Docking of imines, cytotoxicity and DNA interaction studies of metal(II) complexes

Article abstract of DOI:10.1016/j.molstruc.2013.11.038

A new series of eight transition metal [Cu(II), Co(II), Mn(II) and Ni(II)] complexes of Schiff bases were synthesised from pyridine/thiophene carbaldehyde and 2,2-diphenylethanamine and further characterized. The investigation of these complexes confirmed

Full text of DOI:10.1016/j.molstruc.2013.11.038

Journal of Molecular Structure 1059 (2014) 299–308
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Docking of imines, cytotoxicity and DNA interaction studies
of metal(II) complexes
⇑
S. Sujarani, A. Ramu
Department of Inorganic Chemistry, School of Chemistry, Madurai Kamaraj University, Madurai 625 021, Tamil Nadu, India
h i g h l i g h t s
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Synthesis and characterization of pyridine/thiophene based Schiff base by various spectral methods.
M(II) complexes were found to octahedral and square planar geometry, respectively.
The biological studies of cytotoxic studies by using Hep G2 cell lines.
Transition metal complexes are found suitable for designing the anticancer drug.
The docking studies reveal that the imine group serves as Schiff base imine group only.
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of eight transition metal [Cu(II), Co(II), Mn(II) and Ni(II)] complexes of Schiff bases were syn-
thesised from pyridine/thiophene carbaldehyde and 2,2-diphenylethanamine and further characterized.
The investigation of these complexes confirmed that the stability of metal–ligands coordination through
N, N & N, S atoms as bidendate chelates. Docking studies showed that the system binds strongly with
DNA. The metal/ligands ratio of 1:2 was proposed to afford square planar and octahedral geometry for
the complexes. Further, the studies on DNA binding & cleavage characteristics of the ligands & complexes
using spectral and electrophoresis techniques evidenced the groove binding property of DNA with metal
complexes. The synthesized complexes also found to induce cytotoxicity in cell lines.
Received 4 September 2013
Received in revised form 5 October 2013
Accepted 13 November 2013
Available online 23 November 2013
Keywords:
Diphenylethanamine
Metal complexes
Docking with Schiff base imine
DNA interaction studies
Hep G2 cell lines
Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction
osity due to their wide range of applications such as pesticides,
herbicides, insecticides, bactericides and anticonvulsants [13].
Cancer is undoubtedly one of the major health concerns faced
by our society and serves as a primary targets in the field of medic-
inal chemistry. Even though, platinum-based complexes have been
used primarily in research for development of chemotherapeutic
agents [1–3]. In recent years, the interests in this field have shifted
from platinum based agents to non-platinum based agents [4–11]
in order to find different metal complexes with lesser side effects
and/or better cytotoxicity. A wide variety of metal complexes
based on copper, cobalt, manganese and nickel has been inten-
sively studied for replacement of platinum [4–11]. Furthermore,
Cu(II)-based complexes appear to be an attractive and promising
candidates for anticancer therapy that describes the synthesis
and cytotoxic activities of numerous copper(II) complexes [12].
Heterocyclic thiophene/pyridine derivatives are of impressive curi-
Many of drugs that control or kill neoplastic cells are used in che-
motherapy. However, all these drugs showed unpleasant side ef-
fects such as nausea, hair loss and suppression of bone marrow
functions. A chemical modification of existing anticancer agents
to produce novel biomolecules with better therapeutic properties
is highly warranted. The heterocyclic moiety makes them more
interesting because the combinations of the two groups of a spe-
cies with a considerable coordination potential. The existence of
bond between imine and thio group resulted in generation of sta-
ble metal complexes involving five member ring systems [14,15].
Schiff bases are considered as a very important class of organic
compounds, which have extensive applications in many biological
aspects [16]. Transition metal complexes of Schiff bases are one of
the most adaptable and thoroughly studied systems till date. These
complexes have also numerous applications in clinical, analytical
and industrial perspective other than their roles in catalysis and or-
ganic synthesis [17]. Transition metal complexes of those ligands
are studied and exploited because of their potential for therapeutic
⇑
Corresponding author. Tel.: +91 452 2458410; fax: +91 452 2458743; mobile:
+91 9442623835.
E-mail address: ramumku@yahoo.co.in (A. Ramu).
0022-2860/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molstruc.2013.11.038

300
S. Sujarani, A. Ramu / Journal of Molecular Structure 1059 (2014) 299–308
use [18–35]. The bonding modes and the geometry position of the
transition metal complexes in ligand chelation environment serves
as models to enzyme containing metal ion. Hence, in the present
study we synthesized and characterized the heterocycles based
Schiff bases and their complexes and also we investigated their
DNA binding properties and cytotoxic effect on cancer cell lines.
free from protein. The DNA concentration per nucleotide and poly-
nucleotide concentrations were determined by absorption spec-
troscopy using the molar extinction coefficient (6600 Mꢂ1 cmꢂ1
)
at 260 nm. The intrinsic binding constant Kb for the interaction of
these metal complexes with DNA has been calculated from the
absorption spectral changes during the addition of increasing con-
centration of DNA by, the following equation:
½DNAꢄ=ðea
ꢂ
ef Þ ¼ ½DNAꢄ=ðeb
ꢂ
ef Þ þ 1=Kbðeb
ꢂ
ef
Þ
ð1Þ
2. Experimental
where [DNA] is the concentration of DNA in base pairs, the apparent
2.1. Materials and methods
absorption coefficient ea ef and eb correspond to Aobs/[M], the
,
extinction coefficient of the free and the extinction coefficient of
the compound when fully bound to DNA, respectively. Plot of
All chemicals and solvents used in this study were of AR grade.
