Design, synthesis and antiproliferative activity of the new conjugates of E7010 and resveratrol as tubulin polymerization inhibitors

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2015, DOI: 10.1039/C5OB02022K.

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Design, synthesis and antiproliferative activity of new conjugates

of E7010 and resveratrol as tubulin polymerization inhibitors

Received 00th January 20xx,

Accepted 00th January 20xx

1

Ahmed Kamal,*¹Md. Ashraf,¹Shaik Thokhir Basha,¹S. M. Ali Hussaini

,

Shamshair Singh,²MVPS

Vishnuvardhan,¹Boppanna Kiran

,

Balasubramanian Sridhar³

2

DOI: 10.1039/x0xx00000x

www.rsc.org/

A new class of (E)-N-phenyl-3-styrylpyridin-2-amine conjugates were designed and synthesized on the basis of E7010 and

resveratrol scaffolds. These conjugates were evaluated for their antiproliferative activity in four human cancer cell lines

with GI₅₀values ranging from 2.1 µM to 20 µM. Two of the conjugates RSV-1 and RSV-11 were found to possess 13-fold

higher GI₅₀values than resveratrol and 1 to 2 fold higher GI₅₀values than E7010 against human cervical HepG2 cancer line.

They displayed high potency and selectivity in a panel of NCI 60 human cancer cell lines. Based on the GI₅₀values against

panel of 60 NCI cancer cell lines and dock scores from the molecular modelling studies, we selected RSV-1 and RSV-11 for

tubulin polymerization and mechanistic studies. Furthermore, RSV-1 and RSV-11 compounds inhibited the assembly of

tubulin by strongly binding to the colchicine-binding site. G2/M-phase is arrested in HepG2 cells as assessed by flow

cytometry. Structure based studies, western blot and immunofluorescence experiments demonstrated that RSV-1 and

RSV-11 depolymerize microtubules in HepG2 cell line, resulting in an accumulation of G2/M cells.

number of human cancer cell lines including lymphoma,

Introduction

myeloma,

melanoma,

breast,

pancreatic,

colorectal,

hepatocellular and prostate carcinoma.⁷Based on striking

inhibitory effects of resveratrol on cellular actions associated

with cancer initiation, promotion and progression, it is

suggested as a potential anticancer agent.⁸Resveratrol has been

reported to have diverse effects on signalling molecules, such as

down regulation of the expression of angiogenesis-associated

genes, activation of the apoptotic mechanisms⁹and induction of

cell cycle arrest.¹⁰

Antimitotic agents arrest the cells in M phase and some of the

clinically important anticancer drugs, like Vinca alkaloids

(vinblastine

and

vincristine),

taxol,

colchicine

and

combretastatin arrest the cells in M phase by interfering with

the assembly or disassembly of α- and β- tubulin in the

microtubules. At high concentration Vinca alkaloids and other

mitotic agents cause complete depolymerisation of

microtubules, whereas the taxanes cause the polymerization of

microtubules, and some tubulin inhibitors are shown in Fig. 1

Combretastatin is an antimitotic agent isolated from the African

Willow Tree, Combretum caffrum, by Pettit and co-workers

having a cis stilbinoid scaffold.¹It has been found to be a potent

cytotoxic agent against a wide variety of human cancer cell lines,

including multidrug resistant cell lines²and strongly inhibited

the polymerization by binding to the colchicine site of the

tubulin. Resveratrol, a 3,5,4’-trihydroxy-trans-stilbene present in

grapes and other food products also inhibits the tubulin

polymerization.^3-5This trihydroxy trans-stilbene exhibits various

biological

activities

including,

cardioprotective,

anti-

inflammatory and antiaging.⁶Recent studies have shown that

resveratrol has exhibited potential growth inhibitory effect in a

¹Division of Medicinal Chemistry and Pharmacology, Indian Institute of Chemical

Technology, Tarnaka, Hyderabad, 500007, India.

²GVK Biosciences Pvt Ltd, Plot No. 28A, IDA Nacharam, Hyderabad, 500076, India.

³X-ray Crystallography Division, CSIR-Indian Institute of Chemical Technology,

Hyderabad 500007, India.

Fig. 1 Tubulin polymerization inhibitors

†Corresponding author: ahmedkamal@iict.res.in

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E7010 (2-anilinopyridine sulphonamide) is one of the well

known tubulin polymerization inhibitors which binds to the

colchicine site of tubulin and causes cell cycle arrest and

apoptosis in M phase.^11-12HMN-214 (4), a prodrug that is

converted in vivo to the unsubstituted sulfonamide, lacking the

N-acetyl group, inhibits cancer cell growth by interfering with

the action of the polo-like kinase 1 (PLK1).¹³HMN-214, a

synthetic molecule is currently undergoing Phase-1 clinical trials.

Swern’s protocols have been investigated to oxidize the alcohol

(10a-h) as shown Table 1.

It was observed that the oxidizing agents such as DMP and PCC

failed to completely oxidize 2-anilinonicotinol to 2-

anilinonicotinaldehyde and similar results were observed with

Swern’s oxidation. However, IBX gave some better yields (40-

55%) of the aldehydes. Since the yields of the nicotinaldehydes

(9a-h) were not good, we have attempted an alternative

approach to synthesize 9a-h with a view to get better results. To

realize this, 2-anilinonicotinic esters (7a-h) were converted to

Weinerb amide (8a-h) by using trimethylaluminium, N,O-

dimethylamine hydrochloride and CH₂Cl₂as a solvent at 0 °C to

room temperature.²⁰Reduction of Weinreb amides with

diisobutylaluminum hydride afforded 2-anilinonicotinaldehyde

(9a-h) in good yields (70-90%).

Fig. 2 Designing of RSV-1 to RSV-13 conjugates.

The structure-activity relationship (SAR) study of E7010 and its

analogues clarified that the 2-anilinopyridine moiety is essential

for antimitotic and antitumor activity.¹⁴In the recent years,

several efforts have been made in cancer chemotherapy inorder

to identify potential and selective anticancer drugs through the

discovery of a variety of novel drugs. As a part of our ongoing

efforts to discover novel anticancer agents, we have previously

reported 2-anilino linked

aminobenzothiazole,

triazolobenzothiadiazine,¹⁶

sulfonyl hydrazides,¹⁵

2-

1,3,4-

oxadiazole,¹⁷1,2,3-triazole¹⁸conjugates and 2-anilinopyridinyl

dimers¹⁹as potent anticancer agents. On the basis of these

findings, an attempt has been made in the present study to

incorporate 2-anilino pyridyl ring into reveratrol (RSV-1 to RSV-

13) and the strategy employed for design of such conjugates

described in Fig. 2. The conjugates obtained were evaluated for

cytotoxicity, tubulin polymerization and cell-cycle as well as

apoptotic effects.

Scheme 1. Synthesis of substituted 2-anilinonicotinaldehyde (9a-h).

Reagents and conditions. (a) H₂SO₄, ethanol, 80 °C, 2 h; (b) ArNH₂,

ethylene glycol, 180 °C,

6

h; (c) N,O-dimethylhydroxylamine

hydrochloride, AlMe₃, CH₂Cl₂, 0 °C-rt, 8 h.(d) DIBAL-H, CH₂Cl₂, -78 °C, 45

min. (e) IBX, DMSO , 0 °C-rt, 2h (40-55%).

Table 1 Oxidation of 10 (a-h) to 9 (a-h) with various oxidizing reagents

Entry

1

reagent

DMP

solvent

CH₂Cl₂

temp

yield

0-30 °C

<10%

Results and discussion

2

(COCl)₂

TEA, DMSO

PDC

CH₂Cl₂

-60 °C

<6%

Chemistry

3

4

CH₃CN

DMSO

r.t

<10 %

<55 %

The synthetic route to achieve the desired E7010-RSV

conjugates has been outlined in the following Scheme 1.

IBX

The Wittig salts 3,4,5-trimethoxybenzyltriphenylphosphonium

bromide and 3,5-dimethoxyphenyl phosphonium bromides were

prepared from substituted benzaldehydes (11a-b) as shown in

the Scheme 2. The substituted benzaldehydes (11a-b) were

reduced to benzyl alcohol (12a-b) by using sodium borohydride

in methanol to give quantitative yields. This was followed by

bromination of benzylalcohol with phosphorus tribromide in

dichloromethane to afford benzylbromides (13a-b). The Wittig

salts (14a-b)²¹were obtained by the reaction of these

benzylbromides (13a-b) with triphenylphosphine in dry toluene

at elevated temperature.

Commercially available 2-chloronicotinic acid (5) was converted

into ethyl 2-chloronicotinate (6) by refluxing with ethanol and

H₂SO₄. This was followed by the condensation of ethyl 2-

chloronicotinate (6) with various substituted anilines in ethylene

glycol using standard method to provide substituted ethyl 2-

anilinonicotinate (7a-h)¹⁶

.

Initially, the synthesis of aldehyde (9a-h)¹⁸has been attempted

by the reduction of 2-anilinonicotinic ester (7a-h) using reducing

agents such as DIBAL-H, which resulted in 90% of highly reduced

alcohol (10a-h) compared to only 10% of expected controlled

reduced product of aldehyde (9a-h). Therefore, it was

considered to oxidize the alcohol to the desired aldehyde and

for this purpose some selected optimization studies were carried

out. Different oxidizing agents such as DMP, IBX, PCC and

The desired products, RSV-1 to RSV-13 were obtained by the

Wittig reaction between substituted anlinonicotinaldehydes (9a-

h) and various triphenylphosphonium salts (14a-b) by using

sodiumhydride in dry THF.

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Fig.

3 A view of RSV-1, showing the atom-labelling scheme.

Displacement ellipsoids are drawn at the 30% probability level and H

atoms are represented by circles of arbitrary radii.

