Design and Synthesis of Aminostilbene-Arylpropenones as Tubulin Polymerization Inhibitors

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DOI: 10.1002/cmdc.201402256

Design and Synthesis of Aminostilbene–Arylpropenones

as Tubulin Polymerization Inhibitors

Ahmed Kamal,*^{[a, c]}G. Bharath Kumar,^[a]Sowjanya Polepalli,^[b]Anver Basha Shaik,^[a]

Vangala Santhosh Reddy,^[a]M. Kashi Reddy,^[a]Ch. Ratna Reddy,^[a]Rasala Mahesh,^[a]

Jeevak Sopanrao Kapure,^[c]and Nishant Jain^[b]

A series of aminostilbene—arylpropenones were designed and

synthesized by Michael addition and were investigated for

their cytotoxic activity against various human cancer cell lines.

Some of the investigated compounds exhibited significant an-

tiproliferative activity against a panel of 60 human cancer cell

lines of the US National Cancer Institute, with 50% growth in-

hibition (GI₅₀) values in the range from <0.01 to 19.9 mm. One

of the compounds showed a broad spectrum of antiprolifera-

tive efficacy on most of the cell lines, with a GI₅₀value of

<0.01 mm. All of the synthesized compounds displayed cyto-

toxicity against A549 (non-small-cell lung cancer), HeLa (cervi-

cal carcinoma), MCF-7 (breast cancer), and HCT116 (colon carci-

noma) with 50% inhibitory concentration (IC₅₀) values ranging

from 0.011 to 8.56 mm. A cell cycle assay revealed that these

compounds arrested the G2/M phase of the cell cycle. Two

compounds exhibited strong inhibitory effects on tubulin as-

sembly with IC₅₀values of 0.71 and 0.79 mm. Moreover, dot-

blot analysis of cyclin B1 demonstrated that some of the con-

geners strongly induced cyclin B1 protein levels. Molecular

docking studies indicated that these compounds occupy the

colchicine binding site of tubulin.

Introduction

Tubulin binding agents are known to play a major role in

cancer chemotherapy, and well-known tubulin binders such as

paclitaxel, vinca alkaloids, vincristine, and vinblastine are rou-

tinely employed as anticancer agents.^[1]Microtubule, a polymer

of tubulin with a and b subunits, is involved in signal transduc-

tion pathways of mammalian cells, particularly in cell division.^[2]

Hence, antitubulin agents block the transition from interphase

to mitosis^[3]and, thus, effectively block cell-cycle progression,

which results in apoptosis.^[4]Antimitotic agents derived from

natural and synthetic products generally exert their effect as

microtubule stabilizers or polymerizing agents, such as Taxol,

paclitaxel, and docetaxel,^[5]which block microtubule disassem-

bly. They bind at the b-tubulin site in the microtubules and are

used in the treatment of carcinomas, such as lung, breast,

ovarian, and bladder. In contrast, microtubule destabilizers

such as colchicinium,^[6]vinca alkaloids,^[7]and combretastatin A-

4

^[8]bind at the b-tubulin site in microtubules and cause the de-

polymerization of the microtubules. Many of such agents man-

ifest different limitations in their clinical utility owing to drug

resistance and neurotoxicity;^[9]therefore, the development of

new microtubule targeting agents is of significance.

Combretastatin A-4 (CA-4, 1a; Figure 1) is an excellent tubu-

lin polymerization inhibitor that binds to the colchicine bind-

ing site of tubulin and demonstrates cytotoxicity against

a broad spectrum of human cancer cell lines including multi-

drug-resistant cancer cells. However, the in vivo efficiency of

CA-4 is limited owing to its poor pharmacokinetics resulting

from its high lipophilicity and low water solubility. Structural

modification of CA-4 has led to the development of a number

of new CA-4 derivatives that are potent tubulin polymerization

inhibitors,^[10]including combretastatin A-4 phosphate^[11,12](CA-

4P, 1b; Figure 1) and CA-1 disodium phosphate (CA-1P,

Oxi4503) as prodrugs. The water-soluble prodrugs CA-4P and

CA-1P have reached the most advanced stage of preclinical de-

velopment. Currently, CA-4P is under investigation in phase II

trails for ovarian, lung, and anaplastic thyroid cancers, and it

displays selective toxicity towards the vasculature of the

tumor.^[13]However, as its potentiality appears to be uncer-

tain,^[14]many research groups are looking to improve this scaf-

fold by different structural modifications. Some recent studies

have shown that amino-substituted combretastatin derivatives

have potential for further development. In addition, increased

water solubility of a serine prodrug of combretastatin amine

(1d, ombrabulin, AVE-8062) has a stronger effect on tumor

blood flow; this leads to complete blockage of the nutrient

supply to the solid tumor and thereby leads to necrosis.^[15,16]

[a] Dr. A. Kamal, G. B. Kumar, A. B. Shaik, V. S. Reddy, M. K. Reddy, C. R. Reddy,

R. Mahesh

Medicinal Chemistry & Pharmacology

CSIR-Indian Institute of Chemical Technology Hyderabad, 500 007 (India)

E-mail: ahmedkamal@iict.res.in

[b] S. Polepalli, Dr. N. Jain

Centre for Chemical Biology

CSIR-Indian Institute of Chemical Technology Hyderabad, 500 007 (India)

[c] Dr. A. Kamal, J. S. Kapure

Department of Medicinal Chemistry

National Institute of Pharmaceutical Education & Research (NIPER)

Hyderabad-500 037 (India)

Supporting information (¹H NMR, ¹³C NMR, and HRMS spectra of amino-

stilbene–arylpropenones 3a–h, 4a–h, and 5a–f) for this article is avail-

able on the WWW under http://dx.doi.org/10.1002/cmdc.201402256.

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ylpropenones 2a offer excellent

possibilities for hybridization to-

wards the design of improved

tubulin polymerization inhibitors.

In addition, the presence of an

aryl ring in 2a provides an op-

portunity to introduce a privi-

leged indole scaffold in such

a design. We have been previ-

ously involved in the develop-

ment of new heterocyclic scaf-

folds such as combretastatin–

amidobenzothiazole conjugates,

and benzofurans were shown to

be potential inhibitors of tubulin

polymerization with significant

cytotoxic activity.^[23,24]In continu-

ation of our earlier efforts and

on the basis of literature find-

ings, we linked amino-substitut-

ed combretastatin with arylami-

no-2-propenones to generate

aminostilbene–arylpropenone

hybrids. The 3,4,5-trimethoxy-

phenyl unit constitutes the

A ring, which is utilized without

any structural modification in

view of its pharmacophoric im-

portance. The aryl units repre-

sented by the B ring and C ring

are varied to incorporate differ-

ent substituents including the

indole framework in some cases.

Figure 1. Chemical structure of microtubule targeting agents. CA-4 (1a), CA-4P (1b), AVE8063 (1c), AVE8062 (1d),

(Z)-1-(2-bromo-3,4,5-trimethoxyphenyl)-3-[(3-hydroxy-4-methoxyphenyl)amino]prop-2-en-1-one (2a), BPR0L075

(2b), D-24851 (Indibulin, 2c), aminostilbene–arylpropenones 3a–h, 4a–h, and 5a–f.

Structure–activity relationship (SAR) studies of CA-4 analogues

have shown that both hydroxy and amino substituents con- Results and Discussion

tribute to improve the antimitotic activity and provide the op-

portunity to structurally modify this scaffold.^[17]

Chemistry

Recently, a series of antiproliferative compounds based on

the (Z)-1-aryl-3-arylamino-2-propen-1-one scaffold of 2a

(Figure 1) were reported to significantly inhibit multidrug-re-

sistant cancer cells and were also found to arrest the cells in

the G2/M phase of the cell cycle.^[18]A number of tubulin poly-

merization inhibitors containing an indole core nucleus have

been obtained from natural sources and generated through

design and synthesis. Some of the indole-containing com-

pounds exhibit excellent antimitotic activity such as D-64131,

D-24851, BPR0L075, BLF 61-3, and methyl 5-methoxy-3-((3,4,5-

trimethoxyphenyl)thio)-1H-indole-2-carboxylate (ATI ) deriva-

The syntheses of aminostilbene–arylpropenones 3a–h, 4a–h,

and 5a–f described in this study are outlined in Schemes 1

and 2. The final step was performed by Michael condensation

between (Z)-3-substituted-2-methoxy-5-(3,4,5-trimethoxystyry-

l)aniline 12a/12b or (Z)-2-amino-6-methoxy-3-(3,4,5-trimethox-

ystyryl)phenol (21) and substituted phenylprop-2-yn-1-ones

13a–f or N-substituted-3-indolyl prop-2-yn-1-ones 13g/13h in

ethanol. The key intermediates, 12a/12b, were prepared in

five steps. 3,4,5-Trimethoxybenzaldehyde (6) was reduced by

using sodium borohydride in methanol to give (3,4,5-trime-

thoxyphenyl)methanol (7). This was further treated with phos-

phorus tribromide in dichloromethane to produce 5-(bromo-

methyl)-1,2,3-trimethoxybenzene (8), which upon further reac-

tion with triphenylphosphine in toluene gave 3,4,5-trimethoxy-

benzyltriphenylphosphonium bromide (9)^[25]in good yield. The

Witting salt thus formed was treated with substituted benzal-

dehydes 10a/10b in the presence of sodium hydride in di-

chloromethane to produce (Z)-1,2,3-trimethoxy-5-(substituted-

nitrostyryl)benzenes 11 a/11 b and their E isomers in a 1:1 ratio.

tives.^[19]

6-Methoxy-3-(3’,4’,5’-trimethoxybenzoyl)-1H-indole

(BPR0L075, Figure 1), a microtubule depolymerizing agent, ex-

hibits antiangiogenic activities.^[20,21]Indibulin {N-(pyridin-4-yl)-

[1-(4-chlorbenzyl)indol-3-yl]glyoxyl amide, D-24851; Figure 1a}

is a synthetic small molecule with microtubule destabilizing ac-

tivity.^[22]

Thus, it is clear from the background presented above that

the pharmacophores represented by aminostilbene 1c and ar-

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Scheme 1. Reagents and conditions: a) NaBH₄, MeOH, 3 h, 15–208C; b) PBr₃, CH₂Cl₂, 2 h, 15–208C; c) PPh₃, toluene, 12 h, 808C; d) NaH, CH₂Cl₂, 18 h, 15–208C;

e) Zn AcOH, 4 h, RT; f) EtOH, RT, 4 h; g) TBAF (1m in THF), THF 08C, 4 h; h) Zn, ammonium formate, EtOH, 4 h; i) TFA, CH₂Cl₂, 6 h, 0–58C.

Compounds 11 a/11 b were reduced with Zn in acetic acid to

afford 12a/12b.

was further reduced with Zn in acetic acid to give (Z)-2-(tert-

butyldimethylsilyloxy)-3-methoxy-6-(3,4,5-trimethoxystyryl)ani-

line (20). Later, 20 was deprotected by using tetrabutylammo-

nium fluoride (TBAF, 1n in THF) to give derivative 21.^[25]

Aryl/heteroaryl prop-2-yn-1-ones 13a–g were prepared in

two steps, as shown in Scheme 3. Substituted benzaldehydes

22a–g were treated with ethynylmagnesium bromide (0.5n in

THF) to give aryl/heteroaryl prop-2-yn-1-ols 23a–g. These sec-

ondary alcohols were oxidized with 2-iodoxybenzoic acid (IBX)

in dimethyl sulfoxide (DMSO) to produce aryl/heteroaryl prop-

2-yn-1-ones 13a–g in good yields.

Key intermediate 21 was prepared in five sequential steps.

Isovaniline (16) was nitrated with nitronium tetrafluoroborate

to give 2-nitro 3-hydroxybenzaldehyde (17). This was further

treated with tert-butyldimethylsilyl chloride (TBDMSCl) and imi-

dazole in dichloromethane to produce 3-(tert-butyldimethylsily-

loxy)-4-methoxy-2-nitrobenzaldehyde (18). The obtained com-

pound was treated with 3,4,5-trimethoxybenzyltriphenylphos-

phonium bromide (9) in the presence of sodium hydride in di-

chloromethane to produce (Z)-tert-butyl[6-methoxy-2-nitro-3-

(3,4,5-trimethoxystyryl)phenoxy]dimethylsilane (19). Then, 19

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Biological studies

Antiproliferative activity

Congeners 3a–h, 4a–h, and 5a–

f (Scheme 1) and 2 were evaluat-

ed for their cytotoxic activity by

the US National Cancer Institute

(NCI) in

a 60-cell-line screen.

Among the 22 compounds, com-

pounds 3g, 4a, 4 f, 4g, and 4h

were selected for a preliminary

test at a single concentration

(10 mm), and these compounds

exhibited significant growth in-

hibition. Apart from 4h, the

other compounds were further

evaluated in a five-dose screen.

Compounds 3g, 4a, 4 f, and 4g

showed remarkable antiprolifera-

tive activity in a nine cancer

panel of this NCI screen (leuke-

mia, lung, colon, central nervous

system, melanoma, ovary, kidney,

prostate, and breast cancers).

