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International Journal of Medicine and Pharmaceutical Sciences (IJMPS) ISSN 2250-0049 Vol. 3, Issue 3, Aug 2013, 115-132 © TJPRC Pvt. Ltd.

A BIRD’S VIEW ON PHARMACOLOGICAL EVALUATION OF TRANSITION METAL (II) COMPLEXES E. AKILA, M. USHARANI, P. JAYASEELAN, R. ASHOKAN & R. RAJAVEL Department of Chemistry, Periyar University, Salem, Tamil Nadu, India

ABSTRACT A novel bioactive Cu(II), Ni(II) and VO(II) complexes have been synthesized with newly synthesized Schiff base(4, 4′- Bis- [1-ethyl-2-(2-mercapto-phenylimino)-butylideneamino]-biphenyl-3, 3′-diol) derived from 3, 3′dihydroxybenzidine, Benzil and 2-aminothiophenol. The elemental analyses of the complexes are confined to the stoichiometry of the type M2L [M = Cu(II), Ni(II) and VO(II)] respectively, where L is Schiff base ligand. Structures have been proposed from elemental analyses, IR, electronic, 1H NMR, ESR spectral data and magnetic studies. The measured low molar conductance values in DMF indicate that the complexes are non-electrolytes. Spectroscopic studies suggest coordination occurs through azomethine nitrogen, oxygen and sulphur of the ligand with the metal ions. The cleavage products of the complexes analyzed by neutral agarose gel electrophoresis indicated that the interaction of the metal complexes with supercoiled plasmid DNA yielded linear, nicked or degraded DNA. The findings suggest that Cu(II) and VO(II) complexes with potent nucleolytic activity is a good nuclease substitute in the presence of cooxidant. The binding of these Cu(II) complex to DNA has been investigated by viscosity measurements. The experiments indicate that all the compounds can bind to DNA through an intercalative mode. In addition, the antioxidative activity was also determined. The antioxidative activity of the ligand and its complexes demonstrates that, compared to the ligand, the complexes exhibit higher scavenging activity. The Schiff base and its complexes have been screened for their antibacterial (Escherichia coli, Staphylococcus aureus, Klebsilla pneumonia, Bacillus subtilis and Salmonella typhi) and antifungal (Aspergillus niger, Aspergillus flavus, Aspergillus fumigates) activities.

KEYWORDS: DNA Interaction, Antioxidative Activity, Biological Evaluation, Spectroscopic INTRODUCTION The interaction and reaction of metal complexes with DNA has long been the subject of intense investigation in relation to the development of new reagents for biotechnology and medicine (Angamuthu Raja et al., 2005). Design of small molecules that are capable of binding and cleaving DNA at specific sites has been an area of considerable interest. Small molecules that bind to DNA have been used as diagnostic probes for both structural and functional aspects of nucleic acid and in the development of new therapeutic agents (Ramasamy Indumathy et al., 2008). Towards this end, for the past 25 years, increasing attention has been towards polynuclear transition metal complexes. These metal complexes are substitutionally inert; possess rich photophysical and electrochemical properties which render them as useful candidates for applications in the fields of molecular biology, biotechnology and medicine (Kollur Shiva Prasad et al., 2011). Transition metal complexes of N and S-donor ligands such as Schiff bases have inspired researchers due to their potent biological activities including antifungal, antibacterial, anticancer and herbicidal applications. Errors in gene expression can often cause disease and play a secondary role in the outcome and severity of human disease. Medicinal agents can affect gene expression by facilitating, mimicking or inhibiting any one of the properties that exists in typical transcriptional systems (Raman et al., 2012). A large number of evidences indicate the mechanism of action of anticancer


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agents binds through distinctive binding modes to the DNA of cancer infected cell in such a way, that the cell cannot replicate further. This inhibition of replication finally leads to the death of the infected cell. The outstanding criteria for the development of metallodrugs as chemotherapeutic agents are the ability of the metallodrug to provoke DNA cleavage. A large number of transition metal complexes because of their redox properties, have been found to enhance DNA cleavage. A huge number of transition metal complexes have been shown to promote oxidative DNA cleavage in the presence of coreagents (Tan et al., 2009). The pharmacological efficacy of metal complexes depends on the nature of the metal ions and the ligands. It is declared in the literature that synthesized from same ligands with different metal ions possess different biological properties. Considerable effort has been devoted to the preparation and structural characterization of Schiff base metal complexes derived from 3, 3′-dihydroxybenzidine, Benzil and 2-aminothiophenol. However, little attention has been paid to systems in which the Schiff bases are derived from 3, 3′-dihydroxybenzidine have been synthesized and structurally characterized. In continuation of our earlier works on 3, 3′-dihydroxybenzidine, herein we describe a successful synthesis of Cu(II), Ni(II), and VO(II) species chelated by 3, 3′-dihydroxybenzidine, Benzil and 2-aminothiophenol. Their spectral, biological activities, DNA binding and cleaving nature are discussed.

