Configuration change from cis to trans of isothiocyanato groups in nickel(II) species: Experimental verification and theoretical interpretation of reaction consequence and study on their bio-activity

Article abstract of DOI:10.1016/j.poly.2015.03.030

Two new mononuclear Ni(II) complexes, namely [Ni(HL)(SCN)₂(H₂O)] (1) and [Ni(HL)(SCN)₂(4,4′-bipy)] (2) [HL = 2-[(2-piperazin-1-yl-ethylimono)-methyl]phenol] have been synthesized and structurally characterized. The uncoordinated nitrogen atom of the piperazine moiety is protonated to provide electrical neutrality to the system. The configuration of the isothiocyanato ligands is different in the two complexes, cis located in 1 and trans in complex 2. The change of configuration of the isothiocyanato groups on addition of the neutral spacer 4,4-bipyridine to complex 1 has been monitored by conductometric and FTIR spectral studies and the most probable mechanistic pathway has been proposed. DFT calculations have been performed and the outcome corroborates well with the experimental facts. The anticancer activity of the two Ni(II) complexes has been evaluated in human cervical (HeLa) cancer and breast cancer (MCF-7) cell lines. The cytotoxic effects of these complexes were determined by an MTT assay. The fluorescent intensity obtained in HeLa cells reveals the generation of ROS by the complexes using DCFH-DA (2,7-dichlorofluorescein diacetate) dye. The apoptotic cell death was determined by fluorescent staining with acridine orange and ethidium bromide, which confirmed the presence of apoptotic cells. Further, flow cytometry analysis was done, suggesting an arrest of the cell cycle in the S phase of the HeLa cells. Although a detailed molecular mechanism for the anticancer activity of the two complexes was not ascertained, the experimental results suggest that both complexes are effective anticancer agents, with complex 2 seeming to be more promising. The result also indicates that the apoptotic cell death in the cancer cell might be triggered by the process of ROS generation with the complexes. Thermo gravimetric analysis of two complexes hints that NiO particles are the final product after decomposition of the complexes at 600 °C.

Full text of DOI:10.1016/j.poly.2015.03.030

Accepted Manuscript
Configuration change from cis to trans of isothiocyanato groups in nickel(II)
species: experimental verification and theoretical interpretation of reaction con-
sequence and study on their bio-activity
Jaydeep Adhikary, Prateeti Chakraborty, Tanmay Chattopadhyay, Ritika
Prasad, Yair Motro, Biplob Koch, Franz A. Mautner, Yifat Miller, Debasis Das
PII:
S0277-5387(15)00168-0
DOI:
http://dx.doi.org/10.1016/j.poly.2015.03.030
POLY 11253
Reference:
Polyhedron
To appear in:
Received Date:
Accepted Date:
9 December 2014
19 March 2015
Please cite this article as: J. Adhikary, P. Chakraborty, T. Chattopadhyay, R. Prasad, Y. Motro, B. Koch, F.A.
Mautner, Y. Miller, D. Das, Configuration change from cis to trans of isothiocyanato groups in nickel(II) species:
experimental verification and theoretical interpretation of reaction consequence and study on their bio-activity,
Polyhedron (2015), doi: http://dx.doi.org/10.1016/j.poly.2015.03.030
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Configuration change from cis to trans of isothiocyanato groups in nickel(II) species:
experimental verification and theoretical interpretation of reaction consequence and study
on their bio-activity
Jaydeep Adhikary,a Prateeti Chakraborty,a Tanmay Chattopadhyay,b Ritika Prasad,c Yair Motro,d,e
Biplob Koch,*c Franz A. Mautner,* f Yifat Miller,* d,e Debasis Das*a
aDepartment of Chemistry, University of Calcutta, 92 A. P. C. Road, Kolkata-700 009, India
E-mail: dasdebasis2001@yahoo.com
bDepartment of Chemistry, Panchakot Mahavidyalaya,Sarbari, Purulia, Pin-723121, India
cDepartment of Zoology, Faculty of Science, Banaras Hindu University, Varanasi-221005, U.
P., India. E-mail: kochbiplob@gmail.com
dIlse Katz Institute for Nanoscale Science and Technology,Ben-Gurion University of the Negev,
Beér-Sheva 84105, Israel
eDepartment of Chemistry, Ben-Gurion University of the Negev, P.O. Box 653, Be’erSheva
84105, Israel. E-mail: ymiller@bgu.ac.il
fInstitut fuer Physikalische und Theoretische Chemie, Technische Universitaet Graz, Austria
E-mail: mautner@tugraz.at
ABSTRACT
Two new mononuclear Ni(II) complexes, namely [Ni(HL)(SCN)2(H2O)] (1) and
[Ni(HL)(SCN)2(4,4′-bipy)] (2) [HL= 2-[(2-piperazin-1-yl-ethylimono)-methyl]phenol] have
been synthesized and structurally characterized. The uncoordinated nitrogen atom of the
piperazine moiety is protonated to provide electrical neutrality to the system. The configuration
1

of the isothiocyanato ligands is different in the two complexes, cis located in 1 and trans in
complex 2. The change of configuration of the isothiocyanato groups on addition of the neutral
spacer 4,4′-bipyridine to complex 1 has been monitored by conductometric and FTIR spectral
studies and the most probable mechanistic pathway has been proposed. DFT calculations have
been performed and the outcome corroborates well with the experimental facts. The anticancer
activity of the two Ni(II) complexes has been evaluated in human cervical (HeLa) cancer and
breast cancer (MCF-7) cell lines. The cytotoxic effects of these complexes were determined by
an MTT assay. The fluorescent intensity obtained in HeLa cells reveals the generation of ROS by
the complexes using DCFH-DA (2,7-dichlorofluorescein diacetate) dye. The apoptotic cell death
was determined by fluorescent staining with acridine orange and ethidium bromide, which
confirmed the presence of apoptotic cells. Further, flow cytometry analysis was done, suggesting
an arrest of the cell cycle in the S phase of the HeLa cells. Although a detailed molecular
mechanism for the anticancer activity of the two complexes was not ascertained, the
experimental results suggest that both complexes are effective anticancer agents, with complex 2
seeming to be more promising. The result also indicates that the apoptotic cell death in the
cancer cell might be triggered by the process of ROS generation with the complexes. Thermo
gravimetric analysis of two complexes hints that NiO particles are the final product after
decomposition of the complexes at 600 °C.
1. Introduction
Nickel is generally tagged as carcinogenic and toxic, not only in its metallic state but also
in its complexed form, irrespective of its oxidation state. However, recent developments nullify
this tag to some extent and establishes a possible therapeutic interest in some nickel-based
2

