Synthesis, Characterization, DNA Interaction and Pharmacological Studies of Substituted Benzophenone Derived Schiﬀ Base Metal(II) Complexes

Journal of Saudi Chemical Society (2015) 19, 207–216

King Saud University Journal of Saudi Chemical Society

www.ksu.edu.sa www.sciencedirect.com

ORIGINAL ARTICLE Synthesis, characterization, DNA interaction and pharmacological studies of substituted benzophenone derived Schiﬀ base metal(II) complexes

P. Subbaraj a, A. Ramu b, N. Raman c,*, J. Dharmaraja d a Department of Chemistry, Devanga Arts College (Autonomous), Aruppukottai 626 101, Tamil Nadu, India b Department of Inorganic Chemistry, School of Chemistry, Madurai Kamaraj University, Madurai 625 021, Tamil Nadu, India c Research Department of Chemistry, V.H.N.S.N. College(Autonomous), Virudhunagar 626 001, Tamil Nadu, India d Department of Chemistry, Sree Sowdambika College of Engineering, Aruppukottai 626 134, Tamil Nadu, India

Received 11 March 2014; revised 6 May 2014; accepted 15 May 2014 Available online 24 May 2014

KEYWORDS Abstract A new bidentate NO type Schiff base ligand (HL), derived from 2-hydroxy-4-methoxy- Benzophenone; phenyl)phenylmethanone with aniline and its metal(II) [M = Mn, Co, Ni, Cu and Zn] complexes Schiff base; has been synthesized. The synthesized ligand and the metal(II) complexes were structurally charac- 1 Metal(II) complexes; terized by analytical, spectral (FT-IR, UV–vis., H NMR, FAB-Mass, TGA/DTA and EPR) as well XRD, SEM; as molar conductance and magnetic studies. All the complexes are non-electrolytes having 1:2 DNA cleavage stoichiometry. They adopt tetrahedral and octahedral geometry. Thermal behavior of metal(II) complexes (1a–1c) shows loss of coordinated water molecules in the first step followed by the decomposition of ligand moieties in a respective manner and leads to form an air stable metal oxide as the final residue. Micro crystalline nature and the presence of coordinated water molecules have been confirmed by powder XRD, SEM and thermal analyses. The ligand and its complexes have efficient bio-efficacy, DNA binding and cleavage ability. ª 2014 King Saud University. Production and hosting by Elsevier B.V. All rights reserved.

Abbreviations: CT-DNA, calf thymus deoxyribonucleic acid; DPPH, 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl; EB, ethidium bromide; 1. Introduction

