SYNOPSIS
SSYYNNOOPPSSIISS
I
The work carried in the research tenure has been compiled in the form of a
thesis entitled “Synthesis and anticancer activity of heterocycles based on
chalcone and 2-anilinopyridine/pyrazole/isoxazole congeners as tubulin
inhibitors”. The main aim of this work is design and synthesis of biologically active
imidazopyridine-oxindole conjugates, imidazothiadiazole/isoxazole chalcone
derivatives and aryl substituted 2-anilino-pyridine/pyrazole/isoxazole congeners
have been prepared and evaluated for their anticancer activity and inhibition of
tubulin polymerization. The thesis has been divided into four chapters.
� CHAPTER I: This chapter gives the general introduction about cancer
chemotherapy, tubulin polymerization inhibitors, particularly of colchicines,
combretastatin and benzene sulfonamides as microtubule inhibitors and the
objectives of the present work.
� CHAPTER II: This chapter describes the synthesis of imidazopyridine-oxindole
analogues evaluation of their anticancer activity against a human cancer cell lines.
This chapter is mainly focused on the mitochondrial membrane depolarization and
inhibition of tubulin polymerization has been investigated.
�� CHAPTER III (SECTION-A): This chapter deals with the synthesis of new
imidazothiadiazole-chalcones conjugates. This chapter is mainly focused on the
mitochondrial mediated apoptotic pathway responsible for the significant
anticancer activity.
�� CHAPTER III (SECTION-B): This chapter illustrates design, synthesis and
biological evaluation of isoxazole-chalcones as potent anticancer agents. Further,
these compounds have been evaluated for their anticancer activity and cell cycle
effects.
SYNOPSIS
SSYYNNOOPPSSIISS
II
�� CHAPTER IV (SECTION-A): This chapter deals with the new synthesis and
biological evaluation of aryl substituted 2-anilinopridine derivatives as potent
anticancer agents. Further, these compounds have been evaluated for their
antitubulin activity.
�� CHAPTER IV (SECTION-B): This chapter describes the synthesis and
biological evaluation of aryl substituted pyrazole/isoxazole congeners. This present
chapter is mostly focused on the antiproliferative activity and inhibition of tubulin
polymerization.
GENERAL INTRODUCTION
This chapter describes the general introduction about cancer, tubulin
inhibitors and the objectives of the present work..
TUBULIN POLYMERIZATION INHIBITORS
Cancer is a group of diseases characterized by uncontrolled growth or
spread of abnormal cells. Since it involves the conversion of any normal cells to a
cancerous cell showing tandem replication and cell divisions at much faster rate in
comparison to the normal cells and thus provides a potential target area for the
development of chemotherapeutic agents. It is now clear that chemotherapy’s most
effective role in solid tumors is as an adjuvant to initial therapy by surgical or radio
therapeutic procedures. Chemotherapy becomes critical to effective treatment
because only systemic therapy can attack micro metastases. These agents can be
categorized into functional sub groups: alkylating agents, antimetabolites,
antibiotics and antimitotics.
Microtubules are protein biopolymers formed through polymerization of
heterodimers of α and β-tubulin. Tubulin polymerization is reversible and the
CHAPTER-I
SSYYNNOOPPSSIISS
III
dynamic assembly and disassembly of microtubules are involved in a number of
cell functions, including cell division, migration and shape change. There are many
natural synthetic compounds targeting the tubulin−microtubule system was
reported.
On the basis of their effects on microtubule assembly, microtubule-targeted
drugs are generally classified as either a polymerizing agent or a depolymerizing
agent. The class of polymerizing agents includes paclitaxel (Fig. 1), docetaxel, and
polyisoprenyl benzophenones and these promote microtubule assembly and
stabilize microtubules. The class of depolymerizing agents includes, vinblastine,
colchicines, combretastatin A-4 and E7010 etc., inhibits polymerization of tubulin.
Figure 1. Structures of tubulin inhibitors.
SSYYNNOOPPSSIISS
IV
.
“Synthesis and Biological Evaluation of Imidazopyridine-Oxindole Conjugates as
Microtubule Targeting Agents.”
