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An experimental and theoretical approach on kinetics and mechanism for

formation of a four membered (S, S) chelated Pt(II) complex

Article  in  RSC Advances · February 2016

DOI: 10.1039/C5RA21161A

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An experimental and theoretical approach on the

kinetics and mechanism for the formation of a four-
Cite this: RSC Adv., 2016, 6, 18288
membered (S, S) chelated Pt(II) complex†
Venkata P. Reddy B.,a Subhajit Mukherjee,a Ishani Mitra,a Koyel Misra,a
Partha Sarathi Sengupta,b Wolfgang Linert,c J. C. Bose K,d Goutam Kr. Ghosha
and Sankar Ch. Moi*a

The interaction of a chemoprotective agent, diethyldithiocarbamate (DDTC), with cis-[Pt(pic)(H2O)2]2+, 2

Received 12th October 2015
Accepted 3rd February 2016
DOI: 10.1039/c5ra21161a
www.rsc.org/advances
(where pic ¼ 2-aminomethylpyridine), in aqueous medium was investigated experimentally and
theoretically. The equilibrium constant (KE) for the formation of the outer sphere association complex
and the rate constants for both steps have been evaluated experimentally, and were compared with the
theoretically determined values. The activation parameters (DHǂ, DSǂ and DGǂ) for both the steps were
calculated and an associative mechanism is proposed. The bonding modes of the product
[Pt(pic)(DDTC)]+, 3, were confirmed by spectroscopic measurements and supported by Time Dependent
Density Functional Theory (TD-DFT) and Natural Bond Orbital (NBO) calculations. Penta coordinated
platinum transition state geometries for both the steps were fully optimized and confirmed by frequency
analysis and an intrinsic reaction coordinate method molecular docking study has been performed to
understand the binding interaction of complex 2 with B-DNA. The anticancer properties of 2 and 3 were
investigated on a HeLa cell line, and they showed remarkable activity of about 70% compared to cisplatin
at 50 mM concentration.

much interest to investigate how variation in the structures of

Introduction
Cisplatin is the most widely used drug for the treatment of
cancer among the commercially available Pt(II)-based chemo-
therapeutic agents.1–5 The clinical use of cisplatin is curbed by
its inactivity against several types of cancer, acquired resistance
in long term treatment along with high toxicity to normal cells.
Consequently, researchers are aiming to synthesise safer Pt(II)-
based anticancer drugs with reduced toxicity and wider appli-
cation. One of the recognised causes for the development of
resistance is reduced transport of drugs across the cell
membrane. Y. Kidani and co-workers6 noted that cellular
resistance of cisplatin can be overcome by changing the
ammine ligands to 1,2-diaminocyclohexane (DACH). As a result,
certain planned amines were used to synthesize Pt(II)
complexes7,8 and good results were found. It is therefore of
a
Department of Chemistry, National Institute of Technology, Durgapur-713209, W. B.,
India. E-mail: sankarmoi67@yahoo.com
b
Vivekananda Mahavidyalay, Bardhaman, West Bengal, India
c
Institute of Applied Synthetic Chemistry, Vienna University of Technology,
Getreidemarkt, 9/163-AC, 1060, Vienna, Austria
d
Department of Bio-Technology, National Institute of Technology, Durgapur-713209,
W. B., India
† Electronic supplementary information (ESI) available. See
10.1039/c5ra21161a
amine ligands affect the toxicity of platinum complexes. We
have chosen 2-aminomethylpyridine (pic) as the carrier ligand
in the Pt(II) complex [Pt(pic)(H2O)2]2+ 2 due to its s-donor and p-
acceptor properties, which make it suitable for substitution by
biomolecules and kinetic investigations. To tune the activity of
Pt(II)-based anticancer drugs, a new strategy has been developed
to design novel Pt(II) complexes9–12 with N- and S-containing
ligands.
Dithiocarbamate or its substituted derivative dieth-
yldithiocarbamate (DDTC) has versatile uses in the area of bio-
inorganic chemistry as a rescue agent13,14 against cisplatin
toxicity. Another fascinating aspect of the bidentate (S, S)
chelating ligand DDTC is its high selectivity to protect from
gastrointestinal, renal and bone marrow toxicity induced by
Pt(II)-based anticancer drugs without inhibition of the latter’s
antitumor effect.15–18 Few Pt(II) and Pd(II) dithiocarbamate
complexes have been reported for their remarkable cytotoxic
properties, lower cross-resistance and minimum nephrotoxi-
city19 compared to cisplatin. Binding of Pt(II) to dithiocarbamate
sulfur tends to be irreversible in contrast to thio-ether sulfur20–22
and results in a stable adduct, which however does not interfere
with the tumoricidal Pt–DNA interaction. Thus, the protective
action of dithiocarbamate against the toxicity of cisplatin seems
DOI:
to be the formation of its stable platinum dithiocarbamate
complexes. In vitro kinetic and mechanistic studies on complex

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2 with a few thiols23–25 have already been investigated.

Combined experimental and theoretical investigation on the
kinetics and mechanism of the substitution reaction of 2 with
DDTC has not yet been studied in detail. Herein, we have
endeavoured to correlate the experimental and theoretical
kinetic data for the reaction. The kinetic study aims to under-
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stand the drug reservoir mechanism between DDTC and

complex 2. Cell-based assays and DNA binding studies were
done to evaluate the anticancer properties of the complexes
(Fig. 1).