2-pyridine/2-thiophene carbaldehyde (Sigma Aldrich), 2,2-diphen-
ylethanamine (Sigma Aldrich), transition metal ions [Copper(II)
chloride, Cobalt(II)chloride, Nickel(II)chloride and Manga-
nese(II)sulfate] (Merck), Calf thymus DNA (Sigma), Tris–hydrochlo-
ride (SRL) and sodium chloride (SRL) were used as such without
further purification.
Elemental analyses (C, H & N) were performed using an Elemen-
tary Vario EL elemental analyzer CHNS Mode. Metal contents were
determined volumetrically by titration against standard Ethylene
diamine tetra acetic acid (EDTA) solution after complete decompo-
sition of their complexes with concentrated nitric acid. The chlo-
rine content was determined by Volhard test. Molar
conductivities of the metal complexes were determined in Di-
methyl sulphoxide (DMSO ꢁ10ꢂ2 M) at room temperature using
an EI Model 611E digital conductivity meter. The magnetic suscep-
tibilities of complexes were determined on Gouy balance, and the
diamagnetic corrections were made by Pascal’s constant and
CuSO4ꢃ5H2O was used as a calibrant. Electrospray Ionization Mass
Spectrometry (ESI-MS) analyses were recorded in LCQ Fleet (Ther-
mo Fisher Instruments Limited, USA). Nuclear magnetic resonance
[DNA]/(eb
ꢂ
e
f) vs [DNA] gave a straight line with a slope of
f)) and Kb was determined
1/(eb f) and an intercept of 1/Kb(eb
ꢂ
e
ꢂ
e
from the ratio of the slope to intercept.
2.1.2. CD spectra
CD spectra of DNA in presence and absence of all the complexes
were recorded on a JASCO J-810 (163–900 nm) spectropolarimeter
using a quartz cuvette of 1 mm optical path length at increasing
complex/DNA ratio (r = 0.01–0.04). Each sample solution was
scanned in the range of 220–320 nm. Every CD spectrum was col-
lected after averaging over at least four accumulations using a scan
speed of 100 nm minꢂ1 and a 1s response time from, which the
buffer back ground had been subtracted [DNA] = 100 lM.
2.1.3. Gel electrophoresis
The cleavage of DNA in the presence of the activating agent
H2O2 was monitored using agarose gel electrophoresis. In cleavage
reactions, super coiled pUC19 DNA (500 ng) in 10% DMSO; 5 mM
Tris–HCl; 50 mM NaCl buffer at pH 7.2 was treated with Mn(II)
complex. The samples were incubated for 1 h duration at 37 °C. A
loading buffer containing 25% bromo phenol blue, 0.25% xylene
spectroscopic measurements were made on
a Perkin–Elmer
300 MHz spectrometer. Duetrated organic solvents along with tet-
ramethylsilane (TMS) as the internal standard were used. UV–Vis
spectral measurements for the synthesized complexes were made
in DMSO using JASCO double beam recording spectrophotometer
in the range 190–1100 nm. The infrared spectra of all complexes
as well as ligands were recorded using KBr pellets on a JASCO FT-
IR 410 double beam infrared spectrophotometer in the range of
400–4000 cmꢂ1. Electron paramagnetic resonance spectra of the
copper complexes were obtained on a Jeol-300 MHz EPR spectrom-
eter. The spectra were recorded for the complexes as solid forms at
room temperature (RT) and solutions of complexes dissolved in
acetonitrile at 77 K. 2,2-diphenyl-1-picrylhydrazyl (DPPH) was
used as the field marker. Cyclic voltammetric measurements were
carried out on a Bio-Analytical System (BAS) CV-50W model elec-
trochemical analyzer. The three electrodes cell comprising of a ref-
erence Ag/AgCl, counter electrode as platinum wire and working
glassy carbon (GC) electrodes with surface area of 0.07 cm2 were
used. The GC was polished with 0.3 and 0.005 mm alumina before
each experiment and if necessary the electrode was sonicated in
distilled water for 10 min. Dissolved oxygen was removed by purg-
ing pure nitrogen gas into the solution for about 15 min before
each experiment. A cyclic voltammogram has been recorded for a
blank solution to check the purity of the supporting electrolyte
and the solvent.
cyanol and 30% glycerol (3 lL) was added and electrophoresis
was performed at 60 V for 2 h in Tris–acetate–EDTA (TAE) buffer
(40 mM Tris–base; 20 mM acetic acid; 1 mM EDTA) using 1% aga-
rose gel containing 1.0 l
g mLꢂ1 ethidium bromide. The cleavage
products were irradiated at room temperature with a UV lamp
(365 nm, 10 W) and analyzed with a Bio-Rad Model XI computer
controlled electrophoresis power supply (Bio-Rad, USA).
