Tetradentate-arm Schiff base derived from the condensation reaction of 3,3′-dihydroxybenzidine, glyoxal/diacetyl and 2-aminophenol: Designing, structural elucidation and properties of their binuclear metal(II) complexes

2.1 Materials and reagents

Cu(OCOCH₃)·H₂O, Ni(OCOCH₃)·4H₂O, VOSO₄·5H₂O, 3,3′-dihydroxybenzidine, glyoxal, diacetyl and 2-aminophenol were purchased from Aldrich. Ethanol, DMSO and DMF were used as solvents purchased from Loba chemicals. The solvents and reagents of analytical grade were obtained commercially and used without further purification.

2.2 Physical measurements

The elemental analysis was performed using a Carlo-Eraba 1106 instrument. Molar conductance of the complexes in DMF solution was measured with ELICO CM 185 conductivity Bridge. The infrared spectra were recorded on the SHIMADZU model spectrometer using a KBr disk. Electronic absorption spectra in the UV–Visible range were recorded on a Perkin Elmer Lambda-25 between 200 and 800 nm by using DMF as the solvent. ESR spectra were recorded on a Varian JEOL-JES-TE100 ESR spectrophotometer at X-band microwave frequencies for powdered samples at room temperature. Magnetic susceptibility data were collected on powdered sample of the compounds at room temperature with a PAR155 vibrating sample magnetometer. Thermal studies were carried out in the 100–900 °C range using an SDT Q600 V20.9 Build 20 model thermal analyzer. Molecular modeling studies have been carried out using the density functional theory (DFT) calculations with a hybrid functional B3LYP (Becke’s three-parameter hybrid functional using the LYP correlation functional) using the 6-311++G(d,p) basis set (Schlegel, 1982; Peng et al., 1996) and were performed using the Gaussian 03W software package.

2.3 Synthesis of ligand

The tetradentate binuclear Schiff base ligand was prepared according to the literature method. To an ethanolic solution of 3,3′-dihydroxybenzidine, glyoxal/diaceyl and ethanolic solution of 2-aminophenol was added in drop wise. The reaction mixture was kept on water bath for refluxion. It was refluxed for 2 h as shown in Fig. 1. Brown color solid was separated and was filtered off, washed with 5 ml of cold ethanol and then dried in air (Sreedaran et al., 2008).

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Figure. 1 Synthetic root of ligands and binuclear Schiff base complexes.

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2.4 Synthesis of metal complexes

All the new binuclear Schiff base complexes were prepared by the same general procedure with stoichiometric amount of ligand and metal salts in a 1:2 mol ratio. To an ethanolic solution of Schiff base ligand metal salts were added. The reaction mixture was kept on a water bath for refluxing about 3 h as shown in Fig. 1. The separated complexes were collected by filtration, washed with hot methanol and dried under vacuum. The same procedure was followed for the synthesis of diacetyl complexes (Emara et al., 2007).

2.5 Cyclic voltammetry

The voltammetric experiments were performed with a CHI 760 electrochemical analyzer, in single compartmental cells using tetrabutylammonium perchlorate as a supporting electrolyte. The redox behavior of the complexes has been examined in a scan rate of 0.1 V s⁻¹ in the potential range +1.2 to −2.0 V. A three-electrode configuration was used, comprising a glassy carbon electrode as the working electrode, a Pt-wire as the auxiliary electrode and a calomel electrode as the reference electrode. The electrochemical data such as cathodic peak potential (E_pc) and anodic peak potential (E_pa) were measured.

2.6 Gel electrophoresis

The cleavage of pUC18 DNA was determined by agarose gel electrophoresis (Raman et al., 2007a,b). The gel electrophoresis experiments were performed by incubation of the samples containing 40 μM pUC18 DNA, 50 μM metal complexes and 50 μM H₂O₂ in Tris–HCl buffer (pH 7.2) at 37 °C for 2 h. After incubation, the samples were electrophoresed for 2 h at 50 V on 1% agarose gel using Tris–acetic acid–EDTA buffer (pH 7.2). The gel was then stained using 1 μg/cm³ ethidium bromide and photographed under ultraviolet light at 360 nm. All the experiments were performed at room temperature.

