Synthesis, in vitro and in vivo antitumor and antiviral activity of novel 1-substituted benzimidazole derivatives

Abstract

A novel series of 5-nitro-1H-benzimidazole derivatives substituted at position 1 by heterocyclic rings was synthesized. Cytotoxicity and antiviral activity of the new compounds were tested. Compound 3 was more active than doxorubicin against A-549, HCT-116 and MCF-7. However, compound 3 showed no activity against human liver carcinoma Hep G-2 cell line. Compounds 9 and 17b (E) showed potency near to doxorubicin against the four cell lines. The acute toxicity of compound 9 on liver cancer induced in rats was determined in vivo. Interestingly, it showed restoration activity of liver function and pathology towards normal as compared to the cancer-bearing rats induced by DENA. Compounds 17a (Z), 17b (E) and 18a (Z) were the most promising compounds for their antiviral activity against rotavirus Wa strain.

• Antitumor
• antiviral
• benzimidazoles
• cytotoxicity

Introduction

Liver cancer is one of the most common malignancies and the third most deadly cancers in the world1. The major risk factors in liver cancer include hepatitis viral infection, food additives, alcohol, aflatoxins, environmental and industrial toxic chemicals and air and water pollutants2^,3. Diethylnitrosamine (DENA) is a well-known hepatocarcinogenic agent present in tobacco smoke, water, cured and fried meals, cheddar cheese, agriculture chemicals and cosmetics and pharmaceutical products4. DENA is known to induce liver cancer in experimental animal models through inhibition of many enzymes involved in DENA repair mechanism5. In rats, DENA is a potent hepatocarcinogen influencing the initiation stage of carcinogenesis during a period of enhanced cell proliferation accompanied by hepatocellular necrosis and induces DNA carcinogen adducts, DNA-strand breaks and in turn hepatocellular carcinomas without cirrhosis through the development of putative pre-neoplastic focal lesions6. Although there are many strategies for the treatment of liver cancer7, their therapeutic outcome remains very poor. Therefore, prevention seems to be the best strategy for lowering the incidence of this disease. In this regard, many compounds have been tested and proved efficacy against experimentally induced hepatocarcinogenesis.

Benzimidazole heterocycle is an important multifunctional system for medicinal applications in various therapeutic fields. This is due to the presence of this heterocyclic moiety in many bioactive compounds and its interaction with many enzymes and different kinds of proteins. The broad range of activity of benzimidazole derivatives includes their activity as antihypertensive, anti-inflammatory, antibacterial, antifungal, anthelmintic, antiviral, antioxidant, antiulcer, antitumor and pyschoactivity8–16. The 1-substituted-2-methyl-5-nitro benzimidazole derivatives with various heterocycles at position 1 were prepared by our laboratory group. It was found that thiadiazole ring linked at position 1 to benzimidazole ring through a methylene group, I, has cytotoxicity against breast cancer (MCF-7; Figure 1)17. The 1-substituted-2-methyl benzimidazole derivative II was reported to be cytotoxic against non-small lung cancer and breast cancer18. It was evident that 1-(4-bromobenzyl) benzimidazole derivative III was the most potent androgen receptor antagonist for prostate cancer19. This is in addition to our very recent published work in synthesis of novel series of cytotoxic benzimidazole derivatives20–22. Moreover, many antiviral substituted benzimidazoles at position 1 were either clinically applied or evaluated using a wide range of virus strains23^,24. The nucleoside benzimidazole analogs with ribofuranosyl moiety at 1-position, 5,6-dichloro-1-(β-D-ribofuranosyl)benzimidazole (DRB IV) and its derivatives TCRB V, BDCRB VI and maribavir VII are well-known inhibitor of HCMV which inhibits viral RNA synthesis by blocking RNA polymerase II (Figure 1)25–28. The benzimidazole derivative VIII (JNJ 2408068) had an inhibitory activity against RSV at nanomolar concentration and 100 000 times better than that ribavirin29. Japan Tobacco (JTK-109) IX, with cyclohexyl ring at position-1 of benzimidazole, is an important example of benzimidazole which was patented as HCV NS5B RNA-dependent RNA polymerase inhibitors (Figure 1)30^,31.

Figure 1. Benzimidazole structures I–XIV substituted at position 1 as lead potent active compounds for antitumor and antiviral activity.

Depending upon the above-mentioned reasons, we have designed new kinds of 1-substituted benzimidazole derivatives through evaluating their cytotoxicity against various cell lines and evaluating their anticancer effect on diethylnitrosamine-induced hepatocellular carcinoma in rats, hoping to be used either alone or in combination with conventional chemotherapy in treatment of cancer. This study has also been achieved to obtain compounds with superior chemotherapeutic index in terms of increased bioavailability, higher cytotoxicity and lower side effects than the commercial available drugs.

It is clear that the above-mentioned biologically active benzimidazoles, the heterocyclic substituents at position 1 are connected to the benzimidazole core either through linker or directly without linker. Therefore, by taking benzimidazoles I and JNJ 2408068 XIII as lead compounds, our strategy for the synthesis of new benzimidazole derivatives was to construct optimized cyclic or heterocyclic ring at position 1 of benzimidazole moiety that offers the potential to significantly advance out the antitumor activity (in vitro and in vivo) and antiviral activity. The general structures A (with heterocyclic rings connected to benzimidazole moiety at position 1 by methylene or ethylene linker) and B (with heterocyclic rings directly connected to benzimidazole moiety at position 1 without linker) were chosen to be used as the key for the synthesis of various benzimidazole derivatives (Figure 2).

Figure 2. Structural requirements around benzimidazole nucleus for the study.

Experimental

Chemistry

Microanalyses, spectral data of the compounds were performed in the Microanalytical, National Research Centre, Cairo, Egypt. The IR spectra (4000–400 cm⁻¹) were recorded using KBr pellets in a Jasco FT/IR 300E Fourier (Easton, MD) transform infrared spectrophotometer on a Perkin Elmer FT-IR 1650 (spectrophotometer, Waltham, MA). The NMR spectra were recorded using Joel EX-270 MHz and 500 MHz NMR spectrophotometers. Chemical shifts are reported in parts per million (ppm) from the tetramethylsilane resonance in the indicated solvent. Coupling constants are reported in Hertz (Hz); spectral splitting partners are designed as follow: singlet (s); doublet (d); triplet (t); multiplet (m). The mass spectra were carried out using Finnigan mat SSQ 7000 (Thermo. Inst. Sys. Inc., Waltham, MA) spectroscopy at 70 eV. Thin layer chromatography was performed on Merck aluminium sheet TLC Silica gel 60 F254. Column chromatography was carried out using Merck silica gel (230–400 mesh particle size). Detection of the zones was performed using ultraviolet irradiation at 254 nm.

Synthesis

Procedure for the preparation of 1-allyl-2-methyl-5-nitro-1H-benzo[d]imidazole (2)

Allyl bromide (0.98 ml, 28.0 mmol) was slowly added dropwise to a stirred solution of 5-nitro-2-methylbenzimidazole 1 (2 g, 11.3 mmol) and sodium hydride (0.6 g, 28.0 mmol) in dimethyl formamide (30 ml). The reaction was stirred at room temperature for about 8 h. The mixture was then poured onto water and the precipitate was collected by filtration. Yield: 80%. m.p.: 150–152 °C. IR (cm⁻¹): 1618 (C=N), 1517, 1332 (NO₂). ¹H NMR (DMSO-d₆) δ (ppm): 2.55 (s, 3H, CH₃), 4.90–5.16 (m, 2H, CH₂), 5.00 (d, J = 10.0 Hz, 1H, vinyl), 5.16 (d, J = 10.0 Hz, 1H, vinyl), 5.98–6.01 (m, 1H, vinyl CH₂–CH=CH₂), 7.68 (d, J = 9.1 Hz, 1H, H7), 8.09 (dd, J _6–7 = 9.1 Hz, J _6–4 = 2.0 Hz, 1H, H6), 8.39 (s, 1H, H4). ¹³C NMR (DMSO-d₆) δ (ppm): 14.24, 46.50, 107.45, 110.88, 114.84, 117.46, 117.91, 118.89, 133.30, 140.20, 141.99, 142.73, 143.02, 147.54, 157.03. MS: m/z 218, 24.7% (M⁺+ 1); m/z 217, 100% (M⁺); m/z 201, 10.9% (M⁺ – H – CH₃); m/z 190, 2.3% (M⁺ – CH=CH₂); m/z 177, 1.4% (M⁺+ H – CH₂–CH=CH₂); m/z 144, 16.4% (M⁺ – NO₂ – CH=CH₂). Anal. Calcd for C₁₁H₁₁N₃O₂ (217.22): C, 60.82; H, 5.10; N, 19.34. Found: C, 60.77; H, 5.15; N, 19.24.

Procedure for the preparation of 1-(2,3-dibromopropyl)-2-methyl-5-nitro-1H-benzo[d]imidazole (3)

Compound 2 (3 g, 13.8 mmol) was dissolved in 50 ml chloroform. The solution was cooled in ice bath with stirring. Then bromine (1.7 ml, 13.8 ml) was dissolved in 5 ml chloroform and added to the above mixture vigorously in three portions. The mixture was stirred in the ice bath for 2 h. The formed precipitate was filtered, washed with petroleum ether 60–80. Yield: 70%. m.p.: 172–174 °C. IR (cm⁻¹): 1625 (C=N), 1532, 1344 (NO₂). ¹H NMR (DMSO-d₆) δ (ppm): 2.50 (s, 3H, CH₃), 4.12–4.14 (m, 2H, CH₂), 4.80–5.01 (m, 3H, CHBr–CH₂Br), 7.97 (d, J = 9.1 Hz, 1H, H7), 8.28 (d, J = 9.1 Hz, 1H, H6), 8.55 (s, 1H, H4). ¹³C NMR (DMSO-d₆) δ (ppm): 13.66, 36.28, 49.52, 50.96, 112.58, 116.17, 120.04, 132.79, 134.72, 137.60, 144.58, 157.34. MS: m/z 377, 16.4% (M⁺); m/z 201, 2.8% (M⁺ – H – 2Br – CH₃); m/z 144, 100% (M⁺ – NO₂ – CHBr-CH₂Br). Anal. Calcd for C₁₁H₁₁Br₂N₃O₂ (377.03): C, 35.04; H, 2.94; Br 42.39, N, 11.14. Found: C, 35.16; H, 2.77; Br 42.48, N, 11.20.

General procedure for the preparation of compounds (4–6)

A suspension of dibromo derivative 3 (3 g, 8 mmol) in ethanol was treated with potassium carbonate (16 mmol) for 2–3 h. The diamino derivative (8 mmol) was then added to the reaction mixture and refluxed for about 6–8 h. On cooling the reaction mixture, yellow to orange solid products separated out. They were collected by filtration, washed with water and dried.

