nickel(II) complexes

Accepted Manuscript Synthesis, X-ray Crystal Structure, DNA/protein binding, DNA Cleavage and Cytotoxicity Studies of N(4) Substituted Thiosemicarbazone Based Copper(II)/ Nickel(II) Complexes Mathiyan Muralisankar, Jebiti Haribabu, Nattamai S.P. Bhuvanesh, Ramasamy Karvembu, Anandaram Sreekanth PII: DOI: Reference:

S0020-1693(16)30209-2 http://dx.doi.org/10.1016/j.ica.2016.04.043 ICA 17027

To appear in:

Inorganica Chimica Acta

Received Date: Revised Date: Accepted Date:

15 March 2016 22 April 2016 24 April 2016

Please cite this article as: M. Muralisankar, J. Haribabu, N.S.P. Bhuvanesh, R. Karvembu, A. Sreekanth, Synthesis, X-ray Crystal Structure, DNA/protein binding, DNA Cleavage and Cytotoxicity Studies of N(4) Substituted Thiosemicarbazone Based Copper(II)/Nickel(II) Complexes, Inorganica Chimica Acta (2016), doi: http:// dx.doi.org/10.1016/j.ica.2016.04.043

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Synthesis, X-ray Crystal Structure, DNA/protein binding, DNA Cleavage and Cytotoxicity Studies of N(4) Substituted Thiosemicarbazone Based Copper(II)/Nickel(II) Complexes Mathiyan Muralisankar,a Jebiti Haribabu,a Nattamai S. P. Bhuvanesh,b Ramasamy Karvembua and Anandaram Sreekantha* a

Department of Chemistry, National Institute of Technology, Tiruchirappalli - 620 015, India.

b

Department of Chemistry, Texas A & M University, College Station, TX 77842, USA

Abstract N-ethyl-2-(phenyl(pyridin-2-yl)methylene)hydrazinecarbothioamide obtained

from

(CuBpyeTscCl)

4-ethyl-3-thiosemicarbazide and

Ni[BpyeTsc]2Cl2

and

were

2-benzoylpyridine. synthesized

from

(HBpyeTsc) The

was

complexes

HBpyeTsc

and

[CuCl2⋅2H2O]/[NiCl2⋅6H2O]. The synthesized ligand and complexes were characterized by analytical and various spectroscopic techniques. The molecular structure of the ligand and complexes was determined by single crystal X-ray diffraction method. The single crystal X-ray diffraction revealed that CuBpyeTscCl and Ni[BpyeTsc]2Cl2 exhibited square planar and distorted octahedral geometry respectively. All of the synthesized compounds HBpyeTsc, CuBpyeTscCl and Ni[BpyeTsc]2Cl2 were studied for their interaction with calf thymus DNA (CT DNA) and bovine serum albumin (BSA). A DNA cleavage study showed that the complexes cleaved DNA without any external agent. The alterations in the secondary structure of the protein by the ligand and complexes were confirmed by synchronous fluorescence spectroscopic studies. Spectral evidences also showed good binding property of the complexes with the protein. An in vitro cytotoxicity study of the complexes found significant activity against lung cancer cell lines (A549) and less toxicity towards the normal cell lines (L929). The best results for the copper(II) and nickel(II) complexes are the IC50 values of 56.07 and 80.10 µM respectively. Keywords: Cu(II) and Ni(II) complexes; DNA/protein binding property; DNA cleavage; cytotoxicity.

*Corresponding Author E-mail address: [email protected]; Tel: +91 431 2503642

1. Introduction Medicinal inorganic chemistry offers abundant opportunities for the design of therapeutic agents not reachable by organic compounds [1-3]. The wide assortment of coordination numbers and geometries, available redox states, thermodynamic and kinetic characteristics, and intrinsic properties of the cationic metal ion and ligand itself offer the medicinal chemist a large diversity of reactivities to be exploited [4]. The pervasive success of cisplatin in the clinical treatment of various types of neoplasias has placed coordination chemistry of metal-based drugs in the frontline in the fight against cancer [5]. Although highly effective in treating a variety of cancers, the cure with cisplatin is still limited by dose-limiting side effects [6] and inherited or acquired resistance phenomena, only partially revised by employment of new platinum drugs [7-10]. These problems have stimulated a far-reaching search and encouraged chemists to develop alternative strategies, based on different metals, with improved pharmacological properties and aimed at different targets [11]. In this field, copper and nickel complexes showed promising perspectives. Copper-based complexes have been considered on the assumption that endogenous metals may be less toxic for normal cells with respect to cancer cells. Copper is also an essential element for most aerobic organisms, labouring as a structural and catalytic cofactor, and subsequently it is involved in many biological pathways. The bioinorganic chemistry of nickel has also been rapidly expanded due to the increasing interest in nickel complexes that have been shown to act as antiepileptic and anticonvulsant agents or vitamins or have shown antifungal, antimicrobial and anticancer / antiproliferative activity. The real drive towards the development of the coordination chemistry of thiosemicarbazones has been provided by the remarkable biological activity and medicinal property that is related to their metal complexing ability [12-14]. Thiosemicarbazones have been a subject of many reviews wherein different aspects of their coordination behavior leading to excellent anti-tumour, fungicide, antibacterial, anti-inflammatory and anti-viral properties are discussed [15-18]. The biological activity of certain thiosemicarbazones is due to the ability to form terdentate chelates with transition metal ions bonding through NNS and ONS donor atoms [19]. The interaction of metal complexes with DNA has recently gained much attention because it indicates that the complexes may have potential biological activity and their activity depends

on the mode and the affinity of the binding with DNA [20]. Even though many transition metal complexes were reported as antitumour agents and some of them were under clinical trials, developing copper and nickel based metallodrugs is of special importance because of their biocompatibility. Copper and nickel complexes have proved to be the best candidates towards the search of the metal complexes of biological importance [21]. Synthetic copper(II) and nickel(II) complexes have been reported as potential anticancer and cancer inhibiting agents and a number of copper and nickel complexes have been found to be active both in vitro and in vivo [22]. Similarly, protein was also established as one of the main molecular targets in the action of anticancer agents. The interaction between protein and drugs provides useful information on the structural features that determine the therapeutic effectiveness of drugs and also to study the pharmacological response of drugs [23]. Nowadays interactions of the proteins with the metal complexes are important in the search of new drug molecules. Herein we report the synthesis and characterization of copper(II) and nickel(II) complexes containing 2-benzoylpyridine(4)-N(4)(ethyl-1,4-diyl)thiosemicarbazone ligand. The interaction of the copper(II) and nickel(II) complexes with CT-DNA and BSA was studied using spectrometric methods. Copper(II) and nickel(II) complexes were tested for their in vitro cytotoxicity against A549 cancer cells and L929 normal cells. Also DNA cleavage ability of the copper(II) and nickel(II) complexes was estimated using pUC19 DNA.

