THESIS

SYNTHESIS AND ANTICANCER ACTIVITY OF NEW PYRROLO[2,1-C][1,4]

BENZODIAZEPINES

AND COMBRETASTATIN DERIVATIVES

THESIS SUBMITTED
TO

KAKATIYA UNIVERSITY

FOR THE DEGREE OF DOCTOR OF PHILOSOPHY (IN CHEMISTRY) BY

ADLA MALLA REDDY

UNDER THE SUPERVISION OF DR. AHMED KAMAL DIVISION OF ORGANIC CHEMISTRY-I INDIAN INSTITUTE OF CHEMICAL TECHNOLOGY, HYDERABAD JUNE, 2011

THESIS

SYNTHESIS AND ANTICANCER ACTIVITY OF NEW PYRROLO[2,1-C][1,4]

BENZODIAZEPINES

AND COMBRETASTATIN DERIVATIVES

THESIS

SUBMITTED

TO

KAKATIYA UNIVERSITY
OF

FOR THE DEGREE

DOCTOR OF PHILOSOPHY (IN CHEMISTRY) BY

ADLA MALLA REDDY

UNDER THE SUPERVISION OF DR. AHMED KAMAL DIVISION OF ORGANIC CHEMISTRY-I INDIAN INSTITUTE OF CHEMICAL TECHNOLOGY, HYDERABAD JUNE, 2011

THESIS

DECLARATION
I hereby declare that the original research work embodied in the thesis entitled SYNTHESIS AND ANTICANCER ACTIVITY OF NEW PYRROLO[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 work incorporated 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

THESIS

Dedicated to My Beloved Parents

THESIS

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 my indebtedness 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 ChemistryI 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

THESIS 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 brothers 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)

THESIS

GENERAL REMARKS

H 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 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.

THESIS

ABBREVIATIONS

BF3.OEt2 BnBr n-BuLi CaCO3 CCl4 CH3CN CDCl3 Cs2CO3 DCC DCM DIBAL-H DMF DMSO EtSH EDCI

: : : : : : : : : : : : : :

Boron trifluoride diethyletherate Benzylbromide : n-Butyl Lithium Calcium carbonate Carbontetrachloride Acetonitrile Deuterated chloroform Cesium carbonate N, N'-Dicyclohexylcarbodiimide Dichloromethane Diisobutylaluminium hydride N, N'-Dimethylformamide Dimethylsulfoxide Ethanethiol 1-[3-(Dimethylamino)propyl]-3-

ethylcarobodiimidehydrochloride EtOAc HgCl2 HNO3 HOBt H2SO4 HCl IPA K2CO3 KOAc LiBr LiOH : : : : : : : : : : Ethyl acetate Mercuric chloride Nitric acid 1-Hydroxybenzotriazole Sulphuric acid Hydrochloric acid Isopropyl alcohol Potassiumcarbonate Potassium acetate Lithiumbromide : Lithiumhydroxide 8

THESIS MeOH NaBH4 NaClO2 NaOH NaOMe NaH

NH2NH2.H2O

: : : : : : : : : : : : : : : : :

Methylalcohol Sodiumborohydride Sodium chlorite Sodium hydroxide Sodium methoxide Sodium hydride hydrazine Hydrate Stannous chloride Stannic chloride Thionylchloride Tetrabutylammoniumfluoride tert-Butyldimethylchlorosilane Trifluoroacetic acid Chlorotrimethylsilane Tetrahydrofuran Triethyl amine Triphenylphosphine

SnCl2 SnCl4 SOCl2 TBAF TBDMSCl TFA TMSCl THF TEA TPP

CONTENTS
Page No.

THESIS SYNOPSIS CHAPTER-I GENERAL INTRODUCTION Introduction to Cancer 42 62 65 33 12

Current Area of Work Objectives of Present Work References CHAPTER-II

SYNTHESIS AND BIOLOGICAL EVALUATION OF CHALCONEPYRROLOBENZODIAZEPINE CONJUGATES AS ANTICANCER AGENTS Introduction Present Work Biological Activity Experimental References 79 84 87 108

73

CHAPTER-III

SYNTHESIS AND BIOLOGICAL EVALUATION OF COMBRETASTATIN DERIVATIVES AS ANTICANCER AGENTS Introduction Present work Biological Activity Experimental References 152 119 124 129 114

CHAPTER-IV SECTION-A SYNTHESIS AND BIOLOGICAL EVALUATION OF BENZYLIDENE-9(10H)ANTHRACENONE LINKED PYRROLOBENZODIAZEPINES AS ANTICANCER AGENTS Introduction 157

10

THESIS Present work Biological Activity 164 Experimental References SECTION-B SYNTHESIS AND BIOLOGICAL EVALUATION OF PYRROLOBENZODIAZEPINE DIMERS AS ANTICANCER AGENTS Introduction Present work Biological Activity 188 Experimental References LIST
OF

160

166 178 CHALCONE180 185

191 202 207 211

PUBLICATIONS
AND

AND

PATENTS

SYMPOSIUM

CONFERENCES

11

THESIS

SYNOPSIS

The work carried in the research tenure has been compiled in the form of a thesis entitled SYNTHESIS AND ANTICANCER ACTIVITY OF NEW PYRROLO[2,1-C] S [1,4]BENZODIAZEPINES AND COMBRETASTATIN DERIVATIVES. 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

THESIS anticancer activity. The thesis has been divided into four chapters. CHAPTER I gives the introduction about the chemotherapy of cancer, DNA binding ability with particular reference to pyrrolobenzodiazepines. CHAPTER II describes the synthesis of a new class of C8-linked chalcone-pyrrolobenzodiazepine analogues and evaluation of their anticancer activity. CHAPTER III describes II the synthesis and anticancer evaluation of combretastatin derivatives. CHAPTER IV 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 DNAbinding ability and cytotoxicity. CHAPTER I GENERAL INTRODUCTION 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 other parts 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 chemotherapys 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 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 like alkylating agents, antimetabolites, antibiotics, and

THESIS 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 7 OH 8 9 H 11 OCH3 N H 10 1 11a 2 N 5 6 4 3 O Anthramycin H N O N O O N N O HO H3CO O Tomaymycin H N N

CONH2

OCH3 H3CO SJG-136

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 and sequence selectivity.

14

carbinolamine HN H2 N OH H OH H 10 N H 11 N O Anthram ycin

O N N N DNA H3 C NH 2 O O HN OH H H 10 N 11 N O O NH H NH 2 N N N DNA

THESIS

H3C

Figure 2. Mechanism of action of PBDs with DNA. CHAPTER II - SYNTHESIS AND BIOLOGICAL EVALUATION OF CHALCONEPYRROLOBENZODIAZEPINE CONJUGATES AS ANTICANCER AGENTS 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 ,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 shared with, 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-2nitrobenzoyl]proline methylester (7), which is obtained according to the literature method starting from vanillin. This upon selective reduction by 15

THESIS employing DIBAL-H, and protection with TMSCl/EtSH followed by deprotection using BF3.OEt2/EtSH affords the precursor 10 as shown in Scheme 1.
HO MeO 1 O H HO (i) MeO 2 O (iii) BnO MeO 6 (vi) BnO MeO 7 O NO2 N COOMe (vii) BnO MeO 8 O NO2 N CHO O NO2 OH (v) BnO MeO 5 O NO2 OMe (iv) BnO MeO 4 O OMe OH (ii) MeO 3 O HO OMe

(viii) HO MeO 10 O NO2 CH(SEt)2 N (ix) BnO MeO 9 O NO2 N 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, K2CO3, 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. ClaisenSchmidt 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

THESIS alkylation of hydroxyl group with dibromoalkanes by using K2CO3 as a base in dry acetone to afford precursors 14a-f and 17a-c.

O MeO MeO OMe 11 CH3 +

CHO (i) R OH 12a, b MeO MeO OMe

O R OH 13a, b (ii)

14a; R = H, n = 2 14b; R = H, n = 3 14c; R = H, n = 4 14d; R = OMe, n = 2 14e; R = OMe, n = 3 14f; R = OMe, n = 4

O MeO MeO OMe R O Br ( )n

14a-f

Scheme 2. Reagents and conditions: (i) aq.KOH, ethanol, 4 h; (ii) dibromoalkane, acetone, K2CO3, reflux, 24 h.

17

THESIS
O + OMe 15 OH 12b 16 OH CHO (i) O OMe

(ii)

O 17a; n = 2 17b; n = 3 17c; n = 4 OMe O Br ( )n

17a-c
Scheme 3. Reagents and conditions: (i) aq.KOH, ethanol, 4 h; (ii) dibromoalkane, acetone, K2CO3, reflux, 24 h.

Compound 10 has been coupled to compounds 14a-f and 17a-c in the presence of K2CO3 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 the corresponding imines 20a-f and 23a-c (Schemes 4 & 5).

18

THESIS
O MeO MeO OMe 14a-f (i) O MeO MeO OMe R O 18a-f O ( )n MeO O O MeO MeO OMe 19a-f R O O ( )n MeO O (iii) O MeO MeO OMe R O 20a-f O ( )n MeO O
Scheme 4. Reagents and conditions: (i) K2CO3, acetone, 12 h, reflux; (ii) SnCl2.2H2O, MeOH, 4 h, reflux; (iii) HgCl2, CaCO3, MeCN, H2O, (4:1) 12 h, rt.

R O Br ( )n +

HO MeO

NO2 N O 1 0

CH(SEt)2

NO2 N

CH(SEt)2

(ii)

NH2 N

CH(SEt)2

N N

19

THESIS
O OMe O 17a-c (i) O OMe O 21a-c O ( )n MeO O (ii) O OMe O 22a-c O ( )n MeO O (iii) O OMe O 23a-c O ( )n MeO O N H NH2 N CH(SEt)2 NO2 N CH(SEt)2 Br ( )n + MeO O 10 N HO NO2 CH(SEt)2

n = 2, 3, 4

Scheme 5. Reagents and conditions: (i) K2CO3, acetone, 12 h, reflux; (ii) SnCl2.2H2O, MeOH, 4 h, reflux; (iii) HgCl2, CaCO3, MeCN, H2O, (4:1) 12 h, rt.

The thermal denaturation studies show that these conjugates (20af 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 <0.012.7 M, in comparison to adriamycin (GI50, <0.01-14.7 M). According to the in vitro screening data, compound 46b has significant cytotoxicity against all the cancer cell lines with GI50 values ranging from <0.01-0.17 M 0.1-2.19 M and has shown more potency against PC-3 prostate cancer cell line with GI50 <0.01 M.

20

THESIS CHAPTER III: SYNTHESIS AND BIOLOGICAL EVALUATION OF COMBRETASTATIN DERIVATIVES AS ANTICANCER AGENTS 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-4 derivatives with 2-aminobenzothiazoles and these new compounds exhibit potent anticancer activity.
MeO MeO OMe OMe combretastatin A-4 colchicine OH MeO NHCOCH3 MeO OMe O OMe

Figure 3. Potential inhibitors of tubulin polymerization The precursor (Z)-2-(2-methoxy-5-(3,4,5-

trimethoxystyryl)phenoxy)acetic acid 9 has been prepared by employing commercially available isovanillin. Hydroxy group protection of isovanillin 21

THESIS 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 -bromoethyl acetate in the presence of K2CO3 gives the ester 8, which on hydrolysis with LiOH affords the acid 9 (Scheme 1).
CHO (i) OH OMe 1 CHO (ii) OTBDMS OMe 2 CH2OH (iii) OTBDMS OMe 3 CH2Br OTBDMS OMe 4 (iv) CH2PPh3Br OTBDMS OMe 5

MeO MeO OMe 7 (vii)

OH OMe

(vi) MeO MeO OMe 6

OTBDMS (v) OMe

O MeO MeO OMe 8 O OMe OEt (viii) MeO MeO OMe 9 O

O OH

OMe

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, K2CO3, 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 -bromoethyl acetate in the presence of K2CO3 gives the ester compound 12. The ester compound on hydrolysis with LiOH affords the

22

THESIS chalcone acid (E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1enyl)phenoxy)aceticacid 13.

O MeO + MeO OMe 10 OH OMe 1 CHO (i) MeO MeO OMe 11 (ii) O OH OMe

O MeO MeO OMe 13 O

O OH (iii) MeO MeO

O O 12

O OEt

OMe

OMe

OMe

Scheme 2. Reagents and conditions: i) aq. KOH, ethanol, 12h; ii) 2-bromoethyl acetate, K2CO3, 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 bromoethylacetate in the presence of K2CO3 gives the ester compound 15. The ester compound on hydrolysis with LiOH affords pyrazoline acid 2-(5-(1acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)-2-met hoxyphenoxy)acetic acid 16.

23

THESIS
O O MeO MeO OMe 11 OH OMe (i) N MeO MeO OMe 14 N OH OMe

(ii) O O O OMe OMe 16 OH N (iii) MeO MeO OMe 15 N O OMe O OEt

O N MeO MeO N

Scheme 3. Reagents and conditions: i) NH2NH2.H2O, Acetic acid, reflux, 14h; ii) 2bromoethyl acetate, K2CO3, DMF, 12h; iii) LiOH, THF, H2O

O MeO MeO OMe 9 O OMe OH + H2N N S 17 (i) 18a, R = -H 18b, R = -NO2 18c, R = -F 18d, R = -Cl 18e, R = -OMe 18f, R = -OCF3 18g, R = -Me 18h, R = -CF3 18i, R = -OEt R

O MeO MeO OMe O OMe N H

N S 18 a-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

THESIS dichloromethane to give the desired combretastatin-benzothiazole analogues 18a-i.

O MeO MeO OMe 13 O OMe O OH + H2N N S 17 R

(i) 19a 19b 19c 19d 19e 19f 19g 19h 19i R = -H R = -NO2 R = -F R = -Cl R = -OMe R = -OCF3 R = -Me R = -CF3 R = -OEt

O MeO MeO OMe O

O N H

N S

OMe 19a-i

Scheme 5. Reagents and conditions: i) EDCI/HOBT, DCM, 14-16h

The synthesis of chalcone-benzothiazole derivatives 19a-i is outlined in Scheme 5. The chalcone acid 13 undergoes amide bond formation with 2amino benzothiazoles in the presence of EDCI/HOBt in dichloromethane to give the desired chalcone-benzothiazole analogues 19a-i.
O N MeO MeO OMe 16 (i) O N MeO MeO OMe N O OMe 20a-i O N H N S R 20a 20b 20c 20d 20e 20f 20g 20h 20i R = -H R = -NO2 R = -F R = -Cl R = -OMe R = -OCF3 R = -Me R = -CF3 R = -OEt N O OMe O OH + H2N N S 17 R

Scheme 6. Reagents and conditions: i) EDCI/HOBT, DCM, 14-16h

25

THESIS The pyrazolineacid 16 undergoes amide bond formation with 2aminobenzothiazoles 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 M. These compounds have also been evaluated for its tubulin binding activity and some of the compounds exhibited appreciable good tubulin binding activity. CHAPTER IV: (SECTION-A) - SYNTHESIS AND BIOLOGICAL EVALUATION OF BENZYLIDENE( 9(10H)-ANTHRACENONE LINKED PYRROLOBENZODIAZEPINES AS ANTICANCER AGENTS 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

THESIS 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(10H)-anthracenones undergo alkylation of hydroxyl group with dibromoalkanes using K2CO3 as a base in dry acetone to afford precursors 4a-f (Scheme 1).
O + R 1 OH 2a, b 3a, b R (ii) OH CHO (i) O

O 4a; R = H, n = 2 4b; R = H, n = 3 4c; R = H, n = 4 4d; R = OMe, n = 2 4e; R = OMe, n = 3 4f; R = OMe, n = 4

4a-f
R

Br ( )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 K2CO3 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

THESIS Finally the amino compounds upon deportation with HgCl2/CaCO3 cyclized to provide the corresponding imines 8a-f (Scheme-2).
O HO + MeO 4 a-f R O (i) Br ( )n O 5 NO2 CH(SEt)2 N

R O 6a-f O O ( )n MeO O (ii) R O 7a-f O (iii) O ( )n MeO O NH2 CH(SEt)2 N NO2 CH(SEt)2 N

R O 8a-f n = 2, 3, 4 O ( )n MeO O N N H

Scheme 2. Reagents and conditions: (i) K2CO3, acetone, 12 h, reflux; (ii) SnCl2.2H2O, MeOH, 4 h, reflux; (iii) HgCl2, CaCO3, MeCN, H2O, (4:1) 12 h, rt.

These benzylidine anthracenone linked PBD analogues have been tested 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

THESIS

CHAPTER IV: (SECTION-B) - SYNTHESIS AND BIOLOGICAL EVALUATION OF CHALCONE( PYRROLOBENZODIAZEPINE DIMERS AS ANTICANCER AGENTS 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.
O R R
1

CHO CH3 (i) + R4 R

3

O R1 R2 3a, b 3a; R1 = OH, R2 = H, R3 = H, R4 = OH 3b; R1 = H, R2 = OH, R3 = OH, R4 = H R4 R3

1a,b

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 units are 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

THESIS
O R 3a; R = 4,4'-dihydroxy 3b; R = 3,3'-dihydroxy (i) R Br + O ( )n MeO 4a-c O NO2 N CH(SEt)2

(EtS)2HC

O2N N O

( )n

Chalcone

O ( )n MeO

NO2 N O

CH(SEt)2

OMe (ii)

5a-f

(EtS)2HC

H2N N O

( )n

Chalcone

O ( )n MeO

NH2 N O

CH(SEt)2

OMe (iii)

6a-f

H N

( )n

Chalcone

O ( )n MeO

N N O

OMe O 7a-f

O 7a-c; Chalcone = O O O 7d-f; Chalcone = O n = 2,3,4 O

n = 2,3,4

Scheme 2. Reagents and conditions: (i) K2CO3, acetone, 12 h, reflux; (ii) SnCl2.2H2O, MeOH, 4 h, reflux; (iii) HgCl2, CaCO3, MeCN, H2O, (4:1) 12 h, rt.

Compound 4a-c has been coupled to dihydroxychalcones 3a,b in the presence of K2CO3 and dry acetone under reflux conditions to give corresponding nitro compounds 5a-f. These nitro compounds upon reduction

30

THESIS 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 Tm 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 Tm of 0.7
o

C 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

THESIS

CHAPTER-I GENERAL INTRODUCTION

32

THESIS 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, unless effectively 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 persons 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. Persons 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 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. Chemotherapy drugs 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 therapies such as tamoxifen and transplant options such as those done with bone marrow are

33

THESIS 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

THESIS 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.
Thymine O Sugar N O N H H N N Adenine N H N N Sugar Sugar N O H N O H N H N N N Cytosine

H N

N Sugar Guanine

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 and which 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

THESIS 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

THESIS 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), optical rotatory 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,
31

P NMR experiments such

as NOSEY or COSY can be used to locate precisely the ligand on the strand 37

THESIS 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 noncovalent binders, intercalaters and other type of adducts such as coordination complexes and triple helices.10

5' O H2C O

Major groove side

3'

O O P O Adenine O O H2C

NH2 N N N N CC 1065 9-Anthryloxirene Bisfunctional alkylating agents Aflatoxin B1 Mitomycin C (acid activity) Benz[a]pyrene

O HN O

CH2 O H3C O P O O O N O

Thymine

O O P OO H2C O

CH2 O O P O O CH2 O O P O O Cytosine O CH2 O O P O O CH2 O

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

O H2N N N O

O Guanine 1 7N 5 O P O 6 NH 8 O N 4 O 9 3 2 NH2 H2C O O P OO O H2C O O P OO

PBDs Mitomycin C (reducing conditions) Saframycins PAHs (Benz[a]pyrene) N-Hydroxy-2-naphthylamine Dehydroretronecine

Minor groove side

Figure 3. Structure of DNA

38

THESIS

1.4. TYPES 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 (Figure3). 1.4.1. INTERCALATERS Intercalaters consist of a flat, generally -deficient aromatic or heteroaromatic 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).
H3CO HN OMe O N R acridines R' OH H O O H3C
+ OH NH3

NHSO2CH3

OH

O X OH

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

THESIS methane sulphonic acid esters, nitrosoureas, triazenes and platinum complexes (e.g. cisplatin13) (Figure-5).

Cl R N Cl N N

N N N N

H3N Pt H3N

Cl Cl

O O

O S () n O O

nitrogen mustards

triethylenemelamine

cisplatin

methanesulfonates

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).

OH O

HN O

CH3 COOH OCH3

OH O

OH

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

THESIS

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 bisbenzimidazole (Hoecht 33258).18
CH3 O N N H N H CH3 N O N H

O H

CH3 N O N H NH2 + NH2

41

THESIS 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).
OH H N N O

O H2N H3C O

CH2OCONH2 OCH3 N NH

H 3C

OCH3 H CONH2

Mitomycin

Anthramycin

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

THESIS 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. PYRROLO[2,1-C][1,4]BENZODIAZEPINES ANTIBIOTICS AS DNA INTERACTIVE ANTITUMOR

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

tomaymycin,24 sibiromycin,25 chicamycin A,26 neothramycin A, B27 and DC8128 have been isolated from various streptomyces species (Figure-9).
9 8
A

10
B 5

N 11 N

R8

OR9 H N N

OCH3 H R1 CON R2

7 6

11a C

1
2

PBD ring system

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

R8 R7

N N O

H R H3C H3CHN

HO O

OMe H N N O

OH H CH3

Tomaymycin (R7 = OCH3, R8 = OH, R = CH3) Prothracarcin (R7 = R8 = H, R = CH3) Sibanomicine (R8 = H, R7 = sibirosamine pyronoside as in , R = Et)

CH3 O

OH OH

Sibiromycin

HO H3CO

H N

OCH3 H N OH

HO H3CO
8

N N O

R1 R2

O Chicamycin A

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

Figure 9. Naturally occurring PBDs

43

THESIS

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).
O HN H OH H N H N O C(11) (S) carbinolamine CH3 H2N -H2O +H2O HO MeO O C (11)-N(10) imine O HN HO MeO O C(11) (R/S) aminal H N HN N H N N N DNA CH3 N N H N N N DNA CH3

HO MeO

Figure 10. Formation of PBD-DNA covalent adduct

44

THESIS Molecular modeling, solution NMR, fluorimetry and DNA foot printing experiments reveal that the PBDs recognize a three base pair motif with a preference for 5Pu-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).
(h) Bulky substituents at N10 (eg. acetyl) inhibit DNA(a) An imine, carbinolamine binding and cytotoxicity methyl ether required at N10-C11 (g) Electron-donating substituents required at position 7,8 or 9 of A-ring R8 R7 (b) (S)-Stereochemistry required at C11a R9
10

11

N O

11a

1 2

(c) Replacement of C1 with an oxygen maintains cytotoxicity R (d) Endocyclic or exocyclic unsaturation at C2 enhances cytotoxicity and in vivo antitumour activity. Fully unsaturated C-ring leads to complete loss of DNA-binding and cytotoxicity

(f) Sugar moiety at C7 enhances DNA-binding affinity and cytotoxicity in some cell lines

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

45

THESIS Figure 10. Structure activity relationship of PBD ring system

1.6. SYNTHETIC APPROACHES OF PYRROLO[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. KANEKO 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

THESIS
R1 H N N O
1

H N N O
+ 2

S H R3 R1 R2 S H O
4

R1 R2

O H R3

R2
i

N N

ii

SR4 H R3

R1 R2

H N N S
3

R3
iii

R1 H N N O
8

N N O
6 +

H R3
iv

R1 R2

OCH3 H R3

R2
v

R1 R2

H N N O

SR4 H R3

R1 R2

H N N
7

H R3

R1 = H, OH, OBn, OCH3, OAc R2 = H, OCH3 R3 = H, = CH-CH3 (E), OH (a), OAc (b), = CH-COOEt (E) R4 = CH3
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, K2CO3, THF or DMF; (iii) Al-Hg, aq.THF or KH2PO4, 0-5
o

C, 14 h; (iv) 0.1 N methanolic HgCl2, 0

C or SiO2

chromatography, 5 oC; (v) CH3OH.

