Catechol oxidase, phosphatase-like activity, DNA/BSA binding studies of RuII complexes of S-allyldithiocarbazate: synthesis and spectral studies

Article abstract of DOI:10.21577/0103-5053.20160318

New ruthenium(II) complexes with formulae [RuCl(CO)(PPh₃)₂L₁] and [RuCl(CO)(PPh₃)₂L₂] (L₁ = S-allyl-4-methoxy benzylidene hydrazine carbodithioate, L₂ = S-allyl-1-naph

Full text of DOI:10.21577/0103-5053.20160318

http://dx.doi.org/10.21577/0103-5053.20160318
J. Braz. Chem. Soc., Vol. 28, No. 8, 1414-1429, 2017.
Printed in Brazil - ©2017 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00
Article
Catechol Oxidase, Phosphatase-Like Activity, DNA/BSA Binding Studies of RuII
Complexes of S-Allyldithiocarbazate: Synthesis and Spectral Studies
Ponnusamy Selvakumar,a Ramaswamy Narayanasamy,*,b Nanjan Nanjundan,b
Krishnaswamy Velmuruganc and Raju Nandhakumarc
aDepartment of Chemistry, SVS College of Engineering, 642 109 Coimbatore, India
bDepartment of Chemistry, Coimbatore Institute of Technology, 641 014 Coimbatore, India
cDepartment of Chemistry, Karunya University, 641 114 Karunya Nagar Coimbatore, India
New ruthenium(II) complexes with formulae [RuCl(CO)(PPh3)2L1] and [RuCl(CO)(PPh3)2L2]
(L1 = S-allyl-4-methoxy benzylidene hydrazine carbodithioate, L2 = S-allyl-1-naphthylidene
hydrazine carbodithioate) have been synthesized and characterized by elemental analysis, Fourier
transform infrared (FTIR), nuclear magnetic resonance (NMR), UV-Vis, electrospray ionization
(ESI)-mass spectral studies. Both the Schiff base ligands are coordinated to ruthenium through
azomethine nitrogen (C=N) and thiolato sulfur donor atoms in the thiolate form. On the basis of
spectral data obtained, an octahedral structure has been assigned to all the new complexes, satisfied
by coordination of donor atoms N and S. The stability of complexes in solution was determined
by molar conductivity measurements. The interaction of the two complexes with calf thymus (CT)
DNA was investigated by fluorescence spectroscopic and viscometric methods. Bovine serum
albumin (BSA) protein was examined by fluorescence spectroscopic methods. These techniques
indicate that the two metal complexes bind to DNA via intercalation mode and BSA bind with static
interaction. Catecholase and phosphatase like activities promoted by these two complexes under
physiological conditions have been studied. In vitro anticancer activities have been demonstrated by
3,4,5-dimethylthiazolyl-2-2,5-diphenyltetrazolium bromide (MTT) assay, acridine orange/ethidium
bromide (AO/EB) and diamidino-2-phenylindole (DAPI) staining against HeLa cancer cell line.
Keywords: S-allyldithiocarbazate, ruthenium complexes, DNA/BSA binding, kinetics
Introduction
[ImH][trans-RuCl4(Im)2] (KP1019) have progressed
into clinical trials with very promising results.5-7 The
mechanism of above ruthenium complexes has been most
widely studied in order to establish the basis for their
unique properties, whch three relevant main features are:
(i) they have the ability to interact with serum proteins,
such as albumin and transferrin that endows to have
tumor seeking properties;8 (ii) activation of ruthenium(III)
through intracellular reduction to allow generation of toxic
ruthenium(II) species9 and (iii) tumor site reactions prefer
protein binding instead of DNA binding, it gives contrast to
the behavior of platinum(II) complexes, such as cisplatin.10
Recent research on anticancer ruthenium(II) complexes as
drugs bind not only to the primary target DNA, but also with
the serum albumin proteins, such as bovine serum albumin
(BSA) and human serum albumin (has).As serum proteins
perform transport, distribution, accumulation and excretion
properties of drugs in tumor tissue, special consideration
has been given to the study of binding of metallo-drugs
The use of chiral metal complexes to probe the structure
of DNA is an active area of research at the interface of
chemistryandbiology.1 Biologicalimportanceofmetaldrugs
cannot be denied after the approval of platinum anticancer
drugs, such as carboplatin, oxaliplatin, nedaplatin along
with cisplatin.2,3 However, these platinum drugs possess
inherent limitations, such as severe tissue toxicity including
nephrotoxicity, ototoxicity, neurotoxicity, acquisition of
resistance to the treatment, low water solubility and relative
inactivity against gastrointestinal tumors.4 It is well known
that many complexes exert their drug effects through
binding to DNA or proteins. This led us to search for non-
platinum metal complexes, such as ruthenium complexes
as cytotoxic agents. Among ruthenium-based complexes,
[HIm][trans-RuCl4(DMSO)(Im)] (NAMI-A) and
*e-mail: narayanasamycit@gmail.com

