Synthesis, Characterization and Role of Anions (Nitrate, Sulphate, Oxalate and Acetate) \in the Biological Activity of Hydrazine Derived Compounds and Their Metal Chelates

Hydrazine derived compounds and its Co(II), Cu(II) and Ni(II) chelates having the same metal ion but different anions (e.g., nitrate, sulphate, oxalate and acetate) have been synthesised and characterised by their physical, spectral and analytical data. In order to evaluate the participating role of anions in the antibacterial activity, these synthesised ligands and its metal chelates have been screened against bacterial species such as Staphylococcus aureous, pseudomonas aeruginosa, Klebsiella pneumonae and Proteus vulgarus, and the results are reported.


INTRODUCTION
Recently, there has been a considerable interest in the chemistry of hydrazine and hydrazone compounds because of their potential pharmacological applications1-4. Several reports5-8 on the metallic chemistry of acyl-and arylhydrazines have already been published. As a further contribution, we have previously reported9-11 pyrrolyl-, thienyl-, furanyl and benzimidazole-derived hydrazines and their 3d metal chelates which have shown the biological properties. This growing interest between the relationship12-16 of metals and biological processes has drawn attention17-20 of many researchers that metals are assential for life as well as for various biochemical reactions. A number of such biochemical reactions are catalysed17,18 by enzymes containing metals such as zinc, cobalt, iron etc. Also many metal chelates are found5, 6 to be more carcinostatic than the unchelated compounds/drugs and similarly, many antibacterial drugs when are chelated, their biological activity is effectively altered7-o. In understanding the apparent role of metals and their mode of action in biological processes, we have commenced a research program which has already revealed21-26 the significant role of metals in antibacterial activity whereas, the role of anions which stay as a counterpart of the metals in such chelated compounds has been ignored or not studied before. In this effort to highlight the participating role of anions in biological processes, we wish to report the synthesis, structural and biological studies of Co(ll), Cu(ll) and Ni(ll). metal chelates of hydrazine derived compounds having the same metal atom but different an=ons (e.g., nitrate, sulphate, oxalate or acetate).

Material and methods
All chemicals and solvents used were of Analar grade. All the metals were used as their metal(ll) salts. Infrared spectra were recorded on Philips Analytical PU 9800 FTIR and Nicolet FTIR instruments. UV The title compounds (Fig 1) were prepared by the same method reported12 earlier by us. To a hot ethanolic solution (20 mL) of the ligand (0. 02 mol) was added an aqueous solution (10 mL) of the respective metal(ll) (0.01 mol) salt. The mixture was refluxed for h. The resulting mixture was cooled, filtered and reduced nearly half its volume. This concentrated solution was left overnight at room temperature which resulted in the formation of a solid product. The product thus obtained was filtered, washed with ethanol (2x10 mL) then with ether (10 mL) and dried. Crystallisation in hot aqueous ethanol gave the desired complexes 1-24 (Table 2).
Antibacterial studies The synthesised metal chelates and the free ligands were screened for their antibacterial activity against bacterial species, Staphylococcus aureus(a), Pseudomonas aeruginosa(b), Klebsiella pneumonae(c) and Proteus vulgarus(d). The paper disc diffusion method was used for the determination of antibacterial activity. Preparation of discs A ligand/complex (30#g)in DMF (0.01 ml) 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 37oC and then applied on 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 the specific agar 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 120oC and then poured into previously washed and sterilised petri dishes and stored at 40oC for inoculation.

Procedure of inoculation
Inoculation was done with the help of a platinum wire loop which was made red hot on a flame, cooled and then used for the application of bacterial strains. Application of discs A sterilised forcep was used for the application of paper disc on the already inoculated agar plates. When the discs were applied, they were incubated at 37o C for 24 h. The zone of inhibition was then measured (in diameter) around the disc.

RESULTS AND DISCUSSION
Physical properties All the metal chelates were found to be coloured, moisture and air stable solids. They are soluble in DMF, DMSO and water and partially soluble in chloroform, acetone, ethanol and benzene. Their melting behaviour, solubility and crystalline nature suggested that they are all non-polymeric. The low molar conductance values (11-15 ohm-1 cm2 mol-1) of the metal chelates indicated28,29 that they are all non-electrolyte in nature, probably due to the charge neutralisation of the the metal ion. The room temperature magnetic susceptibility measurements (

Infrared spectra
The comparative studies of the important infrared bands of the ligands and its complexes indicated that the ligands are co-ordinated to the metal atom possibly in three ways., a) The (NH2) and (NH) bands appeared at 3215 and 3190 and 3100 cm-1 in the case of the free ligando A lowering in these bands (--10-25 cm-1) is observed in the case of their metal chelates, indicating32 co-ordination through these groups.
b) The azomethine (C=N) stretching vibrations are found at 1625 cm-;this is also lowered (---10-15 cm-1) in the case of metal chelates, suggesting co-ordination through nitrogen of the azomethine linkage.
In view of the above observations, it is proposed that all the metal chelates show an octahedral geometry by accomodating two ligands acting as tridentates and form a stable configuration of the metal chelate.

Antibacterial properties
Our previous studies39-43 in enlightening the role of metal ions give a detailed and systematic description of their antibacterial properties. However, in the present work, elaboration of the participating role of anions in the antibacterial activity is worked out. Table 4 reproduces the result of these studies. It is interesting to note that when the metal chelate having the same metal atom but different anions (nitrate, sulphate, oxalate or acetate) was individually screened for its antibacterial activity, its degree of potency killing the tested bacterial strains varied. For example, cobalt chelate having nitrate anion was found to be more bactericidal than the cobalt chelate having anions sulphate, oxalate and acetate. The identical results were found for the other copper(ll) and nickel(ll) chelates. On comparison of the present data with the data obtained earlier for the same metal chelates of chloride anion, we are now, able to draw the order of potency as to be nitrate > oxalate > acetate > chloride > sulphate which significantly alters the role of metal ions in the biological activity. We are, however, not able to explain at this stage, the possible mechanism of this role of anions. But our in vitro studies are in progress which may help us in determining this mechanism and the exact participating behaviour of anions.