Master Document Thesis Reem

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

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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. Chemistry……………………………………………………………………42

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

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4.1.2.1 Method A………………………………………………………....54 4.1.2.2 Method B…………………………………………………………56

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

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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,

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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: 13C 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-chloroacetyloxybenzaldehyde (4a),………………………………………..………........57 Figure 7: 1 H NMR spectrum of 3-ethoxy-4-chloroacetyloxybenzaldehyde (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: 1HNMR spectrum of 1,7-bis(4-adamantylaminoacetyloxy)- 3-(methoxyphenyl)-1,6-heptadiene-3,5-dione (5a),……………………65 Figure 12: 1H NMR spectrum of 1,7-bis(4-bis(2-chloroethyl)aminoacetyloxy)-3- (methoxyphenyl)-1,6-heptadiene-3,5- dione (5e),……...68 Figure 13: 1H NMR spectrum of 1,7-bis(4-adamantylaminopropionyloxy)-3-(methoxyphenyl)-1,6-heptadiene-3,5-dione (5h),..........................69 Figure 14: Mass spectrum of 1,7-bis(4-adamantylaminopropionyloxy)-3-(methoxyphenyl)- 1,6-heptadiene-3,5-dione (5h),………………70 Figure 15: Mass spectrum of 1,7-bis(4-(methylthiadiazolyl)aminopropionyloxy)-3- (methoxyphenyl)-1,6-heptadiene-3,5- dione (5j),.........72 Figure 16: Mass spectrum of di-O-Adamantoylcurcumin (8a),...............................76 Figure 17: 1H NMR spectrum of di-O-heptanoylethyl curcumin (8d),……………..80

Figure 18: 13C NMR spectrum of di-O-heptanoylethyl curcumin (8d),…………….81 Figure 19: 2D 1H NMR (COSY) spectrum of di-O-heptanoylethyl curcumin (8d),…………………………………………………………..81

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Figure 20: 1H, 13C NMR (C,H correlation) spectrum of di-O-heptanoyl-

ethyl curcumin (8d),……………………... …………………………….82

Figure 21: 1H NMR spectrum of di-O-(2-Thienoyl)curcumin (8e),……..………….84 Figure 22: Mass spectrum of di-O-(2-thienoyl)ethyl curcumin (8f),………………..85

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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

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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

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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

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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.

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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-

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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, 1H-NMR, 13C-NMR and mass spectroscopy.

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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-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-(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-(ethoxyphenyl)-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.

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2. Introduction

2.1. Overview

Curcumin (1), [diferuloylmethane, 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-

heptadiene-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

H3CO OCH3

HO OH

OH O

H3CO OCH3

(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 β-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 β-diketone

moiety in the antioxidant mechanism of curcumin and its derivatives[35,36] . A

hydrogen atom donation from the β-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 β-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 β-diketone moiety alone does not have

antioxidant properties. Apparently, the presence of both β-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 β-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-KB) 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 [3H]arachidonic acid into macrophage lipids by

increasing the secretion of 6-keto-PGF1α [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-

α/lipopolysaccharide (TNF/LPS)-activated HIMECs through blockade of NF-кB 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-KB) 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 β-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].

R1

HO

O O

R2

OH

(1) R1= R2 = OCH3 (curcumin)

(2) R1= H, R2 = OCH3 (desmethoxycurcumin)

(3) R1= R2 = H (bis-desmethoxycurcumin)

Desmethoxycurcumin (2) was recently found to be selectively active against KB

cells (nasopharynx epidermoid carcinoma) with an ED50 value of 1µg/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

OHHO

(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)

OHO

(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).

OCH3

OHHO

H3CO

OCOCH3H3COCO

(10)

OCH3

OHHO

H3CO

O OCOCH3

(11)

H3CO

HO

O

OCH3

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 = Glc6_1Api

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 IC50 of 41.3µg/ml and >50 µg/ml, respectively[115].

HO

OCH3

OH

O HO H

(15)

HO

OCH3

OH

O OCH3

(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 (1ζ)-

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

H3CO

HO

OCH3

OH

OH OH

(17)

H3CO

HO

O O

OCH3

OH

OH

(18)

H3CO

HO

O O

OCH3

OH (19)

HO

OCH3

OH

O OHH3CO

(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 LTB4 biosynthesis or

anti-inflammatory activity.

OH3CO

(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].

H3CO OCH3

O O

H

H3CO OCH3

(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].

H3CO OCH3

O O

OCH3H3CO

(24)

20

H3CO OCH3

O O

OCH3H3CO

CH3

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].

H3CO

H3COCO

O

OCH3

OCOCH3

OH

(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 β-diketone in a cyclic carbon chain appears

to play an important role for cytotoxicity in this class of compounds[65].

OCH3

OHHO

H3CO

O OH

(27)

21

H3CO

HO

OH OH

OCH3

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

SH3C

SCH3

OH

OCH3

HO

OCH3

(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 IC50 = 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

R2

OH

R1

HO

(30)

(a) R1 = R2 = OCH3

(b) R1 = OCH3, R2 = H

(c) R1 = R2 = H

N N

R2

OH

R1

HO

O OH

(31)

(a) R1 = R2 = OCH3

(b) R1 = OCH3, R2 = H

(c) R1 = R2 = H

N NH

OCH3

OH

H3CO

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

OCH3

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.

R3

OR2

OH

R4

R1R2

R3

R1

R4

(36)

R1 R2 R3 R4

(a) H CH3 OCH3 CH3

(b) H C2H5 OH C2H5

(c) H i-C3H7 OH i-C3H7

(d) OCH3 H H H

(e) H H Cl H

(f) H t-C4H9 OH t-C4H9

Furthermore, some curcumin bioconjugates, namely di-O-glycinoyl curcumin

(37), di-O-glycinoyl-C4-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 β-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.

H3CO

H2N-H2C-OC-O

OCH3

O-CO-CH2-NH2

OO

(37)

H3CO

H2N-H2C-OC-O

OCH3

O-CO-CH 2-NH2

OO

CO-CH2-NH2

(38)

H3CO OH

H3CO 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

R4 R4R3

R2

R1 R1

R2

R3

O OH

(41)

R1 R2 R3 R4

(a) F H H H

(b) H F OCH3 H

(c) H CF3 F H

(d) H H OCF3 H

(e) F H F H

(f) F H OCH3 H

(g) F H H OCH3

Glycosidation[128] of curcumin has recently been reported by acetobromoglucose

in the presence of triethylbenzylammonium bromide (Et3BnN+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

H3CO OCH3

OH

O OH

O

HO

HOOH

HOH2C

(42)

O

H3CO OCH3

O

O OH

O

HO

HOOH

HOH2COHO

OH

OH

CH2OH

(43)

27

OHO

R1

OR 2

R1

HO

(44) (a) R1 = OCH3, R2 = Glc-Ac (b) R1 = H, R2 = Glc-Ac (c) R1 = OCH3, R2 = Gal-Ac

O OH

OCH 3

OGlc

H3CO

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 3OH H3CO

X

(46) X = H, OH

(47) X = O

28

OH

H3CO

HO

H3CO

OH

O (48)

OH HO

O

OH OHHO

(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

H3CO

HOH

(50)

29

OHO

H3CO

H OH

(51)

During the course of some studies on bio-active constituents of natural medicines

and medicinal foodstuffs, cyclic diarylheptanoids, namely acerosides B1 (52), B2 (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].

OO

OH

O

H

OH

OH

OH

HO

(52)

OOO

H

OH

OH

OH

HO

OH

(53)

30

O

HO

O

O

O

OOH

OH

O

OHOH

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

β-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].

OHO

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.

H3CO

Glu-O

O

HO

O

O-GluH3CO

H3CO

HO

OH

O

(57) (58)

H3CO

H3CO OH

HOH

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].

H3CO

HO

HO

H3CO

OCH3

OH

OOH

OH O

OH

OCH3 (60)

32

H3CO

O

O

H3CO

OCH3

OH

OOH

OH O

OH

OCH3

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

OO

(62)

R R

R1

O O

(63)

(a) R = H, R1 = H2 (d) R = F R1 = H2

(b) R = Cl, R1 = H2 (e) R = H R1 = Br

(c) R = C(CH3)3 R1 = H2 (f) R = H R1 = NO

33

O O

O O

(64)

O O O

R R

(65)

(a) R = H

(b) R = Cl

The β-diketone (63a) displayed moderate cytotoxicity, whereas the β-triketone

(65a) and the β-tetraketone (64) were inactive. The 4-chlorophenyl derivative (63b)

was also more active than the corresponding triketone (65b). It would appear that the

β-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). β-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 PGE2 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 PGE2 production. It also inhibited significantly the

formation of edema in the carrageenan-induced model of inflammation in mice by the

oral route[136].

O

(H3C)2N

OCH3

OCH3

(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.

R1

O

R3

O

R2 R1

O

R3

OH

R2 (68) (69)

R1

OH

R3

OH

R2 (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].

