Synthesis, molecular structure and electrochemical properties of nickel(ii) benzhydrazone complexes: influence of ligand substitution on DNA/protein interaction, antioxidant activity and cytotoxicity

Article abstract of DOI:10.1039/c5ra19530f

A series of new nickel(ii) benzhydrazone complexes having the general formula [Ni(L)₂] (where L = thiophene aldehyde benzhydrazone) have been synthesized via the reaction of Ni(OAc)₂·4H₂O with 2 equivalents of benzhydrazon

Full text of DOI:10.1039/c5ra19530f

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PAPER
Synthesis, molecular structure and electrochemical
properties of nickel(II) benzhydrazone complexes:
influence of ligand substitution on DNA/protein
interaction, antioxidant activity and cytotoxicity†
Cite this: RSC Adv., 2015, 5, 101932
*
Ramasamy Raj Kumar and Rengan Ramesh
A series of new nickel(II) benzhydrazone complexes having the general formula [Ni(L)2] (where L ¼
thiophene aldehyde benzhydrazone) have been synthesized via the reaction of Ni(OAc)2$4H2O with
2
equivalents of benzhydrazone ligands in a DMF/ethanol medium. The complexes have been
characterized by analytical, spectral (FT-IR, UV-vis, NMR and ESI-mass) and single-crystal X-ray
crystallography methods. All the complexes exhibit quasi-reversible one-electron reduction responses
(NiII–NiI) within the E1/2 range from ꢀ0.71 to ꢀ0.77 V versus SCE. The structure of one of the complexes
has been determined by a single-crystal X-ray diffraction study, which shows that coordination of the
benzhydrazone ligands to the nickel occurs via azomethine nitrogen and imidolate oxygen atoms as
monobasic bidentate donors with two units of the ligand in a square-planar geometry. The DNA-binding
interactions of the complexes with calf thymus DNA have been investigated by absorption, emission,
electrochemical, circular dichroism and viscosity measurements, which revealed that the complexes
could interact with DNA via intercalation. The protein-binding interactions of the complexes with BSA
were investigated by UV-vis, fluorescence and synchronous fluorescence methods, which indicated that
stronger binding of the complexes with BSA and a static quenching mechanism was observed. Moreover,
the potential for free-radical scavenging of all the complexes was also determined using DPPH, hydroxyl
and nitric oxide radicals under in vitro conditions. Furthermore, the cytotoxicity of all the complexes was
examined in vitro on the human cervical cancer cell line HeLa, MCF-7 and the normal mouse embryonic
fibroblast cell line NIH-3T3 under identical conditions and they exhibited good IC50 values. These values
were further supported by a neutral red uptake assay using HeLa cell lines. AO-EB/DAPI staining assays
and flow cytometry analysis revealed that the complexes induce cell death only by apoptosis.
Received 22nd September 2015
Accepted 10th November 2015
DOI: 10.1039/c5ra19530f
www.rsc.org/advances
improvements to cancer therapy. In addition to cisplatin,
several platinum complexes (carboplatin, oxaliplatin, nedapla-
Introduction
tin and lobaplatin) have been approved for current tumour
therapy. Complexes of ruthenium, titanium, and gallium have
already been tested in clinical phase I and phase II studies.7,8
Preclinical research also involves metal complexes containing
other non-platinum metals (e.g., iron, cobalt, and gold).9
The interactions of metal complexes with serum albumins
have received much attention in the scientic community via
studying the pharmacodynamics and structure–activity rela-
tionships of antitumor metallopharmaceuticals.10 Serum
albumins are the most abundant proteins in plasma and have
many physiological functions.11–13 In particular, they
contribute to the control of osmotic blood pressure and the
maintenance of blood pH.14 BSA is the most well-studied serum
albumin due to its structural resemblance to human serum
albumin (HSA).
The interaction of metal complexes with DNA has long been
a subject of great interest in relation to the development of new
reagents for biotechnology and medicine.1–3 A number of bio-
logical experiments have also revealed that DNA is the main
intracellular target of anticancer drugs due to the interaction
between small molecules and DNA, which can cause damage to
DNA in cancer cells, prevent the division of cancer cells and
result in cell death.4 The accidental discovery of the antitumor
properties of cisplatin by Rosenberg et al.5,6 was followed by one
of the most impressive drug success stories ever and signicant
School of Chemistry, Bharathidasan University, Tiruchirappalli – 620 024, Tamil
Nadu, India. E-mail: ramesh_bdu@yahoo.com; rrameshbdu@gmail.com; Fax: +91-
431-2407045/2407020; Tel: +91-431-2407053
† Electronic supplementary information (ESI) available: UV-vis, ESI-mass and
cyclic voltammograms of the complexes. CCDC 1032855. For ESI and
crystallographic data in CIF or other electronic format see DOI:
10.1039/c5ra19530f
Free radicals inside the human body play a pathogenic role
in most chronic degenerative diseases, including inammatory,
101932 | RSC Adv., 2015, 5, 101932–101948
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cancer, autoimmune, cardiovascular and neurodegenerative containing thiophene aldehyde benzhydrazone ligands and their
diseases and aging.15–18 Free radicals can adversely affect lipids, interactions with DNA/protein, antioxidant activity and cytotox-
proteins and DNA and have been implicated in the aging icity. In addition, the effect of substituents in nickel(II) complexes
process and in a number of human diseases. Antioxidants are on the abovementioned properties is also studied in detail.
capable of neutralising these reactive species in terms of
prevention, interception and damage repair.19,20
Experimental section
Hydrazones are an important class of ligands with inter-
Materials and instrumentation
esting ligation properties due to the presence of several coor-
dination sites21 and are widely applied in the elds of
medicines, insecticides and analytical reagents due to their
superior bioactivity.22 The hydrazone unit offers a number of
interesting features such as a degree of rigidity, a conjugated p
system and a NH unit that readily participates in hydrogen
bonding and may be a site of protonation–deprotonation. Some
hydrazone analogs have been investigated as potential oral iron-
chelating drugs for the treatment of genetic disorders such as
thalassemia and have also been proposed as possible metal-
chelating agents for treating neurodegenerative disorders
such as Alzheimer's disease.23–25 Hydrazone ligands create an
environment similar to that present in biological systems,
usually by coordination through oxygen and nitrogen atoms. In
this respect, the formation of metal complexes plays a substan-
tial role in enhancing their biological activity.
Although this was not understood until the 1970s, nickel
plays important roles as a catalytic center in both redox and
non-redox enzymes, wherein it has important consequences in
human health (e.g., urease), energy science (e.g., hydrogenase),
and environment (e.g., carbon monoxide dehydrogenases).26
Nickel(II) compounds, which can be reversibly reduced to
nickel(I) species, have attracted attention as models of redox-
active nickel-containing enzymes27 and as electrocatalysts.28
Complementing their natural biochemical functions,
complexes of nickel also display pharmacological potential.
Nickel(II) complexes are regarded as some of the most prom-
ising alternatives to the traditional cisplatin as anticancer
drugs. This idea is supported by an extensive number of
research articles that describe the synthesis, DNA-binding
and cytotoxic activities of numerous nickel(II) complexes.29–31
The synthesis, structure, DNA-binding properties and anti-
Nickel(II) acetate tetrahydrate, thiophene-2-carboxaldehyde and
benzhydrazide derivatives were purchased from Merck and
Aldrich Chemicals and used as received. Solvents were dried and
freshly distilled prior to use. Calf thymus DNA (CT-DNA) and
ethidium bromide (EtBr) were purchased from Sigma-Aldrich
Chemicals and used as received. Bovine serum albumin (BSA)
was purchased from Himedia. 3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) was purchased from
Sigma-Aldrich and used as received. Human cervical cancer cells
and MCF-7 human breast cancer cells were obtained from the
National Centre for Cell Science (NCCS), Pune, India. The
annexin V-FITC kit (APOAF-20TST) from Sigma-Aldrich was
utilized according to the instructions from the manufacturer. All
other chemicals and reagents used for biological studies were of
high-quality biological grade. Elemental analyses were performed
on a Vario EL III CHNS elemental analyser. Melting points were
measured with an electrical instrument and are uncorrected.
Infrared spectra were obtained in KBr pellets with a JASCO 200
Plus spectrometer. NMR spectra were obtained in DMSO-d6 for
the ligands and complexes with a Bruker 400 MHz instrument
using TMS as the internal reference. Mass spectrometric analysis
was performed using the ESI technique on a Waters Q-TOF Micro
mass spectrometer for all these complexes in solution in DMF.
Electronic spectroscopy was performed with a Cary 300 Bio
(Varian) spectrophotometer using cuvettes with a path length of 1
cm. Emission intensity measurements were carried out using
a Jasco FP-6500 spectrouorometer. Circular dichroism spectra
were obtained using a JASCO J-810 spectropolarimeter equipped
with a Peltier temperature-control device at room temperature
with a quartz cell with a path length of 1 cm. Results for each
sample solution were the average of three accumulations using
a scan speed of 500 nm minꢀ1 at a response time of 1 s. Viscosity
oxidant activity of
a
nickel(II) complex with bis(N-
allylbenzimidazol-2-ylmethyl)benzylamine have been re-
ported.32 The synthesis, characterisation and in vitro phar-
macological evaluation of new water-soluble Ni(II) complexes
of 4N-substituted thiosemicarbazones of 2-oxo-1,2-
dihydroquinoline-3-carboxaldehyde have been described.33
The synthesis, structure and biological activity of nickel(II)
complexes with mefenamat and nitrogen-donor ligands have
been reported.34 Variations in the biomolecular interactions
of nickel(II) hydrazone complexes upon tuning the hydrazide
fragment have been reported.35
¨
measurements were carried out on a Schott Gerate AVS 310
viscometer at 29 ꢁ 0.1 ꢂC in a thermostatic water bath. The
supporting electrolyte tetrabutylammonium perchlorate (TBAP)
was purchased from Aldrich and dried in vacuum prior to use.