2.7 Anti-bacterial activity

The anti-bacterial activity of the complexes of Cu(II), Ni(II) and VO(II) was checked by the disk diffusion technique (Chandra and Kumar, 2004; Chandra and Gupta, 2004). This was done on Gram negative bacteria like Klebsiella pneumoniae, Escherichia coli and Gram positive bacteria like Staphylococcus aureus and Bacillus subtilis at 37 °C. The disk of Whatmann No. 4 filter paper having the diameter 8.00 mm was soaked in the solution of compounds in DMF (1.0 mg cm⁻¹). After drying it was placed on nutrient agar plates. The inhibition areas were observed after 36 h. DMF was used as a control and Streptomycin as a standard.

3.1 Elemental composition

The composition and purity of the coordinative compounds was determined by the C, H, N and metal contents. Analytical results indicate a good purity of the coordinative compounds. The elemental analysis data were compared with that of the formulation, which gives good agreement with the proposed formula. The elemental analysis data of the complexes were produced as shown in Table 1.

3.2 Conductivity studies

The molar conductance of the binuclear complexes was an aid for proposing their formulas. Conductivity measurements were carried out in 10⁻³ mol dm⁻³, DMF at 30 °C. The molar conductance values of the all the metal complexes are in the range of 12.03–15.21 ohm⁻¹ cm² mol⁻¹ (Table 1) which indicate the non-ionic in nature of these complexes and they are considered as non-electrolytes (Mohamed et al., 2010).

3.3 ¹H NMR spectra

The ¹H NMR spectrum of the Schiff base ligands was recorded in CDCl₃ at room temperature. The ligands were prepared by dissolution in CDCl₃ and the chemical shifts were recorded with respect to TMS. The ¹H NMR spectra of the Schiff base ligands exhibited a signal at 8.3–8.5 ppm due to the azomethine protons. The ¹H NMR spectra of the ligands exhibited a broad singlet peak at 7.94–8.85 ppm for the OH protons of the 2-aminophenol and 3,3′-dihydroxybenzidine groups. The peaks observed at range 6.98–8.10 ppm are assignable to the protons of the aromatic rings as multiplet peaks. The ¹H NMR spectrum of C₂₈H₂₂N₄O₄ is shown in Fig. 2.

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Figure. 2 ¹H NMR spectrum of Schiff base ligand-1.

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3.4 IR spectra

A comparative study of the IR spectra between the free ligands and metallic complexes, evinces modifications of the vibration bands characteristic of the azomethinic group, −C⚌N, such as a deformation band outside the O−H, plane and shifting skeletal vibration of the aromatic rings. Deformation bands outside the O−H, plane of the free ligands are missing in the complexes. Over the 400–700 cm⁻¹ domain, the appearance of some new bands, characteristic to the M−O bonding vibration, is observed (Ramachandraiah et al., 1989; Nakamoto, 1963).

A strong band is observed in the free ligands around 1610 and 1612 cm⁻¹, characteristic of the azomethine (−C⚌N) group (Ramesh and Natarajan, 1996). Coordination of the Schiff base to the metal through azomethine nitrogen atom is expected to reduce the electron density in the azomethine link and lower the (−C⚌N) absorption frequency. In the spectra of all new complexes, the band due to (−C⚌N) showed a negative shift at 1598–1610 cm⁻¹, indicating coordination of the azomethine nitrogen to metal atom (Ramesh et al., 2000). Another medium intensity band around 3382 and 3400 cm⁻¹ in the free ligands due to the (OH) was absent in the complexes, indicating deprotonation of the Schiff bases prior to the coordination. The various absorption bands in the region 1472–1494 cm⁻¹ may be assigned due to ν(C⚌C) aromatic stretching vibrations of the 3,3′-dihydroxybenzidine and 2-aminophenol. The spectra of the oxovanadium(IV) complexes show band around 930–990 cm⁻¹ region (Zhan and Yuan, 1999) This band was typical of oxometal species and was assigned to the V⚌O stretching of the vanadyl group (Giovana and Geraldo, 2005).

