Polyhedron 30 (2011) 2667–2677

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Polyhedron

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DNA interaction, superoxide scavenging and cytotoxicity studies on newcopper(II) complexes derived from a tridentate ligand

Kaushik Ghosh a,⇑, Pramod Kumar a, Nidhi Tyagi a, Udai P. Singh a, Nidhi Goel a, Ajanta Chakraborty b,Partha Roy b, Maria Camilla Baratto c

a Department of Chemistry, Indian Institute of Technology, Roorkee, Roorkee 247667, Uttarakhand, Indiab Department of Biotechnology, Indian Institute of Technology, Roorkee, Roorkee 247667, Uttarakhand, Indiac Department of Chemistry, University of Siena, Via Aldo Moro, Siena I-53100, Italy

a r t i c l e i n f o

Article history:Received 6 May 2011Accepted 13 July 2011Available online 28 July 2011

Keywords:Copper complexesCrystal structureSuperoxide dismutaseDNA interactionNuclease activityCytotoxicity

0277-5387/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.poly.2011.07.019

⇑ Corresponding author. Fax: +91 1332 273560.E-mail address: ghoshfcy@iitr.ernet.in (K. Ghosh).

a b s t r a c t

The copper complexes [Cu(Pyimpy)(H2O)](ClO4)2 (1), [Cu(Pyimpy)2](ClO4)2 (2), [Cu(Pyimpy)(Cl)2]�2H2O(3�2H2O), [Cu(Pyimpy)(N3)(ClO4)]2 (4) and [Cu(Pyimpy)(SCN)(ClO4)]2 (5) were synthesized and character-ized by spectroscopic techniques, crystal structures and electrochemical studies (Pyimpy: (2-((2-phenyl-2-(pyridin-2-l)hydrazono)methyl)pyridine)). The superoxide scavenging activity of the two water solublecomplexes 1 and 3 was examined. DNA interaction studies by UV–Vis absorption spectral changes duringa titration experiment indicated the generation of new species. These small molecule SOD mimics exhib-ited excellent DNA cleavage activity in the presence of H2O2 as well as 2-mercaptoethanol. Complexes1–5 exhibited better cytotoxicity compared to CuCl2�2H2O and the ligand Pyimpy, and showed morepotency than cisplatin for MCF-7, PC-3 and HEK-293 cells. Complex 3 exhibited the highest potencyfor MCF-7, PC-3 and HEK-293 cells compared to the other complexes.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Studies on DNA interactions with transition metal complexeshave considerable current interest because of their applications inbiotechnology and medicine [1–3]. The discovery of cis-platin fol-lowed by platinum metal based anti-tumor drugs was importantfor their use as chemotherapeutic agents. However, platinum baseddrugs exhibit some serious side effects and toxicity [4,5]. Hencethere is a continuous quest for metal based drugs that could be usedfor cancer treatment, and a few ruthenium based anti-cancer drugsare in clinical trial [6]. Among first row transition metal complexes,copper complexes possess several interesting properties whichcould be exploited to design and synthesize copper complexesexhibiting anti-tumor activity. Copper is a biologically relevant me-tal ion and it is important for the activity of several metalloen-zymes. This metal ion possesses accessible redox properties incopper complexes under physiological conditions. Moreover copperhas a high affinity for nucleobases [7,8]. Sigman and co-workersdeveloped the first copper based chemical nuclease that effectivelycleaves DNA [9]. Several copper complexes and their DNA interac-tions and nuclease activities have been reported in the literature.Some of them are mononuclear [10,11], some are binuclear [12],and multinuclear [7,8,13] copper complexes also exhibit nuclease

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activity. There are reports in the literature [3,14] where mixed li-gands copper complexes afford interesting results during DNAbinding and cleavage activity. An investigation of the literaturegave rise to two types of DNA interactions with copper complexes.The first type was a non-covalent DNA interaction and the secondone was covalent interaction. In non-covalent interactions, thereare external binding [7], groove binding [8,12] and intercalation[14]. The covalent interaction of copper with DNA could be possiblethrough the N-7 of purine bases and the phosphate group of thepolynucleotide chain [7,8]. However, there are few reports wherecopper interacts with DNA covalently or gives rise to new speciesduring DNA interaction [15–19] and such chemistry has been givenless attention [15]. The covalent interaction of copper with DNAwas evidenced by X-ray crystal structures by Geierstanger et al.[16], Kagawa et al. [17] and Gao et al. [18], however a copper saltinstead of a copper complex was used during crystallization. Onthe other hand, to the best of our knowledge, only few reports[15,19] describe the covalent interactions between DNA and coppercomplexes. Palaniandavar and co-workers found excellent nucleaseactivity for their complexes [15] which interacted covalently withDNA. Marzilli and co-workers, although not reporting nucleaseactivity, have documented excellent biological activity and acovalent interaction for CuPCPH (PCPH = 1-(a-pyridylmethylene)-2-(a-pyridyl)hydrazine) [19]. These data clearly expressed thatnuclease activity would be efficient and biologically the complexwould be very effective if the redox active copper center interacts

2668 K. Ghosh et al. / Polyhedron 30 (2011) 2667–2677

covalently with DNA. To acquire a covalent interaction as well as re-dox properties simultaneously from a chemical nuclease, a differentstrategy has been adopted in some recent reports [20–23]. In thesereports both platinum and copper were bound to the same ligandsand the DNA interaction was studied. Platinum was utilized for thecovalent binding and the redox active copper center was responsi-ble for nuclease activity. Our curiosity originated from the applica-tions of copper complexes in DNA interaction and nuclease activitystudies [24]. Recently we have reported [25] a manganese complexderived from the tridentate ligand Pyimpy, which is similar to thePCPH ligand [19]. Interesting biological activity of CuPCPH via acovalent DNA interaction prompted us to explore the copper chem-istry of Pyimpy. We have recently reported excellent anti-canceractivity of copper complexes derived from Pyimpy [26].

Hence in the present work, we report the synthesis and charac-terization of [Cu(Pyimpy)(H2O)](ClO4)2 (1), [Cu(Pyimpy)2](ClO4)2

(2), [Cu(Pyimpy)(Cl)2]�2H2O (3�2H2O), [Cu(Pyimpy)(N3)(ClO4)]2 (4)and [Cu(Pyimpy)(SCN)(ClO4)]2 (5). The molecular structures oftwo complexes, namely 3�2H2O and 4, were determined by singlecrystal X-ray diffraction. The ligand binding to the copper centerwas also supported by EPR spectral studies. Redox properties ofthe copper(II) centers were examined by electrochemical studies.It has been documented in the literature that small molecule SODmimics exhibit nuclease activity [24,25]. Hence, before studyingthe DNA interaction of the complexes, we examined the superoxidescavenging activity by xanthine–xanthine oxidase nitroblue tetra-zolium assay. The DNA binding and nuclease activity were alsoinvestigated and the results of our mechanistic studies are describedin this communication. The cytotoxicity of the complexes 1–5 withMCF-7, PC-3 and HEK-293 cells was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.

2. Experimental

2.1. Materials

Phenylhydrazine, 2-mercaptoethanol and hydrogen peroxide(S.D. Fine, Mumbai, India), sodium azide (Sigma–Aldrich, Stein-heim, Germany), copper perchlorate hexahydrate (Alfa Aesar, India,Himedia Laboratories Pvt. Ltd., Mumbai, India), ammonium thiocy-anate, ethylenediamine tetraacetic acid and cupric chloride (MerckLimited, Mumbai, India), pyridine-2-aldehyde, sodium hydride, 2-chloropyridine (Acros organics, USA) were used as obtained. Thesupercoiled pBR322 DNA and CT–DNA were purchased from Banga-lore Genei (India) and stored at 4 �C. Agarose (molecular biologygrade) and ethidium bromide were obtained from Sigma–Aldrich.Tris buffer and phosphate buffer was prepared in deionised water.Solvents used for spectroscopic studies were HPLC grade andpurified by standard procedures before use [27]. The ligand Pyimpy(2-((2-phenyl-2-(pyridin-2-yl)hydazono)methyl)pyridine) wassynthesized by the reported procedure [25].

