G150

Benzimidazole Condensed Ring Systems: New Synthesis and Antineoplastic Activity of Substituted 3,4-Dihydro- and 1,2,3,4-Tetrahydro-benzoimidazo[1,2-a]pyrimidine Derivatives

As part of an ongoing effort to develop new antineoplastic agents, a series of substituted 3,4-dihydro- and 1,2,3,4-tetrahydro-benzoimidazo[1,2-a]pyrimidine derivatives (5-19) were synthesized. 1,2,3,4-Tetrahydrobenzoimidazo[1,2-a]pyrimidine-2-one derivatives (5-7) were prepared via a one-pot two-component thermal cyclization reaction of 2-aminobenzimidazole 1 and β-substituted methyl cinnamates (2-4). Vilsmir-Haack formylation of these derivatives (5-7) afforded the 2-chloro-3-carboxaldehyde targets (8-10), followed by nucleophilic displacement of the chloro atom in the 3-carboxaldehyde compounds (8-10) to yield the remaining final targets (11-19). The structures of the synthesized derivatives (5-19) were confirmed by means of IR, 1H NMR, MS, and elemental analyses. The synthesized derivatives (5-19) were subjected to the National Cancer Institute (NCI) in vitro disease human cell screening panel assay. 2-Chloro-4-phenyl-3,4-dihydrobenzoimidazo[1,2-a]pyrimidine-3-carboxyaldehyde (8, NCI 722731) and 4-(4-methoxyphenyl)-2-(4-methylpiperazin-1-yl)-3,4-dihydrobenzoimidazo[1,2-a]pyrimidine-3-carboxaldehyde (18, NCI 722739) showed a variable degree of antineoplastic activity against some of the cell lines tested. 2-Chloro-4-(4-nitrophenyl)-3,4-dihydrobenzoimidazo[1,2-a]pyrimidine-3-carboxyaldehyde (10, NCI 722743) exhibited good in vitro antineoplastic activity with subpanel disease selectivity against all the cell lines tested with log10 GI50 (M), the concentration that inhibits 50% of cell growth, values ranging from -5.08 to < -8.00. Key words: Benzimidazole, Methyl cinnamates, Pyrimido[1,2-a]benzimidazole, Azinazoles, Anticancer, Cytotoxicity INTRODUCTION Azinazoles including pyrido-, pyrimido-, and triazino-benzimidazole heterocycles have played a pivotal role in the development of effective pharmacophores (Frolov, 2004). The benzimidazole nucleus is an essential part of many antineoplastic derivatives (Soliman et al., 1984). The pyrido[1,2-a]benzimidazole ring system has been found to exhibit anticancer properties by Badawy and Kappe (Badawy and Kappe, 1995). We became interested in the pyrimido[1,2-a]benzimidazole framework (A) when we tried to determine the effect of the nature of the condensed heterocycle as part of our continuation of this research. The majority of DNA intercalating antitumor drugs has a common general structure comprising a planar tricyclic and tetracyclic chromophore (Filippatos et al., 1994; Matelli et al., 1988; Kimura et al., 1992; Abadi et al., 1999; Palmer et al., 1988). These structural observations led to the synthesis of a series of substituted 3,4-dihydro- and 