A Simple, Efficient Synthesis of 2-Aryl Benzimidazoles Using Silica Supported Periodic Acid Catalyst and Evaluation of Anticancer Activity

A new, efficient method for the synthesis of 2-aryl substituted benzimidazole by using silica supported periodic acid (H5IO6-SiO2) as a catalyst has been developed. The salient feature of the present method includes mild reaction condition, short reaction time, high yield and easy workup procedure. The synthesized benzimidazoles exhibited potent anticancer activity against MCF7 and HL60 cell lines.


Introduction
The benzimidazole nucleus is commonly present in a large number of natural products as well as pharmacologically active compounds [1]. It shows a wide spectrum of biological and pharmacological properties such as antifungal [2], antimicrobial [3], anthelmintic [4,5], antiviral [6,7], topoisomerase inhibition [8] and anticancer activities [9]. Some of their derivatives are marketed as antifungal drug (Carbendazim) [10], anthelmintic drug (Mebendazole and Thiabendazole) [11], antipsychotic drug (Pimozide) [12] and antiulcer agent (Omeprazole) [13]. Owing to their interesting pharmacological properties, great attention has been paid to the synthesis of benzimidazoles. Two main synthetic methods were well known in the literature. The most common method is direct condensation of 1,2-phenylenediamine and carboxylic acids [14,15] or their derivatives [16], that require strong acidic conditions and sometimes need high temperature or the use of microwave [17]. The other synthetic route involves a two-step procedure that includes the cyclo-dehydrogenation of aniline Schiff 's bases, which are often generated in situ from the condensation of 1,2-phenylenediamines and aldehydes [18], followed by oxidation with stoichiometric amount of oxidants, such as MnO 2 [19], Oxone [20], NaHSO 3 [21,22], I 2 /KI/K 2 CO 3 /H 2 O [23] or catalytic use of CAN [24] and AIKIT-5 [25]. More recently, 2-alkyl substituted benzimidazoles are synthesized by using hexafluorophosphoric acid under microwave condition [26].
There is renewed interest in the silica supported catalyzed reactions [27]. These reactions have relatively shorter reaction time with high yield and cleaner chemistry. Moreover, the catalyst is easily separated from reaction mixture by simple filtration. There are very few reports involving solid supported catalyzed reaction for synthesis of benzimidazole derivatives. Jacob et al. [28] synthesized 1,2-disubstituted benzimidazoles by silica supported ZnCl 2 catalyst that was found to be of poor yield. Patil et al. [29] developed a method for synthesis of 2-alkyl benzimidazoles using silica supported HBF 4 . Paul and Basu [30] described the synthesis of 1,2-disubstituted benzimidazoles by using silica gel soaked with Fe 2 (SO 4 ) 3 ⋅ H 2 O. Recently, Kumar et al. [31] reported silica supported HClO 4 catalyzed synthesis of benzimidazoles.
Periodic acid is an easily available hypervalent iodine reagent which is used in the oxidation of various functional groups [32,33]. However, there are no reported efforts for the synthesis of benzimidazoles by using periodic acid. In this paper, we report an efficient and facile synthesis of 2aryl benzimidazoles by using silica supported periodic acid (H 5 IO 6 -SiO 2 ) as a catalyst (Scheme 1). Further, all synthesized derivatives were screened for anticancer activity against two cancer cell lines, namely, MCF7 and HL60.