H3CO

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'

OXN

O

RR'' 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

R1O OR1

O O

curcumin skeletoncarbonyl group

R2

N-(CH2)xR3

(CH2)x-NR2

R3

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

R1O OR1

O

R2

O

R2


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

CHOR1O

+

OO

B2O3

(BuO)3B/

n-BuNH2

R1O

O O

OR1

OHHO

(1) R1 = CH3 (2) R1 = C2H5

O

Cl (CH2)n

Cl anh. K2CO3/

O

/ RT

R1O

O O

OR1

OO (CH2)n

OO

n(H2C)Cl Cl

(3 a-d)

OO

B2O3/ (BuO)3B/ n-BuNH2

HO

CHOR1O

Cl

O

(CH2)n

Cl+

1N NaOH/ CHCl3 or

O

CHOR1OO

n(H2C)Cl

(4 a-d)anh. K2CO3/

O

Scheme 1

2

2

40

R1O

O O

OR1

OO (CH2)n

OO

n(H2C)Cl Cl

(3 a-d)

Scheme 2

R2R3NH NEt3/

O

NH2- -SO2NHR4

R1OO O

OR1

OO (CH2)n

OO

n(H2C)R3R2N NR2R3

R1OO O

OR1

OO (CH2)n

OO

n(H2C)NH- -SO2NHR4-NHR4NHSO2-

(5 a-n, 6a-n)

(7a-f)

41

R1O

HO

O O

OR 1

OH

Scheme 3

(1), (2)

COCl

R1O

O

O O

OR 1

O-

R1O

O

O O

OR 1

O-CO- CO

(8a, b)

S CO- SOC

(8e, f)

Na2CO3/

O

S COCl

Na2CO3/

O

Na2CO3/

O

CH3(CH 2)5COClR1O

H3C(H 2C)5OCO

O O

OR 1

OCO(CH 2)5CH 3

(8c, d)

42

4. Results and Discussion

4.1. Chemistry

Compounds 3a-d; namely: di-O-chloroacetylcurcumin (3a), di-O-

chloropropionylcurcumin (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

HaRBase

- Ha

O-

R

O

R -

A B

+Ar

O

HB R

O

H HH

O-

Ar - H2OR

O

HAr

H

D EC

+ H+

Equation 1

Variation of the reaction conditions involved also the use of concentrated HCl at

25-30°C[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):

CHORO RO

O O

OR

OHHO HO

NaOH/EtOH, NaOEt/EtOH

or c.HClXXX

O O+

1121

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 1H-NMR.

OR

OCO(CH2)nNHR2

RO

R2HNn(H2C)OCO

O O

CO

(CH2)nNHR2

11

(I)

44

R1O

R2HNOCHNO2SO

O O

OR1

OSO2NHCONHR2SO2

NHCONHR2 (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:

H3CO

O O

OCH3

OHHO

HO

COOHH3CO

HO

H3CO

O

+

H3CO

HO

CHO

+ CH3COCH3

Ferulic acid

Feruloylmethane

acetonevanillin

NaOH

NaOH

Equation 3

Cleavage of the β-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

H3CO

O O

OCH3

OHHO

CH3H3C

OO OB

O

O O

CH3H3C

H3C CH3

O

HHO

OCH3

+ B2O3 + BO2- + H2O

(A)

n-BuNH2

OB

O

O O

CH=CHHC=HC

HC=HC CH=CH

OH

OCH3

HO

OCH3 OCH3

O+H

H3CO

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):

R1O

HO

OR1

OH

O O

R1O

HO

CHO

+

O O

/ B2O3

(BuO)3B/ n-BuNH22

47

(1) R1 = CH3 ; (2) R1 = C2H5

Equation 5

Curcumin (1) was obtained from vanillin in 73% yield and its structure was

confirmed by IR, 1H-NMR and 13C-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 νas and νs C-O-C.

1H-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 δ 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

H3CO OCH3

HO OH

OH O

H3CO OCH3

(a) (b)

(1)

OCH3

OH

H3CO

HO

O OHc

Hb Ha Hb

Hc

H

(1b)

48

The Ha proton resonated as a singlet at δ 6.0618 ppm. Two doublets were assigned

for 2 Hb and 2 Hc protons at δ 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.

HX

HA

HB

The spectrum of curcumin showed 2 doublets arising from ortho couplings with

JAB value of ~ 8 Hz[150]. The expected doublet of doublets for HB proton resulting

from ortho and meta couplings was not observed. The para coupling JAX was also

unresolved and not visible[151]. Therefore, the signal for HX proton was detected as a

singlet at δ 7.5470 ppm.

49

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

In 13C-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-

OCH3, 12 Ar-C and 7 C1-C7 carbons, including characteristic peaks of 2 carbonyl

groups, of the heptadienedione chain.

H3CO

HO

O O

OCH3

OH

7

65

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, 1H-NMR,

13C-NMR and MS.

As for curcumin, its 1H-NMR spectrum (figure 2) lacked the typical doublet of

doublets characteristic for the HB proton in 1,2,4-trisubstitued benzene ring pattern;

para coupling was also unobservable. However, the spectrum showed a triplet and a

quartet at δ 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 Ha, Hb and Hc respectively.

50

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

13C-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: 13C-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

C23 H24O6

M+ 396 (23)

O

O

+

HO C13H17O2

m/z 205 (26)

O

C11H13O2

m/z 177 (48)

O+

+O

O

C10H11O2

m/z 163 (78)

O

O

H

C11H12O2

m/z 176 (42)

+

+

+

C10H11O

m/z 147 (47)

O +

C9H9

m/z 117 (48)

+

m/z 107 (33)

HO HO

+

C7H7O

O O

m/z 123 (38)

C7H7O2

+

OH

m/z 110 (30)

C7H10Om/z 89 (100)

C7H5

++

+

m/z 65 (57)

C5H5

CH3 C O+

m/z 43 (57)

C2H3O

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

H3CO

O O

OCH3

OO

ClCO(CH2)nCl/

Na2CO3

1O

(CH2)n

ClO

(CH2)nCl

(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. H2SO4[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 Na2CO3 was preferred (Scheme

1).

Thus, reacting a cold solution of curcumin in dry acetone containing Na2CO3 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, 1H-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 νas

and νs C-O-C characteristic for curcumin.

1H-NMR spectrum (figure 5) of di-O-chloroacetylcurcumin (3a) showed 3

consecutive singlets assigned for the OCH3, CH2-Cl and Ha protons and 2 doublets

resonating at δ 6.56905 and 7.6116 ppm assigned for Hb and Hc 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 Ha was due to 2 protons.

1H-NMR spectrum of di-O-chloropropionylcurcumin (3b) showed 2 characteristic

triplets at δ 3.1223 and 3.8952 ppm, with coupling constant J ~ 6.3 Hz, assigned for

CH2-Cl and COCH2 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: 1H-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-chloroacyloxybenzaldehyde (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 K2CO3[156] instead of

CHCl3 and NaOH gave the same products with high purity and yield. Compounds 4a-

d were identified by IR and 1H-NMR.

HO

R1O CHO CHO

O

R1O

Cl

O

Cl(CH2)n

+O

(CH2)nCl

1N NaOH/ CHCl3 or

K2CO3/ acetone

4a-d

57

(4a) R1 = CH3 n = 1; (4b) R1 = CH3 n = 2

(4c) R1 = C2H5 n = 1; (4d) R1 = C2H5 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 νas and νs C-O-C functional groups.

1H-NMR spectrum of 3-methoxy-4-chloroacetyloxybenzaldehyde (4a) (figure 6)

showed 2 consecutive singlets at δ 3.8445 and 4.7157 ppm assigned for OCH3 and

CH2Cl protons. Signals on the aromatic region showed coupling patterns due to

1,2,4-trisubstituted benzene ring: HA proton appeared as a doublet due to coupling

with the ortho HB proton with JAB = 8.4 Hz. HB Proton was shown as a doublet of

doublets with J values of 7.65 Hz (ortho coupling with HA) and 1.55 Hz (meta

coupling with HX). HX proton resonated as a doublet and the para coupling, JAX =

1.55 Hz, was well resolved and well visible. A downfield singlet was assigned for the

aldehydic proton at δ 9.9476 ppm.

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

58

1H-NMR spectrum of 3-ethoxy-4-chloroacetyloxybenzaldehyde (4c) (figure 7)

was characterized by the appearance of a triplet and a quartet at δ 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 CH2-Cl protons resonated as a singlet at

its expected chemical shift δ 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. HA proton appeared as a doublet due to coupling with the ortho HB

proton with JAB = 7.98 Hz. HB Proton was shown as a doublet of doublets with J

values of 8.07 Hz (ortho coupling with HA) and 1.65 Hz (meta coupling with HX). HX

proton resonated as a doublet and the para coupling, JAX = 1.63 Hz, was well resolved

and well visible. A downfield singlet was assigned for the aldehydic proton at δ

9.9709 ppm.

CHO

OCH2CH3

ClH2COCO

59

Figure 7: 1H-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-chloroacylcurcumin (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.

R1O

O

OR1

O

O O

R1O

O

CHO

+

O O

/ B2O3

(BuO)3B/ n-BuNH2Cl

(CH2)n

O

Cl(CH2)n

O O

(CH2)nCl

(4a-d)

2

(3a-d)

(3a) R1 = CH3, n = 1; (3b) R1 = CH3, n = 2

(3c) R1 = C2H5, n = 1; (3d) R1 = C2H5, n = 2

Compounds 3a-d were identified by IR, 1H-NMR and by MS for 3c.

IR and 1H-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 νas and νs C-

O-C characteristic for curcumin derivatives.

1H-NMR spectrum (figure 8) of di-O-chloroacetylethyl curcumin (3c) showed a

triplet and a quartet at δ 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 CH2-Cl and Ha protons

and 2 doublets resonating at δ 6.5553 and 7.605 ppm, J = 15.93 Hz, assigned for Hb

and Hc 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 Ha was due to 2 protons.

Figure 8: 1H-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

C27H26O8Cl2

M+ 548, M+ +2 550, M+ +4 552

+

O

O

O

O O

O

O

O

C27H30O8

+

m/z 482 (0.24)

+O

O O

C19H20O3

m/z 296 (0.14)

O

O

O

Cl

O

O

+

O

HO

m/z 242 (0.6), 244 (0.26)

C12H15O2

m/z 191 (0.17)

C10H14O2

m/z 166 (2)

++

C12H15O3Cl

OH O

H

OO+ O

++

or

+

C4H5O2

m/z 85 (69)C4H7O2

m/z 87 (12)

C6H11

m/z 83 (100)

C5H7O

Chart 2: Possible Fragmentation Pattern for compound 3c

62

1H-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 CH2CH2-Cl protons. Protons Ha,

Hb and Hc 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: 1H-NMR spectrum of di-O-chloropropionylethyl curcumin (3d)

63

4.1.3. 2-Chloroethylamine monohydrochloride and bis(2-chloroethyl)-

amine hydrochloride

NH2CH2CH2OHSOCl2/dry C6H6

NH2CH2CH2Cl

SOCl2/dry C6H6HN

CH2CH2OH

CH2CH2OHHN

CH2CH2Cl

CH2CH2Cl

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 (SOCl2), phosphoryl chloride (POCl3),

phosphorus trichloride (PCl3) or phosphorus pentachloride (PCl5). In the present

work, SOCl2 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

OR1

O

R1O

O O

R2R3NH/reflux

(3a-d) Et3N / EtOH

(CH2)n

O O

(CH2)nCl Cl

O

OR1

O

R1O

O O

(CH2)n

O O

(CH2)nR3R2N NR2R3

(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], triethylamine[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 νas and νs 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.

