Synthesis, characterizations, crystal structures, BSA-binding, molecular docking, and cytotoxic activities of nickel(II) and copper(II) coordination complexes with bidentate N,S-chelating ligand

Article abstract of DOI:10.1080/00958972.2019.1602867

In the present investigation, we report the synthesis and characterization of three novel nickel(II) [Ni(L)₂] (1) and copper(II) [Cu(L)₂] (2a) and [Cu(L)₂] (2b) complexes, which are made through the coordination of (E)-1-(

Full text of DOI:10.1080/00958972.2019.1602867

Journal of Coordination Chemistry
ISSN: 0095-8972 (Print) 1029-0389 (Online) Journal homepage: https://www.tandfonline.com/loi/gcoo20
Synthesis, characterizations, crystal structures,
BSA-binding, molecular docking and cytotoxic
activities of nickel(II) and copper(II) coordination
complexes with bidentate N,S-chelating ligand
Noorussabah, Mukesh Choudhary, Achintya Jana, Neeladri Das, B. Mohan, K.
Ahmad, Sangeeta, S. Bharti, M. K. Mishra & S. R. Sharma
To cite this article: Noorussabah, Mukesh Choudhary, Achintya Jana, Neeladri Das, B. Mohan,
K. Ahmad, Sangeeta, S. Bharti, M. K. Mishra & S. R. Sharma (2019): Synthesis, characterizations,
crystal structures, BSA-binding, molecular docking and cytotoxic activities of nickel(II) and
copper(II) coordination complexes with bidentate N,S-chelating ligand, Journal of Coordination
Chemistry, DOI: 10.1080/00958972.2019.1602867
To link to this article: https://doi.org/10.1080/00958972.2019.1602867
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Synthesis, characterizations, crystal structures, BSA-binding, molecular
docking and cytotoxic activities of nickel(II) and copper(II) coordination
complexes with bidentate N,S-chelating ligand
NOORUSSABAH†, MUKESH CHOUDHARY1†, ACHINTYA JANA‡, NEELADRI DAS‡, B. MOHAN†,
K. AHMAD†, SANGEETA†, S. BHARTI†, M. K. MISHRA† and S. R. SHARMA†
†
Department of Chemistry, National Institute of Technology Patna, Patna 800005, India
Department of chemistry, Indian Institute of Technology Patna, Patna 801106, India
‡
In the present investigation, we report the synthesis and characterization of three novel nickel(II)
Ni(L) ] (1) and copper(II) [Cu(L) ] (2a) and [Cu(L) ] (2b) complexes, which are made through
[
2
2
2
the coordination of (E)-1-(2-methoxybenzylidine)-4-phenylethiosemicarbazone (HL) as
bidentate N,S-ligand. Several modern techniques including experimental electronic and
fluorescence spectroscopy, single-crystal X-ray crystallography, molecular docking and BSA-
binding are used to characterize the isolated coordination compounds. X-ray crystallography,
FT-IR and UV-visible spectra agree with the observed crystal structures. The crystallographic
and spectroscopic studies confirmed that these complexes display four-coordinate square planar
trans-[MN S ] coordination geometry, whose central metal(II) atom lies on the center of
2
2
symmetry. Complexes 1 and 2a crystallized in the orthorhombic system of the space group Pbca
whereas 2b crystallized in the monoclinic system of P2 /c. The binding affinity of complexes
1
towards bovine serum albumin (BSA) was determined by UV-visible and fluorescence
spectrophotometric methods. The cytotoxic/antiproliferative potential of the synthesized
compounds on human cell lines was also investigated by MTT assay.
1
Corresponding author. Email: mukesh@nitp.ac.in

Graphical abstract
Keywords: Coordination complexes; BSA Binding studies; Molecular docking; Protein
interaction; Cytotoxic studies
1
. Introduction
The synthesis and study of low molecular weight coordination complexes of nickel(II) and
copper(II) bound by nitrogen and sulfur ligands are very important because of their pivotal roles
in bioinorganic chemistry, potential catalytic applications [1-4] and BSA binding [5, 6]. Over the
last few decades, thiosemicarbazones and their metal complexes have received considerable
attention in view of their structural diversity, variable binding modes, and promising biological
implications [7-9]. Thiosemicarbazone and/or 4-phenylthiosemicarbazide derived Schiff bases
possess a wide range of biological properties and they act as antibacterial, antifungal, antitumor,
anticancer and anti-inflammatory agents [10-12]. The biological activity of thiosemicarbazone is
related to its chelating ability with transition metal ions, and its coordination to metal ions
through nitrogen and sulfur atoms allows it to act as a potent cytotoxic compound and enhance

