Distorted tetrahedral bis-(N,S) bidentate Schiff base complexes of Ni(II), Cu(II) and Zn(II): Synthesis, characterization and biological studies

Article abstract of DOI:10.1016/j.poly.2016.02.049

Transition metal complexes of acetophenone-S-allyldithiocarbazate [H-(Ap-sadtc)] of the general formula [M-(Ap-sadtc)₂] (Ni²⁺, Cu²⁺, Zn²⁺) (1-3) were synthesized and characterized by elemental analysis, UV-Vis,

Full text of DOI:10.1016/j.poly.2016.02.049

Accepted Manuscript
Distorted tetrahedral bis-(N,S) bidentate Schiff base complexes of Ni(II), Cu(II)
and Zn(II): Synthesis, characterization and biological studies
Nanjan Nanjundan, Ramaswamy Narayanasamy, Steven Geib, Krishnaswamy
Velmurugan, Raju Nandhakumar, Manickam Dakshinamoorthi Balakumaran,
Pudupalayam Thangavelu Kalaichelvan
PII:
S0277-5387(16)00144-3
DOI:
http://dx.doi.org/10.1016/j.poly.2016.02.049
POLY 11866
Reference:
To appear in:
Polyhedron
Received Date:
Accepted Date:
4 December 2015
24 February 2016
Please cite this article as: N. Nanjundan, R. Narayanasamy, S. Geib, K. Velmurugan, R. Nandhakumar, M.D.
Balakumaran, P.T. Kalaichelvan, Distorted tetrahedral bis-(N,S) bidentate Schiff base complexes of Ni(II), Cu(II)
and Zn(II): Synthesis, characterization and biological studies, Polyhedron (2016), doi: http://dx.doi.org/10.1016/
j.poly.2016.02.049
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Distorted tetrahedral bis-(N,S) bidentate Schiff base complexes of Ni(II),
Cu(II) and Zn(II): Synthesis, characterization and biological studies
a
a
b
c
Nanjan Nanjundan , Ramaswamy Narayanasamy* , Steven Geib , Krishnaswamy Velmurugan ,
c
d
Raju Nandhakumar , Manickam Dakshinamoorthi Balakumaran , Pudupalayam Thangavelu
d
Kalaichelvan
a
Department of Chemistry, Coimbatore Institute of Technology ,Coimbatore-641 014, India.
b
Department of Chemistry, University of Pittsburgh, PA 15260 USA.
c
Department of Chemistry, Karunya University, Karunya Nagar, Coimbatore - 641 114, India.
d
Centre for Advanced Studies in Botany, School of Life Sciences, University of Madras, Guindy
Campus, Chennai - 600 025, Tamil Nadu, India.
*
To whom correspondence should be addressed, e-mail: narayanasamycit@gmail.com
Fax: + 91-422-2575020.

Abstract
Transition metal complexes of acetophenone-S-allyldithiocarbazate [H-(Ap-sadtc)] of the
2
+
2+
2+
general formula [M-(Ap-sadtc) ] (Ni , Cu , Zn ) (1-3) were synthesized and characterized by
2
1
13
elemental analysis, UV-Vis, FT-IR, H NMR, C NMR and mass spectroscopy. The molecular
structures of the ligand and its complexes 1-3 were confirmed by both theoretical as well as
experimental methods. The interactions of the complexes with biomolecules, such as calf thymus
DNA (CT-DNA) and bovine serum albumin (BSA), were explored using absorption and
emission spectral techniques. All the complexes synthesized were found to be most promising
due to their large binding affinity towards different bio-macromolecules and higher Kcat values in
catechol oxidation (3,5-DTBC) / phosphate hydrolysis (4-NPP) reactions. The in vitro anticancer
activities using MTT and a morphological staining assay indicate that the complexes are active
against the (Hela) cervical cancer cell line, which is consistent with our above mentioned
hypothesis.
Keywords: Schiff base complexes, X-ray/DFT study, DNA/BSA binding activities, catalytic
efficacy, in vitro anticancer activities.

1. Introduction
Transition metal complexes have become of increasing importance in synthetic chemistry,
coordination chemistry, homogenous catalysis and biological chemistry [1]. Platinum based
drugs have been found to be potentially active for their diverse clinical cancer treatment [2-6].
The dose of cisplatin that can be applied to patients is limited by its high toxicity, and the side
effects of cisplatin therapy include damage to the kidney (nephrotoxicity), hearing (ototoxicity),
peripheral neuropathy and neurotoxicity, nausea and vomiting. Therefore, considerable attempts
are being made to replace this drug with suitable alternatives, and numerous transition metal
complexes have been synthesized and tested for their cytotoxicity. In general, Schiff base metal
complexes have widespread applications in electrochemistry, nanotechnology, catalysis, material
science and as potential therapeutic agents. Amongst these, N,S bidentate Schiff base ligands
derived from thiosemicarbazone or dithiocarbazate have attracted considerable interest. The
versatile applications of dithiocarbazate ligands containing metal atoms coordinated with sulfur
and nitrogen donors due to their soft-hard nature are attractive and allow them to show a
structure-activity relationship (SAR) [7]. The first row transition elements play a vital role in the
synthesis of several coordination complexes due their different oxidation states which facilitate
their structural, stereochemical, spectroscopic and electrochemical properties. Among the various
transition metals, the present study is focused on nickel, copper and zinc metal ions based on
their widespread industrial and biological applications [8-11].
The transition metal ions (Ni, Cu and Zn) attached to the donor atoms of the bis(dithiocarbazate)
Schiff base ligand form stable metal ion complexes. Metal-based anticancer drugs exhibit
enhanced selectivity and novel modes of DNA interaction, like non-covalent interactions that
mimic the mode of interaction of biomolecules. The propensity for the interaction of metal
complexes with calf thymus DNA (CT-DNA) is of paramount importance to understand the
mechanism using emission spectral titrations. On the other hand, the design of protein targeting
metal-based anticancer agents with potential in vitro toxicity has gained importance recently
[
12,13]. Changing the ligand environment towards a specific target is one way of tuning the
selectivity of a drug molecule. In another way, a change in the metal environment plays a
significant role in the binding of the metal complex to a biomolecule, such as DNA or protein.
Generally, nickel and copper metal ions have redox nature, which may promote the

