Synthesis , Characterization and Pharmacological Activities of 3 , 6-Disubstituted-1 , 2 , 4-triazolo [ 3 , 4-b ]-1 , 3 , 4-thiadiazoles and their Dihydro Analogues

4-Amino-5-aryl/heteroaryl substituted-3-mercapto-1,2,4triazoles 3(a-d) were prepared from the corresponding aromatic carboxylic acids through a multi-step sequence. Compounds 3(a-d) were made to react with various aromatic/hetero aromatic acids and hetero aromatic aldehydes to give 3,6-disubstituted-1,2,4-triazolo [3,4-b]-1,3,4-thiadizoles and 3,6-disubstituted-5,6-dihydro-1,2,4triazolo [3,4-b]-1,3,4-thiadizoles respectively. Elemental analysis, IR, 1 H NMR and mass spectral data elucidated the structures of all newly synthesized compounds. Synthesized compounds are studied for their antibacterial, antifungal, anti-inflammatory and analgesic activities. Some of the tested compounds showed significant pharmacological activities.

Condensation of the triazoles 3 (a-d) with aromatic acids in the presence of phosphorous oxychloride (Scheme 2) produced a series of triazolo thiadiazoles (4-16); while its condensation with hetero aromatic aldehydes (Scheme 3) afforded a series of 5,6-dihydro triazolo thiadiazoles (17-20).The structure assigned to compounds was substantiated by their analytical and other spectral data.

Experimental
Thin layer chromatography was used to access the completion of the reaction and purity of the compounds synthesized.Melting points were taken in open glass capillary tubes using thiels tube containing liquid paraffin and are uncorrected.IR spectra in KBr were recorded on a Shimadzu-8400 FTIR spectrophotometer, 1 H NMR spectra were recorded on Brucker spectrophotometer (400MHz) in DMSO-d 6 /CDCl 3 using TMS as an internal standard (chemical shifts are expressed in δ ppm), mass spectra were recorded in Finnigan MAT 8230 mass spectrophotometer and elemental analysis were recorded on Thermo Finnigan FLASH EA 1112 CHNS anlyser.The purity of the compounds were checked on silica gel-G coated plates by using ethyl acetate and petroleum ether (1:1) as the eluent and observed in UV light.All the synthesized compounds gave satisfactory elemental analyses.

General procedure for the preparation of Aryl hydrazide. (1)
To the methyl/ethyl esters of substituted aromatic acids (0.1 mol), hydrazine hydrate (0.1mol) was added and refluxed the solution for 30 min.20 mL of ethanol was added to the refluxing mixture as a solvent in order to homogenize solution.The resulting mixture was further allowed to reflux for 6 h.Excess ethanol was distilled out 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 petroleum ether (1:1) as the eluent and observed under UV light.

General procedure for the preparation of 2-aryl substituted-5-mercapto-1,3,4oxadiazole (2)
To a solution of 1 (0.1 mol) in ethanol (30 mL), KOH (0.1 mol) in absolute ethanol (50 mL) and CS 2 (0.2 mol) were added and refluxed for about 5 h till evolution of hydrogen sulfide was ceased.The reaction mixture was cooled at room temperature and diluted with water.
On acidification with dilute hydrochloric acid, the required oxadiazoles was precipitated.It was filtered, thoroughly washed with cold water and recrystallised from ethanol.
General procedure for the preparation of 3-substituted-4-amino-

General method for the synthesis of 3-aryl/heteroaryl-6-(2-substituted-4quinolinyl)-1,2,4-triazolo [3,4-b]-1,3,4-thiadiazoles. 4(a-d)
A mixture of respective triazole (0.02mol), 2-phenyl-quinoline-4-carboxylic acid (0.02mol), and phosphorous oxychloride (10mL) was heated under reflux for 4-6 h.The reaction mixture was cooled to room temperature and the mixture was gradually poured onto crushed ice with stirring.Finely powdered potassium carbonate and the required amount of solid potassium hydroxide were added till the pH of the mixture was raised to 8, to remove the excess of phosphorous oxychloride.Allowed the mixture to stand overnight, solid was separated.It was filtered, washed thoroughly with cold water, dried and recrystallized from hot ethanol.Similarly other compounds were prepared.[3,4-      [3,4-   [3,4-  In the 1 H NMR spectra of synthesized compound, the peaks due to NH 2 and SH, which were present in the amino mercapto triazole were absent that further confirmed the involvement of these functional groups in the cyclization of triazole to triazolo thiadiazoles.Similarly the absence of SH proton and the down fielding of NH 2 protons (integration for one proton) in the 1 H NMR spectra established that -CHO group of the aromatic aldehydes reacted with -SH & -NH 2 groups of triazoles and thus converted to dihydro triazolo thiadiazoles (17-20).The 1 H NMR, mass spectra, IR and elemental analysis supported the structure of various synthesized triazolo thiadiazoles and their dihydro analogues.

3-(3,5-dimethoxy phenyl)-6-[1-(2,4-dichloro phenoxy) ethyl]-1,2,4-triazolo [3,4-b]-1,3,4-thiadiazole (16b)
The antimicrobial results showed that some of the compounds are active against the tested microbes.It can be concluded that none of the prepared compounds were superior to positive controls against various tested microbial strains, but was interesting to note that compounds 8d, 9a, 9b, 9d, 14d & 16d were very sensitive to all the tested organisms comparable to the standards used at the concentration of 30µg/mL.Antimicrobial effects of the some of the synthesized compounds were reported in Table 3 & 4 as zone of inhibition against various bacterial and fungal strains respectively.
Anti-inflammatory and analgesic activity screening indicated that some of the tested compound 10d, 12d, 14a, 14b, 14d, 16a, 16b and 16d showed good anti-inflammatory and analgesic activities.Other compounds had moderate anti-inflammatory and analgesic activities.Anti-inflammatory and analgesic effects of the newly synthesized compounds were reported in Table 1

Anti-inflammatory activity
All the synthesized compounds were evaluated for their anti-inflammatory activity against carrageenan-induced acute paw oedema in albino rats (Wistar strain) weighing 150-200g 15,16 .The animals were weighed and numbered into various groups and each group contained six animals.One group served as control and received 0.1mL of 1% gum acacia suspension orally.Group II served as standard and received phenylbutazone at a dose of 5mg/kg suspension in gum acacia orally.One hour after the administration of test compounds at a dose of 5mg/kg as suspension in gum acacia, 0.1 mL of 1% carrageenan in normal saline was given subcutaneously to the sub plantar region of right hind paw.The paw volume was measured immediately ('0' h) and after 1, 2, 3 and 4 th h respectively by using plethysmograph.The amount of oedema in the drug-treated groups was compared in relation to the control group with the corresponding time intervals.The percentage of inhibition by the drugs was calculated by using the formula, Percentage inhibition = 100 (1-V test / V control ), Where V test = mean increase in paw volume of drug treated group; V control = mean increase in paw volume of control group.The results were expressed as percentage inhibition of oedema over the untreated control group.
The reaction mixture was cooled at room temperature and was neutralized with dilute hydrochloric acid.The solid obtained was filtered, thoroughly washed with cold water and recrystallised from ethanol.

Table 2 .
Analgesic activities of compounds

Table 3 .
Antifungal activities of compounds