1H-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 δ 7.3099 ppm

assigned for 2 NH. It also showed 3 consecutive singlets assigned for CH2-, -OCH3

and Ha protons and 2 doublets resonating at δ 6.7444 and 7.5315 ppm, with J = 15.66

Hz, assigned for Hb and Hc respectively. Signals on the aromatic region showed

coupling patterns typical of 1,2,4-trisubstituted benzene ring. HA Proton appeared as a

doublet due to coupling with the ortho HB proton with JAB = 8.25 Hz. HB Proton was

shown as a doublet of doublets with J values of 8.22 Hz (ortho coupling with HA) and

1.5 Hz (meta coupling with HX). HX Proton resonated as a doublet and the para

coupling, JAX = 0.24 Hz, was not well resolved.

66

Figure 11: 1H 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

C45H54N2O8

+

M+ 750 (absent)

C7H7O

m/z 107 (33)

HN

HN

O O+

O

+

C5H3O C4H3O

m/z 81 (39) m/z 79 (43) m/z 67 (7)

HO + HO

+

C6H5O

m/z 93 (36)

+O

+

C10H15

m/z 135 (100)

O

+ or

+

C6H5

m/z 77 (36)

+

C5H5O

Chart 3: Possible Fragmentation Pattern for compound 5a

1H-NMR spectrum of compound 5b showed a downfield D2O-exchangeable

singlet at δ 9.6925 ppm assigned for NH. It also showed 3 consecutive singlets

assigned for CH2-, -OCH3 and Ha protons and 2 doublets resonating at δ 6.7646 and

7.5466 ppm, with J = 15.66 Hz, assigned for Hb and Hc respectively. Signals on the

aromatic region showed coupling patterns typical of 1,2,4-trisubstituted benzene ring.

HA Proton appeared as a doublet due to coupling with the ortho HB proton with JAB =

8.25 Hz. HB Proton was shown as a doublet of doublets with J values of 8.25 Hz

(ortho coupling with HA) and 1.65 Hz (meta coupling with HX). HX Proton resonated

68

as a doublet and the para coupling, JAX = 1.65 Hz, was not well resolved. Protons on

the benzothiazole rings were highly deshielded and were shown as a multiplet.

1H-NMR spectrum of compound 5c showed 3 consecutive singlets assigned for

COCH2N-, CH3 and -OCH3 respectively. Protons Ha, Hb and Hc 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 δ 9.685 ppm assigned for 2 NH protons which were

D2O-exchangeable.

1H-NMR spectrum of compound 5d showed a high field signal at δ 2.202 ppm

assigned for 2 NH protons. 2 Consecutive triplets were assigned for the 8 protons of

CH2-CH2 moieties. 2 Singlets assigned for COCH2N- and -OCH3 were also shown.

Protons Ha, Hb and Hc 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.

1H-NMR spectrum of compound 5e (figure 12) showed 3 consecutive singlets

assigned for COCH2N-, -OCH3 and Ha protons. It also showed 2 triplets at δ 2.5042

and 3.4841 ppm, with J = 3.57 Hz, and assigned for NCH2CH2-Cl and NCH2CH2-Cl

respectively, the later being overlapped by the solvent signal. Two doublets

resonating at δ 6.7718 and 7.5736 ppm, with J = 15.9 Hz were assigned for Hb and Hc

respectively. Signals on the aromatic region showed coupling patterns typical of

1,2,4-trisubstituted benzene ring. HA proton appeared as a doublet due to coupling

69

with the ortho HB proton with JAB = 8.25 Hz. HB Proton was shown as a doublet of

doublets with J values of 8.37 Hz (ortho coupling with HA) and 1.65 Hz (meta

coupling with HX). HX Proton resonated as a doublet and the para coupling, JAX =

1.38 Hz, was well resolved and well detected.

Figure 12: 1H NMR spectrum of 1,7-bis(4-bis(2-chloroethyl)aminoacetyloxy)-3-

(methoxyphenyl)-1,6-heptadiene-3,5-dione (5e)

1H-NMR spectrum of compound 5f showed 2 new high field singlets assigned for

2 x N(CH3)2 and 2 NH protons, the latter overlapping with OCH2 singlet. 2

Consecutive triplets were assigned for the 8 protons of CH2-CH2 moieties. 2 Singlets

assigned for COCH2N- and -OCH3 were also shown. Protons Ha, Hb and Hc 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

δ 9.7368 ppm was assigned for the enolic OH proton.

1H-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 δ 3.882 ppm, overlapping with singlet of OCH3, assigned for 2 NH. It also

showed 2 consecutive triplets assigned for N-CH2-CH2-CO. A singlet and 2 doublets

resonating downfield were assigned for Ha, Hb and Hc respectively. Signals on the

aromatic region showed coupling patterns typical of 1,2,4-trisubstituted benzene ring.

HA and HB protons appeared as 2 doublets due to coupling with ortho protons with

JAB = 7.8 Hz.

Figure 13: 1H NMR spectrum of 1,7-bis(4-adamantylaminopropionyloxy)-3-

(methoxyphenyl)-1,6-heptadiene-3,5-dione (5h)

71

In 13C-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-OCH3, 12 Ar-C and 7

C1-C7 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 CH2-CH2 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

C47H58N2O8

+

O

O

O

O O

O

O

O

C37H46N2O8

m/z 646 (2.6)

O+

HO

O

OO

HO

m/z 151 (6.8)

C11H14O3

m/z 194 (33.6)

C11H12O2

m/z 164 (27)

++

C9H11O2

+

NH

NH

M+ 778 absent

H2N

O

O

O

O O

O

O

O

C36H42NO8

m/z 616 (3.1)

+

O

O

O

OH OH

O

O

O

C25H35O8

m/z 463 (10.3)

+

C19H23

m/z 251 (9.7)

+

HO

orHN

+H2C

C11H18N

orH2N

C10H17N

HO+

HOC8H7O2

m/z 135 (25.6)

or

C10H15

+

+

H

O

OH

+

CH2O2m/z 46 (44.8)

HO C O+

CHO2

m/z 45 (100)

CH3 C O+

C2H3O

m/z 43 (72)

+

C3H5

m/z 41 (80.5)m/z 77 (20.8)

C6H5

Chart 4: Possible Fragmentation Pattern for compound 5h

73

1H-NMR spectrum of compound 5j showed signals similar to those recorded for

5c. However, 2 triplets were assigned for the CO-CH2-CH2-N- protons.

In 13C-NMR spectrum of compound 5j, chemical shifts assignments were made

for the 2 Ar-OCH3, 12 Ar-C and 7 C1-C7 carbons, including characteristic peaks of 2

carbonyl groups, of the heptadienedione chain. In addition, the spectrum also showed

peaks for 2 x thiadiazole CH3 and the 2 Cs of the ring. Signals for the 4 Cs of the 2 x

CH2-CH2 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


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

C33H34N6O8S2

+

C31H26N6O7S2

m/z 658 (1.1)

O

C10H13O

m/z 149 (35)m/z 142 (10)

+

NH

NH

M+ + 1 707 (0.7)

O

O

C20H19O3

m/z 307 (2.5)

C19H17O3

m/z 293 (3)

HO C O+

CHO2

m/z 45 (18.1)

CH3 C O+

C2H3Om/z 43 (100)

NN

S S

NN

CH3H3C

HO

O

O

O

OH

O

O+

NH

NH

NN

S S

NN

CH3H3C

O O

+O

OH

+

NN

S

NN

SH3C NH

H3C NH2

+ +

C5H8N3S C3H5N3Sm/z 115 (5)

NN

SH3C

+

C3H4N2Sm/z 100 (5.5)

OH O+

+

m/z 73 (17.5) m/z 55 (72.4)C4H9O

C3H3O

Chart 5: Possible Fragmentation Pattern for compound 5j

1H-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 CH2 and 2 NH. It also

showed a triplets and quartet assigned for -OCH2CH3 and OCH2CH3 respectively. Ha

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 δ 6.4541 and

7.5722 ppm, with J = 15.66 Hz, were assigned for Hb and Hc respectively. HA Proton

appeared as a doublet due to coupling with the ortho HB proton with JAB = 8.25 Hz.

75

HB Proton was also shown as a doublet with J values of 8.25 Hz (ortho coupling with

HA) while HX 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

OR1

O

R1O

O O

(3a-d) Et3N / EtOH

(CH2)n

O O

(CH2)nCl Cl

O

OR1

O

R1O

O O

(CH2)n

O O

(CH2)nHN NH SO2NHR4

R4HNO2S

H2N SO2NH4/

(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

OH1, 2+

ClOC

Na2CO3/acetone

reflux

RO

OCO

O O

OR

OCO

7a,b

1 1

11

(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 Na2CO3 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, 1H-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 νas and νs C-O-C. A broad band due to

intramolecularly H-bonded OH was also observed.

1H-NMR spectrum of compound 8a showed a broad hump characteristic for

adamantyl protons and 2 consecutive singlets assigned for -OCH3 and Ha protons.

