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T HESIS
S YNTHESIS AND ANTICANCER ACTIVITY OF NEW P YRROLO[2,1-C][1,4]
BENZODIAZEPINES AND COMBRETASTATIN DERIVATIVES
T HESIS
SUBMITTED TO K AKATIYA UNIVERSITY
FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
(IN CHEMISTRY )
B Y
ADLA MALLA REDDY
UNDER THE SUPERVISION OF
DR. AHMED K AMAL
DIVISION OF ORGANIC CHEMISTRY -IINDIAN INSTITUTE OF CHEMICAL TECHNOLOGY , H YDERABAD
JUNE, 2011
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T HESIS
S YNTHESIS AND ANTICANCER ACTIVITY OF NEW P YRROLO[2,1-C][1,4]
BENZODIAZEPINES AND COMBRETASTATIN DERIVATIVES
T HESIS
SUBMITTED TO K AKATIYA UNIVERSITY
FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
(IN CHEMISTRY )
B Y
ADLA MALLA REDDY
UNDER THE SUPERVISION OF
DR. AHMED K AMAL
DIVISION OF ORGANIC CHEMISTRY -IINDIAN INSTITUTE OF CHEMICAL TECHNOLOGY , H YDERABAD
JUNE, 2011
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T HESIS
DECLARATION
I hereby declare that the original research work embodied in the thesis
entitled “S YNTHESIS AND ANTICANCER ACTIVITY OF NEW P YRROLO[2,1-C][1,4]
BENZODIAZEPINES AND COMBRETASTATIN DERIVATIVES“ submitted to Kakatiya
University for the award of degree of Doctor of Philosophy (Ph.D) in
Chemistry of the faculty of Physical Sciences is the outcome of the
investigation carried out by me under the supervision of Dr. Ahmed Kamal,
Scientist H (Director level), IICT, Hyderabad. I declare that the workincorporated is original and due acknowledgement has been made wherever
it is not so. The same has not been submitted elsewhere for any degree or
diploma.
I also declare that I myself solely responsible for the genuineness of
the findings/observations pertaining to these study in order to complete this
thesis.
Adla Malla Reddy
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T HESIS
Dedicated to My Beloved Parents
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T HESIS
ACKNOWLEDGEMENTS
It gives me an immense pleasure and pride to express my sincere gratitude and respect
for my teacher and guide Dr. Ahmed Kamal , Directors Grade Scientist, Division of Organic
Chemistry-I, IICT, Hyderabad, for his expert and inspiring guidance. I proclaim myindebtedness to him for his constant encouragement along with the useful suggestions and
constructive criticisms during the entire tenure of this work. I consider myself fortunate in that it
would have been impossible to achieve this goal without his support and care.
I am indebted to the director Dr. J. S. Yadav for having given me an opportunity to
carryout the work and allowing me to submit in the form of thesis. It is a great privilege for me
to be associated with Dr. J. M. Rao , Head, Division of Organic Chemistry–I for his kind help
and encouragement.
My heartful thank to Dr. M. Venkateswara Rao and Dr. Manika Pal Bhadra for their
constant support, encouragement and timely advice. I take this opportunity to record my
appreciation to spectroscopic and analytical divisions of IICT, especially to Dr. A. C. Kunwar
and Dr. R. Srinivas.
I am grateful to Prof. G. Venkateshwar Rao , Head, Department of Chemistry, Kakatiya
University, Prof. Ch. Sanjeeva Reddy (Board of studies Chairman, K.U.), Prof. T. Bhasker
Rao (Dean, Faculty of Science, K.U.) and Prof. N. Vasudeva Reddy for their invaluable
suggestions and advices while writing thesis.
My special thanks to Dr. Rajesh V.C.R.N.C. Shetti, Paidakula Suresh, Dr. B. Rajendra
Prasad , Dr. N. Shankaraiah, J.N.S.R.Murthy, N. Sankara Rao and Dr. Janaki Ramaiah for
their cooperation in my research.
I am grateful to my lab mates K.Srinivas Reddy , Devaiah, Laxma reddy , Kaleem ,
P.Praveen, Naseer , Krishnaji , Venkat , Adil , Malik , Ameer , Rajender , Azeez , Bharathi ,
Surendra , Dastagiri , Prabhakar, Venkatreddy , Sreekanth, Vishwanath , Ramakrishna , Kashi
Reddy , Raju, Ratna Reddy, Sheshadri , Subbareddy , Santhosh Reddy, Saidi Reddy, balakrihna,
Bazi, Fazil, Asharf, Srinivas, Narasimha , Swapna , Jaki, Farheen Sulthana, Bharath, Ali,
Premsagar, Subbarao, Imnthiaz and my other labmates Naresh , Somaiah, Venkataiah,
Gourishankar, Markandeya, Jitender and Prasad.
I acknowledge the help received from Usha , Sainadh, Shyam , Chandrashekar , Balaraj ,
and Padma.
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T HESIS
I always feel thankful to have friends like Kota Srinivas, Alli Satish, Udutala Komarelli,
Srinivas yadav, Sattenapalli Narasimha, P. Venkata Raji Reddy, Bala Bhaskar, Pitta Bhaskar
Reddy, Madhuraju, Doma Mahender Reddy, Muppidi Venugopal and Gangarapu Srinivas.
It is also an appropriate time to remember all my teachers and professors who at various
stages of my educational carrier encouraged me to reach this level and it all is the fruit of their
teaching and blessings.
I owe more than myself to my beloved father Sri Indra Reddy and mother Mallakka , who
always dreamt that I reach golden heights and sky is the only limit to my success. To them I
dedicate this thesis. On this occasion my heart goes for my brothers Mr . Srinivas Reddy , Mr .
Damodar Reddy and my elder brother’s children Pooja and Manoj Reddy.
My heartfelt thanks to my life partner Smt . Swapna for her unflinching moral support at
every stage and my sweet kisses to my child baby Ananya.
I take this opportunity to record my appreciation to spectroscopic and analytical division
of IICT.
I also thank National Cancer Institute (NCI), USA, Advanced Center for Treatment,
Research and Education in Cancer (ACTREC), Navi Mumbai and Regional Research
Laboratory (RRL), Jammu for helping in the biological studies.
Financial assistance from the Council of Scientific and Industrial Research (CSIR), New
Delhi in the form of fellowship is gratefully acknowledged. Finally, I thank Director, IICT, for allowing me to submit my work in the form of thesis.
(Adla Malla Reddy )
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T HESIS
GENERAL R EMARKS
1H NMR and 13C NMR spectra are recorded on Varian Gemini 200 or Varian Unity 400 or
Varian Inova 500 or Bruker Avance 300 MHz. Making a solution of samples in
CCl4/CDCl3 (1:1) solvent using tetramethylsilane (TMS) as the internal standard unless
otherwise mentioned, and are given in the δ scale. The standard abbreviations s, d, t, q, m,
dd, dt, ABq, br s refer to singlet, doublet, triplet, quartet, multiplet, doublet of a doublet,
doublet of a triplet, AB quartet and broad singlet respectively.
Mass spectra recorded on CEC-21-110B, Finnigan Mat 1210 or MICROMASS-7070
spectrometers operating at 70eV using a direct inlet system. If necessary, FABMS is
recorded.
Melting points are determined on an Electrothermal melting point apparatus and are
uncorrected.
All reactions are monitored by thin layer chromatography (TLC) carried out on 0.25 mm E.
Merck silica gel plates (60F-254) with UV light, iodine as probing agents. Acme (India)
silica gel (finer than 200 mesh) is used for flash chromatography.
The reactions wherever anhydrous conditions needed are carried out under the positive
pressure of nitrogen atmosphere using dry and freshly distilled solvents.
All solvents and reagents were purified by standard techniques. All evaporation of solvents
was carried out under reduced pressure on Buchi-RE-121 rotary evaporator below 45 °C.
Yield reported are isolated yields of material judged homogeneous by TLC and NMR
spectroscopy.
The names of all compounds given in the experimental section were taken from
ACD/Name, Version 1.0.
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T HESIS
ABBREVIATIONS
BF3
.
OEt2 : Boron trifluoride diethyletherateBnBr : Benzylbromide
n-BuLi : n-Butyl Lithium
CaCO3 : Calcium carbonate
CCl4 : Carbontetrachloride
CH3CN : Acetonitrile
CDCl3 : Deuterated chloroform
Cs2CO3 : Cesium carbonate
DCC : N, N'-Dicyclohexylcarbodiimide
DCM : Dichloromethane
DIBAL-H : Diisobutylaluminium hydride
DMF : N, N'-Dimethylformamide
DMSO : Dimethylsulfoxide
EtSH : Ethanethiol
EDCI : 1-[3-(Dimethylamino)propyl]-3-
ethylcarobodiimidehydrochloride
EtOAc : Ethyl acetate
HgCl2 : Mercuric chloride
HNO3 : Nitric acid
HOBt : 1-Hydroxybenzotriazole
H2SO4 : Sulphuric acid
HCl : Hydrochloric acid
IPA : Isopropyl alcohol
K 2CO3 : Potassiumcarbonate
KOAc : Potassium acetate
LiBr : Lithiumbromide
LiOH : Lithiumhydroxide
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T HESIS
MeOH : Methylalcohol
NaBH4 : Sodiumborohydride
NaClO2 : Sodium chlorite
NaOH : Sodium hydroxide
NaOMe : Sodium methoxide
NaH : Sodium hydride
NH2NH2.H2O : hydrazine Hydrate
SnCl2 : Stannous chloride
SnCl4 : Stannic chloride
SOCl2 : Thionylchloride
TBAF : Tetrabutylammoniumfluor
TBDMSCl : tert-Butyldimethylchlorosilane
TFA : Trifluoroacetic acid
TMSCl : Chlorotrimethylsilane
THF : Tetrahydrofuran
TEA : Triethyl amine
TPP : Triphenylphosphine
CONTENTS
Page No.
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T HESIS
S YNOPSIS 12
CHAPTER-I GENERAL INTRODUCTION
Introduction to Cancer 33
Current Area of Work 42
Objectives of Present Work 62
References 65
CHAPTER-II S YNTHESIS AND BIOLOGICAL EVALUATION OF CHALCONE-P YRROLOBENZODIAZEPINE CONJUGATES AS ANTICANCER AGENTS
Introduction 73
Present Work 79
Biological Activity 84
Experimental 87
References 108
CHAPTER-III S YNTHESIS AND BIOLOGICAL EVALUATION OF COMBRETASTATIN
DERIVATIVES AS ANTICANCER AGENTS
Introduction 114
Present work 119
Biological Activity 124
Experimental 129
References 152
CHAPTER-IV
SECTION-A S YNTHESIS AND BIOLOGICAL EVALUATION OF BENZYLIDENE-9(10H)-ANTHRACENONE LINKED P YRROLOBENZODIAZEPINES AS ANTICANCER AGENTS
Introduction 157
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T HESIS
Present work 160
Biological Activity
164
Experimental 166
References 178
SECTION-B S YNTHESIS AND BIOLOGICAL EVALUATION OF CHALCONE-P YRROLOBENZODIAZEPINE DIMERS AS ANTICANCER AGENTS
Introduction 180
Present work 185
Biological Activity
188
Experimental 191
References 202
LIST OF PUBLICATIONS AND PATENTS 207
S YMPOSIUM AND CONFERENCES 211
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T HESIS
SS YNOPSIS YNOPSIS
The work carried in the research tenure has been compiled in the form
of a thesis entitled “SS YNTHESIS YNTHESIS AANDND AANTICANCERNTICANCER AACTIVITY CTIVITY OOFF NNEWEW PP YRROLO YRROLO[2,1-[2,1-CC]]
[1,4][1,4]BENZODIAZEPINESBENZODIAZEPINES AANDND CCOMBRETASTATINOMBRETASTATIN DDERIVATIVESERIVATIVES“. The main aim of this
work has been to design and synthesize biologically active molecules like
pyrrolobenzodiazepines and combretastatin which are known for potent
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T HESIS
anticancer activity. The thesis has been divided into four chapters. CCHAPTERHAPTER II
gives the introduction about the chemotherapy of cancer, DNA binding ability
with particular reference to pyrrolobenzodiazepines. CCHAPTERHAPTER IIII describes the
synthesis of a new class of C8-linked chalcone-pyrrolobenzodiazepine
analogues and evaluation of their anticancer activity. CCHAPTERHAPTER IIIII describes
the synthesis and anticancer evaluation of combretastatin derivatives.
CCHAPTERHAPTER IVIV comprises of two sections, SECTION-A deals with the synthesis and
biological evaluation of benzylidineanthracenone linked
pyrrolobenzodiazepines as anticancer agents. SECTION-B deals with synthesis
of chalcone- pyrrolobenzodiazepine dimers and evaluation of their DNA-
binding ability and cytotoxicity.
CCHAPTERHAPTER II –– GGENERALENERAL IINTRODUCTIONNTRODUCTION
This chapter describes the general introduction about cancer and
pyrrolobenzodiazepines.. Cancer is one of the leading causes of death in the
industrialized world. Cancer arises when a population of cells within the body
escapes from normal control. It involves the conversion of any normal cell to
a cancerous cell showing tandem replication and cell division at much faster
rate in comparison to the normal cells. Cancer cells often travel to otherparts of the body where they begin to grow and replace normal tissue. This
process is called metastasis, which occurs as the cancer cells get into the
bloodstream or lymph vessels of our body. It is now clear that
chemotherapy’s most effective role in solid tumors is as an adjuvant to the
initial therapy by surgical or radiotherapeutic procedures. Chemotherapy
becomes critical to effective treatment because only systemic therapy can
attack micrometastases. These agents can be categorized into functional
subgroups like alkylating agents, antimetabolites, antibiotics, and
antimitotics.
The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are well known class of
antitumour antibiotics with sequence selective DNA binding ability that are
derived from various Streptomyces species. The first PBD antitumour
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T HESIS
antibiotic anthramycin has been described by Leimgruber and co-workers in
1963 and since then a number of compounds have been developed based
on the PBD ring system leading to some efficient DNA binding ligands. Their
mode of interaction with DNA has been extensively studied and it is
considered unique as they bind within the minor groove of B form DNA.
These compounds exert their biological activity by covalently binding to the
C2-amino group of guanine residue in the minor groove of DNA through the
imine or imine equivalent functionality at N10-C11 of PBD moiety.
H3C
OH
N
HN
O
OCH3
CONH2
Anthramycin
1
2
45
36
7
8 9
10
11
N
N
O
HO
H3CO
Tomaymycin
H
N
NO
H3CO
O
HON
N
O
H
OCH3
SJG-136
H
11a
Figure 1. Biologically important DNA interactive natural/unnatural PBDs.
The molecular modeling studies suggested that C8 would be the
preferred position for attachment of second interacting group to develop the
unsymmetrical DNA cross-linking agents. A number of naturally occurring
and synthetic compounds based on PBD ring system, such as anthramycin,
tomaymycin, DC-81 and its dimers (presently, one of the dimer SJG-136 is
under clinical evaluation), have shown varying degrees of DNA binding
affinity and anticancer activity. In view of the importance of these molecules,
there is considerable interest in the structural modification of PBD's,
particularly at C8 position to improve upon their DNA binding potential andsequence selectivity.
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T HESIS
C
OH
N
HN
H
O
NH2
O
HOH
N NH2NDNA
1110
H3C
OH
N
HN
H
O
NH2
O
NHH
HNN
DNA
11
10
Anthramycin
Figure 2. Mechanism of action of PBDs with DNA.
CCHAPTERHAPTER II - SII - S YNTHESIS YNTHESIS AANDND BBIOLOGICALIOLOGICAL EEVALUATIONVALUATION OOFF CCHALCONEHALCONE--PP YRROLOBENZODIAZEPINE YRROLOBENZODIAZEPINE CCONJUGATESONJUGATES AASS AANTICANCERNTICANCER AAGENTSGENTS
This chapter describes the synthesis and biological activity of
chalcone-pyrrolobenzodiazepine conjugates. Chalcones are a class of
anticancer agents that have shown promising therapeutic efficacy for the
management of human cancers. Chemically they consist of open-chain
flavonoids in which the two aromatic rings are joined by a three-carbon α,β-
unsaturated carbonyl system. Recent studies revealed that these chalcones
had shown a wide variety of anticancer, anti-inflammatory, antiinvasive,
antituberculosis, and antifungal activities. The trimethoxychalcone (13b) is
potential anticancer agent and binding strongly to tubulin at a site sharedwith, or close to, the colchicines binding site. Chalcones have attracted more
interest in recent years because of their diverse pharmacological properties.
The parent molecule of chalcone derivatives and its hydroxyl chalcones have
been reported for their antiproliferative and antitumor activity. In
continuation of efforts towards the design and synthesis of new PBD
analogues, we synthesized chalcone-PBD analogues. The DNA binding
characteristics of these conjugates have been evaluated by thermal
denaturation studies.
Synthesis of these chalcone linked PBD analogues (20a-f and 23a-c)
has been carried out by employing the (2S)-N-[4-benzyloxy-5-methoxy-2-
nitrobenzoyl]proline methylester (7), which is obtained according to the
literature method starting from vanillin. This upon selective reduction by
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T HESIS
employing DIBAL-H, and protection with TMSCl/EtSH followed by deprotection
using BF3.OEt2/EtSH affords the precursor 10 as shown in Scheme 1.
MeO
HO
MeO
HO
MeO
HO
MeO
BnO
MeO
BnO
HOH OMe
OMeOMe
O O O
OO
NO2
MeO
BnO
MeO
HO
N
O
CH(SEt)2NO2
MeO
BnO
OH
O
NO2
(i) (ii)
(iii)
(iv)(v)
(vi)
(vii)
(viii)
(ix)
1 2 3
456
7 8
910
N
NO2
O
COOMe
MeO
BnO
N
NO2
O
CHO
MeO
BnO
N
NO2
O
CH(SEt)2
Scheme 1. Reagents and conditions: (i) NH2SO3H, NaClO2, H2O, rt, 2 h, 90%; (ii) H2SO4,
MeOH, reflux, 4 h, 85%; (iii) benzylbromide, K 2CO3, acetone, reflux, 24 h, 92%; (iv) SnCl4,
fuming HNO3, CH2Cl2, 5 min, -25 oC, 78%; (v) 2N LiOH, MeOH, H2O, THF (1:1:3), rt, 12 h,
83%; (vi) SOCl2, C6H6, L-proline methylester hydrochloride, THF, 1-2 h, rt, 85%; (vii) DIBAL-H,
CH2Cl2, 0.5 -1 h, -78 oC, 65%; (viii) EtSH, TMSCl, CH2Cl2, 8-12 h, rt, 72%; (ix) BF3.OEt2, EtSH,
CHCl3, rt, 8 h, 75%;
The preparation of chalcone intermediates 14a-f and 17a-c has been
carried out by synthetic sequence illustrated in Schemes-2 and 3. Claisen-
Schmidt condensation of trimethoxyacetophenone with benzaldehydes by
using ethanol as solvent in the presence of aqueous KOH gives
trimethoxychalcones 13a,b. The cyclic chalcone 16 has been prepared
under the same reaction conditions by condensing 1-indanone with vaniline
to give indanochalcone. These trimethoxy and indano chalcones undergo
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T HESIS
alkylation of hydroxyl group with dibromoalkanes by using K 2CO3 as a base in
dry acetone to afford precursors 14a-f and 17a-c.
MeO
MeO
OMe
CH3
O CHO
R
OH
+
MeO
MeO
OMe
O
OH
R
MeO
MeO
OMe
O
O
R
Br ( )n
(i)
(ii)
14a-f
11 12a, b13a, b
14a; R = H, n = 214b; R = H, n = 3
14c; R = H, n = 414d; R = OMe, n = 214e; R = OMe, n = 314f ; R = OMe, n = 4
Scheme 2. Reagents and conditions: (i) aq.KOH, ethanol, 4 h; (ii) dibromoalkane, acetone,
K 2CO3, reflux, 24 h.
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T HESIS
17a; n = 217b; n = 317c; n = 4
CHO
OMe
OH
+
(i)
(ii)
17a-c
15 12b
16
O O
OH
OMe
O
O
OMe
Br ( )n
Scheme 3. Reagents and conditions: (i) aq.KOH, ethanol, 4 h; (ii) dibromoalkane, acetone,
K 2CO3, reflux, 24 h.
Compound 10 has been coupled to compounds 14a-f and 17a-c in the
presence of K 2CO3 and dry acetone under reflux conditions to give
corresponding nitro compounds 18a-f and 21a-c. These nitro compounds
upon reduction with SnCl2.2H2O in methanol under reflux conditions give
amino compounds 19a-f and 22a-c. the amino compounds upon
deprotection followed by cyclization with HgCl2/CaCO3 to provide thecorresponding imines 20a-f and 23a-c (Schemes 4 & 5).
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T HESIS
MeO
MeO
OMe
O
O
R
Br ( )n
14a-f
HO
MeO
NO2
O
N
CH(SEt)2
10
+
O
MeO
NO2
O
N
CH(SEt)2O
O
MeO
MeO
OMe
R
( )n
O
MeO
NH2
O
N
CH(SEt)2O
O
MeO
MeO
OMe
R
( )n
O
MeO
O
O
MeO
MeO
OMe
R
( )n
N
N
O
H
18a-f
19a-f
20a-f
(i)
(ii)
(iii)
Scheme 4. Reagents and conditions: (i) K 2CO3, acetone, 12 h, reflux; (ii) SnCl2.2H2O, MeOH,4 h, reflux; (iii) HgCl2, CaCO3, MeCN, H2O, (4:1) 12 h, rt.
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T HESIS
n = 2, 3, 4
HO
MeO
NO2
O
N
CH(SEt)2
10
+
O
MeO
NO2
O
N
CH(SEt)2O
OMe
( )n
O
MeO
NH2
O
N
CH(SEt)2O
OMe
( )n
O
MeO
O
OMe
( )n
N
O
H
21a-c
22a-c
23a-c
(i)
(ii)
(iii)
17a-c
O
O
OMe
Br ( )n
O
O
O
Scheme 5. Reagents and conditions: (i) K 2CO3, acetone, 12 h, reflux; (ii) SnCl2.2H2O, MeOH,4 h, reflux; (iii) HgCl2, CaCO3, MeCN, H2O, (4:1) 12 h, rt.
The thermal denaturation studies show that these conjugates (20a–f
and 23a-c) possess good DNA binding ability compared to DC-81. These
findings suggest that these conjugate agents bind more efficiently to DNA
than DC-81. Compounds 20a-f and 23a-c exhibit significant anticancer
activity against eight cancer cell lines with GI50 values ranging from <0.01-
2.7 μM, in comparison to adriamycin (GI50, <0.01-14.7 μM). According to thein vitro screening data, compound 46b has significant cytotoxicity against all
the cancer cell lines with GI50 values ranging from <0.01-0.17 μM 0.1-2.19 µM
and has shown more potency against PC-3 prostate cancer cell line with GI50
<0.01 μM.
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T HESIS
CCHAPTERHAPTER III: SIII: S YNTHESIS YNTHESIS AANDND BBIOLOGICALIOLOGICAL EEVALUATIONVALUATION OOFF CCOMBRETASTATINOMBRETASTATIN DDERIVATIVESERIVATIVES AASS AANTICANCERNTICANCER AAGENTSGENTS
This chapter describes the design, synthesis, and in vitro cytotoxicity
of novel analogues of combretastatin, and its chalcone, pyrazoline
derivatives with amino benzothiazoles. Tubulin is a heterodimeric protein
consisting of A and B subunits. During cellular division upon binding of GTP,
tubulin polymerizes into microtubules. This formation of microtubule is
essential for chromosome separation and formation of two daughter cells.
When ligands that interact with tubulin are present, a reduction in cellular
division is observed and shows anticancer activity. Tubulin having colchicine
binding site and if any ligand binds to this site prevents the tubulin
polymerization. Colchicine and combretastatin-A-4 [CA-4] are the good
examples as tubulin binding agents. Combretastatin A-4 is a naturally
occurring stilbene and isolated from the African willow tree (Combretum
caffrum). CA-4 shows interesting anticancer potential due to its antitubulin
properties. It strongly binds to the colchicine site of tubulin thus preventing
tubulin polymerization and causes antimitotic effect. In view of the
interesting biological properties exhibited by these molecules it was
considered of interest to synthesize analogues of combretastatin A-4derivatives with 2-aminobenzothiazoles and these new compounds exhibit
potent anticancer activity.
MeO
MeO
OMe
OMe
OH
combretastatin A-4 colchicine
MeO
MeO
OMe
O
OMe
NHCOCH3
Figure 3. Potential inhibitors of tubulin polymerization
The precursor (Z)-2-(2-methoxy-5-(3
trimethoxystyryl)phenoxy)acetic acid 9 has been prepared by employing
commercially available isovanillin. Hydroxy group protection of isovanillin
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T HESIS
with TBDMS-Cl followed by reduction of aldehyde group with NaBH4 gives the
alcohol 3. The benzyl alcohol was converted to benzyl bromide 4 using LiBr
followed by salt formation with PPh3 to give the compound 5. This on Wittig
reaction with trimethoxybenzaldehyde gives TBDMS-protected
combretastatin A-4, which upon deprotection with TBAF gives the compound
combretastatin A-4, 7. This upon etherification with α-bromoethyl acetate in
the presence of K 2CO3 gives the ester 8, which on hydrolysis with LiOH
affords the acid 9 (Scheme 1).
OMe
OH
CHO
OMe
OTBDMS
CHO
OMe
OTBDMS
CH2OH
OMe
OTBDMS
CH2Br
OMe
OTBDMS
CH2PPh3Br
MeO
MeO
OMe
OMe
OTBDMSMeO
MeO
OMe
OMe
OH
(i) (ii) (iii)
(iv)
(v)(vi)
MeO
MeO
OMe
OMe
OOEt
OMeO
MeO
OMe
OMe
OOH
O
(vii)
(viii)
1 2 3 4
56
7
8 9
Scheme 1. Reagents and conditions: i) TBDMS-Cl, TEA, DMF; ii) NaBH4, MeOH ; iii) LiBr,
THF ; iv) PPH3, toluene ; v) n-BuLi, THF,-20 oC, trimethoxybenzaldehyde ; vi) TBAF, THF ; vii)
2-bromoethyl acetate, K 2CO3, DMF ; viii) LiOH, THF, H2O
The preparation of chalcone derivative 13 has been carried out by
synthetic sequence illustrated in Scheme-2. Claisen-Schmidt condensation of
trimethoxyacetophenone 10 with isovaniline by using ethanol as solvent in
the presence of aqueous KOH gives trimethoxychalcones 11. This upon
etherification with α-bromoethyl acetate in the presence of K 2CO3 gives the
ester compound 12. The ester compound on hydrolysis with LiOH affords the
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T HESIS
chalcone acid (E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-
enyl)phenoxy)aceticacid 13.
MeO
MeO
OMe
O
MeO
MeO
OMe
O
OMe
OH
OMe
OH
CHO
+
13
MeO
MeOOMe
O
OMe
OOEt
O
MeO
MeOOMe
O
OMe
OOH
O
10 11
12
(i)
(ii)
(iii)
1
Scheme 2. Reagents and conditions: i) aq. KOH, ethanol, 12h; ii) 2-bromoethyl acetate,
K 2CO3, DMF, 12h; iii) LiOH, THF, H2O
The preparation of pyrazoline derivative 16 has been carried out by
the synthetic sequence illustrated in Scheme-3. Cyclization of
trimethoxychalcone 11 with hydrazine hydrate in acetic acid under reflux
conditions gives pyrazoline derivative 14. This upon etherification with α-
bromoethylacetate in the presence of K 2CO3 gives the ester compound 15.
The ester compound on hydrolysis with LiOH affords pyrazoline acid 2-(5-(1-
acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)-2-met
hoxyphenoxy)acetic acid 16.
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T HESIS
MeO
MeO
OMe
O
OMe
OH MeO
MeO
OMe
N
OMe
OH
N
O
16
MeO
MeO
OMe
N
OMe
O
N
O
OEt
O
MeO
MeO
OMe
N
OMe
O
N
O
OH
O
11 14
15
(i)
(ii)
(iii)
Scheme 3. Reagents and conditions: i) NH2NH2.H2O, Acetic acid, reflux, 14h; ii) 2-
bromoethyl acetate, K 2CO3, DMF, 12h; iii) LiOH, THF, H2O
MeO
MeO
OMe
OMe
OOH
O
9
N
SH2N+
MeO
MeO
OMe
OMe
ONH
O
S
N
17
18a, R = -H18b, R = -NO218c, R = -F18d, R = -Cl18e, R = -OMe18f, R = -OCF318g, R = -Me18h, R = -CF3
18i, R = -OEt
R
R
18 a-i
(i)
Scheme 4. Reagents and conditions: i) EDCI/HOBT, DCM, 14-16h
The synthesis of combretastatin-benzothiazole analogues 18a-i is
outlined in Scheme 4. The combretastatin acid 9 undergoes amide bond
formation with 2-aminobenzothiazoles in the presence of EDCI/HOBt in
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T HESIS
dichloromethane to give the desired combretastatin-benzothiazole analogues
18a-i.
19a R = -H19b R = -NO219c R = -F19d R = -Cl19e R = -OMe19f R = -OCF319g R = -Me19h R = -CF319i R = -OEt
N
SH
2N
+
17
R
13
MeO
MeO
OMe
O
OMe
OOH
O
MeO
MeO
OMe
O
OMe
ONH
O
S
N R
19a-i
(i)
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 2-
amino benzothiazoles in the presence of EDCI/HOBt in dichloromethane to
give the desired chalcone-benzothiazole analogues 19a-i.
20a R = -H20b R = -NO220c R = -F
20d R = -Cl20e R = -OMe20f R = -OCF320g R = -Me20h R = -CF320i R = -OEt
(i)
N
SH2N
+
17
R
20a-i
16
MeO
MeO
OMe
N
OMe
O
N
O
OH
O
MeO
MeO
OMe
N
OMe
O
N
O
NH
O
S
N R
Scheme 6. Reagents and conditions: i) EDCI/HOBT, DCM, 14-16h
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T HESIS
The pyrazolineacid 16 undergoes amide bond formation with 2-
aminobenzothiazoles in the presence of EDCI/HOBt in dichloromethane to
give the desired pyrazoline-benzothiazole analogues 20a-I (Scheme-6). The
anticancer activity of the synthesized compounds was evaluated by the
National Cancer Institute (NCI), USA. Fourteen compounds were selected for
NCI-60 cell line anticancer screening program by National Cancer Institute
(NCI), Bethesda, USA. After preliminary screening on the tumour cell lines,
these compounds were tested for five dose concentration on a panel of 59
human tumour cell lines derived from nine different cancer types: leukaemia,
lung, colon, CNS, melanoma, ovarian, renal, prostate and breast. These
compounds exhibited significant anticancer activity with GI50 values ranging
from 0.019 to 18.6 μM. These compounds have also been evaluated for its
tubulin binding activity and some of the compounds exhibited appreciable
good tubulin binding activity.
CCHAPTERHAPTER IV: (IV: (SECTION-A) - S- S YNTHESIS YNTHESIS AANDND BBIOLOGICALIOLOGICAL EEVALUATIONVALUATION OOFF BBENZYLIDENEENZYLIDENE--9(10H)-A9(10H)-ANTHRACENONENTHRACENONE LLINKEDINKED PP YRROLOBENZODIAZEPINES YRROLOBENZODIAZEPINES AASS AANTICANCERNTICANCER AAGENTSGENTS
In recent years there has been increasing interest in the design of
conjugate molecules that could act in a specific manner on more than one
target. The development of such conjugates lowers the risk of drug-drug
interaction in comparison to cocktails but could also enhance the efficacy as
well as improve the safety aspects in relation to the drugs that interact on a
single target. Several conjugate compounds, in which a known antitumour
compound or some simple active moiety tethered to PBD, have been
designed, synthesized and evaluated for their biological activity. Recently,
Wang and co-workers have synthesized indole-PBD conjugates as potential
antitumour agents and a correlation between antitumour activity and
apoptosis has been well explained. More recently, we have also reported
some of the PBD conjugates that demonstrated potent apoptotic activity
through mitochondrial-mediated pathway.
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T HESIS
In continuation of these efforts, the synthesis and biological evaluation
of benzylidineanthracenone linked pyrrolobenzodiazepines attached through
an alkane spacer is described. The preparation of benzylidineanthracenones
intermediates 4a-f has been carried out by synthetic sequence illustrated in
Scheme-1. The intermediates 3a,b are synthesized by reacting anthrone 1
with different benzaldehydes in the presence of 10% IPA.HCl (isopropyl
alcoholic solution of HCl). These benzylidene-9(10 H )-anthracenones undergo
alkylation of hydroxyl group with dibromoalkanes using K 2CO3 as a base in
dry acetone to afford precursors 4a-f (Scheme 1).
CHO
R
OH
+
(i)
(ii)
4a-f
1 2a, b3a, b
4a; R = H, n = 24b; R = H, n = 34c; R = H, n = 44d; R = OMe, n = 24e; R = OMe, n = 34f ; R = OMe, n = 4
OO
R
OH
O
R
OBr
( )n
Scheme 1. Reagents and conditions: (i) IPA.HCl, 5 h; (ii) dibromoalkane, acetone, K2CO3,
reflux, 24 h.
Compound 5 has been coupled to compounds 4a-f in the presence of
K 2CO3 and dry acetone under reflux conditions to give the corresponding
nitro compounds 6a-f . These nitro compounds upon reduction with
SnCl2.2H2O in methanol under reflux conditions give amino compounds 7a-f.
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T HESIS
Finally the amino compounds upon deportation with HgCl2/CaCO3 cyclized to
provide the corresponding imines 8a-f (Scheme-2).
O
R
OBr
( )n4a-f
HO
MeO
NO2
O
N
CH(SEt)2
5
+
O
MeO
NO2
O
N
CH(SEt)2O
R
( )n
6a-f
7a-f
8a-f
(i)
(ii)
(iii)
O
O
MeO
NH2
O
N
CH(SEt)2O
R
O
( )n
O
MeO
O
R
O
N
N
O
H( )n
n = 2, 3, 4
Scheme 2. Reagents and conditions: (i) K 2CO3, acetone, 12 h, reflux; (ii) SnCl2.2H2O, MeOH,4 h, reflux; (iii) HgCl2, CaCO3, MeCN, H2O, (4:1) 12 h, rt.
These benzylidine anthracenone linked PBD analogues have beentested for their cytotoxicity against different human cancer cell lines that
comprise of Zr-75-1, MCF-7, KB, Gurav, DWD, Colo-205, A-549, Hop-62 and
A-2780 by using the Sulforhodamine B (SRB) method. All the compounds
exhibited significant anticancer activity.
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T HESIS
CCHAPTERHAPTER IV: (IV: (SECTION-B) - S- S YNTHESIS YNTHESIS AANDND BBIOLOGICALIOLOGICAL EEVALUATIONVALUATION OOFF CCHALCONEHALCONE--PP YRROLOBENZODIAZEPINE YRROLOBENZODIAZEPINE DDIMERSIMERS AASS AANTICANCERNTICANCER AAGENTSGENTS
Many molecules based on PBD ring system have been synthesized to
improve their biological profile and in this search C-7 or C-8 linked dimers of
PBD have been prepared, which are capable of sequence selective DNA
interaction and cross-linking. Thurston and co-workers have synthesized C-8
linked PBD dimers by linking C8-position of the A-rings through varying
lengths of alkyl chain to explore DNA-cross linking ability. The results
indicate that DSB-120 is an efficient cross-linking agent and the cross-linking
ability of these PBD dimers after 2h incubation at 37 °C has been found to be
0.01 nm. Furthermore, the in vitro cytotoxicity data in human K562 and
rodent ADJ-PC6 cell lines correlate with both the thermal denaturation data
and the cross-linking efficiencies. Recently, C2/C2’-exo-unsaturated C-8
linked PBD dimers (SJG-136) have been synthesized which exhibit
extraordinary DNA binding affinity and cytotoxicity.
CH3
O CHO
+
O
R3
R4(i)
1a,b 23a, b
3a; R1 = OH, R2 = H, R3 = H, R4 = OH3b; R1 = H, R2 = OH, R3 = OH, R4 = H
R2
R1
R3
R4 R2
R1
Scheme 1. Reagents and conditions: (i) aq.KOH, ethanol, 24 h
This chapter describes the synthesis and biological activity of
chalcone-pyrrolobenzodiazepine dimers. In these dimers the two PBD unitsare linked through a chalcone moiety. The preparation of dihydroxychalcone
intermediates (3a,b) has been carried out by synthetic sequence illustrated
in Scheme-1. Claisen-Schmidt condensation of hydroxyacetophenones with
hydroxybenzaldehydes using ethanol as solvent in the presence of aqueous
KOH gives dihydroxychalcones 3a,b.
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T HESIS
O
3a; R = 4,4'-dihydroxy3b; R = 3,3'-dihydroxy
RR
O
MeO
NO2
O
N
CH(SEt)2
4a-c
+
O
MeO
NO2
O
N
CH(SEt)2O ( )n
5a-f
6a-f
7a-f
(i)
(ii)
(iii)
Br ( )n
OO
OMeN
O
(EtS)2HC O2N( )n
O
MeO
O ( )nOO
OMe
( )n
N
NN
N
O
H
O
H
Chalcone
O
MeO
NH2
O
N
CH(SEt)2O ( )nOO
OMeN
O
(EtS)2HC H2N( )n
Chalcone
Chalcone
O
O O
7a-c; Chalcone =
7d-f; Chalcone =
O
O O
n = 2,3,4
n = 2,3,4
Scheme 2. Reagents and conditions: (i) K 2CO3, acetone, 12 h, reflux; (ii) SnCl2.2H2O, MeOH,4 h, reflux; (iii) HgCl2, CaCO3, MeCN, H2O, (4:1) 12 h, rt.
Compound 4a-c has been coupled to dihydroxychalcones 3a,b in the
presence of K 2CO3 and dry acetone under reflux conditions to give
corresponding nitro compounds 5a-f . These nitro compounds upon reduction
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T HESIS
with SnCl2.2H2O in methanol under reflux conditions give amino compounds
6a-f. Finally the amino compounds upon deprotection followed by cyclization
with HgCl2/CaCO3 provide the corresponding imines 7a-f (Scheme-2).
The cytotoxic activity of these chalcone-PBD dimers (7a-f ) has been
evaluated on a panel of 5 tumor cell lines that comprise HT-29, PC3, A-375,
A-549 and B-16 by using the Sulforhodamine B (SRB) method. These
analogues showed promising activity against PC-3 cell line compared to
other cell lines tested. These PBD dimers elevate the helix melting
temperature of CT-DNA in the range of 3.5-5.4 oC. Compound 7a showed the
highest ΔT m of 4.8 oC at 0 h and increased upto 5.4 oC after 18 h incubation,
whereas the naturally occurring DC-81 exhibits a ΔT m of 0.7 oC after
incubation under similar conditions. These results indicate that the effect on
DNA binding affinity by introducing the chalcone scaffold on PBD moiety
through different alkane spacers at C8-position of the DC-81.
In conclusion, the work carried in the research tenure we designed and
synthesized different series of biologically active molecules like
pyrrolobenzodiazepines and combretastatins which are known for potent
anticancer activity. The conjugates of PBD with chalcones and benzylidene
anthrones showed significant anticancer activity as well as good DNA binding
ability. The combretastatin derivatives with benzothiazoles showed potential
anticancer activity.
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T HESIS
CCHAPTERHAPTER-I-I
GGENERALENERAL IINTRODUCTIONNTRODUCTION
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1. CANCER
Cancer is one of the leading causes of death in the industrialized world.