The human breast, prostate cancer cell lines MCF-7, PC-3 andHEK-293 cells were obtained from the National Center for CellScience (NCCS), Pune, India. All the cell culture reagents were pur-chased from GIBCO (Invitrogen, USA). Penicillin, streptomycin,MTT (3-(4,5-dimethyl-2-thiazolyl-2,5-diphenyltetrazolium bro-mide), cell culture grade DMSO, agarose and all analytical gradechemicals were obtained from Himedia (Mumbai, India). Injectablecisplatin was obtained from Ranbaxy (Mumbai, India).

2.2. Instrumentations

Elemental analysis was carried out on an Elemental model VarioEL-III. Infrared spectra were recorded as KBr pellets on a NicoletNEXUS Aligent 1100 FT-IR spectrometer, using 50 scans and are

reported in cm�1. Electronic spectra were recorded in CH3CN,DMF and phosphate buffer solution with an Evolution 600, Thermoscientific, UV–Vis spectrophotometer using cuvettes of 1 cm pathlength. Fluorescence spectra were recorded by a Varian Cary(Eclipse) fluorescence spectrophotometer. Circular dichroism (CD)spectra were recorded on a Chirascan circular dichroism spectrom-eter, Applied photophysics, UK. Solid state UV–Vis spectra were re-corded by a Shimadzu UV-2450, UV–VIS spectrophotometer. Molarconductivities were determined in dimethylformamide (DMF) at10�3 M at 25 �C with a Systronics 304 conductometer. Magneticsusceptibilities were determined at 294 K with a vibrating samplemagnetometer model 155, using nickel as a standard. Magneticmeasurements were carried out on a powdered sample with aQuantum Design MPMS XL SQUID magnetometer at a temperatureranging from 5 to 300 K under an applied magnetic field of 1000 G.The TG–DTA experiments were performed on a Perkin–Elmer’s(Pyris Diamond) thermogravimetry analyzer under a N2 atmo-sphere. Cyclic voltammetric studies were performed on a CH-600electroanalyser in DMF with 0.1 M tetrabutylammonium perchlo-rate (TBAP) as a supporting electrolyte. The working electrode, ref-erence electrode and auxiliary electrode were glassy carbon, Ag/AgCl and a Pt wire electrode, respectively. The concentration ofthe compounds was of the order of 10�3 M. The ferrocene/ferroce-nium couple occurs at E1/2 = +0.44 (80) V versus Ag/AgCl under thesame experimental conditions.

EPR measurements (CW X-band (9.4 GHz)) were carried outwith a Bruker Elexsys E500 series using the Bruker ER4122 SHQEcavity. The spectra at 70 and 120 K were recorded with an OxfordESR900 helium continuous flow cryostat. The low temperaturespectra were simulated using software for fitting EPR frozen solu-tion spectra that is a modified version of a program written by J.R.Pilbrow (Cusimne) [28]. In addition the Cosmos program, writtenby Basosi and co-workers, was able to cover virtually all motionalconditions from ‘‘fast tumbling’’ to ‘‘incipient slow motion’’ for thecopper complexes [29]. All spectra were recorded with 0.5 mTmodulation amplitude and 2 mW power. The solutions were pre-pared dissolving complexes 1 and 2 in CH3CN, and 3 and 4 inDMF in order to have a final concentration of 50 mM.

Caution: Perchlorate salts of metal complexes with organic li-gands are potentially explosive. Only a small amount of materialshould be prepared and handled carefully.

2.3. Preparation of the complexes

2.3.1. Synthesis of [Cu(Pyimpy)(H2O)](ClO4)2 (1)A 5 mL methanolic solution of the ligand Pyimpy (0.137 g,

0.5 mmol) was added dropwise to a magnetically stirred 5 mlmethanolic solution of Cu(ClO4)2�6H2O (0.185 g, 0.5 mmol). Thesolution turned green immediately and the mixture was stirredin air for 2 h. After the complete evaporation of the methanol,the green solid obtained was washed with toluene and methanol.Slow evaporation of a CH3CN/toluene (3:2) solution of the complexyielded green crystals. Yield: 88%. Anal. Calc. for C17H16N4O9Cl2Cu:C, 36.80; H, 2.91; N, 11.85. Found: C, 36.32; H, 2.95; N, 11.80%. IR(KBr disk, m/cm�1): 1602 (s,mC@N), 1445 (s), 1309 (m), 1240 (m),1108 (s,br,mClO4 ), 775 (m), 703 (m), 623 (m,mClO4 ). UV–Vis [CH3CN;kmax/nm (e/(M�1 cm�1)]: 291 (9720), 300 (9280), 372 (15 650). leff

(296 K): 1.68 lB. Conductivity (KM/X�1 cm2 mol�1) in DMF: 131.

2.3.2. Synthesis of [Cu(Pyimpy)2](ClO4)2 (2)A methanolic solution (10 mL) of Cu(ClO4)2�6H2O (0.185 g,

0.5 mmol) was added dropwise to a magnetically stirred 10 mLmethanolic solution of the ligand Pyimpy (0.274 g, 1 mmol). Themixture was stirred in air for 2 h and a yellow microcrystallinecompound precipitated. The precipitate was filtered off andwashed with methanol. Yellow needle-shaped crystals for X-ray

K. Ghosh et al. / Polyhedron 30 (2011) 2667–2677 2669

crystallography were obtained within 3 days on slow evaporationof 2 in CH3CN/toluene (3:2) solution. Yield: 58%. Anal. Calc. forC34H28N8O8Cl2Cu (powder): C, 50.35; H, 3.48; N, 13.82. Found: C,49.95; H, 3.53; N, 13.99%. IR (KBr disk, m/cm�1): 1597 (s,mC@N),1470 (s), 1308 (s), 1090 (s,br,mClO4 ), 772 (m), 622 (m,mClO4 ). UV–Vis [CH3CN; kmax/nm (e/(M�1 cm�1)]: 292 (18 050), 300 (18 080),347 (27 290). leff (296 K): 1.69 lB. Conductivity (KM/X�1 cm2

mol�1) in DMF: 128.

2.3.3. Synthesis of [Cu(Pyimpy)(Cl)2]�2H2O (3�2H2O)A solution of CuCl2�2H2O (0.171 g, 1.0 mmol) in 5 ml methanol

was added to a 5 ml methanolic solution of ligand Pyimpy(0.274 g, 1 mmol) while stirring. After 2 h stirring, a green precip-itate was filtered off and washed with methanol. Crystals suitablefor X-ray analysis were obtained within three days by recrystalliza-tion of 3 from its methanol/diethyl ether (5:2) solution. Yield: 86%.Anal. Calc. for C17H18N4Cl2O2Cu: C, 45.90; H, 4.08; N, 12.60. Found:C, 45.82; H, 4.13; N, 12.65%. IR (KBr disk, m/cm�1): 1642 (m), 1602(s,mC@N), 1444 (s), 1305 (m), 1239 (m), 778 (m), 702 (m). UV–Vis[CH3CN; kmax/nm (e/(M�1 cm�1)]: 291 (8670), 300 (7850), 372(14 740). leff (296 K): 1.74 lB. Conductivity (KM/X�1 cm2 mol�1)in DMF: 22.

