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Synthesis and cytotoxic activities of novel hybrid compounds of imidazole

scaffold-based 2-substituted benzofurans{

Wen-Jian Song, Xiao-Dong Yang,* Xiang-Hui Zeng, Xiao-Liang Xu, Gao-Lan Zhang and Hong-Bin Zhang*

Received 29th February 2012, Accepted 25th March 2012

DOI: 10.1039/c2ra20376f

A series of novel hybrid compounds between 2-substituted

benzofuran and imidazole have been prepared and evaluated in

vitro against a panel of human tumor cell lines. The results show

that the hybrid compounds were more selective towards an

ovarian carcinoma cell line (Skov-3) and suggest that hybridcompounds bearing 2-substituted benzofuran and benzimidazole

moieties, as well as imidazolium salts, were vital for modulating

cytotoxic activity. The 2-substituted benzofuran imidazole

hybrids 24 and 8 can serve as valuable leads for further structural

modifications.

Cancer is a major burden of disease worldwide and is the leading

cause of human mortality exceeded only by cardiovascular diseases.1

Therefore, development of new anticancer drugs and more effective

treatment strategies for cancer are of the utmost importance.2

Natural products represent a significant source of inspiration for the

design of structural analogues with improved pharmacological

profiles in medicinal chemistry.3

Naturally occurring substitutedbenzofurans are an important class of biologically active oxygen-

containing heterocycles. Natural products possessing the 2-substi-

tuted benzofuran moiety exhibit a broad range of biological and

pharmacological activities such as antimicrobial, antiviral, antiox-

idant, antifungal, antiproliferative, anti-inflamanatory, antifeedant,

anti-HIV and antiplatelet activities.4 Recently, naturally occurring

benzofurans have been identified to possess anti-tumor activity.5 As

exemplified in Scheme 1, 1-(6-hydroxy-2-isopropenyl-1-benzofuran-

5-yl)-1-ethanone6 and calophione A7 are also 2-substituted benzo-

furan derived compounds exhibiting potent cytotoxic activities

against human breast cancer cells and colon cancer cells.6,7

Imidazole and its derivatives have attracted considerable interest in

recent years for their versatile properties in chemistry and

pharmacology. Biological activities of imidazole derivatives have

been reported to include antimicrobial and antifungal, antimuscari-

nic, thromboxane synthetase inhibition, antiinflamatory, antiar-

rhythmic, and plasmid DNA cleavage activities,8 especially anti-

tumor activity.9 For example, two new imidazolium halides

(Scheme 1), Lepidiline A and Lepidiline B, isolated from the roots

ofLepidium meyenii, showed potent cytotoxic activity against human

cancer cell lines.10 Recently, we have reported the synthesis of a series

of novel hybrid compounds of imidazole moieties such as NMIB

(Scheme 1) and their potential anti-tumor activity.11

Molecular hybridization as a drug discovery strategy involves the

rational design of new chemical entities by the fusion of two drugs,

both active compounds and/or pharmacophoric units recognized and

derived from known bioactive molecules.12 Considering the antic-

ancer activities of naturally occurring 2-substituted benzofurans as

well as the potent cytotoxic activities of natural and synthetic

imidazole derivatives, we were interested in synthesizing a number of

new hybrid compounds bearing 2-substituted benzofuran and

imidazole moieties (Scheme 1).

Although benzofurantriazole hybrid molecules through a

heptyloxybenzene chain were synthesized and found to exhibit

CYP26A1 inhibitory activity by Simons,13 to the best of our

knowledge, no reports concerning anti-tumor activity for hybridcompounds between 2-substituted benzofuran and imidazole

have been reported.

In the present research, we have designed and synthesized a series

of novel hybrid compounds of imidazole scaffold-based 2-substituted

benzofurans. The purpose of this study was to investigate the anti-

tumor activity of benzofuranimidazole hybrids, with the ultimate

aim of developing novel potent anti-tumor agents.

As shown in Scheme 2, substituted salicylaldehydes (1) were

condensed with ethyl bromoacetate to afford benzofuran-2-carbox-

ylate compounds (2, 7581% yields).14 The benzofuran 2-carboxylate

compounds 2 were reduced with LiAlH4 to the respective benzofuran

2-methanol compounds (3, 7089% yields).15 Subsequently, the

benzofuran 2-methanol compounds (3) were transformed via themesylate to the respective fifteen 2-substituted benzofuranimidazole

hybrids (418) with various substituted imidazoles by refluxing under

toluene with 5483% yields (two steps).16 Finally, six benzofuran-

based imidazolium salts (1924) were prepared with excellent yields

by reaction of 2-substituted benzofuranimidazole hybrids with the

corresponding alkyl bromides by refluxing under toluene (7592%

yields).17 The structures of hybrid compounds are shown in Table 1.