Vol. 28, No. 8, 2017
Selvakumar et al.
1415
to the proteins.11 Dithiocarbazates constitute an important
class of mixed hard-soft nitrogen-sulfur donor ligands.
There is continuing interest in the coordination chemistry
of heterocyclic bidentate ligands containing NS donor
set.12-14 Although plenty of studies have been done on
metal complexes with dithiocarbazic acid,15 Schiff bases of
S-methyl,16-18 S-benzyl,19 S-acetyl20 and other N-substituted
derivatives of S-methyldithiocarbazate21 have been reported.
A minor change in ligand alters biological character of
metal chelates.22 Influenced by the above facts, we wish to
synthesize S-allyldithiocarbazate, RCH=NNHC(S)SR’and
allyl group in the molecular structure of new complexes
to bring many interesting chemical behaviors and can be
used as a ligand.23-26 Ligands exhibit thionethiol tautomer
(Scheme 1), N and S donor atoms linked to the metal ion to
form six-membered rings. Hopefully, S-allyldithiocarbazate
to ruthenium gives better biological performance.
methods. Double distilled water was used to prepare
buffers. Calf thymus DNA (CT-DNA), protein markers
and bovine serum albumin (BSA) were obtained from
Genei, Bangalore and Himedia, India, respectively.
S-Allyldithiocarbazate ligand was synthesized according
to previously published method with slight modification.28
The starting complex, RuHClCO(PPh3)3 was prepared
according to the literature reports.29
Stock solution of compounds (1 × 10-3 mol L-1 in DMSO)
was stored at 4 °C and the required concentrations were
prepared for all experiments. All the stock solutions were
used after no more than 4 days. Solution of compounds
was freshly prepared one hour prior to the biochemical
evaluations.
Physical measurements
An attempt is made to synthesize ruthenium complexes
with S-allyldithiocarbazate and these ligands are known
to coordinate via extended π-conjugation through ligand
deprotonation.27 In this paper, we have reported two new
ruthenium complexes comprising p-methoxybenzaldehyde
and 1-naphthaldehyde based S-allyldithiocarbazate,
together with their binding ability towards calf thymus (CT)
DNA/BSA protein. Furthermore, their catechol oxidase
activity and catalytic activities (hydrolysis reaction of the
4-nitrophenyl phosphate (4-NPP)) have been evaluated.
Microanalyses of carbon, hydrogen, nitrogen and sulfur
were carried out using Vario EL III Elemental analyzer
at SAIF-Cochin, India. Conductivity measurements were
carried out at 25 °C on an Elico CM-180 model conductivity
meter with dipping type cell. Infrared (IR) spectra of the
ligand and its complexes were obtained as KBr pellets
on a Bruker model spectrophotometer at the range of
4000-400 cm-1. Electronic spectra of the ligand and its
complexes were recorded using a Shimadzu UV-1650 PC
spectrophotometer at 800-200 nm range. 1H nuclear magnetic
resonance (NMR) spectra were recorded in Jeol GSX-400
instrument at room temperature using tetramethylsilane as
the internal standard in dimethyl sulfoxide (DMSO-d6) as
solvent. Electrospray ionization mass spectra (ESI-MS) were
recorded using LC-MS Q-ToF Micro analyzer (Shimadzu) in
SAIF, Punjab University, Chandigarh. All MS results were
given in the form: m/z, assignment.
Experimental
Reagents and materials
All the reagents purchased were AR grade and used
without further purification. RuCl3.3H2O and ethidium
bromide (EB) were purchased from Sigma-Aldrich and
used as received. Solvents were distilled using standard
Melting points were checked on a Technico micro
heating table and uncorrected. Fluorescence spectral
Scheme 1. Thione-thioltautomerism of the bidentate Schiff base ligands.

1416 Catechol Oxidase, Phosphatase-Like Activity, DNA/BSA Binding Studies of RuII Complexes of S-Allyldithiocarbazate
J. Braz. Chem. Soc.
data were performed on a JASCO FP-8200 fluorescence
spectrophotometer at room temperature. Data were
expressed as the mean standard deviation from three
independent experiments.
Synthesis of [RuCl(CO)(PPh3)2L1 (1)
Yield: (0.105 g) 70%; color: red brown; anal. calcd.
for C49H43ClN2O2P2RuS2 requires: C, 61.66; H, 4.54; N,
2.93; S, 6.72%. Found: C, 61.24; H, 4.31; N, 2.85; S,
6.68%. Selected IR data (KBr) ν / cm-1 1596 (C=N), 1085
(N−N), 1019 (C−S−S), 1246 (C−O−C), 1930 (C≡O), 1430
{Ph(P-Ph)}; 1H NMR (400 MHz, DMSO) d 3.83 (2H, CH2),
7.00-8.20 (4H, Ar-H), 8.59 (1H, CH=N), 3.3 (3H, OCH3);
UV-Vis (DMSO) λ / nm 276, 365, 393, 424. ESI-MS
(DMSO) m/z, observed: 919.03, (M − Cl)+ requires: 919.50.
Synthesis of ruthenium(II) Schiff base complexes
The complexes were prepared according to the
following common procedure in Scheme 2. To a boiling
solution of [RuHCl(CO)(PPh3)3] (0.1 g, 0.1 mmol) in
MeOH/CHCl3 (1:1 v/v) (20 mL), corresponding Schiff
base ligand (0.05 or 0.07 g, 0.1 mmol) in MeOH/CHCl3
(1:1 v/v) (20 mL)was added dropwise. The resulting
mixture was heated under reflux for 6 h on a water bath
that resulted in rapid color change from light orange to
dark brown. After refluxing, the resulting solution was
filtered and the filtrate was left undisturbed for the slow
evaporation of solvent. After few days, brown colored
complexes were obtained.
Synthesis of [RuCl(DMSO)3L2] (2)
Yield: (0.10 g) 67%; color: brown; anal. calcd. for
C52H43ClN2OP2RuS2 requires: C, 64.09; H, 4.45; N, 2.87;
S, 6.58%. Found: C, 63.87; H, 4.33; N, 2.75; S, 6.52%. IR
(KBr) ν / cm-1 1647 (C=N), 1081 (N−N), 991 (C−S−S),
1
1924 (C≡O), 1426 {Ph(P-Ph)}; H NMR (400 MHz,
DMSO) d 3.3 (2H, CH2), 7.13-8.10 (7H, Ar-H), 8.65 (1H,
CH=N); UV-Vis (DMSO) λ / nm 278, 362, 393, 410. ESI-
Scheme 2. The synthetic route of ruthenium(II) complexes 1 and 2.