2.3.4. Synthesis of [Cu(Pyimpy)(N3)(ClO4)]2 (4)A 10 mL methanolic solution of Cu(ClO4)2�6H2O (0.185 g,

0.5 mmol) was added dropwise to a magnetically stirred 10 mlmethanolic solution of Pyimpy (0.137 g, 1 mmol). The mixturewas stirred in air for 10 min and the solution became green in col-or. After half an hour of constant stirring a methanolic solutioncontaining 0.0325 g (0.5 mmol) sodium azide was added dropwise.After 2 h stirring, a green precipitate was filtered off and washedwith methanol and diethyl ether and dried overnight in vacuo.Crystals of diffraction quality were obtained by slow evaporationat ambient temperature after 3–4 days from a CH3CN–DMF (6:4)solution of 4. Yield: 78%. Anal. Calc. for C34H28N14O8Cl2Cu2: C,42.60; H, 2.94; N, 20.45. Found: C, 42.91; H, 2.97; N, 20.37%. IR(KBr disk, m/cm�1): 2053 (s,mN3 ), 1598 (s,mC@N), 1564 (m), 1443(s), 1305 (m), 1114 (s,br,mClO4 ), 776 (m), 702 (m), 623 (m,mClO4 ).UV–Vis [CH3CN; kmax/nm (e/(M�1 cm�1)]: 335 (19 100), 383(3450). leff (296 K): 1.81 lB. Conductivity (KM/X�1 cm2 mol�1) inDMF: 61.

2.3.5. Synthesis of [Cu(Pyimpy)(SCN)(ClO4)]2 (5)This complex was prepared by the same procedure as for 4.

Ammonium thiocyanate was used instead of sodium azide. Yield:90%. Anal. Calc. for C36H28N10O8S2Cl2Cu2: C, 43.64; H, 2.98; N,14.14; S, 6.47. Found: C, 43.89; H, 2.85; N, 14.44; S, 6.45%. IR(KBr disk, m/cm�1): 2047 (s,mSCN), 1637 (m), 1603 (s,mC@N), 1444(s), 1307 (m), 1100–1058 (s,d,mClO4 ), 775 (m), 702 (m), 623(m,mClO4 ). UV–Vis [CH3CN; kmax/nm (e/(M�1 cm�1)]: 303 (12 130),346 (12 500), 371 (27 290). leff (296 K): 1.83 lB. Conductivity(KM/X�1 cm2 mol�1) in DMF: 67.

2.4. Superoxide dismutase assay

The superoxide dismutase (SOD) activities of complexes 1 and 3were determined by using the ability to inhibit the reduction ofnitroblue tetrazolium (NBT) by superoxide radical O2

�� generatedby the xanthine/xanthine oxidase method [24]. The reaction sys-tem contained 0.2 mM xanthine, 0.6 mM NBT and 50 mU/mL xan-thine oxidase to start the reaction in 0.1 M phosphate buffer at pH7.8. The extent of NBT reduction was followed spectrophotometri-cally by measuring the absorbance at 555 nm. Each experimentwas performed in duplicate and the SOD activity has been definedas the concentration of the tested compound for the 50% inhibitionof the NBT reduction (IC50 value) by the superoxide produced.

2.5. DNA binding and cleavage experiments

DNA binding experiments were carried out in 0.1 M phosphatebuffer (pH 7.2) using a solution of calf thymus (CT) DNA whichgave a ratio of UV–Vis absorbance at 260 and 280 nm (A260/A280)of ca. 1.8, indicating that the CT–DNA was sufficiently protein free[30]. The concentration of DNA solution was determined by theabsorbance at 260 nm and the extinction coefficient e260 was takento be 6600 cm�1, as reported in the literature [31]. Binding con-stants, (Kb) and Stern–Volmer constants (Ksv) were determinedby absorption titration and ethidium bromide fluorescencequenching experiments, respectively in 0.1 M phosphate buffer(pH 7.2) containing 1–5% DMF solution [24]. Circular dichroism(CD) spectra of CT–DNA in the absence and presence of the coppercomplexes were recorded with a 0.1 cm path-length cuvette after10 min incubation at 25 �C. The concentrations of the complexesand CT–DNA were 50 and 200 lM, respectively.

Cleavage of plasmid DNA was monitored by using agarose gelelectrophoresis. Supercoiled pBR322 DNA (100 ng) in (TBE) tris–boric acid–EDTA buffer (pH 8.2) was treated with the copper(II)complexes (50 lM) and 2–5% DMF (complexes 2, 4 and 5) in thepresence or absence of additives. The oxidative DNA cleavage bythe complexes was studied in the presence of H2O2 (200 lM, oxi-dizing agent) or 2-mercaptoethanol (200 lM, reducing agent)and KI, NaN3, DMSO, urea (2–20 mM) and catalase (200 mU). Thesamples were incubated for 1.5 h at 37 �C, added loading buffer(25% bromophenol blue and 30% glycerol). The agarose gel (0.8%)containing 2 lL (10 mg/mL stock) of ethidium bromide (EB) wasprepared and the electrophoresis of the DNA cleavage productswas performed on it. The gel was run at 60 V for 2 h in TBE bufferand the bands were identified by placing the stained gel under anilluminated UV lamp. The fragments were photographed by using agel documentation system (BIO RAD).

2.6. Cell culture and cytotoxicity assay

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) assay was carried out as described previously [32] usingMCF-7, PC-3 and HEK cell lines. The MCF-7 cells were maintainedin DMEM and PC-3 cells in DMEM-F12 media supplemented with10% fetal bovine serum (heat inactivated) and 1% antibiotic(100 U/ml of penicillin and 100 lg/ml streptomycin) mix at 37 �Cin a humidified atmosphere in a CO2 incubator. All the experimentswere performed using cells below 14 and 32 passages for MCF-7,PC-3 and HEK-293 cells, respectively. In brief, 5 � 103 cells in200 ll of medium were plated in 96-well plates (Griener, Ger-many) and grown under normal conditions. Serial dilutions ofthe family of copper complexes 1–5 ranging from 0.05 to 100 lMin ethanol were added to the monolayer in triplicates. The finalethanol concentration used for all dilutions (0.1%) was used as avehicle control. Cultures were assayed after 24 h by the additionof 50 ll of 5 mg/ml MTT and incubating for 4 h at 37 �C. TheMTT-containing medium was aspirated and 200 ll of DMSO(Himedia, Mumbai, India) and 25 ll of Sorensen glycine buffer(0.1 M glycine and 0.1 M NaCl, pH 10.5) were added to lyse thecells and solubilize the water insoluble formazon. Absorbance val-ues of the lysates were determined on a Fluostar optima (BMG Lab-tech, Germany) microplate reader at 570 nm. The percentageinhibition was calculated as:

Mean OD of vehicle treated cells—mean OD of treated cellsMean OD of vehicle treated cells

� 100

The IC50 values were calculated using graph pad prism, version 5.02software (GraphPad Software Inc., CA, USA).

Fig. 1. Ball and stick diagram of [Cu(Pyimpy)(Cl)2]�2H2O (3�2H2O) showing theatom numbering scheme. Solvent molecules and hydrogen atoms are omitted forclarity.

Fig. 2. Ball and stick diagram of [Cu(Pyimpy)(N3)(ClO4)]2 (4) showing the atomnumbering scheme. Hydrogen atoms are omitted for clarity.

2670 K. Ghosh et al. / Polyhedron 30 (2011) 2667–2677

2.7. X-ray crystallography

The X-ray data collection and processing for 3�2H2O and 4 wereperformed on a Bruker Kappa Apex-II CCD diffractometer usinggraphite monochromated Mo Ka radiation (k = 0.71070 Å) at273 K for 4 and at 296 K for 3�2H2O. The crystal structures weresolved by direct methods. Structure solution, refinement and dataoutput were carried out with the SHELXTL program [33]. All non-hydrogen atoms were refined anisotropically. Hydrogen atomswere placed in geometrically calculated positions and refined usinga riding model. Images were created with the DIAMOND program[34].

3. Results and discussion

3.1. Synthesis

The reaction of the ligand Pyimpy and Cu(ClO4)2�6H2O in meth-anol afforded [Cu(Pyimpy)(H2O)](ClO4)2 (1) when the metal–salt toligand ratio was 1:1, whereas the same reaction but with a ratio of1:2 resulted in [Cu(Pyimpy)2](ClO4)2 (2). The complex [Cu(Pyim-py)(Cl)2]�2H2O (3�2H2O) was synthesized by treating Pyimpy withCuCl2�2H2O. Treatment of complex 1 with NaN3 and NH4SCN gaverise to [Cu(Pyimpy)(N3)(ClO4)]2 (4) and [Cu(Pyimpy)(SCN)(ClO4)]2

(5) respectively. All of the above complexes were isolated with avery good yield of 58–90% and their syntheses are summarizedin Scheme 1.