Cytotoxic activity

Primarily, we performed SRB assay to determine the in vitro

antiproliferative activity of RSV-1 to RSV-13 in a panel of four

cancer cell lines viz., A549 (Lung cancer), HepG2 (Liver cancer),

HeLa (Cervical cancer) and DU145 (prostate cancer). The results

revealed that RSV-1 to RSV-13 inhibited the proliferation with a

GI₅₀values in the range of 2 µM to 42.0 µM wherein E7010 and

resveratrol were used as standards (Table 3). The most potent

compounds like (RSV-1 and RSV-11) demonstrated anti-

proliferative effect superior to that of resveratrol and

comparable to E7010 (GI₅₀3.0 µM) against HepG2 cell line.

Furthermore, some representative compounds were evaluated

against sixty human cancer cell lines derived from nine types of

cancer diseases (lung, leukemia, colon, melanoma, ovarian,

renal, prostate, breast, and CNS) at a single dose at the National

Cancer Institute (NCI; Bethesda, MD, USA). Amongst them RSV-1

was found to be active against this panel of sixty cell lines with

GI₅₀values in the range of 1.0 µM to 16. µM (GI₅₀is the molar

concentration causing 50% cell growth inhibition) as shown in

Table 4. Notably, RSV-1 displayed selectivity on several cell lines

from the panel of lung cancer cells (1.0-4.0 µM).

Scheme 2. Synthesis of RSV-1 to RSV-13 conjugates. Reagents and

conditions. (a) NaBH₄, methanol, rt, 2 h; (b) PBr₃, CH₂Cl₂, rt, 2 h. (c) PPh₃,

toluene, 100 °C, 2 h; (d) 9a-h, NaH, THF, 0°C-rt, 2 h.

Table 2 Percentage of yields of E and Z isomers of RSV-1 to RSV-13

Comp

RSV-1

RSV-2

RSV-3

RSV-4

RSV-5

RSV-6

RSV-7

RSV-8

RSV-9

RSV-10

RSV-11

RSV-12

RSV-13

R₁

H

R₂

R₃

H

X

Y

Z

E/Z (%)

H

OMe OMe OMe 90/10

OMe OMe OMe 94/6

OMe OMe OMe 90/10

OMe OMe OMe 94/6

OMe OMe OMe 93/7

OMe OMe OMe 94/6

OMe OMe OMe 95/5

H

F

H

Cl

H

OH

OMe

F

H

OMe

F

OMe

F

H

OMe

H

OMe 90/10

OMe 92/8

OMe 90/10

OMe 94/6

H

F

H

Cl

H

OMe

H

OMe

Fig. 4 SAR analysis of E7010 and resveratrol conjugates.

X-ray Crystallography

In the terms of SAR analysis of these conjugates, we first

examined the influence of 3,4,5-trimethoxyphenyl group along

with the other substituents at the 2-amino position of the

pyridyl ring (ring A). It was observed that compounds like RSV-4

and RSV-5 that contain electron donating groups viz., 4-methoxy

and 4-hydroxy substitutions on the ring-A showed poor

inhibitory effect (>8.0 µM) and when these groups were

replaced by 3,4-dimethoxy and 3,4,5-trimethoxy groups, there

was not much affect on the potency. However fluoro and chloro

substituents at ring A (RSV-2 and RSV-3) enhanced the cytotoxic

activity (4.0-7.0 µM). Surprisingly, unsubstituted ring-A (RSV-1)

substantially enhanced the potency in all the tested cell lines

with strong inhibitory effect on human liver cancer cell line with

a GI₅₀value of 3.0 µM. An overall SAR analysis of these

conjugates influenced by some important substitutions towards

the tubulin activity is shown in Fig. 4.

X-ray data for the compounds were collected at room

temperature using a Bruker Smart Apex CCD diffractometer with

graphite monochromated MoKα radiation (α=0.71073Å) with ω-

scan method.²²Preliminary lattice parameters and orientation

matrices were obtained from four sets of frames.

Finally, we examined the cytotoxic effect of RSV-9 to RSV-13 by

replacement of 3,4,5-trimethoxy substitutions with 3,5-

dimethoxy substituents at ring-C along with the various

substituents at ring A. Unsubstituted ring A (RSV-9) and 4-fluro

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substituted ring A (RSV-10) were significantly more potent than

RSV-12 and RSV-13 with 4-methoxy and 3,4-dimethoxy at the

ring A respectively. Whereas 4-chloro substituent on the ring A

(RSV-11) is one of the most potent compound having exhibiting

GI₅₀of 2.0 µM against HepG2 cell line.

Table 3 GI₅₀(µM) of compounds against selected cancer cell lines

Comp

RSV-1

RSV-2

RSV-3

RSV-4

RSV-5

RSV-6

RSV-7

RSV-8

RSV-9

RSV-10

RSV-11

RSV-12

RSV-13

RSV

A549^a

HepG2^b

HeLa^c

Du145^d

6.8 ±0.16

6.0 ±0.04

7.2 ±0.18

- e

9.4 ±0.23

17 ±0.07

8.7 ±0.15

- e

3.9 ±0.32

5.6 ±0.14

4.1 ±0.14

5.4 ±0.33

4.8 ±0.22

15 ±0.021

14 ±0.35

- e

9.2 ±0.2

6.5 ±0.06

3 ±0.09

14.5

3.3 ±0.08

4.4 ±0.03

3.8 ±0.15

- e

9.8 ±0.04

- e

4.3 ±0.14

14 ±0.2

13 ±0.49

- e

13 ±0.11

7.9 ±0.14

13 ±0.15

3.6 ±0.03

6.1 ±0.26

3.3±0.05

1.8 ±0.25

15 ±0.12

16 ±0.14

40 ± 0.22

2.8 ± 0.22

- e

8.8 ±0.01

6.3 ±0.22

5.9±0.03

3.9 ±0.16

4.1 ±0.04

21 ±0.18

43 ± 0.53

4.8 ± 0.22

- e

8.2±0.06

5.8 ±0.12

12 ±0.26

12 ±0.08

39 ± 0.23

1.2 ± 0.3

- e

42 ± 0.06

1.8 ± 0.22

E7010

^aA549: human lung adenocarcinoma epithelial cell line. ^bHepG2: human liver cancer cell line ^cHeLa: human cervix

cancer cell line. ^dDU145: human prostate cancer cell line. ^eGI₅₀value not attained at the concentration used.

Table 4 Cytotoxicity of RSV-1 (NSC: 761393/1) in sixty human cancer cell lines

Panel/Cell Line

Leukemia

CCRF-CEM

HL-60(TB)

MOLT

GI₅₀µM

Panel/Cell Line

SF-539

Panel/Cell Line

OVCAR-8

NCI/ADR-RES

SK-OV-3

Renal Cancer

786-0

GI₅₀µM

6.7

1.2

7.1±0.74

8.0±0.92

6.7±0.34

7.0±0.39

4.0±0.70

SNB-75

4.1±0.35

4.5±0.18

1.5±0.09

9.0±1.02

6.0±0.44

5.4±0.34

4.4±0.34

11.1±1.04

Melanoma

LOXIMVI

MALME-3M

M14

RPMI

6.0±1.04

3.0±0.70

5.0±0.84

4.0±0.04

5.0±0.50

5.0±0.34

15.5±2.01

5.0±0.54

SR

A498

Lung Cancer

A549/ATCC

HOP-62

MDA-MB-435

SK-MEL-28

CNS Cancer

SF-295

ACHN

4.0±0.44

1.0±0.10

1.0±0.06

1.0±0.03

2.0±0.34

4.0±0.51

CAKI-1

5.0±0.74

1.0±0.03

4.0±0.47

4.0±0.20

4.0±0.14

8.5±0.07

5.0±0.03

RXF-393

SN12C

NCI-H226

NCI-H23

NCI-H322M

NCI-H460

Colon Cancer

COLO 205

HCT-116

HCT-15

SF-539

TK-10

SNB-75

UO-31

U251

Prostate Cancer

PC-3

SK-MEL-5

UACC-257

UACC-62

Ovarian Cancer

IGROV1

8.0±1.06

6.0±0.84

4.0±0.64

4.0±0.19

5.0±0.34

5.5±0.54

5.0±0.71

DU-145

Breast Cancer

MCF-7

13.0±1.24

7.5±1.01

4.0±0.34

13.0±1.98

14.0±1.764

5.0±0.75

HT29

HS578T

SW-620

OVCAR-3

OVCAR-4

OVCAR-5

10.0±1.14

16.0±1.18

BT-549

CNS Cancer

SF-295

T-47D

5.0±0.50

MDA-MB-468

7.0±1.02

Calculated GI₅₀and TGI values of RSV-1 against a panel of sixty cancer cells line

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Molecular modelling studies

The co-crystal structure of tubulin was downloaded from the

protein data bank with PDB ID: 4O2B²³and resolution of 2.30 Å

to carry out the docking studies. This 4O2B complex consisted of

tubulin alpha-1B chain (A, C), tubulin beta-2B chain (B, D),

stathmin-4 (E) and tubulin-tyrosine ligase (F) subunits.

Colchicine binds at the interface of alpha and beta subunits,

hence both the chain A and B were selected for docking studies

and the docking studies were performed using Glide-SP protocol

(Schrödinger).²⁴

confirmation was 0.6 Å, this validated protocol was used for

docking of E7010 conjugates. Molecular docking studies

revealed that colchicine, RSV-1 and RSV-11 displayed a high

docking score of -8.000, -7.645 and -7.871 kcal/mol respectively.