4-[(2-Methyl-5-nitro-1H-benzo[d]imidazol-1-yl)methyl] imidazolidine-2-thione (4)

Yield: 70%. m.p.: 210–211 °C. IR (cm⁻¹): 3344 (NH), 1616 (C=N), 1520, 1344 (NO₂), 1322 (C=S). ¹H NMR (DMSO-d₆) δ (ppm): 2.55 (s, 3H, CH₃), 3.05–3.15 (m, 1H, CH), 4.34–4.35 (m, 2H, CH₂), 5.19–5.20 (m, 2H, CH₂), 7.64 (d, J = 9.1 Hz, 1H, H7), 8.03 (d, J = 9.1 Hz, 1H, H6), 8.30 (s, 1H, H4). MS: m/z 218, 74.6% (M⁺+ H – NH–CS–NH) Anal. Calcd for C₁₂H₁₃N₅O₂S (291.07): C, 49.47; H, 4.50; N, 24.04; S, 11.01. Found: C, 49.61; H, 4.39; N, 24.16; S, 11.09.

2-Methyl-5-nitro-1-[(piperazin-2-yl)methyl]-1H-benzo[d] imidazole (5)

Yield: 64%. m.p.: 220–222 °C. IR (cm⁻¹): 3244 (NH), 1629 (C=N), 1520, 1322 (NO₂). ¹H NMR (DMSO-d₆) δ (ppm): 2.07–2.20 (m, 2H, CH₂), 2.46 (s, 3H, CH₃), 2.95–3.00 (m, 2H, CH₂), 3.20–3.25 (m, 1H, CH), 4.34–4.35 (m, 2H, CH₂), 5.23–5.24 (m, 2H, CH₂), 7.54 (dd, J _6–7 = 9.1 Hz, J _6–4 = 2 Hz, 1H, H6), 7.98 (d, J = 9.1 Hz, 1H, H7), 8.11 (s, 1H, H4). ¹³C NMR (DMSO-d₆) δ (ppm): 14.38, 51.21, 109.45, 109.68, 115.22, 118.59, 121.48, 126.77, 139.77, 143.14, 141.94, 157.10. MS: m/z 274, 5.5% (M⁺ – 1); m/z 259, 11.0% (M⁺ – H – CH₃). Anal. Calcd for C₁₃H₁₇N₅O₂ (275.31): C, 56.71; H, 6.22; N, 25.44. Found: C, 56.79; H, 6.32; N, 25.31.

1,2,3,4-Tetrahydro-2-[(2-methyl-5-nitro-1H-benzo[d] imidazol-1-yl)methyl]quinoxaline (6)

Yield: 58%. m.p.: 194–196 °C. IR (cm⁻¹): 3438 (NH), 1614 (C=N), 1519, 1337 (NO₂). ¹H NMR (DMSO-d₆) δ (ppm): 2.38 (s, 3H, CH₃), 3.30–3.37 (m, 1H, CH), 3.65–3.69 (m, 2H, CH₂), 4.25–4.27 (m, 2H, CH₂), 7.86–8.00 (m, 5H, aromatic protons + H6 benzimidazole), 8.19 (d, J = 9.0 Hz, 1H, H7), 8.25 (s, 1H, H4). Anal. Calcd for C₁₇H₁₇N₅O₂ (323.35): C, 63.15; H, 5.30; N, 21.66. Found: C, 63.24; H, 5.22; N, 21.75.

General procedure for the preparation of benzimidazole derivatives (7 and 8)

Compound 5 or 6 was added to sodium hydride in DMF (30 ml) and stirred for 15 min. Then acetyl chloride was added dropwise to the reaction mixture and stirred at room temperature for about 8 h. The mixture was poured into ice water and the precipitate was collected by filtration and crystallized from appropriate solvent.

2-Methyl-5-nitro-1-[(1,4-diacetylpiperazin-2-yl) methyl]-1H-benzo[d]imidazole (7)

Yield: 45%. m.p.: 183–184 °C. ¹H NMR (DMSO-d₆) δ (ppm): 2.07 (m, 2H, CH₂), 2.24 (s, 6H, 2 COCH₃), 2.31 (s, 3H, CH₃), 2.95–2.98 (m, 2H, CH₂), 3.22–3.25 (m, 1H, CH), 4.34–4.39 (m, 2H, CH₂), 5.23–5.26 (m, 2H, CH₂), 7.54 (dd, J _6–7 = 9.1 Hz, J _6–4 = 2.0 Hz, 1H, H6), 7.98 (d, J = 9.1 Hz, 1H, H7), 8.11 (s, 1H, H4). Anal. Calcd for C₁₇H₂₁N₅O₄ (359.16): C, 56.82; H, 5.89; N, 19.49. Found: C, 56.91; H, 5.77; N, 19.38.

1,2,3,4-Tetrahydro-2-[(2-methyl-5-nitro-1H-benzo[d] imidazol-1-yl)methyl]1,4-diacetylquinoxaline (8)

Yield: 58%. m.p.: 176–178 °C. ¹H NMR (DMSO-d₆) δ (ppm): 2.37 (s, 6H, 2 COCH₃), 2.46 (s, 3H, CH₃), 3.34–3.37 (m, 1H, CH), 3.69–3.70 (m, 2H, CH₂), 4.25–4.27 (m, 2H, CH₂), 7.86–8.00 (m, 5H, aromatic protons + H6 benzimidazole), 8.19 (d, J = 9.1 Hz, 1H, H7), 8.24 (s, 1H, H4). Anal. Calcd for C₂₁H₂₁N₅O₂ (407.16): C, 61.91; H, 5.20; N, 17.19. Found: C, 61.83; H, 5.27; N, 17.25.

General procedure for the preparation of compounds 9–13

Methyl amine, glycine or dihydroxy derivative was added dropwise to a stirred solution of compound 3 (5 g, 13.3 mmol) and potassium carbonate (26.6 mmol) in acetone (30 ml). The mixture was stirred at 25 °C for about 10 h. Then the mixture was poured onto water. The formed precipitates were collected by filtration and crystallized from appropriate solvent.

N¹,N²-Dimethyl-3-(2-methyl-5-nitro-1H-benzo[d] imidazol-1-yl)propane-1,2-diamine (9)

Yield: 75%. m.p.: 165–167 °C. ¹H NMR (DMSO-d₆) δ (ppm): 2.20 (s, 6H, 2 NHCH₃), 2.37 (s, 3H, CH₃), 3.06–3.10 (m, 1H, CH), 3.95–3.99 (m, 2H, CH₂), 4.82–4.85 (m, 2H, CH₂), 7.56 (d, J = 9.1 Hz, 1H, H7), 7.96 (d, J = 9.1 Hz, 1H, H6), 8.30 (s, 1H, H4), 8.50 (broad band, 2 NH). ¹³C NMR (DMSO-d₆) δ (ppm): 13.13, 99.35, 115.19, 117.82, 120.06, 124.02, 124.36, 125.01, 125.53, 126.51, 126.00, 139.72, 143.48, 146.44, 149.33, 152.76, 159.19. MS: m/z 277, 2.0% (M⁺); m/z 249, 5.0% (M⁺–2CH₃+2H). Anal. Calcd for C₁₃H₁₉N₅O₂ (277.32): C, 56.30; H, 6.91; N, 25.25. Found: C, 56.22; H, 6.99; N, 25.39.

3-(2-Methyl-5-nitro-1H-benzo[d]imidazol-1-yl) propane-1,2-diaminoacetic acid (10)

Yield: 80%. m.p.: 150–152 °C. IR (cm⁻¹): 3438, 3209 (NH + carboxylic OH), 1682 (carboxylic C=O), 1616 (C=N), 1516, 1340 (NO₂). ¹H NMR (DMSO-d₆) δ (ppm): 2.37 (s, 3H, CH₃), 2.43–2.45 (m, 2H, CH₂), 2.59–2.32 (m, 2H, CH₂), 3.05–3.08 (m, 1H, CH), 3.83–3.88 (m, 2H, CH₂), 4.34–4.36 (m, 2H, CH₂), 7.56 (d, J = 9.1 Hz, 1H, H7), 8.01 (d, J = 9.1 Hz, 1H, H6), 8.30 (s, 1H, H4), 8.45 (broad band, 2 NH), 10.48 (s, 2H, 2 COOH). ¹³C NMR (DMSO-d₆) δ (ppm): 13.50, 43.22, 161.06, 166.90, 171.48. MS: m/z 364, 25.0% (M⁺ – 1); m/z 231, 24.5% (M⁺ – H – 2 CH₂COOH – NH), 202, 100% (M⁺ – H – 2 CH₂COOH – 2 NH – CH₂), 173, 88.0% (M⁺ – 2 H – 2 CH₂COOH – 2 NH – CH₂ – CH – CH₃). Anal. Calcd for C₁₅H₁₉N₅O₆ (365.34): C, 49.31; H, 5.24; N, 19.17. Found: C, 49.38; H, 5.17; N, 19.23.

1-[(1,4-Dioxan-2-yl)methyl]-2-methyl-5-nitro-1H-benzo[d] imidazole (11)

Yield: 83%. m.p.: 205–207 °C. IR (cm⁻¹):1621 (C=N), 1519, 1334 (NO₂).¹H NMR (DMSO-d₆) δ (ppm): 2.37 (s, 3H, CH₃), 2.87–2.90 (m, 2H, CH₂), 3.21–3.30 (m, 1H, CH), 3.76–3.85 (m, 2H, CH₂), 4.36–4.38 (m, 2H, CH₂), 4.82–4.87 (m, 2H, CH₂), 7.62 (d, J = 9.1 Hz, 1H, H7), 8.03 (d, J = 9.1 Hz, 1H, H6), 8.30 (s, 1H, H4). MS: m/z 277, 0.6% (M⁺); m/z 191, 58.7% (M⁺+ H – 1,4-dioxane ring); m/z 178, 5.8% (M⁺+ 2H – 1,4-dioxane ring – CH₂); m/z 163, 21.7% (M⁺+ 2H – 1,4-dioxane ring – CH₂ – CH₃). Anal. Calcd for C₁₃H₁₅N₃O₄ (277.28): C, 56.31; H, 5.45; N, 15.15. Found: C, 56.43; H, 5.39; N, 15.21.

1-[(2,3-Dihydrobenzo[b]1,4-dioxin-3-yl)methyl]-2-methyl-5-nitro-1H-benzo[d]imidazole (12)

Yield: 87%. m.p.: 208–210 °C. ¹H NMR (DMSO-d₆) δ (ppm): 2.34 (s, 3H, CH₃), 3.30–3.36 (m, 1H, CH), 3.68–3.69 (m, 2H, CH₂), 4.88–4.91 (m, 2H, CH₂), 7.60–7.80 (m, 4H, aromatic), 7.94 (d, J = 9.1 Hz, 1H, H7), 7.97 (d, J = 9.1 Hz, 1H, H6), 8.07 (s, 1H, H4). Anal. Calcd for C₁₇H₁₅N₃O₄ (325.32): C, 62.76; H, 4.65; N, 12.92. Found: C, 62.85; H, 4.53; N, 12.83.

