40 Original Article

Microwave Assisted Synthesis and Anti-Lipase Activity of Some New Fluorine-Containing Benzimidazoles

Affiliations

Key words

▶ microwave chemistry ● ▶ fluorine chemistry ● ▶ anti lipase activity ● ▶ 2-substituted benzimida●

zoles

▶ Orlistat ●

E. Menteşe1, F. Yilmaz1, S. Ülker2, B. Kahveci3 1

Department of Chemistry,Art and Science Faculty, Recep Tayyip Erdogan University, Rize, Turkey Department of Biology, Art and Science Faculty, Recep Tayyip Erdogan University, Rize, Turkey 3 Department of Nutrition and Dietetics, Faculty of Health Sciences, Karadeniz Technical University, Trabzon, Turkey 2

Abstract



In this study, a new series of fluorine containing benzimidazoles (4a–l) and bisbenzimidazoles (6a– c, 8) were synthesized by the reaction of o-phenylenediamines with iminoester hydrochlorides (3a–l, 7) in methanol under microwave irradiation. The structures of these newly synthesized com-

Introduction



received 08.01.2014 accepted 07.03.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1371890 Published online: April 2, 2014 Drug Res 2015; 65: 40–45 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence E. Menteşe, PhD Department of Chemistry Art and Science Faculty Recep Tayyip Erdoğan University Fener Street Atatürk Road 53100 Rize Turkey Tel.: + 90/464/223 61 26 Fax: + 90/464/223 4019 [email protected]

Benzimidazole is known as a privileged structure and an important pharmacophore in medicinal chemistry [1–3]. It is naturally found in vitamin B12 and shows structural similarity with adenine and guanine [4]. Benzimidazoles have been known for their use in humans for a long time, and thus the synthesis of their derivatives has attracted much attention so that numerous benzimidazole drugs have been described in literature by organic chemists [5, 6]. Over the last few decades, the use of fluorine in drug discovery has expanded by 5–15 % of the launched drugs worldwide. These include Fluoxetine (antidepressant) [7], Atorvastatin (cholesterol-loweringdrug) [8], Ciprofloxacin (antibiotic) [9] and Sitagliptin (antidiabetic agent) [10], which have been in medical use over past 50 years [11]. Moreover, the fluorine atom enhances binding affinity to proteins, increases metabolic stability, changes physicochemical properties and improves some biological activities [12, 13]. This is why the synthesis of fluorine containing compounds has been of great interest to synthetic organic chemists. However, synthesis of fluorine containing benzimidazoles has gained little interest despite their high biological activities [14]. In the literature, the most known procedure for the synthesis of benzimidazole and bisbenzimidazole derivatives is via condensation of o-phenylenediamine derivatives with carboxylic acids in the presence of strong acid (polyphosphoric

Menteşe E et al. Fluorine-containing Benzimidazoles … Drug Res 2015; 65: 40–45

pounds were identified by IR, 1H-NMR, 13C-NMR, mass spectroscopy and elemental analysis data. The synthesized compounds were screened for their pancreatic lipase activities. Our results indicate that the compounds 6a, 6b and 6c can serve as an anti-lipase agent. The compounds 6b and 6c inhibited pancreatic lipase activity by 84.03 % and 97.49 % at a concentration of 3 μg/mL, respectively.

acid (PPA) or mineral acid) under oxidative conditions [15–17]. Also, various procedures have been developed for the synthesis of benzimidazole and bisbenzimidazole derivatives. However, many of these procedures have some disadvantages like harsh reaction conditions (coming from strong acids), long reaction times, using irreversible catalyst and occurence of side products [16, 17]. Therefore, our research has been focused on solving the problems encountered in the synthesis of benzimidazoles and bisbenzimidazoles in recent years. As a result of these research, we have developed a convenient technique for the synthesis of these kinds of compounds by using o-phenylenediamines and iminoester hydrochlorides in methanol under microwave irradiation in good yield [2, 18–20]. In our previous study, we investigated the antilipase activity of some benzimidazole derivatives, which do not contain fluorine, and demonstrated that benzimidazole derivatives had important anti-lipase activity for the first time [18, 19]. Herein, we describe the synthesis of more fluorine containing benzimidazole derivatives by using microwave irradiation and the investigation of their anti-lipase activities.

