Development of Green protocols for the synthesis of 1H-benzo[d]imidazolo aryl amide derivatives and evaluation of their biological activity

Cite this paper: Vietnam J. Chem., 2020, 58(6), 779-784 Article DOI: 10.1002/vjch.202000079 779 Wiley Online Library © 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH Development of Green protocols for the synthesis of 1H-benzo[d]imidazolo aryl amide derivatives and evaluation of their biological activity E. Laxminarayana 1 , B. Srinivasa Reddy 2 , Ch. Venkata Ramana Reddy 3* 1 Sreenidhi Institute of Science and Technology (Autonomous), Ghatkesar, Hyderabad-501 301 Telangana India 2 Mahatma Gandhi Institute of Technology, Kokapet, Gandipet, Hyderabad - 500075 Telangana India 3 Department of Chemistry, Jawaharlal Nehru Technological University Hyderabad Kukatpally, Hyderabad, Telangana, India - 500085 Submitted May 11, 2020; Accepted July 1, 2020 Abstract Benzimidazole scaffold containing chemical derivatives show superior biological activity. In view of this, we synthesized a combination of benzimidazole and aryl amides. The target compounds (6a-j) were synthesized using ethyl 3-iodobenzoate and benzimidazole as starting materials under microwave conditions. The synthesized compounds were screened against gram (+ve) and gram (-ve) bacteria as well as for their anti-oxidant activity. Compound 6a was proved to be most active in inhibiting the DPPH free radical. Keywords. Benzimidazole, aryl amides, anti-bacterial activity, anti-oxidant activity. 1. INTRODUCTION Benzimidazoles exhibits various activities such as antibacterial, [1,2] antioxidant, [3-5] antiviral, [6] antimicrobial, [7-10] anti-HIV, [11] antiulcer, [12] anti- inflammatory, [13] analgesic, [14] antihypertensive, [15] antituberculosis, [16] anticonvulsant, [17] antifungal, [18] anticancer, [19-21] and contains other biological pharmacophores. [22-24] In view of the importance of the benzimidazole nucleus, it was thought that it would be worthwhile to design and synthesize some new benzimidazole derivative that has potential biological importance. Herein, we describe the synthesis of benzimidazole derivatives under green conditions using microwave irradiation and screening against antibacterial and anti-oxidant activities. 2. MATERIALS AND METHODS All chemicals and reagents were of analytical grade. Solvents were used in purified form. For thin‐layer chromatography (TLC) E. Merck AL silica gel 60F254 plates were used and spots were visualized under UV light. IR spectra were recorded on a Perkin Elmer (FT‐IR) spectrometer. Only intense peaks are diagnosed and reported. 1 H NMR and 13 C NMR spectra were recorded using Varian NMR‐400 MHz and 70 MHz instruments respectively. All the chemical shifts were reported in δ (ppm) using TMS as an internal standard. Signals are indicated as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad); and coupling constants in Hz. Mass spectra were recorded with a PESciex model API 3000 mass spectrometer. All the reactions were carried out under the atmosphere of nitrogen gas. Preparation of inoculums By the standard method of inoculation an inoculating loop was touched each of four or five well isolated colonies of the same morphological type and inoculum was inoculated in to 5 mL of nutrient broth. The broth was allowed to incubate at 37 °C for 24 h until a slight visible turbidity appeared. The turbidity of activity