Electrochemical measurements were made using a CH instru-
ment using a glassy carbon working electrode and 0.05 M [(n-
C4H9)4N](ClO4) (TBAP) as the supporting electrolyte. All poten-
tials were referenced to the saturated calomel electrode (SCE) and
the solutions were purged with N2 before each set of experiments.
Therefore, considerable attempts are being made to research
the interaction of nickel(II) complexes with DNA and their cyto-
Preparation of benzhydrazone ligands
toxic activities. Based on the abovementioned facts and consid- Monobasic bidentate benzhydrazone ligands were prepared by
ering the role and activity of nickel and its complexes in biological a reported procedure.36 To a stirred ethanolic solution (10 mL)
systems, along with the signicance of hydrazones in medicine, of 4-substituted benzhydrazide (68–85 mg, 5 mmol), an etha-
we report in this study a systematic study of the synthesis, nolic (10 mL) solution of thiophene-2-aldehyde (0.46 mL, 56–
structure and electrochemical properties of nickel(II) complexes 63 mg, 5 mmol) was added dropwise. The reaction mixture was
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reuxed for 3 h and the solution concentrated to 5 mL and S, 10.94. Found: C, 49.22; H, 2.68; N, 9.50; S, 10.98. IR
cooled to room temperature. The solid that was formed was (KBr, cmꢀ1): 1583 s n(C]N–N]C), 1238 s n(C–O). UV-vis in DMF:
ltered, washed with cold methanol (5 mL) and dried in air.
lmax/nm (3max/dm3 molꢀ1 cmꢀ1) 266(86, 760) (intraligand tran-
Benzoic acid thiophene-2-ylmethylene-hydrazide (HL1). sition), 363(14 308) (LMCT), 424(1086) (forbidden d / d tran-
Colour: cream; yield: 87%; mp: 150 ꢂC; anal. calc. for sition). ESI-MS: m/z 592.57.
C
12H10N2OS (230.29 g molꢀ1): C, 62.58; H, 4.37; N, 12.16; S,
13.92. Found: C, 62.53; H, 4.40; N, 12.10; S, 13.96. IR (KBr,
cmꢀ1): 3254 s n(N–H), 1644 s n(C]N) + n(C]O). 1H NMR (400 MHz,
DMSO-d6) d (ppm): 11.8 (s, 1H, NH), 8.7 (s, 1H, CH]N), 7.2–8.0
(m, 8H, aromatic). UV-vis (DMF): 265, 322 nm.
[Ni(L3)2] (3)
Color: orange; yield: 88.49%; mp: 344 ꢂC; anal. calc. for
C
24H16Br2N4S2O2Ni (675.05 g molꢀ1): C, 42.70; H, 2.38; N, 8.29;
S, 9.50. Found: C, 42.62; H, 2.39; N, 8.20; S, 9.49. IR (KBr, cmꢀ1):
1578 s n(C]N–N]C), 1240 s n(C–O). UV-vis in DMF: lmax/nm
(3max/dm3 molꢀ1 cmꢀ1) 265(80, 720) (intraligand transition),
365(11 252) (LMCT), 426(2559) (forbidden d / d transition).
ESI-MS: m/z 682.70.
4-Chlorobenzoic acid thiophene-2-ylmethylene-hydrazide
(HL2). Colour: cream; yield: 86%; mp: 156 ꢂC; anal. calc. for
C
12H9ClN2OS (264.73 g molꢀ1): C, 54.44; H, 3.42; N, 10.58; S,
12.11. Found: C, 54.48; H, 3.46; N, 10.60; S, 12.14. IR (KBr,
cmꢀ1): 3280 s n(N–H), 1639 s n(C]N) + n(C]O). 1H NMR (400 MHz,
DMSO-d6) d (ppm): 11.9 (s, 1H, NH), 8.7 (s, 1H, CH]N), 7.2–8.0
(m, 7H, aromatic). UV-vis (DMF): 268, 324 nm.
[Ni(L4)2] (4)
4-Bromobenzoic acid thiophene-2-ylmethylene-hydrazide Color: orange; yield: 97.05%; mp: 352 ꢂC; anal. calc. for
(HL3). Colour: cream; yield: 84%; mp: 158 ꢂC; anal. calc. for
26H22N4S2O4Ni (574.41 g molꢀ1): C, 54.36; H, 3.86; N, 9.75; S,
C
C
12H9BrN2OS (309.18 g molꢀ1): C, 46.61; H, 2.93; N, 9.06; S, 11.16. Found: C, 54.30; H, 3.83; N, 9.78; S, 11.14. IR (KBr, cmꢀ1):
10.37. Found: C, 46.60; H, 2.98; N, 9.05; S, 10.25. IR (KBr, cmꢀ1): 1520 s n(C]N–N]C), 1250 s n(C–O). UV-vis in DMF: lmax/nm
3296 s n(N–H), 1636 s n(C]N) + n(C]O). 1H NMR (400 MHz, DMSO- (3max/dm3 molꢀ1 cmꢀ1) 272(80, 470) (intraligand transition),
d6) d (ppm): 11.9 (s, 1H, NH), 8.7 (s, 1H, CH]N), 7.1–7.9 (m, 7H, 367(11 187) (LMCT), 422(2422) (forbidden d / d transition).
aromatic). UV-vis (DMF): 268, 323 nm.
ESI-MS: m/z 577.48.
4-Methoxybenzoic acid thiophene-2-ylmethylene-hydrazide
(HL4). Colour: cream; yield: 87%; mp: 162 ꢂC; anal. calc. for
C
X-ray crystallography
13H12N2O2S (260.31 g molꢀ1): C, 59.98; H, 4.64; N, 10.76; S,
Single crystals of complex 4 that were suitable for X-ray
diffraction analysis were grown by slow evaporation of solu-
tions of the complex in dimethylformamide at room tempera-
ture. Data collection was carried out using a Bruker SMART
APEX II single-crystal X-ray diffractometer using mono-
12.31. Found: C, 60.01; H, 4.63; N, 12.34; S, 12.29. IR (KBr,
cmꢀ1): 3272 s n(N–H), 1642 s n(C]N) + n(C]O). 1H NMR (400 MHz,
DMSO-d6) d (ppm): 11.7 (s, 1H, NH), 8.7 (s, 1H, CH]N), 7.2–8.9
(m, 7H, aromatic), 3.8 (s, 3H, OCH3). UV-vis (DMF): 268, 324 nm.
˚
chromated MoKa radiation (l ¼ 0.71073 A). Absorption correc-
Synthesis of new square planar nickel(II) benzhydrazone
complexes
tions were performed by a multi-scan method using the SADABS
soware.38 Corrections were made for Lorentz and polarization
effects. Structures were solved by SIR92 and rened by full-
matrix least squares on F2 using SHELXL 97.39 All non-
hydrogen atoms were rened anisotropically and the
hydrogen atoms in these structures were located via the differ-
ence Fourier map and constrained to ideal positions in the
renement procedure. The unit cell parameters were deter-
mined by the method of difference vectors using reections
scanned from three different zones of the reciprocal lattice.
Intensity data were measured using u and 4 scans with a frame
width of 0.5ꢂ. Frame integration and data reduction were per-
formed using the Bruker SAINT-Plus (version 7.06a) soware.40
The crystal structure and structure renement parameters for
the complex are given in Table 1.
All reactions were carried out under anhydrous conditions and
the new nickel(II) complexes were prepared according to the
following procedure.37 A hot solution of Ni(CH3COO)2$4H2O
(1 mmol) in ethanol was added to a boiling solution of the
benzhydrazone ligands (2 mmol) (HL1–HL4) in DMF. The
reaction mixtures were heated to reux for 5 h. An orange-
colored mononuclear complex [Ni(L)2] was precipitated. The
solid was separated by ltration, washed with ethanol and
diethyl ether then dried under vacuum.
[Ni(L1)2] (1)
Color: orange; yield: 91.9%; mp: 328 ꢂC; anal. calc. for
C
24H18N4S2O2Ni (517.25 g molꢀ1): C, 55.72; H, 3.50; N, 10.83; S,
12.39. Found: C, 55.80; H, 3.59; N, 10.88; S, 12.38. IR (KBr,
cmꢀ1): 1526 s n(C]N–N]C), 1242 s n(C–O). UV-vis in DMF: l3max/nm DNA binding studies
(3max/dm3 molꢀ1 cmꢀ1) 268(60, 470) (intraligand transition),
Experiments involving the binding of compounds to CT-DNA
361(10 326) (LMCT), 424(1965) (forbidden d / d transition).
ESI-MS: m/z 522.85.
were performed in double-distilled water with Tris (5 mM)
and sodium chloride (50 mM) and the pH was adjusted to 7.2
with hydrochloric acid. A solution of CT-DNA in the buffer gave
a ratio of UV absorbance of about 1.9 at 260 and 280 nm, which
[Ni(L2)2] (2)
Color: orange; yield: 89%; mp: 340 ꢂC; anal. calc. for suggested that the DNA was sufficiently free from protein. The
24H16Cl2N4S2O2Ni (586.14 g molꢀ1): C, 49.17; H, 2.75; N, 9.55; concentration per nucleotide was determined by electronic
C
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Table 1 Crystal data and structure refinement of complex 4
Cyclic voltammetry
Electrochemical studies were performed on a CH Instruments
electrochemical analyser and the experiments were carried out
in a three-electrode system comprising a glassy carbon working
electrode, a platinum wire auxiliary/counter electrode and
a saturated calomel electrode (SCE), which was used as the
reference electrode. Cyclic voltammograms of the complexes
were recorded in DMF solutions and buffer (5 mM Tris–HCl/
Empirical formula
Formula weight
Colour
C26H22N4O4S2Ni
577.29
Orange
296(2)
0.71073
Monoclinic
P21/c
13.5191(10)
5.4599(4)
18.3237(14)
90.00
108.169(4)
90.00
1285.09(17)
2
1.431
1.012
532
0.54 ꢃ 0.09 ꢃ 0.09
1.59–30.39
ꢀ19 # h # 19
ꢀ7 # k # 4
ꢀ25 # l # 26
12 482
3830
3830/0/71
1.041
Temperature (K)
˚
Wavelength (A)
Crystal system
Space group
˚
a (A)
50 mM NaCl, pH 7.2) solutions at a scan rate of 100 mV sꢀ1
.