The infrared spectra show bands in the region 450–500 cm⁻¹ corresponding to ν(M–N) vibrations (Chandra and Gupta, 2004; Chandra and Kumar, 2004). The presence of bands in all the complexes in the region 456–474 cm⁻¹ originates from the ν(M–N) azomethine vibration modes and identifies coordination of azomethine nitrogen and also the presence of bands at 510–524 cm⁻¹ indicates ν(M–O) vibrations (Fraser et al., 1994). The IR spectral data of all the complexes are given in Table 2.

3.5 Electronic spectra

The geometry of the metal complexes has been deduced from the electronic spectra of the complexes. Electronic spectra of all the complexes were recorded in DMF medium in Table 2. Only UV absorption bands corresponding to the π–π⁎ and n–π⁎ transitions in an organic molecule are observed for ligands. Additional bands of the resolved medium intensity d–d transitions in the visible region (500–800 nm) are observed for the complexes. Based on the obtained results on absorption in the visible region and literature data were obtained on the position and intensity of bands of the d–d transitions for the square planar for copper(II), nickel(II) and square pyramidal for oxovanadium(IV) complexes (Manonmani et al., 2001).

The bands obtained in the range of 259–282 nm was assigned to the intra ligand charge transfer transition (π → π^⁎). An intense peak in the range of 442–480 nm was due to ligand-to-metal charge transfer transitions. The bands are indicative of benzene and other chromophore moieties present in the ligand moiety.

The Cu₂(L¹) and Cu₂(L²) complexes show a broad absorption peak at 592 and 550 nm, respectively, arises due to the d–d transition ²B_1g → ²A_1g suggests that the copper ion exhibits a square planar geometry (Akagi et al., 2004). According to the literature data, the principal feature of square planar nickel(II) complex is the presence of two well-defined bands (Ababei et al., 2009). We have also observed two weak intensity bands of the Ni(II) for complexes Ni₂(L¹) and Ni₂(L²) at 562, 631 nm and 621, 742 nm corresponding to ¹A_1g → ¹A_2g, ¹A_1g → ¹B_1g transitions, respectively (Chandra and Sharma, 2002).

Usually, the electronic spectra of the square-pyramidal oxovandium(IV) complexes (d¹) are characterized by the presence of three absorption bands corresponding to d_xy → d_xz, d_yz, ${\text{d}}_{\mathit{xy}}\to {\text{d}}_x^2-{\text{d}}_z^2$ and ${\text{d}}_{\mathit{xy}}\to {\text{d}}_z^2$ transitions (Vanquickenborne and McGlynn, 1968). In the electronic spectra of the VO(L¹) complexes three absorption bands were observed at 545, 609, and 662 nm. Similarly, for VO(L²) complexes three absorption bands were observed at 540, 621, and 759 nm. The observed results indicate that the present oxovandium(IV) complexes are square-pyramidal geometry.

3.6 Cyclic voltammetry

The electrochemical techniques are the most effective and versatile methods available for the mechanistic study of redox systems (Mahalakshmi and Rajavel, 2014). The electrochemical behavior was studied by cyclic voltammetry in DMF containing 10⁻¹ M tetra(n-butyl) ammonium perchlorate over the range of 1.2 to −2.0 V. The electrochemical data of the complexes are summarized in Table 3 (Reduction and Oxidation). In Cu₂(L¹) and Cu₂(L²) complexes, the Cu(II) shows quasireversible reduction waves. Controlled potential electrolysis was carried out at 100 mV s⁻¹ and the experiment reports that each couple corresponds to one electron transfer process. So, the processes are assigned as follows. $${\text{Cu}}^{\text{II}}{\text{Cu}}^{\text{II}}\leftrightarrows {\text{Cu}}^{\text{I}}{\text{Cu}}^{\text{II}}\leftrightarrows {\text{Cu}}^{\text{I}}{\text{Cu}}^{\text{I}}$$

The Cu(II) complexes show a quasireversible oxidation waves, which are assigned as a Cu(II)/Cu(III) couple. The ΔE_p values suggest that each couple was quasireversible. The E_1/2 values indicate that each couple corresponds to one electron transfer process.

