Synthesis and biological evaluation of triphenyl-imidazoles as a new class of antimicrobial agents European Journal of Chemistry 9 (4) (2018) 369-374 European Journal of Chemistry View Journal Online View Article Online Synthesis and biological evaluation of triphenyl-imidazoles as a new class of antimicrobial agents Anupam 1, Mohammed Al-Bratty 2, Hassan Ahmad Alhazmi 2,3, Shamim Ahmad 4, Supriya Maity 1, Md Shamsher Alam 2,* and Waquar Ahsan 2 1 Department of Pharmaceutical Chemistry, Translam Institute of Pharmaceutical Education & Research, Dr. A.P.J. Abdul Kalam Technical University, Mawana Road, Meerut 250001, Uttar Pradesh, India anuchaudhary321@gmail.com (A.), supriyaphd@gmail.com (S.M) 2 Department of Pharmaceutical Chemistry, College of Pharmacy, Jazan University, Jazan 45142, Kingdom of Saudi Arabia malbratty@jazanu.edu.sa (M.A.), haalhazmi@jazanu.edu.sa (H.A.A.), mosalam@jazanu.edu.sa (M.S.A), wmohammad@jazanu.edu.sa (W.A) 3 Substance Abuse Research Centre, Jazan University, Jazan, 45142, Kingdom of Saudi Arabia 4 Department of Pharmacognosy, Translam Institute of Pharmaceutical Education and Research, Dr. A.P.J. Abdul Kalam Technical University, Mawana Road, Meerut 250001, Uttar Pradesh, India shamim2009in@gmail.com (S.A) * Corresponding author at: Department of Pharmaceutical Chemistry, College of Pharmacy, Jazan University, Jazan 45142, Kingdom of Saudi Arabia Tel: +966.017.3342662 Fax: +966.017.3342662 e-mail: mosalam@jazanu.edu.sa (M. S. Alam). 10.5155/eurjchem.9.4.369-374.1785 Received: 01 September 2018 Received in revised form: 13 October 2018 Accepted: 15 October 2018 Published online: 31 December 2018 Printed: 31 December 2018 Newer triphenyl-imidazole derivatives (4a-h) were synthesized in good yields by the reaction of benzil and substituted benzaldehydes in equimolar quantities and refluxing the product with acetyl chloride thereafter. Structures were confirmed by using FT-IR, 1H NMR and 13C NMR spectroscopic methods. All the synthesized compounds were tested for their antimicrobial activity using agar diffusion technique against Gram positive (Staphhylococcus aureus and Bacillus subtilis), Gram negative (Escherichia coli and Pseudomonas aureginosa) as well as Fungal strain (Candida albicans). Interestingly compounds 4a, 4b, 4f and 4h showed significant antibacterial activity, whereas compound 4b was found to have remarkable activity against the fungal strain. The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of most active compounds were determined by broth dilution method and compound 4b emerged to have potent activities against most of the strains having MIC in the range of 25-200 µg/mL. To check the possible toxicities of the most active compounds, they were orally administered in rats and the concentration of liver enzymes serum glutamic-oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT) and alkaline phosphatase (ALKP) were determined. Compound 4h showed significant increase in the enzymes level depicting the hepatotoxicity. The structure- activity relationship studies showed the importance of electron withdrawing groups at the distant phenyl ring at ortho and para positions as the compounds having chloro or nitro at these positions tend to be more active than the compounds with electron releasing groups such as methoxy. These compounds may act as lead compounds for further studies and appropriate modification in their structure may lead to agents having high efficacy with lesser toxicity. Synthesis Imidazole Hepatic toxicity Antifungal activity Enzyme estimation Antimicrobial activity Cite this: Eur. J. Chem. 2018, 9(4), 369-374 Journal website: www.eurjchem.com 1. Introduction In the current world, health system is very much affected by serious systemic bacterial and fungal infections. It is reported that fungal infections are exaggerated