INTRODUCTION:

Decreased or no antibacterial activity shown by existing antibacterial agents including antibiotics is largely confined to resistance developed by the pathogenic bacteria toward these agents. Production of drug inactivating enzymes such as β-lactamase is the common mechanism of resistance to β-lactam antibiotics. Alteration in a single aminoacid of bacterial enzyme is found to make the bacteria resistant and is observed with quinolone antibiotics. Bacterial species including pseudomonas are developing resistance through changing the cell permeability. Some bacteria are resistant to tetracyclines because they have an active transport pump which removes tetracycline from the cell membrane. Resistance developed by S. aureus in mainly confined to alteration in penicillin binding proteins (transpeptidases). Thus increasing bacterial resistance towards the existing antibacterial agent became a major reason for introduction of new and potential drugs to combat various bacterial infections.

Triazole derivatives ( 1H- 1,2,4 and 4H- 1,2,4 ) showed very promising biological activities including anticonvulsant ^[1-2], antifungal ^[3-5], anticancer ^[6-9], anti-inflammatory ^[10-12] , antibacterial ^[13-16] and others.

Several compounds (Figure 1) containing 1, 2, 4-triazole rings are well known as drugs. For example, fluconazole is used as an antimicrobial drug ^[17], while vorozole, letrozole and anastrozole are non-steroidal drugs used for the threatment of cancer ^[18] and loreclezole is used as an anticonvulsant ^[19]The imidazole derivative dacarbazine (as imidazole derivatives) which contains amino-diazo chain exhibit antineoplastic and antibacterial activity. On the basis of above observations it was planned to prepare 4H – 1, 2, 4 – triazole derivatives substituted at position 1 by amino-diazo chain. The presence of this chain on triazole moiety was assumed to possess very potent antimicrobial, analgesic and anti-inflammatory activity

MATERIAL AND METHOD:

All the chemicals required were purchased from the local suppliers and were purified by established methods. The melting points were recorded by open capillary method and are uncorrected. The purity and homogeneity of the synthesized compounds was routinely ascertained by the thin layer chromatography, performed on plates coated with silica gel- G using the solvent system methanol: carbon tetrachloride: acetone in the ratio of 50: 40: 10. The absorption maxima of the synthesized compounds were recorded in methanol/ ethanol (analytical grade, 1mg/100mL). The methanolic/ ethanol solutions of the synthesized compounds were scanned on Shimadzu UV 1700 spectrophotometer, Kyoto, Japan; in the region 200-400 nm. The IR spectra were recorded in KBr disc on FTIR Shimadzu 8400 S Japan. The ¹H-NMR spectra were recorded on Bruker Avance II 400 NMR spectrometer using d6-DMSO as solvent and TMS as an internal solvent. The Mass spectra were recorded on Waters Q –Toff-micro spectrometer. The C, H, N, S analyses were carried out on elemental analyzer Vario H- III. The antimicrobial activity was measured by cup plate method using ciprofloxacin and Fluconazole as standard drug during evolution of antibacterial and antifungal activity respectively.

Table1: Physical Constants of the synthesized derivatives 4 [a-e]

Comp.	R1	R2	R3	Mol.For. [Wt.]	Y [%]	M.P. (⁰C)	Rf	C.%cal. [%fond]	H.%cal. [%fond]	N.%cal. [%fond]	S.%cal. [%fond]
4a	CH₃	H	C₆H₅	C₉H₁₀N₆S [234]	31.11	111-114	0.58	46.15 [46.14]	4.27 [4.28]	35.89 [35.88]	13.67 [13.68]
4b	CH₃	H	C₆H₄-NO_2-p	C₉H₉ N₇ O₂S [279]	65.23	134-136	0.69	38.70 [38.68]	3.52 [3.56]	35.12 [35.14]	11.46 [11.48]
4c	CH₃	H	C₆H₄- CH₃-o	C₁₀H₁₂N₆S [248]	31.47	152-154	0.64	48.38 [48.34]	4.83 [4.85]	33.87 [33.85]	12.90 [12.94]
4d	CH₃	H	C₆H₄-OCH_3-p	C₁₀H₁₂N₆OS [264]	70.93	212-215	0.62	45.45 [45.46]	4.54 [4.56]	31.81 [31.85]	12.12 [12.08]
4e	CH₃	C₆H₅	C₆H₅	C₁₅H₁₄N₆S [310]	73.82	256-258	0.74	58.06 [58.06]	4.51 [4.53]	27.09 [27.07]	10.32 [10.33]

Table 2: Results of in vitro antibacterial activity of the synthesized compounds 4(a-e)

Comp. No.	Gram positive bacteria				Gram negative bacteria
	S. aureus		B. subtilis		E. coli		P. aeruginosa
	ZOI (mm)	% Inhibition	ZOI (mm)	% Inhibition	ZOI (mm)	% Inhibition	ZOI (mm)	% Inhibition
4a	22	91.66	09	37.5	01	4.16	10	40
4b	03	12.5	09	37.5	02	8.33	08	32
4c	04	16.66	05	20.83	01	4.16	05	20
4d	12	50	01	4.16	19	79.16	01	4
4e	13	54.16	07	29.16	14	58.33	10	40
Std.	24	100	24	100	24	100	25	100

Std. = Ciprofloxacin; ZOI (mm) = Zone of inhibition in millimeter, Borer size: 10 mm, Solvent: DMF [Internal solvent]

Table 3: Results of in vitro antifungal activity of the synthesized compounds 4a-e