In a typical cyclic voltammograms of the Ni₂(L¹) and Ni₂(L²), the Ni(II) shows quasireversible reduction waves. The ΔE_p values suggest the existence of a quasireversible couple. The E_1/2 values indicate that each couple corresponds to one electron transfer process. Table 3 represents the electrochemical data of Ni(II) complexes in cathodic and anodic potential, respectively. Controlled potential electrolysis was also carried out and the experimental reports suggest that each couple correspond to one electron transfer process, as follows (Olar et al., 2005). Similarly oxidation process is also quasireversible in nature. $${\text{Ni}}^{\text{II}}{\text{Ni}}^{\text{II}}\leftrightarrows {\text{Ni}}^{\text{I}}{\text{Ni}}^{\text{II}}\leftrightarrows {\text{Ni}}^{\text{I}}{\text{Ni}}^{\text{I}}$$

The cyclic voltammograms for the oxovanadium(IV) complexes in DMF consists of quasireversible reduction waves (Table 3). This irreversible reduction may be attributed to reduction of V^IV to V^III. The V^IV to V^V oxidation in complex is quasireversible in nature. Electrochemical measurements of oxovanadium(IV) complex with tetradentate dibasic Schiff base ligands are reported to show oxidation to V^V and reduction to V^III vs SCE, respectively.

3.7 Thermal analysis

The TG curves of all complexes show that the thermal decomposition of the anhydrous product takes place in several steps as reported in Table 4. It is possible that the different groups in the ligands decrease the stability of all of the complexes. Furthermore, it is known that the electronegativity and atomic radius of the central metal atom also affect the thermal stability (Essa et al., 1994).

The thermogram of ligand with the molecular formula (C₂₈H₂₂N₄O₄) shows two decomposition steps within the temperature range from 120 to 360 °C. The first step occurs within the temperature range 120–290 °C with an estimated mass loss 44.00% (calculated mass loss = 43.28) which is reasonably accounted for the loss of benzidine groups. The second step occurs within the temperature range 290–360 °C with an estimated mass loss of 56.00% (calculated mass loss = 55.78%), which is reasonably accounted for the loss of aromatic ligand groups.

The thermogram of ligand with the molecular formula (C₃₂H₃₀N₄O₄) shows two decomposition steps within the temperature range from 350 to 642 °C. The first step occurs within the temperature range 350–490 °C with an estimated mass loss 40.22% (calculated mass loss = 40.28) which is reasonably accounted for the loss of benzidine groups. The second step occurs within the temperature range 490–642 °C with an estimated mass loss of 56.00% (calculated mass loss = 56.18%), which is reasonably accounted for the loss of aromatic ligand groups.

The TG curves of the Cu(II) complex (Fig. 3) with the molecular formula [Cu₂(C₂₈H₁₈N₄O₄)] are thermally decomposed in a single step in the temperature range from 275 to 340 °C with an estimated mass loss 60.72% (calculated mass loss = 60.76%), which is attributed to the loss of benzidine groups and aromatic ligand groups. The last step did not finish completely. Therefore, last decomposition residue was not determined.

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Figure. 3 Thermal analysis curve for [Cu₂(C₂₈H₁₈N₄O₄)] binuclear Schiff base complex.

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The TG curves of the Ni(II) complex with the molecular formula [Ni₂(C₂₈H₁₈N₄O₄)] are thermally decomposed in a single step in the temperature range from 244 to 407 °C with an estimated mass loss 63.74% (calculated mass loss = 63.70%), which is attributed to the loss of benzidine groups and aromatic ligand groups. The last step did not finish completely. Therefore, last decomposition residue was not determined.

The TG curves of the VO(II) complex with the molecular formula [VO₂(C₂₈H₁₈N₄O₄)] are thermally decomposed in a single step in the temperature range from 200 to 380 °C with an estimated mass loss 65.13% (calculated mass loss = 65.70%), which is attributed to the loss of benzidine groups and aromatic ligand groups.

