2.1. Materials and Methods

All chemicals used in the present work, viz., thiophene-2-carboxaldehyde, o-phenylenediamine, 1, 10-phenanthroline, Copper, Zinc, Cobalt and Nickel chlorides were of analytical reagent grade (Merck, Germany). Common solvents like Ethanol, Methanol, and DMSO used at various stages of this work were purified according to the standard procedures described in Weissenburg series¹⁹ and in quantitative analysis by Vogel²⁰. Elemental analysis (C, H, N and S) were performed using a Carlo Erba 1108 analyzer. The molar conductivity of the complex in DMSO (10^-3mol/dm³) was measured using a Systronic Model-304 digital direct reading conductivity meter. Magnetic susceptibility measurements were carried out by employing the Gouy method at room temperature on powder sample of the complex. Copper sulphate was used as calibrant. Infra-red spectra of the Schiff base and its metal complexes were recorded as KBr discs in the range 400-4000 cm⁻¹ on a Shimadzu spectrophotometer. The electronic absorption spectra of the Schiff base and its metal complexes were recorded on a Shimadzu UV-1601 spectrophotometer. The ¹H NMR spectra of the Schiff base and its zinc complex in DMSO-d₆ were recorded using tetramethylsilane as internal standard. Electrospray ionisation mass spectrometry (ESI-MS) analysis was performed in the positive ion mode on a liquid chromatography-ion trap mass spectrometer.

2.2. Estimation of metal

The metals were estimated gravimetrically as their oxides²¹ by fusion with AnalaR ammonium oxalate. In a typical experiment, about 0.3g of the dried complex was accurately weighed in a previously weighed silica crucible. AnalaR ammonium oxalate, roughly three parts by weight of the complex, was added and the mixture was incinerated slowly at first and then strongly using a Bunsen burner for 3h. It was then cooled in a desiccator and weighed. The procedure was repeated till the final oxide weight was constant. From the weight, the percentage of metal in the complex was calculated.

2.3. Determination of Chloride Content

The chloride present in the complexes was determined gravimetrically as silver nitrate test²².

2.4. Synthesis of ligand (L)

An ethanolic solution of thiophene-2-carboxaldehyde (0.04mol, 3.67ml) was added dropwise to an ethanolic solution of o-phenylenediamine (0.02mol, 2.163g) with continuous stirring and reaction mixture was refluxed for ca. 5h ^23–25. The yellow product obtained was filtered and recrystallized from ethanol and dried in desiccator over anhydrous calcium chloride. Yield = 83%. The schematic representation of the Schiff base is given in figure 1.

Figure 1 Synthesis of the Schiff base. 

2.5. Synthesis of metal complexes

An ethanolic solution of the Schiff base (0.002 mol) was added to the ethanolic solution of metal (II) chlorides (0.002 mol) and refluxed for ca. 3 h. To the above mixture an ethanolic solution of 1,10 - phenanthroline(0.002 mol) was added in a 1:1:1 molar ratio and refluxed for about ca.2 h. The solid product formed was filtered and washed with ethanol.

2.6. Biological Studies

2.7. Catalytic activities

A large number of Schiff base metal complexes exhibit catalytic activities^27–35. The catalytic oxidation of 1° and 2° alcohols to the corresponding carbonyl compounds have been studied by H₂O₂ as oxidant using the complex [MLX]Cl₂ as catalyst. About 0.1 mol H₂O₂ was added to the mixture of 0.1 mol substrate and 0.001 mol [MLX]Cl₂ in CH₂Cl₂ was stirred for 4-5 hrs at room temperature. The progress of the reaction and completion was checked by TLC. The product was separated and purified by column. The percentage yield of the product was noted.

3.1. Infrared Spectral Studies

The IR spectrum of the free ligand shows band at 1660 cm⁻¹ is characteristics of the azomethine moiety. This band is shifted to lower frequency in the complexes which indicates the coordination of metal to the azomethine nitrogen. The absorption band at 848 cm⁻¹ is due to thiophene ring ν_C-S-C. This band shifts to the lower wave number in the complexes. This shows the sulfur in the thiophene is participated in the complexation^36,37. The appearance of two new bands in the regions 510-495 cm ⁻¹ and 421-418 cm ⁻¹ in the spectra of complexes is due to v_M-N and v_M-S stretching vibrations respectively also confirmed the formation of metal complexes ^38,39. The FT IR Spectral data are summarized in Table 2.