RSV-2, RSV-3, RSV-4, RSV-8, RSV-9 and RSV-10 also docked well

into the tubulin active site with similar orientation, however the

dock score was in the range of -4.00 to -6.23 kcal/mol. RSV-5,

RSV-12 and RSV-13 displayed a very low dock score of -2.46, -

2.88 and -2.10 whereas there were no docking poses retrieved

for RSV-6 and RSV-7.

Protein preparation and grid generation

3-Methoxy group of RSV-1 and RSV-11 showed hydrogen

bonding interactions with Cys B: 241 with a distance of 2.20 Å

and 2.41 Å. In case of RSV-11, the nitrogen in the pyridine ring

showed hydrogen bond interactions with Asn A: 101 and Asn B:

258 with a distance of 2.02 Å and 2.41 Å. The nitrogen in the

pyridine ring of the RSV-1 also showed hydrogen bonding with

Asn A: 101 and Asn B: 258 with a distance of 2.31 Å and 2.63 Å.

Pi-sigma interaction was also observed between the pyridine

ring and Lys B: 254 for both RSV-1 and RSV-11. Both dimethoxy

and trimethoxy groups were very well accommodated into the

hydrophobic pocket of chain B surrounded by Ile378, Ala316,

Phe268, Leu255, Leu242 and Leu252 amino acids for both RSV-

11 and RSV-1. An additional halogen bond was observed for

RSV-11 with Asn B: 350 at a distance of 3.50 Å and the predicted

binding conformations of RSV-1 and RSV-11 are given in Fig. 5

and Fig. 6 respectively. Based on the GI₅₀values against a panel

of sixty cancer cell lines (NCI) and the dock scores from the

molecular modelling studies, prompted us to select RSV-1 and

RSV-11 for the effect on tubulin polymerization and detailed

biological studies.

The multi-step Schrödinger’s (Schrödinger L.L.C., USA) protein

preparation tool has been used for the final preparation of the

receptor model.²⁵Water molecules beyond 5 Å from the

heteroatom (crystal ligand) were deleted. Then, this step is

followed by restrained minimization using the OPLS 2005 force

field to RMSD of 0.3 Å. Docking simulations were performed

using Glide-SP protocol. The binding region was defined using a

grid of 10 Å × 14 Å × 14 Å box centred on the centroid of the

crystal ligand on to the 3D structure of the protein, in order to

confine the centroid of the crystal ligand and the default settings

were used for all the remaining parameters.

Ligand preparation

The conjugates were sketched using chemdraw and were

minimized using MacroModel (a module of Schrödinger)

multiminimization. The compounds were minimized and

prepared for docking using Ligprep (a module of Schrödinger)

and all the thirteen compounds were subjected for docking

studies using Glide-SP.

Colchicine was redocked into the tubulin protein and the RMS

deviation between the colchicine docking and crystal

Fig. 5 Predicted binding mode of RSV-1

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Fig. 6 Predicted binding mode of RSV-11.

Tubulin polymerization inhibition

In this study we investigated the ability of these conjugates

(RSV-1 and RSV-11) to inhibit the tubulin assembly as IC₅₀

values. The progression in tubulin polymerization was

investigated by monitoring the increase in fluorescence emission

at 420 nm at 3 µM concentration employing E7010 as the

positive control. These conjugates showed significant activity to

inhibit the tubulin polymerization which is comparable to E7010,

whereas RSV-1 inhibited the tubulin polymerization by 55.2%

while RSV-11 inhibited by 60.3% (Fig. 7). The IC₅₀values of RSV-

1, RSV-11 and E7010 are 3.9 ± 0.24 µM, 3.4±0.26 µM and

3.1±0.15 µM respectively (Table 5).

concentration. B) Percentage of tubulin polymerization inhibition

compared to control.

Cell cycle analysis

Tubulin polymerization inhibitors interfere with the M phase of

the cell cycle by altering the dynamics of tubulin polymerization,

which finally results in the induction of apoptosis within mitosis.

Therefore, it was considered of interest to examine the effect of

RSV-1 and RSV-11 on cell cycle progression by flow cytometry

analysis. HepG2 cells were treated with the conjugates (RSV-1

and RSV-11) at 2 µM and 4 µM incubated for 48 h. The results

demonstrated that untreated HepG2 cells distributed in G1

(5.75%), G1 (80.68%), S ((4.98%) and G2/M (8.59%) phase. When

compared to control, both the compounds (RSV-1 and RSV-11)

arrested most of cells in G2/M phase and at 2 µM concentration

22.55% accumulated in G2/M phase by RSV-1. Upon increasing

the concentration from 2.0 to 4.0 µM, the accumulation of cells

increased by 3.5 folds at the G2/M phase, whereas RSV-11

arrested 13.86% and 41.79% at 2 and 4 µM respectively at the

G2/M phase. (Table 6 and Fig. 8 & 9).

Table 5 IC₅₀values of RSV-1, RSV-11 and E7010 against tubulin

Compod

RSV-1

IC₅₀± SD (µM)a

3.9 ± 0.24

RSV-11

E7010

3.4 ± 0.26

3.6 ± 0.15

^aConcentration of drug to inhibit 50% of tubulin assembly.

Table 6 Distribution of Cells at G1, S and G2/M Phase of Cell Cycle

following Treatment with RSV-1 and RSV-11 in HepG2 Cellsa

Figure 7. Effect of compounds on tubulin polymerization: Tubulin

polymerization was monitored by the increase in fluorescence at 360 nm

(excitation) and 420 nm (emission) for 1 h at 37⁰C. Synthesized

compounds RSV-1 and RSV-11 were included at a final concentration of

Comp

G0

G1

S

G2/M

8.59

control

5.75

2.48

2.64

4.93

7.98

80.68

65.52

14.29

72.68

31.10

4.98

9.45

7.30

8.53

19.12

RSV-1 (2µM)

RSV-1 (4µM)

RSV-11 (2µM)

RSV-11 (4µM)

22.55

75.78

13.86

41.79

Fig.

7 Effect of compounds on tubulin polymerization: Tubulin

polymerization was monitored by the increase in fluorescence at 360 nm

(excitation) and 420 nm (emission) for 1 h at 37⁰C. Synthesized

compounds RSV-1 and RSV-11 were included at a final concentration of

3 µM. E7010 was used as a positive control. A) Real time kinetic graph of

tubulin polymerization: compounds RSV-1, RSV-11 and E7010 at 3 µM

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tubulin in the supernatant fraction showed an increased level of

free tubulin after treatment of RSV-1 and RSV-11 however there

was a steady decrease in the polymerized tubulin in the pellet.

Taxol, which was used as the negative control, stabilized the

tubulin assembly as the amount of tubulin in the pellet was

predominant than the cytosolic fraction. Densitometry analysis

was performed for band intensities obtained from slot blot.

Percentage of supernatant to pellet fractions were calculated

and shown in Fig. 10.

Fig. 8 Distribution of Cells at G1, S and G2/M Phase of Cell Cycle

following treatment with RSV-1 and RSV-11 in HepG2 Cells

Immuno cytochemistry

To validate the effect of these conjugates on cellular tubulin,

immunohistochemistry studies have been carried out to

examine the in situ effects of RSV-1 and RSV-11 on the cellular

microtubules in HepG2 cancer cells. HepG2 cells seeded on

sterile cover slips are treated with the compounds at

a

concentration of 2 µM and E7010 was used as a standard for 48

h.

Fig. 9 FACS analysis of cell cycle distribution of HepG2 cells after

treatment with, RSV-1 and RSV-11 conjugates at 2 µM and 4 µM

concentrations for 48 h. Cell cycle analysis was performed employing

propidium iodide as indicated under Materials and Methods. The

percentage of cells in each phase of cell cycle was quantified by flow

cytometry.

Tubulin western blot Analysis

In order to extend the in vitro effects of the compounds on

tubulin polymerization to the cellular effects, Western blot

analysis of α-tubulin in HepG2 cells was performed by treating

E7010 (1 μM), taxol (1 μM) and RSV-1 and RSV-11 (2 and 4µM)

for 48 h.

Fig. 11 HepG2 cells were treated with compounds RSV-1 and RSV-11 at 2

µM concentration for 48 h followed by staining with α-tubulin antibody.

Microtubule organization was clearly observed by green colour tubulin

network like structures in control cells and was found to be disrupted in

cells treated with compounds RSV-1 and RSV-11. E7010 was used as

positive control.

Fig. 10 Western blot analysis of alpha-tubulin in the soluble and

polymerized fractions of HepG2 cells treated with compounds RSV-1 and

RSV-11 at 2 & 4 µM concentrations and Taxol and E7010 were used at 1

μM for 48 h.

Free and polymerized tubulin was isolated as described in the

experimental procedures. Results indicate that while, the

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In this study, untreated human lung cancer cells displayed the

normal distribution of microtubules. The cells treated with

compounds RSV-1 and RSV-11 showed disrupted microtubule

organization as seen in Fig. 11, thus demonstrating the inhibition

of tubulin polymerization and moreover the standard also

showed disrupted microtubule organization. Thus this

immunofluorescence study showed that the level of tubulin

polymerization inhibition was comparable to that of E7010 for

both RSV-1 and RSV-11.

insight towards the development of tubulin polymerization

inhibitors as potential anticancer agents. The work reported

herein is anticipated to be helpful in the development of newer

anticancer agents.

Acknowledgements

The authors, M.A, T.B.S, S.M.A.H and M.V acknowledge the

Council of Scientific and Industrial Research (CSIR), New Delhi

(India) for the financial support under the 12th Five Year Plan

project “Affordable Cancer Therapeutics (ACT)” (CSC0301). This

project was also supported by King Saud University, Deanship of

Scientific Research, Research Chair.

Caspase assay

To determine the apoptotic activity of active compounds, AFC

conjugated caspase 3 and 9 substrates were used. It is well

known that caspases play a crucial role in the induction of

apoptosis and amongst them caspase-3 happens to be one of

the effector caspase which inturn is activated by caspase-9.

