Metal based SOD mimetic therapeutic agents: Synthesis, characterization and biochemical studies of metal complexes

2.1 Materials

All chemicals used in the present work viz, 4-aminoantipyrine, furfuraldehyde, 2-aminobenzothiazole, 3-nitrobenzaldehyde, Fe(III), Co(II), Ni(II), Cu(II) and Zn(II) chlorides were of analytical reagent grade (Merck). The solvents used in the synthesis of the ligands and metal complexes were distilled before use. Calf thymus DNA was purchased from Genie Biolab, Bangalore, India.

2.2 Instrumentation

The elemental analysis was performed using Elementar Vario EL III Carlo Erba 1108. The amount of metal present in the metal complexes was estimated using ammonium oxalate method (Vogel 1978). IR spectra of the Schiff base ligands and their metal complexes were recorded on Perkin–Elmer FT-IR 783 Spectrophotometer in 4000–300 cm⁻¹ range using KBr disc. ¹H NMR spectra were recorded on a Bruker Avance Dry 300 MHz FT-NMR Spectrometer in DMSO with TMS as the internal reference. The FAB mass spectrum of the Schiff base ligands and their complexes were recorded on a JEOL SX 102/DA-6000 mass spectrometer/data system using argon/xenon (6 kV, 10 mA) as the FAB gas. ESR spectra of the mixed ligand copper complex was recorded on a Varian E 112 EPR Spectrometer in DMSO solution both at room temperature (300 K) and at liquid nitrogen temperature (77 K) using TCNE (tetracyanoethylene) as the g marker. Electronic absorption spectra of the Schiff base ligands and their mixed ligand complexes were recorded in DMSO using a Systronics 2201 double beam UV–Vis., spectrophotometer. Molar conductance of the metal complexes was measured in DMSO solution using a coronation digital conductivity metre. The magnetic susceptibility values were calculated using the relation μ_eff = 2.83(χ_m T)^1/2. The diamagnetic corrections were made by Pascal’s constant and Hg[Co(SCN)₄] was used as a calibrant. Electrochemical experiments were performed on a CHI 604D electrochemical analysis system with a three-electrode system consisting a glassy carbon working electrode, Pt wire auxiliary electrode and Ag/AgCl reference electrode. Tetrabutylammoniumperchlorate (TBAP) was used as the supporting electrolyte. All solutions were purged with N₂ for 30 min prior to each set of experiments. Calf thymus DNA was purchased from Bangalore Genei (India). Tris–HCl buffer solution used for binding studies was prepared using double distilled water.

3.1 Preparation of ligands

The Schiff base ligands (L¹, L²) were prepared according to earlier reports, with slight modifications (Ismail et al., 1997; Vicini et al., 2003)

L¹: The ligand (L¹) was prepared as reported previously.

L²: The Schiff base (L²) was prepared by the condensation of 2-aminobenzothiazole (1 mM) and 3-nitrobenzaldehyde (1 mM) dissolved in 30 mL absolute EtOH, in the presence of few drops of piperidine. The resulting mixture was stirred well, refluxed for 7 h. The mixture was concentrated to its half volume then cooled. Petroleum ether was added to the reaction mixture dropwise until a product began to precipitate. The formed product was filtered off, washed several times with EtOH, recrystallized from EtOH.

3.2 Preparation of metal complexes

An ethanolic solution of metal (M = FeCl₃.6H₂O, CoCl₂.6H₂O, NiCl₂.6H₂O, CuCl₂.2H₂O and ZnCl₂) (1 mM) chlorides was stirred with an ethanolic solution of ligand(s) (L¹/L²) (1 mM) and the resultant mixture was refluxed for ca. 6–8 h. Then, the volume of the solution was reduced to one-third on a water bath. The solid complex precipitated was filtered, washed thoroughly with ethanol and dried in vacuum. The schematic route for the synthesis of Schiff base ligands and their metal complexes is given in Scheme 1.

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Scheme 1

Schematic route for synthesis of Schiff base ligands and its metal complexes.