the biological activity [13, 14]. The deprotonated thiosemicarbazone ligands usually coordinate
to Pt(II), Pd(II), Cu(II), Ru(II) and Os(II) through N,S-donor atoms in their bidentate form or
tridentate form, to form metallic complexes of different molecular geometry [15, 16]. The square
planar Pt(II) and Pd(II) complexes with thiosemicarbazone ligands derived from
phenylacetaldehyde and 2-formylpyridine showed high cytotoxicity in vitro against HL60
leukemia and P388 mouse leukemia cell lines [17], while Pt(II) and Pd(II) complexes with
p-isopropylbenzaldehyde thiosemicarbazone ligands exhibit strong cytotoxic activities on mouse
tumor cell growth inhibition [18]. Jayabalakrishnan et al. [19] reported two 4-phenyl-3-
thiosemicarbazone ligands and its Ru(II) complexes focused on the binding and cleavage
properties with CT-DNA. The interesting observation was that in all the complexes reported by
them the in vitro cytotoxicity of the ligands and complexes assayed against HeLa and MCF-7
cell lines showed higher cytotoxic activity with the lower IC values indicating their efficiency
5
0
in killing the cancer cells even at low concentrations. Casas et al. [20] reported a series of
dimethyl Tl(III) complexes of acetylferrocene thiosemicarbazonates as a N,S-chelating ligand,
forming either a four-membered or a five-membered chelate ring depending on the substituent on
the N(4) atom. The four-membered chelate rings were also observed [21-23] for organometallic
derivatives of group 13 elements (Al, Ga and In), for Pd as well as other dimethyl Tl(III) and
Ru(II) complexes [24].
DNA is the primary target molecule for most anticancer drugs as it regulates many
biochemical processes occurring in the cellular system. Many small molecules exert their
anticancer effect by interfering with DNA-replication leading to cell death through apoptosis.
Binding studies of small molecules to DNA are very important in the development of new
therapeutic reagents and DNA molecular probes [25]. It has been shown that the 훼-(N)-
heterocyclic carbaldehyde thiosemicarbazones act as chelating agents of the transition metals and
some of them exhibit antitumor activity by inhibiting the biosynthesis of DNA, possibly by
blocking the enzyme ribonucleotide diphosphate reductase [26, 27]. In particular, ruthenium(II)
organometallics represent one of the latest trends in metallodrug research [28]. Among these
DNA-targeted guest molecules, copper(II)- and nickel(II)-based compounds have attracted much
attention because of low toxicity and their promising anticancer activity [29-31], DNA-binding
and -cleavage activity [32], metallodrugs [33]. Bovine serum albumin (BSA) has been widely
used as a model protein to study drug protein interactions due to its structural similarity with

human serum albumin (HSA), its low cost, stability, ease of purification, abundance and ligand
binding properties [34]. Schiff base metal complexes can bind to BSA with high affinity, thereby
quenching BSA’s intrinsic fluorescence and inducing conformational changes [35].
Literature reveals that the syntheses and structural studies of Cu(II) and Ni(II) complexes
of 2-methoxybenzylidine derivative of 4-phenylthiosemicarbazide with molecular docking into
the grooves of target DNA- and BSA-binding is missing and, to date, no further investigations
are considered; this impelled us to make the present study. Inspired by recent studies on metal-
coordinated Schiff bases for their BSA- and protein-binding [35, 37] and based on our good
experience on Schiff base Ni(II) and Cu(II) coordinated complexes [38-41], in this paper we plan
to synthesize, characterize as well as to study their molecular docking and BSA-binding of three
novel nickel(II) [Ni(L) ] (1) and copper(II) [Cu(L) ] (2a) and [Cu(L) ] (2b) complexes with
2
2
2
(E)-1-(2-methoxybenzylidine)-4-phenylethiosemicarbazone (HL) as bidentate N,S-ligand. A
solid-state structure is planned to be determined by X-ray analysis. The crystal structure
determination also confirmed that the structures of all complexes are isostructural. The refined
single-crystal structures are further subjected to molecular docking into the grooves of target
DNA and binding affinity of complexes with the DNA and interacting residues of DNA was
evaluated and discussed. Further protein binding constant (K ), quenching constant (K ) and
b
SV
number of binding sites (n) of complexes towards BSA was determined by using fluorescence
spectroscopy. The cytotoxic/antiproliferative potential of the synthesized compounds on human
cell lines was also investigated by MTT assay.
2
2
. Experimental
.1. Materials
Chemicals used were of analAR grade. 4-Phenyl-thiosemicarbazide, 2-methoxy-benzaldehyde,
Ni(OAc) ·4H O, Cu(OAc) ·H O and Cu(NO ) ·3H O were obtained from Sigma. Human
2
2
2
2
3 2
2
embryonic kidney (HEK293), human heptoma cells (HepG2) and human prostate carcinoma
LNCaP) cells were procured from National Centre for Cell Sciences, Pune, India. Dulbecco’s
(
modified eagle’s media (DMEM), RPMI-1640 medium, antibiotic cocktail and fetal bovine
serum (FBS), TrypLE™ Express enzyme solution and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide) were obtained from Gibco-life Technologies, Thermo Fisher
Scientific (USA). Used solvents were dried and purified as reported standard methods [42, 43].