Cancer arises when a population of cells within the body escapes from
normal control mechanisms and continues to increase until, unlesseffectively treated, the host dies. Although there are many kinds of cancer,
they all start because of uncontrolled growth of normal cells. Cancer cells
often travel to other parts of the body where they begin to grow and replace
normal tissue. This process, called metastasis, occurs as the cancer cells get
into the bloodstream or lymph vessels of our body. There are over 200
different types of cancers that can occur anywhere in the body. Cancer
treatment will be entirely based on person’s unique situation. Certain types
of cancer respond very differently to different types of treatment, so
determining the type of cancer is a vital step toward knowing which
treatments will be most effective. The cancer's stage will also determine the
best course of treatment, since early stage cancers respond to different
therapies than later-stage ones. Person’s overall health, lifestyle, and
personal preferences will also play a part in deciding which treatment
options will be best.
The four major types of treatment for cancer are surgery, radiation,
chemotherapy, and biological therapies. Hormone therapies such as
tamoxifen and transplant options such as those done with bone marrow are
also useful for treating certain cancers.
1.1. CHEMOTHERAPY
Chemotherapy is the treatment of cancer with drugs that can destroy
cancer cells by impeding their growth and reproduction. Chemotherapydrugs are given intravenously by injection or by mouth. Chemotherapy is
often used alone or in conjunction with radiation therapy or with surgery.
While surgery and radiation therapy are used to treat localized cancers,
chemotherapy is used to treat cancer cells that have metastasized to other
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T HESIS
parts of the body, because they travel throughout the body in the
bloodstream. Depending on the type of cancer and its stage of development,
chemotherapy can be used to cure cancer, to keep the cancer from
spreading, to slow the cancer's growth, to kill cancer cells that may have
spread to other parts of the body, or to relieve symptoms caused by cancer.
Although chemotherapeutic drugs attack reproducing cells, they
cannot differentiate between cells of normal tissues and cancer cells. The
damage to normal cells can result in side effects. These cells usually repair
themselves after chemotherapy. Several exciting uses of chemotherapy hold
more promise for curing or controlling cancer. New drugs, new combinations
of chemotherapy drugs and new delivery techniques are the expected
advances in the coming years for curing or controlling cancer and improving
the quality of life for people with cancer. Chemotherapeutic drugs are
divided into several categories based on how they affect specific chemical
substances within the cancer cells, which cellular activities or processes the
drug interferes with, and which specific phases of the cell cycle the drug
affects. These include DNA topoisomerase I and II inhibitors, antimitotic
agents, antimetabolites, DNA interactive agents and miscellaneous agents.
1.2. DNA AS A CELLULAR TARGET FOR ANTICANCER DRUGS
DNA is one of the main targets in the design of antineoplastic agents.
Deoxyribonucleic acid (DNA)1-3 is a long molecule that contains coded
instructions for the cells. The monomer units of DNA are nucleotides that
consist of a 5-carbon sugar (deoxyribose), a nitrogen containing base
attached to the sugar and a phosphate group. There are four different types
of nucleotides found in DNA with adenine (A), thymine (T), cytosine (C) and
guanine (G). The particular order of the bases that are arranged along the
sugar-phosphate back bone is called the DNA sequence; the sequence
specifies the exact genetic instructions required to create a particular
organism with its own unique traits. The two DNA strands held together with
weak bonds between the bases on each strand, forming a base pair (bp).
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T HESIS
During cell division the DNA molecule unwinds and weak bonds between the
base pairs break, allowing the strands to separate, and each strand direct
the synthesis of complementary strands, with free nucleotides matching up
with their complementary bases on each of the separated strands.
N
N N
N
O
N
N N
O
Sugar
NH
H
H
Sugar
Guanine
Cytosine
N
N N
N
NNN
O
O
H
H
Sugar
Sugar
Adenine
Thymine H
H
H
Figure-1. Hydrogen bonding between A-T and G-C base pairs of DNA
The base pairs are rotated 36° with respect to each adjacent pair, so
that there are 10 pairs per helical turn, each represented by 3.4 A°. This
gives rise to two well-defined channels known as minor groove and major
groove. The major groove is approximately 24 A° in width and much deeper
than minor groove, which is only 10 A° in width.4 The maintenance of coding
information in DNA stems from its ability to form a complementary double
stranded structure. Complementarily results in a large part from formation of
hydrogen bonds between specific opposing bases. Thus, adenine pairs with
thymine (an A-T pair) and cytosine with guanine (a C-G pair) by the
formation of 2 and 3 hydrogen bonds respectively. The atoms involved in
formation of these specific H-bonds are thus inaccessible unless the helical
structure is disrupted. However, on either side of the planar bases lie
additional H-bond donating and accepting groups specific for each base andwhich protrude into the relatively accessible major and minor grooves of the
helix. Thus in the major groove the C6 amino group of adenine or the C4
amino group of cytosine can act as hydrogen bond donating groups while the
adenine N7, thymine O4, guanine N7, O6 can act as hydrogen bond
accepting groups, and the thymine methyl as a hydrophobic site. Similarly in
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T HESIS
the minor groove the adenine N3, thymine O2, guanine N3, and the cytosine
O2 can act as H-bond acceptors and the C2 amino group of guanine can act
as a donating group. Both grooves therefore carry base dependent
sequences of potential H-bonding atoms that can be used as a target in the
design of sequence specific DNA binding compounds (Fig.1 & 2).
Figure 2. The structure of part of DNA double helix.
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Double stranded DNA can exist in three forms namely A, B and Z.5 The
B-form is most stable under high humid conditions because water molecules
stabilize the structure by forming a spine of hydration in the minor groove.6
Since this form almost exclusively predominates in the biological evaluation,
this B-DNA is used in the design of new DNA-binding antitumor drugs.
1.3. EVALUATION OF DRUG-DNA INTERACTIONS
DNA is believed to be the molecular target of a number of clinically
important antitumour antibiotics. Based upon their diverse structural types,
it is not surprising that these compounds each have been found to react in
quite different ways with DNA and that the biochemical consequences of
DNA are equally diverse, all though all can ultimately produce cell death.
Understanding the interactions between drugs and DNA is a necessary first
step in elucidating the molecular basis for the potent anti-tumor activities of
these compounds.
In recent years, several advances have been made in the elucidation of
drug-DNA interactions. Spectral methods are available to evaluate the extent
of DNA-binding and to know in which sequence the ligand binds. Physical
methods like UV-spectroscopy, fluorescence, circular dichroism (CD), opticalrotatory dispersion (ORD), IR, Raman spectroscopy and viscometry
measurements have been used for the measurement of binding. Thermal
denaturation studies on DNA are common and involve measuring the melting
point of DNA alone and in the presence of a ligand (drugs). Binding will often
stabilize the helix and elevate the melting temperature. However, none of
these physical techniques allows determining the specific location of binding
on a DNA strand. To do this two types of assays are used namely, strand
cleavage assay and affinity cleavage assay.7 Other powerful techniques for
studying DNA binding with short lengths of DNA includes NMR and X-ray
crystallography,8 which can provide precise structural information about
functional groups involved. Three dimensional 1H, 31P NMR experiments such
as NOSEY or COSY can be used to locate precisely the ligand on the strand
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T HESIS
and which can be used in conjugation with computational methods to
generate useful 3-dimensional models of ligand-DNA complexes.9 DNA ‘foot
printing’ is an alternative approach that can be used for covalent and non-
covalent binders, intercalaters and other type of adducts such as co-
ordination complexes and triple helices.10
N
NN
NOH2C
O
PO
O O-
OH2C
O
O
PO O-
O
H2C O
OH2C
O
PO O-
O
PO O-
O
H2C O
O
P O-
O
O
O
O
CH2
O
P
P O-O
O
CH2O
OP O-O
O
CH2O
O
O
P O-O
O
CH2O
O-
OO
OCH2
O
Thymine
CytosineGuanine
Adenine
Major groove side
Minor groove side
PBDsMitomycin C (reducing conditions)SaframycinsPAHs (Benz[a]pyrene)N-Hydroxy-2-naphthylamineDehydroretronecine
CC 10659-Anthryloxirene
NH2
Bisfunctional alkylating agentsAflatoxin B1
Mitomycin C (acid activity)Benz[a]pyrene
Cis-Pt(NH3)2Cl2 (N7, O6 bridge)
3'5'
1
34
56
7
8
N
HN
O
O
N
N
O
H2N
NHN
N
O
NH229
H3C
Figure 3. Structure of DNA
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1.4. T YPES OF DRUGS THAT INTERACT WITH DNA
Based on drug-DNA interactions DNA-interactive agents are
categorized in to four broad classes are known as intercalators, alkylating
agents, DNA strand breakage compounds and groove binders of DNA (Figure-
3).
1.4.1. INTERCALATERS
Intercalaters consist of a flat, generally π -deficient aromatic orheteroaromatic system, which binds to DNA by insertion between the base
pairs of the double helix. Intercalation causes the base pairs to separate
vertically, there by distorting the sugar-phosphate back bone and changing
the degree of rotation between successive base pairs e.g. acridines,11
actinomycins12 (Figure-4).
N
HN
H3CO NHSO2CH3
R'R
O
X
OH
H O
O
NH3+
OH
H3C
OH
OH
O
OOMe
acridines anthracyclines
Figure-4. Structures of DNA Intercalaters
1.4.2. ALKYLATING AGENTS
The DNA alkylaters (irreversible inhibitors) react with the DNA
(enzyme) to form covalent bonds. The important classes of alkylating agents
utilized in cancer chemotherapy are nitrogen mustards, ethylene amides,
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T HESIS
methane sulphonic acid esters, nitrosoureas, triazenes and platinum
complexes (e.g. cisplatin13) (Figure-5).
Cl
ClH3N
H3N
cisplatin
R N
Cl
Cl
nitrogen mustards methanesulfonates
N
N
N
N
N
N
triethylenemelamine
SO
OS
OO
O O
Pt ( )n
Figure 5. Structures of alkylating agents.
1.4.3. DNA STRAND BREAKERS
Some DNA-interactive drugs initially intercalate into DNA but then in
certain conditions, react in such a way as to generate radicals. The reaction
of these radicals with the sugar moieties leads to DNA strand scission. e.g.
bleomycin and the enediyne antitumor antibiotics14 (Figure-6).
HN
CH3
COOH
OCH3
O
OH
OH
OH
O
O
Figure 6. Structures of dynemicin
1.4.4. GROOVE BINDERS
Drugs that bind to DNA may occur on the major groove face, minor
groove face or a combination. The grooves are excellent sites for sequence
specific recognition since there are many potential hydrogen bond donor and
acceptor atoms unique to each base pair combination along the base edges.
The greater width associated with the B-DNA major groove makes the major
groove somewhat more preferable binding groove.
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T HESIS
1.4.4.1. IMPORTANCE OF MINOR GROOVE BINDERS
Groove binding can be via the major or minor groove and covalently or
non-covalently. Most DNA interactive proteins bind in the major groove, while
small molecules of less than 1000 Da, including many antibiotics, binds in
the minor groove. The minor groove represents a vulnerable site of attack in
that it is normally unoccupied, and this is presumably the reason for the
evolution of antibiotics that attack the DNA of competing organisms. Thus,
although at first sight minor groove binders are less attractive as probes in
that they target the less information rich minor groove nevertheless, they
may prove to have several advantages compared with major groove ligands.
The development of sequence-specific probes based on naturally occurring
DNA groove-binding agents is, therefore, an alternative and complementary
approach to the antisense oligonucleotide strategy. The main motive for
synthesizing a large number of analogues and conjugates of naturally
occurring minor groove-binding agents, is to generate new lead compounds
with potential anticancer properties and specific DNA sequence recognition.
1.4.4.2. NON-COVALENT MINOR GROOVE BINDERS
These compounds are typically isohelical with B-DNA and fits snugly
within the minor groove, held in a position by a combination of hydrogen
bonds, vander waal forces and electrostatic interactions. Examples include
distamycin15 (Figure 7), netropsin,16 CC-1065,17 lexitropsins and bis-
benzimidazole (Hoecht 33258).18
N
NH
NHH
OO
CH3
N
NH
O
NH2
NH2
CH3
N
NH
O
CH3
+
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T HESIS
Figure 7. Structures of distiamycin
1.4.4.3. COVALENT BINDING TO MINOR GROOVE OF DNA
Drugs which bind covalently to DNA are used to either add substituents
onto base residues, or to form cross links between different sections of DNA.
The first mechanism results in a base-pairing mismatch during DNA
replication, and the DNA is ultimately fragmented by the enzymes which try
to repair it. The second mechanism binds together the two strands of the
DNA helix, preventing separation during the replication process.
Electrophilic functional groups such as epoxides, aziridines,
carbinolamines, imines and cyclopropanes are found in a variety of synthetic
and natural products capable of covalent interaction with DNA. Examples
include mitomycin, saframycins and pyrrolobenzodiazepines (anthramycin)19
(Figure 8).
N
HN
O
H3C
O
O
H2N
H3C N NH
OCH3
CH2OCONH2
Mitomycin Anthramycin
CONH2
OCH3OH
H
Figure 8. Covalant minor groov binding agents
1.5. CURRENT AREA OF WORK
As discussed previously the chemotherapy of cancer could be done by
DNA binding agents. The ultimate goal is to design and synthesize agents
capable of specifically inhibiting the expression of particular proteins critical
for tumour cell proliferation, metastasis or drug resistance. For complete
biological specificity, such agents must be able to recognize duplex DNA in
order to target individual gene sequences. As the naturally occurring
pyrrolo[2,1-c] [1,4]benzodiazepines have shown promising antitumor activity
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T HESIS
due to the sequence-selective binding in the minor groove of DNA. The aim
of this work is to synthesize and evaluate novel DNA binding
pyrrolobenzodiazepine conjugates as potential anticancer agents.
1.5.1. P YRROLO[2,1-C][1,4]BENZODIAZEPINES AS DNA INTERACTIVE ANTITUMOR ANTIBIOTICS
The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a group of potent,
naturally occurring antitumor antibiotics produced by various Streptomyces
species. To date thirteen structures which include anthramycin,18
mezethramycin,20 porothramycin,21 prothracarcin,22 sibanomycine,23
tomaymycin,24 sibiromycin,25 chicamycin A,26 neothramycin A, B27 and DC-
8128 have been isolated from various streptomyces species (Figure-9).
N
HN
OCON
OR9
R8
OCH3
H
N
N
O
HOH
H3CO
N
N
O
H1
6711a
2
5
8
9
8
Anthramycin (R8 = CH3, R9 = R1 = R2 = H)Mazethramycin (R8 = R1 = CH3, R9 = R2 = H)Porothramycin B (R8 = H, R9 = R1 = R2 = CH3)
1011
N
N
O
R
R8
R7
Tomaymycin (R7 = OCH3, R8 = OH, R = CH3)
Prothracarcin (R7 = R8 = H, R = CH3)
Sibanomicine (R8 = H, R7 = sibirosamine
pyronoside as in , R = Et)
N
H
N
OCH3
HO H
Sibiromycin
N
HN
O
HO
H
H3CO
OCH3
OH
Chicamycin A
R1R2
Neothramycin A ( R1 = H; R2 = OH)
Neothramycin B ( R1 = OH, R2 = H)DC-81 (R1 = R2 = H)
O
O
OH
CH3H3C
H3CHN
3
OH
OH
R1
R2
OMe
A B
C
PBD ring system
H
Figure 9. Naturally occurring PBDs
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1.5.2. PBD-DNA INTERACTIONS
The cytotoxicity and antitumor activity of these agents are attributed
to their property of sequence selective covalent binding to the N2 of guanine
in the minor groove of duplex DNA via acid-labile aminal bond to the
electrophilic imine at N10-C11 position.29 These molecules possess an (S)-
configuration at the chiral C11a-position which provides a right handed twist
when viewed from A-ring towards C-ring. This feature provides the
appropriate three dimensional shape for the isohelicity with the minor groove
of B-form DNA leading to a snug fit at the binding site (Figure-10).
N
HN
CH3
HO
MeOO
HH
OH
N
N
CH3
HO
MeOO
H
HN
N N
N
DNA
O
H2N
N
HN
CH3
HO
MeO
O
H
HN
N N
N
DNA
O
HN
-H2O
+H2O
C(11) (S) carbinolamine C (11)-N(10) imine
C(11) (R/S) aminal
Figure 10. Formation of PBD-DNA covalent adduct
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T HESIS
Molecular modeling, solution NMR, fluorimetry and DNA foot printing
experiments reveal that the PBDs recognize a three base pair motif with a
preference for 5’Pu-G-Pu sequences.30 The PBDs have shown to interfere with
the action of endonuclease enzymes on DNA31 and to block the transcription
by inhibiting DNA polymerase in a sequence specific manner,32 processes
which may be relevant for their biological activity.
1.5.3. STRUCTURE ACTIVITY RELATIONSHIP
Structure activity relationship for PBDs has been derived by Thurston
and coworkers. (S)-Configuration at the C11a position is required for snug fit
in the DNA minor groove. PBD with (R)-configuration at C11a has shown to
be devoid of both DNA binding affinity and in vitro cytotoxicity.33 The
electron donating substituents are required in the aromatic A ring for
biological activity. C2 substituted naturally occurring PBDs exhibit more
cytotoxicity compared to unsubstituted PBDs. Bulky substituents like a sugar
moiety at C7 position enhance the DNA binding affinity and cytotoxicity
(Figure-11).
N
NH
R9
R8
R7
OR
1
2
3
1011
11a
(a) An imine, carbinolaminemethyl ether required at N10-C11
(b) (S)-Stereochemistryrequired at C11a
(c) Replacement of C1 withan oxygen maintainscytotoxicity
(d) Endocyclic or exocyclicunsaturation at C2 enhances
cytotoxicity and in vivoantitumour activity. Fullyunsaturated C-ring leads tocomplete loss of DNA-bindingand cytotoxicity
(e) Small substituents (eg. -OH)tolerated at C3 in fully saturatedC-ring compounds
(f) Sugar moiety at C7enhances DNA-bindingaffinity and cytotoxicityin some cell lines
(g) Electron-donatingsubstituents requiredat position 7,8 or 9of A-ring
(h) Bulky substituents at N10
(eg. acetyl) inhibit DNA-binding and cytotoxicity
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T HESIS
Figure 10. Structure activity relationship of PBD ring system
1.6. S YNTHETIC APPROACHES OF P YRROLO[2,1-C][1,4]BENZODIAZEPINES
The first total synthesis of a carbinolamine containing PBD of
anthramycin has been reported by Leimgruber in 1968.34 Extensive reviews
of the synthetic literature of the PBDs have appeared in 1994, 1998 and
2002.35 Various approaches to the synthesis of PBD antibiotics including
hydride reduction of seven-member cyclic dilactams,36 reductive cyclization
of acyclic nitroaldehydes,37 iminothioether approach38 cyclization of
aminothioacetals,39 deprotective cyclization of the diethylthioacetals via N10
protected precursors,40 oxidation of cyclic secondary amines,41 reductive
cyclizations42 and solid phase approaches43 have been investigated.
1.6.1. K ANEKO APPROACH (IMINOTHIOETHER REDUCTION)
Kaneko and coworkers36a developed a mild method for the reduction of
PBD dilactams to the carbinolamine using aluminium amalgam (Scheme 1). This methodology has been employed for the preparation of bicyclic and
tricyclic analogues of anthramycin and the total synthesis of some naturally
occurring PBDs like chicamycin.38b By using this approach Baraldi and
coworkers have synthesized some heterocyclic PBD analogues in which the A
ring of PBD skelton is replaced with a 1,3 or 1,5-disubstituted pyrazole
nucleus.
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T HESIS
N
HN
O
O
HR1
R2R3
N
HN
S
S
HR1
R2R3
N
HN
O
S
HR1
R2 R3
N
N
O
SR4
HR1
R2 R3
N
HN
O
SR4
HR1
R2R3
N
N
O
HR1
R2R3
N
HN
O
HR1
R2 R3
N
HN
O
HR1
R2R3
OCH3
R1 = H, OH, OBn, OCH3, OAcR2 = H, OCH3
R3 = H, = CH-CH3 (E), OH (a), OAc (b), = CH-COOEt (E)
R4 = CH3
1
2+
3
4
+
5
6
7
8
i ii
iii
ivv
Scheme 1. Reagents and conditions: (i) P2S5, C6H6, 80 oC or P2S5, NaHCO3, CH3CN, 15 min,
or (p-CH3OC6H4PS2)2, C6H6, 80 oC; (ii) Et3OBF4, CH2Cl2, KHCO3 or CH3I, K 2CO3, THF or DMF; (iii)
Al-Hg, aq.THF or KH2PO4, 0-5 oC, 14 h; (iv) 0.1 N methanolic HgCl2, 0 oC or SiO2
chromatography, 5 oC; (v) CH3OH.
1.6.2. THRUSTON’S APPROACH (C YCLIZATION B Y DEPROTECTION OF DIETHYLTHIOACETAL)
Thurston and coworkers39a developed an efficient method for the
synthesis of various PBDs containing carbinolamine moiety by employing
mercuric chloride (HgCl2) and calcium carbonate (CaCO3) in aqueous
acetonitrile at room temperature. In this procedure, the products have been
generally isolated in the imine form and it has been extensively utilized for
the synthesis of a variety of naturally occurring and synthetic PBDs including
DC-81 (Scheme 2), C8-linked DC-81 dimers, A ring modified analogues of
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T HESIS
PBD, PBD-EDTA conjugate, lexitropsin conjugates of PBD, C2 linked PBD
dimers, imine-amide PBD dimers and naphthalimide conjugates of PBD.44
NO2
N
CH(SEt)2
O
NH2
N
CH(SEt)2
O
N
N
O
COOH
RO
H3CO COOH
PhH2CO
H3CO
a R = H
b R = PhCH2
NO2
H3CO
PhH2CO
PhH2CO
H3CO
RO
H3CO
a R = PhCH2
b R = H
i
9 10
1213
iii
v
vi
ii
iv
11
H
Scheme 2. Reagents and conditions: (i) PhCH2Cl, THF, NaOH, H2O, reflux, 48 h; (ii) SnCl4,
HNO3, CH2Cl2, -25 oC, 5 min; (iii) (COCl)2, THF, DMF, 3 h then, pyrrolidine-2-
carboxaldehydediethylthioacetal, Et3N, H2O, 0 oC, 1.5 h; (iv) SnCl2.2H2O, MeOH, reflux, 45
min; (v) HgCl2, CaCO3, CH3CN-H2O, 12 h; (vi) 10% Pd-C, EtOH, cyclohexadiene, 3 h.
1.6.3. FUKUYAMA-T YPE APPROACH
In order to incorporate certain labile functionalities such as C8-epoxide
moiety in the PBD system, the conventional deprotective cyclization of
diethyl thioacetal failed to give results. Therefore, Thurston and coworkers40
have made various attempts to synthesize these newly designed PBDs with
potential DNA binding affinity. In this effort, a Fukuyama-type approach45 has
been attempted wherein 9-fluorenyl methyloxy carbonyl (Fmoc) group can
be used to protect the amine group and which can be easily removed bycleavage with Bu4N+F- (TBAF) at room temperature (Scheme 3).
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T HESIS
O
H3CO N
CH(SEt)2
O
O
H3CO N
CH(SEt)2NH
O
Fmoc
O
H3CO N
N
O
OH
Fmoc
a X = NO2
b X = NH2
O
H3CO N
N
O
OH
FmocO
O
H3CO N
N
O
O
H
H
14
1617
18
i
15
ii
iii
iv
v
X
H
Scheme 3. Reagents and conditions: (i) SnCl2.2H2O, CH3OH, reflux, 3 h; (ii) Na2CO3 (aq),
Fmoc-Cl, 0 oC, 4 h, Dioxane, rt, 16 h; (iii) HgCl2, CaCO3, CH3CN-H2O, rt, 48 h; (iv) m-CPBA,
CH2Cl2, rt, 72 h; (v) TBAF, DMF, rt, 15 min.
Baraldi and coworkers46 synthesized hybrid molecules containing PBD
and minor groove binding oligo-pyrrole carriers, while Hurley and coworkers47
have synthesized AT-groove binding hybrids by using this approach. In the
same manner Suzuki coupling of C7 aryl substituted PBDs have been
synthesized by Thurston and co-workers.48 This B-ring strategy of Fukuyama
and coworkers has also been employed for the synthesis of C2/C2'-exo-
unsaturated PBD dimer, C2-C3/C2'-C3'-endo unsaturated PBD dimer withremarkable covalent DNA binding affinity.49
1.6.4. K AMAL’S APPROACH (OXIDATION OF C YCLIC SECONDARY AMINE)
This approach is based on the oxidation of PBD secondary amine and
has been considered as one of the most attractive methods as mentioned in
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T HESIS
one of the reviews on the synthesis of PBDs.35a Kamal and coworkers41
developed a novel method for the oxidation of PBD secondary amine to the
corresponding imines. Although PBDs with either a secondary amine or
amide functionality at N10-C11 are readily synthesized, the introduction of
imine or carbinolamine at this position is problematic due to the reactivity of
these functional groups. As described in the literature, the cyclic secondary
amine precursors have been readily prepared from corresponding nitro
aldehydes. This upon oxidation with DMSO/(COCl)2 or TPAP (tetra-n-propyl
ammonium perruthenate) gives corresponding imines in good yields
(Scheme 4).
NO2
N
CHO
O
N
HN
S
O
H
N
HN
O
H
19 20
21
iii
iii
N
N
O
H
22
Scheme 4. Reagents and conditions: (i) Pd/C; (ii) Raney Ni; (iii) swern/TPAP
1.6.5. REDUCTIVE C YCLIZATION APPROACH
Miyamoto and coworkers50 reported the first total synthesis of N10-C11
imine containing PBDs via reductive cyclization for neothramycin A and B.
Thurston and coworkers51 have carried out a detailed investigation on the
reductive cyclization of N-(2-aminobenzoyl)pyrrolidine-2-carboxaldehydes.
1.6.6. AZIDO REDUCTIVE C YCLIZATION
In an endeavor to explore new practical methods for the preparation of
PBDs particularly by the azido reductive process has been extensively
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T HESIS
investigated by Kamal and co-workers. This procedure has been examined
by different reagents such as N,N-dimethylhydrazine/catalytic ferric chloride,
ferrous sulphate/ammonia, and samarium iodide (SmI2). The same group has
carried out another interesting study on the enzymatic reduction of aryl
azides to aryl amines by employing baker's yeast. This biocatalytic reductive
methodology has been applied to the chemoenzymatic synthesis of PBDs via
the reductive cyclization of arylazido aldehydes (Scheme 5).52
N
NH
O
N
O
CHO
N
O
CHOR1
R2
R1
R2R3 R3
R1
R2R3
N3 NO2
Reagent used for azide reduction
(i) HMDST(ii) bakers' yeast
(iii)N,N-Dimethyl hydrazine/ ferric chloride(iv) TMSI
(v) SmI2(vi) HI(vii) FeSO4.7 H2O
Reagents used for nitro reduction
(i) Fe/ AcOH(ii)N,N-Dimethyl hydrazine/ ferric chloride
R1= OH, OBn, OCH3
R2 = OCH3; R3 = H, OH
Scheme 5;
1.6.7. SOLID PHASE APPROACH
Combinatorial synthesis has become popular in recent years. By using
this technique a large number of distinct molecules could be synthesized in a
short time and resource effective manner. In this pursuit, Thurston and
coworkers43 have developed a solid phase synthesis of PBD imines on p-
nitrophenylcarbonate Wang resin using a variety of oxidation and cyclization
procedures. Kamal and coworkers53 have developed some new synthetic
strategies on PBD dilactams and PBD imines by using Wang resin (Scheme
6).
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T HESIS
O CCl3
NH
N
HN
OO
O
OH + +Fmoc-N
COOCH3
OH
Fmoc-NO
COOCH3
HNO
COOCH3
N
N3
O
COOCH3
O
R
N
N3
O
CHO
O
R
N
HN
OOH
R
O
N
N
OO
RHH
H
N
N
OOH
RH
R
25 27 28
2930
31 34
35
36
32
33
i
iii
iv
v
vii
vi
vii
vi
ii26
Cl3CCN
Scheme 6. Reagents and conditions: (i) DBU, CH2Cl2; (ii) BF3.OEt2 or CF3SO3H, CH2Cl2; (iii)20% piperidine/DMF; (iv) subistituted 2-azido benzoic acid, DCC, DMAP, CH2Cl2, 0 oC; (v)
DIBAL-H, CH2Cl2, -78 oC; (vi) PPh3, toluene; (vii) TFA/CH2Cl2 (1:3).
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1.7. STRUCTURAL MODIFICATION OF PBDS
In the search for compounds with better antitumor selectivity and DNA
sequence specificity many structurally modified PBD analogues have been
synthesized in an attempt to increase their potency against tumor cells.
1.7.1. A-RING MODIFICATIONS
Baraldi and coworkers54 have synthesized heterocyclic PBD analogues
in which A-ring of the PBD is replaced with a 1,3 or 1,5-di-substituted
pyrazole nucleus and some of these PBD analogues have exhibited
interesting profile of cytotoxicity. Leoni55 prepared a range of N6- and N7-
alkyl substituted derivatives of pyrazolo[4,3-c]pyrrolo[1,2-a]
[1,4]diazepinones. Thurston and coworkers44b have reported pyridine and
pyrimidine A-ring analogues of PBD. It is observed that aromatic A-ring has a
modest influence on the thermal denaturation of DNA.
NN
O
NHN
CH3
H3C
NN
O
NHN
H3C
Cl
N
N
HN
O
H
OCH3
N
N N
N
O
H
H3CO
OCH3
1.7.2. B-RING MODIFICATIONS
Nacci and coworkers56 have reported the synthesis of pyrrolo[2,1-c]
[1,4]benzothiazepine compounds as sulphur containing B-ring modified
analogues of PBD. To investigate the role played by the non-covalent
interactions Robba and co-workers57 synthesized a series of PBDs having
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T HESIS
N10-C11 amidines functionality. Interestingly, these compounds have shown
DNA binding affinity comparable to the DC-81, a natural product that binds
covalently to DNA.
N
S
O
N
S
OCH3
HN
NHNH2
N
S
NNHCONHC6H5
OCOCH3
N
S
NNHCO-p-FC6H4
H
H H
1.7.3. C-RING MODIFICATIONS
The degree of saturation of the C-ring is thought to give significant
effects on biological activity. For example, the completely unsaturated
system is unlikely to exhibit antitumor activity. A number of naturally
occurring PBDs namely anthramycin, tomaymycin, sibiromycin andneothramycin have different type of substitutions in the C-ring. It is
interesting to note that these C-ring modified PBDs appear to provide both
greater differential thermal stabilization of duplex DNA and significantly
enhance kinetic reactivity during covalent adduct formation. Thurston and
coworkers58 have synthesized a series of C2-exo unsaturated PBDs and
C2/C3-endo unsaturated PBDs. Partial unsaturation of C-ring enhances both
the DNA-binding affinity and in vitro cytotoxic potency. This group has also
reported the synthesis of novel C2-aryl 2,3-unsaturated and 1,2-unsaturated
PBDs.59
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N
N
O
H3CO
H3CO N
N
O
H3CO
H3COOCH3
O
N
N
O
H3CO
H3CO
OCH3
H
H
N
N
O
H3CO
H3CO
H
H
Ph
Recently, Kamal and co-workers60 have synthesized a series of C2-
fluorinated PBDs61 and have been screened for in vitro cytotoxicity against a
number of cancer cell lines and also studied for DNA binding affinity.
Through a large number of SAR studies it is now known that the substitution
pattern (particularly at C2) and the degree of unsaturation of the C-ring are
crucial for maximising cytotoxicity and antitumour activity in the PBD family.
In conclusion, these experimental observations permitted us to
hypothesize that an increase of carbinolamine reactivity is detrimental in
terms of cytotoxicity. Therefore, A-ring modifications do not seem to boost
PBD activity as much as C-ring modifications do. Based on the current
knowledge of SARs, the C2-side chains play a major role in increasing the
cytotoxicity. Therefore, combination of heterocyclic A-ring analogues with
side chain at position C2 on the C-ring could permit to obtain new potent
derivatives lacking cardiotoxicity.62
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N
N
O
R
R1
R = F, R1 = H, R2 = H, R3 = HR = H, R1 = F, R2 = H, R3 = HR = F, R1 = F, R2 = H, R3 = HR = F, R1 = H, R2 = OBn, R3 = OCH3
R = F, R1 = H, R2 = OH, R3 = OCH3
HR2
R3
N
N
H3CO
ON
N
O
OCH3
O OFF
(CH2)n HH
n = 3, 4, 5
N
N
H3CO
ON
N
O
OCH3
O OFF
(CH2)n HH
n = 3, 4, 5
N
N
O
R
R1
HR2
R3
1.8. PBD DIMERS
In an attempt to extend the number of base pairs spanned by these
molecules, PBD dimers have been synthesized with the hope that enhanced
sequence selectivity might increase selectivity for tumor cells.
1.8.1. C7-LINKED DIMERS
Suggs and coworkers63 have reported the first PBD dimer comprising
of two PBD units joined through A-C7/A-C7’ positions via alkanediyldioxy
linker (some including nitrogen heteroatoms) or alkanediyldisulfide linkers.
The C7-lilnked dimers have been considered unique among DNA-cross linkers
in their specificity for dG-containing duplex DNA.
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N
N O
O
H
O N
N
O
H
N
CH3
7 7
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 (DSB-
120) has been highly potent. Molecular modeling and NMR studies confirmed
that these dimers span six base pairs in the minor groove of duplex DNA.
O
H3CO N
N
O
HN
N
O
OCH3
O
H
DSB 120
Kamal and coworkers44d have synthesized C8-linked imine-amide mixed
dimers wherein one ring of PBD has imine function and the other has amide
group. One of these dimers elevates helix-melting temperature of CT-DNA bya remarkable 17 oC after incubation for 18 h at 37 oC. These dimers have
shown potent antitumor activity in different cell lines.
O
H3CO N
HN
O
HN
N
O
OCH3
O
H
O
Recently, Thurston and coworkers49 have synthesized C8-linked C2/C2’
exo unsaturated dimers as novel cross-linking agents with remarkable DNA
binding affinity and cytotoxicity. These molecules have shown significant in
vivo potency and have been selected for clinical trials.
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O
H3CO N
N
O
HN
N
O
OCH3
O
H
Kamal and coworkers64 have synthesized C8-linked C2-S and C2-R
fluoro substituted PBD dimers and C2/C2’-exo-difluoromethylene dimers.
These dimers possess in vitro anticancer activity in a number of human
cancer cell lines. The replacement of hydrogen with fluorine atom at the C2-
position of the PBD ring system leads to significant increase in cytotoxicity.
N
N
O
H3CO
N
N
O
OCH3
OF F
OHH
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
NN
N
O ONH
NH
O OHH
1.9. C8-LINKED H YBRIDS OF P YRROLOBENZODIAZEPINES
In the search for compounds with better antitumour activity and DNA
sequence specificity many PBD analogues have been synthesized. In the
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literature, much attention has been given to the substitution in the A-ring
particularly at the C8-position as the structural activity relationships suggest
that the substitution at this position cause immense biological responses.
These compounds are capable of recognizing heterogeneous DNA
sequences.
N
N
O
H
MeO
OR alkanes
HN N
ONH
HN
O
Me
O
N
Cl
O
OH
HN
O
NH
HN
O
N
HN
O
n
N
O
O
NH
HN
O
NH
NN
NMe
O
Figure 14. C8-linked PBD hybrids
R =
O
O
HN O
N
NS
NN
O O S
Me
Some of the interesting C8-linked hybrids of pyrrolo[2,1-c]
[1,4]benzodiazepines are UTA-6026,65a seco-CBI,65b pyrene,65c lexitropsin,65d
naphthalimide,65e indole,65f conjugates (Figure 14). In general, the interaction
with DNA tends to be dominated by the minor groove-binding moiety, i.e. the
conjugates bind to the minor groove with preferential interaction with AT-rich
sequences. It may be noted that, the cytotoxicity of these hybrid derivatives
is much greater than that of the alkylating units alone. However, subsequent
molecular modeling studies suggested that C8 would be the preferred
position for the attachment of second interacting group. Baraldi and
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coworkers46 have linked distamycine and netropsin antibiotics to the C8
position of PBD through linker of varying lengths.
HNH2N
ONHHCl
HN O
O
n
N
N
O
H3CO
H
n = 1-4
N
Recently, Lown and coworkers66 have reported PBD-glycosylated
pyrrole and imidazole polyamide conjugates. These compounds have been
prepared with varying number of pyrrole and imidazole containing
polyamides and incorporating glucose moieties in order to improve the water
solubility of PBD-polyamide conjugates. The water soluble PBD-polyamide
conjugates exhibited a higher level of cytotoxic activity than the natural and
synthetic PBDs.
N
N
O
OCH3
ONH
ON
NH
O
N CH3
CH3
O
H
HO
OH
H
H
HH
OH
OH
H n( )3
Pyrrole Polyamide - PBD Conjugate
Hurley and coworkers47 and Denny and coworkers67 have been
designed and synthesized unsymmetrical DNA cross-linkers by linking the
seco-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indo-4-one (seco-CBI) to PBD
moiety. These compounds have anticipated cross-linking between N3 of adenine and N2 of guanine in the minor groove of DNA. One of these
compounds exhibited IC50 in the pico molar range.
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N
N
O
O
H3CO
N
Cl
OH
O
H( )5
CBI - PBD Conjugate
Kamal and co-workers68 have synthesized a series of PBD conjugates
by linking different DNA interacting ligands such as naphthalimides, poly-
aromatic hydrocarbons (pyrene amine and chrysene amine) and
benzimidazoles by using varying linker length to enhance the DNA binding
affinity and antitumor activity.
N
N
O
H
H3CO
O
O
(CH2)nHN
n = 3-4
N
N
O
O
H3CO
H
n = 3-5
N
HN
O
N
NH3C
( )n
Hurley and co-workers69 have synthesized novel DNA-DNA interstrand
adenine-guanine cross-linking UTA-6026 compound. Preliminary in vitro tests
showed that UTA-6026 has remarkably potent cytotoxicity to several tumour
cell lines (IC50 = 0.28 nM in human breast tumour cell line MCF7, IC50 = 0.047
nM in colon tumour cell line SW-480 and IC50 = 5.1 nM in human lung tumour
cell line A549).
N
N
O
O
H3CO
HN
O
NH
N
NH
H3C
O
O
H
Kamal and co-workers70 have designed and synthesized PBD-
morpholine, N-methyl piperizine and N,N-dimethyl amine hybrids in attempts
to improve the water solubility and cytotoxicity of the PBD compounds.
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Based on the solubility recently Lown and co-workers71 have designed and
synthesized novel PBD-gylcosylated pyrrol and imidazole polyamide
conjugates and described as water insoluble and water-soluble PBD
conjugates. Further, these conjugates have been tested against a panel of 60
human cancer cell lines by NCI and demonstrated that the water-soluble
PBD-polyamide conjugates exhibited a higher level of cytotoxic activity than
the existing natural and synthetic PBDs.
N
N
O
O
H3CO
N H
O
In addition to above derivatives, this group has also reported the
synthesis and DNA binding affinity of quinolone,72 pyrimidine hybrids,87 C2/C8
dimers,74 azepine conjugates75 and methanesulfonate derivatives76 of
pyrrolo[2,1-c][1,4]benzodiazepines.
N
N
O
HO
H3CO
O
nN N
CH3
F
N
N
O
HO
H3CO
NnH3COOC
O
F
F
N
N
O
HO
H3CO
H3CO2SOn
n = 3−5
1.10. OBJECTIVES OF THE PRESENT WORK
The molecular modeling, NMR studies, fluorimetry, and DNA foot
printing experiments have given an insight into the mechanism of action of
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PBDs, thus providing opportunities to synthesize novel PBD conjugates with
both improved binding affinity and modified sequence selectivity or with a
change in the binding mode and also probably reducing their undesirable
side effects.
