DNA binding, antioxidant, cytotoxicity (MTT, lactate dehydrogenase, NO), and cellular uptake studies of structurally different nickel(II) thiosemicarbazone complexes: Synthesis, spectroscopy, electrochemistry, and X-ray crystallography

Article abstract of DOI:10.1007/s00775-012-0969-x

Three new nickel(II) thiosemicarbazone complexes have been synthesized and characterized by analytical, spectral, and single-crystal X-ray diffraction studies. In complex 1, the ligand 2-hydroxy-1-naphthaldehydethiosemicarbazone coordinated as a monobasic tridentate donor, whereas in complexes 2 and 3, the ligands salicylaldehyde-4(N)-ethylthiosemicarbazone and 2-hydroxy-1- naphthaldehyde-4(N)-ethylthiosemicarbazone coordinated as a dibasic tridentate donor. The DNA binding ability of the complexes in calf thymus DNA was explored by absorption and emission titration experiments. The antioxidant property of the new complexes was evaluated to test their free-radical scavenging ability. In vitro cytotoxicity assays were performed for the new complexes in A549 and HepG2 cell lines. The new compounds overcome cisplatin resistance in the A549 cell line and they were also active in the HepG2 cell line. The cellular uptake study showed the accumulation of the complexes in tumor cells depended on the nature of the ligand attached to the nickel ion. Graphical abstract: Structurally different nickel(II) thiosemicarbazone complexes for which DNA binding, antioxidant, cytotoxicity, and cellular uptake studies were performed [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00775-012-0969-x

J Biol Inorg Chem (2013) 18:233–247
DOI 10.1007/s00775-012-0969-x
ORIGINAL PAPER
DNA binding, antioxidant, cytotoxicity (MTT, lactate
dehydrogenase, NO), and cellular uptake studies of structurally
different nickel(II) thiosemicarbazone complexes: synthesis,
spectroscopy, electrochemistry, and X-ray crystallography
•
R. Prabhakaran P. Kalaivani R. Huang
•
•
•
•
•
P. Poornima V. Vijaya Padma F. Dallemer
K. Natarajan
Received: 2 July 2012 / Accepted: 8 December 2012 / Published online: 29 December 2012
Ó SBIC 2012
Abstract Three new nickel(II) thiosemicarbazone com-
plexes have been synthesized and characterized by ana-
lytical, spectral, and single-crystal X-ray diffraction
studies. In complex 1, the ligand 2-hydroxy-1-naphthal-
dehydethiosemicarbazone coordinated as a monobasic tri-
dentate donor, whereas in complexes 2 and 3, the ligands
salicylaldehyde-4(N)-ethylthiosemicarbazone and 2-hyd-
roxy-1-naphthaldehyde-4(N)-ethylthiosemicarbazone coor-
dinated as a dibasic tridentate donor. The DNA binding
ability of the complexes in calf thymus DNA was explored
by absorption and emission titration experiments. The
antioxidant property of the new complexes was evaluated
to test their free-radical scavenging ability. In vitro cyto-
toxicity assays were performed for the new complexes in
A549 and HepG2 cell lines. The new compounds overcome
cisplatin resistance in the A549 cell line and they were also
active in the HepG2 cell line. The cellular uptake study
showed the accumulation of the complexes in tumor cells
depended on the nature of the ligand attached to the nickel
ion.
Keywords Nickel(II) complexes ꢀ Thiosemicarbazones ꢀ
X-ray crystallography ꢀ Electrochemistry ꢀ DNA binding
Introduction
Thiosemicarbazones and their metal complexes have
become an area of intensive study because of their inter-
esting chemical and structural properties and their wide-
ranging biological activities [1–11]. The biological and
medicinal properties of thiosemicarbazones depend on the
chemical nature of the moiety attached to the C=S carbon
atom. The present level of interest in metal complexes of
thiosemicarbazones stems from the fact that the biological
activities are often attributed to the chelation of thiosemi-
carbazones to a transition-metal ion [12–19]. In connection
with the antitumor activity of thiosemicarbazones, pres-
ently there are three main points on which current research
is focusing. First, thiosemicarbazones are inhibitors of
ribonucleotide reductase activity. Ribonucleotide reductase
catalyzes the synthesis of deoxyribonucleotides required
for DNA synthesis. Since deoxyribonucleotides are present
at extremely low levels in mammalian cells, their synthesis
is a crucial and rate-controlling step in the pathway leading
to the biosynthesis of DNA. Mammalian ribonucleotide
reductase is composed of two dissimilar proteins—R1,
which contains polythiols, and R2, which contains non-
heme iron and a free tyrosyl radical. Both the R1 subunit
and the R2 subunit contribute to the active site of the
enzyme [20]. Thiosemicarbazones can destabilize or
damage the non-heme-iron-stabilized tyrosyl free radical
R. Prabhakaran (&) ꢀ P. Kalaivani ꢀ K. Natarajan (&)
Department of Chemistry, Bharathiar University,
Coimbatore 641046, India
e-mail: rpnchemist@gmail.com
K. Natarajan
e-mail: k_natraj6@yahoo.com
R. Huang
Department of Chemistry, Michigan State University,
East Lansing, MI 48824, USA
P. Poornima ꢀ V. Vijaya Padma
Department of Biotechnology, Bharathiar University,
Coimbatore 641046, India
F. Dallemer
Laboratoire Chimie Provence-CNRS UMR6264, Universite
of Aix-Marseille I, II and III-CNRS, Campus Scientifique
´
´ ˆ
de Saint-Jerome, Avenue Escadrille Normandie-Niemen,
13397 Marseille Cedex 20, France
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and thus inhibit the catalytic function of ribonucleotide
reductase. Second, thiosemicarbazones can stabilize
cleavable complexes formed by topoisomerase II and
DNA, leading to apoptosis [21]. Third, more recently they
have been recognized as an inhibitor of ATP-binding cas-
sette transporter proteins, which are responsible for the
expulsion from the cell of exogenous molecules that allow
cells to develop multidrug resistance. Their action consists
in preventing cells from expelling these compounds [22].
The design of compounds able to bind and react with
selective nucleotide sequences is of great importance in
probing biological processes and in developing therapeutic
drugs. Transition-metal compounds that bind to DNA in a
covalent or noncovalent fashion or induce DNA strand
scission have potential applications as tools for probing
biomolecular structure and function and as cytotoxic agents
in cancer chemotherapy [23, 24]. Therefore, DNA-target-
ing drugs remain in the limelight and compounds acting
against cancer cells selectively over healthy cells are
receiving more attention [25–29]. Despite the therapeutic
benefit of platinum-based treatment regimens, the efficacy
of platinum drugs is still limited by side effects and
intrinsic and acquired resistances [30]. Therefore, the
search for new potential platinum drugs is continuing, and
some pioneering strategies have emerged. These strategies
are represented by the synthesis of nonclassical platinum
compounds [31, 32]. With the above-mentioned intention
in mind, we synthesized a few platinum-mimic nickel
complexes containing biologically active thiosemicarba-
zones, and this article deals with their synthesis, charac-
terization, and chelating behavior and DNA binding,
antioxidant, cytotoxicity, and cellular uptake studies.
separated was filtered off and washed thoroughly with
ethanol and then dried in vacuo. The compound was re-
crystallized from hot ethanol. The product is soluble in
warm acetone, methanol, ethanol, dichloromethane, chlo-
roform, dimethylformamide, and dimethyl sulfoxide
(DMSO). Yield: 82 % (4.019 g). Melting point 261 °C.