Chemotherapy is often the treatment of choice for many types of cancer as a
result the search for newer chemotherapeutic agents still plays a major role in the
fight against cancer. 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
conjugates, in which a known antitumor compounds or some simple active
pharmacophore moiety tethered to each other, have been designed, synthesized
and evaluated for their biological activity. Recently, Kamal and co-workers have
synthesized imidazopyridine/imidazopyrimidine- chalcone derivatives as potential
antitumour agents and a correlation between antitumour activity and apoptosis has
been well explained.
In continuation of these efforts, we have synthesized imidazopyridine-
oxindole derivatives. This led to a new library of conjugates that were evaluated for
their anticancer potential. In view of their promising activity, we decided to
investigate their role towards cytotoxicity, cell-cycle effects, and apoptosis by using
the human breast cancer cell lines (MCF-7). Therefore this chapter describes the
synthesis, anticancer activity, mitochondrial membrane depolarization ability and
inhibition percentage of tubulin polymerization of some new analogues. Further
apoptosis associated Western blot analysis of Akt (ser-473) was carried out to
further understand the underlying mechanisms and pathways involved.
CHAPTER-II
SSYYNNOOPPSSIISS
V
SYNTHESIS OF IMIDAZOPYRIDINE-OXINDOLE CONJUGATES
The imidazopyridine-oxindole conjugates (7a-t) were synthesized by
employing Knoevenagel reaction between equimolar mixture of substituted
imidazo[1,2-a]pyridine aldehydes (5a–e) and oxindoles (6a-e). The compound
structures were confirmed by means of 1H NMR, 13C NMR, HRMS and IR spectra.
All the compounds were obtained in pure E- isomeric forms and were in confimity
to the previously reported literature. The key intermediate, imidazo[1,2-a]pyridine
aldehydes (5a–e) were prepared by means of Vilsmeier - Haac reaction on the
corresponding imidazo[1,2-a]pyridine (4a–e) that were inturn obtained by the
reaction between 2-aminopyridine (2) and substituted 2-bromo-1-phenylethanones
(1a-e).The sequential steps of these reactions are outlined in Scheme 1.
Scheme 1. Reagents & conditions: (i) Acetone, reflux, 6-8 h; (ii) 2 N HCl, reflux, 1 h, 85-92%;
(iii) POCl3, DMF, reflux, 1 h, 75-80 %; (iv) piperdine, EtOH, reflux 3-5 h, 50-70%.
SSYYNNOOPPSSIISS
VI
All these conjugates (7a–t) showed significant anticancer activity, with GI50
values ranging from nanomolar to micromolar. Moreover, from the MTT
proliferation assay, the three most promising conjugates 7a, 7e, 7f, 7j and 7k
showed higher cytotoxicity in various human cancer cells. Flow cytometry (FACS)
analysis showed a greater cell population in the G2/M phase, indicating that the
imidazopyridine-oxindole conjugates possess the ability to induce apoptosis.
Compounds 7a, 7e, 7f, 7j and 7k inhibited microtubule assembly as shown in the
tubulin polymerization assay. Interestingly, it was observed that IC50 of compound
7f was similar to that of combretastatin A-4.
To understand the mechanism of cell death, further biological assays suchas
western blot analysis of Akt (ser-473) was carried out. The results indicated that
significantly decreased the Akt phosphorylation upon treatment with conjugates 7e,
7f and 7k. Furthermore, Hoechst staining further confirmed conjugates 7a, 7e, 7f, 7j
and 7k caused significant nuclear condensation in MCF-7 cells.
This investigation reveals that imidazopyridine-oxindole conjugates enhance
anticancer activity. Based on the results of the studies reported herein, it is evident
that conjugates 7f and 7k is a suitable candidate for further detailed studies.
(ChemMedChem. 2013, 8, 2015 and back cover page 2080).
“Synthesis of Imidazothiadiazole-Chalcones as Potential Anticancer Agents with
Apoptosis Inducing Ability.”
Many chemotherapeutics are known to induce apoptosis, which is a
promising strategy for cancer drug discovery. Among the different approaches
available to promote apoptosis, the development of conjugates is one of the main
strategies. In an effort to establish new candidates with improved anticancer
activity and apoptosis inducing ability, imidazothiadiazole-chalcone conjugates
have been synthesized and their anticancer activity has been evaluated. The
CHAPTER-III (SECTION-A)
SSYYNNOOPPSSIISS
VII
cytotoxic studies of the hybrid agents on human cancer cell lines indicate that most
of the conjugates induced higher cytotoxicity. In view of promising activity it has
been considered of interest to investigate their role in cell proliferation and
apoptosis.