1,2,3,4-tetrahydro-benzoimidazo[1,2-a]pyrimidine derivatives (5-19) possessing a bridgehead nitrogen atom with a view of designing new potential anticancer agents. Thermal cyclization to form a new C-N bond is a common synthetic approach to synthesize azinazoles. Thus, the pyrimido[1,2-a]benzimidazoles tricyclic system is formed by the reaction of 2-aminobenzimidazole with α-diketones (Kreutzberger and Leger, 1981), 2-amino-3-ethoxycarbonyl-4,5-dihydrofuranes (Elnagdi and WamHoff, 1981), and carbon suboxide in the presence of aluminium chloride (Bousignore et al., 1992). In the present study, we describe a new and efficient method for synthesizing selected pyrimido[1,2-a]benzimidazole derivatives (5-19) by the one-pot two-component thermal condensation reaction and the evaluation of the anticancer properties of these derivatives. Correspondence to: Atef Abdel-monem Abdel-hafez, Department of Pharmaceutical Medicinal Chemistry, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt Tel: 20-88-0230-3006, Fax: 20-88-0233-2776 E-mail: [email protected] MATERIALS AND METHODS Melting points were determined on an electrothermal melting point apparatus and were uncorrected. Thin layer chromatography (TLC) was carried out on precoated silica gel TLC plates (Merck, 60 F254, 2.5 x 5.0 cm, 0.25 mm layer thickness) and the spots were detected under UV light. IR spectra were recorded as KBr discs on a Shimadzu IR 200-91527 spectrophotometer. 1H-NMR spectra were measured on a JNM-GX 400 FT NMR spectrophotometer (JEOL Co., Tokyo, Japan) and all chemical shifts are given in δ ppm relative to tetramethylsilane (TMS). Electron impact (EI) mass spectra were measured with JEOL-JMS-AX 505 spectrophotometer at an ionization voltage of 70 eV. Elemental analyses were performed at the microanalytical center of Toyama Medical and Pharmaceutical University, Toyama, Japan. All chemicals used are of analytical grade. General Method for the Synthesis of 4-Substituted-1,2,3,4-Tetrahydrobenzoimidazo[1,2-a]pyrimidin-2-one Derivatives (5-7) 2-Aminobenzimidazole 1 (1 g) and 1.1 equivalents of the appropriate methyl cinnamate (2-4) were added to a 100 mL round-bottomed flask equipped with a reflux condenser. The reaction mixture was heated for 10-15 minutes at 180-200°C until fusion. Before solidification, benzene (50 mL) was added to the reaction mixture to form a slurry. The precipitate was filtered, washed with benzene, and the crude material was crystallized from the appropriate solvent. The physical constants are listed in Table I. 4-Phenyl-1,2,3,4-tetrahydrobenzoimidazo[1,2-a]pyrimidin-2-one (5) IR (KBr) ν cm⁻¹: 3405, 2815, 1663, 1629, 1595. 1H-NMR (DMSO-d6): 2.91 (1H, dd, J = 16.6, 3.2 Hz, Ha-3); 3.51 (1H, dd, J = 16.6, 7.3 Hz, Hb-3); 5.93 (1H, dd, J = 7.3, 3.2 Hz, H-4); 6.82-7.46 (8H, m, Ar-H); 11.59 (1H, brs, NH). EI-MS m/z [M⁺] 263. 