Study of Zn(II)-salicylidene-4-(p-chlorophenyl)-2-aminothiazole Complex by Polarographic Method with Its Antibacterial activity

Newly Zn(II)-salicylidene-4-(p-chlorophenyl)-2-aminothiazole (SCAT) ligand was synthesized and studied in DMF media by the polarographic method and produce a DC and DPP polarogram in KCl (supporting electrolyte) with Britton-Robinson buffer. The far FT-IR spectral study show signals at 465 and 412 cm -1 respectively which confirm the metal-ligand bonding. The serial tube dilution method (MIC) was used for investigating the antibacterial activity of this newly synthesized complex and ligand toward pathogenic bacteria, B. subtilis


INTRODUCTION
Schiff bases have received a lot of interest for their synthesis, structure, and reactivity due to their simplicity of synthesis and structural adaptability. However, Schiff bases have shown several clinical features, such as antifungal, antibacterial, and antioxidant, actions [1][2][3][4][5] . The derivatives of salicylaldehyde are coordinated with two donor sites, the O atom of the phenolic group (-OH) and the N atom of the azomethine group (>C=N-). Due to their ability to bind through N, O, and/or S atoms as either bidentate or tridentate chelators, which commonly form four-or six-coordinate complexes, Schiff bases are reported to be the most suitable chelating ligands in coordination chemistry 6,7 . Our interest in salicylidene-4-(p-chlorophenyl)-2aminothiazole (SCAT) ligand is due to the presence of azomethine (>C=N-), -Cl and -OH moiety as they play an important role in chemotherapy and pharmaceutical perspective [8][9][10] . When employing the polarographic method, compound solution-mercury interactions 11 , reflect their oxidation-reduction behavior, which can be useful for both physiological and biological objectives. In recent years, for the treatment of microbial disease, researcher has tried to increase the potentiality of the drug by the addition of a central metal atom 12 . So we have synthesized, and characterized, salicylidene-4-(p-chlorophenyl)-2aminothiazole (SCAT) ligand and its Zn(II) complex for their polarographic behavior and antibacterial activity.

MATERIALS AND METHODS
In this polarographic and antibacterial study, all organic compounds utilized were A . R . g r a d e . 2 -a m i n o t h i a z o l e ( AT ) a n d p-chlorobenzaldehy de (Sigma Aldrich Chemicals Pvt Lt.). After double distillation, absolute methanol (E. Merck) was used, in the formation of the complex, Zn(II) acetate was used as received. Melting points of freshly synthesized chemicals were identified using the melting point apparatus. The purity of the chemicals was regularly examined using thin-layer chromatography. The KBr pellets method was used to record FT-IR spectra from the SHIMADZU FT-IR 01504 spectrophotometer. DC polarogram and DPP were recorded using the Elico DC Polarograph Model CL-362, and a systronic MK VI pH meter was used for pH measurements. A stock solution of the Schiff base and its Zn(II) complex has been synthesized in a DMF with Britton-Robinson (0.04 M) buffer. The polarogram of the (SCAT) ligand and its Zn(II) complex was taken after removing oxygen.
For antibacterial activity, the serial tube dilution method was used and the stock solution of the synthesized compounds under test was prepared by dissolving 15-20 mg/2.5 mL in an appropriate solvent DMF. For antibacterial activity, the solution was properly diluted with sterilized water to get 120, 60, 30, 15, 7.5, and 3.75 µg/mL 10 mL N. Broth was added to each of the glass tubes and autoclaved at 15 lbs/sq. inch pressure for 15 minutes. A properly diluted stock solution of the organic compound under test was added to the glass tubes to 120, 60, 30, 15, 7.5, and 3.75 µg/ mL concentrations. Antimicrobial loopful of log phase cultures of Bacillus subtilis and Escherichia coli were inoculated and the tubes were incubated at 37°C. The MIC was determined after 24 hours.

Synthesis of Salicylidene-4-(p-chlorophenyl)-2aminothiazole (SCAT) Ligand
The salicylidene-4-(p-chlorophenyl)-2aminothiazole (SCAT) ligand was synthesized, according to reports in the literature 13 . To prepare a reaction solution of p-chlorophenyl-2-aminothiazole (4.3 g, 20 mmol) and salicylaldehyde (2.5 g, 20 mmol) in ethanol. The reaction solution was stirred and heated gently for 2 and a half hours, and an orange crystalline precipitate could be observed. The resultant precipitate was recovered from the ethanol and dried at 55 o C which was carefully confirmed by silica gel thin layer chromatography (TLC). The synthesis of the SCAT ligand is represented in Scheme 1. The synthesis of the Zn(II) complex is represented in scheme 2.