Anal. Calcd for C12H11N3OS (%): C, 58.76; H, 4.52; N,
17.13; S, 13.07. Found (%): C, 58.69; H, 4.49; N, 17.05; S,
12.99. IR (cm-1) in KBr: 3,446 (mOH), 1,604 (mC=N), 1,278
1
(mC–O), 815 (mC=S). H NMR (DMSO-d6, ppm): d 11.5 (s,
1H, OH), 10.7 (s, 1H, NHCS), 8.10 (s, 1H, CH=N), 9.2 (s,
–NH2), 7.1–7.8 (m, aromatic protons).
A method similar to that described above was followed
to prepare all the other thiosemicarbazone ligands.
Preparation of H2L2
This was prepared from ethylthiosemicarbazide (2 g,
0.016 mol) and salicylaldehyde (2.05 g, 0.016 mol). Yield:
80 % (2.856 g). Melting point 160 °C. Anal. Calcd for
C10H13N3OS (%): C, 53.79; H, 5.87; N, 18.82; S, 14.36.
Found (%): C, 53.71; H, 5.83; N, 18.76; S, 14.29. IR
(cm-1) in KBr: 3,415 (mOH), 1,621 (mC=N), 1,276 (mC–O),
825 (mC=S). 1H NMR (DMSO-d6, ppm): d 11.1 (s, 1H, OH),
9.65 (s, 1H, NHCS), 7.87 (s, 1H, CH=N), 8.31 (d, terminal
–NH), 3.69 (p, –CH2–), 1.25 (t, –CH3), 6.8–7.7 (m, aro-
matic protons).
Preparation of H2L3
This was prepared from ethylthiosemicarbazide (2.98 g,
0.025 mol) and 2-hydroxy-1-naphthaldehyde (4.3 g,
0.025 mol). Yield: 78 % (5.33 g). Melting point 204 °C.
Anal. Calcd for C14H15N3OS (%): C, 61.51; H, 5.53; N,
15.37; S, 11.73. Found (%): C, 61.48; H, 5.46; N, 15.31; S,
11.66. IR (cm-1) in KBr: 3,418 (mOH), 1,620 (mC=N), 1,282
Materials and methods
1
The ligands 2-hydroxy-1-naphthaldehydethiosemicarba-
zone (H2L1), salicylaldehyde-4(N)-ethylthiosemicarbazone
(H2L2), and 2-hydroxy-1-naphthaldehyde-4(N)-ethylthi-
osemicarbazone (H2L3)—and the nickel precursor
[NiCl2(PPh3)2] were prepared according to standard liter-
ature procedures [33, 34]. All reagents used were Analar
grade and were purified and dried according to the standard
procedure [35].
(mC–O), 815 (mC=S). H NMR (DMSO-d6, ppm): d 11.3 (s,
1H, OH), 10.8 (s, 1H, NHCS), 8.11 (s, 1H, CH=N), 9.18 (d,
terminal –NH), 3.73 (p, –CH2–), 1.25 (t, –CH3), 7.2–7.8
(m, aromatic protons).
Preparation of [Ni(Nap-tsc)(PPh3)]ꢀClꢀH2O (1)
An ethanolic solution (25 ml) of [NiCl2(PPh3)2] (0.200 g;
0.30 mmol) was slowly added to H2L1 (0.075 g;
0.30 mmol) in dichloromethane (25 ml). The mixture was
allowed to stand for 4 days at room temperature. The dark-
red crystals obtained were filtered off and washed with n-
hexane. Yield: 86 % (0.159 g). Melting point 284 °C.
Anal. Calcd for C30H26ClN3O2SNiP (%): C, 58.33; H,
4.24; N, 6.80; S, 5.19. Found (%): C, 58.28; H, 4.19; N,
6.72; S, 5.12. Fourier transform (FT) IR (cm-1) in KBr:
3,160 cm-1 (mN(2)H),1,600 (mC=N), 1,351 (mC–O), 812 (mC=S),
Preparation of H2L1
Two grams (0.02 mol) of thiosemicarbazide was dissolved
in 20 ml hot ethanol and to this was added 3.8 g (0.02 mol)
of 2-hydroxy-1-naphthaldehyde in 10 ml ethanol over a
period of 10 min with continuous stirring. Further, the
mixture was stirred for 5 h at room temperature and a
yellow compound began to separate out. The compound
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235
1,435, 1,094, 697 cm-1 (for PPh3); UV–vis (CH2Cl2), kmax
(nm): 268 (intraligand transition); 328, 380 (ligand-to-
metal charge transfer, LMCT); 417, 686 (forbidden
DMSO at room temperature with a Bruker 400-MHz
instrument, with the chemical shift relative to tetrameth-
ylsilane. Melting points were recorded with Lab India
apparatus. Single-crystal data collections for the new
Ni(II) complexes were performed at 273(2) K with a
Bruker SMART 1000 CCD diffractometer using mono-
1
d ? d transition). H NMR (DMSO-d6, ppm): d 8.07 (d,
(J = 11.2 Hz) 1H, CH=N), 6.4–7.8 (m, aromatic), 9.4 (s,
–HN–C=S), 6.48 (br, –NH2), 6.51 (s, –NH2).
A similar method was followed to synthesize other
complexes.
˚
chromatic Mo Ka (k = 0.71073 A) radiation. The data
were collected and processed using the software program
SAINT and the structure was solved and refined by full-
matrix least squares on F2 using SHELXL-97 [36]. Cyclic
voltammograms were recorded using a CH Instruments
apparatus by using a platinum wire working electrode and
a platinum disc counter electrode. All the potentials were
referenced to the standard Ag/AgCl electrode and ferro-
cene was used as an external standard.
Preparation of [Ni(Sal-etsc)(PPh3)] (2)
This was prepared by the same procedure as described for 1
with H2L2 (0.068 g; 0.30 mmol). The dark-red crystals
obtained were separated and washed with n-hexane and
dried in a vacuum. It was crystallized using ethanol/chlo-
roform. Yield: 75 % (0.116 g). Melting point 230 °C.
