Pharmacological Role of Anions (Sulphate, Nitrate, Oxalate and Acetate) on the Antibacterial Activity of Cobalt(II), Copper(II) and Nickel(II) Complexes With Nicotinoylhydrazine-Derived ONO, NNO and SNO Ligands

Mixed ligands biologically active complexes of cobalt(II), copper(II) and nickel(II) with nicotinoylhydrazine-derived ONO, NNO and SNO donor schiff-base ligands having the same metal ion but different anions such as sulphate, nitrate, oxalate and acetate have been synthesised and characterised on the basis of their physical, analytical and spectral data. In order to evaluate the role of anions on their bioability, these ligands and their synthesised metal complexes with various anions have been screened against bacterial species such as Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus and the title studies have proved a definative role of anions in increasing the biological activity


INTRODUCTION
The relationshipS-5 between metal ions and biological systems is currently obvious and hence, is a sublect of reat interest for researchers. A bulk of literature-13 has drawn attention to this act of tile most significant nature and use of metal ions for life and their role in different biochemical processes. For example, functioning of many enzymes is metal ion dependent4,5. The metal?ion may. induce by coordin.ation a specific 'Loc.l geometry" of the apoprotein metal binding site so that only certain substances are able to become attached to tile t'ramework formed by the result of such interaction. The increasing interest in this area, also attracted us to investigate and enlighten the biological role16-23 of metal ions in chelation and subsequen.tly increasing the p_har.mac210gical p.roperties. Paralleling the same concepts, we have furthermore investigatedtor the first time the possible role24-2 of anions (counter part of metal ions) in biological systems and in extension to the same, now, wish to report this role of anions (sulphate, nitrate, oxalate and acetate) on biologically, active metal(II) complexes of nicotinoylhyffrazine-derived Schiff-base ligands reported7 earlier by us. The.present studies princip.ally consist of the formation of various metal(II) co.m_plexes having the same metal ion but i:lifferent anions represented by a general formula [M(E)2 X2] where M = Co(II),. Cu(II) and Ni(II), L = L, L and L3 (Fig.l) and.X = S.O4, NO3, C204 a.nd CH3CO2, and then investigating the role of anions on the antibacterial activity against bacteri/tl species Escherichia  To a hot n-Outanol solution (2-5 mL) of the ligand (0.004 mol) was added an ethanolic solution (15 mL) of the metal salt (0.002 moI). The mixture was refluxed for 2 h.The resulting mixture was cooled, filtered and reduced to nearly a half of.its volume (20 mL) by an evaporator..The concentrated solution so oOtained was left overniglat at room temperature which resulted in the formation of a solid product. The solid product thus formed was filtered, wa.shed with n-butanol (2x 10 mL) and dried. Crystallisatlon ot the crude product in aquegus n-utanol (50_ %) gave the desired metal complexes 1-36 (Table 2).

Antibacterial Studies
The synt.hesised metal complexes and ligands were screened for their antibacterial activity. against bacterial species Escherichia coli (a), Pseudomonas aeruginosa (b) ani Slaphylococcus aureus (c). The paper .disc .diffuso.n method deviced and reported earlier7,20 _by us was used for the determination ot antibacterial activity.

Preparation of Discs
Metal complexes/ligands (30 pg) i.n DMF (0.01 mL) were applied on a disc prepared fr.om blotting paper (3 mm diameter) with the help of a micropipette. The discs were incubate0 at 3_7oC f6r 48h and then applied on specific bacteria grown agar plates.
Preparation of Agar -Plate Mimmal agar was used for the growth of specific bacterial species. For this purpose different _media were used specifically according to t-he proposed metliod28 of Merck.

Method of Inoculation
A platinum wire loop was used for inoculation which was firs.t made red hot in a flame, cooled in air and then used for th.e ap.plication of specific bacterial strains. The precultur.e .was prepared in 2 mL of nutrient trotfi by selecting a suitable colony of bacteria and tlaen transfering it to the nutrient broth. It was incubated for 2 h at 37oC. Then 500 laL of the culture was spread on the specific agar plate and incubated for 24 h at 37oC. Application of Discs A sterilised forecep was used for the application of disc on already incubated ag.ar, plates.
When the disc was applied, it was then ncubated at 37oC for 2 h. The diameter ot tlae zone of inhibition was measured.

RESULTS AND DISCUSSION
The structure of the title ligands (Table 1) was established as reported:7 earlier and structure of their metal complexes synthesised in the present studies were established with the help of their physical, spectral and analytical data ( Table 2). All the metal complexes were prepared by a stoichiometric reaction of metal(II) salt having different anions and the respective ligands in a molar ratio M:L = 1:2 (Fig 2). All complexes formed are air and moisture stable They are soluble in DMSO, DMF and water and insoluble in other solvents. The conductivity of these complexes (12-20 ohm-cruZ mol-) in DMF shows all of them to be non-electrolytesZ8,29. Their solubility, crystalline nature and melting behaviour suggest that they are all non-polymeric.

Infrared Spectra
The prominent IR frequencies of the complexes are listed in Table 2. The scrutiny of imp.ortant bands exhibited by the ligands and'their complexes show that a.band at 3280_ qm-l in tlae spectra of free ligands assigned to V_(NH) remmns .u.ncha.nged in the spectra ortheir metal comp.lexes thus indicating tlaat ligands are not coordinated through -Nil. However, a strong bani:l in the spectra of llands at 1670 cmis due to V(C=O) stretching distinctly.
shiftel towards lower frequencyby 30-40 cm-l indicative of the participation of tfiis carbonyl group in coordination. IR spectra of all ligands exhibit a band at "1620 cmdue to azomethine moiety which also shiftea towa.rds, lower frequency by 5-10 c.minvariably in all the complexes suggesting respectively, tlaat the ligands are coordinated to tte metal ion via C = 3DN. In the fiir infrared region three bands around 375-382 cm-, 445-450 cmand 510-525 cmare observed for all the complexes, which are not found in the sp.ectrum of the fr.ee The nature of the ligand field around the metal io.n and the geometry of the complexes have been deduced from t-he electronic spectral data and magnetic moment valuesgiven in Table 2  .nitrate anions as .a cou.nter p.art, were touncl to be more bactericidal than the Co(II) complexes having anions oter than nitrate. Simi.'l.arly, the same Co(II) complexes with oxalate anions were observed to t)e more antit)acterial than the Co(II) complexes having acetate and sulphate anions. The identical results were found in Ni(II) and Cu(II) complexes. From the obtained data, it was generally observed that the order of potency in comparison to the metal compl.exes having cl:iloride anigns reported earlier7 and to the present smdi.es haying anions otlaer than chloride against tlae tested bacterial species was founi:l to follow tlae orcter as nitrate > oxalate > acetate > chloride > sulphate In the light of the above observations, it is found that the role of anions in increasing the antibacterial activity of metal complexes is definite.