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T HESIS

S YNTHESIS AND ANTICANCER ACTIVITY OF NEW P YRROLO[2,1-C][1,4]

BENZODIAZEPINES AND COMBRETASTATIN DERIVATIVES

T HESIS

SUBMITTED TO K AKATIYA UNIVERSITY

FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

(IN CHEMISTRY )

B Y

ADLA MALLA REDDY

UNDER THE SUPERVISION OF

DR. AHMED K AMAL

DIVISION OF ORGANIC CHEMISTRY -IINDIAN INSTITUTE OF CHEMICAL TECHNOLOGY , H YDERABAD

JUNE, 2011

1



T HESIS

S YNTHESIS AND ANTICANCER ACTIVITY OF NEW P YRROLO[2,1-C][1,4]

BENZODIAZEPINES AND COMBRETASTATIN DERIVATIVES

T HESIS

SUBMITTED TO K AKATIYA UNIVERSITY

FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

(IN CHEMISTRY )

B Y

ADLA MALLA REDDY

UNDER THE SUPERVISION OF

DR. AHMED K AMAL

DIVISION OF ORGANIC CHEMISTRY -IINDIAN INSTITUTE OF CHEMICAL TECHNOLOGY , H YDERABAD

JUNE, 2011

2



T HESIS

DECLARATION

I hereby declare that the original research work embodied in the thesis

entitled “S YNTHESIS AND ANTICANCER ACTIVITY OF NEW P YRROLO[2,1-C][1,4]

BENZODIAZEPINES AND COMBRETASTATIN DERIVATIVES“ submitted to Kakatiya

University for the award of degree of Doctor of Philosophy (Ph.D) in

Chemistry of the faculty of Physical Sciences is the outcome of the

investigation carried out by me under the supervision of Dr. Ahmed Kamal,

Scientist H (Director level), IICT, Hyderabad. I declare that the workincorporated is original and due acknowledgement has been made wherever

it is not so. The same has not been submitted elsewhere for any degree or

diploma.

I also declare that I myself solely responsible for the genuineness of

the findings/observations pertaining to these study in order to complete this

thesis.

Adla Malla Reddy

3



T HESIS

Dedicated to My Beloved Parents

4



T HESIS

ACKNOWLEDGEMENTS

It gives me an immense pleasure and pride to express my sincere gratitude and respect

for my teacher and guide Dr. Ahmed Kamal , Directors Grade Scientist, Division of Organic

Chemistry-I, IICT, Hyderabad, for his expert and inspiring guidance. I proclaim myindebtedness to him for his constant encouragement along with the useful suggestions and

constructive criticisms during the entire tenure of this work. I consider myself fortunate in that it

would have been impossible to achieve this goal without his support and care.

I am indebted to the director Dr. J. S. Yadav for having given me an opportunity to

carryout the work and allowing me to submit in the form of thesis. It is a great privilege for me

to be associated with Dr. J. M. Rao , Head, Division of Organic Chemistry–I for his kind help

and encouragement.

My heartful thank to Dr. M. Venkateswara Rao and Dr. Manika Pal Bhadra for their

constant support, encouragement and timely advice. I take this opportunity to record my

appreciation to spectroscopic and analytical divisions of IICT, especially to Dr. A. C. Kunwar

and Dr. R. Srinivas.

I am grateful to Prof. G. Venkateshwar Rao , Head, Department of Chemistry, Kakatiya

University, Prof. Ch. Sanjeeva Reddy (Board of studies Chairman, K.U.), Prof. T. Bhasker

Rao (Dean, Faculty of Science, K.U.) and Prof. N. Vasudeva Reddy for their invaluable

suggestions and advices while writing thesis.

My special thanks to Dr. Rajesh V.C.R.N.C. Shetti, Paidakula Suresh, Dr. B. Rajendra

Prasad , Dr. N. Shankaraiah, J.N.S.R.Murthy, N. Sankara Rao and Dr. Janaki Ramaiah for

their cooperation in my research.

I am grateful to my lab mates K.Srinivas Reddy , Devaiah, Laxma reddy , Kaleem ,

P.Praveen, Naseer , Krishnaji , Venkat , Adil , Malik , Ameer , Rajender , Azeez , Bharathi ,

Surendra , Dastagiri , Prabhakar, Venkatreddy , Sreekanth, Vishwanath , Ramakrishna , Kashi

Reddy , Raju, Ratna Reddy, Sheshadri , Subbareddy , Santhosh Reddy, Saidi Reddy, balakrihna,

Bazi, Fazil, Asharf, Srinivas, Narasimha , Swapna , Jaki, Farheen Sulthana, Bharath, Ali,

Premsagar, Subbarao, Imnthiaz and my other labmates Naresh , Somaiah, Venkataiah,

Gourishankar, Markandeya, Jitender and Prasad.

I acknowledge the help received from Usha , Sainadh, Shyam , Chandrashekar , Balaraj ,

and Padma.

5



T HESIS

I always feel thankful to have friends like Kota Srinivas, Alli Satish, Udutala Komarelli,

Srinivas yadav, Sattenapalli Narasimha, P. Venkata Raji Reddy, Bala Bhaskar, Pitta Bhaskar

Reddy, Madhuraju, Doma Mahender Reddy, Muppidi Venugopal and Gangarapu Srinivas.

It is also an appropriate time to remember all my teachers and professors who at various

stages of my educational carrier encouraged me to reach this level and it all is the fruit of their

teaching and blessings.

I owe more than myself to my beloved father Sri Indra Reddy and mother Mallakka , who

always dreamt that I reach golden heights and sky is the only limit to my success. To them I

dedicate this thesis. On this occasion my heart goes for my brothers Mr . Srinivas Reddy , Mr .

Damodar Reddy and my elder brother’s children Pooja and Manoj Reddy.

My heartfelt thanks to my life partner Smt . Swapna for her unflinching moral support at

every stage and my sweet kisses to my child baby Ananya.

I take this opportunity to record my appreciation to spectroscopic and analytical division

of IICT.

I also thank National Cancer Institute (NCI), USA, Advanced Center for Treatment,

Research and Education in Cancer (ACTREC), Navi Mumbai and Regional Research

Laboratory (RRL), Jammu for helping in the biological studies.

Financial assistance from the Council of Scientific and Industrial Research (CSIR), New

Delhi in the form of fellowship is gratefully acknowledged. Finally, I thank Director, IICT, for allowing me to submit my work in the form of thesis.

(Adla Malla Reddy )

6



T HESIS

GENERAL R EMARKS

1H NMR and 13C NMR spectra are recorded on Varian Gemini 200 or Varian Unity 400 or

Varian Inova 500 or Bruker Avance 300 MHz. Making a solution of samples in

CCl4/CDCl3 (1:1) solvent using tetramethylsilane (TMS) as the internal standard unless

otherwise mentioned, and are given in the δ scale. The standard abbreviations s, d, t, q, m,

dd, dt, ABq, br s refer to singlet, doublet, triplet, quartet, multiplet, doublet of a doublet,

doublet of a triplet, AB quartet and broad singlet respectively.

Mass spectra recorded on CEC-21-110B, Finnigan Mat 1210 or MICROMASS-7070

spectrometers operating at 70eV using a direct inlet system. If necessary, FABMS is

recorded.

Melting points are determined on an Electrothermal melting point apparatus and are

uncorrected.

All reactions are monitored by thin layer chromatography (TLC) carried out on 0.25 mm E.

Merck silica gel plates (60F-254) with UV light, iodine as probing agents. Acme (India)

silica gel (finer than 200 mesh) is used for flash chromatography.

The reactions wherever anhydrous conditions needed are carried out under the positive

pressure of nitrogen atmosphere using dry and freshly distilled solvents.

All solvents and reagents were purified by standard techniques. All evaporation of solvents

was carried out under reduced pressure on Buchi-RE-121 rotary evaporator below 45 °C.

Yield reported are isolated yields of material judged homogeneous by TLC and NMR

spectroscopy.

The names of all compounds given in the experimental section were taken from

ACD/Name, Version 1.0.

7



T HESIS

ABBREVIATIONS

BF3

.

OEt2 : Boron trifluoride diethyletherateBnBr : Benzylbromide

n-BuLi : n-Butyl Lithium

CaCO3 : Calcium carbonate

CCl4 : Carbontetrachloride

CH3CN : Acetonitrile

CDCl3 : Deuterated chloroform

Cs2CO3 : Cesium carbonate

DCC : N, N'-Dicyclohexylcarbodiimide

DCM : Dichloromethane

DIBAL-H : Diisobutylaluminium hydride

DMF : N, N'-Dimethylformamide

DMSO : Dimethylsulfoxide

EtSH : Ethanethiol

EDCI : 1-[3-(Dimethylamino)propyl]-3-

ethylcarobodiimidehydrochloride

EtOAc : Ethyl acetate

HgCl2 : Mercuric chloride

HNO3 : Nitric acid

HOBt : 1-Hydroxybenzotriazole

H2SO4 : Sulphuric acid

HCl : Hydrochloric acid

IPA : Isopropyl alcohol

K 2CO3 : Potassiumcarbonate

KOAc : Potassium acetate

LiBr : Lithiumbromide

LiOH : Lithiumhydroxide

8



T HESIS

MeOH : Methylalcohol

NaBH4 : Sodiumborohydride

NaClO2 : Sodium chlorite

NaOH : Sodium hydroxide

NaOMe : Sodium methoxide

NaH : Sodium hydride

NH2NH2.H2O : hydrazine Hydrate

SnCl2 : Stannous chloride

SnCl4 : Stannic chloride

SOCl2 : Thionylchloride

TBAF : Tetrabutylammoniumfluor

TBDMSCl : tert-Butyldimethylchlorosilane

TFA : Trifluoroacetic acid

TMSCl : Chlorotrimethylsilane

THF : Tetrahydrofuran

TEA : Triethyl amine

TPP : Triphenylphosphine

CONTENTS

Page No.

9



T HESIS

S YNOPSIS 12

CHAPTER-I GENERAL INTRODUCTION

Introduction to Cancer 33

Current Area of Work 42

Objectives of Present Work 62

References 65

CHAPTER-II S YNTHESIS AND BIOLOGICAL EVALUATION OF CHALCONE-P YRROLOBENZODIAZEPINE CONJUGATES AS ANTICANCER AGENTS

Introduction 73

Present Work 79

Biological Activity 84

Experimental 87

References 108

CHAPTER-III S YNTHESIS AND BIOLOGICAL EVALUATION OF COMBRETASTATIN

DERIVATIVES AS ANTICANCER AGENTS

Introduction 114

Present work 119

Biological Activity 124

Experimental 129

References 152

CHAPTER-IV

SECTION-A S YNTHESIS AND BIOLOGICAL EVALUATION OF BENZYLIDENE-9(10H)-ANTHRACENONE LINKED P YRROLOBENZODIAZEPINES AS ANTICANCER AGENTS

Introduction 157

10



T HESIS

Present work 160

Biological Activity

164

Experimental 166

References 178

SECTION-B S YNTHESIS AND BIOLOGICAL EVALUATION OF CHALCONE-P YRROLOBENZODIAZEPINE DIMERS AS ANTICANCER AGENTS

Introduction 180

Present work 185

Biological Activity

188

Experimental 191

References 202

LIST OF PUBLICATIONS AND PATENTS 207

S YMPOSIUM AND CONFERENCES 211

11



T HESIS

SS YNOPSIS YNOPSIS

The work carried in the research tenure has been compiled in the form

of a thesis entitled “SS YNTHESIS YNTHESIS AANDND AANTICANCERNTICANCER AACTIVITY CTIVITY OOFF NNEWEW PP YRROLO YRROLO[2,1-[2,1-CC]]

[1,4][1,4]BENZODIAZEPINESBENZODIAZEPINES AANDND CCOMBRETASTATINOMBRETASTATIN DDERIVATIVESERIVATIVES“. The main aim of this

work has been to design and synthesize biologically active molecules like

pyrrolobenzodiazepines and combretastatin which are known for potent

12



T HESIS

anticancer activity. The thesis has been divided into four chapters. CCHAPTERHAPTER II

gives the introduction about the chemotherapy of cancer, DNA binding ability

with particular reference to pyrrolobenzodiazepines. CCHAPTERHAPTER IIII describes the

synthesis of a new class of C8-linked chalcone-pyrrolobenzodiazepine

analogues and evaluation of their anticancer activity. CCHAPTERHAPTER IIIII describes

the synthesis and anticancer evaluation of combretastatin derivatives.

CCHAPTERHAPTER IVIV comprises of two sections, SECTION-A deals with the synthesis and

biological evaluation of benzylidineanthracenone linked

pyrrolobenzodiazepines as anticancer agents. SECTION-B deals with synthesis

of chalcone- pyrrolobenzodiazepine dimers and evaluation of their DNA-

binding ability and cytotoxicity.

CCHAPTERHAPTER II –– GGENERALENERAL IINTRODUCTIONNTRODUCTION

This chapter describes the general introduction about cancer and

pyrrolobenzodiazepines.. Cancer is one of the leading causes of death in the

industrialized world. Cancer arises when a population of cells within the body

escapes from normal control. It involves the conversion of any normal cell to

a cancerous cell showing tandem replication and cell division at much faster

rate in comparison to the normal cells. Cancer cells often travel to otherparts of the body where they begin to grow and replace normal tissue. This

process is called metastasis, which occurs as the cancer cells get into the

bloodstream or lymph vessels of our body. It is now clear that

chemotherapy’s most effective role in solid tumors is as an adjuvant to the

initial therapy by surgical or radiotherapeutic procedures. Chemotherapy

becomes critical to effective treatment because only systemic therapy can

attack micrometastases. These agents can be categorized into functional

subgroups like alkylating agents, antimetabolites, antibiotics, and

antimitotics.

The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are well known class of

antitumour antibiotics with sequence selective DNA binding ability that are

derived from various Streptomyces species. The first PBD antitumour

13



T HESIS

antibiotic anthramycin has been described by Leimgruber and co-workers in

1963 and since then a number of compounds have been developed based

on the PBD ring system leading to some efficient DNA binding ligands. Their

mode of interaction with DNA has been extensively studied and it is

considered unique as they bind within the minor groove of B form DNA.

These compounds exert their biological activity by covalently binding to the

C2-amino group of guanine residue in the minor groove of DNA through the

imine or imine equivalent functionality at N10-C11 of PBD moiety.

H3C

OH

N

HN

O

OCH3

CONH2

Anthramycin

1

2

45

36

7

8 9

10

11

N

N

O

HO

H3CO

Tomaymycin

H

N

NO

H3CO

O

HON

N

O

H

OCH3

SJG-136

H

11a

Figure 1. Biologically important DNA interactive natural/unnatural PBDs.

The molecular modeling studies suggested that C8 would be the

preferred position for attachment of second interacting group to develop the

unsymmetrical DNA cross-linking agents. A number of naturally occurring

and synthetic compounds based on PBD ring system, such as anthramycin,

tomaymycin, DC-81 and its dimers (presently, one of the dimer SJG-136 is

under clinical evaluation), have shown varying degrees of DNA binding

affinity and anticancer activity. In view of the importance of these molecules,

there is considerable interest in the structural modification of PBD's,

particularly at C8 position to improve upon their DNA binding potential andsequence selectivity.

14



T HESIS

C

OH

N

HN

H

O

NH2

O

HOH

N NH2NDNA

1110

H3C

OH

N

HN

H

O

NH2

O

NHH

HNN

DNA

11

10

Anthramycin

Figure 2. Mechanism of action of PBDs with DNA.

CCHAPTERHAPTER II - SII - S YNTHESIS YNTHESIS AANDND BBIOLOGICALIOLOGICAL EEVALUATIONVALUATION OOFF CCHALCONEHALCONE--PP YRROLOBENZODIAZEPINE YRROLOBENZODIAZEPINE CCONJUGATESONJUGATES AASS AANTICANCERNTICANCER AAGENTSGENTS

This chapter describes the synthesis and biological activity of

chalcone-pyrrolobenzodiazepine conjugates. Chalcones are a class of

anticancer agents that have shown promising therapeutic efficacy for the

management of human cancers. Chemically they consist of open-chain

flavonoids in which the two aromatic rings are joined by a three-carbon α,β-

unsaturated carbonyl system. Recent studies revealed that these chalcones

had shown a wide variety of anticancer, anti-inflammatory, antiinvasive,

antituberculosis, and antifungal activities. The trimethoxychalcone (13b) is

potential anticancer agent and binding strongly to tubulin at a site sharedwith, or close to, the colchicines binding site. Chalcones have attracted more

interest in recent years because of their diverse pharmacological properties.

The parent molecule of chalcone derivatives and its hydroxyl chalcones have

been reported for their antiproliferative and antitumor activity. In

continuation of efforts towards the design and synthesis of new PBD

analogues, we synthesized chalcone-PBD analogues. The DNA binding

characteristics of these conjugates have been evaluated by thermal

denaturation studies.

Synthesis of these chalcone linked PBD analogues (20a-f and 23a-c)

has been carried out by employing the (2S)-N-[4-benzyloxy-5-methoxy-2-

nitrobenzoyl]proline methylester (7), which is obtained according to the

literature method starting from vanillin. This upon selective reduction by

15



T HESIS

employing DIBAL-H, and protection with TMSCl/EtSH followed by deprotection

using BF3.OEt2/EtSH affords the precursor 10 as shown in Scheme 1.

MeO

HO

MeO

HO

MeO

HO

MeO

BnO

MeO

BnO

HOH OMe

OMeOMe

O O O

OO

NO2

MeO

BnO

MeO

HO

N

O

CH(SEt)2NO2

MeO

BnO

OH

O

NO2

(i) (ii)

(iii)

(iv)(v)

(vi)

(vii)

(viii)

(ix)

1 2 3

456

7 8

910

N

NO2

O

COOMe

MeO

BnO

N

NO2

O

CHO

MeO

BnO

N

NO2

O

CH(SEt)2

Scheme 1. Reagents and conditions: (i) NH2SO3H, NaClO2, H2O, rt, 2 h, 90%; (ii) H2SO4,

MeOH, reflux, 4 h, 85%; (iii) benzylbromide, K 2CO3, acetone, reflux, 24 h, 92%; (iv) SnCl4,

fuming HNO3, CH2Cl2, 5 min, -25 oC, 78%; (v) 2N LiOH, MeOH, H2O, THF (1:1:3), rt, 12 h,

83%; (vi) SOCl2, C6H6, L-proline methylester hydrochloride, THF, 1-2 h, rt, 85%; (vii) DIBAL-H,

CH2Cl2, 0.5 -1 h, -78 oC, 65%; (viii) EtSH, TMSCl, CH2Cl2, 8-12 h, rt, 72%; (ix) BF3.OEt2, EtSH,

CHCl3, rt, 8 h, 75%;

The preparation of chalcone intermediates 14a-f and 17a-c has been

carried out by synthetic sequence illustrated in Schemes-2 and 3. Claisen-

Schmidt condensation of trimethoxyacetophenone with benzaldehydes by

using ethanol as solvent in the presence of aqueous KOH gives

trimethoxychalcones 13a,b. The cyclic chalcone 16 has been prepared

under the same reaction conditions by condensing 1-indanone with vaniline

to give indanochalcone. These trimethoxy and indano chalcones undergo

16



T HESIS

alkylation of hydroxyl group with dibromoalkanes by using K 2CO3 as a base in

dry acetone to afford precursors 14a-f and 17a-c.

MeO

MeO

OMe

CH3

O CHO

R

OH

+

MeO

MeO

OMe

O

OH

R

MeO

MeO

OMe

O

O

R

Br ( )n

(i)

(ii)

14a-f

11 12a, b13a, b

14a; R = H, n = 214b; R = H, n = 3

14c; R = H, n = 414d; R = OMe, n = 214e; R = OMe, n = 314f ; R = OMe, n = 4

Scheme 2. Reagents and conditions: (i) aq.KOH, ethanol, 4 h; (ii) dibromoalkane, acetone,

K 2CO3, reflux, 24 h.

17



T HESIS

17a; n = 217b; n = 317c; n = 4

CHO

OMe

OH

+

(i)

(ii)

17a-c

15 12b

16

O O

OH

OMe

O

O

OMe

Br ( )n

Scheme 3. Reagents and conditions: (i) aq.KOH, ethanol, 4 h; (ii) dibromoalkane, acetone,

K 2CO3, reflux, 24 h.

Compound 10 has been coupled to compounds 14a-f and 17a-c in the

presence of K 2CO3 and dry acetone under reflux conditions to give

corresponding nitro compounds 18a-f and 21a-c. These nitro compounds

upon reduction with SnCl2.2H2O in methanol under reflux conditions give

amino compounds 19a-f and 22a-c. the amino compounds upon

deprotection followed by cyclization with HgCl2/CaCO3 to provide thecorresponding imines 20a-f and 23a-c (Schemes 4 & 5).

18



T HESIS

MeO

MeO

OMe

O

O

R

Br ( )n

14a-f

HO

MeO

NO2

O

N

CH(SEt)2

10

+

O

MeO

NO2

O

N

CH(SEt)2O

O

MeO

MeO

OMe

R

( )n

O

MeO

NH2

O

N

CH(SEt)2O

O

MeO

MeO

OMe

R

( )n

O

MeO

O

O

MeO

MeO

OMe

R

( )n

N

N

O

H

18a-f

19a-f

20a-f

(i)

(ii)

(iii)

Scheme 4. Reagents and conditions: (i) K 2CO3, acetone, 12 h, reflux; (ii) SnCl2.2H2O, MeOH,4 h, reflux; (iii) HgCl2, CaCO3, MeCN, H2O, (4:1) 12 h, rt.

19



T HESIS

n = 2, 3, 4

HO

MeO

NO2

O

N

CH(SEt)2

10

+

O

MeO

NO2

O

N

CH(SEt)2O

OMe

( )n

O

MeO

NH2

O

N

CH(SEt)2O

OMe

( )n

O

MeO

O

OMe

( )n

N

O

H

21a-c

22a-c

23a-c

(i)

(ii)

(iii)

17a-c

O

O

OMe

Br ( )n

O

O

O


The thermal denaturation studies show that these conjugates (20a–f

and 23a-c) possess good DNA binding ability compared to DC-81. These

findings suggest that these conjugate agents bind more efficiently to DNA

than DC-81. Compounds 20a-f and 23a-c exhibit significant anticancer

activity against eight cancer cell lines with GI50 values ranging from <0.01-

2.7 μM, in comparison to adriamycin (GI50, <0.01-14.7 μM). According to thein vitro screening data, compound 46b has significant cytotoxicity against all

the cancer cell lines with GI50 values ranging from <0.01-0.17 μM 0.1-2.19 µM

and has shown more potency against PC-3 prostate cancer cell line with GI50

<0.01 μM.

20



T HESIS

CCHAPTERHAPTER III: SIII: S YNTHESIS YNTHESIS AANDND BBIOLOGICALIOLOGICAL EEVALUATIONVALUATION OOFF CCOMBRETASTATINOMBRETASTATIN DDERIVATIVESERIVATIVES AASS AANTICANCERNTICANCER AAGENTSGENTS

This chapter describes the design, synthesis, and in vitro cytotoxicity

of novel analogues of combretastatin, and its chalcone, pyrazoline

derivatives with amino benzothiazoles. Tubulin is a heterodimeric protein

consisting of A and B subunits. During cellular division upon binding of GTP,

tubulin polymerizes into microtubules. This formation of microtubule is

essential for chromosome separation and formation of two daughter cells.

When ligands that interact with tubulin are present, a reduction in cellular

division is observed and shows anticancer activity. Tubulin having colchicine

binding site and if any ligand binds to this site prevents the tubulin

polymerization. Colchicine and combretastatin-A-4 [CA-4] are the good

examples as tubulin binding agents. Combretastatin A-4 is a naturally

occurring stilbene and isolated from the African willow tree (Combretum

caffrum). CA-4 shows interesting anticancer potential due to its antitubulin

properties. It strongly binds to the colchicine site of tubulin thus preventing

tubulin polymerization and causes antimitotic effect. In view of the

interesting biological properties exhibited by these molecules it was

considered of interest to synthesize analogues of combretastatin A-4derivatives with 2-aminobenzothiazoles and these new compounds exhibit

potent anticancer activity.

MeO

MeO

OMe

OMe

OH

combretastatin A-4 colchicine

MeO

MeO

OMe

O

OMe

NHCOCH3

Figure 3. Potential inhibitors of tubulin polymerization

The precursor (Z)-2-(2-methoxy-5-(3

trimethoxystyryl)phenoxy)acetic acid 9 has been prepared by employing

commercially available isovanillin. Hydroxy group protection of isovanillin

21



T HESIS

with TBDMS-Cl followed by reduction of aldehyde group with NaBH4 gives the

alcohol 3. The benzyl alcohol was converted to benzyl bromide 4 using LiBr

followed by salt formation with PPh3 to give the compound 5. This on Wittig

reaction with trimethoxybenzaldehyde gives TBDMS-protected

combretastatin A-4, which upon deprotection with TBAF gives the compound

combretastatin A-4, 7. This upon etherification with α-bromoethyl acetate in

the presence of K 2CO3 gives the ester 8, which on hydrolysis with LiOH

affords the acid 9 (Scheme 1).

OMe

OH

CHO

OMe

OTBDMS

CHO

OMe

OTBDMS

CH2OH

OMe

OTBDMS

CH2Br

OMe

OTBDMS

CH2PPh3Br

MeO

MeO

OMe

OMe

OTBDMSMeO

MeO

OMe

OMe

OH

(i) (ii) (iii)

(iv)

(v)(vi)

MeO

MeO

OMe

OMe

OOEt

OMeO

MeO

OMe

OMe

OOH

O

(vii)

(viii)

1 2 3 4

56

7

8 9

Scheme 1. Reagents and conditions: i) TBDMS-Cl, TEA, DMF; ii) NaBH4, MeOH ; iii) LiBr,

THF ; iv) PPH3, toluene ; v) n-BuLi, THF,-20 oC, trimethoxybenzaldehyde ; vi) TBAF, THF ; vii)

2-bromoethyl acetate, K 2CO3, DMF ; viii) LiOH, THF, H2O

The preparation of chalcone derivative 13 has been carried out by

synthetic sequence illustrated in Scheme-2. Claisen-Schmidt condensation of

trimethoxyacetophenone 10 with isovaniline by using ethanol as solvent in

the presence of aqueous KOH gives trimethoxychalcones 11. This upon

etherification with α-bromoethyl acetate in the presence of K 2CO3 gives the

ester compound 12. The ester compound on hydrolysis with LiOH affords the

22



T HESIS

chalcone acid (E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-

enyl)phenoxy)aceticacid 13.

MeO

MeO

OMe

O

MeO

MeO

OMe

O

OMe

OH

OMe

OH

CHO

+

13

MeO

MeOOMe

O

OMe

OOEt

O

MeO

MeOOMe

O

OMe

OOH

O

10 11

12

(i)

(ii)

(iii)

1

Scheme 2. Reagents and conditions: i) aq. KOH, ethanol, 12h; ii) 2-bromoethyl acetate,

K 2CO3, DMF, 12h; iii) LiOH, THF, H2O

The preparation of pyrazoline derivative 16 has been carried out by

the synthetic sequence illustrated in Scheme-3. Cyclization of

trimethoxychalcone 11 with hydrazine hydrate in acetic acid under reflux

conditions gives pyrazoline derivative 14. This upon etherification with α-

bromoethylacetate in the presence of K 2CO3 gives the ester compound 15.

The ester compound on hydrolysis with LiOH affords pyrazoline acid 2-(5-(1-

acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)-2-met

hoxyphenoxy)acetic acid 16.

23



T HESIS

MeO

MeO

OMe

O

OMe

OH MeO

MeO

OMe

N

OMe

OH

N

O

16

MeO

MeO

OMe

N

OMe

O

N

O

OEt

O

MeO

MeO

OMe

N

OMe

O

N

O

OH

O

11 14

15

(i)

(ii)

(iii)

Scheme 3. Reagents and conditions: i) NH2NH2.H2O, Acetic acid, reflux, 14h; ii) 2-

bromoethyl acetate, K 2CO3, DMF, 12h; iii) LiOH, THF, H2O

MeO

MeO

OMe

OMe

OOH

O

9

N

SH2N+

MeO

MeO

OMe

OMe

ONH

O

S

N

17

18a, R = -H18b, R = -NO218c, R = -F18d, R = -Cl18e, R = -OMe18f, R = -OCF318g, R = -Me18h, R = -CF3

18i, R = -OEt

R

R

18 a-i

(i)

Scheme 4. Reagents and conditions: i) EDCI/HOBT, DCM, 14-16h

The synthesis of combretastatin-benzothiazole analogues 18a-i is

outlined in Scheme 4. The combretastatin acid 9 undergoes amide bond

formation with 2-aminobenzothiazoles in the presence of EDCI/HOBt in

24



T HESIS

dichloromethane to give the desired combretastatin-benzothiazole analogues

18a-i.

19a R = -H19b R = -NO219c R = -F19d R = -Cl19e R = -OMe19f R = -OCF319g R = -Me19h R = -CF319i R = -OEt

N

SH

2N

+

17

R

13

MeO

MeO

OMe

O

OMe

OOH

O

MeO

MeO

OMe

O

OMe

ONH

O

S

N R

19a-i

(i)


The synthesis of chalcone-benzothiazole derivatives 19a-i is outlined

in Scheme 5. The chalcone acid 13 undergoes amide bond formation with 2-

amino benzothiazoles in the presence of EDCI/HOBt in dichloromethane to

give the desired chalcone-benzothiazole analogues 19a-i.

20a R = -H20b R = -NO220c R = -F

20d R = -Cl20e R = -OMe20f R = -OCF320g R = -Me20h R = -CF320i R = -OEt

(i)

N

SH2N

+

17

R

20a-i

16

MeO

MeO

OMe

N

OMe

O

N

O

OH

O

MeO

MeO

OMe

N

OMe

O

N

O

NH

O

S

N R


25



T HESIS

The pyrazolineacid 16 undergoes amide bond formation with 2-

aminobenzothiazoles in the presence of EDCI/HOBt in dichloromethane to

give the desired pyrazoline-benzothiazole analogues 20a-I (Scheme-6). The

anticancer activity of the synthesized compounds was evaluated by the

National Cancer Institute (NCI), USA. Fourteen compounds were selected for

NCI-60 cell line anticancer screening program by National Cancer Institute

(NCI), Bethesda, USA. After preliminary screening on the tumour cell lines,

these compounds were tested for five dose concentration on a panel of 59

human tumour cell lines derived from nine different cancer types: leukaemia,

lung, colon, CNS, melanoma, ovarian, renal, prostate and breast. These

compounds exhibited significant anticancer activity with GI50 values ranging

from 0.019 to 18.6 μM. These compounds have also been evaluated for its

tubulin binding activity and some of the compounds exhibited appreciable

good tubulin binding activity.

CCHAPTERHAPTER IV: (IV: (SECTION-A) - S- S YNTHESIS YNTHESIS AANDND BBIOLOGICALIOLOGICAL EEVALUATIONVALUATION OOFF BBENZYLIDENEENZYLIDENE--9(10H)-A9(10H)-ANTHRACENONENTHRACENONE LLINKEDINKED PP YRROLOBENZODIAZEPINES YRROLOBENZODIAZEPINES AASS AANTICANCERNTICANCER AAGENTSGENTS

In recent years there has been increasing interest in the design of

conjugate molecules that could act in a specific manner on more than one

target. The development of such conjugates lowers the risk of drug-drug

interaction in comparison to cocktails but could also enhance the efficacy as

well as improve the safety aspects in relation to the drugs that interact on a

single target. Several conjugate compounds, in which a known antitumour

compound or some simple active moiety tethered to PBD, have been

designed, synthesized and evaluated for their biological activity. Recently,

Wang and co-workers have synthesized indole-PBD conjugates as potential

antitumour agents and a correlation between antitumour activity and

apoptosis has been well explained. More recently, we have also reported

some of the PBD conjugates that demonstrated potent apoptotic activity

through mitochondrial-mediated pathway.

26



T HESIS

In continuation of these efforts, the synthesis and biological evaluation

of benzylidineanthracenone linked pyrrolobenzodiazepines attached through

an alkane spacer is described. The preparation of benzylidineanthracenones

intermediates 4a-f has been carried out by synthetic sequence illustrated in

Scheme-1. The intermediates 3a,b are synthesized by reacting anthrone 1

with different benzaldehydes in the presence of 10% IPA.HCl (isopropyl

alcoholic solution of HCl). These benzylidene-9(10 H )-anthracenones undergo

alkylation of hydroxyl group with dibromoalkanes using K 2CO3 as a base in

dry acetone to afford precursors 4a-f (Scheme 1).

CHO

R

OH

+

(i)

(ii)

4a-f

1 2a, b3a, b

4a; R = H, n = 24b; R = H, n = 34c; R = H, n = 44d; R = OMe, n = 24e; R = OMe, n = 34f ; R = OMe, n = 4

OO

R

OH

O

R

OBr

( )n

Scheme 1. Reagents and conditions: (i) IPA.HCl, 5 h; (ii) dibromoalkane, acetone, K2CO3,

reflux, 24 h.

Compound 5 has been coupled to compounds 4a-f in the presence of

K 2CO3 and dry acetone under reflux conditions to give the corresponding

nitro compounds 6a-f . These nitro compounds upon reduction with

SnCl2.2H2O in methanol under reflux conditions give amino compounds 7a-f.

27



T HESIS

Finally the amino compounds upon deportation with HgCl2/CaCO3 cyclized to

provide the corresponding imines 8a-f (Scheme-2).

O

R

OBr

( )n4a-f

HO

MeO

NO2

O

N

CH(SEt)2

5

+

O

MeO

NO2

O

N

CH(SEt)2O

R

( )n

6a-f

7a-f

8a-f

(i)

(ii)

(iii)

O

O

MeO

NH2

O

N

CH(SEt)2O

R

O

( )n

O

MeO

O

R

O

N

N

O

H( )n

n = 2, 3, 4


These benzylidine anthracenone linked PBD analogues have beentested for their cytotoxicity against different human cancer cell lines that

comprise of Zr-75-1, MCF-7, KB, Gurav, DWD, Colo-205, A-549, Hop-62 and

A-2780 by using the Sulforhodamine B (SRB) method. All the compounds

exhibited significant anticancer activity.

28



T HESIS

CCHAPTERHAPTER IV: (IV: (SECTION-B) - S- S YNTHESIS YNTHESIS AANDND BBIOLOGICALIOLOGICAL EEVALUATIONVALUATION OOFF CCHALCONEHALCONE--PP YRROLOBENZODIAZEPINE YRROLOBENZODIAZEPINE DDIMERSIMERS AASS AANTICANCERNTICANCER AAGENTSGENTS

Many molecules based on PBD ring system have been synthesized to

improve their biological profile and in this search C-7 or C-8 linked dimers of

PBD have been prepared, which are capable of sequence selective DNA

interaction and cross-linking. Thurston and co-workers have synthesized C-8

linked PBD dimers by linking C8-position of the A-rings through varying

lengths of alkyl chain to explore DNA-cross linking ability. The results

indicate that DSB-120 is an efficient cross-linking agent and the cross-linking

ability of these PBD dimers after 2h incubation at 37 °C has been found to be

0.01 nm. Furthermore, the in vitro cytotoxicity data in human K562 and

rodent ADJ-PC6 cell lines correlate with both the thermal denaturation data

and the cross-linking efficiencies. Recently, C2/C2’-exo-unsaturated C-8

linked PBD dimers (SJG-136) have been synthesized which exhibit

extraordinary DNA binding affinity and cytotoxicity.

CH3

O CHO

+

O

R3

R4(i)

1a,b 23a, b

3a; R1 = OH, R2 = H, R3 = H, R4 = OH3b; R1 = H, R2 = OH, R3 = OH, R4 = H

R2

R1

R3

R4 R2

R1

Scheme 1. Reagents and conditions: (i) aq.KOH, ethanol, 24 h

This chapter describes the synthesis and biological activity of

chalcone-pyrrolobenzodiazepine dimers. In these dimers the two PBD unitsare linked through a chalcone moiety. The preparation of dihydroxychalcone

intermediates (3a,b) has been carried out by synthetic sequence illustrated

in Scheme-1. Claisen-Schmidt condensation of hydroxyacetophenones with

hydroxybenzaldehydes using ethanol as solvent in the presence of aqueous

KOH gives dihydroxychalcones 3a,b.

29



T HESIS

O

3a; R = 4,4'-dihydroxy3b; R = 3,3'-dihydroxy

RR

O

MeO

NO2

O

N

CH(SEt)2

4a-c

+

O

MeO

NO2

O

N

CH(SEt)2O ( )n

5a-f

6a-f

7a-f

(i)

(ii)

(iii)

Br ( )n

OO

OMeN

O

(EtS)2HC O2N( )n

O

MeO

O ( )nOO

OMe

( )n

N

NN

N

O

H

O

H

Chalcone

O

MeO

NH2

O

N

CH(SEt)2O ( )nOO

OMeN

O

(EtS)2HC H2N( )n

Chalcone

Chalcone

O

O O

7a-c; Chalcone =

7d-f; Chalcone =

O

O O

n = 2,3,4

n = 2,3,4


Compound 4a-c has been coupled to dihydroxychalcones 3a,b in the

presence of K 2CO3 and dry acetone under reflux conditions to give

corresponding nitro compounds 5a-f . These nitro compounds upon reduction

30



T HESIS

with SnCl2.2H2O in methanol under reflux conditions give amino compounds

6a-f. Finally the amino compounds upon deprotection followed by cyclization

with HgCl2/CaCO3 provide the corresponding imines 7a-f (Scheme-2).

The cytotoxic activity of these chalcone-PBD dimers (7a-f ) has been

evaluated on a panel of 5 tumor cell lines that comprise HT-29, PC3, A-375,

A-549 and B-16 by using the Sulforhodamine B (SRB) method. These

analogues showed promising activity against PC-3 cell line compared to

other cell lines tested. These PBD dimers elevate the helix melting

temperature of CT-DNA in the range of 3.5-5.4 oC. Compound 7a showed the

highest ΔT m of 4.8 oC at 0 h and increased upto 5.4 oC after 18 h incubation,

whereas the naturally occurring DC-81 exhibits a ΔT m of 0.7 oC after

incubation under similar conditions. These results indicate that the effect on

DNA binding affinity by introducing the chalcone scaffold on PBD moiety

through different alkane spacers at C8-position of the DC-81.

In conclusion, the work carried in the research tenure we designed and

synthesized different series of biologically active molecules like

pyrrolobenzodiazepines and combretastatins which are known for potent

anticancer activity. The conjugates of PBD with chalcones and benzylidene

anthrones showed significant anticancer activity as well as good DNA binding

ability. The combretastatin derivatives with benzothiazoles showed potential

anticancer activity.

31



T HESIS

CCHAPTERHAPTER-I-I

GGENERALENERAL IINTRODUCTIONNTRODUCTION

32



T HESIS

1. CANCER

Cancer is one of the leading causes of death in the industrialized world.

Cancer arises when a population of cells within the body escapes from

normal control mechanisms and continues to increase until, unlesseffectively treated, the host dies. Although there are many kinds of cancer,

they all start because of uncontrolled growth of normal cells. Cancer cells

often travel to other parts of the body where they begin to grow and replace

normal tissue. This process, called metastasis, occurs as the cancer cells get

into the bloodstream or lymph vessels of our body. There are over 200

different types of cancers that can occur anywhere in the body. Cancer

treatment will be entirely based on person’s unique situation. Certain types

of cancer respond very differently to different types of treatment, so

determining the type of cancer is a vital step toward knowing which

treatments will be most effective. The cancer's stage will also determine the

best course of treatment, since early stage cancers respond to different

therapies than later-stage ones. Person’s overall health, lifestyle, and

personal preferences will also play a part in deciding which treatment

options will be best.

The four major types of treatment for cancer are surgery, radiation,

chemotherapy, and biological therapies. Hormone therapies such as

tamoxifen and transplant options such as those done with bone marrow are

also useful for treating certain cancers.

1.1. CHEMOTHERAPY

Chemotherapy is the treatment of cancer with drugs that can destroy

cancer cells by impeding their growth and reproduction. Chemotherapydrugs are given intravenously by injection or by mouth. Chemotherapy is

often used alone or in conjunction with radiation therapy or with surgery.

While surgery and radiation therapy are used to treat localized cancers,

chemotherapy is used to treat cancer cells that have metastasized to other

33



T HESIS

parts of the body, because they travel throughout the body in the

bloodstream. Depending on the type of cancer and its stage of development,

chemotherapy can be used to cure cancer, to keep the cancer from

spreading, to slow the cancer's growth, to kill cancer cells that may have

spread to other parts of the body, or to relieve symptoms caused by cancer.

