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Chem Biol Drug Des 2014; 84: 473–488 Research Article Synthesis, Molecular Docking, and Biological Evaluation of Some Novel Hydrazones and Pyrazole Derivatives as Anti-inflammatory Agents Khaled O. Mohammed1 and Yassin M. Nissan2,* 1 Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr Elini Street, Cairo 11562, Egypt 2 Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr Elini Street, Cairo 11562, Egypt *Corresponding author: Yassin M. Nissan, [email protected] 2-Hydrazinyl-N-(4-sulfamoylphenyl)acetamide 3 was the key intermediate for the synthesis of novel hydrazones 4–10 and pyrazole derivatives 11–17. All compounds were tested for their in vivo anti-inflammatory activity and their ability to inhibit the production of PGE2 in serum samples of rats. IC50 values for the most active compounds for inhibition of COX-1 and COX-2 enzymes were determined in vitro, and they were also tested for their ulcerogenic effect. Molecular docking was performed on the active site of COX-2 to predict their mode of binding to the amino acids. Most of the synthesized compounds showed good antiinflammatory activity especially compounds 3, 4, 8, 9, 15, and 17 which showed better activity than diclofenac as the reference drug. Compounds 3, 8, 9, 13, and 15–17 were less ulcerogenic than indomethacine as the reference drug. Most of the synthesized compounds interacted with Tyr 385 and Ser 530 in molecular docking study with additional hydrogen bond for compound 17. Compound 17 showed good selectivity index value of 11.1 for COX-1/COX-2 inhibition in vitro. Key words: 4-benzenesulfonamide, anti-inflammatory, hydrazone, pyrazole Received 22 February 2014, revised 26 March 2014 and accepted for publication 3 April 2014 Although of the high effectiveness of the steroidal antiinflammatory drugs, their serious side-effects limit their use in common inflammation and as pain killers (1). The search for safer drugs launched with the development of nonsteroidal anti-inflammatory drugs. In spite of their great safety over the steroidal drugs, they still have some sideeffects, the most common one is the development of peptic and duodenal ulcer on long-term use (2). Moreover, ª 2014 John Wiley & Sons A/S. doi: 10.1111/cbdd.12336 some kidney and liver malfunctions were observed (2). The rise of selective COX-2 inhibitors reduced the risk of some of these side-effects (3), but other side-effects were also observed, and the most serious one was the capability of some drugs of causing cardiac arrest in some patients (4). But although high selectivity is demanded to reduce the gastrointestinal side-effects, it was discussed recently that COX-2 high selectivity could be the major cause of cardiac and renal problems (5,6), and hence, moderate selectivity could reduce the risk of both gastrointestinal and cardiac side-effects (7). Therefore, seeking for new molecules with anti-inflammatory activity and diminished side-effects is still a goal for medicinal chemists all over the world. In our seek for novel anti-inflammatory agents with less side-effects and better activity, we have focused in the current research of merging more than one active moiety known for their presence in several anti-inflammatory agents. The first moiety was phenylacetamide, a wellknown moiety in several pain killers especially in paracetamol and phenacetin (8–10). Literature describes the usage of phenylacetamide in the synthesis of new anti-inflammatory agents (11–14). The second moiety was sulfamoyl moiety in para position of phenylacetamide. The antiinflammatory effect of sulfonamides was reported in many researches (14, 15). The presence of 4-benezensulfonamide in the structure of celecoxib, one of the highly selective anti-inflammatory drugs (16), and valdecoxib is also encouraging to use such moiety in our design for new anti-inflammatory agents. H2 NO 2S H 2NO2 S N N O CF3 Celecoxib N Valdecoxib 473 Mohammed and Nissan On the other hand, several hydrazone derivatives were synthesized and evaluated for their anti-inflammatory activity as well as COX-2 selectivity (17–19). It was interesting also to synthesize a series of pyrazole derivatives as pyrazole moiety is still a target for many researchers in their seek for new and selective anti-inflammatory agents (20–23). Based on the aforementioned and in our seek to develop new anti-inflammatory agents, we have designed and synthesized novel hydrazones and pyrazole derivatives merging phenylacetamide, sulfamoyl and hydrazone or pyrazole moieties in two series of compounds (A & B) hoping to reach new agents with better activity and less side-effects. All the synthesized compounds were subjected for in vivo biological evaluation through rat paw edema, in vitro assay for both COX-1 and COX-2 inhibitions, evaluating their ulcerogenic activity and their ability to inhibit PGE2 production in rat serum samples. Moreover, molecular docking on the active site of COX-2 was performed to predict their binding mode to the amino acids of the active site of the enzyme. H N H 2NO2 S N H O A N H N Ar H2 NO2 S N N Ph O Ar B Experimental General chemistry Melting points were determined on Electro thermal Stuart 5MP3 digital melting point apparatus and were uncorrected. Elemental microanalyses were performed at the micro analytical centre, Al-Azhar University, Cairo, Egypt. IR spectra were recorded on a Bruker Fourier transform (FT)-IR spectrophotometer as KBr disks. NMR spectra (in DMSO-d6) were recorded on Bruker AC-300 Ultra Shield NMR spectrometer (Bruker, Flawil, Switzerland, d ppm) at 400 MHz using TMS as internal standard and peak multiplicities are designed as follows: s, singlet; d, doublet; t, triplet; and m, multiplet. Silica gel used for column chromatography was obtained from Fluka, and 70—230 mesh thin layer chromatography was carried out on silica gel TLC plates with fluorescence indicator (F254). 