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European Journal of Medicinal Chemistry 45 (2010) 3770e3779

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European Journal of Medicinal Chemistry

journal homepage: http: / /www.elsevier .com/locate/ejmech

Original article

Synthesis and reactivity studies on new copper(II) complexes: DNA binding,generation of phenoxyl radical, SOD and nuclease activities

Kaushik Ghosh a,*, Pramod Kumar a, Nidhi Tyagi a, Udai P. Singh a, Vaibhave Aggarwal a,Maria Camilla Baratto b

aDepartment of Chemistry, Indian Institute of Technology, Roorkee, Roorkee-247667, Uttarakhand, IndiabDepartment 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 11 January 2010Received in revised form11 May 2010Accepted 11 May 2010Available online 19 May 2010

Keywords:Copper complexesElectrochemistryGalactose oxidaseSuperoxide dismutaseDNA bindingNuclease activity

* Corresponding author. Tel.: þ91 1332285547; faxE-mail address: ghoshfcy@iitr.ernet.in (K. Ghosh).

0223-5234/$ e see front matter � 2010 Elsevier Masdoi:10.1016/j.ejmech.2010.05.026

a b s t r a c t

Tridentate ligand PhimpH binds to Cu(II) centre after deprotonation affording a new family of complexes[Cu(Phimp)(H2O)]2(ClO4)2 (1), [Cu(Phimp)2] (2) and [Cu(Phimp)(L)] (3e5) where L are CH3COO�, SCN�

and NO2� respectively. The molecular structures of complexes 1$CH3CN and 3 were determined by X-ray

crystallography. Electrochemical studies depicted Cu(II)/Cu(I) couple in the range of �0.50 to �0.65 V vs.Ag/AgCl. EPR spectral studies for complexes 4 and 5 indicated the order of covalent character in 4 > 5with dx2�y2 ground state. The phenoxyl radical complexes have been generated in situ by the oxidation ofthese complexes. Superoxide dismutase activity and DNA binding studies were examined. Thesecomplexes exhibited nuclease activity and the mechanism of DNA cleavage was investigated.

� 2010 Elsevier Masson SAS. All rights reserved.

1. Introduction

Interaction of DNA with transition metal complexes has gainedconsiderable current interest due to its various applications incancer research and nucleic acid chemistry [1]. cis-Diamminedi-chloroplatinum (II) (also known as DDP or cisplatin) and carbo-platin are used effectively as chemotherapeutic agent but they aremost effective for a particular type of cancerous cells [2]. Forexample, cisplatin is most effective for testicular and ovariancancerous cells, however, it exhibits nephrotoxicity and ototoxicside effects [2,3]. Hence, in the field of metallopharmaceuticalresearch there has been a continuous quest for new metal-basedantitumor drugs having lesser toxic effects and wider range ofapplicability for different cancerous cells. With a long-term goal formetal-based drug discovery, DNA interaction studies on severaltransition metal complexes [4e10] other than platinum complexesare finding a lot of interest and few ruthenium complexes are inclinical trial [11]. Among the first row transition metal complexes,copper complexes have several important features in this regard.Firstly, copper is a biologically relevant metal and hence its

: þ91 1332 273 5601.

son SAS. All rights reserved.

management is easier for the cells. Secondly, copper complexespossess accessible redox properties and thirdly, copper has highaffinity to the nucleobases [12].

Our interest originated from the nuclease activity of coppercomplexes having ligands containing phenolato donors. Such typeof complexes having copperephenolato bond(s) is not only efficientin hydrolytic cleavage of DNA [13] but also in oxidative cleavage[14] and in photolytic cleavage [15] of DNA. Moreover, Peralta et al.reported oxidative as well as photolytic cleavage of DNA withcopper complexes having ligands with phenolato donors [16]. DNAinteraction studies with polyphenol type ligands were also exam-ined by Tan et al. and Ghosh et al. [17,18]. Interestingly, Reedijk andhis coworkers found a copper complex [CuII(pyrimol)Cl], whichcleaves DNA by self activation where the ligand contains phenolatofunctional group [19]. Hence versatile role of copper complexescontaining ligands having phenolato donor(s) as chemical nucle-ases prompted us to examine the DNA interaction and nucleaseactivity of copper complexes. We designed ligand PhimpH (shownin Scheme 1) having two imines and one phenolato donors. Theligand was synthesized by using phenyl hydrazine as startingmaterial. This type of ligand could provide us the possible oppor-tunity of modification(s) to any one of the six-membered ringspresent in the ligand by using substituted phenyl hydrazine,substituted 2-chloropyridine and different compounds having

OH

HC

N

N N

PhimpH

Scheme 1. Schematic drawing of tridentate ligand, PhimpH.

K. Ghosh et al. / European Journal of Medicinal Chemistry 45 (2010) 3770e3779 3771

aldehyde functional group. Such modification(s) could give us thestructural and mechanistic insights of the DNA binding event. Onthe other hand, binding of tridentate ligand to copper centre mayprovide us with the possibility of synthesizing mixed ligandcomplexes to get a new family of copper complexes for DNAinteraction studies [14,15].

In the study of reactivity, presence of phenolato donor in theligand framewill help us to explore ligand centered oxidation of theresultant copper complexes. In relevance to the structural andfunctional modeling of active site of galactose oxidase (GO)enzyme, this chemistry is well known and important [20,21].Moreover, this chemistry may be exploited to develop novel cata-lysts and functional materials [22].

Herein we report the synthesis and characterization of a newfamily of copper complexes with this tridentate ligand, PhimpH.Molecular structures of two representative complexes [Cu(Phimp)(H2O)]2(ClO4)2∙CH3CN (1∙CH3CN) and [Cu(Phimp)(CH3COO)] (3)were determined by single crystal X-ray diffraction. The binding ofligand to copper centre was also supported by EPR spectral studies.Electrochemical measurements were investigated to find out thestabilization of copper(II) centre in the complexes and we havegenerated phenoxyl radical complexes upon oxidation of thecomplexes reported here.

It has been documented in the literature that the small moleculesuperoxide dismutase (SOD) mimics as well as SOD enzymesexhibit nuclease activity [23]. Several copper and manganesecomplexes, which mimic SOD activity, were shown to possessantitumor activity and have been proposed as a new class ofpotential anticancer agents [24]. Recently, we communicateda group of manganese complexes which exhibited both SOD andDNA cleavage activities [25,26]. Herein we studied SOD activity aswell as DNA interaction studies and nuclease activities with thesecomplexes and investigated the mechanism.

