Synthesis of Some Benzimidazole Derivatives Bearing 1,3,4-Oxadiazole Moiety as Anticancer Agents

Abstract

In an effort to establish new benzimidazole related structural leads with improved anticancer activity, several new benzimidazole derivatives (5a–i) with 1,3,4-oxadiazole scaffold incorporated were synthesized and studied for their anticancer activity. The anticancer screening against MDA-MB-231 breast cancer cell lines showed that compound (5c) exhibited moderate cytotoxicity.

Introduction

Benzimidazole derivatives are structural isosters of naturally occurring nucleotides, thus they can interact with biological macromolecules such as proteins, enzymes and receptors. The unique structural features and a wide range of biological activities of benzimidazole made it privileged structure in drug discovery^1–4. Apart from benzimidazoles, the five membered azole heterocycles, especially, oxadiazole has been introduced into drug discovery for several purposes. Molecular modeling and pharmacokinetic studies have demonstrated that incorporating oxadiazole moiety to drug-like molecules change their polarity, flexibility as well as metabolic profile and ability to engage in hydrogen bonding. Hence, oxadiazoles have been widely employed as bioesteric replacement of carbonyl compounds, such as carbamates, hydroxamic esters, amides and esters in a number of biological targets^5–9.

The above mentioned considerations prompted us to design a new series of benzimidazole derivatives with 1,3,4-oxadiazole moieties incorporated into benzimidazole nuclei. Here in, we report the synthesis (Scheme 1), characterization and cytotoxic screening of a new class of 1, 3, 4-oxadazolylbenzimidazole.

Scheme 1.

Reagents and condition (a) N₂H₄H₂O, Ethanol, Reflux; (b) CS₂, KOH, Ethanol; (c) K₂CO₃, D-glucose in DMF or NaOAc, Ethanol, XCH₂Ph; (d) NaH,

Experimental

Reagents and solvents were purchased from Sigma-Aldrich Chemical Company Inc. and used as received. Melting points were determined in open capillaries on a Gallenkamp digital melting point apparatus and were uncorrected. The Infrared spectra were recorded in KBr discs using Shimadzu FT-IR 8000 spectrometer. ¹H NMR (DMSO-d₆) and ¹³C NMR spectra were recorded using Bruker 300 MHz spectrometer using TMS as internal standard. Thin-layer chromatography was performed using precoated silica gel plates (silica gel 0.25 mm, 60G F₂₅₄).

General procedure for compounds 3a–i

Synthesized by a method reported in literature¹⁰. To a solution of substituted benzohydrazide (2a–i) (0.01 mol) in ethanol (25 mL) at 0 °C, carbon disulphide (2 mL) and potassium hydroxide (0.6 g) were added and the reaction mixture was refluxed until the evolution of H₂S gas ceased (3–4 h). Excess solvents were evaporated under reduced pressure and the residue was dissolved in water and then acidified with dilute hydrochloric acid (10%) to pH 5. The precipitate was filtered off, dried and crystallized from ethanol.

5-(4-Methoxyphenyl)-1,3,4-oxadiazole-2-thiol (3b)

White solid, Yield: 76%; mp: 169–171 °C; ¹H NMR (300 MHz, CDCl₃) δ: 3.75 (s, 3H), 7.08–7.21 (m, 3H, Ar–H), 7.59–7.64 (m, 1H, Ar–H), 7.70 (d, 1H, J = 7.0, Ar–H), 7.82 (d, 1H, J = 7.0, Ar–H), 7.95 (s, 1H, Ar–H).

General procedure for the synthesis of 4a–i

A mixture of 3 (0.01 mol), K₂CO₃ (0.012 mol), D-glucose (0.004 mol), 2-chloromethylbenzimidazole (0.01 mol) and DMF (35 mL) was stirred at room temperature for 2 h. At the end of this period, the reaction mixture was poured into ice-water. The separated product was filtered, washed with water, dried and recrystallized from ethyl acetate to obtain pure 4.

2-(5-Phenyl-1,3,4-oxadiazol-2-ylthio)methyl)-1H-benzimidazole (4a)

White solid, yield: 76%; mp: 168 170 °C; ¹H NMR (300 MHz, DMSO-d₆): δ 4.80 (s, 2H, -CH₂-S-), 7.10–8.00 (m, 9H, Ar-H), 12.60 (s, 1H, D₂O exch., –NH).

2-((5-(4-Methoxyphenyl)-1,3,4-oxadiazol-2-ylthio)methyl)-1H-benzimidazole (4b)

Light yellow crystal, yield: 53.9%, mp: 120–121 °C. ¹H NMR (300 MHz, DMSO-d₆) δ: 3.85 (s, 3H), 4.58 (s, 2H), 7.13 (d, J = 8.8 Hz, 2H), 7.51 (d, J = 8.2 Hz, 1H), 7.88 (d, J = 8.8 Hz, 2H), 7.98 (d, J = 8.2 Hz, 1H), 8.50 (s, 1H).