very much due to excessive use of broad spectrum antibiotics, anticancer drugs and immunosuppressive agents [1]. Most of the existing medicines to combat these deadly microbes are getting microbial resistance and became an important concern in antimicrobial therapy [2]. Although, many compounds have been synthesized to control these infections but their clinical use are restricted due to high toxicities and microbial resistance [3]. In clinical medicine, imidazole derivatives possess exten- sive medicinal applications and this stimulated the researchers to develop a big number of newer therapeutic agents. There is evidence confirming that numerous pharmacologically active derivatives hold nitrogen containing five membered rings [4]. In the last few decades, imidazole rings having active hydrogen atom have been emerged as potential pharmacophore posses- sing numerous biological activities [5] and proved to be remarkable significance in the field of medical science. Azole antifungal drugs with imidazole ring have been found to destroy the lipid bilayer membranes of fungi by preventing the accumulation of methylated sterols. Cell wall of fungus is mainly composed of erogesterol, which plays crucial role in membrane permeability, enzyme activity as well as cell cycle functioning [6]. At high concentrations, imidazole drugs directly exert inhibitory effects on the membranes without intervention with the sterols [7,8]. ABSTRACT RESEARCH ARTICLE KEYWORDS European Journal of Chemistry ISSN 2153-2249 (Print) / ISSN 2153-2257 (Online) – Copyright © 2018 The Authors – Atlanta Publishing House LLC – Printed in the USA. This work is published and licensed by Atlanta Publishing House LLC – CC BY NC – Some Rights Reserved. http://dx.doi.org/10.5155/eurjchem.9.4.369-374.1785 http://dx.doi.org/10.5155/eurjchem.9.4.369-374.1785 https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.9.4.369-374.1785&domain=pdf&date_stamp=2018-12-31 http://www.eurjchem.com/ http://dx.doi.org/10.5155/eurjchem.9.4.369-374.1785 mailto:anuchaudhary321@gmail.com mailto:supriyaphd@gmail.com mailto:malbratty@jazanu.edu.sa mailto:haalhazmi@jazanu.edu.sa mailto:mosalam@jazanu.edu.sa mailto:wmohammad@jazanu.edu.sa mailto:shamim2009in@gmail.com mailto:mosalam@jazanu.edu.sa http://www.eurjchem.com/ https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.9.4.369-374.1785&domain=pdf&date_stamp=2018-12-31� 370 Anupam et al. / European Journal of Chemistry 9 (4) (2018) 369-374 N N Cl Clotrimazole H N N H N Cl Cl Clonidine H N N NO2 Azomycin O N N Cl Cl Cl Cl Miconazole N N O O O Cl Cl N N O Ketoconazole Figure 1. Structures of marketed drugs having imidazole ring. In last few years, a great attention has been given to imidazole heterocyclics due to significant pharmacological activities such as antitubercular, antifungal, antibacterial, anti- inflammatory and antitumor [9]. Numerous well reported drugs in the market with remarkable therapeutic activities like clotrimazole, clonidine, azomycine, miconazole, moxonidine, ketoconazole, cimetidine and etomidate have potential imidazole ring (Figure 1) [10,11]. These numerous advantages of imidazole ring motivated us to discover novel derivatives of imidazole and study against well-known Gram (+ve) and Gram (-ve) bacteria as well as fungus C. albicans. In our present work, newer imidazole derivatives have been designed and screened for antibacterial activity against Gram negative bacteria (E. coli and P. aureginosa), Gram posi- tive bacteria (S. aureus and B. subtilis) and fungus C. albicans using reference drugs ampicillin and griseofulvin. Further- more, toxicity evaluation by liver enzyme estimation was performed for the selected most potent compounds to evaluate any hepatotoxicity caused by them. Promising results of the present study would form the basis of further preclinical and clinical investigation to develop newer imidazole deriva- tives as potential antimicrobial agents. 