growing broth cultures wad then adjusted with broth to obtained a half of MC Farland standard (1108 to 108 cfu/ml) this was used as a starting inoculums for the assay. Antimicrobial assay by well diffusion method The antimicrobial assay was carried out by the well Vietnam Journal of Chemistry Ch. Venkata Ramana Reddy et al. © 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 780 diffusion method. [25] A standardized 1 to 2×10 7 cfu/mL 0.5 MC Farland standards was introduced on to the surface of sterile agar plate and evenly distributed the inoculums by using a sterile glass spreader. Simultaneously, 8-mm wells were cut from the plate using a sterile cork borer. 60 µL of the pigment at 10 mg/ml was introduced in to each well. The agar plates were incubated aerobically at 37 °C. After 24 hrs the inhibition zones were measured with a ruler and compared with the control well containing only DMSO and 10 mg/mL of Gentamycin as standard. Antioxidant Activity The antioxidant activity was performed using DPPH radical scavenging method [26] where BHT was used as a positive control for comparison. LiOH.H2O Ar-NH2,DIPEA, HATU, DMF O 0C- RT, 18 h I OEt O + N H N MeHN NHMe CuI, K3PO4, Toluene Microwawe, 120 0C, 30 min., N N OEt O EtOH:Water (3:1) N N OH O Micowave, 100 0C, 1 h N N N H O 6(a-j) 1 2 3 4 ArNH2 (5a-5j):= NH2 NH2 NH2 N NH2 Ar NO2 OMe Br N N H2N H2N H2N H2N OMe H NH2 a b c d e f g h i j 5(a-j) N N H2N NO2 Scheme 1: Synthesis of Benzimidazole derivatives Synthesis of ethyl 3-(1H-benzo[d]imidazol-1- yl)benzoate (3): To a degassed stirred solution of ethyl 3-iodobenzoate 1 (8.54 mL, 50.78 mmol), in toluene (160 mL) at room temperature was added copper (I) iodide (804 mg, 4.23 mmol), N,N’- dimethylethylenediamine (0.88 mL, 8.46 mmol), benzimidazole 2 (5g, 42.32 mmol) and potassium phosphate (12.6 g, 88.87 mmol) and stirred at 120 0 C for 30 min. in microwave irradiation. Reaction was monitored by TLC, after completion of reaction, Cooled the reaction mixture to room temperature and diluted with EtOAc, filtered the reaction mixture through celite pad and filtrate was concentrated under reduced pressure to get crude product. Obtained crude product was purified by column chromatography to afford ethyl 3-(1H- benzo[d]imidazol-1-yl) benzoate 3 (11 g, 82 %) as brown colour liquid. IR (KBr): υmax 3066 (C-H), 2981 (C-H), 1719 (C=O) cm -1 ; 1 H-NMR (400 MHz, CDCl3): 8.41 (s, 1H), 8.21 (d, J = 8 Hz, 1H), 8.03 (s, 1H), 7.86 (d, J = 8 Hz, 2H), 7.76 (d, J = 7.6, 2H), 7.56 (d, J = 7.2, 2H), 4.35 (q, J = 6.8 Hz, 2H), 1.40 (t, J = 7.2 Hz, 3H). ESI-MS: m/z, 267.17 (M+H). Synthesis of 3-(1H-benzo[d]imidazol-1-yl)benzoic acid (4): To a stirred solution of ethyl 3-(1H- benzo[d]imidazol-1-yl)benzoate 3 (8 g, 29.96 mmol) in a EtOH: water (80 mL; 3:1) at room temperature was added LiOH.H2O (2.5 g, 59.92 mmol) and stirred the reaction at 100 o C for 1 h in microwave irradiation. Reaction was monitored by TLC, after completion of reaction, the reaction mixture was cooled to room temperature and diluted with EtOAc as drop wise and stirred the reaction at room temperature for 18h. After completion of reaction, the reaction mixture was poured in to water (70 mL), extracted with diethyl ether (2×70 mL) to remove impurities and the combined aqueous layer was acidified with concentrated 4N HCl (pH = 2), resulting precipitate was filtered, washed the precipitate with water, pentane and