1.6.2. THRUSTONS APPROACH (CYCLIZATION BY Thurston and coworkers

39a

DEPROTECTION

OF DIETHYLTHIOACETAL)

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

THESIS PBD, PBD-EDTA conjugate, lexitropsin conjugates of PBD, C2 linked PBD dimers, imine-amide PBD dimers and naphthalimide conjugates of PBD.44
RO H3CO 9 i aR=H b R = PhCH2 RO H3CO O 13 vi a R = PhCH2 b R=H N N H v PhH2CO H3CO O 12 COOH PhH2CO H3CO 10 NO2 COOH PhH2CO H3CO O 11 iv NH2 N NO2 CH(SEt) 2 N

ii

iii

CH(SEt)2

Scheme 2. Reagents and conditions: (i) PhCH2Cl, THF, NaOH, H2O, reflux, 48 h; (ii) SnCl4, HNO3, CH2Cl2, -25
o

C,

min;

(iii)

(COCl)2,

THF,

DMF,

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-TYPE

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 approach 45 has been attempted wherein 9-fluorenyl methyloxy carbonyl (Fmoc) group can be used to protect the amine group and which can be easily removed by cleavage with Bu4N+F- (TBAF) at room temperature (Scheme 3).

48

THESIS
O H3CO O 14 a X = NO2 b X = NH2 O H3CO O 17 v O O H3CO O 18
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) HgCl 2, CaCO3, CH3CN-H2O, rt, 48 h; (iv) m-CPBA, CH2Cl2, rt, 72 h; (v) TBAF, DMF, rt, 15 min.

CH(SEt)2 N

ii

O H3CO

Fmoc NH

CH(SEt)2

N O 15 iii Fmoc OH N H N O 16

i O

Fmoc OH N H N

O iv H3CO

N N

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'-exounsaturated PBD dimer, C2-C3/C2'-C3'-endo unsaturated PBD dimer with remarkable covalent DNA binding affinity.49 1.6.4. KAMALS APPROACH (OXIDATION OF CYCLIC
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

THESIS 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).
H N N O 20 ii H N O 21 iii O 22 N N H S H

NO2 CHO N 19 O i H N

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

1.6.5. REDUCTIVE CYCLIZATION 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 CYCLIZATION In an endeavor to explore new practical methods for the preparation of PBDs particularly by the azido reductive process has been extensively

50

THESIS 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
R1 R2 O N3 CHO N R3 R1 R2 N N H R3 R1 R2 O NO2 CHO N R3

O R1= OH, OBn, OCH3 R2 = OCH3; R3 = H, OH

Scheme 5; Reagent used for azide reduction (i) HMDST (ii) bakers' yeast (iii) N,N-Dimethyl hydrazine/ ferric chloride (iv) TMSI (v) SmI 2 (vi) HI (vii) FeSO4.7 H 2O Reagents used for nitro reduction (i) Fe/ AcOH (ii) N,N-Dimethyl hydrazine/ ferric chloride

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 pnitrophenylcarbonate 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

THESIS

52

THESIS

OH 25

Cl3CCN 26

CCl3 NH

COOCH3 + Fmoc-N 28 OH

27

ii

COOCH3 iii HN 30 iv N3 R N 31 O vi H N R N 32 O vii H N R N 33 O O H R OH O H O R O Fmoc-N 29

COOCH3 O

COOCH3 O

N3 R N 34 O vi

CHO O

N N 35 O vii

H O

N N 36 O

H OH

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

THESIS 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 N7alkyl 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.
H3C N N CH3 O H3C H N Cl N N O

N N

H N N O

OCH3 H H3CO

OCH3 N N O N

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

THESIS 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.
NHNH2 H N S

S N O

OCH3 H

N N S

NHCONHC6H5 H OCOCH3

N N S

NHCO-p-FC6H4 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 and neothramycin 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

THESIS
H3CO H3CO O N N H H3CO H3CO O N N H O OCH3

H3CO H3CO

N N O

H3CO H3CO OCH3

N N O

Ph

Recently, Kamal and co-workers60 have synthesized a series of C2fluorinated 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

THESIS
R2 R3 O N N H R R1 R3 O R2 N N H R R1

R = F, R1 = H, R2 = H, R3 = H R = H, R1 = F, R2 = H, R3 = H R = F, R1 = F, R2 = H, R3 = H R = F, R1 = H, R2 = OBn, R3 = OCH3 R = F, R1 = H, R2 = OH, R3 = OCH3 N N O n = 3, 4, 5

H F

(CH2)n

N N O

H F

OCH3 H3CO

H F N

(CH2)n

N N O

H F

OCH3 H3CO O n = 3, 4, 5

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

THESIS
H N O N
7 O

CH3 N

N O 7 O N

1.8.2. C8-LINKED DIMERS 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 (DSB120) has been highly potent. Molecular modeling and NMR studies confirmed that these dimers span six base pairs in the minor groove of duplex DNA.
H N O DSB 120 N O OCH3 O H3CO O N N H

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 by a remarkable 17 oC after incubation for 18 h at 37 oC. These dimers have shown potent antitumor activity in different cell lines.
N N O H N N O O H

O OCH3

O H3CO

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

THESIS
N N O O OCH3 O H3CO O N N

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 C2position of the PBD ring system leads to significant increase in cytotoxicity.
H F N O N O O N N O H F

OCH3 H3CO

1.8.3. C2-LINKED DIMERS 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 N O N N O

H O N H N H O

1.9. C8-LINKED HYBRIDS OF PYRROLOBENZODIAZEPINES In the search for compounds with better antitumour activity and DNA sequence specificity many PBD analogues have been synthesized. In the

59

THESIS 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.
R

alkanes

O MeO

N N O

Cl N O OH H N O

R= HN

Me N O O N H

H N O

HN N H N O Me N N N O N H O O N H

O N O O HN O O O N N H

HN O

S N N

N Me

Figure 14. C8-linked PBD hybrids

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

THESIS coworkers46 have linked distamycine and netropsin antibiotics to the C8 position of PBD through linker of varying lengths.
H N H2N NH HCl H N O N n n = 1-4 O O H3CO O N 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.
H OH HO HO H H OH O ( )3 OH N N H H O N H N CH3 CH3

H N

OCH3 O 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. compounds exhibited IC50 in the pico molar range. One of these

61

THESIS
Cl O N ( )5 O N N O CBI - PBD Conjugate H

H3CO OH

Kamal and co-workers68 have synthesized a series of PBD conjugates by linking different DNA interacting ligands such as naphthalimides, polyaromatic hydrocarbons (pyrene amine and chrysene amine) and benzimidazoles by using varying linker length to enhance the DNA binding affinity and antitumor activity.

H N O

H N (CH2)n O H3CO n = 3-4 O N N H H3C N N N O ()

O n

N N O

H3CO n = 3-5

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).
H N N H O H3CO O N

H3C N H

N O O

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

THESIS 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.
O N O H3CO O N N H

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.
F O N N CH3 O n N N O F F O H3COOC N O n N N O H n = 35 H H3CO2SO O n N N O H

H3CO

H3CO

H3CO

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

THESIS 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 , -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 colchicinebinding site of tubulin. A water-soluble prodrug, combretastatin A-4phosphate 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 potent inhibitor 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

THESIS 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 linked activities we have synthesized The benzylideneanthrone pyrrolobenzodiazepine conjugates.

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

THESIS

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66

THESIS 17. Hurley, L. H.; Renolds, V. L.; Swernson, D. H.; Petzold, G. L.; Scahill, T. A. Science 1984, 226, 843. 18. Karlovsky, P.; deCock, A. W. Anal Biochem. 1991, 194, 192. 19. Dwyer, P. J.; Shoemaker, D.; Zaharko, D. S.; Grieshaber, C.; Plowman. Cancer Chemother. Pharmacol. 1987, 19, 6. 20. (a) Kunimoto, S.; Masuda, T.; Kanbayashi, M.; Hamada, M.; Umazawa, H. J. Antibiot. 1980, 33, 665. (b) Takeuchi, T.; Miyamoto, M.; Ishizuka, M.; Naganawa, H.; Kondo, S.; Hamada, M.; Umezawa, H. J. Antibiot. 1976, 29, 93. 21. Shimizu, K. I.; Kawamoto, I.; Tomita, F.; Morimoto, M.; Fuzimoto, K. J. Antibiot. 1982, 35, 972. 22. (a) Hara, M.; Tamaoki, T.; Yashida, M.; Morimoto, M.; Xlakano, J. Antibiot. 1988, 41, 702. (b) Langley, D. R.; Thurston, D. E. J. Org. Chem. 1987, 52, 91. 23. (a) Itoh, J.; Watanabe, H. O.; Ishii, S.; Gomi, S.; Nagasawa, M. and Yamamoto, A. J. Antibiot. 1988, 41, 1281. (b) Leber, J. D.; Hooner, J. R. C.; Holden, K. G.; Johnson, R. R.; Hed, S. M. J. Am. Chem. Soc. 1988, 110, 299. 24. Hurley, L. H.; Reck, T.; Thurston, D. E.; Langley, D. R.; Holder, K. G.; Hertzberg, R. P.; Hwover, J. R. E.; Gallegher, G.; Faucette, L. F. Jr.; Mong, S. M.; Johnson, R. K. Chem. Res. Toxicol. 1988, 1, 258. 25. Arima, K.; Kohsaka, M.; Tamura, G.; Imanaka, H.; Sakai, H. J. Antibiot. 1972, 25, 437. 26. Konishi, M.; Ohkuma, H.; Naruse, N.; Kawaguchi, H. J. Antibiot. 1984, 37, 200. 27. Tsunkawa, M.; Kamei, H.; Konishi, M.; Miyaki, T.; Oki, T.; Kawaguchi, H. J. Antibiot. 1988, 41, 1366.

67

THESIS 28. (a) Kunimoto, M.; Masuda, T.; Kanbayashi, N.; Hamada, M.; Naganawa, H.; Miyamoto, M.; Takeuchi, T.; Unezawa, H. J. Antibiot. 1980, 33, 665. (b) Thurston, D.E.; Murthy, V. S.; Langley, D. R.; Jones, G. B. Synthesis 1990, 1, 81. (c) Bose, D. S.; Jones, G. B.; Thurston, D. E. Tetrahedron 1992, 48, 751. 29. (a) Thurston, D. E. Advances in the Study of Pyrrolo[2,1-c]

[1,4]benzodiazepine (PBD) Antitumor Antibiotics. Molecular Aspects of Anticancer Drug-DNA Interactions; The Macmillan Press Ltd.; London, U.K., 1993; pp 54-88. (b) Petrusek, R. L.; Uhlenhopp, E. L.; Duteau, N.; Hurley, L. H. J. Biol. Chem. 1982, 257, 6207. 30. (a) Hurley, L. H.; Reck, T.; Thurston, D. E.; Langley, D. R.; Holden, K. G.; Hertzberg, R. P.; Hoover, J. R. E.; Gallagher, G. Jr.; Faucette, L. F.; Mong, S. M.; Johnson, R. K. Chem. Res. Toxicol. 1988, 1, 258. (b) Boyd, F. L.; Stewart, D.; Remers, W. A.; Barkley, M. D.; Hurley, L. H. Biochemistry 1990, 29, 2387. 31. Puvvada, M. S.; Hartley, J. A.; Jenkins, T. C.; Thurston, D. E. Nucleic Acids Res. 1993, 21, 3671. 32. Puvvada, M. S.; Forrow, S. A.; Hartley, J. A.; Stephenson, P.; Gibson, I.; Jenkins, T. C.; Thurston, D. E. Biochemistry 1997, 36, 2478. 33. Kopka, M. L.; Goodsell, D. S.; Baikalov, I.; Grzeskowiak, K.; Cascio, D.; Dickerson, R. E. Biochemistry 1994, 33, 13593. 34. Leimgruber, W.; Batcho, A. D.; Czajkowski, R. C. J. Am. Chem. Soc. 1968, 90, 5641. 35. (a) Thurston, D. E.; Bose, D. S. Chem. Rev. 1994, 94, 433. (b) Kamal, A.; Rao, M. V.; Reddy, B. S. N. Khim. Getero. Soed. (Chemistry of Heterocyclic Compounds), 1998, 1588. (c) Kamal, A.; Rao, M. V.; Laxman, N.; Ramesh, G.; Reddy, G. S. K. Current Medicinal Chemistry Anti-Cancer Agents 2002, 2, 215.

68

THESIS 36. (a) Kaneko, T.; Wong, H.; Doyle, T. W. Tetrahedron Lett. 1983, 24, 5165. (b) Suggs, J. W.; Wang, Y. S.; Lee, K. S. Tetrahedron Lett. 1985, 26, 4871. 37. Lown, J. W.; Joshua, A. V. Biochem. Pharmacol. 1979, 28, 2017. 38. (a) Langlois, N.; Favre, F.; Rojas, A. Tetrahedron Lett. 1993, 34, 4635. (b) Kaneko, T.; Wong, H.; Doyle, T. W.; Rose, W. C.; Bradner, W. T. J. Med. Chem. 1985, 28, 388. 39. (a) Langley, D. R.; Thurston, D. E. J. Org. Chem. 1987, 52, 91. (b) Courtney, S. M.; Thurston, D. E. Tetrahedron Lett. 1993, 34, 5327. (c) Bose, D. S.; Jones, G. B.; Thurston, D. E. Tetrahedron 1992, 48, 751. 40. Wilson, S. C.; Howard, P. W.; Forrow, S. M.; Hartley, J. A.; Adams, L. J.; Jekins, T. C.; Kelland, L. R.; Thurston, D. E. J. Med. Chem. 1999, 42, 4028. 41. (a) Kamal, A.; Rao, N. V. Chem. Commun. 1996, 385. (b) Kamal, A.; Howard, P. W.; Reddy, B. S. N.; Reddy, B. S. P.; Thurston, D. E. Tetrahedron 1997, 53, 3223. (c) Kraus, G. A.; Melekhov, A. Tetrahedron 1998, 54, 11749. 42. Kamal, A.; Laxman, E.; Laxman, N.; Rao, N. V. Bioorg. Med. Chem. Lett. 2000, 10, 2311. 43. (a) Berry, J. M.; Howard, P. W.; Thurston, D. E. Tetrahedron Lett. 2000, 41, 6171. (b) Kamal, A.; Reddy, G. S. K.; Raghavan, S. Bioorg. Med. Chem. Lett. 2001, 41, 387. 44. (a) Bose, D. S.; Thompson, A. S.; Ching, J.; Hartley, J. A.; Berardini, M. D.; Jenkins, T. C.; Neidle, S.; Hurley, L. H.; Thurston, D. E. J. Am. Chem. Soc. 1992, 114, 4939. (b) Bose, D. S.; Thompson, A. S.; Smellie, M.; Berardini, M. D.; Hartley, J. A.; Jenkins, T. C.; Neidle, S.; Thurston, D. E. J. Chem. Soc. Chem. Commun. 1992, 1518. (c) Reddy, B. S. P.; Damayanthi; Y.; Reddy, B. S. N.; Lown, J. W. Anti-Cancer Drug Design

69

THESIS 2000, 15, 225. (d) Kamal, A.; Laxman, N.; Ramesh, G.; Neelima, K.; Kondapi, A. K. Chem. Commun. 2001, 437. (e) Kamal, A.; Laxman, N.; Ramesh, G.; Srinivas, O.; Ramulu, P. Bioorg. Med. Chem. Lett. 2002, 12, 1917. (f) Kamal, A.; Reddy, B. S. N.; Reddy, G. S. K.; Ramesh, G. Bioorg. Med. Chem. Lett. 2002, 12, 1933. 45. Fukuyama, T.; Liu, G.; Linton, S. D.; Lin, S. C.; Nishino, H. Tetrahedron Lett. 1993, 34, 2577. 46. 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. 47. Zou, Q.; Duan, W.; Simmons, D.; Shyo, Y.; Raymond, M. A.; Dorr, R. T.; Hurley, L. H. J. Am. Chem. Soc. 2001, 123, 4865. 48. Cooper, N.; Hagan, D. R.; Tiberghien, A.; Ademefun, T.; Matthews, C. S.; Howard, P. W. and Thurston, D. E. Chem. Commun. 2002, 1764. 49. (a) Gregson, S. J.; Howard, P.W.; Hartley, J. A.; Brooks, N. A.; Adams, L. J.; Jenkins, T. C.; Kelland, L. R.; Thurston, D. E. J. Med. Chem. 2001, 44, 737. (b) Gregson, S. J.; Howard, P.W.; Corcoran, K. E.; Jenkins, T. C.; Kelland, L. R.; Thurston, D. E. Bioorg. Med. Chem. Lett. 2001, 11, 2859. 50. Miyamoto, M.; Kondo, S.; Naganawa, H.; Maeda, K.; Ohno, M.; Umezawa, H. J. Antibiot. 1973, 30, 340. 51. Thurston, D. E.; Langley, D. R. J. Org. Chem. 1986, 51, 705. 52. (a) Kamal, A.; Reddy, B. S. P.; Reddy, B. S. N. Tetrahedron Lett. 1996, 37, 6803. (b) Kamal, A.; Laxman, E.; Laxman, N.; Rao, N. V. Bioorg. Med. Chem. Lett. 2000, 10, 2311. (b) Kamal, A.; Laxman, E.; Arifuddin, M. Tetrahedron Lett. 2000, 41, 7743. (c) Kamal, A.; Laxman, E.; Reddy, P. S. M. M. Tedrahedron Lett. 2000, 41, 8631.

70

THESIS 53. (a) Kamal, A.; Reddy, G. S. K.; Reddy, K. L. Tedrahedron Lett. 2001, 42, 6969. (b) Kamal, A.; Reddy, G. S. K.; Reddy, K. L.; Raghavan, S. Tetrahedron Lett. 2002, 43, 2103. 54. Baraldi, P. G.; Leoni, A.; Cacciari, B.; Manfreini, S.; Simoni, D.; Bergomi, M.; Menta, E.; Spinelli, S. J. Med. Chem. 1994, 37, 4329. 55. Leoni, A. Ph.D. Thesis, University of Ferrara, Italy, 1992. 56. (a) Garofalo, A.; Balconi, G.; Botta, M.; Corelli, F.; DIncalci, M.; Fabrizi, G.; Fiorini, I.; Lamba, D.; Nacci, V. Eur. J. Med. Chem. 1993, 28, 213. (b) Nacci, V.; Garofalo, A.; Anzini, M.; Campiani, G. J. Heterocycl. Chem. 1988, 25, 1007. 57. Foloppe, M. P.; Rault, S.; Thurston, D. E.; Jenkins, T. C.; Robba, M. Eur. J. Med. Chem. 1996, 31, 407. 58. (a) Gregson, S. J.; Howard, P. W.; Corcoran, K. E.; Barcella, S.; Yasin, M. M.; Hurst, A. A.; Jenkins, T. C.; Kelland, L. R.; Thurston, D. E. Bioorg. Med. Chem. Lett. 2000, 10, 1845. (b) Gregson, S. J.; Howard, P. W.; Barcella, S.; Nakamya, A.; Jenkins, T. C.; Kelland, L. R.; Thurston, D. E. Bioorg. Med. Chem. Lett. 2000, 10, 1849. 59. (a) Kang, G. D.; Howard, P. W.; Thurston, D. E. Chem. Commun. 2003, 1688. (b) Tiberghien, A. C.; Hagan, D.; Howard, P. W.; Thurston, D. E. Bioorg. Med. Chem. Lett. 2004, 14, 5041. 60. Kamal, A.; Reddy, K. L.; Reddy, G. S. K.; Reddy, B. S. N. Tetrahedron Lett. 2004, 45, 3499. 61. ONeil, I. A.; Thompson, S.; Kalindjian, S. B.; Jenkins, T. C. Tetrahedron Lett. 2003, 44, 7809. 62. Baraldi, P. G.; Cacciari, B.; Guiotto, A.; Romagnoli, R.; Spalluto, G. Recent Res. Dev. In. Org. Chem. 1998, 2, 567. 63. (a) Farmer, J. D.; Rudnicki, S. M.; Suggs, J. W. Tetrahedron Lett. 1988, 29, 5105. (b) Farmer, J. D.; Gustafson, G. R.; Conti, A.; Zimmt, M. B.; Suggs, J. W. Nucleic acids Res. 1991, 19, 899.

71

THESIS 64. Kamal, A.; Reddy, P. S. M. M.; Reddy, D. R. S. Bioorg. Med. Chem. Lett. 2004, 14, 2669. 65. (a) Zou, Q.; Duan, W.; Simmons, D.; Shyo, Y.; Raymond, M. A.; Dorr, R. T.; Hurley, L. H. J. Am. Chem. Soc. 2001, 123, 4865; (b) Tercel, M.; Stribbling, S. M.; Shephard, H.; Siim, B. G.; Wu, K.; Pullen, S. M.; Bottin, K. J.; Wilson, W. R.; Denny, W. A. J. Med. Chem. 2003, 46, 2132; (c) Kamal, A.; Ramesh, G.; Srinivas, O.; Ramulu, P. Bioorg. Med. Chem. Lett. 2004, 14, 471; (d) Reddy, B. S. P.; Damayanthi; Y.; Reddy, B. S. N.; Lown, J. W. Anti-Cancer Drug Design 2000, 15, 225; (e) Kamal, A.; Reddy, B. S. N.; Reddy, G. S. K; Ramesh, G. Bioorg. Med. Chem. Lett. 2002, 12, 1933; (f) 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; 66. Kumar, R.; Lown, J. W. Org. Biomol. Chem. 2003, 1, 3327. 67. Tercel, M.; Stribbling, S. M.; Shephard, H.; Siim, B. G.; Wu, K.; Pullen, S. M.; Bottin, K. J.; Wilson, W. R.; Denny, W. A. J. Med. Chem. 2003, 46, 2132. 68. (a) Kamal, A.; Ramesh, G.; Ramulu, P.; Srinivas, O.; Rehana, T.; Sheelu, G. Bioorg. Med. Chem. Lett. 2003, 13, 3451. (b) Kamal, A.; Ramesh, G.; Srinivas, O.; Ramulu, P. Bioorg. Med. Chem. Lett. 2004, 14, 471. (c). Kamal, A.; Ramulu, P.; Srinivas, O.; Ramesh, G.; Kumar, P. P. Bioorg. Med. Chem. Lett. 2004, 14, 4791. 69. Zou, Q.; Duan, W.; Simmons, D.; Shyo, Y.; Raymond, M. A.; Dorr, R. T.; Hurley, L. H. J. Am. Chem. Soc. 2001, 123, 4865. 70. Kamal, A.; Laxman, N.; Ramesh, G.; Srinivas, O.; Ramulu, P. Bioorg. Med. Chem. Lett. 2002, 12, 1917. 71. Kumar, R.; Lown, J. W. Org. Biomol. Chem. 2003, 1, 3327. 72. Kamal, A.; Devaiah, V.; Reddy, K. L.; Kumar, M. S. Bioorg. Med. Chem. 2005, 13, 2021. 73. Kamal, A.; Reddy, K. L.; Devaiah, V.; Shankaraiah, N.; Kumar, M. S.; Reddy, G. S. K. Lett. Drug Des. Discov. 2005, 1, 55.

72

THESIS 74. Kamal, A.; Srinivas, O.; Ramulu, P.; Ramesh, G.; Kumar, P. P.; Kumar, M. S. Bioorg. Med. Chem. 2004, 12, 4337. 75. Kamal, A.; Reddy, D. R.; Reddy, P. S. M. M. Rajendar. Bioorg. Med. Chem. Lett. 2006, 16, 1160. 76. Kamal, A.; Ramulu, P.; Srinivas, O.; Ramesh, G. Bioorg. Med. Chem. Lett. 2003, 13, 3517.