2
2
.2. Preparations
.2.1. Synthesis of (E)-1-(2-methoxybenzylidine)-4-phenylethiosemicarbazone (HL). The
ligand HL was synthesized according to an analogous method reported earlier [44, 45]. HL, a
bidentate N,S-ligand, was synthesized by refluxing equimolar mixtures of
4
-phenylthiosemicarbazide (10 mmol, 1.67 g) and 2-methoxybenzaldehyde (10 mmol, 1.36 g) in
dry methanol at 55-60 C for 3 h. A colorless solution was obtained and was left overnight. The
colorless crystalline product of HL was obtained, filtered off, washed with ethanol, and stored in
a desiccator over CaCl . The Schiff base HL gave satisfactory elemental analysis. Yield: 2.48 g
2
(
82%). Color: colorless, melting point: 178 C. Anal. Calc. for C H N OS, HL (%): C, 63.11;
1
5
15
3
H, 5.29; N, 14.70. Found (%): C, 63.13; H, 5.30; N, 14.73. FAB-mass (m/z): Obs. (Calcd):
2
85.09 (286).
2
.2.2. Synthesis of [Ni(L) ] (1). HL (2.0 mmol, 0.570 g) was dissolved in ethanol (20 mL) in the
2
presence of triethylamine (3.0 mmol, 40 µL) and was combined with Ni(OAc) ·4H O (1.0 mmol,
2
2
0
.249 g) with constant stirring and heating (60 C). On cooling the solution to room temperature,
green crystals suitable for single-crystal X-ray diffraction were recovered from mother liquor
which were filtered off, washed with ethanol and stored in a desiccator over CaCl . Yield: 0.57g
2
(
70%). Color: green, melting point: 240 C. Anal. Calc. for C H NiN O S (1) (%): C, 57.40;
3
0
28
6
2 2
H, 4.47; N, 13.37. Found: C, 57.43; H, 4.50; N, 13.39. FAB-mass (m/z): Obs. (Calcd): 627.41
628).
(
2
.2.3. Synthesis of [Cu(L) ] (2a). HL (2.0 mmol, 0.570 g) was dissolved in ethanol (20 mL) in
2
the presence of triethylamine (3.0 mmol, 40 µL) and was combined with Cu(OAc) ·H O
2
2
(1.0 mmol, 0.1998 g) with constant stirring and heating (70 C). On cooling the solution to room
temperature, light-brown crystals separated and were filtered off, washed with MeOH and stored
in a CaCl desiccator. Complex 2a gave satisfactory elemental analysis. Yield: 0.524 g (76%).
2
Color: light blue, melting point: 208 C, Anal. Calc. for C H CuN O S (2a) (%): C, 56.95; H,
3
0
28
6
2 2
4
.45; N, 13.27. Found: C, 56.99; H, 4.46; N, 13.29. FAB-mass (m/z): Obs. (Calcd): 632.25(633).

2
.2.4. Synthesis of [Cu(L) ] (2b). To an ethanol solution (20 mL) of Cu(NO ) ·3H O (1.0 mmol,
2 3 2 2
0
.241 g) was stirred with ethanol solution of HL (2.0 mmol, 0.570 g) continuously for 3 h after
adding 2 drops of triethylamine. To the above reaction mixture, ammonium thiocyanate
2.0 mmol, 0.116 g) in MeOH (20 mL) was added with constant stirring. One minute later, pink
(
color turned to pink-red and the odor of ammonium thiocyanate was detected. The pink-red
crystals suitable for single-crystal X-ray diffraction were recovered from mother liquor and were
filtered off, washed with MeOH, and stored in a desiccator over CaCl . Complex 2b gave same
2
elemental analysis as 2a. The basic difference between the structures of 2a and 2b was that 2a
was crystallized in the orthorhombic system of space group Pbca whereas 2b crystallized in the
monoclinic system of the space group P21/c.
2
.3. Physical measurements
Elemental analyses were made on an ElementarVario EL III Carlo Erba 1108 analyzer. FAB
mass spectra were recorded on a JEOL SX 102/DA 6000 mass spectrometer using xenon (6 kV,
1
0 mA) as the FAB gas. The accelerating voltage was 10 kV and the spectra were recorded at
room temperature (RT) with m-nitro-benzoyl alcohol as the matrix. Magnetic susceptibility
measurements of powder samples of complexes were made on a Gouy balance using a
-6
mercury(II) tetrathiocynato cobaltate(II) as calibrating agent (χ = 16.44×10 c.g.s. units). All
g
the experimental data were corrected for diamagnetic contributions, which were estimated from
Pascal’s tables, and temperature-independent paramagnetism (TIP). The molar ion exchange was
-3
measured using a systronics digital conductivity meter (TDS-308) using a 10 M solution in
DMSO. FT-IR spectra were recorded on a Shimadzu IRAffinity-1S Fourier Transform Infrared
-1
Spectrophotometer from 4000-400 cm on KBR pellets. UV-visible spectra were obtained on a
Thermo scientific Evolution-300 spectrophotometer. Fluorescence spectroscopic data were
measured on a Fluoromax-4 Spectro-fluorometer. Single-crystal X-ray structure determination
was carried out on a Bruker Smart 1000 CCD area detector diffractometer.
2
.4. X-ray crystallographic analysis
Crystals suitable for single-crystal X-ray analysis for 1, 2a and 2b were grown by slow
evaporation of the reaction mixture at room temperature. Single-crystals suitable for single-
crystal X-ray analysis were mounted on glass fibers and data were collected on a Bruker Smart

CCD detector using graphite monochromated Mo Kα radiation (λ = 0.71073 Å) at 296 K. The
structures were solved by direct methods using SHELXS-97 [46] and refined by full-matrix
2
least-square techniques [47] against F using SHELXL-97. All non-hydrogen atoms were refined
using anisotropic thermal parameters. Hydrogens were geometrically fixed and allowed to refine
isotropically. Crystallographic data for the structures reported in this paper have been deposited
with the Cambridge Crystallographic Data Centre (CCDC) as supplementary publication
numbers CCDC 1565755, 1836258 and 1837668 for 1, 2a and 2b, respectively.
2
.5. Protein-binding studies
The protein binding of 1 and 2a with BSA was studied by fluorescence spectra recorded at a
fixed excitation wavelength corresponding to BSA at 280 nm and monitoring the emission at
3
48 nm. The excitation and emission slot widths and scan rates were maintained constant for all
the experiments [48]. Stock solution of BSA was prepared in 50 µM phosphate buffer (pH = 7.2)
and stored in the dark at 4 C for further use. Concentrated stock solutions of each test
compound were prepared by dissolving them in DMF-Phosphate buffer (5:95) and diluted with
phosphate buffer to get required concentrations. 2.5 mL of BSA solution was titrated by
-6
successive addition of a 10 M stock solution of the complexes using a micropipette. In UV-
visible absorption experiment, a fixed concentration of BSA was titrated with increasing
concentration of complexes.
2
.6. Molecular docking studies
Molecular docking investigations of 1 and 2a have highlighted the accurate and preferred
orientation of the compounds at the binding sites of the DNA. These dimensional coordinates of
target DNA (duplex of sequence d(CGCGAATTCGCG)2 dodecamer) were taken from protein
Data Bank with PDB ID IBNA. The given compounds were used for docking studies with DNA
to predict the bound confirmations the binding affinities. AutoDock4 [49] and Auto Dock Vina
+
[
[
50] were used for docking purpose. PyMol [51], Discovery Studio Visualizer [52] and LigPlot
53] were used for visualization purpose to visualize the different interactions between the target
and given compounds.

