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

Article abstract of DOI:10.1016/j.ejmech.2014.03.043

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 by in vit

Full text of DOI:10.1016/j.ejmech.2014.03.043

European Journal of Medicinal Chemistry 78 (2014) 281e293
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Binuclear copper complexes: Synthesis, X-ray structure and
interaction study with nucleotide/protein by in vitro biochemical
and 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, India
b Department of Chemistry, Texas A&M University, College Station, TX 77843, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
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 by in vitro biochemical 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,
Received 4 July 2013
Received in revised form
17 February 2014
Accepted 14 March 2014
Available online 19 March 2014
Keywords:
particularly with IC50 as low as 0.7
m
M against HeLa cell line.
Ó 2014 Elsevier Masson SAS. All rights reserved.
Binuclear copper complex
Nucleotide and protein interaction
Cytotoxicity
1. Introduction
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
efficiency or DNA interaction than the mononuclear complexes
[12e17]. Based on these facts, several reports are published on the
synthesis of copper(II) complexes along with their interactions
with DNA [18e20].
Consequent to the discovery and extensive use of cis-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 of cis-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 efficacious, less toxic, target
specific 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 significance 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
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: dharmaraj@buc.edu.in (N. Dharmaraj).
http://dx.doi.org/10.1016/j.ejmech.2014.03.043
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

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M. Alagesan et al. / European Journal of Medicinal Chemistry 78 (2014) 281e293
great deal of attention. Binuclear Cu(II) complexes have also been
reported as bio-inspired efficient catalysts in phosphoester hydro-
lysis [23].
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.
Proteins are important biomolecules with considerable signifi-
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 efficacy of drugs, etc. Over the past few years, much
research has been focused on the determinant factors that influ-
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 metal
ions 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-
fied 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 significant to gain some insight
into the influence 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 efficient
The IR spectra of the binuclear copper(II) complexes showed
bands in the region 3200e3260 cmꢀ1, indicating the presence of
NH groups. The peak at 1600e1630 cmꢀ1 due to
n
(C]O) and peak
for
n
(C]N) in the region 1460e1490 cmꢀ1 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] and
ligand 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 1 and the selected bond
lengths and bond angles are listed in Table 2. In crystal structure,
both the copper(II) ions are five 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,
bipyramid (TBP,
¼ 1) topologies were analyzed for both com-
plexes [33], the value obtained was
s
¼ 0) and trigonal-
(0.7
mM) to arrest the growth of cancer cells (HeLa) and a detailed
s
probe into the mechanism of anticancer activity would provide
some valuable information on the future prospects of these novel
binuclear copper complexes for biological evaluations.
s
¼ 0.141 and 0.100 for 1 and 2
respectively which clearly indicates that the environment around
the copper(II) ions is close to the SP topology. The non-bonded Cue
ꢀ
ꢀ
Cu 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 4.82ꢁ (1) and 4.51ꢁ (2) between the mean
planes of the five 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 in
both 1 and 2. The bond distances and bond angles of the complex
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
in Scheme 1. The structure of the binuclear complexes 1 and 2 were
characterized by various physico-chemical techniques and finally
confirmed by single crystal X-ray studies. The binuclear structure of
R
H
R
N
N
R
H
CHO
Cl
N
O
5h, reflux
EtOH
H
[CuCl2(DMSO)2]
N
Cu
+
O
N
H2N
O
DMF/MeOH
OH
Cu
N
O
O
OH
O
Cl
R = H or CH3
N
H
R
Scheme 1. Synthesis of ligands and binuclear copper(II) complexes.

