Efficient Electrochemical Synthesis, Antimicrobial and Antiinflammatory Activity of 2–amino-5-substituted- 1,3,4-oxadiazole Derivatives

An efficient electrochemical method for the preparation of 2-amino-5-substituted-1,3,4-oxadiazoles (4a-k) at platinum anode through the electrooxidation of semicarbazone (3a-k) at controlled potential electrolysis has been reported in the present study. The electrolysis was carried out in the acetic acid solvent and lithium perchlorate was used as supporting electrolyte. The products were characterized by IR,1H-NMR,13C-NMR, mass spectra and elemental analysis. The synthesized compounds were screened for their in vitro growth inhibiting activity against different strains of bacteria viz., Klebsilla penumoniae, Escherichia coli, Bassilus subtilis and Streptococcus aureus and antifungal activity against Aspergillus niger and Crysosporium pannical and results have been compared with the standard antibacterial streptomycin and antifungal griseofulvin. Compounds exhibits significant antibacterial activity and antifungal activity. Compounds 4a and g exhibited equal while 4c, d, i and j slightly less antibacterial activity than standard streptomycin. Compounds 4a and g exhibited equal while 4b, c, d, f and i displayed slightly less antifungal activity than standard griseofulvins.

In the context of green chemistry, some 5-substituted-2-amino-1,3,4-oxadiazoles (4) have been synthesized by electrooxidative cyclization of semicarbazone (3)  as a new general environmentally benign synthetic method.The development of ecofriendly synthetic methods are the need of the hour.In this respect, organic synthesis involving multi-component reactions under reagent-free conditions is a basic protocol because multistep conventional synthesis produces considerable amounts of environmentally unfavorable wastes, mainly due to a series of complex isolation procedures often involving expensive, toxic and hazardous solvents and reagents after each step.The application of electricity as a non conventional energy source for activation of reactions in suitable solvents has now gained popularity over the usual homogeneous and heterogeneous reactions, as it provides chemical processes with special attributes, such as enhanced reaction rates, better selectivity, higher yield of pure products and several eco-friendly advantages.These reactions do not require oxidizing reagents and can be performed at ordinary room temperature.

General experimental procedure:
Column chromatography was carried out by using Merck silica gel 60.The purity of the synthesized compounds were ascertained by TLC on precoated silica gel plates in various solvent systems using iodine vapors and UV light as detecting agent.The melting points were recorded on an electrothermal apparatus and were uncorrected.Infra red spectra were recorded on a Shimadzu 8201 PC IR spectrophotometer in KBr pellets and reported in cm -1 . 1 H NMR and 13 C NMR spectra were measured on Bruker DRX 300 MHz FT spectrometer instruments using DMSO-d 6 as solvent with TMS and CDCl 3 as internal standards (chemical shift in δ ppm).Carbon multiplities were assigned by DEPT techniques.The structures of the newly synthesized compounds were assigned on the basis of elemental analysis and were recorded on a Elementar Vario EL III.Carbon, hydrogen and nitrogen analyses were within ±0.4% of the theoretical values.Mass spectra were taken out on a Jeol SX 102/DA-6600 mass spectrometer using Argon/Xenon (6 KV, 10 mA) as the FAB gas.All the chemicals used were of synthetic and AR grade and was procured from Agros-Organics, USA, S. D. Fine Chem.Ltd., Mumbai and Merck, Mumbai, India.

Electrolysis:
Preparative scale controlled potential electrolysis [26][27][28][29][30] were performed at room temperature in 250 ml three-electrode cell assembly with platinum plate as working as well as counter electrode and saturated calomel electrode as reference electrode.Magnetic stirrer was used for the proper mixing of reaction mixture.All the electrolysis experiments were carried out at their corresponding oxidation potentials and were completed in 3 to 5 h.After which no oxidation product was seen to diffuse in the bulk.All the products were solid and coloured and entirely different from the starting compound.The current potential data was recorded with the help of potentiostat at the interval of 15 min and has been depicted in Table 1.Approximately 4-6.5 Fmol -1 of electricity was passed for the electrolysis which is very small in comparison to energy used in other conventional methods.The physical and analytical data of the synthesized compounds (4a-k) has been depicted in the Table 2.The spectral (IR, 1 H NMR, 13 C NMR and MS) and analytical data are in good agreement with their structures.

