH
O OH
OHO
OH
OCH
3

(60)
32
H
3
CO
O
O
H
3
CO
OCH
3
OH
O OH
OHO
OH
OCH
3
O
O

(61)

2.3.2.3. Non-heptanoid Derivatives

Recently, some 1,3-diaryl-1,3-diketopropane (62, 63), butane (64) or pentane (65),
structurally related to curcumin, were prepared and screened for cytotoxicity effect
against the human tumor cell line panel
[65]
.
N N
N
O O


(62)

R R
R
1
O O


(63)

(a) R = H, R
1
= H
2
(d) R = F R
1
= H
2

(b) R = Cl, R
1
= H
2
(e) R = H R
1
= Br
(c) R = C(CH
3
)
3
R
1
= H
2
(f) R = H R
1
= NO

33
O O
O O

(64)

O O O
R R

(65)
(a) R = H
(b) R = Cl

The -diketone (63a) displayed moderate cytotoxicity, whereas the -triketone
(65a) and the -tetraketone (64) were inactive. The 4-chlorophenyl derivative (63b)
was also more active than the corresponding triketone (65b). It would appear that the
-diketone moiety enhances the cytotoxic properties. The 4-tert-butylated phenyl
derivative (63c) was more potent than the unsubstituted derivative (63a) and against
1A9 (ovarian cancer) cells selectively. Thus, introducing an electron-donating
substituent, as the tert-butyl group, on the phenyl ring led to increased cytotoxicity
against 1A9 cell line. Replacing the hydrogen atom with an electron-withdrawing
substituent as fluorine, at the para position on the benzene rings gave compound
(63d) with increased activity against 1A9 cell compared with the unsubstituted
derivative (63a) and 4-chlorophenyl substituted compound (63b). -Bromination
between the keto groups (63e) led to enhanced activity compared with the
unsubstituted compounds (63a-d) against 1A9 cell. However, nitroso (63f)
substitution at this position abolished activity
[65]
. In addition, replacing the phenyl
groups in compound (63e) with thiophene moieties provided compound (66) with
increased cytotoxicity against HOS and 1A9 cell lines
[65]
.
34
S
S
Br
O O

(66)

Moreover, a series of 1,3-diarylpropenones was synthesized and screened as
potential inhibitors of NO and PGE
2
production in the RAW 264.7 macrophage cell
line. 4-Dimethylamino-2',5'-dimethoxychalcone (67) was found to be the most potent
and dual inhibitor of NO and PGE
2
production. It also inhibited significantly the
formation of edema in the carrageenan-induced model of inflammation in mice by the
oral route
[136]
.
O
(H
3
C)
2
N
OCH
3
OCH
3

(67)

Afterwards, a novel series of dibenzoylmethane derivatives (68-70) having both
sunscreen and cytotoxic activity has been obtained by derivatizing commercial
dibenzoylmethanes
[137]
. Many of the prepared compounds showed antimelanoma
activity.

R
1
O
R
3
O
R
2

R
1
O
R
3
OH
R
2

(68) (69)
R
1
OH
R
3
OH
R
2

(70)
35
Cyclocur (71), a cyclic curcumin analog, was prepared from curcumin
[93]
. Its
effect was examined on the proliferation of MCF-7 human breast tumor cells and
found to be less inhibitory than curcumin
[66]
.
H
3
CO
HO
O
OCH
3
OH
O

(71)

Recently, a new series of 3,5-bis(substituted benzylidene)-4-piperidones (72), 2,7-
bis(substituted benzylidene)cycloheptanones (73), 1,5-bis(substituted phenyl)-1,4-
pentadien-3-ones (74), 1,1-bis(substituted cinnamoyl)cyclopentanes (75) and 1,1-
bis(substituted cinnamoyl)cyclohexanes (76) has been synthesized and compounds
were tested for their antioxidant activity
[138]
. Some of the synthesized compounds
exhibited high free radical scavenger activity.
R'
XO
R'
OX
N
O
R
R'' R''

R'
XO
R'
OX
O
R'' R''


(72) (73)

R'
XO
R'
OX
O
R'' R''

(74)
36


O
R' R'
O
XO
R" R"
OX

(75)

O
R' R'
O
XO
R" R"
OX


(76)




37
3. Research Objectives

The introductory part revealed that curcumin, its derivatives as well as its
modified arylheptanoids constitute a wide variety of biologically active agents
covering diverse pharmacologic activities. However, the poor absorption of curcumin
through the intestinal wall on oral intake needs to be improved in order to achieve
significant concentration inside the cells for appropriate activity. One of the most
easily accessible approaches is to make bioconjugates of curcumin to promote its
entry into the target cells.
Curcumin has an interesting structure with phenolic groups and an active
methylene function which are potential for its activity. The phenolic groups are sites
involved in enzymatic activity at the receptor sites while double bonds are essential
for proper conformational flexibility of the molecule
[139]
. Structural modification of
curcumin has been reported to enhance its activity.
Accordingly, it became of interest to synthesize some new bioconjugates with
various functionalities supported on the curcumin skeleton to be evaluated for
anticancer activity. Curcumin bioconjugates can serve a dual purpose of systemic
delivery as well as therapeutic agents against cancer diseases. This design would
allow enzyme-mediated transformation of the bioconjugate within the target organ.
The selected moieties involved in such structural modification featured sulfonamide,
alkyl, cycloalkyl and heterocyclic amino functionalities attached to curcumin or ethyl
curcumin through an acetyl or propionyl bridge to investigate the effect of molecular
modification on the biological activity of compounds (A).





38

O
O O
O
R
1
O OR
1
O
O
curcumin skeleton
carbonyl group
R
2
N-(CH
2
)
x
R
3
(CH
2
)
x
-N
R
2
R
3
methyl or ethyl bridge
alkyl, cycloalkyl, heterocyclic amine or sulfonamide.


(A)

Furthermore, we were motivated to select adamantoyl chloride, heptanoyl chloride
and 2-thienoyl chloride and directly attach them through an ester function to the
curcumin and ethyl curcumin core to furnish compounds (B).

O
O O
O
R
1
O OR
1
O
R
2
O
R
2
curcumin skeleton
carbonyl group
Adamantyl or thienyl

or hexyl
(B)
The conjugate bonds reported herein are ester or amino ester linkages which are
enzyme sensitive to produce the expected systemic delivery.
The synthesis of the target compounds is outlined in Schemes 1, 2 and 3.
Recently, curcumin, having a planar topology, has been shown to inhibit
topoisomerase II in a similar fashion to the antineoplastic agent etoposide
[140-142]
.
Results pointed to DNA damage induced by topoisomerase II poisoning as a possible
mechanism by which curcumin initiated apoptosis. With the hope to go a step forward
in the field of anticancer agents, the synthesized compounds were screened for their
cytotoxic activity as well as topoisomerase inhibitory activity.
39
HO
CHO
R
1
O
+
O O
B
2
O
3
(BuO)
3
B/
n-BuNH
2
R
1
O
O O
OR
1
OH HO
(1) R
1
= CH
3
(2) R
1
= C
2
H
5
O
Cl (CH
2
)
n
Cl anh. K
2
CO
3
/
O
/ RT
R
1
O
O O
OR
1
O O (CH
2
)
n
O O
n
(H
2
C)
Cl Cl
(3 a-d)
O O
B
2
O
3
/
(BuO)
3
B/ n-BuNH
2
HO
CHO
R
1
O
Cl
O
(CH
2
)
n
Cl
+
1N NaOH/ CHCl
3
or
O
CHO
R
1
O
O
n
(H
2
C)
Cl
(4 a-d)
anh. K
2
CO
3
/
O
Scheme 1
2
2

40
R
1
O
O O
OR
1
O O (CH
2
)
n
O O
n
(H
2
C)
Cl Cl
(3 a-d)
Scheme 2
R
2
R
3
NH NEt
3
/
O
NH
2
- -SO
2
NHR
4
R
1
O
O O
OR
1
O O (CH
2
)
n
O O
n
(H
2
C)
R
3
R
2
N NR
2
R
3
R
1
O
O O
OR
1
O O (CH
2
)
n
O O
n
(H
2
C)
NH-
-SO
2
NHR
4 -NH R
4
NHSO
2
-
(5 a-n, 6a-n)
(7a-f)

41
R
1
O
HO
O O
OR
1
OH
Scheme 3
(1), (2)
COCl
R
1
O
O
O O
OR
1
O-
R
1
O
O
O O
OR
1
O- CO- CO
(8a, b)
S
CO-
S
OC
(8e, f)
Na
2
CO
3
/
O
S
COCl
Na
2
CO
3
/
O
Na
2
CO
3
/
O
CH
3
(CH
2
)
5
COCl
R
1
O
H
3
C(H
2
C)
5
OCO
O O
OR
1
OCO(CH
2
)
5
CH
3
(8c, d)

42
4. Results and Discussion

4.1. Chemistry

Compounds 3a-d; namely: di-O-chloroacetylcurcumin (3a), di-O-
chloropropionyl- curcumin (3b), di-O-chloroacetylethyl curcumin (3c) and di-O-
chloropropionylethyl curcumin (3d), were the key intermediates for the preparation of
our target compounds. To achieve this, Scheme 1 was adopted.

4.1.1. 1,7-Bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (1)
and 1,7-bis(4-hydroxy-3-ethoxyphenyl)-1,6-heptadiene-3,5-dione (2)

The chemical structure of curcumin was elucidated
[143]
after its isolation in 1870
and was subsequently confirmed by synthesis. Vanillin and acetylacetone were used
in this synthesis which required eight reaction steps. However, the yield was very
poor, and therefore this synthesis had little practical value
[99]
.
In an attempt to prepare curcumin and ethyl curcumin, search of the literature
demonstrated that chalcones, in general, are known to be prepared via the classic
enolate condensation reaction which is an acyl addition reaction of a nucleophilic
enolate to an electrophilic carbonyl carbon
[144]
. In such case, generation of the enolate
anion (B) can be obtained by reacting an aliphatic ketone (A) with the aromatic
aldehyde (C) in 40% KOH solution at RT
[145]
, alcoholic NaOH
[136,138]
at RT or
NaOMe in refluxing EtOH
[138]
to afford the expected aldolate (D) which eliminated
water under the reaction conditions to give the Claisen-Schmidt product, the
conjugated ketone (E)
[146]
(Equation 1):

43
O
H
a
R
Base
- H
a
O
-
R
O
R
-
A B
+
Ar
O
H
B
R
O
H
H
H
O
-
Ar - H
2
O
R
O
H
Ar
H
D E
C
+ H
+

Equation 1

Variation of the reaction conditions involved also the use of concentrated HCl at
25-30C
[138,147,148]
. Accordingly, trials to prepare curcumin and ethyl curcumin (2) by
the previously mentioned methods using vanillin or ethyl vanillin and acetylacetone in
conc. HCl, NaOH/EtOH or NaOEt/EtOH was fruitless (Equation 2):
CHO RO
RO
O O
OR
OH HO
HO
NaOH/EtOH, NaOEt/EtOH
or c.HCl
XXX
O O
+
1
1
2
1

Equation 2

In the same way, it was also designed to prepare some bioconjugates substituted
at the methylene group in position 4 of the heptadienedione moiety as well as at the
phenolic groups; however, attempts to prepare such compounds (I and II) through the
formation of a sodium salt gave nothing but decomposition products in addition to the
unreacted starting material as revealed by TLC and
1
H-NMR.
OR
OCO(CH
2
)
n
NHR
2
RO
R
2
HN
n
(H
2
C)OCO
O O
CO
(CH
2
)
n
NHR
2
1
1

(I)
44
R
1
O
R
2
HNOCHNO
2
SO
O O
OR
1
OSO
2
NHCONHR
2
SO
2
NHCONHR
2

(II)

In 1973, Roughley and Whiting
[107]
reported the instability of curcumin in alkaline
medium and explained its alkaline degradation as shown in equation 3:
H
3
CO
O O
OCH
3
OH
HO
HO
COOH H
3
CO
HO
H
3
CO
O
+
H
3
CO
HO
CHO
+ CH
3
COCH
3
Ferulic acid
Feruloylmethane
acetone
vanillin
NaOH
NaOH

Equation 3

Cleavage of the -diketone function in curcumin gave ferulic acid (4-hydroxy-3-
methoxycinnamic acid) and feruloylmethane; the latter formed vanillin and acetone
by a retro-aldol fission. Later, Sardjiman and co-workers
[148]
and Nurfina and co-
workers
[99]
reassured the instability of curcumin at pHs higher than 6.5 which is
caused by the active methylene group as previously mentioned above.
However, in the present investigation, the synthetic method was completely
different from the previously mentioned methods and involved a one step
condensation of the appropriate aromatic aldehyde with acetylacetone-boric oxide
complex. This method was originally reported by Pabon
[143]
and modified by
Roughley and co-worker
[107]
. The method found large application because of its
45
versatility, wide application for the preparation of symmetrical and unsymmetrical
curcuminoids and other diketones, high yield and purity
[65,99,129,149]
.
Pabon
[143]
, at first, mentioned the synthesis of curcumin by heating vanillin,
acetylacetone and boric anhydride (2:1:2) for 30 minutes and claimed a yield of 10%
in this one step procedure. Then, he improved this procedure by using tributyl borate
and piperidine as catalysts and the freshly prepared complex of acetylacetone and
boric anhydride (yield 25%)
[143]
. In addition, he synthesized some curcumin
derivatives, using vanillin (or benzaldehyde derivatives), tributyl borate, ethyl acetate,
the complex of acetylacetone and boric anhydride, and butylamine at RT.
The main features in Pabon procedure for the synthesis of curcumin are (Equation
4):
a) The protection of the active methylene group by reacting acetylacetone with boric
anhydride in order to produce the acetylacetone-boric anhydride complex (A).
b) The less reactive methyl terminals of this complex will react with the aldehydic
group of vanillin in order to give curcumin in the form of the complex with boron (B).
c) The complex is then decomposed by using either dilute acids or bases. Dilute acid
is preferable since curcumin itself is unstable under alkaline conditions (C).

46
H
3
CO
O O
OCH
3
OH
HO
CH
3 H
3
C
O O
O
B
O
O O
CH
3
H
3
C
H
3
C CH
3
O
H
HO
OCH
3
+ B
2
O
3
+ BO
2
-
+ H
2
O
(A)
n-BuNH
2
O
B
O
O O
CH=CH HC=HC
HC=HC CH=CH
OH
OCH
3
HO
OCH
3
OCH
3
O
+
H
H
3
CO
H
+
O
(B)
2
(C)
HCl
4
Cl
-
Cl
-
2


Equation 4

Guided by the considerable evidence accumulated in the literature demonstrating
the inablicability of the normal Claisen-Schmidt condensation for the preparation of
various curcuminoids and the efficacy of the acetylacetone-boric anhydride complex
method, the latter method
[99, 107, 143]
was adopted successfully for the preparation of
the starting materials curcumin (1) and ethyl curcumin (2) (Equation 5):
R
1
O
HO
OR
1
OH
O O
R
1
O
HO
CHO
+
O O
/ B
2
O
3
(BuO)
3
B/ n-BuNH
2
2

47

(1) R
1
= CH
3
; (2) R
1
= C
2
H
5


Equation 5

Curcumin (1) was obtained from vanillin in 73% yield and its structure was
confirmed by IR,
1
H-NMR and
13
C-NMR.
IR spectrum of curcumin showed a strong and broad hydroxyl absorption band at
3468.5 cm
-1
. The spectrum also showed stretching absorption bands for C=O, C=C
conjugated and
as
and
s
C-O-C.

1
H-NMR spectrum (figure 1) indicated that the diketone existed entirely in the
enolic form (b) as shown below. Interchange between equivalent enols is presumably
rapid. Thus, the spectrum showed an upfield singlet assigned for the 6 protons of the 2
methoxyl groups and 2 downfield singlets for the 2 phenolic OH and the enol OH
protons at 9.7411 and 10.1312 ppm respectively. Methine protons of the heptatriene
chain resonated at their expected chemical shifts and were coinciding with those
previously reported
[99,107,121,138]
:

HO OH
O O
H
3
CO OCH
3

HO OH
OH O
H
3
CO OCH
3

(a) (b)
(1)
OCH
3
OH
H
3
CO
HO
O O H
c
H
b
H
a
H
b
H
c
H

(1b)

48
The H
a
proton resonated as a singlet at 6.0618 ppm. Two doublets were assigned
for 2 H
b
and 2 H
c
protons at 6.711 and 7.5470 ppm respectively.
The 1,2,4-trisubstitued benzene rings pattern common in aromatic compounds was
not observed in case of curcumin. However, it was recognized in many of the spectra
as will be explained later.
H
X
H
A
H
B


The spectrum of curcumin showed 2 doublets arising from ortho couplings with
J
AB
value of ~ 8 Hz
[150]
. The expected doublet of doublets for H
B
proton resulting
from ortho and meta couplings was not observed. The para coupling J
AX
was also
unresolved and not visible
[151]
. Therefore, the signal for H
X
proton was detected as a
singlet at 7.5470 ppm.