2-Methyl-1-[(5,7-dimethyl-1,4-dioxepan-2-yl) methyl]-5-nitro-1H-benzo[d]imidazole (13)

Yield: 77%. m.p.: 240–242 °C. IR (cm⁻¹):1621 (C=N), 1520, 1337 (NO₂). ¹H NMR (DMSO-d₆) δ (ppm): 1.09 (s, 6H, 2 CH₃), 1.90 (s, 3H, CH₂), 2.49 (s, 3H, CH₃), 3.21–3.24 (m, 3H, 3 CH), 4.48–4.52 (m, 2H, CH₂), 4.93–4.97 (m, 2H, CH₂), 7.64 (d, J = 9.1 Hz, 1H, H7), 8.01 (d, J = 9.1 Hz, 1H, H6), 8.30 (s, 1H, H4). MS: m/z 319, 1.0% (M⁺). Anal. Calcd for C₁₆H₂₁N₃O₄ (319.36): C, 60.17; H, 6.63; N, 13.16. Found: C, 60.25; H, 6.54; N, 13.26.

Procedure for the preparation of 2-(2-methyl-5-nitro-1H-benzo[d]imidazol-1-yl)-1-phenylethanone (14)

A solution of 5-nitro-2-methylbenzimidazole 1 (5 g, 28 mmol) and sodium hydride (0.6 g, 28 mmol) in dimethyl formamide (30 ml) was stirred at room temperature. Then phenacyl bromide (5.6 g, 28 mmol) was added dropwise to the reaction mixture. The reaction was allowed to stirring at room temperature for about 8 h. The mixture was then poured onto water and the precipitate was collected by filtration. Yield: 84%. m.p.: 146–147 °C. IR (cm⁻¹): 1690 (C=O), 1617 (C=N), 1516, 1334 (NO₂). ¹H NMR (DMSO-d₆) δ (ppm): 2.50 (s, 3H, CH₃), 6.23 (s, 2H, CH₂), 7.55–7.85 (m, 5H, aromatic), 8.00–8.25 (m, 3H, H7 + H6 + H4). MS: m/z 295, 22.5% (M⁺); m/z 279, 4.0% (M⁺ – H – CH₃); m/z 190, 3.4% (M⁺ – C₆H₅ – CO). Anal. Calcd for C₁₆H₁₃N₃O₃ (295.10): C, 65.08; H, 4.44; N, 14.23. Found: C, C, 65.16; H, 4.32; N, 14.30.

General procedure for the preparation of 2-(2-methyl-5-nitro-1H-benzo[d]imidazol-1-yl)-3-aryl-1-phenylprop-2-en-1-one (15–18)

A mixture of compound 14 (6.7 mmol), aromatic aldehydes (6.7 mmol) and 10% aqueous sodium hydroxide (10 ml) in ethanol (30 ml) was stirred at room temperature for about 3 h. The resulting solid was washed, dried and crystallized from ethanol. The two stereoisomers (E and Z) were separated into individual pure components 15a(Z)–18a(Z) as major components and 15b(E)–18b(E) as minor components using column chromatography on silica gel (200 × 30 mm, n-hexane, n-hexane/ethylacetate 10:1, 3:1, 2:1, 1:1).

(Z)-2-(2-Methyl-5-nitro-1H-benzo[d]imidazol-1-yl)-1-phenyl-3-p-tolylprop-2-en-1-one (15a)

Yield: 72%. m.p.: 150–152 °C. IR (cm⁻¹): 1647 (C=O), 1602 (C=C), 1517, 1338 (NO₂). ¹H NMR (DMSO-d₆) δ (ppm): 2.19 (s, 3H, CH₃ p-tolyl), 2.33 (s, 3H, CH₃ benzimidazole), 6.86 (d, J = 8.0 Hz, 2H, p-tolyl AB-system), 7.07 (d, J = 8.0 Hz, 2H, p-tolyl AB-system), 7.38 (d, J = 9.0 Hz, 1H, H7), 7.60 (t, J = 7.5 Hz, 2H, aromatic), 7.70 (t, J = 7.5 Hz, 1H, aromatic), 7.94 (s, 1H, CH=C), 7.95 (d, J = 7.5 Hz, 2H, aromatic), 8.04 (d, J = 9.0 Hz, 1H, H6), 8.51 (s, 1H, H4). MS: m/z 397, 4.0% (M⁺). Anal. Calcd for C₂₄H₁₉N₃O₃ (397.14): C, 72.53; H, 4.82; N, 10.57. Found: C, 72.61; H, 4.73; N, 10.44.

(E)-2-(2-Methyl-5-nitro-1H-benzo[d]imidazol-1-yl)-1-phenyl-3-p-tolylprop-2-en-1-one (15b)

Yield: 15%. m.p.: 135–136 °C. IR (cm⁻¹): 1650 (C=O), 1518, 1330 (NO₂). ¹H NMR (DMSO-d₆) δ (ppm): 2.20 (s, 3H, CH₃ p-tolyl), 2.35 (s, 3H, CH₃ benzimidazole), 6.87 (d, J = 8.0 Hz, 2H, p-tolyl AB-system), 7.07 (d, J = 8.0 Hz, 2H, p-tolyl AB-system), 7.61 (t, J = 7.5 Hz, 2H, aromatic), 7.71 (t, J = 7.5 Hz, 1H, aromatic), 7.84 (d, J = 9.0 Hz, 1H, H7), 7.92 (s, 1H, CH=C), 7.99 (d, J = 7.5 Hz, 2H, aromatic), 8.07 (d, J = 1.7 Hz, 1H, H4), 8.12 (d, J = 9.0 Hz, 1H, H6). MS: m/z 397, 75.0% (M⁺); m/z 382, 12.1%, (M⁺ – CH₃); m/z 292, 14.1% (M⁺ – C₆H₅CO); m/z 246, 41.2% (M⁺ – C₆H₅CO– NO₂). Anal. Calcd for C₂₄H₁₉N₃O₃ (397.14): C, 72.53; H, 4.82; N, 10.57. Found: C, 72.65; H, 4.71; N, 10.49.

(Z)-3-(2-Methoxyphenyl)-2-(2-methyl-5-nitro-1H-benzo[d] imidazol-1-yl)-1-phenylprop-2-en-1-one (16a)

Yield: 68%. m.p.: 190–192 °C. IR (cm⁻¹): 1636 (C=O), 1595 (C=C), 1522, 1343 (NO₂). ¹H NMR (DMSO-d₆) δ (ppm): 2.37 (s, 3H, CH₃), 3.69 (s, 3H, OCH₃), 6.49 (d, J = 7.5 Hz, 1H, aromatic), 6.69 (m, 1H, aromatic), 7.05 (d, J = 7.5 Hz, 1H, aromatic), 7.30 (d, J = 9.0 Hz, 1H, H7), 7.33–7.4 (m, 1H, aromatic), 7.59–7.66 (m, 2H, aromatic), 7.72–7.75 (m, 1H, aromatic), 7.93 (d, J = 7.5 Hz, 2H, aromatic), 8.02 (d, J = 9.0 Hz, 1H, H6), 8.07 (s, 1H, CH=C), 8.47 (s, 1H, H4). ¹³C NMR (DMSO-d₆) δ (ppm): 13.67, 55.99, 110.58, 111.95, 114.75, 118.34, 119.81, 120.85, 129.40, 129.47, 133.09, 133.44, 136.60, 139.08, 139.43, 142.01, 143.16, 155.82, 157.84, 191.67. MS: m/z 413, 35.6% (M⁺). Anal. Calcd for C₂₄H₁₉N₃O₄ (413.43): C, 69.72; H, 4.63; N, 10.16. Found: C, 69.65; H, 4.70; N, 10.23.

(E)-3-(2-Methoxyphenyl)-2-(2-methyl-5-nitro-1H-benzo[d] imidazol-1-yl)-1-phenylprop-2-en-1-one (16b)

Yield: 17%. m.p.: 115–116 °C. IR (cm⁻¹): 1653 (C=O), 1597 (C=C), 1521, 1338 (NO₂). ¹H NMR (DMSO-d₆) δ (ppm): 2.34 (s, 3H, CH₃), 3.74 (s, 3H, OCH₃), 6.56 (d, J = 7.5 Hz, 1H, aromatic), 6.66–6.75 (m, 1H, aromatic), 7.03 (d, J = 7.5 Hz, 1H, aromatic), 7.31–7.35 (m, 1H, aromatic), 7.60–7.65 (m, 2H, aromatic), 7.71–7.76 (m, 1H, aromatic), 7.82 (d, J = 7.5 Hz, 1H, aromatic), 7.94 (s, 1H, aromatic), 7.95 (s, 1H, CH=C), 7.98 (d, J = 9 Hz, 1H, H7), 8.04 (d, J = 9 Hz, 1H, H6), 8.07 (s, 1H, H4). ¹³C NMR (DMSO-d₆) δ (ppm): 14.44, 14.16, 56.48, 107.10, 111.03, 112.37, 115.22, 118.79, 120.59, 121.36, 128.66, 129.38, 133.47, 135.23, 137.14, 139.62, 140.40, 142.51, 143.70, 156.35, 157.90, 158.31, 192.07. MS: m/z 413, 9.4% (M⁺); m/z 382, 4.2%, (M⁺ – OCH₃); m/z 226, 36.3% (M⁺ – C₆H₅CO). Anal. Calcd for C₂₄H₁₉N₃O₄ (413.43): C, 69.72; H, 4.63; N, 10.16. Found: C, 69.68; H, 4.71; N, 10.25.

(Z)-2-(2-Methyl-5-nitro-1H-benzo[d]imidazol-1-yl)-3-(5-methylfuran-2-yl)-1-phenylprop-2-en-1-one (17a)

Yield: 70%. m.p.: 178–180 °C. IR (cm⁻¹): 1614 (C=O), 1519, 1341 (NO₂). ¹H NMR (DMSO-d₆) δ (ppm): 2.35 (s, 3H, CH₃ furan), 2.47 (s, 3H, CH₃ benzimidazole), 6.23 (d, J = 2.5 Hz, 1H, furan), 6.55 (d, J = 2.5 Hz, 1H, furan), 7.34 (d, J = 9.0 Hz, 1H, H7), 7.39–7.48 (m, 1H, aromatic), 7.59 (m, 2H, aromatic), 7.72 (s, 1H, CH=C), 7.87–7.90 (m, 2H, aromatic), 8.07 (d, J = 9.0 Hz, 1H, H6), 8.50 (s, 1H, H4). ¹³C NMR (DMSO-d₆) δ (ppm): 13.90, 14.02, 22.66, 51.08, 110.99, 111.19, 115.05, 118.65, 119.02, 124.14, 129.30, 129.57, 132.26, 137.55, 143.45, 146.31, 149.87, 159.41, 191.70. MS: m/z 387, 87.5% (M⁺). Anal. Calcd for C₂₂H₁₇N₃O₄ (387.12): C, 68.21; H, 4.42; N, 10.85. Found: C, 68.33; H, 4.35; N, 10.92.