SYNTHESIS OF IMIDAZOTHIADIAZOLE-CHALCONE DERIVATIVES
The imidazothiadiazole-chalcone derivatives (7a-m) were prepared by the
Claisen-Schmidt condensation of appropriate substituted acetophenones (6a-d)
upon treatment with imidazothiadiazole aldehydes (5a-d) in the presence of NaOH
(10%). The imidazothiadiazole aldehydes were obtained by means of the Vilsmeier-
Hacc reaction on the corresponding imidazothiadiazole (4a-d), in which (4a-c) were
prepared from the appropriate 5-methyl-1,3,4-thiadiazol-2-amine (2) and
bromoketones (1). Similarly, (4d) was prepared from 5-methyl-1,3,4-thiadiazol-2-
amine (2 ) and 2-bromoacetic acid as illustrated in Scheme 2.
Further, imidazothiadiazole-chalcone derivatives (11a-d) were obtained by
the Claisen-Schmidt condensation of appropriate substituted imidazothiadiazole
ethanones (9a-d) upon treatment with 3,4,5-trimethoxy benzaldehyde (10) in the
presence of NaOH (10%) as shown in Scheme 3. Imidazothiadiazole ethanones (9a-
d) were prepared by Grignard reaction on corresponding imidazothiadiazole
aldehydes (5a-d) with methyl magnesium bromide and followed by oxidation with
IBX in DMSO.
SSYYNNOOPPSSIISS
VIII
R2
O
Br +
NHN
S NH
NN
S NH
O
R2
N
N
N
S
R2
N
N
N
S
R2
CHO
+R1 CH3
O
R1
O
N
NS
N
R2
1 2 3
4a-d
5a-d6a-d
7a-m
7a : R1 = 3,4,5-trimethoxyphenyl; R2 = 4-methoxyphenyl
7b : R1 = 3,4,5-trimethoxyphenyl; R2 = 4-fluorophenyl
7c : R1 = 3,4,5-trimethoxyphenyl; R2 = 4-chlorophenyll
7d : R1 = 3,4,5-trimethoxyphenyl; R2 = chloro
7e : R1 = 2-(6-(3,4,5-
trimethoxyphenyl))pyridinyl; R2 = 4-methoxyphenyl
7f : R1 = 2-(6-(3,4,5-
trimethoxyphenyl))pyridinyl; R2 = 4-fluorophenyl
7g : R1 = 2-(6-(3,4,5-
trimethoxyphenyl))pyridinyl; R2 = 4-chlorophenyl
7h : R1 = 2-(6-(3,4,5-
trimethoxyphenyl))pyridinyl; R2 = chloro
7i : R1 = 3,4-dimethoxyphenyl; R2 = 4-methoxyphenyl
7j : R1 = 3,4-dimethoxyphenyl; R2 = 4-fluorophenyl
7k : R1 = 3,4-dimethoxyphenyl; R2 = 4-chlorophenyl
7l : R1 = 3,4-dimethoxyphenyl; R2 = chloro
7m : R1 = 4-fluorophenyl; R2 = 4-fluorophenyl
(i)(ii)/ for 4a-c
(v)
.HBr
6a R1= 3,4,5-trimethoxyphenyl
6b R1= 3,4-dimethoxyphenyl
6c R1= 4-Flourophenyl
6d R1= 2-(6-(3,4,5-
trimethoxyphenyl))pyridinyl
N
S
HN
NH
2
+ CH2COOH
Br (iii)/ for 4d(iv)
Scheme 2. Reagents & Conditions: (i) Acetone, reflux, 4-5 h; (ii) 6N HCl, reflux, 1 h, 87-
90%; (iii) EtOH, POCl3, reflux, 7 h, 20% NH3, 69%; (iv) POCl3, DMF, reflux for 8-12 h, 70-80%
(v) 10% aq. NaOH, 1-2 h, rt, 75-85%.
