Synthesis, Characterization, Spectral Studies and Antifungal Activity of Mn(II), Fe(II), Co(II), Ni(II), Cu(II) and Zn(II) Complexes with 2-(4- Sulphophenylazo)-1,8-Dihydroxy-3,6-Napthalene Disulphonic Acid Trisodium Salt

Complexes of the type Na6[M(HL)2(H2O)2], where M= Mn(II), Fe(II), Co(II), Ni(II), Cu(II) and Zn(II) and Na3H2L= 2-(4-sulphophenylazo)-1,8-dihydroxy 3,6 naphthalene disulphonic acid trisodium salt, have been synthesized and characterized by physico-chemical (elemental analyses, solubility, electrolytic conductance, magnetic susceptibility measurement) and spectral (UV-Visible, IR, ESR, powder x-ray diffraction) techniques for their structure and studied for their antifungal activity against ten fungi. The anionic 1:2 metal:ligand complexes show octahedral geometry around M(II), a significant antifungal activity against Curvularia lunata and Alternaria triticina and a moderate activity against Alternaria brassicicola, Alternaria brassicae, Alternaria solanae, Curvularia species, Helminthosporium oryzae, Collectotrichum capsici, Aspergillus niger, Aspergillus flavus and Fusarium udum.


Synthesis of Complexes
To an aqueous solution of the ligand, 2(4-sulphophenylazo)-l,8,dihydroxy-3,6-napthalene disulphonic G.Pandey and K.K. Narang Bio&organic Chemistry and Applications acid trisodium salt, Na3H2L (ca 2 m mol, 1.14082 g), aqueous solutions of metal salts, (ca 1 m mol, 0.197 g of MnC12.4H20, 0.2780 g of FeSO4.7H20, 0.281 g of COSO4.7H20, 0.2809 g of NiSO4.7H20, 0.2497 g of CuSOa'5H20 and 0.2870 g of ZnSOa.7H20) were respectively added with constant stirring. These solution mixtures were then digested on water bath with constant stirring at about 95 C. The solutions were made alkaline (~8-9 pH) by adding NaOH solutions and further refluxed for about 2 hours. The solutions were concentrated up to half of the volume by heating on water bath and then about 10 ml absolute ethanol/methanol was added. The reaction mixtures were further refluxed for ca two hours. Shinning microcrystals, thus obtained, were filtered, washed with ethanol, several times, and dried over PO5 under reduced pressure. Recrystalization was. carried out from ethanol solution. Their and physico-chemical and elemental data are presented in Table-1. Room temperature magnetic susceptibility measurements were carried out on a Faraday type balance (Cahn-electronic balance 75570) using catena-tetrathiocyanatocobalt(II) mercury(II), [CoHg(SCN)4] as standard. Experimental magnetic susceptibility (M) was corrected for diamagnetism using the procedure of Figgis and Lewis/23/. Electronic spectra (1500-200 nm) of the ligand and complexes were recorded on a CARY-2390 UV-Visible spectrophotometer in double distilled water using the technique of Lee et al/24/. IR spectra between 4000-400 cm were recorded on a JASCO FT/IR-5300 spectrophotometer in nujol mulls.
The ESR spectra of Cu(II) complex of NaH2L was recorded on an X-band spectrometer model Varian-E-11 at liquid nitrogen temperature and room temperature in solid state using DPPH (ca-diphenyl, 13-picryl hydrazyl) as <g> marker. The field setup was 3200 G and scan range was 2000G. Powder X-ray diffraction data were analysed by Ito's method /25/. Molar conductance of all the water-soluble complexes was measured at room temperature on a WTW conductivity bridge fitted with Philips magic eye.

Antifungal activity
The fungi were isolated on PDA (250 g peeled potato, 20 g dextrose, 15 g agar powder and 1L distilled water) medium from their respective hosts collected from the experimental farm of Banaras Hindu University and incubated for one week at room temperature. The cultures were purified by single spore isolation on PDA slants.
Aqueous solutions of each metal salt, MnCb_.4H_O, FeSO4.7H20, COSO4.7H20, NiSO,.7H20, CuSO.5H_O, and ZnSO4.7HO, were prepared by dissolving the required amounts in double distilled water thereby making concentrations of 1000 ppm. Aqueous solutions of NaHzL was prepared by dissolving the required amount in double distilled water to make concentrationsof 1000 ppm. Also complexes of metal ions (Mn(II), Fe(II), Co(II), Ni(II), Cu(II) and Zn(II)) with the ligands NaHzL were dissolved in the required amount of double distilled water to make concentrations up to 1000 ppm. These solutions were diluted further by addition of double distilled water for preparing the required sets of metal mixture solutions, namely, A, A, Az, B, B, Bz, 1, 2, 3, 4 etc. Antifungal activity of the above metal complexes was studied on various fungi, namely, Alternaria brassicicola, A lternaria brassicae, A lternaria solanae, A lternaria triticina, Curvularia species, Curvularia lunata, Helminthosporium oryzae, Collectotrichum capsici, Aspergillus niger, Aspergillus flavus, and Fusarium udum, by using the spore germination technique/26/. For comparison, antifungal activity of the ligands was also determined on the same fungi. The antifungal activity of the above water-soluble complexes, metal salts and ligands was also studied by poisoned food technique on the fungi, Curvularia species, Curvularia lunala, Alternaria triticina, Alternaria brassicicola and Fusariam udum /27/. G. Pandey and K.K. Narang