˚
b (A)
˚
c (A)
TBAP and buffer solution were the supporting electrolytes.
Oxygen was eliminated by purging the solutions with pure
nitrogen gas, which had been previously saturated with solvent
vapours. The electrode surfaces were freshly polished with
alumina powder and washed with double-distilled water aer
each polishing step. Allꢂ electrochemical measurements were
performed at 25.0 ꢁ 0.2 C.
a (ꢂ)
b (ꢂ)
g (ꢂ)
3
˚
Volume (A )
Z
Dcal (mg mꢀ3
)
Absorption coefficient (mmꢀ1
F(000)
)
Crystal size (mm)
Theta range (ꢂ)
Circular dichroism
Limiting indices
Circular dichroism is a useful technique for determining how
the conformation of a CT-DNA chain is altered by a bound
complex. A solution of CT-DNA displays a positive band
(275 nm) from base-stacking interactions and a negative band
(245 nm) from the right-handed helicity of DNA.41 Conforma-
tional changes were measured upon the addition of complexes
Reections collected
Independent reections
Data/restraints/parameters
Goodness-of-t (GOF) on F2
Final R indices [I > 2s(I)]
R indices (all data)
R1 ¼ 0.0545, wR2 ¼ 0.1512
R1 ¼ 0.1151, wR2 ¼ 0.1159 (20 mM) at a DNA concentration of 60 mM in a Tris–HCl buffer
Rint
0.0463
0.656 and ꢀ0.778
medium. The spectra of control DNA and the complexes were
Largest diff. peak and hole eAꢀ3
monitored from 220 to 320 nm. Classical intercalation reactions
tend to increase the intensities of both bands due to strong
base-stacking interactions of stable DNA conformations (right-
spectroscopy using a molar extinction coefficient of 6600 handed B conformations of CT-DNA), whereas simple groove
Mꢀ1 cmꢀ1 at 260 nm. The complexes were dissolved in binding and electrostatic interactions with small molecules
a combined solvent of 5% DMF and 95% Tris–HCl buffer for all yield small perturbations or no perturbation in the base-
experiments. A stock solution of CT-DNA was stored at 277 K stacking and helicity bands.42
and used aer no more than 4 days. Electronic absorption
titration experiments were performed by keeping a xed
concentration of the metal complex constant (25 mM) and
varying the nucleotide concentration (0–60 mM). However, when
obtaining the absorption spectra, equal amounts of DNA were
added to both complex and reference solutions to eliminate the
absorbance of DNA itself. Samples were equilibrated before
obtaining each spectrum.
Further support for the binding of complexes to DNA via
intercalation was obtained using uorescence spectral tech-
niques in order to nd out whether a complex can displace EB
from a DNA–EB complex. Ethidium bromide displacement
experiments were carried out by adding solutions of the
complexes to a Tris–HCl buffer solution (pH 7.2) of a DNA/EB
mixture. DNA was pretreated with ethidium bromide at
a [DNA]/[EB] ratio of 10 for 30 min at 27 ꢂC, then a test solution
was added to this mixture of EB–DNA and the change in uo-
rescence intensity was measured. The excitation wavelength
was xed at 545 nm for EB bound to DNA. Emissions were
recorded with increasing concentrations of nickel(II) complexes
and the emission range was adjusted before measurements.
Metal complexes (0–60 mM) were then added to the mixture and
their effect on the emission intensity was measured.
Viscosity experiments
Viscosity experiments were performed using an Ubbelohde-type
viscometer at a constant temperature of 29.0 ꢁ 0.1 ꢂC in
a thermostatic water bath. Calf thymus DNA sample solutions
were prepared by sonication in order to minimize complexities
arising from the exibility of DNA.43 CT-DNA solutions (5 mM)
were titrated with nickel(II) benzhydrazone complexes (0.5–
5 mM), monitoring the variation in the viscosity in each case.
The ow time was measured with a digital stopwatch; each
sample was measured at least three times and an average ow
time was calculated. Data are presented as (h ꢀ ho)1/3 versus the
binding ratio, where h is the viscosity of DNA in the presence of
the complex and ho is the viscosity of DNA alone. Relative
viscosity values were determined according to the equation h ¼
(t ꢀ to)/to, where to is the total ow time for the buffer and t is
the ow time that was observed for DNA in the presence and
absence of the complex.44
Protein binding studies
The protein-binding interactions of nickel(II) hydrazone
complexes with bovine serum albumin (BSA) were investigated
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using uorescence spectra, which were obtained with an exci- iron–EDTA solution (ferrous ammonium sulphate (0.331 mM)
tation wavelength of 280 nm and an emission wavelength of and EDTA (0.698 mM)), 0.5 mL EDTA solution (0.048 mM) and
345 nm corresponding to those of free bovine serum albumin 1.0 mL DMSO (10.08 mM DMSO (v/v) in 0.1 M phosphate buffer,
(BSA). The excitation and emission slit widths and scan rates pH 7.4) was sequentially added to test tubes containing the test
were maintained at a constant for all experiments. Samples compounds at different concentrations in the range of 10–
were thoroughly degassed using pure nitrogen gas for 50 mM. The reaction mixture contained EDTA (0.1 mM), Fe3+
15 minutes using quartz cells (4 ꢃ 1 ꢃ 1 cm) with high-vacuum (167 mM), DMSO (33 mM) in phosphate buffer (50 mM, pH 7.4),
Teon stopcocks. Stock solutions of BSA were prepared in and the tested compounds at various concentrations in the
50 mM phosphate buffer (pH 7.2) and stored in the dark at 4 ꢂC range of 10–50 mM. The reaction was initiated by adding 0.5 mL
for additional use. Concentrated stock solutions of metal ascorbic acid (1.25 mM) and incubated at 80–90 ꢂC for 15 min in
complexes were prepared by dissolving them in DMF : phos- a water bath. Aer incubation, the reaction was terminated by
phate buffer (5 : 95) and diluted suitably with phosphate buffer the addition of 1.0 mL ice-cold trichloroacetic acid (TCA)
to obtain appropriate concentrations. A 2.5 mL solution of BSA (107 mM). Subsequently, 3.0 mL Nash reagent was added to
(1 mM) was titrated by consecutive additions of a 25 mL stock each tube, which was le at room temperature for 15 min. The
solution of a complex (10ꢀ3 M) using a micropipette. Synchro- reaction mixture without the sample was used as a control. The
nous uorescence spectra were also obtained using the same intensity of the colour that was formed was measured spectro-
concentrations of BSA and complexes as mentioned above with photometrically at 412 nm against a reagent blank.
two different values of Dl (difference between the excitation and
emission wavelengths of BSA) such as 15 and 60 nm.
Nitric oxide (NO) radical-scavenging activity was determined
based on the reported method, in which sodium nitroprusside
in an aqueous solution at physiological pH spontaneously
generates nitric oxide, which interacts with oxygen to produce
nitrate ions that can be estimated using the Griess reagent.48
For this experiment, sodium nitroprusside (10 mM) in phos-
phate buffered saline was mixed with the test compounds at
different concentrations in the range of 10–50 mM and incu-
bated at room temperature for 150 min. The reaction mixture
without the sample but with equivalent amount of solvent
served as a control. Aer the incubation period, 0.5 mL Griess
reagent containing sulphanilamide (5.8 mM), H3PO4 (20 mM)
and N-(1-naphthyl)ethylenediamine dihydrochloride (0.39 mM)
were added and mixed before standing for 30 min at 25 ꢂC. The
absorbance of the pink-coloured chromophore that was formed
during diazotization was measured at 546 nm.
For the abovementioned three assays, all tests were run in
triplicate and various concentrations of the complexes were
used to establish a concentration at which the complexes dis-
played around 50% activity. The percentage activity was calcu-
lated using the formula: % suppression ratio ¼ [(Ao ꢀ Ac)/Ao] ꢃ
100, where Ao and Ac represent the absorbance in the absence
and presence of the test compounds, respectively. The
concentration that corresponds to 50% activity (IC50) can be
Protein cleavage experiments
Protein cleavage experiments were performed by incubating BSA
(20 mM) with 1–4 in Tris–HCl buffer for 4 h at 37 ꢂC according to
the literature.45 The samples were dissolved in a loading buffer
(24 mL) containing SDS (7% w/v), glycerol (4% v/v), Tris–HCl
buffer (50 mM, pH 7.2), mercaptoethanol (2% v/v) and bromo-
phenol blue (0.01% w/v). The protein solutions were then dena-
tured by heating to boil for 3 min. The samples were then loaded
onto a 3% polyacrylamide (stacking) gel. Gel electrophoresis was
carried out beginning at 60 V until the dye passed into the
separating gel (12% polyacrylamide) from the stacking (3%) gel,
followed by setting the voltage to 110 V for 1.5 h. Staining was
performed with a Coomassie Brilliant Blue R-250 solution (acetic
acid–methanol–water ¼ 1 : 2 : 7 v/v) and destaining was carried
out with a water–methanol–acetic acid mixture (5 : 4 : 1 v/v) for 4
h. The gels, aer destaining, were scanned with a PrecisionScan
LTX scanner and the images were further processed using the
Adobe Photoshop 7.0 soware package.