Although chemotherapeutic drugs attack reproducing cells, they

cannot differentiate between cells of normal tissues and cancer cells. The

damage to normal cells can result in side effects. These cells usually repair

themselves after chemotherapy. Several exciting uses of chemotherapy hold

more promise for curing or controlling cancer. New drugs, new combinations

of chemotherapy drugs and new delivery techniques are the expected

advances in the coming years for curing or controlling cancer and improving

the quality of life for people with cancer. Chemotherapeutic drugs are

divided into several categories based on how they affect specific chemical

substances within the cancer cells, which cellular activities or processes the

drug interferes with, and which specific phases of the cell cycle the drug

affects. These include DNA topoisomerase I and II inhibitors, antimitotic

agents, antimetabolites, DNA interactive agents and miscellaneous agents.

1.2. DNA AS A CELLULAR TARGET FOR ANTICANCER DRUGS

DNA is one of the main targets in the design of antineoplastic agents.

Deoxyribonucleic acid (DNA)1-3 is a long molecule that contains coded

instructions for the cells. The monomer units of DNA are nucleotides that

consist of a 5-carbon sugar (deoxyribose), a nitrogen containing base

attached to the sugar and a phosphate group. There are four different types

of nucleotides found in DNA with adenine (A), thymine (T), cytosine (C) and

guanine (G). The particular order of the bases that are arranged along the

sugar-phosphate back bone is called the DNA sequence; the sequence

specifies the exact genetic instructions required to create a particular

organism with its own unique traits. The two DNA strands held together with

weak bonds between the bases on each strand, forming a base pair (bp).

34



T HESIS

During cell division the DNA molecule unwinds and weak bonds between the

base pairs break, allowing the strands to separate, and each strand direct

the synthesis of complementary strands, with free nucleotides matching up

with their complementary bases on each of the separated strands.

N

N N

N

O

N

N N

O

Sugar

NH

H

H

Sugar

Guanine

Cytosine

N

N N

N

NNN

O

O

H

H

Sugar

Sugar

Adenine

Thymine H

H

H

Figure-1. Hydrogen bonding between A-T and G-C base pairs of DNA

The base pairs are rotated 36° with respect to each adjacent pair, so

that there are 10 pairs per helical turn, each represented by 3.4 A°. This

gives rise to two well-defined channels known as minor groove and major

groove. The major groove is approximately 24 A° in width and much deeper

than minor groove, which is only 10 A° in width.4 The maintenance of coding

information in DNA stems from its ability to form a complementary double

stranded structure. Complementarily results in a large part from formation of

hydrogen bonds between specific opposing bases. Thus, adenine pairs with

thymine (an A-T pair) and cytosine with guanine (a C-G pair) by the

formation of 2 and 3 hydrogen bonds respectively. The atoms involved in

formation of these specific H-bonds are thus inaccessible unless the helical

structure is disrupted. However, on either side of the planar bases lie

additional H-bond donating and accepting groups specific for each base andwhich protrude into the relatively accessible major and minor grooves of the

helix. Thus in the major groove the C6 amino group of adenine or the C4

amino group of cytosine can act as hydrogen bond donating groups while the

adenine N7, thymine O4, guanine N7, O6 can act as hydrogen bond

accepting groups, and the thymine methyl as a hydrophobic site. Similarly in

35



T HESIS

the minor groove the adenine N3, thymine O2, guanine N3, and the cytosine

O2 can act as H-bond acceptors and the C2 amino group of guanine can act

as a donating group. Both grooves therefore carry base dependent

sequences of potential H-bonding atoms that can be used as a target in the

design of sequence specific DNA binding compounds (Fig.1 & 2).

Figure 2. The structure of part of DNA double helix.

36



T HESIS

Double stranded DNA can exist in three forms namely A, B and Z.5 The

B-form is most stable under high humid conditions because water molecules

stabilize the structure by forming a spine of hydration in the minor groove.6

Since this form almost exclusively predominates in the biological evaluation,

this B-DNA is used in the design of new DNA-binding antitumor drugs.

1.3. EVALUATION OF DRUG-DNA INTERACTIONS

DNA is believed to be the molecular target of a number of clinically

important antitumour antibiotics. Based upon their diverse structural types,

it is not surprising that these compounds each have been found to react in

quite different ways with DNA and that the biochemical consequences of

DNA are equally diverse, all though all can ultimately produce cell death.

Understanding the interactions between drugs and DNA is a necessary first

step in elucidating the molecular basis for the potent anti-tumor activities of

these compounds.

In recent years, several advances have been made in the elucidation of

drug-DNA interactions. Spectral methods are available to evaluate the extent

of DNA-binding and to know in which sequence the ligand binds. Physical

methods like UV-spectroscopy, fluorescence, circular dichroism (CD), opticalrotatory dispersion (ORD), IR, Raman spectroscopy and viscometry

measurements have been used for the measurement of binding. Thermal

denaturation studies on DNA are common and involve measuring the melting

point of DNA alone and in the presence of a ligand (drugs). Binding will often

stabilize the helix and elevate the melting temperature. However, none of

these physical techniques allows determining the specific location of binding

on a DNA strand. To do this two types of assays are used namely, strand

cleavage assay and affinity cleavage assay.7 Other powerful techniques for

studying DNA binding with short lengths of DNA includes NMR and X-ray

crystallography,8 which can provide precise structural information about

functional groups involved. Three dimensional 1H, 31P NMR experiments such

as NOSEY or COSY can be used to locate precisely the ligand on the strand

37



T HESIS

and which can be used in conjugation with computational methods to

generate useful 3-dimensional models of ligand-DNA complexes.9 DNA ‘foot

printing’ is an alternative approach that can be used for covalent and non-

covalent binders, intercalaters and other type of adducts such as co-

ordination complexes and triple helices.10

N

NN

NOH2C

O

PO

O O-

OH2C

O

O

PO O-

O

H2C O

OH2C

O

PO O-

O

PO O-

O

H2C O

O

P O-

O

O

O

O

CH2

O

P

P O-O

O

CH2O

OP O-O

O

CH2O

O

O

P O-O

O

CH2O

O-

OO

OCH2

O

Thymine

CytosineGuanine

Adenine

Major groove side

Minor groove side

PBDsMitomycin C (reducing conditions)SaframycinsPAHs (Benz[a]pyrene)N-Hydroxy-2-naphthylamineDehydroretronecine

CC 10659-Anthryloxirene

NH2

Bisfunctional alkylating agentsAflatoxin B1

Mitomycin C (acid activity)Benz[a]pyrene

Cis-Pt(NH3)2Cl2 (N7, O6 bridge)

3'5'

1

34

56

7

8

N

HN

O

O

N

N

O

H2N

NHN

N

O

NH229

H3C

Figure 3. Structure of DNA

38



T HESIS

1.4. T YPES OF DRUGS THAT INTERACT WITH DNA

Based on drug-DNA interactions DNA-interactive agents are

categorized in to four broad classes are known as intercalators, alkylating

agents, DNA strand breakage compounds and groove binders of DNA (Figure-

3).

1.4.1. INTERCALATERS

Intercalaters consist of a flat, generally π -deficient aromatic orheteroaromatic system, which binds to DNA by insertion between the base

pairs of the double helix. Intercalation causes the base pairs to separate

vertically, there by distorting the sugar-phosphate back bone and changing

the degree of rotation between successive base pairs e.g. acridines,11

actinomycins12 (Figure-4).

N

HN

H3CO NHSO2CH3

R'R

O

X

OH

H O

O

NH3+

OH

H3C

OH

OH

O

OOMe

acridines anthracyclines

Figure-4. Structures of DNA Intercalaters

1.4.2. ALKYLATING AGENTS

The DNA alkylaters (irreversible inhibitors) react with the DNA

(enzyme) to form covalent bonds. The important classes of alkylating agents

utilized in cancer chemotherapy are nitrogen mustards, ethylene amides,

39



T HESIS

methane sulphonic acid esters, nitrosoureas, triazenes and platinum

complexes (e.g. cisplatin13) (Figure-5).

Cl

ClH3N

H3N

cisplatin

R N

Cl

Cl

nitrogen mustards methanesulfonates

N

N

N

N

N

N

triethylenemelamine

SO

OS

OO

O O

Pt ( )n

Figure 5. Structures of alkylating agents.

1.4.3. DNA STRAND BREAKERS

Some DNA-interactive drugs initially intercalate into DNA but then in

certain conditions, react in such a way as to generate radicals. The reaction

of these radicals with the sugar moieties leads to DNA strand scission. e.g.

bleomycin and the enediyne antitumor antibiotics14 (Figure-6).

HN

CH3

COOH

OCH3

O

OH

OH

OH

O

O

Figure 6. Structures of dynemicin

1.4.4. GROOVE BINDERS

Drugs that bind to DNA may occur on the major groove face, minor

groove face or a combination. The grooves are excellent sites for sequence

specific recognition since there are many potential hydrogen bond donor and

acceptor atoms unique to each base pair combination along the base edges.

The greater width associated with the B-DNA major groove makes the major

groove somewhat more preferable binding groove.

40



T HESIS

1.4.4.1. IMPORTANCE OF MINOR GROOVE BINDERS

Groove binding can be via the major or minor groove and covalently or

non-covalently. Most DNA interactive proteins bind in the major groove, while

small molecules of less than 1000 Da, including many antibiotics, binds in

the minor groove. The minor groove represents a vulnerable site of attack in

that it is normally unoccupied, and this is presumably the reason for the

evolution of antibiotics that attack the DNA of competing organisms. Thus,

although at first sight minor groove binders are less attractive as probes in

that they target the less information rich minor groove nevertheless, they

may prove to have several advantages compared with major groove ligands.

The development of sequence-specific probes based on naturally occurring

DNA groove-binding agents is, therefore, an alternative and complementary

approach to the antisense oligonucleotide strategy. The main motive for

synthesizing a large number of analogues and conjugates of naturally

occurring minor groove-binding agents, is to generate new lead compounds

with potential anticancer properties and specific DNA sequence recognition.

1.4.4.2. NON-COVALENT MINOR GROOVE BINDERS

These compounds are typically isohelical with B-DNA and fits snugly

within the minor groove, held in a position by a combination of hydrogen

bonds, vander waal forces and electrostatic interactions. Examples include

distamycin15 (Figure 7), netropsin,16 CC-1065,17 lexitropsins and bis-

benzimidazole (Hoecht 33258).18

N

NH

NHH

OO

CH3

N

NH

O

NH2

NH2

CH3

N

NH

O

CH3

+

41



T HESIS

Figure 7. Structures of distiamycin

1.4.4.3. COVALENT BINDING TO MINOR GROOVE OF DNA

Drugs which bind covalently to DNA are used to either add substituents

onto base residues, or to form cross links between different sections of DNA.

The first mechanism results in a base-pairing mismatch during DNA

replication, and the DNA is ultimately fragmented by the enzymes which try

to repair it. The second mechanism binds together the two strands of the

DNA helix, preventing separation during the replication process.

Electrophilic functional groups such as epoxides, aziridines,

carbinolamines, imines and cyclopropanes are found in a variety of synthetic

and natural products capable of covalent interaction with DNA. Examples

include mitomycin, saframycins and pyrrolobenzodiazepines (anthramycin)19

(Figure 8).

N

HN

O

H3C

O

O

H2N

H3C N NH

OCH3

CH2OCONH2

Mitomycin Anthramycin

CONH2

OCH3OH

H

Figure 8. Covalant minor groov binding agents

1.5. CURRENT AREA OF WORK

As discussed previously the chemotherapy of cancer could be done by

DNA binding agents. The ultimate goal is to design and synthesize agents

capable of specifically inhibiting the expression of particular proteins critical

for tumour cell proliferation, metastasis or drug resistance. For complete

biological specificity, such agents must be able to recognize duplex DNA in

order to target individual gene sequences. As the naturally occurring

pyrrolo[2,1-c] [1,4]benzodiazepines have shown promising antitumor activity

42



T HESIS

due to the sequence-selective binding in the minor groove of DNA. The aim

of this work is to synthesize and evaluate novel DNA binding

pyrrolobenzodiazepine conjugates as potential anticancer agents.

1.5.1. P YRROLO[2,1-C][1,4]BENZODIAZEPINES AS DNA INTERACTIVE ANTITUMOR ANTIBIOTICS

The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a group of potent,

naturally occurring antitumor antibiotics produced by various Streptomyces

species. To date thirteen structures which include anthramycin,18

mezethramycin,20 porothramycin,21 prothracarcin,22 sibanomycine,23

tomaymycin,24 sibiromycin,25 chicamycin A,26 neothramycin A, B27 and DC-

8128 have been isolated from various streptomyces species (Figure-9).

N

HN

OCON

OR9

R8

OCH3

H

N

N

O

HOH

H3CO

N

N

O

H1

6711a

2

5

8

9

8

Anthramycin (R8 = CH3, R9 = R1 = R2 = H)Mazethramycin (R8 = R1 = CH3, R9 = R2 = H)Porothramycin B (R8 = H, R9 = R1 = R2 = CH3)

1011

N

N

O

R

R8

R7

Tomaymycin (R7 = OCH3, R8 = OH, R = CH3)

Prothracarcin (R7 = R8 = H, R = CH3)

Sibanomicine (R8 = H, R7 = sibirosamine

pyronoside as in , R = Et)

N

H

N

OCH3

HO H

Sibiromycin

N

HN

O

HO

H

H3CO

OCH3

OH

Chicamycin A

R1R2

Neothramycin A ( R1 = H; R2 = OH)

Neothramycin B ( R1 = OH, R2 = H)DC-81 (R1 = R2 = H)

O

O

OH

CH3H3C

H3CHN

3

OH

OH

R1

R2

OMe

A B

C

PBD ring system

H

Figure 9. Naturally occurring PBDs

43



T HESIS

1.5.2. PBD-DNA INTERACTIONS

The cytotoxicity and antitumor activity of these agents are attributed

to their property of sequence selective covalent binding to the N2 of guanine

in the minor groove of duplex DNA via acid-labile aminal bond to the

electrophilic imine at N10-C11 position.29 These molecules possess an (S)-

configuration at the chiral C11a-position which provides a right handed twist

when viewed from A-ring towards C-ring. This feature provides the

appropriate three dimensional shape for the isohelicity with the minor groove

of B-form DNA leading to a snug fit at the binding site (Figure-10).

N

HN

CH3

HO

MeOO

HH

OH

N

N

CH3

HO

MeOO

H

HN

N N

N

DNA

O

H2N

N

HN

CH3

HO

MeO

O

H

HN

N N

N

DNA

O

HN

-H2O

+H2O

C(11) (S) carbinolamine C (11)-N(10) imine

C(11) (R/S) aminal

Figure 10. Formation of PBD-DNA covalent adduct

44



T HESIS

Molecular modeling, solution NMR, fluorimetry and DNA foot printing

experiments reveal that the PBDs recognize a three base pair motif with a

preference for 5’Pu-G-Pu sequences.30 The PBDs have shown to interfere with

the action of endonuclease enzymes on DNA31 and to block the transcription

by inhibiting DNA polymerase in a sequence specific manner,32 processes

which may be relevant for their biological activity.

1.5.3. STRUCTURE ACTIVITY RELATIONSHIP

Structure activity relationship for PBDs has been derived by Thurston

and coworkers. (S)-Configuration at the C11a position is required for snug fit

in the DNA minor groove. PBD with (R)-configuration at C11a has shown to

be devoid of both DNA binding affinity and in vitro cytotoxicity.33 The

electron donating substituents are required in the aromatic A ring for

biological activity. C2 substituted naturally occurring PBDs exhibit more

cytotoxicity compared to unsubstituted PBDs. Bulky substituents like a sugar

moiety at C7 position enhance the DNA binding affinity and cytotoxicity

(Figure-11).

N

NH

R9

R8

R7

OR

1

2

3

1011

11a

(a) An imine, carbinolaminemethyl ether required at N10-C11

(b) (S)-Stereochemistryrequired at C11a

(c) Replacement of C1 withan oxygen maintainscytotoxicity

(d) Endocyclic or exocyclicunsaturation at C2 enhances

cytotoxicity and in vivoantitumour activity. Fullyunsaturated C-ring leads tocomplete loss of DNA-bindingand cytotoxicity

(e) Small substituents (eg. -OH)tolerated at C3 in fully saturatedC-ring compounds

(f) Sugar moiety at C7enhances DNA-bindingaffinity and cytotoxicityin some cell lines

(g) Electron-donatingsubstituents requiredat position 7,8 or 9of A-ring

(h) Bulky substituents at N10

(eg. acetyl) inhibit DNA-binding and cytotoxicity

45



T HESIS

Figure 10. Structure activity relationship of PBD ring system

1.6. S YNTHETIC APPROACHES OF P YRROLO[2,1-C][1,4]BENZODIAZEPINES

The first total synthesis of a carbinolamine containing PBD of

anthramycin has been reported by Leimgruber in 1968.34 Extensive reviews

of the synthetic literature of the PBDs have appeared in 1994, 1998 and

2002.35 Various approaches to the synthesis of PBD antibiotics including

hydride reduction of seven-member cyclic dilactams,36 reductive cyclization

of acyclic nitroaldehydes,37 iminothioether approach38 cyclization of

aminothioacetals,39 deprotective cyclization of the diethylthioacetals via N10

protected precursors,40 oxidation of cyclic secondary amines,41 reductive

cyclizations42 and solid phase approaches43 have been investigated.

1.6.1. K ANEKO APPROACH (IMINOTHIOETHER REDUCTION)

Kaneko and coworkers36a developed a mild method for the reduction of

PBD dilactams to the carbinolamine using aluminium amalgam (Scheme 1). This methodology has been employed for the preparation of bicyclic and

tricyclic analogues of anthramycin and the total synthesis of some naturally

occurring PBDs like chicamycin.38b By using this approach Baraldi and

coworkers have synthesized some heterocyclic PBD analogues in which the A

ring of PBD skelton is replaced with a 1,3 or 1,5-disubstituted pyrazole

nucleus.

46



T HESIS

N

HN

O

O

HR1

R2R3

N

HN

S

S

HR1

R2R3

N

HN

O

S

HR1

R2 R3

N

N

O

SR4

HR1

R2 R3

N

HN

O

SR4

HR1

R2R3

N

N

O

HR1

R2R3

N

HN

O

HR1

R2 R3

N

HN

O

HR1

R2R3

OCH3

R1 = H, OH, OBn, OCH3, OAcR2 = H, OCH3

R3 = H, = CH-CH3 (E), OH (a), OAc (b), = CH-COOEt (E)

R4 = CH3

1

2+

3

4

+

5

6

7

8

i ii

iii

ivv

Scheme 1. Reagents and conditions: (i) P2S5, C6H6, 80 oC or P2S5, NaHCO3, CH3CN, 15 min,

or (p-CH3OC6H4PS2)2, C6H6, 80 oC; (ii) Et3OBF4, CH2Cl2, KHCO3 or CH3I, K 2CO3, THF or DMF; (iii)

Al-Hg, aq.THF or KH2PO4, 0-5 oC, 14 h; (iv) 0.1 N methanolic HgCl2, 0 oC or SiO2

chromatography, 5 oC; (v) CH3OH.

1.6.2. THRUSTON’S APPROACH (C YCLIZATION B Y DEPROTECTION OF DIETHYLTHIOACETAL)

Thurston and coworkers39a developed an efficient method for the

synthesis of various PBDs containing carbinolamine moiety by employing

mercuric chloride (HgCl2) and calcium carbonate (CaCO3) in aqueous

acetonitrile at room temperature. In this procedure, the products have been

generally isolated in the imine form and it has been extensively utilized for

the synthesis of a variety of naturally occurring and synthetic PBDs including

DC-81 (Scheme 2), C8-linked DC-81 dimers, A ring modified analogues of

47



T HESIS

PBD, PBD-EDTA conjugate, lexitropsin conjugates of PBD, C2 linked PBD

dimers, imine-amide PBD dimers and naphthalimide conjugates of PBD.44

NO2

N

CH(SEt)2

O

NH2

N

CH(SEt)2

O

N

N

O

COOH

RO

H3CO COOH

PhH2CO

H3CO

a R = H

b R = PhCH2

NO2

H3CO

PhH2CO

PhH2CO

H3CO

RO

H3CO

a R = PhCH2

b R = H

i

9 10

1213

iii

v

vi

ii

iv

11

H

Scheme 2. Reagents and conditions: (i) PhCH2Cl, THF, NaOH, H2O, reflux, 48 h; (ii) SnCl4,

HNO3, CH2Cl2, -25 oC, 5 min; (iii) (COCl)2, THF, DMF, 3 h then, pyrrolidine-2-

carboxaldehydediethylthioacetal, Et3N, H2O, 0 oC, 1.5 h; (iv) SnCl2.2H2O, MeOH, reflux, 45

min; (v) HgCl2, CaCO3, CH3CN-H2O, 12 h; (vi) 10% Pd-C, EtOH, cyclohexadiene, 3 h.

1.6.3. FUKUYAMA-T YPE APPROACH

In order to incorporate certain labile functionalities such as C8-epoxide

moiety in the PBD system, the conventional deprotective cyclization of

diethyl thioacetal failed to give results. Therefore, Thurston and coworkers40

have made various attempts to synthesize these newly designed PBDs with

potential DNA binding affinity. In this effort, a Fukuyama-type approach45 has

been attempted wherein 9-fluorenyl methyloxy carbonyl (Fmoc) group can

be used to protect the amine group and which can be easily removed bycleavage with Bu4N+F- (TBAF) at room temperature (Scheme 3).

48



T HESIS

O

H3CO N

CH(SEt)2

O

O

H3CO N

CH(SEt)2NH

O

Fmoc

O

H3CO N

N

O

OH

Fmoc

a X = NO2

b X = NH2

O

H3CO N

N

O

OH

FmocO

O

H3CO N

N

O

O

H

H

14

1617

18

i

15

ii

iii

iv

v

X

H

Scheme 3. Reagents and conditions: (i) SnCl2.2H2O, CH3OH, reflux, 3 h; (ii) Na2CO3 (aq),

Fmoc-Cl, 0 oC, 4 h, Dioxane, rt, 16 h; (iii) HgCl2, CaCO3, CH3CN-H2O, rt, 48 h; (iv) m-CPBA,

CH2Cl2, rt, 72 h; (v) TBAF, DMF, rt, 15 min.

Baraldi and coworkers46 synthesized hybrid molecules containing PBD

and minor groove binding oligo-pyrrole carriers, while Hurley and coworkers47

have synthesized AT-groove binding hybrids by using this approach. In the

same manner Suzuki coupling of C7 aryl substituted PBDs have been

synthesized by Thurston and co-workers.48 This B-ring strategy of Fukuyama

and coworkers has also been employed for the synthesis of C2/C2'-exo-

unsaturated PBD dimer, C2-C3/C2'-C3'-endo unsaturated PBD dimer withremarkable covalent DNA binding affinity.49

1.6.4. K AMAL’S APPROACH (OXIDATION OF C YCLIC SECONDARY AMINE)

This approach is based on the oxidation of PBD secondary amine and

has been considered as one of the most attractive methods as mentioned in

49



T HESIS

one of the reviews on the synthesis of PBDs.35a Kamal and coworkers41

developed a novel method for the oxidation of PBD secondary amine to the

corresponding imines. Although PBDs with either a secondary amine or

amide functionality at N10-C11 are readily synthesized, the introduction of

imine or carbinolamine at this position is problematic due to the reactivity of

these functional groups. As described in the literature, the cyclic secondary

amine precursors have been readily prepared from corresponding nitro

aldehydes. This upon oxidation with DMSO/(COCl)2 or TPAP (tetra-n-propyl

ammonium perruthenate) gives corresponding imines in good yields

(Scheme 4).

NO2

N

CHO

O

N

HN

S

O

H

N

HN

O

H

19 20

21

iii

iii

N

N

O

H

22

Scheme 4. Reagents and conditions: (i) Pd/C; (ii) Raney Ni; (iii) swern/TPAP

1.6.5. REDUCTIVE C YCLIZATION APPROACH

Miyamoto and coworkers50 reported the first total synthesis of N10-C11

imine containing PBDs via reductive cyclization for neothramycin A and B.

Thurston and coworkers51 have carried out a detailed investigation on the

reductive cyclization of N-(2-aminobenzoyl)pyrrolidine-2-carboxaldehydes.

1.6.6. AZIDO REDUCTIVE C YCLIZATION

In an endeavor to explore new practical methods for the preparation of

PBDs particularly by the azido reductive process has been extensively

50



T HESIS

investigated by Kamal and co-workers. This procedure has been examined

by different reagents such as N,N-dimethylhydrazine/catalytic ferric chloride,

ferrous sulphate/ammonia, and samarium iodide (SmI2). The same group has

carried out another interesting study on the enzymatic reduction of aryl

azides to aryl amines by employing baker's yeast. This biocatalytic reductive

methodology has been applied to the chemoenzymatic synthesis of PBDs via

the reductive cyclization of arylazido aldehydes (Scheme 5).52

N

NH

O

N

O

CHO

N

O

CHOR1

R2

R1

R2R3 R3

R1

R2R3

N3 NO2

Reagent used for azide reduction

(i) HMDST(ii) bakers' yeast

(iii)N,N-Dimethyl hydrazine/ ferric chloride(iv) TMSI

(v) SmI2(vi) HI(vii) FeSO4.7 H2O

Reagents used for nitro reduction

(i) Fe/ AcOH(ii)N,N-Dimethyl hydrazine/ ferric chloride

R1= OH, OBn, OCH3

R2 = OCH3; R3 = H, OH

Scheme 5;

1.6.7. SOLID PHASE APPROACH

Combinatorial synthesis has become popular in recent years. By using

this technique a large number of distinct molecules could be synthesized in a

short time and resource effective manner. In this pursuit, Thurston and

coworkers43 have developed a solid phase synthesis of PBD imines on p-

nitrophenylcarbonate Wang resin using a variety of oxidation and cyclization

procedures. Kamal and coworkers53 have developed some new synthetic

strategies on PBD dilactams and PBD imines by using Wang resin (Scheme

6).

51



T HESIS

52



T HESIS

O CCl3

NH

N

HN

OO

O

OH + +Fmoc-N

COOCH3

OH

Fmoc-NO

COOCH3

HNO

COOCH3

N

N3

O

COOCH3

O

R

N

N3

O

CHO

O

R

N

HN

OOH

R

O

N

N

OO

RHH

H

N

N

OOH

RH

R

25 27 28

2930

31 34

35

36

32

33

i

iii

iv

v

vii

vi

vii

vi

ii26

Cl3CCN

Scheme 6. Reagents and conditions: (i) DBU, CH2Cl2; (ii) BF3.OEt2 or CF3SO3H, CH2Cl2; (iii)20% piperidine/DMF; (iv) subistituted 2-azido benzoic acid, DCC, DMAP, CH2Cl2, 0 oC; (v)

DIBAL-H, CH2Cl2, -78 oC; (vi) PPh3, toluene; (vii) TFA/CH2Cl2 (1:3).

53



T HESIS

1.7. STRUCTURAL MODIFICATION OF PBDS

In the search for compounds with better antitumor selectivity and DNA

sequence specificity many structurally modified PBD analogues have been

synthesized in an attempt to increase their potency against tumor cells.

1.7.1. A-RING MODIFICATIONS

Baraldi and coworkers54 have synthesized heterocyclic PBD analogues

in which A-ring of the PBD is replaced with a 1,3 or 1,5-di-substituted

pyrazole nucleus and some of these PBD analogues have exhibited

interesting profile of cytotoxicity. Leoni55 prepared a range of N6- and N7-

alkyl substituted derivatives of pyrazolo[4,3-c]pyrrolo[1,2-a]

[1,4]diazepinones. Thurston and coworkers44b have reported pyridine and

pyrimidine A-ring analogues of PBD. It is observed that aromatic A-ring has a

modest influence on the thermal denaturation of DNA.

NN

O

NHN

CH3

H3C

NN

O

NHN

H3C

Cl

N

N

HN

O

H

OCH3

N

N N

N

O

H

H3CO

OCH3

1.7.2. B-RING MODIFICATIONS

Nacci and coworkers56 have reported the synthesis of pyrrolo[2,1-c]

[1,4]benzothiazepine compounds as sulphur containing B-ring modified

analogues of PBD. To investigate the role played by the non-covalent

interactions Robba and co-workers57 synthesized a series of PBDs having

54



T HESIS

N10-C11 amidines functionality. Interestingly, these compounds have shown

DNA binding affinity comparable to the DC-81, a natural product that binds

covalently to DNA.

N

S

O

N

S

OCH3

HN

NHNH2

N

S

NNHCONHC6H5

OCOCH3

N

S

NNHCO-p-FC6H4

H

H H

1.7.3. C-RING MODIFICATIONS

The degree of saturation of the C-ring is thought to give significant

effects on biological activity. For example, the completely unsaturated

system is unlikely to exhibit antitumor activity. A number of naturally

occurring PBDs namely anthramycin, tomaymycin, sibiromycin andneothramycin have different type of substitutions in the C-ring. It is

interesting to note that these C-ring modified PBDs appear to provide both

greater differential thermal stabilization of duplex DNA and significantly

enhance kinetic reactivity during covalent adduct formation. Thurston and

coworkers58 have synthesized a series of C2-exo unsaturated PBDs and

C2/C3-endo unsaturated PBDs. Partial unsaturation of C-ring enhances both

the DNA-binding affinity and in vitro cytotoxic potency. This group has also

reported the synthesis of novel C2-aryl 2,3-unsaturated and 1,2-unsaturated

PBDs.59

55



T HESIS

N

N

O

H3CO

H3CO N

N

O

H3CO

H3COOCH3

O

N

N

O

H3CO

H3CO

OCH3

H

H

N

N

O

H3CO

H3CO

H

H

Ph

Recently, Kamal and co-workers60 have synthesized a series of C2-

fluorinated PBDs61 and have been screened for in vitro cytotoxicity against a

number of cancer cell lines and also studied for DNA binding affinity.

Through a large number of SAR studies it is now known that the substitution

pattern (particularly at C2) and the degree of unsaturation of the C-ring are

crucial for maximising cytotoxicity and antitumour activity in the PBD family.

In conclusion, these experimental observations permitted us to

hypothesize that an increase of carbinolamine reactivity is detrimental in

terms of cytotoxicity. Therefore, A-ring modifications do not seem to boost

PBD activity as much as C-ring modifications do. Based on the current

knowledge of SARs, the C2-side chains play a major role in increasing the

cytotoxicity. Therefore, combination of heterocyclic A-ring analogues with

side chain at position C2 on the C-ring could permit to obtain new potent

derivatives lacking cardiotoxicity.62

56



T HESIS

N

N

O

R

R1

R = F, R1 = H, R2 = H, R3 = HR = H, R1 = F, R2 = H, R3 = HR = F, R1 = F, R2 = H, R3 = HR = F, R1 = H, R2 = OBn, R3 = OCH3

R = F, R1 = H, R2 = OH, R3 = OCH3

HR2

R3

N

N

H3CO

ON

N

O

OCH3

O OFF

(CH2)n HH

n = 3, 4, 5

N

N

H3CO

ON

N

O

OCH3

O OFF

(CH2)n HH

n = 3, 4, 5

N

N

O

R

R1

HR2

R3

1.8. PBD DIMERS

In an attempt to extend the number of base pairs spanned by these

molecules, PBD dimers have been synthesized with the hope that enhanced

sequence selectivity might increase selectivity for tumor cells.

1.8.1. C7-LINKED DIMERS

Suggs and coworkers63 have reported the first PBD dimer comprising

of two PBD units joined through A-C7/A-C7’ positions via alkanediyldioxy

linker (some including nitrogen heteroatoms) or alkanediyldisulfide linkers.

The C7-lilnked dimers have been considered unique among DNA-cross linkers

in their specificity for dG-containing duplex DNA.

57



T HESIS

N

N O

O

H

O N

N

O

H

N

CH3

7 7


Thurston and coworkers44a have synthesized A-C8/A-C8’ dimers by

linking two PBD monomers at their C8 position through varying lengths of

alkyl chain. These dimers form an irreversible interstand cross-link between

two guanine bases with the minor groove. Thermal denaturation and

cytotoxic studies exhibited that the dimer linked with propane chain (DSB-

120) has been highly potent. Molecular modeling and NMR studies confirmed

that these dimers span six base pairs in the minor groove of duplex DNA.

O

H3CO N

N

O

HN

N

O

OCH3

O

H

DSB 120

Kamal and coworkers44d have synthesized C8-linked imine-amide mixed

dimers wherein one ring of PBD has imine function and the other has amide

group. One of these dimers elevates helix-melting temperature of CT-DNA bya remarkable 17 oC after incubation for 18 h at 37 oC. These dimers have

shown potent antitumor activity in different cell lines.

O

H3CO N

HN

O

HN

N

O

OCH3

O

H

O

Recently, Thurston and coworkers49 have synthesized C8-linked C2/C2’

exo unsaturated dimers as novel cross-linking agents with remarkable DNA

binding affinity and cytotoxicity. These molecules have shown significant in

vivo potency and have been selected for clinical trials.

58



T HESIS

O

H3CO N

N

O

HN

N

O

OCH3

O

H

Kamal and coworkers64 have synthesized C8-linked C2-S and C2-R

fluoro substituted PBD dimers and C2/C2’-exo-difluoromethylene dimers.

These dimers possess in vitro anticancer activity in a number of human

cancer cell lines. The replacement of hydrogen with fluorine atom at the C2-

position of the PBD ring system leads to significant increase in cytotoxicity.

N

N

O

H3CO

N

N

O

OCH3

OF F

OHH


Lown and coworkers43c reported PBD dimers in which two monomers

have been joined tail to tail (C-ring) at C2 position through alkyl amide linker.

These compounds exhibited moderate to promising cytotoxic potency

against different cancer cells.

N

NN

N

O ONH

NH

O OHH

1.9. C8-LINKED H YBRIDS OF P YRROLOBENZODIAZEPINES

In the search for compounds with better antitumour activity and DNA

sequence specificity many PBD analogues have been synthesized. In the

59



T HESIS

literature, much attention has been given to the substitution in the A-ring

particularly at the C8-position as the structural activity relationships suggest

that the substitution at this position cause immense biological responses.

These compounds are capable of recognizing heterogeneous DNA

sequences.

N

N

O

H

MeO

OR alkanes

HN N

ONH

HN

O

Me

O

N

Cl

O

OH

HN

O

NH

HN

O

N

HN

O

n

N

O

O

NH

HN

O

NH

NN

NMe

O

Figure 14. C8-linked PBD hybrids

R =

O

O

HN O

N

NS

NN

O O S

Me

Some of the interesting C8-linked hybrids of pyrrolo[2,1-c]

[1,4]benzodiazepines are UTA-6026,65a seco-CBI,65b pyrene,65c lexitropsin,65d

naphthalimide,65e indole,65f conjugates (Figure 14). In general, the interaction

with DNA tends to be dominated by the minor groove-binding moiety, i.e. the

conjugates bind to the minor groove with preferential interaction with AT-rich

sequences. It may be noted that, the cytotoxicity of these hybrid derivatives

is much greater than that of the alkylating units alone. However, subsequent

molecular modeling studies suggested that C8 would be the preferred

position for the attachment of second interacting group. Baraldi and

60



T HESIS

coworkers46 have linked distamycine and netropsin antibiotics to the C8

position of PBD through linker of varying lengths.

HNH2N

ONHHCl

HN O

O

n

N

N

O

H3CO

H

n = 1-4

N

Recently, Lown and coworkers66 have reported PBD-glycosylated

pyrrole and imidazole polyamide conjugates. These compounds have been

prepared with varying number of pyrrole and imidazole containing

polyamides and incorporating glucose moieties in order to improve the water

solubility of PBD-polyamide conjugates. The water soluble PBD-polyamide

conjugates exhibited a higher level of cytotoxic activity than the natural and

synthetic PBDs.

N

N

O

OCH3

ONH

ON

NH

O

N CH3

CH3

O

H

HO

OH

H

H

HH

OH

OH

H n( )3

Pyrrole Polyamide - PBD Conjugate

Hurley and coworkers47 and Denny and coworkers67 have been

designed and synthesized unsymmetrical DNA cross-linkers by linking the

seco-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indo-4-one (seco-CBI) to PBD

moiety. These compounds have anticipated cross-linking between N3 of adenine and N2 of guanine in the minor groove of DNA. One of these

compounds exhibited IC50 in the pico molar range.

61



T HESIS

N

N

O

O

H3CO

N

Cl

OH

O

H( )5

CBI - PBD Conjugate

Kamal and co-workers68 have synthesized a series of PBD conjugates

by linking different DNA interacting ligands such as naphthalimides, poly-

aromatic hydrocarbons (pyrene amine and chrysene amine) and

benzimidazoles by using varying linker length to enhance the DNA binding

affinity and antitumor activity.

N

N

O

H

H3CO

O

O

(CH2)nHN

n = 3-4

N

N

O

O

H3CO

H

n = 3-5

N

HN

O

N

NH3C

( )n

Hurley and co-workers69 have synthesized novel DNA-DNA interstrand

adenine-guanine cross-linking UTA-6026 compound. Preliminary in vitro tests

showed that UTA-6026 has remarkably potent cytotoxicity to several tumour

cell lines (IC50 = 0.28 nM in human breast tumour cell line MCF7, IC50 = 0.047

nM in colon tumour cell line SW-480 and IC50 = 5.1 nM in human lung tumour

cell line A549).

N

N

O

O

H3CO

HN

O

NH

N

NH

H3C

O

O

H

Kamal and co-workers70 have designed and synthesized PBD-

morpholine, N-methyl piperizine and N,N-dimethyl amine hybrids in attempts

to improve the water solubility and cytotoxicity of the PBD compounds.

62



T HESIS

Based on the solubility recently Lown and co-workers71 have designed and

synthesized novel PBD-gylcosylated pyrrol and imidazole polyamide

conjugates and described as water insoluble and water-soluble PBD

conjugates. Further, these conjugates have been tested against a panel of 60

human cancer cell lines by NCI and demonstrated that the water-soluble

PBD-polyamide conjugates exhibited a higher level of cytotoxic activity than

the existing natural and synthetic PBDs.

N

N

O

O

H3CO

N H

O

In addition to above derivatives, this group has also reported the

synthesis and DNA binding affinity of quinolone,72 pyrimidine hybrids,87 C2/C8

dimers,74 azepine conjugates75 and methanesulfonate derivatives76 of

pyrrolo[2,1-c][1,4]benzodiazepines.

N

N

O

HO

H3CO

O

nN N

CH3

F

N

N

O

HO

H3CO

NnH3COOC

O

F

F

N

N

O

HO

H3CO

H3CO2SOn

n = 3−5

1.10. OBJECTIVES OF THE PRESENT WORK

The molecular modeling, NMR studies, fluorimetry, and DNA foot

printing experiments have given an insight into the mechanism of action of

63



T HESIS

PBDs, thus providing opportunities to synthesize novel PBD conjugates with

both improved binding affinity and modified sequence selectivity or with a

change in the binding mode and also probably reducing their undesirable

side effects.

Cancer drug discovery is one of the most rapidly changing areas of

pharmaceutical research. The search for new drugs in the field of oncology

has refocused on natural products. Among the currently identified antitumor

agents, Chalcones, constitute an important group of natural products and

serve as precursors for the synthesis of different classes of flavonoids, which

are common substances in plants. Chalcones are open-chain flavonoids in

which two aromatic rings are joined by a three carbon α, β -unsaturated

carbonyl system (1,3-diphenyl-2-propen-1-ones). The remarkable biological

potential of these chalcones is due to their possible interactions with various

proteins related to cell apoptosis and proliferation. Recent studies have

shown that these chalcones induce apoptosis in a variety of cell types,

including breast cancers. Therefore, in the present chapter a series of

chalcone-PBD conjugates linked through simple alkane spacers have been

synthesized and evaluated for their biological activity (Chapter-II).

The combretastatin A-4 (CA-4) is a natural product found to have

potent anticancer activity against a number of human cancer cell lines

including multidrug resistant cancer cell lines and binds to the colchicine-

binding site of tubulin. A water-soluble prodrug, combretastatin A-4-

phosphate is now in clinical trials for thyroid cancer and in patients with

advanced cancer. The cis configuration only of CA-4 is biologically active,

with the trans form showing little or no activity. The amino derivative of CA-4

is also in clinical trials as a water-soluble aminoacid prodrug.

Being a potentinhibitor of colchicine binding, CA-4 is also shown to inhibit the growth and

development of blood vessels, angiogenesis. Various structural modifications

to CA-4 have been reported including variation of the A- and B-ring

constituents. The chalcone and pyrazoline derivatives of CA-4 also showed

64



T HESIS

potent anticancer activity. In view of potent anticancer activity exhibited by

CA-4 derivatives, we synthesized amidobenzothiazole analogues of CA-4 and

evaluated for its anticancer activity. The synthesized analogues exhibited

significant anticancer activity (Chapter-III).