49


Figure 1:
1
H NMR spectrum of curcumin (1)

In
13
C-NMR spectrum, assignments for the carbon atoms of curcumin were made
by comparison with those reported in the literature for curcumin
[93]
as well as for
similar compounds
[93,110,129]
. Similar chemical shifts were observed for the 2 Ar-
OCH
3
, 12 Ar-C and 7 C
1
-C
7
carbons, including characteristic peaks of 2 carbonyl
groups, of the heptadienedione chain.
H
3
CO
HO
O O
OCH
3
OH
7
6
5
4
2
1
3
2'
3'
4'
5'
6'
5'
2'
4'
6'
1' 3'
1'

Following the same procedure, ethyl curcumin (2) was obtained from 4-hydroxy-
3-ethoxybenzaldehyde in 68% yield. Its structure was also confirmed by IR,
1
H-NMR,
13
C-NMR and MS.
As for curcumin, its
1
H-NMR spectrum (figure 2) lacked the typical doublet of
doublets characteristic for the H
B
proton in 1,2,4-trisubstitued benzene ring pattern;
para coupling was also unobservable. However, the spectrum showed a triplet and a
quartet at 1.358 and 4.094 ppm with coupling constant J = 7 Hz assigned for the
methyl and methylene protons of the ethyl group respectively. The heptadienedione
protons resonated at their expected chemical shifts showing consecutively 1 singlet
and 2 doublets with J = 16 Hz, for H
a
, H
b
and H
c
respectively.

50


Figure 2:
1
H NMR spectrum of ethyl curcumin (2)


13
C-NMR spectrum (figure 3) of compound 2 supported its structure showing
signals at chemical shifts similar to those of curcumin. In addition it showed signals
assignable to the 2 methyl and 2 methylene carbons of the ethoxyl group.
51



Figure 3:
13
C-NMR spectrum of ethyl curcumin (2)

MS of 2 (figure 4) showed the M
+
at 396 and the base peak at m/z 89
corresponding to hept-3-ene-1,6-diyne cation. The possible fragmentation pattern is
illustrated in chart 1.

52


Figure 4: MS of ethyl curcumin (2)














53
O
HO
O O
OH
O
C
23
H
24
O
6
M
+
396 (23)
O
O
+
HO
C
13
H
17
O
2
m/z 205 (26)
O
C
11
H
13
O
2
m/z 177 (48)
O
+
+
O
O
C
10
H
11
O
2
m/z 163 (78)
O
O
H
C
11
H
12
O
2
m/z 176 (42)
+
+
+
C
10
H
11
O
m/z 147 (47)
O +
C
9
H
9
m/z 117 (48)
+
m/z 107 (33)
HO HO
+
C
7
H
7
O
O O
m/z 123 (38)
C
7
H
7
O
2
+
OH
m/z 110 (30)
C
7
H
10
O
m/z 89 (100)
C
7
H
5
+
+
+
m/z 65 (57)
C
5
H
5
CH
3
C O
+
m/z 43 (57)
C
2
H
3
O


Chart 1: Possible Fragmentation Pattern for compound 2


54
4.1.2. Di-O-Chloroacetylcurcumin (3a) and di-O-chloropropionylcurcumin (3b)
Di-O-Chloroacetylethyl curcumin (3c) and di-O-chloropropionylethyl
curcumin (3d)

4.1.2.1. Method A

H
3
CO
O O
OCH
3
O
O
ClCO(CH2)
n
Cl/
Na
2
CO
3
1
O
(CH
2
)
n
Cl
O
(CH
2
)
n
Cl


(3a) n = 1 ; (3b) n = 2

Esterification of phenols is very common in organic synthesis. Phenolic esters are
formed by treating appropriate phenolic compounds with acid chlorides or acid
anhydrides in presence of alkali hydroxides
[152]
, pyridine
[153-155]
or anhydrous alkali
carbonates
[156]
in various solvents. It can also be performed with acid anhydrides in
conc. H
2
SO
4
[157]
or by treating phenols with esters in acid medium
[158]
.


In the present investigation, the use of NaOH or pyridine was excluded due to the
ease of degradation of curcumin under these conditions. Trials with triethylamine or
alkali carbonates gave better results and anhydrous Na
2
CO
3
was preferred (Scheme
1).
Thus, reacting a cold solution of curcumin in dry acetone containing Na
2
CO
3
with
2.5 molar equivalents of either chloroacetyl chloride or chloropropionyl chloride
proceeded smoothly over 3 days. Work up of the reaction mixture produced the
products 3a,b in fair yield which were purified by chromatography on a column of
silica gel. Their structure was assessed by IR,
1
H-NMR and, for compound 3b, by
MS.
55
IR spectra of 3a,b showed the presence of the intramolecularly H-bonded OH of
the enol group and 2 stretching absorption bands due to the ester and ketone carbonyl
groups. In addition, it showed stretching absorption bands for C=C conjugated and
as

and
s
C-O-C characteristic for curcumin.

1
H-NMR spectrum (figure 5) of di-O-chloroacetylcurcumin (3a) showed 3
consecutive singlets assigned for the OCH
3
, CH
2
-Cl and H
a
protons and 2 doublets
resonating at 6.56905 and 7.6116 ppm assigned for H
b
and H
c
respectively. A
downfield multiplet was integrated for the 6 aromatic protons. The spectrum lacked
the deshielded signal assigned for the enol OH proton present in its precursor. This
evidenced that the singlet for H
a
was due to 2 protons.

1
H-NMR spectrum of di-O-chloropropionylcurcumin (3b) showed 2 characteristic
triplets at 3.1223 and 3.8952 ppm, with coupling constant J ~ 6.3 Hz, assigned for
CH
2
-Cl and COCH
2
protons respectively. In addition, the 6 protons of the
heptadienedione chain resonated at their expected chemical shifts. Signals on the
aromatic region were unresolved and difficult to assign.

56

Figure 5:
1
H-NMR spectrum of di-O-chloroacetylcurcumin (3a)
MS of compound 3b did not show the molecular ion peak (M
+
) at m/z 549.45 but
showed a fragment at m/z 284 corresponding to 7-phenyl-1-(4-hydroxyphenyl)-
heptane-3,5-dione cation. The base peak was shown at m/z 73 corresponding to butan-
2-ol cation.

4.1.2.2. Method B

Method B aimed at the preparation of the target intermediates through an indirect
way. These intermediates 3a-d, were synthesized by a 2-step reaction involving the
treatment of 3-alkoxy-4-chloroacyloxy- benzaldehyde (4a-d) with acetylacetone
under Pabon

conditions
[99, 107, 143]
(Scheme 1).
The first step i.e. the synthesis of compounds 4a-d, a typical ester formation, was
achieved by treating a cold solution of the appropriate phenolic aldehyde and 1N
NaOH
[129]
in chloroform with a chloroformic solution of chloroacetyl chloride or
chloropropionyl chloride. Work up of the reaction mixture provided the required
products 4a-d.
Repeating the same reaction using acetone and anhydrous K
2
CO
3
[156]
instead of
CHCl
3
and NaOH gave the same products with high purity and yield. Compounds 4a-
d were identified by IR and
1
H-NMR.

HO
R
1
O
CHO
CHO
O
R
1
O
Cl
O
Cl
(CH
2
)
n
+
O
(CH
2
)n
Cl
1N NaOH/ CHCl
3
or
K
2
CO
3
/ acetone
4a-d

57
(4a) R
1
= CH
3
n = 1; (4b) R
1
= CH
3
n = 2
(4c) R
1
= C
2
H
5
n = 1; (4d) R
1
= C
2
H
5
n = 2
IR spectra of compounds 4a-d lacked the stretching absorption band characteristic
for OH group. It showed stretching absorption bands for C=O ester, C=O aldehyde,
C=C aromatic and
as
and
s
C-O-C functional groups.


1
H-NMR spectrum of 3-methoxy-4-chloroacetyloxybenzaldehyde (4a) (figure 6)
showed 2 consecutive singlets at 3.8445 and 4.7157 ppm assigned for OCH
3
and
CH
2
Cl protons. Signals on the aromatic region showed coupling patterns due to
1,2,4-trisubstituted benzene ring: H
A
proton appeared as a doublet due to coupling
with the ortho H
B
proton with J
AB
= 8.4 Hz. H
B
Proton was shown as a doublet of
doublets with J values of 7.65 Hz (ortho coupling with H
A
) and 1.55 Hz (meta
coupling with H
X
). H
X
proton resonated as a doublet and the para coupling, J
AX
=
1.55 Hz, was well resolved and well visible. A downfield singlet was assigned for the
aldehydic proton at 9.9476 ppm.


Figure 6:
1
H NMR spectrum of 3-methoxy-4-chloroacetyloxybenzaldehyde (4a)
58


1
H-NMR spectrum of 3-ethoxy-4-chloroacetyloxybenzaldehyde (4c) (figure 7)
was characterized by the appearance of a triplet and a quartet at 1.3138 and 4.15575
ppm with coupling constant J = 6.87 Hz assigned for the methyl and methylene
protons of the ethyl group respectively. The CH
2
-Cl protons resonated as a singlet at
its expected chemical shift 4.7413 ppm. Signals for the aromatic region showed
coupling patterns due to 1,2,4-trisubstituted benzene ring in the same manner as for
compound 4a. H
A
proton appeared as a doublet due to coupling with the ortho H
B

proton with J
AB
= 7.98 Hz. H
B
Proton was shown as a doublet of doublets with J
values of 8.07 Hz (ortho coupling with H
A
) and 1.65 Hz (meta coupling with H
X
). H
X

proton resonated as a doublet and the para coupling, J
AX
= 1.63 Hz, was well resolved
and well visible. A downfield singlet was assigned for the aldehydic proton at
9.9709 ppm.



CHO
OCH
2
CH
3
ClH
2
COCO

59
Figure 7:
1
H-NMR spectrum of 3-ethoxy-4-chloroacetyloxybenzaldehyde (4c)
The second step in this synthesis used Pabon reaction conditions
[99, 107, 143]
for the
preparation of the target intermediates namely di-O-chloroacyl- curcumin (3a,b) and
di-O-chloroacylethyl curcumin (3c,d). Thus, tri-(n-butyl)borate was added to the
appropriate 3-alkoxy-4-chloroacyloxybenzaldehyde (4a-d) followed by addition of a
previously prepared complex, formed by stirring acetylacetone with boric anhydride,
and n-butylamine.
R
1
O
O
OR
1
O
O O
R
1
O
O
CHO
+
O O
/ B
2
O
3
(BuO)
3
B/ n-BuNH
2
Cl
(CH
2
)
n
O
Cl
(CH
2
)
n
O O
(CH
2
)
n
Cl
(4a-d)
2

(3a-d)

(3a) R
1
= CH
3
, n = 1; (3b) R
1
= CH
3
, n = 2
(3c) R
1
= C
2
H
5
, n = 1; (3d) R
1
= C
2
H
5
, n = 2

Compounds 3a-d were identified by IR,
1
H-NMR and by MS for 3c.
IR and
1
H-NMR spectra of compounds 3a,b were coinciding with those
previously obtained from method A.
IR spectra of 3c,d showed the intramolecularly H-bonded OH of the enol group
and 2 stretching absorption bands due to the ester and ketone carbonyl groups. In
addition, it showed stretching absorption bands for C=C conjugated and
as
and
s
C-
O-C characteristic for curcumin derivatives.

1
H-NMR spectrum (figure 8) of di-O-chloroacetylethyl curcumin (3c) showed a
triplet and a quartet at 1.4164 and 4.1049 ppm with coupling constant J = 6.87 Hz
assigned for the methyl and methylene protons of the ethyl group respectively. The
60
spectrum also showed 2 consecutive singlets assigned for the CH
2
-Cl and H
a
protons
and 2 doublets resonating at 6.5553 and 7.605 ppm, J = 15.93 Hz, assigned for H
b

and H
c
respectively. A downfield multiplet was integrated for the 6 aromatic protons.
The spectrum lacked the deshielded signal assigned for the enol OH proton present in
its precursor. This evidenced that the singlet for H
a
was due to 2 protons.


Figure 8:
1
H-NMR spectrum of di-O-chloroacetylethyl curcumin (3c)

MS of 3c (figure 9) showed M
+
, M
+
+2, M
+
+4 at 529, 531 and 533 respectively,
due to the contribution of the chlorine isotopes. It also showed the base peak at m/z 83
corresponding to 1-hexene cation or pent-1-en-3-one cation. The possible
fragmentation pattern is illustrated in chart 2.
61


Figure 9: MS of di-O-chloroacetylethyl curcumin (3c)

O
O
O
Cl
O O
O
O
O
Cl
C
27
H
26
O
8
Cl
2
M
+
548, M
+
+2 550, M
+
+4 552
+
O
O
O
O O
O
O
O
C
27
H
30
O
8
+
m/z 482 (0.24)
+
O
O O
C
19
H
20
O
3
m/z 296 (0.14)
O
O
O
Cl
O
O
+
O
HO
m/z 242 (0.6), 244 (0.26)
C
12
H
15
O
2
m/z 191 (0.17)
C
10
H
14
O
2
m/z 166 (2)
+
+
C
12
H
15
O
3
Cl
OH O
H
O
O
+
O
+
+
or
+
C
4
H
5
O
2
m/z 85 (69)
C
4
H
7
O
2
m/z 87 (12)
C
6
H
11
m/z 83 (100)
C
5
H
7
O

Chart 2: Possible Fragmentation Pattern for compound 3c
62

1
H-NMR spectrum (figure 10) of di-O-chloropropionylethyl curcumin (3d)
showed the characteristic triplet and quartet assigned for the methyl and methylene
protons of the ethyl group respectively at their expected chemical shifts. The spectrum
also showed 2 consecutive triplets assigned for the CH
2
CH
2
-Cl protons. Protons H
a
,
H
b
and H
c
of the heptadienedione chain resonated as singlet and 2 doublets
respectively at their expected chemical shifts. Signals on the aromatic region showed
coupling patterns due to 1,2,4-trisubstituted benzene ring and the aromatic protons
were well resolved and appeared as 2 doublets, with ortho coupling, and a singlet.
The spectrum also showed a deshielded signal assigned for the enol OH proton.



Figure 10:
1
H-NMR spectrum of di-O-chloropropionylethyl curcumin (3d)








63
4.1.3. 2-Chloroethylamine monohydrochloride and bis(2-chloroethyl)-
amine hydrochloride

NH
2
CH
2
CH
2
OH
SOCl
2
/dry C
6
H
6
NH
2
CH
2
CH
2
Cl
SOCl
2
/dry C
6
H
6
HN
CH
2
CH
2
OH
CH
2
CH
2
OH
HN
CH
2
CH
2
Cl
CH
2
CH
2
Cl


Chlorination of alcohols, to produce an alkyl chloride, has been extensively
studied
[159]
and many reports achieved such reaction by the use of various chlorinating
agents including dry HCl gas, thionyl chloride (SOCl
2
), phosphoryl chloride (POCl
3
),
phosphorus trichloride (PCl
3
) or phosphorus pentachloride (PCl
5
). In the present
work, SOCl
2
was chosen as the chlorinating agent. The required compounds were
identified by comparison of their melting points with those reported in the
literature
[160]
.

64
4.1.4. 1,7-Bis(4-Alkyl(cycloalkyl or heteroaryl)aminoacyloxy)-3-(methoxy-
phenyl)-
1,6-heptadiene-3,5-dione (5a-n) and 1,7-bis(4-alkyl (cycloalkyl or
heteroaryl)-
aminoacyloxy)-3-(ethoxyphenyl)-1,6-heptadiene-3,5-dione (6a-n)

O
OR
1
O
R
1
O
O O
R
2
R
3
NH/reflux
(3a-d)
Et3N / EtOH
(CH
2
)n
O O
(CH
2
)n
Cl
Cl
O
OR
1
O
R
1
O
O O
(CH
2
)n
O O
(CH
2
)n
R
3
R
2
N
NR
2
R
3

(5a-n), ( 6a-n)

Amination of halogenated compounds was shown to proceed via a nucleophilic
substitution reaction to give an amine product
[161]
. The displaced halogen anion
liberated during the reaction was always trapped in alkaline medium. Different
reaction conditions including the use of various solvents and basic media have been
developed to influence the rate and maximize the yield of such substitution reaction.
These include the use of sodium alkoxide
[162,163]
, NaH
[162,164]
, aqueous alkali
hydroxides
[163]
, triethyl- amine
[162,164-166]
or alkali carbonates
[164,165,167,168]
, in protic or
aprotic solvents.
In the present work, several reaction conditions were tried and the best method
producing pure materials in good yields was selected. Thus, two series of our target
compounds were synthesized by heating under reflux an ethanolic solution of di-O-
chloroacylcurcumin (3a,b) or di-O-chloroacylethyl curcumin (3c,d) and the
appropriate alkyl, cycloalkyl or heteroarylamine in presence of triethylamine (Scheme
65
2). IR spectra of compounds 5a-n and 6a-n showed stretching absorption bands due
to NH, C=O of ester and of ketone, conjugated C=C and
as
and
s
C-O-C. A band
assigned for OH intramolecularly H-bonded was overlapping the NH band. A C=N
vibrational band appeared mixed the conjugated C=C in compounds containing
aromatic heterocyclic nucleus.