Experimental
Instrumentation
Elemental analysis of the complexes was carried out on a Per-
kin-Elmer 2400 series-II CHNS/O Analyser. Conductivity
measurements were performed with a Systronics conductivity
meter (Model 308), where the cell constant was calibrated with
standard buffer solution. The pH measurements were done
with the help of a EUTECH digital pH meter (pH Tutor) with an
accuracy of 0.01 U, calibrated with a standard phosphate
buffer solution (KH2PO4/Na2HPO4). Fourier transform infrared
(FTIR) spectra of the complexes were recorded on a Nicolet-iS-10
spectrometer (KBr disk, in the range of 4000–400 cm1). The
kinetic measurements were conducted at pH 4.0 on a Shimadzu
UV 1800 spectrophotometer attached to a thermoelectric cell
temperature controller (Model TCC-100, accuracy 0.1  C). 1H
NMR spectra were recorded with a Bruker Avance-400 (400
MHz) spectrometer using D2O or DMSO-d6 as the solvent. ESI
mass spectra were carried out on a Waters Q-TOF Micro YA263
Mass spectrometer in water. The pKa values of complex 2 were
determined by a Metrohm 888 Titrando titro-processor. A Stat
Fax™® 2100 Microplate (USA) Enzyme-linked immunosorbent
assay (ELISA) plate reader was used for the MTT assay.
Reagents and methods
All chemicals used for the kinetic and bioactivity study were of
the highest purity available. The starting compounds K2PtCl4
(99%), 2-aminomethylpyridine (pic) (97%), and sodium dieth-
yldithiocarbamate (NaDDTC) were purchased from Sigma
Aldrich. AgClO4 (97%), AgNO3 (99%), K2HPO4 and NaClO4 were
purchased from Merck. All these reagents were used without
further purication. For kinetic purposes, double distilled water
was used to prepare all kinds of solutions and molecular biology
grade water was used for the bioactivity study. All solvents used
were of analytical grade. Cisplatin (Cis-gland) was purchased
Fig. 2 pH vs. volume (mL) of 0.05 M KOH solution.
from Gland Pharma Limited. Culture medium (DMEM) and FBS
were purchased from Hi-Media.
Synthesis of complexes 1–3
Complex 1 was prepared according to the literature method26
with some modication. K2PtCl4 (250 mg, 0.6023 mmol) was
dissolved in 7.5 mL water and heated at 40–45  C followed by
dropwise addition of an aqueous solution of 2-aminomethyl
pyridine (pic) (65 mg, 0.6023 mmol). The reaction mixture was
heated at 50–60  C with constant stirring for two hours. A
greenish yellow coloured precipitate was obtained and the
solution became colourless aer a few hours of stirring indi-
cating the completion of the reaction. The precipitate was
ltered and subsequently washed with water, acetone and
diethyl ether. It was then dried in a desiccator until a constant
weight was obtained (yield ¼ 175 mg, 78%). Elemental analysis
(%): C6H8N2Cl2Pt (calc. in parentheses): C 18.8 (19.2), H 2.0
(2.1), N 7.4 (7.5) and Cl 9.5 (9.7).
The diaqua complex 2 was prepared in solution using the
method of Hay and Basak.27 The chloro complex was converted
into the diaqua analogue in solution by adding two equivalents
of AgClO4. The solution was kept in the dark overnight, and the
AgCl precipitate was removed by ltration through a 0.1 mm
pore size membrane lter. Great care was taken to ensure that

Fig. 3UV-vis (A, black) and simulated TD-DFT (B, red) spectra of
Fig. 1 Structure of ligand and complexes. complex 3.

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Table 1 TD-DFT and experimental band position of [Pt(pic)(DDTC)]

Experimental band TD-DFT peaks (oscillator

(3 dm3 mol1 cm1) strength) Major contribution

310 316 (0.1103) H1 / LUMO (86%) H1 / L+1 (5%)

262 258 (0.1673) H1 / L+3 (91%)
214 216 (0.005) HOMO / L+7 (66%), H7 / L+2 (11%), H7 / L+3 (14%)
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the resulting solution was free of Ag+ ions and that the chloro
complex had been converted completely into the corresponding
diaqua species. For the bioactivity study, another aliquot of the
diaqua [Pt(pic)(H2O)2](NO3)2 complex 2 solution was prepared
from [Pt(pic)Cl2] as the NO3 salt using AgNO3 solution (the
same method as for AgClO4).
Job’s method of continuous variation was carried out to
conrm the metal : ligand (M : L) ratio for the reaction between
complex 2 with DDTC at pH 4.0. Complex 2 and the ligand were
Scheme 1 Resonance structures of diethyldithiocarbamate (DDTC).
mixed according to their probable ratios, like 1 : 1, 1 : 2, 2 : 1
etc. and heated to about 50  C for a few hours and the absor-
bances were measured until a constant maximum was reached.
The exact M : L ratio was found with proper calculation and
shows a bell shaped curve when absorbance vs. [M]/[M] + [L] was
plotted (ESI Fig. S1†). The development of a characteristic peak
in the product complex 3 at 262 nm was monitored as a function
of time at different xed temperatures. Complex 2 and the
ligand DDTC were mixed in a 1 : 1 molar ratio at pH 4.0 in
accordance with Job’s result to obtain the solid product by slow
evaporation.

