Association of late transition metal complexes with ethyl 2-(2-(4-chlorophenylcarbamothioyl)hydrazono)propanoate: Design, synthesis and in vitro anticancer studies

Article abstract of DOI:10.1016/j.ica.2015.03.013

Abstract A newly synthesized chelating agent ethyl 2-(2-(4-chlorophenylcarbamothioyl)hydrazono)propanoate with SNO donor sites is employed for the synthesis of Co(II), Ni(II), Cu(II) and Zn(II) complexes. Structure of ligand is determined by single crysta

Full text of DOI:10.1016/j.ica.2015.03.013

Inorganica Chimica Acta 430 (2015) 216–224
Contents lists available at ScienceDirect
Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Association of late transition metal complexes with ethyl
2-(2-(4-chlorophenylcarbamothioyl)hydrazono)propanoate:
Design, synthesis and in vitro anticancer studies
Aishakhanam H. Pathan a, Asha K. Ramesh b, Raghavendra P. Bakale a, Ganesh N. Naik a, H.G. Rohit Kumar b,
Christopher S. Frampton c, Gopal M. Advi Rao b, Kalagouda B. Gudasi a,
⇑
a Department of Chemistry, Karnatak University, Dharwad 580 003, Karnataka, India
b Department of Biochemistry, Davangere University, Davangere 577002, Karnataka, India
c SAFC Pharmorphix, A Sigma-Aldrich Company, 250 Cambridge Science Park, Milton Road, Cambridge CB4 0WE, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
A newly synthesized chelating agent ethyl 2-(2-(4-chlorophenylcarbamothioyl)hydrazono)propanoate
with SNO donor sites is employed for the synthesis of Co(II), Ni(II), Cu(II) and Zn(II) complexes.
Structure of ligand is determined by single crystal X-ray diffraction analysis. Octahedral geometry is
assigned to all the complexes based on the physical and spectral data. The copper complex has shown
redox responses in the applied voltage range, whereas the ligand and other complexes are electrochemi-
cally inactive. The new complexes synthesized have been evaluated for in vitro antiproliferative activity
against human cancer cells of different origin such as COLO-205 and K-562. Among the compounds tested
Received 15 October 2014
Received in revised form 24 December 2014
Accepted 5 March 2015
Available online 20 March 2015
Keywords:
Ethyl pyruvate
Thiosemicarbazone
Metal complexes
X-ray structure
Apoptosis
for their antiproliferative activity, IC50 value of copper complex is 15.16 and 8.65
lM for K-562 and COLO-
205, respectively. Time dependent significant fall in mitochondrial membrane potential (
DWm) without
generating reactive oxygen species (ROS) is observed in COLO-205 cells treated with copper complex,
whereas its antiproliferative effect mediated by the induction of apoptotic cell death is evidenced by
the sub-G0/G1 cells accumulation, oligonucleosomal DNA fragmentation, caspase-3 activation and the
nuclear condensation. Further results confirmed subsequent apoptosis induction through activation of
intrinsic apoptotic pathway.
Caspase-3 activity
Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction
Thiosemicarbazone of ethyl pyruvate (EP) derivatives have
been a subject of interest to researchers of different profiles
Metal based drugs such as organoplatinum compounds are
extensively used in cancer chemotherapy [1–3]. The clinical utility
of these compounds is limited to a relatively narrow range of
tumors (sarcomas, small cell lung cancer, ovarian cancer, lym-
phomas and germ cell tumors) [4,5], by dose-limiting side effect
and acquired resistance due to prolonged administration. To cir-
cumvent these problems, much attention has been focused on
designing unconventional metallo-organic compounds which
greatly facilitate the interaction of the biomolecules in biological
systems [6–9]. Recently it is shown that cupredoxin azurin
preferentially enter cancer cells rather than normal cells and form
complex with the tumor suppressor protein PS3, stabilizing it and
increasing its intracellular level, thereby inducing apoptosis via
corpus mediated mitochondrial pathway [10].
including pharmacologists and coordination chemists [11–15].
These derivatives have shown promising antitumor activity
against various cancer cell lines [16,17]. Their biological activities
are explained on the basis of various mechanisms including
intercalation properties, inhibition of DNA and RNA, breaking of
DNA strands, alteration of cell membrane functions and free radi-
cal mediated alkylation [18,19]. Thus, a combination of such a
small molecule (EP) with potent cytotoxic thiosemicarbazide
pharmacophore may act in an additive or synergistic mode,
showing effect on the DNA-binding properties of the parental
moiety. Metallo-organic compounds of such derivatives tend to
be strongly mutagenic and have shown promising chemothera-
peutic activities. Elevated intracellular metal levels in particular
copper levels predispose cancerous cells to ionophoric copper
sensitivity. Several copper coordinating lipophilic thiosemicar-
bazone compounds are being investigated as potential anticancer
therapeutics [20].
⇑
Corresponding author. Tel.: +91 836 2215286 (O); fax: +91 836 2771275.
E-mail address: kbgudasi@gmail.com (K.B. Gudasi).
http://dx.doi.org/10.1016/j.ica.2015.03.013
0020-1693/Ó 2015 Elsevier B.V. All rights reserved.

A.H. Pathan et al. / Inorganica Chimica Acta 430 (2015) 216–224
217
Therefore, in the present work, as a part of our search for new
molecules with anticancer activity, we have focused our attention
toward the synthesis of ethyl pyruvate derived thiosemicarbazone
ligand (E)-ethyl 2-(2-(4-chlorophenylcarbamothioyl)hydrazono)-
propanoate and its cobalt (CoL2), nickel (NiL2), copper (CuL) and zinc
(ZnL2) complexes. These were characterized thoroughly and
systematically tested for their anticancer activity. Among all the
complexes tested, copper complex was found to be more active on
colon adenocarcinoma. Using varied lines of experimental strate-
gies, it is shown that copper complex induces cell death through
apoptotic mode. Thus the present results might open a new insight
in the design of a new type of metal based therapeutic agent.
Ni(II), Cu(II) and Zn(II) (1 mmol) in methanol (5 mL) and the mix-
ture was stirred at room temperature for 4 h. The precipitates
obtained were filtered, washed thoroughly with methanol, then
with ether and dried in vacuo. Attempts to grow single crystals
were unsuccessful. The proposed structures of the complexes are
given in Scheme 2.
2.1.2.1. C24H26Cl2N6O4S2Zn (ZnL2). Anal. Calc. C, 43.48; H, 3.95; Cl,
10.70; N, 12.68; O, 9.65; S, 9.67; Zn, 9.86%. Found: C, 43.47; H,
3.95; Cl, 10.76; N, 12.65; S, 9.69; Zn, 9.80%. IR (cmÀ1). NH; 3325,
Phenyl NH; 1651, C@O; 1593, C@N. 1H NMR (DMSO-d6) d: 10.23 (s,
1H, NH phenyl), 4.21–4.24 (m, 2H, CH2), 2.17 (s, 3H, N@C@CH3),
1.26 (t, 3H, CH3), Molar conductivity (ohmÀ1 cm2 molÀ1): 21.33. leff.