EXPERIMENTAL PROTOCOLS Reagents and Instruments The reagents and chemicals were obtained from commercial sources (Sigma–Aldrich, USA, Merck and Loba chemicals, India,). Metal salts, 3, 3′-dihydroxybenzidine, Benzil and 2-aminothiophenol were obtained from Aldrich and used as received. Ethanol, DMSO and DMF were used as solvents purchased from Merck and Loba chemicals. Elemental analysis was carried out on a Carlo Erba Model 1106 elemental analyzer. IR spectra were recorded on a Thermo Nicolet, Avatar 370 model spectrophotometer on KBr disks in the range 4000– 400 cm-1. Molar conductivity was measured by using an ELICO CM 185 conductivity Bridge using freshly prepared solution of the complexes in DMF solution. Electronic spectra were recorded at 300 K on a Perkin-Elmer Lambda 40(UV-Vis) spectrometer using DMF in the range 200-800 nm. Magnetic susceptibility measurements were carried out by employing the Gouy method at room temperature. EPR spectra were recorded on an E-112 ESR spectrometer at X-band microwave frequencies for powdered samples. The 1

H NMR spectra were recorded in DMSO-d6 on a BRUKER ADVANCED III 400 MHz spectrophotometer using TMS as

an internal reference. Synthesis of 4, 4′- Bis- [1-Ethyl-2-(2-Mercapto-Phenylimino)-Butylideneamino]-Biphenyl-3, 3′-Diol The Schiff base ligand was synthesized by adding 3, 3ꞌ-dihydroxybenzidine (1 mM) in 10 mL of ethanol, Benzil (2 mM) and 2-aminothiophenol (2 mM) in 20 mL of ethanol were mixed and heated at reflux for 2 hrs as shown in Figure 1. The resulting yellow color solution was allowed to cool. The yellow color product was obtained and dried in desiccator using silica gel as drying agent. Yield: 85%. M.p:172 ⁰C. Anal. Calc. For C36H38N4O2S2: C, 69.35; H, 6.10; N, 8.99. Found: C, 68.37; H, 8.98; N, 8.98%. UV–Vis [λ (nm), ɛ (M-1 cm-1), (dmf)]: 280 (28, 000) (π→ π*), 365 (36,500)( n→ π*). IR (KBr, cm-1): 1609 ν(C=N), 3360 ν (O-H), 730 ν(C-S). Methodology for the Synthesis of Cu2 [C36H34N4O2S2] The Schiff base 4, 4′- Bis- [1-ethyl-2-(2-mercapto-phenylimino)-butylideneamino]-biphenyl-3, 3′-diol (1 mM) dissolved in boiling absolute ethanol (20 ml) was added to a hot solution of Copper(II) acetate salts (2mM) in ethanol. The mixture was heated with continuous stirring for 20 min and refluxed for 3 hrs on water bath. The mixture was then allowed


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to stand overnight whereupon a dark coloured precipitate obtained was filtered, washed with ethanol and dried in vacuo. Yield: 75%. M.p:>200 ⁰C. Elemental analyses, Anal. Calc. For Cu2[C36H34N4O2S2] : C, 57.91 ; H, 4.55; N, 7.50. Found: C, 57.90; H, 4.51; N, 7.52%. UV–Vis [λ(nm),ɛ(M-1 cm-1),(dmf)]: 275 (27,500)( π→ π*), 355 (35,500)( n→ π*), 480 (48,000) )( L→ M), 590 (59,000) (d→d). IR (KBr, cm-1): 1605 ν(C=N), 710 ν(C-S), 460 ν(M-N), 530 ν(M-O). Methodology for the Synthesis of Ni2 [C36H34N4O2S2] 20 ml ethanolic solution of 4, 4′- Bis- [1-ethyl-2-(2-mercapto-phenylimino)-butylideneamino]-biphenyl-3, 3′-diol (1 mM) was added to a absolute ethanolic (20 ml) hot solution of Nickel(II) acetate salts (2mM). The mixture was heated with continuous stirring for 20 min and refluxed for 3 hrs on water bath. The mixture was then allowed to stand overnight whereupon a brownish black coloured precipitate obtained was filtered, washed with ethanol and dried in vacuo. Yield: 75%. M.p:>200 ⁰C. Elemental analyses, Anal. Calc. For Ni2[C36H34N4O2S2]: C, 58.68; H, 4.61 ; N, 7.60. Found: C, 58.69; H, 4.60; N, 7.61%. UV–Vis [λ(nm),ɛ(M-1 cm-1),(dmf)]: 280 (28,000)( π→ π*), 370 (37,000)( n→ π*), 475 (47,500) )( L→ M), [560 (56,000), 632(63,200)] (d→d). IR (KBr, cm-1): 1607 ν(C=N), 744 ν(C-S), 454 ν(M-N), 544 ν(M-O). Methodology for the Synthesis of VO2 [C36H34N4O2S2] 4, 4′- Bis- [1-ethyl-2-(2-mercapto-phenylimino)-butylideneamino]-biphenyl-3, 3′-diol (1 mM) dissolved in boiling absolute ethanol (20 ml) was added to a hot solution of Vanadium(II) acetate salts (2mM) in ethanol. The mixture was heated with continuous stirring for 20 min and refluxed for 3 hrs on water bath. The mixture was then allowed to stand overnight whereupon a dark coloured precipitate obtained was filtered, washed with ethanol and dried in vacuo. Yield: 70%. M.p:>200 ⁰C. Elemental analyses, Anal. Calc. For VO2 [C36H34N4O2S2]: C, 57.39; H, 4.51; N, 7.43. Found: C, 57.38; H, 4.50; N, 7.40%. UV–Vis [λ(nm),ɛ(M-1 cm-1),(dmf)]: 273 (273,00)( π→ π*), 380 (380,00)( n→ π*), 485 (485,00) )( L→ M), [522 (522,00), 580 (580,00), 630 (630,00)] (d→d). IR (KBr, cm-1): 1595 ν(C=N), 752 ν(C-S), 468 ν(M-N), 560 ν(M-O), 990 ν(V=O). C2 H5 H2 N