(E)-2-(2-Methyl-5-nitro-1H-benzo[d]imidazol-1-yl)-3-(5-methylfuran-2-yl)-1-phenylprop-2-en-1-one (17b)

Yield: 17%. m.p.: 137–139 °C. IR (cm⁻¹): 1621 (C=O), 1520, 1337 (NO₂). ¹H NMR (DMSO-d₆) δ (ppm): 1.92 (s, 3H, CH₃ furan), 2.47 (s, 3H, CH₃ benzimidazole), 6.24 (d, J = 2.5 Hz, 1H, furan), 6.55 (d, J = 2.5 Hz, 1H, furan), 7.58–7.61 (m, 2H, aromatic), 7.67–7.77 (m, 1H, aromatic), 7.70 (s, 1H, CH=C), 7.82 (d, J = 9.0 Hz, 1H, H7), 7.90–7.95 (m, 2H, aromatic), 8.09 (d, J = 9.0 Hz, 1H, H6), 8.11 (s, 1H, H4). ¹³C NMR (DMSO-d₆) δ (ppm): 13.86, 14.16, 51.09, 107.02, 111.20, 118.30, 119.25, 123.92, 124.01, 129.25, 129.67, 132.59, 132.92, 135.26, 137.60, 143.26, 146.37, 148.14, 158.26, 159.33, 191.67. MS: m/z 386, 9.4% (M⁺ – 1); m/z 292, 4.2%, (M⁺+ H – methylfuryl – CH₃); m/z 262, 36.3% (M⁺ – C₆H₅ – NO₂ – 2H); m/z 249, 36.3% (M⁺ – C₆H₅ – NO₂ – CH₃). Anal. Calcd for C₂₂H₁₇N₃O₄ (387.12): C, 68.21; H, 4.42; N, 10.85. Found: C, 68.37; H, 4.33; N, 10.94.

(Z)-2-(2-Methyl-5-nitro-1H-benzo[d]imidazol-1-yl)-1-phenyl-3-(thiophen-2-yl)prop-2-en-1-one (18a)

Yield: 68%. m.p.: 188–190 °C. IR (cm⁻¹): 1643 (C=O), 1522, 1338 (NO₂). ¹H NMR (DMSO-d₆) δ (ppm): 2.38 (s, 3H, CH₃), 7.14 (m, 1H, H′3 thiophene), 7.61 (m, 2H, H′2 + H′4 thiophene), 7.70–7.78 (m, 1H, aromatic), 7.79–7.86 (m, 2H, aromatic), 7.86 (d, J = 9.0 Hz, 1H, H7), 7.97–7.99 (m, 2H, aromatic), 8.12 (d, J = 9.0 Hz, 1H, H6), 8.18 (s, 1H, H4), 8.37 (s, 1H, CH=C). MS: m/z 393, 5.0% (M⁺+ 2H + 2H). Anal. Calcd for C₂₁H₁₅N₃O₃S (389.43): C, 64.77; H, 3.88; N, 10.79l; S, 8.23. Found: C, 64.65; H, 3.96; N, 10.64; S, 8.31.

(E)-2-(2-Methyl-5-nitro-1H-benzo[d]imidazol-1-yl)-1-phenyl-3-(thiophen-2-yl)prop-2 -en-1-one (18b)

Yield: 17%. m.p.: 125–127 °C. IR (cm⁻¹): 1648 (C=O), 1521, 1338 (NO₂). ¹H NMR (DMSO-d₆) δ (ppm): 2.38 (s, 3H, CH₃), 7.15–7.25 (m, 1H, H′3 thiophene), 7.60–7.72 (m, 2H, H′2 + H′4 thiophene), 7.69–7.75 (m, 1H, aromatic), 7.80–7.87 (m, 2H, aromatic), 7.87 (d, J = 9.0 Hz, 1H, H7), 7.96–7.99 (m, 2H, aromatic), 8.14 (d, J = 9.0 Hz, 1H, H6), 8.19 (s, 1H, H4), 8.39 (s, 1H, CH=C). ¹³C NMR (DMSO-d₆) δ (ppm): 14.01, 106.97, 118.68, 119,55, 125.84, 128.70, 129.24, 129.77, 133.01, 134.42, 134.45, 136.37, 139.06, 140.27, 143.59, 148.49, 157.96, 191.59. MS: m/z 390, 0.3% (M⁺+ 1); m/z 260, 1.2%, (M⁺ – thiophene – NO₂); m/z 226, 18.0% (M⁺ – C₆H₅ – thiophene – 3H). Anal. Calcd for C₂₁H₁₅N₃O₃S (389.43): C, 64.77; H, 3.88; N, 10.79; S, 8.23. Found: C, 64.62; H, 3.93; N, 10.69; S, 8.28.

General procedure for the preparation of 1-(2,3,6,7-tetrahydro-7-aryl-5-phenyl-1H-1,4-diazepin-6-yl)-2-methyl-5-nitro-1H-benzo[d]imidazole derivatives (19–22); 5,6-dihydro-5-(2-methyl-5-nitro-1H-benzo[d] imidazol-1-yl)-6-aryl-4-phenylpyrimidine-2(1H)-thione derivatives (23–26)

Compounds 15, 16, 17 or 18 (4 mmol) were added to ethylenediamine or thiourea (4 mmol). Then, Sodium hydroxide (10 mmol) in ethanol (25 ml) was added. The reaction mixture was refluxed in for 8 h. The solution was poured onto ice water. The precipitate was filtered and crystallized from ethanol.

1-((Z)-2,3,6,7-Tetrahydro-5-phenyl-7-p-tolyl-1H-1,4-diazepin-6-yl)-2-methyl-5-nitro-1H-benzo[d]imidazole (19)

Yield: 60%. m.p.: 220–222 °C. IR (cm⁻¹): 1615 (C=N), 1517, 1332 (NO₂). ¹H NMR (DMSO-d₆) δ (ppm): 2.07 (s, 3H, CH₃ p-tolyl), 2.46 (s, 3H, CH₃ benzimidazole), 2.96 (s, 2H, CH₂–NH), 4.32 (s, 2H, CH₂-N), 5.23 (d, J = 1.5 Hz 1H, CH), 6.12 (d, J = 1.5 Hz, 1H, CH), 6.81 (d, J = 8.0 Hz, 2H, p-tolyl AB-system), 7.02 (d, J = 8.0 Hz, 2H, p-tolyl AB-system), 7.26 (m, 3H, aromatic), 7.54 (m, 3H, 2H aromatic + H7), 7.98 (d, J = 9.0 Hz, 1H, H6), 8.11 (s, 1H, H4). MS: m/z 440, 5.5% (M⁺+ H). Anal. Calcd for C₂₆H₂₅N₅O₂ (439.20): C, 71.05; H, 5.73; N, 15.93. Found: C, 71.13; H, 5.64; N, 15.86.

1-((Z)-3,4,5,6-Tetrahydro-5-(2-methoxyphenyl)-7-phenyl-2H-1,4-diazepin-6-yl)-2-methyl-5-nitro-1H-benzo[d]imidazole (20)

Yield: 71%. m.p.: 165–166 °C. IR (cm⁻¹): 1598 (C=N), 1510, 1334 (NO₂). ¹H NMR (DMSO-d₆) δ (ppm): 2.37 (s, 3H, CH₃), 3.53 (s, 2H, CH₂–NH), 3.69 (s, 3H, OCH₃), 3.98 (s, 2H, CH₂–N), 4.72 (d, J = 1.5 Hz 1H, CH), 5.64 (d, J = 1.5 Hz, 1H, CH), 6.48 (d, J = 7.5 Hz, 1H, aromatic), 6.67–6.69 (m, 1H, aromatic), 7.05 (d, J = 7.5 Hz, 1H, aromatic), 7.30–7.35 (m, 2H, aromatic), 7.61–7.65 (m, 2H, aromatic), 7.75–7.81 (m, 1H, aromatic), 7.94–7.99 (m, 2H, 1H aromatic + H7), 8.07 (d, J = 9.0 Hz, 1H, H6), 8.10 (s, 1H, H4). ¹³C NMR (DMSO-d₆) δ (ppm): 16.00, 30.90, 56.15, 63.33, 72.60, 112.91, 120.84, 128.12, 136.2. MS: m/z 413, 18.75% (M⁺ – H – NH(CH₂)₂N + O). Anal. Calcd for C₂₆H₂₅N₅O₃ (455.20): C, 68.56; H, 5.53; N, 15.37. Found: C, 68.63; H, 5.45; N, 15.46.

1-((Z)-3,4,5,6-Tetrahydro-5-(5-methylfuran-2-yl)-7-phenyl-2H-1,4-diazepin-6-yl)-2-methyl-5-nitro-1H-benzo[d]imidazole (21)

Yield: 50%. m.p.: 180–182 °C. IR (cm⁻¹): 1624 (C=N), 1516, 1336 (NO₂). ¹H NMR (DMSO-d₆) δ (ppm): 2.43 (s, 3H, CH₃ furan), 2.50 (s, 3H, CH₃ benzimidazole), 3.93 (s, 2H, CH₂–NH), 4.30 (s, 2H, CH₂–N), 4.70 (d, J = 1.5 Hz 1H, CH), 5.36 (d, J = 1.5 Hz, 1H, CH), 6.12 (d, J = 2.5 Hz, 1H, furan), 6.39 (d, J = 2.5 Hz, 1H, furan), 7.29–7.60 (m, 5H, aromatic), 7.75 (d, J = 9.0 Hz, 1H, H7), 7.97 (d, J = 9.0 Hz, 1H, H6), 8.29 (s, 1H, H4). MS: m/z 429, 0.06% (M⁺). Anal. Calcd for C₂₄H₂₃N₅O₃ (429.18): C, 67.12; H, 5.40; N, 16.31. Found: C, 67.23; H, 5.29; N, 16.40.