Synthesis safety note

Metal perchlorate salts should be handled carefully as these are
explosive and hazardous upon strong heating. Appropriate
safety precautions were taken while handling the salt. No
Fig. 4 A typical kinetic plot of ln(Aa  At) versus time: [complex 2] ¼
explosion event was encountered in the present study.
2.43  104 mol dm3; [DDTC] ¼ 2.43  103 mol dm3;
temperature ¼ 25  C. Inset: plot of ln D versus time. pKa value determination of complex 2
The pKa values of complex 2 were determined by the potentio-
metric method using the tiamo 2.4 soware package. The
electrode and titro processor were calibrated with standard
buffer solutions prepared according to NBS specications. pH
versus volume titration (Fig. 2) of complex 2 (2.05  103 M)
with standard 0.05 M KOH solution was done at a constant ionic
Scheme 2
strength (0.1 M NaClO4) and at 25  C. pH was plotted against the
volume of base consumed. The relationship pH  p[H] ¼ 0.05
Table 2 Rate constants for the reaction of 2 with DDTC at pH 4.0 and was observed. Equivalent points EP1 and EP2 were observed in
with 0.1 M NaClO4 the titration curve corresponding to the pKa values HP1 and
HP2. The pKa1 and pKa2 values of complex 2 were found to be
DDTC
4.76 and 6.83 respectively, which are comparable with the re-
Temp. ( C) 103k1 (M1 s1) KE1 (dm3 mol1) 105k2 (s1) ported pKa values of the almost similar system
[Pt(MAMP)(H2O)2]2+.7
25 3.33  0.08 261  0.13 2.15  0.01
30 4.54  0.05 300  0.22 2.80  0.03
35 6.66  0.12 349  0.16 3.79  0.03 Spectroscopic study
40 9.09  0.09 398  0.21 4.94  0.01 Electronic absorption spectrum of 1 in DMSO, lmax (3/M1
45 14.28  0.05 448  0.32 6.45  0.03
cm1): 308 nm (5012). Selected IR frequencies (ESI Fig. S2†)
(KBr disk, cm1): 3200–3170(b), 3110(s), 2924(s), 1616(s),

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Table 3 Experimental activation parameters for the reaction of complex 2 with DDTC

System Step 1 DH1ǂ (kJ mol1) DS1ǂ (J K mol1) DG1ǂ (kJ mol1)
Complex 2/DDTC 53.87  1.46 107.43  1.13 85.83  1.80
Step 2 DH2ǂ (kJ mol1) DS2ǂ (J K mol1) DG2ǂ (kJ mol1)
40.98  1.17 193.67  1.81 100.18  1.71
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Fig. 5 Optimized structure of complex 3.

1559(m), 1449(m), 1288(s), 1162(m) and 425(s). 1H NMR (400

MHz, DMSO-d6, d in ppm, J in Hz) of 1 (ESI Fig. S3†): d 8.91 (d,
J ¼ 6.9, 1Ha), d 8.25 (t, J ¼ 1.9, 8.4, 1Hc), d 7.81 (d, J ¼ 10.8, 1Hd),
d 7.46 (t, J ¼ 9.9, 7.6, 1Hb), d 6.8 (s, 2Hg), d 4.40 (t, J ¼ 7.5, 7.5,
2Hf), d 3.3 (water in DMSO), and d 2.49 (solvent DMSO).
13
C NMR of 1 (ESI Fig. S4†) (400 MHz, d in ppm, DMSO-d6):
d Ce 150.20, Ca 141.64, Cc 124.74, Cd 122.9, Cb 41.56 and Cf
39.52.
Electronic spectrum of DDTC substituted product 3 displays
bands at different lmax values in aqueous medium with hyp-
sochromic as well as hyperchromic shi as compared to
complex 2 (ESI Fig. S5†).
Fig. 6 HOMO and LUMO and their energy difference of complex 3.
The important bands in the IR spectra of 3 (ESI Fig. S6†) are:
3400–3380(br) cm1, 2974(br) cm1, 2823(m) cm1, 1684(s)
cm1, 1637(s) cm1, 1541 cm1, 1522 cm1, 1285–1080(br),
940(s) cm1, 627(s) cm1 and 476(s) cm1.
1
H NMR of 3 (ESI Fig. S7†) (400 MHz, DMSO-d6, d in ppm, J in
Hz) of complex 3: d 8.5 (d, J ¼ 6.4, 1Ha), d 8.29 (t, J ¼ 2.8, 2.0,
1Hc), d 7.74 (t, J ¼ 7.6, 8.0, 1Hd), 7.56 (d, J ¼ 6.4, 1Hb), 4.40 (d, J ¼
0 0
5.6, 2Hf), 2.92 (m, 4Hx & x ), and 1.15 (t, 6Hy & y ).
The important peaks of the IR spectrum of the free ligand
DDTC (ESI Fig. S8†) are: 3500–3200(br) cm1, 2978(s) cm1,
2923(s) cm1, 2075(br) cm1, 1671(s) cm1, 1617(s) cm1,
1590(s) cm1, 1434(s) cm1, 1262(s) cm1, 1204(s) cm1, 1131
cm1, 1063 cm1, 936 cm1 and 911 cm1.
The ESI mass spectrum of product complex 3 (ESI Fig. S9†)
shows a molecular ion peak m/z at 451.08 (100%) in water.