Diamagnetic.
2. Experimental protocols
2.1.2.2. C24H26Cl2CoN6O4S2 (CoL2). Anal. Calc. C, 43.91; H, 3.99; Cl,
10.80; Co, 8.98; N, 12.80; S, 9.77%. Found: C, 43.93; H, 3.97; Cl,
10.80; Co, 9.05; N, 12.77; S, 9.80%. IR (cmÀ1). 3303, Phenyl NH;
1652, C@O; 1590, C@N. Molar conductivity (ohmÀ1 cm2 molÀ1):
3.78. leff. 4.33 BM.
2.1. Materials and methods
All the reagents are commercially available (Aldrich or Merck)
and used as supplied. Solvents were purified and dried according
to standard procedures. Thiosemicarbazone was prepared accord-
ing to the earlier reports with slight modifications [21]. Metal chlo-
rides used were in the hydrated form. Metal content was
determined according to the standard methods. Melting points
(m.p.) were determined in open capillaries and are uncorrected.
The molar conductivity was measured on ELICO-CM-82
Conductivity Bridge. Infrared spectra were recorded in KBr discs
in the region 4000–400 cmÀ1 on a Nicolet 170 SX FT-IR spectropho-
tometer. 1H and 13C spectra of the ligand were recorded in
(CD3)2SO on a BRUKER 500 MHz spectrometer. All the compounds
were analyzed for Carbon, Hydrogen, Nitrogen and Sulfur by
Thermo quest elemental analyzer. Electronic spectra of all the
compounds in DMSO were recorded on a UV–Vis spectrophotome-
ter (Varian Cary 50 Bio). Magnetic susceptibilities at room
2.1.2.3. C24H26Cl2N6NiO4S2 (NiL2). Anal. Calc. C, 43.93; H, 3.99; Cl,
10.81; N, 12.81; Ni, 8.94; S, 9.77%. Found: C, 43.91; H, 4.02; Cl,
10.82; N, 12.79; Ni, 9.01; S, 9.81%. IR (cmÀ1). 3314, Phenyl NH;
1654, C@O; 1590, C@N. Molar conductivity (ohmÀ1 cm2 molÀ1):
6.93. leff. 3.12 BM.
2.1.2.4. C12H19Cl2CuN3O5S (CuL). Anal. Calc. C, 31.90; H, 4.24; Cl,
15.69; Cu, 14.06; N, 9.30; S, 7.10%. Found: C, 31.96; H, 4.26; Cl,
15.74; Cu, 14.01; N, 9.59; S, 7.10%. IR (cmÀ1). 3310, Phenyl NH;
1639, C@O; 1594, C@N. kmax (nm), 440–460, 670. kmax (nm): 306
and 367. Molar conductivity (ohmÀ1 cm2 molÀ1): 19.78. leff.
1.86 BM.
temperature were determined using
a Gouy balance with
2.2. Crystal structure analysis of ethyl 2-(2-(4-
chlorophenylcarbamothioyl)hydrazono)propanoate [LH]
Hg[Co(SCN)4] as the reference compound and magnetic moments
were calculated making the necessary diamagnetic corrections.
Thermal analysis of the metal complexes were carried out in nitro-
gen atmosphere on universal V2.4F TA Instrument keeping final
temperature at 1000 °C with a heating rate of 10 °C/min. The cyclic
voltammetric experiments were carried out with a three electrode
A selected crystal of LH was mounted on the tip of a 200 lm
Mitagen loop with perfluorinated oil and cooled rapidly to 100 K
in a stream of cold nitrogen. Data were collected on an Oxford
Diffraction, (Agilent Technologies), SuperNova X-ray diffractome-
ter equipped with an Oxford Cryosystems Cobra Low temperature
apparatus using
a CHI1110A electrochemical analyzer (USA).
The EPR spectra of copper complex were recorded on a Varian
E-4 X-band spectrometer using TCNE as g-marker.
device using Cu K
a radiation (a = 154.178 pm) from a SuperNova
Cu X-ray micro source and focusing mirror optics. The structures
were solved by direct methods and refined against F2 by full-
matrix least-squares using the program SHELXTL [23]. Full data col-
lection and refinement details are given in Table 1.
2.1.1. Preparation of ethyl 2-(2-(4-
chlorophenylcarbamothioyl)hydrazono)propanoate (LH)
The ligand (LH) was prepared in two steps. First, 4-(4-
chlorophenyl)thiosemicarbazide was obtained and purified by
the literature method [22]. It (0.201 g, 1 mmol) was then refluxed
with methanolic solution (20 mL) of ethyl pyruvate (0.116 g,
1 mmol) for 2 h. Partial evaporation of the solvent yielded crystals
suitable for X-ray single crystal analysis which were filtered off,
washed with methanol and dried in vacuum (Scheme 1).
Yield: 86%; mp: 133–135 °C.
C12H14ClN3O2S: Anal. Calc. C, 48.07; H, 4.71; N, 14.02; S, 10.70%.
Found: C, 48.10; H, 4.77; N, 13.98; S, 10.73%. IR (cmÀ1). 3249,
Hydrazine NH; 3290, Phenyl NH; 1685, C@O; 1627, C@N. 1H
NMR (DMSO-d6) ppm: 11.01 (s, 1H, Hydrazine NH), 10.00 (s, 1H,
NH phenyl), 4.20–4.23 (m, J = 5, 2H, CH2), 2.17 (s, 3H, N@C@CH3),
1.26 (t, J = 5, 3H, CH3), 13C NMR (DMSO-d6) d: 177.71 (C@S),
164.14 (C@O), 140.07 (C@N), 61.25 (CH2), 14.02 (CH3).
2.3. Cell lines and culture conditions
Human chronic myelogenous leukemia (K-562) was obtained
from Prof. Kondaiah, Indian Institute of Science (Bangalore,
India). Colon adenocarcinoma (COLO-205) and non-tumor cell line
(VERO, renal, green monkey) was purchased from National Centre
for Cell Science (Pune, India). K-562 and COLO-205 cell lines were
cultured in RPMI-1640 supplemented with 10% FCS (Invitrogen,
USA), penicillin G (100 IU/mL) and streptomycin (100 lg/mL) at
37 °C in a humidified 5% CO2 atmosphere. VERO cell lines were
maintained in MEM medium.