NH2

C 2 H5

NH2

+

2

+ O

HO

2

O

SH

OH

Benzil

3, 3' -Dihydroxybenzidine

2-aminothiophenol

Reflux for 2hrs C 2 H5

C 2 H5

C 2 H5

N

C2 H5

N

N

N

OH

HO SH

HS

4,4'-Bis-[1-ethyl-2-(2-mercapto-phenylimino)-butylideneamino]-biphenyl-3,3'-diol Metal salts Stirred for 20 min C2 H5

C2 H 5

N

Reflux for 3hrs

C2 H5

N

N

N

M

M S

C2 H 5

O

O

S

M=Cu(II), Ni(II) & VO(II)

Figure 1: Synthesis Pathway for Schiff Base Ligand and its Binuclear Schiff Base Metal Complexes


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Antibacterial Antifungal Screening In vitro antimicrobial screening is performed by the agar disc diffusion method (Moamen S. Refata et al., 2012). The species used in the screening are; Bacteria as: Escherichia coli, Staphylococcus aureus, Klebsilla pneumonia, Bacillus subtilis and Salmonella typhi. Fungi as: Aspergillus niger, Aspergillus flavus, Aspergillus fumigates. Stock cultures of the tested organisms are maintained on nutrient agar media by sub culturing in Petri dishes. The media are prepared by adding the components as per manufacturer’s instructions and sterilized in the autoclave at 121 ⁰C and atmospheric pressure for 15 min. Each medium is cooled to 45–60 ⁰C and 20 ml of it, is poured into a Petri dish and allowed to solidify. After solidification, petri plates with media are spread with 1.0 ml of bacterial or fugal suspension prepared in sterile distilled water. The wells are bored with cork borer and the agar plugs are removed. To each agar well, unique concentration of 100 µg for each compound in DMF (75 µl) were applied to the corresponding well (6 mm). All the plates are incubated at 37 ⁰C for 24 h and they are observed for the growth inhibition zones. The presence of clear zones around the wells indicates that the ligand and its complexes are active. The diameter of zone of inhibition is calculated in millimeters. The well diameter is deducted from the zone diameter and the values are tabulated (Ekamparam Akila et al., 2013). Antioxidant Activity The antioxidant activity assay employed is a technique depending on measuring the consumption of stable free radicals i.e. evaluate the free radical scavenging activity of the investigated component. The methodology assumes that the consumption of the stable free radical (X′) will be determined by reactions as follows: X′+ YH → XH +Y′ The rate or the extent of the process measured in terms of the decrease in X′ concentration would be related to the ability of the added compounds to trap free radicals. The decrease in colour intensity of the free-radical solution due to scavenging of the free radical by the antioxidant material is measured at a specific wavelength. DPPH Free Radical Scavenging Activity The hydrogen atom or electron donation ability of the corresponding compounds was measured from the bleaching of purple colored of the DMF solution of DPPH. This spectrophotometric assay uses the stable radical diphenylpicrylhydrazyl (DPPH) as a reagent (Kavitha et al., 2013). Different concentrations (200, 400, 600, 800, 1000 µg/ mL) of the chemical compounds were dissolved in DMF to determine IC50 (concentration makes 50% inhibition of DPPH color). Fifty microliters of various sample concentrations were added to 5 ml of 0.004% DMF solution of DPPH. After a 60 min of incubation at dark, the absorbance was read against a blank at 517 nm. ABTS Free Radical Scavenging Activity The advantage of ABTS-derived free radical method over other methods is that the produced color remains stable for than one hour, and the reaction is stoichiometric (Charikleia Tolia et al., 2013). For each of the investigated compounds with different concentrations (200, 400, 600, 800, 1000 µg/ mL), 2 ml of ABTS solution (60 mM) was added to 3 ml MnO2 solution (25 mg/ml), all prepared in 5 ml aqueous phosphate buffer solution (pH 7, 0.1 M). The mixture was shaken, centrifuged, filtered and the absorbance of the resulting green–blue solution (ABTS radical solution) at 734 nm was adjusted to approx. 0.5. Then, 50 ml of (2 mM) solution of the tested compound in spectroscopic grade MeOH/ phosphate buffer (1:1) was added. The absorbance was measured, and the reduction in color intensity was expressed as inhibition percentage. L-ascorbic acid was used as a standard antioxidant (positive control).