2. Experimental section

2.1. Reagents and material

All the chemicals were purchased from Sigma, S. D. Fine, Spec-trochem India, Acros, Himedia, Merck, and SRL. Phenyl hydrazine,2-mercaptoethanol and hydrogen peroxide (S. D. Fine, Mumbai,India), salicylaldehyde (SRL, Mumbai, India), sodium nitrite (SigmaAldrich, Steinheim, Germany), copper perchlorate hexahydrate(Alfa Aesar, India), ammonium thiocyanate, ethylenediamine tet-raacetic acid (Merck Limited, Mumbai, India), sodium hydride and2-chloropyridine (Acros organics, USA) were used as obtained.Xanthine, nitro blue tetrazolium (NBT) (Himedia, Mumbai, India)and xanthine oxidase (XO) from bovine milk. The supercoiled

pBR322 DNA and calf thymus DNA (CT-DNA) were purchased fromBangalore Genei (India). Agarose (molecular biology grade) andethidium bromide (EB) were obtained from Sigma Aldrich. Tris andphosphate buffer were prepared in deionised water. Solvent usedfor spectroscopic studies were HPLC grade and purified by standardprocedures before use [25].

2.2. Methods and instrumentation

Elemental analyses were carried out on Elementor model VarioEL-III. Infrared spectra were recorded as KBr pellets on a NicoletNEXUS Aligent 1100 FT-IR Spectrometer, using 50 scans and werereported in cm�1. Electronic spectra were recorded in, DMSO,methanol, dichloromethane and phosphate buffer solution withEvolution 600, Thermo scientific, UVevisible spectrophotometer.Fluorescence spectra were recorded by Varian Cary (Eclipse) fluo-rescence spectrophotometer. Circular dichroism (CD) spectra wererecorded on Chirascan circular dichroism spectrometer, Appliedphotophysics, UK. Magnetic susceptibilities were determined at294 K with vibrating sample Magnetometer model 155, using nickelas a standard. Cyclic voltammetric studies were performed on a CH-600 electroanalyser in DMF with 0.1 M tetrabutylammoniumperchlorate (TBAP) as supporting electrolyte. Theworking electrode,reference electrode and auxiliary electrode were glassy carbonelectrode, Ag/AgCl electrode and Pt wire respectively. The concen-tration of the compounds was in the order of 10�3 M. The ferrocene/ferrocenium couple occurs at (E1/2)¼þ0.45 (80) V vs. Ag/AgCl underthe same experimental conditions. Magnetic measurements werecarried out on powdered sample with a Quantum Design MPMS XLSQUIDmagnetometer at temperature ranging from 5 to 300 K underan applied magnetic field of 1000 G.

Caution! Perchlorate salts of metal complexes with organicligands are potentially explosive. Only a small amount of materialshould be prepared and handled with caution.

2.3. Preparation of complexes

2.3.1. [Cu(Phimp)(H2O)]2(ClO4)2 (1)A batch (0.145 g, 0.5 mmol) of PhimpH was dissolved in 8 mL

toluene, and the solution was stirred with (0.012 g, 0.5 mmol)sodium hydride for 30 min. Solution of Cu(ClO4)2$6H2O (0.185 g,0.5 mmol) in 4 mL of acetone was added to it and the reactionmixture was stirred for 2 h. A green turbidity appeared andmicrocrystalline compound was filtered and washed with tolueneand diethyl ether. Single crystals of the complex for X-ray crystal-lography were obtained within 2e3 days on slow evaporation ofacetonitrile/toluene (3:2) mixture. Yield: 95%, Anal. calcd. forC19H17.5N3.5ClO6Cu (489.8): C, 46.59; H, 3.60; N, 10.01. Found: C,46.43; H, 3.52; N, 10.12. IR (KBr disk, nmax cm�1): 1611 (nC]N), 1564,1497, 1350, 1310, 1090, 750, 629 cm�1. UVevisible (methanol): lmax(nm) (˛ (M�1 cm�1)): 392 (7840), 331 (9630), 246 (16 980). LM(DMF): 70 U�1 cm2 mol�1.

2.3.2. [Cu(Phimp)2] (2)A batch (0.289 g, 1.0 mmol) of PhimpH was dissolved in 8 mL

acetonitrile and the solution was stirred with (0.024 g, 1.0 mmol)sodium hydride for 30 min. A solution of Cu(ClO4)2$6H2O (0.111 g,0.3 mmol) in 3 mL acetonitrile was added dropwise to the depro-tonated ligand solution. After 15 min of stirring microcrystallinegreenish-yellow compound was precipitated. The solid wasseparated by filtration after 3 h of stirring, washed with acetoni-trile and diethyl ether and then dried in vacuo. Yield: 73%, Anal.calcd. for C36H28N6O2Cu (640.1): C, 67.54; H, 4.41; N, 13.13. Found:C, 68.21; H, 4.43; N, 13.38. IR (KBr disk, nmax cm�1): 1608 (nC]N),1595, 1465, 1436, 1335, 1306, 1151, 764, 698 cm�1. UVevisible

K. Ghosh et al. / European Journal of Medicinal Chemistry 45 (2010) 3770e37793772

(dichloromethane): lmax (nm) (˛ (M�1 cm�1)): 466 (1780), 404(17600), 338 (51580), 309 (40 000), 278 (35 650). meff (296 K): 1.89BM. LM (DMF): 15 U�1 cm2 mol�1.

2.3.3. [Cu(Phimp)(CH3COO)] (3)A batch of PhimpH (0.145 g, 0.5 mmol) was dissolved in 8 mL

acetonitrile and the solution was stirred with (0.012 g, 0.5 mmol)sodium hydride for 30 min. A solution of Cu(ClO4)2$6H2O (0.185 g,0.5 mmol) in acetonitrile was added into it. After half an hour ofstirring a methanolic solution containing sodium acetate (0.041 g,0.5 mmol) was added dropwise. After 2 h stirring green solutionwas generated. Evaporating the solvent, microcrystalline greencompound was precipitated out and the solid was separated byfiltration. Single crystals of the complex for X-ray crystallographywere obtained within 3e4 days upon slow evaporation of meth-anol/diethyl ether (3:2) solution. Yield: 75%, Anal. calcd. forC20H17N3O3Cu (410.9): C, 58.46; H, 4.17; N, 10.23. Found: C, 59.04;H, 4.20; N, 10.35. IR (KBr disk, nmax cm�1): 1610 (nC]N), 1563, 1488,1444, 1308, 1201, 753, 567 cm�1. UVevisible (methanol): lmax (nm)(˛ (M�1 cm�1)): 496 (530), 481 (530), 390 (11 040), 331 (12 970),246 (24 500). meff (296 K): 1.72 BM. LM (DMF): 14 U�1 cm2 mol�1.

2.3.4. [Cu(Phimp)(SCN)] (4)This complex was prepared in a similar procedure as for 3. Yield:

62%, Anal. calcd. for C19H14N4SOCu (409.9): C, 55.67; H, 3.44; N,13.67; S, 7.82. Found: C, 54.90; H, 3.26; N, 13.36; S, 7.37. IR (KBr disk,nmax cm�1): 2080 (nSCN), 1611 (nC]N), 1565, 1497, 1351, 753,702 cm�1. UVevisible (DMSO): lmax (nm) (˛ (M�1 cm�1)): 405(7650), 333 (7960), 325 (7950), 264 (11 710). meff (296 K): 1.79 BM.LM (DMF): 34 U�1 cm2 mol�1.