Vol. 28, No. 8, 2017
Selvakumar et al.
1417
MS (DMSO) m/z, observed: 939.06, (M − Cl)+ requires:
939.51.
the reference solution to eliminate the absorbance of
the CT-DNA itself and TrisHCl buffer was subtracted
through base line correction. Emissive intensity of
CT-DNA was determined by fluorescence spectrometry
and the excitation wavelength was adjusted before taking
measurements. Emissive titration was performed with
a fixed concentration of metal complexes (25 µmol).
While gradually increasing the concentration of DNA
(0-10 µmol),the emission intensities were recorded in
the range of 360-500 nm at room temperature (298 K).
Titrations were manually done by using a micropipette
for the addition of CT-DNA. It is noteworthy that DNA in
double- distilled water does not show any luminescence.
DNA binding studies
Stability study
Stability is the major requirement for the biological
evaluation of DNA/BSA binding interaction study, so
checking the stability of the complexes in solution is very
important.30 DMSO is the most widely preferred solvent
to prepare stock solutions for biophysical and biological
testing. For many transition metal complexes, DMSO acts
as a good ligand, coordinating with the metal center through
either the sulfur or oxygen atom. Therefore, it is imperative
to establish the stability of organometallic compounds
in DMSO before performing biological experiments. To
check the stability of the ruthenium complexes 1 and 2,
electronic spectra have been recorded in DMSO at four
different time points (0 min, 1, 6 and 12 h) through the UV
absorption method. The UV-visible spectral results did not
reveal any appreciable changes in either the intensity or the
position of the absorption bands in DMSO solution. Further
confirmations of the stability of the complexes were given
by conductivity measurements, the molar conductivity
values for the ruthenium(II) complexes (1 mmol) in DMSO
showed low value 0.29-0.36 ohm−1 cm2 mol−1 due to their
non-electrolytic nature. This confirms stability of the
complexes in solution.
Ethidium bromide displacement study
Further support of DNA binding of the complexes was
measured by the fluorescence-based ethidium bromide
(EB) displacement. A typical assay was carried out as
follows. EB displacement experiments were performed
by monitoring changes in the fluorescence intensity on
the emission wavelength (λem) 604 nm and the excitation
wavelength (λex) 550 nm, after aliquot addition of tested
compounds to an aqueous solution of EB-DNA. EB alone
showed that the minimal and maximum fluorescence were
achieved due to the formation of intercalative EB-DNA
adduct. Addition of increasing amounts of ruthenium
complex to the DNA-EB adduct quenched the fluorescence.
For the emission quenching experiments, CT-DNA was
pretreated with EB in the ratio [DNA]/[EB] = 10 for
30 minutes at 37 °C. Then, the titration compounds were
added to this mixture of EB-DNA and the change in
fluorescence intensity was measured.
Fluorescence emission titration study
In the evaluation of antitumor property of
any compound, DNA binding is predominant in
the pharmacology and hence interaction between
DNA and ruthenium(II) complexes has significant
importance.31 The binding mode and tendency of binding
of ruthenium(II) complexes to CT-DNA have been studied
by physicochemical methods at room temperature. DNA
binding experiments includes luminescence measurement
to clarify the binding affinity of ruthenium(II) complexes
by emissive titration and viscosity measurements at room
temperature. The reported compounds were insoluble in
water under titration conditions. The complexes were
soluble in DMSO and, to diminish the negative control
of the solvent, stock solutions were prepared. The stock
solutions of selected test compounds were dissolved in
a mixed solvent of 5% DMSO and 95% TrisHCl buffer
(5 mM Tris-HCl/50 mM NaCl buffer for pH 7.2) for all
the experiments and stored at 4 °C for further use. Now,
the solvent medium has no influence on the compounds.
While measuring the emissive spectra, an equal amount
of DNA was added to the test compound solutions and
Viscosity experiment
Viscosity measurements were carried out using an
Ubbelodhe viscometer maintained at a constant temperature
of 27.0 0.1 °C in a thermostatic bath. DNA samples of
approximately 200 base pairs in length were prepared by
sonication in order to minimize complexities arising from
CT-DNA flexibility. The flow time was measured three
times, after 5 min of incubation, with each addition of
the ligand and their complexes and the average flow time
was taken for calculation of relative viscosity. Relative
viscosities for CT-DNA in the presence and absence of the
compound were calculated from the relation g = (t − to) / to,
where t is the observed flow time of DNA-containing
solution and to is the flow time of Tris-HCl buffer alone.
Data were presented as (η/η0)1/3 versus binding ratio
(R = [Complex] / [DNA] = 0.0-1.2), where η is the viscosity
of CT-DNA in the presence of the compound and η0 is the
viscosity of CT-DNA alone.

1418 Catechol Oxidase, Phosphatase-Like Activity, DNA/BSA Binding Studies of RuII Complexes of S-Allyldithiocarbazate
J. Braz. Chem. Soc.
Protein binding studies
solution of 3,5-DTBC. The reaction with complexes was
monitored after the addition of compounds to DMSO
solution of 3,5-DTBC for 4 h. Initially, complexes showed
bands at λ = 400 nm. Upon addition of 3,5-DTBC, spectra
exhibit the immediate increments. Thus, the experiment
unambiguously proves that oxidation of 3,5-DTBC to
3,5-DTBQ is catalyzed by the ruthenium complexes, as it
is well-established that shows a maximum at λ = 400 nm
in pure DMSO.
Several different transport proteins exist in blood
plasma, but only albumin is able to bind wide diversity
of ligands reversibly with high affinity. The interaction of
metal ions has led to our interest in studying the interaction
of transition metal complexes with proteins. The majority
of intrinsic fluorescence of BSA, when excited at 290 nm
is provided by tryptophan residues alone. The intensity of
emission depends on the exposure of tryptophan residues
to nearby polar environment and to quenching groups of
complexes through molecular interaction. Quenching
of tryptophan residues of BSA was performed using
new ruthenium complexes 1 and 2 as quenchers. The
excitation wavelength of BSA at 290 nm and emission at
339 nm was monitored for the protein binding studies. The
excitation and emission slit width’s rate and scan rate were
maintained constant for all the experiments. Samples were
carefully purged using pure nitrogen gas for 15 minutes.
Quartz cells (4 × 1 × 1 cm) with high vacuum Teflon
stopcocks were used for degassing. Stock solution of BSA
was prepared in 50 mmol Tris-HCl buffer (pH 7.2), stored
in dark at 4 °C for further use. Titrations were manually
done by using a micropipette for the addition of ruthenium
complexes 1 and 2.
Phosphate hydrolysis
Kinetic experiments for the hydrolysis of 4-nitro
phenyl phosphate by ruthenium(II) complexes were
measured by emissive titration at room temperature. To
study the phosphate activity of ruthenium complexes 1
and 2, disodium salt of 4-NPP was a preferred choice of
substrate, represented in Scheme 4. The hydrolytic tendency
of the complexes was measured by monitoring time
evaluation of p-nitrophenolate in DMSO (λem = 469 nm)
through a wavelength scan from 200 to 800 nm over 2 h.
20 equivalents of the substrate were used relative to the
catalyst. The excitation wavelength was fixed and adjusted
before measurements. Emissive titration was performed
with increasing concentrations of ruthenium(II) complexes
(0-25 µmol) that quenched the fluorescence. Titrations were
manually done by using a micropipette for the addition of
complexes 1 and 2.
Catechol oxidase studies
Catechol oxidase, a type-3 copper protein, can bind
oxygen reversibly at room temperature and so it can be
used to oxidize catechols to respective quinones. As a
model of the enzyme here we have taken ruthenium(II)
complexes. To study the catalytic performance of
ruthenium(II) complexes, 3,5-DTBC is employed as a
substrate because its low reduction potential makes it
easy to oxidize and the bulky tert-butyl groups prevent
further over oxidation, such as ring opening and it shows
a low quinine-catechol reduction potential (Scheme 3).
Further, the oxidation product 3,5-DTBQ (3,5-di-
tert-butylquinone) is highly stable and gives emission
maximum at λem 434 nm in DMSO. The catalytic
activity of the complexes was screened by treating a
1 × 10-4 mol L-1 solution of complex with a 1 × 10-2 mol L-1
Scheme 4. Catalytic hydrolysis of 4-NPP to 4-nitrophenolate in DMSO
by RuII complexes.
In vitro cytotoxicity studies
Maintenance of cancer cell lines
HeLa cancer cell line was obtained from National
Centre for Cell Science (NCCS), Pune, India, and cell
viability was assessed by MTT (3,4,5-dimethylthiazolyl-
2-2,5-diphenyltetrazolium bromide) method. HeLa cells
were maintained in a humidified atmosphere containing
5% CO2 at 37 °C in DMEM medium supplemented with
100 units of penicillin, 100 µg mL−1 of streptomycin, and
10% fetal bovine serum. Briefly, HeLa cells with a density
of 1 × 104 cells per well were precultured in 96-well
microtiter plates for 48 h under 5% CO2.
Scheme 3. Catalytic oxidation of 3,5-DTBC to 3,5-DTBQ in DMSO in
the presence of oxygen.