Cancer drug discovery is one of the most rapidly changing areas of
pharmaceutical research. The search for new drugs in the field of oncology
has refocused on natural products. Among the currently identified antitumor
agents, Chalcones, constitute an important group of natural products and
serve as precursors for the synthesis of different classes of flavonoids, which
are common substances in plants. Chalcones are open-chain flavonoids in
which two aromatic rings are joined by a three carbon α, β -unsaturated
carbonyl system (1,3-diphenyl-2-propen-1-ones). The remarkable biological
potential of these chalcones is due to their possible interactions with various
proteins related to cell apoptosis and proliferation. Recent studies have
shown that these chalcones induce apoptosis in a variety of cell types,
including breast cancers. Therefore, in the present chapter a series of
chalcone-PBD conjugates linked through simple alkane spacers have been
synthesized and evaluated for their biological activity (Chapter-II).
The combretastatin A-4 (CA-4) is a natural product found to have
potent anticancer activity against a number of human cancer cell lines
including multidrug resistant cancer cell lines and binds to the colchicine-
binding site of tubulin. A water-soluble prodrug, combretastatin A-4-
phosphate is now in clinical trials for thyroid cancer and in patients with
advanced cancer. The cis configuration only of CA-4 is biologically active,
with the trans form showing little or no activity. The amino derivative of CA-4
is also in clinical trials as a water-soluble aminoacid prodrug.
Being a potentinhibitor of colchicine binding, CA-4 is also shown to inhibit the growth and
development of blood vessels, angiogenesis. Various structural modifications
to CA-4 have been reported including variation of the A- and B-ring
constituents. The chalcone and pyrazoline derivatives of CA-4 also showed
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potent anticancer activity. In view of potent anticancer activity exhibited by
CA-4 derivatives, we synthesized amidobenzothiazole analogues of CA-4 and
evaluated for its anticancer activity. The synthesized analogues exhibited
significant anticancer activity (Chapter-III).
Benzylideneanthrones are class of compounds known to exert potential
antitumor properties. The antitumor activity of these 10-substituted
benzylideneanthrones shows through inhibition of tubulin polymerization.
Helge Prinz and co-workers have been synthesized and reported the potent
in vitro antitumor activity and inhibition of tubulin polymerization of different
anthracenone analogues. In an attempt to establish new conjugates of PBDs
with improved anticancer activities we have synthesized
benzylideneanthrone linked pyrrolobenzodiazepine conjugates. The
synthesized compounds have exhibited significant DNA-binding ability.
(Chapter-IV/Section-A).
Chalcones represent an important group of natural products belonging
to the flavonoids family. Natural and synthetic chalcones have been reported
to posses strong antiproliferative effects in primary as well as established
ovarian cancer cells and in gastric cancer (HGC-27) cells. Recent studies
have shown that these chalcones induce apoptosis in a variety of cell types,
including breast cancers. Previous references reveals that the dimers of
PBDs posses promising anticancer activity. In this context, new analogues of
dimers of pyrrolobenzodiazepines with chalcone have been prepared and
evaluated for anticancer activity (Chapter-IV/Section-B).
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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.
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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,
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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.
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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,
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P. S. M. M. Tedrahedron Lett . 2000, 41, 8631.
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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.; D’Incalci, 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. O’Neil, I. A.; Thompson, S.; Kalindjian, S. B.; Jenkins, T. C. Tetrahedron
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62. Baraldi, P. G.; Cacciari, B.; Guiotto, A.; Romagnoli, R.; Spalluto, G.
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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.
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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.
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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.
CCHAPTERHAPTER-II-II
SS YNTHESIS YNTHESIS AANDND BBIOLOGICALIOLOGICAL EEVALUATIONVALUATION OOFF CCHALCONEHALCONE--PP YRROLOBENZODIAZEPINE YRROLOBENZODIAZEPINE CCONJUGATESONJUGATES AASS AANTICANCERNTICANCER AAGENTSGENTS
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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).
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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. DNA-
binding ability has been observed by thermal denaturation studies with CT-
DNA (∆ T m > 15.1 °C for a 5:1 ratio of DNA:PBD at 37 °C for 18 h incubation)
and the cross-linking efficiency has been investigated by using an agarose
gel electrophoresis assay. The results indicate that DSB-120 is an efficient
cross-linking agent and the cross-linking ability of these PBD dimers after 2 h
incubation at 37 °C has been found to be 0.01 nm. Furthermore, the in vitro
cytotoxicity data in human K562 and rodent ADJ-PC6 cell lines correlate with
both the thermal denaturation data and the cross-linking efficiencies.
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N
N
O
HOH
H3CO
R1R2
N
HN
O
HH3C
OR
CONH2
OCH3
N
N
O
CH3
HO
H3CO
H
N
HN
O
CH3
HOH
O
OHOMe
N
HN
O
HOH
H3CO
OCH3
OH
O
OH
CH3H3C
H3CHN
OH
N
N
O
HOH
H3CO
( R1 = H; R2 = OH) (5)( R1 = OH, R2 = H) (6)
chicamycin
(1)
R= OH or OCH3
anthramycin sibiromycin
tomamycin
neothramycin A and BDC-81
(3)
(7)
(4)
(2)
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 α,β-unsaturated carbonyl
system. Chalcones, considered as the precursor of flavonoids and
isoflavonoids, are abundant in edible plants. However, most of the chalcones
are particularly attractive since it specifically generates the (E)-isomer from
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substituted benzaldehydes and acetophenones. Recent studies revealed that
these chalcones had shown a wide variety of anticancer,10-17 anti-
inflammatory,18-20 antiinvasive,21 antituberculosis,22 and antifungal23 activities.
Chalcones have shown promising anticancer therapeutic efficacy for the
management of human cancers. Recently, different chalcone analogues have
been synthesized and they have been screened for in vitro cytotoxicity
against a number of cancer cell lines.
The substituted chalcones have shown potential anticancer activity.
Ducki and co-workers have synthesized and reported trimethoxy substituted
chalcones24 (8) and (9), that possess potential anticancer activity and bind
strongly to tubulin at a site shared with, or close to, the colchicines binding
site.25-26 The anticancer activity and tubulin binding property of these
chalcones is comparable with combretastatin A-4 (CA-4). The IC50 value of
compound SD400 (9) against the K562 human chronic myelogenous
leukemia cell line is 0.21 nM whereas combretastatin A-4 (CA-4) shows the
IC50 is 2.0 nM. Presently phosphate prodrugs of these compounds (8) and (9)
are under preclinical evaluation. The compound (8) inhibits cell growth at
low concentrations (IC50, P388 murine leukaemia cell line 2.6 nM) and shares
many structural features common to other tubulin-binding agents27 (Figure
2).
MeO
MeO
O
OMe
OMe
OH MeO
MeO
O
OMeOMe
OH
9 (SD400)8
Figure 2. Structures of potential anticancer chalcones
The anticancer activity of certain chalcones is believed to be a result of
binding to tubulin and preventing it from polymerizing into microtubules.
Tubulin is a protein that exists as a heterodimer of two homologous α- and
β -subunits. Many molecules based on a chalcone scaffold have been
synthesized to improve their biological profile, including their capability as
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T HESIS
sequence selective DNA interactive and cross-linking agents.
Trihydroxychalcone (10) represent a new class of tyrosinase inhibitors. The
ease of synthesis of chalcones from substituted benzaldehydes and
acetophenones, makes them an attractive scaffold. Chalcones have
attracted more interest in recent years because of their diverse
pharmacological properties.28 Among these properties, their cytotoxicity
effects have been extensively examined. Some of the natural chalcones
have been found in a variety of plant sources. These natural compounds
have served as valuable leads for further design and synthesis of more
active analogues.29 A chalcone compound (11) has been reported for its
antiproliferative and antitumor activity 30(Figure 3).
HO
O OCH3
OH
OH
H3CO
O
H3CO
OCH3
OH
1110
Figure 3
Further, in this trimethoxy chalcone series different analogues havebeen synthesized by different groups and evaluated for their cytotoxicity.
These compounds have shown promising activity against different cancer
cell lines31 (Figure 4).
MeO
MeO
O
OMe
NO2
MeO
MeO
O
OMe
OMe
B(OH)2
MeO
MeO
O
OMe
OMe
12 13
14
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T HESIS
Figure 4
Recently different series of chalcone analogues with potent anticancer
activity has been reported. Dalip Kumar and co-workers have synthesized
and reported indolylchalcones (15) that are most potent and selective
anticancer agents with IC50 values 0.03 and 0.09 µM, against PaCa-2 cell
line.32 Lawrence and co-workers reported new chalcone derivative (16) which
possess good anticancer activity33 (Figure 5).
NH
O
OMe
OMe
OMe
O
OMe
N
OMe
16 (MDL)15
Figure 5
Curcumin, a polyphenolic natural compound (17) derived from dietary
spice turmeric, possesses diverse pharmacological effects including
anticancer, anti-inflammatory, antioxidant, and antiangiogenic activities.34 A
series of of chalcone dimers has been reported as potent inhibitors of various
cancer cells at very low concentrations. The compound 3,5-bis(2-
fluorobenzylidene)-4-piperidone (18, also known as EF24) is a synthetic
analog of curcumin that was first reported by Adams.35 Other analogues of
3,5-bis(benzylidene)-4-piperidones (19, also known as CLEFMA) and (20) are
have been advanced as synthetic analogs of curcumin for anti-cancer
activity and anti-inflammatory properties and these dimers have shown
promising antiproliferative activity against various cancer cell lines36 (Figure
6).
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T HESIS
NH
O FF
N
O ClCl
O
OH
O19 (CLEFMA)
18 (EF24)
N
O
H3CO
HO
OCH3
OH
CH3
20
HO OH
OMeMeO
OOH
17 (curcumin)
Figure 6. Structures of bis-chalcones
The cyclic chalcone analogues, E-2-arylmethylene-1-indanones, E-2-
arylmethylene-1-tetralones and E-2-arylmethylene-1-benzosuberones have
been synthesized and their cytotoxicity has been determined against
different cancer cell lines.37 Amongst these cyclic chalcones, compounds
(21a, 21b, 22a and 22b) have shown potential anticancer activity against
human cancer cell lines. These compounds inhibit RNA and protein syntheses
and induced apoptosis which are likely major mechanisms whereby
cytotoxicity is mediated. The active compound (22b) in these cyclic
chalcones declines the mitochondrial function as well as mitochondrial DNA
damage. Compound (21b) also showed good activity in targeting
Alzheimer’s disease by inhibition of (acetylcholinesterase) AChE-induced Ab
aggregation38 (Figure 7).
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T HESIS
O
N
MeO NO2
O
21a 21b
22b
O
OMe
22a
Figure 7
2.2. PRESENT WORK In the past few years, several hybrid compounds, in which a known
antitumour compound or some simple active moiety tethered to PBD, have
been designed, synthesized and evaluated for their biological activity.39−41
Recently, Wang and co-workers have synthesized indole-PBD conjugates
(23) as potential antitumour agents and a correlation between antitumour
activity and apoptosis has been well explained.42 For the last few years, we
have been involved in the development of new synthetic strategies for thepreparation of PBD ring system43,44 and also in the design as well as synthesis
of structurally modified PBDs and their conjugates.45-48 More recently, we
have also reported some of the PBD conjugates (24) that demonstrated
potent apoptotic activity through mitochondrial-mediated pathway49 (Figure
8).
O
MeO
N
N
O
O
F
Cl
N
N H
O
NH
NH
O
MeO N
NO
O
H
23
24
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Figure 8. Examples for apoptotic inducing PBDs
In view of the interesting biological activities exhibited by PBD
conjugates, there has been considerable interest in structural modifications
apart from the development of new synthetic strategies in this laboratory forsuch compounds. Based on the diverse biological activities of the chalcones
and the pyrrolo[2,1-c][1,4]benzodiazepines, A series of novel compounds
have been designed and synthesized that have both the chalcone as well as
pyrrolo[2,1-c][1,4]benzodiazepine moieties linked through varying length of
alkane spacers and have been evaluated for their antitumour activity and
DNA-binding affinity.
The present work describes the design, synthesis, DNA binding affinity
and in vitro cytotoxicity of novel chalcones linked to a PBD moiety at the C8-
position of the PBD ring through different alkane spacers. Therefore this
chapter describes the synthesis, DNA-binding ability and anticancer activity
of some new PBD conjugates.
2.2.1. S YNTHESIS OF PBD PRECURSORS
The precursor (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]pyrolidine-
2-carboxaldehydediethylthioacetal 33 have been prepared by employing
commercially available vanillicacid. Esterification of vanillicacid 25 followed
by benzylation by literature method50 afford benzylated methyl ester 27. This
upon nitration followed by hydrolysis gives nitro acid 29. This has been
further coupled to L-proline methyl ester to afford the compound 30, which
upon reduction with DIBAL-H produces the corresponding aldehyde 31. The
aldehyde group of compound 31 has been protected with EtSH/TMSCl affords
32. Compound 32 upon debenzylation affords (2S)-N-[4-hydroxy-5-methoxy-
2-nitrobenzoyl]pyrolidine-2-carboxaldehydediethylthioacetal (33) (Scheme
1).
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T HESIS
BnO
MeO
NO2
OH
O
BnO
MeO
NO2
O
N
COOMe
BnO
MeO
NO2
O
N
CHO BnO
MeO
NO2
O
N
CH(SEt)2
HO
MeO
NO2
O
N
CH(SEt)2
2829
31 32 33
(vi)
(vii)
(iv)(v)
HO
H3COOH
O
HO
H3COOCH3
O
BnO
H3COOCH3
O
ii
iii
26 27
i
25
BnO
H3COOCH3
O
NO2
(viii)
30
Scheme 1. Reagents and conditions: i) H2SO4, MeOH, 24 h; ii) Benzyl bromide, K 2CO3,
acetone, 24 h; iii) HNO3-H2SO4, SnCl4, CH2Cl2, -25 oC, 5 min; iv) 2N LiOH, THF, 12 h; (v) SOCl2,
C6H6, L-proline methyl ester, THF, 6h; (vi) DIBAL-H, CH2Cl2, -70 oC, 30 min; (vii) EtSH, TMSCl,
CH2Cl2, 18h; (viii) EtSH, BF3-OEt2, CH2Cl2, 12h;
2.2.2. SYNTHESIS OF CHALCONE DERIVATIVES
The preparation of chalcone intermediates 37a-f and 40a-c has been
carried out by synthetic sequence illustrated in Scheme-2 & 3. Claisen-
Schmidt condensation of trimethoxy acetophenone with benzaldehydes
using ethanol as solvent in the presence of aqueous KOH gives
trimethoxychalcones 36a,b. The cyclic chalcone (39) have been prepared
using piperidine as base in ethanol solvent under reflux by condensing 1-
indanone with vanillin. Using aqueous KOH for preparation of indanochalcone taking longer reaction time and the yield is very less (20%). These
trimethoxy and indanochalcones undergo etherification of hydroxyl group
with dibromoalkanes by using K 2CO3 as base in dry acetone afford precursors
37a-f and 40a-c.
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T HESIS
MeO
MeO
OMe
CH3
O CHO
R
OH
+
MeO
MeO
OMe
O
OH
R
MeO
MeO
OMe
O
O
R
Br ( )n
(i)
(ii)
37a-f
34 35a, b 36a, b
37a; R = H, n = 237b; R = H, n = 337c; R = H, n = 437d; R = OMe, n = 237e; R = OMe, n = 337f ; R = OMe, n = 4
Scheme 2. Reagents and conditions: (i) aq.KOH, ethanol, 4h; (ii) dibromoalkane, acetone,
K 2CO3, reflux, 24h.
40a; n = 240b; n = 340c; n = 4
CHO
OMe
OH
+
(i)
(ii)
40a-c
38 3539
O O
OH
OMe
O
O
OMe
Br ( )n
Scheme 3. Reagents and conditions: (i) ethanol/piperidine, reflux, 10 h; (ii) dibromoalkane,acetone, K 2CO3, reflux, 24 h.
2.2.3. S YNTHESIS OF C8-LINKED CHALCONE-PBD H YBRIDS
Compound 33 has been coupled to compounds 37a-f in the presence
of K 2CO3 and dry acetone under reflux gives corresponding nitro compounds
41a-f . These nitro compounds upon reduction with SnCl2.2H2O in methanol
under reflux give amino compounds 42a-f . The amino compounds on
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T HESIS
deprotection with HgCl2/CaCO3 afford corresponding imines 43a-f (Scheme-
4).
MeO
MeO
OMe
O
O
R
Br ( )n
37a-f
HO
MeO
NO2
O
N
CH(SEt)2
33
+
O
MeO
NO2
O
N
CH(SEt)2O
O
MeO
MeO
OMe
R
( )n
O
MeO
NH2
O
N
CH(SEt)2O
O
MeO
MeO
OMe
R
( )n
O
MeO
O
OMeO
MeO
OMe
R
( )n
N
N
O
H
41a-f
42a-f
43a-f
i
ii
iii
Scheme 4. Reagents and conditions: (i) K 2CO3, acetone, 24 h, reflux; (ii) SnCl2.2H2O, MeOH,5 h, reflux; (iii) HgCl2, CaCO3, MeCN, H2O, (4:1) 12 h, rt.
Compound 33 has been coupled to compounds 40a-c in the presence
of K 2CO3 and dry acetone under reflux gives corresponding nitro compounds
44a-c. These nitro compounds upon reduction with SnCl2.2H2O in methanol
under reflux give amino compounds 45a-c. The amino compounds on
deprotection with HgCl2/CaCO3 afford corresponding imines 46a-c (Scheme-
5).
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T HESIS
HO
MeO
NO2
O
N
CH(SEt)2
33
+
O
MeO
NO2
O
N
CH(SEt)2O
OMe
( )n
O
MeO
NH2
O
N
CH(SEt)2O
OMe
( )n
O
MeO
O
OMe
( )n
N
N
O
H
44a-c
45a-c
46a-c
i
ii
iii
40a-c
O
O
OMe
Br ( )n
O
O
O
Scheme 5. Reagents and conditions: (i) K 2CO3, acetone, 12 h, reflux; (ii) SnCl2.2H2O, MeOH,
4 h, reflux; (iii) HgCl2, CaCO3, MeCN, H2O, (4:1) 12 h, rt.
2.3. BIOLOGICAL ACTIVITY
2.3.1. DNA BINDING AFFINITY : THERMAL DENATURATION STUDIES
The DNA binding affinity of these new C8-linked chalcone-PBD
conjugates (43a-f and 46a-c) has been evaluated through thermal
denaturation studies with duplex-form of calf thymus DNA (CT-DNA) by using
modified reported procedure.51 The DNA-PBD solutions are incubated at 37
οC for 0 h and 18 h prior to analysis. Samples are monitored at 260 nm using
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T HESIS
a Beckman DU-7400 spectrophotometer fitted with high performance
temperature controller and heated at 1οC/min in the range of 40-95
οC. DNA
helix-coil transition temperatures are given by: ∆ T m = T m(DNA+PBD)–T m(DNA
alone), where the T m value for the PBD-free CT-DNA is 69.8± 0.01. Thesestudies were carried out at PBD/DNA molar ratio 1:5. The increase in melting
temperature (∆ T m) for each compound is examined at 0 h and 18 h of
incubation at 37οC. Melting studies show that these compounds stabilize the
thermal helix coil or melting stabilization for the CT-DNA duplex at pH 7.0,
and incubated at 37οC with ligand/DNA molar ratio of 1:5. The increase in
the helix melting temperature (∆ T m) for each compound has been examined
at 0 h and 18 h incubation at 37 ο C.
Interestingly, all the chalcone-PBD conjugates elevate the helix melting
temperature of CT-DNA in the range of 4.0-8.6 oC. The compound 46b
showed highest ΔT m of 7.9 oC at 0 h and increased upto 8.6 oC after 18 h
incubation, whereas the naturally occurring DC-81 exhibits a ΔT m of 0.7 oC
after incubation under similar conditions (Table 1). These results indicate
that the effect on DNA binding affinity by introducing the chalcone scaffold
on PBD moiety through different alkane spacers at C8-position of the DC-81.
Table 1.Thermal denaturation data for chalcone-PBD conjugates with calf thymus (CT)-DNA
Compound[PBD]:[DNA]
molar ratiob
ΔT m (oC)a after incubation at 37 oCfor
0 h 18 h
43a 1:5 5.1 5.8
43b 1:5 5.2 6.1
43c 1:5 4.6 5.0
43d 1:5 5.3 5.9
43e 1:5 4.0 4.5
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T HESIS
43f 1:5 4.0 4.3
46a 1:5 7.5 8.2
46b 1:5 7.9 8.6
46b 1:5 7.1 8.4DC-81 1:5 0.3 0.7
a For CT-DNA alone at pH 7.00 ± 0.01, T m = 68.5 0C Δ 0.01 (mean value from 10 separate
determinations), all ΔT m values are ± 0.1 - 0.2 0C. b For a 1:5 molar ratio of [PBD]/[DNA],
where CT-DNA concentration = 100 μM and ligand concentration = 20 μM in aqueous
sodium phosphate buffer [10 mM sodium phosphate + 1 mM EDTA, pH 7.00 ± 0.01].
2.3.2. ANTICANCER ACTIVITY
Compounds (43a-f and 46a-c) have been evaluated for their in vitrocytotoxicity in selected human cancer cell lines of barest, ovarian, colon,
prostate, cervix, lung and oral cancer using Sulforhodamine B (SRB)
method.52 The in vitro cytotoxicity results of these compounds expressed in
GI50 values which carried out the experiments at 10-4 to 10-7 M concentrations
and the data is illustrated in Table 2. The results from these experiments
reveal that compounds 43a-f and 46a-c showed GI50 values in the range of
<0.01-2.7 μM, while the positive controls, DC-81 and adriamycin exhibited
the GI50 in the range of 0.1-0.17 μM and <0.01-14.7 μM respectively. The
synthesized chalcone-PBD conjugates exhibited significant anticancer
activity against PC-3 human prostate cancer cell line (GI50 range, <0.01−0.22
μM) compared to other cell lines tested.
The active compound 46b which is an indano chalcone analogue of
PBD exhibited strong effect against all cell lines tested (GI50, <0.01-0.17 μM)
and it showed a GI50 value of <0.01 against PC-3 cell line. In
trimethoxychalcone-PBD analogues the compounds 43b and 43d showed
promising activity against different cancer cell lines. Among the chalcone-
PBD conjugates synthesized, the compounds with indano chalcone moiety
exhibited superior activity compared to the compounds with trimethoxy
chalcone moiety.
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Table 2. GI50 valuesa (in μM) for compounds 43a-f and 46a-c in selectedhuman cancer cell lines.
Compound
GI50 values (μM)
Breast Ovarian Colon Prostate Cervix Lung Oral
MCF-7 A2780 Colo205 PC-3 SiHa A 549 Hop-62 KB
43a 0.11 0.14 0.16 0.14 0.12 0.14 0.15 0.15
43b 0.05 0.03 0.15 0.16 1.8 1.88 0.2 2.1
43c 0.29 0.1 0.15 0.16 0.17 0.1 0.13 0.1
43d 0.07 0.028 0.16 0.22 2.7 -- -- 2.6
43e 0.11 0.14 0.17 0.1 0.12 0.14 0.1 0.14
43f 0.14 0.12 0.14 0.15 0.15 0.16 0.16 0.17
46a 0.1 0.12 0.14 0.08 0.14 0.1 0.14 0.18
46b 0.01 0.013 0.14 <0.01 0.17 0.17 0.0160.01
6
46c 0.17 0.14 0.17 0.16 0.16 0.11 0.17 0.14
DC-81 0.16 0.13 0.1 -- 0.16 -- 0.11 0.17
ADR<0.0
10.02 14.7 <0.01 0.19 13 <0.01 0.16
a 50% Growth inhibition and the values are mean of four determinationsADR, adriamycin.
2.4. CONCLUSION
In conclusion, we have synthesized a series of novel C8-linked chalcone-
PBD conjugates (43a-f and 46a-c). For synthesized compounds
anticancer activity has been evaluated against eight human cancer cell
lines (barest, ovarian, colon, prostate, cervix, lung and oral cancer). All
the compounds exhibited significant anticancer activity. Moreover these
compounds exhibited significant DNA binding ability.
2.5. EXPERIMENTAL SECTION
Methyl-4-hydroxy-3-methoxy benzoate (26)
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The compound 4-hydroxy-3-methoxy benzoic acid 25 (168 mg, 1 mmol) was
dissolved in methanol (10 mL) and to this was added concentrated H2SO4 (2
mL) and the reaction mixture was stirred for 24 h at room temperature. After
completion of the reaction as indicated by TLC, methanol was evaporated
under reduced pressure and the residue was neutralized with saturated
NaHCO3 solution and extracted with ethyl acetate, dried over Na2SO4 and
concentrated to give the crude product. This was further purified by column
chromatography using hexane: ethyl acetate (2:8) as a solvent system to
obtain the pure product 26 (178 mg, 98% yield).
1H NMR (200 MHz, CDCl3): δ 3.88 (s, 3H), 3.95 (s, 3H), 6.62 (bs, 1H), 7.17 (d,
1H, J = 9.2 Hz ), 7.60 (d, 1H), 7.78 (s, 1H);
EIMS: m/z 182 [M]+.
Methyl-4-benzyloxy-3-methoxybenzoate (27)
To the solution of compound 26 (182 mg, 1 mmol) in acetone (20 mL)
was added, anhydrous K 2CO3 (553 mg, 4 mmol) and benzyl bromide (256 mg,
1.5 mmol), the mixture was refluxed in an oil bath for 24 h. The reaction was
monitored by TLC using EtOAc-hexane (2:8) and K 2CO3 was removed by
filtration, solvent was evaporated under reduced pressure. The crude thus
obtained was purified by column chromatography (10% EtOAc-hexane) to
afford compound 27 as white solid (250 mg, 92%);
Mp: 116−118οC.
1H NMR (200 MHz, CDCl3): δ 3.94 (s, 3H), 3.98 (s, 3H), 5.2 (s, 2H), 6.88 (d,
1H, J = 8.2 Hz), 7.20−7.50 (m, 6H), 7.65 (d, 1H, J = 8.8 Hz);
EI MS: m/z 272 [M]+
Methyl-4-benzyloxy-5-methoxy-2-nitrobenzoate (28)
A freshly prepared mixture of stannic chloride (301 mg, 1.2 mmol) and
fuming nitric acid (98 mg, 1.56mmol) in dichloromethane was added drop
wise over 5 minutes with stirring to a solution of methyl-4-benzyloxy-3-
methoxybenzoate 27 (272 mg, 1 mmol) in dichloromethane (30 mL) at –78
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T HESIS
οC. The mixture was maintained at –78
οC for a further 5 minutes, quenched
with water (20 mL) and then allowed to return room temperature. The
organic layer was separated and the aqueous layer was extracted with
dichloromethane (2x20 mL). The combined organic phase dried over Na2SO4,evaporated in vacuum and purified by column chromatography (20% EtOAc-
hexane) affords 28 as a yellow solid (247 mg, 78%). Mp 128−130οC;
1H NMR (200 MHz, CDCl3): δ 7.65 (d, 1H, J = 8.8 Hz), 7.45−7.2 (m, 6H), 6.78
(d, 1H, J = 8.2 Hz), 5.2 (s, 2H), 3.95 (s, 3H), 3.89 (s, 3H);
EIMS: m/z 317 (M)+.
4-Benzyloxy-5-methoxy-2-nitrobenzoic acid (29)
2N Lithium hydroxide monohydrate solution (1.22 mL) was added to a
solution of methyl-4-benzyloxy-5-methoxy-2-nitrobenzoate 28 (317 mg, 1
mmol) in THF-H2O-MeOH (4:1:1) and the mixture stirred at room temperature
for 12 h. After completion reaction THF and methanol were evaporated, the
aqueous phase was acidified with dilute HCl to pH 7 and reextracted with
CH2Cl2 to give a 4-benzyloxy-5-methoxy-2-nitrobenzoic acid 29 as a pale
yellow solid (251 mg, 83%);
Mp: 180−182οC;
1H NMR (200 MHz, CDCl3): δ 7.44−7.22 (m, 6H), 7.2 (s, 1H), 5.25 (s, 2H), 3.98
(s, 3H);
EIMS: m/z 303 (M)+.
Methyl-(2S)-N-[4-benzyloxy-5-methoxy-2-nitrobenzoyl]prrolidine-2-
carboxylate (30)
To a suspension of compound 29 (303 mg, 1 mmol) and thionyl chloride (476
mg, 4.0 m mol) in dry benzene (15 mL) was added few drops of DMF and
stirred for 6 hr. The toluene was evaporated in vacuum and the resultant oil
was dissolved in dry THF (20 ml) and added drop wise over a period of 30
mins to a cooled suspension of L-proline methyl ester hydrochloride (248 mg,
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T HESIS
1.5 mmol), triethyl amine (303 mg, 3 mmol) in THF (20 mL). After completion
of addition the reaction mixture was brought to ambient temperature and
stirred for additional hours. The THF was evaporated under vacuum and the
aqueous layer was extracted with ethyl acetate, washed with water followed
by brine solution. The organic layer was dried over Na2SO4 evaporated under
vacuum and was purified by column chromatography using EtOAc-hexane
(3:7) to afford compound 30 as yellow solid. Yield (352 mg, 70%).
Mp: 152-154 ºC;
1H NMR (300 MHz, CDCl3): δ 7.72 (s, 1H), 7.3–7.48 (m, 6H), 5.2 (s, 2H), 4.68–
4.75 (m, 1H), 3.92 (s, 3H), 3.8 (s, 3H), 3.26–3.4 (m, 1H), 3.1–3.25 (m, 1H),
2.2–2.45 (m, 1H), 1.81–2.12 (m, 3H);
ESIMS: m/z 414 (M) +.
(2S)-N-[4-Benzyloxy-5-methoxy–2-nitrobenzoyl]pyrrolidine-2-
carboxaledhyde (31)
Diisobutylaluminiumhydride solution (2.5 ml of 1.0 M solution in toluene) was
added drop wise to a stirred solution of the compound 30 (414 mg, 1mmol)
in anhydrous dichloromethane (10 mL) under nitrogen atmosphere at –78 oC.
After addition the mixture was stirred for 30 mins. After completion of reaction it was decomposed by slow addition of methanol (2 mL) followed by
5% HCl (2 mL). The resultant mixture was allowed to warm to room
temperature. The reaction mixture layer was extracted with chloroform
(4X20 mL), the combined organic layers were dried over Na2SO4 and solvent
was evaporated under vacuum to afford the crude aldehyde 31. Yield (254
mg 65%)
1H NMR (300 MHz, CDCl3): δ 9.8 (s, 1H), 9.25 (s, 1H), 7.75 (s, 1H), 7.25–7.5
(m, 5H), 6.56 (s, 1H), 5.2 (s, 2H), 4.7 (m, 1H), 4.02 (m, 1H), 3.18–3.38 (m,
2H), 1.88–2.4 (m, 4H);
ESIMS: m/z 355 (M-CHO) +
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T HESIS
(2S)-N-[4-Benzyloxy-5-methoxy–2-nitrobenzoyl]pyrrolidine-2-carboxaledhyde diethylthioacetal (32)
Ethanethiol (278 mg, 4.4 mmol) was added to a stirred solution of
nitroaldehyde 31 (384 mg, 1 mmol) in dry dichloromethane (25 mL) under
nitrogen atmosphere. The mixture was stirred for further 10 mins followed by
the addition of trimethylsilly chloride (540 mg, 5 mmol). The resulting
mixture was stirred at room temperature for about 18 hrs. After completion
of the reaction, the reaction mixture was neutralized with sodium
bicarbonate solution and extracted with chloroform (2x15 mL). The combined
organic phases were dried over Na2SO4 and evaporated under vacuum to
afford the crude diethylthioacetal (32), which was purified by column
chromatography by using EtOAc-hexane (6:4) to afford pure compound 32
as viscous oil. Yield (335 mg, 75%).
1H NMR (300 MHz, CDCl3): δ 7.75 (s, 1H), 7.32–7.48 (s, 5H), 6.85 (s, 1H), 5.2
(s, 2H), 4.85 (d, 1H), 4.66–4.75 (m, 1H), 4 (s, 3H), 3.2–3.35 (m, 2H), 2.65–
2.96 (m, 4H), 1.21–1.42 (m, 6H);
ESIMS: m/z 490 (M+H)+.
(2S)-N-[4-Hydroxy-5-methoxy–2-nitrobenzoyl]pyrrolidine-2-
carboxaledhyde diethylthioacetal (33) To a stirred solution of EtSH of (1.91 gm, 20 mmol) and BF3OEt2 (1.41 gm, 10
mmol) in dichloromethane was added drop wise to a solution of the
compound 32 (0.49 gm, 1mmol) in dichloromethane (10 mL) at room
temperature. Stirring was continued until TLC indicated complete of the
reaction. The reaction mixture was quenched with bicarbonate solution and
the extracted with chloroform (3x25 mL). The combined organic phases were
washed with brine solution (1x25 mL), dried over Na2SO4 and the solvent
removed under vacuum to afford the crude product. This was further purified
by column chromatography using ethyl acetate-hexane (7:3) as eluant to
afford pure compound 33. Yield (300 mg, 75%).
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1H NMR (300 MHz, CDCl3): δ 7.6 (s, 1H), 6.75 (s, 1H), 4.85 (d, 1H), 4.6–4.7 (m,
1H), 3.9 (s, 3H) 3.2–3.32 (m, 2H), 2.7–2.88 (m, 4H), 1.75–2.35 (m, 4H), 1.2–
1.4 (m, 6H);
ESIMS: m/z 400 (M) +.
(E)-3-(4-hydroxyphenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one
(36a)
To a stirred mixture of 3,4,5-trimethoxy acetophenone (210 mg, 1 mmol)
and 4-hydroxybenzaldehyde (122 mg, 1 mmol) in ethanol (10 mL) was added
50% aqueous solution of potassium hydroxide (1 ml) and stirred for 4 h at
room temperature. After completion of the reaction checked by TLC, the
solvent was evaporated, neutralized with dilute HCl and extracted with
ethylacetate (2x50 ml). The combined organic fractions were washed with
water followed by brain, dried over Na2SO4 and purified by column
chromatography using (30% EtOAC:hexane) to obtain the pure product 36a
as yellow solid. Yield (285 mg, 90%).
Mp: 136-138 ºC;
1H NMR (300 MHz, CDCl3): δ 7.79 (d, 1H, J = 15.1 Hz), 7.57 (d, 2H, J = 9 Hz),
7.36 (d, 1H, J = 15.1 Hz), 7.27 (s, 2H), 6.91 (d, 2H, J = 8.3 Hz), 6.05 (bs, 1H),3.94 (s, 6H), 3.92 (s, 3H);
ESIMS: m/z 315 (M+H)+.
(E)-3-(4-hydroxy-3-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)prop-
2-en-1-one (36b)
The compound 36b was prepared according to the method described for
compound 36a by employing compound 3,4,5-trimethoxy acetophenone
(210 mg, 1mmol), and 3-methoxy-4-hydroxy benzaldehyde (152 mg, 1
mmol). Yield (310 mg, 90%)
Mp: 127-129 ºC;
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T HESIS
1H NMR (300 MHz, CDCl3): δ 7.68 (d, 1H, J = 15.8 Hz), 7.27 (d, 1H, J = 15.8
Hz), 7.16–7.23 (m, 3H), 7.05 (d, 1H, J = 2.2 Hz), 6.89 (d, 1H, J = 8.3 Hz),
6.18 (bs, 1H), 3.96 (s, 9H), 3.89 (s, 3H);
ESIMS: m/z 345 (M+H)+.
(E)-3-(4-(3-bromopropoxy)phenyl)-1-(3,4,5-trimethoxyphenyl)prop-
2-en-1-one (37a)
To a solution of compound 36a (314 mg, 1 mmol) in dry acetone (15 mL)
was added, anhydrous K 2CO3 (274 mg, 2 mmol), 1,3-dibromopropane (605
mg, 3 mmol) and the mixture was stirred at reflux temperature for 24 hours.
The reaction was monitored by TLC using ethyl acetate-hexane (3:7). After
completion of the reaction as indicated by the TLC, K 2CO3 was removed by
filtration and the solvent evaporated under reduced pressure, diluted with
water and extracted with ethyl acetate(2X20 ml). The combined organic
phases were dried over Na2SO4 and evaporated under vacuum. The residue,
thus obtained was purified by column chromatography using ethyl acetate
and hexane (2:8) to afford pure compound 37a as viscous liquid. Yield (418
mg, 95%)
1H NMR (300 MHz, CDCl3): δ 7.78 (d, 1H, J = 15.8 Hz), 7.62 (d, 2H, J = 9 Hz),7.36 (d, 1H, J = 15.8 Hz), 7.25 (s, 2H), 6.92 (d, 2H, J = 8.3 Hz), 4.05 (t, 2H, J
= 6, 5.2 Hz), 3.95 (s, 6H), 3.91 (s, 3H), 3.42 (t, 2H, J = 6.7, 6 Hz), 2.04-2.16
(m, 2H);
ESIMS: m/z 436 (M+H)+.
(E)-3-(4-(4-bromobutoxy)phenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-
en-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%).
1H NMR (300 MHz, CDCl3): δ 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
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T HESIS
= 6, 5.2 Hz), 3.95 (s, 6H), 3.9 (s, 3H), 3.47 (t, 2H, J = 6.7, 6 Hz), 1.92–2.13
(m, 4H);
ESIMS: m/z 451 (M+H)+.
(E)-3-(4-(5-bromopentoxy)phenyl)-1-(3,4,5-trimethoxyphenyl)prop-
2-en-1-one (37c)
The compound 37c was prepared according to the method described for
compound 37a by employing compound 36a (314 mg, 1 mmol), and 1,5
dibromopentane (689 mg, 3 mmol). Yield (422 mg, 91%)
1H NMR (300 MHz, CDCl3): δ 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), 1.59–1.71 (m, 2H);
ESIMS: m/z 464 (M+H)+.
(E)-3-(4-(3-bromopropoxy)-3-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (37d)
The compound 37d was prepared according to the method described for
compound 37a by employing compound 36b (344 mg, 1 mmol), and 1,3
dibromopropane (605 mg, 3 mmol). Yield (400 mg, 86%)1H NMR (200 MHz, CDCl3): δ 7.77 (d, 1H, J = 15.8 Hz), 7.33 (d, 1H, J = 15.8
Hz), 7.21−7.28 (m, 3H), 7.16 (d, 1H, J = 2.2 Hz), 6.90 (d, 1H, J = 8.3 Hz), 4.08
(t, 2H, J = 6.7, 6 Hz), 3.95 (s, 6H), 3.94 (s, 6H), 3.43 (t, 2H, J = 6.7 Hz),
1.82−1.97 (m, 2H);
ESIMS: m/z 466 (M+H)+.
(E)-3-(4-(4-bromobutoxy)-3-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (37e)
The compound 37e was prepared according to the method described for
compound 37a by employing compound 36b (344 mg, 1 mmol), and 1,4-
dibromobutane (647 mg, 3 mmol). Yield (425 mg, 88%)
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1H NMR (200 MHz, CDCl3): δ 7.7 (d, 1H, J = 15.8 Hz), 7.27 (d, 1H, J = 15.8
Hz), 7.17–7.24 (m, 3H), 7.1 (d, 1H, J = 1.5 Hz), 6.84 (d, 1H, J = 8.3 Hz), 4.07
(t, 2H, J = 6, 5.2 Hz), 3.95 (s, 6H), 3.91 (s, 3H), 3.9 (s, 3H), 3.5 (t, 2H, J =
6.79, 6.04 Hz), 1.94–2.17 (m, 4H);
ESIMS: m/z 483 (M+H)+.
(E)-3-(4-(3-bromopropoxy)-3-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (37f)
The compound 37f was prepared according to the method described for
compound 37a by employing compound 36b (344 mg, 1 mmol), and 1,5
dibromopentane (689 mg, 3 mmol). Yield (450 mg, 91%)
1H NMR (400 MHz, CDCl3): δ 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-3-
methoxy benzaldehyde (152 mg, 1 mmol) in ethanol (10 ml) was added few
drop of piperidine and refluxed for 10 h. After completion of the reaction
checked by TLC, the solvent was evaporated and extracted with ethyl
acetate (2x50 ml). The combined organic fractions were washed with water
followed by brain, dried over Na2SO4 and purified by column chromatography
using (30% EtOAC:hexane) to obtain the pure product 39. Yield (200 mg,
75%).