2.3.5. [Cu(Phimp)(NO2)] (5)This complex was prepared in a similar procedure as for 3. Yield:

65%, Anal. calcd. for C18H14N4O3Cu (410.9): C, 54.34; H, 3.55; N,14.08. Found C, 54.87; H, 3.90; N, 13.56. IR (KBr disk, nmax cm�1):1614 (nC]N), 1563, 1497, 1364, 1351, 1168, 753, 702 cm�1. UVevisible(DMSO): lmax (nm) (˛ (M�1 cm�1)): 509 (560), 497 (590), 401(12 340), 333 (13 660), 262 (19 540). meff (296 K): 1.82 BM. LM(DMF): 10 U�1 cm2 mol�1.

2.4. X-ray crystallography

The X-ray data collection and processing for (1$CH3CN) and 3were performed on Bruker Kappa Apex-II CCD diffractometer byusing graphite monochromated Mo-Ka radiation (l ¼ 0.71070 Å) at293 and 273 K respectively. Crystal structures were solved by directmethods. Structure solution, refinement and data output werecarried out with the SHELXTL program [27,28]. All non-hydrogenatomswere refined anisotropically. Hydrogen atomswere placed ingeometrically calculated positions and refined using a ridingmodel.Images were created with the DIAMOND program [29].

2.5. EPR spectral studies

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 K were recorded with an Oxford ESR900helium continuous flow cryostat. The low temperature spectrawere simulated using software for fitting EPR frozen solutionspectra that is a modified version of a program written by J.R. Pil-brow (Cusimne) [30]. In addition the Cosmos program, written byBasosi and coworkers, was able to cover virtually all motionalconditions from “fast tumbling” to “incipient slow motion” forcopper complexes [31]. All spectra were recorded with 0.5 mTmodulation amplitude and 2 mW powers. All solutions were

prepared by dissolving the complex in the DMF in order to havea final concentration of 50 mM.

2.6. Generation of the phenoxyl radical complexes

The phenoxyl radical species of the copper complexes weregenerated in situ by adding (NH4)2[CeIV(NO3)6] (CAN, 2.0� 10�4 M)into a CH3CN solution of these phenolate complexes (1.0 � 10�4 M)in a UV cell at 0 �C.

2.7. Superoxide dismutase assay

SOD activity of the complexes 1 and 3were determined by usingthe ability to inhibit the reduction of nitro blue tetrazolium (NBT)by superoxide radical O2�

� generated by the xanthine/xanthineoxidase method [32]. The reaction system contained 0.2 mMxanthine, 0.6 mM NBT and 70 mU/mL xanthine oxidase to start thereaction in 0.1 M phosphate buffer at pH 7.8. The extent of NBTreduction was followed spectrophotometrically by measuring theabsorbance at 560 nm. Each experiment was performed in dupli-cate and the SOD activity has been defined as the concentration ofthe tested compound for the 50% inhibition of the NBT reduction(IC50 value) by superoxide produced.

2.8. DNA binding and cleavage experiments

These experiments were carried out in 0.1 M phosphate buffer(pH 7.2) using a solution of calf thymus DNA (CTeDNA) which gavea ratio of UVevisible absorbance at 260 and 280 nm (A260/A280) ofca.1.8, indicating that the CT-DNAwas sufficiently protein free [33].The concentration of DNA solution was determined by UV absor-bance at 260 nm and the extinction coefficient e260 was taken6600 cm�1 as reported in the literature [34]. Absorbance titrationexperiments were carried out with complex concentration of50 mM varying the CT-DNA concentration from 0e170 mM in 0.1 Mphosphate buffer (pH 7.2) containing 5% DMF. The bindingconstants Kb were determined from a plot of [DNA]/(ea � ef) vs.[DNA] using the equation: [DNA]/(ea � ef) ¼ [DNA]/(eb � ef) þ [Kb(eb � ef)]�1, where [DNA] is the concentration of DNAin base pairs. The apparent absorption coefficients ea, ef and ebcorrespond to Aobs/[Complex], the extinction coefficient of the freecomplex and the extinction coefficient of the copper complexes inthe fully bound form respectively.

All the complexes were dissolved in phosphate buffer (pH 7.2)used for DNA interaction studies and kept for one week. A smallchange in absorbance without any l shifting was observed inUVevisible spectrum which predicts the stability of the complexesin the above buffer solution.

Fluorescence quenching experiments were carried out by thesuccessive addition of complexes 1, 2 and 3 to the DNA (25 mM)solutions containing 5 mM ethidium bromide (EB) in 0.1 M phos-phate buffer (pH 7.2). For better solubility of complex 2 we use 5%DMF. These samples were excited at 250 nm and emissions wereobserved between 500 and 700 nm. SterneVolmer quenchingconstants were calculated using the given equation.

I0=I ¼ 1þ KsvQ ;

Where I0 and I are the fluorescence intensities in the absence andpresence of complex and Q is the concentration of quencher(complexes 1, 2 and 3). Ksv is a linear SterneVolmer constant givenby the ratio of slope to intercept in the plot of I0/I versus Q.

Circular dichroism (CD) spectra of CTeDNA in absence andpresence of the copper complexeswere recordedwith a 0.1 cmpath-

K. Ghosh et al. / European Journal of Medicinal Chemistry 45 (2010) 3770e3779 3773

length cuvette after 10min incubation at 25 �C. The concentration ofthe complexes and CT-DNAwere 50 and 200 mM respectively.

Cleavage of plasmid DNA was monitored by using agarose gelelectrophoresis. Supercoiled pBR322 DNA (100 ng) was incubatedwith complexes 1e5 for 2 h at 37 �C. The oxidative DNA cleavage bythe complexes were studied in the presence of H2O2 (200e400 mM,oxidizing agent) or 2-mercaptoethanol (BME) (200e400 mM,reducing agent). Cleavage of pBR322 DNA in presence of standardradical scavengers and reaction inhibitors were analyzed. Reactiveoxygen species (ROS) scavengers like DMSO, D2O, urea, NaN3(20 mM) and catalase (200 mU) were added to the reactionmixture. After incubation, added loading buffer (25% bromophenolblue and 30% glycerol). The agarose gel (0.8%) containing 0.4 mg/mLof ethidium bromide (EB) was prepared and the electrophoresis ofthe DNA cleavage products was performed on it. The gel was run at60 V for 2 h in Triseboric acide EDTA (TBE) buffer and the bandswere identified by placing the stained gel under an illuminated UVlamp. The fragments were photographed by using gel documen-tation system (BIO RAD).

3. Results and discussion

3.1. Syntheses and general properties

Ligand PhimpH was prepared according to the reported proce-dure [25]. Reaction of the deprotonated ligand (Phimp�) and Cu(ClO4)2$6H2O in toluene: acetone (2:1) afforded [Cu(Phimp)(H2O)]2(ClO4)2 (1) when metal-salt to ligand ratio was 1:1. On theother hand, the reaction with 1:3 of metal-salt to ligand ratioresulted [Cu(Phimp)2] (2). Treatment of complex 1with CH3COONa,NH4SCN and NaNO2 gave rise to complexes [Cu(Phimp)(CH3COO)](3), [Cu(Phimp)(SCN)] (4) and [Cu(Phimp)(NO2)] (5) respectively.Complexes were isolated with a very good yield of 60e95% andtheir syntheses were summarized in Scheme 2.