2. Experimental 2.1. Instrumentations and materials The melting points of the synthesized derivatives were estimated by the open capillary method and were uncorrected. FT-IR Spectra (KBr) was taken on Jasco FT/IR 410 spectro- meter. NMR spectra of triphenyl-imidazoles were recorded on Bruker 400 Ultra shield NMR spectrometer operating at 400 MHz to record 1H NMR and 100 MHz for 13C NMR in CDCl3 solvent with tetramethylsilane (TMS) as internal standard [12,13]. Log P was determined by octanol: phosphate buffer method. Retention factor (Rf ) was calculated through thin layer Chromatography (TLC) using solvent system of benzene: acetone (60:40, v:v). Elemental analyses were performed on a Perkin-Elmer model 240c analyzer (Perkin Elmer, USA). 2.2. Synthesis of 2-(3-substituted phenyl)- 4,5-diphenyl -1H- imidazoles (3a-h) Benzil (1 mol) (1), ammonia solution (5 mL) and substi- tuted benzaldehdyes (1 mol) (2) were mixed with 50 mL glacial acetic acid in a 100 mL round bottom flask (RBF) and refluxed for 2-3 h on heating mantle. Completion of the reaction was monitored by using Thin Layer Chromatography (TLC). After completion of reaction, the reaction mixture was poured onto 300 mL cold water and neutralized with 5% ammonium hydroxide solution. The mixture was then kept in fridge overnight. The precipitated product was filtered and recrystallized with absolute ethanol to obtain colorless or pale yellow crystalline solid (Scheme 1) [14-16]. 2.3. Synthesis of 1-(2-(2-substituted phenyl)-4, 5-diphenyl - 1H-imidazol-1-yl) ethanones (4a-h) 0.5 g of substituted imidazole derivatives (3a-h) and freshly prepared 2.5 mL acid chloride solution were taken in round bottom flask with benzene (30 mL) as a solvent, pyridine as a catalyst and was refluxed for 4-5 h. TLC with solvent system of benzene: acetone (60:40, v:v) was utilized to check the reaction status. Mixture was cooled at room temperature, poured into ice and kept overnight in refrige- rator. The compounds were filtered and recrystallized by ethanol (Scheme 1). 1-(2-(2-Chlorophenyl)-4, 5-diphenyl-1H-imidazol-1-yl)etha- none (4a): Color: Colorless. Yield: 48 %. M.p.: 248-250 °C. FT- IR (KBr, ν, cm-1): 3362 (Ar-CH), 1635 (C=O), 1568 (C=C), 1438 (C=N), 758 (C-Cl). 1H NMR (400 MHz, CDCl3, δ, ppm): 2.20 (s, 3H, CH3), 7.22-7.37 (m, 10H, CH, benzil), 7.38-7.48 (m, 4H, CH, chlorobenzene). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 25 (CH3), 122 (C=C, Imidazole), 167.8 (C=O), 127.4, 127.5, 128.8, 128.9, 129.3, 129.4, 130.2, 132.3, 133.3, 138.5 (C, Ar), 143.5 (C=N, Imidazole). Rf value: 0.71 (Benzene: acetone, 60:40). Log P: 5.72. 1-(2-(4-Chlorophenyl)-4, 5-diphenyl-1H-imidazol-1-yl) etha- none (4b): Color: Colorless. Yield: 45 %. M.p.: 246-248 °C. FT- IR (KBr, ν, cm-1): 3300 (Ar- CH), 1630 (C=O), 1530 (C=C), 1420 (C=N), 740 (C-Cl). 1H NMR (400 MHz, CDCl3, δ, ppm): 2.21 (s, 3H, CH3), 7.22-7.40 (m, 10H, CH, benzil), 7.41-7.48 (m, 4H, CH, chlorobenzene). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 24.5 (CH3), 122.4 (C=C, Imidazole), 166.6 (C=O), 128.3, 127.3, 128.5, 128.7, 129.4, 129.2, 130.6, 132.5, 133.7, 138.2 (C, Ar), 144.5 (C=N, Imidazole). Rf value: 0.77 (Benzene: acetone, 60:40). Log P :5.70. 1-(2-(2-Methoxyphenyl)-4, 5-diphenyl-1H-imidazol-1-yl) et- hanone (4c): Color: Colorless. Yield: 35 %. M.p.: 165-167 °C. FT-IR (KBr, ν, cm-1): 3100 (Ar- CH), 1540 (C=O), 1510 (C=C), 1410 (C=N), 1022(C-O-C). 1H NMR (400 MHz, CDCl3, δ, ppm): 2.26 (s, 3H, CH3), 3.73 (s, 3H, OCH3), 6.83- 7.37 (m, 4H, CH, methoxybenzene), 7.38-7.48 (m, 10H, CH, benzil). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 25.1 (CH3), 56.2 (OCH3), 121.5 (C=C, Imidazole), 167.2 (C=O), 114.3, 127.2, 127.4, 128.5, 128.7, 129.6, 129.7, 130.3, 132.4, 133.2, 138.3 (C, Ar), 143.2 (C=N, Imidazole). Rf value: 0.83 (Benzene: acetone, 60:40). Log P: 5.04. 