Compound 3g with a methoxy

Scheme 2. Reagents and conditions: a) Nitronium tetrafluoroborate, CH₃NO₂/CH₂Cl₂(6:4), À408C, 8 h; b) TBDMSCl,

imidazole, CH₂Cl₂, 3 h, 15–208C; c) NaH, CH₂Cl₂, 18 h, 15–208C; d) Zn AcOH, 4 h, RT; e) EtOH, RT, 4 h; f) TBAF (1m

in THF), THF 08C, 4 h; g) Zn, ammonium formate, EtOH, 4 h.

group in the B ring and an indole moiety in the C ring

emerged as the most promising in this series. Notably, 3g

demonstrated

a 50% growth inhibition (GI₅₀) value of

<0.01 mm in most of the cell lines of the 60-cell-line panel;

thus, it possesses a broad spectrum of cytotoxicity. Com-

pounds 4a, 4 f, and 4g have a common dimethoxy group in

the B ring as a substituent, and presumably, the presence of

trimethoxy (for 4a), 4-trifluoromethoxy (for 4 f), and indole (for

4g) groups in the C rings of these molecules resulted in cyto-

toxic activity in the NCI screen (see Table 1). Therefore, all of

the compounds of this series were evaluated for their antiproli-

ferative activities. Interestingly, similar to the NCI screening re-

sults, compounds 3a–h (Figure 2) possessing a methoxy group

in the B ring showed profound cytotoxic activity with a 50%

inhibitory concentration (IC₅₀) value range of 0.011–4.68 mm.

Among them, compound 3e, which possess meta-amine and

para-methoxy groups on the C ring, inhibited the growth of

A549 cells with a IC₅₀value of 11 nm, as shown in Table 2.

Moreover, compounds 4a–h with meta,para-dimethoxy-substi-

tuted Brings also inhibited cell growth with an IC₅₀value in

the range from 0.09 to 7.59 mm. Notably, 4e with meta-amino

and para-methoxy substitutions in the C ring inhibited growth

of A549 cells with an IC₅₀value of 90 nm. In addition, hydroxy

and methoxy substituents in the B rings of 5a–h resulted in

less-potent cytotoxic activities with IC₅₀values of 0.91–7.33 mm.

Compound 5e with meta-amino and para-methoxy groups in

the C ring showed potent cytotoxic activity against MCF-7 cells

with an IC₅₀value of 0.91 mm. Taken together, these results sug-

gest that congeners with methoxy or dimethoxy moieties in

the B ring and indole or meta-amino and para-methoxy substi-

tutions in the C ring exhibit potent antiproliferative activities.

Moreover, on the basis of the five-dose results of the NCI, we

Scheme 3. Reagents and conditions: a) Ethynylmagnesium bromide (0.5n in

THF), THF, 8 h, 15–208C; b) IBX, DMSO, 15–208C, 2 h.

Final compounds 3e, 4e, and 5e were prepared by the re-

duction of 3d, 4d, and 5d, respectively, with Zn and ammoni-

um formate in methanol. Compounds 14a, 14b, and 14c

upon deprotection with TBAF (1n in THF) afforded 3c, 4c, and

5c, respectively. Compounds 3g and 4g were prepared from

15a and 15b by deprotection of the tert-butoxycarbonyl (Boc)

group by using trifluoroacetic acid (TFA) in dichloromethane.

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Table 1. In vitro cytotoxic effect of conjugates 3g, 4a, 4 f, 4g, and 1a against a panel of 60 human cancer cells.

Cell line

GI₅₀[mm]^[a]

Cell line

GI₅₀[mm]^[a]

3g^[b]

4a^[c]

4 f^[d]

4g^[e]

1a^[f]

3g^[b]

4a^[c]

4 f^[d]

4g^[e]

1a^[f]

Leukemia

CCRF-CEM

HL60(TB)

K-562

MOLT-4

RPMI-8226

SR

Ovarian

IGROV1

<0.01

2.93

3.24

3.38

4.62

3.04

2.03

3.55

2.80

2.93

3.99

3.66

2.75

1.37

–

1.54

2.53

0.63

0.46

0.002

–

0.002

0.003

<0.01

6.06

2.31

2.91

3.64

4.70

3.12

2.91

7.07

3.48

7.54

8.20

4.59

3.04

5.38

1.23

1.72

1.52

2.45

3.33

3.49

12.3

0.015

0.001

0.015

0.1

0.003

0.001

0.063

[h]

OVCAR-3

OVCAR-4

OVCAR-5

OVCAR-8

NCI/ADR-RES

SK-OV-3

Non-small-cell lung

A549/ATCC

HOP-62

HOP-92

NCI-H226

NCI-H23

NCIH322M

NCI-H460

NCI-H522

Renal

786-0

A498

ACHN

CAKI-1

RXF 393

SN12C

TK-10

UO-31

<0.01

0.01

5.14

2.46

1.14

5.45

4.22

7.71

3.93

4.54

7.85

12.6

19.9

4.38

1.10

3.34

1.96

14.5

11.6

1.50

7.28

3.75

1.20

5.60

1.76

0.015

0.002

0.003

0.007

0.001

<0.01

3.76

2.40

3.99

3.37

4.34

1.83

5.56

4.96

2.18

4.85

4.79

5.42

2.04

1.32

1.75

1.58

1.69

2.44

1.86

0.32

0.1

0.006

0.025

0.002

0.006

0.1

0.02

[h]

<0.01

–

[g]

–

0.02

4.03

5.93

2.63

[g]

<0.01

–

[h]

–

<0.01

0.019

Colon

Prostate

PC-3

DU-145

[h]

COLO-205

HCC-2998

HCT-116

HCT-15

HT29

<0.01

2.31

8.92

2.78

3.08

3.12

3.21

4.12

2.98

3.71

3.58

3.42

3.07

3.69

4.09

1.51

3.34

1.76

2.32

2.28

3.69

1.63

0.1

<0.01

2.89

3.64

–

3.44

2.06

0.001

0.063

0.003

0.1

0.005

0.003

4.11

Breast

MCF7

MDA-MB-31/ATCC

HS 578T

BT-549

T-47D

MDA-MB-435

<0.01

2.34

4.01

2.76

4.08

3.00

6.44

5.90

4.16

3.16

0.72

1.73

2.21

1.64

1.55

1.28

0.005

0.001

10

KM12

SW-620

[h]

–

[g]

Melanoma

LOX IMVI

MALME-3M

M14

MDA-MB-435

SK-MEL-2

SK-MEL-28

SK-MEL-5

UACC-257

UACC-62

–

1.68

1.88

10

[g]

<0.01

5.00

<0.01

1.59

3.54

2.91

2.53

1.22

4.54

4.28

1.69

5.95

14.7

3.46

1.84

0.44

1.27

2.87

2.43

2.21

1.86

3.14

3.16

1.39

0.003

0.019

0.002

0.001

0.003

0.007

0.003

0.005

<0.01

–

[g]

CNS

[h]

–

SF-268

SF-295

SF-539

SNB-19

SNB-75

U251

<0.01

6.04

2.40

1.92

4.49

1.39

3.84

8.90

4.12

2.26

4.97

2.65

3.46

3.57

7.94

1.70

3.92

1.47

3.84

0.006

0.003

0.002

0.003

0.007

10.4

3.73

4.33

6.86

<0.01

[g]

–

4.38

3.11

[a] Data are reported as the GI₅₀value (concentration required to cause 50% inhibition of cell growth after an incubation time of 48 h). [b] NSC777174.

[c] NSC777180. [d] NSC773189. [e] NSC777183. [f] NSC613729. [g] Not active. [h] Not tested.

merization and arrest cells at the G2/M phase of the cell

cycle.^[26]As the majority of the molecules in this series exhibit

significant cytotoxicity, we tested their ability to induce cy-

clin B1 protein, a marker for mitosis. To test this possibility,

A549 cells were treated with various congeners at 1 mm con-

centrations for 24 h. Subsequently, dot-blot analysis for cy-

clin B1 indicated that most of the congeners strongly induced

Figure 2. Structure–activity relationship of aminostilbene–arylpropenones.

cyclin B1 protein levels. Notably, 3e, 3g, 4e, and 5e robustly

activated cyclin B1. These results suggest that the congeners

indeed arrest cells at the G2/M phase of the cell cycle; actin

hypothesize that these compounds inhibit a common target in

all cell lines.

was employed as a loading control (Figure 3).

Effect on cell-cycle arrest

Dot-blot analysis for cyclin B1

To investigate the effect of potential compounds 3e, 3g, and

4e on the cell-cycle distribution of A549 cells, flow cytometry

analysis was performed at a concentration of 1 mm for 24 h.

Given that the congeners contain a combretastatin moiety,

these molecules are expected to interfere with tubulin poly-

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In contrast, vehicle- or DMSO-treated cells showed the majority

of the population in the G1 phase (67.7%) of the cell cycle.^[27]

Table 2. In vitro cytotoxic effect of aminostilbene–arylpropenones 3a–h,

4a–h, and 5a–f.

Compd

IC₅₀[mm]^[a]

A549^[b]

HeLa^[c]

MCF-7^[d]

HCT116^[e]

Inhibition of tubulin polymerization

3a

3b

3c

3d

3e

3 f

3g

3h

4a

4b

4c

4d

4e

4 f

4g

4h

5a

5b

5c

5d

5e

5 f

1.68Æ0.12

3.51Æ0.3

0.77Æ0.04

3.81Æ0.51

0.26Æ0.03

0.25Æ0.02

0.23Æ0.012

2.21Æ0.09

0.27Æ0.03

2.95Æ0.78

1.59Æ0.2

4.68Æ0.47

1.51Æ0.02

3.98Æ0.8

0.83Æ0.05

1.29Æ0.14

To further investigate whether the antiproliferative activities of

compounds 3e, 3g, and 4e were derived from interaction

with tubulin, they were evaluated for their inhibition of tubulin

polymerization (Table 3). The tubulin assembly assays revealed

that these molecules showed potent inhibition of tubulin poly-

merization with IC₅₀values of 0.71 mm for 3e and 0.79 mm for

3g; these values are comparable to that for CA-4 (1a, Table 3).

0.011Æ0.001 0.052Æ0.013 0.025Æ0.001 0.028Æ0.004

2.69Æ0.23

0.26Æ0.024

1.32Æ0.09

2.95Æ0.3

0.015Æ0.003 0.053Æ0.001 0.031Æ0.001 0.033Æ0.001

0.67Æ0.04

6.25Æ0.24

0.28Æ0.02

0.92Æ0.01

4.15Æ0.09

0.09Æ0.001

5.21Æ0.019

8.56Æ0.02

0.14Æ0.015

2.25Æ0.024

7.08Æ0.56

1.58Æ0.09

7.33Æ0.21

1.31Æ0.02

1.31Æ0.4

0.46Æ0.02

3.72Æ0.35

1.58Æ0.07

0.91Æ0.016

1.2Æ0.08

1.07Æ0.049

3.08Æ0.1

0.37Æ0.05

4.32Æ0.021

0.3Æ0.01

5.67Æ0.27

2.09Æ0.04

1.55Æ0.31

0.11Æ0.08

4.75Æ0.07

3.02Æ0.05

3.39Æ0.2

3.24Æ0.13

4.79Æ0.25

0.19Æ0.08

7.59Æ0.49

2.48Æ0.21

1.95Æ0.6

Effect of 3e, 3g, and 4e on microtubule network

0.19Æ0.045

2.86Æ0.06

1.67Æ0.05

0.89Æ0.03

1.56Æ0.014

2.78Æ0.38

4.17Æ0.14

2.66Æ0.42

2.51Æ0.08

7.08Æ0.042

The presence of anomalous spindle fibers owing to altered mi-

crotubule dynamics is a hallmark of cells treated with microtu-

bule inhibitors.^[28]These aminostilbene–arylpropenones signifi-

cantly decreased cell growth, inhibited tubulin assembly, and

stalled cells in the G2/M phase of the cell cycle. Therefore, we

were prompted to evaluate their ability to disrupt microtubule

networks in the cells.^[29]Consequently, A549 cells were treated

with 3e, 3g, and 4e at 1 mm concentrations for 24 h. Immuno-

fluorescence analysis revealed that the cells exhibited rounded

morphology typical of a mitotic-arrest population, and more-

over, chromatin was condensed in the nuclei, which suggests

metaphase arrest (Figure 5).

2.23Æ0.15

3.19Æ0.4

3.63Æ0.41

2.69Æ0.32

6.76Æ0.54

3.98Æ0.73

1.75Æ0.026

1.94Æ0.02

2.2Æ0.16

2.6Æ0.02

0.91Æ0.04

2.12Æ0.063

1a (CA-4) 0.031Æ0.001 0.058Æ0.007 0.058Æ0.003 0.398Æ0.46

[a] Concentration required to inhibit 50% cell growth following 48 h

treatment with the tested drug. Values represent meanÆstandard devia-

tion from three different experiments performed in triplicate. [b] A549:

non-small cell lung cancer. [c] HeLa: cervix cancer. [d] MCF-7: breast

cancer. [e] HCT116: colon cancer cell line.