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3.3 DNA binding experiments

Interaction of the complex with calf thymus DNA has been studied by recording electronic absorption spectra. A solution of CT-DNA in 5 mM Tris–HCl/50 mM NaCl (pH 7.0) gave a ratio of UV absorbance at 260 and 280 nm (A₂₆₀/A₂₈₀) of 1.8–1.9, indicating that the DNA is sufficiently free of proteins. A concentrated stock solution of DNA was prepared in 5 mM Tris–HCl/50 mM NaCl in water at pH 7.0 and the concentration of CT-DNA was determined per nucleotide by taking the absorption coefficient (6600 dm³ mol⁻¹ cm⁻¹) at 260 nm. Stock solutions are stored at 4 °C and were used after no more than 4 days. Doubly distilled water was used to prepare buffer solutions. Solutions were prepared by mixing the complex and CT-DNA in DMF medium. After the equilibrium reached ca. 5 min the spectra were recorded against an analogous blank solution containing the same concentration of DNA. UV spectral data were fitted into Eq. (1) to obtain the intrinsic binding constant (K_b)

3.4 Antimicrobial activity

The in vitro evaluation of antimicrobial activity was carried out. The prepared compounds were tested against some fungi and bacteria to provide the minimum inhibitory concentration (MIC) for each compound. MIC is the lowest concentration of the solution to inhibit the growth of a test organism. The in vitro biological screening effects of the investigated compounds were tested against the bacterial species Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Salmonella typhi and Pseudomonas aeruginosa and fungal species Aspergillus niger, Rhizopus stolonifer, Aspergillus flavus, Rhizoctonia bataicola and Candida albicans by disc diffusion method. One day prior to the experiment, the bacterial and fungal cultures were inoculated in nutrient broth (inoculation medium) and incubated overnight at 37 °C. Inoculation medium containing 24 h grown culture was added aseptically to the nutrient medium and mixed thoroughly to get uniform distribution. This solution was poured (25 ml in each dish) into petri dishes and then allowed to attain room temperature. Wells (6 mm in diameter) were cut in the agar plates using proper sterile tubes. Then, the wells were filled up to the surface of agar with 0.1 ml of the test compounds dissolved in DMSO (200 μg/ml). The plates were allowed to stand for an hour in order to facilitate the diffusion of the drug solution. Then the plates were incubated at 37 °C for 24 h for bacteria and 48 h for fungi and the diameter of the inhibition zones was measured. Minimum inhibitory concentrations (MICs) were detected by the serial dilution method. The lowest concentration (μg/ml) of compounds, which inhibits the growth of bacteria after 24 h incubation at 37 °C, and of fungi after 72 h incubation at 37 °C was taken as the MIC.

3.5 Superoxide dismutase (SOD) activity

In vitro SOD activity was measured using alkaline DMSO as a source of superoxide radical ion ( ${\text{O}}_2^{·-}$ ) and nitrobluetetrazolium (NBT) as ${\text{O}}_2^{·-}$ scavenger (Bhirud et al., 1990, 1991). In general, 400 μL of the sample to be assayed was added to a solution containing 2.1 mL of 0.2 M potassium phosphate buffer (pH 7.8) and 1 mL of 56 μM NBT. The tubes were kept in ice for 20 min and then 1.5 mL of alkaline DMSO solution was added while stirring. The absorbance was then monitored at 540 nm against a sample prepared under a similar condition except that NaOH was absent in DMSO. The measure of SOD activity, expressed as IC₅₀ is the concentration of the substrate or complex which causes 50% inhibition of the reduction of NBT.

4.1 4.1.¹H NMR Spectra

The ligand (L¹) shows the following signals and their assignments are given below: phenyl multiplet at 7.0–7.5 δ (5H), –CH⚌N at 7.8 (due to furfuryl moiety), –C–CH₃ at 2.2 δ, N–CH₃ at 1.5 δ. The ligand (L²) shows the following signals and their assignments are given below: phenyl multiplet at 7.5–8.1 δ (8H), –CH⚌N at 7.9 δ (due to phenyl moiety). The azomethine proton (–CH⚌N) signal in the spectrum of zinc complex is shifted down field (7.6 and 7.7 ppm) compared to the free ligands, suggesting deshielding of the azomethine group due to the coordination with metal ion. All protons were found to be in similar regions.