Compound no.	*A. fumigatus*		*C. albicans*
Compound no.	Zone of Inhibition (mm)	% Inhibition	Zone of Inhibition (mm)	% Inhibition
4a	03	75	14	56
4b	01	25	01	4
4c	02	50	01	4
4d	02	50	01	4
4e	01	25	12	48
Std.	04	100	25	100

EXPERIMENTAL WORK:

1. SYNTHESIS^{20- 22}: Compounds 3(a-b) were synthesized by established methods and were obtained as per the reported yield. The compounds 3 (a-b) were diazotized as per the standard procedure. The corresponding solutions of diazotized product in 10% NaOH were prepared and cooled by addition of crushed ice. To the cooled preparations, cold solution of different amines in. 10% aq. NaOH were added and were stirred vigorously to obtain the titled compound 4 (a-e). All the synthesized compounds were recrystallized with rectified spirit. Good results (highest yields) were obtained when the diazotized compound and amines were taken in equimolar concentration. The physical constants of all the synthesized compounds are given in Table 1.

2. ANTIMICROBIAL ACTIVITY^23-25: All the synthesized compounds 4(a-e) were evaluated for in vitro antimicrobial activity against the pathogens S. aureus, B. subtilis, P. aeruginosa, E. coli, A. niger and C. albicans. as per the literature methods. The method used for this purpose was cup – plate method. Dimethyl formamide (DMF) was used as solvent. Ciprofloxacin and fluconazole were used as standard reference compound for antibacterial and antifungal activity respectively. Test and standard compounds were administered at concentration of 100 μg/ ml. The antibacterial and antifungal activity was expressed as zone of inhibition (ZOI, millimeter) and % inhibition and is presented in Table 2 and Table 3 respectively.

RESULT: - The characterization data of all the synthesized compounds are follows:

4a. λ _max 355nm (methanol) ; IR (KBr, V max, cm^-1): 3382.91 (-NH), 3203.54 (-CH, Ar), 1602.74 (-N=N), 1199.64 (-C=S,triazole), 750.26 (-CH),763.76 (out of plane, Ar, C-H), ¹H-NMR (DMSO-d6, δ ppm): 2.03 [s, 3H, CH₃] ,4.67 [s, 1H, NH], 6.61-6.99 [m, 5H, Ar-ring], 11.41 [s, SH], MS: m/z 234 [M⁺] ,

4b. λ _max 385nm (methanol) ; IR (KBr, V max, cm^-1): 3377.12 (-NH), 3271.05 (-CH, Ar), 1596.95 (-N=N), 1245.93 (-C=S,triazole), 754.12 (out of plane, Ar, C-H), ¹H-NMR (DMSO-d6, δ ppm): 2.08 [s, 3H, CH₃] ,4.67 [s, 1H, NH], 6.54-6.57 [m, 4H, C₆H₄], 11.41 [s, SH], MS: m/z 278 [M⁺]

4c. λ _max 356nm (ethanol) ;: IR (KBr, V max, cm^-1): 3386.77 (-NH), 2916.17 (-CH, Ar), 1600.81 (-N=N), 1147.57 (-C=S,triazole), 752.19 (out of plane, Ar, C-H), ¹H-NMR (DMSO-d6, δ ppm): 2.06 [s, 6H, 2CH₃] ,4.64 [s, 1H, NH], 6.58-6.62 [m, 4H, C₆H₄], 11.39 [s, SH], MS: m/z 248 [M⁺]

4d. λ _max 338nm (ethanol) ;: IR (KBr, V max, cm^-1): 3168.83 (-NH), 2995.25 (-CH, Ar), 1602.74 (-N=N), 1159.14 (-C=S,triazole), 754.12, 730.97 (out of plane, Ar, C-H), ; ¹H-NMR (DMSO-d6, δ ppm): 1.82 [s, 3H, -CH₃] ,2.02 [s, 3H, - OCH₃], 4.62[s,NH], 6.52-6.58 [m, 4H, -C₆H₄], ; MS: m/z 263 [M⁺]

4e. λ _max 354nm (ethanol) ;: IR (KBr, V max, cm^-1): 3382.91 (-NH), 3041.53 (-CH, Ar), 1596.95 (-N=N), 1172.64 (-C=S,triazole), 744.47 (out of plane, Ar, C-H), ; ¹H-NMR (DMSO-d6, δ ppm): 2.11 [s, 3H, CH₃] ,6.62-6.98 [m, 10H, Ar-ring], MS: m/z 310 [M⁺]

DISCUSSION AND CONCLUSION:

The physical constants and characterization data of all the synthesized compounds reveals their successful synthesis. At concentration of 100 μg/ ml, all the synthesized compounds 4 (a-e) exhibited antimicrobial activities against tested microorganism to different strength. Among the tested compound, the compound 4a exhibited highest activity (91.66%) against S. aureus. Against E. coli, 4d showed 79.16% inhibition. Compound 4a showed 75% activity against A. fumigatus. From the above findings it can be concluded that the titled compounds substituted either with phenyl ring or phenyl rings substituted with strong electron releasing groups (like -OCH₃) showed highest antimicrobial activity.

ACKNOWLEDGEMENT:

Authors are thankful to Dr. Kanika Sharma, Associate Professor, College of Science, Mohan Lal Sukhadia University, Udaipur (Raj.) for providing necessary facilities during evaluation of antimicrobial activity of the synthesized compounds

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Received on 08.04.2010 Modified on 15.05.2010

Asian J. Research Chem. 3(2): April- June 2010; Page 355-358