complexes [1-9]. Surprisingly the largest congener of nickel, i.e. platinum, has brought about a
revolution in the field of medicine, in particularly in chemotherapy with the serendipitous
discovery of the amazing anticancer activity of cis-platin, a purely inorganic compound.
Interestingly, trans-platin has shown virtually no activity and thus establishes a myth that the
configuration of the species may control the reactivity. It is well known to us that the
coordination chemistry of Ni(II) is very fascinating owing to its inherent ability to adopt various
coordination geometries, which often interconvert depending on certain factors, like the nature of
the ligands, solvent, temperature etc [10-22], and thus as far as geometrical diversity is
concerned Ni(II) may exhibit more diversity in its complexes compared to Pd(II)/Pt(II). It is well
documented that the combination of isothiocyanate and nickel(II) in the presence of a diamine or
a Schiff-base as a co-ligand may generate cis-/trans or linkage isomers or polymeric species
[23-33]. It is also very interesting and challenging to convert one isomeric product to another or
to convert the configuration of isothiocyanato ligands from cis to trans or vice versa. With those
aims in mind, in the present study we have selected 2-[(2-piperazin-1-yl-ethylimino)-
methyl]phenol (HL), a Schiff-base, as a co-ligand which on reaction with Ni(ClO4)2 followed by
NaSCN produces exclusively one product, namely [Ni(HL)(SCN)2(H2O)] (1) having two
isothiocyanato groups in the cis position. Then we treated complex 1 with several neutral spacers
and/or ligands like pyridine-N-oxide, imidazole, 2,2′-bipyridine, 4,4′-bipyridine etc. We were
successful in making a new complex only when 4,4′-bipyridine was used and crystal structure
analysis reveals that the configuration of the two isothiocyanato groups changes to trans
(Scheme 1). The reaction pathways associated with the configurational change of the
isothiocyanato groups have been established experimentally and rationalized by DFT
3

calculations. Here, it is noteworthy that even in the presence of excess of water complex 2 never
reverts back to 1, which has also been authenticated by DFT calculations.
The apparently diverse activity of the two geometrical isomers of Pt(NH3)2Cl2, as stated
above, inspired us to investigate the preliminary activity of our two complexes, having
isothiocyanato ligands in cis and trans orientations, as antitumor agents. We have thoroughly
investigated the cytotoxicity of our complexes on Hela and MCF-7 cells and explored the origin
of the antitumor activity of our complexes where interestingly the trans-species shows slightly
higher activity. Ultra-pure NiO was prepared using the pyrolytic decomposition of complexes 1
and 2 for 2 h, a technique used earlier [34,35].
Scheme 1. Synthetic route to complexes 1 and 2.
2. Experimental
2.1. Materials and method
All the chemicals salicylaldehyde, N-(2-aminoethyl)piperazine, nickel(II) perchlorate
hexahydrate, sodium thiocyanate and 4,4´-bipyridine were obtained from commercial sources
4

and used as received. All other chemicals are of AR grade. Solvents were dried according to
standard procedures and distilled prior to use. Dulbecco’s modified eagle medium (DMEM) was
purchased from Cellclone, (Genetix Biotech Asia Pvt. Ltd.), Fetal bovine serum (FBS), 1000 IU
of penicillin, 10 mg/mL streptomycin and the MTT reagent from Himedia, India. DMSO
(dimethylsulphoxide) and RNAse were obtained from GeNie, Merck, India. Ethidium bromide,
Triton X-100 and other chemicals of analytical grade were purchased from Lobachemie Pvt.
Ltd., India. Acridine orange was obtained from SRL Pvt. Ltd., India. Agarose and 2´,7´-
dichlorodihydrofluorescein diacetate (DCFH-DA) were purchased from Sigma, USA, whilst
propidium iodide (PI) was obtained from EMD Millipore-Calbiochem, USA.
Elemental analyses (carbon, hydrogen and nitrogen) were performed using a Perkin-Elmer 240C
analyzer. Infrared spectra (4000-400 cm-1) were recorded at 28 °C on a Shimadzu FTIR-8400S
spectrophotometer using KBr as the medium. Electronic spectra (1400–200 nm) were obtained at
27 °C using a Shimadzu UV-3101PC spectrophotometer with methanol and acetonitrile as the
solvent and reference. The conductance of methanolic solutions of the complexes was measured
using a SYSTRONICS 306 conductivity meter. Thermal analyses (TG-DTA) were carried out on
a Mettler Toledo (TGA/SDTA 851) thermal analyzer in flowing nitrogen at a rate of 30 cm3 min-
1. X-ray powder diffraction (XRPD) was performed on a XPERT-PRO diffractometer with
monochromated Cu-Kα radiation (40.0 kV, 30.0 mA) at room temperature. Colorimetric analysis
for the MTT was analyzed in a microplate reader Microscan (MS5608A). The fluorescence
image analysis was done under a fluorescence microscope (Nikkon E800), whereas the cell cycle
phase distribution was analyzed on FAC Scan machine (Becton Dickinson).
2.2. Synthesis
5