transformation of biomolecules through an oxidation pathway [14]. In addition, the redox
inactive zinc metal ion behaves in a different way [15]. Thus, these three mononuclear metal
complexes have unique structural features which justify their selection for the present
investigation.
Many studies have been carried out to establish a relationship between copper-mediated substrate
oxidation and structural parameters, which will influence catecholase activities. Crucial factors,
such as metal–metal distance, electrochemical properties, exogenous bridging ligand, ligand
structure and pH of the medium, do play a major role in the catecholase activity [16-21].
Recently, it was documented that the presence of a positive charge center close to the metal
center might enhance activity. Therefore, much attention has been focused on biomimetic and
bioinspired catalysts of transition metal-based complexes containing hard and soft donor atoms
[
22,23]. Among the metal complexes, M(II) complexes with N,O and N,S-donor ligands have
profound catalytic efficacy, particularly in catalyzing 3,5-DTBC to 3,5-DTBQ. Furthermore,
active research on hydrolytic catalysis of phosphoric and carboxylic acid esters involving
biochemical processes have gained utmost importance. The backbones of DNA, RNA and ATP
macromolecules show significant biochemical behavior involving the formation or cleavage of
P-O bonds [24-34]. The hydrolyses of phosphate monoester bonds in model complexes, such as
4
-nitrophenyl phosphate (4-NPP), exhibit specific reactions which indicate the reactivity of
metalloenzymes with DNA. When compared to other transition metals, Ni, Cu and Zn ions
possess very favorable catalytic activity from the reactant and product release viewpoint [35-45].
Influenced by the above facts, it was considered worthwhile to undertake the synthesis and
characterization of the ligand [H-(Ap-sadtc)] and the mononuclear complexes [Ni-(Ap-sadtc)
2
]
(
1), [Cu-(Ap-sadtc) ] (2) and [Zn-(Ap-sadtc) ] (3), which confirm the presence of quite
2
2
appreciable tetrahedral distortions about the metal centers. The ligand and its complexes 1-3 are
envisaged by applying density functional theory (DFT) calculations that determine the quantum
mechanical (QM) reactivity descriptors, viz. the highest occupied molecular orbital (HOMO) and
lowest unoccupied molecular orbital (LUMO). A comparative study of the biomolecular
interactions (DNA/BSA) of the metal complexes were conducted along with their related anti-
cancer activities. The results of the present investigation confirm that the metal complexes could
bind to DNA in the intercalation mode and BSA exhibiting the static quenching mechanism.

These three structurally similar complexes exhibit good catecholase and hydrolytic activities.
Moreover, the cytotoxicity of the metal complexes against the HeLa cell line was assessed by an
MTT assay and cell death analysis by AO/EB and DAPI staining methods.
2. Experimental
2
.1. Reagents
Potassium hydroxide pellets, hydrazine hydrate, carbon-disulfide, acetophenone, nickel(II)
acetate tetrahydrate, copper(II) acetate monohydrate and zinc(II) acetate dihydrate high purity
reagents were purchased from Merck and used as such. Calf thymus DNA (CT-DNA) and
bovine serum albumin (BSA) were obtained from Genei, Bangalore and Himedia, India
respectively. Ethidium bromide (EB), 3,5-tris(hydroxymethyl) amino methane, di-tert-
butylcatechol (3,5-DTBC), 4-nitrophenyl phosphate disodium salt hexahydrate (4-NPP) and allyl
bromide were purchased from Sigma-Aldrich and used as received. All solvents were dried and
used freshly, distilled unless otherwise stated. Double distilled water was used to prepare buffers.
2
.2. Instrumental methods
Melting points were measured on an Electro-thermal model 9200. C, H, N and S elemental
analyses of the compounds were performed on a CHNS-O model EA 2000 elemental analyzer.
Electronic spectra of the ligand/complexes 1-3 were recorded in DMSO using a Shimadzu UV-
1
650 PC spectrophotometer in the 800-200 nm range. Fourier Transform Infrared spectra of the
synthesized compounds were carried out with a Bruker Scientific 600 Infrared
-1
1
13
spectrophotometer in the spectral range 4000-400 cm using KBr pellets. H and C NMR
spectra were obtained on a Bruker High Resolution console (300.13 MHz, CDCl ). Emission
3
spectra were measured with a Jasco FP 6600 spectrofluorometer. ESI-MS spectra were recorded
using an LC-MS Q-TOF micro analyzer (Shimadzu) in the SAIF, Punjab University,
Chandigarh. The X-ray intensity data were measured on a Bruker Apex II CCD system equipped
with a Cu IMuS micro-focus (λ = 1.54178 Å). The frames were integrated with the Bruker
SAINT software package using a narrow-frame algorithm. Geometry optimization by density
functional theory (DFT) method was performed using the GAUSSIAN09 (B3LYP) package. All
-3
reactions were carried out in an oxygen atmosphere. Stock solutions of complexes 1-3 (1.0×10
o
M in DMSO) were stored at 4 C and the required concentrations were prepared for all

experiments. All the stock solutions were used after no more than four days. Solutions of the
compounds were freshly prepared 1 hour prior to biochemical evaluation. Data are expressed as
the mean ± standard deviation from three independent experiments.
2
.3.1. Synthesis of [H-(Ap-sadtc)]
[
H-(Ap-sadtc)] was synthesized according to the previously published procedure [46,47]. To an
ethanolic solution (30 mL) of 5 mL (0.1 mol) hydrazine hydrate and 5.6 g (0.1 mol) KOH,
carbon disulfide 6.1 mL (0.1 mol) was added dropwise in an ice bath. After 30 min, 8.6 mL (0.1
mol) of allyl bromide was added and the solution was stirred at 5 °C for 1 h. An ethanolic
solution (25 mL) of acetophenone 11.7 mL (0.1 mol) was added to this mixture and heated under
stirring. After 15 mins, a yellow product was separated by filtration, washed with water and
vacuum dried. The product was recrystalised from hot ethanol and single crystals for X-ray
structure analysis were obtained by the slow evaporation method. Yield: 75-80%. Colour:
yellow. MP: 145 °C. Anal. Calc. for C12
H
14
N
2
S : C, 57.64; H, 5.64; N, 11.19; S, 25.61; Found:
2
-1
C, 57.49; H, 5.54; N, 11.06; S, 25.48%. UV-vis (DMSO), λ max (nm): 368. FT-IR (ν cm , KBr):
1
1
596 ν(C=N); 3057 ν(N-H); 1043 ν(C=S); 982 ν(C-S); 680 ν(C-HAllyl); 1515, 1546, 1561, 1627,
1
693, 1677 ν(C Ar). H NMR (300.13, MHz, CDCl
3
, 25 °C) δ (ppm): 5.20 (m, 2H, -CH
); 7.40-7.93 (d, 5H, CAr). C
); 130.62 (-CH); 37.45 (-CH2);
); 127.09-138.88 (CAr). ESI-MS (Calc., found, m/z):
2
); 5.94
1
3
(
m, H, -CH); 3.88 (d, H, -CH); 9.9 (s, H, -NH); 2.39 (s, 3H, -CH
NMR (300.13, MHz, CDCl , 25 °C) δ (ppm): 119.27 (-CH
00.06 (C=S); 158.19 (C=N); 15.51 (CH
3
3
2
2
2
3
50.38, 250.37.
2
.3.2. Synthesis of complexes 1-3
[
Ni-(Ap-sadtc)
2
] (1)
A boiling methanolic solution (20mL) of Ni(CH
3
COO)
2
.4H O (0.248 g, 1 mmol) was added
2
dropwise to a methanol solution (10 mL) of [H-(Ap-sadtc)] (0.5 g, 2 mmol) with an immediate
colour change from yellow to brownish black. The solution was then refluxed for 1 hour. After
cooling, it was filtered and the filtrate was kept at an ambient temperature for slow evaporation.
After two days, the brownish-black needle-shaped diffraction quality crystals that deposited were
collected by filtration, washed with small amounts of methanol and air dried. Yield: 88%.
o
Colour: brownish black. MP: 175-178 C. Anal. Calc. for C24
H
26
N
4
NiS : C, 51.71; H, 4.70; N,
4