Vol. 28, No. 8, 2017
Selvakumar et al.
1419
Cell viability assay
and 1-naphthaldehyde. Successively, complexes 1 and
2 were prepared via equimolar amounts of ligand with
[RuHCl(CO)(PPh3)3]. In this preparation, the ligand
acted as a monobasic bidentate ligand with NS fashion
replacing a hydride and one triphenylphosphine ligand
from the corresponding starting complex. The prepared
complexes are air stable in solution and in the solid
state at room temperature, soluble in common organic
solvents, such as dimethyl sulfoxide, dimethyl formamide,
dichloromethane. The complexes have been characterized
by elemental analysis, IR, 1H NMR, UV-Vis and ESI-MS.
The elementary analysis gave satisfactory results, which
supports the proposed molecular formulae [RuCl(CO)
(PPh3)2L1] and [RuCl(CO)(PPh3)2L2].
The complexes 1 and 2 were dissolved in 0.1% DMSO
(the concentration of DMSO did not exceed 0.1% v/v) to
obtain a solution of 1 mmol each. The samples were then
diluted to 100 µmol in PBS solution and filter-sterilized
using a 0.22 µm syringe filter. This 100 µmol solution in
PBS was further used in cell cytotoxicity studies. Then,
each well was loaded with 10 µL MTT solution (5 mg mL−1
in PBS pH 7.4) for 4 h at 37 °C. The insoluble formazan
was dissolved in 100 µL of 4% DMSO and the cell viability
was determined by measuring the absorbance of each well
at 570 nm using Bio-Rad 680 microplate reader. All the
experiments were performed in triplicate and the percentage
of cell viability was calculated according to the equation 1.
Inhibition rate (%) = OD (control) − OD (Drug treated
IR spectra
cells) / OD (control) × 100
(1)
The IR spectra of Schiff bases and corresponding
ruthenium complexes were compared in the region
4000-400 cm-1 (Figure S1). The IR spectra do not show any
υ(SH) band at 2700 cm-1, but the presence of broad υ(NH)
band in the region 3107-3120 cm-1 suggests that the free
ligands are in keto form in solid state.33 The strong bands
observed at 1610, 1660 cm-1 and 848, 828 cm-1 confirm
the formation of ligands with υ(C=N), υ(C=S), but IR
spectra of complexes show reduction in the frequency
of azomethine group and the new band appeared at 1596
and 1647 cm-1 indicating the coordination of azomethine
group to ruthenium.34 Absence of band due to υ(C=S) in
the complexes confirms the coordination of Schiff bases
to ruthenium and the disappearance is due to negative
coordination of ligand in the form of υ(C−S) and the
new band appeared in the complexes 1 and 2 at 829,
800 cm-1 concludes tautomerism of (−NH−C=S) group
and coordination of thiolatesulfur after deprotanation.35
The complexes display a medium to strong band in the
region 1930-1924 cm-1 which is attributed to the terminally
coordinated carbonyl group υ(C≡O) and is observed at a
slightly higher frequency than in the precursor complexes.36
After 48 h, the cells were observed with an inverted
phase contrast microscope, photographed with a Nikon
FM 10 camera.
Acridine orange/ethidium bromide (AO/EB) and diamidino-
2-phenylindole (DAPI) fluorescent assay for cell death
AO/EB and DAPI staining analysis was performed as
described earlier.32 HeLa cells were cultured separately
in 6-well plates and treated with IC50 concentration of
the complex for 24 h, when DMSO (0.02%) was used as
solvent control. The cells were harvested and washed with
ice-cold phosphate-buffered saline (PBS) and 40 µL of
AO/EB solution (1 part of 100 µg mL−1 ofAO in PBS; 1 part
of 100 µg mL−1 of EB in PBS) was added and examined in a
fluorescent microscope (Carl Zeiss, Jena, Germany), using
an UV filter (450-490 nm). 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 DAPI
(1 µg mL−1) for 45 min at room temperature in the dark.
Three hundred cells per sample were counted, in triplicate,
for each time point and scored as viable or dead, and if dead,
whether by apoptosis or necrosis as judged from the nuclear
morphology and cytoplasmic organization. Morphological
features of interest were photographed.
1H NMR spectra
1H NMR spectral data are incorporated in Experimental
section because it gives useful supporting information.
NMR spectra of the complexes do not register any signal
corresponding to NH; it supports IR results that the
ligand adopts thione-thioltautomerism and coordination
of enolization by the thiolatesulfur of ligand under the
anionic form upon deprotonation at N2 (Figure S2).37
In addition to this, the complexes showed singlet for
azomethine (−H−C=N) proton at 8.59 and 8.65 ppm for
Results and Discussion
We have relied on the use of stable starting material,
[RuHCl(CO)(PPh3)3], during the synthesis of ruthenium(II)
carbonyl complexes. The S-allyldithiocarbazate ligands
were synthesized via typical condensation route from their
parent S-allyldithiocarbazate with p-methoxybenzaldehyde