2-Hydrazinyl-N-(4-sulfamoylphenyl)acetamide 3 A mixture of the chloro derivative 2 (0.01 mol, 2.48 g) with hydrazine hydrate (0.01 mol, 0.5 g) was refluxed in absolute ethanol (20 mL) in the presence of catalytic amount of anhydrous sodium acetate (0.5 g) for 3 h. The precipitate formed was filtered, dried, washed with hot ethanol, and crystallized from DMF/H2O to give white powder of 3. Yield: 80%; mp 208–210 °C; IR (KBr) (cm1): 3358–3332 (2NH2), 3280–3196 (2NH), 3100–3052 (arom.CH), 2924 (aliph.CH), 1685(C=O); 1HNMR(400 MHz, DMSOd6) d ppm: 3.43(s, 2H, NH2, D2O exchangeable), 4.37(s, 2H, 474 CH2),7.266 (s, 2H, SO2NH2, D2O exchangeable), 7.74– 7.87 (m, 4H, Ar H), 7.89 (s, 1H, NH, D2O exchangeable), 10.39 (s, 1H, NH, D2O exchangeable), 13CNMR (100.63 MHz, DMSO-d6) d ppm: 64.18(1), 119.52(2), 127.12(2), 138.86(1), 142.22(1), 169.72(1); Anal. Calcd. for C8H12N4O3S: C, 39.34; H, 4.95; N, 22.94; Found: C, 39.41; H, 4.97; N, 23.01. General procedure for preparation of compounds 4-10 To a solution of the hydrazino derivative 3 (0.01 mol, 2.44 g) in absolute ethanol (20 mL), the appropriate aromatic aldehyde was added (0.01 mol) and the solution was refluxed for 3–4 h, cooled and the formed precipitate was filtered off, dried, and crystallized from ethanol to give the corresponding hydrazone derivatives 4–10, respectively. 2-(2-Benzylidenehydrazinyl)-N-(4-sulfamoylphenyl) acetamide 4 Yield: 72%; mp 198–200 °C; IR (KBr) (cm1): 3373–3332 (NH2), 3250–3194 (2NH) 3131–3057 (arom. CH), 2954 (aliph. CH), 1689 (C=O); 1HNMR (400 MHz, DMSO-d6) d ppm: 4.46 (s, 2H, CH2), 6.93–7.22 (m, 3H, ArH), 7.27 (s, 2H, SO2NH2, D2O exchangeable),7.28–7.32 (m, 2H, ArH),7.47–7.85(m, 4H, ArH),7.88 (s, 1H, NH, D2O exchangeable), 8.72 (s, 1H, N=CH), 10.89 (s, 1H, NH, D2O exchangeable); 13CNMR (100.63 MHz, DMSO-d6) d ppm: 58.98(1), 119.39(2), 125.91(1), 127.32(2), 128.83(1), 129.00(1), 129.40(1), 131.59(1), 131.85(1), 136.40(1), 139.22(1), 141.95(1), 169.86(1); Anal. Calcd. for C15H16N4O3S: C, 54.20; H, 4.85; N, 16.86; Found: C, 54.28; H, 4.90; N, 16.94. 2-(2-(2-Fluorobenzylidene)hydrazinyl)-N-(4sulfamoylphenyl)acetamide 5 Yield: 70%; mp 202–204 °C; IR (KBr) (cm1): 3338–3329 (NH2), 3267–3225 (2NH), 3107–3051 (arom. CH), 2924 (aliph. CH), 1701 (C=O); 1HNMR (400 MHz, DMSO-d6) d ppm: 4.45 (s, 2H, CH2), 7.11–7.13 (m, 2H, ArH) 7.15–7.23 (m, 2H, ArH),7.27 (s, 2H, SO2NH2,D2O exchangeable), 7.51–7.83 (m, 4H, ArH), 7.84 (s, 1H, NH, D2O exchangeable),8.85 (s, 1H, N=CH), 10.83 (s, 1H, NH, D2O exchangeable); 13CNMR (100.63 MHz, DMSO-d6) d ppm: 58.94(1), 115.81(1), 116.03(1), 119.38(2), 127.31(2), 127.68(1), 127.76(1), 130.49(1), 133.07(1), 139.22(1), 141.94(1), 160.82(1), 169.80(1); Anal. Calcd for C15H15FN4O3S: C, 51.42; H, 4.32; N, 15.99; Found: C, 51.49; H, 4.30; N, 16.17. 2-(2-(4-Bromobenzylidene)hydrazinyl)-N-(4sulfamoylphenyl)acetamide 6 Yield: 65%; mp 188–190 °C; IR (KBr) (cm1): 3404–3354 (NH2), 3261–3186 (2NH), 3101–3049 (arom. CH), 2924 Chem Biol Drug Des 2014; 84: 473–488 Novel Hydrazones and Pyrazole Derivatives (aliph. CH), 1689 (C=O); 1HNMR (400 MHz, DMSO-d6) d ppm: 4.46 (s, 2H, CH2), 7.15–7.23 (m, 2H, ArH),7.27 (s, 2H, SO2NH2, D2O exchangeable), 7.43–7.50 (m, 2H, ArH),7.78–7.81 (m, 4H, ArH), 7.82 (s, 1H, NH, D2O exchangeable), 8.70 (s, 1H, N=CH), 10.81 (s, 1H, NH, D2O exchangeable); 13CNMR (100.63 MHz, DMSO-d6) d ppm: 58.90(1), 119.381(2), 120.71(1), 127.31(4), 127.75 (1), 130.17(1), 131.92(1), 135.79(1), 139.24(1), 141.92(1), 169.62(1); Anal. Calcd for C15H15BrN4O3S: C, 43.81; H, 3.68; N, 13.62; Found: C, 43.39; H, 3.65; N, 13.71. 2-(2-(4-Chlorobenzylidene)hydrazinyl)-N-(4sulfamoylphenyl)acetamide 7 Yield: 70%; mp 192–194 °C; IR (KBr) (cm1): 3404–3354 (NH2), 3261–3186 (2NH), 3097–3049 (arom CH), 2954 (aliphatic CH), 1689 (C=O); 1HNMR (400 MHz, DMSO-d6) d ppm: 4.46 (s, 2H, CH2), 7.21 (s, 2H, SO2NH2, D2O exchangeable), 7.34–7.36 (m, 2H, ArH),7.49–7.52 (m, 2H, ArH), 7.79–7.81(m, 4H, ArH), 7.82 (s, 1H, NH, D2O exchangeable), 8.74 (s, 1H, N=CH), 10.82 (s, 1H, NH, D2O exchangeable); 13CNMR (100.63 MHz, DMSO-d6) d ppm: 58.92(1), 119.39(2), 127.31(4), 127.44(1), 129.02(1), 130.11(1), 132.14(1), 135.44(1), 139.22(1), 141.96(1), 169.65(1); Anal. Calcd for C15H15ClN4O3S: C, 49.11; H, 4.12; N, 15.27; Found: C, 49.22; H, 4.14; N, 15.38. C15H15FN4O3S: C, 51.42; H, 4.32; N, 15.99; Found: C, 51.58; H, 4.31; N, 16.12. 2-(2-(4-Methoxybenzylidene)hydrazinyl)-N-(4sulfamoylphenyl)acetamide 10 Yield: 60%; mp 178–180 °C; IR (KBr) (cm1): 3442–3352 (NH2), 3263–3205 (2NH), 3105–3049 (arom. CH), 30082954 (aliph. CH), 1693 (C=O); 1HNMR (400 MHz, DMSOd6) d ppm: 3.73 (s, 3H, OCH3), 4.41(s, 2H, CH2) 6.86– 6.89 (m, 2H, ArH), 7.27 (s, 2H, SO2NH2, D2O exchangeable), 7.41–7.44 (m, 2H, ArH), 7.79–7.821(m, 4H, ArH), 7.83 (s, 1H, NH, D2O exchangeable), 8.65 (s, 1H, N=CH), 10.80 (s, 1H, NH, D2O exchangeable); 13CNMR (100.63 MHz, DMSO-d6) d ppm: 55.58(1), 59.07(1), 116.43(1), 114.49(2), 119.37(2), 127.31(4), 129.13(1), 139.21(1), 141.95(1), 159.44(1), 170.05(1); Anal. Calcd for C16H18N4O4S: C, 53.03; H, 5.01; N, 15.46; Found: C, 53.17; H, 5.04; N, 15.53. General procedure for preparation of compounds 11–17 To a solution of the hydrazino derivative 3 (0.01 mol, 2.44 g) in glacial acetic acid (20 mL), the appropriate aromatic chalcone derivative (0.01 mol) was added and the solution was refluxed in oil bath for 6–8 h. The formed precipitate was filtered, washed, dried, and crystallized from DMF/H2O to give the pyrazole derivatives 11-17, respectively. 2-(2-(4-(Dimethylamino)benzylidene)hydrazinyl)-N(4-sulfamoylphenyl)acetamide 8 Yield: 75%; mp 162–164 °C; IR (KBr) (cm1): 3367–3329 (NH2), 3246–3178 (2NH), 3097–3039 (arom. CH), 3005– 2920 (aliph. CH), 1701 (C=O); 1HNMR (400 MHz, DMSOd6) d ppm: 2.88 (s, 6H, 2CH3), 4.37 (s, 2H, CH2), 6.64–6.66 (m, 2H, ArH), 7.27 (s, 2H, SO2NH2, D2O exchangeable), 7.30–7.32 (m, 2H, ArH), 7.79–7.83 (m, 4H, ArH), 7.85 (s, 1H, NH, D2O exchangeable), 8.50 (s, 1H, N=CH), 10.80 (s, 1H, NH, D2O exchangeable); 13CNMR (100.63 MHz, DMSO-d6) d ppm: 40.20(2), 59.24(1), 112.48(1), 119.36(2), 124.37(1), 127.11(1), 127.31(4), 133.14(1), 139.19(1), 141.97(1), 150.52(1), 170.29(1); Anal. Calcd for C17H21N5O3S: C, 54.38; H, 5.64; N, 18.65; Found: C, 54.48; H, 5.69; N, 18.79. 2-(3,5-Diphenyl-1H-pyrazol-1-yl)-N-(4sulfamoylphenyl)acetamide 11 Yield: 50%; mp 223–225 dec°C; IR (KBr) (cm1): 3355–3331 (NH2), 3276 (NH), 3103–3085 (arom. CH), 2856 (aliph. CH), 1687 (C=O); 1HNMR (400 MHz, DMSO-d6) d ppm: 4.57 (s, 2H, CH2), 6.62 (s, 1H, C4 pyrazole), 7.21 (s, 2H, SO2NH2, D2O exchangeable), 7.28–7.30 (m, 4H, ArH), 7.72–7.74 (m, 2H, ArH), 7.81–7.85 (m, 8H, ArH), 10.79(s, 1H, NH, D2O exchangeable); 13CNMR (100.63 MHz, DMSO-d6) d ppm: 54.60(1), 119.37(2), 119.43(4), 127.00(2), 127.35(4), 138.53 (2), 139.32(2), 141.82(2), 142.34(2), 162.86(1), 168.25(1); Anal. Calcd for C23H20N4O3S: C, 63.87; H, 4.66; N, 12.95; Found C, 63.64; H, 5.18; N, 13.11. 