Anal. Calcd for C28H26N3OSNiP (%): C, 62.00; H, 4.83; N,
7.75; S, 5.91. Found (%): C, 61.96; H, 4.78; N, 7.72; S,
5.86. FT-IR (cm-1) in KBr: 1,614 (mC=N), 1,348 (mC–O), 753
(mC–S), 1,432, 1,088, 694 (for PPh3). UV–vis (CH2Cl2),
kmax (nm): 207 (intraligand transition); 303, 382 (LMCT);
DNA binding study
Calf thymus DNA (CT-DNA) solutions of various con-
centrations (0.05–0.5 lM) dissolved in a phosphate buffer
(pH 7) were added to the metal complexes (10 lM dis-
solved in a DMSO/H2O mixture). Absorption spectra were
recorded after equilibrium had been attained at 20 °C for
10 min. The intrinsic binding constant Kb was determined
using the Stern–Volmer equation [37, 38]:
1
420, 440 (forbidden d ? d transition). H NMR (DMSO-
d6, ppm): d 9.3 [d, (J = 13.2 Hz) terminal –NH], 8.4 [d,
(J = 11.0 Hz) –HC=N], 6.9–7.8 (m, aromatic protons),
3.32 (q, –CH2) 1.5 (t, 3H, –CH3).
½DNAꢁ=½ea ꢂ efꢁ ¼ ½DNAꢁ=½eb ꢂ efꢁ þ 1=Kb½eb ꢂ efꢁ:
ð1Þ
Preparation of [Ni(Nap-etsc)(PPh3)] (3)
where the absorption coefficients ea, ef, and eb correspond
to Aobsd/[DNA], the extinction coefficient for the free
complex, and the extinction coefficient for the complex in
the fully bound form, respectively. The slope and the
intercept of the linear fit of the plot of [DNA]/(ea - ef)
versus [DNA] give 1/(ea - ef) and 1/Kb(eb - ef), respec-
tively. The intrinsic binding constant Kb was obtained from
the ratio of the slope to the intercept (Table 5) [37].
Emission measurements were conducted using a JASCO
FP-6600 spectrofluorometer. Tris(hydroxymethyl)amino-
methane buffer was used as a blank to make preliminary
adjustments. The excitation wavelength was fixed and the
emission range was adjusted before measurements. All
measurements were made at 20 °C. For emission spectrum
titrations, the concentration of the complex was maintained
at 10 lM and the concentration of DNA was varied from
0.05 to 0.5 lM. The emission enhancement factors were
measured by comparing the intensities at the emission
spectrum maxima under similar conditions. Ethidium bro-
mide (EB)–DNA fluorescence quenching experiments were
conducted by adding 0.1 lM solutions of the complexes
(10 ll each time) to samples containing 0.5 lM EB,
1.0 lM DNA, and tris(hydroxymethyl)aminomethane
buffer (pH 7.2). Before the measurements, the system was
shaken and incubated at room temperature for approxi-
mately 5 min. The emission was recorded at 530–750 nm.
This was prepared by same the procedure as described for 1
with H2L3 (0.086 g; 0.30 mmol). The dark-red crystals
obtained were filtered off and washed with n-hexane.
Yield: 84 % (0.149 g). Melting point 284 °C. Anal. Calcd
for C32H28N3OSNiP (%): C, 64.89; H, 4.76; N, 7.09; S,
5.41. Found (%): C, 64.84; H, 4.70; N, 7.01; S, 5.35. FT-IR
(cm-1) in KBr: 1,595 (mC=N), 1,349 (mC–O), 745 (mC–S),
1,437, 1,093, 697 (for PPh3); UV–vis (CH2Cl2), kmax (nm):
357, 375, 407 (LMCT); 418 (forbidden d ? d transition).
1H NMR (DMSO-d6, ppm): d 9.35 [d, (J = 11.0 Hz) ter-
minal –NH], 8.5 [d, (J = 8.6 Hz) –HC=N], 7.0–8.1 (m,
aromatic protons), 3.3 (q, –CH2), 1.65 (t, 3H, –CH3).
Measurements
IR spectra were measured using KBr pellets with a
Nicolet instrument between 400 and 4,000 cm-1. Ele-
mental analysis of carbon, hydrogen, nitrogen, and sulfur
was conducted using a Vario EL III CHNS instrument at
the Department of Chemistry, Bharathiar University,
Coimbatore, India. The electronic spectra of the com-
plexes were recorded in dichloromethane using a JASCO
V-630 spectrophotometer in the 200–800-nm range.
Emission spectra were recorded using a JASCO FP-6600
spectrofluorometer. 1H NMR spectra were recorded in
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Diphenyl-1-picrylhydrazyl radical scavenging assay
of 0.4 N sodium hydroxide was added to all the tubes. The
intensity of the color developed was measured at 420 nm
with a Shimadzu UV–vis spectrophotometer. The amount
of LDH released was expressed as a percentage.
The potential antioxidant activity of the new complexes (1–
3) was evaluated by diphenyl-1-picrylhydrazyl (DPPH)
radical scavenging assay according to the procedure
described previously with a slight modification [39]. DPPH
free radicals are used for rapid analysis of antioxidants.
While scavenging the free radicals, the antioxidants donate
hydrogen and form a stable DPPH molecule. This reaction
involves a color change from purple to yellow that can be
measured spectroscopically. Briefly, the complex
(25–350 lM) was added to 2 ml DPPH (0.1 mM in
methanol) and was mixed rapidly. The radical scavenging
capacity was measured every 10 min using a spectropho-
tometer by monitoring the decrease in absorbance at
517 nm. The percent DPPH decolorization of the sample
was calculated. L-Ascorbic acid was used as a positive
control.
Nitric oxide assay
The amount of nitrite was determined by the method of
Stueher and Marletta [42]. Nitrite reacts with Griess
reagent to give a colored complex which can be measured
at 540 nm. To 100 ll of the medium, 50 ll of Griess
reagent I was added, mixed, and allowed to react for
10 min. This was followed by addition of 50 ll of Griess
reagent II and the reaction mixture was mixed well and
incubated for another 10 min at room temperature. The
intensity of pink color developed was measured at 540 nm
with a MicroQuant plate reader (BioTek Instruments).
Cellular uptake study
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide assay
Cellular uptake of the ligands and the complexes (1–3) was
quantified according to the literature method with a slight
modification [43]. Briefly, the lung cancer cells (A549) and
liver cancer cells (HepG2) were treated with different
concentrations of the ligands and complexes for 4, 12, 24,
and 48 h (for A549 cells, 17 lM H2L1, 20 lM H2L2,
15 lM H2L3, 10 lM 1, 8 lM 2, 10 lM 3; for and HepG2
cells, 15 lM H2L1, 19 lM H2L2, 14 lM H2L3, 8 lM 1,
7 lM 2, 8 lM 3). The medium was aspirated and cells
were washed three times with ice-cold PBS. Then the cells
were lysed with PBS containing 1 % Triton X-100. The
concentration of the complexes in the cell lysates was
measured with a florescence spectrophotometer (JASCO
FP-6600) at their maximum excitation/emission wave-
lengths of 382 nm/482 nm, 340 nm/395 nm, and 432 nm/
499 nm, respectively, for complexes 1–3. To offset the
background florescence from the cellular components,
separate standardization curves were prepared using cel-
lular lysates containing a series of known concentrations of
different complexes, and the intracellular concentrations
were found using the standard curve.
