Iraqi J Pharm Sci, Vol.21(2) 2012 Nitrate derivatives of mefenamic acid 24 Design and Synthesis of Some Nitrate Derivatives of Mefenamic Acid with Expected Nitric Oxide Release Ahmed A. Sadon *,1 and Ahlam J. Qasir * * Department of Pharmaceutical Chemistry,College of Pharmacy,University of Baghdad,Baghdad, Iraq Abstract This study include design and synthesis of 2 derivatives of compounds consisting of mefenamic acid, glycine and organic nitrates (2-nitrooxy ethanol or 1,3-dinitrooxy-2-propanol). Nitric oxide NO has been reported to support many of the same mucosal protection mechanisms as prostaglandins and is sufficient for acute gastroprotection and ulcer healing. So we suppose these 2 compounds would reduce non-steroidal anti- inflammatory drugs NSAIDs gastrointestinal side effect. Key words: Non-Steroidal anti-inflammatory drugs (NSAIDs), Glycine, Mefenamic acid, Organic nitrates, Nitric oxide (NO). اهيك هع تىقع تحرر ألوكسيذ الٌتريكبعض هشتقاث الٌتراث لحاهض الويفيٌ تصوين وتخليق عبذ الىاحذ سعذوى احوذ *،1 قصيرو احالم جويل * . *فشع انكيمياء انصيذالويت ، كهيت انصيذنت ، انجامعت انمستىصشيت ، بغذاد ، انعشاق الخالصة مع وتشاث انميفيىامك مع انكاليسيه حامط يه جذيذيه هما عباسة عه مشتقاث تتضمه انذساست تصميم و تخهيك مشكب . انعذيذ مه انبحىث انحذيثت تؤيذ انذوس انمشابه بيه أوكسيذ بشوباوىل(-٢-دايىايتشوكسي-١,٣و أ وايتشوكسي إيثاوىل-٢) عضىيت ىاءا عهى رنك وفتشض بأن هزيه انمشتقيه انجذيذيه انىايتشيك و انبشوستىكالوذيه في حمايت انقىاة انمعىيت و معانجت تقشحاتها. ب ستيشويذيت انمضادة نإلنتهاب عهى انقىاة انمعىيت. انغيش قهيم األعشاض انجاوبيت نألدويت سيساهمان في ت عضىيت، اوكسيذ الٌتريك حاهض الويفيٌاهيك ،ًتراث ، كاليسيي ( ، NSAIDsلتهاباث الغيرستيرويذيت ) لال االدويت الوضادة الكلواث الوفتاحيت : (NO . ) Introduction Nonsteroidal anti-inflammatory drugs (NSAIDs) are still one of the first choices in the treatment of inflammatory conditions and among the most commonly prescribed drugs (1) . However, they are not devoid of adverse effects, gastrointestinal (GI) ulceration and renal damage being the most common, and even life-threatening (2) . Several strategies have been followed to reduce these adverse effects, including enteric coating, parenteral administration, and formulation of prodrugs that require hepatic metabolism for the cyclooxygenase (COX) activity to be unmasked and coadministration of either suppressors of acid secretion or exogenous prostaglandins (PGs), without the desired results (3) . A more recent and promising approach is that of COX-2 inhibitors (4) . But these drugs may lead to increased cardiovascular events (5) . Another approach to reduce the toxicity of NSAIDs is the linking of a nitric monoxide (NO) releasing moiety to these compounds, and thus, a new category of anti-inflammatory agents is emerging, the NO- NSAIDs (6) . Nitric oxide is a small diatomic radical that plays an important physiological role in nervous, cardiovascular, and immune systems (7) . Its generation is controlled by the three isoforms of NO synthase (NOS), neuronal and endothelial NOS (nNOS and eNOS), which are constitutive and produce nanomolar amounts of NO important for normal cell function and tissue protection, and inducible NOS (iNOS) which generates high amounts of NO responsible for the destruction of invading pathogens as part of an overall inflammatory response (8) . The endogenous tissue NO, generated constitutively by GI eNOS and nNOS, appears to play a key role in the chronic maintenance of gastrointestinal tissue integrity and in adaptive cytoprotection to injurious stimuli, perhaps acting synergistically with other cytoprotective prostaglandins (9) . NO promotes several gastric