Material and Methods



General All the chemicals were supplied from Merck (Darmstadt, Germany), Aldrich and Fluka (Buchs

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Authors

Original Article 41

General procedure for the synthesis of compound 2 [21] Pd/C (5 mmol) catalyst and hydrazine hydrate (50 mmol) were added to the solution of compound 1 (10 mmol) in ethanol, and the reaction mixture was irradiated in microwave in a closed vessel with the pressure control at 105 °C for 15 min (hold time) at 300 W maximum power. The progress of the reaction was monitored by TLC, ethylacetate:Hexane, 3:1. Then, the catalyst was separated by filtration and the combined filtrate poured into water. The obtained orange solid was recrystallized from ethanol-water (1:3). Yield: 95 %, mp: 92–93 °C.

General procedure for the synthesis of compounds 4a–l A mixture of 4-fluoro-1,2-phenylenediamine (2) (0.010 mol) and corresponding iminoester hydrochlorides (0.013 mol) (3a–l) in dry methanol (15 mL) was irradiated in microwave in a closed vessel with the pressure control at 65 °C for 11 min (hold time) at 300 W maximum power. After the completion of the reaction, (monitored by TLC, ethylacetate:Hexane, 3:1), the mixture was poured onto water. The precipitate formed was filtered and recrystallized from ethanol-water (1:3) to give pure 4a–l.

2-Benzyl-6(5)-fluoro-1H-benzimidazole (4a): White crystal; yield: 85 %; mp: 169–170 °C; 1H NMR (400 MHz, DMSO-d6): δ 4.17 (s, 2 H, CH2), 6.95–7.01 (m, 1 H, Ar-H), 7.12–7.33 (m, 6 H, Ar-H), 7.45–7.49 (m, 1 H, Ar-H), 12.42 (brs, 1 H, NH); 13C NMR (100 MHz, DMSO-d6): δ 35.4 (CH2), 109.6 (d, JC,F = 12.5 Hz, Ar-C), 121.7, 127.0 (2C), 128.9 (2C), 129.2 (2C), 137.9 (Ar-C), 155.5 (C = N), 158.7 (d, JC,F = 232 Hz, C-F); IR (KBr) cm − 1: 1 628 (C = N), 1 131 (C-F); Anal. Calcd. for C14H11FN2: C, 74.32; H, 4.90; N, 12.38. Found: C, 74.29; H, 4.86; N, 12.33; LC-MS (ESI): m/z ( %): 227.01 (100) [M + ]. 2-(2-Chlorobenzyl)-6(5)-fluoro-1H-benzimidazole (4b): White crystal; yield: 83 %; mp: 203–204 °C; 1H NMR (400 MHz, DMSOd6): δ 4.30 (s, 2 H, CH2), 6.96–7.00 (m, 1 H, Ar-H), 7.12–7.39 (m, 4 H, Ar-H), 7.40–7.45 (m, 1 H, Ar-H), 7.46–7.48 (m, 1 H, Ar-H), 12.41 (brs, 1 H, NH); 13C NMR (100 MHz, DMSO-d6): δ 33.2 (CH2), 109.7 (d, JC,F = 25 Hz, Ar-C), 121.8, 127.8, 129.2, 129.7, 131.9, 133.7, 135.4 (Ar-C), 154.1 (C = N), 158.7 (d, JC,F = 233 Hz, C-F); IR (KBr) cm − 1: 1 627 (C = N), 1 138 (C-F); Anal. Calcd. for C14H10ClFN2: C, 64.50; H, 3.87; N, 10.75. Found: C, 64.39; H, 3.80; N, 10.67; LC-MS (ESI): m/z ( %): 260.87 (100) [M + ], 262.98 (33) [M + 2].