The effect of the ligands and the complexes (1–3) on the
viability of human lung cancer cells (A549) and liver
cancer cells (HepG2) was assayed by 3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
[40]. The cells were seeded at a density of 10,000 cells per
well in 200 ll RPMI 1640 medium and were allowed to
attach overnight in a CO2 incubator, and then the com-
plexes (1–3) dissolved in DMSO were added to the cells at
a final concentration of 1, 10, 25, and 50 lM in the cell
culture medium. After 48 h, the wells were treated with
20 ll MTT (5 mg/ml phosphate-buffered saline, PBS) and
incubated at 37 °C for 4 h. The purple formazan crystals
formed were dissolved in 200 ll DMSO and read at
570 nm in a microplate reader.
Release of lactate dehydrogenase
Lactate dehydrogenase (LDH) activity for the ligands and
the complexes was determined by the linear region of a
pyruvate standard graph using regression analysis and was
expressed as the percentage leakage as described previ-
ously [41]. Briefly, to a set of tubes, 1 ml of buffered
substrate (lithium lactate) and 0.1 ml of the medium or cell
extract were added and the tubes were incubated at 37 °C
for 30 min. After 0.2 ml of NAD solution had been added,
the incubation was continued for another 30 min. The
reaction was then arrested by adding 0.1 ml 2,4-dinitro-
phenylhydrazine and the tubes were incubated for a further
period of 15 min at 37 °C. After this, 0.1 ml of medium or
cell extract was added to blank tubes after the reaction had
bee arrested with 2,4-dinitrophenylhydrazine. Then, 3.5 ml
Results and discussion
The thiosemicarbazone ligands (H2L1–H2L3) were reacted
with an equimolar amount of [NiCl2(PPh3)2] in 1:1 ethanol/
dichloromethane and this resulted in the formation of new
complexes (1–3) (Scheme 1), where the substituted thio-
semicarbazones acted as a dibasic/monobasic tridentate
ONS ligand. The analytical data confirmed the stoichiom-
etry of complexes 1–3. The structures of complexes 1–3
were confirmed by X-ray crystallography. The new
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237
Scheme 1 Preparation of new
nickel(II) complexes
[NiCl2(PPh3)2]
CH2Cl2 / Ethanol
Room temp.
S
S
S
N
N
N
NH2
N
N
N
N
H
N
H
H
H
H
PPh3
S
PPh3
S
PPh3
O
O
O
S
Ni
Ni
Ni
.Cl. H2O
NH2
NH
NH
N
N
N
N
N
N
H
2
1
3
complexes are soluble in common organic solvents such as
dichloromethane, chloroform, benzene, acetonitrile, etha-
nol, methanol, dimethylformamide, and DMSO.
characteristic absorption bands due to triphenylphosphine
were also present in the expected region [49]. The elec-
tronic spectra of the new nickel(II) complexes displayed
four to five bands in the region around 207–686 nm. The
band at 207–268 nm has been assigned to an intraligand
transition, the band at 303–407 nm has been assigned to an
LMCT (s ? d), and the shoulder at 417–686 nm has been
assigned to a forbidden d ? d transition [50, 51].
IR and electronic absorption spectra
The IR spectra of the thiosemicarbazone ligands (H2L1–
H2L3) showed a sharp band at 1,604–1,621 cm-1 corre-
sponding to mC=N of the azomethine group. In the IR
spectra of their corresponding complexes (1–3), this band
was shifted to lower frequency (1,595–1,614 cm-1), indi-
cating the coordination of the azomethine nitrogen atom
[44, 45]. A broad band corresponding to mO–H appeared in
the region from 3,415 to 3,446 cm-1 for the ligands, and
this disappeared completely after complexation with the
nickel(II) ions, showing deprotonation prior to coordina-
tion through the oxygen atom in all three complexes. This
was further corroborated with the increase in the phenolic
C–O stretching frequency (1,348–1,351 cm-1) [46]. The
ligand can exist in thione–thiol tautomerization since it
contains a thioamide (–NH–C=S) functional group. The
absence of the mS–H stretching in the region from 2,500 to
2,600 cm-1 and presence of mN–H stretching around
3,140–3,300 cm-1 in the IR spectra of the ligands indicate
the presence of the thione form in the solid state. This is
further inferred from the presence of a strong band in the
region from 815 to 825 cm-1 due to the mC=S stretching
completely disappearing in the spectra of new complexes 2
and 3, and the appearance of a new band at 745–753 cm-1
due to mC–S indicated the coordination of the NH–C=S
group and subsequent coordination through the sulfur atom
[14, 47]. However, in complex 1, the presence of mN–H
stretching at 3,053 cm-1 and the absence of the mS–H)
stretching frequency evidenced the thione sulfur coordi-
nation to the nickel ion [47, 48]. Moreover, the
1H NMR spectra
1
The H NMR spectra of the ligands (H2L1–H2L3) and the
corresponding complexes (1–3) recorded in DMSO showed
all the expected signals. The sharp singlet observed at d
11.1–11.5 ppm in the spectra of the free ligands due to the
phenolic–OH proton completely disappeared in the spectra
of all three complexes, confirming the involvement of
phenolic oxygen in coordination. The spectra of H2L2 and
H2L3 showed a singlet at d 9.65–10.8 ppm corresponding
to the N(2)H–C=S proton [52], but in complexes 2 and 3
there was no resonance attributable to N(2)H, indicating
coordination of the ligand in the anionic form after de-
protonation at N(2). The spectrum of H2L1 showed a sharp
singlet at d 10.7 ppm corresponding to the N(2)H–C=S
group, but this disappeared and a new singlet appeared at d
9.4 ppm in complex 1, indicating the coordination of the
thionic form of the ligand to the nickel ion [53]. In com-
plexes 2 and 3, two doublets observed at d 8.4–8.5 ppm
and d 9.3–9.35 ppm were assigned to azomethine and
terminal –NH group protons [51, 54]. In complex 1, a
doublet corresponding to an azomethine proton was
observed at d 8.07 ppm and two broad singlets appeared at
d 6.48 ppm and d 6.51 ppm, which were assigned to NH2
group protons. The doublets observed for azomethine and
terminal –NH group protons may be due to the coupling
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with a phosphorus atom of the triphenylphosphine and the
restricted rotation of the C–N bond of the ligand, respec-
tively [18, 54]. Further, the spectra of all three complexes
showed a series of overlapping multiplets for aromatic
protons at d 6.4–8.1 ppm [55]. In addition, a quartet was
observed around d 3.30–3.32 ppm corresponding to –CH2–
group protons and a triplet was observed at d 1.5–1.65 ppm
due to CH3 group protons of the ligands in complexes 2
and 3 [56].
Description of the crystal structures
The crystal structures of complexes 1–3 along with their
numbering schemes and their hydrogen-bonding diagrams
are given in Figs. 1, 2, and 3. Crystallographic data and bond
parameters are given in Tables 1 and 2. Complexes 1 and 3
crystallized in monoclinic space group P21/c and complex 2
crystallized in orthorhombic space group P212121.