Result and Discussion
Herein, we used unprecedented silica supported periodic acid (H 5 IO 6 -SiO 2 ) as catalyst for synthesis of 2-aryl benzimidazole derivatives. In our initial experiments, we choose 1,2-phenylenediamine (1 mmol) and m-nitrobenzaldehyde (1 mmol) as a model reaction for optimization of catalyst and reaction conditions. The results are summarized in Table 1. The use of 20 mol% of H 5 IO 6 catalyst supported on silica resulted in 95% of desired product, 5 g in 15 min at room temperature (  (Table 1, entries 5 and 10) at room temperature and 60 ∘ C even after prolonged time (10 h). Further, the reactions were carried out with only H 5 IO 6 without silica support at room temperature and 60 ∘ C ( Table 1, entries 4 and 9) where the yield was not found to be more than 35%. These results confirmed that H 5 IO 6 supported on silica significantly increased the efficacy of catalysts which may be attributed to the increase in available surface area. Thus, we found optimized conditions as the 1,2-phenylenediamine (1 mmol), aldehyde (1 mmol) and H 5 IO 6 (0.20 mmol supported on silica) in acetonitrile (ACN) at room temperature.
Feasibility of the methodology was examined for a series of aryl/heteroaryl aldehydes bearing electron donating as well as electron withdrawing groups under the optimized reaction conditions and corresponding products were obtained in good to excellent yields ( Table 2). Presence of electron withdrawing group in aldehyde system fastened the reaction (entries 5f-5h) while opposite effect was observed for electron donating substituents and hindered aldehydes (entries 5b and 5h). We have not observed any remarkable change in the reaction time for the different substituted diamines. The reaction underwent smoothly even with aldehyde bearing two functional groups (entries 5c, 5h, 6c & 7c) and afforded corresponding products in good yields (entry 5h). The reaction was carried out with substituted 1,2-phenylenediamine (entries 6a-6e and 7a-7c) and afforded 2,5-substituted benzimidazoles in moderate to good yields. Using these reaction conditions exclusively formation of 2-substituted benzimidazoles was observed. The products were characterized by their physical and spectral data. Thus, Table 2 illustrates generality and efficiency of this method for the synthesis of benzimidazoles.
Although the exact mechanism is not clear, a proposed mechanism for the formation of benzimidazole is shown in

Anticancer Activity
All the synthesized benzimidazoles were tested for their anticancer activity against two cell lines MCF7 (human breast adenocarcinoma) and HL60 (human promyelocytic leukemia) by MTT colorimetric assay using cisplatin as a standard anticancer drug. The results are expressed as Scheme 3: Plausible mechanism towards the formation of 2-aryl benzimidazole. and summarized in Table 2. Anticancer activity varies with substitution at 5-position of benzimidazole ring. The benzoyl substituted benzimidazole (7a-7c) showed the highest potency against two cell lines, while carboxyl substituted compounds (6a and 6b) were moderately potent (with the exception of 6c), as compared to unsubstituted benzimidazole (5a-5c). Dichloro derivatives (5c and 6c) exhibited more activity as compared to monochloro derivatives (5d and 6d) against MCF7 and HL60. Substitution of nitro group (5e-5g) showed moderate and mostly similar effect for the given cell lines. Compound 5b with phenolic −OH group (IC 50 27.63 M for MCF7 and 28.68 M for HL60) was comparable to 5h (IC 50 29.67 M for MCF7 and 26.52 M for HL60) which has additional methoxy group at p-position. Replacing ring carbon of benzene ring with nitrogen atom as in 5i (IC 50 30.42 M) showed better activity against MCF7 when compared with 5a (IC 50 35.67 M), but same compound did not show substantial difference in activity against HL60. Bisbenzimidazole (8) was found to be more active for MCF7 (IC 50 17.45 M) than HL60 (IC 50 30.69 M). All the tested compounds are found to be more effective against both cell lines as compared to cisplatin.

Conclusion
We have developed a short and efficient method for the synthesis of 2-aryl benzimidazoles from 1,2-phenylenediamines and aryl aldehydes using H 5 IO 6 -SiO 2 as catalyst. The mild reaction condition, low cost, easy workup procedure and good to excellent yields as well as the scope for using wide substrates make our methodology a valuable contribution to the existing processes for synthesis of benzimidazole derivatives. Among 18 derivatives, newly synthesized 5-substituted derivatives exhibited excellent activity against MCF7 and HL60 cell lines. The overall activities for all the derivatives tested were found in micromolar range. The current study provides better insight into the designing of more potent anticancer agents in the future.

