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.

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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

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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))

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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.