In complex 1, the ligand (H2L1) is coordinated to nickel
by using its phenolic oxygen, N1 nitrogen, and thione
sulfur atoms and behaved as a monobasic tridentate donor
by forming a six-membered ring and a five-membered ring
with a bite angle of 88.16(5)° (S–Ni–N). The presence of a
chloride ion outside the coordination sphere compensated
for the charge of nickel as Ni2?, resulting in the formation
Fig. 2 ORTEP diagram of [Ni(Sal-etsc)(PPh3)] (2)
1
of ionic compound 1. From the H NMR spectral studies,
there was a detectable proton signal at d 9.4 ppm corre-
sponding to the N(2)H–C=S group. However, the position
of this proton (hydrogen) could not be locates in the X-ray
crystallographic analysis. While dealing the hydrogen-
bonding interaction, we found the donor–acceptor distance
Fig. 3 ORTEP diagram of [Ni(Nap-etsc)(PPh3)] (3)
˚
(2.763 A) corresponding to the N(14)ꢀꢀꢀO(35) bond
between the imine nitrogen and the oxygen atom of the
water which came through the solvent of crystallization.
The water molecule may be from the ethanol which was
used for the preparation of the complex. The presence of
four hydrogen bonds creates a 2D network in the complex:
the first one between one of the hydrogen atoms of the
Fig. 1 ORTEP diagram of [Ni(H-Nap-tsc)(PPh3)]ꢀClꢀH2O (1)
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J Biol Inorg Chem (2013) 18:233–247
239
Table 1 Crystal data of the new nickel(II) thiosemicarbazone complexes
[Ni(H-Nap-tsc)(PPh3)]ꢀClꢀH2O (1)
[Ni(Sal-etsc)(PPh3)] (2)
[Ni(Nap-etsc)(PPh3)] (3)
Empirical formula
C30H26ClN3NiO2PS
617.73
C28H26N3NiOPS
542.26
C32H28N3NiOPS
592.31
Formula weight
Temperature
173(2) K
173(2) K
173(2) K
´
˚
˚
0.71073 A
˚
0.71073 A
Wavelength
0.71073 A
Crystal system
Monoclinic
Orthorhombic
Monoclinic
Space group
P21/c
P212121
P21/c
Unit cell dimensions
´
˚
´
˚
˚
12.755(3) A
a
10.565(2) A
13.292(3) A
´
˚
´
˚
˚
22.037(4) A
b
13.185(3) A
11.542(2) A
´
˚
´
˚
˚
10.309(2) A
c
18.174(4) A
19.039(4) A
a
90°
90°
90°
90°
90°
b
97.19(3)°
90°
2,874.8(10) A
108.05(3)°
90°
2,777.2(10) A3
c
3
3
˚
˚
Volume
2,531.6(9) A
Z
4
4
4
Density (calculated)
Absorption coefficient
F(000)
1.427 Mg m-3
0.929 mm-1
1,276
1.423 Mg m-3
0.939 mm-1
1,128
1.417 Mg m-3
0.863 mm-1
1,232
h range for data collection
Index ranges
1.61–28.23°
-16 B h B 16, -29 B k B 29,
-13 B l B 13
1.91–28.19°
-13 B h B 13, -16 B k B 16,
-22 B l B 23
1.61–28.25°
-17 B h B 17, -15 B k B 15,
-25 B l B 25
Collected/unique reflections
Completeness to h
33,759/6,922 (Rint = 0.0265)
27,751/5,831 (Rint = 0.0262)
28.19, 96.3 %
Full-matrix least squares on F2
28,619/6,667 (Rint = 0.0250)
28.25, 96.9 %
Full-matrix least squares on F2
28.23, 97.6 %
Full-matrix least squares on F2
6,922/0/359
Refinement method
Data/restraints/parameters
Goodness of fit on F2
5,831/0/324
6,667/0/356
0.828
0.567
1.356
Final R indices [I [ 2r(I)]
R indices (all data)
R1 = 0.0404, wR2 = 0.0962
R1 = 0.0289, wR2 = 0.1061
0.357 and -0.270 e- A-3
R1 = 0.0285, wR2 = 0.0670
R1 = 0.0229, wR2 = 0.0714
0.228 and -0.149 e- A-3
R1 = 0.0469, wR2 = 0.1616
R1 = 0.0408, wR2 = 0.1646
0.848 and -0.516 e- A-3
Largest diffraction peak and hole
˚
amine nitrogen and the chloride ion, the second one
between the chloride ion and one of the hydrogen atoms
(H35A) of the water molecule, the third one between the
oxygen atom of the water molecule and the imine nitrogen,
and the fourth one between H35(B) of the water molecule
and the second chloride ion (Table 3). The S(1)–Ni–O(1)
[177.46(4)°] and P(1)–Ni–N(13) [176.32(4)°] bond angles
deviate significantly from the ideal angle of 180°, causing
significant distortion in the square plane.
Table 2 Selected bond lengths (A) and angles (°) of the new nickel
(II) thiosemicarbazone complexes
1
2
3
Bond lengths
Ni–O
1.8471(13)
1.8734(14)
2.1528(6)
2.2114(6)
1.8347(14)
1.8848(15)
2.1426(6)
2.2103(6)
1.8445(19)
1.8848(15)
2.1426(6)
2.2000(7)
Ni–N
Ni–S
Ni–P
The ligand H2L2 coordinated to the metal by losing two
protons from its tautomeric thiol form and phenolic –OH in
complex 2 and acts as a dibasic tridentate ligand via the
thiolate sulfur, phenolic oxygen, and hydrazinic nitrogen
atoms by forming a six-membered ring and a five-mem-
bered ring with bite angle of 87.08(5)° (S–Ni–N). In this
complex, the S(1)–Ni–O(1) [174.30(5)°] and P(1)–Ni–N(1)
Bond angles
O–Ni–N
S–Ni–P
N–Ni–S
O–Ni–P
N–Ni–P
O–Ni–S
93.26(6)
94.20(3)
88.16(5)
84.49(5)
176.32(4)
177.47(4)
94.98(6)
91.59(2)
87.09(5)
87.00(5)
172.84(5)
174.30(6)
95.20(8)
92.95(3)
87.80(7)
84.13(6)
174.68(6)
176.91(6)
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J Biol Inorg Chem (2013) 18:233–247
125 mV, respectively. Generally, the redox processes are
defined by the coordination number, stereochemistry, and
hard/soft donor character of the ligands. However, owing to
inherent difficulties in relating the coordination number
and stereochemistry of the species present in solution, the
redox process is generally described in terms of the nature
of the ligands present [62]. Patterson and Holm [63] have
shown that soft donor ligands tend to show more positive
E1/2 values and hard donor ligands tend to show more
negative E1/2 values. For this complex, the E1/2 values
observed at 0.7734 and -0.3422 V for the oxidation and
reduction processes, respectively, are similar to the values
observed for other nickel(II) complexes [64]. In addition, it
showed a quasi-reversible ligand reduction at -1.3482 V
with a peak-to-peak separation of 545 mV. The cyclic
voltammogram of 3 showed a one-electron reversible
reduction at E1/2 of -0.2122 V [65]. The quasi-reversible
ligand reduction was observed at E1/2 of -1.0424 V with a
peak-to-peak separation of 453 mV (Fig. 4) [66]. In addi-
tion, the complex exhibited an irreversible oxidation at E1/2
of 0.774 V.
