Designing and synthesis of antifungal active macrocyclic ligand and its complexes derived from diethylphthalate and benzidine.

Three novel complexes of Cu(II), Co(II) and Zn(II) using a macrocyclic ligand derived by the condensation of diethylphthalate and benzidine have been designed,synthesized and characterized by UV-Vis.,IR,Mass and Elemental analyses data in order to find out their antifungal activities. The stoichiometry of the complexes has been found to be 1: 1(Metal : Ligand). The analytical data indicate that the complexes exhibit square-planar geometry. The antifungal activity of the macrocyclic ligand and its metal complexes has been screened in vitro against fungi such as Aspergillus niger, A. flavus, Trichoderma harizanum, T. viridae and Rhizoctonia solani.

In recent, much interest is focused on the synthesis of macrocyclic complexes with potential medicinal application (Kong et al., 1999). It is well known that various organic ligands possess strong fungicidal, insecticidal, herbicidal and antibacterial properties (Ashu Chaudhary and Singh, 2001; Chitra Gupta and Goutam, 2002; Glory et al., 2006; Biyala et al., 2006; Singh et al., 2003; Venkateswar Rao and Venkata Narasaiah, 2003). It has been reported that the activity of biometals is very often altered through the formation of chelates with different bioligands. In recent years, an extensive literature has been published in the field of chelate compounds with antimicrobial activities. Metal coordination complexes have been widely studied for their antimicrobial (Kamalakannan et al., 2002) and anticancer (Treshchalina et al., 1979) properties. Many drugs demonstrated modified pharmacological and toxicological properties when administered in the form of metallic complexes. Transition metals form some specific and important drugs. These drugs showed antiserothenine, antihistaminic, anticonvulsant and antifungal activities. It has been well established that certain platinum and palladium complexes demonstrated carcinostatic activity (Biyala et al., 2004). It is suggested that the compounds exert antimicrobial activity either by killing the microbe or by inhibiting the growth of the microbe. The antimicrobial activity of these compounds depends upon the nature of the microorganisms. This prompted us to design and synthesize antifungal active macrocylic complexes formed by the condensation of diethylphthalate and benzidine using metal salts. The in vitro antifungal activities of the investigated compounds were tested against fungi such as Aspergillus niger, A. flavus, Trichoderma harizanum, T. viridae and Rhizoctonia solani.

Materials and Methods

All chemical reagents were purchased from Merck. Lithium perchlorate (LiClO4) was purchased from Sigma. Anhydrous grade ethanol and DMSO were obtained from Fisher Scientific Company. Micro analytical data 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 Shimadzu FTIR-8400S spectrophotometer in 4,000~200 cm-1 range using KBr as solvent. The UV-Vis. spectra were recorded on a Shimadzu UV-1601 spectrophotometer. Magnetic susceptibility measurements of the complexes were carried out by Guoy balance using copper sulphate as the calibrant. The values were corrected for diamagnetism by applying Pascal's constants. The molar conductance of the complexes was measured using a Systronic conductivity bridge.

Antifungal activity

NCCLS approved standard potato dextrose agar medium was used for antifungal activity by well diffusion method (Irobi et al., 1996). DMSO was used as the solvent and fluconazole (antifungal agent) as control. The PDA medium was prepared and inoculation was done inside the Laminar Air Flow. A well was made on the agar medium inoculated with microorganisms. The well was filled with the test solution, covered with petri plates and they were incubated at 35℃ for 72 h. During the incubation period, the solution was diffused and affected the growth of the inoculated microorganisms. The inhibition zone was developed, at which the concentration was noted. The minimum inhibitory concentration (MIC) values of the compounds against the growth of microorganisms are summarized in Table 3.

Table 3

Antifungal activity of the synthesized compounds MIC (minimum inhibitory concentration×10-4 M) [standard error: ± 0.00001 M]

Isolation and identification

The organisms (A. niger, A. flavus, T. harizanum, T. viridae and Rhizoctonia solani) were isolated by primary selection from a sample of naturally contaminated cassava waste by serial dilution and courplate technique. The pure cultures were identified by their pharmacology and colony characteristics. The organisms were maintained on PDA slant (potato dextrose agar) stored at 4℃. The plants were freshly made once a month.