2. Experimental 2.1. Materials and methods All the chemicals were purchased from Sigma Aldrich / Merck and used as received. Solvents were purified by distillation and retained under inert atmosphere. The melting points were determined on Lab India instrument and are uncorrected. The elemental analyses were performed using a Vario EL−III CHNS analyzer. Magnetic susceptibilities were measured using Sherwood Scientific auto magnetic susceptibility balance. FT-IR spectra were recorded in the range of 4000−400 cm-1 (KBr pellets) and far-IR spectra were recorded in the range of 400−30 cm-1 (polyethylene pellets) using a PerkinElmer Frontier FT-IR/FIR spectrometer. UV−visible spectra were recorded in the range of 800-250 nm using a PG Instruments T90+UV−visible spectrophotometer in DMF solution. Emission spectra were measured on a Jasco V−630 spectrophotometer using 5% DMF in buffer as the solvent. NMR spectra were recorded in CDCl3 by using TMS as an internal standard on a Bruker 400 MHz spectrometer. The

electronspray ionization mass (ESI-MS) spectra of the ligand and complexes were recorded on a THERMO exactive orbitab mass spectrometer. EPR spectrum was recorded on a JEOL EPR spectrometer at liquid nitrogen temperature, operating at X−band frequency (9.1 GHz). 2.2.

Synthesis

of

N-ethyl-2-(phenyl(pyridin-2-yl)methylene)hydrazinecarbothioamide

(HBpyeTsc) The ligand HBpyeTsc was prepared by refluxing a mixture of 4-ethyl-3thiosemicarbazide (0.119 g, 0.001 mol) and 2-benzoylpyridine (0.183 g, 0.001 mol) in 40 mL of absolute ethanol in the presence of 3 drops of conc. acetic acid refluxed for 5 h. The progress of the reaction was monitored by TLC. The reaction mixture was kept for 10 days where the light yellow colored crystals of HBpyeTsc began to separate. X-ray quality single crystals of the compound were grown from a solution of the compound HBpypTsc in chloroform layered with methanol in 6 days. Yield: 81%. Yellow solid. m.p.: 186 oC. Anal. Calc. C15H16N4S (%): C, 63.35; H, 5.67; N, 19.07; S, 11.28. Found: C, 63.24; H, 5.60; N, 19.03; S, 11.22. UV-Vis (CH3OH): λmax, nm (ε, dm3 mol-1cm-1) 262 (22666), 335 (27333). FT-IR (KBr): ʋ, cm-1 3187 (N– H), 1592 (C=N), 1322 (C=S), 1114 (N-N). 1H NMR (400 MHz, CDCl3): δ, ppm 13.67 (s, 1H), 8.81-8.83 (d, J = 8.0 Hz, 1H), 7.79-7.73 (m, 1H), 7.50-7.49 (t, J = 4.0 Hz, 3H), 7.45-7.38 (t, J = 4.0 Hz , 4H ), 7.37-7.27 (m, 1H), 3.80-3.77 (q, J = 8.0 Hz, 2H), 1.30-1.27 (t, J = 4.0 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ, ppm 178.1 (C=S), 152.4 (C=N), 148.6, 142.4, 137.7, 137.1, 129.1, 128.5, 126.1, 124.1 (aromatic carbons), 39.1 and 14.4 (aliphatic carbons). ESI-MS m/z = 285.11 [L+1]+. 2.3. Synthesis of copper(II) and nickel(II) complexes 2.3.1. CuBpyeTscCl CuCl2·2H2O (0.170 g, 0.001 mol) was dissolved in 10 mL of methanol to which 20 mL methanol solution of HBpyeTsc (0.284 g, 0.001 mol) was added. The reaction mixture was stirred for 4 h at room temperature, and then the brown-black precipitate was filtered off and washed with cold methanol, and dried in a vacuum. The suitable crystals for X-ray diffraction were grown from a CH3OH/CH3CN mixture (1:1). Yield: 80 %. Light brown solid. m.p.: 225 °C. Anal. Calc.: C, 47.12; H, 3.95; N, 14.65; S, 8.39. Found: C, 47.03; H, 3.86; N, 14.60; S, 8.31. UV-Vis (CH3OH): λmax, nm (ε, dm3mol-1cm-1) 266 (24000), 306 (19333), 420 (21333). FT-IR (KBr): ʋ, cm-1 1529 (C=N), 1245 (C=S), 1158 (N-N) and far-IR (Polyethylene): ʋ, cm-1 399 (Cu–

Nazo), 351 (Cu–S), 304 (Cu–Cl), 274 (Cu–Npy). ESI−MS m/z = 383.10 [Cu(L)Cl + H]+. EPR (LNT): g, 2.216, 2.046. 2.3.2. Ni[BpyeTsc]2Cl2 NiCl2⋅6H2O (0.238 g, 0.001 mol) in 20 mL of chloroform was taken and 20 mL methanol solution of HBpyeTsc (0.284 g, 0.001 mol) was added to the former. The reaction mixture was stirred for 6 h at room temperature, then the brown precipitate was filtered off, washed with cold methanol, and dried in a vacuum. The suitable crystals for X-ray diffraction were grown from a CH3OH/CH3CN mixture (1:1). Yield: 76%. Brown solid. m.p.: 260 °C. Anal. Calc.: C, 51.60; H, 4.62; N, 16.05; S, 9.18. Found: C, 51.73; H, 4.57; N, 16.01; S, 9.29. UV−Vis (CH3OH): λmax, nm (ε, dm3mol-1cm-1) 265 (26666), 318 (18666), 415 (26000). FT−IR (KBR): ʋ, cm-1 1561 (C=N), 1215 (C=S), 1167 (N–N) and far-IR (Polyethylene): ʋ, cm-1 467 (Ni–Nazo), 354 (Ni–S), 230 (Ni–Npy). ESI−MS m/z = 626.22 [Ni(HL)2]2+). µ = 2.17 BM. 2.4. Single crystal X-ray crystallography A Bruker APEX2 or Bruker GADDS X-ray (three-circle) diffractometer was employed for crystal screening, unit cell determination, and data collection. The X-ray radiation employed was generated from a Mo sealed X-ray tube (Kα = 0.70173 Å; 40 kV, 40 mA) for HBpyeTsc and CuBpyeTscCl, and a Cu sealed X-ray tube (Kα= 1.54178 Å; 40 kV, 40 mA) for Ni[BpyeTsc]2Cl2 fitted with a graphite monochromator. Sixty data frames were taken at widths of 0.5°. These reflections were used in the auto-indexing procedure to determine the unit cell. A suitable cell was found and refined by nonlinear least squares and Bravais lattice procedures. The unit cell was verified by examination of the h k l overlays on several frames of data by comparing with both the orientation matrices. No super-cell or erroneous reflections were observed. After careful examination of the unit cell, a standard data collection procedure was initiated using omega scans. Integrated intensity information for each reflection was obtained by reduction of the data frames with the program APEX2 [53]. The integration method employed a three dimensional profiling algorithm and all data were corrected for Lorentz and polarization factors, as well as for crystal decay effects. Finally, the data were merged and scaled to produce a suitable data set. The absorption correction program SADABS [54] was employed to correct the data for absorption effects. Systematic reflection conditions and statistical tests of the data suggested the space group. Solution was obtained readily using SHELXTL (XS) [55]. Hydrogen atoms were placed in idealized positions and were set riding on the respective parent atoms. All non-hydrogen