4-(4-Methoxyphenyl)-1,2,3,4-tetrahydrobenzoimidazo[1,2-a]pyrimidin-2-one (6) IR (KBr) ν cm⁻¹: 3408, 2805, 1665, 1627, 1590. 1H-NMR (DMSO-d6): 2.89 (1H, dd, J = 16.6, 4.0 Hz, Ha-3); 3.43 (1H, dd, J = 16.4, 6.8 Hz, Hb-3); 3.71 (3H, s, OCH3); 5.86 (1H, dd, J = 16.4, 6.8 Hz, H-4); 6.84-7.45 (8H, m, Ar-H); 11.70 (1H, brs, NH). EI-MS m/z [M⁺] 293. 4-(4-Nitrophenyl)-1,2,3,4-tetrahydrobenzoimidazo[1,2-a]pyrimidin-2-one (7) IR (KBr) ν cm⁻¹: 3415, 2835, 1670, 1630, 1600. 1H-NMR (DMSO-d6): 2.97 (1H, dd, J = 17.0, 1.7 Hz, Ha-3); 3.62 (1H, dd, J = 16.6, 7.3 Hz, Hb-3); 6.16 (1H, m, H-4); 7.0-8.2 (8H, m, Ar-H); 11.87 (1H, brs, NH). EI-MS m/z [M⁺] 308. General Method for Synthesis of 2-Chloro-4-Substituted-3,4-Dihydrobenzoimidazo[1,2-a]pyrimidin-3-carboxaldehyde Derivatives (8-10) Phosphorus oxychloride (0.017 mole) was gradually added to a stirred suspension of the appropriate benzoimidazo[1,2-a]pyrimidin-2-one derivatives 5-7 (0.0075 mole) in dimethylformamide (20 mL), and the reaction mixture was stirred at 80°C for 2-3 hours. After cooling and the addition of water, the product was filtered, washed with water, and the crude material was crystallized from the appropriate solvent. The physical constants are listed in Table I. 2-Chloro-4-phenyl-3,4-dihydrobenzoimidazo[1,2-a]pyrimidine-3-carboxaldehyde (8) IR (KBr) ν cm⁻¹: 3435, 2860, 1695, 1638, 1606, 1580. 1H-NMR (DMSO-d6): 3.78 (1H, d, J = 7.08 Hz, H-3); 6.53 (1H, brs, H-4); 7.09-8.24 (8H, m, Ar-H); 9.62 (1H, s, -CHO). EI-MS m/z [M⁺] 309. 2-Chloro-4-(4-methoxyphenyl)-3,4-dihydrobenzoimidazo[1,2-a]pyrimidine-3-carboxaldehyde (9) IR (KBr) ν cm⁻¹: 3450, 2875, 1697, 1640, 1605, 1578. 1H-NMR (DMSO-d6): 3.94 (1H, brs, H-3); 3.75 (3H, s, OCH3); 6.52 (1H, brs, H-4); 7.60-8.53 (8H, m, Ar-H); 9.62 (1H, s, -CHO). EI-MS m/z [M⁺] 339. 2-Chloro-4-(4-nitrophenyl)-3,4-dihydrobenzoimidazo[1,2-a]pyrimidine-3-carboxaldehyde (10) IR (KBr) ν cm⁻¹: 3460, 2885, 1708, 1645, 1610, 1590. 1H-NMR (DMSO-d6): 3.94 (1H, brs, H-3); 6.60 (1H, brs, H-4); 7.06-8.16 (9H, m, Ar-H); 9.63 (1H, s, -CHO). EI-MS m/z [M⁺] 354. General Method for Synthesis of Substituted-3,4-Dihydrobenzoimidazo[1,2-a]pyrimidin-3-carboxaldehyde Derivatives (11-19) A mixture of the targeted 2-chloro-4-substituted-3,4-dihydrobenzoimidazo[1,2-a]pyrimidine-3-carboxaldehyde derivative 8-10 (0.004 mole) with the appropriate secondary amine (0.010 mole) in dimethylformamide (10 mL) was stirred at 60°C for 1 hour. After cooling, the product was filtered, washed with water, and the crude material was crystallized from the appropriate solvent. The physical constants are listed in Table I. 4-Phenyl-2-(piperidin-1-yl)-3,4-dihydrobenzo[4,dazo[1,2-a]pyrimidine-3-carboxaldehyde (11) IR (KBr) ν cm⁻¹: 3425, 2920, 1693, 1650, 1625, 1605. 