INTRODUCTION:

Thiadiazole is a versatile moiety that exhibits a wide variety of biological activities. Thiadiazole moiety acts as “hydrogen binding domain” and “two-electron donor system”. Many drugs containing thiadiazole nucleus are available in the market such as acetazolamide, methazolamide, sulfamethazole, etc. Thiadiazole can act as the bio-isosteric replacement of the thiazole moiety. So it acts like third and fourth generation cephalosporins, hence can be used in antibiotic preparations. Thiadiazole is a 5-membered ring system containing two nitrogen and one sulphur atom. Members of this ring system have found their way into such diverse application as pharmaceuticals, oxidation inhibitors, cyanine dyes, & metal complexing agents.1,3,4 thiadiazole derivatives having antibacterial^1,8, antifungal², nticonvulsant³,antihelmentic⁴,antinflammatory^5-7 ,cytotoxic activity⁹ antileishmenial¹⁰ analgesic^11, activity¹².

MATERIAL AND METHODS:

Experimental:

The purity of the synthesized compounds were ascertained by thin layer chromatography on silica gel G in various solvent systems, the most common solvent system used were chloroform, ethyl acetate, and Methanol in the ratio of 5:4:1 , benzene and acetone in two ratios 5:1 and 4:1 and using iodine vapors and UV chamber as detecting agent. Compounds were detected by column chromatography. Melting points were determined by open capillary boiling point determination method and are uncorrected. Elemental analyses were done using Infra-red spectra were recorded on Bruker alpha Spectrophotometer in. Proton NMR spectra were recorded in deuterated chloroform (CDCl₃) on Bruker Advance 400-NMR Spectrometer using tetramethylsilane as internal standard. The MASS spectrum was recorded on TOF MS ES+ Mass spectrometer.

General procedure:

Synthesis of thiosemicarbazone:

Thiosemicarbazide (0.01 M) and Crystalline sodium acetate (0.02 M) were taken in RB Flask, and 0.5 gm of aromatic acid was added slowly with continuous stirring. The mixture was turbid so added methanol until clear solution obtained shake mixture for few minutes and allowed to stand. Now this mixture is refluxed for 3-4 hours at 60-70^OC with continuous stirring. After refluxing, thiosemicarbazone precipitated from the cold solution. Other derivatives were prepared similarly (_1-5).

Synthesis of 2-amino 5- aryl 1, 3, 4- thiadiazole :

Thiosmicarbazone 2a (0.01 м) and Sodium acetate (0.02 м) were dissolved in 30-40 ml of glacial acetic acid taken in a round-bottom flask equipped with a separating funnel for the addition of bromine. Bromine (0.7 ml in 5 ml glacial acetic acid) was added slowly to it, while stirring magnetically. Now this solution was refluxed for 5-6 hours with magnetic stirrer at the temp. 75-80 C. Then this solution is poured on crushed ice precipitate which got separated is dried and crystallized. Other compounds (_1-5) were prepared similarly and data were collected.

Where, R= Phenyl, p-chloro-phenyl, p-nitro-phenyl, m-nitro-phenyl, o-chloro-phenyl.

Compound 1:

The mixture of thiosemicarbazide, sodium acetate and benzoic acid were added with methanol to get clear solution. This mixture was refluxed for 3-4 hour at 60-70◦c.prepared thiosemicarbazone and sodium acetate dissolved in glacial acetic acid with addition of bromine. This solution mixture was refluxed for 5-6 hours at 75-80^OC. the compound was extracted out with CHCl₃under reduced pressure.

FTIR(KBr)1640,1493,1458,1076,681(thiadiazole),3369.63 (NH),1670,1640(C=O).1HNMR(DMSOds,400MHz):4.1 (1H,s,J=3.935),3.605(4H,m,J=3.595),1.98(2H,d,J=1.977), Mass(CPS):m/z(%) calculated 177, found 176.

Compound 2:

The mixture of thiosemicarbazide, sodium acetate and 4-chloro benzoic acid were added with methanol to get clear solution. This mixture was refluxed for 3-4 hour at 60-70◦c.prepared thiosemicarbazone and sodium acetate dissolved in glacial acetic acid with addition of bromine.this solution mixture was refluxed for 5-6 hours at 75-80^oC. The compound was extracted out with CHCl₃under reduced pressure.

FTIR:1637.41,1490.30,1398.30,1180.5,757.53(thiadiazole),3320.16(NH),1589.90(C=O).¹HNMR(DMSOds,400MHz): 3.65(1H,s,J=3.577),3.24(5H,m,J=3.288),2.09(2H,d,J=2.079), Mass(CPS):m/z(%) calculated 211, found 211.2.

Compound 3:

The mixture of thiosemicarbazide, sodium acetate and 4-nitro benzoic acid were added with methanol to get clear solution. This mixture was refluxed for 3-4 hour at 60-70◦c.prepared thiosemicarbazone and sodium acetate dissolved in glacial acetic acid with addition of bromine. This solution mixture was refluxed for 5-6 hours at 75-80^OC. the compound was extracted out with CHCl₃under reduced pressure.

