Biologically Active Transition Metal Chelates of Ni(II), Cu(II) and Zn(II) With 2-Aminothiazole-Derived Schiff-bases: Their Synthesis, Characterization and the Role of Anions (NO3, SO42-, C2O42-and CH3CO2-) on Their Antibacterial Properties

Biologically active nickel(ll), copper(ll) and zinc(ll) chelates with thiazole-derived nitro- and chlorosalicylaldehyde Schiff-bases having the same metal ion but different anions, e.g. nitrate, sulfate, oxalate and acetate have been synthesized and characterized on the basis of their physical, spectral and analytical data. In order to evaluate the possible participating role of anions on the antibacterial properties, these ligands and their synthesized metal chelates with various anions have been screened against bacterial species Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus.


INTRODUCTION
Various thiazole compounds have been found to be associated with diverse antibacterial , antifungal2, antitumour3, anticancer 4 and anthelmintic 5 activities. Many studies 6-1 have described the biological role of metals and chelation and have established the fact that those anticancer drugs as viable ligands exhibit increased anticancer activity when administered as their metal chelates. The connected metal centers in such biologically active molecules may involve different functions such as oxygen transport, DNA inhibitor, enzymatic activity, electron transfer and lock geometry. All these observations related to the essential role of metals linkage attracted our attention to commence a systematic reseamh program -s to investigate the factors responsible for this relationship. During the course of previous investigations thiazole-derived nitro-and chlorosalicylaldehyde Schiff-bases and their various transition metal chelates were prepared 6 as antibacterial agent and studied for this relationship of metal chelation and the subsequent antibacterial properties. In continuation to the same, during the present work we wish to extend the same studies hitherto the less investigated possible participating biological role of anions on these prepared chelates, which stay as the counterpart of the complexes. For this purpose thiazole-derived nitro-and chlorosalicylaldehyde Schiff-bases (HL and HL2) ( All chemicals and solvents used were of Analar grade. All metal were used as their nitrates, sulfates, oxalates and acetates. IR spectra were recorded on a Philips Analytical PU 9800 FTIR Spectrophotometer. UV-Visible spectra were obtained on a Hitachi U-2000 double-beam spectrophotometer. Conductance of the metal complexes was determined in DMF on a YSI-32 model conductometer. Magnetic measurements were done on solid complexes using the Gouy method. Melting points were recorded on a Gallenkamp apparatus and are uncorrected. The antibacterial studies were carried out with the help of the Department of Pathology, Quaid-e-Azam Medical College, Bahawalpur, Pakistan. with 2-Aminothiazole-Derived Schiff-Base Ligands Preparation of Schiff-bases The ligands were prepared by adopting the same procedure as reported earlier16.

Preparation of Metal Complexes
An ethanol solution of appropriate metal(ll) salt (1 mmol, 20 mL) was added to a stirred hot ethanol solution of the respective Schiff-base (2 mmol, 30 mL). The resulting mixture was refluxed for 3 h.

Antibacterial Studies
The synthesized metal chelates in comparison to the free ligand were screened for their antibacterial activity against bacterial species Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. The paper disc diffusion method was used for the determination of the antibacterial activity. Preparation of Discs.
The ligand/complex (30 Ig) in DMF (0.01mL) was applied on a paper disc, [prepared from blotting paper (3 mm diameter)] with the help of a micropipette. The discs were left in an incubator for 48 h at 37 C and then applied on the bacteria grown agar plates. Preparation of Agar Plates. Minimal agar was used for the growth of specific bacterial species. For the preparation of agar plates for Escherichia coil, MacConkey agar (50 g), obtained from Merck Chemical Company, was suspended in freshly distilled water (1 L). It was allowed to soak for 15 minutes and then boiled on a water bath until the agar was completely dissolved. The mixture was autoclaved for 15 minutes at 120 C and then poured into previously washed and sterilized Petri dishes and stored at 40 C for inoculation. Procedure of Inoculation.
Inoculation was done with the help of a platinum wire loop which was made red hot in a flame, cooled and then use for the application of bacterial strains. Application of Discs.
A sterilized forceps was used for the application of paper disc on the already inoculated agar plates. When the discs were applied, they were incubated at 37 C for 24 h. The zone of inhibition was then measured (in diameter) around the disc.