Two doublets resonating at δ 6.5539 and 7.6111 ppm, with J = 15.9 Hz were assigned

for Hb and Hc respectively. Signals on the aromatic region showed coupling patterns

typical of 1,2,4-trisubstituted benzene ring. HA proton appeared as a doublet due to

77

coupling with the ortho HB proton with JAB = 8.25 Hz. HB Proton was shown as a

doublet of doublets with J values of 8.04 Hz (ortho coupling with HA) and 1.65 Hz

(meta coupling with HX). HX proton resonated as a singlet at 7.0974 ppm while the

para coupling was not resolved and could not be detected.

In 13C-NMR spectrum of compound 8a, chemical shifts assignments were made

for the 2 Ar-OCH3, 12 Ar-C and 7 C1-C7 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

C43H48O8

+

O

C10H12O3m/z 180 (4.8)

M+ 692 (2.1)

O

O

C32H38O6

m/z 518 (2.75)

C13H16O3

m/z 220 (1.7)

C10H15

C11H15O

or

C11H16O2

+OC6H5O

m/z 93 (29)

HO C O+

CHO2

m/z 45 (31.1)

CH3 C O+

C2H3Om/z 43 (50)

+

C3H5

m/z 41 (92)

O

O

O+

m/z 57 (22)

C3H5O

O

O

O

O

O

O

O

C43H45O7

m/z 673 (3.7)

O

O

O

O

O

C42H48O5

+

m/z 632 (3.2)

+

O

O

O

OH

+

HO

OH

+

O

H

+ +O

OHO+

m/z 163 (5.6)

+

m/z 135 (100)

79

Chart 6: Possible Fragmentation Pattern for compound 8a

1H-NMR spectrum of compound 8b showed a broad hump characteristic for

adamantyl protons overlapping with the triplet of OCH2CH3. A quartet at δ

4.1611ppm was assigned for OCH2CH3 protons of the ethyl group with J = 6.97 Hz.

A signal at δ 5.7825 ppm, integrated for only 1 proton, was assigned to Ha proton.

This was evidenced by the appearance of a deshielded proton at δ 9.8134 ppm

assigned for the enol OH proton. Two doublets resonating at δ 6.4541 and 7.5722

ppm, with J = 15.66 Hz were assigned for Hb and Hc respectively. Signals on the

aromatic region did not show coupling patterns typical of 1,2,4-trisubstituted benzene

ring. HA Proton appeared as a doublet due to coupling with the ortho HB proton with

JAB = 8.25 Hz. The spectrum lacked the typical doublets of doublet characteristic for

the HB proton but resonated as a doublet at δ 7.1065 ppm with JAB value of 8.25 Hz

(ortho coupling with HA). HX Proton resonated as a singlet at δ 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

OR1

O

R1O

O O

O O

HO

OR1

OH

R1O

O O

+ C6H13COClNa2CO3/ 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 Na2CO3[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, 1H-NMR and MS.

1H-NMR spectrum of compound 8c showed 2 triplets resonating high field

assigned for the terminal CH3 protons and the CO-CH2- of the heptanoyl group. A

broad hump characteristic for aliphatic (CH2)4 protons was shown at high field. The

spectrum also showed 2 consecutive singlets assigned for -OCH3 and Ha protons at

their expected chemical shifts. Two doublets resonating at δ 6.5468 and 7.6184 ppm,

with J = 15.75 Hz were assigned for Hb and Hc respectively. Signals on the aromatic

region showed coupling patterns typical of 1,2,4-trisubstituted benzene ring. HA

Proton appeared as a doublet due to coupling with the ortho HB proton with JAB =

8.04 Hz. HB Proton was shown as a doublet of doublets with J values of 9 Hz (ortho

coupling with HA) and 1.8 Hz (meta coupling with HX). HX Proton resonated as a

singlet at 7.6184 ppm while the para coupling was not resolved and could not be

detected.

1H-NMR spectrum of compound 8d (figure 17) showed signals characteristic for

the (CH2)5-CH3 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

OCH2CH3 group. A signal at δ 5.8384 ppm was assigned to Ha protons. Two doublets

resonating at δ 6.5400 and 7.0383 ppm, were assigned for Hb and Hc respectively.

Signals on the aromatic region did not show coupling patterns typical of 1,2,4-

trisubstituted benzene ring. HA Proton appeared as a doublet due to coupling with the

ortho HB proton with JAB = 7.5 Hz. The spectrum lacked the typical doublet of

doublets characteristic for the HB proton but resonated as a doublet at δ 7.1134 ppm

81

with JAB value of 7.5 Hz (ortho coupling with HA). HX Proton resonated as a singlet at

δ 7.2551 ppm while the para coupling was not resolved and could not be detected.

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

In 13C-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 1H-NMR (COSY) (figure 19) and

1H,13C-NMR (H,C correlation) (figure 20).

82

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

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

83

Figure 20: 1H, 13C 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

OH1, 2+ Na2CO3/acetone

RO

-OCO

O O

OR

OCO-

7c,d

RTS

-COCl

SS

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, 1H-NMR and MS.

1H-NMR spectrum of 8e (figure 21) showed 2 singlets assigned for OCH3 and Ha

protons and 2 doublets resonating at δ 7.051 and 7.697 ppm, with J = 16 Hz, assigned

for Hb and Hc respectively. The aromatic protons resonated as well-resolved signals

showing a doublet of doublet for HB due to ortho and meta coupling, a doublet for HA

and a singlet for HX protons. Signals for protons on the thienyl groups were the most

deshielded and resonated as 2 doublets and doublets of doublet.

85

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

1H-NMR spectrum of 8f lacked the typical doublets of doublet characteristic for

the HB proton in 1,2,4-trisubstituted benzene ring pattern; para coupling was also

unobservable. It showed a singlet assigned for HX and 2 doublets with coupling

constant J = 8.52 Hz for HA and HB protons. However, the spectrum showed a triplet

and a quartet at δ 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 Ha, Hb and Hc

respectively. Protons on the thienyl rings were resolved and resonated as 3 doublets,

distorted in some cases, at δ 7.5690, 8.02645 and 8.1015 ppm and were assigned for

thienyl-H5, -H4 and -H3 protons respectively[169]. The coupling constant for H4 and H3

was consistent with that previously reported[150], J = 3.57 Hz.

86

In 13C-NMR spectrum of compound 8f, chemical shifts assignments were made

for the 2 Ar-OCH2CH3, 12 Ar-C and 7 C1-C7 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


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

87

O

O

O

O O

O

O

O

C33H28O8S2

+

M+ + 1 617 (0.7)

C7H7O

m/z 107 (9)

m/z 44 (68.6)

CH3 C O+

C2H3Om/z 43 (100)

S

S

O

O

O

OH O

O

O

O

C32H28O8S2

+

m/z 604 (1.6)

S

S

O O

O+

O

O

C24H17O5S

+

m/z 417 (2.6)

S

HO

OH

OH

O

O

C16H16O5S

+

m/z 320 (1.6)

S O

O

S

+

C11H7O2S

m/z 203 (2.3)

O O+

O+

O

+

C7H9O2 C5H7O C4H5O

m/z 125 (8) m/z 83 (26.6) m/z 69 (15)

HO + HO

+

C6H5O

m/z 93 (11.5)

+O

CH3CHOSO+

C5H3OSm/z 111 (13)

S

C4H3S

m/z 83 (26.6)

+CO2 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 K2CO3 in refluxing acetone, NEt3 in DMF or pyridine at

room temperature or under reflux were unsuccessful.

H3CO

HO

O O

OCH3

OH

+ ClSO2NCO XXX

Acetone/ K2CO3

or DMF/ NEt3 or pyridine

H3CO

OCNO2SO

O O

OCH3

OSO2NCO

RNH2

1

H3CO

O

O O

OCH3

OO2S SO2

NH 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

HO

H3C O

HN

NCH3

O

HN N

CH3

HN

O

NH2

NH2+ Cl-

(a)

Distamycin A

O

HN

NCH3

O

HN N

CH3

HN

O

NH2

NH2+

2 Cl-

HN

NH2+

H2N

(b)

Netropsin

NO

HN

HN

O

HN

HN

SO2CH3

OCH3

N

O

NCH3

HNNH2

O

CH3 (c)

NetAmsa

N

HNR

CH3

HN

O

NH3C

CH3O

n (d)

91

N

HN

S

CH3

HN

O

NH3C

CH3

n

OO

O

O

(e)

N

O

O

OH3C

OCH3

OCH3

O

N HN

N

OHGlc

OH

OHCHN

OO

(f) (g)

N

N

O

O

OOH

O

O

O

OH

H3CO OCH3

O

HOO

OH3C

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

R1O OR1

O O


R2

N-(CH2)xR3

(CH2)x-NR2

R3

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

R1O OR1

O

R2

O

R2


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

OOCH3

OH

OH

OH

O

COCH2OH

O

NH2OH

CH3

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 β-

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)aminoacetyloxy)-3-(ethoxyphenyl)-1,6-heptadiene-3,5-dione (6b) and 1,7-bis(4-

(2-diethylaminoethyl)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 IC50 = 4.75 µM followed by compound 5e with an IC50 =

7 µM. Compounds 5c and 5j were also cytotoxic to the same cell line with IC50 = 7.5

96

µM while compounds 5g had IC50 = 8.5 µM and 6g had IC50 = 11.5 µM. 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 IC50

= 4.25, 6.75, 8.75 and 9 µM 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 IC50 = 10 µM. 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 IC50 of 11 and 11.5 µM 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 IC50 = 11.5 µM (Chart 9). Regarding SK-OV-3

cell line (Chart 11), compound 6b was the most cytotoxic among these series with

IC50 = 2.8 µM. It was also not cytotoxic to any of the noncancer cells tested up to 25

µM. 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

H3CO

O

OCH3

O

O

CH2

ClCl O

CH2 3a

H3CO

O O

OCH3

OO CH2

OO

H2CHNN

NS

CH3

NH

N N

S CH3

5c

H3CO

O O

OCH3

OO CH2

OO

H2CNN

Cl

Cl

Cl

Cl

5e

H3CO

O O

OCH3

OO CH2

OO

H2C

HNH

N NNEt

Et

Et

Et

5g

99

H3CO

O O

OCH3

OO (CH2)2

OO

(H2C)2

N

NS

CH3

NH

N N

S CH3NH

5j

H3CO

O O

OCH3

OO (CH2)2

OO

(H2C)2

NHNHClCl

5k

O

O O

O

OO CH2

OO

H2CNHHNN

S N

S

OCH3

OCH3

6b

O

O O

O

OO CH2

OO

H2CNHHNN

NS N N

S CH3

CH3

6c

O

O O

O

OO CH2

OO

H2C

HNH

N

ClCl

6d

100

O

O O

O

OO CH2

OO

H2CNN

Cl

Cl

Cl

Cl

6e

O

O O

O

OO CH2

OO

H2C

HNH

N NNH3C

H3C

CH3

CH3

6f

O

O O

O

OO CH2

OO

H2C

HNH

N NNEt

Et

Et

Et

6g

O

OCH3

O

H3CO

O O

CH2

O O-O2SHN NHSO2-CH2

H2NNH2

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, ν (cm-1) on

Perkin Elmer 1430 spectrophotometer.