2-(2-(4-Fluorobenzylidene)hydrazinyl)-N-(4sulfamoylphenyl)acetamide 9 Yield: 80%; mp 185–187 °C; IR (KBr) (cm1): 3356–3320 (NH2), 3263–3220 (2NH), 3105–3051 (arom. CH), 2954 (aliph. CH), 1687 (C=O); 1HNMR (400 MHz, DMSO-d6) d ppm: 4.45 (s, 2H, CH2) 7.11–7.23 (m, 2H, ArH), 7.26 (s, 2H, SO2NH2, D2O exchangeable), 7.51–7.55 (m, 2H, ArH), 7.72–7.82 (m, 4H, ArH), 7.85 (s, 1H, NH, D2O exchangeable), 8.70 (s, 1H, N=CH), 10.81 (s, 1H, NH, D2O exchangeable); 13CNMR (100.63 MHz, DMSO-d6) d ppm: 58.94(1), 116.43(1), 119.38(2), 127.31(4), 127.76(1), 130.49(1), 133.03(1), 139.22(1), 141.94(1), 160.83(1), 169.79(1); 2-(5-(2-Fluorophenyl)-3-phenyl-1H-pyrazol-1-yl)-N(4-sulfamoylphenyl)acetamide 12 Yield: 43%; mp 217–219 °C; IR (KBr) (cm1): 3353–3325 (NH2), 3278 (NH), 3105–3055 (arom. CH), 2954 (aliph. CH), 1685 (C=O); 1HNMR (400 MHz, DMSO-d6) d ppm: 4.57 (s, 2H, CH2), 6.62 (s, 1H, C4 pyrazole), 7.21 (s, 2H, SO2NH2, D2O exchangeable),7.28–7.30 (m, 3H, ArH), 7.72–7.74 (m, 2H, ArH), 7.81–7.85 (m, 8H, ArH), 10.77(s, 1H, NH, D2O exchangeable); 13CNMR (100.63 MHz, DMSO-d6) d ppm: 58.72(1), 119.39(2), 119.45(4), 123.51 (1), 127.02(2), 127.36(4), 138.54(1), 139.32(2), 141.82(2), 142.33(1), 162.87(2), 168.32(1); Anal. Calcd for Chem Biol Drug Des 2014; 84: 473–488 475 Mohammed and Nissan C23H19FN4O3S: C, 61.32; H, 4.25; N, 12.44; Found: C, 61.17; H, 4.66; N, 12.49. 2-(5-(4-Bromophenyl)-3-phenyl-1H-pyrazol-1-yl)-N(4-sulfamoylphenyl)acetamide 13 Yield: 55%; mp 223–225 °C; IR (KBr) (cm1): 3354–3332 (NH2), 3278 (NH), 3103–3050 (arom. CH), 2955 (aliph. CH), 1685 (C=O); 1HNMR (400 MHz, DMSO-d6) d ppm: 4.57 (s, 2H, CH2), 6.63 (s, 1H, C4 pyrazole), 7.27 (s, 2H, SO2NH2, D2O exchangeable),7.28–7.32 (m, 3H, ArH), 7.72–7.88 (m, 10H, ArH), 10.80 (s, 1H, NH, D2O exchangeable); 13CNMR (100.63 MHz, DMSO-d6) d ppm: 54.58(1), 119.23(2), 119.27(2), 119.44(4), 126.96(1), 127.24(4), 127.35(2), 138.53(2), 142.11(2), 163.50(2), 169.67(1); Anal. Calcd for C23H19BrN4O3S: C, 54.02; H, 3.74; N, 10.96; Found: C, 53.93; H, 4.17; N, 11.05. 2-(5-(4-Chlorophenyl)-3-phenyl-1H-pyrazol-1-yl)-N(4-sulfamoylphenyl)acetamide 14 Yield: 80%; mp 208–210 °C; IR (KBr) (cm1): 3356–3332 (NH2), 3280 (NH), 3104–3053 (arom. CH), 2956 (aliph. CH), 1684 (C=O); 1HNMR (400 MHz, DMSO-d6) d ppm: 4.58 (s, 2H, CH2), 6.62 (s, 1H, C4 pyrazole), 7.22 (s, 2H, SO2NH2, D2O exchangeable), 7.25–7.32 (m, 3H, ArH), 7.72–7.86 (m, 10H, ArH), 10.82 (s, 1H, NH, D2O exchangeable); 13CNMR (100.63 MHz, DMSO-d6) d ppm: 59.65(1), 119.38(2), 119.44(4), 123.49(1), 127.01(2), 127.35(4), 138.53(1), 139.31(2), 141.84(2), 142.34(1), 162.87(2), 168.31(1); Anal. Calcd for C23H19ClN4O3S: C, 59.16; H, 4.10; N, 12.00; Found: C, 58.98; H, 4.49; N, 12.03. 2-(5-(4-(Dimethylamino)phenyl)-3-phenyl-1Hpyrazol-1-yl)-N-(4-sulfamoylphenyl)acetamide 15 Yield: 58%; mp 168–170 °C; IR (KBr) (cm1): 3356–3334 (NH2), 3273 (NH), 3103–3059 (arom. CH), 2924 (aliph. CH), 1697 (C=O); 1HNMR (400 MHz, DMSO-d6) d ppm: 2.88 (s, 6H, 2CH3), 4.37 (s, 2H, CH2), 6.64 (s, 1H, C4 pyrazole), 7.22 (s, 2H, SO2NH2, D2O exchangeable),7.27– 7.32 (m, 3H, ArH), 7.68–7.89 (m, 10H, ArH), 10.80 (s, 1H, NH, D2O exchangeable); 13CNMR (100.63 MHz, DMSOd6) d ppm: 40.40(2), 59.25(1), 111.54(1), 112.15(1), 112.48 (1), 119.37(1), 122.00(4), 124.36(1), 126.96(1), 127.12(1), 127.24(4), 129.96(1), 133.14(1), 139.06(1), 142.05(1), 150.52(1), 163.51(1), 168.29(1); Anal. Calcd for C25H25N5O3S: C, 63.14; H, 5.30; N, 14.73; Found: C, 62.96; H, 5.74; N, 14.72. 2-(5-(4-Fluorophenyl)-3-phenyl-1H-pyrazol-1-yl)-N(4-sulfamoylphenyl)acetamide 16 Yield: 55%; mp 224–226 °C; IR (KBr) (cm1): 3357–3332 (NH2), 3280 (NH), 3107–3054 (arom. CH), 2950 (aliph. CH), 1687 (C=O); 1HNMR (400 MHz, DMSO-d6) d ppm: 4.57 (s, 2H, CH2), 6.63 (s, 1H, C4 pyrazole), 7.22 (s, 2H, SO2NH2, 476 D2O exchangeable), 7.26–7.31 (m, 3H, ArH), 7.75–7.82 (m, 10H, ArH), 10.79 (s, 1H, NH, D2O exchangeable); 13CNMR (100.63 MHz, DMSO-d6) d ppm: 58.77(1), 119.39(2), 119.45(3), 123.51(1), 127.01(2), 127.36(4), 138.54(2), 139.33(2), 141.82(2), 142.33(1),162.87(2), 168.32(1); Anal. Calcd for C23H19FN4O3S: C, 61.32; H, 4.25; N, 12.44; Found: C, 61.13; H, 4.75; N, 12.39. 2-(5-(4-Methoxyphenyl)-3-phenyl-1H-pyrazol-1-yl)N-(4-sulfamoylphenyl)acetamide 17 Yield: 52%; mp 225–227 °C; IR (KBr) (cm1): 3358–3332 (NH2), 3278 (NH), 3099–3055 (arom. CH), 2954 (aliph. CH), 1685 (C=O); 1HNMR (400 MHz, DMSO-d6) d ppm: 3.60 (s, 3H, OCH3), 4.57(s, 2H, CH2), 6.92 (s, 1H, C4 pyrazole), 7.21 (s, 2H, SO2NH2, D2O exchangeable), 7.23– 7.26 (m, 3H, ArH), 7.71–7.88 (m, 10H, ArH), 10.29 (s, 1H, NH, D2O exchangeable); 13CNMR (100.63 MHz, DMSOd6) d ppm: 59.25(1), 64.20(1), 119.22(2), 119.49(4), 127.09 (2), 127.15(4), 127.24(2), 138.60(1), 138.92(2), 142.10(2), 142.30(1), 162.50(1), 169.67(1); Anal. Calcd for C24H22N4O4S: C, 62.32; H, 4.79; N, 12.11; Found C, 62.17; H, 5.27; N, 12.15. Molecular docking All the molecular modeling studies were carried out on an Intel Pentium 1.6 GHz processor, 512 MB memory with Windows XP operating system using Molecular Operating Environment (MOE, 10.2008) software. All the minimizations were performed with MOE until a RMSD gradient of 0.05 kcal/mol Ao1 with MMFF94X force field and the partial charges were automatically calculated. The X-ray crystallographic structure of COX-2 enzyme with its cocrystallized ligand (Diclofenac) in the file (PDB ID: 1PXX) was obtained from the Protein Data Bank. The enzyme was prepared for docking studies where: (i) Ligand molecule was removed from the enzyme active site; (ii) hydrogen atoms were added to the structure with their standard geometry; (iii) MOE Alpha Site Finder was used for the active sites search in the enzyme structure, and dummy atoms were created from the obtained alpha spheres; (iv) the obtained model was then used in predicting the ligand–enzyme interactions at the active site. Biological screening Carrageenan-induced rat paw edema assay Male Wistar rats, each weighing 120–180 g, were purchased from the animal breeding unit of the