In IR, azomethine (eHC ¼ Ne) characteristic band in free ligandwas observed at 1615 cm�1. Coordination of the nitrogen with themetal centre was expected to reduce the electron density in theazomethine moiety and thus lower the nC]N [35]. Decrease instretching frequencies for nC]N in complexes 1e5 clearly indicate theligationof azomethinenitrogen tometal centre. Complex1 showed IRbands near 1118 and1080 cm�1 together with a band at 628 cm�1

(Fig. S1). The splitting of these two bands suggested the presence ofcoordinatedperchlorate ion to themetal centre [36]. Incomplex4, thestrong band at 2080 cm�1 showed the presence of the N-coordinatedthiocynate (Fig. S2) [37]. The two bands at 1364 and 1168 cm�1 incomplex 5 (Fig. S3) assigned the presence of nitrite group bound tocopper centre and the absence of d (ONO) mode (approximately at840e890 cm�1) suggested that the nitrite ligand was monodentate[38]. The absorption band 388e406 nm for all complexes in differentsolvents shows a strong charge transfer transition. These bands areprobably due to phenolate / Cu(II) ligand-to-metal charge transfer(LMCT) [14]. The molar conductivity measurement of complex 1 in

PhimpH [Cu(Phimp)(H2O)]

2(C

[Cu(Phimp)2]

Cu(ClO4)2• 6H2O

1:13:1

(2)

Cu(ClO4)2• 6H2O

Scheme 2. Synthetic proced

DMF solution (ca.10�3M)was found to be 70U�1 cm2mol�1 at 25 �Cindicating unieuni valent (1:1) electrolyte behaviors (vide infra). Onthe other hand the conductivity values of complexes 2e5were in therange of 10e35 U�1 cm2 mol�1confirming neutral electrolyticbehavior [39].

3.2. Description of structures

In order to confirm mode of coordination of the ligand PhimpH,crystals were grown for complex (1∙CH3CN) in acetonitrile/toluene(3:2), for complex 3 in methanol/diethyl ether (3:2) solution andstructures were determined by X-ray diffraction studies. We werehaving difficulties with perchlorate anions during the structuresolution of complex 1∙CH3CN. This is most probably due to thelarge thermal motion of perchlorate anions at room temperature.This could be avoided by collecting the data at low temperature.However, we continued our studies with the structure derived fromthese data. A ball and stick representation of themetal coordinationenvironment in complexes 1∙CH3CN and 3 were displayed in Fig. 1and Fig. 2 respectively. Table 1 summarizes the experimentalcrystal data for complexes 1∙CH3CN and 3 and their selected bondlengths and bond angles are listed in Table 2.

Phenolato oxygen (OPh), azomethine nitrogen (NIm) and pyri-dine nitrogen (NPy) bind to the metal centre for both complexes inmeridional fashion. Fig. 1 is a view of [Cu(Phimp)(H2O)]2(ClO4)2∙CH3CN in complex 1∙CH3CN. In case of 1∙CH3CN, the halves of themolecule are related to a crystallographic mirror plane whichbisects the bridging perchlorate units and each copper(II) centre isfive coordinated. The equatorial plane of the coordination sphereconsists of the phenolato oxygen (OPh), azomethine nitrogen (NIm),pyridine nitrogen (NPy) and H2O molecule while the axial positionsare occupied by perchlorate ions. In complex 3 the Cu(II) centreadopted a distorted square planar geometry where copper centre iscoordinated to OPh, NIm and NPy atoms of the ligand along with Oatom of acetate ion (Fig. 2) respectively. The ligand biting angles atthe metal centre are nearly 81.7� (N4eCu2eN5) and nearly 93.6�

(N5eCu2eO2) in complex (1∙CH3CN) and the other two angleswith H2O are 90.26� and 94.58�. These biting angles have similarvalues of 81.3� (N1eCu1eN2) and 92.2� (N2eCu1eO2) for complex3 and the monodentate CH3COO� ion makes 90.6� and 95.6� at themetal centre. In complex 1∙CH3CN the bridging perchlorate anionoccupies the apical position between two copper centres withCueOClO4 bond lengths of 2.49 Å and 2.51 Å in the crystal lattice.The distance is slightly longer than the distance (2.46 Å) reportedby Nair and coworkers [40]. The perchlorate bridged Cu/Cudistance is 4.51 Å which is less than the reported value [41]. TheCueOPh distances in both the complexes are consistent with thestructures reported by Ghosh and coworkers [42] however, slightlylower than the values reported in the literature [43]. In complex1∙CH3CN, CueNIm distance (1.94 Å) is similar however, in complex3 the CueNIm distance (1.99 Å) is longer to the crystal data reported[43,44]. Stronger coordination of NPy to Cu(II) centre was obtained

lO4)2

[Cu(Phimp)(NO2)]

[Cu(Phimp)(SCN)]

NaNO2

NH4SCN(1)

(5)

(4)

[Cu(Phimp)(OAc)] (3)

CH3COONa

ures of complexes 1e5.

Table 1Summary of crystal data and data collection parameters for [Cu(Phimp)(H2O)]2(ClO4)2 $CH3CN (1$CH3CN) and [Cu(Phimp)(CH3COO)] (3).

Empirical formula C38H35Cl2Cu2N7O12 C20H17CuN3O3

Formula weight(gmol�1)

979.71 410.92

Temperature/K 293(2) 273(2)l (Å) (MoeKa) 0.71073 0.71073Crystal system Monoclinic TriclinicSpace group P21/C P-1a (Å) 17.972(5) 8.324(10)b (Å) 12.236(5) 10.935(14)c (Å) 18.883(5) 11.580(15)a (�) 90.000(5) 65.91(4)g (�) 90.000(5) 68.39(4)b (�) 93.302(5) 74.90(4)V (Å3) 4146(2) 887(2)Z 4 2rcalc (gcm�3) 1.570 1.538Crystal size (mm) 0.24 � 0.19 � 0.17 0.23 � 0.19 � 0.17F(000) 1928 423Theta range for

data collection2.01e26.86 1.94e27.02

Index ranges �22 < h < 16, �15< k < 15, �24 < l < 23

�10 < h < 10, �13< k < 13, �14 < l < 14

Refinement method Full matrixleast-squares on F2

Full matrix least-squareson F2

Data/restraints/parameters 8705/26/563 3876/0/245GOFa on F2 1.025 0.833R1b [I > 2s(I)] 0.0598 0.0724R1[all data] 0.1100 0.1374wR2c [I > 2s(I)] 0.1659 0.1821wR2 [all data] 0.2009 0.2496

a GOF ¼ [S[w(Fo2 � Fc2)2]/M � N]1/2 (M ¼ number of reflections, N ¼ number of

parameters refined).b R1 ¼ SkFoj � jFcjSjFoj.c wR2 ¼ [S[w(Fo2 � Fc

2)2]/S [(Fo2)2]]1/2.

