Synthesis, Characterization and Acute Anti-inflammatory Evaluation of New Mefenamic Acid Derivatives Having 4-Thiazolidinone Nucleus

138 Synthesis, Characterization and Acute Anti-inflammatory Evaluation of New Mefenamic Acid Derivatives Having 4-Thiazolidinone Nucleus Mustafa H. Ali Alsafi *,1 and Muthanna S. Farhan ** * Ministry of Health and Environment , Baghdad , Iraq. Department of Pharmaceutical Chemistry, College of Pharmacy, University of Baghdad, Baghdad, Iraq. Abstract Mefenamic acid (MA) is one of the non-steroidal anti-inflammatory drugs, it is widely used probably due to having both anti-inflammatory and analgesic activity, the main side effects of mefenamic acid include gastrointestinal tract (GIT) disturbance mainly diarrhea, peptic ulceration, and gastric bleeding. The analgesic effects of NSAIDs are probably linked to COX-2 inhibition, while COX-1 inhibition is the major cause of this classic adverse effects. Introduction of thiazolidinone may lead to the increase in the bulkiness leads to the preferential inhibition of COX-2 rather than COX-1 enzyme. The study aimed to synthesize derivatives of mefenamic acid with more potency and to decrease the drug's potential side effects, new series of 4-thiazolidinone derivatives of mefenamic acid were synthesized IVa-g. The synthetic procedures for target compounds and their intermediates are designed to be as follows: acylation of secondary amine of mefenamic acid by chloroacetylchloride to produce compound (I), then reaction between compound (I) and hydrazine hydrate to form hydrazine derivative of mefenamic acid (compound II). After that, Schiff base formation by addition of seven benzaldehyde derivatives and finally, cyclization in presence of thioglycolic acid to form 4-thiazolidinone heterocyclic ring. The characterization of the titled compounds has been established on the basis of their spectral FTIR, HNMR data, and by measurements of their physical properties. In vivo acute anti-inflammatory effect of the synthesized compounds was evaluated in rats using egg-white induced edema model of inflammation. The tested compounds and the reference drug produced significant reduction of paw edema with respect to the effect of dimethyl sulfoxide 10%v/v (control group). Compound IVe showed more potent effect than mefenamic acid at 240-300 min, while at time 300 min, compounds IVa and IVd exhibit more potent anti-inflammatory effect than mefenamic acid (50mg/kg, i.p.) as they reduced paw edema significantly more than mefenamic acid at mentioned intervals (p<0.05) . On the other hand compound IVc exhibited lower anti-inflammatory effect.


Introduction
NSAIDs have become important as an analgesic, antipyretic, and anti-inflammatory medications throughout the world. The main mechanism of NSAIDs action is inhibition both isoforms of the enzyme cyclooxygenase COX-1 and COX-2 which catalyzes the synthesis of PGH2 from arachidonic acid (1,2) . Since the gastrointestinal (GI) toxicity, as well as renal toxicity, were identified as the main adverse effect of the chronic use of the known (NSAIDs), which were mediated by COX-1 enzyme inhibition, attempt was made to develop COX-2 selective inhibitors (coxibs), which possess the therapeutic effect devoid of gastrointestinal (GI) and renal toxicity. (1)(2)(3)(4)(5)(6) 3-Dimensional structures analysis for the two isomers of COX enzyme demonstrated differences in the hydrophobic channel structure, as well as a rather larger orifice and the presence of an additional pocket lying away from the catalytic site, this makes the selective COX-2 inhibitors to require longer time to fit the COX-2 active site, but when they bind the enzyme, their bond may become permanent. They also may become competitive COX-1 inhibitors when given at high doses (7) . Well-known NSAIDs modification into more selective COX-2 inhibitors is considered an interesting maneuver. Yet, no general line is followed as a procedure for this purpose (8,9) . Thiazolidinones are thiazolidine derivatives possessing at position 1 a sulfur atom, at position 3 a nitrogen atom and at position 2, 4, or 5 a carbonyl group .
Derivatives of this compound constitute members of the widely investigated agents, and their presence was first documented in penicillin. Similarly, 1,3-thiazolidin-4-ones are heterocyclic nucleus that contain a sulfur atom and nitrogen at position 1 and 3, respectively and a carbonyl group at position 4, it has been studied extensively in recent years (10) .The 4-thiazolidinone skeleton is very flexible and has included in a variety of clinically important medications such as some antitubercular, antimicrobial, anti-inflammatory and antiviral especially anti-HIV agents (11) .Zarghi et al. reported a novel series of 2,3-diaryl-1,3thiazolidine-4-ones showing COX-2 inhibition properties which was more selective than celecoxib (9) .
The aim of this work is synthesize and preliminary anti-inflammatory evaluation of 4thiazolidinone derivatives of mefenamic acid in order to obtain a more potent mefenamic acid analogues.