M. Alagesan et al. / European Journal of Medicinal Chemistry 78 (2014) 281e293
283
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.
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
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 of HL2 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 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 N2 and Cl atom of
1and 2.
2.2. DNA binding properties
the compounds intercalate to the base pairs of DNA, the
the intercalated compounds could couple with orbitals of the
base pairs, thus decreasing the * transition energies, resulted
p* orbital of
p
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, fluores-
cence emission using ethidium bromide, cyclic voltammetry and
circular dichroism.
p
/ p
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 affirm
quantitatively the affinity of the compounds bound to DNA, the
intrinsic binding constants (Kb) of the compounds with DNA was
obtained by using the following equation [38]
ꢀ
ꢁ
ꢀ
ꢁ
ꢀ
ꢁ
2.2.1. Absorption spectral studies
½DNAꢂ= εa ꢀ εf ¼ ½DNAꢂ= εb ꢀ εf þ 1=Kb εb ꢀ εf
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 of
CT-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 extent of 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].
Where, [DNA] is the concentration of DNA in base pairs and the
apparent molar extinction coefficients εa, εf, and εb correspond to
Aobs/[compound], the extinction coefficient of the free compound,
and the extinction coefficient of the compound when fully bound to
DNA, respectively. The plot of [DNA]/(εa ꢀ εf) versus [DNA] gave a
slope and intercept which are equal to 1/(εb ꢀ εf) and 1/Kb(εb ꢀ εf),
respectively, Kb is the ratio of the slope to the intercept.
The magnitude of intrinsic binding constants (Kb) were calcu-
lated as 2.3 ꢃ 104 Mꢀ1 (HL1) and 1.13 ꢃ 104 Mꢀ1 (HL2) for the li-
gands and 3.46 ꢃ 105 Mꢀ1 (1) and 1.96 ꢃ 105 Mꢀ1 (2) for the
complexes. The observed values of Kb revealed that the ligands and
the Cu(II) complexes bind to DNA via intercalative mode [39] and
the corresponding plot is given in Fig. 3. From the results obtained,
it has been found that both complexes showed higher affinity to
binding with DNA. In order to clarify whether hypochromism and
The electronic absorption spectra of the free ligands HL1 and
HL2 as well as the complexes, 1 and 2 measured as a function of

