SYNTHESIS, CHARACTERIZATION AND ANTIMICROBIAL ACTIVITY OF TRANSITION METAL COMPLEXES OF 4-[(2- HYDROXY-4-METHOXYPHENYL)METHYLENEAMINO]-2,4- DIHYDRO-3H-1,2,4-TRIAZOLE-3-THIONE

A novel nitrogen containing 4-[(2-hydroxy-4-methoxyphenyl)methyleneamino]-2,4-dihydro-3H-1,2,4-triazole-3-thione ligand (H2L) was synthesized by using an equimolar ratio of 4-amino-1,2,4-triazole-3-thione and 2-hydroxy-4-methoxybenzaldehyde. A series of Mn(II), Co(II), Ni(II), Cu(II), Zn(II) and Cd(II) complexes was synthesized by using the ligand. The synthesized ligand and transition metal complexes were characterized by IR, 1H NMR, 13C NMR, Mass spectrometry, UV, XRD and TGA investigation methods. Spectral data suggests that the ligand acts as a tridentate SNO donor. Further, the synthesized H2L ligand and their metal complexes were screened for antimicrobial activity. The results of biological activities showed that the metal complexes have higher antifungal as well as antibacterial activity as compared to the parent H2L ligand against the tested microbes.


INTRODUCTION
Schiff base and their metal complexes play an important role in the biological systems due to their ability to bind to biologically active sites. 1,2,4-triazole derivatives show various pharmaceutical potential 1-6 including antitubercular 7,8 antimicrobial, 9 antifungal 10,11 anticancer, 12 cytotoxic 13 and antioxidant 14 activities. Due to multidentate nature, they are chelate-forming ligands and can form various transition metal complexes. 15 Various triazole moieties containing drugs such as vorozole, anastrozole and letrozole ( Figure 1) are commercially available in the market. 16

Figure 1. Some commercially marketed azole drugs
Schiff bases derived from vanillin are well-known in the literature for a wide range of biological activities. [17][18][19] An important benefit of using 2-hydroxy-4-methoxy benzaldehyde instead of vanillin is the availability of chelating position of -OH group, which can easily deprotonate and bind to metal ions.

EXPERIMENTAL
All the used chemicals were AR grade and purchased from Spectrochem or SD Fine Ltd. and used without purification. The 4-amino-1,2,4-triazole-3-thione was prepared according to the literature method. 20 1 H and 13 C NMR spectra were recorded in DMSO-d6 on Bruker NMR, IR spectra on Perkin IR spectrometer, UV analyses were performed on a Shimadzu UV-VIS instrument and TGA results were obtained on a Shimadzu thermogravimetric analyzer. XRD measurements were done on a Philips Bragg--Brentano parafocusing goniometer (CuKα) at 298 K.
The physical and analytical data of the synthesized compound are reported in Table 1.

General procedure for preparation of metal complexes (6a-6f)
A methanolic solution of metal salt was added dropwise to a boiling methanolic solution of ligand H2L in an equimolar ratio (1:1) with constant stirring. The p H of the reaction mixture was adjusted to 7.5 to 8.5 by adding 10 % ethanolic ammonia solution and the contents were refluxed for about 7-8 hr.
The precipitated solid metal complex was filtered and washed several times with absolute methanol and dried (Scheme 3).

Scheme 3. Synthesis of metal complexes (6a-6f)
Physical and analytical data of the synthesized metal complexes is given in Table 1. The analogous reaction with iron(II) salts resulted in a complex mixture containing an iron(II) and two iron(III) complexes as detected by Mössbauer. Efforts to grow single crystals were unsuccessful. The synthesized complexes were proved to be X-ray amorphous except Mn(II), Zn(II) and Cd(II) complexes. These complexes look like to be monoclinic, and the lattice constants could not be determined exactly because of small particle size and wide bands of compounds.

H NMR spectra
In the 1 H NMR spectrum of the ligand, the azomethine (-CH=N-) proton appearing as a singlet at 9.14 δ ppm was shifted to δ 9.18 ppm in Zn complex. The signal for azomethine proton in zinc complex was observed slightly downfield, which is due to the transfer of electrons from azomethine nitrogen to zinc metal. There is formation of a coordination bond between azomethine nitrogen and zinc (Zn←N) [21]. The characteristic proton signal of triazole (-SH) and phenolic (-OH) disappeared in Zn complex, indicating that -S -,-Obindings form during complex formation. The coordinated water peak appeared at δ 3.04 ppm in the spectrum of Zn complex.

Mass spectra
The LCMS spectrum of ligand (H2L) showed a peak at 251.1 amu corresponding to M+H confirming the suggested formula (C10H10N4O2S). The mass spectrum of metal complexes displayed peaks at 361.0 and 331.2 amu for nickel (II) and zinc(II) complexes, respectively, corresponding to the molecular ion peak (M+H). The molecular ion peaks of metal complexes confirm the proposed molecular formula C10H14N4NiO5S for nickel and C10H10N4O3SZn for zinc complex. The mass spectra of metal complexes reveal the evidence of equimolar ratio of metal and ligand (1:1) as presented in Scheme 3.