defense mechanisms by increasing mucus and bicarbonate secretion in the GI tract, increasing mucosal blood flow, and inhibiting the pro- inflammatory activities of neutrophils and platelets (10) . Additionally, NO may reduce inflammation connected to oxidative stress by scavenging reactive oxygen species, which can adversely increase mucosal permeability and kill cells (8) . Furthermore, it is found that NO and NO-derived reactive nitrogen species interact with peroxidases (11) and lipoxygenases (12) , altering the generation of prostaglandins and leukotrienes, which are signaling molecules involved in inflammation (13) . 1 Corresponding author E- mail : ahlmedsadon78@yahoo.com Received : 24/12/2011 Accepted : 20/5/2012 Iraqi J Pharm Sci, Vol.21(2) 2012 Nitrate derivatives of mefenamic acid 25 Several NO-NSAIDs, such as NO-aspirin, NO- diclofenac, NO-flurbiprofen, and NO- ketoprofen, have already been synthesized and clinical trials for some of them revealed protective activity against acute myocardial infarction (14) . These compounds appear to have a safe GI profile and can be effective in a variety of diseases including cardiovascular, rheumatological, lung diseases, Alzheimer’s disease and cancer. There is also evidence to suggest that these compounds release NO in a metabolic, and not spontaneous, way and therefore, they do not interfere with blood pressure regulation (15) . Bearing in mind the above studies, we designed new derivatives of NO-releasing NSAIDs (scheme-1). Two derivatives of mefenamic acid-glycine esterified with 2-bromoethanol or 1,3- dibromo-2-propanol where they are then converted to nitrooxy derivatives upon treatment with silver nitrate in acetonitrile. SOCl2 CH3OH O O H N CH3 H Cl O OH H2N Glycine + NH CH3 CH 3 R O OH DCC DMAP NMM Coupling R O 1 N N a O H D e- e st er if ic a ti o n O OH H NR O O O H2N CH3 DCC DMAPSteglich esterification Br O H NR O O O H NR O O Br HO DCC DMAP Steglich esterification OH Br Br O H NR O O O N+ O O  O N+ O O O N+ O O  O H NR O O Br Br Compound (1) Compound (2) Compound (3) Compound (4) Compound (5) AgNO3 Compound (I) AgNO3 Compound (II) R= · Scheme 1 : Chemical synthesis of compounds I and II. Iraqi J Pharm Sci, Vol.21(2) 2012 Nitrate derivatives of mefenamic acid 26 Experimental Chemicals 1,3-dibromo-2-propanol, 4-dimethyl amino pyridine (DMAP), N-methylmorpholine (NMM) (Aldrich), 2-bromoethanol, glycine, N,N′-dicyclohexylcarbodiimide (DCC) (Fluka), mefenamic acid (SDI), silver nitrate (Riedel-Dehaën), thionyl chloride (Merck), all solvents were of AnalaR type and used without further purification. Detection Melting points (uncorrected) were determined by capillary tube method on Barnstead/Electrothermal (UK) and FT-IR spectra were recorded on FT-IR spectrophotometer Shimadzu (Japan) at the college of science/ department of chemistry (Al-Mustansyria University). The CHNO analysis was carried out using Carlo-Erba 1106 Elemental analyzer (France). Ascending thin layer chromatography (TLC) was run on silica gel 60 F254 pre-coated aluminum sheets, Merck (Germany) to check the purity and the reaction's progress. The detection of derivatives was done using UV 254 nm lamp and the chromatograms were eluted by two solvent system: A\ Ethanol: water (7:3) (16) . B\ Acetone: chloroform: acetic acid: water (3:2:1:4) (17) . Synthesis Synthesis of compound (1); (methyl-2- aminoacetate hydrochloride) (18) : H Cl O O H 2 N CH 3· Thionyl chloride (3.25ml, 45 mmol) was slowly added by dripping to methanol (40 ml) kept on a temperature at (−10) to (−5°C); then glycine (3gm, 40 mmol) was added. The stirred mixture