untitled European Journal of Chemistry 3 (3) (2012) 356‐358 European Journal of Chemistry ISSN 2153‐2249 (Print) / ISSN 2153‐2257 (Online)  2012 EURJCHEM DOI:10.5155/eurjchem.3.3.356‐358.621 European Journal of Chemistry Journal homepage: www.eurjchem.com Anticancer activity studies of some cyclic benzimidazole derivatives Ali El‐Shekeil*, Abeer Omer Obeid and Sama Al‐Aghbari Chemistry Department, Faculty of Science, Sana’a University, 2463, Sana’a, Yemen *Corresponding author at: Chemistry Department, Faculty of Science, Sana’a University, 2463, Sana’a, Yemen. Tel.: +967.7.33215234; fax: +967.1.464484. E‐mail address: shekeil2000@yahoo.com (A. El‐Shekeil). ARTICLE INFORMATION ABSTRACT Received: 23 April 2012 Received in revised form: 01 June 2012 Accepted: 17 June 2012 Online: 30 September 2012 KEYWORDS New benzimidazole derivatives, namely, (N‐(4‐methoxyphenyl)methylene]‐1H‐benzimidazol‐ 2‐amine (2a), (N‐(3,4‐dimethoxyphenyl)methylene]‐1H‐benzimidazol‐2‐amine (2b), and (N‐ (3,4,5‐trimethoxyphenyl)methylene]‐1H‐benzimidazol‐2‐amine (2c) were synthesized by reaction of a Schiff base with malononitrile in absolute ethanol. Structures of compounds have been confirmed by IR, 1H NMR and elemental analysis. Compounds 2a‐c were screened for their in vitro anticancer potential using HeLa and PC3 cells. All compounds showed limited cytotoxicity except compound 2a that showed a moderate cytotoxic effect towards HeLa cells. PC3 cells Hela cells Cytotoxicity Anti‐cancer activity Benzimidazole derivatives 4‐Amino‐2‐(3,4,5‐ trimethoxyphenyl)benzo[4,5]imidazo[1,2‐ a]pyrimidine‐3‐carbonitrile 1. Introduction Cancer is the biggest health issue in the world. Cancer developed resistance against many existing anticancer drugs. This keeps a research window open in search for newer anticancer molecules. However, it is rather difficult to come up with a molecule that can selectively inhibit the proliferation of abnormal cells only with least or no effect on normal cells. Many authors worldwide have studied benzimidazole. A potential of antitumor, anti‐proliferative or anticancer has been reported [1‐4]. Benzimidazole nucleus has been confirmed as an important pharmacophore in drug discovery [5]. Its derivatives have been reported to exhibit antitumor [6], antibacterial [7], anti‐ inflammatory [8] and promising anticancer activities [9‐10]. Moreover, benzimidazoles have been used as biomimetic of guanine residues [11] and they selectivity inhibit endothelial cell growth and suppress angiogenesis in vivo and in vitro [12]. Due to a broad spectrum of activities noted so far and in continuation of our research interest on the synthesis of bioactive heterocycles, we describe here the synthesis of cyclic benzimidazole derivatives and study their anticancer effect. 2. Experimental 2.1. Chemicals 2‐Aminobenzimidazole, 4‐methoxybenzaldehyde, 3,4,5‐ trimethoxybenzaldehyde, 3,4‐dimethoxybenzaldehyde, piperi‐ dine and malononitrile were obtained commercially from Aldrich and Fluka Chemicals. Solvents were reagent grade and were used as received: dry absolute ethanol, petroleum ether, n‐hexane and ethyl acetate. Progress of reactions was monitored by thin layer chromatography (TLC) using precoated aluminum sheet silica gel; Merck 60 F254 and was visualized by UV lamp. 2.2. Instrumentations Melting points were measured on an electrothermal melting point apparatus and were not corrected. Fourier‐ transform infrared spectra were recorded using the KBr disc technique on a JASCO 410 FTIR spectrophotometer. Elemental (CHN) analysis was performed using an Exeter CE‐440 elemental analyzer. 