2.3. Cytotoxic activity
2.3.1. Evaluation of in vitro anticancer activity
Cytotoxicity studies of the complexes along with cyclophospha-
mide (control) were carried out on human liver cancer (HepG2)
cells and normal cells, which were obtained from the National Cen-
tre for Cell Science, Pune, India. Cell viability was assessed by MTT
assay. HepG2 cells were grown in Eagle’s minimum essential med-
ium (EMEM) containing 10% fetal bovine serum (FBS), whereas NIH
3T3 fibroblasts were grown in Dulbecco’s modified eagle’s medium
(DMEM) containing 10% FBS. For the screening, the cells were
seeded into 96-well plates in 1 ꢅ 105 cells/well of the respective
medium containing 10% FBS and incubated at 37 °C under condi-
tions of 5% CO2, 95% air and 100% relative humidity for 24 h prior
to the addition of the compounds. The compounds were dissolved
in DMSO and diluted in the respective medium containing 1% FBS.
After 24 h, the medium was replaced with the respective medium
with 1% FBS containing the compounds at various concentrations
and incubated at 37 °C under conditions of 5% CO2, 95% air and
100% relative humidity for 48 h. All the experiments were per-
2.2. DNA interaction studies
2.1.1. Electronic absorption spectra
The DNA binding experiments of the metal complexes with CT-
DNA were performed in Tris buffer (5 mM, pH 7.1). A solution of
CT-DNA in the buffer gave a ratio of UV–Vis absorbance at 260
and 280 nm of about 1.9:1, indicating that the DNA was sufficiently
formed in triplicates. After 48 h, 10 l
L of MTT (5 mg mLꢂ1) in phos-
phate buffered saline (PBS) was added to each well and incubated
at 37 °C for 4 h. The extracellular medium containing MTT was
then removed and the formazoan crystals were dissolved in

S. Sujarani, A. Ramu / Journal of Molecular Structure 1059 (2014) 299–308
301
100 lL of DMSO. The absorbance was then measured at 570 nm
3. Results and discussion
using a micro-plate reader (ELISA). The IC50 values were calculated
and a graph was plotted with the time variation vs. IC50 values in
In the present study, new Schiff base ligands (DNTE and DNPE)
and their metal complexes have been investigated. The modern
spectral techniques such as IR, UV–Vis, 1H &13C NMR, EPR & mass,
microanalytical and cyclic voltammetry were used for character-
ization of ligands and complexes. Elemental data analysis and
physical characteristics of Schiff’s base ligands and the complexes
undertaken are summarized in Table S1. (Supplementary data
files). The observed molar conductance of the complex in DMSO
(10ꢂ2 M solution at RT) are consistent with Cu(II) and Ni(II) elec-
trolytic in nature and other complexes are non-electrolytic nature.
lM.
2.4. Docking studies
2.4.1. Computational methods
Docking simulations were performed using the Lamarckian ge-
netic algorithm (LGA) and the Solis & Wets local search method
[36]. Initial position, orientation, and torsions of the ligand mole-
cules were set randomly. Each docking experiment was derived
from 10 different runs that were set to terminate after a maximum
of 250,000 energy evaluations. The population size was set to 150.
During the search, a translational step of 0.2 Å, and quaternion and
torsion steps of 5 were applied.
3.1. 1H NMR spectra
The 1H NMR spectra of the synthesised ligands (DNPE & DNTE)
showed the characteristic NMR signals and the data tabulated in
Table 1 (Fig. S1). (Supplementary data files). Signals of the aromatic
protons appeared in the range of 7–7.9 ppm and 6.7–8.1 ppm. The
presence of signal at d 4.3, d 4.2 ppm (DNPE) and d 4.5 ppm, d
4.3 ppm (DNTE) are due to CAH and CAH2 protons, respectively
of ethanamine moiety. Formation of diphenylethanamine Schiff
base has been confirmed by the appearance of CH@N azomethine
proton signal at d 8.3 ppm and d 8.2 ppm.
2.5. Synthesis of pyridine/thiophene based ligands
Diphenylethanamine (0.005 M) and appropriate heterocyclic
aldehyde (2-thiophene/2-pyridine carbaldehyde) (0.005 M) were
dissolved in dichloromethane (20 ml) in presence of anhydrous so-
dium sulfate and the reaction mixture was refluxed in water bath
at 40 °C for 3 h until the yellow color homogeneous liquid solution
obtained and the product was monitored by TLC and subsequently
washed thrice with dichloromethane and evaporated to dryness
followed by recrystallization using ethanol (60%) (Scheme 1).
3.2. 13C NMR spectra
2.6. Synthesis of pyridine/thiophene based Schiff base M(II) complexes
The 13C NMR (Fig. S2) (Supplementary data files) spectra of
DNPE and DNTE ligands consist of sharp signals at d 51.8 ppm; d
52.2 ppm and 65.8 ppm; d 64.2 ppm are due to CH2 and CH carbon
atoms of the ethanamine moiety, respectively. The signals at d
116–132 ppm may be attributed to the phenyl carbon atoms and
the signals CH@N carbon assigned at d 162.7 ppm; d 160.97 ppm
were assigned as azomethine carbons of Schiff bases, confirm the
structure of the ligands.
The metal complexes were synthesized by mixing 0.01 M of
corresponding transition metal chlorides in ethanol with 0.02 M
of the Schiff’s base. The reaction mixture was refluxed at 60 °C
for 6 h. The mixture was allowed to cool to room temperature.
The solid complexes obtained were filtered; washed with ethanol;
recrystallised from ethanol and dried in a vacuum (see Scheme 2).
Scheme 1. (a) DNPE & (b) DNTE.