kept on 40-45°C for 2 hrs and then was refluxed for 4 h at 60-70°C under continuous stirring. Then excess thionyl chloride and solvent were removed under reduced pressure giving crude glycine methyl ester hydrochloride, which was treated with an 8 ml portion of cold diethyl ether at 0°C. The resulting solid product was collected and dried under vacuum. It was re-crystallized from hot methanol by slow addition of an 8 ml of diethyl ether followed by cooling at 0°C. The crystals were collected on the following day and washed twice with a (diethyl ether /methanol) mixture (5:1 ratio) followed by pure diethyl ether and dried under vacuum to give pure glycine methyl ester hydrochloride. The percent of yield is 95%. The physical appearance, melting point and the Rf value are listed in table (1) the characteristic IR bands are listed in table(1) the characteristic IR bands are listed in table(3). Synthesis of compound (2); (methyl 2-(2- ((2,3-dimethylphenyl) amino) benzamido) acetate) (19) : O O O H N NH CH3 CH3 CH3 To a stirred solution of compound 1 (1.883 gm, 15 mmol) in (120ml) of dimethyl formamide DMF, (3.3 ml, 30 mmol) of N- methylmorpholine (NMM) was added with stirring for 10 minutes, later on (3.62 gm, 15 mmol) of mefenamic acid was added, and the mixture was cooled down to (−10C), then (0.183 gm, 1.5 mmol) of 4-dimethyl amino pyridine (DMAP) and (3.4 gm, 16.5 mmol) of N,N′-dicyclohexylcarbodiimide (DCC) were added with stirring which was continued for 2 days at 0C and then at room temperature for 3 days. The reaction mixture was subjected to evaporation under vacuum to get rid of DMF, re-dissolved in 150 ml of chloroform from which the N,N′-Dicyclohexyl urea (DCU) was filtered off. The clear filtrate was washed with 0.5 N NaOH (3x150 ml), 1N HCl (3x75 ml), saturated sodium chloride solution (3x75 ml) and water (2x75 ml). The chloroform layer was dried over anhydrous sodium sulphate and the solvent was evaporated under vacuum. The percent of yield is 83.5%. The physical appearance, melting point and the Rf value are listed in table (1) the characteristic IR bands are listed in table(3). Synthesis of compound (3); (2-(2-((2,3- dimethylphenyl) amino) benzamido) acetic acid) (20) : O OH O N H NH CH3 CH3 To a stirred solution of compound 2 (3.123 gm, 10 mmol) in 60 ml acetone, kept on 18-22°C, an aqueous solution of 1 N NaOH (25 ml) was added drop wise over a period of 30 minutes where the stirred reaction mixture was continued for additional 8 hours. The mixture was treated with 1 N HCl to get pH 1. Iraqi J Pharm Sci, Vol.21(2) 2012 Nitrate derivatives of mefenamic acid 27 A precipitate was appeared which was separated by filtration, washed on the filter paper with water, and re-crystallized from n- hexane:acetone (4:3) mixture. The percent of yield is 53.5%. The physical appearance, melting point and the Rf value are listed in table (1), the characteristic IR bands are listed in table(3). Synthesis of compound (4); (2-bromoethyl 2- (2-((2,3-dimethylphenyl) amino) benzamido) acetate) (21) : O O O H N NH CH3 CH3 Br To a stirred solution of compound 3 (1.49 gm, 5 mmol), 2-bromoethanol (0.71 ml, 10 mmol) and DMAP (0.305 gm, 2.5 mmol) in (30ml) of dichloromethane (DCM) kept at −10C, DCC (1.14 gm, 5.5 mmol) was added. The stirring reaction mixture was continued for 2 days at 0C and then at room temperature for 3 days.The reaction mixture was subjected to evaporation under vacuum to get rid of DCM, re-dissolved in 50 ml ethyl acetate from which the N,N′-Dicyclohexyl urea (DCU) was filtered off. The clear filtrate washed with 5% sodium bicarbonate solution (3x25 