Fig. 1. Ball-and-stick representation of the crystal structure of [Cu(Phimp)(H2O)]2(ClO4)2 $CH3CN (1∙CH3CN). Atoms are shown as spheres of arbitrary diameter.

Table 2Selected bond lengths (Å) and angles (�) for [Cu(Phimp)(H2O)]2(ClO4)2 $CH3CN(1$CH3CN) and [Cu(Phimp)(CH3COO)] (3).

K. Ghosh et al. / European Journal of Medicinal Chemistry 45 (2010) 3770e37793774

in complexes 1∙CH3CN and 3 because CueNPydistances observedfor these two complexes were smaller than the distances reportedin the literature (close to 2 Å) [45,46]. All these distances confirmthe binding of Phimp� to Cu(II) metal centre.

In 1∙CH3CN, water molecule is bound more tightly to the metalcentre with CueOH2 distance of 1.94 Å which is lower than thereported data (1.99e2.05 Å) [44]. The CueO(CH3COO�) distance(1.99 Å) in complex 3 is longer than values available in the literature[34]. The distance of other oxygen of the carboxylate group (shownas dotted line in Fig. 2) is 2.74 Å which is longer than the reportedbidentate carboxylate ligand [46]. Hence, binding of acetate ion hasbeen considered as monodentate for complex 3. All the bonddistances CueOPh, CueNIm and CueNPy in complex 1∙CH3CN arelower than complex 3. Hence, in complex 1∙CH3CN the tridentateligand is more tightly bound as compared to complex 3. The ligandhas three six-membered rings; among them, the pyridine and thephenolato ring are in the same plane whereas the other phenyl ringis roughly perpendicular (88.93� for complex 1∙CH3CN and 86.82�

for complex 3) to the ligand binding plane.p-Stacking interactions with aryl hydrogen and hydrogen

bonding network are important in supramolecular chemistry and

Fig. 2. Ball-and-stick representation of the crystal structure of [Cu(Phimp)(CH3COO)](3). Atoms are shown as spheres of arbitrary diameter.

crystal engineering [47]. Non-covalent interactions found in thecrystal structures of 1∙CH3CN and 3 are described in supportinginformation. There are three types of non-covalent interactionsobserved in the packing diagram of complex 3. Firstly, arylhydrogen-phenolato oxygen hydrogen bonding (distance 2.58 Å)and secondly, aryl hydrogen-carboxylato oxygen (distance 2.34 Å)interaction described in Fig. S5. Thirdly, the aryl hydrogen-pyridinering (CeH/p) weak interaction (distance 2.96 Å) shown in Fig. S6.The distance between two copper centers in complex 3 is 11.49 Åprecluding any possibility of direct Cu/Cu interaction, whereas the

Bond length (Å) Bond angles (�)

[Cu(Phimp)(H2O)]2(ClO4)2$CH3CN (1$CH3CN)Cu(1)eO(1) 1.889(3) O(1)eCu(1)eN(1) 174.69(14)Cu(1)eN(1) 1.957(3) O(1)eCu(1)eN(2) 93.48(14)Cu(1)eO(1w) 1.957(3) O(1)eCu(1)eO(1w) 90.28(14)Cu(1)�N(2) 1.940(4) N(1)eCu(1)eN(2) 81.64(14)Cu(1)eO(11) 2.516 N(1)eCu(1)eO(1w) 94.57(15)Cu(2)eO(2) 1.888(3) N(2)eCu(2)eO(1w) 176.16(14)Cu(2)eN(4) 1.964(4) O(2)eCu(2)eN(4) 174.74(14)Cu(2)eO(2w) 1.964(3) O(2)eCu(2)eN(5) 93.66(14)Cu(2)eN(5) 1.939(4) O(2)eCu(2)eO(1w) 90.00(15)Cu(2)eO(12) 2.493 N(4)eCu(2)eN(5) 81.79(15)

N(4)eCu(2)eO(1w) 94.57(15)N(5)eCu(2)eO(1w) 176.33(15)

[Cu(Phimp)(CH3COO)] (3)Cu(1)eO(4) 1.990(8) O(2)eCu(1)eN(1) 173.3(3)Cu(1)eN(1) 1.987(8) O(2)eCu(1)eO(4) 90.6(3)Cu(1)eN(2) 1.990(8) O(2)eCu(1)eN(2) 92.2(3)Cu(1)eO(2) 1.896(7) O(4)eCu(1)eN(2) 171.0(4)O(4)eCu(2) 94.51(14) N(1)eCu(1)eN(2) 81.3(3)O(3)eCu(1) 93.59(13) N(1)eCu(1)eO(4) 95.6(3)

4

252 294 336 378

5

Magnetic field (mT)

Fig. 3. 70 K X-band EPR spectra of complexes (4) and (5) in DMF (blue or exp) paired totheir best fit simulations (pink or sim). (For interpretation of the references to colour inthis figure legend, the reader is referred to the web version of this article.)

0 100 200 3000.0

0.2

0.4

0.6

0.8

mχmc(

T3

lom

1-)

K

T (K)

Fig. 4. Thermal dependence of cmT for complex 1.

Table 4Cyclic voltammetric redox potentials for complexes 1e5 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 w10�3 M.

K. Ghosh et al. / European Journal of Medicinal Chemistry 45 (2010) 3770e3779 3775

same distance in complex 1∙CH3CN is 4.51 Å. From the structuralevidences for complex 1∙CH3CN it has been found out that one ofthe perchlorate anions is bridged between two copper centers. Thisprompted us to determine the variable temperature magneticmoment (vide infra).

3.3. EPR spectral studies

The magnetic moment data and the crystal structure confirmthe stabilization of Cu(II) centre in all metal complexes reported inthis study. To get more insight into the one-electron paramagneticcentre we have investigated complexes 4 and 5 by EPR at lowtemperature. The X-band EPR spectra paired to their best fitsimulations for complexes 4 and 5 in frozen solution at 70 K areshown in Fig. 3 and magnetic parameters are reported in Table 3.The EPR spectra of the complexes 4 and 5 in DMF exhibit a set ofthree well resolved transitions in the low field region, a typical caseof axial geometry.

From Table 3 it is clear that gk > gt for complexes 4 and 5which indicates that the unpaired electron predominantly lies in

Table 3Magnetic parameters and empirical data for complexes 4 and 5.

Complex gk gt Ak(mT)(�0.1)

At(mT)(�0.1)

G a2 f(cm)

4 2.242(�1) 2.055(�2) 17.8 1.1 4.548 0.524 1265 2.245(�1) 2.062(�2) 17.8 1.0 4.065 0.532 126

the dx2�y2 orbital as a ground state [48]. gk values for complexes 4and 5 are less than 2.3 suggesting covalent character of the metal-ligand bond [49].