284
M. Alagesan et al. / European Journal of Medicinal Chemistry 78 (2014) 281e293
Table 1
that forms soluble complex with nucleic acids and emits intense
Experimental data for crystallographic analysis.
fluorescence 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 fluorescence 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 fluores-
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 fluorescence has been observed, nor
new peak in the spectra appeared. The fluorescence 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 significant decrease of the
fluorescence intensity of the emission band of the DNA-EB system
(Fig. 4) indicate that the strong binding affinity of the compounds
with DNA over DNA-EB.
1
2
Empirical formula
Formula weight
Wavelength (A)
Crystal system
Space group
C28H22Cl2Cu2N4O4
676.48
1.54178
Triclinic
P-1
C30H26Cl2Cu2N4O4
704.53
1.54178
Monoclinic
P2(1)/c
ꢀ
Unit cell dimensions
ꢀ
A (A)
6.3713 (7)
8.8192 (10)
11.8050 (11)
86.780 (8)
88.816 (9)
79.462 (6)
651.07 (12)
1
12.4800 (11)
6.4162 (6)
17.4813 (17)
90
93.177 (7)
90
1397.6 (2)
2
1.674
ꢀ
b (A)
ꢀ
c (A)
a
b
g
(ꢁ)
(ꢁ)
(ꢁ)
3
ꢀ
Volume (A )
Z
Density (calculated)
1.725
(Mg/m3)
Absorption coefficient 4.279
(mmꢀ1
F (000)
4.013
)
342
716
6.03e59.99ꢁ
Theta range for
data collection
Index ranges
3.75e59.96ꢁ
ꢀ7 ꢄ h ꢄ 7, ꢀ9 ꢄ k ꢄ 9, ꢀ14 ꢄ h ꢄ 14, ꢀ7 ꢄ k ꢄ 6,
ꢀ13 ꢄ l ꢄ 13
ꢀ19 ꢄ l ꢄ 19
Independent
reflections
Max. and min.
transmission
Data/restraints/
parameters
1869 [R (int) ¼ 0.0367]
2047 [R (int) ¼ 0.0522]
0.8824 and 0.7259
1869/0/181
0.9241 and 0.6897
2047/0/191
The observed quenching of DNA-EB fluorescence 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 of I0/I versus [Q], Kq is given by the ratio of the
slope to the intercept. The Kq values for HL1, HL2, 1 and 2 were
Goodness-of-fit on F2 1.113
0.947
Final R indices [I >
2sigma(I)]
R indices (all data)
R1 ¼ 0.0406,
R1 ¼ 0.0256, wR2 ¼ 0.0731
wR2 ¼ 0.1064
R1 ¼ 0.0425,
R1 ¼ 0.0294, wR2 ¼ 0.0746
0.267 and ꢀ0.298
wR2 ¼ 0.1079
0.998 and ꢀ0.345
Largest diff. peak
found to be 2.4 ꢃ 103
M
ꢀ1, 5.6 ꢃ 103
M
ꢀ1, 5.1 ꢃ 104 Mꢀ1 and
ꢀꢀ3
and hole (e.A
)
3.1 ꢃ 104 Mꢀ1 respectively. Further, the binding constant (Kapp
)
value obtained for the compounds using the following equation,
red shift of absorption band can be used as positive criterions for
DNA binding modes, titration experiments of EBeDNA system have
been performed.
KEB½EBꢂ ¼ Kapp½compoundꢂ
KEB ¼ 1.0 ꢃ 107 Mꢀ1 and [EB] ¼ 2.5
m
M were 3.47 ꢃ 104 Mꢀ1
,
5.98 ꢃ 104
M
ꢀ1, 2.05 ꢃ 105 Mꢀ1and 1.01 ꢃ 105 Mꢀ1 for HL1, 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.
2.2.2. EB-DNA quenching studies
To find 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 fluorescence experiments. The fluo-
rescence based competitive binding serves as an indirect evidence
to understand the DNA-complex binding mode. EB, a phenan-
thridine fluorescence dye, is a typical indicator of intercalation [40]
Moreover, complex
1 showed higher DNA binding affinity
compared to complex 2.
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
UVevisible and fluorescence spectroscopies. The cyclic voltam-
mogram of binuclear copper complexes 1 and 2 in the absence and
presence of CT-DNA is shown in Fig. 6. In the CV of complex 1, 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
Table 2
ꢁ
ꢀ
Selected bond lengths (A) and angles ( ) of the complexes.
1
2
Cu(1) e O(1)
Cu(1) e N(1)
Cu(1) e O(2)
Cu(1) e Cl(1)
1.952 (3)
1.947 (3)
1.970 (3)
2.498 (11)
1.992 (3)
1.964 (16)
1.939 (2)
1.984 (16)
2.515 (7)
1.981 (16)
Cu(1) e O1(1) bridge
potentials (DEp) are calculated as 73 and 68 mV, and the ratio of
N(1)-Cu(1)-O(1)
N(1)-Cu(1)-O(2)
O(1)-Cu(1)-O(2)
N(1)-Cu(1)-O(1) bridge
O(1)-Cu(1)-O(1) bridge
O(2)-Cu(1)-O(1) bridge
N(1)-Cu(1)-Cl(1)
O(1)-Cu(1)-Cl(1)
O(2)-Cu(1)-Cl(1)
90.82 (12)
80.99 (12)
164.97 (12)
156.46 (13)
79.10 (11)
103.63 (11)
98.81 (10)
101.19 (9)
92.55 (9)
91.09 (7)
80.56 (7)
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 E1/2 respectively,
165.33 (7)
159.31 (8)
79.08 (7)
104.78 (7)
98.93 (6)
106.34 (5)
86.97 (5)
101.28 (5)
and a change in
anodic peak currents ipa/ipc, are 1.8 and 1.5. The separations of the
anodic and cathodic peak potentials for the complex 2 ( Ep) 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
DE of about 80 and 58 mV. The ratio of cathodic to
D
,
O(1) bridge-Cu(1)-Cl(1)
103.96 (9)