1H and 13C 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 δ 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

R1O

HO

OR1

OH

(1) R1= CH3 (2) R1 = C2H5

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 H2O, dried over anhydrous MgSO4 (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) ν (cm-1): 3468.5 (br, OH), 1627.8 (C=O),

1604.4, 1508.1 (C=C, Ar), 1282.4, 1027.3 (νas and νs C-O-C); 1H-NMR of 1 (DMSO-

103

d6) δ (ppm) (300 MHz) (figure 1): 3.8366 (s, 6H, 2 x OCH3), 6.0618 (s, 1H, Ha),

6.711 (d, 2H, 2 x Hb, J = 16.6 Hz), 6.8227 (d, 2H, 2 x HA, JAB = 8.22 Hz, ortho

coupling), 7.1569 (d, 2H, 2 x HB, JAB = 8.03 Hz, ortho coupling), 7.3273 (s, 2H, 2 x

HX), 7.5470 (d, 2H, 2 x Hc, J = 15.35 Hz), 9.7411 (s, 2H, 2 x phenolic OH), 10.1312

(s, 1H, enol OH); 13C-NMR of 1 (DMSO-d6) δ ppm (300 MHz): 56.2179 (2 x OCH3),

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-ethoxybenzaldehyde in 68%

yield; m.p. = 143○C (reported m.p. = 158○-160○C)[138]. IR of ethyl curcumin (2) ν (cm-

1): ; 1H-NMR of 2 (DMSO-d6) δ ppm (500 MHz) (figure 2): 1.358 (t, 6H, 2 x

OCH2CH3, J = 7 Hz), 4.094 (q, 4H, 2 x CH2CH3, J = 7 Hz), 6.050 (s, 2H, Ha), 6.746

(d, 2H, 2 x Hb, J = 16 Hz), 6.850 (d, 2H, 2 x HA, JAB = 8 Hz, ortho coupling), 7.1495

(d, 2H, 2 x HB, JAB = 8.5 Hz, ortho coupling), 7.305 (s, 2H, 2 x HX), 7.552 (d, 2H, 2 x

Hc, J = 16 Hz), 9.607 (s, 2H, 2 x phenolic OH); 13C-NMR of 2 (DMSO-d6) δ ppm

(500 MHz) (figure 3): 15.16 (2 x OCH2CH3), 64.36 (2 x OCH2CH3), 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

H3CO

O

OCH3

O

O

(CH2)nClCl

(CH2)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 MgSO4 (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) ν (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 (ν as and ν s C-O-C). 1H-NMR of 3a (CDCl3) δ ppm (300 MHz) (figure

5): 3.876 (s, 6H, 2 x OCH3), 4.1003 (s, 4H, 2 x CH2), 5.8557(s, 2H, Ha), 6.56905 (d,

dist, 2H, 2Hb, J = 15.9 Hz), 7.0754-7.2613 (m, 6H, 2 x 3 Ar-H), 7.6116 (d, dist, 2H,

2Hc, J = 15.7 Hz).

IR of di-O-chloropropionylcurcumin (3b) ν (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 (ν as and ν s C-O-C). 1H-NMR of 3b (DMSO-d6) δ ppm (300 MHz):

105

3.1223 (t, 4H, 2 x CH2-Cl, J = 6.1 Hz), 3.8403 (s, 6H, 2 x OCH3), 3.8952 (t, 4H, 2 x

COCH2, J = 6.33 Hz), 6.2156 (s, 2H, Ha), 7.0095 (d, dist, 2H, 2 x Hb, J = 15.9 Hz),

7.1524-7.5351 (m, 6H, 2 x 3 Ar-H), 7.6569 (d, dist, 2H, 2 x Hc, 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

C25H22Cl2O8

136

43

O O

H3CO OCH3

OCOCH2ClClH2COCO

3a

549

C27H26Cl2O8

143

38

O O

H3CO OCH3

OCOCH2CH2ClClH2CH2COCO

3b

5.2.2.2. Method B

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

CHO

O

O

(CH2)n

Cl

R1O

(4a) R1 = CH3 n = 1; (4b) R1 = CH3 n = 2

(4c) R1 = C2H5 n = 1; (4d) R1 = C2H5 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 MgSO4 (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 K2CO3.

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 ν (cm-1): 2933.4, 2847 (CHO), 1765 (C=O ester), 1698 (C=O aldehyde),

1600, 1505 (C=C aromatic), 1180, 1150, 1100, 1020 (νas and νs C-O-C); 1H- NMR of

4a (DMSO-d6) δ ppm (500 MHz) (figure 6): 3.8445 (s, 3H, OCH3), 4.7157 (s, 2H,

CH2-Cl), 7.3897 (d, 1H, HA, JAB = 8.40, ortho coupling), 7.5647, 7.5800 (dd, 1 H, HB,

J = 1.55 Hz and 7.65 Hz, meta and ortho coupling), 7.6075 (d, 1H, HX, JAX = 1.55 Hz,

para coupling), 9.9476 (s, 1H, CHO).

IR of 4b ν (cm-1): 3073, 2940 (CHO), 1760 (C=O ester), 1698 (C=O aldehyde),

1601, 1495 (C=C aromatic), 1154, 1120, 1068, 1025 (νas and νs C-O-C).

1H- NMR of 4c (DMSO-d6) δ ppm (300 MHz) (figure 7): 1.3138 (t, 3H,

OCH2CH3, J = 6.87 Hz), 4.15575 (q, 2H, OCH2CH3, J = 6.87 Hz), 4.7413 (s, 2H,

CH2Cl), 7.4136 (d, 1H, HA, JAB = 7.98 Hz, ortho coupling), 7.5762, 7.6051 (dd, 1H,

HB, J = 1.65 Hz and 8.07 Hz, meta and ortho coupling), 7.6285 (d, 1H, HX, JAX = 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 2COCO

3

24

43

78

68-71

C10H9ClO4

228.5

4b

OCH3

CHO

ClH2CH2COCO

24

3

41

72

65-69

C11H11ClO4

242.5

4c

OCH 2CH3

CHO

ClH 2COCO

24

3

42

67

61-64

C11H11ClO4

242.5

4d

OCH2CH3

CHO

ClH2CH2COCO

24

3

39

61

55-59

C12H13ClO4

256.5

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

curcumin (3c,d)

O

OR1

O

R1O

O O

(CH2)nn(H2C)

O OClCl

(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 H2O, dried over

anhydrous MgSO4 (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 1H-NMR spectra of compounds 3a,b were coinciding with those obtained

from method A.

IR of 3c ν (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 (ν as and ν s C-O-

C); 1H- NMR of 3c (CDCl3) δ ppm (300 MHz) (figure 8): 1.4164 (t, 6H, 2 x

OCH2CH3, J = 6.87 Hz), 4.1049 (q, 4H, 2 x OCH2CH3, J = 6.87 Hz), 4.3329 (s, 4H, 2

x CH2-Cl), 5.8457(s, 2H, Ha), 6.5553 (d, 2H, 2 x Hb, J = 15.93 Hz), 7.0764-7.1762

(m, 6H, 2 x 3 Ar-H), 7.605 (d, 2H, 2 x Hc, 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).

1H- NMR of 3d (CDCl3) δ ppm (500 MHz) (figure 10): 1.442 (t, 6H, 2 x

OCH2CH3, J = 7 Hz), 3.105 (t, 4H, 2 x COCH2, J = 7Hz), 3.904 (t, 4H, 2 x CH2-Cl, J

= 7 Hz), 4.131 (q, 4H, 2 x OCH2CH3, J = 7 Hz), 5.871 (s, 1H, Ha), 6.577 (d, 2H, 2 x

Hb, J = 16 Hz), 7.098 (d, 2H, 2 x HA, JAB = 8 Hz, ortho coupling), 7.1735 (d, 2H, 2 x

109

HB, JAB = 8.5 Hz, ortho coupling), 7.286 (s, 2H, 2 x HX), 7.559 (d, 2H, 2 x Hc, J = 16

Hz), 9.965 (s, 1H, enol OH).

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

O

OR1

O

R1O

O O

(CH2)nn(H2C)

O OClCl

(3a-d)

M. wt M. Formula m.p. ○C

Yield %

n R1 No.

521

C25H22Cl2O8

136

61

1

CH3

3a

549

C27H26Cl2O8

143

56

2

CH3

3b

549

C27H26Cl2O8

130

59

1

C2H5

3c

577

C29H30Cl2O8

145

54

2

C2H5

3d

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

hydrochloride

NH2CH2CH2Cl . HCl NH

CH2CH2Cl

CH2CH2Cl

. 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 ½ 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-heptadiene-3,5-dione (6a-n)

OR1

O

R1O

O O

n(H2C)

O O

(CH2)nR3R2N NR2R3

(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 CHCl3 (15ml) giving compounds 5a-n and 6a-n (Table

4).