3.8 Magnetic moment studies

The magnetic susceptibility measurements provide information regarding the structure of the metal complexes. Magnetic susceptibility was determined using a magnetic susceptibility balance. The magnetic susceptibility measurements show that the copper(II) and oxovanadium(IV) complexes are paramagnetic at ambient temperature (Djebbar-Sid et al., 1998). The magnetic moments of the complexes were measured at room temperature. On the basis of the magnetic and spectral evidence the copper(II) complexes have binuclear structures in which the copper(II) cation has an approximately square-planar environment (Khan et al., 2007).

The measured magnetic moment of the mononuclear copper(II) complex was 1.73 B.M. The magnetic moment of binuclear [Cu₂(L¹)] and [Cu₂(L²)] complexes is 1.72 B.M and 1.70 B.M. Magnetic susceptibility measurements show that these complexes are paramagnetic, which corresponds to the +2 oxidation state of copper(II) complexes. The values are almost equal spin only value. This indicates that the two metal centers are equivalent and there is no interaction between the two metal centers (Venkatachalam et al., 2008).

On the basis of the magnetic data, the oxovanadium(IV) complexes have a binuclear structure. The magnetic moments of the VO(L¹) and VO(L²) complexes are 1.74 B.M and 1.72 B.M range. It is possible that the oxovanadium(IV) complexes have square-pyramidal geometry (Tümer et al., 1998). The values are almost equal spin only value. This indicates that the two metal centers are equivalent and there is no interaction between the two metal centers.

The magnetic moment of the Ni(II) complex is diamagnetic in nature and have square planar geometry around the metal atom and there is no metal–metal interaction in Ni(II) centers.

3.9 EPR spectra

The EPR spectra of complexes provide information about the importance in studying the metal ion environment. The EPR spectra of the [Cu₂(C₂₈H₁₈N₄O₄)], [Cu₂(C₃₂H₂₆N₄O₄)], [VO₂(C₂₈H₁₈N₄O₄)] and [VO₂(C₃₂H₂₆N₄O₄)] Schiff base complexes were recorded on powder samples at room temperature, on X-band at frequency 9.3 GHz under the magnetic field strength 4000 G.

The EPR spectrum of the [Cu₂(C₂₈H₁₈N₄O₄)] (Fig. 4) and [Cu₂(C₃₂H₂₆N₄O₄)] complexes shows a broad signal with g_iso at 1.9998 and 1.9986 which is consistent with an square planar geometry. Isotropic lines are usually the results of either intermolecular spin exchange, which can broaden the lines or occupancy of the unpaired electron in a degenerate orbital in square planar geometry. This indicates that the two paramagnetic centers are equivalent and there is no considerable exchange interaction in the complex (Venkatachalam et al., 2008).

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Figure. 4 The EPR spectrum of the [Cu₂(C₂₈H₁₈N₄O₄)] complex at room temperature, frequency = 9.41266 GHz.

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The EPR spectrum of [VO₂(C₂₈H₁₈N₄O₄)] and [VO₂(C₃₂H₂₆N₄O₄)] complexes at room temperature gives a broad but partially resolved eight line EPR spectrum (Kannan and Ramesh, 2006). The spectral pattern and the values of g_iso = 2.0001 and 1.9999 are used to assign the binuclear structure. The eight-line isotropic spectrum of mononuclear species (Vanadium) changes into a broad structured rhombic spectrum with a partial resolution of parallel and perpendicular lines centered at 313.42 mT. The g_iso values indicate that the two paramagnetic centers are equivalent and there is no super exchange interaction between the two metal centers (Upadhyay et al., 1992). So, that the complexes are of square-pyramidal geometry around the central metal atom (Raman et al., 2005; Patel and Kolawole, 1982).