Benzylideneanthrones are class of compounds known to exert potential

antitumor properties. The antitumor activity of these 10-substituted

benzylideneanthrones shows through inhibition of tubulin polymerization.

Helge Prinz and co-workers have been synthesized and reported the potent

in vitro antitumor activity and inhibition of tubulin polymerization of different

anthracenone analogues. In an attempt to establish new conjugates of PBDs

with improved anticancer activities we have synthesized

benzylideneanthrone linked pyrrolobenzodiazepine conjugates. The

synthesized compounds have exhibited significant DNA-binding ability.

(Chapter-IV/Section-A).

Chalcones represent an important group of natural products belonging

to the flavonoids family. Natural and synthetic chalcones have been reported

to posses strong antiproliferative effects in primary as well as established

ovarian cancer cells and in gastric cancer (HGC-27) cells. Recent studies

have shown that these chalcones induce apoptosis in a variety of cell types,

including breast cancers. Previous references reveals that the dimers of

PBDs posses promising anticancer activity. In this context, new analogues of

dimers of pyrrolobenzodiazepines with chalcone have been prepared and

evaluated for anticancer activity (Chapter-IV/Section-B).

65



T HESIS

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Lett. 2003, 13, 3517.

CCHAPTERHAPTER-II-II

SS YNTHESIS YNTHESIS AANDND BBIOLOGICALIOLOGICAL EEVALUATIONVALUATION OOFF CCHALCONEHALCONE--PP YRROLOBENZODIAZEPINE YRROLOBENZODIAZEPINE CCONJUGATESONJUGATES AASS AANTICANCERNTICANCER AAGENTSGENTS

73



T HESIS

2.1. INTRODUCTION

Naturally occurring pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) have

attracted the attention of many researchers largely because of the potent

anticancer activity exhibited by most of these compounds bearing this ring

system. Some of the compounds of this class have undergone clinical

studies.1,2 Apart from their anticancer activity, PBDs are of considerable

interest due to their ability to recognize and subsequently form covalent

bonds to specific base sequences of double strand DNA. They are

monofunctional alkylating agents, and have potential as gene regulators,

probes and as tools in molecular biology.3-5 The pyrrolo[2,1-c]

[1,4]benzodiazepines (PBDs) are a family of antitumour antibiotics derived

from various Streptomyces species6 and are generally referred to as the

anthramycin family, which comprise of some representative members like

anthramycin (1), sibiromycin (2), tomaymycin (3), chicamycin A (4),

neothramycin A (5), and B (6), and DC-81 (7) (figure 1).

74



T HESIS




interaction and cross-linking. Thurston and co-workers7 have synthesized C-8

linked PBD dimers by linking at their C8-position of the A-rings through

varying lengths of alkyl chain to explore their DNA-cross linking ability. DNA-

binding ability has been observed by thermal denaturation studies with CT-

DNA (∆ T m > 15.1 °C for a 5:1 ratio of DNA:PBD at 37 °C for 18 h incubation)

and the cross-linking efficiency has been investigated by using an agarose

gel electrophoresis assay. The results indicate that DSB-120 is an efficient

cross-linking agent and the cross-linking ability of these PBD dimers after 2 h

incubation at 37 °C has been found to be 0.01 nm. Furthermore, the in vitro

cytotoxicity data in human K562 and rodent ADJ-PC6 cell lines correlate with

both the thermal denaturation data and the cross-linking efficiencies.

75



T HESIS

N

N

O

HOH

H3CO

R1R2

N

HN

O

HH3C

OR

CONH2

OCH3

N

N

O

CH3

HO

H3CO

H

N

HN

O

CH3

HOH

O

OHOMe

N

HN

O

HOH

H3CO

OCH3

OH

O

OH

CH3H3C

H3CHN

OH

N

N

O

HOH

H3CO

( R1 = H; R2 = OH) (5)( R1 = OH, R2 = H) (6)

chicamycin

(1)

R= OH or OCH3

anthramycin sibiromycin

tomamycin

neothramycin A and BDC-81

(3)

(7)

(4)

(2)


Recently, C2/C2’-exo-unsaturated C-8 linked PBD dimers (SJG-136)have been synthesized which exhibit extraordinary DNA binding affinity and

cytotoxicity.8 In recent years, a large number of hybrid molecules containing

the PBD ring system have been synthesized leading to novel sequence

selective DNA cross-linking agents.9 It is belived that interactions in a manner

different from those of other tubulin-binding antimitotic agents.

2.1.1. INTRODUCTION OF CHALCONES

Chemically chalcones comprise of open-chain flavonoids in which the

two aromatic rings are joined by a three-carbon α,β-unsaturated carbonyl

system. Chalcones, considered as the precursor of flavonoids and

isoflavonoids, are abundant in edible plants. However, most of the chalcones

are particularly attractive since it specifically generates the (E)-isomer from

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T HESIS

substituted benzaldehydes and acetophenones. Recent studies revealed that

these chalcones had shown a wide variety of anticancer,10-17 anti-

inflammatory,18-20 antiinvasive,21 antituberculosis,22 and antifungal23 activities.

Chalcones have shown promising anticancer therapeutic efficacy for the

management of human cancers. Recently, different chalcone analogues have

been synthesized and they have been screened for in vitro cytotoxicity

against a number of cancer cell lines.

The substituted chalcones have shown potential anticancer activity.

Ducki and co-workers have synthesized and reported trimethoxy substituted

chalcones24 (8) and (9), that possess potential anticancer activity and bind

strongly to tubulin at a site shared with, or close to, the colchicines binding

site.25-26 The anticancer activity and tubulin binding property of these

chalcones is comparable with combretastatin A-4 (CA-4). The IC50 value of

compound SD400 (9) against the K562 human chronic myelogenous

leukemia cell line is 0.21 nM whereas combretastatin A-4 (CA-4) shows the

IC50 is 2.0 nM. Presently phosphate prodrugs of these compounds (8) and (9)

are under preclinical evaluation. The compound (8) inhibits cell growth at

low concentrations (IC50, P388 murine leukaemia cell line 2.6 nM) and shares

many structural features common to other tubulin-binding agents27 (Figure

2).

MeO

MeO

O

OMe

OMe

OH MeO

MeO

O

OMeOMe

OH

9 (SD400)8

Figure 2. Structures of potential anticancer chalcones

The anticancer activity of certain chalcones is believed to be a result of

binding to tubulin and preventing it from polymerizing into microtubules.

Tubulin is a protein that exists as a heterodimer of two homologous α- and

β -subunits. Many molecules based on a chalcone scaffold have been

synthesized to improve their biological profile, including their capability as

77



T HESIS

sequence selective DNA interactive and cross-linking agents.

Trihydroxychalcone (10) represent a new class of tyrosinase inhibitors. The

ease of synthesis of chalcones from substituted benzaldehydes and

acetophenones, makes them an attractive scaffold. Chalcones have

attracted more interest in recent years because of their diverse

pharmacological properties.28 Among these properties, their cytotoxicity

effects have been extensively examined. Some of the natural chalcones

have been found in a variety of plant sources. These natural compounds

have served as valuable leads for further design and synthesis of more

active analogues.29 A chalcone compound (11) has been reported for its

antiproliferative and antitumor activity 30(Figure 3).

HO

O OCH3

OH

OH

H3CO

O

H3CO

OCH3

OH

1110

Figure 3

Further, in this trimethoxy chalcone series different analogues havebeen synthesized by different groups and evaluated for their cytotoxicity.

These compounds have shown promising activity against different cancer

cell lines31 (Figure 4).

MeO

MeO

O

OMe

NO2

MeO

MeO

O

OMe

OMe

B(OH)2

MeO

MeO

O

OMe

OMe

12 13

14

78



T HESIS

Figure 4

Recently different series of chalcone analogues with potent anticancer

activity has been reported. Dalip Kumar and co-workers have synthesized

and reported indolylchalcones (15) that are most potent and selective

anticancer agents with IC50 values 0.03 and 0.09 µM, against PaCa-2 cell

line.32 Lawrence and co-workers reported new chalcone derivative (16) which

possess good anticancer activity33 (Figure 5).

NH

O

OMe

OMe

OMe

O

OMe

N

OMe

16 (MDL)15

Figure 5

Curcumin, a polyphenolic natural compound (17) derived from dietary

spice turmeric, possesses diverse pharmacological effects including

anticancer, anti-inflammatory, antioxidant, and antiangiogenic activities.34 A

series of of chalcone dimers has been reported as potent inhibitors of various

cancer cells at very low concentrations. The compound 3,5-bis(2-

fluorobenzylidene)-4-piperidone (18, also known as EF24) is a synthetic

analog of curcumin that was first reported by Adams.35 Other analogues of

3,5-bis(benzylidene)-4-piperidones (19, also known as CLEFMA) and (20) are

have been advanced as synthetic analogs of curcumin for anti-cancer

activity and anti-inflammatory properties and these dimers have shown

promising antiproliferative activity against various cancer cell lines36 (Figure

6).

79



T HESIS

NH

O FF

N

O ClCl

O

OH

O19 (CLEFMA)

18 (EF24)

N

O

H3CO

HO

OCH3

OH

CH3

20

HO OH

OMeMeO

OOH

17 (curcumin)

Figure 6. Structures of bis-chalcones

The cyclic chalcone analogues, E-2-arylmethylene-1-indanones, E-2-

arylmethylene-1-tetralones and E-2-arylmethylene-1-benzosuberones have

been synthesized and their cytotoxicity has been determined against

different cancer cell lines.37 Amongst these cyclic chalcones, compounds

(21a, 21b, 22a and 22b) have shown potential anticancer activity against

human cancer cell lines. These compounds inhibit RNA and protein syntheses

and induced apoptosis which are likely major mechanisms whereby

cytotoxicity is mediated. The active compound (22b) in these cyclic

chalcones declines the mitochondrial function as well as mitochondrial DNA

damage. Compound (21b) also showed good activity in targeting

Alzheimer’s disease by inhibition of (acetylcholinesterase) AChE-induced Ab

aggregation38 (Figure 7).

80



T HESIS

O

N

MeO NO2

O

21a 21b

22b

O

OMe

22a

Figure 7

2.2. PRESENT WORK In the past few years, several hybrid compounds, in which a known

antitumour compound or some simple active moiety tethered to PBD, have

been designed, synthesized and evaluated for their biological activity.39−41

Recently, Wang and co-workers have synthesized indole-PBD conjugates

(23) as potential antitumour agents and a correlation between antitumour

activity and apoptosis has been well explained.42 For the last few years, we

have been involved in the development of new synthetic strategies for thepreparation of PBD ring system43,44 and also in the design as well as synthesis

of structurally modified PBDs and their conjugates.45-48 More recently, we

have also reported some of the PBD conjugates (24) that demonstrated

potent apoptotic activity through mitochondrial-mediated pathway49 (Figure

8).

O

MeO

N

N

O

O

F

Cl

N

N H

O

NH

NH

O

MeO N

NO

O

H

23

24

81



T HESIS

Figure 8. Examples for apoptotic inducing PBDs

In view of the interesting biological activities exhibited by PBD

conjugates, there has been considerable interest in structural modifications

apart from the development of new synthetic strategies in this laboratory forsuch compounds. Based on the diverse biological activities of the chalcones

and the pyrrolo[2,1-c][1,4]benzodiazepines, A series of novel compounds

have been designed and synthesized that have both the chalcone as well as

pyrrolo[2,1-c][1,4]benzodiazepine moieties linked through varying length of

alkane spacers and have been evaluated for their antitumour activity and

DNA-binding affinity.

The present work describes the design, synthesis, DNA binding affinity

and in vitro cytotoxicity of novel chalcones linked to a PBD moiety at the C8-

position of the PBD ring through different alkane spacers. Therefore this

chapter describes the synthesis, DNA-binding ability and anticancer activity

of some new PBD conjugates.

2.2.1. S YNTHESIS OF PBD PRECURSORS

The precursor (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]pyrolidine-

2-carboxaldehydediethylthioacetal 33 have been prepared by employing

commercially available vanillicacid. Esterification of vanillicacid 25 followed

by benzylation by literature method50 afford benzylated methyl ester 27. This

upon nitration followed by hydrolysis gives nitro acid 29. This has been

further coupled to L-proline methyl ester to afford the compound 30, which

upon reduction with DIBAL-H produces the corresponding aldehyde 31. The

aldehyde group of compound 31 has been protected with EtSH/TMSCl affords

32. Compound 32 upon debenzylation affords (2S)-N-[4-hydroxy-5-methoxy-

2-nitrobenzoyl]pyrolidine-2-carboxaldehydediethylthioacetal (33) (Scheme

1).

82



T HESIS

BnO

MeO

NO2

OH

O

BnO

MeO

NO2

O

N

COOMe

BnO

MeO

NO2

O

N

CHO BnO

MeO

NO2

O

N

CH(SEt)2

HO

MeO

NO2

O

N

CH(SEt)2

2829

31 32 33

(vi)

(vii)

(iv)(v)

HO

H3COOH

O

HO

H3COOCH3

O

BnO

H3COOCH3

O

ii

iii

26 27

i

25

BnO

H3COOCH3

O

NO2

(viii)

30

Scheme 1. Reagents and conditions: i) H2SO4, MeOH, 24 h; ii) Benzyl bromide, K 2CO3,

acetone, 24 h; iii) HNO3-H2SO4, SnCl4, CH2Cl2, -25 oC, 5 min; iv) 2N LiOH, THF, 12 h; (v) SOCl2,

C6H6, L-proline methyl ester, THF, 6h; (vi) DIBAL-H, CH2Cl2, -70 oC, 30 min; (vii) EtSH, TMSCl,

CH2Cl2, 18h; (viii) EtSH, BF3-OEt2, CH2Cl2, 12h;

2.2.2. SYNTHESIS OF CHALCONE DERIVATIVES

The preparation of chalcone intermediates 37a-f and 40a-c has been

carried out by synthetic sequence illustrated in Scheme-2 & 3. Claisen-

Schmidt condensation of trimethoxy acetophenone with benzaldehydes

using ethanol as solvent in the presence of aqueous KOH gives

trimethoxychalcones 36a,b. The cyclic chalcone (39) have been prepared

using piperidine as base in ethanol solvent under reflux by condensing 1-

indanone with vanillin. Using aqueous KOH for preparation of indanochalcone taking longer reaction time and the yield is very less (20%). These

trimethoxy and indanochalcones undergo etherification of hydroxyl group

with dibromoalkanes by using K 2CO3 as base in dry acetone afford precursors

37a-f and 40a-c.

83



T HESIS

MeO

MeO

OMe

CH3

O CHO

R

OH

+

MeO

MeO

OMe

O

OH

R

MeO

MeO

OMe

O

O

R

Br ( )n

(i)

(ii)

37a-f

34 35a, b 36a, b

37a; R = H, n = 237b; R = H, n = 337c; R = H, n = 437d; R = OMe, n = 237e; R = OMe, n = 337f ; R = OMe, n = 4

Scheme 2. Reagents and conditions: (i) aq.KOH, ethanol, 4h; (ii) dibromoalkane, acetone,

K 2CO3, reflux, 24h.

40a; n = 240b; n = 340c; n = 4

CHO

OMe

OH

+

(i)

(ii)

40a-c

38 3539

O O

OH

OMe

O

O

OMe

Br ( )n

Scheme 3. Reagents and conditions: (i) ethanol/piperidine, reflux, 10 h; (ii) dibromoalkane,acetone, K 2CO3, reflux, 24 h.

2.2.3. S YNTHESIS OF C8-LINKED CHALCONE-PBD H YBRIDS

Compound 33 has been coupled to compounds 37a-f in the presence

of K 2CO3 and dry acetone under reflux gives corresponding nitro compounds

41a-f . These nitro compounds upon reduction with SnCl2.2H2O in methanol

under reflux give amino compounds 42a-f . The amino compounds on

84



T HESIS

deprotection with HgCl2/CaCO3 afford corresponding imines 43a-f (Scheme-

4).

MeO

MeO

OMe

O

O

R

Br ( )n

37a-f

HO

MeO

NO2

O

N

CH(SEt)2

33

+

O

MeO

NO2

O

N

CH(SEt)2O

O

MeO

MeO

OMe

R

( )n

O

MeO

NH2

O

N

CH(SEt)2O

O

MeO

MeO

OMe

R

( )n

O

MeO

O

OMeO

MeO

OMe

R

( )n

N

N

O

H

41a-f

42a-f

43a-f

i

ii

iii


Compound 33 has been coupled to compounds 40a-c in the presence

of K 2CO3 and dry acetone under reflux gives corresponding nitro compounds

44a-c. These nitro compounds upon reduction with SnCl2.2H2O in methanol

under reflux give amino compounds 45a-c. The amino compounds on

deprotection with HgCl2/CaCO3 afford corresponding imines 46a-c (Scheme-

5).

85



T HESIS

HO

MeO

NO2

O

N

CH(SEt)2

33

+

O

MeO

NO2

O

N

CH(SEt)2O

OMe

( )n

O

MeO

NH2

O

N

CH(SEt)2O

OMe

( )n

O

MeO

O

OMe

( )n

N

N

O

H

44a-c

45a-c

46a-c

i

ii

iii

40a-c

O

O

OMe

Br ( )n

O

O

O

Scheme 5. Reagents and conditions: (i) K 2CO3, acetone, 12 h, reflux; (ii) SnCl2.2H2O, MeOH,

4 h, reflux; (iii) HgCl2, CaCO3, MeCN, H2O, (4:1) 12 h, rt.

2.3. BIOLOGICAL ACTIVITY

2.3.1. DNA BINDING AFFINITY : THERMAL DENATURATION STUDIES

The DNA binding affinity of these new C8-linked chalcone-PBD

conjugates (43a-f and 46a-c) has been evaluated through thermal

denaturation studies with duplex-form of calf thymus DNA (CT-DNA) by using

modified reported procedure.51 The DNA-PBD solutions are incubated at 37

οC for 0 h and 18 h prior to analysis. Samples are monitored at 260 nm using

86



T HESIS

a Beckman DU-7400 spectrophotometer fitted with high performance

temperature controller and heated at 1οC/min in the range of 40-95

οC. DNA

helix-coil transition temperatures are given by: ∆ T m = T m(DNA+PBD)–T m(DNA

alone), where the T m value for the PBD-free CT-DNA is 69.8± 0.01. Thesestudies were carried out at PBD/DNA molar ratio 1:5. The increase in melting

temperature (∆ T m) for each compound is examined at 0 h and 18 h of

incubation at 37οC. Melting studies show that these compounds stabilize the

thermal helix coil or melting stabilization for the CT-DNA duplex at pH 7.0,

and incubated at 37οC with ligand/DNA molar ratio of 1:5. The increase in

the helix melting temperature (∆ T m) for each compound has been examined

at 0 h and 18 h incubation at 37 ο C.

Interestingly, all the chalcone-PBD conjugates elevate the helix melting

temperature of CT-DNA in the range of 4.0-8.6 oC. The compound 46b

showed highest ΔT m of 7.9 oC at 0 h and increased upto 8.6 oC after 18 h

incubation, whereas the naturally occurring DC-81 exhibits a ΔT m of 0.7 oC

after incubation under similar conditions (Table 1). These results indicate

that the effect on DNA binding affinity by introducing the chalcone scaffold

on PBD moiety through different alkane spacers at C8-position of the DC-81.

Table 1.Thermal denaturation data for chalcone-PBD conjugates with calf thymus (CT)-DNA

Compound[PBD]:[DNA]

molar ratiob

ΔT m (oC)a after incubation at 37 oCfor

0 h 18 h

43a 1:5 5.1 5.8

43b 1:5 5.2 6.1

43c 1:5 4.6 5.0

43d 1:5 5.3 5.9

43e 1:5 4.0 4.5

87



T HESIS

43f 1:5 4.0 4.3

46a 1:5 7.5 8.2

46b 1:5 7.9 8.6

46b 1:5 7.1 8.4DC-81 1:5 0.3 0.7

a For CT-DNA alone at pH 7.00 ± 0.01, T m = 68.5 0C Δ 0.01 (mean value from 10 separate

determinations), all ΔT m values are ± 0.1 - 0.2 0C. b For a 1:5 molar ratio of [PBD]/[DNA],

where CT-DNA concentration = 100 μM and ligand concentration = 20 μM in aqueous

sodium phosphate buffer [10 mM sodium phosphate + 1 mM EDTA, pH 7.00 ± 0.01].

2.3.2. ANTICANCER ACTIVITY

Compounds (43a-f and 46a-c) have been evaluated for their in vitrocytotoxicity in selected human cancer cell lines of barest, ovarian, colon,

prostate, cervix, lung and oral cancer using Sulforhodamine B (SRB)

method.52 The in vitro cytotoxicity results of these compounds expressed in

GI50 values which carried out the experiments at 10-4 to 10-7 M concentrations

and the data is illustrated in Table 2. The results from these experiments

reveal that compounds 43a-f and 46a-c showed GI50 values in the range of

<0.01-2.7 μM, while the positive controls, DC-81 and adriamycin exhibited

the GI50 in the range of 0.1-0.17 μM and <0.01-14.7 μM respectively. The

synthesized chalcone-PBD conjugates exhibited significant anticancer

activity against PC-3 human prostate cancer cell line (GI50 range, <0.01−0.22

μM) compared to other cell lines tested.

The active compound 46b which is an indano chalcone analogue of

PBD exhibited strong effect against all cell lines tested (GI50, <0.01-0.17 μM)

and it showed a GI50 value of <0.01 against PC-3 cell line. In

trimethoxychalcone-PBD analogues the compounds 43b and 43d showed

promising activity against different cancer cell lines. Among the chalcone-

PBD conjugates synthesized, the compounds with indano chalcone moiety

exhibited superior activity compared to the compounds with trimethoxy

chalcone moiety.

88



T HESIS

Table 2. GI50 valuesa (in μM) for compounds 43a-f and 46a-c in selectedhuman cancer cell lines.

Compound

GI50 values (μM)

Breast Ovarian Colon Prostate Cervix Lung Oral

MCF-7 A2780 Colo205 PC-3 SiHa A 549 Hop-62 KB

43a 0.11 0.14 0.16 0.14 0.12 0.14 0.15 0.15

43b 0.05 0.03 0.15 0.16 1.8 1.88 0.2 2.1

43c 0.29 0.1 0.15 0.16 0.17 0.1 0.13 0.1

43d 0.07 0.028 0.16 0.22 2.7 -- -- 2.6

43e 0.11 0.14 0.17 0.1 0.12 0.14 0.1 0.14

43f 0.14 0.12 0.14 0.15 0.15 0.16 0.16 0.17

46a 0.1 0.12 0.14 0.08 0.14 0.1 0.14 0.18

46b 0.01 0.013 0.14 <0.01 0.17 0.17 0.0160.01

6

46c 0.17 0.14 0.17 0.16 0.16 0.11 0.17 0.14

DC-81 0.16 0.13 0.1 -- 0.16 -- 0.11 0.17

ADR<0.0

10.02 14.7 <0.01 0.19 13 <0.01 0.16

a 50% Growth inhibition and the values are mean of four determinationsADR, adriamycin.

2.4. CONCLUSION

In conclusion, we have synthesized a series of novel C8-linked chalcone-

PBD conjugates (43a-f and 46a-c). For synthesized compounds

anticancer activity has been evaluated against eight human cancer cell

lines (barest, ovarian, colon, prostate, cervix, lung and oral cancer). All

the compounds exhibited significant anticancer activity. Moreover these

compounds exhibited significant DNA binding ability.

2.5. EXPERIMENTAL SECTION

Methyl-4-hydroxy-3-methoxy benzoate (26)

89



T HESIS

The compound 4-hydroxy-3-methoxy benzoic acid 25 (168 mg, 1 mmol) was

dissolved in methanol (10 mL) and to this was added concentrated H2SO4 (2

mL) and the reaction mixture was stirred for 24 h at room temperature. After

completion of the reaction as indicated by TLC, methanol was evaporated

under reduced pressure and the residue was neutralized with saturated

NaHCO3 solution and extracted with ethyl acetate, dried over Na2SO4 and

concentrated to give the crude product. This was further purified by column

chromatography using hexane: ethyl acetate (2:8) as a solvent system to

obtain the pure product 26 (178 mg, 98% yield).

1H NMR (200 MHz, CDCl3): δ 3.88 (s, 3H), 3.95 (s, 3H), 6.62 (bs, 1H), 7.17 (d,

1H, J = 9.2 Hz ), 7.60 (d, 1H), 7.78 (s, 1H);

EIMS: m/z 182 [M]+.

Methyl-4-benzyloxy-3-methoxybenzoate (27)

To the solution of compound 26 (182 mg, 1 mmol) in acetone (20 mL)

was added, anhydrous K 2CO3 (553 mg, 4 mmol) and benzyl bromide (256 mg,

1.5 mmol), the mixture was refluxed in an oil bath for 24 h. The reaction was

monitored by TLC using EtOAc-hexane (2:8) and K 2CO3 was removed by

filtration, solvent was evaporated under reduced pressure. The crude thus

obtained was purified by column chromatography (10% EtOAc-hexane) to

afford compound 27 as white solid (250 mg, 92%);

Mp: 116−118οC.

1H NMR (200 MHz, CDCl3): δ 3.94 (s, 3H), 3.98 (s, 3H), 5.2 (s, 2H), 6.88 (d,

1H, J = 8.2 Hz), 7.20−7.50 (m, 6H), 7.65 (d, 1H, J = 8.8 Hz);

EI MS: m/z 272 [M]+

Methyl-4-benzyloxy-5-methoxy-2-nitrobenzoate (28)

A freshly prepared mixture of stannic chloride (301 mg, 1.2 mmol) and

fuming nitric acid (98 mg, 1.56mmol) in dichloromethane was added drop

wise over 5 minutes with stirring to a solution of methyl-4-benzyloxy-3-

methoxybenzoate 27 (272 mg, 1 mmol) in dichloromethane (30 mL) at –78

90



T HESIS

οC. The mixture was maintained at –78

οC for a further 5 minutes, quenched

with water (20 mL) and then allowed to return room temperature. The

organic layer was separated and the aqueous layer was extracted with

dichloromethane (2x20 mL). The combined organic phase dried over Na2SO4,evaporated in vacuum and purified by column chromatography (20% EtOAc-

hexane) affords 28 as a yellow solid (247 mg, 78%). Mp 128−130οC;

1H NMR (200 MHz, CDCl3): δ 7.65 (d, 1H, J = 8.8 Hz), 7.45−7.2 (m, 6H), 6.78

(d, 1H, J = 8.2 Hz), 5.2 (s, 2H), 3.95 (s, 3H), 3.89 (s, 3H);

EIMS: m/z 317 (M)+.

4-Benzyloxy-5-methoxy-2-nitrobenzoic acid (29)

2N Lithium hydroxide monohydrate solution (1.22 mL) was added to a

solution of methyl-4-benzyloxy-5-methoxy-2-nitrobenzoate 28 (317 mg, 1

mmol) in THF-H2O-MeOH (4:1:1) and the mixture stirred at room temperature

for 12 h. After completion reaction THF and methanol were evaporated, the

aqueous phase was acidified with dilute HCl to pH 7 and reextracted with

CH2Cl2 to give a 4-benzyloxy-5-methoxy-2-nitrobenzoic acid 29 as a pale

yellow solid (251 mg, 83%);

Mp: 180−182οC;

1H NMR (200 MHz, CDCl3): δ 7.44−7.22 (m, 6H), 7.2 (s, 1H), 5.25 (s, 2H), 3.98

(s, 3H);

EIMS: m/z 303 (M)+.

Methyl-(2S)-N-[4-benzyloxy-5-methoxy-2-nitrobenzoyl]prrolidine-2-

carboxylate (30)

To a suspension of compound 29 (303 mg, 1 mmol) and thionyl chloride (476

mg, 4.0 m mol) in dry benzene (15 mL) was added few drops of DMF and

stirred for 6 hr. The toluene was evaporated in vacuum and the resultant oil

was dissolved in dry THF (20 ml) and added drop wise over a period of 30

mins to a cooled suspension of L-proline methyl ester hydrochloride (248 mg,

91



T HESIS

1.5 mmol), triethyl amine (303 mg, 3 mmol) in THF (20 mL). After completion

of addition the reaction mixture was brought to ambient temperature and

stirred for additional hours. The THF was evaporated under vacuum and the

aqueous layer was extracted with ethyl acetate, washed with water followed

by brine solution. The organic layer was dried over Na2SO4 evaporated under

vacuum and was purified by column chromatography using EtOAc-hexane

(3:7) to afford compound 30 as yellow solid. Yield (352 mg, 70%).

Mp: 152-154 ºC;

1H NMR (300 MHz, CDCl3): δ 7.72 (s, 1H), 7.3–7.48 (m, 6H), 5.2 (s, 2H), 4.68–

4.75 (m, 1H), 3.92 (s, 3H), 3.8 (s, 3H), 3.26–3.4 (m, 1H), 3.1–3.25 (m, 1H),

2.2–2.45 (m, 1H), 1.81–2.12 (m, 3H);

ESIMS: m/z 414 (M) +.

(2S)-N-[4-Benzyloxy-5-methoxy–2-nitrobenzoyl]pyrrolidine-2-

carboxaledhyde (31)

Diisobutylaluminiumhydride solution (2.5 ml of 1.0 M solution in toluene) was

added drop wise to a stirred solution of the compound 30 (414 mg, 1mmol)

in anhydrous dichloromethane (10 mL) under nitrogen atmosphere at –78 oC.

After addition the mixture was stirred for 30 mins. After completion of reaction it was decomposed by slow addition of methanol (2 mL) followed by

5% HCl (2 mL). The resultant mixture was allowed to warm to room

temperature. The reaction mixture layer was extracted with chloroform

(4X20 mL), the combined organic layers were dried over Na2SO4 and solvent

was evaporated under vacuum to afford the crude aldehyde 31. Yield (254

mg 65%)

1H NMR (300 MHz, CDCl3): δ 9.8 (s, 1H), 9.25 (s, 1H), 7.75 (s, 1H), 7.25–7.5

(m, 5H), 6.56 (s, 1H), 5.2 (s, 2H), 4.7 (m, 1H), 4.02 (m, 1H), 3.18–3.38 (m,

2H), 1.88–2.4 (m, 4H);

ESIMS: m/z 355 (M-CHO) +

92



T HESIS

(2S)-N-[4-Benzyloxy-5-methoxy–2-nitrobenzoyl]pyrrolidine-2-carboxaledhyde diethylthioacetal (32)

Ethanethiol (278 mg, 4.4 mmol) was added to a stirred solution of

nitroaldehyde 31 (384 mg, 1 mmol) in dry dichloromethane (25 mL) under

nitrogen atmosphere. The mixture was stirred for further 10 mins followed by

the addition of trimethylsilly chloride (540 mg, 5 mmol). The resulting

mixture was stirred at room temperature for about 18 hrs. After completion

of the reaction, the reaction mixture was neutralized with sodium

bicarbonate solution and extracted with chloroform (2x15 mL). The combined

organic phases were dried over Na2SO4 and evaporated under vacuum to

afford the crude diethylthioacetal (32), which was purified by column

chromatography by using EtOAc-hexane (6:4) to afford pure compound 32

as viscous oil. Yield (335 mg, 75%).

1H NMR (300 MHz, CDCl3): δ 7.75 (s, 1H), 7.32–7.48 (s, 5H), 6.85 (s, 1H), 5.2

(s, 2H), 4.85 (d, 1H), 4.66–4.75 (m, 1H), 4 (s, 3H), 3.2–3.35 (m, 2H), 2.65–

2.96 (m, 4H), 1.21–1.42 (m, 6H);

ESIMS: m/z 490 (M+H)+.

(2S)-N-[4-Hydroxy-5-methoxy–2-nitrobenzoyl]pyrrolidine-2-

carboxaledhyde diethylthioacetal (33) To a stirred solution of EtSH of (1.91 gm, 20 mmol) and BF3OEt2 (1.41 gm, 10

mmol) in dichloromethane was added drop wise to a solution of the

compound 32 (0.49 gm, 1mmol) in dichloromethane (10 mL) at room

temperature. Stirring was continued until TLC indicated complete of the

reaction. The reaction mixture was quenched with bicarbonate solution and

the extracted with chloroform (3x25 mL). The combined organic phases were

washed with brine solution (1x25 mL), dried over Na2SO4 and the solvent

removed under vacuum to afford the crude product. This was further purified

by column chromatography using ethyl acetate-hexane (7:3) as eluant to

afford pure compound 33. Yield (300 mg, 75%).

93



T HESIS

1H NMR (300 MHz, CDCl3): δ 7.6 (s, 1H), 6.75 (s, 1H), 4.85 (d, 1H), 4.6–4.7 (m,

1H), 3.9 (s, 3H) 3.2–3.32 (m, 2H), 2.7–2.88 (m, 4H), 1.75–2.35 (m, 4H), 1.2–

1.4 (m, 6H);

ESIMS: m/z 400 (M) +.

(E)-3-(4-hydroxyphenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one

(36a)

To a stirred mixture of 3,4,5-trimethoxy acetophenone (210 mg, 1 mmol)

and 4-hydroxybenzaldehyde (122 mg, 1 mmol) in ethanol (10 mL) was added

50% aqueous solution of potassium hydroxide (1 ml) and stirred for 4 h at

room temperature. After completion of the reaction checked by TLC, the

solvent was evaporated, neutralized with dilute HCl and extracted with

ethylacetate (2x50 ml). The combined organic fractions were washed with

water followed by brain, dried over Na2SO4 and purified by column

chromatography using (30% EtOAC:hexane) to obtain the pure product 36a

as yellow solid. Yield (285 mg, 90%).

Mp: 136-138 ºC;

1H NMR (300 MHz, CDCl3): δ 7.79 (d, 1H, J = 15.1 Hz), 7.57 (d, 2H, J = 9 Hz),

7.36 (d, 1H, J = 15.1 Hz), 7.27 (s, 2H), 6.91 (d, 2H, J = 8.3 Hz), 6.05 (bs, 1H),3.94 (s, 6H), 3.92 (s, 3H);


(E)-3-(4-hydroxy-3-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)prop-

2-en-1-one (36b)

The compound 36b was prepared according to the method described for

compound 36a by employing compound 3,4,5-trimethoxy acetophenone

(210 mg, 1mmol), and 3-methoxy-4-hydroxy benzaldehyde (152 mg, 1

mmol). Yield (310 mg, 90%)

Mp: 127-129 ºC;

94



T HESIS

1H NMR (300 MHz, CDCl3): δ 7.68 (d, 1H, J = 15.8 Hz), 7.27 (d, 1H, J = 15.8

Hz), 7.16–7.23 (m, 3H), 7.05 (d, 1H, J = 2.2 Hz), 6.89 (d, 1H, J = 8.3 Hz),

6.18 (bs, 1H), 3.96 (s, 9H), 3.89 (s, 3H);


(E)-3-(4-(3-bromopropoxy)phenyl)-1-(3,4,5-trimethoxyphenyl)prop-

2-en-1-one (37a)

To a solution of compound 36a (314 mg, 1 mmol) in dry acetone (15 mL)

was added, anhydrous K 2CO3 (274 mg, 2 mmol), 1,3-dibromopropane (605

mg, 3 mmol) and the mixture was stirred at reflux temperature for 24 hours.

The reaction was monitored by TLC using ethyl acetate-hexane (3:7). After

completion of the reaction as indicated by the TLC, K 2CO3 was removed by

filtration and the solvent evaporated under reduced pressure, diluted with

water and extracted with ethyl acetate(2X20 ml). The combined organic

phases were dried over Na2SO4 and evaporated under vacuum. The residue,

thus obtained was purified by column chromatography using ethyl acetate

and hexane (2:8) to afford pure compound 37a as viscous liquid. Yield (418

mg, 95%)

1H NMR (300 MHz, CDCl3): δ 7.78 (d, 1H, J = 15.8 Hz), 7.62 (d, 2H, J = 9 Hz),7.36 (d, 1H, J = 15.8 Hz), 7.25 (s, 2H), 6.92 (d, 2H, J = 8.3 Hz), 4.05 (t, 2H, J

= 6, 5.2 Hz), 3.95 (s, 6H), 3.91 (s, 3H), 3.42 (t, 2H, J = 6.7, 6 Hz), 2.04-2.16

(m, 2H);


(E)-3-(4-(4-bromobutoxy)phenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-

en-1-one (37b)


compound 37a by employing compound 36a (314 mg, 1 mmol), and 1,4

dibromobutane (647 mg, 3 mmol). Yield (403 mg, 90%).


7.31 (d, 1H, J = 15.8 Hz), 7.23 (s, 2H), 6.88 (d, 2H, J = 8.3 Hz), 4.04 (t, 2H, J

95



T HESIS

= 6, 5.2 Hz), 3.95 (s, 6H), 3.9 (s, 3H), 3.47 (t, 2H, J = 6.7, 6 Hz), 1.92–2.13

(m, 4H);


(E)-3-(4-(5-bromopentoxy)phenyl)-1-(3,4,5-trimethoxyphenyl)prop-

2-en-1-one (37c)

The compound 37c was prepared according to the method described for

compound 37a by employing compound 36a (314 mg, 1 mmol), and 1,5

dibromopentane (689 mg, 3 mmol). Yield (422 mg, 91%)


7.35 (d, 1H, J = 15.8 Hz), 7.25 (s, 2H), 6.91 (d, 2H, J = 8.3 Hz), 4.04 (t, 2H, J

= 6, 5.2 Hz), 3.94 (s, 6H), 3.91 (s, 3H), 3.41 (t, 2H, J = 6.7, 6 Hz), 1.82-2.03

(m, 4H), 1.59–1.71 (m, 2H);


(E)-3-(4-(3-bromopropoxy)-3-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (37d)

The compound 37d was prepared according to the method described for

compound 37a by employing compound 36b (344 mg, 1 mmol), and 1,3

dibromopropane (605 mg, 3 mmol). Yield (400 mg, 86%)1H NMR (200 MHz, CDCl3): δ 7.77 (d, 1H, J = 15.8 Hz), 7.33 (d, 1H, J = 15.8

Hz), 7.21−7.28 (m, 3H), 7.16 (d, 1H, J = 2.2 Hz), 6.90 (d, 1H, J = 8.3 Hz), 4.08

(t, 2H, J = 6.7, 6 Hz), 3.95 (s, 6H), 3.94 (s, 6H), 3.43 (t, 2H, J = 6.7 Hz),

1.82−1.97 (m, 2H);


(E)-3-(4-(4-bromobutoxy)-3-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (37e)

The compound 37e was prepared according to the method described for

compound 37a by employing compound 36b (344 mg, 1 mmol), and 1,4-

dibromobutane (647 mg, 3 mmol). Yield (425 mg, 88%)

96



T HESIS


Hz), 7.17–7.24 (m, 3H), 7.1 (d, 1H, J = 1.5 Hz), 6.84 (d, 1H, J = 8.3 Hz), 4.07

(t, 2H, J = 6, 5.2 Hz), 3.95 (s, 6H), 3.91 (s, 3H), 3.9 (s, 3H), 3.5 (t, 2H, J =

6.79, 6.04 Hz), 1.94–2.17 (m, 4H);


(E)-3-(4-(3-bromopropoxy)-3-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (37f)

The compound 37f was prepared according to the method described for




Hz), 7.21−7.29 (m, 3H), 7.16 (d, 1H, J = 2.2 Hz), 6.90 (d, 1H, J = 8.3 Hz), 4.09

(t, 2H, J = 6.7, 6 Hz), 3.96 (s, 6H), 3.94 (s, 6H), 3.45 (t, 2H, J = 6.7 Hz),

1.84−2.02 (m, 4H), 1.58−1.73 (m, 2H);


(E)-2-(4-hydroxy-3-methoxybenzylidene)-2,3-dihydroinden-1-one(39)

To

a stirred mixture of 1-indanone (132 mg, 1 mmol) and 4-hydroxy-3-

methoxy benzaldehyde (152 mg, 1 mmol) in ethanol (10 ml) was added few

drop of piperidine and refluxed for 10 h. After completion of the reaction

checked by TLC, the solvent was evaporated and extracted with ethyl

acetate (2x50 ml). The combined organic fractions were washed with water

followed by brain, dried over Na2SO4 and purified by column chromatography

using (30% EtOAC:hexane) to obtain the pure product 39. Yield (200 mg,

75%).