2.4. Preparation of working stock
Solutions were freshly prepared by dissolving the prepared
compounds in DMSO to a concentration of 50 mM and further
diluted with RPMI-1640 for the experiments. Each experiment
contained two controls: cell control (untreated cells) and
2.1.2. Preparation of the complexes
To a hot solution of LH (1 mmol) in methanol (20 mL) was
slowly added, a hot solution of the metal chlorides Viz: Co(II),

218
A.H. Pathan et al. / Inorganica Chimica Acta 430 (2015) 216–224
H
N
H
N
O
NH2
NH3, CS2, NH2NH2.H2O
EtOH, Sodium chloroacetate
NH2
O
H3C
+
S
Cl
4-Chloro anilline
Cl
O
4-Chlorothiosemicarbazide
Ethyl pyruvate
CH3
H
N
H
N
O
N
S
O
Cl
ethyl 2-(2-(4-chlorphenylcarbamothioyl)hydrazono)propanoate(LH)
Scheme 1. Synthetic route for the preparation of ligand LH.
CH3
H
N
N
O
CH3
N
H
N
N
O
S
O
S
N
Cl
M
.H2O
Cl
S
O
O
Cu
Cl
Cl
N
OH2
OH2
O
N
N
H
CH3
M=Co(II), Ni(II), Zn(II)
Scheme 2. Proposed structures of the complexes.
additional control (cells treated with maximal concentration of
used DMSO solution-less than 0.01%).
Table 1
Crystal data and crystal refinements of LH.
2.5. In vitro cytotoxicity evaluation
Crystal data
LH
Empirical formula
Formula mass
Crystal size (mm)
Crystal system
Space group
a (pm)
C
12H14ClN3O2S
2.5.1. MTT assay
299.77
MTT assay was carried out as described earlier [24,25], with
slight modifications. Briefly, exponentially growing cells were
seeded at a density of 5 Â 104 cells/mL, incubated for 24 h and
treated with various concentrations of CoL2, CuL and NiL2 (0.5–
0.40 Â 0.40 Â 0.20
monoclinic
P21/c
1063.99(4)
1256.02(4)
1073.88(5)
106.142(4)
1378.55(9)
4
b (pm)
c (pm)
250
added and incubated for 4 h. Formazan crystals formed were dis-
solved by adding 100 L of DMSO and absorbance were measured
lM). After 48 h of incubation, MTT reagent (1 mg/mL) was
b (°)
V (pm3 Â 106)
Z
l
at 570 nm using microplate reader (Bio-Rad, USA). A graph of con-
centration versus percentage cell viability was plotted and IC50 val-
ues were calculated.
Dcalc (g cmÀ3
l
)
1.444
0.430
100
(cmÀ1) (Mo K
a)
T (K)
2h(max) (°)
52.74
Index range
2.5.2. LDH release assay
h
k
l
À13 ? 9
À12 ? 15
À13 ? 12
6196
2808
2480
182
0.0290
0.0774
0.339, À0.244
Release of lactate dehydrogenase (LDH) is an indicator of loss of
membrane integrity and hence cell injury. This assay was designed
to estimate the number of nonviable cells present in a mixed pop-
ulation of living and dead cells, using standard protocols [26].
Reflections collected
Unique reflections (Rint
Reflections with Fo > 4
Number of parameters
)
r(Fo)
5 Â 104 cells/mL were seeded and treated with 20
lM of copper
complex (CuL) for 24, 48 and 72 h. The percentage of LDH released
upon treatment was analyzed. The percentage of LDH released
was calculated as: (LDH activity in media) = (LDH activity in
media + intracellular LDH activity) Â 100.
R1
wR2 (all data)
Maximum and minimum residual density
((e pmÀ3) Â 10À6
)

A.H. Pathan et al. / Inorganica Chimica Acta 430 (2015) 216–224
219
2.6. Measurement of mitochondrial membrane potential (
intracellular ROS
D
W
m) and
2.10. Cell cycle analysis
Cells were cultured and treated with the different concentration
of CuL (5, 10, and 20 M). After 48 h of treatment, cells were har-
The effect of CuL treatment on mitochondrial membrane poten-
tial was measured using JC-1 dye (Molecular Probes, USA) as per
standard protocol [27]. Cellular ROS levels were evaluated with
the redox sensitive dye CM-H2DCFDA (Molecular Probes, USA).
For both the experiments, 5 Â 104 cells/mL were treated with
l
vested, washed and fixed at 4 °C in 70% ethanol overnight. Fixed
cells were washed and incubated at 37 °C with RNase (20 mg/
mL) for 4 to 5 h. After RNase treatment, propidium iodide (PI,
1 mg/mL) was added and incubated in dark at room temperature
for 30 min before acquiring the flow cytometric reading (FACS cal-
ibur using Cell Quest Pro software, BD Biosciences, USA). The per-
centage of cells in sub-G0/G1, G0/G1, S and G2/M phase were
determined. At least 10000 cells were sorted per FACS experiment.
15 lM concentration of CuL and incubated for different time peri-
ods at 37 °C. After respective treatment period, cells were washed
and labeled for 10 min with dye at 37 °C. Cells were then washed,
resuspended in PBS, and fluorescence were measured using fluo-
rescence spectrophotometer (Varian, The Netherlands) [27]. JC-1
was excited at 490 nm, and emission was monitored at 530 and
590 nm. CM-H2DCFDA fluorescence emission was measured at
530 nm by exciting at 490 nm.
2.11. Qualitative assessment of apoptosis by performing confocal
imaging
Annexin V/PI labeling assay was performed as per the manufac-
turer instructions (Invitrogen, USA). Briefly, 1 Â 106 cells were
2.7. Measurement of caspase-3 activity
treated with CuL (20 lM) for 48 h, washed with ice cold PBS and
resuspended in binding buffer. Then the cells were stained with
The activity of caspase-3 in cell lysates was performed using
caspase-3 colorimetric assay kit (Sigma, USA), following the manu-
facturer instructions. 1 Â 106 cells/mL were treated with CuL
annexin V conjugated to FITC (25 g/mL) and PI (100 g/mL) for
l
l
15 min in dark. Subsequently, cells were washed and resuspended
in annexin V binding buffer, spread over a slide and observed
under an inverted confocal laser scanning microscope (LSM 510
META; Zeiss). Imaging was performed to visualize the early and
late apoptotic cells.
(5 lM) for 48 h and washed with PBS. Thereafter, pellet was sus-
pended in lysis buffer and incubated on ice for 15 min, followed
by centrifugation at 20000g for 15 min at 4 °C. The supernatant
was collected and samples were mixed with assay buffer with or
without caspase-3 inhibitor (Ac-DEVD-CHO). Finally, caspase-3
substrate (Ac-DEVD-pNA) was added to all the samples and incu-
bated at 37 °C for 90 min. Absorbance was recorded at 405 nm.