Table 4 Important bond lengths (Å) and bond angles ( ) of the final product [Pt(pic)(DDTC)] using different protocols in the gas and aqueous
phases

B3LYP MPW1PW91 PBEPBE

Method
Medium Gas Aqueous Gas Aqueous Gas Aqueous Ref. 52–54

Pt–N1 2.094 2.085 2.066 2.059 2.070 2.065 2.026(3)

Pt–N2 2.140 2.120 2.110 2.089 2.130 2.112 2.015(3)
Pt–S1 2.365 2.377 2.340 2.349 2.356 2.364 2.356(3)
Pt–S2 2.359 2.377 2.334 2.351 2.348 2.363 2.349(3)
C7–N3 1.320 1.320 1.314 1.315 1.330 1.330 —
N1–Pt–N2 79.18 79.42 79.60 79.90 79.64 79.84 —
N1–Pt–S2 103.25 103.44 102.95 103.31 102.82 102.07 —
S1–Pt–S2 74.58 74.28 74.89 74.64 74.88 74.64 —
N2–Pt–S1 102.99 102.86 102.56 102.16 102.67 102.45 —

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Fig. 7 Optimized structures and selected structural parameters for both the steps of the substitution reaction on complex 2 with DDTC for paths
A and B. Distance and bond angles are in Å and degrees ( ) respectively.

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Fig. 8 Energy profile diagram for paths A and B.
Kinetic investigation
The usual mixing technique was followed and pseudo-rst-
order reaction conditions were employed throughout the
kinetic runs for the reactions. The progress of the reaction was
followed by measuring the increase in absorbance for DDTC
with complex 2 at 310 nm, where the spectral differences
between complex 2 and the product complex 3 are at
a maximum. The pH and ionic strength were maintained at 4.0
and 0.1 mol dm3 (with NaClO4) respectively. The method of
Weyh and Hamm28 was adopted to calculate the rate constants
for both the kinetic studies. The rate data, represented as an
average of duplicate runs, were reproducible to within 4%.
Computational details
All calculations were performed with the Gaussian09 (ref. 29)
programme. For optimisation of complex 3 in both the gas and
aqueous phases, we have used three density functional theory
methods. The rst is the gradient-corrected DFT level using the
three parameter t of exchange and correlation functionals of
Becke (B3LYP)30 which includes the correlation functional of
Lee, Yang and Parr (LYP).31 The second is MPW1PW91 (ref. 32
and 33) which utilizes a modication of the gradient-corrected
Perdew–Wang exchange functional, PW91, combined with the
non-local Perdew–Wang correlation functional and the last one
Table 5 Activation free energy and rate constants based on the computational study using the B3LYP/6-31g(d)/LANL2DZ protocol
Path A
Complex 2/DDTC DGǂ (kJ mol1)
First step 69.3
Second step 96.8
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is PBEPBE.34,35 The relativistic effective core potential (ECP) and
associated valence double x (zeta) basis set of Hay and Wadt36,37
(LANL2DZ) were employed for Pt. This comprises of electrons in
the 6s, 6p, and 5d orbitals. The standard split valence basis set 6-
31G(d)38,39 was applied for carbon, oxygen, nitrogen, hydrogen and
sulfur. For the studied reaction, all the stationary points located on
the PES were characterized as minima (all positive frequencies) or
rst-order transition states (one imaginary frequency) through
harmonic frequency calculations at the B3LYP/6-31G(d)/LANL2DZ
level of theory. The vibrational normal modes have been assigned
on the basis of the potential energy distribution (PED) calculated
with the use of the VEDA program.40
For every transition state, the corresponding structures were
conrmed by the intrinsic reaction coordinate (IRC) method41,42
implemented in Gaussian 09. Thermal contribution of the
energetic properties was also considered at the standard state,
namely, at 298.15 K and 1 atm. The rate constants (k) were
calculated within the transition state theory according to the
Eyring equation:43
ǂ
k(T) ¼ (KBT/h)eDG /KT (1)
where T is the absolute temperature, kB is the Boltzmann
constant, and h is Planck’s constant. DGǂ is the free energy of
activation for each step. The standard concentration (c0 ¼ 1 mol
dm3) was considered in the theoretical calculations.
Natural bond orbital (NBO) analysis44 at the B3LYP/LANL2DZ/
6-31G(d) level was calculated to understand the orbital interac-
tions and charge delocalization during the course of reaction. In
the NBO analysis, NBOs are rst dened for each covalent bond,
lone pair, and anti-bonding orbital using the molecular orbitals
obtained by quantum chemical calculations, and the orbital
interaction energies are consequently calculated for all possible
interactions between electron-donor Lewis-type NBOs (i), and
acceptor non-Lewis NBOs (j). For each electron-donor NBO (i)
and acceptor NBO (j), the stabilization energy Eij associated with
the delocalization i / j is estimated by eqn (2):
Eij ¼ qif(i,j)2/3j  3i (2)
where qi is the donor orbital occupancy, 3i and 3j are the orbital
energies and Eij is the off-diagonal NBO Fock matrix
element.45,46
Molecular docking of complex 2 with B-DNA (PDB ID:1BNA)
was done using HEX 8.0.0 soware.47 HEX calculates DNA–
complex binding, using the knowledge of 3D shape only by
assuming that the complex is rigid and that it superposes pairs
of molecules using the knowledge of 3D shape only. It uses
a spherical polar Fourier correlation to accelerate the
Path B
Rate (M1 s1) DGǂ (kJ mol1) Rate (s1)
4.43 90.34 8.82  104
0.68  105 67.11 10.86
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Table 6 Charges and electronic configuration for the complexes 3 (S, S) and 30 (S, N)

Complex 3 (S, S) Complex 30 (S, N)

Atom Charge Natural electronic conguration Charge Natural electronic conguration

Pt 0.15537 [core]6S(0.58)5d(8.91)6p(0.34)6d(0.02) 0.31241 [core]6S(0.58)5d(8.83)6p(0.27)6d(0.02)