1H NMR (300 MHz, DMSO D6): δ 9.77 (s, 1H), 7.79 (d, 1H, J = 7.3 Hz), 7.67–

7.74 (m, 2H), 7.44–7.56 (m, 2H), 7.37 (s, 1H), 7.3 (d, 1H, J = 8 Hz), 6.92 (d,

1H, J = 8 Hz), 4.12 (s, 2H), 3.90 (s, 3H);

ESIMS: m/z 289 (M+Na)+.

97



T HESIS

(E)-2-(4-(3-bromopropoxy)-3-methoxybenzylidene)-2,3-

dihydroinden-1-one (40a)

The compound 40a was prepared according to the method described for

compound 37a by employing compound 39 (266 mg, 1 mmol), and 1,3-dibromopropane (605 mg, 3 mmol). Yield (320 mg, 82%)

1H NMR (300 MHz, CDCl3): δ 7.87 (d, 1H, J = 7.5 Hz), 7.48−7.61 (m, 3H),

7.40 (t, 1 H, J = 7.5, 6.7 Hz), 7.21−7.27 (m, 1H), 7.13 (d, 1H, J = 1.5 Hz),

6.93 (d, 1H, J = 8.3 Hz), 4.19 (t, 2H, J = 6 Hz), 3.99 (s, 2H), 3.92 (s, 3H),

3.63 (t, 2H, J = 6.7, 6 Hz), 2.33−2.44 (m, 2H);


(E)-2-(4-(4-bromobutoxy)-3-methoxybenzylidene)-2,3-dihydroinden-

1-one (40b)


compound 37a by employing compound 39 (266 mg, 1 mmol), and 1,4-


1H NMR (300 MHz, CDCl3): δ 7.88 (d, 1H, J = 8 Hz), 7.48–7.63 (m, 3H), 7.35–

7.46 (m, 1H), 7.24 (dd, 1H, J = 8, 2.2 Hz), 7.14 (d, 1H, J = 1.4 Hz), 6.89 (d,

1H, J = 8.8 Hz), 4.09 (t, 2H, J = 5.8 Hz), 4 (s, 2H), 3.93 (s, 3H), 3.5 (t, 2H, J

= 6.6 Hz), 1.94–2.21 (m, 4H);

ESIMS: m/z 401 (M)+.

(E)-2-(4-(5-bromopentoxy)-3-methoxybenzylidene)-2,3-

dihydroinden-1-one (40c)


compound 37a by employing compound 39 (266 mg, 1 mmol), and 1,5-


1H NMR (300 MHz, CDCl3): δ 7.87 (d, 1H, J = 7.5 Hz), 7.48−7.61 (m, 3H), 7.4

(t, 1 H, J = 7.5, 6.7 Hz), 7.23 (dd, 1H, J = 8.3, 1.5 Hz), 7.13 (d, 1H, J = 2.2

98



T HESIS

Hz), 6.87 (d, 1H, J = 8.3 Hz), 4.05 (t, 2H, J = 6.7, 6 Hz), 3.99 (s, 2H), 3.92 (s,

3H), 3.43 (t, 2H, J = 6.7 Hz), 1.83−2.03 (m, 4H), 1.60−1.74 (m, 2H);


2S)-N-{4-(3-[(E)-3-(4-phenoxy)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one]propyl)oxy-5-methoxy-2-nitrobenzoyl}-pyrrolidine-2-carboxaldehydediethyl thioacetal (41a)

To a solution of (2S)-N-[4-hydroxy-5-methoxy–2-nitrobenzoyl]pyrrolidine-2-

carboxal dehydediethylthioacetal (33) (400 mg, 1 mmol) in dry acetone (15

mL) was added, anhydrous K 2CO3 (276 mg, 2 mmol), (E)-3-(4-(3-

bromopropoxy) phenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (37a)

(435 mg, 1 mmol) and the mixture was stirred at reflux temperature for 24

hours. The reaction was monitored by TLC using ethyl acetate-hexane (1:1).

After completion of the reaction as indicated by the TLC, K 2CO3 was removed

by filtration and the solvent evaporated under reduced pressure, diluted with

water and extracted with ethyl acetate. The organic phase was dried over

Na2SO4 and evaporated under vacuum. The residue, thus obtained was

purified by column chromatography using ethyl acetate and hexane (1:1) to

afford compound 41a as yellow solid. Yield (680 mg, 90%)

Mp: 176-178 ºC;1H NMR (300 MHz, CDCl3): δ 7.77 (d, 1H, J = 15.8 Hz), 7.68 (s, 1H), 7.61 (d,

1H, J = 15.1 Hz), 7.25 (s, 2H), 6.91 (d, 2H, J = 8.3 Hz), 6.77 (s, 1H), 4.82 (d,

1H, J = 3.7 Hz), 4.62–4.71 (m, 1H), 4.1–4.23 (m, 4H), 3.95 (s, 6H), 3.91 (s,

6H), 3.17–3.31 (m, 2H), 2.62–2.85 (m, 4H), 2.2–2.36 (m, 1H), 2.02–2.16 (m,

3H), 1.91–1.98 (m, 1H), 1.74–1.86 (m, 1H), 1.31–1.41 (m, 6H);


2S)-N-{4-(4-[(E)-3-(4-phenoxy)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one]butyl)oxy-5-methoxy-2-nitrobenzoyl}-pyrrolidine-2-carboxaldehydediethyl thioacetal (41b)


compound 41a by employing (2S)-N-[4-hydroxy-5-methoxy–2-

nitrobenzoyl]pyrrolidine-2-carboxal dehydediethylthioacetal (33) (400 mg,

99



T HESIS

1mmol), and (E)-3-(4-(4-bromobutoxy)phenyl)-1-(3,4,5-

trimethoxyphenyl)prop-2-en-1-one (37b) (449 mg, 1 mmol). Yield (705 mg,

91%)

Mp: 175-177 ºC;

1H NMR (200 MHz, CDCl3): δ 7.73 (d, 1H, J = 15.8 Hz), 7.63 (s, 1H), 7.57 (d,

1H, J = 15.1 Hz), 7.24 (s, 2H), 6.88 (d, 2H, J = 8.3 Hz), 6.76 (s, 1H), 4.81 (d,

1H, J = 3.7 Hz), 4.62–4.7 (m, 1H), 4.08–4.22 (m, 4H), 3.95 (s, 6H), 3.9 (s,

6H), 3.16–3.3 (m, 2H), 2.62–2.86 (m, 4H), 2.19–2.36 (m, 1H), 2.01–2.16 (m,

5H), 1.90–1.99 (m, 1H), 1.74–1.87 (m, 1H), 1.30–1.39 (m, 6H);


2S)-N-{4-(5-[(E)-3-(4-phenoxy)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one]pentyl)oxy-5-methoxy-2-nitrobenzoyl}-pyrrolidine-2-carboxaldehydediethylthioacetal (41c)


compound 41a by employing (2S)-N-[4-hydroxy-5-methoxy–2-

nitrobenzoyl]pyrrolidine-2-carboxaldehydediethylthioacetal (33) (400 mg,

1mmol), and (E)-3-(4-(5-bromopentoxy)phenyl)-1-(3,4,5-

trimethoxyphenyl)prop-2-en-1-one (37c) (463 mg, 3 mmol). Yield (721 mg,

92%)Mp: 172-174 ºC;


1H, J = 15.1 Hz), 7.24 (s, 2H), 6.9 (d, 2H, J = 8.3 Hz), 6.77 (s, 1H), 4.82 (d,

1H, J = 3.7 Hz), 4.61–4.71 (m, 1H), 4.1–4.22 (m, 4H), 3.96 (s, 6H), 3.91 (s,

6H), 3.17–3.31 (m, 2H), 2.61–2.85 (m, 4H), 2.2–2.35 (m, 1H), 2.02–2.16 (m,

5H), 1.9–1.98 (m, 1H), 1.72–1.82 (m, 3H), 1.31–1.4 (m, 6H);


2S)-N-{4-(3-[(E)-3-(4-[3-methoxyphenoxy])-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one]propyl)oxy-5-methoxy-2-nitrobenzoyl}-pyrrolidine-2-carboxaldehydediethyl thioacetal (41d)


compound 41a by employing (2S)-N-[4-hydroxy-5-methoxy–2-nitrobenzoyl]

100



T HESIS

pyrrolidine-2-carboxal dehyde diethylthioacetal (33) (400 mg, 1 mmol), and

(E)-3-(4-(3-bromopropoxy)-3-methoxyphenyl)-1-(3,4,5-

trimethoxyphenyl)prop-2-en-1-one (37d) (465 mg, 3 mmol). Yield (712 mg,

90%)

Mp: 165-167 ºC;


1H, J = 15.6 Hz), 7.14–7.25 (m, 3H), 7.11 (s, 1H), 6.83 (d, 1H, J = 7.8 Hz),

6.76 (s, 1H), 4.82 (d, 1H, J = 3.9 Hz), 4.60–4.74 (m, 1H), 4.0–4.15 (m, 4H),

3.95 (s, 6H), 3.93 (s, 3H), 3.90 (s, 3H), 3.16–3.31 (m, 2H), 2.61–2.88 (m,

4H), 2.16–2.38 (m, 1H), 1.79–2.14 (m, 5H), 1.79–2.14 (m, 5H), 1.28–1.42

(m, 6H);


2S)-N-{4-(4-[(E)-3-(4-[3-methoxyphenoxy])-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one]butyl)oxy-5-methoxy-2-nitrobenzoyl}-pyrrolidine-2-carboxaldehydediethyl thioacetal (41e)


compound 41a by employing (2S)-N-[4-hydroxy-5-methoxy–2-nitrobenzoyl]

pyrrolidine-2-carboxal dehydediethylthioacetal (33) (400 mg, 1mmol), and

(E)-3-(4-(4-bromobutoxy)-3-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (37e) (479 mg, 3 mmol). Yield (751 mg, 93%)

Mp: 165-167 ºC;


1H, J = 15.8 Hz), 7.27 (s, 2H), 7.21−7.26 (dd, 1H, J = 7.5, 1.5 Hz), 7.16 (d,

1H, J = 1.5 Hz), 6.92 (d, 1H, J = 8.3 Hz), 6.82 (s, 1H), 4.88 (d, 1H, J = 3.7

Hz), 4.65−4.75 (m, 1H), 4.15−4.27 (m, 4H), 3.96 (s, 6H), 3.94 (s, 6H), 3.92

(s, 3H), 3.17−3.34 (m, 2H), 2.64−2.89 (m, 4H), 2.20−2.38 (m, 1H), 2.03−2.17

(m, 5H), 1.90−2.02 (m, 1H), 1.73−1.87 (m, 1H), 1.30–1.40 (m, 6H);


101



T HESIS

2S)-N-{4-(5-[(E)-3-(4-[3-methoxyphenoxy])-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one]pentyl)oxy-5-methoxy-2-nitrobenzoyl}-pyrrolidine-2-carboxaldehydediethyl thioacetal (41f)


compound 41a by employing (2S)-N-[4-hydroxy-5-methoxy–2-nitrobenzoyl]pyrrolidine-2-carboxal dehydediethylthioacetal (33) (400 mg, 1mmol), and

(E)-3-(4-(5-bromopentoxy)-3-methoxyphenyl)-1-(3,4,5-

trimethoxyphenyl)prop-2-en-1-one (37f) (493 mg, 3 mmol). Yield (765 mg,

94%)

Mp: 164-166 ºC;


1H, J = 15.8 Hz), 7.27 (s, 2H), 7.22−7.25 (dd, 1H, J = 7.9, 1.9 Hz), 7.16 (d,1H, J = 2.2 Hz), 6.91 (d, 1H, J = 7.9 Hz), 6.83 (s, 1H), 4.88 (d, 1H, J = 3.9

Hz), 4.68−4.74 (m, 1H), 4.08−4.17 (m, 4H), 3.95 (s, 6H), 3.94 (s, 6H), 3.93

(s, 3H), 3.19−3.33 (m, 2H), 2.68−2.86 (m, 4H), 2.22−2.32 (m, 1H), 2.07−2.14

(m, 1H), 1.92−2.02 (m, 5H), 1.76−1.86 (m, 1H), 1.67−1.75 (m, 2H), 1.31–1.39

(m, 6H);


7-Methoxy-8-[3-{(E)-3-(4-phenoxy)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one} propoxy]-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (43a)

To the compound 41a (754 mg, 1 mmol) in methanol (20 mL) was added

SnCl2.2H2O (1.12 g, 5 mmol) and reflux for 5 hrs and checked TLC indicated

the reaction was completed. The methanol was evaporated under vacuum

and the reaction mass was neutralized with 10% NaHCO3 solution and the

extracted with ethyl acetate and chloroform (2x30mL and 2x30mL). The

combined organic phases was dried over Na2SO4 and evaporated under

vacuum to afford the crude aminodiethylthioacetal 42a (650 mg, 89%),

which was used directly in the next step due to its potential stability

problem.

102



T HESIS

A solution of 42a (724 mg, 1.0 mmol), HgCl2 (677 mg, 2.5 mmol) and CaCO3

(250 mg, 2.5 mmol) in acetonitrile-water (4:1) was stirred slowly at room

temperature overnight until complete consumption of starting material as

indicated by the TLC. The clear organic supernatant liquid was extracted with

ethyl acetate and washed with saturated 5% NaHCO3 (20 mL), brine (20 mL)

and the combined organic phase was dried over Na2SO4. The organic layer

was evaporated in vacuum to afford a white solid, which was purified by

column chromatography with MeOH-CHCl3 (1:20) to obtain the pure product

43a. Yield (325 mg, 54%).

Mp: 122-123 ºC;


Hz), 7.62 (d, 2H, J = 9 Hz), 7.51 (s, 1H), 7.38 (d, 1H, J = 15.1 Hz), 7.28 (s,

2H), 6.93 (d, 2H, J = 9 Hz), 6.82 (s, 1H), 4.08–4.18 (m, 4H), 3.95 (s, 6H),

3.93 (s, 3H), 3.92 (s, 3H), 3.71–3.84 (m, 2H), 3.52–3.64 (m, 1H), 2.28–2.38 (m,

2H), 1.83–2.05 (m, 4H);


7-Methoxy-8-[4-{(E)-3-(4-phenoxy)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one} butoxy]-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c]

[1,4]benzodiazepin-5-one (43b)

This compound was prepared according to the method described for the

compound 43a employing 41b (768 mg, 1.0 mmol) which reduction with

SnCl2.2H2O (1.12 g, 5 mmol) gives amino compound 42b. Deprotection

followed by cyclization of 42b (738 mg, 1.0 mmol) with HgCl2 (677 mg, 2.5

mmol), CaCO3 (250 mg, 2.5 mmol) in acetonitrile-water (4:1) to obtain the

pure product 43b. Yield (330 mg, 53%).

Mp: 121-123 ºC;


Hz), 7.61 (d, 2H, J = 9 Hz), 7.52 (s, 1H), 7.37 (d, 1H, J = 15.8 Hz), 7.28 (s,

2H), 6.94 (d, 2H, J = 9 Hz), 6.82 (s, 1H), 4.06–4.17 (m, 4H), 3.96 (s, 6H),

3.94 (s, 3H), 3.93 (s, 3H), 3.78–3.85 (m, 1H), 3.69–3.77 (m, 1H), 3.53–3.65

(m, 1H), 2.28–2.38 (m, 2H), 1.94–2.16 (m, 6H);

103



T HESIS


7-Methoxy-8-[5-{(E)-3-(4-phenoxy)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one} pentoxy]-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (43c)


compound 43a employing 41c (782 mg, 1.0 mmol) which reduction with

SnCl2.2H2O (1.12 g, 5 mmol) gives amino compound 42c. Deprotection

followed by cyclization of 42c (752 mg, 1.0 mmol) with HgCl2 (677 mg, 2.5


pure product 43c. Yield (342 mg, 54%).

Mp: 120-122 ºC;

1H NMR (200 MHz, CDCl3): δ 7.77 (d, 1H, J = 15.1 Hz), 7.68 (d, 1H, J = 4.5 Hz),

7.62 (d, 2H, J = 9 Hz), 7.52 (s, 1H), 7.37 (d, 1H, J = 15.1 Hz), 7.27 (s, 2H),

6.93 (d, 2H, J = 9 Hz), 6.81 (s, 1H), 4.07–4.16 (m, 4H), 3.95 (s, 6H), 3.93 (s,

3H), 3.92 (s, 3H), 3.72–3.85 (m, 2H), 3.52–3.65 (m, 1H), 2.28–2.38 (m, 2H),

1.9–2.13 (m, 6H), 1.61−1.72 (m, 2H);


7-Methoxy-8-[3-{(E)-3-(4-[3-methoxyphenoxy])-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one}propoxy]-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (43d)


compound 43a employing 41d (784 mg, 1.0 mmol) which reduction with

SnCl2.2H2O (1.12 g, 5 mmol) gives amino compound 42d. Deprotection

followed by cyclization of 42d (754 mg, 1.0 mmol) with HgCl2 (677 mg, 2.5


pure product 43d. Yield (361 mg, 57%).

Mp: 115-117 ºC;


Hz), 7.52 (s, 1H), 7.33 (d, 1H, J = 15.4 Hz), 7.27 (s, 2H), 7.24 (dd, 1H, J =

8.1, 1.7 Hz), 7.15 (d, 1H, J = 1.8 Hz), 6.90 (d, 1H, J = 8.3 Hz), 6.81 (s, 1H),

4.03–4.13 (m, 4H), 3.95 (s, 6H), 3.94 (s, 6H), 3.93 (s, 3H), 3.78–3.87 (m,

104



T HESIS

1H), 3.69–3.76 (m, 1H), 3.69–3.76 (m, 1H), 3.53–3.64 (m, 1H), 2.27–2.38

(m, 1H), 2.0–2.11 (m, 1H), 1.84–1.98 (m, 4H);


7-Methoxy-8-[4-{(E)-3-(4-[3-methoxyphenoxy])-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one}butoxy]-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (43e)


compound 43a employing 41e (798 mg, 1.0 mmol) which reduction with

SnCl2.2H2O (1.12 g, 5 mmol) gives amino compound 42e. Deprotection

followed by cyclization of 42e (768 mg, 1.0 mmol) with HgCl2 (677 mg, 2.5


pure product 43e. Yield (362 mg, 57%).

Mp: 115-117 ºC;



8.1, 1.7 Hz), 7.15 (d, 1H, J = 1.8 Hz), 6.91 (d, 1H, J = 8.3 Hz), 6.82 (s, 1H),

4.05–4.16 (m, 4H), 3.96 (s, 6H), 3.94 (s, 6H), 3.93 (s, 3H), 3.76–3.85 (m,

1H), 3.69–3.75 (m, 1H), 3.53–3.65 (m, 1H), 2.26–2.38 (m, 2H), 1.92–2.07

(m, 4H), 1.61–1.79 (m, 2H);ESIMS: m/z 645 (M+H)+.

7-Methoxy-8-[5-{(E)-3-(4-[3-methoxyphenoxy])-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one}pentoxy]-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (43f)


compound 43a employing 41f (812 mg, 1.0 mmol) which reduction with

SnCl2.2H2O (1.12 g, 5 mmol) gives amino compound 42f . Deprotection

followed by cyclization of 42f (782 mg, 1.0 mmol) with HgCl2 (677 mg, 2.5


pure product 43f. Yield (345 mg, 52%).

Mp: 113-114 ºC;

105



T HESIS



8.1, 1.7 Hz), 7.14 (d, 1H, J = 1.8 Hz), 6.91 (d, 1H, J = 8.3 Hz), 6.81 (s, 1H),

4.07–4.15 (m, 4H), 3.95 (s, 6H), 3.94 (s, 6H), 3.93 (s, 3H), 3.77–3.86 (m,

1H), 3.68–3.76 (m, 1H), 3.52–3.64 (m, 1H), 2.27–2.38 (m, 2H), 2–2.11 (m,

1H), 1.9–2.08 (m, 6H), 1.62−1.71 (m, 2H);


2S)-N-{4-(3-[(E)-2-(4-{3-methoxybenzylidene}oxy)-2,3-dihydroinden-1-one]propyl) oxy-5-methoxy-2-nitrobenzoyl}-pyrrolidine-2-carboxaldehydediethylthioacetal (44a)

The compound 44a was prepared according to the method described for

compound 41a by employing 2S)-N-[4-hydroxy-5-methoxy–2-nitrobenzoyl]


(E)-2-(4-(3-bromopropoxy)-3-methoxybenzylidene)-2,3-dihydroinden-1-one

(40a) (387 mg, 1 mmol). Yield (655 mg, 92%)

1H NMR (200 MHz, CDCl3): δ 7.88 (d, 1H, J = 7.5 Hz), 7.70 (s, 1H), 7.49−7.61

(m, 3H), 7.41 (t, 1 H, J = 7.5, 6.7 Hz), 7.23 (dd, 1H, J = 8.3, 2.2 Hz), 7.14 (d,

1H, J = 2.2 Hz), 6.93 (d, 1H, J = 8.3 Hz), 6.76 (s, 1H), 4.82 (d, 1H, J = 3.77

Hz), 4.61−4.71 (m, 2H), 4.31(t, 2H, J = 6 Hz), 4.27 (t, 2H, J = 6 Hz), 4 (s,

2H), 3.92 (s, 6H), 3.13−3.32 (m, 2H), 2.6−2.85 (m, 4H), 2.35−2.47 (m, 2H),

2.19−2.34 (m, 2H), 1.88−2.01 (m, 1H), 1.73−1.87 (m, 1H), 1.28–1.41 (m, 6H);


2S)-N-{4-(4-[(E)-2-(4-{3-methoxybenzylidene}oxy)-2,3-dihydroinden-1-one]butyl)oxy-5-methoxy-2-nitrobenzoyl}-pyrrolidine-2-carboxaldehydediethylthioacetal (44b)




(E)-2-(4-(4-bromobutoxy)-3-methoxybenzylidene)-2,3-dihydroinden-1-one

(40b) (401 mg, 1 mmol). Yield (660 mg, 91%)

106



T HESIS

1H NMR (200 MHz, CDCl3): δ 7.86 (d, 1H, J = 7.5 Hz), 7.64 (s, 1H), 7.5–7.61

(m, 3H), 7.39 (t, 1H, J = 6.7, 7.5 Hz), 7.2 (d, 1H, J = 8.3 Hz), 7.13 (s, 1H),

6.87 (d, 1H, J = 8.3 Hz), 6.7 (s, 1H), 4.78 (d, 1H, J = 3.7 Hz), 4.6–4.7 (m,

1H), 4.12–4.3 (m, 4H), 4 (s, 2H), 3.87 (s, 6H), 3.15–3.3 (m, 2H), 2.61–2.82

(m, 4H), 2.19–2.35 (m, 1H), 2.01–2.18 (m, 4H), 2.18–2.2 (m, 1H), 1.64–1.84

(m, 2H), 1.26–1.39 (m, 6H);


2S)-N-{4-(5-[(E)-2-(4-{3-methoxybenzylidene}oxy)-2,3-dihydroinden-1-one]pentyl)oxy-5-methoxy-2-nitrobenzoyl}-pyrrolidine-2-carboxaldehydediethylthioacetal (44c)




(E)-2-(4-(5-bromopentoxy)-3-methoxybenzylidene)-2,3-dihydroinden-1-one

(40c) (315 mg, 3 m mol). Yeild (686 mg, 93%)

1H NMR (300 MHz, CDCl3): δ 7.87 (d, 1H, J = 7.5 Hz), 7.62 (s, 1H), 7.49−7.61

(m, 3H), 7.4 (t, 1 H, J = 7.5, 6.7 Hz), 7.23 (dd, 1H, J = 8.3, 1.5 Hz), 7.14 (d,

1H, J = 2.2 Hz), 6.88 (d, 1H, J = 8.3 Hz), 6.77 (s, 1H), 4.82 (d, 1H, J = 3.7

Hz), 4.61−4.71 (m, 2H), 4.05−4.17 (m, 4H), 4 (s, 2H), 3.93 (s, 3H), 3.92 (s,

3H), 3.15−3.31 (m, 2H), 2.62−2.87 (m, 4H), 2.19–2.35 (m, 1H), 1.88−2.16

(m, 6H), 1.65−1.86 (m, 3H), 1.29–1.41 (m, 6H);


7-Methoxy-8-[3-{(E)-2-(4-{3-methoxybenzylidene}oxy)-2,3-dihydroinden-1-one} propoxy]-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one(46a)

This compound was prepared according to the method described for thecompound 43a employing 44a (706 mg, 1.0 mmol) which reduction with

SnCl2.2H2O (1.12 g, 5 mmol) gives amino compound 45a. Deprotection

followed by cyclization of 45a (676 mg, 1.0 mmol) with HgCl2 (677 mg, 2.5

107



T HESIS


pure product 46a. Yield (285 mg, 51%).

Mp: 105-107 ºC;

1H NMR (300 MHz, CDCl3): δ 7.9 (d, 1H, J = 7.2 Hz), 7.55–7.71 (m, 4H), 7.51

(s, 1H), 7.38–7.47 (m, 1H), 7.3 (dd, 1H, J = 8, 1.4 Hz), 7.2 (d, 1H, J = 1.4

Hz), 6.98 (d, 1H, J = 8 Hz), 6.82 (s, 1H), 4.09–4.25 (m, 4H), 4.04 (s, 2H),

3.93 (s, 6H), 3.65–3.86 (m, 2H), 3.48–3.63 (m, 1H), 2.23–2.41 (m, 2H),

1.96–2.18 (m, 4H);


7-Methoxy-8-[4-{(E)-2-(4-{3-methoxybenzylidene}oxy)-2,3-dihydroinden-1-one} butoxy]-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one(46b)


compound 43a employing 44b (720 mg, 1.0 mmol) which reduction with

SnCl2.2H2O (1.12 g, 5 mmol) gives amino compound 45b. Deprotection

followed by cyclization of 45b (690 mg, 1.0 mmol) with HgCl2 (677 mg, 2.5



Mp: 106-107 ºC;1H NMR (200 MHz, CDCl3): δ 7.92 (d, 1H, J = 7.2 Hz), 7.56–7.72 (m, 4H),

7.52 (s, 1H), 7.38–7.48 (m, 1H), 7.29 (dd, 1H, J = 8, 1.4 Hz), 7.19 (d, 1H, J =

1.4 Hz), 6.97 (d, 1H, J = 8 Hz), 6.82 (s, 1H), 4.11–4.28 (m, 4H), 4.03 (s, 2H),

3.93 (s, 6H), 3.67–3.86 (m, 2H), 3.49–3.66 (m, 1H), 2.25–2.39 (m, 2H),

1.94–2.19 (m, 5H), 1.65–1.8 (m, 1);

13C NMR (75 MHz, CDCl3): δ194.28, 150.02, 149.35, 149.28, 138.12, 134.33, 134.15,

132.45, 130.85, 128.75, 128.28, 127.55, 126.04, 124.55, 124.23, 113.84, 112.5, 111.43,110.32, 105.57, 68.49, 65.5, 55.96, 53.66, 46.63, 33.28, 32.31, 31.51, 30.48, 30.27, 29.54,

25.79, 24.11;


108



T HESIS

7-Methoxy-8-[5-{(E)-2-(4-{3-methoxybenzylidene}oxy)-2,3-dihydroinden-1-one} pentoxy]-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one(46c)


compound 43a employing 44c (734 mg, 1.0 mmol) which reduction withSnCl2.2H2O (1.12 g, 5 mmol) gives amino compound 45c. Deprotection

followed by cyclization of 45c (714 mg, 1.0 mmol) with HgCl2 (677 mg, 2.5


pure product 46c. Yield (341 mg, 58%).

Mp: 106-108 ºC;

1H NMR (300 MHz, CDCl3): δ 7.91 (d, 1H, J = 7.2 Hz), 7.56–7.71 (m, 4H),

7.52 (s, 1H), 7.38–7.48 (m, 1H), 7.31 (dd, 1H, J = 8, 1.4 Hz), 7.19 (d, 1H, J =

1.4 Hz), 6.98 (d, 1H, J = 8 Hz), 6.81 (s, 1H), 4.1–4.26 (m, 4H), 4.03 (s, 2H),

3.93 (s, 6H), 3.65–3.85 (m, 2H), 3.49–3.65 (m, 1H), 2.23–2.39 (m, 2H),

1.92–2.13 (m, 6H), 1.63−1.75 (m, 2H);


2.6. THERMAL DENATURATION STUDIES

The compounds 43a-f and 46a-c were subjected to DNA thermal

melting (denaturation) studies using duplex form calf thymus DNA (CT-DNA)

using modification reported procedure.51 Working solutions were produced by

appropriate dilution in aqueous buffer (10 mM NaH2PO4/NaH2PO4, 1 mM

Na2EDTA, pH 7.00±0.01) containing CT-DNA, (100 µ M in phosphate) and the

PBD (20 µ M) were prepared by addition of concentrated PBD solutions in

methanol to obtain a fixed [PBD]/[DNA] molar ratio of 1:5. The DNA-PBD

solutions were incubated at 37οC for 0 h prior to analysis sample were

monitored a 260 nm using a Beckman DU-7400 spectrophotometer fitted

with high performance temperature controller. Heating was applied at a rate

of 1οC min-1 in the 40−90

οC range. DNA helix-coil transition temperatures

(T m) were determined from the maxima in the d(A260)/dT derivative plots.

Results for each compound are shown as mean ± standard derivation from

109



T HESIS

the least three determinations and are corrected for the effects of methanol

co-solvent using a linear correction term. Ligand-induced alteration in DNA

melting behavior are given by ∆ T m = T m(DNA+PBD)−T m(DNA alone), where

the T m value for the PBD free CT-DNA is 69.8 ± 0.001. The fixed [PBD]/[DNA]ratio used did not result in binding saturation of the host DNA duplex for any

compound examined.

2.7. ANTICANCER ACTIVITY SEREENING

The synthesized compounds (43a-f and 46a-c) were evaluated for

their in vitro anticancer activity in selected human cancer cell lines. A

protocol of 48 h continuous drug exposure and a Sulforhodamine B (SRB)

protein assay was used to estimate cell viability or growth. The cell lines

were grown in RPMI 1640 medium containing 10% fetal bovine serum and

2 mML-glutamine, and were inoculated into 96-well microtiter plates in 90

µL at plating densities depending on the doubling time of individual cell

lines. The microtiter plates were incubated at 37οC, 5% CO2, 95% air and

100% relative humidity for 24 h prior to addition of experimental drugs.

Aliquots of 10 µL of the drug dilutions were added to the appropriate

microtiter wells already containing 90 µL of cells, resulting in the required

final drug concentrations. Each compound was evaluated for four

concentrations (0.1, 1, 10 and 100 µM) and each was done in triplicate

wells. Plates were incubated further for 48 h, and assay was terminated by

the addition of 50 µL of cold trichloro acetic acid (TCA) (final concentration,

10% TCA) and plates were again incubated for 60 min at 4οC. The plates

were washed five times with tap water and air-dried. Sulforhodamine B

(SRB) solution (50 µL) at 0.4% (w/v) in 1% acetic acid was added to each of

the wells, and plates were incubated for 20 min at room temperature. The

residual dye was removed by washing five times with 1% acetic acid. The

plates were air-dried. Bound stain was subsequently eluted with 10 mM

trizma base, and the absorbance was read on an ELISA plate reader at a

110



T HESIS

wavelength of 540 nm with 690 nm reference wavelengths. Percent growth

was calculated on a plate-by-plate basis for test wells relative to control

wells. The above determinations were repeated three times.

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L.;Balzarini, J.; Clercq, E. D.; Stables, J. P. J. Med. Chem. 2002, 45,

3103; (b) Dimmock, J. R.; Kandepu, N. M.; Nazarali, A. J.; Kowalchuk, T.

P.; Motaganahalli, N.; Quail, J. W.; Mykytiuk, P. A.; Audette, G. F.; Prasad,

L.; Perjesi, P.; Allen, T. M.; Santos, C. L.; Szydlowski, J.; Clercq, E. D.;

Balzarinir, J. J. Med. Chem. 1999, 42, 1358; (c) Kubalkova1, J.;

Tomeckova, V.; Perjesi, P.; Guzy, J. Cent. Eur. J. Biol. 2009, 4, 90.

38. Rizzo, S.; Bartolini, M.; Ceccarini, L.; Piazzi, L.; Gobbi, S.; Cavalli, A.;

Recanatini, M.; Andrisano, V.; Rampa, A. Bioorg. Med. Chem. 2010 , 18,

1749.

39. Tercel, M.; Stribbling, S. M.; Sheppard, H.; Siim, B. G.; Wu, K.; Pullen, S.M.; Botting, K. J.; Wilson, W. R.; Denny, W. A. J. Med. Chem. 2003, 46,

2132.

40. Baraldi, P. G.; Balboni, G.; Cacciari, B.; Guiotto, A.; Manfredini, S.;

Romagnoli, R.; Spalluto, G.; Thurston, D. E.; Howard, P. W.; Bianchi, N.;

Rutigiiano, C.; Mischiati, C.; Gambari, R. J. Med. Chem. 1999, 42, 5131.

115



T HESIS

41. Damayanthi, Y.; Reddy, B. S. P.; Lown, J. W. J. Org.Chem. 1999, 64,

290..

42. Wang, J. J.; Shen, Y. K.; Hu, W. P.; Hsieh, M. C.; Lin, F. L.; Hsu, M. K.; Hsu,

M. H. J. Med. Chem. 2006, 49, 1442

43. Kamal, A.; Laxman, E.; Reddy, P. S. M. M. Tetrahedron Lett . 2000, 41,

7743.

44. Kamal, A.; Shankaraiah, N.; Devaiah, V.; Reddy, K. L. Tetrahedron Lett .

2006, 47, 6553.

45. Kamal, A.; Devaiah, V.; Reddy, K. L.; Kumar, M. S. Bioorg. Med. Chem.

2005, 13, 2021.

46. Kamal, A.; Naseer, A. K.; Reddy, S.; Ahmed, S. K.; Kumar, M. S.; Juvekar,

A.; Sen, S.; Zingde, S. Bioorg. Med. Chem. Lett . 2007, 19, 5345.

47. Kamal, A.; Kumar, P. P.; Seshadri, B. N.; Srinivas, O.; Kumar, M. S.; Sen,

S.; Kurian, N.; Juvekar, A.; Zingde, S. Bioorg. Med. Chem. 2008, 16,

3895.

48. Kamal, A.; Shankaraiah, N.; Devaiah, V.; Reddy, K. L.; Juvekar, A.;

Kurian, N.; Zingde, S. Bioorg. Med. Chem. Lett . 2008, 18, 1468.

49. Kamal, A.; Bharathi, E. V.; Ramaiah, M. J.; Dastagiri, D.; Reddy, J. S.;

Viswanath, A.; Sultana, F.; Pushpavalli, S. N. C. V. L.; Bhadra, M. P.;

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Med. Chem. 2010, 18, 526.

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1990, 82, 1107.

116



T HESIS

CCHAPTERHAPTER-III-III

S YNTHESIS AND BIOLOGICAL EVALUATION OF COMBRETASTATIN DERIVATIVES AS

ANTICANCER AGENTS

117



T HESIS

3.1. INTRODUCTION

Microtubules are cytoskeletal structures that are formed by self

assembly of α and β tubulin heterodimers and are involved in many cellular

functions.1 Their most important role is the formation of the mitotic spindle,

and they are essential to the mitotic division of cells. Tubulin is an α,β

heterodimeric protein that is the main constituent of microtubules (Figure 1).

Tubulin is the target of numerous small molecule antiproliferative ligands

that act by interfering with microtubule dynamics.2 These ligands can be

broadly divided into two categories: those that inhibit the formation of the

mitotic spindle such as colchicine (1)3,4 and vinblastine5 and those that

inhibit the disassembly of the mitotic spindle once it has formed, such as

paclitaxel.6

Figure 1. 3-D model of microtubule

The three characterized binding sites of tubulin are the taxane domain,

the vinca domain, and the colchicine domain, and many compounds interact

with tubulin at these known sites. Many tubulin binding compounds, such as

118



T HESIS

paclitaxel and vinblastine, are in clinical use for various types of cancer2

(Figure 2).

MeO

MeO

OMe

OMe

O

NH

O

Colchicine (1)

O

OO

O

OH

MeO

OMe

OMe

podophylotoxin (3)

MeO

MeO

OMe

OMe

OH

Combretastatin A-4 (2a)

Figure 2. Examples of tubulin binding agents

3.2. COMBRETASTATINS

Antimitotic agents are one of the major class of cytotoxic drugs for

cancer treatment, and microtubules are an important target for many natural

product anticancer agents such as combretastatin A-4 (2a)7 and

podophyllotoxin (3).2,8 The combretastatins are a group of diarylstilbenes

isolated from the stem wood of the South African tree Combretum caffrum.9

Compound 2a was found to have potent anticancer activity against a

number of human cancer cell lines including multidrug resistant cancer cell

lines and binds to the colchicine-binding site of tubulin.10

A water-solubleprodrug, combretastatin A-4-phosphate 2b, is now in clinical trials for thyroid

cancer11-13 and in patients with advanced cancer.14 Compound 2b induces

vascular shutdown within tumors at doses less than one-tenth of the

maximum tolerated dose (MTD) and without detectable morbidity, assuming

119



T HESIS

a MTD of 1000mg/kg.7 Hydrolysis in vivo by endogenous nonspecific

phosphatases under physiological conditions affords 2b15,16 (Figure 3).

MeO

MeO

OMe

OMe

R

4a, R = NH24b, R = NH-CO-CH-(NH2)(CH2OH)

MeO

MeO

OMe

OMe

OPO3Na2

Combretastatin A-4P (2b)

Figure 3

The amino derivative of combretastatin, 4a (AC7739) is also in clinical

trials as a water-soluble amino acid prodrug (4b).17 In contrast to colchicine,

the antivascular effects of compound 2a in vivo are apparent well below the

maximum tolerated dose, offering a wide therapeutic window. The

compound 2a as well as being a potent inhibitor of colchicines binding is also

shown to inhibit the growth and development of blood vessels,

angiogenesis.5,18-21 The cis configuration only of 2a is biologically active, with

the trans form showing little or no activity.22 The active cis double bond in 2a

is readily converted to the more stable trans isomer during storage ormetabolism, resulting in a dramatic decrease in antitumor activity.23,24

CA-4 is an exceptionally strong inhibitor of tubulin polymerization and

is potently cytotoxic against murine lymphocytic leukemia and against

human ovarian and colon cancer cell lines.25-27 Its mechanism of action is

thought to be related to the tubulin binding properties that result in rapid

tumour endothelial cell damage, neovascular shutdown, and subsequent

haemorrhagic necrosis.28,29 It has been recently demonstrated that CA-4P, a

combretastatin-A4 prodrug, induces cell death primarily through mitotic

catastrophe in a panel of human B-lymphoid tumors.30 Mitotic catastrophe

appears to be a cell death modality different from apoptosis. Indeed, it has

been reported that CA-4 was able to activate caspase-9, but the inhibition of

caspase-9 by the use of the specific inhibitor Z -LEHDfmk did not inhibit

120



T HESIS

combretastatin-induced cell death. This indicates that apoptosis is a

secondary mechanism of death in a small proportion of cells treated with CA-

4.30

NH2

HO

OH

OH

Arotinoids (5) Trans- Resveratol (6)

Figure 4

The apoptotic activity of natural and synthetic stilbene arotinoids (5)

structurally related to vitamin A.31,32 Because some derivatives demonstrated

potent apoptotic activity in both normal and multidrug-resistant (MDR) cell

lines, it would be informative to explore novel stilbenoids as a logical starting

point in the quest for anticancer chemotherapeutics. The amino derivative

AC7739 (4a)33 and compounds structurally related to trans-resveratrol (6)34

possess potent apoptosis-inducing activity. The cis or trans stereochemistry

of the double bond of biologically active stilbenes was the major

discriminating factor affecting their apoptotic activity. CA-4 kills cells with a

modality different from apoptosis (Figure 4).

Various structural modifications to 2a have been reported including

variation of the A- and B-ring substituents.35-37 Many modifications of the B-

ring result in decreased bioactivity; however, substitution of the 3′-OH with

an amino group results in potent bioactivity and good water solubility.38 The

3,4,5-trimethoxy substituted pattern in ring A, resembling the trimethoxyaryl

ring of colchicine, is optimal for bioactivity of 2a.24

From the previouscomparative studies of the combretastatin it appears that the cis orientation

of the two aromatic rings plays an important key role in cytotoxicity.

However, during storage and administration cis combretastatin analogues

tend to isomerize to trans forms which show a dramatic decrease in their

121



T HESIS

inhibitory effects on cancer cell growth and tubulin polymerization.39

Structural alteration of the stilbene motif of CA-4 can be extremely effective

in producing potent apoptosis-inducing agents by activating both the

intrinsic and the extrinsic pathways. Accordingly, a number of cis-restricted

analogues of CA-4 were prepared using 1,2-substituted five-membered

heterocycles such as imidazole,40 oxazole,40 pyrazole,40,41 triazole,41 tetrazole,41

thiazole,41 furanone,42 dioxolane,43 and furazan44 to avoid the stability

problem. Many of them showed potent cytotoxicity against various cancer

cells compared to CA-4.