1
0.05; S, 23.01; Found: C, 51.68; H, 4.59; N, 9.98; S, 22.89%. UV-vis (Tris-HCL buffer), λ max
-1
(
nm): 236, 260, 387, 479. FT-IR (ν cm , KBr): 1532 ν(C=N); 923 ν(C-S-S); 688 ν(C-HAllyl);
1
3
1
1
5
516, 1546, 1561, 1626, 1677, 1693 ν(CAr). C NMR (300.13, MHz, CDCl
19.02 (-CH ); 178.88 (C-S); 22.80 (-CH ); 129.96-139.50 (CAr); ESI-MS (Calc., found, m/z):
57.44, 557.1.
3
, 25 °C) δ (ppm):
2
3
2
.3.3. [Cu-(Ap-sadtc) ] (2)
2
A methanolic solution of Cu(CH
3
COO)
2
.H O (0.199 g, 1 mmol) was added dropwise to a hot
2
acetonitrile solution of [H-(Ap-sadtc)] (0.5 g, 2 mmol). The resulting greenish-black mixture
was refluxed for 1 hour. After cooling, the clear solution was filtered and the filtrate was kept in
a CaCl
filtered and washed with a small amount of petroleum ether/acetonitrile. Yield: 85%. Colour:
greenish black. MP: 190-195 °C. Anal. Calc. for C24 : C, 51.26; H, 4.66; N, 9.96; S,
2.81; Found: C, 51.07; H, 4.50; N, 9.81; S, 22.68%. UV-vis (Tris-HCL buffer), λ max (nm): 238,
2
desiccator in the dark. After one week, square-shaped X-ray quality crystals were
4 4
H26CuN S
2
3
1
-1
14, 393, 493. FT-IR (ν cm , KBr): 1532 ν(C=N); 922 ν(C-S-S); 691 ν(C-HAllyl); 1516, 1546,
561, 1626, 1677, 1693 ν(CAr). ESI-MS (Calcd, found, m/z): 562.3; 562.27.
2
.3.4. [Zn-(Ap-sadtc) ] (3)
2
An ethanolic solution of Zn(CH
3
COO)
2
.2H O (0.219 g, 1 mmol) was added to a hot
2
dichloromethane solution of [H-(Ap-sadtc)] (0.5 g, 2 mmol), then heated on a water bath for 5
minutes, filtered and kept in a refrigerator. After 15 days, X-ray quality yellow crystals were
obtained, filtered and washed with a small amount of petroleum ether/ethanol. Yield: 82%.
o
Color: Yellow. MP: 160-165 C. Anal. Calc. for C24
H
26
N
4
4
S Zn: C, 51.10; H, 4.65; N, 9.93; S,
2
2
1
2.74; Found: C, 51.15; H, 4.51; N, 9.81; S, 22.60%; UV-vis (Tris-HCL buffer), λ max (nm): 238,
-1
60, 398, 497. FT-IR (ν cm , KBr): 1533 ν(C=N); 917 ν(C-S-S); 690 ν(C-HAllyl); 1516, 1546,
1
561, 1626, 1677, 1693 ν(CAr); H NMR (300.13, MHz, CDCl
3
, 25 °C) δ (ppm): 5.15 (m, 2H, -
1
3
CH
2
); 5.87 (m, H, -CH; 2.30 (m, 3H, -CH
, 25 °C) δ (ppm): 119.88 (-CH ); 171.48 (C-S); 161.04 (C-CH
37.32 (CAr). ESI-MS (Calcd, found, m/z): 564.14, 564.10.
3
); 7.41-7.51 (m, 5H, CAr). C NMR (300.13, MHz,
CDCl
3
2
3
); 21.32 (CH ); 127.97-
3
1
2
.4. Crystal structure characterization

Single crystal X-ray data of [H-(Ap-sadtc)] and the three mononuclear complexes 1-3 were
collected by a Bruker Apex II CCD diffractometer using CuK radiation (λ= 1.54178 Å). The
α
linear absorption coefficients, scattering factors for the atoms and the anomalous dispersion
corrections were referred from the International Tables for X-ray Crystallography. The data
integration and reduction were worked out with SAINT software. The structures were solved by
2
direct methods using SHELXTL-2014 and refined on F by full-matrix least-squares using the
SHELXTL-2014 program package. All the non-H atoms were treated anisotropically. The H
atoms attached to carbon atoms were positioned geometrically and treated as riding atoms using
SHELXL-2014 default parameters. The structures of the ligand and the three new mononuclear
metal complexes 1-3 are shown in Fig. 1. Details of the crystallographic data, structure
refinement parameters and selected bond lengths and angles are listed in Tables 1 and 2.
2
.5. Computational methods
The initial geometry for the DFT calculations was based on the measured X-ray diffraction
structure. All theoretical computations were done by using the Gaussian09 program package
[
48]. Geometry optimizations for the thione tautomeric form of the ligand and metal complexes
were performed using the DFT method with Becke’s three parameters hybrid exchange-
correlation functional B3LYP/6-311G. Visualizations of the DFT optimized structures and the
frontier molecular orbitals (HOMO and LUMO) of the ligand and complexes 1-3 were
performed using the chemcraft program package (http://www.chemcraftprog.com). To ascertain
stationary points, a further frequency test was performed. Also, a theoretical investigation of the
natural charge for each atom, QM descriptors, were calculated using the same basic set.
2
.6. DNA binding emission spectral studies
The interaction of the complexes with CT-DNA was investigated by dissolving the complexes 1-
in a solvent mixture of 5% DMSO and 95%Tris-HCl buffer (5 mM Tris-HCl/50 mMNaCl
3
buffer, pH = 7.2) the solutions were stored at 4 °C for further experiments and used within 4
days. Emissive titrations were performed with a fixed concentration of the metal complexes 1-3
(
25µM) but by varying the nucleotide (CT-DNA) concentration from 0 to 10 µM. Before taking
measurements, the mixture was shaken up and incubated at room temperature for 30 min. In
addition, a proper wavelength with CT-DNA was fixed by the emission range and adjusted.

2
.7. Competitive binding fluorescence measurements
Further, to support the competitive binding of complexes 1-3 to DNA via intercalation,
fluorescence emission quenching experiments were performed. The fluorescence quenching
experiments were carried out by adding different concentrations of complexes 1-3 to the EB-
bound CT-DNA solution in 5% DMSO and 95% Tris-HCl buffer. The excitation (550 nm) and
emission (605 nm) fluorescence intensities were recorded at room temperature. EB alone showed
minimal fluorescence and the fluorescence was enhanced greatly with the gradual addition of
CT-DNA until a maximum fluorescence was achieved due to the formation of an intercalative
DNA-EB adduct. Addition of increasing amounts of the complexes 1-3 to the DNA-EB adduct
quenched the fluorescence. For the emission quenching experiments, CT-DNA was pretreated
o
with EB in a ratio [DNA]/[EB] = 10 for 30 minutes at 37 C. The titrants were then added to this
mixture of EB-DNA and the change in fluorescence intensity was measured.
2
.8. BSA binding studies
The fluorescence spectra were recorded at an emission wavelength of 342 nm using a 4mL
quartz cell on a JASCO F6500 spectrofluorometer after each addition of the quencher. Stern–
Volmer and Scatchard equations and graphs were used in order to study the interaction of the
quencher with BSA. Preparation of samples for fluorescence spectra: five 10 mL clean and dried
-6
sample test tubes were taken, Tris–HCl buffer 2.0 mL (pH 7.20), BSA 1.0 x 10 M and the
-6
concentrations of the complexes 1-3 were 10, 20, 30, 40 and 50 x 10 M for each of the five test
tubes. The sixth test tube, containing only BSA solution at pH 7.2, was marked as a “control”.
After mixing, the solutions were allowed to stand for 15 min for maximum binding of the
complexes to BSA.
2
.9. Catechol oxidase activity
In the majority of the investigations dedicated to the study of potential catecholase activity of
biomimicking coordination compounds, 3,5-di-tert-butylcatechol(3,5-DTBC) is chosen as the
model substrate because its low redox potential makes it easily oxidized to the corresponding
quinone (3,5-DTBQ). Since, it prevents over oxidation, such as ring opening, and being stable, it
shows a maximum emission at 435 nm in pure DMSO. Before proceeding into a detailed kinetic