dried product under vacuum to afford 3-(1H-benzo[d]imidazol-1- yl)benzoic acid 4 (6.65 g, 95 %) as off white solid. IR (KBr): υmax 2923 (C-H), 1718 (C=O) cm -1 ; 1 H-NMR (400 MHz, CDCl3): 10.09 (brs, 1H), 8.58 (d, J = 1.6 Hz, 1H), 8.30 (d, 1H), 8.11 (s, 1H), 7.94 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 7.2 Hz, 2H), 7.64 (d, J = 6.8 Hz, 2H); ESI-MS: m/z, 238.9 (M+H). Vietnam Journal of Chemistry Development of Green protocols for the © 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 781 General procedure Synthesis of 3-(1H-benzo[d]imidazol-1-yl)-N-aryl benzamide derivatives 6(a-j): To a stirred solution of 3-(1H-benzo[d]imidazol-1-yl)benzoic acid 4 (5 mg, 2.1 mmol) in Dimethylfarmamide (DMF) (5 mL) N,N-Diisopropylethylamine (DIPEA) (5 mg, 4.2 mmol), 1-[Bis(dimethylamino)methylene]-1H- 1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) (1.59 g, 4.2 mmol) were added and stirred the reaction at room temperature for 18 h after the addition of aryl amine 5(a-j) (2.5 mmol). After completion of reaction, reaction mixture was poured into ice cold water extracted with EtOAc, combined extracts were washed with water, brine solution, dried the extracts over anhy. Na2SO4 and evaporated solvent to afford crude product. Obtained crude product was purified by column chromatography to afford pure compounds 6(a-j) and yields of the products varied between 80-95 %. By adapting this procedure the compounds 6(a-j) were synthesized. 3-(1H-benzo[d]imidazol-1-yl)-N- phenylbenzamide (6a): Yield: 92 %; IR (KBr): υmax 3124 (C-H), 1686 (C=O) cm -1 ; 1 H-NMR (400 MHz, CDCl3): 8.09 (s, 1H), 8.02 (brs, 1H), 7.91 (s, J = 7.1 Hz, 1H), 7.89 (d, J = 8.2 Hz, 1H), 7.75 (d, J = 6.2 Hz, 2H), 7.65 (d, J = 6.4 Hz, 2H), 7.52 (d, J = 6.2 Hz, 1H), 7.41(t,1H), 7.26-7.18 (m, 2H), 7.24 (d, 2H), 7.07 (t, 1H); ESI-MS: m/z, 314 (M+H). 3-(1H-benzo[d]imidazol-1-yl)-N-(4- nitrophenyl)benzamide (6b): Yield: 85 %; 1 H-NMR (400 MHz, CDCl3): 8.46 (brs, 1H), 8.12 (s, 1H), 8.2 (d, 2H), 7.92 (s, J = 7.2 Hz, 1H), 7.95 (d, J = 8.2 Hz, 2H), 7.81 (d, J = 6.2 Hz,1H), 7.64 (d, J = 6.4 Hz, 2H), 7.56 (d, J = 6.2 Hz, 1H), 7.40 (t, 1H), 7.28-7.14 (m, 2H). ESI-MS: m/z, 359 (M+H). 3-(1H-benzo[d]imidazol-1-yl)-N-(4- methoxyphenyl)benzamide (6C): Yield: 84 %; 1 H-NMR (400 MHz, CDCl3): 8.44 (brs, 1H), 8.11 (s, 1H), 8.02 (s, 1H), 7.95 (d, J = 8.0 Hz,1H), 7.74 (d, J = 6.0 Hz, 2H), 7.62 (d, J = 6.2 Hz, 1H), 7.54 (d, J = 6.4 Hz, 2H), 7.41 (t, 1H), 7.25 (m, 2H), 6.82 (d, 2H), 3.62 (s, 3H); ESI-MS: m/z, 344 (M+H). 3-(1H-benzo[d]imidazol-1-yl)-N-(4- bromophenyl)benzamide (6d): Yield: 78 %; 1 H-NMR (400 MHz, CDCl3): 8.3 (brs, 1H), 8.1 (s, 1H), 8.02 (s, 1H), 7.92 (d, J = 8.2 Hz,1H), 7.85 (d, J = 6.1 Hz, 2H), 7.63 (d, J = 6.0 Hz,2H), 7.55 (d, J = 6.0 Hz, 2H), 7.51 (d, J = 6.4 Hz, 1H), 7.42 (t, 1H), 7.24-7.12 (m, 2H) ESI-MS: m/z, 392 (M+H), 393 (M+2H), 3-(1H-benzo[d]imidazol-1-yl)-N-(pyridin-3- yl)benzamide (6e): Yield: 86 %; 1 H-NMR (400 MHz, CDCl3): 8.63 (s, 1H), 8.31 (d, 1H), 8.05 (s, 1H), 8.02 (brs, 1H), 7.94 (s, 1H), 7.85 (d, J = 8.0 Hz,1H), 7.71 (d, J = 6.4 Hz,2H), 7.56 (d, J = 6.2 Hz,1H), 7.46 (t, 1H), 7.4 (t, 1H), 7.23-7.18 (m, 3H) ESI-MS: m/z, 315 (M+H). 