1H NMR spectra of the compounds were recorded on a Varian Gemini‐200 spectrometer (200 MHz) using DMSO‐d6 as solvent and TMS as internal reference. Anticancer activity was evaluated at the International Center for Chemical Sciences and Dr. Panjwani Center for Molecular Medicine and Drug Research, University of Karachi, Pakistan. 2.3. Synthesis of Schiff base ligands Syntheses of Schiff base compounds 1a‐c were carried out through the method described by Nawrocka et al. [13] involving the condensation of an equimolar mixture of 2‐ aminobenzimidazole (0.01 mol) and the aldehyde as 3,4,5‐ trimethoxybenzaldehyde (0.01 mol), 3,4‐dimethoxybenz‐ aldehyde (0.01 mol) and 4‐methoxybenzaldehyde (0.01 mol), respectively, in ethanol (25 mL). The mixture was refluxed for 24 hours under nitrogen atmosphere. Schiff base compounds formed were isolated by crystallization from a suitable solvent. 2.4. Synthesis of cyclic benzimidazole Schiff base compounds (0.01 mol) were reacted with malanonitrile (0.01 mol) in ethanol with a catalytic amount of piperidine (0.2 mL). The reaction mixture was heated under reflux for 4 hrs. El‐Shekeil et al. / European Journal of Chemistry 3 (3) (2012) 356‐358 357 N N H NH2 + R1 R2 R3 C N N H N R1 R2 R3 NC CN N N N R1 R2 R3 CN H2N a H OCH3 H c OCH3 OCH3 OCH3 Piperidine Ethanol 1a-c 2a-ca-c R1 R2 R3 b H OCH3 OCH3 O H Scheme 1 Then the reaction mixture was poured onto crushed ice and neutralized by hydrochloric acid to give a crude product which was filtered off, washed several times with cold water, dried and crystallized (Scheme 1). 4‐Amino‐2‐(4‐methoxyphynyl)benzo[4,5]imidazo[1,2‐a] pyrimidine‐3‐carbonitrile (2a): Yellow crystals, recrystallized from ethanol. Yield: 35%. M.p.: 103‐105 oC. FT‐IR (KBr, , cm‐1): 3367 (NH2) (amine), 2222 (CN) (cyano), 1645, 1605 (C=N) (pyrimidine and imidazole), 1571 (C=C) (aromatic). 1H NMR (200 MHz, DMSO d6, δ, ppm): 3.90 (s, 3H, OCH3,), 6.90‐8.40 (m, 8H, Ar‐H, NH2). Anal. calcd. for C18H13N5O: C, 68.6; H, 4.2; N, 22.2. Found: C, 68.9; H, 3.9; N, 21.9%. 4‐Amino‐2‐(3,4‐dimethoxyphenyl)benzo[4,5]imidazo[1,2‐a] pyrimidine‐3‐carbonitrile (2b): Dark Yellow crystals, recrystallized from ethanol. Yield: 30%. M.p.: 143‐144 oC. FT‐IR (KBr, , cm‐1): 3372 (NH2) (amine), 2222 (CN) (cyano), 1642, 1600 (C=N) (pyrimidine and imidazole), 1581 (C=C) (aromatic). 1H NMR (200 MHz, DMSO d6, δ, ppm): 3.80 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 7.15‐8.16 (m, 9H, Ar‐H, NH2). Anal. calcd. for C19H15N5O2: C, 66.1; H, 4.4; N, 20.3. Found: C, 62.9; H, 4.3; N, 19.8%. 4‐Amino‐2‐(3,4,5‐trimethoxyphenyl)benzo[4,5]imidazo[1,2‐ a] pyrimidine‐3‐carbonitrile (2c): Yellow crystals, recrystallized from n‐hexane‐ethyl acetate. Yield: 35%. M.p.: 117‐120 oC. FT‐ IR (KBr, , cm‐1): 3397 (NH2) (amine), 2224 (CN) (cyano), 1647, 1600 (C=N) (pyrimidine and imidazole), 1582 (C=C) (aromatic). 1H NMR (200 MHz, DMSO‐ d6, δ, ppm): 3.99 (s, 6H, OCH3), 3.82 (s, 3H, OCH3,) 6.87‐8.43 (m, 8H, Ar‐H, NH2). Anal. calcd. for C20H17N5O3: C, 64.0; H, 4.6; N; 18.7. Found: C, 62.4; H, 4.5; N, 19.5%. 