EDS, energy dispersive spectrometry; Kb, intrinsic binding constant; pUC-19 DNA, plasmid University of California 19 deoxyribonucleic Recently in the treatment of cancer with a chemotherapeutic acid; SEM, scanning electron microscopy; TGA, thermogravimetric approach DNA is the target molecule. Cisplatin and its analysis. derivatives are widely used as anticancer drugs but they create * Corresponding author. Tel.: +91 92451 65958; fax: +91 4562 several side effects such as anemia, diarrhea, alopecia, pete- 281338. E-mail address: [email protected] (N. Raman). chia, fatigue nephrotoxicity, emetogenesis, ototoxicity, and neurotoxicity. As a result, bioinorganic chemists are focussing Peer review under responsibility of King Saud University. their attention on the design and synthesis of novel dynamic metal complexes from bioactive ligands. The Schiff base ligands are derived by the condensation of aldehyde/ketone Production and hosting by Elsevier with a primary amine, which play a pivotal role in human http://dx.doi.org/10.1016/j.jscs.2014.05.002 1319-6103 ª 2014 King Saud University. Production and hosting by Elsevier B.V. All rights reserved. 208 P. Subbaraj et al. welfare and these are the essential ligands in modern coordina- of the free ligand and its zinc(II) complex in DMSO-d6 were tion and medicinal chemistry [15,34]. The azomethine recorded in a Bruker Advance DRX 300 FTNMR spectrome- (AHC‚NA) linkage present in Schiff base ligand(s) and its ter using tetramethylsilane as internal standard. VGZAB-HS metal(II) complexes show a wide range of biocidal activities spectrometer was used to record the FAB-mass spectra of the such as antibacterial [33,36], antifungal [31,35], herbicidal, ligand and its complexes using 3-nitrobenzylalcohol as matrix. anti-inflammatory [21], anticancer, anti-diabetic [25] and EPR spectrum of copper(II) complex in DMSO was recorded antitumor [27] activities. Benzophenone derivatives are very in both RT and LNT from VARIAN E-112 ESR spectrometer important compounds due to their various biological and using DPPH as internal standard. Perkin Elmer (TGS-2 model) physicochemical properties such as electrochemical, spectro- thermal analyzer in dynamic N2 atmosphere (flow rate 20 mL/ scopic, metal complexation, adsorptive and crystallographic min) with a heating rate of 10 C/min was used for thermal properties [2,12,17] among others. The most important biolog- analysis. X-ray diffraction (XRD) patterns were recorded with ical property of benzophenone derivatives is their ability to a Bruker AXS D8 Advance powder X-ray diffractometer absorb a broad range of UV radiation (200 to 350 nm). (X-ray source: Cu, wavelength 1.5406 A˚ ). Si(Li)PSD was used Due to this property, the benzophenone derivatives like as detector. Scanning electron micrography with energy disper- 2-hydroxy-4-methoxybenzophenone and 2-hydroxy-4-meth- sive spectrometry associated (SEM/EDS) was used for mor- oxy-40-methoxybenzophenone were used as raw materials in phological evaluation. Oxidative DNA cleavage activities the manufacture of sunscreen creams [4]. These creams are were monitored by agarose gel electrophoresis method. helpful to avoid photosensitization, phototoxicity or allergic reactions of patients with various medicinal treatments [18]. 2.2. Synthesis of Schiff base ligand (HL) This fact tempted us to synthesize a benzophenone derivative. Hence, herein, we report the synthesis and characterization of Equimol (10 mmol) quantity of 2-hydroxy-4-methoxybenz- a Schiff base ligand (HL = 5-methoxy-2-(phenyl(phenylimi- ophenone and aniline was dissolved in 20 mL of ethanol and no)-methyl)phenol) which is derived from the condensation the solution was refluxed for 4 h under constant stirring. This of 2-hydroxy-4-methoxybenzophenone with aniline and its condensation reaction was carried out by using acid catalyst metal(II) complexes (1a–1e) [M = Mn(II), Co(II), Ni(II), (few drops of HCl). The formed water was removed from Cu(II) and Zn(II)]. The synthesized ligand and its Schiff base the reaction mixture using sodium sulfate (dehydrating agent). metal(II) complexes were characterized with the help of vari- After completion of the reaction, the mixture was reduced to 1 ous spectral (IR, UV–vis., H NMR, EPR, FAB-MS, powder half of its original volume using a water bath and kept aside XRD and SEM analysis) studies. DNA cleavage ability of the at room temperature. Yellow crystals of 5-methoxy-2-(phenyl synthesized complexes has been explored by gel electrophoresis (phenylimino)methyl)phenol (Scheme 1) were obtained from method under oxidative (H2O2) condition. Moreover, we have slow evaporation (Yield : 87%). accounted the biological activities against few pathogenic bacterial and fungal strains. 2.3. Synthesis of Schiff base metal(II) complexes (1a–1e)

2. Experimental Schiff base metal(II) complexes (1a–1e) were synthesized from Schiff base (10 mmol/10 mL ethanol) with corresponding All reagents and chemicals were purchased from extra pure metal(II) salts (5 mmol/10 mL ethanol) in a 2:1 ratio and Sigma products and used as such. Standard methods [30] were refluxed for 5–6 h (Scheme 2). The resulting solution was used to purify the solvents. pUC-19 DNA was purchased from reduced to half by a water bath and kept aside. On standing, Genei, Bangalore (India). Agarose and ethidium bromide were the obtained solid products were filtered by vacuum filtration, obtained from Sigma (USA). Tris–HCl [(hydroxymethyl) washed several times with water, ethanol, diethyl ether and aminomethane-HCl] buffer solution was prepared using deion- finally dried in vacuo over anhydrous CaCl2 (Yield: 60–70%). ized and triple distilled water using a quartz water distillation setup. 2.4. Pharmacological studies

2.1. Physical measurements 2.4.1. Biological activity In vitro biological activities of Schiff base ligand and its meta- Open capillary method was used to determine the melting l(II) complexes in DMSO medium were screened against few points of all the synthesized ligands and its metal complexes bacterial and fungal strains using agar as the medium by a well and are uncorrected. Microanalysis of free Schiff base and its diffusion method [28]. All the experiments were made in three metal complexes was performed by a CHN analyzer Carlo Erba replicates for each compound and the detailed procedure for 1108, Heraeus. Elico conductivity bridge (Model No. CM 180) measuring the zone of inhibition as described in our earlier was used to measure the molar conductance of synthesized work [32,35]. The obtained zone of inhibition (in mm) of the complexes in 103 M solution using DMSO as solvent. Per- Schiff base metal(II) complexes was compared with commer- kin–Elmer 783 FTIR spectrophotometer was used to record cially available controls. the IR spectra of the ligand and its complexes in the range of 400–4000 cm1. Magnetic susceptibility measurement on pow- 2.4.2. Antioxidant activity der samples were carried out by the Gouy balance method [10]. The synthesised Schiff base ligand (HL) and its metal(II) com- The electronic absorption spectra of the ligand and its plexes (1a–1e) have been screened for their in vitro antioxidant complexes were recorded in the range of 200–1100 nm using a activities by DPPH free radical scavenging model according to Shimadzu UV-1601 spectrophotometer. Proton NMR spectra the method of Blois [3] at 37 C. 500 lM stock solutions of the Synthesis, characterization and studies of Schiff base metal(II) complexes 209

controlhi and the percentage of the free radical scavenging activity ¼ ðAcontrolAsampleÞ 100 was calculated and the results Acontrol were compared with standard control.