Materials and Methods
Chemicals used during the synthesis were supplied by hyper-chem (China). Completion of reactions and the purity of compounds were monitored by thin-layer chromatography (TLC), using silica gel GF254 (type 60) pre-coated aluminum sheets, Merck (Germany) exposed to UV-254 nm light, five solvent systems were used which include: ethyl acetate:hexane(4:6) (12,13) , ethyl acetate:hexane (6:4) , ethyl acetate: hexane (3:7),ethyl acetate: petroleum ether(5:5) and ethyl acetate: petroleum ether(4:6). Melting points were detected by using Stuart SMP3 melting point apparatus in open capillary tubes, and are uncorrected. The infrared spectra were performed in thin film techginqe, (υ, cm -1 ), on Shimadzu FTIR spectrophotometer, (Japan). 1 HNMR spectra were obtained on BRUKER model Ultra shield 300 MHz spectrophotometer, and BRUKER model Ultra shield 400 MHz spectrophotometer, using deuterated DMSO-d6 as solvents and TMS as an internal standard, at Al-albayt University, Amman-Jordan. (14,15) Mefenamic acid (2.0 g, 8.29 mmol) was dissolved in dichloromethane (DCM) 20 ml and in the presence of triethylamine (1.4 mL, 9.97 mmol) the mixture were stirred at 0 o C for10 min. Then Chloroacetylchloride (1.4 mL,17.6 mmol) was added dropwise. After that stirring at 0 o C for 15 min and at room temperature for one hour ,The end of reaction was detected by TLC , After end of reaction aqueous K2CO3( 2 eq.)( 2.5 g) was added to the mixture and the mixture were stirred for 30 min was washed with Dichloromethane and water. The aqueous layer was extracted with 3 × 30 mL of CH2Cl2. The organic layers were collected and concentrated. The remaining residue was purified by recrystallization from benzene. The percent yield and physical data are given in Table (2

-(N-(2,3-dimethylphenyl)-2hydrazineylacetamido)benzoic acid (II). (16)
A mixture of compound (I) (0.98 g, 3.1 mmol) and hydrazine hydrate 80% (9.4 mmol) in ethanol (20 mL) was heated under reflux for six hours, then cooled and poured on to crushed ice (30 g), then the solid product which formed was filtered off, washed with water and recrystallized from ethanol to give compound (II) The percent yield and physical data are given in Table (

-(2-(2benzylidenehydrazineyl)-N-(2,3-dimethyl phenyl)acetamido)benzoic acid)compound III a-g . (17)
A mixture of 1.9 mmol of Compound II and 1.9 mmol of the corresponding aldehyde derivatives that mentioned in table (1) in 20 ml of absolute ethanol was stirred at room temperature for 0.5 to 1h, with addition of two to three drops of hydrochloric acid as a catalyst. The end of the reaction was observed by TLC, and the benzyledine hydrazines were isolated by concentrating of the crude product at reduced pressure, the resulting precipitate was filtered, washed with 10 ml water and recrystallized from ethanol. The percent yield and physical data are given in Table (2)

Preliminary pharmacological studies Anti-inflammatory evaluation study
The inflammatory model that used to evaluate final compounds (IVa-g) for the In-vivo acute anti-inflammatory effects exploited egg-white induced rat paw edema, for comparison with antiinflammatory activity of mefenamic acid. The decrease of paw thickness is the basis of screening of the newly synthesized compounds for their antiinflammatory activity.

