Short Communication

Synthesis and biological evaluation of di‑ and tri‑substituted imidazoles as safer anti‑inflammatory‑antifungal agents

Asif Husain, Sushma Drabu, Nitin Kumar, M. M. Alam, Sandhya Bawa

Department of ABSTRACT Pharmaceutical Chemistry, PURPOSE: In view of the potential pharmacophoric nature of imidazole nucleus, two series of imidazole derivatives, Faculty of Pharmacy, Jamia Hamdard, 2,4‑disubstituted‑1 H‑imidazoles (2a‑m) and 1,2,4‑trisubstituted‑1 H‑imidazoles (3a‑m), were synthesized with an New Delhi, India aim of obtaining dual acting compounds i.e., anti‑inflammatory and antifungal agents. MATERIALS AND METHODS: The title compounds were synthesized from 4‑methoxyphenyl glyoxal (1) following multistep synthesis, and their Address for correspondence: structures were established on the basis of modern analytical techniques (IR, NMR and MS). The synthesized Dr. Asif Husain, imidazoles were tested for their in vivo anti‑inflammatory activity. In addition to that, some compounds were also E‑mail: drasifhusain@yahoo. evaluated for their analgesic and ulcerogenic effects. The compounds were also evaluated for their in vitro antifungal com activity. RESULTS: Di‑ and tri‑substituted imidazole derivatives (2a‑m and 3a‑m) were successfully synthesized. In in vivo anti‑inflammatory test, six compounds (2 h, 2 l, 3 g, 3 h, 3 l and 3 m) exhibited good anti‑inflammatory activity (49.58 to 58.02% inhibition) with minimal GI irritation (severity index; 0.17 to 0.34). These compounds were also tested for their analgesic activity and showed appreciable protection (40.53 to 49.60% protection) against saline‑induced writhing test. Indomethacin was used as standard drug for comparison. In antifungal test, two compounds (3 h and 3 l) displayed appreciable antifungal activity (MIC; 12.5 µg mL‑1) against the fungal strains tested. CONCLUSION: Two compounds, 2‑(4‑nitrophenyl)‑4‑(4‑methoxyphenyl)‑1‑phenyl‑1H‑imidazole (3 h) and 2,4‑di‑(4‑methoxyphenyl)‑1‑phenyl‑1H‑imidazole (3 l), emerged as lead compounds having dual biological activities; good anti‑inflammatory as well as antifungal effect with lesser GI irritation. Received : 09-04-12 Review completed : 25-05-12 Accepted : 14-06-12 KEY WORDS: Analgesic, antifungal, anti‑inflammatory, imidazole, ulcerogenicity

olypharmacy or multiple drug therapy for the treatment as anti‑inflammatory,[3,4] antimicrobial,[5,6] anticonvulsant[7,8] P of anti‑inflammatory conditions associated with and anticancer[9] activities. Many drugs have imidazole nucleus microbial infections is a common practice. Monotherapy of in their structure [Figure 1], like dacarbazine as anticancer, drug having dual inhibition i.e., both anti‑inflammatory and metronidazole as antifungal, cimetidine as antihistaminics and antimicrobial effect, help a patient not only economically but flumazenil as benzodiazepine antagonist.[3] also in the minimization of side effects.[1] The commonly used non‑steroidal anti‑inflammatory drugs (NSAIDs) are associated Imidazole‑containing antifungal drug, ketoconazole, was also [2] with GI side effects, thus there is still need for safer NSAIDs. found to have anti‑inflammatory activity in addition to its antifungal activity.[10] Flutrimazole, another imidazole‑based Imidazoles are an important class of heterocyclic compounds due wide spectrum antifungal agent, was found to be a good topical to their multiple therapeutic and pharmacological actions, such anti‑inflammatory agent.[11] Therefore, imidazole nucleus seems to be an important pharmacophore for designing new drug Access this article online Quick Response Code: candidates [Figure 2]. Website: www.jpbsonline.org Owing to the versatility of imidazole nucleus, in view of above‑mentioned reports, in continuation to our work on [12] DOI: imidazole derivatives and in search of compounds as dual [13] 10.4103/0975-7406.111822 inhibitors, we have synthesized twenty six new di‑and tri‑substituted imidazole derivatives that might act as dual

How to cite this article: Husain A, Drabu S, Kumar N, Alam MM, Bawa S. Synthesis and biological evaluation of di- and tri-substituted imidazoles as safer anti- inflammatory-antifungal agents. J Pharm Bioall Sci 2013;5:154-61.

 154 Journal of Pharmacy and Bioallied Sciences April-June 2013 Vol 5 Issue 2 Husain, et al.: Studies on imidazoles anti‑inflammatory‑antifungal agents with minimal GI side effects.

Materials and Methods

All the chemicals and regents used in this study were purchased from Merck (India), Sigma Aldrich, S.D. fine and Sisco Research Laboratories Ltd. Melting points were determined in open capillary tubes and are uncorrected. Purity of the compounds was checked by thin layer chromatography (TLC) on silica gel G Plates (Merck No. 5544) using toluene:ethyl acetate:formic acid (5:4:1) and benzene:acetone (8:2) as solvent system and the spots were located either under ultra violet light or through exposure to iodine vapors.

The IR spectra were recorded using KBr pellets of the Figure 1: Imidazole containing drugs compounds on Bruker alpha‑T spectrophotometer.[1] H‑NMR spectra were recorded on DPX‑300 NMR spectrometer or TM BRUKER‑400 Ultra Shield spectrometer in DMSO‑d6 with tetramethylsilane (TMS) as an internal standard; chemical shifts (d) are reported in parts per million (ppm) downfield from TMS. Mass spectra were recorded on Jeol JMS‑D 300 instrument. Elemental analyses were performed on Perkin‑Elmer model 240 analyzer (C, H, N) and found within range of ± 0.4% of theoretical values. The compounds were prepared by reported method[12] with some modifications. The protocol for synthesis of title compounds is represented in Scheme 1.