1H NMR (300 MHz, DMSO D6): δ 9.77 (s, 1H), 7.79 (d, 1H, J = 7.3 Hz), 7.67–
7.74 (m, 2H), 7.44–7.56 (m, 2H), 7.37 (s, 1H), 7.3 (d, 1H, J = 8 Hz), 6.92 (d,
1H, J = 8 Hz), 4.12 (s, 2H), 3.90 (s, 3H);
ESIMS: m/z 289 (M+Na)+.
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T HESIS
(E)-2-(4-(3-bromopropoxy)-3-methoxybenzylidene)-2,3-
dihydroinden-1-one (40a)
The compound 40a was prepared according to the method described for
compound 37a by employing compound 39 (266 mg, 1 mmol), and 1,3-dibromopropane (605 mg, 3 mmol). Yield (320 mg, 82%)
1H NMR (300 MHz, CDCl3): δ 7.87 (d, 1H, J = 7.5 Hz), 7.48−7.61 (m, 3H),
7.40 (t, 1 H, J = 7.5, 6.7 Hz), 7.21−7.27 (m, 1H), 7.13 (d, 1H, J = 1.5 Hz),
6.93 (d, 1H, J = 8.3 Hz), 4.19 (t, 2H, J = 6 Hz), 3.99 (s, 2H), 3.92 (s, 3H),
3.63 (t, 2H, J = 6.7, 6 Hz), 2.33−2.44 (m, 2H);
ESIMS: m/z 388 (M+H)+.
(E)-2-(4-(4-bromobutoxy)-3-methoxybenzylidene)-2,3-dihydroinden-
1-one (40b)
The compound 40b was prepared according to the method described for
compound 37a by employing compound 39 (266 mg, 1 mmol), and 1,4-
dibromobutane (647 mg, 3 mmol). Yield (351 mg, 87%)
1H NMR (300 MHz, CDCl3): δ 7.88 (d, 1H, J = 8 Hz), 7.48–7.63 (m, 3H), 7.35–
7.46 (m, 1H), 7.24 (dd, 1H, J = 8, 2.2 Hz), 7.14 (d, 1H, J = 1.4 Hz), 6.89 (d,
1H, J = 8.8 Hz), 4.09 (t, 2H, J = 5.8 Hz), 4 (s, 2H), 3.93 (s, 3H), 3.5 (t, 2H, J
= 6.6 Hz), 1.94–2.21 (m, 4H);
ESIMS: m/z 401 (M)+.
(E)-2-(4-(5-bromopentoxy)-3-methoxybenzylidene)-2,3-
dihydroinden-1-one (40c)
The compound 40c was prepared according to the method described for
compound 37a by employing compound 39 (266 mg, 1 mmol), and 1,5-
dibromopentane (689 mg, 3 mmol). Yield (372 mg, 89%)
1H NMR (300 MHz, CDCl3): δ 7.87 (d, 1H, J = 7.5 Hz), 7.48−7.61 (m, 3H), 7.4
(t, 1 H, J = 7.5, 6.7 Hz), 7.23 (dd, 1H, J = 8.3, 1.5 Hz), 7.13 (d, 1H, J = 2.2
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T HESIS
Hz), 6.87 (d, 1H, J = 8.3 Hz), 4.05 (t, 2H, J = 6.7, 6 Hz), 3.99 (s, 2H), 3.92 (s,
3H), 3.43 (t, 2H, J = 6.7 Hz), 1.83−2.03 (m, 4H), 1.60−1.74 (m, 2H);
ESIMS: m/z 416 (M+H)+.
2S)-N-{4-(3-[(E)-3-(4-phenoxy)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one]propyl)oxy-5-methoxy-2-nitrobenzoyl}-pyrrolidine-2-carboxaldehydediethyl thioacetal (41a)
To a solution of (2S)-N-[4-hydroxy-5-methoxy–2-nitrobenzoyl]pyrrolidine-2-
carboxal dehydediethylthioacetal (33) (400 mg, 1 mmol) in dry acetone (15
mL) was added, anhydrous K 2CO3 (276 mg, 2 mmol), (E)-3-(4-(3-
bromopropoxy) phenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (37a)
(435 mg, 1 mmol) and the mixture was stirred at reflux temperature for 24
hours. The reaction was monitored by TLC using ethyl acetate-hexane (1:1).
After completion of the reaction as indicated by the TLC, K 2CO3 was removed
by filtration and the solvent evaporated under reduced pressure, diluted with
water and extracted with ethyl acetate. The organic phase was dried over
Na2SO4 and evaporated under vacuum. The residue, thus obtained was
purified by column chromatography using ethyl acetate and hexane (1:1) to
afford compound 41a as yellow solid. Yield (680 mg, 90%)
Mp: 176-178 ºC;1H NMR (300 MHz, CDCl3): δ 7.77 (d, 1H, J = 15.8 Hz), 7.68 (s, 1H), 7.61 (d,
1H, J = 15.1 Hz), 7.25 (s, 2H), 6.91 (d, 2H, J = 8.3 Hz), 6.77 (s, 1H), 4.82 (d,
1H, J = 3.7 Hz), 4.62–4.71 (m, 1H), 4.1–4.23 (m, 4H), 3.95 (s, 6H), 3.91 (s,
6H), 3.17–3.31 (m, 2H), 2.62–2.85 (m, 4H), 2.2–2.36 (m, 1H), 2.02–2.16 (m,
3H), 1.91–1.98 (m, 1H), 1.74–1.86 (m, 1H), 1.31–1.41 (m, 6H);
ESIMS: m/z 755 (M+H)+.
2S)-N-{4-(4-[(E)-3-(4-phenoxy)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one]butyl)oxy-5-methoxy-2-nitrobenzoyl}-pyrrolidine-2-carboxaldehydediethyl thioacetal (41b)
The compound 41b was prepared according to the method described for
compound 41a by employing (2S)-N-[4-hydroxy-5-methoxy–2-
nitrobenzoyl]pyrrolidine-2-carboxal dehydediethylthioacetal (33) (400 mg,
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T HESIS
1mmol), and (E)-3-(4-(4-bromobutoxy)phenyl)-1-(3,4,5-
trimethoxyphenyl)prop-2-en-1-one (37b) (449 mg, 1 mmol). Yield (705 mg,
91%)
Mp: 175-177 ºC;
1H NMR (200 MHz, CDCl3): δ 7.73 (d, 1H, J = 15.8 Hz), 7.63 (s, 1H), 7.57 (d,
1H, J = 15.1 Hz), 7.24 (s, 2H), 6.88 (d, 2H, J = 8.3 Hz), 6.76 (s, 1H), 4.81 (d,
1H, J = 3.7 Hz), 4.62–4.7 (m, 1H), 4.08–4.22 (m, 4H), 3.95 (s, 6H), 3.9 (s,
6H), 3.16–3.3 (m, 2H), 2.62–2.86 (m, 4H), 2.19–2.36 (m, 1H), 2.01–2.16 (m,
5H), 1.90–1.99 (m, 1H), 1.74–1.87 (m, 1H), 1.30–1.39 (m, 6H);
ESIMS: m/z 791 (M+Na)+.
2S)-N-{4-(5-[(E)-3-(4-phenoxy)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one]pentyl)oxy-5-methoxy-2-nitrobenzoyl}-pyrrolidine-2-carboxaldehydediethylthioacetal (41c)
The compound 41c was prepared according to the method described for
compound 41a by employing (2S)-N-[4-hydroxy-5-methoxy–2-
nitrobenzoyl]pyrrolidine-2-carboxal- dehydediethylthioacetal (33) (400 mg,
1mmol), and (E)-3-(4-(5-bromopentoxy)phenyl)-1-(3,4,5-
trimethoxyphenyl)prop-2-en-1-one (37c) (463 mg, 3 mmol). Yield (721 mg,
92%)Mp: 172-174 ºC;
1H NMR (200 MHz, CDCl3): δ 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), 4.61–4.71 (m, 1H), 4.1–4.22 (m, 4H), 3.96 (s, 6H), 3.91 (s,
6H), 3.17–3.31 (m, 2H), 2.61–2.85 (m, 4H), 2.2–2.35 (m, 1H), 2.02–2.16 (m,
5H), 1.9–1.98 (m, 1H), 1.72–1.82 (m, 3H), 1.31–1.4 (m, 6H);
ESIMS: m/z 783 (M+H)+.
2S)-N-{4-(3-[(E)-3-(4-[3-methoxyphenoxy])-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one]propyl)oxy-5-methoxy-2-nitrobenzoyl}-pyrrolidine-2-carboxaldehydediethyl thioacetal (41d)
The compound 41d was prepared according to the method described for
compound 41a by employing (2S)-N-[4-hydroxy-5-methoxy–2-nitrobenzoyl]
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T HESIS
pyrrolidine-2-carboxal dehyde diethylthioacetal (33) (400 mg, 1 mmol), and
(E)-3-(4-(3-bromopropoxy)-3-methoxyphenyl)-1-(3,4,5-
trimethoxyphenyl)prop-2-en-1-one (37d) (465 mg, 3 mmol). Yield (712 mg,
90%)
Mp: 165-167 ºC;
1H NMR (300 MHz, CDCl3): δ 7.7 (d, 1H, J = 15.6 Hz), 7.61 (s, 1H), 7.29 (d,
1H, J = 15.6 Hz), 7.14–7.25 (m, 3H), 7.11 (s, 1H), 6.83 (d, 1H, J = 7.8 Hz),
6.76 (s, 1H), 4.82 (d, 1H, J = 3.9 Hz), 4.60–4.74 (m, 1H), 4.0–4.15 (m, 4H),
3.95 (s, 6H), 3.93 (s, 3H), 3.90 (s, 3H), 3.16–3.31 (m, 2H), 2.61–2.88 (m,
4H), 2.16–2.38 (m, 1H), 1.79–2.14 (m, 5H), 1.79–2.14 (m, 5H), 1.28–1.42
(m, 6H);
ESIMS: m/z 785 (M+H)+.
2S)-N-{4-(4-[(E)-3-(4-[3-methoxyphenoxy])-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one]butyl)oxy-5-methoxy-2-nitrobenzoyl}-pyrrolidine-2-carboxaldehydediethyl thioacetal (41e)
The compound 41e was prepared according to the method described for
compound 41a by employing (2S)-N-[4-hydroxy-5-methoxy–2-nitrobenzoyl]
pyrrolidine-2-carboxal dehydediethylthioacetal (33) (400 mg, 1mmol), and
(E)-3-(4-(4-bromobutoxy)-3-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (37e) (479 mg, 3 mmol). Yield (751 mg, 93%)
Mp: 165-167 ºC;
1H NMR (200 MHz, CDCl3): δ 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), 1.30–1.40 (m, 6H);
ESIMS: m/z 799 (M+H)+.
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T HESIS
2S)-N-{4-(5-[(E)-3-(4-[3-methoxyphenoxy])-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one]pentyl)oxy-5-methoxy-2-nitrobenzoyl}-pyrrolidine-2-carboxaldehydediethyl thioacetal (41f)
The compound 41f was prepared according to the method described for
compound 41a by employing (2S)-N-[4-hydroxy-5-methoxy–2-nitrobenzoyl]pyrrolidine-2-carboxal dehydediethylthioacetal (33) (400 mg, 1mmol), and
(E)-3-(4-(5-bromopentoxy)-3-methoxyphenyl)-1-(3,4,5-
trimethoxyphenyl)prop-2-en-1-one (37f) (493 mg, 3 mmol). Yield (765 mg,
94%)
Mp: 164-166 ºC;
1H NMR (400 MHz, CDCl3): δ 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), 1.31–1.39
(m, 6H);
ESIMS: m/z 813 (M+H)+.
7-Methoxy-8-[3-{(E)-3-(4-phenoxy)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one} propoxy]-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (43a)
To the compound 41a (754 mg, 1 mmol) in methanol (20 mL) was added
SnCl2.2H2O (1.12 g, 5 mmol) and reflux for 5 hrs and checked TLC indicated
the reaction was completed. The methanol was evaporated under vacuum
and the reaction mass was neutralized with 10% NaHCO3 solution and the
extracted with ethyl acetate and chloroform (2x30mL and 2x30mL). The
combined organic phases was dried over Na2SO4 and evaporated under
vacuum to afford the crude aminodiethylthioacetal 42a (650 mg, 89%),
which was used directly in the next step due to its potential stability
problem.
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A solution of 42a (724 mg, 1.0 mmol), HgCl2 (677 mg, 2.5 mmol) and CaCO3
(250 mg, 2.5 mmol) in acetonitrile-water (4:1) was stirred slowly at room
temperature overnight until complete consumption of starting material as
indicated by the TLC. The clear organic supernatant liquid was extracted with
ethyl acetate and washed with saturated 5% NaHCO3 (20 mL), brine (20 mL)
and the combined organic phase was dried over Na2SO4. The organic layer
was evaporated in vacuum to afford a white solid, which was purified by
column chromatography with MeOH-CHCl3 (1:20) to obtain the pure product
43a. Yield (325 mg, 54%).
Mp: 122-123 ºC;
1H NMR (300 MHz, CDCl3): δ 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), 4.08–4.18 (m, 4H), 3.95 (s, 6H),
3.93 (s, 3H), 3.92 (s, 3H), 3.71–3.84 (m, 2H), 3.52–3.64 (m, 1H), 2.28–2.38 (m,
2H), 1.83–2.05 (m, 4H);
ESIMS: m/z 601 (M+H)+.
7-Methoxy-8-[4-{(E)-3-(4-phenoxy)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one} butoxy]-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c]
[1,4]benzodiazepin-5-one (43b)
This compound was prepared according to the method described for the
compound 43a employing 41b (768 mg, 1.0 mmol) which reduction with
SnCl2.2H2O (1.12 g, 5 mmol) gives amino compound 42b. Deprotection
followed by cyclization of 42b (738 mg, 1.0 mmol) with HgCl2 (677 mg, 2.5
mmol), CaCO3 (250 mg, 2.5 mmol) in acetonitrile-water (4:1) to obtain the
pure product 43b. Yield (330 mg, 53%).
Mp: 121-123 ºC;
1H NMR (200 MHz, CDCl3): δ 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), 4.06–4.17 (m, 4H), 3.96 (s, 6H),
3.94 (s, 3H), 3.93 (s, 3H), 3.78–3.85 (m, 1H), 3.69–3.77 (m, 1H), 3.53–3.65
(m, 1H), 2.28–2.38 (m, 2H), 1.94–2.16 (m, 6H);
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T HESIS
ESIMS: m/z 615 (M+H)+.
7-Methoxy-8-[5-{(E)-3-(4-phenoxy)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one} pentoxy]-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (43c)
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 ºC;
1H NMR (200 MHz, CDCl3): δ 7.77 (d, 1H, J = 15.1 Hz), 7.68 (d, 1H, J = 4.5 Hz),
7.62 (d, 2H, J = 9 Hz), 7.52 (s, 1H), 7.37 (d, 1H, J = 15.1 Hz), 7.27 (s, 2H),
6.93 (d, 2H, J = 9 Hz), 6.81 (s, 1H), 4.07–4.16 (m, 4H), 3.95 (s, 6H), 3.93 (s,
3H), 3.92 (s, 3H), 3.72–3.85 (m, 2H), 3.52–3.65 (m, 1H), 2.28–2.38 (m, 2H),
1.9–2.13 (m, 6H), 1.61−1.72 (m, 2H);
ESIMS: m/z 629 (M+H)+.
7-Methoxy-8-[3-{(E)-3-(4-[3-methoxyphenoxy])-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one}propoxy]-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (43d)
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 ºC;
1H NMR (200 MHz, CDCl3): δ 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),
4.03–4.13 (m, 4H), 3.95 (s, 6H), 3.94 (s, 6H), 3.93 (s, 3H), 3.78–3.87 (m,
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T HESIS
1H), 3.69–3.76 (m, 1H), 3.69–3.76 (m, 1H), 3.53–3.64 (m, 1H), 2.27–2.38
(m, 1H), 2.0–2.11 (m, 1H), 1.84–1.98 (m, 4H);
ESIMS: m/z 631 (M+H)+.
7-Methoxy-8-[4-{(E)-3-(4-[3-methoxyphenoxy])-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one}butoxy]-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (43e)
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 ºC;
1H NMR (300 MHz, CDCl3): δ 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),
4.05–4.16 (m, 4H), 3.96 (s, 6H), 3.94 (s, 6H), 3.93 (s, 3H), 3.76–3.85 (m,
1H), 3.69–3.75 (m, 1H), 3.53–3.65 (m, 1H), 2.26–2.38 (m, 2H), 1.92–2.07
(m, 4H), 1.61–1.79 (m, 2H);ESIMS: m/z 645 (M+H)+.
7-Methoxy-8-[5-{(E)-3-(4-[3-methoxyphenoxy])-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one}pentoxy]-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (43f)
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 ºC;
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1H NMR (200 MHz, CDCl3): δ 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),
4.07–4.15 (m, 4H), 3.95 (s, 6H), 3.94 (s, 6H), 3.93 (s, 3H), 3.77–3.86 (m,
1H), 3.68–3.76 (m, 1H), 3.52–3.64 (m, 1H), 2.27–2.38 (m, 2H), 2–2.11 (m,
1H), 1.9–2.08 (m, 6H), 1.62−1.71 (m, 2H);
ESIMS: m/z 659 (M+H)+.
2S)-N-{4-(3-[(E)-2-(4-{3-methoxybenzylidene}oxy)-2,3-dihydroinden-1-one]propyl) oxy-5-methoxy-2-nitrobenzoyl}-pyrrolidine-2-carboxaldehydediethylthioacetal (44a)
The compound 44a was prepared according to the method described for
compound 41a by employing 2S)-N-[4-hydroxy-5-methoxy–2-nitrobenzoyl]
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%)
1H NMR (200 MHz, CDCl3): δ 7.88 (d, 1H, J = 7.5 Hz), 7.70 (s, 1H), 7.49−7.61
(m, 3H), 7.41 (t, 1 H, J = 7.5, 6.7 Hz), 7.23 (dd, 1H, J = 8.3, 2.2 Hz), 7.14 (d,
1H, J = 2.2 Hz), 6.93 (d, 1H, J = 8.3 Hz), 6.76 (s, 1H), 4.82 (d, 1H, J = 3.77
Hz), 4.61−4.71 (m, 2H), 4.31(t, 2H, J = 6 Hz), 4.27 (t, 2H, J = 6 Hz), 4 (s,
2H), 3.92 (s, 6H), 3.13−3.32 (m, 2H), 2.6−2.85 (m, 4H), 2.35−2.47 (m, 2H),
2.19−2.34 (m, 2H), 1.88−2.01 (m, 1H), 1.73−1.87 (m, 1H), 1.28–1.41 (m, 6H);
ESIMS: m/z 707 (M+H)+.
2S)-N-{4-(4-[(E)-2-(4-{3-methoxybenzylidene}oxy)-2,3-dihydroinden-1-one]butyl)oxy-5-methoxy-2-nitrobenzoyl}-pyrrolidine-2-carboxaldehydediethylthioacetal (44b)
The compound 44b was prepared according to the method described for
compound 41a by employing 2S)-N-[4-hydroxy-5-methoxy–2-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%)
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1H NMR (200 MHz, CDCl3): δ 7.86 (d, 1H, J = 7.5 Hz), 7.64 (s, 1H), 7.5–7.61
(m, 3H), 7.39 (t, 1H, J = 6.7, 7.5 Hz), 7.2 (d, 1H, J = 8.3 Hz), 7.13 (s, 1H),
6.87 (d, 1H, J = 8.3 Hz), 6.7 (s, 1H), 4.78 (d, 1H, J = 3.7 Hz), 4.6–4.7 (m,
1H), 4.12–4.3 (m, 4H), 4 (s, 2H), 3.87 (s, 6H), 3.15–3.3 (m, 2H), 2.61–2.82
(m, 4H), 2.19–2.35 (m, 1H), 2.01–2.18 (m, 4H), 2.18–2.2 (m, 1H), 1.64–1.84
(m, 2H), 1.26–1.39 (m, 6H);
ESIMS: m/z 721 (M+H)+.
2S)-N-{4-(5-[(E)-2-(4-{3-methoxybenzylidene}oxy)-2,3-dihydroinden-1-one]pentyl)oxy-5-methoxy-2-nitrobenzoyl}-pyrrolidine-2-carboxaldehydediethylthioacetal (44c)
The compound 44c was prepared according to the method described for
compound 41a by employing 2S)-N-[4-hydroxy-5-methoxy–2-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%)
1H NMR (300 MHz, CDCl3): δ 7.87 (d, 1H, J = 7.5 Hz), 7.62 (s, 1H), 7.49−7.61
(m, 3H), 7.4 (t, 1 H, J = 7.5, 6.7 Hz), 7.23 (dd, 1H, J = 8.3, 1.5 Hz), 7.14 (d,
1H, J = 2.2 Hz), 6.88 (d, 1H, J = 8.3 Hz), 6.77 (s, 1H), 4.82 (d, 1H, J = 3.7
Hz), 4.61−4.71 (m, 2H), 4.05−4.17 (m, 4H), 4 (s, 2H), 3.93 (s, 3H), 3.92 (s,
3H), 3.15−3.31 (m, 2H), 2.62−2.87 (m, 4H), 2.19–2.35 (m, 1H), 1.88−2.16
(m, 6H), 1.65−1.86 (m, 3H), 1.29–1.41 (m, 6H);
ESIMS: m/z 735 (M+H)+.
7-Methoxy-8-[3-{(E)-2-(4-{3-methoxybenzylidene}oxy)-2,3-dihydroinden-1-one} propoxy]-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one(46a)
This compound was prepared according to the method described for thecompound 43a employing 44a (706 mg, 1.0 mmol) which reduction with
SnCl2.2H2O (1.12 g, 5 mmol) gives amino compound 45a. Deprotection
followed by cyclization of 45a (676 mg, 1.0 mmol) with HgCl2 (677 mg, 2.5
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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 ºC;
1H NMR (300 MHz, CDCl3): δ 7.9 (d, 1H, J = 7.2 Hz), 7.55–7.71 (m, 4H), 7.51
(s, 1H), 7.38–7.47 (m, 1H), 7.3 (dd, 1H, J = 8, 1.4 Hz), 7.2 (d, 1H, J = 1.4
Hz), 6.98 (d, 1H, J = 8 Hz), 6.82 (s, 1H), 4.09–4.25 (m, 4H), 4.04 (s, 2H),
3.93 (s, 6H), 3.65–3.86 (m, 2H), 3.48–3.63 (m, 1H), 2.23–2.41 (m, 2H),
1.96–2.18 (m, 4H);
ESIMS: m/z 553 (M+H)+.
7-Methoxy-8-[4-{(E)-2-(4-{3-methoxybenzylidene}oxy)-2,3-dihydroinden-1-one} butoxy]-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one(46b)
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 ºC;1H NMR (200 MHz, CDCl3): δ 7.92 (d, 1H, J = 7.2 Hz), 7.56–7.72 (m, 4H),
7.52 (s, 1H), 7.38–7.48 (m, 1H), 7.29 (dd, 1H, J = 8, 1.4 Hz), 7.19 (d, 1H, J =
1.4 Hz), 6.97 (d, 1H, J = 8 Hz), 6.82 (s, 1H), 4.11–4.28 (m, 4H), 4.03 (s, 2H),
3.93 (s, 6H), 3.67–3.86 (m, 2H), 3.49–3.66 (m, 1H), 2.25–2.39 (m, 2H),
1.94–2.19 (m, 5H), 1.65–1.8 (m, 1);
13C NMR (75 MHz, CDCl3): δ194.28, 150.02, 149.35, 149.28, 138.12, 134.33, 134.15,
132.45, 130.85, 128.75, 128.28, 127.55, 126.04, 124.55, 124.23, 113.84, 112.5, 111.43,110.32, 105.57, 68.49, 65.5, 55.96, 53.66, 46.63, 33.28, 32.31, 31.51, 30.48, 30.27, 29.54,
25.79, 24.11;
ESIMS: m/z 567 (M+H)+.
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7-Methoxy-8-[5-{(E)-2-(4-{3-methoxybenzylidene}oxy)-2,3-dihydroinden-1-one} pentoxy]-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one(46c)
This compound was prepared according to the method described for the
compound 43a employing 44c (734 mg, 1.0 mmol) which reduction withSnCl2.2H2O (1.12 g, 5 mmol) gives amino compound 45c. Deprotection
followed by cyclization of 45c (714 mg, 1.0 mmol) with HgCl2 (677 mg, 2.5
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 ºC;
1H NMR (300 MHz, CDCl3): δ 7.91 (d, 1H, J = 7.2 Hz), 7.56–7.71 (m, 4H),
7.52 (s, 1H), 7.38–7.48 (m, 1H), 7.31 (dd, 1H, J = 8, 1.4 Hz), 7.19 (d, 1H, J =
1.4 Hz), 6.98 (d, 1H, J = 8 Hz), 6.81 (s, 1H), 4.1–4.26 (m, 4H), 4.03 (s, 2H),
3.93 (s, 6H), 3.65–3.85 (m, 2H), 3.49–3.65 (m, 1H), 2.23–2.39 (m, 2H),
1.92–2.13 (m, 6H), 1.63−1.75 (m, 2H);
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 7.00±0.01) containing CT-DNA, (100 µ M in phosphate) and the
PBD (20 µ M) were prepared by addition of concentrated PBD solutions in
methanol to obtain a fixed [PBD]/[DNA] molar ratio of 1:5. The DNA-PBD
solutions were incubated at 37οC for 0 h prior to analysis sample were
monitored a 260 nm using a Beckman DU-7400 spectrophotometer fitted
with high performance temperature controller. Heating was applied at a rate
of 1οC min-1 in the 40−90
οC range. DNA helix-coil transition temperatures
(T m) were determined from the maxima in the d(A260)/dT derivative plots.
Results for each compound are shown as mean ± standard derivation from
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the least three determinations and are corrected for the effects of methanol
co-solvent using a linear correction term. Ligand-induced alteration in DNA
melting behavior are given by ∆ T m = T m(DNA+PBD)−T m(DNA alone), where
the T m value for the PBD free CT-DNA is 69.8 ± 0.001. The fixed [PBD]/[DNA]ratio used did not result in binding saturation of the host DNA duplex for any
compound examined.
2.7. ANTICANCER ACTIVITY SEREENING
The synthesized compounds (43a-f and 46a-c) were evaluated for
their in vitro anticancer activity in selected human cancer cell lines. A
protocol of 48 h continuous drug exposure and a Sulforhodamine B (SRB)
protein assay was used to estimate cell viability or growth. The cell lines
were grown in RPMI 1640 medium containing 10% fetal bovine serum and
2 mML-glutamine, and were inoculated into 96-well microtiter plates in 90
µL at plating densities depending on the doubling time of individual cell
lines. The microtiter plates were incubated at 37οC, 5% CO2, 95% air and
100% relative humidity for 24 h prior to addition of experimental drugs.
Aliquots of 10 µL of the drug dilutions were added to the appropriate
microtiter wells already containing 90 µL of cells, resulting in the required
final drug concentrations. Each compound was evaluated for four
concentrations (0.1, 1, 10 and 100 µM) and each was done in triplicate
wells. Plates were incubated further for 48 h, and assay was terminated by
the addition of 50 µL of cold trichloro acetic acid (TCA) (final concentration,
10% TCA) and plates were again incubated for 60 min at 4οC. The plates
were washed five times with tap water and air-dried. Sulforhodamine B
(SRB) solution (50 µL) at 0.4% (w/v) in 1% acetic acid was added to each of
the wells, and plates were incubated for 20 min at room temperature. The
residual dye was removed by washing five times with 1% acetic acid. The
plates were air-dried. Bound stain was subsequently eluted with 10 mM
trizma base, and the absorbance was read on an ELISA plate reader at a
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wavelength of 540 nm with 690 nm reference wavelengths. Percent growth
was calculated on a plate-by-plate basis for test wells relative to control
wells. The above determinations were repeated three times.
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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.
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41. Damayanthi, Y.; Reddy, B. S. P.; Lown, J. W. J. Org.Chem. 1999, 64,
290..
42. Wang, J. J.; Shen, Y. K.; Hu, W. P.; Hsieh, M. C.; Lin, F. L.; Hsu, M. K.; Hsu,
M. H. J. Med. Chem. 2006, 49, 1442
43. Kamal, A.; Laxman, E.; Reddy, P. S. M. M. Tetrahedron Lett . 2000, 41,
7743.
44. Kamal, A.; Shankaraiah, N.; Devaiah, V.; Reddy, K. L. Tetrahedron Lett .
2006, 47, 6553.
45. Kamal, A.; Devaiah, V.; Reddy, K. L.; Kumar, M. S. Bioorg. Med. Chem.
2005, 13, 2021.
46. Kamal, A.; Naseer, A. K.; Reddy, S.; Ahmed, S. K.; Kumar, M. S.; Juvekar,
A.; Sen, S.; Zingde, S. Bioorg. Med. Chem. Lett . 2007, 19, 5345.
47. Kamal, A.; Kumar, P. P.; Seshadri, B. N.; Srinivas, O.; Kumar, M. S.; Sen,
S.; Kurian, N.; Juvekar, A.; Zingde, S. Bioorg. Med. Chem. 2008, 16,
3895.
48. Kamal, A.; Shankaraiah, N.; Devaiah, V.; Reddy, K. L.; Juvekar, A.;
Kurian, N.; Zingde, S. Bioorg. Med. Chem. Lett . 2008, 18, 1468.
49. Kamal, A.; Bharathi, E. V.; Ramaiah, M. J.; Dastagiri, D.; Reddy, J. S.;
Viswanath, A.; Sultana, F.; Pushpavalli, S. N. C. V. L.; Bhadra, M. P.;
Srivastava, H. K.; Sastry, G. N.; Juvekar, A.; Sen, S.; Zingde, S. Bioorg.
Med. Chem. 2010, 18, 526.
50. Thurston, D. E.; Murthy, V. S.; Langley, D. R.; Jones, G. B. Synthesis.
1990, 81.
51. Puvvada, M. S.; Hartley, J. A.; Jenkins, T. C.; Thurston, D. E. Nucleic Acids
Res. 1993, 21, 3671.
52. 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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T HESIS
CCHAPTERHAPTER-III-III
S YNTHESIS AND BIOLOGICAL EVALUATION OF COMBRETASTATIN DERIVATIVES AS
ANTICANCER AGENTS
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3.1. INTRODUCTION
Microtubules are cytoskeletal structures that are formed by self
assembly of α and β tubulin heterodimers and are involved in many cellular
functions.1 Their most important role is the formation of the mitotic spindle,
and they are essential to the mitotic division of cells. Tubulin is an α,β
heterodimeric protein that is the main constituent of microtubules (Figure 1).
Tubulin is the target of numerous small molecule antiproliferative ligands
that act by interfering with microtubule dynamics.2 These ligands can be
broadly divided into two categories: those that inhibit the formation of the
mitotic spindle such as colchicine (1)3,4 and vinblastine5 and those that
inhibit the disassembly of the mitotic spindle once it has formed, such as
paclitaxel.6
Figure 1. 3-D model of microtubule
The three characterized binding sites of tubulin are the taxane domain,
the vinca domain, and the colchicine domain, and many compounds interact
with tubulin at these known sites. Many tubulin binding compounds, such as
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T HESIS
paclitaxel and vinblastine, are in clinical use for various types of cancer2
(Figure 2).
MeO
MeO
OMe
OMe
O
NH
O
Colchicine (1)
O
OO
O
OH
MeO
OMe
OMe
podophylotoxin (3)
MeO
MeO
OMe
OMe
OH
Combretastatin A-4 (2a)
Figure 2. Examples of tubulin binding agents
3.2. COMBRETASTATINS
Antimitotic agents are one of the major class of cytotoxic drugs for
cancer treatment, and microtubules are an important target for many natural
product anticancer agents such as combretastatin A-4 (2a)7 and
podophyllotoxin (3).2,8 The combretastatins are a group of diarylstilbenes
isolated from the stem wood of the South African tree Combretum caffrum.9
Compound 2a was found to have potent anticancer activity against a
number of human cancer cell lines including multidrug resistant cancer cell
lines and binds to the colchicine-binding site of tubulin.10
A water-solubleprodrug, combretastatin A-4-phosphate 2b, is now in clinical trials for thyroid
cancer11-13 and in patients with advanced cancer.14 Compound 2b induces
vascular shutdown within tumors at doses less than one-tenth of the
maximum tolerated dose (MTD) and without detectable morbidity, assuming
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a MTD of 1000mg/kg.7 Hydrolysis in vivo by endogenous nonspecific
phosphatases under physiological conditions affords 2b15,16 (Figure 3).
MeO
MeO
OMe
OMe
R
4a, R = NH24b, R = NH-CO-CH-(NH2)(CH2OH)
MeO
MeO
OMe
OMe
OPO3Na2
Combretastatin A-4P (2b)
Figure 3
The amino derivative of combretastatin, 4a (AC7739) is also in clinical
trials as a water-soluble amino acid prodrug (4b).17 In contrast to colchicine,
the antivascular effects of compound 2a in vivo are apparent well below the
maximum tolerated dose, offering a wide therapeutic window. The
compound 2a as well as being a potent inhibitor of colchicines binding is also
shown to inhibit the growth and development of blood vessels,
angiogenesis.5,18-21 The cis configuration only of 2a is biologically active, with
the trans form showing little or no activity.22 The active cis double bond in 2a
is readily converted to the more stable trans isomer during storage ormetabolism, resulting in a dramatic decrease in antitumor activity.23,24
CA-4 is an exceptionally strong inhibitor of tubulin polymerization and
is potently cytotoxic against murine lymphocytic leukemia and against
human ovarian and colon cancer cell lines.25-27 Its mechanism of action is
thought to be related to the tubulin binding properties that result in rapid
tumour endothelial cell damage, neovascular shutdown, and subsequent
haemorrhagic necrosis.28,29 It has been recently demonstrated that CA-4P, a
combretastatin-A4 prodrug, induces cell death primarily through mitotic
catastrophe in a panel of human B-lymphoid tumors.30 Mitotic catastrophe
appears to be a cell death modality different from apoptosis. Indeed, it has
been reported that CA-4 was able to activate caspase-9, but the inhibition of
caspase-9 by the use of the specific inhibitor Z -LEHDfmk did not inhibit
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T HESIS
combretastatin-induced cell death. This indicates that apoptosis is a
secondary mechanism of death in a small proportion of cells treated with CA-
4.30
NH2
HO
OH
OH
Arotinoids (5) Trans- Resveratol (6)
Figure 4
The apoptotic activity of natural and synthetic stilbene arotinoids (5)
structurally related to vitamin A.31,32 Because some derivatives demonstrated
potent apoptotic activity in both normal and multidrug-resistant (MDR) cell
lines, it would be informative to explore novel stilbenoids as a logical starting
point in the quest for anticancer chemotherapeutics. The amino derivative
AC7739 (4a)33 and compounds structurally related to trans-resveratrol (6)34
possess potent apoptosis-inducing activity. The cis or trans stereochemistry
of the double bond of biologically active stilbenes was the major
discriminating factor affecting their apoptotic activity. CA-4 kills cells with a
modality different from apoptosis (Figure 4).
Various structural modifications to 2a have been reported including
variation of the A- and B-ring substituents.35-37 Many modifications of the B-
ring result in decreased bioactivity; however, substitution of the 3′-OH with
an amino group results in potent bioactivity and good water solubility.38 The
3,4,5-trimethoxy substituted pattern in ring A, resembling the trimethoxyaryl
ring of colchicine, is optimal for bioactivity of 2a.24
From the previouscomparative studies of the combretastatin it appears that the cis orientation
of the two aromatic rings plays an important key role in cytotoxicity.
However, during storage and administration cis combretastatin analogues
tend to isomerize to trans forms which show a dramatic decrease in their
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T HESIS
inhibitory effects on cancer cell growth and tubulin polymerization.39
Structural alteration of the stilbene motif of CA-4 can be extremely effective
in producing potent apoptosis-inducing agents by activating both the
intrinsic and the extrinsic pathways. Accordingly, a number of cis-restricted
analogues of CA-4 were prepared using 1,2-substituted five-membered
heterocycles such as imidazole,40 oxazole,40 pyrazole,40,41 triazole,41 tetrazole,41
thiazole,41 furanone,42 dioxolane,43 and furazan44 to avoid the stability
problem. Many of them showed potent cytotoxicity against various cancer
cells compared to CA-4.
In earlier study, chalcone derivatives and pyrazoline derivatives of
combretastatin with two aromatic rings of CA-4 have shown an attractive
profile of cytotoxicity and apoptosis inducing activity. Their ability to block
most cells in the G2 phase of the cell cycle suggests that these compounds
could act on targets different from the mitotic spindle. This may confer on
these molecules a wider spectrum of action than the parent CA-4, which
arrests cells in the M phase of the cell cycle.
MeO
MeO
OMe
O
OMe
OH MeO
MeO
OMe
O
OMe
OH
7 8 (SD400)
Figure 5
The chalcone derivatives of combretastatin have showed exciting
potential as anticancer agents. Sylvie Ducki and co-workers synthesized
trimethoxy substituted chalcones
45
7 and 8, which possess potentialanticancer activity and binding strongly to tubulin at a site shared with, or
close to, the colchicines binding site.46,47 The anticancer activity and tubulin
binding property of these chalcones is comparable with combretastatin A-4
(CA-4). The IC50 value of compound SD400 (8) against the K562 human
chronic myelogenous leukemia cell line is 0.21 nM whereas for CA-4 it is 2.0
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T HESIS
nM. Presently phosphate prodrugs of these compounds 7 and 8 are under
preclinical evaluation. The compound 7 inhibits cell growth at low
concentrations (IC50, P388 murine leukaemia cell line 2.6 nM) and shares
many structural features common to other tubulin binding agents48 (Figure
5).
MeO
MeO
OMe
N
OMe
OH
9a, R = H9b, R = Acetyl
NR
Figure 6 Johnson and coworkers49 synthesized N-acetylated and non-acetylated 3,4,5-
tri- or 2,5-dimethoxypyrazoline analogs 9a and 9b of combretastatin-A4. A
non-acetylated derivative (9a) with the same substituents as in CA-4 (2a)
was the most active compound in the series, with IC50 values of 2.1 and 0.5
µM in B16 and L1210 cell lines respectively. A cell-based assay indicated that
compound 9a caused extensive microtubule depolymerization with EC50
value of 7.1 µM in A-10 cells. Molecular modeling studies showed that these
compounds possess a twisted conformation similar to CA-4 (2a) (Figure 6).
3.3. PRESENT WORK
The present work describes the design, synthesis, and anticancer
evaluation of novel analogues of combretastatin, and its chalcone, pyrazoline
derivatives with amino benzothiazoles. In view of the interesting biological
activities exhibited by combretastatin derivatives, there has been
considerable interest in structural modification of these molecules and
development of new synthetic strategies in the laboratory.
These compounds have been prepared by coupling of different 2-
aminobenzothiazoles with combretastatin and its chalcone, pyrazoline
derivatives with amide bond with a view to evaluate more potent anticancer
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T HESIS
molecules. In view of diverse biological activities of the combretastatin
derivatives, we have been designed, synthesized novel compounds with
aminobenzothiazoles and evaluated for their antitumour activity and these
compounds exhibited potential anticancer activity.
3.3.1. S YNTHESIS OF COMBRETASTATIN AND ITS DERIVATIVES
The precursor (Z)-2-[(2-methoxy-5-(3
trimethoxystyryl)]phenoxy)acetic acid 18 has been prepared by employing
commercially available isovanillin. Hydroxy group protection of isovanillin 10
with TBDMS-Cl followed by reduction of aldehyde group with NaBH4 gives
alcohol 12. The benzyl alcohol 12 was converted to benzyl bromide 13 by
using LiBr followed by salt formation with PPh3 affords compound 14.