1
H-NMR spectrum of compound 5a (figure 11) was characterized by a broad
hump typical for protons in cycloalkyl ring systems. This hump was assigned for 30 H
on the adamantyl moieties. The spectrum also showed a singlet at 7.3099 ppm
assigned for 2 NH. It also showed 3 consecutive singlets assigned for CH
2
-, -OCH
3

and H
a
protons and 2 doublets resonating at 6.7444 and 7.5315 ppm, with J = 15.66
Hz, assigned for H
b
and H
c
respectively. Signals on the aromatic region showed
coupling patterns typical of 1,2,4-trisubstituted benzene ring. H
A
Proton appeared as a
doublet due to coupling with the ortho H
B
proton with J
AB
= 8.25 Hz. H
B
Proton was
shown as a doublet of doublets with J values of 8.22 Hz (ortho coupling with H
A
) and
1.5 Hz (meta coupling with H
X
). H
X
Proton resonated as a doublet and the para
coupling, J
AX
= 0.24 Hz, was not well resolved.
66


Figure 11:
1
H NMR spectrum of 1,7-bis(4-adamantylaminoacetyloxy)-3-
(methoxyphenyl)-1,6-heptadiene-3,5-dione (5a)
MS of compound 5a did not show the molecular ion peak (M
+
) at m/z 750. The
base peak was shown at m/z 135 corresponding to adamantyl cation fragment
obtained by cleavage of the side chain at nitrogen. The possible fragmentation pattern
is illustrated in chart 3.
67
O
O
O
O O
O
O
O
C
45
H
54
N
2
O
8
+
M
+
750 (absent)
C
7
H
7
O
m/z 107 (33)
H
N
H
N
O
O
+
O
+
C
5
H
3
O
C
4
H
3
O
m/z 81 (39) m/z 79 (43) m/z 67 (7)
HO
+ HO
+
C
6
H
5
O
m/z 93 (36)
+
O
+
C
10
H
15
m/z 135 (100)
O
+
or
+
C
6
H
5
m/z 77 (36)
+
C
5
H
5
O

Chart 3: Possible Fragmentation Pattern for compound 5a


1
H-NMR spectrum of compound 5b showed a downfield D
2
O-exchangeable
singlet at 9.6925 ppm assigned for NH. It also showed 3 consecutive singlets
assigned for CH
2
-, -OCH
3
and H
a
protons and 2 doublets resonating at 6.7646 and
7.5466 ppm, with J = 15.66 Hz, assigned for H
b
and H
c
respectively. Signals on the
aromatic region showed coupling patterns typical of 1,2,4-trisubstituted benzene ring.
H
A
Proton appeared as a doublet due to coupling with the ortho H
B
proton with J
AB
=
8.25 Hz. H
B
Proton was shown as a doublet of doublets with J values of 8.25 Hz
(ortho coupling with H
A
) and 1.65 Hz (meta coupling with H
X
). H
X
Proton resonated
68
as a doublet and the para coupling, J
AX
= 1.65 Hz, was not well resolved. Protons on
the benzothiazole rings were highly deshielded and were shown as a multiplet.

1
H-NMR spectrum of compound 5c showed 3 consecutive singlets assigned for
COCH
2
N-, CH
3
and -OCH
3
respectively. Protons H
a
, H
b
and H
c
of the
heptadienedione chain resonated as a singlet and 2 doublets respectively at their
expected chemical shifts. Signals on the aromatic region showed coupling patterns
due to 1,2,4-trisubstituted benzene ring and the aromatic protons were well resolved
and appeared as 2 doublets, with ortho coupling, and a singlet. The spectrum also
showed a deshielded singlet at 9.685 ppm assigned for 2 NH protons which were
D
2
O-exchangeable.

1
H-NMR spectrum of compound 5d showed a high field signal at 2.202 ppm
assigned for 2 NH protons. 2 Consecutive triplets were assigned for the 8 protons of
CH
2
-CH
2
moieties. 2 Singlets assigned for COCH
2
N- and -OCH
3
were also shown.
Protons H
a
, H
b
and H
c
of the heptadienedione chain resonated as a singlet and 2
doublets respectively at their expected chemical shifts. Signals on the aromatic region
showed coupling patterns due to 1,2,4-trisubstituted benzene ring and the aromatic
protons were well resolved and appeared as 2 doublets, with ortho coupling, and a
singlet.

1
H-NMR spectrum of compound 5e (figure 12) showed 3 consecutive singlets
assigned for COCH
2
N-, -OCH
3
and H
a
protons. It also showed 2 triplets at 2.5042
and 3.4841 ppm, with J = 3.57 Hz, and assigned for NCH
2
CH
2
-Cl and NCH
2
CH
2
-Cl
respectively, the later being overlapped by the solvent signal. Two doublets
resonating at 6.7718 and 7.5736 ppm, with J = 15.9 Hz were assigned for H
b
and H
c

respectively. Signals on the aromatic region showed coupling patterns typical of
1,2,4-trisubstituted benzene ring. H
A
proton appeared as a doublet due to coupling
69
with the ortho H
B
proton with J
AB
= 8.25 Hz. H
B
Proton was shown as a doublet of
doublets with J values of 8.37 Hz (ortho coupling with H
A
) and 1.65 Hz (meta
coupling with H
X
). H
X
Proton resonated as a doublet and the para coupling, J
AX
=
1.38 Hz, was well resolved and well detected.


Figure 12:
1
H NMR spectrum of 1,7-bis(4-bis(2-chloroethyl)aminoacetyloxy)-3-
(methoxyphenyl)-1,6-heptadiene-3,5-dione (5e)


1
H-NMR spectrum of compound 5f showed 2 new high field singlets assigned for
2 x N(CH
3
)
2
and 2 NH protons, the latter overlapping with OCH
2
singlet. 2
Consecutive triplets were assigned for the 8 protons of CH
2
-CH
2
moieties. 2 Singlets
assigned for COCH
2
N- and -OCH
3
were also shown. Protons H
a
, H
b
and H
c
of the
heptadienedione chain resonated as a singlet and 2 doublets respectively at their
expected chemical shifts. Signals on the aromatic region showed coupling patterns
70
due to 1,2,4-trisubstituted benzene ring and the aromatic protons were well resolved
and appeared as 2 doublets, with ortho coupling, and a singlet. A down field singlet at
9.7368 ppm was assigned for the enolic OH proton.

1
H-NMR spectrum of compound 5h (figure 13) was characterized by a broad
hump assigned for 30 H on the adamantyl moieties. The spectrum also showed a
singlet at 3.882 ppm, overlapping with singlet of OCH
3
, assigned for 2 NH. It also
showed 2 consecutive triplets assigned for N-CH
2
-CH
2
-CO. A singlet and 2 doublets
resonating downfield were assigned for H
a
, H
b
and H
c
respectively. Signals on the
aromatic region showed coupling patterns typical of 1,2,4-trisubstituted benzene ring.
H
A
and H
B
protons appeared as 2 doublets due to coupling with ortho protons with
J
AB
= 7.8 Hz.

Figure 13:
1
H NMR spectrum of 1,7-bis(4-adamantylaminopropionyloxy)-3-
(methoxyphenyl)-1,6-heptadiene-3,5-dione (5h)
71
In
13
C-NMR spectrum of compound 5h, assignments for the carbon atoms of the
curcumin skeleton were made by comparison with those reported previously for
curcumin. Chemical shifts assignments were made for the 2 Ar-OCH
3
, 12 Ar-C and 7
C
1
-C
7
carbons, including characteristic peaks of 2 carbonyl groups, of the
heptadienedione chain. In addition, the spectrum also showed peaks for the 20Cs of
the 2 adamantyl groups and the 4 Cs of the 2 x CH
2
-CH
2
moieties of the propionyl
functionality.
MS of 5h (figure 14) did not show the M
+
peak at m/z 778 but showed a fragment
at m/z 719 corresponding to 1,7-bis(4-adamantylaminopropionyloxy)phenyl-1,6-
hepta-diene-5-hydroxy-3-one cation. The base peak was shown at m/z 45
corresponding to COOH cation. The possible fragmentation pattern is illustrated in
chart 4.


Figure 14: MS of 1,7-bis(4-adamantylaminopropionyloxy)-3-(methoxyphenyl)-
1,6-heptadiene-3,5-dione (5h)

72

O
O
O
O O
O
O
O
C
47
H
58
N
2
O
8
+
O
O
O
O O
O
O
O
C
37
H
46
N
2
O
8
m/z 646 (2.6)
O
+
HO
O
O
O
HO
m/z 151 (6.8)
C
11
H
14
O
3
m/z 194 (33.6)
C
11
H
12
O
2
m/z 164 (27)
+
+
C
9
H
11
O
2
+
N
H
N
H
M
+
778 absent
H
2
N
O
O
O
O O
O
O
O
C
36
H
42
NO
8
m/z 616 (3.1)
+
O
O
O
OH OH
O
O
O
C
25
H
35
O
8
m/z 463 (10.3)
+
C
19
H
23
m/z 251 (9.7)
+
HO
or
HN
+
H
2
C
C
11
H
18
N
or
H
2
N
C
10
H
17
N
HO
+
HO
C
8
H
7
O
2
m/z 135 (25.6)
or
C
10
H
15
+
+
H
O
OH
+
CH
2
O
2
m/z 46 (44.8)
HO C O
+
CHO
2
m/z 45 (100)
CH
3
C O
+
C
2
H
3
O
m/z 43 (72)
+
C
3
H
5
m/z 41 (80.5)
m/z 77 (20.8)
C
6
H
5


Chart 4: Possible Fragmentation Pattern for compound 5h

73

1
H-NMR spectrum of compound 5j showed signals similar to those recorded for
5c. However, 2 triplets were assigned for the CO-CH
2
-CH
2
-N- protons.
In
13
C-NMR spectrum of compound 5j, chemical shifts assignments were made
for the 2 Ar-OCH
3
, 12 Ar-C and 7 C
1
-C
7
carbons, including characteristic peaks of 2
carbonyl groups, of the heptadienedione chain. In addition, the spectrum also showed
peaks for 2 x thiadiazole CH
3
and the 2 Cs of the ring. Signals for the 4 Cs of the 2 x
CH
2
-CH
2
moieties of the propionyl functionality were also assigned.
MS of 5j (figure 15) showed the M
+
+1 peak at m/z 707. The base peak was
shown at m/z 43 corresponding to acetyl cation. The possible fragmentation pattern is
illustrated in chart 5.


Figure 15: MS of 1,7-bis(4-(3-methylthiadiazolyl)aminopropionyloxy)-3-
(methoxyphenyl)-1,6-heptadiene-3,5-dione (5j)





74
O
O
O
O O
O
O
O
C
33
H
34
N
6
O
8
S
2
+
C
31
H
26
N
6
O
7
S
2
m/z 658 (1.1)
O
C
10
H
13
O
m/z 149 (35)
m/z 142 (10)
+
N
H
N
H
M
+
+ 1 707 (0.7)
O
O
C
20
H
19
O
3
m/z 307 (2.5)
C
19
H
17
O
3
m/z 293 (3)
HO C O
+
CHO
2
m/z 45 (18.1)
CH
3
C O
+
C
2
H
3
O
m/z 43 (100)
N
N
S S
N
N
CH
3
H
3
C
HO
O
O
O
OH
O
O
+
N
H
N
H
N
N
S S
N
N
CH
3
H
3
C
O O
+O
OH
+
N N
S
N N
S
H
3
C N
H
H
3
C NH
2
+ +
C
5
H
8
N
3
S C
3
H
5
N
3
S
m/z 115 (5)
N N
S
H
3
C
+
C
3
H
4
N
2
S
m/z 100 (5.5)
OH
O
+
+
m/z 73 (17.5)
m/z 55 (72.4)
C
4
H
9
O
C
3
H
3
O


Chart 5: Possible Fragmentation Pattern for compound 5j


1
H-NMR spectrum of 1,7-bis(4-adamantylaminoacetyloxy)-3-(ethoxyphenyl)-1,6-
heptadiene-3,5-dione (6a) was characterized by a broad hump typical for protons in
cycloalkyl ring systems. This hump was assigned for 30 H on the adamantyl moieties.
The spectrum also showed 2 upfield singlets assigned for CH
2
and 2 NH. It also
showed a triplets and quartet assigned for -OCH
2
CH
3
and OCH
2
CH
3
respectively. H
a

Proton was integrated for 1 H and the enol form was confirmed by the appearance of a
downfield signal assigned for the enol OH. 2 Doublets resonating at 6.4541 and
7.5722 ppm, with J = 15.66 Hz, were assigned for H
b
and H
c
respectively. H
A
Proton
appeared as a doublet due to coupling with the ortho H
B
proton with J
AB
= 8.25 Hz.
75
H
B
Proton was also shown as a doublet with J values of 8.25 Hz (ortho coupling with
H
A
) while H
X
proton resonated as a singlet.

4.1.5. 1,7-Bis(4-(4-substituted sulfanilamido)acyloxy)-3-(methoxyphenyl)-1,6-
heptadiene-3,5-dione (7a,b) and 1,7-bis(4-(4-substituted
sulfanilamido)acyl-
oxy)-3-(ethoxyphenyl)-1,6-heptadiene-3,5-dione (7c-f)

O
OR
1
O
R
1
O
O O
(3a-d)
Et
3
N / EtOH
(CH
2
)n
O O
(CH
2
)n
Cl
Cl
O
OR
1
O
R
1
O
O O
(CH
2
)n
O O
(CH
2
)n
HN
NH SO
2
NHR
4
R
4
HNO
2
S
H
2
N SO
2
NH
4
/

(7a-f)

Using the same general amination procedure as for the preparation of compounds
5a-n and 6a-n, the reaction of the chloroesters 3a-d with various sulfonamides
produced the required compounds 7a-f (Scheme 2).










76



4.1.6. Di-O-Adamantoylcurcumin (8a) and di-O-adamantoylethyl
curcumin (8b)

RO
HO
O O
OR
OH
1, 2
+
ClOC
Na
2
CO
3
/acetone
reflux
RO
OCO
O O
OR
OCO
7a,b
1
1
1
1

(8a,b)


Esterification of curcumin (1) or ethyl curcumin (2) was carried out as previously
mentioned before
[156]
(Scheme 3). A solution of either 1 or 2 in acetone containing
anhydrous Na
2
CO
3
was treated with 2 molar equivalents of adamantoyl chloride and
heated under reflux for 9 h (Scheme 3). Work up of the reaction mixture afforded the
required esters 8a,b which were identified by IR,
1
H-NMR and MS.
IR spectra of compounds 8a,b showed stretching absorption bands due to C=O of
ester and of ketone, conjugated C=C and
as
and
s
C-O-C. A broad band due to
intramolecularly H-bonded OH was also observed.

1
H-NMR spectrum of compound 8a showed a broad hump characteristic for
adamantyl protons and 2 consecutive singlets assigned for -OCH
3
and H
a
protons.
Two doublets resonating at 6.5539 and 7.6111 ppm, with J = 15.9 Hz were assigned
for H
b
and H
c
respectively. Signals on the aromatic region showed coupling patterns
typical of 1,2,4-trisubstituted benzene ring. H
A
proton appeared as a doublet due to
77
coupling with the ortho H
B
proton with J
AB
= 8.25 Hz. H
B
Proton was shown as a
doublet of doublets with J values of 8.04 Hz (ortho coupling with H
A
) and 1.65 Hz
(meta coupling with H
X
). H
X
proton resonated as a singlet at 7.0974 ppm while the
para coupling was not resolved and could not be detected.
In
13
C-NMR spectrum of compound 8a, chemical shifts assignments were made
for the 2 Ar-OCH
3
, 12 Ar-C and 7 C
1
-C
7
carbons, including characteristic peaks of 2
carbonyl groups, of the heptadienedione chain and 2 carbonyls of the ester groups. In
addition, the spectrum also showed peaks for the 20 Cs of the 2 adamantyl groups.
MS of 8a (figure 16) showed the M
+
+1 peak at m/z 692. The base peak was
shown at m/z 135 corresponding to adamantyl cation The possible fragmentation
pattern is illustrated in chart 6.


Figure16: MS of di-O-Adamantoylcurcumin (8a)



78




O
O
O
O O
O
O
O
C
43
H
48
O
8
+
O
C
10
H
12
O
3
m/z 180 (4.8)
M
+
692 (2.1)
O
O
C
32
H
38
O
6
m/z 518 (2.75)
C
13
H
16
O
3
m/z 220 (1.7)
C
10
H
15
C
11
H
15
O
or
C
11
H
16
O
2
+
O
C
6
H
5
O
m/z 93 (29)
HO C O
+
CHO
2
m/z 45 (31.1)
CH
3
C O
+
C
2
H
3
O
m/z 43 (50)
+
C
3
H
5
m/z 41 (92)
O
O
O
+
m/z 57 (22)
C
3
H
5
O
O
O
O
O
O
O
O
C
43
H
45
O
7
m/z 673 (3.7)
O
O
O
O
O
C
42
H
4
8O
5
+
m/z 632 (3.2)
+
O
O
O
OH
+
HO
OH
+
O
H
+
+ O
OH
O
+
m/z 163 (5.6)
+
m/z 135 (100)

79

Chart 6: Possible Fragmentation Pattern for compound 8a

1
H-NMR spectrum of compound 8b showed a broad hump characteristic for
adamantyl protons overlapping with the triplet of OCH
2
CH
3
. A quartet at
4.1611ppm was assigned for OCH
2
CH
3
protons of the ethyl group with J = 6.97 Hz.
A signal at 5.7825 ppm, integrated for only 1 proton, was assigned to H
a
proton.
This was evidenced by the appearance of a deshielded proton at 9.8134 ppm
assigned for the enol OH proton. Two doublets resonating at 6.4541 and 7.5722
ppm, with J = 15.66 Hz were assigned for H
b
and H
c
respectively. Signals on the
aromatic region did not show coupling patterns typical of 1,2,4-trisubstituted benzene
ring. H
A
Proton appeared as a doublet due to coupling with the ortho H
B
proton with
J
AB
= 8.25 Hz. The spectrum lacked the typical doublets of doublet characteristic for
the H
B
proton but resonated as a doublet at 7.1065 ppm with J
AB
value of 8.25 Hz
(ortho coupling with H
A
). H
X
Proton resonated as a singlet at 7.0315 ppm while the
para coupling was not resolved and could not be detected.