1-((Z)-2,3,6,7-Tetrahydro-5-phenyl-7-(thiophen-2-yl)-1H-1,4-diazepin-6-yl)-2-methyl-5-nitro-1H-benzo[d]imidazole (22)

Yield: 65%. m.p.: 188–190 °C. IR (cm⁻¹): 1628 (C=O), 1516, 1334 (NO₂). ¹H NMR (DMSO-d₆) δ (ppm): 2.38 (s, 3H, CH₃), 2.96 (s, 2H, CH₂–NH), 3.72 (s, 2H, CH₂–N), 4.93 (d, J = 1.5 Hz 1H, CH), 5.98 (d, J = 1.5 Hz, 1H, CH),7.14–7.17 (m, 1H, H′3 thiophene), 7.60 (m, 2H, H′2 + H′4 thiophene), 7.69–7.72 (m, 1H, aromatic), 7.79–7.86 (m, 2H, aromatic), 7.87 (d, J = 9.0 Hz, 1H, H7), 7.97–8.00 (m, 2H, aromatic), 8.13 (d, J = 9.0 Hz, 1H, H6), 8.19 (s, 1H, H4). ¹³C NMR (DMSO-d₆) δ (ppm): 15.15, 30.90, 41.33, 41.42, 41.61, 63.26, 79.20, 124.18, 126.80, 128.55, 129.11, 129.24, 129.37, 136.32, 138.89, 142.30, 143.52, 144.45, 184.61. MS: m/z 431, 0.2% (M⁺); m/z 350, 0.4%, (M⁺ – thiophene + 2H); m/z 268, 2.0% (M⁺ – C₆H₅ – thiophene – 3H), m/z 177, 80% (M⁺+ H – 2,3,6,7-tetrahydro-5-phenyl-7-(thiophen-2-yl)-1H-1,4-diazepin-6-yl). Anal. Calcd for C₂₃H₂₁N₅O₂S (431.14): C, 64.02; H, 4.91; N, 16.23; S, 7.43. Found: C, 64.13; H, 4.82; N, 16.18; S, 7.35.

5,6-Dihydro-5-(2-methyl-5-nitro-1H-benzo[d]imidazol-1-yl)-4-phenyl-6-p-tolylpyrimidine-2(1H)-thione (23)

Yield: 78%. m.p.: 210–212 °C. IR (cm⁻¹): 1643 (C=N), 1521, 1338 (NO₂), 1303 (C=S). ¹H NMR (DMSO-d₆) δ (ppm): 2.20 (s, 3H, CH₃ p-tolyl), 2.35 (s, 3H, CH₃ benzimidazole), 4.52 (d, J = 1.5 Hz 1H, CH), 5.90 (d, J = 1.5 Hz, 1H, CH), 6.87 (d, J = 8.0 Hz, 2H, p-tolyl AB-system), 7.05 (d, J = 8.0 Hz, 2H, p-tolyl AB-system), 7.59–7.65 (m, 3H, aromatic), 7.70–7.75 (m, 1H, aromatic), 7.85 (d, J = 9.0 Hz, 1H, H7), 7.97–7.99 (m, 1H, aromatic), 8.07 (s, 1H, H4), 8.10 (d, J = 9.0 Hz, 1H, H6). MS: m/z 422, 4.0% (M⁺ – SH). Anal. Calcd for C₂₅H₂₁N₅O₂S (455.14): C, 65.92; H, 4.65; N, 15.37; S, 7.04. Found: C, 65.85; H, 4.74; N, 15.27; S, 7.14.

5,6-Dihydro-6-(2-methoxyphenyl)-5-(2-methyl-5-nitro-1H-benzo[d]imidazol-1-yl)-4-phenylpyrimidine-2(1H)-thione (24)

Yield: 91%. m.p.: 198–200 °C. IR (cm⁻¹): 1598 (C=N), 1510, 1334 (NO₂). ¹H NMR (DMSO-d₆) δ (ppm): 2.34 (s, 3H, CH₃), 3.71 (s, 3H, OCH₃), 4.97 (d, J = 1.5 Hz 1H, CH), 5.86 (d, J = 1.5 Hz, 1H, CH), 6.56 (d, J = 7.5 Hz, 1H, aromatic), 6.67 (m, 1H, aromatic), 7.04 (d, J = 7.5 Hz, 1H, aromatic), 7.29–7.33 (m, 1H, aromatic), 7.60–7.65 (m, 2H, aromatic), 7.72–7.74 (m, 1H, aromatic), 7.79–7.83 (m, 1H, aromatic), 7.94 (d, J = 9.0 Hz, 1H, H7), 7.98–8.00 (m, 1H, aromatic), 8.04 (d, J = 9.0 Hz, 1H, H6), 8.07 (s, 1H, H4). MS: m/z 468, 0.01% (M⁺ – 3H). Anal. Calcd for C₂₅H₂₁N₅O₃S (471.14): C, 63.68; H, 4.49; N, 14.85; S, 6.80. Found: C, 63.76; H, 4.57; N, 14.75; S, 6.72.

5,6-Dihydro-5-(2-methyl-5-nitro-1H-benzo[d]imidazol-1-yl)-6- (5-methylfuran-2-yl)-4-phenylpyrimidine-2(1H)-thione (25)

Yield: 80%. m.p.: 180–182 °C. ¹H NMR (DMSO-d₆) δ (ppm): 1.92 (s, 3H, CH₃ furan), 2.35 (s, 3H, CH₃ benzimidazole), 4.34 (d, J = 1.5 Hz 1H, CH), 5.09 (d, J = 1.5 Hz, 1H, CH), 6.23 (d, J = 2.5 Hz, 1H, furan), 6.54 (d, J = 2.5 Hz, 1H, furan), 7.58–7.80–7.82 (m, 4H, aromatic), 7.91–7.93 (m, 2H, 1H aromatic + H7), 8.10–8.12 (m, 2H, H6 + H4). ¹³C NMR (DMSO-d₆) δ (ppm): 15.37, 22.71, 31.24, 62.93, 117.70, 129.20, 129.99, 142.68. MS: m/z 444, 3.0% (M⁺ – 1); m/z 418, 0.5%, (M⁺– 2 CH₃ + 3H). Anal. Calcd for C₂₃H₁₉N₅O₃S (445.50): C, 62.01; H, 4.30; N, 15.72; S, 7.20. Found: C, 62.13; H, 4.43; N, 15.82; S, 7.13.

5,6-Dihydro-5-(2-methyl-5-nitro-1H-benzo[d]imidazol-1-yl)-4-phenyl-6-(thiophen-2-yl)pyrimidine-2(1H)-thione (26)

Yield: 68%. m.p.: 205–207 °C. IR (cm⁻¹): 1622 (C=N), 1520, 1338 (NO₂), 1266 (C=S). ¹H NMR (DMSO-d₆) δ (ppm): 2.38 (s, 3H, CH₃), 4.72 (d, J = 1.5 Hz 1H, CH), 5.45 (d, J = 1.5 Hz, 1H, CH), 7.14–7.22 (m, 1H, H′3 thiophene), 7.60–7.64 (m, 2H, H′2 + H′4 thiophene), 7.70–7.74 (m, 1H, aromatic), 7.79–7.83 (m, 2H, aromatic), 7.87 (d, J = 9.0 Hz, 1H, H7), 7.96–8.00 (m, 2H, aromatic), 8.15 (d, J = 9.0 Hz, 1H, H6), 8.19 (s, 1H, H4). ¹³C NMR (DMSO-d₆) δ (ppm): 31.23, 40.67, 128.70, 131.35, 148.43, 155.89, 155.89, 206.92. MS: m/z 447, 0.03% (M⁺); m/z 434, 0.1%, (M⁺ – CH₃ + 2H). Anal. Calcd for C₂₂H₁₇N₅O₂S₂ (447.53): C, 59.04; H, 3.83; N, 15.65; S, 14.33. Found: C, 59.12; H, 3.73; N, 15.76; S, 14.24.

General procedure for the preparation of 1-(4,5-dihydro-5-aryl-3-phenyl-1H-pyrazol-4-yl)-2- methyl-5-nitro-1H-benzo[d]imidazole derivatives (27–30)

Compounds 15, 16, 17 or 18 (21 mmol) were stirred with hydrazine hydrate (99.9% sol., 26 mmol) in ethanol (20 ml) for 3 h. The reaction was stirred at room temperature and monitored by TLC. The solvent was evaporated under reduced pressure and the solids obtained were crystallized from diethyl ether.

1-(4,5-Dihydro-3-phenyl-5-p-tolyl-1H-pyrazol-4-yl)-2-methyl-5-nitro-1H-benzo[d]imidazole (27)

Yield: 66%. m.p.: 212–214 °C. IR (cm⁻¹): 1683 (C=N), 1516, 1340 (NO₂), 1299 (C=S). ¹H NMR (DMSO-d₆) δ (ppm): 2.07 (s, 3H, CH₃ p-tolyl), 2.46 (s, 3H, CH₃ benzimidazole), 5.23 (d, J = 1.5 Hz 1H, CH), 6.49 (d, J = 1.5 Hz, 1H, CH), 6.83 (d, J = 8.0 Hz, 2H, p-tolyl AB-system), 7.02 (d, J = 8.0 Hz, 2H, p-tolyl AB-system), 7.20–7.27 (m, 2H, aromatic), 7.54–7.57 (m, 3H, aromatic),), 8.00 (dd, J _6–7 = 9.2 Hz, J _6–4 = 2.2 Hz, 1H, H6), 8.11 (d, J = 2.3 Hz, 1H, H4), 8.69 (d, J = 9.2 Hz, 1H, H7). MS: m/z 234, 100%, (M⁺ – H – 2-methyl-5-nitro-1H-benzo[d]imidazole part); m/z 177, 32%, (M⁺+ H – 4,5-dihydro-3-phenyl-5-p-tolyl-1H-pyrazol-4-yl part). Anal. Calcd for C₂₄H₂₁N₅O₂ (411.17): C, 70.06; H, 5.14; N, 17.02; S, 7.78. Found: C, 70.13; H, 5.20; N, 17.09; S, 7.64.

1-(4,5-Dihydro-5-(2-methoxyphenyl)-3-phenyl-1H-pyrazol-4-yl)-2-methyl-5-nitro-1H-benzo[d]imidazole (28)

Yield: 56%. m.p.: 195–197 °C. IR (cm⁻¹): 1600 (C=N), 1520, 1338 (NO₂). ¹H NMR (DMSO-d₆) δ (ppm): 2.35 (s, 3H, CH₃), 3.68 (s, 3H, OCH₃), 4.65 (d, J = 1.5 Hz 1H, CH), 6.17 (d, J = 1.5 Hz, 1H, CH), 6.66–6.72 (m, 2H, aromatic), 7.03–7.07 (m, 1H, aromatic), 7.31–7.36 (m, 1H, aromatic), 7.60–7.65 (m, 3H, aromatic), 7.70–7.76 (m, 1H, aromatic), 7.80–7.84 (m, 1H, aromatic), 7.94 (d, J = 9.0 Hz, 1H, H6), 7.97 (d, J = 9.0 Hz, 1H, H7), 8.10 (s, 1H, H4). MS: m/z 411, 0.05% (M⁺ – CH₃ – H). Anal. Calcd for C₂₄H₂₁N₅O₃ (427.46): C, 67.44; H, 4.95; N, 16.38. Found: C, 67.56; H, 4.84; N, 16.27.