Antioxidant activity
The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical-scavenging calculated using the percentage activity.
activity of the compounds was measured according to the
method described by Blois.46 The DPPH radical is a stable free
radical having a lmax of 517 nm. Various concentrations (10–
In vitro anticancer activity: maintenance of cell lines
50 mM) of the test compounds were added to a solution of DPPH Cell viability was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-
(125 mM, 2 mL) in methanol and the nal volume was made up 2,5-diphenyltetrazolium bromide (MTT) assay, which deter-
to 4 mL with double-distilled water. The solution was incubated mines the metabolic activity of mitochondrial cells.49 Cells were
at 37 ꢂC for 30 min in the dark. The decrease in the absorbance plated in a growth medium at a density of 5000 cells per well in
of DPPH was measured at 517 nm. The same experiment carried 96 at-bottomed well plates at a plating density of 10 000 cells
out without the test compounds served as a control.
per well and incubated to allow cell attachment at 37 ꢂC under
Hydroxyl radicals that were produced by the Fe3+/ascorbic conditions of 5% CO2, 95% air and 100% relative humidity.
acid system were detected according to the method of Nash.47 Aer 24 h, the cells were treated with serial concentrations of
The detection of hydroxyl radicals was carried out by measuring the test samples. The compounds were initially dissolved in
the amount of formaldehyde that was produced from an neat DMF and an aliquot sample solution was diluted to twice
oxidation reaction with DMSO. Formaldehyde produced was the desired nal maximum test concentration with serum-free
detected spectrophotometrically at 412 nm. A mixture of 1.0 mL medium. Four additional serial dilutions were performed to
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provide a total of ve sample concentrations. Aliquots of 100 mL lter (450–490 nm). Three hundred cells per sample were
of these different sample dilutions were added to the appro- counted in triplicate for each dose point. Cells were scored as
priate wells, which already contained 100 mL medium, resulting viable, apoptotic or necrotic judging by the staining, nuclear
in the required nal sample concentrations. Following addition morphology and membrane integrity. Morphological changes
of the samples, the plates were incubated for an additional 48 h were also observed and photographed.
at 37 ꢂC, 5% CO2, 95% air and 100% relative humidity. The
medium without samples served as a control and triplicate
measurements were made for all concentrations.
DAPI staining method
Staining with DAPI (40,6-diamidino-2-phenylindole) was carried
out using the following procedure: 5 ꢃ 105 cells were treated
with the complex (100 mg mLꢀ1) for 24 h in a six-well culture
plate and xed with 4% paraformaldehyde followed by per-
meabilization with 0.1% Triton X-100. The cells were then
stained with 50 mg mLꢀ1 DAPI for 30 min at room temperature.
The cells that were undergoing apoptosis, represented by
morphological changes in apoptotic nuclei, were observed and
imaged by ten views at 20ꢃ magnication under a LSM 710 laser
scanning confocal microscope (Zeiss).
MTT assay
3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
(MTT) is a yellow water-soluble tetrazolium salt. A mitochondrial
enzyme in living cells, succinate dehydrogenase, cleaves the
tetrazolium ring, converting MTT into an insoluble purple for-
mazan. Therefore, the amount of formazan that is produced is
directly proportional to the number of viable cells. Aer 48 h
incubation, 15 mL MTT (5 mg mLꢀ1) in phosphate buffered saline
(PBS) was added to each well and incubated at 37 ꢂC for 4 h.
The quantity of formazan formed gave a measure of the
number of viable cells. HeLa, MCF-7, and NIH 3T3 cells were
used for the MTT assay. The absorbance was monitored at
570 nm (measurement) and 630 nm (reference) using a 96-well
plate reader (Bio-Rad, Hercules, CA, USA). Data were collected
for four replicate measurements each and used to calculate the
respective means. The percentage inhibition was calculated
from this data using the formula:
Apoptosis evaluation – ow cytometry
Cells were grown in six-well plates and exposed to three
different concentrations of nickel(II) benzhydrazone complex 4
for 48 h. The annexin V-FITC kit uses annexin V conjugated to
uorescein isothiocyanate (FITC) to label phosphatidylserine
sites on the membrane surface of apoptotic cells. In brief, the
cells were trypsinised, washed with annexin-binding buffer,
incubated with annexin V FITC and PI for 30 minutes and
immediately analysed using a FACSAria-II ow cytometer. The
results were analysed using DIVA soware and the percentage of
positive cells was calculated.
% cell inhibition ¼ 1 ꢀ Abs (sample)/Abs (control) ꢃ 100
The IC50 value was calculated as the concentration of the
complex that was required to reduce the absorbance to half that
of the control.
Results and discussion
Benzhydrazone ligand derivatives were prepared in high yield
by the condensation of thiophene aldehydes with substituted
benzhydrazides in an equimolar ratio. These ligands were
allowed to react with (CH3COO)2Ni$4H2O in a 1 : 2 molar ratio
in a DMF/ethanol medium under reux for 5 h to afford new
square-planar nickel(II) benzhydrazone complexes of the
formula [Ni(L)2] (Scheme 1). In this reaction, the acetate anion
acted as a base and promoted the formation of these complexes.
The oxidation state of nickel remained unchanged during the
formation of the complex. All the complexes were orange in
colour, air-stable in both solid and liquid states at room
temperature and non-hygroscopic. The synthesized nickel(II)
benzhydrazone complexes were sparingly soluble in solvents
such as chloroform, dichloromethane or acetonitrile and
readily soluble only in solvents such as dimethylformamide
(DMF) and dimethyl sulphoxide (DMSO), producing intensely
orange-coloured solutions. The analytical data of all the nick-
el(II) benzhydrazone complexes are in good agreement with the
Neutral red uptake assay
This test was performed according to well-known standard
methods.50 Cells were plated in 96-well plates (40 000 cells per
well) and incubated in DMEM + 10% FBS for 24 hours at 37 ꢂC
and 5% CO2. The complexes were then added at different
concentrations for an additional 24 hours. The cells were then
washed with PBS, aer which 200 mL of a 0.625 mg mLꢀ1
solution of neutral red was added. Aer 3 h, the cells were
again washed with PBS to remove the remaining dye. The
addition of 200 mL ethanol/acetic acid (50/1) resulted in
release of the dye from the cells, which were placed in
a shaking bath until a homogeneous colour was formed
(approx. 1 h). The optical density was measured with a spec-
trometer at 540 nm.
Fluorescent double-staining experiment
Staining with acridine orange and ethidium bromide (AO and proposed molecular structures.
EB) was performed as follows: a cell suspension of each sample
The IR spectra of the free ligands displayed a medium to
containing 5 ꢃ 105 cells was treated with 25 mL AO/EB solution strong band in the region of 3254–3296 cmꢀ1, which is char-
(1 part 100 mg mLꢀ1 of AO in PBS; 1 part of mg mLꢀ1 of EB in acteristic of the N–H functional group. The free ligands also
PBS) and examined at 20ꢃ magnication under a LSM 710 laser displayed nC]N and nC]O absorptions in the region of 1636–
scanning confocal microscope (Carl Zeiss, Germany) using a UV 1644 cmꢀ1, which indicate that the ligands exist in the amide
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spectrometric measurements were carried out under positive/
negative-ion ESI conditions with different cone voltages using
DMF as the solvent and mobile phase. The ESI spectra of
complexes 1 and 2 exhibit base peaks at m/z 522.85 and m/z
592.57, respectively, whereas complexes 3 and 4 display base
peaks at m/z 682.70 and m/z 577.48, respectively.
Single-crystal X-ray crystallography
Scheme 1 Synthesis of nickel(II) benzhydrazone complexes.
Attempts were made to grow single crystals for X-ray diffraction
to conrm the molecular structures and geometry of all the
synthesised complexes. However, we could not succeed in
obtaining single crystals except for complex 4 [Ni(L4)2], owing to
solubility problems. The crystal structure of complex 4 is shown
in Fig. 1 and relevant crystallographic data are listed in Table 1.
The complex crystallizes in the P21/c space group. The nickel ion
is tetracoordinated in the square-planar geometry by two
benzhydrazone ligand molecules that act as monoanionic
bidentate N,O-donors via the azomethine nitrogen and depro-
tonated amide oxygen in the benzhydrazone fragment, forming
two fused ve-membered chelate rings. The ligands are in the
trans position with respect to the C–N bonds and the complex is
centrosymmetric around the nickel center. The nickel atom
therefore sits in a N2O2 coordination environment, which is
square-planar in nature as indicated by all the bond parameters
around the nickel atom. In the complex, the benzhydrazone
ligands bind to the metal center via N and O forming two ve-
membered chelate rings with bite angles N(1)–Ni(1)–O(1) of
96.1(1)ꢂ and O(1)–Ni(1)–N(1) of 83.9(1)ꢂ. The bond lengths of
form in the solid state. Bands that are due to nN–H and nC]O
stretching vibrations were not observed with the complexes,
which indicates that the ligands underwent tautomerization
and subsequent coordination of the imidolate enolate form
during complexation. Coordination of the ligand to the nick-
el(II) ion through an azomethine nitrogen is expected to reduce
the electron density in the azomethine link and thus lower the
absorption frequency aer complexation (1520–1583 cmꢀ1),
which indicates the coordination of azomethine nitrogen to the
nickel(II) ion. The presence of a new band in the region of
440–470 cmꢀ1, which is due to n(Ni–N), is another indication of
the involvement of the nitrogen of the azomethine group in
coordination.52 The band in the region of 1238–1250 cmꢀ1 is
due to the imidolate oxygen, which is coordinated to the
metal.53 The IR spectra of all the complexes therefore conrm
the mode of coordination of the benzhydrazone ligand to the
nickel(II) ion via the azomethine nitrogen and imidolate
oxygen.