4.2 IR spectra

In order to study the bonding mode of the Schiff base to the metal complexes, the IR spectra of the free ligands were compared with the spectrum of the complexes. In the ligand L¹, the absorption band at 1647 cm⁻¹ may be assigned to ν(C⚌O). The absorption band is shifted to a lower wave number 20–32 cm⁻¹ in the spectra of all complexes suggesting the involvement of the pyrazolone oxygen in chelation.

The strong absorption bands located at 1602 and 1629 cm⁻¹ in the spectrum of the free ligands L¹ and L² are attributed to ν(–CH⚌N) vibrations. These bands are shifted (by ∼10–40 cm⁻¹) towards lower frequencies in the spectra of all complexes, which clearly suggested that complexation has taken place through the nitrogen atom of the azomethine group.

In the case L², the bands at 1458 and 1155 cm⁻¹ are characteristic of the ν(C–S) of the thiazole ring. This band remains at the same position in the spectra of the Schiff base as well as in the case of its complexes revealing that sulphur atom of the thiazole ring is not taking part in the coordinate bond formation with the metal ions.

The IR spectra of all complexes show another band in the region 374–378 cm⁻¹, which may be due to M–Cl stretching vibrations. Furthermore, the IR spectra of the Fe(III) show another band in the region 351 cm⁻¹, which may be due to υ(Fe–Cl) vibrations. The IR spectra of the metal complexes also showed some new bands in the region 478–453 and 447–410 cm⁻¹ which may be assigned to ν(M–O) and ν(M–N) bands, respectively. IR spectra of the ligands (L¹ and L²) and metal complexes are presented in Table 2.

4.3 Electronic absorption spectra

The electronic absorption spectra of the ligands and their complexes were recorded in DMSO and presented in Table 3. In the present study, the copper complex exhibited bands at 25, 935 and 20,823 cm⁻¹ which are assigned to ${}^2{\text{B}}_{1\text{g}}\to {}^2{\text{A}}_{1\text{g}}$ transitions characteristic of square planar geometry with $d_{x^2{y}^2}$ ground state (Iskander et al., 2000; Reddy et al., 2000; Lever, 1984 Dutta et al., 1992). The magnetic moment value of 1.82 BM suggested the square-planar geometry around Cu(II) ion.

The absorption spectrum of the nickel complex shows two d-d bands at 20,213 and 24,662 cm⁻¹ which are assigned as ${}^1{\text{A}}_{1\text{g}}\to {}^1{\text{B}}_{1\text{g}}$ and ${}^1{\text{A}}_{1\text{g}}\to {}^1{\text{B}}_{2\text{g}}$ transitions, indicating square planar geometry (Xu et al., 2003). This complex shows the diamagnetic behaviour suggesting the square planar environment around the Ni(II) ion.

The electronic spectrum of Fe(III) complex exhibits bands at 18,832 and 19,064 cm⁻¹ which may be due to ${}^6{\text{A}}_{1\text{g}}\to {}^4{\text{T}}_{1\text{g}}$ (G) and ${}^6{\text{A}}_{1\text{g}}\to {}^4{\text{T}}_{1\text{g}}$ (G) transitions, respectively. These transitions are assigned for octahedral Fe(III) complexes. The electronic transitions together with the magnetic moment value of 5.8 BM suggested the octahedral geometry for the Fe(III) complex.

The spectrum for Co(II) complex shows d–d bands at 17,648 and 32,751 cm⁻¹ may be assigned to ${}^1{\text{A}}_{1\text{g}}\to {}^1{\text{B}}_{1\text{g}}$ and n–π^∗ respectively, in square planar stereochemistry. The magnetic moment value of 4.1 BM indicated the square planar geometry for the Co(II) ion.

Zn(II) is a d¹⁰ metal ion, no band is expected in the visible region and is also found as a diamagnetic complex, as expected. However, a strong band observed at 563 nm is assignable to the L → M charge transfer transition (Temel et al., 2002) which is compatible with this complex having a square planar geometry.