2.5. Cytotoxicity Cytotoxic activity of compounds was evaluated in 96‐well flat‐bottomed microplates by using the standard MTT (3‐[4,5‐ dimethylthiazole‐2‐yl]‐2,5‐diphenyl‐tetrazolium bromide) colorimetric assay [14]. For this purpose, PC‐3 cells (Prostate Cancer) and HeLa cells were cultured in Dulbecco’s Modified Eagle’s Medium, and Minimal Essential Medium (MEM), supplemented with 5% of fetal bovine serum (FBS), 100 IU/mL of penicillin and 100 µg/mL of streptomycin in 25 cm3 flask, and kept in 5% CO2 incubator at 37 oC. Exponentially growing cells were harvested, counted with haemocytometer and diluted with a particular medium. Cell culture with the concentration of 1x105 cells/mL was prepared and introduced (100 µL/well) into 96‐well plates. After overnight incubation, medium was removed and 200 µL of fresh medium was added with different concentrations of compounds (1‐100 µM). After 48 h, 50 µL MTT (2 mg/mL) was added to each well and incubated further for 4 hours. Subsequently, 100 µL of DMSO was added to each well. The extent of MTT reduction to formazan within cells was calculated by measuring the absorbance at 570 nm, using a micro plate reader (Spectra Max plus, Molecular Devices, Ca, USA). The cytotoxicity was recorded as concentration causing 50% growth inhibition (IC50). 3. Results and discussion 3.1. Synthesis and characterization Schiff base compounds (1a‐c) was prepared by reaction of 2‐aminobenzimidazole with 4‐methoxybenzaldehyde, 3,4‐ dimethoxybenzaldehyde and 3,4,5‐trimethoxybenzaldehyde, respectively, in refluxing solvent (Scheme 1). Schiff base compounds 1a‐c (0.01 mol) were reacted with malononitrile (0.01 mole) to give 2a‐c compounds (Scheme 1). Structures of compounds were confirmed on the basis of their elemental analysis and spectral data. IR spectra of compounds 2a‐c showed absorption bands characteristic for NH2 at 3367, 3372 and 3397 cm‐1 and CN at 2222, 2222 and 2224 cm‐1, respectively. 1H NMR spectra showed multiple signals around 7.00‐8.40, 7.18‐8.16, and 6.80‐ 8.40 ppm, ascribed to aromatic and NH2 protons. 3.2. Anticancer activity Results of in vitro anticancer activity of the tested compounds 2a‐c were evaluated for cytotoxicity against PC3 cells and Hela cells of humans in comparison with doxorubicin as a positive control. All tested compounds showed a limited cytotoxic activity except compound 2a that showed moderate cytotoxic effect towards Hela cells. Table 1 represents the cytotoxic activity of the tested compounds. Table 1. Cytotoxic activity of compounds 2a‐c against HeLa Cells and PC3 Cells. Compound PC3 HeLa IC50 ± SD* (μM) IC50 ± SD (μM) 2a >50 22.57 ± 0.16 2b >50 >50 2c >50 >50 Doxorubicin (as control) 0.912 ± 0.120 3.10 ± 0.20 * SD: Standard deviation. Acknowledgements The authors would like to acknowledge the continuous support of Sana’a University and especially the Faculty of Science and the Chemistry Department. Thanks are due to The University of Science and Technology, Sana’a, as represented by the Rector, Prof. Dr. Hameed Aqlan for supporting Sama Al‐ Aghabari in her study for Ph.D. The authors would like also to thank Mr. Mohammed Abdelqawi Ha‐el from Ha‐el Saeed Corporation for financial support. 358 El‐Shekeil et al. / European Journal of Chemistry 3 (3) (2012) 356‐358 4. Conclusion The present work describes the synthesis and in vitro anticancer evaluation of some cyclic benzimidazole derivatives 2a‐c. Most of the tested compounds showed limited cytotoxic activity except compound 2a that showed a moderate cytotoxic effect towards Hela cells. References [1]. 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