˚
Ni(1)–N(1) and Ni(1)–O(1) are 1.850(2) and 1.838(2) A, respec-
The absorption spectra of all the nickel(II) benzhydrazone
complexes 1–4 were obtained in dimethylformamide solution in
the range of 200–800 nm at room temperature and all the
complexes exhibited three bands with absorption maxima in
the region of 265–426 nm, as shown in Fig. S1–S4 (ESI†). The
electronic spectrum of the hydrazone ligand in DMF solution
displayed two broad absorption bands at 268 and 323 nm,
which correspond to p–p* and n–p* transitions, respectively.
The former band is due to p–p* transitions within the aromatic
rings and remains almost unchanged in the spectra of the metal
complexes, whereas the second band, which is due to n–p*
transitions within the pC]N–N chromophore, is shied to
a longer wavelength as a consequence of coordination when
binding to the metal atom, which conrms both the formation
of the metal complexes and the fact that the azomethine
nitrogen is involved in coordination.54 The UV-vis spectra of the
nickel(II) complexes exhibited bands in the region of 265–
272 nm, which correspond to ligand-centered (LC) transitions.
The other high-intensity band, which was observed in the
region of 361–365 nm, is due to a nitrogen-metal charge transfer
(LMCT) absorption band. The longer-wavelength band in the
region of 421–426 nm corresponds to d–d transitions of d8
low-spin NiII with slightly distorted square-planar geometry.
tively. The bond lengths and bond angles as given in Table 2 are
in good agreement with reported data on related nickel(II)
complexes with square-planar geometry.55 Because all four
nickel(II) complexes exhibit similar spectral properties, the
other three complexes are considered to have a similar structure
to that of complex 4. X-ray determination conrms the structure
that was proposed on the basis of spectroscopic data, which is
consistent with the bivalency of the metal and the monoionic
nature of the ligand in the complexes.
1
The H NMR spectra of the complexes in DMSO gave very
broad signals, which suggest that ligand exchange took place in
the DMSO solution. However, we obtained mass spectra for all
the complexes that conrm the formation of nickel(II) benzhy-
Fig. 1 Molecular structure of complex 4 (hydrogen atoms are omitted
for clarity; displacement parameters are drawn at 50% probability
drazone complexes, as shown in Fig. S5–S8 (ESI†). Mass level).
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Table 3 Cyclic voltammetry data of the nickel(II) benzhydrazone
Electron-transfer properties
complexesa
Electrochemical studies were carried out for all the free ligands
and nickel(II) benzhydrazone complexes in DMF solution under
an atmosphere of nitrogen in the potential range from +1.4 to
ꢀ1.4 V. Tetrabutylammonium perchlorate (TBAP) (0.05 M) was
used as the supporting electrolyte and the concentration of the
complexes was ꢄ10ꢀ3 M. The potentials of all the nickel(II)
benzhydrazone complexes are summarized in Table 3 and cyclic
voltammograms of all the complexes are shown in Fig. S9–S12
(ESI†). We did not observe any redox waves within the potential Epc
limits from +1.4 to ꢀ1.4 V for free ligands and therefore all the
responses that were observed within these potential limits were
due to the metal centre only. All the complexes displayed
a quasi-reversible reduction (NiII / NiI) at a scan rate of 100
mV sꢀ1. The single-electron nature of the voltammograms was
conrmed by a comparison of the current heights for the
complexes and that for a simple [Fe(bipy)3]2+ complex under
identical conditions.56 All the complexes exhibited well-dened
reduction waves with E1/2 in the range from ꢀ0.71 to ꢀ0.77 V.
The redox processes were quasi-reversible in nature and char-
acterized by a rather large peak-to-peak separation (DEp) of 210–
310 mV.57 The redox potentials were virtually independent of the
scan rates, which indicates quasi-reversibility.58 In general, the
reason for quasi-reversible electron transfer in the above
complexes may either be due to slow electron transfer or the
deposition of the complex on the electrode surface.
Complexes
Epc (V)
Epa (V)
E1/2 (V)
DEp (mV)
1
2
3
4
ꢀ0.87
ꢀ0.88
ꢀ0.89
ꢀ0.87
ꢀ0.58
ꢀ0.67
ꢀ0.62
ꢀ0.56
ꢀ0.73
ꢀ0.77
ꢀ0.76
ꢀ0.71
290
210
270
310
a
Solvent ¼ dimethylformamide; [complex] ¼ 1 ꢃ 10ꢀ3 M; supporting
electrolyte: [Bu4N](ClO4) (0.05 M); scan rate: 100 mV sꢀ1; Epa and
¼
anodic and cathodic peak potentials, respectively; E1/2
0.5(Epc + Epa); DEp ¼ (Epc ꢀ Epa); all potentials are referenced to SCE.
¼
the electron-withdrawing character of R. A plot of formal
potential E1/2 versus s (where s is the Hammett substituent
constant59 of R; s values for R: OCH3 ¼ ꢀ0.27, H ¼ 0.00, Cl ¼
+0.23, Br ¼ +0.23) is found to be linear for the reduction
couples, as shown in Fig. 2. The slope of this line, which is
known as the reaction constant, r, is a measure of the sensitivity
of E1/2 to the substituent (R) for the NiII/NiI couple. This shows
that the nature of the para-substituent R on the thiophene
aldehyde benzhydrazone ligands can inuence the potentials of
metal centers in a predictable manner.
DNA-binding studies
DNA is the primary pharmacological target of many antitumor
compounds. DNA–metal complex interactions are of paramount
importance in understanding the mechanism of tumour inhi-
bition in the treatment of cancer. The interaction of nickel(II)
benzhydrazone complexes 1–4 with CT-DNA was studied by
a number of techniques such as absorption spectral titration,
uorescence spectroscopy, cyclic voltammetry, circular
dichroism and viscosity measurements.
The NiII/NiI reduction potential has been found to be
sensitive to the nature of the substituent R in the thiophene
aldehyde benzhydrazone ligand, which perturbs the reduction
of the metal. The potential increases linearly with an increase in
Table 2 Selected bond lengths (A˚) and bond angles (ꢂ) for complex 4
Complex
4
UV-vis absorption studies
Ni(1)–O(1)
1.838(2) Electronic absorption spectroscopy is commonly employed to
Ni(1)–N(1)
O(1)–C(1)
O(2)–C(5)
O(1)–C(8)
1.850(2)
1.306(3)
1.358(3)
1.420(5)
determine the binding ability of metal complexes with a DNA
helix. Complexes that bind to DNA by intercalation are char-
acterised by a change in absorbance (hypochromism) and
S(1)–C(10)
S(2)–C(11)
N(1)–N(2)
N(1)–C(9)
1.724(2)
1.703(4)
1.379(3)
1.300(4)
1.317(4)
180.0(9)
180.0(1)
83.9(1)
N(2)–C(1)
O(1)–Ni(1)–O(1)
N(1)–Ni(1)–N(1)
O(1)–Ni(1)–N(1)
N(1)–Ni(1)–O(1)
N(1)–N(2)–C(1)
S(1)–C(10)–C(13)
Ni(1)–O(1)–C(1)
Ni(1)–N(1)–N(2)
N(1)–C(9)–H(9)
N(2)–N(1)–C(9)
N(2)–C(1)–C(2)
O(1)–C(1)–N(2)
S(1)–C(10)–C(9)
Ni(1)–N(1)–C(9)
96.1(1)
108.2(2)
110.8(2)
110.9(2)
114.4(2)
116.3(3)
116.6(2)
119.1(3)
122.6(3)
125.6(2)
Fig. 2 Least-squares plot of E1/2 values for the NiII/NiI reduction
potential vs. s.
129.1(2)
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bathochromic shi in wavelength, which are due to a strong [3b ꢀ 3f], respectively. Kb was calculated from the ratio of the
stacking interaction between the aromatic chromophore of the slope to the intercept.
test complex and DNA base pairs.60 The extent of hypochromism
Values of the intrinsic binding constant (Kb) were calculated
is commonly related to the strength of intercalation.61 The using the abovementioned equation and found to be 1.60(ꢁ0.10)
interaction of the complexes with CT-DNA was monitored by ꢃ 105 Mꢀ1, 2.84(ꢁ0.06) ꢃ 105 Mꢀ1, 2.34(ꢁ0.08) ꢃ 105 Mꢀ1 and
obtaining the UV-visible spectra of the system. The experiment 3.55(ꢁ0.02) ꢃ 105 Mꢀ1, corresponding to complexes 1–4. From
was carried out by keeping the concentration of the nickel(II) this experimental result, it is very clearly shown that an increase
benzhydrazone complexes (25 mM) constant and changing the in the electron-donating ability of the substituent present in the
concentration of CT-DNA (0–60 mM). The absorption bands at ligand increases the DNA-binding ability of the complex. The
273 nm and 321 nm for the complexes were studied for corre- presence of electron-withdrawing groups in complexes 2 and 3
sponding changes in absorptivity upon the incremental addi- results in almost comparable values of the binding constant.
tion of CT-DNA.