Experimental Section

Chemistry

Hence, HepG2 cells were treated with

2 μM & 4 µM

concentrations of RSV-1 and RSV-11 along with the positive

control E7010 (1 μM) and examined the activation of caspase-9

as well as -3 at 48 h as described in the procedure. The results

indicate that there is nearly 2 to 4 fold induction in the activity

of the caspases for the cells treated by RSV-11 and E7010 also

induced caspase activity under similar conditions.

All chemicals and reagents were obtained from Aldrich (Sigma–

Aldrich, S t. Louis, MO, USA), Lancaster (AlfaAesar, Johnson

Matthey Company, Ward Hill, MA, USA) or Spectrochem Pvt. Ltd

(Mumbai, India) and were used without further purification.

Reactions were monitored by TLC, performed on silica gel glass

plates containing 60 G F -254, and visualization on TLC was

achieved by UV light or iodine indicator. Column

chromatography was performed with Merck 60–120 mesh silica

gel. 1H spectra were recorded on AVANS-300 (300 MHz) or

JCAMP: (300/500 MHz) instruments. Chemical shifts (δ) were

reported in ppm downfield from internal TMS standard. EI-MS

spectra were recorded on Micro mass, Quattro LC using ES I+

software with capillary voltage 3.98 kV and ESI mode positive

ion trap detector. High -resolution mass spectra (HRMS) were

recorded on QSTAR XL Hybrid MS /MS mass spectrometer. The

purity of tested compounds was determined by HPLC performed

on a Shimadzu LCMS-2020 apparatus equipped with a SPD-

M20A diode array detector and a Shimadzu SIL-20AC auto

injector using C18 column (X-Bridge BEH 2.5 μm C18, 4.6 mm X

100 mm column). Elution conditions: mobile phase A 0.05%

Formic acid in water; mobile phase B 0.05% Formic acid in

Acetonitrile. The flow rate was 1.0 mL/min and the injection

volume was 3.5 μL at 30 °C and detection at 200-400 nm.

16000

Caspase-3

Caspase-9

14000

12000

10000

8000

6000

4000

2000

0

Control R1 2µM R1 4µM R11 2µMR11 4µM E7010

Fig. 12 Effect of compounds RSV-1 (R1) and RSV-11 (R11) (at

a

concentration of 2 ꢀM and 4 ꢀM) on caspase-9, and -3 activity in HepG2

cells determined by fluorimetry at 48 h (E7010 is used as the positive

control).

CONCLUSION

General method for the synthesis of substituted Ethyl 2-

chloronicotinate (6): To the 2-chloronicotinic acid (20 gm) in

ethanol (50 mL) was added cat. amount of conc. sulphuric acid

(10 mL) at 0 °C. The reaction mixture was shifted on oil bath and

stirred at 80 °C. After 2 h the reaction mixture was cooled to

room temperature and neutralized with saturated solution of

NaHCO₃(150 mL), extracted with ethyl acetate. The combined

organic layers were dried over Na₂SO₄and evaporated to give

crude product which was purified by column chromatography

eluting with ethyl acetate and hexane (1:9), afford pure

compound as liquid.

These conjugates (RSV-1 to RSV-13) have been designed,

synthesized and evaluated for their antiproliferative activities

against four human cancer cell lines (A549, HeLa, DU-145,

HepG2). These compounds exhibited potent cytotoxicity with

GI₅₀values ranging from 2.0- 45.0 μM. Among these compounds

RSV-1 and RSV-11 exhibited potent activity in HepG2 cell lines

(human lung adenocarcinoma epithelial cell line) with GI₅₀

values in the range of 2.0 μM and 3.0 μM. RSV-1 was also

evaluated in a panel of sixty cell lines of the NCI that showed

promising GI₅₀values in certain cell lines. Both RSV-1 and RSV-

11 inhibited the tubulin polymerization by binding to the

colchicine binding site of the tubulin. Further, the cell cycle

analysis in HepG2 cells revealed that they showed cell cycle

arrest at G2/M phase and these studies have provided better

General method for the synthesis of substituted Ethyl 2-

(phenylamino)nicotinate (7a-h): The mixture of ethyl 2-

chloronicotinate (6, 2 gm, 1 eq.) and corresponding anilines (0.9

eq.) in ethylene glycol (5 mL) was stirred at 160 °C for 6 h. The

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reaction mixture was cooled to room temperature and poured

into water. The aqueous layer was extracted with ethyl acetate.

The combined organic layer was washed with brine, dried over

MgSO₄and concentrated in vacuo. The crude product was

purified by column chromatography eluting with EtOAc/hexane

(1:9), afford substituted ethyl 2-(phenylamino)nicotinate (7a-h).

(m, 2H), 6.80 (q, J = 3.01, 4.89 Hz, 1H), 4.38 (q, J = 7.15 Hz, 2H),

1.42 (t, J = 7.15 Hz, 3H); EI MS m/z 296 [M]⁺.

General method for the synthesis of substituted (8a-h): A flame

dried flask (2 neck 100 mL) fitted with one addition funnel and a

N₂inlet tube was charged with N,O-dimethylhydroxylamine

hydrochloride (3 eq.) in CH₂Cl₂(10 mL) and reaction mixture was

stirred at -5 °C. A solution of trimethylaluminum (3 eq.) in

toluene was added drop-wise over 30 min to the N,O-dimethyl

hydroxyamine.HCl solution. To this solution was added the

substituted ethyl 2-(phenylamino)nicotinate (1 g, 1 eq.) as a

solution in CH₂Cl₂(5 mL) over 10 min. The mixture was allowed

to warm to room temperature and then stirred at samne

temperature for 8 h. The reaction mixture was hydrolyzed with

aqueous NH₄Cl, extracted with chloroform. The combined

organic layers were dried over Na₂SO₄. Evaporation of the

solvent left a foamy material, which was purified by column

chromatography on silica gel with AcOEt/hexane to provide 8a-h

as a yellow liquid. Yield: 60-78%.

Synthesis of Ethyl 2-(phenylamino)nicotinate (7a): Yield: 85%;

m.p.: 62-64 °C; ¹H NMR (300 MHz, CDCl₃+DMSO-d₆): δ 10.22 (bs,

1H), 8.34 (dd, J = 4.7, 1.5 Hz, 1H), 8.20 (dd, J = 7.9, 2.3 Hz, 1H),

7.68 (m, 2H), 7.30 (m, 2H), 7.00 (d, J = 7.1, 1.6 Hz, 1H), 6.66 (dd, J

= 7.8, 4.7 Hz, 1H), 4.38 (q, J = 7.0 Hz, 2H), 1.43 (t, J = 7.0 Hz, 3H);

EI MS m/z 243 [M+H]⁺.

Ethyl 2-((4-fluorophenyl)amino)nicotinate (7b): Yield: 76%; m.p:

1

67-69 °C; H NMR (300 MHz, CDCl₃+DMSO-d₆): δ 10.19 (bs, 1H),

8.32 (dd, J = 4.6, 2.0 Hz, 1H), 8.22 (dd, J = 8.0, 2.0 Hz, 1H), 7.65

(m, 2H), 7.00 (m, 2H), 6.70 (dd, J = 7.3, 4.6 Hz, 1H), 4.40 (q, J =

7.3 Hz, 2H), 1.44 (t, J = 7.3 Hz, 3H); EI MS m/z 260 [M]⁺.

Ethyl 2-((4-chlorophenyl)amino)nicotinate (7c): Yield 72%; m.p:

98-100 °C; ¹H NMR (300 MHz, CDCl₃+DMSO-d₆): δ 10.26 (bs, 1H),

8.34 (q, J = 2.6 Hz, 1H), 8.20 (dd, J = 8.3, 2.6, Hz 1H), 7.67 (d, J =

9.0 Hz, 2H), 7.24 (d, J = 9.0 Hz, 2H), 6.70 (dd, J = 7.5, 4.5 Hz, 1H),

4.38 (q, J = 7.5 Hz, 2H), 1.42 (t, J = 7.5 Hz, 3H); EI MS m/z 277

[M+H]⁺.

N-Methoxy-N-methyl-2-(phenylamino)nicotinamide (8a): Yield:

1

70%; H NMR (300 MHz, CDCl₃) δ 8.92 (s, 1H), 8.29 (dd, J = 3,0,

1.5 Hz, 1H), 7.87 (dd, J = 5.2, 2.2 Hz, 1H), 7.60 (d, J = 7.5 Hz, 2H),

7.31 (t, J =8.3 Hz, 2H), 7.00 (t, J = 7.5 Hz, 2H) 6.72 (dd, J = 4.5, 3.0

Hz, 1H), 3.57 (s, 3H), 3.38 (s, 3H); MS (ESI): m/z 258 [M+H]⁺.

Ethyl 2-((4-hydroxyphenyl)amino)nicotinate (7d): Yield: 75%;

m.p: 143-145 °C; ¹H NMR (CDCl₃, 500 MHz): δ 10.01 (s, 1H), 8.31

(dd, J = 3.6, 1.5 Hz, 1H), 8.20 (dd, J = 7.8, 1.5 Hz, 1H), 7.52 (dd, J =

7.0, 2.2 Hz, 2H), 6.87 (dd, J = 7.0, 2.3 Hz, 2H), 6,64 (dd, J = 7.8,

4.6 Hz, 1H), 5.9 (s, 1H), 4.22 (q, 2H), 1.43 (t, J = 7.0 Hz, 3H); EI MS

m/z 258 [M]⁺.