3.2. Description of molecular structures

The molecular structures of complexes 3�2H2O and 4 are de-picted in Figs. 1 and 2, respectively. The matrix parameters are de-scribed in Table 1 and selected bond distances and bond angles aredescribed in Table 2. In the solution of the crystal structure ofcomplex 2 we ended up with a disorder in the solvent (toluene)molecules in the crystal lattice, however, the description of themolecular structure and the coordination around the copper centerwas clear from the crystallographic data. In the crystal structuresof 3�2H2O and 4, two pyridine nitrogen (NPy) donors and one azo-methine nitrogen (NIm) bind to the metal center in a meridionalfashion. The ligand has three six-membered rings, among them,two pyridine rings are in the same plane whereas the other phenylring is roughly perpendicular (75.59� for 3�2H2O; 78.45� for 4) tothe ligand binding plane.

3.2.1. Structure of [Cu(Pyimpy)(Cl)2]�2H2O (3�2H2O)To describe the geometry around the copper center we calcu-

lated the structural index parameter (s) [35] for this complex. De-tails are described in supporting information. The value of s clearlyindicates a distorted square pyramidal stereochemistry around the

Pyimpy[Cu(Pyimpy)(H2O)](ClO4)2

[Cu(Pyimp

[Cu(Pyimp

CuCl2 2H2O

Cu(ClO4)2 6H2O

1:1

2

(1)Cu(ClO4)2 6H2O

Scheme 1. Synthetic rout

metal centers in 3�2H2O. The square plane consists of two NPy andone NIm donors along with a Cl� ligand, whereas the other Cl�

ligand acts as the axial ligand in the molecular structure of3�2H2O. The copper center is 0.30 Å above the plane generated byN1, N2, N3 and Cl1. The Cu–NPy distances, Cu–N1 and Cu–N3, are2.0210(19) and 2.0027(19) Å, respectively, and the Cu–Nim

distance, Cu–N2, is 1.9707(19) Å, which is consistent with thereported data [24]. The equatorial Cu–Cl bond distance is2.2326(6) Å, which is close to the literature values [15,36]. In thecomplex 3�2H2O, the axial Cu–Cl bond distance, 2.5204(7) Å, islonger than the equatorial Cu–Cl distance due to the squarepyramidal geometry [15].

[Cu(Pyimpy)(N3)(ClO4)]2

[Cu(Pyimpy)(SCN)(ClO4)]2y)2](ClO4)2

y)(Cl)2]

Cu(ClO4)2NaN3

NH4SCN

6H2O

:1

(2)

(4)

(5)

(3)

Cu(ClO4)2 6H2O

1.

2.

1.

2.

es to complexes 1–5.

Table 1Summary of crystal data and data collection parameters for 3�2H2O and 4.

3�2H2O 4

Empiricalformula

C17H14Cl2CuN4O2 C34H28N14O8Cl2Cu2

Formula weight(g mol�1)

440.77 958.70

T (K) 296(2) 273(2)k (Å) (Mo Ka) 0.71073 0.71073Crystal system triclinic monoclinicSpace group P�1 P21/ca (Å) 8.1324(3) 12.495(4)b (Å) 14.7220(7) 9.810(2)c (Å) 16.6591(8) 16.598(5)a (�) 70.539(2) 90.00c (�) 85.454(2) 90.00b (�) 86.695(2) 109.454(12)V (Å3) 1873.61(14) 1918.4(9)Z 4 2qcalc (g cm�3) 1.563 1.660Crystal size (mm) 0.35 � 0.27 � 0.17 0.26 � 0.23 � 0.20F(0 0 0) 892 972h (�) 1.30–28.25 2.45–27.17Index ranges �10 6 h 6 10,

�19 6 k 6 19, �21 6 l 6 22�16 6 h 6 15,�11 6 k 6 12, �21 6 l 6 21

Data/restraints/parameters

9071/0/469 4252/0/271

Goodness-of-fit(GOF)a

1.058 0.822

R1b [I > 2r(I)] 0.0345 0.0520

R1 [all data] 0.0446 0.0758wR2

c [I > 2r(I)] 0.1174 0.1168wR2 [all data] 0.1362 0.1731

a GOF = [R[w(Fo2 � Fc

2)2]/M–N]1/2 (M = number of reflections, N = number ofparameters refined).

b R1 = R||Fo| � |Fc||/R|Fo|.c wR2 = [R[w(Fo

2 � Fc2)2]/R[(Fo

2)2]]1/2.

Table 2Selected bond lengths (Å) and angles (�) for 3�2H2O and 4.

Bond length (Å) Bond angles (�)

3�2H2OCu(1)�N(1) 2.0210(19) N(1)�Cu(1)�N(2) 79.31(8)Cu(1)�N(2) 1.9707(19) N(1)�Cu(1)�N(3) 157.16(8)Cu(1)�N(3) 2.0027(19) N(1)�Cu(1)�Cl(1) 99.05(6)Cu(1)�Cl(1) 2.2326(6) N(1)�Cu(1)�Cl(2) 90.15(6)Cu(1)�Cl(2) 2.5204(7) N(2)�Cu(1)�N(3) 79.32(8)

N(2)�Cu(1)�Cl(1) 161.24(6)N(2)�Cu(1)�Cl(2) 93.52(6)N(3)�Cu(1)�Cl(1) 98.59(6)N(3)�Cu(1)�Cl(2) 99.11(6)Cl(1)�Cu(1)�Cl(2) 105.20(2)

4Cu(1)�N(1) 1.929(5) N(1)�Cu(1)�N(4) 102.6(2)Cu(1)�N(4) 2.022(5) N(1)�Cu(1)�N(5) 169.9(2)Cu(1)�N(5) 1.960(4) N(1)�Cu(1)�N(6) 98.3(2)Cu(1)�N(6) 2.002(5) N(4)�Cu(1)�N(5) 79.89(19)

N(4)�Cu(1)�N(6) 59.12(19)N(5)�Cu(2)�N(6) 79.60(19)

K. Ghosh et al. / Polyhedron 30 (2011) 2667–2677 2671

3.2.2. Structure of [Cu(Pyimpy)(N3)(ClO4)]2 (4)The structure determination revealed that complex 4 is a cen-

trosymmetric dimeric copper complex having two copper centersbridged by two azide ions in an end-on fashion [37]. Each coppercenter is found to be coordinated to a tridentate ligand and twobridging azide and one ClO4

� anion. In this case, the halves ofthe molecule are related by a crystallographic mirror plane whichbisects the side-on bridging azide units, and each copper(II) hasone coordinated perchlorate anion. The distances from the bridgingazide to the copper centers are not equal and one distance is short-er than the other one for a particular copper center. The ligand and

the bridging N3� with a shorter Cu–NN3 distance constitute the

equatorial plane. The bridging N3� with a longer Cu–NN3 distance

and the ClO4� anion act as trans ligands, remaining in axial posi-

tions. The Cu–NPy or Cu–NIm distances for the ligands are similarto the distances found in the case of 3�2H2O. The Cu—OClO4 distanceof 2.52 Å is consistent with the reported values [38]. The equatorialCu–NN3 distance is 1.9229(5) Å whereas the axial Cu–NN3 distanceis 2.61 Å. These data are consistent with other copper dimer com-plexes bridged with azide anions in an end-on fashion with a Cu–N–Cu angle of 84.49�. The ligand biting angles at the metal centerare 79.89(19)� (N4–Cu1–N5) and 79.60(19)� (N5–Cu1–N6) in com-plex 4. The Cu� � �Cu distance of 3.394 Å is close to these types ofazide bridged dinuclear copper complexes [37,39]. This distancewill predict the probable interaction of the two copper centers.These data prompted us to study the variable temperature mag-netic susceptibility and to carry out EPR spectral studies (videinfra).