3.2. Electronic absorption Spectra and magnetic measurement

The UV-Vis. spectra of the ligand and its mixed ligand metal complexes were recorded in DMSO. The ligand shows the two bands at 42918 and 33113cm⁻¹ assigned for π → π*, n → π* electronic transitions respectively⁴⁰. The extension of the absorption spectra from UV region to visible region is due to ligand to metal charge transfer and d-d transition bands of the metal in the complexes. The electronic spectrum of the Cu(II) complex showed three bands at 18868,16367 and 13004cm⁻¹ assigned for ²B_1g → ²B_2g, ²B_1g → ²E_g, ²B_1g, → ²A_2g electronic transition which suggested the distorted octahedral geometry of the metal complex. It had a magnetic moment value of 1.98 B.M which also confirmed the possibility of an octahedral geometry. Zn(II) complex shows two bands at 32258, 34483cm⁻¹ assigned for π → π*, n → π* electronic transition^39,41. Ni(II) complex showed three bands at 20833, 16103 and 12987cm⁻¹ assigned for ³A_2g(F) →³T_lg(P) ³A_2g(F) → ³T_lg(F), ³A_2g(F) → ³T_2g(F) electronic transitions suggest the octahedral geometry which was further supported by its magnetic moment value of 3.12 B.M.^42–44. Co(II) complex shows two band at 16667, 14556cm⁻¹ assigned for ⁴T_1g(F) → ⁴T_1g(Ρ) electronic transitions suggest the octahedral geometry ⁴¹. The electronic absorption spectrum of the ligand and its Cobalt complex are given in figure 2 and 3.

Figure 2 UV Visible spectra of Ligand. 

Figure 3 UV Visible spectra of [CoLX]Cl₂ 

3.3. Proton Magnetic Resonance Spectra

The Proton Magnetic Resonance Spectra of ligand and its diamagnetic Zn (II) complex was recorded in DMSO-d₆. The thiophene proton appeared as a singlet at 6.9 ppm. The iminic proton appeared as a singlet at 7.9 ppm and the phenyl multiplet at 7.5-7.7 ppm. In the ¹H-NMR spectrum of Zn (II) complex thiophene proton appeared as a singlet at 6.9-7.2 ppm. The iminic Proton appeared as a singlet at 8.4 ppm and phenyl multiplet at 7.5-7.7 ppm. The singlet at 7.3-7.6 ppm was assigned to phenanthroline proton.

3.4. Mass Spectra

The ESI mass spectra of the ligand and its Zn (II) complex were recorded and the obtained molecular ion peak confirmed the proposed formulae. The mass spectrum of the ligand exhibits peak at 296(M+) with 100% abundance which was also confirmed by ‘nitrogen rule’. The mass spectrum of the inc complex shows peak at 542 (M⁺) with 100% abundance confirms the stoichiometry of metal complexes as [MLX] Cl₂ type.

Based on the elemental analysis, molar conductance, magnetic moments, IR, UV-Vis., proton NMR and Mass spectral data, the proposed structure of the complexes are given in figure 2.

Figure 2 Structure of synthesised complexes. 

3.5. Biological Studies

3.5.1. Antioxidant Studies

DPPH (2,2-diphenyl-1-picrylhydrazyl) is a stable radical in organic solvents; in alcoholic solution it appears purple and has a single electron. This radical is able to react with any compound that releases a hydrogen atom or an electron, resulting in a color change from purple to yellow. The degree of discoloration indicates the scavenging potential of the samples in terms of hydrogen donating ability. Figure 3 shows the comparative effect of metal complexes on DPPH radical. From the IC₅₀ value (table 4) it is inferred that Cobalt (II) complexes (IC₅₀ = 0.601 μΜ) exhibit potent antioxidant activity when compared to other complexes. The increased activity of the complexes can be attributed to the electron withdrawing effect of Zn (II), Co (II), Ni(II) and Cu (II) ions which facilitate the release of hydrogen to reduce the DPPH radical.

Figure 3 Antioxidant activity of mixed ligand complexes 

Table 4 Antioxidant activity of [MLX]Cl₂ 

Concentration	% of radical scavenging activity
	[CuLX] Cl2	[ZnLX] Cl2	[NiLX] Cl2	[CoLX] Cl2
1000 μg/ml	60.8	52.4	55.3	62.7
500 μg/ml	48.3	43.7	45.0	56.8
250 μg/ml	41.6	36.9	37.4	47.3
125 μg/ml	35.0	29.3	35.6	40.4
IC50 (μΜ)	1.061	1.601	1.401	0.601

3.6. Catalytic Activities

The catalytic oxidation of 1° and 2° alcohols with H₂O₂ was carried out in CH₂Cl₂ in the presence of mixed ligand complex as catalyst ⁴⁵. During the course of the reaction water is produced only as a byproduct. The result shows that the metal complexes effectively catalyse the oxidation of alcohols using H₂O₂ as oxidant and is evident from Table 6.