FTIR:1642,1376.92,1277.04,719.17(thiadiazole),3392(NH),1712 (C=O). ¹HNMR (DMSOds, 400MHz): 8.365(1H,s, J=8.361), 8.275(4h,m,J=8.271) ,8.035(2H,d, J=8.032)

Mass (CPS):m/z(%) calculated 222, found 221.

Compound 4:

The mixture of thiosemicarbazide, sodium acetate and 3-nitro benzoic acid were added with methanol to get clear solution. This mixture was refluxed for 3-4 hour at 60-70^◦C.prepared thiosemicarbazone and sodium acetate dissolved in glacial acetic acid with addition of bromine. This solution mixture was refluxed for 5-6 hours at 75-80^◦C. the compound was extracted out with CHCl₃under reduced pressure.

FTIR:1642.62,1460,1375,1281,720.08(thiadiazole),3394,2920(NH),1711.43(C=O).¹HNMR(DMSOds,400MHz):8.785 (1H,s,J=8.7865),8.49(4H,m,J=8.521),8.442(1H,d,J=8.444),Mass(CPS):m/z(%) calculated 222, found 220.8.

Compound 5:

The mixture of thiosemicarbazide, sodium acetate and 2-chloro benzoic acid were added with methanol to get clear solution. This mixture was refluxed for 3-4 hour at 60-70◦c.prepared thiosemicarbazone and sodium acetate dissolved in glacial acetic acid with addition of bromine. This solution mixture was refluxed for 5-6 hours at 75-80◦C. the compound was extracted out with CHCl₃under reduced pressure.

FTIR1644.25,1458.31,1376.99,1186.21,715(thiadiazole),3345(NH),1663(C=O).¹HNMR(DMSOds,400MHz): 8.035 (2H,d,J=8.0295),7.55(5H,t,J=7.5475)Mass (CPS): m/z (%) calculated211,found 211.3.

Purification of compounds:

All the derivatives were purified by column chromatography using silica gel (60-120 mess). The actual fraction was collected by regular checking of TLC. Finally the compounds were further purified by preparative TLC. Solvent system used were chloroform: ethyl acetate: Methanol in the ratio of 5:4:1, benzene : acetone( 5:1).

Table:1-Physical properties of synthesize compounds:

compds	R	Molecular formula	Molecular weight	Melting point( ^OC)	% yield
*C1.*	-H	C₈H₇N₃S	177	180	52
*C2.*	-4Cl	C₈H₇N₃SCl	211	250	67
*C3.*	-4NO₂	C₈H₇N₃SNO₂	222	245	64
*C4.*	-3NO₂	C₈H₇N₃SNO₂	222	330	58
*C5.*	-2Cl	C₈H₇N₃SCl	211	245	62

Table:2

Zone of inhibition in mm
Compounds	*S. aureus*	% of inhibition	*B. subtilis*	% of inhibition	*E .coli*	% of inhibition	*P. aeruginosa*	% of inhibition
1	14	87.50	13	76.47	14	87.5	14	77.78
2	13	81.25	15	88.23	15	93.75	16	88.88
3	15	93.75	14	82.35	12	75.00	15	83.33
4	12	75.00	15	88.23	13	81.25	13	72.22
5	14	87.50	16	94.11	14	87.50	14	77.77
Ofloxacine	16	100.00	17	100.00	16	100.00	18	100.00

RESULT AND DISCUSSION:

All synthesized compounds 5 were evaluated for their antimicrobial activity against Escherichia coli representing Gram–negative bacteria, Bacillus subtilis. The results of antimicrobial effect of all tested compounds were reported as zone of inhibition in mm and are shown in Table 1. The result showed that most of newly synthesized compounds exhibited potent antibacterial activity. Generally the test compounds showed good activity against Gram– positive bacteria as compared to ciprofloxacin. Other compound showed moderate activity against Gram– positive bacteria. Compounds showed good activity against Gram–negative bacteria as compared to ciprofloxacin(Table-2) and all synthesized show moderate anti-inflammatory activity(Table-3)..

Table-3:

Compounds	Dose mg/kg	Inhibition of paw oedema after 3 hrs	Inhibition of paw oedema after 6 hrs(%)
1	90	29.89	57.46
2	90	32.34	59.26
3	90	29.54	57.87
4	90	30.12	58.45
5	90	29.00	57.12
Control	-	-	-
Ibuprofen	40	40.29	66.44

CONCLUSION:

After the experiment it is concluded that the compounds which are synthesized in the project having good yield value. The synthesize Thiadiazole compounds identified and characterize by 1HNMR, MASS and FTIR spectra. After it the Pharmacological activity was done. The entire compound gives good response for antibacterial and anti-inflammatory activity.

ACKNOWLEDGEMENT:

The authors would like to express their gratitude and thanks to the Head, Dept. of Pharmaceutical Chemistry, K.N.I.M.T. Sultanpur, C.D.R.I. Lucknow for necessary facilities for this research Work.

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Received on 25.01.2013 Modified on 08.02.2013

Asian J. Research Chem. 6(3): March 2013; Page 209-211