National Ophthalmology Institute, Egypt. They were housed under appropriate conditions of controlled humidity, temperature, and light. The animals were allowed free access to water and were fed a standard pellet rat diet. The animals were kept at an ambient temperature of 22 °C 2 °C and a humidity of 65–70%. The study was conducted according to the guidelines for animal experiments set by Faculty of Chem Biol Drug Des 2014; 84: 473–488 Novel Hydrazones and Pyrazole Derivatives Pharmacy, Cairo University, in accordance with the international guidelines. A 1% suspension of carrageenan was prepared by sprinkling 1 g carrageenan powder in small amounts over the surface of 100 mL saline (NaCl 0.9% w/v), and the particles allowed to soak between additions. The suspension was then left at 37 °C for 2–3 h before use. Right paw is marked with ink at the level of lateral malleolus; basal paw volume is measured plethysmographically by volume displacement method using plethysmometer (UGO Basile 7140) by immersing the paw till the level of lateral malleolus. 0.05 mL of this suspension was injected into the subplantar surface of the right hind paw of the rat. The paw volume is measured again at 1, 2, 3, & 4 h after challenge. The increase in paw volume is calculated as percentage compared with the basal volume. The animals were randomized and divided into groups consists of six animals per group. The tested compounds and the reference drug (diclofenac) were given orally (5 mg/kg) 30 min prior to the intraplatar injection of carrageenan. The difference of average values between treated animals and control group is calculated for each time interval and evaluated statistically. The percent inhibition is calculated using the formula as follows: %edema inhibition ¼ ½1 ðVt=VcÞ 100 Vt and Vc are edema volume in the drug-treated and control groups, respectively (24). PGE2 inhibition in rat serum samples All synthesized compounds as well as diclofenac as the reference drug were given orally to male albino rats, and blood samples were taken after 2 h of administration. Serum samples were prepared by centrifugation where PGE2 concentrations were estimated using rat-specific immunoassay kit (25) using diclofenac as the reference drug. The assay was performed following the instructions in the leaflet of the kit (26). In vitro COX-1 and COX-2 inhibition assay COX-1 and COX-2 inhibitory activity was determined using COX (ovine) inhibitor screening assay enzyme-immuno assay (EIA) kit according to manufacturer’s instructions (27). COX catalyzes the first step in the biosynthesis of arachidonic acid to PGH2. The PGF2a produced from PGH2 by reduction with stannous chloride is measured by EIA. The compounds were dissolved in dimethylsulfoxide (DMSO). The enzymes COX-1 and COX-2 (10 lL), heme (10 lL), and samples (20 lL) were added to the supplied reaction buffer solution (950 lL, 0.1 M Tris–HCl, pH 8 containing 5 mM ethylenediaminetetraacetate (EDTA), and 2 mM phenol). The mixture of these solutions were incubated for a period of 10 min at 37 °C, after that COX reactions were initiated by adding arachidonic acid (10 lL, Chem Biol Drug Des 2014; 84: 473–488 making final concentration 100 lM) solution. The COX reactions were stopped by addition of HCl (1 M, 50 lL) after 2 min, and then saturated stannous chloride (100 lL) was added and again incubated for 5 min at room temperature. The PGF2a formed in the samples by COX reactions was quantified by EIA. The precoated 96-well plate was incubated with samples for 18 h at room temperature. After incubation, the plate was washed to remove any unbound reagent, and then Ellman’s reagent (200 lL), which contains substrate to acetylcholinesterase, was added and incubated for 60–90 min (until the absorbance of Bo well is in the range 0.3–0.8 A.U.) at room temperature. The plate was then read by an ELISA plate reader at 410 nm. The IC50 of inhibition of COX-1 and COX-2 was calculated by the comparison of the sample-treated incubations with control incubations. Celecoxib was used as reference standard in the study. Ulcerogenic effect Male albino rats (100–120 g) were fasted for 12 h prior to the administration of the compounds. The animals were divided into groups, each of six animals. The control group received 1% Tween-80 orally. Other groups received indomethacin or the tested compounds orally in two equal doses at 0 and 12 h for three successive days at a dose of 20 mg/kg per day. Animals were killed by diethyl ether 6 h after the last dose, and the stomach was removed. An opening at the greater curvature was made, and the stomach was cleaned by washing with cold saline and inspected with a 3 A magnifying lens for any evidence of hyperemia, hemorrhage, definite hemorrhagic erosion, or ulcer. An arbitrary scale was used to calculate the ulcer index which indicates the severity of the stomach lesions. Ulcers were classified into levels: level I, in which the ulcer area is <1 mm2; level II, in which the ulcer area is in the range from 1 to 3 mm2; and level III, in which the ulcer area equals 3 mm2. The ulcer index was calculated as 1 9 (number of ulcers of level I) + 2 9 (number of ulcers of level II) + 3 9 (number of ulcers of level III) (28). Results and discussion Chemistry Sulfanilamide 1 (CAS No. 63-74-1) was reacted with choloroacetylchloride in DMF in the presence of catalytic amounts of anhydrous sodium acetate to afford 2-chloroN-(4-sulfamoylphenyl)acetamide 2. Structure of compound 2 was verified by its reported melting point = 217 °C (29). Compound 2 was then reacted with hydrazine hydrate in absolute ethanol in the presence of catalytic amounts of anhydrous sodium acetate to obtain 2-hydrazinyl-N-(4-sulfamoylphenyl)acetamide 3. The structure of compound 3 was confirmed by elemental and spectral analyses. IR of compound 3 revealed bands at 3358 and 3280 cm1 corresponding to NH–NH2 groups and 1685 cm1 for 477 Mohammed and Nissan C=O. 1H-NMR spectrum in (DMSO-d6) of 3 exhibited a singlet signal at 3.43 ppm exchangeable by D2O corresponding to NH2 and another singlet at 7.89 ppm exchangeable by D2O corresponding to NH group. Condensation of compound 3 with different aromatic aldehydes in absolute ethanol led to the formation of the corresponding hydrazone derivatives 4–10. The structures of compounds 4–10 were confirmed by spectral and elemental analyses. 