INTRODUCTION:

IN recent year, the chemistry of 1-2-4 triazoles and their fused heterocyclic derivative has received considerable attention owing to their synthetic and effective biological importance. Heterocyclic bearing a symmetrical triazole are reported to show a broad spectrum of pharmacological properties such as antimicrobial^1,6, antithyroid^7,8, antitubercular⁹, anti-inflammatory¹⁰, analgesic^11,12 and anticonvulsant activities¹³. A large number of heterocyclic compounds containing the 1-2-4 triazolo ring are associated with diverse pharmacological properties such as anti-inflammatory, fungicidal antimicrobial and antiviral activity. Thiosemicarbazide compound are exhibit thee anti microbial activity that has been widely used in many developing countries. Thiosemicarbazones have had lengthy history as potential prophylactic therapeutics for human disease.

In this paper we reported the synthesis of some potential thiosemicarbazide derivatives which shows the appreciable antimicrobial, anti-inflammatory and analgesic activity.

Prompted by the observed biological activities of the some thiosemicarbazide triazolo derivatives in continuation of our ongoing studies on novel biologically and pharmacologically active molecules, we have designed and synthesized some new- (3E)-N-(4-amino-5-sulfanyl-4, 5-dihydro-3H-1, 2, 4-triazol-3-yl)-3-(4-methylbenzylidene) triazane-1-carboxamide derivatives as potential antimicrobial, anti-inflammatory and analgesic agents. The results of this study are discussed in this paper.

EXPERIMENTAL WORK:

The preparation of the target compounds is outlined in scheme. The melting point were determining in open capillary tube and are uncorrected. Thin layer chromatography (TLC) was carried out using silica gel (Merck). The FT-IR spectra were recorded in KBr Pellets on Perkin-Elmer 882, FT-IR spectrometer. ¹H NMR spectra were recorded (in DMSO-d₆/CDCl3 or mixture of them) on DPX 300 NMR spectrometer and BRUKER-400. Chemical shift are expressed in ppm relative to tetramethylsilane. The physical constant and spectral data of the synthesized compound are presented.

The compound III and their respective hydrazide derivatives. These compounds were obtained by using different aromatic acid and the method reported in the literature.

Synthesis of (2E)-2-(4-methoxybenzylidene) hydrazinecarboxamide (I) Equivalent weight of 4 methoxy benzaldehyde (0.01) and thiosemicarbazide (0.01) taken in to the 5oo rbf. Then the mixture kept for reflux condesation for 4 hours in presence of 60ml dry ethanol. The above mixture pours in to the cold water with continuous stirring, until it gives the precipitate. The crystals dried at 60⁰ C^{18, 19}.

Synthesis of (3E)-N-(4-amino-5-sulfanyl-4, 5-dihydro-3H-1, 2, 4-triazol-3-yl)-3-(4- Methylbenzylidene) triazane-1-carboxamide (III) ¹⁴

To a continuously stirred solution of potassium hydroxide (8.4g, 0.15 moles) and the 4-methoxy benz-1thiosemicarbazide (18.5g, 0.1 moles) in absolute ethanol (100ml), carbon disulphide (11.2g, 0.15mole) was added drop wise. After the complete addition the mixture was diluted with absolute ethanol (75ml) and agitated for 14hrs. It was then diluted with dry ether (100ml) and the precipitate solid was collected by filtration, washed with ether and dried at 65^oc. Then obtain crude suspension of potassium dithiocarbazinate (II).

To above comp (II) suspension hydrazine hydrate (10ml, 0.2mol) was added and the mixture was refluxed for an hour. The colour of the reaction mixture changed to green with the evaluation of hydrogen sulphide and the homogeneous mass was obtained. It was then cooled and diluted with cold water (100ml). The cold mixture was acidified with concentrated hydrochloric acid. The sold separated was filtered, washed with water, dried and recristallysed from ethanol.

Synthesis of respective derivatives of Synthesis of (3E)-N-(4-amino-5-sulfanyl-4,5-dihydro-3H-1,2,4-triazol-3-yl)-3-(4- methylbenzylidene)triazane-1-carboxamide (III): (R¹-R¹⁰) The equivalent weight of (3E)-N-(4-amino-5-sulfanyl-4,5-dihydro-3H-1,2,4-triazol-3-yl)-3-(4- methylbenzylidene)triazane-1-carboxamide (III) and aromatic aldehyde reflux for 4 hre in presence of glacial acetic acid and dry ethanol . The refluxed reaction mixture poured into the cold water and separates the formed precipitate. Collect the precipitate and recrystallised it.