2-((4-Fluorophenyl)amino)-N-methoxy-N-methylnicotinamide

(8b): Yield: 78%; ¹H NMR (300 MHz, CDCl₃) δ 9.02 (s, 1H), 8.28

(dd, J = 2.26 and 3,02, 1H), 7.91 (dd, J = 2.26 and 5.28, 1H), 7.55-

7.59 (m, 2H), 7.23-7.28 (m, 2H), 6.75 (dd, J = 3.02, 4.53, 1H) 3.58

(s, 3H), 3.39 (s, 3H); MS (ESI): m/z 276 [M+H]⁺.

2-((4-Chlorophenyl)amino)-N-methoxy-N-methylnicotinamide

(8c): Yield: 75%; ¹H NMR (300 MHz, CDCl₃) δ 9.01 (s, 1H), 8.25

(dd, J = 3.0, 2.2 Hz, 1H), 7.90 (dd, J = 2.28, 5.22 Hz, 1H), 7.60-7.55

(m, 2H), 7.29-7.22 (m, 2H), 6.78 (dd, J = 4.5, 3.0 Hz, 1H) 3.56 (s,

3H), 3.38 (s, 3H); MS (ESI): m/z 292 [M+H]⁺.

Ethyl 2-((4-methoxyphenyl)amino)nicotinate (7e): Yield: 80%;

m.p: 88-90 °C; ¹H NMR (300 MHz, CDCl₃+DMSO-d₆): δ 10.00 (s,

1H), 8.32 (dd, J = 4.6, 2.3 Hz, 1H), 8.20 (dd, J = 7.8, 2.3 Hz, 1H),

7.52 (dd, J = 7.0, 2.3 Hz, 2H), 6.88 (dd, J = 7.0, 2.3 Hz, 2H), 6,64

(dd, J = 7.8, 4.6 Hz, 1H), 4.38 (q, J = 7.0 Hz, 2H), 3.82 (s, 3H), 1.43

(t, J = 7.0 Hz, 3H); EI MS m/z 273 [M+H]⁺.

N-Methoxy-2-((4-hydroxyphenyl)amino)-N-methylnicotinamide

(8d): Yield: 68%. ¹H NMR (300 MHz, CDCl₃) δ 9.0 (s, 1H), 8.18

(dd, J = 3,0, 2.1 Hz, 1H), 7.90 (dd, J = 5.2, 2.1 Hz, 1H), 7.59-7.55

(m, 2H), 7.28-7.23 (m, 2H), 6.77 (dd, J = 4.5, 3.0 Hz, 1H), 3.58 (s,

3H), 3.36 (s, 3H); MS (ESI): m/z 274 [M+H]⁺.

Ethyl 2-((3,4-dimethoxyphenyl)amino)nicotinate (7f): Yield:

1

80%; m.p: 88-90 °C; H NMR (300 MHz, CDCl₃): δ 10.02 (s, 1H),

8.33 (dd, J = 2.6, 1.8 Hz, 1H), 8.23 (dd, J = 6.0, 1.8 Hz, 1H), 7.27

(d, J = 2.2 Hz, 1H), 7.18 (dd, J = 6.0, 2.6 Hz, 1H), 6.86 (d, J = 8.65

Hz, 1H), 6.67 (dd, J = 4.8, 3.0 Hz, 1H), 4.38 (q, 2H), 3.89 (s, 3H),

3.87 (s, 3H), 1.41 (t, 3H); EI MS m/z 302.

N-Methoxy-2-((4-methoxyphenyl)amino)-N-

1

methylnicotinamide (8e): Yield: 80%. H NMR (300 MHz, CDCl₃)

δ 9.05 (s, 1H), 8.28 (dd, J = 3,2, 2.2 Hz, 1H), 7.91 (dd, J = 5.1, 2.2

Hz, 1H), 7.59-7.45 (m, 2H), 7.28-7.21 (m, 2H), 6.75 (dd, J = 4.0,

3.1 Hz, 1H), 3.89 (s, 3H), 3.58 (s, 3H), 3.39 (s, 3H); MS (ESI): m/z

288 [M+H]⁺.

Ethyl 2-((3,4,5-trimethoxyphenyl)amino)nicotinate (7g): Yield:

75%; m.p: 143-145 °C; ¹H NMR (CDCl₃, 500 MHz): δ 10.14 (bs,

1H), 8.37 (dd, J = 5.3, 2.3 Hz, 1H), 8.25 (dd, J = 7.6, 2.3 Hz, 1H),

7.00 (s, 2H), 6.71 (dd, J = 8.3, 5.3 Hz, 1H), 4.38 (q, J = 7.5 Hz, 2H),

3.88 (s, 6H), 3.83 (s, 3H), 1.43 (t, J = 7.5 Hz, 3H); MS (EI): m/z 333

[M+H]⁺.

N-Methyl-2-((3,4-dimethoxyphenyl)amino)-N-

methylnicotinamide (8f): Yield: 77%; ¹H NMR (300 MHz, CDCl₃) δ

8.79 (S, 1H), 8.25 (dd, J =3.0, 2.2 Hz, 1H), 7.87 ( dd, J = 6.0, 1.5

Hz, 1H), 7.18 ( d, J =3.0 Hz, 1H), 7.12 ( dd, J = 6.0, 2.2 Hz, 1H),

6.85 (d, J = 8.30 Hz, 1H), 6.68 ( dd, J = 4.5, 3.0 Hz, 1H), 3.88 (s,

Ethyl 2-(3,4,5 trifluoroanilino)nicotinate (7h): Yield: 76%;

m.p:106-108 °C; ¹H NMR (500 MHz, CDCl₃): δ 10.33 (bs, 1H), 8.39

(dd, J = 1.88, 3.01 Hz, 1H), 8.27 (dd, J = 1.88, 6.02 Hz, 1H), 7.48

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3H), 3.86 (s, 3H), 3.59 (s, 3H), 3.39 (s, 3H); MS (ESI): m/z 318

[M+H]⁺.

2-((3,4-Dimethoxyphenyl)amino)nicotinaldehyde (9f): Yield:

1

95%; 98-100 °C; H NMR (300 MHz, CDCl₃) δ 10.06 (s, 1H), 8.36

(dd, J = 2.8, 1.8 Hz, 1H), 8.29 (dd, J = 6.0, 1.8 Hz, 1H), 7.42 (m,

1H), 7.28 (m, 1H), 6.98-7.06 (m, 2H), 3.98 (s, 3H), 3.97 (s, 3H);

MS (ESI): m/z 259 [M+H]⁺.

N-Methoxy-N-methyl-2-((3,4,5-

trimethoxyphenyl)amino)nicotinamide (8g): Yield: 68%; ¹H NMR

(300 MHz, CDCl₃) δ 8.77 (s, 1H), 8.26 (dd, J = 3.0, 2.2 Hz 1H), 7.86

( dd, J = 6.0, 1.5 Hz, 1H), 7.22 (s, 2H), 6.87 (dd, J = 6.0, 2.2 Hz 1H),

3.89 (s, 6H), 3.86 (s, 3H), 3.59 (s, 3H), 3.37 (s, 3H); MS (ESI): m/z

348 [M+H]⁺.

2-((3,4,5-Trimethoxyphenyl)amino)nicotinaldehyde (9g): Yield:

88%; m.p: 112-114 °C; ¹H NMR (300 MHz, CDCl₃) δ 10.38 (s, 1H),

9.88 (s, 1H), 8.41 (dd, J = 3.0 2.2 Hz, 1H), 7.86 (dd, J = 5.2, 2.2 Hz,

1H), 7.04 (s, 2H), 6.87-6.83 (m, 1H), 3.89 (s, 6H), 3.84 (s, 3H); MS

(ESI): m/z 289 [M+H]⁺.

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

trifluorophenyl)amino)nicotinamide (8h): Yield: 76%. ¹H NMR

(300 MHz, CDCl₃) δ 9.23 (s, 1H), 8.36 (dd, J = 3.1, 2.2 Hz, 1H),

7.89 ( dd, J = 6.0, 1.5 Hz, 1H), 7.32 (s, 2H), 6.87 (dd, J =6.0, 2.2

Hz, 1H), 3.59 (s, 3H), 3.37 (s, 3H); MS (ESI): m/z 312[M+H]⁺.

2-((3,4,5-Trifluorophenyl)amino)nicotinaldehyde (9h): Yield:

88%; m.p: 112-114 °C; ¹H NMR (300 MHz, CDCl₃) δ 10.48 (s, 1H),

9.89 (s, 1H), 8.45 (dd, J = 2.6, 1.8 Hz, 1H), 7.92 (dd, J = 5.6, 1.8

Hz, 1H), 7.55 (dd, J = 6.4 Hz, 2H), 6.94 (dd, J = 5.2, 2.6 Hz, 1H);

MS (ESI): m/z 253 [M+H]⁺.

General method for the synthesis of substituted 2-

aminonicotinaldehydes (9a-h): To a stirred solution of N-

methoxy-N-methyl-2-(phenylamino)nicotinamide (8a-h, 1 g 1

eq.) in DCM (15 mL) was added di-iso-butylaluminum hydride

(1.5 eq.) at -78 °C inert condition. After stirring for 45 min, the

solution was quenched with methanol. The reaction mixture was

extracted with CH₂Cl₂and dried over MgSO₄. After evaporation

of the solvent, the residue was purified by silica-gel column

chromatography to give corresponding aldehydes (9a-h). Yields:

80-90%.

General procedure for the synthesis of substituted benzylalcols

(12a-b): To a stirred solution of substituted benzalldehydes

(11a-b, 5 g, 1 eq.) in methanol (30 mL) was added NaBH₄(1.5

eq.)) at 0 °C. The reaction mixture was stirred at rt for 3 h. After

completion of reaction, methanol was evaporated under

reduced pressure and diluted with water and ethyl acetate. The

organic layer was extracted, dried over Na₂SO₄and

concentrated in vacuum to give crude compound which was

purified by column chromatography eluting with ethyl

acetate/hexane to give pure compound as white solid (12a-b).