302
S. Sujarani, A. Ramu / Journal of Molecular Structure 1059 (2014) 299–308
3.3. IR spectra
frequency (1621 and 1612 cmꢂ1) in the metal complexes suggests
the coordination of metal ion through nitrogen atom of azome-
thine group [38]. The exhibited broad bands in the range of
3382–3450 cmꢂ1, indicating the presence of coordinated water
molecules [37]. The absence of new bands in the spectrum of li-
The IR spectra provide valuable information regarding the coor-
dination sites of the aldemine moiety. The bands at 1641 cmꢂ1
(DNPE) and 1633 cmꢂ1 (DNTE) for the ligands, shifted to lower
Scheme 2. Synthesis of pyridine/thiophene based Schiff base complexes (a1) (DNPE)2Cu complex, (b1) (DNTE)2Cu complex, (a2) (DNPE) of Co(II) & Mn(II) (b2) (DNTE)2Mn, (b3)
(DNTE)2Co (a3) (DNPE)2Ni and (b4) (DNTE)2Ni.

S. Sujarani, A. Ramu / Journal of Molecular Structure 1059 (2014) 299–308
303
Fig. 7 (continued)
Scheme 3. Mass fragmentation pattern of DNPE.
Table 1
NMR spectra of ligands.
Schiff bases
DNPE
d/ppm 1H NMR
Splittingꢆ
d/ppm 13C NMR
Type of proton
8.3
4.3
4.5
s
d
t
162.7
51.8
65.8
CH@N
ACH2
ACH
7–7.9
m
121–154
Aromatic proton
DNTE
8.2
4.2
4.3
s
d
t
160.97
52.2
64.2
CH@N
ACH2
ACH
6.7–8.1
m
116–142
Aromatic proton
gands, which are present in the spectra of complexes, lies in the
range of 538–588 cmꢂ1, (DNPE) due to
(M–N) [39,40]. Similarly,
(M–N) 524–549 cmꢂ1 and (M–S) 441–470 cmꢂ1 (DNTE) vibra-
tions support the involvement of N and S atoms in the formation
of complex with metal ions were under investigation [41]. Finally,
the IR spectral studies confirmed both the ligands and their coordi-
nation with metal ions. The results are tabulated in Table S2 and
shown in Fig. S3 (Supplementary data files).
c
c
c

304
S. Sujarani, A. Ramu / Journal of Molecular Structure 1059 (2014) 299–308
3.4. UV spectra
The UV absorbance at 27,027 and 38,167 cmꢂ1 and 27,322 and
37,593 cmꢂ1 for the free ligands, DNPE and DNTE are characteristic
of p–
pꢆ and n–pꢆ transition, which could be due to the presence of
phenyl and imine groups, which confirms the formation of Schiff
bases. The characteristic absorbance band for the Cu(II) complexes
15,197 cmꢂ1 and 14,619 cmꢂ1, also confirm the Square planar
geometry. Similarly, the other six metal(II) complexes viz., Co(II),
Mn(II) and Ni(II) show d–d bands containing the octahedral/square
planar geometry and the results are tabulated in Table 2 spectra
are shown in Fig. S4 (Supplementary data files).
3.5. Electrochemical properties
The electrochemical characteristics of all the complexes have
been determined by cyclic voltammetry and the results are repre-
sented in Fig. 1 and Table S3 (Supplementary data files). The elec-
trochemical data of the copper(II) complex showed one well
defined redox couple corresponding to Cu(II)/(I). The cathodic peak
appearing in 59 mV, corresponds to one electron copper and the
corresponding anodic peak appears in the 282 mV. The measured
D
Ep = ꢂ223 mV, clearly indicate that these redox couples are quasi
reversible one electron reaction [47a,47b] Similarly, the (DNTE)2Co
complex showed one well defined redox couple corresponding to
Co(II)/(I). The cathodic peak appearing in the ꢂ1120 mV and the
corresponding anodic peak appears at ꢂ786 mV of Co(III)/Co(II) re-
dox couple. The
D
Ep is ꢂ334 mV. From this data, it can be ac-
counted as quasi reversible one electron process. However, the
(DNPE)2Mn complex showed two well defined redox couple corre-
sponding to Mn(II/I) as expected. The cathodic peak appearing in
the ꢂ841 mV corresponds to one electron reduction of Mn(I/II)
and the corresponding anodic peak appears in the ꢂ619 mV. The
measured
DEp = 222 mV clearly indicate that these Mn(II) reduc-
Fig. 1. Cyclic voltammogram of (a) (DNPE) and (b) (DNTE) complexes.
tion to Mn(I) is limited through the formation of the Mn(II) com-
plex. The electrochemical data of other two Ni(II) complexes
showed well defined redox couple corresponding to Ni(I)/(II). The
cathodic peak appearing in the ꢂ1153 mV, ꢂ997 mV corresponds
to one electron reduction of Ni(II)/(I) and the corresponding
anodic peak appears in the ꢂ607 mV, ꢂ474 mV. The measured
3.7. EPR spectra
The EPR spectrum of (DNPE)2Cu and (DNTE)2Cu complexes
anisotropy (Fig. S6) (Supplementary data files) exhibits axial type
signals with two ‘g’ values 2.13 and 2.02, corresponding to g|| and
g\ are tabulated in (Table 3) and the ordering of g values
(g|| > g\ > 2.0023) observed for Cu(II) complex indicates that the
unpaired electron is localized in the dx2 ꢂ y2 ground state in a
square planar geometry of the cu(II) ion [48].