1H-NMR spectra in (DMSO-d6) of compounds 4–10 revealed singlet signals in the range of 8.50–8.74 ppm corresponding to N=CH proton, which is the result of the condensation reactions. The 1H-NMR spectra of compounds 4–10 were devoid of the singlet signals at 3.43 for the reacted NH2, and instead singlet signals appeared in the range of 7.82–7.88 ppm which was exchangeable by D2O for NH groups. Compounds 11–17 were synthesized through the reaction between the hydrazinyl derivative 3 with different aromatic chalcones in glacial acetic acid. The aromatic pyrazole derivatives were obtained rather than the pyrazoline derivatives as the reaction was conducted in glacial acetic acid not in absolute ethanol (30). The structures of compounds 11–17 were confirmed by elemental and spectral analyses. 1HNMR spectra in (DMSO-d6) of compounds 11–17 revealed singlet signals in the range of 6.62–6.92 ppm corresponding to CH at C4 of pyrazole ring. The spectra also lack the signals which correspond to NH–NH2 groups. There was no doublet or triplet signals in 1HNMR spectra in (DMSO-d6) of compounds 11–17, confirming the formation of aromatic pyrazole cyclization rather than pyrazoline formation (Scheme 1). Molecular docking In 2003, it was discovered that a series of anti-inflammatory drugs inhibits PGE2 synthesis by interacting with Tyr 385 and Ser 530 in the active site of COX-2 enzyme and compounds that could interact with such amino acids can exhibit anti-inflammatory activity (31). Interactions with Arg 120 may play also a role in the inhibitory activity (31). Tyr-385 is essential for the cyclooxygenase activity of PGH synthase and that nitration of this residue can be prevented by indomethacin. We conclude that Tyr 385 is at or near the cyclooxygenase active site of PGH synthase and the tyrosine residue could be involved in the first step of the cyclooxygenase reaction, which is the abstraction of the 13-proS hydrogen from arachidonate (32). Acetylation of Ser 530 of sheep prostaglandin endoperoxide (PGG/H) synthase by aspirin causes irreversible inactivation of cyclooxygenase enzyme. Thus, Ser 530 does lie in a highly conserved region, probably involved in cyclooxygenase catalysis (33). Arg 120 is located near the mouth of the hydrophobic channel that forms the cyclooxygenase active site of prostaglandin endoperoxide H synthases (PGHSs)-1 and -2. Arg 120 forms an ionic bond with the carboxylate group of arachidonate, and this interaction is an important contributor to the overall strength of 478 arachidonate binding to PGHS-1(34). The structure of arachidonic acid (substrate) co-crystallized with the active site of COX-2 is displayed in (Figures 1 and 2). In our attempts to predict the mechanism of action of the synthesized compounds as well as to evaluate their binding mode to the amino acids of the active site of COX-2 enzyme, molecular docking of all of the synthesized compounds was performed on the active site of COX-2 enzyme. The Protein Data Bank file with the code 1PXX was used for this purpose. The file contains COX-2 enzyme co-crystallized with diclofenac. All docking procedures were achieved by MOE (Molecular Operating Environment) software 10.2008 provided by chemical computing group, Canada. To perform accurate validation of the docking protocol, docking of the co-crystallized ligand (Diclofenac) should be carried out to study the scoring energy (S), root mean standard deviation (rmsd), and amino acid interactions. Docking was performed using London dG force, and refinement of the results was carried out using force-field energy. Diclofenac is fitted in the active site pocket with S = 11.9408 Kcal/mol and rsmd= 0.3682. Diclofenac interacts with the two amino acids Tyr 385 and Ser 530 by three hydrogen bonds, the bond length of the two hydrogen bonds with Ser 530 was 2.7 Ao and 2.9 A while that with Tyr 385 was 2.7 Ao as displayed in (Figures 3 and 4). Following the aforementioned docking protocol, all the synthesized compounds were docked on the active site of COX-2 enzyme. All the synthesized compounds were fit in the active site of the enzyme. Docking scores, amino acid interactions as well as hydrogen bond lengths were summarized in Table 1. All compounds were fit on the active site of COX-2 enzyme with an energy score ranging between 11.8629 and 13.6435 kcal/mol comparable with that of diclofenac. All of the synthesized compounds interacted with Ser 530 and/or Tyr 385 with one or more hydrogen bonds except for compounds 6, 7, and 11. The sulfonamide group which represents an acidic moiety for interaction was the most interacting group in all compounds except for compounds 6, 7, and 11. Compounds 9 and 17 showed the best docking score values with 13.4546 and 13.6435 Kcal/mol, respectively, while compound 11 showed the worst docking score value with 11.8629 Kcal/mol. Regarding the amino acid interactions between the synthesized compounds and the amino acids of the active site of COX-2 enzyme, the sulfonamide moiety interacted with Tyr 385 by one hydrogen bond in the case of compounds 3, 4, 8, 9, 10, 12, 13, and 16, while compounds 5, 14, 15, and 17 interacted with the same amino acid by two hydrogen bonds. Studying the interChem Biol Drug Des 2014; 84: 473–488 Novel Hydrazones and Pyrazole Derivatives Scheme 1: Reagents and solvents (a) anhydrous sodium acetate, absolute ethanol. (b) anhydrous sodium acetate, hydrazine hydrate, absolute ethanol. (c) absolute ethanol. (d) glacial acetic acid. action of the synthesized compounds with Ser 530, it was observed that compounds 4, 8, 10, 12, 15, 16, and 17 interacted with Ser 530 by one hydrogen bond while compounds 5 and 9 interacted with Ser 530 by two hydrogen bonds. Arg 120 interactions were observed in case of compounds 3 with two hydrogen bonds, compounds 6 and 7 with one hydrogen bond. Compound 11 was the only compound to interact with Tyr 355 with one hydrogen bond. Finally, an additional hydrogen bond was observed in case of compound 17 between the p-methoxy group and Tyr 115. Based on the pattern of diclofenac interactions with the amino acids of the active site of COX-2, good to moderate