Synthesis of Some Novel Heterocyclic 1 , 2 , 4-triazolo [ 3 , 4-b ] [ 1 , 3 , 4 ] Thiadiazole Derivatives as Possible Antimicrobial Agents

Article history: Received on: 23/10/2013 Revised on: 29/11/2013 Accepted on: 12/12/2013 Available online: 27/02/2014 A series of 6-aryl-3(3,4–dialkoxyphenyl) -[1,2,4]triazole [3,4-b][1,3,4] thiadiazole (7a-7o) were synthesized by condensing 4-amino-5-(3,4-dialkoxyphenyl)-4H-[1,2,4]triazole-3-thiol (6) with various aromatic carboxylic acids in the presence of phosphorous oxychloride through one-pot reaction. The structures of these newly synthesized compounds were confirmed on the basis of IR, H NMR and mass spectral studies. All the synthesized compounds were screened for their antimicrobial activity against a variety of microorganisms.


Chemistry
Melting points were determined in open glass capillary tube and are uncorrected.Purity of the compounds was checked by thin layer chromatography (TLC) on silica gel G coated plates using ethyl acetate and n hexane (1:1, v/v); iodine chamber and observed in UV light.IR spectra were recorded on BRUKER 375-FTIR Spectrometer. 1 H NMR spectra recorded on a Bruker BioSpin Avance III 700MHz FT-NMR spectrometer using TMS as an internal standard.Chemical shifts are reported in parts per million (d) and signals are described as singlet (s), doublet (d), triplet (t), and multiplet (m).The mass spectra were recorded on a Q-TRAP 1400 spectrophotometer on Turbo spray mode.Solvents and reagents were purchased from the commercial vendors in the appropriate grade and were used without purification.Reaction sequence employed for the synthesis of title compounds is shown in scheme 1.

General method for the synthesis of 3,4-dialkoxy benzoic acid (2)
To a solution of substituted benzaldehyde (0.176 mol) in 30 ml water, a solution of potassium permanganate (0.191 mol) in 60 ml water was added at 70-80°C over a period of 2 h.The reaction mass was stirred at same temperature for 2 h and filtered.The filtrate was acidified using conc.hydrochloric acid at 0-5°C.The product obtained was filtered, washed with water and dried.The crude product was recrystallized from methanol (Mathew et al., 2006).

General method for the synthesis of 3,4-dialkoxy benzoate (3)
To a solution of substituted benzoic acid in methanol (absolute), conc.sulphuric acid was added slowly at 0-5°C over a period of 30 min and refluxed for 2 h.After quenching into cold water (in some reactions bicarbonates also added), precipitated solid was filtered, washed with water and dried.The completion of reaction was monitored by using TLC (Mathew et al., 2006).

General method for the synthesis of 3,4-dialkoxyphenyl carbohydrazide (4)
The methyl esters of substituted aromatic acids (0.1 M), in 30 ml of methanol were dissolved and hydrazine hydrate (0.1 M) was added drop wise to the mixture with Ar' Scheme 1. Synthesis of substituted triazolothiadiazoles stirring.The resulting mixture was allowed to reflux for 6 h and the contents were allowed to cool.The crystals formed was filtered, washed thoroughly with water and dried.The completion of the reaction was monitored on TLC by using silica gel-G coated plates by using ethyl acetate and n Hexane (1:1) as the eluent and observed in UV light (Mathew et al., 2006).
The color of the reaction mixture changed to green with the evolution of hydrogen sulfide gas (lead acetate paper and odor).A homogenous reaction mixture was obtained during the reaction process.The reaction mixture was cooled to room temperature and diluted with water (100 ml).On acidification with concentrated hydrochloric acid, the corresponding triazole was precipitated which was recrystallized with methanol (Mathew et al., 2006).
Solid residue was precipitated with evolution of gas.It was collected after 12 hours standing, was filtered, washed with cold water, dried and recrystallized from ethanol (7a-7o).

Antibacterial activity
The newly synthesized compounds were evaluated for their antibacterial activity against Staphylococcus aureus (MTCC No 3160), Bacillus cereus (MTCC No 9786), Escherichia coli (MTCC No 1698) and Pseudomonas aeruginosa (MTCC No 4673) bacterial strains by serial plate dilution method (Barry, 1991;Karegoudar et al., 2008).Serial dilutions of the drug in Muller-Hinton broth were taken in tubes and their pH was adjusted to 5.0 using phosphate buffer.Standardized suspension of the test bacterium was inoculated and incubated for 16-18 h at 37°C.The minimum inhibitory concentration (MIC) was noted by observing the lowest concentration of the drug at which there was no visible growth.A number of antimicrobial discs are placed on the agar for the sole purpose of producing zones of inhibition in the bacterial lawn.Agar media were poured into each petri dish.Excess of suspension was decanted and placing in incubator at 37 °C for 1 h dried the plates.Using an agar punch, wells were made on these seeded agar plates and minimum inhibitory concentrations of the test compounds in dimethylsulfoxide (DMSO) were added into each labelled well.A control was also prepared for the plates in the same way using solvent DMSO.The petri dishes were prepared in triplicate and maintained at 37 °C for 3-4 days.Antibacterial activity was determined by measuring the diameter of inhibition zone.Activity of each compound was compared with Gentamycin as standard.Zone of inhibition was determined for (7a-7o) and the results are summarized in table no 2.

Antifungal activity
Newly prepared compounds were screened for their antifungal activity against Candida albicans (MTCC No 1637) and Aspergillus niger (MTCC No 10180), in DMSO by serial plate dilution method (Verma, 1998;Karegoudar et al., 2008).Agar media were prepared by dissolving peptone (1 g), D-glucose (4 g) and agar (2 g) in distilled water (100 ml) and adjusting the pH to 5.7.Normal saline was used to make a suspension of spore of fungal strains for lawning.A loopful of particular fungal strain was transferred to 3 ml saline to get a suspension of corresponding species.Agar media of 20 ml were poured into each petri dish.Excess of suspension was decanted and plates were dried by placing in an incubator at 37°C for 1 h.Using an agar punch each labelled well were made on these seeded agar plates and minimum inhibitory concentrations of the test compounds in dimethylsulfoxide (DMSO) were added into each labelled well.A control was also prepared for the plates in the same way using solvent DMSO.The petri dish were prepared in triplicate and maintained at 37 °C for 3-4 days.Antifungal activity was determined by measuring the diameter of the inhibition zone.Activity of each compound was compared with Miconazole as standard.Zones of inhibition were determined for (7a-7o) and the results are summarized in table no 3.

Antibacterial activity
The antibacterial activity of the newly synthesized compounds 7a-7o were reported as minimum inhibitory concentration (MIC) at the concentration range, 1.56 -25 μg/ml against Staphylococcus aureus , Bacillus cereus (Gram-positive bacteria) and Escherichia coli, Pseudomonas aeruginosa (Gramnegative bacteria) using gentamycin as standard and the results are summarized in table no 2. Compounds 7a, 7d and 7m showed comparatively good activity against S. aureus and B. cereus, Compound 7g showed moderate to good activity against B. cereus and Compound 7m exhibited moderate to good activity against E. coli.

Antifungal activity
The antifungal data of the synthesized compounds 7a-7o were reported as minimum inhibitory concentration (MIC) at the concentration range, 1.56 -25 μg/ml against Candida albicans and Aspergillus niger using miconazole as standard and the results are summarized in table no 3.The compound 7a exihibited highest activity against all tested fungi.Compound 7d showed good activity against C. albicans while Compounds 7m, 7n and 7o exhibited moderate to good activity against A. niger.