M. Alagesan et al. / European Journal of Medicinal Chemistry 78 (2014) 281e293
285
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 (0e20 mM).
Epa and Epc, is 0.15 and 0.016 V in the absence of DNA. The presence
of DNA in the solution at the same concentration of binuclear
recorded after the addition of different concentration of binuclear
copper complexes 1 and 2 caused an increase in the intensity of
both positive and negative bands of DNA due to an intercalative
mode of interaction between them.
complex causes shift in E1/2 of 0.15 and 0.023 V and a decrease in
DE
of 4 and 14 mV. The ratio of cathodic to anodic peak currents ipa/ipc
,
are 0.30 and 0.047, the value of ipa/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 anodic and cathodic peak potentials shift more
positive values, but ipc/ipa value decreases with the increase of the
DNA concentration. The more decrease of the peak currents
observed for 1 than that of 2 upon addition of CT-DNA indicated
that the binding affinity of the former to DNA is stronger than that
of the latter.
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, 1 and 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 2.5
m
M to 10
mM, the extent of NC form
2.2.4. Circular dichroism (CD) studies
of DNA was also increased. Moreover, at 10
mM concentration, 1
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 reflected as significant changes in intrinsic CD spec-
trum. In Fig. 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 [46e48]. The CD spectra of DNA
showed more cleavage efficiency than 2 to 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 efficiency of complexes, 1 and
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

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M. Alagesan et al. / European Journal of Medicinal Chemistry 78 (2014) 281e293
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 fluores-
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 fluorescence 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, fluores-
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 (0e8
mM).
The fluorescence 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.
Fig. 3. Plots of [DNA]/(εa ꢀ εf) versus [DNA] for the compounds with CT-DNA.
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 as
anaerobic conditions and thereby confirmed that the cleavage did
not follow oxidative mechanism.
Addition of the above compounds to the solution of BSA resulted
in a significant decrease in the fluorescence intensity of BSA at
345 nm, up to 51%, 57%, 89% and 78% of the initial fluorescence
intensity of BSA for HL1, HL2, 1 and 2 respectively. The observed
blue shift of 5 and 6 nm for 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 definite interaction of the compounds with
the BSA protein. Quenching may occur by different mechanism
usually classified as dynamic quenching and static quenching. Dy-
namic quenching refers to a process in which the fluorophore and
2.4. Protein binding studies
Bovine serum albumin (BSA) and human serum albumin (HSA)
are the most widely investigated proteins [51,52]. Among them,
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] ¼ 10
[compound] ¼ 0e16 M.
mM, [EB] ¼ 10
mM, and
m

M. Alagesan et al. / European Journal of Medicinal Chemistry 78 (2014) 281e293
287
absorption spectrum of fluorophores, reveals quenching of BSA by
the compounds are static quenching processes [53]. To study the
quenching process further, fluorescence quenching data were
analyzed with the SterneVolmer and Scatchard equations.
The values of Kq and Kbin for the ligands and the binuclear Cu(II)
complexes suggested that the complexes interact with BSA more
strongly than ligands. The quenching constants (Kq ¼ 9.5 ꢃ 106 (1),
7.2 ꢃ 106 Mꢀ1 (2), 1.1 ꢃ 104 Mꢀ1 (HL1), 1.7 ꢃ 104 Mꢀ1 (HL2)) have
been calculated from the plot of I0/I versus [Q] (Fig. 11B). Based on
the plot of log (I0 ꢀ I)/I versus log [Q] (Fig. 11A), binding constant
(Kbin 1.8 ꢃ 106 Mꢀ1 (1), 1.1 ꢃ 106 Mꢀ1 (2), 1.2 ꢃ 104 Mꢀ1 (HL1),
1.3 ꢃ 104 Mꢀ1 (HL2)) have been obtained. The value of ‘n’ indicates
the existence of a single binding site in BSA for the complex or
ligand. The larger values of Kq and Kbin indicate a strong interaction
between the BSA and the complex over the ligands.
2.4.1. Characteristics of the synchronous fluorescence spectra
Synchronous fluorescence spectra provide information on the
molecular micro-environment particularly in the vicinity of the
fluorophore functional groups [55]. The fluorescence of BSA is due
to presence of tyrosine and tryptophan residues. Among them,
tryptophan is the most dominant fluorophore, 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 find out the conformational changes around tryptophan and
tyrosine region. According to Miller, the difference between the
Fig. 5. SterneVolmer plots of the fluorescence titration of the ligands and the
complexes.
the quencher come into contact during the transient existence of
the excited state. Static quenching refers to the fluorophoree
quencher 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
excitation and emission wavelengths (Dl
¼
lem
ꢀ
lex) reflects the
spectra of a different nature of chromophores [56]. If the Dl value is
15 nm, the synchronous fluorescence of BSA is characteristic of a
tyrosine residue, whereas a larger Dl value (60 nm) is characteristic
Fig. 6. Cyclic voltammogram of complexes in the absence and presence (inner line) of DNA (10 m .
M). Scan rate: 100 mV sꢀ1
Fig. 7. Circular dichroic spectra of CT-DNA (10 mM) with the addition of different concentration of binuclear copper complexes 1 and 2 (10 mM).

