binuclear copper complexes-interaction study with proteins

8/11/2019 Binuclear Copper Complexes-Interaction Study With Proteins

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Original article

Binuclear copper complexes: Synthesis, X-ray structure andinteraction study with nucleotide/protein by in vitro biochemicaland electrochemical analysis

M. Alagesan a, N.S.P. Bhuvanesh b, N. Dharmaraj a,*

a Inorganic and Nanomaterials Research Laboratory, Department of Chemistry, Bharathiar University, Coimbatore 641 046, Indiab Department of Chemistry, Texas A&M University, College Station, TX 77843, USA

a r t i c l e i n f o

Article history:

Received 4 July 2013

Received in revised form

17 February 2014

Accepted 14 March 2014

Available online 19 March 2014

Keywords:

Binuclear copper complex

Nucleotide and protein interaction

Cytotoxicity

a b s t r a c t

Two new, binuclear copper(II) hydrazone complexes have been synthesized and characterized by various

physico-chemical techniques including single crystal X-ray diffraction. Interaction of these complexes

with nucleotide and protein were analyzed byin vitrobiochemical and electrochemical analysis. Both the

complexes exhibited intercalative mode of binding with DNA. Further, gel electrophoresis assay

demonstrated the ability of the complexes to cleave the supercoiled pBR322 plasmid DNA to nicked

circular DNA form. Cytotoxicity of the complexes performed against a panel of cancer cell lines and a

normal cell line proved that these complexes are potentially cytotoxic against the cancerous cell lines,

particularly with IC50 as low as 0.7 mM against HeLa cell line.

2014 Elsevier Masson SAS. All rights reserved.

1. Introduction

Consequent to the discovery and extensive use ofcis-platin as an

anticancer drug, synthesis of novel, bio-active metal complexes is

one of the pioneering topics in medicinal inorganic chemistry[1].

But, multifactorial resistance (intrinsic or acquired) and inherent

limitations such as serious side effects and general toxicity has

limited the use ofcis-platin. Therefore, considerable attempts are

being made to replace this drug with suitable alternatives by syn-

thesizing numerous transition metal complexes and tested for their

anticancer activities [2e7]. So more efcacious, less toxic, target

specic and non-covalent DNA binding anticancer drugs are to be

developed. On the other hand, copper, being a bio-essential

element and the one that accumulates in tumors due to the se-

lective permeability of cancer cell membranes as well as its com-plexes attained more signicance in nucleic acid chemistry as

compared to the heavier transition elements.

Generally, anticancer agents that are approved for clinical use

are molecules which damage DNA, block DNA synthesis indirectly

through inhibition of nucleic acid precursor biosynthesis or disrupt

hormonal stimulation of cell growth [7]. Therefore, considerable

effort has been now focused on the development of new anticancer

drugs based on transition metal complexes, particularly, bio-compatible copper(II) complexes, that bind to and cleave DNA un-

der physiological conditions [8]. Additionally, copper complexes are

also shown to up-regulate DNA-binding, a pivotal molecule in the

regulation of cell progression, cell survival and apoptosis [9].

An understanding on the binding modes to DNA would give

insights into the understanding of the biochemical mechanism of

action of the metal complexes. The chemistry of binuclear copper

complexes with ligands of biological relevance and with metal

centers at close proximity is one of the central themes of current

research [10] due to their interesting structural, electrochemical

and magnetic properties[11]and also because of their relevance to

the active sites of several metalloenzymes and greater cleaving

efciency or DNA interaction than the mononuclear complexes

[12e17]. Based on these facts, several reports are published on thesynthesis of copper(II) complexes along with their interactions

with DNA[18e20].

The continuing investigation on binuclear and polynuclear

metal complexes stems from the interest of researchers to under-

stand biological processes such as hydroxylation, oxygen transport,

electron transfer and catalytic oxidation and they give opportunity

to study the intramolecular binding, magnetic exchange in-

teractions, multi-electron redox reactions and possible activation of

small substrate molecules. Many metalloenzymes contain two

copper ions in their active site that operate cooperatively [21,22]

and consequently, complexes with two metal centers drawn a* Corresponding author.

E-mail address:[email protected](N. Dharmaraj).

Contents lists available atScienceDirect

European Journal of Medicinal Chemistry

j o u r n a l h o m e p a g e : h t t p : / / w w w . e l se v i e r . c o m / l o c a t e / ej m e c h

http://dx.doi.org/10.1016/j.ejmech.2014.03.043

0223-5234/

2014 Elsevier Masson SAS. All rights reserved.

European Journal of Medicinal Chemistry 78 (2014) 281e293
mailto:[email protected]://www.sciencedirect.com/science/journal/02235234http://www.elsevier.com/locate/ejmechhttp://dx.doi.org/10.1016/j.ejmech.2014.03.043http://dx.doi.org/10.1016/j.ejmech.2014.03.043http://dx.doi.org/10.1016/j.ejmech.2014.03.043http://dx.doi.org/10.1016/j.ejmech.2014.03.043http://dx.doi.org/10.1016/j.ejmech.2014.03.043http://dx.doi.org/10.1016/j.ejmech.2014.03.043http://www.elsevier.com/locate/ejmechhttp://www.sciencedirect.com/science/journal/02235234http://crossmark.crossref.org/dialog/?doi=10.1016/j.ejmech.2014.03.043&domain=pdfmailto:[email protected]


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great deal of attention. Binuclear Cu(II) complexes have also been

reported as bio-inspired efcient catalysts in phosphoester hydro-

lysis[23].

