Synthesis and spectral characterization of antifungal sensitive schiff base transition metal complexes.

New N2O2 donor type Schiff base has been designed and synthesized by condensing acetoacetanilido-4-aminoantipyrine with 2-aminobenzoic acid in ethanol. Solid metal complexes of the Schiff base with Cu(II), Ni(II), Co(II), Mn(II), Zn(II), VO(IV), Hg(II) and Cd(II) metal ions were synthesized and characterized by elemental analyses, magnetic susceptibility, molar conduction, fast atom bombardment (FAB) mass, IR, UV-Vis, and (1)H NMR spectral studies. The data show that the complexes have the composition of ML type. The UV-Vis. and magnetic susceptibility data of the complexes suggest a square-planar geometry around the central metal ion except VO(IV) complex which has square-pyramidal geometry. The in vitro antifungal activities of the compounds were tested against fungi such as Aspergillus niger, Aspergillus flavus, Rhizopus stolonifer, Candida albicans, Rhizoctonia bataicola and Trichoderma harizanum. All the metal complexes showed stronger antifungal activities than the free ligand. The minimum inhibitory concentrations (MIC) of the metal complexes were found in the range of 10~31 µg/ml.

Drug resistance has become a growing problem in the treatment of infectious diseases caused by bacteria, fungi, parasite and virus. Infectious diseases like diarrhoea, dysentery, tuberculosis, acute respiratory tract infections, AIDS and recently SARS are global threat and their incidences are increasing significantly day by day. Although a number of chemotherapeutic agents are available in market places, the pathogenic organisms are developing resistance to these agents. So, it is important to find out safer, more effective and inexpensive chemotherapeutic agents. An extensive literature has developed in recent years in the field of chelate compounds with special reference to their antimicrobial activities (Al-Sha'alan et al., 2007).

Recent years have witnessed a great deal of interest in the synthesis and characterization of transition Schiff base metal chelates of pyrazolone derivatives. Among these derivatives, 4-aminoantipyrine is a remarkable reagent due to its importance in biological, pharmacological, clinical and analytical applications (Raman et al., 2007). Studies of a new kind of chemotherapeutic Schiff bases are now attracting the attention of biochemists. In particular, heterocyclic Schiff base ligands containing an additional imine function could be applied variously. They have been of great importance due to their synthetic flexibility, selectivity and sensitivity towards the metal ions. The heterocyclic ring containing sulphur, nitrogen and oxygen import special biological activity to these Schiff bases and their metal complexes (Hankare et al., 2001). Hence, in this paper we describe the synthesis and characterization of antifungal sensitive transition metal complexes of N2O2 donor type tetradentate Schiff base formed by condensing Acetoacetanilido-4-aminoantipyrine with 2-aminobenzoic acid. The in vitro antifungal activities of the investigated compounds were tested against fungi such as Aspergillus niger, Aspergillus flavus, Rhizopus stolonifer, Candida albicans, Rhizoctonia bataicola and Trichoderma harizanum.

Materials and Methods

Apparatus and reagents

All reagents 4-aminoantipyrine, aceto-acetanilide, 2-aminobenzoic acid, vanadyl sulphate and various metal(II) chlorides were of Merck products and used as supplied. Anhydrous grade methanol and DMSO were purified according to standard procedures. Microanalytical data, 1H NMR and FAB Mass spectra of the compounds were recorded at the Regional Sophisticated Instrumentation Center, Central Drug Research Institute (RSIC, CDRI), Lucknow. The FAB mass spectrum of the complex was recorded on a JEOL SX 102/DA-6000 mass spectrometer/data system using argon/xenon (6 kV, 10 mA) as the FAB gas. The accelerating voltage was 10 kV and the spectra were recorded at room temperature using m-nitrobenzylalcohol (NBA) as the matrix. The IR spectra of the samples were recorded on a Perkin-Elmer 783 spectrophotometer in 4000~200 cm-1 range using KBr pellet. The UV-Vis. spectra were recorded on a Shimadzu UV-1601 spectrophotometer using DMF as solvent. Magnetic susceptibility measurements of the complexes were carried out by Guoy balance using copper sulphate as the calibrant. The molar conductance of the complexes was measured using a Systronic conductivity bridge.