288
M. Alagesan et al. / European Journal of Medicinal Chemistry 78 (2014) 281e293
cancer cells. From the results obtained, it is clear that coordination of
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 required concentration 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 in Table 4. The IC50 values presented in
Table 4 showed that the new complexes, 1 and 2 are significantly
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 significant activity on the above
Fig. 8. Cleavage of supercoiled pBR322 DNA by the copper(II) complexes in a buffer
containing 5 mM TriseHCl and 50 mM NaCl. Lane1, DNA control; lane 2, DNA þ2.5
mM
complex 1; lane 3, DNA þ5
m
M complex 1; lane 4, DNA 10
m
M complex 1; lane 5,
DNA þ2.5 M complex 2; lane 6, DNA þ5
m
m
M complex 2; lane 7, DNA þ10
mM complex
2. Forms I and II are supercoiled and nicked circular of DNA, respectively.
cancer cells (IC50 ¼ 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
of tryptophan [57]. To investigate the structural changes that
occurred in BSA upon the addition of our compounds, synchronous
fluorescence spectra of BSA were measured before and after the
addition of test compounds. The synchronous fluorescence spectra
of BSA with various concentrations of test compounds were
recorded at Dl ¼ 15 nm and Dl ¼ 60 nm and are shown in Figs. 12
and 13, respectively. In the synchronous fluorescence spectra of BSA
at Dl ¼ 15, the addition of the compounds to the solution of BSA
resulted in a small decrease in the fluorescence intensity of BSA at
302 nm up to 30, 39, 56 and 56% of the initial fluorescence 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 fluorescence spectra of BSA at Dl ¼ 60, the addition of
compounds to the solution of BSA significantly decreased the
fluorescence 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 fluorescence spectral
studies suggested that the fluorescence intensity of both tyrosine
and tryptophan residues were affected by increasing the concen-
tration of compounds, but the significant decrease along with a
blue shift of the fluorescence 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 significantly 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.
the extended planar structure induced by the
resulting from the chelation of the Cu(II) ion with ligand.
p e p* conjugation
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 vitro anticancer 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 order
HL2 < HL1 < 2 < 1 and the corresponding values are 1.13 ꢃ 104 Mꢀ1
,
2.3 ꢃ 104
M
ꢀ1, 1.96 ꢃ 105 Mꢀ1 and 3.46 ꢃ 105 respectively. Hypo-
chromism in the absorption band of the complexes upon the
addition of DNA determined by UVevisible absorption and fluo-
rescence emission titration methods suggested an intercalative
mode of interaction between them. Significant changes observed in
the CD signature of CT-DNA at the helical and base stack regions
further supported the winding of DNA upon intercalation by the
complexes 1 and 2. 2.5e10 mM solutions of the complexes cleaved
supercoiled DNA into nicked circular form without any external
agents such as an oxidant/reductant or laser/UVevisible light. The
in vitro anti cancer activity of complexes 1 and 2 demonstrated that
they are significantly toxic to HeLa, EAC and DAL cancerous cells.
Particularly, cytotoxicity of complex 1 is promising (IC50 ¼ 0.7
mM)
in comparison with reported binuclear complexes and hence, a
suitable candidate for further studies of Cu-based chemothera-
peutic agents.
4. Experimental
2.5. Evaluation of in vitro anticancer activity
4.1. Materials and methods
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.
Reagent grade chemicals were used without further purification
in all the synthetic work. Solvents were purified by standard
methods. Triply distilled water was used to prepare buffer solutions
and biological experiments. CuCl2ꢅ2H2O, benzhydrazide, p-tol-
uichydrazide, salicylaldehyde, ethidium bromide, calf-thymus DNA
(CT-DNA), trypan blue and 3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) were purchased from
SigmaeAldrich, Chemie and Alfa Aesar. Human cervical cancer cell
line (HeLa), normal mouse embryonic fibroblasts cell line (NIH
3T3), were obtained from National Centre for Cell Science (NCCS),
Pune, India. Dalton’s ascites lymphoma (DAL) and Ehrlich ascites
carcinoma (EAC) cell lines were obtained from Amala Cancer
Research Center, Thrissur, India. All other chemicals and reagents
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]. The number of dead cells can be easily calculated
by counting stained cells through microscope. The results of in 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-
ton’s ascites lymphoma cell lines, whereas the ligand and
[CuCl2(DMSO)2] did not show any significant activity on all the