3.10 Molecular modeling

Theoretical calculations (Geary, 1971; Allen et al., 1991; Rappe and Casewit, 1997) have paid a considerable attention to the characterization and inferences of geometrical optimization of the prepared [Cu₂(C₂₈H₁₈N₄O₄)] (Fig. 5) and [Cu₂(C₃₂H₂₆N₄O₄)] complexes, therefore we could obtain the optimized geometry for these complex by computing the theoretical physical parameters, such as, bond lengths and bond angles using the Gaussian 03W software package. The bond angles ( $\acute{\AA }$ ) and bond lengths ( $\acute{\AA }$ ) obtained from the energy minimum optimized structures are given as below.In [Cu₂(C₂₈H₁₈N₄O₄)] complex, the central atom is an approximately square planar geometry with N₂O₂. The Cu atom surrounded by two azomethine N atoms and two phenolic oxygen atoms, with Cu₃₁–O₄₂ and O₂₉–Cu₃₁ bond lengths are 1.7908(Ǻ) and 1.813(Ǻ), Cu₃₁–N₃₅ is 1.7971(Ǻ) and N₁₅–Cu₃₁ is 1.8149(Ǻ). Similarly the adjacent Cu atom shows, O₁₄–Cu₁₇ and Cu₁₇–O₂₈ bond lengths are 1.8137(Ǻ) and 1.7914(Ǻ), N₁₃–Cu₁₇ is 1.8154(Ǻ) and Cu₁₇–N₂₁ is 1.7979(Ǻ). The binuclear metal complexes as a whole consist of two parallel approximately planar segments each consists of square planar geometry with four-member chelate ring. The bond angle around Cu metal ion, N₁₅–Cu₃₁–O₂₉, N₁₅–Cu₃₁–N_35, N₁₅–Cu₃₁–O₄₂, O₂₉–Cu₃₁–N₃₅, O₂₉–Cu₃₁–O₄₂ and N₃₅–Cu₃₁–O₄₂ are 88.0337 ( $\acute{\AA }$ ), 85.6241 ( $\acute{\AA }$ ), 163.2518 ( $\acute{\AA }$ ), 166.3778 ( $\acute{\AA }$ ), 100.8902 ( $\acute{\AA }$ ) and 88.3534 ( $\acute{\AA }$ ). The adjacent Cu atom shows N₁₃–Cu₁₇–O₁₄, N₁₃–Cu₁₇–N₂₁, N₁₃–Cu₁₇–O₂₈, O₁₄–Cu₁₇–N₂₁, O₁₄–Cu₁₇–O₂₈ and N₂₁–Cu₁₇–O₂₈ with bond angles 87.9927 ( $\acute{\AA }$ ), 85.6278 ( $\acute{\AA }$ ), 163.0533 ( $\acute{\AA }$ ), 166.6582 ( $\acute{\AA }$ ), 100.8962 ( $\acute{\AA }$ ) and 88.3349 ( $\acute{\AA }$ ). Similarly in [Cu₂(C₃₂H₂₆N₄O₄)] complex, the Cu atom consist of square planar geometry with the tetradentate binucleating Schiff base ligand. The bond lengths of the two Cu atoms in dimeric structure, N₁₃–Cu₁₇, O₁₄–Cu₁₇, N₁₅–Cu₃₁, Cu₁₇–N₂₁, Cu₁₇–O₂₈, O₂₉–Cu₃₁, Cu₃₁–O₄₂ and C₃₁–N₃₅ are 1.8157( $\acute{\AA }$ ), 1.807( $\acute{\AA }$ ), 1.8158( $\acute{\AA }$ ), 1.8045( $\acute{\AA }$ ), 1.7917( $\acute{\AA }$ ), 1.807( $\acute{\AA }$ ), 1.7917( $\acute{\AA }$ ) and 1.8046( $\acute{\AA }$ ). The bond angle which is around the Cu²⁺ ion shows in the range of N₁₅–Cu₃₁–O₂₉, N₁₅–Cu₃₁–N₃₅, N₁₅–Cu₃₁–O₄₂, O₂₉–Cu₃₁–N₃₅, O₂₉–Cu₃₁–O₄₂ and N₃₅–Cu₃₁–O₄₂ are 88.5698 ( $\acute{\AA }$ ), 86.1152 ( $\acute{\AA }$ ), 160.4615 ( $\acute{\AA }$ ), 163.3774 ( $\acute{\AA }$ ), 101.1117 ( $\acute{\AA }$ ) and 88.8251 ( $\acute{\AA }$ ). For the adjacent atom, the bond angle in the range of N₁₃–Cu₁₇–O₁₄, N₁₃–Cu₁₇–N₂₁, N₁₃–Cu₁₇–O₂₈, O₁₄–Cu₁₇–N₂₁, O₁₄–Cu₁₇–O₂₈ and N₂₁–Cu₁₇–O₂₈ are 88.5705 ( $\acute{\AA }$ ), 86.1211 ( $\acute{\AA }$ ), 160.4509 ( $\acute{\AA }$ ), 163.402 ( $\acute{\AA }$ ), 101.0965 ( $\acute{\AA }$ ) and 88.8305 ( $\acute{\AA }$ ). The bond angles ( $\acute{\AA }$ ) and bond lengths (Å) as obtained from molecular modeling studies reveal a square planar geometry for these complexes.