In vitro antifungal activity

In vitro antifungal activity assay was performed by well diffusion method. DMSO was used as a solvent and nystatin (antifungal agent) as control. The Schiff base and its complexes were tested against fungi such as A. niger, A. flavus, R. stolonifer, C. albicans, R. bataicola and T. harizanum, cultured on potato dextrose agar as medium. In a typical procedure, a well was made on the agar medium inoculated with fungi. The well was filled with the test solution using a micropipette and the plate was incubated at 30℃ for 72 h. During this period, the test solution diffused and the growth of the inoculated fungi was affected. The inhibition zone developed on the plate was measured. The MIC of the complexes was determined by serial dilution technique (Reiner, 1982).

Synthesis of Schiff base

Acetoacetanilido-4-aminoantipyrine was prepared by the reported method (Raman et al., 2001). Using this, the following Schiff base has been synthesized.

Acetoacetanilido-4-aminoantipyrinyl-2-aminobenzoic acid (H2L)

An ethanolic (50 ml) solution of acetoacetanilido-4-aminoantipyrine (3.62 g, 0.01 mol) and 2-amino benzoic acid (1.37 g 0.01 mol) were mixed at room temperature (27℃) and reflux for 36 h after the addition of 1 g of anhydrous potassium carbonate. The potassium carbonate was filtered off from the reaction mixture and the resulting solution was concentrated to 20 ml on a water bath. The solid product formed was separated by filtration and washed thoroughly with ethanol and then dried in vacuo.

Syntheses of metal complexes

A solution of CuCl2/NiCl2/CoCl2/MnCl2/ZnCl2/VOSO4/HgCl2 and CdCl2 (5mmol) and the Schiff base H2L (5 mmol) in ethanol (50 ml) was boiled under reflux for 1 h. The resulting solution was concentrated to 15 ml and the mixture was cooled to 0℃ for ca. 12 h. The solid complex formed was removed by filtration, washed with ethanol and dried in vaccuo. Vanadyl complex was synthesized by the same procedure but in the presence of 5% aqueous sodium acetate solution (5 ml).

Results and Discussion

The analytical data for the Schiff base and its complexes together with some physical properties are summarized in Table 1. The analytical data of the complexes correspond well with the general formula ML where M = Cu(II), Ni(II), Co(II), Zn(II), Hg(II), Cd(II), VO(IV) and Mn(II); L = C28H27N5O3. The formulation of the complexes was based on IR data, molar conductivity values, elemental analyses, which were in close agreement with the values calculated for the assigned molecular formulae and on FAB mass spectral studies. The elemental analytical data were in good agreement with the molecular formulae arrived for the ligand and its complexes. The molar conductance values of the complexes in DMF lie in the range 0.8 to 2.36 ohm- cm2 mol-1 which is quite lower than that expected for an electrolyte and reveal their non-electrolytic nature. The magnetic susceptibilities of the complexes at room temperature are consistent with square-planar geometry around the central metal ion, except for the VO(IV) complex which exhibits square-pyramidal geometry.

Table 1

Physical characterization, analytical, molar conductance, and magnetic susceptibility data of the Schiff base ligand and its complexes

Cacld = Calculated values (Theoretical); Found = Experimental values; M =Metal, C = Carbon, H = Hydrogen and N = Nitrogen; BM = Bohr magnetron (unit of magnetic susceptibility).

Mass spectra

The FAB mass spectra of the ligand (H2L) and its copper complex [CuL] were recorded and they are used to compare their stoichiometry composition. The Schiff base shows a molecular ion peak at m/z 481. The molecular ion peak for the copper complex, observed at m/z 542 confirms the stoichiometry of metal chelates as ML type. This composition is also supported by the mass spectra of other complexes. It is in good agreement with the microanalytical data.