Polarographic Study of Zn(II) Complex
Zn(II) and its complex with SCAT ligand both occur in a two-electron reduction wave in 40% DMF with B-R buffer at pH 6.5±0.01 (Fig. 4). In the polarographic study when the progressive increase in the concentration of Schiff base (depolarizer), changes in the potential to a more negative value and a decrease in the height of the diffusion current, indicated complexes formation 18 . A linear straight line (Fig. 5) was obtained by plotting E 1/2 against log Cx (concentration of the Schiff base in logarithmic form) indicating the formation of the most stable complex in solution 19 . The stability constant of the complex was calculated using the difference in E 1/2 /Ep between the free metal ion and the complex ion [20][21][22][23] . The polarographic data was treated with Lingane equation 24 to produce a stoichiometric 1:2 (M:L) ratio with a stability constant log β 2 =4.4 of the Zn(II) complex. streptomycin (standard drug) and we obtained enough results when Zn(II) ion have been binding with SCAT, through oxygen and nitrogen donor sites. The SCAT ligand enhanced the antimicrobial activity when binding with the metal ion.
Results also show that Zn(II) complex has strong antibacterial properties due to the presence of electron-withdrawing 32 (azomethine) groups connected to the aromatic system. The fact that when N and O atoms are present in the SCAT ligand provides extra support with enhanced biological activity 33,34 . As reported previously, chelation 35,36 enhances the ligand's antibacterial potency and effectiveness compared to either metal ions or uncoordinated ligands.

IR Spectroscopic Characterization Salicylidene-4-(p-chlorophenyl)-2-aminothiazole (SCAT) ligand and its Zn (II) Complex
The signal at 3547 cm -1 in the FT-IR spectra of the SCAT ligand is due to the phenolic OH group25. This signal is disappearing in Zn(II) complex indicating the phenolic OH involved in coordination 26 . The medium signal seen in the complex 1613 cm -1 frequency range was characterized as corresponding to the (C=N) mode and lower frequency indicates that the azomethine nitrogen atom is involved in the complexation 25 . The lower (C=N) frequency indicates stronger Zn-N bonding. In the IR spectra of the complex, a signal was observed at 465 and 412 cm -1 which is due to the (Zn-O) and (Zn-N) stretching vibrations, respectively 27,28 . Confirmed the metal-ligand bonding.

Antibacterial activity
The synthesized SCAT ligand and its Zn(II) complex were investigated for their in vitro antibacterial activity against the specified microorganisms Gram-positive (B. subtilis) and Gram-negative (E. coli) by the serial tube dilution method [29][30][31] . The results of the antimicrobial studies were presented in Fig. 6 and Fig. 7 (A, B, C, and  D ). The novel salicylidene-4-(p-chlorophenyl)-2-aminothiazole (SCAT) ligand and its Zn(II) complex were tested for their efficacy in inhibiting microorganisms at a minimum concentration (µg/ mL). The antibacterial activity of SCAT against B. subtilis and E. coli were found to be good, with MIC values revealed to be 60 and 65 µg/mL ( Fig. 6 and Fig. 7 (A), (B)), which is advantageous from a clinical and pharmaceutical viewpoint. In the newly synthesized Zn(II) complex, MIC values were found to be 20 µg/mL and 25 µg/mL ( Fig. 6 and Fig. 7 (C), (D)), against pathogenic bacteria B. subtilis and E. coli. The antibacterial activity of SCAT ligand and its Zn(II) complex have been compared with

CONCLUSION
In the present research work, the polarographic and antibacterial activity of the salicylidene-4-(p-chlorophenyl)-2-aminothiazole (SCAT) ligand and its Zn(II) complex were studied. The formation of the complex between Zn(II) ion and SCAT ligand was confirmed by stability constant (log β 2 ) with stoichiometric 1:2 (M:L) ratio and FT-IR Spectroscopy. The polarographic method is used for the calculation of stability constants of complex, which is useful in the drug industry for the treatment of toxic metals 37 and the binding mechanism of Zn(II) ion in biological systems 38 . The antibacterial activity results exhibited that the SCAT ligand enhanced its bacterial activity when binding with Zn(II) ion and formed a complex.

ACKNOwLEDGEMENT
We would like to express our thanks of gratitude to the head of the chemistry department, Dr. Harisingh Gour University, Sagar for his support with the polarographic studies. The author Manohar Solanki is thankful to UGC, New Delhi for providing the JRF.