2.2.1. Preparation of [Ni(HL)(SCN)2(H2O)] (1)
A methanolic solution (5 mL) of N-(2-aminoethyl)piperazine (0.258 g, 2 mmol) was
added dropwise to a hot methanolic solution (10 mL) of salicylaldehyde (0.244 g, 2 mmol) and
the resulting solution was refluxed for 1 h. The mixture was then cooled to room temperature. A
methanolic solution (10 mL) of Ni(ClO4)2.6H2O (0.731 g, 2 mmol) was added to the Schiff base
solution and the resulting solution was stirred for 3-4 h. The solution was then cooled, followed
by the addition of an aqueous solution (5 mL) of NaSCN (0.324 g, 4 mmol). The solution was
allowed to stir further for 2 h. The resulting green solution was filtered, kept in a CaCl2
desiccator in the dark and after a few days crystals suitable for X-ray data analysis were
obtained. Yield 79%. Anal. Calc. for C15H21N5O2S2Ni: C, 42.25; H, 4.92; N, 16.42. Found: C,
42.29; H, 4.98; N, 16.67%. IR (KBr, cm-1): ν(C=N) 1644; ν(skeletal vibration) 1596; ν(SCN-)
2078, 2091; ν(H2O) 3270. λMax/nm(solid, reflectance:) 627, 1013.
2.2.2. Preparation of [Ni(HL)(SCN)2(4,4′-bipy)] (2)
Complex 2 was prepared by following a similar procedure as mentioned for the
preparation of complex 1. Here a 5 mL methanolic solution of 4,4´-bipyridine (0.156 g, 1 mmol)
was added in at the end and the resulting solution was stirred again for 2 h. The final solution
was filtered and kept in a CaCl2 desiccator. After 7 days, light green coloured crystals suitable
for X-ray data analysis were obtained. Yield 56%. Anal. Calc. for C25H27N7O1S2Ni: C, 53.15; H,
4.78; N, 17.36. Found: C, 53.19; H, 4.83; N, 17.31%. IR (KBr, cm-1): ν(C=N) 1647; ν(skeletal
vibration) 1597; ν(SCN-) 2086. λmax/nm (solid, reflectance): 594, 898, 1189.
Caution! Transition metal perchlorate complexes are potentially explosive and should be
handled in small amounts and with proper precautions [36].
6

2.3. X-ray structural determination
The X-ray crystallographic data of the two compounds were collected on a Bruker-AXS
SMART CCD diffractometer at 100(2) K. The crystallographic data, conditions retained for the
intensity data collection and some features of the structure refinements are listed in Table 1. The
intensities were collected with Mo-Kα radiation (λ = 0.71073 Å). Data processing, Lorentz-
polarization and absorption corrections were performed using SAINT, SMART and the
SADABS computer programs [37]. The structures were solved by direct methods and refined by
full-matrix least-squares methods on F2, using the SHELXTL program package [38]. All non-
hydrogen atoms were refined anisotropically. The hydrogen atoms were located from difference
Fourier maps, assigned with isotropic displacement factors and included in the final refinement
cycles by use of HFIX (H bonded to parent C atom) or DFIX (H bonded to parent N or O atom)
of the SHELXTL program. In the case of 2, the electron density of disordered lattice water
molecules was excluded by applying the SQUEEZE procedure [39]. Molecular plots were
performed with the Mercury program [40].
Table 1
Crystallographic data and refinement parameters for complexes 1 and 2
1
2
Formula
C15H21N5NiO2S2
426.20
C25H27N7NiOS2
564.37
Formula Weight
Crystal System
Monoclinic
Monoclinic
7

Space group
a [Å]
P21/n (No. 14)
7.9483(8)
20.843(2)
11.0975(12)
90
C2/c (No. 15)
37.403(2)
9.7786(11)
16.1493(14)
90
b [Å]
c [Å]
α [deg]
β [deg]
98.861(15)
106.416(14)
90
90
γ [deg]
V [Å3]
1816.5(3)
4
5665.8(10)
8
Z
D(calc) [g/cm3]
Mu(MoKa) [/mm ]
F(000)
1.558
1.317
888
1.323
0.862
2352
Crystal Size [mm]
Temperature [K]
Radiation [Å]
0.10 x 0.15 x 0.18 0.20 x 0.23 x 0.30
100
100
MoKα 0.71073
2.0, 26.3
14262, 3690,
0.043
MoKα 0.71073
2.2, 26.4
22062, 5789,
0.030
Θ Min-Max [Deg]
Tot., Uniq. Data, R(int)
Observed data [I > 2.0 σ(I)] 3299
5344
Refinement Nref, Npar
R, wR2, S
3690, 238
5789, 333
0.0483, 0.1272,
1.11
0.0468, 0.1055,
1.17
8

2.4. Computational details
All the initial structures were first constructed using the Spartan package [41]. Initial
optimizations for the constructed structures were performed with the PM3 semi-empirical
method using the Spartan package [41], then density functional theory (DFT) calculations were
carried out using the GAMESS package [42,43]. Gradient corrected geometry optimizations
were performed using the Steven-Basch-Karuss-Jasien-Cundari (SBKJC) [44-46] and the 6-31G
basis sets. The structures were calculated by the code in GAMESS using the Tao-Perdew-
Staroverov-Scuseria functional (TPSS) [47]. Vibrational harmonic frequency analyses of the
optimized structures were computed to examine the minima structures with no imaginary
frequencies. The optimized structures for the two complexes were compared to the crystal
structures. Furthermore, intermediates along the isomerization process were predicted and
optimized with the aim of understanding the mechanism of the process.
2.5. Bioactivity
2.5.1. Cell culture
HeLa (human cervical cancer cell line) and MCF-7 (human breast cancer cell line) cells
were maintained in Dulbecco’s modified eagle medium (DMEM), supplemented with 10% fetal
bovine serum (FBS), antibiotic solution (1000 IU of penicillin and 10mg/mL streptomycin), at
37 °C in a humidified atmosphere of 5% CO2.
2.5.2. Cytotoxicity assay
Cell cytotoxicity was assessed by an MTT assay using the MTT reagent 3-(4,5-
dimethylthiazol-2-yl)-2,5 diphenyltetrazolium-bromide dye as described by Mossman [48].
Briefly, 2×104 HeLa and MCF-7 cells were seeded in 96-well culture plates with various
9