2-((5-(4-Nitrophenyl)-1,3,4-oxadiazol-2-yl)thiomethyl)-1H-benzimidazole (4c)

White solid, yield: 76%; mp: 168 170 °C; ¹H NMR (300 MHz, DMSO-d₆): δ 4.90 (s, 2H, -CH₂-S-), 7.30–8.40 ( m, 8H, Ar-H), 12.92 (s, 1H, D₂O exch., –NH).

2-((5-(4-Methyphenyl)l-1,3,4-oxadiazol-2-yl)thiomethyl)-1H-benzimidazole(4d)

White solid, yield: 76%; mp: 202 205 °C; IR (KBr): ¹H-NMR (300 MHz, DMSO-d₆): δ 2.30 (s, 3H, Ar-CH₃), 4.80 (s, 2H, -CH₂-S-), 7.00–8.00 (m, 8H, Ar-H), 12.50(s, 1H, D₂O exch., –NH).

2-((5-(4-Tert-Butoxyphenyl)-1,3,4-oxadiazol-2-ylthio)methyl)-1H-benzimidzole(4e)

White solid, yield: 51.4%; mp: 195 197 °C; ¹H NMR (300MHz, DMSO-d₆) δ 1.24 (s, 9H), 7.36 (s, 2H, NH₂), 7.61–7.81 (m, 9H, Ar-H)

2-((5-(4-Fluorophenyl)-1,3,4-oxadiazol-2-ylthio)methyl)-1H-benzoimidazole(4f)

Light yellow powder, yield: 56.2%, mp: 132–133 °C. ¹H NMR (300 MHz, DMSO-d₆) δ: 4.61 (s, 2H), 7.45–8.04 (m, 3H), 8.53 (s, 1H).

4-(5-((1H-Benzimidazol-2-yl)methylthio)-1,3,4-oxadiazol-2-yl)-benzenamine(4g)

White solid, yield: 76%; mp: 168 170 °C; ¹H NMR (300 MHz, DMSO-d₆): δ:4.90 (s, 2H, - CH₂-S-), 5.97 (s, 2H, -NH₂), 7.30–8.40 ( m, 8H, Ar-H), 12.92 (s, 1H, D₂O exch., –NH).

2-((5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-ylthio)methyl)-1H-benzimidazole(4h)

White solid, yield: 76%; mp: 145 147 °C; ¹H NMR (300 MHz, DMSO-d₆): δ 4.68 (s, 2H, -CH₂-S-), 7.20–7.90 (m, 8H, Ar-H), 12.60 (s, 1H, D₂O exch., NH).

2-((5-(4-Bromophenyl)-1,3,4-oxadiazol-2-ylthio)methyl)-1H-benzimidazole(4i)

White powder, yield: 76%; mp: 145 147 °C; ¹H NMR (300 MHz, DMSO-d₆): δ 4.68 (s, 2H, -CH₂-S-), 7.20–7.90 (m, 8H, Ar-H), 12.60 (s, 1H, D₂O exch., NH).

General procedure for compounds 5a–i

To a solution of compound 4 (0.01 mol) in dimethylformamide (25 mL), sodium hydride (0.0 mol) was added gradually under ice cooling. The reaction mixture was stirred for 30 min and then the appropriate halide (0.01 mol) was added portion wise. The whole mixture was warmed to room temperature and stirred for 4 h, poured onto ice-water with continuous stirring. The precipitated product was filtered off and purified by silica gel column-chromatography.

2-((5-Phenyl-1,3,4-oxadiazol-2-ylthio)methyl)-1-benzylbenzimidazole (5a)

White solid, yield:76%; mp: 168 170 °C; ¹H NMR (300 MHz, DMSO-d₆): δ 4.80 (s, 2H, - CH₂-S-), 5.13 (s,2H, CH₂), 7.06–7.88 (m, 14H, Ar-H); 13C-NMR (DMSO d₆) δ 20.3 (-CH₂-S-), 122.1, 122.9, 126.4, 129.3, 132.0, 138.1, 152.9, 161.5, 165.6 (Ar-H); Anal. Calcd. For C₂₃H₁₈N₄OS: C, 69.32; H, 4.55; N, 14.06. Found:C, 69.30; H, 4.92; N, 14.19.