Complex 1 has a lower binding constant, which may be due to the
The absorption spectra of complexes 1–4 in the absence and presence of H as a substituent. Electronic absorption titration
presence of CT-DNA are shown in Fig. 3. From the electronic studies reveal that all the complexes interact with DNA via the
absorption spectral data, it is clear that with an increase in the intercalative mode and complex 4 binds to CT-DNA more strongly
concentration of DNA added to the nickel(II) complexes 1–4 all than the other complexes. The binding mode needs to be proved
the abovementioned absorption bands exhibited hypochrom- by further experiments.
ism of 34.14%, 34.82%, 34.05% and 41.92%, respectively,
accompanied by bathochromic shis of 2–3 nm, which shows
that the nickel(II) benzhydrazone complexes were bound
strongly to CT-DNA via the intercalative mode. The observed
hypochromism was due to stacking interactions between the
aromatic chromophores of the complexes and DNA base pairs,
which is consistent with the intercalative mode of binding.
These observations are similar to those that were reported
earlier for various metallointercalators.62 To compare the
binding strengths of the complexes with CT-DNA, the intrinsic
binding constant Kb was determined from eqn (1):
Emission quenching titration
EB is a general uorescent probe for DNA structures and has
been used in examinations of the mode and process of the
binding of metal complexes to DNA. The uorescence emis-
sions of EB bound to DNA in the presence of nickel(II) benzhy-
drazone complexes are shown in Fig. 4. The intensity of the
emissions of the EB–DNA system (l ¼ 595 nm) apparently
decreased as the concentration of the complexes increased,
which indicates that the complexes replaced EB in the DNA–EB
system. The resulting decrease in uorescence was caused by EB
moving from a hydrophobic environment to an aqueous
environment.
[DNA]/[3a ꢀ 3f] ¼ [DNA]/[3b ꢀ 3f] + 1/Kb[3b ꢀ 3f]
(1)
The quenching constant was calculated from the Stern–
Volmer eqn (2):
where [DNA] is the concentration of DNA in base pairs, 3a is the
extinction coefficient of the complex at a given DNA concen-
tration, 3f is the extinction coefficient of the complex in free
solution and 3b is the extinction coefficient of the complex when
fully bound to DNA. A plot of [DNA]/[3b ꢀ 3f] versus [DNA] gave
a slope and intercept that were equal to 1/[3b ꢀ 3f] and (1/Kb)
I0/I ¼ 1 + Kq[Q]
(2)
where I0 is the emission intensity in the absence of the quencher,
I is the emission intensity in the presence of the quencher, Kq is
Fig. 3 Electronic absorption spectra of complexes bound to DNA: 1 Fig. 4 Fluorescence quenching curves of EtBr bound to DNA: 1 (A), 2
(A), 2 (B), 3 (C) and 4 (D). [DNA] ¼ 0–60 mM, [complex] ¼ 25 mM (inset: (B), 3 (C) and 4 (D). [DNA] ¼ 10 mM, [EB] ¼ 10 mM, [complex] ¼ 0–60 mM
plot of [DNA]/(3a ꢀ 3b) vs. [DNA]).
(inset: plot of I0/I vs. [Q]).
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the quenching constant, and [Q] is the quencher concentration.
Kq is the slope that is obtained from a plot of I0/I versus [Q]
(shown as insets in Fig. 4). The quenching plots illustrate that
the quenching of EtBr bound to CT-DNA by the nickel(II) benz-
hydrazone complexes is in good agreement with the linear
Stern–Volmer equation. The Kq values that were obtained from
the experiments are 1.01(ꢁ0.14) ꢃ 105 Mꢀ1, 1.03(ꢁ0.10) ꢃ
105 Mꢀ1, 1.03(ꢁ0.12) ꢃ 105 Mꢀ1 and 1.04(ꢁ0.08) ꢃ 105 Mꢀ1
,
respectively. Furthermore, the apparent DNA-binding constant
(Kapp) was calculated from the following eqn (3):
KEtBr[EtBr] ¼ Kapp[complex]
(3)
Fig. 5 Cyclic voltammetry measurements at a scan rate of 100 mV sꢀ1
[Complex] ¼ 10 mM and [CT-DNA] addition (0–30 mM).
.
where the concentration of the complex has the value that
corresponds to a 50% reduction in the uorescence intensity of
EtBr, KEtBr ¼ 1.0 ꢃ 107 Mꢀ1 and [EtBr] ¼ 10 mM, and was found
to be 5.26 ꢃ 105 Mꢀ1, 5.35 ꢃ 105 Mꢀ1, 5.81 ꢃ 105 Mꢀ1, and
6.25 ꢃ 105 Mꢀ1 for complexes 1–4, respectively.
of the nickel(II) hydrazone complexes is indicative of an inter-
calative binding mode with DNA molecules.66
The uorescence quenching spectra of DNA-bound EtBr in
the presence of complexes 1–4 shown in Fig. 4 illustrate that as
the concentration of the complexes increased, the emission
band at 595 nm (545 nm excitation) exhibited hypochromism of
up to 44.85%, 44.53%, 45.73% and 46.20% with a hypsochromic
shi of 2–3 nm in the initial uorescence intensity for
complexes 1–4, respectively. The observed decrease in uores-
cence intensity clearly reveals that EtBr molecules were dis-
placed from their DNA binding sites by the complexes under
investigation.63
Circular dichroism spectral study
CD has been effectively used to interpret the conformational
changes of DNA upon binding to a metal complex. The circular
dichroism spectrum of DNA displays a positive band at 275 nm
(UV-vis: lmax 258 nm), which is due to base stacking, and
a negative band at 245 nm, which is due to the helicity of B-type
DNA.67 Simple groove binding and electrostatic interaction of
the molecules give rise to little or no perturbation in base
stacking and helicity, although intercalation increases the
intensities of both the positive and negative bands. The CD
spectrum of CT-DNA (60 mM) in the presence of complex 1
(20 mM) is shown in Fig. 6. From the experimental results, we
observed that the addition of the complexes to the DNA system
increased the intensity of both the positive and negative bands
of free DNA, which is clear evidence of intercalation between the
nickel(II) benzhydrazone complexes and CT-DNA. The CD
spectra indicate that the binding of complexes 1–4 to CT-DNA
results in signicant increases in the intensities of both posi-
tive and negative bands without any shi in peak positions,
which shows that the binding of complexes does not lead to any
signicant change in the conformation of CT-DNA.61
Cyclic voltammetry
The different modes of interaction of metal complexes with
DNA can be studied not only by electronic absorption spectral
studies but also by cyclic voltammetry. In order to investigate
the mode of interaction between the nickel(II) hydrazone
complexes and DNA, a cyclic voltammetry experiment was
carried out. In general, the electrochemical potential of a small
molecule will shi in a positive direction when it intercalates
into a DNA double helix and in a negative direction in the case
of electrostatic interaction with DNA.64,65
In CV titration, both the concentration and volume of the
analyte were kept constant while changing the concentration of
DNA in the solution. The typical CV behaviour of the nickel(II)
hydrazone complexes [Ni(L1)2] in the absence and presence of
Viscosity studies
different concentrations of CT-DNA is shown in Fig. 5. When CT- To further investigate the binding of the nickel(II) benzhy-
DNA was added to a solution of a complex, both anodic and drazone complexes, viscosity measurements were carried out on
cathodic peak current heights of the complex decreased in the calf thymus DNA by varying the concentration of the added
same manner during the addition of increasing amounts of complex. Optical photophysical probes generally provide clues
DNA. Furthermore, during the addition of DNA, the anodic peak that are necessary, but not sufficient to support a binding
potential (Epa), cathodic peak potential (Epc) and E1/2 (calculated model. Viscosity measurements that are sensitive to changes in
as the average of Epc and Epa) all exhibited positive shis. These length are regarded as the least ambiguous and most critical
positive shis are considered as evidence of the intercalation of tests of binding model in a solution in the absence of crystal-
the complex into DNA, because this type of interaction is due to lographic structural data or NMR.68 A classical intercalation
hydrophobic interaction. From another point of view, if a mole- model results in a lengthening of the DNA helix, as base pairs
cule binds electrostatically to the negatively charged deoxyribose are separated to accommodate the binding ligand, which leads
phosphate backbone of DNA, a negative peak potential should to an increase in the viscosity of DNA. However, partial and/or
be detected. Therefore, the positive shi in the CV peak potential non-classical intercalation of a ligand may bend or kink the
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(1 mM) and uorescence spectra were obtained in the range of
300–450 nm upon excitation at 280 nm. The effects of the
complexes on the uorescence emission spectrum of BSA are
given in Fig. 8. Upon addition of the nickel(II) benzhydrazone
complexes to a solution of BSA at an emission wavelength of 345
nm, decreases of up to 40.12%, 35.84%, 34.64% and 43.06%
from the initial uorescence intensity of BSA accompanied by
a hypsochromic shi of 3–5 nm for complexes 1–4, were
observed, respectively.
The blue shi that was observed was mainly due to the fact
that the active site of the protein is buried in a hydrophobic
environment. These results suggest a strong interaction of all
the complexes with the BSA protein. Fluorescence quenching is
described by the Stern–Volmer relation:
Fig. 6 Circular dichroism spectrum of CT-DNA (60 mM) in the pres-
ence of complex 1 (20 mM).