Units of caspase-3 were determined from a standard curve.
2.12. Statistical analysis
All the results have been expressed as the mean S.E. for data
obtained from at least three separate experiments. Data were ana-
lyzed for statistical significance by the paired t test and p < 0.05 val-
ues were considered to indicate statistically significant differences.
2.8. DNA fragmentation assay
The DNA fragmentation assay was performed as described pre-
3. Results and discussion
viously with slight modifications [28]. 5 Â 105 cells per well were
cultured for 48 h with 5 and 25 lM of CuL. The cells were washed
3.1. Molar conductivity measurements
twice with PBS (4 °C, pH 7.4) and collected by centrifugation at
250g for 5 min. The pellet was then treated with 0.5 mL of lysis
buffer (1 M Tris–HCl, pH 7.4, 0.5 M EDTA, 10% sucrose) for over-
night at room temperature. Supernatants were collected, treated
Molar conductance values of complexes in DMSO at concentra-
tion 10À3 M fall in the range 1.29–21.33 mho cm2 molÀ1. These
values are much less than that expected for 1:1 electrolytes
(65–90 mho cm2 molÀ1) and hence are non-electrolytic in nature
[30]. The analytical and physicochemical data of the complexes
are summarized in experimental section.
with 20% SDS and 100 lg/mL RNase, and incubated at 56 °C for
8 h. After incubation, Proteinase K was added and further incu-
bated over night at 56 °C. DNA was precipitated using 10 M ammo-
nium acetate and absolute alcohol, stored at À80 °C over night, and
washed with 70% ethanol at 13000 rpm. DNA was then collected
and dissolved in TE buffer (10 mM Tris pH 8.0, EDTA 1 mM).
Purity of extracted DNA was checked by performing dipheny-
3.2. IR spectral studies
Comparison of the IR spectra of the metal complexes with that
of the free ligand revealed the tridentate behavior of the ligand
coordinating through >C@O, >C@N and >C@S groups. As there are
two five-membered coordination rings adjacent to each other, it
lamine method. For analysis, 10–20 lL of DNA was loaded on a
1.5% agarose gel containing 10 mg/mL ethidium bromide.
Electrophoresis was performed in 0.5Â Tris borate–EDTA buffer
(pH 8.0) at 100 V for 1 h, and fragmented DNA was visualized
and photographed using gel documentation system (DNR, Israel).
would impose considerable strain. The negative shift of
m(>C@O)
and (>C@N) bonds of the ligand on coordination suggests its
m
association with metal ions through carbonyl oxygen and azome-
thine nitrogen. The bands observed for free LH at 3290 and
3249 cmÀ1 may be assigned to the symmetric vibrations of the
2.9. Acridine orange–ethidium bromide (AO/EB) double staining
The morphological changes in COLO-205 cells upon treatment
with CuL due to apoptosis were detected by performing fluores-
cence microscopy using acridine orange (AO) and ethidium
bromide (EB) staining [29]. Briefly, 1 Â 104 cells/mL were seeded
on to cover slip, and treated with CuL for 48 h. After treatment,
cells were washed with PBS and stained with AO/EB (working
m
(NH). The absence of a band at 2500 cmÀ1 indicates the thioketo
nature of free LH in the solid state [31]. The
m(>C@S) band,
observed at 752 cmÀ1 in the spectrum of the free LH, shifts by
60 67 cmÀ1 to lower wave numbers in the complexes as a result
of enolization and consequent coordination to the metal through
the deprotonated thiolate [21]. This is also supported by the
concentration of 10
lg/mL) dye for 10 min. The cells were then
disappearance of the band at 3249 cmÀ1 assigned to
m(NH) in the
washed twice with PBS, mounted and immediately observed under
spectrum of ligand. Thus the ligand apparently coordinates
fluorescence microscope (Olympus, Japan) using blue filter.
through the N, O and S atoms.

220
A.H. Pathan et al. / Inorganica Chimica Acta 430 (2015) 216–224
3.3. NMR spectral studies
3.6. Electrochemical studies
The ligand LH displays two sharp singlets at 11.01 and
10.00 ppm attributed to the thiohydrazinic and phenyl NH, respec-
tively. The first signal disappeared on complexation with Zn(II) ion,
indicating the thioenolisation and subsequent coordination of sul-
fur via deprotonation. Further no peak corresponding to @SH was
observed in the spectrum indicating the sulfur coordination
through deprotonation. The aromatic protons observed in the
region 7.44–7.64 ppm show small shifts on coordination, due to
variation in electron density and steric constraints brought about
by chelation. A multiplet at 4.21 and a triplet at 1.26 ppm are
assigned to CH2 and CH3 protons of ester, respectively. The CH3
proton signal observed at 2.17 ppm in ligand shifts downfield
due to the involvement of the adjacent nitrogen of azomethine
group >C@N in bonding with the Zn(II) ion, as a result of which
the CH3 protons become less shielded.
The redox activity of ligand and its complexes were studied in
the range +0.2 to À0.8 V versus the ferrocene–ferrocenium redox
system in DMSO (0.1 M tetrabutylammonium bromide supporting
electrolyte). No peak corresponding to the reduction has been
observed down to À0.8 V, as the ligand is shown to be electro-
chemically inactive in the potential range studied. Further the
nickel, cobalt and zinc complexes were also found to be electro-
chemically inactive. The analysis of cyclic voltammetric responses
of copper complex with the scan rate varying from 0.05 to
0.15 vsÀ1 gives the evidence for a quasi-reversible one electron
redox process. At a scan rate of 0.05 vsÀ1, the copper complex exhi-
bits a pair of cathodic and anodic peak potentials at À0.043 V and
+0.305 V, respectively, representing the Cu(II)/Cu(I) couple. The
negative shift of
DEp > 59 mV (DEp is the difference in the anodic
and cathodic peak potentials) and Epc (cathodic peak potential)
with increasing scan rate suggest a quasi-reversible nature of the
system [38].
13C NMR spectrum of ligand show a signal at 177.71 ppm corre-
sponds to the thioamide carbon (HN@C@S). The resonances at
164.14 and 140.07 are assigned to >C@N and >C@O, respectively,
the methylene and methyl carbon signals of ester group are
observed at 61.25 and 14.02, respectively. The 13C NMR spectrum
of Zn(II) complex showed a downfield shift of signal corresponding
to carbonyl, azomethine and thioamide carbon suggesting the
coordination through carbonyl oxygen, azomethine nitrogen and
thioamide sulfur.