In earlier study, chalcone derivatives and pyrazoline derivatives of

combretastatin with two aromatic rings of CA-4 have shown an attractive

profile of cytotoxicity and apoptosis inducing activity. Their ability to block

most cells in the G2 phase of the cell cycle suggests that these compounds

could act on targets different from the mitotic spindle. This may confer on

these molecules a wider spectrum of action than the parent CA-4, which

arrests cells in the M phase of the cell cycle.

MeO

MeO

OMe

O

OMe

OH MeO

MeO

OMe

O

OMe

OH

7 8 (SD400)

Figure 5

The chalcone derivatives of combretastatin have showed exciting

potential as anticancer agents. Sylvie Ducki and co-workers synthesized

trimethoxy substituted chalcones

45

7 and 8, which possess potentialanticancer activity and binding strongly to tubulin at a site shared with, or

close to, the colchicines binding site.46,47 The anticancer activity and tubulin

binding property of these chalcones is comparable with combretastatin A-4

(CA-4). The IC50 value of compound SD400 (8) against the K562 human

chronic myelogenous leukemia cell line is 0.21 nM whereas for CA-4 it is 2.0

122



T HESIS

nM. Presently phosphate prodrugs of these compounds 7 and 8 are under

preclinical evaluation. The compound 7 inhibits cell growth at low

concentrations (IC50, P388 murine leukaemia cell line 2.6 nM) and shares

many structural features common to other tubulin binding agents48 (Figure

5).

MeO

MeO

OMe

N

OMe

OH

9a, R = H9b, R = Acetyl

NR

Figure 6 Johnson and coworkers49 synthesized N-acetylated and non-acetylated 3,4,5-

tri- or 2,5-dimethoxypyrazoline analogs 9a and 9b of combretastatin-A4. A

non-acetylated derivative (9a) with the same substituents as in CA-4 (2a)

was the most active compound in the series, with IC50 values of 2.1 and 0.5

µM in B16 and L1210 cell lines respectively. A cell-based assay indicated that

compound 9a caused extensive microtubule depolymerization with EC50

value of 7.1 µM in A-10 cells. Molecular modeling studies showed that these

compounds possess a twisted conformation similar to CA-4 (2a) (Figure 6).

3.3. PRESENT WORK

The present work describes the design, synthesis, and anticancer

evaluation of novel analogues of combretastatin, and its chalcone, pyrazoline

derivatives with amino benzothiazoles. In view of the interesting biological

activities exhibited by combretastatin derivatives, there has been

considerable interest in structural modification of these molecules and

development of new synthetic strategies in the laboratory.

These compounds have been prepared by coupling of different 2-

aminobenzothiazoles with combretastatin and its chalcone, pyrazoline

derivatives with amide bond with a view to evaluate more potent anticancer

123



T HESIS

molecules. In view of diverse biological activities of the combretastatin

derivatives, we have been designed, synthesized novel compounds with

aminobenzothiazoles and evaluated for their antitumour activity and these

compounds exhibited potential anticancer activity.

3.3.1. S YNTHESIS OF COMBRETASTATIN AND ITS DERIVATIVES

The precursor (Z)-2-[(2-methoxy-5-(3

trimethoxystyryl)]phenoxy)acetic acid 18 has been prepared by employing

commercially available isovanillin. Hydroxy group protection of isovanillin 10

with TBDMS-Cl followed by reduction of aldehyde group with NaBH4 gives

alcohol 12. The benzyl alcohol 12 was converted to benzyl bromide 13 by

using LiBr followed by salt formation with PPh3 affords compound 14.

OMe

OH

CHO

OMe

OTBDMS

CHO

OMe

OTBDMS

CH2OH

OMe

OTBDMS

CH2Br

OMe

OTBDMS

CH2PPh3Br MeO

MeO

OMe

OMe

OTBDMSMeO

MeO

OMe

OMe

OH

(i) (ii) (iii)

(iv)

(v)(vi)

MeO

MeO

OMe

OMe

OOEt

OMeO

MeO

OMe

OMe

OOH

O

(vii)

(viii)

10 11 12 13

1415

16

17 18

Scheme 1. Reagents and conditions: i) TBDMS-Cl, TEA, DMF, 1h; ii) NaBH4, MeOH, 2h; iii)

LiBr, TMS-Cl, THF, 1h; iv) PPH3, toluene, 8h; v) n-BuLi, THF,-20 oC, trimethoxybenzaldehyde,

30 min; vi) TBAF, THF, 20 min; vii) 2-bromoethyl acetate, K 2CO3, DMF, 12h; viii) LiOH.H2O,

THF, H2O, 12h.

124



T HESIS

The compound 14 on Wittig reaction with trimethoxybenzaldehyde

gives TBDMS-protected combretastatin A-4 15. This compound upon

deprotection with TBAF gives combretastatin A-4, 16. This upon

etherification with α-bromo ethyl acetate in the presence of K 2CO3 affords the

ester compound 17. The ester compound on hydrolysis with LiOH affords (Z)-

2-(2-methoxy-5-(3,4,5-trimethoxystyryl) phenoxy)acetic acid 18 (Scheme 1).

3.3.2. S YNTHESIS OF CHALCONE DERIVATIVE OF COMBRETASTATIN

The preparation of chalcone derivative 22 was carried out by synthetic

sequence illustrated in Scheme-2. Claisen-Schmidt condensation of

trimethoxy acetophenone 19 with 4-methoxy-3-hydroxybenzaldehyde

(isovaniline) using ethanol as solvent in the presence of aqueous KOH gives

trimethoxychalcones 20. This upon etherification with α-bromo ethyl acetate

in the presence of K 2CO3 gives ester compound 21. The ester compound on

hydrolysis with LiOH affords chalcone acid, (E)-2-(2-methoxy-5-(3-oxo-3-

(3,4,5-trimethoxyphenyl)prop-1-enyl) phenoxy)aceticacid 22 (Scheme 2).

MeO

MeO OMe

O

MeO

MeO OMe

O

OMe

OH

OMeOH

CHO

+

22

MeO

MeO

OMe

O

OMe

OOEt

O

MeO

MeO

OMe

O

OMe

OOH

O

19 20

21

(i)

(ii)

(iii)

Scheme 2. Reagents and conditions: i) aq. KOH, ethanol, 6h; ii) 2-bromoethyl acetate,K 2CO3, DMF, 12h; iii) LiOH. H2O, THF, H2O, 14h

3.3.3. S YNTHESIS OF P YRAZOLINE DERIVATIVE OF COMBRETASTATIN

The preparation of pyrazoline derivative 25 has been carried out by

synthetic sequence illustrated in Scheme-3. Cyclization of trimethoxy

125



T HESIS

chalcone 20 with hydrazine hydrate in acetic acid under reflux gives

pyrazoline derivative 23. This upon etherification with α-bromoethyl acetate

in the presence of K 2CO3 gives the ester 24. The ester compound on

hydrolysis with LiOH affords pyrazoline acid 2-(5-(1-acetyl-3-(3,4,5-

trimethoxyphenyl)-4,5-dihydro-1H-pyrazol -5-yl)-2-

methoxyphenoxy)aceticacid 25 (Scheme 3).

MeO

MeO

OMe

O

OMe

OH MeO

MeO

OMe

N

OMe

OH

N

O

25

MeO

MeO

OMe

N

OMe

O

N

O

OEt

O

MeO

MeO

OMe

N

OMe

O

N

O

OH

O

20 23

24

(i)

(ii)

(iii)

Scheme 3. Reagents and conditions: i) NH2NH2.H2O, Acetic acid, reflux, 14h; ii) 2-

bromoethyl acetate, K 2CO3, DMF, 12h; iii) LiOH.H2O, THF, H2O

3.3.4. S YNTHESIS OF COMBRETASTATIN-BENZOTHIAZOLE ANALOGUES

The synthesis of combretastatin-benzothiazole derivatives (27a-i) is

outlined in Scheme 4. Combretastatin acid 18 undergoes amide bond

formation with different 2-amino benzothiazoles in the presence of

EDCI/HOBT in dichloromethane affords combretastatin-benzothiazole

analogues 27a-i (Scheme-4).

The synthesis of chalcone-benzothiazole derivatives (28a-i) is outlined

in Scheme 5. Chalcone acid 22 undergoes amide bond formation with

different 2-amino benzothiazoles in the presence of EDCI/HOBT in

126



T HESIS

dichloromethane affords chalcone-benzothiazole analogues 28a-i (Scheme-

5).

MeO

MeO

OMe

OMe

OOH

O

18

N

SH2N+

MeO

MeO

OMe

OMe

ONH

O

S

N

26

27a, R = -H27b, R = -NO227c, R = -F27d, R = -Cl27e, R = -OMe27f , R = -OCF327g, R = -Me27h, R = -CF327i, R = -OEt

R

R

27 a-i

(i)


28a, R = -H28b, R = -NO228c, R = -F28d, R = -Cl28e, R = -OMe28f , R = -OCF328g, R = -Me

28h, R = -CF328i, R = -OEt

N

SH2N

+

26

R

22

MeO

MeO

OMe

O

OMe

OOH

O

MeO

MeO

OMe

O

OMe

ONH

O

S

N R

28

(i)


The synthesis of pyrazoline-benzothiazole derivatives (29a-i) is

outlined in Scheme 6. Pyrazoline acid 25 undergoes amide bond formation

with different 2-amino benzothiazoles in the presence of EDCI/HOBT in

127



T HESIS

dichloromethane affords pyrazoline-benzothiazole analogues 29a-i (Scheme-

6).

29a, R = -H29b, R = -NO229c, R = -F29d, R = -Cl29e, R = -OMe29f , R = -OCF329g, R = -Me29h, R = -CF329i, R = -OEt

N

SH2N

+

26

R

29

25

MeO

MeO

OMe

N

OMe

O

N

O

OH

O

MeO

MeO

OMe

N

OMe

O

N

O

NH

O

S

N R

(i)


3.4. BIOLOGICAL EVALUATION

3.4.1. ANTICANCER ACTIVITY

The anticancer activity of the synthesized compounds has been

evaluated by the National Cancer Institute (NCI), USA and determined using

the sulforhodamine B (SRB) assay50. Thirteen compounds have been selected

for preliminary screening which anticancer activity evaluation was performed

at 10 µΜ concentration. After preliminary screening on tumor cell lines,

active compounds were tested for five dose concentration on a panel of 60

human tumor cell lines derived from nine different cancer types: leukaemia,

lung, colon, CNS, melanoma, ovarian, renal, prostate and breast. Out of

thirteen compounds tested in preliminary screening eleven compounds

(27a, 27c, 27e, 27h, 27f, 28a, 28c, 28e, 28h, 28f and 29h) were

selected for five dose testing on a panel of 60 human cancer cell lines. The

results expressed as GI50 values for the test compounds are illustrated in

128



T HESIS

Table 1. All the compounds exhibited potential anticancer activity with GI50

values ranging 0.019-30.7 µΜ.

The combretastatin-benzothiazole analogues (27a, 27c, 27e, 27h and

27f) exhibited potential anticancer activity with GI50 values ranging 0.019-18.6 µΜ. The most active compound in this series 27a showed good

activity against all cell lines tested and the GI50 values are in the range of

0.019–11µ M. This compound was found to show highest activity against

MDA-MB-435 (melanoma) cancer cell line with a GI50 value of 0.019µ M. The

trimethoxychalcone-benzothiazole analogues (28a, 28c, 28e, 28h and 28f)

also exhibited significant anticancer activity with GI50 values ranging 0.3-

30.7 µΜ. The anticancer activity (GI50) of the compound 28a is in the rangeof 0.3–6.42µ M. This compound was found to show highest activity against

MDA-MB-435 (melanoma) cancer cell line with a GI50 value of 0.3µ M. The

compound 28f which having trifluoromethoxy group at 6-position of

benzothiazole ring also showed significant activity (GI50, 0.3–9.52µ M).

Table 1. Anticancer activity of compounds 27a, 27c, 27e, 27h, 27f, 28a,

28c, 28e, 28h, 28f and 29h against the NCI human cancer cell lines

Panel/CellLine

a

GI50 values (µM)

27a 27c 27e 27h 27f 28a 28c 28e 28h28f

29h

Leukemia

CCRF-CEM

K-562

MOLT-4

RPMI-8226

SR

0.07

8

0.04

2

0.34

0.15

0.03

7

3.70

3.62

3.07

6.36

2.19

3.87

3.6

3.87

5.43

3.69

4.27

3.3

3.75

5.48

3.35

4.12

3.43

4.21

5.83

3.55

2.09

0.44

3.03

1.09

0.38

5.73

4.32

29.9

7.35

3.17

3.55

3.52

3.5

3.02

2.53

3.37

3.67

4.31

3.84

0.85

2.5

0.4

3

2.8

9

2.0

2

0.3

9

na

na

na

2.21

na

Non-small cell lung

A549/ATCC

EKVX

0.34

0.03

5.99

5.72

5.19

5.11

4.46

6.25

6

7.89

2.63

5.91

23.2

na

11.7

na

14.3

30.3

1.9

9

2.16

4.1

129



T HESIS

HOP-62

HOP-92

NCI-H226

NCI-H23

NCI-H322M

NCI-H460

NCI-H522

8

na

0.03

9

0.27

0.11

na

0.05

2

0.07

9

5.70

5.73

5.60

4.26

na

4.36

2.92

23

6.28

4.51

3.91

na

3.73

3.09

5.22

2.88

3.17

3.9

5.38

3.55

2.89

7.09

2.74

4.3

4.39

na

4.11

3.16

6.11

1.33

2.77

3.4

4.26

2.64

1.32

11.7

2.39

21.1

11

na

13.5

3.2

4.11

2.62

na

5.06

7.16

3.32

2.16

6.57

4.1

21.5

3.68

6.73

4.06

2

5.1

1

2.1

2

2.14

3.3

5

2.1

5

2.7

7

2.4

3

0.6

7

2.2

1.7

2.22

3.76

3.67

na

2.71

Colon

COLO-205

HCC-2998

HCT-116

HCT-15

HT29

KM12

SW-620

2.83

0.37

0.04

6

0.04

8

3.16

0.05

8

na

7.78

>10

0

4.48

8.19

9.63

4.78

65

3.89

7.34

3.7

2.89

3.34

3.12

5.1

17.1

4.23

3.29

3.24

14.9

3.14

4.65

43.3

17.1

3.92

3.23

na

4.48

5.65

5.47

2.14

2.88

0.49

3.49

0.36

0.62

26.8

33

15.7

3.96

na

2.97

3.84

na

19.3

3.98

3.34

na

2.4

4.07

na

21.3

3.83

2.68

21.5

1.77

3.82

6.3

7

6.1

8

1.4

3

0.4

3.6

6

1.5

1

1.0

8

3.94

na

2.39

9.26

na

na

na

CNS

SF-268

SF-295

SF-539

SNB-19

SNB-75

U251

0.18

0.22

0.04

5

na

0.04

7.23

4.29

4.51

na

4.39

4.33

4.31

1.84

2.84

6.3

1.91

4

3.85

3.36

2.66

5.03

1.58

3.67

6.01

3.7

2.28

7.08

1.56

3.37

0.95

3.41

0.34

2.81

0.53

0.96

3.78

18.8

2.49

17.3

1.67

6.08

4.16

6.68

2.17

5.61

2.04

3.08

3.28

8.87

2.1

6.01

2.2

3.27

1.2

2.2

1

0.2

9

1.5

3.84

3.06

1.75

11

1.55

3.08

130



T HESIS

6

0.04

8

8

0.3

4

0.7

Melanoma

LOX IMVI

MALME-3M

M14

MDA-MB-435

SK-MEL-2SK-MEL-28

SK-MEL-5

UACC-257

UACC-62

0.06

7

1.99

0.07

2

0.01

9

0.18

na

0.03

6

11

0.06

3

3.23

5.22

6.82

0.33

8.46na

1.76

8.69

4.05

4.17

na

4.33

1.16

4.074.41

2.68

na

5.93

4.01

na

3.58

1.84

3.983

2.84

40

3.9

4.77

na

4.36

1.93

3.294.57

3.26

na

5.11

0.52

6.42

3.29

0.3

5.394.61

1.76

1.37

7.87

3.37

27.7

10.7

1.93

na24.1

4.49

na

33.6

2.59

9.62

3.28

1.51

24.214.4

4.56

20.4

14.5

2.88

8.75

3.31

1.16

309.47

2.88

na

22.7

0.4

7

2.6

9

1.7

2

0.3

4.9

73.3

1

2.0

3

8.0

8

9.5

2

3.36

6.44

na

3.08

na3.56

na

3.77

na

Ovarian

IGROV1

OVCAR-3

OVCAR-4

OVCAR-5

OVCAR-8

NCI/ADR-RES

SK-OV-3

0.21

0.04

3

0.37

na

0.25

0.03

3

0.34

4.17

9.99

7.30

na

7.15

2.55

8

7

2.17

5.31

5.89

5.28

2.3

4.69

4.48

2.11

4.66

5.98

3.86

2.12

3.29

5.64

3.37

5.34

3.12

5

2.7

8.42

3.6

0.48

3.2

3.54

2.77

0.48

2.54

16.2

3.4

14.7

na

10.2

2.43

15.2

7.73

2.6

11.2

na

3.49

1.89

7.32

15.6

1.95

4.37

na

3.85

1.73

5.08

2.5

4

1.5

2

2.0

7

4.7

6

1.5

4

0.4

9

2.1

6

5.3

1.84

2.9

28.2

3.36

3.13

5.75

131



T HESIS

Renal

786-0A498

ACHN

CAKI-1

RXF 393

SN12C

TK-10

UO-31

7.3

0.36

0.09

3

0.41

0.05

0.37

na

0.58

7.126.25

3.33

2.78

6.29

4.61

9.89

2.56

7.523.5

7.77

3.2

2.5

5.75

7.54

4.31

6.962.81

4.82

3.1

2.27

4.43

7.03

3.59

na3.61

8.31

4.6

2.65

6.42

18.6

4.37

4.1222.6

3.46

3.78

1.83

3.02

3.3

3.07

24.7na

12.4

30.7

8.5

17.4

20.9

6.83

19.4na

4.94

25.1

2.37

5.19

16.4

5.31

24.6na

7.48

10.9

2.6

5.41

15.6

7.8

2.2

6

7.4

1.62

2.2

1.5

8

1.7

4

2.0

8

1.6

1

3.582.78

2.48

2.17

2.09

3.31

2.85

1.93

Prostate

PC-3

DU-145

0.18

0.1

6.74

8.97

4.45

2.04

4.32

2.51

5.12

4.11

3.63

1.94

22.9

4.05

12.2

2.49

12

2.62

3.1

4

1.5

6

2.92

1.82

Breast

MCF7

MDA-MB-231

HS 578T

BT-549

T-47D

MDA-MB-468

0.05

1

0.19

na

0.41

1.0

0.1

2.3

9.34

>10

0

16.7

3.39

3.68

2.88

5.4

4.15

na

4.03

2.29

3.16

3.38

4.18

5.23

4.79

2.16

2.89

5.16

4.33

10.5

3.95

2.3

1.05

2.69

4.75

4.82

4.97

2.3

3.7

12.6

10.5

4.82

na

13.1

3.02

5.82

3.72

8.38

14.7

10.5

3.05

7.14

4.81

11.2

15

13.5

1.3

22.0

1

3.0

8

2.7

1

3.0

8

3.5

2

4.02

3.11

2.48

3.55

2.75

6.12

a Values are reported as GI50, the µ M concentration of the compound required tocause 50% inhibition of cell growth.na = not active(GI50 >50µ M )

The pyrazoline-benzothiazole analogue (29h) exhibited promising

anticancer activity with GI50 values ranging 1.55-28.2 µΜ. This compound

132



T HESIS

was found to show highest activity against SNB-75 (CNS) cancer cell line with

a GI50 value of 1.55 µ M. In comparison, the anticancer activity exhibited by

combretastatin and its chalcone, pyrazoline derivatives attached to

aminobenzothiazole, the combretastatin moiety attached to benzothiazoleanalogues are more active than chalcone moiety, than pyrazoline moiety.

3.4.2. INHIBITION OF TUBULIN POLYMERIZATION

Since these synthesized new compounds has structural resemblance to

combretastatin, it has been considered of interest to investigate their effect

on tubulin polymerization. One of the possible explanations of compounds

showing anticancer activity is the inhibition of tubulin polymerization to

functional microtubules as it is observed with antimitotic agents such as

podophyllotoxin and combretastatin. As tubulin subunits heterodimerize and

self-assemble to form microtubules in a time dependent manner, the

progression of tubulin polymerization has been investigated by monitoring

the increase in fluorescence emission at 460 nm (excitation wavelength is

360 nm) in 384 well plate for 1 h at 37 oC with and without the compounds at

3 µM concentration.

Real time kinetic graph of tubulin polymerization at 3 µM concentration

-0.8

1.2

3.2

5.2

7.2

9.2

11.2

13.2

15.2

1 4 7 1 0

1 3

1 6

1 9

2 2

2 5

2 8

3 1

3 4

3 7

4 0

4 3

4 6

4 9

5 2

5 5

5 8

Time (min)

F l u o r e s c e n c e u n i t s

Control

27f

28a

28c

28f

28h

Noco

Podo

133



T HESIS

Effect of compounds on tubulin polymerization at 3 µM concentration

-10

0

10

20

30

40

50

60

70

Control 27a 27b 27c 27d 27e 27f 27g 27h 27i 28a 28b 28c 28d 28e 28f 28g 28h Nocod Podo

Compounds

% I n h i b i t i o n

( T u b u l i n p o l y m e r i z a t i o n )

Figure 6: Effect of compounds on tubulin polymerization: Tubulin polymerization wasmonitored by the increase in fluorescence at 340 nm (excitation) and 460 nm (emission) for1 hrs at 37 oC. All the compounds were included at a final concentration of 3 μM.Podophyllotoxin and Nocodazole were used as positive control.

Among the seventeen compounds examined, 28a, 28c, 28f, 28h and

27f inhibited tubulin polymerization to 59.1%, 59.7%, 52.5, 54.1 and 55%

respectively compared to control and a similar pattern of inhibition has been

observed with the positive controls, podophyllotoxin (55.7%), Nocodazole

(49.6%) as shown in Figure 6.

3.5. CONCLUSION

In conclusion, we have synthesized different analogues of novel

combretastatin derivatives with amino benzothiazoles (27a-i, 28a-i and

29a-i). For synthesized compounds anticancer activity has been

evaluated by the National Cancer Institute (NCI), USA, against nine

human cancer cell lines (leukaemia, lung, colon, CNS, melanoma,

ovarian, renal, prostate and breast). All the compounds exhibited good

anticancer activity. Some of synthesized compounds exhibited good

inhibition of tubulin polymerization.

3.6. EXPERIMENTAL SECTION

3-(tert-butyldimethylsilyloxy)-4-methoxybenzaldehyde (11)

The compound 3-hydroxy-4-methoxy benzaldehyde 10 (152 mg, 1 mmol)

was dissolved in DMF (10 mL) and to this was added Triethylamine (2 mL)

134



T HESIS

and cooled to 5-10 oC. Then add the TBDMS-C l(165 mg, 1.1 mmol) to the

reaction mass. The reaction was stirred for 1 h at room temperature. After

completion of the reaction as indicated by TLC, bicarbonate solution was

added to reaction mass and extracted with ethyl acetate. The organic layer

was dried over Na2SO4 and concentrated to give the crude product. This was

further purified by column chromatography using hexane: ethyl acetate (1:9)

as a solvent system to obtain the pure product 11 as oil. Yield (221 mg,

82%).

1H NMR (300 MHz, CDCl3): δ 9.71 (s, 1H), 7.31–7.38 (dd, 1H, J = 8.3, 1.5 Hz),

7.24 (d, 1H, J = 1.5 Hz), 6.84 (d, 1H, J = 8.3 Hz), 3.82 (s, 3H), 0.93 (s, 9H),

0.1 (s, 6H);


(3-(tert-butyldimethylsilyloxy)-4-methoxyphenyl)methanol(12)

The compound 3-(tert-butyldimethylsilyloxy)-4-methoxybenzaldehyde (11)

(266 mg, 1 mmol) was dissolved in methanol and cooled to 10 oC. Then

NaBH4 (111 mg, 3 mmol) was added in portion wise to the cooled solution.

Stirred the reaction mixture for two hour and checked the TLC for completion

of reaction.ice was added to reaction mixture after completion of reaction toquench the excess reagent and concentrated the reaction mass, extracted

with ethyl acetate. The organic layer was dried over Na2SO4 and

concentrated to give the crude product. This was further purified by column

chromatography using hexane: ethyl acetate (2:8) as a solvent system to

obtain the pure product 12. Yield (246 mg, 90%).

1H NMR (200 MHz, CDCl3): δ 6.78–6.85 (m, 2H), 6.75 (d, 1H, J = 2.4 Hz), 4.49

(s, 2H), 3.78 (s, 3H), 0.99 (s, 9H), 0.12 (s, 6H);


(5-(bromomethyl)-2-methoxyphenoxy)(tert-butyl)dimethylsilane(13)

To a solution of anhydrous LiBr (172 mg, 2 mmol) in dry THF (15 mL) was

added TMS-Cl (270 mg, 2.5 mmol) and stirred for 10 minutes. Then added

135



T HESIS

the compound 12 (268 mg, 1 mmol) and the mixture was stirred at room

temperature for one hour. The reaction was monitored by TLC using ethyl

acetate-hexane (3:7). After completion of the reaction as indicated by the

TLC, the reaction mixture was quenched with ice water and the solvent

evaporated under reduced pressure, diluted with water and extracted with

ethyl acetate (2X20 ml). The combined organic phases were given washing

with 1N solution of NaOH, dried over Na2SO4 and evaporated under vacuum

to afford crude product of 13. The residue, thus obtained was taken to next

step without purification because of stability problem. Yield (300 mg, 90%).

3-{[1-(tert -butyl)-1,1-dimethylsilyl]oxy}-4-methoxybenzyltriphenylphosphonium bromide (14)

To a solution of PPh3 (262 mg, 1 mmol) in dry toluene (15 mL) was added

compound 13 (331 mg, 1 mmol) and the reaction mixture was refluxed for 6

hours. The reaction was monitored by TLC using ethyl acetate-hexane (1:1).

After completion of the reaction as indicated by the TLC, the toluene was

evaporated to half volume and stirred the reaction mixture at room

temperature for 16 hours to precipitate the product 14. The precipitated

product was filtered, washed with cooled toluene solvent and recrystalised

the crude product in toluene solvent and dried to obtain pure product 14.

Yield (450 mg, 75%).

1H NMR (200 MHz, CDCl3): δ 7.74–8.05 (m, 15H), 6.97–7.11 (m, 1H), 6.83 (d,

1H, J = 8.1 Hz), 6.5 (d, 1H, J = 2.2 Hz), 5.35 (d, 2H, J = 13.2 Hz), 3.93 (s, 3H),

1.05 (s, 9H), 0.17 (s, 6H);

(Z)-tert-butyl(2-methoxy-5-(3,4,5-

trimethoxystyryl)phenoxy)dimethylsilane(15)

To a solution of compound 14 (593 mg, 1 mmol) in dry THF (15 mL) was

cooled to –30 oC temperature and added n-BuLi (1.6 M solution in hexane)

(0.7 ml, 1.1 mmol) slowly. After addition stirred the reaction mixture for 20

minutes and added the solution of compound trimethoxybenzaldehyde (196

136



T HESIS

mg, 1 mmol) in dry THF. The mixture was stirred at –30 oC temperature for

30minutes. The reaction was monitored by TLC using ethyl acetate-hexane

(3:7). After completion of the reaction as indicated by the TLC, the reaction

mixture was quenched with ammonium chloride solution and extracted with

ethyl acetate (2X20 ml). The combined organic phases were given washing

with water followed by brain solution, dried over Na2SO4 and evaporated

under vacuum to afford crude product of 15. The crude product was mixture

of Z - and E- isomers. The Z - isomer was separated by flash column

chromatography using hexane:ethyl acetate (19:1) as a solvent system to

obtain the pure product 12 as colour less oil. Yield (180 mg, 41%).

1H NMR (300 MHz, CDCl3): δ 6.76–6.82 (dd, 1H, J = 8.3, 2.2 Hz), 6.73 (d, 1H, J

= 2.2 Hz), 6.69 (d, 1H, J = 8.3 Hz), 6.39–6.46 (m, 3H), 6.36 (d, 1H, J = 12 Hz),

3.79 (s, 3H), 3.77 (s, 3H), 3.69 (s, 6H), 0.92 (s, 9H), 0.04 (s, 6H);


(Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenol (16)

To a solution of acompound 15 (430 mg, 1 mmol) in dry THF (15 mL) was

added 1M TBAF solution (1 ml, 1 mmol) under cooling conditions and stirred

for 20 minutes at 5-10 oC. The reaction was monitored by TLC using ethylacetate-hexane (6:4). After completion of the reaction as indicated by the

TLC, the reaction mixture was quenched with bicarbonate solution and

extracted with ethyl acetate (2X20 ml). The combined organic phases were

given washing with water followed by brine solution, dried over Na2SO4 and

evaporated under vacuum to afford crude product of 13. This was further

purified by column chromatography using hexane: ethyl acetate (2:8) as a

solvent system to obtain the pure product12.

Yield (250 mg, 79% yield).

Mp: 115-116 ºC;

1H NMR (200 MHz, CDCl3): δ 6.92 (d, 1H, J = 2.2 Hz), 6.77–6.83 (dd, 1H, J =

8.3, 1.5 Hz), 6.73 (d, 1H, J = 8.3 Hz), 6.53 (s, 2H), 6.47 (d, 1H, J = 12 Hz),

6.41 (d, 1H, J = 12 Hz), 5.57 (bs, 1H), 3.86 (s, 3H), 3.84 (s, 3H), 3.7 (s, 6H);


137



T HESIS

(Z)-ethyl 2-(2-methoxy-5-(3,4,5-

trimethoxystyryl)phenoxy)acetate( 17)

To a solution of compound 16 (316 mg, 1 mmol) in dry DMF (15 mL) was

added, anhydrous K 2CO3 (276 mg, 2 mmol), α-bromoethylacetate (183 mg,1.1 mmol) and the mixture was stirred at room temperature for 24 hours.



filtration, diluted with water and extracted with dichloromethane (2X20 ml).

The combined organic phases were washed with water followed by brine

solution, dried over Na2SO4 and evaporated under vacuum. The residue, thus

obtained was purified by column chromatography using ethyl acetate and

hexane (5:5) to afford pure compound 17 as sticky mass. Yield (355 mg,

88%)

Mp: 108-109 ºC;

1H NMR (300 MHz, CDCl3): δ 6.83–6.89 (dd, 1H, J = 8.3, 1.5 Hz), 6.75 (d, 1H, J

= 8.3 Hz), 6.7 (d, 1H, J = 1.5 Hz), 6.4–6.46 (m, 3H), 6.47 (d, 1H, J = 12 Hz),

4.44 (s, 2H), 4.11–4.2 (q, 2H), 3.85 (s, 3H), 3.8 (s, 3H), 3.69 (s, 6H), 1.24 (t,

3H, J = 7.5, 6.7 Hz);


(Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)acetic acid (18)

To a solution of compound 17 (402 mg, 1 mmol) in THF (15 mL) and water (2

ml) was added, LiOH (48 mg, 2 mmol) and the mixture was stirred at room

temperature for 12 hours. The reaction was monitored by TLC using ethyl

acetate. After completion of the reaction as indicated by the TLC, the solvent

was removed under vacuum and neutralized with dilute HCl up to pH 7. After

neutralization the reaction mixture was extracted with dichloromethane

(2X20 ml). The combined organic phases were washed with water followed

by brine solution, dried over Na2SO4 and evaporated under vacuum to obtain

138



T HESIS

compound 18. This crude compound was purified by recrystalization by

using ethyl acetate as solvent to obtain the pure product 18 as white solid.

Yield (300 mg, 81%)

Mp: 154-156 ºC;

1H NMR (200 MHz, CDCl3): δ 7.96 (bs, 1H), 6.84–6.9 (dd, 1H, J = 8.3, 1.5 Hz),

6.77 (d, 1H, J = 8.3 Hz), 6.72 (d, 1H, J = 1.5 Hz), 6.31–6.49 (m, 4H), 4.47 (s,

2H), 3.86 (s, 3H), 3.8 (s, 3H), 3.69 (s, 6H);


(E)-3-(3-hydroxy-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)prop-

2-en-1-one (20)

To a stirred mixture of 3,4,5-trimethoxyacetophenone (210 mg, 1 mmol) and

3-hydroxy-4-methoxybenzaldehyde (152 mg, 1 mmol) in ethanol (10 ml) was

added 50% aqueous solution of potassium hydroxide (1 ml) and stirred for 6

h at room temperature. After completion of the reaction checked by TLC, the




chromatography using (30% EtOAC:hexane) to obtain the pure product 20.

Yield (300 mg, 86%).

Mp: 133-134 ºC;

1H NMR (300 MHz, CDCl3): δ 7.76 (d, 1H, J = 16 Hz), 7.36 (d, 1H, J = 16 Hz),

7.25−7.32 (m, 3H), 7.14 (dd, 1H, J = 8.7, 2.1 Hz), 6.89 (d, 1H, J = 8.7 Hz),

5.74 (bs, 1H), 3.96 (s, 9H), 3.94 (s, 3H);


(E)-Ethyl-2-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-enyl)phenoxy) acetate (21)


added, anhydrous K 2CO3 (276 mg, 2 mmol), α-bromoethylacetate (183 mg,

1.1 mmol) and the mixture was stirred at room temperature for 12 hours.

139



T HESIS







hexane (6:4) to afford pure compound 21 as yellow solid. Yield (395 mg,

91%)

Mp: 129-130 ºC;


Hz), 7.27−7.33 (m, 3H), 7.14−7.18 (dd, 1H, J = 7.8, 1.5 Hz), 6.94 (d, 1H, J =

7.8 Hz), 4.74 (s, 2H), 4.24−4.32 (q, 2H), 3.96 (s, 9H), 3.94 (s, 3H), 1.31 (t, 3H,

J = 7 Hz);


(E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-enyl)phenoxy)aceticacid (22)


ml) was added, LiOH.H2O (48 mg, 2 mmol) and the mixture was stirred at

room temperature for 14 hours. The reaction was monitored by TLC using

ethyl acetate. After completion of the reaction as indicated by the TLC, the

solvent was removed under vacuum and neutralized with dilute HCl up to pH

7. After neutralization the reaction mixture was extracted with

dichloromethane (2X20 ml). The combined organic phases were washed with

water followed by brine solution, dried over Na2SO4 and evaporated under

vacuum to obtain compound 22. This crude compound was purified by

recrystalization by using ethyl acetate as solvent to obtain the pure product

22 as yellow solid. Yield (351 mg, 87%)

Mp: 170-172 ºC;

140



T HESIS

1H NMR (500 MHz, CDCl3+DMSO D6): δ 8.24 (bs, 1H), 7.74 (d, 1H, J = 15.8

Hz), 7.77 (d, 1H, J = 15.8 Hz), 7.46−7.5 (m, 2H), 7.42 (s, 2H), 7.08 (d, 1H, J =

7.9 Hz 4.79 (s, 2H), 3.96 (s, 6H), 3.92 (s, 3H), 3.85 (s, 3H);

ESIMS: m/z 403 (M+H)

+

.

1-(5-(3-hydroxy-4-methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihydropyrazol-1-yl)ethanone (23)

To a stirred mixture of compound (20) (344 mg, 1 mmol) in acetic acid (10

ml) was added hydrazine hydrate (150 mg, 3 mmol) and stirred for 14 h at

reflux temperature. After completion of the reaction as checked by TLC, the

reaction mass was poured in to ice water and filtered the compound

precipitated. Air dried the filtered compound and purified by recrystalization

from ethanol to obtain the pure product 23 as white solid. Yield (320 mg,

79%).

Mp: 143-145 ºC;

1H NMR (300 MHz, CDCl3): δ 6.95 (s, 2H), 6.68−6.84 (m, 3H), 5.45−5.57 (dd,

1H, J = 11.7, 4.4 Hz), 3.91 (s, 6H), 3.89 (s, 3H), 3.85 (s, 3H), ), 3.6−3.79 (dd,

1H, J = 17.6, 11.2 Hz), ), 3.01−3.22 (dd, 1H, J = 17.6, 4.4 Hz), 2.42 (s, 3H);


Ethyl-2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)-2-methoxyphenoxy)acetate (24)


added, anhydrous K 2CO3 (276 mg, 2 mmol), α-bromoethylacetate (183 mg,

1.1 mmol) and the mixture was stirred at room temperature for 12 hours.







hexane (9:1) to afford pure compound 24 as white solid. Yield (401 mg, 82%)

141



T HESIS

Mp: 137-139 ºC;

1H NMR (500 MHz, CDCl3): δ δ 6.92 (s, 2H), 6.78−6.86 (m, 2H), 6.71 (s, 1H),

5.44−5.5 (dd, 1H, J = 11.8, 3.9 Hz), 4.61 (s, 2H), 4.17 (q, 2H), 3.9 (s, 6H),

3.87 (s, 3H), 3.83 (s, 3H), ), 3.62−3.71 (dd, 1H, J = 17.8, 11.8 Hz), 3.06−3.13(dd, 1H, J = 17.8, 3.9 Hz), 2.38 (s, 3H), 1.24 (t, 3H)


2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)-2-methoxyphenoxy)acetic acid (25)


ml) was added, LiOH.H2O (48 mg, 2 mmol) and the mixture was stirred at

room temperature for 14 hours. The reaction was monitored by TLC usingethyl acetate. After completion of the reaction as indicated by the TLC, the

solvent was removed under vacuum and neutralized with dilute HCl up to pH

7. After neutralization the reaction mixture was extracted with

dichloromethane (2X20 ml). The combined organic phases were washed with

water followed by brine solution, dried over Na2SO4 and evaporated under

vacuum to obtain the crude compound 25. This crude compound was

purified by recrystalization by using ethyl acetate as solvent to obtain the

pure product 25 as white solid. Yield (375 mg, 81%)

Mp: 175-177 ºC;

1H NMR (200 MHz, CDCl3): δ 7.81 (bs, 1H), 7.03 (s, 2H), 6.87−6.95 (m, 2H),

6.69 (s, 1H), 5.38−5.52 (dd, 1H, J = 11.6, 3.9 Hz), 4.54 (s, 2H), 3.85 (s, 6H),

3.83 (s, 3H), 3.82 (s, 3H), 3.61−3.7 (dd, 1H, J = 17.9, 11.6 Hz), 3.05−3.13 (dd,

1H, J = 17.8, 3.9 Hz), 2.39 (s, 3H);


(Z)-N-(benzo[d]thiazol-2-yl)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy) acetamide (27a)

To a solution of 2-aminobenzothiazole 26a (150 mg, 1.0 mmol) in

dichloromethane (20 mL) was added 1-(3-Dimethylaminopropyl)-3-

ethylcarbodiimide hydrochloride (EDCI.HCl) (191 mg, 1 mmol) and 1-

142



T HESIS

hydroxy-1,2,3-benzotriazole (HOBt) (13.5 mg, 0.1 mmol). Then added (Z)-2-

(2-methoxy-5-(3,4,5-trimethoxystyryl) phenoxy)acetic acid (18) (374 mg, 1

mmol) and the reaction mixture was stirred at temperature temperature for

24h and the reaction was monitored by TLC. Then water is added and

extracted with dichloromethane. The solvent was evaporated under vacuum

to afford the crude product. This was further purified by column

chromatography using ethyl acetate and hexane as solvent system to obtain

the pure product (27a) (395mg, 80% yield).