2018 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.9.4.369-374.1785 Anupam et al. / European Journal of Chemistry 9 (4) (2018) 369-374 371 O O + HO N H N CH3COOH + SOCl2 CH3COCl + SO2 + HCl N N H3C O Reflux for 4-5 hrs Reflux for 2-3 hours Reflux for 2-3 hrs Ammonia solution and acetic acid 80°C (3a-h) R= 2-Cl; 4-Cl; 2-OCH3; 3-OCH3; 4-OCH3, 2-NO2; 3-NO2; 4-NO2 R R R (4a-h) (1) (2) N H N (3a-h) R Scheme 1. Synthetic route to the titled compounds 4a-h. 1-(2-(3-Methoxyphenyl)-4, 5-diphenyl-1H-imidazol-1-yl) et- hanone (4d): Color: Colorless. Yield: 34 %. M.p.: 166-168 °C. FT-IR (KBr, ν, cm-1): 3450 (Ar-CH), 1650 (C=O), 1530 (C=C), 1360 (C=N), 1023 (C-O-C). 1H NMR (400 MHz, CDCl3, δ, ppm): 2.14 (s, 3H, CH3), 3.43 (s, 3H, OCH3), 6.46-7.11 (m, 4H, CH, methoxybenzene), 7.43-7.79 (m, 10H, CH, benzil). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 25.4 (CH3), 55.8 (OCH3), 122.4 (C=C, Imidazole), 167.2 (C=O), 114.3, 127.2, 127.4, 128.6, 128.7, 129.4, 129.5, 130.5, 132.6, 133.7, 138.3 (C, Ar), 143.4 (C=N, Imidazole). Rf value: 0.80 (Benzene: acetone, 60:40). Log P: 5.02. 1-(2-(4-Methoxyphenyl)-4, 5-diphenyl-1H-imidazol-1-yl) et- hanone (4e): Color: Colorless. Yield: 32 %. M.p.: 167-169 °C. FT-IR (KBr, ν, cm-1): 3230 (Ar- CH), 1610 (C=O), 1570 (C=C), 1390 (C=N), 1043 (C-O-C). 1H NMR (400 MHz, CDCl3, δ, ppm): 2.28 (s, 3H, CH3), 3.52 (s, 3H, OCH3), 6.56- 7.10 (m, 4H, CH, methoxybenzene), 7.15-7.46 (m, 10H, CH, benzil). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 24.6 (CH3), 55.2 (OCH3), 122.3 (C=C, Imidazole), 167.1 (C=O), 114.2, 127.1, 127.4, 128.4, 128.8, 129.4, 129.5, 130.6, 132.2, 133.5, 138.6 (C, Ar), 142.5 (C=N, Imidazole). Rf value: 0.79 (Benzene: acetone, 60:40). Log P: 5.0. 1-(2-(2-Nitrophenyl)-4, 5-diphenyl-1H-imidazol-1-yl) etha- none (4f): Color: Pale yellow. Yield: 71 %. M.p.: 168-170 °C. FT- IR (KBr, ν, cm-1): 3120 (Ar- CH), 1670 (C=O), 1540 (C=C), 1480(-NO2), 1260 (C=N). 1H NMR (400 MHz, CDCl3, δ, ppm): 2.20 (s, 3H, CH3), 7.22-7.48 (m, 10H, CH, benzil), 7.71- 8.25 (m, 4H, CH, nitrobenzene). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 24.7 (CH3), 55.2 (OCH3), 122.3 (C=C, Imidazole), 167.3 (C=O), 114.4, 127.2, 127.5, 128.5, 128.7, 129.3, 129.6, 130.5, 132.3, 133.4, 138.3 (C, Ar), 142.3 (C=N, Imidazole). Rf value: 0.68 (Benzene: acetone, 60:40). Log P: 4.98. 1-(2-(3-Nitrophenyl)-4, 5-diphenyl-1H-imidazol-1-yl) etha- none (4g): Color: Pale yellow. Yield: 70.58 %. M.p.: 166-168 °C. FT-IR (KBr, ν, cm-1): 3110 (Ar- CH), 1650 (C=O), 1510 (C=C), 1450(-NO2), 1250 (C=N). 1H NMR (400 MHz, CDCl3, δ, ppm): 2.10 (s, 3H, CH3), 7.10-7.88 (m, 10H, CH, benzil), 7.91- 8.25 (m, 4H, CH, nitrobenzene). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 24.6 (CH3), 55.8 (OCH3), 121.3 (C=C, Imidazole), 168.3 (C=O), 113.4, 127.4, 127.6, 128.1, 128.6, 129.4, 129.7, 130.2, 132.7, 133.3, 138.5 (C, Ar), 141.3 (C=N, Imidazole). Rf value: 0.68 (Benzene: acetone, 60:40). Log P: 4.96. 1-(2-(4-Nitrophenyl)-4, 5-diphenyl-1H-imidazol-1-yl) etha- none (4h): Color: Pale yellow. Yield: 72 %. M.p.: 168-170 °C. FT-IR (KBr, ν, cm-1): 3510 (Ar- CH), 1610 (C=O), 1560 (C=C), 1410(-NO2), 1230 (C=N). 1H NMR (400 MHz, CDCl3, δ, ppm): 2.60 (s, 3H, CH3), 7.11-7.56 (m, 10H, CH, benzil), 7.60- 8.15 (m, 4H, CH, nitrobenzene). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 24.3 (CH3), 55.7 (OCH3), 121.1 (C=C, Imidazole), 167.3 (C=O), 112.4, 126.4, 127.3, 128.3, 128.5, 129.6, 129.8, 130.6, 132.4, 133.6, 138.4 (C, Ar), 140.3 (C=N, Imidazole). Rf value: 0.65 (Benzene: acetone, 60:40). Log P: 4.90. 