3.7. Thermal studies
Thermogravimetric analysis of the complexes has been carried
out in nitrogen atmosphere with a heating rate of 10 °C/min. The
thermogram of [CuLCl(H2O)2]*H2O complex exhibits several ther-
mal events. The first weight loss of 3.81% (cal 3.98%) in the tem-
perature range of 60–100 °C corresponds to the loss of a lattice
water molecule. The second weight loss of 8.12% (cal 7.96%) in
160–230 °C corresponds to two coordinated water molecules. The
corresponding DTA peak at 190 °C in the complex signifies the
endothermic process. Further decomposition of 74.81% (73.70%)
above 250 °C corresponds to the combined loss of coordinated
chloride and the ligand. Whereas the thermograms of Co(II) and
Ni(II) shows no weight loss in the region 30–200 °C indicating
the absence of lattice or coordinated water molecules. Weight loss
of 87.4% and 89.16%, in the temperature range 200–400 °C, is due
to the loss of ligand in the respective metal complexes. The plateau
obtained above 700 °C corresponds to the formation of stable
respective metal oxides.
3.4. Mass spectral studies
The mass spectrum of the ligand shows a molecular ion peak
m/z at 299 (M+) corresponds to the molecular weight of the ligand
and some prominent signals corresponding to its various frag-
ments and isotopes.
3.5. Magnetic properties, electronic and EPR spectral studies
The electronic spectrum of ligands show bands around 276, 310
and 360 nm. The first band ꢀ276 nm is assigned to a ligand
p ?
p⁄
transition, while the band around 300–310 nm to n ? p⁄ transition
associated with imine function of thiosemicarbazone. This
band shifts to lower energies upon complexation. Another band
observed at 330–360 nm region is assigned to n ? p⁄ transition
originating from the thioamide function of thiosemicarbazone
[32].
3.8. Crystallographic studies of LH
The ligand LH (CCDC 978614) crystallized as yellow colored
crystals in monoclinic crystal system having space group C2/c
with a = 10.639 Å, b = 12.5602 Å, c = 10.7388 Å,
a = c = 90.00°,
b = 106.142°, V = 1378.55 Å3 and Z = 4.00. Details of the data collec-
tion and refinements are given in Table 1. Selected bond lengths,
bond angles and torsion angles are given in Tables 2–4, respec-
tively. The ORTEP diagram is shown in Fig. 1.
The UV–Vis spectrum of Cu(II) complex exhibits bands around
455 nm and 670 nm attributable to C.T. transition of S ? Cu(II)
[33] and the d–d transition, 2E ? 2T2, respectively, corresponding
to octahedral structure [33]. The magnetic moment of 1.86 BM
for Cu(II) supports octahedral structure for the complex [34]. The
broad isotropic peak observed with giso 2.01 and 2.03 in the X-band
EPR spectra at RT and LNT suggest the absence of spin–spin inter-
action in the complex [21]. In the electronic spectrum of nickel
complex, three d–d bands were observed at 913, 617, and
The bond angles around C8, viz. N3–C8–C9 = 117.31(13)°, N3–
C8–C10 = 123.38(13)°, C9–C8–C10 = 119.31(13)° clearly reveals
the sp2 hybridized state of C8. The bond angles around N2 viz.,
N3–N2–C7 = 120.10(13)°, N3–N2–H2B = 119.3(13)°, and C7–N2–
371 nm attributable to spin-allowed transitions 3A2g ? 3T2g
,
3A2g ? 3T1g and 3A2g ? 3T1g (P) respectively, representing octahe-
dral geometry for the complex [35]; the magnetic moment
3.12 BM obtained for the complex suggest the same [33]. The elec-
tronic spectrum of Co(II) complex shows absorption at ꢀ370 nm
and is assigned to LMCT transition [36]. The peaks around 438–
Table 2
Selected bond lengths (Å) of LH.
Atoms
Bond distances (Å)
Atoms
Bond distances (Å)
Cl1–C4
S1–C7
O1–C10
N1–C7
N1–C1
N2–N3
N3–C8
C1–C6
C1–C2
1.7414(15)
1.6720(15)
1.2182(18)
1.3440(19)
1.4157(18)
1.3629(17)
1.2893(19)
1.390(2)
C8–C9
C8–C10
C9–H9A
C9–H9B
C11–C12
C11–H11A
C12–H12A
C12–H12B
C12–H12C
1.498(2)
1.500(2)
0.9800
0.9800
1.506(2)
0.9900
0.9800
0.9800
0.9800
4
450 and 640–670 nm were attributed to T1g(F) ? 4T1g (P) and
4T1g ? 4A2g assigning an octahedral structure for the complex with
the magnetic moment value 4.33 BM [21,37]. The diamagnetic zinc
complexes show absorptions only in the higher frequency region
with high extinction coefficient values attributed to the ligand
electron transitions.
1.395(2)

A.H. Pathan et al. / Inorganica Chimica Acta 430 (2015) 216–224
221
Table 3
antitumor activity compared to ligand alone (Table 5). Also copper
alone did not induced any significant cytotoxicity on the cell lines
tested. The enhanced activity of copper complex is mainly attribu-
ted to redox active metal ion and the ligand attached. Interestingly,
cytotoxicity of these molecules on a non tumor cell line (VERO cell
Selected bond angles of LH (°).
Atoms
Bond angles (°)
Atoms
Bond angles (°)
C10–O2–C11
C7–N1–C1
C7–N1–H1A
C1–N1–H1A
N3–N2–C7
C7–N2–H2B
C8–N3–N2
C6–C1–C2
115.86(11)
130.32(13)
113.5(13)
116.0(13)
120.10(13)
120.6(13)
120.22(13)
119.83(13)
123.94(13)
C2–C1–N1
C3–C2–C1
116.18(13)
120.46(14)
124.25(14)
124.24(13)
110.3
O1–C10–O2
O1–C10–C8
O2–C11–H11A
O2–C11–H11B
N1–C7–S1
line) showed IC50 value higher than 250 lM, which might suggest
these compounds have a low toxicity to normal cells.
