Synthesis and Antimicrobial Activity of Some Novel Schiff Bases Containing 1 , 2 , 4-Triazole-3-thione

A series of Schiff bases (4a-j) were prepared by reacting 4-amino-5(1-phenylethyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione and substituted aromatic aldehydes. The chemical structures were confirmed by means of UV, H NMR, IR, MS(API-ES) spectral data and elemental analysis. All the synthesized compounds were screened for their antimicrobial activity. Among the tested compounds, Schiff bases with 4-OH and 4-NO2 substituent exhibited remarkable antimicrobial activity against all the tested microorganism.


Introduction
The compounds carrying azomethine functional group -C=N-which are known as Schiff bases gain importance in medicinal and pharmaceutical field due to the most versatile organic synthetic intermediates and also showing a broad range of biological activities such as antituberculosis 1,2 , anticancer 3,4 , analgesic and antiinflamatory 5,6 , anticonvulsant [7][8][9] , antibacterial and antifungal 10,11 activities.
1,2,4-Triazoles have also been found to possess a wide spectrum of pharmacological activities [12][13][14][15][16][17][18] .The 1,2,4-triazole nucleus has been incorporated in to a wide variety of therapeutically interesting molecules to transform them in to better drugs.The drugs such as fluconazole and itraconazole are the best examples for potent antifungal molecules possessing triazole nucleus 19 .
The development of antimicrobial resistance has increased in this century and there is a need of synthesis of new antimicrobial agents which will be more selective, potent and less toxic compared to the used drugs in clinical treatment.These observations prompted us to synthesize a series of Schiff bases containing 1,2,4-triazole ring system on the same molecule due to the importance of triazole and Schiff bases in biological systems by means of antimicrobial activity.

Experimental
All the chemicals and solvents were purchased from Merck, Aldrich, and Fluka.Melting points were determined with Kleinfeld SMP-II and were uncorrected.UV spectra were recorded on a Schimadzu UV 2100S spectrophotometer.The IR spectra were recorded on a Schimadzu FTIR 8400S spectrometry. 1H NMR spectra were recorded in DMSO on a Bruker Avance-DPX-400 spectrometer in DMSO-d 6 and chemical shifts were given in δ ppm with tetramethylsilane (TMS).The splitting patterns of 1 H NMR were designed as follows: s: singlet, d: doublet, t: triplet, q: quarlet, m. multiplet.Elemental analysis was performed on Leco CHNS-932 analyzer.

4-{[(4-Benzyloxyphenyl)methylidene]amino}-5-(1-phenylethyl)-2,4-dihydro-3H-1,2, 4-triazole-3-thione (4j)
UV: λ maks (nm) , (logε) : 203 (4,45); 327 (4,48).IR v maks (cm -1 ) 3200 (N-H), 1166 (C=S). 1 20,21 .Mueller-Hinton broth for bacteria, RPMI-1640 medium for yeast strain were used as the test medium.Serial two-fold dilutions ranging from 5000µg/ml to 4.9 µg/mL were prepared in medium.The inoculum were prepared using a 4-6 h broth culture of each bacteria and 24 h culture of yeast strains adjusted to a turbidity equivalent to a 0.5 Mc Farland standard, diluted in broth media to give a final concentration of 5x105 cfu/mL for bacteria and 0.5x103 to 2.5x103 cfu/mL for yeast in the test tray.The trays were covered and placed in plastic bags to prevent evaporation.The trays containing Mueller-Hinton broth were incubated at 35 o C for18-20 h and the trays containing RPMI-1640 medium were incubated at 35 o C for 46-50 h.The MIC was defined as the lowest concentration of compound giving complete inhibition of visible growth.The compounds were isolated in satisfactory yields (50-82%) and purified by recrystallisation, using ethanol.The purities of the synthesized compounds were checked using HPLC.The structures of the synthesized compounds (4a-j) were supported by elemental analysis and by the spectral data achieved from UV, IR, 1 H-NMR and mass spectroscopy which were in agreement with the proposed structures.The characterization data of all the new compounds are summarized in Table 1.(7.10)In the 1 H NMR spectrum, the azomethine protons of compounds (4a-j) resonated at 9.22-10.27ppm as expected.Chemical shifts of phenolic protons of compounds 4d, 4g and 4h were detected at 9.43, 8.55 and 9.99 ppm respectively.The triazole NH signal of all synthesized compounds appeared as a singlet at 13.70-14.04ppm.The aromatic protons were also observed at the expected regions.
The mass spectroscopic fragmentation of compound 4b and 4e were studied under atmospheric pressure electrospray ionization (API-ES).The API-ES spectra of these compounds displayed molecular ions which were confirmed their molecular weights..

Antibacterial and antifungal activity
The minimum inhibitory concentration (MIC) of the synthesized compounds was determined against the bacteria and the yeast C. albicans using a standard broth dilution technique.All the MIC results are presented in Table 2.The obtained data reported that compounds were able to inhibit the growth of the selected microorganisms in vitro showing MIC values between 9 and 625 µg/mL.None of them showed any inhibition against Pseudomonas aeruginosa ATCC 27853 and their results were not shown in Table 2.In general Schiff bases and presence of pharmacophores like -NO 2 , -Br, phenolic -OH and -Cl are reported to posses antimicrobial activities 22 .Among the tested compounds 4d and 4e containing 4-OH and 4-NO 2 substituted phenyl ring, were found to be the most active derivatives at an MIC value of 9 µg/mL against C. albicans.The compound 4e showed same MIC value (9 µg/mL) as reference compound cefuroxime against S. Epidermidis.

Conclusion
A series of novel Schiff bases have been synthesized and screened for their antimicrobial activities.The antimicrobial screening indicated that among the tested compounds Schiff bases with 4-OH and 4-NO 2 substituent exhibited remarkable antimicrobial activity against all the tested microorganism.Thus, the compound 4d and 4e having OH and NO 2 substituent could be lead compounds for further molecular modifications in the search for novel antibacterial agents.

Table 2 .
Antimicrobial activity data of synthesized compounds a