2-(Phenylamino)nicotinaldehyde (9a): Yield: 92%; m.p: 75-77 °C

¹H NMR (300 MHz, CDCl₃) δ 10.44 (S, 1H), 9.80 (s, 1H), 8.41 (dd, J

= 2.6, 1.8 Hz, 1H), 7.86 (dd, J = 2.6, 1.8 Hz, 1H), 7.74 (d, J = 7.9

Hz, 2H), 7.36 (t, J = 8.2, 7.5 Hz, 2H), 7.09 (m, 1H), 6.83 (dd, J =

4.89, 2.63 Hz, 1H); MS (ESI): m/z 199 [M+H]⁺.

(3,4,5-Trimethoxyphenyl)methanol (12a): Yield: 91%; m.p: 45-

1

46 °C; H NMR (CDCl₃, 400 MHz): δ 6.92 (s, 2H), 4.64 (d, J=5.12

Hz, 2H), 3.90 (s, 3H), 3.88 (s, 6H). EI MS: m/z 199 [M+H]⁺.

(3,5-Dimethoxyphenyl)methanol (12b): Yield: 96%; m.p: 52-54

C; ¹H NMR (CDCl₃, 400 MHz): δ 6.50 (s, 2H). 6.36 (s, 1H), 4.59-

4.60 (d, J=4.01 Hz, 2H), δ 3.78 (s, 6H).EI MS: m/z 169 [M+H]⁺.

2-((4-Fluorophenyl)amino)nicotinaldehyde (9b): Yield: 85%; 79-

81 °C ¹H NMR (300 MHz, CDCl₃) δ 10.36 (s, 1H), 9.87 (s, 1H), 8.39

(d, J = 2.2 Hz, 1H), 7.87 (dd, J = 5.2, 2.2 Hz, 1H), 7.63-7.70 (m,

2H), 7.01-7.09 (m, 2H), 6.83 (dd, J = 4.5, 3.0 Hz, 1H); MS (ESI):

m/z 216 [M]⁺.

General procedure for the synthesis of (13a-b): To a stirred

solution of substituted benzylalcohols (12a-b, 4 g, 1 eq.) in

CH₂Cl₂(30 mL) was added PBr₃(1 eq.) at 0 °C. The reaction

mixture was stirred at rt for 4 h. After completion of reaction,

reaction mixture poured into ice cold water and extracted with

DCM, dried over Na₂SO₄and concentrated in vacuum to give

pure compound as a solid (13a-b).

2-((4-Chlorophenyl)amino)nicotinaldehyde (9c): Yield: 85%; 82-

84 °C; ¹H NMR (300 MHz, CDCl₃) δ 10.35 (s, 1H), 9.86 (s, 1H),

8.37 (d, J = 2.2 Hz, 1H), 7.85 (dd, J = 5.2, 2.2 Hz, 1H), 7.64-7.70

(m, 2H), 7.00-7.09 (m, 2H), 6.83 (dd, J = 4.5, 3.0 Hz, 1H); MS

(ESI): m/z 232[M]⁺.

5-(Bromomethyl)-1,2,3-trimethoxybenzene (13a): 93%; m.p: 73-

75 °C; ¹H NMR (CDCl₃, 400 MHz) δ 6.58 (s, 2H), 4.42 (s, 2H), 3.82

(s, 9H). EI MS: m/z 261 [M+H]+.

2-((4-Hydroxyphenyl)amino)nicotinaldehyde (9d): Yield: 71%;

m.p: 88-91 °C ¹H NMR (300 MHz, CDCl₃): δ 10.25 (s, 1H), 9.87 (s,

1H), 8.35 (dd, J = 3.0, 1.5 Hz, 1H), 7.82 (dd, J = 5.6, 1.5 Hz, 1H),

7.58 (d, J = 8.6 Hz, 2H), 6.92-6.89 (m, 2H), 6.76 (dd, J = 4.8, 2.6

Hz, 1H); MS (ESI): m/z 215 [M+H]⁺.

1-(Bromomethyl)-3,5-dimethoxybenzene (13b): Yield: 95%; m.p:

1

76-78 °C; H NMR (400 MHz, CDCl3): δ 6.54 (s, 2H), 6.39 (s, 1H),

4.42 (s, 2H), 3.79 (s, 6H). EI MS: m/z 231 [M+H]+.

General procedure for the synthesis of substituted

Benzyltriphenylphosphonium bromide (14a-b): To a stirred

solution of substituted benzylbromides (13a-b, 3 g, 1 eq.) in

toluene (20 mL) was added PPh₃(1.3 eq.) at 0 °C. The reaction

mixture was stirred at 100 °C for 6 h. After completion of

reaction, solid was filtered through Buckner funnel and washed

with hexane to give pure compound as a white solid (14a-b).

2-((4-Methoxyphenyl)amino)nicotinaldehyde (9e): Yield: 92%;

m.p: 88-90°C; ¹H NMR (300 MHz, CDCl₃) δ 10.26 (s, 1H), 9.86 (s,

1H), 8.37 (dd, J = 3.0, 1.3 Hz, 1H), 7.84 (dd, J = 5.6, 1.8 Hz, 1H),

7.58 (d, J = 8.6 Hz, 2H), 6.92 (d, J = 8.6 Hz, 2H), 6.76 (dd, J = 4.8,

2.6 Hz, 1H) 3.81 (s, 3H); MS (ESI): m/z 229 [M]⁺.

10 | J. Name., 2012, 00, 1-3

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General method for the synthesis of desired E-R conJugates

(RSV-1 to RSV-13): To a stirred solution of bromotriphenyl(3,4,5-

trimethoxybenzyl)phosphorane (792 mg, 1.5 eq.) in THF (10 mL)

was added sodium hydride (1.5 eq.) at 0 °C. After 10 min 2-

(phenylamino)nicotinaldehyde (200 mg, 1.01 mmol) was added

to the reaction mixture at 0 °C. Ice bath was removed and then

reaction was allowed for stirring at room temperature for 4 h.

After 4h, reaction mixture was neutralised with NH₄Cl and then

aqueous layer was extracted with ethyl acetate (4x20 mL), the

combined organic layer was dried over Na₂SO₄and the solvent

was evaporated under vacuum to afford the crude product

which was purified by column chromatography on silica gel with

AcOEt/hexane to provide RSV-1 to RSV-13 in good yields.

found 379.16486 [M+H]⁺. Anal. Calcd for C₂₂H₂₂N₂O₄: C, 69.83; H,

5.86; N, 7.40; Found: C, 69.78; H, 5.99; N, 7.94. HPLC: tR 5.6 min,

purity 97.9%.

(E)-N-(4-Methoxyphenyl)-3-(3,4,5-trimethoxystyryl)pyridin-2-

1

amine (RSV-5): Yield: 75%; m.p: 186-188 °C; H NMR (300 MHz,

CDCl₃): δ 8.12 (dd, J = 3.3, 1.5 Hz, 1H), 7.64 (dd, J = 6.0, 1.6 Hz,

1H), 7.32 (d, J = 8.7 Hz, 2H), 6.93 (s, 2H), 6.89 (d, J = 8.7 Hz, 2H),

6.76-6.79 (m, 1H), 6.68 (s, 2H), 6.34 (s, 1H), 3.89 (s, 6H), 3.87 (s,

3H), 3.78 (s, 3H). ¹³C NMR (125 MHz, CDCl₃): δ 155.3, 153.2,

146.9, 138.1, 134.9, 133.6, 132.5, 132.3, 122.5, 122.3, 119.7,

115.1, 114.1, 103.4, 60.8, 55.9, 55.4. MS (ESI): [M+H]⁺= 393.

HRMS (ESI) for C₂₃H₂₅N₂O₄, calcd 393.18088, found 393.18057

[M+H]⁺. Anal. Calcd for C₂₃H₂₅N₂O₄: C, 70.39; H, 6.16; N, 7.14;

Found: C, 70.78; H, 6.39; N, 7.27. HPLC: tR 5.2 min, purity 96.9%.

(E)-N-Phenyl-3-(3,4,5-trimethoxystyryl)pyridin-2-amine (RSV-1):

Yield: 72%; m.p: 161-163 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.17

(d, J = 3.9 Hz, 1H), 7.67 (d, J = 6.0 Hz, 1H), 7.43 (d, J = 7.9 Hz, 2H),

7.3 (m, 2H), 7.00 (m, 1H), 6.93 (s, 2H), 6.82-6.85 (m, 1H), 6.66 (s,

2H), 6.49 (s, 1H), 3.88 (s, 6H), 3.86 (s, 3H). ¹³C NMR (125 MHz,

CDCl₃): δ 160.9, 152.6, 147.0, 140.6, 138.7, 135.2, 132.8, 128.8,

123.2, 122.1, 120.3, 119.6, 115.9, 104.5, 100.3, 55.3. MS (ESI):

[M+H]⁺= 363. HRMS (ESI) for C₂₂H₂₂N₂O₃, calcd 363.17032,

found363.17007 [M+H]⁺. Anal. Calcd for C₂₂H₂₂N₂O₃: C, 72.91; H,

6.12; N, 7.73; Found: C, 72.66; H, 6.61; N, 7.11. HPLC: tR 4.9 min,

purity 98.9%.