1H-NMR (DMSO-d6): 1.67 (6H, m, -CH2-CH2-CH2- of piperidino); 3.11 (4H, m, -CH2-N-CH2- of piperidino); 3.54 (1H, dd, J = 16.60, 7.32 Hz, H-3); 5.87 (1H, dd, J = 7.36, 3.68 Hz, H-4); 7.08-7.71 (9H, m, Ar-H); 9.03 (1H, brs, CHO). EI-MS m/z [M⁺] 358. 4-(Methoxyphenyl)-2-(piperidin-1-yl)-3,4-dihydrobenzoimidazo[1,2-a]pyrimidine-3-carboxaldehyde (12) IR (KBr) ν cm⁻¹: 3440, 2945, 1700, 1660, 1625, 1610. 1H-NMR (DMSO-d6): 1.41 (2H, m, -CH2-CH2-CH2- of piperidino); 1.53 (4H, m, -CH2-CH2-CH2- of piperidino); 3.32 (4H, m, -CH2-N-CH2- of piperidino); 3.52 (1H, dd, J = 16.60, 7.32 Hz, H-3); 3.71 (3H, s, CH3); 5.64 (1H, dd, J = 7.08, 3.68 Hz, H-4); 7.11-7.79 (8H, m, Ar-H); 9.12 (1H, brs, CHO). EI-MS m/z [M⁺] 388. 4-(Nitrophenyl)-2-(piperidin-1-yl)-3,4-dihydrobenzoimidazo[1,2-a]pyrimidine-3-carboxaldehyde (13) IR (KBr) ν cm⁻¹: 3485, 2940, 1705, 1658, 1629, 1610. 1H-NMR (DMSO-d6): 1.43 (2H, m, -CH2-CH2-CH2- of piperidino); 1.56 (4H, m, -CH2-CH2-CH2- of piperidino); 3.38 (4H, m, -CH2-N-CH2- of piperidino); 3.62 (1H, dd, J = 16.60, 7.32 Hz, H-3); 5.93 (1H, dd, J = 7.36, 3.68 Hz, H-4); 7.11-8.24 (8H, m, Ar-H); 9.54 (1H, brs, CHO). EI-MS m/z [M⁺] 403. 2-Morpholino-4-phenyl-3,4-dihydrobenzoimidazo[1,2-a]pyrimidine-3-carboxaldehyde (14) IR (KBr) ν cm⁻¹: 3428, 2935, 1695, 1645, 1630, 1600. 1H-NMR (DMSO-d6): 3.47 (4H, t, J = 16.42 Hz, -CH2-N-CH2- of morpholino); 3.55 (1H, dd, J = 16.42, 7.32 Hz, H-3); 3.88 (4H, t, J = 16.42 Hz, -CH2-O-CH2- of morpholino); 5.87 (1H, dd, J = 7.12, 3.68 Hz, H-4); 7.09-7.74 (8H, m, Ar-H); 9.02 (1H, brs, CHO). EI-MS m/z [M⁺] 360. 2-Morpholino-4-(4-methoxyphenyl)-3,4-dihydrobenzoimidazo[1,2-a]pyrimidine-3-carboxaldehyde (15) IR (KBr) ν cm⁻¹: 3450, 2950, 1705, 1640, 1628, 1605. 1H-NMR (DMSO-d6): 3.31 (4H, t, J = 16.42 Hz, -CH2-N-CH2- of morpholino); 3.51 (1H, dd, J = 16.42, 7.32 Hz, H-3); 3.72 (3H, s, CH3); 3.73 (4H, t, J = 16.42 Hz, -CH2-O-CH2- of morpholino); 5.67 (1H, dd, J = 7.08, 3.68 Hz, H-4); 7.14-7.82 (8H, m, Ar-H); 9.13 (1H, brs, CHO). EI-MS m/z [M⁺] 390. 2-Morpholino-4-(4-nitrophenyl)-3,4-dihydrobenzoimidazo[1,2-a]pyrimidine-3-carboxaldehyde (16) IR (KBr) ν cm⁻¹: 3480, 2935, 1710, 1648, 1630, 1610. 1H-NMR (DMSO-d6): 3.35 (4H, t, J = 16.42 Hz, -CH2-N-CH2- of morpholino); 3.63 (1H, dd, J = 16.42, 7.32 Hz, H-3); 3.77 (4H, t, J = 16.42 Hz, -CH2-O-CH2- of morpholino); 5.98 (1H, dd, J = 7.12, 3.68 Hz, H-4); 7.28-8.15 (8H, m, Ar-H); 9.62 (1H, brs, CHO). EI-MS m/z [M⁺] 405. 2-(4-Methylpiperazin-1-yl)-4-phenyl-3,4-dihydrobenzoimidazo[1,2-a]pyrimidine-3-carboxaldehyde (17) IR (KBr) ν cm⁻¹: 3430, 2930, 1690, 1635, 1625, 1600. 1H-NMR (DMSO-d6): 2.32 (3H, s, N-CH3); 2.49 (4H, t, J = 16.54 Hz, -CH2-N(CH3)-CH2-); 3.13 (4H, t, J = 16.54 Hz, -CH2-N-CH2-); 3.55 (1H, dd, J = 16.48, 7.32 Hz, H-3); 5.86 (1H, dd, J = 7.32, 3.68 Hz, H-4); 7.07-7.70 (8H, m, Ar-H); 9.02 (1H, brs, CHO). EI-MS m/z [M⁺] 373. 