concentrations of complexes 1 and 2 (stock solutions were prepared in DMSO and the final
working stock solutions were prepared by diluting in culture medium; the final concentration of
DMSO used in the case of the IC50 value is 0.1% v/v and 0.15% v/v for complexes 2 and 1
respectively). After 24 h incubation at 37 ºC and 5% CO2, 10 µL of MTT solution (5 mg/mL)
were added to each well, and the samples were incubated further for 2 h at 37 ºC. The purple
formazan crystals formed were finally dissolved in 100 µL of DMSO. Plates were then analyzed
on a microplate reader at 570 nm. At least three independent experiments were carried out in
triplicate. The IC50 value was determined by calculating the percentage (%) cell inhibition.
%inhibition = (Control abs − sample abs) / (Control abs) × 100
2.5.3. Reactive oxygen species (ROS) measurement
Reactive oxygen species (ROS) measurement was performed using the non-fluorescent
dye 2,7-dichlorofluorescein diacetate (DCFH-DA). It diffuses into the cells and gets deacetylated
by esterase to form the non-fluorescent 2,7-dichlorofluorescein (DCFH). In the presence of
reactive oxygen species, DCFH reacts with ROS to form the fluorescent product DCF and which
gets entrapped in the cells. In a 6-well plate, 2 x 105 HeLa cells were grown on cover slips and
allowed to adhere overnight. 234 and 352 µM of complex 1 and 142 and 177µM of complex 2
were added for 24 h. Briefly, the cells were exposed to 10µM DCFH-DA for 30 min at 37 °C.
The cells were then mounted directly in PBS, and were subjected to fluorescence microscopy
[49]. DCF induced fluorescence was detected (excitation 485 nm; emission 530 nm) and
photographed using a fluorescence microscope (Nikkon E800, Japan).
2.5.4. Apoptotic assay
10

Morphological alterations indicating apoptosis were examined by acridine orange (AO)
and ethidium bromide (EB) staining. AO tends to permeate both live and dead cells and stains
the nuclei green, whereas EB is taken up by dead cells when the membrane integrity is lost and
the nucleus stains orange/red. 2 × 105 Hela cells were seeded on cover slips in a 6-well culture
plate. Complexes 1 and 2 treated HeLa cells, as indicated above, were incubated at 37 °C and 5%
CO2 for 24 h. The cells were washed twice and then stained with EB (100 µg/mL) for 2.5 min
and with AO (100 µg/mL) for 1 min at room temperature as modified from a previously
described method [50]. The cells were once again washed with PBS, mounted and then observed
under a fluorescence microscope (Nikkon E800, Japan) and photographed.
2.5.5. Cell cycle analysis
Flow cytometry analysis to check the cell cycle phase distribution of HeLa cells on treatment
with nickel complexes 1 and 2 was carried out after staining fixed HeLa cells with propidium
iodide (PI). For this, 4 × 105 HeLa cells were seeded in a 6-well culture plate in DMEM. After 24
h the cells were exposed to the complexes with concentrations that corresponded to the IC50
value and one concentration lower than the IC50 value for 24 h. After treatment, the cells were
collected by trypsinization, washed with PBS and fixed overnight at -20 °C in 70% ethanol. On
fixation, the cells were washed again with ice cold PBS and incubated in 500 µL propidium
iodide (PI) - RNAse solution [1 mg/mL PI solution, Triton X-100 (0.1% v/v) and 10 mg/mL
RNAse] for 30 min at 37 °C as discussed [51]. The cell cycle phase distribution was analyzed by
FAC Scan using Cell Quest software (Becton Dickinson).
3. Results and discussion
11

3.1. Syntheses and characterization
The complex 1 was prepared by adopting the template synthesis technique by treating a
methanolic solution of nickel(II) perchlorate with the Schiff-base ligand HL, formed in situ via
condensation between salicylaldehyde and N-(2-aminoethyl)-piperazine, followed by addition of
an excess of sodium thiocyanate. Complex 2 was prepared by adding 4,4΄-bipyridine to the
methanolic solution of complex 1. Interestingly, no new species was observed to be generated
when other neutral ligands, like imidazole, pyridine-N-oxide or 2,2′-bipyridine, were added to
the methanolic solution of complex 1. The two complexes were characterized by routine
physicochemical techniques as well as by X-ray single crystal structure analyses (vide infra).
Both complexes exhibit bands due to a C=N stretch in the range 1644-1647 cm-1 and skeletal
vibrations in the range 1596-1597 cm-1. Complex 1 shows strong and sharp bands at 2091 and
2078 cm-1 due to the presence of isothiocyanato ligands, most probably in two different
environments, whereas complex 2 exhibits only one very strong and sharp band at 2086 cm-1
suggesting that both coordinated isothiocyanato ligands are in the same environment [52,53].
Complex 1 also shows a broad band at 3270 cm-1 assigned to the O-H symmetry stretching
vibrational mode of a water molecule coordinated with the metal. (Fig.S1-S2 in the Supporting
Information). The ambient temperature (298 K) magnetic susceptibility measurements (µeff = 3.2
B.M for 1 and 3.3 B.M. for 2) suggest that in both complexes Ni(II) possesses an octahedral
configuration. An electronic spectral study in methanol also supports an octahedral geometry
around Ni(II) in both cases (Fig. S3 in the Supporting Information) [54,55]. A solid state
electronic spectral study has also been performed up to the near-IR region. Complex 1 does not
show a splitting of the d-d band in the range 800-1300 nm, whereas complex 2 exhibits further
splitting of the d-d band in that region (Fig. S4 in the Supporting Information). Those data
12

suggest that complex 1 should have a cis-configuration, whereas complex 2 most probably
adopts a trans-configuration [31,53,55,56].
3.2. Description of the crystal structures of complexes 1 and 2
To determine the structures of complexes 1 and 2 at the atomic level we used crystal X-
ray diffraction. The molecular structures that have been obtained for complexes 1 and 2 are
presented in Fig. 1. Selected bond distances and angles for complexes 1 and 2 are collected in
Table 2. One can see from Figure 1 that in both of the neutral and monoclinic complexes the
Ni(II) centres are octahedral, ligated by O(1), N(1) and N(2) donor atoms of the Schiff base
ligand in a meridonial conformation. The NiN4O2 octahedron in complex 1 is completed by the
N(4) and N(5) atoms of two terminal isothiocyanato groups in the cis position and the O(2) atom
of an aqua ligand. In complex 2, the NiN5O octahedron is completed by the N(4) and N(5) atoms
of two terminal NCS groups in the trans position and the N(6) atom of the bipyridine ligand. The
Ni-N/O bond distances in both complexes are in the range 2.014(2) to 2.313(2) Å. The N(4)-
Ni(1)-N(5) bond angles of the NCS ligands are 88.28(10) and 175.56(9)° for 1 and 2,
respectively. The Ni-N-C bond angles are 141.0(2) and 154.7(2)° in complex 1, and 153.4(2) and
163.2(2)° in complex 2. The N-C-S bond angles are in the range 177.9(3)-179.2(3)°. In complex
1 the N(1)-C(8)-C(9)-N(2) torsion angle is -58.7(3)°, while in complex 2 it is +54.6(3)°. Finally,
the “piperazine” moiety of the Schiff base has a chair conformation in both complexes. In
complex 1 the Ni(1)-N(2) bond is 2.313(2) Å and it is in an axial position with respect to the
chair of the “piperazine” ring, whereas in complex 2 the shorter Ni(1)-N(2) bond of 2.244(2) Å is
in the equatorial position.
13