Antibacterial activity

All the newly synthesized compounds were initially screened for their in vitro antibacterial activity against Escherischia coli, Pseudomonas areuginosa, Staphylococcus aureus and bacillus subtilis by serial dilution method. The compound was tested at a concentration of 200 µg/ml in dimethylformamide against all organisms. Furacine was used as standard drug for comparison of antibacterial activity and solvent control was kept. The minimum inhibitory concentration (MIC values) of the compound were determined, the result of antibacterial screening studies are reported. A stock solution of concentration 100 µg/ml was prepared by dissolving 1mg of the test compound in 10 ml of dimethylformamide. One loopful of an 18hr broth culture was inoculated into 5ml nutrient broth and this was incubated at 37⁰ C.

The nutrient broth (0.5ml) was taken in tubes with the labelled the number a₁-a₁₀ and the test compound were added according to the dilution and labelled it according to the no. This process was repeated serially to obtain the quantity indicates in each of test tube. The 11 test tube kept as the control group. Drop of diluted broth culture of the test organisation (approximately 0.05) was added to all test tube using sterilised Pasteur pipette. The solution mixed gently and incubation was carried out at 37^o c for 16 to 18 hrs. Furacine was dissolved in dimethylformamide and was used as standard drug for comparison. The minimum inhibitory concentration at which there was no turbidity was taken as the minimum inhibitory concentration (MIC values) ^15,16.

The antibacterial screening result indicates that the entire tested compounds were found to posses lesser degree of activity against all the tested organisms compared to the standard. The result of studies are given in Table III

Anti-inflammatory activity:

Paw edema inhibition test was used on rats. Carrageenan (0.1ml of a suspension in saline) was injected into the sub planar tissue of the right hind paw, 30 min after the oral administration (100mg/kg) of the test compound. Volume was measured by using mercury Plethysmometer (U Basile, Italy) before and 2and 4 h after the injection of the irritant.

The increase in volume of the paw was adopted as a measure of the edema. Swelling in treated animals was calculated as a percentage of inhibition comparison to control^17,18. The difference of average value between treated animal and control group is calculated for each time interval and statically evaluated. The difference at the various time intervals gives some hints for the duration of the anti-inflammatory effect. The ED₅₀ value can be determined. The results are revealed in the table no. I

Analgesic activity:

The phenylqunone writhing test was used in mice, according to the method of Siegmund modified by Hendershot and Forsaith. 0.1ml/10g of a hydroalcoholic solution of 0.02% phenylquinone were injected i.p 20 min after administration of the test compound (100mg/kg sc). The analgesic activity was evaluated by the number of writhes between the 5^th and 15^th min following the injection of phenylquinone. Results were expressed as percentage of protection with respect to the control group^17,18,19. The Results were revealed in the table no. II

RESULT AND DISCUSSION:

The synthesis followed is outlined in scheme. The derivatives synthesized and subjected it for its antimicrobial and pharmacological evaluation. The antimicrobial screening followed according to the literature survey and it mentioned in table no.III.

TABLE NO. I: Characterisation data of compounds R₁to R₁₀:

Comp. No	Ar	Yield (%)	Nature of comp.	M.P	Solvent for recrystallisation	Mol. Formula
R₁	C₈H₈O₂	66.6%	yellow powder	220-25	Ethanol	C₁₉H₂₂N₈0₃S
R₂	C₇H₅OBr	74%	white crystals	184-86	Ethanol	C₁₈H₁₉BrN₈0₂S
R₃	C₇H₆O₂	84%	yellow powder	175-78	Methanol	C₁₈H₂₀N₈O₃S
R₄	C₇H₆O	94%	brown crystals	176-79	Methanol	C₁₈H₂₀N₈O₂S
R₅	C₈H₈O₂	62%	yellowish green powder	195-98	Ethanol	C₁₉H₂₂N₈O₃S
R₆	C₇H₆OCl	72%	white crystallised	195-20	Ethanol	C₁₈H₁₉ClN₈O₂S
R₇	C₈H₈O	89%	brown powder	215-17	Methanol	C₁₉H₂₂N₈O₂S
R₈	C₈H₈O	87%	Yellow powder	167-70	Methanol	C₁₉H₂₂N₉O₄S
R₉	C₇H₅O₃N	58%	Yellow crystal	210-15	Methanol	C₁₈H₁₉N₉O₄S
R₁₀	C₇H₇NO	64%	Yellow crystals	220-22	Ethanol	C₁₈H₂₁N₉O₂S