2.5. Nucleases studies

2.5.1. Binding studies The interaction between metal complexes and DNA was stud- ied using electronic absorption and ﬂuorescence techniques.

2.5.1.1. Electronic absorption study. The concentration of CT Scheme 1 Synthesis of Schiff base ligand (HL). DNA was determined from its UV absorbance at 260 nm (e = 6600 M1 cm1). Absence of any protein contamination with CT DNA was conﬁrmed from the absorption ratio at 260 and 280 nm which was found to be 1.87 [24] and the stock solutions were kept at 4 C. To prepare buffer and other solutions redistilled water free from CO2 was used. The electronic absorption spectra of synthesized complexes were recorded in the presence and absence of CT-DNA in 0.5% DMSO medium. These titrations were performed to keep the concentration of the complexes constant and by varying the DNA concentration (0–50 lM). Binding experiments were done in 0.5 % DMSO solution using Tris–HCl/NaCl buffer (5 mM Tris–HCl, 5 mM NaCl, pH = 7.2). Absorption spectra were recorded after successive addition of CT-DNA to the complex solution. The intrinsic binding constant (Kb) was calculated according to the following equation [38] ½DNA ½DNA 1 ¼ þ ðea efÞ ðeb efÞ Kbðeb efÞ

where ef, eb and ea are the molar extinction coefﬁcients of the free complex in solution, complex in the completely bound form with CT-DNA and complex bound to DNA at a deﬁnite concentration respectively. In the plot of [DNA]/(ea ef) vs. [DNA], Kb was calculated.

2.5.1.2. Fluorescence study. Fluorescence spectra were recorded at room temperature with excitation at 520 nm and emission at 1600 nm. Fluorescence spectra of the complexes were recorded before and after the addition of DNA in the presence of 5 mM Scheme 2 General outline for the synthesis of Schiff base Tris–HCl/50 mM NaCl buffer. A ﬁxed concentration value of metal(II) complexes. the complex (103 M) was titrated with increasing amounts of DNA [39] over the range of 20–100 lM and 1.0 · 10–5 M ethidium bromide (EB). ligand and its complexes in ethanol (25 mL) were prepared. From the stock solution, different (10, 20, 30, 40 and 50 lM) concentrations of Schiff base ligand and the title complexes 2.6. DNA cleavage study were prepared by serial dilutions. 1 mL of each complex solution having different concentrations was taken in different test pUC-19 DNA at pH 7.5 in Tris–HCl buffered solution was tubes and 4 mL of a 0.1 mmol ethanolic DPPH solution was used to perform the gel electrophoresis technique. Oxidative added and shaken vigorously for about 2–3 min, then incu- cleavage of DNA was examined keeping the concentration of bated in a dark room for 30 min at room temperature. A blank the complexes as 30 lM and 2 lLofpUC-19 DNA was added DPPH solution was used for the baseline correction because and the volume was made up to 16 lL using 5 mM of the odd electron in the DPPH gave a strong absorption Tris–HCl/5 mM NaCl buffer solution. The resulting solution maximum at 517 nm (by using a Shimadzu UV-1601 spectro- was incubated at 37 C for 2 h after the addition of DNA. photometer). After incubation, the absorbance values of each 2 lL of 25 % bromophenol blue was added to quench the solution were measured at 517 nm and there was a change reaction. Samples were electrophoresed for 2 h at 50 V in (decrease) in the absorbance values indicating that the Tris-acetate-EDTA (TAE) buffer using 1% agarose gel con- complexes showed moderate free radical scavenging activity. taining 1.0 lg/mL ethidium bromide (EB) and photographed Ascorbic acid (AA) was used as the reference or positive under UV light. 210 P. Subbaraj et al.