Table 2
Selected bond distances (Å) and bond angles (°) for complexes 1 and 2.
Distances
(1) (X= O(2))
2.019(2)
2.039(2)
2.048(3)
2.071(3)
2.127(2)
2.313(2)
1.329(4)
1.283(4)
1.167(4)
1.643(3)
1.167(4)
1.649(3)
(2) (X= N(6))
2.014(2)
2.017(2)
2.096(2)
2.066(2)
2.117(2)
2.244(2)
1.315(3)
1.275(3)
1.159(3)
1.652(3)
1.156(4)
1.625(3)
Ni(1)-N(1)
Ni(1)-O(1)
Ni(1)-N(4)
Ni(1)-N(5)
Ni(1)-X
Ni(1)-N(2)
O(1)-C(1)
N(1)-C(7)
N(4)-C(14)
C(14)-S(1)
N(5)-C(15)
C(15)-S(2)
Angles
N(1)-Ni(1)-O(1)
N(1)-Ni(1)-N(4)
O(1)-Ni(1)-N(4)
N(1)-Ni(1)-N(5)
O(1)-Ni(1)-N(5)
N(4)-Ni(1)-N(5)
N(1)-Ni(1)-X
88.95(9)
89.60(8)
88.79(9)
90.64(9)
86.77(9)
89.07(9)
175.56(9)
176.87(9)
173.34(10)
89.00(9)
98.16(10)
93.25(10)
88.28(10)
88.44(9)
14

O(1)-Ni(1)-X
90.69(9)
85.25(9)
172.37(10)
81.09(9)
170.02(9)
100.98(10)
87.51(10)
89.74(9)
154.7(2)
179.2(3)
141.0(2)
177.9(3)
92.40(8)
93.59(9)
90.85(9)
82.69(8)
172.28(8)
89.38(8)
90.31(9)
95.30(8)
163.2(2)
179.1(2)
153.4(2)
178.6(3)
N(4)-Ni(1)-X
N(5)-Ni(1)-X
N(1)-Ni(1)-N(2)
O(1)-Ni(1)-N(2)
N(4)-Ni(1)-N(2)
N(5)-Ni(1)-N(2)
N(2)-Ni(1)-X
C(14)-N(4)-Ni(1)
N(4)-C(14)-S(1)
C(15)-N(5)-Ni(1)
N(5)-C(15)-S(2)
Table 3
Hydrogen bonding system for complex 1 and complex 2
D-H..Aa
Symmetry of A
D..A (Å)
D-H..A (°)
Compound 1
N(3)-H(90)…O(1)
N(3)-H(91)…S(2)
O(2)-H(92)…S(1)
O(2)-H(93)…S(2)
[x+1/2,-y+1/2,z-1/2]
[x+1,y,z]
2.635(3)
3.434(3)
169(4)
170(3)
172(3)
175(3)
[x+1/2,-y+1/2,z+1/2] 3.184(2)
[x+1/2,-y+1/2,z+1/2] 3.384(2)
15

Compound 2
N(3)-H(90)...S(1)
N(3)-H(91)...N(7)
[x,-y,z-1/2]
[-x,-y,-z]
3.290(2)
2.813(3)
146(2)
172(3)
Fig. 1 (a) ORTEP view of complex 1 and (b) ORTEP view of complex 2.
In both complexes, the N(3) atom of the “piperazine” moiety does not coordinate to the
Ni(II) centre, but acts as a donor for hydrogen bonds (see Table 3). In addition, the aqua ligand
in complex 1 is also involved in hydrogen bonding interactions. Therefore, in the crystal
structure of complex 1 the Ni complex molecules are linked by hydrogen bonding interactions
between the aqua ligands and the N(3) atom of the “piperazine”. In complex 2 the pyridine rings
of the bipyridine ligand form an interplanar angle of 24.3° and thus form polymeric layers. Fig.
S5 (see Supporting Information) demonstrates the packing of the 3D polymeric chains in
complexes 1 and 2.
3.3. Conductometric and IR titration study
In order to visualize the change in orientation of the two coordinated isothiocyanato
ligands and thereby the reaction pathway associated with the transformation of 1 to 2, we have
resorted to conductometric and solution phase FTIR spectral studies. First of all the molar
conductance of 1 in a methanolic solution has been determined as 52 Ω-1 cm2 mol-1, indicating its
non-electrolytic nature. Then a methanolic solution of 4,4′-bipyridine was added to the solution
of 1 with constant slow stirring and the change in molar conductance value was monitored with
time. The conductance value was observed to increase and attained a maximum value of 92
16

Ω-1 cm2 mol-1 after 30 min of mixing. Then gradually it decreased in value and attained a
constant value of 26 Ω-1 cm2 mol-1 after 2 h (Fig. S6, Supporting Information). The data clearly
hints that initially after addition of 4,4′-bipyridine, one thiocyanato ligand undergoes dissociation
and giving a 1:1 electrolyte, then after 2 h the dissociated SCN again ligates to produce 2, which
is a non-electrotyte.
Fig. 2(a) depicts the solid state overlapped FT-IR spectra of 1 and 2 in the range 1900-
2250 cm-1, the fingerprint region for the SCN stretching frequency. In 1 the SCN groups are in
the cis position, whereas in 2 they have a trans orientation. A solution phase ATR study has been
carried out according to the following manner. First, the FT-IR spectrum of 1 in methanol was
recorded [Fig. 2 (b)(i)], which shows two bands centered at 2107 and 2010 cm-1, characteristic of
cis-thiocyanato groups. Then 4,4′-bipyridine was added to the solution of 1 with stirring and
spectra were recorded after 30 min [Fig. 2 (b)(ii)], 1 h [Fig. 2 (b)(iii)] and 2 h [Fig. 2 (b)(iv)]. It
is very clear from those spectra that after 2 h the complete transformation of the coordinated
isothiocyanato ligands from the cis to trans orientation takes place, as is evident from the
generation of a new band at 2045 cm-1, with the earlier two bands completely vanishing.
We have also performed an experiment where complex 2 is treated with excess water, with the
view to understand whether complex 1 could be regenerated from complex 2 on dilution. We did
not get any change in the FT-IR spectral pattern on dilution and thereby nullify that possibility.
This fact also implies that complex 2 is more stable than 1 and that view has further been
supported by DFT calculations (vide infra).
Fig.2 (a) Overlapped FT-IR spectra of complexes 1 and 2 in the range 1900-2250 cm-1 in the
solid state. (b) FT-IR spectra of the transformation of the cis to trans arrangement of the
17