2-(3-Chlorobenzyl)-6(5)-fluoro-1H-benzimidazole (4c): White crystal; yield: 82 %, mp: 186–187 °C; 1H NMR (400 MHz, DMSOd6): δ 4.19 (s, 2 H, CH2), 6.98–7.13 (m, 1 H, Ar-H), 7.28–7.38 (m, 5 H, Ar-H), 7.42 (s, 1 H, Ar-H), 12.44 (s, 1 H, NH); 13C NMR (100 MHz, DMSO-d6): δ 34.8 (CH2), 98.0 (d, JC,F = 27 Hz, Ar-C), 104.4 (d, JC,F = 23 Hz, Ar-C), 109.3, 109.9, 112.0, 119.5, 127.0, 128.1, 129.2, 130.8, 133.5, 140.3 (Ar-C), 155.0 (C = N), 158.7 (d, JC,F = 232 Hz, C-F); IR (KBr) cm − 1: 1 633 (C = N), 1 132 (C-F); Anal. Calcd. for C14H10ClFN2: C, 64.50; H, 3.87; N, 10.75. Found: C, 64.45; H, 3.82; N, 10.71; LC-MS (ESI): m/z ( %): 260.90 (100) [M + ], 262.99 (32) [M + 2]. 2-(4-Chlorobenzyl)-6(5)-fluoro-1H-benzimidazole (4d): White crystal; yield: 85 %; mp: 166–167 °C; 1H NMR (400 MHz, DMSOd6): δ 4.17 (s, 2 H, CH2), 6.95–7.01 (m, 1 H, Ar-H), 7.27–7.48 (m, 6 H, Ar-H), 12.38 (s, 1 H, NH); 13C NMR (100 MHz, DMSO-d6): δ 34.6 (CH2), 109.8 (d, JC,F = 25 Hz, Ar-C), 121.9, 128.9 (2C), 131.2 (2C), 131.8, 136.8 (2C) (Ar-C), 155.1 (C = N), 158.7 (d, JC,F = 233 Hz, C-F); IR (KBr) cm − 1: 1 630 (C = N), 1 137 (C-F); Anal. Calcd. for C14H10ClFN2: C, 64.50; H, 3.87; N, 10.75. Found: C, 64.42; H, 3.80; N, 10.68. LC-MS (ESI): m/z ( %): 260.88 (100) [M + ], 262.98 (32) [M + 2]. 2-(2-Bromobenzyl)-6(5)-fluoro-1H-benzimidazole (4e): White powder; yield: 84 %; mp: 213–214 °C; 1H NMR (400 MHz, DMSOd6): δ 4.30 (s, 2 H, CH2), 6.96–7.00 (m, 1 H, Ar-H), 7.21–7.38 (m, 5 H, Ar-H), 7.63–7.65 (m, 1 H, Ar-H), 12.40 (s, 1 H, NH); 13C NMR (100 MHz, DMSO-d6): δ 35.8 (CH2), 104.2 (d, JC,F = 25 Hz, Ar-C), 109.8 (d, JC,F = 26 Hz, Ar-C), 122.2, 124.5, 128.4, 129.3 (d, JC,F = 7 Hz, Ar-C), 132.0, 133.0, 137.1 (Ar-C), 154.7 (C = N), 159.1 (d, JC,F = 235 Hz, C-F); IR (KBr) cm − 1: 1 626 (C = N), 1 136 (C-F); Anal. Calcd. for C14H10BrFN2: C, 55.10; H, 3.30; N, 9.18. Found: C, 55.03; H, 3.22; N, 9.09; LC-MS (ESI): m/z ( %): 304.81 (87) [M + ], 306.17 (100) [M + 2]. 2-(4-Bromobenzyl)-6(5)-fluoro-1H-benzimidazole (4f): White powder; yield: 87 %; mp: 165–166 °C; 1H NMR (400 MHz, DMSOd6): δ 4.15 (s, 2 H, CH2), 6.98–7.11 (m, 1 H, Ar-H), 7.13–7.53 (m, 6 H, Ar-H), 12.42 (s, 1 H, NH); 13C NMR (100 MHz, DMSO-d6): δ 34.7 (CH2), 98.11, 104.4 (d, JC,F = 27 Hz, Ar-C), 109.6, 109.9, 110.2, 119.6, 131.5 (2C), 131.8 (2C), 137.3, 144.3 (Ar-C), 154.9 (C = N), 158.7 (d, JC,F = 232 Hz, C-F); IR (KBr) cm − 1: 1 631 (C = N), 1 135 (C-F); Anal. Calcd. for C14H10BrFN2: C, 55.10; H, 3.30; N, 9.18. Found: C, 55.06; H, 3.25; N, 9.13; LC-MS (ESI): m/z ( %): 304.82 (85) [M + ], 306.18 (100) [M + 2]. 2-(4-Methylbenzyl)-6(5)-fluoro-1H-benzimidazole (4g): Pale brown powder; yield: 81 %; mp: 183–184 °C; 1H NMR (400 MHz, DMSO-d6): δ 2.25 (s, 3 H, CH3), 4.11 (s, 2 H, CH2), 6.93–7.00 (m, 1 H, Ar-H), 7.12 (d, J = 8 Hz 2 H, Ar-H), 7.20 (d, J = 8 Hz 2 H, Ar-H), 7.31–7.51 (m, 1 H, Ar-H), 12.30 (s, 1 H, NH); 13C NMR (100 MHz, DMSO-d6): δ 21.1 (CH3), 35.0 (CH2), 97.99, 104.3 (d, JC,F = 23 Hz, Ar-C), 109.4 (d, JC,F = 28 Hz, Ar-C), 111.9, 119.4, 129.1, 131.5, 134.8, 135.2, 144.2 (Ar-C), 154.2 (C = N), 158.5 (d, JC,F = 232 Hz, C-F); IR (KBr) cm − 1: 1 629 (C = N), 1 134 (C-F); Anal. Calcd. for C15H13FN2: C, 74.98; H, 5.45; N, 11.16. Found: C, 74.93; H, 5.41; N, 11.10; LC-MS (ESI): m/z ( %): 240.93 (100) [M + 1], 242.05 (17) [M + 2]. 2-(2-Fluorobenzyl)-6(5)-fluoro-1H-benzimidazole (4h): Pale brown powder; yield: 86 %; mp: 179–180 °C; 1H NMR (400 MHz, DMSO-d6): δ 4.21 (s, 2 H, CH2), 6.96–7.00 (m, 1 H, Ar-H), 7.13– 7.46 (m, 6 H, Ar-H), 12.43 (s, 1 H, NH); 13C NMR (100 MHz, Menteşe E et al. Fluorine-Containing Benzimidazoles … Drug Res 2015; 65: 40–45