LDH is a cytoplasmic enzyme that is constantly expressed in all
cells, enables its use in assessing plasma membrane integrity by
determining the amount of LDH in extracellular space [40]. LDH
release assay was performed as a means of cytotoxicity in terms
of cellular damage on CuL treated COLO-205 cells. A significant
(p < 0.01) time dependent increase in the release of LDH into the
surrounding media was noticed upon treatment with CuL
110.3
127.51(12)
106.93(12)
118.00(11)
O2–C11–C12
N2–C7–S1
C6–C1–N1
H2B = 120.6(13)° also illustrate the sp2 hybridized state of N2. The
bond distance of 1.3629(17) Å, 1.3687(19) Å confirms the single
bond character of the N2–N3 and N2–C7 linkage. The C–C bond
distances and C–C–C bond angles of phenyl ring are in accordance
with the values reported in literature [21,39,40]. The bond dis-
tances C@O (C2@O1@1.231 Å) and C@N (C8@N3@1.2893(19) Å)
are in conformity with the double bond character. The torsional
angle À178.25(13)° exhibited by N3–C8–C10–O2 and 2.0(2)° by
N3–C8–C10–O1 indicates that N3 and O2 atom are trans to one
another and N3 and O1 are cis to each other. The torsional angle
for N3–N2–C7–S1 = 174.31(10)° clearly shows that S1 and N3 are
trans to each other. The molecular packing diagram shows two lay-
ers of molecules. In each molecule, presence of intra molecular
hydrogen bonding stabilizes the crystal packing. The two strong
intramolecular hydrogen bonds between N1H and N3 [N1–
H. . .N3 = 2.171(18) Å] and N2H and O1 [N2–H2B. . .O1 = 1.97(2) Å]
are also present. It gives a significant contribution to the planarity
of the thiosemicarbazide moiety.
(20 lM) compared to control (Fig. 2b). This may be due to intense
cellular damage caused by CuL complex upon treatment. Results
from LDH assay are consistent with cytotoxic MTT assay results
and confirm the potential cytotoxic effect of CuL complex.
3.10. Copper complex alters
DWm without generating ROS
To supplement the encouraging cytotoxicity results of CuL, it
was thought to explicate its molecular basis. The changes in the
D
Wm after treating COLO-205 cells with 15 lM of CuL were mea-
sured. A time dependent significant (p < 0.01) fall in the membrane
potential was observed from 6 h of treatment and continued as
time increased (Fig. 2c). Pre-incubation with 50
teine (NAC) did not reversed the fall in
lM N-acetyl cys-
D
Wm.
To elucidate the possible pathway involved in the copper com-
plex induced cell death, ROS levels were monitored at different
time points by treating COLO-205 cells with 15 lM CuL.
However, significant increase in the ROS level was not observed
in CuL treated cells compared to control cells (Fig. 2d). Cells pre-
treated with NAC also did not show any changes in ROS level.
3.9. Evaluation of cytotoxicity and possible mechanism of cell death
Hence, comparing the close correspondence between the
and absence of ROS in CuL treated cells, one can rule out the
involvement of ROS in altering the Wm. Since fall in Wm has
direct relationship with caspase mediated cell death [42], it was
proved that mitochondrion plays an important role in the cell
death process. This result further confirms the potential of CuL to
destroy integrity of mitochondrial membrane.
DWm fall
To evaluate the role of synthesised metal complexes (CoL2, NiL2
and CuL) as antiproliferative agents, they were tested on two dif-
ferent human cell lines, K-562 and COLO-205. Both the cells were
treated with different concentrations of CoL2, NiL2 and CuL for
48 h and subjected to MTT assay, with cisplatin as a positive con-
trol [39]. All tested complexes showed noticeable antiproliferative
effect (Fig. 2a) and IC50 values (Table 5). Among the panel of com-
plexes, CuL exhibited strong dose dependent antiproliferative
D
D
activity against COLO-205 cell line (IC50 value 8.65
parable with positive control cisplatin (IC50 value of 7.24
against more resistant [41] cell line K-562 (IC50 of 15.16
the other hand, cobalt (CoL2) and nickel (NiL2) complexes exerted
weak activity against K-562 (IC50 > 250) and showed compara-
tively more activity against COLO-205. As copper complex (CuL)
showed higher antiproliferative activity among all tested com-
plexes, CuL is selected further for mechanistic studies. Further, it
is noticed that ethyl pyruvate derived thiosemicarbazone ligand
coordinated with copper ion has a synergetic effect on the
l
l
M) com-
M), than
M). On
3.11. Measurement of caspase-3 activity and DNA fragmentation
l
To check whether CuL induced cell death is mediated by apop-
tosis; caspase-3 activity and DNA fragmentation assay were per-
formed. CuL induced apoptosis was first demonstrated by
caspase-3 activity assay. Changes in the caspase-3 activity upon
treatment (5 lM) of CuL in COLO-205 cells were measured using
colorimetric assay kit. A significant (p < 0.01) time dependent
increase in the caspase-3 activity was observed upon treatment,
confirming the apoptotic mode of cell death (Fig. 2e). Activation
of caspase-3 often leads to the activation of caspase activated
DNase (ICAD) through removal of its inhibitor site, inhibitor of cas-
pase activated DNase (ICAD) [43] thereby playing important role in
the CAD dependent induction of apoptosis [44]. Such activated CAD
cleaves the DNA at internucleosomal region and leads to DNA frag-
mentation in apoptotic cell. Therefore, we analyzed the DNA frag-
mentation in cells upon CuL treatment. In our studies, COLO-205
Table 4
Torsional angles of LH.
Atoms
Bond angles (°)
Atoms
Bond angles (°)
C1–N1–C7–N2
C1–N1–C7–S1
N3–N2–C7–N1
N3–N2–C7–S1
N3–C8–C10–O2
C9A–C8–C10–O1
C2–C3–C4–F1
178.06(13)
À0.8(2)
C7–N2–N3–C8
C7–N1–C1–C6
C7–N1–C1–C2
C6–C1–C2–C3
N1–C1–C2–C3
Cl1–C4–C5–C6
C3–C4–C5–C6
178.71(13)
24.2(2)
cells treated for 48 h with 5 and 25 lM CuL showed typical DNA
À4.69(19)
174.31(10)
À178.25(13)
2.0(2)
À158.36(15)
ladder conformation upon gel electrophoresis (Fig. 2f), which is
the major biochemical hallmark of apoptosis. A notable increase
in the percent DNA damage was observed with increasing concen-
trations of CuL, with control cells showing no laddering of DNA.
À1.4(2)
À178.93(13)
179.74(11)
À0.8(2)
178.51(12)

222
A.H. Pathan et al. / Inorganica Chimica Acta 430 (2015) 216–224
Fig. 1. ORTEP diagram of LH.
Fig. 2. Effects of metal complexes of ethyl 2-(2-(4-chlorophenylcarbamothioyl)hydrazono)propanoate on cancer cell lines. (a) Cytotoxic effect of test complexes on K-562 and
COLO-205 cell lines after 48 h treatment was assessed by performing MTT assay. (b) LDH release was assayed after treating COLO-205 cells with 20 M of CuL for 24, 48 &
72 h treatment. (c) Time-dependent measurement of Wm upon 15 M CuL treatment on COLO-205 cells with or without 30 min pre-incubation with 50 M NAC was
measured using JC-1 dye. (d) Intracellular ROS level was measured using CM-H2DCFDA. COLO-205 cells were treated with 15 M CuL with or without pre-treatment of NAC
for 30 min and were stained and fluorescence was measured. (e) Caspase-3 activity was determined in COLO-205 cells treated with 5 M of CuL for 24 and 48 h, using DEVD-
pNA as substrate at 405 nm. Activity was measured in terms of concentration of pNA formed per min per ml of cell lysate. (f) CuL induces internucleosomal DNA
fragmentation. DNA separated from COLO-205 cells treated for 48 h with 5 and 25 M CuL, were subjected to electrophoresis in 1.5% agarose. Lane 1, 200 bp DNA ladder; Lane
M CuL. Data presented are representative of two experiments and the results were same. The data represents Mean SE
l
D
l
l
l
l
l
2, DMSO control; Lane 3, 5 lM CuL; Lane 4, 25 l
(n = 3). ⁄p < 0.05, ⁄⁄p < 0.01 compared with the control group.