Non-covalent interactions like p-stacking interactions with arylhydrogens and hydrogen bonding networks are important insupramolecular chemistry and crystal engineering [40]. The non-covalent interactions derived from crystal structures of 3�2H2Oand 4 are described in Supporting information.

3.3. General properties

In the IR spectrum, the azomethine (–HC@N–) characteristicband for the free ligand was observed at 1600 cm�1. A small de-crease in stretching frequencies for mC@N in complexes 1–5 indi-cated the ligation of the azomethine nitrogen to the metal center[24]. Thermal analysis data indicated the presence of coordinatedwater molecules, and the presence of bands near 1090 and 623cm�1 in infrared spectra expressed the presence of the uncoordi-nated perchlorate anion in 1. An intense band near 2052 cm�1 forcomplex 4 (Fig. S1) indicated the presence of a bridged azide ligandbetween the two copper centers [37,41]. In complex 5, the strongband at 2047 cm�1 showed the presence of the coordinated thiocy-anate (Fig. S2) [42]. The lack of splitting of the band near1090 cm�1 in complex 1 suggested the presence of a non-coordi-nated perchlorate ion however, complexes 4 and 5 afforded a splitsignal at 1090 cm�1, predicting the presence of a metal coordi-nated perchlorate ion [24]. In the UV–Vis spectra, the intense elec-tronic bands observed near 335 nm for complexes 1–5 wereassignable to p–p⁄ intraligand transitions. The absorption bandsnear 372 nm for 1 and 3–5, along with a band near 355 nm for 2,were of a charge transfer type. According to the TG curve(Fig. S4, blue line) the first weight loss of �3.2% between 120.0and 150.0 �C corresponds to the release of one coordinated watermolecule. Magnetic susceptibility measurements at room temper-ature gave indicate a one-electron paramagnetic metal center forall the complexes. The molar conductivity measurement of com-plexes 1 and 2 in DMF solution (ca. 10�3 M) was found to be130 X�1 cm2 mol�1 at 25 �C, indicating bi–univalent electrolyticbehaviors. On the other hand, the values for complexes 4 and 5were in the range 61–67 X�1 cm2 mol�1 at 25 �C, showing thecomplexes to be uni–univalent electrolytes, and complex 3 affor-ded a conductivity value of 22 X�1 cm2 mol�1, confirming neutralelectrolytic behavior [43].

The molecular structure of 3�2H2O is consistent with the con-ductivity value, however we found that 4 and 5 are uni–univalentelectrolytes in solution. This prompted us to examine the solidstate electronic spectral studies. The solid state absorption spectrarecorded in a BaSO4 matrix for complexes 1, 4 and 5 are shown inFig. S3. The UV–Vis spectra in solution and in the solid state for 1are similar; however these spectra for 4 and 5 are different. It isimportant to note that both the azide bridged complex 4 and thethiocyanate bridged complex 5 show a broad band in the range

2500 2750 3000 3250 3500 3750Magnetic Field (G)

Fig. 3. X-band EPR spectra of complex 4 in DMF at 120 K with experimental (black)and simulated (red) spectral lines. (For interpretation of the references to color inthis figure legend, the reader is referred to the web version of this paper.)

Table 3Magnetic parameters and empirical data for complexes 1, 2, 3 and 4.

T (K) g|| g\ A||(G) A\(G) G a2 f (cm)

1 70 2.233 2.076 180 10 3.13 0.513 1242 120 2.258 2.084 155 8 3.12 0.567 1453 120 2.253 2.070 154 19 3.70 0.572 1464 120 2.246 2.057 162 12 4.45 0.537 138

2672 K. Ghosh et al. / Polyhedron 30 (2011) 2667–2677

600–800 nm, along with some characteristic features near 420 nm.Hence the probable dissociation of 4 in solution to the complexcation [Cu(Pyimpy)(N3)]+ and the ClO4

� anion is proposed andthe possible dissociation into [Cu2(Pyimpy)2(N3)2]2+ and 2ClO4

�

was discarded. This was also supported by the lack of d–d transi-tions in the solution spectra of the azide bridged complex[44,45]. The thiocyanate bridged dimeric copper complexes exhibitthe same characteristic bands near to 630 nm due to d–d transi-tions [46]. However we were unable to predict the mode of SCN�

bridging from only IR and UV–Vis data. Conductivity measurement,IR and solid state UV–Vis spectral studies of complex 5 show itprobably has similar structural features to 4 in solution and inthe solid state. This indicates the probable dissociation of 5 tothe complex cation [Cu(Pyimpy)(SCN)]+and the ClO4

� anion. Theabove studies indicate that all the complexes have similar mono-nuclear behavior in solution.

3.4. EPR spectra and magnetic properties

The magnetic moment data and crystal structures confirm thestabilization of the Cu(II) center in all the copper complexes 1–5reported in this study. To get more insight into the one electronparamagnetic center, we have investigated the low temperatureEPR spectral studies for complexes 1, 2, 3 and 4. The X-band EPRspectra, paired to their best fit simulations, are shown in Fig. 3for complex 4 at 120 K and in Fig. S10 for complex 1 at 70 K andfor complexes 2 and 4 at 120 K. The magnetic parameters for thesecomplexes are described in Table 3.

From Table 3 it is clear that g|| > g\ for all the complexes whichindicates that the unpaired electron predominantly lies in thedx2—y2 orbital as the ground state [47]. The g|| values for complexes1, 2, 3 and 4 are less than 2.3, suggesting the covalent character ofthe metal–ligand bond [48]. The magnitude of g|| and A|| dependson the electron donating power of a ligand to the metal center,weak coordination gives high g|| and low A|| values [49]. In ourcomplexes we found the order of g|| is 2 > 3 > 4 > 1 and the orderof A|| is 1 > 4 > 2 > 3. These data indicate that the coordination ofthe ligands in complex 1 and 4 is weaker as compared to 2 and3. Conversely, smaller g|| values are attributed to increased delocal-ization of the unpaired spin density away from the copper nucleusand are often interpreted in terms of increased covalency of themetal–ligand bond [50]. The high g|| value for complex 2 showsthe lower covalent character as compared to complexes 1, 3 and 4.

The axial symmetry parameter G is defined as [51]:

G ¼ gjj � 2:0023=g? � 2:0023

and it predicts the exchange interaction in the complexes. From Ta-ble 3 the G value for complex 4 is greater than 4, indicating no ex-change interaction between the copper centers in DMF at 120 K.However, the G values for complexes 1, 2 and 3 were less than 4,confirming considerable exchange interactions in these complexes[52]. The factor a2 is the covalency parameter [53] and it is evalu-ated by the expression:

a2 ¼ Ajj=pþ ðgjj � 2:0023Þ þ 3=7ðg? � 2:0023Þ þ 0:004

where the dipolar term p is estimated from the expression:

p ¼ ðAjj � A?Þ=ðgjj � 2Þ � 5=4ðg? � 2Þ � 6=7

The a2 value decreases with increasing covalency to a minimumtheoretical value of 0.5, and increases up to a maximum value of1.0 for a completely ionic copper–ligand bond. In our case, the a2

values are close to 0.5, indicating little ionic character in all thecomplexes [54].

The empirical quotient f = g||/A|| may be considered as a diagnos-tic parameter of the stereochemistry of complexes. At given values

of magnetic parameters, f increases with increasing tetrahedraldistortion and the range reported for square planar complexes is105–135 cm [55]. The f value for complex 1 is in the range for asquare planar geometry, however for complexes 2, 3 and 4 this va-lue is higher than 135 cm, indicating a considerable tetrahedraldistortion around the copper site, which could be due to the rigid-ity of the metal center.

The molecular structure determination of 4 indicates azidebridged dimeric copper centers. This prompted us to examine thevariable temperature magnetic susceptibility, and data were re-corded on a SQUID magnetometer using the powder form of thesample in the range 5–300 K. The plot of vm versus T is shown inFig. S11. The molar susceptibility value increases continuouslywhen the temperature decreases to 5 K, without any maximum.The value of vm at 300–60 K increased to 0.014 cm3 mol�1. On low-ering the temperature, there is a rapid increase in vm to0.109 cm3 mol�1 at 5 K. These features indicated very a weak inter-molecular antiferromagnetic interaction which is consistent withtwo copper(II) centers at a distance of 3.394 Å with a three-atomsbridge [56].