Chemistry Figure 2: Antifungal drugs having anti‑inflammatory activity Synthesis of 4‑methoxyphenyl glyoxal (1)

A mixture of pure selenium dioxide (0.5 mol), water (10 mL) and dioxane (300 mL) were heated in a round bottom flask at 50-55°C with stirring till clear solution was obtained. To the above solution, 4‑methoxy‑acetophenone (0.5 mol) was added in one lot. The mixture was further refluxed with stirring for 4 h, during refluxing small amount of selenium was precipitated. The reaction mixture was decanted to remove the precipitate. The clear solution so obtained was distilled to remove excess dioxane and water. A yellow liquid was found pure on TLC examination (toluene:ethyl acetate:formic acid, 1 5:4:1, v/v/v). Yield: 58%; mp 67-68°C; Rf: 0.45; H‑NMR (d, ppm):

3.86 (s, 3 H, OCH3), 6.98 and 7.46 (d, each, A2B2, p‑anisyl ring), 9.73 (s, 1 H, CHO); Mass (m/z): 164 (M+).

General method for the preparation of 2,4‑disubstituted‑1 Scheme 1: Schematic representation of synthesis of imidazoles H‑imidazole (2a‑m)

A mixture of compound 1 (0.025 mol), aryl aldehyde (0.025 4‑(4‑methoxyphenyl)‑2‑Phenyl‑1 H‑imidazole (2a) mol) and ammonium acetate (10 g) in glacial acetic ‑1 acid (50 mL) was refluxed in a round bottom flask for 5 h. Yield: 46%; m.p. 161-162°C; Rf 0.59; IR (cm ): 3462 (N‑H), After refluxing, the mixture was cooled to room temperature 3021 (C‑H), 1590 (C = N), 1441 (C = C), 1328 (C‑N); 1H‑NMR (d, and then poured into cold water (200 mL). A precipitate ppm): 3.85 (s, 3 H, OCH3), 6.93 and 7.49 (d, each, A2B2, p‑anisyl was separated out, which was filtered, washed, dried and ring), 7.14-7.33 (m, 3 H, H‑3,4,5, phenyl), 7.38-7.54 (m, 2 H, crystallized from acetone. The compound was found pure H‑2,6, phenyl), 7.75 (d, 1 H, H‑5, imidazole), 10.43 (bs, 1 H, NH); + on TLC (toluene:ethyl acetate: formic acid, 5:4:1, v/v/v) MS (m/z): 250 [M ]. Anal. Calcd. for C16H14N2O (%): C, 76.78; examination. H, 5.64; N, 11.19. Found: C, 76.52; H, 5.65; N, 11.16.

Journal of Pharmacy and Bioallied Sciences April-June 2013 Vol 5 Issue 2 155  Husain, et al.: Studies on imidazoles

2‑(2‑chlorophenyl)‑4‑(4‑methoxyphenyl)‑1 H‑imidazole (2b) 2‑(4‑nitrophenyl)‑4‑(4‑methoxyphenyl)‑1 H‑imidazole (2 h)

‑1 ‑1 Yield: 52%; m.p. 137-138°C; Rf 0.69; IR (cm ): 3435 (N‑H), Yield: 58%; m.p. 131-132°C; Rf 0.71; IR (cm ): 3427 (N‑H), 3018 (C‑H), 1584 (C = N), 1436 (C = C), 1322 (C‑N), 708 (C‑Cl); 3016 (C‑H), 1568 (C = N),C 1426 ( = C), 1330 (C‑N); 1 1 H‑NMR (d, ppm): 3.87 (s, 3 H, OCH3), 6.97 and 7.53 (d, each, H‑NMR (d, ppm): 3.88 (s, 3 H, OCH3), 6.98 and 7.53 (d, each,

A2B2, p‑anisyl ring), 7.23-7.42 (m, 2 H, H‑4,5, phenyl), 7.65- A2B2, p‑anisyl ring), 7.46 and 8.27 (d, each, A2B2, p‑substituted 7.78 (m, 3 H, H‑3,6, phenyl + H‑5, imidazole), 10.27 (bs, 1 H, phenyl), 7.93 (d, 1 H, H‑5, imidazole), 10.31 (bs, 1 H, NH); + + NH); MS (m/z): 285 [M ]. Anal. Calcd. for C16H13ClN2O (%): MS (m/z): 295 [M ]. Anal. Calcd. for C16H13N3O3 (%): C, 65.08; C, 67.49; H, 4.60; N, 9.84. Found: C, 67.33; H, 4.59; N, 9.82. H, 4.44; N, 10.23. Found: C, 65.18; H, 4.45; N, 10.26.

2‑(3‑chlorophenyl)‑4‑(4‑methoxyphenyl)‑1 H‑imidazole (2c) 2‑(2‑hydroxyphenyl)‑4‑(4‑methoxyphenyl)‑1 H‑imidazole (2i)

‑1 ‑1 Yield: 44%; m.p. 147-148°C; Rf 0.74; IR (cm ): 3431 (N‑H), Yield: 46%; m.p. 139°C; Rf 0.63; IR (cm ): 3432 (N‑H), 3314 (O‑H), 3022 (C‑H), 1568 (C = N),C 1435 ( = C), 1327 (C‑N), 3016 (C‑H), 1578 (C = N), 1431 (C = C), 1318 (C‑N); 1H‑NMR (d, 1 712 (C‑Cl); H‑NMR (d, ppm): 3.86 (s, 3 H, OCH3), 6.95 and ppm): 3.85 (s, 3 H, OCH3), 6.87 and 7.45 (d, each, A2B2, p‑anisyl

7.50 (d, each, A2B2, p‑anisyl ring), 7.21-7.85 (m, 5 H, H‑2,4,5,6, ring), 7.16-7.32 (m, 2 H, H‑4,5, phenyl), 7.51-7.83 (m, 2 H, H‑3,6, phenyl + H‑5, imidazole), 10.36 (bs, 1 H, NH); MS (m/z): phenyl), 7.91 (d, 1 H, H‑5, imidazole), 10.28 (bs, 1 H, NH); + + 285 [M ]. Anal. Calcd. for C16H13ClN2O (%): C, 67.49; H, 4.60; MS (m/z): 266 [M ]. Anal. Calcd. for C16H14N2O2 (%): C, 72.17; N, 9.84. Found: C, 67.37; H, 4.60; N, 9.81. H, 5.30; N, 10.52. Found: C, 72.33; H, 5.29; N, 10.55.