4-(4-Methoxyphenyl)-2-(4-methylpiperazin-1-yl)-3,4-dihydrobenzo[4,dazo[1,2-a]pyrimidine-3-carboxaldehyde (18) IR (KBr) ν cm⁻¹: 3445, 2940, 1700, 1640, 1625, 1600. 1H-NMR (DMSO-d6): 2.18 (3H, s, N-CH3); 2.30 (4H, t, J = 16.54 Hz, -CH2-N(CH3)-CH2-); 3.43 (4H, t, J = 16.54 Hz, -CH2-N-CH2-); 3.48 (1H, dd, J = 16.48, 7.32 Hz, H-3); 5.85 (1H, dd, J = 7.08, 3.68 Hz, H-4); 7.06-7.69 (8H, m, Ar-H); 9.12 (1H, brs, CHO). EI-MS m/z [M⁺] 403. 2-(4-Methylpiperazin-1-yl)-4-(4-nitrophenyl)-3,4-dihydrobenzo[4,dazo[1,2-a]pyrimidine-3-carboxaldehyde (19) IR (KBr) ν cm⁻¹: 3480, 2930, 1700, 1645, 1625, 1605. 1H-NMR (DMSO-d6): 2.41 (3H, s, N-CH3); 2.50 (4H, t, J = 16.54 Hz, -CH2-N(CH3)-CH2-); 3.50 (4H, t, J = 16.54 Hz, -CH2-N-CH2-); 3.58 (1H, dd, J = 16.48, 7.32 Hz, H-3); 5.90 (1H, dd, J = 7.32, 3.68 Hz, H-4); 7.08-8.11 (8H, m, Ar-H); 9.56 (1H, brs, CHO). EI-MS m/z [M⁺] 418. RESULTS AND DISCUSSION Chemistry The synthetic strategy for the synthesis of the target derivatives (5-19) is depicted in Scheme 1. Two-component condensation reactions are powerful tools for the synthesis of organic compounds, since the products are formed in a single step and diversity can be achieved by simply varying each component. In recognition of the interest in developing new approaches to the synthesis of a variety of heterocycles incorporating a benzimidazole moiety from readily obtainable inexpensive starting materials, 2-aminobenzimidazole 1 and β-substituted methyl cinnamates (2-4) were utilized as building blocks for a possible new route to design the pyrimido[1,2-a]benzimidazole tricyclic system. 1,2,3,4-Tetrahydrobenzoimidazo[1,2-a]pyrimidine-2-one derivatives (5-7) were prepared via a thermal condensation reaction of 2-aminobenzimidazole 1 and β-substituted methyl cinnamates (2-4) at 180-200°C. Vilsmir-Haack formylation of these derivatives (5-7) using two molar equivalent amounts of phosphorus oxychloride afforded the 2-chloro-3-carboxaldehyde targets (8-10). Nucleophilic displacement of the chloro atom in the 3-carboxaldehyde compounds (8-10) using secondary amines yielded the remaining final targets (11-19). The structure of the synthesized derivatives (5-19) was confirmed by means of IR, 1H-NMR, MS, and elemental analyses. The IR spectra of azinazoles (5-19) contain stretching absorption bands of C=O, C=N, and C=C bonds. The 1H-NMR spectrum of compounds (8-19) exhibited a signal at δ 9.02-9.63 recognized as arising from aldehydic protons, in addition to characteristic signals for H-3 and H-4 protons. A broad singlet at δ 11.59-11.87 assigned to NH proton appeared at 1H-NMR spectra of 1,2,3,4-tetrahydrobenzoimidazo[1,2-a]pyrimidine-2-one derivatives (5-7). The synthesized benzoimidazo[1,2-a]pyrimidine derivatives (5-19) were characterized by IR, ^1H-NMR, MS, and elemental analyses, confirming their proposed structures. The IR spectra