ml), 1N HCl (3x25 ml) and water (2x25 ml). The ethyl acetate layer was dried over anhydrous sodium sulphate and evaporated under vacuum. The product was dissolved in ethanol (20 ml) with stirring. To the stirred solution about 20 ml of distilled water was added slowly from a dripping funnel. The precipitated solid was separated by filtration, dried and kept in a desiccator. The percent of yield is 82%. The physical appearance, melting point and the Rf value are listed in table (1) the characteristic IR bands are listed in table(3). Synthesis of compound (5); (1,3- dibromopropan-2-yl 2-(2-((2,3- dimethylphenyl) amino) benzamido) acetate) (21) : O O O H N NH CH3 CH3 Br Br To a stirred solution of compound 3 (1.49 gm, 5 mmol), 1,3-dibromo-2-propanol (1.02 ml, 10 mmol) and DMAP (0.061 gm, 0.5 mmol) in (30ml) of DCM kept at −10C, DCC (1.14 gm, 5.5 mmol) was added.The reaction mixture was worked up as in synthesis of intermediate 4. The percent of yield is 77%. The physical appearance, melting point and the Rf value are listed in table (1) the characteristic IR bands are listed in table(3). Synthesis of compound (I); (2-(nitrooxy) ethyl 2-(2-((2,3-dimethylphenyl) amino) benzamido) acetate) (22) : O O O H N NH CH3 CH3 O N+ O O- A solution of compound 4 (0.81 gm, 2 mmol) in dry acetonitrile (10 ml) was treated portion wise with a solution of AgNO3 (1.36 gm, 8 mmol) in dry acetonitrile (20 ml) and the whole stirred mixture was refluxed for 48h in the dark. At the end of the reflux, the inorganic solid (black coloured silver bromide) was filtered off. The filtrate was subjected to evaporation under vacuum till dryness; the residue was dissolved in 20 ml DCM and washed with water (3x40 ml), saturated sodium chloride solution (2x40 ml) and finally water (2x40 ml). The organic layer was dried over anhydrous sodium sulfate, evaporated under vacuum and the obtained residue was re- crystallized from absolute methanol and triturated with petroleum ether (40-60C). The percent of yield is 41%. The physical appearance, melting point and the Rf value are listed in table (1), the elemental microanalysis results are presented in table(2), and the characteristic IR bands are listed in table (3) . Synthesis of compound (II); (1,3-bis (nitrooxy) propan-2-yl-2-(2((2,3- dimethylphenyl) amino) benzamido) acetate) (22) : O O O H N NH CH3 CH3 O N+ O O- O N+ O O- A solution of compound 5 (0.996 gm, 2 mmol) in dry acetonitrile (10 ml) was treated portion wise with a solution of AgNO3 (2.72 gm, 16 mmol) in dry acetonitrile (40 ml) and the whole stirred mixture was worked up as in synthesis of compound I. The percent of Iraqi J Pharm Sci, Vol.21(2) 2012 Nitrate derivatives of mefenamic acid 28 yield is 38%. The physical appearance, melting point and the Rf value are listed in table (1) , the elemental microanalysis results are presented in table(2), and the characteristic IR bands are listed in table (3) . Result and Discussion The synthesis of the designed compounds has been successfully achieved. And the purity and structural formulas of the synthesized compounds were characterized by their melting points, Rf values, FT-IR spectroscopy and elemental microanalysis.We expect these two compounds I and II would reduce gastrointestinal side effect largely. So we recommend for their biological evaluation for safe gastrointestinal profile. Table 1 : The characterization and physical data of the synthesized compounds. Compounds Empirical formula Description Melting point o C Rf value Solvent system Compound 1 C3H8ClNO2 White powder 175-176 0.34 A Compound 2 C18H20N2O3 Pale yellow powder 126-128 0.68 