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Ferric Reducing Antioxidant Power Assay Ferric reducing antioxidant power (FRAP) was measured by a modified method of Benzie and Strain (Benziea et al., 1996). The antioxidant potentials of the compounds were estimated as their power to reduce the TPTZ-Fe(III) complex to TPTZ-Fe(II) complex (FRAP assay), which is simple, fast, and reproducible. FRAP working solution was pre-pared by mixing a 25.0 ml, 10 mm TPTZ solution in 40 mm HCl, 20 mm FeCl 3.6H2O and 25 ml, 0.3 M acetate buffer at pH 3.6. A mixture of 40.0 ml, 0.5 mm sample solution with different concentrations (200, 400, 600, 800, 1000 µg/ mL) and 1.2 ml FRAP reagent was incubated at 37°C for 15 mins. Absorbance of intensive blue colour [Fe(II)-TPTZ] complex was measured at 593 nm. The ascorbic acid was used as a standard antioxidant com-pound. The results are expressed as ascorbic equivalent (mmol/100 g of dried compound). All the tests were run in triplicate and are expressed as the mean and standard deviation (SD). Agarose Gel Electrophoresis Agarose gel electrophoresis was used to study the DNA cleavage activity of the complexes. pUC18 plasmid was cultured, isolated and used as DNA for the experiment. The gel electrophoresis experiments were performed by incubation of the samples containing 40 μM pUC18 DNA, 50 μM metal complexes and 50 μM H 2O2 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 acidEDTA buffer (pH 7.2). The gel was then stained using 1 μg cm-3 ethidium bromide (EB) and photographed under ultraviolet light at 360 nm. All the experiments were performed at room temperature (Nahid Shahabadi et al., 2010). DNA-Binding Viscosity Measurements Viscosity experiments were carried on an Ostwald viscometer, immersed in a thermostated water-bath maintained at a constant temperature at 30.0±0.1 ⁰C. CT DNA samples of approximately 0.5mM were prepared by sonicating in order minimize complexities arising from CT DNA flexibility (Raman et al., 2010). Flow time was measured with a digital stopwatch three times for each sample and an average flow time was calculated. Data were presented as (η/ηo)1/3 versus the concentration of the metal(II) complexes, where η is the viscosity of CT DNA solution in the presence of complex, and ηo is the viscosity of CT DNA solution in the absence of complex. Viscosity values were calculated after correcting the flowtime of buffer alone (t0), η = (t−t0)/t0.

RESULTS AND DISCUSSIONS The Schiff base ligand and its Cu(II), Ni(II) and VO(II) complexes have been synthesized and characterized by spectral and elemental analytical data of the complexes are summarized in Table 1. They are found to be air stable. The ligand is soluble in common organic solvents but the complexes are soluble only in DMF and DMSO. The analytical data of the complexes correspond well with the general formula M 2L where M= Cu (II), Ni (II), and VO (II); L = ligand. The bimeric nature of the complexes was confirmed from their magnetic susceptibility data. The lower conductance values of the complexes support their non electrolytic nature and are consistent with other related complexes. Table 1: Analytical Data of the Schiff Base Ligand and its Binuclear Metal Complexes Compounds

Molecular Formula

L

C36H38N4O2S2

[Cu2L]

Cu2[C36H34N4O2S2]

Yield %

Melting Point (º)

Yellow

85

172

Greenish blue

75

>200

Color

Calculated (Found) (%) C H N Metal 69.35 6.10 8.99 -(68.37) (6.08) (8.98) 57.91 4.55 7.50 17.03 (57.90) (4.51) (7.52) (17.02)

scm2 mol-1 -112


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Table 1: Contd., [Ni2L]

Ni2[C36H34N4O2S2]

[VO2L]

VO2[C36H34N4O2S2]

Brownish black Blackish blue

75

>200

70

>200

58.68 (58.69) 57.39 (57.38)

4.61 (4.60) 4.51 (4.50)

7.60 (7.61) 7.43 (7.40)

15.94 (15.92) 15.65 (15.63)

134 150

Molar Conductivity Measurements The chelates were dissolved in DMF and the molar conductivities of their 10 -3 M solutions at 25 ⁰C were measured. The molar conductance values of the complexes are given in the Table 1. The molar conductivity values of Cu(II), Ni(II) and VO(II) complexes were found to be in the range of 112- 150 scm2 mol-1. These relatively low values indicate the non-electrolytic nature of these complexes (Moamen et al., 2012). The neutrality of the complexes can be accounted by both the deprotonated nature of the ligand with most complexes. IR Spectroscopy The significant IR absorption bands of the prepared ligand and its Cu(II), Ni(II) and VO(II) complexes are given in the Table 2. The characteristic absorption bands of the ligand are shifted on complex formation and new characteristic vibrational bands of the complexes appear. The IR spectrum of the ligand did not display a characteristic band for a C=O group after the condensation reaction of Benzil with 3, 3′-dihydroxybenzidine and 2-aminothiophenol. This result indicates that the formation of Schiff base was completed. The medium band located in the range between 1609 cm-1 is assigned to the υ(C=N) stretching vibration of the azomethine group of the ligand. This band is shifted by 5–10 cm-1 to lower wavenumber after complexation. These shifts to lower wavenumbers support the participation of the azomethine group of this ligand in binding to the metal ion (Umemura et al., 2008). This can be explained by the donation of electrons from the nitrogen to the empty d-orbitals of the metal atoms. In the FT-IR spectrum of the ligand, a broad band at 3350 cm-1 was assigned to the O–H stretching vibration of the 4, 4′- Bis- [1-ethyl-2-(2-mercapto-phenylimino)-butylideneamino]-biphenyl-3, 3′-diol. The deprotonation of the O–H is indicated by the absence of a band in the metal complexes at 3350 cm-1. It has been reported that oxovanadium complexes with coordination number 5 have υ(V=O) values higher than and lower than about 992 cm−1, respectively (Akbar Ali et al., 1978). The coordination mode of the ligand with the metal ion is further supported by new frequencies occurring in the 530–560 and 454–468 cm−1 ranges, which have been assigned tentatively to υ(M–O) and υ(M–N), respectively (Emam et al., 2012; Sartaj Tabassum et al., 2012). Table 2: Infrared Spectroscopic Data of the Schiff Base Ligand and its Binuclear Metal Complexes Compounds