study, the ability of complexes 1-3 were evaluated for the oxidation 3,5-DTBC to 3,5-DTBQ at
-4
room temperature in an oxygen atmospheric condition. For this purpose, 10 M DMSO solutions
-2
of these three metal complexes were treated with 10 M (100 equivalents) of 3,5-DTBC.
Maintaining the complex concentration as a constant, by adding the different substrate
-2
concentrations (10-50 x 10 M), the immediate reaction progress was monitored by fluorescence
spectrophotometer every 15 minutes over a reaction period of 2 hours, which was indicative for
the formation of the corresponding quinone 3,5-DTBQ.
2
.10. NPP hydrolysis
The rate of hydrolysis of 4-NPP was measured by monitoring the emission spectra at 485nm due
to the release of the P-nitrophenolate anion. In a typical experiment, 2.0 mL of freshly prepared
-2
-3
4
-NPP (10-50 x 10 mol dm ) in dimethyl sulfoxide (DMSO) was transferred into a cuvette in
the sample compartment of the spectrometer. A similar solution was placed in a reference
compartment to correct the hydrolysis in the absence of catalyst. 1.0 mL of Tris-HCl buffer(pH =
7
.2) was added to the reference cuvette to bring the total volume to 3.0 mL and the cuvette was
-3
-3
equilibrated at room temperature for 10 min. The complexes 1-3 (10 mol dm ) were mixed
with 4-NPP separately and the emission spectra were recorded immediately and at regular time
intervals of 15 mins until completion of the reaction.
2
.11. Cell lines and cultural conditions
HeLa and normal Vero cell lines were cultured in RPMI-1640 medium with 10% FBS, 2mML-
glutamine and 1% penicillin/streptomycin under a fully humidified atmosphere 5% CO at 37 °C.
2
2
.11.1. In vitro cytotoxicity by MTT assay
The effect of the synthesized compounds on the viability of the HeLa cell line was determined by
an MTT (3-[4,5-dimethyl thiozole-2-yl]-2-5-diphenyl tetrazolium bromide) assay. 100µL of the
cell suspensions in growth medium were plated in a 96-well microtitre plate at concentrations of
4
1
x 10 cells/well and incubated for 48 h at 37 °C in a humidified incubator. After 48 hours of
incubation, the cells attained confluence; they were incubated again in the presence of various
concentrations in 0.1% DMSO for 72 h at 37 °C. After removal of the sample solution, they were
washed with phosphate buffered saline (pH 7.2), then 20µL of MTT (5mg/mL) was added to

each well of the plate. The plate was then incubated for 4 h at 37 °C. The solution in each well
including MTT was aspirated and 100 µL of buffered DMSO was added to dissolve the
formazone crystals. The plates were shaken for 5 mins and the optical density was measured on a
microplate ELISA reader at 540 nm with cis-platin as a control. The cytotoxicity was obtained
by comparing the absorbance between the samples and the control. The percentage inhibition
was calculated as follows:
%
inhibition = [Abs (Control) – Abs (Test)/Abs (Control)] x100
IC50 was calculated from the dose-response curve using GraphPad Prism 6 software.
2
.11.2. Apoptotic assays by fluorescence microscopy
6
HeLa cells were seeded onto chamber slides in six-well plates at a density of 1 x 10 cells per
well and incubated for 24 h. The cells were cultured in RPMI 1640 supplemented with 10% fetal
bovine serum (FBS) and incubated at 37 °C in 5% CO . The medium was removed and replaced
2
with a medium (final DMSO concentration, 0.05% v/v) containing the complexes (50 µM) for 24
h. The medium was removed again and the cells were washed with ice-cold phosphate buffer
saline (PBS) and fixed with formalin (4%, w/v). Cell nuclei were counterstained with acridine
-1
-1
orange (AO) and ethidium bromide (EB) (AO: 100 mg mL , EB: 100 mg mL ) for 10 min. For
DAPI staining, the treated cells were fixed with 80% ethanol at room temperature for 30 min.
The fixative was removed and the cells were washed with PBS 3 times, and then incubated with
−
1
DAPI (1 µg mL ) for 45 min at room temperature in the dark. Both techniques were used to
distinguish viable cells, early apoptotic cells with blebbing and necrotic cells. Acridine orange
intercalates into DNA and gives a green fluorescence and thus the viable cells appear with a
green nucleus, while early apoptotic cells have condensed or fragmented nuclei. EB is taken up
only by the non-viable cells, giving a bright orange nucleus of the dead cells overwhelming the
acridine orange stain. DAPI dye is effective for fixed-cell staining and quantification of DNA
content. HeLa cells were mounted on a slide and the images were observed under a fluorescent
microscope in a green/blue filter on excitation at 350 nm and emission at 460 nm. The cells were
observed and imaged with a fluorescence microscope (Nikon, Yokohama, Japan).
3. Results and discussion
3
.1. Synthesis and characterization

The three novel mononuclear complexes 1-3, formed in a high yield of up to 80% from a mixture
of [H-(Ap-sadtc)] and the metal(II) acetate in a 2:1 molar ratio, are shown in Scheme 1. The
complexes are pure, crystalline in nature and completely soluble in ethanol, methanol,
acetonitrile, chloroform, dichloromethane, DMF and DMSO, but are insoluble in hexane,
petroleum ether and diethyl ether. The complexes were characterized by elemental analysis, UV-
1
13
Vis, infrared, H NMR, C NMR and mass spectroscopy, and by single crystal X-ray diffraction
studies.
3
.1.1. Infrared spectra
-1
The FTIR spectrum of the ligand did not show any band at 2570 cm (-SH), which indicates a
solid state thione form. Two strong bands appeared for the ligand at 3057 (-NH) and 1043 (C=S)
-1
cm , which were absent after complexation indicating that these two bands disappeared due to
deprotonation of the ligand and chelation of the thiolate sulfur atom. It suggests that the thione
form has been converted into the thiolate form, so that complexation can occur (Scheme 2)
-1
[
49,50]. A strong vibration observed at 1596 cm for the ligand, corresponding to the C=N band,
-1
was shifted to 1532-1533 cm in the metal complexes due to the interaction of the azomethine
nitrogen atom during bonding [51,52]. The investigated and calculated frequencies of the
dithiocarbazate ligand and its complexes 1-3 are given in Table S1.
3
.1.2. Electronic spectra
The electronic spectral absorption of [H-(Ap-sadtc)] in DMSO solution at 368 nm confirms an
intra-ligand transition, which is shown in Fig. S1. The ligand exhibits an n→π* transition, which
is due to coordination between the nitrogen atom and dithiocarbazate moieties. The spectra of
complexes 1-3 were recorded in Tris-HCl buffer and bands appeared at 236, 260, 238 and 314
nm due to a strong absorption of azomethine nitrogen atom, as shown in Figs. S2-S4. Also,
LMCT broad bands appeared at 389, 393 and 398 nm for the respective complexes 1-3, which
were mainly due to an S→M(II) charge transfer. All the complexes were expected to show the
1
1
1
1
1
transitions A
1
g→ A
2
g, A
1
g→ B
1
g and A g→ Eg in the visible region, but the prepared
1
complexes showed only a single (d-d) band at 479, 493 and 497 nm, respectively for the
1
1
A g→ Eg transition as the presence of sulfur covered the other bands.
1