CHAPTER-II SYNTHESIS AND BIOLOGICAL EVALUATION OF CHALCONEPYRROLOBENZODIAZEPINE CONJUGATES AS ANTICANCER AGENTS

73

THESIS

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

THESIS 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-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. DNAbinding ability has been observed by thermal denaturation studies with CTDNA ( Tm > 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

THESIS
H3C OR H N N O R= OH or OCH3 anthramycin (1) CONH2 H3C H3CHN CH3 O OH OH OCH3 H OMe H N N O sibiromycin (2) OCH3 H N O chicamycin (4) HO H3CO O DC-81 (7) N N OH OH H CH3

HO O

HO H3CO

N N O

H CH3

HO H3CO

H N

tomamycin (3) HO H3CO O N N R1 R2 H

( R1 = H; R2 = OH) (5) ( R1 = OH, R2 = H) (6) neothramycin A and B

Figure 1. Naturally occurring PBDs 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 ,-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 76

THESIS substituted benzaldehydes and acetophenones. Recent studies revealed that these chalcones had shown a wide variety of anticancer,10-17 antiinflammatory,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).
O MeO MeO OMe 8 OH OMe MeO MeO OMe 9 (SD400) OH OMe O

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 - and -subunits. Many molecules based on a chalcone scaffold have been synthesized to improve their biological profile, including their capability as 77

THESIS 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, attracted more makes interest them in an attractive years scaffold. because Chalcones of their have recent 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
OH O OCH3 H3CO HO 10 OH H3CO OCH3 11
30

(Figure 3).
O OH

Figure 3 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 lines31 (Figure 4).
O MeO MeO OMe 12 NO2 MeO MeO OMe 13 O OMe

O MeO MeO OMe 14 B(OH)2 OMe

78

THESIS 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 M, against PaCa-2 cell line.32 Lawrence and co-workers reported new chalcone derivative (16) which possess good anticancer activity33 (Figure 5).
O OMe N H 15 OMe OMe OMe 16 (MDL) N OMe O

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(2fluorobenzylidene)-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 lines 36 (Figure 6).

79

THESIS
OH O MeO HO 17 (curcumin) Cl O Cl H3CO N O O OH 19 (CLEFMA) HO N CH3 20 OMe OH N H 18 (EF24) O OCH3 OH F O F

Figure 6. Structures of bis-chalcones The cyclic chalcone analogues, E-2-arylmethylene-1-indanones, E-2arylmethylene-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 Alzheimers disease by inhibition of (acetylcholinesterase) AChE-induced Ab aggregation38 (Figure 7).

80

THESIS
O NO2 O Me N 21a O OMe 21b

22a

22b

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 the preparation 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 N H F Cl N N 24 O O O MeO O N N H N H 23 O MeO O N N H

81

THESIS 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 for such 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 C8position 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. SYNTHESIS

OF

PBD PRECURSORS

The precursor (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]pyrolidine2-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-methoxy2-nitrobenzoyl]pyrolidine-2-carboxaldehydediethylthioacetal (33) 1). (Scheme

82

THESIS
HO H3CO 25 O OH i HO H3CO 26 O OCH3 ii BnO H3CO 27 O iii (v) (iv) BnO H3CO O 28 OCH3

BnO MeO 30

NO2 N O (vi)

COOMe

BnO MeO

NO2 OH O 2 9

NO2 OCH3

BnO MeO 3 1

NO2 N O

CHO

(vii)

BnO MeO

NO2 N O 3 2

CH(SEt)2 (viii) HO MeO

NO2 N O 33

CH(SEt)2

Scheme 1. Reagents and conditions: i) H2SO4, MeOH, 24 h;

o

ii) Benzyl bromide, K2CO3,

acetone, 24 h; iii) HNO3-H2SO4, SnCl4, CH2Cl2, -25 C, 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. ClaisenSchmidt 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 1indanone with vanillin. Using aqueous KOH for preparation of indano chalcone taking longer reaction time and the yield is very less (20%). These trimethoxy and indanochalcones undergo etherification of hydroxyl group with dibromoalkanes by using K2CO3 as base in dry acetone afford precursors 37a-f and 40a-c.

83

THESIS
O MeO MeO OMe 34 OH 35a, b CH3 + R MeO OMe 36a, b (ii) OH CHO (i) MeO O R

37a; R = H, n = 2 37b; R = H, n = 3 37c; R = H, n = 4 37d; R = OMe, n = 2 37e; R = OMe, n = 3 37f; R = OMe, n = 4

O MeO MeO OMe 37a-f R O Br ( )n

Scheme 2. Reagents and conditions: (i) aq.KOH, ethanol, 4h; (ii) dibromoalkane, acetone, K2CO3, reflux, 24h.

O +

CHO (i) OMe

O OMe OH 39

38

OH 35

(ii)

O 40a; n = 2 40b; n = 3 40c; n = 4 40a-c

Scheme 3. Reagents and conditions: (i) ethanol/piperidine, reflux, 10 h; (ii) dibromoalkane, acetone, K2CO3, reflux, 24 h.

OMe O Br ( )n

2.2.3. SYNTHESIS OF C8-LINKED CHALCONE-PBD HYBRIDS

Compound 33 has been coupled to compounds 37a-f in the presence of K2CO3 and dry acetone under reflux gives corresponding nitro compounds 41a-f. These nitro compounds upon reduction with SnCl 2.2H2O in methanol under reflux give amino compounds 42a-f. The amino compounds on 84

THESIS deprotection with HgCl2/CaCO3 afford corresponding imines 43a-f (Scheme4).

O MeO MeO OMe 37a-f i O MeO MeO OMe R O 41a-f O ( )n MeO O O MeO MeO OMe 42a-f R O O ( )n MeO O iii O MeO MeO OMe R O 43a-f O ( )n MeO O
Scheme 4. Reagents and conditions: (i) K2CO3, acetone, 24 h, reflux; (ii) SnCl2.2H2O, MeOH, 5 h, reflux; (iii) HgCl2, CaCO3, MeCN, H2O, (4:1) 12 h, rt.

R O Br ( )n +

HO MeO

NO2 N O 33

CH(SEt)2

NO2 N

CH(SEt)2

ii

NH2 N

CH(SEt)2

N N

Compound 33 has been coupled to compounds 40a-c in the presence of K2CO3 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 (Scheme5).

85

THESIS
O OMe O 40a-c i O OMe O 44a-c O ( )n MeO O ii O OMe O 45a-c O ( )n MeO O iii O OMe O 46a-c O ( )n MeO O N N H NH2 N CH(SEt)2 NO2 N CH(SEt)2 Br ( )n + MeO O 33 N HO NO2 CH(SEt)2

Scheme 5. Reagents and conditions: (i) K2CO3, 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

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

86

THESIS a Beckman DU-7400 spectrophotometer fitted with high performance temperature controller and heated at 1 C/min in the range of 40-95 C. DNA helix-coil transition temperatures are given by: Tm = Tm(DNA+PBD)Tm(DNA alone), where the Tm 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 ( Tm) for each compound is examined at 0 h and 18 h of incubation at 37 C. 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 37

C with ligand/DNA molar ratio of 1:5. The increase in

the helix melting temperature ( Tm) for each compound has been examined at 0 h and 18 h incubation at 37 C. Interestingly, all the chalcone-PBD conjugates elevate the helix melting temperature of CT-DNA in the range of 4.0-8.6
o

C. The compound 46b

showed highest Tm 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 Tm 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 Tm (oC)a after incubation at 37 oC [PBD]:[DNA] for Compound molar ratiob 0h 18 h 43a 43b 43c 43d 43e 1:5 1:5 1:5 1:5 1:5 5.1 5.2 4.6 5.3 4.0 87 5.8 6.1 5.0 5.9 4.5

THESIS 43f 46a 46b 46b DC-81

a

1:5 1:5 1:5 1:5 1:5

4.0 7.5 7.9 7.1 0.3

0 b

4.3 8.2 8.6 8.4 0.7

For CT-DNA alone at pH 7.00 0.01, Tm = 68.5 C 0.01 (mean value from 10 separate For a 1:5 molar ratio of [PBD]/[DNA],

determinations), all Tm values are 0.1 - 0.2 0C.

where CT-DNA concentration = 100 M and ligand concentration = 20 M 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 vitro cytotoxicity 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 M, while the positive controls, DC-81 and adriamycin exhibited the GI50 in the range of 0.1-0.17 M and <0.01-14.7 M respectively. The synthesized chalcone-PBD conjugates exhibited significant anticancer activity against PC-3 human prostate cancer cell line (GI50 range, <0.010.22 M) 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 M) 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 chalconePBD conjugates synthesized, the compounds with indano chalcone moiety exhibited superior activity compared to the compounds with trimethoxy chalcone moiety.

88

THESIS Table 2. GI50 valuesa (in M) for compounds 43a-f and 46a-c in selected human cancer cell lines. GI50 values (M)
Compound Breast MCF-7 Ovarian A2780 Colon Colo205 Prostate PC-3 Cervix SiHa Lung A 549 Hop-62 Oral KB

43a 43b 43c 43d 43e 43f 46a 46b 46c DC-81 ADR
a

0.11 0.05 0.29 0.07 0.11 0.14 0.1 0.01 0.17 0.16 <0.0 1

0.14 0.03 0.1 0.028 0.14 0.12 0.12 0.013 0.14 0.13 0.02

0.16 0.15 0.15 0.16 0.17 0.14 0.14 0.14 0.17 0.1 14.7

0.14 0.16 0.16 0.22 0.1 0.15 0.08 <0.01 0.16 -<0.01

0.12 1.8 0.17 2.7 0.12 0.15 0.14 0.17 0.16 0.16 0.19

0.14 1.88 0.1 -0.14 0.16 0.1 0.17 0.11 -13

0.15 0.2 0.13 -0.1 0.16 0.14 0.016 0.17 0.11 <0.01

0.15 2.1 0.1 2.6 0.14 0.17 0.18 0.01 6 0.14 0.17 0.16

50% Growth inhibition and the values are mean of four determinations ADR, adriamycin.

2.4. CONCLUSION
In conclusion, we have synthesized a series of novel C8-linked chalconePBD 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

THESIS 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).
1

H NMR (200 MHz, CDCl3): 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 K2CO3 (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 K2CO3 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: 116118 C.
1

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

1H, J = 8.2 Hz), 7.207.50 (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-3methoxybenzoate 27 (272 mg, 1 mmol) in dichloromethane (30 mL) at 78 90

THESIS

C. The mixture was maintained at 78 C 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% EtOAchexane) affords 28 as a yellow solid (247 mg, 78%). Mp 128 130 C;
1

H NMR (200 MHz, CDCl3): 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: 180 182 C;
1

H NMR (200 MHz, CDCl3): 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-2carboxylate (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

THESIS 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 C;
1

H NMR (300 MHz, CDCl3): 7.72 (s, 1H), 7.37.48 (m, 6H), 5.2 (s, 2H), 4.68

4.75 (m, 1H), 3.92 (s, 3H), 3.8 (s, 3H), 3.263.4 (m, 1H), 3.13.25 (m, 1H), 2.22.45 (m, 1H), 1.812.12 (m, 3H); ESIMS: m/z 414 (M) +. (2S)-N-[4-Benzyloxy-5-methoxy2-nitrobenzoyl]pyrrolidine-2carboxaledhyde (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 Na 2SO4 and solvent was evaporated under vacuum to afford the crude aldehyde 31. Yield (254 mg 65%)
1

H NMR (300 MHz, CDCl3): 9.8 (s, 1H), 9.25 (s, 1H), 7.75 (s, 1H), 7.257.5

(m, 5H), 6.56 (s, 1H), 5.2 (s, 2H), 4.7 (m, 1H), 4.02 (m, 1H), 3.183.38 (m, 2H), 1.882.4 (m, 4H); ESIMS: m/z 355 (M-CHO) +

92

THESIS (2S)-N-[4-Benzyloxy-5-methoxy2-nitrobenzoyl]pyrrolidine-2carboxaledhyde 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%).
1

H NMR (300 MHz, CDCl3): 7.75 (s, 1H), 7.327.48 (s, 5H), 6.85 (s, 1H), 5.2

(s, 2H), 4.85 (d, 1H), 4.664.75 (m, 1H), 4 (s, 3H), 3.23.35 (m, 2H), 2.65 2.96 (m, 4H), 1.211.42 (m, 6H); ESIMS: m/z 490 (M+H)+. (2S)-N-[4-Hydroxy-5-methoxy2-nitrobenzoyl]pyrrolidine-2carboxaledhyde 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

THESIS
1

H NMR (300 MHz, CDCl3): 7.6 (s, 1H), 6.75 (s, 1H), 4.85 (d, 1H), 4.64.7 (m,

1H), 3.9 (s, 3H) 3.23.32 (m, 2H), 2.72.88 (m, 4H), 1.752.35 (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 C;
1

H NMR (300 MHz, CDCl3): 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); ESIMS: m/z 315 (M+H)+. (E)-3-(4-hydroxy-3-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)prop2-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 C;

94

THESIS
1

H NMR (300 MHz, CDCl3): 7.68 (d, 1H, J = 15.8 Hz), 7.27 (d, 1H, J = 15.8

Hz), 7.167.23 (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); ESIMS: m/z 345 (M+H)+. (E)-3-(4-(3-bromopropoxy)phenyl)-1-(3,4,5-trimethoxyphenyl)prop2-en-1-one (37a) To a solution of compound 36a (314 mg, 1 mmol) in dry acetone (15 mL) was added, anhydrous K2CO3 (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, K2CO3 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%)
1

H NMR (300 MHz, CDCl3): 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); ESIMS: m/z 436 (M+H)+. (E)-3-(4-(4-bromobutoxy)phenyl)-1-(3,4,5-trimethoxyphenyl)prop-2en-1-one (37b) The compound 37b was prepared according to the method described for compound 37a by employing compound 36a (314 mg, 1 mmol), and 1,4 dibromobutane (647 mg, 3 mmol). Yield (403 mg, 90%).
1

H NMR (300 MHz, CDCl3): 7.74 (d, 1H, J = 15.8 Hz), 7.57 (d, 2H, J = 9 Hz),

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

THESIS = 6, 5.2 Hz), 3.95 (s, 6H), 3.9 (s, 3H), 3.47 (t, 2H, J = 6.7, 6 Hz), 1.922.13 (m, 4H); ESIMS: m/z 451 (M+H)+. (E)-3-(4-(5-bromopentoxy)phenyl)-1-(3,4,5-trimethoxyphenyl)prop2-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%)
1

H NMR (300 MHz, CDCl3): 7.77 (d, 1H, J = 15.8 Hz), 7.62 (d, 2H, J = 9 Hz),

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), 1.591.71 (m, 2H); ESIMS: m/z 464 (M+H)+. (E)-3-(4-(3-bromopropoxy)-3-methoxyphenyl)-1-(3,4,5trimethoxyphenyl)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%)
1

H NMR (200 MHz, CDCl3): 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); ESIMS: m/z 466 (M+H)+. (E)-3-(4-(4-bromobutoxy)-3-methoxyphenyl)-1-(3,4,5trimethoxyphenyl)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 1,4dibromobutane (647 mg, 3 mmol). Yield (425 mg, 88%)

96

THESIS
1

H NMR (200 MHz, CDCl3): 7.7 (d, 1H, J = 15.8 Hz), 7.27 (d, 1H, J = 15.8

Hz), 7.177.24 (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), 1.942.17 (m, 4H); ESIMS: m/z 483 (M+H)+. (E)-3-(4-(3-bromopropoxy)-3-methoxyphenyl)-1-(3,4,5trimethoxyphenyl)prop-2-en-1-one (37f) The compound 37f was prepared according to the method described for compound 37a by employing compound 36b (344 mg, 1 mmol), and 1,5 dibromopentane (689 mg, 3 mmol). Yield (450 mg, 91%)
1

H NMR (400 MHz, CDCl3): 7.76 (d, 1H, J = 15.8 Hz), 7.33 (d, 1H, J = 15.8

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); ESIMS: m/z 494 (M+H)+. (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-3methoxy 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%).
1

H NMR (300 MHz, DMSO D6): 9.77 (s, 1H), 7.79 (d, 1H, J = 7.3 Hz), 7.67

7.74 (m, 2H), 7.447.56 (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

THESIS (E)-2-(4-(3-bromopropoxy)-3-methoxybenzylidene)-2,3dihydroinden-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 1,3dibromopropane (605 mg, 3 mmol). Yield (320 mg, 82%)
1

H NMR (300 MHz, CDCl3): 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); ESIMS: m/z 388 (M+H)+. (E)-2-(4-(4-bromobutoxy)-3-methoxybenzylidene)-2,3-dihydroinden1-one (40b) The compound 40b was prepared according to the method described for compound 37a by employing compound 39 (266 mg, 1 mmol), and 1,4dibromobutane (647 mg, 3 mmol). Yield (351 mg, 87%)
1

H NMR (300 MHz, CDCl3): 7.88 (d, 1H, J = 8 Hz), 7.487.63 (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), 1.942.21 (m, 4H); ESIMS: m/z 401 (M)+. (E)-2-(4-(5-bromopentoxy)-3-methoxybenzylidene)-2,3dihydroinden-1-one (40c) The compound 40c was prepared according to the method described for compound 37a by employing compound 39 (266 mg, 1 mmol), and 1,5dibromopentane (689 mg, 3 mmol). Yield (372 mg, 89%)
1

H NMR (300 MHz, CDCl3): 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

THESIS 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); ESIMS: m/z 416 (M+H)+. 2S)-N-{4-(3-[(E)-3-(4-phenoxy)-1-(3,4,5-trimethoxyphenyl)prop-2en-1-one]propyl)oxy-5-methoxy-2-nitrobenzoyl}-pyrrolidine-2carboxaldehydediethyl thioacetal (41a) To a solution of (2S)-N-[4-hydroxy-5-methoxy2-nitrobenzoyl]pyrrolidine-2carboxal dehydediethylthioacetal (33) (400 mg, 1 mmol) in dry acetone (15 mL) was added, anhydrous K2CO3 (276 mg, 2 mmol), (E)-3-(4-(3(37a) bromopropoxy) phenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one

(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 C;
1

H NMR (300 MHz, CDCl3): 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), 4.624.71 (m, 1H), 4.14.23 (m, 4H), 3.95 (s, 6H), 3.91 (s, 6H), 3.173.31 (m, 2H), 2.622.85 (m, 4H), 2.22.36 (m, 1H), 2.022.16 (m, 3H), 1.911.98 (m, 1H), 1.741.86 (m, 1H), 1.311.41 (m, 6H); ESIMS: m/z 755 (M+H)+. 2S)-N-{4-(4-[(E)-3-(4-phenoxy)-1-(3,4,5-trimethoxyphenyl)prop-2en-1-one]butyl)oxy-5-methoxy-2-nitrobenzoyl}-pyrrolidine-2carboxaldehydediethyl thioacetal (41b) The compound 41b was prepared according to the method described for compound 41a by employing (2S)-N-[4-hydroxy-5-methoxy2nitrobenzoyl]pyrrolidine-2-carboxal dehydediethylthioacetal (33) (400 mg, 99

THESIS 1mmol), 91%) Mp: 175-177 C;

1

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,

H NMR (200 MHz, CDCl3): 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), 4.624.7 (m, 1H), 4.084.22 (m, 4H), 3.95 (s, 6H), 3.9 (s, 6H), 3.163.3 (m, 2H), 2.622.86 (m, 4H), 2.192.36 (m, 1H), 2.012.16 (m, 5H), 1.901.99 (m, 1H), 1.741.87 (m, 1H), 1.301.39 (m, 6H); ESIMS: m/z 791 (M+Na)+. 2S)-N-{4-(5-[(E)-3-(4-phenoxy)-1-(3,4,5-trimethoxyphenyl)prop-2en-1-one]pentyl)oxy-5-methoxy-2-nitrobenzoyl}-pyrrolidine-2carboxaldehydediethylthioacetal (41c) The compound 41c was prepared according to the method described for compound 1mmol), 92%) Mp: 172-174 C;
1

41a and

by

employing

(2S)-N-[4-hydroxy-5-methoxy2-

nitrobenzoyl]pyrrolidine-2-carboxal- dehydediethylthioacetal (33) (400 mg, (E)-3-(4-(5-bromopentoxy)phenyl)-1-(3,4,5trimethoxyphenyl)prop-2-en-1-one (37c) (463 mg, 3 mmol). Yield (721 mg,

H NMR (200 MHz, CDCl3): 7.76 (d, 1H, J = 15.8 Hz), 7.68 (s, 1H), 7.6 (d,

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), 4.614.71 (m, 1H), 4.14.22 (m, 4H), 3.96 (s, 6H), 3.91 (s, 6H), 3.173.31 (m, 2H), 2.612.85 (m, 4H), 2.22.35 (m, 1H), 2.022.16 (m, 5H), 1.91.98 (m, 1H), 1.721.82 (m, 3H), 1.311.4 (m, 6H); ESIMS: m/z 783 (M+H)+. 2S)-N-{4-(3-[(E)-3-(4-[3-methoxyphenoxy])-1-(3,4,5trimethoxyphenyl)prop-2-en-1-one]propyl)oxy-5-methoxy-2nitrobenzoyl}-pyrrolidine-2-carboxaldehydediethyl thioacetal (41d) The compound 41d was prepared according to the method described for compound 41a by employing (2S)-N-[4-hydroxy-5-methoxy2-nitrobenzoyl] 100

THESIS pyrrolidine-2-carboxal dehyde diethylthioacetal (33) (400 mg, 1 mmol), and (E)-3-(4-(3-bromopropoxy)-3-methoxyphenyl)-1-(3,4,5trimethoxyphenyl)prop-2-en-1-one (37d) (465 mg, 3 mmol). Yield (712 mg, 90%) Mp: 165-167 C;
1

H NMR (300 MHz, CDCl3): 7.7 (d, 1H, J = 15.6 Hz), 7.61 (s, 1H), 7.29 (d,

1H, J = 15.6 Hz), 7.147.25 (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), 4.604.74 (m, 1H), 4.04.15 (m, 4H), 3.95 (s, 6H), 3.93 (s, 3H), 3.90 (s, 3H), 3.163.31 (m, 2H), 2.612.88 (m, 4H), 2.162.38 (m, 1H), 1.792.14 (m, 5H), 1.792.14 (m, 5H), 1.281.42 (m, 6H); ESIMS: m/z 785 (M+H)+. 2S)-N-{4-(4-[(E)-3-(4-[3-methoxyphenoxy])-1-(3,4,5trimethoxyphenyl)prop-2-en-1-one]butyl)oxy-5-methoxy-2nitrobenzoyl}-pyrrolidine-2-carboxaldehydediethyl thioacetal (41e) The compound 41e was prepared according to the method described for compound 41a by employing (2S)-N-[4-hydroxy-5-methoxy2-nitrobenzoyl] pyrrolidine-2-carboxal dehydediethylthioacetal (33) (400 mg, 1mmol), and (E)-3-(4-(4-bromobutoxy)-3-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)prop2-en-1-one (37e) (479 mg, 3 mmol). Yield (751 mg, 93%) Mp: 165-167 C;
1

H NMR (200 MHz, CDCl3): 7.77 (d, 1H, J = 15.8 Hz), 7.69 (s, 1H), 7.34 (d,

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), 1.301.40 (m, 6H); ESIMS: m/z 799 (M+H)+.