3.12. Morphological evaluation of apoptosis
membrane. In contrast, CuL treated cells exhibited apoptotic mor-
phological changes including condensed chromatin and frag-
mented nuclei. A stronger apoptosis signal was observed with
treatment of the CuL, strongly suggesting the apoptotic mode of
cell death, since chromatin condensation, loss of membrane integ-
rity, formation of apoptotic bodies and formation of membrane
blebbing are the major events involved in apoptotic mode of cell
death [45].
3.12.1. Acridine orange–ethidium bromide (AO/EB) double staining
Nuclear morphology of cells undergoing apoptosis was
phenotypically evidenced by staining the cells with AO/EB.
COLO-205 cells treated for 48 h with 15 lM of CuL were stained
and observed under fluorescent microscope (Fig. 3). Cells treated
with DMSO stained green, represent viable cells with intact

A.H. Pathan et al. / Inorganica Chimica Acta 430 (2015) 216–224
223
Table 5
3.12.2. Cell cycle analysis and annexin V-FITC/PI staining
In vitro antiproliferative activity of the complexes evaluated in human cancer cell
lines after 48 h exposure. Results are mean of three independent experiments.
To further support our findings, we checked the effect of CuL
complex on cell cycle progression by flow cytometry. COLO-205
cells incubated with different concentrations of CuL for 48 h
showed significant sub-G0/G1 peaks indicating apoptosis, and the
percentage of apoptotic cells presented a dose-dependent increase
Complexes
IC50
(
l
M)
VERO
COLO-205
K-562
Cu-L
8.65
15.16
120.15
>250.00
>250.00
>250.00
ND
from 5 to 20 lM (sub G0/G1: 4.98–20.64%) compared to control
Co-L2
Ni-L2
LH
99.94
94.75
230.00
7.87
>250.00
>250.00
>250.00
5.99
(Fig. 4a–d). The histograms of CuL treated cells did not show any
significant change in cell cycle profile, except sub G0/G1 pop-
ulation, suggesting the inefficiency of the complex to induce cell
cycle arrest at any phase of the cell cycle. Put together, cell cycle
analysis data clearly indicate that the mechanism of cytotoxicity
Cisplatin
ND – Not determined.
Fig. 3. Detection of apoptosis by fluorescence microscopy. Acridine orange–ethidium bromide double staining. Morphological evidence of apoptosis was obtained by
performing AO/EB double staining. COLO-205 cells were treated with 15
shrinkage and nuclear granulation.
lM of CuL for 48 h and fluorescence microscopic imaging was done. Arrow heads indicate cellular
Fig. 4. Flow cytometric determination of effect of metal complexes on cell cycle and apoptosis. (a–d) Impact of CuL on cell cycle of COLO-205 cells after the treatment for 48 h.
Cell cycle distribution was analyzed by flow cytometry. Histograms are representative of three independent experiments. (e, f) Confocal micrographs of CuL treated COLO-205
cells, stained with Annexin V/PI. (e) Cells treated with DMSO. (f) Cells treated with CuL showing both Annexin V/PI and Annexin V (late and early of stage apoptosis) positive
cells.

224
A.H. Pathan et al. / Inorganica Chimica Acta 430 (2015) 216–224
by CuL may primarily involve apoptotic mode of cell death
induction.
References
[1] C. Deegan, B. Coyle, M. McCann, M. Devereux, D. Egan, Chem. Biol. Interact. 164
(2006) 115.
[2] Z. Afrasiabi, E. Sinn, J. Chen, Y. Ma, A.L. Rheingold, L.N. Zakharov, N. Rath, S.
Padhye, Inorg. Chim. Acta 357 (2004) 271.
[3] R.A. Alderden, M.D. Hall, T.W. Hambley, J. Chem. Educ. 83 (2006) 728.
[4] P.N. Yadav, M.A. Demertzis, D. Kovala-Demertzi, S. Skoulika, D.X. West, Inorg.
Chim. Acta 349 (2003) 30.
[5] E. Wong, C.M. Giandomenico, Chem. Rev. 99 (1999) 2451.
[6] T.W. Hambley, Coord. Chem. Rev. 166 (1997) 181.
[7] B. Stordal, N. Pavlakis, R. Davey, Cancer Treat. Rev. 33 (2007) 688.
[8] I. Gojo, M.L. Tidwell, J. Greer, N. Takebe, K. Seiter, M.F. Pochron, B. Johnson, M.
Sznol, J.E. Karp, Leuk. Res. 31 (2007) 1165.
These conclusions were furthermore verified by observing the
annexin V-FITC/PI stained cells under a confocal microscope.
After 48 h of treatment with CuL, most of the control cells were
negative for both annexin V-FITC and PI (Fig. 4e). Upon CuL treat-
ment, cells were stained with either annexin V-FITC (green color)
alone or by both annexin V-FITC (green color) and PI (red color),
suggesting presence of both early apoptotic and late apoptotic cells
with complete membrane damage (Fig. 4f). These results clearly
suggest that apoptosis could be the mechanism of cell death
induced by the CuL.
[9] A.G. Quiroga, J.M. Perez, I. Lopez-Solera, E.I. Montero, J.R. Masaguer, C. Alonso,
C.N. Ranninger, J. Inorg. Biochem. 69 (1998) 275.
[10] A.M. Fialho, A.M. Chakrabarty, Recent Pat. Anticancer Drug Discov. 2 (2007)
224.
[11] A. Diaz, R. Cao, A. Garcia, Monatsh. Chem. 125 (1994) 825.
[12] K. Grancharov, G.J. Krauss, N. Spassovska, J. Miersch, L. Maneva, J. Mladenova,
E. Golovinsky, Pharmazie 40 (1985) 574.
[13] S. Chandra, K.K. Sharma, Transition Met. Chem. 9 (1984) 1.
[14] S. Hayami, K. Hashiguchi, K. Inoue, Y. Maedaa, J. Nucl. Radiochem. Sci. 5 (2004)
N1.