2.4. Antimicrobial activity 2.4.1. Determination of zone of inhibition All the newly synthesized imidazole derivatives (4a-h) were evaluated for antibacterial and antifungal activities on different strains of microbes (Table 1 and 2). Different cultures of bacteria such as P. aeruginosa (NCIM 2242), B. subtilis (NCIM 2708), S. aureus (NCIM 2079), E. coli (NCIM 2685) and one fungal strain of Candida albicans were brought from Pune, India. These microorganisms were conserved by sub-culturing in nutrient agar medium. Antimicrobial assay of these compounds were studied through agar diffusion technique by determining zone of inhibition [17-19]. Prior to activity, petri dishes were sterilized at 160 °C for 1.5 h in hot air oven. 1% of inoculums were introduced in sterilized agar media. Three bores of 6 mm diameter was made in agar nutrient medium and 1×10 CFU/mL concentration of bacterial culture was used. For antimicrobial studies, 500 and 600 µg/mL concentrations of synthesized compounds were used and zone of inhibition was calculated and compared with the results obtained by 500 µg/mL solution of standard drugs ampicillin and griseofulvin in DMF. 2.4.2. Determination of minimum inhibitory concentration and minimum bactericidal/fungicidal concentration MIC is the minimum concentration that inhibits the significant growth of the microorganisms. DMSO was used as diluent to prepare a concentration of 20 mg/mL of the synthesized compounds. 2018 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.9.4.369-374.1785 372 Anupam et al. / European Journal of Chemistry 9 (4) (2018) 369-374 Table 1. Antimicrobial activity of synthesized compounds 4a-h. Compound Concentration (µg/mL) Zone of inhibition (mm), (Mean±SEM) a Gram negative bacteria Gram positive bacteria Fungus E. coli (NCIM 2685) P. aureginosa (NCIM 2242) S. aureus (NCIM 2079) B. subtilis (NCIM 2708) C. albicans (ATCC 60193) 4a 500 18.3±0.27** 17.2±0.31** 17.8±0.27** 16.0±0.27*** 13.7±0.13 600 19.1±0.16** 18.5±0.431** 20.3±0.21*** 17.0±0.19*** 14.9±0.13** 4b 500 18.3±0.27** 17±0.31** 20.3±0.21*** 16.0±0.27*** 14.3±0 .13** 600 19±0.16*** 17.9±0.32** 21.3±0.31*** 17.0±0.19*** 15.7±0.21*** 4c 500 13.4±0.28 15.6±0.21 13.5±0.31 14.4±0.33 13.2±0.31 600 14.8±0.23 16.3±0.07** 14.7±0.19 14.8±0.13 13.7±0.21 4d 500 13.2±0.25 15.1±0.22 13.7±0.37 14.5±0.36 13.1±0.32 600 14.6±0.22 16.1±0.02** 14.8±0.18 14.7±0.14 13.8±0.22 4e 500 17.0±0.31** 15.6±0.21 13.9±0.31 14.7±0.33 14.7±0.21** 600 17.9±0.32** 15.9±0.07 14.3±0.19 15.3±0.13** 15.1±0.01** 4f 500 19.1±0.21** 15.6± 0.31 15.9±0.54** 14.2±0.33 13.6±0.22 600 19.9 ±0.27*** 15.67± 0.22 16.8±0.41** 14.3±0.32 14.1±0.32 4g 500 14.3±0.21 15.8± 0.25 15.1±0.71** 14.3±0.46 13.5±0.31 600 15.0±0.11** 15.9±0.13 16.5±0.32** 14.4±0.36 14.5±0.22 4h 500 17.7±0.21** 15.5±0.45 18.3±0.32** 14.2± 0.26 13.6±0.35 600 18.9 ±0.27*** 16.5±0.45 19.9±0.21*** 14.4±0.16 14.1±0.52 Ampicillin 500 20.0±.27*** 19.3±0.26*** 23.0±0.21*** 17.1±0.19*** - b Griseofulvin 500 NT c NT NT NT 16±0.27*** DMSO - - - - - - a Data are presented with mean±SEM using one way ANOVA. Extremely significant and significant values are represented as ***p < 0.001 and **p < 0.01 respectively. b No significant inhibitory activity (< 5 mm). c Not Tested. Table 2. Minimum inhibitory concentration and minimum bactericidal concentration of selected compounds. Organism Compound MIC (µg/mL) MBC (µg/mL) E. coli NCIM 2685 4b 25 50 4f 50 50 4h 25 50 Ampicillin 6.25 12.5 P. aureginosa NCIM 2242 4b 100 200 4f 100 200 4h 300 400 Ampicillin > 500 > 500 S. aureus NCIM 2079 4b 50 50 4f 100 200 4h 100 200 Ampicillin 6.25 12.5 B. subtilis NCIM 2708 4b 200 300 4f 300 300 4h 300 400 Ampicillin 6.25 6.25 C. albicans ATCC 60193 4b 100 300 4f 200 300 4h 200 300 Griseofulvin 6.25 6.25 MFC a a MIC: Minimum inhibitory concentration, MFC: Minimum fungicidal concentration. On the basis of the antimicrobial activity shown in agar diffusion technique, three most active compounds were selected for MIC determination. Broth dilution method was employed in this study using 96 wells micro-titre plate [20- 22]. Dilutions