1
3
.1.3. H NMR spectra
The thione and thiol forms of the Schiff base may also exist as either the E or Z conformational
isomer, or as a mixture of both isomers in solution. However, the X-ray crystallographic
structural determination of the ligand shows that in the solid state, it exists solely as the thione
1
form. The H NMR spectrum of the ligand in CDCl
3
exhibits the thione form, as indicated by the
–
NH resonance at 9.9 ppm, showing that in solution, it exists only as the Z isomer [53-55].
1
However, the thiol form may also exist in a CDCl solution of [H-(Ap-sadtc)], the H NMR
3
spectrum displaying a sharp signal for -SH at 4 ppm. The methylene proton of the ligand and
complex 3, appearing at 3.88 and 3.72 ppm respectively, indicates that the allyl sulfur atom is not
involved in coordination. The -CH
2
and -CH protons in the allyl group show multiple peaks at
5
.94-5.96 and 5.17-5.20 ppm, respectively. The aromatic protons appear as doublets within the
range 7.40–7.93 ppm for [H-(Ap-sadtc)] and similar signal peaks are observed for the metal
1
complexes. The -NH and –SH peaks in the H NMR spectrum of the ligand disappeared upon
complexation, which confirms the fact that the N and S atoms are involved in the formation of
the metal complexes.
13
3
.1.4. C NMR spectra
1
3
The C NMR spectra of [H-(Ap-sadtc)] showed signals at 200.06 and 158.19 ppm, which are
attributed to the C=S and C=N groups, respectively. However, in the formation of the metal
complexes 1 and 3, the C=S group experienced a downfield shift at 171.48 and 178.88 ppm, and
the C=N group an upfield shift at 161.04 and 173.94 ppm. These considerable shifts with respect
to [H-(Ap-sadtc)] indicate the involvement of thione sulfur and azomethine nitrogen atoms in
coordination. In addition, C-S carbon atom in [H-(Ap-sadtc)] and complexes 1 and 3 showed
signals at 37.45, 38.58 and 35.59 ppm, respectively. The peaks due to the presence of aromatic
carbons and the methyl group in the ligand and complexes 1 and appeared in the ranges 125.09–
1
39.50 and 15.5-22.80 ppm respectively.
3
.1.5. Crystal structure description of [H-(Ap-sadtc)]
A suitable single crystal was grown from the required amount of hot ethanol by the slow
evaporation method over a time period of one week. A specimen of C12 of approximate
dimensions 0.020 x 0.120 x 0.140 mm was used for the X-ray crystallographic analysis. The
H
14
N S
2 2

final cell constants were based upon the refinement of XYZ-centroids of reflections above 20
σ(I). The calculated minimum and maximum transmission coefficients (based on crystal size)
2
were 0.6370 and 0.9330. The final anisotropic full-matrix least-square refinement on F showed
that [H-(Ap-sadtc)] crystallized in the space group P-1 in the triclinic crystal system. The crystal
structure of the ligand also confirms that it crystallizes as the thione tautomeric form, as
previously predicted by spectroscopic techniques; with the thione sulfur and azomethine nitrogen
atoms in this conformation, the ligand acts as an N,S binding agent. The C=S bond length (1.667
Å) indicates that is a double bond and the C-N bond (1.294 Å) is intermediate between a single
and a double bond, indicating electron delocalization in the N-C-S thioamide group. The N−N
bond distance varied from 1.376(4) to 1.4186(19), 1.396(3) and 1.403(2) Å in the Schiff base to
the complexes, showing that the bond was shorter than a single bond and also indicating
significant π-delocalization of the dithiocarbazate occurred in the C-N-N-C moiety. However,
the rotation of the S-C-S fragment by 180° about the C-N bond orients the donor atom in the
correct position for bidentate coordination.
3
.1.6. Crystal structure description of the metal complexes 1-3
X-ray quality single crystals were isolated from the mother liquors by the slow evaporation
method. Specimens of C24 , of approximate dimensions 0.080 x
.090 x 0.170 and 0.120 x 0.140 x 0.160 mm were used for the X-ray crystallographic analysis.
H26NiN
4
S
4
, and C24
4 4
H26CuN S
0
The ORTEP view of complexes 1-3 at the 25% probability level with the atom numbering
scheme is shown in Fig 1. The calculated minimum and maximum transmission coefficients of
the nickel and copper complexes (based on crystal size) were 0.5360, 0.5530 and 0.7300, 0.6330.
The non-hydrogen atoms were refined anisotropically by applying the full-matrix least-square
2
method on F . The centrosymmetric Ni(II) and Zn(II) complexes crystallized in the monoclinic
space group P121/n1, whereas the Cu(II) complex crystallized in the orthorhombic space group
P
21212, in which the two ligands were symmetrically related to each other and had the same bond
angles and distances. The central metal ion was bis-chelated by the bidentate ligand through the
azomethine nitrogen (N2) and thiolate sulfur (S2) atoms.
The conjugate system of the ligand was influenced by coordination with the metals, as shown by
the N1−N2 bond distance of ligand (1.376(4) Å), with a slight lengthening of the analogous bond
in the complexes (1.4186(19), 1.396(3) and 1.403(2) Å). In addition, the azomethine C9−N2

