Biological Role of Anions (Sulfate, Nitrate , Oxalate and Acetate) on the Antibacterial Properties of Cobalt (II) and Nickel(II) Complexes With Pyrazinedicarboxaimide Derived, Furanyl and Thienyl Compounds

A number of biologically active complexes of cobalt(II) and nickel(II) with pyrazinedicarboxaimido derived thienyl and furanyl compounds having the same metal ion but different anions such as sulphate, nitrate, oxalate and acetate have been synthesized and characterized on the basis of their physical, spectral and analytical data. In order to evaluate the role of anions on their antibacterial properties, these ligands and their synthesized metal complexes with various anions have been screened against bacterial species Escherichia coil,Pseudomonas aeruginosa and Staphylococcus aureus. The title studies have proved a definitive role of anions in increasing the antibacterial properties.


INTRODUCTION
Several reviews and studies in the literaturehave discussed the role of metal ions in biological systems.
There is no longer any controversy in researches [6][7][8][9] that certain types of cancers are viruses associated. The potentiality and affectivity of anticancer drugs have strongly shown their direct relationship [1][2][3][4][5][6][7][8][9][10][11][12][13] with the metal ions. Such a biological role of metal ions is, also, obvious and well known 14"16 in many antibacterial drugs. Our previous studies 172 have systematically elaborated this relationship of metal ions in subsequently increasing the activity of bactericidal compounds. This relationship of metal ions and the biochemical processes ought to gain awareness of those factors, which are responsible in enhancing the biological activity. In exploration of these factors, we, wish to report in the preceding paper the possible role of anions (sulfate, nitrate, oxalate and acetate) which stay as a counter part of the metal ion (cation) in the complex.

Preparation of Ligands
The ligands were prepared by adopting the same procedure as reported earlier 3 by us. The structural determination of these ligands was done with the help of their IR, H-NMR, 3C-NMR and microanalytical data.

Preparation of Metal Complexes
To a hot n_-butanol solution (30 mL) of the ligand (0.02 mol) was added an ethanolic solution (25 mL) of the respective metal(II) salt (0.01 mol). The mixture was refluxed for 2 h. The resulting mixture was cooled, filtered and reduced to half of its volume (20 mL). The concentrated solution so obtained was left overnight at room temperature which resulted in the formation of a solid product. The product thus formed was filtered, washed with n-butanol (2x10 mL), then with ethanol (2xl 0 mL) followed by ether (2x20 mL) and dried. Crystallization in hot aqueous n-butanol (50 %) gave 1 (68 %), 2 (65 %), 3  The ligand/complex (30 lag) 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 37C 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 coli, 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 120C and then poured into previously washed and sterilized Petri dishes and stored at 40C 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 37C for 24 h. The zone of inhibition was then measured (in diameter) around the disc.

RESULTS AND DISCUSSION
All the metal complexes were synthesized by a stoichiometric reaction of metal(II) salt having different anions and the respective ligands in a molar ratio 2 M L (metal ligand). All complexes are air and moisture stable solids, soluble in DMF, DMSO and water and partially soluble in ethanol, acetone and benzene. The conductivity measurements (20-28 ohmcm mol-) of these complexes in DMF "1 ndlcated them to be non-electrolytes. Infrared Spectra The bonding of the ligand to the metal ion was investigated by mainly comparing the infrared spectra of the free iigands with the spectra of their metal complexes. The infrared spectra show a positive shift of bands at 1510 cm due to skeletal modes of the pyrazine ring, indicating 23 co-ordination through pyrazine ring nitrogen. Also, an insignificant shift of v(C=O) at 1670 cm indicated that it is not co-ordinated to the metal atom. The spectra of all complexes ( Table 2) also indicated that a band at 1640 cm due to azomethine v(C=N) linkage was shifted towards lower frequency by 5-10 cm -, respectively, suggesting that the ligands are involved in co-ordination to the metal atom via the azomethine nitrogen. The new bands appearing in the spectra of metal complexes and not observed in the spectra of the ligands within 442-455 cm and 375-386 cm assigned to M-O and M-S modes, respectively, gave a definitive clue 24 that the heteroatoms X (Fig 1) are also involved in the co-ordination. Magnetic Moment The room temperature magnetic susceptibility measurements (Table 1)  Co(lI) and Ni(ll) complexes. assigned 27 to 4Tlg 4T2g (F), 4Tlg ---) 4A2g (F) and 4Tlg ---) 4T 28 lg (P) transitions, respectively, and are suggestive of octahedral geometry around the cobalt ion. Three bands observed at 9315-10312 cm , 15870-17125 cmand 26355-27550 cm l in the spectra of the nickel(II) chelates are due to spin-allowed transitions from 3Azg 3Tzg (F), 3A2g --+ 3Ag (F) and 3Azg --+ 3yg (P), transitions, respectively, in an octahedral environment Based on the above evidences, it is proposed that cobalt(II) and nickel(II) complexes have octahedral geometry in which the ligands behave as tridentate and accommodate themselves in such a way that a stable chelate ring is formed around the metal atom thus attaining a stable configuration.  The title ligands in comparison to their metal complexes were screened against bacterial species Escherichia coil, Pseudomonas aeruginosa and Staphylococcus aureus in order to determine their antibacterial properties. The antibacterial activity was tested at a concentration 30tg/0/01 mL in DMF using paper disc diffusion method. The results of these studies reproduced in Table 3 showed that ligands and all their metal complexes are biologically active against one or more bacterial species and the metal complexes having different anions have been shown to be more antibacterial than the simple uncomplexed parent ligands.
These results clearly indicated that when the same metal chelate having different anions was individually screened, the degree of bactericidal activity varied. For example, the Co(II) complex having nitrate as anion was more bactericidal than the Co(II) complex with sulphate, oxalate or acetate anions. Similarly, the Co(II) complex of oxalate anion was more antibacterial active than the complex with acetate, chloride or sulphate anions. The same results were found for Ni(II) complexes. From the obtained data, it was generally observed that the order of potency in comparison to the metal complexes having chloride anions evaluated and reported earlier 3 and to the results of the present studies for the anions sulphate, nitrate, oxalate and acetate against the tested bacterial species was found to follow the order as: NO > C204 > CH3CO > CI > SO 4.
On the basis of these results, we strongly claim that different anions do effect the biological behavior of the metal chelates. However, at this stage, we are not aware of the actual mechanism. It is however, suspected that factors such as solubility, conductivity, dipole moment and cell permeability mechanism (influenced by the presence of anions) may be the possible reasons for effecting the activity.