Accepted Article

Received Date : 22-Feb-2014 Revised Date : 26-Mar-2014 Accepted Date : 03-Apr-2014 Article type

: Research Article

Synthesis, Molecular Docking and Biological Evaluation of Some Novel Hydrazones and Pyrazole Derivatives as Anti-inflammatory Agents

Khaled Omar Mohammed1 and Yassin Mohammed Nissan*2 1

Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo University.

2

Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University.

Kasr Elini St., Cairo 11562, Egypt.

*Corresponding author: Y. M. Nissan, Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr Elini Street., Cairo 11562, Egypt. Tel.: +201000848844; E-mail address: [email protected].

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/cbdd.12336 This article is protected by copyright. All rights reserved.

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Abstract 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 COX1/ COX-2 inhibition in vitro. Keywords: 4-benzenesulfonamide, hydrazone, pyrazole, anti-inflammatory

Introduction Although of the high effectiveness of the steroidal anti-inflammatory 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 non-steroidal anti-inflammatory drugs. In spite of their great safety over the steroidal drugs they still have some side effects, the most common one is the development of peptic and duodenal ulcer on long term use 2. Moreover, 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

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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 well known 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 anti-inflammatory 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. H2NO 2S

H 2NO2 S

N

O

N CF3

Celecoxib

N

Valdecoxib

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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 inhibition, 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 in order to predict their binding mode to the amino acids of the active site of the enzyme.

H N

N

H N

Ar

N

N H O

H 2NO2 S

N Ph

O

H2 NO 2S

Ar

A

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

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Fourier transform (FT)- IR spectrophotometer as KBr discs. NMR spectra (in DMSO-d6) were recorded on Bruker AC-300 Ultra Shield NMR spectrometer (Bruker, Flawil, Switzerland, δ ppm) at 400 MHz using TMS as internal Standard and peak multiplicities are designed as follows: s, singlet; d, doublet; t, triplet; m, multiplet. Silica gel used for column chromatography was obtained from Fluka, 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 choloro 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 three hours. 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) (cm-1): 3358-3332 (2NH2), 3280-3196 (2NH), 3100-3052 (arom.CH), 2924 (aliph.CH), 1685(C=O); 1HNMR(400MHz, DMSOd6) δ ppm: 3.43(s, 2H, NH2, D2O exchangeable), 4.37(s, 2H, 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) δ 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

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hours, 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) (cm-1): 3373-3332 (NH2), 3250-3194 (2NH) 3131-3057 (arom. CH), 2954 (aliph. CH), 1689 (C=O); 1HNMR (400 MHz, DMSO-d6) δ ppm: 4.46 (s, 2H, CH2), 6.93-7.22 (m, 3H, ArH), 7.27 (s, 2H, SO2NH2, D2Oexchangeable),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);

13

CNMR (100.63 MHz, DMSO-d6) δ 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-(4-sulfamoylphenyl)acetamide 5 Yield: 70 %; mp 202-204 °C; IR (KBr) (cm-1): 3338-3329 (NH2), 3267-3225 (2NH), 3107-3051 (arom. CH), 2924 (aliph. CH), 1701 (C=O); 1HNMR (400 MHz, DMSO-d6) δ 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);

13

CNMR (100.63 MHz, DMSO-d6) δ 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.

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2-(2-(4-Bromobenzylidene)hydrazinyl)-N-(4-sulfamoylphenyl)acetamide 6 Yield: 65 %; mp 188-190 °C; IR (KBr) (cm-1): 3404-3354 (NH2), 3261-3186 (2NH), 3101-3049 (arom. CH), 2924 (aliph. CH), 1689 (C=O); 1HNMR (400 MHz, DMSO-d6) δ ppm: 4.46 (s, 2H, CH2), 7.15-7.23 (m, 2H, ArH),7.27 (s, 2H, SO2NH2, D2Oexchangeable), 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) δ 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-(4-sulfamoylphenyl)acetamide 7 Yield: 70 %; mp 192-194 °C; IR (KBr) (cm-1): 3404-3354 (NH2), 3261-3186 (2NH), 3097-3049 (arom CH), 2954 (aliphatic CH), 1689 (C=O); 1HNMR (400 MHz, DMSO-d6) δ ppm: 4.46 (s, 2H, CH2), 7.21 (s, 2H, SO2NH2, D2Oexchangeable), 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);

13

CNMR (100.63 MHz, DMSO-d6) δ 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.

2-(2-(4-(Dimethylamino)benzylidene)hydrazinyl)-N-(4-sulfamoylphenyl)acetamide 8 Yield: 75 %; mp 162-164 °C; IR (KBr) (cm-1): 3367-3329 (NH2), 3246-3178 (2NH), 3097-3039 (arom. CH), 3005-2920 (aliph. CH), 1701 (C=O); 1HNMR (400 MHz, DMSO-d6) δ ppm: 2.88 (s,

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6H, 2CH3), 4.37 (s, 2H, CH2), 6.64-6.66 (m, 2H, ArH), 7.27 (s, 2H, SO2NH2, D2Oexchangeable), 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) δ 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-(2-(4-Fluorobenzylidene)hydrazinyl)-N-(4-sulfamoylphenyl)acetamide 9 Yield: 80 %; mp 185-187 °C; IR (KBr) (cm-1): 3356-3320 (NH2), 3263-3220 (2NH), 3105-3051 (arom. CH), 2954 (aliph. CH), 1687 (C=O); 1HNMR (400 MHz, DMSO-d6) δ 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);

13

CNMR (100.63 MHz, DMSO-d6) δ 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); 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-(4-sulfamoylphenyl)acetamide 10 Yield: 60 %; mp 178-180 °C; IR (KBr) (cm-1): 3442-3352 (NH2), 3263-3205 (2NH), 3105-3049 (arom. CH), 3008- 2954 (aliph. CH), 1693 (C=O); 1HNMR (400 MHz, DMSO-d6) δ 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) δ 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),

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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 prepration oc 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 hours. The formed preciptate was filtered, washed, dried and crystallized from DMF/H2O to give the pyrazole derivatives 11-17, respectively.

2-(3,5-Diphenyl-1H-pyrazol-1-yl)-N-(4-sulfamoylphenyl)acetamide 11 Yield: 50 %; mp 223-225 dec °C; IR (KBr) (cm-1): 3355-3331 (NH2), 3276 (NH), 3103-3085 (arom. CH), 2856 (aliph. CH), 1687 (C=O); 1HNMR (400 MHz, DMSO-d6) δ 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 ); 13

CNMR (100.63 MHz, DMSO-d6) δ 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-(5-(2-Fluorophenyl)-3-phenyl-1H-pyrazol-1-yl)-N-(4-sulfamoylphenyl)acetamide 12 Yield: 43 %; mp 217-219 °C; IR (KBr) (cm-1): 3353-3325 (NH2), 3278 (NH), 3105-3055 (arom. CH), 2954 (aliph. CH), 1685 (C=O); 1HNMR (400 MHz, DMSO-d6) δ 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), 7.727.74 (m, 2H, ArH), 7.81-7.85 (m, 8H, ArH), 10.77(s, 1H, NH, D2O exchangeable ); 13CNMR

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(100.63 MHz, DMSO-d6) δ 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 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) (cm-1): 3354-3332 (NH2), 3278 (NH), 3103-3050 (arom. CH), 2955 (aliph. CH), 1685 (C=O); 1HNMR (400 MHz, DMSO-d6) δ 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, DMSOd6) δ 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) (cm-1): 3356-3332 (NH2), 3280 (NH), 3104-3053 (arom. CH), 2956 (aliph. CH), 1684 (C=O); 1HNMR (400 MHz, DMSO-d6) δ 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, DMSOd6) δ 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.

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2-(5-(4-(Dimethylamino)phenyl)-3-phenyl-1H-pyrazol-1-yl)-N-(4sulfamoylphenyl)acetamide 15 Yield: 58 %; mp 168-170 °C; IR (KBr) (cm-1): 3356-3334 (NH2), 3273 (NH), 3103-3059 (arom. CH), 2924 (aliph. CH), 1697 (C=O); 1HNMR (400 MHz, DMSO-d6) δ 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 );

13

CNMR

(100.63 MHz, DMSO-d6) δ 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) (cm-1): 3357-3332 (NH2), 3280 (NH), 3107-3054 (arom. CH), 2950 (aliph. CH), 1687 (C=O); 1HNMR (400 MHz, DMSO-d6) δ ppm: 4.57 (s, 2H, CH2), 6.63 (s, 1H, C4 pyrazole), 7.22 (s, 2H, SO2NH2, 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, DMSOd6) δ 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) (cm-1): 3358-3332 (NH2), 3278 (NH), 3099-3055 (arom. CH), 2954 (aliph. CH), 1685 (C=O); 1HNMR (400 MHz, DMSO-d6) δ ppm: 3.60 (s, 3H, OCH3),

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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 );

13

CNMR

(100.63 MHz, DMSO-d6) δ 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-1Ao-1with MMFF94X force field and the partial charges were automatically calculated. The X-ray crystallographic structure of COX-2 enzyme with its co-crystallized 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 gm, 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

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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 Pharmacy, Cairo University in accordance with the international guidelines. A 1 % suspension of carrageenan was prepared by sprinkling 1g 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 hours 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 sub plantar surface of the right hind paw of the rat. The paw volume is measured again at 1, 2, 3 & 4 hours 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 6 animals per group. The tested compounds and the reference drug (Diclofenac) were given orally (5 mg/ Kg) 30 minutes 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 = [1- (Vt / Vc)] X 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 2hr of administration. Serum samples

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were prepared by centrifugation where PGE2 concentrations were estimated using rat specific immunoassay kit

25

using Diclofenac as the reference drug. Based on experimental

trials, no dilution prior to the assay of serum PGE2. 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 EIA kit according to manufacturer’s instructions 27. COX catalyzes the first step in the biosynthesis of arachidonic acid to PGH2. The PGF2α produced from PGH2 by reduction with stannous chloride is measured by EIA. The compounds were dissolved in dimethylsulfoxide (DMSO). The enzyme COX-1 and COX-2 (10 µL), heme (10 µL) and samples (20 µL) were added to the supplied reaction buffer solution (950 µL, 0.1 M Tris–HCl, pH 8 containing 5 mM ethylenediamine tetra acetate (EDTA) and 2 mM phenol). The

mixture of these

solutions were incubated for a period of 10 min at 37 oC, after that COX reactions were initiated by adding arachidonic acid (10 µL, making final concentration 100 µM) solution. The COX reactions were stopped by addition of HCl (1 M, 50 µL) after 2 min and then saturated stannous chloride (100 µL) was added and again incubated for 5 min at room temperature. The PGF2α formed in the samples by COX reactions was quantified by EIA. The pre-coated 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 µL), which contains substrate to acetyl cholinesterase, 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 to control incubations. Celecoxib was used as reference standard in the study.

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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 6 animals. The control group received 1% teween 80 orally. Other groups received Indomethacin or the tested compounds orally in 2 equal doses at 0 and 12 h for 3 successive days at a dose of 20 mg/kg per day. Animals were sacrificed 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 Å 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 less than 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 × (number of ulcers of level I) + 2 × (number of ulcers of level II) + 3 × (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-chloro-N-(4sulfamoylphenyl)acetamide 2. Structure of compound 2 was verified by its reported melting point = 217 oC. 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 cm-1

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corresponding to NH-NH2 group and 1685 cm-1 for 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. 1H-NMR 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 1H-NMR 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

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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 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 & 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

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and refinement of the results was done 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 two hydrogen bonds lengths with Ser 530 were 2.7 Ao and 2.9 A while that with Tyr 385 was 2.7 Ao as displayed in (Figures 3 & 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 acids 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 to -13.6435 Kcal/mol comparable to 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 acids 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 bonds 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 bond. Studying the interaction 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

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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 pmethoxy 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 317 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 carboxylate 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 description 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 experiments 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

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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)±S.E. as well as the percentage edema inhibition. Considering the fact that carrageenaninduced 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 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 four hours 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 to 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.

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 anti-inflammatory 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

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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 were achieved in case of p-bromo derivative 6 and p-chloro 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 to 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.

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 to that of Diclofenc with edema inhibition percentage value of 61% while the p-N,N-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 analogues 11-17, It was obvious that the anti-inflammatory activity of the p-bromo pyrazole

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derivative 13 and p-chloro pyrazole derivative 14 were much better than that of their hydrazone derivative analogues 6 and 7 as the edema inhibition percentage values reaches 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 analogue 10. However, this was not the case for the rest of pyrazole derivatives compared to their hydrazone derivative analogues 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 analogue 4.

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 pre-coated 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.

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All synthesized compounds 3-17 were given orally to male albino rats and blood samples were taken after 2hr 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 ± S.E. as well as percentage of inhibition are listed in Table 5. Compound 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 anti-inflammatory activity of the newly synthesized compounds.

In vitro COX-1 and COX-2 inhibition assay In order 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 by using enzyme-immuno assay (EIA) kit (Cayman Chemical Company, USA). The COX-1 and COX-2 inhibitory activity 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

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was calculated for each compound. IC50 values for COX-1 and COX-2 inhibition as well as selectivity index were listed in Table 6. 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 µM for COX-1 and in the range of 1.73-11.74 µM with selectivity index (SI) ranging between 4.0-11.1. By studying the results in table 6 we can notice that the selectivity index (SI) for the hydrazone derivative derivatives 5, 8, 9 and 10 were 4.9, 8.7, 7.6 and 7.0, respectively. These values have improved with their pyrazole analogues 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 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 Indomethacin was used as the reference standard. The results were expressed by the number of ulcers±S.E. as well as ulcer index±S.E 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 to Indomethacin while compounds 3, 8, 9, 13, 14, 15, 16 and 17 were much less ulcerative than 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.

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Comparing the ulcerative effect of the tested hydrazone derivative compounds 5, 8-10 with the ulcerative effect of their pyrazole analogues 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 analogue 17 as the ulcer index was decreased to 2.79.

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 antiinflammatory 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 to 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.

Acknowledgement The authors thanks Dr. Aymen El-Sahar, assistant lecturer of Pharmacology, Faculty of Pharmacy, Cairo University for his efforts in performing in vivo biological screening and Dr.

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Waleed Ali, associate professor of Biochemistry, Faculty of Medicine, Cairo University for his work in performing in vitro assays.

Conflict of interest The authors declare that they have no conflict of interset

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34- Rieke C., Mulichak A., Garavito R., Smith W. (1999) The Role of Arginine 120 of Human Prostaglandin Endoperoxide H Synthase-2 in the Interaction with Fatty Acid Substrates and Inhibitors. J Biol Chem; 274:17109-17114. 35- Gautam R., Karkhile K.V., Bhutani K.K., Jachak S.M. (2010) Anti-inflammatory, cyclooxygenase (COX)-2, COX-1 inhibitory, and free radical scavenging effects of Rumex nepalensis. Planta Med; 76:1564-1569.

Captions Figure 1. Arachidonic acid 2D interaction description with active site of COX-2 Figure 2. Arachidonic acid 3D interaction description with active site of COX-2 Figure 3. Diclofenac 2D interaction description with active site of COX-2 Figure 4. Diclofenac 2D interaction description with active site of COX-2 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 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 Table 1. Binding scores and amino acid interactions of the docked compounds on the active site of COX-2 enzyme. Table 2. Rat paw edema volume and percentage of inhibition Table 3. The anti-inflammatory activity of compounds 3-10 in descending order Table 4. The anti-inflammatory activity of compounds 11-17 in descending order Table 5. Concentration and inhibition percentage of PGE2 in serum samples

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Table 6. COX-1 and COX-2 inhibition IC50 and selectivity index (SI) Table 7. Ulcer numbers and index for the most active compounds 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.

Table 1. Binding scores and amino acid interactions of the docked compounds on the active site of COX-2 enzyme.

Compound No.

S

Amino acid interactions

Interacting groups

H bond length

Kcal/Mol

Ao 3

4

-12.5870

-12.7512

Tyr 385

SO2NH2

2.95

Arg 120

NH, NH2

2.92, 3.02

Tyr 385

SO2NH2

2.67

Ser 530

SO2NH2

2.98

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5

-12.1160

Tyr 385

SO2NH2

2.65, 2.72

Ser 530

SO2NH2

1.48, 2.98

6

-11.9050

Arg 120

C=NH

2.88

7

-12.0361

Arg 120

C=NH

2.75

8

-13.2910

Tyr 385

SO2NH2

2.39

Ser 530

SO2NH2

1.50

Tyr 385

SO2NH2

2.28

Ser 530

SO2NH2

2.10, 2.35

Tyr 385

SO2NH2

2.47

Ser 530

SO2NH2

1.51

9

10

-13.4546

-12.5816

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11

-11.8629

Tyr 355

C=O

3.00

12

-12.5105

Tyr 385

SO2NH2

1.96

Ser 530

SO2NH2

2.34

13

-11.9204

Tyr 385

SO2NH2

1.90

14

-12.0730

Tyr 385

SO2NH2

2.36, 2.74

15

-12.8598

Tyr 385

SO2NH2

2.78, 2.29

Ser 530

SO2NH2

3.12

Tyr 385

SO2NH2

2.88

Ser 530

SO2NH2

1.79

Tyr 385

SO2NH2

2.38, 2.98

Ser 530

SO2NH2

2.23

16

17

-12.2798

-13.6435

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Tyr 115

OCH3

2.82

Table 2. Rat paw edema volume and percentage of inhibition

Volume of edema (mL) Compou 1hr

2 hr

3 hr

4 hr

nd (Edema inhibition %) Control

35.26±2.707

66.3±2.357

83.51±2.523

91.37±3.092

Diclofena

35.03±1.809

53.29*±1.835

50.21*±3.206

34.28*±1.617

c

(1%)

(20%)

(40%)

(62%)

3

30.19±3.141

36.37*@±2.476

44.1*±2.389

29.24*±3.21

(14%)

(45%)

(47%)

(68%)

14.48*@±3.192

32.57*@±3.524

25.95*±2.967

(78%)

(60%)

(71%)

28.91±2.168

44.6*±4.377

47.97*±4.824

36.96*±3.325

(18%)

(33%)

(43%)

(60%)

31.22±2.63

49.45*±3.667

54.6*±3.423

62.82*@±4.507

(11%)

(25%)

(35%)

(31%)

9.653*@±3.3 4

37 (72%)

5

6

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31.07±2.671

37.73*@±1.695

59.57*±1.483

51.12*@±1.701

(12%)

(43%)

(29%)

(44%)

37.55*@±1.826

36.61*@±1.814

30.25*±1.6

(43%)

(56%)

(67%)

37.21*@±3.446

26.52*@±5.994

14.33*@±1.948

(44%)

(68%)

(84%)

25*@±1.587

44.88*±2.219

37.16*±1.665

(62%)

(46%)

(60%)

41.33±1.168

59.2±1.514

58.28*±3.589

68.65*@±1.115

(0%)

(11%)

(30%)

(25%)

39.3±3.181

40.84*@±1.89

45.35*±2.41

39.55*±2.759

(0%)

(38%)

(45%)

(57%)

26.19±3.395

32.63*@±3.106

53.63*±3.049

45.52*@±3.255

(25%)

(50%)

(36%)

(50%)

17.02*@±2.3

21.7*@±2.356

26.78*@±2.509

39.63*±2.474

(51%)

(67%)

(67%)

(57%)

23.71*@±1.522

28.68*@±2.235

28.42*±2.519

(64%)

(65%)

(68%)

24.4*@±1.022

44.14*±1.111

34.97*±2.065

7

22.1*@±1.26 8

2 (37%) 19.07*@±2.0

9

19 (45%) 18.86*@±1.0

10

41 (46%)

11

12

13

14

24.49*@±2.5 15

33 (30%)

16

20.5*@±1.47

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7

(63%)

(47%)

(61%)

14.99*@±2.084

19.48*@±1.519

16.75*@±2.82

(77%)

(76%)

(81%)

(41%) 9.925*@±2.5 17

74 (71%)

Values were expressed as mean ± SEM of 6 rats. * Significantly different from control at P