I0/I ¼ 1 + Ksv[Q]
(4)
DNA helix, resulting in a decrease in its effective length and
consequently its viscosity.69
where I0 is the emission intensity in the absence of the
quencher, I is the emission intensity in the presence of the
quencher, Ksv is the Stern–Volmer quenching constant, and [Q]
is the quencher concentration. Ksv is the slope that is obtained
from a plot of I0/I versus [Q] (shown in Fig. 9A) and was found to
be 1.02(ꢁ0.04) ꢃ 106 Mꢀ1, 1.01(ꢁ0.07) ꢃ 106 Mꢀ1, 1.01(ꢁ0.18)
ꢃ 105 Mꢀ1 and 1.02(ꢁ0.15) ꢃ 106 Mꢀ1, corresponding to
complexes 1–4. It is assumed that the binding of compounds to
BSA occurs with an equilibrium binding constant and can be
analysed according to the Scatchard equation:
The effects of the nickel(II) complexes on the viscosity of
CT-DNA are shown in Fig. 7. Values of (h ꢀ ho)1/3, where h and ho
are the specic viscosities of DNA in the presence and absence of
the complexes, are plotted against [complex]/[DNA]. The relative
viscosity of CT-DNA exhibits a considerable increase in the
presence of increasing amounts of the complexes. Such behav-
iour indicates that the complexes are inserted between the DNA
bases, thus resulting in an intercalative mode of binding
between DNA and each complex. The ability of the complexes to
increase the viscosity of DNA follows the order 1 < 3 < 2 < 4.
log[(FO ꢀ F)/F] ¼ log K + n log[Q]
(5)
Protein-binding studies
where K and n are the binding constant and number of binding
sites, respectively. A plot of log[(FO ꢀ F)/F] versus log[Q] can be
used to determine the values of both K and n and values that
were calculated for complexes 1–4 from Fig. 9 are listed in Table
4. From the values of n, it is clearly shown that there is only one
independent class of binding sites for the complexes on BSA
and also a direct relation between the binding constant and the
number of binding sites.
Fluorescence quenching of BSA by nickel(II) benzhydrazone
complexes. It is well known that the transport of drugs through
the bloodstream is affected by the interaction of drugs with
blood plasma proteins, in particular serum albumin. Analysis of
the binding of chemical compounds to BSA is commonly
undertaken by examining uorescence spectra. The binding of
BSA to the nickel(II) benzhydrazone complexes was studied by
uorescence measurements at room temperature. Various
concentrations of complexes 1–4 were added to solutions of BSA
Fig. 7 Effect of increasing amounts of complexes 1–4 on the relative
viscosity of CT-DNA at 29.0 ꢁ 0.1 ꢂC. [DNA] ¼ 5 mM and [complexes] ¼ Fig.
8
Emission spectra of BSA at various concentrations of
0–5 mM.
complexes 1–4. [BSA] ¼ 1 mM and [complexes] ¼ 0–70 mM.
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quenching is UV-visible spectroscopy. Fig. 10 shows the UV-vis
spectra of BSA in the presence and absence of the complexes,
which indicates that the absorption intensity of BSA increased
as the complexes were added and there was a small blue shi of
about 2 nm for all the complexes. This revealed the existence of
a static interaction between BSA and the complexes.60
Characteristics of synchronous uorescence spectra
Synchronous uorescence spectra provide information on the
molecular environment, in particular in the vicinity of the
functional groups of a uorophore.70 It is well known that the
uorescence of a protein may be due to the presence of tyrosine,
tryptophan and phenylalanine residues and therefore spectro-
scopic methods are usually applied to investigate conforma-
tional changes of a serum protein. According to Miller,71 in
synchronous uorescence spectroscopy, the difference between
the excitation and emission wavelengths (Dl ¼ lemi ꢀ lexc) is
indicative of the spectra of different types of chromophores;
a small Dl value such as 15 nm is characteristic of tyrosine
residues and a large Dl value such as 60 nm is characteristic of
tryptophan residues. In order to study the structural changes of
BSA in the presence of nickel(II) hydrazone complexes, we ob-
tained synchronous uorescence spectra on the addition of
complexes 1–4.
The synchronous uorescence spectra of BSA with nickel(II)
hydrazone complexes 1–4 were obtained at both Dl ¼ 15 nm
and Dl ¼ 60 nm and are shown in Fig. 11 and 12. In the
synchronous uorescence spectra of BSA at Dl ¼ 15 nm,
increasing the concentration of the complexes in the solution of
BSA resulted in decreases in the uorescence intensity of BSA at
302 nm of 30.84%, 32.78%, 33.18% and 34.71% of the initial
uorescence intensity of BSA and a red shi of 1–2 nm for
complexes 1–4, respectively. However, in the case of the
synchronous uorescence spectra of BSA at Dl ¼ 60 nm, an
increase in the concentration of the complexes in the solution
of BSA resulted in signicant decreases in the uorescence
intensity at 345 nm of up to 51.40%, 37.71%, 34.20% and
51.75% of the initial uorescence intensity of BSA accompanied
by a blue shi of 1–2 nm for complexes 1–4.
Fig. 9 Stern–Volmer plots (A) and Scatchard plots (B) of the fluores-
cence titration of complexes 1–4 with BSA.
Larger values of Kbin and Kq reveal that strong interaction
occurs between BSA and the complexes. The calculated value of
n is around 1 for all the complexes, which indicates the exis-
tence of a single binding site in BSA for all the complexes. The
results of the binding constant values indicate that the
complexes bind to BSA in the order of 4 > 2 > 3 > 1. Complex 4
can interact with the active site by making it more hydrophobic.
Among the four nickel(II) benzhydrazone complexes, complex 4
exhibits a stronger interaction with BSA than the other nickel(II)
benzhydrazone complexes.
These experimental results indicate that nickel(II) benzhy-
drazone complexes do not affect the conformation with resi-
dues led to a decrease in the polarity of the uorophore by
UV-vis absorption studies
Commonly, quenching occurs via either static or dynamic
quenching. Dynamic quenching is a process in which a uo-
rophore and a quencher come into contact during the transient
existence of an excited state, whereas static quenching refers to
the formation of a uorophore–quencher complex in the
ground state. A simple method to determine the type of
Table 4 Binding constants and number of binding sites for the
interactions of nickel(II) benzhydrazone complexes 1–4 with BSA
System
K (Mꢀ1
)
n
BSA + complex 1
BSA + complex 2
BSA + complex 3
BSA + complex 4
1.07(ꢁ0.08) ꢃ 106
1.51(ꢁ0.04) ꢃ 106
1.08(ꢁ0.23) ꢃ 106
2.40(ꢁ0.06) ꢃ 106
1.01(ꢁ0.01)
0.82(ꢁ0.05)
1.02(ꢁ0.15)
0.84(ꢁ0.10)
Fig. 10 Absorption spectra of BSA (1 ꢃ 10ꢀ5 M) in the presence of
complexes 1–4 (5 mM).
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increasing the hydrophobicity in its environment. Hence, the
strong interaction between the complexes and BSA demon-
strates that these complexes can easily be stored in the protein
and can be released at desired targets. The characteristics of the
synchronous uorescence measurements show that conforma-
tional changes occurred in BSA upon interaction with nickel(II)
benzhydrazone complexes.
BSA cleavage studies
The ability of the complexes to cleave protein peptide bonds72
was studied using BSA as a substrate. The experiment was
carried out using BSA (20 mM) in 5 mM Tris–HCl-50 mM NaCl
buffer at pH ¼ 7.2. In the absence of an activator such as
hydrogen peroxide, none of the complexes give rise to any
protein cleavage even at a concentration of 500 mM. When the
cleavage experiment was performed at a concentration of the
complex of 200 mM in the presence of H2O2 (100 mM) at pH 7.2
(Fig. 13), all the complexes produce signicant smearing or
fading of the BSA band, which suggests that the complexes bind
non-specically to the protein and cleave BSA into very small
fragments. All the complexes are capable of cleaving the protein
but without any sequence specicity.
Fig. 12 Synchronous fluorescence spectra of BSA (1 mM) as a function
of the concentration of complexes 1 (A), 2 (B), 3 (C), and 4 (D) (0–
60 mM) with a difference in wavelength of Dl ¼ 60 nm.
antioxidant activity in the order of 4 > 2 > 3 > 1 ligand >
precursors in all the experiments. Complexes 1–4 displayed
almost comparable free-radical-scavenging activity with respect
to the standard antioxidant (BHA). From the IC50 values, it can
be concluded that the nickel complexes possess higher antiox-
idant activity when compared with the free ligands and metallic
precursors, which is due to their chelation with metal ions.
Among the complexes, complex 4 exhibits good radical-
scavenging activity, which might be due to the greater
electron-donating nature of the methoxy substituent and the
planarity of the phenyl group. The lowest activity was observed
for complex 1. Hence, we strongly consider that the present
metal hydrazone complexes can be further evaluated as suitable
candidates leading to the development of new potential anti-
oxidants and therapeutic reagents for some diseases.
Antioxidant activity
In order to determine the ability of the nickel(II) benzhydrazone
complexes to act toward different reactive species, we studied
some radical-scavenging assay methods, in particular DPPH,
OH and NO radical-scavenging assays (Fig. 14). The hydrazone
ligands, metallic precursors and nickel(II) benzhydrazone
complexes were tested in the range of 10–50 mM. It is to be noted
that no signicant radical-scavenging activity was observed for
the ligands and metallic precursors under identical experi-
mental conditions. From the experimental results, the IC50
values of the complexes with respect to the DPPH, OH and NO
radical assays were found to be 41.6, 40, 40.8, and 34.5 mM, 41.6,
40.81, 40, and 35.0 mM, and 44.1, 43, 41.8 and 38.1 mM,
respectively. The IC50 values show that the complexes exhibit
Cytotoxicity
Preliminary up-to-date results are remarkably positive, sup-
porting our facts and conrming the signicant potential of
Fig. 13 SDS-PAGE diagram of the cleavage of bovine serum albumin
(BSA) by complexes 1–4. [BSA] ¼ 20 mM, [complexes] ¼ 200 mM in
5 mM Tris–HCl-50 mM NaCl buffer at pH ¼ 7.2 and in the presence of
Fig. 11 Synchronous fluorescence spectra of BSA (1 mM) as a function hydrogen peroxide (H2O2, 100 mM) at 50 ꢂC with an incubation time of
of the concentration of complexes 1 (A), 2 (B), 3 (C), and 4 (D) (0– 3 h. Lane 1 (control), BSA + H2O2; lane 2, BSA + H2O2 + 1; lane 3, BSA +
60 mM) with a difference in wavelength of Dl ¼ 15 nm.