2‑(4‑chlorophenyl)‑4‑(4‑methoxyphenyl)‑1 H‑imidazole (2d) 2‑(3‑hydroxyphenyl)‑4‑(4‑methoxyphenyl)‑1 H‑imidazole (2j)

‑1 ‑1 Yield: 51%; m.p. 157-158°C; Rf 0.79; IR (cm ): 3429 (N‑H), Yield: 40%; m.p. 128-130°C; Rf 0.71; IR (cm ): 3425 (N‑H), 3014 (C‑H), 1576 (C = N),C 1429 ( = C), 1331 (C‑N), 3309 (O‑H), 3007 (C‑H), 1572 (C = N), 1426 (C = C), 1 1 719 (C‑Cl); H‑NMR (d, ppm): 3.87 (s, 3 H, OCH3), 6.99 and 1311 (C‑N); H‑NMR (d, ppm): 3.83 (s, 3 H, OCH3), 6.85

7.48 (d, each, A2B2, p‑anisyl ring), 7.42 and 7.57 (d, each, A2B2, and 7.42 (d, each, A2B2, p‑anisyl ring), 7.12-7.98 (m, 5 H, p‑substituted phenyl), 7.88 (d, 1 H, H‑5, imidazole), 10.23 (bs, 1 phenyl + H‑5, imidazole), 10.25 (bs, 1 H, NH); MS (m/z): + + H, NH); MS (m/z): 285 [M ]. Anal. Calcd. for C16H13ClN2O (%): 266 [M ]. Anal. Calcd. for C16H14N2O2 (%): C, 72.17; H, 5.30; C, 67.49; H, 4.60; N, 9.84. Found: C, 67.34; H, 4.60; N, 9.82. N, 10.52. Found: C, 72.32; H, 5.25; N, 10.44.

2‑(2‑fluorophenyl)‑4‑(4‑methoxyphenyl)‑1 H‑Imidazole (2e) 2‑(4‑hydroxyphenyl)‑4‑(4‑methoxyphenyl)‑1 H‑imidazole (2k)

‑1 ‑1 Yield: 52%; m.p. 109-10°C; Rf: 0.53; IR (cm ): 3418 (NH), Yield: 56%; m.p. 150°C; Rf 0.61; IR (cm ): 3428 (N‑H), 3027 (CH), 1563 (C = N), 1424 (C = C), 1326 (C‑N); 3312 (O‑H), 3015 (C‑H), 1566 (C = N), 1431 (C = C), 1 1 H‑NMR (d, ppm): 3.85 (s, 3 H, OCH3), 7.01 and 7.54 (d, each, 1323 (C‑N); H‑NMR (d, ppm): 3.86 (s, 3 H, OCH3), 6.82 and

A2B2, p‑anisyl ring), 7.38 (d, 2 H, H‑2,6, phenyl), 7.69 (t, 2 H, 7.46 (d, each, A2B2, p‑anisyl ring), 7.43 and 7.88 (d, each, A2B2, H‑3,5, phenyl), 7.83 (d, 1 H, H‑5, imidazole), 10.38 (bs, 1 H, p‑substituted phenyl), 7.92 (d, 1 H, H‑5, imidazole), 10.32 (bs, 1 + + NH); Mass (m/z): 268 (M ). Anal. Calcd. for C16H13FN2O: C, H, NH); MS (m/z): 266 [M ]. Anal. Calcd. for C16H14N2O2 (%): 71.63; H, 4.88; N, 10.44. Found: C, 71.73; H, 4.87; N, 10.42. C, 72.17; H, 5.30; N, 10.52. Found: C, 72.32; H, 5.28; N, 10.56.

2‑(2‑nitrophenyl)‑4‑(4‑methoxyphenyl)‑1 H‑imidazole (2 f) 2,4‑Di‑(4‑methoxyphenyl)‑1 H‑imidazole (2 l)

‑1 ‑1 Yield: 63%; m.p. 161-162°C; Rf 0.59; IR (cm ): 3424 (N‑H), Yield: 49%; m.p. 183-184°C; Rf 0.69; IR (cm ): 3422 (N‑H), 3019 (C‑H), 1571 (C = N),C 1430 ( = C), 1318 (C‑N); 3010 (C‑H), 1571 (C = N),C 1428 ( = C), 1331 (C‑N); 1 1 H‑NMR (d, ppm): 3.87 (s, 3 H, OCH3), 7.03 and 7.52 (d, each, H‑NMR (d, ppm): 3.84 and 3.91 (s, each, 2 × OCH3), 6.86, 7.12,

A2B2, p‑anisyl ring), 7.31-7.47 (m, 2 H, H‑4,5, phenyl), 7.69- 7.44 and 7.63 (d, each, 2 × A2B2, 2x p‑anisyl ring), 7.86 (d, 1 H, 8.36 (m, 3 H, H‑3,6 + H‑5, imidazole), 10.28 (bs, 1 H, NH); H‑5, imidazole), 10.29 (bs, 1 H, NH); MS (m/z): 280 [M+]. Anal. + MS (m/z): 295 [M ]. Anal. Calcd. for C16H13N3O3 (%): C, 65.08; Calcd. for C17H16N2O2 (%): C, 72.84; H, 5.75; N, 9.99. Found: H, 4.44; N, 10.23. Found: C, 65.23; H, 4.45; N, 10.25. C, 72.57; H, 5.56; N, 9.84.