Proteins are important biomolecules with considerable signi-

cance in biochemistry and their interactions with various mole-

cules such as drugs, dyes and metal complexes have aroused great

interest because of their importance in the explanation of the

structure and function of proteins[24,25]. It is known that serum

albumin is the main protein in blood plasma that acts as a trans-

porter and disposition of many drugs and has been frequently used

as a model protein for investigating the protein folding and ligand-

binding mechanism. The drugeprotein interaction may result in

the formation of a stable proteinedrug complex, which has

important effect on the distribution, free concentration, the meta-

bolism and the efcacy of drugs, etc. Over the past few years, much

research has been focused on the determinant factors that inu-

ence on the protein structures and functions [26e30]. In this re-

gard, bovine serum albumin (BSA) has been studied extensively,

partly because of its structural homology with human serum al-

bumin (HSA). Also, some metal ions present in blood plasma affect

the binding between drugs and serum albumins and could partic-

ipate in many biochemical processes. The phenomenon of confor-

mational alteration of serum albumin molecule caused by metalions is also well known.

Though considerable volume of research is constantly being

undertaken by several research group on the chemistry and bio-

molecular interactions of copper(II) complexes containing diversi-

ed ligand systems, corresponding studies on the binuclear

complexes appear to be limited[23,25]. Hence, the present inves-

tigation on the synthesis of two new binuclear copper(II) hydra-

zone complexes with single crystal structure determination and

interactions with DNA/protein is signicant to gain some insight

into the inuence of structural variation of the coordinated ligand

(hydrazones) on binding properties. The cytotoxicity assay per-

formed using the above binuclear copper complexes is also inter-

esting due to the fact that the former complex is very efcient

(0.7mM) to arrest the growth of cancer cells (HeLa) and a detailedprobe into the mechanism of anticancer activity would provide

some valuable information on the future prospects of these novel

binuclear copper complexes for biological evaluations.

2. Results and discussion

2.1. Synthesis and characterization

The ligands, benzoic acid (2-hydroxy-benzylidine)-hydrazone

(HL1) and 4-methyl-benzoic acid (2-hydroxy-benzylidine)-hydra-

zone (HL2) and the copper(II) complexes [CuCl(L1)]2 (1) and

[CuCl(L2)]2(2) were synthesized according to the reactions shown

inScheme 1.The structure of the binuclear complexes1and2were

characterized by various physico-chemical techniques and nallyconrmed by single crystal X-ray studies. The binuclear structure of

the complexes is stable in the solid phase and in solution medium

also which has been proved through electronic absorption spectral

and cyclic voltametric studies. The copper complexes are very

much soluble in DMF and DMSO, moderately soluble in methanol

and acetonitrile, and practically insoluble in carbon tetrachloride,

chloroform and benzene. The binucleating ligands, HL1 and HL2

coordinate to metal ions via carbonyl oxygen atoms, imine nitrogen

without undergoing enolization and hydroxyl oxygen of salicy-

laldehyde to form penta coordinated square planar binuclear cop-

per(II) complexes.

The IR spectra of the binuclear copper(II) complexes showed

bands in the region 3200e3260 cm1, indicating the presence of

NH groups. The peak at 1600e1630 cm1 due ton(C]O) and peak

forn(C]N) in the region 1460e1490 cm1 indicates the coordina-

tion of hydrazone ligand to copper metal which is in agreement

with previous report[31]. In order to obtain further structural in-

formation electronic spectra of all the complexes were recorded in

DMSOeTris buffer medium. The complexes 1 and 2 exhibit an ab-

sorption maximum in the range 550e610 nm and a shoulder band

in the range 755e785 nm, due to ded transition of the copper(II)

ion [31], indicating that the geometry around the copper ion is

square-pyramidal. The bands found in the range 245e290 nm and

320e360 nm is assigned to an intraligand transition band [32]andligand to metal charge transfer (LMCT) transition, respectively. The

ORTEP diagrams of binuclear complexes,1 and 2 are shown in Fig.1.

Crystallographic data are given in Table 1and the selected bond

lengths and bond angles are listed inTable 2. In crystal structure,

both the copper(II) ions are ve coordinated. The basal plane of

both the complexes is made of O, N and O atoms of the ligand in a

mononegative tridentate form and the fourth coordination site of

the basal plane is occupied by the oxygen atom of symmetric

[CuCl(L)] unit, and one chloride ion occupies axial position in both

the complexes. The [CuCl(L)] unit, which bridged through the ox-

ygen atom of the salicylaldehyde moiety of the ligand resulted in

centrosymmetric [CuCl(L)]2 dimer. The distortion of the coordina-

tion polyhedron from square pyramid (SP, s 0) and trigonal-

bipyramid (TBP, s 1) topologies were analyzed for both com-plexes[33], the value obtained was s 0.141 and 0.100 for1 and 2

respectively which clearly indicates that the environment around

the copper(II) ions is close to the SP topology. The non-bonded CueCu distance was found to be 3.041 A (1) and 3.043 A (2). The cop-

per(II) ion lies at about 0.301 A (1) and 0.277 A (2) above the

average basal plane towards the axial Cl atom in the complex. A

small dihedral angle of 4.82 (1) and 4.51 (2) between the mean

planes of theve member chelation ring and the six member one

ensures that the planarity of square is appreciable. Both the

hydrazone molecules in the complex are coplanar. The hydrazone

moiety possesses hydrogen bond donors and acceptors it provides

possibility of forming hydrogen bond in the crystal. The complexes

are stabilized by hydrogen bonds involving hydrogen from N2 ni-

trogen of the hydrazone ligand and coordinated chlorine atom inboth1 and 2. The bond distances and bond angles of the complex

HN

N

O

OCu

NH

N

O OCu

Cl

Cl

N

HN

OH

O

CHO

OH H2N

HN

O

+ [CuCl2(DMSO)2]

R = H or CH3

5h, reflux

EtOHDMF/MeOH

R

R

R

R

Scheme 1. Synthesis of ligands and binuclear copper(II) complexes.