Free-OH

C36H38N4O2S2 Cu2[C36H34N4O2S2] Ni2[C36H34N4O2S2] VO2[C36H34N4O2S2]

3350 ----

(C=N) (cm−1) 1609 1605 1601 1595

(C-S) (cm−1) 730 710 744 752

(V=O) (cm−1) 992

(M−N) (cm−1) -460 454 468

(M−O) (cm−1) -530 544 560

Electronic Absorption Spectra The electronic absorption spectra can often provide quick and reliable information about the ligand arrangement in transition metal complexes. It also serves as a useful tool to distinguish among the square-planar, octahedral or tetrahedral geometries of the complexes. The absorptions in the ultraviolet region are attributed to transitions within the ligand orbital and those in the visible region are probably due to allowed metal-to-ligand charge transfer transitions. The electronic absorption spectra of the ligand and its Cu(II), Ni(II) and VO(II) complexes were recorded in DMSO at 300K respectively. The bands appearing at the lower energy side are attributable to n→π * transitions associated with the


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azomethine chromophores. The bands at higher energy arise from π→π* transition within the aromatic rings. The absorption bands of the complexes are shifted to longer wavelength region compared to those of the ligand. A moderative intensive band observed in the region of 475- 485 nm is attributable to the LMCT transitions of ligand, complexes respectively. This shift may be attributed to the donation of the lone pairs of electron on the nitrogen atoms of the Schiff base to the metal ion (M←N). In the electronic absorption spectrum of the present copper complex shows two d–d transitions at 590 nm which can be assigned to 2B1g → 2A1g transition respectively. It reveals that the copper(II) complex exists in square planar geometry (Raman et al., 2012). The diamagnetic complex Ni(II) displayed two bands at 560 and 632 nm regions assigned to 1A1g → 1A2g and 1A1g → 1B1g transitions, respectively, consistent with a square planar Ni(II) complex (Claudio Mendicute Fierro et al., 2011). The d-d bands of VO(II)complexes in the visible region at 522- 630 nm, which according to the scheme of Ballhausen and Gray are assigned to dxy→dxz, dyz, dxy→dx2−dz2 and dxy→dz2 (Syamal et al., 1977). The observed results (Table 3) indicate that the present oxovandium(IV) complexes are square-pyramidal geometry. Table 3: UV–Visible Data of Schiff Base and its Binuclear Metal Complexes Compounds C36H38N4O2S2 Cu2[C36H34N4O2S2] Ni2[C36H34N4O2S2] VO2[C36H34N4O2S2] 1

π→ π* 280 275 280 273

Absorption nm n→ π* L→ MCT 365 -355 480 370 475 380 485

d-d -590 560, 632 522, 580, 630

Geometry of the Complex -Square planar Square pyramidal

H NMR Spectra of Organic Compounds A literature survey reveals that the NMR spectroscopy has been proved useful in establishing the structure and

nature of many ligand. The 1H NMR spectra of 4, 4′- Bis- [1-ethyl-2-(2-mercapto-phenylimino)-butylideneamino]biphenyl-3, 3′-diol were recorded in d6-dimethylsulfoxide (DMSO-d6) solution using Me4Si (TMS) as internal standard. The aromatic protons at δ(6.30–8.70) shifted downfield in the complexes. The signal at δ (12.70) (s, 1H) is assigned to thiophenolic proton of (–SH–) group and the signal at δ (8.16) (s, 1H) is assigned to azomethine proton, respectively, in the Schiff’s base ligand. Thus, the 1H NMR result supports the assigned geometry (Mustafa Dolaz et al., 2009). Magnetochemistry Magnetic susceptibility measurements of the complexes were carried out in the solid state at room temperature and provide information regarding their structures. These measurements in the solid state show that the binuclear Cu (II) and VO (II) complexes are paramagnetic and Ni (II) complex is diamagnetic at ambient temperature. Copper complex show µeff value in the range 1.72 B.M. which is close to the spin only value of 1.73 B.M. The lower value of magnetic moment at room temperature is consistent with square planar geometry around the metal ions (Jane N. Mugo et al., 2010). The nickel complexes are diamagnetic in nature due to square planar (Ahmed A. El-Asmy et al., 2010) geometry around the metal ion. The room temperature value of VO (II) ion for the complex is 1.70 B.M. It is possible that the oxovanadium(IV) complexes have square-pyramidal geometry (Leelavathy et al., 2009). 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. Electronic Spin Resonance Spectral Studies The ESR spectrum of Cu (II) complex provides information about hyperfine and super hyperfine structures which are important in studying the metal ion environment, i.e. the geometry, nature of the ligation sites from the Schiff bases to the metal and the degree of covalency of the metal ligand bonds.