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SG, Switzerland). Melting points were determined on capillary tubes on a Büchi oil-heated melting point apparatus (Essen, Germany) and uncorrected. 1H-NMR and 13C-NMR spectra were performed on the Varian-Mercury 400 MHz spectrophotometer (Varian, Darmstadt, Germany) in DMSO-d6 using TMS as internal. Mass spectra were recorded on Thermo Scientific Quantum Access max LC-MS spectrofotometer. A Monomode CEM-Discover Microwave instrument was used in the standard configuration as delivered, including proprietary software. All experiments were carried out in microwave process vials (30 ml) with infrared temperature control sensor. The temperature was computer monitored and maintained constant by a discrete modulation of delivered microwave power. After completion of the reaction, the vial was cooled to 60 ° by air jet cooling. All reactions were monitored by TLC using precoated aluminum sheets (silica gel 60 F 2.54 0.2 mm thickness).

42 Original Article

DMSO-d6): δ 28.6 (d, JC,F = 3 Hz, CH2), 104.2 (d, JC,F = 25 Hz, Ar-C), 110.1 (d, JC,F = 28 Hz, Ar-C), 112.1, 115.7 (d, JC,F = 21 Hz, Ar-C), 119.5, 124.6 (d, JC,F = 15 Hz, Ar-C), 124.9, 129.4 (d, JC,F = 8 Hz, Ar-C), 131.8, 131.9 (Ar-C), 155.0 (C = N), 158.9 (d, JC,F = 232 Hz, C-F), 160.8 (d, JC,F = 243 Hz, C-F); IR (KBr) cm − 1: 1 631 (C = N), 1 134 (C-F); Anal. Calcd. for C14H10F2N2: C, 68.85; H, 4.13; N, 11.47. Found: C, 68.80; H, 4.05; N, 11.41. LC-MS (ESI): m/z ( %): 244.98 (100) [M + ], 245.90 (17) [M + 1].

(400 MHz, DMSO-d6): δ 2.50 (s, 3 H, CH3), 7.01–7.66 (m, 5 H, Ar-H), 8.18–8.12 (m, 2 H, Ar-H), 13.00 (s, 1 H, NH); 13C NMR (100 MHz, DMSO-d6): δ 14.73 (SCH3), 98.1 (d, JC,F = 28 Hz, Ar-C), 104.5, 110.5 (d, JC,F = 89 Hz, Ar-C), 112.3, 119.9, 124.9, 126.6, 127.3, 130.3, 132.1, 135.5, 141.3, 144.2 (Ar-C), 153.2 (C = N), 159.3 (d, JC,F = 235 Hz, C-F); IR (KBr) cm − 1: 1 626 (C = N), 1 140 (C-F); Anal. Calcd. for C14H11FN2S: C, 65.10; H, 4.29; N, 10.84. Found: C, 65.02; H, 4.23; N, 10.76; LC-MS (ESI): m/z ( %): 258.85 (100) [M + 1], 259.90 (18) [M + 2].