Experimental Section
All reactions were performed in open atmosphere with unpurified reagents and distilled solvents. Periodic acid was purchased from Spectrochem. Acetonitrile and silica (230-400) were purchased from Sigma Aldrich. Thin-layer chromatography (TLC) was performed using 0.25 mm silica gel coated plates. Column chromatography was performed using the hexane: ethylacetate solvent and silica gel (60-120 meshes). 1 H NMR (300 MHz) and 13 C NMR (75 MHz) spectra were recorded on Varian Mercury instrument with DMSO-6 or D 2 O as the solvents. Chemical shifts were reported in unit (ppm) with reference to TMS as an internal standard, and values were given in Hertz. Melting points were determined on Thomas Hoover capillary melting point apparatus and are uncorrected. IR spectra were recorded on a Shimadzu FTIR 8400 spectrophotometer in KBr disc and expressed in cm −1 . Elemental analysis was carried out with Thermo-Electron Corporation CHNS analyzer Flash-EA 1112.

Cell
Culture. Two cancer cell lines, MCF7 (human breast adenocarcinoma) and HL60 (human promyelocytic leukemia), were obtained from National Center for Cell Sciences, India. MCF7 was cultured in DMEM medium [35] while HL60 cells were cultured in a humidified atmosphere (37 ∘ C, 5% CO 2 ) in RPMI1640 medium supplemented with 10% fetal bovine serum.

MTT Assay.
Test compounds were evaluated for anticancer activity against two cancer cell lines using cisplatin as standard anticancer drug. The compounds were evaluated in vitro at a concentration range of 10 M to 100 M. The MTT colorimetric assay was used to determine growth inhibition. 100 L of cell suspension (5 × 10 6 cells) were plated in 96well plates and allowed to attach for 24 h. The compounds were dissolved in 0.5% DMSO. Cells were exposed in triplicate wells to these derivatives at various concentrations for 48 h. After 48 h, 20 L MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution (5 mg/mL) was added to each well. After 1 h of incubation, the solution was centrifuged for 5 min under 4000 rpm, and the medium was discarded carefully. The formazan precipitate was dissolved in DMSO (200 L), then shaken by oscillator. The absorbance at 570 nm was determined on a microplate reader (Bio-Rad Model 3350, Japan). The absorbance values were used to calculate % inhibition at various concentrations and IC 50 values.

Procedure for Synthesis of Silica Supported H 5 IO 6
Catalyst (H 5 IO 6 -SiO 2 ). H 5 IO 6 (2.50 g, 10.96 mmol) was dissolved in 15 mL of hot water (70 ∘ C) in a 50 mL round-bottomed flask. To the hot solution was added silica gel (230-400 meshes, 10 g) with vigorous stirring. The resultant H 5 IO 6 (resultant mixture contains 20 wt% of H 5 IO 6 ) supported with silica gel was dried in oven at 100 ∘ C for 12 h to obtain a white free flow powder. The reagent can be stored for 4 months with negligible loss of activity. Benzimidazoles (5a-5i,  6a-6e and 7a-7c). A typical procedure is as follows. A mixture of 1,2-phenylenediamine (108 mg, 1.0 mmol), m-nitrobenzaldehyde (151 mg, 1.0 mmol) in acetonitrile (3.0 mL) was taken, and H 5 IO 6 (45 mg, 20 mmol% supported on silica 210 mg) was added at room temperature. The reaction was stirred at room temperature for 15 minutes. After completion 4 ISRN Organic Chemistry of the reaction (monitored by TLC), filter the reaction mixture over celite. The filtrate was evaporated under vacuum and subsequently dried to afford crude product which was purified by column chromatography using hexane/ethylacetate as eluent to afford pure benzimidazole 5g (227 mg, 95%). (8). A mixture of 1,2-phenylenediamine (216 mg, 2.0 mmol), p-phthalaldehyde (134 mg, 1.0 mmol) in acetonitrile (3.0 mL) was taken, and H 5 IO 6 (90 mg, 40 mol% supported on silica 420 mg) was added at room temperature. The reaction was stirred at room temperature for 35 minutes. After completion of the reaction (monitored by TLC), the reaction mixture was filtered over celite. The filtrate was evaporated under vacuum and subsequently dried to afford crude product which was purified by column chromatography using hexane/ethylacetate as eluent to afford pure benzimidazole 8 (250 mg, 80%). The spectral data are in full agreement with data reported in the literature. Spectral data of compounds are given below.