Effect of 3e, 3g, and 4e on cytosolic tubulin

Given that the aminostilbene–arylpropenones markedly inhibit-

ed in vitro tubulin polymerization, we investigated their ability

to inhibit endogenous tubulin assembly. Microtubules exist in

a dynamic equilibrium between free and polymerized tubu-

lin,^[30]and the amount of free tubulin increases in cells chal-

lenged with microtubule inhibitors. To test this possibility,

A549 cells were treated with 3e, 3g, and 4e at 1 mm concen-

trations for 24 h. Subsequently, the cells were permeabilized

with detergents, and soluble (free tubulin) and polymerized

fractions were collected. Immunoblot analysis suggested that

the DMSO-treated cells contain fairly equal amounts of tubulin

in both fractions (Figure 6.). In comparison, the compound-

treated cells showed a significant amount of tubulin in the

soluble fraction. Thus, these results suggest that the congeners

strongly inhibit tubulin assembly in in vitro assays as well as in

cell-based assays.

Figure 3. Dot-blot analysis of cyclin B1: A549 cells were treated with 1 mm

concentrations of aminostilbene–arylpropenones for 24 h. Later, cells were

harvested and whole-cell lysates were blotted on nitrocellulose membranes.

The blotted proteins were probed with antibodies against cyclin B1. Actin

was employed as loading control.

Molecular modeling studies

A molecular modeling study was performed to elucidate the

binding characters of conjugates 3e, 3g, and 4e with tubulin.

Coordinates of the protein structure of tubulin–colchicine was

obtained from the Protein Data Bank (PDB ID 3E22).^[32]Docking

studies of all compounds were accomplished into the colchi-

cine binding site of tubulin by using AutoDock 4.2 software.^{[ 33]}

Congeners 3a–h, 4a–h, and 5a–f possess two cis-olefinic

units, and they were expected to occupy the colchicine bind-

The results indicate that they caused G2/M cell-cycle arrest

with a large accumulation of cells. Cells treated with 3e at

1 mm showed 76.4% arrest of cells in the G2/M phase, whereas

A549 cells treated with 3g and 4e at 1 mm showed arrest of

cells in the G2/M phase by 73 and 71%, respectively (Figure 4).

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of the B ring showed hydropho-

bic interactions with bLeu254,

bLeu255, and bAla354. The car-

bonyl group on the indole ring

established a hydrophobic inter-

action with bLeu248. Conjugate

4e established

a

hydrogen-

bonding interaction between

the methoxy group on aniline

and bLys254. The carbonyl

group on the methoxyaniline

moiety showed

a

hydrogen-

with

bonding interaction

aThr179 and a hydrophobic in-

teraction with aSer178. The me-

thoxyaniline group showed hy-

drophobic interactions with

aTyr224, bGly247, and aGln11.

The trimethoxyphenyl group

(A ring) was buried in the hydro-

phobic pockets of bLeu255,

bLys254, bLeu248, and bAla250.

Figure 4. Antimitotic effects of 3e, 3g, and 4e by fluorescence-activated cell sorting (FACS) analysis. Induction of

cell-cycle G2/M arrest by compounds 3e, 3g, and 4e. A549 cells were harvested after treatment at 1 mm for 24 h.

Untreated cells and DMSO-treated cells served as controls. The percentage of cells in each phase of the cell cycle

was quantified by flow cytometry.

Table 3. Antitubulin activity of aminostilbene–arylpropenones 3e, 3g,

and 4e.^[a]

Compd

IC₅₀[mm]

3e

3g

4e

1a (CA-4)

0.71Æ0.01

0.79Æ0.04

1.68Æ0.43

0.91Æ0.12

[a] Inhibition of tubulin polymerization (IC₅₀) values for 3e, 3g, and 4e

were determined from tubulin polymerization assays.

ing site of tubulin. Conjugate 3e exhibited a hydrogen-bond-

ing interaction between the oxygen atom of the carbonyl

group on the methoxyaniline ring with bLys254. Hydrophobic

interactions were found between the methoxyaniline ring of

3e with aAsn101 and bAsn249. The amine group of methoxya-

niline was involved in a hydrogen-bonding interaction with

aAla250 and in a hydrophobic interaction with bAsn250. The

A ring (trimethoxyphenyl) of 3e was buried in the hydrophobic

pockets of bLys254, bAsn258, bVal238, bLeu242, bAla250, and

bLeu255 in a manner similar to that of the trimethoxyphenyl

group of colchicine. Some hydrophobic interactions were

formed by the B ring in both the a- and b-tubulin interface

with aSer178, aThr179, bLys352, and bAsn258. Conjugate 3g

showed a hydrogen-bonding interaction between indole and

aGlu183, and this indole ring was buried in hydrophobic inter-

actions with aTyr224, aGly142, and aAla180.

Figure 5. Effect of 3e, 3g, and 4e on microtubules and nuclear condensa-

tion. A549 cells were independently treated with 3e, 3g, and 4e at 1 mm for

24 h. Following treatment, cells were fixed and stained for tubulin by using

4’,6-diamidino-2-phenylindole (DAPI) as a counterstain. Photographs were

taken by using an Olympus confocal fluorescence microscope equipped

with fluorescein isothiocyanate (FITC) and DAPI filter settings. Cells stained

for tubulin and DAPI from the same field of views are represented.

The trimethoxyphenyl group (A ring) showed hydrophobic

interactions with bLeu255, bLeu248, bAla250, bLys254, and

bLys255, and the methoxy group of the A ring established a hy-

drogen-bonding interaction with bLeu255. The methoxy group

The dimethoxyphenyl group (B ring) showed hydrophobic in-

teractions with bMet259, aAla180, bAla316, bLeu255, and

bLys352. Conjugates 3e, 3g, and 4e all interacted with the

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sized by using the Michael addition reaction, and the com-

pounds were evaluated for their cytotoxic activity against

human cancer cell lines. Representative congeners 3g, 4a, 4 f,

and 4g were evaluated in a 60-cell-line panel of the NCI five-

dose screen. Among them, 3g, which possesses a para-me-

thoxy group in the B ring and an indole moiety as the C ring,

exhibited excellent cytotoxicity in most human cancer cell

lines with GI₅₀values <0.01 mm. Compounds 3a–h, 4a–h, and

5a–f all displayed antiproliferative activity against four human

cancer cell lines, including A549 (non-small cell lung cancer),

HeLa (cervical carcinoma), MCF-7 (breast cancer), and HCT116

(colon carcinoma) with IC₅₀values ranging from 0.011 to

8.56 mm, and some of these values are comparable to that of

CA-4 (1a) The cell-cycle assays revealed that congeners 3e,

3g, and 4e caused cell-cycle arrest and accumulated cells in

the G2/M phase. Furthermore, these compounds effectively in-

hibited microtubule assembly and induced cyclin B1 protein

expression. As hypothesized, lead compounds 3e, 3g, and 4e

exert their cytotoxic activity by inhibition of tubulin polymeri-

Figure 6. Distribution of tubulin in polymerized versus soluble fractions ana-

lyzed by immunoblotting in A549 cells treated with 3e, 3g, and 4e. A549

cells were treated with 1 mm of 3e, 3g, and 4e for 24 h. The fractions con-

taining soluble and polymerized tubulin were collected and separated by

sodium dodecyl sulfate polyacrylamide gel electrophoresis. Tubulin was de-

tected by immunoblot analysis. S=soluble, P=polymerized.

a,b-tubulin interface in the colchicine binding pocket

(Figure 7).

Conclusions

A library of novel aminostilbene–arylpropenones (i.e., 3a–h,

4a–h, and 5a–f) comprising an A,B,C ring system was synthe-

Figure 7. Interaction of 3e, 3g, 4e, and 1a (CA-4) with the colchicine binding site of tubulin. Probable hydrogen bonds are shown in red. This figure was

generated by using the PyMOL software.

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RT, and the mixture was stirred for 6 h. The progress of the reac-

tion was monitored by TLC (EtOAc/hexane=1:1). The mixture was

basified with saturated NaHCO₃solution and then extracted with

EtOAc. The organic layer was separated and washed with brine,

dried with anhydrous Na₂SO₄, and concentrated under reduced

pressure to afford the crude compound, which was purified by

column chromatography (25% EtOAc/hexanes). Yield: 1.4 g, 77%;

¹H NMR (300 MHz, CDCl₃): d=6.91 (d, J=2.26 Hz, 1H), 6.80 (dd, J=

2.26 Hz, 8.31 Hz, 1H), 6.73 (d, J=8.31 Hz, 1H), 6.42–6.36 (m, 2H),

6.33 (dd, J=1.51 Hz, 9.82 Hz, 2H), 3.86 (s, 3H), 3.81 (s, 6H),

3.65 ppm (s, 3H); MS (ESI): m/z: 316 [M+H]⁺.

zation, with IC₅₀values of 0.71, 0.79, and 1.68 mm, respectively.

The binding of these conjugates is at the colchicine binding

site of tubulin, as indicated by molecular modeling studies.

These results demonstrate that aminostilbene–arylpropenones

are potent inhibitors of tubulin polymerization, and they are

also amenable for further structural modifications in the dis-

covery and development of effective chemotherapeutics for

the treatment of cancer.

Experimental Section

(Z)-2,3-Dimethoxy-5-(3,4,5-trimethoxystyryl)aniline (12b): Com-

pound 12b was prepared according to the method described for

compound 12a by employing 11 b (2.0 g, 5.32 mmol) and zinc

(1.34 g, 0.0213 mol) to obtain pure 12b as a yellow solid. Yield:

Chemistry

General: All chemicals and reagents were obtained from Sigma–Al-

drich (St. Louis, MO, USA), Lancaster (Alfa Aesar, 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 glass plates coated with silica gel

containing 60 GF254, and visualization was performed by UV light

or iodine indicator. Column chromatography was performed with

1

1.5 g, 82%; H NMR (300 MHz, CDCl₃): d=6.70 (d, J=1.51 Hz, 1H),

6.68 (s, 2H), 6.55 (s, 1H), 6.45 (d, J=12.1 Hz, 1H), 6.36 (d, J=

12.1 Hz, 1H), 3.85 (s, 3H), 3.83 (s, 3H), 3.70 ppm (s, 3H); MS (ESI):

m/z: 346 [M+H]⁺.

(Z)-3-({2-Methoxy-5-[(Z)-3,4,5-trimethoxystyryl]phenyl}amino)-1-

(3,4,5-trimethoxyphenyl)prop-2-en-1-one (3a): 1-(3,4,5-Trimethox-

yphenyl)prop-2-yn-1-one (13a; 69.8 mg, 0.317 mmol) was added to

a stirred solution of 12a (100 mg, 0.317 mmol) in ethanol (5 mL).

The mixture was stirred at 25–358C for 4 h, and the progress of

the reaction was monitored by TLC (hexane/EtOAc=6:4). Then,

water was added to the mixture. A yellow color solid appeared,

which was filtered and washed with ethanol. Yield: 120 mg, 71%;

m.p. 61–638C; ¹H NMR (500 MHz, CDCl₃): d=12.09 (d, J=12.5 Hz,

1H), 7.23–7.21 (m, 1H), 7.20 (s, 2H), 7.15 (d, J=1.8 Hz, 1H), 6.96

(dd, J=6.56, 1.8 Hz, 1H), 6.82 (d, J=8.3 Hz, 1H),6.54 (s, 2H), 6.49

(q, J=12.2 Hz, 2.44 Hz, 2H), 5.92 (d, J=7.9 Hz, 1H), 3.96 (s, 3H),

3.93 (s, 6H), 3.90 (s, 3H), 3.85 (s, 3H), 3.71ppm (s, 6H); ¹³C NMR

(75 MHz, CDCl₃): d=189.7, 152.9, 152.8, 147.5, 142.9, 141.0, 137.0,

134.6, 132.7, 129.9, 129.2, 129.1, 124.6, 113.3, 110.6, 105.7, 104.5,

94.1, 60.8, 56.16, 55.98, 55.9 ppm; IR (KBr): n˜_max=3452, 2934, 2859,

1630, 1588, 1553, 1505, 1472, 1429, 1408, 1363, 1326, 1275, 1199,

1166, 1125, 1002, 914, 845, 769, 580 cm^À1; MS (ESI): m/z: 536

[M+H]⁺; HRMS (ESI): m/z: calcd for C₃₀H₃₄O₈N: 536.22789 [M+H]⁺;

found: 536.22690.

1

Merck 60–120 mesh silica gel. H NMR spectra were recorded with

a Bruker UXNMR/XWIN-NMR (300 MHz) or Inova Varian VXR-Unity

(400 MHz) instrument. Chemical shifts (d) are reported in ppm

downfield from internal (CH₃)₄Si standard. ESI-MS data were record-

ed with a Micromass Quattro LC instrument by using ESI+ soft-

ware with a capillary voltage of 3.98 kV and an ESI mode positive

ion trap detector. HRMS data were recorded with a QSTAR XL

Hybrid MS–MS mass spectrometer. Melting points were deter-

mined with an Electrothermal melting point apparatus.