1420 Catechol Oxidase, Phosphatase-Like Activity, DNA/BSA Binding Studies of RuII Complexes of S-Allyldithiocarbazate
J. Braz. Chem. Soc.
complexes 1 and 2.38 The aromatic protons in complexes 1
and 2 are observed as multiplets around 7.0-8.1 ppm. The
methylene protons of complexes 1 and 2 appeared at 3.8
and 3.3 ppm, respectively, indicating the non-participation
of S-allylsulfur in coordination.39
From the emissive titration spectra (Figure 1), It is
clearly seen that upon addition of increasing concentration
of CT-DNA (0-10 µmol) to a solution of complexes 1 and
2 resulted in hypochromism 50.09 and 46.10% at 410 nm
along with a small red shift (ca. 2 nm). The resulted
hypochromism is due to an intercalative mode of binding,
involving a strong stacking interaction between extending
aromaticity of ligand and the base pairs of DNA. Usually,
the hypochromism is consistent with the strength of
intercalative interaction. The decrease in intensity of bands
from the original intensities was based on DNA binding
enhancement. To compare the affinity of tested compounds
towards CT-DNA quantitatively, the intrinsic binding
constant (Kb) has been determined using the following
Scatchard equation:44
Electronic spectra
The electronic spectra of complexes were recorded
in DMSO in the range of 200-800 nm, which showed
intense absorption in near ultraviolet region and the
displayed four bands in the region around 262-430 nm
(Figure S3). The complexes 1 and 2 exhibited bands at
276 nm, which can be assigned to intra-ligand transitions
in the complexes. The lowest energy absorption maxima
located in the 340-420 nm range, which probably assigned
to S(pπ) → Ru (dπ) LMCT transitions,40 caused by the
promotion of the electron from the full HOMO of the
ligand, of primarily sulfur pπ character, to the empty
LUMO of ruthenium dπ character, are commonly seen in
other metal-dithiocarbazate complexes.41 Further, bands
at 425-438 nm can be designated as (d→d) transition
bands of a spin paired d6 species with octahedral structure
and the spectra give similarities of other octahedral RuII
complexes.42 The emission spectra of complexes 1 and 2
recorded in DMSO showed resemblance shoulder peak at
450 nm when excited at 427 nm.
CF = CT[(I / Io) − P] / [1 − P]
(2)
where CT is the concentration of the probe (complex)
added; CF is the concentration of the free probe and
Io and I are its emission intensities in the absence and
in the presence of DNA, respectively. P is the ratio of
observed emission quantum yield of the bound probe to
the free probe. The value of P was obtained from a plot of
I / Io versus 1 / [DNA], such that the limiting emission yield
was given by the y-intercept. The amount of bound probe
(Cb) at any concentration was equal to CT − CF. The obtained
Scatchard plots of r / CF versus r for tested compounds
with increasing concentration of CT-DNA were showed
in Figure 1. A plot of r / CF versus r (= CB / [DNA]) gives
intrinsic binding constant (Kb) values. The intrinsic binding
constant (Kb) values for the interaction of complexes 1
and 2 with CT-DNA are listed in Table 1. The results
from emissive titration experiment suggest that all the
compounds (1 and 2) can bind to DNA. The Kb values
suggest that a DNA-binding affinity of complex 1 is
stronger than the complex 2.
ESI mass spectra
ESI mass spectra of the ruthenium(II) complexes 1
and 2 were also recorded to support the information of
other spectral studies. ESI-MS data analysis of ruthenium
complexes 1 and 2 showed major peaks at m/z = 919.50 and
939.51, respectively (Figure S4), which were assigned to
[M − Cl]+ ions, revealing that the identity of the complexes
were retained in solution.
DNA binding studies by fluorescence spectra
Ethidium bromide study
In the development of anticancer drugs, investigation
of binding of metal complexes to DNA has a prime
importance. It is a commonly used technique to study
potential interactions (and their likely nature) between DNA
and metal complexes. The emissive titration studies have
been performed by recording changes in emission intensity
by aliquot addition of DNA.43 The intercalation between
metal complexes and DNA usually results in hypochromism
with or without red/blue shift; alternatively, on the other
hand, non-intercalative/electrostatic interaction causes
hyperchromism.
Further, to support competitive binding of the
ruthenium(II) complexes 1 and 2 to DNA via intercalation,
fluorescence emission quenching experiments were
performed. Fluorescence quenching experiments
were carried out by adding different concentrations of
ruthenium(II) complexes 1 and 2 to EB-bound CT-DNA
solution in 5% DMSO and 95% Tris-HCl/NaCl buffer. EB
is a DNA intercalating agent that fluoresces when bound to
polynucleotide molecule (actually the fluorescence intensity
of EB increases by almost 20-fold after binding to DNA).45
The fluorescence of EB increases after intercalating into

Vol. 28, No. 8, 2017
Selvakumar et al.
1421
Figure 1. Emission spectra (400-430 nm) and Scatchard plots of complexes 1 (left) and 2 (right), (25 × 10-6 mol L-1) in the presence of increasing
concentrations of CT DNA (0-10 × 10-6 mol L-1), obtained in Tris-HCl buffer (pH 7.2) with λex = 404 nm.
Table 1. CT-DNA binding constant (Kb), quenching constant (Ksv) and
apparent binding constant (Kapp) values for complexes 1 and 2
decreases rapidly with increasing concentration of the
compounds. This illustration is given as the concentration of
complexes 1 and 2 increases, the emission band at 604 nm
Compound
Kb / mol-1
5.0 × 103
3.0 × 103
Ksv / mol-1
8.0 × 103
7.0 × 103
Kapp / mol-1
9.35 × 105
1.071 × 104
for EB exhibited hypochromism up to 29.65 and 27.03%
with slight red shift from the initial fluorescence intensity,
respectively. Results in Figure 2 indicate that ruthenium(II)
complexes compete with EB to bind with DNA through
intercalation. Notably, the fluorescence quenching constant
has been evaluated using Stern-Volmer equation.47
1
2
DNA. If the metal complex intercalates into DNA, it leads
to decrease in binding sites of DNA available for EB,
resulting in decrease in the fluorescence intensity of the
EB-DNA system.46 The fluorescence intensity of EB-DNA
Fo / F = 1 + Ksv[Q]
(3)