Mp: 132-134 ºC;

1H NMR (200 MHz, CDCl3): δ 10.64 (bs, 1H), 7.74–7.82 (m, 2H), 7.37–7.44

(m, 1H), 7.29 (d, 1H, J = 8.3 Hz), 6.96–7.01 (d, 1H, J = 8.3, 2.2 Hz), 6.93 (d,

1H, J = 2.2 Hz), 6.82 (d, 1H, J = 8.3 Hz), 6.46 (d, 1H, J = 12 Hz), 6.38–4.3 (m,

3H), 4.6 (s, 2H), 4.02 (s, 3H), 3.83 (s, 3H), 3.69 (s, 6H);

13C NMR (75 MHz, CDCl3): δ 167.54, 156.73, 152.98, 149.06, 148.47, 146.6, 137.29,

132.45, 132.2, 130.43, 129.7, 128.61, 126.19, 124.99, 124.05,121.36, 121.18, 118.14, 111.62,

105.81, 70.4, 60.92, 55.9;


(Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)-N-(6-nitrobenzo[d]thiazol-2-yl) acetamide (27b)

This compound was prepared according to the method described for

compound 27a by employing (Z)-2-(2-methoxy-5-(3,4,5-

trimethoxystyryl)phenoxy)aceticacid (18) (374 mg, 1 mmol), and 2-amino-6-

nitrobenzothiazole 26b (195 mg, 1 mmol) to obtain the pure product 27b as

white solid. Yield (442 mg, 80%)

Mp: 145-146 ºC;

1H NMR (300 MHz, CDCl3): δ 11.03 (s, 1H), 8.77 (d, 1H, J = 2.1 Hz), 8.32–8.36

(dd, 1H, J = 8.7, 2.1 Hz), 7.88 (d, 1H, J = 8.7 Hz), 7.03–7.07 (dd, 1H, J = 8, 2.1

Hz), 6.97 (d, 1H, J = 2.1 Hz), 6.87 (d, 1H, J = 8 Hz), 6.52 (d, 1H, J = 12.4 Hz),

6.49 (s, 2H), 6.46 (d, 1H, J = 12.4 Hz), 4.68 (s, 2H), 4.02 (s, 3H), 3.86 (s, 3H),

3.71 (s, 6H);

143



T HESIS


(Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)-N-(6-fluorobenzo[d]thiazol-2-yl)acetamide (27c)

This compound was prepared according to the method described forcompound 27a by employing (Z)-2-(2-methoxy-5-(3,4,5-


fluorobenzothiazole 26c (168 mg, 1 mmol) to obtain the pure product 27c as


Mp: 137-139 ºC;

1H NMR (300 MHz, CDCl3): δ 10.66 (bs, 1H), 7.71–7.76 (m, 1H), 7.47–7.53

(dd, 1H, J = 8.1, 2.4 Hz), 7.13–7.20 (m, 1H), 7.00–7.04 (dd, 1H, J = 8.1, 1.6

Hz), 6.93 (d, 1H, J = 1.6 Hz), 6.83 (d, 1H, J = 8.1 Hz), 6.46–6.51 (m, 3H), 6.44

(d, 1H, J = 12.2 Hz), 4.63 (s, 2H), 3.98 (s, 3H), 3.85 (s, 3H), 3.7 (s, 6H);

13C NMR (75 MHz, CDCl3): δ 167.63, 161.26, 158.04, 156.41, 152.99, 149.04, 146.6,

144.91, 137.24, 132.47, 130.49, 129.75, 128.58, 125.07, 121.99, 118.25, 114.77, 111.66, 107.47,

105.81, 70.46, 60.9, 55.89;


(Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)-N-(6-chlorobenzo[d]thiazol-2-yl)acetamide (27d)




chlorobenzothiazole 26d (184 mg, 1 mmol) to obtain the pure product 27d

as white solid. Yield (385 mg, 71%)

Mp: 136-138 ºC;

1H NMR (200 MHz, CDCl3): δ 10.61 (bs, 1H), 7.8 (d, 1H, J = 2.2 Hz), 7.73 (d,

1H, J = 9 Hz), 7.38–7.44 (dd, 1H, J = 8.3, 2.2 Hz), 7–7.06 (dd, 1H, J = 8.3, 2.2

Hz), 6.94 (d, 1H, J = 2.2 Hz), 6.84 (d, 1H, J = 9 Hz), 6.42–6.54 (m, 4H), 4.64

(s, 2H), 3.98 (s, 3H), 3.85 (s, 3H), 3.7 (s, 6H);

144



T HESIS

13C NMR (75 MHz, CDCl3): δ 167.94, 160.37, 153, 149.02, 146.53, 145.93, 137.31,

132.85, 132.43, 130.53, 129.94, 129.72, 128.65, 127.22, 125.03, 121.72, 121.08, 118.1, 111.76,

105.96, 70.21, 60.91, 56;


(Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)-N-(6-methoxybenzo[d]thiazol-2-yl)acetamide (27e)




methoxybenzothiazole 26e (180 mg, 1 mmol) to obtain the pure product

27e as white solid. Yield (415 mg, 77%)

Mp: 139-140 ºC;

1H NMR (400 MHz, CDCl3): δ 7.71 (d, 1H, J = 9 Hz), 7.3 (d, 1H, J = 2.2 Hz),

6.99–7.1 (m, 2H), 6.94 (d, 1H, J = 2.2 Hz), 6.84 (d, 1H, J = 8.3 Hz), 6.43–6.53

(m, 4H), 4.63 (s, 2H), 3.98 (s, 3H), 3.88 (s, 3H), 3.85 (s, 3H), 3.7 (s, 6H);

13C NMR (75 MHz, CDCl3): δ 186.55, 176.15, 174.09, 172.2, 168.29, 165.82, 161.62,

156.45, 152.54, 151.72, 149.63, 148.92, 147.88, 144.14, 140.9, 137.24, 134.54, 130.86, 125.04,

123.38, 89.5, 80.16, 75.15, 75.02, 48.87;


(Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)-N-(6-trifluoromethoxybenzo[d] thiazol-2-yl)acetamide (27f)




trifluoromethoxybenzothiazole 26f (234 mg, 1 mmol) to obtain the pure

product 27f as white solid. Yield (470 mg, 80%)

Mp: 141-143 ºC;

1H NMR (200 MHz, CDCl3): δ 10.63 (bs, 1H), 7.8 (d, 1H, J = 8.8 Hz), 7.69 (s,

1H), 7.29–7.36 (dd, 1H, J = 8.4, 2.2 Hz), 7.01–7.06 (dd, 1H, J = 8.4, 1.7 Hz),

145



T HESIS

6.95 (d, 1H, J = 1.7 Hz), 6.85 (d, 1H, J = 8.3 Hz), 6.42–6.53 (m, 4H), 4.65 (s,

2H), 3.99 (s, 3H), 3.85 (s, 3H), 3.70 (s, 6H); );

13C NMR (75 MHz, CDCl3): δ 167.82, 157.64, 153.03, 149.08, 147.14, 146.66, 145.5,

137.29, 133.09, 132.5, 130.57, 129.82, 128.59, 125.2, 121.96, 120.16, 118.42, 114.21, 111.7,105.8, 103.38, 70.59, 60.96, 55.95;


(Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)-N-(6-methylbenzo[d]thiazol-2-yl)acetamide (27g)




methylbenzothiazole 26g (180 mg, 1 mmol) to obtain the pure product 27g


Mp: 137-139 ºC;


1H, J = 8.3 Hz), 7.35–7.42 (dd, 1H, J = 8.3, 2.2 Hz), 7.02–7.09 (dd, 1H, J =

8.3, 2.2 Hz), 6.91 (d, 1H, J = 2.2 Hz), 6.84 (d, 1H, J = 8.3 Hz), 6.43–6.55 (m,

4H), 4.65 (s, 2H), 3.98 (s, 3H), 3.86 (s, 3H), 3.7 (s, 6H), 2.46 (s, 3H)

13C NMR (75 MHz, CDCl3): δ 167.48, 166.1, 153.13, 152.23, 148.33, 146.41, 136.45,

135.61, 131.95, 130.34, 129.7, 128.85, 128.43, 123.36, 120.89, 117.13, 116.34, 112.64, 111.37,

105.4, 69.39, 60.02, 55.46, 55.24, 19.55;


(Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)-N-(6-trifluoromethylbenzo [d]thiazol-2-yl)acetamide (27h)




trifluoromethylbenzothiazole 26h (218 mg, 1 mmol) to obtain the pure

product 27h as white solid. Yield (470 mg, 79%)

Mp: 142-144 ºC;

146



T HESIS

1H NMR (200 MHz, CDCl3): δ 10.64 (bs, 1H), 8.12 (s, 1H), 7.93 (d, 1H, J = 8.4

Hz), 7.69–7.75 (m, 1H), 7.01–7.06 (dd, 1H, J = 8.3, 1.7 Hz), 6.95 (d, 1H, J =

1.7 Hz), 6.85 (d, 1H, J = 8.3 Hz), 6.43–6.53 (m, 1H), 4.68 (s, 2H), 3.99 (s, 3H),

3.83 (s, 3H), 3.70 (s, 6H); );13C NMR (75 MHz, CDCl3): δ 168.02, 1549.3, 153.02, 150.77, 149.07, 146.66, 137.32,

132.48, 132.3, 130.59, 129.8, 128.56, 126.01, 125.19, 123.29, 121.35, 119.1, 118.46, 111.72,

105.89, 103.43, 70.6, 60.91, 55.9;


(Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)-N-(6-ethoxybenzo[d]thiazol-2-yl)acetamide (27i)




ethoxybenzothiazole 26i (194 mg, 1 mmol) to obtain the pure product 27i as


Mp: 138-140 ºC;


1H, J = 2.2 Hz), 6.99–7.15 (m, 2H), 6.93 (d, 1H, J = 2.2 Hz), 6.85 (d, 1H, J =

8.3 Hz), 6.44–6.53 (m, 4H), 4.65 (s, 2H), 4.12 (q, 2H), 3.97 (s, 3H), 3.87 (s,

3H), 3.84 (s, 3H), 3.7 (s, 6H), 1.28 (t, 3H);


(E)-N-(benzo[d]thiazol-2-yl)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-enyl)phenoxy)acetamide (28a)


compound 27a by employing (E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-

trimethoxyphenyl)prop-1-enyl) phenoxy) aceticacid (22) (402 mg, 1 mmol),

EDCI.HCl (191 mg, 1 mmol), HOBt (13.5 mg, 0.1 mmol) and 2-

aminobenzothiazole 26a (150 mg, 1 mmol) to obtain the pure product 28a

as yellow solid. Yield (416 mg, 77%)

Mp: 143-145 ºC;

147



T HESIS

1H NMR (200 MHz, CDCl3): δ 11.93 (bs, 1H), 7.81–7.84 (dd, 1H, J = 7.8, 1.9

Hz), 7.72–7.75 (dd, 1H, J = 7.8, 1.9 Hz), 7.67 (d, 1H, J = 15.6 Hz), 7.52–7.6

(m, 2H), 7.38–7.43 (m, 1H), 7.34–7.37 (dd, 1H, J = 7.8, 1.9 Hz), 7.31 (s, 2H),

7.25–7.29 (m, 1H), 7.01 (d, 1H, J = 7.8 Hz), 4.74 (s, 2H), 3.98 (s, 3H), 3.91 (s,

6H), 3.85 (s, 3H);

13C NMR (75 MHz, DMSO D6): δ 187.79, 167.51, 156.35, 152.74, 151.32,

147.42, 147.3, 143.84, 141.69, 133.14, 131.03, 129.83, 127.41, 126.56,

124.13, 123.55, 119.91, 119.01, 113.94, 112.19, 105.97, 67.12, 60.06,

56.01, 55.7;


(E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-enyl)phenoxy)-N-(6-nitrobenzo[d]thiazol-2-yl)acetamide (28b)




EDCI.HCl (191 mg, 1 mmol), HOBt (13.5 mg, 0.1 mmol) and 2-amino-6-

nitrobenzothiazole 26b (195 mg, 1 mmol) to obtain the pure product 28b as

yellow solid. Yield (452 mg, 78%)

Mp: 149-150 ºC;

1H NMR (200 MHz, CDCl3): δ 10.98 (bs, 1H), 8.77 (d, 1H, J = 2.1 Hz), 8.31–

8.38 (dd, 1H, J = 8.3, 2.1 Hz), 7.79 (d, 1H, J = 15.6 Hz), 7.72 (d, 1H, J = 8.3

Hz), 7.28–7.34 (m, 2H), 7.25 (d, 1H, J = 15.6 Hz), 7.23 (s, 2H), 6.95 (d, 1H, J

= 8.3 Hz), 4.83 (s, 2H), 4.01 (s, 3H), 3.92 (s, 6H), 3.9 (s, 3H);


(E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-enyl)phenoxy)-N-(6-fluorobenzo[d]thiazol-2-yl)acetamide (28c)





148



T HESIS

fluorobenzothiazole 26c (168 mg, 1 mmol) to obtain the pure product 28c as


Mp: 145-146 ºC;

1H NMR (300 MHz, CDCl3): δ 10.57 (bs, 1H), 7.65–7.77 (m, 2H), 7.44–7.53 (m,

2H), 7.31–7.39 (m, 2H), 7.25 (s, 2H), 7.24 (d, 1H, J = 15.4 Hz), 7.12 (d, 1H, J

= 8.3 Hz), 4.82 (s, 2H), 4.08 (s, 3H), 3.96 (s, 6H), 3.92 (s, 3H);


(E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-enyl)phenoxy)-N-(6-chlorobenzo[d]thiazol-2-yl)acetamide (28d)





chlorobenzothiazole 26d (184 mg, 1 mmol) to obtain the pure product 28d


Mp: 143-145 ºC;

1H NMR (400 MHz, CDCl3): δ 10.59 (bs, 1H), 7.76–7.81 (m, 1H), 7.71 (d, 1H, J

= 15.1 Hz), 7.65–7.69 (m, 2H), 7.46 (d, 1H, J = 2 Hz), 7.37–7.41 (dd, 1H, J =

8.4, 2 Hz), 7.25 (s, 2H), 7.21 (d, 1H, J = 15.1 Hz), 6.98 (d, 1H, J = 8.3 Hz),

4.83 (s, 2H), 4.07 (s, 3H), 3.95 (s, 6H), 3.93 (s, 3H);


(E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-enyl)phenoxy)-N-(6-methoxybenzo[d]thiazol-2-yl)acetamide (28e)





methoxybenzothiazole 26e (180 mg, 1 mmol) to obtain the pure product

28e as yellow solid. Yield (463 mg, 81%)

Mp: 146-147 ºC;

149



T HESIS


1H, J = 8.3 Hz), 7.34–7.4 (m, 2H), 7.28–7.34 (m, 2H), 7.23 (d, 1H, J = 15.6

Hz), 7.22 (s, 2H), 6.89 (d, 1H, J = 8.3 Hz), 4.82 (s, 2H), 4.02 (s, 3H), 3.96 (s,

6H), 3.95 (s, 3H), 3.88 (s, 3H);


(E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-enyl)phenoxy)-N-(6-trifluoromethoxybenzo[d]thiazol-2-yl)acetamide(28f)





trifluoromethoxybenzothiazole 26f (234 mg, 1 mmol) to obtain the pure

product 28f as yellow solid. Yield (505 mg, 81%)

Mp: 148-150 ºC;

1H NMR (200 MHz, CDCl3): δ 10.62 (bs, 1H), 7.77 (d, 1H, J = 9 Hz), 7.66–7.73

(m, 2H), 7.27–7.39 (m, 3H), 7.24 (d, 1H, J = 15.1 Hz), 7.22 (s, 2H), 6.98 (d,

1H, J = 8.3 Hz), 4.83 (s, 2H), 4.07 (s, 3H), 3.95 (s, 6H), 3.91 (s, 3H);

13C NMR (75 MHz, DMSO D6): δ 187.81, 168.2, 160.57, 152.77, 151.32,

151.18, 147.26, 143.92, 141.69, 133.16, 131.98, 127.41, 126.28, 124.24,

122.95, 122.66, 121.01, 119.87, 113.75, 112.21, 105.97, 67.02, 60.1, 56.05,

55.74;


(E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-enyl)phenoxy)-N-(6-methylbenzo[d]thiazol-2-yl)acetamide (28g)

This compound was prepared according to the method described forcompound 27a by employing (E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-



150



T HESIS

methylbenzothiazole 26g (164 mg, 1 mmol) to obtain the pure product 28g


Mp: 142-144 ºC;


7.63 (s, 1H), 7.31–7.43 (m, 2H), 7.23–7.30 (m, 4H), 7.01 (d, 1H, J = 8.4 Hz),

4.84 (s, 2H), 4.04 (s, 3H), 3.96 (s, 3H), 3.94 (s, 3H), 2.48 (s, 3H);


(E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-enyl)phenoxy)-N-(6-trifluoromethylbenzo[d]thiazol-2-yl)acetamide(28h)





trifluoromethylbenzothiazole 26h (218 mg, 1 mmol) to obtain the pure

product 28h as yellow solid. Yield (512 mg, 84%)

Mp: 147-149 ºC;

1H NMR (200 MHz, CDCl3): δ 10.74 (bs, 1H), 8.11 (s, 1H), 7.87 (d, 1H, J = 8.3

Hz), 7.64–7.74 (m, 2H), 7.28–7.40 (m, 3H), 7.22 (s, 2H), 6.99 (d, 1H, J = 8.3

Hz), 4.84 (s, 2H), 4.09 (s, 3H), 3.95 (s, 6H), 3.91 (s, 3H);

13C NMR (75 MHz, DMSO D6): δ 187.77, 167.94, 158.71, 152.75, 151.32,

147.25, 144.08, 143.86, 141.71, 133.14, 132.6, 128.5, 127.39, 124.23,

121.54, 119.83, 118.33, 114.94, 113.77, 112.2, 105.98, 67.06, 60.06, 56.03,

55.71;


(E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-enyl)phenoxy)-N-(6-ethoxybenzo[d]thiazol-2-yl)acetamide (28i)




151



T HESIS


ethoxybenzothiazole 26i (194 mg, 1 mmol) to obtain the pure product 28i as


Mp: 145-147 ºC;


7.36–7.41 (m, 2H), 7.29–7.34 (m, 2H), 7.25–7.28 (m, 3H), 7.01 (d, 1H, J = 8.3

Hz), 4.84 (s, 2H), 4.06–4.15 (q, 2H), 4.04 (s, 3H), 3.96 (s, 6H), 3.95 (s, 3H),

1.46 (t, 3H);


2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)-2-methoxy phenoxy)-N-(benzo[d]thiazol-2-yl)acetamide (29a)


compound 27a by employing 2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-

dihydro-1H-pyrazol-5-yl)-2-methoxyphenoxy)acetic acid (25) (458 mg, 1

mmol), EDCI.HCl (191 mg, 1 mmol), HOBt (13.5 mg, 0.1 mmol) and 2-

aminobenzothiazole 26a (150 mg, 1 mmol) to obtain the pure product 29a


Mp: 151-153 ºC;

1H NMR (400 MHz, CDCl3): δ 11.03 (bs, 1H), 7.73–7.81 (m, 2H), 7.40 (t, 1H),

7.29 (d, 1H, J = 7.3 Hz), 6.92–6.97 (m, 1H), 6.85–6.91 (m, 4H), 5.44–5.53 (dd,

1H, J = 11.7, 4.5 Hz), 4.69 (s, 2H), 3.99 (s, 3H), 3.90 (s, 6H), 3.86 (s, 3H),

3.64–3.76 (dd, 1H, J = 11.8, 17.3 Hz), 3.04–3.14 (dd, 1H, J = 17.3, 4.5 Hz),

2.40 (s, 3H);


2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)-2-methoxy phenoxy)-N-(6-nitrobenzo[d]thiazol-2-yl)acetamide(29b)




152



T HESIS

mmol), EDCI.HCl (191 mg, 1 mmol), HOBt (13.5 mg, 0.1 mmol) and 2-amino-

6-nitrobenzothiazole 26b (195 mg, 1 mmol) to obtain the pure product 29b


Mp: 158-160 ºC;

1H NMR (300 MHz, CDCl3): δ 11.02 (bs, 1H), 8.7 (d, 1H, J = 2.2 Hz), 8.32–8.37

(dd, 1H, J = 9, 2.2 Hz), 7.87 (d, 1H, J = 9 Hz), 6.99–7.04 (dd, 1H, J = 8.3, 2.2

Hz), 6.94–6.98 (m, 3H), 6.93 (d, 1H, J = 2.2 Hz), 5.5–5.58 (dd, 1H, J = 11.3,

4.5 Hz), 4.81 (s, 2H), 4 (s, 3H), 3.92 (s, 6H), 3.90 (s, 3H), 3.7–3.82 (dd, 1H, J

= 17.3, 12.8 Hz), 3.08–3.18 (dd, 1H, J = 17.3, 4.5 Hz), 2.43 (s, 3H);


2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)-2-methoxy phenoxy)-N-(6-fluorobenzo[d]thiazol-2-yl)acetamide(29c)





6-fluorobenzothiazole 26c (168 mg, 1 mmol) to obtain the pure product 29c

as white solid. Yield (503 mg, 82%)Mp: 153-154 ºC;

1H NMR (200 MHz, CDCl3): δ 10.62 (bs, 1H), 7.71–7.77 (dd, 1H, J = 9, 4.5 Hz),

7.49–7.53 (dd, 1H, J = 8.3, 3 Hz), 7.14–7.22 (m, 1H), 6.97–7.01 (dd, 1H, J =

8.3, 2.2 Hz), 6.96 (s, 2H), 6.88–6.94 (m, 2H), 6.5–6.57 (dd, 1H, J = 11.3, 4.5

Hz), 4.76 (s, 2H), 3.97 (s, 3H), 3.91 (s, 6H), 3.9 (s, 3H), 3.69–3.8 (dd, 1H, J =

173, 11.3 Hz), 3.08–3.17 (dd, 1H, J = 17.3, 4.5 Hz), 2.43 (s, 3H);

13

C NMR (75 MHz, CDCl3+DMSO D6): δ 167.44, 167.22, 156.49, 153.27,152.55, 148.45, 146.87, 144.68, 139.15, 134.49, 132.41, 126.2, 121.33,

121.22, 119.41, 113.79, 113.47, 111.83, 107.24, 106.89, 103.39, 68.34,

59.98, 58.79, 55.51, 41.86, 21.39;


153



T HESIS

2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)-2-methoxy phenoxy)-N-(6-chlorobenzo[d]thiazol-2-yl)acetamide(29d)


compound 27a by employing 2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)-2-methoxyphenoxy)acetic acid (25) (458 mg, 1


6-chlorobenzothiazole 26d (184 mg, 1 mmol) to obtain the pure product 29d


Mp: 153-155 ºC;


1H, J = 9 Hz), 7.34–7.39 (dd, 1H, J = 9, 2.2 Hz), 6.92–6.97 (dd, 1H, J = 8.3,

2.2 Hz), 6.89–6.91 (m, 1H), 5.44–5.52 (dd, 1H, J = 12, 4.5 Hz), 4.74 (s, 2H), 4

(s, 3H), 3.9 (s, 6H), 3.86 (s, 3H), 3.65–3.77 (dd, 1H, J = 17.3, 12 Hz), 3.05–

3.14 (dd, 1H, J = 17.3, 4.5 Hz), 2.4 (s, 3H);


2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)-2-methoxy phenoxy)-N-(6-methoxybenzo[d]thiazol-2-yl)acetamide (29e)





6-methoxybenzothiazole 26e (180 mg, 1 mmol) to obtain the pure product

29e as white solid. Yield (532 mg, 85%)

Mp: 156-157 ºC;

1H NMR (300 MHz, CDCl3): δ 10.45 (bs, 1H), 7.69 (d, 1H, J = 8.3 Hz), 7.29 (d,1H, J = 2 Hz), 7.03–7.07 (dd, 1H, J = 9.3, 3.1 Hz), 6.97–7.00 (dd, 1H, J = 8.3,

2 Hz), 6.96 (s, 2H), 6.91 (d, 1H, J = 8.3 Hz), 6.87 (d, 1H, J = 2 Hz), 5.51–5.55

(dd, 1H, J = 11.4, 4.1 Hz), 4.75 (s, 2H), 3.96 (s, 3H), 3.91 (s, 3H), 3.89 (s, 3H),

154



T HESIS

3.88 (s, 3H), 3.7–3.78 (dd, 1H, J = 7.6, 12.4 Hz), 3.09–3.14 (dd, 1H, J = 7.6,

4.1 Hz), 2.4 (s, 3H);


2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)-2-methoxy phenoxy)-N-(6-trifluoromethoxybenzo[d]thiazol-2-yl)acetamide (29f)





6-trifluoromethoxybenzothiazole 26f (234 mg, 1 mmol) to obtain the pure

product 29f as white solid. Yield (547 mg, 81%)

Mp: 159-161 ºC;


1H, J = 1.5 Hz), 7.29–7.34 (dd, 1H, J = 9, 1.5 Hz), 6.98–7.02 (dd, 1H, J = 8.3,

1.5 Hz), 6.96 (s, 2H), 6.89–6.94 (m, 2H), 5.5–5.58 (dd, 1H, J = 11.3, 4.5 Hz),

4.77 (s, 2H), 3.98 (s, 3H), 3.91 (s, 6H), 3.9 (s, 3H), 3.69–3.81 (dd, 1H, J =

18.1, 12 Hz), 3.08–3.17 (dd, 1H, J = 17.3, 4.5 Hz), 2.43 (s, 3H);

13C NMR (75 MHz, CDCl3+DMSO D6): δ 167.76, 167.4, 157.53, 135.15,

152.58, 148.66, 146.8, 144.41, 139.32, 134.46, 132.42, 126.1, 121.22,

120.02, 119.2, 113.87, 113.52, 111.78, 103.31, 69.14, 60.1, 58.76, 55.51,

55.36, 41.79, 21.35;


2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)-2-methoxy phenoxy)-N-(6-methylbenzo[d]thiazol-2-yl)acetamide

(29g) This compound was prepared according to the method described for




155



T HESIS

6-methylbenzothiazole 26g (164 mg, 1 mmol) to obtain the pure product

29g as white solid. Yield (498 mg, 82%)

Mp: 153-154 ºC;

1H NMR (200 MHz, CDCl3): δ 10.52 (bs, 1H), 7.69 (d, 1H, J = 8.3 Hz), 7.62 (s,

1H), 7.24–7.29 (m, 1H), 6.94–7 (m, 3H), 6.87–6.93 (m, 2H), 5.5–5.57 (dd, 1H,

J = 12, 4.5 Hz), 4.75 (s, 2H), 3.96 (s, 3H), 3.91 (s, 6H), 3.89 (s, 3H), 3.68–3.8

(dd, 1H, J = 17.3, 11.3 Hz), 3.07–3.17 (dd, 1H, J = 17.3, 4.5 Hz), 2.48 (s, 3H),

2.43 (s, 3H);


2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)-2-methoxy phenoxy)-N-(6-trifluoromethylbenzo[d]thiazol-2-yl)acetamide (29h)





6-trifluoromethylbenzothiazole 26h (218 mg, 1 mmol) to obtain the pure

product 29h as white solid. Yield (568 mg, 86%)

Mp: 162-163 ºC;1H NMR (400 MHz, CDCl3): δ 10.82 (bs, 1H), 8.11 (s, 1H), 7.88 (d, 1H, J = 7.9

Hz), 7.67–7.7 (m, 1H), 6.98–7.02 (dd, 1H, J = 7.9, 1.6 Hz), 6.96 (s, 2H), 6.9–

6.94 (m, 2H), 5.51–5.56 (dd, 1H, J = 11.9, 3.9 Hz), 4.79 (s, 2H), 3.99 (s, 3H),

3.91 (s, 6H), 3.89 (s, 3H), 3.71–3.79 (dd, 1H, J = 11.9, 17.5 Hz), 3.1–3.15 (dd,

1H, J = 17.5, 4.7 Hz), 2.43 (s, 3H);

ESIMS: m/z 659 (M+1)+.

2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)-2-methoxy phenoxy)-N-(6-ethoxybenzo[d]thiazol-2-yl)acetamide(29i)




156



T HESIS


6-ethoxybenzothiazole 26i (194 mg, 1 mmol) to obtain the pure product 29i


Mp: 158-159 ºC;


1H, J = 3.1 Hz), 7.02–7.06 (dd, 1H, J = 8.3, 2 Hz), 6.95–7 (m, 3H), 6.9 (d, 1H, J

= 8.3 Hz), 6.87 (d, 1H, J = 2.08 Hz), 5.5–5.56 (dd, 1H, J = 12.4, 4.1 Hz), 4.75

(s, 2H), 4.06– 4.12 (q, 2H), 3.95 (s, 6H), 3.91 (s, 6H), 3.89 (s, 3H), 3.7–3.78

(dd, 1H, J = 17.6, 12.4 Hz), 3.09–3.15 (dd, 1H, J = 17.6, 4.1 Hz), 2.43 (s, 3H),

1.45 (t, 3H);


3.7. TUBULIN POLYMERIZATION ASSAY

A fluorescence based In vitro tubulin polymerization assay was

performed according to the manufacturer’s protocol (BK011, Cytoskeleton,

Inc.). Briefly, the reaction mixture in a total volume of 10 µl contained PEM

buffer, GTP (1 mM) in the presence or absence of test compounds (final

concentration of 3 µM). Tubulin polymerization was followed by a time

dependent increase in fluorescence due to the incorporation of a

fluorescence reporter into microtubules as polymerization proceeds.

Fluorescence emission at 460 nm (excitation wavelength is 360 nm) was

measured by using a Varioscan multimode plate reader (Thermo scientific

Inc.). Podophyllotoxin was used as positive control in each assay.

157



T HESIS

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CCHAPTERHAPTER-IV/S-IV/SECTIONECTION-A-A

SS YNTHESIS YNTHESIS AANDND BBIOLOGICALIOLOGICAL EEVALUATIONVALUATION OOFF BBENZYLIDENEENZYLIDENE-9(10H)--9(10H)-AANTHRACENONENTHRACENONE LLINKEDINKED PP YRROLOBENZODIAZEPINES YRROLOBENZODIAZEPINES AASS AANTICANCERNTICANCER AAGENTSGENTS

163



T HESIS

4.1. I4.1. INTRODUCTIONNTRODUCTION

Cancer represents one of the largest health threats of mankind, and

therefore considerable efforts are undertaken regarding the development of

new chemotherapeutic agents for more potent and more specific anti-cancertherapy. The cells of each organism are in exact regulated mechanism of

equilibrium between growth (proliferation), differentiation (cellular

specialization) and programmed cell death (apoptosis). The most obvious

and medical, most important feature of cancer cells is their uncontrolled

growth. The cellular proliferation is a result of the cell division or mitosis.

Tubulin as well as its isoforms forms the major constituent of the

microtubulins and exists its part as heterodimer of the two globular

polypeptides [α] - and [β] – Tubulin.1 Microtubulins possess a prominent

importance for most diverse functions of the cell, among them the mitosis,

the movement of the cell and the cell formation.2-3 The importance of the

microtubulins for the cellular life and particular for the cell division and thus

linked for the induction and the progression of the apoptosis make it one of

the most attractive therapeutic targets for the development of new

compounds as cancer chemotherapeutics.

4.1.1. I4.1.1. INTRODUCTIONNTRODUCTION OOFF BBENZYLIDENEANTHRACENONESENZYLIDENEANTHRACENONES

Anthracenones are known for biological properties like tubulin binding,

antiproliferation and antipsoriatic4 action. It was found that certain 10-

benzyliden-9(10H)anthracenones are potent inhibitors of the tubulin

polymerization, which accompanying with an elevated cellular proliferation

there by effective anti-cancer compounds. Prinz and co-workers5 have

prepared several structurally new 10-benzylidene and 10-phenylmethyl-9(10H)-anthracenones. These compounds were evaluated for their

antiproliferative activity against K562 leukemia cells and various other

human cancer cell lines. In this series the lead compounds (1 and 2)

interacts with the colchicine site, causing G2/M arrest and induces apoptotic

cell death. There was no growth inhibitory effect in the cell cycle arrested

164



T HESIS

cells. In addition, these compounds (1 and 2) were found to be equally

potent toward parental tumor cell lines and multidrug resistant cells. The

most active compound 1, showed the anticancer activity against K562 cells

in nanomolar range (IC50: 20 nM) (Figure 1).

O

OMe

OH

O

OMe

OH1 2

OMe

O

3

O

OMe

Figure 1

The same group6 synthesized new analogues of 10-(2-oxo-2-

phenylethylidene)-10H-anthracen-9-ones, that were evaluated for

interactions with tubulin as well as antiproliferative activity against a panel

of human and rodent tumor cell lines. The 4-methoxy analogue (3) was most

potent, displaying IC50 values ranging from 40 to 80 nM, even on multidrug

resistant phenotypes, and possesses excellent activity as an inhibitor of tubulin polymerization (IC50: 0.52 μM).

Zuse and coworkers7 synthesized and reported some novel 9-

benzylidene- naphtho[2,3-b]thiophen-4-one derivatives (4 and 5). These

compounds were identified as agents with highest cell growth inhibiting

activity against a broad spectrum of cancer cell lines, including a panel of

cells with multi-drug resistant phenotypes. Amongst them 9-[(4-hydroxy-3,5-

dimethoxy)-benzylidene]-naphtho [2,3-b]thiophen-4-one (4) was the most

active compound in this series. It not only showed high antiproliferative

activity towards various cancer cell lines (IC50 K562 cells 50 nM) but also

strong tubulin polymerization inhibiting activity (IC50: 0.38 μM) (Figure 2).

165



T HESIS

OMe

OH

OH

OMeMeO

5

Figure 2

Recently some of anthracenone series of compounds have also been

synthesized and evaluated for their anticancer property.8-9 They include

anthracenone-based oxime ethers, oxime esters like 6 and 7, and 1,5- or

1,8-disubstituted-10-benzylidene-10H-anthracen-9-ones and 10-(2-oxo-2-

phenylethy lidene)-10H-anthracen-9-ones (8 and 9) (Figure 3).

OCl Cl

8 OH

OMe

OMe

O

NO

OMe

O

NO

OMe

OH

6 7

OCl Cl

9 OMe

OH

Figure 3

Apart from anticancer potential, these anthracenone analoguespossess antipsoriatic properties. Anthralin (10) is the most widely used drug

for psoriasis10. Antipsoriatic anthracenones exhibit three principal cellular

effects, which include interaction with DNA, inhibition of various enzyme

166



T HESIS

systems associated with cell proliferation and redox reactions that result in

alteration of mitochondrial functions and destruction of membrane lipids.

OOH OH

N

12

OOH OHOOH OH

O

OMe

1011

OOH OH

13

O

O

Cl Cl

14

OH

OMe

Figure 4

Klaus Mtiller and coworkers described the synthesis11-14 and

antipsoriatic activity of different anthracenones 11, 12, 13 and 14. These

compounds were evaluated for their ability to inhibit the growth of the

human keratinocyte cell line (HaCaT) and the 5-1ipoxygenase enzyme in

bovine polymorphonuclear leukocytes (Figure 4).

4.1.2. P4.1.2. PRESENTRESENT WWORK ORK


and in vitro cytotoxicity of novel benzylidene-9(10H)-anthracenone-PBD

conjugates linked by a suitable alkane spacers of different lengths (3, 4, 5).

These compounds have been prepared by coupling of benzylidene-9(10H)-

anthracenones by linking through alkane spacers to the C8 position of the

PBD scaffold with a view to combine both the tubulin polymerization and

DNA-binding properties in the same molecule. Based on the diverse

167



T HESIS

biological activities of the benzylidene-9(10H)-anthracenones and the

pyrrolo[2,1-c][1,4] benzodiazepines, there have been considerable efforts in

structural modification of PBDs and development of new synthetic strategies

in this laboratory. In this endeavor a series of new PBD conjugates (22a-f)

that comprise of both moieties designed, synthesized and with varying

alkane spacers have been evaluated for their antitumour activity and DNA-

binding ability.

4.1.2.1. S4.1.2.1. S YNTHESIS YNTHESIS OOFF PBD PPBD PRECURSORSRECURSORS

The precursor (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]pyrolidine-

2-carboxaldehydediethylthioacetal (15) have been prepared from

commercially available vanillin. The synthesis of compound 15 was

discussed in chapter-II.

HO

MeO

NO2

O

N

CH(SEt)2

15

4.1.2.2. S4.1.2.2. S YNTHESIS YNTHESIS OOFF BBENZYLIDENEENZYLIDENE-9(10H)--9(10H)-ANTHRACENONESANTHRACENONES

The preparation of 9-benzylidineanthracenones intermediates (19a-f)

has been carried out by the synthetic sequence illustrated in Scheme-1. The

synthesis of these intermediates is performed by reacting anthrone (16) with

different benzaldehydes in the presence of 10% IPA.HCl (isopropyl alcoholic

solution of HCl). These Benzylidene-9(10H)-anthracenones upon alkylation of

the hydroxyl group by dibromoalkanes using K 2CO3 as a base in dry acetone

affords the required precursors (19a-f) as illustrated in Scheme 1.

168



T HESIS

CHO

R

OH

+(i)

(ii)

19a-f

16 17a, b18a, b

19a; R = H, n = 219b; R = H, n = 319c; R = H, n = 419d; R = OMe, n = 2

19e; R = OMe, n = 319f ; R = OMe, n = 4

OO

R

OH

O

R

OBr

( )n

Scheme 1. Reagents and conditions: (i) IPA.HCl, 5 h; (ii) dibromoalkane, acetone, K 2CO3,

reflux, 24h.

4.1.2.3. S4.1.2.3. S YNTHESIS YNTHESIS OOFF C8-C8-LINKEDLINKED BBENZYLIDENEENZYLIDENE-9(10H)--9(10H)-ANTHRACENONEANTHRACENONE-PBD-PBD CCONJUGATESONJUGATES

Compound 15 has been coupled to compounds 19a-f in the presence

of K 2CO3 and dry acetone under reflux give corresponding nitro compounds

(20a-f). These nitro compounds upon reduction with SnCl2.2H2O in methanol

under reflux give amino compounds (21a-f). The amino compounds upon

deprotection with HgCl2/CaCO3 afford the corresponding imines (22a-f) as

shown in Scheme-2.

169



T HESIS

O

R

OBr

( )n

19a-f

HO

MeO

NO2

O

N

CH(SEt)2

15

+

O

MeO

NO2

ON

CH(SEt)2O

R

( )n

20a-f

21a-f

22a-f

(i)

(ii)

(iii)

O

O

MeO

NH2

O

N

CH(SEt)2O

R

O

( )n

O

MeO

O

R

O

N

N

O

H( )n

Scheme 2. Reagents and conditions: (i) K 2CO3, acetone, 12 h, reflux; (ii) SnCl2.2H2O, MeOH,

4 h, reflux; (iii) HgCl2, CaCO3, MeCN, H2O, (4:1) 12 h, rt.

170



T HESIS

4.1.3. BIOLOGICAL ACTIVITY

4.1.3.1. DNA BINDING AFFINITY : THERMAL DENATURATION STUDIES

The DNA binding affinity of these new C8-linked benzylidene-9(10H)-

anthracenone-PBD conjugates (22a-f ) has been evaluated through thermal

denaturation studies with duplex-form of calf thymus DNA (CT-DNA) by using

modified reported procedure.15 The DNA-PBD solutions are incubated at 37

οC for 0 h and 18 h prior to analysis. Samples are monitored at 260 nm using

a Beckman DU-7400 spectrophotometer fitted with high performance

temperature controller and heated at 1οC/min in the range of 40-95

οC. DNA

helix-coil transition temperatures are given by: ∆ T m = T m(DNA+PBD)–T m(DNA

alone), where the T m value for the PBD-free CT-DNA is 69.8± 0.01. These

studies were carried out at PBD/DNA molar ratio 1:5. The increase in melting

temperature (∆ T m) for each compound is examined at 0 h and 18 h of

incubation at 37οC. Melting studies show that these compounds stabilize the

thermal helix coil or melting stabilization for the CT-DNA duplex at pH 7.0,

and incubated at 37οC with ligand/DNA molar ratio of 1:5. The increase in

the helix melting temperature (∆ T m) for each compound has been examined

at 0 h and 18 h incubation at 37οC.

Interestingly, all the benzylidene-9(10H)-anthracenone-PBD conjugates

elevate the helix melting temperature of CT-DNA in the range of 3.0-5.1 oC.

Compound 22b showed the highest ΔT m of 4.6 oC at 0 h and increased upto

5.1 oC after 18 h incubation, whereas the naturally occurring DC-81 exhibits

a ΔT m of 0.7 oC after incubation under similar conditions (Table 1). These

results indicate that the effect on DNA binding affinity by introducing the

benzylidene-9(10H)-anthracenone scaffold on PBD moiety through different

alkane spacers at C8-position of the DC-81.