D
Ep = ꢂ546 mV, ꢂ523 mV clearly indicate that these redox couples
are quasi reversible process.
3.6. Mass spectra
Mass spectra of the DNPE and (DNPE)2Cu complex [Fig. S5(a)
and (b)] (Supplementary data files) have been recorded and com-
pared for their stoichiometric composition. In the mass spectra of
the ligand, the peak at m/z = 286 amu corresponds to the molecular
ion peak. The peak observed at m/z = 287 amu possibly corre-
sponds to the C21H19NO+ moiety produced. The fragmentation pro-
cesses are carried out in the layout at Scheme 3.
3.8. Thermogravimetric analysis
Thermogravimetric curve (Fig. S7) (Supplementary data files) of
the complex (DNPE)2Cu & (DNTE)2Mn, showed a loss in weight be-
tween 200–380 °C and 180 °C, respectively, indicating the elimina-
tion of chlorine and water molecules from the complex. The results
Table 2
Electronic spectral data and assignments of heterocyclic based derivatives and their transition metal complexes.
Compounds
t1 (cmꢂ1
)
t2 (cmꢂ1
)
n–pꢆ (cmꢂ1
)
p–
pꢆ (cmꢂ1
)
Transition
Geometry
DNPE
–
38,167
38,022
37,735
37,878
35,971
27,027
28,735
27,777
27,173
29,239
(DNPE)2Cu
(DNPE)2Co
(DNPE)2Mn
(DNPE)2Ni
15,197
16,286
13,297
16,501
2T2g 2E2g
?
Square planar [42,43]
Octahedral [44]
Octahedral [45]
Square planar
4
14,705
4T1g(F) ? A2g(F),4T2g(F) & T1g(P)
5E ? 5T2
1A1g
2T2g
?
?
1Bꢆ1g
2E2g
DNTE
–
37,593
37,878
37,73
38,022
38,167
27,322
27,027
26,737
27,624
27,700
(DNTE)2Cu
(DNTE)2Co
(DNTE)2Mn
(DNTE)2Ni
14,619
16,181
18,726
16,155
Square planar
Octahedral
Octahedral [46]
Square planar
4
4
14,792
4T1g(F) ? 4A2g(F), T1g(F) & T1g(P)
6A1
1A1g
?
4T1(G)
1Bꢆ1g
?

S. Sujarani, A. Ramu / Journal of Molecular Structure 1059 (2014) 299–308
305
Table 3
In the present study, an attempt has been made to unravel the
DNA binding and nuclease activity of the transition metal [Cu(II),
Co(II), Ni(II) and Mn(II)] complexes. The absorption spectra ob-
tained in our study was similar to that of the spectra observed ear-
lier for other complexes. The binding constants values are
appreciable and less than 106 Mꢂ1, which indicates the partial
intercalation of DNA in the complexes. The (DNTE)2Ni complexes
serves as a better DNA binding agents with efficient nucleases
activity compared with that of the other complexes, which might
due to the additional oxygen atom in the complexes that enable
efficient DNA binding interaction through groove binding.
The order of intrinsic binding constants Kb obtained for the
complexes are as follow: (DNTE)2Ni > (DNPE)2Cu > (DNPE)2-
Co > (DNPE)2Ni > (DNPE)2Mn > (DNTE)2Co > (DNTE)2Cu > (DNTE)2-
Mn. The proposed mechanism of complexes binding with the DNA
is due to presence of more phenyl and heterocyclic moiety.
EPR spectral parameters of the copper(II) complexes at room temperature and at 77 K.
Compound
At RT
g||
At 77 K
g||
g\
g\
(DNPE)2Cu
(DNTE)2Cu
2.14
2.15
2.09
2.03
2.130
2.155
2.02
2.07
indicated that the ligands decompose gradually to the correspond-
ing metal oxide at higher temperature. These results are in consis-
tent with the composition of the complex [49].
4. DNA binding studies
4.1. Absorption spectral studies
Increase in the amount of CT-DNA (Fig. 2) showed an increase in
molar absorption of the complexes. The absorption bands with a
small red shift from 2 to 10 nm indicate their DNA binding prop-
erty of the complexes. The binding constant values of all the com-
plexes with CT-DNA (Table 4) indicated that there was a finite
interaction between these complexes and CT-DNA. All these results
are consistent with the results observed for classical intercalates
and CT-DNA intercalating metal complexes [50].
4.2. CD spectra
The characteristic CD spectrum of CT-DNA consists of a positive
band at 272 nm, which indicates base stacking, whereas negative
band at 243 nm indicates helicity. These features are the character-
istics of DNA in right-handed B form [51]. The CD spectrum of DNA
in the presence of nickel complexes (R = [DNA]/[Ni]2) undergoes
Fig. 2. Absorption spectra of (a) (DNPE) and (b) (DNTE) of copper complexes in Tris–HCl buffer pH 7.1 in the absence (R = 0) and presence (R = 2, 4, 6,. . ., 8) of increasing
amounts of DNA. R = [DNA]/[complex]. Arrow mark indicates the absorbance changes upon increasing DNA concentration and binding constant plot.