M. Alagesan et al. / European Journal of Medicinal Chemistry 78 (2014) 281e293282


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agree very well with those that are reported in related copper(II)

hydrazone complexes[34]. The distance between the bridged ox-

ygen atom and copper atoms in 1 is 1.953(3) A and 1.992(2) A and

the distance between the carbonyl oxygen and copper atom is

1.970(3)A whereas the distance between bridged oxygen atom and

copper atoms in complex 2 is 1.981(1) A and 1.964(2) A and the

distance between the carbonyl oxygen atom is 1.984(2) A.

The distance between copper and chlorine atoms is found to be

2.499(1) and 2.515(7)A in 1 and 2, respectively. Crystal lattice of the

complexes showed a two-dimensional array in which each unit of

the complex is hydrogen bonded to the other through N2 and Cl

atoms. Molecular packing suggested that the stabilization of lattice

was due to hydrogen bonds, mainly involving the N2and Cl atom of

1and2.

2.2. DNA binding properties

DNA is the primary pharmacological target of many antitumor

compounds, and hence, the interaction between DNA and metal

complexes is of paramount importance in understanding the

mechanism. Thus, the mode and propensity for binding of free li-

gands and binuclear copper(II) complexes to CT-DNA were studied

with multiple techniques such as UVevisible absorption, uores-

cence emission using ethidium bromide, cyclic voltammetry and

circular dichroism.

2.2.1. Absorption spectral studies

Interaction of metal complexes with DNA has been character-

ized through absorption titrations. The UVevisible spectra of the

investigated compounds in the absence as well as the presence ofCT-DNA (nucleotides) were obtained in DMSO:TriseHCl buffer

(5 mmol, pH 7.2) containing 50 mmol NaCl solutions. Binding of

free ligands and the corresponding binuclear copper(II) complexes

to DNA helix has been studied through the changes in intensity

absorption and shift in wavelength. Usually, a compound that

bound with DNA through intercalation exhibits hypochromism,

due to the strong stacking interaction between the planar aromatic

chromophore and the base pairs of DNA [35]. The extentof shift and

hypochromism are commonly found to correlate with the inter-

calative binding strength. But, metal complexes which bind non-

intercalatively or electrostatically with DNA may result in hyper-

chromism or hypochromism [33,36,37].

The electronic absorption spectra of the free ligands HL1 and

HL2 as well as the complexes,1 and 2 measured as a function of

increasing concentration of CT-DNA is shown in Fig. 2. Upon in-

cremental additions of DNA to the test compounds, the absorption

bands of HL1 observed at 323 and 295 nm exhibited a hypo-

chromism of about 4.1% and 3.9% without any shift in the above

band positions. Similarly, absorption bands ofHL2 found at 324 and

296 nm exhibited a hypochromism of 9% and 8.9% without any shift

in the wavelength of absorption. This fact accounts that there is an

interaction between the ligands and DNA through intercalation or

some other mode of binding. However, complex 1 exhibited a

hypochromism of about 19.7% and 19.5% with a hypsochromic shift

of 1 and 2 nm at 374 and 320 nm and the absorption bands of

complex 2 at 381 and 319 nm exhibited the same phenomenon of

hypochromism of about 26.1% and 23.4% respectively with a hyp-

sochromic shift of about 2 nm. These results suggested an intimate

association of the complexes,1 and 2 with CT-DNA, and it is also

likely that they bind to the DNA helix via intercalation [34,37]. After

the compounds intercalate to the base pairs of DNA, thep* orbital ofthe intercalated compounds could couple with p orbitals of the

base pairs, thus decreasing thep/ p* transition energies, resulted

hypochromism [1]. The complexes, 1 and 2 showed more hypo-

chromicity with red shift than the ligands (HL1 and HL2), indi-

cating that the binding strength of the copper(II) complexes is

much stronger than that of the free ligands. In order to afrm

quantitatively the afnity of the compounds bound to DNA, the

intrinsic binding constants (Kb) of the compounds with DNA was

obtained by using the following equation[38]

DNA=

a f

DNA=

b f

1=Kb

b f

Where, [DNA] is the concentration of DNA in base pairs and theapparent molar extinction coefcients a, f, and b correspond to

Aobs/[compound], the extinction coefcient of the free compound,

and the extinction coefcient of the compound when fully bound to

DNA, respectively. The plot of [DNA]/(a f) versus [DNA] gave a

slope and intercept which are equal to 1/(b f) and 1/Kb(b f),

respectively, Kbis the ratio of the slope to the intercept.

The magnitude of intrinsic binding constants (Kb) were calcu-

lated as 2.3 104 M1 (HL1) and 1.13 104 M1 (HL2) for the li-

gands and 3.46 105 M1 (1) and 1.96 105 M1 (2) for the

complexes. The observed values ofKbrevealed that the ligands and

the Cu(II) complexes bind to DNA via intercalative mode[39]and

the corresponding plot is given inFig. 3. From the results obtained,

it has been found that both complexes showed higher afnity to

binding with DNA. In order to clarify whether hypochromism and

Fig. 1. An ORTEP view of copper(II) complexes 1 and 2 with the atom numbering scheme and thermal ellipsoids drawn at 50% probability level.

M. Alagesan et al. / European Journal of Medicinal Chemistry 78 (2014) 281e293 283


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red shift of absorption band can be used as positive criterions for

DNA binding modes, titration experiments of EBeDNA system havebeen performed.