[15] D.K. Chand, H.J. Schneider, J.A. Aguilar, F. Escarti, E. Garcia-Esspara, S.V. Luis,
Inorg. Chim. Acta 316 (2001) 71.
[16] F.L. Lee, E.J. Gabe, L.E. Khoo, W.H. Leong, G. Eng, F.E. Smith, Inorg. Chim. Acta
166 (1989) 257.
[17] L. Savini, P. Massarelli, L. Chiasserini, C. Nencini, C. Pellerano, Farmaco 52
(1997) 609.
[18] H. Kappus, Biochem. Pharmacol. 35 (1986) 1174.
[19] T.W. Gant, D.N.R. Rao, R.P. Mason, G.M. Cohen, Chem. Biol. Interact. 65 (1988)
157.
[20] M.A. Cater, H.B. Pearson, K. Wolyniec, P. Klaver, M. Bilandzic, B.M. Paterson, A.I.
Bush, P.O. Humbert, S.L. Fontaine, P.S. Donnelly, Y. Haupt, ACS Chem. Biol. 8
(2013) 1621.
[21] A.H. Pathan, R.P. Bakale, G.N. Naik, C.S. Frampton, K.B. Gudasi, Polyhedron 34
(2012) 149.
4. Conclusion
The ligand LH acts as tridentate monobasic chelate with N, O
and S as the donating sites toward Co(II), Ni(II), Cu(II) and Zn(II)
ions leading to octahedral geometries. Antiproliferative activity of
panel of complexes synthesized was evaluated. Among the com-
plexes tested, CuL showed potent antiproliferative activity with
its activity comparable to that of standard drug, cisplatin. The
CuL antiproliferative effect was mainly exerted through the induc-
tion of apoptotic cell death as confirmed by flow cytometric sub
G0/G1 cells accumulation and confocal imaging. Possible mecha-
nism of cell death induction was through caspase dependent
intrinsic mitochondrial mediated apoptotic pathway.
Presence of greater number of nucleophilic sites (H2O) seems to
have a greater influence on the overall biological activity of the
complex. The biological activity has been augmented specifically
by redox active Cu and presence of water.
[22] S.B. Himara, K.A. Parikh, B.C. Merja, H.H. Parekh, Indian J. Chem., Sect B 42
(2003) 1172.
[23] G.M. Sheldrick, SHELXTL. Version 5.0, Bruker AXS Inc., Madison, Wisconsin, USA,
2001.
[24] T. Mosmann, J. Immunol. Methods 65 (1983) 55.
[25] K.K. Chiruvella, S.K. Sankaran, M. Singh, M. Nambiar, S.C. Raghavan, ICFAI J.
Biotechnol. 1 (2007) 7.
Acknowledgments
[26] C.V. Kavitha, M. Nambiar, Biochem. Pharmacol. 77 (2009) 348.
[27] S. Shenoy, V.S. Vasania, M. Gopal, A. Mehta, Toxicol. Appl. Pharmacol. 222
(2007) 80.
[28] N. Li, K. Ragheb, G. Lawler, J. Sturgis, B. Rajwa, J.A. Melendez, J.P. Robinson, J.
Biol. Chem. 278 (2003) 8516.
[29] D. Ribble, N.B. Goldstein, D.A. Norris, Y.G. Shellman, BMC Biotechnol. 5 (2005)
12.
[30] M.A. Affan, F.S. Wan, Z. Ngaini, M. Shamsuddin, Anal. Sci. 13 (2009) 63.
[31] A.H. Pathan, K.B. Gudasi, Med. Chem. Res. 22 (2013) 1504.
[32] Z. Afrasiabi, E. Sinn, S. Padhye, S. Dutta, S. Padhye, C. Newton, C.E. Anson, A.K.
Powell, J. Inorg. Biochem. 95 (2003) 306.
[33] H. Okawa, M. Tadokoro, Y. Aratake, M. Ohba, K. Shindo, M. Mitsumi, M.
Tomono, D.E. Funton, J. Chem. Soc., Dalton Trans. (1993) 253.
[34] F.A. Cotton, G. Wilkinson, Adv. Inorg. Chem., fifth ed., Wiley, New York, 1988.
[35] J.C. Bailar, H.J. Emeleus, S.R. Nyholm, A.F.T. Dickenson, Comprehensive
Inorganic Chemistry, Pergamon, Oxford, UK, 1973.
The authors thank the USIC, Karnatak University, Dharwad for
providing the spectral facilities. Recording 1H and 13C Spectral
studies (MU London) and ESR spectra (IIT Bombay) are gratefully
acknowledged. Dr. Aishakhanam H. Pathan is thankful to
University Grants Commission (India) for providing Research
fellowship in Science for Meritorious Students. Miss Asha K.
Ramesh is thankful to Indian Council of Medical Research (India)
for financial support. Authors also thank Prof. Kondaiah P, Indian
Institute of Science and C-CAMP, NCBS, Bangalore for
providing FACS facility. Authors also thank Prof. M.V. Kulkarni for
language corrections. Dr. K.B. Gudasi is thankful to the
Association of Commonwealth Universities (United Kingdom), for
Commonwealth fellowship.
[36] S. Adsule, V. Barve, D. Chen, F. Ahmed, Q.P. Dou, S. Padhye, F.H. Sarkar, J. Med.
Chem. 49 (2006) 7242.
[37] M.B. Ferrari, F. Bisceglie, G. Pelosi, P. Tarasconi, R. Albertini, S. Pinelli, J. Inorg.
Biochem. 87 (2001) 137.
[38] J. Wiecek, V. Dokorou, Z. Ciunik, D. Kovala-Demertzi, Polyhedron 28 (2009)
3298.
Appendix A. Supplementary material
[39] F.E. Smith, Science 113 (1951) 751.
CCDC 978614 contains the supplementary crystallographic data
for LH. These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.
uk/data_request/cif. Supplementary data associated with this arti-
cle can be found, in the online version, at http://dx.doi.org/10.
1016/j.ica.2015.03.013.
[40] C. Korzeniewski, D.M. Callewaert, J. Immunol. Methods 64 (1983) 313.
[41] K.K. Palanichamy, N. Sreejayan, A.C. Ontko, J. Inorg. Biochem. 106 (2012) 32.
[42] A. Strasser, L.O. Connor, V.M. Dixit, Annu. Rev. Biochem. 69 (2000) 217.
[43] B.B. Wolf, M. Schuler, F. Echeverri, D.R. Green, J. Biol. Chem. 274 (1999) 30651.
[44] S. Elmore, Toxicol. Pathol. 35 (2007) 495.
[45] P. Mukhopadhyay, M. Rajesh, G. Hasko, B.J. Hawkins, M. Madesh, P. Pacher,
Nat. Protoc. 2 (2007) 2295.