3. Results and discussion show a broad band around 3300–3500 cm1 and two weaker bands in the region 820–850 and 707–722 cm1 due to mOH 3.1. Micro analysis and molar conductance rocking and wagging mode of vibrations respectively [1,11,26] which suggest the presence of coordinated water (OH ) molecules in metal(II) complexes (1a–1c). All the synthesized complexes are quite air stable, non- 2 hygroscopic and insoluble in water but readily soluble in 3.3. Electronic absorption spectra DMF and DMSO. Micro analytical data, molar conductance and magnetic moment values are described in Table 1. From the analytical and molar conductance data, the complexes have The electronic absorption spectra of free Schiff base ligand a 1:2 stoichiometry (metal:Schiff base ligand) and are non- (HL) and its metal(II) complexes were recorded in DMSO 3 electrolytic in nature [13]. No characteristic precipitate was solution (10 M) in the range 200–1100 nm at room temperature (Table 2). Schiff base ligand shows three bands at 40,825, formed during the addition of AgNO3 in HNO3 which con- 1 * firms the absence of the chloride ion in complexes. 34,400 and 29,375 cm corresponding to p fi p transition of aromatic benzene moiety and n fi p* transition of azomethine C‚N band influence [8,22]. Mn(II) complex shows a broad 3.2. Infrared spectra band centered at 16,460 cm1 which corresponds to 6 4 A1g fi T1g (4G) transition and its magnetic moment value To determine the way of chelation of Schiff base ligand to (5.8 BM) is very close to the theoretically calculated metal(II) ion, the IR spectrum of free Schiff base ligand is (6.0 BM) value. This indicates an octahedral environment compared with IR spectra of its metal(II) complexes. The com- around the Mn(II) ion [8]. Co(II) complex exhibits three bands 1 4 plexes (1a–1e) show a significant change of mC‚N (azome- at 9735, 13,310 and 19,640 cm which corresponds to T1g 1 4 4 4 4 4 thine) band which is shifted from higher (1602 cm )to (F) fi T2g (F), T1g (F) fi A2g (F) and T1g (F) fi T1g (P) lower (1585–1578 cm1) frequencies and also the phenolic respectively and the observed magnetic moment value mOH band is shifted from 1250 to 1265–1275 cm1. These shif- (4.86 BM) indicates that it has an octahedral environment. tings indicate that the Schiff base ligand coordinates to the The nickel(II) complex shows four bands at 10,243, 15,925, 1 4 metal(II) atom via azomethine-N and deprotonated phenolic- 24,563 and 29,870 cm which are assigned to T1g 4 4 4 4 4 O atoms [1,26]. It is further confirmed by the formation of (F) fi T2g (F), T1g (F) fi A2g (F), T1g (F) fi T1g (P) and two new bands in far infrared regions around 425–500 and L fi M charge transfer respectively and the observed magnetic 570–585 cm1 which correspond to m(M–O) and m(M–N) moment (3.17 BM) which confirms the octahedral geometry chelation modes [1,11,26] and these peaks are absent in the free [22]. Copper(II) complex shows two bands, one at ligand. All the complexes (except Cu(II) and Zn(II) complexes) 26,255 cm1 corresponds to the LMCT band and another,

Table 1 Elemental and physical data of Schiff base ligand (HL) and its metal(II) complexes (1a–1e).

Complex Empirical Formula Color M.P. Yield Elemental analysis found (Calc.) % KM formula weight ( C) (%) (S cm2 mol1) MC H N

HL C20H17NO2 303.35 Yellow 87 82 – 79.19 (78.96) 5.65 (5.62) 4.62 (4.65) – 1a C40H36MnN2O6 695.66 Reddish brown 275 70 7.92 (8.05) 69.02 (69.32) 5.22 (5.15) 4.03 (3.95) 12.1 1b C40H36CoN2O6 699.16 Pink 280 65 8.42 (8.31) 68.67 (68.45) 5.19 (5.23) 4.00 (4.05) 15.4 1c C40H36NiN2O6 699.42 Pale green >280 60 8.39 (8.34) 68.69 (68.53) 5.19 (5.12) 4.01 (3.96) 16.7 1d C40H32CuN2O4 667.17 Brown >280 68 9.51 (9.45) 71.89 (71.73) 4.83 (4.65) 4.19 (4.07) 19.3 1e C40H32ZnN2O4 670.17 Pale yellow >280 62 9.76 (9.62) 71.7 0 (71.56) 4.81 (4.73) 4.18 (4.11) 20.1

Table 2 Electronic absorption spectral data (in DMSO) and magnetic susceptibility value of Schiff base metal(II) complexes (1a–1e). 1 Complex kmax (cm ) Band assignments Geometry leff (BM) HL 40,825 INCT (p fi p*)– – 34,400 INCT (p fi p*) 29,375 LMCT (n fi p*) 6 4 1a 16,460 A1g fi T1g (4G) Distorted octahedral 5.8 4 4 1b 9735 T1g (F) fi T2g (F) Distorted octahedral 4.86 4 4 13,310 T1g (F) fi A2g (F) 4 4 19,640 T1g (F) fi T1g (P) 4 4 1c 10,243 T1g (F) fi T2g (F) Distorted octahedral 3.17 4 4 15,925 T1g (F) fi A2g (F) 4 4 24,563 T1g (F) fi T1g (P) 29,870 LMCT (n fi p*) 1d 26,255 d–d envelop Distorted tetrahedral 1.94 11,390 LMCT (n fi p*) 1e 26,293 LMCT (N fi M) Tetrahedral Dia. Synthesis, characterization and studies of Schiff base metal(II) complexes 211 the broad band, centered at 11,390 cm1 is due to d–d elec- hyperfine spectrum and no features characteristic for a dinucle- tronic transition. These bands confirm its distorted tetrahedral ar complex. g-tensor value follows the order as: g||(2.23) > geometry. The observed magnetic moment (1.94 BM) supports g^(2.02) > ge (2.0023) which reveals that the unpaired electron 9 its monomeric nature and geometry of the complex. Zn(II) is predominantly localized in dx2y2 orbital with 3d configura- complex shows only one broad band centered at 26,293 cm1 tion. Cu–Cu interactions are ruled out due to the absence of in the UV region which is due to L fi M charge transfer. From any signal in the half field region [7,16] which is further sup- its analytical and spectral data, by analogy a tetrahedral geom- ported by the observed G (3.59) value and also the exchange etry is also assigned to the Zn(II) complex [22]. of interaction is misaligned. The calculated empirical ratio (g||/A|| = 171 cm) value confirms the distorted tetrahedral 3.4. 1H NMR spectra environment.

