Synthesis and crystal structure of new monometallic and bimetallic copper(II) complexes with N-substituted isatin thiosemicarbazone ligands: Effects of the complexes on DNA/protein-binding property, DNA cleavage study and in vitro anticancer activity

Article abstract of DOI:10.1016/j.poly.2016.06.017

A novel series of N-substituted isatin thiosemicarbazone ligands (L1–L3) and their copper(II) complexes [Cu(II)(ITSC)] were synthesized and characterized by elemental analyses and UV–Vis, FT-IR,¹H &¹³C NMR/EPR and mass spectroscopic

Full text of DOI:10.1016/j.poly.2016.06.017

Accepted Manuscript
Synthesis and crystal structure of new monometallic and bimetallic copper(II)
complexes with N-substituted isatin thiosemicarbazone ligands: Effects of the
complexes on DNA/protein-binding property, DNA cleavage study and in vi-
tro anticancer activity
Mathiyan Muralisankar, Surendran Sujith, Nattamai S.P. Bhuvanesh,
Anandaram Sreekanth
PII:
S0277-5387(16)30250-9
DOI:
http://dx.doi.org/10.1016/j.poly.2016.06.017
POLY 12057
Reference:
Polyhedron
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 May 2016
8 June 2016
9 June 2016
Please cite this article as: M. Muralisankar, S. Sujith, N. S.P. Bhuvanesh, A. Sreekanth, Synthesis and crystal
structure of new monometallic and bimetallic copper(II) complexes with N-substituted isatin thiosemicarbazone
ligands: Effects of the complexes on DNA/protein-binding property, DNA cleavage study and in vitro anticancer
activity, Polyhedron (2016), doi: http://dx.doi.org/10.1016/j.poly.2016.06.017
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Synthesis and crystal structure of new monometallic and bimetallic copper(II)
complexes with N-substituted isatin thiosemicarbazone ligands: Effects of the
complexes on DNA/protein-binding property, DNA cleavage study and in vitro
anticancer activity
Mathiyan Muralisankar,a Surendran Sujith,a Nattamai S. P. Bhuvaneshb and Anandaram
Sreekantha*
a Department of Chemistry, National Institute of Technology, Tiruchirappalli - 620 015, India.
b Department of Chemistry, Texas A & M University, College Station, TX 77842, USA

Synthesis and crystal structure of new monometallic and bimetallic copper(II)
complexes with N-substituted isatin thiosemicarbazone ligands: Effects of the
complexes on DNA/protein-binding property, DNA cleavage study and in vitro
anticancer activity
Mathiyan Muralisankar,a Surendran Sujith,a Nattamai S. P. Bhuvaneshb and Anandaram
Sreekantha*
Abstract
A novel series of N-substituted isatin thiosemicarbazone ligands (L1–L3) and their
copper(II) complexes [Cu(II)(ITSC)] were synthesized and characterized by elemental analyses
1
and UV Visible, FT-IR, H & 13C NMR / EPR and mass spectroscopic techniques. The
molecular structures of L1, L2, L3, 2 and 3 were confirmed by single crystal X-ray
crystallography. The X-ray diffraction studies of the complexes 2 and 3 reveal the square planar
and square pyramidal geometry. The binding affinity and binding mode of the monometallic and
bimetallic complex toward calf thymus DNA (CT-DNA) and bovine serum albumin (BSA) were
determined by UV-Vis and fluorescence spectrophotometric methods. Spectral evidences show
intercalative mode of DNA binding with the copper(II) complexes. Complexes (1, 2 and 3)
cleaved the pUC19 plasmid DNA in the absence of an external agent. An in vitro cytotoxicity
study of the complex 3 found significant activity against human breast (MCF7) and lung (A549)
cancer cell lines.
Keywords: monometallic and bimetallic copper(II) complexes; DNA/protein binding property;
DNA cleavage; cytotoxicity.
*Corresponding Author E-mail address: sreekanth@nitt.edu; Tel: +91 431 2503642

1. INTRODUCTION
Metals have a vital role in biological systems and their role in cellular, subcellular
functions are invariably recognized. The importance of metals is inevitable because they are the
essential part of enzymes which involved in the metabolic or biochemical process. These
elements required only in trace amount for cellular uptake and also it act as a catalyst [1]. The
variable oxidation state of transition metal complexes made them as significant in bioinorganic
chemistry and redox enzyme systems. The multiple reactive sites and the site specific
substructures, stimulate the metal complexes as a promising candidate for artificial
metallonucleases [2]. Nowadays the designing and study of new drugs which contain metals are
one of the embryonic areas of research [3] In 1969 Barnett rosenbrg and co-workers discovered
the antitumor activity of cis-platin and still now it is the widely used metal-based anticancer
drugs against the cancers of ovaries, testicles etc. Even though it have some serious side effect
like nephrotoxicity, cytotoxicity, vomiting, loss of hair etc [4]. The substantial side effect of cis-
platin is mainly arise from its binding mode with DNA, were cisplatin form a covalent crosslink
which inhibited the medicinal successes of cis-platin [5]. There for, the chemist has been paid
much interest on design and synthesis of more biocompatible metal-based drugs, which are less
toxic, high pharmacological effect, target-specific and non-covalent DNA binding mode. One of
the most suitable alternatives is copper, because of its biocompatibility and versatility in
coordination. The assumption that copper like endogenous metals may less toxic for normal cells
with respect to cancer cells [8]. Investigation of literature shows that there are much copper
contain complexes which show potential antitumor, anticancer activities [9-12]. The
pharmacological effect of mononuclear copper complexes are extensively studied, but studies on
binuclear copper complexes are very few till to date. There is a number of proteins which
holding binuclear centers and they have a vital role in biological functioning [13-15] this fact
stimulated us to synthesis and compare between mono nuclear and binuclear copper complexes
of thiosemicarbazone ligands. Many metal-based anticancer drugs exert their pharmacological
effect through binding to DNA or protein. So the study of the interaction between metal
complexes with DNA or protein is momentous in the current scenario. The shape and the
chemical structure of DNA provide a number of opportunities for interaction with metal
complexes. The study interactions of drugs with proteins are very significant because human