atoms were refined with anisotropic thermal parameters. The structure was refined (weighted least squares refinement on F2) to convergence [56]. Olex2 was employed for the final data presentation and structure plots [55]. 2.5. DNA binding studies The UV−visible absorption spectroscopic studies and the DNA binding experiments were performed at room temperature. CT-DNA samples were dissolved in 50 mM NaCl/5 mM Tris HCl (pH 7.2) solution. CT DNA solution displayed a UV absorbance ratio at 260 and 280 nm (A260/A280) of 1.89, indicating that the CT DNA was sufficiently in protein free form. The concentration of the nucleic acid solutions was determined by UV absorbance at 260 nm after 1:100 dilutions. The extinction coefficient at 260 nm was taken as 6600 M-1cm-1 [57]. Stock solutions were stored at 4 °C and used within 4 days. 0−40 µM of CT DNA was added to the complex solution. The spectra were recorded after equilibration for 3 minutes, allowing the compounds to bind to the CT DNA. Concentrated stock solutions were prepared by dissolving calculated amounts of the compounds (HBpyeTsc, CuBpyeTscCl and Ni[HBpyeTsc]2Cl2) in a 5% DMF/5 mM Tris−HCl/50 mM NaCl buffer to required concentrations for all the experiments. The competitive binding of each complex with EB has been investigated by fluorescence spectroscopic technique in order to examine whether the complex can displace EB from its CT DNA-EB complex. EB solution was prepared using Tris HCl/NaCl buffer (pH 7.2). The test solution was added in aliquots of 2.5 µM concentration to DNA-EB and the change in fluorescence intensities at 596 nm (450 nm excitation) was noted down. 2.6. Protein binding studies Quenching of the emission of tryptophan residues of BSA was performed using the compounds (HBpyeTsc, CuBpyeTscCl and Ni[BpyeTsc]2Cl2) as quenchers. To a solution of BSA in phosphate buffer (pH 7.2), increments of the quenchers were added, and the emission signals at 341 nm (280 nm excitation) were recorded after each addition of the quencher. The excitation and emission slit widths and scan rates were constantly maintained for all the experiments. Stock solutions of the test compounds were prepared by dissolving them in DMF and diluted suitably with phosphate buffer to get required concentrations. 2.5 mL of BSA solution (1 µM) was titrated by successive additions of a 10−6 M stock solution of the test compounds (10-4 M) using a micropipette. Synchronous fluorescence spectra measurements also

recorded using the same concentration of BSA and the test compounds as mentioned above with two different ∆λ (difference between the excitation and emission wavelengths of BSA) values such as 15 and 60 nm. 2.7. DNA cleavage studies A mixture of Tris buffer (5mMTris−HCl/50mMNaCl buffer, pH 7.2), pUC19 plasmid DNA (150 µg mL-1) and different amounts of the compounds were incubated for 3 h at 37 oC. A dye solution (0.05% bromophenol blue and 5% glycerol) was added to the mixture prior to electrophoresis. The samples were then analyzed by 1.5% agarose gel electrophoresis [Tris HCl/boric acid/EDTA (TBE) buffer, pH 8.0] for 2 h at 60 mV. The gel was stained with 0.5 µg mL-1 ethidium bromide, visualized by UV light and photographed. The extent of cleavage of pUC19 was determined by measuring the intensity of the bands using Alpha Imager HP instrument. 2.8. Cytotoxic study using MTT assay Cytotoxicity of the complexes was carried out on lung cancer (A549) and L929 normal cell lines. Cell viability was carried out using the MTT assay method [60]. The non-small lung adenocarcinoma cells A549 and L929 normal cells were plated separately in 96 well plates at a concentration of 1 × 105 cells/well. Complexes (CuBpyeTscCl and Ni[BpyeTsc]2Cl2) of concentration ranging from 1-500 µM dissolved in DMSO were seeded to the wells. DMSO was used as the control. After 24 h, the wells were treated with 20 µL MTT [5 mg/ml phosphate buffered saline (PBS)] and incubated at 37 °C for 4 h. The purple formazan crystals formed were dissolved in 200 µL DMSO. The absorbance of the solution was measured at a wavelength of 570 nm using a Beckmann Coulter Elisa plate. Triplicate samples were analyzed for each experiment. The percentage inhibition was calculated using the formula.
    

   

 −
   

   × 
    

   



3. Results and discussion 3.1. Synthesis Thiosemicarbazone (HBpyeTsc) was synthesized by a condensation reaction between substituted heterocyclic based ketone and ethyl thiosemicarbazide (Scheme 1). The copper(II) complex was synthesized by equimolar reaction between CuCl2 ⋅2H2O and the ligand (Scheme 2). The nickel(II) complex was synthesized using NiCl2⋅6H2O as a precursor (Scheme 3). The ligand and its complexes were characterized by elemental analyses and various spectroscopic techniques. The molecular structure of HBpyeTsc, CuBpyeTscCl and Ni[BpyeTsc]2Cl2 was confirmed by single crystal X-ray diffraction study.