M. Alagesan et al. / European Journal of Medicinal Chemistry 78 (2014) 281e293
289
Fig. 9. The emission spectrum of BSA (1
m
M; lex ¼ 280 nm, lem ¼ 345 nm) in the presence of increasing amounts of the ligand HL1(a) and HL2(b) and the complexes 1(c) and 2(d)
(0e8 M). The arrow shows the decreases in the emission intensity upon increasing the concentration of the compounds.
m
used for the biological studies were of high quality and procured
commercially from the reputed suppliers.
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 a
counter 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.
4.2. 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 cmꢀ1. 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 spectrofluorometer respectively, at
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 modifications [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 refluxed on awater bath for 5 h andpoured into
crushed ice. The corresponding hydrazone formed was filtered and
washed several times with distilled water and recrystallized from
ethanol with 85% yield. The purity of the ligands 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. 10. Electronic absorption spectra of BSA (5
(10 M).
mM) with ligands and complexes
m

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M. Alagesan et al. / European Journal of Medicinal Chemistry 78 (2014) 281e293
Fig. 11. Scatchard plots (A) and SterneVolmer plots (B) of fluorescence titration of the ligands and the complexes with BSA.
HL1(0.344 mM, 0.082 g) and refluxed 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: <300 ꢁC. Elemental Analysis: Found
(calculated) (%) for C28H22Cl2Cu2N4O4: C, 49.7 (49.72); H, 3.2 (3.23);
N, 8.28 (8.31). UVevisible (solvent: TriseHCl buffer, nm): (ε,
Mꢀ1 cmꢀ1): 374 (23,800), 319 (25,800), 253 (29,000). IR: nmax
4.6. DNA binding experiments
All experiments involving CT-DNA were performed in tris-
(hydroxymethyl)aminomethaneeHCl (TriseHCl) buffer solution
(pH ¼ 7.2). Stock solution of CT-DNA was stored at 277 K and used
only for a period of 4 days. The concentration of CT-DNA was
determined by UV absorbance at 260 nm and its molar absorption
coefficient was taken as 6600 Mꢀ1 cmꢀ1 [67]. Solutions of CT-DNA
in TriseHCl buffer gave a ratio of UV absorbance at 260 and 280 nm
(A260/A280), of approximately 1.8e1.9, indicating that the DNA was
sufficiently free of protein [68,69]. Concentrated stock solution of
the ligands and binuclear copper(II) complexes (HL1, HL2, 1 and 2)
were prepared in dimethyl sulfoxide (DMSO) and diluted with
TriseHCl buffer to required concentrations. Absorption spectral
titration was performed by keeping the concentration of the test
compounds constant while varying the CT-DNA concentration.
Equal volume of CT-DNA solution was added to the reference so-
lution to eliminate the absorbance of CT-DNA itself. In the ethidium
bromide (EB) fluorescence displacement experiment, 1 mM of the
EB TriseHCl solution was added to CT-DNA solution (at saturated
binding levels [70], stored in the dark for 2 h). Then the solution of
the test compounds was titrated against DNA/EB mixture. Before
measurements, the mixture was shaken up and recorded. The
fluorescence spectra EB bound to CT-DNA was obtained at an
