Synthesis, Antimicrobial and Antiinflammatory Activity of 2,5-Disubstituted-1,3,4-oxadiazoles

In the present study, 2,5-disubstituted-1,3,4-oxadiazoles (3a-o) have been synthesized by the condensation of 4-methoxybenzohydrazide (1) with different aromatic acids (2a-o) in presence of phosphoryl chloride. The structural assignment of this compound (3a-o) has been made on the basis of elemental analysis, UV, IR, 1H NMR and mass spectral data. The synthesized compounds were screened for their in vitro growth inhibiting activity against different strains of bacteria and fungi viz., Staphylococcus aureus, Bacillus subtilis, Bacillus megaterium, Escherichia coli, Pseudomonas aeruginosa, Shigella dysenteriae, Candida albicans, Aspergillus niger and Aspergillus flavus were compared with the standard antibiotics such as chloramphenicol (50 μg/ml) and griseofulvin (50 μg/ml) using well agar diffusion technique. Compounds 3e, 3g, 3h and 3m exhibits highest antibacterial activity and compounds 3d, 3g and 3h showed better antifungal activity. The synthesized compounds (3a-o) were screened for their in vitro antiinflammatory activity against carrageenan-induced rat paw oedema. Compounds 3f and 3i were found to be most active compound of this series, which shows 46.42% and 50% inflammation inhibitory activity, whereas standard drug phenylbutazone exhibit 53.57% antiinflammatory activity at a dose of 50 mg/kg po.

The major side effects in the use of aryl alkanoic acids is their gastric irritancy, which is partly due to the corrosive nature of carboxylic acid group present in them. In order to reduce or mask the side effects of carboxylic moiety we planned to synthesize various 2,5-disubstituted-1,3,4-oxadiazole via the condensation of 4-methoxybenzohydrazide with various aromatic acids in presence of phosphoryl chloride respectively in the hope of getting potent biodynamic agents and evaluate their antimicrobial and antiinfl ammatory activity.

MATERIALS AND METHODS
The identification and purity of the products were checked by TLC (Merck Silica-60F 254 ) with Ethyl acetate: acetone (9:1) using iodine vapours and UV light as detecting agents and the Rf value were given below. Melting points were measured on open capillaries in a liquid paraffin bath and are uncorrected. The absorbance maxima (λmax) were determined on a Systronics UV-Visible double beam spectrophotometer (2201) in ethanol. IR Spectra were taken on a Perkin Elmer Spectrum RX I, FTIR Spectrophotometer using potassium bromide pellets. 1 H NMR spectra were recorded in DMSO-d 6 on AMX-400, NMR spectrometer using TMS as an internal standard (chemical shift in δ ppm). FAB mass spectra were taken out on a JEOL SX102/ DA-6600 mass spectrometer using Argon/Xenon (6 kV, 10 mA) as the FAB gas. Elemental analysis was obtained on a Carlo Erba 1108 Heraeus elemental analyzer. All the chemicals used were of synthetic and AR grade and were procured from Alfa Aesar (4-methoxybenzohydrazide), USA, S.D. Fine Chem. Ltd and Merck, Mumbai, India.
General procedure for the synthesis of 2,5disubstituted-1,3,4-oxadiazoles (3a-o): A mixture of different aromatic acid(s) (0.01 mol) with 4-methoxybenzohydrazide (1.6617 g, 0.01 mol) in phosphoryl chloride (15 ml) was refluxed over a steam bath for 5-6 h. The progress of the reaction was monitored by TLC (Merck Silica-60F 254 ) using ethyl acetate: acetone (9:1) as eluent. The reaction mixture was cooled and poured on to crushed ice (∼200 g) with continuous stirring. The solid mass separated was neutralized with sodium bicarbonate solution (10% w/v). The resulting solid thus obtained was collected by filtration, washed well with cold water, dried in vacuum and recrystallized from absolute ethanol (95%) and analyzed. Adopting the above procedure fi fteen different 2,5-disubstituted-1,3,4-oxadiazoles (3a-o) were synthesized and their characterization data are presented in Table 1. Yield and melting point of the product(s) were determined and summarized below.         The compounds were tested at a concentration of 100 μg/ml (or) 0.00001 nanomoles were prepared in dimethylformamide (DMF). The petridishes used for antibacterial screening were incubated at 37 ± 1º for 24 h, while those used for antifungal activity were incubated at 28º for 48-72 h. The diameters of zone of inhibition (mm) surrounding each of the wells were recorded.