were made to obtain different concentrations of 600, 500, 400, 200, 100, 50, 25, 12.5, and 6.25 µg/mL. Culture for bacterial and fungal strains were prepared by adding equal volumes and incubating at 37 °C for 24 h and 30 °C for 48 h, respectively. Bacterial growth was indicated by appearance of turbidity and ELISA reader at 590 nm was used to measure the absorbance. The well with zero absorbance was measured to be the MIC. DMSO (1-16%) was used as control. All examina- tions were implemented in triplicates. Minimum Bactericidal concentration (MBC) was examined by sub culturing the solutions, which did not show any indication of growth, in neat as well as in dilutions of 1:10 and 1:100. MBC was indicated by 99.9% decline in the growth of original inoculum. 2.5. Toxicity evaluation/liver enzyme estimation Experimental design for biochemical estimation of most potent synthesized compounds on albino rat of either sex (150g-200 g) had been approved by Institutional animal ethical committee (IAEC) of Translam Institute of Pharma- ceutical Education & Research, Meerut, Uttar Pradesh, INDIA (Registration number 1207/PO/c/2008/CPCSEA). To assess the liver toxicity, biochemical parameters such as serum glutamic-oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT) and alkaline phosp- hatase (ALKP) enzyme levels were measured from the serum of treated rats using Roche assay kits [23-26]. The experiment was conducted as per NIH guidelines. These enzymes catalyze the reversible reaction of α-ketoglutaric acid and amino acids. GPT enzyme is mainly present in hepatocytes and its elevated level in blood is used to diagnose any liver toxicity whereas GOT enzyme is present in kidney, heart and liver tissues therefore, is helpful in diagnosis of toxicities of these organs. Furthermore, alkaline phosphatase is another liver enzyme that hydrolyzes para nitrophenyl phosphate to yellow color para nitro phenol during estimation and is used to estimate alkaline phosphatase level in blood sample. 2.6. Statistical analysis The investigations were done in triplicate. Results were statistically presented as mean±standard error of mean (SEM). 2018 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.9.4.369-374.1785 Anupam et al. / European Journal of Chemistry 9 (4) (2018) 369-374 373 Table 3. Toxicity evaluation of selected compounds a. Compounds Serum glutamate pyruvate transaminase (SGPT), IU/L Serum glutamate oxaloacetate transaminase (SGOT), IU/L Alkaline phosphatase (ALKP), IU/L Control 46.14±0.42 41.12±0.35 14.77±0.12 4b 49.24±0.32 * 40.45±0.45 15.76±0.24 4f 43.32±0.58 * 42.27±0.34 19.46±0.26 * 4h 54.52±0.49 ** 55.26±0.27 ** 28.37±0.13 ** a Mean±SEM values (n = 6). Significantly different from control: * p < 0.05, ** p < 0.01. Statistical values p < 0.001 and p < 0.01 were considered as extremely significant and significant respectively using Graph prism software. 3. Results and discussion 3.1. Synthesis In the present research work, newer imidazole derivatives (4a-h) were synthesized by the reaction of benzil and substituted benzaldehydes that afforded compounds 3a-h. These imidazole derivatives (3a-h) were refluxed with freshly prepared acetyl chloride in presence of benzene or pyridine to undergo acetylation. Synthetic route to the titled compounds 4a-h is shown as Scheme 1. All the synthesized compounds were characterized through FT-IR, 1H NMR and 13C NMR spectroscopic methods. Physicochemical properties and spectral characterization supported the structures of the synthesized compounds. FT-IR spectra showed the characteristic bands for Ar-CHstr, C=Ostr, C=Cstr, C=Nstr, NO2str, C-O-Cstr and C-Clstr to be in the range of wavenumber 3510-3100, 1670-1610, 1570-1510, 1438-1230, 1480-1410, 1043-1022 and 758-740 cm-1, respect- tively. The 1H NMR spectra was confirmed for -CH3 and –OCH3 groups by detecting