2-(4-Fluorobenzyl)-6(5)-fluoro-1H-benzimidazole (4i): Pale brown powder; yield: 89 %, mp: 164–165 °C; 1H NMR (400 MHz, DMSO-d6): δ 4.16 (s, 2 H, CH2), 6.98–7.52 (m, 7 H, Ar-H), 12.41 (s, 1 H, NH); 13C NMR (100 MHz, DMSO-d6): δ 34.5 (CH2), 98.1, 104.2 (d, JC,F = 25 Hz, Ar-C), 109.6 (d, JC,F = 64 Hz, Ar-C), 110.1, 112.0, 115.5 (d, JC,F = 12 Hz, Ar-C), 115.7 (d, JC,F = 12 Hz, Ar-C), 119.5, 130.7 (d, JC,F = 7 Hz, Ar-C), 134.0, 144.1 (Ar-C), 155.0 (C = N), 158.9 (d, JC,F = 280 Hz, C-F), 164.0 (d, JC,F = 280 Hz, C-F); IR (KBr) cm − 1: 1 630 (C = N), 1 132 (C-F); Anal. Calcd. for C14H10F2N2: C, 68.85; H, 4.13; N, 11.47. Found: C, 68.78; H, 4.01; N, 11.40; LC-MS (ESI): m/z ( %): 244.99 (100) [M + ], 245.90 (16) [M + 1].

General procedure for the synthesis of compounds 6a–c

2,2'-Bis(2-fluorobenzyl)-1H,1'H-5,5'-bibenzimidazole (6a): White powder; yield: 93 %; mp: 198–200 °C; 1H NMR (400 MHz, DMSOd6): δ 4.25 (s, 4 H, CH2), 6.90–7.16 (m, 2 H, Ar-H), 7.17–7.72 (m, 12 H, Ar-H), 12.47 (s, 2 H, NH); 13C NMR (100 MHz, DMSO-d6): δ 28.60 (d, JC,F = 10, CH2), 111.23, 112.9, 113.8, 115.5 (d, JC,F = 15 Hz, Ar-C), 115.8 (d, JC,F = 13 Hz, Ar-C), 120.7, 121.8, 124.8 (d, JC,F = 15 Hz, Ar-C), 129.3, 131.2, 132.5, 134.4, 135.5, 135.8, 136.1 (Ar-C), 153.4 (C = N), 160.4 (d, JC,F = 243 Hz, C-F); IR (KBr) cm − 1: 1 624 (C = N), 1 230 (C-F); Anal. Calcd. for C28H20F2N4: C, 74.65; H, 4.47; N, 12.44. Found: C, 74.58; H, 4.39; N, 12.37; LC-MS (ESI):m/z ( %): 451.00 (40) [M + 1], 452.05 (15) [M + 2], 225.95 (100) [symmetric division signal].

2-(2,4-Dichlorobenzyl)-6-fluoro-1H-benzimidazole (4j): White crystal; yield: 80 %; mp: 169–170 °C; 1H NMR (400 MHz, DMSOd6): δ 4.29 (s, 2 H, CH2), 6.99–7.00 (m, 1 H, Ar-H), 7.26–7.49 (m, 4 H, Ar-H), 7.64 (s, 1 H, Ar-H), 12.41 (s, 1 H, NH); 13C NMR (100 MHz, DMSO-d6): δ 32.7 (CH2), 99.0, 104.7 (d, JC,F = 23 Hz, Ar-C), 109.5, 110.4, 119.7, 127.9, 129.20, 132.8, 133.5, 134.7 (d, JC,F = 20 Hz, Ar-C), 138.8, 141.5 (Ar-C), 155.0 (C = N), 159.0 (d, JC,F = 233 Hz, C-F); IR (KBr) cm − 1: 1 631 (C = N), 1 133 (C-F), 798 (C-Cl); Anal. Calcd. for C14H9Cl2FN2: C, 56.97; H, 3.07; N, 9.49. Found: C, 56.90; H, 3.02; N, 9.44; LC-MS (ESI): m/z ( %): 294.88 (100) [M + ], 296.83 (65) [M + 2]. 2-(3,4-Dichlorobenzyl)-6-fluoro-1H-benzimidazole (4k): White crystal; yield: 85 %; mp: 170–171 °C; 1H NMR (400 MHz, DMSOd6): δ 4.20 (s, 2 H, CH2), 6.99–7.13 (m, 1 H, Ar-H), 7.27–7.35 (m, 2 H, Ar-H), 7.43–7.62 (m, 2 H, Ar-H), 7.68 (s, 1 H, Ar-H), 12.44 (s, 1 H, NH); 13C NMR (100 MHz, DMSO-d6): δ 34.3 (CH2), 98.0, 104.4 (d, JC,F = 22 Hz, Ar-C), 109.5, 110.3, 119.6, 129.8, 131.0, 131.4, 131.5, 138.9, 141.3 (Ar-C), 155.0 (C = N), 158.9 (d, JC,F = 233 Hz, C-F); IR (KBr) cm − 1: 1 631 (C = N), 1 132 (C-F), 798 (C-Cl); Anal. Calcd. for C14H9Cl2FN2: C, 56.97; H, 3.07; N, 9.49. Found: C, 56.88; H, 3.00; N, 9.42; LC-MS (ESI): m/z ( %): 294.88 (100) [M + ], 296.84 (64) [M + 2].