isothiocyanate ligands in solution (i) before addition of 4,4′-bipyridine; (ii) 30 min after addition
of 4,4′-bipyridine; (iii) 1 h after addition of 4,4′-bipyridine and (iv) 2 h after addition of 4,4′-
bipyridine.
3.4. Geometry optimization and computed IR spectra of complexes 1 and 2
The cis-orientation of the two isothiocyanato ligands in complex 1 changes into a trans
one after addition of 4,4΄-bipyridine. To rationalize the fact via DFT calculations, first we
optimized complexes 1 and 2 (see Fig. 3). The optimized results suggest that 2 is more stable
than 1 by almost 43 kcal/mol. To get a more clear picture we optimized an analogous molecule
to complex 2, but where the two SCN- ligands are in a cis fashion and the bipyridine ligand is in
an axial position, namely cis-[Ni(HL)(NCS)2(4,4′-bipyridine] (see Fig. S7 in the Supporting
Information). This hypothetical molecule has ~60 kcal/mol higher energy than complex 2. Thus,
the energy calculations clearly hints why complex 2 is formed from 1 on addition of 4,4΄-
bipyridine instead of cis-[Ni(HL)(NCS)2(4,4′-bipyridine].
Fig. 3 Optimized geometries of complexes 1 and 2.
To examine the minima of the complexes, the vibrational frequencies were computed and they
showed no imaginary frequencies. We have summarized the computed bonds and angles to
illustrate the comparison between the computed values and experimental values for complexes 1
and 2 (see Table S1-S2 in Supporting Information). From these tables it is evident that the
computed values are in good agreement with the experimental results. The averaged deviations
18

for the distances in complexes 1 and 2 are ~ 4% and ~ 5% and for the angles they are ~ 4 % and
~ 15%, respectively.
We further compared the calculated vibrational frequencies to the experimental
vibrational frequencies. Tables 4 and 5 summarize the selected vibrational frequencies of
complexes 1 and 2, respectively. Here also the calculated vibrational frequencies are in excellent
agreement with the observed vibrational frequencies.
Table 4
Selected vibrational frequencies of complex 1.
Vibrational mode
description
Calc. frequency
(cm-1)
Exp. frequency
(cm-1)
1596
Benzene ring mode
Stretching of C=N
Stretching of SCN
1590
1639
1644
1998
2016
2130
2107
H2O
3283
3270
Table 5
Selected vibrational frequencies of complex 2.
Vibrational mode
description
Calc. frequency
Exp. frequency
(cm-1)
(cm-1)
1536
1636
Benzene ring mode
Stretching of C=N
1597
1647
19

Stretching of SCN
2103
2044
From the results of the conductometic and FTIR titrations we proposed a probable mechanistic
pathway of the observed interesting transformation (Scheme 2) and tried to correlate them using
energy minimization. To understand the mechanism of the formation of trans-[Ni(HL)
(NCS)2(4,4′-bipyridine] from cis-[Ni(HL)(SCN)2(H2O)] we predicted more than ten optional
intermediates (not all shown here), considering the various ligands that might replace the aqua
ligand and the two NCS- ligands of the complexes. Our calculations predicted three intermediates
along the transformation process. It should be noted here that we did not calculate the activation
energies, the transition states or the barriers. Here, we present only the energy differences
between the intermediates along the proposed mechanistic pathway. Fig. 4 illustrates the energy
associated with the complexes and the probable intermediates in the proposed mechanism.
20

Scheme. 2 Probable mechanism of the formation of trans-[Ni(HL) (NCS)2(4,4′-bipyridine] from
cis-[Ni(HL) (SCN)2(H2O)].
Fig. 4 Energy profile diagram of the formation of complex 2 from complex 1.
Our calculations suggest that in the first step the aqua ligand “leaves” the Ni(II) centre
from complex 1 to form intermediate I, which has trans located SCN groups. This process is
endothermic and probably occurs when complex 1 is solvated in methanol. The cis orientation of
the two SCN groups is less preferred, since the cis complex without the aqua ligand showed an
energetically less stable structure (by ~ 12 kcal/mol). In the next step methanol “replaces” the
aqua ligand to form a cis or trans complex, i.e., intermediates II or III. The energy difference
21

between these two intermediates is ~ 0.31 kcal/mol (with the cis complex being slightly more
stable than the trans complex), thus we suggest that an equilibrium between these two
intermediates occurs in this step. This step, which is exothermic, is likely to be very crucial in the
transformation process, since it determines whether the bipyridine ligand will lead to the cis
complex or the trans complex. As noted above, the trans complex is energetically more stable
than the cis complex by 60 kcal/mol, thus after adding bipyridine to the solution, trans-
[Ni(HL)(NCS)2(4,4′-bipyridine] is formed.
We also computed the IR spectra of these three intermediates by following the νSCN
vibrational frequency. Intermediate I exhibited two vibrational frequencies at 2120 and 2036 cm-
1, intermediates II and III also showed two vibrational frequencies: Intermediate II at 2135 and
2008 cm-1 and intermediate III at 2117 and 1999 cm-1. However, the final product trans-[Ni(HL)
(NCS)2(4,4′-bipyridine] revealed only one vibrational frequency at 2044 cm-1. In summary,
bipyridine plays role in stabilizing the complex and in forming a complex that is characterized by
only one band for νSCN in the IR spectrum.
3.5. Bio-activity study
3.5.1. Cytotoxicity assay
The cytotoxic potential of complexes 1 and 2 was tested on HeLa and MCF-7 cells.
Treatment of HeLa and MCF-7 cells with the complexes for 24 h resulted in growth inhibition
(Fig. 5). Complex 2 showed a cytotoxic effect at IC50 = 177 µM (100 µg/mL), while complex 1
showed cytotoxic activity at IC50 = 351 µM (150 µg/mL) for HeLa cells. At the same time both
complexes showed quite a low cytotoxic effect on MCF-7 cells, with IC50 values around 410 µM
(175 µg/mL) for complex 1 and 355 µM (200 µg/mL) for complex 2. The percentage of cell
22