4.1.7. Di-O-Heptanoylcurcumin (8c) and di-O-heptanoylethyl curcumin (8d)

O
OR
1
O
R
1
O
O O
O O
HO
OR
1
OH
R
1
O
O O
+ C
6
H
13
COCl
Na
2
CO
3
/ acetone
(1,2)
RT

(8c,d)

80
The title compounds 8c,d were prepared by esterification of curcumin 1 or ethyl
curcumin 2 in acetone containing anhydrous Na
2
CO
3
[156]
with 2 molar equivalents of
2-heptanoyl chloride at RT (Scheme 3). Work up of the reaction mixture afforded the
required esters 8c,d which were identified by IR,
1
H-NMR and MS.

1
H-NMR spectrum of compound 8c showed 2 triplets resonating high field
assigned for the terminal CH
3
protons and the CO-CH
2
- of the heptanoyl group. A
broad hump characteristic for aliphatic (CH
2
)
4
protons was shown at high field. The
spectrum also showed 2 consecutive singlets assigned for -OCH
3
and H
a
protons at
their expected chemical shifts. Two doublets resonating at 6.5468 and 7.6184 ppm,
with J = 15.75 Hz were assigned for H
b
and H
c
respectively. Signals on the aromatic
region showed coupling patterns typical of 1,2,4-trisubstituted benzene ring. H
A

Proton appeared as a doublet due to coupling with the ortho H
B
proton with J
AB
=
8.04 Hz. H
B
Proton was shown as a doublet of doublets with J values of 9 Hz (ortho
coupling with H
A
) and 1.8 Hz (meta coupling with H
X
). H
X
Proton resonated as a
singlet at 7.6184 ppm while the para coupling was not resolved and could not be
detected.

1
H-NMR spectrum of compound 8d (figure 17) showed signals characteristic for
the (CH
2
)
5
-CH
3
protons and were assigned in a similar manner as for compound 8c. A
triplet and quartet were also detected for the methyl and methylene protons of the
OCH
2
CH
3
group. A signal at 5.8384 ppm was assigned to H
a
protons. Two doublets
resonating at 6.5400 and 7.0383 ppm, were assigned for H
b
and H
c
respectively.
Signals on the aromatic region did not show coupling patterns typical of 1,2,4-
trisubstituted benzene ring. H
A
Proton appeared as a doublet due to coupling with the
ortho H
B
proton with J
AB
= 7.5 Hz. The spectrum lacked the typical doublet of
doublets characteristic for the H
B
proton but resonated as a doublet at 7.1134 ppm
81
with J
AB
value of 7.5 Hz (ortho coupling with H
A
). H
X
Proton resonated as a singlet at
7.2551 ppm while the para coupling was not resolved and could not be detected.


Figure 17:
1
H NMR spectrum of di-O-heptanoylethyl curcumin (8d)

In
13
C-NMR spectrum of compound 8d (figure 18), assignments for the carbon
atoms of the ethyl curcumin skeleton were made by comparison with those reported
previously for ethyl curcumin. Chemical shifts assignments were also made for the 2
carbonyls of the ester groups and for the 2 x 6Cs of the 2 hexyl side chains. Further
confirmation of the structure was made by 2D
1
H-NMR (COSY) (figure 19) and
1
H,
13
C-NMR (H,C correlation) (figure 20).
82


Figure 18:
13
C NMR spectrum of di-O-heptanoylethyl curcumin (8d)




Figure 19: 2D
1
H NMR (COSY) spectrum of di-O-heptanoylethyl curcumin (8d)

83


Figure 20:
1
H,
13
C NMR (C,H correlation) spectrum of di-O-heptanoylethyl
curcumin (8d)
















84
4.1.8. Di-O-(2-Thienoyl)curcumin (8e) and di-O-(2-thienoyl)ethyl curcumin (8f)

RO
HO
O O
OR
OH
1, 2
+
Na
2
CO
3
/acetone
RO
-OCO
O O
OR
OCO-
7c,d
RT
S
-COCl
S S
1
1
1
1

(8e,f)

Following the previous procedure, compounds 8e,f were prepared by esterification
of curcumin or ethyl curcumin with thiophene-2-carbonyl chloride (Scheme 3). The
obtained esters were identified by IR,
1
H-NMR and MS.

1
H-NMR spectrum of 8e (figure 21) showed 2 singlets assigned for OCH
3
and H
a

protons and 2 doublets resonating at 7.051 and 7.697 ppm, with J = 16 Hz, assigned
for H
b
and H
c
respectively. The aromatic protons resonated as well-resolved signals
showing a doublet of doublet for H
B
due to ortho and meta coupling, a doublet for H
A

and a singlet for H
X
protons. Signals for protons on the thienyl groups were the most
deshielded and resonated as 2 doublets and doublets of doublet.
85


Figure 21:
1
H NMR spectrum of di-O-(2-Thienoyl)curcumin (8e)


1
H-NMR spectrum of 8f lacked the typical doublets of doublet characteristic for
the H
B
proton in 1,2,4-trisubstituted benzene ring pattern; para coupling was also
unobservable. It showed a singlet assigned for H
X
and 2 doublets with coupling
constant J = 8.52 Hz for H
A
and H
B
protons. However, the spectrum showed a triplet
and a quartet at 1.2351 and 4.1412 ppm with coupling constant J = 7.04 Hz assigned
for the methyl and methylene protons of the ethyl group respectively. The
heptadienone protons resonated at their expected chemical shifts showing
consecutively 1 singlet and 2 doublets, with J = 15.93 Hz, for H
a
, H
b
and H
c

respectively. Protons on the thienyl rings were resolved and resonated as 3 doublets,
distorted in some cases, at 7.5690, 8.02645 and 8.1015 ppm and were assigned for
thienyl-H
5
, -H
4
and -H
3
protons respectively
[169]
. The coupling constant for H
4
and H
3

was consistent with that previously reported
[150]
, J = 3.57 Hz.
86
In
13
C-NMR spectrum of compound 8f, chemical shifts assignments were made
for the 2 Ar-OCH
2
CH
3
, 12 Ar-C and 7 C
1
-C
7
carbons, including characteristic peaks
of 2 carbonyl groups, of the heptadienedione chain and 2 carbonyls of the ester
groups. In addition, the spectrum also showed peaks for the 2 x 2 4Cs of the 2 thienyl
groups.
MS of 8f (figure 22) showed the M
+
+1 peak at m/z 617. The base peak was
shown at m/z 43 corresponding to acetyl cation The possible fragmentation pattern is
illustrated in chart 7.



Figure 22: MS of di-O-(2-thienoyl)ethyl curcumin (8f)


87
O
O
O
O O
O
O
O
C
33
H
28
O
8
S
2
+
M
+
+ 1 617 (0.7)
C
7
H
7
O
m/z 107 (9)
m/z 44 (68.6)
CH
3
C O
+
C
2
H
3
O
m/z 43 (100)
S
S
O
O
O
OH O
O
O
O
C
32
H
28
O
8
S
2
+
m/z 604 (1.6)
S
S
O O
O
+
O
O
C
24
H
17
O
5
S
+
m/z 417 (2.6)
S
HO
OH
OH
O
O
C
16
H
16
O
5
S
+
m/z 320 (1.6)
S
O
O
S
+
C
11
H
7
O
2
S
m/z 203 (2.3)
O O
+
O
+
O
+
C
7
H
9
O
2
C
5
H
7
O
C
4
H
5
O
m/z 125 (8) m/z 83 (26.6) m/z 69 (15)
HO
+ HO
+
C
6
H
5
O
m/z 93 (11.5)
+
O
CH
3
CHO
S
O
+
C
5
H
3
OS
m/z 111 (13)
S
C
4
H
3
S
m/z 83 (26.6)
+
CO
2 or
+
+


Chart 7: Possible FragmentationPattern for compound 8f
88
4.1.9. Attempt Reacting Curcumin with Chlorosulfonyl isocyanate

As a part of our program, it was also designed to prepare some curcumin
derivatives containing substituted urea moieties attached to the curcumin skeleton
through a sulfonate ester group. Trials to prepare the sulfonate ester under various
reaction conditions including K
2
CO
3
in refluxing acetone, NEt
3
in DMF or pyridine at
room temperature or under reflux were unsuccessful.
H
3
CO
HO
O O
OCH
3
OH
+ ClSO
2
NCO
XXX
Acetone/ K
2
CO
3
or DMF/ NEt
3
or pyridine
H
3
CO
OCNO
2
SO
O O
OCH
3
OSO
2
NCO
RNH
2
1
H
3
CO
O
O O
OCH
3
O
O
2
S SO
2
N
H
N
H
O
RHN
O
NHR
















89
4.2. Anticancer Screening

4.2.1. Introduction

DNA topoisomerases are enzymes essential for the maintenance of chromatin
structure, DNA replication, and mitosis/meiosis in eukaryotic cells. Topoisomerases
mediate sequential breakage and religation of either one (topoisomerase I, Topo I) or
both (topoisomerase II, Topo II) DNA strands, as well as strand passing associated
with such breakage thereby changing the linking number of DNA. These changes are
essential various DNA processes including replication, transcription, and repair.
Among the different types of topoisomerases, Topo I and Topo II have been
established as targets of many chemotherapeutic drugs currently in clinical usage
[170]
.
Inhibition of either enzyme can result in aberrant mitosis in cancer cells hence leading
to mitotic catastrophe, which has been characterized as the primary form of cell death
caused by inhibitors of Topo I and Topo II
[171,172]
.
Therefore, creating DNA-binding compounds which recognize specific sequences
is a central goal in the development of DNA-targeted drugs
[173,174]
. Some highly
interesting lead compounds are the naturally occurring antibiotics distamycin A (a)
and netropsin (b)
[175,176]
which owe their cytotoxic activity to their ability to bind in
the minor groove of the DNA with high AT base selectivity
[177]
or to inhibit the
catalytic activity of topoisomerases. Among other compounds, some promising
candidates (c-g) have been also described
[178-180]
:
90
N
HN
H
O
H
3
C
O
H
N
N
CH
3
O
H
N
N
CH
3
HN
O
NH
2
NH
2
+ Cl
-

(a)
Distamycin A

O
H
N
N
CH
3
O
H
N
N
CH
3
HN
O
NH
2
NH
2
+
2 Cl
-
HN
NH
2
+
H
2
N

(b)
Netropsin
N
O
H
N
H
N
O
HN
H
N
SO
2
CH
3
OCH
3
N
O
N
CH
3
HN
NH
2
O
CH
3

(c)
NetAmsa
N
H
N
R
CH
3
H
N
O
N
H
3
C
CH
3
O
n

(d)
91
N
H
N
S
CH
3
H
N
O
N
H
3
C
CH
3
n
O
O
O
O

(e)
N
O
O
O H
3
C
OCH
3
OCH
3
O

N
H
N
N
OH
Glc
OH
OHCHN
O
O

(f) (g)

N
N
O
O
O
OH

O
O
O
OH
H
3
CO
OCH
3
O
HO
O
O H
3
C
OH

(h) (e)
camptothecin etoposide

In addition, the natural product camptothecin (h), initially discovered because of
its potent antitumor activity
[181]
has been shown to target topo I by binding to the
covalent topo I-DNA complex
[182-184]
. Camptothecin, specifically inhibits relegation
and causes the reversible accumulation of topo I-DNA adducts in vitro and in
vivo
[182,185]
. Topo 1 inhibitors that bind to the covalent complex are termed "poisons",
since they convert an essential enzyme into a DNA-damaging agent
[186]
. The cytotoxic
effects of topo I poisons are roughly proportional to their capacity to stabilize the
92
covalent enzyme-DNA complex
[187]
. In rapidly dividing cells, the DNA replication
fork is thought to collide with the "trapped" topo I-DNA complex, resulting in double
strand breaks and ultimately apoptotic cell death
[188]
.
Accordingly, a systematic study in the curcumin chemistry was designed in which
the diarylheptenedione skeleton has been modified by conjugation with different
functionalities at both ends. The in vitro cytotoxic activity and the inhibition of the
topoisomerases I and II, as possible molecular targets, of some of the newly
synthesized compounds were carried out in order to explain the biological mechanism
of action of these potential curcumin bioconjugates.
The selected moieties involved in such structural modification featured
sulfonamide, alkyl, cycloalkyl and heterocyclic amino functionalities attached to
curcumin or ethyl curcumin through an acetyl or propionyl bridge to investigate the
effect of such molecular modification on the biological activity of compounds (A).
This aminoacetyloxy and aminopropionyloxy groups should represent a structural
equivalent of the amido group present in the previously mentioned lead compounds a-
e with the hope to go a step forward in the field of anticancer agents.


O
O O
O
R
1
O OR
1
O
O
curcumin skeleton
carbonyl group
R
2
N-(CH
2
)
x
R
3
(CH
2
)
x
-N
R
2
R
3
methyl or ethyl bridge
alkyl, cycloalkyl ,heterocyclic amine or sulfonamide.


(A)

93
Furthermore, adamantoyl chloride, heptanoyl chloride and 2-thienoyl chloride
were directly attached through an ester function to the curcumin and ethyl curcumin
core to furnish compounds (B). The conjugate bonds reported herein are ester
linkages which are enzyme sensitive to produce an expected systemic delivery.
O
O O
O
R
1
O OR
1
O
R
2
O
R
2
curcumin skeleton
carbonyl group
Adamantyl or thienyl

or hexyl
(B)

4.2.2. Discussion of the Results

The overall results (Charts 9-11, Tables 7,8, Experimental Part) demonstrated
that, in general, some of the synthesized compounds showed cytotoxic activity against
the tested cancerous cell lines. Although curcumin was documented
[140-142]
to damage
DNA by catalytic topo II poisoning, many of the tested compounds were more active
than curcumin 1 and ethyl curcumin 2 as cytotoxic agents. The most active compound
6b was 10-fold less potent than the reference DNA-intercalating antineoplastic drug
doxorubicin (formerly adriamycin) in various cancer cell lines. Doxorubicin, probably
the most important anticancer drug available, because of its relatively broad spectrum
of activity, has a significant role in the treatment of solid tumors such as carcinoma of
the breast, lung, thyroid and ovary, as well as soft tissue sarcomas. Doxorubicin, a
member of the anthracyclines (rhodomycins) antiobiotics, has a planar anthraquinone
nucleus attached to an amino sugar. While the cytotoxic mechanism of doxorubicin
remains somewhat controversial, there is substantial evidence to suggest that the
94
following events play a role
[189]
: (1) intercalation and alkylation of DNA, (2)
induction of topo II mediated strand breaks, (3) interference with DNA unwinding
and helicase activity, (4) lipid peroxidation, and (5) direct membrane effects at low
concentrations resulting in modification of membrane function and related
cytotoxicity
[190]
.
While there remains debate as to the cytotoxic mechanism, DNA has clearly
emerged as a target. Because of its planar ring structure, doxorubicin can intercalate
between the base pairs in a DNA double helix, cause single-stranded DNA breaks and
impair DNA repair
[191]
. When doxorubicin intercalates with DNA, the planar ring
structure is inserted approximately perpendicularly to the long axis of the DNA
double helix. The polar amino sugar, the quinone function and the phenolic hydroxyl
groups appear to confer added stability to the binding through its interaction with the
sugar phosphate backbone of DNA. Evidences suggest that this DNA binding is
necessary for inhibition of nucleic acid synthesis in tumor cells and for cytotoxic and
antitumor activities
[191]
.
Recent literature reports suggest that doxorubicin chelates iron to catalytically
produce formaldehyde for use in DNA alkylation
[192]
.