2.2.2. EB-DNA quenching studies

Tond the ability of the compounds utilized in the above study

to displace EB from EB-DNA complex, a competitive EB binding

study was undertaken with uorescence experiments. The uo-

rescence based competitive binding serves as an indirect evidence

to understand the DNA-complex binding mode. EB, a phenan-

thridine uorescence dye, is a typical indicator of intercalation[40]

that forms soluble complex with nucleic acids and emits intense

uorescence in the presence of CT-DNA due to intercalation of the

planar phenanthridine ring between adjacent base pairs on the

double helix. The changes observed in the spectra of EB on its

binding to CT-DNA are often used for study between DNA and other

compounds, such as metal complexes. As the free ligands and the

Cu(II) complexes show no uorescence at room temperature in

solution or in the presence of CT-DNA, and their binding to DNA

cannot be directly predicted through the emission spectra. How-

ever, competitive EB binding studies could be used in order to

examine the binding of each compound with DNA. EB does not

show any appreciable emission in buffer solution due to uores-

cence quenching of the free EB by the solvent molecules. Upon

addition of the ligand or complex to a solution containing EB,

neither quenching of free EB uorescence has been observed, nor

new peak in the spectra appeared. The uorescence intensity is

highly enhanced upon addition of CT-DNA, due to its strong inter-

calation with DNA base pairs. Addition of a second molecule, which

may bind to DNA more strongly than EB, results in a decrease in the

DNA-induced EB emission due to the replacement of EB[41]. The

addition of the compounds results in a signicant decrease of the

uorescence intensity of the emission band of the DNA-EB system

(Fig. 4) indicate that the strong binding afnity of the compoundswith DNA over DNA-EB.

The observed quenching of DNA-EB uorescence for the ligand

or complex suggested that they displace EB from the DNA-EB

complex and they can interact with CT DNA probably by the

intercalative mode [42]. The quenching plots (Fig. 5) illustrated that

the quenching of EB bound to DNA by the complexes and free

ligand is in good agreement with the linear SterneVolmer equa-

tion. In the plots ofI0/Iversus [Q], Kq is given by the ratio of the

slope to the intercept. The Kq values for HL1, HL2, 1 and 2 were

found to be 2.4 103 M1, 5.6 103 M1, 5.1 104 M1 and

3.1 104 M1 respectively. Further, the binding constant (Kapp)

value obtained for the compounds using the following equation,

KEBEB Kappcompound

KEB 1.0107 M1 and [EB] 2.5 mM were 3.47 104 M1,

5.98 104 M1, 2.05 105 M1and 1.01 105 M1 forHL1,HL2, 1

and 2 respectively. The data showed that the interaction of the

Cu(II) complexes with DNA is stronger than that of the free ligand,

which is consistent with the electronic absorption spectral results.

Moreover, complex 1 showed higher DNA binding afnity

compared to complex2.

2.2.3. Cyclic voltammetry

Application of electrochemical methods to study metallo-

intercalation and binding of transitional metal complexes to DNA

is a useful complement to other investigation methods, such as

UVe

visible and uorescence spectroscopies. The cyclic voltam-mogram of binuclear copper complexes1 and2in the absence and

presence of CT-DNA is shown inFig. 6. In the CV of complex1, the

anodic (Epa) and cathodic responses (Epc) are found at 0.156,

0.168 V and 0.083, 0.100 V respectively, a characteristic of binu-

clear complex. The separations of the anodic and cathodic peak

potentials (DEp) are calculated as 73 and 68 mV, and the ratio of

cathodic to anodic peak currents ipa/ipc, are 1.9 and 1.6. The pres-

ence of DNA in the solution at the same concentration of binuclear

Cu(II) complex causes shift of 0.040 and 0.029 V in E1/2respectively,

and a change inDEof about 80 and 58 mV. The ratio of cathodic to

anodic peak currentsipa/ipc, are 1.8 and 1.5. The separations of the

anodic and cathodic peak potentials for the complex 2 (DEp) are 310

and 33 mV, and the ratio of cathodic to anodic peak currents ipa/ipc,

are 0.13 and 1.52. The formal potential (E1/2), taken as the average of

Table 1

Experimental data for crystallographic analysis.

1 2

Empirical formula C28H22Cl2Cu2N4O4 C30H26Cl2Cu2N4O4Formula weight 676.48 704.53

Wavelength (A) 1.54178 1.54178

Crystal system Triclinic Monoclinic

Space group P-1 P2(1)/c

Unit cell dimensionsA(A) 6.3713 (7) 12.4800 (11)

b(A) 8.8192 (10) 6.4162 (6)

c(A) 11.8050 (11) 17.4813 (17)

a() 86.780 (8) 90

b() 88.816 (9) 93.177 (7)

g() 79.462 (6) 90

Volume (A3) 651.07 (12) 1397.6 (2)

Z 1 2

Density (calculated)

(Mg/m3)

1.725 1.674

Absorption coefcient

(mm1)

4.279 4.013

F(000) 342 716

Theta range for

data collection

3.75e59.96 6.03e59.99

Index ranges 7 h 7, 9 k 9,

13

l

13

14 h 14, 7 k 6,

19

l

19

Independent

reections

1869 [R(int) 0.0367] 2047 [R(int) 0.0522]

Max. and min.

transmission

0.8824 and 0.7259 0.9241 and 0.6897

Data/restraints/

parameters

1869/0/181 2047/0/191

Goodness-of-t onF2 1.113 0.947

FinalR indices [I>

2sigma(I)]

R1 0.0406,

wR2 0.1064

R1 0.0256, wR2 0.0731

Rindices (all data) R1 0.0425,

wR2 0.1079

R1 0.0294, wR2 0.0746

Largest diff. peak

and hole (e.A3)

0.998 and 0.345 0.267 and 0.298

Table 2

Selected bond lengths (A) and angles () of the complexes.