(Z)-1,2,3-Trimethoxy-5-(4-methoxy-3-nitrostyryl)benzene (11a): A

solution of 4-methoxy-3-nitrobenzaldehyde (10a; 2.0 g, 0.011 mol)

and triphenyl(3,4,5-trimethoxybenzyl)phosphonium bromide (9;

6.34 g, 0.012 mol) in dry CH₂Cl₂(100 mL) was stirred under a nitro-

gen atmosphere. Sodium hydride (1.06 g, 0.044 mol) was added to

the mixture at 08C. Then, the temperature of the mixture was

slowly increased to RT, and the mixture was stirred for 18 h. The

progress of the reaction was monitored by TLC (EtOAc/hexane=

3:7), and water was added after completion of reaction (until foam-

ing stopped). The organic layer was separated, and the aqueous

layer was extracted with CHCl₃. The organic layer was washed with

brine, dried with anhydrous Na₂SO₄, and concentrated under re-

duced pressure to afford the crude compound, which was purified

by column chromatography (15% EtOAc/hexanes). Yield: 1.8 g,

42%; ¹H NMR (300 MHz, CDCl₃): d=7.79 (d, J=2.26 Hz, 1H), 7.42

(dd, J=2.26 Hz, 8.3 Hz, 1H), 6.94 (d, J=8.3 Hz, 1H), 6.58 (d, J=

12.09 Hz, 1H), 6.47 (s, 2H), 6.46 (d, J=12.09 Hz, 1H), 3.93 (s, 3H),

3.85 (s, 3H), 3.71 ppm (s, 6H); MS (ESI): m/z: 346 [M+H]⁺.

(Z)-1-(2-Bromo-3,4,5-trimethoxyphenyl)-3-({2-methoxy-5-[(Z)-

3,4,5-trimethoxystyryl]phenyl}amino)prop-2-en-1-one (3b): Com-

pound 3b was prepared according to the method described for

compound 3a by employing 1-(2-bromo-3,4,5-trimethoxyphenyl)-

prop-2-yn-1-one (13b; 94.9 mg, 0.317 mmol) to obtain pure 3b as

a

yellow solid. Yield: 150 mg, 77%; m.p. 54–578C; ¹H NMR

(300 MHz, CDCl₃): d=11.92 (d, J=12.8 Hz, 1H), 7.28–7.19 (m, 1H),

7.14 (s, 1H), 6.97 (d, J=8.3 Hz, 1H), 6.87 (s, 1H), 6.81 (d, J=8.3 Hz,

1H), 6.49 (d, J=9.06 Hz,4H), 5.64 (d, J=8.30 Hz, 1H), 3.94 (s, 3H),

3.91 (s, 3H), 3.90 (s, 3H), 3.87 (s, 3H), 3.84 (s, 3H), 3.70 ppm (s, 6H);

¹³C NMR (75 MHz, CDCl₃): d=192.2, 152.9, 152.6, 147.5, 144.0,

142.8, 138.3, 137.13, 132.6, 130.01, 129.4, 129.1, 125.9, 124.8, 113.6,

110.6, 107.9, 106.1, 105.7, 98.4, 61.09, 60.91, 60.91, 56.08, 55.99,

55.91 ppm; IR (KBr): n˜_max=3418, 2933, 2839, 2359, 1629, 1581,

1501, 1465, 1424, 1328, 1379, 1202, 1163, 1125, 1102, 1007, 928,

850, 793, 743 cm^À1; MS (ESI): m/z: 614 [M+H]⁺; HRMS (ESI): m/z:

calcd for C₃₀H₃₃O₈NBr: 614.13841 [M+H]⁺; found: 614.13838.

(Z)-5-(3,4-Dimethoxy-5-nitrostyryl)-1,2,3-trimethoxybenzene

(11b): Compound 11 b was prepared according to the method de-

scribed for compound 11 a by employing 4,5-dimethoxy 3-nitro-

benzaldehyde (10b; 2.0 g, 9.5 mmol), triphenyl(3,4,5-trimethoxy-

benzyl)phosphonium bromide (9; 5.45 g, 0.0104 mol), and sodium

hydride (0.910 g, 0.038 mol) to obtain pure 11 b as a yellow solid.

1

Yield: 1.5 g, 42%; H NMR (300 MHz, CDCl₃): d=7.21 (s, 1H), 6.93 (s,

1H), 6.56 (d, J=11.8 Hz, 1H), 6.43 (s, 2H), 6.40 (d, J=11.8 Hz, 1H),

3.91 (s, 3H), 3.79 (s, 3H), 3.71 (s, 6H), 3.66 ppm (s, 3H); MS (ESI): m/

z: 376 [M+H]⁺.

(Z)-1-[3-(tert-Butyldimethylsilyloxy)-4-methoxyphenyl]-3-({2-me-

thoxy-5-[(Z)-3,4,5-trimethoxystyryl]phenyl}amino)prop-2-en-1-

one (14a): Compound 14a was prepared according to the method

described for compound 3a by employing 1-[3-(tert-butyldimethyl-

(Z)-2-Methoxy-5-(3,4,5-trimethoxystyryl)aniline

(12a):

Zinc

(1.46 g, 0.023 mol) was added to a stirred solution of 11 a (2.0 g,

5.78 mmol) in acetic acid (20 mL) under a nitrogen atmosphere at

08C. Then, the temperature of the mixture was slowly increased to

silyloxy)4-methoxyphenyl]prop-2-yn-1-one

(13c;

92.07 mg,

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0.317 mmol) to obtain pure 14a as a yellow solid. Yield: 150 mg,

78%.

878, 854, 776, 657, 591 cm^À1; MS (ESI): m/z: 491 [M+H]⁺; HRMS

(ESI): m/z: calcd for C₂₈H₃₁O₆N₂: 491.21766 [M+H]⁺; found:

491.21770.

(Z)-1-(3-Hydroxy-4-methoxyphenyl)-3-({2-methoxy-5-[(Z)-3,4,5-

trimethoxystyryl]phenyl}amino)prop-2-en-1-one (3c): 1m TBAF in

THF (0.3 mL, 0.272 mmol) was added to a stirred solution of 14a

(150 mg, 0.247 mmol) in THF (15 mL) at 10–158C. Then, the tem-

perature of the mixture was slowly increased to RT, and the mix-

ture was stirred for 6 h. The progress of the reaction was moni-

tored by TLC (hexane/EtOAc=1:1). Upon completion of the reac-

tion, THF was evaporated, and the mixture was partitioned be-

tween water and EtOAc. The compound was purified by column

chromatography (hexane/EtOAc=6:4). Yield: 100 mg, 82%; m.p.

70–728C; ¹H NMR (300 MHz, CDCl₃): d=12.06 (d, J=12.1 Hz, 1H),

7.50 (d, J=6.7 Hz, 2H), 7.24–7.18 (m, 1H), 7.12 (s, 1H), 6,96–6.87

(m, 2H), 6.79 (d, J=8.3 Hz, 1H), 6.49 (d, J=13.6 Hz, 4H), 5.91 (d,

J=8.3 Hz, 1H), 5.68 (s, 1H), 3.94 (s, 6H), 3.85 (s, 3H), 3.71 ppm (s,

6H); ¹³C NMR (75 MHz, CDCl₃): d=187.5, 152.9, 147.3, 140.3, 137.4,

133.8, 132.2, 130.02, 129.8, 129.5, 128.9, 126.3, 123.4, 122.7, 122.4,

121.7, 112.7,110.4, 109.4, 105.79, 96.4, 60.9, 55.93 ppm; IR (KBr):

n˜_max=3431, 2992, 2945, 2843, 1628, 1614, 1581, 1533, 1501, 1475,

1439, 1412, 1348, 1323, 1272, 1241, 1151, 1124, 1081, 1045, 1008,

871 cm^À1; MS (ESI): m/z: 492 [M+H]⁺; HRMS (ESI): m/z: calcd for

C₂₈H₃₀O₇N: 492.20168 [M+H]⁺; found: 492.20145.

(Z)-3-({2-Methoxy-5-[(Z)-3,4,5-trimethoxystyryl]phenyl}amino)-1-

[4-(trifluoromethoxy)phenyl]prop-2-en-1-one (3 f): Compound 3 f

was prepared according to the method described for compound

3a by employing 1-[4-(trifluoromethoxy)phenyl]prop-2-yn-1-one

(13e, 67.88 mg, 0.317 mmol) to obtain pure 3 f as a yellow solid.

Yield: 120 mg, 72%; m.p. 103–1058C; ¹H NMR (300 MHz, CDCl₃):

d=12.15 (d, J=13.4 Hz, 1H), 7.99 (d, J=7.17 Hz, 2H), 7.26 (s, 3H),

7.14 (s, 1H), 6.97 (d, J=8.49 Hz, 1H), 6.82 (d, J=8.49 Hz, 1H), 6.50

(d, J=10.09 Hz, 4H), 5.95 (d, J=7.55 Hz, 1H), 3.96 (s, 3H), 3.85 (s,

3H), 3.71 ppm (s, 6H); ¹³C NMR (75 MHz, CDCl₃): d=189.03, 153.0,

147.6, 143.6, 137.6, 132.7, 130.0, 129.9, 129.4, 129.1, 129.0, 124.8,

120.3, 113.6, 110.7, 105.8, 93.9, 60.9, 55.9, 56.0 ppm; IR (KBr): n˜_max

=

3432, 2935, 2833, 1628, 1592, 1558, 1508, 1472, 1454, 1434, 1416,

1363, 1325, 1298, 1279, 1257, 1180, 1129, 1037, 1015, 1006, 963,

866, 844, 795, 779, 723 cm^À1; MS (ESI): m/z: 530 [M+H]⁺; HRMS

(ESI): m/z: calcd for C₂₈H₂₇O₆NF₃: 530.17850 [M+H]⁺; found:

530.17740.

tert-Butyl-3-[(Z)-3-({2-methoxy-5-[(Z)-3,4,5-trimethoxystyryl]phe-

nyl}amino)acryloyl]-1H-indole-1-carboxylate (15a): Compound

15a was prepared according to the method described for com-

pound 3a by employing tert-butyl 3-propioloyl-1H-indole-1-carbox-

ylate (13 f; 85.4 mg, 0.317 mmol) to obtain pure 15a as a yellow

solid.

(Z)-1-(4-Methoxy-3-nitrophenyl)-3-({2-methoxy-5-[(Z)-3,4,5-trime-

thoxystyryl]phenyl}amino)prop-2-en-1-one (3d): Compound 3d

was prepared according to the method described for compound

3a by employing 1-(4-methoxy-3-nitrophenyl)prop-2-yn-1-one

(13d; 65.05 mg, 0.317 mmol) to obtain pure 3d as a yellow solid.

Yield: 130 mg, 79%; m.p. 164–1668C; ¹H NMR (300 MHz, CDCl₃):

d=12.15 (d, J=12.8 Hz, 1H), 8.44 (s, 1H), 8.14 (d, J=9.06 Hz, 1H),

7.32–7.26 (m, 1H), 7.12 (d, J=8.13 Hz, 2H), 6.98 (d, J=8.3 Hz, 1H),

6.81 (d, J=9.06 Hz, 1H), 6.50 (d, J=8.3 Hz, 4H), 5.91 (d, J=7.55 Hz,

1H), 4.02 (s, 3H), 3.96 (s, 3H), 3.85 (s, 3H), 3.70 ppm (s, 6H);

¹³C NMR (75 MHz, CDCl₃): d=186.9, 155.0, 152.9, 147.6, 143.8,

139.2, 137.15, 133.0, 132.6, 131.6, 130.0, 129.4, 129.07, 128.9, 125.0,

124.9, 113.7, 113.0, 110.7, 105.7, 93.2, 60.9, 55.9,56.7, 56.03 ppm; IR

(KBr): n˜_max=3427, 2934, 2837, 1630, 1612, 1579, 1531, 1503, 1473,

1437, 1414, 1350, 1325, 1270, 1237, 1149, 1183, 1125, 1085, 1047,

1012, 879, 682 cm^À1; MS (ESI): m/z: 521 [M+H]⁺; HRMS (ESI): m/z:

calcd for C₂₈H₂₉O₈N₂: 521.19184 [M+H]⁺; found: 521.19148.

(Z)-1-(1H-indol-3-yl)-3-({2-methoxy-5-[(Z)-3,4,5-trimethoxystyryl]-

phenyl}amino)prop-2-en-1-one (3g): TFA (0.01 mL, 0.188 mmol)

was added to a stirred solution of 15a (100 mg, 0.171 mmol) in

CH₂Cl₂at 08C. Then, the temperature of the mixture was slowly in-

creased to RT, and the mixture was stirred for 2 h. The progress of

the reaction was monitored by TLC (EtOAc/hexane=7:3). The mix-

ture was neutralized by adding a saturated solution of NaHCO₃.

The mixture was filtered through Celite, and the aqueous layer was

extracted with CHCl₃. The organic layer was washed with brine,

dried with anhydrous Na₂SO₄, and concentrated under reduced

pressure to afford the crude compound. Yield: 60 mg, 72%; m.p.