Scheme 1. Synthesis of heterocyclic based thiosemicarbazone (HBpyeTsc)

Scheme 2. Synthesis of copper(II) complex [CuBpyeTscCl]

Scheme 3. Synthesis of nickel(II) complex [Ni[BpyeTsc]2Cl2]

3.2. Spectroscopy Electronic spectra of the ligand (HBpyeTsc) showed two strong absorption bands in the regions 262 and 335 nm, which were assigned to π → π* and n → π* transitions respectively. The spectra of CuBpyeTscCl exhibited three bands. Two bands were observed at 266 and 306 nm, which correspond to intraligand transitions. The band at 420 nm was attributed to d → d transition. Two bands were observed in the spectrum of Ni[BpyeTsc]2Cl2 at 265 and 318 nm, correspond to intraligand transitions. The broad band at 415 nm corresponds to d → d transition [24]. In the solid state HBpypTsc remains in the thione form as indicated by a band at 1322 cm-1 attributable to ʋ(C=S); asymmetric bending frequency of thiocarbonyl is also observed at 845 cm-1. A broad absorption band at 3412 cm-1 is assigned to the ʋ(N-H) of the thiosemicarbazone. Azomethine ʋ(C=N) stretching frequency is observed at 1592 cm-1 and a sharp band observed at 1114 cm-1 is assignable to ʋ(N-N) [25]. The region below 1300 cm-1 is rich in peaks assignable to a combination of the ring breathing and streaking of the heterocyclic ring and phenyl ring. On complexation, azomethine ʋ(C=N) stretching frequency is shifted to a lower region and observed at 1529 and 1561 cm-1 in CuBpyeTscCl and Ni[BpyeTsc]2Cl2 respectively. Furthermore, a band due to ʋ(N-N) in CuBpyeTscCl and Ni[BpyeTsc]2Cl2 has been shifted to 1158 and 1167 cm-1 respectively, indicating the involvement of one of the nitrogen atoms of N-N moiety in bonding with the metals. In the spectra of CuBpyeTscCl and Ni[BpyeTsc]2Cl2, ʋ(C=S) is shifted to a lower value of 1243 and 1215 cm-1 respectively, indicating the involvement of S in coordination. The stretching frequency due to Cu–N

(azomethine nitrogen) is observed at 399 cm-1 and that of Cu–N (pyridyl nitrogen) is observed at 274 cm-1 [26]. The presence of a new band at 351 cm-1 (Cu–S) is an indication for sulfur coordination [27]. A strong band at 304 cm-1 in the spectrum of CuBpyeTscCl is suggestive for terminally bonded chlorine (Cu–Cl) [28]. The Ni–N stretching frequency for the azomethine nitrogen and pyridyl nitrogen is observed at 467 and 230 cm-1 respectively [29]. The presence of a band at 354 cm-1 (Ni–S) in the spectra of the Ni complex suggests the involvement of sulfur in coordination. A sharp singlet observed at 13.67 ppm in the 1H NMR spectrum of the ligand (HBpyeTsc) is assigned to H−N−C=S. The hydrogen attached to the terminal nitrogen shows a doublet 8.82 ppm. The aliphatic protons, CH2 and CH3, are observed as a quartet and triplet at 3.80-3.77 and 1.30-1.27 ppm, respectively. Chemical shift of all other aromatic protons of the pyridine and phenyl rings is observed in the expected regions [30].

13

C NMR spectrum of

HBpyeTsc shows well defined peaks at 178 and 152 ppm due to the thiocarbonyl (C=S) and azomethine (C=N) carbons respectively. Other aromatic and aliphatic carbons exhibited signals in the appropriate regions. X-band EPR spectrum of Cu(II) complex recorded at liquid nitrogen temperature exhibits three well resolved peaks. The axial symmetry parameter (G) is related by the expression,  =  || − 2 /  − 2  It measures the extent of exchange interaction between the copper centers in polycrystalline solid. Since G > 4, the exchange interaction between Cu–Cu ions in the complex is ruled out [31,32]. From the spectral data it is found that g|| (2.216) > g⊥ (2.046) > 2.003 and the value of exchange interaction term (G > 4.0) suggested that the unpaired electron of Cu(II) ion is present in the dx2-y2, which is consistent with square planar geometry of the Cu(II) system (Fig. 1). This is confirmed by single crystal XRD technique. The extent of geometrical distortion is measured by the g|| /A|| ratio [33]. It is known that this ratio for square planar complex falls between 105 and 135 cm-1 and for tetragonally distorted complex it is 135-250 cm-1 [34]. The complex CuBpyeTscCl has g|| /A|| = 127 cm-1, showing square planar geometry. The Ni[BpyeTsc]2Cl2 complex showed the magnetic moment of 2.17 B.M, which is attributable to the octahedral environment around the Ni(II) center (d8, S = 1) [35]. This is also confirmed by single crystal XRD technique. 3.3. Single crystal X-ray crystallographic study

The HBpyeTsc crystallizes in orthorhombic lattice with Pna21 space group symmetry. The experimental details and crystallographic refinement parameters are presented in Table 1. Fig. 2 confirms the molecular structure of the ligand. The structure reveals the fact that two aromatic rings are slightly tilted from the plane of the ring, existing in a propeller like fashion. The molecule exists in Z conformation around the N2−N3 bond. The structure reveals quasi coplanarity in the plane of the thiosemicarbazone but both the aromatic rings are bifurcated and exist in thione form. Table 2 lists the selected bond lengths and angles. The compound has two double bonds in the thiosemicarbazone moiety viz. N2−C6 [1.298(4) Å] and S1−C13 [1.684(3) Å], which are consistent with the previously reported cases [36]. A torsion angle of 176.1(2)° corresponding to the N2−N3−C13−S1 moiety confirms the trans configuration of the thiocarbonyl S1 atom [37]. Considerable amount of the ring bifurcation from the plane of the thiosemicarbazone is indicated by the dihedral angles C(13)−N(3)−N(2)−C(6) [-178.8(3)°] and C2−C1−C6−C7 [15.0(5)°]. The thiocarbonyl S1 and pyridyl N1 atoms are in Z configuration with respect to C1−N2 bond. The complex CuBpyeTscCl crystallizes in monoclinic lattice with P21/n space group symmetry. The experimental details and crystallographic refinement parameters are presented in Table 1 and selected bond lengths and angles are listed in Table 2. Fig. 3 presents the molecular structure of CuBpyeTscCl. Coordination extends the thiosemicarbazone moiety's S(1)−C(13) bond length from 1.684(3) Å to 1.745(2) Å [38,39]. Copper ion is in the same plane along with the coordinating atoms Cl(1), N(1), N(3), and S(1) as indicated by the bond angles N(1)−Cu(1)−Cl(1) [176.05(5)°], N(3)−Cu(1)−Cl(1) [97.57(5)°], N(1)−Cu(1)−N(3) [80.63(7)°] and N(1)−Cu(1)−S(1) [84.79(5)°]. The bond angles are quite far from a perfect square planar geometry around copper(II). The Cu−N and Cu−S bond lengths are relatively smaller (~ 2.021 and 2.236 Å) indicating the domination of thiosemicarbazone moiety in the bonding. It is found that the copper ion is closer to the thiosemicarbazone moiety than to the chloride. The complex Ni[BpyeTsc]2Cl2 (Fig. 4) crystallizes in monoclinic lattice with the space group, C2/c. The structural refinement parameters are given in Table 1 and selected bond distances and angles are given in Table 2. The binding nature of the crystallographically independent molecules in the asymmetric lattice is almost identical. In Ni[BpyeTsc]2Cl2, two molecules of the neutral ligand HBpyeTsc are coordinated in a meridional fashion using pairs of