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Figure. 5 Optimization structure and atoms numbering of [Cu₂(C₂₈H₁₈N₄O₄)].

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3.11 Cleavage of plasmid pUC18 DNA

There are a number of agents, which exert their effect by inhibiting enzymes that act upon DNA. These inhibition results from binding of agents to the enzyme’s site of interaction on the DNA and by not direct enzyme inactivation. Transition metals have been seen to inhibit DNA repair enzymes. When DNA is run on horizontal gel using electrophoresis, the fastest migration will be observed for the open circular form (Form I). If one strand is cleaved, the supercoils will relax to produce a slower-moving open circular form (Form II). If both strands are cleaved, a linear form (Form III) will be generated that migrates in between (Jayaseelan et al., 2016). Fig. 6 illustrates the gel electrophoresis experiments showing the cleavage of plasmid pUC18 DNA induced by the ligand and metal complexes. The control experiments did not show any apparent cleavage of DNA (lanes 1 and 2). [Cu₂(L¹)] and [Ni₂(L¹)] complexes in the presence of H₂O₂ (lanes 3 and 4) at higher concentration (50 μM) show cleavage activity in which supercoiled DNA (Form I) cleaved and supercoiled form converted into open circular form (Form II). [VO₂(L¹)] complex in the presence of H₂O₂ (lane 5) at higher concentration (50 μM) shows the conversion of supercoiled form (Form I) into open circular form (Form II). Lanes 6 and 7 reveal that [Cu₂(L²)] and [Ni₂(L²)] complexes in the presence of H₂O₂ at higher concentration (50 μM) show the conversion of supercoiled form (Form I) into open circular form (Form II) and lane 8 reveals that [VO₂(L²)] complex in the presence of H₂O₂ at higher concentration (50 μM) shows the conversion of supercoiled form (Form I) into linear form (Form III). From the observed results we concluded that Cu(II) complex and VO(II) complex cleave DNA as compared to control DNA and Ni(II) complexes. Probably this may be due to the formation of redox couple of the metal ions and its behavior. It is also thought that most cleavage cases are caused by Cu(II) ions reacting with H₂O₂ to produce the diffusible hydroxyl radical (OH^•) or molecular oxygen which may damage DNA. These results indicated the important role of metal ions in isolated DNA cleavage reaction. As the compound was observed to cleave the DNA, it can be concluded that, the compound inhibits the growth of the pathogenic organism by cleaving the genome.

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Figure. 6 Agarose gel showing the results of electrophoresis of pUC18 DNA with the Schiff base complexes: Lane 1, DNA alone; Lane 2, DNA alone + H₂O₂; Lane 3, DNA + [Cu₂(C₂₈H₁₈N₄O₄)] complex + H₂O₂; Lane 4, DNA + [Ni₂(C₂₈H₁₈N₄O₄)] complex + H₂O₂; Lane 5, DNA + [VO₂(C₂₈H₁₈N₄O₄)] complex + H₂O₂; Lane 6, DNA + [Cu₂(C₃₂H₂₆N₄O₄)] complex + H₂O₂; Lane 7, DNA + [Ni₂(C₃₂H₂₆N₄O₄)] complex + H₂O₂; Lane 8, DNA + [VO₂(C₃₂H₂₆N₄O₄)] complex + H₂O₂.