3-(1H-benzo[d]imidazol-1-yl)-N-(4- nitrobenzyl)benzamide (6f): Yield: 88 %; 1 H-NMR (400 MHz, CDCl3): 8.46 (brs, 1H), 8.12 (s, 1H), 8.03 (d, J = 8 Hz, 2H), 7.90 (s, 1H), 7.95 (d, J = 7.2 Hz, 1H), 7.64 (d, J = 8.0 Hz, 2H), 7.56 (d, J = 6.0 Hz, 1H), 7.40 (t, 6.4, 1H), 7.26- 7.14 (m, 2H), 7.06 (d, J = 6.4 Hz, 2H), 4.40 (s, 2H); 13 C-NMR (70 MHz, CDCl3): 166.2, 148.1, 141.6, 136.2, 137.2, 133.1, 132.6, 130.2, 128.1, 126.3, 123.4, 121.1, 119.8, 119.2, 118.1, 115.1, 110.3, 51.1; ESI-MS: m/z, 373 (M+H) 3-(1H-benzo[d]imidazol-1-yl)-N-((pyridin-4- yl)methyl)benzamide (6g): Yield: 84 %; 1 H-NMR (400 MHz, CDCl3): 8.2 (s, 1H), 8.10 (d, 2H), 8.00 (s, 1H), 8.02 (brs, 1H), 7.94 (d, J = 7.4 Hz, 1H), 7.71 (d, J = 8.0 Hz,2H), 7.56 (d, J = 6.2 Hz, 1H), 7.45 (t, 1H), 7.41 (d, 2H), 7.25-7.13 (m, 2H), 4.76 (s, 2H); ESI-MS: m/z, 329 (M+H). N-(4-methoxybenzyl)-3-(1H-benzo[d]imidazol-1- yl)benzamide (6h): Yield: 87 %; 1 H-NMR (400 MHz, CDCl3): 8.46 (brs, 1H), 8.12 (s, 1H), 8.03 (d, 1H), 7.90 (s, 1H), 7.64 (d, J = 6.4 Hz, 1H), 7.56 (d, J = 6.4 Hz,2H), 7.31 (t, 1H), 7.24-7.16 (m, 2H), 6.91 (d, J = 7.4 Hz 2H), 6.66 (d, J = 8.0 Hz 2H), 4.56 (s, 2H), 3.62 (s, 3H); ESI-MS: m/z, 358 (M+H). 3-(1H-benzo[d]imidazol-1-yl)-N-((pyridin-2- yl)methyl)benzamide (6i): Yield: 91 %; 1 H-NMR (400 MHz, CDCl3): 8.63 (d, 1H), 8.36 (brs, 1H), 8.11 (s, 1H), 8.05 (s, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.89 (t, 1H), 7.72 (d, J = 6.0 Hz, 2H), 7.56 (d, J = 6.4 Hz, 1H), 7.54 (d, J = 6.0 Hz, 1H), 7.4 (t, 1H), 7.3 (t, 1H), 7.26 (m, 2H), 4.74 (s, 2H); ESI-MS: m/z, 329 (M+H). 3-(1H-benzo[d]imidazol-1-yl)-N-((pyrimidin-6- yl)methyl)benzamide (6 j): Yield: 93 %; 1 H-NMR (400 MHz, CDCl3): 9.09 (s, 1H), 8.59 (d, 1H), 8.08 (s, 1H), 8.01 (brs, 1H), 7.5 (s, 1H), 7.90 (d, J = 8.2 Hz,1H), 7.70 (d, J = 6.0 Hz, Vietnam Journal of Chemistry Ch. Venkata Ramana Reddy et al. © 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 782 2H), 7.52 (d, J = 6.4 Hz, 1H), 7.41 (t, 1H), 7.29 (d, 1H), 7.21 (m, 2H), 4.49 (s, 2H); ESI-MS: m/z, 330 (M+H). 3. RESULTS AND DISCUSSION 3.1. Chemistry The target compounds 6(a-j) were prepared as outlined in Schemes 1. The compound ethyl 3- iodobenzoate 1 was reacted with benzimidazole 2 in presence of N,N’-dimethylethylenediamine, cupper(1) iodide and potassium phosphate in toluene at 120 o C for 30 min. in microwave irradiation to afford ethyl 3-(1H-benzo[d]imidazol-1-yl)benzoate 3 as 82% yield. The resulting product was treated with LiOH.H2O at 100 o C for 1 h in microwave irradiation to afford pure 3-(1H-benzo[d]imidazol-1- yl)benzoic acid 4 in 95 % yield as shown in the Scheme 1. The acid intermediate 4 was coupled with different substituted amines (5a-j) in DMF, DIPEA and HATU at room temperature for 18 h to afford pure amide compounds 6(a-j) in excellent yield. The 1 H NMR spectrum of compound 6f showed a broad singlet peak at δ 8.46 due to –NH- group of secondary amine, singlet at 7.90 and doublets at 8.03, 7.95, 7.64, and 7.56 corresponding to aromatic protons. In the 13 C NMR spectrum of compound 6f peak at δ 166.2 indicated the presence of carbonyl carbon (C=O). Further support was also obtained from the mass spectrum which showed peak at m/z = 373 corresponding to (M+H) of the compound 6f. 3.2. Antibacterial activity The result of each compounds are shown in table 1 and figure 1. Control inhibition zone (which indicates inhibition zone of solvent) was subtracted from inhibition zone of compounds which gives actual inhibition zone of compounds. Perusal of the Table 1 reveals that the derivatives having pyridine and pyrimidene derivatives 6e, 6i and 6j are more toxic towards B. cereus (Gram positive) and P. vulgaris (Gram negative) bacteria. This indicates the presence of pyridine and pyrimidine increases the toxic nature. Compounds 6a, 6b, 6c, 6d and 6f are less toxic towards both Gram positive and Gram negative bacteria. 