The proton NMR spectrum of the Schiff base ligand shows a 3.7. Thermal analysis singlet peak at 12.1 ppm which corresponds to the phenolic- OH group which is absent in the spectrum of Zn(II) complex Thermogravimetric (TGA/DTA) spectra of Ni(II) and Cu(II) (1e)(Fig. 1). This indicates that the deprotonated phenolic-O complexes (1c and 1d) were recorded in the temperature rang- atom is involved in chelation. Both the ligand and Zn(II) com- ing from room temperature to 900 C. Thermogram of complex show a group of multiplet (6.5–7.7 ppm) and singlet plex (1c)(Fig. 2) shows three stages of weight loss whereas (3.8 ppm) peaks due to aromatic protons and the methoxy Cu(II) complex (1d) shows only two stages of weight loss. By group respectively. The absence of a peak in the region comparison, the Ni(II) complex shows an initial weight loss 4.69–4.82 ppm reveals the absence of coordinated water at 110–180 C which corresponds to the coordinated water molecules in the complex. molecules [9] whereas Cu(II) complex shows no weight loss at this temperature range confirming the absence of these coordinated water molecules. Both the complexes show a weight 3.5. Mass spectra loss (36–40%) around 350–415 C corresponding to continu- ous sublimation of organic ligand moieties and the formation Fast atomic bombardment mass spectra (FAB-MS) of the syn- of an air stable metal oxide as the end product in the range of thesized ligand and its metal(II) complexes was recorded and 600–810 C. The results well agreed with the composition of the obtained molecular ion (m/z) peaks confirm the proposed the metal complexes. formulae and geometry. Schiff base ligand shows (m/z) peak + at 304 corresponding to the [C20H17NO2] ion, the other 3.8. XRD and SEM analysis molecular ion peaks at 274, 185 and 104 corresponding to [C19H15N1O2], [C13H13N] and [C7H7N] fragments respectively [35]. This fragmentation pattern confirmed the proposed struc- Powder X-ray diffractograms of free Schiff base ligand and ture of the ligand (HL). Ni(II) complex (1c) exhibits (m/z)a Ni(II) complex show sharp peaks which indicate their crystal- peak at 701 with [M+2] pattern which reinforces the data line nature. On comparing the X-ray diffractogram of free obtained by microanalysis. Schiff base ligand (HL) with its Ni(II) complex (1c)(Fig. 3), the Ni(II) complex shows few new peaks which suggest the formation of metal chelates. Using Scherre’s equation [6,14] 3.6. EPR spectral study (D = 0.9 k/b cosh) the crystal size for Ni(II) complex is 38 nm. The SEM pictograph of Ni(II) and Mn(II) complexes EPR spectra of Cu(II) complex (1d) was recorded in DMSO at (Fig. 4) show a homogeneous matrix with an ideal shape of RT and LNT. At LNT, the complex shows a four well resolved uniform phase material. A bundle of irregularly broken straw

Figure 1 Proton NMR spectrum of Zn(II) complex (1e). 212 P. Subbaraj et al.

Figure 2 Thermogram of Ni(II) complex (1c).

Figure 3 Powder X-ray diffractogram pattern of Schiff base ligand (HL) and its Ni(II) complex (1c). Synthesis, characterization and studies of Schiff base metal(II) complexes 213

Figure 4 SEM photographs of (a) Mn(II) and (b) Ni(II) Schiff base complexes (1a and 1c).

Figure 5 Proposed structures of the Schiff base metal complexes (1a–1e). like shape is observed in the Mn(II) complex with the particle size of 12 lm. Particle size of 10 lm with broken ice piece like shape is observed in the Ni(II) complex. Based on the above analytical and spectral studies, it is con- ﬁrmed that the synthesized Schiff base Mn(II), Co(II) and Ni(II) complexes (1a–1c) have octahedral geometry whereas the Cu(II) and Zn(II) complexes have tetrahedral geometry and the structures of the complexes are given in Fig. 5.