UV-Visible spectra
Electronic spectra of the synthesized metal complexes were recorded in DMSO solvent at room temperature. The results can be seen in Table 3. The bands found for d 10 metal-containing complexes (Zn(II), Cd(II) may contain only CT and ligand n⟶π* bands, while ligand absorptions may correspond to n⟶π* and π ⟶π* transitions. [23][24][25] Table 3. UV data for the synthesized metal complexes As can be seen from Table 3, the geometry around Zn(II) and Cd(II) might be different, probably due to the difference in coordination numbers of central ions in these complexes. The UV-Vis intensities of octahedral Mn(II) complexes are very low as a consequence of their doubly forbidden nature and easily be masked by the bands of the organic ligand. Over the ligand bands, the d-d bands of octahedral Co(II) and Ni(II) complexes could be detected ( Table 3). The visible range bands at 552 for Co(II) and at 800 and 613 for Ni(II) probably belongs to the 4 T1g→ 4 T1g(P) and the 3 A2g→ 1 Eg and 3 A2g→ 3 T 1g transitions, respectively. As usual, the distorted tetrahedral Cu(II) complex has only one broadband in the visible region.

IR spectra and bonding modes
Infrared spectroscopy gives information regarding the type of functional groups (-SH, -OH, -N=CH-) attached to the corresponding metal ions. IR spectrum of ligand displays a band corresponding to -N=CH-linkage at 1598 cm -1 , which confirms the formation of Schiff base. Similarly, the band observed around 1608 cm -1 corresponds to the same -N=CH-linkage coordinated to the metals. [26][27][28] It is supported by the appearance of metal-nitrogen ν(M-N) bands between 441 and 466 cm -1 . In the IR spectra of ligand and complexes, a broad band at 2758 and 1109 cm -1 arise due the ν(S-H) and ν(C=S), respectively, while in the complexes, an ionic nature C-S band appears at 720 cm -1 . 27 The broad band in the spectra of complexes in the 3310-3330 cm -1 region indicates the presence of coordinated water. The broad band observed at 3232 cm -1 corresponds to -O-H stretching mode in the ligand, which disappears in the complexes indicating deprotonation of phenolic-OH during the complex formation. In IR spectra, -S-H stretching vibration was absent in metal complex due to deprotonation of thiol group and thiol sulfur coordinated to the metal ion. 29 Some selected IR spectral data are summarized in Table 4.

Sample
Absorption maxima, λmax, nm  From the IR data of the ligand and the corresponding metal complexes, it is clear that the ligand acts as a tridentate ligand. The coordinating atoms are the azomethine nitrogen, the thiol-sulfur and phenolate oxygen.
On the basis of the above spectral observations, we conclude that the ligand coordinates dinegative and tridentate around the metal ions.

Thermogravimetric analysis
Thermal data was recorded in the temperature range between 25-1000 o C under N2 and the results are summarized in Table 5. The primary decomposition intermediates were proved to be X-ray amorphous, based on the mass loss, these are expected to be the apropriate oxides of the central metal ions. Table 5. Thermogravimetric analysis of the synthesized complexes

Antimicrobial study
The synthesized compounds (ligand and metal complexes) were explored for antifungal and antibacterial activities. These activities were performed on Petri-plate containing 30 mL potato dextrose agar and nutrient agar medium. The plates were incubated for 24-48 h and 20-24 h culture of fungal and bacterial strains, respectively and the results were measured in terms of zone of inhibition in mm. Two fungal (Aspergillus niger, Alternaria alternata) and one bacterial (S. Aureus) species at 250 ppm concentration were used for studying antimicrobial activities. Results were compared with the standard drug i. e. carbendazim for fungal and streptomycin for bacterial as positive control reference drugs ( Table 6).
The results of antifungal activity suggested that among the tested compounds, only the cobalt complex was active against Aspergillus niger and copper, cadmium complexes against Alternaria alternate comparable with the standard drug. The antibacterial screening performed against Streptococcus Aureus indicates that all complexes were less potent than the standard drug -streptomycin. Selected images for the antimicrobial activity are shown in Figure 5.

CONCLUSION
The synthesized ligand acts as tridentate around the central metal(II) ions. On the basis of different techniques, the complexes of Mn(II), Co(II), Ni(II) and Cd(II) showed octahedral geometry, but Cu(II) complex exhibits square planar and Zn(II) form tetrahedral geometry. Results of antimicrobial activity indicate that the metal complexes show greater activity than the parent ligand. The ligand and metal complexes showed very less activity against S. aureus, which could be due to azomethine linkage and hetero atoms present in these compounds.