Synthesis, crystal structure, biomolecular interactions and anticancer properties of Ni(II), Cu(II) and Zn(II) complexes bearing S-allyldithiocarbazate

Article abstract of DOI:10.1016/j.ica.2016.10.035

A new series of bivalent Ni(II), Cu(II) and Zn(II) metal complexes containing Schiff base ligand [H-emk-sadtc] [(E)-allyl 2-(butan-2-ylidene) hydrazine carbodithioate)] were synthesized and characterized by different spectroscopic techniques. The molecula

Full text of DOI:10.1016/j.ica.2016.10.035

Accepted Manuscript
Research paper
Synthesis, crystal structure, biomolecular interactions and anticancer properties
of Ni(II), Cu(II) and Zn(II) complexes bearing S-allyldithiocarbazate
Nanjan Nanjundan, Ramaswamy Narayanasamy, Ray Jay Butcher, Jerry P.
Jasinski, Krishnaswamy Velmurugan, RajuNandhakumar, Manickam
Dakshinamoorthi Balakumaran, Pudupalayam Thangavelu Kalaichelvan,
Vettaikaranpudur G. Gnanasoundari
PII:
S0020-1693(16)30758-7
http://dx.doi.org/10.1016/j.ica.2016.10.035
ICA 17328
DOI:
Reference:
To appear in:
Inorganica Chimica Acta
Received Date:
Revised Date:
Accepted Date:
18 June 2016
27 September 2016
23 October 2016
Please cite this article as: N. Nanjundan, R. Narayanasamy, R. Jay Butcher, J.P. Jasinski, K. Velmurugan,
RajuNandhakumar, M. Dakshinamoorthi Balakumaran, P. Thangavelu Kalaichelvan, V.G. Gnanasoundari,
Synthesis, crystal structure, biomolecular interactions and anticancer properties of Ni(II), Cu(II) and Zn(II)
complexes bearing S-allyldithiocarbazate, Inorganica Chimica Acta (2016), doi: http://dx.doi.org/10.1016/j.ica.
2
016.10.035
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1
Synthesis, crystal structure, biomolecular interactions and anticancer properties
of Ni(II), Cu(II) and Zn(II) complexes bearing S-allyldithiocarbazate
a
a
b
c
Nanjan Nanjundan , Ramaswamy Narayanasamy* , Ray Jay Butcher , Jerry P. Jasinski
d
e
f
Krishnaswamy Velmurugan , RajuNandhakumar , Manickam Dakshinamoorthi Balakumaran ,
g
h
Pudupalayam Thangavelu Kalaichelvan , Vettaikaranpudur G. Gnanasoundari
a
Department of Chemistry, Coimbatore Institute of Technology ,Coimbatore-641 014, India.
b
Department of Inorganic and Structural Chemistry, Howard University, Washington DC 20059.
c
Department of Chemistry, Keene State College, Keene, NH 03435-2001, USA.
d,e
Department of Chemistry, Karunya University, Karunya Nagar, Coimbatore - 641 114, India.
f ,g
Centre for Advanced Studies in Botany, School of Life Sciences, University of Madras, Guindy
Campus, Chennai - 600 025, India.
h
Department of Chemistry, CBM college of Arts and Science college, Coimbatore – 641042,
India.
*
To whom correspondence should be addressed, e-mail: narayanasamycit@gmail.com
Fax: + 91-422-2575020.

2
Abstract
A new series of bivalent Ni(II), Cu(II) and Zn(II) metal complexes containing Schiff base ligand
[
H-emk-sadtc] [(E)-allyl 2-(butan-2-ylidene) hydrazine carbodithioate)] were synthesized and
characterized by different spectroscopic techniques. The molecular structure of the ligand and its
complexes 1 [(E)-allyl 2-(butan-2-ylidene) hydrazine carbodithioato nickel(II)]
,
complex 2 [(E)-
allyl 2-(butan-2-ylidene) hydrazine carbodithioato copper(II)] complex 3 [(E)-allyl 2-(butan-2-
,
ylidene) hydrazine carbodithioato zinc(II)] were confirmed by single crystal X-ray
crystallography. XRD data of the complexes had revealed a distorted square planar geometry
around the metal ion, satisfied by N
2
2
S fashion. Density functional calculations of all the
compounds in gas phase were performed by using DFT (B3LYP) with 6-311G basis set. The
calculated FT-IR and structural data are in good agreement with the experimental results and
confirmed by the experimental one. The interactions of calf thymus (CT) DNA and bovine serum
albumin (BSA) with complexes 1-3 were explored by emission spectra. Catecholase and
Phosphatase-like activities of all the complexes were also investigated. An in vitro cytotoxicity
study of the complexes had shown significant activity against human cervical cancer cell line
(
HeLa) with the best results of complex 2, which was found to be more toxic to cancer cells with
a few micromolar concentrations, as the IC50 value, but significantly less toxic to normal cell
lines. Additionally, the cell death analysis was carried out by AO/EB and DAPI staining
methods.
Key words: S- allyl dithiocarbazate, Ni(II), Cu(II) and Zn(II) complexes, Single crystal
Study, Biomolecular interaction, Catalytic activity

3
1
. Introduction
Developments of coordination chemistry of transition metal complexes have gained
significant importance because of their fascinating structures and enormous potential
applications in pharmacy, biochemistry, industry and so on. During the last few decades, Schiff
base metal complexes have been extensively studied. Schiff bases are an interesting group of
organic compounds, having an azomethine linkage (C=N) and are very useful due to their
stability, chelating properties and biological applications. Various transition metal complexes of
Schiff bases derived from the dithiocarbazate compounds, containing nitrogen, sulphur and/or
oxygen as ligand atoms have been found to display a variety of pharmacological properties [1-3].
In recent years, study of dithiocarbazate-derived Schiff base metal complexes has received much
attention [4-6]. Apart from studying different biological processes induced by metal ions, many
new molecules have also been developed over the years, showing interesting properties.
Whereas, the transition metal ions perform a pivotal role in terms of structural organisation and
overall functionality.
In the field of nickel, copper and zinc complexes acting as DNA targeting drugs and
anticarcinogens, the complexes derived from dithiocarbazate ligands played increasingly
important roles, because of the strong coordination ability with metals, resulting in favorable
interaction with DNA, mainly including intercalation and groove binding. On the other hand,
Bovine serum albumins (BSA) are most extensively studied, as they play important role in
bio-regulatory functions like maintenance of colloidal osmotic blood pressure, blood pH and the
deposition of a wide variety of endogenous and exogenous substances in blood. Further BSA
studies are useful for their effective role in drug delivery and binding properties with transition
metal complexes.
Catalytic properties of dithiocarbazate Schiff base metal complexes have been rarely
investigated. Moreover, the coordination compounds of nickel(II), copper(II) and zinc(II) with
Schiff base ligands have been widely studied because, such complexes play an important role in
redox enzyme systems and may provide the basis of models for active sites of biological systems
or can act as catalysts [7-13]. As expected from their hard/soft nature, Schiff base ligands have
found their principal applications in oxidation/reductive reactions, as well as catalysts for
hydrolysis reactions [14,15].Recently, we have reported the synthesis and X-ray structures of