2
.7. Cell culture and cytotoxicity studies
HEK293 and HepG2 cells were maintained and cultured in DMEM media and LNCaP cells were
cultured in RPMI-1640 medium (supplemented with 10% heat inactivated FBS and 1%
penicillin, streptomycin solution) in a humidified incubator (5% CO ) at 37 °C. The cultures of
2
each cell line were maintained and trypsinized not more than 30 passages, after the revival of
cryostocks. The cytotoxicity of each compound was evaluated with the help of MTT assay as
described earlier [54, 55]. Briefly, the cells were plated (9000-10000 cells/well) in a 96-well cell
culture plate and grown overnight. Next day, to each cell line the treatment were given with
increasing concentrations (0–200 μg/mL) of the compounds for 72 h at 37 C in a CO incubator.
2
After 72 h, the mixture of cell culture medium and compounds were pipetted out and cells were
washed three times using phosphate buffer saline (pH 7.4). Subsequently, 20 µL of MTT
solution (from 5 mg/mL stock solution in PBS) and 100 µL of cell culture medium were added to
each well and incubated for 4-5 h at 37 C in the CO incubator. After 4-5 h incubation, the
2
supernatant was removed and the purple formazan crystals were dissolved by adding 150 µL
DMSO to each well. The absorbance was measured at 570 nm on a multi-plate ELSIA reader
(BioRad). The percentage of cell viability was calculated and plotted as a function of
concentration of compound. For anticancer studies paclitaxel was taken as positive control.
3
3
. Results and discussion
.1. Synthesis and characterization
The main idea of the present work is to synthesize, characterize as well as to study their
biological investigations of three novel nickel(II) [Ni(L) ] (1) and copper(II) [Cu(L) ] (2a),
2
2
[
Cu(L) ] (2b) complexes, which are formed through the combination of (E)-1-(2-
2
methoxybenzylidine)-4-phenylethiosemicarbazone (HL) as bidentate N,S-ligand. In the first step,
Schiff base HL was prepared by the reaction between 4-phenylthiosemicarbazide and
2
-methoxybenzaldehyde in a ratio of 1:1 (scheme 1). Finally, the Schiff base (HL) reacted with
Ni(OAc) ·4H O, Cu(OAc) ·H O, Cu(NO ) ·3H O in a ratio of 2:1 to afford 1, 2a and 2b,
2
2
2
2
3 2
2
respectively. The protocol used for the synthesis of HL and its complexes is given in scheme 1.
Suitable crystals were selected for single-crystal X-ray analysis. The synthesis and
characterization of HL and its complexes are still rare, which indicates the novelty of the present
work. The obtained complexes were microcrystalline solids and they were stable in air with

melting points above 200 C. They were insoluble in water and methanol but soluble in organic
solvents such as acetonitrile, chloroform, DMF and DMSO. These complexes displayed a four-
coordinate tetrahedral or square planar trans-[MN S ] coordination geometry whose central
2
2
metal(II) atoms lie on the center of symmetry. The elemental analysis and crystal structures were
in agreement with the chemical formula proposed for all complexes. The observed molar
-3
conductance value of the complexes in acetonitrile (1×10 M) suggest a non-electrolyte nature
−
1
2
−1
[
56] due to the low conductivity values [Λ = 18 Ω cm mol : 150 for 2a and 2b] except 1
M
which was electrolyte in nature. Complex 1 behaves as 1:2 electrolyte in MeCN solution (Λ =
M
-1
2
-1
2
20 Ω cm mol ), indicating the thiosemicarbazone ligand act as neutral N,S-donors in this
complex. Room temperature magnetic moment measurements of the complexes at 298 K
indicate they are paramagnetic. As 1 was found to be paramagnetic, what excludes a square
8
planar configuration? The general rule that in Ni(II) d chemistry, tetrahedral (or nearly
tetrahedral) complexes have temperature-dependent magnetic moments which are usually higher
than the spin-only value while the square-planar complexes are diamagnetic, therefore the value
of magnetic moment would be zero. At 298 K, the calculated value of magnetic moment (μeff)
was 2.35 BM for 1 and 1.68 BM for 2a and 2b. These magnetic moment values lie within the
range normally found for other four-coordinate tetrahedral nickel(II) complexes [57] and/or
square-planar copper(II) complexes [58].