(E)-N-(3,4-Dimethoxyphenyl)-3-(3,4,5-trimethoxystyryl)pyridin-

2-amine (RSV-6): Yield: 71%; m.p: 210-212 °C; ¹H NMR (300

MHz, CDCl₃): δ 8.13 (dd, J = 3.3, 1.6 Hz, 1H), 7.65 (dd, J = 6.0, 1.6

Hz, 1H) 7.10 (d, J =2.2 Hz, 1H), 6.90-7.05 (m, 2H), 6.78-6.85 (m,

2H), 6.61 (d, J = 2.2 Hz, 2H), 6.43 (s, 2H), 3.87 (s, 3H), 3.86 (s,

3H), 3.82 (s, 9H). ¹³C NMR (125 MHz, CDCl₃): δ 160.1, 153.7,

150.6, 149.0, 139.6, 138.4, 136.3, 135.7, 135.0, 133.4, 127.5,

116.3, 114.5, 113.5, 112.8 106.0, 1038, 56.4, 60.9. MS (ESI):

[M+H]⁺= 423. HRMS (ESI) for C₂₄H₂₆N₂O₅, calcd 423.28402,

found 423.28067 [M+H]⁺. Anal. Calcd for C₂₄H₂₆N₂O₅: C, 68.23; H,

6.20; N, 6.63; Found: C, 68.56; H, 6.48; N, 6.36. HPLC: tR 6.1 min,

purity 95.9%.

(E)-N-(4-Fluorophenyl)-3-(3,4,5-trimethoxystyryl)pyridin-2-

1

amine (RSV-2): Yield: 70%; m.p: 167-169 °C; H NMR (300 MHz,

CDCl₃): δ 8.17 (dd, J = 3.3, 1.5 Hz, 1H), 7.68 (dd, J = 6.0, 1.5 Hz,

1H), 7.42 (d, J = 8.8 Hz, 2H), 7.25 (d, J = 3.2 Hz, 2H), 6.93 (s, 2H),

6.84-6.89 (m, 1H), 6.69 (s, 2H), 6.45 (s, 1H), 3.90 (s, 6H), 3.88 (s,

3H). ¹³C NMR (125 MHz, CDCl₃): δ 161.2, 152.7, 147.2, 139.1,

138.5, 135.3, 133.2, 128.7, 126.9, 123.9, 120.6, 116.4, 104.6,

100.3, 55.3. MS (ESI): [M+H]⁺=381. HRMS (ESI) for C₂₂H₂₁FN₂O₃,

calcd, 381.16090 found 381.16061 [M+H]+. Anal. Calcd for

(E)-N-(3,4,5-Trimethoxyphenyl)-3-(3,4,5-

trimethoxystyryl)pyridin-2-amine (RSV-7): Yield: 65%; m.p: 240-

242 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.19 (d, J = 3.5 Hz, 1H), 7.68

(d, J = 7.1 Hz, 1H), 6.96 (d, J = 2.2 Hz, 2H), 6.86 (dd, J = 5.0, 2.2

Hz, 1H), 6.72 (d, J = 10.8 Hz, 4H), 6.36 (s, 1H), 3.90 (s, 6H), 3.87

(s, 3H), 3.84 (s, 6H), 3.81 (s, 3H). ¹³C NMR (125 MHz, CDCl₃): δ

153.5, 152.7, 146.6, 140.7, 135.5, 132.7, 132.5, 122.3, 120.8,

119.7, 116.0, 115.8, 103.7, 98.1, 60.1, 56.1. MS (ESI): [M+H]⁺=

453. HRMS (ESI) for C₂₃H₃₀N₂O₆Na, calcd 453.19961, found

453.20038 [M+ Na]⁺. Anal. Calcd for C₂₃H₃₀N₂O₆: C, 66.36; H,

6.24; N, 6.19; Found: C, 67.00; H, 6.67; N, 6.23. HPLC: tR 5.1 min,

purity 98.9%.

C₂₂H₂₁FN₂O₃: C, 69.46; H, 5.56; N, 7.36; Found: C, 69.57; H, 5.69;

N, 7.08. HPLC: tR 5.9 min, purity 97.7%.

(E)-N-(4-Chlorophenyl)-3-(3,4,5-trimethoxystyryl)pyridin-2-

amine (RSV-3): Yield: 68%m.p: 172-174 °C; ¹H NMR (300 MHz,

CDCl₃): δ 8.17 (dd, J = 3.0, 1.5 Hz, 1H), 7.68 (dd, J = 6.0, 1.5 Hz,

1H), 7.42 (d, J = 8.8 Hz, 2H), 7.25-7.28 (m, 2H), 6.93 (s, 2H), 6.84-

6.89 (m, 1H), 6.69 (s, 2H), 6.45 (s, 1H), 3.90 (s, 6H), 3.88 (s, 3H).

¹³C NMR (125 MHz, CDCl₃): δ 160.0, 152.3, 147.0, 139.2, 138.6,

135.3, 133.2, 128.7, 126.7, 122.9, 120.4, 116.2, 104.6, 100.3,

55.3. MS (ESI): [M+H]⁺= 397. HRMS (ESI) for C₂₂H₂₁ClN₂O₂, calcd

397.17031, found 397.17016 [M+H]⁺. Anal. Calcd for

C₂₂H₂₁ClN₂O₂: C, 66.58; H, 5.33; N, 7.06; Found: C, 66.77; H, 5.19;

N, 7.14. HPLC: tR 5.3 min, purity 97.3%.

(E)-N-(3,4,5-Trifluorophenyl)-3-(3,4,5-trimethoxystyryl)pyridin-

2-amine (RSV-8): Yield: 65%; m.p: 245-247 °C; ¹H NMR (300

MHz, DMSO-d₆): δ 8.19 (d, J = 3.5 Hz, 1H), 7.67 (d, J = 7.1 Hz,

1H), 7.20-7.23 (m, 2H), 6.90-6.93 (m, 3H), 6.69 (s, 2H), 6.53 (s,

1H). ¹³C NMR (125 MHz, DMSO-d₆): δ 151.1, 147.3, 140.3, 136.2,

134.7, 131.2, 122.0, 116.2, 114.6, 104.3, 101.6, 101.3, 58.4, 54.4.

MS (ESI): [M+H]⁺417. HRMS (ESI) for C₂₂H₂₀F₃N₂O₃, calcd

417.14205, found 417.14099 [M+H]⁺. Anal. Calcd for

C₂₂H₁₉F₃N₂O₃: C, 63.46; H, 4.60; N, 6.73;Found: C, 63.50; H, 4.87;

N, 6.21. HPLC: tR 5.6 min, purity 97.3%.

(E)-4-((3-(3,4,5-Trimethoxystyryl)pyridin-2-yl)amino)phenol

1

(RSV-4): Yield: 70%; m.p: 185-187 °C; H NMR (300 MHz, CDCl₃):

δ 8.10 (dd, J =3.0, 1.6 Hz, 1H), 7.66 (dd, J = 6.0, 1.6 Hz, 1H), 7.14

(d, J = 8.68 Hz, 2H), 6.94-7.02 (m, 2H), 6.76-6.81 (m, 1H), 6.69 (d,

J = 5.6 Hz, 4H), 6.31 (s, 1H), 3.90 (s, 6H), 3.87 (s, 3H). ¹³C NMR

(125 MHz, CDCl₃): δ 153.1, 152.9, 146.7, 138.4, 133.9, 133.0,

131.2, 131.0, 122.8, 12.1, 119.7, 115.4, 113.6, 103.4, 55.3. MS

(ESI): [M+H]⁺= 379. HRMS (ESI) for C₂₂H₂₃N₂O₄, calcd 379.16523,

(E)-3-(3,5-Dimethoxystyryl)-N-phenylpyridin-2-amine (RSV-9):

Yield: 75%; m.p: 144-146 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.18

(dd, J = 3.0, 1.6 Hz, 1H), 7.68 (dd, J = 6.0, 1.6 Hz, 1H), 7.46 (d, J =

7.55 Hz, 2H), 7.31 (m, 2H), 6.91-7.05 (m, 3H), 6.82-6.87 (m, 1H),

6.6 (d, J =2.2 Hz, 2H), 6.55 (s, 1H), 6.43 (t, J = 2.2 Hz, 1H), 3.82 (s,

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Journal Name

6H). ¹³C NMR (125 MHz, CDCl₃): δ 160.9, 152.6, 147.0, 140.6,

138.7, 135.2, 132.8, 128.8, 123.2, 122.1, 120.3, 119.6, 115.9,

104.5, 100.3, 55.3. MS (ESI): 333. HRMS (ESI) for C₂₁H₂₁N₂O₂,

calcd 333.15975, found 333.15836 [M+H]⁺. Anal. Calcd for

C₂₁H₂₀N₂O₂: C, 75.88; H, 6.06; N, 8.43; Found: C, 75.60; H, 6.96;

N, 8.71. HPLC: tR 5.5 min, purity 97.8%.

In vitro antitumour screening: The synthesized compounds

(RSV1-RSV13) have been evaluated for their in vitro cytotoxicity

in four different human cancer cell lines (A549, HeLa, HepG2 and

DU-145). A protocol of 48 h continuous drug exposure has been

used and a sulforhodamine B (SRB) protein assay has been

employed to estimate cell viability or growth as described earlier

The cell lines were grown in Dulbecco’s modified Eagle’s

medium containing 10% fetal bovine serum and 2mM L-

glutamine and were seeded into 96-well plates in 100 µL at

plating densities depending on the doubling time of individual

cell lines. The plates were incubated at 37o C, 5% CO2, 95% air,

and 100% relative humidity for 24 h prior to addition of

experimental drugs. Aliquots of 3 µL of the drug dilutions were

added to cells resulting in the required final drug

concentrations. For each compound four concentrations (0.1, 1,

5 and 10 µM) were evaluated and each were done in duplicate

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 incubated for 60 min at

4 °C. The plates were washed five times with 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 wavelength of 560 nm. Percent growth was

calculated on a plate-by-plate basis for test wells relative to

control wells. The above determinations were repeated three

times. Percentage growth was expressed as the (ratio of average

absorbance of the test well to the average absorbance of the

(E)-3-(3,5-Dimethoxystyryl)-N-(4-fluorophenyl)pyridin-2-amine

(RSV-10): Yield: 77%; m.p: 151-152 °C; ¹H NMR (300 MHz,

CDCl₃): δ 8.21 (dd, J = 3.0, 1.5 Hz, 1H), 7.73 (dd, J = 6.0, 1.5 Hz,

1H), 7.41 (s, 1H), 7.38 (s, 1H), 7.28 (s,1H), 7.26 (s, 1H), 6.95 (d, J =

1.5 Hz, 2H), 6.88 (m, 1H) 6.77 ( bs, 1H), 6.59 (d, J = 1.5 Hz, 2H),

6.43 (m, 1H), 3.82 (s, 6H). ¹³C NMR (125 MHz, CDCl₃): δ 161.1,

153.3, 147.0, 140.2, 139.6, 135.3, 135.2, 128.7, 126.7, 122.9,

120.4, 116.2, 104.6, 100.3, 55.3. MS (ESI): 351 [M+H]⁺. HRMS

(ESI) for C₂₁H₂₀FN₂O₂, calcd 351.15974, found 351.15836 [M+H]⁺.