Acute toxicity study:
Acute toxicity study was carried out by "Stair case" method 19 . Swiss mice of either sex were injected with a particular dose, say 100 mg/kg and observed for a period of 24 h for any mortality. The subsequent doses are then increased by a factor 1.5 if the dose was tolerated, and decreased by a factor 0.7 if it was lethal. The LD 50 of the drug was found to be 500 mg/kg body wt. One tenth of this dose was selected as the therapeutic dose for evaluation (i.e. 50 mg/kg).

Antiinflammatory activity against carrageenaninduced rats paws oedema:
Antiinflammatory activity was determined by carrageenan-induced rat paw method of winter et al. 20 . Male Wistar rats (120-150 g) was used for the experiment. They were fed with standard pellet diet and water was given ad libitum. Acute edema was induced in the right hind paw of rats by injecting 0.1 ml of freshly prepared 1% w/v of aqueous solution of carrageenan (Sigma, USA) in the subplanter region of right hind paw. After the carrageenan injection the paw volume was measured before and after 1, 2 and 3 h by plethysmometer (UGO-Basile, Italy). The difference between the left and right paw was taken as a measure of oedema. Any signifi cant reduction in the volume of the paw compared to the control group was considered as anti-inflammatory response 21 . Percent inhibition of inflammation after 3 h was calculated by applying Newbould formula. % inhibition = 100 [1 − a − x/ b − y], where, x = mean paw volume of rats before the administration of carrageenan injection in the test and the standard groups, y = mean paw volume of rats before the administration of carrageenan injection in the control group, a = mean paw volume of rats after the administration of carrageenan and test compound  3a  10  12  13  14  12  12  3b  20  20  17  19  16  15  3c  11  15  13  13  13  10  3d  16  16  15  20  15  14  3e  23  19  21  22  17  15  3f  14  14  15  14  13  12  3g  22  17  18  24  17  20  3h  21  14  22  22  20  16  3i  16  14  15  17  14  14  3j  24  15  16  20  20  12  3k  15   or standard compound, b = mean paw volume of rats after the administration of carrageenan injection in control group. The results are presented in Table 4.

RESULTS AND DISCUSSION
2,5-Disubstituted-1,3,4-oxadiazole (3a-o) was synthesized by the condensation of 4-methoxybenzohydrazide with various aromatic acids in presence of phosphoryl chloride (Scheme 1). The physical and analytical data of the compounds (3a-o) were collected and presented in Table 1. The yields of 3a-o fall in the range of 66-88%. The spectral (IR, 1 H NMR and MS) and analytical data are in good agreement with their structures.
In the toxicity study, LD 50 of the drug was found to be 500 mg/kg body wt. The therapeutic dose of the drug is considered as 1/10 th of the LD 50 value. Screening results of antimicrobial activity reveal ( The results in Table 4 indicate that the compounds 3f and 3i are active (p < 0.001) with the standard. Moreover, compounds 3d, 3g, 3j and 3o show less significant anti-inflammatory activity (p < 0.01). Carrageenan-induced paw edema was taken as a prototype of exudative phase of inflammation. The development of edema has been described as biphasic.
The initial phase is due to the release of histamine, serotonins, 5-hydroxy tryptamine and kinins in the first hour after injection of carrageenan. More pronounced second phase is related to the release of prostaglandin [22][23][24] like substances in 2-3 h. Hence, the signifi cant anti-infl ammatory effect may be due to an inhibitory effect exerted predominantly on the mediators of inflammation induced by phlogogenic stimuli.