The cytotoxic potential of all newly synthesized hybrid compounds

was evaluated in vitro against a panel of human tumor cell lines

according to procedures described in the literature.18

The panel

consisted of ovarian carcinoma (Skov-3), myeloid leukaemia

(HL-60), and breast carcinoma (MCF-7). Cisplatin (DDP) was used

Key Laboratory of Medicinal Chemistry for Natural Resource (YunnanUniversity), Ministry of Education, School of Chemical Science andTechnology, Yunnan University, Kunming, 650091, P. R. China.E-mail: zhanghbyd@gmail.com or xdyang@ynu.edu.cn;Fax: +86-871-5035538; Tel: +86-871-5031119{ Electronic supplementary information (ESI) available: scan spectral data ofthe novel hybrid compounds. See DOI: 10.1039/c2ra20376f.

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as the reference drug. The results are summarized in Table 1 (IC50value, defined as the concentrations corresponding to 50% growth

inhibition).

As shown in Table 1, all 2-substituted benzofuranimidazole

hybrids lacked activity against the HL-60 and MCF-7 tumor cell

lines investigated at a concentration of 40 mg ml21 (except

compounds 8 and 24). However, the hybrid compounds were more

selective towards ovarian carcinoma cell line (Skov-3).

As for Skov-3 cell lines, the structures of the hybrid compounds

have an obvious influence on the cytotoxic activities. In terms of the

benzofuran ring and imidazole ring, the 2-substituted benzofuran

imidazole hybrids 418 with no substituent or a methoxy or an allyl

group at the benzofuran ring, as well as with an imidazole ring or

alkyl substituted-imidazole ring (methyl or ethyl) were almost

inactive (IC50 . 40 mg ml21). However, when a benzimidazole ring

was used instead of a imidazole ring, hybrid compounds displayed

Scheme 2 Synthesis of hybrid compounds 421.

Scheme 1 Design of novel hybrid compounds.

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similar cytotoxic activity in vitro compared with DDP (with an IC50value of 9.5 mg ml21, 7.9 mg ml21 and 9.3 mg ml21 for compounds 8,

16 and 24, except compound 12).

Compared with the above hybrid compounds 418, benzofuran-

based imidazolium salts 1924 exhibited higher cytotoxic activity.

Most of this kind of derivatives showed moderate activity. Similarly,

the hybrid salt with a benzimidazole ring (compound 24, with IC50value of 9.1 mg ml21) exhibited a higher cytotoxic activity than the

hybrid salts with an imidazole ring or alkyl substituted-imidazole

ring (compounds 1923). Compared with compounds 2123 bearing

the same substituents at position 1 and 2 of the imidazole ring, the

cytotoxic activity of the hybrid salt with a benzyl substituent at

position 3 of the imidazole ring (R4 group) was higher than that of

the hybrid compounds with the other alkyl substituents.

The results suggest that 2-substituted benzofuranimidazole

hybrids bearing benzimidazole moieties, as well as imidazolium salts

at the imidazolyl-3 position with a benzyl group, were vital for

modulating cytotoxic activity. The structureactivity relationship

(SAR) results were summarized in Scheme 3.

In conclusion, a number of novel hybrid compounds between

2-substituted benzofuran and imidazole have been prepared in this

research and evaluated in vitro against a panel of human tumor cell

lines. The results show that the hybrid compounds were more

selective towards ovarian carcinoma cell lines (Skov-3) and suggest

that hybrid compounds bearing 2-substituted benzofuran and

benzimidazole moieties, as well as imidazolium salts, were vital for

modulating cytotoxic activity. The 2-substituted benzofuranimida-

zole hybrids 24 and 8 can be considered promising leads for further

structural modifications guided by the valuable information

derivable from our detailed SARs.

Acknowledgements

This work was supported by grants (30960460, 21062026,

2010GA014 and 2009CB522300) from the National Natural

Science Foundation of China, Yunnan Province and the

National Basic Research Program of China (973 Program).

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Table 1 Structures and cytotoxic activities of hybrid compounds 424 in vitrob (IC50, mg ml21a)

Compound R1 R2 Imidazole ring R4 Skov-3 HL-60 MCF-7

4 H H Imidazole .40 .40 .405 H H 2-Methyl-imidazole .40 .40 .406 H H 2-Ethyl-imidazole .40 .40 .407 H H 4-Methyl-imidazole .40 .40 .408 H H Benzimidazole 9.5 8.4 11.89 OMe H Imidazole .40 .40 .40

