Biological Role of Cobalt(II), Copper(II) and Nickel(II) Metal Ions on the Antibacterial Properties of Some Nicotinoyl-Hydrazine Derived Compounds

Several cobalt(II), copper(II) and nickel(II) complexes of nicotinoylhydrazine-derived compounds were prepared and characterised by physical, spectral and analytical data. These compounds and their complexes have proven to be antibacterial. The screening data show the metal complexes to be more potential/bactericidal than the uncomplexed compounds against one or more bacterial species.


INTRODUCTION
The increasing interest in the chemistry of hydrazines and hydrazones because of their potential biological applications 1-4 have drawn a considerable attention during the past few years. Many reportsS-9 have indicated that biologically active compounds/drugs become more carcinostatic and bacteriostatic upon coordination with the metal ions. As a part of our ongoing research programme in elaborating more this pronounced biological role of metal ions we have previously reported and as a further contribution, we have prepared some more biologically active nicotinoylhydrazine derived compounds L I-L4 (Fig. 1) and their Co(II), Cu(II) and Ni(II) complexes alad hence, wish to report the role of metal ions on their biological activity. These synthesised ligands and their complexes have been characterised on the basis of conductance and magnetic measurements, elemental analysis and IH-NMR, IR and electronic spectral data. These ligands and their complexes have been screened for their possible antibacterial activity against bacterial strains of Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. The antibacterial activity data of the ligands are known to be substantially increased upon complexation against one or more bacterial species. All chemicals used in this work were of reagent grade. Co(II), Cu(II), and Ni(II) were used as their chlorides.
Conductance and magnetic measurements were made on a YSI model-32 conductivity bridge and Gouy balance respectively. Infrared spectra were recorded on a Rl0 Hitachi spectrophotometer. IH-NMR spectra were obtained on a Rl0 Perkin-Elmer spectrometer. Electronic spectra were studied on a Hitachi double-beam U-2000 model spectrophotometer using glass cells of cm thickness. Elemental analysis of C, H and N were determined on a Coleman automatic analyser. All melting points were taken on a Gallenkamp melting point apparatus and were uncorrected.
Antibacterial studies were carried out with the help of the Microbiology Laboratory, Department of Pathology, Qaid-e-Azam Medical College, Bahawalpur. These studies were done on wild pathogenic bacterial species collected from the urine and blood samples of infected patients admitted in Bahawal Victoria Hospital, Bahawalpur.

Antibacterial Studies
Antibacterial activity of the prepared ligands and their complexes was tested against bacterial species, obtained from the different patients carrying these bacteria, Escherichia coli, Pseudornonas aeruginosa and Staphylococcus aureus using the paper disc diffusion method.

Preparation of Discs
A ligand/complex (30 g) DMF solution (0.01 mL) was applied on a paper disc prepared from blotting paper (3 mm size) with the help of a micropipett. The discs were left in an incubator for 48 h at 37C and then applied on bacteria grown agar plates.
Preparation of Agar Plates For this purpose minimal agar was used for the growth of specific bacterial species. For Staphylococcus species, a blood agar base with low pH was used. The blood agar base (40 g) was first suspended in cold distilled water (1L) and heated to boiling. It was then sterilised at 120C for 15 minutes and later allowed to cool at 50C. Then 5% sterile defibrinated cow blood was added to it and the mixture was poured into previously washed and sterilised Petri dishes which were stored at 4C for inoculation. For the preparation of agar plates for Escherichia coli and Pseudomonas species, Mac Conkey agar (50 g), obtained from Merck Chemical Company, was suspended in freshly distilled water (1L). It was allowed to soak for 15 minutes and then boiled with constant shaking in 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 sterilised Petri dishes and stored at 4C for inoculation.

Procedure of Inoculation
Inoculation was done with the help of a platinum wire loop which was made red hot on a flame, allowed to cool in air and then used for the application of previously described bacterial strains. The preculture was first prepared in 2 mL of a nutrient broth by selecting a suitable bacterial colony and later on transfered to a nutrient broth which was incubated for 2 h at 37C. Then 500 L of the culture was spread on the specific agar plates, which was incubated for 24 h at 37C. Application of Disc A sterilised forecep was used for the application of paper disc on the already inoculated agar plates. When the disc was applied, it was then incubated at 37C for 24 h. The the zone of inhibition in diameter was measured.

RESULTS AND DISCUSSION
The structural determination of ligands was done on the basis of their IR, IH-NMR and elemental analysis data (Table 1). It is known that benzoyl or nicotinoyl hydrazines exhibit keto-enol tautomerism and as such they can exist in one of these forms. If the ligands exist in the keto form the (NH) and (C=O) absorption bands will appear in the infrared spectra, whereas the absence of these two absorptions may be indicative of enol form. All these ligands show a broad band at 3440 cm-l and a medium intensity band in the rang.e 3280-3390 cm-l attributed to (OH) and (NH). A strong band at 1670 cm-1 is assigned to (C=O) absorption. An intense band in the range 1645-1650 cm-l is assigned21, 22 to (C-N). The presence of these bands thus confirm that the ligands exist in the keto form. The IH-NMR spectra of the ligands (Table 1) also display signals assignable to the azomethine (CH=N) and amide (NH) protons and all other expected protons. Also, the microanalytical data (Table 1) of the ligands was found to be in agreement with the molecular structures of the title ligands.

Metal-Based Drugs
Antibacterial Studies The synthesised ligands and their metal complexes were tested for their antibacterial activity against bacterial species Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. The antibacterial activity of these compounds was tested at a concentration of 30 g/0.01 mL in DMF using paper disc diffusion method as described previouslyl0-12. The same method was applied for assessing the activity, the results of which are reported in Table 3.  The results of these studies clearly indicate that the ligands and their complexes are all potent and biologically active against one or more testing bacterial strains. More so, the metal complexes have shown to be more antibacterial against the same testing species than the simple uncomplexed ligand. This, in turn, has confirmed our all previous studies 0-18 that the metal chelation increases the potency/biological activity of such compounds/drugs which have bactericidal characteristic properties.