body have a number of proteins which all are assigned to different biological functions. Among
the serum albumin proteins, the exploration of the interaction of copper complexes with bovine
serum albumin (BSA) is very significant because of it is structurally homologous to human
serum albumin (HSA). Examination of drug-protein interaction has also another impact that it
influence the distribution, absorption, and metabolism of the drug [16-17].
We choose thiosemicarbazone derivatives as our ligands. The versatility in coordination
and their capabilities such as intramolecular hydrogen bonding, bulkier coligand , and π-π
stacking interactions inspire as to choose it [18,19]. An interesting attribute of the
thiosemicarbazone is that in the solid state, they predominantly exist in the thione form, whereas
in a solution state, they exhibit a thione- thiol tautomerism. The thiol form predominates in the
solution state, and deprotonation at the thiolatc sulfur render the thioscmicarbazonc an anionic
ligand, which can effectively coordinate to a metal atom. In addition, it should be noted that in
the thiol form, there is an effective conjugation along the thiosemicarbazone skeleton thus giving
rise to an efficient electron delocalization along the moiety. Also, literature shows that
thiosemicarbazone based compounds show variety of medicinal properties such as anticancer,
antitumour, antifungal, antibacterial, antiviral, antimalarial, antifilarial and anti-HIV activities
[20-27]. Hence, we are amalgamate copper metal with thiosemicarbazone ligands and we
compare the biological activity of mono and binuclear complexes. In the present work, we are
synthesized and characterized the thiosemicarbazone based mono and bimetallic copper
complexes. The interaction of complexes with DNA and protein were studied, also, the
biological activity examined.
2. Experimental
2.1. Materials and methods
All the chemicals were purchased from Sigma Aldrich / Merck and used as received.
Solvents were purified by distillation and retained under inert atmosphere. The melting points
were determined on Lab India instrument and are uncorrected. The elemental analyses were
performed using a Vario EL−III CHNS analyzer. Magnetic susceptibilities were measured using
Sherwood scientific auto magnetic susceptibility balance. FT-IR spectra were recorded in the
range of 400-4000 cm-1 (KBr pellets) and far-IR spectra were recorded in the range of 400-30
cm-1 (polyethylene pellets) using a PerkinElmer Frontier FT-IR/FIR spectrometer. UV-Visible

spectra were recorded in the range of 900-250nm using a PG Instruments T90+UV-visible
spectrophotometer in DMF solution. Emission spectra were measured on a Jasco V-630
spectrophotometer using 5% DMF in buffer as the solvent. NMR spectra were recorded in
CDCl3 by using TMS as an internal standard on a Bruker 400 MHz spectrometer. EPR spectra
were recorded on a JEOL EPR spectrometer at liquid nitrogen temperature, operating at X-band
frequency (9.1 GHz).
2.2. Synthesis of ligands (L1- L3).
Step 1: Ethanolic solution of cyclohexyl isothiocyanate (0.706 g, 0.005 mole) and
hydrazine hydrate (0.250 g, 0.005 mole) were mixed with constant stirring. The stirring was
continued for one more hour and the white product, N(4)-cyclohexylthiosemicarbazide formed
was filtered, washed, dried and recrystallized from ethanol. Yield: 91%
Step 2: N(4)-cyclohexylthiosemicarbazide (0.173 g, 0.001 mole) was dissolved in
methanol (20 mL) and was added to the appropriate ketone’s [1-(prop-2-yn-1-yl)indoline-2,3-
dione (0.185 g, 0.001 mole); 1-allylindoline-2,3-dione (0.187 g, 0.001 mole) and 1-
benzylindoline-2,3-dione (0.237 g, 0.001 mole)] dissolved in methanol (20 mL), reflux
continuously for 6 h after a few drops of acetic acid. The yellow product formed was filtered off,
washed with cold ethanol and ether, and dried in vacuo. It was recrystallized from
CH3OH/C2H3N mixture (1:1) to get yellow colored crystals suitable for X-ray studies were
obtained from slow evaporation of the reaction mixture for 10-15 days in all three cases.
2.2.1 N-cyclohexyl-2-(2-oxo-1-(prop-2-yn-1-yl)indolin-3-ylidene)hydrazinecarbothioamide
(L1)
N(4)-cyclohexylthiosemicarbazide (0.173 g, 0.001 mole), 1-(prop-2-yn-1-yl)indoline-2,3-
dione (0.185 g, 0.001 mole) were used. Yield: 82%. Yellow. 2 oC. Anal. Calc. C18H20N4OS (%):
C, 63.50; H, 5.92; N, 16.46; S, 9.42. Found: C, 63.22; H, 5.68; N, 16.41; S, 9.41. UV−Vis
(DMF): λmax, nm (ε, dm3mol-1cm-1) 268 (53600), 366 (73200). FT-IR (KBr): ʋ, cm-1 3344 (N–
1
H), 3223 (N–H), 1689 (C=O), 1526 (C=N), 1353 (C=S). H NMR (400 MHz, CDCl3): δ, ppm
12.56 (s, 1H), 7.66 – 7.59 (m, 2H), 7.40 (td, J = 7.8, 1.2 Hz, 1H), 7.14 (td, J = 7.6, 0.8 Hz, 1H),
7.09 (d, J = 7.9 Hz, 1H), 4.53 (d, J = 2.5 Hz, 2H), 4.36 – 4.25 (m, 1H), 2.26 (t, J = 2.5 Hz, 1H),
2.14 (dd, J = 12.0, 3.0 Hz, 2H), 1.80 – 1.72 (m, 2H), 1.71 – 1.62 (m, 1H), 1.52 – 1.41 (m, 2H),
1.32 (dd, J = 11.4, 8.4 Hz, 2H), 1.21 (ddd, J = 15.3, 8.1, 3.4 Hz, 1H). 13C NMR (100 MHz,
CDCl3): δ, ppm 176.4 (C=S), 160.1 (C=O), 141.7 (C=N), 131.1, 130.0, 123.5, 120.6, 119.7,