cis pyridyl nitrogen, trans azomethine nitrogen and cis thiocarbonyl sulphur, resulting in two bicyclic chelate rings. The bond distances and angles obtained from single crystal XRD reveal that the independent units of the molecule exist in a distorted octahedral geometry around the Ni(II). The bond lengths Ni−Nazo, Ni−Npy and Ni-S increase in that order, as in similar compounds [40]. The Ni−Npy bond lengths are 2.092(3) and 2.080(3) Å. The Ni−Nazo bond lengths [2.030(3) and 2.033(3) Å] are less compared to Ni−Npy, indicating higher strength of former bond than the latter. The Ni−S bond lengths in the complex are 2.425(10) and 2.424 (10) Å. Further, the C(13)−S(1) and C(28)−S(2) bond lengths in the complex are 1.687(3) and 1.693 (3) Å respectively, which are intermediate between C-S (1.82 Å) and C=S (1.56 Å). Similarly C(13)−N(3) [1.370(4) Å] and C(28)−N(7) [1.363(4) Å] bond lengths are intermediate between C-N (1.47 Å) and C=N (1.29 Å). The same trend is followed in the bond lengths of N(1)−N(3) and N(5)−N(7). Thus, electrons resonate between C−S, C−N and N−N bonds in Ni[BpyeTsc]2Cl2, which are responsible for partial double bond character in turn strongly implies coordination behavior of HBpyeTsc.

3.4. DNA binding studies 3.4.1. UV-Vis absorption spectra The value of intrinsic binding constant (Kb) was calculated from the absorption spectral titration data using the equation [DNA]/(εa−εf) = [DNA]/(εb−εf) + 1/Kb (εb−εf), where [DNA] is the concentration of DNA in base pairs, εa is the apparent extinction coefficient value found by calculating A(observed)/[complex], εf is the extinction coefficient for the free compound, and εb is the extinction coefficient for the compound in the fully bound form [41]. The value of Kb was found to be 1.24 × 104, 2.07 × 104 and 1.99 × 104 M-1 for the interaction of HBpyeTsc, CuBpyeTscCl and Ni[BpyeTsc]2Cl2, respectively with CT DNA (Table 3). The slope and the intercept of the linear fit of [DNA]/[εa−εf] versus [DNA] plot give 1/[εa−εf] and 1/Kb[εb−εf], respectively. When HBpyeTsc was exposed to CT DNA, the intensity of its intra ligand transition band at 269 nm decreases without any shift (Fig. 5). The electronic absorption spectrum of complex CuBpyeTscCl exhibited three absorption bands at 331, 403 and 426 nm which are assigned to intra ligand transitions, ligand to metal charge transfer (LMCT) and d-d transition respectively. Hypochromism (∆ε, 35-45%) with a small red shift around 3-5 nm was observed after the incremental addition of DNA to the complex. During the incremental addition

of CT DNA to Ni[BpyeTsc]2Cl2, the bands due to intra ligand transitions at 292 and 390 nm exhibited a hypochromism (∆ε, 40-34%) and significant red shift (2-5 nm). The results derived from the UV-Vis titration experiments suggest that all the compounds can bind to DNA. However the complexes interact with CT DNA strongly than the thiosemicarbazone ligand. The Kb value of the compounds follows the order, CuBpyeTscCl > Ni[BpyeTsc]2Cl2 > HBpyeTsc (Fig. 6). 3.4.2. Ethidium bromide (EB) displacement study In order to confirm the mode of binding of the compounds (HBpyeTsc, CuBpyeTscCl and Ni[BpyeTsc]2Cl2) with CT DNA, emission intensity of EB bound DNA (EB-DNA) is monitored as a function of concentration of the compounds. Due to the intercalative binding of EB with DNA, the EB-DNA showed enhancement in the emission spectrum. While adding the compounds to the EB-DNA, the emission intensity is reduced due to the replacement of EB by the compounds. The quenching of fluorescence of EB-DNA by the compounds is shown in Fig. 8. The relative binding propensity of the compounds with CT DNA was determined by the standard Stern-Volmer equation [42,43], Fo/F = 1 + Kq [Q] where Fo is the emission intensity in the absence of quencher, F is the emission intensity in the presence of quencher, Kq is the quenching constant and [Q] is the concentration of the compound. The plot of Fo/F versus [Q] gives Kq (Fig. 7). The values of apparent DNA binding constant (Kapp) are calculated using the equation, KEB[EB] = Kapp[compound] where [compound] is the compound concentration at 50 % reduction in the fluorescence intensity of EB, KEB = 1.0 × 107 M-1 and [EB] = 5 µM. The Kq and Kapp values are listed in Table 3. The DNA binding ability of the compounds follows the order CuBpyeTscCl > Ni[BpyeTsc]2Cl2 > HBpyeTsc, which is supported by the results obtained from absorption spectral studies. The observed quenching and binding constants of the compounds suggest that the interaction of the tested compounds with CT DNA should be of intercalation [44]. 3.5. Protein binding studies 3.5.1. UV-Vis absorption spectra The absorption spectra of BSA and the compounds are measured to find the type of quenching. For a dynamic quenching appreciable change in absorption spectra of the fluorophore is not expected; in contrast, static quenching usually leads to perturbation of the fluorophore [45]. Addition of the compounds to BSA lead to an increase in BSA absorption intensity without

affecting the position of the absorption band. This indicates that the type of interaction between the compounds and BSA is mainly a static quenching [46]. The representative absorption titration spectrum is shown in Fig. 9. The values of the binding constant are comparable with the reported data [47]. 3.5.2. Fluorescence spectra Ability of newly synthesized compounds HBpyeTsc, CuBpyeTscCl and Ni[BpyeTsc]2Cl2 to interact with the most abundant blood protein serum albumin has been the spotlight due their structural homology with human serum albumin. Tyrosine, tryptophan and phenylalanine residues are mainly responsible for the fluorescence property of BSA [48].