CH3
O
H
N
(
i) Ni(OAc) .4H O
N
2
2
N
(ii) Triethyamine
S
(
iii) 1:2 molar ratio
Ni
S
N
N
N
H
O
H
H
N
N
NH2
CH3
S
[
Ni(L) ](1)
CH3
O
2
4
-phenylthiosemicarbazide
CH3
Refluxed in dry
MeOH for 3 hrs
O
O
H
N
N
N
2-methoxy benzaldehyde
S
CH3
(i) Cu(OAc
) .H O
2
2
Cu
S
(ii)Triethyamine
O
N
H
N
H
(iii) 1:2 molar ratio
N
N
N
H
N
O
S
CH3
(E)-1-(2-methoxybenzylidene)-4-
phenylthiosemicarbazone(HL)
[Cu(L)2](2a)
CH3
O
H
N
N
N
S
(
i) Cu(NO ) .3H O
3
2
2
Cu
S
(
ii) Triethyamine
iii) Ammonium thiocyanate
iv)1:2 molar ratio
N
(
N
N
H
(
O
CH3
[Cu(L) ](2b)
2
Scheme 1. Synthetic route for HL and its mononuclear Ni(II) and Cu(II) complexes.
3
.2. Crystal structure description
The molecular structure of 1, 2a and 2b along with the atomic numbering scheme is given in
figures 1-3, respectively. Crystal data and some selected bond lengths and angles are
summarized in tables 1 and 2, respectively. Single-crystal X-ray diffraction reveals that the
complexes are mononuclear. The crystal structure determinations also confirm that the structures

of 1, 2a and 2b are isostructural. The single-crystal X-ray diffraction study reveals that 1
crystallized in the orthorhombic crystal system with space group Pbca [a = 15.4786(11) Å, b =
7
.6422(5) Å, c = 24.613(2) Å, α = 90, β = 90, γ = 90, Z = 4]. Here the E-configuration of HL
in the solid state is illustrated for its coordination with the metals as monobasic bidentate with N-
and S-donor groups suitably oriented for forming five-membered chelate ring. The symmetry
around nickel(II) in 1 can be described as square planar, the nickel(II) ion is bonded to
monobasic bidentate N,S-donor ligand forming a type of tetradentate N S -donor bis-Schiff base
2
2
complex, thus constructing two five-membered chelate rings [1.Ni(1)-N(3)-N(2)-C(9)-S(1a),
.Ni(1)-N(3a)-N(2a)-C(9a)-S(1)]. The Ni-N(imine) bond distances are 1.91 Å and 2.17 Å in 1,
2
which is very close to the reported Ni-N(imine) bond distances for Ni(II) complexes having
perfect square planar geometry [59-61]. The Ni(II) of 1 has crystallographic inversion symmetry
(symmetry code: -x, -y, -z) to offer a square planar geometry and axial positions are vacant. As
shown in figure 1, the perfect geometry may conveniently be measured by the trans angles that
are ideally 180 for 1; N(3)-Ni-N(3a) = 180 and S(1)-Ni-S(1a) = 180. Similar observations
have been made by Kundu et al. [62].
Complexes 2a and 2b afford a four-coordinate square planar trans-[MN S ] coordination
2
2
geometry whose central metal atom lies on the center of symmetry. The X-ray structural studies
of 2a and 2b reveal that the metal complex has 2:1 ligand-to-metal stoichiometry, crystallized in
orthorhombic and monoclinic system in the space group Pbca and P21/c, respectively [a =
1
1
5.507(3) Å, b = 7.5988(16) Å, c = 24.876(5) Å, α = 90, β = 90, γ = 90, Z = 4 for 2a and a =
9.141(7) Å, b = 7.667(3) Å, c = 22.470(9) Å, α = 90, β = 104.803, γ = 90, Z = 4 for 2b] and
consist of a Cu(II) and two monobasic bidentate chelating ligand L. Both the N and the S atoms
of L are at special positions on the perpendicular axes, and the point of intersection of
coordinated axes is the Cu(II) ions. The basal plane of the square plane is bound by two N atoms
from C=N groups, N(3), N(3a) for 2a, N(3), N(6) for 2b and two S atoms from thiol group S(1),
S(1a) for 2a and S(1), S(2) for 2b, forming a type of tetradentate N S -donor, bis-Schiff base
2
2
complex, thus constructing two five-membered chelate rings [1. Cu(1)-S(1a)-C(1a)-N(2a)-N(3a),
.Cu(1)-S(1)-C(1)-N(2)-N(3)] for 2a and [1.Cu(1)-S(1)-C(2a)-N(2)-N(3), 2.Cu(1)-S(2)-C(1)-
2
N(4)-N(5) for 2b. As shown in figures 2 and 3, 2a and 2b have similar solid structure, and the
metal centers were coordinated to two imine-N and bounded to two thiol-S atoms being
deprotonated in trans-confirmation whereas the metals are situated on inversion centers to form a