H2O2 + 2; lane 4, BSA + H2O2 + 3; lane 5, BSA + H2O2 + 4.
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The IC50 values are much higher than those previously reported
for other nickel complexes.74
Neutral red uptake assay
The IC50 values that were obtained from the MTT assay were
further evaluated by a neutral red uptake assay using HeLa cell
lines. The neutral red uptake assay provides a quantitative
estimation of the number of viable cells in a culture. It is based
on the ability of viable cells to incorporate and bind the
supravital dye neutral red in their lysosomes. Dying cells have
an altered membrane potential and therefore can no longer take
up neutral red. The dye is applied to cells in different concen-
trations to allow the determination of the IC50 concentration
(50% reduction of uptake) by measuring OD540
.
Fig. 14 Trends in the inhibition of DPPH, OH and NO radicals by
complexes at various concentrations.
HeLa cells were exposed to different concentrations of the
nickel(II) benzhydrazone complexes 1–4 for 48 h of incubation.
The results show IC50 values of 50.0 ꢁ 1.2, 33.2 ꢁ 1.0, 36.5 ꢁ 0.9
and 20.1 ꢁ 1.0 mM for complexes 1–4, respectively, and these
values were found to be very close to those obtained from the
MTT assay.
this class of nickel complexes as anticancer agents.73 Nickel
hydrazone complexes 1–4 were evaluated for their cytotoxicity
against HeLa, MCF-7 and NIH-3T3 cell lines using a MTT assay
aer 48 hours of inhibition. For comparison, the cytotoxicity of
the well-known anticancer drug cisplatin against all the
abovementioned cell lines is shown in Table 5. The results were
studied by means of cell viability curves and expressed as
values in the studied concentration range from 0.1 to 100 mM.
The activities that correspond to inhibition of cancer cell
growth at a maximum level, expressed as IC50 values, which
relate to inhibition of cancer cell growth at the 50% level, are
noted.
It is to be noted that the precursor and ligand did not display
any inhibition of cell growth, even at concentrations of up to
100 mM, which clearly indicates that chelation of the ligand with
the metal ion is responsible for the observed cytotoxic proper-
ties of the complexes. The results of MTT assays revealed that
the complexes exhibited notable activity against both HeLa and
MCF-7 cell lines with respect to IC50 values, as represented in
Table 5. The cytotoxic activity of complex 4 was found to be
superior when compared with other complexes. The observed
higher efficiency of complex 4 is related to the nature of the
substitution of the benzhydrazone ligand that is coordinated to
the nickel ion. Higher cytotoxicity is observed for complex 4,
which contains an electron-donating methoxy group that
consequently increases the lipophilic character of the metal
complex, which favours its permeation through the lipid layer of
a cell membrane. Between the two different cell lines that were
used in the study, the proliferation of the MCF-7 cell line was
arrested to a greater extent than that of HeLa cells by the
complexes. Although the abovementioned complexes were
active against the cell lines in in vitro cytotoxicity experiments,
none of the complexes could attain the effectiveness shown by
the standard drug cisplatin (IC50 values 16.20 and 13.86 mM,
respectively). The in vitro cytotoxic activity test also shows that
Morphological changes in AO/EB double staining by confocal
study
Upon exposure to cytotoxic agents, cell death may take place via
several modes; among these, apoptosis and necrosis are very
common. Apoptosis or programmed cell death is characterized
by cell shrinkage, blebbing of the plasma membrane, and
chromatin condensation. To investigate morphological
changes, the acridine orange/ethidium bromide (AO/EB)
double-staining technique is frequently used. In order to
investigate the mechanistic aspects of cell death and to deter-
mine changes in nuclear morphology, AO/EB double staining of
MCF-7 cell lines that were treated with complex 4 (10 mM) was
carried out and is shown in Fig. 15. The experiment was based
on the discrimination of live cells from dead cells on the basis of
morphological changes. Acridine orange passes through the
plasma membrane and stains the DNA of live cells with the
appearance of green uorescence. On the other hand, EB is
excluded from cells that have an intact plasma membrane and
stains the DNA of dead cells, producing orange uorescence.
Cells that were incubated with complex 4 for 24 h and irradiated
with visible light exhibited a signicant reddish-orange emis-
sion, which is characteristic of apoptotic cells. The controls,
Table 5 Cytotoxic activity of the complexes, IC50 (mM)a
Complex
HeLa
MCF-7
NIH-3T3
1
2
3
4
52.3 ꢁ 1.5
32.1 ꢁ 1.8
37.1 ꢁ 2.1
18.1 ꢁ 1.9
16.20 ꢁ 0.70
22.4 ꢁ 2.1
18.2 ꢁ 1.1
17.8 ꢁ 1.9
16.3 ꢁ 0.9
13.86 ꢁ 0.5
235.60 ꢁ 0.5
238.72 ꢁ 1.4
242.32 ꢁ 2.0
245.64 ꢁ 1.1
240.52 ꢁ 0.6
the IC50 value of the complex against NIH-3T3 (non-cancerous Cisplatin
cells) was found to be above 235 mM, which conrms that the
complex is very specic for cancer cells compared with cisplatin.
a
IC50 ¼ concentration of the drug that is required to inhibit the growth
of 50% of the cancer cells (mM).
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which were incubated in the dark, displayed only a predomi-
nant green emission. Hence, the observed morphological
changes reveal that complex 4 is able to induce cell death only
via apoptosis.
Nuclear DAPI staining experiment by confocal study
To further conrm the mode of apoptosis, DAPI staining was
also carried out. DAPI is a uorescent nuclear stain that is
excited by ultraviolet light and exhibits strong blue uorescence
when bound to DNA. Control cells and cells treated with
complex 4 (100 mg mLꢀ1) were stained with DAPI and observed
under a confocal microscope (Fig. 16). The control cells, which
were permeabilized with detergent (0.1% Triton X-100),
exhibited light, evenly stained contours of the nuclei, in
contrast to the treated cells, which displayed typical character-
istics of cells undergoing apoptosis. The treated cells were seen
to possess fragmented or highly condensed nuclei, whereas
bright-eld images provide evidence of cell shrinkage and
membrane blebbing, which were attributed to typical features
of apoptotic cells. Necrotic nuclei were not observed with DAPI
staining. Hence, DAPI staining indicates an apoptotic mode of
cell death with the complex.
Fig. 16 Morphological assessment of complex 4 and MCF-7 cells for
24 h. Nuclei were stained with DAPI and observed under a confocal
microscope at 458 nm.
Evaluation of apoptosis-ow cytometry
The ability of nickel(II) benzhydrazone complexes to induce
apoptosis was further investigated with the aid of ow cytom-
etry, using the annexin V-FITC apoptosis detection kit to
perform double staining with propidium iodide and annexin
V-FITC.51 Annexin V, which is a Ca2+-dependent phospholipid-
binding protein with a high affinity for the membrane phos-
pholipid phosphatidylserine (PS), is quite helpful for identi-
fying apoptotic cells with exposed PS. Propidium iodide is
a standard ow cytometry viability probe used to distinguish
viable from non-viable cells. MCF-7 cells were treated with
complex 4 at three different concentrations for 48 h: see Fig. 17.
The annexin V+/PI+ (Q2) population represents cells undergoing
apoptosis, which increased from 30.7% and 48.6% to 52.7% for
50 mM, 100 mM and 200 mM concentrations of the complexes.
This observation is in good agreement with the results obtained
from uorescence staining methods.
Fig. 17 Apoptosis assays: propidium iodide and annexin-positive (Q2)
and propidium iodide-positive (Q4); flow cytometry results of
untreated MCF-7 control cells and cells treated with complex 4 at
50 mM, 100 mM and 200 mM concentrations for 48 h, respectively.
Conclusion
The present study focuses on the synthesis and characterization
of four new square-planar nickel(II) complexes with thiophene
aldehyde benzhydrazone ligands. The characterization of the
complexes was accomplished by analytical and spectral
methods. All the complexes were found to have a metal : ligand
molar ratio of 1 : 2. All the complexes exhibit a quasi-reversible
one-electron reduction response versus SCE. The DNA-binding
interactions of the complexes with CT-DNA show that the
binding mode is essentially non-covalent via intercalation. The
binding constants showed that the DNA-binding ability
increased in the order of 4 > 2 > 3 > 1. Competitive binding
Fig. 15 AO/EB double staining of MCF-7 cells treated with complex 4
(10 mM) showing changes in nuclear morphology at 488–600 nm. The
scale bar corresponds to 50 mm.
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Acknowledgements
R. R. K. thanks the DST-INSPIRE Program for nancial assis-
tance as a JRF (IF130075). We thank UGC-SAP and DST-India
(FIST programme) for the use of Bruker 400 MHz NMR,
UV-visible and uorescence spectral facilities at the School of
Chemistry, Bharathidasan University. We thank Prof. A. Ramu,
Madurai Kamaraj University and Prof. N. Thajuddin, Bhar-
athidasan University, for providing CD and confocal instru-
mental facilities. We thank Dr Sanjib Karmakar, SAIF,
Department of Instrumentation & USIC, Gauhati University,
Guwahati for collecting single-crystal X-ray diffraction data. We
also thank the CSIR, New Delhi for the electrochemical analyser
through research project Ref. No.: 02(0142)/13/EMR-II.
27 L. Signor, C. Knuppe, R. Hug, B. Schweizer, A. Pfaltz and
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