3.9. In vitro biological studies

In vitro antimicrobial activity of the ligand (HL) and its metal complexes were screened against few bacterial (Escherichia coli, Staphylococcus saphyphiticus, Staphylococcus aureus, Pseudomonas aeruginosa) and fungal strains (Aspergillus niger, Enterobacter species, Candida albicans) by a well diffusion method using agar as nutrient. Measurement of zone of inhibition against the growth of bacteria and fungi for the ligand (HL) and its metal complexes is shown in Fig. 6. All the complexes show a considerable signiﬁcant activity at higher concentration (50 lg) against micro organisms (Table 3). With the help of Overtones concept and the Tweedy’s chelation the- ory [5,37], Cu(II) complex shows a remarkable antimicrobial activity than the other complexes and free ligand (HL) and they follow the order as: Control > (1d) > (1c) > (1b) > Figure 6 Biological activity of Schiff base ligand (HL) and its (1e) > (1a) > HL. The higher activity of the Cu(II) complex metal(II) complexes (1a–1e). 214 P. Subbaraj et al.

may be due to the greater electronegativity and lower atomic radius. Moreover, the metal(II) complexes inhibit the growth of micro organisms following any one of the paths by (i) dis-

bicans turbing the respiration process and (ii) blocking the synthesis of proteins.

3.10. Antioxidant activity

In vitro antioxidant activities of the synthesised Schiff base ligand (HL) and its metal(II) complexes (1a–1e) were tested by DPPH free radical scavenging methods and ascorbic acid was used as the reference or positive control. All the analyses were done in three replicates and their representative graph is shown in Fig. 7. Reduction capability of free radical (DPPH) is determined by the decrease in its absorbance value at 517 nm (blank) which can be induced by antioxidant. From the results, it is seen that the Schiff base metal(II) complexes have higher activities than the free Schiff base ligand (HL) due to the presence of the metal(II) moieties in the complexes.

3.11. DNA binding studies

3.11.1. Absorption and viscosity studies Electronic absorption spectroscopy is a signiﬁcant method to examine the binding modes of the synthesized metal(II) complexes with CT-DNA. The binding of intercalative ligand to DNA has been well characterized classically through absorption titration, following the hypochromism and red shift associated with the binding of the colored complex to the helix, ).

1e due to the intercalative mode involving a strong stacking inter- – action between an aromatic chromophore and the DNA base 1a pairs. The magnitudes of the hypochromism and red shift are commonly found to depend on the strength of the intercalative interaction [29]. In the UV region, Cu(II) complex shows three bands at 225, 290 and 343 nm which can be attributed to ) and its metal(II) complexes ( HL 24 h 48 h5 72 457 h6 6 24 h7 711 89– 48 612 h 8 5 99 8 – 11 9 72 h – 10 8 14 – 9 10 6810121113––– 13 24 10 h – 11 48 h 10 10 15 72 h 13 – 13 12 24 h 13 12 48 h 9 12 – 14 72 h 11 11 11 14 – 24 h 14 12 48 10 h 17 – 72 h – 14 8 24 13 h 11 48 h 12 – 14 10 72 h 10 12 24 h 16 10 – 12 48 h 13 72 h – 8 12 – – – 9 13 – – 14 – 1011 – – – 15 9 – 13 – 16 10 12 18 – – 13 131516 17 – 14 – 18 14 – – 20 15 – – – Escherichia coli Staphylococcus saphyphiticus Staphylococcus aureus Pseudomonas aeruginosa Aspergillus niger Enterobacter species Candida al Antimicrobial activity of Schiff base ligand (