anti-inflammatory activity could be observed with compounds 3–17 except for compounds 6, 7, and 11. This conclusion could be postulated on the fact that the sulfonamide group which is the acidic moiety replacing carboxylChem Biol Drug Des 2014; 84: 473–488 ate in diclofenac should interact with the amino acids Tyr 385 and Ser 530, and as this is achieved in all compounds except 6, 7, and 11, weak or no anti-inflammatory activity could be observed for these compounds. On the other hand, good anti-inflammatory activity could be observed with compounds 5 and 17 as they interact with the amino acids of the active site of COX-2 enzyme with four and five hydrogen bonds, respectively. The 2D and 3D interaction descriptions of compounds 5 and 17 with the active site amino acids of COX-2 enzyme are displayed in (Figures 5–8), respectively. Biological screening In vivo anti-inflammatory activity All synthesized compounds 3–17 were evaluated for their in vivo anti-inflammatory activity by carrageenan-induced rat paw edema method (24). The protocol of animal exper479 Mohammed and Nissan involves prostaglandins as the mediators for the inflammation, any anti-inflammatory activity in the second phase of this biphasic event can be attributed to the inhibition of prostaglandin synthesis (24). All the synthesized compounds showed good to moderate anti-inflammatory activity especially after 4 h of administration except compounds 6, 7, and 11 with edema inhibition percentage values of 31%, 44%, and 25%, respectively. Compounds 5, 10, and 16 showed comparable activity with that of diclofenac with edema inhibition percentage values of 60%, 60%, and 61%, respectively. Compounds 12, 13, and 14 were slightly less active than diclofenac with edema inhibition percentage values of 57%, 50%, and 57%, respectively. On the other hand, compounds 3, 4, 8, 9, 15, and 17 were more active than diclofenac with edema inhibition percentage values of 68%, 71%, 67%, 84%, 68%, and 81%, respectively. Figure 1: Arachidonic acid 2D interaction description with active site of COX-2. iments was approved by ethical committee. Each test compound was dosed orally (5 mg/kg body weight) 30 min prior to induction of inflammation by carrageenan injection. Diclofenac was used as a reference anti-inflammatory drug, and anti-inflammatory activity was calculated at hourly intervals up to 4 h after injection and presented in Table 2 as the mean paw volume (mL) SE as well as the percentage edema inhibition. Considering the fact that carrageenan-induced paw edema assay is a biphasic event (25) involving the release of histamine and serotonin as the mediators of inflammation in the first phase which normally lasts for about 2 h after the carrageenan injection followed by the second phase which generally operates between 2 and 4 h after the carrageenan injection and It was obvious from the above results that the adapted design for the synthesis of novel anti-inflammatory agents has succeeded in the light of the biological results for the in vivo evaluation of anti-inflammatory activity for the synthesized compounds. The starting hydrazinyl derivative 3 showed better antiinflammatory activity than that of diclofenac with rising edema inhibition percentage value from 62% for diclofenac to 68% for the hydrazino derivative 3. Upon condensation with different aromatic aldehydes, the formed hydrazone derivatives 4–10 gave also active compounds, especially for the unsubstituted derivative 4 with edema inhibition percentage value 71%, p-N,N-dimethyl derivative 8 with edema inhibition percentage value of 67%, and the p-flouro derivative 9 with edema inhibition percentage value of 84% which was the best activity observed in this series. However, less active compounds Figure 2: Arachidonic acid 3D interaction description with active site of COX-2. 480 Chem Biol Drug Des 2014; 84: 473–488 Novel Hydrazones and Pyrazole Derivatives Figure 3: Diclofenac 2D interaction description with active site of COX-2. Figure 4: Diclofenac 2D interaction description with active site of COX-2. were achieved in case of p-bromo derivative 6 and pchloro derivative 7 with edema inhibition percentage values of 31% and 44%, respectively, while the o-flouro derivative 5 and the p-methoxy derivative 10 showed comparable activity with that of diclofenac with edema inhibition percentage value of 60%. The order of anti-inflammatory activity for hydrazone derivatives with its starting hydrazino derivative 3 could be summarized in Table 3. Chem Biol Drug Des 2014; 84: 473–488 The second series in this research was the pyrazole derivatives 11–17. In this series, the observed anti-inflammatory activities were good to moderate activities except for the unsubstituted derivative 11 with edema inhibition percentage value of 25%. Moderate activity was observed for o-flouro derivative 12, p-bromo derivative 13, and the p-chloro derivative 14 with edema inhibition percentage values of 57%, 50%, and 57%, respectively. The p-flouro derivative 16 showed comparable activity with that of diclofenc with edema inhibition percentage value of 61%, while the p-N,N481 Mohammed and Nissan Table 1: Binding scores and amino acid interactions of the docked compounds on the active site of COX-2 enzyme Compound No. S Kcal/Mol Amino acid interactions Interacting groups H bond length Ao 3 12.5870 4 12.7512 5 12.1160 6 7 8 11.9050 12.0361 13.2910 9 13.4546 10 12.5816 11 12 11.8629 12.5105 13 14 15 11.9204 12.0730 12.8598 16 12.2798 17 13.6435 Tyr 385 Arg 120 Tyr 385 Ser 530 Tyr 385 Ser 530 Arg 120 Arg 120 Tyr 385 Ser 530 Tyr 385 Ser 530 Tyr 385 Ser 530 Tyr 355 Tyr 385 Ser 530 Tyr 385 Tyr 385 Tyr 385 Ser 530 Tyr 385 Ser 530 Tyr 385 Ser 530 Tyr 115 SO2NH2 NH, NH2 SO2NH2 SO2NH2 SO2NH2 SO2NH2 C=NH C=NH SO2NH2 SO2NH2 SO2NH2 SO2NH2 SO2NH2 SO2NH2 C=O SO2NH2 SO2NH2 SO2NH2 SO2NH2 SO2NH2 SO2NH2 SO2NH2 SO2NH2 SO2NH2 SO2NH2 OCH3 2.95 2.92, 2.67 2.98 2.65, 1.48, 2.88 2.75 2.39 1.50 2.28 2.10, 2.47 1.51 3.00 1.96 2.34 1.90 2.36, 2.78, 3.12 2.88 1.79 2.38, 2.23 2.82 3.02 2.72 2.98 2.35 2.74 2.29 2.98 dimethyl derivative 15 and p-methoxy derivative 17 showed better activity than diclofenac with edema inhibition percentage values of 68% and 81%, respectively. The order of anti-inflammatory activity for