O
O OCH
3
OH
OH
OH
O
COCH
2
OH
O
NH
2
OH
CH
3

Doxorubicin

95
The structure of our curcumin analogs could explain the cytotoxicity of some of
the newly synthesized compounds. Like doxorubicin, curcumin analogs possess a
planar conjugated structure with two etherified phenolic groups and an enolisable -
diketone structure. These structural similarities to doxorubicin may contribute to the
interaction of these compounds with DNA and to their cytotoxic activity. Not
surprisingly, the synthesized compounds are less toxic than doxorubicin in non-
cancerous cell lines, an effect that can be attributed to the well-documented
antioxidant activity
[23-25]
and the reduction of free radicals
[78]
of curcumin and
curcumin analogs. Our presumption of the importance of the aminoacetyloxy and
aminopropionyloxy moieties for the cytotoxicity properties of the synthesized
curcumin analogs has been also confirmed in these series of compounds.
Curcumin showed only moderate activity against BT-549 while being completely
nontoxic to the noncancerous Vero and LLC-PK1 cells.
Some of the synthesized compounds, namely 1,7-bis(4-(5-methylthiadiazol-2-yl)-
aminoacetyloxy)-3-(methoxyphenyl)-1,6-heptadiene-3,5-dione (5c), 1,7-bis(4-(bis(2-
chloroethyl)aminoacetyloxy)-3-(methoxyphenyl)-1,6-heptadiene-3,5-dione (5e), 1,7-
bis(4-(2-diethylaminoethyl)aminoacetyloxy)-3-(methoxyphenyl)-1,6-heptadiene-3,5-
dione (5g), 1,7-bis(4-(5-methylthiadiazol-2-yl)aminopropionyloxy)-3-
(methoxyphenyl)-1,6-heptadiene-3,5-dione (5j), 1,7-bis(4-(6-methoxybenzothiazol-2-
yl)aminoacetyl- oxy)-3-(ethoxyphenyl)-1,6-heptadiene-3,5-dione (6b) and 1,7-bis(4-
(2-diethylamino- ethyl)aminoacetyloxy)-3-(ethoxyphenyl)-1,6-heptadiene-3,5-dione
(6g) showed cytotoxic activity against SK-MEL cancerous cell lines (Chart 8) with
no or little effect on the noncancerous cells. Compound 6b was the most active
against SK-MEL cells with IC
50
= 4.75 M followed by compound 5e with an IC
50
=
7 M. Compounds 5c and 5j were also cytotoxic to the same cell line with IC
50
= 7.5
96
M while compounds 5g had IC
50
= 8.5 M and 6g had IC
50
= 11.5 M. Compounds
5j, 5e, 5c and 1,7-bis(4-(2-chloroethyl)aminoacetyloxy)-3-(ethoxyphenyl)-1,6-
heptadiene-3,5-dione (6d) exhibited cytotoxic activity against BT-549 cells with IC
50

= 4.25, 6.75, 8.75 and 9 M respectively (Chart 10). Di-O-chloroacetylcurcumin
(3a), 6g and 1,7-bis(4-(4-sulfanilamido)acetyloxy)-3-(methoxyphenyl)-1,6-
heptadiene-3,5-dione (7a) were almost as active as curcumin against BT-549
exhibiting IC
50
= 10 M. 1,7-Bis(4-(bis(2-chloroethyl)aminoacetyloxy)-3-
(ethoxyphenyl)-1,6-heptadiene-3,5-dione (6e) and 1,7-bis(4-(2-
dimethylaminoethyl)aminoacetyloxy)-3-(ethoxyphenyl)-1,6-heptadiene-3,5-dione (6f)
were slightly less active than curcumin exhibiting IC
50
of 11 and 11.5 M respectively
(Chart 10). Moreover, 1,7-bis(4-(2-chloroethyl)aminopropionyloxy)-3-
(methoxyphenyl)-1,6-heptadiene-3,5-dione (5k), 1,7-bis(4-(5-methylthiadiazol-2-yl)-
aminoacetyloxy)-3-(ethoxyphenyl)-1,6-heptadiene-3,5-dione (6c) and 6g were the
most cytotoxic against KB cells with IC
50
= 11.5 M (Chart 9). Regarding SK-OV-3
cell line (Chart 11), compound 6b was the most cytotoxic among these series with
IC
50
= 2.8 M. It was also not cytotoxic to any of the noncancer cells tested up to 25
M. All other compounds were inactive against this type of cancer cells. It was also
noted that none of the tested compounds having an adamantoyl, heptanoyl and
thienoyl functions directly esterified to the phenolic hydroxyl groups (compounds 8a-
f), showed any activity against all cell lines used indicating that this linkage is not
appropriate for activity. It is also worth-mentioning that none of the tested compounds
showed any inhibition of the topoisomerase I. Some compounds exhibited very low
inhibition of the catalytic activity of topoisomerase II which does not correlate with
the cytotoxic effect indicating that the cytotoxicity of these compounds should be
explained through a mechanism of action different from inhibition of topoisomerases.
97
Similar to doxorubicin, the cytotoxicity of these compounds, especially for compound
6b, may be due to their ability to bind to DNA.

4.2.3. Conclusion

In summarizing all data available (Charts 8-11, Tables 7,8), it becomes obvious
that our starting compounds, curcumin and ethyl curcumin, did not exhibit the
expected cytotoxicity effect. Besides, most of the newly synthesized compounds were
more active than curcumin and ethyl curcumin but were less cytotoxic than the
reference compound doxorubicin. Surprisingly, many of these compounds were not
cytotoxic to noncancer cells. Within our series, compounds 5c, 5e, 5g, 5j, 6b and 6g
having 5-methylthiadiazole, 6-methoxybenzothiazole, diethylaminoethyl and the
usual alkylating bis(2-chloroethyl)amino moieties showed the highest cytotoxic
activity against SK-MEL cancer cells (Chart 8). Compounds 5k, 6c and 6g were less
cytotoxic to KB cancer cells (Chart 9). Moreover, compounds 5c, 5e, 5j, 5k, 6d, 6e,
6f and 6g showed cytotoxicity against BT-549 cancer cells (Chart 10) with 5j being
the most active compound. Curcumin and our intermediate di-O-
chloroacetylcurcumin (3a) were also cytotoxic against the same cell line but were less
active than the synthesized compounds. Finally, compound 6b was the only one
exhibiting cytotoxicity against SK-OV-3 cancer cells (Chart 11). Therefore, these
compounds and especially compound 6b should be very promising candidates for
further studies.





98




O O
H
3
CO
O
OCH
3
O
O
CH
2
Cl
Cl
O
CH
2

3a

H
3
CO
O O
OCH
3
O O CH
2
O O
H
2
C
HN
N
N
S
CH
3
NH
N
N
S
CH
3

5c


H
3
CO
O O
OCH
3
O O CH
2
O O
H
2
C
N
N
Cl
Cl
Cl
Cl

5e



H
3
CO
O O
OCH
3
O O CH
2
O O
H
2
C
H
N
H
N
N N
Et
Et
Et
Et


5g




99
H
3
CO
O O
OCH
3
O O (CH
2
)
2
O O
(H
2
C)
2
N
N
S
CH
3
NH
N
N
S
CH
3
NH

5j


H
3
CO
O O
OCH
3
O O (CH
2
)
2
O O
(H
2
C)
2
NH NH
Cl
Cl


5k


O
O O
O
O O CH
2
O O
H
2
C
NH
HN
N
S
N
S
OCH
3
OCH
3

6b


O
O O
O
O O CH
2
O O
H
2
C
NH
HN
N
N
S
N
N
S
CH
3
CH
3

6c

O
O O
O
O O CH
2
O O
H
2
C
H
N
H
N
Cl
Cl

6d
100
O
O O
O
O O CH
2
O O
H
2
C
N
N
Cl
Cl
Cl
Cl

6e


O
O O
O
O O CH
2
O O
H
2
C
H
N
H
N
N N
H
3
C
H
3
C
CH
3
CH
3


6f

O
O O
O
O O CH
2
O O
H
2
C
H
N
H
N
N N
Et
Et
Et
Et


6g



O
OCH
3
O
H
3
CO
O O
CH
2
O O
-O
2
SHN
NHSO
2
-
CH
2
H
2
N
NH
2


7a


101
5. Experimental Part
5.1. General
Melting points were determined in open glass tubes on a Branstead/Electrothermal
IA9100 melting point apparatus and are uncorrected.
Infrared (IR) spectra were recorded, for potassium bromide discs, (cm
-1
) on
Perkin Elmer 1430 spectrophotometer.

1
H and
13
C nuclear magnetic resonance (NMR) spectra were determined on Jeol
(300 MHz, 500 MHz) and Ultrashield Bruker Biospin (500 MHz) spectrometers.
Chemical shifts are expressed as values (ppm) using tetramethylsilane (TMS) as
internal reference. Signals are indicated by the following letters: s = singlet, d =
doublet, t = triplet, q = quartet, m = multiplet, br = broad.
Mass spectra (MS) were obtained on GC/MS QP 5000 (Ver. 2), Class-5000 (Ver.
1.2) Shimadzu apparatus.
Follow up of the reaction and checking the homogeneity of the compounds were
made by ascending thin layer chromatography (TLC) run on pre-coated (0.25 mm)
(GF 254) silica gel plates. The ratio of the solvent systems used as eluents were
volume to volume. Visualization of the spots was performed by exposure to UV lamp
at 254 nm. Silica gel (60-230 mesh E. Merck) was employed for routine column
chromatography separations.
5.2. Chemical Procedures

According to Scheme 1, the key starting materials, commercially available,
curcumin (1) and ethyl curcumin (2) were either purchased from Sigma-Aldrich (St.
Louis, MO) or prepared according to previously reported methods
[99,107,122, 138]
.

102
5.2.1. 1,7-Bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione
(1)
(65,99,107,122,126,138,148)
and 1,7-bis(4-hydroxy-3-ethoxyphenyl)-1,6-
heptadiene
3,5-dione (2)
(138)

O O
R
1
O
HO
OR
1
OH


(1) R
1
= CH
3
(2) R
1
= C
2
H
5


Tri-(n-butyl) borate (21 ml, 0.08 mol) was added to a solution of the appropriate
aromatic aldehyde (0.04 mol) in dry ethyl acetate (20 ml). A previously prepared
complex, formed by stirring acetylacetone (2 g, 0.02 mol) with boric anhydride (1 g,
0.014 mol) for 30 min at RT, was also added and the reaction mixture was stirred for
5 min. n-Butylamine (0.1 ml) was added dropwise while stirring at 10 min intervals
(total amount = 0.4 ml) and stirring was continued for further 4 h, after which the
mixture was allowed to stand overnight. 0.4 N HCl (30 ml, 60

C) was then added and


the mixture was stirred for 1 h. The organic layers were separated and the aqueous
fraction was extracted with EtOAc (3 x 25 ml). The combined organic layers were
washed with H
2
O, dried over anhydrous MgSO
4
(5 g) and evaporated to ~ 15 ml.
MeOH (10 ml) was then added and the mixture was cooled in the refrigerator for 3h
to give an orange precipitate of curcumin (1) or ethyl curcumin (2). The product was
filtered, washed with cold MeOH and dried.
Curcumin (1) was obtained from vanillin in 73% yield; m.p. = 175

C (reported
m.p. = 181

-183

C
[65]
, 184

-185

C
[107]
, 184

-186

C
[122]
, 180

-182

C
[126]
, 175

-177

C

[136]
, 182

-183

C
[148]
). IR of curcumin (1) (cm
-1
): 3468.5 (br, OH), 1627.8 (C=O),
1604.4, 1508.1 (C=C, Ar), 1282.4, 1027.3 (
as
and
s
C-O-C);
1
H-NMR of 1 (DMSO-
103
d
6
) (ppm) (300 MHz) (figure 1): 3.8366 (s, 6H, 2 x OCH
3
), 6.0618 (s, 1H, H
a
),
6.711 (d, 2H, 2 x H
b
, J = 16.6 Hz), 6.8227 (d, 2H, 2 x H
A
, J
AB
= 8.22 Hz, ortho
coupling), 7.1569 (d, 2H, 2 x H
B
, J
AB
= 8.03 Hz, ortho coupling), 7.3273 (s, 2H, 2 x
H
X
), 7.5470 (d, 2H, 2 x H
c
, J = 15.35 Hz), 9.7411 (s, 2H, 2 x phenolic OH), 10.1312
(s, 1H, enol OH);
13
C-NMR of 1 (DMSO-d
6
) ppm (300 MHz): 56.2179 (2 x OCH
3
),
101.4313 (C-4), 111.8205 (2 x C-2'), 116.2327 (2 x C-5'), 121.6219 (2 x C-6'),
123.7059 ( 2 x C-1'), 126.8662 (C-2 + C-6), 141.2783 (C-1 + C-7), 148.5301 (2 x C-
3'), 149.8889 (2 x C-4'), 183.7664 (C-3 + C-5).
Ethyl curcumin (2) was obtained from 4-hydroxy-3-ethoxy- benzaldehyde in 68%
yield; m.p. = 143

C (reported m.p. = 158

-160

C)
[138]
. IR of ethyl curcumin (2) (cm
-
1
): ;
1
H-NMR of 2 (DMSO-d
6
) ppm (500 MHz) (figure 2): 1.358 (t, 6H, 2 x
OCH
2
CH
3
, J = 7 Hz), 4.094 (q, 4H, 2 x CH
2
CH
3
, J = 7 Hz), 6.050 (s, 2H, H
a
), 6.746
(d, 2H, 2 x H
b
, J = 16 Hz), 6.850 (d, 2H, 2 x H
A
, J
AB
= 8 Hz, ortho coupling), 7.1495
(d, 2H, 2 x H
B
, J
AB
= 8.5 Hz, ortho coupling), 7.305 (s, 2H, 2 x H
X
), 7.552 (d, 2H, 2 x
H
c
, J = 16 Hz), 9.607 (s, 2H, 2 x phenolic OH);
13
C-NMR of 2 (DMSO-d
6
) ppm
(500 MHz) (figure 3): 15.16 (2 x OCH
2
CH
3
), 64.36 (2 x OCH
2
CH
3
), 101.37 (C-4),
112.88 (2 x C-2'), 116.25 (2 x C-5'), 121.49 (2 x C-6'), 123.47 (2 x C-1'), 126.79 (C-2
+ C-6), 141.20 (C-1 + C-7), 147.59 (2 x C-3'), 150.05 (2 x C-4'), 183.67 (C-3 + C-5);
MS of 2 (figure 4) m/z (% relative abundance): M
+
396 (23), 378 (17), 349 (11), 300
(8), 246 (10), 231 (14), 231 (14), 217 (11), 206 (11), 205 (26), 204 (47), 191 (70), 178
(11), 177 (43), 176 (42), 165 (15), 163 (78), 162 (9), 159 (24), 148 (17), 147 (47), 146
(16), 145 (69), 143 (8), 136 (41), 134 (39), 131 (56), 123 (38), 119 (14), 117 (48), 115
(24), 110 (30), 107 (33), 105 (22), 103 (44), 95 (7), 94 (7), 91 (65), 90 (20), 89 (100),
81 (12), 79 (37), 78 (41), 77 (88), 69 (41), 67 (12), 66 (13), 65 (57), 43 (57).

104
5.2.2. Di-O-Chloroacetylcurcumin (3a) and di-O-chloropropionyl-
curcumin (3b)
O O
H
3
CO
O
OCH
3
O
O
(CH
2
)n
Cl
Cl
(CH
2
)n
O


(3a) n = 1 (3b) n = 2

5.2.2.1. Method A

Sodium carbonate (2 g) was added to a stirred ice cold solution of curcumin (1)
(3.68g, 0.01 mol) in dry acetone (50 ml) for 15 min. Chloroacetyl chloride or
chloropropionyl chloride (0.025 mol) was dropwise added and the mixture was stirred
for 3 days at RT. The reaction mixture was filtered, evaporated and extracted with
EtOAc (3 x 30 ml). The organic layer was dried over anhydrous MgSO
4
(5 g) and
evaporated under reduced pressure to give an orange oil. Addition of EtOH (20 ml)
gave a yellow precipitate which was filtered off. The product was purified by column
chromatography using toluene/MeOH (97.5: 2.5 v/v) as eluent (Table 1).
IR of di-O-chloroacetylcurcumin (3a) (cm
-1
): 3414.8 (OH, intramolecularly H-
bonded), 1768.7 (C=O, ester), 1635.7 (C=O, ketone), 1599.9, 1507.6 (C=C Ar),
1254.8, 1122.4 (
as
and
s
C-O-C).
1
H-NMR of 3a (CDCl
3
) ppm (300 MHz) (figure
5): 3.876 (s, 6H, 2 x OCH
3
), 4.1003 (s, 4H, 2 x CH
2
), 5.8557(s, 2H, H
a
), 6.56905 (d,
dist, 2H, 2H
b
, J = 15.9 Hz), 7.0754-7.2613 (m, 6H, 2 x 3 Ar-H), 7.6116 (d, dist, 2H,
2H
c
, J = 15.7 Hz).
IR of di-O-chloropropionylcurcumin (3b) (cm
-1
): 3414.2 (OH, intramolecularly
H-bonded), 1746.6, (C=O, ester), 1635.1 (C=O, ketone), 1607.4, 1506.4 (C=C Ar),
1253, 1129.3 (
as
and
s
C-O-C).
1
H-NMR of 3b (DMSO-d
6
) ppm (300 MHz):
105
3.1223 (t, 4H, 2 x CH
2
-Cl, J = 6.1 Hz), 3.8403 (s, 6H, 2 x OCH
3
), 3.8952 (t, 4H, 2 x
COCH
2
, J = 6.33 Hz), 6.2156 (s, 2H, H
a
), 7.0095 (d, dist, 2H, 2 x H
b
, J = 15.9 Hz),
7.1524-7.5351 (m, 6H, 2 x 3 Ar-H), 7.6569 (d, dist, 2H, 2 x H
c
, J = 15.9 Hz); MS of
3b: m/z (% relative abundance): M
+
549.5 (absent), 284 (14.28), 241 (8.92), 227
(3.57), 199 (4.46), 185 (16.07), 171 (6.25), 143 (7.14), 129 (37.5), 111 (14.28), 97
(31.25), 83 (37.5), 73 (100), 69 (57.25).

Table 1: Physicochemical Data of di-O-chloroacetylcurcumin (3a) and
di-O-chloropropionylcurcumin (3b)

M. wt Mol. Formula

m.p.