1H-NMR of 5a (DMSO-d6) δ ppm (300 MHz) (figure 11): 1.1609-1.9951 (hump,

br, 30H, 2 x Ad-H), 2.0858 (s, 4H, 2 x CO-CH2), 3.8311 (s, 6H, 2 x OCH3), 6.0334 (s,

br, 2H, Ha), 6.7444 (d, 2H, 2x Hb, J = 15.66 Hz), 6.8016 (d, 2H, 2 x HA, JAB = 8.25

Hz, ortho coupling), 7.1295, 7.1569 (dd, dist, 2H, 2 x HB, 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 HX, JAX =

0.24 Hz, para coupling), 7.5315 (d, 2H, 2 x Hc, 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 ν (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 (νas and νs C-O-C); 1H-NMR of 5b (DMSO-d6) δ

ppm (300 MHz): 2.0876 (s, 4H, 2 x CH2 ), 3.8384 (s, 12H, 4 x OCH3), 6.0599 (s, 2H,

Ha), 6.7646 (d, 2H, 2 x Hb, J = 15.66 Hz), 6.8236 (d, 2H, 2 x HA, JAB = 8.25 Hz, ortho

coupling), 7.1413, 7.1688 (dd, 2H, 2 x HB, J = 1.65 Hz and 8.25 Hz, meta and ortho

coupling), 7.32815 (d, 2H, HX, JAX = 1.65 Hz, para coupling), 7.5466 (d, 2H, 2 x Hc,

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, D2O-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 ν (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 (νas and νs C-O-C); 1H-NMR of 5c (DMSO-d6) δ ppm (300 MHz): 2.5033 (s,

4H, 2 x CH2), 3.3522 (s, 6H, 2 x CH3), 3.835 (s, 6H, 2 x OCH3), 6.0590 (s, 2H, Ha),

6.7631 (d, 2H, 2 x Hb, J = 15.93 Hz), 6.8218 (d, 2H, 2 x HA, JAB = 8.22 Hz, ortho

coupling), 7.1409, 7.1676 (dd, 2H, 2 x HB, J = 1.65 Hz and 8.01 Hz, meta and ortho

coupling), 7.3272 (d, 2H, 2 x HX, JAX = 1.65 Hz), 7.5457 (d, 2H, 2 x Hc, J = 15.66

Hz), 9.6852 (s, 2H, 2 x NH, D2O-exchangeable).

1H-NMR of 5d (CD3OD) δ ppm (500 MHz): 2.292 (s, 2H, 2 x NH), 2.305 (t, dist,

4H, 2 x CH2-N), 3.230 (t, 4H, 2 x CH2-Cl, J = 3.5 Hz), 3.331 (s, 4H, 2 x COCH2-

N), 3.918 (s, 6H, 2 x OCH3), 4.903 (s, 2H, 2 x Ha), 6.643 (d, 2H, 2 x Hb, J = 15.5 Hz),

6.839 (d, 2H, 2 x HA, JAB = 8 Hz, ortho coupling), 7.119 (d, 2H, 2 x HB, 7.7 Hz, ortho

coupling), 7.227 (s, 2H, 2 x HX), 7.584 (d, 2H, 2 x Hc, J = 15.5 Hz).

112

IR of 5e ν (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 (νas and νs C-O-

C); 1H-NMR of 5e (DMSO-d6) δ ppm (300 MHz) (figure 12): 2.0867 (s, 4H, 2 x

COCH2-N), 2.5042 (t, 8H, 4 x N-CH2CH2-Cl, J = 3.57 Hz), 3.4841 (t, 8H, 4 x

NCH2CH2-Cl, overlapping with solvent signal), 3.8384 (s, 6H, 2 x OCH3), 6.0599 (s,

1H, Ha), 6.7718 (d, 2H, 2 x Hb, J = 15.9 Hz), 6.8245 (d, 2H, 2 x HA, JAB = 8.25 Hz,

ortho coupling), 7.1409, 7.1688 (dd, 2H, 2 x HB, J = 1.65 Hz and 8.37 Hz, meta and

ortho coupling), 7.3277 (d, 2H, 2 x HX, JAX = 1.38 Hz, para coupling), 7.5736 (d, 2H,

2 x Hc, J = 15.9 Hz), 9.6953 (s, br, 1H, enol OH).

1H-NMR of 5f (DMSO-d6) δ ppm (300 MHz): 3.1748 (s, 12H, 2 x N(CH3)2),

3.5387 (t, dist, 4H, 2 x CH2-N), 3.6657 (t, 4H, 2 x CH2-NH, J = 5.49 Hz), 3.8012 (s,

dist, 2H, 2 x NH, overlapping with OCH2), 3.8343 (s, 4H, 2 x COCH2-N), 4.0602 (s,

6H, 2 x OCH3), 6.0595 (s, 1H, Ha), 6.7586 (d, 2H, 2 x Hb, J = 15.75 Hz), 6.8337 (d,

2H, 2 x HA, JAB = 8.07 Hz, ortho coupling), 7.1513 (d, 2H, 2 x HB, J = 8.07 Hz, ortho

coupling), 7.3235 (s, 2H, 2 x HX), 7.5427 (d, 2H, 2 x Hc, J = 15.75 Hz), 9.7368 (s, 1H,

enol H).

1H-NMR of 5h (CD3OD) δ ppm (500 MHz) (figure 13): 1.184-2.172 (hump, br,

30H, 2 x Ad-H), 2.631 (t, dist, 4H, 2 x CH2-N, J = 6.2 Hz), 3.064 (t, 4H, 2 x CO-

CH2, J = 6.5 Hz), 3.882 (s, 2H, 2 x NH overlapping with OCH3 signal), 3.921 (s, 6H,

2 x OCH3), 4.936 (s, 2H, Ha), 6.632 (d, 2H, 2x Hb, J = 15.7 Hz), 6.835 (d, 2H, 2 x HA,

JAB = 8 Hz, ortho coupling), 7.111 (d, 2H, 2 x HB, J = 7.8 Hz, ortho coupling), 7.218 (

d, 2H, 2 x HX), 7.577 (d, 2H, 2 x Hc, J = 15.8 Hz); 13C-NMR of 5h (CD3OD) δ ppm

(500 MHz): 39.51 (2 x CH2N), 40.48 (2 x CH2CO), 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 OCH3), 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 ν (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 (νas and νs C-O-C); 1H-NMR of 5j (CD3COCD3) δ ppm

(500 MHz): 3.612 (s, 6H, 2 x CH3), 3.908 (s, 6H, 2 x OCH3), 4.084 (t, dist, 4H, 2 x

CH2-N), 4.609 (t, dist, 4H, 2 x CH3-CO), 5.791 (s, br, 2H, Ha), 6.701 (d, 2H, 2x Hb, J

= 15.5 Hz), 6.898-7.676 (m, 6H, 2 x Ar-H), 7.744 (d, 2H, 2 x Hc, J = 15.5 Hz), 8.317

(s, 2H, 2 x NH); 13C-NMR of 5j (CD3COCD3) δ ppm (500 MHz): 45.35 (2 x CH2N),

47.25 (2 x CH2CO), 60.85 (2 x OCH3), 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

1H-NMR of 6a (CDCl3) δ ppm (300 MHz): 1.2516-2.3596 ((hump, br, 30H, 2 x Ad-

H), 1.4805 (t, 6H, 2 x OCH2CH3, J = 6.87 Hz), 2.3504 (s, 4H, 2 x CH2CO), 2.6306 (s,

2H, 2 x NH), 4.1516 (q, 4H, 2 x OCH2CH3, J = 6.87 Hz), 5.7825 (s, 1H, Ha), 6.4541

(d, 2H, 2 x Hb, J = 15.66 Hz), 6.9307 (d, 2H, 2 x HA, JAB = 8.25 Hz, ortho coupling),

7.0315 (s, 2H, 2 x HX), 7.10655 (d, 2H, 2 x HB, J = 8.25 Hz, ortho coupling), 7.5722

(d, 2H, 2 x Hc, J = 15.66 Hz), 9.8134 (s, 1H, enol OH).

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

OR1

O

R1O

O O

n(H2C)