4.4 ESR spectra

ESR spectrum of the copper complex was recorded in DMSO at 300 and 77 K. The observed trend of g_||(2.28) > g_⊥(2.05) > g_e(2.0023) describes the axial symmetry with the unpaired electron residing in the $d_{x^2{y}^2}$ orbital (Hathaway et al., 1970). The value of g_|| < 2.3 in the present copper complex gives a clear indication of a covalent character of the metal–ligand bond and delocalization of the unpaired electron into the ligand.

Researchers reported a good correlation between f factor (f = g_||/A_||) and SOD like activity of copper complexes. The f value for CuZn SOD is 160 cm indicating a tetrahedral distortion from square planar geometry (f value is above 135 cm) and one of the features that enhances the catalytic activity of the enzyme. From the above EPR spectral data, the f values for copper complex is observed at 150 cm. Therefore, our synthesized copper complex exhibits appreciable square planar distortion that is expected to show high SOD like activity. The EPR parameters g_||, g_⊥, A_|| and the energies of d–d transition were used to evaluate the bonding parameters α², β², γ² which may be regarded as measures of the covalency of the in-plane σ bonds, in-plane π bonds and out-of-plane π bonds.

Molecular orbital coefficients α² (covalent inplane σ-bonding), β² (covalent in- plane π- bonding) and γ² (out-plane π -bonding) were calculated using the following equations:

The empirical factor f = g_///A_// cm⁻¹ is an index of tetragonal distortion. Values of this factor may vary from 105 to 135 for small to extreme distortions in square planar complexes and it depends on the nature of the coordinated atoms (Pogni et al., 2000). The f values of complexes are found to be in the range 137–154, indicating significant distortion from planarity. These values are shown in Table 4.

4.5 Mass spectra

The FAB mass spectra of the Schiff base and its metal complexes are used to compare the stoichiometry compositions. The Schiff base ligands L¹ and L² show a molecular ion peak at m/z = 282 and 284. The molecular ion copper complex was observed at m/z = 701 which shows the stoichiometry of the complexes as supported by the FAB mass spectra of other complexes. Elemental analysis values are in close agreement with the values calculated from the molecular form of these complexes, which is further supported by the FAB-mass studies of representative complexes.

4.6 DNA binding studies

4.6.2

4.6.2 Electro chemical studies

In the cyclic voltammetric (CV) study, mixed ligand complexes were performed in the presence and absence of CT DNA and are shown in Fig. 1. From the CV results, it could be found that mixed ligand complexes exhibited a pair of redox peaks for one electron transfer couple of Cu(II)/Cu(I) at the scan rate of +2 to −2 V. The ratio of oxidation peak current to reduction peak current (Ipa/Ipc) of 0.5 and the peak-to-peak separation (ΔEp) of 0.26 suggested the characteristic of the electro-transfer process, and this was fairly common for Cu(II)/Cu(I) couple because of the reorganization of the coordination sphere (Palaniandavar et al., 1995). After interaction with CT DNA, the value of ΔEp was decreased to 0.20 V, suggesting that the reversibility of the electron-transfer process of the copper complex changed better. Both the oxidation and the reduction peak potentials underwent positive shifts accompanied by the decreases of the redox peak currents. The electrochemical potential of the small molecules would shift positively when it intercalated into DNA double helix, and if it is bound to DNA by electrostatic interaction, the potential would shift in a negative direction (Carter et al., 1989). So, the positive shift of the redox potentials implied that the present mixed ligand complex bind to DNA via an intercalation mode. The electrochemical parameters of mixed ligand complexes are shown in Table 5.

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Figure 1

Cyclic voltammogram of [CuL¹L²Cl]Cl] in buffer pH = 7.2 at 25 °C in the presence of increasing amount of DNA.

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Table 5 Electrochemical parameters for the mixed ligand complexes on interaction with CT DNA.