95–978C; ¹H NMR (300 MHz, CDCl₃): d=11.87 (d, J=12.3 Hz, 1H),

8.62 (s, 1H), 8.45–8.42 (m, 1H), 7.80 (d, J=2.8 Hz, 1H), 7.42–7.39

(m, 1H), 7.30–7.27 (m, 1H), 7.13–7.05 (m, 2H), 6.9 (dd, J=8.3 Hz,

1.7 Hz, 1H), 6.82 (d, J=8.3 Hz, 1H), 6.56 (s, 2H), 6.50 (d, J=4.7 Hz,

2H), 5.85 (d, J=7.9 Hz, 1H), 3.95(s, 3H), 3.86 (s, 3H), 3.71 ppm (s,

6H); ¹³C NMR (75 MHz, CDCl₃): d=188.1, 152.9, 147.3, 140.6, 136.5,

132.8, 129.86, 129.83, 129.5, 128.9, 125.5, 123.6, 122.9, 122.0, 121.7,

118.7, 112.8, 111.5, 110.5, 105.8, 96.4, 60.9, 55.9, 55.8 ppm; IR (KBr):

n˜_max=3388, 2953, 2859, 1628, 1580, 1513, 1453, 1428, 1366, 1324,

1276, 1241, 1157, 1126, 1025, 1000, 966, 864, 790, 769, 758, 656,

579 cm^À1; MS (ESI): m/z: 485 [M+H]⁺; HRMS (ESI): m/z: calcd for

C₂₉H₂₉O₅N₂: 485.20711 [M+H]⁺; found: 485.20611.

(Z)-1-(4-Amino-3-methoxyphenyl)-3-({2-methoxy-5-[(Z)-3,4,5-tri-

methoxystyryl]phenyl}amino)prop-2-en-1-one

(3e):

Zinc

(77.12 mg, 1.22 mmol) and ammonium formate (78.34 mg,

1.22 mmol) were added to a stirred solution of 3d (150 mg,

0.306 mmol) in ethanol (10 mL) at RT. The mixture was stirred for

4 h, and the progress of the reaction was monitored by TLC

(EtOAc/hexane=7:3). The mixture was then filtered through a bed

of Celite, and the filtrate was evaporated under reduced pressure.

Then, water (10 mL) and EtOAc (30 mL) were added to the concen-

trate. The organic layer was separated, dried with anhydrous

Na₂SO₄, and concentrated to afford the crude product, which was

further purified by column chromatography (EtOAc/hexane=6:4)

to obtain a pure yellow solid. Yield: 110 mg, 78%; m.p. 71–738C;

¹H NMR (300 MHz, CDCl₃): d=12.04 (d, J=12.04 Hz, 1H), 7.36–7.32

(m, 2H), 7.21–7.13 (m, 1H), 7.12 (d, J=1.51 Hz, 1H), 6.95–6.91(dd,

J=1.51 Hz, 8.3 Hz, 1H), 6.82–6.78 (m, 2H), 6.54 (s, 2H), 6.48 (d, J=

2.26 Hz, 2H), 5.91 (d, J=7.55 Hz, 1H), 3.94 (s, 3H), 3.90 (s, 3H),

3.85(s, 3H),3.70 ppm (s, 6H); ¹³C NMR (75 MHz, CDCl₃): d=190.2,

152.9, 150.1, 147.4, 142.1, 137.08, 135.9, 132.7, 132.3, 129.8, 129.5,

129.3, 129.1, 124.6, 118.8, 113.6, 113.1, 110.5, 109.3, 105.7, 94.2,

60.9, 55.9, 55.5 ppm; IR (KBr): n˜_max=3444, 3341, 2934, 2836, 1629,

1587, 1551, 1505, 1474, 1363, 1325, 1275, 1204, 1177, 1127, 1022,

(Z)-3-({2-Methoxy-5-[(Z)-3,4,5-trimethoxystyryl]phenyl}amino)-1-

(1-methyl-1H-indol-3-yl)prop-2-en-1-one (3h): Compound 3h was

prepared according to the method described for compound 3a by

employing

1-(1-methyl-1H-indol-3-yl)prop-2-yn-1-one

(13g;

58.1 mg, 0.317 mmol) to obtain pure 3h as a yellow solid. Yield:

115 mg, 73%; m.p. 122–1248C; ¹H NMR (300 MHz, CDCl₃): d=

11.87(d, J=12.1 Hz, 1H), 8.45–8.41 (m, 1H), 7.67(s, 1H), 7.35–7.23

(m, 3H), 7.11–7.03 (s, 2H), 6.92 (d, J=8.3 Hz, 1H), 6.78 (d, J=

8.3 Hz, 1H), 6.58 (s, 2H), 6.49 (qt, J=12.1 Hz, 5.3 Hz, 2H), 5.79 (d,

J=7.55 Hz, 1H), 3.96 (s, 3H), 3.83 (s, 6H), 3.70 ppm (s, 6H);

¹³C NMR (75 MHz, CDCl₃): d=187.5, 152.9, 147.3, 140.3, 137.4,

133.2, 132.8, 130.0, 129.8, 129.5, 128.9, 126.3, 123.4, 122.7, 122.4,

121.7, 112.7, 110.4, 109.4, 105.7, 96.4, 61.0, 60.9, 55.9 ppm; IR (KBr):

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n˜_max=3434, 2929, 2835, 1631, 1604, 1578, 1524, 1488, 1469, 1433,

1415, 1368, 1327, 1277, 1217, 1187, 1146, 1126, 1087, 1026, 1005,

947, 877, 857, 783, 752 cm^À1; MS (ESI): m/z: 499 [M+H]⁺; HRMS

(ESI): m/z: calcd for C₃₀H₃₁O₅N₂: 499.22275 [M+H]⁺; found:

499.22339.

(Z)-3-({2,3-Dimethoxy-5-[(Z)-3,4,5-trimethoxystyryl]phenyl}ami-

no)-1-(4-methoxy-3-nitro phenyl)prop-2-en-1-one (4d): Com-

pound 4d was prepared according to the method described for

compound 3a by employing 12b (100 mg, 0.289 mmol) and 13d

(59.3 mg, 0.289 mmol) to obtain pure 4d as a yellow solid. Yield:

1

120 mg, 75%; m.p. 144–1478C; H NMR (300 MHz, CDCl₃): d=12.21

(Z)-3-({2,3-Dimethoxy-5-[(Z)-3,4,5-trimethoxystyryl]phenyl}ami-

no)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (4a): Compound

4a was prepared according to the method described for com-

pound 3a by employing 12b (100 mg, 0.289 mmol) and 13a

(63.8 mg, 0.289 mmol) to obtain pure 4a as a yellow solid. Yield:

120 mg, 73%; m.p. 61–638C; ¹H NMR (300 MHz, CDCl₃): d=12.12

(d, J=12.1 Hz, 1H), 7.30–7.23 (m, 1H), 7.19 (s, 2H), 6.79 (s, 1H),

6.58–6.49 (m, 5H), 5.93 (d, J=8.3 Hz, 1H), 3.96 (s, 3H), 3.94 (s, 6H),

3.90 (s, 3H), 3.84 (s, 3H), 3.82 (s, 3H), 3.72 ppm (s, 6H); ¹³C NMR

(75 MHz, CDCl₃): d=189.7, 152.7, 152.5, 142.9, 140.8, 137.05, 136.6,

134.64, 134.6, 133.6, 133.2, 130.1, 129.2, 128.4, 127.4, 107.6, 106.2,

105.7, 104.4, 103.3, 94.06, 60.7, 60.6, 56.9, 55.75, 55.58 ppm; IR

(KBr): n˜_max=3424, 2935, 2832, 1629, 1585, 1554, 1503, 1470, 1425,

1367, 1327, 1277, 1235, 1200, 1165, 1125, 1075, 1000, 913, 862,

838, 772, 734, 659, 585 cm^À1; MS (ESI): m/z: 566 [M+H]⁺; HRMS

(ESI): m/z: calcd for C₃₁H₃₆O₉N: 566.23846 [M+H]⁺; found:

566.23749.

(d, J=12.8 Hz, 1H), 8.43 (s, 1H), 8.14 (d, J=9.06 Hz, 1H), 7.37–7.30

(m, 1H), 7.12 (d, J=9.06 Hz, 1H), 6.77 (s, 1H), 6.57–6.47 (m, 5H),

5.92 (d, J=7.5 Hz, 1H), 4.03 (s, 3H), 3.97 (s, 3H), 3.84 (s, 3H),

3.74 ppm (s, 9H); ¹³C NMR (75 MHz, CDCl₃): d=187.1, 155.0, 152.9,

143.9, 139.1, 137.2, 136.9, 133.5, 133.4, 133.1, 131.6, 130.4, 129.2,

125.06, 113.06, 108.0, 106.5, 105.8, 93.3, 60.9, 60.8, 56.7, 55.9,

55.7 ppm; IR (KBr): n˜_max=3433, 2963, 2937, 2838, 1636, 1592, 1556,

1503, 1464, 1429, 1420, 1324, 1295, 1282, 1235, 1128, 1075, 1013,

999, 926 cm^À1; MS (ESI): m/z: 551 [M+H]⁺; HRMS (ESI): m/z: calcd

for C₃₀H₃₁O₉N₂: 551.2029 [M+H]⁺; found: 551.2031.

(Z)-1-(3-Amino-4-methoxyphenyl)-3-({2,3-dimethoxy-5-[(Z)-3,4,5-

trimethoxystyryl]phenyl}amino)prop-2-en-1-one (4e): Compound

4e was prepared according to the method described for com-

pound 3e by employing 4d (150 mg, 0.288 mmol), zinc (72.71 mg,

1.15 mmol), and ammonium formate (73.67 mg, 1.15 mmol) to

obtain pure 4e as a yellow solid. Yield: 111 mg, 78%; m.p. 68–

1

718C; H NMR (300 MHz, CDCl₃): d=12.1 (d, J=12.1 Hz, 1H), 7.34

(Z)-1-(2-Bromo-3,4,5-trimethoxyphenyl)-3-({2,3-dimethoxy-5-[(Z)-

3,4,5-trimethoxystyryl]phenyl}amino)prop-2-en-1-one (4b): Com-

pound 4b was prepared according to the method described for

compound 3a by employing 12b (100 mg, 0.289 mmol) and 13b

(86.5 mg, 0.289 mmol) to obtain pure 4b as a yellow solid. Yield:

(d, J=7.4 Hz, 2H), 7.23–7.19 (m, 1H), 6.81 (d, J=8.49 Hz, 1H), 6.75

(s, 1H), 6.53–6.51 (m, 5H), 5.96 (d, J=7.9 Hz, 1H), 3.95 (s, 3H), 3.90

(s, 3H), 3.83 (s, 3H), 3.72 ppm (s, 9H); ¹³C NMR (75 MHz, CDCl₃): d=

190.4, 152.9, 152.7, 150.1, 142.2, 137.1, 136.6, 135.9, 134.1, 133.3,

132.4, 132.3, 130.1, 129.4, 118.8, 113.6, 109.2, 107.2, 106.3, 105.8,

94.4, 60.8, 60.7, 55.9, 55.7, 55.5 ppm; IR (KBr): n˜_max=3444, 3371,

2933, 2834, 1630, 1584, 1552, 1504, 1469, 1428, 1367, 1326, 1276,

1204, 1176, 1146, 1126, 1076, 999, 919, 854, 774 cm^À1; MS (ESI): m/

z: 521 [M+H]⁺; HRMS (ESI): m/z: calcd for C₂₉H₃₃O₇N₂: 521.22823

[M+H]⁺; found: 521.22722.

1

130 mg, 69.7%; m.p. 55–578C; H NMR (300 MHz, CDCl₃): d=12.04

(d, J=12.8 Hz, 1H), 7.31–7.24 (m, 1H), 6.87 (s, 1H), 6.76 (d, J=

6.7 Hz, 1H), 6.57 (s, 1H), 6.52–6.47 (m, 4H), 5.67 (d, J=7.55 Hz, 1H),

3.96 (s, 3H), 3.90 (d, J=3.02 Hz, 9H), 3.87 (s, 3H), 3.71 ppm (s, 9H);

¹³C NMR (75 MHz, CDCl₃): d=192.4, 152.9, 152.7, 150.8, 144.1,

142.9, 138.3, 136.8, 133.6, 133.4, 132.3, 130.4, 129.3, 108.0, 107.9,

106.5, 106.1, 105.9, 98.6, 61.12, 60.8, 60.9, 55.7, 55.9, 56.1 ppm; IR

(KBr): n˜_max=3434, 2935, 2833, 1630, 1582, 1505, 1466, 1423, 1381,

1329, 1276, 1239,1203, 1166, 1126, 1104, 1075, 1005, 932, 864, 850,

794, 657 cm^À1; MS (ESI): m/z: 644 [M+H]⁺; HRMS (ESI): m/z: calcd

for C₃₁H₃₅O₉NBr: 644.14897 [M+H]⁺; found: 644.14957.