Table 3 Hydrogen bonds for complex 1
D–HꢀꢀꢀA
d(D–H) d(HꢀꢀꢀA) d(DꢀꢀꢀA) \(D–H–A)
˚
(A)
˚
(A)
˚
(A)
(°)
N16(A)–H16(B)ꢀꢀꢀCl1(A) 0.985
O35(A)–H35(B)ꢀꢀꢀCl1(B) 0.860
O35(A)–H35(A)ꢀꢀꢀCl1(A) 0.936
2.245
2.359
2.219
–
3.227
3.216
3.113
2.763
174.78
175.22
159.41
–
N14(A)ꢀꢀꢀO(35)
–
Symmetry operation: (x, y, z); (-x, ?1/2 ? y, 1/2 - z); (-x, -y, -z);
(x, 1/2 - y, 1/2 ? z)
A acceptor, D donor
[172.84(5)°] bond angles deviate considerably from the
ideal angle of 180°. The variation in the bonding parame-
ters indicates that there is considerable distortion in the
NiSNOP core around nickel. The C–S bond distance of
´
˚
1.753(2) A indicates that the ligand is bound to nickel in
the thiolate form [57]. In complex 3, the ligand (H2L3)
coordinated to the metal in an ONS fashion and acted as a
dibasic tridentate ligand coordinating through thiolate
sulfur, phenolic oxygen, and hydrazinic nitrogen atom, by
forming a six-membered ring and a five-membered ring
with a bite angle of 87.80(7)° (S–Ni–N). The S(1)–Ni–O(1)
(176.9°) and P(1)–Ni–N(1) (174.68°) bond angles showed
significant distortion around the nickel atom. The C–S
DNA binding studies
˚
bond distance of 1.7490 A found for the complex indicates
Absorption titration experiments were performed to study
the DNA binding property of the new nickel(II) complexes
(1–3). The absorption spectra of the complexes at constant
concentration (10 lM) in the presence of different con-
centrations of CT-DNA (0.05–0.50 lM) are given in
Fig. 5. The absorption spectra of complex 1 consist of two
resolved bands centered at 271 nm (intraligand transition)
and 325 nm (LMCT). As the DNA concentration is
increased, hyperchromism (A = 0.2223–1.4465) with a
blueshift of 10 nm (up to 261 nm) was observed in the
intraligand band. The LMCT band at 325 nm showed
modest hypochromism (A = 0.1858–0.1431) with negli-
gible shifts in the wavelength. The binding behavior of
complexes 2 and 3 is also quite similar. Complex 2
exhibited hyperchromism in the intraligand band at 269 nm
(A = 0.1945–1.5374) with 8-nm blueshift and hypochro-
mism at 325 nm (LMCT) (A = 0.1840–0.1100). The
spectra of complex 3 consist of two resolved bands cen-
tered at 273 nm (intraligand transition) and 379 nm
(LMCT). As the DNA concentration is increased, hyper-
chromism (A = 0.2835–2.2745) with a blueshift of 13 nm
was observed in the intraligand band. The LMCT band at
379 nm (A = 0.1421–0.1171) showed hypochromism with
negligible shifts in the wavelength. The observed hyper-
chromic effect with a blueshift suggested that the new
complexes bind to CT-DNA by external contact, possibly
due to electrostatic binding [67, 68].
that the ligand bound to nickel in the thiolate form. In all
three complexes, the observed Ni–N bond distance of
˚
1.8734(14)–1.885(2) A is a bit shorter compared with the
bond distances in already reported nickel(II) complexes,
which may be due to the strong p interaction between the
imine nitrogen and nickel. All other observed bond lengths
are within the range of those of already reported complexes
[58–61]. The variation in the bonding parameters indicates
that there is considerable distortion from a square planar
geometry in the NiSNOP core around nickel.
Electrochemistry
Electron transfer properties of the new Ni(II) complexes
were studied by cyclic voltammetry using dichlomethane
as a solvent, platinum wire as the working electrode, and a
platinum disc as the counter electrode and 0.1 M tetrabu-
tylammonium perchlorate as the supporting electrolyte at a
scan rate of 100 mV s-1. All potentials were referenced to
the Ag/AgCl electrode. Ferrocene was used as an external
standard. These complexes were electroactive in the
2.0 V sweep range. Complex 1 showed a quasi-reversible
oxidation at 0.503 V with peak-to-peak separation of
602 mV and reversible ligand reduction at -1.6575 V with
peak-to-peak separation of 75 mV. It also exhibited irre-
versible ligand oxidation at 0.920 V (Table 4). Complex 2
showed reversible oxidation and quasi-reversible reduction
on both sides with peak-to-peak separations of 59 and
The intrinsic binding constant Kb is a useful tool to
monitor the magnitude of the strength with which
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J Biol Inorg Chem (2013) 18:233–247
241
Table 4 Electrochemical data of the new nickel(II) complexes
Complex NiII–NiIII oxidation
NiII–NiI reduction
Ligand reduction
Epa Epc
(mV) (V) (V)
Ligand oxidation
Epa Epc E
‘
Epa Epc
(V) (V)
E
‘
(V)
DEp
Epa
(mV) (V)
Epc
(V)
E
‘
(V)
DEp
E
‘
(V)
DEp
DEp
(mV) (V) (V) (V) (mV)
1
2
3
0.202 0.804 0.503 602
0.744 0.803 0.774 59
–
–
–
–
-1.695 -1.620 -0.6575 75
0.920
–
–
–
–
–
–
–
-0.405 -0.280 -0.342 125
-0.262 -0.162 -0.212 100
-1.621 -1.076 -1.348
-1.269 -0.816 -1.042
545
453
–
–
–
–
intensity when bound intercalatively to DNA. When
complexes 1–3 were added to DNA pretreated with EB, the
DNA-induced emission intensity at 604 nm was decreased
(Fig. 7). This indicated that the complexes could replace
EB from the EB–DNA system. Such a characteristic
change is often observed in intercalative DNA interactions.
The Stern–Volmer quenching constant, KSV, obtained as
the slope of I0/I versus the concentration of the quencher
was evaluated for complexes 1–3 and was found to be
9.52 9 103, 3.35 9 103, and 8.27 9 103, respectively. A
large decrease in emission intensity suggests stronger
binding to CT-DNA [70].
DPPH radical scavenging assay
The free-radical scavenging activity of new nickel(II)
complexes 1–3 was tested by the ability to bleach the stable
radical DPPH. This assay provided information on the
reactivity of the compounds with a stable free radical.
Because of the odd electron, DPPH shows a strong
absorption band at 517 nm [14]. As this electron becomes
paired off in the presence of a free-radical scavenger, the
absorption vanishes, and the resulting decolorization is
stoichiometric with respect to the number of electrons
taken up. The new complexes exhibited a significant DPPH
radical scavenging effect greater than that of well-estab-
lished vitamin C, a reference drug established elsewhere
[71, 72]. The free-radical scavenging activity of the com-
plexes increased with increase in the concentration of the
complexes, and 59, 73, and 53.3 % activity was shown at
250 lg/ml concentration of complexes 1, 2, and 3,
respectively (Fig. 8). The observed antioxidant activity of
the nickel complex may be due to the neutralization of the
free-radical character of DPPH by transfer of either an
electron or a hydrogen atom [73]. It could also be due to
the inhibition of lipid peroxidation as indicated earlier [74].