2‑(3‑nitrophenyl)‑4‑(4‑methoxyphenyl)‑1 H‑imidazole (2 g) 2‑(4‑hydroxy‑3‑methoxyphenyl)‑4‑(4‑methoxyphenyl)‑1 H‑imidazole (2 m) ‑1 Yield: 51%; m.p. 127-128°C; Rf 0.55; IR (cm ): 3430 (N‑H), ‑1 3021 (C‑H), 1566 (C = N),C 1433 ( = C), 1329 (C‑N); Yield: 53%; m.p. 144°C; Rf 0.78; IR (cm ): 3431 (N‑H), 1 H‑NMR (d, ppm): 3.87 (s, 3 H, OCH3), 7.05 and 7.55 (d, each, 3018 (C‑H), 1563 (C = N),C 1433 ( = C), 1326 (C‑N); 1 A2B2, p‑anisyl ring), 7.28-8.33 (m, 5 H, H‑2,4,5,6, phenyl + H‑5, H‑NMR (d, ppm): 3.87 and 3.95 (s, each, 2 × OCH3), 6.89 and + imidazole), 10.35 (bs, 1 H, NH); MS (m/z): 295 [M ]. Anal. 7.45 (d, each, A2B2, p‑anisyl ring), 7.03 (d, 1 H, H‑5, phenyl),

Calcd. for C16H13N3O3 (%): C, 65.08; H, 4.44; N, 10.23. Found: 7.17 (d, 1 H, H‑2, phenyl), 7.32 (dd, 1 H, H‑6, phenyl), 7.82 (d, C, 65.10; H, 4.42; N, 10.28. 1 H, H‑5, imidazole), 10.26(bs, 1 H, NH); MS (m/z): 296 [M+].

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+ Anal. Calcd. for C17H16N2O3: C, 68.91; H, 5.44; N, 9.45. Found: 1 H, H‑5, imidazole); MS (m/z): 344 [M ]. Anal. Calcd. for

C, 68.88; H, 5.46; N, 9.17. C22H17FN2O (%): C, 76.73; H, 4.98; N, 8.13. Found: C, 76.56; H, 5.02; N, 8.05. General method for the preparation of 1,2,4‑trisubstituted‑1 H‑imidazoles (3a‑m) 2‑(2‑nitrophenyl)‑4‑(4‑methoxyphenyl)‑1‑phenyl‑1 H‑imidazole (3 f) Compound 2a‑m (0.01 mol) was suspended in ‑1 tetrahydrofuran (20 mL) and was refluxed with Yield: 48%; m.p. 178°C; Rf 0.61; IR (cm ): 3020 (C‑H), chlorobenzene (2 mL) in the presence of 2-3 drops of triethylamine 1572 (C = N), 1438 (C = C), 1329 (C‑N); 1H‑NMR (d, ppm): for 8 h. The completion of reaction was determined by TLC. After 3.84 (s, 3 H, OCH3), 6.92-7.55 (m, 13 H, 3x phenyl ring), 7.95 (s, refluxing, acetone was added to the reaction mixture and it was 1 H, H‑5, imidazole); MS (m/z): 371 [M+]. Anal. Calcd. for kept at room temperature overnight. Later a precipitate formed C22H17N3O3 (%): C, 71.15; H, 4.61; N, 11.31. Found: C, 71.32; which was filtered and recrystallized from ethanol. The compound H, 4.52; N, 11.29. was found pure on TLC examination (benzene: acetone, 8:2, v/v). 2‑(3‑nitrophenyl)‑4‑(4‑methoxyphenyl)‑1‑Phenyl‑1 4‑(4‑methoxyphenyl)‑1,2‑diphenyl‑1 H‑imidazole (3a) H‑imidazole (3 g)

‑1 ‑1 Yield: 47%; m.p. 176-178°C; Rf 0.63; IR (cm ): 3455 (N‑H), Yield: 43%; m.p. 161-162°C; Rf 0.68; IR (cm ): 3015 (C‑H), 3024 (C‑H), 1579 (C = N),C 1440 ( = C), 1333 (C‑N); 1575 (C = N), 1433 (C = C), 1326 (C‑N); 1H‑NMR (d, ppm): 1 H‑NMR (d, ppm): 3.83 (s, 3 H, OCH3), 6.87 and 7.42 (d, each, 3.86 (s, 3 H, OCH3), 6.90-7.52 (m, 13 H, 3x phenyl ring), 7.97 (s, + A2B2, p‑anisyl ring), 7.08-7.71 (m, 10 H, 2 × phenyl), 7.95 (s, 1 1 H, H‑5, imidazole); MS (m/z): 371 [M ]. Anal. Calcd. for + H, H‑5, imidazole), 10.41 (bs, 1 H, NH); MS (m/z): 326 [M ]. C22H17N3O3 (%): C, 71.15; H, 4.61; N, 11.31. Found: C, 71.08;

Anal. Calcd. for C22H18N2O (%): C, 80.96; H, 5.56; N, 8.58. H, 4.56; N, 11.32. Found: C, 80.74; H, 5.48; N, 8.37. 2‑(4‑nitrophenyl)‑4‑(4‑methoxyphenyl)‑1‑Phenyl‑1 2‑(2‑Chlorophenyl)‑4‑(4‑methoxyphenyl)‑1‑phenyl‑1 H‑imidazole (3 h) ‑1 H‑imidazole (3b) Yield: 54%; m.p. 156°C; Rf 0.71; IR (cm ): ‑1 3013 (C‑H), 1581 (C = N),C 1443 ( = C), 1325 (C‑N), Yield: 50%; m.p. 157-58°C; Rf 0.78; IR (cm ): 3023 (C‑H), 1 1 708 (C‑Cl); H‑NMR (d, ppm): 3.85 (s, 3 H, OCH3), 6.81- 1582 (C = N), 1440 (C = C), 1333 (C‑N); H‑NMR (d, ppm):