bond in the nickel complex was found to be 1.282(2) Å, and the C1-N2 bonds in the copper and
zinc complexes were found to be 1.287(3) and 1.285(3) Å, which are slightly shorter than in the
ligand (1.343(5) Å), clearly indicating that the complexation involves deprotonation at the N1
atom. The C4-S2 bond is strongly affected in the nickel (1.7397(18) and 1.7420(17) Å) copper
(
1.739(2) Å) and zinc complexes (1.741(2) and 1.737(2) Å), being longer than the analogous
bond in the Schiff base (1.667(3) Å). So, the C4-S2 bond involved in complexation is a single
bond. The Ni−S (2.1548(5), 2.1583(5) Å), Cu-S (2.2362(7), 2.2363(7) Å), Zn-S (2.2814(5),
2
2
.2817(5) and Ni−N (1.9320(13), 1.9254(12) Å), Cu-N (2.0011(18) Å), Zn-N (2.0546(15),
.0674(15) Å bond lengths are similar to in the bis-chelated four coordinate mononuclear
complexes 1-3. The Ni(II), Cu(II) and Zn(II) central ions have bite angles of 100.91(5),
1
1
01.62(11) and 108.48(6)° for N3-M-N1, while the S3-M-S1 bond angles are 90.97(18),
01.98(4) and 115.13(2)° respectively. The geometrical parameters suggest that none of the
complexes show an ideal tetrahedral geometry due to the restricted bite angles of the chelating
dithiocarbazate ligands. The metal ion occupies a position on a twofold axis and adopts a
distorted tetrahedral geometry. From the above facts, the geometry of complex 1 is more
distorted than complexes 2 and 3.
3
.1.7. DFT calculations
The density functional theory calculations were carried out in the gas phase to optimize the
structure of [H-(Ap-sadtc)] and its metal complexes 1-3, using coordinates of the crystal
structure from the CIF file. The optimized geometries of [H-(Ap-sadtc)] and the mononuclear
complexes 1-3 are depicted in Fig 2. Selected bond lengths and bond angles of both the ligand
and the complexes are given in Table 2. The calculated values are in good agreement with the
crystallographic data in all cases. The distance for the C=S bond (1.706 Å) in the ligand is
lengthened in the complexes to 1.739, 1.740, 1.805, 1.758, 1.789, and 1.779 Å, suggesting that
the sulfur atom coordinates with the metal complexes. Further, the C-N bond lengths varied from
the ligand to the complexes, indicating that deprotonation occurs on complex formation. The
optimized structures were used in the DFT calculations of vibrational frequencies. The predicted
FT-IR spectra using the B3LYP/6-311G level were correlated with an optimal scaling factor of
0
.9613 [56]. The calculated FT-IR spectra present a strong correlation and are in good agreement
with the experimental data [57]. The frontier molecular orbitals play an important role in the

electric and optical properties, as well as in UV-Vis spectra. The energies of the HOMO and
LUMO orbitals and their orbital energy gap were calculated using the DFT method and pictorial
illustrations of these frontier orbitals for the compounds are shown in Fig 2.
The highest occupied molecular orbital (HOMO) represents the outermost orbital filled by
electrons and behaves as an electron donor, while the lowest unoccupied molecular orbital
(
LUMO) is the first empty innermost orbital unfilled by electrons and behaves as an electron
acceptor. These orbitals are also called the frontier molecule orbitals (FMOs). Positive and
negative regions are shown by red and blue colours, respectively. In the ligand, almost all of the
molecules is localized in the HOMO and LUMO, except the allyl (–CH2-CH=CH2) moiety. The
energy gap between the HOMO and LUMO characterizes the chemical stability of the molecule.
The energy gaps in the case of the title ligand and compounds 1-3 are found to be 4.08, 1.48,
0
.75 and 2.42 eV, respectively. Since, a lower energy gap implies more interaction and less
stability, complex 2 has a higher chemical activity than the other compounds. This observation
suggests that the photo-excitation of the complexes could result in facile electronic transitions
from the HOMO, centered on the dithiocarbazate moiety, to the LUMO, spread over the M(II)
ion and the N, S atoms. It can also explain the remarkable photo-cytotoxicity of the
dithiocarbazate complexes. Additionally, a variety of quantum mechanical (QM) reactivity
descriptors, like the electronic chemical potential (µ) chemical hardness (ƞ) and electrophilicity
(
Ѡ) were calculated with the help of the following equations:
ƞ = (I – A)/ 2
µ = - (I +A)/2
2
Ѡ = µ /2 ƞ
where I = - EHOMO , A = - ELUMO
.
The calculated QM reactivity descriptors in Table 3 also determine the binding strength trends of
the complexes. The chemical hardness is quite useful to rationalize the relative stability and
reactivity of chemical species. Hard species, having a large HOMO–LUMO gap, will be more
stable and less reactive than soft species, having small HOMO–LUMO gap; however, a smaller
HOMO–LUMO gap requires less excitation energy and so the absorption bands of molecules are
shifted towards the visible region. This suggests that the bonding of the metal(II) ion modifies
the charge distribution of the π-electrons of the free ligand. This lower energy gap implies that
the delocalization of π-electrons from the metal complexes will result in good non-linear optical

properties in the order 2 > 1 > 3 > L. The natural charge analysis indicates electrovalent bonding
between the ligand and the corresponding metal complexes as listed in Table S2. The negative
charge of the ligand is mainly localized on the S1, N1 and N2 atoms. Upon coordination of S1 to
the metal center, the electron cloud of the C=S bond is displaced towards the S atom, which is
partially compensated by electron displacement. The calculated charges on the metals were
1
.1434, 1.0069 and 1.5728, comparably lower than the formal charge of +2 for the electronic
8
10
configurations of complexes 1, 2 (d ) and 3 (d ). This confirms electron transmission of the
donor atoms toward the central metal.
3
3
.2. CT-DNA binding studies
.2.1. Fluorescence emission spectra
The binding studies of small molecules with calf thymus DNA were carried out by using
fluorescence emission spectroscopy. It is well known that any antitumor drug relies on its
binding character, binding mode, selectivity toward specific DNA base pairs and kinetic
procedure followed during its reaction with DNA. An important class of compounds for cancer
chemotherapy involves non-covalent interactions between CT-DNA and the metal complexes,
such as intercalation, groove binding and electrostatic binding [81-84]. Through strong stacking,
non-covalent intercalations usually result in hypochromism/hyperchromism, with or without
blue/red shifts in the fluorescence emission spectra.
In the presence of increasing amounts of CT-DNA (0-10µM) at a constant metal complex
concentration (25µm), the emission spectral technique was used to determine the intrinsic
binding constant (k ). The interactions of complexes 1-3 with DNA have shown that for the
b
emission wavelength region of 412-429 nm, the intensity of the bands decreased from their
original intensities, based on DNA binding enhancement. It suggests that the metal complexes
are electron-acceptors and the DNA molecule is an electron-donor. All three metal complexes
show hypochromism of 27.3, 58.1 and 41%, and a small blue shift, which may be observed when
the DNA duplex is stabilized. A blue shift may also be attributed to improper coupling (due to
conformational changes) of the π* orbital of the intercalated ligand with the π orbital of the base
pairs. The intrinsic binding constant (K
b
) has been determined using the following Scatchard
equation [58]:
C
F
= C
T
[(I/I - P)/1 - P]
0