Anal. Calcd for C₂₁H₁₉FN₂O₂: C, 71.98; H, 5.47; N, 8.00; Found: C,

71.45; H, 5.99; N, 8.51. HPLC: tR 5.5 min, purity 98.1%.

(E)-N-(4-Chlorophenyl)-3-(3,5-dimethoxystyryl)pyridin-2-amine

(RSV-11): Yield: 75%; m.p: 163-165 °C; ¹H NMR (300 MHz,

CDCl₃): δ 8.16 (dd, J = 3.0, 1.6 Hz, 1H), 7.70 (dd, J = 6.0, 1.6 Hz,

1H), 7.41 (d, J = 7.4 Hz, 2H), 7.25-7.28 (m, 2H), 6.94 (d, J = 1.5 Hz,

2H), 6.86-6.90 (m, 1H), 6.77 (s, 1H), 6.59 (d, J = 1.5 Hz,2H), 6.43

(t, J =2.2 Hz, 1H), 3.82 (s, 6H). ¹³C NMR (125 MHz, CDCl₃): δ

161.0, 152.3, 147.0, 139.2, 138.6, 135.3, 133.2, 128.7, 126.7,

122.9, 120.4, 116.2, 104.6, 100.3, 55.3. MS (ESI); 367 [M+H]⁺.

HRMS (ESI) for C₂₁H₂₀ClN₂O₂, calcd 367.17032, found 367.17007

[M+H]⁺. Anal. Calcd for C₂₁H₁₉ClN₂O₂: C, 68.76; H, 5.22; N, 7.64;

Found: C, 68.23; H, 5.29; N, 7.01. HPLC: tR 5.7 min, purity 97.3%.

(E)-3-(3,5-Dimethoxystyryl)-N-(4-methoxyphenyl)pyridin-2-

amine (RSV-12): Yield: 78%; m.p: 182-184 °C; ¹H NMR (300 MHz,

CDCl₃): δ 8.16 (dd, J = 3.6, 1.5 Hz, 1H), 7.67 (dd, J = 6.0, 1.5 Hz,

1H), 7.10 (d, J = 2.2 Hz, 2H), 6.90-7.05 (m, 3H), 6.78-6.85 (m, 2H),

control wells) 100. Growth inhibition of 50% (GI₅₀

) was

calculated from [(T0 - T)/(C - T0)] * 100 = 50, which is the drug

concentration resulting in a 50% reduction in the net protein

increase (as measured by SRB staining) in control cells during the

drug incubation. Where, T0 = Optical density at time zero, OD of

control = C, and OD of test growth in the presence of drug = T.

6.62 (d, J =2.2 Hz, 2H), 6.43 (s, 2H), 3.88 (s, 3H), 3.82 (s, 6H). ¹³

C

NMR (125 MHz, CDCl₃): δ 161.5, 160.1, 153.3, 148.0, 140.5,

135.0, 133.4, 133.2, 121.7, 116.2, 115.1, 113.3, 105.5, 100.9,

55.8, 55.1. MS (ESI) 363 (M+H)⁺. HRMS (ESI) for C₂₂H₂₂N₂O₃,

calcd 363.19065, found 363.19053 [M+H]⁺. Anal. Calcd for

C₂₂H₂₂N₂O₃: C, 72.91; H, 6.12; N, 7.73; Found: C, 72.84; H, 6.54;

N, 7.71. HPLC: tR 6.2 min, purity 98.7%.

Tubulin polymerization assay: An in vitro assay for monitoring

the time dependent polymerization of tubulin to microtubules

was performed employing

a fluorescence-based tubulin

polymerization assay kit (BK011P, Cytoskeleton, Inc.) according

to the manufacturer’s protocol. The reaction mixture in a final

volume of 10 µL in PEM buffer (80 MM PIPES, 0.5 mm EGTA,

2mM MgCl₂, Ph 6.9) contained, 2 mg/mL porcine brain tubulin,

fluorescent reporter, 1 mM GTP in the presence or absence of

test compounds (RSV1-RSV13) (3 µM) at 37 °C. Tubulin

polymerization was followed by monitoring the fluorescence -

(E)-N-(3,4-Dimethoxyphenyl)-3-(3,5-dimethoxystyryl)pyridin-2-

1

amine (RSV-13): Yield: 75%; m.p:190-192 °C; H NMR (300 MHz,

CDCl₃): δ 8.15 (dd, J = 3.7, 1.5 Hz, 1H), 7.67 (dd, J = 6.0, 1.5 Hz,

1H), 7.10 (d, J = 2.2 Hz, 1H), 6.90-7.05 (m, 3H), 6.78-6.85 (m, 2H),

6.62 (d, J =2.2 Hz, 2H), 6.43 (s, 2H), 3.87 (s, 3H), 3.86 (s, 3H), 3.82

(s, 6H). ¹³C NMR (125 MHz, CDCl₃): δ 161.0, 153.2, 149.1, 147.1,

144.9, 138.8, 135.2, 134.1, 132.7, 123.3, 119.8, 115.3, 112.7,

111.7, 105.9, 104.6, 100.3, 56.2, 55.8, 55.3. MS (ESI): 393

[M+H]⁺. HRMS (ESI) for C₂₃H₂₅N₂O₄, calcd 393.18088, found

393.18054 [M+H]⁺. Anal. Calcd for C₂₃H₂₄N₂O₄: C, 70.39; H, 6.16;

N, 7.14; Found: C, 70.98; H, 6.20; N, 7.09. HPLC: tR 6.7 min,

purity 96.8%.

enhancement due to the incorporation of

a fluorescence

reporter into microtubules as polymerization proceeds.

Fluorescence emission at 420 nm (excitation wavelength is 360

nm) was measured for 1 h at 1 min interval in a multimode plate

reader (Tecan multimode reader). Nocodazole was used as a

positive control under similar experimental conditions.

The IC₅₀value was defined as the drug concentration required to

inhibit 50% of tubulin assembly compared to controls. The

Biological evaluation:

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reaction mixture for these experiments include: 10 µL in PEM

buffer (80 MM PIPES, 0.5 mM EGTA, 2 mM MgCl₂, Ph 6.9)

contained, 2 mg/mL porcine brain tubulin, fluorescent reporter,

1 mM GTP in the presence or absence of test compounds RSV1,

RSV11 at 2.5 mM, 5 mM, 10 mM, and 15 mM concentrations,

different concentrations of E7010 was used as positive control in

this study. Polymerization was monitored by increase in the

fluorescence emission at 420 nm (excitation wavelength is 360

nm) was measured for 1 h at 1 min interval in a multimode plate

reader (Tecan multimode reader)

Caspase assay: HepG2 cells were plated in 6 well plates, grown

to 60-80% confluence, and treated with either no drug or at a

concentrations (2 µM & 4µM) of test compounds RSV1 and

RSV11. E7010 was used as positive control. After 48 hours cells

were collected by scraping and washed with PBS, centrifuged to

collect pellet. The cells were lysed in 200 µL of 1X lysis buffer by

purging at least through an insulin syringe followed by

incubation on ice for 10-20min. The lysate was centrifuged at

13,200 rpm for 20min at 4 °C and transferred the supernatant to

fresh tubes. In a 96 well black polystyrene plate, 50 µL of 2x

assay buffer, 50 µL cell lysate and 2 µL of caspase-3 & 9

substrates were taken. The reaction was allowed to take place

for 1 h. The fluorescence generated by the release of the

fluorogenic group AFC on cleavage by caspase-3 & 9 was

measured by excitation at 400 nm and emission at 505 nm for

every 5 min over 1 h. Protein was estimated by Bradford’s

method and normalized accordingly using Tecan multimode

reader.

Cell cycle analysis: human liver carcinoma cell line (HepG2) in 6

well plates were incubated for 48 h in the presence or absence

of tested compounds RSV1 and RSV11 (2 & 4 µM). Cells were

harvested with Trypsin-EDTA, fixed with ice-cold 70% ethanol at

4 °C for 30 min, ethanol was removed by centrifugation and cells

were stained with 250 mL of DNA staining solution [10mg of

Propidium Iodide (PI), 0.1mg of trisodium citrate, and 0.03 mL of

Triton X-100 were dissolved in 100mL of sterile water] at room

temperature for 30 min in the dark. The DNA contents of 20,000

events were measured by flow cytometry (DAKO CYTOMATION,

Beckman Coulter, Brea, CA). Histograms were analyzed using

Summit Software.

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Design, synthesis and antiproliferative activity of the new conjugates of E7010 and resveratrol as tubulin polymerization inhibitors

DOI: 10.1039/c5ob02022k

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