N1 0.45201 [core]2S(1.28)2p(4.15)3p(0.01) 0.44872 [core]2S(1.28)2p(4.15)3p(0.01)
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N2 0.82678 [core]2S(1.40)2p(4.41)3p(0.01) 0.84424 [core]2S(1.40)2p(4.42)3p(0.01)

S1 0.05890 [core]3S(1.71)3p(4.20)3d(0.02)4p(0.01) 0.16600 [core]3S(1.70)3p(4.10)3d(0.02)4p(0.01)
S2 0.05940 [core]3S(1.71)3p(4.20)3d(0.02)4p(0.01) 0.10555 [core]3S(1.74)3p(4.13)4S(0.01)3d(0.01)4p(0.01)
N3 0.38469 [core]2S(1.41)2p(4.41)3p(0.02) 0.49922 [core]2S(1.32)2p(4.17)3p(0.01)

Fig. 9Molecular docking model illustrating the interaction between

complex 2 and B-DNA.
Fig. 10 % of growth inhibition of HeLa cells in the presence of
[Pt(pic)(H2O)2](NO3)2 and the substituted complex [Pt(pic)(DDTC)] NO3
from 5 mM to 50 mM concentration compared with cisplatin.

Scheme 3 Proposed mechanism for the reaction of DDTC with

complex 2 at pH 4.0.

calculations. In order to run the docking protocol in HEX, rst
the DNA and ligand in PDB format were loaded. By clicking on
the control option in the main menu, docking was selected and
activated. The parameters which were used in the docking
process are listed in the ESI, Table S1.† The results obtained
aer the docking process were analysed. The E-total values of
the complex against DNA were calculated. To validate the
procedure and results, we ran the same protocol three times
and obtained almost the same results. RMSD values were
calculated and the RMSD is lower than 2 Å.
Fig. 11 Microscopic view of (a) untreated HeLa cells, (b) treated with
complex 2 (10 mM), (c) treated with complex 3 (10 mM) and (d) treated
with cisplatin (10 mM).
Bioactivity
Anticancer properties, cells and culture conditions. HeLa
cells were used for investigation of the anticancer properties of
the complexes. The cells were cultured in DMEM containing
10% FBS and 1% of penicillin/streptomycin (50 IU mL1 and