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The EPR spectrum pattern of the solid copper (II) complex at room temperature exhibits an axial type symmetry, where g|| > g┴ > 2.0023, indicating d(x2−y2) ground state for square planar copper(II) complex. The spectrum of the Cu (II) complex shows a well-defined g┴ and g║ values at 2.472 and 2.092, respectively. The geometric parameter G, which is a measure of the exchange interaction between the copper centres, is calculated using the equation: G = (g|| -2)/ (g┴ –2). If G < 4.0 considerable exchange interaction is indicated in the solid complex and the value is greater than 4 which is indicative of absence of exchange interaction between the Cu(II) ions (Ekamparam Akila et al., 1983). In the copper (II) complexes g|| > g┴ > 2.0023 and G values are consistent with a dx2-y2 ground state. These observation leads to the confirmation of square planar geometry around the metal ion. Molecular Modelling The modern foremost advances in the computational chemistry tools provides an alternative, approximate, approach for obtaining the 3D dimensional structures of the complexes in the case of absence of X-ray crystal structure. The optimized geometry of the Binuclear Schiff base Cu2 [C36H34N4O2S2] complex is represented (Figure 2) with some of selected structural parameters (bond length and bond angles). The values of the bond length (Å) and angles (˚) are shown in table 4 & 5. Four NNOS donor atoms from the approximately planar Schiff base ligand (binding via two imine nitrogen, thiophenolic sulphur and phenolic oxygen) and it forms tetradentate environment around the two Cu atoms. According to these parameters in the table we could determine each Cu atom is surrounded by a square planar formed by the two azomethine nitrogen, one thiophenolic sulphur and one phenolic oxygen group respectively (Maurya et al., 2008).

Figure 2: 3D Structure of Compound of [Cu2 (C34H30N4O2S2] Complex Table 4: Various Bond Lengths of [Cu2 (C34H30N4O4)] Complex Atoms N(8)-Cu(45) N(14)-Cu(46) N(19)-Cu(45) N(20)-Cu(46) S(33)-Cu(45) O(34)-Cu(45) O(35)-Cu(46) S(36)-Cu(46)

Actual Bond Length 1.846 1.846 1.846 1.846 1.884 1.872 1.797 2.202

Table 5: Various Bond Angles of [Cu2 (C34H30N4O4)] Complex Atoms C(2)-N(8)-Cu(45) C(16)-N(8)-Cu(45)

Actual Bond Angle 111.000 111.000

Atoms N(8)-Cu(45)-N(19) N(8)-Cu(45)-S(33)

Actual Bond Angle 104.501 99.066


A Bird’s View on Pharmacological Evaluation of Transition Metal (II) Complexes

C(11)-N(14)-Cu(46) C(15)-N(14)-Cu(46) C(18)-N(19)-Cu(45) C(25)-N(19)-Cu(45) C(17)-N(20)-Cu(46) C(27)-N(20)-Cu(46) C(24)-S(33)-Cu(45) C(3)-O(34)-Cu(45) C(10)-O(35)-Cu(46) C(28)-S(36)-Cu(46)

Table 5: Contd., 111.000 N(8)-Cu(45)-O(34) 111.000 N(19)-Cu(45)-S(33) 111.000 N(19)-Cu(45)-O(34) 111.000 S(33)-Cu(45)-O(34) 111.000 N(14)-Cu(46)-N(20) 111.000 N(14)-Cu(46)-O(35) 94.985 N(14)-Cu(46)-S(36) 110.227 N(20)-Cu(46)-O(35) 114.324 N(20)-Cu(46)-S(36) 90.815 O(35)-Cu(46)-S(36)

123

87.405 90.012 139.938 126.333 104.499 84.671 160.085 119.008 80.707 109.942

Antibacterial Antifungal Activity The biological activity for H2L ligand and its Cu (II), Ni (II) and VO (II) complexes are tested against different organisms as gram-negative, gram positive and fungi using different concentrations. The zone of inhibition against the growth of microorganisms for the compounds is shown in the Figure 3, 4, 5, 6, 7, 8 & 9. It has been suggested that the ligand with the N, O and S donor system might have inhibited enzyme production, since enzymes which require free hydroxyl groups for their activity appearance to the especial susceptibility to deactivation by the ions of the complexes. The complexes facilitate their diffusion through the lipid layer of spore membranes to the site of action ultimately killing them by combining with OH groups of certain cell enzymes. The variation in the effectiveness of different biocidal agents against different organisms depends on the impermeability of the cell. Chelation reduces the polarity of the central metal atom, mainly because of partial sharing of its positive charge with the ligand. Also, the normal cell process may be affected by the formation of hydrogen bond, through the azomethine nitrogen atom with the active centers of cell constituents. The chosen compounds used for this investigation are as example only for this study. Also, the absence of bulkiness observed around the metal ions which may permit their interaction easily with the cell enzymes is considered the main cause for the choice. From the results, it is clear that Cu(II)–H2L and VO(II)–H2L complex exhibits inhibition towards all the studied microorganisms and however Ni(II)–H2L complex exhibits less inhibition. From structure point of view of the prepared compounds with their effects on microbial test. It is clear that the formation of the chelate derived 2:1 molar ratio (M: L) sometimes increase the biological activities of the complex compared to the free ligand. This is may be attributed to the presence of N, O and S groups around the two central atoms arising the inhibition activity of the central atoms which is not the only responsible for the biological activity of its complex. Some metal complexes can enhance the activity and others can reduce this activity with respect to the parent ligand (Chinnasamy Jayabalakrishnan et al., 2002; Azza A.A. AbouHussein et al., 2012).