distorted square planar [63]. As they are crystallographically centrosymmetric, the perfect
symmetry may conveniently be measured by trans angles [S(1)-Cu(1)-S(1) = 180, N(3)-Cu(1)-
N(3) = 180 for 2a and N(3)-Cu(1)-N(6) = 159.6, S(1)-Cu(1)-S(2) = 156.32 for 2b] that are
ideally 180 for square planar complexes [64]. The Cu-N bond distances were 2.008 Å in 2a and
1
.993(4) Å in 2b which are located in the range of known values for Cu-N(imine) bonds in
analogous square planar Cu(II) species [65]. While formation of 2a required use of
Cu(OAc) ·H O and triethylamine, the formation of 2b required use of Cu(NO ) ·3H O,
2
2
3 2
2
triethylamine and ammonium thiocynate. The yield of 2a and 2b was the same with molecular
formula and the only difference is their crystallographic space groups [orthorhombic with Pbca
for 2a and monoclinic with P2 /c for 2b], therefore 2a and 2b are highly isostructural. Further, it
1
-
is assumed that counter anion and SCN ions have no significant role in the complexation of 2b.
As shown in figures S1-S3, in crystal structures of complexes, there are no remarkable
intermolecular hydrogen bonds and intermolecular π---π interaction within van der Waals radii.
Thus, the crystal packing may be dominated by non-covalent and weak van der Waals forces.
The molecular arrangement and anti-parallel orientation of the molecular planes shows one-
dimensional chains along the crystallographic c-axis. The unit cell packing diagrams of the
complexes are graphically presented in figures S1-S3, along with c-axis.
3
.3. FT-IR and UV-visible spectroscopy
The FT-IR spectra of Schiff base (HL) and its complexes [Ni(L) ] (1), [Cu(L) ] (2a) and
2
2
-1
[
Cu(L) ] (2b) were recorded from 400 to 4000 cm at room temperature. Characteristic IR bands
2
for HL are different from those of the complexes providing significant information regarding the
bonding sites of the thiosemicarbazone ligand to the metals. Some representative IR spectra of
HL and its complexes are given in the Supplementary Material (figures S4-S6). In the IR
-1
-1
-1
spectrum of HL the highest frequency bands at 3277 cm , 3142 cm and 2999 cm may be
assigned to the asymmetric v(N-H) vibrations and symmetric v(N-H) vibrations of the imino- and
amino-groups, respectively [66]. These v(N-H) vibrations shifted to higher frequencies (3379-
-1
3
380 cm ) in all the complexes at the same positions compared to that of HL indicates that free
-1
v(N-H) vibrations do not participate in coordination. A band appeared at 1249 cm for HL due to
vibration of the C=S double bond which disappeared in the spectra of the complexes and a new
-1
-1
band appeared at 1244 cm for 1, 1242 cm for 2a and 2b, indicating that the other coordination

is through thiolate sulphur after enolization followed by deprotonating on sulphur [67, 68]. Free
-1
HL shows a sharp intense band at 1593 cm assigned to the v(C=N) of azomethine group. This
-1
band shifted to higher frequencies at 1597 cm in all the complexes, indicating coordination of
azomethine nitrogen of ligand to the metal ion [69]. The IR data of HL and its complexes 1, 2a
and 2b showed that HL was coordinated to the metal ion in a monobasic bidentate-N,S manner
(
L). The infrared spectra of HL and their complexes agree with the observed crystal structures
shown in figures 1-3.
The nature of ligand and field around the metal ion was deducted from the electronic
spectra. UV-visible absorption spectra of HL and its complexes 1, 2a and 2b were recorded from
00-800 nm using DMSO as solvent at 25 C. Representative UV-vis spectra of HL and its
2
complexes are depicted in figure 4. The UV-visible spectra of thiosemicarbazone (HL) showed
two absorption bands 325 and 342 nm due to π-π* and n-π* transitions, respectively. The
electronic spectrum of 1 displayed two bands of 359 nm and 576 nm, which are assigned to
1
1
1
1
A → E (n ) and A → B (n ) transitions, respectively. This complex is paramagnetic in
g
g
1
1g
2g
2
nature; therefore, four-coordinate tetrahedral geometry [70] is suggested. The electronic
spectrum of 2a of HL shows two bands at 270 and 574 nm, which was assigned to the
2
2
2
2
B → B and B → Eg transitions, respectively. Since the value of magnetic moment was
1
g
2g
1g
1
.68 BM, a four-coordinate square planar geometry [71] was suggested for 2a. Similar electronic
spectrum was observed for 2b of HL.
3
.4. Protein binding and molecular docking studies
The absorption spectra of protein (BSA) were obtained in presence of metal complexes 1 and 2a
to predict the type of quenching process. Some representative fluorescence curve of BSA in the
presence and absence of complexes is graphically presented in figures 5, S7 and S8. Quenching
mainly occurs through either static or dynamic modes. Dynamic quenching refers to the process
in which quencher and fluorophore come in contact during excited state, whereas in static
quenching they come in contact during ground state. The obtained electronic spectra showed that
the absorption intensity of BSA was enhanced upon addition of 1 and 2a. This result suggested
that the binding of metal complexes with BSA was through a static quenching mode [72]. The
interaction of 1 and 2a and BSA were also observed through fluorescence spectral method. A
solution of BSA (1 µM) was titrated with various concentrations of metal complexes (0 µM –

2
5 mM) and the spectra were obtained in the range of 300-400 nm upon excitation at 280 nm.
With the increase in concentrations of metal complexes, the characteristic fluorescence emission
band of BSA at 335 nm was quenched in a consistent manner, along with a bathochromic shift of
around 2-5 nm. The observed bathochromic shift is due to the fact that the active site in BSA is
suppressed in a hydrophobic environment [73]. The above results suggested that a strong
interaction occurred between the metal complexes and BSA. To assess the interaction between
the metal complexes and BSA, Stern-Volmer (figure S9) and Scatchard plots (figure S10) were
used. The Stern-Volmer quenching relation is given by following equation:
I /I = K [Q] + 1
(1)
0
SV
where I is the emission intensity in the absence of the quencher, I is the emission intensity in the
0
presence of the quencher, [Q] is the concentration of quencher and KSV is the Stern-Volmer
quenching constant. The quenching constant (K ) can be calculated using the plot of I /I versus
SV
0
[
Q] (figure S9). When a small drug molecule binds to a set of equivalent sites on a
macromolecule, the equilibrium between free and bound molecules is represented by the
Scatchard equation [74]:
log [(I -I)/I] = log K + n log[Q]
(2)
0
b
where K is the binding constant of the complex with BSA and n is the number of binding sites.
b
From the plot of log [(I -I)/I] versus log[Q] (figure S10), the binding constant (K ) and the
0
b
number of binding sites (n) have been obtained. The binding constant (K ), quenching constant
b
(
K ), and the number of binding sites (n) for the interaction of 1 and 2a with BSA are given in
SV
table 3. The observed results clearly indicate that 1 and 2a have only one binding site to interact
with BSA and the binding constant values for 1 is more than that of 2a.
Molecular docking study gives the accurate and preferred orientations of the chemical
compound at the binding site of the receptor. Here, in molecular docking studies, we found that 1
and 2a were docked into the grooves of target DNA and resulted docked conformations were
selected on the basis of obtained interaction energy parameters and scoring functions (table 4).
Conformations of docked compounds with DNA are shown in figures 6 and 7, showing the
interactions between complexes and target DNA. Complex 1 shows a little higher free energy of