2,2'-Bis(3-fluorobenzyl)-1 H,1'H-5,5'-bibenzimidazole (6b): White powder; yield: 93 %; mp: 350 °C (dec.); 1H NMR (400 MHz, DMSO-d6): δ 4.53 (s, 4 H, CH2), 6.91–7.20 (m, 8 H, Ar-H), 7.46– 7.80 (m, 6 H, Ar-H), 12.48 (s, 2 H, NH); 13C NMR (100 MHz, DMSO-d6): δ 38.70 (CH2), 113.2, 115.2 (d, JC,F = 15 Hz, Ar-C), 116.7 (d, JC,F = 14 Hz, Ar-C), 116.88, 121.1, 124.8 (d, JC,F = 15 Hz, Ar-C), 131.5, 133.4, 134.8, 135.7, 136.1 (Ar-C), 153.8 (C = N), 169.9 (d, JC,F = 242 Hz, C-F); IR (KBr) cm − 1: 1 616 (C = N), 1 147 (C-F); Anal. Calcd. for C28H20F2N4: C, 74.65; H, 4.47; N, 12.44. Found: C, 74.60; H, 4.42; N, 12.41; LC-MS (ESI): m/z ( %): 451.01 (40) [M + 1], 452.07 (17) [M + 2], 225.96 (100) [symmetric division signal].

2-(4-(Methylthio)phenyl)-6(5)-fluoro-1H-benzimidazole (4l): Orange powder; yield: 80 %, mp: 215–216 °C; 1H NMR NO2

F

1

NH2

NH2

MW, Pd/C

NH.HCl

+ NH2

F

2

N MW, MeOH

R

EtO

3

R: 4a: -CH2C6H5

4f: -CH2C6H4-Br(p)

4k: -CH2C6H3-Cl,Cl(3.4)

4b: -CH2C6H4-Cl(o)

4g: -CH2C6H4-CH3 (p)

4l: -C6H4-SCH3 (p)

4c: -CH2C6H4-Cl(m)

4h: -CH2C6H4-F(o)

4d: -CH2C6H4-Cl(p)

4i: -CH2C6H4-F(p)

4e: -CH2C6H4-Br(o)

4j: -CH2C6H3-Cl,Cl(2.4)

Menteşe E et al. Fluorine-containing Benzimidazoles … Drug Res 2015; 65: 40–45

R N H

F

4 a-l

Fig. 1 The synthetic route for the compounds 4a–l.

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A mixture of 3,3’-diaminobenzidine (5) (0.010 mol) and corresponding iminoester hydrochlorides (0.026 mol) in dry methanol (15 mL) was irradiated in microwave in a closed vessel at 60 ° for 11 min, at 300 W maximum power. After the completion of the reaction, (monitored by TLC, ethylacetate:Hexane, 3:1), the mixture was poured onto H2O. The precipitate formed was filtered and recrystallized from ethanol-water (1:2) to give pure compounds 6a–c.

Original Article 43

NH2

R

H N

H2N

NH2

EtO

R

Fig. 2 The synthesis of compounds 6a–c.

N

NH.HCl

N H

R N

H2N

5

6a-c

R:

NH2

6b: -CH2C6H4-F(m)

NH.HCl

OEt +

F

NH2 2

6c: -CH2C6H4-F(p)