110.1 (aromatic C), 76.1, 73.0 (propa ), 53.4, 28.9, 25.5, 24.8 (Cyclohexane C). ESI-MS
m/z=341.14 [M+1]+
2.2.2 2-(1-allyl-2-oxoindolin-3-ylidene)-N-cyclohexylhydrazinecarbothioamide (L2)
N(4)-cyclohexylthiosemicarbazide (0.173 g, 0.001 mole), 1-allylindoline-2,3-dione
o
(0.187 g, 0.001 mole) were used. Yield: 80%. Yellow. m.p.: 180 C. Anal. Calc. C18H22N4OS
(%): C, 63.13; H, 6.48; N, 16.36; S, 9.36. Found: C, 63.10; H, 6.42; N, 16.32; S, 9.35. UV−Vis
(DMF): λmax, nm (ε, dm3mol-1cm-1) 265 (53000), 367 (79400). FT-IR (KBr): ʋ, cm-1 3349 (N–
1
H), 3258 (N–H), 1684 (C=O), 1521 (C=N), 1344 (C=S). H NMR (500 MHz, CDCl3): δ, ppm
12.57 (s, 1H), 7.65 (d, J = 7.3 Hz, 1H), 7.62 (d, J = 7.4 Hz, 1H), 7.41 (t, J = 7.6 Hz, 1H), 7.15 (t,
J = 7.4 Hz, 1H), 7.10 (d, J = 7.8 Hz, 1H), 4.54 (s, 2H), 4.37 – 4.28 (m, 1H), 2.28 (t, J = 7.2 Hz,
1H), 2.15 (d, J = 10.3 Hz, 2H), 1.78 (d, J = 12.6 Hz, 3H), 1.67 (d, J = 12.4 Hz, 2H), 1.46 (dd, J =
13
24.1, 12.1 Hz, 3H), 1.35 (dt, J = 20.8, 10.6 Hz, 2H), 1.31 – 1.21 (m, 1H). C NMR (100 MHz,
CDCl3): δ, ppm 176.5 (C=S), 160.7 (C=O), 142.8 (C=N), 131.0, 130.8, 123.1, 120.0, 119.6,
118.3 (aromatic C), 130.5, 109.9 ( ), 53.3, 32.5, 25.6, 24.8 (Cyclohexane C). ESI-MS
m/z=343.15 [M+1]+
2.2.3. 2-(1-benzyl-2-oxoindolin-3-ylidene)-N-cyclohexylhydrazinecarbothioamide (L3)
N(4)-cyclohexylthiosemicarbazide (0.173 g, 0.001 mole), 1-benzylindoline-2,3-dione
o
(0.237 g, 0.001 mole) were used. Yield: 83%. Yellow. m.p.: 168 C. Anal. Calc. C22H24N4OS
(%): C, 67.32; H, 6.16; N, 14.27; S, 8.17. Found: C, 67.28; H, 6.08; N, 14.22; S, 8.15. UV−Vis
(DMF): λmax, nm (ε, dm3mol-1cm-1) 281 (56200), 369 (73800). FT-IR (KBr): ʋ, cm-1 3343 (N–
1
H), 3229 (N–H), 1687 (C=O), 1523 (C=N), 1348 (C=S). H NMR (500 MHz, CDCl3): δ, ppm
12.75 (s, 1H), 7.67 (d, J = 7.9 Hz, 1H), 7.60 (d, J = 7.5 Hz, 1H), 7.35 – 7.24 (m, 6H), 7.08 (t, J =
7.6 Hz, 1H), 6.79 (d, J = 7.9 Hz, 1H), 4.94 (s, 2H), 4.39 – 4.29 (m, 1H), 2.16 (d, J = 10.8 Hz,
1H), 1.78 (d, J = 13.2 Hz, 2H), 1.72 – 1.63 (m, 2H), 1.47 (dd, J = 24.5, 12.1 Hz, 2H), 1.36 (dd, J
13
= 22.2, 10.8 Hz, 2H), 1.26 (dd, J = 24.2, 12.0 Hz, 1H). C NMR (100 MHz, CDCl3): δ, ppm
176.5 (C=S), 161.1 (C=O), 142.8 (C=N), 135.1, 131.0, 130.5, 129.0, 128.1, 127.5, 123.2, 120.6,
119.7, 110.1 (aromatic C), 53.3, 32.5, 25.6, 24.8 (Cyclohexane C). ESI-MS m/z=393.17 [M+1]+
2.3 Synthesis of copper (II) complexes (1, 2 and 3)
Step 3: The chloroformic solution of CuCl2.2H2O (0.001 mole) was added into the
solution of an appropriate substituted isatin thiosemicarbazone ligands (0.001 mole) in methanol.
The reaction mixture was stirred for 2 hours at room temputure, and then the precipitate formed

was filtered and washed with chloroform. The suitable crystals for X-ray diffraction were grown
from CH3OH/C2H3N mixture (1:1) to get blackish brown colored crystals suitable for X-ray
studies were obtained from slow evaporation of the reaction mixture for 20 days in complexes 2
and 3.
2.3.1 2-(2-oxo-1-(prop-2-yn-1-yl)indolin-3-ylidene)-N-cyclohexyl-hydrazinecarbothioamide
copper(II) (1)
CuCl2.2H2O (0.170 g, 0.001 mole), L1 (0.340 g, 0.001 mole) were used. Yield: 75%.
Light brown block solid. m.p.: 239 °C. Anal. Calcd. For C18H19ClCuN4OS: C, 49.31; H, 4.37; N,
12.78; S, 7.31. Found: C, 49.36; H, 4.35; N, 12.76; S, 8.56. UV−Vis (DMF): λmax, nm (ε,
dm3mol-1cm-1) 266 (51466), 359 (15400), 433 (18933). FT-IR (KBr): ʋ, cm-1 3245 (N–H), 1640
(C=O), 1503 (C=N), 1245 (C= S) and far-IR (Polyethylene) ʋ, cm-1 362 (Cu–Nazo), 348 (Cu-S),
316 (Cu-Cl). Epr (LNT): ‘g’ values 2.25, 2.04
2.3.2 2-(1-allyl-2-oxoindolin-3-ylidene)-N-cyclohexylhydrazinecarbothioamide copper(II)
(2)
CuCl2.2H2O (0.170 g, 0.001 mole), L2 (0.342 g, 0.001 mole) were used. Yield: 79%.
Light brown block solid. m.p.: 243 °C. Anal. Calcd. For C18H21ClCuN4OS: C, 49.08; H, 4.81; N,
12.72; S, 7.28. Found: C, 49.61; H, 4.82; N, 12.68; S, 7.22. UV−Vis (DMF): λmax, nm (ε,
dm3mol-1cm-1) 265 (50666), 356 (26000), 424 (32066). FT-IR (KBr): ʋ, cm-1 3257 (N–H), 1634
(C=O), 1508 (C=N), 1263 (C= S) and far-IR (Polyethylene) ʋ, cm-1 369 (Cu–Nazo), 328 (Cu-S),
302 (Cu-Cl). Epr (LNT): ‘g’ values 2.22, 2.05
2.3.3 2-(1-benzyl-2-oxoindolin-3-ylidene)-N-cyclohexylhydrazinecarbothioamide copper(II)
(3)
CuCl2.2H2O (0.170 g, 0.001 mole), L3 (0.392 g, 0.001 mole) were used. Yield: 61%.
Light brown block solid. m.p.: 252 °C. Anal. Calcd. For C44H46Cl2Cu2N8O2S2: C, 53.87; H, 4.73;
N, 11.42; S, 6.54. Found: C, 53.91; H, 4.74; N, 11.38; S, 6.51. UV−Vis (DMF): λmax, nm (ε,
dm3mol-1cm-1) 265 (46533), 373 (25800), 423 (21733). FT-IR (KBr): ʋ, cm-1 3221 (N–H), 1634
(C=O), 1508 (C=N), 1274 (C= S) and far-IR (Polyethylene) ʋ, cm-1 371 (Cu–Nazo), 348 (Cu-S),
316 (Cu-Cl). Epr (LNT): ‘g’ values 2.04, 2.00
2.4. Single crystal X-ray diffraction studies