binding with DNA (-8.3 kcal/mol) as compared to 2a, which shows -8 kcal/mol binding free
energy. Both 1 and 2a bind to the DNA showing little difference in their binding affinities due to
the differences between the metals by which these complexes are linked [75]. Complex 1 was
found to interact with C3, G4, A5, A6 and T7 of strand A, G16, A17, A18, T19 and T20 of
strand B of DNA and forming a hydrogen bond with A5 of strand A. Complex 2a shows several
close interactions with C9, G10, C11 and G12 of strand A, and G14, C15, G16, A17 and A18 of
strand B of target DNA, where three hydrogen bonds are forming with C11 of strand A and A17,
A18 of strand B of the DNA.
3
.5. Cytotoxicity studies
To study the cytotoxic/antiproliferative potential of synthesized compounds on human cell lines,
MTT assay was used as it is a widely used method to evaluate the number of viable cells in a
particular format of treatment. This assay is based on the observation that viable cells having
active metabolism can convert MTT into purple formazan having absorbance maxima at 570 nm,
however, dead cells do not have the capability to convert MTT into formazan [76]. The
compounds were screened in the concentration range of 0-200 µg/mL, and treatments were given
for 72 h. Interestingly, the treatment of these compounds does not affect the viability of HEK293
cells, but in the case of LNCaP and HepG2 cells they inhibited the cell viability in concentration
dependent manner (figure 8). These results suggested that these compounds are less effective
against HepG2 cells compared to LNCaP cells (figure 8). For LNCaP cells, the estimated IC50
values of for present complexes were 44.12 µg/mL, 31.67 µg/mL and 71.38 µg/mL, respectively.
The investigated compounds are more cytotoxic than HL and these results reveal that the
cytotoxic activity increases when ligand are coordinated to the metal ion [77]. Probably, the high
cytotoxicity of the compounds may be related to the intercalation of each metal complex with
nitrogen bases of DNA tumor cells, causing greater conformational changes in the double-helix
of DNA and then producing cell death [78]. On the other hand, these compounds were more
cytotoxic compared with the toxic activity shown by cis-platin assayed in (HL60) human
leukemia cells [17] and to Pt(II) and Pd(II) complexes with phenylacetaldehyde
thiosemicarbazone ligands assayed in the K562 human chronic myelogenous leukemia cell line
[
79]. Cell viability results speculated that synthesized compounds may be further optimized and
employed as promising anticancer agents.

4
. Conclusion
We have synthesized and characterized three nickel(II) [Ni(L) ] (1), and copper(II) [Cu(L) ] (2a)
2
2
and [Cu(L) ] (2b) complexes which are formed through coordination of (E)-1-(2-
2
methoxybenzylidine)-4-phenylethiosemicarbazone (HL) as bidentate N,S-ligand. Single-crystal
analysis of synthesized complexes display a distorted tetrahedral and/or square planar trans-
[
MN S ] coordination whose M(II) atoms lie on the center of symmetry. The Schiff base (HL)
2 2
was prepared by the condensation of 4-phenyl thiosemicarbazide and 2-methoxybenzaldehyde in
:1 molar ratio. Protein binding study of metal complexes indicated that a strong interaction has
1
occurred between the metal complexes and BSA. Complex 1 shows a little higher free binding
energy (-8.3 kcal/mol) with docked target DNA as compared to 2a, which shows -8 kcal/mol
binding free energy.
Supplementary materials
CCDC 1565755, 1836258 and 1837668 contain the supplementary crystallographic data for 1, 2a
and 2b, respectively. Copies of this information can be obtained free of charge via
http://www.ccdc.cam.ac.uk/conts/retrieving.html/datarequest/cif, or from the Cambridge
Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; Fax: (+44)-1223-
3
36033 or E-mail: deposit@ccdc.cam.ac.uk.
Acknowledgements
Authors thank the Sophisticated Analytical Instrument Facility (SAIF), STIC Cochin for single
crystal data collection of [Ni(L) ] (1), and SAIF IIT Patna for [Cu(L) ] (2a) and [Cu(L) ] (2b).
2
2
2
Financial assistance received from the Council of Scientific and Industrial Research (CSIR),
New Delhi, India (Grant No. 01(2856)/16/EMR-II) is gratefully acknowledged. B. Mohan and
Noorussabah acknowledge CSIR and TEQIP-II, NIT Patna, respectively, for their fellowships.