3.5. Cyclic voltammetric studies

The redox behavior of all the Cu(II) complexes was investigatedby cyclic voltammetry at a glassy carbon electrode using Ag/AgClas a reference electrode. Table 4 describes the electrochemical datafor all the complexes in DMF solution at 298 K and representativevoltammograms for 3 and 4 are shown in Fig. 4 (the remaining vol-tammograms are displayed in Supporting information). Examina-tion of the cyclic voltammograms indicates two types of curvesfor the complexes reported in this paper. Complexes 1, 2, 4 and 5gave rise to voltammograms of type A and complex 3 afforded acurve of type B. The type A curves clearly indicate a quasireversibleCuII/CuI couple having E1/2 values in the range �0.033 to 0.065 Vversus the Ag/AgCl electrode. The cyclic voltammograms of typeA afforded sharp anodic peaks in the range +70 to +140 mV, which

Table 4Cyclic voltammetric redox potentials for complexes 1–5 in DMF at 298 K. Conditions:solvent DMF; supporting electrolyte, TBAP (0.1 M); working electrode glassy-carbon;reference electrode, Ag/AgCl; scan rate 0.1 V/s, concentration �10�3 M.

E1/2[Cu(II)Cu(I)]a,V (DEp, V)b

ECu(I)/Cu(0)

(V)E1/2[Cu(II)Cu(I)],V (DEp, V)

ECu(I)/Cu(0)

(V)

1 �0.061 (�0.76) +0.140 4 �0.061 (�0.76) +0.0712 �0.033 (�0.138) +0.123 5 �0.065 (�0.100) +0.0943 �0.003 (�0.092) +0.495

a E1/2 = 0.5(Epa + Epc) where Epa Epc are anodic and cathodic peak potential,respectively.

b DEp = (Epa � Epc).

-0.52 0.00 0.52

(a)μA

0.52 0.00 0.52

V vs Ag+/ AgCl

8 μM

(b)

Fig. 4. Cyclic voltammograms of a 10�3 M solution of (a) 4 (b) 3 in DMF in presenceof 0.1 M TBAP as a supporting electrolyte, glassy-carbon as a working electrode andAg/AgCl is reference electrode; scan rate 0.1 V/s.

2 4 6 8 10

0

20

40

60

13

% In

hibi

tion

Complex (μM)

Fig. 5. Dependence of the inhibition of NBT reduction by superoxide on theconcentration of 1 and 3. Experimental condition: incubation time = 5 min,NBT = 600 lM, xanthine = 200 lM, xanthine oxidase = 50 mU.

K. Ghosh et al. / Polyhedron 30 (2011) 2667–2677 2673

were possibly due to the CuI/Cu0 couple [44,57]. Complex 3showed a quasireversible cyclic voltammogram (type B) and theE1/2 value of the CuII/CuI redox potential for 3 was found to be near�0.003 V. The anodic peak at +0.495 V for 3 was possibly due to theCuI/Cu0 couple [44,57].

3.6. Superoxide dismutase assay

We have examined the SOD activity of 1 and 3 using the xan-thine/xanthine oxidase assay due to their solubility in 0.1 M phos-phate buffer (pH 7.8). Complexes 1 and 3 showed IC50 values of7.41 and 4.92 lM, respectively and the inhibition curves are shownin Fig. 5. These values are better than the values obtained for CuSO4

and [Cu(salicylate)2], and are even better than the values for [Cu(-Phimp)(H2O)]2(ClO4)2 and [Cu(Phimp)(CH3COO)] [24].

3.7. DNA interaction studies

3.7.1. Stability of the complexes in bufferWe have examined the stability of complexes 1 and 3 in buffer

for all the biological activity studies which afforded the stability ofthe complexes in different buffer solutions (details are described inSupporting information). DNA binding studies were investigatedby absorption spectral studies, ethidium bromide displacement as-say and circular dichroism.

3.7.2. Absorption spectral studiesElectronic absorption spectroscopic techniques were used to

investigate the binding of DNA with the copper complexes. Toachieve this, the absorption spectra of the complexes in the ab-sence and presence of calf thymus DNA (CT–DNA) at different con-centrations were measured. The changes in absorbance with anisosbestic point at 358 nm for complexes 2 and 3 are shown inFig. 6 (the absorption spectra obtained for complexes 1, 4 and 5are shown in Supporting information). The binding constants Kb

for all the above complexes are described in Table 5. The intrinsicbinding constant Kb values for the complexes 1–5 have been deter-mined from the plot of DNA/(ea–eb) versus DNA and were found tobe in the range 4.0 � 103–8.8 � 103 M�1. The observed bindingconstants were similar to other complexes [58], but smaller thanclassical intercalators and metallointercalators where the bindingconstants were reported to be of the order of 107 M�1 [59]. Duringthe titration with CT–DNA we found some interesting spectralchanges. We observed a 10 nm blue shift of the CT band(345 nm) for complex 2 and the concomitant formation of a peakposition of the CT band and an isosbestic point at same wavelength(358 nm) to that of 2. These types of spectral changes are possibleonly in the case of metal DNA covalent bond formation or due tothe generation of new species during the DNA interaction[15,19]. In non-covalent interactions, such typical changes are gen-erally not observed [7,8,12,14]. In fact, at the end of the titrationwe ended up with the same spectra with a single peak at 335 nmfor all the complexes (1–5). It may be possible that the coppercomplexes became attached to the nucleic acid bases because oftheir high affinity [7,8] and hence the generation of new specieshas been suggested.

Following the procedure described by Marzilli and co-workers[19], we observed no changes for complexes 1 and 3 in the pHrange 3–10. Moreover mimicking the DNA bases, we titrated thecomplexes with pyridine and imidazole in proper buffer condi-tions. No change was observed even at high concentrations(0.5 M) of pyridine and imidazole. The absorption spectra of com-plex 3 in H2O (MilliQ) instead of tris or phosphate buffer in the

300 350 400 450

0.00

0.25

0.50

0.75

1.00A

bsor

banc

e

Wavelength (nm)

isosbestic point at 358 nm

300 350 400 4500.0

0.3

0.6

0.9

Abs

orba

nce

Wavelength (nm)

isosbestic point at 358 nm

(a)

(b)

Fig. 6. Absorption spectra of complexes 2 and 3 in 0.1 M phosphate buffer (pH 7.2)containing 4% DMF in the presence of increasing amounts of DNA. (a) [2] = 17 lM,[DNA] = 0–116.5 lM. (b) [3] = 56.7 lM, [DNA] = 0–172.3 lM.

Table 5Selected absorption binding constants (Kb) and Stern–Volmer quenching constants(Ksv) for complexes 1–5.

Complex Kb (M�1) Ksv (M�1)

1 4.5 � 103 2.75 � 104

2 8.8 � 103 7.33 � 104

3 4.0 � 103 2.65 � 104

4 5.7 � 103 –5 5.9 � 103 –Pyimpy – 1.80 � 104

550 600 650 7000

235

470

705

Inte

nsity

Wavelength (nm)

Fig. 7. Fluorescence emission spectra of EB–DNA in 0.1 M phosphate buffer (pH 7.2)in the absence (dashed line) and presence (solid line) of complex 3. [EB] = 10 lM,[DNA] = 25 lM, [3] = 0–39.2 lM, kex = 250 nm and kem = 602 nm.

2674 K. Ghosh et al. / Polyhedron 30 (2011) 2667–2677

presence of increasing amounts of DNA afforded similar spectralchanges (Fig. S16a). This eliminates the possible interaction ofPO4

� with the copper center. Moreover we examined a reversetitration experiment where we titrated DNA with the copper com-plex and investigated the spectral change (Fig. S16b). Initially wefound the formation of a peak at 335 nm, most probably due tothe generation of new species. Addition of an excess amount of 3afforded a peak at 372 nm for complex 3. These data also sup-ported the formation of the same new species. At this stage weare unable to predict the composition of the new species, howeverwe extended our DNA binding studies and the results will be de-scribed hereafter.