where C
and I are the emission intensities in the absence and in the presence of DNA, respectively. The
value of P is obtained from a plot of I/I versus 1/[DNA], such that the limiting emission yield is
given by the y-intercept. The amount of bound probe (C ) at any concentration is equal to C
. The slopes of the obtained Scatchard plots of r/ C versus r for the tested compounds with an
increasing concentration of CT-DNA (Fig. 3) gives the intrinsic binding constant (K ) values, as
listed in Table 4.
T
and C
F
are the concentrations of the probe metal complex (25µm) and the free probe, I
0
0
B
T
−
C
F
F
b
This observation suggests strong intercalation of the complexes to DNA, with complex 2 having
a comparatively higher intercalation than complexes 1 and 3, also being higher than the observed
values reported earlier [59]. The presence of transition metal ions in the complexes also accounts
for the higher binding extent of the complexes with CT-DNA. It has been reported earlier that a
transition metal complex ultimately leads to an effective binding with the helical structure of
DNA and exposes the embedded base pairs to the helix exterior via normal intercalation [60].
From the results of the binding constants, it is concluded that the complexes 1-3 have a strong
binding affinity with CT-DNA [61].
3
.2.2. EB-DNA displacement study
Ethidium bromide (EB) is a fluorescent dye and also a typical indicator of intercalation, since
upon the formation of soluble complexes with nucleic acids, EB emits intense fluorescence in the
presence of CT DNA due to intercalation of its aromatic ring between adjacent base pairs on the
double helix. The changes observed in the spectra of EB upon its binding to CT-DNA are often
used for the interaction study between DNA and other compounds, such as metal complexes.
Before any measurements were taken, the mixture was shaken up and recorded. The fluorescence
spectra of DNA bound EB were obtained at excitation and the emission wavelengths of 541 and
6
05 nm, respectively.
The quenching is due to the reduction of the number of binding sites on DNA that are available
to EB. The fluorescence emission intensity of the DNA–EB system in the absence and presence
of compounds added to DNA pre-treated with EB causes an appreciable reduction in the
emission intensity with increasing complex concentrations. This indicates that complexes 1-3 can
compete with EB, binding stronger to DNA. Furthermore, the quenching data were analyzed

according to the Stern–Volmer equation and Ksv values were obtained from the slope of the plot
of I /I versus [Q]. The quenching plots illustrate that the quenching of EB bound to CT-DNA by
0
complexes 1-3 are in good agreement with the linear Stern–Volmer equation. The Kapp values
were measured from the extent of reduction of the EB emission intensity by the complexes. The
apparent binding constant values were obtained for the compounds using the following equation
[
62-64]:
KEB x [EB] = Kapp [M50%]
-
7
-1
where KEB = 1.0 × 10 M is the DNA binding constant of EB, [EB] is the concentration of EB
(
7.5µM) and [M50%] is the concentration of the compound used to obtain a 50% reduction in
fluorescence intensity of DNA pretreated with EB. The Stern–Volmer constant Kapp values of the
complexes are consistent with the emission titration results. On the basis of spectroscopic
studies, it is concluded that complexes 1-3 bind to CT-DNA in an intercalative mode and
complex 2 binds to CT-DNA more strongly than the other two complexes.
3
3
.3. BSA interaction studies
.3.1. Fluorescence quenching study
The binding affinity of complexes 1-3 with BSA were examined by the fluorescence spectral
technique. BSA has a strong fluorescence emission band around 342 nm, fixing the excitation
wavelength at 290 nm, and the fluorescence intensity of BSA was quenched drastically when the
concentrations of the complexes 1-3 were increased. Generally, the fluorescence quenching can
be illustrated by the well-known Stern–Volmer equation [65]:
F
0
q o
/F = 1+ KSV [Q] = 1+ K τ [Q]
where F
0
and F are the fluorescence intensities of BSA in the absence and presence of the
complexes, respectively, KSV is the linear Stern–Volmer quenching constant, kq is the quenching
-8
rate constant of the biomolecule, τ
without the quencher and [Q] is the concentration of the quenching complex. The value of Ksv is
obtained from the slope of a linear plot of F /F vs [Q]. Different mechanisms occur during
quenching, which are usually classified as dynamic quenching and static quenching, in which the
o
is the average fluorescence lifetime (10 S) [66] of BSA
0

transient existence of an exited state of the fluorophore and the quencher is referred to as a
dynamic quenching process, while static quenching is due to the formation of a ground-state
complex between the fluorophore and the quencher. In both cases, the fluorescence intensity is
mainly related to the concentration of the quencher. Therefore, the quenched fluorophore can
serve as an indicator for the quenching agent. This linear Stern–Volmer plot indicates that more
than one process contributes to the overall quenching of BSA. It has been reported in many cases
that the fluorophores can be quenched by both collision (dynamic quenching) and by complex
formation with the same quencher (static quenching). This complex formation is facilitated
through hydrogen bonding and hydrophobic interactions. The quenching constant K
q
of the
complexes 1-3 follows the order: 2 > 1 > 3, which is in good agreement with the trend in the
1
0
DNA binding affinities. The maximum possible value for dynamic quenching is (2.0 x 10 L
-1
-1
mol S ), which is comparably lower than the quenching constant of the complexes 1-3. The
1
3
14
3
-1 -1
Stern–Volmer plots show that the high quenching constant values of 10 -10 dm mol s
indicate that strong ground state complex formation contributes significantly to the overall
quenching of fluorescence intensity of BSA [67].
3
.3.2. Determination of binding parameters and binding number
Fluorescence quenching is often a result of energy transfer from a donor (Trp) to an acceptor
metal complex). However, in all the systems and combinations studied, the quenching of Trp
(
fluorescence is static and Forster’s long-range energy transfer is not taking place. When a static
quenching interaction occurs, if it is assumed that the complex binds independently to a set of
equivalent binding sites in BSA, the binding parameters can be determined according to the
Scatchard equation [68,69]:
log (F
0
-F/F) = log K′ + n log[Q]
b
where K′ is the binding constant for the binding of the complex with BSA and n is the number
b
of binding sites per BSA molecule. This gives a straight line in the plots of log(F
0
-F/F) vs
log[Q], as shown in Fig. 6.
The values of n indicate the existence of a single binding site in BSA for complexes 1-3. The
observed values of K , Kbin and ‘n’ indicate a strong interaction between the BSA protein and the
complexes, and the values follow the order: 2 > 1 > 3.
q

3
.4.1. Kinetic studies for catechol oxidase activity
Detailed reactivity and kinetic studies of complexes 1-3 were performed in dimethyl sulfoxide
DMSO) because of their good solubility along with that of the substrate 3,5-DTBC and its
(
product 3,5-DTBQ, as depicted in Scheme 3.
All the complexes show efficient catecholase activity at (pH 7.2) because of the de-protonation
of 3,5-DTBC and subsequent binding to the metal centre before its oxidation. Upon addition of
3
,5-DTBC to the metal complexes, spectral studies were carried out which showed an
immediately increment of the emission bands at 435 nm, proving the oxidation of catechol to
quinone (see Fig. 7). The kinetic studies used the method of initial rate by monitoring the growth
-4
of the quinone band. The oxidation rates and various kinetic parameters were determined by 10
M solutions of the complexes with different concentrations of 3,5-DTBC under aerobic
conditions. In all cases, a first order kinetic dependence was observed at low concentrations of
3
,5-DTBC, whereas at higher concentrations, saturation kinetics were recorded, as shown in
Figs. S15-S17. The observed data were analyzed on the basis of the Michaelis-Menten approach
for the enzymatic kinetic model, which seemed to be appropriate. The values of the Michaelis
binding constant (K ), maximum velocity (Vmax) and the rate constant (Kcat) for the dissociation
M
substrates were calculated for the complexes from graphs of 1/V versus 1/[S] (Fig. 7), known as
the Lineweaver-Burk (double reciprocal) graph, using the equation and the kinetic parameters as
listed in Table 6.
1
/V = { K / Vmax}{1/[S]} + 1/ Vmax
M
Among the three mononuclear complexes reported herein, the highest catecholase activity is
observed for complex 2. If the reduction potential is too negative, the complex shows decreased
catalytic activity due to more difficulty in reduction to copper(I), a more positive reduction
potential of the complex gives higher catalytic activity. The donor atom on the dithiocarbazate
moiety may also help to facilitate the substrate-catalyst interaction by forming a positive channel,
showing better catalytic activity [70].
On account of the proposed mechanistic steps of the catecholase oxidase activity of M(II)
complexes, a plausible mechanistic pathway is suggested, as depicted in Scheme 4. Upon
addition of 3,5-DTBC to the metal complexes 1-3, immediately the catechol molecule