Compound	Redox couple	${}^{\text{a}}{\text{E}}_{1/2}$ (V)		bΔEp(V)		Ipa/Ipc
		Free	Bound	Free	Bound
[CuL1L2Cl]Cl	Cu(II) → Cu(I)	0.301	0.270	0.261	0.213	1.15
[NiL1L2Cl] Cl	Ni(II) → Ni(I)	−0.331	−0.348	−0.435	−0.437	1.09
[CoL1L2Cl] Cl	Co(III) → Co(II)	−0.242	−0.351	0.447	0.501	1.28
[FeL1L2 (Cl)3]	Fe (III) → Fe(II)	−0.218	−0.310	0.439	0.486	1.26
[ZnL1L2Cl] Cl	Zn(II) → Zn(0)	−0.347	−0.353	0.338	−0.310	0.86

4.7 Superoxide dismutase (SOD) mimic activities

Transition metal complexes have received much attention in the development of superoxide dismutase (SOD) mimic activities for scavenging of the superoxide free radical ions ( ${\text{O}}_2^{-·}$ ) (Patel et al., 2006; 2007; 2009). The SOD activity for the complexes was tested using NBT assay. The SOD activities of these complexes have been compared with those of the well known native enzyme (Table 6) (Siddiqi et al., 2009, 2010, 2011; Sorenson, 1984). The IC₅₀ values for Cu(II), Ni(II), Co(II), Fe(III) and Zn(II) complexes are found to be 98, 96, 87, 93, 85 μM respectively, indicating that the copper(II) complexes showed good activity. These values may be due to the strong field created by the Schiff base ligand which opposes the interaction of coordinated copper with ${\text{O}}_2^{-·}$ radical. A greater interaction between superoxide ion and Cu(II) complex is induced due to the stronger axial bond, which results in an increased catalytic activity. In addition azomethine ligands containing electron withdrawing substituent stabilizes the Cu(I) complex formed during superoxide dismutation reaction which further reacts with superoxide ion to give hydrogen peroxide. The difference in reactivities of the synthesized complexes may be attributed to the coordination environment and the redox potential of the couple in metal complexes during the catalytic cycle. It has been reported that the redox potential of copper(II) ions is affected by the preset ligand system. The present Cu(II) complexes have a distorted square planar geometry encouraging a greater accessibility of the superoxide radical anion ( ${\text{O}}_2^{-·}$ ). Generally, metal-catalyzed superoxide dismutation is described by the following two equations:

4.8 Antimicrobial activity

The in vitro biological screening effects of the investigated compounds were tested against the bacterial species and fungal species by the disc diffusion method. The minimum inhibitory concentration (MIC) values of the synthesized compounds are summarized in Tables 6 and 7. A comparative study of the metal salts, ligands and their complexes (MIC values) indicates that complexes exhibit higher antimicrobial activity than the free ligands. The enhanced activity of the complexes can be explained on the basis of Overtone’s concept (Sabrina Belaida et al., 2008) and Tweedy’s Chelation theory (Dharamaraj et al., 2001). According to Overtone’s concept of cell permeability, the lipid membrane that surrounds the cell favours the passage of only the lipid soluble materials which makes liposolubility as an important factor, and which controls the antimicrobial 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 of п-electrons over the whole chelate ring and enhances the lipophilicity of the complexes (Tumer et al., 1999).

The antimicrobial activity of the metal salts was also investigated. It was found that the metal salts exhibit antimicrobial activity at the concentration range used to assay the activity of the complexes but can be toxic. The toxic nature was reduced by the incorporation of suitably substituted ligands instead of chloride ion in the metal salts. This increased lipophilicity enhances the permeation 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 and as a result microorganisms die. The increased activity of the complexes may also be explained on the basis of their high solubility, fitness of the particles, size of the metal ion and the presence of the bulkier organic moieties.

Furthermore, the mode of action of the compounds may involve the formation of hydrogen bonds through the azomethine(C⚌N) group of complexes with the active centres of cell constituents resulting in the interference with normal cell processes. The different lipophilic behaviour of the aromatic residues such as antipyrine, and furfuraldehyde is involved in the biological activity mechanisms. There are other factors which also increase the activity are solubility, conductivity and bond length between the metal and ligand (Mohamed et al., 2009; Mahajan et al., 2009; chohan et al., 2001).