3.7.3. Ethidium bromide displacement assayWe have examined the competitive binding of ethidium bro-

mide (EthBr) versus our complexes with DNA using fluorescencespectral studies to get better insight into the DNA binding events.The fluorescence quenching spectra of the complexes 1, 2 and 3were recorded and a representative spectrum for complex 3 is pre-sented in Fig. 7 (spectra for complexes 1, 2 and Pyimpy are shownin Supporting information). The Stern–Volmer constant Ksv wascalculated by the plot of I0/I versus [complex] (Fig. S17b). The con-stant Ksv for the ligand Pyimpy is 1.8 � 104 and for complexes 1, 2and 3 the Ksv values are 2.75 � 104, 2.65 � 104 and 7.33 � 104,respectively (Table 5) and these are less than the values for metal-lointercalators [60]. The Ksv values indicate that only the ligand hassome quenching due to planarity. Replacement of EthBr by com-plex 2 was higher due to the presence of two ligands.

3.7.4. Circular dichroism spectral studiesThe CD spectroscopic technique is useful in monitoring the con-

formational changes of DNA in solution. The CD spectrum of CT–DNA was recorded in the range 225–300 nm and it has been foundthat there is one positive band at 278 nm due to base stacking andone negative band at 246 nm due to helicity. An examination ofFig. 8 indicates that there is a small change in both the negativeand positive bands for complexes 1 and 3. Complex 2 results in adecrease in both the positive and negative ellipticity bands. Thissuggests that the DNA interaction of these complexes does notmuch affect the conformational changes of DNA [61]. These dataindicate a better interaction of complex 2 as compared to com-plexes 1 and 3 with CT–DNA and this is consistent with our dataderived from absorption and fluorescence spectral studies.

A comparison of absorption titration, fluorescence spectral andcircular dichroism spectral data between copper complexes andzinc complexes [62] derived from Pyimpy afforded a similar modeof interaction with DNA.

3.8. Nuclease activity

An investigation of the literature afforded efficient DNA cleav-age activity for complexes which interact with DNA covalentlyand the metal center is redox active [15,19–23]. In our complexeswe found the generation of new species, probably via covalentinteraction with DNA, and we extended our DNA interaction stud-ies by examining the nuclease activity of these complexes. Thecleavage of supercoiled pBR322 DNA (100 ng) by the complexesand concomitant formation of nicked (NC) and linear (LC) DNA

240 260 280 300

-2

0

2

2

3

1DNA

θ (m

deg

)

Wavelength (nm)

Fig. 8. Circular dichroism spectra of CT–DNA and its interaction with complexes 1,2 and 3 in 0.1 M phosphate buffer (pH 7.2) after 10 min incubation at 25 �C.

1 2 3 4 5 6 7 8 9 10 11 12

K. Ghosh et al. / Polyhedron 30 (2011) 2667–2677 2675

were studied by gel electrophoresis in tris–boric acid–EDTA buffer(TBE). Due to constraints in solubility, we performed the nucleaseactivity in 5% DMF for complexes 2, 4 and 5.

DNA cleavage experiments were performed in the presence ofan oxidizing agent (H2O2) and a reducing agent (2-mercap-toethanol or BME). Complexes 1 and 3 cleaved the DNA in the pres-ence of H2O2 as well as 2-mercaptoethanol and did not exhibit(may be negligible) cleavage activity in the absence of any oxidiz-ing or reducing agents until a concentration of 80 lM (Fig. 9a, lanes4 and 10). Interestingly, the whole DNA got converted to the NCform as well as the LC form in the presence of H2O2 or BME at aminimum of 50 lM concentration of these complexes (Fig. 9a,lanes 7 and 13). However, we did not observe any LC form ofDNA in the case of complexes 2, 4 and 5, although they showednuclease activity in the presence of H2O2 or BME (Fig. 9b). The var-iation of H2O2 and BME with a fixed concentration of 3 (50 lM)indicated the conversion of the SC form to NC and LC forms wasmore dependent on the complex concentration rather than theH2O2 and BME concentrations (Fig. S20). In the control experiment,H2O2 and 2-mercaptoethanol did not exhibit any cleavage in theabsence of the copper complexes (Fig. 9a, lanes 2 and 3). Henceefficient nuclease activity was afforded by all the complexes inthe presence of oxidizing or reducing agents.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

1 2 3 4 5 6 7 8 9 10 11 12

NC

LC

SC

NC

LC

SC

(a)

(b)

Fig. 9. Gel electrophoresis separations showing the cleavage of supercoiled pBR322DNA (100 ng) by complexes 1 and 3 in the presence of H2O2 (200 lM) and BME(200 lM). Incubated at 37 �C for 1.5 h. (a) Key: lane 1, DNA control; lane 2,DNA + H2O2; lane 3, DNA + BME; lane 4, DNA + 3 (80 lM); lane 5, DNA + 3(10 lM) + H2O2; lane 6, DNA + 3 (25 lM) + H2O2; lane 7, DNA + 3 (50 lM) + H2O2;lane 8, DNA + 3 (80 lM) + H2O2; lane 9, DNA + 3 (80 lM) + BME; lane 10, DNA + 1(80 lM); lane 11, DNA + 1 (10 lM) + H2O2; lane 12, DNA + 1 (25 lM) + H2O2; lane13, DNA + 1 (50 lM) + H2O2; lane 14, DNA + 1 (80 lM) + H2O2; lane 15, DNA + 1(80 lM) + BME. (b) Key: lane 1, DNA control; lane 2, DNA + H2O2; lane 3,DNA + BME; lane 4, DNA + 2 (50 lM); lane 5, DNA + 2 (50 lM) + H2O2; lane 6,DNA + 2 (50 lM) + BME; lane 7, DNA + 4 (50 lM); lane 8, DNA + 4 (50 lM) + H2O2;lane 9, DNA + 4 (50 lM) + BME; lane 10, DNA + 5 (50 lM); lane 11, DNA + 5(50 lM) + H2O2; lane 12, DNA + 5 (50 lM) + BME.

We have examined the activity of the complexes by varying theincubation time during our gel electrophoresis experiments. Plotsof the DNA cleavage activity against incubation time for complexes1 and 3 (50 lM) in the presence of H2O2 (200 lM) are depicted inFig. 10. The reactions were monitored over a period of 15–90 minat 37 �C. Interestingly, the incubation time increased the conver-sion of the SC form to the NC and LC forms of pBR322 DNA. A15 min incubation yielded only the NC form without the formationof any linear DNA. An incubation time of 90 min was enough forcomplete conversion of the SC form to the NC and LC forms ofsupercoiled pBR322 DNA (lanes 8 and 12). The ligand Pyimpyshowed negligible nuclease activity in the presence of H2O2 or 2-mercaptoethanol (Fig. 10).

The presence of diffusible radical species can be diagnosed bymonitoring the quenching of DNA cleavage in the presence of rad-ical scavengers in solution [24]. Standard radical scavengers wereadded to the reaction of complex 3 with H2O2 or BME during theDNA cleavage experiments by gel electrophoresis. The individualaddition of DMSO, D2O, NaN3, urea (2 mM) and catalase(200 mU) had little effect on the DNA cleavage (Fig. S21). High con-centrations (20 mM) of radical scavengers did not produce any ma-jor inhibition of nuclease activity (Fig. 11). However a closer look atthe data obtained from the gel electrophoresis studies indicated avery small inhibition of nuclease activity in the presence of DMSO(Fig. 11, lane 6) and NaN3 (Fig. 11, lanes 12 and 13). D2O (whichshowed lifetime enhancement of singlet oxygen) did not enhancethe nuclease activity (Fig. 11, lanes 10 and 11), rather it showeda little inhibition of DNA cleavage.