Figure 3: Difference between the Anti-Bacterial Activities of the Schiff Base and its Binuclear Metal Complexes against Staphylococcus aureus


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Figure 4: Difference between the Anti-Bacterial Activities of the Schiff Base and its Binuclear Metal Complexes against Bacillus subtilis

Figure 5: Difference between the Anti-Bacterial Activities of the Schiff Base and its Binuclear Metal Complexes against Escherichia coli

Figure 6: Difference between the Anti-Bacterial Activities of the Schiff Base and its Binuclear Metal Complexes against Klebsilla pneumonia


A Bird’s View on Pharmacological Evaluation of Transition Metal (II) Complexes

Figure 7: Difference between the Anti-Bacterial Activities of the Schiff Base and its Binuclear Metal Complexes against Salmonella typhi

Figure 8: Difference between the Anti-Fungal Activities (against Aspergillus niger) of the Schiff Base and its Binuclear Metal Complexes against Fungi. Standard = Clotrimazole Inhibition Zone in mm, Concentration 100 µg/mL

Figure 9: Difference between the Anti-Fungal Activities (against Aspergillus flavus) of the Schiff Base and its Binuclear Metal Complexes against Fungi. Standard = Clotrimazole Inhibition Zone in mm, Concentration 100 µg/mL

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Figure 10: Difference between the Anti-Fungal Activities (against Aspergillus fumigates) of the Schiff Base and its Binuclear Metal Complexes against Fungi. Standard = Clotrimazole Inhibition Zone in mm, Concentration 100 µg/mL Antioxidant Activity DPPH Radical Scavenging Activity The antioxidant activity of the ligand and its complexes have attracted increasing interests and been substantially investigated (Akila Ekamparam et al., 2013). Figure 11 shows the plots of DPPH● free radical scavenging activity for the ligand and its Cu(II), Ni(II) and VO(II) complexes. It is obvious that the scavenging activity increases with increasing sample concentration in the range tested. As shown in the Figure, the free ligand L has less scavenging activity than the complexes within the investigated concentration range due to the OH groups which can react with DPPH radical by the typical H-abstraction reaction to form a stable radical. As shown in Figure 11 Cu(II) complex possess more efficient activity in quenching DPPH• radical than the free ligand L consistence with previously reported data for other elements. When the ligand L interacts with the positively charged metal ions, electron density is drawn from the oxygen. This flow of electron density causes the O–H bond to become more polarized; as a result, the H atom have a greater tendency to ionize than those in the free ligand L not bound to the metal ions in DMF and thus increases the possibility of scavenging ability. Cu(II) and VO(II) complexes displayed the highest scavenging activities than the Ni(II) complex may be due to the ionic size effect. This indicates that the DPPH● scavenging activity of the metal complexes depends on the central ion.

Figure 11: DPPH Scavenging Activity of 4, 4′- Bis- [1-Ethyl-2-(2-Mercapto-Phenylimino)Butylideneamino]-Biphenyl-3, 3′-Diol and its Complexes


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Figure 12: DPPH Scavenging Capacities (IC50) of Metal Complexes FRAP Assay A capacity to transfer a single electron i.e. the antioxi- dant power of all compounds was determined by a FRAP assay. The FRAP value was expressed as an equivalent of standard antioxidant ascorbic acid (mmol/100g of dried compound). FRAP values indicate that all the compounds have a ferric reducing antioxidant power (Ketan S. Patel et al., 2012). The compounds Cu (II) and VO (II) showed relatively high antioxidant activity than the Ni(II) complex (Figure 13).

Figure 13: FRAP Assay of 4, 4′- Bis- [1-Ethyl-2-(2-Mercapto-Phenylimino)Butylideneamino]-Biphenyl-3, 3′-Diol and its Complexes ABTS Radical Scavenging Activity The scavenging activity of the ABTS radical cation (ABTS•+) may be related to the magnitude of the total antioxidant activity of the compounds (Charikleia Tolia et al., 2013). The ABTS radical scavenging activity of the compounds was moderate to high in comparison to that of the reference compound Gallic acid. All complexes show higher scavenging activity of ABTS•+ than free 4, 4′- Bis- [1-ethyl-2-(2-mercaptophenylimino)-butylideneamino]-biphenyl-3, 3′-diol. These results revealing that compounds Cu (II) and VO (II) exhibits the highest scavenging activity of the ABTS radical cation among the complexes.