4
new distorted tetrahedral nickel(II), copper(II) and zinc(II) complexes with Acetophenone
appended Schiff base ligand.
As a continuation of our research work neutral mononuclear Ni(II), Cu(II) and Zn(II)
complexes with the ligand (H-emk-sadtc = Ethyl methyl ketone with S-allyldithiocarbazate) of
general formulae 1 Ni[(H-emk-sadtc)
2
], 2 Cu[(H-emk-sadtc)
2
] and 3 Zn[(H-emk-sadtc) ] were
2
synthesized and characterized. All these compounds were structurally characterized by single
crystal X-ray diffractometry. The interaction of complexes 1-3 with CT-DNA and BSA were
studied using fluorescence spectroscopy. Furthermore, compound 2 showed interesting catalytic
activity towards catechol oxidation and phosphate hydrolysis as reported here. The anticancer
activity of all the complexes was tested with HeLa cancer cell line. The mode of cell death was
studied with AO/EB and DAPI staining assays.
2
.
Experimental
2
.1. Materials and Instrumentation
All the reagents used in this study were of analytical grade and used without further
purification. Solvents were purified and dried according to the standard procedures. Double
distilled water was used to prepare buffers. Calf thymus DNA (CT-DNA), agarose, protein
markers and Bovine serum albumin (BSA) were obtained from Genei, Bangalore and Himedia,
India respectively. Ethidium bromide (EB), Methylene blue and Tris(hydroxymethyl) amino
methane were purchased from Sigma-Aldrich and used as received.
Elemental analyses (C, H, N and S) were carried out on a Vario EL III CHNS analyzer.
Infrared spectra were recorded (as KBr pellets) on Perkin-Elmer FT-IR spectrophotometer in the
-1
1
13
range of 4000-400 cm . H and C NMR spectra were recorded on Bruker Ultra Shield at 300
MHz, using CDCl as a solvent and TMS as an internal reference. Mass spectra for the
3
complexes were recorded on advanced Q-TOF Micro™ mass spectrometer, using electro spray
ionization probe. Electronic spectra were obtained on JASCO V-570 Spectrophotometer.
Fluorescence spectral data were performed on a JASCO FP-8200 fluorescence
spectrophotometer at room temperature. Single crystal X-ray diffraction data collections were
recorded at 173 K on a SHELXL-2014/7 diffractometer, equipped with liquid nitrogen cryostat.
The melting points were checked on a Technico micro heating apparatus and were uncorrected.
-3
o
Stock solutions of M(II) complexes (1.0×10 M in DMSO) were stored at 4 C and the required

5
concentrations were prepared for all the experiments. All the stock solutions were used within
four days. Solutions of the compounds were freshly prepared 1 hour prior to the biochemical
evaluation. Data were expressed as the mean ± standard deviation from three independent
experiments.
2
.2. Synthetic procedure for the ligand [H-emk-sadtc]
The ligand [H-emk-sadtc] was prepared according to literature procedure [16]. The light
yellow precipitate was dissolved in acetonitrile/ethanol (70:30) mixture (20 mL) and kept for
slow evaporation. After few days, light yellow needle-shaped single crystals isolated were
washed with small amount of cold ethanol and pet-ether and dried in vacuum over anhydrous
CaCl
Characterization data: Yield: 60-65%; Color: yellow; M.P: 150 C; Anal. Cal: C
Calculated: C, 47.49; H, 6.97; N, 13.84; S, 31.69. Obtained: C, 46.94; H, 6.84; N, 13.79; S,
2
.
o
8
H
14
N
2
2
S ;
-1
3
1.74; UV-Vis (DMSO), λmax (nm): 276, 368. FT-IR (νcm , KBr): 1635 ν(C=N); 1033 ν(C=S);
1
0
H NMR (CDCl
s, 2H, S-CH
7.7 (S-CH ), 77.10 (C-CH
found, m/z) = 202.3, 202.03.
3
, 25 C, ppm): 9.87 (s, 1H, NH), 2.33 (s, 2H, CH
2
), 1.14- 2.35 (s, 3H, CH
, 25 C, ppm): 199.3 (C=S), 157.55 (C=N),
), 16.5 (CH3), 118.9 (CH=CH ).ESI-MS (Calcd,
3
), 3.95
1
3
0
(
2
), 1.96 (s, H, CH); C NMR (CDCl
3
3
2
2
), 32.53 (CH-CH
3
2
2
.3. Synthesis of the Complex 1 Ni[(H-emk-sadtc)
Nickel(II) acetate tetrahydrate (0.248 g, 1 mmol) in 20 mL of ethanol and the ligand
H-emk-sadtc] (0. 404 g, 2 mmol) in 20 mL of hot ethanol was added dropwise, the colour
2
]
[
changes from light green to brown . The volume of the solution was reduced to 15 mL and kept
for slow evaporation. Dark brown square-shaped single crystals were obtained after one week.
The crystals were washed with ethanol and pet-ether and dried in vacuum over anhydrous CaCl .
2
o
Characterization data: Yield: 80-85%; Color: Dark brown; M.P: 155 C; Anal. Cal:
NiS ; Calculated: C, 41.65; H, 5.68; N, 12.14; S, 27.80. Obtained: C, 40.64; H, 5.58; N,
1.89; S, 27.74; UV-Vis (DMSO), λmax (nm): 368, 396, 456, 507. FT-IR (νcm , KBr): 1589
C
16
H
26
N
4
4
-1
1
1
0
ν(C=N); H NMR (CDCl
3
, 25 C, ppm): 2.95 (s, 2H, CH
2
), 1.10- 1.50 (s, 3H, CH
, 25 C, ppm): 170.07 (C-S), 37.65 (S-CH
), 118.2 (CH=CH
3
), 3.97 (s, 2H,
1
3
0
S-CH
CH ), 28.54 (CH-CH
61.36, 462.94
2
), 3.01 (s, H, CH); C NMR (CDCl
3
2
), 77.48 (C-
+
2
3
), 14.47 (CH
3
2
).ESI-MS (Calcd, found(M+H) , m/z) =
4