7.56 (m, 13 H, 3x phenyl ring), 7.93 (s, 1 H, H‑5, imidazole); 3.83 (s, 3 H, OCH3), 6.87-7.47 (m, 13 H, 3x phenyl ring), 7.92 (s, + + MS (m/z): 361 [M ]. Anal. Calcd. for C22H17ClN2O (%): C, 1 H, H‑5, imidazole); MS (m/z): 371 [M ]. Anal. Calcd. for

73.23; H, 4.75; N, 7.76. Found: C, 73.35; H, 4.68; N, 7.54. C22H17N3O3 (%): C, 71.15; H, 4.61; N, 11.31. Found: C, 71.11; H, 4.58; N, 11.36. 2‑(3‑chlorophenyl)‑4‑(4‑methoxyphenyl)‑1‑phenyl‑1 H‑imidazole (3c) 2‑(2‑hydroxyphenyl)‑4‑(4‑methoxyphenyl)‑1‑Phenyl‑1 H‑imidazole (3i) ‑1 Yield: 43%; m.p. 135-36°C; Rf 0.81; IR (cm ): 3008 (C‑H), ‑1 1574 (C = N), 1437 (C = C), 1329 (C‑N), 715 (C‑Cl); Yield: 58%; m.p. 168°C; Rf 0.78; IR (cm ): 3319 (O‑H), 1 H‑NMR (d, ppm): 3.84 (s, 3 H, OCH3), 6.87-7.55 (m, 13 H, 3x 3015 (C‑H), 1561 (C = N),C 1433 ( = C), 1324 (C‑N); + 1 phenyl ring), 7.95 (s, 1 H, H‑5, imidazole); MS (m/z): 361 [M ]. H‑NMR (d, ppm): 3.85 (s, 3 H, OCH3), 6.81-7.49 (m, 13 H, 3x + Anal. Calcd. for C22H17ClN2O (%): C, 73.23; H, 4.75; N, 7.76. phenyl ring), 7.95 (s, 1 H, H‑5, imidazole); MS (m/z): 342 [M ].

Found: C, 73.16; H, 4.72; N, 7.79. Anal. Calcd. for C22H18N2O2 (%): C, 77.17; H, 5.30; N, 8.18. Found: C, 77.04; H, 5.32; N, 8.21. 2‑(4‑chlorophenyl)‑4‑(4‑methoxyphenyl)‑1‑phenyl‑1 H‑imidazole (3d) 2‑(3‑hydroxyphenyl)‑4‑(4‑methoxyphenyl)‑1‑Phenyl‑1 H‑imidazole (3j) ‑1 Yield: 41%; m.p. 140°C; Rf 0.86; IR (cm ): 3010 (C‑H), ‑1 1578 (C = N), 1439 (C = C), 1328 (C‑N), 708 (C‑Cl); Yield: 54%; m.p. 157-58°C; Rf 0.79; IR (cm ): 3313 (O‑H), 1 H‑NMR (d, ppm): 3.85 (s, 3 H, OCH3), 6.84-7.51 (m, 13 H, 3x 3016 (C‑H), 1555 (C = N),C 1431 ( = C), 1326 (C‑N); + 1 phenyl ring), 7.92 (s, 1 H, H‑5, imidazole); MS (m/z): 361 [M ]. H‑NMR (d, ppm): 3.86 (s, 3 H, OCH3), 6.85-7.51 (m, 13 H, 3x + Anal. Calcd. for C22H17ClN2O (%): C, 73.23; H, 4.75; N, 7.76. phenyl ring), 8.02 (s, 1 H, H‑5, imidazole); MS (m/z): 342 [M ].

Found: C, 73.17; H, 4.68; N, 7.81. Anal. Calcd. for C22H18N2O2 (%): C, 77.17; H, 5.30; N, 8.18. Found: C, 77.03; H, 5.26; N, 8.20. 2‑(4‑fluorophenyl)‑4‑(4‑methoxyphenyl)‑1‑phenyl‑1 H‑imidazole (3e) 2‑(4‑Hydroxyphenyl)‑4‑(4‑Methoxyphenyl)‑1‑Phenyl‑1 H‑Imidazole (3k) ‑1 Yield: 65%; m.p. 146-148°C; Rf: 0.62; IR (cm ): 3016 (C‑H), 1 ‑1 1566 (C = N), 1441 (C = C), 1332 (C‑N); H‑NMR (d, ppm): Yield: 41%; m.p. 175°C; Rf 0.64; IR (cm ): 3316 (O‑H),

3.81 (s, 3 H, OCH3), 6.82-7.38 (m, 13 H, 3x phenyl ring), 7.83 (s, 3013 (C‑H), 1553 (C = N),C 1438 ( = C), 1317 (C‑N);

Journal of Pharmacy and Bioallied Sciences April-June 2013 Vol 5 Issue 2 157  Husain, et al.: Studies on imidazoles

1 H‑NMR (d, ppm): 3.84 (s, 3 H, OCH3), 6.78-7.64 (m, 13 H, injection. Thus the edema volume in control group (Vc) and

3 × phenyl ring), 8.11 (s, 1 H, H‑5, imidazole); MS (m/z): edema volume in groups treated with test compounds (Vt) was + 342 [M ]. Anal. Calcd. for C22H18N2O2 (%): C, 77.17; H, 5.30; measured and the percentage inhibition of edema was calculated N, 8.18. Found: C, 77.28; H, 5.31; N, 8.14. using the formula:

2,4‑Di‑(4‑methoxyphenyl)‑1‑phenyl‑1 H‑imidazole (3 l) Anti‑inflammatory activity (% inhibition) = {(Vc ‑ Vt)/Vc} × 100

‑1 Analgesic activity Yield: 52%; m.p. 163-64°C; Rf 0.71; IR (cm ): 3009 (C‑H), 1546 (C = N), 1432 (C = C), 1318 (C‑N); 1H‑NMR (d, ppm): The compounds which showed anti‑inflammatory activity above 3.82 and 3.86 (s, each, 2x OCH3), 6.89-7.68 (m, 13 H, 3 × phenyl ring), 7.98 (s, 1, H‑5, imidazole); MS (m/z): 356 [M+]. Anal. 70% of that of the Indomethacin were screened for analgesic activity. Analgesic activity was evaluated by acetic acid induced Calcd. for C23H20N2O2 (%): C, 77.51; H, 5.66; N, 7.86. Found: C, 77.33; H, 5.61; N, 7.82. writhing method.[15]

2-(4-Hydroxy-3-methoxyphenyl)-4-(4-methoxyphenyl)-1- Swiss albino mice (25-30 g) of either sex were divided into group of six in each. A 1% aqueous sodium chloride solution (i.p. phenyl-1 H‑imidazole (3 m) Yield: 58%; m.p. 172°C; Rf 0.63; IR (cm‑1): 3322 (O‑H), 3014 (C‑H), 1553 (C = N), 1427 (C = C), injection in a volume of 0.1 mL) was used as writhing induced 1 agent. Mice were kept individually in the test cage, before acetic 1324 (C‑N); H‑NMR (d, ppm): 3.87 (s, 3 H, OCH3), 6.88‑7.72 (m, 13 H, 3 × phenyl ring), 8.15 (s, 1H, H‑5, imidazole); MS acid injection and habituated for 30 min. Screening of analgesic (m/z): 372 [M+]. Anal. Calcd. for C H N O (%):C, 74.18; H, activity was performed after p.o. administration of test drugs at a 23 20 2 3 ‑1 5.41; N, 7.52. Found: C, 74.02; H, 5.43; N, 7.45. dose of 10 mgkg . Group I was taken as control and received CMC suspension only, group II received reference drug Indomethacin Pharmacology and rest of the groups were treated with test drugs suspended in 1.0% CMC. After 1 h of drug administration 0.10 mL of 1% acetic acid solution was given to mice intraperitoneally. Stretching All the animals used in the present study were kept in the movements consisting of arching of the back, elongation of animal house in accordance with the University Animal body and extension of hind limbs were counted for 5-15 min of Ethical Committee of Jamia Hamdard, which applies sodium chloride injection. The analgesic activity was expressed the guidelines and rules laid down by the Committee for in terms of percentage protection. the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Analgesic activity (% protection) = {(n n′)/n} × 100 Empowerment, Government of India. Albino rats of Wistar ‑ strain of either sex, weighing 180-200 g and Albino mice of where, n = mean number of writhes of control group, n′ = either sex weighing 22-25 g, were used for pharmacological mean number of writhes of test group. activities. The animals were housed in groups of six and acclimatized to room conditions for at least 2 days before the Ulcerogenic activity experiments. Food and water were freely available up to the time of experiments except during the short time they were Acute ulcerogenesis test was done according to the reported removed from the cages for testing. method.[2] Albino rats (150-200 g) were divided into different groups consisting of six animals in each group. Ulcerogenic Anti‑inflammatory activity activity evaluated after p.o. administration of test compounds or Indomethacin at the dose of 30 mg kg‑1. Control rats received p.o. The synthesized compounds were evaluated for their in vivo administration of vehicle (suspension of 1% methyl cellulose). anti‑inflammatory activity using carrageenan‑induced Food but not water was removed 24 h before administration of rat paw edema method[14] using Indomethacin as the test compounds. After the drug treatment the rats were fed standard. The paw volume was measured by using digital with normal diet for 17 h and then sacrificed. The stomach was Plethysmometer (Panlab). removed and opened along the greater curvature, washed with distilled water and cleaned gently by dipping in normal saline. The The rats were randomly divided into groups of six. One group mucosal damage was examined by means of a magnifying glass. was kept as control, and received only 0.5% carboxymethyl For each stomach the mucosal damage was assessed according cellulose (CMC) solution. Another group was kept as standard to the following scoring system: 0.5: redness, 1.0: spot ulcers, 1.5: and received Indomethacin (10 mgkg‑1 p.o.), other groups hemorrhagic streaks, 2.0: ulcers >3 but <5, 3.0: ulcers >5. The received test compounds (10 mgkg‑1 p.o.). Carrageenan mean score of each treated group minus the mean score of control solution (0.1% in sterile 0.9% NaCl solution) in a volume of group was regarded as severity index of gastric mucosal damage. 0.1 mL was injected subcutaneously into the sub‑plantar region of the right hind paw of each rat, 1 h after the administration Antifungal activity of the test compounds and standard drugs. The paw volume was measured by saline displacement shown on screen of The newly prepared compounds were screened for their digital Plethysmometer (Panlab) at 2 and 3 h after carrageenan antifungal activity against Candida albicans, Rhizopus oryza,