B Compound 3 C17H18N2O3 Yellow powder 150-151 0.31 B Compound 4 C19H21BrN2O3 Red crystal 156-158 0.75 B Compound 5 C20H22Br2N2O3 Red crystal 168-170 0.83 B Compound I C19H21N3O6 Brown powder 172-173 0.49 B Compound II C20H22N4O9 Brown powder 198-201 0.34 B Table 2 : Elemental microanalysis of the compounds I and II. Compound Empirical formula Molecular weight Elemental analysis % Element Calculated Observed I C19H21N3O6 387.387 C 58.91 58.13 H 5.46 5.71 N 10.85 10.33 O 24.78 25.83 II C20H22N4O9 462.41 C 51.95 50.37 H 4.8 5.06 N 12.12 12.59 O 31.14 31.97 Table 3 : Characteristic FT-IR absorptions bands of the synthesized compounds. Compound Band (cm -1 ) Interpretation Compound 1 2500-3300 1751 1585, 1504 1261 Asymmetrical and symmetrical NH3 + stretching C=O stretching of ester Asymmetrical and symmetrical bending of NH3 + Asymmetrical C-O stretching of ester Compound 2 3377 3311 3063, 3037 2931 2856 1749 1629 1581 1510 1276 1228 N-H stretching of 2º aromatic amine N-H stretching of 2º amide Aromatic C-H stretching Asymmetrical C-H stretching of CH3 and CH2 Symmetrical C-H stretching of CH3 and CH2 C=O stretching of ester C=O stretching of 2º amide (amide I) N-H bending of 2º amide (amide II) N-H bending of 2º aromatic amine C-N stretching of 2º aromatic amine Asymmetrical C-O stretching of ester Compound 3 3423 3367 2750-3200 3034 2937 1741, 1707 1626 1579 1512 1278 1211 N-H stretching of 2º aromatic amine N-H stretching of 2º amide OH stretching of carboxyl group Aromatic C-H stretching Asymmetrical C-H stretching of CH2 C=O stretching of acid C=O stretching of 2º amide (amide I) N-H bending of 2º amide (amide II) N-H bending of 2º aromatic amine C-N stretching of 2º aromatic amine Asymmetrical C-O stretching of acid Iraqi J Pharm Sci, Vol.21(2) 2012 Nitrate derivatives of mefenamic acid 29 Compound 4 3308 3059 2931 2856 1747, 1705 1639 1583 1516 1276 1224 N-H stretching of 2º amide Aromatic C-H stretching Asymmetrical C-H stretching of CH3 and CH2 Symmetrical C-H stretching of CH3 and CH2 C=O stretching of ester C=O stretching of 2º amide (amide I) N-H bending of 2º amide (amide II) N-H bending of 2º aromatic amine C-N stretching of 2º aromatic amine Asymmetrical C-O stretching of ester Compound 5 3275 3055 2929 2856 1745, 1710 1631 1581 1514 1265 1224 N-H stretching of 2º amide Aromatic C-H stretching Asymmetrical C-H stretching of CH3 and CH2 Symmetrical C-H stretching of CH3 and CH2 C=O stretching of ester C=O stretching of 2º amide (amide I) N-H bending of 2º amide (amide II) N-H bending of 2º aromatic amine C-N stretching of 2º aromatic amine Asymmetrical C-O stretching of ester Compound I 3286 3061 2931 2856 1747, 1710 1635 1581 1516 1278 1226 N-H stretching of 2º amide Aromatic C-H stretching Asymmetrical C-H stretching of CH3 and CH2 Symmetrical C-H stretching of CH3 and CH2 C=O stretching of ester Overlapped C=O stretching of 2º amide (amide I) and N=O asymmetrical stretching of nitrate N-H bending of 2º amide (amide II) N-H bending of 2º aromatic amine C-N stretching of 2º aromatic amine Asymmetrical C-O stretching of ester Compound II 3277 3059 2929 2854 1745, 1714 1637 1579 1512 1278 1226 N-H stretching of 2º amide Aromatic C-H stretching Asymmetrical C-H stretching of CH3 and CH2 Symmetrical C-H stretching of CH3 and CH2 C=O stretching of ester Overlapped C=O stretching of 2º amide (amide I) and N=O asymmetrical stretching of nitrate N-H bending of 2º amide (amide II) N-H bending of 2º aromatic amine C-N stretching of 2º aromatic amine Asymmetrical C-O stretching of ester References 1. 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