the pyrazole derivatives could be summarized in Table 4. Comparing the anti-inflammatory activity of hydrazone derivatives 4–10 and their pyrazole analogs 11–17, it was obvious that the anti-inflammatory activity of the p-bromo pyrazole derivative 13 and p-chloro pyrazole derivative 14 was much better than that of their hydrazone derivative analogs 6 and 7 as the edema inhibition percentage values reach 57% and 50% for 13 and 14, respectively, instead of 31% and 44% for 6 and 7, respectively. In case of p-methoxy pyrazole derivative 17, the anti-inflammatory was remarkably increased with edema inhibition percentage of 81% which was better than that of diclofenac instead of 60% for its hydrazone derivative analog 10. However, this was not the case for the rest of pyrazole derivatives compared with their hydrazone derivative analogs as the activity decreased especially for the unsubstituted pyrazole derivative 11 with edema inhibition percentage value of 25% instead of 71% for its hydrazone derivative analog 4. 482 Evaluation of PGE2 inhibition in rat serum samples After in vivo evaluation of anti-inflammatory activity, it was interesting to measure the inhibition of PGE2 production that could contribute to the inhibition of COX-1 and COX-2 enzymes (25). The assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody (specific for rat PGE2) has been precoated into a micro plate. Standards, controls, and samples are pipetted into the wells, and any rat PGE2 present in the solutions is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked monoclonal antibody specific for PGE2 [PGE2 conjugate] is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells. The enzyme reaction yields a blue product that turns yellow when the stop solution is added. The intensity of the color measured is in proportion to the amount of rat PGE2 bound in the initial step. The sample values are then read off the standard curve. All synthesized compounds 3–17 were given orally to male albino rats, and blood samples were taken after 2 h of administration. Serum samples were prepared by centrifugation where PGE2 concentrations were estimated using rat-specific immunoassay kit (26) using diclofenac as the reference drug. Results of PGE2 concentrations SE as well as percentage of inhibition are listed in Table 5. Compounds 9 and 17 showed the highest inhibition percentage of PGE2 production in serum samples with inhibition percentage values of 78.12% and 76.49%, respectively. These compounds were also the most active compounds in rat paw edema evaluation with edema inhibition percentage values of 84% and 81%, respectively. On the other hand, the least active compounds in rate paw edema test such as compounds 6, 7, and 11 showed low percentage inhibition of PGE2 production with values of 16.12%, 37.22%, and 17.85%, respectively. From these results, we can assume that COX enzyme inhibition could be the mechanism of action for the antiinflammatory activity of the newly synthesized compounds. In vitro COX-1 and COX-2 inhibition assay To study the mechanism of action of the newly synthesized compounds and whether their anti-inflammatory effect is due to COX enzyme inhibition, in vitro assay for COX-1 and COX-2 enzymes inhibition was conducted. The most active compounds were screened using EIA kit (Cayman Chemical Company, Ann Arbor, MI, USA). The COX-1 and COX-2 inhibitory activities of compounds were evaluated by method as described by Gautam et al. (35). IC50 values for the most active compounds were determined using celecoxib as the reference drug. Furthermore, selectivity index for COX-1/COX-2 inhibition was calculated for each compound. IC50 values for COX-1 and COX-2 inhibitions as well as selectivity index were listed in Table 6. Chem Biol Drug Des 2014; 84: 473–488 Novel Hydrazones and Pyrazole Derivatives Figure 5: Compound 5 2D interaction description with active site of COX-2. Figure 6: Compound 5 3D interaction description with active site of COX-2. Chem Biol Drug Des 2014; 84: 473–488 483 Mohammed and Nissan Figure 7: Compound 17 2D interaction description with active site of COX-2. Figure 8: Compound 17 3D interaction description with active site of COX-2. All the tested compounds showed inhibitory effect on both COX-1 and COX-2 enzymes with IC50 values in the range of 13.26–47.96 lM for COX-1 and in the range of 1.73– 11.74 lM with selectivity index (SI) ranging between 4.0 and 11.1. By studying the results in Table 6, we can notice that the selectivity index (SI) for the hydrazone derivatives 5, 8, 9, and 10 were 4.9, 8.7, 7.6, and 7.0, respectively. These val484 ues have improved with their pyrazole analogs 12, 15, 16, and 17 to rise to 6.5, 8.8, 9.4, and 11.1, respectively. The selectivity index of the active synthesized compounds cannot be compared with that of the reference drug (celecoxib), but although high selectivity is demanded to reduce the gastrointestinal side-effects, it was discussed recently that COX-2 high selectivity could be the major cause of cardiac and renal problems (5, 6) and hence Chem Biol Drug Des 2014; 84: 473–488 Novel Hydrazones and Pyrazole Derivatives Table 2: Rat paw edema volume and percentage of inhibition Volume of edema (mL) 1h 2h Compound (Edema inhibition %) Control Diclofenac 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 35.26 35.03 30.19 9.653*,** 28.91 31.22 31.07 22.1*,** 19.07*,** 18.86*,** 41.33 39.3 26.19 17.02*,** 24.49*,** 20.5*,** 9.925*,** 2.707 1.809 (1) 3.141 (14) 3.337 (72) 2.168 (18) 2.63 (11) 2.671 (12) 1.262 (37) 2.019 (45) 1.041 (46) 1.168 (0) 3.181 (0) 3.395 (25) 2.3 (51) 2.533 (30) 1.477 (41) 2.574 (71) 3h 66.3 53.29* 36.37*,** 14.48*,** 44.6* 49.45* 37.73*,** 37.55*,** 37.21*** 25*,** 59.2 40.84*,** 32.63*,** 21.7*,** 23.71*,** 24.4*,** 14.99*,** 2.357 1.835 (20) 2.476 (45) 3.192 (78) 4.377 (33) 3.667 (25) 1.695 (43) 1.826 (43) 3.446 (44) 1.587 (62) 1.514 (11) 1.89 (38) 3.106 (50) 2.356 (67) 1.522 (64) 1.022 (63) 2.084 (77) 4h 83.51 50.21* 44.1* 32.57*,** 47.97* 54.6* 59.57* 36.61*,** 26.52*,** 44.88* 58.28* 45.35* 53.63* 26.78*,** 28.68*,** 44.14* 19.48*,** Values were expressed as mean SEM of six rats. Statistical analysis was performed by *Significantly different from control at p < 0.05. **Significantly different from diclofenac at p < 0.05. ANOVA 2.523 3.206 (40) 2.389 (47) 3.524 (60) 4.824 (43) 3.423 (35) 1.483 (29) 1.814 (56) 5.994 (68) 2.219 (46) 3.589 (30) 2.41 (45) 3.049 (36) 2.509 (67) 2.235 (65) 1.111 (47) 1.519 (76) 91.37 34.28* 29.24* 25.95* 36.96* 62.82*,** 51.12*,** 30.25* 14.33*** 37.16* 68.65*,** 39.55* 45.52*,** 39.63* 28.42* 34.97* 16.75*,** 3.092 1.617 (62) 3.21 (68) 2.967 (71) 3.325 (60) 4.507 (31) 1.701 (44) 1.6 (67) 1.948 (84) 1.665 (60) 1.115 (25) 2.759 (57) 3.255 (50) 2.474 (57) 2.519 (68) 2.065 (61) 2.82 (81) followed by Tukey’s post hoc test. Table 3: The anti-inflammatory activity of compounds 3–10 in descending order Compound No. H N N H O H 2NO2 S N H H 2NO2 S O N H N moderate selectivity could reduce the risk of both gastrointestinal and cardiac side-effects (7). Ulcerogenic effect The most active compounds 3-5, 8, 9, and 12–17 were evaluated for their ulcerogenic effects in rats, and indoChem Biol Drug Des 2014; 84: 473–488 4-F 84 4 H 71 3 R H N 9 NH 2 O H2 NO 2S Inhibition % R N H N R 8 5 10 7 6 68 4-N(CH3)2 2-F 4-OCH3 4-Cl 4-Br 67 60 60 44 31 methacin was used as the reference standard. The results were expressed by the number of ulcers SE as well as ulcer index SE (28) as shown in Table 7. All of the tested compounds were less ulcerative than indomethacin. Compounds 4, 5, 10, 12, and 14 were ulcerative in a comparable manner with indomethacin, while compounds 3, 8, 9, 13, 14, 15, 16, and 17 were much less ulcerative than 485 Mohammed and Nissan Table 4: The anti-inflammatory activity of compounds 11–17 in descending order Compound H N H 2NO2 S N N Ph O No. R Inhibition % 17 15 16 12 14 13 11 4-OCH3 4-N(CH3)2 4-F 2-F 4-Cl 4-Br H 81 68 61 57 57 50 25 R Table 5: Concentration and inhibition percentage of PGE2 in serum samples Group PGE2 (Pg/mL) Normal control (1% Tween-80) Carrageenan (1%Tween-80) Diclofenac 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1.98 49.81*,** 11.8*,** 22.31*,** 23.91*,** 30.21*,** 41.78*,** 31.27*,** 25.78*,** 10.9*,** 28.97*,** 40.92* 30.21*,** 29.35*,** 31.75*,** 21.32*,** 24.19*,** 11.71*,** 0.50 2.58 1.81 2.97 2.98 3.51 3.51 3.09 3.18 1.93 3.21 4.15 3.25 2.99 3.02 2.27 1.99 1.18 IC50 (lM) Inhibition (%) 76.31 55.21 52.00 39.35 16.12 37.22 48.24 78.12 41.84 17.85 39.35 41.08 36.26 57.20 51.44 76.49 Values were expressed as mean SEM of six rats. Statistical analysis was performed by ANOVA followed by Tukey’s post hoc test. *Significantly different from normal control at p < 0.05. **Significantly different from carrageenan at p < 0.05. indomethacin. These less ulcerative compounds showed good selectivity index (SI) of COX -1/COX-2 inhibition with values of 7.4, 8.7, 7.6, 8.3, 7.0, 8.8, 9.4, and 11.1, respectively. Comparing the ulcerative effect of the tested hydrazone derivative compounds 5, 8–10 with the ulcerative effect of their pyrazole analogs 12, 15–17 may led to the conclusion that cyclization to pyrazole did not much decrease the ulcerative effect except for compound 10 with an ulcer index value of 12.4 which has markedly decrease in case of its pyrazole analog 17 as the ulcer index was decreased to 2.79. 486 Table 6: COX-1 and COX-2 inhibition IC50 and selectivity index (SI) Compound COX-1 COX-2 Selectivity index 3 4 5 8 9 10 12 13 14 15 16 17 Celecoxib 15.89 24.06 28.34 19.91 13.26 18.70 24.01 20.59 47.69 14.86 21.09 19.61 >30 2.12 3.51 5.68 2.26 1.73 2.65 3.64 2.46 11.74 1.68 2.22 1.76 0.22 7.4 6.8 4.9 8.7 7.6 7.0 6.5 8.3 4.0 8.8 9.4 11.1 >200 Conclusion In the light of biological results, we can conclude that the hydrazino derivative 3 synthesized hydrazone derivatives 4–10 with the exception of 5 & 6 and the synthesized pyrazole derivatives 12–17 could represent good candidates for the search for novel anti-inflammatory agents urged by their good anti-inflammatory activity in vivo and in vitro assays and the low ulcerative effect of most of the synthesized compounds. Compound 17 has edema inhibition percentage value of 81% better than that of diclofenac, percentage of inhibition of PGE2 production value of 76.49% with slight increase than that of diclofenac, the best selectivity index among the synthesized compounds with a value of 11.1 and much less ulcerative effect with ulcer index value of 2.79 compared with 12.82 for indomethacin. Molecular docking study for compound 17 showed good energy score and interactions with Tyr 385 and Ser 530 by three hydrogen bonds and additional hydrogen bond with Tyr 115 with the methoxy group. Chem Biol Drug Des 2014; 84: 473–488 Novel Hydrazones and Pyrazole Derivatives Table 7: Ulcer numbers and index for the most active compounds Compound Normal control (1% Tween-80) Ulcer control (indomethacin 20 mg/kg) 3 4 5 8 9 10 12 13 14 15 16 17 Ulcer number (mean SE) U.I. (mean SE) 0.00 0.00 0.00 0.00 8.62* 0.50 12.82* 0.29 3.56*,** 8.29* 8.35* 1.88** 1.92** 8.22* 8.3* 3.60*,** 8.12* 1.85*,** 3.65*,** 2.47*,** 0.31 0.81 0.73 0.29 0.21 0.31 0.56 0.33 0.59 0.19 0.32 0.43 3.7*,** 12.27* 12.31* 3.39*,** 3.45*,** 12.4* 12.29* 3.71*,** 12.3* 3.85*,** 3.69*,** 2.79*,** 0.27 0.99 1.03 0.31 0.37 0.70 1.1 0.24 0.47 0.31 0.33 0.29 Values were expressed as mean SEM of six rats. Statistical analysis was performed by ANOVA followed by Tukey’s post hoc test. *Significantly different from normal control at p < 0.05. **Significantly different from ulcer control (indomethacin 20 mg/kg) at p < 0.05. Acknowledgments The authors thank Dr. Aymen El-Sahar, assistant lecturer of Pharmacology, Faculty of Pharmacy, Cairo University, for his efforts in performing in vivo biological screening and Dr. Waleed Ali, associate professor of Biochemistry, Faculty of Medicine, Cairo University, for his work in performing in vitro assays. 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Figure S9. 13 C-NMR spectrum of compound 6. Figure S10. IR spectrum of compound 9. Figure S11. 1H-NMR spectrum of compound 9. Figure S12. 13 C-NMR spectrum of compound 9. Figure S13. 1H-NMR spectrum of compound 12. Figure S14. 13 C-NMR spectrum of compound 12. Figure S15. 1H-NMR spectrum of compound 14. Figure S16. IR spectrum of compound 16. Figure S17. 1H-NMR spectrum of compound 16. Figure S18. 13 C-NMR spectrum of compound 16. Figure S19. IR spectrum of compound 14. Chem Biol Drug Des 2014; 84: 473–488