Fluorescence

quenching reflects conformational and dynamic changes in BSA. The interaction between BSA and the compounds is studied by fluorescence measurements at room temperature. The solution of BSA (1 µM) was titrated with the different concentration of the compounds (0-20 µM) in the range 290-500 nm (λex 280 nm, Fig. 12). The fluorescence intensity of BSA at 349 nm is quenched by HBpyeTsc, CuBpyeTscCl and Ni[BpyeTsc]2Cl2 with red or blue shift (61 %, 4 nm, HBpyeTsc; 86 %, 6 nm, CuBpyeTscCl; and 73 %, 5 nm, Ni[BpyeTsc]2Cl2). The observed red or blue shift is due to the fact that the interactive sites of the protein are present in a hydrophobic environment. Results reveal that interaction takes place between the compounds and BSA at hydrophobic sites [49]. The fluorescence quenching constant (Kq) was found by Stern−Volmer equation using F0/F versus [Q] plot (Fig. 10). Further, the equilibrium binding constant was evaluated using the equation log[(F0−F)/F] = log Kb + n log [Q] where Kb is binding constant of the compound with BSA and n is the number of binding sites. From the log [(F0−F)/F] versus log [Q] plot (Fig. 11), the binding constant and the number of binding sites have been calculated. The evaluated values of Kq, Kb and n are gathered in Table 4. The estimated value of n (∼1) strongly supports the existence of a single binding site in BSA. The values of Kb for these compounds further suggested that CuBpyeTscCl interacts with BSA rather strongly relative to other HBpyeTsc and Ni[BpyeTsc]2Cl2 under investigation [50]. 3.5.3. Synchronous fluorescence spectra A synchronous fluorescence spectrum obtains sufficient information about the molecular micro-environment, particularly in the vicinity of the fluorophore functional groups [51]. According to Miller, the difference between the excitation and emission wavelengths (∆λ) reflects the nature of the chromophore. While a large ∆λ value such as 60 nm is characteristic of

tryptophan residue, a lower ∆λ value such as 15 nm is characteristic of tyrosine. To investigate the structural changes occurred in BSA due to the addition of the compounds, we have measured synchronous fluorescence spectra of BSA with the compounds (Figs. 13 and 14). In the synchronous fluorescence spectra of BSA at ∆λ = 15, addition of the compounds to the solution of BSA resulted in a decrease of the fluorescence intensity of BSA (at 307 nm) in the magnitude of 61.8, 74.4 and 68.9 % for HBpyeTsc, CuBpyeTscCl and Ni[BpyeTsc]2Cl2, respectively without any shift in the emission wavelength. On the other hand, in the case of synchronous fluorescence spectra of BSA at ∆λ = 60, the addition of the compounds to the solution of BSA resulted in a significant decrease of the fluorescence intensity of BSA (at 348 nm) up to 63.3, 86.0 and 71.6 % respectively for HBpyeTsc, CuBpyeTscCl and Ni[BpyeTsc]2Cl2, respectively, without any change in the position of the emission band. The results suggest that the interaction of the CuBpyeTscCl with BSA affects the conformation of both tyrosine and tryptophan; the effect is more pronounced toward tryptophan than tyrosine. 3.6. DNA cleavage studies The cleaving efficiency of the ligand and complexes has been assessed by their ability to convert supercoiled pUC19 DNA from form I to form II by agarose gel electrophoresis. As shown in Fig. 15, no distinct DNA cleavage was observed for the control in which the complex was absent (lane 1); however, fixed concentration of HBpyeTsc, CuBpyeTscCl and Ni[BpyeTsc]2Cl2 cleave SC (Form I) DNA into nicked circular (NC) (Form II) DNA. Hence, the complexes exhibited DNA cleavage activity in the absence of any external agent. Additionally, the amount of helical unwinding induced by the complex bound to SC DNA provides evidence for the intercalation mode of interaction between the compounds and DNA [52]. The DNA cleavage activity of HBpyeTsc, CuBpyeTscCl and Ni[BpyeTsc]2Cl2 can be estimated from the percentage of cleavage (C) (Table 5). 3.7. Cytotoxicity In vitro cytotoxicity of the complexes is performed on the A549 cancer (human lung cancer cells) and L929 (normal cells) cells by using MTT assay and compared with cisplatin [IC50 = 18.0 µM (A549) and 6.0 µM (L929)]. Figs. 16 and 17 show the cytotoxicity of the Cu(II) and Ni(II) complexes after 24 h incubation on A549 cancer and L929 cell lines, respectively. CuBpyeTscCl and Ni[BpyeTsc]2Cl2 exhibit cytotoxicity with IC50 values of 56.07 and 80.10 µM against A549 cancer cell line. Both the complexes are less toxic towards the normal cell (L929)

as it is evident from the high IC50 values (above 600−800 µM). The Cu(II) complex has shown significant cytotoxic activity but less than cisplatin. IC50 values of both the complexes are listed in Table 6. 4. Conclusion The thiosemicarbazone ligand (HBpyeTsc) and its complexes (CuBpyeTscCl and Ni[BpyeTsc]2Cl2) are characterized by analytical and spectral (Mass, EPR/NMR, FT-IR and UVVis) techniques. The structure of the ligand and the complexes has been confirmed by single crystal X-ray diffraction study.