Bioinorganic Chemistry and Applications
A drop of metal-complex solution was placed on a grease-free glass slide and 200-300 spores of the test fungi were placed with the help of a sterilized inoculation needle on the solution. The slides were then placed in a moist chamber and incubated at 25 + 2C, for 24 h. After incubation the spores were fixed and stained with lectophenol cotton blue, and spore germination was observed under a light microscope. Similar spore numbers of each fungus were mixed in sterilized distilled water, which served as control. For measurement of inhibition, the percentage germination was subtracted by a hundred to get percentage inhibition. All the experiments were conducted in triplicate. The data were subjected to students 't' test for statistical significance.
Mycellial growth of five fungi, with or without chemicals, was observed by taking dry weight of fungi grown in 150 ml conical flask. All the conical flasks were filled with 50 ml potato dextrose broth. Required amounts of the chemicals were then added to the broth to get the desired concentrations (250, 500 and 750 ppm) individually and in the mixture and dissolved and mixed thoroughly by shaking the flasks. After autoclaving for 15 min (at 121C) the broth was allowed to cool down and a 5 mm disc of fungal mycelium was taken from the border of an actively growing fungal colony and inoculated into the broth. The flasks were incubated at 25 + 2C for one week.. Potato dextrose broth without the chemicals served as control.
After one week, the broth with the fungal colony was filtered on previously weighed filter papers and dried at 80C for 24 h. The dry weight of the fungal colony was determined by deducting the weight of the filter paper from the total weight of filter paper and mycelium. All the experiments were conducted in triplicate. The data were subjected to student 't' test for statistical significance. Antifungal activity measured by these methods is presented in the Tables (8,9,10,11,12).

Statistical analysis
The data recorded for different concentrations of metal-complex solutions were subjected to the following statistical analysis.

Analysis of variance (ANOVA)
The analysis of variance was carried out separately tbr each fungus against all the compounds at various concentrations according to the procedure of Randomized Block Design Analysis/28/.

IR Spectra
Aromatic t-hydroxy azo compounds, such as the present ligand, undergo fast intramolecular proton transfer between the enol-azo form and keto-hydrazone form. This equilibrium has been studied by a variety of spectroscopic techniques/8/.
A weak band observed at ca. 490-440 cm"1, in the spectra of the complexes was assigned to the v (M-N) vibration/31/.
A broad band centered between 3500-3400 cm "1 in all the complexes arises due to v(OH) of the coordinated H20. Besides, rocking (9r) and twisting (9t) H20 modes between 912-823 cm and around 679-590 cm -, respectively, are observed in the complexes indicating coordinated water molecules/32/. Upon heating, above 150 C, the colour of the complexes changed and the IR spectra showed disappearance of the H20 vibrations. v(S-O) in Na31-t2L at about 1020 cm " remains unaffected in the complexes, indicating that the-SO3 groups remain ionic even in complexes/33/. Synthesis, Characterization, Spectral Studies and Antifungal Activity

Magnetic moments
The effective magnetic moments (ta) have been calculated from the room temperature magnetic susceptibility measurements (Xg), assuming Curie Law behaviour of the complexes and using literature values /38/ for diamagnetic corrections for the ions and also using directly measured corrections for the ligand/39/. The magnetic moments of Mn(II), Co(II), Fe(II), Ni(II), and Cu(ll) complexes have been found in the range as expected for Oh complexes (Table-l). The ta. value, 6.12 BM for Mn(II) complex lies well within the range for high spin Oh complexes and 4.97 BM value for Fe(II) complex is diagnostic for d 6 system with some antiferromagnetic exchange interaction/40/. The ta. values for Co(II), Ni(II), Cu(II) have Vol. 3, Nos. [3][4]2005 Synthesis, Characterization, Spectral Studies and Antifungal Activity been found to be 4.59, 2.9 and 1.9 BM respectively, suggesting that all these ions have octahedral arrangements/41/( Figure 5).  Table 4 and the spectrum is illustrated in Figures 6 and 7  Four lines have been observed in the LNT spectra, which remain unresolved at room temperature suggesting that a single copper ion is present in a molecule, or the complex .is a monomer. G values are related by the expression, G gll- 2 and related to the exchange interaction between the copper centers, are g+/--2 greater than four, indicating negligible interaction between the two copper centers/49/. G.Pandey and K.K. Narang Bioinorganic Chemistry and Applications