1-(4,5-Dihydro-5-(5-methylfuran-2-yl)-3-phenyl-1H-pyrazol-4-yl)-2-methyl-5-nitro-1H-benzo[d]imidazole (29)

Yield: 70%. m.p.: 182–184 °C. IR (cm⁻¹): 1598 (C=N), 1522, 1339 (NO₂). ¹H NMR (DMSO-d₆) δ (ppm): 1.92 (s, 3H, CH₃ furan), 2.37 (s, 3H, CH₃ benzimidazole), 4.62 (d, J = 1.5 Hz 1H, CH), 5.76 (d, J = 1.5 Hz, 1H, CH), 6.23 (d, J = 2.5 Hz, 1H, furan), 6.55 (d, J = 2.5 Hz, 1H, furan), 7.56–7.68 (m, 4H, aromatic), 7.82–7.85 (m, 1H, aromatic), 7.91 (d, J = 9.0 Hz, 1H, H7), 8.10–8.12 (m, 2H, H6 + H4). ¹³C NMR (DMSO-d₆) δ (ppm): 63.73, 72.89, 79.71, 159.95. MS: m/z 318, 16.47% (M⁺ – H – 5-methylfuryl). Anal. Calcd for C₂₂H₁₉N₅O₃ (401.15): C, 65.83; H, 4.77; N, 17.45. Found: C, 65.75; H, 4.65; N, 17.34.

1-(4,5-Dihydro-3-phenyl-5-(thiophen-2-yl)-1H-pyrazol-4-yl)-2-methyl-5-nitro-1H-benzo[d]imidazole (30)

Yield: 68%. M.p.: 203–205 °C. IR (cm⁻¹): 1618 (C=N), 1520, 1339 (NO₂). ¹H NMR (DMSO-d₆) δ (ppm): 2.38 (s, 3H, CH₃), 4.85 (d, J = 1.5 Hz 1H, CH), 5.60 (d, J = 1.5 Hz, 1H, CH), 7.14–7.18 (m, 1H, H′3 thiophene), 7.61–7.76 (m, 2H, H′2 + H′4 thiophene), 7.71–7.76 (m, 1H, aromatic), 7.79–7.84 (m, 2H, aromatic), 7.87 (d, J = 9.0 Hz, 1H, H7), 7.98–8.13 (m, 2H, aromatic), 8.14 (d, J = 9.0 Hz, 1H, H6), 8.19 (s, 1H, H4). MS: m/z 297, 0.55% (M⁺+ H – Ph – CH₃ – NH). Anal. Calcd for C₂₁H₁₇N₅O₂S (403.11): C, 62.52; H, 4.25; N, 17.36; S, 7.95. Found: C, 62.65; H, 4.13; N, 17.45; S, 7.88.

Antitumor activity

Materials and methods of in vitro studies

The MTT assay developed by Mosmann32 was modified by Miura33 and used to determine the in vitro inhibitory effects of test compounds on cell growth. Briefly, medium containing 10 × 10³ cells (Hep-G2, HCT-116, MCF-7 or A-549 cells) in fresh complete growth medium was seeded into each well of a 96-well microplate, with the compound solution added simultaneously to triplicate wells, before the final volume was made up to 100 µl. The plate was incubated at 37 °C for 72 h in a humidified atmosphere of 5% CO₂ using a water jacketed carbon dioxide incubator (Sheldon, TC2323, Cornelius, OR). Media was aspirated, fresh medium (without serum) was added and cells were incubated either alone (negative control) or with different concentrations of sample to give a final concentration of (100, 50, 25, 12.5, 6.25, 3.125, 1.56 and 0.78 µg/ml). Cells were suspended in RPMI 1640 medium for (Hep-G2, MCF-7 and HCT-116), and DMEM for (A549), 1% antibiotic–antimycotic mixture (10 000 U/ml potassium penicillin, 10 000 µg/ml streptomycin sulfate and 25 µg/ml amphotericin B) and 1% l-glutamine in 96-well flat bottom microplate at 37 °C under 5% CO₂. After 48 h of incubation, medium was aspirated, 200 μl of 10% sodium dodecyl sulphate (SDS) in de-ionized water was added to each well and further incubated overnight at 37 °C under 5% CO₂. A 200 μl of 10% SDS in de-ionized water was added to each well to stop the reaction and to solubilize any MTT-formazan that had formed and then incubated overnight at 37 °C. Doxorubicin, which is known natural cytotoxic agent, was used as positive control in 100 µg/ml and gave 100% lethality under the same conditions34. A 100 µl of 0.02N HCl/50% N,N-dimethyl formamide/20% SDS was added to solubilize any MTT-formazan that had formed. The optical density of each well was measured at 575 nm (OD₅₇₅) using a microplate multi-well reader (Bio-Rad Laboratories Inc., model 3350, Hercules, CA) and the inhibition of cell growth (%) was calculated as (1 – T/C) × 100, where C is the mean OD₅₇₅ of the control group and T is that of the treated group. The IC₅₀ was determined from the dose-response curve. A statistical significance was tested between samples and negative control (cells with vehicle) using independent t-test by SPSS 11 program (Chicago, IL).

Materials and methods of in vivo study

Animals

The animal care and handling was done according to the guidelines set by the World Health Organization, Geneva, Switzerland, and according to approval from the ethics committee for animals care at the National Research Centre, Egypt (ethic No. 10-230). Adult male Sprague–Dawley rats (180 ± 20 g, body weight), were purchased from the animal house of National Research Centre, Egypt. They were kept for a week under environmentally controlled conditions (constant temperature 25–27 °C, with 12 h light/dark cycle) for 1 week prior to starting the experiments. The mice were kept as 10 animals per cage, and they were provided with tap water and commercial diets

Materials

Diethylnitrosamine (DENA) and carbon tetrachloride (CCl₄) were purchased from Sigma Chemical Co. (St. Louis, MO). DENA was dissolved in saline and injected in a single dose (200 mg/kg, i.p.) to initiate hepatic carcinogenesis, while CCl4 was used in a single dose (2 ml/kg) by gavage as 1:1 dilution in corn oil to stimulate liver cell proliferation and regeneration35. Aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP) and total bilirubin (Bili) kits were obtained from Biomerieux (France).

Experimental design

In vivo assay of acute toxicity of the compounds

The acute toxicity of the synthesized compounds was determined in vivo according to Prieur et al.36 and Ghosh37. Briefly, animals were divided into subgroups (10 rats each) administrated i.v. in a single dose with gradually doses ranged from 0 to 2500 µg/kg. Control animals received the vehicle alone (DMSO). Mortality of the animals was observed up to one month post-treatment. LD₅₀ (the median lethal doses) of each compound was determined as (the dose resulted in 50% mortality of the animals) was calculated using a computer program for Probit analysis. The compounds which exhibited acute cytotoxicity will be further examined for its protective effect on DENA-induced liver cancer in rats.

In vivo antitumor activity of the compounds on liver cancer induced by DENA

Adult male Sprague–Dawley rats were divided into three groups with eight animals in each group. Group 1 (untreated control group): animals were fed on a standard diet and given water throughout the course of the experiment. Group 2 (DENA treated group): Rats were injected with a single dose of DENA (200 mg/kg, i.p.) and 2 weeks later received a single dose of CCl₄ (2 ml/kg) by oral gavage at 1:1 dilution in corn oil for 32 weeks. Group 3 (DENA and compound 9 group): Rats-bearing cancer from Group 2 were treated with compound 9 by intravenous administration at dose of 1/10 of the LD₅₀ value and continued for 10 consecutive days and the experiment was ended after 30 days from last injection.

At the end of the treatment protocol, animals were anesthetized with ether and blood samples were drawn from the orbital venous plexus. Serum was separated by centrifugation for 5 min at 1500 × g and stored at −20 °C until analysis. This was then used to determine liver function by assaying ALT, AST and ALP activities and total bilirubin spectrophotometrically according to the manufacturer's instructions, using reagent kits obtained from Roche Diagnostics (Mannheim, Germany).

Histopathological examination

For histopathological examination, animals were sacrificed by decapitation and their livers were rapidly excised, weighed, washed with saline, portions of liver were fixed in 10% formalin in saline, then embedded in paraffin wax, serially sectioned and stained according to Conn et al.38 using a standard method of hematoxylin and eosin (H&E).

Statistical analysis

The results are reported as mean ± standard error (SE) of eight rats. Statistical differences were analyzed according to followed by one way ANOVA test followed by student's t-test wherein the differences were considered to be significant at p < 0.05.

Antiviral activity

Cytotoxicity test

It was done according to Simoes et al.39 as well as Walum et al.40. Briefly, all samples (100 mg) were dissolved in 500 μl of DMSO. Decontamination of samples was done by adding 12 μl of 100× of antibiotic–antimycotic mixture to 500 μl of each sample. Then, bi-fold dilutions were done to 100 μl of original dissolved samples and 100 μl of each dilutions were inoculated in Hep-2 and MA104 cell lines (obtained from the holding company for Biological Products & Vaccines VACSERA, Egypt) previously cultured in 96 multi-well plates (Greiner-Bio One, Germany) to estimate the non-toxic dose of the tested samples. Cytotoxicity assay was done using cell morphology evaluation by inverted light microscope and cell viability test applying trypan blue dye exclusion method.

Cell morphology evaluation by inverted light microscopy

Hep-2 and MA104 cell cultures (2 × 10⁵cells/ml) were prepared in 96-well tissue culture plates (Greiner-Bio One, Germany). After 24 h incubation at 37 °C in a humidified 5% (v/v) CO₂ atmosphere cell monolayers were confluent, the medium was removed from each well and replenished with 100 μl of bi-fold dilutions of different samples tested prepared in DMEM (GIBCO BRL). For cell controls, 100 μl of DMEM without samples was added. All cultures were incubated at 37 °C in a humidified 5% (v/v) CO₂ atmosphere for 72 h. Cell morphology was observed daily for microscopically detectable morphological alterations, such as loss of confluence, cell rounding and shrinking and cytoplasm granulation and vacuolization. Morphological changes were scored (Simoes et al.)39.

Cell viability assay

It was done by trypan blue dye exclusion method (Walum et al.)40. Hep-2 and MA104 cell cultures (2 × 10⁵cells/ml) were grown in 12-well tissue culture plates Greiner-Bio One, Germany). After 24 h incubation, the same assay described above for tested samples cytotoxicity was followed by applying 100 μl of tested samples dilutions (bi-fold dilutions) per well. After 72 h, the medium was removed, cells were trypsinized and an equal volume of 0.4% (w/v). Trypan blue dye aqueous solution was added to cell suspension. Viable cells were counted under the phase contrast microscope.