6
2
.4. Synthesis of the Complex 2 Cu[(H-emk-sadtc)
2
]
A hot ethanolic solution of ligand [H-emk-sadtc] (0.404 g, 2 mmol) in 20 mL was added
to copper(II) acetate monohydrate (0.199 g, 1 mmol) in 20 mL of chloroform and the solution
0
was allowed to reduce to 15 mL on a water bath at 50 C. The resulted brownish black solution
was cooled and filtered. The filtered solution was kept for slow evaporation at room temperature.
After 10 days, brownish - black single crystals were collected by filtration, washed with
chloroform/ethanol mixture (50:50) and dried over CaCl
Characterization data: Yield: 80-85%; Color: Brownish black; M.P: 155 C; Anal. Cal:
; Calculated: C, 41.22; H, 5.62; N, 12.02; S, 27.51. Obtained: C, 40.84; H, 5.48;
2
.
o
C
16
4 4
H26CuN S
-1
N, 11.92; S, 27.64; UV-Vis (DMSO), λmax (nm): 363, 389, 406, 624. FT-IR (νcm , KBr): 1604
ν(C=N).ESI-MS (Calcd, found, m/z) = 466.21, 466.93.
2
.5. Synthesis of the Complex 3 Zn[(H-emk-sadtc)
2
]
Zinc(II) acetate dihydrate (0.219 g, 1 mmol) dissolved in 20 mL of methanol was added
to the ligand [H-emk-sadtc] solution (0.404 g, 2 mmol) of 20 mL of acetonitrile with constant
stirring for 10 minutes. Then, the resulting mixture was refluxed on a water bath for 30 mins and
reduced upto 15 mL, cooled and filtered. The filtrate was kept in refrigerator over a period of 15
days and the yellow colored needle - shaped crystals were isolated from the mother liquor and
dried in CaCl
Characterization data: Yield 70%; Color; Yellow; M.P: 176 C; Anal. Formula: C16
Calculated: C, 41.06; H, 5.60; N, 11.97; S, 27.40; Obtained: C, 41.28; H, 5.41; N, 10.98; S,
2
desiccator.
o
H
26
N
4
4
S Zn
-1
1
2
2.59; UV-Vis (Tris-HCL buffer), λmax (nm): 236, 341. FT-IR (νcm , KBr): 1612 ν(C=N); H
0
NMR (CDCl
3
, 25 C, ppm): 2.32 (s, 2H, CH
2
), 1.06- 2.31 (s, 3H, CH
, 25 C, ppm): 177.91 (C-S), 177.23 (C-N), 35.53 (S-CH
), 19.76 (CH ), 117.9 (CH=CH ).
3
), 3.82 (s, 2H, S-CH
2
),
),
1
3
0
2
7
.43 (s, H, CH); C NMR (CDCl
3
2
7.22 (C-CH ), 33.43 (CH-CH
2
3
3
2
2
.6. Crystallographic measurements
Crystals of the ligand [H-emk-sadtc] and its complexes 1-3 of suitable size selected from
the mother liquor were mounted on the tip of a glass fiber and cemented using epoxy resin. The
crystallographic data for all the compounds were collected on a SHELXL-2014/7 diffractometer
equipped with a CCD-area detector on Oxford Diffraction X-Calibur Eos Gemini System. X-ray
radiation employed was generated from a Mo sealed X-ray tube (Kα = 0.70173 Å), fitted with a

7
graphite monochromator. Data analysis was carried out using CrysAlis program. Unit-cell
dimensions and intensity data for all the compounds were measured at 173(2) K. The unit cell
was verified by the examination of h k l overlays on several frames of data by comparing with
both the orientation matrices. After careful examination of the unit cell, a standard data
collection procedure was initiated using omega scans. All the non-hydrogen atoms were refined
with anisotropic thermal parameters. The structure was refined (weighted least squares
2
refinement on F ) to convergence. Absorption corrections were carried out using multi-scan
method. The MERCURY was employed for the final data presentation and structure plots.
Crystal data and additional details of data collection and refinement of the structures are
presented in Table 1.
Insert Table 1
2
.7. Theoretical calculations
All the computational calculations were performed with Gaussian 09 program using
Density Functional Theory (DFT) with a combination of 6-311G basis set [19]. The initial
coordinates obtained from the crystal structure of the ligand and its complexes were used for
optimization. Visualization of the DFT optimized structures and frontier molecular orbitals
(
HOMO and LUMO) of the ligand and its complexes 1-3 were performed with the chemcraft
program package (http://www.chemcraftprog.com). The vibrational frequency calculations were
performed to ensure the optimized geometries that represent local minima on the potential
energy surface with only positive eigen-values. The minimum energy structures were ensured by
the absence of any imaginary frequency. Also, the theoretical investigation of the natural charge
for each atom and QM descriptors were calculated using the same basic set.
2
.8. DNA and EB-DNA binding experiments
DNA and EB-DNA binding experimental studies were carried out by employing the
procedure reported by us previously [16].
2
.9. Tryptophan fluorescence quenching experiments
Tryptophan fluorescence quenching studies were carried out as described previously [20].

8
2
.10. Catalytic oxidation of 3,5-DTBC
A study of catecholase activity was performed by the widely used 3,5-di-tert-
butylcatechol (3,5-DTBC) as the substrate, because its low redox potential makes it easily
oxidized to the corresponding quinone (3,5-DTBQ) [21]. Since, it prevents over oxidation
reaction, such as ring opening because of its highly stability, it shows a maximum emission at
4
35 nm in pure DMSO. The increase in emission at 435 nm with the increased oxidized species
of 3,5-tert-butyl benzoquinone showed that the complexes 1-3 were oxidized 3,5-DTBC
effectively under the same reaction conditions. Hence, a detailed kinetic study of DTBC was
performed, before we started to evaluate the catalytic ability of the complexes 1-3 to oxidize 3,5-
DTBC to 3,5-DTBQ at room temperature in oxygen atmospheric condition (Scheme 1).
Insert Scheme 1
-4
-2
For this purpose, 10 M DMSO solutions of the metal complexes were treated with 10 M (100
equivalents) 3,5-DTBC. Maintaining the concentration of the complex as constant and by adding
-2
different substrate concentrations (10 - 50 X 10 M), the immediate reaction progress was
monitored by the fluorescence spectrometer at 15 minutes time interval over the reaction period
of 2 hours, which was indicative of the formation of the corresponding quinone 3,5-DTBQ and
was analyzed by Michaelis–Menten equation, as well as, Lineweaver–Burk plot (Fig. 5), which
provided a turnover number.
2
.11. Phosphatase hydrolysis activity
In order to study the hydrolytic activity of the complexes 1-3, (10 M) solutions in pure
-4
-2
dimethyl sulphoxide (DMSO) were treated with 100 equiv (10 M) of disodium (4-nitrophenyl)
phosphate hexahydrate (4-NPP) under aerobic conditions at the room temperature (Scheme 2).
Insert Scheme 2
To determine the dependence of rate on the substrate concentrations and various kinetic
-4
parameters, (10 M) solution of the complexes 1-3 were treated with 10, 20, 30, 40, 50 and 60
equivalents of the substrate. The emission versus wavelength scans of the solution was recorded
at regular time interval of 15 min in the wavelength range of 350-650 nm. The reaction was