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3.12 Anti-bacterial activity

The main aim of production and synthesis of anti-bacterial compound is to inhibit the pathogenic bacteria without any side effects on the patients. Also, it is worthy to stress here on the basic idea of applying any chemotherapeutic agent which depends essentially on the specific control of only one biological function and not multiple ones. Cu(II), Ni(II) and VO(II) Schiff base complexes show a remarkable anti-bacterial activity against different types of Gram-positive (G+) bacteria like S. aureus, B. subtilis and Gram-negative (G–) bacteria like E. coli, K. pneumoniae. The comparison of biological activities of the ligand and its complexes shows the following results as reported in Figs. 7–10. Streptomycin was used as standard antibiotics. The metal complexes show pronounced activity because of chelation (Raman et al., 2007a,b). The minimum inhibitory concentration (MIC) values of the investigated compounds indicate that most complexes have higher antibacterial activity than the free ligand. Such increased activity of the metal chelates can be explained on the basis of chelation theory. 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 of π-electrons over the whole chelate ring and enhances the penetration of the complexes into lipid membranes and blocking of the metal binding sites in the enzymes of micro-organisms. These complexes also disturb the respiration process of the cell and thus block the synthesis of proteins, which restricts further growth of the organism (Dharmaraj et al., 2001).

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Figure. 7 Difference between the anti-bacterial activities of the Schiff base and its binuclear metal complexes against Escherichia coli. 1, Ligand-1; 2, [Cu₂(C₂₈H₁₈N₄O₄)]; 3, [Ni₂(C₂₈H₁₈N₄O₄)]; 4, [VO₂(C₂₈H₁₈N₄O₄)]; 5, Ligand-2; 6, [Cu₂(C₃₂H₂₆N₄O₄)]; 7, [Ni₂(C₃₂H₂₆N₄O₄)]; 8, [VO₂(C₃₂H₂₆N₄O₄)]; 9, Streptomycin.

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Figure. 8 Difference between the anti-bacterial activities of the Schiff base and its binuclear metal complexes against Staphylococcus aureus. 1, Ligand-1; 2, [Cu₂(C₂₈H₁₈N₄O₄)]; 3, [Ni₂(C₂₈H₁₈N₄O₄)]; 4, [VO₂(C₂₈H₁₈N₄O₄)]; 5, Ligand-2; 6, [Cu₂(C₃₂H₂₆N₄O₄)]; 7, [Ni₂(C₃₂H₂₆N₄O₄)]; 8, [VO₂(C₃₂H₂₆N₄O₄)]; 9, Streptomycin.

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Figure. 9 Difference between the anti-bacterial activities of the Schiff base and its binuclear metal complexes against Klebsiella pneumoniae. 1, Ligand-1; 2, [Cu₂(C₂₈H₁₈N₄O₄)]; 3, [Ni₂(C₂₈H₁₈N₄O₄)]; 4, [VO₂(C₂₈H₁₈N₄O₄)]; 5, Ligand-2; 6, [Cu₂(C₃₂H₂₆N₄O₄)], 7, [Ni₂(C₃₂H₂₆N₄O₄)]; 8, [VO₂(C₃₂H₂₆N₄O₄)]; 9, Streptomycin.

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Figure. 10 Difference between the anti-bacterial activities of the Schiff base and its binuclear metal complexes against Bacillus subtilis. 1, Ligand-1; 2, [Cu₂(C₂₈H₁₈N₄O₄)]; 3, [Ni₂(C₂₈H₁₈N₄O₄)]; 4, [VO₂(C₂₈H₁₈N₄O₄)]; 5, Ligand-2; 6, [Cu₂(C₃₂H₂₆N₄O₄)]; 7, [Ni₂(C₃₂H₂₆N₄O₄)]; 8, [VO₂(C₃₂H₂₆N₄O₄)]; 9, Streptomycin.

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