TABLE NO. II

Compound	Anti-inflammatory activity 2 h 4 h		Analgesic
R₁	35.7	24.6	28.4
R₂	-	-	28
R₃	39.2	14.2	26
R₄	37.9	7.1	26.4
R₅	23.6	50.0**	23.7
R₆	24.1	15.0	24.7
R₇	-	-	13.0
R₈	13.0	22.2	19.0
R₉	-	14	15.4
R₁₀	10.8	27.9**	19.5

Acetylslicilic acid 60.0* 78.7*

Phenylbutazone 61.0*

Compounds were administered at the dose of 100mg/kg sc or po. The significance vs. Control was p< 0.05 and **p < 0.01

**: Compound shows the significant activity compare to standard drug

TABLE NO.III

Comp. No.	Antibacterial activity data of the newly synthesized compound Minimum inhibitory Concentration (µ/ml)
Comp. No.	*E.coli*	*P. aeruginosa*	*S. aureus*	*B. subtilis*
R₁	10	20	20	<10
R₂	100	10	-	100
R₃	100	-	100	100
R₄	-	-	20	-
R₅	100	-	-	100
R₆	10	20	10	10
R₆	20	10	10	20
R₇	100	-	100	100
R₈	20	50	50	20
R₉	10	20	20	10
R₁₀	100	200	50	100
Furacin	6	12.5	12.5	12.5

Index:

Diameter of the disc - 5mm

Amount of the sample – 100µg/mL

Control (solvent) - DMF (Dimethyl formamide ) .

CHARACTERISATION DATA OF COMPOUND:

R₁ = (3E)-3-(3-methoxybenzylidene)-N-(4-{[(E)-phenylmethylidene]amino}-5-sulfanyl-4,5-dihydro-3H-1,2,4-triazol-3-yl)triazane-1-carboxamide,IR(KBr)3299(NHamide),3296-3152(NH-str.),1628-1465(C=N) 1646(NHCONH),898(Ar-CH).¹H NMR δ( DMSO d6) 6.32(S,H,NH),7.24(ArH) 8.21(d,1H,CH=N), 11.13(S,1H,NH). m/z 445

R₂ = (3E)-N-(4-{[(E)-(4-bromophenyl)methylidene]amino}-5-sulfanyl-4,5-dihydro-3H-1,2,4-triazol-3-yl)-3-(4-methoxybenzylidene)triazane-1-carboxamide.IR(KBr) bond3297(NH amide),3030(C-H str.) 1627-1462(C=N) 1648(NHCONH),748(Ar-CH).¹H NMR δ ( DMSO d6 ) 5.4(S,2H,OCH₃),6.31(S,2H,NH₂) 7.6-7.88(S,1H,NH). m/z 458, 459(m+1), 460(m⁺²).

R₃ = (3E)-N-(4-{[(E)-(3-hydroxyphenyl)methylidene]amino}-5-sulfanyl-4,5-dihydro-3H-1,2,4-triazol-3-yl)-3-(4-methoxybenzylidene)triazane-1-carboxamide.IR(KBr)3032(CHstr),1628 1460(C=H),1645(NHCONH),809(ArCH).¹HNMRδ(DMSOd6),5.3(S,2H,OCH₃)6.31(S,H,NH₂),7.22(Ar-CH),8.20(d,1H,CH=N)10.8310.9(S,2H,OH),11.14(S,1H,NH). m/z 428.

R₄ = (3E)-3-(benzylidene)-N-(4-{[(E)-phenylmethylidene]amino}-5-sulfanyl-4,5-dihydro-3H-1,2,4-triazol-3-yl)triazane-1-carboxamide.IR(KBr)3033(C-Hstr)1640(NHCONH),1628-1463(C=N),898(Ar-Hbond).¹HNMRδ(DMSOd6)5.4(S,2H,OCH₃),7.21(Ar-H),6.30(S,H,NH₂),8.24(d,1H,CH=N), 11.12(S,1H,NH). m/z 412, m⁺¹ 413.