9
followed spectrophotometrically by monitoring the increase in emission at 485 nm (4-NP band
maximum) as a function of time as depicted in Fig 6. From this figure, it is evident that the
observed spectral changes upon the addition of complexes 1-3 with increasing concentration of
4
-NPP demonstrates active hydrolysis reaction. The dependency of rate on the concentration of
substrate was analyzed by the Michaelis−Menten approach of enzymatic kinetics to obtain
Lineweaver−Burk plot and the values of kinetic parameters, Vmax, K , and Kcat [23,24].
M
2
2
.12. Antiproliferative activity
.12.1. Cell culture
The human cervical cancer cell line (HeLa) was obtained from the National Center for
Cell Science (NCCS), Pune, India. The cells were cultured in RPMI 1640 medium (Biochrom
AG, Berlin, Germany), supplemented with 10% fetal bovine serum (Sigma), cisplatin (Getwell
pharmaceuticals, India), and 100 µg/mL penicillin and 100 µg/mL streptomycin, as antibiotics
(
Himedia, Mumbai, India) in 96-well culture plates at 37°C under a humidified atmosphere of
% CO in a CO incubator (Heraeus, Hanau, Germany). All the experiments were performed
using cells from the passage 15 or less.
2
2
2
2
.12.2. Cell viability assay
The in vitro growth inhibitory effect of the complexes 1–3 towards tumour cell line was
evaluated by the MTT (tetrazolium salt reduction) assay on the human cervical cancer cell line
HeLa) and normal Vero cell line. Cell viability was carried out using MTT assay method as
(
prescribed previously [25, 26]. The complexes 1-3 in the concentration range of 10-100 µM were
dissolved in DMSO and in all the experiments, the percentage of DMSO was maintained in the
range of 0.1-1%. DMSO by itself was found to be non-toxic to the cells upto 1% concentration.
After 24 h, the medium was replaced with respective medium of 1% FBS containing the
compounds at various concentrations and incubated at 37 °C under the conditions of 5% CO ,
2
−
1
9
5% air and 100% relative humidity for 24 h. After 24 h, 10 µL of MTT (5 mg mL ) in
phosphate buffered saline (PBS) was added to each well and the plates were wrapped with
aluminum foil and incubated at 37 °C for 4 h. The medium with MTT was then discarded and the
formed formazan crystals were dissolved in 100 µL of DMSO. The absorbance was monitored at
5
70 (measurement) and 630 nm (reference) using a 96-well plate reader (Bio-Rad, Hercules, CA)
to 50% of those in the untreated control wells and a graph was plotted with the percentage of cell

10
inhibition versus concentration. Data were collected for three replicates each, and used to
calculate the mean. The percentage inhibition was calculated from this data, using the formula
[
27, 28].
Inhibition rate (IR %) = OD (control) - OD Drug treated cells / OD (control) × 100
The corresponding IC50 (concentration of drug that inhibits cell growth by 50%) value was
determined by the nonlinear regression analysis using Origin 6.0 software.
3
. Results and Discussion
3
.1. Synthesis and characterization
Three new mononuclear complexes were synthesized from straight forward reactions of
the ligand mixed with metal acetates in 2:1 ligand: metal ratio. Complexes 1-3 were obtained in
good yields and found soluble in ethanol, methanol, chloroform, dichloromethane, DMSO and
DMF. The complexes were stable at room temperature and analytically pure microanalytical data
confirmed to the proposed molecular formula (data given in the Experimental section).
Insert Scheme 3
3
.1.1. FT - IR spectra
The ligand has a proton adjacent to the thione group. It has been stated that the thione
group [-NH-C(=S)-] is relatively unstable in monomeric form and tends to form a more stable C-
S single bond by enethiolization, if there is at least one hydrogen atom adjacent to the thione
υ (C=S) bond [29]. FT-IR spectral assignments for the ligand and its complexes 1-3 are listed in
the experimental section and also in Table S1. FT-IR spectrum of the ligand does not exhibit a
-1
υ(S–H) band at around 2,500–2,700 cm , suggesting that in the solid state, the thione form
-1
predominates [30]. The strong band at 3178.6 cm of the free ligand is assigned to -NH and lack
of this band in the complexes indicates that the nitrogen atom is deprotonated during
coordination. This provides a strong evidence for the coordination of the ligand with M(II) ion in
the thiolate form [31,32]. In addition, a strong band assigned to azomethine υ(C=N) vibration
-1
was observed at 1635 cm in the spectra of the ligands that experienced a downward shift of 23-
-1
4
6 cm , indicating the coordination of ligands to M(II) ion via azomethine nitrogen atom. The
-1
hydrazinic υ(N-N) stretching vibration band of free ligands at 1303.3 cm is also shifted to the

11
-1
higher region 70-116 cm upon metal complexation, to further support the coordination of
-1
nitrogen atom to the metal centre. Another strong band at 1033.8 cm for the free ligand is
assigned to υ(C=S), which is also disappeared in the complexes 1-3, suggesting that the sulphur
atom is coordinated through the metal centre in thiolate form [33]. The υ(C – S-S) strong band of
-1
-1
the uncoordinated free ligand at 956.6 cm is also shifted to higher wave number at 23cm in the
spectra of the respective complexes, coordinated via the thione/thiolate sulphur atom. A new
-1
band that is observed for the metal complexes at 586.3 cm is attributed to υ(M-N) and the
-1
complexes exhibit medium intensity band at 424.3 – 416.6 cm , which may be assigned to υ(M-
S).
3
.1.2. Emission Spectra
The electronic spectral data of the ligand showed bands at 222, 304 and 335 nm. The
intensity of the band at 222 nm is due to (π → π*, CH=N) and 304 nm band for (π → π*,
dithiocarbazate moiety). Complexes 1 and 2 possess similar characteristic spectral features that
are depicted in Fig S1. The spectral data were summarized in the experimental section. The
complexes exhibited intra-ligand transitions in the range of 360-397 nm, which are mostly due to
non-bonding electrons (n → π*, dithiocarbazate moiety) present in the nitrogen atom of
azomethine group in Schiff base complexes. In addition, the complexes showed bands at around
4
00 nm that might be assigned to S → M charge transfer transition, indicating the coordination
of Schiff base to the metal atom via thiolate sulfur. While, Ni(II) and Cu(II) complexes (1 and 2)
showed very broad weak d-d band around 660-680 nm region attributed to distorted square
planar geometry. The diamagnetic complexes showed absorption bands corresponding to
1
1
1
1
1
1
A
1g→ A2g, A1g→ B1g and A1g→ E transitions [34]. The absorption observed for Zn(II)
g
1
0
complex (3) is consistent with d metal ion configuration and shows no d-d transitions as well.
1
3
.1.3. H NMR spectra
In the presence of transition metal ions, Schiff base ligands are converted into thiol
tautomeric forms to facilitate the formation of centric neutral bis-ligand metal complexes. Both
thiolo sulfur and azomethine nitrogen, like other kinds of these ligands, coordinate to the metal
atom. The spectra of free ligand and its complexes 1 and 3 showed well-defined signals that
1
indicate no fluxionality in solution (Fig. S4–S6). The integration of the H resonances confirms