C


Yield
(%)
Structure
Cpd
No

521

C
25
H
22
Cl
2
O
8


136

43
O O
H3CO OCH3
OCOCH2Cl ClH2COCO


3a


549

C
27
H
26
Cl
2
O
8


143

38
O O
H3CO OCH3
OCOCH2CH2Cl ClH2CH2COCO



3b



5.2.2.2. Method B

a) 3-Alkoxy-4-chloroacyloxybenzaldehyde (4a-d)

CHO
O
O
(CH
2
)n
Cl
R
1
O


(4a) R
1
= CH
3
n = 1; (4b) R
1
= CH
3
n = 2
(4c) R
1
= C
2
H
5
n = 1; (4d) R
1
= C
2
H
5
n = 2

a.1. The appropriate acid chloride (0.01 mol) in chloroform (20 ml) was added
dropwise over a period of 5 min to an ice-cold stirred solution of the aldehyde (0.01
106
mol) and 1N NaOH (2 ml) in chloroform (5 ml). The mixture was stirred at RT for 1
day and then evaporated to dryness. The residue was extracted (3 x 20 ml) with
EtOAc and 0.1 N NaOH. The combined organic layers were washed with water (3 x
20 ml), dried over anhydrous MgSO
4
(5 g) and removed under reduced pressure to
give a colorless oil. Trituration of the crude product with EtOH (20 ml) gave colorless
crystals of 4a-d which were crystallized from EtOH (Table 2).

a.2. The selected acid chloride (0.01 mol) was dropwise added to an ice cold stirred
solution of the appropriate aldehyde (0.01mol) in dry acetone and anhydrous K
2
CO
3
.
The mixture was further stirred in an ice-bath for 3 h, filtered and concentrated.
Dilution with EtOH (20 ml) precipitated colorless crystals of 4a-d which were filtered
and crystallized from EtOH (Table 2).
IR of 4a (cm
-1
): 2933.4, 2847 (CHO), 1765 (C=O ester), 1698 (C=O aldehyde),
1600, 1505 (C=C aromatic), 1180, 1150, 1100, 1020 (
as
and
s
C-O-C);
1
H- NMR of
4a (DMSO-d
6
) ppm (500 MHz) (figure 6): 3.8445 (s, 3H, OCH
3
), 4.7157 (s, 2H,
CH
2
-Cl), 7.3897 (d, 1H, H
A
, J
AB
= 8.40, ortho coupling), 7.5647, 7.5800 (dd, 1 H, H
B
,
J = 1.55 Hz and 7.65 Hz, meta and ortho coupling), 7.6075 (d, 1H, H
X
, J
AX
= 1.55 Hz,
para coupling), 9.9476 (s, 1H, CHO).
IR of 4b (cm
-1
): 3073, 2940 (CHO), 1760 (C=O ester), 1698 (C=O aldehyde),
1601, 1495 (C=C aromatic), 1154, 1120, 1068, 1025 (
as
and
s
C-O-C).

1
H- NMR of 4c (DMSO-d
6
) ppm (300 MHz) (figure 7): 1.3138 (t, 3H,
OCH
2
CH
3
, J = 6.87 Hz), 4.15575 (q, 2H, OCH
2
CH
3
, J = 6.87 Hz), 4.7413 (s, 2H,
CH
2
Cl), 7.4136 (d, 1H, H
A
, J
AB
= 7.98 Hz, ortho coupling), 7.5762, 7.6051 (dd, 1H,
H
B
, J = 1.65 Hz and 8.07 Hz, meta and ortho coupling), 7.6285 (d, 1H, H
X
, J
AX
= 1.63
Hz, para coupling), 9.9709 (s, 1H, CHO).
107
Table 2: Physicochemical Data of the Synthesized Compounds 4a-d



Cpd
No

Structure
Method
A
Reaction
Time(h)
Method
B
Reaction
Time(h)
Yield
%
A
Yield
%
B

m.p.

C

Molecular
Formula

M.wt


4a

OCH
3
CHO
ClH
2
COCO




3



24



43


78


68-
71




C
10
H
9
ClO
4



228.5


4b

OCH
3
CHO
ClH
2
CH
2
COCO



24


3


41


72


65-
69


C
11
H
11
ClO
4



242.5


4c

OCH
2
CH
3
CHO
ClH
2
COCO



24


3


42



67


61-
64


C
11
H
11
ClO
4



242.5


4d

OCH
2
CH
3
CHO
ClH
2
CH
2
COCO



24



3


39


61



55-
59



C
12
H
13
ClO
4



256.5

b) Di-O-Chloroacylcurcumin (3a,b) and di-O-chloroacylethyl
curcumin (3c,d)

O
OR
1
O
R
1
O
O O
(CH
2
)n n(H
2
C)
O O
Cl
Cl

(3a-d)

To a stirred solution of the appropriate 3-alkoxy-4-chloroacyloxybenzaldehyde
(4a-d) (0.04 mol) in dry EtOAc (20 ml) was added tri-(n-butyl) borate (21 ml, 0.08
mol) and the mixture was stirred at RT for 10 min. The previously prepared complex
formed by stirring for 1 h acetylacetone (2 g, 0.02 mol) with boric anhydride (1 g,
108
0.014 mol) was then added to this solution. After stirring for 5 min at RT, n-
butylamine (0.1 ml) was dropwise added every 10 min (total amount 0.4 ml) and the
reaction mixture was stirred at RT for an overnight. 0.4 N HCl (30 ml, 60

C) was then
added and the mixture was further stirred for 2 h followed by extraction with EtOAc
(3 x 25 ml). The combined organic layers were washed with H
2
O, dried over
anhydrous MgSO
4
(5 g) and concentrated to ~ 15 ml. MeOH (15 ml) was added and
the mixture was allowed to stand in the refrigerator for 3 h to give a yellow
precipitate of 3a-d which was filtered off, washed with cold MeOH and dried (Table
3).
IR and
1
H-NMR spectra of compounds 3a,b were coinciding with those obtained
from method A.
IR of 3c (cm
-1
): 3435.6 (OH, intramolecularly H-bonded), 1746 (C=O, ester),
1630.9 (C=O, ketone), 1558.1, 1496.3 (C=C aromatic), 1270, 1157 (
as
and
s
C-O-
C);
1
H- NMR of 3c (CDCl
3
) ppm (300 MHz) (figure 8): 1.4164 (t, 6H, 2 x
OCH
2
CH
3
, J = 6.87 Hz), 4.1049 (q, 4H, 2 x OCH
2
CH
3
, J = 6.87 Hz), 4.3329 (s, 4H, 2
x CH
2
-Cl), 5.8457(s, 2H, H
a
), 6.5553 (d, 2H, 2 x H
b
, J = 15.93 Hz), 7.0764-7.1762
(m, 6H, 2 x 3 Ar-H), 7.605 (d, 2H, 2 x H
c
, J = 15.93 Hz); MS of 3c (figure 9) m/z (%
relative abundance): M
+
548 (0.24), M
+
+2 550 (0.21), M
+
+4 552, 533 (0.23), 531
(0.28), 529 (0.29), 482 (0.24), 471 (0.21), 412 (0.15), 377 (0.13), 373 (0.3), 343
(0.21), 323 (0.12), 296 (0.14), 274 (0.14), 244 (0.26), 242 (0.6), 227 (0.15), 216
(0.24), 191 (0.17), 166 (2), 137 (2), 120 (2), 87 (12), 85 (69), 83 (100).

1
H- NMR of 3d (CDCl
3
) ppm (500 MHz) (figure 10): 1.442 (t, 6H, 2 x
OCH
2
CH
3
, J = 7 Hz), 3.105 (t, 4H, 2 x COCH
2
, J = 7Hz), 3.904 (t, 4H, 2 x CH
2
-Cl, J
= 7 Hz), 4.131 (q, 4H, 2 x OCH
2
CH
3
, J = 7 Hz), 5.871 (s, 1H, H
a
), 6.577 (d, 2H, 2 x
H
b
, J = 16 Hz), 7.098 (d, 2H, 2 x H
A
, J
AB
= 8 Hz, ortho coupling), 7.1735 (d, 2H, 2 x
109
H
B
, J
AB
= 8.5 Hz, ortho coupling), 7.286 (s, 2H, 2 x H
X
), 7.559 (d, 2H, 2 x H
c
, J = 16
Hz), 9.965 (s, 1H, enol OH).

Table 3: Physicochemical Data of the Synthesized Compounds 3a-d
O
OR
1
O
R
1
O
O O
(CH
2
)n n(H
2
C)
O O
Cl
Cl

(3a-d)

M. wt M. Formula m.p.

C


Yield
%
n R
1
No.

521

C
25
H
22
Cl
2
O
8


136

61

1

CH
3


3a


549

C
27
H
26
Cl
2
O
8


143

56

2

CH
3


3b


549

C
27
H
26
Cl
2
O
8


130

59

1

C
2
H
5


3c


577

C
29
H
30
Cl
2
O
8


145

54

2

C
2
H
5


3d



5.2.3. 2-Chloroethylamine monohydrochloride and bis(2-chloroethyl)amine
hydrochloride

NH
2
CH
2
CH
2
Cl . HCl

NH
CH
2
CH
2
Cl
CH
2
CH
2
Cl
. HCl


Thionyl chloride (30 ml) was added dropwise to a solution of ethanolamine or
diethanolamine (10 ml) in dry benzene (70 ml) in an ice bath. The mixture was heated
under reflux for h and cooled to RT. The oily residue obtained was separated and
crystallized from EtOH and diethyl ether (charcoal). m.p. = 143-146

C as reported
[160]

110
for 2-chloroethylamine monohydrochloride and 212-214

C as reported
[160]
for bis(2-
chloroethyl)amine hydrochloride.
5.2.4. 1,7-Bis(4-Alkyl(cycloalkyl or heteroaryl)aminoacyloxy)-3-
(methoxyphenyl)-
1,6-heptadiene-3,5-dione (5a-n) and 1,7-bis(4-alkyl (cycloalkyl or
heteroaryl)-
aminoacyloxy)-3-(ethoxyphenyl)-1,6-hepta- diene-3,5-dione (6a-n)

OR
1
O
R
1
O
O O
n(H
2
C)
O O
(CH
2
)n
R
3
R
2
N
NR
2
R
3


(5a-n, 6a-n)

Triethylamine (5 drops) were added to a solution of di-O-chloroacylcurcumin
3a,b or di-O-chloroacylethyl curcumin 3c,d (0.005 mol) in EtOH (30 ml). The
appropriate amine (0.01 mol) was then added and the mixture was heated under reflux
for the specified time (Table 4). EtOH was evaporated under reduced pressure and the
residue was crystallized from CHCl
3
(15ml) giving compounds 5a-n and 6a-n (Table
4).



1
H-NMR of 5a (DMSO-d
6
) ppm (300 MHz) (figure 11): 1.1609-1.9951 (hump,
br, 30H, 2 x Ad-H), 2.0858 (s, 4H, 2 x CO-CH
2
), 3.8311 (s, 6H, 2 x OCH
3
), 6.0334 (s,
br, 2H, H
a
), 6.7444 (d, 2H, 2x H
b
, J = 15.66 Hz), 6.8016 (d, 2H, 2 x H
A
, J
AB
= 8.25
Hz, ortho coupling), 7.1295, 7.1569 (dd, dist, 2H, 2 x H
B
, J = 1.5 Hz and 8.22 Hz,
meta and ortho coupling), 7.3099 (s, dist, 2H, 2 x NH), 7.1734 ( d, 2H, 2 x H
X
, J
AX
=
0.24 Hz, para coupling), 7.5315 (d, 2H, 2 x H
c
, J = 15.66 Hz); MS of 5a m/z (%
abundance): M
+
750 (absent), 135 (100), 107 (33.03), 93 (35.71), 83 (30.35), 81
(39.28), 79 (42.85) 77 (35.71).
111
IR of 5b (cm
-1
): 3550, 3414.4 (NH + OH intramolecular H-bonded), 1733.7
(C=O, ester), 1637.4 (C=O, ketone), 1618 (C=N mixed with C=C aromatic), 1510.2
(C=C, aromatic), 1272.1 1122.9 (
as
and
s
C-O-C);
1
H-NMR of 5b (DMSO-d
6
)
ppm (300 MHz): 2.0876 (s, 4H, 2 x CH
2
), 3.8384 (s, 12H, 4 x OCH
3
), 6.0599 (s, 2H,
H
a
), 6.7646 (d, 2H, 2 x H
b
, J = 15.66 Hz), 6.8236 (d, 2H, 2 x H
A
, J
AB
= 8.25 Hz, ortho
coupling), 7.1413, 7.1688 (dd, 2H, 2 x H
B
, J = 1.65 Hz and 8.25 Hz, meta and ortho
coupling), 7.32815 (d, 2H, H
X
, J
AX
= 1.65 Hz, para coupling), 7.5466 (d, 2H, 2 x H
c
,
J = 15.66 Hz, overlapping with benzothiazole multiplet), 7.525-7.610 (m, 6H, 2 x 3
benzothiazole-H), 9.6925 (s, 2H, 2 x NH, D
2
O-exchangeable); MS of 5b m/z (%
abundance): M
+
808 (absent), 163 (53.57), 161 (35.71), 127 (41.07), 125 (100), 97
(62.5), 83 (37.5), 79 (67.85), 77 (71.42).
IR of 5c (cm
-1
): 3425 (NH + OH intramolecularly H-bonded), 1727.3 (C=O,
ester), 1630 (C=O, ketone mixed with C=N), 1575, 1506 (C=C aromatic), 1272.5,
1120.7 (
as
and
s
C-O-C);
1
H-NMR of 5c (DMSO-d
6
) ppm (300 MHz): 2.5033 (s,
4H, 2 x CH
2
), 3.3522 (s, 6H, 2 x CH
3
), 3.835 (s, 6H, 2 x OCH
3
), 6.0590 (s, 2H, H
a
),
6.7631 (d, 2H, 2 x H
b
, J = 15.93 Hz), 6.8218 (d, 2H, 2 x H
A
, J
AB
= 8.22 Hz, ortho
coupling), 7.1409, 7.1676 (dd, 2H, 2 x H
B
, J = 1.65 Hz and 8.01 Hz, meta and ortho
coupling), 7.3272 (d, 2H, 2 x H
X
, J
AX
= 1.65 Hz), 7.5457 (d, 2H, 2 x H
c
, J = 15.66
Hz), 9.6852 (s, 2H, 2 x NH, D
2
O-exchangeable).

1
H-NMR of 5d (CD
3
OD) ppm (500 MHz): 2.292 (s, 2H, 2 x NH), 2.305 (t, dist,
4H, 2 x CH
2
-N), 3.230 (t, 4H, 2 x CH
2
-Cl, J = 3.5 Hz), 3.331 (s, 4H, 2 x COCH
2
-
N), 3.918 (s, 6H, 2 x OCH
3
), 4.903 (s, 2H, 2 x H
a
), 6.643 (d, 2H, 2 x H
b
, J = 15.5 Hz),
6.839 (d, 2H, 2 x H
A
, J
AB
= 8 Hz, ortho coupling), 7.119 (d, 2H, 2 x H
B
, 7.7 Hz, ortho
coupling), 7.227 (s, 2H, 2 x H
X
), 7.584 (d, 2H, 2 x H
c
, J = 15.5 Hz).
112
IR of 5e (cm
-1
): 3414.8 (OH intramolecularly H- bonded), 1728.4 (C=O, ester),
1637.6 (C=O, ketone), 1618.1, 1511 (C=C aromatic), 1272.6, 1122.9 (
as
and
s
C-O-
C);
1
H-NMR of 5e (DMSO-d
6
) ppm (300 MHz) (figure 12): 2.0867 (s, 4H, 2 x
COCH
2
-N), 2.5042 (t, 8H, 4 x N-CH
2
CH
2
-Cl, J = 3.57 Hz), 3.4841 (t, 8H, 4 x
NCH
2
CH
2
-Cl, overlapping with solvent signal), 3.8384 (s, 6H, 2 x OCH
3
), 6.0599 (s,
1H, H
a
), 6.7718 (d, 2H, 2 x H
b
, J = 15.9 Hz), 6.8245 (d, 2H, 2 x H
A
, J
AB
= 8.25 Hz,
ortho coupling), 7.1409, 7.1688 (dd, 2H, 2 x H
B
, J = 1.65 Hz and 8.37 Hz, meta and
ortho coupling), 7.3277 (d, 2H, 2 x H
X
, J
AX
= 1.38 Hz, para coupling), 7.5736 (d, 2H,
2 x H
c
, J = 15.9 Hz), 9.6953 (s, br, 1H, enol OH).

1
H-NMR of 5f (DMSO-d
6
) ppm (300 MHz): 3.1748 (s, 12H, 2 x N(CH
3
)
2
),
3.5387 (t, dist, 4H, 2 x CH
2
-N), 3.6657 (t, 4H, 2 x CH
2
-NH, J = 5.49 Hz), 3.8012 (s,
dist, 2H, 2 x NH, overlapping with OCH
2
), 3.8343 (s, 4H, 2 x COCH
2
-N), 4.0602 (s,
6H, 2 x OCH
3
), 6.0595 (s, 1H, H
a
), 6.7586 (d, 2H, 2 x H
b
, J = 15.75 Hz), 6.8337 (d,
2H, 2 x H
A
, J
AB
= 8.07 Hz, ortho coupling), 7.1513 (d, 2H, 2 x H
B
, J = 8.07 Hz, ortho
coupling), 7.3235 (s, 2H, 2 x H
X
), 7.5427 (d, 2H, 2 x H
c
, J = 15.75 Hz), 9.7368 (s, 1H,
enol H).