singlets at δ 2.14-2.60 and 3.43-3.73 ppm, respectively. On the other hand, CH-methoxybenzene, CH- benzil, CH-nitrobenzene and CH-chlorobenzene groups were confirmed by showing multiplet at δ 6.46-7.46, 7.10-7.88, 7.60-8.25 and 7.38-7.48 ppm, respectively. The 13C NMR spectra confirmed the presence of -CH3, –OCH3, C=C, imidazole and benzene ring. 3.2. Antimicrobial activity All the synthesized compounds (4a-h) were tested for their antimicrobial activity using agar diffusion technique against Gram +ve (Staphhylococcus aureus and Bacillus subtilis), Gram –ve (Escherichia coli and Pseudomonas auregi- nosa) as well as Fungal strain (Candida albicans). The anti- microbial study revealed promising results for most of the compounds of the series. It was noticed that compounds 4a, 4b and 4c were effective (**p < 0.01) against P. aureginosa at 600 µg/mL concentration, whereas, compounds 4b, 4f and 4h were found to be significantly (***p < 0.001) active against Gram negative E coli. Interestingly, compounds 4a and 4b also displayed significant inhibition (***p < 0.001) against S. aureus and B subtilis. These results were comparable to that of standard drug ampicillin. In screening results for antifungal activity, compound 4b was found to be significantly (***p < 0.001) active against C. albicans at 600 µg/mL concentration and was comparable to standard drug griseofulvin. The data for antimicrobial activities are shown in Table 1. Minimum inhibitory concentration and minimum bacte- ricidal concentration of most active compounds 4b, 4f and 4h were examined using broth dilution method against bacterial strains S. aureus (NCIM 2079), B. subtilis (NCIM 2708), E. coli (NCIM 2685) and P. aureginosa (NCIM 2242) as well as fungal strain C. albicans (ATCC 60193). The results obtained were in close comparison to the standard antimicrobial drugs ampi- cillin and griseofulvin. The results of MICs and MBCs for the selected compounds 4b, 4f and 4h are summarized in Table 2 and were found to be in the range of 25-50 µg/mL for E. coli and were lesser than the standard drug ampicillin (6.25 µg/mL). When tested against P. aureginosa, MICs were obtained to be in the range of 100-300 µg/mL. Against other bacterial strains S. aureus and B. subtilis, the MICs for the selected compounds were found to be in higher range (50-300 µg/mL) as compared to ampicillin (6.25 µg/mL). Similar results were obtained when these compounds were tested against fungal species C. albicans as the MIC values were found to be in the range of 100-200 µg/mL as compared to standard drug griseofulvin (6.25 µg/mL). In the present study, compound 4b emerged to have most potent activities against most of the strains having MIC in the range of 25-200 µg/mL. 3.3. Toxicity evaluation/liver enzyme estimation Toxicity evaluation was performed for the most active compounds 4b, 4f and 4h to check any toxicity on the liver by estimating the liver enzymes and the data are shown in Table 3. The results were found to be comparable to control and the compounds were devoid of hepatotoxic effects except compound 4h that showed raised levels of SGOT, SGPT and ALP. 4. Conclusion In the present study, newer triphenyl-imidazole deriva- tives (4a-h) were synthesized and tested for their antimicro- bial activity using agar diffusion technique against Gram +ve, Gram –ve as well as fungal strains. From the antimicrobial activity results, it was observed that compounds 4a, 4b, 4f and 4h with electron withdrawing group at ortho or para positions (2-Cl, 4-Cl, 2-NO2 and 4-NO2) substituted at distant phenyl ring showed highest activities against Gram positive and Gram negative bacterial stains. Whereas, electron releasing group – OCH3 at ortho, meta or para position at distal phenyl ring as in compounds 4c, 4d and 4e showed moderate results. Com- pound with electron