2-((5-(4-Methoxyphenyl)-1,3,4-oxadiazol-2-yl]thiomethyl}-1-benzylbenzimidazole (5b)

Pale brown solid, yield: 66%; mp: 168 170 °C; ¹H NMR (300 MHz, DMSO-d₆): δ s, 3H, -OCH₃), 4.24 (s, 2H, -CH₂-S-), 5.04 (s, 2H, CH₂), 7.10–8.00 (m, Ar-H); Anal. Calcd. For C₂₄H₂₀N₄O₂S: C, 67.27; H, 4.70, N, 13.07. Found:C, 67.30; H, 4.92; N, 13.19.

2-((5-(4-Nitrophenyl)-1,3,4-oxadiazol-2-yl)thiomethyl)-1-benzylbenzimidazole (5c)

Yellow solid, yield: 52%; mp: 168 170 °C; ¹H NMR (300 MHz, DMSO-d₆): δ 4.20 (s, 2H, - CH₂-S-), δ 5.54 (s, 2H, CH₂), 7.30–8.40 ( m, 13H, Ar-H);. Anal. Calcd. for C₂₃H₁₇N₅O₃S: C, 62.29; H, 3.86; N, 15.79. Found:C, 62.36; H, 3.15; N, 15.80.

2-((5-(4-Methylphenyl)-1,3,4-oxadiazol-2-yl)thiomethyl}-1-benzylbenzimidazole (5d)

White solid, yield: 68%; mp: 202 205 °C; ¹H NMR (300 MHz, DMSO-d₆): δ .32 (s, 3H, Ar-CH₃), 4.28 (s, 2H, -CH₂-S-),5.04 (s, 2H, CH₂), 7.00–7.98 (m, Ar-H); Anal. Calcd. for C₂₄H₂₀N₄OS: C, 69.88; H, 4.89; N, 13.58. Found: C, 69.30; H, 4.86; N, 13.37.

2-((5-(4-Tert-butylphenyl)-1,3,4-oxadiazol-2-yl)thiomethyl}-1-benzylbenzimidazole (5e)

White solid, yield: 56%; mp: 202 205 °C; ¹H NMR (300 MHz, DMSO-d₆): δ .32 (s, 9H, -C(CH₃)₃, 4.20 (s, 2H, -CH₂-S-), 5.02 (s, 2H, CH₂), 6.98 – 7.78 (m, Ar-H); Anal. Calcd. for C₂₇H₂₆N₄OS: C, 71.34; H, 5.76; N, 12.32. Found:C, 71.55; H, 5.02; N, 12.46.

2-((5-(4-Fluorophenyl)-1,3,4-oxadiazol-2-yl)thiomethyl)-1-benzylbenzimidazole (5f)

Light yellow crystal, yield: 48%, mp: 132–133 °C. ¹H NMR (300 MHz, DMSO-d₆) δ: 4.54 (s, 2H, -CH₂-S-), 4.61 (s, 2H), 6.85–7.53 (m, Ar-H), Anal. Calcd. for C₂₃H₁₇FN₄OS: C, 66.33; H, 4.11; N, 13.45. Found: C, 66.38; H, 4.77; N, 13. 01.

4-(5-((-Benzylbenzimidazol-2-yl)methylthio)-1,3,4-oxadiazol-2-yl)benzenamine (5g)

Yellow solid, yield: 76%; mp: 202 205 °C; ¹H NMR (300 MHz, DMSO-d₆): δ 3.80 (s, 2H, -NH₂), 4.20 (s, 2H, -CH₂-S-), 5.02 (s, 2H, CH₂), 6.58 – 7.50 (m, 13H, Ar-H); Anal. Calcd. for C₂₃H₁₉N₅OS: C, 66.81; H, 4.63; N, 16.94. Found: C, 66.55; H, 4.02; N, 16.46.

2-((5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl)thiomethyl)-1-benzylbenzimidazole (5h)

Light yellow crystal, yield: 56.2%, mp: 152–155 °C. ¹H NMR (300 MHz, DMSO-d₆) δ:4.54 (s, 2H, -CH₂-S-), 4.61 (s, 2H), 6.36–7.53 (m, 13H, Ar-H), Anal. Calcd. for C₂₃H₁₇ClN₄OS: C, 66.33; H, 4.11; N, 13.45. Found: C, 66.38; H, 4.77; N, 13. 01.

2-((5-(4-Bromophenyl)-1,3,4-oxadiazol-2-yl)thiomethyl)-1-benzylbenzimidazole (5i)

Light yellow crystal, yield: 56.2%, mp: 122–125 °C. ¹H NMR (300 MHz, DMSO-d₆) δ:4.58 (s, 2H, -CH₂-S-), 4.57 (s, 2H), 6.85–7.53 (m,13H, Ar-H), Anal. Calcd. for C₂₃H₁₇BrN₄OS: C, 57.87; H, 3.59; N, 11.74. Found: C, 57.38; H, 3.59; N, 11. 01.