1 2

Cu(1) eO(1) 1.952 (3) 1.964 (16)

Cu(1) eN(1) 1.947 (3) 1.939 (2)

Cu(1) eO(2) 1.970 (3) 1.984 (16)

Cu(1) eCl(1) 2.498 (11) 2.515 (7)

Cu(1) eO1(1) bridge 1.992 (3) 1.981 (16)

N(1)-Cu(1)-O(1) 90.82 (12) 91.09 (7)

N(1)-Cu(1)-O(2) 80.99 (12) 80.56 (7)

O(1)-Cu(1)-O(2) 164.97 (12) 165.33 (7)

N(1)-Cu(1)-O(1) bridge 156.46 (13) 159.31 (8)

O(1)-Cu(1)-O(1) bridge 79.10 (11) 79.08 (7)

O(2)-Cu(1)-O(1) bridge 103.63 (11) 104.78 (7)

N(1)-Cu(1)-Cl(1) 98.81 (10) 98.93 (6)

O(1)-Cu(1)-Cl(1) 101.19 (9) 106.34 (5)

O(2)-Cu(1)-Cl(1) 92.55 (9) 86.97 (5)

O(1) bridge-Cu(1)-Cl(1) 103.96 (9) 101.28 (5)



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EpaandEpc, is 0.15 and 0.016 V in the absence of DNA. The presence

of DNA in the solution at the same concentration of binuclearcomplex causes shift in E1/2 of 0.15 and 0.023 V and a decrease inDE

of 4 and 14 mV. The ratio of cathodic to anodic peak currents ipa/ipc,

are 0.30 and 0.047, the value ofipa/ipc also decreases with the in-

crease of the DNA concentration. The decrease in peak currents can

be explained in terms of an equilibrium mixture of free and DNA-

bound copper(II) complex to the electrode surface [43,44]. Upon

addition of DNA, the anodicand cathodic peak potentials shift more

positive values, but ipc/ipavalue decreases with the increase of the

DNA concentration. The more decrease of the peak currents

observed for 1 than that of2 upon addition of CT-DNA indicated

that the binding afnity of the former to DNA is stronger than that

of the latter.

2.2.4. Circular dichroism (CD) studies

Circular dichroic spectral analysis provides valuable information

on the binding mode of metal complexes with DNA[45]. Generally,

structural alterations of DNA caused by interaction with com-

pounds are reected as signicant changes in intrinsic CD spec-

trum. InFig. 7,the CD spectrum of free DNA showed a positive peak

at approximately 278 nm and a negative peak at 247 nm which

corresponds to B-DNA. These bands are caused by stacking in-

teractions between the bases and the helical supra structure of the

polynucleotide that provides an asymmetric environment for the

bases[41]. Simple groove binding and electrostatic interaction of

molecules show less or no perturbation on the base stacking and

helicity, while intercalation decreases or increases the intensities of

both positive and negative bands[46e

48]. The CD spectra of DNA

recorded after the addition of different concentration of binuclear

copper complexes 1 and 2 caused an increase in the intensity ofboth positive and negative bands of DNA due to an intercalative

mode of interaction between them.

2.3. Cleavage of pBR322 plasmid DNA by copper(II) complexes

To assess the DNA cleavage ability of the new Cu(II) complexes

by gel electrophoresis, supercoiled (SC) pBR322 DNA was incubated

with three different concentrations of copper complexes in 5 mM

TriseHCl/50 mM NaCl buffer (pH 7.2). Both the complexes, 1and 2

exhibited concentration-dependent nuclease activity during which

SC DNA was converted in to nicked circular (NC) DNA (Fig. 8, lanes

3e8) without any reductant. Upon increasing the concentration of

the complex solution from 2.5mM to 10mM, the extent of NC formof DNA was also increased. Moreover, at 10 mM concentration, 1

showed more cleavage efciency than2to convert SC DNA (Form I)

to NC DNA (Form II) revealing the superior performance of the

former. However, free ligands and precursor

complex [CuCl2(DMSO)2] did not exhibit any cleavage activity un-

der the same experimental conditions. Thus, the cleavage proper-

ties of the present compounds are attributed to the coordination

geometries and the proximity of the DNA-bound complexes to the

deoxyl ribose rings, as understood from the spectral and electro-

chemical properties. Similar observation was made by Yingying

Kou et al.[49],. Further, the cleavage efciency of complexes, 1and

2 remains unaffected in the presence of scavengers of hydroxyl

radicals (DMSO and mannitol), singlet oxygen (sodium azide and L-

histidine), and superoxide radical scavengers (SOD). This indicates

Fig. 2. Changes in the electronic absorption spectra of the ligands HL1 (a), HL2 (b) and complexes 1(c) and 2(d) (25 mM) with increasing concentration of CT-DNA (0e20mM).



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that the cleavage of DNA probably follows a hydrolytic cleavage

mechanism[39,49,50]. Moreover, inhibition or promotion of DNA

cleavage was not appreciably altered under aerobic as well asanaerobic conditions and thereby conrmed that the cleavage did

not follow oxidative mechanism.

2.4. Protein binding studies

Bovine serum albumin (BSA) and human serum albumin (HSA)

are the most widely investigated proteins [51,52]. Among them,

BSA is the major soluble protein that has many physiological

functions, such as maintaining the osmotic pressure and pH of

blood and as carriers transporting a great number of endogenous

and exogenous compounds such as fatty acids, amino acids, drugs

and pharmaceuticals[53]. A useful feature of the intrinsic uores-

cence of proteins is the high sensitivity of tryptophan and its local

environment. Changes in the emission spectra of tryptophan are

common in response to protein conformational transitions, subunit

associations, substrate binding, or denaturation[54]. Therefore, the

intrinsic uorescence of proteins can provide considerable infor-

mation on their structure and dynamics and is often utilized in the

study of protein folding and association reactions. Hence, uores-

cence quenching is an important technique to study the interaction

of metal complexes with BSA because of its accuracy, sensitivity,

rapidity and convenience of usage. A solution of BSA (1 mM) was

titrated with various concentrations of the compounds (0e8mM).

The uorescence of BSA at around 345 nm was gradually quenched

upon increasing the concentration of ligands and complexes with a

little blue shift of the emission maximum wavelength as shown in

Fig. 9.