Figure 7 In vitro antioxidant activities of Schiff base ligand (HL) and its metal(II) complexes by DPPH free radical scavenging assay Table 3 ControlHL 1a 131b 1c 141d 151e 15[–] = Less active. 16 17 16 17 18 15 16 17 16 17 18 19 21 22 18 20 21 Complexes Zone of Inhibition (in mm) method at different concentrations (10–50 lM). Synthesis, characterization and studies of Schiff base metal(II) complexes 215 p fi p* transitions of the coordinated ligand. By varying the concentration of CT-DNA, the intensity of the peak decreases (hypochromism) and kmax value is shifted toward the red region (bathochromism). These spectral results suggest that the Cu(II) complex interacts with DNA through stacking interaction between the aromatic chromophore and the base pair of DNA. The p* orbital of intercalated ligand couples with the p orbital of the base pair which leads to a decrease in the p fi p* transition energy resulting in the bathochromism. On the other hand, the coupling p orbital is partially filled by electrons, thus decreasing the transition probabilities and concomitantly resulting in hypochromism. The intrinsic binding constant (Kb) values of complexes were determined from the plot of [DNA] vs. [DNA]/(ea ef) using the absorption at 343 nm [19]. The intrinsic binding constant (Kb) value for Cu(II) complex is 1.0 · 104 M–1 [20]. This value is lower than the standard ethidium bromide. It shows that the present complexes are optimum intercalators than the standard ethidium bromide [EB] whose binding constant is in the range 1.4 · 106 M1. To further confirm the interaction mode of the complexes with DNA, a viscosity study was carried out. As known, the viscosity of DNA is sensitive to changes occurring on the length of the DNA helix mainly upon interaction of DNA with Figure 8 Fluorescence emission spectra of the CT-DNA-EB a compound. In the case of classic intercalation such as ethi- system (1.0 · 10–5 mol L1 EB, 1.0 · 104 mol L1 CT-DNA) in dium bromide, the compound inserts in between the DNA the absence ( ) of 1.0 · 103 mol L1 Cu(II) complex (20 lL base pairs leading to an increase in the separation of base pairs per scan). at intercalation sites in order to host the bound compound [23] and the length of the DNA helix and subsequently, the DNA viscosity will exhibit an increase, which is usually proportional to the strength of the interaction. On the other hand, in the existence of a partial and/or non-classic intercalation of a compound to DNA grooves, a bend or kink in the DNA helix should appear resulting in a slight reduction of its effective length with the DNA viscosity showing a slight decrease or remaining unchanged. It is found that the viscosity of the DNA solution is increased with increasing ratio of the complexes to DNA. As expected, the known DNA-intercalator EB increased the relative viscosity of DNA due to its strong intercalation. The ligand HL exhibits a pathetic intercalation Figure 9 Changes in the agarose gel electrophoresis pattern of as compared with EB. Moreover, the synthesized complexes pUC-19 DNA induced by free Schiff base ligand and its metal(II) exhibit a minor increase in the relative viscosity of CT-DNA, complexes in the presence of the oxidant (H2O2) [Lane 1: DNA suggesting an intercalation mode between the complexes and alone, Lane 2: Mn(II) complex, Lane 3: Ni(II) complex, Lane 4: DNA. This result further suggests an intercalating binding Cu(II) complex, Lane 5: Zn(II) complex and Lane 6: Schiff base mode of the complexes with DNA and also parallels the above ligand]. spectroscopic results, such as hypochromism and red shift of the complexes in the presence of DNA. Thus, the viscosity studies provide a strong evidence for intercalation. The results show that the fluorescence intensity of DNA-EB decreases remarkably with the addition of the complex which 3.11.2. Fluorescence studies indicate that the complex binds to DNA by intercalation or Fluorescence spectra are also used to study the interaction partial intercalation replacing EB from the DNA structure. between the synthesized Schiff base metal(II) complexes with DNA by measuring the emission intensity of ethidium bromide 3.12. Oxidative DNA cleavage study (EB) bound to DNA. EB is weakly fluorescent and it can emit intense fluorescent light in the presence of DNA due to its The cleavage efficiency of synthesized Schiff base metal(II) intercalative binding to DNA. However, this enhanced fluores- complexes with pUC-19 DNA has been examined by the gel cence could be quenched or partially quenched by the addition electrophoresis method in the presence of oxidant (H2O2). of a second molecule that can replace the bound EB or break Control experiment using pUC-19 DNA alone does not show the secondary structure of DNA. So, EB can be used as a any significant cleavage even on longer exposure time. From probe for the determination of DNA structure. In this study, the observed results, it is concluded that Ni(II) and Cu(II) the emission spectrum of EB bound to DNA in the absence complexes (1c and 1d) cleave the DNA significantly as com- and presence of Cu(II) complex (1d) was recorded (Fig. 8). pared to the other complexes as shown in Fig. 9. 216 P. Subbaraj et al.