OMe
OH
CHO
OMe
OTBDMS
CHO
OMe
OTBDMS
CH2OH
OMe
OTBDMS
CH2Br
OMe
OTBDMS
CH2PPh3Br MeO
MeO
OMe
OMe
OTBDMSMeO
MeO
OMe
OMe
OH
(i) (ii) (iii)
(iv)
(v)(vi)
MeO
MeO
OMe
OMe
OOEt
OMeO
MeO
OMe
OMe
OOH
O
(vii)
(viii)
10 11 12 13
1415
16
17 18
Scheme 1. Reagents and conditions: i) TBDMS-Cl, TEA, DMF, 1h; ii) NaBH4, MeOH, 2h; iii)
LiBr, TMS-Cl, THF, 1h; iv) PPH3, toluene, 8h; v) n-BuLi, THF,-20 oC, trimethoxybenzaldehyde,
30 min; vi) TBAF, THF, 20 min; vii) 2-bromoethyl acetate, K 2CO3, DMF, 12h; viii) LiOH.H2O,
THF, H2O, 12h.
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The compound 14 on Wittig reaction with trimethoxybenzaldehyde
gives TBDMS-protected combretastatin A-4 15. This compound upon
deprotection with TBAF gives combretastatin A-4, 16. This upon
etherification with α-bromo ethyl acetate in the presence of K 2CO3 affords the
ester compound 17. The ester compound on hydrolysis with LiOH affords (Z)-
2-(2-methoxy-5-(3,4,5-trimethoxystyryl) phenoxy)acetic acid 18 (Scheme 1).
3.3.2. S YNTHESIS OF CHALCONE DERIVATIVE OF COMBRETASTATIN
The preparation of chalcone derivative 22 was carried out by synthetic
sequence illustrated in Scheme-2. Claisen-Schmidt condensation of
trimethoxy acetophenone 19 with 4-methoxy-3-hydroxybenzaldehyde
(isovaniline) using ethanol as solvent in the presence of aqueous KOH gives
trimethoxychalcones 20. This upon etherification with α-bromo ethyl acetate
in the presence of K 2CO3 gives ester compound 21. The ester compound on
hydrolysis with LiOH affords chalcone acid, (E)-2-(2-methoxy-5-(3-oxo-3-
(3,4,5-trimethoxyphenyl)prop-1-enyl) phenoxy)aceticacid 22 (Scheme 2).
MeO
MeO OMe
O
MeO
MeO OMe
O
OMe
OH
OMeOH
CHO
+
22
MeO
MeO
OMe
O
OMe
OOEt
O
MeO
MeO
OMe
O
OMe
OOH
O
19 20
21
(i)
(ii)
(iii)
Scheme 2. Reagents and conditions: i) aq. KOH, ethanol, 6h; ii) 2-bromoethyl acetate,K 2CO3, DMF, 12h; iii) LiOH. H2O, THF, H2O, 14h
3.3.3. S YNTHESIS OF P YRAZOLINE DERIVATIVE OF COMBRETASTATIN
The preparation of pyrazoline derivative 25 has been carried out by
synthetic sequence illustrated in Scheme-3. Cyclization of trimethoxy
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T HESIS
chalcone 20 with hydrazine hydrate in acetic acid under reflux gives
pyrazoline derivative 23. This upon etherification with α-bromoethyl acetate
in the presence of K 2CO3 gives the ester 24. The ester compound on
hydrolysis with LiOH affords pyrazoline acid 2-(5-(1-acetyl-3-(3,4,5-
trimethoxyphenyl)-4,5-dihydro-1H-pyrazol -5-yl)-2-
methoxyphenoxy)aceticacid 25 (Scheme 3).
MeO
MeO
OMe
O
OMe
OH MeO
MeO
OMe
N
OMe
OH
N
O
25
MeO
MeO
OMe
N
OMe
O
N
O
OEt
O
MeO
MeO
OMe
N
OMe
O
N
O
OH
O
20 23
24
(i)
(ii)
(iii)
Scheme 3. Reagents and conditions: i) NH2NH2.H2O, Acetic acid, reflux, 14h; ii) 2-
bromoethyl acetate, K 2CO3, DMF, 12h; iii) LiOH.H2O, THF, H2O
3.3.4. S YNTHESIS OF COMBRETASTATIN-BENZOTHIAZOLE ANALOGUES
The synthesis of combretastatin-benzothiazole derivatives (27a-i) is
outlined in Scheme 4. Combretastatin acid 18 undergoes amide bond
formation with different 2-amino benzothiazoles in the presence of
EDCI/HOBT in dichloromethane affords combretastatin-benzothiazole
analogues 27a-i (Scheme-4).
The synthesis of chalcone-benzothiazole derivatives (28a-i) is outlined
in Scheme 5. Chalcone acid 22 undergoes amide bond formation with
different 2-amino benzothiazoles in the presence of EDCI/HOBT in
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T HESIS
dichloromethane affords chalcone-benzothiazole analogues 28a-i (Scheme-
5).
MeO
MeO
OMe
OMe
OOH
O
18
N
SH2N+
MeO
MeO
OMe
OMe
ONH
O
S
N
26
27a, R = -H27b, R = -NO227c, R = -F27d, R = -Cl27e, R = -OMe27f , R = -OCF327g, R = -Me27h, R = -CF327i, R = -OEt
R
R
27 a-i
(i)
Scheme 4. Reagents and conditions: i) EDCI/HOBT, DCM, 14-16h
28a, R = -H28b, R = -NO228c, R = -F28d, R = -Cl28e, R = -OMe28f , R = -OCF328g, R = -Me
28h, R = -CF328i, R = -OEt
N
SH2N
+
26
R
22
MeO
MeO
OMe
O
OMe
OOH
O
MeO
MeO
OMe
O
OMe
ONH
O
S
N R
28
(i)
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
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T HESIS
dichloromethane affords pyrazoline-benzothiazole analogues 29a-i (Scheme-
6).
29a, R = -H29b, R = -NO229c, R = -F29d, R = -Cl29e, R = -OMe29f , R = -OCF329g, R = -Me29h, R = -CF329i, R = -OEt
N
SH2N
+
26
R
29
25
MeO
MeO
OMe
N
OMe
O
N
O
OH
O
MeO
MeO
OMe
N
OMe
O
N
O
NH
O
S
N R
(i)
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 µΜ concentration. After preliminary screening on tumor cell lines,
active compounds were tested for five dose concentration on a panel of 60
human tumor cell lines derived from nine different cancer types: leukaemia,
lung, colon, CNS, melanoma, ovarian, renal, prostate and breast. Out of
thirteen compounds tested in preliminary screening eleven compounds
(27a, 27c, 27e, 27h, 27f, 28a, 28c, 28e, 28h, 28f and 29h) were
selected for five dose testing on a panel of 60 human cancer cell lines. The
results expressed as GI50 values for the test compounds are illustrated in
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T HESIS
Table 1. All the compounds exhibited potential anticancer activity with GI50
values ranging 0.019-30.7 µΜ.
The combretastatin-benzothiazole analogues (27a, 27c, 27e, 27h and
27f) exhibited potential anticancer activity with GI50 values ranging 0.019-18.6 µΜ. The most active compound in this series 27a showed good
activity against all cell lines tested and the GI50 values are in the range of
0.019–11µ M. This compound was found to show highest activity against
MDA-MB-435 (melanoma) cancer cell line with a GI50 value of 0.019µ M. The
trimethoxychalcone-benzothiazole analogues (28a, 28c, 28e, 28h and 28f)
also exhibited significant anticancer activity with GI50 values ranging 0.3-
30.7 µΜ. The anticancer activity (GI50) of the compound 28a is in the rangeof 0.3–6.42µ M. This compound was found to show highest activity against
MDA-MB-435 (melanoma) cancer cell line with a GI50 value of 0.3µ M. The
compound 28f which having trifluoromethoxy group at 6-position of
benzothiazole ring also showed significant activity (GI50, 0.3–9.52µ M).
Table 1. Anticancer activity of compounds 27a, 27c, 27e, 27h, 27f, 28a,
28c, 28e, 28h, 28f and 29h against the NCI human cancer cell lines
Panel/CellLine
a
GI50 values (µM)
27a 27c 27e 27h 27f 28a 28c 28e 28h28f
29h
Leukemia
CCRF-CEM
K-562
MOLT-4
RPMI-8226
SR
0.07
8
0.04
2
0.34
0.15
0.03
7
3.70
3.62
3.07
6.36
2.19
3.87
3.6
3.87
5.43
3.69
4.27
3.3
3.75
5.48
3.35
4.12
3.43
4.21
5.83
3.55
2.09
0.44
3.03
1.09
0.38
5.73
4.32
29.9
7.35
3.17
3.55
3.52
3.5
3.02
2.53
3.37
3.67
4.31
3.84
0.85
2.5
0.4
3
2.8
9
2.0
2
0.3
9
na
na
na
2.21
na
Non-small cell lung
A549/ATCC
EKVX
0.34
0.03
5.99
5.72
5.19
5.11
4.46
6.25
6
7.89
2.63
5.91
23.2
na
11.7
na
14.3
30.3
1.9
9
2.16
4.1
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HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
8
na
0.03
9
0.27
0.11
na
0.05
2
0.07
9
5.70
5.73
5.60
4.26
na
4.36
2.92
23
6.28
4.51
3.91
na
3.73
3.09
5.22
2.88
3.17
3.9
5.38
3.55
2.89
7.09
2.74
4.3
4.39
na
4.11
3.16
6.11
1.33
2.77
3.4
4.26
2.64
1.32
11.7
2.39
21.1
11
na
13.5
3.2
4.11
2.62
na
5.06
7.16
3.32
2.16
6.57
4.1
21.5
3.68
6.73
4.06
2
5.1
1
2.1
2
2.14
3.3
5
2.1
5
2.7
7
2.4
3
0.6
7
2.2
1.7
2.22
3.76
3.67
na
2.71
Colon
COLO-205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
2.83
0.37
0.04
6
0.04
8
3.16
0.05
8
na
7.78
>10
0
4.48
8.19
9.63
4.78
65
3.89
7.34
3.7
2.89
3.34
3.12
5.1
17.1
4.23
3.29
3.24
14.9
3.14
4.65
43.3
17.1
3.92
3.23
na
4.48
5.65
5.47
2.14
2.88
0.49
3.49
0.36
0.62
26.8
33
15.7
3.96
na
2.97
3.84
na
19.3
3.98
3.34
na
2.4
4.07
na
21.3
3.83
2.68
21.5
1.77
3.82
6.3
7
6.1
8
1.4
3
0.4
3.6
6
1.5
1
1.0
8
3.94
na
2.39
9.26
na
na
na
CNS
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
0.18
0.22
0.04
5
na
0.04
7.23
4.29
4.51
na
4.39
4.33
4.31
1.84
2.84
6.3
1.91
4
3.85
3.36
2.66
5.03
1.58
3.67
6.01
3.7
2.28
7.08
1.56
3.37
0.95
3.41
0.34
2.81
0.53
0.96
3.78
18.8
2.49
17.3
1.67
6.08
4.16
6.68
2.17
5.61
2.04
3.08
3.28
8.87
2.1
6.01
2.2
3.27
1.2
2.2
1
0.2
9
1.5
3.84
3.06
1.75
11
1.55
3.08
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T HESIS
6
0.04
8
8
0.3
4
0.7
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
0.06
7
1.99
0.07
2
0.01
9
0.18
na
0.03
6
11
0.06
3
3.23
5.22
6.82
0.33
8.46na
1.76
8.69
4.05
4.17
na
4.33
1.16
4.074.41
2.68
na
5.93
4.01
na
3.58
1.84
3.983
2.84
40
3.9
4.77
na
4.36
1.93
3.294.57
3.26
na
5.11
0.52
6.42
3.29
0.3
5.394.61
1.76
1.37
7.87
3.37
27.7
10.7
1.93
na24.1
4.49
na
33.6
2.59
9.62
3.28
1.51
24.214.4
4.56
20.4
14.5
2.88
8.75
3.31
1.16
309.47
2.88
na
22.7
0.4
7
2.6
9
1.7
2
0.3
4.9
73.3
1
2.0
3
8.0
8
9.5
2
3.36
6.44
na
3.08
na3.56
na
3.77
na
Ovarian
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
0.21
0.04
3
0.37
na
0.25
0.03
3
0.34
4.17
9.99
7.30
na
7.15
2.55
8
7
2.17
5.31
5.89
5.28
2.3
4.69
4.48
2.11
4.66
5.98
3.86
2.12
3.29
5.64
3.37
5.34
3.12
5
2.7
8.42
3.6
0.48
3.2
3.54
2.77
0.48
2.54
16.2
3.4
14.7
na
10.2
2.43
15.2
7.73
2.6
11.2
na
3.49
1.89
7.32
15.6
1.95
4.37
na
3.85
1.73
5.08
2.5
4
1.5
2
2.0
7
4.7
6
1.5
4
0.4
9
2.1
6
5.3
1.84
2.9
28.2
3.36
3.13
5.75
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T HESIS
Renal
786-0A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
7.3
0.36
0.09
3
0.41
0.05
0.37
na
0.58
7.126.25
3.33
2.78
6.29
4.61
9.89
2.56
7.523.5
7.77
3.2
2.5
5.75
7.54
4.31
6.962.81
4.82
3.1
2.27
4.43
7.03
3.59
na3.61
8.31
4.6
2.65
6.42
18.6
4.37
4.1222.6
3.46
3.78
1.83
3.02
3.3
3.07
24.7na
12.4
30.7
8.5
17.4
20.9
6.83
19.4na
4.94
25.1
2.37
5.19
16.4
5.31
24.6na
7.48
10.9
2.6
5.41
15.6
7.8
2.2
6
7.4
1.62
2.2
1.5
8
1.7
4
2.0
8
1.6
1
3.582.78
2.48
2.17
2.09
3.31
2.85
1.93
Prostate
PC-3
DU-145
0.18
0.1
6.74
8.97
4.45
2.04
4.32
2.51
5.12
4.11
3.63
1.94
22.9
4.05
12.2
2.49
12
2.62
3.1
4
1.5
6
2.92
1.82
Breast
MCF7
MDA-MB-231
HS 578T
BT-549
T-47D
MDA-MB-468
0.05
1
0.19
na
0.41
1.0
0.1
2.3
9.34
>10
0
16.7
3.39
3.68
2.88
5.4
4.15
na
4.03
2.29
3.16
3.38
4.18
5.23
4.79
2.16
2.89
5.16
4.33
10.5
3.95
2.3
1.05
2.69
4.75
4.82
4.97
2.3
3.7
12.6
10.5
4.82
na
13.1
3.02
5.82
3.72
8.38
14.7
10.5
3.05
7.14
4.81
11.2
15
13.5
1.3
22.0
1
3.0
8
2.7
1
3.0
8
3.5
2
4.02
3.11
2.48
3.55
2.75
6.12
a Values are reported as GI50, the µ M concentration of the compound required tocause 50% inhibition of cell growth.na = not active(GI50 >50µ M )
The pyrazoline-benzothiazole analogue (29h) exhibited promising
anticancer activity with GI50 values ranging 1.55-28.2 µΜ. This compound
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T HESIS
was found to show highest activity against SNB-75 (CNS) cancer cell line with
a GI50 value of 1.55 µ M. In comparison, the anticancer activity exhibited by
combretastatin and its chalcone, pyrazoline derivatives attached to
aminobenzothiazole, the combretastatin moiety attached to benzothiazoleanalogues are more active than chalcone moiety, than pyrazoline moiety.
3.4.2. INHIBITION OF TUBULIN POLYMERIZATION
Since these synthesized new compounds has structural resemblance to
combretastatin, it has been considered of interest to investigate their effect
on tubulin polymerization. One of the possible explanations of compounds
showing anticancer activity is the inhibition of tubulin polymerization to
functional microtubules as it is observed with antimitotic agents such as
podophyllotoxin and combretastatin. As tubulin subunits heterodimerize and
self-assemble to form microtubules in a time dependent manner, the
progression of tubulin polymerization has been investigated by monitoring
the increase in fluorescence emission at 460 nm (excitation wavelength is
360 nm) in 384 well plate for 1 h at 37 oC with and without the compounds at
3 µM concentration.
Real time kinetic graph of tubulin polymerization at 3 µM concentration
-0.8
1.2
3.2
5.2
7.2
9.2
11.2
13.2
15.2
1 4 7 1 0
1 3
1 6
1 9
2 2
2 5
2 8
3 1
3 4
3 7
4 0
4 3
4 6
4 9
5 2
5 5
5 8
Time (min)
F l u o r e s c e n c e u n i t s
Control
27f
28a
28c
28f
28h
Noco
Podo
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T HESIS
Effect of compounds on tubulin polymerization at 3 µM concentration
-10
0
10
20
30
40
50
60
70
Control 27a 27b 27c 27d 27e 27f 27g 27h 27i 28a 28b 28c 28d 28e 28f 28g 28h Nocod Podo
Compounds
% I n h i b i t i o n
( T u b u l i n p o l y m e r i z a t i o n )
Figure 6: Effect of compounds on tubulin polymerization: Tubulin polymerization wasmonitored by the increase in fluorescence at 340 nm (excitation) and 460 nm (emission) for1 hrs at 37 oC. All the compounds were included at a final concentration of 3 μM.Podophyllotoxin and Nocodazole were used as positive control.
Among the seventeen compounds examined, 28a, 28c, 28f, 28h and
27f inhibited tubulin polymerization to 59.1%, 59.7%, 52.5, 54.1 and 55%
respectively compared to control and a similar pattern of inhibition has been
observed with the positive controls, podophyllotoxin (55.7%), Nocodazole
(49.6%) as shown in Figure 6.
3.5. CONCLUSION
In conclusion, we have synthesized different analogues of novel
combretastatin derivatives with amino benzothiazoles (27a-i, 28a-i and
29a-i). For synthesized compounds anticancer activity has been
evaluated by the National Cancer Institute (NCI), USA, against nine
human cancer cell lines (leukaemia, lung, colon, CNS, melanoma,
ovarian, renal, prostate and breast). All the compounds exhibited good
anticancer activity. Some of synthesized compounds exhibited good
inhibition of tubulin polymerization.
3.6. EXPERIMENTAL SECTION
3-(tert-butyldimethylsilyloxy)-4-methoxybenzaldehyde (11)
The compound 3-hydroxy-4-methoxy benzaldehyde 10 (152 mg, 1 mmol)
was dissolved in DMF (10 mL) and to this was added Triethylamine (2 mL)
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T HESIS
and cooled to 5-10 oC. Then add the TBDMS-C l(165 mg, 1.1 mmol) to the
reaction mass. The reaction was stirred for 1 h at room temperature. After
completion of the reaction as indicated by TLC, bicarbonate solution was
added to reaction mass and extracted with ethyl acetate. The organic layer
was dried over Na2SO4 and concentrated to give the crude product. This was
further purified by column chromatography using hexane: ethyl acetate (1:9)
as a solvent system to obtain the pure product 11 as oil. Yield (221 mg,
82%).
1H NMR (300 MHz, CDCl3): δ 9.71 (s, 1H), 7.31–7.38 (dd, 1H, J = 8.3, 1.5 Hz),
7.24 (d, 1H, J = 1.5 Hz), 6.84 (d, 1H, J = 8.3 Hz), 3.82 (s, 3H), 0.93 (s, 9H),
0.1 (s, 6H);
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 toquench the excess reagent and concentrated the reaction mass, extracted
with ethyl acetate. The organic layer was dried over Na2SO4 and
concentrated to give the crude product. This was further purified by column
chromatography using hexane: ethyl acetate (2:8) as a solvent system to
obtain the pure product 12. Yield (246 mg, 90%).
1H NMR (200 MHz, CDCl3): δ 6.78–6.85 (m, 2H), 6.75 (d, 1H, J = 2.4 Hz), 4.49
(s, 2H), 3.78 (s, 3H), 0.99 (s, 9H), 0.12 (s, 6H);
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
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T HESIS
the compound 12 (268 mg, 1 mmol) and the mixture was stirred at room
temperature for one hour. The reaction was monitored by TLC using ethyl
acetate-hexane (3:7). After completion of the reaction as indicated by the
TLC, the reaction mixture was quenched with ice water and the solvent
evaporated under reduced pressure, diluted with water and extracted with
ethyl acetate (2X20 ml). The combined organic phases were given washing
with 1N solution of NaOH, dried over Na2SO4 and evaporated under vacuum
to afford crude product of 13. The residue, thus obtained was taken to next
step without purification because of stability problem. Yield (300 mg, 90%).
3-{[1-(tert -butyl)-1,1-dimethylsilyl]oxy}-4-methoxybenzyltriphenylphosphonium bromide (14)
To a solution of PPh3 (262 mg, 1 mmol) in dry toluene (15 mL) was added
compound 13 (331 mg, 1 mmol) and the reaction mixture was refluxed for 6
hours. The reaction was monitored by TLC using ethyl acetate-hexane (1:1).
After completion of the reaction as indicated by the TLC, the toluene was
evaporated to half volume and stirred the reaction mixture at room
temperature for 16 hours to precipitate the product 14. The precipitated
product was filtered, washed with cooled toluene solvent and recrystalised
the crude product in toluene solvent and dried to obtain pure product 14.
Yield (450 mg, 75%).
1H NMR (200 MHz, CDCl3): δ 7.74–8.05 (m, 15H), 6.97–7.11 (m, 1H), 6.83 (d,
1H, J = 8.1 Hz), 6.5 (d, 1H, J = 2.2 Hz), 5.35 (d, 2H, J = 13.2 Hz), 3.93 (s, 3H),
1.05 (s, 9H), 0.17 (s, 6H);
(Z)-tert-butyl(2-methoxy-5-(3,4,5-
trimethoxystyryl)phenoxy)dimethylsilane(15)
To a solution of compound 14 (593 mg, 1 mmol) in dry THF (15 mL) was
cooled to –30 oC temperature and added n-BuLi (1.6 M solution in hexane)
(0.7 ml, 1.1 mmol) slowly. After addition stirred the reaction mixture for 20
minutes and added the solution of compound trimethoxybenzaldehyde (196
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T HESIS
mg, 1 mmol) in dry THF. The mixture was stirred at –30 oC temperature for
30minutes. The reaction was monitored by TLC using ethyl acetate-hexane
(3:7). After completion of the reaction as indicated by the TLC, the reaction
mixture was quenched with ammonium chloride solution and extracted with
ethyl acetate (2X20 ml). The combined organic phases were given washing
with water followed by brain solution, dried over Na2SO4 and evaporated
under vacuum to afford crude product of 15. The crude product was mixture
of Z - and E- isomers. The Z - isomer was separated by flash column
chromatography using hexane:ethyl acetate (19:1) as a solvent system to
obtain the pure product 12 as colour less oil. Yield (180 mg, 41%).
1H NMR (300 MHz, CDCl3): δ 6.76–6.82 (dd, 1H, J = 8.3, 2.2 Hz), 6.73 (d, 1H, J
= 2.2 Hz), 6.69 (d, 1H, J = 8.3 Hz), 6.39–6.46 (m, 3H), 6.36 (d, 1H, J = 12 Hz),
3.79 (s, 3H), 3.77 (s, 3H), 3.69 (s, 6H), 0.92 (s, 9H), 0.04 (s, 6H);
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 ethylacetate-hexane (6:4). After completion of the reaction as indicated by the
TLC, the reaction mixture was quenched with bicarbonate solution and
extracted with ethyl acetate (2X20 ml). The combined organic phases were
given washing with water followed by brine solution, dried over Na2SO4 and
evaporated under vacuum to afford crude product of 13. This was further
purified by column chromatography using hexane: ethyl acetate (2:8) as a
solvent system to obtain the pure product12.
Yield (250 mg, 79% yield).
Mp: 115-116 ºC;
1H NMR (200 MHz, CDCl3): δ 6.92 (d, 1H, J = 2.2 Hz), 6.77–6.83 (dd, 1H, J =
8.3, 1.5 Hz), 6.73 (d, 1H, J = 8.3 Hz), 6.53 (s, 2H), 6.47 (d, 1H, J = 12 Hz),
6.41 (d, 1H, J = 12 Hz), 5.57 (bs, 1H), 3.86 (s, 3H), 3.84 (s, 3H), 3.7 (s, 6H);
ESIMS: m/z 317 (M+H)+.
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T HESIS
(Z)-ethyl 2-(2-methoxy-5-(3,4,5-
trimethoxystyryl)phenoxy)acetate( 17)
To a solution of compound 16 (316 mg, 1 mmol) in dry DMF (15 mL) was
added, anhydrous K 2CO3 (276 mg, 2 mmol), α-bromoethylacetate (183 mg,1.1 mmol) and the mixture was stirred at room temperature for 24 hours.
The reaction was monitored by TLC using ethyl acetate-hexane (6:4). After
completion of the reaction as indicated by the TLC, K 2CO3 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 ºC;
1H NMR (300 MHz, CDCl3): δ 6.83–6.89 (dd, 1H, J = 8.3, 1.5 Hz), 6.75 (d, 1H, J
= 8.3 Hz), 6.7 (d, 1H, J = 1.5 Hz), 6.4–6.46 (m, 3H), 6.47 (d, 1H, J = 12 Hz),
4.44 (s, 2H), 4.11–4.2 (q, 2H), 3.85 (s, 3H), 3.8 (s, 3H), 3.69 (s, 6H), 1.24 (t,
3H, J = 7.5, 6.7 Hz);
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
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T HESIS
compound 18. This crude compound was purified by recrystalization by
using ethyl acetate as solvent to obtain the pure product 18 as white solid.
Yield (300 mg, 81%)
Mp: 154-156 ºC;
1H NMR (200 MHz, CDCl3): δ 7.96 (bs, 1H), 6.84–6.9 (dd, 1H, J = 8.3, 1.5 Hz),
6.77 (d, 1H, J = 8.3 Hz), 6.72 (d, 1H, J = 1.5 Hz), 6.31–6.49 (m, 4H), 4.47 (s,
2H), 3.86 (s, 3H), 3.8 (s, 3H), 3.69 (s, 6H);
ESIMS: m/z 375 (M+H)+.
(E)-3-(3-hydroxy-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)prop-
2-en-1-one (20)
To a stirred mixture of 3,4,5-trimethoxyacetophenone (210 mg, 1 mmol) and
3-hydroxy-4-methoxybenzaldehyde (152 mg, 1 mmol) in ethanol (10 ml) was
added 50% aqueous solution of potassium hydroxide (1 ml) and stirred for 6
h at room temperature. After completion of the reaction checked by TLC, the
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 ºC;
1H NMR (300 MHz, CDCl3): δ 7.76 (d, 1H, J = 16 Hz), 7.36 (d, 1H, J = 16 Hz),
7.25−7.32 (m, 3H), 7.14 (dd, 1H, J = 8.7, 2.1 Hz), 6.89 (d, 1H, J = 8.7 Hz),
5.74 (bs, 1H), 3.96 (s, 9H), 3.94 (s, 3H);
ESIMS: m/z 345 (M+H)+.
(E)-Ethyl-2-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-enyl)phenoxy) acetate (21)
To a solution of compound 20 (344 mg, 1 mmol) in dry DMF (15 mL) was
added, anhydrous K 2CO3 (276 mg, 2 mmol), α-bromoethylacetate (183 mg,
1.1 mmol) and the mixture was stirred at room temperature for 12 hours.
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T HESIS
The reaction was monitored by TLC using ethyl acetate-hexane (6:4). After
completion of the reaction as indicated by the TLC, K 2CO3 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 ºC;
1H NMR (300 MHz, CDCl3): δ 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-1-enyl)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 Na2SO4 and evaporated under
vacuum to obtain compound 22. This crude compound was purified by
recrystalization by using ethyl acetate as solvent to obtain the pure product
22 as yellow solid. Yield (351 mg, 87%)
Mp: 170-172 ºC;
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1H NMR (500 MHz, CDCl3+DMSO D6): δ 8.24 (bs, 1H), 7.74 (d, 1H, J = 15.8
Hz), 7.77 (d, 1H, J = 15.8 Hz), 7.46−7.5 (m, 2H), 7.42 (s, 2H), 7.08 (d, 1H, J =
7.9 Hz 4.79 (s, 2H), 3.96 (s, 6H), 3.92 (s, 3H), 3.85 (s, 3H);
ESIMS: m/z 403 (M+H)
+
.
1-(5-(3-hydroxy-4-methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihydropyrazol-1-yl)ethanone (23)
To a stirred mixture of compound (20) (344 mg, 1 mmol) in acetic acid (10
ml) was added hydrazine hydrate (150 mg, 3 mmol) and stirred for 14 h at
reflux temperature. After completion of the reaction as checked by TLC, the
reaction mass was poured in to ice water and filtered the compound
precipitated. Air dried the filtered compound and purified by recrystalization
from ethanol to obtain the pure product 23 as white solid. Yield (320 mg,
79%).
Mp: 143-145 ºC;
1H NMR (300 MHz, CDCl3): δ 6.95 (s, 2H), 6.68−6.84 (m, 3H), 5.45−5.57 (dd,
1H, J = 11.7, 4.4 Hz), 3.91 (s, 6H), 3.89 (s, 3H), 3.85 (s, 3H), ), 3.6−3.79 (dd,
1H, J = 17.6, 11.2 Hz), ), 3.01−3.22 (dd, 1H, J = 17.6, 4.4 Hz), 2.42 (s, 3H);
ESIMS: m/z 401 (M+H)+.
Ethyl-2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)-2-methoxyphenoxy)acetate (24)
To a solution of compound 23 (400 mg, 1 mmol) in dry DMF (15 mL) was
added, anhydrous K 2CO3 (276 mg, 2 mmol), α-bromoethylacetate (183 mg,
1.1 mmol) and the mixture was stirred at room temperature for 12 hours.
The reaction was monitored by TLC using ethyl acetate-hexane (6:4). After
completion of the reaction as indicated by the TLC, K 2CO3 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%)
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Mp: 137-139 ºC;
1H NMR (500 MHz, CDCl3): δ δ 6.92 (s, 2H), 6.78−6.86 (m, 2H), 6.71 (s, 1H),
5.44−5.5 (dd, 1H, J = 11.8, 3.9 Hz), 4.61 (s, 2H), 4.17 (q, 2H), 3.9 (s, 6H),
3.87 (s, 3H), 3.83 (s, 3H), ), 3.62−3.71 (dd, 1H, J = 17.8, 11.8 Hz), 3.06−3.13(dd, 1H, J = 17.8, 3.9 Hz), 2.38 (s, 3H), 1.24 (t, 3H)
ESIMS: m/z 487 (M+H)+.
2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)-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 usingethyl acetate. After completion of the reaction as indicated by the TLC, the
solvent was removed under vacuum and neutralized with dilute HCl up to pH
7. After neutralization the reaction mixture was extracted with
dichloromethane (2X20 ml). The combined organic phases were washed with
water followed by brine solution, dried over Na2SO4 and evaporated under
vacuum to obtain the crude compound 25. This crude compound was
purified by recrystalization by using ethyl acetate as solvent to obtain the
pure product 25 as white solid. Yield (375 mg, 81%)
Mp: 175-177 ºC;
1H NMR (200 MHz, CDCl3): δ 7.81 (bs, 1H), 7.03 (s, 2H), 6.87−6.95 (m, 2H),
6.69 (s, 1H), 5.38−5.52 (dd, 1H, J = 11.6, 3.9 Hz), 4.54 (s, 2H), 3.85 (s, 6H),
3.83 (s, 3H), 3.82 (s, 3H), 3.61−3.7 (dd, 1H, J = 17.9, 11.6 Hz), 3.05−3.13 (dd,
1H, J = 17.8, 3.9 Hz), 2.39 (s, 3H);
ESIMS: m/z 459 (M+H)+.
(Z)-N-(benzo[d]thiazol-2-yl)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy) acetamide (27a)
To a solution of 2-aminobenzothiazole 26a (150 mg, 1.0 mmol) in
dichloromethane (20 mL) was added 1-(3-Dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (EDCI.HCl) (191 mg, 1 mmol) and 1-
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T HESIS
hydroxy-1,2,3-benzotriazole (HOBt) (13.5 mg, 0.1 mmol). Then added (Z)-2-
(2-methoxy-5-(3,4,5-trimethoxystyryl) phenoxy)acetic acid (18) (374 mg, 1
mmol) and the reaction mixture was stirred at temperature temperature for
24h and the reaction was monitored by TLC. Then water is added and
extracted with dichloromethane. The solvent was evaporated under vacuum
to afford the crude product. This was further purified by column
chromatography using ethyl acetate and hexane as solvent system to obtain
the pure product (27a) (395mg, 80% yield).
Mp: 132-134 ºC;
1H NMR (200 MHz, CDCl3): δ 10.64 (bs, 1H), 7.74–7.82 (m, 2H), 7.37–7.44
(m, 1H), 7.29 (d, 1H, J = 8.3 Hz), 6.96–7.01 (d, 1H, J = 8.3, 2.2 Hz), 6.93 (d,
1H, J = 2.2 Hz), 6.82 (d, 1H, J = 8.3 Hz), 6.46 (d, 1H, J = 12 Hz), 6.38–4.3 (m,
3H), 4.6 (s, 2H), 4.02 (s, 3H), 3.83 (s, 3H), 3.69 (s, 6H);
13C NMR (75 MHz, CDCl3): δ 167.54, 156.73, 152.98, 149.06, 148.47, 146.6, 137.29,
132.45, 132.2, 130.43, 129.7, 128.61, 126.19, 124.99, 124.05,121.36, 121.18, 118.14, 111.62,
105.81, 70.4, 60.92, 55.9;
ESIMS: m/z 507 (M+H)+.
(Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)-N-(6-nitrobenzo[d]thiazol-2-yl) acetamide (27b)
This compound was prepared according to the method described for
compound 27a by employing (Z)-2-(2-methoxy-5-(3,4,5-
trimethoxystyryl)phenoxy)aceticacid (18) (374 mg, 1 mmol), and 2-amino-6-
nitrobenzothiazole 26b (195 mg, 1 mmol) to obtain the pure product 27b as
white solid. Yield (442 mg, 80%)
Mp: 145-146 ºC;
1H NMR (300 MHz, CDCl3): δ 11.03 (s, 1H), 8.77 (d, 1H, J = 2.1 Hz), 8.32–8.36
(dd, 1H, J = 8.7, 2.1 Hz), 7.88 (d, 1H, J = 8.7 Hz), 7.03–7.07 (dd, 1H, J = 8, 2.1
Hz), 6.97 (d, 1H, J = 2.1 Hz), 6.87 (d, 1H, J = 8 Hz), 6.52 (d, 1H, J = 12.4 Hz),
6.49 (s, 2H), 6.46 (d, 1H, J = 12.4 Hz), 4.68 (s, 2H), 4.02 (s, 3H), 3.86 (s, 3H),
3.71 (s, 6H);
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T HESIS
ESIMS: m/z 552 (M+H)+.
(Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)-N-(6-fluorobenzo[d]thiazol-2-yl)acetamide (27c)
This compound was prepared according to the method described forcompound 27a by employing (Z)-2-(2-methoxy-5-(3,4,5-
trimethoxystyryl)phenoxy)aceticacid (18) (374 mg, 1 mmol), and 2-amino-6-
fluorobenzothiazole 26c (168 mg, 1 mmol) to obtain the pure product 27c as
white solid. Yield (430 mg, 81%)
Mp: 137-139 ºC;
1H NMR (300 MHz, CDCl3): δ 10.66 (bs, 1H), 7.71–7.76 (m, 1H), 7.47–7.53
(dd, 1H, J = 8.1, 2.4 Hz), 7.13–7.20 (m, 1H), 7.00–7.04 (dd, 1H, J = 8.1, 1.6
Hz), 6.93 (d, 1H, J = 1.6 Hz), 6.83 (d, 1H, J = 8.1 Hz), 6.46–6.51 (m, 3H), 6.44
(d, 1H, J = 12.2 Hz), 4.63 (s, 2H), 3.98 (s, 3H), 3.85 (s, 3H), 3.7 (s, 6H);
13C NMR (75 MHz, CDCl3): δ 167.63, 161.26, 158.04, 156.41, 152.99, 149.04, 146.6,
144.91, 137.24, 132.47, 130.49, 129.75, 128.58, 125.07, 121.99, 118.25, 114.77, 111.66, 107.47,
105.81, 70.46, 60.9, 55.89;
ESIMS: m/z 525 (M+H)+.
(Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)-N-(6-chlorobenzo[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-(3,4,5-
trimethoxystyryl)phenoxy)aceticacid (18) (374 mg, 1 mmol), and 2-amino-6-
chlorobenzothiazole 26d (184 mg, 1 mmol) to obtain the pure product 27d
as white solid. Yield (385 mg, 71%)
Mp: 136-138 ºC;
1H NMR (200 MHz, CDCl3): δ 10.61 (bs, 1H), 7.8 (d, 1H, J = 2.2 Hz), 7.73 (d,
1H, J = 9 Hz), 7.38–7.44 (dd, 1H, J = 8.3, 2.2 Hz), 7–7.06 (dd, 1H, J = 8.3, 2.2
Hz), 6.94 (d, 1H, J = 2.2 Hz), 6.84 (d, 1H, J = 9 Hz), 6.42–6.54 (m, 4H), 4.64
(s, 2H), 3.98 (s, 3H), 3.85 (s, 3H), 3.7 (s, 6H);
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13C NMR (75 MHz, CDCl3): δ 167.94, 160.37, 153, 149.02, 146.53, 145.93, 137.31,
132.85, 132.43, 130.53, 129.94, 129.72, 128.65, 127.22, 125.03, 121.72, 121.08, 118.1, 111.76,
105.96, 70.21, 60.91, 56;
ESIMS: m/z 541 (M+H)+.
(Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)-N-(6-methoxybenzo[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-(3,4,5-
trimethoxystyryl)phenoxy)aceticacid (18) (374 mg, 1 mmol), and 2-amino-6-
methoxybenzothiazole 26e (180 mg, 1 mmol) to obtain the pure product
27e as white solid. Yield (415 mg, 77%)
Mp: 139-140 ºC;
1H NMR (400 MHz, CDCl3): δ 7.71 (d, 1H, J = 9 Hz), 7.3 (d, 1H, J = 2.2 Hz),
6.99–7.1 (m, 2H), 6.94 (d, 1H, J = 2.2 Hz), 6.84 (d, 1H, J = 8.3 Hz), 6.43–6.53
(m, 4H), 4.63 (s, 2H), 3.98 (s, 3H), 3.88 (s, 3H), 3.85 (s, 3H), 3.7 (s, 6H);
13C NMR (75 MHz, CDCl3): δ 186.55, 176.15, 174.09, 172.2, 168.29, 165.82, 161.62,
156.45, 152.54, 151.72, 149.63, 148.92, 147.88, 144.14, 140.9, 137.24, 134.54, 130.86, 125.04,
123.38, 89.5, 80.16, 75.15, 75.02, 48.87;
ESIMS: m/z 537 (M+H)+.
(Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)-N-(6-trifluoromethoxybenzo[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-(3,4,5-
trimethoxystyryl)phenoxy)aceticacid (18) (374 mg, 1 mmol), and 2-amino-6-
trifluoromethoxybenzothiazole 26f (234 mg, 1 mmol) to obtain the pure
product 27f as white solid. Yield (470 mg, 80%)
Mp: 141-143 ºC;
1H NMR (200 MHz, CDCl3): δ 10.63 (bs, 1H), 7.8 (d, 1H, J = 8.8 Hz), 7.69 (s,
1H), 7.29–7.36 (dd, 1H, J = 8.4, 2.2 Hz), 7.01–7.06 (dd, 1H, J = 8.4, 1.7 Hz),
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6.95 (d, 1H, J = 1.7 Hz), 6.85 (d, 1H, J = 8.3 Hz), 6.42–6.53 (m, 4H), 4.65 (s,
2H), 3.99 (s, 3H), 3.85 (s, 3H), 3.70 (s, 6H); );
13C NMR (75 MHz, CDCl3): δ 167.82, 157.64, 153.03, 149.08, 147.14, 146.66, 145.5,
137.29, 133.09, 132.5, 130.57, 129.82, 128.59, 125.2, 121.96, 120.16, 118.42, 114.21, 111.7,105.8, 103.38, 70.59, 60.96, 55.95;
ESIMS: m/z 591 (M+H)+.
(Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)-N-(6-methylbenzo[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-(3,4,5-
trimethoxystyryl)phenoxy)aceticacid (18) (374 mg, 1 mmol), and 2-amino-6-
methylbenzothiazole 26g (180 mg, 1 mmol) to obtain the pure product 27g
as white solid. Yield (471 mg, 89%)
Mp: 137-139 ºC;
1H NMR (300 MHz, CDCl3): δ 10.59 (bs, 1H), 7.5 (d, 1H, J = 2.3 Hz), 7.71 (d,
1H, J = 8.3 Hz), 7.35–7.42 (dd, 1H, J = 8.3, 2.2 Hz), 7.02–7.09 (dd, 1H, J =
8.3, 2.2 Hz), 6.91 (d, 1H, J = 2.2 Hz), 6.84 (d, 1H, J = 8.3 Hz), 6.43–6.55 (m,
4H), 4.65 (s, 2H), 3.98 (s, 3H), 3.86 (s, 3H), 3.7 (s, 6H), 2.46 (s, 3H)
13C NMR (75 MHz, CDCl3): δ 167.48, 166.1, 153.13, 152.23, 148.33, 146.41, 136.45,
135.61, 131.95, 130.34, 129.7, 128.85, 128.43, 123.36, 120.89, 117.13, 116.34, 112.64, 111.37,
105.4, 69.39, 60.02, 55.46, 55.24, 19.55;
ESIMS: m/z 521 (M+H)+.
(Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)-N-(6-trifluoromethylbenzo [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-(3,4,5-
trimethoxystyryl)phenoxy)aceticacid (18) (374 mg, 1 mmol), and 2-amino-6-
trifluoromethylbenzothiazole 26h (218 mg, 1 mmol) to obtain the pure
product 27h as white solid. Yield (470 mg, 79%)
Mp: 142-144 ºC;
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1H NMR (200 MHz, CDCl3): δ 10.64 (bs, 1H), 8.12 (s, 1H), 7.93 (d, 1H, J = 8.4
Hz), 7.69–7.75 (m, 1H), 7.01–7.06 (dd, 1H, J = 8.3, 1.7 Hz), 6.95 (d, 1H, J =
1.7 Hz), 6.85 (d, 1H, J = 8.3 Hz), 6.43–6.53 (m, 1H), 4.68 (s, 2H), 3.99 (s, 3H),
3.83 (s, 3H), 3.70 (s, 6H); );13C NMR (75 MHz, CDCl3): δ 168.02, 1549.3, 153.02, 150.77, 149.07, 146.66, 137.32,
132.48, 132.3, 130.59, 129.8, 128.56, 126.01, 125.19, 123.29, 121.35, 119.1, 118.46, 111.72,
105.89, 103.43, 70.6, 60.91, 55.9;
ESIMS: m/z 597 (M+Na)+.
(Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)-N-(6-ethoxybenzo[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-(3,4,5-
trimethoxystyryl)phenoxy)aceticacid (18) (374 mg, 1 mmol), and 2-amino-6-
ethoxybenzothiazole 26i (194 mg, 1 mmol) to obtain the pure product 27i as
white solid. Yield (485 mg, 88%)
Mp: 138-140 ºC;
1H NMR (300 MHz, CDCl3): δ 10.61 (bs, 1H), 7.72 (d, 1H, J = 8.3 Hz), 7.4 (d,
1H, J = 2.2 Hz), 6.99–7.15 (m, 2H), 6.93 (d, 1H, J = 2.2 Hz), 6.85 (d, 1H, J =
8.3 Hz), 6.44–6.53 (m, 4H), 4.65 (s, 2H), 4.12 (q, 2H), 3.97 (s, 3H), 3.87 (s,
3H), 3.84 (s, 3H), 3.7 (s, 6H), 1.28 (t, 3H);
ESIMS: m/z 551 (M+H)+.
(E)-N-(benzo[d]thiazol-2-yl)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-enyl)phenoxy)acetamide (28a)
This compound was prepared according to the method described for
compound 27a by employing (E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-
trimethoxyphenyl)prop-1-enyl) phenoxy) aceticacid (22) (402 mg, 1 mmol),
EDCI.HCl (191 mg, 1 mmol), HOBt (13.5 mg, 0.1 mmol) and 2-
aminobenzothiazole 26a (150 mg, 1 mmol) to obtain the pure product 28a
as yellow solid. Yield (416 mg, 77%)
Mp: 143-145 ºC;
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1H NMR (200 MHz, CDCl3): δ 11.93 (bs, 1H), 7.81–7.84 (dd, 1H, J = 7.8, 1.9
Hz), 7.72–7.75 (dd, 1H, J = 7.8, 1.9 Hz), 7.67 (d, 1H, J = 15.6 Hz), 7.52–7.6
(m, 2H), 7.38–7.43 (m, 1H), 7.34–7.37 (dd, 1H, J = 7.8, 1.9 Hz), 7.31 (s, 2H),
7.25–7.29 (m, 1H), 7.01 (d, 1H, J = 7.8 Hz), 4.74 (s, 2H), 3.98 (s, 3H), 3.91 (s,
6H), 3.85 (s, 3H);
13C NMR (75 MHz, DMSO D6): δ 187.79, 167.51, 156.35, 152.74, 151.32,
147.42, 147.3, 143.84, 141.69, 133.14, 131.03, 129.83, 127.41, 126.56,
124.13, 123.55, 119.91, 119.01, 113.94, 112.19, 105.97, 67.12, 60.06,
56.01, 55.7;
ESIMS: m/z 535 (M+H)+.
(E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-enyl)phenoxy)-N-(6-nitrobenzo[d]thiazol-2-yl)acetamide (28b)
This compound was prepared according to the method described for
compound 27a by employing (E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-
trimethoxyphenyl)prop-1-enyl) phenoxy) aceticacid (22) (402 mg, 1 mmol),
EDCI.HCl (191 mg, 1 mmol), HOBt (13.5 mg, 0.1 mmol) and 2-amino-6-
nitrobenzothiazole 26b (195 mg, 1 mmol) to obtain the pure product 28b as
yellow solid. Yield (452 mg, 78%)
Mp: 149-150 ºC;
1H NMR (200 MHz, CDCl3): δ 10.98 (bs, 1H), 8.77 (d, 1H, J = 2.1 Hz), 8.31–
8.38 (dd, 1H, J = 8.3, 2.1 Hz), 7.79 (d, 1H, J = 15.6 Hz), 7.72 (d, 1H, J = 8.3
Hz), 7.28–7.34 (m, 2H), 7.25 (d, 1H, J = 15.6 Hz), 7.23 (s, 2H), 6.95 (d, 1H, J
= 8.3 Hz), 4.83 (s, 2H), 4.01 (s, 3H), 3.92 (s, 6H), 3.9 (s, 3H);
ESIMS: m/z 580 (M+H)+.
(E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-enyl)phenoxy)-N-(6-fluorobenzo[d]thiazol-2-yl)acetamide (28c)
This compound was prepared according to the method described for
compound 27a by employing (E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-
trimethoxyphenyl)prop-1-enyl) phenoxy) aceticacid (22) (402 mg, 1 mmol),
EDCI.HCl (191 mg, 1 mmol), HOBt (13.5 mg, 0.1 mmol) and 2-amino-6-
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fluorobenzothiazole 26c (168 mg, 1 mmol) to obtain the pure product 28c as
yellow solid. Yield (487 mg, 81%)
Mp: 145-146 ºC;
1H NMR (300 MHz, CDCl3): δ 10.57 (bs, 1H), 7.65–7.77 (m, 2H), 7.44–7.53 (m,
2H), 7.31–7.39 (m, 2H), 7.25 (s, 2H), 7.24 (d, 1H, J = 15.4 Hz), 7.12 (d, 1H, J
= 8.3 Hz), 4.82 (s, 2H), 4.08 (s, 3H), 3.96 (s, 6H), 3.92 (s, 3H);
ESIMS: m/z 553 (M+H)+.
(E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-enyl)phenoxy)-N-(6-chlorobenzo[d]thiazol-2-yl)acetamide (28d)
This compound was prepared according to the method described for
compound 27a by employing (E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-
trimethoxyphenyl)prop-1-enyl) phenoxy) aceticacid (22) (402 mg, 1 mmol),
EDCI.HCl (191 mg, 1 mmol), HOBt (13.5 mg, 0.1 mmol) and 2-amino-6-
chlorobenzothiazole 26d (184 mg, 1 mmol) to obtain the pure product 28d
as yellow solid. Yield (475 mg, 78%)
Mp: 143-145 ºC;
1H NMR (400 MHz, CDCl3): δ 10.59 (bs, 1H), 7.76–7.81 (m, 1H), 7.71 (d, 1H, J
= 15.1 Hz), 7.65–7.69 (m, 2H), 7.46 (d, 1H, J = 2 Hz), 7.37–7.41 (dd, 1H, J =
8.4, 2 Hz), 7.25 (s, 2H), 7.21 (d, 1H, J = 15.1 Hz), 6.98 (d, 1H, J = 8.3 Hz),
4.83 (s, 2H), 4.07 (s, 3H), 3.95 (s, 6H), 3.93 (s, 3H);
ESIMS: m/z 570 (M+H)+.
(E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-enyl)phenoxy)-N-(6-methoxybenzo[d]thiazol-2-yl)acetamide (28e)
This compound was prepared according to the method described for
compound 27a by employing (E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-
trimethoxyphenyl)prop-1-enyl) phenoxy) aceticacid (22) (402 mg, 1 mmol),
EDCI.HCl (191 mg, 1 mmol), HOBt (13.5 mg, 0.1 mmol) and 2-amino-6-
methoxybenzothiazole 26e (180 mg, 1 mmol) to obtain the pure product
28e as yellow solid. Yield (463 mg, 81%)
Mp: 146-147 ºC;
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1H NMR (300 MHz, CDCl3): δ 10.62 (bs, 1H), 7.75 (d, 1H, J = 15.6 Hz), 7.71 (d,
1H, J = 8.3 Hz), 7.34–7.4 (m, 2H), 7.28–7.34 (m, 2H), 7.23 (d, 1H, J = 15.6
Hz), 7.22 (s, 2H), 6.89 (d, 1H, J = 8.3 Hz), 4.82 (s, 2H), 4.02 (s, 3H), 3.96 (s,
6H), 3.95 (s, 3H), 3.88 (s, 3H);
ESIMS: m/z 565 (M+H)+.
(E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-enyl)phenoxy)-N-(6-trifluoromethoxybenzo[d]thiazol-2-yl)acetamide(28f)
This compound was prepared according to the method described for
compound 27a by employing (E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-
trimethoxyphenyl)prop-1-enyl) phenoxy) aceticacid (22) (402 mg, 1 mmol),
EDCI.HCl (191 mg, 1 mmol), HOBt (13.5 mg, 0.1 mmol) and 2-amino-6-
trifluoromethoxybenzothiazole 26f (234 mg, 1 mmol) to obtain the pure
product 28f as yellow solid. Yield (505 mg, 81%)
Mp: 148-150 ºC;
1H NMR (200 MHz, CDCl3): δ 10.62 (bs, 1H), 7.77 (d, 1H, J = 9 Hz), 7.66–7.73
(m, 2H), 7.27–7.39 (m, 3H), 7.24 (d, 1H, J = 15.1 Hz), 7.22 (s, 2H), 6.98 (d,
1H, J = 8.3 Hz), 4.83 (s, 2H), 4.07 (s, 3H), 3.95 (s, 6H), 3.91 (s, 3H);
13C NMR (75 MHz, DMSO D6): δ 187.81, 168.2, 160.57, 152.77, 151.32,
151.18, 147.26, 143.92, 141.69, 133.16, 131.98, 127.41, 126.28, 124.24,
122.95, 122.66, 121.01, 119.87, 113.75, 112.21, 105.97, 67.02, 60.1, 56.05,
55.74;
ESIMS: m/z 619 (M+H)+.
(E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-enyl)phenoxy)-N-(6-methylbenzo[d]thiazol-2-yl)acetamide (28g)
This compound was prepared according to the method described forcompound 27a by employing (E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-
trimethoxyphenyl)prop-1-enyl) phenoxy) aceticacid (22) (402 mg, 1 mmol),
EDCI.HCl (191 mg, 1 mmol), HOBt (13.5 mg, 0.1 mmol) and 2-amino-6-
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methylbenzothiazole 26g (164 mg, 1 mmol) to obtain the pure product 28g
as yellow solid. Yield (478 mg, 81%)
Mp: 142-144 ºC;
1H NMR (300 MHz, CDCl3): δ 7.76 (d, 1H, J = 15.6 Hz), 7.70 (d, 1H, J = 8.3 Hz),
7.63 (s, 1H), 7.31–7.43 (m, 2H), 7.23–7.30 (m, 4H), 7.01 (d, 1H, J = 8.4 Hz),
4.84 (s, 2H), 4.04 (s, 3H), 3.96 (s, 3H), 3.94 (s, 3H), 2.48 (s, 3H);
ESIMS: m/z 549 (M+H)+.
(E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-enyl)phenoxy)-N-(6-trifluoromethylbenzo[d]thiazol-2-yl)acetamide(28h)
This compound was prepared according to the method described for
compound 27a by employing (E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-
trimethoxyphenyl)prop-1-enyl) phenoxy) aceticacid (22) (402 mg, 1 mmol),
EDCI.HCl (191 mg, 1 mmol), HOBt (13.5 mg, 0.1 mmol) and 2-amino-6-
trifluoromethylbenzothiazole 26h (218 mg, 1 mmol) to obtain the pure
product 28h as yellow solid. Yield (512 mg, 84%)
Mp: 147-149 ºC;
1H NMR (200 MHz, CDCl3): δ 10.74 (bs, 1H), 8.11 (s, 1H), 7.87 (d, 1H, J = 8.3
Hz), 7.64–7.74 (m, 2H), 7.28–7.40 (m, 3H), 7.22 (s, 2H), 6.99 (d, 1H, J = 8.3
Hz), 4.84 (s, 2H), 4.09 (s, 3H), 3.95 (s, 6H), 3.91 (s, 3H);
13C NMR (75 MHz, DMSO D6): δ 187.77, 167.94, 158.71, 152.75, 151.32,
147.25, 144.08, 143.86, 141.71, 133.14, 132.6, 128.5, 127.39, 124.23,
121.54, 119.83, 118.33, 114.94, 113.77, 112.2, 105.98, 67.06, 60.06, 56.03,
55.71;
ESIMS: m/z 603 (M+H)+.
(E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-enyl)phenoxy)-N-(6-ethoxybenzo[d]thiazol-2-yl)acetamide (28i)
This compound was prepared according to the method described for
compound 27a by employing (E)-2-(2-methoxy-5-(3-oxo-3-(3,4,5-
trimethoxyphenyl)prop-1-enyl) phenoxy) aceticacid (22) (402 mg, 1 mmol),
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EDCI.HCl (191 mg, 1 mmol), HOBt (13.5 mg, 0.1 mmol) and 2-amino-6-
ethoxybenzothiazole 26i (194 mg, 1 mmol) to obtain the pure product 28i as
yellow solid. Yield (443 mg, 76%)
Mp: 145-147 ºC;
1H NMR (200 MHz, CDCl3): δ 7.75 (d, 1H, J = 15.8 Hz), 7.70 (d, 1H, J = 9 Hz),
7.36–7.41 (m, 2H), 7.29–7.34 (m, 2H), 7.25–7.28 (m, 3H), 7.01 (d, 1H, J = 8.3
Hz), 4.84 (s, 2H), 4.06–4.15 (q, 2H), 4.04 (s, 3H), 3.96 (s, 6H), 3.95 (s, 3H),
1.46 (t, 3H);
ESIMS: m/z 579 (M+H)+.
2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)-2-methoxy phenoxy)-N-(benzo[d]thiazol-2-yl)acetamide (29a)
This compound was prepared according to the method described for
compound 27a by employing 2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-
dihydro-1H-pyrazol-5-yl)-2-methoxyphenoxy)acetic acid (25) (458 mg, 1
mmol), EDCI.HCl (191 mg, 1 mmol), HOBt (13.5 mg, 0.1 mmol) and 2-
aminobenzothiazole 26a (150 mg, 1 mmol) to obtain the pure product 29a
as white solid. Yield (477 mg, 80%)
Mp: 151-153 ºC;
1H NMR (400 MHz, CDCl3): δ 11.03 (bs, 1H), 7.73–7.81 (m, 2H), 7.40 (t, 1H),
7.29 (d, 1H, J = 7.3 Hz), 6.92–6.97 (m, 1H), 6.85–6.91 (m, 4H), 5.44–5.53 (dd,
1H, J = 11.7, 4.5 Hz), 4.69 (s, 2H), 3.99 (s, 3H), 3.90 (s, 6H), 3.86 (s, 3H),
3.64–3.76 (dd, 1H, J = 11.8, 17.3 Hz), 3.04–3.14 (dd, 1H, J = 17.3, 4.5 Hz),
2.40 (s, 3H);
ESIMS: m/z 591 (M+H)+.
2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)-2-methoxy phenoxy)-N-(6-nitrobenzo[d]thiazol-2-yl)acetamide(29b)
This compound was prepared according to the method described for
compound 27a by employing 2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-
dihydro-1H-pyrazol-5-yl)-2-methoxyphenoxy)acetic acid (25) (458 mg, 1
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mmol), EDCI.HCl (191 mg, 1 mmol), HOBt (13.5 mg, 0.1 mmol) and 2-amino-
6-nitrobenzothiazole 26b (195 mg, 1 mmol) to obtain the pure product 29b
as white solid. Yield (535 mg, 84%)
Mp: 158-160 ºC;
1H NMR (300 MHz, CDCl3): δ 11.02 (bs, 1H), 8.7 (d, 1H, J = 2.2 Hz), 8.32–8.37
(dd, 1H, J = 9, 2.2 Hz), 7.87 (d, 1H, J = 9 Hz), 6.99–7.04 (dd, 1H, J = 8.3, 2.2
Hz), 6.94–6.98 (m, 3H), 6.93 (d, 1H, J = 2.2 Hz), 5.5–5.58 (dd, 1H, J = 11.3,
4.5 Hz), 4.81 (s, 2H), 4 (s, 3H), 3.92 (s, 6H), 3.90 (s, 3H), 3.7–3.82 (dd, 1H, J
= 17.3, 12.8 Hz), 3.08–3.18 (dd, 1H, J = 17.3, 4.5 Hz), 2.43 (s, 3H);
ESIMS: m/z 636 (M+H)+.
2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)-2-methoxy phenoxy)-N-(6-fluorobenzo[d]thiazol-2-yl)acetamide(29c)
This compound was prepared according to the method described for
compound 27a by employing 2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-
dihydro-1H-pyrazol-5-yl)-2-methoxyphenoxy)acetic acid (25) (458 mg, 1
mmol), EDCI.HCl (191 mg, 1 mmol), HOBt (13.5 mg, 0.1 mmol) and 2-amino-
6-fluorobenzothiazole 26c (168 mg, 1 mmol) to obtain the pure product 29c
as white solid. Yield (503 mg, 82%)Mp: 153-154 ºC;
1H NMR (200 MHz, CDCl3): δ 10.62 (bs, 1H), 7.71–7.77 (dd, 1H, J = 9, 4.5 Hz),
7.49–7.53 (dd, 1H, J = 8.3, 3 Hz), 7.14–7.22 (m, 1H), 6.97–7.01 (dd, 1H, J =
8.3, 2.2 Hz), 6.96 (s, 2H), 6.88–6.94 (m, 2H), 6.5–6.57 (dd, 1H, J = 11.3, 4.5
Hz), 4.76 (s, 2H), 3.97 (s, 3H), 3.91 (s, 6H), 3.9 (s, 3H), 3.69–3.8 (dd, 1H, J =
173, 11.3 Hz), 3.08–3.17 (dd, 1H, J = 17.3, 4.5 Hz), 2.43 (s, 3H);
13
C NMR (75 MHz, CDCl3+DMSO D6): δ 167.44, 167.22, 156.49, 153.27,152.55, 148.45, 146.87, 144.68, 139.15, 134.49, 132.41, 126.2, 121.33,
121.22, 119.41, 113.79, 113.47, 111.83, 107.24, 106.89, 103.39, 68.34,
59.98, 58.79, 55.51, 41.86, 21.39;
ESIMS: m/z 609 (M+H)+.
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2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)-2-methoxy phenoxy)-N-(6-chlorobenzo[d]thiazol-2-yl)acetamide(29d)
This compound was prepared according to the method described for
compound 27a by employing 2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)-2-methoxyphenoxy)acetic acid (25) (458 mg, 1
mmol), EDCI.HCl (191 mg, 1 mmol), HOBt (13.5 mg, 0.1 mmol) and 2-amino-
6-chlorobenzothiazole 26d (184 mg, 1 mmol) to obtain the pure product 29d
as white solid. Yield (518 mg, 82%)
Mp: 153-155 ºC;
1H NMR (400 MHz, CDCl3): δ 10.74 (bs, 1H), 7.76 (d, 1H, J = 2.2 Hz), 7.67 (d,
1H, J = 9 Hz), 7.34–7.39 (dd, 1H, J = 9, 2.2 Hz), 6.92–6.97 (dd, 1H, J = 8.3,
2.2 Hz), 6.89–6.91 (m, 1H), 5.44–5.52 (dd, 1H, J = 12, 4.5 Hz), 4.74 (s, 2H), 4
(s, 3H), 3.9 (s, 6H), 3.86 (s, 3H), 3.65–3.77 (dd, 1H, J = 17.3, 12 Hz), 3.05–
3.14 (dd, 1H, J = 17.3, 4.5 Hz), 2.4 (s, 3H);
ESIMS: m/z 626 (M+H)+.
2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)-2-methoxy phenoxy)-N-(6-methoxybenzo[d]thiazol-2-yl)acetamide (29e)
This compound was prepared according to the method described for
compound 27a by employing 2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-
dihydro-1H-pyrazol-5-yl)-2-methoxyphenoxy)acetic acid (25) (458 mg, 1
mmol), EDCI.HCl (191 mg, 1 mmol), HOBt (13.5 mg, 0.1 mmol) and 2-amino-
6-methoxybenzothiazole 26e (180 mg, 1 mmol) to obtain the pure product
29e as white solid. Yield (532 mg, 85%)
Mp: 156-157 ºC;
1H NMR (300 MHz, CDCl3): δ 10.45 (bs, 1H), 7.69 (d, 1H, J = 8.3 Hz), 7.29 (d,1H, J = 2 Hz), 7.03–7.07 (dd, 1H, J = 9.3, 3.1 Hz), 6.97–7.00 (dd, 1H, J = 8.3,
2 Hz), 6.96 (s, 2H), 6.91 (d, 1H, J = 8.3 Hz), 6.87 (d, 1H, J = 2 Hz), 5.51–5.55
(dd, 1H, J = 11.4, 4.1 Hz), 4.75 (s, 2H), 3.96 (s, 3H), 3.91 (s, 3H), 3.89 (s, 3H),
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3.88 (s, 3H), 3.7–3.78 (dd, 1H, J = 7.6, 12.4 Hz), 3.09–3.14 (dd, 1H, J = 7.6,
4.1 Hz), 2.4 (s, 3H);
ESIMS: m/z 621 (M+H)+.
2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)-2-methoxy phenoxy)-N-(6-trifluoromethoxybenzo[d]thiazol-2-yl)acetamide (29f)
This compound was prepared according to the method described for
compound 27a by employing 2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-
dihydro-1H-pyrazol-5-yl)-2-methoxyphenoxy)acetic acid (25) (458 mg, 1
mmol), EDCI.HCl (191 mg, 1 mmol), HOBt (13.5 mg, 0.1 mmol) and 2-amino-
6-trifluoromethoxybenzothiazole 26f (234 mg, 1 mmol) to obtain the pure
product 29f as white solid. Yield (547 mg, 81%)
Mp: 159-161 ºC;
1H NMR (400 MHz, CDCl3): δ 10.71 (bs, 1H), 7.79 (d, 1H, J = 8.3 Hz), 7.69 (d,
1H, J = 1.5 Hz), 7.29–7.34 (dd, 1H, J = 9, 1.5 Hz), 6.98–7.02 (dd, 1H, J = 8.3,
1.5 Hz), 6.96 (s, 2H), 6.89–6.94 (m, 2H), 5.5–5.58 (dd, 1H, J = 11.3, 4.5 Hz),
4.77 (s, 2H), 3.98 (s, 3H), 3.91 (s, 6H), 3.9 (s, 3H), 3.69–3.81 (dd, 1H, J =
18.1, 12 Hz), 3.08–3.17 (dd, 1H, J = 17.3, 4.5 Hz), 2.43 (s, 3H);
13C NMR (75 MHz, CDCl3+DMSO D6): δ 167.76, 167.4, 157.53, 135.15,
152.58, 148.66, 146.8, 144.41, 139.32, 134.46, 132.42, 126.1, 121.22,
120.02, 119.2, 113.87, 113.52, 111.78, 103.31, 69.14, 60.1, 58.76, 55.51,
55.36, 41.79, 21.35;
ESIMS: m/z 675 (M+H)+.
2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)-2-methoxy phenoxy)-N-(6-methylbenzo[d]thiazol-2-yl)acetamide
(29g) This compound was prepared according to the method described for
compound 27a by employing 2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-
dihydro-1H-pyrazol-5-yl)-2-methoxyphenoxy)acetic acid (25) (458 mg, 1
mmol), EDCI.HCl (191 mg, 1 mmol), HOBt (13.5 mg, 0.1 mmol) and 2-amino-
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6-methylbenzothiazole 26g (164 mg, 1 mmol) to obtain the pure product
29g as white solid. Yield (498 mg, 82%)
Mp: 153-154 ºC;
1H NMR (200 MHz, CDCl3): δ 10.52 (bs, 1H), 7.69 (d, 1H, J = 8.3 Hz), 7.62 (s,
1H), 7.24–7.29 (m, 1H), 6.94–7 (m, 3H), 6.87–6.93 (m, 2H), 5.5–5.57 (dd, 1H,
J = 12, 4.5 Hz), 4.75 (s, 2H), 3.96 (s, 3H), 3.91 (s, 6H), 3.89 (s, 3H), 3.68–3.8
(dd, 1H, J = 17.3, 11.3 Hz), 3.07–3.17 (dd, 1H, J = 17.3, 4.5 Hz), 2.48 (s, 3H),
2.43 (s, 3H);
ESIMS: m/z 605 (M+H)+.
2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)-2-methoxy phenoxy)-N-(6-trifluoromethylbenzo[d]thiazol-2-yl)acetamide (29h)
This compound was prepared according to the method described for
compound 27a by employing 2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-
dihydro-1H-pyrazol-5-yl)-2-methoxyphenoxy)acetic acid (25) (458 mg, 1
mmol), EDCI.HCl (191 mg, 1 mmol), HOBt (13.5 mg, 0.1 mmol) and 2-amino-
6-trifluoromethylbenzothiazole 26h (218 mg, 1 mmol) to obtain the pure
product 29h as white solid. Yield (568 mg, 86%)
Mp: 162-163 ºC;1H NMR (400 MHz, CDCl3): δ 10.82 (bs, 1H), 8.11 (s, 1H), 7.88 (d, 1H, J = 7.9
Hz), 7.67–7.7 (m, 1H), 6.98–7.02 (dd, 1H, J = 7.9, 1.6 Hz), 6.96 (s, 2H), 6.9–
6.94 (m, 2H), 5.51–5.56 (dd, 1H, J = 11.9, 3.9 Hz), 4.79 (s, 2H), 3.99 (s, 3H),
3.91 (s, 6H), 3.89 (s, 3H), 3.71–3.79 (dd, 1H, J = 11.9, 17.5 Hz), 3.1–3.15 (dd,
1H, J = 17.5, 4.7 Hz), 2.43 (s, 3H);
ESIMS: m/z 659 (M+1)+.
2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)-2-methoxy phenoxy)-N-(6-ethoxybenzo[d]thiazol-2-yl)acetamide(29i)
This compound was prepared according to the method described for
compound 27a by employing 2-(5-(1-acetyl-3-(3,4,5-trimethoxyphenyl)-4,5-
dihydro-1H-pyrazol-5-yl)-2-methoxyphenoxy)acetic acid (25) (458 mg, 1
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mmol), EDCI.HCl (191 mg, 1 mmol), HOBt (13.5 mg, 0.1 mmol) and 2-amino-
6-ethoxybenzothiazole 26i (194 mg, 1 mmol) to obtain the pure product 29i
as white solid. Yield (502 mg, 78%)
Mp: 158-159 ºC;
1H NMR (300 MHz, CDCl3): δ 10.47 (bs, 1H), 7.68 (d, 1H, J = 9.3 Hz), 7.28 (d,
1H, J = 3.1 Hz), 7.02–7.06 (dd, 1H, J = 8.3, 2 Hz), 6.95–7 (m, 3H), 6.9 (d, 1H, J
= 8.3 Hz), 6.87 (d, 1H, J = 2.08 Hz), 5.5–5.56 (dd, 1H, J = 12.4, 4.1 Hz), 4.75
(s, 2H), 4.06– 4.12 (q, 2H), 3.95 (s, 6H), 3.91 (s, 6H), 3.89 (s, 3H), 3.7–3.78
(dd, 1H, J = 17.6, 12.4 Hz), 3.09–3.15 (dd, 1H, J = 17.6, 4.1 Hz), 2.43 (s, 3H),
1.45 (t, 3H);
ESIMS: m/z 635 (M+H)+.
3.7. TUBULIN POLYMERIZATION ASSAY
A fluorescence based In vitro tubulin polymerization assay was
performed according to the manufacturer’s protocol (BK011, Cytoskeleton,
Inc.). Briefly, the reaction mixture in a total volume of 10 µl contained PEM
buffer, GTP (1 mM) in the presence or absence of test compounds (final
concentration of 3 µM). Tubulin polymerization was followed by a time
dependent increase in fluorescence due to the incorporation of a
fluorescence reporter into microtubules as polymerization proceeds.
Fluorescence emission at 460 nm (excitation wavelength is 360 nm) was
measured by using a Varioscan multimode plate reader (Thermo scientific
Inc.). Podophyllotoxin was used as positive control in each assay.
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3.8. REFERENCES
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3. Bhattacharyya, B.; Panda, D.; Gupta, S.; Banerjee, M. Antimitotic
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6. Jordan, M. A.; Wilson, L. Nat. Rev. Cancer. 2004, 4, 253.
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8. Pandit, B.; Sun, Y.; Chen, P.; Sackett, D. L.; Hu, Z.; Rich, W.; Li, C.;
Lewis, A.; Schaefer, K.; Li, P. K. Bioorg. Med. Chem. 2006, 14, 6492.
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10. Cragg, G. M., Kingston, D. G., Newman, D. J. Anticancer Agents
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11. Cooney, M. M.; Ortiz, J.; Bukowski, R. M.; Remick, S. C. Curr.
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12. Young, S. L.; Chaplin, D. J. Expert Opin. Invest. Drugs. 2004, 13,
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16. Bohle, A. S.; Leuschner, I.; Kalthoff, H.; Henne-Bruns, D. Int. J.
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17. Ohsumi,K.;Hatanaka, T.;Nakagawa,R.; Fukuda,Y.; Morinaga, Y.;
Suga, Y.;Nihei,Y.; Ohishi,K.; Akiyama,Y.; Tsuji, T. Anti-Cancer Drug
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18. Kanthou, C.; Tozer, G. M. Int. J. Exp. Pathol. 2009, 90, 284.
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22. Pettit, G. R.; Rhodes, M. R.; Herald, D. L.; Hamel, E.; Schmidt, J.
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29. Chaplin, D. J.; Pettit, G. R.; Parkins, C. S.; Hill, S. A.; Br. J. Cancer
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30. Nabha, S. M.; Mohammed, R. M.; Dandashi, M. H.; Coupaye-
Gerard, B.; Aboukameel, A.; Petit, G. R.; Al-Katib, A. M. Clin. Cancer Res.
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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.; D’Alessandro, 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,
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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. Tron,G. C.; Pirali, T.; Sorba,G.; Pagliai, F.; Busacca, S.;Genazzani,
A. A. J. Med. Chem. 2006, 49, 3033.
36. Nam, N. H. Curr. Med. Chem. 2003, 10, 1697.
37. Liou, J. P.; Chang, Y. L.; Kuo, F. M.; Chang, C. W.; Tseng, H. Y.;
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. Cushman, M.; Nagarathnam, D.; Gopal, D.; Chakraborti, A. K.; Lin,
C. M.; Hamel, E. J. Med. Chem. 1991, 34, 2579.
40. Wang, L.; Woods, K. W.; Li, Q.; Barr, K. J.; McCroskey, R. W.;
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. Kim, Y.; Nam, N.-H.; You, Y.-J.; Ahn, B.-Z. Bioorg.Med. Chem. Lett.
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43. Shirai, R.; Takayama, H.; Nishikawa, A.; Koiso, Y.; Hashimoto, Y.
Bioorg. Med. Chem. Lett. 1998, 8, 1997.
44. Tron, G. C.; Pagliai, F.; Grosso, E. D.; Genazzani, A. A.; Sorba, G. J. Med. Chem. 2005, 48, 3260.
45. (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.
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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. Pettit, G. R.; Singh, S. B.; Hamel, E.; Lin, C. M.; Alberts, D. S.;
Garcia-Kendall, D. Experientia 1989, 45, 209.
47. Lin, C. M.; Ho, H. H.; Pettit, G. R.; Hamel, E. Biochemistry 1989,
28, 6984.
48. Jordan, A.; Hadfield, J. A.; Lawrence, N. J.; McGown, A. T. Med.
Res. Rev . 1998, 18, 259.
49. Johnson, M.; Younglove, B.; Lee, L.; LeBlanc, R.; Holt, H.; Hills, P.;
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.
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CCHAPTERHAPTER-IV/S-IV/SECTIONECTION-A-A
SS YNTHESIS YNTHESIS AANDND BBIOLOGICALIOLOGICAL EEVALUATIONVALUATION OOFF BBENZYLIDENEENZYLIDENE-9(10H)--9(10H)-AANTHRACENONENTHRACENONE LLINKEDINKED PP YRROLOBENZODIAZEPINES YRROLOBENZODIAZEPINES AASS AANTICANCERNTICANCER AAGENTSGENTS
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4.1. I4.1. INTRODUCTIONNTRODUCTION
Cancer represents one of the largest health threats of mankind, and
therefore considerable efforts are undertaken regarding the development of
new chemotherapeutic agents for more potent and more specific anti-cancertherapy. The cells of each organism are in exact regulated mechanism of
equilibrium between growth (proliferation), differentiation (cellular
specialization) and programmed cell death (apoptosis). The most obvious
and medical, most important feature of cancer cells is their uncontrolled
growth. The cellular proliferation is a result of the cell division or mitosis.
Tubulin as well as its isoforms forms the major constituent of the
microtubulins and exists its part as heterodimer of the two globular
polypeptides [α] - and [β] – Tubulin.1 Microtubulins possess a prominent
importance for most diverse functions of the cell, among them the mitosis,
the movement of the cell and the cell formation.2-3 The importance of the
microtubulins for the cellular life and particular for the cell division and thus
linked for the induction and the progression of the apoptosis make it one of
the most attractive therapeutic targets for the development of new
compounds as cancer chemotherapeutics.
4.1.1. I4.1.1. INTRODUCTIONNTRODUCTION OOFF BBENZYLIDENEANTHRACENONESENZYLIDENEANTHRACENONES
Anthracenones are known for biological properties like tubulin binding,
antiproliferation and antipsoriatic4 action. It was found that certain 10-
benzyliden-9(10H)anthracenones are potent inhibitors of the tubulin
polymerization, which accompanying with an elevated cellular proliferation
there by effective anti-cancer compounds. Prinz and co-workers5 have
prepared several structurally new 10-benzylidene and 10-phenylmethyl-9(10H)-anthracenones. These compounds were evaluated for their
antiproliferative activity against K562 leukemia cells and various other
human cancer cell lines. In this series the lead compounds (1 and 2)
interacts with the colchicine site, causing G2/M arrest and induces apoptotic
cell death. There was no growth inhibitory effect in the cell cycle arrested
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T HESIS
cells. In addition, these compounds (1 and 2) were found to be equally
potent toward parental tumor cell lines and multidrug resistant cells. The
most active compound 1, showed the anticancer activity against K562 cells
in nanomolar range (IC50: 20 nM) (Figure 1).
O
OMe
OH
O
OMe
OH1 2
OMe
O
3
O
OMe
Figure 1
The same group6 synthesized new analogues of 10-(2-oxo-2-
phenylethylidene)-10H-anthracen-9-ones, that were evaluated for
interactions with tubulin as well as antiproliferative activity against a panel
of human and rodent tumor cell lines. The 4-methoxy analogue (3) was most
potent, displaying IC50 values ranging from 40 to 80 nM, even on multidrug
resistant phenotypes, and possesses excellent activity as an inhibitor of tubulin polymerization (IC50: 0.52 μM).
Zuse and coworkers7 synthesized and reported some novel 9-
benzylidene- naphtho[2,3-b]thiophen-4-one derivatives (4 and 5). These
compounds were identified as agents with highest cell growth inhibiting
activity against a broad spectrum of cancer cell lines, including a panel of
cells with multi-drug resistant phenotypes. Amongst them 9-[(4-hydroxy-3,5-
dimethoxy)-benzylidene]-naphtho [2,3-b]thiophen-4-one (4) was the most
active compound in this series. It not only showed high antiproliferative
activity towards various cancer cell lines (IC50 K562 cells 50 nM) but also
strong tubulin polymerization inhibiting activity (IC50: 0.38 μM) (Figure 2).
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T HESIS
OMe
OH
OH
OMeMeO
5
Figure 2
Recently some of anthracenone series of compounds have also been
synthesized and evaluated for their anticancer property.8-9 They include
anthracenone-based oxime ethers, oxime esters like 6 and 7, and 1,5- or
1,8-disubstituted-10-benzylidene-10H-anthracen-9-ones and 10-(2-oxo-2-
phenylethy lidene)-10H-anthracen-9-ones (8 and 9) (Figure 3).
OCl Cl
8 OH
OMe
OMe
O
NO
OMe
O
NO
OMe
OH
6 7
OCl Cl
9 OMe
OH
Figure 3
Apart from anticancer potential, these anthracenone analoguespossess antipsoriatic properties. Anthralin (10) is the most widely used drug
for psoriasis10. Antipsoriatic anthracenones exhibit three principal cellular
effects, which include interaction with DNA, inhibition of various enzyme
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T HESIS
systems associated with cell proliferation and redox reactions that result in
alteration of mitochondrial functions and destruction of membrane lipids.
OOH OH
N
12
OOH OHOOH OH
O
OMe
1011
OOH OH
13
O
O
Cl Cl
14
OH
OMe
Figure 4
Klaus Mtiller and coworkers described the synthesis11-14 and
antipsoriatic activity of different anthracenones 11, 12, 13 and 14. These
compounds were evaluated for their ability to inhibit the growth of the
human keratinocyte cell line (HaCaT) and the 5-1ipoxygenase enzyme in
bovine polymorphonuclear leukocytes (Figure 4).
4.1.2. P4.1.2. PRESENTRESENT WWORK ORK
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
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T HESIS
biological activities of the benzylidene-9(10H)-anthracenones and the
pyrrolo[2,1-c][1,4] benzodiazepines, there have been considerable efforts in
structural modification of PBDs and development of new synthetic strategies
in this laboratory. In this endeavor a series of new PBD conjugates (22a-f)
that comprise of both moieties designed, synthesized and with varying
alkane spacers have been evaluated for their antitumour activity and DNA-
binding ability.
