New Ternary Transition Metal Complexes of 2-{ [ ( 2-aminophenyl ) imino ] methyl } Phenol and Metformin : Synthesis , Characterization and Antimicrobial Activity

Complexes of Co(II), Ni(II) and Cu(II) were synthesized from Schiff base 2-{[(2-aminophenyl)imino]methyl}phenol and metformin. The authenticity of the transition metal complexes were characterized by elemental analyses, conductance and magnetic susceptibility measurements, as well as spectroscopic (IR, electronic) and thermal studies. IR spectral studies revealed the existence of the ligands in the amine form in the solid state. The magnetic and electronic spectral studies suggest an octahedral geometry for all the complexes. The metformin acts as a bidentate ligand and Schiff base of o-phynelendiamine and salicylaldehyde acts as a tridentate ligand. Antimicrobial screening of the Schiff base, metformin and transition metal complexes were determined against the bacteria Escherichia coli and Bacillus megaterium.


Introduction
Metformin hydrochloride (N,N-dimethyl-imido-dicarbonimidic diamide hydrochloride) is a strongly basic bisusbstituted guanidine derivative with short side chains.It is an orally administered biguanide that has been widely used in the treatment and management of noninsulin dependent diabetes mellitus (NIDDM).Metformin lowers both basal and postprandial elevated blood glucose in patients with NIDDM, when hyperglycemia cannot be satisfactorily managed on diet alone [1][2][3][4] .Schiff bases offer a versatile and flexible series of ligands capable to bind with various metal ions to give complexes with suitable properties for theoretical and/or practical applications.The chemistry of metal complexes containing salentype Schiff-base ligands derived from condensation of aldehydes and amines is of enduring significance, since they have common features with metalloporphyrins with respect to their electronic structures and catalytic activities that mimic enzymatic hydrocarbon oxidation 5 .Catalytic activity of such metal complexes has been highlighted in the past few decades 6 .The presence of transition metals in human blood plasma indicates their importance in the mechanism for accumulation, storage and transport of transition metals in living organisms [7][8][9] .

Experimental
The compounds o-phenylenediamine, salicylaldehyde, metformin and transition metal salts were obtained from S. d. fine chemicals Ltd.India and Escherichia coli; MTCC 1687, Bacillus megaterium; MTCC 428 were collected from MTCC Bangalore, India.

Preparation of Schiff base and transition metal complexes
The Schiff base under investigation was prepared by mixing an ethanolic solution (50 cm 3 ) of (1.22 g) 0.01 mole of salicylaldehyde with (1.08 g) 0.01 mole of o-phenylenediamine in the same volume of ethanol.Few drops of 10% NaOH were added to adjust pH 7 and the obtained mixture then refluxed with stirring for two hours.The obtained precipitate was collected by filtration through Buchhner funnel, recrystallized from ethanol and dried at room temperature.The yield was 65% and its melting point obtained was195 ºC.
The complexes under investigation were prepared by mixing 50 cm 3 ethanolic solution of 0.01 M Schiff base, 0.01 M metal salt and 0.01 M metformin.The obtained mixture was refluxed with continues stirring for four hours.The resulted mixture was filtered; the product was collected and then washed several times with hot ethanol until the filtrate became clear.The complexes were dried in desiccator over anhydrous CaCl 2 under vacuum.The yield ranged from 60-75% and the melting points of all complexes was above 350 ºC.

Analysis and physical measurements
Elements like C, H, O and N were analyzed with a Perkin-Elmer 2400 series II elemental analyzer.Magnetic susceptibilities were measured at room temperature on a Gouy 10 balance using Hg[Co(CNS)] 4 as calibrate.The IR spectra were recorded on a Perkin-Elmer Lamda-983 spectrometer with samples prepared as KBr pellets and UV Visible reflectance spectra were obtained on a Beckman DK-2A spectrophotometer using MgO as reference.Thermal measurements were carried out using Perkin-Elmer TGA-7DSC-PYRIS-1-DTA-7 thermal analyzer maintained at a 10 o C min -1 heating rate.

Antimicrobial assay
The experiments were designed so as to test the effect of the presence of the ligand and their metal chelates in liquid culture media.2x10 -3 M of ligands and their metal chelates Co(II), Ni(II) and Cu(II) were supplemented in nutrient broth.The flasks were inoculated with 5% (v/v) actively growing inoculums and incubated for 24 hours on rotary shaker adjusted at 120 rpm and 37 0 C.After the incubation growth was measured spectrophotometrically at 660 nm.The % growth inhibition was calculated with reference to growth in the respective medium without any inhibitory compounds.

Results and Discussion
The analytical data of the complexes is presented in Table 1  All synthesized complexes are colored and possess high decomposition points.All are amorphous and stable in air.The complexes are partially soluble in methanol and insoluble in water and other organic solvents.

Conductance measurements
The conductivity of complexes was measured in 1:1 mixture of methanol and water at room temperature.All the complexes showed the molar conductance values for 10 -3 M concentration in range 8 to 14 ohm -1 cm 2 mol -1 .It is suggesting that all complexes have non electrolyte nature 11 .The molar conductance values of the complexes are listed in Table 1.
Table 1.Analytical data and some physical properties of the metal complexes

Infrared spectra
There is strong coupling among the IR bands of ternary complexes and hence, quantitative interpretation of the bands in the IR spectra is not possible without normal coordinate analysis.Important IR frequencies of the complexes are listed in Table 2 along with their suggested assignments.

Antimicrobial activity
The highest activity was reported with Cu(II) complexes with 11.57mm and 12.29 mm growth inhibition for E. coli and B. megaterium respectively.The least activity has been reported for the Co(II) complexes (Table 7).All synthesized metal complexes have shown more inhibitory activity against bacteria as compared to parental ligands.Similar correlation and increase in antibacterial activity of ligands on complexation with reference to parent ligands has been well cited [15][16][17] .Such increased activity of the metal chelates can be explained on the basis of overtone's concept and chelation theory.According to Overtone's concept of cell permeability, the lipid membrane that surrounds the cell favors the passage of only lipid soluble materials due to which liposolubility is an important factor that controls antimicrobial activity.On chelation, the polarity of metal ion is reduced to a greater extent due to the overlap of the ligand orbital and partial sharing of the positive charge of the metal ion with donor groups.Further, it increases the delocalization of π-electrons over the whole chelates ring and enhances the lipophilicities of the complex.The increased lipophilicities of complexes permit easy penetration into lipid membranes of organisms and facilitates as blockage of metal binding sites in enzymes 18 .

Conclusion
On the basis of elemental analyses, IR, thermogravimetric analyses, UV Visible reflectance spectra, molar conductance and magnetic properties, it is possible to assign octahedral geometry to the all metal complexes as shown in Figure 1.The calculated values of β are favorable for assigned structure of accounted for covalent character of complexes.We are optimistic that future studies on biological properties of these complexes of Schiff base, metformin and there derivatives may lead to the development of a new class of specific and effective pharmaceutical agents.
Cu(II) complexes have shown promising antibacterial activity against E. coli and B. megaterium.Least activity is shown for Co(II) complexes because Co(II) act as cofactor in many systems.The antimicrobial activity was explored on the basis of overtone concept of cell permeability.
indicates 1:1:1 stoichiometry.The general equation for the formation of the complexes is shown as below: M 2+ + HA + HL MAL + 2H + Where M 2+ = Co(II) or Ni(II) or Cu(II), A = Schiff base and L = Metformin.

Table 5 .
Electronic spectra and magnetic moment data for the complexes

Table 7 .
Effect of ligand and metal complexes on the growth of bacteria