showed characteristic absorption bands corresponding to carbonyl (C=O), imine (C=N), and aromatic (C=C) bonds. In the ^1H-NMR spectra of compounds 8-19, a distinctive aldehydic proton signal appeared between δ 9.02 and 9.63 ppm, alongside signals attributed to the H-3 and H-4 protons of the heterocyclic ring. For the 1,2,3,4-tetrahydro derivatives (5-7), a broad singlet at δ 11.59–11.87 ppm was observed, corresponding to the NH proton. The synthetic approach employed a one-pot two-component thermal condensation reaction between 2-aminobenzimidazole and β-substituted methyl cinnamates, yielding the 1,2,3,4-tetrahydrobenzoimidazo[1,2-a]pyrimidine-2-one derivatives (5-7). Subsequent Vilsmir-Haack formylation with phosphorus oxychloride converted these into 2-chloro-3-carboxaldehyde derivatives (8-10). These intermediates underwent nucleophilic substitution with various secondary amines to afford the final substituted 3,4-dihydrobenzoimidazo[1,2-a]pyrimidine-3-carboxaldehyde derivatives (11-19). The biological evaluation of these compounds was conducted through the National Cancer Institute (NCI) in vitro human tumor cell line screening panel. Among the tested compounds, 2-chloro-4-(4-nitrophenyl)-3,4-dihydrobenzoimidazo[1,2-a]pyrimidine-3-carboxaldehyde (compound 10) demonstrated significant antineoplastic activity with selective cytotoxicity across various cancer cell lines, showing log GI50 values ranging from -5.08 to less than -8.00, indicating potent growth inhibition at low micromolar to submicromolar concentrations. Other compounds, such as 2-chloro-4-phenyl-3,4-dihydrobenzoimidazo[1,2-a]pyrimidine-3-carboxaldehyde (8) and 4-(4-methoxyphenyl)-2-(4-methylpiperazin-1-yl)-3,4-dihydrobenzoimidazo[1,2-a]pyrimidine-3-carboxaldehyde (18), exhibited variable degrees of antineoplastic activity against selected cell lines, suggesting that substitution patterns on the phenyl ring and the nature of the amine substituent influence the biological activity. The results indicate that the benzoimidazo[1,2-a]pyrimidine scaffold, particularly with electron-withdrawing substituents such as nitro groups, and appropriate amine substitutions, holds promise as a framework for developing novel anticancer agents. The study provides a new synthetic route to access these heterocyclic compounds efficiently and highlights their potential in anticancer drug discovery. In conclusion, the one-pot two-component thermal cyclization followed by Vilsmir-Haack formylation and nucleophilic substitution constitutes an effective synthetic strategy for benzoimidazo[1,2-a]pyrimidine derivatives. The biological screening results underscore the importance of structural modifications for optimizing antineoplastic activity, warranting further investigation into these compounds as potential G150 chemotherapeutic agents.