The axial symmetry parameter G is defined as [50]

G ¼ gk � 2:0023=gt � 2:0023

and it predicts the exchange interaction in the complexes. FromTable 3 the G values for both the complexes 4 and 5 are greater than4, indicating no exchange interaction between the copper centersin solid sample. The factor a2 is a covalency parameter, whichdescribes the in-plane sigma bonding, arises from the dipoleedi-pole interaction between magnetic moments associated with thespin motion of the electron and the nucleus [51]. The parameter a2

for the metal-ligand bond is evaluated by the expression

a2 ¼ Ak=pþ�gk � 2:0023

�þ 3=7ðgt � 2:0023Þ þ 0:004

Where the dipolar term p is estimated from the expression

p ¼�Ak � At

�.�gk � 2

�� 5=4ðgt � 2Þ � 6=7

a2 value decreases with increasing covalency to a minimum theo-retical values of 0.5 up to a maximum of 1.0 for a completely ioniccopper-ligand bond. In our case a2 are decreasing as 5 > 4, socomplex 5 have more ionic character [52].

The empirical quotient f ¼ gk/Ak may be considered as a diag-nostic parameter of the stereochemistry of the complexes. At givenvalues of magnetic parameters f increases with increasing tetra-hedral distortion and the range reported for square planarcomplexes is 105e135 cm [53]. The f values for complexes 4 and 5are in the range of square planar.

Complex E1/2[Cu(II)eCu(I)],a

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

V (DEp, mV)

1 �0.50 (87) 4 �0.55 (138)2 �0.61 (177) 5 �0.56 (102)3 �0.65 (137) e

a E1/2 ¼ 0.5(Epa þ Epc) where Epa Epc are anodic and cathodic peak potentialrespectively.

b DEp ¼ (Epa � Epc).

O

HC

N

N N

Cu OAc

O

HC

N

N N

Cu X

2 eq. [(NH4)2Ce(NO3)6]

0 C

O

X = CH3COO or NO

3

.

Scheme 3. Schematic drawing for generation of phenoxyl radical complexes.

Table 5UVevisible data of phenoxyl radical of complexes 1e5 in acetonitrile.

Complex lmax

1 415, 490, 7152 404, 550, 7003 405, 480, 7204 400, 6505 395, 675

K. Ghosh et al. / European Journal of Medicinal Chemistry 45 (2010) 3770e37793776

3.4. Magnetic properties

The one-electron paramagnetic complexes (2e5) have copperatom in þ2 oxidation state implicated by magnetic susceptibilitymeasurements at room temperature [54]. However, molecularstructure determination of complex 1∙CH3CN indicated perchlorateanion bridged dimeric copper centres. This prompted us to examinethe variable temperature magnetic susceptibility data for complex1∙CH3CN. Data were recorded on SQUID magnetometer using thepowder formofsample in the rangeof5e300K.TheplotofcmTversusT is shown in Fig. 4. The valueofcmTat 5Kwas0.054 cm3mol�1 K andincreased gradually to a value of 0.75 cm3mol�1 K at 90 K. This valueof cmT remains approximately constant (0.75 cm3mol�1 K) from90Kto room temperature. Such solid state magnetic behavior is trulycharacteristic of weak antiferromagnetic interaction [55] which wasconsistentwith two copper(II) centres at a longdistance (4.51Å)witha three e atoms bridge. However, the behavior of this complex insolution is under progress and we suspect that most probablya monomeric species is generated.

3.5. Cyclic voltammetric studies

The redox behavior of all Cu(II) complexes was investigated bycyclic voltammetry at a glassy carbon electrode using Ag/AgClreference electrode. Table 4 describes the electrochemical data forall the complexes in DMF solution at 298 K (shown in supportinginformation).

All the complexes show quasi-reversible cyclic voltammogramhaving DEp values in between 87e200 mV [54]. Repeated scans, as

Fig. 5. UVevisible spectral change upon the oxidation of complex 3 (1.0 � 10�5 M)with CAN (2.0 � 10�5 M) in CH3CN at 0 �C. Inset: time course of absorption change at720 nm due to the decomposition of phenoxyl radical complex of 3.

well as different scan rates, showed that dissociation does not takeplace in these complexes. The E1/2 values for CuII/CuI couple werefound to be in the potential range �0.50 to �0.65 V vs. Ag/AgCl.Phenolato oxygen stabilizes higher oxidation states and the nega-tive potentials are consistent with the data reported by Tolman andcoworkers [56]. Complexes 2 and 3 have more negative E1/2 valuesand complex 4 have less negative E1/2 values which are expectedfrom simple hard/soft and acid/base considerations. The borderlinebase NO2

� in complex 5 has E1/2 value (�0.56 V) which is in betweenthe above two classes. It has been reported that E1/2 value shifts tomore negative potential due to the replacement of neutral solventligand by anionic ligands [57]. Interestingly, consistent data wereobtained in our investigation.

3.6. Generation of phenoxyl radical complexes

Generation of phenoxyl radical complexes through the oxida-tion of phenolato copper(II) complexes with (NH4)2[CeIV(NO3)6](CAN) and relevant chemistry are important for the active sitemodeling of GO [58,59]. Addition of two equivalents of the CANafforded the corresponding phenoxyl radical complexes at 0 �C(Scheme 3). During our studies on the synthesis of phenoxyl radicalcomplexes we found that the radicals were unstable and graduallydecomposed within very short time (w1 min) because of theparticipation of oxygen or solvent for the regeneration of theprecursor complex. The formation of the phenoxyl radicalcomplexes showed greenisheyellow color and have beenconfirmed by the characteristic peaks in UVevisible spectra for GOenzyme [20,21]. Upon addition of CAN, the LMCT band for thecomplex 3 at 390 nm readily disappeared together with theconcomitant appearance of the new bands at 405, 480 and 720 nmwhich indicated the formation of phenoxyl radical complex (Fig. 5).A similar absorption spectrum (415, 490 and 715 nm) was alsoobtained during the oxidation of 1 in the same oxidation procedure(Fig. S13). Complex 2 containing two phenolato donors affordedtwo peaks at 550 and 700 nm upon oxidation by CAN (Fig. S14).These changes in absorption spectrawere obtained in the oxidationof the complexes 4 and 5 by CAN under the same experimentalcondition as shown in Fig. S15 and Fig. S16 respectively. Table 5shows all the characteristic peaks in absorption spectra of phe-noxyl radical complexes 1e5.There was no change in LMCT bandand no peaks at 480e490 nm were observed in absorption spectraof radical complexes 4 and 5 however peaks at 650 nm for 4 and

Table 6SOD activity of complexes 1 and 3, [Cu(salicylate)2], [Cu(NA/Sal)] (NA/Sal¼ nicotinic-salicylic acid), CuSO4 and the native CuZnSOD as assessed by the NBTassay.

Complex IC50 (mM)

1 11.203 8.31[Cu(salicylate)2] 16[Cu(NA/Sal)] 42.79CuSO4 30Native CuZnSOD 0.04

0 10 20 30 40

20

40

60

80

Complex 3Complex 1

)%(

noitibihnI

Concentration ( M)μ

Fig. 6. Effect of complexes 1 and 3 on the inhibition of the reduction. Incubation timewas 5 min.