Antifungal activity
Copper(II) sulphate and Na3HL tested individually as well as in mixture showed antifungal activity against all the fungi tested (Tables 8, 9, 10), though copper sulphate was much better than the Na3H_L, where spore germination of all the fungi was lower than Na3H2L at 259 ppm concentration. Copper sulphate did not effect germination of Curvularia lunata significantly in its diluted form (Table 8) and similar results were obtained in case of Na3H2L also (Table 9). Moreover, Na3H2L in its diluted from B (142 ppm) also did not effect spore germination of A lternaria triticina significantly. In all the other concentrations of the two compounds spore germination was affected significantly (Table 8,9,10). But the two compounds A (copper sulphate) and B (ligand) in their mixture (1:2) form showed more efficacy in inhibiting spore germination of Curvularia lunata and A. triticina at concentration 2(233 ppm) whereas at slightly higher concentration A(259 ppm) and B(285 ppm) the effect of individual compounds was not significant. The effect of mixed compounds and the individual compounds at the above concentrations showed almost similar results for the rest of the fungi (Tables 8,9,10).
When M(II) and ligands were mixed in ratio of 1:2, an excellent synergistic effect was noticed in the case of two of the tested fungi, viz., Curvularia lunata and Alternaria triticina. At concentration A (129 ppm) and B(285 ppm), the effect of individual compounds was not significant against Curvularia lunata whereas when the two compounds were mixed even at the lowest concentration 4(58 ppm), there was significant reduction in spore germination. Similarly at B(142 ppm) spore germination of Alternaria triticina was not significantly reduced. But the two compounds in mixture inhibited spore germination significantly even at the lowest concentration 4(58 ppm). Similar results were obtained when 5 selected fungi were taken for their mycelial growth on potato dextrose broth supplemented with the chemicals. The spores which showed sensitivity against the chemicals also showed a similar trend in the production of mycelial dry weight. Out of the 5 tested fungi, Alternaria triticina showed maximum sensitivity when the chemicals were mixed, followed by Alternaria brassicae and Fusarium udurn ( Table 11).
The result of the present experiment showed the probable synergistic effect of the two compounds in the mixture. Such compounds may inhibit development of resistance since they have multisite action majority in comparison to widely used fungicides with single site of action. Further experimentation with these compounds in glasshouse and under field conditions is suggested for practical application of plant disease control.
The effect of ligand Na3HzL was not very significant on the fungus Altarnaria solanae but it was very effective in case of Fe(II), Co(II), Ni(II), and Zn(II) complexes with Na3HzL where the inhibition (percent) G.Pandey and K.K. Narang   Vol. 3, Nos. [3][4]2005 Synthesis, Characterization, Spectral Studies and Antifungal Activity      Vol. 3, Nos. [3][4]2005 Synthesis, Characterization, Spectral Studies and Antifungal Activity   (Table 12). In case of Collectrotrichun capscici, Aspergillus niger and Fusarium udum the effects of ligand, Na3HzL, as well as Mn(II), Fe(II), Co(II), Ni(II), Cu(II) and Zn(II) complexes with Na3HzL, were very significant showing inhibition up-to 100 percent in many of these cases.
From an overall study of the effect of the different metal salts, ligand Na3HzL, and its Mn(II), Fe(II), Co(II), Ni(II), Cu(II), and Zn(II) complexes, it is inferred that the metal complexes are better fungicidal agents than their parent ligands. Again in certain cases the complexes (metal salts + ligands) are more effective i.e., show more fungi-toxicity in comparison to the individual metal salts or the ligands, thereby indicating a good synergistic effect or the effect of complexation in solution.
For practical utility of these compounds, the inhibiting capacity of the complexes was compared with commercially available fungicides, e.g. dithane-M-45 (a broad fungicide) which is used in the inhibition of spore germination in the 0.1 0.2% in the field condition limit for many fungi. It was found that in the case of Na6[Ni(HL)2(HO)2] against Curvularia species, Colletotricum capsici, Alternaria brassicicola and Alternaria solanae and Na6[Cu(HL)z(H20)2] against Curvularia species, Colletotricum capsici, Fusarium udum, etc., the effect of the complexes was found to be better than that of commercially available fungicides e. g. dithane M-45. This observation is quite significant and opens up a new field of research where metal complexes of organic molecules are better fungi-toxic than commercial products, showing greater possibility of applicability of these complexes under field conditions. A detailed mechanistic study may be desirable in future.