12
that [H-emk-sadtc] ratio is in good agreement with the structural composition of cis complexes
1
1
-3 (Fig. S4–S6). H NMR data for the ligand and its corresponding complexes are summarized
in the experimental section. The ligand shows signal at 9.87 ppm, which is assigned to N-H
proton and it exists, predominantly as E form of the isomer [35]. The lack of this signal in
complexes confirms that azomethine nitrogen is coordinated to the metal centre. This is
inconsistent with the presence of thione form in solution [36]. For further confirmation, it is
1
noted that H NMR spectra of the ligand in CDCl
3
do not show any signal to S-H proton in
solution, which is a strong evidence that it also remains solely in thione tautomeric form. In
contrast, Schiff bases derived from S-alkyl/aryl dithiocarbazates have always been shown
deprotonation, while coordinating to the metal ion, yielding complexes of deprotonated thiolate
form of the ligand [37, 38]. The methylene protons, ligands and their complexes do not show any
changes. It remains that S-benzyl/allyl sulphur do not take part in coordination.
13
3
.1.4. C NMR spectra
1
13
Due to paramagnetic nature of the complex 2, H and C NMR spectra could not be
1
3
studied. C NMR spectra of the ligand and its complexes were recorded in CDCl
signals obtained are in good agreement with the probable structure. The ligand shows signals at
99.30 and 157.55 ppm, which are due to thioamide carbon (C=S) and azomethine carbon
C=N), respectively. The low intensity is due to the deshielding effect of nitrogen and two
3
and the
1
(
sulphur atoms, adjacent to carbon. These electronegative elements produce a large downfield
shift, since they are directly attached to the carbon atom. In the complexes, (C=S) peak is shifted
significantly upfield to maximum 13-22 ppm, as compared to the ligand, which confirms
1
3
bonding through thione sulphur atom. However, comparison of C NMR spectra of the ligand
with that of the complexes 1 and 3 show that there is only a very little shift of the signals due to
the azomethine and thioamide carbon atoms, indicating the fact that the coordination does not
affect the electronic environment around those atoms, because of delocalization of electrons in
the dithiocarbazate moiety (Fig. S7 – S9).
3
.1.5. Description of Molecular structure [H-emk-sadtc]
The molecular structure of [H-emk-sadtc] with its atom numbering is shown in Fig.
. The selected bond lengths and angles are listed in Table 2. Schiff base is obtained as
1

13
monoclinic crystals with a space group of P2 /n. Schiff base is in thione form with C5=S1 bond
1
distance 1.6603(17) is intermediate between a pure single bond ca. 1.81 Å and the double bond
ca. 1.58 Å, possibly due to extensive conjucation over C5-N1-N2-C1 chain and other
intermolecular interactions [39]. The N1-N2 distance, 1.3865(19) is similar to that found in S-
benzyldithiocarbazate [40] and S- methyldithiocarbazate [41] although, a little but significant
contraction of the bond may be attributed to the extension of a π- electronic system along with
dithiocarbazate backbone [42]. The cis-trans isomerism is exhibited in Schiff base around
thioamide moiety [43]. As observed that the ethyl methyl ketone chain is trans with respect to
terminal thione S1 atom about the azomethine nitrogen atom N2, which is similar to the free,
unsubstituted thiosemicarbazides and S-alkyldithiocarbazates [44]. In contrast, S-allyl group is
cis with respect to the terminal C5=S1 about the C5-S2 bond. The bond angles are approximately
0
2
closer to 120 in consistent with sp hybridization. Both ethyl methyl ketone and S-allyl moieties
are not essentially planar, but exist with twisted conformations as shown from their dihedral
angles.
Insert Fig. 1
The N2 - N1(H)-C5=S1 and N1(H)-C5-S2-C6 chains adopt trans conformation with
o
o
torsion angles -178.67(12) [ ] and -178.4(6) [ ], respectively whereas, S1-C5-S2-C6 chain adopts
o
cis-conformation about C1=N2 with torsion angle 1.7 [ ] that shows E configuration. However,
0
in solution, 180 rotations of the fragment S2-C5-S1 about C5-N1 bond orients two donor atoms
N, S that are in the correct position for bidentate coordination.
3
.1.6. Molecular structural characterization of complexes 1-3
Insert Fig.2
Crystal structures of complexes 1-3 reveal four - coordinate geometries with two
symmetric Schiff bases bonded to the metal ions as shown in Fig. 2. In complexes 1-3,
deprotonation of the ligand leads to tautomerization to the iminothiolate. In each case, the
metal(II) center is coordinated by two N,S-bidentate ligand and exists within a cis-O2S2 donor
set that defines a square planar geometry. While coordinating in iminothiolate form, the negative

14
charge generated on sulphur atom is delocalized in the C=N – N=C chain, as indicated by the
intermediate C5-N2 bond lengths in complexes 1(1.280), 2 (1.287, 1.277) and 3 (1.283, 1.291).
Insert Table 2
In all the complexes, M-N bond distances are comparably shorter than M-S
distances. This indicates that the coordinated ligand is in thiolato form. The sum of the angles
around metal centre for complexes 1-3 (364.4, 380.3, 409.60) indicate that complex 3 is more
distorted than complexes 1 and 2 as a regular square planar geometry.
3
.1.7. DFT geometrical analysis and calculations
The geometries of ligand and its complexes 1-3 were optimized using B3LYP/6-
11G level of calculations in the singlet ground state. The input files were prepared from
3
crystallographic coordinates obtained from X-ray measurements. The optimized structures of the
compounds were depicted in Fig. S13 and contour plots of the compounds were shown in Fig
S14. The important calculated bond-lengths and bond-angles of all the compounds are in good
agreement with experimentally observed single crystal X-ray data measurements as listed in
Table 2. FT-IR spectroscopic analyzes were carried out to investigate vibrational responses of
the functional groups of all compounds. The vibrational assignments of FT-IR intensities and
normal mode descriptions of compounds are listed in the supplementary material (Table S1) and
the obtained spectra of all compounds are given in Fig S3.
-
1
FT-IR spectrum of NH stretching band is usually observed above 3200 cm .
However, this band is shifted to lower frequency region due to intra- and inter-molecular
-1
hydrogen bonding by the NH band that is observed at 3178 cm of the IR spectrum. From the
-
1
computed frequencies, a strong peak at 3156 cm corresponding to NH stretching vibration of
-1
the ligand is observed. The C=N band in free ligand occurred at 1695 cm , upon complexation,
-1
is shifted to 1535, 1538 cm , which confirms the coordination of ligand through imine nitrogen.
The absence of C=S band in complexes, is confirmed by the thiolic sulphur is coordinated to the
metal ion. The theoretical calculations are strongly correlated with the experimental values and
confirmed that the ligand is coordinated through N
2
2
S mode.
Since the experimental and theoretical data can be used for the identification of
compounds, we have followed these data to confirm the structure of complexes. A difference