1
H-NMR of 5h (CD
3
OD) ppm (500 MHz) (figure 13): 1.184-2.172 (hump, br,
30H, 2 x Ad-H), 2.631 (t, dist, 4H, 2 x CH
2
-N, J = 6.2 Hz), 3.064 (t, 4H, 2 x CO-
CH
2
, J = 6.5 Hz), 3.882 (s, 2H, 2 x NH overlapping with OCH
3
signal), 3.921 (s, 6H,
2 x OCH
3
), 4.936 (s, 2H, H
a
), 6.632 (d, 2H, 2x H
b
, J = 15.7 Hz), 6.835 (d, 2H, 2 x H
A
,
J
AB
= 8 Hz, ortho coupling), 7.111 (d, 2H, 2 x H
B
, J = 7.8 Hz, ortho coupling), 7.218 (
d, 2H, 2 x H
X
), 7.577 (d, 2H, 2 x H
c
, J = 15.8 Hz);
13
C-NMR of 5h (CD
3
OD) ppm
(500 MHz): 39.51 (2 x CH
2
N), 40.48 (2 x CH
2
CO), 47.10, 47.27, 47.44, 47.61, 47.78,
47.90, 47.95, 48.07, 48.12, 48.24 (2 x 10 Ad-C), 55.07 (2 x OCH
3
), 101.37 (C-4),
110.27 (2 x C-2'), 115.04 (2 x C-5'), 120.79 (2 x C-6'), 122.77 (2 x C-1'), 127.07 (C-2
113
+ C-6), 129.77 (C-1 + C-7), 140.94 (2 x C-3'), 148.06 (2 x C-4'), 149.25 (C-3 + C-5),
183.23 (2 x CO); MS of 5h (Figure 14) m/z (% relative abundance): M
+
at 778
(absent), 719 (2.4), 705 (1.8), 698 (2.6), 692 (2), 646 (2.3), 616 (3.1), 611 (2.7), 581
(3), 575 (3.6), 562 (2.8), 530 (2.3), 463 (10.3), 442 (1.3), 430 (2.2), 422 (1.8), 392
(1.4), 340 (1.2), 338 (1.7), 318 (1.2), 305 (1.4), 289 (1), 281 (1.4), 273 (1.4), 251
(9.7), 237 (1.1), 195 (10.2), 194 (33.6), 180 (5), 164 (27), 151 (6.8), 135 (25.6), 121
(7), 119 (2.7), 116 (6.2), 107 (11.3), 106 (74.4), 94 (49.7), 93 (15.2), 91 (13.8), 81 (8),
79 (12.9), 77 (20.8), 67 (13.9), 65 (8.8), 57 (22), 55 (34.8), 53 (19), 46 (44.8), 45
(100), 44 (30.5), 43 (72), 41 (80.5).
IR of 5j (cm
-1
): 3435.7 (NH + OH intramolecularly H-bonded), 1790.6 (C=O,
ester), 1658.9 (C=O, ketone), 1558 (C=N mixed with C=C aromatic), 1498.8 (C=C
aromatic), 1273.2, 1169.9 (
as
and
s
C-O-C);
1
H-NMR of 5j (CD
3
COCD
3
) ppm
(500 MHz): 3.612 (s, 6H, 2 x CH
3
), 3.908 (s, 6H, 2 x OCH
3
), 4.084 (t, dist, 4H, 2 x
CH
2
-N), 4.609 (t, dist, 4H, 2 x CH
3
-CO), 5.791 (s, br, 2H, H
a
), 6.701 (d, 2H, 2x H
b
, J
= 15.5 Hz), 6.898-7.676 (m, 6H, 2 x Ar-H), 7.744 (d, 2H, 2 x H
c
, J = 15.5 Hz), 8.317
(s, 2H, 2 x NH);
13
C-NMR of 5j (CD
3
COCD
3
) ppm (500 MHz): 45.35 (2 x CH
2
N),
47.25 (2 x CH
2
CO), 60.85 (2 x OCH
3
), 105.75 (C-4), 111.80 (2 x C-2'), 116.85 (2 x
C-5'), 120.0 (2 x C-6'), 123.75 (2 x C-1'), 128.82 (C-2 + C-6), 134.93 (C-1 + C-7),
142.00 (2 x C-3'), 147.85 (2 x C-4'), 148.95 (C-3 + C-5), 170.40 (2 x thiazole C-2),
180.85 (2 x thiazole C-5), 192.75 (2 x CO); MS of 5j (figure 15) m/z (% relative
abundance): M
+
+1 707 (0.7), 664 (0.9), 658 (1.1), 618 (0.6), 561 (0.7), 496 (0.6), 491
(0.9), 387 (0.7), 307 (2.5), 293 (3), 268 (2), 242 (11), 171 (4), 167 (7), 150 (5), 149
(35), 142 (10), 115 (5), 100 (5.5), 99 (4.1), 83 (6.4), 73 (17.5), 63 (12), 55 (72.4), 45
(18.1), 43 (100).
114
1
H-NMR of 6a (CDCl
3
) ppm (300 MHz): 1.2516-2.3596 ((hump, br, 30H, 2 x Ad-
H), 1.4805 (t, 6H, 2 x OCH
2
CH
3
, J = 6.87 Hz), 2.3504 (s, 4H, 2 x CH
2
CO), 2.6306 (s,
2H, 2 x NH), 4.1516 (q, 4H, 2 x OCH
2
CH
3
, J = 6.87 Hz), 5.7825 (s, 1H, H
a
), 6.4541
(d, 2H, 2 x H
b
, J = 15.66 Hz), 6.9307 (d, 2H, 2 x H
A
, J
AB
= 8.25 Hz, ortho coupling),
7.0315 (s, 2H, 2 x H
X
), 7.10655 (d, 2H, 2 x H
B
, J = 8.25 Hz, ortho coupling), 7.5722
(d, 2H, 2 x H
c
, J = 15.66 Hz), 9.8134 (s, 1H, enol OH).

Table 4: Physicochemical Data of the Synthesized Compounds 5a-n and 6a-n.
OR
1
O
R
1
O
O O
n(H
2
C)
O O
(CH
2
)n
R
3
R
2
N
NR
2
R
3


(5a-n, 6a-n)


Cp
d
No

R
1


n

R
2
R
3

Reactio
n
Time
h
Yiel
d
%
m.p.
C

M. Formula
M.
wt


5a


-CH
3



1


NH




12


86


110
-15


C
45
H
54
N
2
O
8



75
0


5b


-CH
3



1

N
S
NH
H
3
CO





12


59


160
-
63


C
41
H
36
N
4
O
10
S
2



80
8


5c


-CH
3



1


N N
S
NH
H
3
C




24


63


170
-73


C
31
H
30
N
6
O
8
S
2



67
8

5d

-CH
3


1


HN
Cl





6


83


87-
93


C
29
H
32
Cl
2
N
2
O
8



60
7
















115
5e -CH
3

1

N
Cl
Cl


4-5

95 85-
90
C
33
H
38
Cl
4
N
2
O
8

73
2


5f


-CH
3



1


NCH
2
CH
2
NH
H
3
C
H
3
C




1


69


160
-65


C
33
H
44
N
4
O
8



62
4


5g


-CH
3



1


NCH
2
CH
2
NH
C
2
H
5
C
2
H
5




1


62


150
-55


C
37
H
52
N
4
O
8



68
0


5h


-CH
3



2


NH



12


69



140


C
47
H
58
N
2
O
8




77
8


5i


-CH
3



2

N
S
NH
H
3
CO



36


47


170
-74



C
43
H
40
N
4
O
10
S
2



83
6


5j


-CH
3



2


N N
S
NH
H
3
C



36


43


120
-23


C
33
H
34
N
6
O
8
S
2



70
6


5k


-CH
3



2


HN
Cl



36


71


90-
95


C
31
H
36
Cl
2
N
2
O
8



63
5


5l


-CH
3



2


N
Cl
Cl



36


74


87-
93


C
35
H
42
Cl
4
N
2
O
8



76
0


5m


-CH
3



2


NCH
2
CH
2
NH
H
3
C
H
3
C




3-4


66


160
-65


C
35
H
48
N
4
O
8



65
2


5n


-CH
3



2


NCH
2
CH
2
NH
C
2
H
5
C
2
H
5



3-4


69


155
-60


C
39
H
56
N
4
O
8



70
8

116

6a

CH
2
CH
3



1

NH



4-5

71.5


115
-20

C
47
H
58
N
2
O
8


77
8



6b


CH
2
CH
3




1

N
S
NH
H
3
CO



12


66


137
-40


C
43
H
40
N
4
O
10
S
2



83
6


6c


CH
2
CH
3




1

N N
S
NH
H
3
C



12


72


105
-10



C
33
H
34
N
6
O
8
S
2



70
6

6d

CH
2
CH
3



1


HN
Cl



9

56

107
-10

C
31
H
36
Cl
2
N
2
O
8


63
5



6e


CH
2
CH
3




1


N
Cl
Cl




9




65


95-
100


C
35
H
42
Cl
4
N
2
O
8



76
0


6f


CH
2
CH
3




1


NCH
2
CH
2
NH
H
3
C
H
3
C




6


67


115
-20


C
35
H
48
N
4
O
8



65
2


6g


CH
2
CH
3




1


NCH
2
CH
2
NH
C
2
H
5
C
2
H
5




6


69


105
-
110


C
39
H
56
N
4
O
8



70
8


6h


CH
2
CH
3




2


NH




6



67



85-
90


C
49
H
62
N
2
O
8



80
6


6i


CH
2
CH
3




2

N
S
NH
H
3
CO



16


58


165
-70


C
45
H
44
N
4
O
10
S
2



86
4


6j


CH
2
CH
3




2

N N
S
NH
H
3
C



36


79


105
-
110



C
35
H
38
N
6
O
8
S
2



73
4


117

6k

CH
2
CH
3



2
HN
Cl


12

91

115
-
120

C
33
H
40
Cl
2
N
2
O
8


66
3


6l


CH
2
CH
3




2


N
Cl
Cl



12


64


125
-
130


C
37
H
46
Cl
4
N
2
O
8




78
8


6m


CH
2
CH
3




2

NCH
2
CH
2
NH
H
3
C
H
3
C




3-4


89


155
-60


C
37
H
52
N
4
O
8



68
0


6n


CH
2
CH
3




2


NCH
2
CH
2
NH
C
2
H
5
C
2
H
5




3-4


81


155
-60


C
41
H
60
N
4
O
8



73
6


5.2.5. 1,7-Bis(4-(4-substituted sulfanilamido)acyloxy)-3-(methoxyphenyl)-1,6-
heptadiene-3,5-dione (7a,b) and 1,7-bis(4-(4-substituted sulfanilamido)-
acyloxy)-3-(ethoxyphenyl)-1,6-heptadiene-3,5-dione (7c-f)

O
OR
1
O
R
1
O
O O
(CH
2
)n
O O
(CH
2
)n
HN
NH
R
4
HNO
2
S
SO
2
NHR
4

(7a-f)

Following the same procedure as for the preparation of compounds 5a-n and 6a-n,
the appropriate sulfonamide derivative (0.01 mol) was added to a solution of di-O-
chloroacylcurcumin 3a,b or di-O-chloroacylethyl curcumin 3c,d (0.005 mol) in EtOH
(30 ml) containing triethylamine (5 drops). The mixture was heated under reflux for
the specified time (Table 5), EtOH was evaporated under reduced pressure and the
residue was purified by elution on a column of silica gel using a mixture of toluene-
EtOH (97:3 v/v) to give compounds 7a-f (Table 5).


118
Table 5: Physicochemical Data of the Synthesized Compounds (7a-f)


O
OR
1
O
R
1
O
O O
(CH
2
)n
O O
(CH
2
)n
HN
NH
R
4
HNO
2
S
SO
2
NHR
4


(7a-f)


Cpd
No
R
1
n R
4
Reaction
Time
h
Yield
%
m.p.
C
M. Formula M. wt

7a

CH
3


1

H

16

69

155-60


C
37
H
36
N
4
O
12
S
2


792


7b


CH
3



1

N
N




12


50


175-78



C
45
H
40
N
8
O
12
S
2



948

7c

C
2
H
5


1

H

18

63

143-48


C
39
H
40
N
4
O
12
S
2


820


7d


C
2
H
5



1

N
N




24


60


185-90



C
47
H
44
N
8
O
12
S
2



976

7e

C
2
H
5


2

H

18

78

115-20


C
41
H
44
N
4
O
12
S
2


848


7f


C
2
H
5



2

N
N




24


57


115-20



C
49
H
48
N
8
O
12
S
2



1004


5.2.6. Di-O-Adamantoylcurcumin (8a) and di-O-adamantoylethyl curcumin (8b)

-OCO
O O
OCO-
R
1
O OR
1

(8a) R
1
= CH
3

(8b) R
1
= C
2
H
5


119
Adamantoyl chloride (0.03 mol) dissolved in the least amount of EtOH (10 ml)
was added to a mixture of curcumin (1) or ethyl curcumin (2) (0.01 mol) and Na
2
CO
3
(2 g) in acetone (50 ml). The mixture was heated under reflux for 9-19 hrs, filtered
and evaporated to dryness. The residue was extracted with EtOAc (3 x 25 ml). The
combined organic layer was dried over anhydrous MgSO
4
(5 g), filtered, evaporated
and crystallized from EtOH (15 ml) to give a yellow precipitate of 8a,b ( Table 6).
IR of 8a (cm
-1
): 3435.8 (OH intramolecularly H-bonded), 1750.4 (C=O, ester),
1630.9 (C=O, ketone), 1599, 1507.4 (C=C aromatic), 1255.2, 1122.8 (
as
and
s
C-O-
C);
1
H-NMR of 8a (CDCl
3
) ppm (300 MHz): 1.2342-2.1929 (hump, br, 30H, 2 x
Ad-H), 3.8467 (s, 6H, 2 x OCH
3
), 5.8475 (s, 2H, H
a
), 6.5539 (d, 2H, 2 x H
b
, J = 15.9
Hz), 7.0076 (d, 2H, 2 x H
A
, J
AB
= 8.25 Hz, ortho coupling), 7.0974 (s, 2H, 2 x H
X
),
7.1340, 7.1608 (dd, 2H, 2 x H
B
, J = 1.65 Hz and 8.04 Hz, meta and ortho coupling),
7.6111 (d, 2H, 2 x H
c
, J = 15.9 Hz);
13
C-NMR of 8a (CDCl
3
) ppm (500 MHz):
56.01 (2 x OCH
3
), 27.88, 27.94, 36.48, 38.73, 38.81, 41.14, 76.79, 77.04, 77.29 (2 x
Ad-C), 101.75 (C-4), 111.48 (2 x C-2'), 121.16 (2 x C-5'), 123.32 (2 x C-6'), 124.03 (2
x C-1'), 129.18 (C-2 + C-6), 140.13 (C-1 + C-7), 141.95 (2 x C-3'), 151.52 (2 x C-4'),
175.60 (C-3 + C-5), 183.16 (2 x CO); MS of 8a (figure 16) m/z (% relative
abundance): M
+
692 (2.1), 673 (3.7), 657 (2.4), 648 (2.3), 632 (3.2), 626 (2.1), 618
(2.3), 567 (2), 532 (2.17), 518 (2.75), 513 (2), 509 (2.5), 502 (2.4), 497 (1.7), 475
(2.1), 474 (2.2), 459 (2.3), 453 (4.3), 437 (2), 429 (1.34), 408 (1.3), 394 (1.6), 386
(1.35), 364 (1.26), 359 (3), 329 (1.2), 305 (1), 278 (1.2), 264 (1.5), 262 (2), 220 (1.7),
196 (3.4), 180 (4.8), 177 (1.5), 163 (5.6), 152 (2), 135 (100), 123 (5), 119 (2.9), 107
(9.1), 105 (5), 95 (3.2), 93 (29), 91 (15), 81 (9.1), 79 (36), 77 (16.2), 69 (8.7), 65
(9.1), 57 (22), 55 (29), 45 (31.1), 44 (26.4), 43 (50), 41 (92).
120

1
H-NMR of 8b (CDCl
3
) ppm (300 MHz): 1.2516-2.3596 (hump, br, 30H, 2 x Ad-
H overlapping with t of OCH
2
CH
3
), 1.4805 (t, 6H, 2 x OCH
2
CH
3
, J = 6.87 Hz),
4.1611 (q, 4H, 2 x OCH
2
CH
3
, J = 6.97 Hz), 5.7825 (s, 1H, H
a
), 6.4541 (d, 2H, 2 x H
b
,
J = 15.66 Hz), 6.9307 (d, 2H, 2 x H
A
, J
AB
= 8.25 Hz, ortho coupling), 7.0315 (s, 2H, 2
x H
X
), 7.1065 (d, 2H, 2 x H
B
, J
AB
= 8.25 Hz, ortho coupling), 7.5722 (d, 2H, 2 x H
c
, J
= 15.66 Hz); 9.8134 (s, 1H, enol OH).
5.2.7. Di-O-Heptanoylcurcumin (8c) and di-O-heptanoylethyl curcumin (8d)

O
OR
1
O
R
1
O
O O
O O



(8c) R
1
= CH
3

(8d) R
1
= C
2
H
5



Heptanoyl chloride (0.03 mol) was added dropwise to an ice cold mixture of
curcumin (1) or ethyl curcumin (2) (0.015 mol) and Na
2
CO
3
(2 g) in acetone (50 ml).
The mixture was stirred at room temperature for 4-6 hrs, evaporated to dryness and
the residue was extracted with EtOAc (2 x 20 ml). The combined organic extracts
were dried over anhydrous MgSO
4
, evaporated and crystallized from EtOH to give a
yellow precipitate of 8c,d (Table 6).
IR of 8c (cm
-1
): 1768 (C=O, ester), 1629 (C=O, ketone), 1598, 1512 (C=C
aromatic), 1120, 1010 (
as
and
s
C-O-C);
1
H-NMR of 8c (CDCl
3
) ppm (300 MHz):
0.9068 (t, 6H, 2 x (CH
2
)
5
-CH
3
, J = 6.96 Hz), 1.3049-1.7861 (m, 16H, 2 x (CH
2
)
4
),
2.5849 (t, 4H, 2 x CH
2
CO, J = 7.32 Hz), 3.8672 (s, 6H, 2 x OCH
3
), 5.8543 (s, 2H,
H
a
), 6.5468 (d, 2H, 2 x H
b
, J = 15.75 Hz), 7.0475 (d, 2H, 2 x H
A
, J
AB
= 8.04 Hz, ortho
coupling), 7.11405, 7.146 (dd, 2H, 2 x H
B
, J = 1.8 Hz and 9 Hz, meta and ortho
coupling), 7.2563 (s, 2H, 2 x H
X
), 7.6184 (d, 2H, 2 x H
c
, J = 15.75 Hz).
121
IR of 8d (cm
-1
): 1769 (C=O, ester), 1629 (C=O, ketone), 1590, 1500 (C=C
aromatic), 1115, 1020 (
as
and
s
C-O-C);
1
H-NMR of 8d (CDCl
3
) ppm (300 MHz)
(figure 17): 0.90441 (t, 6H, 2 x (CH
2
)
5
-CH
3
, J = 6.96 Hz), 1.3269-1.5724 (m, 16H, 2
x (CH
2
)
4
), 1.7666 (t, 6H, 2 x OCH
2
CH
3
, J = 7.3 Hz), 2.5727 (t, 4H, 2 x CH
2
-CO, J =
7.3 Hz), 4.0865 (q, 4H, 2 x OCH
2
CH
3
, J = 6.96 Hz), 5.8384 (s, 2H, H
a
), 6.5400 (d,
2H, 2 x H
b
, J = 15.75 Hz), 7.0383 (d, 2H, 2 x H
A
, J
AB
= 8.4 Hz, ortho coupling),
7.1134 (d, 2H, 2 x H
B
, J
AB
= 7.5 Hz, ortho coupling), 7.2551 (s, 2H, 2 x H
X
), 7.6013
(d, 2H, 2 x H
c
, J = 15.75 Hz);
13
C-NMR of 8d (CDCl
3
) ppm (300 MHz) (figure 18):
14.164, 14.813, 22.592, 25.157, 28.874 (5 peaks for 5Cs of side chain), 31.569 (2 x
OCH
2
CH
3
), 34.157 (2 x -OCOCH
2
C
5
H
11
), 64.454 (2 x OCH
2
CH
3
), 101.874 (C-4),
112.477 (2 x C-2'), 121.034 (2 x C-5'), 123.316 (2 x C-6'), 124.133 (2 x C-1'),
133.812 (C-2 + C-6), 140.171 (C-1 + C-7), 141.728 (2 x C-3'), 150.911 (2 x C-4'),
171.743 (C-3 + C-5), 183.216 (2 x CO); Examination of 2D
1
H-NMR (COSY)
(CDCl
3
) (figure 19) and
1
H,
13
C-NMR (C,H correlation) (CDCl
3
) (figure 20)
provided further confirmation for the structure of compound 8d.