1422 Catechol Oxidase, Phosphatase-Like Activity, DNA/BSA Binding Studies of RuII Complexes of S-Allyldithiocarbazate
J. Braz. Chem. Soc.
where Fo and F are fluorescence intensities in the absence
and the presence of quencher, respectively. Kq is the
linear Stern-Volmer quenching constant. The value Kq is
obtained with a slope from the plot of Fo / F versus [Q].
Stern-Volmer plots of Fo / F versus [Q] was shown in
Figure 2. The quenching constant (Kq) values are obtained
from the slope, which indicate that the complexes strongly
bind to DNA. The quenching constant (Kq) values are
obtained from the slopes, which are 8.0 × 103, 7.0 × 103
for complexes 1 and 2, respectively. The selected test
compounds can quench the EB emission by either
competing with EB for binding sites or by accepting
the excited state photon. It is unlikely that ruthenium
complexes can compete with EB for intercalative binding
sites. Further, the apparent DNA binding constant (Kapp)
values are also calculated using the equation 4:48
in Table 1. DNA binding ability of complex 1 is stronger
than complex 2, which is consistent with the results
obtained from the above fluorescence spectral studies.49
The quenching and binding constants of ruthenium(II)
complexes indicate that the interaction of tested compounds
with CT-DNA should be of intercalation.
DNA-binding study with viscosity measurements
DNA viscosity is sensitive to DNA length change;
therefore, its measurement upon the addition of a compound
is often concerned as the least ambiguous method to
clarify the interaction mode of a compound with DNA
and provides reliable evidence for the intercalative binding
mode. In general, the viscosity of CT-DNA increases
when a compound binds DNA in an intercalating mode,
but remains unchanged when a compound binds DNA in
an electrostatic mode. If the groove binding mode occurs,
there is a little effect on the viscosity of DNA. The length
of the DNA helix increases upon intercalation as base pairs
are separated to accommodate the binding complex, which
results in the increased viscosity of the DNA. The viscosity
measurements of compounds on the relative viscosities of
CT-DNA are shown in Figure 3.
KEB[EB] = Kapp[M50%
]
(4)
where KEB = 1.0 × 10-7 mol-1 is the DNA-binding constant
of EB; [EB] is the concentration of EB (7.5 µmol) and
[M50%] is the concentration of the compound used to obtain
50% reduction in fluorescence intensity of DNA pretreated
with EB. The Kapp values for complexes 1 and 2 are given
Figure 2. Fluorescence spectra (550-610 nm) and Stern-Volmer plots of complexes 1 (left) and 2 (right) (0-50 µmol) with EB bound CT-DNA. Arrows
indicates decreasing emission intensity with increasing concentration of complexes with λex = 550 nm.

Vol. 28, No. 8, 2017
Selvakumar et al.
1423
is monitored using complexes 1 and 2 as quenchers with
increasing complex concentration. The Io / I versus [complex]
plot are constructed using the corrected fluorescence data
taking into account the effect of dilution. Fluorescence
spectra of BSA with various concentrations of ruthenium(II)
complexes are obtained from 280-590 nm. Subsequently,
the change of fluorescence intensity is related to both the
concentration and nature of the quencher. Therefore, the
quenched fluorophore serves as an indicator to evaluate the
ability of quenching agent.
An examination of the spectra showed a noteworthy
decrease in fluorescence intensity at ca. 335 nm between
the test compounds and BSA protein. The examined red
shift is mainly due to the fact that the active site in protein
is buried in a hydrophobic environment. The data reveal
certain interactions taking place between the complexes
and BSA protein. To understand the quenching progression
deeply, quenching constant (Kq) is evaluated by following
Stern-Volmer and Scatchard equation53
Figure 3. Effect of ruthenium(II) complexes (1 and 2) on the viscosity
of CT-DNA.
When ruthenium(II) complexes 1 and 2 were treated
with CT-DNA (200 µM) and the concentrations of
complexes (0-120 µM) are increased from a ratio of
R = 0-1.2 (1 / R = [Complex] / [DNA]), the relative viscosity
of DNA increases steadily. This increase suggested that
selected compounds can deeply intercalate into DNA base
pairs and increase DNA viscosity similar to some known
intercalation.50 This result is consistent with our above
mentioned hypothesis.
log [(Io − I) / I] = log Kbin + nlog [Q]
(5)
where Kbin is the binding constant of the compounds with
BSA and n is the number of binding sites. The number of
n and Kbin have been found from the plot of log (Io − I) / I
versus log [Q] (Figure 4).
BSA interaction studies
The calculated Kq, Kbin and n values are given in Table 2.
The value of n indicates the existence of two binding sites
in BSA for the complexes.13 The larger values of Kq and
Kbin indicate strong interaction between BSA protein and
the complexes used in this study.
The protein binding study was performed in most
abundant blood protein serum albumin (BSA). The
interactions between BSA and metal complexes have
attracted immense current interest because of their structural
homology with human serum albumin. It constitutes about
55% of total plasma protein and plays a pivotal role in
transport of drug and its metabolism.51 BSA exhibits intrinsic
fluorescence because of the presence of aromatic amino acids
phenylalanine, tyrosine, and tryptophan. The fluorescence of
tyrosine is completely quenched when it is ionized, whereas,
phenylalanine has very low quantum yield. This reveals that
the intrinsic fluorescence of BSA is due to tryptophan alone.
The relative ratio of fluorescence intensity for three amino
acids (tryptophan, tyrosine and phenylalanine) residues is
(100:9:0.5) and thus, the intrinsic fluorescence intensity of
BSA when excited at 295 nm mainly comes from tryptophan
residues, such as, Trp-134 and Trp-212 more exposed to the
environment.52 Understandably, fluorescence behavior of
BSA can provide significant information about the structure,
dynamics and protein folding. Fluorescence quenching
experiments have been done using a solution of BSA
(5 µmol) as the substrate in TrisHCl/50 mmol NaCl buffer
at pH 7.2. Quenching of the emission intensity of tryptophan
residues of BSA at 335 nm (excitation wavelength at 295nm)
Catechol activity
To study the catalytic performance of ruthenium(II)
complexes, 3,5-DTBC is employed as a substrate because
its low reduction potential makes it easy to oxidize and
the bulky tert-butyl groups prevent further over oxidation,
such as ring opening and it shows a low quinine-catechol
reduction potential (Scheme 3).54 The reactions were
performed at room temperature and monitored by
fluorescence spectroscopic technique. 3,5-DTBC forms
oxidation product 3,5-DTBQ, which is highly stable and
exhibits a characteristic emission intensity at λemis = 437 nm
(λex = 401 nm) in pure DMSO. The reaction is monitored
by treating 1 × 10-4 mol L-1 solution of selected compounds
with 100 equivalents of 3,5-DTBC in which upon oxidation
of the catecholic substrate, new band started (Figure 5)
to gradually appear at about 437 nm in time due to the
formation of the oxidized product 3,5-DTBQ. Further, the
experiment clearly shows that the oxidation of 3,5-DTBC