6g and 6h have shown moderate toxic nature, when compared to slandered drug. On the other hand, 6j (16 mm) displayed the highest activity against P. vulgaris. 3.3. Antioxidant activity The results of antioxidant activity of the compounds 6a-j are shown in table 2 and figure 2. The activity was assessed by measuring its electron donating ability to DPPH which was indicated by changes in absorbance of the solution at 517 nm. The result of the radical scavenging was expressed in terms of half-inhibition concentration (IC50) which denotes the concentration required to scavenge 50 % of DPPH radicals. Series 6a-j is found to be more potent as depicted in table 2. A compound 6d was found to be most potent with IC50 value 18.52 μg/mL due to presence of –Br group as electron releasing group. The study reveals that electron with drawing groups increase the antioxidant potential which may be due to the intensification of positive charge on -NH of amide associated with negative inductive effect of these groups. The positive charge intensification may lead to free radical quenching. Moreover, electron withdrawing groups are themselves good free radical quenchers. On the contrary, substitution with electron releasing groups was found to decrease the radical scavenging potential possibly due to their positive inductive effect. Table 1: Antibacterial activities of compounds 6a-j 6a 6b 6c 6d 6e 6f 6g 6h 6i 6j Gentamycin Zone of inhibition in mm Gram- positive B. subtitis 4 4 5 4 8 9 2 7 8 9 19 B. cereus 6 2 3 2 12 5 7 8 9 10 14 S. aureus 3 4 8 5 7 6 5 7 11 12 16 Gram- negative E. coli 7 2 7 5 8 8 9 4 11 11 13 P. vulgaris 5 1 4 3 12 8 6 5 10 16 20 K. pneumonia 3 4 6 4 9 6 5 7 10 12 15 Vietnam Journal of Chemistry Development of Green protocols for the © 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 783 Figure 1: Antibacterial activity of benzimidazoles 6a-j Table 2: Antioxidant activity of benzimidazoles 6a-j Compound IC50 (µg/mL) 6a 4.03 6b 8.21 6c 11.23 6d 18.52 6e 6.21 6f 12.45 6g 11.65 6h 9.25 6i 6.26 6j 4.52 % Inhibition of DPPH free radical at different concentrations Figure 2: Antioxidant activity of benzimidazoles 6a-j 4. CONCLUSIONS In conclusion, we have successfully achieved in the synthesis of a series of title compounds via a multi- step synthetic strategy and all the compounds were screened for anti bacterial activity results showing that compound 6e, 6i and 6j showed highest inhibition zone. The antioxidant studies showing that 6g has highest IC50 values. These results suggested that the synthesized molecules are potent anti- bacterial agents. Acknowledgements. The authors are thankful to TEQIP-III, JNTUH (JNTUH/TEQIP- III/CRS/2019/Chemistry/01) for providing financial assistance to carry out this work. The authors also would like to thank to Vietnam Academy of Science and Technology (VAST) for supporting this research. REFERENCES 1. R. Srivastava, S. K. Gupta, F. Naaz, A. Singh, V. K. Singh, R. Verma, N. Singh, R. K. Singh. Synthesis, antibacterial activity, synergistic effect, cytotoxicity, docking and molecular dynamics of benzimidazole analogues, Comput. Biol. Chem., 2018, 76, 1-16. 2. K. 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