4.1.2.1. S4.1.2.1. S YNTHESIS YNTHESIS OOFF PBD PPBD PRECURSORSRECURSORS
The precursor (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]pyrolidine-
2-carboxaldehydediethylthioacetal (15) have been prepared from
commercially available vanillin. The synthesis of compound 15 was
discussed in chapter-II.
HO
MeO
NO2
O
N
CH(SEt)2
15
4.1.2.2. S4.1.2.2. S YNTHESIS YNTHESIS OOFF BBENZYLIDENEENZYLIDENE-9(10H)--9(10H)-ANTHRACENONESANTHRACENONES
The preparation of 9-benzylidineanthracenones intermediates (19a-f)
has been carried out by the synthetic sequence illustrated in Scheme-1. The
synthesis of these intermediates is performed by reacting anthrone (16) with
different benzaldehydes in the presence of 10% IPA.HCl (isopropyl alcoholic
solution of HCl). These Benzylidene-9(10H)-anthracenones upon alkylation of
the hydroxyl group by dibromoalkanes using K 2CO3 as a base in dry acetone
affords the required precursors (19a-f) as illustrated in Scheme 1.
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T HESIS
CHO
R
OH
+(i)
(ii)
19a-f
16 17a, b18a, b
19a; R = H, n = 219b; R = H, n = 319c; R = H, n = 419d; R = OMe, n = 2
19e; R = OMe, n = 319f ; R = OMe, n = 4
OO
R
OH
O
R
OBr
( )n
Scheme 1. Reagents and conditions: (i) IPA.HCl, 5 h; (ii) dibromoalkane, acetone, K 2CO3,
reflux, 24h.
4.1.2.3. S4.1.2.3. S YNTHESIS YNTHESIS OOFF C8-C8-LINKEDLINKED BBENZYLIDENEENZYLIDENE-9(10H)--9(10H)-ANTHRACENONEANTHRACENONE-PBD-PBD CCONJUGATESONJUGATES
Compound 15 has been coupled to compounds 19a-f in the presence
of K 2CO3 and dry acetone under reflux give corresponding nitro compounds
(20a-f). These nitro compounds upon reduction with SnCl2.2H2O in methanol
under reflux give amino compounds (21a-f). The amino compounds upon
deprotection with HgCl2/CaCO3 afford the corresponding imines (22a-f) as
shown in Scheme-2.
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O
R
OBr
( )n
19a-f
HO
MeO
NO2
O
N
CH(SEt)2
15
+
O
MeO
NO2
ON
CH(SEt)2O
R
( )n
20a-f
21a-f
22a-f
(i)
(ii)
(iii)
O
O
MeO
NH2
O
N
CH(SEt)2O
R
O
( )n
O
MeO
O
R
O
N
N
O
H( )n
Scheme 2. Reagents and conditions: (i) K 2CO3, acetone, 12 h, reflux; (ii) SnCl2.2H2O, MeOH,
4 h, reflux; (iii) HgCl2, CaCO3, MeCN, H2O, (4:1) 12 h, rt.
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4.1.3. BIOLOGICAL ACTIVITY
4.1.3.1. DNA BINDING AFFINITY : THERMAL DENATURATION STUDIES
The DNA binding affinity of these new C8-linked benzylidene-9(10H)-
anthracenone-PBD conjugates (22a-f ) has been evaluated through thermal
denaturation studies with duplex-form of calf thymus DNA (CT-DNA) by using
modified reported procedure.15 The DNA-PBD solutions are incubated at 37
οC for 0 h and 18 h prior to analysis. Samples are monitored at 260 nm using
a Beckman DU-7400 spectrophotometer fitted with high performance
temperature controller and heated at 1οC/min in the range of 40-95
οC. DNA
helix-coil transition temperatures are given by: ∆ T m = T m(DNA+PBD)–T m(DNA
alone), where the T m value for the PBD-free CT-DNA is 69.8± 0.01. These
studies were carried out at PBD/DNA molar ratio 1:5. The increase in melting
temperature (∆ T m) for each compound is examined at 0 h and 18 h of
incubation at 37οC. Melting studies show that these compounds stabilize the
thermal helix coil or melting stabilization for the CT-DNA duplex at pH 7.0,
and incubated at 37οC with ligand/DNA molar ratio of 1:5. The increase in
the helix melting temperature (∆ T m) for each compound has been examined
at 0 h and 18 h incubation at 37οC.
Interestingly, all the benzylidene-9(10H)-anthracenone-PBD conjugates
elevate the helix melting temperature of CT-DNA in the range of 3.0-5.1 oC.
Compound 22b showed the highest ΔT m of 4.6 oC at 0 h and increased upto
5.1 oC after 18 h incubation, whereas the naturally occurring DC-81 exhibits
a ΔT m of 0.7 oC after incubation under similar conditions (Table 1). These
results indicate that the effect on DNA binding affinity by introducing the
benzylidene-9(10H)-anthracenone scaffold on PBD moiety through different
alkane spacers at C8-position of the DC-81.
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Table 1.Thermal denaturation data for benzylidene-9(10H)-anthracenone-PBD conjugates with calf thymus (CT)-DNA
Compound[PBD]:[DNA]
molar ratiob
ΔT m (oC)a after incubation at 37 oCfor
0 h 18 h
22a 1:5 4.3 4.9
22b 1:5 4.6 5.1
22c 1:5 4.1 4.6
22d 1:5 3.6 4.0
22e 1:5 3.0 3.5
22f 1:5 4.0 4.3
DC-81 1:5 0.3 0.7a For CT-DNA alone at pH 7.00 ± 0.01, T m = 68.5 0C Δ 0.01 (mean value from 10 separate
determinations), all ΔT m values are ± 0.1 - 0.2 0C. b For a 1:5 molar ratio of [PBD]/[DNA],
where CT-DNA concentration = 100 μM and ligand concentration = 20 μM in aqueous
sodium phosphate buffer [10 mM sodium phosphate + 1 mM EDTA, pH 7.00 ± 0.01].
4.1.3.2. ANTICANCER ACTIVITY
Compounds (22a-f ) have been evaluated for their in vitro cytotoxicity
in selected human cancer cell lines of barest, ovarian, colon, prostate, cervix,
lung and oral by using Sulforhodamine B (SRB) method.16 The in vitro
cytotoxicity results of these compounds expressed in GI50 values which
carried out the experiments at 10-4 to 10-7 M concentrations and the data is
illustrated in Table 2. The results from these experiments reveal that
compounds 22a-f showed GI50 values in the range of 0.08-2.5 μM, while thepositive controls, DC-81 and adriamycin exhibited the GI50 in the range of
0.1-0.17 μM and <0.01-14.7 μM respectively, in the cell lines employed. The
synthesized novel benzylidene-9(10H)-anthracenone-PBD conjugates
exhibited significant anticancer activity against MCF-7 human breast cancer
cell line (GI50 range, 0.08−0.14 μM) compared to other cell lines tested. The
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active compound 22e exhibited strong effect against all cell lines tested
(GI50, 0.08-1.8 μM) and it showed a GI50 value of 0.08 against MCF-7 cell line.
Table 2. GI50 valuesa (in μM) for compounds 22a-f in selected human cancercell lines.
Compound
GI50 values (μM)
Breast Ovarian ColonProstat
eCervix Lung Oral
MCF-7 A2780 Colo205 PC-3 SiHaA
549Hop-62 KB
22a 0.12 0.18 0.17 1.3 2.1 1.53 0.34 2.1
22b 0.14 0.16 0.11 2.2 2.5 0.15 0.2 2.4
22c 0.15 0.12 1.2 0.2 --- 0.16 0.261.82
22d 0.12 0.14 0.11 0.18 2.2 1.76 0.190.19
22e 0.08 0.15 0.12 0.18 -- 1.8 0.18 0.2
22f 0.1 0.15 -- 0.15 2.2 1.94 0.170.18
DC-81 0.16 0.13 0.1 -- 0.16 -- 0.110.17
ADR <0.01 0.02 14.7 <0.01 0.19 13 <0.010.16
a 50% Growth inhibition and the values are mean of four determinationsADR, adriamycin.
4.1.4. CONCLUSION
In conclusion, we have synthesized a series of novel C8-linked
benzylidene-9(10H )-anthracenone-PBD conjugates ( 22a-f ). For
synthesized compounds anticancer activity has been evaluated
against eight human cancer cell lines (barest, ovarian, colon,
prostate, cervix, lung and oral). All the compounds exhibited
significant anticancer activity. Moreover these compounds exhibited
significant DNA binding ability .
4.1.5. EXPERIMENTAL SECTION
10-(4-hydroxybenzylidene)anthracen-9(10H)-one (18a)
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To a stirred mixture of anthrone 16 (194 mg, 1 mmol) and 4-hydroxy
benzaldehyde 17a (122 mg, 1 mmol) was added isopropylalcoholic.HCl
solution (IPA.HCl, 10%solution) (5 ml) under nitrogen atmosphere with
maintaining cooling conditions. The temperature raised to room temperature
and stirring continued for 5 h. After completion of the reaction checked by
TLC, the solvent was evaporated, neutralized with saturated bicarbonate
solution and extracted with chloroform (2x50 ml). The combined organic
fractions were washed with water followed by brain, dried over Na2SO4 and
purified by column chromatography using (20% EtOAC:hexane) to obtain the
pure, yellow colored solid product 18a. Yield (210 mg, 70%). mp: 106-108
ºC;
1H NMR (300 MHz, CDCl3): δ 9.84 (bs, 1H), 8.31 (dd, 1H, J = 7.8, 1.5 Hz),
8.13–8.23 ( m, 2H), 7.72–7.86 ( m, 3H), 7.39–7.66 (m, 3H), 7.29 (d, 2H, J =
8.6 Hz), 6.79 (d, 2H, J = 7.8 Hz);
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), and3-methoxy-4-hydroxy benzaldehyde 17b (152 mg, 1 mmol). Yield (241 mg,
72%). Mp: 102-104 ºC;
1H NMR (300 MHz, CDCl3): δ 8.2–8.28 (m, 2H), 7.97 (d, 1H, J = 8.3 Hz), 7.66
(d, 1H, J = 8.3 Hz), 7.56–7.63 (m, 1H), 7.43–7.5 (m, 2H), 7.36–7.42 (m, 1H),
7.26–7.31 (dd, 1H, J = 8.3, 1.5 Hz), 6.87–6.91 (dd, 1H, J = 8.3, 1.5 Hz), 6.86
(s, 1H), 6.75 (d, 1H, J = 1.5 Hz), 5.59 (bs, 1H), 3.69 (s, 3H);
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
K 2CO3 (276 mg, 2 mmol), 1,3 dibromopropane (605 mg, 3 mmol) and the mixture was stirred at
reflux temperature for 24 hours. The reaction was monitored by TLC using ethyl acetate-hexane
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(1:9). After completion of the reaction as indicated by the TLC, K 2CO3 was removed by filtration
and the solvent evaporated under reduced pressure, diluted with water and extracted with ethyl
acetate (2X20 ml). The combined organic phases were dried over Na2SO4 and evaporated under
vacuum. The residue, thus obtained was purified by column chromatography using ethyl acetate
and hexane (1:9) to afford pure compound 19a as liquid. Yield (352 mg, 83%)
1H NMR (200 MHz, CDCl3): δ 8.16–8.29 (m, 2H), 7.95 (d, 1H, J = 7.5 Hz),
7.53–7.63 (m, 2H), 7.41–7.51 (m, 2H), 7.38 (t, 1H, J = 7.5 Hz), 7.16–7.28
(m, 3H), 6.77 (d, 2H, J = 8.3 Hz), 3.98 (t, 2H, J = 6, 5.2 Hz), 3.5 (t, 2H, J =
6.7, 6 Hz), 2.24–2.45 (m, 2H);
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%)
1H NMR (300 MHz, CDCl3): δ 8.16–8.30 (m, 2H), 7.96 (d, 1H, J = 7.5 Hz),
7.54–7.63 (m, 2H), 7.42–7.5 (m, 2H), 7.39 (t, 1H, J = 7.5 Hz), 7.16–7.29 (m,
3H), 6.77 (d, 2H, J = 8.3 Hz), 3.99 (t, 2H, J = 6, 5.2 Hz), 3.47 (t, 2H, J = 6.7,
6 Hz), 1.89–2.05 (m, 4H);ESIMS: m/z 434 (M+H)+.
10-(4-(5-bromopentoxy)benzylidene)anthracen-9(10H)-one (19c)
The compound 19c was prepared according to the method described for compound 19a by
employing compound 18a (298 mg, 1 mmol), and 1,5 dibromopentane (689 mg, 3 mmol). Yield
(380 mg, 84%)
1H NMR (200 MHz, CDCl3): δ 8.16–8.31 (m, 2H), 7.96 (d, 1H, J = 7.5 Hz),
7.53–7.63 (m, 2H), 7.42–7.51 (m, 2H), 7.39 (t, 1H, J = 7.5 Hz), 7.15–7.28
(m, 3H), 6.78 (d, 2H, J = 8.3 Hz), 3.99 (t, 2H, J = 6, 5.2 Hz), 3.28 (t, 2H, J =
6.7, 6 Hz), 1.81–2.03 (m, 4H), 1.63−1.75 (m, 2H);
ESIMS: m/z 448 (M+H)+
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10-(4-(3-bromopropoxy)-3-methoxybenzylidene)anthracen-9(10H)-one (19d)
The compound 19d was prepared according to the method described for compound 19a by
employing compound 18b (328 mg, 1 mmol), and 1,3 dibromopropane (605 mg, 3 mmol). Yield
(411 mg, 91%)1H NMR (300 MHz, CDCl3): δ 8.22 (t, 2H, J = 7.5 Hz), 7.95 (d, 1H, J = 7.5
Hz), 7.63 (d, 1H, J = 8.3 Hz), 7.53–7.6 (m, 1H), 7.33–7.48 ( m, 3H), 7.22–
7.31 (m, 1H), 6.85 (d, 1H, J = 8.3 Hz), 6.74–6.82 (m, 2H), 4.13 (t, 2H, J = 6
Hz), 3.57–3.68 (m, 5H), 2.25–2.42 (m, 2H);
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 1,4-
dibromobutane (647 mg, 3 mmol). Yield (415 mg, 89%)
1H NMR (400 MHz, CDCl3): δ 8.25 (t, 2H, J = 8.3 Hz), 7.98 (d, 1H, J = 7.5
Hz), 7.57–7.67 (m, 2H), 7.36–7.50 ( m, 3H), 7.23–7.31 (m, 1H), 6.88 (d, 1H,
J = 8.3 Hz), 6.76–6.82 (m, 2H), 4.05 (t, 2H, J = 6 Hz), 3.64 (s, 3H), 3.5 (t,
2H, J = 6.7, 6 Hz), 1.94–2.17 (m, 4H);
ESIMS: m/z 463 (M)+.
10-(4-(5-bromopentoxy)-3-methoxybenzylidene)anthracen-9(10H)-one (19f)
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%)
1H NMR (300 MHz, CDCl3): δ 8.23 (t, 2H, J = 7.5 Hz), 7.96 (d, 1H, J = 7.5
Hz), 7.62 (d, 1H, J = 8.3 Hz), 7.51–7.61 (m, 1H), 7.32–7.48 (m, 3H), 7.22–7.3 (m, 1H), 6.84 (d, 1H, J = 8.3 Hz), 6.74–6.81 (m, 2H), 4.11 (t, 2H, J = 6
Hz), 3.65 (s, 3H), 3.44 (t, 2H, J = 6.7, 6 Hz), 1.82–2.06 (m, 4H), 1.61–1.76
(m, 2H);
ESIMS: m/z 478 (M+H)+.
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(2S)-N-{4-(3-[4-[(10-oxo-9,10-dihydro-9-anthracenyliden)methyl]phenoxy]propyl)oxy-5-methoxy-2-nitrobenzoyl}-pyrrolidine-2-carboxaldehydediethylthioacetal (20a)
To a solution of (2S)-N-[4-hydroxy-5-methoxy–2-nitrobenzoyl]pyrrolidine-2-
carboxal dehydediethylthioacetal (15) (400 mg, 1 mmol) in dry acetone (15mL) was added, anhydrous K 2CO3 (276 mg, 2 mmol), 10-(4-(3-
bromopropoxy)benzylidene)anthracen-9(10H)-one (19a) (419 mg, 1 mmol)
and the mixture was stirred at reflux temperature for 12 hours. The reaction
was monitored by TLC using ethyl acetate-hexane (1:1). After completion of
the reaction as indicated by the TLC, K 2CO3 was removed by filtration and the
solvent evaporated under reduced pressure, diluted with water and
extracted with ethyl acetate. The organic phase was dried over Na2SO4 and
evaporated under vacuum. The residue, thus obtained was purified by
column chromatography using ethyl acetate and hexane (1:1) to afford
compound 20a as yellow solid. Yield (672 mg, 90%).
Mp: 124-126 ºC;
1H NMR (300 MHz, CDCl3): δ 8.19–8.3 (m, 2H), 7.97 (d, 1H, J = 7.3 Hz),
7.53–7.66 (m, 3H), 7.34–7.5 (m, 3H), 7.19–7.32 (m, 3H), 6.71–6.83 (m, 3H),
4.8 (d, 1H, J = 3.6 Hz), 4.61-4.7 (m, 1H), 4.19 (t, 2H, J = 5.1 Hz), 4.09 (t,
2H, J = 5.1 Hz), 3.92 (s, 3H), 3.17–3.29 (m, 2H), 2.69-2.88 (m, 4H), 2.18–
2.35 (m, 2H), 1.94–2.14 (m, 3H), 1.73–1.90 (m, 1H), 1.26–1.39 (m, 6H);
ESIMS: m/z 739 (M+H)+.
(2S)-N-{4-(4-[4-[(10-oxo-9,10-dihydro-9-anthracenyliden)methyl]phenoxy]butyl)oxy-5-methoxy-2-nitrobenzoyl}-pyrrolidine-2-carboxaldehydediethylthioacetal (20b)
The compound 20b was prepared according to the method described for
compound 20a by employing (2S)-N-[4-hydroxy-5-methoxy–2-nitrobenzoyl]pyrrolidine-2-carboxal dehydediethylthioacetal (15) (400 mg, 1 mmol), and
10-(4-(4-bromobutoxy) benzylidene)anthracen-9(10H)-one (19b) (433 mg, 1
mmol). Yield (700 mg, 92%).
Mp: 125-126 ºC;
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1H NMR (200 MHz, CDCl3): δ 8.18–8.3 (m, 2H), 7.98 (d, 1H, J = 7.3 Hz),
7.54–7.67 (m, 3H), 7.34–7.51 (m, 3H), 7.18-7.3 (m, 3H), 6.7–6.84 (m, 3H),
4.81 (d, 1H, J = 3.6 Hz), 4.59–4.71 (m, 1H), 4.17 (t, 2H, J = 5.1 Hz), 4.07 (t,
2H, J = 5.1 Hz), 3.91 (s, 3H), 3.16–3.29 (m, 2H), 2.67–2.85 (m, 4H), 2.19–
2.35 (m, 1H), 1.9–2.15 (m, 6H), 1.72–1.9 (m, 1H), 1.26–1.4 (m, 6H);
ESIMS: m/z 775 (M+Na)+.
(2S)-N-{4-(5-[4-[(10-oxo-9,10-dihydro-9-anthracenyliden)methyl]phenoxy]pentyl)oxy-5-methoxy-2-nitrobenzoyl}-pyrrolidine-2-carboxaldehydediethylthioacetal (20c)
The compound 20c was prepared according to the method described for compound 20a by
employing (2S )- N -[4-hydroxy-5-methoxy–2-nitrobenzoyl] pyrrolidine-2-carboxal
dehydediethylthioacetal (15) (400 mg, 1 mmol), and 10-(4-(5-bromopentoxy)
benzylidene)anthracen-9(10H)-one (19c) (447 mg, 1 mmol). Yield (715 mg, 93%).
Mp: 124-125 ºC;
1H NMR (400 MHz, CDCl3): δ 8.18–8.31 (m, 2H), 7.98 (d, 1H, J = 7.3 Hz),
7.53–7.67 (m, 3H), 7.33–7.51 (m, 3H), 7.19–7.31 (m, 3H), 6.72–6.84 (m,
3H), 4.81 (d, 1H, J = 3.6 Hz), 4.6–4.71 (m, 1H), 4.18 (t, 2H, J = 5.1 Hz), 4.07
(t, 2H, J = 5.1 Hz), 3.91 (s, 3H), 3.18-3.28 (m, 2H), 2.66–2.85 (m, 4H), 2.17–
2.31 (m, 1H), 1.92–2.16 (m, 6H), 1.76–1.98 (m, 3H), 1.26–1.41 (m, 6H);ESIMS: m/z 767 (M+H)+.
(2S)-N-{4-(3-[2-methoxy-4-[(10-oxo-9,10-dihydro-9-anthracenyliden)methyl] phenoxy]propyl)oxy-5-methoxy-2-nitrobenzoyl}-pyrrolidine-2-carboxaldehyde diethylthioacetal(20d)
The compound 20d was prepared according to the method described for compound 20a by
employing (2S )- N -[4-hydroxy-5-methoxy–2-nitrobenzoyl]pyrrolidine-2-carboxal
dehydediethylthioacetal (15) (400 mg, 1 mmol), and 10-(4-(3-bromopropoxy)-3-
methoxybenzylidene)anthracen-9(10H)-one (19d) (449 mg, 1 mmol). Yield (700 mg, 91%).
Mp: 120-121 ºC;
1H NMR (300 MHz, CDCl3): δ 8.18–8.27 (m, 2H), 7.95 (d, 1H, J = 8 Hz), 7.7
(s, 1H), 7.53–7.65 (m, 2H), 7.32–7.49 (m, 3H), 7.21–7.32 (m, 1H), 6.72–6.88
(m, 4H), 4.80 (d, 1H, J = 3.6 Hz), 4.58–4.73 (m, 1H), 4.33 (t, 2H, J = 5.8 Hz),
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4.22 (t, 2H, J = 5.8 Hz), 3.92 (s, 3H), 3.64 (s, 3H), 3.14–3.26 (m, 2H), 2.63–
2.86 (m, 4H), 2.29–2.47 (m, 2H), 2.02–2.29 (m, 2H), 1.73–1.98 (m, 2H),
1.25–1.4 (m, 6H);
ESIMS: m/z 769 (M+H)+.
(2S)-N-{4-(4-[2-methoxy-4-[(10-oxo-9,10-dihydro-9-anthracenyliden)methyl] phenoxy]butyl)oxy-5-methoxy-2-nitrobenzoyl}-pyrrolidine-2-carboxaldehydediethyl thioacetal(20e)
The compound 20e was prepared according to the method described for compound 20a by
employing (2S )- N -[4-hydroxy-5-methoxy–2-nitrobenzoyl] pyrrolidine-2-carboxal
dehydediethylthioacetal (15) (400 mg, 1 mmol), and 10-(4-(4-bromobutoxy)-3-
methoxybenzylidene)anthracen-9(10H)-one (19e) (463 mg, 1 mmol). Yield (720 mg, 91%).
Mp: 119-122 ºC;
1H NMR (400 MHz, CDCl3): δ 8.23–8.3 (m, 2H), 8.02(d, 1 H, J = 8.1 Hz), 7.69
(s, 1 H), 7.6–7.68 (m, 2H), 7.45–7.54 (m, 2H), 7.38–7.44 (t, 1H, J = 6.7 Hz),
7.26–7.31 (m, 1H), 6.9–6.94 (m, 1H), 6.8–6.86 (m, 3H), 4.88 (d, 1H, J = 3.7
Hz), 4.67–4.75 (m,1H), 4.22 (t, 2H, J = 5.6 Hz), 4.14 (t, 2H, J = 5.6 Hz), 3.92
(s, 3H), 3.65 (s, 3H), 3.2–3.32 (m, 2H), 2.68–2.87 (m, 4H), 2.21–2.34 (m,
1H), 2.05–2.15 (m, 5H), 1.9–2.02 (m, 1H), 1.74–1.86 (m, 1H), 1.29–1.39 (m,
6H);ESIMS: m/z 783 (M+H)+.
(2 S )- N -{4-(5-[2-methoxy-4-[(10-oxo-9,10-dihydro-9-anthracenyliden)methyl]
phenoxy]pentyl)oxy-5-methoxy-2-nitrobenzoyl}-pyrrolidine-2-carboxaldehyde
diethylthioacetal(20f)
The compound 20f was prepared according to the method described for compound 20a by
employing (2S )- N -[4-hydroxy-5-methoxy–2-nitrobenzoyl] pyrrolidine-2-carboxal
dehydediethylthioacetal (15) (400 mg, 1 mmol), and 10-(4-(5-bromopentoxy)-3-
methoxybenzylidene)anthracen-9(10H)-one (19f) (477 mg, 1 mmol). Yield (751 mg, 94%).
Mp: 118-120 ºC;
1H NMR (300 MHz, CDCl3): δ 8.21–8.26 (m, 2H), 7.96 (d, 1H, J = 8.3 Hz),
7.57–7.65 (m, 3H), 7.37–7.48 (m, 3H), 7.23–7.28 (m, 1H), 6.85–6.91 (m,
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1H), 6.76–6.78 (m, 3H), 4.81 (d, 1H, J = 3.7 Hz), 4.63–4.69 (m, 1H), 4.02–
4.14 (m, 4H), 3.93 (s, 3H), 3.64 (s, 3H), 3.16–3.3 (m, 2H), 2.63–2.87 (m,
4H), 2.16–2.38 (m, 2H), 1.87–2.14 (m, 5H), 1.68–1.87 (m, 3H), 1.28–1.39
(m, 6H);
ESIMS: m/z 797 (M+H)+.
7-Methoxy-8-[3-{4-[(10-oxo-9,10-dihydro-9-anthracenyliden)methyl]phenoxy} propoxy]-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (22a)
To the compound 20a (738 mg, 1 mmol) in methanol (20 mL) was added
SnCl2.2H2O (1.12 g, 5 mmol) and reflux for 5 hrs and checked TLC indicated
the reaction was completed. The methanol was evaporated under vacuum
and the reaction mass was neutralized with 10% NaHCO3 solution and the
extracted with chloroform (2x30 mL). The combined organic phases was
dried over Na2SO4 and evaporated under vacuum to afford the crude
aminodiethylthioacetal 21a (652 mg, 91%), which was used directly in the
next step due to its potential stability problem.
A solution of 21a (708 mg, 1 mmol), HgCl2 (677 mg, 2.5 mmol) and
CaCO3 (250 mg, 2.5 mmol) in acetonitrile-water (4:1) was stirred slowly at
room temperature overnight until complete consumption of starting materialas indicated by the TLC. The clear organic supernatant liquid was extracted
with chloroform and washed with saturated 5% NaHCO3 (20 mL), brine (20
mL) and the combined organic phase was dried over Na2SO4. The organic
layer was evaporated in vacuum to afford crude solid, which was purified by
column chromatography with MeOH-CHCl3 (1:20) to obtain the pure product
22a. Yield (306 mg, 51%).
Mp: 107-109 ºC;
1H NMR (200 MHz, CDCl3): δ 8.19–8.3 (m, 2H), 7.98 (d, 1H, J = 8.3 Hz),
7.54–7.67 (m, 3H), 7.33–7.52 (m, 4H), 7.17–7.3 (m, 3H), 6.72–6.86 (m, 3H),
4.03–4.19 (m, 4H), 3.92 (s, 3H), 3.77–3.85 (m, 1H), 3.68–3.74 (m, 1H),
3.53–3.62 (m, 1H), 2.24–2.37 (m, 2H), 1.96–2.18 (m, 4H);
ESIMS: m/z 585 (M+H)+.
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7-Methoxy-8-[4-{4-[(10-oxo-9,10-dihydro-9-anthracenyliden)methyl]phenoxy}butoxy]-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (22b)
This compound was prepared according to the method described for the
compound 22a employing 20b (752 mg, 1 mmol) which reduction withSnCl2.2H2O (1.12 g, 5 mmol) gives amino compound 21b. Deprotection of
21b (722 mg, 1 mmol) with HgCl2 (677 mg, 2.5 mmol), CaCO3 (250 mg, 2.5
mmol) in acetonitrile-water (4:1) gives the pure product 22b. Yield (320 mg,
53%).
Mp: 106-108 ºC;
1H NMR (300 MHz, CDCl3): δ 8.18–8.29 (m, 2H), 7.98 (d, 1H, J = 8.3 Hz),
7.55–7.66 (m, 3H), 7.35–7.53 (m, 4H), 7.17–7.29 (m, 3H), 6.73–6.84 (m,
3H), 4.04–4.19 (m, 4H), 3.93 (s, 3H), 3.77–3.86 (m, 1H), 3.69–3.74 (m, 1H),
3.53–3.63 (m, 1H), 2.14–2.22 (m, 2H), 1.85–21.97 (m, 6H);
ESIMS: m/z 599 (M+H)+.
7-Methoxy-8-[5-{4-[(10-oxo-9,10-dihydro-9-anthracenyliden)methyl]phenoxy} pentoxy]-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (22c)
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 ºC;
1H NMR (300 MHz, CDCl3): δ 8.19–8.31 (m, 2H), 7.98 (d, 1H, J = 8.3 Hz),
7.55–7.67 (m, 3H), 7.34–7.53 (m, 4H), 7.16–7.3 (m, 3H), 6.73–6.86 (m, 3H),4.04–4.19 (m, 4H), 3.92 (s, 3H), 3.76–3.85 (m, 1H), 3.68–3.74 (m, 1H),
3.52–3.62 (m, 1H), 2.24–2.36 (m, 2H), 1.85–2.14 (m, 6H), 1.64−1.76 (m,
2H);
ESIMS: m/z 613 (M+H)+.
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T HESIS
7-Methoxy-8-[3-{2-methoxy-4-[(10-oxo-9,10-dihydro-9-anthracenyliden) methyl]phenoxy}propoxy]-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4] benzodiazepin-5-one(22d)
This compound was prepared according to the method described for the
compound 22a employing 20d (768 mg, 1 mmol) which reduction withSnCl2.2H2O (1.12 g, 5 mmol) gives amino compound 21d. Deprotection of
21d (738 mg, 1 mmol) with HgCl2 (677 mg, 2.5 mmol), CaCO3 (250 mg, 2.5
mmol) in acetonitrile-water (4:1) obtains the pure product 22d. Yield (335
mg, 54%).
Mp: 102-103 ºC;
1H NMR (200 MHz, CDCl3): δ 8.19–8.25 (m, 2H), 7.97 (d, 1H, J = 8.3 Hz),
7.55–7.67 (m, 3H), 7.32–7.51 (m, 4H), 7.24–7.3 (m, 1H), 6.83–6.91 (m, 1H),
6.72–6.81 (m, 3H), 4.08–4.13 (m, 4H), 3.91 (s, 3H), 3.76–3.85 (m, 1H),
3.68–3.72 (m, 1H), 3.63 (s, 3H), 3.52–3.6 (m, 1H), 2.26–2.36 (m, 2H), 1.99–
2.18 (m, 4H);
ESIMS: m/z 615 (M+H)+.
7-Methoxy-8-[4-{2-methoxy-4-[(10-oxo-9,10-dihydro-9-anthracenyliden) methyl]phenoxy}butoxy]-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4] benzodiazepin-5-one(22e)
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 ºC;
1H NMR (200 MHz, CDCl3): δ 8.21–8.26 (m, 2H), 7.96 (d, 1H, J = 8.3 Hz),7.55–7.66 (m, 3H), 7.31–7.48 (m, 4H), 7.23–7.31 (m, 1H), 6.83–6.9 (m, 1H),
6.74–6.8 (m, 3H), 4.06–4.13 (m, 4H), 3.92 (s, 3H), 3.76–3.86 (m, 1H), 3.66–
3.71 (m, 1H), 3.62 (s, 3H), 3.52–3.58 (m, 1H), 2.27–2.36 (m, 2H), 1.98–2.14
(m, 6H);
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13C NMR (75 MHz, CDCl3): δ 184.95, 189.9, 162.46, 148.95, 148.55, 140.67,
136.43, 133.11, 132.63, 132.43, 130.66, 129.25, 128.14, 127.49, 126.98,
126.79, 122.86, 122.5, 112.52, 111.46, 68.57, 68.36, 56.09, 55.69, 53.66,
46.95, 46.65, 33.25, 29.66, 25.91, 25.77, 24.16,
ESIMS: m/z 629 (M+H)+.
7-Methoxy-8-[5-{2-methoxy-4-[(10-oxo-9,10-dihydro-9-anthracenyliden) methyl]phenoxy}pentoxy]-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4] benzodiazepin-5-one(22f)
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 ºC;
1H NMR (200 MHz, CDCl3): δ 8.19–8.26 (m, 2H), 7.97 (d, 1H, J = 8.3 Hz),
7.54–7.67 (m, 3H), 7.31–7.51 (m, 4H), 7.24–7.31 (m, 1H), 6.84–6.91 (m,
1H), 6.73–6.81 (m, 3H), 4.07–4.15 (m, 4H), 3.91 (s, 3H), 3.76–3.86 (m, 1H),
3.68–3.71 (m, 1H), 3.63 (s, 3H), 3.52–3.61 (m, 1H), 2.26–2.35 (m, 2H),
1.96–2.15 (m, 6H), 1.64–1.75 (m, 2H);13C NMR (75 MHz, CDCl3): δ
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 7.00±0.01) containing CT-DNA, (100 µ M in phosphate) and the
PBD (20 µ M) were prepared by addition of concentrated PBD solutions in
methanol to obtain a fixed [PBD]/[DNA] molar ratio of 1:5 The DNA-PBD
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T HESIS
solutions were incubated at 37οC for 0 h prior to analysis sample were
monitored a 260 nm using a Beckman DU-7400 spectrophotometer fitted
with high performance temperature controller. Heating was applied at a rate
of 1
ο
C min-1 in the 40−90
ο
C range. DNA helix-coil transition temperatures(T m) were determined from the maxima in the d (A260)/dT derivative plots.
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 ∆ T m = T m (DNA+PBD)- T m (DNA alone), where
the T m value for the PBD free CT-DNA is 69.8 ± 0.001 the fixed [PBD]/[DNA]
ratio used did not result in binding saturation of the host DNA duplex for any
compound examined.
4.1.7. ANTICANCER ACTIVITY SCREENING
The synthesized compounds (22a-f ) were evaluated for their in vitro
anticancer activity in selected human cancer cell lines. A protocol of 48 hcontinuous drug exposure and a sulforhodamine B (SRB) protein assay was
used to estimate cell viability or growth. The cell lines were grown in RPMI
1640 medium containing 10% fetal bovine serum and 2 mML-glutamine,
and were inoculated into 96-well microtiter plates in 90 µL at plating
densities depending on the doubling time of individual cell lines. The
microtiter plates were incubated at 37οC, 5% CO2, 95% air and 100%
relative humidity for 24 h prior to addition of experimental drugs. Aliquots
of 10 µL of the drug dilutions were added to the appropriate microtiter
wells already containing 90 µL of cells, resulting in the required final drug
concentrations. Each compound was evaluated for four concentrations (0.1,
1, 10 and 100 µM) and each was done in triplicate wells. Plates were
incubated further for 48 h, and assay was terminated by the addition of 50
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T HESIS
µL of cold trichloro acetic acid (TCA) (final concentration, 10% TCA) and
plates were again incubated for 60 min at 4οC. The plates were washed
five times with tap water and air-dried. Sulforhodamine B (SRB) solution
(50 µL) at 0.4% (w/v) in 1% acetic acid was added to each of the wells, andplates were incubated for 20 min at room temperature. The residual dye
was removed by washing five times with 1% acetic acid. The plates were
air-dried. Bound stain was subsequently eluted with 10 mM trizma base,
and the absorbance was read on an ELISA plate reader at a wavelength of
540 nm with 690 nm reference wavelengths. Percent growth was
calculated on a plate-by-plate basis for test wells relative to control wells.
The above determinations were repeated three times.
4.1.8. REFERENCES:
1. Honore, S.; Pasquier, E.; Braguer, D. Understanding microtubule dynamics
for improved cancer therapy. Cell. Mol. LifeSci. 2005, 62, 3039.2. Pellegrini, F.; Budman, D. R. Review: tubulinfunction, actionofantitubulin
drugs, and newdrug development. Cancer Invest . 2005, 23, 264.
3. Hadfield, J. A.; Ducki, S.; Hirst, N.; McGown, A. T. Progress in Cell Cycle
Research, 2003, 5, 309.
4. Keme´ny, L.; Ruzicka, T.; Braun-Falco, O. Dithranol. Skin Pharmacol.
1990, 3, 1-20
5. Prinz, H., Ishii, Y., Hirano, T., Stoiber, T., Camacho Gomez, J.A., Schmidt,
P., Düssmann, H., Burger, A.M., Prehn, J.H., Günther, E.G., Unger, E.,
Umezawa, K. J. Med. Chem. 2003, 46, 3382.
6. Prinz, H.; Schmidt, P.; Bohm, K. J.; Baasner, S.; Muller, K.; Unger, E.;
Gerlach, M.; Gunther, E. G. J. Med. Chem. 2009, 52, 1284.
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T HESIS
7. Zuse, A., Schmidt, P., Baasner, S., Bohm, K.J., Muller, K., Gerlach, M.,
Gunther, E.G., Unger, E., Prinz, H., J. Med. Chem. 2006, 49, 7816.
8. Surkau, G.; Böhmb, K.; Müller, K.; Prinz, H. Eur. J. Med. Chem., 2010, 45,
3354.
9. Nickel, H. C.; Schmidt, P.; Böhmb, K. J.; Baasner, S.; Müller, K.; Gerlach, M.;
Unger, E.; Günther, E. G.; Prinz, H. Eur. J. Med. Chem., 2010, 45, 3420.
10. Keme´ny, L.; Ruzicka, T.; Braun-Falco, O. Dithranol, Skin Pharmacol.
1990, 3, 1-20.
11. Mtiller, K.; Reindl, H.; Gawlik, I. Eur. J. Med. Chem.1998, 33, 969.
12. Mtiller, K.; Sellmer, A.; Prinz, H. Eur J Med Chem, 1997, 32, 895.
13. Mtiller, K.; Altmannb, R.; Prinz, H. Eur J. Med. Chem. 1998, 33, 209.
14. Kratz, U.; Prinz, H, Müller, K. Eur J. Med. Chem. 2010, 45, 5278.
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.
CCHAPTERHAPTER-IV/S-IV/SECTIONECTION-B-B
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T HESIS
SS YNTHESIS YNTHESIS AANDND BBIOLOGICALIOLOGICAL EEVALUATIONVALUATION OOFF CCHALCONEHALCONE--PP YRROLOBENZODIAZEPINE YRROLOBENZODIAZEPINE DDIMERSIMERS AASS AANTICANCERNTICANCER AAGENTSGENTS
4.2. INTRODUCTION
Naturally occurring pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) have
attracted the attention of many researchers largely because of the potent
anticancer activity exhibited by most of these compounds bearing this ring
system. Some of the compounds of this class have undergone clinical
studies1,2, Apart from their anticancer activity, PBDs are of considerable
interest due to their ability to recognize and subsequently form covalent
bonds to specific base sequences of double strand DNA. They are
monofunctional alkylating agents, and have potential as gene regulators,
probes and as tools in molecular biology.3-5 The pyrrolo[2,1-c]
[1,4]benzodiazepines (PBDs) are a family of antitumour antibiotics derived
from various Streptomyces species6 and are generally referred to as the
anthramycin family, which comprise of some representative members like
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T HESIS
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. DNA-
binding ability has been observed by thermal denaturation studies with CT-
DNA (∆ T m > 15.1 °C for a 5:1 ratio of DNA:PBD at 37 °C for 18 h incubation).