101

THESIS 2S)-N-{4-(5-[(E)-3-(4-[3-methoxyphenoxy])-1-(3,4,5trimethoxyphenyl)prop-2-en-1-one]pentyl)oxy-5-methoxy-2nitrobenzoyl}-pyrrolidine-2-carboxaldehydediethyl thioacetal (41f) The compound 41f was prepared according to the method described for compound 41a by employing (2S)-N-[4-hydroxy-5-methoxy2-nitrobenzoyl] pyrrolidine-2-carboxal dehydediethylthioacetal (33) (400 mg, 1mmol), and (E)-3-(4-(5-bromopentoxy)-3-methoxyphenyl)-1-(3,4,5trimethoxyphenyl)prop-2-en-1-one (37f) (493 mg, 3 mmol). Yield (765 mg, 94%) Mp: 164-166 C;
1

H NMR (400 MHz, CDCl3): 7.76 (d, 1H, J = 15.8 Hz), 7.68 (s, 1H), 7.33 (d,

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), 1.311.39 (m, 6H); ESIMS: m/z 813 (M+H)+. 7-Methoxy-8-[3-{(E)-3-(4-phenoxy)-1-(3,4,5-trimethoxyphenyl)prop2-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

THESIS 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 Na 2SO4. 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 C;
1

H NMR (300 MHz, CDCl3): 7.77 (d, 1H, J = 15.1 Hz), 7.67 (d, 1H, J = 4.5

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), 4.084.18 (m, 4H), 3.95 (s, 6H), 3.93 (s, 3H), 3.92 (s, 3H), 3.713.84 (m, 2H), 3.523.64 (m, 1H), 2.282.38 (m, 2H), 1.832.05 (m, 4H); ESIMS: m/z 601 (M+H)+. 7-Methoxy-8-[4-{(E)-3-(4-phenoxy)-1-(3,4,5-trimethoxyphenyl)prop2-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 C;
1

H NMR (200 MHz, CDCl3): 7.79 (d, 1H, J = 15.1 Hz), 7.68 (d, 1H, J = 4.5

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), 4.064.17 (m, 4H), 3.96 (s, 6H), 3.94 (s, 3H), 3.93 (s, 3H), 3.783.85 (m, 1H), 3.693.77 (m, 1H), 3.533.65 (m, 1H), 2.282.38 (m, 2H), 1.942.16 (m, 6H); 103

THESIS ESIMS: m/z 615 (M+H)+. 7-Methoxy-8-[5-{(E)-3-(4-phenoxy)-1-(3,4,5-trimethoxyphenyl)prop2-en-1-one} pentoxy]-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c] [1,4]benzodiazepin-5-one (43c) This compound was prepared according to the method described for the 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 mmol), CaCO3 (250 mg, 2.5 mmol) in acetonitrile-water (4:1) to obtain the pure product 43c. Yield (342 mg, 54%). Mp: 120-122 C;
1

H NMR (200 MHz, CDCl3): 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), 4.074.16 (m, 4H), 3.95 (s, 6H), 3.93 (s, 3H), 3.92 (s, 3H), 3.723.85 (m, 2H), 3.523.65 (m, 1H), 2.282.38 (m, 2H), 1.92.13 (m, 6H), 1.61 1.72 (m, 2H); ESIMS: m/z 629 (M+H)+. 7-Methoxy-8-[3-{(E)-3-(4-[3-methoxyphenoxy])-1-(3,4,5trimethoxyphenyl)prop-2-en-1-one}propoxy]-(11aS)-1,2,3,11atetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (43d) This compound was prepared according to the method described for the 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 mmol), CaCO3 (250 mg, 2.5 mmol) in acetonitrile-water (4:1) to obtain the pure product 43d. Yield (361 mg, 57%). Mp: 115-117 C;
1

H NMR (200 MHz, CDCl3): 7.76 (d, 1H, J = 15.4 Hz), 7.67 (d, 1H, J = 4.3

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), 4.034.13 (m, 4H), 3.95 (s, 6H), 3.94 (s, 6H), 3.93 (s, 3H), 3.783.87 (m, 104

THESIS 1H), 3.693.76 (m, 1H), 3.693.76 (m, 1H), 3.533.64 (m, 1H), 2.272.38 (m, 1H), 2.02.11 (m, 1H), 1.841.98 (m, 4H); ESIMS: m/z 631 (M+H)+. 7-Methoxy-8-[4-{(E)-3-(4-[3-methoxyphenoxy])-1-(3,4,5trimethoxyphenyl)prop-2-en-1-one}butoxy]-(11aS)-1,2,3,11atetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (43e) This compound was prepared according to the method described for the 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 mmol), CaCO3 (250 mg, 2.5 mmol) in acetonitrile-water (4:1) to obtain the pure product 43e. Yield (362 mg, 57%). Mp: 115-117 C;
1

H NMR (300 MHz, CDCl3): 7.77 (d, 1H, J = 15.4 Hz), 7.68 (d, 1H, J = 4.3

Hz), 7.51 (s, 1H), 7.34 (d, 1H, J = 15.4 Hz), 7.27 (s, 2H), 7.23 (dd, 1H, J = 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), 4.054.16 (m, 4H), 3.96 (s, 6H), 3.94 (s, 6H), 3.93 (s, 3H), 3.763.85 (m, 1H), 3.693.75 (m, 1H), 3.533.65 (m, 1H), 2.262.38 (m, 2H), 1.922.07 (m, 4H), 1.611.79 (m, 2H); ESIMS: m/z 645 (M+H)+. 7-Methoxy-8-[5-{(E)-3-(4-[3-methoxyphenoxy])-1-(3,4,5trimethoxyphenyl)prop-2-en-1-one}pentoxy]-(11aS)-1,2,3,11atetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (43f) This compound was prepared according to the method described for the 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 mmol), CaCO3 (250 mg, 2.5 mmol) in acetonitrile-water (4:1) to obtain the pure product 43f. Yield (345 mg, 52%). Mp: 113-114 C;

105

THESIS
1

H NMR (200 MHz, CDCl3): 7.76 (d, 1H, J = 15.4 Hz), 7.66 (d, 1H, J = 4.3

Hz), 7.51 (s, 1H), 7.32 (d, 1H, J = 15.4 Hz), 7.28 (s, 2H), 7.23 (dd, 1H, J = 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), 4.074.15 (m, 4H), 3.95 (s, 6H), 3.94 (s, 6H), 3.93 (s, 3H), 3.773.86 (m, 1H), 3.683.76 (m, 1H), 3.523.64 (m, 1H), 2.272.38 (m, 2H), 22.11 (m, 1H), 1.92.08 (m, 6H), 1.62 1.71 (m, 2H); ESIMS: m/z 659 (M+H)+. 2S)-N-{4-(3-[(E)-2-(4-{3-methoxybenzylidene}oxy)-2,3dihydroinden-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-methoxy2-nitrobenzoyl] pyrrolidine-2-carboxal dehyde diethylthioacetal (33) (400 mg, 1 mmol), and (E)-2-(4-(3-bromopropoxy)-3-methoxybenzylidene)-2,3-dihydroinden-1-one (40a) (387 mg, 1 mmol). Yield (655 mg, 92%)
1

H NMR (200 MHz, CDCl3): 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), 1.281.41 (m, 6H); ESIMS: m/z 707 (M+H)+. 2S)-N-{4-(4-[(E)-2-(4-{3-methoxybenzylidene}oxy)-2,3dihydroinden-1-one]butyl)oxy-5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehydediethylthioacetal (44b) The compound 44b was prepared according to the method described for compound 41a by employing 2S)-N-[4-hydroxy-5-methoxy2-nitrobenzoyl] pyrrolidine-2-carboxal dehyde diethylthioacetal (33) (400 mg, 1 mmol), and (E)-2-(4-(4-bromobutoxy)-3-methoxybenzylidene)-2,3-dihydroinden-1-one (40b) (401 mg, 1 mmol). Yield (660 mg, 91%)

106

THESIS
1

H NMR (200 MHz, CDCl3): 7.86 (d, 1H, J = 7.5 Hz), 7.64 (s, 1H), 7.57.61

(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), 4.64.7 (m, 1H), 4.124.3 (m, 4H), 4 (s, 2H), 3.87 (s, 6H), 3.153.3 (m, 2H), 2.612.82 (m, 4H), 2.192.35 (m, 1H), 2.012.18 (m, 4H), 2.182.2 (m, 1H), 1.641.84 (m, 2H), 1.261.39 (m, 6H); ESIMS: m/z 721 (M+H)+. 2S)-N-{4-(5-[(E)-2-(4-{3-methoxybenzylidene}oxy)-2,3dihydroinden-1-one]pentyl)oxy-5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehydediethylthioacetal (44c) The compound 44c was prepared according to the method described for compound 41a by employing 2S)-N-[4-hydroxy-5-methoxy2-nitrobenzoyl] pyrrolidine-2-carboxal dehyde diethylthioacetal (33) (400 mg, 1 mmol), and (E)-2-(4-(5-bromopentoxy)-3-methoxybenzylidene)-2,3-dihydroinden-1-one (40c) (315 mg, 3 m mol). Yeild (686 mg, 93%)
1

H NMR (300 MHz, CDCl3): 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), 2.192.35 (m, 1H), 1.88 2.16 (m, 6H), 1.65 1.86 (m, 3H), 1.291.41 (m, 6H); ESIMS: m/z 735 (M+H)+. 7-Methoxy-8-[3-{(E)-2-(4-{3-methoxybenzylidene}oxy)-2,3dihydroinden-1-one} propoxy]-(11aS)-1,2,3,11a-tetrahydro-5Hpyrrolo[2,1-c][1,4]benzodiazepine-5-one(46a) This compound was prepared according to the method described for the compound 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

THESIS mmol), CaCO3 (250 mg, 2.5 mmol) in acetonitrile-water (4:1) to obtain the pure product 46a. Yield (285 mg, 51%). Mp: 105-107 C;
1

H NMR (300 MHz, CDCl3): 7.9 (d, 1H, J = 7.2 Hz), 7.557.71 (m, 4H), 7.51

(s, 1H), 7.387.47 (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), 4.094.25 (m, 4H), 4.04 (s, 2H), 3.93 (s, 6H), 3.653.86 (m, 2H), 3.483.63 (m, 1H), 2.232.41 (m, 2H), 1.962.18 (m, 4H); ESIMS: m/z 553 (M+H)+. 7-Methoxy-8-[4-{(E)-2-(4-{3-methoxybenzylidene}oxy)-2,3dihydroinden-1-one} butoxy]-(11aS)-1,2,3,11a-tetrahydro-5Hpyrrolo[2,1-c][1,4]benzodiazepine-5-one(46b) This compound was prepared according to the method described for the 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 mmol), CaCO3 (250 mg, 2.5 mmol) in acetonitrile-water (4:1) to obtain the pure product 46b. Yield (316 mg, 55%). Mp: 106-107 C;
1

H NMR (200 MHz, CDCl3): 7.92 (d, 1H, J = 7.2 Hz), 7.567.72 (m, 4H),

7.52 (s, 1H), 7.387.48 (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), 4.114.28 (m, 4H), 4.03 (s, 2H), 3.93 (s, 6H), 3.673.86 (m, 2H), 3.493.66 (m, 1H), 2.252.39 (m, 2H), 1.942.19 (m, 5H), 1.651.8 (m, 1);
13

C NMR (75 MHz, CDCl3): 194.28, 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; ESIMS: m/z 567 (M+H)+.

108

THESIS 7-Methoxy-8-[5-{(E)-2-(4-{3-methoxybenzylidene}oxy)-2,3dihydroinden-1-one} pentoxy]-(11aS)-1,2,3,11a-tetrahydro-5Hpyrrolo[2,1-c][1,4]benzodiazepine-5-one(46c) This compound was prepared according to the method described for the compound 43a employing 44c (734 mg, 1.0 mmol) which reduction with SnCl2.2H2O (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 mmol), CaCO3 (250 mg, 2.5 mmol) in acetonitrile-water (4:1) to obtain the pure product 46c. Yield (341 mg, 58%). Mp: 106-108 C;
1

H NMR (300 MHz, CDCl3): 7.91 (d, 1H, J = 7.2 Hz), 7.567.71 (m, 4H),

7.52 (s, 1H), 7.387.48 (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), 4.14.26 (m, 4H), 4.03 (s, 2H), 3.93 (s, 6H), 3.653.85 (m, 2H), 3.493.65 (m, 1H), 2.232.39 (m, 2H), 1.922.13 (m, 6H), 1.63 1.75 (m, 2H); ESIMS: m/z 581 (M+H)+.

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 7.000.01) containing CT-DNA, (100 M in phosphate) and the PBD (20 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 37

C 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 1 C min-1 in the 40 90

C range. DNA helix-coil transition temperatures

(Tm) were determined from the maxima in the d(A260)/dT derivative plots. Results for each compound are shown as mean standard derivation from

109

THESIS 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 Tm = Tm(DNA+PBD) m(DNA alone), where T the Tm 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 L at plating densities depending on the doubling time of individual cell lines. The microtiter plates were incubated at 37 C, 5% CO2, 95% air and 100% relative humidity for 24 h prior to addition of experimental drugs. Aliquots of 10 L of the drug dilutions were added to the appropriate microtiter wells already containing 90 L of cells, resulting in the required final drug concentrations. Each compound was evaluated for four concentrations (0.1, 1, 10 and 100 M) and each was done in triplicate wells. Plates were incubated further for 48 h, and assay was terminated by the addition of 50 L of cold trichloro acetic acid (TCA) (final concentration, 10% TCA) and plates were again incubated for 60 min at 4

C. The plates

were washed five times with tap water and air-dried. Sulforhodamine B (SRB) solution (50 L) 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

THESIS 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.

2.8. 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 (Cancer Chemother.) 1982, 9, 924.

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THESIS 2. 3. 4. 5. 6. Tsugaya, M.; Washida, H.; Hirao, N.; Hachisuka, Y.; Sakagami, H.; Iwase, Y. Hinyokika Kiya. 1986, 32, 1443. Dervan, P. B. Science 1986, 232, 464. Thurston, D. E.; Thompson, A. S. Chem. Br. 1990, 26, 767. White, S.; Swezyk, J. W.; Turner, J. M.; Baird, E. E.; Dervan, P. B. Nature 1998, 391, 468. Thurston, D. E. Advances in the study of Pyrrolo[2,1-c] [1,4]benzodiazepine (PBD) Antitumour Antibiotics. In Molecular Aspects of Anticancer Drug DNA Interactions; Eds, Neidle, S and Waring, M. J., Eds.; The Macmillan Press Ltd.: London, 1993, Vol. 1, 54. 7. Thurston, D. E.; Bose, D. S.; Thompson, A. S.; Howard, P. W.; Leoni, A.; Croker, S. J.; Jenkins, T. C.; Neidle, S.; Hartley, J. A.; Hurley, L. H. J. Org. Chem. 1996, 61, 8141. 8. 9. 10. Gregson, S. J.; Howard, P. W.; Jenkins, T. C.; Kelland, L. R.; Thurston, D. E. Chem. Commun. 1999, 797. Thurston, D. E.; Morris, S. J.; Hartley, J. A. Chem. Commun. 1996, 563. (a) Modzelewska, A.; Pettit, C.; Achanta, G.; Davidson, N. E.; Huang, P.; Khan, S. R. Bioorg. Med. Chem. 2006, 14, 3491; (b) Lawrence, N. J.; Patterson, R. P.; Ooi, L-. L.; Cook, D.; Ducki, S. Bioorg. Med. Chem. Lett. 2006, 14, 3491; (c) LeBlanc, R.; Dickson, T.; Brown, T.; Stewart, M.; Pati, H. N.; VanDerveer, D.; Arman, H.; Harris, J.; Pennington, W.; Holt, Jr-. H. L.; Lee, M. Bioorg. Med. Chem. 2005, 13, 6025; (d) Koteswara Rao, Y.; Fang, S-. H.; Tzeng, Y-. M. Bioorg. Med. Chem. 2004, 12, 2679; (e) Lawrence, N. J.; Rennison, D.; Woo, M.; McGown, A. T.; Hadfield, J. A. Bioorg. Med. Chem. Lett. 2001, 11, 51; (f) Ducki, S.; Forrest, R.; Hadfield, J. A.; Kendall, A.; Lawrence, N. J.; McGown, A. T.; Rennison, D. Bioorg. Med. Chem. Lett. 1998, 8, 1051. 11. Park, E. J.; Park, H. R.; Lee, J. S.; Kim, J. Planta Med. 1998, 64, 464.

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THESIS 12. DeVincenzo, R.; Scambia, G.; Panici, B. P.; Ranelletti, F. O.; Bonanno, G.; Ercoli, A.; Monache, D. F.; Ferrari, F.; Piantelli, M.; Mancuso, S. Anticancer Drug Des. 1995, 10, 481. 13. 14. 15. Rui, H. J. Cell. Biochem. 1997, 67, 7. Claude, A. C.; Jean, C. L.; Patric, T.; Christelle, P.; Gerard, H.; Albert, J. C.; Jean, L. D. Anticancer Res. 2001, 21, 3949. Rosa, D. V.; Cristiano, F.; Mariagrazia, D.; Cristiana, G.; Antonella, R.; Ezio, B.; Paolo, M.; Piero, V.; Federica, B.; Franco, O. R.; Salvatore, M.; Giovanni, S. Cancer Cheomother. Pharmacol. 2000, 46, 305. 16. 17. 18. 19. 20. 21. Kumar, S. K.; Erin, H.; Catherine, P.; Halluru, G.; Davidson, N. E.; Khan, S. R. J. Med. Chem. 2003, 46, 2813. Anto, R. J.; Sukumaran, K.; Kuttan, G.; Rao, M. N. A.; Subbaraju, V.; Kuttan, R. Cancer Lett. 1995, 97, 33. Ko, H. H.; Tsao, L. T.; Yu, K. L.; Liu, C. T.; Wang, J. P.; Lin, C. N. Bioorg. Med. Chem. 2003, 11, 105. Matsuda, H.; Morikawa, T.; Ando, S.; Iwao, T.; Masayuki, Y. Bioorg. Med. Chem. 2003, 11, 1995. Hsieh, H. K.; Tsao, L. T.; Wang, J. P.; Lin, C. N. J. Pharm. Pharmacol. 2000, 52, 163. Parmer, V. S.; Sharma, N. K.; Husain, M.; Watterson, A. C.; Kumar, J.; Samuelson, L. A.; Ashok, L. C.; Prasad, A. K.; Kumar, A.; Malhotra, S.; Kumar, N.; Jha, A.; Singh, A.; Singh, I.; Himanshu; Vats, A.; Shakil, N. A.; Trikha, S.; Mukherjee, S.; Sharma, S. K.; Singh, S. K.; Kumar, A.; Jha, H. N.; Olsen, C. E.; Stove, C. P.; Bracke, M. E.; Mareel, M. M. Bioorg. Med. Chem. 2003, 11, 913. 22. 23. Lin, Y. M.; Zhou, Y.; Flavin, M. T.; Zhou, L. M.; Nie, W.; Chen, F. C. Bioorg. Med. Chem. 2002, 10, 2795. Lopez, S. N.; Castelli, M. V.; Zacchino, S. A.; Dominguez, J. N.; Lobo, G.; Jaime, C. C.; Cortes, J. C. G.; Ribas, J. C.; Devia, C.; Ana, M. R.; Ricardo, D. E. Bioorg. Med. Chem. 2001, 9, 1999.

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THESIS 24. (a) Lawrence, N. J.; Patterson, R. P.; Ooi, L.; Cook, D.; Ducki, S. Bioorg. Med. Chem. Lett. 2006, 16, 5844; (b) Ducki, S.; Forrest, R.; Hadfield, J. A.; Kandall, A.; Lawrence, N. J.; McGown, A. T.; Rennison, D. Bioorg. Med. Chem. Lett. 1998, 8, 1051; (c) Ducki, S.; Woo, D. R. M.; Kendall, A.; Chabert, J. F. D.; McGown, A. T.; Lawrence, N. J. Bioorg. Med. Chem. 2009, 17, 7698. 25. 26. 27. 28. Pettit, G. R.; Singh, S. B.; Hamel, E.; Lin, C. M.; Alberts, D. S.; GarciaKendall, D. Experientia 1989, 45, 209. Lin, C. M.; Ho, H. H.; Pettit, G. R.; Hamel, E. Biochemistry 1989, 28, 6984. Jordan, A.; Hadfield, J. A.; Lawrence, N. J.; McGown, A. T. Med. Res. Rev. 1998, 18, 259. (a) Lin, Y. M.; Zhou, Y. Flavin, M. T.; Zhou, L.M.; Nie, W. PCT Int. Appl. 2003, 121; (b) Soloway, A. H.; Tjarks, W.; Barnum, B. A.; Rong, F. G.; Barth, R. F.; Codogni, I. M.; Wilson, G. Chem. Rev. 1998, 98, 1515; (c) Sedlacek, H. H.; Czech, J.; Naik, R.; Kaur, G.; Worland, P.; Losiewicz, M.; Parker, B.; Carlson, B.; Smith, A.; Senderowicz, A.; Sausville, E. Int. J. Oncol. 1996, 9, 1143; (d) Cushman, M.; Zhu, H.; Geahlen, R. L.; Kraker, A. J. J. Med. Chem. 1994, 37, 3353; (e) Campbell, D. R.; Kurzer, M. S. J. Steroid Biochem. Mol. Biol. 1993; (f) Ibrahim, A. R.; Abul-Haji, Y. J. J. Steroid Biochem. Mol. Biol. 1990, 37, 257. 29. 30. 31. Kumar, S. K.; Hager, E.; Pettit, C.; Gurulingappa, H.; Davidson, N. E.; Khan, S. R. J. Med. Chem. 2003, 46, 2813. Myung, J.; Kim, K. B.; Crews, C. M. Med. Res. Rev. 2001, 21, 245. (a) Bandgar, B. P.; Gawande, S. S.; Bodade, R. G.; Totre, J. V.; Khobragade, C. N. Bioorg. Med. Chem. 2010, 18, 1364; (b) Rao, Y. K.; Fang, S.; Tzeng, Y. Bioorg. Med. Chem. 2009 17, 7909; (c) Kong, Y.; Wang, K.; Edler, M. C.; Hamel, E.; Mooberry, S. L.; Paige, M. A.; Brown, M. L. Bioorg. Med. Chem. 2010, 18, 971. 32. Kumar, D.; Kumar, N. M.; Akamatsu, K.; Kusaka, E.; Harada, H.; Ito, T. Bioorg. Med. Chem. Lett. 2010, 20, 3916. 114

THESIS 33. (a) Lawrence, N. J.; McGown, A. T.; Ducki, S.; Hadfield, J. A. Anti-Cancer Drug Des. 2000, 15, 135; (b) Peyrot, V.; Leynadier, D.; Sarrazin, M.; Briand, C.; Menendez, M.; Laynez, J.; Andreu, J. M. J. M. Biochemistry. 1992, 31, 11125 34. (a) Aggarwal, B. B.; Kumar, A; Bharti, A. C. Anticancer Res. 2003, 23, 363; (b) Aggarwal, B. B.; Sundaram, C; Malani, N; Ichikawa, H. AdV. Exp. Med. Biol. 2007, 595, 1. 35. Adams, B. K.; Ferstl, E. M.; Davis, M. C.; Herold, M.; Kurtkaya, S.; Camalier, R. F.; Hollingshead, M. G.; Kaur, G.; Sausville, E. A.; Rickles, F. R.; Snyder, J. P.; Liotta, D. C.; Shoji, M. Bioorg. Med. Chem. 2004, 12, 3871. 36. (a) Modzelewska, A.; Pettit, C.; Achanta, G.; Davidson, N. E.; Huang P.; Khan S. R. Bioorg. Med. Chem. 2006, 14, 3491; (b) Lagisetty, P.; Vilekar, P.; Sahoo, K.; Anant, S.; Awasthi, V. Bioorg. Med. Chem. 2010, 18, 6109. 37. (a) Dimmock, J. R.; Zello, G. A.; Oloo, E. O.; Quail, J. W.; Kraatz, H. B.; Perjesi, P.; Aradi, F.; Takacs-Novak, K.; Allen, T. M.; Santos, C. 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

THESIS 41. 42. 43. 44. 45. 46. 47. Damayanthi, Y.; Reddy, B. S. P.; Lown, J. W. J. Org.Chem. 1999, 64, 290.. 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 Kamal, A.; Laxman, E.; Reddy, P. S. M. M. Tetrahedron Lett. 2000, 41, 7743. Kamal, A.; Shankaraiah, N.; Devaiah, V.; Reddy, K. L. Tetrahedron Lett. 2006, 47, 6553. Kamal, A.; Devaiah, V.; Reddy, K. L.; Kumar, M. S. Bioorg. Med. Chem. 2005, 13, 2021. 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. 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. 49. Kamal, A.; Shankaraiah, N.; Devaiah, V.; Reddy, K. L.; Juvekar, A.; Kurian, N.; Zingde, S. Bioorg. Med. Chem. Lett. 2008, 18, 1468. 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.; Srivastava, H. K.; Sastry, G. N.; Juvekar, A.; Sen, S.; Zingde, S. Bioorg. Med. Chem. 2010, 18, 526. 50. 51. 52. Thurston, D. E.; Murthy, V. S.; Langley, D. R.; Jones, G. B. Synthesis. 1990, 81. Puvvada, M. S.; Hartley, J. A.; Jenkins, T. C.; Thurston, D. E. Nucleic Acids Res. 1993, 21, 3671. 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.