Synthesis of macrocyclic ligands

An ethanolic solution of diethylphthalate (10 mmol) was added to the ethanolic solution of benzidine (10 mmol) and refluxed for 3h. Then the solution was reduced to one-third on a water bath. The solid complex precipitated was filtered and washed thoroughly with ethanol and dried in vacuo.

Synthesis of metal complexes

A solution of macrocyclic ligand (5 mmol) in ethanol (20 ml) was added to a solution of MCl2 (5 mmol) in ethanol (10 ml) and the mixture was refluxed for 3 h and concentrated to one-third volume on a water bath after cooling. The solid product formed was filtered, washed with ethanol and dried in vacuo.

Results and Discussion

All the complexes are stable at room temperature, insoluble in water and partially soluble in acetonitrile but soluble in DMF and DMSO. The physical properties and analytical data of the complexes are enlisted in Table 1. The elemental analysis data of the complexes are in good agreement with theoretical values. These complexes showed high conductance values (105~136 ohm-1 cm2 mol-1) indicating their electrolytic nature. High molar conductance values of all the complexes in DMSO indicate that the chloride ions are present outside of the coordination sphere which is confirmed by silver nitrate test. The magnetic moment (Table 1) of the Cu(II) complex under the present study was 1.81 B.M. at room temperature, suggesting a square-planar geometry around the copper ion (Harikumaran Nair et al., 2005).

Table 1

Physical characterization, analytical, molar conductance and magnetic susceptibility data of the complexes

Mass spectra

A fast atom bombardment mass spectrum was obtained for the macrocyclic ligand (L). In this spectrum was seen the ligand molecular ion peak at m/z 628, as expected for a monomeric formulation of the respective ring. Also the fast atom bombardment mass spectrum of copper [Cu(L)]Cl2 complex shows molecular ion peak at m/z 761 [M+], which confirms the 1 : 1 stoichiometric ratio of the complex formation. Further, the peak at m/z 71 is due to the presence of two chloride ions which reinforces the conclusions drawn from the conductance studies.

IR spectra

The infrared spectra gave some important information regarding to the skeleton of the complexes. The IR spectra of the macrocyclic ligands show characteristic bands for ν (N-H) at 3327 cm-1 and amide (C = O) at 1619 cm-1. In the complexes, ν (N-H) bands were shifted to lower frequencies (104 cm-1), due to coordination of the NH groups. On the other hand, the stretching vibration of ν (C = O) was not affected in all the complexes, which indicates that the carbonyl groups are not involved in coordination to the metal cation. The coordination of nitrogen to the metal atom is supported by the appearance of a new band in the region 436 cm-1 assignable to ν (M-N) vibration.

Electronic spectra

The electronic absorption spectra of ligand and its complexes were recorded at 300 K using suitable solvent. The solvent, absorption region, assignment of the absorption bands and the proposed geometry of the complexes are given in Table 2. From the table, we concluded that all the complexes are having square-planar geometry.

Table 2

Electronic absorption spectral data of the complexes at 300 K

Based on the above spectral data, the proposed structure of the macrocyclic ligand and its metal complexes is shown in Fig. 1 and 2.

Fig. 1

Structure of macrocyclic ligand.

Fig. 2

Structure of macrocyclic complexes.

Disc diffusion method

The in vitro antifungal activity of the compounds was tested against filamentous fungi such as Aspergillus niger, Aspergillus flavus, Trichoderma harizanum, Trichoderma viridae and Rhizoctonia solani, by serial Dilution method. The minimum inhibitory concentration (MIC) values of the compounds against the growth of microorganisms are summarized in Table 3. A comparative study of the ligand and its complexes (MIC values) indicates that most of the metal chelates show higher antibacterial activity than the free ligand. (Mishra and Singh, 1993; Dharmaraj et al., 2001). The increased activity of the metal chelates is due to increased lipophilicity of the metalions.

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 (Srivastava, 1981). According to Overtone's concept of cell permeability, the lipid membrane that surrounds the cell favours the passage of only the lipid-soluble 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.