Synthesis of new heterocyclic compounds containing benzimidazole moiety as inhibitors of breast cancer cell growth

Chemistry

The target compounds were synthesized as depicted in Scheme 1 and Equations 1–2. The key intermediate, 1-(1H-benzimidazol-2-yl)-2-bromoethanone (1), was synthesized as previously reported [24]. Treatment of 1 with the appropriate substituted anilines gave the corresponding products 2–5 (Scheme 1). The reaction of 1 with sodium benzoate or its 2-hydroxy derivative yielded the respective benzoate 6 or 7. In addition, the desired hydrazides 8 and 9 were synthesized by treatment of the substrate 1 with the appropriate hydrazides. By contrast, the reaction of 1 with 1,2-phenylenediamine or 2-aminophenol gave 3-(1H-benzimidazol-2-yl)-1,2-dihydroquinoxaline (10) and 3-(1H-benzimidazol-2-yl)-2H-benzo[b][1,4]oxazine (11), respectively. All compounds 2–11 were fully characterized by spectroscopic methods and elemental analysis.

Known 2-acetylimidazoles 12a,b [25] were allowed to react with isatins to furnish the desired 2-(1H-benzimidazole-2-yl)-6-substituted quinoline-4-carboxylic acids 13–15 (Equation 1).

Finally, the substrates 16a [26–28] and 16b were used in the preparation of benzimidazo[1,2-d][1,2,4]triazine-4-carbonitriles 17–24 (Equation 2). Again, the given structures of the synthesized compounds 12–24 were fully consistent with the spectroscopic and elemental analysis data.

Biology

All compounds 2–15 and 17–24 were evaluated for their in vitro antitumor activity against human MCF7 cell line (breast cancer) using a one dose primary anticancer in vitro assay [29, 30]. The results are presented in Table 1. The requirement for antitumor activity set by the National Cancer Institute is that the fraction of surviving tumor cells is 30% or less, which corresponds to inhibition of 70% or more. According to this definition it may be concluded that compounds 13–15 and 24 are active. The IC₅₀ values for the active compounds 13–15 and 24 are also given in Table 1.

Conclusion

A series of benzimidazoles 2–11, 13–15 and 17–24 were synthesized and evaluated for their in vitro antitumor activity against MFC7 cell line. Compounds 13–15 and 24 exhibit significant activity.

General

Unless otherwise noted, all materials were obtained from commercial suppliers (Aldrich and Merck companies) and used without further purification. Melting points were recorded using an Electrothermal C14500 apparatus and were uncorrected. Microanalyses were performed at the microanalytical unit, Cairo University. IR spectra were recorded on a Mattson 5000 FT-IR spectrometer using KBr disks. ¹H NMR (400 MHz) and ¹³C NMR (100 MHz) spectra were recorded on a Bruker Avance-400 spectrometer in DMSO-d₆ at Georgia State University, Atlanta, GA, USA and the Korea Institute of Science and Technology, Republic of Korea. Mass spectral analyses were performed on a JOEL JMS-600H spectrometer at Cairo University. Reaction progress was monitored using TLC on silica gel plates 60 F₂₄₅ E. Merck, and the spots were visualized by UV light at 366 nm or 245 nm. Substrates 1 [24], 12a,b [25] and 16a [26–28] were synthesized according to reported methods.

1-(1H-Benzimidazol-2-yl)-2-[(substituted phenyl)amino]ethanones 2–5

A mixture of 1-(1H-benzimidazol-2-yl)-2-bromoethanone 1 [24] (0.5 g, 0.002 mol), a substituted aniline (0.002 mol) and sodium bicarbonate (1.55 g) in anhydrous ethanol (50 mL) was heated under reflux for 4 h. Then the mixture was allowed to cool to room temperature and the resultant precipitate was filtered, washed thoroughly with water, dried and crystallized from absolute ethanol to afford the title compound 2–5.

1-(1H-Benzimidazol-2-yl)-2-oxoethyl benzoates 6, 7

Sodium benzoate or sodium 2-hydroxybenzoate (0.002 mol) was added at room temperature to a solution of 1-(1H-benzimidazol-2-yl)-2-bromoethanone 1 [24] (0.5 g, 0.002 mol) in DMF. Stirring at room temperature was continued for another 8 h. Then the mixture was poured into water and the precipitated product was filtered, washed thoroughly with water, dried and crystallized from DMF to afford the title compound 6, 7.

N’-[2-(1H-Benzimidazol-2-yl)-2-oxoethyl]benzohydrazides 8, 9

A mixture of 1-(1H-benzimidazol-2-yl)-2-bromoethanone 1 [24] (0.5 g, 0.002 mol), an acid hydrazide (0.002 mol) and sodium bicarbonate (1.55 g) in absolute ethanol (50 mL) was heated under reflux for 6 h. Then the mixture was allowed to cool to room temperature and the resulting precipitate was filtered, washed thoroughly with water, dried and crystallized from absolute ethanol to afford the title compound 8, 9.