A Bruker APEX2 or Bruker GADDS X-ray (three-circle) diffractometer was employed
for crystal screening, unit cell determination, and data collection. The X-ray radiation employed
was generated from a Mo sealed X-ray tube (Kα = 0.70173 Å; 40 kV, 40 mA) for L1, L2, L3, 2
and 3 fitted with a graphite monochromator (175 mm collimator with 0.5 mm pinholes). Sixty
data frames were taken at widths of 0.5°. These reflections were used in the auto-indexing
procedure to determine the unit cell. A suitable cell was found and refined by nonlinear least
squares and Bravais lattice procedures. The unit cell was verified by examination of the h k l
overlays on several frames of data by comparing with both the orientation matrices. No super-
cell or erroneous reflections were observed. After careful examination of the unit cell, a standard
data collection procedure was initiated using omega scans. Integrated intensity information for
each reflection was obtained by reduction of the data frames with the program APEX2 [28]. The
integration method employed a three dimensional profiling algorithm and all data were corrected
for Lorentz and polarization factors, as well as for crystal decay effects. Finally, the data were
merged and scaled to produce a suitable data set. The absorption correction program SADABS
[29] was employed to correct the data for absorption effects. Systematic reflection conditions and
statistical tests of the data suggested the space group. Solution was obtained readily using XT/XS
in APEX2 [30]. Hydrogen atoms were placed in idealized positions and were set riding on the
respective parent atoms. All non-hydrogen atoms were refined with anisotropic thermal
parameters. The structure was refined (weighted least squares refinement on F2) to convergence
[31]. Olex2 was employed for the final data presentation and structure plots [30].
2.5. DNA binding studies
The binding of metal complexes with CT DNA was carried out in Tris HCl/NaCl buffer
(pH 7.2). The bulk solution of CT DNA was prepared by diluting the CT DNA using Tris
HCl/NaCl buffer followed by stirring at 4 °C for three days, and kept at 4 °C for not more than a
week. The stock solution of CT DNA gave a ratio of UV absorbance at 260 and 280 nm
(A260/A280) of 1.9, indicating that the DNA was sufficiently free of proteins. The bulk DNA
solution was further diluted to 10 folds to show maximum absorbance at 260 nm. The absorption
coefficient of CT DNA was 6600 cm-1 M-1 per nucleotide [32]. Cu(II) complexes of required
concentration were prepared by dissolving the calculated amount of the complexes in 5%
DMF/Tris HCl/NaCl. Complex solution of concentration 15 µM was taken in cuvette and CT

DNA of equivalent concentration (0–50 µM) was added each time and the significant absorbance
change was noted.
The competitive binding of each complex with EB has been investigated by fluorescence
spectroscopic technique in order to examine whether the complex can displace EB from its CT
DNA-EB complex. Ethidium bromide solution was prepared using Tris HCl/NaCl buffer (pH
7.2). The test solution was added in aliquots of 2.5 µM concentration to DNA-EB and the change
in fluorescence intensities at 607 nm (520 nm excitation) was noted down.
Viscosity experiments were carried out using a semi micro viscometer maintained at 27
°C in a thermostatic water bath. DNA samples (0.5 µM) were prepared by sonication in order to
minimize complexities arising from DNA flexibility. Flow time was measured three times for
each sample and an average flow time was calculated. The values of relative specific viscosity
(η/η0), where η is the relative viscosity of DNA in the presence of the complex and η0 I s the
relative viscosity of DNA alone, were plotted against 1/R (1/R = [compound]/[DNA]). Relative
viscosity (η0) values were calculated from the observed flow time of the DNA solution (t)
corrected for the flow time of the buffer alone (t0), using the expression η0 = (t − t0)/t0 [33].
2.6. Protein binding studies
The binding of Cu(II) complexes (1, 2 and 3) with BSA was studied using fluorescence
spectra recorded at a fixed excitation wavelength corresponding to BSA at 280 nm and
monitoring the emission at 335 nm. The excitation and emission slit widths and scan rates were
constantly maintained for all the experiments. Stock solution 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
solutions of each test compound were prepared by dissolving them in DMF–phosphate buffer
(5:95) and diluted with phosphate buffer to get required concentrations. 2.5 ml of BSA solution
was titrated by successive additions of a 10−6 M stock solution of the complexes using a
micropipette. For synchronous fluorescence spectra measurements, the same concentration of
BSA and the complexes were used and the spectra were measured at two different ∆λ (difference
between the excitation and emission wavelengths of BSA) values of 15 and 60 nm.
2.7. DNA cleavage studies
A mixture of Tris buffer (5mMTris–HCl/50mMNaCl buffer, pH7.2), pUC19 plasmid
DNA (150 µg mL-1) and different amounts of the complexes (1–3) were incubated for 3 h at 37
oC. A dye solution (0.05% bromophenol blue and 5% glycerol) was added to the mixture prior to

electrophoresis. The samples were then analyzed by 1.5% agarose gel electrophoresis [Tris–
HCl/boric acid/EDTA (TBE) buffer, pH 8.0] for 2 h at 60 mV. The gel was stained with 0.5 µg
mL-1 ethidium bromide, visualized by UV light and photographed. The extent of cleavage of
pUC19 was determined by measuring the intensity of the bands using AlphaImager HP
instrument.
2.8. Cytotoxic activity
Cytotoxicity of complexes (1, 2 and 3) was evaluated by using the MTT [3- (4,5 dimethyl
thiazole-2-yl)-2,5-diphenyltetrazolium] assay [34]. The non-small lung adenocarcinoma cells
(A549 cells) and human breast cancer cells (MCF-7) were plated separately in 96 well plates at a
concentration of 1 × 105 cells/well. Complexes (1-3) of concentration ranging from 10-250 µM
dissolved in DMSO were seeded to the wells. DMSO was used as the control. After 24 h, the
wells were treated with 20 µL MTT [5 mg/ml phosphate buffered saline (PBS)] and incubated at
37 °C for 4 h. This colorimetric test is based on the selective ability of viable cells to reduce the
tetrazolium component of MTT into purple coloured formazan crystals formed were dissolved in
200 µL DMSO. The absorbance of the solution was measured at a wavelength of 570 nm using a
Beckmann Coulter Elisa plate. Triplicate samples were analyzed for each experiment. The
percentage inhibition was calculated using the formula.
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3. Results and discussion
3.1. Synthesis
Cyclohexylhydrazinecarbothioamide were synthesized using the standard procedure [35]
(Scheme 1). The substituted heterocyclic based ligands (L1–L3) were synthesized by a
condensation reaction from substituted heterocyclic based ketone and N(4) cyclohexylthio
semicarbazide (Scheme 2). The Cu(II) complexes were synthesized using CuCl2.2H2O as the
precursor (Scheme 3). All the ligands and their Cu(II) complexes were characterized by
elemental analyses and/or various spectroscopic techniques. The molecular structures of L1, L2,
L3, 2 and 3 were confirmed by single crystal X-ray diffraction studies.