withdrawing group chloro at para posi- tion to phenyl ring was found to be significantly effective against C. albicans fungal strain. These results indicate the influence of the type and position of electron withdrawing groups such as chloro and nitro on antimicrobial activities. Also, three hydrophobic phenyl rings attached to the hetero- cyclic imidazole in the synthesized compounds increased the lipophilicity and formed a large non-polar structure which could act as a substrate of lanosterol-α-demethylase enzyme and therefore could result into the inhibition of ergosterol synthesis and might be the mechanism behind the antifungal activity of the compounds. Moreover, the present study would form the basis of further preclinical and clinical investigations to develop newer imidazole derivatives as potential antimic- robial agents. Acknowledgements Authors are thankful to College of Pharmacy, Jazan University, Saudi Arabia and TIPER, Meerut, U.P. India for providing necessary facilities. 2018 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.9.4.369-374.1785 374 Anupam et al. / European Journal of Chemistry 9 (4) (2018) 369-374 Disclosure statement Conflict of interests: Authors have no any conflict of interest. Author contributions: All authors contributed equally to this work. Ethical approval: All ethical guidelines have been adhered. ORCID Anupam http://orcid.org/0000-0002-8135-2774 Mohammed Al-Bratty http://orcid.org/0000-0001-8687-0573 Hassan Ahmad Alhazmi http://orcid.org/0000-0002-9647-9006 Shamim Ahmad http://orcid.org/0000-0003-2540-9542 Supriya Maity http://orcid.org/0000-0003-4043-1131 Md Shamsher Alam http://orcid.org/0000-0001-9127-5023 Waquar Ahsan http://orcid.org/0000-0002-5987-2933 References [1]. Gabera, H. M.; Bagley, M. C. Eur. J. Chem. 2011, 2, 214-222. [2]. Desai, N. C.; Dodiya, A.; Shihory N. J. Saudi Chem. Soc. 2013, 17, 259- 267. [3]. Verma, B. 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This is an open access article distributed under the terms and conditions of the CC BY NC License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited without any further permission from Atlanta Publishing House LLC (European Journal of Chemistry). No use, distribution or reproduction is permitted which does not comply with these terms. Permissions for commercial use of this work beyond the scope of the License (http://www.eurjchem.com/index.php/eurjchem/pages/view/terms) are administered by Atlanta Publishing House LLC (European Journal of Chemistry). 2018 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.9.4.369-374.1785 http://orcid.org/0000-0002-8135-2774 http://orcid.org/0000-0001-8687-0573 http://orcid.org/0000-0002-9647-9006 http://orcid.org/0000-0003-2540-9542 http://orcid.org/0000-0003-4043-1131 http://orcid.org/0000-0001-9127-5023 http://orcid.org/0000-0002-5987-2933 http://www.eurjchem.com/index.php/eurjchem/pages/view/terms http://creativecommons.org/licenses/by-nc/4.0 http://www.eurjchem.com/index.php/eurjchem/pages/view/terms 1. Introduction 2. Experimental 2.1. Instrumentations and materials 2.2. Synthesis of 2-(3-substituted phenyl)- 4,5-diphenyl -1H-imidazoles (3a-h) 2.3. Synthesis of 1-(2-(2-substituted phenyl)-4, 5-diphenyl -1H-imidazol-1-yl) ethanones (4a-h) 2.4. Antimicrobial activity 2.4.1. Determination of zone of inhibition 2.4.2. Determination of minimum inhibitory concentration and minimum bactericidal/fungicidal concentration 2.5. Toxicity evaluation/liver enzyme estimation 2.6. Statistical analysis 3. Results and discussion 3.1. Synthesis 3.2. Antimicrobial activity 3.3. Toxicity evaluation/liver enzyme estimation 4. Conclusion Disclosure statement ORCID References PrintField10: PrintField11: PrintField12: PrintField13: PrintField14: PrintField15: PrintField20: PrintField21: PrintField22: PrintField23: PrintField24: PrintField25: