Iraqi Journal Of Pharmaceutical Sciences Iraqi J.Pharm.Sci., Vol.17 (1) ,2008 Mefenamic acid derivatives 7 Synthesis and Preliminary Pharmacological Evaluation of Aminobenzensulfonamides Derivatives of Mefenamic Acid as a Potential Anti-inflammatory Agents Monther F. Mahdi*, 1 * Department of Pharmaceutical Chemistry, College of Pharmacy, University of Baghdad. , Baghdad , Iraq Abstract A group of amino derivatives [4-aminobenzenesulfonamide,4-amino-N¹ methylbenzenesulfonamide, or N¹-(4-aminophenylsulfonyl)acetamide] bound to carboxyl group of mefenamic acid a well known nonsteroidal anti-inflammatory drugs (NSAIDs) were designed and synthesized for evaluation as a potential anti-inflammatory agent. In vivo acute anti-inflammatory activity of the final compounds (9, 10 and 11) was evaluated in rat using egg-white induced edema model of inflammation in a dose equivalent to (7.5mg/Kg) of mefenamic acid. All tested compounds produced a significant reduction in paw edema with respect to the effect of propylene glycol 50% v/v (control group). Moreover, the 4- amino-N-methylbenzenesulfonamide derivative (compound 10) exhibited comparable anti- inflammatory activity to diclofenac (3mg/Kg) at times 180-300 minute with the same onset of action. The results of this study indicate that the incorporation of the 4-aminobenzenesulfonamide pharmacophore and its derivatives in to mefenamic acid maintain its anti-inflammatory activity. Key ward: benzenesulfonamide, anti-inflammatory, paw edema, NSAIDs, mefenamic acid ةصالخلا - عجممنمجزم4- عجمم- --عنجزمفمس تمزنبمن ع - , -)-نا ,ديامانوفلس نيزنب ليثم-نا-ونيما-)-نا ,ديامانوفلس نيزنب ليثم-نا-ونيما-4 - عجممفمس تمزنبمن ع - ,4 [عنشماممعتم تشمىا نم ةعجمجم م ت- فمرجغم ترىجغ -سم تشيغ ءموج- مmefenamic acid ) ) معىس- مفشنشمامم تل سفمبرجزمتنشجب نشج م زج-م ] زنبمنجز( زجى ع - نفم تنرام تسف , وغسمواججام تبي تجمم تش ق متلتىا مم . بش قمتلتىا م ,ا-ممششهم ي غنمتىاججشا مبش ق نمام ممتلتىا مم متنشجب نشج م زج-م( نفم تنغحمف زىل- تمتةةم تنجلمعرىس- مم حعمم تىا فجمموسهم تنن-مفنغاممعل ن م11 ,10 ,9تنشغبن نم تما مجم) (propylene glycol%50عنلا/بلا(.بزم تشغبن نم تشلىنغ م نىنهم نلب معف غ متنمحعممف تشا سنممعكم تنغ فنجتمبل لمة )(7.5 ( ااغمني تجممع ق متلتىا ممعا سنمم10 عغبم( - عجمم- --عنجزمفمس تمزنبمن ع -4بشنشماممو فمم.ال مانامحت معمىك . نىجنممنا م ت-س زممومجغم تام -م ن-ع .ممتن- لنمنجم لمعكمنبام تبي تجمم ةفى- مجممقاجام300_ 180نفم ا نعنلا/بلا( 3تن- لنمنجم لم) - عجممفمس تمزنبمن ع -م عمىا ونمعكم تشجب نشج م زج-مي نامانامني تجىنم تش ق .4 تنسفم تيا اجغسم Introduction Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used to treat acute or chronic inflammation and offer symptomatic pain relief(1,2). Conventional NSAIDs act by non selective inhibition of cyclooxygenase (COX) enzymes, which catalyze the formation of prostaglandins (PGs) from arachidonic acid(3, 4). There are three isoenzymes of COX (COX-1, COX-2 and COX-3) have been identified(5,6). COX-1 is expressed in most tissues of the body and largely governs the homeostatic production of arachidonic acid metabolites necessary to maintain physiologic integrity(7). COX-2 is highly induced in settings of inflammation by cytokines and inflammatory mediators or physiological stress(8,9). COX-3 activity in human has not been confirmed(10), but it may be implicated in fever(11). All classic NSAIDs inhibit COX-2 as well as COX-1 to varying degrees; thus they can be considered nonspecific(12,13). All classical NSAIDs are associated with an increased risk of gastrointestinal (GI) ulcers and serious upper GI complications, including GI hemorrhage, perforation, and obstruction(14,15). In contrast many of the selective COX-2 inhibitors containing benzene-sulfonamide derivative, like valdecoxib(I) (16) , celecoxib(II) (17),or benzene-N-methyl sulfonamide like compound (III) (18) and benzene methylsulfonyl 1 Corresponding author : E-mail : dmfalameri@yahoo.com Received : 29 /10 / 2007 Accepted : 15 /3/ 2008 Iraqi J.Pharm.Sci., Vol.17 (1) ,2008 Mefenamic acid derivatives 8 O SH3C O O O O SH3C O O O SO2NH COCH3 SO2NHCOCH3 SO2NHCH3 O O derivative, like Rofecoxib (IV) exert anti- inflammatory and analgesic activity in the clinic with markedly less GI toxicity than traditional NSAIDs(19). In a recent study, it was shown that the incorporation of a para-N- acetylsulfonamido substitute on the C-3 phenyl ring of the Rofecoxib regioisomer provided a highly potent and selective COX-2 inhibitor (compound V) that has the potential to acetylate the COX-2 isozyme(20). The improved GI tolerance of COX-2 selective inhibitors not withstanding, there is evidence to suggest that COX-2 selective inhibitors may inhibit COX-1 and induce GI irritation or ulceration with long term use or at higher doses(21,22). Preclinical cardiovascular and renal liabilities of at least some COX-2 selective inhibitors have also been reported(23). Thus there is still a need for new anti-inflammatory agents with an improved safety profile. Valdecoxib (I) (III) Rofecoxib(IV) Celecoxib (II) (V) NN H3C SH2N O O CF3 O N SH2N O O CH3 Iraqi J.Pharm.Sci., Vol.17 (1) ,2008 Mefenamic acid derivatives 9 In the view of this background, the present study was conducted to design, synthesize and preliminarily evaluate new mefenamic acid derivatives as potential NSAIDs. [Future study: to measure their selectivity’sمonمCOX-2 enzyme.] Chemistry The general routes outlined in schemes 1 and 2 were used to synthesize all compounds described here. 4-aminobenzene-sulfonamide (4) and 4-amino-N-methylbenzene sulfonamide (6) was prepared as described by Vogel (24) starting from acetanilide as shown in scheme 1.Their characterization and physical data are presented in the table1. HN C CH3 O2S Cl HN C CH3 O O (1) ACETANILIDE (2) ClSO3H O2S NH2 HN C CH3 O O2S NH2 NH2 O2S NHCH3 HN C CH3 O O2S NHCH3 NH2 HCl/ref luxH2O + CH3COOH (4) (3) CH3-NH2/heat (5) (6) HCl/ref luxH2O + CH3COOH Scheme 1: Synthesis of 4-aminobenzene sulf onamide (4) & 4-amino-N1-methylbenzene sulfonamide (6) NH3 solution/heat + HCl + HCl Iraqi J.Pharm.Sci., Vol.17 (1) ,2008 Mefenamic acid derivatives 10 Scheme 2: Synthesis of compounds 9, 10, and 11 NH CH3 CH3 COOH (7) 2 THF DCC NH CH3 C O O C O NH CH3 CH3 + DCU (8) (6) ref lux CH3 CH3 NH C O N H (11) (4) ref lux CH3 CH3 NH C O N H H2NO2S (9) AC2O pyridineCH3 CH3 NH C O N H SO 2NHCOCH3 (10) CH3 H3CHNO2S + (7) + (7) + CH3COOH DCU: dicyclohexyl urea Iraqi J.Pharm.Sci., Vol.17 (1) ,2008 Mefenamic acid derivatives 11 Table (1): The characterization and physical data of the compounds (3-6 and 8-11). Solvent system: Methanol: Acetic acid: Ether: Benzene (2:18:60:20) Experimental All reagents and anhydrous solvents were of analar type and generally used as received from the commercial supplier(Merk- Germany,Reidel-Dehean-Germany ,Sigma- Aldrich-Germany and BDH- England).Mefenamic acid was supplied from Micro Company - Indian.Melting points were determined by capillary method on Thomas Hoover apparatus (England) and ascending thin layer chromatography (TLC) was run on DC-Kartan SI Alumina 0.2 mm to check the purity and progress of reaction. The identification of compounds was done using iodine vapor and the chromatograms were eluted by: Methanol: Acetic acid: Ether: Benzene (2:18:60:20). IR spectra were recorded on model 500 scientific IR spectrophotometer, Buck Company (USA) as a KBr film.CHN microanalysis has been done using exter TE micro-analyzer (Germany).The analysis was done in the micro analytical center faculty of science –University of Cairo. Synthesis of 2-(2, 3-dimethylphenylamino) benzoic anhydride (8): Mefenamic acid (comp.7) (5g, 20.7mmol) was dissolved in THF (30ml), and then DCC (2.12g, 10.35mmol) was added. The reaction mixture was continuously stirred at room temperature for 4 hours. A white precipitate of DCU was formed which then removed by filtration. The solvent was evaporated under vacuum to give comp.8(26) . The percent yield, physical data and Rf value were given in table (1). IR 3330(NH) of secondary amine 1814 and 1743 (C=O) of anhydride, 1618, 1515 and 1488 ( C C st.v.), 1274, 1215 and 1172[C - (C=O) – O-(C=O) –C] cm-1 of anhydride. Synthesis of 2-(2, 3-dimethylphenylamino)-N- (4-sulfamoylphenyl) benzamide (9): Compound 8 (2.5g, 5.4mmol), compound 4 (0.93g, 5.4mmol), zinc dust (6mg), glacial acetic acid (0.5ml, 8.75mmol) and dioxane (20ml) were placed in a flask, equipped with refluxed condenser, boiling stones were added. The reaction mixture was refluxed gently for 90 minutes. The solvent was evaporated under vacuum, the residue was dissolved in ethyl acetate, washed with NaHCO3 (10%, 3*10ml), HCl (1N, 3*10ml) and distilled water (3*10ml), filtered over anhydrous magnesium sulfate. The filtrate is evaporated under vacuum to give the product. The crystallization is carried out by dissolving the compound in ethyl acetate and petroleum ether (80-100 oC) is added to the filtrate until turbidity take place and it is kept in cold place over night. The mixture is filtered while it is cold and the precipitate is collected to give comp.9(27) . The percent yield, physical data and Rf value were given in table (1). IR 3376and3304 (N-H) of primary sulfonamide, 3227 (N-H) of secondary amine, 1660 (C=O) of secondary amide, 1598and1530( C C st.v.), 1327and1157 (SO2) cm-1 . Compound Empirical formula Molecular weight Description % yield Melting point Observed reported Rf value 3 C8H10N2O3S1 214 Faint yellow crystals 53 213-214 216 (25) 0.45 4 C6H8N2O2S1 172 White crystals 51 160-161 163-164 (24) 0.75 5 C9H12N2O3S1 228 White crystals 62 179-181 0.52 6 C7H10N2O2S1 186 White powder 44 107-108 0.68 8 C30H28N2O3 464 White powder 80 141-143 0.69 9 C21H21N3O3S1 395 White crystals 40 198-199 0.82 10 C23H23N3O4S1 437 White powder 48 169-171 0.76 11 C22H23N3O3S1 409 White crystals 35 180-181 0.8 Iraqi J.Pharm.Sci., Vol.17 (1) ,2008 Mefenamic acid derivatives 12 CHN Calculated (C21H21N3O3S1): C, 63.78; H, 5.35; N, 10.36; S, 8.11. Found: C, 62.55; H, 5.44; N, 10.51; S, 8.25. Synthesis of N-(4-(N-acetylsulfamoyl)-2-(2, 3-dimethylphenylamino) benzamide (10): Acetic anhydride (0.6ml, 6mmol), was added to a solution of compound 9 (0.79g, 2mmol) in pyridine (10ml) and the reaction was allowed to proceed then at 25 oC with stirring for 6 hours. Ethyl acetate (100ml) was added and this solution was washed successively with saturated aqueous ammonium chloride (2x20ml) followed by distilled water (2x20ml). The organic fraction was dried with anhydrous magnesium sulfate and the solvent was removed in vacuum to give comp.10 (28) .The percent yield, physical data and Rf value were given in table (1). IR 3350and3292 (N-H) of secondary amide andsulfonamide respectively, 1670 (C=O) of secondary amide, 1595, 1533, and1450( C C st.v.) and1332 and1157(SO2) cm-1 . CHN Calculated (C23H23N3O4S1): C, 63.14; H, 5.30; N, 9.60; S, 7.33. Found: C, 62.25; H, 5.40; N, 9.83; S, 7.48. Synthesis of 2-(2, 3dimethylphenylamino)-N- (4-(N-methylsulfamoyl) benzamide (11): Compound 8 (2.5g, 5.4mmol), compound 6 (1g, 5.4mmol), zinc dust (6mg), glacial acetic acid (0.5ml, 8.75mmol) and dioxane (25ml) were placed in flask, equipped with reflux condenser, boiling stones were added. The reaction mixture was refluxed gently for 90 minutes, and then it was worked up as prescribed in section3.2 to liberate comp.11. The percent yield, physical data and Rf value were given in table (1). IR 3334and3201 (N-H) of secondary amide andsulfonamide, 1664 (C=O) of secondary amide, 1591, 1529 and 1496 ( C C st.v.) and1321 and1159(SO2) cm-1 . CHN Calculated (C22H23N3O3S1): C, 64.53; H, 5.66; N, 10.26; S, 7.83. Found: C, 65.20; H, 5.58; N, 10.45; S, 8.01. Pharmacology: Albino rats of either sex weighing (150 ± 10 g) were supplied by the National Center for Quality Control and Drug Research and were housed in the animal house of the College of Pharmacy, University of Baghdad under standardized conditions (12 light-12 dark cycle) for 7 days for acclimatization. Animals were fed commercial chaw and had free access to water ad libitum. Animals were brought 1 hour before the experiment to the laboratory, and were divided into five groups (each group consist of 6 rats) as follows: group A: served as control and treated with the vehicle (propylene glycol 50% v/v in water); group B: treated with sodium diclofenac (reference agent) in a dose of 3mg/kg suspended in propylene glycol (29); group C, D and E: treated with tested compounds 9, 10 and 11 respectively in a dose equivalent to 7.5 mg/kg of mefenamic acid as finely homogenized suspension in 50% v/v propylene glycol(30) . Anti-inflammatory activity: The anti-inflammatory activity of the tested compounds was studied using egg-white induced edema model (31). The drugs or the vehicle were administered i.p. at time zero and acute inflammation was induced by a subcutaneous injection of 0.05ml of undiluted egg-white into the planter side of the left hind paw of the rats at time 15 minutes. The paw thickness was measured by vernier at eight time intervals (0, 15, 30, 60, 120, 180, 240 and 300 minutes) after vehicle or drugs administration. The data are expressed as mean ± S.E.M. and results were analyzed for statistical significance using Student t-test (Two-Sample Assuming Equal Variances) for comparisons between mean values. While comparisons between different groups were made using ANOVA: Two-Factor Without Replication. Probability (P) value of less than 0.05 was considered significant. Results and Discussion The most widely used primary test to screen new anti-inflammatory agents is based on the ability of a compound to reduce local edema induced in the rat paw following injection of an irritant agent (32). When egg- white is injected into the paw of rats, a substantial induction of COX-2 is observed at 2 hours coinciding with enhanced PGs and local edema (33). Tables 2 and 3 show the effect of tested compounds on egg-white induced edema as an indicator for their anti- inflammatory activity. The intraplanter injection of egg-white into rat hind paw induces a progressive edema, which was reached maximum (measured by millimeter) after 2 hours of injection. Table 2 showed the effect of tested compounds (9, 10 and11) in respect to control group. All tested compounds were effectively limited the increase in paw edema, with the effect of compounds 9 and 10 started at time 30 minute (significantly difference compared to control), while compound 11 started at time 120 minute. However, the effect of all tested compounds continued till the end of the experiment with statistically significant (p >0.05) reduction in paw edema. The differences among the Iraqi J.Pharm.Sci., Vol.17 (1) ,2008 Mefenamic acid derivatives 13 compounds started at time 30 minute in which the compounds 9 and 10 significantly difference at time 30 and 60 minute compared to compound 11. However, the differences among the compounds continued from the time 180 to 300 minute with statistically significant (p >0.05) reduction in paw edema in the following orders 10, 11, and 9 respectively. Table 2: Effect of Control and Compounds 9, 10 and 11 on egg-white induced paw edema in rats. Treatment groups Time (min) Control (n=6) Compound9 (n=6) Compound 10 (n=6) Compound11 (n=6) P aw th ic kn es s (m m ) 0 4.46 ± 0.16 4.39±0.10 4.41±0.08 4.38±0.13 15 5.41 ± 0.18 5.45±0.07 5.42±0.12 5.35±0.11 30 6.05 ± 0.16 5.80±0.05*ª 5.76±0.13*ª 6.01±0.10 b 60 6.35 ± 0.07 6.00±0.05*ª 6.00±0.13*ª 6.33±0.09 b 120 6.50 ± 0.09 5.73±0.05*ª 5.66±0.08*ª 5.70±0.10*ª 180 5.93 ± 0.11 5.40±0.05*ª 5.09±0.05*b 5.30±0.07*c 240 5.38 ± 0.09 5.13±0.05*ª 4.86±0.07*b 4.95±0.07*c 300 5.20 ± 0.10 5.05±0.04*ª 4.56±0.08*b 4.68±0.05*c Non-identical superscripts (a, b, and c) among different groups are considered significantly different (P<0.05). * significantly different compared to control (P<0.05). Table 3 shows the effect of tested compounds (9, 10 and11) with respect to the reference group (diclofenac). As seen in this table; at time 0 and 15 minute there are no differences among different groups; at time 30, only compound 11 is significantly different than diclofenac; at time 60 and 120 all compounds are significantly different than diclofenac; while at time 180 to 300 compounds 9 and 11 are significantly different than diclofenac. The differences among the compounds started at time 30 minute in which the compounds 9 and 10 significantly difference at time 30 and 60 minute compared to compound 11 while at time 120 compound 10 is significantly different than compounds 9 and 11. However, the differences among the compounds continued from the time 180 to 300 minute with statistically significant (p > 0.05) reduction in paw edema in the following orders 10, 11, and 9 respectively. Iraqi J.Pharm.Sci., Vol.17 (1) ,2008 Mefenamic acid derivatives 14 Table 3: Effect of Diclofenac and Compounds 9, 10 and 11 on egg-white induced paw edema in rats. Treatment groups Time (min) Diclofenac (n=6) Compound9 (n=6) Compound 10 (n=6) Compound11 (n=6) P aw th ic kn es s (m m ) 0 4.38±0.14 4.39±0.10 4.41±0.08 4.38±0.13 15 5.37±0.41 5.45±0.07 5.42±0.12 5.35±0.11 30 5.78±0.11 5.80±0.05 ª 5.76±0.13 ª 6.01±0.10*b 60 5.60± 0.10 6.00±0.05*ª 6.00±0.13*ª 6.33±0.09*b 120 5.35±0.10 5.73±0.05*ª 5.66±0.08*b 5.70±0.10*ª 180 5.07±0.10 5.40±0.05*ª 5.09±0.05 b 5.30±0.07*c 240 4.87±0.10 5.13±0.05*ª 4.86±0.07 b 4.95±0.07*c 300 4.61±0.10 5.05±0.04*ª 4.56±0.08 b 4.68±0.05*c Non-identical superscripts (a, b, and c) among different groups are considered significantly different (P<0.05). * Significantly different compared to control (P<0.05). Conclusion The in vivo anti-inflammatory study showed that the incorporation of 4- aminobenzenesulfon- amide, 4-amino-N-methylbenzenesulfonamide, or N-(4-aminophenylsulfonyl) acetamide into well known anti-inflammatory drug (mefenamic acid) maintains its anti- inflammatory activity. 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