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Table 1. Crystal data and structure refinement of 1, 2a and 2b.
1
2a
2b
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
Space group
Unit cell dimensions
a (Å)
C H NiN O S
C H CuN O S
C H CuN O S
3
0
28
6
2 2
30 28
6
2 2
30 28
6 2 2
627.41
296(2)
632.25
632.25
150
293
0.71073
0.71073
0.71073
Orthorhombic
Pbca
Orthorhombic
Pbca
Monoclinic
P21/c
15.4786(11)
7.6422(5)
24.613(2)
90
15.507(3)
19.141(7)
7.667(3)
22.470(9)
90
b (Å)
7.5988(16)
c (Å)
24.876(5)
α (°)
90

(°)
γ (°)
Volume (Å )
90
90
104.803(11)
90
90
90
3
2911.4(4)
1.431
2931.3(11)
3188(2)
1.317
3
Dcalc. (Mg/m )
Z
1.433
4
4
4
-
1
Absorption coefficient (mm )
F (000)
0.849
0.926
0.857
1304
1308
1308
Crystal size (mm)
0.30 x 0.20 x 0.20
1.65 to 28.38
-20<=h<=16
0.20 x 0.20 x 0.15
0.20 x 0.25 x 0.10
θ Range for data collection (°)
Limiting index
2.627 to 28.394
-19<=h<=20
-10<=k<=10
-33<=l<=33
2.500 to 28.388
-25<=h<=25
-10<=k<=10
-29<=l<=29
-
-
10<=k<=10
32<=l<=31
Reflections collected/unique [Rint] 21911/3632[0.0416]
54434/3663[0.0794] 69403/7921[0.1405]
Completeness to 
28.38 - 99.6%
28.394 - 99.8%
28.388 - 99.0%
Absorption correction
Semi-empirical
Semi-empirical
Semi-empirical
from equivalents
from equivalents
from equivalents
Max. and min. transmission
Refinement method
0.8486 and 0.7848
0.929 and 0.887
0.746 and 0.680
Full-matrix
Full-matrix
Full-matrix
least-squares
least-squares
least-squares
Data / restraints / parameters
3632 / 0 / 189
1.138
3663 / 0 / 201
1.043
7921 / 0 / 372
1.071
2
Goodness-of-fit on F
Final R indices [I>2 (I)]
R
1
= 0.0407
R
1
= 0.0403
R
1
= 0.0934
wR2 = 0.1035
wR2 = 0932
wR2 = 0.1857

Table 2. Selected bond lengths (Å) and angles (°) for 1, 2a and 2b.
1
2a
2b
Bond length
Bond angles
Ni(1)-N(3)
1.9127(17)
2.1708(6)
2.1708(6)
1.9128(17)
180.00(10)
85.28(6)
94.72(6)
94.72(6)
85.28(6)
180.0
S(1)-Cu(1)
2.2556
2.008
S(1)-Cu(1)
2.225(2)
1.987(3)
2.235(2)
1.993(4)
85.2 (1)
156.32(6)
100.1(1)
97.8(1)
Ni(1)-S(1)#1
N(3)-Cu(1)
N(3)-Cu(1)
Ni(1)-S(1)
S(1)-C(1)
1.741(2)
1.364(3)
83.91
S(2)-Cu(1)
Ni(1)-N(3)#1
N(3)-Ni(1)-N(3)#1
N(3)-Ni(1)-S(1)#1
N(3)#1-Ni(1)-S(1)#1
N(3)-Ni(1)-S(1)
N(3)#1-Ni(1)-S(1)
S(1)#1-Ni(1)-S(1)
O(1)-C(9)
N(6)-Cu(1)
S(1)-Cu(1)-N(3)
S(1)-Cu(1)-S(1)
S(1)-Cu(1)-N(3)
N(3)-Cu(1)-S(1)
N(3)-Cu(1)-N(3)
S(1)-Cu(1)-N(3)
S(1)-Cu(1)-N(3)
S(1)-Cu(1)-S(2)
S(1)-Cu(1)-N(6)
N(3)-Cu(1)-S(2)
N(3)-Cu(1)-N(6)
S(2)-Cu(1)-N(6)
180.00
96.09
96.09
180.00
83.91
159.6(2)
85.2(1)
Symmetry transformations used to generate equivalent atoms: #1 -x,-y+2,-z+1 for 1.

Table 3. Protein binding constant (K ), quenching constant (K ) and number of binding sites
b
SV
(n) of 1 and 2a.
-1
-1
Complex
K (M )
K (M )
n
b
SV
5
2
5
5
1
2.38 × 10
7.73 × 10
0.015 × 10
0.005 × 10
1.245
0.709
2
a
2
5

Table 4. Binding affinity of 1 and 2a with the DNA generated from molecular docking, and
th
interacting residues of DNA (Note: C9(A) means, the cytosine at 9 position of strand A).
Affinity
kcal/mol)
Complex
Interacting residues
(
1
-8.3
C3(A), G4(A), A5(A), A6(A), T7(A), G16(B), A17(B), A18(B),
T19(B) and T20(B)
2
a
-8.0
C9(A), G10(A), C11(A), G12(A), G14(B), C15(B), G16(B),
A17(B) and A18(B)
2
6

Figure 1. ORTEP structure of 1 with 50% thermal ellipsoid and with atom numbering scheme.
2
7

Figure 2. ORTEP structure of 2a with 30% thermal ellipsoid and with atom numbering scheme.
2
8

Figure 3. ORTEP structure of 2b with 50% thermal ellipsoid and with atom numbering scheme.
2
9

Figure 4. (a) The UV-visible absorption spectra of HL and its complexes 1 and 2a; (b) UV-
visible absorption spectra of HL and its complexes with expanded the y-axis from 300-800 nm.
3
0