Determination of adenovirus type 7 and rotavirus Wa strain titers using plaque assay

Non-toxic dilutions were mixed (100 μl) with 100 μl of different doses of adenovirus type 40 (1 × 10⁵, 1 × 10⁶, 1 × 10⁷) and the same doses of rotavirus Wa strain. The infectivities of the rotavirus stocks were activated with 10 μg/ml trypsin for 1/2 h at 37 °C. The mixture was incubated for 1/2 h in 37 °C. The inoculation of (100 μl) 10-fold dilutions of treated and untreated Adenovirus type 7 and rotavirus Wa strain was carried out into Hep 2 and MA 104 cell lines for Adenovirus type 7 and rotavirus Wa strain, respectively, in 12 multi-well plates. After 1 h of incubation for adsorption at 37 °C in a 5% CO₂-water vapor atmosphere without constant rocking. The plates were rocked intermittently to keep the cells from drying. After adsorption, 1 ml of 2× media [Dulbecco's Modified Eagle Medium (DMEM), Gibco-BRL] plus 1 ml 1% agarose was added to each well, 0.5 μg/ml was added to the media-agarose mixture in the case of rotavirus Wa strain and the plates were incubated at 37 °C in a 5% CO₂-water vapor atmosphere. After the appropriate incubation period, the cells were stained with 0.4% crystal violet after formalin fixation, and the number of plaques was counted. The viral titers were then calculated, and expressed as plaque-forming units per milliliter (pfu/ml).

Results and discussion

Chemistry

2-Methyl-5-nitro-1H-benzimidazole 1, previously described by our group17^,18^,20^,41, was used as a starting material to prepare benzimidazole derivatives. During transformation of compound 1 into 2, alkylation occurred using allyl bromide with stirring at room temperature in DMF and sodium hydride. Addition of bromine at the double bond of the allyl group was accomplished in chloroform to produce compound 3. Compound 3 was reacted with thiourea, ethylene diamine and o-phenylene diamine in ethanol and triethylamine to form the benzimidazole derivatives 4, 5 and 6, respectively, with different heterocyclic rings (imidazolidine-2-thione ring in 4, piperazine ring in 5, and 1,2,3,4-tetrahydroquinoxaline ring in 6) at position-1 of the benzimidazole nucleus (Scheme 1)42. Diacetylation of 5 and 6 was performed using acetyl chloride to form the diacetyl benzimidazole derivatives 7 and 8, respectively. Next, Compound 3 reacted with two equivalents of methyl amine or glycine in the presence of potassium carbonate in acetone to achieve the diamino derivatives 9 and 10 respectively (Scheme 1). Scheme 2 represents the formation of three novel benzimidazole derivatives having heterocyclic rings containing oxygen via the reaction of 3 with the dihydroxy compounds. Compound 3 reacted with ethylene glycol, benzene-1, 2-diol, or pentan-2,4-diol in the presence of potassium carbonate in acetone to form the benzimidazole derivatives 11, 12 and 13, respectively, with different oxygen containing heterocyclic rings (1,4-dioxane ring in 11, 2,3-dihydro-benzo[1,4]dioxine ring in 12, and 5,7-dimethyl-[1,4]dioxepane ring in 13) at position-1 of the benzimidazole nucleus in good to excellent yields (Scheme 2).

Scheme 1. Synthesis of novel benzimidazole derivative 3–10 connected at position 1 via methylene linker.

Scheme 2. Synthesis of benzimidazole derivatives 11–13 with heterocyclic rings connected at position 1 via methylene linker (using different diols).

Indeed, 2-(2-methyl-5-nitro-1H-benzo[d]imidazol-1-yl)-1-phenylethanone 14, which is considered as an important starting material for synthesis of various benzimidazole derivatives with antiviral and anticancer activity, was prepared by the reaction of 2-methyl-5-nitro-1H-benzimidazole 1 with phenacyl bromide with stirring at room temperature in DMF and sodium hydride for 8 h. Condensation reaction of 14 with various functionalized aromatic and heterocyclic aldehydes was performed under stirring at room temperature in 10% NaOH and ethanol as a solvent to form the benzimidazole derivatives, 2-(2-methyl-5-nitro-1H-benzo[d]imidazol-1-yl)-3-aryl-1-phenylprop-2-en-1-one, 15–18, with α,β-unsaturated ketones (Scheme 3)43. Compounds 15–18 reacted with ethylene diamine44, thiourea44 and hydrazine hydrate45 in ethanol to form the benzimidazole derivatives, 1-(2,3,6,7-tetrahydro-7-aryl-5-phenyl-1H-1,4-diazepin-6-yl)-2-methyl-5-nitro-1H-benzo[d]imidazole derivatives 19–22, 5,6-dihydro-5-(2-methyl-5-nitro-1H-benzo[d]imidazol-1-yl)-6-aryl-4-phenylpyrimidine-2(1H)-thione 23–26 derivatives and 1-(4,5-dihydro-5-aryl-3-phenyl-1H-pyrazol-4-yl)-2-methyl-5-nitro-1H-benzo[d]imidazole derivatives 27–30 with seven, six and five-membered heterocyclic rings, respectively, at the position 1 of the benzimidazole nucleus in good to excellent yields (Scheme 3). Interestingly, the two stereoisomers (E and Z) were separated successfully into individual pure components [15a(Z)–18a(Z) as major components and 15b(E)–18b(E) as minor components] by column chromatography on silica gel and characterized by combination of ¹H, ¹³C NMR, IR and Mass Spectrometry. The structures of all the new products were established also by ¹H, ¹³C NMR, IR and Mass Spectrometry. The ¹H, ¹³C NMR spectra as well as the X-ray structure analysis allowed the complete assignment of all signals of the stereoisomers. As the chemical shift is an electronic environment dependent, a similar degree of conjugation between the aromatic ring and the olefinic α,β-unsaturated conjugated carbonyl system provides a closed chemical shift values for the (E) and (Z) isomers. However, the significant differences between the isomers appear in the vinyl proton and the aromatic region in the ¹H NMR. The olefinic proton (CH=C) of the (Z) isomers was appeared at more downfield shift than in the case of the olefinic proton (CH=C) of the (E) isomer. This is attributed to the direction of the carbonyl group which is in the same side of the olefinic double bond in the case of the (Z) isomer (Scheme 3). Therefore, the carbonyl group causes de-shielding of the olefinic proton in (Z) isomers. This behavior was observed in the most cases of the (E) and (Z) isomers. For example, in 3-(2-Methoxyphenyl)-2-(2-methyl-5-nitro-1H-benzo[d]imidazol-1-yl)-1-phenylprop-2-en-1-one 16a (Z) the olefinic proton was appeared at δ 8.07 ppm whereas in 16b (E) was appeared at δ 7.95 ppm. This downfield shift in the case of the olefinic proton of 16a (Z) is due to the existence of the carbonyl group and the olefinic double bond at the same side. The ¹³C NMR signals leads to the observation that the chemical shifts of the carbon nuclei change by about 1–3 ppm between the two (E) and (Z) isomers. The ¹³C NMR spectra of both (E) and (Z) isomers display CH₃ carbon of benzimidazole at position 2 at 13–15 ppm, olefinic carbon–carbon double bond at 130–157 ppm and carbonyl carbon at 190–193 ppm. Interestingly, structure of 2-(2-methyl-5-nitro-1H-benzo[d]imidazol-1-yl)-1-phenyl-3-p-tolylprop-2-en-1-one; 15a(Z); was established by X-ray crystal structure analysis (Figure 3). The crystallographic data, collection, refinement and the geometric parameters (bond length, bond angle and torsion angle) of 15a(Z) are represented in the Supplementary Material. It is clear from X-ray structure that the (Z) isomer reduces the possible steric interaction between tolyl unit and the phenyl ring adjacent to the carbonyl group. This could much favor the direction of the reaction toward the preference of formation of the favorable (Z) isomer as a more stable conformer. This reason also explains the high yields of the (Z) isomers as the major product compared to the (E) isomers.

Figure 3. X-ray structure of 2-(2-methyl-5-nitro-1H-benzo[d]imidazol-1-yl)-1-phenyl-3-p-tolylprop-2-en-1-one; 15a(Z).

Scheme 3. Synthesis of benzimidazole derivatives 14–30 with heterocyclic rings directly connected at position 1.

Antitumor activity

In vitro cytotoxicity studies

The in vitro cytotoxic screening of the synthesized compounds against four different cell lines was reported in Table 1. From these results, the following important points can be detected. In the case of in vitro cytotoxic activity against human lung adenocarcinoma cells (A-549), three of the tested compounds 3, 9 and 17b (E) (IC₅₀: 28.91, 134.90, 123.70 µM, respectively) showed anticancer activity comparable to doxorubicin (84.10 µM). On the other hand, the other compounds show no appreciable cytotoxic activity against A-549 cell line. For the in vitro cytotoxic activity against human colorectal carcinoma cells (HCT-116), the same three compounds 3, 9 and 17 b (E) (IC₅₀: 97.60, 175.90, 135.90 µM, respectively) from all the tested compounds were found to possess anticancer activity as compared to doxorubicin (111.70 µM). While in the case of the in vitro cytotoxic activity against human hepatocellular carcinoma (Hep G-2), three of the tested compounds, 9, 16a (Z) and 17b (E) exhibited less cytotoxic activity (IC₅₀: 141.70, 135.00, 89.40 µM, respectively) than the standard (IC₅₀: 63.60 µM; Table 1). In the case of in vitro cytotoxic activity against human breast cancer cells (MCF-7), four of the tested compounds showed significant activity which are 3, 9, 13 and 17b (E) (IC₅₀: 142.45, 329.40, 196.00, 161.40 µM, respectively). Compound 3 and 17b (E) were the most active against MCF-7 than doxorubicin (IC₅₀: 163.80 µM). However, the four compounds exhibited less cytotoxic activity than the standard (Table 1).

Table 1. % Growth inhibitory activity at 300 µM of the synthesized compounds, indicating IC₅₀ (µg/ml) between brackets..

		% Inhibitory activity against tumor cell lines at 300 µM
Compound	R	A-549	HCT-116	Hep-G2	MCF-7
2		42.6	13.2	71.9	35.2
3		100 [28.9]*(±2.1)	98.4 [97.6]* (±9.8)	68.6	90.6 [142.4]* (±6.9)
4		11.9	3.0	22.9	33.4
5		23.8	32.2	55.7	55.3
6		51.4	59.9	34.4	34.5
7		–	–	–	–
8		–	–	–	–
9		97.5 [134.9]* (±12.5)	93.6 [175.9]* (±6.6)	57.2 [141.7]* (±15.1)	82.4 [329.4]* (±21.4)
10		3.8	16.5	20.7	18.4
11		30.8	36.1	59.5	52.7
12		22.1	76.0	29.3	23.5
13		12.7	27.8	74.5	89.9 [196.0]* (±15.8)
14		48.5	64.3	54.1	70.1
15a (Z)	15a (Z)	8.1	64.2	55.4	59.2
16a (Z)		30.4	66.2	89.9 [135.0]* (±5.7)	74.4
17a (Z)		0.0	23.3	52.1	35.1
18a (Z)		19.1	38.5	64.0	69.6
15b (E)		–	–	–	–
16b (E)		10.4	0.0	22.2	35.4
17b (E)		94.6 [123.7]* (±11.5)	95.1 [135.9]* (±12.5)	97.1 [89.4]* (±3.7)	83.9 [161.4]* (±18.9)
18b (E)		0.0	5.0	42.6	49.8
19		0.0	0.0	28.4	17.6
20		0.0	10.7	43.3	15.6
21		0.0	3.0	32.0	38.3
22		0.0	34.8	40.3	50.6
23		0.0	24.4	25.3	32.1
24		0.0	24.1	31.1	55.7
25		8.5	0.0	13.9	24.6
26		12.8	77.0	41.2	38.2
27		7.2	81.1	40.0	35.1
28		38.3	65.5	60.8	49.4
29		0.0	35.3	41.7	39.5
30		48.8	49.9	62.4	59.6
	Doxorubicin	[84.1]* (±12.6)	[111.7]* (±20.5)	[63.6]* (±9.4)	[163.8]* (±10.1)

*IC₅₀ (µM), (±X): standard error; “–”: no activity.