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Penicillium citrum and Aspergillus niger fungal strains at a spectral data. The synthesized compounds gave M+ peak in concentration of 100 µgmL‑1 by cup‑plate method[16] using reasonable intensities. In case of compounds having phenyl ketoconazole as standard. Compounds inhibiting growth of rings with chloro‑substituents (2b, 2c, 2d, 3b, 3c and 3d) the one or more of the above microorganisms were further tested molecular ion peak or their fragments having chloro‑group for minimum inhibitory concentration (MIC).[17] Sabouraud appeared as cluster of peaks. agar media was prepared by dissolving peptone (1 g), D‑glucose (4 g) and agar (2 g) in distilled water (100 mL) Pharmacology and adjusting pH to 5.7. Normal saline was used to make a suspension of spore of fungal strain for lawning. A loopful of The synthesized compounds were evaluated for anti‑inflammatory particular fungal strain was transferred to 3 mL saline to get activity along with analgesic and ulcerogenic effect. The selected a suspension of corresponding species. Agar media (20 mL) compounds which showed good anti‑inflammatory effect (70% was poured into each petridish. Excess of suspension was to that of the standard drug) were also tested for their antifungal decanted and the plates were dried by placing in an incubator effect. at 37°C for 1 h. Wells were made using an agar punch and, each well was labeled accordingly. A control was also prepared In vivo anti‑inflammatory activity of the title compounds was in triplicate and maintained at 37°C for 3-4 days. The determined by carrageenan‑induced rat paw edema method[14] at antifungal activity of the compounds was compared with the dose level of 10 mg kg‑1 of body weight. Six of the twenty tested the standard drug; ketoconazole. The nutrient broth, which compounds showed statistically significant anti‑inflammatory contained logarithmic serially two fold diluted amount of test activity with respect to standard drug indomethacin. Compounds, compound and controls, was inoculated with approximately 2‑(4‑nitrophenyl) ‑4‑(4‑methoxyphenyl)‑1‑phenyl‑1 H‑imidazole 1.6 × 104‑6 × 104 c.f.u. mL‑1. The cultures were incubated (3 h) and 2,4‑di‑(4‑methoxyphenyl)‑1‑phenyl‑1 H‑imidazole for 48 h at 37°C and the growth was monitored. The lowest (3 l) having p‑nitro and p‑methoxy substitution, were concentration (highest dilution) required to inhibit the growth found to be the most active compounds with 58.02 and of fungus was regarded as MIC. 56.17% inhibition, respectively. In addition, four more compounds, 2‑(4‑nitrophenyl)‑4‑(4‑methoxyphenyl)‑1 Results and Discussion H‑imidazole (2 h) 2,4‑di‑(4‑methoxyphenyl)‑1 H‑imidazole (2L), 2‑(3‑nitrophenyl)‑4‑(4‑methoxyphenyl)‑1‑phenyl‑1 H‑imidazole Chemistry (3 g) and 2-(4-hydroxy-3-methoxyphenyl)-4-(4-methoxyphenyl)- 1-phenyl-1 H‑imidazole (3 m) also showed appreciable activity Synthesis of the intermediates and target compounds (2a‑m with 51.02, 49.79, 49.58 and 52.88% inhibition, respectively. and 3a‑m) was accomplished as outlined in Scheme I. The standard drug indomethacin exhibited 70.78% inhibition The tri‑substituted imidazoles, 1,2,4‑trisubstituted‑1 in rat paw edema [Table 1]. After analyzing the results, following H‑imidazoles (3a‑m), were synthesized by refluxing remarks could be made regarding the SAR: 2,4‑disubstituted‑1 H‑imidazoles (2a‑m) with chlorobenzene • The anti‑inflammatory activity was found to be better in in tetrahydrofuran (THF) using triethylamine as catalyst. tri‑substituted imidazoles (3a‑m) as compared to that of 2,4‑Disubstituted‑1 H‑imidazoles (2a‑m) were synthesized by the di‑substituted imidazoles (2a‑m). refluxing 4‑methoxyphenyl glyoxal (1) with aryl aldehyde in • Compounds with electropositive groups like methoxy, nitro, glacial acetic acid using ammonium acetate as catalyst. The etc., (2 h, 2 l, 3 h and 3 l) were found to be more active required intermediate compound 4‑methoxyphenyl glyoxal (1) than the compounds with electronegative group like chloro, was synthesized from 4‑methoxy acetophenone using selenium fluoro, etc., (2d, 2e, 3d and 3e). dioxide. The structures were established on the basis of modern • p‑Substituted derivatives were found to be better in analytical techniques (IR, NMR and MS). their anti‑inflammatory action than o‑ or m‑substituted derivatives (h was more active than g; k was more active In general, the IR spectra of the compounds showed than i). characteristic band around 3430 cm‑1 for N‑H of di‑substituted imidazoles (2a‑m). Disappearance of this band from the The compounds (2 h, 2 l, 3 g, 3 h, 3 l and 3 m) that exhibited spectra of tri‑substituted imidazoles (3a‑m) indicated the above 70% of anti‑inflammatory activity of indomethacin substitution of hydrogen of NH of the di‑substituted imidazole were further evaluated for their analgesic effects using sodium ring by phenyl to give tri‑substituted imidazoles. Other chloride induced writhing method[15] and ulcerogenic effect by bands were observed at appropriate places corresponding to reported method.[2] the structure. In[1] H‑NMR spectral data, all the compounds showed characteristic peaks at appropriate d‑values. Presence The results of analgesic activity [Table 2] indicated that of a broad singlet around d 10.35 (NH) and a doublet around d compounds 3 h and 3 l showed 49.99 and 47.14% protection, 7.75 (imidazole, H‑5) indicated the formation of di‑substituted respectively, against sodium chloride induced writhings test. imidazole ring. Similarly, disappearance of broad singlet of The standard drug indomethacin was found to have 59.60% NH of imidazole confirmed the conversion of di‑substituted protection at the same dose level, 10 mgkg‑1 p.o. Compounds imidazoles (2a‑m) to tri‑substituted imidazoles (3a‑m). The 2 h, 2 l, 3 g and 3 m also showed appreciable analgesic activity structures of the compounds were further supported by mass with 40.53, 42.74, 41.79 and 44.83% protection, respectively.