4. Conclusion [13] W.J. Geary, Coord. Chem. Rev. 7 (1971) 81–122. [14] A. Guinier, X-Ray Diffraction in Crystals, Imperfect Crystals and Amorphous Bodies, W.H. Freeman and Company, San The synthesized Schiff base ligand and its metal(II) complexes Francisco, USA, 1963. were characterized by microanalytical and various spectral [15] M.S. Habibi, E. Askari, Synth. React. Inorg. Met. Org. Nano- studies. The observed molar conductance, magnetic suscepti- Met. Chem. 43 (2013) 406–411. bility and spectral data confirm tetrahedral/octahedral envi- [16] B.J. Hathaway, A.A.G. Tomlinson, Coord. Chem. Rev. 5 (1970) ronment around the M(II) ions. The presence of coordinated 1–43. water molecules in all the complexes (except Cu(II) and Zn(II) [17] P. Jandera, M. Skavrada, L. Andel, D. Komers, G. Guiochon, J. complexes) has been further confirmed from thermal analysis. Chromatogr. 908A (2001) 3–17. The powder X-ray diffraction pattern and SEM show sharp [18] A.K. Kadry, C.S. Okereke, M.S. Abdel-Rahman, M.A. crystalline peaks with microcrystalline and homogeneous Friedman, R.A. Davis, J. Appl. Toxicol. 15 (1995) 97–102. [19] S. Kashanian, J.E.N. Dolatabadi, DNA Cell Biol. 28 (10) (2009) particle nature. In vitro antimicrobial activity of the Cu(II) 535–540. complex reveals higher activity than the other complexes [20] S. Kashanian, M.M. Khodaei, P. Pakravan, DNA Cell Biol. 29 against different microorganisms. Cu(II) and Ni(II) complexes (10) (2010) 639–646. show moderate DNA binding and oxidative cleavage activities [21] S. Kumar, D.N. Dhar, P.N. Saxena, J. Sci. Ind. Res. 68 (2009) than the other complexes. 181–187. [22] A.B.P. Lever, Inorganic Electronic Spectroscopy, Elsevier, New Acknowledgements York, 1971. [23] J. Liu, H. Zhang, C. Chen, H. Deng, T. Lu, L. Li, Int. J. Inorg. Chem. (2003) 114–119. The authors communicate their sincere thanks to the [24] J. Marmur, J. Mol. Biol. 3 (1961) 208–218. Managing Board, Principal and Head of the Department of [25] S. Misra, K.B. Pandeya, A.K. Tiwari, A.Z. Ali, T. Saradamani, the Devanga Arts College, Aruppukottai and the VHNSN S.B. Agawane, K. Madhusudana, Int. J. Nutr. Metab. 4 (1) College, Virudhunagar for their moral support. They express (2012) 11–18. their gratitude to SAIF, IIT–Madras, IIT Bombay and the [26] K. Nakamoto, Infrared Spectra of Inorganic and Coordination Punjab University for recording NMR, EPR and FAB–Mass Compounds, Part B, Fifth ed., Wiley Interscience, New York, spectra respectively and STIC, Cochin for TGA/DTA and 1971. SEM studies. [27] A.A. Osowole, R. Kempe, R. Schobert, Int. Res. J. Pure Appl. Chem. 2 (2) (2012) 105–129. [28] M.J. Pelczar, E.C.S. Chan, N.R. Krieg, Microbiology, Fifth ed., References Tata McGraw Hill Publishing Co., Ltd, New Delhi, 1998. [29] A.M. Pyle, J.P. Rehmann, R. Meshoyrer, C.V. Kumar, N.J. [1] L.J. Bellamy, The Infrared Spectra of Complex Molecules, Turro, J.K. Barton, J. Am. Chem. Soc. 111 (1989) 3051–3059. Second ed., Chapman & Hall, Methuen, London, 1958. [30] D.D. Perrin, W.L.F. Armarego, D.R. Perrin, Purification of [2] S.E. Blanco, J.J. Silber, F.H. Ferretti, J. Mol. Struct. Laboratory Chemicals, Pergamo Press, Oxford, 1980. (Theochem). 582 (2002) 91–105. [31] N. Raman, S. Thalamuthu, J. Dhaveethuraja, M.A. [3] M.S. Blois, Nature 181 (1958) 1199–1200. Neelakandan, S. Banerjee, J. Chil. Chem. Soc. 53 (2008) 1439– [4] P. Collins, J. Ferguson, Br. J. Dermatol. 131 (1994) 124–129. 1443. [5] B.S. Creaven, B. Duff, D.A. Egan, K. Kavanagh, G. Rosair, [32] N. Raman, R. Jeyamurugan, A. Sakthivel, L. Mitu, V.R. Thangella, M. Wals, Inorg. Chim. Acta 363 (2010) 4048– Spectrochim. Acta 75A (2010) 88–97. 4058. [33] N. Raman, S. Sobha, A. Thamaraichelvan, Spectrochim. Acta [6] B.D. Cullity, Elements of X–ray Diffraction, Second ed., 78 (2011) 888–898. Addison-Wesley Publishing Company Inc, Reading, Ma, 1978. [34] N. Raman, S. Sobha, L. Mitu, J. Saudi Chem. Soc. 17 (2) (2013) [7] R.S. Drago, M.J. Desmond, B.B. Corden, K.A. Miller, J. Am. 151–159. Chem. Soc. 105 (1983) 2287–2296. [35] P. Subbaraj, A. Ramu, N. Raman, J. Dharmaraja, Int. J. Emerg. [8] T.M. Dunn, The Visible and Ultraviolet Spectra of Complex Sci. Technol. 1 (7) (2013) 79–84. Compounds in Modern Coordination Chemistry, Wiley [36] P. Subbaraj, A. Ramu, N. Raman, J. Dharmaraja, Spectrochim. Interscience, New York, 1960. Acta 117A (2014) 65–71. [9] C. Duval, Inorganic Thermogravimetric Analysis, Second ed., [37] B.G. Tweedy, Phytopathology 55 (1964) 910–914. Elsevier, Amsterdam, 1963. [38] A. Wolfe, G.H. Shimer, T. Meehan, Biochemistry 26 (20) (1987) [10] A. Earnshaw, Introduction to Magneto Chemistry, Academic 6392–6396. press, New York, 1968. [39] S. Zhang, J. Zhou, J. Coord. Chem. 61 (15) (2008) 2488–2498. [11] J.R. Ferraro, Low-Frequency Vibrations of Inorganic and Coordination Compounds, Plenum press, New York, 1971. [12] S. Fukushima, Y. Ito, H. Hosomi, S. Ohba, Acta Crystallogr. Sect. B Struct. Sci. 54 (6) (1998) 895–906.