SSYYNNOOPPSSIISS
IX
N
N
N
S
R2
CHO
+
R1 H
O
N
NS
N
R2
5a-d
10
11a-d
N
N
N
S
R2
8a-d
HO
N
N
N
S
R2
9a-d
O
O
R1
(i) (ii)
(iii)
11a : R1 = 3,4,5-trimethoxyphenyl; R2 = 4-methoxyphenyl
11b : R1 = 3,4,5-trimethoxyphenyl; R2 = 4-fluorophenyl
11c : R1 = 3,4,5-trimethoxyphenyl; R2 = 4-chlorophenyl
11d : R1 = 3,4,5-trimethoxyphenyl; R2 = chloro
Scheme 3. Reagents & Conditions: (i) CH3MgBr, THF, 0-5 oC, 5-6 h, 62-71%; (ii) IBX,
DMSO, 0 oC, 2-3 h, 71-85%; (iii) 10 % aq. NaOH, 1-2 h, rt, 68-80%.
The anticancer activity of these conjugates (7a-m and 11a-d) was carried out
in selected human cancer cell lines. These compounds have exhibited significant
anticancer activity against various cancer cell lines with low IC50 values. According
to the in vitro screening data, compounds 11a and 11b possess significant
cytotoxicity against all the cancer cell lines. These representative compounds are
inducing apoptosis at G1 phase of cell cycle through chromatin condensation.
Further, role of the cell- cycle regulatory proteins provide the mechanism for the cell
growth inhibition of these compounds. Interestingly, compounds 11a and 11b have
shown promising anticancer activity more than doxorubicin and both these
compounds are suitable lead candidates for detailed biological studies in the
treatment of cancer.
SSYYNNOOPPSSIISS
X
“Synthesis of Isoxazole-Chalcones as Tubulin Polymerization Inhibitors.”
In an effort to establish new candidates with improved anticancer activity,
chalcone derivatives bearing isoxazole moiety synthesized and their cytotoxic
studies of these conjugates on human cancer cell lines indicate most of the
conjugates induced higher cytotoxicity.
SYNTHESIS OF ISOXAZOLE-CHALCONE CONJUGATES
The synthesis of isoxazole-chalcone (8a-m) derivatives is sketched in Scheme
4, which were obtained by the Claisen-Schmidt condensation of suitable substituted
acetophenones with isoxazole aldehydes (6a-d). The key intermediates 5-
substituted phenyl-isoxazol-3-carbaldehydes (6a-d) were prepared in four
sequential steps. Initially substituted acetophenones (1a-d) reacted with diethyl
oxalate (2) in the presence of sodium ethanolate in ethanol obtained ethyl 2,4-dioxo-
4-(substituted phenyl)butanoates (3a-d). This was further cyclised with
hydroxylamine hydrochloride in ethanol to produce ethyl 5-substituted phenyl-
isoxazol-3-carboxylates (4a-d) in good yields. The obtained carboxylates were
reduced to (5-substitutedphenyl-isoxazol-3-yl) methanol (5a-d) with LiAlH4 in THF.
These were selectively oxidized to 5-substituted phenyl-isoxazol-3-carbaldehydes
(6a-d) by IBX in DMSO.
CHAPTER-III ((SSEECCTTIIOONN--BB))
SSYYNNOOPPSSIISS
XI
Scheme 4. Reagents & Conditions: (i) Na metal, EtOH, rt; 4-5 h 85-90%; (ii) NH2OH.HCl,
EtOH, reflux, 2-3 h, 73-81%; (iii) LAH, THF, rt, 2-3 h, 70-80%; (iv) IBX, DMSO, 0 oC, 2-3 h,
76-85%; (v) 10% aq. NaOH, 1-2 h, rt, 70-84%.
Further, the isoxazole chalcones (12a-b) were prepared by reaction of 1-(5-(4-
methoxyphenyl) isoxazol-3-yl)ethanone 10 with corresponding aldehydes (11a-b).
The intermediate 10 was obtained by Grignard reaction of 5-(4-
methoxyphenyl)isoxazole-3-carbaldehyde with methyl magnesium bromide,
followed by oxidation with IBX in DMSO (Scheme 5).
SSYYNNOOPPSSIISS
XII
Scheme 5. Reagents & Conditions: (i) CH3MgBr, THF, 5-6 h, 0oC; (ii) IBX, DMSO, 0 oC, 2-3
h, 66%; (iii) 10% aq. NaOH, 1-2 h, rt, 74-79%.