15
between experimental and theoretical studies is that the computations were done in gas phase
while X-ray data were obtained in solid phase for all the compounds. While some calculated
values are smaller than the experimental, some values are larger. Some of bond angles showed
large deviations from experimental ones. In the free ligand C=N, C=S bond lengths of 1.359 and
1
.707Å are elongated in complexes. The calculated geometry also proves distorted square planar
geometry around metal atom.
The atomic charge distributions on donor–acceptor atoms for the complexes under
investigation were obtained from NBO analysis. Electronegativity (χ), chemical potential (µ),
global hardness (η), global softness (S) and electrophilicity index (ω) are global reactivity
descriptors, which are highly successful in predicting global reactivity trends. On the basis of
Koopmans’s theorem [45], global reactivity descriptors are calculated from the energies of
frontier molecular orbitals εHOMO, εLUMO as, listed in Table S2.
Χ = -1/2(εHOMO + εLUMO
)
µ = - χ = 1/2(εHOMO + εLUMO
)
η = 1/2(εHOMO - εLUMO
)
S = 1/2η
2
ω = µ /2η
Depending upon the value of electrophilicity index, the molecule can act as an electrophile or a
nucleophile. Based on the above calculations, the electrophilicity index of the free ligand and its
complexes are found to be 1.717, 2.293, 5.564 and 2.066 eV. Thus, the complex 2 comparably
has higher global electrophilicity index (ω) and hence better is electrophilic character. The
kinetic stability, chemical reactivity and color of the transition metal complexes in solutions are
governed by HOMO–LUMO energy differences. The HOMO–LUMO energy gap of compounds
are 4.513, 3.698, 1.836 and 3.516, where a ligand can be excited easily whereas, a higher energy
gap results in higher kinetic stability, but lower chemical reactivity of the molecule. Further, the
energy of HOMO is directly related to ionization potential and LUMO energy is directly related
to electron affinity. Thus, greater the EHOMO value greater will be, the electron donating
capability and smaller the ELUMO value will be smaller the resistance to accept electrons.
The net atomic charge for atoms of the title compound calculated by Natural Population
Analysis (NPA) was presented in Table S3. The calculated charge value for nitrogen atoms are
in contrast to the expected one (-1 a.u.), which indicates electron transfer from these atoms to

16
metal center. Furthermore, the decrease of formal charges of sulfur and chlorine from the
expected values of -2 and -1 a.u. is a reason for the migration of electrons to metal centre.
Compared to atomic charges it can be seen that electron charge density on metal atoms in their
respective complexes has increased, i.e., before complexation, charge on metal cation is +2, and
after complexation charge of metal atoms are 1.26330, 1.07996 and 1.48052 respectively. This
indicates that the ligands transfer their negative charges to the respective metal ions upon
complex formation.
3
.2. Emission spectra titration and CT-DNA binding evaluation
DNA binding is an important parameter for the chemical and photo-induced DNA
cleavage activity of metal complexes. Therefore, interaction of metal complexes with CT-DNA
can be conveniently monitored by fluorescence spectroscopy and also the changes in emission
and shift in wavelength maxima. Binding of complexes to DNA through intercalation generally
leads to hypochromism and bathochromism of emission band. This shift is observed due to
strong stacking interaction between aromatic chromophore of the ligand and base pairs of DNA.
Minor groove binding of the complexes to DNA results in hyperchromism in absorption
intensity, resulting in the unwinding of DNA double helix as well as, its unstacking and
concomitant exposure of the bases.
Insert Fig. 3
The observed emission spectra of complexes in the presence and absence of CT-
DNA exhibited a hypochromic shift, which indicates that there is interaction between the
complexes and CT-DNA, which is an evidence for intercalative binding mode as depicted in Fig.
3
. In order to quantify binding affinity between complexes 1-3 and CT-DNA, the binding
5
-1
constant (K ) values are listed in Table 3. The complex 2(2.556 x 10 M ) shows higher binding
b
propensity compared to other complexes. All the complexes have shown binding affinity
5
-1
yielding (K
.3. Emission titration in the presence of Ethidium bromide
The fluorescence spectral method using Ethidium bromide (EB) as a reference was
b
) values (~10 M ) in the order of 2 >1 >3.
3
used to determine relative binding of complexes 1-3 in Tris-HCl buffer (pH=7.2) solution of
EB/DNA mixture. Before starting measurements, the mixture was shaken up and recorded. EB

17
was non-emissive in Tris-HCl buffer (pH=7.2) but, in the presence of DNA, EB showed
enhanced intensity due to intercalative binding to DNA. Fluorescence intensities at 605 nm
(
excited at 540 nm) were measured at different complex concentrations (10-50 µM). The relative
binding tendency of metal complexes to CT-DNA was determined by the comparison of slope of
the line in fluorescence intensity versus complex concentration. Stern-Volmer quenching
constant, Ksv was calculated using the equation [46, 47]
0
I /I = 1 + Ksv[Q]
where, Io and I are fluorescence intensities of EB-DNA in the absence and presence of complex
respectively. [Q] is quencher concentration, Ksv is a linear Stern- Volmer quenching constant
slope obtained from linear fit plot of I
0
/I vs [complex]. Further, the apparent binding constant
(
Kapp) was calculated from the following equation:
KEB [EB] = Kapp [complex]
where, the complex concentration was obtained from the value at a 50% reduction of
7
-1
fluorescence intensity of EB, KEB = 1.0×10 M and[EB] = 7.5 µM.
Insert Fig. 4
3
.4. Competitive interaction of the complexes with EB-DNA
To check whether the binding of complexes 1-3 with DNA is of the groove or
intercalative nature, we have performed a comparative binding study with EB by adding the
complexes as quenchers. EB is a planar cationic dye that can intercalate to CT-DNA and soluble
complexes with nucleic acids. EB in buffer medium with stacking shows interaction of planar
phenanthiridium ring between the adjacent base pairs [48, 49]. The enormous quenching in the
emission intensity of EB in the DNA environment can be rationalized by considering the
intercalative binding of EB with DNA [50].
The emission spectra of DNA bound EB in the presence of varying concentrations of
the complex is shown in (Fig. 4). The plot illustrates that the quenching EB bound DNA with
complexes are in good agreement with linear Stern-Volmer equation [51, 52]. Stern-Volmer
5
-1
quenching constant (KSV) 2.3-3.2 x 10 M and the apparent binding constant (Kapp) 1.59-1.74 x
5
-1
1
0 M values are listed in Table 3.