Addition of the above compounds to the solution of BSAresulted

in a signicant decrease in the uorescence intensity of BSA at

345 nm, up to 51%, 57%, 89% and 78% of the initial uorescenceintensity of BSA for HL1, HL2,1 and 2 respectively. The observed

blueshiftof 5 and 6 nmfor 1 and 2 is mainly due to the fact that the

active site in the protein is buried in a hydrophobic environment.

This result suggested a denite interaction of the compounds with

the BSA protein. Quenching may occur by different mechanism

usually classied as dynamic quenching and static quenching. Dy-

namic quenching refers to a process in which the uorophore and

Fig. 3. Plots of [DNA]/(a f) versus [DNA] for the compounds with CT-DNA.

Fig. 4. Fluorescence quenching curves of ethidium bromide bound to DNA: ligands HL1(a) and HL2(b) and the complexes 1(c) and 2 (d). [DNA] 10 mM, [EB] 10 mM, and

[compound]

0e

16mM.



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the quencher come into contact during the transient existence of

the excited state. Static quenching refers to the uorophoreequencher complex formation in the ground state. A simple method

to explore the type of quenching is UVevisible absorption spec-

troscopy. UVevisible spectra of BSA in the absence and presence of

the compounds (Fig. 10) showed that the absorption intensity of

BSA was enhanced as the compounds were added and there

observed a slight blue shift of about 2 and 3 nm for the ligand and

binuclear Cu(II) complexes, respectively. Hence, change in the

absorption spectrum ofuorophores, reveals quenching of BSA by

the compounds are static quenching processes[53]. To study the

quenching process further, uorescence quenching data were

analyzed with the SterneVolmer and Scatchard equations.

The values ofKqandKbinfor the ligands and the binuclear Cu(II)

complexes suggested that the complexes interact with BSA more

strongly than ligands. The quenching constants (Kq 9.5 106 (1),

7.2 106 M1 (2), 1.1 104 M1 (HL1), 1.7 104 M1 (HL2)) have

been calculated from the plot ofI0/Iversus [Q](Fig. 11B). Based on

the plot of log (I0 I)/Iversus log [Q](Fig. 11A), binding constant

(Kbin 1.8 106 M1 (1), 1.1 106 M1 (2), 1.2 104 M1 (HL1),

1.3 104 M1 (HL2)) have been obtained. The value of nindicates

the existence of a single binding site in BSA for the complex or

ligand. The larger values ofKqandKbinindicate a strong interaction

between the BSA and the complex over the ligands.

2.4.1. Characteristics of the synchronousuorescence spectra

Synchronous uorescence spectra provide information on the

molecular micro-environment particularly in the vicinity of the

uorophore functional groups[55]. The uorescence of BSA is due

to presence of tyrosine and tryptophan residues. Among them,

tryptophan is the most dominant uorophore, located at the sub-

strate binding sites. Most of the drugs bind to the protein in the

active binding sites. Hence, synchronous method is usually applied

to nd out the conformational changes around tryptophan and

tyrosine region. According to Miller, the difference between the

excitation and emission wavelengths (Dl lem lex) reects the

spectra of a different nature of chromophores [56]. If the Dl value is

15 nm, the synchronous uorescence of BSA is characteristic of a

tyrosine residue, whereas a larger Dl value (60 nm) is characteristic

Fig. 5. SterneVolmer plots of the uorescence titration of the ligands and the

complexes.

Fig. 6. Cyclic voltammogram of complexes in the absence and presence (inner line) of DNA (10 mM). Scan rate: 100 mV s1.

Fig. 7. Circular dichroic spectra of CT-DNA (10 mM) with the addition of different concentration of binuclear copper complexes 1 and 2 (10mM).



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of tryptophan [57]. To investigate the structural changes that

occurred in BSA upon the addition of our compounds, synchronous

uorescence spectra of BSA were measured before and after the

addition of test compounds. The synchronous uorescence spectra

of BSA with various concentrations of test compounds were

recorded atDl

15 nm andDl

60 nm and are shown in Figs. 12and 13, respectively. In the synchronousuorescence spectra of BSA

at Dl 15, the addition of the compounds to the solution of BSA

resulted in a small decrease in the uorescence intensity of BSA at

302 nmup to30,39, 56 and 56% of the initial uorescence intensity

of BSA for the ligands and the complexes respectively, with no shift

in their emission wavelength maxima. But, in the case of the syn-

chronous uorescence spectra of BSA at Dl 60, the addition of

compounds to the solution of BSA signicantly decreased the

uorescence intensity of BSA at 342 nm, up to 16, 42, 88 and 74.1%

accompanied with a blue shift of 1 and 2 nm for the ligands and

complexes, respectively. Thus, synchronous uorescence spectral

studies suggested that the uorescence intensity of both tyrosine

and tryptophan residues were affected by increasing the concen-

tration of compounds, but the signicant decrease along with a

blue shift of the uorescence intensity of tryptophan has been

observed. These results suggested that the interaction of the ligand

and the complex with BSA affects the conformation of tryptophan

much than the tyrosine micro-region. The binding strength of the

binuclear Cu(II) complexes with BSA is signicantly higher than

that of the ligands, which can be explained by the fact that the

hydrophobicity of the complex is greater than that of the ligand. So,

the strong interaction between the compounds and BSA suggested

that these compounds can easily be stored in protein and can be

released to desired targets. Hence, we took interest to study the

cytotoxicity of the compounds.

2.5. Evaluation of in vitro anticancer activity

Cytotoxicity of the compounds were tested against a series of

cancer cell lines and a normal cell line by two different methods

such as Trypan blue dye exclusion and MTT method.