inhibition with respect to complexes 1 and 2 against both the cell lines are shown in Fig. 5A and
5B. Our results indicate that complex 2 is more efficient in inhibiting the growth of HeLa cells in
comparison to complex 1 after 24 h. However, in the case of MCF-7 cells, both complexes
exhibited a low cytotoxic activity as compared to HeLa cells. It is important to note that the
concentration of DMSO is so low that it will not show any toxicity, as the cytotoxicity of DMSO
is more than 1% in the case of normal cells. In 2013, a study by Milenkovi et al also showed the
cytotoxic activity of three square-planar complexes of Ni(II) with ethyl (2E)-2-[2
(diphenylphosphino)benzylidene]hydrazinecarboxylate and monodentate pseudohalides, the
appropriate ligand, and a nickel salt by MTT assay after 48 h treatment in six tumor cell lines
(A549, MDA-MB-361, Hela, FemX, LS-174 and K562) [57]. They reported that all the
complexes showed cytotoxicity towards all cell lines and the activity was increased compared to
the ligand and also the complexes showed more cytotoxicity towards K562 cells in comparison
to other cell lines. Similarly, the antiproliferative activities of three complexes, Mn(II), Ni(II)
and Cu(II), of demethylcantharate and 2-aminopyridine and [Na2(DCA)] against human
hepatoma cells (SMMC- 7721) and human gastric cancer cells (MGC80-3) also showed that the
complexes had a strong antiproliferative effect against the two types of cancer cells [58].
Fig. 5 The cytotoxicity profile of Ni complexes 1 and 2 against HeLa [A] and MCF-7 [B] cells
at different concentrations was estimated by an MTT assay. Data are expressed as a percentage
of control SEM from three independent experiments.
3.5.2. ROS measurement
23

Based on the results obtained from the cell cytotoxicity assay, we found that the
nickel(II) complexes showed more activity towards HeLa than MCF-7 cells, therefore the HeLa
cell line was selected for further investigations related to anticancer activity. To examine the
generation of ROS in HeLa cells on treatment with the complexes, DCFH-DA dye was used.
HeLa cells were exposed to DCFH-DA after treatment with complexes 1 and 2, along with a
control, for 24 h. Both the complexes induced ROS generation in a concentration dependent
manner after 24 h, as reflected by the increase in fluorescence intensity (Fig. 6). A higher amount
of ROS was generated at their IC50 values [complex 1 - 150 µg/mL (351 µM) and complex 2 -
100 µg/mL (177 µM)] than at a concentration lower than the IC50 value in comparison to the
control (Fig. 6). ROS is found to trigger apoptosis in cells [59]. Some other nickel and copper
derivatives [Ni(II) and Cu(II) N(4)-ethylmorpholine citronellal thiosemicarbazonate] also
triggered ROS generation in the HT29 cell line, showing a remarkable and rapid ROS increase,
and it was proposed that perhaps the ROS could be mediating the process of cellular damage
[60]. Similarly, changes in the intracellular ROS were detected using fluorescence spectrometry
and the fluorescence intensity was measured by DCF after treatment of HeLa cells for 6 h with
the Cu(II) complex of the ethyl 2-[bis (2-pyridylmethyl) amino] propionate ligand and the results
showed that the concentration of the ROS increased in a dose- and time-dependent manner
compared to a control group [61].
On the basis of our results, we suggest that both the Ni complexes could induce apoptosis
in cancer cells, as a ROS is generated which may lead to cellular damage. To validate the above
statement, we performed fluorescent staining with acridine orange and ethidium bromide to
analyse the apoptosis.
24

Fig. 6 Detection of a ROS by measuring the fluorescence intensity in HeLa cells using a DCFH-
DA probe under a fluorescence microscope. Cells were treated with 234 and 351 µM of complex
1 and 142 and 177 µM of complex 2 for 24 h.
3.5.3. Apoptotic assay
During apoptosis various structural changes take place, like nuclear and cytoplasmic
condensation, breaking up of the cell into membrane bound fragments and formation of apoptotic
bodies which are engulfed and undergo autolysis within phagosomes, and are rapidly degraded
by lysosomal enzymes [62]. The AO/EB staining of HeLa cells was done to determine apoptosis
on treatment with complexes 1 and 2 (Fig. 7) and the image obtained shows the presence of
apoptotic cells. The cells with green fluorescence and an intact green nucleus represent the live
cells. The apoptotic morphology of cells in the treated samples shows cellular shrinkage,
membrane blebbing, nuclear fragmentation and chromatin condensation. At 24 h of incubation of
HeLa cells with both complexes, we observed that at a lower concentration they showed an early
stage of apoptosis with presence of membrane blebbing and some cells with nuclear
fragmentation, but at higher concentration, that is the IC50 concentration, they showed later
stages of apoptosis with most of the cells undergoing nuclear fragmentation (Fig. 7). Similar
results were also reported by Luying Li, et al in 2013, to identify the possible involvement of
apoptosis in HeLa cells treated with the Cu(II) complexes CuPDTP, CuADTP and CuBFDTP for
24 h, on staining with AO and EB. Fluorescence images of the AO/EB-stained HeLa cell showed
apoptotic cells as well as some of them even showing secondary necrosis. On treatment of the
cells with significant amounts of the Cu(II) complexes, the cells exhibited condensed orange
25