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THESIS

CHAPTER-III SYNTHESIS AND BIOLOGICAL EVALUATION OF COMBRETASTATIN DERIVATIVES AS ANTICANCER AGENTS

117

THESIS

3.1. INTRODUCTION
Microtubules are cytoskeletal structures that are formed by self assembly of and 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 , 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

THESIS paclitaxel and vinblastine, are in clinical use for various types of cancer2 (Figure 2).
MeO MeO OMe O OMe Colchicine (1) OH O O O O MeO OMe podophylotoxin (3) OMe O NH MeO OMe OMe Combretastatin A-4 (2a) OH MeO

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-soluble prodrug, 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

THESIS 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 Combretastatin A-4P (2b) OPO3Na2 MeO MeO OMe OMe 4a, R = NH2 4b, R = NH-CO-CH-(NH2)(CH2OH) R

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 shown to tolerated inhibit dose, the offering and a wide therapeutic of window. blood The compound 2a as well as being a potent inhibitor of colchicines binding is also growth development 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 or metabolism, 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

THESIS combretastatin-induced cell death. This indicates that apoptosis is a secondary mechanism of death in a small proportion of cells treated with CA4.30
OH NH2 HO

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 Bring 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 previous comparative 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

THESIS 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.
O MeO MeO OMe 7 OH OMe MeO MeO OMe 8 (SD400) O OH OMe

Figure 5 The chalcone derivatives of combretastatin have showed exciting potential as anticancer agents. Sylvie Ducki and co-workers synthesized trimethoxy substituted chalcones45 7 and 8, which possess potential anticancer 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

THESIS 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).
N MeO MeO OMe 9a, R = H 9b, R = Acetyl N R OH OMe

Figure 6 Johnson and coworkers49 synthesized N-acetylated and non-acetylated 3,4,5tri- 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 M 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 M 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 2aminobenzothiazoles with combretastatin and its chalcone, pyrazoline derivatives with amide bond with a view to evaluate more potent anticancer

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THESIS 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. SYNTHESIS OF COMBRETASTATIN AND ITS DERIVATIVES

The precursor (Z)-2-[(2-methoxy-5-(3,4,5-

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.
CH2OH (ii) OTBDMS OMe 11 (iii) OTBDMS OMe 12

CHO (i) OH OMe 10

CHO

CH2Br OTBDMS OMe 13 (iv) CH2PPh3Br OTBDMS OMe 14

MeO MeO OMe 16 (vii)

OH OMe

(vi) MeO MeO OMe 15

OTBDMS (v) OMe

O MeO MeO OMe 17 O OMe OEt (viii) MeO MeO OMe 18 O

O OH

OMe

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, K2CO3, DMF, 12h; viii) LiOH.H2O, THF, H2O, 12h.

124

THESIS 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 -bromo ethyl acetate in the presence of K2CO3 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. SYNTHESIS OF CHALCONE DERIVATIVE OF COMBRETASTATIN

The preparation of chalcone derivative 22 was carried out by synthetic sequence trimethoxy illustrated in Scheme-2. 19 with Claisen-Schmidt condensation of acetophenone 4-methoxy-3-hydroxybenzaldehyde

(isovaniline) using ethanol as solvent in the presence of aqueous KOH gives trimethoxychalcones 20. This upon etherification with -bromo ethyl acetate in the presence of K2CO3 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).
O MeO + MeO OMe 19 OH OMe CHO (i) MeO MeO OMe 20 (ii) O OH OMe

O MeO MeO OMe 22 O

O OH (iii) MeO MeO

O O 21

O OEt

OMe

OMe

OMe

Scheme 2. Reagents and conditions: i) aq. KOH, ethanol, 6h; ii) 2-bromoethyl acetate, K2CO3, DMF, 12h; iii) LiOH. H2O, THF, H2O, 14h

3.3.3. SYNTHESIS OF PYRAZOLINE DERIVATIVE OF COMBRETASTATIN

The preparation of pyrazoline derivative 25 has been carried out by synthetic sequence illustrated in Scheme-3. Cyclization of trimethoxy 125

THESIS chalcone 20 with hydrazine hydrate in acetic acid under reflux gives pyrazoline derivative 23. This upon etherification with -bromoethyl acetate in the presence of K2CO3 gives the ester 24. The ester compound on hydrolysis with LiOH affords pyrazoline acid 2-(5-(1-acetyl-3-(3,4,5-5-yl)-2O O MeO MeO OMe 20 OH OMe (i) MeO MeO OMe 23 N N OH OMe

trimethoxyphenyl)-4,5-dihydro-1H-pyrazol methoxyphenoxy)aceticacid 25 (Scheme 3).

(ii)

O N MeO MeO OMe 25 N O OMe O OH (iii) N MeO MeO OMe

O N O OMe 24 O OEt

Scheme 3. Reagents and conditions: i) NH2NH2.H2O, Acetic acid, reflux, 14h; ii) 2bromoethyl acetate, K2CO3, DMF, 12h; iii) LiOH.H2O, THF, H2O

3.3.4. SYNTHESIS OF COMBRETASTATIN-BENZOTHIAZOLE ANALOGUES

The synthesis of combretastatin-benzothiazole derivatives (27a-i) is outlined in Scheme 4. Combretastatin acid 18 undergoes amide bond formation EDCI/HOBT with in different 2-amino benzothiazoles affords in the presence of dichloromethane 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

THESIS dichloromethane affords chalcone-benzothiazole analogues 28a-i (Scheme5).

O MeO MeO OMe 18 (i) 27a, R = -H 27b, R = -NO2 27c, R = -F 27d, R = -Cl 27e, R = -OMe 27f, R = -OCF3 27g, R = -Me 27h, R = -CF3 27i, R = -OEt O OMe OH + H2N N S 26 R

O MeO MeO OMe O OMe N H

N S 27 a-i

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

O MeO MeO OMe 22 O

O OH + H2N N S 26 R

OMe

(i) 28a, R = -H 28b, R = -NO2 28c, R = -F 28d, R = -Cl 28e, R = -OMe 28f, R = -OCF3 28g, R = -Me 28h, R = -CF3 28i, R = -OEt

O MeO MeO OMe 28 O

O N H

N S

OMe

Scheme 5. Reagents and conditions: i) EDCI/HOBT, DCM, 14-16h

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

THESIS dichloromethane affords pyrazoline-benzothiazole analogues 29a-i (Scheme6).

O N MeO MeO OMe 25 (i) O N MeO MeO OMe 29 N O OMe O N H N S R 29a, R = -H 29b, R = -NO2 29c, R = -F 29d, R = -Cl 29e, R = -OMe 29f, R = -OCF3 29g, R = -Me 29h, R = -CF3 29i, R = -OEt N O OMe O OH + H2N N S 26 R

Scheme 6. Reagents and conditions: i) EDCI/HOBT, DCM, 14-16h

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 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

THESIS Table 1. All the compounds exhibited potential anticancer activity with GI50 values ranging 0.019-30.7 . The combretastatin-benzothiazole analogues (27a, 27c, 27e, 27h and 27f) exhibited potential anticancer activity with GI50 values ranging 0.01918.6 . 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 0.01911 M. This compound was found to show highest activity against MDA-MB-435 (melanoma) cancer cell line with a GI50 value of 0.019 M. The trimethoxychalcone-benzothiazole analogues (28a, 28c, 28e, 28h and 28f) also exhibited significant anticancer activity with GI50 values ranging 0.330.7 . The anticancer activity (GI50) of the compound 28a is in the range of 0.36.42 M. This compound was found to show highest activity against MDA-MB-435 (melanoma) cancer cell line with a GI50 value of 0.3 M. The compound 28f which having trifluoromethoxy group at 6-position of benzothiazole ring also showed significant activity (GI50, 0.39.52 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/Cell Line
a

GI50 values (M)

27f 28a 28c 28e 28h 28 f 2.5 0.4 29h

27a

27c

27e

27h

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

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

THESIS
5.1 1 8 na HOP-62 HOP-92 NCI-H226 NCI-H23 NCI-H322M NCI-H460 NCI-H522 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 2.1 2 2.1 4 3.3 5 2.1 5 2.7 7 2.4 3 0.6 7 6.3 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 7 6.1 8 1.4 3 0.4 3.6 6 1.5 1 1.0 8 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 3.94 na 2.39 9.26 na na na 2.2 1.7 2.22 3.76 3.67 na 2.71

130

THESIS
6 0.04 8 8 0.3 4 0.7 0.4 0.06 Melanoma LOX IMVI MALME-3M M14 MDA-MB-435 SK-MEL-2 SK-MEL-28 SK-MEL-5 UACC-257 UACC-62 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 8.46 na 1.76 8.69 4.05 4.17 na 4.33 1.16 4.07 4.41 2.68 na 5.93 4.01 na 3.58 1.84 3.98 3 2.84 40 3.9 4.77 na 4.36 1.93 3.29 4.57 3.26 na 5.11 0.52 6.42 3.29 0.3 5.39 4.61 1.76 1.37 7.87 3.37 27.7 10.7 1.93 na 24.1 4.49 na 33.6 2.59 9.62 3.28 1.51 24.2 14.4 4.56 20.4 14.5 2.88 8.75 3.31 1.16 30 9.47 2.88 na 22.7 7 2.6 9 1.7 2 0.3 4.9 7 3.3 1 2.0 3 8.0 8 9.5 2 2.5 4 Ovarian IGROV1 OVCAR-3 OVCAR-4 OVCAR-5 OVCAR-8
NCI/ADR-RES

3.36 6.44 na 3.08 na 3.56 na 3.77 na

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

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

SK-OV-3

131

THESIS
2.2 6 Renal 786-0 A498 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 7.4 7.12 6.25 3.33 2.78 6.29 4.61 9.89 2.56 7.52 3.5 7.77 3.2 2.5 5.75 7.54 4.31 6.96 2.81 4.82 3.1 2.27 4.43 7.03 3.59 na 3.61 8.31 4.6 2.65 6.42 18.6 4.37 4.12 22.6 3.46 3.78 1.83 3.02 3.3 3.07 24.7 na 12.4 30.7 8.5 17.4 20.9 6.83 19.4 na 4.94 25.1 2.37 5.19 16.4 5.31 24.6 na 7.48 10.9 2.6 5.41 15.6 7.8 1.6 2 2.2 1.5 8 1.7 4 2.0 8 1.6 1 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 1.3 2 Breast MCF7
MDA-MB-231

3.58 2.78 2.48 2.17 2.09 3.31 2.85 1.93

2.92 1.82

0.05 1 0.19 na 0.41 1.0 0.1

2.3 9.34 >10 0 16.7 3.39 3.68

2.0 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 3.0 8 2.7 1 3.0 8 3.5 2 4.02 3.11 2.48 3.55 2.75 6.12

HS 578T BT-549 T-47D MDA-MB-468

Values are reported as GI50, the M concentration of the compound required to cause 50% inhibition of cell growth. na = not active(GI50 >50 M )
a

The pyrazoline-benzothiazole analogue (29h) exhibited promising anticancer activity with GI50 values ranging 1.55-28.2 . This compound 132

THESIS was found to show highest activity against SNB-75 (CNS) cancer cell line with a GI50 value of 1.55 M. In comparison, the anticancer activity exhibited by combretastatin and its chalcone, pyrazoline derivatives attached to aminobenzothiazole, the combretastatin moiety attached to benzothiazole analogues 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 M concentration.
15.2 13.2
Fluorescence units

Real time kinetic graph of tubulin polymerization at 3 M concentration

Control 27f 28a 28c 28f 28h Noco

11.2 9.2 7.2 5.2 3.2 1.2 13 22 28 43 49 58 1 4 7 10 16 19 25 31 34 37 40 46 52

Time (min)

55

-0.8

Podo

133

THESIS
70 %Inhibition (Tubulin polymerization) 60 50 40 30 20 10 0 -10
Control 27a 27b 27c 27d 27e 27f 27g 27h 27i 28a 28b 28c 28d 28e 28f 28g 28h Nocod Podo

Effect of compounds on tubulin polymerization at 3 M concentration

Compounds

Figure 6: Effect of compounds on tubulin polymerization: Tubulin polymerization was monitored by the increase in fluorescence at 340 nm (excitation) and 460 nm (emission) for 1 hrs at 37 oC. All the compounds were included at a final concentration of 3 M. 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

THESIS 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%).
1

H NMR (300 MHz, CDCl3): 9.71 (s, 1H), 7.317.38 (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); ESIMS: m/z 267 (M+H)+. (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 to quench the excess reagent and concentrated the reaction mass, extracted with ethyl acetate. The organic layer was dried over Na 2SO4 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%).
1

H NMR (200 MHz, CDCl3): 6.786.85 (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); ESIMS: m/z 269 (M+H)+. (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

THESIS 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}-4methoxybenzyltriphenylphosphonium 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%).
1

H NMR (200 MHz, CDCl3): 7.748.05 (m, 15H), 6.977.11 (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-(3,4,5trimethoxystyryl)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

THESIS 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%).
1

H NMR (300 MHz, CDCl3): 6.766.82 (dd, 1H, J = 8.3, 2.2 Hz), 6.73 (d, 1H, J

= 2.2 Hz), 6.69 (d, 1H, J = 8.3 Hz), 6.396.46 (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); ESIMS: m/z 431 (M+H)+. (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 ethyl acetate-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 product 12. Yield (250 mg, 79% yield). Mp: 115-116 C;
1

H NMR (200 MHz, CDCl3): 6.92 (d, 1H, J = 2.2 Hz), 6.776.83 (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); ESIMS: m/z 317 (M+H)+. 137

THESIS (Z)-ethyl trimethoxystyryl)phenoxy)acetate( 17) To a solution of compound 16 (316 mg, 1 mmol) in dry DMF (15 mL) was added, anhydrous K2CO3 (276 mg, 2 mmol), -bromoethylacetate (183 mg, 1.1 mmol) and the mixture was stirred at room temperature for 24 hours. The reaction was monitored by TLC using ethyl acetate-hexane (6:4). After completion of the reaction as indicated by the TLC, K2CO3 was removed by 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 C;
1

2-(2-methoxy-5-(3,4,5-

H NMR (300 MHz, CDCl3): 6.836.89 (dd, 1H, J = 8.3, 1.5 Hz), 6.75 (d, 1H, J

= 8.3 Hz), 6.7 (d, 1H, J = 1.5 Hz), 6.46.46 (m, 3H), 6.47 (d, 1H, J = 12 Hz), 4.44 (s, 2H), 4.114.2 (q, 2H), 3.85 (s, 3H), 3.8 (s, 3H), 3.69 (s, 6H), 1.24 (t, 3H, J = 7.5, 6.7 Hz); ESIMS: m/z 403 (M+H)+.

(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

THESIS 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 C;
1

H NMR (200 MHz, CDCl3): 7.96 (bs, 1H), 6.846.9 (dd, 1H, J = 8.3, 1.5 Hz),

6.77 (d, 1H, J = 8.3 Hz), 6.72 (d, 1H, J = 1.5 Hz), 6.316.49 (m, 4H), 4.47 (s, 2H), 3.86 (s, 3H), 3.8 (s, 3H), 3.69 (s, 6H); ESIMS: m/z 375 (M+H)+. (E)-3-(3-hydroxy-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)prop2-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 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 20. Yield (300 mg, 86%). Mp: 133-134 C;
1

H NMR (300 MHz, CDCl3): 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); ESIMS: m/z 345 (M+H)+. (E)-Ethyl-2-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1enyl)phenoxy) acetate (21) To a solution of compound 20 (344 mg, 1 mmol) in dry DMF (15 mL) was added, anhydrous K2CO3 (276 mg, 2 mmol), -bromoethylacetate (183 mg, 1.1 mmol) and the mixture was stirred at room temperature for 12 hours.

139

THESIS The reaction was monitored by TLC using ethyl acetate-hexane (6:4). After completion of the reaction as indicated by the TLC, K2CO3 was removed by 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 (6:4) to afford pure compound 21 as yellow solid. Yield (395 mg, 91%) Mp: 129-130 C;
1

H NMR (300 MHz, CDCl3): 7.73 (d, 1H, J = 15.6 Hz), 7.36 (d, 1H, J = 15.6

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); ESIMS: m/z 431 (M+H)+. (E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1enyl)phenoxy)aceticacid (22) To a solution of compound 21 (431 mg, 1 mmol) in THF (15 mL) and water (2 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 Na 2SO4 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 C;

140

THESIS
1

H NMR (500 MHz, CDCl3+DMSO D6): 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)-4,5dihydropyrazol-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 C;
1

H NMR (300 MHz, CDCl3): 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); ESIMS: m/z 401 (M+H)+. Ethyl-2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1Hpyrazol-5-yl)-2-methoxyphenoxy)acetate (24) To a solution of compound 23 (400 mg, 1 mmol) in dry DMF (15 mL) was added, anhydrous K2CO3 (276 mg, 2 mmol), -bromoethylacetate (183 mg, 1.1 mmol) and the mixture was stirred at room temperature for 12 hours. The reaction was monitored by TLC using ethyl acetate-hexane (6:4). After completion of the reaction as indicated by the TLC, K2CO3 was removed by 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 (9:1) to afford pure compound 24 as white solid. Yield (401 mg, 82%) 141

THESIS Mp: 137-139 C;

1

H NMR (500 MHz, CDCl3): 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) ESIMS: m/z 487 (M+H)+. 2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5yl)-2-methoxyphenoxy)acetic acid (25) To a solution of compound 24 (486 mg, 1 mmol) in THF (15 mL) and water (2 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 Na 2SO4 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 C;
1

H NMR (200 MHz, CDCl3): 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); ESIMS: m/z 459 (M+H)+. (Z)-N-(benzo[d]thiazol-2-yl)-2-(2-methoxy-5-(3,4,5trimethoxystyryl)phenoxy) acetamide (27a) To a solution of 2-aminobenzothiazole 26a (150 mg, 1.0 mmol) in dichloromethane (20 mL) was added 1-(3-Dimethylaminopropyl)-3ethylcarbodiimide hydrochloride (EDCI.HCl) (191 mg, 1 mmol) and 1142

THESIS 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 C;
1

H NMR (200 MHz, CDCl3):

10.64 (bs, 1H), 7.747.82 (m, 2H), 7.377.44

(m, 1H), 7.29 (d, 1H, J = 8.3 Hz), 6.967.01 (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), 6.384.3 (m, 3H), 4.6 (s, 2H), 4.02 (s, 3H), 3.83 (s, 3H), 3.69 (s, 6H);
13

C NMR (75 MHz, CDCl3):

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; ESIMS: m/z 507 (M+H)+. (Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)-N-(6nitrobenzo[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-(3,4,5trimethoxystyryl)phenoxy)aceticacid (18) (374 mg, 1 mmol), and 2-amino-6nitrobenzothiazole 26b (195 mg, 1 mmol) to obtain the pure product 27b as white solid. Yield (442 mg, 80%) Mp: 145-146 C;
1

H NMR (300 MHz, CDCl3): 11.03 (s, 1H), 8.77 (d, 1H, J = 2.1 Hz), 8.328.36

(dd, 1H, J = 8.7, 2.1 Hz), 7.88 (d, 1H, J = 8.7 Hz), 7.037.07 (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

THESIS ESIMS: m/z 552 (M+H)+. (Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)-N-(6fluorobenzo[d]thiazol-2-yl)acetamide (27c) This compound was prepared according to the method described for compound 27a by employing (Z)-2-(2-methoxy-5-(3,4,5trimethoxystyryl)phenoxy)aceticacid (18) (374 mg, 1 mmol), and 2-amino-6fluorobenzothiazole 26c (168 mg, 1 mmol) to obtain the pure product 27c as white solid. Yield (430 mg, 81%) Mp: 137-139 C;
1

H NMR (300 MHz, CDCl3):

10.66 (bs, 1H), 7.717.76 (m, 1H), 7.477.53

(dd, 1H, J = 8.1, 2.4 Hz), 7.137.20 (m, 1H), 7.007.04 (dd, 1H, J = 8.1, 1.6 Hz), 6.93 (d, 1H, J = 1.6 Hz), 6.83 (d, 1H, J = 8.1 Hz), 6.466.51 (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);
13

C NMR (75 MHz, CDCl3):

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; ESIMS: m/z 525 (M+H)+. (Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)-N-(6chlorobenzo[d]thiazol-2-yl)acetamide (27d) This compound was prepared according to the method described for compound 27a by employing (Z)-2-(2-methoxy-5-(3,4,5trimethoxystyryl)phenoxy)aceticacid (18) (374 mg, 1 mmol), and 2-amino-6chlorobenzothiazole 26d (184 mg, 1 mmol) to obtain the pure product 27d as white solid. Yield (385 mg, 71%) Mp: 136-138 C;
1

H NMR (200 MHz, CDCl3): 10.61 (bs, 1H), 7.8 (d, 1H, J = 2.2 Hz), 7.73 (d,

1H, J = 9 Hz), 7.387.44 (dd, 1H, J = 8.3, 2.2 Hz), 77.06 (dd, 1H, J = 8.3, 2.2 Hz), 6.94 (d, 1H, J = 2.2 Hz), 6.84 (d, 1H, J = 9 Hz), 6.426.54 (m, 4H), 4.64 (s, 2H), 3.98 (s, 3H), 3.85 (s, 3H), 3.7 (s, 6H);

144

THESIS
13

C NMR (75 MHz, CDCl3):

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; ESIMS: m/z 541 (M+H)+. (Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)-N-(6methoxybenzo[d]thiazol-2-yl)acetamide (27e) This compound was prepared according to the method described for compound 27a by employing (Z)-2-(2-methoxy-5-(3,4,5trimethoxystyryl)phenoxy)aceticacid (18) (374 mg, 1 mmol), and 2-amino-6methoxybenzothiazole 26e (180 mg, 1 mmol) to obtain the pure product 27e as white solid. Yield (415 mg, 77%) Mp: 139-140 C;
1

H NMR (400 MHz, CDCl3): 7.71 (d, 1H, J = 9 Hz), 7.3 (d, 1H, J = 2.2 Hz),

6.997.1 (m, 2H), 6.94 (d, 1H, J = 2.2 Hz), 6.84 (d, 1H, J = 8.3 Hz), 6.436.53 (m, 4H), 4.63 (s, 2H), 3.98 (s, 3H), 3.88 (s, 3H), 3.85 (s, 3H), 3.7 (s, 6H);
13

C NMR (75 MHz, CDCl3):

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; ESIMS: m/z 537 (M+H)+. (Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)-N-(6trifluoromethoxybenzo[d] thiazol-2-yl)acetamide (27f) This compound was prepared according to the method described for compound 27a by employing (Z)-2-(2-methoxy-5-(3,4,5trimethoxystyryl)phenoxy)aceticacid (18) (374 mg, 1 mmol), and 2-amino-6trifluoromethoxybenzothiazole 26f (234 mg, 1 mmol) to obtain the pure product 27f as white solid. Yield (470 mg, 80%) Mp: 141-143 C;
1

H NMR (200 MHz, CDCl3):

10.63 (bs, 1H), 7.8 (d, 1H, J = 8.8 Hz), 7.69 (s,

1H), 7.297.36 (dd, 1H, J = 8.4, 2.2 Hz), 7.017.06 (dd, 1H, J = 8.4, 1.7 Hz),

145

THESIS 6.95 (d, 1H, J = 1.7 Hz), 6.85 (d, 1H, J = 8.3 Hz), 6.426.53 (m, 4H), 4.65 (s, 2H), 3.99 (s, 3H), 3.85 (s, 3H), 3.70 (s, 6H); );
13

C NMR (75 MHz, CDCl3):

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; ESIMS: m/z 591 (M+H)+. (Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)-N-(6methylbenzo[d]thiazol-2-yl)acetamide (27g) This compound was prepared according to the method described for compound 27a by employing (Z)-2-(2-methoxy-5-(3,4,5trimethoxystyryl)phenoxy)aceticacid (18) (374 mg, 1 mmol), and 2-amino-6methylbenzothiazole 26g (180 mg, 1 mmol) to obtain the pure product 27g as white solid. Yield (471 mg, 89%) Mp: 137-139 C;
1

H NMR (300 MHz, CDCl3): 10.59 (bs, 1H), 7.5 (d, 1H, J = 2.3 Hz), 7.71 (d,

1H, J = 8.3 Hz), 7.357.42 (dd, 1H, J = 8.3, 2.2 Hz), 7.027.09 (dd, 1H, J = 8.3, 2.2 Hz), 6.91 (d, 1H, J = 2.2 Hz), 6.84 (d, 1H, J = 8.3 Hz), 6.436.55 (m, 4H), 4.65 (s, 2H), 3.98 (s, 3H), 3.86 (s, 3H), 3.7 (s, 6H), 2.46 (s, 3H)
13

C NMR (75 MHz, CDCl3):