18
Insert Table 3
3
.5. Tryptophan Quenching Experiments
Serum albumin is the most abundant protein in the circulatory system. The most
important physiological function of serum albumin is to maintain osmotic pressure, pH of blood
and transport a wide variety of endogenous and exogenous compounds, including fatty acids,
metal, amino acids, steroids and drugs. Because of these extraordinary characteristics, albumins
from the various sources have gained extensive biomedical and industrial applications, as well
as, research interest [53]. Fluorescence spectroscopy is an effective method to examine
interaction between metal complexes and BSA. Here, we have investigated the binding affinity
of complexes 1–3 with BSA under physiological conditions.
Insert Fig. 5
Fig. 5 shows the effect of increasing concentrations of the added complexes 1-3, on fluorescence
emission of BSA. The intensity of broad emission band at 342 nm decreases largely with the
increasing concentration of complexes, which confirmed the fact that the interaction between the
complexes and BSA have occurred.
Insert Fig. 6
The extent of quenching of fluorescence intensity, as expressed by the value of Stern-
Volmer constant (Ksv), is a measure of protein binding affinity of the complexes. It is calculated
from the equation [54, 55].
0
F /F = 1 + KSV[Q]
where, F
respectively. KSV is Stern-Volmer quenching constant and [Q] is the concentration quencher. The
plot of F /F vs [Q] (Fig.6) shows the value of KSV. According to the equation.
0
and F are steady-state fluorescence intensities in the absence and presence of quencher,
0
q 0
KSV = K τ
where, K
quencher and the value of τ
binding of complexes 1-3 with BSA as listed in Table 4.The values of quenching rate constant
q
is quenching rate constant and τ
0
is fluorescence life - time of protein in the absence of
−
8
0
is considered to be 10 s. KSV and K values are obtained from the
q

19
1
0
-1
q
(K ) is 1000 times higher than optical collision rate constant, 2 x 10 M [56]. These results
suggest that all the complexes interact strongly with BSA and the probable quenching
mechanism is not initiated by dynamic quenching, but by a static one.
3
.6. Binding Analysis
When the small molecules bind independently to a set of equivalent sites on a
macromolecule, the equilibrium between free and bound molecules is given by Scatchard
equation [57, 58].
0
log (F - F)/F = log K + n log [Q]
Where, K and ‘n’ are the binding constant and number of binding sites, respectively. The plot of
Log [F -F/F] versus log [Q] (Fig. 5.) can be used to determine the values of both K as well as,
n’ and such values are calculated for the complexes, 1-3 and listed in Table 4.
0
‘
Insert Table 4
The values of ‘n’ are approximately equal to 1, indicating that there is one class of binding site to
complex in BSA. Fluorescence spectral results clearly show that the complexes are having the
binding constant values that follows the order 2>1>3. The higher binding affinity of 2 is
enhanced due to copper metal centre and the efficacy may be increased by the phenyl group.
3
.7. Kinetic Studies for Catecholase Activity
Proteins are the most important building blocks of a living cell. The entire collection
of proteins of a living cell is also called as proteome. Metalloproteins containing Cu perform four
basic functions: i) metal ion storage, transport and uptake ii) electron transfer iii) dioxygen
storage, transport and uptake and iv) catalysis.
Insert Fig. 7
Catechol oxidase is a type III copper protein, containing a binuclear active site
actively involved in the oxidation of catechols to the highly reactive quinones that undergo auto-
polymerization and transform into a brown pigment, melanin. Moreover, the oxidation product,
3
,5-DTBQ is highly stable and a characteristic emission band is appeared at 435 nm in pure

20
DMSO solvent system. Before going into the detailed kinetics, we have examined the 3,5-DTBC
to the corresponding 3,5-DTBQ in DMSO solvent. We have observed that by using, the
complexes as catalyst and treated with 100 equivalents of 3,5-DTBC at room temperature under
aerobic conditions. The progress of the reaction is followed by recording fluorescence spectra of
the mixture at 15 minutes time interval. The gradual increase of an emission band corresponds to
quinone.
In each cases, first-order kinetics is observed at a lower concentrations of 3,5-DTBC,
whereas, higher concentrations led to the saturation kinetics (Fig. S15). The observed rate versus
concentration of the substrate data are then analyzed on the basis of Michaelis−Menten approach
of enzymatic kinetics to get Lineweaver−Burk (double reciprocal) plot and the values of kinetic
parameters, Vmax, K
M
, and Kcat. The curves of observed rate vs [substrate] and Lineweaver−Burk
plots for complexes 1-3 are shown in Fig. 7.
1
/V = { KM/ Vmax}{1/[S]} + 1/ Vmax
-4
-1
The various kinetic parameters, such as Michaelis-Menten constant, Kcat (in 10 h ),
maximum velocity Vmax and Michaelis binding constant (K ) for complexes 1-3 are shown in
M
Table 5.These results clearly show the ability of complex 2 to effectively catalyze 3,5-DTBC.
Insert Table 5
3
.8. Phosphate Ester Cleavage Study and Kinetics
There are many reports cited in literature in which, the labile coordination site of
mononuclear transition metal complexes assist the hydrolysis/ transesterification of phosphate
esters. To study Phosphatase activity of complexes 1-3, a disodium salt of (4-nitrophenyl)-
phosphate hexahydrate was chosen as substrate. The catalytic efficiency of these complexes was
determined by the method of pseudo first-order rate constant (kobs) by monitoring the growth of
4
-nitrophenolate emission band (485 nm), as a function of time. Their hydrolytic tendency was
detected spectrophotometrically by monitoring time evolution of p-nitrophenolate anion (4-NP),
through a wavelength scan from 350 to 650 nm in pure DMSO. After the addition of substrate to
solutions of complexes 1-3, the immediate spectral change corresponding to 4-NP anion for all
the complexes showed promising catalytic activity towards 4-NPP. The course of a typical
reaction with complexes 1-3 is shown in Fig. 8.