All the compounds (8a-m) and (12a-b) are showing significant anticancer
activity against various human cancer cell lines. Among the compounds,
compounds 8a, 8b, 8e, 8i, 8j and 8k are showing more potential cytotoxicity. In cell
cycle analysis, the major populations of cells treated with compounds 8e and 8i
accumulated in G2/M phase with 72.73 and 71.19%, respectively. Apoptosis is an
essential continuous process of destruction of unwanted cells during development
or homeostasis in multi-cellular organisms. In apoptosis chromatin condensation
takes place and it was found that these compounds (8a, 8b, 8c, 8e and 8i) upon
treatment at 3 µM concentration for 24 h caused significant nuclear condensation in
DU-145 cells. Among the five compounds tested for the caspase activity,
compounds 8e and 8i have shown a significant elevation in caspase -3 activity at 24
h. Although, compounds 8a, 8b, and 8c showed moderate elevation in caspase 3 at
24 h.This data reveals that caspase dependent mode of apoptosis induced by these
compounds.
SSYYNNOOPPSSIISS
XIII
“Synthesis of Aryl Substituted 2-Anilinopyridine Derivatives as Potent
Antimitotic Agents.”
We have been designed and synthesized a new class of E-7010 derivatives as
potent antimitotic agents (Z)-3-(arylamino)-1-(2-(arylamino) pyridin-3-yl) prop-2-
en-1-one. Further, these compounds evaluated their anticancer activity against
various human cancer cell lines apart from their effect on tubulin polymerization.
SYNTHESIS OF (Z)-3-(ARYLAMINO)-1-(2-ARYLAMINO)PYRIDIN-3-YL)PROP-
2-EN-1-ONE
Preparation of the (Z)-3-(arylamino)-1-(2-(arylamino)pyridin-3-yl)prop-2-en-
1-one conjugates (9a-q) are depicted in Scheme 6. The esterification of 2-
chloronicotinic acid (1) in presence of sulphuric acid catalyst provided ethyl 2-
chloronicotinate (2), then upon nucleophilic substitution with substituted aromatic
amines (3a-d) provided ethyl 2-(arylamino)nicotinates (4a-d) in quantitative yield.
This is followed by reduction with LAH in THF to afford (2-(arylamino)pyridin-3-
yl)methanol intermediates (5a-d), which upon oxidation with IBX produce the
aldehydes (6a-d). These aldehydes (6a-d) were treated with ethynyl magnesium
bromide in THF provide intermediates (7a-d) followed by oxidation with IBX afford
the corresponding precursors (8a-d). Consequently, the target conjugates (9a-q)
were formed by reaction between the corresponding precursors (8a-d) and aryl
amines in ethanol.
CHAPTER-IV ((SSEECCTTIIOONN--AA))
SSYYNNOOPPSSIISS
XIV
N Cl
COOH
N Cl
COOEt N NH
COOEt
R1
R2
R3
N NH
CH2OH
R1
R2
R3
N NH
CHO
R1
R2
R3
N NH
R1
R2
R3
OH
N NH
R1
R2
R3
O
N NH
R1
R2
R3
O HNAr
+
R1
R2
R3
NH2
(i) (ii)
(iii)
(iv)(v)
(vi)
(vii)
1 2
3a-d4a-d
5a-d6a-d7a-d
8a-d 9a-q
Scheme 6. Reagents & conditions: (i) Conc. H2SO4, ethanol, reflux, 4 h, 85%; (ii)
ethylene glycol, 120 oC, 5-6 h, 73-81% (iii) LAH, THF, rt, 3-4 h, 70-79%; (iv) IBX,
DMSO, 0 oC, 2 h, 72-81%; (v) ethynyl magnesium bromide, THF, 0 oC, rt, 3-4 h, 61-
70%; (vi) IBX, DMSO, 0 oC, 2-3 h, 70-81%; (vii) aryl amines, EtOH, 4-5 h, rt, 65-81%.