OEt

HCl.NH

F N H

7

2,2'-Bis(4-fluorobenzyl)-1 H,1'H-5,5'-bibenzimidazole (6c): White powder; yield: 95 %, mp: 215–217 °C; 1H NMR (400 MHz, DMSOd6): δ 4.19 (s, 4 H, CH2), 6.57–6.86 (m, 2 H, Ar-H), 7.14–7.18 (m, 4 H, Ar-H), 7.27–7.70 (m, 8 H, Ar-H), 12.35 (s, 2 H, NH); 13C NMR (100 MHz, DMSO-d6): δ 34.5 (CH2), 104.2 (d, JC,F = 25 Hz, Ar-C), 113.5, 115.4 (d, JC,F = 20 Hz, Ar-C), 115.8, 116.4, 121.6, 131.0, 131.1 (d, JC,F = 3 Hz, Ar-C), 134.2 (d, JC,F = 3 Hz, Ar-C), 134.3, 134.5, 134.7, 135.7 (Ar-C), 154.5 (C = N), 161.5 (d, JC,F = 240 Hz, C-F); IR (KBr) cm − 1: 1 617 (C = N), 1 145 (C-F); Anal. Calcd. for C28H20F2N4: C, 74.65; H, 4.47; N, 12.44. Found: C, 74.61; H, 4.41; N, 12.40; LC-MS (ESI): m/z ( %): 451.00 (38) [M + 1], 452.05 (17) [M + 2], 225.95 (100) [symmetric division signal].

General procedure for the synthesis of 2-(4-((6-fluoro1H-benzimidazol-2-yl)methyl)benzyl)-6-fluoro-1Hbenzimidazole 8 A mixture of 4-fluoro-1,2-phenylenediamine (2) (0.020 mol) and compound 7 (0.014 mol) in dry methanol (15 mL) was irradiated in microwave in a closed vessel at 65 °C for 11 min (hold time) at 300 W maximum power. After the completion of the reaction, (monitored by TLC, ethylacetate:Hexane, 3:1), the mixture was poured onto water. The precipitate formed was filtered and recrystallized from ethanol-water (1:3) to give pure compound 8. White powder; yield: 90 %; mp: 285–286 °C; 1H NMR (400 MHz, DMSO-d6): δ 4.13 (s, 4 H, CH2), 6.94–6.99 (m, 2 H, Ar-H), 7.26– 7.43 (m, 6 H, Ar-H), 7.45–7.47 (m, 2 H, Ar-H), 12.35 (brs, 2 H, NH); 13C NMR (100 MHz, DMSO-d6): δ 35.0 (CH2), 109.6 (d, JC,F = 25 Hz, Ar-C), 121.7, 129.4 (2C), 136.1, 136.2, 131.0, 136.4 (Ar-C), 155.5 (C = N), 158.7 (d, JC,F = 233 Hz, C-F); IR (KBr) cm − 1: 1 632 (C = N), 1 137 (C-F); Anal. Calcd. for C22H16F2N4: C, 70.58; H, 4.31; N, 12.96. Found: C, 74.50; H, 4.23; N, 12.89; LC-MS (ESI): m/z ( %): 374.93 (100) [M + ], 376.05 (41) [M + 1].

Anti-lipase activity assay The lipase inhibitory effects of those compounds were evaluated against Porcine Pancreatic Lipase (obtained from Applichem, Germany) (15 ng/mL). Lipase activity assay were done according to Kurihara et al. [22]. The lipase activity was measured using 4-methylumbelliferyl oleate (4-MU oleate) as a substrate. Briefly, compounds were mixed with PPL 1:2 (v/v) and incubated for 30 min. The microtiter plates containing, 50 μL 0.1 mM 4-MU oleate, 25 μL diluted compound-lipase solution, 25 μL dH2O and assay buffer (13 mMTris-HCl, 150 mMNaCl, and 1.3 mM CaCl2, pH

Fig. 3 Synthesis of compound 8.

H N

N N

F

8

Table 1 Inhibitory effects of selected synthesized compounds (at final concentration of 3 μg/mL). Orlistat was used as positive control.

6a 6b 6c 4k Orlistat

% Inhibition

IC50 (μg/mL)

74.85 ± 0.60 84.03 ± 4.15 97.49 ± 1.15 35.58 ± 11.21 92.7 ± 0.05

2.14 ± 0.005 2.01 ± 0.247 1.50 ± 0.015 3.25 ± 0.997 0.21 ± 0.005

8.0) were incubated at 37 °C for 20 min. After incubation, in order to stop the reaction, 0.1 mL 0.1 M citrate buffer was added reaction mixture. The amount of 4-methylumbelliferone released by the lipase was measured by using a spectrofluorometer (SpectraMax M5, Molecular Devices) at an excitation wavelength of 355 nm and an emission wavelength of 460 nm. The inhibitory activity of those compounds and Orlistat (Xenical, Hoffman, La Roche, Segrate, Italy), a positive control against pancreatic lipase were measured at various concentration. Residual activities were calculated by comparing to control without inhibitor. The assays were done in triplicate. The IC50 value was determined as the concentration of compound that give 50 % inhibition of maximal activity.