(Z)-3-({2,3-Dimethoxy-5-[(Z)-3,4,5-trimethoxystyryl]phenyl}ami-

no)-1-[4-(trifluoromethoxy)phenyl]prop-2-en-1-one (4 f): Com-

pound 4 f was prepared according to the method described for

compound 3a by employing 12b (100 mg, 0.289 mmol) and 13e

(61.9 mg, 0.289 mmol) to obtain pure 4 f as a yellow solid. Yield:

1

120 mg, 74%; m.p. 107–1108C; H NMR (300 MHz, CDCl₃): d=12.21

(Z)-1-[3-(tert-Butyldimethylsilyloxy)-4-methoxyphenyl]-3-({2,3-di-

methoxy-5-[(Z)-3,4,5-trimethoxystyryl]phenyl}amino)prop-2-en-

1-one (14b): Compound 14b was prepared according to the

method described for compound 3a by employing 12b (100 mg,

0.289 mmol) and 13c (83.9 mg, 0.289 mmol) to obtain pure 14b as

a yellow solid. Yield: 150 mg, 81%.

(d, J=12.8 Hz, 1H), 7.98 (d, J=8.3 Hz, 2H), 7.35–7.31 (m, 1H), 7.27–

7.25 (m, 3H), 6.77 (s, 1H), 6.57–6.46 (m, 4H), 5.93 (d, J=8.3 Hz,

1H), 3.96 (s, 3H), 3.83 (s, 3H), 3.71 ppm (s, 9H); ¹³C NMR (75 MHz,

CDCl₃): d=182.9, 152.9, 152.7, 151.4, 143.7, 137.6, 137.2, 136.9,

133.6, 133.4, 132.3, 132.0, 130.3, 129.3, 129.1, 128.0, 120.9, 107.9,

106.5, 105.9, 94.03, 60.89, 60.8, 55.9, 55.7 ppm; IR (KBr): n˜_max=3432,

2947, 2842, 1621, 1534, 1514, 1501, 1450, 1411, 1321, 1315, 1292,

1255, 1189, 1165, 1117, 1052, 1001, 925, 845, 818, 789 cm^À1; MS

(ESI): m/z: 560 [M+H]⁺; HRMS (ESI): m/z: calcd for C₂₉H₂₉O₇NF₃:

560.18906 [M+H]⁺; found: 560.18821.

(Z)-3-({2,3-Dimethoxy-5-[(Z)-3,4,5-trimethoxystyryl]phenyl}ami-

no)-1-(3-hydroxy-4-methoxyphenyl)prop-2-en-1-one (4c): Com-

pound 4c was prepared according to the method described for

compound 3c by employing 14b (150 mg, 0.236 mmol) and TBAF

(1n in THF) (0.3 mL, 0.26 mmol) to obtain pure 4c as a yellow

tert-Butyl 3-[(Z)-3-({2,3-dimethoxy-5-[(Z)-3,4,5-trimethoxystyryl]-

phenyl}amino)acryloyl]-1H-indole-1-carboxylate (15b): Com-

pound 15b was prepared according to the method described for

compound 3a by employing 13 f (77.8 mg, 0.289 mmol) to obtain

pure 15b as a yellow solid.

1

solid. Yield: 99 mg, 80%; m.p. 64–668C; H NMR (300 MHz, CDCl₃):

d=12.09 (d, J=12.8 Hz, 1H), 7.53 (d, J=6.7 Hz, 2H), 7.29–7.22 (m,

1H), 6.89 (d, J=9.06 Hz, 1H), 6.75 (s, 1H), 6.53–6.51 (m, 5H), 5.94

(d, J=8.3 Hz, 1H), 5.78 (s, 1H), 3.95 (s, 3H), 3.94 (s, 3H), 3.83 (s,

3H), 3.71 ppm (s, 9H), ¹³C NMR (75 MHz, CDCl₃): d=189.9, 152.9,

152.7, 149.4, 145.2, 142.6, 137.2, 136.7, 134.1, 133.3, 132.9, 132.4,

130.2, 129.4, 120.4, 113.6, 109.8, 107.4, 106.4, 105.9, 94.2, 60.8, 60.7,

55.9, 55.7 ppm; IR (KBr): n˜_max=3331, 2990, 2965, 2935, 2837, 1632,

1580, 1546, 1504, 1464, 1435, 1370, 1328, 1282, 1203, 1178, 1162,

1127, 1074, 1054, 1026, 999, 974,945, 853, 775, 691 cm^À1; MS (ESI):

m/z: 522 [M+H]⁺; HRMS (ESI): m/z: calcd for C₂₉H₃₂O₈N: 522.21224

[M+H]⁺; found: 522.21105.

(Z)-3-({2,3-Dimethoxy-5-[(Z)-3,4,5-trimethoxystyryl]phenyl}ami-

no)-1-(1H-indol-3-yl)prop-2-en-1-one (4g): Compound 4g was

prepared according to the method described for compound 3g by

employing 15b (100 mg, 0.163 mmol) and TFA (0.01 mL,

0.178 mmol) to obtain pure 4g as a yellow solid. Yield: 62 mg,

74%; m.p. 88–908C; ¹H NMR (300 MHz, CDCl₃): d=11.96 (d, J=

11.9 Hz, 1H), 8.47 (s, 1H), 8.37–8.36 (m, 1H), 7.82 (d, J=2.89 Hz,

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1H), 7.44–7.39 (m, 2H), 7.32–729 (m, 1H), 7.15 (dd, J=4.1 Hz,

8.1 Hz, 1H), 6.75 (d, J=1.2 Hz, 1H), 6.53 (d, J=12.1 Hz, 4H), 5.86

(d, J=8.1 Hz, 1H), 3.96 (s, 3H), 3.84 (s, 3H), 3.72 (s, 3H), 3.71 ppm

(s, 6H); IR (KBr): n˜_max=3415, 2931, 2839, 1627, 1580, 1516, 1427,

1372, 1310, 1275, 1239, 1158, 1125, 1078, 1036, 1007, 997, 889,

771 cm^À1; MS (ESI): m/z: 515 [M+H]⁺; HRMS (ESI): m/z: calcd for

C₃₀H₃₁O₆N₂: 515.21766 [M+H]⁺; found: 515.21847.

method described for compound 12a by employing 19 (900 mg,

1.89 mmol) and zinc (484.9 mg, 7.57 mmol) to obtain pure 20 as

a light brown solid. Yield: 700 mg, 83%; H NMR (300 MHz, CDCl₃):

d=6.70 (d, J=8.3 Hz, 1H), 6.51 (s, 2H), 6.47 (d, J=4.5 Hz, 2H), 6.27

(d, J=8.3 Hz, 1H), 3.81 (s, 3H), 3.76 (s, 3H), 3.63 (s, 6H), 1.02 (s,

9H), 0.17 ppm (s, 6H); MS (ESI): m/z: 446 [M+H]⁺.

1

(Z)-2-Amino-6-methoxy-3-(3,4,5-trimethoxystyryl)phenol

(21):

(Z)-3-({2,3-Dimethoxy-5-[(Z)-3,4,5-trimethoxystyryl]phenyl}ami-

no)-1-(1-methyl-1H-indol-3-yl)prop-2-en-1-one (4h): Compound

4h was prepared according to the method described for com-

pound 3a by employing 13g (52.9 mg, 0.289 mmol) to obtain

pure 4h as a yellow solid. Yield: 112 mg, 73%; m.p. 112–1158C;

¹H NMR (300 MHz, CDCl₃): d=12.01 (d, J=12.1 Hz, 1H), 8.50 (s,

1H), 7.73 (s, 1H), 7.37 (s, 3H), 7.24–7.17 (m, 1H), 6.8 (s, 1H), 6.59 (d,

J=12.1 Hz, 5H), 5.90 (d, J=7.55 Hz, 1H), 4.05 (s, 3H), 3.92 (s, 3H),

3.87 (s, 3H), 3.72 ppm (s, 9H); ¹³C NMR (75 MHz, CDCl₃): d=187.5,

153.02, 140.5, 137.5, 134.8, 133.3, 133.1, 132.5, 132.0, 129.6, 126.4,

122.7, 122.4, 121.8, 117.8, 109.4, 107.0, 106.3, 106.1, 96.7, 61.03,

60.9, 60.7, 56.02, 55.8 ppm; IR (KBr): n˜_max=3432, 2931, 2832, 1628,

1528, 1522, 1505, 1487, 1469, 1425, 1366, 1326, 1276, 1216, 1147,

1125, 1087, 999, 947, 859, 791, 770 cm^À1; MS (ESI): m/z: 529

[M+H]⁺; HRMS (ESI): m/z: calcd for C₃₁H₃₃O₆N₂: 529.23331 [M+H]⁺;

found: 529.23274.

Compound 21 was prepared according to the method described

for compound 3c by employing 20 (100 mg, 0.224 mmol) to

obtain pure 21 as a yellow liquid. ¹H NMR (300 MHz, CDCl₃): d=

6.67 (d, J=8.3 Hz, 1H), 6.53–6.43 (m, 4H), 6.32 (d, J=8.3 Hz, 1H),

3.85 (s, 3H), 3.81 (s, 3H), 3.63 ppm (s, 6H); MS (ESI): m/z: 332

[M+H]⁺.

(Z)-3-({2-Hydroxy-3-methoxy-6-[(Z)-3,4,5-trimethoxystyryl]pheny-

l}amino)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (5a): Com-

pound 5a was prepared according to the method described for

compound 3a by employing 21 (100 mg, 0.301 mmol) and 13a

(66.2 mg, 0.301 mmol) to obtain pure 5a as a yellow solid. Yield:

123 mg, 73%; m.p. 61–638C; ¹H NMR (300 MHz, CDCl₃): d=11.93

(d, J=12.2 Hz, 1H), 7.84–7.75 (m, 1H), 7.18 (s, 2H), 6.80 (d, J=

8.12 Hz, 1H), 6.67 (d, J=11.8 Hz, 1H), 6.61–6.55 (m, 2H), 6.44 (s,

2H), 5.85–5.81 (m, 2H), 3.92 (s, 6H), 3.91 (s, 3H), 3.90 (s, 3H), 3.81

(s, 3H), 3.58 ppm (s, 6H); ¹³C NMR (75 MHz, CDCl₃): d=189.6, 152.8,

152.6, 149.8, 146.3, 137.2, 137.0, 135.0, 132.3, 132.0, 125.6, 125.2,

122.3, 121.1, 106.1, 105.9, 104.5, 92.7, 60.89, 60.8, 56.2, 56.1,

55.6 ppm; IR (KBr): n˜_max=3411, 2953, 2925, 2852, 1625, 1583, 1550,

1496, 1463, 1410, 1327, 1286, 1201, 1166, 1125, 1088, 1002, 919,

855, 833, 773 cm^À1; MS (ESI): m/z: 552 [M+H]⁺; HRMS (ESI): m/z:

calcd for C₃₀H₃₄O₉N: 552.22281 [M+H]⁺; found: 552.22399.

3-Hydroxy-4-methoxy-2-nitrobenzaldehyde (17): Nitromethane

(20 mL) and nitronium tetrafluoroborate (9.2 g, 0.069 mol) were

added to a stirred solution of isovaniline (16; 10 g, 0.066 mol) in

CH₂Cl₂(30 mL) at À408C. Then, the mixture was stirred for 6 h. The

progress of the reaction was monitored by TLC (EtOAc/hexane=

2:8). Water was added to the mixture at RT. The mixture was then

concentrated, and the aqueous layer was extracted with diethyl

ether (3ꢁ). The organic layer was washed with water and brine,

dried with anhydrous Na₂SO₄, and concentrated under reduced

pressure to afford the crude compound, which was purified by

column chromatography (EtOAc/hexane=3:7). Yield: 6.0 g, 46%;

¹H NMR (300 MHz, CDCl₃): d=9.79 (s, 1H), 7.39 (d, J=8.3 Hz, 1H),

7.08 (d, J=8.4 Hz, 1H), 4.02 ppm (s, 3H); MS (ESI): m/z: 220

[M+Na]⁺.

(Z)-1-(2-Bromo-3,4,5-trimethoxyphenyl)-3-({2-hydroxy-3-me-

thoxy-6-[(Z)-3,4,5-trimethoxystyryl]phenyl}amino)prop-2-en-1-

one (5b): Compound 5b was prepared according to the method

described for compound 3a by employing 13b (89.6 mg,

0.301 mmol) to obtain pure 5b as a yellow solid. Yield: 135 mg,

74%; m.p. 54–568C; ¹H NMR (300 MHz, CDCl₃): d=11.86 (d, J=

12.6 Hz, 1H), 7.93–7.84 (m, 1H), 6.85–6.80 (m, 2H), 6.68 (d, J=

11.89 Hz, 1H), 6.61–6.55 (m, 2H), 6.46 (s, 2H), 5.89 (s, 1H), 5.54 (d,

J=7.55 Hz, 1H), 3.90 (s, 6H), 3.89 (s, 3H), 3.87 (s, 3H), 3.81 (s, 3H),

3.63 ppm (s, 6H); ¹³C NMR (75 MHz, CDCl₃): d=199.1, 152.5, 150.8,

149.6, 146.3, 143.9, 138.4, 136.9, 132.6, 131.9, 125.3, 124.9, 122.2,

121.1, 108.0, 106.1, 106.0, 97.04, 61.1, 60.9, 60.8, 56.3, 56.1,

55.7 ppm; IR (KBr): n˜_max=3425, 2957, 2935, 2836, 1625, 1580, 1504,

1461, 1423, 1381, 1330, 1285, 1204, 1164, 1126, 1104, 1006, 850,

790 cm^À1; MS (ESI): m/z: 630 [M+H]⁺; HRMS (ESI): m/z: calcd for

C₃₀H₃₃O₉NBr: 630.13332 [M+H]⁺; found: 630.13385.