Scheme 1 Synthesis of N(4)-cyclohexylthiosemicarbazide.
Scheme 2 Synthesis of heterocyclic based thiosemicarbazone.
Scheme 3 Synthesis of copper(II) complexes.

3.2. Spectroscopy
Electronic spectra of the ligands (L1, L2 and L3) showed two strong absorption bands
around 265-281 and 366-369 nm, which were assigned to π → π* and n → π* transitions
respectively. The spectra of the complexes (1, 2 and 3) exhibited three bands. The high intensity
bands in the regions 264-266 nm correspond to intra ligand transitions. A less intense band at
358-373 nm has been assigned to ligand to metal charge transfer (LMCT) transitions
respectively. The charge transfer transitions are observed in the region 421-432 nm. All
complexes, taken in DMF at same concentration i.e. for fresh solution day one and after three
days. There was no distinct changes observed in the UV-visible bands which clearly shows that
the copper(II) complexes are stable in test solution. However, the Cu(ll) complexes appear to
exist in a wide range of stereochemistry making it difficult to use electronic spectroscopy alone
as a definitive tool for identifying the geometry. Similar assignments have been made for other
copper(II) square planar and square pyramidal complexes [36,37].
In the FT-IR spectra of the solid state ligands (L1, L2 and L3), two bands were observed
for the stretching of N-H groups in the range of 3343-3349 and 3223-3258 cm-1, which was
absent in the complexes indicating deprotonation of the N-H group. The cyclohexene attached N-
H groups appeared in the region 1321-1357 cm-1. A strong band observed in the region 1684-
1689 cm-1 in the FT-IR spectra of ligands was assigned to the C=O stretching vibration. This
band was shifted to the lower frequency range of 1634-1640 cm-1 in the spectra of the complexes
(1, 2 and 3), consistent with the coordination of O to the metal ion. The characteristic band for
C=S appeared in the region 1344-1353 cm-1 in the spectra of L1, L2 and L3 were shifted to
lower frequency 1245-1274 cm-1 on complexation, indicating involvement of S coordination.
The C=N stretching frequency of the ligands was observed at 1521-1526 cm-1. During
complexation there is a decrease in azomethine stretching frequency 1503-1514 cm-1, which
suggested that N coordinate to a metal ion. The Cu-N stretching frequencies for azomethine
nitrogen are observed around 362-371 cm-1. The presence of a new band in the 328-348 cm-1
range, which is assignable to Cu-S is another indication of the involvement of sulphur
coordination. Bands at 302-316 cm-1 for the complexes are due to Cu-Cl stretching suggestive
for terminally bonded chlorine [38].

1
A sharp singlet observed at around 12.56−12.75 ppm in the H NMR spectrum of the
ligand L1-L3 is assigned to H−N−C=S. The signals of cyclohexane protons in the all ligands
appeared around 2.28−1.26 ppm. The signals around 7.67−6.79 ppm corresponding to aromatic
protons of the ligands (L1−L3). In the ligand L1 and L2 the CH2−C≡CH and CH2−CH2=CH2
protons shows signal around 4.53, 4.36−4.25 and 4.54, 4.37−428 ppm respectively. The signal at
4.94 ppm corresponding to benzyl CH2 protons of the ligand L3. 13C NMR spectra of the ligands
showed resonances due to thiocarbonyl (C=S), carbonyl carbon (C=O) and imine carbon (C=N)
in the regions 176.4−176.5, 160.1−161.1 and 141.7−142.8 ppm respectively. The ligands L1
(CH2-C≡CH) and L2 (CH2−CH2=CH2), shows the carbon signals at 53.4, 76.1, 73.0 and 53.3,
130.5, 109.2 ppm respectively. The signal regions 53.3 ppm corresponding to benzyl carbon of
L3. The cyclohexane carbons appeared around 42.0−43.5, 28.9−32.5, 25.5−25.6 and 24.8 ppm
respectively. Chemical shift of all other aromatic and aliphatic protons/carbons was observed in
the expected region [39].
The oxidation state of the central copper atom in the complexes was confirmed by the
measurements of EPR spectroscopy. X-band CW EPR spectra of the Cu(II) complexes were
recorded at LNT. For complexes 1, 2 and 3 are EPR active due to the presence of an unpaired
ꢖ
electronꢄꢑꢕ = 1 2ꢒ and the spin angular momentum quantum number can have valuesꢄꢑꢗꢘ =
ꢖ
ꢄ± 1 2ꢒ, which in the absence of external magnetic field the spin state of electrons are
degenerate state. In the presence of magnetic field degeneracy no more present. The low energy
sate has the spin magnetic moment aligned with the field and correspond to the quantum
ꢖ
ꢖ
numberꢄꢑꢗꢘ = ꢄ− 1 2ꢒ, while the high energy stateꢄꢑꢗꢘ = ꢄ+ 1 2ꢒ has its moment opposed to
the filed. Each spin state further spilt into four levels due to hyperfine splitting with nuclear spin
of Cu(II) (I = 3/2) [40,41]. The transition between this split levels take place according
toꢄꢑꢙꢄꢗꢚ = 0). All the complexes (1, 2 and 3) showed well resolved quartet hyperfine splitting
typical of square planar and square pyramidal Cu(II) system (Fig. 1 and S1), which was
confirmed by single crystal XRD technique. For all the complexes g|| > g⊥ suggesting that the
system is axial. The trend in the g value (g|| > g⊥> 2.00) and the value of exchange interaction
term (G > 4.0) suggested that the unpaired electron of Cu(II) ion is present in the dx2−y2 orbital
(Table 1).
3.3. Single crystal X-ray crystallographic studies