SSYYNNOOPPSSIISS
XV
Table 1. Structures of compounds 9a−−−−q melting points and their yields
Comp Ar R1 R2 R3 Melting point oC Yield % (mg)
9a 5-indolyl H OCH3 H 162−164 68% (93)
9b 6-indolyl H OCH3 H 227−229 72% (99)
9c 3,5-dimethoxyphenyl H OCH3 H 238−240 80% (116)
9d 4-methoxyphenyl H OCH3 H 260−262 79% (106)
9e 4-trifluoromethylphenyl H OCH3 H 234−236 74% (109)
9f 5-indolyl OCH3 OCH3 OCH3 139−141 81% (127)
9g 6-indolyl OCH3 OCH3 OCH3 156−158 69% (108)
9h 4-methoxyphenyl OCH3 OCH3 OCH3 197−199 70% (107)
9i 3,4,5-trimethoxyphenyl OCH3 OCH3 OCH3 242−244 79% (138)
9j 5-indolyl H F H 134−136 76% (106)
9k 3,5-dimethoxyphenyl H F H 196−198 65% (95)
9l 4-methoxyphenyl H F H 290−292 81% (110)
9m 3,4,5-trimethoxyphenyl H F H 291−293 80% (127)
9n 4-trifluoromethylphenyl H F H 279−281 78% (117)
9o 5-indolyl H H H 278−280 75% (119)
9p 6-indolyl H H H 152−154 79% (126)
9q 3,4,5-trimethoxyphenyl H H H 165−167 71% (129)
Compounds 9a-q were evaluated for their in vitro anticancer activity in
selected human cancer cell lines of MCF-7 (breast), A-549 (lung), HCT-116 (colon)
and HeLa (cervix) by MTT assay. The compounds exhibiting cytotoxic activity IC50
≤ 10-5M (10 µM) are considered to be active on the respective cell lines. The
SSYYNNOOPPSSIISS
XVI
concentration causing 50% cell inhibition (IC50) compared with the control was
calculated. Among the compounds, 9a and 9k are more potent against A-549 cell
line than E7010 and compound 9h is showing significant activity against MCF-7 cell
line. Compounds 9a, 9h and 9k have been examined about the cell cycle alterations
in A-549 lung cancer cells to understand the phase distribution and these are
arresting cells at G2/M phase. These compounds are inhibiting tubulin
polymerization. Particularly, compound 9a has shown significant antitubulin
activity than a known inhibitor of tubulin polymerization, E-7010.
“Synthesis of Aryl Substituted Pyrazole/Isoxazole Congeners as a New Class
Inhibitors of Tubulin Polymerization.”
In continuation to our efforts on the design of new anticancer agents (Z)-3-
(arylamino)-1-(5-(substituted phenyl)-1H-pyrazol-3-yl)prop-2-en-1-one and (Z)-3-
(arylamino)-1-(5-(substituted phenyl)isoxazol-3-yl)prop-2-en-1-one derivatives have
been synthesized. These new pyrazole/isoxazole congeners have been evaluated
for their anticancer activity. Further, some detailed biological studies related to the
mechanism aspects have been carried out for some of the representative
compounds.
SYNTHESIS OF (Z)-3-ARYLAMINO-1-(5-ARYL-1H-PYRAZOL-3-YL)PROP-2-
EN-1-ONE AND (Z)-3-ARYLAMINO-1-(5-ARYL ISOXAZOL-3-YL)PROP-2-EN-
1-ONE DERIVATIVES
Synthesis of (Z)-3-Arylamino-1-(5-aryl-1H-pyrazol-3-yl)prop-2-en-1-one and
(Z)-3-Arylamino-1-(5-aryl isoxazol-3-yl)prop-2-en-1-one conjugates (9a-t) is as
shown in Scheme 7. Diethyloxalate have been treated with substituted
acetophenones (1a-d) in presence of base gave compounds (3a-d). These
CHAPTER-IV ((SSEECCTTIIOONN--BB))
SSYYNNOOPPSSIISS
XVII
intermediates (3a-d) were cyclised with hydrazine hydrochloride dihydrochloride
(for 4a-c) and hydroxylamine hydrochloride (for 4d-g) to obtain compounds (4a-g).
Reduction of compounds (4a-g) with LAH in dry THF to afford alcohols (5a-g),
followed by oxidation with IBX gave substituted isoxazole/pyrazole aldehydes
(6a-g). These intermediate aldehydes (6a-g) were treated with ethynyl magnesium
bromide in THF to obtain compounds (7a-g). The precursor compounds (8a-g) were
obtained by oxidation of intermediates (7a-g) with IBX in DMSO. Finally, we
achieved desired conjugates (9a-t) by the reaction of appropriate the compounds
(8a-g) and aryl amines in ethanol.