3-(tert-Butyldimethylsilyloxy)-4-methoxy-2-nitrobenzaldehyde

(18): Imidazole (0.789 g, 0.0116 mol) and tert-butylchlorodimethylsi-

lane (1.68 g, 0.0116 mol) were added to a stirred solution of 17

(2.0 g, 0.0101 mol) in CH₂Cl₂(30 mL) at 08C. The temperature was

slowly increased to RT, and the mixture was stirred for 3 h. The

progress of the reaction was monitored by TLC (EtOAc/hexane=

2:8). Upon completion of the reaction, water was added to mix-

ture, which was extracted with CH₂Cl₂. The organic layer was

washed with water and brine and dried with anhydrous Na₂SO₄. Fi-

nally, the solvent was evaporated under reduced pressure to afford

the pure compound. Yield: 2.9 g, 91%; ¹H NMR (300 MHz, CDCl₃):

d=9.56 (s, 1H), 7.26 (d, J=8.31 Hz, 1H), 6.82 (d, J=8.31 Hz, 1H),

3.74 (s, 3H), 0.93 (s, 9H), 0.09 ppm (s, 6H); MS (ESI): m/z: 312

[M+H]⁺.

(Z)-1-[3-(tert-Butyldimethylsilyloxy)-4-methoxyphenyl]-3-({2-hy-

droxy-3-methoxy-6-[(Z)-3,4,5-trimethoxystyryl]phenyl}amino)-

prop-2-en-1-one (14c): Compound 14c was prepared according

to the method described for compound 3a by employing 13c

(82.4 mg, 0.301 mmol) to obtain pure 14c as a yellow solid. Yield:

138 mg, 73%.

(Z)-tert-Butyl[6-methoxy-2-nitro-3-(3,4,5-trimethoxystyryl)phe-

noxy]dimethylsilane (19): Compound 19 was prepared according

to the method described for compound 11 a by employing 18

(3.69 g, 7.06 mmol) to obtain pure 19 as a yellow solid. Yield: 2.7 g,

(Z)-3-({2-Hydroxy-3-methoxy-6-[(Z)-3,4,5-trimethoxystyryl]pheny-

l}amino)-1-(3-hydroxy-4-methoxyphenyl)prop-2-en-1-one

(5c):

Compound 5c was prepared according to the method described

for compound 3c by employing 14c (100 mg, 0.161 mmol) to

obtain pure 5c as a yellow solid. Yield: 63 mg, 76%; m.p. 65–678C;

¹H NMR (300 MHz, CDCl₃): d=11.95 (d, J=12.08 Hz, 1H), 7.82–7.73

(m, 1H), 7.52–7.48 (m, 2H), 6.86 (d, J=8.3 Hz, 1H), 6.79 (d, J=

8.3 Hz, 1H), 6.67 (d, J=12.08 Hz, 1H), 6.58–6.54 (m, 2H), 6.44 (s,

2H), 5.84–5.81 (m, 2H), 5.64 (s, 1H), 3.95 (s, 3H), 3.94 (s, 3H), 3.90

(s, 3H), 3.81 (s, 3H), 3.59 ppm (s, 6H), ¹³C NMR (75 MHz, CDCl₃): d=

1

48%; H NMR (300 MHz, CDCl₃): d=6.77 (s, 2H), 6.61 (d, J=12.0 Hz,

1H), 6.41 (s, 2H), 6.38 (d, J=12.0 Hz, 1H), 3.82 (s, 3H), 3.81 (s, 3H),

3.67 (s, 6H), 0.94 (s, 9H), 0.21 ppm (s, 6H). MS (ESI, m/z): 476

[M+1]⁺.

(Z)-2-(tert-Butyldimethylsilyloxy)-3-methoxy-6-(3,4,5-trimethox-

ystyryl)aniline (20): Compound 20 was prepared according to the

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189.6, 152.5, 149.4, 146.3, 145.2, 137.05, 136.9, 132.9, 132.1, 132.07,

125.8, 125.2, 122.1, 120.9, 120.2, 113.6, 109.8, 105.9, 105.8, 92.6

60.7, 56.2, 55.8, 55.6 ppm; IR (KBr): n˜_max=3417, 2959, 2935, 2837,

1626, 1608, 1587, 1548, 1496, 1462, 1421, 1327, 1265, 1125, 1008,

1020, 849, 775 cm^À1; MS (ESI): m/z: 508 [M+H]⁺; HRMS (ESI): m/z:

calcd for C₂₈H₃₀O₈N: 508.19659 [M+H]⁺; found: 508.19559.

and concentrated under reduced pressure to afford pure 23a–g.

Compound 23a–g (1 mmol) was dissolved in DMSO, and a solution

of IBX (1.1 mmol) in DMSO (10 mL) was added at 10–158C. Then,

the temperature of the mixture was slowly increased to RT, and

the mixture was stirred for 4–6 h. The progress of the reaction was

monitored by TLC (EtOAc/hexane=3:7). Water was added, and the

mixture was filtered through Celite. The aqueous layer was extract-

ed with EtOAc. The organic layer was washed with water and

brine, dried with anhydrous Na₂SO₄, and concentrated under re-

duced pressure to afford the crude product, which was purified by

column chromatography (EtOAc/hexane=3:7).

(Z)-3-({2-Hydroxy-3-methoxy-6-[(Z)-3,4,5-trimethoxystyryl]pheny-

l}amino)-1-(4-methoxy-3-nitrophenyl)prop-2-en-1-one (5d): Com-

pound 5d was prepared according to the method described for

compound 3a by employing 13d (61.7 mg, 0.301 mmol) to obtain

pure 5d as a yellow solid. Yield: 124 mg, 76%; m.p. 120–1248C;

¹H NMR (500 MHz, CDCl₃): d=11.98 (d, J=12.5 Hz, 1H), 8.42 (d, J=

2.1 Hz, 1H), 8.14–8.12 (m, 1H), 7.88–7.84 (m, 1H), 7.11 (d, J=

8.85 Hz, 1H), 6.81 (d, J=8.85 Hz, 1H), 6.69 (d, J=11.9 Hz, 1H), 6.61

(d, J=8.3 Hz, 1H), 6.56 (d, J=11.7 Hz, 1H), 6.43 (s, 2H), 5.88 (s, 1H),

5.80 (d, J=7.7 Hz, 1H), 4.01 (s, 3H), 3.91 (s, 3H), 3.80 (s, 3H),

3.59 ppm (s, 6H); ¹³C NMR (75 MHz, CDCl₃): d=186.6, 154.8, 152.6,

150.6, 146.3, 139.1, 137.2, 137.0, 133.0,132.6, 131.9, 125.3, 124.9,

122.4, 121.12, 112.9, 106.4, 105.9, 91.9, 60.8, 56.6, 56.3, 55.6 ppm; IR

(KBr): n˜_max=3400, 2955, 2937, 2863, 1627, 1592, 1556, 1496, 1463,

1421, 1328, 1258, 1163, 1126, 1087, 1041, 1013, 963, 922, 855, 776,

736 cm^À1; MS (ESI): m/z: 537 [M+H]⁺; HRMS (ESI): m/z: calcd for

C₂₈H₂₉O₉N₂: 537.18676 [M+H]⁺; found: 537.18637.

1-(3,4,5-Trimethoxyphenyl)prop-2-yn-1-one (13a): Compound

13a was prepared according to the method described for com-

pound 13 by employing 1-(3,4,5-trimethoxyphenyl)prop-2-yn-1-ol

(23a; 750 mg, 3.38 mmol) and IBX (1.04 g, 3.7 mmol) to obtain

pure 13a as a light brown solid. Yield: 650 mg, 88%; m.p. 123–

1

1258C; H NMR (300 MHz, CDCl₃): d=7.43 (s, 2H), 3.95 (s, 3H), 3.93

(s, 6H), 3.43 ppm (s, 1H); MS (ESI): m/z: 221 [M+H]⁺.

1-(2-Bromo-3,4,5-trimethoxyphenyl)prop-2-yn-1-one (13b): Com-

pound 13b was prepared according to the method described for

compound 13 by employing 1-(2-bromo-3,4,5-trimethoxyphenyl)-

prop-2-yn-1-ol (23b; 750 mg, 2.49 mmol) and IBX (767 mg,

2.74 mmol) to obtain pure 13b as a brown solid. Yield: 640 mg,

1

(Z)-1-(3-Amino-4-methoxyphenyl)-3-({2-hydroxy-3-methoxy-6-

[(Z)-3,4,5-trimethoxystyryl]phenyl}amino)prop-2-en-1-one (5e):

Compound 5e was prepared according to the method described

for compound 3e by employing 5d (100 mg, 0.186 mmol) to

obtain pure 5e as a yellow solid. Yield: 70 mg, 74%; m.p. 75–788C;

¹H NMR (300 MHz, CDCl₃): d=12.08 (d, J=12.08 Hz, 1H), 7.36–7.33

(m, 2H), 7.23–7.19 (m, 1H), 6.82–6.75 (m, 2H), 6.51 (d, J=5.28 Hz,

6H), 5.95 (d, J=7.93 Hz, 1H), 3.95 (s, 3H), 3.90 (s, 3H), 3.83 (s, 3H),

3.71 ppm (s, 6H); IR (KBr): n˜_max=3426, 2995, 2938, 2837, 1610,

1579, 1532, 1505, 1462, 1421, 1353, 1327, 1283, 1237, 1185, 1126,

1089, 1009, 855, 827, 774 cm^À1; MS (ESI): m/z: 507 [M+H]⁺; HRMS

(ESI): m/z: calcd for C₂₈H₃₁O₇N₂: 507.21258 [M+H]⁺; found:

507.21359.

86%; m.p. 79–808C; H NMR (300 MHz, CDCl₃): d=7.46 (s, 1H), 3.98

(s, 3H), 3.93 (s, 3H), 3.89 (s, 3H), 3.50 ppm (s, 1H); MS (ESI): m/z:

298 [M+H]⁺.

1-[4-(tert-Butyldimethylsilyloxy)-3-methoxyphenyl]prop-2-yn-1-

one (13c): Compound 13c was prepared according to the method

described for compound 13 by employing 1-[3-(tert-butyldimethyl-

silyloxy)-4-methoxyphenyl]prop-2-yn-1-ol (23c; 750 mg, 2.57 mmol)

and IBX (790 mg, 2.82 mmol) to obtain pure 13c as a brown solid.

1

Yield: 620 mg, 83%; m.p. 80–828C; H NMR (300 MHz, CDCl₃): d=

7.67 (dd, J=6.41 Hz, 0.92 Hz, 1H), 7.43 (dd, J=0.92 Hz, 1.52 Hz,

1H), 6.73 (d, J=8.54 Hz, 1H), 3.71 (s, 3H), 3.19 (s, 1H), 0.83 (s, 9H),

0.12 ppm (s, 6H); MS (EI): m/z: 291 [M+H]⁺.

1-(3-Methoxy-4-nitrophenyl)prop-2-yn-1-one (13d): Compound

13d was prepared according to the method described for com-

pound 13 by employing 1-(4-methoxy-3-nitrophenyl)prop-2-yn-1-ol

(23d; 750 mg, 3.62 mmol) and IBX (1.12 g, 3.98 mmol) to obtain

pure 13d as a brown solid. Yield: 500 mg, 74%; m.p. 120–1238C;

¹H NMR (500 MHz, CDCl₃): d=8.61 (d, J=2.0 Hz, 1H), 8.31 (dd, J=

2.0 Hz, 8.85 Hz, 1H), 7.19 (d, J=8.85 Hz, 1H), 4.06 (s, 3H), 3.51 ppm

(s, 1H). MS (EI): m/z: 206 [M+H]⁺.