The molecular structure of ligands (L1, L2 and L3) and the complexes (2 and 3) with the
atomic labelling schemes are shown in the Fig. 2–6. Crystal data and some selected inter-atomic
bond lengths and angles are given in the Tables 2–5. Ligands (L1, L2 and L3) exists in the
thione form as confirmed by the C=S bond length of 1.6723(16), 1.6696(18) and 1.6723 (14) Ǻ.
The crystal structures of the ligands showed the existence of an intra-molecular hydrogen bond
between N–H and the carbonyl oxygen. An intra-molecular hydrogen bond between the N(3)–
H(3) proton and O1 is present in ligands (L1, L2 and L3) [2.769, 2.7850 and 2.7441 Å], creating
a six-membered ring and favouring the Z-isomer with respect to the imine C=N bond. This
hydrogen bond renders the isatin and TSc (N2–N3–C12/16–S1–N4) moieties essentially
coplanar.
The complex 2 crystallized in monoclinic P – 1 space group with one molecules of the
ligand per copper. The copper (II) adopts a square planar geometry with binding of the ligands as
monobasic tridentate (ONS) donors. The S1−C12 bond length (1.731 Å) in 2 is higher than that
of L2 [42]. There is an increase in the C–O and C–N bond lengths (involved in coordination) in
2 compared to L2. The Cu−S bond is longer than the Cu–Cl, Cu–O and Cu–N bonds
[Cu(1)−S(1) 2.236 Å, Cu(1)−Cl(1) 2.192 Å, Cu(1)−O(1) 2.072 Å and Cu(1)−N(1) 1.970 Å],
which are in the expected range for thiosemicarbazone complexes. The torsional angles in 2 are -
2.4 [Cu(1) –S(1) –C(12) –N(2)], 176.7 [Cu(1) –S(1) –C(12) –N(4)], 179.0 [Cu(1)–O(1) –C(2) –
N(3)].
The complex 3 crystallizes in monoclinic lattice with P 1 21/n 1 space group symmetry.
Coordination extends the thiosemicarbazone moiety's S(1)−C(13) bond length from 1.684(3) Å
(L3) to 2.2821(5) Å (3). The asymmetric unit is formed by one half of the molecule and the
other half is related by a center of inversion in the Cu(1)−S(1)−Cu(1) −S(1)#1 ring. The
coordination geometry at each Cu(II) is square pyramidal. The ligand 3 coordinates to the metal
in a tridentate manner through its carbonyl oxygen, azomethine nitrogen and thiolate sulfur, after
enolization and deprotonation, to form the basal plane along with one chlorine atom. The sulfur
atom takes the axial position of the adjacent monomer like its counterpart does to form two
distorted square pyramids. The Cu−Cu separation is found to be 3.541 Å. The τ value of 0.137 at
metal centres indicates slight distortion from perfect square pyramidal geometry [43]. Thus, the
two metal centres are bridged via sulfur atoms resulting in the formation of four five membered
rings in which one copper atom is shared by two fused rings. A four membered ring involving

the copper atoms and the sulfur atoms is also formed. The five membered ring Cu(1), O(1), C(2),
C(1), is approximately planar with a maximum deviation of −3.6(2) Å for C(2), but Cu(1), S(1),
C(16), N(2) ring is slightly distorted as evidenced by the maximum deviation of −4.31(16) Å;
likewise for their counterpart rings. The four membered ring Cu(1)−S(1)−Cu(1)#1 makes an
angle of 88.283(17) Å with the mean plane of bicyclic chelate system O(1), C(2), C(1), N(1),
N(2), C(16), S(l), Cu(1) which is slightly distorted from planarity. The present compound
contains the shortest apical Cu–S distance reported to date for square-pyramidal Cu(II)
complexes of N4–substituted isatin thiosemicarbazones [44]. The free ligand (L3) exists as the
thione tautomer and coordinates to the Cu(II) atom in the deprotonated thiolate form, thus
rendering a single-bond character for the C−S bond. Similarly, coordination of the azomethine N
atom and carbonyl O atom to the central Cu(II) atom results in a redistribution of the electron
density along the thiosemicarbazone chain, giving rise to changes in the bond distances along the
moiety compared with those of the uncoordinated thiosemicarbazone. There is increase in the
C=N and C=O bond distance (involved in coordination) in complex 3 compared to ligand L3.
The Cu−S bond is longer than the Cu–Cl, Cu–O and Cu–N bonds [Cu(1)−S(1) 2.2821(13) Å,
Cu(1)−Cl 2.2140(5) Å, Cu(1)−O(1) 2.1069(13) Å and Cu(1)−N(1) 1.9812(16) Å.
3.4. DNA binding studies
For evaluating the potentials of antitumor activities of any new complexes, DNA binding
is the significant footstep for chemical nuclease activity of the metal complexes. The
understanding of the interaction between DNA and a complexes to be investigated is of most
importance. Hence the mode and tendency for the binding of complexes to CT DNA were
studied with different methods.
3.4.1. Electronic absorption titration
Electronic absorption spectroscopic studies are one of most reliable techniques have been used to
investigate the interactions of metal complexes with DNA. When metal complexes are bound to
a DNA, there should be different types of binding modes occur, non-covalent binding, metal
coordination to the nucleobases, intercalation, phosphate backbone binding. From the change in
absorbance and shift in wavelengths, we can understand the binding nature of metal complexes
with DNA. In general, the intercalative mode of binding due to the strong stacking interaction
between an aromatic chromophore and the base pairs of DNA, usually results in hypochromism
along with or without a small red or blue shift [45]. The extent of shift and hypochromism

correlate with the intercalative binding strength. The absorption spectrum of the complexes 1, 2
and 3 in the absence and presence of CT DNA are shown in Fig. 7 and S2. Upon increasing
concentration of DNA, the absorption band of the complexes 1, 2 and 3 exhibited a
hypochromism (∆ε, 23, 28 and 38%) with a red shift of (1, 2 and 5) nm respectively. These
results suggested that all the new copper complexes bind to the DNA helix via intercalation, due
to stacking interaction between the planar aromatic chromophore and the base pairs of DNA.
Monometallic complexes of 1 and 2 showed less hypochromicity than the bimetallic complex 3,
indicating that the binding strength of bimetallic complexes is much stronger than the other
complexes. The binding constant of the complexes with CT DNA (Kb) was obtained from the
ratio of slope to intercept in plots [DNA]/(εa − εf) versus [DNA] according to the equation [46]
[DNA]/(εa−εf) = [DNA]/(εb−εf) + 1/Kb (εb−εf) where [DNA] is the concentration of DNA in base
pairs, εa is the apparent extinction coefficient value found by calculating A(observed)/[complex],
εf is the extinction coefficient for the free compound, and εb is the extinction coefficient for the
compound in the fully bound form. Each set of data, when fitted into the above equation, gave a
straight line with a slope of 1/(εb − εf) and an y-intercept of 1/Kb(εb − εf) and the value of Kb was
determined from the ratio of slope to intercept (Fig. 8). The magnitudes of intrinsic binding
constants (Kb) are shown in Table 6. The observed values of Kb revealed that the Cu(II)
complexes bind to DNA via intercalative mode [47]. The Kb values were found to be in the range
of 1.49 – 1.72 × 105 M-1. Complex 3 showed better DNA binding affinity compared to the other
complexes. Which is due to the geometry of the bimetallic copper complex bridged with sulphur
plays profound effect on DNA binding abilities, which cause the considerable difference in the
Kb value of bimetallic copper complex 3 over than mono metallic copper complexes 1 and 2.
3.4.2. Viscosity measurements
The nature of binding of the metal complexes to the CT DNA was further investigate by
viscosity measurements. For intercalation bond between a copper(II) complexes and the base
pairs of DNA forces these base pairs away from each other and therefore, unwinding the double
helix and lengthening a given amount of DNA which in turn, increases the viscosity of the DNA
solution Fig .12. The increase in the viscosity of DNA follows the order 3> 2 > 1, but lower than
that for the standard DNA intercalator EB. Thus, all these observations suggest that the N-