In vivo toxicity and antitumor study

In vivo assay of acute toxicity

The result revealed that although the prepared compounds which gave positive in vitro were tested for the acute toxicity in healthy animals, compound 9 was the only compound that exhibited acute toxicity in healthy animals. The concentrations required by compound 9 for 50% mortality of the animals was found to be 640 µg/kg body weight as shown in Figure 4.

Figure 4. In vivo assay of acute toxicity LD₅₀ of the synthesized compound 9 µg/kg body weight.

Hepatoprotective effect of the synthesized compound 9 against liver cancer induced by DENA

As shown in Table 2, DENA-treated rats showed significant (p < 0.05) increase in serum AST, ALT and ALP activities, along with significant (p < 0.05) increase in total bilirubin level compared to control. The treatment with compound 9 at a dose of 1/10 of the LD₅₀ values in the rats resulted in normalization in AST, ALT and ALP activities as well as the total bilirubin level compared to DENA-treated (Table 2). This means that compound 9 is a potent anticancer agent and has effect on liver cancer induced in rats.

Table 2. Liver function test of normal, DENA- and compound 9-treated DENA rats.

Parameter	Control	DENA	9
AST (U/l)	56.00 ± 5.2	110.00 ± 7.8*	64.00 ± 6.10*†
ALT (U/l)	38.00 ± 3.7	95.00 ± 7.0*	46.00 ± 5.00†
ALP (U/l)	110.00 ± 9.3	215.00 ± 15.7*	121.00 ± 14.80†
Bili (mg/dl)	1.40 ± 0.11	2.90 ± 0.20*	1.72 ± 0.19†

Data are expressed as Mean ± SE (n = 8), * and † indicated Significance different from normal control and from DENA-treated rats at p < 0.05, respectively.

In this study, serum obtained from tumor bearing rats showed significant increase in activities of AST, ALT, ALP along with significant increase in bilirubin level compared to control animals. The elevation of these enzyme activities was indicative of the toxic effect of DENA on the liver tissue. It is known that N-nitroso compounds act as strong carcinogens in various mammals including primates46. DENA has been shown to be metabolized by cytochrome P-450 IIE1 (CYP 2E1) to its active ethyl radical metabolite, which could interact with DNA causing mutation and carcinogenesis47. Administration of compound 9 to DENA treated rats showed restoration activity of AST, ALT, ALP and bilirubin level towards normal. Such reverse in serum enzyme activities could be attributed to the ability of 9 to inhibit CYP 2E1 activity, presumably by serving as a competitive inhibitor, leading to a decrease in the formation and/or bioactivation of these nitrosamines.

Histopathological examination

The microscopic examinations of liver sections from different groups were summarized in Figure 5. In this study, histological examination of rat liver sections was consistent with the results obtained from biochemical studies. Liver of control animals as presented in (Figure 5A) revealed normal architecture characterized by polyhedral shaped hepatocytes and cytoplasm granulated with small uniform nuclei. Hepatocytes were arranged in well-organized hepatic cords and separated by narrow blood sinusoids.

Figure 5. Liver section of untreated normal control rats [A] showed unremarkable pathological changes. Liver section from rats treated with DENA showed: hyperchromatism, hyperplasia, proliferating hepatocytes [B]; loss architecture, both hepatic and portal with significant tumor thrombi within portal vessels, slightly larger tumor cells having more irregular nuclei [C]; malignant nuclei [D]; megalocytosis, hyperchromatic nuclei, nuclear vacuolization and prominence [E]; dissolution of hepatic cords appeared as empty vacuoles aligned by strands of necrotic hepatocytes [F]; disarrangement of normal hepatic cells with intense centrilobular necrosis [G]. Liver of the DENA-rats treated with compound 9 showed normal hepatic lobule architecture [H] (H&E, magnification 400×).

Liver of rats treated with DENA alone showed distortion in the tissue organization with hyperchromatism, hyperplasia, proliferating hepatocytes (Figure 5B), both hepatic and portal with significant tumor thrombi within portal vessels, tumor cells are slightly larger have more irregular nuclei and numerous mitotic figures (Figure 5C) with malignant nuclei (Figure 5D). Some section showed megalocytosis, hyperchromatic nuclei as well as nuclear vacuolization and nuclear prominence (Figure 5E) dissolution of hepatic cords which appeared as empty vacuoles aligned by strands of necrotic hepatocytes (Figure 5F). Also, disarrangement of normal hepatic cells with intense centrilobular necrosis were observed induced cancer liver (Figure 5G). Theses marked changes in the hepatic architecture could be explained based on that DENA treatment manifested its toxic effects through the generation of ROS. The resulting effect was the production of elevated amounts of malondialdehyde and conjugated dienes, which caused deleterious effects on the membranous components of hepatocytes.

Liver of the DENA-rats treated with compound 9 showed improvement in the hepatic pattern with more or less normal hepatic lobule architecture (Figure 5H). The histological results confirmed the biochemical results which improved that compound 9 is a potent anticancer agent.

Taken together, from the biochemical and histological data, our results demonstrate that the synthesized compound 9 may be potent anticancer agents for inclusion in modern clinical trials.

Antiviral activity

Table 3 represented the non-toxic doses of tested materials on MA104 and Hep2 cell lines. Similar sensitivity of the two types of cell lines to toxic materials in tested samples was observed. However, slight high resistance to toxic materials was observed with Hep-2 cell line. Considerable antiviral activity against rotavirus Wa strain which percentage of reduction of infectious rotavirus particles was 66.7, 70 and 63.3% for materials 18a (Z), 17b (E) and 17a (Z), respectively (Table 4). Less percentage of reduction was observed with adenovirus type 7 which was 56.7 and 50% for materials 17b (E) and 17a (Z), respectively (Table 5). The higher resistance to effect of tested materials for adenovirus than rotavirus may return to the nature of the genome of the two viruses. Adenovirus is a DNA virus while rotavirus is RNA virus. The activity of the rest of the materials is weak which ranged from 10% to 20% on rotavirus Wa strain and 0% to 20% on adenovirus type 7 (data not shown).

Table 3. Non-toxic doses of tested materials on MA104 and Hep2 cell lines.

Material number	Non-toxic dose on MA104 cell line (µg/ml)	Non-toxic dose on Hep2 cell line (µg/ml)
3	40	40
4	40	50
5	30	30
10	40	40
15a (Z)	40	50
17a (Z)	30	30
17b (E)	30	30
18a (Z)	30	30
19	30	30
20	30	30
21	30	30
22	40	40
23	30	40
24	40	40
26	40	50
29	30	30
30	30	30

Table 4. Anti-rotavirus Wa strain activity of non-toxic doses from tested materials.

Tested material	Initial viral titre	Final viral titre	% of reduction	Mean % of reduction
18a (Z)	1 × 10^4	3 × 10^3	70%	66.7%
	1 × 10^5	3 × 10^4	70%
	1 × 10^6	4 × 10^5	60%
17b (E)	1 × 10^4	3 × 10^3	70%	70%
	1 × 10^5	3 × 10^4	70%
	1 × 10^6	3 × 10^5	70%
17a (Z)	1 × 10^4	3 × 10^3	70%	63.3%
	1 × 10^5	4 × 10^4	60%
	1 × 10^6	4 × 10^5	60%

Table 5. Anti-adenovirus type 7 strain activity of non-toxic doses from tested materials.

Tested material	Initial viral titre	Final viral titre	% of reduction	Mean % of reduction
17b (E)	1 × 10^5	4 × 10^4	60%	56.7%
	1 × 10^6	4 × 10^5	60%
	1 × 10^7	5 × 10^6	50%
17a (Z)	1 × 10^5	5 × 10^4	50%	50%
	1 × 10^6	5 × 10^5	50%
	1 × 10^7	5 × 10^6	50%

Structure–activity relationship

The best substitution at position 1 of benzimidazole was found to be the 2,3-dibromopropyl substituent in compound 3 to obtain the best cytotoxicity. This may be due to the flexible aliphatic chain substituted with the voluminous electron donating two bromine atoms making the best fitting with the receptor. Also, the propyl-1,2-diamine was a favored substitution at position 1 of benzimidazole in compound 9 as it also possesses both flexibility as well as the electronegative two amino groups. It was noticed that 9 was more active than 10 having the diglycinyl groups, as the presence of the two carboxylic groups demolishes the activity. Substitution at position 1 with the 2-propen-1-one chain having 2-methylfuryl ring in the E configuration was favored due to steric factors as well as the possible hydrophobic interaction of the 2-methylfuryl ring with the active site. Similarly compound 17b was found to be the most active among the series against rotavirus Wa strain, which can be attributed to the same previously mentioned effects. Compounds 17a and 18a were active against the virus but to a lesser extent than 17b. The activity of both compounds may be also due to the hydrophobic interaction of the 2-methyl furyl and of the thienyl ring with the receptor. The antitumor activity was abolished by the presence of heterocyclic rings directly linked or linked through a methylene group to position 1.

Conclusion

A series of novel benzimidazole derivatives substituted at position 1 by heterocyclic ring systems were synthesized and evaluated for their cytotoxicity against A-549, HCT-116, Hep-G2 and MCF-7 cell lines (in vitro and in vivo) and antiviral activity (against rotavirus Wa strain and adenovirus type 7). The in vitro cytotoxic screening of the new compounds against four different cell lines was achieved. Compound 3 was more active than doxorubicin against A-549, HCT-116 and MCF-7. Compound 9 and 17b (E) showed potency near to doxorubicin against the four cell lines. The acute toxicity of compound 9 on liver cancer induced in rats was determined in vivo. Interestingly, it showed restoration activity of liver function and pathology towards normal as compared to the cancer-bearing rats induced by DENA. In addition, compounds 18a (Z), 17b (E) and 17a (Z) were the most promising antiviral compounds against rotavirus Wa strain, while 17b (E) and 17a (Z) showed considerable antiviral activity against adenovirus type 7.

Supplementary material available online.

Declaration of interest

The authors report no conflicts of interest.