Synthesis, Characterization and Antimicrobial Activity of d8-10 Metal Complexes of some 2-Substituted-1H-Benzimidazoles

The metal complexes of nine 2-substituted-1H-benzimidazoles (I-IX) with Ni(II), Pd(II), Cu(II), Ag(I), Zn(II) salts were synthesized. The compounds were characterized by melting point, analytical data, IR spectroscopy and magnetic susceptibility. The antimicrobial activity of the compounds was determined by the disk diffusion method in Mueller-Hinton Agar on Staphylococcus aureus ATCC 6538, Staphylococcus epidermidis ATCC 12228, Escherichia coli ATCC 8739, Klebsiella pneumoniae ATCC 4352, Pseudomonas aeruginosa ATCC 1539, Salmonella typhi, Shigella flexneri, Proteus mirabilis, Candida albicans ATCC 10231. Cu(II)and Ag(I)complexes of II, III and IV showed considerable activity against S. aureus, S. epidermidis, Ps. aeruginosa, S. typhi, Sh. flexneri and C. albicans microorganisms, the ligands themselves having no effect.

In this research, the microbiological activity of the complexes were examined and compared with that of the ligands by means of the disk diffusion method.

MATERIALS AND METHODS Preparation of the ligands
The ligands I-IV, were prepared by modifying slightly the literature method[l] and V-IX according to the Phillips method [26]. Only ligand VI was obtained by using the melting process [27].

Metal-Based Drugs
Preparation of metal complexes in ethanol/water A 5 mL aqueous solution of 0.04 M metal salts (0.2 mmole; e.g. 50 mg NiC12.6H20) and 0.4 mmole ligand (e.g. 80 mg II) in 25 mL of ethanol in a reaction tube was stirred vigorously. The solution mixture was refluxed with stirring for about 4 hours. The metal complexes with AgNO3 were obtained for one hour at 50+5C. The mixture was then allowed to stand at room temperature overnight to give a solid product. This was then filtered, washed with water and ethanol, dried under vacuo over anhydrous CaCI2.
Compositions and purity of all the ligands and metal complexes were checked by using microanalysis, IR, TLC and melting points.  Antimicrobial activity of the compounds The disk diffusion method was used for determining the antimicrobial activity. The antibacterial activity against Staphylococcus aureus A TCC 6538, S. epidermidis A TCC 12228, Escherichia coli A TCC 8739, Klebsiella pneumoniae ATCC 4352, Pseudomonas aeruginosa ATCC 1539, Salmonella typhL Shigella flexneri and the antifungal activity against Candida albicans ATCC 10231 were investigated.
Mueller-Hinton agar (Difco, Detroit, USA) was melted at 100C and then cooled to 56C, was poured into plates of 9 cm diameter in quantities of 20 mL, and let on a flat surface to solidify and the surface of the medium was dried at 37C. Then, cultures of each bacteria and yeast strain, after being kept in Mueller-Hinton broth (Difco) at 37C for 18-24 hours and diluted with Mueller-Hinton broth to 10 cfu/mL, were pipetted into the Mueller-Hinton agar plates prepared as described above. The surface of the medium was allowed to dry. The 10 mg/mL (in DMSO, E. Merck)compound imPoregnated disks were applied to the surface of inoculated plates. The plates were placed in an incubator at 3 7 C. After 18-24 hours of incubation, the plates were examined and the compounds which were found effective against the strains were selected in order to determine the minimum inhibitory concentrations.
Determination of MIC The minimum inhibitory concentrations were determined by the microbroth dilution technique using Mueller-Hinton broth. Serial two-fold dilutions ranging from 5000 to 4.8 mcg/mL were prepared in Mueller-Hinton broth. The inoculum was prepared with a 4-6 hours broth culture of each strain adjusted to a turbidity' equivalen to 0.5 McFarland standard, diluted in Mueller-Hinton broth to give a f'mal concentration of 5x10 cfu/L in the test tray. The trays were covered and placed in plastic bags to prevent drying; incubation was at 37C for 18-20 hours. The MIC was defined as the lowest concentration of compound giving complete inhibition of visible growth. MIC values of compounds are given Table 3.

RESULTS AND DISCUSSIONS
Some selected IR bands of the complexes are shown in Table 2. According to literature, the coordinations occur through the C=N nitrogen atom of imidazole in all the complexes [28]. The v(C=N) bands of imidazole and the v(C=N) bands of pyridine of the ligands II, III and IV can be clearly seen in the IR spectra. Imidazole v(C=N) band in all the metal complexes and both v(C=N) bands in the metal complexes of II-IV are shifted to higher frequencies compared with those of the free ligands by 5-40 cm-. Similarly, the v(C-O) band frequencies of phenol and alcohol groups in the ligands increase on complexation. In addition to this, the characteristic acetate, nitrate and perchlorate bands confirm the suggested tentative formula of the complexes.

Antimicrobiai activity of the ligands and their metal complexes
The antimicrobial activity (MIC, tg/mL) of the compounds are shown in Table 3. From Table 3, the complexes of II! with Cu(ll)and Ag(I) have various antimicrobial activities. Cu(II) salts and II and III ligands which are not effective alone to two (S. azreus and S. epideridis) of the nine bacteria, however, they show some effect in their Cu(II)-complex form. This effect can be logically explained by the fact that the benzimidazole derivatives must be activated by Cu(II) ions in some way (maybe synergetic activity). However, we think that the activity was revealed as a result of the independent characteristic of the metal complexes. We presume that this effect is due to the formation of an "enzyme-Cu(II)-benzimidazole" mixed complex, by the bonding of metal complexes through the metal atom to the microorganism's cell enzymes. The ternary complexes, composed of enzyme, metal ion and organic ligand, may be likely proposed considering the results in the some biological studies[29-32]. The benzimidazole derivative, which is the organic portion of this complex structure, is responsible for the ineffectiveness of the said Cu(lI) complexes to the other bacteria except S. aureus and S. epidermidis. The expected activity, in the form of Cu(II) complexes, will be obtained by appropriate benzimidazole derivatives, even though they may not be active alone by themselves.