The

interaction of HBpyeTsc,

CuBpyeTscCl and

Ni[BpyeTsc]2Cl2 with CT DNA and BSA protein was investigated using UV-Visible and fluorescence spectroscopic methods. Absorption and emission spectral studies indicate that the complexes CuBpyeTscCl and Ni[BpyeTsc]2Cl2 interact with CT DNA and BSA protein more strongly than the ligand. DNA unwinding study of CuBpyeTscCl and Ni[BpyeTsc]2Cl2 suggested intercalation mode of binding of the complexes with DNA. Both the complexes exhibited good cytotoxicity against A549 cancer cell line and less toxicity against L929 normal cell line. Electronic supplementary information (ESI) available: Crystallographic data for the structures reported in this paper have been deposited with the Cambridge Crystallographic Data Centre (CCDC) as supplementary publication numbers (CCDC 1418909, CCDC 1418907, and CCDC 1418908 for HBpyeTsc, CuBpyeTscCl and Ni[BpyeTsc]2Cl2 respectively). Copies of the data can be obtained free of charge from the CCDC (12 Union Road, Cambridge CB2 1EZ, UK; Tel.: + 44-1223-336408; Fax: + 44-1223-336003; e-mail: [email protected]; Web site http://www.ccdc.cam.ac.uk).

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Fig. 1. EPR spectrum of CuBpyeTscCl in frozen DMF solution. Microwave power, 0.98 mW; microwave frequency, 9.1 GHz.

Fig. 2. Thermal ellipsoid plot of HBpyeTsc with displacement ellipsoids drawn at the 50% probability level.

Fig. 3. Thermal ellipsoid plot of CuBpyeTscCl with displacement ellipsoids drawn at the 50% probability level.

Fig. 4. Thermal ellipsoid plot of Ni[BpyeTsc]2Cl2 with displacement ellipsoids drawn at the 50% probability level.

Fig. 5. Absorbance titrations of HBpyeTsc, CuBpyeTscCl and Ni[BpyeTsc]2Cl2 (5 µM) with CT DNA (0-35 µM).

Fig. 6 Plots of [DNA]/(εa − εf) versus [DNA] for the titration of HBpyeTsc, CuBpyeTscCl and Ni[BpyeTsc]2Cl2with CT DNA.

Fig. 7. Stern-Volmer plots of fluorescence titrations of HBpyeTsc, CuBpyeTscCl and Ni[BpyeTsc]2Cl2with CT DNA.

Fig. 8. Fluorescence titrations of HBpyeTsc, CuBpyeTscCl and Ni[BpyeTsc]2Cl2 (0-35 µM) with EB bound CT DNA (5µM).

Fig. 9. Absorbance titrations of HBpyeTsc, CuBpyeTscCl and Ni[BpyeTsc]2Cl2 with BSA.

Fig. 10. Stern-Volmer plots of the fluorescence titrations of HBpyeTsc, CuBpyeTscCl and Ni[BpyeTsc]2Cl2 with BSA.

Fig. 11. Plot of log [Q] vs. log[(F0 − F)/F].

Fig. 12. Fluorescence titrations of HBpyeTsc, CuBpyeTscCl and Ni[BpyeTsc]2Cl2 (0-14 µM) with BSA (1 µM).

Fig. 13. Synchronous spectra of BSA (1 µM) as a function of concentration of HBpyeTsc, CuBpyeTscCl and Ni[BpyeTsc]2Cl2 (0-14 µM) with ∆λ = 60 nm.

Fig. 14. Synchronous spectra of BSA (1µM) as a function of concentration of HBpyeTsc, CuBpyeTscCl and Ni[BpyeTsc]2Cl2 (0-14 µM) with ∆λ = 15 nm.

Fig. 15. Cleavage of supercoiled pUC19 DNA (30 µM) by HBpyeTsc, CuBpyeTscCl and Ni[BpyeTsc]2Cl2 in a buffer containing 5% DMF/5 mM Tris–HCl/50 mM NaCl at pH = 7.2 and 37 oC with an incubation time of 3 h. Lane 1, DNA control; lane 2, DNA + HBpyeTsc (150 µM); lane 3, DNA + CuBpyeTscCl (150 µM); lane 4, DNA + Ni[BpyeTsc]2Cl2 (150 µM). Forms SC and NC are supercoiled and nicked circular DNA, respectively.

Fig. 16. Cytotoxicity of CuBpyeTscCl and Ni[BpyeTsc]2Cl2 after 24 h incubation on A549 cell lines.

Fig. 17. Cytotoxicity of CuBpyeTscCl and Ni[BpyeTsc]2Cl2 after 24 h incubation on L929 cell lines.

Table 1 Crystal data and structure refinement for HBpyeTsc, CuBpyeTscCl and Ni[BpyeTsc]2Cl2 Empirical formula Formula weight Temperature (K) Wavelength (Å) Crystal system Space group Unit cell dimensions

HBpyeTsc C15 H16 N4 S

CuBpyeTscCl

Ni[BpyeTsc]2Cl2 C30 H32 Cl2N8 Ni S2

P na21

C15 H15 Cl Cu N4 S 382.36 150.15 0.71073 Monoclinic P 1 21/c 1

a (Å)

17.571(8)

9.399(3)

37.5439(19)

b (Å)

9.348(4)

16.391(5)

10.9795(6)

c (Å) α (°) β (°) γ (°)

17.747(8) 90 90 90

10.670(3) 90 105.008(3) 90

16.7001(9) 90 95.631(4) 90

284.38 110.15 0.71073 Orthorhombic

680.64 110.15 1.54178 Monoclinic C 1 2/c 1

2915(2) 8 1.296

1587.7(8)

0.218 1200

1.675 780

3.289

0.57 × 0.54 × 0.29

0.25 × 0.2 × 0.07

0.05 × 0.03 × 0.02

2.295 to 27.706

2.243 to 27.564

2.365 to 62.293

Reflections collected

-22<=h<=22, -12<=k<=12, -23<=l<=23 32149

-12<=h<=12, -21<=k<=21, -13<=l<=13 18227

-42<=h<=42, -12<=k<=11, -19<=l<=18 76186

Independent reflections [R(int)]

6736 (0.0796)

3648 (0.0342)

5282 (0.0628)

Completeness to theta = 27.50°

100.0 % Semi-empirical from equivalents

99.8 % Semi-empirical from equivalents 0.7456 and 0.5938

85.2 % Semi- empirical from equivalents

Volume (Å3) Z Density Mg/m3 Absorption coefficient (mm-1) F(000) Crystal size (mm3) Theta range for data collection (°) Index ranges

Absorption correction Max. and min. transmission

4 1.600

0.7456 and 0.5223 Full-matrix leastsquares on F2

Refinement method Data / restraints / parameters Goodness-of-fit on F2 Final R indices [I>2sigma(I)] R indices (all data) Largest diff. peak and hole (e.Å-3)

6850.8(6) 8 1.320

6736 / 1 / 363 1.036 R1 = 0.0445, wR2 = 0.0973 R1 = 0.0550, wR2 = 0.1037 0.596 and -0.331