In vitro anticancer screening

Hanks balanced salt solution, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES), ethanol, 96 well plates, general reagents and supplies, were all purchased from Sigma-Aldrich Co. (St. Louis, MO) and VWR International (Radnor, PA). Imaging probes were supplied by Life Technologies Grand Island, NY.

Cell culture

MDA-MB-231 (ATCC® HTB-26^™) human breast cancer cells were obtained from ATCC (Manassas, VA). MDA-MB-231 cells were brought up in ATCC-formulated Leibovitz’s L-15 Medium [Catalog No. 30-2008], supplemented with 10% FBS and penicillin/streptomycin (100 U/0.1 mg/mL). After confluence, the cells were sub-cultured and grown in DMEM containing phenol red, 10% FBS, 4 mM L-glutamine, 20 μM sodium pyruvate and penicillin/streptomycin (100 U /0.1 mg/mL). Culture conditions were maintained [37 °C in 5% CO₂/atmosphere] and every 2–5 days, the media was replaced and cells sub-cultured.

Proliferation and toxicity screening

Compounds were dissolved in DMSO, vortexed and stored at −20 °C in the dark. A stock solution for each experimental compound was prepared in HBSS+5 mM (N-[2-hydroxyethyl-piperazine]-N′-[2-ethanesulfonic acid]) (HEPES), adjusted to a pH of 7.4. An initial screening was conducted at 5 concentrations within the test range of 0.416 mg/mL to 0. 0208 mg/mL for toxicity and mitostatic potential. Briefly, for cell proliferation studies, 96 well plates contained a low cell plating density [0.04×10⁶ / well] and full growth media ± experimental compounds. A 72 hour cell count was evaluated relative to positive and negative (paclitaxel) controls. The toxic effects of each compound were evaluated at 24 hours, where cells were plated in low serum media at higher plating cell plating density [0.4×10⁶ / well] ± experimental compounds with positive and negative (paclitaxel) controls.

Results and Discussion

Chemistry

The synthetic strategies adopted for the synthesis of the target compounds are depicted in Scheme 1. Condensation of 3 with 2-chloromethylbenzimidazole in presence of potassium hydroxide in ethanol afforded compound 4. The reaction was comprised of two steps; the first step is the formation of a salt and in the second step the salt was treated with 2-chloromethyl-benzimidazole afforded 4 in fair to good yields. N-benzylation of 4 with benzyl bromide in presence of sodium hydride in DMF afforded 5 in fair to good yields.

The structures of the newly synthesized compounds were established on the basis IR, ¹H NMR and ¹³C NMR spectrum. In general, IR spectra of the compounds showed peaks at 3326 and 2557 cm⁻¹ for NH and SH, respectively. In ¹H NMR spectra, there appeared a singlet at around δ 11.9 indicative of ring H-N and another singlet at δ 13.4 for S-H, both disappeared by addition of D₂O as confirmation for these groups. The 1H NMR of 4 showed disappearance of broad singlet of SH and corresponding appearance of the singlet at δ 4.35–4.15 ppm integrating for two protons due to S-CH₂groupforming a linker through which benzimidazole nuclei attached with oxadiazole nuclei. The chemical shift in ¹³C NMR spectra at δ 173.56 and 156.24 could be accounted for C=O and C=N. All target compounds showed a singlet at chemical shift around δ 5.0 in ¹H NMR spectra and ¹³C NMR spectra showed at around δ 30.9 which could be accounted for N-CH₂ group. The multiplet peaks shown at around δ 7.2–6.9 accounted for aromatic protons.

Anticancer assay

The synthesized compounds were screened for their in vitro anticancer potential against a panel of breast cancer cell lines: MDA-MB-231 using paclitaxel as control. The relationship between surviving fraction of cells and drug concentration was plotted and the response parameter IC₅₀ was calculated. IC₅₀ value corresponds to the concentration required for 50% inhibition of cell viability. The IC₅₀ value of the test compounds are shown in Table 1. Compound 5c exhibited moderate inhibition potency (< 50 μM) as described in Table 1.

Table 1.

IC₅₀ value of the test compounds

Entry	Structure	IC50(μM)
5a		MDA-MB-231 >1098.4
5b		>1296.0
5c		>17.5
5d		>1245.7
5e		>1134.3
5f		>616.7
5g		>1238.6
5h		>201.1

Conclusion

The present work, through simple synthetic approaches, led to the development of a hybrid of benzimidazoles containing 1,3,4-oxadiazolepharmacophore that exhibited moderate cytotoxic activities against breast tumor cell lines. The activity could be attributed to some sort of synergism between benzimidazole coupled with 1,3,4-oxadiazole molecule.