21
Insert Fig. 8
Initially, a first-order dependence of substrate concentration was observed (Fig. S16). At
higher concentrations, saturation kinetics was found and first-order rate kinetics was gradually
deviated from unity for all the active complexes. The dependence of rate on the substrate
concentration had suggested a catalyst−substrate binding to be an initial step in the catalytic
mechanism. A treatment on the basis of Michaelis−Menten approach, originally developed for
enzyme kinetics was, therefore, applied and linearized by means of Lineweaver−Burk plot (Fig.
8
.) to calculate various kinetic parameters, such as Michaelis−Menten constant (K
M
)and
maximum initial rate (Vmax).
Insert Table 6
Kcat values can be calculated by dividing Vmax values by the concentration of the
corresponding complexes. These calculations are same as catecholase oxidation. The data
obtained from Lineweaver−Burk plot model were used for the comparison of catalytic activity
towards the hydrolysis of 4-NPP, as shown in Table 6. It might be stated that the complex 2
belong to the highly efficient catalyst group, where order of their activity is 2>1>3.
3
3
.9. Cytotoxicity in HeLa cells
.9.1. MTT assay
Cytotoxicity of the complexes 1-3 was tested against human cervical cancer cell line
HeLa) and normal Vero cell line using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
(
bromide (MTT) assay. Cisplatin was used as reference compound to evaluate the cytotoxic
activity.
For the cytotoxic activity, different concentrations of the complexes, 10 -100µM
were tested in triplicate for 48 h in two independent experiments. The results were expressed as
the average ± standard deviation of two independent experiments. From the percentage cell
growth inhibition versus complex concentration plot, a dose-dependent cell death inducing the
ability of complexes has been observed. This indicates that the metal ion does play a role in the
observed cytotoxicity of metal complexes. All the complexes are active against the tested cell
lines, but do not show any damage to normal cells with respect to the standard drug cisplatin.

22
When compared to inhibitory effect with respect to IC50 values, complex 2 showed higher
activity than other complexes (Table 7). Also, the cytotoxicity of complexes 1-3 follows the
order 2>1>3>, which is consistent with the strong DNA binding of the complexes.
Insert Table 7
3
.9.2. Dual-staining Assay
In order to assess the type of cell death induced by complexes in HeLa cells, the
morphological changes after double staining with Acridine Orange–Ethidium Bromide (AO–EB)
were investigated by fluorescence microscopy (Fig. 9). Four types of cytological changes were
observed from fluorescence emission and the morphological features of chromatin condensation
in AO/EB stained nuclei [59, 60]. (i) viable cells having uniform green fluorescing nuclei with
highly organized structure; (ii) early apoptotic cells (which still have intact membranes, but have
started undergoing DNA fragmentation) having green fluorescing nuclei, but peri-nuclear
chromatin condensation is visible as bright green patches or fragments; (iii) late apoptotic cells
having orange to red fluorescing nuclei with condensed or fragmented chromatin; and (iv)
necrotic cells, swollen to large sizes, having uniformly orange to red fluorescing nuclei with no
indication of chromatin fragmentation. These morphological changes are observed for complex
2
, which suggested the fact that the cells are committed to more efficient apoptotic cell death
[
61]. The mechanism of action is still not completely elucidated. Efforts are on to establish the
mechanism of action. Inhibition of cancer cells and induction of apoptosis might be the efficient
ways of treating cancer. Apoptosis eliminates cells, exposing the organism to danger. For
example, virally infected cells or cells with damaged DNA will be removed by apoptosis [62]. In
several proposed mechanisms, the two important types of apoptosis are: death receptor–
dependent pathway, also called as extrinsic pathway and mitochondrial–dependent or intrinsic
pathway. The positively charged metal complexes have the potential to act as effective metal-
based anticancer drugs.
3
.9.3. DAPI staining
To understand nuclear morphology and to determine cell death mechanism, DAPI
staining test was carried out for its complexes 1-3. Chromatin condensation during the process of

23
apoptosis (Type I programmed cell death) is a characterizing marker of nuclear alteration. HeLa
cells were treated with 50 µM concentration of the complexes 1-3. The cells were incubated for
2
4 h before DAPI nuclear staining. Cells were examined under fluorescent microscope, fitted
with a DAPI filter. Control cells (treated with 10% (v/v) DMSO) hardly showed sort of
condensation in comparison with the treated cells, as shown in (Fig. 10).
Insert Fig. 9
After cell fixation, apoptotic nuclei were identified by the reduced nuclear size and
gathering of condensed chromatin at the periphery of nuclear membrane, besides, the fragmented
morphology of nuclear bodies. Furthermore, the normal cells are of uniform morphology without
any nuclear condensation. The result clearly shows that as the concentration of the metal
complex increases, the percentage of cells undergoing apoptosis also increases. Besides showing
nuclear change, the treated cells exhibited a shrinking morphology, which is another important
hallmark of apoptosis.
4
. Conclusion
Three important potentially new anticancerous complexes derived from
S-allyldithiocarbazate derivative had been synthesized and characterized. The characterization of
the complexes was accomplished by elemental analyses and various spectral techniques. The
solid state structure of the ligand and complexes 1-3 were confirmed by single-crystal X-ray
crystallography. The binding behaviour of complexes 1-3 with DNA had revealed the existence
of intercalative mode of interaction. From the results of fluorescence quenching experiments of
BSA, it is concluded that the complexes have strong binding affinity in the static mode.
Furthermore, the complexes had shown effective bio - catalytic activity for the oxidation of 3,5-
DTBC and hydrolysis of 4-NPP. In addition, the complexes had shown widespread anti-tumor
potency with low IC50 values tested by MTT assay against normal Vero cell line and HeLa
cancer cell line. In particular, complex 2 exhibited comparatively higher cytotoxicity than other
complexes. Finally, the characteristics of apoptosis in cell morphology have been observed using
AO/EB and DAPI staining. The results obtained from the present compounds are of importance
for the development of metal-based agents for anti-cancer applications in the future.

24
Acknowledgement
We are sincerely thankful to the SAIF Punjab University, Chandigarh for ESI mass facility and
The Director, CAS in Botany, School of Life Sciences, University of Madras for providing
laboratory facilities to perform the cell line studies. JPJ acknowledges the NSF-MRI program
(
Grant No.CHE-1039027) for funds to purchase the X-ray diffractometer.
Electronic supplementary information (ESI) available
1
UV Spectra of compounds (Fig S1), FTIR Spectra of compounds (Fig S2-S3), H NMR Spectra
1
3
of compounds (Fig S4-S6), C NMR Spectra of compounds (Fig S7-S9), ESI Mass Spectrum of
compounds (Fig S10-S12), Optimized structures (Fig S13). FMO diagrams (Fig S14). Kinetic
Plot (3,5-DTBC) Rate vs Concentration of complexes (Fig S15), Kinetic Plot (4-NPP) Rate vs
Concentration of complexes (Fig S16), DFT and Experimental values of IR spectra (Table S1).
Electronic properties of [H-emk-sadtc)] and its complexes 1–3 in B3LYP/6-311G level (Table
S2). Natural charge and electronic configuration (Table S3). CCDC reference numbers 1484684-
1
484687 for compounds.

25
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29
Fig. 1. Molecular structure of [H-emk-sadtc] with 50% probability level.