Results and Discussion



Chemistry Obesity is widely recognized as a major public health problem. Pancreatic lipase inhibitors, such as Orlistat, are used as therapeutic agent for curing obesity [23]. Therefore, the need for the synthesis of new drugs is highly essential so as to eradicate overweight problems. In our previous paper, we reported some benzimidazole derivatives containing chloro and bromo atoms as lipase inhibition agents. In this study, we have synthesized fluorine containing benzimidazole derivatives starting from iminoesterhydrochloride via microwave heating and their pancreatic lipase inhibitory activities were investigated. The results indicated that the fluorine containing bisbenzimidazole derivatives are effective agents for lipase inhibition agents. We firstly synthesized iminoester hydrochlorides (3a–l, 7) according to the Pinner method [24]. In order to synthesize the compound 2 [21], compound 1 reacted with Pd/C catalyst and hydrazine hydrate, under microwave irradiation. Then, iminoesterhydrochlorides (3a–l, 7) were reacted with 4-fluoro-1,2Menteşe E et al. Fluorine-Containing Benzimidazoles … Drug Res 2015; 65: 40–45

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6a: -CH2C6H4-F(o)

44 Original Article

Fig. 4 Dose-dependent inhibitory effect of the synthesized compounds. Orlistat was used as positive control. All compounds and Orlistat were measured at final concentrations of 0.116–9.325 μg/mL. Residual activities of compounds were expressed as the mean ± S.D. in triplicate.

130 120 110

Residual Activity %

100 90 80 70 60 50 40 30 20 10 0 –10 1

2

3

6a

4 5 6 Conc (µg/mL) 6b

6c

7

4k

phenylenediamine (2) to obtain the corresponding benzimidazole ▶ Fig. 1). derivatives (4a–l) (● Synthesis of the compounds 6a–c was performed from the reaction of 3,3’-diaminobenzidine (5) and iminoester hydrochlorides of phenylacetic acids as starting materials. The synthetic path of ▶ Fig. 2. the compounds 6a–c is shown in ● The compound 8 was obtained from the reaction of 1,4-bisiminoester hydrochloride (7) with 4-fluoro-1,2-phenylenediamine ▶ Fig. 3). (2) under microwave irradiation (●

Anti-lipase activity All compounds were evaluated with regard to pancreatic lipase activity and 6a, 6b, 6c and 4k showed anti-lipase activity at various concentrations. No significant inhibitory effect was detected for other compounds. Among the tested compounds, 6b and 6c showed the most significant anti-lipase activity. These compounds inhibited pancreatic lipase activity by 84.03 % and ▶ Table 1). 97.49 % at a concentration of 3 μg/mL, respectively (● Orlistat, known pancreatic lipase inhibitor used as anti-obesity drug, showed inhibitory effect by 99.1 % at the same concentration. Compound 6c and 6b IC50 values were calculated as 1.50 μg/mL and 2.01 μg/mL, respectively. Also compounds 6a and 4k IC50 values were determined as 2.14, and 3.25, respectively. Dose ▶ Fig. dependent inhibitory effects of compounds were depicted ● 4. Orlistat is the only approved anti-obesity medication [23] but it has some side effects, such as fecal incontinence, flatulence, and steatorrhea [21, 25, 26]. Synthesized compounds 6a, 6b and 6c can be alternatives to Orlistat.

Conclusion



We have successfully synthesized some flourine containing benzimidazole derivarives via microwave heating and investigated their pancreatic lipase inhibition activities. Our data demonstrate that the compounds 6a–c may be a suitable or superior alternative to the anti-obesity drug Orlistat. Furthermore, these

Menteşe E et al. Fluorine-containing Benzimidazoles … Drug Res 2015; 65: 40–45

8

9

10

Orlistat

results could be inspirational for further investigation of potential bioactive fluorine containing heterocyclic compounds.

Conflict of Interest



We declared that we have no conflict of interest with respect to this study.

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Menteşe E et al. Fluorine-Containing Benzimidazoles … Drug Res 2015; 65: 40–45

Microwave assisted synthesis and anti-lipase activity of some new fluorine-containing benzimidazoles.

In this study, a new series of fluorine containing benzimidazoles (4a-l) and bisbenzimidazoles (6a-c, 8) were synthesized by the reaction of o-phenyle...
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