substituted aromatic ring of complex 3 showed enhanced binding ability compared to other
complexes.
3.4.3. Ethidium bromide displacement study
The absorption titration results indicate that the new copper(II) complexes effectively
bind to DNA, In order to get further evidences for binding mode and to compare their binding
affinities, ethidium bromide displacement experiments were carried out. Usually, intense
fluorescent light is emitted from EB in the presence of CT DNA due to the strong intercalation
between adjacent DNA base pairs in the double helix; EB is considered to be a typical indicator
of intercalation [48]. Hence, EB displacement technique can be used to obtain indirect evidence
for the DNA binding mode. The displacement technique is based on the decrease of fluorescence
intensity due to the displacement of EB from a DNA sequence by a quencher and the quenching
is due to the reduction of the number of binding sites on the DNA that is available to the EB. The
changes observed in the fluorescence quenching spectra of DNA-bound EB in the presence of
copper(II) complexes (0-50 µM) are shown in Fig. 10 and S3 which illustrate that as copper(II)
complexes intercalate into DNA, it leads to a decrease in the binding sites of DNA available for
EB, resulting in a decrease in the fluorescence intensity of the CT DNA-EB system.
Fluorescence quenching is explained by the Stern-Volmer equation [49] Fo/F = 1 + Kq [Q] where
Fο and F are the fluorescence intensities in the absence and presence of complex respectively, Kq
is a linear Stern-Volmer quenching constant, and [Q] is the concentration of complex. The slope
of the plot of Fo/F versus [Q] gave Kq (Fig. 9). The apparent DNA binding constant (Kapp) values
was calculated by using the equation KEB [EB] = Kapp [complex] where [complex] is the complex
concentration at 50% reduction in the fluorescence intensity of EB, KEB = 1.0 × 107 M-1 and [EB]
= 5 µM. The quenching constant Kq and Kapp values follows order 3> 2> 1, which follows the
same order as discussed in the absorption titration and values are listed in Table 6.
3.4.4 Electrochemical titration
The cyclic voltammetric technique can be used to investigate the interaction between the
metal complexes and DNA, which also provides an important complement to the previously used
spectral experiments [50]. The understanding of the interaction between DNA and a complexes,
the cyclic voltammetric technique was carried out and the results are given in Fig. 11 and S4.
Obviously, in the absence of CT DNA, both of the monometallic and bimetallic complexes (1, 2
and 3) have a couple of waves corresponding to Cu(II)/Cu(I) with the cathodic (EPc) and anodic

peak potential (EPa) being −1.03 and V −0.75 for the complex 1, −1.04 V and −0.77 V for the
complex 2 and -1.06 V and −0.76 for the complex 3, respectively. The separations of anodic and
cathodic peaks (∆Ep) are found to be 0.28 V (1), 0.27 V (2) and 0.30 V (3). The formal potentials
o
of the Cu(I)/Cu(II) couple in free form (Ef ), which are taken as the average of EPc and EPa, are
−0.89, −0.90 and −0.91 V for the complexes 1, 2 and 3. On the addition of CT DNA with
complexes, the voltammetric peak currents decreased apparently, indicating that there exist
interactions between the complexes and CT DNA [51]. The drop of the voltammetric currents in
the presence of CT DNA may be attributed to slow diffusion of the complexes (1, 2 and 3) bound
to DNA. The cathodic (EPc) and anodic peak (EPa) potentials are found to be −1.01 and −0.72 V
for the complex 1, −1.06 and −0.75 V for the complex 2 and −0.99 and −0.67 V for the complex
3. It can be seen that the peak-to-peak separation becomes larger, as ∆Ep = 0.29, 0.31 and 0.32 V
for the complexes 1, 2 and 3 respectively, suggesting that in the presence of CT DNA the
electron-transfer process becomes less reversible for the complexes. The formal potentials of the
o
Cu(I)/ Cu(II) couple in binding form (Eb ) are −0.86, -0.91 and −0.83 V for complexes 1, 2 and 3
respectively. Clearly, the values of the three complexes are shifted towards positive region by
0.03, 0.01 and 0.08 V for the complexes 1, 2 and 3 respectively, suggesting that the complexes
o
o
could intercalate to DNA [52]. The separation between Eb and Ef can be used to estimate the
ratio of binding constants for the reduced and oxidized forms to DNA using the equation as
follows:
ꢛꢜ − ꢛꢝ = 0.059ꢄꢞꢟꢠꢄ[ꢡꢢꢣꢑꢚꢒꢄ/ꢄꢡꢢꢣ ꢚꢚ
]
ꢒ
ꢑ
Where KCu(I) and KCu(II) are the binding constant of Cu(I) and Cu(II) forms to DNA, respectively.
The KCu(I)/KCu(II) values were estimated to be 3.22, 1.46 and 3.32 for the complexes 1, 2 and 3
respectively. The shift in the anodic and cathodic peak potentials towards more positive side and
the stronger affinity for less positively charged redox form indicating that the reduced forms of
the complexes can interact more strongly than the oxidized ones. And this agreed with the
intercalation mode of binding over electrostatic interaction mode. In the case of intercalation +1
form copper bound more strongly than +2 form of copper, probably because of the importance of
hydrophobic interactions.
3.5. Protein binding studies
3.5.1. Absorbance and fluorescence studies

UV-visible absorption measurement is a simple method to investigate the structural
changes and to know which type of quenching occurs either dynamic or static quenching. The
dynamic quenching refers to a process in which the fluorophore and the quencher come into
contact during the transient existence of the excited state while static quenching refers to
fluorophore–quencher complex formation in the ground state. The absorption band obtained for
BSA at 278 nm in the absence of quencher when addition of the complexes (1, 2 and 3) to BSA
lead to an increase in BSA absorption intensity without affecting the position of absorption band.
This indicates that the type of interaction between Cu(II) complexes and BSA was mainly a
static quenching process [53]. The representative absorption titration spectra is shown in Fig. 13.
Fluorescence emission spectroscopy is usually employed to determine the interaction
ability of metal complexes with human serum albumin, which is the most abundant protein in
plasma. To understand the mechanism of interaction between the complexes (1, 2 and 3) and
BSA, fluorescence quenching experiments have been carried out. Generally, the fluorescence
property of protein is caused by three intrinsic characteristics of the protein, mainly due to the
presence of tryptophan, tyrosine and phenylalanine residues [54]. Fig. 14 and S5 shows the
fluorescence emission spectra of BSA after the addition of complexes (1, 2 and 3). When
increasing amount of complex solution was added to a fixed quantity of BSA, fluorescence
intensity at 345 nm decreases upto 54.1, 69.2 and 81.8% for complexes respectively, with
hypochromic shift of 16, 19 and 12 nm for complexes 1, 2 and 3 respectively. The blue shift
primarily arises due to the presence of the active site of the protein in a hydrophobic
environment. It suggested that interaction is taking place between the compounds and BSA [55].
The fluorescence quenching is described by the Stern-Volmer relation [56,57] Fo/F = 1 + Kq [Q]
where Fo and F demonstrate the fluorescence intensities in the absence and presence of quencher,
respectively. Kq is a linear Stern-volmer quenching constant, and [Q] is the quencher
concentration. The quenching constant (Kq) can be calculated using the plot of log (Fo/F) versus
log [Q] (Fig. 15). When small molecules bind independently to a set of equivalent site, on a
macromolecule, the equilibrium between free and bound molecules is represented by the
Scatchard equation. log[(Fo−F)/F] = log Kb + n log[Q] where Kb is the binding constant of the
complex with BSA and n is the number of binding sites. From the plot of log[(Fo−F)/F] versus
log[Q] (Fig.16), the number of binding sites (n) and the binding constant (Kb) values have been
obtained. The quenching constant (Kq), binding constant (Kb) and number of binding sites (n) for