R CH3
O
+
O
OEt
O
EtOR
O
COOEt
O
R XN
COOEt
R XN
CH2OH
R XN
CHO
1a-d 23a-d
4a-g
5a-g6a-g
R XN
HO
R XN
O
X N
O HNAr
7a-g
8a-g 9a-t
(i) (ii)
(iii)
(iv)(v)
(vi)
(vii)
R1
R2
R3
Scheme 7. Reagents & Conditions: (i) Na metal, EtOH, rt; 4-5 h, 85-90%; (ii)
NH2NH2.2HCl for 4a-c, NH2OH.HCl for 4d-g, EtOH, reflux, 2-3 h, 71-81%; (iii)
LAH, THF, rt, 2-3 h, 70-80%; (iv) IBX, DMSO, 0 oC, 2-3 h, 71-85%; (v) ethynyl
magnesium bromide, THF, 0 oC, rt, 4-5 h, 59-69%; (vi) IBX, DMSO, 0 oC, 2-3 h, 73-
86%; (vii) arylamines, EtOH, 4 h, rt, 69-82%.
SSYYNNOOPPSSIISS
XVIII
Table 2. Structures of compounds 9a−−−−t melting points and their yields
Comp Ar X R1 R2 R3 Melting point oC
Yield %
(mg)
9a 5-indolyl NH OCH3 OCH3 OCH3 162−164 75% (120)
9b 6-indolyl NH
OCH3 OCH3 OCH3 227−229 78% (125)
9c 4-trifluoromethylphenyl NH OCH3 OCH3 OCH3 238−240 76% (131)
9d 4-methoxyphenyl NH
OCH3 OCH3 OCH3 260−262 80% (126)
9e 3, 4, 5-trimethoxyphenyl NH
OCH3 OCH3 OCH3 234−236 77% (139)
9f 5-indolyl NH
H OCH3 OCH3 139−141 82% (137)
9g 3, 4, 5-trimethoxyphenyl NH H F H 197−199 80% (163)
9h 5-indolyl O OCH3 OCH3 OCH3 242−244 77% (127)
9i 6-indolyl O
OCH3 OCH3 OCH3 134−136 72% (116)
9j 4-methoxyphnyl O
OCH3 OCH3 OCH3 290−292 75% (118)
9k 3, 4, 5-trimethoxyphenyl O
OCH3 OCH3 OCH3 291−293 72% (130)
9l 6-indolyl O
H OCH3 OCH3 278−280 79% (131)
9m 3, 4, 5-trimethoxyphenyl O
H OCH3 OCH3 152−154 76% (143)
9n 5-indolyl O
H OCH3 H 165−167 72% (125)
9o 6-indolyl O
H OCH3 H 192-194 78% (135)
9p 4-trifluoromethylphenyl O
H OCH3 H 227-229 69% (130)
9q 4-methoxyphenyl O
H OCH3 H 161-163 71% (120)
9r 3, 4, 5-trimethoxyphenyl O H OCH3 H 159-161 76% (151)
9s 5-indolyl O
H F H 183-185 79% (140)
9t 3, 4, 5-trimethoxyphenyl O
H F H 171-172 71% (144)
SSYYNNOOPPSSIISS
XIX
A new class of (Z)-3-(arylamino)-1-(5-(substituted phenyl)-1H-pyrazol-3-
yl)prop-2-en-1-one and (Z)-3-(arylamino)-1-(5-(substituted phenyl)isoxazol-3-
yl)prop-2-en-1-one conjugates were synthesized and tested for their in vitro
anticancer activities against various human cancer cell lines MCF-7 (breast), A549
(lung), HCT116 (colon) and HeLa (cervix). All the compounds showed moderate to
good anticancer potency against most of the tested cancer cell lines. Particularly,
compounds 9a, 9b and 9f have shown significant anticancer activity against A-549
cell line, in which 9a, 9b are more potent than E-7010 with lesser IC50 values 0.72
µM and 1.21 µM, respectively. Further, these compounds 9a, 9b and 9f were
arrested A-549 cells at G2/M phase in FACS analysis. Moreover, compounds 9a and
9b exhibited superior inhibition of tubulin assembly with IC50 values as 1.28 and
0.28 µM respectively; however, under similar experimental conditions, E-7010
showed antitubulin activity with IC50 value 2.64 µM. Interestingly, compounds 9a
and 9b which demonstrated strong inhibition of tubulin assembly, had the lowest
IC50 values than known tubulin assembly inhibitor, E-7010.