Among the three complexes, complex 2 showed the best
activity.
Fig. 4 Cyclic voltammogram of complex 3
compounds bind with CT-DNA (Table 5). It can be
determined by monitoring the changes in the absorbance in
the intraligand band at the corresponding kmax with
increasing concentration of DNA and is given by the ratio
of slope to the y intercept in plots of [DNA]/(ea - ef)
versus [DNA] (insets in Fig. 5).
In the emission spectra, complex 1 exhibited fluores-
cence at 299 nm (Fig. 6). On addition of CT-DNA to
this complex, the fluorescence intensity decreased
(I = 177.71–83.84) without any shift in wavelength. Sim-
ilarly, complexes 2 and 3 showed hypochromism at
430 nm (I = 113.66–82.11) with a 9-nm blueshift and
at 442 nm (I = 377.70–184.61) with a 4 nm redshift,
respectively. The marked decreases in the fluorescence
intensity of the new complexes indicate the intercalative
binding mode of DNA.
To further confirm the interaction between complexes
1–3 and CT-DNA, competitive binding studies using EB (a
planar intercalating nonemissive molecule which fluoresces
intensely when bound to DNA) were conducted. The
emission intensity of EB is used as a structural probe as
EB shows reduced emission intensity in buffer solution
because of solvent quenching or because of a photoelectron
transfer mechanism [69] and enhancement of emission
MTT assay
The ligands, complexes 1–3, and the standard cisplatin
were evaluated for their cytotoxicity in two different
human tumor cell lines (A549 and HepG2) by means of a
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J Biol Inorg Chem (2013) 18:233–247
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
1.6
1.2
0.8
0.4
0.0
2.5
2.0
1.5
1.0
0.5
0.0
2.5
2.0
1.5
1.0
0.5
0.0
0.0 0.1 0.2 0.3 0.4 0.5
0.0 0.1 0.2 0.3 0.4 0.5
[DNA]
[DNA]
300
400
500
600
300
400
500
Wavelength (nm)
Wavelength (nm)
2.4
2.0
1.6
1.2
0.8
0.4
0.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0.0 0.1 0.2 0.3 0.4 0.5
[DNA]
300
400
500
600
700
Wavelength (nm)
Fig. 5 Absorption titration spectra of complexes 1–3 with increasing concentrations (0.05–0.5 lM) of calf thymus DNA (CT-DNA; phosphate
buffer, pH 7). The insets show binding isotherms with CT-DNA
complexes in the cell lines tested, and it exhibited much
better activity than cisplatin in the A549 cell line. Between
cell lines, there is a weakly discernible trend in the activ-
ities the complexes showed against the liver cancer cell
line (HepG2). By comparing the cytotoxicity with that of
the conventional standard cisplatin, we found that the
complexes exhibited excellent activity in the lung cancer
cell line (A549). In the liver cancer cell line the complexes
exhibited higher activity than cisplatin but not the maxi-
mum observed as in the lung cancer cell line. In addition,
the activities of the complexes were much higher than
those of their parent ligands.
Table 5 Binding constant for interaction of nickel(II)complexes with
calf thymus DNA (CT-DNA)
System
Kb (9105 M-1
)
CT-DNA ? 1
CT-DNA ? 2
CT-DNA ? 3
2.22343
8.79644
1.12006
colorimetric assay (MTT assay) which measures mito-
chondrial dehydrogenase activity as an indication of cell
viability, and the cells were evaluated after 48 h. All the
ligands and complexes showed activity and their corre-
sponding IC50 values, corresponding to inhibition of cancer
cell growth at the 50 % level, are shown in Fig. 9. All the
complexes have cytotoxic potencies, with IC50 values
generally in the low micromolar range. As a general
observation, complex 2 is more active than the other
LDH release
LDH is a membrane-bound enzyme and its presence is a
measure of cell membrane integrity. Leakage of LDH into
123

J Biol Inorg Chem (2013) 18:233–247
243
150
100
50
200
150
100
50
0
350
400
450
500
550
295
300
305
310
315
320
Wavelength (nm)
Wavelength (nm)
400
350
300
250
200
150
100
400
450
500
Wavelength (nm)
Fig. 6 Changes in the emission spectra of complexes 1–3 with increasing concentrations (0.05–0.5 lM) of CT-DNA (phosphate buffer, pH 7)
the culture medium is often associated with membrane
damage and therefore serves as a means to detect apopto-
sis. In this study, significant levels of LDH leakage were
observed in the cell culture medium of A549 and HepG2
cell lines when they are treated with the respective IC50
concentrations (for A549, 17 lM H2L1, 20 lM H2L2,
15 lM H2L3, 10 lM 1, 8 lM 2, 10 lM 3; for HepG2,
15 lM H2L1, 19 lM H2L2, 14 lM H2L3, 8 lM 1, 7 lM 2,
8 lM 3) for 48 h (Fig. 10). There was a significant varia-
tion in the LDH release caused by the ligands and the
complexes when comparing among them and with the
control. However, there was no appreciable difference
among the complexes on rupturing the cell membrane. The
LDH release further confirmed the higher impact of the
complexes on lung and liver cancer cell lines by comparing
them with the ligands and the control.
Nitric oxide assay
Nitric oxide has been shown to directly inhibit methionine
adenosyl transferase, leading to glutathione depletion, and its
reaction with superoxide generates the strong oxidant per-
oxynitrite, which can initiate lipid peroxidation or cause
direct inhibition of the mitochondrial respiratory chain [75].
Hence the presence of nitric oxide is also an important
measure of cytotoxicity. In this study it is obvious that the
treatment with the ligands and their complexes significantly
increased nitric oxide levels in the human lung and liver
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1.35
1.30
1.25
1.20
1.15
1.10
1.05
1.00
1.16
1.14
1.12
1.10
1.08
1.06
1.04
1.02
1.00
0.98
120
100
80
60
40
20
0
700
600
500
400
300
200
100
0
0
5
10 15 20 25 30 35 40
[Q] ( M)
μ
0
10
20
[Q] (
30
40
μ
M)
550
600
650
700
750
550
600
650
700
750
Wavelength (nm)
Wavelength (nm)
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1.0
600
500
400
300
200
100
0
0
10 20 30 40 50 60
[Q] ( M)
μ
550
600
650
700
750
Wavelength (nm)
Fig. 7 Emission spectra of ethidium bromide bound to DNA in the
presence of complexes 1–3 in tris(hydroxymethyl)aminomethane–
HCl buffer (pH 7.2). Arrows indicate the intensity changes upon
increasing the concentration of the complexes. Inset fluorescence
quenching curve of DNA-bound ethidium bromide with the
complexes
100
90
80
70
60
50
40
30
20
10
0
Fig. 9 The IC50 values (50 % inhibition of cell growth for 48 h) for
the ligands, complexes 1–3, and cisplatin. MTT 3-(4,5-dimethylthi-
azol-2-yl)-2,5-diphenyltetrazoliumbromide
0
100
200
300
Concentration of the complex (μM)
1
2
3
Ascorbic acid
cancer cell lines tested (A549 and HepG2) when compared
with the control group (Fig. 11). The results indicate the very
poor radical scavenging activity of the ligands.