10 OMe H 2-Methyl-imidazole .40 .40 .4011 OMe H 2-Ethyl-imidazole .40 .40 .4012 OMe H Benzimidazole .40 .40 .4013 H Allyl Imidazole .40 .40 .4014 H Allyl 2-Methyl-imidazole .40 .40 .4015 H Allyl 2-Ethyl-imidazole .40 .40 .4016 H Allyl Benzimidazole 7.9 .40 .4017 OMe Allyl Imidazole 36.2 .40 .4018 OMe Allyl Benzimidazole 9.3 .40 .4019 H H Imidazole Benzyl 20.8 .40 .4020 H H 2-Methyl-imidazole Benzyl 25.4 .40 .4021 H H 2-Ethyl-imidazole Benzyl 23.2 .40 .4022 H H 2-Ethyl-imidazole 2-Bromobenzyl 35.1 .40 .4023 H H 2-Ethyl-imidazole Butyl 39.5 .40 .4024 H H Benzimidazole Benzyl 9.1 7.4 12.5

DDP 8.9 5.5 13.0

a Cytotoxicity as IC50 for each cell line, is the concentration of compound which reduced by 50% the optical density of treated cells with respect tountreated cells using the MTT assay. b Data represent the mean values of three independent determinations.

Scheme 3 Structureactivity relationship of hybrid compounds.

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imidazole hybrids 418. To a solution of benzofuran 2-methanolcompound 3 (1 mmol) in dichloromethane (50 mL) was addedmethanesulfonyl chloride (1.2 mmol) and triethylamine (2 mmol) at0 uC. The resulting mixture was stirred at room temperature for 2 h. Afterquenching the reaction with water (50 mL), the layers were separated. The

organic phase was dried over anhydrous Na2SO4 and concentrated, andused for the next synthetic step. A mixture of the previous methanesulfo-nate and imidazole or substituted imidazole (3 mmol) was stirred in toluene(20 ml) at reflux for 2448 h (monitored by TLC). After cooling to roomtemperature, the solvent was concentrated, and the residue was dilutedwith EtOAc (20 mL). The organic layer was washed with water (20 mL)and brine (20 mL), dried over anhydrous Na2SO4 and concentrated. Theresidue was purified by column chromatography (silica gel, petroleumether 6090 uC, ethyl acetate) to afford 418 in 5483% yield (two steps).Compound 8: white powder, yield 78%, mp 128130uC (CHCl3).

1H NMR(300 MHz, CDCl3) d 7.96 (1H, s), 7.847.81 (1H, m), 7.477.37 (3H, m),7.287.17 (4H, m), 6.56 (1H, s), 5.32 (2H, s). 13C NMR (75 MHz, CDCl3)d 155.08 (C), 151.07 (C), 143.82 (C), 143.02 (CH), 133.66 (C), 127.69 (C),124.86 (CH), 123.24 (CH), 122.42 (CH), 121.20 (CH), 120.47 (CH), 111.32(CH), 109.77 (CH), 105.51 (CH), 42.18 (CH2). HR-ESI-MS m/z Calcd forC16H12N2O 248.0950, Found 248.0944. Anal. Calcd for C16H12N2O: C,

77.40; H, 4.87; N, 11.28. Found: C, 77.33; H, 4.86; N 10.95.17 General procedure for the preparation of 2-substituted benzofuranimidazolium bromides 1924. A mixture of 2-substituted benzofuranimidazole hybrids 418 (1 mmol) and alkyl bromides (1.2 mmol) wasstirred in toluene (10 ml) at reflux for 816 h. A white solid was formed.After completion of the reaction as indicated by TLC, the precipitatewas filtered through a small pad of Celite, and washed with toluene (3 610 ml), then dried to afford 1924 in 7592% yield. Pure samples wereobtained after recrystallization from an appropriate solvent (acetone ormethanol). Compound 24: white powder, yield 89%, mp 218220 uC(MeOH). 1H NMR (300 MHz, MeOD) d 8.39 (1H, s), 6.60 (1H, d, J =7.6 Hz), 6.37 (1H, d, J = 7.6 Hz), 6.196.10 (3H, m), 6.015.91 (6H, m),5.805.73 (2H, m), 5.70 (s, 1H), 4.52 (s, 2H), 4.27 (s, 2H). 13C NMR(75 MHz, MeOD) d 148.74 (C), 142.01 (C), 133.02 (C), 131.42 (C), 129.04(CH), 128.91 (CH), 128.04 (CH), 127.57 (C), 127.20 (CH), 127.09 (CH),125.19 (CH), 123.12 (CH), 121.37 (CH), 113.67 (CH), 113.48 (CH), 110.81(CH), 107.80 (CH), 50.77 (CH2), 43.86 (CH2). HR-ESI-MS m/z Calcd for

C23H19N2O [M-Br]+ 339.1492, Found 339.1483. Anal. Calcd forC23H19BrN2O: C, 65.88; H, 4.57; N, 6.68. Found: C, 66.29; H, 4.57; N6.31.

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