emission wavelength of 602 nm. The electrochemical titration ex-
periments were performed by keeping the concentration of the test
compounds constant while varying the CT-DNA concentration us-
ing TriseHCl buffer as solvent. Circular dichroic experiments were
carried out by keeping the concentration of CT-DNA constant but
varying the concentration of binuclear copper(II) complexes, by
monitoring the changes in the positive peak and negative peak of
CT-DNA.
(cmꢀ1):
nC]O: 1623, nC]N: 1480.
4.4.2. Synthesis of [CuCl(L2)]2 (2)
Complex was prepared by following the procedure as
2
described for complex 1 with HL2 (0.344 mM, 0.088 g) as the ligand
and [CuCl2(DMSO)2] as starting precursors. Transparent, green
crystals were obtained on slow evaporation of the reaction mixture
over a period of 15e20 days.
Yield: 49%. Melting point: <300 ꢁC. Elemental Analysis: Found
(calculated) (%) for C30H26Cl2 Cu2N4O4: C, 51.1 (51.4); H, 3.71 (3.76);
N, 8.25 (8.28). UVevisible (solvent: 5% DMSO and 95% TriseHCl
buffer nm): (ε, Mꢀ1 cmꢀ1) 375 (5000), 320 (5360), 254 (6120). IR:
nmax (cmꢀ1):
nC]O: 1626, nC]N: 1478.
4.5. X-ray crystallography
BRUKER GADDS X-ray (three-circle) diffractometer was
A
employed for crystal screening, unit cell determination and data
collection. The goniometer was controlled using the FRAMBO
software, v.4.1.05 [62]. The sample was optically centered with the
aid of a video camera such that no translations were observed as
the crystal was rotated through all positions. 180 data frames were
taken at widths of 0.5ꢁ. These reflections were used to determine
the unit cell using Cell Now [63]. Data reduction, structure solution,
and refinement integrated intensity information for each reflection
was obtained by reduction of the data frames with APEX2 [64]. The
integration method employed a three dimensional profiling algo-
rithm and all data were corrected for Lorentz and polarization
factors, as well as for crystal decay effects. Finally the data was
merged and scaled to produce a suitable data set. SADABS [65] was
used to correct the data for absorption effects. A solution was ob-
tained readily SHELXTL [66] while hydrogen atoms bound to car-
bon were placed in idealized positions hydrogen atom attached to
N were found from difference Fourier map and were set riding on
the parent atoms. All non-hydrogen atoms were refined with
anisotropic thermal parameters. The structure was refined
(weighted least squares refinement on F2) to convergence [66].
4.7. DNA cleavage experiment
The cleavage of DNA was monitored using agarose gel electro-
phoresis. Reactions using supercoiled pBR322 plasmid DNA in Trise
HCl buffer (50 mM) with 50 mM NaCl (pH, 7.2) was treated with the
compounds HL1, HL2, 1 and 2 (2.5e10
TriseHCl buffer to a total volume of 20
m
m) by dilution with the
mL. The samples were
incubated for 0.5 h at 37 ꢁC. A loading buffer containing 25% bro-
mophenol blue, 0.25% xylene cyanol, 30% glycerol was added and
electrophoresis performed at 40 V for 3 h in TriseAcetateeEDTA
(TAE) buffer using 1% agarose gel containing 1.0 mg/mL ethidium
bromide. Agarose gel electrophoresis of plasmid DNA was visual-
ized by photographing the fluorescence of intercalated ethidium