O O

(CH2)nR3R2N NR2R3

(5a-n, 6a-n) Cpd No

R1

n

R2 R3

Reaction Time h

Yield %

m.p. °C

M. Formula

M. wt

5a

-CH3

1

NH

12

86

110-15

C45H54N2O8

750

5b

-CH3

1

N

S

NH

H3CO

12

59

160- 63

C41H36N4O10S2

808

5c

-CH3

1

NN

SNHH3C

24

63

170-73

C31H30N6O8S2

678

5d

-CH3

1

HN

Cl

6

83

87-93

C29H32Cl2N2O8

607

115

5e -CH3 1

N

Cl

Cl

4-5

95 85-90

C33H38Cl4N2O8

732

5f

-CH3

1

NCH2CH2NH

H3C

H3C

1

69

160-65

C33H44 N4O8

624

5g

-CH3

1

NCH2CH2NH

C2H5

C2H5

1

62

150-55

C37H52N4O8

680

5h

-CH3

2

NH

12

69

140

C47H58N2O8

778

5i

-CH3

2

N

S

NH

H3CO

36

47

170-74

C43H40N4O10S2

836

5j

-CH3

2

NN

SNHH3C

36

43

120-23

C33H34N6O8S2

706

5k

-CH3

2

HN

Cl

36

71

90-95

C31H36Cl2N2O8

635

5l

-CH3

2

N

Cl

Cl

36

74

87-93

C35H42Cl4N2O8

760

5m

-CH3

2

NCH2CH2NH

H3C

H3C

3-4

66

160-65

C35H48N4O8

652

5n

-CH3

2

NCH2CH2NH

C2H5

C2H5

3-4

69

155-60

C39H56N4O8

708

116

6a

CH2CH3

1

NH

4-5

71.5

115-20

C47H58N2O8

778

6b

CH2CH3

1

N

S

NH

H3CO

12

66

137-40

C43H40N4O10S2

836

6c

CH2CH3

1

NN

SNHH3C

12

72

105-10

C33H34N6O8S2

706

6d

CH2CH3

1

HN

Cl

9

56

107-10

C31H36Cl2N2O8

635

6e

CH2CH3

1

N

Cl

Cl

9

65

95-100

C35H42Cl4N2O8

760

6f

CH2CH3

1

NCH2CH2NH

H3C

H3C

6

67

115-20

C35H48N4O8

652

6g

CH2CH3

1

NCH2CH2NH

C2H5

C2H5

6

69

105-110

C39H56N4O8

708

6h

CH2CH3

2

NH

6

67

85-90

C49H62N2O8

806

6i

CH2CH3

2

N

S

NH

H3CO

16

58

165-70

C45H44N4O10S2

864

6j

CH2CH3

2

NN

SNHH3C

36

79

105-110

C35H38N6O8S2

734

117

6k

CH2CH3

2 HN

Cl

12

91

115-120

C33H40Cl2N2O8

663

6l

CH2CH3

2

N

Cl

Cl

12

64

125-130

C37H46Cl4N2O8

788

6m

CH2CH3

2

NCH2CH2NH

H3C

H3C

3-4

89

155-60

C37H52N4O8

680

6n

CH2CH3

2

NCH2CH2NH

C2H5

C2H5

3-4

81

155-60

C41H60N4O8

736

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

OR1

O

R1O

O O

(CH2)n

O O

(CH2)nHN NHR4HNO2S SO2NHR4

(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

OR1

O

R1O

O O

(CH2)n

O O

(CH2)nHN NHR4HNO2S SO2NHR4

(7a-f) Cpd No

R1 n R4 Reaction Time h

Yield %

m.p. °C

M. Formula M. wt

7a

CH3

1

H

16

69

155-60

C37H36N4O12S2

792

7b

CH3

1

N

N

12

50

175-78

C45H40N8O12S2

948

7c

C2H5

1

H

18

63

143-48

C39H40N4O12S2

820

7d

C2H5

1

N

N

24

60

185-90

C47H44N8O12S2

976

7e

C2H5

2

H

18

78

115-20

C41H44N4O12S2

848

7f

C2H5

2

N

N

24

57

115-20

C49H48N8O12S2

1004

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

-OCO

O O

OCO-

R1O OR1

(8a) R1 = CH3

(8b) R1 = C2H5

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 Na2CO3

(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 MgSO4 (5 g), filtered, evaporated

and crystallized from EtOH (15 ml) to give a yellow precipitate of 8a,b ( Table 6).

IR of 8a ν (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 (νas and νs C-O-

C); 1H-NMR of 8a (CDCl3) δ ppm (300 MHz): 1.2342-2.1929 (hump, br, 30H, 2 x

Ad-H), 3.8467 (s, 6H, 2 x OCH3), 5.8475 (s, 2H, Ha), 6.5539 (d, 2H, 2 x Hb, J = 15.9

Hz), 7.0076 (d, 2H, 2 x HA, JAB = 8.25 Hz, ortho coupling), 7.0974 (s, 2H, 2 x HX),

7.1340, 7.1608 (dd, 2H, 2 x HB, J = 1.65 Hz and 8.04 Hz, meta and ortho coupling),

7.6111 (d, 2H, 2 x Hc, J = 15.9 Hz); 13C-NMR of 8a (CDCl3) δ ppm (500 MHz):

56.01 (2 x OCH3), 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

1H-NMR of 8b (CDCl3) δ ppm (300 MHz): 1.2516-2.3596 (hump, br, 30H, 2 x Ad-

H overlapping with t of OCH2CH3), 1.4805 (t, 6H, 2 x OCH2CH3, J = 6.87 Hz),

4.1611 (q, 4H, 2 x OCH2CH3, J = 6.97 Hz), 5.7825 (s, 1H, Ha), 6.4541 (d, 2H, 2 x Hb,

J = 15.66 Hz), 6.9307 (d, 2H, 2 x HA, JAB = 8.25 Hz, ortho coupling), 7.0315 (s, 2H, 2

x HX), 7.1065 (d, 2H, 2 x HB, JAB = 8.25 Hz, ortho coupling), 7.5722 (d, 2H, 2 x Hc, J

= 15.66 Hz); 9.8134 (s, 1H, enol OH).

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

O

OR1

O

R1O

O O

O O

(8c) R1 = CH3

(8d) R1 = C2H5

Heptanoyl chloride (0.03 mol) was added dropwise to an ice cold mixture of

curcumin (1) or ethyl curcumin (2) (0.015 mol) and Na2CO3 (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 MgSO4, evaporated and crystallized from EtOH to give a

yellow precipitate of 8c,d (Table 6).

IR of 8c ν (cm-1): 1768 (C=O, ester), 1629 (C=O, ketone), 1598, 1512 (C=C

aromatic), 1120, 1010 (νas and νs C-O-C); 1H-NMR of 8c (CDCl3) δ ppm (300 MHz):

0.9068 (t, 6H, 2 x (CH2)5-CH3, J = 6.96 Hz), 1.3049-1.7861 (m, 16H, 2 x (CH2)4),

2.5849 (t, 4H, 2 x CH2CO, J = 7.32 Hz), 3.8672 (s, 6H, 2 x OCH3), 5.8543 (s, 2H,

Ha), 6.5468 (d, 2H, 2 x Hb, J = 15.75 Hz), 7.0475 (d, 2H, 2 x HA, JAB = 8.04 Hz, ortho

coupling), 7.11405, 7.146 (dd, 2H, 2 x HB, J = 1.8 Hz and 9 Hz, meta and ortho

coupling), 7.2563 (s, 2H, 2 x HX), 7.6184 (d, 2H, 2 x Hc, J = 15.75 Hz).

121

IR of 8d ν (cm-1): 1769 (C=O, ester), 1629 (C=O, ketone), 1590, 1500 (C=C

aromatic), 1115, 1020 (νas and νs C-O-C); 1H-NMR of 8d (CDCl3) δ ppm (300 MHz)

(figure 17): 0.90441 (t, 6H, 2 x (CH2)5-CH3, J = 6.96 Hz), 1.3269-1.5724 (m, 16H, 2

x (CH2)4), 1.7666 (t, 6H, 2 x OCH2CH3, J = 7.3 Hz), 2.5727 (t, 4H, 2 x CH2-CO, J =

7.3 Hz), 4.0865 (q, 4H, 2 x OCH2CH3, J = 6.96 Hz), 5.8384 (s, 2H, Ha), 6.5400 (d,

2H, 2 x Hb, J = 15.75 Hz), 7.0383 (d, 2H, 2 x HA, JAB = 8.4 Hz, ortho coupling),

7.1134 (d, 2H, 2 x HB, JAB = 7.5 Hz, ortho coupling), 7.2551 (s, 2H, 2 x HX), 7.6013

(d, 2H, 2 x Hc, J = 15.75 Hz); 13C-NMR of 8d (CDCl3) δ 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

OCH2CH3), 34.157 (2 x -OCOCH2C5H11), 64.454 (2 x OCH2CH3), 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 1H-NMR (COSY)

(CDCl3) (figure 19) and 1H, 13C-NMR (C,H correlation) (CDCl3) (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-

R1O OR1

S S (8e) R1 = CH3

(8f) R1 = C2H5

Compounds 8e,f (Table 6) were prepared by adding thiophene-2-carbonyl

chloride (0.03 mol) adopting the previously mentioned procedure.

IR of 8e ν (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 (νas and νs C-O-

122

C); 1H-NMR of 8e (DMSO) δ ppm (500 MHz) (figure 21): 3.861 (s, 6H, 2 x OCH3),

6.232 (s, 2H, Ha), 7.051 (d, 2H, 2 x Hb, J = 16 Hz), 7.32, 7.336 (dd, 2H, 2 x HB, J =

3.5 Hz and 7.8 Hz, meta and ortho coupling), 7.396 (d, 2H, 2 x HA, JAB = 7.5 Hz,

ortho coupling), 7.588 (s, 2H, 2 x HX), 7.697 (d, 2H, 2 x Hc, 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 ν (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 (νas and νs C-

O-C); 1H-NMR of 8d (DMSO-d6) δ ppm (300 MHz): 1.2351 (t, 6H, 2 x 3 OCH2CH3,

J = 7.00 Hz), 4.1412 (q, 4H, 2 x OCH2CH3, J = 7.04 Hz), 6.2138 (s, 2H, Ha), 7.0269

(d, 2H, 2 x Hb, J = 15.93 Hz), 7.3222 (d, 2H, 2 x HA, JAB = 8.52 Hz, ortho coupling),

7.3364 (s, 2H, 2 x HX), 7.67525 (d, 2H, 2 x Hc, J = 15.93 Hz), 7.3845 (d, dist, 2H, 2 x

HB, JAB = 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); 13C-

NMR of 8f (DMSO) δ ppm (500 MHz): 40.13 (2 x CH3), 64.95 (2 X OCH2), 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

OR1

O

R1O

O O

R2R2

O O

(8a-f) Cpd No

R1 R2 Reaction Time (h)

Yield (%)

m.p. M. Formula M.wt

8a

CH3

9

47

215

C43H48O8

692

8b

C2H5

16

36

220

C45H52O8

720

8c

CH3

C6H13

4-5

63

99.5

C35H44O8

606

8d

C2H5

C6H13

4-5

77

128-30

C37H48O8

620

8e

CH3 S

4

81.5

199-203

C31H24O8S2

588

8f

C2H5 S

6

51

217-19

C33H28O8S2

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 (µM)* 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 µM

NA = not active up to 25 µM. NC = not cytotoxic up to 25 µM

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

doxo

rubic

in 1 2 3a 3c 3d 5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 6a 6b 6c 6d 6e 6f 6g 6h 6m 6n 7 8a 8c 8d 8e 8f

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

doxo

rubic


KB

Compound number

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




IC 50

129

0

5

10

15

20

25

30

doxo

rubic


BT-549

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




IC 50

130

0

5

10

15

20

25

30

doxo

rubic


SK-OV-3

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




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 37oC 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 37oC 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 (IC50) 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 MgCl2, 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 (µM) 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 µM.