306
S. Sujarani, A. Ramu / Journal of Molecular Structure 1059 (2014) 299–308
Table 4
Ligand-based absorption spectral properties of transition metal complexes, bounda to CT-DNA.
Complexes
kmax (nm)
Hypochromism H (%)
Red shift
D
k (nm)
Binding constant Kb (Mꢂ1
)
(DNPE)2Cu
(DNPE)2Co
(DNPE)2Mn
(DNPE)2Ni
(DNTE)2Cu
(DNTE)2Co
(DNTE)2Mn
(DNTE)2Ni
262
262
264
278
264
266
264
266
14
15
7
14
19
14
13
24
2
2
2
10
2
2
0
2
6.23 ꢅ 104
5.82 ꢅ 104
4.00 ꢅ 104
4.91 ꢅ 104
2.16 ꢅ 104
3.70 ꢅ 104
1.15 ꢅ 104
7.25 ꢅ 104
a
Measurements made at different R values, where R = [DNA]/[complex], concentrations of solutions of Cu(II), Co(II), Mn(II) and Ni(II) complexes in pH 7.1 in 10% DMSO-
buffer solutions.
Fig. 3. Circular dichroism spectra of CT DNA in the absence (r = 0) and in presence of complex (a) (DNPE)2Ni and (b) (DNTE)2Ni (r = 0 and 0.1); [CT DNA] = 100 lM. Cell path
length = 1 mm. Arrow mark indicates the molar ellipticity change upon increasing complex concentration.
Table 5
Cytotoxic potency (IC50, lM) of the ligand and their metal complexes 1–10 on the
Hep-G2 Cell lines tested.
Compound
IC50
,
l
M (24 h)
IC50, lM (48 h)
DNPE
2443
1852
2270
3224
2204
2245
3416
1446
1317
1361
3441
2798
1343
1513
2319
1432
4254
1335
1696
1214
1282
3162
(DNPE)2Cu
(DNPE)2Co
(DNPE)2Mn
(DNPE)2Ni
DNTE
(DNTE)2Cu
(DNTE)2Co
(DNTE)2Mn
(DNTE)2Ni
Control
Fig. 4. DNA cleavage studies of (DNTE)2Ni. Lane C: Control DNA. Lane 1:
DNA + 2 M; Lane 2: DNA + 4 M; Lane 3: DNA + 6 M; in the different concentra-
tion of complex.
l
l
l
changes in both the positive and negative bands. The complex
[Ni(II)] showed a decrease in intensity in the negative band and
an increase in intensity of the positive band with shifts toward
longer wavelengths of about 5 nm (Fig. 3 and Table S4) (Supple-
mentary data files). These spectral changes observed for DNA on
interaction with Ni(II) have been associated with the conforma-
tional change from B to A form [52], Thus, the CD spectral results
for the complex [Ni(II)] implicates that there is a conformational
changes from B to A form DNA. Interestingly, (DNTE)2Cu [53] and
(DNTE)2Mn [54] complexes were found to induce conformational
change from B to A form. It is evident from the CD spectral results,
an increase and a decrease in intensities of the positive and nega-
tive bands, respectively. The characteristic red shift revealed that
the transition of DNA conformation from B to more A-like form
on binding to (DNTE)2Ni complexes. The complexes with more
phenyl moieties and their hydrophobic nature also evidenced the
conformational changes B to A on DNA binding.
4.3. DNA cleavage studies
The stronger DNA binding affinity and the transition of DNA
conformation by the (DNTE)2Ni complex has been investigated
by circular dichorism. Furthermore, there has been a substantial
and continuing interest in DNA cleavage reactions, activated by
metal ions. The interaction of Ni(II) complex with super coiled
pUC19 DNA in 10% DMSO-5 mM Tris–HCl buffer pH 7.2 was stud-
ied by agarose gel electrophoresis. For this, the DNA was incubated
with various concentration of (DNTE)2Ni for 1 h and then subjected
to agarose gel electrophoresis. Relatively fast migration of DNA
was observed for the super coiled form (Form I). DNA cleavage
was not observed for controls, in which complex was absent (Lane
1). With increasing concentration of (DNTE)2Ni complex

S. Sujarani, A. Ramu / Journal of Molecular Structure 1059 (2014) 299–308
307
Fig. 5. Docking of (a) DNTE and (b) DNPE with DNA.
(lanes 2–4), the intensity of Form I of pUC19 DNA diminishes grad-
5. Conclusions
ually with concomitant increase of nicked form (Form II) shown in
Fig. 4.
ꢀ Two series of new pyridine/thiophene based Schiff’s base metal
complexes have been successfully prepared and characterized
by using various spectral & analytical techniques.
4.4. Studies on the anticancer activity of complexes
ꢀ The geometry and the magnetic characteristics of synthesised
complexes were confirmed by the IR & UV spectroscopy and
confirmed that the ligands are coordinated through the central
metal ions through N, N & N, S bonding modes.
ꢀ The biological studies such as DNA interaction absorption, CD
spectroscopic technique showed the positive results and the
results were compared with cytotoxic effect of the complexes
on Hep G2 cell lines.