2.5.1. Trypan Blue assay

Trypan Blue, a blue acid dye with two azochromophoric groups

will not enter into the live cell, but into dead cell and makes it blue

color stain[58,59]. Thenumber of dead cells can be easily calculated

by counting stained cells through microscope. The results ofin vitro

cytotoxicity test were shown in Table 3 as their IC50 values. The

result obtained showed that both the binuclear copper(II) com-

plexes are highly toxic towards Ehrlich ascites carcinoma and Dal-

tons ascites lymphoma cell lines, whereas the ligand and

[CuCl2(DMSO)2] did not show any signi

cant activity on all the

cancer cells. From the results obtained,it is clear that coordinationof

copper atom enhanced the cytotoxic potential of free ligands[60].

2.5.2. MTT assay

The potential toxicity of the compounds towards the HeLa

(cancer cell line) and NIH 3T3 (normal cells) was further studied by

MTT assay. The complexes were dissolved in DMSO and diluted to

the requiredconcentration and same volume of DMSO was taken as

control to balance solvent activity in the cytotoxicity experiment.

The results were analyzed by means of cell inhibition expressed as

IC50 values and are given inTable 4. The IC50 values presented in

Table 4showed that the new complexes,1 and 2 are signicantly

active against HeLa, with less toxicity to normal cells. However, it is

to be noted that both the ligands and precursor complex

[CuCl2(DMSO)2] did not show any signicant activity on the above

cancer cells (IC50 above 100 mM). Hence, it is concluded that

chelation of the ligand with Cu(II) ion only responsible for the

observed cytotoxic properties of the new Cu(II) complexes. The

better cytotoxic activity of the Cu(II) complex may be attributed to

the extended planar structure induced by the p ep* conjugation

resulting from the chelation of the Cu(II) ion with ligand.

3. Conclusion

In this study, we describe the synthesis and single crystal

structure of two new, binuclear copper(II) hydrazone complexes.

Interaction of the complexes and free ligands with biomolecules

such as DNA/BSA and in vitroanticancer activity versus cancer and

normal cells were also presented. The magnitude of binding con-

stant of the test compounds with CT-DNA decreased in the orderHL2< HL1


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used for the biological studies were of high quality and procured

commercially from the reputed suppliers.

4.2. Physical measurements

Elemental analyses (C, H and N) were performed on Vario EL III

Elemental analyzer instrument. IR spectra of the samples were

recorded as KBr pellets on a Nicolet Avatar instrument in the fre-

quency range of 400e4000 cm1. Melting points were determined

with a Lab India instrument. Absorption and emission spectra were

recorded in DMSO-buffer solution on a Jasco V-630 spectropho-

tometer and Jasco FP 6600 spectrouorometer respectively, at

room temperature. Electrochemical measurements were per-

formed in a conventional two compartment three electrode cell

with a mirror polished GCE as a working electrode, Pt wire as acounter electrode and NaCl saturated Ag/AgCl as a reference elec-

trode. The electrochemical measurements were carried out with

CHI electrochemical workstation (Model 643B, Austin, TX, USA). All

the electrochemical measurements were carried out under nitro-

gen atmosphere at room temperature. Induced Circular Dichroism

spectra were recorded on JASCO J-810 spectropolarimeter with

PMT detector in DMSO-buffer solution.

4.3. Synthesis of benzoic acid (2-hydroxy-benzylidine)-hydrazone

(HL1) and 4-methyl-benzoic acid (2-hydroxy-benzylidine)-

hydrazone(HL2) ligands

The ligands were prepared according to the literature method

with slight modi

cations[32,61]. The hydrazone ligands (HL1) and(HL2) were synthesized by mixing equimolar amounts of salicy-

laldehyde (0.122 g; 1 mM) with benzhydrazide(1 mM) or p-tol-

uichydrazide (0.150 g; 1 mM) in ethanol (50 mL) respectively. The

reaction mixturewas reuxedonawaterbathfor5handpouredinto

crushed ice. The corresponding hydrazone formed was ltered and

washed several times with distilled water and recrystallized from

ethanol with 85% yield. Thepurity of theligands was checked by TLC

and melting point has been compared with the literature[61].

4.4. Synthesis of metal complexes

4.4.1. Synthesis of [CuCl(L1)]2(1)

A warm DMF solution (20 mL) containing [CuCl2(DMSO)2]

(0.344 mM, 0.1 g) was added to a methanolic solution of

Fig. 9. The emission spectrum of BSA (1mM;lex 280 nm, lem 345 nm) in the presence of increasing amounts of the ligandHL1(a) andHL2(b) and the complexes1(c) and2(d)

(0e8 mM). The arrow shows the decreases in the emission intensity upon increasing the concentration of the compounds.

Fig. 10. Electronic absorption spectra of BSA (5 mM) with ligands and complexes

(10mM).



10/13

HL1(0.344 mM, 0.082 g) and reuxed for an hour. Green single

crystals suitable for X-ray studies were obtained on slow evapo-

ration of the reaction mixture over a period of 15e20 days.

Yield: 52%. Melting point:


11/13

bromide under a UV illuminator. The cleavage efciency was

determined based on the ability of the complex to convert the

supercoiled DNA (SC) to nicked circular form (NC).