1424 Catechol Oxidase, Phosphatase-Like Activity, DNA/BSA Binding Studies of RuII Complexes of S-Allyldithiocarbazate
J. Braz. Chem. Soc.
Figure 4. Fluorescence titrations of BSA with the complexes 1 (left) and 2 (right). Arrow shows the change upon increasing complex concentration. Stern-
Volmer plots and Scatchard plots of the fluorescence titration of complexes 1 and 2.
Table 2. Quenching constant (Kq), binding constant (Kbin), and number of binding sites (n) for the interactions of complexes with BSA
Compound
Kb / mol-1
8.3 × 105
6.16 × 105
Ksv / mol-1
1.156 × 106
1.060 × 106
Kq / mol-1
1.156 × 1014
1.060 × 1014
n value
2.306
2.264
1
2
to 3,5-DTBQ is catalyzed by the selected compounds, as
it is well established that 3,5-DTBQ shows maximum at
provided in Experimental section). The catalytic behavior
showed saturation kinetics and a treatment based on
Michaelis-Menten model seemed to be appropriate under
excess substrate conditions.55 The observed rate constants
(Ki) were obtained by the initial rate method. Plots of Ki
vs. [DTBC] gave a non-linear curve of decreasing slope
(Figure 5) which was well explained by the following
equations 6-8.
λemis = 437 nm in pure DMSO.
Kinetic study of the catacholase activity
To understand kinetic aspects of catalysis for selected
compounds, the rate constant for compounds as catalyst
was determined by initial rate methods (detailed description

Vol. 28, No. 8, 2017
Selvakumar et al.
1425
dissociation of substrates S (i.e. turnover number, Kcat) were
calculated from the graphs of 1 / V vs. 1 / [S] (Figure 5)
known as Lineweaver-Burk graph by using above equations
and all these parameters are listed in Table 3. These results
state that complex 1 is a highly efficient catalyst group than
the complex 2.
(6)
(7)
(8)
Table 3. Kinetic parameters for complexes 1 and 2 in DMSO solution
Catalyst
Km / (mol L-1) Vmax / (mol S-1)
Kcat / h-1
105
The Michaelis-Menten constant (Km) and maximum
initial rate (Vmax) were determined by linearization using
Lineweaver-Burk plots (Figure 5). The rate constants for
1
2
105.08 × 10-4
104.64 × 10-4
5.58 × 10-4
2.46 × 10-4
104
Figure 5. Time resolved emission spectra of the reaction and Lineweaver-Burk plot for complexes 1 (left) and 2 (right) with 3,5-DTBC after the addition
of complexes in 15 minutes time interval.