The cytotoxicity of the M(II) complexes (DNPE and DNTE) on
Hep-G2 human liver cancer cell line have been evaluated in com-
parison with the widely used drug cyclophosphamide as control
under standard conditions by using MTT assay. Table 5 indicates
the IC50 values of the complexes on Hep-G2 cell line. The results re-
vealed that the anticancer activity of the complexes is time-depen-
dent and varies with the mode and extent of their interaction with
DNA. The IC50 values for the ligands and complexes indicate that
the anticancer activity was due to the complex species rather than
the dissociated ligands. (DNTE)2Ni and (DNTE)2Mn exhibited
appreciable cytotoxic effects on Hep-G2 cell line. All these results
suggested that the DNA binding activity of the complexes through
N–S interactions could be attributed for the cytotoxic effect of
complexes on Hep-G2 cell line. The cytotoxic nature of the
(DNTE)2Ni, (DNTE)2Mn and (DNPE)2Cu complexes [55] has open
up the possibility for its applications as anticancer drugs.
ꢀ The docking studies reveal that the imine group serves as Schiff
base imine group for the binding.
ꢀ To conclude, all these results suggested that the cytotoxic activ-
ity of the transition metal complexes could be exploited for the
design of novel anticancer drug employing reported ligands and
metal combinations.
Acknowledgements
We gratefully acknowledge the financial support received from
JRF meritorious fellowship Ref. No. F4-1/2006(BSR)/7-119/
2007(BSR) UGC, New Delhi for carrying out this research work.
4.5. Docking studies
The synthetic route used to synthesize title compounds is out-
lined in Scheme 1. DNTE and DNPE were prepared by treatment
of corresponding 2,2-diphenylethanamine with pyridine-2-carbal-
dehyde and thiophene-2-carbaldehyde in the presence of dichloro-
methane. The structure of synthesized compound was assigned on
the basis of their spectral studies. In the present study, we con-
structed a DNA-ligands complex model with an X-ray crystal struc-
ture and the ligands were observed in crystal complex structures
using MMFF94 simulation and by Gasteiger partial charges to gain
better enrichment in virtual screening. Docking simulations were
performed using the Lamarckian genetic algorithm (LGA) and the
Solis & Wets local search method [39]. The result gives the total en-
ergy of binding modes is shown in Table S5 (Supplementary data
files). Calculated free energy of binding for compound in the inhib-
itor binding site (IBS) was ꢂ3.79 kcal/mol and ꢂ4.07 kcal/mol in
the best pose (Fig. 5).
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.molstruc.20
13.11.038.
References
[1] B. Lippert, Wiley Interscience, 1999.
[2] A.S. Abu-Surrah, M. Kettunen, Curr. Med. Chem. 13 (2006) 13371357.
[3] C.S. Allardyce, P.J. Dyson, Platinum Met. Rev. 45 (2001) 6269.
[4] I. Ott, R. Gust, Arch. Pharm. Chem. Life 340 (2007) 117126.
[5] M.A. Jakupec, M. Galanski, V.B. Arion, C.G. Hartinger, B.K. Keppler, Dalton
Trans. (2008) 183194.
[6] P. Yang, M. Guo, Coord. Chem. Rev. 189 (1999) 185186.
[7] S.K. Hadjikakou, N. Hadjiliadis, Coord. Chem. Rev. 253 (2009) 235249.

308
S. Sujarani, A. Ramu / Journal of Molecular Structure 1059 (2014) 299–308
[8] K. Strohfeldt, M. Tacke, Chem. Soc. Rev. 37 (2008) 11741187.
[35] C.Y. Duan, Y.P. Tian, X.Z. You, T.C.W. Mak, Polyhedron 16 (1997) 40974103.
[36] F.J. Solis, R.J.B. Wets, Minimization by random search techniques, Math. Oper.
Res. 6 (1981) 19–30.
[37] B. Jezowska, J. Lisowski, Polyhedron 7 (1988) 337343.
[38] J.M. Sece, M. Quiros, M.J.G. Garmendia, Polyhedron 19 (2000) 10051009.
[39] M. Thomas, M.K.M. Nair, P.K. Radhakrishnan, Synth. React. Inorg. Met. Org.
Chem. 25 (1995) 471479.
[9] P.M. Abeysinghe, M.M. Harding, Dalton Trans. 32 (2007) 34743482.
[10] R. Gust, D. Posselt, K. Sommer, J. Med. Chem. 47 (2004) 58375846.
[11] C.G. Hartinger, P.J. Dyson, Chem. Soc. Rev. 38 (2009) 391401.
[12] P. Yang, M. Guo, Coord. Chem. Rev. 185 (1999) 189211.
[13] Z.A. Kaplancikli, G. Turan-Zitouni, A. Ozdemir, G. Revial, Eur. J. Med. Chem. 43
(2008) 155159.
[14] D.H. Boschelli, D.T. Connor, D.A. Barnemeir, J. Med. Chem. 36 (1993) 18021809.
[15] S.H. Gaikwad, T.N. Lokhande, M.A. Anuse, Ind. J. Chem 44A (2005) 16251630.
[16] B.S. Tovrog, D.J. Kitko, R.S. Dragom, J. Am. Chem. Soc. 98 (1976) 51445153.
[17] H. Sharghi, M.A. Nasseri, Bull. Chem. Soc. 76 (2003) 137142.
[18] M.A. Ali, M.T.H. Tarafder, J. Inorg. Nucl. Chem. 39 (1977) 17851788.
[19] M.A. Ali, C.M. Haroon, M. Nazimuddin, S.M.M.H. Majumder, M.T.H. Tarafder,
M.A. Khair, Transit. Met. Chem. 17 (1992) 133136.