(Z)-3-({2-Hydroxy-3-methoxy-6-[(Z)-3,4,5-trimethoxystyryl]pheny-

l}amino)-1-[4-(trifluoromethoxy)phenyl]prop-2-en-1-one

(5 f):

Compound 5 f was prepared according to the method described

for compound 3a by employing 13e (64.4 mg, 0.301 mmol) to

obtain pure 5 f as a yellow solid. Yield: 130 mg, 78%; m.p. 97–

1

998C; H NMR (300 MHz, CDCl₃): d=12.01 (d, J=12.8 Hz, 1H), 7.96

(d, J=8.3 Hz, 2H), 7.87–7.80 (m, 1H), 7.23 (d, J=8.3 Hz, 3H), 6.79

(d, J=8.3 Hz, 1H), 6.65 (d, J=11.3 Hz, 1H), 6.60–6.51 (m, 2H), 6.43

(s, 2H), 5.79 (d, J=7.5 Hz, 1H), 3.85 (s, 3H), 3.80 (s, 3H), 3.56 ppm

(s, 6H); ¹³C NMR (75 MHz, CDCl₃): d=188.7, 152.5, 151.2, 150.5,

146.3, 137.8, 137.2, 137.1, 132.5, 132.01, 129.02, 125.4, 125.05,

122.4, 122.01, 121.07, 120.23, 106.3, 105.9, 105.7, 92.5, 60.7, 56.2,

55.6 ppm; IR (KBr): n˜_max=3429, 3311, 2935, 2898, 2837, 1625, 1606,

1550, 1503, 1462, 1421, 1327, 1284, 1257, 1176, 1126, 1088, 1007,

855, 775 cm^À1; MS (ESI): m/z: 546 [M+H]⁺; HRMS (ESI): m/z: calcd

for C₂₈H₂₇O₇NF₃: 546.1739 [M+H]⁺; found: 546.1766.

1-[3-(Trifluoromethoxy)phenyl]prop-2-yn-1-one

(13e):

Com-

pound 13e was prepared according to the method described for

compound 13 by employing 1-[4-(trifluoromethoxy)phenyl]prop-2-

yn-1-ol (23e; 750 mg, 3.47 mmol) and IBX (1.07 g, 3.82 mmol) to

obtain pure 13e as a brown solid. Yield: 600 mg, 80%; m.p. 97–

1

998C; H NMR (500 MHz, CDCl₃): d=7.83 (d, J=8.3 Hz, 2H), 7.21 (d,

J=8.3 Hz, 2H), 3.15 ppm (s, 1H); MS (EI): m/z: 215 [M+H]⁺.

tert-Butyl 3-propioloyl-1H-indole-1-carboxylate (13 f): Compound

13 f was prepared according to the method described for com-

pound 13 by employing tert-butyl 3-(1-hydroxyprop-2-yn-1-yl)-1H-

indole-1-carboxylate (23 f; 750 mg, 2.76 mmol) and IBX (851 mg,

3.04 mmol) to obtain pure 13 f as a brown solid. Yield: 620 mg,

83%; m.p. 150–1528C; ¹H NMR (300 MHz, CDCl₃): d=8.43 (s, 1H),

8.32 (d, J=8.1 Hz, 1H), 8.12 (s, J=8.1 Hz, 1H), 7.41–7.35 (m, 2H),

3.27 (s, 1H), 1.72 ppm (s, 9H); MS (EI): m/z: 291 [M+H]⁺.

General procedure for the preparation of 1-arylprop-2-yn-1-one

(13): 0.5 n Ethynylmagnesium bromide in THF (1.5 mmol) was

added to a stirred solution of substituted benzaldehyde 22a–g

(1 mmol) in THF at 08C. Then, the temperature of the mixture was

slowly increased to RT, and the mixture was stirred for 6–8 h. The

progress of the reaction was monitored by TLC (EtOAc/hexane=

1:1). A solution of saturated ammonium chloride was added, and

the mixture was concentrated and extracted with EtOAc. The or-

ganic layer was washed with brine, dried with anhydrous Na₂SO₄,

1-(1-Methyl-1H-indol-3-yl)prop-2-yn-1-one (13g): Compound 13g

was prepared according to the method described for compound

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13 by employing 1-(1-methyl-1H-indol-3-yl)prop-2-yn-1-ol (23g;

800 mg, 4.32 mmol) and IBX (1.33 g, 4.75 mmol) to obtain pure

13g as a brown solid. Yield: 800 mg, 73%; m.p. 180–1838C;

¹H NMR (300 MHz, CDCl₃): d=8.38–8.35 (m, 1H), 7.95 (s, 1H), 7.36

(t, J=2.83 Hz, 5.09 Hz, 2H), 7.33–7.32 (m, 1H), 3.88 (s, 3H),

3.16 ppm (s,1H); MS (EI): m/z: 184 [M+H]⁺.

of vacuum. The blot was blocked with 5% blotto for 1 h at RT. Im-

munoblot analysis was performed as described previously by using

UVP, biospectrum 810 imaging system.^[27]

Tubulin polymerization assay: An in vitro assay for monitoring the

time-dependent polymerization of tubulin to microtubules was

performed by employing a fluorescence-based tubulin polymeri-

zation assay kit (BK011, Cytoskeleton, Inc.) according to the manu-

facturer’s protocol. The reaction mixture in a final volume of 10 mL

in PEM buffer [80 mm piperazine-N,N’-bis(2-ethanesulfonic acid)

(PIPES), 0.5 mm ethylene glycol tetraacetic acid (EGTA), 2 mm MgCl₂,

pH 6.9] in 384-well plates contained bovine brain tubulin

(2 mgmL^À1), 10 mm fluorescent reporter, 1 mm guanosine-5’-tri-

phosphate (GTP) in the presence or absence of test compounds at

378C. Tubulin polymerization was followed by monitoring the fluo-

rescence enhancement resulting from the incorporation of a fluo-

rescence reporter into the microtubules as the polymerization pro-

ceeded. Fluorescence emission at l=420 nm (l_exc=360 nm) was

measured for 1 h at 1 min intervals in a multimode plate reader

(Tecan M200). To determine the IC₅₀values of the compounds

against tubulin polymerization, the compounds were preincubated

with tubulin at varying concentrations (0.01, 0.1, 1, 10, and

100 mm). Assays were performed under conditions similar to those

employed for polymerization assays as described above.^[29]

Biology

Cell cultures, maintenance, and antiproliferative evaluation: All the

cell lines used in this study were purchased from the American

Type Culture Collection (ATCC, United States). A549, MCF-7, HeLa,

and HCT116 were grown in Dulbecco’s modified Eagle’s medium

(containing 10% fetal bovine serum in a humidified atmosphere of

5% CO₂at 378C). Cells were trypsinized when subconfluent from

T25 flasks/60 mm dishes and seeded in 96-well plates. The amino-

stilbene–arylpropenones were evaluated for their in vitro antiproli-

ferative activity in four different human cancer cell lines. A protocol

of 48 h continuous drug exposure was used, and a sulforhodami-

ne B (SRB) cell proliferation assay was employed to estimate cell vi-

ability or growth. The cell lines were grown in their respective

media containing 10% fetal bovine serum and were seeded into

96-well microtiter plates in 200 mL aliquots at plating densities de-

pending on the doubling time of individual cell lines. The microtit-

er plates were incubated at 378C, 5% CO₂, 95% air, and 100% rela-

tive humidity for 24 h prior to the addition of the experimental

drugs. Aliquots of 2 mL of the test compounds were added to the

wells already containing 198 mL of cells, which resulted in the re-

quired final drug concentrations. For each compound, four concen-

trations (0.1, 1, 10, and 100 mm) were evaluated, and each was

done in triplicate wells. Plates were incubated for another 48 h,

and the assay was terminated by the addition of 100 mL of 10%wt/

vol cold trichloroacetic acid and incubated at 48C for 1 h. The su-

pernatant was discarded. The plate was washed with tap water

(4ꢁ) and was allowed to air dry. The cells were then stained with

0.057% SRB dissolved in 1% acetic acid for 30 min at RT. Unbound

SRB was washed away with four washes of 1% acetic acid. The

plate was again allowed to air dry, and the bound SRB stain, repre-

senting surviving cells, was dissolved in Tris base (10 mm, 50 mL).

The optical density was determined at l=510 nm by using a micro-

plate reader (Enspire, PerkinElmer, USA). Percent growth was calcu-

lated on a plate-by-plate basis for test wells relative to control

wells. The above determinations were repeated thrice. The growth

inhibitory effects of the compounds were analyzed by generating

dose response curves as a plot of the percentage surviving cells

versus compound concentration. The sensitivity of the cancer cells

to the test compound was expressed in terms of IC₅₀, a value de-

fined as the concentration of compound that produced 50% re-

duction relative to the control absorbance. IC₅₀values are indicated

as meansÆstandard deviations of three independent experi-

ments.^[27]

Analysis of cell cycle: A549 cells in 60 mm dishes were incubated

for 24 h in the presence or absence of test compounds 3e, 3g,

and 4e at 1 mm concentration. Cells were harvested with trypsin–

EDTA and fixed with ice-cold 70% ethanol at 48C for 30 min; then,

ethanol was removed by centrifugation, and the cells were stained

with DNA staining solution [1 mL; 0.2 mg of propidium iodide (PI),

and 2 mg RNase A] for 30 min as described earlier. The DNA con-

tents of 20000 events were measured by flow cytometer (BD FACS-

Canto II). Histograms were analyzed by using FCS express 4 plus.

Immunohistochemistry of tubulin and analysis of nuclear morpholo-

gy: A549 cells were seeded on glass cover slip and incubated for

24 h in the presence or absence of test compounds at a concentra-

tion of 1 mm. Cells grown on coverslips were fixed in 3.5% formal-

dehyde in phosphate-buffered saline (PBS) pH 7.4 for 10 min at RT.

Cells were permeabilized for 6 min in PBS containing 0.5% Triton

X-100 (Sigma) and 0.05% Tween-20 (Sigma). The permeabilized

cells were blocked with 2% bovine serum albumin (Sigma) in PBS

for 1 h. Later, the cells were incubated with the primary antibody

for tubulin (Sigma) at 1:200 diluted in blocking solution for 4 h at

RT. Subsequently, the antibodies were removed, and the cells were

washed with PBS (3ꢁ). Cells were then incubated with fluorescein

isothiocyanate labeled anti-mouse secondary antibody (1:500) for

1 h at RT. Cells were washed with PBS (3ꢁ) and mounted in

medium containing 4’,6-diamidino-2-phenylindole. Images were

captured by using the Olympus confocal microscope FLOW VIEW

FV 1000 series and analyzed with FV10ASW 1.7 series software.

Western blot analysis of soluble versus polymerized tubulin: Cells

were seeded in 12-well plates at 1ꢁ10⁵cells per well in complete

growth medium. Following individual treatment of cells with com-

pounds 3e, 3g, and 4e for 24 h, the cells were washed with PBS

and subsequently soluble and insoluble tubulin fractions were col-

lected. To collect the soluble tubulin fractions, cells were permeabi-

lized with prewarmed lysis buffer [200 mL; 80 mm PIPES-KOH

(pH 6.8), 1 mm MgCl₂, 1 mm EGTA, 0.2% Triton X-100, 10% glycerol,

0.1% protease inhibitor cocktail (Sigma–Aldrich)] and incubated for

3 min at 308C. The lysis buffer was gently removed and mixed

with 3ꢁLaemmli’s sample buffer [100 mL; 180 mm Tris-Cl pH 6.8,

6% sodium dodecyl sulfate (SDS), 15% glycerol, 7.5% b-mercap-

Dot-blot assay: A549 cells were treated with 1 mm 3a–h, 4a–h, and

5a–f for 24 h. Subsequently, cells were harvested and proteins

were quantified by using Amido Black followed by densitometry

analysis. Equal amounts of protein were blotted on a nitrocellulose

membrane by using a Bio-Dot SF microfiltration apparatus (Bio-

Rad). Briefly, the nitrocellulose membrane and three filters papers

(Whatmann 3) were soaked in 1ꢁ Tris-buffered saline (TBS) solution

for 10 min. Later, the filter papers and membrane were arranged in

the apparatus and connected to a vacuum pump (Millipore). The

membranes were rehydrated by using 1ꢁ TBS (100 mL) by vacuum

filtration. Subsequently, the samples (50 mL) were blotted on the

membranes and washed with 1ꢁ TBS (200 mL) through application

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Keywords: antiproliferation

· cell-cycle arrest · dot-blot

analyses · molecular docking · polymerization

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Received: June 23, 2014

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Published online on && &&, 0000

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FULL PAPERS

A. Kamal,* G. B. Kumar, S. Polepalli,

A. B. Shaik, V. S. Reddy, M. K. Reddy,

C. R. Reddy, R. Mahesh, J. S. Kapure,

N. Jain

Sittin’ on the docking site of tubulin:

A series of aminostilbene–arylprope-

nones are designed, synthesized, and

evaluated for their cytotoxic activity.

These compounds inhibit microtubule

assembly and induce cyclin B1 protein

expression. Docking experiments reveal

that these compounds bind with the

colchicine binding site of tubulin.

&& – &&

Design and Synthesis of

Aminostilbene–Arylpropenones as

Tubulin Polymerization Inhibitors

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Article Doi

Design and Synthesis of Aminostilbene-Arylpropenones as Tubulin Polymerization Inhibitors

DOI: 10.1002/cmdc.201402256

Source and publish data:

Authors:

Article abstract of DOI:10.1002/cmdc.201402256

Full text of DOI:10.1002/cmdc.201402256

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