1H NMR spectrum

The 1H NMR spectrum of H2L in CDCl3 solution shows the following signals: C6H5 as multiplet at δH 6.9~7.7, -C-CH3 at δH 2.1, =N-CH3 at δH 3.1, Ph-NH- at δH 7.8, -C-CH at δH 4.8. The peaks observed at δH 11.2 and δH 10.2 are attributable to the acidic -OH group present in the 2-aminobenzoic acid and enolic -HC=O group present in acetoacetanilide moiety, respectively. The absence of these two peaks in the zinc complex indicates the deprotonation of the acidic -OH and enolic -HC=O group of acetoacetanilide moiety of the Schiff base on chelation. The azomethine proton signal in the zinc complex is shifted to down field compared to the free ligand, suggesting deshielding of the azomethine group due to the coordination with metal ion. There is no change in all of the other signals of this complex.

IR spectra

In order to study the binding mode of the Schiff base to the metal ion of the complexes, the IR spectrum of the free ligand was compared with the spectra of the complexes. The IR spectrum of the ligand shows a weak broad band in the region 2900~3400 cm-1 assignable to intramolecular hydrogen bonding between enolisable -HC=O group of acetylacetone and acidic -OH group. Absence of this band in complexes indicates the deprotonation of the intramolecular hydrogen bonded enolic and acidic -OH groups upon complexation. The ligand shows its characteristic =C=N- bands at ca. 1640~1600 cm-1, which are also shifted to lower frequencies in the spectra of the complexes (1620~1580 cm-1). The metal chelates show some new bands in the region 480~450 cm-1 and 400~350 cm-1 which are due to the formation of M-O and M-N bonds respectively (Tabassum et al., 2004). Hence, it is concluded that the coordination to the metal ion occurs through the enolisable carbonyl group of acetoacetanilide moiety, acidic group of 2-aminobenzoic acid and the azomethine nitrogen atoms of the Schiff base. In addition to other bands, the vanadyl complex shows an additional band at 990~970 cm-1 attributed to V=O frequency (Mishra et al., 2005).

Electronic absorption spectra

The electronic absorption spectra of the Schiff base and its Cu(II), Ni(II), Co(II) and VO(IV) complexes were recorded at 300 K. The absorption region, assigned and the proposed geometry of the complexes are given in Table 2. These values are comparable with that of the other reported complexes (Tabassum et al., 2004; Lever, 1968; Mishra et al., 2005) and consistent with square-planar geometry except VO(IV) complex which is having square-pyramidal geometry (Sharma et al., 2003).

Table 2

Electronic absorption spectral data of the compounds

EtOH = Ethanol, DMF = Dimethylformide, INCT = Intra-ligand charge transfer transition.

Antifungal activity

The in vitro antifungal activities of the compounds were tested against A. niger, A. flavus, R. stolonifer, C. albicans, R. bataicola and T. harizanum by the serial dilution method. The minimum inhibitory concentration (MIC) values of the compounds are summarized in Table 3. A comparative study of the ligand and its complexes (MIC values) indicates that complexes exhibit higher antifungal activity than the free ligand. From the MIC values, it was found that the compound VOL is more potent among the other investigated complexes and the Schiff base. Such increased activity of the complexes can be explained on the basis of Overtone's concept (Anjaneyula and Rao, 1986) and Tweedy's Chelation theory (Dharmaraj et al., 2001). According to Overtone's concept of cell permeability, the lipid membrane that surrounds the cell favours the passage of only the lipidsoluble materials due to which liposolubility is an important factor, which controls the antifungal activity. On chelation, the polarity of the metal ion will be 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 π-electrons over the whole chelate ring and enhances the lipophilicity of the complexes. The increased lipophilicity enhances the penetration of the complexes into lipid membranes and blocking of the metal binding sites in the enzymes of microorganisms. These complexes also disturb the respiration process of the cell and thus block the synthesis of the proteins that restricts further growth of the organism. Furthermore, the mode of action of the compound may involve formation of a hydrogen bond through the azomethine group with the active centre of cell constitutes resulting in interference with the normal cell process.

Table 3

Minimum inhibitory concentration of the synthesized compounds against growth of six fungi (µg/ml)