coordinates to the complexes and forms an intermediate substrate-complex adduct 1a. This result
indicates a change in coordination environment of the M(II) centre from tetra to penta-
coordinate [71], which may favor electron transfer and equilibration between M(II)-(3,5-DTBC)
and the semiquinone intermediate M(I)-(3,5-DTBQ) (1b, 1c). Its subsequent reaction with
dioxygen may also result in electron reduction, leading to the oxidation of the M(I) ion and
release the quinone molecule along with hydrogen peroxide as a byproduct, 1d [72-75]. After the
quinone molecule is released, the catalyst is regenerated and the catalytic cycle continues [76].
3
.4.2. Kinetics of NPP hydrolysis
The hydrolytic reaction in enzyme-catalysis involves metal ions that are assumed to activate a
water molecule, which forms a hydroxyl group as a nucleophile in the reaction system [77]. The
-
M(II)-bound OH then acts as a nucleophile to attack the phosphate atom of 4-NPP ester, which
leads to hydrolysis (Fig. 8), shown by emission increases at 485 nm due to the formation of 4-
nitrophenolate ions over time (Scheme 5). Since, the catalyst concentration was essentially
constant during measurements, the initial first order rates were measured at different
concentrations of the substrate at pH 7.2 and room temperature. The initial rates obtained for a
range of 4-NPP concentrations were fitted to the Michaelis-Menten equation (Figs. S18-S20) and
linearized by means of the Lineweaver–Burk method, i.e. 1/rate vs. 1/[4-NPP] by changing the
concentration of the substrate (Fig. 8). The kinetic parameters of K , Vmax and the catalytic
M
constant Kcat values are summarized in Table 7. Hydrolysis of 4-nitrophenylphosphate by the
Cu(II), Ni(II) and Zn(II) complexes show that the Cu(II) complex has a higher catalytic activity
than the Ni(II) and Zn(II) complexes, similar to earlier reports [78,79]. All the complexes have
shown better efficacy than catecholase activity.
3
.5.1. In vitro anticancer activity evaluation by an MTT assay
In the present study, the antitumor efficacy of complexes 1–3 was assayed by determining the
viability of HeLa cells using an MTT assay and cis-platin was used as a positive control to assess
the cytotoxicity of the tested mononuclear complexes. The results were analyzed by means of
cell inhibition, expressed as IC50 values which are listed in Table 8. It is notable that complex 2
possesses the most potent inhibitory effect against the HeLa cell line. The IC50 value is well

known for the anticancer drug cisplatin, as shown in Fig. 9. The results of the MTT assay in
Table 8 shows that complexes 1-3 inhibit the growth of cells in a concentration dependent
manner. A low IC50 value implies cytotoxicity at low drug concentrations. The significant
inhibitory activity of the complexes and their binding affinity to CT-DNA/BSA clearly indicates
that the incorporation of the complexes has a marked effect on the cytotoxicity. In addition, an in
vitro cytotoxicity assay conducted on a HeLa cancer cell line versus normal vero cell line has
demonstrated that complexes 1-3 show profound anticancer activity.
3
.5.2. AO-EB/DAPI staining assay
Cell apoptosis is an autonomic ordered programmed cell death in order to maintain homeostasis,
which is controlled by serial genes. The morphological changes of HeLa cells have been
observed under a light microscope. The design of chemotherapeutic drugs, in order to understand
the complexities of apoptosis evolved by cancer cells and the development of strategies to
selectively induce apoptosis in cancer cells, have turned into a unique target in cancer drug
development [80]. The morphological changes induced by complexes 1-3 were evaluated using
AO/EB and DAPI-stained HeLa cancer cells. After treatment of HeLa with 12.5 µM of 1-3,
apoptotic cells with apoptotic features, such as nuclear shrinkage and chromatin condensation,
were found, as shown in Fig. 10. In this figure, yellow arrows show early apoptotic HeLa cells
with membrane blebbing, which is seen at a fixed concentration of complexes 1-3, and blue
arrows exhibit late apoptotic cells with chromatin aggregation, that is highly condensed
chromatin. Necrotic cells having uniform orange to red nuclei with a condensed structure are
observed. The overall results indicate that complexes 1-3 induce cell death by necrosis, which is
in good agreement with the above toxicity results.
4. Conclusions
In conclusion, we have synthesized an N,S donor Schiff base and its mononuclear Ni(II), Cu(II)
and Zn(II) complexes. The characterization of these compounds was carried out using elemental
1
13
analysis, FT-IR, UV-vis, H NMR, C NMR and mass spectrometry. Structural determination
reveals that the complexes are discrete four coordinated, with a slightly distorted tetrahedral
geometry. DFT calculations on all the complexes are in good agreement with the structural
results obtained by X-ray crystallography. The CT-DNA/protein binding properties of the

complexes were examined by fluorescence spectra. All the fluorescence experiments show that
the complexes 1-3 interact strongly with CT-DNA by an intercalative mode and BSA confirmed
that the complexes have a greater binding affinity via the static mode. In this connection, the
catecholase biomimetic catalytic activities of complexes 1-3 have been investigated. The results
obtained show that all the complexes catalyze the aerobic oxidation of catechol to the
corresponding o-quinone. Interestingly, all the three mononuclear complexes provide good
phosphate-hydrolysis catalytic activity that is relevant to the binding of phosphate esters to the
active site of an enzyme that catalyzes the phosphoryl transfer reaction. The cytotoxicity study
shows that the complexes exhibit good cytotoxic activity against HeLa cell lines. Additionally,
apoptotic tests indicate that the complexes show an apoptotic effect on HeLa cells. Based on the
observed results, further studies are planned with these compounds against lung, bladder and
breast cell lines.
Acknowledgement
The author N. N would like to thank P. Sugumar, Centre of Advanced Studies in Crystallography
and Biophysics, University of Madras for his help in the valuable crystallographic discussions.
We are sincerely thankful to the SAIF Punjab University, Chandigarh for the ESI mass facility
and the Director, CAS in Botany, School of Life Sciences, University of Madras for providing
laboratory facilities to perform the cell lines studies.
Electronic supplementary information (ESI) available
1
UV spectra of the compounds (Figs. S1-S4), FTIR spectra of the compounds (Figs. S5-S8), H
1
3
NMR spectra of the compounds (Figs. S9-S10), C NMR spectra of compounds (Figs. S11-
S13), ESI Mass spectrum of complex 1 (Fig. S14), kinetic Plot (3,5-DTBC) rate vs concentration
of the complexes (Figs. S15-S17), kinetic plot (4-NPP) rate vs concentration of the complexes
(
Figs. S18-S20), DFT and experimental values of IR spectra (Table S1). The natural charge and
electronic configuration (Table S2). CCDC reference numbers 1418965-1418968 for the
compounds. For ESI data in CIF or other electronic format see DOI:

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[
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[
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