Methods Animals
Albino rats of either sex weighing (250 ± 50 gm) were supplied by Biotechnology Research Center, AL-Nahrain University, and were housed under standardized conditions in the Biotechnology Research Center, AL-Nahrain University animal house. Commercial chaw was used to feed the animals and they had free access to water ad libitum. Animals were brought to the laboratory, one hour before the experiment; animals were divided into nine groups (six rats per group) as follow: Group A: injected with the vehicle (dimethyl sulfoxide 10% v/v) and served as a control group. Group B: injected with mefenamic acid as reference substance with a dose of 50mg/kg (19) , dissolved in dimethyl sulfoxide 10% (v/v). Group C-I: injected with the tested compounds (IVag) by doses that are determined below. (dissolved in dimethyl sulfoxide 10% v/v). (20) Dose of reference compound /Mwt. of reference compound = dose of tested compound/ Mwt. of tested compound.

Experimental design
Egg albumin was used to induce rat paw edema as acute inflammatory model for studying of the final compounds activity (21) . 0.05mL of undiluted ovalbumin was subcutaneously injected into the rats' planter side of the hind paw; preceded by a half hour of intraperitoneal injection of the drugs or their vehicle. Electronic LCD Digital vernier caliper gauge stainless steel ruler was used for measuring paw thickness at 7 time periods (0, 30, 60, 120, 180, 240, and 300 minutes) after the Compounds injection.

Statistical analysis
The mean ± SEM was used to report data of this work then student t-test (Two Sample Assuming Equal Variances) used to calculate data statistical significance between means. ANOVA (Two factors without Replication) is used to compare between different groups. P-value < 0.05 was assumed significant.

Results and Discussion
The anti-inflammatory activity of the tested compounds has been evaluated in comparison with their vehicle (control group) and mefenamic acid. Table (4) explains the effect of tested compounds (IVa-g) in comparison to control and mefenamic acid. The tested compounds and the reference drug produced significant reduction of paw edema with respect to the effect of dimethyl sulfoxide 10%v/v (control group). All tested compounds significantly limited the inflammation in paw edema, the onset of mefenamic acid and compounds IVa,c,e started at time 120 min but IVc became comparable to control at 180 min until the end of the study. While compound IVg started at 180 min and Compounds IVb,f started at 240 min. compound IVe exhibited more potent anti-inflammatory effect than mefenamic acid (50mg/kg, i.p.) at 240-300 min., while compounds IVa,d exhibited higher antiinflammatory effect at time 300 min. However, the effect of all tested compound continued till the end of experiment with statistically significant (P<0.05) reduction in paw edema thickness as shown in figure (1).

Comparative analysis
The comparison explains that at 0-60 min., there are no differences among all groups. Compounds (IV a,d,e,f) at time 120-240 minutes show comparable effect to mefenamic acid except IV e which becomes more potent at 240-300 min. at time 300 min compounds IV a,d exhibit more potent effect than mefenamic acid, while IVf remains comparable until the end of the study. Although; compounds IVb,g significantly limited the increase in paw edema in comparison to control group, but they are significantly less effective than mefenamic acid and other tested compounds at interval of 120 minutes and after that they exhibit comparable effect to mefenamic acid until the end of the experiment.

Conclusions
A new derivatives of mefenamic acid were successfully synthesized by conventional method and tested for anti-inflammatory activity, acute antiinflammatory study using egg white induced edema model of inflammation revealed that the incorporation of 4-thiazolidinone moiety into a mefenamic acid maintained or enhanced its antiinflammatory activity and also the antiinflammatory study show that compounds (IVa , IVd , IVe) contain [OH,F,N(CH3)2] groups respectively which are electron donating groups showed superior anti-inflammatory activity to mefenamic acid probably due to formation of hydrogen bonding with the target receptor in the body .