[20] M.A. Ali, M.H. Kadir, M. Nazimuddin, S.M.M.H. Majumder, M.T.H. Tarafder,
M.A. Khair, Ind. J. Chem. 27A (1988) 10641067.
[21] M.A. Ali, M. Uddin, M.N. Uddin, D.A. Chowdhury, M.T.H. Tarafder, Ind. J. Chem.
25A (1986) 238.
[40] K. Nakamoto, Infrared and Raman Spectra of inorganic and coordination comp
Edition Wile, New York, 1997.
[41] A. Jain, R. Goyal, D.D. Agarwal, J. Inorg. Nuclear Chem. 43 (1981) 20052009.
[42] N. Mondal, D.K. Dey, S. Mitra, K.M.A. Malik, Polyhedron 19 (2000) 27072711.
[43] M.M. Omar, G.G. Mohamed, Spectrochim. Acta Part A Mol. Biomol. Spectrosc.
61 (2005) 929936.
[44] C.H. Krishna, C.M. Mahapatra, K.C. Dush, J. Inorg. Nucl. Chem. 39 (1977)
12531258.
[45] K. Gudasi, S.A. Patil, R. Vadavi, V. Rashmi, R. Shenoy, M.S. Patil, J. Serb. Chem.
Soc. 71 (2006) 357372.
[46] A.D. Garnovskii, I.S. Vasilchenko, D.A. Garnovskii, B.I. Kharisov, J. Coord. Chem.
62 (2009) 151204.
[47] P. Tharmaraj, D. Kodimunthiri, C.D. Sheela, P. Prakash, J. Coord. Chem. 62
(2009) 13471355;
[22] M.A. Ali, S.M.M.H. Majumder, R.J. Butcher, J.P. Jasinski, J.M. Jasinski,
Polyhedron 16 (1997) 27492754.
[23] M.T.H. Tarafder, M. Rahim, Ind. J. Chem 28A (1989) 1105.
[24] Y.P. Tian, C.Y. Duan, X.Z. You, Transit. Met. Chem. 23 (1998) 17.
[25] Y.P. Tian, C.Y. Duan, Z.L. Lu, X.Z. You, Polyhedron 15 (1996) 22632271.
[26] X.H. Zhu, S.H. Liu, Y.J. Liu, J. Ma, C.Y. Duan, X.Z. You, Y.P. Tian, F.X. Xie, S.S. Ni,
Polyhedron 19 (1998) 181196.
(a) S. Chandra, N. Gupta, L.K. Gupta, Synth. React. Inorg. Met. Org. Chem. 4
(2004) 919;
(b) A.A. Isse,
20912096.
A Gennaro, E. Vianello, J. Chem. Soc., Dalton Trans. (1993)
[27] M.T.H. Tarafder, M.A. Ali, D.J. Wee, K. Azahari, S. Silong, K.A. Crouse, Transition
Met. Chem. 25 (2000) 456460.
[48] V.T. Kasumov, A. Bulut, F. Koksal, M. Aslanoglu, I. Ucar, C. Kazak, Polyhedron 25
(2006) 11331141.
[28] M.T.H. Tarafder, M.A. Ali, N. Saravanan, W.Y. Weng, S. Kumar, N. Umar, Transit.
Met. Chem. 25 (2000) 295298.
[29] M.A. Ali, N.E. Ibrahim, R.J. Butcher, J.P. Jasinski, J.M. Jasinski, J.C. Bryan,
Polyhedron 17 (1998) 18031809.
[30] M.A. Ali, K.K. Dey, M. Nazimuddin, F.E. Smith, R.J. Butcher, J.P. Jasinski, J.M.
Jasinski, Polyhedron 15 (1996) 33313339.
[31] M.A. Ali, M. Nazimuddin, R. Shaha, R.J. Butcher, J.C. Bryan, Polyhedron 17
(1998) 39553961.
[49] T.H. Rakha, K.M. Ibrahim, M.I. Khalıfa, Thermochim. Acta 144 (1989) 53.
[50] S. Chandra, L.K. Gupta, J. Saudi Chem. Soc. 8 (2004) 7784.
[51] M.T. Carter, M. Rodriguez, A.J. Bard, J. Am. Chem. Soc. 111 (1989) 89018911.
[52] D.L. Boger, S.R. Fink, S.R. Brunette, W.C. Tse, M.P. Hedrick, J. Am. Chem. Soc. 123
(2001) 58785891.
[53] V.F. Ivanov, L.E. Minchenkova, A.K. Schyolkina, A.I. Poletayer, Biopolymers 12
(1973) 405421.
[54] N. Magne, J.L. Fischel, J.L. Dubreuil, A. Formento, S. Marcie, J.L. Lagrange, G.
Milano, Br. J. Cancer 86 (2002) 819827.
[32] M.A. Ali, S.G. Teoh, J. Inorg. Nucl. Chem. 40 (1978) 20132018.
[33] S. Bhattacharjee, R. Bhattacharyya, J. Chem. Soc., Dalton Trans. (1992)
13571364.
[55] S. Komeda, M. Lutz, A.L. Spek, M. Chikuma, J. Reedijk, J. Inorg. Chem. 39 (2000)
42304236.
[34] A.G. Bingham, H. Bogge, A. Muller, E.W. Ainscough, A.M. Brodie, J. Chem. Soc.,
Dalton Trans. (1987) 493.