the interaction of the Cu(II) complexes with BSA are shown in Table 7. In all the complexes,
only one binding site is available to interact with BSA. Results showed that complexes 3 interact
strongly with BSA compared to 2 and 1.
3.5.2. Characteristics of synchronous fluorescence spectra
Synchronous fluorescence spectroscopy provides sound information about the molecular
microenvironment, particularly in the vicinity of the fluorophore functional groups, the structural
changes occurring to BSA upon the addition of the Cu(II) complexes. It is well-know that the
fluorescence of BSA is normally, due to the presence of tyrosine, tryptophan and phenylalanine
residues are responsible for the fluorescence property of BSA. According to miller, the
difference between the excitation and emission wavelength (∆λ = λem - λexc ) reflects the spectra
of a different nature of chromophores, [58] with large ∆λ value, such as 60 nm, is characteristic
of tryptophan residue and a small ∆λ value, such as 15 nm, is characteristic of tyrosine. The
synchronous fluorescence spectra of BSA with various concentrations of Cu(II) complexes (1, 2
and 3) were recorded at ∆λ = 15 nm and ∆λ = 60 nm. On addition of the complexes, the
fluorescence intensity of tryptophan residue at 340 nm decreased in the magnitude of 70.3, 73.8
and 83.3% for complexes 1, 2 and 3 respectively (Fig. 17 and S6). Similarly, there was also
decrease in the intensity of tyrosine residue at 300 nm. The magnitude of decrease was 43.3, 65.3
and 70.8% for complexes 1, 2 and 3 respectively (Fig. 18 and S7). The synchronous fluorescence
spectral studies clearly suggested that the fluorescence intensities of both the tryptophan and
tyrosine were affected with increasing concentration of the complexes. The results indicate that
the interaction of complexes with BSA affects the conformation of both tryptophan and tyrosine
micro-region [59].
3.6. DNA cleavage
The cleaving efficacy of complexes (1, 2 and 3) has been assessed by their ability to
convert supercoiled pUC19 DNA from form I to form II by agarose gel electrophoresis. As
shown in (Fig. 19), no distinct DNA cleavage was observed for the control in which the complex
was absent (lane 1); however, with fixed concentration of the complexes (1, 2 and 3), cleave SC
(Form I) DNA into nicked circular (NC) (Form II) DNA, Hence, the complexes exhibited DNA
cleavage activity in the absence of an external agent [60]. Additionally, the amount of helical
unwinding induced by the complex bound to SC DNA provides evidence for the intercalation

mode of interaction between the complexes and DNA. The DNA cleavage activity of the
complexes (1, 2 and 3) can be estimated from the percentage of cleavage (C) (Table 8).
3.7. In vitro cytotoxic activity evaluation by MTT assays
The cytotoxicity of the monometallic and bimetallic complexes toward MCF7 (human
breast cancer cells) and A549 (human lung cancer cells) cells has been examined by using MTT
assay [61] and compared with cyclophosphamide [IC50 = 6.58 µM (MCF7) and 22.36 µM
(A549)] under identical conditions. Figs. 20 and 21 show the cytotoxicity of the complexes (1-3)
after 24 h incubation on MCF7 and A549 cancer cell lines, respectively. Complexes 2 and 3
exhibited moderate cytotoxicity with IC50 values of 88.59 and 82.56 µM against A549
respectively. The same complexes showed half inhibition concentration of 120.68 and 110.34
µM against MCF7 cell line respectively. Complex 1 showed only least activity. The enhanced
activity of complex 3 compared to complexes 1 and 2 may be due to its bimetallic structure. The
results of the cytotoxicity coincides well with the DNA and protein binding ability of the
complexes. The IC50 values of all the complexes are listed in Table 9.
4. Conclusion
Two monometallic and one bimetallic copper(II) complexes with N-alkylated isatin based
thiosemicarbazone ligands have been synthesized and characterized. Single crystal X-ray
diffraction studies revealed that the 2 have square planar and 3 have square pyramidal geometry.
The DNA binding affinity of the complexes were valuated using spectrophotometric methods.
The results supported the interaction of the complexes with CT DNA through intercalation.
Complexes 3 have better DNA cleaving ability compared to the other complexes. Fluorescence
quenching experiments of BSA confirms the binding ability of the complexes and the mode of
binding is static. In addition, the synchronous spectral investigations revealed that the complexes
induced a small change in the secondary structure of the protein at the studied conditions. All
complexes exhibited good cytotoxicity against cancer cell line A549and MCF7.
Electronic supplementary information (ESI) available: Crystallographic data for the
structures reported in this paper have been deposited with the Cambridge Crystallographic Data
Centre (CCDC) as supplementary publication numbers (CCDC 1418911, CCDC 1418910,
CCDC 1418912, CCDC 1470836 and CCDC 1470837 for L1, L2, L3, 2 and 3 respectively

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Fig. 1 EPR spectrum of complex 3 in frozen DMF solution. Microwave power, 0.98 mW;
microwave frequency, 9.1 GHz.

Fig. 2 The molecular structure of ligand L1, with displacement ellipsoids drawn at the 50%
probability level.
Fig. 3 The molecular structure of ligand L2, with displacement ellipsoids drawn at the 50%
probability level.
Fig. 4 The molecular structure of ligand L3, with displacement ellipsoids drawn at the 50%
probability level.

Fig. 5 The molecular structure of complex 2, with displacement ellipsoids drawn at the 50%
probability level.
Fig. 6 The molecular structure of complex 3, with displacement ellipsoids drawn at the 50%
probability level.

Fig. 7 Absorption spectra of complex 3 in Tris-HCl buffer upon addition of CT DNA. [Complex]
= 1.5 × 10−5 M, [DNA] = 0-50 µM. Arrow shows that the absorption intensities decrease upon
increasing DNA concentration.
Fig. 8 Stern-Volmer plots of absorbance titrations of the complexes with CT DNA.
Fig. 9 Stern-Volmer plots of fluorescence titrations of the complexes with CT DNA.