Cross-linking efficiency has been investigated by using an agarose gel
electrophoresis assay. The results indicate that DSB-120 is an efficient cross-
linking agent. Furthermore, the in vitro cytotoxicity data in human K562 and
rodent ADJ-PC6 cell lines correlate with both the thermal denaturation data
and the cross-linking efficiencies. Recently, C2/C2’-exo-unsaturated C-8
linked PBD dimers
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T HESIS
N
N
O
HOH
H3CO
R1R2
N
HN
O
HH3C
OR
CONH2
OCH3
N
N
O
CH3
HO
H3CO
H
N
HN
O
CH3
HOH
O
OHO
N
HN
O
HOH
H3CO
OCH3
OH
O
OH
CH3H3C
H3CHN
OH
CH3
N
N
O
HOH
H3CO
5 ( R1 = H; R2 = OH)6 ( R1 = OH, R2 = H)
Figure 1. Naturally occurring PBDs
3
2
chicamycin
7
1R= OH or OCH3
anthramycin sibiromycin
tomamycin
4
neothramycin A and BDC-81
(SJG-136) have been synthesized which exhibit extraordinary DNA bindingaffinity and cytotoxicity.8 In recent years, a large number of hybrid molecules
containing the PBD ring system have been synthesized leading to novel
sequence selective DNA cross-linking agents.9 It is belived that interactions in
a manner different from those of other tubulin-binding antimitotic agents.
4.2.1. CHALCONES
Chemically chalcones comprise of open-chain flavonoids in which the
two aromatic rings are joined by a three-carbon α,β-unsaturated carbonyl
system. Chalcones, considered as the precursor of flavonoids and
isoflavonoids, are abundant in edible plants. However, most of the chalcones
are particularly attractive since it specifically generates the (E)-isomer from
substituted benzaldehydes and acetophenones. Recent studies revealed that
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T HESIS
these chalcones had shown a wide variety of anticancer,10-17 anti-
inflammatory,18-20 antiinvasive,21 antituberculosis,22 and antifungal23 activities.
Chalcones have shown promising anticancer therapeutic efficacy for the
management of human cancers. Recently, different chalcone analogues have
been synthesized and they have been screened for in vitro cytotoxicity
against a number of cancer cell lines.
The substituted chalcones have shown potential anticancer activity.
Ducki and co-workers have synthesized and reported trimethoxy substituted
chalcones24 (8) and (9), that possess potential anticancer activity and bind
strongly to tubulin at a site shared with, or close to, the colchicines binding
site.25-26 The anticancer activity and tubulin binding property of these
chalcones is comparable with combretastatin A-4 (CA-4). The IC50 value of
compound SD400 (9) against the K562 human chronic myelogenous
leukemia cell line is 0.21 nM whereas combretastatin A-4 (CA-4) shows the
IC50 is 2.0 nM. Presently phosphate prodrugs of these compounds (8) and (9)
are under preclinical evaluation. The compound (8) inhibits cell growth at
low concentrations (IC50, P388 murine leukaemia cell line 2.6 nM) and shares
many structural features common to other tubulin-binding agents27 (Figure
2).
MeO
MeO
O
OMe
OMe
OH MeO
MeO
O
OMeOMe
OH
9 (SD400)8
Figure 2
The anticancer activity of certain chalcones is believed to be a result of
binding to tubulin and preventing it from polymerizing into microtubules.
Tubulin is a protein that exists as a heterodimer of two homologous α- and
β -subunits. Many molecules based on a chalcone scaffold have been
synthesized to improve their biological profile, including their capability as
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T HESIS
sequence selective DNA interactive and cross-linking agents. The ease of
synthesis of chalcones from substituted benzaldehydes and acetophenones,
makes them an attractive scaffold. Chalcones have attracted more interest
in recent years because of their diverse pharmacological properties.28 Among
these properties, their cytotoxicity effects have been extensively examined.
Some of the natural chalcones have been found in a variety of plant sources.
These natural compounds have served as valuable leads for further design
and synthesis of more active analogues.29
Further, in this trimethoxy chalcone series different analogues have
been synthesized by different groups and evaluated for their cytotoxicity.
These compounds have shown promising activity against different cancer
cell lines.30 Curcumin, a polyphenolic natural compound (10) derived from
dietary spice turmeric, possesses diverse pharmacological effects including
anticancer, anti-inflammatory, antioxidant, and antiangiogenic activities.31 A
series of of chalcone dimers has been reported as potent inhibitors of various
cancer cells at very low concentrations. The compound 3,5-bis(2-
fluorobenzylidene)-4-piperidone (11, also known as EF24) is a synthetic
analog of curcumin that was first reported by Adams.32 Other analogues of
3,5-bis(benzylidene)-4-piperidones (12, CLEFMA) and (13) are have been
advanced as synthetic analogs of curcumin for anti-cancer activity and anti-
inflammatory properties and these dimers have shown promising
antiproliferative activity against various cancer cell lines33 (Figure 3).
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T HESIS
NH
O FF
N
O ClCl
O
OH
O12 (CLEFMA)
11 (EF24)
N
O
H3CO
HO
OCH3
OH
CH3
13
HO OH
OMeMeO
OOH
10 (curcumin)
Figure 3 The cyclic chalcones34, compounds (14, 15, 16 and 17) have been
shown potential anticancer activity against human cancer cell lines. These
compounds inhibit RNA and protein syntheses and induced apoptosis which
are likely major mechanisms whereby cytotoxicity is mediated. The active
compound (17) in these cyclic chalcones declines the mitochondrial function
as well as mitochondrial DNA damage (Figure 4).
O
N
MeO NO2
O
14 15
17
O
OMe
16
Figure 4
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T HESIS
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 andPBD entities with varying alkane spacers and have been evaluated them for
their antitumour activity and DNA-binding ability.
4.2.2.1. S YNTHESIS OF PBD PRECURSORS
The precursor (2S)-N-[4-(n-Bromoalkooxy)-5-methoxy-2-nitrobenzoyl]
pyrrolidine-2-carboxaldehydediethylthioacetal 19a-c have been prepared by
employing (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]pyrolidine-2-
carboxaldehydediethylthio acetal 18 which was prepared from commercially
available vanillic acid. Compound 18 undergoes etherification with
dibromoalkanes in the presence of K 2CO3 in acetone gives corresponding
compounds 19a-c (Scheme 1).
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T HESIS
HO
MeO
NO2
O
N
CH(SEt)2
18
O
MeO
NO2
O
N
CH(SEt)2Br ( )n
19a-c
n = 2-4
(i)
SCHEME 1. REAGENTS AND CONDITIONS: I) DIBROMOALKANE, K 2CO3, ACETONE, REFLUX,
14H
4.2.2.2. S YNTHESIS OF CHALCONE INTERMEDIATES
The preparation of dihydroxychalcone intermediates 22a,b has been
carried out by synthetic sequence illustrated in Scheme-2. Claisen-Schmidt
condensation of hydroxyacetophenones 20a,b with hydroxybenzaldehydes
21a,b by using ethanol as solvent in the presence of aqueous KOH gives
dihydroxychalcones 22a,b.
CH3
O CHO
+
O
R3
R4(i)
20a,b 21a,b22a,b
22a; R1 = H, R2 = OH, R3 = OH, R4 = H22b; R1 = OH, R2 = H, R3 = H, R4 = OH
R2
R1
R3
R4 R2
R1
20a; R1 = H, R2 = OH
20b; R1 = OH, R2 = H
21a; R3 = H, R4 = OH
21b; R3 = OH, R4 = H
SCHEME 2. REAGENTS AND CONDITIONS: I) AQ.KOH, ETHANOL, 24 H
4.2.2.3. S YNTHESIS OF C-8 LINKED CHALCONE-PBD DIMERS
Compound 19a-c has been coupled to dihydroxychalcones 22a,b in
the presence of K 2CO3 and dry acetone under reflux affords corresponding
nitro compounds 23a-f . These nitro compounds upon reduction with
SnCl2.2H2O in methanol under reflux give amino compounds 24a-f . The
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T HESIS
amino compounds on deprotection with HgCl2/CaCO3 provide corresponding
imines 25a-f (Scheme-3).
O
O O25a-c; Chalcone =
25d-f; Chalcone =
O
O O
n = 2-4
n = 2-4
O
22a; R = 4,4'-dihydroxy22b; R = 3,3'-dihydroxy
O
MeO
NO2
O
N
CH(SEt)2
19a-c
+
O
MeO
NO2
O
N
CH(SEt)2O ( )n
23a-f
24a-f
25a-f
(i)
(ii)
(iii)
Br ( )n
OO
OMeN
O
(EtS)2HC O2N( )n chalcone
O
MeO
NH2
O
N
CH(SEt)2O ( )nOO
OMeN
O
(EtS)2HC H2N( )n chalcone
O
MeO
O ( )nOO
OMe
( )n chalconeN
N N
N
O
H H
O
RR
Scheme 3. Reagents and conditions: (i) K 2CO3, acetone, reflux, 12h; (ii) SnCl2.2H2O, MeOH, 4 h,reflux; (iii) HgCl2, CaCO3, MeCN, H2O, (4:1) rt, 12 h
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T HESIS
4.2.3. BIOLOGICAL ACTIVITY
4.2.3.1. DNA BINDING AFFINITY : THERMAL DENATURATION STUDIES
The DNA binding affinity of these new C8-linked chalcone-PBD dimmers
(25a-f ) has been evaluated through thermal denaturation studies with
duplex-form of calf thymus DNA (CT-DNA) by using modified reported
procedure.35 The DNA-PBD solutions are incubated at 37οC for 0 h and 18 h
prior to analysis. Samples are monitored at 260 nm using a Beckman DU-
7400 spectrophotometer fitted with high performance temperature controller
and heated at 1ο
C/min in the range of 40-95ο
C. DNA helix-coil transitiontemperatures are given by: ∆ T m = T m(DNA+PBD)–T m(DNA alone), where the
T m value for the PBD-free CT-DNA is 69.8± 0.01. These studies were carried
out at PBD/DNA molar ratio 1:5. The increase in melting temperature (∆ T m)
for each compound is examined at 0 h and 18 h of incubation at 37οC.
Melting studies show that these compounds stabilize the thermal helix coil or
melting stabilization for the CT-DNA duplex at pH 7.0, and incubated at 37οC
with ligand / DNA molar ratio of 1:5. The increase in the helix melting
temperature (∆ T m) for each compound has been examined at 0 h and 18 h
incubation at 37οC.
Interestingly, all the PBD dimers elevate the helix melting temperature
of CT-DNA in the range of 3.5-5.4 oC. Compound 25a showed the highest ΔT m
of 4.8 oC at 0 h and increased upto 5.4 oC after 18 h incubation, whereas the
naturally occurring DC-81 exhibits a ΔT m of 0.7 oC after incubation under
similar conditions (Table 1). These results indicate that the effect on DNAbinding affinity by introducing the chalcone scaffold on PBD moiety through
different alkane spacers at C8-position of the DC-81.
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T HESIS
Table 1.Thermal denaturation data for chalcone-PBD dimers withcalf thymus (CT)-DNA
Compound[PBD]:[DNA]
molar ratiob
ΔT m (oC)a after incubation at 37 oCfor
0 h 18 h
25a 1:5 4.8 5.4
25b 1:5 4.6 4.9
25c 1:5 4.7 5.1
25d 1:5 4.1 4.8
25e 1:5 3.5 3.9
25f 1:5 4.5 4.7
DC-81 1:5 0.3 0.7
a For CT-DNA alone at pH 7.00 ± 0.01, T m = 68.5 0C Δ 0.01 (mean value from 10 separate
determinations), all ΔT m values are ± 0.1 - 0.2 0C. b For a 1:5 molar ratio of [PBD]/[DNA],
where CT-DNA concentration = 100 μM and ligand concentration = 20 μM in aqueous
sodium phosphate buffer [10 mM sodium phosphate + 1 mM EDTA, pH 7.00 ± 0.01].
4.2.3.2. ANTICANCER ACTIVITY
Compounds 25a-f have been evaluated for their in vitro cytotoxicity in
selected human cancer cell lines of colon, prostate, melanoma and lung by
using MTT assay method. The in vitro cytotoxicity results of these
compounds expressed in IC50 values which carried out the experiments at 10-
4 to 10-7 M concentrations and the data is illustrated in Table 2. The results
from these experiments reveal that compounds 25a-f showed IC50 values in
the range of 0.008-29.1 μM whereas DC-81 showed IC50 values in the range
of 2-26.2 μM. The synthesized novel chalcone-PBD dimers exhibited
significant anticancer activity against PC-3 human prostate cancer cell line
(IC50 range, 0.008−8.3 μM) compared to other cell lines HT-29, A-375, A-549
and B-16. Compound 25a exhibited strong effect against PC-3 (IC50, 0.008
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T HESIS
μM) and A-375 cell linec (IC50, 0.01 μM). The compound 25c also showed
significant activity against PC-3 cell line (IC50, 0.007 μM). Among the chalcone-
PBD dimers synthesized, the compounds having 4,4’- bonding of chalcone
showed superior activity compared to the compounds with 3,3’- bonding of
chalcone.
Table 2. IC50 valuesa (in μM) for compounds 25a-f in selected human cancercell lines.
CompoundIC50 values (μM)
HT-29b PC-3c A-375d A 549e B-16f
25a 10.4 0.008 0.01 19.0 29.1
25b 0.97 0.51 3.05 7.3 19.2
25c 15.8 0.007 0.97 8.9 18.6
25d 2.02 0.11 3 28.5 2.69
25e 26.4 5.1 8.3 -- 22.9
25f 23.5 8.3 27.8 11.7 --
DC-81 8.1 2 26.2 -- 21.1a 50% Growth inhibition and the values are mean of three determinations, b colon cancer, c
prostate cancer, d skin cancer, e lung cancer, f Mouse macrophages cell line.
4.2.4. CONCLUSION
A series of novel C8-linked chalcone-PBD dimers have been
synthesized and evaluated for its anticancer activity. These new analogues
exhibited significant anticancer activity against different cancer cell lines.
The DNA binding ability of these compounds carried out by thermal
denaturation of calf thymus (CT)-DNA. The thermal denaturation studies
have shown that these conjugates have better DNA binding ability when
compared to DC-81. Among these hybrids the compound 25a show superioranticancer activity as well as high DNA-binding ability (5.4
οC).
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4.2.5. EXPERIMENTAL SECTION
(2S)-N-[4-(3-Bromopropoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxa- ldehydediethylthioacetal (19a)
To a solution of compound 18 (400 mg, 1 mmol) in dry acetone (15 ml)
was added, anhydrous K 2CO3 (553 mg, 4 mmol), 1,3-dibromopropane (242
mg, 1.2 mmol) and the mixture was stirred at reflux temperature for 14 h.
The reaction was monitored by TLC using EtOAc-hexane (1:1), K 2CO3 was
removed by filtration and the solvent was evaporated under the vacuum,diluted with water and extracted with ethyl acetate. The combined organic
phases were dried (Na2SO4) and evaporated under vacuum and the residue
was purified by column chromatography (40% EtOAc-hexane) to afford
compound 19a as yellow liquid (418 mg, 94%).
1H NMR (200 MHz, CDCl3) δ 7.7 (s, 1H), 6.8 (s, 1H), 4.82-4.87 (d, 1H, J = 4.6
Hz), 4.63-4.75 (m, 1H), 3.98-4.25 (t, 2H, J = 6.8 Hz), 3.95 (s, 3H), 3.62-3.68
(t, 2H, J = 6.6 Hz), 3.2-3.35 (m, 2H), 2.6-2.9 (m, 4H), 1.7-2.5 (m, 6H), 1.2-1.4(m, 6H);
FABMS: 521 [M]+.
(2S)-N-[4-(4-Bromobutoxy-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxaldehyde diethylthioacetal (19b)
The compound 19b was prepared according to the method described
for compound 19a by employing compound 18 (400 mg, 1 mmol),
anhydrous K 2CO3 (553 mg, 4 mmol) and 1, 4-dibromopropane (256 mg, 1.2
mmol). Yield (440 mg, 96%).
1H NMR (200 MHz, CDCl3) δ 7.65 (s, 1H), 6.8 (s, 1H), 4.82-4.87 (d, 1H, J = 4.6
Hz), 4.6-4.71 (m, 1H), 3.98-4.1 (t, 2H, J = 6.7 Hz), 3.95 (s, 3H), 3.52-3.59 (t,
2H, J = 6.8 Hz), 3.15-3.3 (m, 2H), 2.6-2.9 (m, 4H), 1.7-2.4 (m, 8H), 1.21-1.45
(m, 6H);
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FABMS: 535 [M]+.
(2S)-N-[4-(5-Bromopentyloxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxaldehydediethylthioacetal (19c)
The compound 19c was prepared according to the method described
for compound 19a by employing compound 18 (400 mg, 1 mmol),
anhydrous K 2CO3 (553 mg, 4 mmol) and 1, 5-dibromopropane (270 mg, 1.2
mmol). Yield (440 mg, 96%).
1H NMR (200 MHz, CDCl3) δ 7.65 (s, 1H), 6.8 (s, 1H), 4.82-4.87 (d, 1H, J =
4.45 Hz), 4.6-4.71 (m, 1H), 3.98-4.15 (t, 2H, J = 6.57 Hz), 3.95 (s, 3H), 3.52-
3.6 (t, 2H, J = 6.38 Hz), 3.15-3.3 (m, 2H), 2.65-2.85 (m, 4H), 1.6-2.4 (m, 8H),
1.21-1.4 (m, 6H);
FABMS: 549 [M]+
(E)-1,3-bis(4-hydroxyphenyl)prop-2-en-1-one (22a)
To a stirred mixture of 4-hydroxyacetophenone 20a (136 mg, 1 mmol) and4-hydroxybenzaldehyde 21a (122 mg, 1 mmol) in ethanol (10 ml) was added
50% aqueous solution of potassium hydroxide (1 ml) and stirred for 24 h at
room temperature. After completion of the reaction checked by TLC, the
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: 178–179 oC
1H NMR (200 MHz, CDCl3): δ 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)+.
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(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: 181–183 oC
1H NMR (300 MHz, CDCl3 + DMSOd6): δ 9.37 (s, 1H), 9.24 (s, 1H), 7.77 (s,
1H), 7.56 (d, 1H, J = 15.67 Hz), 7.32−7.45 (m, 2H), 7.24 (t, 1H, J = 7.93, 7.55
Hz), 7.15 (t, 1H, J = 7.43, 7.43 Hz), 701−7.09 (m, 2H), 6.93−7 (dd, 1H, J = 8.3,
2.4 Hz), 6.76−7.83 (dd, 1H, J = 8.3, 1.88 Hz);
ESIMS: m/z 241 (M+H)+.
(E)-1,3-bis[4-{3-[(2S)-N-(4-oxy-5-methoxy-2-aminobenzoyl)pyrrolidine-2-carboxaldehydediethylthioacetal]propoxy}phenoxy]prop-2-en-1-one(23a)
To a solution of (2S)-N-[4-(3-Bromopropoxy)-5-methoxy-2-nitrobenzoyl]
pyrrolidine-2-carboxaldehydediethylthioacetal (19a) (1.145 g, 2.1 mmol) in
dry acetone (15 mL) was added, anhydrous K 2CO3 (552 mg, 4 mmol), (E)-1,3-
bis(4-hydroxyphenyl)prop-2-en-1-one (22a) (240 mg, 1 mmol) and themixture was stirred at reflux temperature for 12 hours. The reaction was
monitored by TLC using ethyl acetate-hexane (2:1). After completion of the
reaction as indicated by the TLC, K 2CO3 was removed by filtration and the
solvent evaporated under reduced pressure, diluted with water and
extracted with ethyl acetate. The organic phase was dried over Na2SO4 and
evaporated under vacuum. The residue, thus obtained was purified by
column chromatography using ethyl acetate and hexane (2:1) to afford
compound 23a as yellow solid. Yield (851 mg, 75%)
Mp: 207–209 oC
1H NMR (200 MHz, CDCl3): δ 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),
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4.65−4.76 (m, 2H), 4.09−4.22 (m, 8H), 3.91 (s, 6H), 3.16−3.32 (m, 4H),
2.64−2.87 (m, 8H), 2.21−2.34 (m, 2H), 2.08−2.19 (m, 6H), 1.91−2.03 (m, 2H),
1.72−1.86 (m, 2H), 1.29−1.41 (m, 12H);
ESIMS: m/z 1122 (M+H)+
.
(E)-1,3-bis[4-{4-[(2S)-N-(4-oxy-5-methoxy-2-aminobenzoyl)pyrrolidine-2-carboxaldehydediethylthioacetal]butoxy}phenoxy]prop-2-en-1-one(23b)
The compound 23b was prepared according to the method described
for compound 23a by employing (2S)-N-[4-(4-Bromobutoxy)-5-methoxy-2-
nitrobenzoyl] pyrrolidine-2-carboxaldehydediethylthioacetal (19b) (1.175 g,
2.1 mmol), and (E)-1,3-bis(4-hydroxyphenyl)prop-2-en-1-one (22a) (240 mg,
1 mmol). Yield (912 mg, 79%)
Mp: 205–207 oC
1H NMR (200 MHz, CDCl3): δ 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), 1.90−2 (m, 2H),
1.72−1.87 (m, 2H), 1.29−1.39 (m, 12H); ESIMS: m/z 1149 (M+H)+.
(E)-1,3-bis[4-{5-[(2S)-N-(4-oxy-5-methoxy-2-aminobenzoyl)pyrrolidine-2-carboxaldehydediethylthioacetal]pentoxy}phenoxy]prop-2-en-1-one (23c)
The compound 23c was prepared according to the method described for
compound 23a by employing (2S)-N-[4-(5-Bromopentoxy)-5-methoxy-2-
nitrobenzoyl] pyrrolidine-2-carboxaldehydediethylthioacetal (19c) (1.21 g,
2.1 mmol), and (E)-1,3-bis(4-hydroxyphenyl)prop-2-en-1-one (22a) (240 mg,
1 mmol). Yield (930 mg, 78%)
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Mp: 208–209 oC
1H NMR (200 MHz, CDCl3): δ 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-2-aminobenzoyl)pyrrolidine-2-carboxaldehydediethylthioacetal]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-2-
nitrobenzoyl] pyrrolidine-2-carboxaldehydediethylthioacetal (19a) (1.145 g,
2.1 mmol), and (E)-1,3-bis(3-hydroxyphenyl)prop-2-en-1-one (22b) (240 mg,
1 mmol). Yield (850 mg, 75%)
Mp: 214–216 oC
1H NMR (500 MHz, CDCl3): δ 7.67−7.74 (m, 3H), 7.57 (d, 1H, J = 7.9 Hz), 7.55
(d, 1H, J = 14.8 Hz), 7.46 (d, 1H, J = 14.8 Hz), 7.37 (t, 1H, J = 7.9 Hz), 7.29
(t, 1H, J = 7.9 Hz), 7.17 (d, 1H, J = 1.9 Hz), 7.08−7.13 (dd, 1H, J = 7.9, 1.9
Hz), 6.91−6.96 (dd, 1H, J = 7.9, 1.9 Hz), 6.77 (s, 1H), 6.76 (s, 1H); 4.81 (d, 2H,
J = 3.9 Hz), 4.62−4.7 (m, 2H), 4.19−4.36 (m, 8H), 3.92 (s, 6H), 3.13−3.29 (m,
4H), 2.64−2.86 (m, 8H), 2.31−2.41 (m, 4H), 2.21−2.3 (m, 2H), 2.02−2.12 (m,
2H), 1.87−1.99 (m, 2H), 1.28−1.4 (m, 12H);
ESIMS: m/z 1121 (M+H)+.
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(E)-1,3-bis[3-{4-[(2S)-N-(4-oxy-5-methoxy-2-aminobenzoyl)pyrrolidine-2-carboxaldehydediethylthioacetal]butoxy}phenoxy]prop-2-en-1-one(23e)
The compound 23e was prepared according to the method described for
compound 23a by employing (2S)-N-[4-(4-Bromobutoxy)-5-methoxy-2-
nitrobenzoyl] pyrrolidine-2-carboxaldehydediethylthioacetal (19b) (1.175 g,
2.1mmol), and (E)-1,3-bis(3-hydroxyphenyl)prop-2-en-1-one (22b) (240 mg,
1 mmol). Yield (910 mg, 79%)
Mp: 215–217 oC
1H NMR (400 MHz, CDCl3): δ 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), 6.91−6.95 (dd, 1H, J = 7.9, 1.9 Hz), 6.76 (s, 6H), 4.82 (d, 2H, J = 3.9 Hz),
4.62−4.71 (m, 2H), 4.17−4.34 (m, 8H), 3.91 (s, 6H), 3.13−3.28 (m, 4H),
2.64−2.81 (m, 8H), 2.22−2.34 (m, 4H), 2.01−2.13 (m, 8H), 1.91−2 (m, 2H),
1.73−1.87 (m, 2H), 1.28−1.39 (m, 12H);
ESIMS: m/z 1150 (M+H)+.
(E)-1,3-bis[3-{5-[(2S)-N-(4-oxy-5-methoxy-2-aminobenzoyl)pyrrolidine-2-carboxaldehydediethylthioacetal]pentoxy}phenoxy]prop-2-en-1-one(23f)
The compound 23f was prepared according to the method described for
compound 23a by employing (2S)-N-[4-(5-Bromopentoxy)-5-methoxy-2-
nitrobenzoyl] pyrrolidine-2-carboxaldehydediethylthioacetal (19c) (1.21 g,
2.1 mmol), and (E)-1,3-bis(3-hydroxyphenyl)prop-2-en-1-one (22b) (240 mg,
1 mmol). Yield (925 mg, 77%)Mp: 213–215 oC
1H NMR (200 MHz, CDCl3): δ 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
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Hz), 6.9−6.96 (dd, 1H, J = 7.9, 1.9 Hz), 6.75 (s, 6H), 4.82 (d, 2H, J = 3.9 Hz),
4.63−4.72 (m, 2H), 4.15−4.34 (m, 8H), 3.92 (s, 6H), 3.13−3.29 (m, 4H),
2.63−2.8 (m, 8H), 2.21−2.35 (m, 4H), 2.04−2.12 (m, 8H), 1.89−1.95 (m, 2H),
1.72−1.86 (m, 6H), 1.27−1.39 (m, 12H);
ESIMS: m/z 1178 (M+H)+.
(E)-1,3-bis[4-{3-[7-Methoxy-8-oxy-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4] benzodiazepin-5-one]propyloxy}phenoxy]prop-2-en-1-one (25a)
To the compound 23a (1121 mg, 1 mmol) in methanol (20 mL) was
added SnCl2.2H2O (2.24, 10 mmol) and reflux for 5 hrs and checked TLC
indicated the reaction was completed. The methanol was evaporated under
vacuum and the reaction mass was neutralized with 10% NaHCO3 solution
and the extracted with ethyl acetate and chloroform (2x30mL and 2x30mL).
The combined organic phases was dried over Na2SO4 and evaporated under
vacuum to afford the crude aminodiethylthioacetal 24a (960 mg, 90%),
which was used directly in the next step due to its potential stability
problem.
A solution of 24a (1061 mg, 1.0 mmol), HgCl2 (1.35 g, 5 mmol) and
CaCO3 (500 mg, 5 mmol) in acetonitrile-water (4:1) was stirred slowly at
room temperature overnight until complete consumption of starting
material as indicated by the TLC. The clear organic supernatant liquid was
extracted with ethyl acetate and washed with saturated 5% NaHCO3 (20
mL), brine (20 mL) and the combined organic phase was dried over Na2SO4.
The organic layer was evaporated in vacuum to afford a yellow solid, which
was purified by column chromatography with MeOH-CHCl3 (1:20) to obtain
the pure product 25a. Yield (411 mg, 51%).
Mp: 191–193 oC
1H NMR (200 MHz, CDCl3): δ 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
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(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-5H-pyrrolo[2,1-c][1,4] benzodiazepin-5-one]butyloxy}phenoxy]prop-2-en-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: 190–192 oC
1H NMR (200 MHz, CDCl3): δ 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);
13C NMR (75 MHz, CDCl3): δ 188.68, 164.55, 162.38, 160.85, 150.65, 147.74, 143.73,
140.5, 131.17, 130.61, 130.03, 127.65, 120.17, 119.42, 114.79, 114.16, 111.54, 110.4, 68.46,
67.6, 56.05, 53.65, 46.61, 29.61, 25.88, 25.56, 24.1, 22.56
ESIMS: m/z 841 (M+H)+.
(E)-1,3-bis[4-{5-[7-Methoxy-8-oxy-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4] benzodiazepin-5-one]pentyloxy}phenoxy]prop-2-en-1-one (25c)
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.
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Deprotection followed by cyclization of 24c (1117 mg, 1 mmol) with HgCl2
(1.35 g, 5 mmol), CaCO3 (500 mg, 5 mmol) in acetonitrile-water (4:1) to
obtain the pure product 25c. Yield (490 mg, 56%).
Mp: 191–192o
C1H NMR (200 MHz, CDCl3): δ 8 (d, 2H, J = 9.06 Hz), 7.72 (d, 1H, J = 15.1 Hz),
7.61 (d, 2H, J = 4.53 Hz), 7.57 (d, 2H, J = 9.06 Hz), 7.45 (s, 2H), 7.39 (d, 1H, J
= 15.1 Hz), 6.92 (d, 2H, J = 9.06 Hz), 6.87 (d, 2H, J = 9.06 Hz), 6.74 (s, 2H),
4.07−4.23 (m, 8H), 3.92 (s, 6H), 3.75−3.85 (m, 2H), 3.67−3.73 (m, 2H),
3.52−3.62 (m, 2H), 2.24−2.37 (m, 4H), 1.93−2.14 (m, 12H), 1.63−1.75 (m, 4H);
ESIMS: m/z 869 (M+H)+.
(E)-1,3-bis[3-{3-[7-Methoxy-8-oxy-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4] benzodiazepin-5-one]propoxy}phenoxy]prop-2-en-1-one (25d)
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: 187–189 oC
1H NMR (300 MHz, CDCl3): δ 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), 6.91−6.96 (dd, 1H, J = 7.9, 2.1 Hz), 6.79 (s, 2H),
4.07−4.23 (m, 8H), 3.92 (s, 6H), 3.73−3.84 (m, 2H), 3.65−3.72 (m, 2H),3.51−3.63 (m, 2H), 2.24−2.39 (m, 4H), 1.98−2.13 (m, 8H);
13C NMR (75 MHz, CDCl3): δ 190.13, 181.9, 164.57, 162.43, 159.11, 150.59, 147.75,
144.71, 140.49, 139.46, 136.17, 129.91, 129.56, 122.27, 121.25, 121.09, 120.28, 119.59, 116.76,
114.05, 113.62, 111.55, 110.55, 65.36, 64.44, 56.09, 53.67, 46.64, 29.64, 28.97, 24.14,
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T HESIS
ESIMS: m/z 813 (M+H)+.
(E)-1,3-bis[3-{4-[7-Methoxy-8-oxy-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4] benzodiazepin-5-one]butoxy}phenoxy]prop-2-en-1-one (25e)
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: 186–188 oC
1H NMR (200 MHz, CDCl3): δ 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), 6.91−6.95 (dd, 1H, J = 7.9, 1.9 Hz), 6.76 (s, 6H),
4.06−4.23 (m, 8H), 3.93 (s, 6H), 3.76−3.85 (m, 2H), 3.66−3.74 (m, 2H),
3.52−3.63 (m, 2H), 2.24−2.38 (m, 4H), 1.94−2.15 (m, 12H);
ESIMS: m/z 841 (M+H)+.
(E)-1,3-bis[3-{5-[7-Methoxy-8-oxy-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4] benzodiazepin-5-one]pentoxy}phenoxy]prop-2-en-1-one (25f)
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: 187–188 oC
1H NMR (300 MHz, CDCl3): δ 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
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T HESIS
(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), 6.9−6.96 (dd, 1H, J = 7.9, 1.9 Hz), 6.75 (s, 6H),
4.07−4.23 (m, 8H), 3.92 (s, 6H), 3.75−3.85 (m, 2H), 3.67−3.73 (m, 2H),
3.52−3.62 (m, 2H), 2.24−2.37 (m, 4H), 1.93−2.14 (m, 12H), 1.63−1.75 (m, 4H);
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 7.00±0.01) containing CT-DNA, (100 µ M in phosphate) and the
PBD (20 µ M) were prepared by addition of concentrated PBD solutions in
methanol to obtain a fixed [PBD]/[DNA] molar ratio of 1:5 The DNA-PBD
solutions were incubated at 37οC for 0 h prior to analysis sample were
monitored a 260 nm using a Beckman DU-7400 spectrophotometer fitted
with high performance temperature controller. Heating was applied at a rate
of 1
ο
C min-1 in the 40−90
ο
C range. DNA helix-coil transition temperatures(T m) were determined from the maxima in the d (A260)/dT derivative plots.
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 ∆ T m = T m (DNA+PBD)- T m (DNA alone), where
the T m value for the PBD free CT-DNA is 69.8 ± 0.001 the fixed [PBD]/[DNA]
ratio used did not result in binding saturation of the host DNA duplex for any
compound examined.
4.2.6. PROCEDURE FOR MTT-ASSAY
Toxicity of test compound in cells was determined by MTT assay based
on mitochondrial reduction of yellow MTT tetrazolium dye to a highly colored
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T HESIS
blue formazan product. 1x104 Cells (counted by Trypan blue exclusion dye
method) in 96-well plates were incubated with compounds with series of
concentrations tested for 48 hrs at 37 oC in RPMI/DMEM/MEM with 10% FBS
medium. Then the above media was replaced with 90 µl of fresh serum free
media and 10 µl of MTT reagent (5mg/ml) and plates were incubated at 37 oC
for 4 h, there after the above media was replaced with 200 µl of DMSO and
incubated at 37 oC for 10 min. The absorbance at 570 nm was measured on a
spectrophotometer (spectra max, Molecular devices) IC50 values were
determined from plot: % inhibition (from control) versus concentration.
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T HESIS
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23. Lopez, S. N.; Castelli, M. V.; Zacchino, S. A.; Dominguez, J. N.; Lobo, G.;
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Kendall, D. Experientia 1989, 45, 209.
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31. (a) Aggarwal, B. B.; Kumar, A; Bharti, A. C. Anticancer potential of
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T HESIS
LIST OF PUBLICATIONS AND PATENTS
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T HESIS
LIST OF PUBLICATIONS
1. Synthesis of a new 4-aza-2,3-didehydropodophyllotoxin analogues as
potent cytotoxic and antimitotic agents.
Ahmed Kamal, Paidakula Suresh, Adla Malla Reddy, Banala Ashwini
Kumar, Papagari Venkat Reddy, Paidakula Raju, Jaki R. Tamboli,
Thokhir B. Shaik, Nishant Jain, Shasi V. Kalivendi
Bioorganic & Medicinal Chemistry , 19, (2011) 2349-2358.
2. Synthesis of new 4β -Acrylamidopodophyllotoxin Congeners as DNA
Strands Breakage Agents.
Ahmed Kamal,*, Paidakula Suresh, M. Janaki Ramaiah, Adla Malla
Reddy, Banala Ashwini Kumar, Paidakula Raju, Vinay Gopal, S.N.C.V L.
Pushpavalli, Pranjal Sarma, Manika Pal-Bhadra,*
Bioorganic & Medicinal Chemistry (accepted)
3. Anti-tubercular agents. Part 5: Synthesis and biological evaluation of
benzothiadiazine 1,1-dioxide based congeners.
Ahmed Kamal*, Rajesh V.C.R.N.C. Shetti, Shaik Azeeza, S. Kaleem
Ahmed, P. Swapna, A. Malla Reddy, Inshad Ali Khan, Sandeep
Sharma, Sheikh Tasduq Abdullah
European Journal of Medicinal Chemistry 45 (2010) 4545-4553.
4. Sulfamic acid as an efficient and recyclable catalyst for the ring
opening of epoxides with amines and anilines: An easy synthesis of -
amino alcohols under solvent-free conditions.
Ahmed Kamal *, B. Rajendra Prasad, A. Malla Reddy, M. Naseer A.
Khan
Catalysis Communications 8 (2007) 1876–1880
5. Synthesis and Anticancer Activity of Chalcone-pyrrolobenzodiazepine
Conjugates Linked via 1,2,3-triazole Ring Side-armed with Alkane
Spacers.
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T HESIS
Ahmed Kamal, S. Prabhakar, M. Janaki Ramaiah, P. Venkat Reddy, Ch.
Ratna Reddy, A. Mallareddy, Nagula Shankaraiah, T. Lakshmi
Narayan Reddy, S.N.C.V.L. Pushpavalli, Manika Pal-Bhadra
European Journal of Medicinal Chemistry (in press)
6. Carbazole-pyrrolo[2,1-c][1,4]benzodiazepine conjugates: Design,
synthesis and biological evaluation.
Ahmed Kamal*, Rajesh VCRNC Shetti, M.Janaki Ramaiah, P.Swapna,
M.P.Narasimha Rao, A.Malla Reddy, S.N.C.V.L. Pushpavalli, Manika
Pal-Bhadra.
Medicinal Chemistry Communications (accepted).
7. An efficient approach for the preparation of bioactive moleculesemploying Paal-Knorr protocol.
Ahmed Kamal, Rajesh.V.C. R. N. C. Shetti, P. Swapna, M. P. Narasimha
Rao, A. Malla Reddy, M. Rafiq H. Siddiqui, Abdullah Alarifi
(communicated to Synlett )
8. Design, Synthesis and Anticancer Evaluation of Carbazole-
Benzothiazole Conjugates.
Ahmed Kamal*, Rajesh VCRNC Shetti, M.Janaki Ramaiah, P.Swapna,M.P.Narasimha Rao, A.Malla Reddy, H.K.Srivastava, G.Narahari
Sastry, Manika Pal-Bhadra (to be communicated to Arch. de Pharm).
LIST OF PATENTS
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T HESIS
1. Benzylidineanthracenone linked pyrrolobenzodiazepine hybrids useful
as anti cancer agents and the process for preparation thereof.
Ahmed Kamal, A Malla Reddy, P Suresh and Rajesh VCRNC Shetti
Indian patent application no. 2886/DEL/2010
USA patent application no. 13/048248
2. Benzothiazole hybrids useful as potential anticancer agents and
process for the preparation thereof
Ahmed Kamal, A Malla Reddy, P Suresh, Rajesh V C R N C Shetti,
Harish Chandra Pal and Ajit Kumar Saxena
Indian patent application no. 270/DEL/2011.
3. Amidobenzothiazole analogues useful as potential anticancer agentsand process for the preparation thereof
Ahmed Kamal, A Malla Reddy, P Suresh, N Sankara Rao and Rajesh
VCRNC Shetti.
Indian patent application no. 266/DEL/08.
PCT application no. PCT/IN2011/000187
4. Synthesis of new benzothiazole derivatives as potential anti-tubercular
agentsAhmed Kamal, Rajesh V C R N C Shetti, P Swapna, Shaik Azeeza, A
Malla Reddy, Inshad Ali Khan, Sheikh Tasduq Abdulla, Sandeep
Sharma and Nitin pal Kalia
Indian patent application no. 2179/DEL/2010
5. Biological evaluation of 4-aza-2,3-didehydropodophyllotoxin analoguespossessing potent antitumour activity
Ahmed Kamal, P Suresh, B Ashwini Kumar, A Malla Reddy, P. VenkatReddy and Jaki Rasheed Tamboli
Indian patent application no. 2887/DEL/2010
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T HESIS
6. Pyrrolo[2,1-c][1,4] benzodiazepine conjugates linked through
piperazine moiety as potential antitumour agent and process for the
preparation thereof
Ahmed Kamal, Rajesh V C R N C Shetti, K Srinivasa Reddy, A Malla
Reddy and P Swapna
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 PVenkat 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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T HESIS
S YMPOSIUM AND CONFERENCES ATTENDED
1. Participated in 12th CRSI National Symposium in Chemistry & 4th CRSI-
RSC Symposium in Chemistry from 4-7 of February, 2010 at NIPER &
IICT, Hyderabad, India.
2. Participated in 13th ISCB International Conference on “Interplay of Chemical and
Biological Sciences: Impact on Health and Environment” at New Delhi, India on 26 th