In the lower concentration range we have found similar behav-ior of these radical scavengers on the nuclease activity (Fig. S21).Probable participation of hydrogen peroxide was also excludeddue to the results of addition of catalase during the cleavage reac-tion (Fig. 11, lanes 14–15). Hence we speculate that the hydroxylradical and singlet oxygen may have minor role in the nucleaseactivity. It is known in the literature [9] that some copper centeredradicals, which are not easily reached by radical scavengers, some-times are responsible for nuclease activity. A metal bound speciesformulated as [CuOH]2+, [CuOOH]+ and [CuO]+ have some possibil-ity for DNA strand scission [9,13,63–65]. We would like to mention

NC

LC

SC

Fig. 10. Gel electrophoresis separations showing the cleavage of supercoiledpBR322 DNA (100 ng) by complexes 1 and 3 in the presence of H2O2 (200 lM)and BME (200 lM). Incubated at 37 �C for 1.5 h. Key: lane 1, DNA; lane 2,DNA + Pyimpy; lane 3, DNA + Pyimpy (50 lM) + H2O2; lane 4, DNA + Pyimpy(50 lM) + BME; lanes 5–8, DNA + 3 (50 lM) + H2O2 + incubation time 15, 30, 60and 90 min, respectively; lanes 9–12, DNA + 1 (50 lM) + H2O2 + incubation time 15,30, 60 and 90 min, respectively.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

NC

LC

SC

Fig. 11. Gel electrophoresis separations showing the cleavage of supercoiledpBR322 DNA (100 ng) by complex 3 (50 lM) in the presence of H2O2 (200 lM)and BME (200 lM). Incubated at 37 �C for 1.5 h. Key: lane 1, DNA; lane 2,DNA + H2O2; lane 3, DNA + BME; lane 4, DNA + 3 + H2O2; lane 5, DNA + 3 + BME;lanes 6, 8, 10, 12, 14, DNA + 3 + H2O2 + DMSO, urea, D2O, NaN3, catalase, respec-tively; lanes 7, 9, 11, 13, 15, DNA + 3 + BME + DMSO, urea, D2O, NaN3, catalase,respectively.

Table 6IC50 values of cell viability assay for MCF-7, PC-3 and HEK-293 cells. The values arethe mean ± SEM of three independent experiments.

Complex IC50 (lM)

MCF-7 PC-3 HEK-293

1 14.75 ± 1.81 7.99 ± 0.88 18.58 ± 3.292 11.03 ± 0.58 10.69 ± 0.35 14.34 ± 2.003 4.29 ± 0.42 6.34 ± 0.58 5.32 ± 0.384 16.54 ± 1.04 11.10 ± 0.50 13.08 ± 2.155 7.58 ± 0.61 13.68 ± 2.59 10.51 ± 1.37CuCl2 >100 >100 >100Pyimpy 89.57 ± 0.27 96.66 ± 2.02 >100Cisplatin 64.13 ± 3.98 80.00 ± 5.19 78.30 ± 3.55

2676 K. Ghosh et al. / Polyhedron 30 (2011) 2667–2677

here that the nuclease activity studies on zinc complexes derivedfrom the same ligand did not exhibit any DNA cleavage activity,in the presence of either H2O2 or 2-mercaptoethanol [62].

3.9. Cytotoxicity assays

DNA interaction and nuclease activity studies on complexes 1–5afforded interesting data and these data prompted us to determinethe cytotoxiciy assays for the above complexes. The anti-canceractivities of 1–5 against MCF-7, PC-3 and HEK-293 have beeninvestigated in comparison with the widely used anti-cancer drugcisplatin under identical conditions by using MTT assay. Theresults (IC50 values) of our cell viability assays are shown in Table 6.The starting material CuCl2�2H2O afforded IC50 values greater than100 lM for MCF-7, PC-3 and HEK-293 cells. The ligand Pyimpy hasIC50 values of 89, 96 lM and greater than 100 lM for MCF-7, PC-3and HEK-293 cells, respectively. Interestingly, the complexes 1–5exhibited more cytotoxicity for MCF-7, PC-3 and HEK-293 cellsas compared to CuCl2�2H2O and the ligand Pyimpy. All the com-plexes 1–5 exhibited potency approximately 4–18 times more thancisplatin for MCF-7, PC-3 and HEK-293 cells. Complex 3 has thehighest potency for these three types of cells. This indicated thatthe cytotoxicities of complexes 1–5 vary from cell to cell and showdifferent potency for different cells (MCF-7, PC-3 and HEK-293).Our complexes showed better efficacy in terms of cytotoxicity ascompared to cisplatin since the IC50 values of those complexeswere at least four times lower than cisplatin in all the cell linestested. Even if the time period of 72 h had been done keeping inmind the activity time of cisplatin, the initial concentration ofthe complexes could be further reduced into the nM range and stillbe better than cisplatin. The remaining results on this biologicalactivity have been published elsewhere [26].

4. Conclusions

The synthesis and characterization of novel copper complexesderived from the tridentate ligand Pyimpy have been reported.Molecular structure determination revealed mononuclear as wellas dinuclear complexes, however solution behavior indicated thepresence of mononuclear species in solution. The complexes werecharacterized by EPR spectral data, and their redox properties wereexamined. The magnetic properties of the dinuclear complex 4afforded weak antiferromagnetically coupling between the twocopper centers. The two water soluble complexes 1 and 3 exhibitedSOD activity, affording IC50 values of 7.41 and 4.92 lM, respec-tively, and the values are better than our previous report [24].Investigation of DNA binding studies by absorption spectra indi-cated formation of a new species during DNA interaction, probablyvia covalent bond formation. To the best of our knowledge, such atype of interaction is only in very few reports available in the liter-ature [15,19]. We found out excellent nuclease activity for all of

these complexes in the presence of H2O2 and BME. Hence this fam-ily of complexes is another set of examples where small moleculeSOD mimics exhibit SOD activity as well as nuclease activity.Appreciable inhibition of nuclease activity was not observed inthe presence of radical scavengers. We speculate that the reactiveoxygen species did not have a major role in the DNA cleavage activ-ity. Some copper centered radicals which were not easily reachedby the radical scavengers or some metal bound species formulatedas [CuOH]2+ [CuOOH]+ and [CuO]+ possibly cause the DNA strandscission. Complexes 1–5 exhibited more cytotoxicity for MCF-7,PC-3 and HEK-293 cells as compared to CuCl2�2H2O and the free li-gand Pyimpy. All the complexes 1–5 showed approximately 4–18times more potency than cisplatin. Detail biological activity studiesare under progress and part of our in vitro anti-cancer activitystudies were published recently [26].

Acknowledgments

K.G. is thankful to DST, New Delhi, India for SERC FAST Trackproject (SR/FTP/CS-44/2006) for financial support. P.K. and N.T.are thankful to CSIR, India for financial assistance. We are thankfulto IITR for instrumental facilities. M.C.B. is thankful to PRIN Miur2007 and PAR 2007, University of Siena, Italy. We acknowledgeProf. Riccardo Basosi (Department of Chemistry, University of Sie-na, Italy) for giving us the possibility to use the facilities present inhis research laboratory (EPR equipment and simulation programs)and for the fruitful discussions on the results. U.P.S. is thankful toIIT Roorkee for the single crystal X-ray facility.

Appendix A. Supplementary data

Additional characterization data, packing diagrams, DNA bind-ings and nuclease experiments (Table S1–S2 and Fig. S1–S21).CCDC 773742 and 113743 contain the supplementary crystallo-graphic data for 3 and 4. These data can be obtained free of chargevia http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from theCambridge Crystallographic Data Centre, 12 Union Road, Cam-bridge CB2 1EZ, UK; fax: (+44) 1223 336 033; or e-mail: depos-it@ccdc.cam.ac.uk. CCDC reference numbers 773742 and 113743contain the supplementary. Supplementary data associated withthis article can be found, in the online version, at doi:10.1016/j.poly.2011.07.019.

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