4.8. Protein binding studies

Protein-binding studies were performed by uorescence

quenching experiments using bovine serum albumin (BSA). The

uorescence spectra were recorded with an excitation at 280 nm

andcorrespondingemission at 345 nm assignable tothat of BSA and

the ligands/complexes as quenchers with increasing concentration

[71]. The excitation and emission slit widths and scan rates were

maintained constant forall the experiments. A stocksolution of BSA

was prepared in 50 mM phosphate buffer (pH 7.2) and stored in

the dark at 4 C for further use. Concentrated stock solution of the

ligands and its copper complexes were prepared by dissolving them

in DMSO and diluted to required concentrations with phosphate

buffer (5:95). BSA solution (1 mM) was titrated by successive addi-

tions of test solutions HL1, HL2, 1 and 2 using micropipettes for all of

the experiments. Synchronous uorescence spectra was also

recorded using the same concentration of BSA and compounds as

mentioned above with two different Dl (difference between the

excitation and emission wavelengths of BSA) values such as 15 and

60 nm. Thequenching constant (Kq) canbe calculated using the plot

ofI0/Iversus [Q]. If it is assumed that thebinding of compoundswith

BSA occurs at equilibrium, the equilibrium binding constant can be

analyzed according to the Scatchard equation:

logI0I=I logKbinnlogQ

Where,Kbinis the binding constant of compound with BSA and nis

the number of binding sites. From the plot of log(I0I)/Iversus log

[Q], the number of binding sites (n) and the binding constant (Kbin)was calculated.

4.9. Evaluation of cytotoxicity

Cytotoxicity of the binuclear complexes1 and 2 as well as free

ligands HL1 and HL2 was determined by Trypan blue and MTT

assays as given below.

4.9.1. Trypan Blue assay

Trypan Blue Assay was performed against Daltons ascites

lymphoma (DAL) and Ehrlich ascites carcinoma (EAC) cells and

these cells were grown in the peritoneal cavity of mice by serial

transplantation. For the experiment, the cells were aspirated from

the peritoneal cavity of tumor bearing mice, pelleted by centrifu-gation and maintained by intraperitoneal inoculation of 10 6 cells/

mouse. The cells were washed thrice with phosphate buffer saline

and made up to a concentration of 10 million/mL. The cells

(1 million) were incubated with different concentration of drugs in

a total volume of 0.9 mL with PBS at 37 C for 3 h. After incubation,

0.1 mL of Trypan blue (1%) was added and in vitrocell viability is

measured by trypan blue exclusion test based on the ability of

trypan blue to stain dead cells. The percentage of inhibition was

calculated using the following equation.

%inhibition No: of dead cells=No: of dead cells

No of live cells 100

Fig. 12. Synchronous spectra of BSA (1 mM) in the presence of increasing amounts of the ligands HL1(a) and HL2(b) and the complexes1(c) and 2(d) (0e8 mM) at a wavelength

difference ofDl 15 nm. The arrow shows the decreases in the emission intensity upon increasing concentration of the compounds.



12/13

4.9.2. MTT assay

The growth inhibitory effect of the same complexes and li-

gands were also assessed versus a cancer (HeLa), and normal (NIH

3T3) by means of MTT assay[72]. For the screening experiments,

the cells were seeded into 96-well plates in 100 mL of the

respective medium containing 10% FBS, at a plating density of

10,000 cells/well. The cells were incubated at 37 C in 5% CO2and

95% air at a relative humidity of 100% for 24 h prior to the

addition of the test compounds. The compounds were dissolved

in DMSO and diluted in the respective medium containing 1% FBS.

The maximum concentration DMSO used in the experiment is

10mL. After 24 h, the medium was replaced with the respective

medium with 1% FBS containing the compounds at various con-

centrations and incubated at 37 C under conditions of 5% CO2,

95% air, and 100% relative humidity for 48 h. Triplicate cultures

were established for each treatment, and the medium not con-

taining the compounds served as the control. After 48 h, 10 mL of

MTT (5 mg/mL) in phosphate buffered saline (PBS) was added to

each well and incubated at 37 C for 4 h. The medium with MTT

was then icked off, and the formed formazan crystals were

dissolved in 100mL of DMSO. Mean absorbance for each drug dose

was expressed as a percentage of the control untreated wellabsorbance and plotted versus drug concentration. IC50 values

represent the drug concentrations that reduced the mean absor-

bance at 570 nm to 50% of those in the untreated control wells

and a graph was plotted with the percentage of cell inhibition

versus concentration. From this, the IC50 value was calculated

[73].

Fig. 13. Synchronous spectra of BSA (1 mM) in the presence of increasing amounts of the ligandsHL1(a) and HL2(b) and the complexes 1(c) and 2(d) (0e8 mM) at a wavelength

difference ofDl 60 nm. The arrow shows the decreases in the emission intensity upon increasing concentration of the compounds.

%inhibition mean OD of untreated cellscontrol=mean OD of treated cells 100

Table 3

Cytotoxicity of the complexes determined by Trypan Blue dye exclusion method.

Compound IC50 values (mM)

EAC DLA

Complex 1 4 0.50 4.1 0.57

Complex 2 5 0.86 4.1 0.61

Table 4

Cytotoxicity of the complexes determined by MTT assay.

Compound IC50values (mM)

HeLa NIH 3T3

Complex 1 0.7 0.98 22 0.54

Complex 2 18.6 0.98 54 0.98



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Supplementary material

Crystallographic data for the structure reported in this paper

have been deposited with the Cambridge Crystallographic Data

Centre (CCDC) as supplementary CCDC reference numbers of the

complexes are 852728 and 852729, respectively. Copies of the data

can be obtained free of charge from the CCDC (12 Union Road,

Cambridge CB2 1EZ, UK; Tel.: 44-1223-336408; Fax: 44-1223-

336003; e-mail: [email protected]; Web site http://www.

ccdc.cam.ac.uk/).

Acknowledgment

The corresponding author of the manuscript (N. D) acknowl-

edges the Council of Scientic and Industrial Research (CSIR),

Ministry of Human Resources Development (MHRD), Government

of India, New Delhi, for the nancial support in the form of a major

research project (CSIR sanction letter No. 01(2684)/12/EMReII

dated 03. 10. 2012). We thank prof. A. Ramu, School of Chemistry,

Madurai Kamaraj University, India, for his help to record CD spectra

and Dr. S. Abraham John, Department of Chemistry, Gandhigram

Rural Institute eDeemed University, Gandhigram, India, for elec-

trochemical measurement of samples.

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