Table 7Selected binding constants (Kb) from absorption titration and SterneVolmerquenching constants (Ksv) from fluorescence quenching for the complexes 1e4.

Complex Kb (M�1) Ksv (M�1)

[Cu(Phimp)(H2O)]2 (ClO4)2 (1) 1.43 � 104 3.01 � 104

[Cu(Phimp)2] (2) 1.99 � 104 9.00 � 104

[Cu(Phimp)(OAc)] (3) 1.06 � 104 4.61 � 104

[Cu(Phimp)(SCN)] (4) 0.94 � 104 e

K. Ghosh et al. / European Journal of Medicinal Chemistry 45 (2010) 3770e3779 3777

675 nm for 5 were observed. It is reported in the literature thatstability of the phenoxyl radical complexes increase with theincrease in l value of the lowest energy transition. Itoh andcoworkers reported [60] stable phenoxyl radical complexes derivedfrom eSMe substituted ligand. Those complexes showed lowestenergy transition at 870 nm in UVevisible spectra. On the otherhand, McMaster and coworkers [22] got the same band at around700e800 nm because their ligands did not possess eSMe group.The stability order of the phenoxyl radical complexes was3 > 1 > 2 > 5 > 4 which also follows the order of their lowestenergy transition.

3.7. Superoxide dismutase assay

Wehaveexamined theSODactivityof1and3using thexanthine/xanthine oxidase assay due to their conducive solubility. SODactivity wasmonitored by reduction of nitro blue tetrazolium (NBT)with O2�

� generated by xanthine/xanthine oxidase system. As thereaction proceeds, the farmazan color is developed and a colorchange fromyellow to blue appeared which was associated with an

300 350 400 450 5000.00

0.25

0.50

0.75

ecnabrosbA

Wavelength (nm)

Complex 1

Fig. 7. Absorption spectra of complexes 1 in 0.1 M phosphate buffer (pH 7.2) in thepresence of increasing amounts of DNA. [Complex] ¼ 50 mM, [DNA] ¼ 0e170 mM.Arrows show the absorbance changes upon increasing DNA concentration.

increase in the absorbance at 560 nm. The rate of absorption changewas determined and the concentration required to produce 50%inhibition (IC50) be obtained by graphing the rate of NBT reduction(along Y-axis) versus the concentration of the test solution (along X-axis). Table 6 shows the IC50 values for complexes 1 and 3 alongwiththe IC50 values for [Cu(salicylate)2] [61], [Cu(NA/Sal)] (NA/Sal ¼ nicotinic-salicylic acid) [62], CuSO4 [63] and the native CuZn-SOD [23] as assessed by NBT assay. The complexes 1 and 3 showedIC50 values of 11.20 and 8.31 respectively (Fig. 6). The IC50 values forour copper complexes were similar to the value reported for [Cu(salicylate)2] and better than the values reported for [Cu(NA/Sal)]and CuSO4.

3.8. DNA binding and cleavage experiment

3.8.1. Absorption studiesElectronic absorption spectroscopic technique was used to

investigate the binding of DNA with copper complexes. To achievethis, the absorption spectra of complexes in the absence andpresence of calf thymus DNA (CTeDNA) at different concentrationswere measured. The change in absorbance with an isosbestic pointat 358 nm for complex 1 are shown in Fig. 7 (absorption spectraobtained for complexes 2, 3 and 4 are shown in Fig. S17eS19). Uponaddition of DNA, a considerable decrease in absorptivity wasobserved without appreciable change in the wavelength of CTtransition for complexes 1, 2, 3 and 4.The binding constants Kb forall the above complexes are described in Table 7. Hypochromicityfor CT transition without bathochromic shift showed interactionbetween the surface of DNA and copper complexes [64]. Theintrinsic binding constant Kb for each complex has been deter-mined from the plot of [DNA]/(ea � eb) vs [DNA] and values were

Fig. 8. Fluorescence emission spectra of the EB-DNA in 0.1 M phosphate buffer (pH 7.2)in the presence of complex 3. [EB] ¼ 5 mM, [DNA] ¼ 25 mM, [Cu(Phimp)(OAc)] ¼ 0e28.40 mM, lex ¼ 250 and lem ¼ 605 nm. Inset: SterneVolmer plot forquenching by complex 3.

Fig. 9. Gel electrophoresis separations showing the cleavage of supercoiled pBR322 DNA (100 ng) by complexes 1 and 3 in presence of H2O2 and BME. Incubated at 37 �C for 2 h (A)DNA control (lane 1); DNA þ 1 (100 mM) (lane 2); DNA þ 1 (50 mM) þ H2O2 (lane 3); DNA þ 1 (50 mM) þ BME (200 mM) (lane 4); DNA þ 1 (100 mM) þ BME. (400 mM) (lane 5);DNA þ 3 (100 mM) (lane 6); DNA þ 3 (50 mM) þ H2O2 (lane 7); DNA þ 3 (50 mM) þ BME (200 mM) (lane 8); DNA þ 3 (100 mM) þ BME (400 mM) (lane 9). (B) DNA (lane 1); DNA þ 3(50 mM) (lane 2); DNA and 15, 25, 50, 75 and 100 mM of 3with BME (lane 3e7). (C) DNA (lane 1); DNA þ 3 (50 mM) (lane 2); DNA þ 3 (50 mM) þ BME (200 mM) þ Incubation time 15,30, 60 and 120 min respectively (lane 3e6).

K. Ghosh et al. / European Journal of Medicinal Chemistry 45 (2010) 3770e37793778

found to be in the range 0.94 � 104e1.99 � 104 M�1. The observedbinding constants are similar to many copper complexes [65] butsmaller than the classical intercalators and metallointercalatorswhere binding constant was reported to be in the order of107 M�1[66]. The Kb value for complex 2 is nearly 1.5 to 2 timesmore than that of the complexes 1, 3 and 4. This shows that 2 bindsmore strongly to DNA as compared to other complexes.

3.8.2. Fluorescence studiesAbsorption spectra prompted us to examine the competitive

binding of ethidium bromide vs. our complexes with DNA usingfluorescence spectral studies to get better insight of DNA binding.Ethidium bromide (EB) emits intense fluorescence in the presenceof DNA due to its strong intercalation between the DNA base pairs.The enhanced fluorescence can be quenched by the addition of thecomplexes to the EB-DNAmixture due to reduction in the emissionintensity indicating the competitive binding with EB [67]. Thefluorescence quenching curves of ethidium bromide bound to DNAby complex 3 and SterneVolmer plot (inset) are shown in Fig. 8. Forcomplexes 1 and 2 fluorescence quenching curves and theirSterneVolmer plots are shown in Supporting information (Figs. S20and S21 respectively). The SterneVolmer quenching constant, Ksvvalues were obtained from the slope of the regression curve andlisted in Table 7. These Ksv values are less than the quencher(10.0� 104 M�1) [68]. The extent of ethidium bromide quenching ismore significant for 2 than the other complexes 1 and 3. These dataare consistent with the above absorption spectral studies.