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; ESIMS: m/z 521 (M+H)+. (Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)-N-(6trifluoromethylbenzo [d]thiazol-2-yl)acetamide (27h) This compound was prepared according to the method described for compound 27a by employing (Z)-2-(2-methoxy-5-(3,4,5trimethoxystyryl)phenoxy)aceticacid (18) (374 mg, 1 mmol), and 2-amino-6trifluoromethylbenzothiazole 26h (218 mg, 1 mmol) to obtain the pure product 27h as white solid. Yield (470 mg, 79%) Mp: 142-144 C;

146

THESIS
1

H NMR (200 MHz, CDCl3): 10.64 (bs, 1H), 8.12 (s, 1H), 7.93 (d, 1H, J = 8.4

Hz), 7.697.75 (m, 1H), 7.017.06 (dd, 1H, J = 8.3, 1.7 Hz), 6.95 (d, 1H, J = 1.7 Hz), 6.85 (d, 1H, J = 8.3 Hz), 6.436.53 (m, 1H), 4.68 (s, 2H), 3.99 (s, 3H), 3.83 (s, 3H), 3.70 (s, 6H); );
13

C NMR (75 MHz, CDCl3):

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; ESIMS: m/z 597 (M+Na)+. (Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)-N-(6ethoxybenzo[d]thiazol-2-yl)acetamide (27i) This compound was prepared according to the method described for compound 27a by employing (Z)-2-(2-methoxy-5-(3,4,5trimethoxystyryl)phenoxy)aceticacid (18) (374 mg, 1 mmol), and 2-amino-6ethoxybenzothiazole 26i (194 mg, 1 mmol) to obtain the pure product 27i as white solid. Yield (485 mg, 88%) Mp: 138-140 C;
1

H NMR (300 MHz, CDCl3): 10.61 (bs, 1H), 7.72 (d, 1H, J = 8.3 Hz), 7.4 (d,

1H, J = 2.2 Hz), 6.997.15 (m, 2H), 6.93 (d, 1H, J = 2.2 Hz), 6.85 (d, 1H, J = 8.3 Hz), 6.446.53 (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); ESIMS: m/z 551 (M+H)+. (E)-N-(benzo[d]thiazol-2-yl)-2-(2-methoxy-5-(3-oxo-3-(3,4,5trimethoxyphenyl)prop-1-enyl)phenoxy)acetamide (28a) This compound was prepared according to the method described for compound EDCI.HCl 27a (191 mg, by 1 employing mmol), HOBt (E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5(13.5 mg, 0.1 mmol) and 2trimethoxyphenyl)prop-1-enyl) phenoxy) aceticacid (22) (402 mg, 1 mmol), aminobenzothiazole 26a (150 mg, 1 mmol) to obtain the pure product 28a as yellow solid. Yield (416 mg, 77%) Mp: 143-145 C;

147

THESIS
1

H NMR (200 MHz, CDCl3): 11.93 (bs, 1H), 7.817.84 (dd, 1H, J = 7.8, 1.9

Hz), 7.727.75 (dd, 1H, J = 7.8, 1.9 Hz), 7.67 (d, 1H, J = 15.6 Hz), 7.527.6 (m, 2H), 7.387.43 (m, 1H), 7.347.37 (dd, 1H, J = 7.8, 1.9 Hz), 7.31 (s, 2H), 7.257.29 (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);
13

C NMR (75 MHz, DMSO D6):

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; ESIMS: m/z 535 (M+H)+. (E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1enyl)phenoxy)-N-(6-nitrobenzo[d]thiazol-2-yl)acetamide (28b) This compound was prepared according to the method described for compound 27a by employing (E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5trimethoxyphenyl)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-amino-6nitrobenzothiazole 26b (195 mg, 1 mmol) to obtain the pure product 28b as yellow solid. Yield (452 mg, 78%) Mp: 149-150 C;
1

H NMR (200 MHz, CDCl3): 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), 7.287.34 (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); ESIMS: m/z 580 (M+H)+. (E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1enyl)phenoxy)-N-(6-fluorobenzo[d]thiazol-2-yl)acetamide (28c) This compound was prepared according to the method described for compound 27a by employing (E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5trimethoxyphenyl)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-amino-6-

148

THESIS fluorobenzothiazole 26c (168 mg, 1 mmol) to obtain the pure product 28c as yellow solid. Yield (487 mg, 81%) Mp: 145-146 C;
1

H NMR (300 MHz, CDCl3): 10.57 (bs, 1H), 7.657.77 (m, 2H), 7.447.53 (m,

2H), 7.317.39 (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); ESIMS: m/z 553 (M+H)+. (E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1enyl)phenoxy)-N-(6-chlorobenzo[d]thiazol-2-yl)acetamide (28d) This compound was prepared according to the method described for compound 27a by employing (E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5trimethoxyphenyl)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-amino-6chlorobenzothiazole 26d (184 mg, 1 mmol) to obtain the pure product 28d as yellow solid. Yield (475 mg, 78%) Mp: 143-145 C;
1

H NMR (400 MHz, CDCl3): 10.59 (bs, 1H), 7.767.81 (m, 1H), 7.71 (d, 1H, J

= 15.1 Hz), 7.657.69 (m, 2H), 7.46 (d, 1H, J = 2 Hz), 7.377.41 (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); ESIMS: m/z 570 (M+H)+. (E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1enyl)phenoxy)-N-(6-methoxybenzo[d]thiazol-2-yl)acetamide (28e) This compound was prepared according to the method described for compound 27a by employing (E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5trimethoxyphenyl)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-amino-6methoxybenzothiazole 26e (180 mg, 1 mmol) to obtain the pure product 28e as yellow solid. Yield (463 mg, 81%) Mp: 146-147 C;

149

THESIS
1

H NMR (300 MHz, CDCl3): 10.62 (bs, 1H), 7.75 (d, 1H, J = 15.6 Hz), 7.71 (d,

1H, J = 8.3 Hz), 7.347.4 (m, 2H), 7.287.34 (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); ESIMS: m/z 565 (M+H)+. (E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1enyl)phenoxy)-N-(6-trifluoromethoxybenzo[d]thiazol-2-yl)acetamide (28f) This compound was prepared according to the method described for compound 27a by employing (E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5trimethoxyphenyl)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-amino-6trifluoromethoxybenzothiazole 26f (234 mg, 1 mmol) to obtain the pure product 28f as yellow solid. Yield (505 mg, 81%) Mp: 148-150 C;
1

H NMR (200 MHz, CDCl3): 10.62 (bs, 1H), 7.77 (d, 1H, J = 9 Hz), 7.667.73

(m, 2H), 7.277.39 (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);
13

C NMR (75 MHz, DMSO D6):

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; ESIMS: m/z 619 (M+H)+. (E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1enyl)phenoxy)-N-(6-methylbenzo[d]thiazol-2-yl)acetamide (28g) This compound was prepared according to the method described for compound 27a by employing (E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5trimethoxyphenyl)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-amino-6-

150

THESIS methylbenzothiazole 26g (164 mg, 1 mmol) to obtain the pure product 28g as yellow solid. Yield (478 mg, 81%) Mp: 142-144 C;
1

H NMR (300 MHz, CDCl3): 7.76 (d, 1H, J = 15.6 Hz), 7.70 (d, 1H, J = 8.3 Hz),

7.63 (s, 1H), 7.317.43 (m, 2H), 7.237.30 (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); ESIMS: m/z 549 (M+H)+. (E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1enyl)phenoxy)-N-(6-trifluoromethylbenzo[d]thiazol-2-yl)acetamide (28h) This compound was prepared according to the method described for compound 27a by employing (E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5trimethoxyphenyl)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-amino-6trifluoromethylbenzothiazole 26h (218 mg, 1 mmol) to obtain the pure product 28h as yellow solid. Yield (512 mg, 84%) Mp: 147-149 C;
1

H NMR (200 MHz, CDCl3): 10.74 (bs, 1H), 8.11 (s, 1H), 7.87 (d, 1H, J = 8.3

Hz), 7.647.74 (m, 2H), 7.287.40 (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);
13

C NMR (75 MHz, DMSO D6):

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; ESIMS: m/z 603 (M+H)+. (E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1enyl)phenoxy)-N-(6-ethoxybenzo[d]thiazol-2-yl)acetamide (28i) This compound was prepared according to the method described for compound 27a by employing (E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5trimethoxyphenyl)prop-1-enyl) phenoxy) aceticacid (22) (402 mg, 1 mmol),

151

THESIS EDCI.HCl (191 mg, 1 mmol), HOBt (13.5 mg, 0.1 mmol) and 2-amino-6ethoxybenzothiazole 26i (194 mg, 1 mmol) to obtain the pure product 28i as yellow solid. Yield (443 mg, 76%) Mp: 145-147 C;
1

H NMR (200 MHz, CDCl3): 7.75 (d, 1H, J = 15.8 Hz), 7.70 (d, 1H, J = 9 Hz),

7.367.41 (m, 2H), 7.297.34 (m, 2H), 7.257.28 (m, 3H), 7.01 (d, 1H, J = 8.3 Hz), 4.84 (s, 2H), 4.064.15 (q, 2H), 4.04 (s, 3H), 3.96 (s, 6H), 3.95 (s, 3H), 1.46 (t, 3H); ESIMS: m/z 579 (M+H)+. 2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5yl)-2-methoxy phenoxy)-N-(benzo[d]thiazol-2-yl)acetamide (29a) This compound was prepared according to the method described for compound 27a by employing 2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5dihydro-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 2aminobenzothiazole 26a (150 mg, 1 mmol) to obtain the pure product 29a as white solid. Yield (477 mg, 80%) Mp: 151-153 C;
1

H NMR (400 MHz, CDCl3): 11.03 (bs, 1H), 7.737.81 (m, 2H), 7.40 (t, 1H),

7.29 (d, 1H, J = 7.3 Hz), 6.926.97 (m, 1H), 6.856.91 (m, 4H), 5.445.53 (dd, 1H, J = 11.7, 4.5 Hz), 4.69 (s, 2H), 3.99 (s, 3H), 3.90 (s, 6H), 3.86 (s, 3H), 3.643.76 (dd, 1H, J = 11.8, 17.3 Hz), 3.043.14 (dd, 1H, J = 17.3, 4.5 Hz), 2.40 (s, 3H); ESIMS: m/z 591 (M+H)+. 2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5yl)-2-methoxy phenoxy)-N-(6-nitrobenzo[d]thiazol-2-yl)acetamide (29b) This compound was prepared according to the method described for compound 27a by employing 2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5dihydro-1H-pyrazol-5-yl)-2-methoxyphenoxy)acetic acid (25) (458 mg, 1

152

THESIS mmol), EDCI.HCl (191 mg, 1 mmol), HOBt (13.5 mg, 0.1 mmol) and 2-amino6-nitrobenzothiazole 26b (195 mg, 1 mmol) to obtain the pure product 29b as white solid. Yield (535 mg, 84%) Mp: 158-160 C;
1

H NMR (300 MHz, CDCl3): 11.02 (bs, 1H), 8.7 (d, 1H, J = 2.2 Hz), 8.328.37

(dd, 1H, J = 9, 2.2 Hz), 7.87 (d, 1H, J = 9 Hz), 6.997.04 (dd, 1H, J = 8.3, 2.2 Hz), 6.946.98 (m, 3H), 6.93 (d, 1H, J = 2.2 Hz), 5.55.58 (dd, 1H, J = 11.3, 4.5 Hz), 4.81 (s, 2H), 4 (s, 3H), 3.92 (s, 6H), 3.90 (s, 3H), 3.73.82 (dd, 1H, J = 17.3, 12.8 Hz), 3.083.18 (dd, 1H, J = 17.3, 4.5 Hz), 2.43 (s, 3H); ESIMS: m/z 636 (M+H)+. 2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5yl)-2-methoxy phenoxy)-N-(6-fluorobenzo[d]thiazol-2-yl)acetamide (29c) This compound was prepared according to the method described for compound 27a by employing 2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5dihydro-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-amino6-fluorobenzothiazole 26c (168 mg, 1 mmol) to obtain the pure product 29c as white solid. Yield (503 mg, 82%) Mp: 153-154 C;
1

H NMR (200 MHz, CDCl3): 10.62 (bs, 1H), 7.717.77 (dd, 1H, J = 9, 4.5 Hz),

7.497.53 (dd, 1H, J = 8.3, 3 Hz), 7.147.22 (m, 1H), 6.977.01 (dd, 1H, J = 8.3, 2.2 Hz), 6.96 (s, 2H), 6.886.94 (m, 2H), 6.56.57 (dd, 1H, J = 11.3, 4.5 Hz), 4.76 (s, 2H), 3.97 (s, 3H), 3.91 (s, 6H), 3.9 (s, 3H), 3.693.8 (dd, 1H, J = 173, 11.3 Hz), 3.083.17 (dd, 1H, J = 17.3, 4.5 Hz), 2.43 (s, 3H);
13

C NMR (75 MHz, CDCl3+DMSO D6):

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; ESIMS: m/z 609 (M+H)+.

153

THESIS 2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5yl)-2-methoxy phenoxy)-N-(6-chlorobenzo[d]thiazol-2-yl)acetamide (29d) This compound was prepared according to the method described for compound 27a by employing 2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5dihydro-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-amino6-chlorobenzothiazole 26d (184 mg, 1 mmol) to obtain the pure product 29d as white solid. Yield (518 mg, 82%) Mp: 153-155 C;
1

H NMR (400 MHz, CDCl3): 10.74 (bs, 1H), 7.76 (d, 1H, J = 2.2 Hz), 7.67 (d,

1H, J = 9 Hz), 7.347.39 (dd, 1H, J = 9, 2.2 Hz), 6.926.97 (dd, 1H, J = 8.3, 2.2 Hz), 6.896.91 (m, 1H), 5.445.52 (dd, 1H, J = 12, 4.5 Hz), 4.74 (s, 2H), 4 (s, 3H), 3.9 (s, 6H), 3.86 (s, 3H), 3.653.77 (dd, 1H, J = 17.3, 12 Hz), 3.05 3.14 (dd, 1H, J = 17.3, 4.5 Hz), 2.4 (s, 3H); ESIMS: m/z 626 (M+H)+. 2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5yl)-2-methoxy phenoxy)-N-(6-methoxybenzo[d]thiazol-2yl)acetamide (29e) This compound was prepared according to the method described for compound 27a by employing 2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5dihydro-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-amino6-methoxybenzothiazole 26e (180 mg, 1 mmol) to obtain the pure product 29e as white solid. Yield (532 mg, 85%) Mp: 156-157 C;
1

H NMR (300 MHz, CDCl3): 10.45 (bs, 1H), 7.69 (d, 1H, J = 8.3 Hz), 7.29 (d,

1H, J = 2 Hz), 7.037.07 (dd, 1H, J = 9.3, 3.1 Hz), 6.977.00 (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), 5.515.55 (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

THESIS 3.88 (s, 3H), 3.73.78 (dd, 1H, J = 7.6, 12.4 Hz), 3.093.14 (dd, 1H, J = 7.6, 4.1 Hz), 2.4 (s, 3H); ESIMS: m/z 621 (M+H)+. 2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5yl)-2-methoxy phenoxy)-N-(6-trifluoromethoxybenzo[d]thiazol-2yl)acetamide (29f) This compound was prepared according to the method described for compound 27a by employing 2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5dihydro-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-amino6-trifluoromethoxybenzothiazole 26f (234 mg, 1 mmol) to obtain the pure product 29f as white solid. Yield (547 mg, 81%) Mp: 159-161 C;
1

H NMR (400 MHz, CDCl3): 10.71 (bs, 1H), 7.79 (d, 1H, J = 8.3 Hz), 7.69 (d,

1H, J = 1.5 Hz), 7.297.34 (dd, 1H, J = 9, 1.5 Hz), 6.987.02 (dd, 1H, J = 8.3, 1.5 Hz), 6.96 (s, 2H), 6.896.94 (m, 2H), 5.55.58 (dd, 1H, J = 11.3, 4.5 Hz), 4.77 (s, 2H), 3.98 (s, 3H), 3.91 (s, 6H), 3.9 (s, 3H), 3.693.81 (dd, 1H, J = 18.1, 12 Hz), 3.083.17 (dd, 1H, J = 17.3, 4.5 Hz), 2.43 (s, 3H);
13

C NMR (75 MHz, CDCl3+DMSO D6):

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; ESIMS: m/z 675 (M+H)+. 2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5yl)-2-methoxy phenoxy)-N-(6-methylbenzo[d]thiazol-2-yl)acetamide (29g) This compound was prepared according to the method described for compound 27a by employing 2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5dihydro-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-amino-

155

THESIS 6-methylbenzothiazole 26g (164 mg, 1 mmol) to obtain the pure product 29g as white solid. Yield (498 mg, 82%) Mp: 153-154 C;
1

H NMR (200 MHz, CDCl3): 10.52 (bs, 1H), 7.69 (d, 1H, J = 8.3 Hz), 7.62 (s,

1H), 7.247.29 (m, 1H), 6.947 (m, 3H), 6.876.93 (m, 2H), 5.55.57 (dd, 1H, J = 12, 4.5 Hz), 4.75 (s, 2H), 3.96 (s, 3H), 3.91 (s, 6H), 3.89 (s, 3H), 3.683.8 (dd, 1H, J = 17.3, 11.3 Hz), 3.073.17 (dd, 1H, J = 17.3, 4.5 Hz), 2.48 (s, 3H), 2.43 (s, 3H); ESIMS: m/z 605 (M+H)+. 2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5yl)-2-methoxy phenoxy)-N-(6-trifluoromethylbenzo[d]thiazol-2yl)acetamide (29h) This compound was prepared according to the method described for compound 27a by employing 2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5dihydro-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-amino6-trifluoromethylbenzothiazole 26h (218 mg, 1 mmol) to obtain the pure product 29h as white solid. Yield (568 mg, 86%) Mp: 162-163 C;
1

H NMR (400 MHz, CDCl3): 10.82 (bs, 1H), 8.11 (s, 1H), 7.88 (d, 1H, J = 7.9

Hz), 7.677.7 (m, 1H), 6.987.02 (dd, 1H, J = 7.9, 1.6 Hz), 6.96 (s, 2H), 6.9 6.94 (m, 2H), 5.515.56 (dd, 1H, J = 11.9, 3.9 Hz), 4.79 (s, 2H), 3.99 (s, 3H), 3.91 (s, 6H), 3.89 (s, 3H), 3.713.79 (dd, 1H, J = 11.9, 17.5 Hz), 3.13.15 (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-5yl)-2-methoxy phenoxy)-N-(6-ethoxybenzo[d]thiazol-2-yl)acetamide (29i) This compound was prepared according to the method described for compound 27a by employing 2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5dihydro-1H-pyrazol-5-yl)-2-methoxyphenoxy)acetic acid (25) (458 mg, 1 156

THESIS mmol), EDCI.HCl (191 mg, 1 mmol), HOBt (13.5 mg, 0.1 mmol) and 2-amino6-ethoxybenzothiazole 26i (194 mg, 1 mmol) to obtain the pure product 29i as white solid. Yield (502 mg, 78%) Mp: 158-159 C;
1

H NMR (300 MHz, CDCl3): 10.47 (bs, 1H), 7.68 (d, 1H, J = 9.3 Hz), 7.28 (d,

1H, J = 3.1 Hz), 7.027.06 (dd, 1H, J = 8.3, 2 Hz), 6.957 (m, 3H), 6.9 (d, 1H, J = 8.3 Hz), 6.87 (d, 1H, J = 2.08 Hz), 5.55.56 (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), 3.73.78 (dd, 1H, J = 17.6, 12.4 Hz), 3.093.15 (dd, 1H, J = 17.6, 4.1 Hz), 2.43 (s, 3H), 1.45 (t, 3H); ESIMS: m/z 635 (M+H)+. 3.7. TUBULIN POLYMERIZATION ASSAY A fluorescence based In vitro tubulin polymerization assay was

performed according to the manufacturers protocol (BK011, Cytoskeleton, Inc.). Briefly, the reaction mixture in a total volume of 10 l contained PEM buffer, GTP (1 mM) in the presence or absence of test compounds (final concentration of 3 M). Tubulin polymerization was followed by a time dependent fluorescence increase reporter in fluorescence into due to as the incorporation of a microtubules 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

THESIS

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C.; Hamel, E.; Hackl, T.; Lee, K.-H. J. Med. Chem. 1998, 41, 1155. Nat. Prod. 2000, 63, 969. 1996, 27, 586. Gerard, B.; Aboukameel, A.; Petit, G. R.; Al-Katib, A. M. Clin. Cancer Res. 2002, 8, 2733. 31. Simoni, D.; Roberti, M.; Invidiata, F. P.; Rondanin, R.; Baruchello, R.; Malagutti, C.; Mazzali, A.; Rossi, M.; Grimaudo, S.; Dusonchet, L.; Meli, M.; Raimondi, M. V.; Alessandro, N.; Tolomeo, M. Bioorg. Med. Chem. Lett. 2000, 10, 2669. 32. Simoni, D.; Roberti, M.; Invidiata, F. P.; Rondanin, R.; Baruchello, R.; Malagutti, C.; Mazzali, A.; Rossi, M.; Grimaudo, S.; Capone, F.; Dusonchet, L.; Meli, M.; Raimondi, M. V.; Landino, M.; DAlessandro, N.; Tolomeo, M.; Arindam, D.; Lu, S.; Benbrook, D. M. J. Med .Chem. 2001, 44, 2308. 33. Grimaudo, S.; Tolomeo, M.; Capone, F.; Pagliaro, M.; Rondanin, R.; Baruchello, R.; Simoni, D.; Mariani, G. Blood 2001, 98, 445.

160

THESIS 34. Roberti, M.; Pizzirani, D.; Simoni, D.; Rondanin, R.; Baruchello, R.;

Bonora, C.; Buscemi, F.; Grimaudo, S.; Tolomeo, M. J. Med. Chem. 2003, 46, 3546. 35. 36. 37. Tron,G. C.; Pirali, T.; Sorba,G.; Pagliai, F.; Busacca, S.;Genazzani, Nam, N. H. Curr. Med. Chem. 2003, 10, 1697. Liou, J. P.; Chang, Y. L.; Kuo, F. M.; Chang, C. W.; Tseng, H. Y.; A. A. J. Med. Chem. 2006, 49, 3033.

Wang, C. C.; Yang, Y. N.; Chang, J. Y.; Lee, S. J.; Hsieh, H. P. J. Med. Chem. 2004, 47, 4247. 38. Ohsumi, K.; Nakagawa, R.; Fukuda, Y.; Hatanaka, T.; Morinaga, Y.; Nihei, Y.; Ohishi, K.; Suga, Y.; Akiyama, Y.; Tsuji, T. J. Med. Chem. 1998, 41, 3022. 39. 40. Cushman, M.; Nagarathnam, D.; Gopal, D.; Chakraborti, A. K.; Lin, Wang, L.; Woods, K. W.; Li, Q.; Barr, K. J.; McCroskey, R. W.; C. M.; Hamel, E. J. Med. Chem. 1991, 34, 2579. Hannick, S. M.; Gherke, L.; Credo, R. B.; Hui, Y.-H.; Marsh, K.; Warner, R.; Lee, J. Y.; Zielinski-Mozng,N.; Frost, D.; Rosenberg, S. H.; Sham, H. L. J. Med.Chem. 2002, 45, 1697. 41. Ohsumi, K.; Hatanaka, T.; Fujita, K.; Nakagawa, R.; Fukuda, Y.; Nihei, Y.; Suga, Y.; Morinaga, Y.; Akiyama, Y.; Tsuji, T. Bioorg. Med. Chem. Lett. 1998, 8, 3153. 42. 43. 44. 45. Kim, Y.; Nam, N.-H.; You, Y.-J.; Ahn, B.-Z. Bioorg.Med. Chem. Lett. Shirai, R.; Takayama, H.; Nishikawa, A.; Koiso, Y.; Hashimoto, Y. Tron, G. C.; Pagliai, F.; Grosso, E. D.; Genazzani, A. A.; Sorba, G. (a) Lawrence, N. J.; Patterson, R. P.; Ooi, L.; Cook, D.; Ducki, S. 2002, 12, 719. Bioorg. Med. Chem. Lett. 1998, 8, 1997. J. Med. Chem. 2005, 48, 3260. Bioorg. Med. Chem. Lett. 2006, 16, 5844; (b) Ducki, S.; Forrest, R.; Hadfield, J. A.; Kandall, A.; Lawrence, N. J.; McGown, A. T.; Rennison, D.