R₅ = (3E)-3-(4-methoxybenzylidene)-N-(4-{[(E)-phenylmethylidene]amino}-5-sulfanyl-4,5-dihydro-3H-1,2,4-triazol-3-yl)triazane-1carboxamide.IR(KBr)3031(CH),1640(NHCONH),1628(C=N)709(Ar Hband),2922(CH2).¹HNMRδ(DMSO)d6,5.3(S,2H,O-CH₃),6.30(S,H,NH),7.20(Ar H),8.23(d,1H,CH=N),11.14(S,1H,NH).m/z 442.

R₆ = (3E)-N-(4-{[(E)-(3-chlorophenyl)methylidene]amino}-5-sulfanyl-4,5-dihydro-3H-1,2,4-triazol-3-yl)-3-(4-methoxybenzylidene)triazane-1-carboxamide. IR(KBr)3035(CHstr),1638(NHCONH),1630(C=N),748(Ar-H),746cm^-1(C-Cl str).¹HNMRδ(DMSO)d6,5.4(S,2H,OCH₂),6.30(S,H,NH₂),7.18(Ar-H),8.24(d,1H,CH=N), 11.10(S,1H,NH). m/z 446, 447.5, 448.

R₇= (3E)-3-(4-methylbenzylidene)-N-(4-{[(E)-phenylmethylidene]amino}-5-sulfanyl-4,5-dihydro-3H-1,2,4-triazol-3-yl)triazanecarboxamide.IR(KBr)3032(CHstr),1640(NHCONH)1628(C=N),748(Ar-H). ¹HNMRδ(DMSO)d63.91(S,2H,CH₃),5.4(S,2H,OCH₂),6.30(S,H,NH),7.0-8.4(m,Ar-H).m/z 426.

R₈ = (3E)-3-(3-methylbenzylidene)-N-(4-{[(E)-phenylmethylidene]amino}-5-sulfanyl-4,5-dihydro-3H-1,2,4-triazol-3-yl)triazane-1-carboxamide.IR(KBr)3030(C Hstr),1642(NHCONH),1628(C=N),748(Ar-H),3010-894(C-Haromatic).¹HNMRδ(DMSO)d63, 3.92(S,H,CH₃),5.6(S,2H,OCH₂),6.35(S,2H,NH₂),7.0-8.4(m,Ar-H). M/z 472.

R₉ = (3E)-3-(3-nitrobenzylidene)-N-(4-{[(E)-phenylmethylidene]amino}-5-sulfanyl-4,5-dihydro-3H-1,2,4-triazol-3-yl)triazane-1-carboxamide.IR(KBr),3032(C-Hstr),1646(NHCONH),1628 1462(C=N),1154-326(CN),898(Ar-Hbond).¹HNMRδ(DMSO)d6,3.9(S,3H,OCH₃),6.32(S,H,NH), 8.26(d,1H,CH=N),7.0-8.5(H,Ar-H).m/z 458.

R₁₀ = (3E)-3-(4-aminobenzylidene)-N-(4-{[(E)-phenylmethylidene]amino}-5-sulfanyl-4,5-dihydro-3H-1,2,4-triazol-3-yl)triazane-1-carboxamide.IR(KBr)3030(C-Hstr),1642(NHCONH),1628-1462(C=N),1155-326(C-N),897(Ar-H).¹HNMRδ(DMSO)d6,3.9(S,3H,OCH₃),6.35(S,H,NH),8.25(1H,CH=N),12.6(2H,NH₂).m/z 428.

Scheme

The synthesized compound R₈, R₉ and R₁₀ exhibit the appreciable activity, while R₁, R₂, R₅ exhibit the moderate inhibition of the microbial growth. The tested compound shows the result which comparable to the standard gives less value. The R₉ and R₁₀ compounds attached the electron windrowing group, this compound exhibit the satisfying activity. The result obtained from the animal screening mentioned in the table no. I, the compound R₅ and R₁₀posses the good anti-inflammatory activity, the tested compound result compared with the standard drug which is mentioned in the procedure, while all compound having the moderate analgesic activity.

ACKNOWLEDGEMENT:

We are also thankful to the Principal Prof Dr. N. V. Kalyane and Management of B.L.D.E.A college of Pharmacy, Bijapur for providing the necessary facilities to carry out this work.

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Received on 19.03.2011 Modified on 14.04.2011

Research J. Pharm. and Tech. 4(9): Sept. 2011; Page 1400-1403