2828

Full-matrix leastsquares on F2

0.7519 and 0.5495 Full-matrix leastsquares on F2

3648 / 0 / 200 1.063 R1 = 0.0293, wR2 = 0.0682 R1 = 0.0352, wR2 = 0.0707 0.593 and -0.370

5282 / 0 / 387 1.092 R1 = 0.0463, wR2 = 0.1354 R1 = 0.0618, wR2 = 0.1418 0.761 and -0.945

Table 2 Selected bond lengths (Å) and bound angles (°) HBpyeTsc

CuBpyeTscCl

Ni[BpyeTsc]2Cl2

S(1)–C(13)

1.684(3)

Cu(1)–N(1)

1.9753(17)

Ni(1)–S(1)

2.4259(10)

N(1)–C(1)

1.349(4)

Cu(1)–N(3)

2.0021(17)

Ni(1)–S(2)

2.4241(10)

N(1)–C(5)

1.330(4)

Cu(1)–S(1)

2.2361(7)

Ni(1)–N(1)

2.030(3)

N(3)–N(2)

1.363(4)

Cu(1)–Cl(1)

2.2330(7)

Ni(1)–N(2)

2.092(3)

N(3)–C(13)

1.365(4)

N(2)–C(13)

1.321(3)

Ni(1)–N(5)

2.033(3)

N(4)–C(13)

1.322(4)

N(1)–N(2)

1.364(2)

Ni(1)–N(6)

2.080(3)

N(4)–C(14)

1.467(4)

N(4)–C(13)

1.341(3)

S(1)–C(13)

1.687(3)

C(14)–C(15)

1.504(6)

S(1)–C(13)

1.745(2)

S(2)–C(28)

1.693(3)

N(2)–N(3)–C(13)

119.2(3)

N(1)–Cu(1)–N(3)

80.63(7)

S(2)–Ni(1)–S(1)

95.72(3)

N(1)–C(1)–C(6)

117.9(3)

N(1)–Cu(1)–S(1)

84.79(5)

N(1)–Ni(1)–S(1)

82.12(8)

N(1)–C(1)–C(2)

121.3(3)

N(2)–N(1)–Cu(1)

122.77(13)

N(1)–Ni(1)–S(2)

92.83(8)

N(1)–C(5)–C(4)

123.7(3)

N(2)–C(13)–S(1)

125.68(15)

N(1)–Ni(1)–N(5)

173.37(10)

N(2)–C(6)–C(1)

127.2(3)

N(2)–C(13)–N(4)

116.93(18)

N(2)–Ni(1)–S(1)

159.02(8)

N(2)–C(6)–C(7)

113.3(3)

Cl(1)–Cu(1)–S(1)

96.70(3)

N(2)–Ni(1)–S(2)

91.69(8)

N(2)–N(3)–H(3)

120.4

N(1)–Cu(1)–Cl(1)

176.05(5)

N(5)–Ni(1)–S(1)

93.63(8)

N(3)–C(13)–S(1)

118.2(2)

N(3)–Cu(1)–S(1)

164.72(5)

N(5)–Ni(1)–S(2)

82.50(8)

N(4)–C(13)–S(1)

125.0(3)

N(3)–Cu(1)–Cl(1)

97.57(5)

N(5)–Ni(1)–N(2)

106.82(11)

N(4)–C(14)–C(15)

112.5(3)

N(3)–C(6)–C(5)

122.6(2)

N(6)–Ni(1)–S(1)

91.57(8)

C(5)–N(1)–C(1)

113.3(3)

N(4)–C(13)–S(1)

117.40(15)

N(6) –Ni(1)–S(2)

159.35(8)

C(6)–N(2)–N(3)

120.3(3)

N(4)–C(14)–C(15)

112.92(18)

N(6)–Ni(1)–N(2)

88.26(11)

C(1)–C(6)–C(7)

119.4(3)

C(1)–N(1)–Cu(1)

116.76(13)

C(13)–S(1)–Ni(1)

95.89(12)

Table 3 DNA binding constant (Kb), Stern-Volmer constant (Kq) and the apparent binding constant (Kapp) values Compound

Kb (M-1)

Kq (M-1)

Kapp (M-1)

HBpyeTsc

1.24×104

2.01×104

1.01×106

CuBpyeTscCl

2.07×104

3.93×104

1.97×106

Ni[BpyeTsc]2Cl2

1.99×104

2.79×104

1.39×106

Table 4 Protein binding constant (Kb), quenching constant (Kq) and number of binding sites (n) Compound

Kb (M-1)

Kq (M-1)

n

HBpyeTsc

3.04×104

1.00×105

0.90

CuBpyeTscCl

3.49×106

4.05×105

1.22

Ni[BpyeTsc]2Cl2

6.74×105

2.00×105

1.11

Table 5 Self-activated cleavage data of SC pUC19 DNA (30 µM) by HBpyeTsc, CuBpyeTscCl and Ni[BpyeTsc]2Cl2 (150 µM) for an incubation time of 3 h Lane No

DNA control

Percentage of cleavage (C) (%) SC

NC

1

DNA

100

0

2

DNA + 1 (150 µM)

93.61

6.39

3

DNA + 2 (150 µM)

49.27

50.73

4

DNA + 3 (150 µM)

49.78

50.22

Table 6 In vitro cytotoxic studies of the complexes against A549and L929 cancer cell lines Complex

IC50 (µM) A549

L929

CuBpyeTscCl

56.07

>800

Ni[BpyeTsc]2Cl2

80.10

>600

Cisplatin

18

6

Synthesis, X-ray Crystal Structure, DNA/protein binding, DNA Cleavage and Cytotoxicity Studies of N(4) Substituted Thiosemicarbazone Based Copper(II)/Nickel(II) Complexes Graphical abstract Copper(II) and Nickel(II) complexes containing thiosemicarbozone ligand have been synthesized and evaluated for its biological applications like DNA/protein binding, DNA cleavage and cytotoxicity studies.

Research highlights 1. Novel Cu(II) and Ni(II) complexes have been prepared and characterized by various spectroscopy techniques. 2. Cu(II) and Ni(II) complexes structure were confirmed by XRD studies. 3. Cu(II) and Ni(II) complexes interacted with DNA/BSA with appreciable binding constant. 4. A DNA cleavage study showed that the complexes cleaved DNA without any external agents. 5. Cytotoxicity study of the complexes found significant activity against lung (A549) cancer cell lines and less toxic towards the normal cell lines (L929).