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500 mg L1) and cultured for two days at 37  C in a 5% CO2
incubator.
In vitro cytotoxicity assay. Cytotoxicity was investigated by
MTT colorimetric assay.48,49 Approximately 10 000–12 000 cells
were added to each well of a 96-well plate containing DMEM
medium. The cells were treated with the desired concentrations
(5–50 mM) of complex 2 and 3. Cisplatin (5–50 mM) was used as
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the positive control50 and the untreated cells were used as the
negative control. The plates were incubated for 24 hours. 20 mL
MTT (5 mg mL1 in PBS) solution was then added to each well
and incubation was done for another 3 hours. 150 mL of DMSO
was added to each well to dissolve the blue formazan product.
The absorbance of this product was measured at 540 nm, using
an ELISA plate reader.51
Microscopic observation of complex-treated HeLa cells. The
HeLa cells were allowed to grow in 100 mm culture plates
containing DMEM with 10% FBS and an appropriate amount of
penicillin/streptomycin. To acquire around 70% conuence,
they were incubated at 37  C in a 5% CO2 incubator. These cells
were treated with a 10 mM concentration of complex 2, 3 and
cisplatin, and incubated for 12 hours. The negative control plate
was also maintained, where the cells remained untreated. These
plates were washed with 1 PBS and used to capture images by
an inverted microscope with 100 magnication (Ziess Axio
Observer.Z1).
Results and discussion
Spectroscopic analyses
Electronic spectra and TD-DFT. The UV-vis spectrum of
product 3 (Fig. 3) shows that there are three absorption peaks at
214, 262 and 310 nm, which are due to p–p* and n–p* transi-
tions in different MOs of the ligands. Complex 3 also shows
a hyperchromic and slight red shi as compared to complex 2.
To evaluate the nature of the orbitals involved in the transitions
related to the experimental observations, a theoretical approach
Time Dependent Density Functional Theory (TD-DFT) compu-
tational method was applied. The theoretical calculations
predict that each molecular orbital is formed by the combina-
tion of a large number of atomic orbitals. The bands in the
simulated spectrum appear at 216, 258 and 316 nm. The
experimental and theoretical lmax values corresponding to the
bands observed for the electronic transitions are given in Table
1, which support the experimental results. The major contri-
bution of the molecular orbitals involved in the electronic
transitions is shown in the ESI, Fig. S10.†
FTIR spectra. The ligand DDTC has three prospective donor
centres (S, S and N) and FTIR has been used to conrm the
bonding nature of the ligand in complex 3. The important
bands in the FTIR spectrum of 3 have been assigned by
comparison with the spectral data of DDTC and complex 2. The
IR peak at 2075 cm1 in DDTC has shied to 2025 cm1 in 3.
This is most probably due to the partial multiple bond nature
between carbon and sulfur (–SgCNEt2), which is quite similar to
the behaviour of SCN when coordinated to Pt(II). The peak is
broad, possibly due to delocalisation of charge between both
the sulfur donor centres (Scheme 1) and their coordination to
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the metal centre. A Pt–S stretch should appear below 400 cm1
and was obtained theoretically at 317 cm1. A strong band at
1541 cm1 and a medium band at 940 cm1 can be assigned to
the (CgN) and (CgS) partial double bond str. frequency
respectively. The position of these bands in complex 3 reveals
that DDTC binds to Pt(II) as a bidentate ligand through the
sulfur centres.
1
H NMR spectra. The d values of the aromatic protons in 3
are consistent with those in complex 2. The chemical shi
position of the ethyl protons is also unaffected when compared
to that in DDTC, which indicates that the amine nitrogen is not
a coordinating site to the metal.
ESI mass spectra. The product complex 3 obtained from the
reaction mixture of a 1 : 1 molar ratio of complex 2 and DDTC
shows a molecular ion peak m/z at 451.08, which corresponds to
the exact molecular mass of product 3 as suggested in the
mechanism.
Conductivity measurement
Conductivity measurement of complex 3 in conductivity water
shows 1 : 1 electrolytes, which clearly indicates that the complex
is carrying one unit of positive charge.
Kinetic study (experimental)
The pKa value52 of the ligand DDTC is 3.37 at 25  C, thus at pH
4.0, the ligand DDTC exists in its anionic form which is involved
in the kinetic process. Both the sulfur centres are reactive due to
negative charge delocalisation between them as shown in
Scheme 1.
The pKa values of complex 2 were obtained at 4.76 and 6.83.
However, the pKa1 value was found to be different from the
previously reported26 value (5.82). The pKa values were deter-
mined using tiamo soware, which is more reliable and accu-
rate than the formerly used methods. We can assume that at pH
4.0 the reactant complex 2 exists in its diaqua form. At
a particular temperature, pH 4.0 and a xed concentration of
complex 2, the plots of ln(Aa  At) versus time (where Aa and At
are the absorbances at innite time, i.e. aer the completion of
the reaction and at time t) for various ligand concentrations
were curved at the initial stage and subsequently were of
a constant slope (Fig. 4). This indicates that the reaction
proceeds through a two-step consecutive process and we
suggest that in the rst step one aqua ligand is replaced in 2 by
one sulfur atom of DDTC. The second step is slower, where
another aqua molecule is substituted by the other sulfur atom
of the dithiocarbamate group and ring closure takes place.
The rate constants of the reaction for such a two-step process
can be evaluated by assuming the following:
k1 k2
A !B !C
where A is the diaqua complex 2, B is the sulfur bonded one
aqua ligand substituted intermediate, and C is the nal product
3. Formation of C from B is predominant aer some time has
passed, i.e. when the reaction is complete.
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Evaluation of k1
The rst step substitution process, the A / B step, is dependent
on the incoming ligand concentration. At each temperature, the
rate constant k1(obs) values were evaluated using the method of
Weyh and Hamm28 from the plots of ln D (the variation of D is
shown in Fig. 4, inset) versus time (where t is small) and are
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collected in the ESI, Table S2.† Using the usual consecutive rate
law:
(Aa  At) ¼ a1 exp(k1(obs)t) + a2 exp(k2(obs)t) (4)
where a1 and a2 are constants that depend on the rate constant
and extinction coefficient, the values of [(Aa  At)  a2-
exp(k2(obs)t)] were obtained from the parameter X–Y at
different times.
ln D ¼ constant  k1(obs)t (5)
where D is the difference between the observed and extrapo-
lated part of the linear portion of (Aa  At) versus time (when t is
small). Hence, k1(obs) is derived from the slope of the plot of ln D
versus t (correction coefficient is 0.9879).
The same procedure was followed in the range of ligand
concentration from 2.43  103 mol dm3 to 7.29  103 mol
dm3, at a constant complex 2 concentration (2.43  104 mol
dm3) and at different temperatures (25, 30, 35, 40 and 45  C).
The reaction rate increases with the increase in ligand
concentration up to a limiting rate (ESI Fig. S11†). The depen-
dency of the k1(obs) values on ligand concentration can be
explained in terms of rapid formation of the inner sphere
association complex between the reactant complex 2 and DDTC
in the step A / B. This is followed by inner sphere association
through sulfur coordination via displacement of one water
molecule resulting in the intermediate B. The following Scheme
2 can be proposed for the reaction:
Based on the above equations, a rate expression (6) can be
derived for the A / B step:
d[B]/dt ¼ k1KE[2][DDTC]/(1 + KE[DDTC]) (6)
d[B]/dt ¼ k1(obs)[2]T (7)
where the subscript T stands for the total concentration of the
complex (2), thus:
1/k1(obs) ¼ 1/k1 + 1/k1KE[DDTC] (8)
where k1 is the rate constant for the formation of B and KE is the
outer sphere association equilibrium constant. A plot of 1/k1(obs)
versus 1/[DDTC] was found to be linear (ESI Fig. S12†) with an
intercept of 1/k1 and a slope of 1/k1KE. This equation is appli-
cable to all the temperatures under the kinetic study. The rst
step rate constant k1 and KE values were obtained from the
intercept (1/k1) and from the slope-to-intercept ratios (1/k1KE).
The k1 and KE values (Table 2) thus obtained are dependent on
the ligand concentration.
18296 | RSC Adv., 2016, 6, 18288–18299
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Paper
Evaluation of k2
The second step B / C involves displacement of another water
molecule accompanied by ring closure through another sulfur
atom of the dithiocarbamate group of DDTC (Scheme 2).
According to the HSAB principle, formation of a Pt(II)–sulfur
four-membered ring is more favourable than a platinum–
nitrogen four-membered chelate. In the literature, few four-
membered metal chelates have been reported with so metal
ions like Pt(II)53 and Pd(II)54,55 where dithiocarbamate moieties
use both of their S-donor centres to chelate the metal ion.
Consequently, we expect that DDTC would show a similar type
of bonding nature with Pt(II) in complex 3 and the same has
been conrmed from spectroscopic data.
At each temperature, the k2(obs) values were calculated from
the limiting linear portion (when t is large) of the plot of ln(Aa 
At) versus t (Fig. 4) and the values are collected in the ESI, Table
S3.† Unlike k1, the k2 values were found to be independent of
ligand concentration at all the temperatures studied. The
derived rate equation of the reaction based on kinetic obser-
vations is as follows:
kE  k1 ½DDTC
Rate ¼ ½complex 2 þ k2 ½complex 2 (9)
1 þ kE ½DDTC
where KE is the outer sphere association equilibrium constant.
Effect of temperature
The reaction was studied at ve different temperatures at
different ligand concentrations and the substitution rate
constants for both the steps (k1 and k2) are given in Table 2. The
activation parameters calculated from the Eyring plots (ESI
Fig. S13a and b†) (R2 for k1 is 0.9795 and R2 for k2 is 0.9865) are
given in Table 3. The Gibbs free energy of activation was derived
from the thermodynamic relation DGǂ ¼ DHǂ  TDSǂ. The
activation parameters DHǂ and DSǂ are compared with analo-
gous systems in the ESI, Table S4.†
Computational study
A list of bond distances (Å) and angles (degrees) obtained from
the optimized geometry of complex 3 (Fig. 5) is presented in
Table 4 and compared with the reported data of closely related
structures.56–58 The energies of the most signicant orbitals
(HOMO, LUMO, HOMO1, and LUMO+1) of complex 3 and
their energy differences are shown in Fig. 6.
Kinetic investigation by DFT
The reaction of complex 2 with DDTC is a second-order nucle-
ophilic substitution (SN2) process. In a general SN2 reaction
path, ve stationary states e.g., the reactant (R), reactant inter-
mediate (RI), TS, product intermediate (PI) and product (P) are
found.59,60 The reaction may proceed in two different paths (A
and B) both having two steps. In path A, the aqua molecule trans
to the ammine (NH2) group of the carrier ligand pic leaves rst
through a trigonal bipyramidal (tbp) transition state resulting
in a mono aqua [Pt(pic)(H2O)–S–DDTC] complex (B). In the
second step, the aqua molecule trans to the pyridine nitrogen
This journal is © The Royal Society of Chemistry 2016