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Table 1: Anti‑inflammatory activity of the title compounds Compounds % Inhibition±SEMa Compounds % Inhibition±SEMa After 2 h After 3 h After 2 h After 3 h Indomethacin 61.55±0.937 70.78±1.707 3 a 20.23±1.666** 29.62±2.208** 2 a 24.22±3.144** 32.71±3.215** 3 b 21.34±1.886** 32.30±2.027** 2 b 22.66±2.459** 31.27±2.483** 3 d 26.67±1.333** 39.09±0.882** 2 d 27.55±1.675** 35.18±1.380** 3 e 22.23±1.602** 31.89±1.077** 2 e 21.12±2.049** 29.63±1.656** 3 g 33.78±1.710** 49.58±0.741** 2 g 33.78±1.710** 45.88±1.330** 3 h 37.78±2.377** 58.02±1.886** 2 h 34.45±3.793** 51.02±4.103** 3 i 24.67±1.213** 38.89±1.093** 2 i 22.45±1.517** 36.21±1.646** 3 k 26.89±1.261** 41.15±0.823** 2 k 25.56±1.666** 38.89±1.380** 3 l 34.45±1.437** 56.17±1.224** 2 l 29.34±2.177** 49.79±2.442** 3 m 33.78±2.522** 52.88±1.475** 2 m 32.67±2.058** 48.76±1.138** - - - **P<0.01, aRelative to their respective standard and data were analyzed by one‑way ANOVA followed by Dunnett multiple comparison test for n=6

The results indicated that the compounds with p‑nitro Table 2: Analgesic effects along with ulcerogenic effect of substitution and p‑methoxy substitution were found to be a some selected imidazole derivatives good anti‑inflammatory agent with high analgesic activity. The Compounds Severity Analgesic activity a activity was found to increase by the replacement of N‑proton index No. of writhes/30 mina % Protectionb of imidazole ring by phenyl group. Control 0.00±0.00 41.67±1.68 ‑ Indomethacin 0.84±0.10** 16.67±0.61** 59.60 The selected compounds were also screened for their acute 2 h 0.34±0.17ns 24.67±0.67** 40.53** ulcerogenic activity and found to be low in ulcerogenic 2 l 0.25±0.11ns 23.67±0.49** 42.74** action; severity index ranging from 0.17 to 0.34 whereas the 3 g 0.25±0.11ns 24.17±0.74** 41.79** ns standard drug indomethacin showed high severity index of 3 h 0.17±0.10 20.67±0.49** 49.99* 3 l 0.17±0.10ns 21.84±0.31** 47.14** 0.84. 2‑(4‑Nitrophenyl)‑4‑(4‑methoxyphenyl)‑1‑phenyl‑1 3 m 0.34±0.17ns 22.84±0.60** 44.83** H‑imidazole (3 h) and 2,4‑di‑(4‑methoxyphenyl)‑1‑phenyl‑1 ns a H‑imidazole (3 l) showed maximum reduction in ulcerogenic **P<0.01, *P< 0.05, P>0.05, Relative to the control and data were analyzed by one‑way ANOVA followed by Dunnett multiple comparison activity (severity index = 0.17). The other tested compounds test for n=6, bRelative to their respective standard (Indomethacin) and also exhibited better GI safety profile as compared to that of data were analyzed by one‑way ANOVA followed by Dunnett multiple the standard drug indomethacin. comparison test for n=6

The results of pharmacological activities indicate Table 3: Antifungal activity (minimum inhibitory ‑1 that the synthesized compounds not only have good concentration; µgmL ) of some selected imidazole derivatives anti‑inflammatory‑analgesic activity but also have less GI Compounds Candida Rhizopus Penicillium Aspergillus irritation. The results are presented in Tables 1 and 2. albicans oryza citrum niger 2 a 50 >100 >100 50 All the synthesized compounds (2a‑m and 3a‑m) were tested 2 b 50 >100 50 >100 ‑1 2 c >100 >100 50 50 for their antifungal effect initially at 100 µg mL by cup‑plate 2 d 25 50 50 50 method and the compounds inhibiting the growth of one 2 e 25 50 25 50 or more strains were further tested for their MIC [Table 3]. 2 f 50 >100 >100 >100 Two compounds, 2-(4-nitrophenyl)-4-(4-methoxyphenyl)-1- 2 g >100 50 >100 50 phenyl-1 H-imidazole (3 h) and 2,4-di-(4-methoxyphenyl)-1- 2 h 50 50 >100 >100 2 i >100 >100 >100 >100 phenyl-1 H-imidazole (3 l), were found to be the most active 2 j 50 >100 >100 >100 - among all the tested twenty compounds with MIC of 12.5 µgmL 2 k 50 >100 >100 50 1 against all the tested four fungal strains. Two more compounds, 2 l >100 >100 >100 >100 24 -( -hydroxy-3-methoxyphenyl)-4-(4-methoxyphenyl)-1 2 m 12.5 25 50 25 H- imidazole (2 m) and 2-(4-hydroxy-3-methoxyphenyl)-4- 3 a >100 >100 >100 50 (4-methoxyphenyl)-1-phenyl-1H-imidazole (3 m), were also 3 b 50 >100 >100 >100 3 c >100 >100 >100 50 good in their antifungal action. Rests of the compounds were 3 d 50 >100 50 50 moderate to low in their action. 3 e 50 50 25 >100 3 f >100 >100 >100 >100 Conclusion 3 g 25 50 25 50 3 h 12.5 12.5 12.5 12.5 3 i >100 >100 >100 >100 To sum up, two compounds among the synthesized twenty 3 j 50 >100 >100 50 imidazole derivatives, 2-(4-nitrophenyl)-4-(4-methoxyphenyl)- 3k 50 >100 >100 50 1-phenyl-1 H-imidazole (3 h) and 2,4-di-(4-methoxyphenyl)- 3 l 12.5 12.5 12.5 12.5 1 -phenyl-1 H- imidazole (3 l), emerged as lead compounds 3 m 25 50 50 50 Ketoconazole 6.25 6.25 6.25 6.25 with good anti-inflammatory as well as antifungal actions.

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