*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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146

العربي ملخصال

و هـو المـادة الفعالـة فـي ,من الموثق جيدا في المراجـع أن الكركـومين

لـه فعاليـة ,رموريـك المسـتخلص مـن ريزومـة الكركومـا لونجـا تمسحوق ال

ـدة و أثـره تو من أكثر هذه الفعاليا. المجال بيولوجية واسعة ـادة لألكس كمـواد مض

ـية الـى جانـب الديدانالفطريات وااللتهابات و السرطان و الميكروبات و ـات و الحساس و الفيروس

ـ . من األمراض السرطانية واقيفعاليته ك ـة الت ـبه فـي وقد وجد أن لكثير من المـواد الطبيعي ي تش

ـة الكركومين تركيبها الكيميائي ـال .فعاليات بيولوجيـة مختلف ـبيل المث ـذكر علـي س المـواد : ون

ـة سـوا ,المحلقنةو حورةالم الهبتانويد أريل على ثنائي ةحتويالم ـر هبتانودي ـتبدالت غي ءكذلك مس

.المستخلصة من النباتات أو المحضرة كيميائيا

ـة ا ابن ـات الكيميائي ـميم بعـض المركب ـم تص ـد ت لتـي ءا على ما سبق من المعلومات فق

ـة ,تحتوي على مجموعات األلكايل أمينو ـر المتجانس ـات غي ـيكلوألكايل أمينـو و أمينـو الحلق الس

ـد . األسايل بمجموعة الفينول الموجـودة فـي مركـب الكركـومين قنطرةترتبط من خالل التي و ق

.تصميم بعض استرات الكركومين كذلك تم

ـرق و ـة فـي المراجـع لط ـية الموثق يشمل البحث أيضا مناقشة بعض النظريات األساس

ـير بعـض ا . تحضير المركبات المصممة لمـواد و لقد استعرض البحث المراجع التي تخـص تحض

ـير . مثل األسيالت و األمينات و األسترات ـتعرض البحـث الطريقـة المتميـزة لتحض ـذلك اس و ك

ـيد البوريـك مـع مختلـف ـيتون المـرتبط بأكس ـيتايل أس بعض المركبات الوسيطة بتفاعل األس

.ر الكركومين وايثايل الكركومين االبتدائيان بهذه الطريقةيحضت تم و لقد .األلدهيدات

147

ـم تم تحضير و ـدة و ه ـيطة جدي ـات وس أربعين مركبا نهائيا جديدا الى جانب ثمانية مركب

:كالتالي

: المركبات االبتدائية -

الكركومين �

ايثايل الكركومين �

:المركبات الوسيطة -

و )ب3(كلورو بروبيونايل الكركومين و ثنائي أوكسو )ا3(سو كلورو أسيتايل الكركومين ثنائي أوك �

و ثنائي أوكسو كلورو بروبيونايل ايثايل ) ج3(تايل ايثايل الكركومين ثنائي أوكسو كلورو أسي

).د3(الكركومين

بيونايل كلوروبرو- 4- ميثوكسي- 3و )ا4(أسيتايل أوكسي البنزالدهايد كلورو- 4- ميثوكسي- 3 �

- 3 و) ج4(أسيتايل أوكسي البنزالدهايد كلورو- 4- ايثوكسي- 3 و) ب4(أوكسي البنزالدهايد

).د4(وبيونايل أوكسي البنزالدهايد كلوروبر- 4- ايثوكسي

:المركبات النهائية -

ميثوكسي (- 3- )أمينوأسيتايل أوكسي)سيكلوألكايل أو الحلقة غير المتجانسة(ألكايل- 4(ثنائي- 7, 1 �

).ز- ا 5(دايون - 5, 3- هيبتادايين- 6, 1- )فينايل

- 3- )ونايل أوكسيأمينوبروبي)سيكلوألكايل أو الحلقة غير المتجانسة(ألكايل- 4(ثنائي- 7, 1 �

).ن- ح 5(دايون - 5, 3- هيبتادايين- 6, 1- )ميثوكسي فينايل(

ايثوكسي (- 3- )أمينوأسيتايل أوكسي)سيكلوألكايل أو الحلقة غير المتجانسة(ألكايل- 4(ثنائي- 7, 1 �

).ز - أ 5(دايون - 5, 3- هيبتادايين- 6, 1- )فينايل

- 3- )أمينوبروبيونايل أوكسي)نسةسيكلوألكايل أو الحلقة غير المتجا(ألكايل- 4(ثنائي- 7, 1 �

).ن- ح 6(دايون - 5, 3- هيبتادايين- 6, 1- )ايثوكسي فينايل(

ميثوكسي (- 3- )أمينوأسيتايل أوكسي)سيكلوألكايل أو الحلقة غير المتجانسة(ألكايل- 4(ثنائي- 7, 1 �

).ز 5(دايون - 5, 3- هيبتادايين- 6, 1- )فينايل

148

- 6, 1- )ميثوكسي فينايل(- 3- )أسيتايل أوكسي)مستبدالت السلفونايل أميدو- 4(- 4(ثنائي- 7, 1 �

).- أ 7(دايون - 5, 3- هيبتادايين

- 6, 1- )ايثوكسي فينايل(- 3- )أسيتايل أوكسي)مستبدالت السلفونايل أميدو- 4(- 4(ثنائي- 7, 1 �

).- ب 7(دايون - 5, 3- هيبتادايين

).ب 8(و ثنائي أوكسي أدامانتويل ايثايل الكركومين ) أ 8(ين ثنائي أوكسي أدامانتويل الكركوم �

).د 8(و ثنائي أوكسي هيبتانويل ايثايل الكركومين ) ج 8(ثنائي أوكسي هيبتانويل الكركومين �

8(ايثايل الكركومين )ثيينويل- 2(- و ثنائي أوكسي) هـ 8(الكركومين ) ثيينويل- 2(- ثنائي أوكسي �

).و

ـائل الطيفيـة التـي تم اثبات التركيب الب و نائي للمركبات المحضرة بواسطة مختلـف الوس

ـرنين ـدروجين النـووي تشمل األشعة تحت الحمـراء و ال ـرنين المغناطيسـي للهي النـووي و ال

.و كذلك طيف الكتلة 13و الكربون الثنائي األبعاد المغناطيسي للهيدروجين

ـا ـيم فعاليته ـرة لتقي ـات المحض كمـواد قاتلـة للـألورام و لقد تم اختيار بعـض المركب

ـار الكركـومين و ايثايـل الكركـومين و ـاتج مـن اختب السرطانية و مقارنة هذا التأثير مع مثيله الن

ـرطانية ـا الس ـم و. كذلك مركب دوكسوروبيسين المعروفين بنشاطهم القاتل للخالي ـا ت بعـض راختي

ـا مثـل تا السرطانية لالخالي ـا خالالعيين تأثير المركبات المختارة عليه ـرطان الي ـ (ية س االميالنوم SK-

MEL (, سرطان الجلد)اابيدرمال كارسينوم KB (, ـرطان خاليا ـدد س ـدى غ ـال (اللبنيـة الث داكت

BT-459 كارسينوما ـيض ) ـرطان المب ـا س و كذلك خالي )SK-OV-3 ( . ـتخدمت انسـجة ـد اس و لق

ـرة كلـى ال ـا الفيرو المأخوذة من الخاليا الليفية لكلى القرد و كذلك خاليا قش ـة للخالي ـر كأمثل خنزي

.غير السرطانية

ـن مركباتال بعضو لقد تم اثبات فعالية ـائي - 7, 1 :همم ـازول - 5(- 4(ثن - 2- ميثايـل ثيادي

- 7, 1و ) ج 5(دايـون - 5, 3- هيبتادايين- 6, 1- )ميثوكسي فينايل(- 3- )أمينوأسيتايل أوكسي)آيل

- 5, 3- هيبتادايين- 6, 1- )وكسي فينايلميث(- 3- )أمينوأسيتايل أوكسي)كلورو ايثايل- 2(ثنائي- 4(ثنائي

ـازول - 5(- 4(ثنائي- 7, 1و ) هـ 5(دايون - 3- )أمينوبروبيونايـل أوكسـي )آيـل - 2- ميثايل ثيادي

ـادايين - 6, 1- )ميثوكسي فينايل( ـائي - 7, 1و ) ى 5(دايـون - 5, 3- هيبت ميثوكسـي - 6(- 4(ثن

6(دايـون - 5, 3- يبتادايينه- 6, 1- )ايثوكسي فينايل(- 3- )أمينوأسيتايل أوكسي)آيل- 2- بنزوثيازول

149

ـر خدمالخاليا السرطانية المستلبعض كمواد قاتلة ) ب ـا غي ة في االختبار مع عدم تأثيرها على الخالي

.السرطانية

للخاليا السرطانية بواسطة لمواد المحضرة كمضاداتا و تم محاولة التعرف إلى ميكانيكية عمل

يلمركبات المحضرة أللم يثبت و .2و 1أيزوميريز اختبار قدرة هذه المواد على تثبيط انزيم التوبو

المواد كمواد قاتلةهذه مما يثبت أن ميكانيكية عمل 2و أ 1تأثير على تثبيط انزيم التوبوأيزوميريز

.لخاليا السرطانية قد تم من خالل ميكانيكية أخرىل

150

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