171



T HESIS

Table 1.Thermal denaturation data for benzylidene-9(10H)-anthracenone-PBD conjugates with calf thymus (CT)-DNA

Compound[PBD]:[DNA]

molar ratiob


0 h 18 h

22a 1:5 4.3 4.9

22b 1:5 4.6 5.1

22c 1:5 4.1 4.6

22d 1:5 3.6 4.0

22e 1:5 3.0 3.5

22f 1:5 4.0 4.3

DC-81 1:5 0.3 0.7a For CT-DNA alone at pH 7.00 ± 0.01, T m = 68.5 0C Δ 0.01 (mean value from 10 separate




4.1.3.2. ANTICANCER ACTIVITY

Compounds (22a-f ) have been evaluated for their in vitro cytotoxicity

in selected human cancer cell lines of barest, ovarian, colon, prostate, cervix,

lung and oral by using Sulforhodamine B (SRB) method.16 The in vitro

cytotoxicity results of these compounds expressed in GI50 values which

carried out the experiments at 10-4 to 10-7 M concentrations and the data is

illustrated in Table 2. The results from these experiments reveal that

compounds 22a-f showed GI50 values in the range of 0.08-2.5 μM, while thepositive controls, DC-81 and adriamycin exhibited the GI50 in the range of

0.1-0.17 μM and <0.01-14.7 μM respectively, in the cell lines employed. The

synthesized novel benzylidene-9(10H)-anthracenone-PBD conjugates

exhibited significant anticancer activity against MCF-7 human breast cancer

cell line (GI50 range, 0.08−0.14 μM) compared to other cell lines tested. The

172



T HESIS

active compound 22e exhibited strong effect against all cell lines tested

(GI50, 0.08-1.8 μM) and it showed a GI50 value of 0.08 against MCF-7 cell line.

Table 2. GI50 valuesa (in μM) for compounds 22a-f in selected human cancercell lines.

Compound

GI50 values (μM)

Breast Ovarian ColonProstat

eCervix Lung Oral

MCF-7 A2780 Colo205 PC-3 SiHaA

549Hop-62 KB

22a 0.12 0.18 0.17 1.3 2.1 1.53 0.34 2.1

22b 0.14 0.16 0.11 2.2 2.5 0.15 0.2 2.4

22c 0.15 0.12 1.2 0.2 --- 0.16 0.261.82

22d 0.12 0.14 0.11 0.18 2.2 1.76 0.190.19

22e 0.08 0.15 0.12 0.18 -- 1.8 0.18 0.2

22f 0.1 0.15 -- 0.15 2.2 1.94 0.170.18

DC-81 0.16 0.13 0.1 -- 0.16 -- 0.110.17

ADR <0.01 0.02 14.7 <0.01 0.19 13 <0.010.16

a 50% Growth inhibition and the values are mean of four determinationsADR, adriamycin.

4.1.4. CONCLUSION

In conclusion, we have synthesized a series of novel C8-linked

benzylidene-9(10H )-anthracenone-PBD conjugates ( 22a-f ). For

synthesized compounds anticancer activity has been evaluated

against eight human cancer cell lines (barest, ovarian, colon,

prostate, cervix, lung and oral). All the compounds exhibited

significant anticancer activity. Moreover these compounds exhibited

significant DNA binding ability .

4.1.5. EXPERIMENTAL SECTION

10-(4-hydroxybenzylidene)anthracen-9(10H)-one (18a)

173



T HESIS

To a stirred mixture of anthrone 16 (194 mg, 1 mmol) and 4-hydroxy

benzaldehyde 17a (122 mg, 1 mmol) was added isopropylalcoholic.HCl

solution (IPA.HCl, 10%solution) (5 ml) under nitrogen atmosphere with

maintaining cooling conditions. The temperature raised to room temperature

and stirring continued for 5 h. After completion of the reaction checked by

TLC, the solvent was evaporated, neutralized with saturated bicarbonate

solution and extracted with chloroform (2x50 ml). The combined organic

fractions were washed with water followed by brain, dried over Na2SO4 and

purified by column chromatography using (20% EtOAC:hexane) to obtain the

pure, yellow colored solid product 18a. Yield (210 mg, 70%). mp: 106-108

ºC;

1H NMR (300 MHz, CDCl3): δ 9.84 (bs, 1H), 8.31 (dd, 1H, J = 7.8, 1.5 Hz),

8.13–8.23 ( m, 2H), 7.72–7.86 ( m, 3H), 7.39–7.66 (m, 3H), 7.29 (d, 2H, J =

8.6 Hz), 6.79 (d, 2H, J = 7.8 Hz);


10-(4-hydroxy-3-methoxybenzylidene)anthracen-9(10H)-one (18b)


compound 18a by employing compound anthrone 16 (194 mg, 1 mmol), and3-methoxy-4-hydroxy benzaldehyde 17b (152 mg, 1 mmol). Yield (241 mg,

72%). Mp: 102-104 ºC;

1H NMR (300 MHz, CDCl3): δ 8.2–8.28 (m, 2H), 7.97 (d, 1H, J = 8.3 Hz), 7.66

(d, 1H, J = 8.3 Hz), 7.56–7.63 (m, 1H), 7.43–7.5 (m, 2H), 7.36–7.42 (m, 1H),

7.26–7.31 (dd, 1H, J = 8.3, 1.5 Hz), 6.87–6.91 (dd, 1H, J = 8.3, 1.5 Hz), 6.86

(s, 1H), 6.75 (d, 1H, J = 1.5 Hz), 5.59 (bs, 1H), 3.69 (s, 3H);


10-(4-(3-bromopropoxy)benzylidene)anthracen-9(10H)-one (19a)

To a solution of compound 18a (298 mg, 1 mmol) in dry acetone (15 mL) was added, anhydrous

K 2CO3 (276 mg, 2 mmol), 1,3 dibromopropane (605 mg, 3 mmol) and the mixture was stirred at

reflux temperature for 24 hours. The reaction was monitored by TLC using ethyl acetate-hexane

174



T HESIS

(1:9). After completion of the reaction as indicated by the TLC, K 2CO3 was removed by filtration

and the solvent evaporated under reduced pressure, diluted with water and extracted with ethyl

acetate (2X20 ml). The combined organic phases were dried over Na2SO4 and evaporated under

vacuum. The residue, thus obtained was purified by column chromatography using ethyl acetate

and hexane (1:9) to afford pure compound 19a as liquid. Yield (352 mg, 83%)

1H NMR (200 MHz, CDCl3): δ 8.16–8.29 (m, 2H), 7.95 (d, 1H, J = 7.5 Hz),

7.53–7.63 (m, 2H), 7.41–7.51 (m, 2H), 7.38 (t, 1H, J = 7.5 Hz), 7.16–7.28

(m, 3H), 6.77 (d, 2H, J = 8.3 Hz), 3.98 (t, 2H, J = 6, 5.2 Hz), 3.5 (t, 2H, J =

6.7, 6 Hz), 2.24–2.45 (m, 2H);


10-(4-(4-bromobutoxy)benzylidene)anthracen-9(10H)-one (19b)

The compound 19b was prepared according to the method described for compound 19a by

employing compound 18a (298 mg, 1 mmol), and 1,4 dibromobutane (647 mg, 3 mmol). Yield

(365 mg, 84%)


7.54–7.63 (m, 2H), 7.42–7.5 (m, 2H), 7.39 (t, 1H, J = 7.5 Hz), 7.16–7.29 (m,

3H), 6.77 (d, 2H, J = 8.3 Hz), 3.99 (t, 2H, J = 6, 5.2 Hz), 3.47 (t, 2H, J = 6.7,

6 Hz), 1.89–2.05 (m, 4H);ESIMS: m/z 434 (M+H)+.

10-(4-(5-bromopentoxy)benzylidene)anthracen-9(10H)-one (19c)

The compound 19c was prepared according to the method described for compound 19a by

employing compound 18a (298 mg, 1 mmol), and 1,5 dibromopentane (689 mg, 3 mmol). Yield

(380 mg, 84%)


7.53–7.63 (m, 2H), 7.42–7.51 (m, 2H), 7.39 (t, 1H, J = 7.5 Hz), 7.15–7.28

(m, 3H), 6.78 (d, 2H, J = 8.3 Hz), 3.99 (t, 2H, J = 6, 5.2 Hz), 3.28 (t, 2H, J =

6.7, 6 Hz), 1.81–2.03 (m, 4H), 1.63−1.75 (m, 2H);

ESIMS: m/z 448 (M+H)+

175



T HESIS

10-(4-(3-bromopropoxy)-3-methoxybenzylidene)anthracen-9(10H)-one (19d)

The compound 19d was prepared according to the method described for compound 19a by

employing compound 18b (328 mg, 1 mmol), and 1,3 dibromopropane (605 mg, 3 mmol). Yield

(411 mg, 91%)1H NMR (300 MHz, CDCl3): δ 8.22 (t, 2H, J = 7.5 Hz), 7.95 (d, 1H, J = 7.5

Hz), 7.63 (d, 1H, J = 8.3 Hz), 7.53–7.6 (m, 1H), 7.33–7.48 ( m, 3H), 7.22–

7.31 (m, 1H), 6.85 (d, 1H, J = 8.3 Hz), 6.74–6.82 (m, 2H), 4.13 (t, 2H, J = 6

Hz), 3.57–3.68 (m, 5H), 2.25–2.42 (m, 2H);


10-(4-(4-bromobutoxy)-3-methoxybenzylidene)anthracen-9(10H)-one (19e)


compound 19a by employing compound 18b (328 mg, 1 mmol), and 1,4-


1H NMR (400 MHz, CDCl3): δ 8.25 (t, 2H, J = 8.3 Hz), 7.98 (d, 1H, J = 7.5

Hz), 7.57–7.67 (m, 2H), 7.36–7.50 ( m, 3H), 7.23–7.31 (m, 1H), 6.88 (d, 1H,

J = 8.3 Hz), 6.76–6.82 (m, 2H), 4.05 (t, 2H, J = 6 Hz), 3.64 (s, 3H), 3.5 (t,

2H, J = 6.7, 6 Hz), 1.94–2.17 (m, 4H);

ESIMS: m/z 463 (M)+.

10-(4-(5-bromopentoxy)-3-methoxybenzylidene)anthracen-9(10H)-one (19f)




1H NMR (300 MHz, CDCl3): δ 8.23 (t, 2H, J = 7.5 Hz), 7.96 (d, 1H, J = 7.5

Hz), 7.62 (d, 1H, J = 8.3 Hz), 7.51–7.61 (m, 1H), 7.32–7.48 (m, 3H), 7.22–7.3 (m, 1H), 6.84 (d, 1H, J = 8.3 Hz), 6.74–6.81 (m, 2H), 4.11 (t, 2H, J = 6

Hz), 3.65 (s, 3H), 3.44 (t, 2H, J = 6.7, 6 Hz), 1.82–2.06 (m, 4H), 1.61–1.76

(m, 2H);


176



T HESIS

(2S)-N-{4-(3-[4-[(10-oxo-9,10-dihydro-9-anthracenyliden)methyl]phenoxy]propyl)oxy-5-methoxy-2-nitrobenzoyl}-pyrrolidine-2-carboxaldehydediethylthioacetal (20a)

To a solution of (2S)-N-[4-hydroxy-5-methoxy–2-nitrobenzoyl]pyrrolidine-2-

carboxal dehydediethylthioacetal (15) (400 mg, 1 mmol) in dry acetone (15mL) was added, anhydrous K 2CO3 (276 mg, 2 mmol), 10-(4-(3-

bromopropoxy)benzylidene)anthracen-9(10H)-one (19a) (419 mg, 1 mmol)

and the mixture was stirred at reflux temperature for 12 hours. The reaction

was monitored by TLC using ethyl acetate-hexane (1:1). After completion of

the reaction as indicated by the TLC, K 2CO3 was removed by filtration and the

solvent evaporated under reduced pressure, diluted with water and

extracted with ethyl acetate. The organic phase was dried over Na2SO4 and

evaporated under vacuum. The residue, thus obtained was purified by

column chromatography using ethyl acetate and hexane (1:1) to afford

compound 20a as yellow solid. Yield (672 mg, 90%).

Mp: 124-126 ºC;


7.53–7.66 (m, 3H), 7.34–7.5 (m, 3H), 7.19–7.32 (m, 3H), 6.71–6.83 (m, 3H),

4.8 (d, 1H, J = 3.6 Hz), 4.61-4.7 (m, 1H), 4.19 (t, 2H, J = 5.1 Hz), 4.09 (t,

2H, J = 5.1 Hz), 3.92 (s, 3H), 3.17–3.29 (m, 2H), 2.69-2.88 (m, 4H), 2.18–

2.35 (m, 2H), 1.94–2.14 (m, 3H), 1.73–1.90 (m, 1H), 1.26–1.39 (m, 6H);


(2S)-N-{4-(4-[4-[(10-oxo-9,10-dihydro-9-anthracenyliden)methyl]phenoxy]butyl)oxy-5-methoxy-2-nitrobenzoyl}-pyrrolidine-2-carboxaldehydediethylthioacetal (20b)


compound 20a by employing (2S)-N-[4-hydroxy-5-methoxy–2-nitrobenzoyl]pyrrolidine-2-carboxal dehydediethylthioacetal (15) (400 mg, 1 mmol), and

10-(4-(4-bromobutoxy) benzylidene)anthracen-9(10H)-one (19b) (433 mg, 1

mmol). Yield (700 mg, 92%).

Mp: 125-126 ºC;

177



T HESIS


7.54–7.67 (m, 3H), 7.34–7.51 (m, 3H), 7.18-7.3 (m, 3H), 6.7–6.84 (m, 3H),

4.81 (d, 1H, J = 3.6 Hz), 4.59–4.71 (m, 1H), 4.17 (t, 2H, J = 5.1 Hz), 4.07 (t,

2H, J = 5.1 Hz), 3.91 (s, 3H), 3.16–3.29 (m, 2H), 2.67–2.85 (m, 4H), 2.19–

2.35 (m, 1H), 1.9–2.15 (m, 6H), 1.72–1.9 (m, 1H), 1.26–1.4 (m, 6H);


(2S)-N-{4-(5-[4-[(10-oxo-9,10-dihydro-9-anthracenyliden)methyl]phenoxy]pentyl)oxy-5-methoxy-2-nitrobenzoyl}-pyrrolidine-2-carboxaldehydediethylthioacetal (20c)

The compound 20c was prepared according to the method described for compound 20a by

employing (2S )- N -[4-hydroxy-5-methoxy–2-nitrobenzoyl] pyrrolidine-2-carboxal

dehydediethylthioacetal (15) (400 mg, 1 mmol), and 10-(4-(5-bromopentoxy)

benzylidene)anthracen-9(10H)-one (19c) (447 mg, 1 mmol). Yield (715 mg, 93%).

Mp: 124-125 ºC;


7.53–7.67 (m, 3H), 7.33–7.51 (m, 3H), 7.19–7.31 (m, 3H), 6.72–6.84 (m,

3H), 4.81 (d, 1H, J = 3.6 Hz), 4.6–4.71 (m, 1H), 4.18 (t, 2H, J = 5.1 Hz), 4.07

(t, 2H, J = 5.1 Hz), 3.91 (s, 3H), 3.18-3.28 (m, 2H), 2.66–2.85 (m, 4H), 2.17–

2.31 (m, 1H), 1.92–2.16 (m, 6H), 1.76–1.98 (m, 3H), 1.26–1.41 (m, 6H);ESIMS: m/z 767 (M+H)+.

(2S)-N-{4-(3-[2-methoxy-4-[(10-oxo-9,10-dihydro-9-anthracenyliden)methyl] phenoxy]propyl)oxy-5-methoxy-2-nitrobenzoyl}-pyrrolidine-2-carboxaldehyde diethylthioacetal(20d)

The compound 20d was prepared according to the method described for compound 20a by

employing (2S )- N -[4-hydroxy-5-methoxy–2-nitrobenzoyl]pyrrolidine-2-carboxal

dehydediethylthioacetal (15) (400 mg, 1 mmol), and 10-(4-(3-bromopropoxy)-3-

methoxybenzylidene)anthracen-9(10H)-one (19d) (449 mg, 1 mmol). Yield (700 mg, 91%).

Mp: 120-121 ºC;

1H NMR (300 MHz, CDCl3): δ 8.18–8.27 (m, 2H), 7.95 (d, 1H, J = 8 Hz), 7.7

(s, 1H), 7.53–7.65 (m, 2H), 7.32–7.49 (m, 3H), 7.21–7.32 (m, 1H), 6.72–6.88

(m, 4H), 4.80 (d, 1H, J = 3.6 Hz), 4.58–4.73 (m, 1H), 4.33 (t, 2H, J = 5.8 Hz),

178



T HESIS

4.22 (t, 2H, J = 5.8 Hz), 3.92 (s, 3H), 3.64 (s, 3H), 3.14–3.26 (m, 2H), 2.63–

2.86 (m, 4H), 2.29–2.47 (m, 2H), 2.02–2.29 (m, 2H), 1.73–1.98 (m, 2H),

1.25–1.4 (m, 6H);


(2S)-N-{4-(4-[2-methoxy-4-[(10-oxo-9,10-dihydro-9-anthracenyliden)methyl] phenoxy]butyl)oxy-5-methoxy-2-nitrobenzoyl}-pyrrolidine-2-carboxaldehydediethyl thioacetal(20e)

The compound 20e was prepared according to the method described for compound 20a by


dehydediethylthioacetal (15) (400 mg, 1 mmol), and 10-(4-(4-bromobutoxy)-3-

methoxybenzylidene)anthracen-9(10H)-one (19e) (463 mg, 1 mmol). Yield (720 mg, 91%).

Mp: 119-122 ºC;

1H NMR (400 MHz, CDCl3): δ 8.23–8.3 (m, 2H), 8.02(d, 1 H, J = 8.1 Hz), 7.69

(s, 1 H), 7.6–7.68 (m, 2H), 7.45–7.54 (m, 2H), 7.38–7.44 (t, 1H, J = 6.7 Hz),

7.26–7.31 (m, 1H), 6.9–6.94 (m, 1H), 6.8–6.86 (m, 3H), 4.88 (d, 1H, J = 3.7

Hz), 4.67–4.75 (m,1H), 4.22 (t, 2H, J = 5.6 Hz), 4.14 (t, 2H, J = 5.6 Hz), 3.92

(s, 3H), 3.65 (s, 3H), 3.2–3.32 (m, 2H), 2.68–2.87 (m, 4H), 2.21–2.34 (m,

1H), 2.05–2.15 (m, 5H), 1.9–2.02 (m, 1H), 1.74–1.86 (m, 1H), 1.29–1.39 (m,

6H);ESIMS: m/z 783 (M+H)+.

(2 S )- N -{4-(5-[2-methoxy-4-[(10-oxo-9,10-dihydro-9-anthracenyliden)methyl]

phenoxy]pentyl)oxy-5-methoxy-2-nitrobenzoyl}-pyrrolidine-2-carboxaldehyde

diethylthioacetal(20f)

The compound 20f was prepared according to the method described for compound 20a by


dehydediethylthioacetal (15) (400 mg, 1 mmol), and 10-(4-(5-bromopentoxy)-3-

methoxybenzylidene)anthracen-9(10H)-one (19f) (477 mg, 1 mmol). Yield (751 mg, 94%).

Mp: 118-120 ºC;


7.57–7.65 (m, 3H), 7.37–7.48 (m, 3H), 7.23–7.28 (m, 1H), 6.85–6.91 (m,

179



T HESIS

1H), 6.76–6.78 (m, 3H), 4.81 (d, 1H, J = 3.7 Hz), 4.63–4.69 (m, 1H), 4.02–

4.14 (m, 4H), 3.93 (s, 3H), 3.64 (s, 3H), 3.16–3.3 (m, 2H), 2.63–2.87 (m,

4H), 2.16–2.38 (m, 2H), 1.87–2.14 (m, 5H), 1.68–1.87 (m, 3H), 1.28–1.39

(m, 6H);


7-Methoxy-8-[3-{4-[(10-oxo-9,10-dihydro-9-anthracenyliden)methyl]phenoxy} propoxy]-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (22a)

To the compound 20a (738 mg, 1 mmol) in methanol (20 mL) was added

SnCl2.2H2O (1.12 g, 5 mmol) and reflux for 5 hrs and checked TLC indicated

the reaction was completed. The methanol was evaporated under vacuum

and the reaction mass was neutralized with 10% NaHCO3 solution and the

extracted with chloroform (2x30 mL). The combined organic phases was

dried over Na2SO4 and evaporated under vacuum to afford the crude

aminodiethylthioacetal 21a (652 mg, 91%), which was used directly in the

next step due to its potential stability problem.

A solution of 21a (708 mg, 1 mmol), HgCl2 (677 mg, 2.5 mmol) and

CaCO3 (250 mg, 2.5 mmol) in acetonitrile-water (4:1) was stirred slowly at

room temperature overnight until complete consumption of starting materialas indicated by the TLC. The clear organic supernatant liquid was extracted

with chloroform and washed with saturated 5% NaHCO3 (20 mL), brine (20

mL) and the combined organic phase was dried over Na2SO4. The organic

layer was evaporated in vacuum to afford crude solid, which was purified by

column chromatography with MeOH-CHCl3 (1:20) to obtain the pure product

22a. Yield (306 mg, 51%).

Mp: 107-109 ºC;


7.54–7.67 (m, 3H), 7.33–7.52 (m, 4H), 7.17–7.3 (m, 3H), 6.72–6.86 (m, 3H),

4.03–4.19 (m, 4H), 3.92 (s, 3H), 3.77–3.85 (m, 1H), 3.68–3.74 (m, 1H),

3.53–3.62 (m, 1H), 2.24–2.37 (m, 2H), 1.96–2.18 (m, 4H);


180



T HESIS

7-Methoxy-8-[4-{4-[(10-oxo-9,10-dihydro-9-anthracenyliden)methyl]phenoxy}butoxy]-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (22b)


compound 22a employing 20b (752 mg, 1 mmol) which reduction withSnCl2.2H2O (1.12 g, 5 mmol) gives amino compound 21b. Deprotection of

21b (722 mg, 1 mmol) with HgCl2 (677 mg, 2.5 mmol), CaCO3 (250 mg, 2.5

mmol) in acetonitrile-water (4:1) gives the pure product 22b. Yield (320 mg,

53%).

Mp: 106-108 ºC;


7.55–7.66 (m, 3H), 7.35–7.53 (m, 4H), 7.17–7.29 (m, 3H), 6.73–6.84 (m,

3H), 4.04–4.19 (m, 4H), 3.93 (s, 3H), 3.77–3.86 (m, 1H), 3.69–3.74 (m, 1H),

3.53–3.63 (m, 1H), 2.14–2.22 (m, 2H), 1.85–21.97 (m, 6H);


7-Methoxy-8-[5-{4-[(10-oxo-9,10-dihydro-9-anthracenyliden)methyl]phenoxy} pentoxy]-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (22c)


compound 22a employing 20c (766 mg, 1 mmol) which reduction with

SnCl2.2H2O (1.12 g, 5 mmol) gives amino compound 21c. Deprotection of

21c (736 mg, 1 mmol) with HgCl2 (677 mg, 2.5 mmol), CaCO3 (250 mg,

2.5mmol) in acetonitrile-water (4:1) affords the pure product 22c. Yield (355

mg, 57%).

Mp: 107-108 ºC;


7.55–7.67 (m, 3H), 7.34–7.53 (m, 4H), 7.16–7.3 (m, 3H), 6.73–6.86 (m, 3H),4.04–4.19 (m, 4H), 3.92 (s, 3H), 3.76–3.85 (m, 1H), 3.68–3.74 (m, 1H),

3.52–3.62 (m, 1H), 2.24–2.36 (m, 2H), 1.85–2.14 (m, 6H), 1.64−1.76 (m,

2H);


181



T HESIS

7-Methoxy-8-[3-{2-methoxy-4-[(10-oxo-9,10-dihydro-9-anthracenyliden) methyl]phenoxy}propoxy]-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4] benzodiazepin-5-one(22d)


compound 22a employing 20d (768 mg, 1 mmol) which reduction withSnCl2.2H2O (1.12 g, 5 mmol) gives amino compound 21d. Deprotection of

21d (738 mg, 1 mmol) with HgCl2 (677 mg, 2.5 mmol), CaCO3 (250 mg, 2.5

mmol) in acetonitrile-water (4:1) obtains the pure product 22d. Yield (335

mg, 54%).

Mp: 102-103 ºC;


7.55–7.67 (m, 3H), 7.32–7.51 (m, 4H), 7.24–7.3 (m, 1H), 6.83–6.91 (m, 1H),

6.72–6.81 (m, 3H), 4.08–4.13 (m, 4H), 3.91 (s, 3H), 3.76–3.85 (m, 1H),

3.68–3.72 (m, 1H), 3.63 (s, 3H), 3.52–3.6 (m, 1H), 2.26–2.36 (m, 2H), 1.99–

2.18 (m, 4H);


7-Methoxy-8-[4-{2-methoxy-4-[(10-oxo-9,10-dihydro-9-anthracenyliden) methyl]phenoxy}butoxy]-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4] benzodiazepin-5-one(22e)


compound 22a employing 20e (782 mg, 1 mmol) which reduction with

SnCl2.2H2O (1.12 g, 5 mmol) gives amino compound 21e. Deprotection of

21e (752 mg, 1 mmol) with HgCl2 (677 mg, 2.5 mmol), CaCO3 (250 mg, 2.5

mmol) in acetonitrile-water (4:1) gives the pure product 22e. Yield (365 mg,

58%).

Mp: 101-103 ºC;

1H NMR (200 MHz, CDCl3): δ 8.21–8.26 (m, 2H), 7.96 (d, 1H, J = 8.3 Hz),7.55–7.66 (m, 3H), 7.31–7.48 (m, 4H), 7.23–7.31 (m, 1H), 6.83–6.9 (m, 1H),

6.74–6.8 (m, 3H), 4.06–4.13 (m, 4H), 3.92 (s, 3H), 3.76–3.86 (m, 1H), 3.66–

3.71 (m, 1H), 3.62 (s, 3H), 3.52–3.58 (m, 1H), 2.27–2.36 (m, 2H), 1.98–2.14

(m, 6H);

182



T HESIS

13C NMR (75 MHz, CDCl3): δ 184.95, 189.9, 162.46, 148.95, 148.55, 140.67,

136.43, 133.11, 132.63, 132.43, 130.66, 129.25, 128.14, 127.49, 126.98,

126.79, 122.86, 122.5, 112.52, 111.46, 68.57, 68.36, 56.09, 55.69, 53.66,

46.95, 46.65, 33.25, 29.66, 25.91, 25.77, 24.16,


7-Methoxy-8-[5-{2-methoxy-4-[(10-oxo-9,10-dihydro-9-anthracenyliden) methyl]phenoxy}pentoxy]-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4] benzodiazepin-5-one(22f)


compound 22a employing 20f (796 mg, 1 mmol) which reduction with

SnCl2.2H2O (1.12 g, 5 mmol) gives amino compound 21f . Deprotection of 21f

(766 mg, 1 mmol) with HgCl2 (677 mg, 2.5 mmol), CaCO3 (250 mg, 2.5 mmol)

in acetonitrile-water (4:1) affords the pure product 22f. Yield (370 mg, 57%).

Mp: 101-103 ºC;


7.54–7.67 (m, 3H), 7.31–7.51 (m, 4H), 7.24–7.31 (m, 1H), 6.84–6.91 (m,

1H), 6.73–6.81 (m, 3H), 4.07–4.15 (m, 4H), 3.91 (s, 3H), 3.76–3.86 (m, 1H),

3.68–3.71 (m, 1H), 3.63 (s, 3H), 3.52–3.61 (m, 1H), 2.26–2.35 (m, 2H),

1.96–2.15 (m, 6H), 1.64–1.75 (m, 2H);13C NMR (75 MHz, CDCl3): δ


4.1.6. THERMAL DENATURATION STUDIES

The compounds 22a-f were subjected to DNA thermal melting

(denaturation) studies using duplex form calf thymus DNA (CT-DNA) using

modification reported procedure. Working solutions were produced by




methanol to obtain a fixed [PBD]/[DNA] molar ratio of 1:5 The DNA-PBD

183



T HESIS




of 1

ο

C min-1 in the 40−90

ο

C range. DNA helix-coil transition temperatures(T m) were determined from the maxima in the d (A260)/dT derivative plots.




melting behavior are given by ∆ T m = T m (DNA+PBD)- T m (DNA alone), where

the T m value for the PBD free CT-DNA is 69.8 ± 0.001 the fixed [PBD]/[DNA]

ratio used did not result in binding saturation of the host DNA duplex for any

compound examined.

4.1.7. ANTICANCER ACTIVITY SCREENING

The synthesized compounds (22a-f ) were evaluated for their in vitro

anticancer activity in selected human cancer cell lines. A protocol of 48 hcontinuous drug exposure and a sulforhodamine B (SRB) protein assay was

used to estimate cell viability or growth. The cell lines were grown in RPMI

1640 medium containing 10% fetal bovine serum and 2 mML-glutamine,

and were inoculated into 96-well microtiter plates in 90 µL at plating

densities depending on the doubling time of individual cell lines. The

microtiter plates were incubated at 37οC, 5% CO2, 95% air and 100%

relative humidity for 24 h prior to addition of experimental drugs. Aliquots

of 10 µL of the drug dilutions were added to the appropriate microtiter

wells already containing 90 µL of cells, resulting in the required final drug

concentrations. Each compound was evaluated for four concentrations (0.1,

1, 10 and 100 µM) and each was done in triplicate wells. Plates were

incubated further for 48 h, and assay was terminated by the addition of 50

184



T HESIS

µL of cold trichloro acetic acid (TCA) (final concentration, 10% TCA) and

plates were again incubated for 60 min at 4οC. The plates were washed

five times with tap water and air-dried. Sulforhodamine B (SRB) solution

(50 µL) at 0.4% (w/v) in 1% acetic acid was added to each of the wells, andplates were incubated for 20 min at room temperature. The residual dye

was removed by washing five times with 1% acetic acid. The plates were

air-dried. Bound stain was subsequently eluted with 10 mM trizma base,

and the absorbance was read on an ELISA plate reader at a wavelength of

540 nm with 690 nm reference wavelengths. Percent growth was

calculated on a plate-by-plate basis for test wells relative to control wells.

The above determinations were repeated three times.

4.1.8. REFERENCES:

1. Honore, S.; Pasquier, E.; Braguer, D. Understanding microtubule dynamics

for improved cancer therapy. Cell. Mol. LifeSci. 2005, 62, 3039.2. Pellegrini, F.; Budman, D. R. Review: tubulinfunction, actionofantitubulin

drugs, and newdrug development. Cancer Invest . 2005, 23, 264.

3. Hadfield, J. A.; Ducki, S.; Hirst, N.; McGown, A. T. Progress in Cell Cycle

Research, 2003, 5, 309.

4. Keme´ny, L.; Ruzicka, T.; Braun-Falco, O. Dithranol. Skin Pharmacol.

1990, 3, 1-20

5. Prinz, H., Ishii, Y., Hirano, T., Stoiber, T., Camacho Gomez, J.A., Schmidt,

P., Düssmann, H., Burger, A.M., Prehn, J.H., Günther, E.G., Unger, E.,

Umezawa, K. J. Med. Chem. 2003, 46, 3382.

6. Prinz, H.; Schmidt, P.; Bohm, K. J.; Baasner, S.; Muller, K.; Unger, E.;

Gerlach, M.; Gunther, E. G. J. Med. Chem. 2009, 52, 1284.

185



T HESIS

7. Zuse, A., Schmidt, P., Baasner, S., Bohm, K.J., Muller, K., Gerlach, M.,

Gunther, E.G., Unger, E., Prinz, H., J. Med. Chem. 2006, 49, 7816.

8. Surkau, G.; Böhmb, K.; Müller, K.; Prinz, H. Eur. J. Med. Chem., 2010, 45,

3354.

9. Nickel, H. C.; Schmidt, P.; Böhmb, K. J.; Baasner, S.; Müller, K.; Gerlach, M.;

Unger, E.; Günther, E. G.; Prinz, H. Eur. J. Med. Chem., 2010, 45, 3420.

10. Keme´ny, L.; Ruzicka, T.; Braun-Falco, O. Dithranol, Skin Pharmacol.

1990, 3, 1-20.

11. Mtiller, K.; Reindl, H.; Gawlik, I. Eur. J. Med. Chem.1998, 33, 969.

12. Mtiller, K.; Sellmer, A.; Prinz, H. Eur J Med Chem, 1997, 32, 895.

13. Mtiller, K.; Altmannb, R.; Prinz, H. Eur J. Med. Chem. 1998, 33, 209.

14. Kratz, U.; Prinz, H, Müller, K. Eur J. Med. Chem. 2010, 45, 5278.


Res. 1993, 21, 3671.

16. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.;

Waerren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst .

1990, 82, 1107.

CCHAPTERHAPTER-IV/S-IV/SECTIONECTION-B-B

186



T HESIS

SS YNTHESIS YNTHESIS AANDND BBIOLOGICALIOLOGICAL EEVALUATIONVALUATION OOFF CCHALCONEHALCONE--PP YRROLOBENZODIAZEPINE YRROLOBENZODIAZEPINE DDIMERSIMERS AASS AANTICANCERNTICANCER AAGENTSGENTS

4.2. INTRODUCTION

Naturally occurring pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) have

attracted the attention of many researchers largely because of the potent

anticancer activity exhibited by most of these compounds bearing this ring

system. Some of the compounds of this class have undergone clinical

studies1,2, Apart from their anticancer activity, PBDs are of considerable

interest due to their ability to recognize and subsequently form covalent

bonds to specific base sequences of double strand DNA. They are

monofunctional alkylating agents, and have potential as gene regulators,

probes and as tools in molecular biology.3-5 The pyrrolo[2,1-c]

[1,4]benzodiazepines (PBDs) are a family of antitumour antibiotics derived

from various Streptomyces species6 and are generally referred to as the

anthramycin family, which comprise of some representative members like

187



T HESIS

anthramycin (1), sibiromycin (2), tomaymycin (3), chicamycin A (4),

neothramycin A (5), and B (6), and DC-81 (7) (Figure 1).




interaction and cross-linking. Thurston and co-workers7 have synthesized C-8

linked PBD dimers by linking at their C8-position of the A-rings through

varying lengths of alkyl chain to explore their DNA-cross linking ability. DNA-

binding ability has been observed by thermal denaturation studies with CT-

DNA (∆ T m > 15.1 °C for a 5:1 ratio of DNA:PBD at 37 °C for 18 h incubation).

Cross-linking efficiency has been investigated by using an agarose gel

electrophoresis assay. The results indicate that DSB-120 is an efficient cross-

linking agent. Furthermore, the in vitro cytotoxicity data in human K562 and

rodent ADJ-PC6 cell lines correlate with both the thermal denaturation data

and the cross-linking efficiencies. Recently, C2/C2’-exo-unsaturated C-8

linked PBD dimers

188



T HESIS

N

N

O

HOH

H3CO

R1R2

N

HN

O

HH3C

OR

CONH2

OCH3

N

N

O

CH3

HO

H3CO

H

N

HN

O

CH3

HOH

O

OHO

N

HN

O

HOH

H3CO

OCH3

OH

O

OH

CH3H3C

H3CHN

OH

CH3

N

N

O

HOH

H3CO

5 ( R1 = H; R2 = OH)6 ( R1 = OH, R2 = H)


3

2

chicamycin

7

1R= OH or OCH3

anthramycin sibiromycin

tomamycin

4

neothramycin A and BDC-81

(SJG-136) have been synthesized which exhibit extraordinary DNA bindingaffinity and cytotoxicity.8 In recent years, a large number of hybrid molecules

containing the PBD ring system have been synthesized leading to novel

sequence selective DNA cross-linking agents.9 It is belived that interactions in

a manner different from those of other tubulin-binding antimitotic agents.

4.2.1. CHALCONES

Chemically chalcones comprise of open-chain flavonoids in which the

two aromatic rings are joined by a three-carbon α,β-unsaturated carbonyl

system. Chalcones, considered as the precursor of flavonoids and

isoflavonoids, are abundant in edible plants. However, most of the chalcones

are particularly attractive since it specifically generates the (E)-isomer from

substituted benzaldehydes and acetophenones. Recent studies revealed that

189



T HESIS

these chalcones had shown a wide variety of anticancer,10-17 anti-

inflammatory,18-20 antiinvasive,21 antituberculosis,22 and antifungal23 activities.

Chalcones have shown promising anticancer therapeutic efficacy for the

management of human cancers. Recently, different chalcone analogues have

been synthesized and they have been screened for in vitro cytotoxicity

against a number of cancer cell lines.

The substituted chalcones have shown potential anticancer activity.

Ducki and co-workers have synthesized and reported trimethoxy substituted

chalcones24 (8) and (9), that possess potential anticancer activity and bind

strongly to tubulin at a site shared with, or close to, the colchicines binding

site.25-26 The anticancer activity and tubulin binding property of these

chalcones is comparable with combretastatin A-4 (CA-4). The IC50 value of

compound SD400 (9) against the K562 human chronic myelogenous

leukemia cell line is 0.21 nM whereas combretastatin A-4 (CA-4) shows the

IC50 is 2.0 nM. Presently phosphate prodrugs of these compounds (8) and (9)

are under preclinical evaluation. The compound (8) inhibits cell growth at

low concentrations (IC50, P388 murine leukaemia cell line 2.6 nM) and shares

many structural features common to other tubulin-binding agents27 (Figure

2).

MeO

MeO

O

OMe

OMe

OH MeO

MeO

O

OMeOMe

OH

9 (SD400)8

Figure 2

The anticancer activity of certain chalcones is believed to be a result of

binding to tubulin and preventing it from polymerizing into microtubules.

Tubulin is a protein that exists as a heterodimer of two homologous α- and

β -subunits. Many molecules based on a chalcone scaffold have been

synthesized to improve their biological profile, including their capability as

190



T HESIS

sequence selective DNA interactive and cross-linking agents. The ease of

synthesis of chalcones from substituted benzaldehydes and acetophenones,

makes them an attractive scaffold. Chalcones have attracted more interest

in recent years because of their diverse pharmacological properties.28 Among

these properties, their cytotoxicity effects have been extensively examined.

Some of the natural chalcones have been found in a variety of plant sources.

These natural compounds have served as valuable leads for further design

and synthesis of more active analogues.29

Further, in this trimethoxy chalcone series different analogues have

been synthesized by different groups and evaluated for their cytotoxicity.

These compounds have shown promising activity against different cancer

cell lines.30 Curcumin, a polyphenolic natural compound (10) derived from

dietary spice turmeric, possesses diverse pharmacological effects including

anticancer, anti-inflammatory, antioxidant, and antiangiogenic activities.31 A

series of of chalcone dimers has been reported as potent inhibitors of various

cancer cells at very low concentrations. The compound 3,5-bis(2-

fluorobenzylidene)-4-piperidone (11, also known as EF24) is a synthetic

analog of curcumin that was first reported by Adams.32 Other analogues of

3,5-bis(benzylidene)-4-piperidones (12, CLEFMA) and (13) are have been

advanced as synthetic analogs of curcumin for anti-cancer activity and anti-

inflammatory properties and these dimers have shown promising

antiproliferative activity against various cancer cell lines33 (Figure 3).

191



T HESIS

NH

O FF

N

O ClCl

O

OH

O12 (CLEFMA)

11 (EF24)

N

O

H3CO

HO

OCH3

OH

CH3

13

HO OH

OMeMeO

OOH

10 (curcumin)

Figure 3 The cyclic chalcones34, compounds (14, 15, 16 and 17) have been

shown potential anticancer activity against human cancer cell lines. These

compounds inhibit RNA and protein syntheses and induced apoptosis which

are likely major mechanisms whereby cytotoxicity is mediated. The active

compound (17) in these cyclic chalcones declines the mitochondrial function

as well as mitochondrial DNA damage (Figure 4).

O

N

MeO NO2

O

14 15

17

O

OMe

16

Figure 4

192



T HESIS

4.2.2. PRESENT WORK


and in vitro cytotoxicity of novel chalcones-PBD dimers by a suitable alkane

spacer (3, 4, and 5). These compounds have been prepared by coupling of

chalcone with alkane spacers to the C8 position of the PBD with a view to

combine both the pharmacophores chalcone and PBD in the same molecule.

Based on the diverse biological activities of the chalcones and the

pyrrolo[2,1-c][1,4]benzodiazepines there has been considerable interest in

structural modification of PBDs and development of new synthetic strategies

in the laboratory. In this endeavor we have designed and synthesized a

series of novel compounds of dimers (25a-f) that have both chalcone andPBD entities with varying alkane spacers and have been evaluated them for

their antitumour activity and DNA-binding ability.

4.2.2.1. S YNTHESIS OF PBD PRECURSORS

The precursor (2S)-N-[4-(n-Bromoalkooxy)-5-methoxy-2-nitrobenzoyl]

pyrrolidine-2-carboxaldehydediethylthioacetal 19a-c have been prepared by

employing (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]pyrolidine-2-

carboxaldehydediethylthio acetal 18 which was prepared from commercially

available vanillic acid. Compound 18 undergoes etherification with

dibromoalkanes in the presence of K 2CO3 in acetone gives corresponding

compounds 19a-c (Scheme 1).