3.8.3. Circular dichroismInvestigation of DNA interaction through circular dichroism

spectroscopic technique (shown in Fig. S22) gave rise to a smallchange in negative bands with a very little shift in lmax and thepositive band at 278 nm showed decrease in molar ellipticity (q278)without any change in wavelength for complexes 1 and 3. Complex2 afforded a decrease in both the positive and negative ellipticitybands. This suggests that the DNA interaction of these complexesdo not affect the conformational changes of DNA [69]. These dataindicate a better interaction of complex 2 as compared tocomplexes 1 and 3 with CTeDNA and consistent with our dataderived from absorption spectral and fluorescence quenchingstudies.

Metal complex and DNA interaction depends on the size, chargeand shape of the complex. Absorption and circular dichroismspectral studies indicated external binding of the complexes;however, fluorescence studies showed that the Ksv values werehigher than the external binder but lower than the putative inter-calators. Hence, there may be a partial intercalation of thecomplexes with DNA. Moreover in terms of DNA interaction, it hasbeen found that complex 2wasmore effective as compared to othercomplexes because of higher Kb and Ksv values and circulardichroism spectra.

3.8.4. Nuclease activityThe cleavage of supercoiled pBR322 DNA by the complexes was

studied by gel electrophoresis in Triseboric acideEDTA buffer.Complexes 1 and 3 are water soluble and stable in buffer. For bettersolubility of complexes 2, 4 and 5we used 5% acetonitrile in buffer.The strand scissions of plasmid pBR322 were assayed in the pres-ence of H2O2 or 2-mercaptoethanol. The incubation of variousconcentration of complexes 1e5 and H2O2 or 2-mercaptoethanolwith pBR322 (100 ng) resulted supercoiled form (SC form) yieldingnicked form (NC form). However, linearized DNAwas not observedunder these conditions. In the control experiment, H2O2 and 2-mercaptoethanol showed very little cleavage (Fig. 9, Lane 2, 3).There was no cleavage of supercoiled pBR322 in presence of 5%acetonitrile, CuCl2∙2H2O and ligand PhimpH (Fig.S23, Lane 2, 3, 4).Complexes 1e5 did not show cleavage of DNA without additives.Fig. 9(A) and Fig. S23 show the cleavage of complexes 1e5 inpresence of H2O2 or 2-mercaptoethanol.

Fig. 9(B) shows the concentration dependent cleavage reactionsafter 2 h incubation (pH 7.2, 37 �C) for complex 3 with 2-mercap-toethanol (200 mM). The amount of NC form of DNA increased withthe increase of complex concentration (15e100 mM) and noappearance of linear form occurred at higher concentration. Thetime dependence of the cleavage reaction was examined and theDNA cleavage against time for complex 3 (50 mM) in presence of 2-mercaptoethanol (200 mM) is depicted in Fig. 9(C). The reactionswere monitored over a period of 15e120 min at 37 �C and theincubation time increased the conversion of SC form to NC form.

The presence of diffusible radical species can be diagnosed bymonitoring the quenching of DNA cleavage in the presence ofradical scavengers in solution. Standard radical scavengers wereadded to the reaction of complex 3 with H2O2 or 2-mercaptoe-thanol. The effect of individual addition of DMSO, D2O, NaN3, ureaand catalase are shown in Fig. S24. The addition of hydroxyl radicalscavengers such as urea don’t inhibit the conversion of SC form toNC form (lane 12,13) and high concentration of DMSO has littleeffect on DNA cleavage (lane 6,7). These results suggest thathydroxyl radicals may not be the reactive species involved in thecleavage process. The addition of D2O increase the conversion of SCform to NC form (lane 8,9) and the addition of NaN3 (lane 10, 11)blocks the DNA cleavage induced by the complex which suggestthat 1O2 or any other singlet oxygen- like entity participate in theDNA strand scission. As for the catalase, it stop the breakdown ofDNA (lane 14,15), indicating that hydrogen peroxide participate inDNA cleavage. This confirmed that species exerting DNA cleavagewere the reactive oxygen species (ROS) [14,70].

4. Conclusion

A new family of mononuclear copper complexes derived fromligand PhimpH have been synthesized and characterized. Investiga-tions of UVevisible, IR spectral studies and magnetic moment data

K. Ghosh et al. / European Journal of Medicinal Chemistry 45 (2010) 3770e3779 3779

confirm the formation of this family of complexes 1e5. X-ray crystalstructural investigations of complexes 1 revealed a perchloratebridged dimer having copper centrewith distorted square pyramidalgeometry. Investigation of magnetic properties afforded weaklyantiferromagnetically coupled copper centers at a separation of4.51 Å. In molecular structure of 3, copper was coordinated to theligand and the acetate ion in a distorted square planar geometry. Inboth complexes, deprotonated ligand (Phimp�) was bound to themetal centre meridionally. The phenolato ring and the pyridine ringwere in the meridional plane whereas the other phenyl ring of theligand is roughly perpendicular to the above plane. EPR spectral dataof 4 and 5 supported the binding of Phimp� and other monodentateligands.Thesedataalsopredicted thepresenceofunpairedelectron indx2�y2 orbital and the covalency order was 4 > 5. Electrochemicalinvestigation predicted the stability of Cu(II) centre in the environ-ment of Phimp� and Cu(II) centre wasmore stabilized in presence ofCH3COO� ligand. We examined the ligand centered oxidation ofcomplexes 1e5 in relevance with the active site modeling of GO andphenoxyl radical complexes were generated in solution at 0 �C.Complexes1 and3 exhibited superoxide scavengingactivitywith IC50valuesof11.20and8.31mMrespectively inxanthine/xanthineoxidasenitro blue tetrazolium (NBT) assay. DNA interaction studies with thehelp of absorbance spectral, fluorescence quenching and circulardichroism spectral studies indicated an external binding pathway forDNA binding. Investigation of nuclease activity predicted that all thecomplexes cleave pBR322 DNA in presence of 2-mercaptoethanol orH2O2. Mechanistic studies showed the participation of reactiveoxygen species (ROS) for the nuclease activity. Hence this family ofmononuclear copper complexes is another set of exampleswhere thecomplexes exhibited SOD as well as nuclease activity.

Acknowledgements

KG is thankful to DST, New Delhi, India for SERC FAST Trackproject support and CSIR for financial support. PK and NT arethankful to CSIR, India for financial assistance. We are thankful toIITR for instrumental facilities. MCB is thankful to PRIN Miur 2007and PAR 2007, University of Siena, Italy. We acknowledge Prof.Riccardo Basosi (Department of Chemistry, University of Siena,Italy) for giving the possibility to use the facilities present in hisresearch laboratory (EPR equipment and simulation programs) andfor the fruitful discussion on results. UPS is thankful to Head,Instrumentation Centre, IIT Roorkee for single crystal X-ray facility.

Appendix. Supporting information

Supplementary data associated with this article can be found inthe online version, at doi:10.1016/j.ejmech.2010.05.026.

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