5.2.8. Di-O-(2-Thienoyl)curcumin (8e) and di-O-(2-thienoyl)ethyl curcumin (8f)
-OCO
O O
OCO-
R
1
O OR
1
S S

(8e) R
1
= CH
3

(8f) R
1
= C
2
H
5


Compounds 8e,f (Table 6) were prepared by adding thiophene-2-carbonyl
chloride (0.03 mol) adopting the previously mentioned procedure.
IR of 8e (cm
-1
): 3339.6 (OH intramolecularly H-bonded), 1746.7 (C=O, ester),
1672.9 (C=O, ketone), 1606.8, 1494.5 (C=C aromatic), 1278.3, 1122 (
as
and
s
C-O-
122
C);
1
H-NMR of 8e (DMSO) ppm (500 MHz) (figure 21): 3.861 (s, 6H, 2 x OCH
3
),
6.232 (s, 2H, H
a
), 7.051 (d, 2H, 2 x H
b
, J = 16 Hz), 7.32, 7.336 (dd, 2H, 2 x H
B
, J =
3.5 Hz and 7.8 Hz, meta and ortho coupling), 7.396 (d, 2H, 2 x H
A
, J
AB
= 7.5 Hz,
ortho coupling), 7.588 (s, 2H, 2 x H
X
), 7.697 (d, 2H, 2 x H
c
, J = 16 Hz), 7.856 (d,
2H, 2 x thienyl-5-H, J = 8 Hz), 8.037, 8.059 (dd, 2H, 2 x thienyl-4-H), J = 1 Hz and 4
Hz ortho and meta coupling), 8.112 (d, 2H, 2 x thienyl-3-H, J = 4 Hz).
IR of 8f (cm
-1
): 3431.1 (OH intramolecularly H-bonded), 1727 (C=O, ester),
1626.5 (C=O, ketone), 1596.7, 1508.7 (C=C aromatic), 1250.7, 1117.2 (
as
and
s
C-
O-C);
1
H-NMR of 8d (DMSO-d
6
) ppm (300 MHz): 1.2351 (t, 6H, 2 x 3 OCH
2
CH
3
,
J = 7.00 Hz), 4.1412 (q, 4H, 2 x OCH
2
CH
3
, J = 7.04 Hz), 6.2138 (s, 2H, H
a
), 7.0269
(d, 2H, 2 x H
b
, J = 15.93 Hz), 7.3222 (d, 2H, 2 x H
A
, J
AB
= 8.52 Hz, ortho coupling),
7.3364 (s, 2H, 2 x H
X
), 7.67525 (d, 2H, 2 x H
c
, J = 15.93 Hz), 7.3845 (d, dist, 2H, 2 x
H
B
, J
AB
= 8.52 Hz, ortho coupling), 7.5690 (d, dist, 2H, 2 x thienyl-H-4), 8.0264 (br,
2H, 2 x thienyl-H-5, J = 3.57 Hz), 8.1015 (d, 2H, 2 x thienyl-H-3, J = 3.57 Hz);
13
C-
NMR of 8f (DMSO) ppm (500 MHz): 40.13 (2 x CH
3
), 64.95 (2 X OCH
2
), 102.31
(C-4), 113.79 (2 x C-2'), 121.94 (2 x C-5'), 123.94 (2 x C-6'), 125.22 (2 x C-1'),
129.26 (C-2 + C-6), 131.84 (thienyl-C-4), 134.46 (thienyl-C-3), 135.72 (thienyl-C-5),
135.83 (thienyl-C-2), 140.30 (C-1 + C-7), 141.37 (2 x C-3'), 151.02 (2 x C-4'),
159.88 (C-3 + C-5), 183.69 (2 x CO); MS of 8f (figure 22) m/z (% relative
abundance): M
+
616 (0.7), 604 (1.6), 588 (1.3), 561 (0.8), 510 (1.3), 490 (0.6), 458
(0.9), 431 (0.9), 417 (2.6), 399 (0.9), 371 (0.5), 344 (1.2), 320 (1.6), 314 (3.5), 295
(0.9), 262 (0.6), 247 (1.3), 219 (1), 203 (2.3), 193 (1.7), 173 (1.7), 163 (3), 161 (3),
143 (57), 138 (11), 125 (8), 121 (6), 119 (7), 111 (13), 107 (9), 105 (8.5), 99 (4), 97
(3), 95 (13), 93 (11.5), 91 (11.5), 91 (13), 85 (29), 83 (26.6), 81 (8.5), 79 (10), 77
(6.3), 69 (15), 67 (16.2), 45 (18.4), 44 (68.6), 43 (100).
123

Table 6: Physicochemical Data of the Synthesized Compounds 8a-f

O
OR
1
O
R
1
O
O O
R
2
R
2
O O


(8a-f)

Cpd
No
R
1
R
2
Reaction
Time (h)
Yield
(%)
m.p. M. Formula M.wt

8a

CH
3




9

47

215

C
43
H
48
O
8


692

8b

C
2
H
5




16

36

220

C
45
H
52
O
8


720

8c

CH
3


C
6
H
13


4-5

63

99.5

C
35
H
44
O
8


606

8d

C
2
H
5


C
6
H
13



4-5

77

128-30

C
37
H
48
O
8


620

8e

CH
3

S



4

81.5

199-203

C
31
H
24
O
8
S
2


588

8f

C
2
H
5

S



6

51

217-19

C
33
H
28
O
8
S
2


616


124
5.3. Anticancer Screening

5.3.1. Materials and Methods

5.3.1.1. Cytotoxicity to Mammalian Cells

The cytotoxic activity of the tested compounds 1, 2, 3a, 3c, 3d, 5a-k, 6a-h, 6m,n,
7a, 8a, 8c-f was determined against human cancer cell line SK-MEL (malignant,
melanoma), KB (epidermal carcinoma, oral), BT-459 (ductal carcinoma, breast), and
SK-OV-3 (ovary carcinoma). Vero cells, derived from monkey kidney fibroblasts,
and LLC-PK1, from pig kidney epithelial tissue, were used representing noncancerous
cells. The assay was performed in 96-well tissue culture treated microplates according
to the neutral red staining procedure as modified by Borenfreund and Peurner
[193]
. The
results are reported in Table 7 and represented in Charts 8-11.
125
Table 7: Cytotoxicity of the Synthesized Compounds to Mammalian
Cells

IC 50 value (M)*
Compd No
CANCER CELLS
NONCANCER
CELLS

SK-MEL KB BT-549 SK-OV-3 VERO LLC-PK1
1 13.75 >25 10.5 NA NC NC
2 16 >25 18.0 NA NC NC
3a 12.5 >25 10.0 NA NC NC
3c NA NA NA NA NC NC
3d NA NA NA NA NC NC
5a 15.0 >25 19.4 NA NC NC
5b 15.0 23 18.1 NA NC 22.5
5c 7.5 22.5 8.75 NA 23.0 22.5
5d NA NA NA NA NC NC
5e 7.0 19.5 6.75 NA >25 20.0
5f 15.0 NA 20.0 22.5 NA NC
5g 8.5 24.0 16.3 15.5 19.5 NC
5h 18.5 >25 20.50 NA NC NC
5i 23.8 >25 23.75 NA NC NC
5j 7.5 16.3 4.25 NA 15.0 15.5
5k 15.0 11.5 11.00 23.8 12.5 16.8
6a NA NA NA NA NC NC
6b 4.75 NA NA 2.8 NC NC
6c 12.5 11.5 12.00 15.8 11.5 13.0
6d 13.3 12.5 9.00 NA 11.0 6.8
6e 14.5 21.5 11.0 13.5 15.0 13.8
6f NA NA 11.50 NA NC 22.0
6g 11.5 11.5 10.00 14.8 5.0 5.8
6h NA 15 NA NA NC NC
6m NA NA NA NA NC NC
6n NA NA NA NA NC NC
7a 18.8 14 10.5 22.5 11 15.75
8a NA NA NA NA NC NC
8c NA NA NA NA NC NC
8d NA NA NA NA NC NC
8e NA NA NA NA NC NC
8f NA NA NA NA NC NC
Doxorubicin
0.55 <0.55 <0.55 0.8 >5.0 0.75

*Stock solution = 5mM in DMSO; Test concentrations = 25, 8.33, 2.78 M
NA = not active up to 25 M.
NC = not cytotoxic up to 25 M

126
SK-MEL = Human malignant, Melanoma
KB = Human Epidermal Carcinoma, Oral
BT-549 = Ductal Carcinoma, Breast
SK-OV -3 = Human Ovary carcinoma
Vero = Monkey Kidney Fibroblasts
LLC-PK1 = Pig Kidney Epithelial




































127
0
5
10
15
20
25
30
d
o
x
o
r
u
b
i
c
i
n 1 2
3
a
3
c
3
d
5
a
5
b
5
c
5
d
5
e
5
f
5
g
5
h 5
i
5
j
5
k
6
a
6
b
6
c
6
d
6
e
6
f
6
g
6
h
6
m
6
n 7
8
a
8
c
8
d
8
e
8
f
SK-MEL


Compound number

Chart 8: Cytotoxic Effect of the Synthesized Compounds to SK-Mel Cells

: Most Cytotoxic Compounds
: Less cytotoxic Compounds
: Inactive Compounds















IC
50

128
0
5
10
15
20
25
30
d
o
x
o
r
u
b
ic
in 1 2
3
a
3
c
3
d
5
a
5
b
5
c
5
d
5
e
5
f
5
g
5
h 5
i
5
j
5
k
6
a
6
b
6
c
6
d
6
e
6
f
6
g
6
h
6
m
6
n 7
8
a
8
c
8
d
8
e
8
f
KB

Compound number


Chart 9: Cytotoxic Effect of the Synthesized Compounds to KB Cells

: Most Cytotoxic Compounds
: Less cytotoxic Compounds
: Inactive Compounds





















IC
50

129
0
5
10
15
20
25
30
d
o
x
o
r
u
b
ic
in 1 2
3
a
3
c
3
d
5
a
5
b
5
c
5
d
5
e
5
f
5
g
5
h 5
i
5
j
5
k
6
a
6
b
6
c
6
d
6
e
6
f
6
g
6
h
6
m
6
n 7
8
a
8
c
8
d
8
e
8
f
BT-549

Compound number

Chart 10: Cytotoxic Effect of the Synthesized Compounds to BT-549 Cells

: Most Cytotoxic Compounds
: Less cytotoxic Compounds
: Inactive Compounds















IC
50

130

0
5
10
15
20
25
30
d
o
x
o
r
u
b
ic
in 1 2
3
a
3
c
3
d
5
a
5
b
5
c
5
d
5
e
5
f
5
g
5
h
5
i
5
j
5
k
6
a
6
b
6
c
6
d
6
e
6
f
6
g
6
h
6
m
6
n 7
8
a
8
c
8
d
8
e
8
f
SK-OV-3

Compound number


Chart 11: Cytotoxic Effect of the Synthesized Compounds to SK-OV-3 Cells

: Most Cytotoxic Compounds
: Less cytotoxic Compounds
: Inactive Compounds






IC
50

131
5.3.1.2. Interaction with Topoisomerases

Measurement of the catalytic activity of topoisomerase I (topo I) was based on the
conversion of supercoiled DNA to relaxed DNA. Cleavage complex stabilization was
assayed by measuring the nicked DNA produced in the presence of the tested
compounds. Human topo I and the topo I drug kit were purchased from Topogen Inc.
(Columbus, Ohio). A supercoiled plasmid DNA (pHOT1) with a high affinity topo I
recognition element was used as substrate. Enzyme activity was assayed in a total
volume of 20 l containing 250 ng of DNA, test compound, 2 to 4 U of purified
enzyme, 10 mM EDTA, 0.15 mM NaCl, 0.1% bovine serum albumin (BSA), 0.1 mM
spermidine, and 5% glycerol. The reaction mixture was incubated at 37
o
C for 30 min.
Reactions were then terminated by the addition of 1% sodium dodecyl sulfate (SDS)
followed by treatment with proteinase K (50 g/ml) at 37
o
C for 30 min. DNA was
extracted with chloroform-isoamyl alcohol (24:1 v/v) and analyzed by electrophoresis
on a 1% agarose gel in TAE buffer (40 mM Tris acetate, 2 mM EDTA, pH 8.5). The
gel was stained with ethidium bromide, destained in water, and photographed on a
UV transilluminator followed by a densitometric analysis using NIH image software
1.52. Enzyme activity was measured as a percentage of substrate DNA converted to
product. The concentration of test compound that prevented 50% of the substrate
from being converted into product (IC
50
) was calculated.
For the determination of cleavage complex formation activity with topo I, the
assay was performed with a minimum of 4U of purified enzyme as described above,
except that ethidium bromide was included in both the agarose gel and running buffer
to resolve nicked DNA from supercoiled or relaxed species. Drug-induced
stabilization of the cleaved complex was determined in terms of the percent nicked
DNA produced.
132
The relaxation activity of Topo II was analyzed in the same manner, except that
the reaction mixture contained 10 mM Tris-HCl (pH 7.9), 50 mM NaCl, 50 mM KCl,
5 mM MgCl
2
, 0.1 mM EDTA, 15 g/mL bovine serum albumin (BSA), 1 mM ATP
and 4 units Topo IIa. Decatenation of kDNA (TopoGEN Inc.) was performed in the
same condition except that supercoiled plasmid DNA was substituted by 400 ng
kDNA.
All compounds tested 1, 2, 3a, 3c, 3d, 5a-k, 6a-e, 6m,n, 7a, 8a, 8c-f were
dissolved in DMSO provided that the DMSO concentration did not exceed 2.5% in all
the assays and a DMSO control was always included. The results are reported in
Table 8.


























133
Table 8: Anticancer Activity of the Synthesized Compound (Inhibition
of Topoisomerase Activity)

IC 50 value (M)
Sample
Topoisomerase I Topoisomerase II

cleavage* catalytic** cleavage* catalytic**
1 NA NA ND 15
2 NA NA ND 7.5
3a NA NA ND 20
3c NA NA ND 12
3d NA NA ND 20
5a NA NA ND 4
5b NA NA ND 15
5c NA NA ND 15
5d NA NA ND 0.35
5e NA NA ND 5
5f NA NA ND 16
5g NA NA ND 3
5h NA NA ND 18
5i NA NA ND 19
5j NA NA ND 3
5k NA NA ND 2.9
6a NA NA ND 3.4
6b NA NA ND 12
6c NA NA ND 2.7
6d NA NA ND 4.1
6e NA NA ND 3.4
6f NA NA ND 4
6g NA NA ND 10
6h NA NA ND 2.1
6m NA NA ND NA
6n NA NA ND 7.1
7a NA NA ND 3
8a NA NA ND 4.5
8c NA NA ND 2.9
8d NA NA ND 3
8e NA NA ND 8
8f NA NA ND 23

ND = not determined NA = not active up to 25 M.
*Cleavage complex stabilization activity (similar to camptothecin for topo I and
etoposide for topo II).
**Inhibition of catalytic activity of topoisomerase i.e. relaxation of
supercoiled DNA by topoisomerase I and decatenation of KDNA by
topoisomerase II.
134
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