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Figure 14: ABTS Free Radical Scavenging Assay of 4, 4â&#x20AC;˛- Bis- [1-Ethyl-2-(2-MercaptoPhenylimino)-Butylideneamino]-Biphenyl-3, 3â&#x20AC;˛-Diol and its Complexes DNA Cleavage Studies The representative Cu (II), Ni (II) and VO (II) complexes were studied for their DNA cleavage activity by the agarose gel electrophoresis method, as shown in Figure 15. Control experiments clearly revealed that the untreated DNA and DNA with H2O2 do not show any cleavage, indicating non-involvement of the hydroxyl radical in the cleavage reaction, whereas the complexes exhibit cleavage activity on DNA (Vivian Chagas da Silveir et al., 2008). Compound [Ni2L] showed less effect through the incubation period, whereas compounds 3 and 5 exhibited a degradation effect on the DNA, as judged by the rate of DNA migration in the gel. Probably this may be due to the formation of a redox couple of the metal ion and its behaviour. The observed lower cleavage activity of [Ni2L] could be related to their poor binding ability to DNA. The results indicate the important role of metal ions in isolated DNA cleavage reactions. The metal complexes were able to convert supercoiled DNA into open circular DNA. The proposed general oxidative mechanisms and account of DNA cleavage by hydroxyl radicals via abstraction of a hydrogen atom from sugar units, which predict the release of specific residues arising from the transformed sugars, depend on the position from which the hydrogen atom is removed. The cleavage is inhibited by free radical scavengers implying that the hydroxyl radical or peroxy derivatives mediate the cleavage reaction. The reaction is modulated by a metallo complex bound hydroxyl radical or a peroxo species generated from the co-reactant H2O2. According to these results, we infer that complexes [Cu2L] and [VO2L] act as a potent nuclease agents. As the compounds were observed to cleave DNA, it can be concluded that the compounds inhibit the growth of the pathogenic organism by cleaving the genome. We have observed a significant effect of conjugation in the sulphur containing Schiff base, reducing the cleavage efficiency. The results are of importance in designing metal-based complexes containing sulfur containing ligands for pharmacological applications (Neelakantan et al., 2008).

Figure 15: Changes in the Agarose Gel Electrophoretic Pattern of pUC18DNA Induced by H 2O2 and Metal Complexes: Lane 1, DNA Alone; Lane 2, DNA Alone + H2O2; Lane 3, DNA + Cu2 [C36H34N4O2S2] Complex + H2O2; Lane 4, DNA + Ni2 [C36H34N4O2S2] ComplexH2O2; Lane 5, DNA + VO2 [C36H34N4O2S2] Complex + H2O2


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DNA Binding Viscosity Measurements To further clarify the binding modes of the [Cu2L] with DNA, viscosity measurements were carried out. Hydrodynamic measurements that are sensitive to length change (i.e., viscosity) are regarded as the least ambiguous and the most critical tests of the binding model in solution. A classical intercalation model resulted in the lengthening of the DNA helix as the base pairs were separated to accommodate the binding complex, leading to an increase in DNA viscosity. In contrast, a partial, non-classical intercalation could bend (or kink) the DNA helix and reduce its effective length and, concomitantly, its viscosity. There is a marked effect of [Cu2L] on the viscosity of CT-DNA as shown in Figure 6. With an increasing amount of DNA with different concentration (20µM, 40 µM, 60 µM and 80 µM) increases the effective length of DNA which supports that [Cu2L] bind through intercalation mode but with different affinity, i.e., also show some affinity for binding with grooves of DNA through hydrogen bonding. However, strong binding is presumably due to intercalation with DNA (Kollur Shiva Prasad et al., 2011).

Figure 16: Viscosity Measurements: The Effect of the Increasing Amount of [Cu2 (L)] Complex on the Relative Viscosity of DNA at 27±0.1°C (20 µM, 40 µM, 60 µM, 80 µM)

CONCLUSIONS On the basis of various physico-chemical and spectral data presented and discussed above, the complexes may tentatively be suggested to have square planar geometry except VO(II) which was proposed to square-pyramidal geometry. The therapeutic promise of the investigated metal (II) complexes were found to exhibit higher antimicrobial activity than the ligand. The DNA-binding properties revealed that the [Cu2L] complex effectively bind with DNA and suggested an intercalative binding mode of the complex. The interaction of these complexes with CT-DNA was investigated by gel electrophoresis and concluded that VO (II) and Cu (II) complexes cleave DNA in the presence of H2O2, whereas the control DNA and Ni (II) complex are less effective. Moreover, the ligand and its complexes were screened for antioxidant activity using DPPH, FRAP and ABTS assay. The relation between the structure of the studied compounds and their activity was discussed. The compounds are ordered as: H2L > Ni (II) complex > VO (II) complex > Cu (II) complex for the antioxidant activity using DPPH, FRAP and ABTS comparable to ascorbic acid.

ACKNOWLEDGEMENTS The authors owe a favour to our Supervisor for his encouragement and support. This work was supported by a Grant from the DST (Department of Science and Technology), New Delhi.


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