M. Alagesan et al. / European Journal of Medicinal Chemistry 78 (2014) 281e293
291
Fig. 12. Synchronous spectra of BSA (1
mM) in the presence of increasing amounts of the ligands HL1(a) and HL2(b) and the complexes 1(c) and 2(d) (0e8
mM) at a wavelength
difference of Dl ¼ 15 nm. The arrow shows the decreases in the emission intensity upon increasing concentration of the compounds.
bromide under a UV illuminator. The cleavage efficiency was
determined based on the ability of the complex to convert the
supercoiled DNA (SC) to nicked circular form (NC).
Where, Kbin is the binding constant of compound with BSA and n is
the number of binding sites. From the plot of log(I0 ꢀ I)/I versus log
[Q], the number of binding sites (n) and the binding constant (Kbin
)
was calculated.
4.8. Protein binding studies
4.9. Evaluation of cytotoxicity
Protein-binding studies were performed by fluorescence
quenching experiments using bovine serum albumin (BSA). The
fluorescence spectra were recorded with an excitation at 280 nm
and corresponding emission at 345 nm assignable to that of BSA and
the ligands/complexes as quenchers with increasing concentration
[71]. The excitation and emission slit widths and scan rates were
maintained constant for all the experiments. A stock solution of BSA
was prepared in 50 mM phosphate buffer (pH ¼ 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
Cytotoxicity of the binuclear complexes 1 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 Dalton’s 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 106 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 vitro cell 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.
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 fluorescence 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. The quenching constant (Kq) can be calculated using the plot
of I0/I versus [Q]. If it is assumed that the binding of compounds with
BSA occurs at equilibrium, the equilibrium binding constant can be
analyzed according to the Scatchard equation:
%inhibition ¼ No: of dead cells=ðNo: of dead cells
þ No of live cellsÞ ꢃ 100
log½ðI0 ꢀ IÞ=Iꢂ ¼ log Kbin þ nlog½Qꢂ

292
M. Alagesan et al. / European Journal of Medicinal Chemistry 78 (2014) 281e293
Fig. 13. Synchronous spectra of BSA (1
mM) in the presence of increasing amounts of the ligands HL1(a) and HL2(b) and the complexes 1(c) and 2(d) (0e8 mM) at a wavelength
difference of Dl ¼ 60 nm. The arrow shows the decreases in the emission intensity upon increasing concentration of the compounds.
the cells were seeded into 96-well plates in 100
mL of the
Table 3
respective medium containing 10% FBS, at a plating density of
10,000 cells/well. The cells were incubated at 37 ꢁC in 5% CO2 and
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
Cytotoxicity of the complexes determined by Trypan Blue dye exclusion method.
Compound
IC50 values (
mM)
EAC
DLA
Complex 1
Complex 2
4 ꢆ 0.50
5 ꢆ 0.86
4.1 ꢆ 0.57
4.1 ꢆ 0.61
10 mL. 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-
Table 4
Cytotoxicity of the complexes determined by MTT assay.
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 flicked off, and the formed formazan crystals were
Compound
IC50 values (
mM)
HeLa
NIH 3T3
Complex 1
Complex 2
0.7 ꢆ 0.98
22 ꢆ 0.54
54 ꢆ 0.98
dissolved in 100 mL of DMSO. Mean absorbance for each drug dose
18.6 ꢆ 0.98
was expressed as a percentage of the control untreated well
absorbance 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
%inhibition ¼ ½mean OD of untreated cellsðcontrolÞ=ðmean OD of treated cellsꢂ ꢃ 100
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,
and a graph was plotted with the percentage of cell inhibition
versus concentration. From this, the IC50 value was calculated
[73].

M. Alagesan et al. / European Journal of Medicinal Chemistry 78 (2014) 281e293
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The corresponding author of the manuscript (N. D) acknowl-
edges the Council of Scientific and Industrial Research (CSIR),
Ministry of Human Resources Development (MHRD), Government
of India, New Delhi, for the financial 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 e Deemed University, Gandhigram, India, for elec-
trochemical measurement of samples.
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