nuclei, which is a hallmark of late apoptotic cells; however, the necrotic cells displayed
structurally intact nuclei with orange staining [63].
We therefore suggest that the results obtained in our case substantiate that the complexes
induce apoptotic cell death in cancer cells that might be triggered by the process of ROS
generation by the complexes.
Fig. 7 (A-E) showing AO/EB dual staining of HeLa cells to identify changes in the nuclear
morphology. Figure (A) represent control cells with green fluorescence, figures (B - C) and (D -
E) represent complexes 1 and 2 treated cells respectively. The blue arrows show live cells, red
arrows show apoptotic cells and red dotted arrows represent the apoptotic cells with membrane
blebbing.
3.5.4. Cell cycle analysis
To identify whether the complexes have any effect on cell cycle phase distribution or not,
cell cycle analysis was done by flow cytometry after exposure of HeLa cells to complexes 1 and
2 for 24 h. The results of the FACS analysis have been presented in the form of a histogram
(Figure 8) which shows that both the complexes induced a shift of cell population in the cell
cycle phases compared to the control. Treatment at the IC50 value for both complexes 1 (351 µM)
and 2 (177 µM) showed a pronounced effect on the cell cycle, as it induced an increase of the
percentage of the cells in the S phase, but a decrease in the percentage of cells at the G1 and G2
phase, in comparison to the control, suggesting an arrest of the cell cycle in the S phase. In the
case of complex 1 about 15% of cells are in the S phase at its IC50 value, and in the case of
complex 2 double the number of cells, that is 18%, are in the S phase at its IC50 value, as
26

compared to the control. In the same manner, Milenkovi et al. in 2013 also reported three square-
planar complexes of Ni(II) with ethyl (2E)-2-[2-(diphenylphosphino) benzylidene]
hydrazinecarboxylate and monodentate pseudohalides that caused changes in the cell cycle
distribution. They showed that all the investigated nickel complexes induced an increase of the
percentage of cells in the sub-G1 phase and a decrease of the percentage of cells in the G1 phase
compared to the control, while two of their compounds induced an increase in the percentage of
cells in the S and G2 phase only at the highest concentration [57].
Fig. 8 Analysis of the cell cycle distribution of HeLa cells on treatment with complexes 1 and 2
after 24 h by flow cytometry analysis. Control (a), and after 24 h treatment with nickel
complexes 1 (b-c) and 2 (d-e).
3.6. Solid state thermal studies of complexes 1 and 2
The thermal stabilities of complexes 1 and 2 have been investigated using thermo-
gravimetric analysis (TGA) in the temperature range 30-700 °C in a single run in a dynamic
nitrogen environment. The TGA results for complexes 1 and 2 are shown in S8 (see Supporting
Information). The TGA results for complexes 1 and 2 indicate that the complexes are stable up to
80 and 136 °C, respectively. For complex 1, the first step weight loss is 4.5% (calc. 4.88%) in
the temperature range 80-160 °C, which corresponds to the loss of one coordinated water
molecule. In the IR spectrum, the broad band at around 3270 cm-1 that was observed for complex
1 is due to an OH stretching vibration, and this band was absent in the dehydrated species,
confirming the elimination of the water molecule on heating. For complex 2, the weight loss
starts at 136 °C. One can see that in the case of complex 2, two steps of decomposition occur:
27

one appears at 136 °C and second after heating over 136 °C. Finally, for both complexes 1 and
2, NiO is obtained as the thermally stable final product, which is further characterised by PXRD
(see S9 in Supporting Information file). For complex 1 the total experimental weight loss is
82.37% (calc. 82.6%) at 609 °C and for complex 2 the total experimental weight loss is 87.3%
(calc. 86.7% ) at 559 °C.
4. Conclusions
On reaction of the Schiff base ligand 2-[(2-piperazin-1-yl-ethylimono)-methyl]phenol
(HL), prepared via condensation of salicylaldehyde and N-(2-aminoethyl)-piperazine, with
nickel(II) perchlorate followed by sodium thiocyanate, complex 1, [Ni(HL)(SCN)2(H2O)] is
obtained. X-ray single crystal structural analysis reveals that the nitrogen atom of the piperazine
moiety is protonated, which is responsible for the coordination of two isothiocyanato ligands to
attain electroneutrality and the two isothiocyanato ligands are in the cis position. Complex 1 on
reaction with 4,4΄-bipyridine (4,4΄-bpy) yields another mononuclear complex,
[Ni(HL)(SCN)2(4,4΄-bpy)] (2), with the configuration of the isothiocyanato ligands changing
from cis to trans. This configuration change has been monitored by IR titration and
conductometric studies, and a reaction mechanism has been proposed. Detailed DFT
calculations, which reveal the higher stability of the trans species by ~43 kcal mol-1 over the cis
one, have been performed to rationalize the proposed mechanism. The bioactivity of the two well
characterized complexes as antitumor agents has been investigated extensively. The synthesized
nickel complexes show good anticancer activity. Both the complexes exhibited cytotoxicity
towards the tested cancer cell lines, having a preference to the HeLa cell line over the MCF-7
cell line. The activity of both complexes was shown from the cell cycle distribution (increase of
28

the percentage of cells in the S-phase and decrease of the percentage of cells in the G1 phase)
and the property to induce generation of ROS, possibly leading to apoptotic cell death. Both the
complexes are found to be active, though complex 2 seems to be more promising. Further
detailed investigation, which is underway in our laboratory, is necessary to find out the
applicability of the tarns-variety as an antitumor agent and to find out the reason, most likely
different from the operative mechanism of cis-platin, behind the better activity of the trans
species.
Appendix A. Supplementary data
CCDC 971172 and 971173 contain the supplementary crystallographic data for complexes 1 and
2.
These
data
can
be
obtained
free
of
charge
via
http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic Data
Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-
mail: deposit@ccdc.cam.ac.uk. Supplementary data associated with this article can be found in
the online version.
Acknowledgements
This work was supported by the Council of Industrial and Scientific Research, New Delhi [CSIR
project no. 01(2464)/11/EMR-II dated 16-05-2011 to DD]. The authors wish to thank the Head,
Department of Zoology, Faculty of Science, Banaras Hindu University for extending laboratory
facilities and the Coordinator, Interdisciplinary school of life sciences, Banaras Hindu University
for providing the FACS facility.
References
29