Paper
leaves the platinum coordination zone resulting in the product
complex 3. This is also true for path B, where the aqua molecule
trans to the pyridine nitrogen leaves the coordination zone rst
through tbp to the mono aqua complex [Pt(pic)(H2O)–S–DDTC]
complex (B0 ). The next step is formation of the (S, S) coordinated
complex (C). All the intermediate structures found in this
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reaction are stabilized through H bonds since the interaction
with the Pt(II) atom is negligible (Pt–S distances being above
3.365 Å as compared to 2.356 Å.57,58 All stationary states (Fig. 7),
i.e. the four reactant intermediates (RI1, RI10 , RI2 and RI20 ),
transition states (TS1, TS10 , TS2, and TS20 ) and four product
intermediates (PI1, PI10 , PI2 and PI20 ), located on the PES are
conrmed by frequency analysis. The coordinates of all the
stationary states and negative frequencies of the corresponding
rst-order saddle point are shown in the ESI, Table S5.† The
total energy prole diagrams (energies are in kJ mol1) for the
studied reaction in the different possible paths (A and B) are
shown in Fig. 8. Rate constants and activation parameters for
the reaction calculated for both the possible paths (A and B) are
shown in Table 5, in which the relative energy change in path A
is reasonable compared to path B. The theoretically calculated
rst step rate constant (k1 ¼ 4.43 M1 s1) is found to be higher
than the experimentally determined rst step rate constant
(k1 ¼ 3.33  103 M1 s1). It might be explained on the basis of
the fact that the outer sphere association equilibrium (KE)
constant and rst step reaction rate constant are not distin-
guishable or separable in the computational method. The
association equilibrium (KE) constant and actual rst step rate
constant (k1) are collectively shown as a theoretical rst step rate
constant, which is greater than the experimental rate constant
at 25  C. In the case of the second step, the theoretical rate
constant (k2 ¼ 0.68  105 s1) is closer to the experimental
value (k2 ¼ 2.15  105 s1) at 25  C. As a result, the theoreti-
cally calculated and experimentally determined rate constants
are reasonably comparable.
Natural bond orbital (NBO) analysis
The coordinating sites to Pt(II) were conrmed by natural bond
orbital (NBO) analysis, calculated at the B3LYP/LANL2DZ/6-
31G(d) level. NBO analysis provides detailed insight into the
nature of electronic structure and bonding in the probable
complexes 3 and 30 with DDTC by considering61 chelation
through (S, S) or (S, N) respectively in the substitution reaction.
The calculated atomic charge and electronic congurations for
complex 3 (S, S) (Fig. 5) and the other probable complex 30 (S, N)
(ESI Fig. S14†) are listed in Table 6. The formal charge of Pt in
both complexes 3 and 30 is +2.0 a.u. The calculated natural
charge on Pt(II) in complex 3 (0.1553 a.u.) is lower than that in 30
(0.31241 a.u.) which implies that there is more charge donation
to the Pt(II) centre from (S, S) in 3 than from (S, N) in 30 .
Moreover, the formal charge on both (S, S) in complex 3
(0.05890 and 0.05940 a.u.) is less than that on (S, N) in complex
30 (0.10555 and 0.49922 a.u.). The formation of complex 3
through (S, S) coordination can hence be conrmed by
considering better charge transfer from the donor centres (S, S)
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to Pt(II) rather than (S, N), which is in concurrence with the
spectroscopic results and also the SHAB principle.
Molecular docking with B-DNA
Molecular docking studies of complex 2 with the DNA duplex of
sequence d(CGCGAATTCGCG)2 dodecamer (PDB ID:1BNA)
were performed in order to predict the chosen binding site.
Complex 2 is exible enough to adopt a conformation which is
complementary to the minor groove62 of the DNA (Fig. 9). The
almost planar structure of complex 2 ts into the minor groove
of B-DNA in a parallel way with respect to the DNA backbone.
The platinum complex forms two hydrogen bonds with the
guanine (2.8 Å) and adenine (1.9 Å) bases of the 30 to 50 poly-
nucleotide chain in DNA. The resulting binding energy of the
docked metal complex was found to be 34.77 kcal mol1.
Reaction mechanism
The experimental kinetic study shows a rapid equilibrium due
to outer sphere association between complex 2 and the ligand
DDTC. DDTC substitutes two water molecules of complex 2 and
gives the substituted product 3 through (S, S) coordination. The
leaving group H2O trans to the primary amine is substituted
rst rather than the one trans to the pyridine nitrogen due to its
more labile nature. The second step is slower than the rst, and
is also independent of the ligand concentration. The four-
membered Pt–S bonded complex is quite unnatural in Pt(II)
chemistry and should be unstable. However, here it is stabilised
by simultaneous electromeric and inductive effects of the ethyl
groups attached to the nitrogen in DDTC. A 1 : 1 metal–ligand
ratio obtained from Job’s method of continuous variation also
corroborates with our proposition. The IR spectrum of 3 also
conrms that DDTC behaves as a bidentate ligand (Scheme 3).
Theoretical investigations suggest that the reaction proceeds
through path A instead of path B. A theoretical study has been
performed to verify our proposed mechanism, which is estab-
lished based on experimental work and good agreement was
found between them.
Bioactivity
Anticancer property and IC50 value of complexes 2 and 3.
Inhibition of cell proliferation was investigated by incubating
HeLa cells with various concentrations (5.0–50 mM) of complex
2, 3 and cisplatin by comparing with the negative control
(untreated cells) under the same experimental conditions. A
graphical plot (Fig. 10) of the percentage of growth inhibition of
the HeLa cells versus the complex concentrations reveals that
the rate of growth inhibition increases with the increasing
concentrations of the complexes. This indicates that the
complexes show potential inhibition of HeLa cell proliferation
in a concentration-dependent treatment. Complexes 2 and 3
thus have potent anticancer activity comparable with that of the
established standard metallodrug, cisplatin. The half maximal
inhibitory concentration (IC50) values of complexes 2 and 3 were
calculated and found to be 36  0.6 and 30  0.4 mM respec-
tively, as compared to 12 mM of cisplatin42 for HeLa cells. The
microscopic images of HeLa cells treated with a 10 mM
RSC Adv., 2016, 6, 18288–18299 | 18297