Fig. 8 Diphenyl-1-picrylhydrazyl (DPPH) scavenging activity of
new Ni(II) complexes 1–3
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J Biol Inorg Chem (2013) 18:233–247
245
Fig. 11 Nanomoles of nitrite released by the human cancer cell lines
A549 and HepG2 after an incubation period of 48 h with the ligands
(for A549, 17 lM H2L1, 20 lM H2L2, 15 lM H2L3; for HepG2,
15 lM H2L1, 19 lM H2L2, 14 lM H2L3) and complexes 1–3 (for
A549, 10 lM 1, 8 lM 2, 10 lM 3; for HepG2, 8 lM 1, 7 lM 2,
8 lM 3). Error bars represent the standard error of the mean (n = 6)
Fig. 10 Percentage of lactate dehydrogenase (LDH) released by the
human cancer cell lines A549 and HepG2 after an incubation period
of 48 h with the ligands (for A549,17 lM H2L1, 20 lM H2L2, 15 lM
H2L3; for HepG2, 15 lM H2L1, 19 lM H2L2, 14 lM H2L3) and
complexes 1–3 (IC50 for A549, 10 lM 1, 8 lM 2, 10 lM 3; IC50 for
HepG2, 8 lM 1, 7 lM 2, 8 lM 3). Error bars represent the standard
error of the mean (n = 6)
Intracellular Uptake
A549
80
HepG2
70
60
50
40
30
20
10
0
Cellular uptake study
To explain the significant difference in the cytotoxicities,
uptake studies were performed in A549 and HepG2 cell
lines. The amount of compounds which were taken up by
both liver and lung carcinoma cells in the different incuba-
tion periods (4, 12, 24, and 48 h) was investigate in triplicate
as shown in Figs. 12 and 13. The intracellular concentration
of the test compounds was found using the method described
in ‘‘Materials and methods.’’ The intracellular concentration
of a specific drug is vital in the design of a therapeutic drug.
The percentage of cellular uptake was higher in complex 3,
followed by complexes 1 and 2 and then the ligands. The
lower percentage of cellular uptake of the ligands reflects the
meager uptake of the compounds into the cells. Moreover,
the intracellular uptake of the complexes was investigated in
the presence of glutathione as an antioxidant, and significant
reduction in the cellular uptake was observed. This may be
due to the displacement of the complexes by glutathione in
the cells [76]. The greater electron-withdrawing effect of the
chelating ligand in 3 increases the cellular uptake level by
increasing the lipophilic character of the central metal atom,
which subsequently favors permeation through the lipid
layer of the cell membrane [77]. Moreover, it was found that
the cytotoxicity determined by MTT assay was not dispro-
portionately influenced by the complexes having different
cellular uptake levels.
1
H2L1
2
H2L2
3
H2L3
Fig. 12 Percentage of intracellular uptake of the ligands (for A549,
17 lM H2L1, 20 lM H2L2, 15 lM H2L3; for HepG2, 15 lM H2L1,
19 lM H2L2, 14 lM H2L3) and complexes 1–3 (for A549, 10 lM 1,
8 lM 2, 10 lM 3: for HepG2, 8 lM 1, 7 lM 2, 8 lM 3) by human
cancer cell lines A549 and HepG2 after an incubation period of 4 h.
Error bars represent the standard error of the mean (n = 6)
and X-ray crystallographic techniques. From the X-ray
analysis, it was found that in complexes 2 and 3, the ligand
coordinated as a dibasic tridentate donor by forming a
stable five-membered ring and a six-membered ring
through phenolic oxygen, azomethine nitrogen, and thio-
late sulfur atoms. However, in complex 1, the ligand
coordinated as a monobasic tridentate donor with phenolic
oxygen, azomethine nitrogen, and thione sulfur atoms. The
new complexes were subjected to studies of their potential
biological properties such as cytotoxicity (MTT assay,
LDH release, and nitric oxide assay) and cellular uptake
along with free-radical scavenging and DNA binding
studies. The complexes exhibited better binding with DNA,
which was inferred from the greater magnitude of the
binding constants. The cytotoxicity of the complexes in
MTT assay showed that the complexes exhibited higher
activity than their parent ligands and the conventional
standard cisplatin in both cell lines used for the investi-
gation. Correlating the activity of the complexes with their
Conclusion
The newly synthesized nickel(II) thiosemicarbazones were
structurally characterized by various analytical, spectral,
123

246
J Biol Inorg Chem (2013) 18:233–247
Fig. 13 Percentage of
intracellular uptake of
Intracellular uptake
80
70
60
50
40
30
20
10
0
complexes 1–3 (for A549,
10 lM 1, 8 lM 2, 10 lM 3; for
HepG2, 8 lM 1, 7 lM 2, 8 lM
3) by human cancer cell lines
A549 and HepG2 after
incubation periods of 4, 12, 24,
and 48 h. Error bars represent
the standard error of the mean
(n = 6)
A5491
A5492
4h
A5493
24h
HepG21
48h
HepG22
HepG23
12h
4h+GSH
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(Tokyo) 40:195–200
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structures, we found that the increase in electron density on
the metal increases the degree of cytotoxicity. In vivo
cytotoxicity studies are under way to explore further the
biological activities of the complexes.
Supplementary material
12. Campbell MJ (1975) Coord Chem Rev 15:279–319
13. Prabhakaran R, Huang R, Karvembu R, Jayabalakrishnan C,
Natarajan K (2007) Inorg Chim Acta 360:691–694
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Kalaivani P, Karvembu R, Dharmaraj N, Bertagnolli H, Natarajan
K (2011) Spectrochim Acta 78:844–853
Crystallographic data for 1, 2, and 3 have been deposited
with the Cambridge Crystallographic Data Centre (CCDC)
as supplementary publication nos. CCDC-678416, CCDC-
678415, and CCDC-678417. The data can be obtained
free of charge from http://www.ccdc.cam.ac.uk/conts/
retrieving.html or from the CCDC (12 Union Road, Cam-
bridge CB2 1EZ, UK; Fax: ?44-1223-336033; e-mail:
deposit@ccdc.cam.ac.uk).
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Acknowledgments The authors gratefully acknowledge the Council
of Scientific and Industrial Research (CSIR), New Delhi, India, and
Department of Science and Technology (DST), India, for financial
assistance. The reviewers are thanked for their valuable comments
and suggestions enabling us to present this article in its current form.
19. Prabhakaran R, Kalaivani P, Poornima P, Dallemer F, Paramag-
uru G, Vijaya Padma V, Renganathan R, Natarajan K (2012)
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