Synthesis, characterization and biological evaluation of 2,5-di-substituted 1,3,4-oxadiazole derivatives

We have reported some novel 1,3,4-oxadiazole synthesized by conventional method .The reaction of 5-bromothiophene-2-carbohydrazide and different benzoic acid derivatives reflux in toluene using phosphorus oxychloride as a catalyst, yielded a series of 2,5-di-substituted 1,3,4-oxadiazole HM-2a to HM-2t. The newly synthesized 2,5-di-substituted 1,3,4-oxadiazole were purified by column chromatography and characterized by IR, Mass, 1H NMR, 13C NMR spectroscopy and elemental analysis. All synthesized compounds were screened for antimicrobial activity using cup plate method. All the compounds showed moderate to good antimicrobial activity and anti fungal activity.


EXPERIMENTAL
All chemicals and solvents were purchased from Spectrochem Pvt Ltd., Mumbai of AR grade and were used without further purification. Melting points were taken in open capillary method and are uncorrected. IR spectra were recorded on FTIR-8400 spectrophotometer (Shimadzu, Kyoto, Japan), using DRS probe KBr pallet. 1 H-NMR spectra of the synthesized compounds were recorded on a Bruker-Avance-II (400 MHz) DMSO-d6 solvent. Chemical shifts are expressed in δ ppm downfield from TMS as an internal standard. Mass spectra were determined using direct inlet probe on a GCMS-QP 2010 mass spectrometer (Shimadzu, Kyoto, Japan). Physical constants of the synthesized compounds are shown in Table 1.

1. General synthesis of methyl 5-bromothiophene-2-carboxylate (Int 1)
To a well stirred 5-bromothiophene-2-carboxylic acid (0.01 mol) in methanol (25 ml), add concentrated sulphuric acid (0.01 mol) at 0-15 °C and then reflux the reaction mass at 65-70 °C for 6-7 hours. Then after completion of the reaction cool the reaction mixture at 15 °C and add cold water and stir for 3 hours at room temperature. Separated solid washed with water and collected by filtration.

General synthesis of 5-bromothiophene-2-carbohydrazide (Int 2)
Methyl 5-bromothiophene-2-carboxylate (0.01 mol) in methanol was refluxed with hydrazine hydrate (0.1 mol) for 5 to 6 hours. After completion of the reaction checked by TLC, the reaction mixture was cooled to room temperature. The separated solid was filtered, was with cold methanol and crystallized from methanol.

3. General synthesis of Oxadiazole (Int HM-2a-t)
A mixture of 5-bromothiophene-2-carbohydrazide (2.17 g, 0.01 mol) and different aryl acids (0.01 mol) in phosphorous oxychloride (10 ml), was refluxed with continuous stirring. After completion the reaction (6-7 hour monitoring by TLC), the content was cooled to room temperature then add ice cooled water and neutralized with sodium        International Letters of Chemistry, Physics and Astronomy Vol. 34

1. 1. Well Diffusion/Agar Cup Method
In vitro affectivity of antimicrobial agents can be demonstrated by observing their capacity to inhibit bacterial growth on suitable media. The production of a zone depends on two factors namely bacterial growth and concentration of antimicrobial agent. The hole/well punch method was first used by Bennett. This diffusion method has proved more effective than many other methods. According to Lt. General Raghunath the well technique is 5-6 times more sensitive than using disk method.

1. 2. Principle
When antimicrobial substance is added in agar cup (made in a medium previously inoculated with test organism) the redial diffusion of an antimicrobial agent through the agar, produces a concentration gradient. The test organism is inhibited at the minimum inhibitory concentration (MIC), giving rise to a clear zone of inhibition.  Greseofulvin ----500 100 100

CONCLUSION
We have developed a novel 2,5-disubstituted-oxadiazole by the reaction of different aromatic acid and hydrazide in high yield and purity. The reaction of various aromatic acid and 5-bromothiophene-2-carbohydrazide was carried out by refluxing in POCl 3 . All the synthesized compounds were evaluated for their antimicrobial activity. The investigation of antibacterial and antifungal screening data revealed that, the compounds HM-2h, HM-2m, HM-2r, and HM-2t shows good activity against bacterial stain, while HM-2h, HM-2j, HM-2k, HM-2r, and HM-2t shows comparatively good activity against fungal stain.