161

THESIS Bioorg. Med. Chem. Lett. 1998, 8, 1051; (c) Ducki, S.; Woo, D. R. M.; Kendall, A.; Chabert, J. F. D.; McGown, A. T.; Lawrence, N. J. Bioorg. Med. Chem. 2009, 17, 7698. 46. 47. 48. 49. Pettit, G. R.; Singh, S. B.; Hamel, E.; Lin, C. M.; Alberts, D. S.; Lin, C. M.; Ho, H. H.; Pettit, G. R.; Hamel, E. Biochemistry 1989, Jordan, A.; Hadfield, J. A.; Lawrence, N. J.; McGown, A. T. Med. Johnson, M.; Younglove, B.; Lee, L.; LeBlanc, R.; Holt, H.; Hills, P.; Garcia-Kendall, D. Experientia 1989, 45, 209. 28, 6984. Res. Rev. 1998, 18, 259. Mackay, H.; Brown, T.; Mooberry, S. L.; Lee, M. Bioorg. Med. Chem. Lett. 2007, 17, 5897. 50. (a) Boyd, R. B. The NCI In Vitro Anticancer Drug Discovery Screen. In anticancer Drug Development Guide: Preclinical Screening, Clinical Trials, and Approval; Teicher, B., Ed.; Humana Press Inc.: Totowa, NJ, 1997, p23; (b) Kehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D. J. Natl. Cancer Inst. 1990, 82, 1107.

162

THESIS

CHAPTER-IV/SECTION-A SYNTHESIS AND BIOLOGICAL EVALUATION OF BENZYLIDENE-9(10H)ANTHRACENONE LINKED PYRROLOBENZODIAZEPINES AS ANTICANCER AGENTS

163

THESIS

4.1. INTRODUCTION
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-cancer therapy. 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 [] - and [] 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. INTRODUCTION OF BENZYLIDENEANTHRACENONES

Anthracenones are known for biological properties like tubulin binding, antiproliferation and antipsoriatic4 action. It was found that certain 10benzyliden-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-phenylmethyl9(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

THESIS 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 O

OH 1 OMe 2 OMe

OMe OH 3

OMe

Figure 1 The same group6 synthesized new analogues were of 10-(2-oxo-2for phenylethylidene)-10H-anthracen-9-ones, that evaluated

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 M). Zuse and coworkers7 synthesized and reported some novel 9benzylidene- 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-3,5dimethoxy)-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 M) (Figure 2).

165

S OMe 4 OMe OH 5 MeO

THESIS
OMe

OH

Figure 2 Recently some of anthracenone series of compounds have also been synthesized and evaluated for their anticancer property.8-9 1,8-disubstituted-10-benzylidene-10H-anthracen-9-ones
O

They include 10-(2-oxo-2-

anthracenone-based oxime ethers, oxime esters like 6 and 7, and 1,5- or and phenylethy lidene)-10H-anthracen-9-ones (8 and 9) (Figure 3).
O

N 6

O OH

7 OMe OMe Cl O Cl

Cl

Cl

OMe 8 OMe OH 9

OH OMe

Figure 3 Apart from anticancer potential, these anthracenone analogues possess 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

THESIS systems associated with cell proliferation and redox reactions that result in alteration of mitochondrial functions and destruction of membrane lipids.
OH O OH

OH O

OH

OH O

OH

O 10 11 OMe OH O OH O 12

O 13

Cl

Cl OH

14

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. PRESENT WORK

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

THESIS 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 binding ability. with varying alkane spacers have been evaluated for their antitumour activity and DNA-

4.1.2.1. SYNTHESIS OF PBD PRECURSORS

The precursor (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]pyrolidine2-carboxaldehydediethylthioacetal discussed in chapter-II.
HO MeO O 15 NO2 N

(15)

have

been

prepared

from

commercially available vanillin. The synthesis of compound 15 was

CH(SEt)2

4.1.2.2. SYNTHESIS OF BENZYLIDENE-9(10H)-ANTHRACENONES

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 K2CO3 as a base in dry acetone affords the required precursors (19a-f) as illustrated in Scheme 1.

168

THESIS
O + 16 R OH 17a, b CHO (i) O

18a, b R (ii)

OH

O 19a; R = H, n = 2 19b; R = H, n = 3 19c; R = H, n = 4 19d; R = OMe, n = 2 19e; R = OMe, n = 3 19f; R = OMe, n = 4 19a-f O Br ( )n

R Scheme 1. Reagents and conditions: (i) IPA.HCl, 5 h; (ii) dibromoalkane, acetone, K2CO3,
reflux, 24h.

4.1.2.3. SYNTHESIS OF C8-LINKED BENZYLIDENE-9(10H)-ANTHRACENONE-PBD CONJUGATES

Compound 15 has been coupled to compounds 19a-f in the presence of K2CO3 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

THESIS
O HO + MeO O 19a-f R O (i) O Br ( )n 15 N NO2 CH(SEt)2

R O 20a-f O O ( )n MeO O (ii) R O 21a-f O (iii) R O 22a-f O ( )n MeO O

Scheme 2. Reagents and conditions: (i) K2CO3, acetone, 12 h, reflux; (ii) SnCl2.2H2O, MeOH, 4 h, reflux; (iii) HgCl2, CaCO3, MeCN, H2O, (4:1) 12 h, rt.

NO2 N

CH(SEt)2

O ( )n MeO

NH2 N O

CH(SEt)2

N N

170

THESIS

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

C 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 1 C/min in the range of 40-95 C. DNA helix-coil transition temperatures are given by: Tm = Tm(DNA+PBD)Tm(DNA alone), where the Tm 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 ( Tm) for each compound is examined at 0 h and 18 h of incubation at 37 C. 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 37

C with ligand/DNA molar ratio of 1:5. The increase in

the helix melting temperature ( Tm) for each compound has been examined at 0 h and 18 h incubation at 37 C. 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 Tm 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 Tm 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

THESIS

Table 1.Thermal denaturation data for benzylidene-9(10H)-anthracenonePBD conjugates with calf thymus (CT)-DNA [PBD]:[DNA] molar ratiob 1:5 1:5 1:5 1:5 1:5 1:5 1:5 Tm (oC)a after incubation at 37 oC for 0h 4.3 4.6 4.1 3.6 3.0 4.0 0.3
0 b

Compound 22a 22b 22c 22d 22e 22f DC-81

a

18 h 4.9 5.1 4.6 4.0 3.5 4.3 0.7

For CT-DNA alone at pH 7.00 0.01, Tm = 68.5 C 0.01 (mean value from 10 separate For a 1:5 molar ratio of [PBD]/[DNA],

determinations), all Tm values are 0.1 - 0.2 0C.

where CT-DNA concentration = 100 M and ligand concentration = 20 M in aqueous sodium phosphate buffer [10 mM sodium phosphate + 1 mM EDTA, pH 7.00 0.01].

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 M, while the positive controls, DC-81 and adriamycin exhibited the GI 50 in the range of 0.1-0.17 M and <0.01-14.7 M 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, 0.080.14 M) compared to other cell lines tested. The 172

THESIS active compound 22e exhibited strong effect against all cell lines tested (GI50, 0.08-1.8 M) and it showed a GI50 value of 0.08 against MCF-7 cell line. Table 2. GI50 valuesa (in M) for compounds 22a-f in selected human cancer cell lines. GI50 values (M)
Compound

Breast MCF-7

Ovarian A2780

Colon Colo205

Prostat e PC-3

Cervix SiHa A 549

Lung Hop-62

Oral KB

22a 22b 22c 22d 22e 22f DC-81 ADR

a

0.12 0.14 0.15 0.12 0.08 0.1 0.16 <0.01

0.18 0.16 0.12 0.14 0.15 0.15 0.13 0.02

0.17 0.11 1.2 0.11 0.12 -0.1 14.7

1.3 2.2 0.2 0.18 0.18 0.15 -<0.01

2.1 2.5 --2.2 -2.2 0.16 0.19

1.53 0.15 0.16 1.76 1.8 1.94 -13

0.34 0.2 0.26 0.19 0.18 0.17 0.11 <0.01

2.1 2.4 1.8 2 0.1 9 0.2 0.1 8 0.1 7 0.1 6

50% Growth inhibition and the values are mean of four determinations ADR, 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

THESIS 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 C;
1

H NMR (300 MHz, CDCl3): 9.84 (bs, 1H), 8.31 (dd, 1H, J = 7.8, 1.5 Hz),

8.138.23 ( m, 2H), 7.727.86 ( m, 3H), 7.397.66 (m, 3H), 7.29 (d, 2H, J = 8.6 Hz), 6.79 (d, 2H, J = 7.8 Hz); ESIMS: m/z 299 (M+H)+. 10-(4-hydroxy-3-methoxybenzylidene)anthracen-9(10H)-one (18b) The compound 18b was prepared according to the method described for compound 18a by employing compound anthrone 16 (194 mg, 1 mmol), and 3-methoxy-4-hydroxy benzaldehyde 17b (152 mg, 1 mmol). Yield (241 mg, 72%). Mp: 102-104 C;
1

H NMR (300 MHz, CDCl3): 8.28.28 (m, 2H), 7.97 (d, 1H, J = 8.3 Hz), 7.66

(d, 1H, J = 8.3 Hz), 7.567.63 (m, 1H), 7.437.5 (m, 2H), 7.367.42 (m, 1H), 7.267.31 (dd, 1H, J = 8.3, 1.5 Hz), 6.876.91 (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); ESIMS: m/z 329 (M+H)+. 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 K2CO3 (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

THESIS (1:9). After completion of the reaction as indicated by the TLC, K2CO3 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%)
1

H NMR (200 MHz, CDCl3): 8.168.29 (m, 2H), 7.95 (d, 1H, J = 7.5 Hz),

7.537.63 (m, 2H), 7.417.51 (m, 2H), 7.38 (t, 1H, J = 7.5 Hz), 7.167.28 (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), 2.242.45 (m, 2H); ESIMS: m/z 420 (M+H)+. 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%)
1

H NMR (300 MHz, CDCl3): 8.168.30 (m, 2H), 7.96 (d, 1H, J = 7.5 Hz),

7.547.63 (m, 2H), 7.427.5 (m, 2H), 7.39 (t, 1H, J = 7.5 Hz), 7.167.29 (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), 1.892.05 (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%)
1

H NMR (200 MHz, CDCl3): 8.168.31 (m, 2H), 7.96 (d, 1H, J = 7.5 Hz),

7.537.63 (m, 2H), 7.427.51 (m, 2H), 7.39 (t, 1H, J = 7.5 Hz), 7.157.28 (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), 1.812.03 (m, 4H), 1.63 1.75 (m, 2H); ESIMS: m/z 448 (M+H)+

175

THESIS 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%)
1

H NMR (300 MHz, CDCl3): 8.22 (t, 2H, J = 7.5 Hz), 7.95 (d, 1H, J = 7.5

Hz), 7.63 (d, 1H, J = 8.3 Hz), 7.537.6 (m, 1H), 7.337.48 ( m, 3H), 7.22 7.31 (m, 1H), 6.85 (d, 1H, J = 8.3 Hz), 6.746.82 (m, 2H), 4.13 (t, 2H, J = 6 Hz), 3.573.68 (m, 5H), 2.252.42 (m, 2H); ESIMS: m/z 450 (M+H)+. 10-(4-(4-bromobutoxy)-3-methoxybenzylidene)anthracen-9(10H)-one (19e) The compound 19e was prepared according to the method described for compound 19a by employing compound 18b (328 mg, 1 mmol), and 1,4dibromobutane (647 mg, 3 mmol). Yield (415 mg, 89%)
1

H NMR (400 MHz, CDCl3): 8.25 (t, 2H, J = 8.3 Hz), 7.98 (d, 1H, J = 7.5

Hz), 7.577.67 (m, 2H), 7.367.50 ( m, 3H), 7.237.31 (m, 1H), 6.88 (d, 1H, J = 8.3 Hz), 6.766.82 (m, 2H), 4.05 (t, 2H, J = 6 Hz), 3.64 (s, 3H), 3.5 (t, 2H, J = 6.7, 6 Hz), 1.942.17 (m, 4H); ESIMS: m/z 463 (M)+. 10-(4-(5-bromopentoxy)-3-methoxybenzylidene)anthracen-9(10H)-one (19f) The compound 19f was prepared according to the method described for compound 19a by employing compound 18b (328 mg, 1 mmol), and 1,5 dibromopentane (689 mg, 3 mmol). Yield (432 mg, 90%)
1

H NMR (300 MHz, CDCl3): 8.23 (t, 2H, J = 7.5 Hz), 7.96 (d, 1H, J = 7.5

Hz), 7.62 (d, 1H, J = 8.3 Hz), 7.517.61 (m, 1H), 7.327.48 (m, 3H), 7.22 7.3 (m, 1H), 6.84 (d, 1H, J = 8.3 Hz), 6.746.81 (m, 2H), 4.11 (t, 2H, J = 6 Hz), 3.65 (s, 3H), 3.44 (t, 2H, J = 6.7, 6 Hz), 1.822.06 (m, 4H), 1.611.76 (m, 2H); ESIMS: m/z 478 (M+H)+.

176

THESIS (2S)-N-{4-(3-[4-[(10-oxo-9,10-dihydro-9anthracenyliden)methyl]phenoxy]propyl)oxy-5-methoxy-2nitrobenzoyl}-pyrrolidine-2-carboxaldehydediethylthioacetal (20a) To a solution of (2S)-N-[4-hydroxy-5-methoxy2-nitrobenzoyl]pyrrolidine-2carboxal dehydediethylthioacetal (15) (400 mg, 1 mmol) in dry acetone (15 mL) was added, anhydrous K2CO3 (276 mg, 2 mmol), 10-(4-(3bromopropoxy)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, K2CO3 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 C;
1

H NMR (300 MHz, CDCl3): 8.198.3 (m, 2H), 7.97 (d, 1H, J = 7.3 Hz),

7.537.66 (m, 3H), 7.347.5 (m, 3H), 7.197.32 (m, 3H), 6.716.83 (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), 3.173.29 (m, 2H), 2.69-2.88 (m, 4H), 2.18 2.35 (m, 2H), 1.942.14 (m, 3H), 1.731.90 (m, 1H), 1.261.39 (m, 6H); ESIMS: m/z 739 (M+H)+. (2S)-N-{4-(4-[4-[(10-oxo-9,10-dihydro-9anthracenyliden)methyl]phenoxy]butyl)oxy-5-methoxy-2nitrobenzoyl}-pyrrolidine-2-carboxaldehydediethylthioacetal (20b) The compound 20b was prepared according to the method described for compound 20a by employing (2S)-N-[4-hydroxy-5-methoxy2-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 C;

177

THESIS
1

H NMR (200 MHz, CDCl3): 8.188.3 (m, 2H), 7.98 (d, 1H, J = 7.3 Hz),

7.547.67 (m, 3H), 7.347.51 (m, 3H), 7.18-7.3 (m, 3H), 6.76.84 (m, 3H), 4.81 (d, 1H, J = 3.6 Hz), 4.594.71 (m, 1H), 4.17 (t, 2H, J = 5.1 Hz), 4.07 (t, 2H, J = 5.1 Hz), 3.91 (s, 3H), 3.163.29 (m, 2H), 2.672.85 (m, 4H), 2.19 2.35 (m, 1H), 1.92.15 (m, 6H), 1.721.9 (m, 1H), 1.261.4 (m, 6H); ESIMS: m/z 775 (M+Na)+. (2S)-N-{4-(5-[4-[(10-oxo-9,10-dihydro-9anthracenyliden)methyl]phenoxy]pentyl)oxy-5-methoxy-2nitrobenzoyl}-pyrrolidine-2-carboxaldehydediethylthioacetal (20c) The compound 20c was prepared according to the method described for compound 20a by employing (2S)-N-[4-hydroxy-5-methoxy2-nitrobenzoyl] (15) (400 mg, 1 mmol), and pyrrolidine-2-carboxal 10-(4-(5-bromopentoxy) dehydediethylthioacetal Mp: 124-125 C;
1

benzylidene)anthracen-9(10H)-one (19c) (447 mg, 1 mmol). Yield (715 mg, 93%). H NMR (400 MHz, CDCl3): 8.188.31 (m, 2H), 7.98 (d, 1H, J = 7.3 Hz),

7.537.67 (m, 3H), 7.337.51 (m, 3H), 7.197.31 (m, 3H), 6.726.84 (m, 3H), 4.81 (d, 1H, J = 3.6 Hz), 4.64.71 (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), 2.662.85 (m, 4H), 2.17 2.31 (m, 1H), 1.922.16 (m, 6H), 1.761.98 (m, 3H), 1.261.41 (m, 6H); ESIMS: m/z 767 (M+H)+. (2S)-N-{4-(3-[2-methoxy-4-[(10-oxo-9,10-dihydro-9anthracenyliden)methyl] phenoxy]propyl)oxy-5-methoxy-2nitrobenzoyl}-pyrrolidine-2-carboxaldehyde diethylthioacetal(20d) The compound 20d was prepared according to the method described for compound 20a by employing dehydediethylthioacetal Mp: 120-121 C;
1

(2S)-N-[4-hydroxy-5-methoxy2-nitrobenzoyl]pyrrolidine-2-carboxal (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%). H NMR (300 MHz, CDCl3): 8.188.27 (m, 2H), 7.95 (d, 1H, J = 8 Hz), 7.7

(s, 1H), 7.537.65 (m, 2H), 7.327.49 (m, 3H), 7.217.32 (m, 1H), 6.726.88 (m, 4H), 4.80 (d, 1H, J = 3.6 Hz), 4.584.73 (m, 1H), 4.33 (t, 2H, J = 5.8 Hz),

178

THESIS 4.22 (t, 2H, J = 5.8 Hz), 3.92 (s, 3H), 3.64 (s, 3H), 3.143.26 (m, 2H), 2.63 2.86 (m, 4H), 2.292.47 (m, 2H), 2.022.29 (m, 2H), 1.731.98 (m, 2H), 1.251.4 (m, 6H); ESIMS: m/z 769 (M+H)+. (2S)-N-{4-(4-[2-methoxy-4-[(10-oxo-9,10-dihydro-9anthracenyliden)methyl] phenoxy]butyl)oxy-5-methoxy-2nitrobenzoyl}-pyrrolidine-2-carboxaldehydediethyl thioacetal(20e) The compound 20e was prepared according to the method described for compound 20a by employing (2S)-N-[4-hydroxy-5-methoxy2-nitrobenzoyl] (15) (400 mg, 1 mmol), and pyrrolidine-2-carboxal 10-(4-(4-bromobutoxy)-3dehydediethylthioacetal Mp: 119-122 C;
1

methoxybenzylidene)anthracen-9(10H)-one (19e) (463 mg, 1 mmol). Yield (720 mg, 91%). H NMR (400 MHz, CDCl3): 8.238.3 (m, 2H), 8.02(d, 1 H, J = 8.1 Hz), 7.69

(s, 1 H), 7.67.68 (m, 2H), 7.457.54 (m, 2H), 7.387.44 (t, 1H, J = 6.7 Hz), 7.267.31 (m, 1H), 6.96.94 (m, 1H), 6.86.86 (m, 3H), 4.88 (d, 1H, J = 3.7 Hz), 4.674.75 (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), 3.23.32 (m, 2H), 2.682.87 (m, 4H), 2.212.34 (m, 1H), 2.052.15 (m, 5H), 1.92.02 (m, 1H), 1.741.86 (m, 1H), 1.291.39 (m, 6H); ESIMS: m/z 783 (M+H)+. (2S)-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 employing (2S)-N-[4-hydroxy-5-methoxy2-nitrobenzoyl] (15) (400 mg, 1 mmol), and pyrrolidine-2-carboxal 10-(4-(5-bromopentoxy)-3dehydediethylthioacetal Mp: 118-120 C;
1

methoxybenzylidene)anthracen-9(10H)-one (19f) (477 mg, 1 mmol). Yield (751 mg, 94%). H NMR (300 MHz, CDCl3): 8.218.26 (m, 2H), 7.96 (d, 1H, J = 8.3 Hz),

7.577.65 (m, 3H), 7.377.48 (m, 3H), 7.237.28 (m, 1H), 6.856.91 (m,

179

THESIS 1H), 6.766.78 (m, 3H), 4.81 (d, 1H, J = 3.7 Hz), 4.634.69 (m, 1H), 4.02 4.14 (m, 4H), 3.93 (s, 3H), 3.64 (s, 3H), 3.163.3 (m, 2H), 2.632.87 (m, 4H), 2.162.38 (m, 2H), 1.872.14 (m, 5H), 1.681.87 (m, 3H), 1.281.39 (m, 6H); ESIMS: m/z 797 (M+H)+. 7-Methoxy-8-[3-{4-[(10-oxo-9,10-dihydro-9anthracenyliden)methyl]phenoxy} propoxy]-(11aS)-1,2,3,11atetrahydro-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 material as 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 Na 2SO4. 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 C;
1

H NMR (200 MHz, CDCl3): 8.198.3 (m, 2H), 7.98 (d, 1H, J = 8.3 Hz),

7.547.67 (m, 3H), 7.337.52 (m, 4H), 7.177.3 (m, 3H), 6.726.86 (m, 3H), 4.034.19 (m, 4H), 3.92 (s, 3H), 3.773.85 (m, 1H), 3.683.74 (m, 1H), 3.533.62 (m, 1H), 2.242.37 (m, 2H), 1.962.18 (m, 4H); ESIMS: m/z 585 (M+H)+. 180

THESIS 7-Methoxy-8-[4-{4-[(10-oxo-9,10-dihydro-9anthracenyliden)methyl]phenoxy}butoxy]-(11aS)-1,2,3,11atetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (22b) This compound was prepared according to the method described for the compound 22a employing 20b (752 mg, 1 mmol) which reduction with SnCl2.2H2O (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 C;
1

H NMR (300 MHz, CDCl3): 8.188.29 (m, 2H), 7.98 (d, 1H, J = 8.3 Hz),

7.557.66 (m, 3H), 7.357.53 (m, 4H), 7.177.29 (m, 3H), 6.736.84 (m, 3H), 4.044.19 (m, 4H), 3.93 (s, 3H), 3.773.86 (m, 1H), 3.693.74 (m, 1H), 3.533.63 (m, 1H), 2.142.22 (m, 2H), 1.8521.97 (m, 6H); ESIMS: m/z 599 (M+H)+. 7-Methoxy-8-[5-{4-[(10-oxo-9,10-dihydro-9anthracenyliden)methyl]phenoxy} pentoxy]-(11aS)-1,2,3,11atetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (22c) This compound was prepared according to the method described for the 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 C;
1

H NMR (300 MHz, CDCl3): 8.198.31 (m, 2H), 7.98 (d, 1H, J = 8.3 Hz),

7.557.67 (m, 3H), 7.347.53 (m, 4H), 7.167.3 (m, 3H), 6.736.86 (m, 3H), 4.044.19 (m, 4H), 3.92 (s, 3H), 3.763.85 (m, 1H), 3.683.74 (m, 1H), 3.523.62 (m, 1H), 2.242.36 (m, 2H), 1.852.14 (m, 6H), 1.64 1.76 (m, 2H); ESIMS: m/z 613 (M+H)+.