RESULT AND DISCUSSIONS Physical Properties
Schiff-bases ( Fig. 1) were prepared by reacting equimolar amounts of nitro-or chlorosalicylaldehyde and 2-aminothiazole respectively in ethanol. The crystallized products after characterization were reported earlier 16 and now used further for the preparation of its Ni(ll), Cu(ll) and Zn(ll) complexes having different anions as NO3, SO42-, C2042and CH3CO2".
All the prepared complexes are crystalline solids and melt with decomposition above 245 C without showing sharp melting points. The complexes are soluble in DMF, DMSO and benzene. Their melting behavior, solubility and crystalline nature suggested that they are non-polymeric. Elemental analysis data (Table 1) indicated 1:2 (metal:ligand) stoichiometry. Low conductance values (11-18 ohm -1 cm 2 mo1-1) indicated that the complexes are non-electrolytes 17,18.

Infrared Spectra
The detailed IR spectra of the ligands 16 show a weak band at 2900 cm -1 instead of a broad medium band at 3100 cm-1. This might be due to intramolecular hydrogen bonding between the phenolic (-OH) hydrogen and the nitrogen of the azomethine group9. The absence of vOH in the complexes suggested the deprotonation of the phenolic-OH group of the Schiff-base and its co-ordination through oxygen atom. Moreover, the vC-O and vC=N modes which appeared at 1280 and 1630 cmrespectively in the Schiff-bases were shifted in the complexes. This shifting of vC-O towards higher frequency (~1330-1345 cm-1) and lowering of vC=N (~1590-1620 cm-1) suggested that the co-ordination of the Schiff-bases occurred through the deprotonated oxygen of the phenolic-OH group and nitrogen of the azomethine (HC=N) groups19-21.
Further conclusive evidence of the co-ordination of the ligand with the metal was confirmed by the appearance of weak low frequency bands at 485-520 and 355-370 cm -1 (Table 2) due to metaloxygen and metal-nitrogen stretching vibrations 22 in the metal complexes and not observed in the spectra of Schiff-bases.

UV-Visible spectra and Magnetic moments
The electronic spectra of the Ni(ll) complexes exhibited four bands at 7915-8955, 14225-14875, 24225-24760 and 29550-30125 cm-. The first three bands were assigned to the spin-allowed transitions 3A2g(v)-->3Tg, 3Ag(v)3T9(F) and 3Ag(v3)-3Tg(P respectively. The fourth band at 29550-30125 cmwas of high intensity band due to ligand-metal charge-transfer. The occurrence of three spin-allowed transitions supported an octahedral geometry for the Ni(ll) complexes23. The magnetic moments of Ni(ll) complexes also lie in the range 2.9-3.2 B.M (Table 1) and confirm the octahedral geometry4.  In the light of the above discussion an octahedral structure for the Ni(ll) and Zn(ll) chelates arid a distorted octahedml structure for the Cu(ll) chelates is proposed and it is tentatively proposed that the Schiff-bases coordinate through the nitrogen of the azomethine group, nitrogen of thiazole ring and deprotonated oxygen of phenolic group forming a stable chelate ring (Fig 2).

Antibacterial Studies
The antibacterial activity of Schiff-bases in comparison to its metal chelates having the same metal atom but different anions was studied against bacterial species Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. Paper disc diffusion method reported 29-31 earlier was adopted for screening. The Schiffobases and their complexes individually exhibited varying degrees of inhibitory effects on the growth of the tested bacterial species. The antibacterial results reproduced in Table 3 evidently show that the activity of the Schiff-bases became more R M=Ni(II), Cu(ll) or Zn(ll) Proposed Structure of the Metal(ll) chelate pronounced when co-ordinated to these metals. When the same metal chelate having different anions was individually screened the degree of bactericidal activity/potency also varied. From the with 2-Aminothiazole-Derived Schiff-Base Ligands obtained data, it was generally observed that the order of potency in comparison to the metal complexes having chloride anions evaluated and reported earlier as and to the results of the present studies against the same tested bacterial species under the same conditions were found to follow the order as: NO3 > C204 > CH3002 > CI > SO4 On the basis of these results, it is strongly claimed that different anions dominantly effect the biological behavior of the metal chelates. It is however suspected that factors such as solubility, conductivity, dipole moment and cell permeability mechanisms are certainly influenced by the presence of these anions in the chelate and may cause in increasing this activity/potency. Further in vivo studies are in progress, which would help the authors to establish the real mechanism and role of anions in increasing this biological activity.