Screening for Antimicrobial activity:
All the synthesized compounds were tested for antimicrobial activity by adopting the experimental method of Benson [31] .Whatman No.1 filter paper discs of 6 mm diameter, placed in a Petri dish, were autoclaved.The test compounds in measured quantities (1.0 mg, 0.5 mg) were dissolved in 5 ml dimethylformamide to produce 200 ppm and 100 ppm solutions, respectively.The filter paper discs were allowed to dry and the amount of the substance per disc was taken as 500 and 250 μg.The bacterial (24 h) and fungal (48 h) cultures from the slants were diluted with sterile water and mixed thoroughly to prepare a clear homogeneous suspension.These suspensions were uniformly spread on solidifi ed agar (nutrient and potato dextrose agar) medium.The fi lter paper discs prepared from dimethylformamide medium were carefully placed over the spread cultures and incubated at 37º for 24 h for bacteria and at 28-30º for 48 h for fungi.Paper discs treated with dimethylformamide alone served as control.After the incubation period the plates were examined for inhibition zones.The diameters of inhibition zones (including the diameter of the disc) were measured.All determinations were made in triplicate for each of the compounds and the average value was taken.
The antibacterial and antifungal screening results were presented in Tables 3 and 4.

Antiinflammatory activity against carrageenaninduced rat paws oedema:
Antiinflammatory activity was determined by carrageenan-induced rat paw method of Winter [32] .Male Wistar rats (125-150 g) were used for the experiment.They were fed with standard pellet diet and water was given ad libitum.The animals were acclimatized for one week under laboratory conditions before performing the test.mg/kg body weight po, suspended in 1% w/v of CMC in water).The control group received corresponding amount of vehicle (1% w/v of CMC).All the test compounds and standard drug were administered 30 min prior to carrageenan injection.
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.Percent inhibition of infl ammation 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 or standard compound, b = mean paw volume of rats after the administration of carrageenan injection in control group.The results are presented in Table 5.

RESULTS AND DISCUSSION
It is obvious that an environmentally benign synthetic method for the synthesis of 2-amino-5-substituted-1,3,4-oxadiazoles would be highly attractive.Keeping this objective in the mind, we have synthesized 2-amino-5-substituted-1,3,4-oxadiazole 4 derivatives  through the electrooxidation of semicarbazone 3 at a platinum electrode in controlled potential electrolysis in acetic acid (Scheme 1).This electrochemical cyclization evolves the oxadiazoles without requirement of any hazardous reagents.We have used acetic acid as a solvent and lithium perchlorate (LiClO 4 ) as an electrolyte that can be handled very easily without major precautions.
The fi rst step of the mechanism (Scheme 2) represents the deprotonation of 3 to form an anion 3a, which rearranges itself into 3b and evolves a free radical 3c after one electron oxidation.Subsequent second electron oxidation from the free radical 3c gives a carbocation 3d.The formation of carbon oxygen bond completes the ring.On loosing a proton in the last step 2-amino-5-substituted-1,3,4-oxadiazole 4 was obtained.
The antimicrobial activity indicated that compounds 4a, b, c, d, f, g and i were found to be active against  better antifungal activity against Aspergillus niger and C. pannical along with the standard fungicide Griseofulvins.The screening results revealed that compounds 4a and g showed equal antifungal activity when compared with the griseofulvins.
The antimicrobial activity of the compounds varied upon the type and position of the substituents at 5-substituted-2-amino-1,3,4-oxadiazole moiety.It can be concluded from the antimicrobial screening results that when 5-substituted-2-amino-1,3,4-oxadiazoles were substituted with aryl halide the antimicrobial activity was altered to an appreciable extent.
The results of antiinflammatory activity (Table 5) shows that the compounds 4d and 4h were active (p<0.01)with the standard.Moreover, compounds 4c, d, f and g showed signifi cant antiinfl ammatory activity (p<0.01).Carrageenan-induced paw edema was taken as a prototype of exudative phase of infl ammation.The development of edema has been described as biphasic.The initial phase was due to the release of histamine, serotonins, 5-hydroxy tryptamine and kinins in the fi rst hour after injection of carrageenan.The second phase was related to the release of prostaglandin [33][34][35] 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.