181

THESIS 7-Methoxy-8-[3-{2-methoxy-4-[(10-oxo-9,10-dihydro-9anthracenyliden) methyl]phenoxy}propoxy]-(11aS)-1,2,3,11atetrahydro-5H-pyrrolo[2,1-c][1,4] benzodiazepin-5-one(22d) This compound was prepared according to the method described for the compound 22a employing 20d (768 mg, 1 mmol) which reduction with SnCl2.2H2O (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 C;
1

H NMR (200 MHz, CDCl3): 8.198.25 (m, 2H), 7.97 (d, 1H, J = 8.3 Hz),

7.557.67 (m, 3H), 7.327.51 (m, 4H), 7.247.3 (m, 1H), 6.836.91 (m, 1H), 6.726.81 (m, 3H), 4.084.13 (m, 4H), 3.91 (s, 3H), 3.763.85 (m, 1H), 3.683.72 (m, 1H), 3.63 (s, 3H), 3.523.6 (m, 1H), 2.262.36 (m, 2H), 1.99 2.18 (m, 4H); ESIMS: m/z 615 (M+H)+. 7-Methoxy-8-[4-{2-methoxy-4-[(10-oxo-9,10-dihydro-9anthracenyliden) methyl]phenoxy}butoxy]-(11aS)-1,2,3,11atetrahydro-5H-pyrrolo[2,1-c][1,4] benzodiazepin-5-one(22e) This compound was prepared according to the method described for the 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 C;
1

H NMR (200 MHz, CDCl3): 8.218.26 (m, 2H), 7.96 (d, 1H, J = 8.3 Hz),

7.557.66 (m, 3H), 7.317.48 (m, 4H), 7.237.31 (m, 1H), 6.836.9 (m, 1H), 6.746.8 (m, 3H), 4.064.13 (m, 4H), 3.92 (s, 3H), 3.763.86 (m, 1H), 3.66 3.71 (m, 1H), 3.62 (s, 3H), 3.523.58 (m, 1H), 2.272.36 (m, 2H), 1.982.14 (m, 6H);

182

THESIS
13

C NMR (75 MHz, CDCl3): 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, ESIMS: m/z 629 (M+H)+. 7-Methoxy-8-[5-{2-methoxy-4-[(10-oxo-9,10-dihydro-9anthracenyliden) methyl]phenoxy}pentoxy]-(11aS)-1,2,3,11atetrahydro-5H-pyrrolo[2,1-c][1,4] benzodiazepin-5-one(22f) This compound was prepared according to the method described for the 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 C;
1

H NMR (200 MHz, CDCl3): 8.198.26 (m, 2H), 7.97 (d, 1H, J = 8.3 Hz),

7.547.67 (m, 3H), 7.317.51 (m, 4H), 7.247.31 (m, 1H), 6.846.91 (m, 1H), 6.736.81 (m, 3H), 4.074.15 (m, 4H), 3.91 (s, 3H), 3.763.86 (m, 1H), 3.683.71 (m, 1H), 3.63 (s, 3H), 3.523.61 (m, 1H), 2.262.35 (m, 2H), 1.962.15 (m, 6H), 1.641.75 (m, 2H);
13

C NMR (75 MHz, CDCl3):

ESIMS: m/z 643 (M+H)+.

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 appropriate dilution in aqueous buffer (10 mM NaH2PO4/NaH2PO4, 1 mM Na2EDTA, pH 7.000.01) containing CT-DNA, (100 M in phosphate) and the PBD (20 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

183

THESIS solutions were incubated at 37

C 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 1 C min-1 in the 40 90

C range. DNA helix-coil transition temperatures

(Tm) were determined from the maxima in the d (A260)/dT derivative plots. Results for each compound are shown as mean standard derivation from 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 Tm = Tm (DNA+PBD)- Tm (DNA alone), where the Tm 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 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 L at plating densities depending on the doubling time of individual cell lines. The microtiter plates were incubated at 37

C, 5% CO2, 95% air and 100%

relative humidity for 24 h prior to addition of experimental drugs. Aliquots of 10 L of the drug dilutions were added to the appropriate microtiter wells already containing 90 L of cells, resulting in the required final drug concentrations. Each compound was evaluated for four concentrations (0.1, 1, 10 and 100 M) and each was done in triplicate wells. Plates were incubated further for 48 h, and assay was terminated by the addition of 50 184

THESIS L of cold trichloro acetic acid (TCA) (final concentration, 10% TCA) and plates were again incubated for 60 min at 4

C. The plates were washed

five times with tap water and air-dried. Sulforhodamine B (SRB) solution (50 L) 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 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. Kemeny, 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., Dssmann, H., Burger, A.M., Prehn, J.H., Gnther, 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

THESIS 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.; Bhmb, K.; Mller, K.; Prinz, H. Eur. J. Med. Chem., 2010, 45, 3354. 9. Nickel, H. C.; Schmidt, P.; Bhmb, K. J.; Baasner, S.; Mller, K.; Gerlach, M.; Unger, E.; Gnther, E. G.; Prinz, H. Eur. J. Med. Chem., 2010, 45, 3420. 10. Kemeny, 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, Mller, K. Eur J. Med. Chem. 2010, 45, 5278. 15. Puvvada, M. S.; Hartley, J. A.; Jenkins, T. C.; Thurston, D. E. Nucleic Acids 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.

CHAPTER-IV/SECTION-B

186

THESIS

SYNTHESIS AND BIOLOGICAL EVALUATION OF CHALCONEPYRROLOBENZODIAZEPINE DIMERS AS ANTICANCER AGENTS

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

THESIS anthramycin (1), sibiromycin (2), tomaymycin (3), chicamycin A (4), neothramycin A (5), and B (6), and DC-81 (7) (Figure 1). 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-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. DNAbinding ability has been observed by thermal denaturation studies with CTDNA ( Tm > 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 crosslinking 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

THESIS
H3C OR H N N 1 R= OH or OCH3 anthramycin O CONH2 H3C H3CHN CH3 O OH OH OCH3 H CH3O HO O H N N O 2 sibiromycin OCH3 H N O 4 chicamycin HO H3CO O 7 DC-81 Figure 1. Naturally occurring PBDs N N OH OH H CH3

HO H3CO

N N

H CH3

HO H3CO

H N

O 3 tomamycin HO H3CO O N N R1 R2 H

5 ( R1 = H; R2 = OH) 6 ( R1 = OH, R2 = H) neothramycin A and B

(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.

4.2.1. CHALCONES
Chemically chalcones comprise of open-chain flavonoids in which the two aromatic rings are joined by a three-carbon ,-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

THESIS 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).
O OH OMe OMe 8 MeO MeO OMe 9 (SD400) OH OMe

O MeO MeO

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 - and -subunits. Many molecules based on a chalcone scaffold have been synthesized to improve their biological profile, including their capability as 190

THESIS 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(2fluorobenzylidene)-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 inflammatory properties and (12, CLEFMA) and (13) are have been dimers have shown promising advanced as synthetic analogs of curcumin for anti-cancer activity and antithese antiproliferative activity against various cancer cell lines33 (Figure 3).

191

THESIS
OH O MeO HO 10 (curcumin) Cl O Cl H3CO N O O OH 12 (CLEFMA) HO N CH3 13 OMe OH N H 11 (EF24) O OCH3 OH F O F

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 NO2 O Me N 14 O OMe 15

16

17

Figure 4

192

THESIS

4.2.2. PRESENT WORK

The present work describes the design, synthesis, DNA binding affinity 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 and PBD entities with varying alkane spacers and have been evaluated them for their antitumour activity and DNA-binding ability.

4.2.2.1. SYNTHESIS OF PBD PRECURSORS

The precursor (2S)-N-[4-(n-Bromoalkooxy)-5-methoxy-2-nitrobenzoyl] pyrrolidine-2-carboxaldehydediethylthioacetal 19a-c have been prepared by employing available vanillic (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]pyrolidine-2acid. Compound 18 undergoes etherification with carboxaldehydediethylthio acetal 18 which was prepared from commercially dibromoalkanes in the presence of K2CO3 in acetone gives corresponding compounds 19a-c (Scheme 1).

193

THESIS
HO MeO O 18 n = 2-4 19a-c SCHEME 1. REAGENTS AND CONDITIONS: I) DIBROMOALKANE, K2CO3, ACETONE, REFLUX, 14H NO2 N CH(SEt)2 (i) Br O ( )n MeO O NO2 N CH(SEt)2

4.2.2.2. SYNTHESIS 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.
O R R
1

CHO CH3 (i) + R4 R

3

O R
1

R4 R3 22a,b

R2

20a,b

21a,b

20a; R1 = H, R2 = OH 20b; R1 = OH, R2 = H

22a; R1 = H, R2 = OH, R3 = OH, R4 = H 22b; R1 = OH, R2 = H, R3 = H, R4 = OH

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. SYNTHESIS OF C-8 LINKED CHALCONE-PBD DIMERS

Compound 19a-c has been coupled to dihydroxychalcones 22a,b in the presence of K2CO3 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

THESIS amino compounds on deprotection with HgCl2/CaCO3 provide corresponding imines 25a-f (Scheme-3).
O R 22a; R = 4,4'-dihydroxy 22b; R = 3,3'-dihydroxy (i) R Br + O ( )n MeO 19a-c O NO2 N CH(SEt)2

(EtS)2HC

O 2N N O

( )n

chalcone

O ( )n MeO

NO2 N O

CH(SEt)2

OMe 23a-f (ii)

(EtS)2HC

H2N N O

( )n

chalcone

O 24a-f

O ( )n MeO

NH2 N O

CH(SEt)2

OMe (iii)

H N

( )n

chalcone

O ( )n MeO

N N O

OMe O 25a-f

O 25a-c; Chalcone =

O O

n = 2-4

25d-f; Chalcone =

O n = 2-4

Scheme 3. Reagents and conditions: (i) K2CO3, acetone, reflux, 12h; (ii) SnCl2.2H2O, MeOH, 4 h, reflux; (iii) HgCl2, CaCO3, MeCN, H2O, (4:1) rt, 12 h

195

THESIS

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 37 C for 0 h and 18 h prior to analysis. Samples are monitored at 260 nm using a Beckman DU7400 spectrophotometer fitted with high performance temperature controller and heated at 1

C/min in the range of 40-95

C. DNA helix-coil transition

temperatures are given by: Tm = Tm(DNA+PBD)Tm(DNA alone), where the Tm 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 ( Tm) for each compound is examined at 0 h and 18 h of incubation at 37

C.

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 37 C with ligand / DNA molar ratio of 1:5. The increase in the helix melting temperature ( Tm) for each compound has been examined at 0 h and 18 h incubation at 37 C. 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 Tm 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 Tm 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.

196

THESIS

Table 1.Thermal denaturation data for chalcone-PBD dimers with calf thymus (CT)-DNA Tm (oC)a after incubation at 37 oC [PBD]:[DNA] for Compound molar ratiob 0h 18 h 25a 25b 25c 25d 25e 25f DC-81
a

1:5 1:5 1:5 1:5 1:5 1:5 1:5

4.8 4.6 4.7 4.1 3.5 4.5 0.3

b

5.4 4.9 5.1 4.8 3.9 4.7 0.7

For CT-DNA alone at pH 7.00 0.01, Tm = 68.5 0C 0.01 (mean value from 10 separate For a 1:5 molar ratio of [PBD]/[DNA],

determinations), all Tm values are 0.1 - 0.2 0C.

where CT-DNA concentration = 100 M and ligand concentration = 20 M in aqueous sodium phosphate buffer [10 mM sodium phosphate + 1 mM EDTA, pH 7.00 0.01].

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 104

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 M whereas DC-81 showed IC50 values in the range of 2-26.2 M. The synthesized novel chalcone-PBD dimers exhibited significant anticancer activity against PC-3 human prostate cancer cell line (IC50 range, 0.008 8.3 M) 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

THESIS M) and A-375 cell linec (IC50, 0.01 M). The compound 25c also showed significant activity against PC-3 cell line (IC50, 0.007 M). Among the chalconePBD 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 M) for compounds 25a-f in selected human cancer cell lines. Compound 25a 25b 25c 25d 25e 25f DC-81
a

IC50 values (M) HT-29b 10.4 0.97 15.8 2.02 26.4 23.5 8.1 PC-3c 0.008 0.51 0.007 0.11 5.1 8.3 2 A-375d 0.01 3.05 0.97 3 8.3 27.8 26.2 A 549e 19.0 7.3 8.9 28.5 -11.7 -B-16f 29.1 19.2 18.6 2.69 22.9 -21.1
b

50% Growth inhibition and the values are mean of three determinations, prostate cancer, d skin cancer, e lung cancer, f Mouse macrophages cell line.

colon cancer,

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 superior anticancer activity as well as high DNA-binding ability (5.4 C).

198

THESIS

4.2.5. EXPERIMENTAL SECTION

(2S)-N-[4-(3-Bromopropoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2carboxa- ldehydediethylthioacetal (19a) To a solution of compound 18 (400 mg, 1 mmol) in dry acetone (15 ml) was added, anhydrous K2CO3 (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), K2CO3 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%).
1

H NMR (200 MHz, CDCl3) 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-2carboxaldehyde diethylthioacetal (19b) The compound 19b was prepared according to the method described for compound 19a by employing compound 18 (400 mg, 1 mmol), anhydrous K2CO3 (553 mg, 4 mmol) and 1, 4-dibromopropane (256 mg, 1.2 mmol). Yield (440 mg, 96%).
1

H NMR (200 MHz, CDCl3) 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

THESIS FABMS: 535 [M]+.

(2S)-N-[4-(5-Bromopentyloxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine2-carboxaldehydediethylthioacetal (19c) The compound 19c was prepared according to the method described for compound 19a by employing compound 18 (400 mg, 1 mmol), anhydrous K2CO3 (553 mg, 4 mmol) and 1, 5-dibromopropane (270 mg, 1.2 mmol). Yield (440 mg, 96%).
1

H NMR (200 MHz, CDCl3) 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), 3.523.6 (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) and 4-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 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 22a. Yield (185 mg, 77%). Mp: 178179 oC
1

H NMR (200 MHz, CDCl3): 7.98 (d, 2H, J = 9 Hz), 7.72 (d, 1H, J = 15.1 Hz),

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

THESIS (E)-1,3-bis(3-hydroxyphenyl)prop-2-en-1-one (22b) The compound 22b was prepared according to the method described for compound 22a by employing compound 3-hydroxyacetophenone 20b (136 mg, 1mmol), and 3-hydroxybenzaldehyde 21b (122 mg, 1 mmol). Yield (190 mg, 78%). Mp: 181183 oC
1

H NMR (300 MHz, CDCl3 + DMSOd6): 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 (dd, 1H, J = 8.3, 7 2.4 Hz), 6.76 7.83 (dd, 1H, J = 8.3, 1.88 Hz); ESIMS: m/z 241 (M+H)+. (E)-1,3-bis[4-{3-[(2S)-N-(4-oxy-5-methoxy-2aminobenzoyl)pyrrolidine-2carboxaldehydediethylthioacetal]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 K2CO3 (552 mg, 4 mmol), (E)-1,3bis(4-hydroxyphenyl)prop-2-en-1-one (22a) (240 mg, 1 mmol) and the mixture 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, K2CO3 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: 207209 oC
1

H NMR (200 MHz, CDCl3): 8.02 (d, 2H, J = 9 Hz), 7.78 (d, 1H, J = 15.8 Hz),

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

THESIS 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-2aminobenzoyl)pyrrolidine-2carboxaldehydediethylthioacetal]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-2nitrobenzoyl] 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: 205207 oC
1

H NMR (200 MHz, CDCl3): 8.03 (d, 2H, J = 8.9 Hz), 7.78 (d, 1H, J = 15.1 Hz),

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 (m, 2H), 2 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-2aminobenzoyl)pyrrolidine-2-carboxaldehyde diethylthioacetal]pentoxy}phenoxy]prop-2-en-1-one (23c) The compound 23c was prepared according to the method described for compound 23a by employing (2S)-N-[4-(5-Bromopentoxy)-5-methoxy-2nitrobenzoyl] 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

THESIS Mp: 208209 oC

1

H NMR (200 MHz, CDCl3): 8.03 (d, 2H, J = 8.9 Hz), 7.77 (d, 1H, J = 15.5 Hz),

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-2aminobenzoyl)pyrrolidine-2carboxaldehydediethylthioacetal]propoxy}phenoxy]prop-2-en-1-one (23d) The compound 23d was prepared according to the method described for compound 23a by employing (2S)-N-[4-(3-Bromopropoxy)-5-methoxy-2nitrobenzoyl] 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: 214216 oC
1

H NMR (500 MHz, CDCl3): 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), 6.916.96 (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

THESIS (E)-1,3-bis[3-{4-[(2S)-N-(4-oxy-5-methoxy-2aminobenzoyl)pyrrolidine-2carboxaldehydediethylthioacetal]butoxy}phenoxy]prop-2-en-1-one (23e) The compound 23e was prepared according to the method described for compound 23a by employing (2S)-N-[4-(4-Bromobutoxy)-5-methoxy-2nitrobenzoyl] 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: 215217 oC
1

H NMR (400 MHz, CDCl3): 7.66 7.74 (m, 3H), 7.56 (d, 1H, J = 7.9 Hz), 7.55

(d, 1H, J = 15.1 Hz), 7.47 (d, 1H, J = 15.1 Hz), 7.36 (t, 1H, J = 7.9 Hz), 7.29 (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), 6.916.95 (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 (m, 2H), 2 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-2aminobenzoyl)pyrrolidine-2carboxaldehydediethylthioacetal]pentoxy}phenoxy]prop-2-en-1-one (23f) The compound 23f was prepared according to the method described for compound 23a by employing (2S)-N-[4-(5-Bromopentoxy)-5-methoxy-2nitrobenzoyl] 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: 213215 oC
1

H NMR (200 MHz, CDCl3): 7.66 7.74 (m, 3H), 7.57 (d, 1H, J = 7.9 Hz), 7.54

(d, 1H, J = 15.1 Hz), 7.47 (d, 1H, J = 15.1 Hz), 7.36 (t, 1H, J = 7.9 Hz), 7.28 (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

THESIS Hz), 6.96.96 (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), 1.891.95 (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-5Hpyrrolo[2,1-c][1,4] benzodiazepin-5-one]propyloxy}phenoxy]prop-2en-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 Na 2SO4. 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: 191193 oC
1

H NMR (200 MHz, CDCl3): 8.01 (d, 2H, J = 9 Hz), 7.73 (d, 1H, J = 15.8 Hz),

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

THESIS (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); ESIMS: m/z 813 (M+H)+. (E)-1,3-bis[4-{4-[7-Methoxy-8-oxy-(11aS)-1,2,3,11a-tetrahydro-5Hpyrrolo[2,1-c][1,4] benzodiazepin-5-one]butyloxy}phenoxy]prop-2en-1-one (25b) This compound was prepared according to the method described for the 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 pure product 25b. Yield (450 mg, 53%). Mp: 190192 oC
1

H NMR (200 MHz, CDCl3): 7.99 (d, 2H, J = 9 Hz), 7.72 (d, 1H, J = 15.8 Hz),

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);
13

C NMR (75 MHz, CDCl3): 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 ESIMS: m/z 841 (M+H)+.

(E)-1,3-bis[4-{5-[7-Methoxy-8-oxy-(11aS)-1,2,3,11a-tetrahydro-5Hpyrrolo[2,1-c][1,4] benzodiazepin-5-one]pentyloxy}phenoxy]prop-2en-1-one (25c) This compound was prepared according to the method described for the compound 25a employing 23c (1177 mg, 1 mmol) which reduction with SnCl2.2H2O (2.24 mg, 10 mmol) gives amino compound 24c. 206

THESIS 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: 191192 oC
1

H NMR (200 MHz, CDCl3): 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); ESIMS: m/z 869 (M+H)+. (E)-1,3-bis[3-{3-[7-Methoxy-8-oxy-(11aS)-1,2,3,11a-tetrahydro-5Hpyrrolo[2,1-c][1,4] benzodiazepin-5-one]propoxy}phenoxy]prop-2en-1-one (25d) This compound was prepared according to the method described for the 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 mmol), CaCO3 (500 mg, 5 mmol) in acetonitrile-water (4:1) to obtain the pure product 25d. Yield (415 mg, 51%). Mp: 187189 oC
1

H NMR (300 MHz, CDCl3): 7.65 7.71 (m, 3H), 7.62 (d, 2H, J = 4.5 Hz), 7.56

(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), 6.916.96 (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);
13

C NMR (75 MHz, CDCl3): 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

THESIS ESIMS: m/z 813 (M+H)+. (E)-1,3-bis[3-{4-[7-Methoxy-8-oxy-(11aS)-1,2,3,11a-tetrahydro-5Hpyrrolo[2,1-c][1,4] benzodiazepin-5-one]butoxy}phenoxy]prop-2-en1-one (25e) This compound was prepared according to the method described for the 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 mmol), CaCO3 (500 mg, 5 mmol) in acetonitrile-water (4:1) to obtain the pure product 25e. Yield (455 mg, 53%). Mp: 186188 oC
1

H NMR (200 MHz, CDCl3): 7.65 7.74 (m, 3H), 7.61 (d, 2H, J = 4.5 Hz), 7.56

(d, 1H, J = 7.9 Hz), 7.55 (d, 1H, J = 15.1 Hz), 7.47 (d, 1H, J = 15.1 Hz), 7.36 (t, 1H, J = 7.9 Hz), 7.29 (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), 6.916.95 (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); ESIMS: m/z 841 (M+H)+. (E)-1,3-bis[3-{5-[7-Methoxy-8-oxy-(11aS)-1,2,3,11a-tetrahydro-5Hpyrrolo[2,1-c][1,4] benzodiazepin-5-one]pentoxy}phenoxy]prop-2en-1-one (25f) This compound was prepared according to the method described for the 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 mmol), CaCO3 (500 mg, 5 mmol) in acetonitrile-water (4:1) to obtain the pure product 25f. Yield (485 mg, 55%). Mp: 187188 oC
1

H NMR (300 MHz, CDCl3): 7.66 7.73 (m, 3H), 7.62 (d, 2H, J = 4.5 Hz), 7.57

(d, 1H, J = 7.9 Hz), 7.53 (d, 1H, J = 15.1 Hz), 7.46 (d, 1H, J = 15.1 Hz), 7.36 208

THESIS (t, 1H, J = 7.9 Hz), 7.28 (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 Hz), 6.96.96 (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); ESIMS: m/z 869 (M+H)+.

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 appropriate dilution in aqueous buffer (10 mM NaH2PO4/NaH2PO4, 1 mM Na2EDTA, pH 7.000.01) containing CT-DNA, (100 M in phosphate) and the PBD (20 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 37

C 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 1 C min-1 in the 40 90

C range. DNA helix-coil transition temperatures

(Tm) were determined from the maxima in the d (A260)/dT derivative plots. Results for each compound are shown as mean standard derivation from 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 Tm = Tm (DNA+PBD)- Tm (DNA alone), where the Tm 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

THESIS 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 l of fresh serum free media and 10 l of MTT reagent (5mg/ml) and plates were incubated at 37 oC for 4 h, there after the above media was replaced with 200 l 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

THESIS

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THESIS

LIST OF PUBLICATIONS AND PATENTS

215

THESIS

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 4 -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) 18761880 5. Synthesis and Anticancer Activity of Chalcone-pyrrolobenzodiazepine Conjugates Linked via 1,2,3-triazole Ring Side-armed with Alkane Spacers. 216

THESIS 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 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 molecules employing 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 Carbazoleconjugates: Design,

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

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THESIS 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 agents and process for the preparation thereof Ahmed Kamal, A Malla Reddy, P Suresh, N Sankara Rao and Rajesh VCRNC Shetti. Indian patent application no. 266/DEL/08. PCT application no. PCT/IN2011/000187 4. Synthesis of new benzothiazole derivatives as potential anti-tubercular agents Ahmed 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 Indian patent application no. 2179/DEL/2010 5. Biological evaluation of 4-aza-2,3-didehydropodophyllotoxin analogues possessing potent antitumour activity Ahmed Kamal, P Suresh, B Ashwini Kumar, A Malla Reddy, P. Venkat Reddy and Jaki Rasheed Tamboli Indian patent application no. 2887/DEL/2010

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THESIS 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 Indian patent application no. 683/DEL/10 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 Indian patent application no. 678/DEL/10 8. Chalcone linked pyrrolo[2,1-C][1,4] benzodiazepine hybrids as potential anticancer agents and process for the preparation thereof Ahmed Kamal, B Rajendra Prasad and A Malla Reddy Indian patent application no. 537/DEL/08. 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 P Venkat Reddy Indian patent application no. 2697/DEL/10 10. Quinazoline linked pyrrolo[2,1-C][1,4] benzodiazepine hybrids as

potential anticancer agents and process for the preparation thereof Ahmed Kamal, B Rajendra Prasad and A Malla Reddy Indian patent application no. 518/DEL/08. 11. Benzophenone-piperazine linked pyrrolo[2,1-c][1,4]

benzodiazepine hybrids as potential anticancer agents and process for the preparation there of Ahmed Kamal, B Rajendra Prasad and A Malla Reddy Indian patent application no. 787/DEL/08.

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THESIS

SYMPOSIUM AND CONFERENCES

ATTENDED

1. Participated in 12th CRSI National Symposium in Chemistry & 4th CRSIRSC 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 26th February -1st March 2009.

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