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concentration of complex 2, 3 and cisplatin show adequate
cellular growth inhibition in the order of (d) > (c) > (b) as
compared with the untreated cells (a) (Fig. 11).
The result of the MTT assay and the microscopic images
depict that complex 3 has better anticancer properties than
complex 2. The higher cell growth inhibition activity observed
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for complex 3 might be explained on the basis of its highly
strained four-membered ring structure.
Conclusions
Complex 2 reacts with DDTC at pH 4.0 and proceeds through
two consecutive steps, to form complex 3. The rate constants (k1
and k2) of the two step-process were evaluated experimentally
and were comparable to the theoretically calculated ones.
Among the three potential donor centres (S, S and N) of DDTC,
only the (S, S) donor pair chelates the metal and forms a stable
four-membered complex 3, which is unusual in Pt(II) chemistry.
DFT study and the simulated TD-DFT spectrum of complex 3
also support the formation of the four-membered complex.
NBO analysis also clearly reveals that the (S, S) donor pair of
DDTC chelates the metal rather than the (S, N) donor pair. The
plausible associative mechanism is conrmed by theoretical
investigation. The activation parameters (low DH1ǂ and DH2ǂ
values and negative DS1ǂ and DS2ǂ values) for the rst and
second steps suggest an associative mode of activation for the
substitution process, with a good degree of ligand participation
in the transition state.
Four reactant intermediates (RI1, RI10 , RI2 and RI20 ) and four
product intermediates (PI1, PI10 , PI2 and PI20 ) are observed
corresponding to the rst and second step of reaction for both
paths A and B. Molecular docking of the Pt(II) complex 2 with B-
DNA reveals minor groove binding with a total binding free
energy of 34.77 kcal mol1. Complexes 2 and 3 have accept-
able water solubility and are stable in aqueous solution. A
potent in vitro anticancer activity (70% as compared to
cisplatin) against HeLa cells is observed at higher concentra-
tions for complexes 2 and 3. Hence, there is a fair chance to
employ these Pt(pic) complexes to treat tumour proliferations
aer clinical trials.
Acknowledgements
The authors are thankful to the National Institute of Technology
Durgapur-713209 and Department of Science and Technology
(DST), Government of India (Project No.: SB/EMEQ-028/2013)
for providing the necessary assistance and nancial support
for carrying out this work. We would like to thank the referees
for their valuable comments and suggestions.
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