3-(1H-Benzimidazol-2-yl)-1,2-dihydroquinoxaline (10) and 3-(1H-benzimidazol-2-yl)-2H-benzo[b][1,4]oxazine (11)

A mixture of 1-(1H-benzimidazol-2-yl)-2-bromoethanone 1 [24] (0.01 mol), 1,2-phenylenediamine or 2-aminophenol (0.011 mol), and ethanol (20 mL) was heated under reflux for 3 h. After cooling, the separated solid was filtered and washed with ethanol. The precipitate was then suspended in water, stirred with saturated sodium bicarbonate solution (30 mL, 5%), filtered, washed thoroughly with water, dried and crystallized from ethyl acetate/ethanol (1:1) to afford the title compound 10 or 11.

2-(1H-Benzimidazol-2-yl)quinoline-4-carboxylic acids 13–15

A solution of potassium hydroxide (1.02 g, 0.018 mol) in water (5 mL) was added dropwise to the appropriate isatin derivatives (0.003 mol) in ethanol (10 mL) over 15 min. The appropriate benzimidazole derivatives 12a,b [25] (0.003 mol) were added and the reaction mixture was heated under reflux for 18 h, then cooled to room temperature and the solvent was removed under vacuum. The resulting solid was dissolved in water, washed with diethyl ether, cooled in ice-cold water and acidified with acetic acid. The separated solid was filtered and recrystallized from acetic acid to afford the title compounds 13–15.

Ethyl 2-cyanomethyl-5-methyl-1H-benzimidazole-1-carboxylate (16b)

A solution of 5-methyl-2-cyanomethylbenzimidazole [27, 28] (0.01 mol) in pyridine (20 mL) was stirred in an ice bath and treated dropwise with ethyl chloroformate (2.7 g, 0.025 mol). Stirring was continued for an additional 10 min and then the mixture was poured on cold water (400 mL). The resultant precipitate of 16b was collected by filtration, dried and crystallized from ethanol: yellow compound; yield 74%; mp 76–78°C; IR: 2220 (CN), 1715 (CO), 1620 cm^-1 (C=N); ¹H NMR: δ 1.25 (t, J = 7 Hz, 3H, CH₂CH₃), 2.34 (s, 3H, CH₃), 3.67 (s, 2H, CH₂-CN), 4.17 (q, J = 7 Hz, 2H, CH₂CH₃), 7.12 (d, J = 8.7 Hz, 1H), 7.33 (s, 1H), 7.54 (d, J = 8.7 Hz, 1H); MS: m/z 243 [M⁺]. Anal. Calcd for C₁₃H₁₃N₃O₂: C, 64.19; H, 5.39; N, 17.27. Found: C, 64.24; H, 5.45; N, 17.33.

1-Oxo-2-(substituted phenyl)-1,2-dihydrobenzimidazo[1,2-d][1,2,4]triazine-4-carbonitriles 17–24

To a solution of 16a [26] or 16b (0.01 mol) in pyridine (20 mL) in an ice bath, a solution of a diazonium hydrochloride [amine (0.01 mol), 36% HCl (3 mL), ice water (10 mL) and NaNO₂ (0.7 g, 0.01 mol)] was added with stirring. After 24 h, the mixture was diluted with ice-cold water (200 mL), and the resultant precipitate of 17–24 was collected by filtration, dried and crystallized.

Biology

All materials were obtained from Sigma Chemical Co. (USA). Human tumor cell lines were obtained frozen in liquid nitrogen (-180°C) from the American Type Culture Collection. The tumor cell lines were maintained in the National Cancer Institute, Cairo, Egypt, by serial subculturing. In vitro antitumor activity against human MCF7 (breast cancer cell line) was determined using the Sulforhodamine B assay [29, 30]. Cells were plated in a 96-multiwell plate (10⁴ cells/well) for 24 h before treatment with the compounds to allow attachment of the cell to the wall of the plate. Monolayer cells were incubated with the compounds for 48 h at 37°C in a humidified incubator with 5% CO₂. Cells were fixed with trichloroacetic acid and stained for 30 min with 0.4% (wt/vol) Sulforhodamine B (SRB) stain dissolved in 1% acetic acid. Unbound dye was washed with 1% acetic acid and protein bound dye was extracted with Tris EDTA. The optical density (OD) of each well was measured spectrophotometrically at 564 nm with an ELISA microplate reader (Meter tech. Σ 960, USA). Cell survival was calculated as follows: survival fraction = OD (treated cells)/OD (control cells) (Table 1). For determination of IC₅₀, different concentrations of the compound under test (0, 1, 2.5, 5 and 10 μg/mL) were added to the cell monolayer wells which were prepared for each individual dose. The absorbance of each well was determined by an ELISA reader. The relation between surviving fraction and compound concentration was plotted to obtain the survival curve of the tumor cell line after the specified compound (Table 1).