193



T HESIS

HO

MeO

NO2

O

N

CH(SEt)2

18

O

MeO

NO2

O

N

CH(SEt)2Br ( )n

19a-c

n = 2-4

(i)

SCHEME 1. REAGENTS AND CONDITIONS: I) DIBROMOALKANE, K 2CO3, ACETONE, REFLUX,

14H

4.2.2.2. S YNTHESIS OF CHALCONE INTERMEDIATES

The preparation of dihydroxychalcone intermediates 22a,b has been

carried out by synthetic sequence illustrated in Scheme-2. Claisen-Schmidt

condensation of hydroxyacetophenones 20a,b with hydroxybenzaldehydes

21a,b by using ethanol as solvent in the presence of aqueous KOH gives

dihydroxychalcones 22a,b.

CH3

O CHO

+

O

R3

R4(i)

20a,b 21a,b22a,b

22a; R1 = H, R2 = OH, R3 = OH, R4 = H22b; R1 = OH, R2 = H, R3 = H, R4 = OH

R2

R1

R3

R4 R2

R1

20a; R1 = H, R2 = OH

20b; R1 = OH, R2 = H

21a; R3 = H, R4 = OH

21b; R3 = OH, R4 = H

SCHEME 2. REAGENTS AND CONDITIONS: I) AQ.KOH, ETHANOL, 24 H

4.2.2.3. S YNTHESIS OF C-8 LINKED CHALCONE-PBD DIMERS

Compound 19a-c has been coupled to dihydroxychalcones 22a,b in

the presence of K 2CO3 and dry acetone under reflux affords corresponding

nitro compounds 23a-f . These nitro compounds upon reduction with

SnCl2.2H2O in methanol under reflux give amino compounds 24a-f . The

194



T HESIS

amino compounds on deprotection with HgCl2/CaCO3 provide corresponding

imines 25a-f (Scheme-3).

O

O O25a-c; Chalcone =

25d-f; Chalcone =

O

O O

n = 2-4

n = 2-4

O

22a; R = 4,4'-dihydroxy22b; R = 3,3'-dihydroxy

O

MeO

NO2

O

N

CH(SEt)2

19a-c

+

O

MeO

NO2

O

N

CH(SEt)2O ( )n

23a-f

24a-f

25a-f

(i)

(ii)

(iii)

Br ( )n

OO

OMeN

O

(EtS)2HC O2N( )n chalcone

O

MeO

NH2

O

N

CH(SEt)2O ( )nOO

OMeN

O

(EtS)2HC H2N( )n chalcone

O

MeO

O ( )nOO

OMe

( )n chalconeN

N N

N

O

H H

O

RR

Scheme 3. Reagents and conditions: (i) K 2CO3, acetone, reflux, 12h; (ii) SnCl2.2H2O, MeOH, 4 h,reflux; (iii) HgCl2, CaCO3, MeCN, H2O, (4:1) rt, 12 h

195



T HESIS

4.2.3. BIOLOGICAL ACTIVITY

4.2.3.1. DNA BINDING AFFINITY : THERMAL DENATURATION STUDIES

The DNA binding affinity of these new C8-linked chalcone-PBD dimmers

(25a-f ) has been evaluated through thermal denaturation studies with

duplex-form of calf thymus DNA (CT-DNA) by using modified reported

procedure.35 The DNA-PBD solutions are incubated at 37οC for 0 h and 18 h

prior to analysis. Samples are monitored at 260 nm using a Beckman DU-

7400 spectrophotometer fitted with high performance temperature controller

and heated at 1ο

C/min in the range of 40-95ο

C. DNA helix-coil transitiontemperatures are given by: ∆ T m = T m(DNA+PBD)–T m(DNA alone), where the

T m value for the PBD-free CT-DNA is 69.8± 0.01. These studies were carried

out at PBD/DNA molar ratio 1:5. The increase in melting temperature (∆ T m)

for each compound is examined at 0 h and 18 h of incubation at 37οC.

Melting studies show that these compounds stabilize the thermal helix coil or

melting stabilization for the CT-DNA duplex at pH 7.0, and incubated at 37οC

with ligand / DNA molar ratio of 1:5. The increase in the helix melting

temperature (∆ T m) for each compound has been examined at 0 h and 18 h

incubation at 37οC.

Interestingly, all the PBD dimers elevate the helix melting temperature

of CT-DNA in the range of 3.5-5.4 oC. Compound 25a showed the highest ΔT m

of 4.8 oC at 0 h and increased upto 5.4 oC after 18 h incubation, whereas the

naturally occurring DC-81 exhibits a ΔT m of 0.7 oC after incubation under

similar conditions (Table 1). These results indicate that the effect on DNAbinding affinity by introducing the chalcone scaffold on PBD moiety through

different alkane spacers at C8-position of the DC-81.

196



T HESIS

Table 1.Thermal denaturation data for chalcone-PBD dimers withcalf thymus (CT)-DNA

Compound[PBD]:[DNA]

molar ratiob


0 h 18 h

25a 1:5 4.8 5.4

25b 1:5 4.6 4.9

25c 1:5 4.7 5.1

25d 1:5 4.1 4.8

25e 1:5 3.5 3.9

25f 1:5 4.5 4.7

DC-81 1:5 0.3 0.7

a For CT-DNA alone at pH 7.00 ± 0.01, T m = 68.5 0C Δ 0.01 (mean value from 10 separate




4.2.3.2. ANTICANCER ACTIVITY

Compounds 25a-f have been evaluated for their in vitro cytotoxicity in

selected human cancer cell lines of colon, prostate, melanoma and lung by

using MTT assay method. The in vitro cytotoxicity results of these

compounds expressed in IC50 values which carried out the experiments at 10-

4 to 10-7 M concentrations and the data is illustrated in Table 2. The results

from these experiments reveal that compounds 25a-f showed IC50 values in

the range of 0.008-29.1 μM whereas DC-81 showed IC50 values in the range

of 2-26.2 μM. The synthesized novel chalcone-PBD dimers exhibited

significant anticancer activity against PC-3 human prostate cancer cell line

(IC50 range, 0.008−8.3 μM) compared to other cell lines HT-29, A-375, A-549

and B-16. Compound 25a exhibited strong effect against PC-3 (IC50, 0.008

197



T HESIS

μM) and A-375 cell linec (IC50, 0.01 μM). The compound 25c also showed

significant activity against PC-3 cell line (IC50, 0.007 μM). Among the chalcone-

PBD dimers synthesized, the compounds having 4,4’- bonding of chalcone

showed superior activity compared to the compounds with 3,3’- bonding of

chalcone.

Table 2. IC50 valuesa (in μM) for compounds 25a-f in selected human cancercell lines.

CompoundIC50 values (μM)

HT-29b PC-3c A-375d A 549e B-16f

25a 10.4 0.008 0.01 19.0 29.1

25b 0.97 0.51 3.05 7.3 19.2

25c 15.8 0.007 0.97 8.9 18.6

25d 2.02 0.11 3 28.5 2.69

25e 26.4 5.1 8.3 -- 22.9

25f 23.5 8.3 27.8 11.7 --

DC-81 8.1 2 26.2 -- 21.1a 50% Growth inhibition and the values are mean of three determinations, b colon cancer, c

prostate cancer, d skin cancer, e lung cancer, f Mouse macrophages cell line.

4.2.4. CONCLUSION

A series of novel C8-linked chalcone-PBD dimers have been

synthesized and evaluated for its anticancer activity. These new analogues

exhibited significant anticancer activity against different cancer cell lines.

The DNA binding ability of these compounds carried out by thermal

denaturation of calf thymus (CT)-DNA. The thermal denaturation studies

have shown that these conjugates have better DNA binding ability when

compared to DC-81. Among these hybrids the compound 25a show superioranticancer activity as well as high DNA-binding ability (5.4

οC).

198



T HESIS

4.2.5. EXPERIMENTAL SECTION

(2S)-N-[4-(3-Bromopropoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxaldehydediethylthioacetal (19a)

To a solution of compound 18 (400 mg, 1 mmol) in dry acetone (15 ml)

was added, anhydrous K 2CO3 (553 mg, 4 mmol), 1,3-dibromopropane (242

mg, 1.2 mmol) and the mixture was stirred at reflux temperature for 14 h.

The reaction was monitored by TLC using EtOAc-hexane (1:1), K 2CO3 was

removed by filtration and the solvent was evaporated under the vacuum,diluted with water and extracted with ethyl acetate. The combined organic

phases were dried (Na2SO4) and evaporated under vacuum and the residue

was purified by column chromatography (40% EtOAc-hexane) to afford

compound 19a as yellow liquid (418 mg, 94%).

1H NMR (200 MHz, CDCl3) δ 7.7 (s, 1H), 6.8 (s, 1H), 4.82-4.87 (d, 1H, J = 4.6

Hz), 4.63-4.75 (m, 1H), 3.98-4.25 (t, 2H, J = 6.8 Hz), 3.95 (s, 3H), 3.62-3.68

(t, 2H, J = 6.6 Hz), 3.2-3.35 (m, 2H), 2.6-2.9 (m, 4H), 1.7-2.5 (m, 6H), 1.2-1.4(m, 6H);

FABMS: 521 [M]+.

(2S)-N-[4-(4-Bromobutoxy-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxaldehyde diethylthioacetal (19b)

The compound 19b was prepared according to the method described

for compound 19a by employing compound 18 (400 mg, 1 mmol),

anhydrous K 2CO3 (553 mg, 4 mmol) and 1, 4-dibromopropane (256 mg, 1.2


1H NMR (200 MHz, CDCl3) δ 7.65 (s, 1H), 6.8 (s, 1H), 4.82-4.87 (d, 1H, J = 4.6

Hz), 4.6-4.71 (m, 1H), 3.98-4.1 (t, 2H, J = 6.7 Hz), 3.95 (s, 3H), 3.52-3.59 (t,

2H, J = 6.8 Hz), 3.15-3.3 (m, 2H), 2.6-2.9 (m, 4H), 1.7-2.4 (m, 8H), 1.21-1.45

(m, 6H);

199



T HESIS

FABMS: 535 [M]+.

(2S)-N-[4-(5-Bromopentyloxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxaldehydediethylthioacetal (19c)

The compound 19c was prepared according to the method described

for compound 19a by employing compound 18 (400 mg, 1 mmol),

anhydrous K 2CO3 (553 mg, 4 mmol) and 1, 5-dibromopropane (270 mg, 1.2


1H NMR (200 MHz, CDCl3) δ 7.65 (s, 1H), 6.8 (s, 1H), 4.82-4.87 (d, 1H, J =

4.45 Hz), 4.6-4.71 (m, 1H), 3.98-4.15 (t, 2H, J = 6.57 Hz), 3.95 (s, 3H), 3.52-

3.6 (t, 2H, J = 6.38 Hz), 3.15-3.3 (m, 2H), 2.65-2.85 (m, 4H), 1.6-2.4 (m, 8H),

1.21-1.4 (m, 6H);

FABMS: 549 [M]+

(E)-1,3-bis(4-hydroxyphenyl)prop-2-en-1-one (22a)

To a stirred mixture of 4-hydroxyacetophenone 20a (136 mg, 1 mmol) and4-hydroxybenzaldehyde 21a (122 mg, 1 mmol) in ethanol (10 ml) was added

50% aqueous solution of potassium hydroxide (1 ml) and stirred for 24 h at

room temperature. After completion of the reaction checked by TLC, the




chromatography using (30% EtOAC:hexane) to obtain the pure product 22a.

Yield (185 mg, 77%).

Mp: 178–179 oC


7.56 (d, 2H, J = 8.3 Hz), 7.37 (d, 1H, J = 15.1 Hz), 6.92 (d, 2H, J = 9 Hz),

6.87 (d, 2H, J = 9 Hz); ESIMS: m/z 241 (M+H)+.

200



T HESIS

(E)-1,3-bis(3-hydroxyphenyl)prop-2-en-1-one (22b)


compound 22a by employing compound 3-hydroxyacetophenone 20b (136

mg, 1mmol), and 3-hydroxybenzaldehyde 21b (122 mg, 1 mmol). Yield (190

mg, 78%).

Mp: 181–183 oC

1H NMR (300 MHz, CDCl3 + DMSOd6): δ 9.37 (s, 1H), 9.24 (s, 1H), 7.77 (s,

1H), 7.56 (d, 1H, J = 15.67 Hz), 7.32−7.45 (m, 2H), 7.24 (t, 1H, J = 7.93, 7.55

Hz), 7.15 (t, 1H, J = 7.43, 7.43 Hz), 701−7.09 (m, 2H), 6.93−7 (dd, 1H, J = 8.3,

2.4 Hz), 6.76−7.83 (dd, 1H, J = 8.3, 1.88 Hz);


(E)-1,3-bis[4-{3-[(2S)-N-(4-oxy-5-methoxy-2-aminobenzoyl)pyrrolidine-2-carboxaldehydediethylthioacetal]propoxy}phenoxy]prop-2-en-1-one(23a)

To a solution of (2S)-N-[4-(3-Bromopropoxy)-5-methoxy-2-nitrobenzoyl]

pyrrolidine-2-carboxaldehydediethylthioacetal (19a) (1.145 g, 2.1 mmol) in

dry acetone (15 mL) was added, anhydrous K 2CO3 (552 mg, 4 mmol), (E)-1,3-

bis(4-hydroxyphenyl)prop-2-en-1-one (22a) (240 mg, 1 mmol) and themixture was stirred at reflux temperature for 12 hours. The reaction was

monitored by TLC using ethyl acetate-hexane (2:1). After completion of the

reaction as indicated by the TLC, K 2CO3 was removed by filtration and the

solvent evaporated under reduced pressure, diluted with water and

extracted with ethyl acetate. The organic phase was dried over Na2SO4 and

evaporated under vacuum. The residue, thus obtained was purified by

column chromatography using ethyl acetate and hexane (2:1) to afford

compound 23a as yellow solid. Yield (851 mg, 75%)

Mp: 207–209 oC


7.68 (s, 2H), 7.61 (d, 2H, J = 9 Hz), 7.41 (d, 1H, J = 15.8 Hz), 6.97 (d, 2H, J

= 8.3 Hz), 6.92 (d, 2H, J = 8.3 Hz), 6.81 (s, 2H), 4.85 (d, 2H, J = 3.8 Hz),

201



T HESIS

4.65−4.76 (m, 2H), 4.09−4.22 (m, 8H), 3.91 (s, 6H), 3.16−3.32 (m, 4H),

2.64−2.87 (m, 8H), 2.21−2.34 (m, 2H), 2.08−2.19 (m, 6H), 1.91−2.03 (m, 2H),

1.72−1.86 (m, 2H), 1.29−1.41 (m, 12H);

ESIMS: m/z 1122 (M+H)+

.

(E)-1,3-bis[4-{4-[(2S)-N-(4-oxy-5-methoxy-2-aminobenzoyl)pyrrolidine-2-carboxaldehydediethylthioacetal]butoxy}phenoxy]prop-2-en-1-one(23b)

The compound 23b was prepared according to the method described

for compound 23a by employing (2S)-N-[4-(4-Bromobutoxy)-5-methoxy-2-

nitrobenzoyl] pyrrolidine-2-carboxaldehydediethylthioacetal (19b) (1.175 g,

2.1 mmol), and (E)-1,3-bis(4-hydroxyphenyl)prop-2-en-1-one (22a) (240 mg,

1 mmol). Yield (912 mg, 79%)

Mp: 205–207 oC


7.69 (s, 2H), 7.6 (d, 2H, J = 8.9 Hz), 7.4 (d, 1H, J = 15.1 Hz), 6.97 (d, 2H, J =

8.2 Hz), 6.93 (d, 2H, J = 8.2 Hz), 6.82 (s, 2H), 4.88 (d, 2H, J = 3.4 Hz),

4.67−4.75 (m, 2H), 4.07−4.23 (m, 8H), 3.92 (s, 6H), 3.17−3.33 (m, 4H),

2.65−2.87 (m, 8H), 2.22−2.34 (m, 2H), 2.01−2.15 (m, 10H), 1.90−2 (m, 2H),

1.72−1.87 (m, 2H), 1.29−1.39 (m, 12H); ESIMS: m/z 1149 (M+H)+.

(E)-1,3-bis[4-{5-[(2S)-N-(4-oxy-5-methoxy-2-aminobenzoyl)pyrrolidine-2-carboxaldehydediethylthioacetal]pentoxy}phenoxy]prop-2-en-1-one (23c)


compound 23a by employing (2S)-N-[4-(5-Bromopentoxy)-5-methoxy-2-

nitrobenzoyl] pyrrolidine-2-carboxaldehydediethylthioacetal (19c) (1.21 g,

2.1 mmol), and (E)-1,3-bis(4-hydroxyphenyl)prop-2-en-1-one (22a) (240 mg,

1 mmol). Yield (930 mg, 78%)

202



T HESIS

Mp: 208–209 oC


7.69 (s, 2H), 7.62 (d, 2H, J = 8.9 Hz), 7.4 (d, 1H, J = 15.5 Hz), 6.98 (d, 2H, J =

8.3 Hz), 6.92 (d, 2H, J = 8.3 Hz), 6.82 (s, 2H), 4.87 (d, 2H, J = 3.4 Hz),

4.66−4.75 (m, 2H), 4.08−4.23 (m, 8H), 3.91 (s, 6H), 3.17−3.32 (m, 4H),

2.65−2.86 (m, 8H), 2.22−2.35 (m, 2H), 2.05−2.19 (m, 10H), 1.9−2.01 (m, 2H),

1.65−1.88 (m, 6H), 1.29−1.39 (m, 12H);

ESIMS: m/z 1178 (M+H)+.

(E)-1,3-bis[3-{3-[(2S)-N-(4-oxy-5-methoxy-2-aminobenzoyl)pyrrolidine-2-carboxaldehydediethylthioacetal]propoxy}phenoxy]prop-2-en-1-one(23d)


compound 23a by employing (2S)-N-[4-(3-Bromopropoxy)-5-methoxy-2-

nitrobenzoyl] pyrrolidine-2-carboxaldehydediethylthioacetal (19a) (1.145 g,

2.1 mmol), and (E)-1,3-bis(3-hydroxyphenyl)prop-2-en-1-one (22b) (240 mg,

1 mmol). Yield (850 mg, 75%)

Mp: 214–216 oC

1H NMR (500 MHz, CDCl3): δ 7.67−7.74 (m, 3H), 7.57 (d, 1H, J = 7.9 Hz), 7.55

(d, 1H, J = 14.8 Hz), 7.46 (d, 1H, J = 14.8 Hz), 7.37 (t, 1H, J = 7.9 Hz), 7.29

(t, 1H, J = 7.9 Hz), 7.17 (d, 1H, J = 1.9 Hz), 7.08−7.13 (dd, 1H, J = 7.9, 1.9

Hz), 6.91−6.96 (dd, 1H, J = 7.9, 1.9 Hz), 6.77 (s, 1H), 6.76 (s, 1H); 4.81 (d, 2H,

J = 3.9 Hz), 4.62−4.7 (m, 2H), 4.19−4.36 (m, 8H), 3.92 (s, 6H), 3.13−3.29 (m,

4H), 2.64−2.86 (m, 8H), 2.31−2.41 (m, 4H), 2.21−2.3 (m, 2H), 2.02−2.12 (m,

2H), 1.87−1.99 (m, 2H), 1.28−1.4 (m, 12H);

ESIMS: m/z 1121 (M+H)+.

203



T HESIS

(E)-1,3-bis[3-{4-[(2S)-N-(4-oxy-5-methoxy-2-aminobenzoyl)pyrrolidine-2-carboxaldehydediethylthioacetal]butoxy}phenoxy]prop-2-en-1-one(23e)


compound 23a by employing (2S)-N-[4-(4-Bromobutoxy)-5-methoxy-2-

nitrobenzoyl] pyrrolidine-2-carboxaldehydediethylthioacetal (19b) (1.175 g,

2.1mmol), and (E)-1,3-bis(3-hydroxyphenyl)prop-2-en-1-one (22b) (240 mg,

1 mmol). Yield (910 mg, 79%)

Mp: 215–217 oC



(t, 1H, J = 7.9 Hz), 7.18 (d, 1H, J = 1.9 Hz), 7.06−7.11 (dd, 1H, J = 7.9, 1.9

Hz), 6.91−6.95 (dd, 1H, J = 7.9, 1.9 Hz), 6.76 (s, 6H), 4.82 (d, 2H, J = 3.9 Hz),

4.62−4.71 (m, 2H), 4.17−4.34 (m, 8H), 3.91 (s, 6H), 3.13−3.28 (m, 4H),

2.64−2.81 (m, 8H), 2.22−2.34 (m, 4H), 2.01−2.13 (m, 8H), 1.91−2 (m, 2H),

1.73−1.87 (m, 2H), 1.28−1.39 (m, 12H);

ESIMS: m/z 1150 (M+H)+.

(E)-1,3-bis[3-{5-[(2S)-N-(4-oxy-5-methoxy-2-aminobenzoyl)pyrrolidine-2-carboxaldehydediethylthioacetal]pentoxy}phenoxy]prop-2-en-1-one(23f)


compound 23a by employing (2S)-N-[4-(5-Bromopentoxy)-5-methoxy-2-

nitrobenzoyl] pyrrolidine-2-carboxaldehydediethylthioacetal (19c) (1.21 g,

2.1 mmol), and (E)-1,3-bis(3-hydroxyphenyl)prop-2-en-1-one (22b) (240 mg,

1 mmol). Yield (925 mg, 77%)Mp: 213–215 oC



(t, 1H, J = 7.9 Hz), 7.18 (d, 1H, J = 1.9 Hz), 7.07−7.12 (dd, 1H, J = 7.9, 1.9

204



T HESIS

Hz), 6.9−6.96 (dd, 1H, J = 7.9, 1.9 Hz), 6.75 (s, 6H), 4.82 (d, 2H, J = 3.9 Hz),

4.63−4.72 (m, 2H), 4.15−4.34 (m, 8H), 3.92 (s, 6H), 3.13−3.29 (m, 4H),

2.63−2.8 (m, 8H), 2.21−2.35 (m, 4H), 2.04−2.12 (m, 8H), 1.89−1.95 (m, 2H),

1.72−1.86 (m, 6H), 1.27−1.39 (m, 12H);

ESIMS: m/z 1178 (M+H)+.

(E)-1,3-bis[4-{3-[7-Methoxy-8-oxy-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4] benzodiazepin-5-one]propyloxy}phenoxy]prop-2-en-1-one (25a)

To the compound 23a (1121 mg, 1 mmol) in methanol (20 mL) was

added SnCl2.2H2O (2.24, 10 mmol) and reflux for 5 hrs and checked TLC

indicated the reaction was completed. The methanol was evaporated under

vacuum and the reaction mass was neutralized with 10% NaHCO3 solution

and the extracted with ethyl acetate and chloroform (2x30mL and 2x30mL).

The combined organic phases was dried over Na2SO4 and evaporated under

vacuum to afford the crude aminodiethylthioacetal 24a (960 mg, 90%),

which was used directly in the next step due to its potential stability

problem.

A solution of 24a (1061 mg, 1.0 mmol), HgCl2 (1.35 g, 5 mmol) and

CaCO3 (500 mg, 5 mmol) in acetonitrile-water (4:1) was stirred slowly at

room temperature overnight until complete consumption of starting

material as indicated by the TLC. The clear organic supernatant liquid was

extracted with ethyl acetate and washed with saturated 5% NaHCO3 (20

mL), brine (20 mL) and the combined organic phase was dried over Na2SO4.

The organic layer was evaporated in vacuum to afford a yellow solid, which

was purified by column chromatography with MeOH-CHCl3 (1:20) to obtain

the pure product 25a. Yield (411 mg, 51%).

Mp: 191–193 oC


7.61 (d, 2H, J = 4.5 Hz), 7.57 (d, 2H, J = 9 Hz), 7.44 (s, 2H), 7.39 (d, 1H, J =

15.8 Hz), 6.93 (d, 2H, J = 9 Hz), 6.87 (d, 2H, J = 9 Hz), 6.74 (s, 2H), 4.08−4.24

205



T HESIS

(m, 8H), 3.92 (s, 6H), 3.75−3.84 (m, 2H), 3.66−3.73 (m, 2H), 3.52−3.63 (m,

2H), 2.24−2.38 (m, 4H), 1.98−2.12 (m, 8H);


(E)-1,3-bis[4-{4-[7-Methoxy-8-oxy-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4] benzodiazepin-5-one]butyloxy}phenoxy]prop-2-en-1-one (25b)


compound 25a employing 23b (1149 mg, 1 mmol) which reduction with

SnCl2.2H2O (2.24 mg, 10 mmol) gives amino compound 24b. Deprotection

followed by cyclization of 24b (1089 mg, 1 mmol) with HgCl2 (1.35 g, 5

mmol), CaCO3 (500 mg, 5 mmol) in acetonitrile-water (4:1) to obtain the


Mp: 190–192 oC


7.62 (d, 2H, J = 4.5 Hz), 7.56 (d, 2H, J = 9 Hz), 7.45 (s, 2H), 7.39 (d, 1H, J =

15.8 Hz), 6.93 (d, 2H, J = 9 Hz), 6.88 (d, 2H, J = 9 Hz), 6.75 (s, 2H), 4.06−4.23

(m, 8H), 3.93 (s, 6H), 3.76−3.85 (m, 2H), 3.66−3.74 (m, 2H), 3.52−3.63 (m,

2H), 2.24−2.38 (m, 4H), 1.94−2.15 (m, 12H);

13C NMR (75 MHz, CDCl3): δ 188.68, 164.55, 162.38, 160.85, 150.65, 147.74, 143.73,

140.5, 131.17, 130.61, 130.03, 127.65, 120.17, 119.42, 114.79, 114.16, 111.54, 110.4, 68.46,

67.6, 56.05, 53.65, 46.61, 29.61, 25.88, 25.56, 24.1, 22.56


(E)-1,3-bis[4-{5-[7-Methoxy-8-oxy-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4] benzodiazepin-5-one]pentyloxy}phenoxy]prop-2-en-1-one (25c)


the compound 25a employing 23c (1177 mg, 1 mmol) which reduction

with SnCl2.2H2O (2.24 mg, 10 mmol) gives amino compound 24c.

206



T HESIS

Deprotection followed by cyclization of 24c (1117 mg, 1 mmol) with HgCl2

(1.35 g, 5 mmol), CaCO3 (500 mg, 5 mmol) in acetonitrile-water (4:1) to

obtain the pure product 25c. Yield (490 mg, 56%).

Mp: 191–192o

C1H NMR (200 MHz, CDCl3): δ 8 (d, 2H, J = 9.06 Hz), 7.72 (d, 1H, J = 15.1 Hz),

7.61 (d, 2H, J = 4.53 Hz), 7.57 (d, 2H, J = 9.06 Hz), 7.45 (s, 2H), 7.39 (d, 1H, J

= 15.1 Hz), 6.92 (d, 2H, J = 9.06 Hz), 6.87 (d, 2H, J = 9.06 Hz), 6.74 (s, 2H),

4.07−4.23 (m, 8H), 3.92 (s, 6H), 3.75−3.85 (m, 2H), 3.67−3.73 (m, 2H),

3.52−3.62 (m, 2H), 2.24−2.37 (m, 4H), 1.93−2.14 (m, 12H), 1.63−1.75 (m, 4H);


(E)-1,3-bis[3-{3-[7-Methoxy-8-oxy-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4] benzodiazepin-5-one]propoxy}phenoxy]prop-2-en-1-one (25d)


compound 25a employing 23d (1121 mg, 1 mmol) which reduction with

SnCl2.2H2O (2.24 g, 10 mmol) gives amino compound 24d. Deprotection

followed by cyclization of 24d (1061 mg, 1 mmol) with HgCl2 (1.35 g, 5


pure product 25d. Yield (415 mg, 51%).

Mp: 187–189 oC


(d, 1H, J = 7.9 Hz), 7.54 (d, 1H, J = 15.1 Hz), 7.47 (d, 1H, J = 15.1 Hz), 7.37

(t, 1H, J = 7.9 Hz), 7.3 (t, 1H, J = 7.9 Hz), 7.17 (d, 1H, J = 2.1 Hz), 7.08−7.13

(dd, 1H, J = 7.9, 2.1 Hz), 6.91−6.96 (dd, 1H, J = 7.9, 2.1 Hz), 6.79 (s, 2H),

4.07−4.23 (m, 8H), 3.92 (s, 6H), 3.73−3.84 (m, 2H), 3.65−3.72 (m, 2H),3.51−3.63 (m, 2H), 2.24−2.39 (m, 4H), 1.98−2.13 (m, 8H);

13C NMR (75 MHz, CDCl3): δ 190.13, 181.9, 164.57, 162.43, 159.11, 150.59, 147.75,

144.71, 140.49, 139.46, 136.17, 129.91, 129.56, 122.27, 121.25, 121.09, 120.28, 119.59, 116.76,

114.05, 113.62, 111.55, 110.55, 65.36, 64.44, 56.09, 53.67, 46.64, 29.64, 28.97, 24.14,

207



T HESIS


(E)-1,3-bis[3-{4-[7-Methoxy-8-oxy-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4] benzodiazepin-5-one]butoxy}phenoxy]prop-2-en-1-one (25e)


compound 25a employing 23e (1149 mg, 1 mmol) which reduction with

SnCl2.2H2O (2.24 g, 10 mmol) gives amino compound 24e. Deprotection

followed by cyclization of 24e (1089 mg, 1 mmol) with HgCl2 (1.35 g, 5


pure product 25e. Yield (455 mg, 53%).

Mp: 186–188 oC




(dd, 1H, J = 7.9, 1.9 Hz), 6.91−6.95 (dd, 1H, J = 7.9, 1.9 Hz), 6.76 (s, 6H),

4.06−4.23 (m, 8H), 3.93 (s, 6H), 3.76−3.85 (m, 2H), 3.66−3.74 (m, 2H),

3.52−3.63 (m, 2H), 2.24−2.38 (m, 4H), 1.94−2.15 (m, 12H);


(E)-1,3-bis[3-{5-[7-Methoxy-8-oxy-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4] benzodiazepin-5-one]pentoxy}phenoxy]prop-2-en-1-one (25f)


compound 25a employing 23f (1177 mg, 1 mmol) which reduction with

SnCl2.2H2O (2.24 g, 10 mmol) gives amino compound 24f . Deprotection

followed by cyclization of 24f (1117 mg, 1 mmol) with HgCl2 (1.35 g, 5


pure product 25f. Yield (485 mg, 55%).

Mp: 187–188 oC



208



T HESIS


(dd, 1H, J = 7.9, 1.9 Hz), 6.9−6.96 (dd, 1H, J = 7.9, 1.9 Hz), 6.75 (s, 6H),

4.07−4.23 (m, 8H), 3.92 (s, 6H), 3.75−3.85 (m, 2H), 3.67−3.73 (m, 2H),

3.52−3.62 (m, 2H), 2.24−2.37 (m, 4H), 1.93−2.14 (m, 12H), 1.63−1.75 (m, 4H);


4.2.5. THERMAL DENATURATION STUDIES

The compounds 43a-f and 46a-c were subjected to DNA thermal

melting (denaturation) studies using duplex form calf thymus DNA (CT-DNA)

using modification reported procedure.51 Working solutions were produced by




methanol to obtain a fixed [PBD]/[DNA] molar ratio of 1:5 The DNA-PBD




of 1

ο

C min-1 in the 40−90

ο

C range. DNA helix-coil transition temperatures(T m) were determined from the maxima in the d (A260)/dT derivative plots.




melting behavior are given by ∆ T m = T m (DNA+PBD)- T m (DNA alone), where

the T m value for the PBD free CT-DNA is 69.8 ± 0.001 the fixed [PBD]/[DNA]

ratio used did not result in binding saturation of the host DNA duplex for any

compound examined.

4.2.6. PROCEDURE FOR MTT-ASSAY

Toxicity of test compound in cells was determined by MTT assay based

on mitochondrial reduction of yellow MTT tetrazolium dye to a highly colored

209



T HESIS

blue formazan product. 1x104 Cells (counted by Trypan blue exclusion dye

method) in 96-well plates were incubated with compounds with series of

concentrations tested for 48 hrs at 37 oC in RPMI/DMEM/MEM with 10% FBS

medium. Then the above media was replaced with 90 µl of fresh serum free

media and 10 µl of MTT reagent (5mg/ml) and plates were incubated at 37 oC

for 4 h, there after the above media was replaced with 200 µl of DMSO and

incubated at 37 oC for 10 min. The absorbance at 570 nm was measured on a

spectrophotometer (spectra max, Molecular devices) IC50 values were

determined from plot: % inhibition (from control) versus concentration.

210



T HESIS

4.2.7. REFERENCES:

1. Kimura, K.; Ogawa, M.; Oguro, M.; Koyama, Y.; Saito, T.; Furue, H.; Ota,

K.; Yamada, K.; Hoshino, A.; Nakumura, T.; Masaoka, T.; Taguchi, T.;

Kimura, I.; Hattori, T. Gan to Kagaku Ryoho ( J. Cancer Chemother .)

1982, 9, 924.

2. Tsugaya, M.; Washida, H.; Hirao, N.; Hachisuka, Y.; Sakagami, H.; Iwase,

Y. Hinyokika Kiya. 1986, 32, 1443.

3. Dervan, P. B. Science 1986, 232, 464.

4. Thurston, D. E.; Thompson, A. S. Chem. Br . 1990, 26, 767.

5. White, S.; Swezyk, J. W.; Turner, J. M.; Baird, E. E.; Dervan, P. B. Nature

1998, 391, 468.6. Thurston, D. E. Advances in the study of Pyrrolo[2,1-c]

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LIST OF PUBLICATIONS AND PATENTS

215



T HESIS

LIST OF PUBLICATIONS

1. Synthesis of a new 4-aza-2,3-didehydropodophyllotoxin analogues as

potent cytotoxic and antimitotic agents.

Ahmed Kamal, Paidakula Suresh, Adla Malla Reddy, Banala Ashwini

Kumar, Papagari Venkat Reddy, Paidakula Raju, Jaki R. Tamboli,

Thokhir B. Shaik, Nishant Jain, Shasi V. Kalivendi

Bioorganic & Medicinal Chemistry , 19, (2011) 2349-2358.

2. Synthesis of new 4β -Acrylamidopodophyllotoxin Congeners as DNA

Strands Breakage Agents.

Ahmed Kamal,*, Paidakula Suresh, M. Janaki Ramaiah, Adla Malla

Reddy, Banala Ashwini Kumar, Paidakula Raju, Vinay Gopal, S.N.C.V L.

Pushpavalli, Pranjal Sarma, Manika Pal-Bhadra,*

Bioorganic & Medicinal Chemistry (accepted)

3. Anti-tubercular agents. Part 5: Synthesis and biological evaluation of

benzothiadiazine 1,1-dioxide based congeners.

Ahmed Kamal*, Rajesh V.C.R.N.C. Shetti, Shaik Azeeza, S. Kaleem

Ahmed, P. Swapna, A. Malla Reddy, Inshad Ali Khan, Sandeep

Sharma, Sheikh Tasduq Abdullah

European Journal of Medicinal Chemistry 45 (2010) 4545-4553.

4. Sulfamic acid as an efficient and recyclable catalyst for the ring

opening of epoxides with amines and anilines: An easy synthesis of -

amino alcohols under solvent-free conditions.

Ahmed Kamal *, B. Rajendra Prasad, A. Malla Reddy, M. Naseer A.

Khan

Catalysis Communications 8 (2007) 1876–1880

5. Synthesis and Anticancer Activity of Chalcone-pyrrolobenzodiazepine

Conjugates Linked via 1,2,3-triazole Ring Side-armed with Alkane

Spacers.

216

http://www.sciencedirect.com/science/article/pii/S0223523411004302?_alid=1770046426&_rdoc=11&_fmt=high&_origin=search&_docanchor=&_ct=17&_zone=rslt_list_item&md5=039a3a41148829d12eb4407b16aed475








T HESIS

Ahmed Kamal, S. Prabhakar, M. Janaki Ramaiah, P. Venkat Reddy, Ch.

Ratna Reddy, A. Mallareddy, Nagula Shankaraiah, T. Lakshmi

Narayan Reddy, S.N.C.V.L. Pushpavalli, Manika Pal-Bhadra

European Journal of Medicinal Chemistry (in press)

6. Carbazole-pyrrolo[2,1-c][1,4]benzodiazepine conjugates: Design,

synthesis and biological evaluation.

Ahmed Kamal*, Rajesh VCRNC Shetti, M.Janaki Ramaiah, P.Swapna,

M.P.Narasimha Rao, A.Malla Reddy, S.N.C.V.L. Pushpavalli, Manika

Pal-Bhadra.

Medicinal Chemistry Communications (accepted).

7. An efficient approach for the preparation of bioactive moleculesemploying Paal-Knorr protocol.

Ahmed Kamal, Rajesh.V.C. R. N. C. Shetti, P. Swapna, M. P. Narasimha

Rao, A. Malla Reddy, M. Rafiq H. Siddiqui, Abdullah Alarifi

(communicated to Synlett )

8. Design, Synthesis and Anticancer Evaluation of Carbazole-

Benzothiazole Conjugates.

Ahmed Kamal*, Rajesh VCRNC Shetti, M.Janaki Ramaiah, P.Swapna,M.P.Narasimha Rao, A.Malla Reddy, H.K.Srivastava, G.Narahari

Sastry, Manika Pal-Bhadra (to be communicated to Arch. de Pharm).

LIST OF PATENTS

217



T HESIS

1. Benzylidineanthracenone linked pyrrolobenzodiazepine hybrids useful

as anti cancer agents and the process for preparation thereof.

Ahmed Kamal, A Malla Reddy, P Suresh and Rajesh VCRNC Shetti

Indian patent application no. 2886/DEL/2010

USA patent application no. 13/048248

2. Benzothiazole hybrids useful as potential anticancer agents and

process for the preparation thereof

Ahmed Kamal, A Malla Reddy, P Suresh, Rajesh V C R N C Shetti,

Harish Chandra Pal and Ajit Kumar Saxena

Indian patent application no. 270/DEL/2011.

3. Amidobenzothiazole analogues useful as potential anticancer agentsand process for the preparation thereof

Ahmed Kamal, A Malla Reddy, P Suresh, N Sankara Rao and Rajesh

VCRNC Shetti.


PCT application no. PCT/IN2011/000187

4. Synthesis of new benzothiazole derivatives as potential anti-tubercular

agentsAhmed Kamal, Rajesh V C R N C Shetti, P Swapna, Shaik Azeeza, A

Malla Reddy, Inshad Ali Khan, Sheikh Tasduq Abdulla, Sandeep

Sharma and Nitin pal Kalia


5. Biological evaluation of 4-aza-2,3-didehydropodophyllotoxin analoguespossessing potent antitumour activity

Ahmed Kamal, P Suresh, B Ashwini Kumar, A Malla Reddy, P. VenkatReddy and Jaki Rasheed Tamboli


218



T HESIS

6. Pyrrolo[2,1-c][1,4] benzodiazepine conjugates linked through

piperazine moiety as potential antitumour agent and process for the

preparation thereof

Ahmed Kamal, Rajesh V C R N C Shetti, K Srinivasa Reddy, A Malla

Reddy and P Swapna


7. Carbazole linked pyrrolo [2,1-c][1,4] benzodiazepine hybrids as

potential anticancer agents and process for the preparation thereof

Ahmed Kamal, Rajesh V C R N C Shetti, K Srinivasa Reddy and A Malla

Reddy


8. Chalcone linked pyrrolo[2,1-C][1,4] benzodiazepine hybrids as


Ahmed Kamal, B Rajendra Prasad and A Malla Reddy


9. Synthesis of new 4-acrylamidopodophyllotoxin congeners as

antitumour antibiotics and the process for preparation thereof.

Ahmed Kamal, P Suresh, B Ashwini Kumar, A MallaReddy and PVenkat Reddy


10. Quinazoline linked pyrrolo[2,1-C][1,4] benzodiazepine hybrids as




11. Benzophenone-piperazine linked pyrrolo[2,1-c][1,4]

benzodiazepine hybrids as potential anticancer agents and process for

the preparation there of



219



T HESIS

S YMPOSIUM AND CONFERENCES ATTENDED

1. Participated in 12th CRSI National Symposium in Chemistry & 4th CRSI-

RSC Symposium in Chemistry from 4-7 of February, 2010 at NIPER &

IICT, Hyderabad, India.

2. Participated in 13th ISCB International Conference on “Interplay of Chemical and

Biological Sciences: Impact on Health and Environment” at New Delhi, India on 26 th

copy (2) of malla raeddy thesis

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