1426 Catechol Oxidase, Phosphatase-Like Activity, DNA/BSA Binding Studies of RuII Complexes of S-Allyldithiocarbazate
J. Braz. Chem. Soc.
Phosphate hydrolysis
inhibition expressed as IC50 values, which were listed in
Table 5. It is notable that complex 1possesses the most potent
inhibitory effect against HeLa cell line, and its IC50 value is
well known for the anticancer drug (cisplatin). The results
of MTT assay in Table 5 showed that the complexes 1 and
2 inhibit the growth of cells in a concentration-dependent
manner. It is due to the fact that ruthenium complexes have
a capacity to reduce the energy status in tumors as well
as to enhance tumor hypoxia, which also influences their
antitumor activities. The high cytotoxic behavior of the
complex 1 is similar to that of DNA/BSA binding affinities
as discussed previously. Upon the confirmation of excellent
cytotoxicity properties of the complexes, further staining
assay were encouraged against HeLa liver cancer cell line
to understand their mechanism of action of drugs.
Many hydrolytic processes in enzyme-catalysis involve
metal ions in close proximity to the active site. The selected
compounds meet the requisites for use as a functional model
system for hydrolytic enzymes. To study the phosphatase
activity of complexes, disodium salt of 4-NPP (Scheme 4)
is the preferred choice of substrate.56 The hydrolysis of
4-NPP was detected emissive intensity by monitoring the
time evolution of p-nitrophenolate ion (4-NP) in DMSO at
λ
emis = 469 nm (λex = 423 nm) through a wavelength scan of
400-800 nm over 2 h and 40 equivalent of substrate were
used relatively to the catalyst.
Kinetic study of the phosphate hydrolysis
The kinetic studies of compounds were performed by
the initial slope method by monitoring the increase rate in
emissive intensity band at 469 nm, which corresponds to the
increase in 4-nitrophenolate concentration.57 The dependence
of rate constants was determined by using five different
substrate concentrations. The catalytic behavior showed
saturation kinetics and a treatment based on Michaelis-
Menten model seemed to be appropriate under excess
substrate conditions.58 The observed rate constants (Ki)
were extracted by initial rate method. Plots of the Michaelis-
Menten constant (Km) and maximum initial rate (Vmax) were
determined by linearization using Lineweaver-Burk plots
(Figure 6). The rate constants for dissociation of substrates S
(i.e. turnover number, Kcat) were calculated from the graphs
of 1 / V vs. 1 / [S] (Figure 6) known as Lineweaver-Burk
graph by using above equation 8 and all these parameters
are listed in Table 4. These results state that complex 1 is a
efficiently better catalyst group than the complex 2.
On the basis of kinetic data, Lewis acid activation
mechanism is proposed for the metal catalyzed 4-NPP
cleavage. The high P−O bond cleavage rate of compounds
can be attributed to the presence of H2O groups in the
framework, which could act as a nucleophile in hydrolytic
reaction.
Staining assay
The apoptotic morphologies induced by 1 and 2 have
been investigated by using AO/EB staining and adopting
fluorescence microscopy.60 Apoptotic pathways are
important targets that should be considered in the design
of potential anticancer agents. The cytological changes
observed were classified into four types: (i) viable cells;
(ii) early apoptotic cells; (iii) late apoptotic cells and
(iv) necrotic cells. In Figure 7 (1A and 2A), blue arrows
exhibited late apoptotic cells with chromatin aggregation,
that is, highly condensed chromatin which is seen at a
fixed concentration of complexes 1 and 2 and necrotic
cells having uniform orange to red nuclei with condensed
structure were also observed. These morphological changes
were observed for complex 1, which suggested that the
cells are devoted to induced cell death via necrotic and the
resulting images of the control and treated HeLa cells are
depicted in Figure 7.
Conclusion
Two new ruthenium(II) complexes, containing
p-anisaldehyde-S-allylditiocarbazate (L1), 1-napthaldehyde-
S-allylditiocarbazate (L2) with general formulae [RuCl(CO)
(PPh3)2L1] and [RuCl(CO)(PPh3)2L2] have been synthesized
and characterized by elemental analysis, IR, NMR,
electronic and ESI-MS analyses. Elemental analysis
supports well with the proposed molecular formula. On the
basis of spectral studies, an octahedral geometry is proposed
for all the ruthenium complexes. The binding strength of
the complexes with CT-DNA is calculated by fluorescence
spectroscopic titrations and it showed that the complex 1
exhibited higher Kb value than the complex 2. However,
competitive DNA binding studies suggested the potential
of the compounds to displace EB. From the binding
In vitro anticancer studies
MTT assay
The positive results obtained from the biomolecules
interaction studies encouraged us to further determine the
cytotoxicity of the complexes against a HeLa liver cancer
cell line. The cell viability assay for the newly synthesized
ruthenium(II) complexes (1 and 2) was evaluated using
the method of MTT assay. Cisplatin was used as a positive
control.59 The results were analyzed by means of cell

Vol. 28, No. 8, 2017
Selvakumar et al.
1427
Figure 6. Phosphate hydrolysis emission spectrum and Lineweaver-Burk plot for complexes 1 (left) and 2 (right).
Table 4. Phosphate hydrolysis kinetic parameters for complexes 1 and 2
Table 5. In vitro cytotoxicity of the complexes in normal and HeLa
cancer cell line
Catalyst
Km / (mol L-1) Vmax / (mol S-1)
Kcat / h-1
3117
IC50 / µMa
1
2
155.89 × 10-3
52.07 × 10-3
1.605 × 10-3
1.919 × 10-3
Complex
Vero
52.31 2.41
54.24 3.65
−
HeLa
1041
1
18.74 2.14
22.34 2.42
13.00 2.01
2
Cisplatinb
experiments, all the complexes exhibited intercalation
mode of the interaction with DNA. Furthermore, protein
binding properties of complexes examined by fluorescence
spectra suggested that the binding affinity of complex 1 was
stronger than that of complex 2. In addition, the complexes
exhibited promising catalytic activity in the oxidation of
aFifty percent inhibitory concentration after exposure for 24 h in the MTT
assay; bdata from reference 58.
3,5-DTBC and the hydrolysis of 4-NPP with high TON
values. In addition, the complexes had shown widespread

1428 Catechol Oxidase, Phosphatase-Like Activity, DNA/BSA Binding Studies of RuII Complexes of S-Allyldithiocarbazate
J. Braz. Chem. Soc.
Figure 7. Images of HeLa cancer cells with complexes after AO/EB (1A, 2A) and DAPI (1B, 2B) staining at 24 h. Blue arrows show late apoptosis and
red arrows show necrotic cells.
anti-tumor potency with low IC50 values tested by MTT
assay against normal Vero cell line and HeLa cancer
cell line. Finally, the characteristics of apoptosis in cell
morphology have been observed using AO/EB and DAPI
staining. The results obtained from the present complexes
are of importance for the development of metal-based
chemotherapeutic agents in the future.
3. Wheaten, N. J.; Walker, S.; Craig, G. E.; Oun, R.; Dalton Trans.
2010, 39, 8113.
4. Barry, N. P. E.; Sadler, P. J.; Chem. Commun. 2013, 49, 99.
5. Bruijnincx, P. C.A.; Sadler, P. J.; Curr. Opin. Chem. Biol. 2008,
12, 197.
6. Suess-Fink, G.; Dalton Trans. 2010, 39, 1673.
7. Hotze, A. C. G.; Kariuki, B. M.; Hannon, M. J.; Angew. Chem.
2006, 118, 4957.
Supplementary Information
8. Ang, W. H.; Dyson, P. J.; Eur. J. Inorg. Chem. Soc. 2006, 20,
4003.
Supplementary information (IR, NMR, UV-Vis, ESI-
MS) is available free of charge at http://jbcs.sbq.org.br as
PDF file.
9. Schluga, P.; Hartinger, C. G.; Egger, A.; Reisner, E.; Galanski,
M.; Jakupec, M. A.; Keppler, B. K.; Dalton Trans. 2006, 1796.
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Acknowledgments
11. Timerbaev,A. R.; Hartinger, G.;Alekseenko, S. S.; Keppler, B.
K.; Chem. Rev. 2006, 106, 2224.
We are sincerely thankful to the SAIF Punjab University,
Chandigarh for ESI Mass and NMR facility and the director,
CAS in botany, School of Life Sciences, University of
Madras, for providing laboratory facilities to perform the
cell lines studies. This work is supported by the Principal
Dr T. Kannan, SVS College of Engineering, Coimbatore.
12. AkbarAli, M.; Mirza, A. H.; Fereday, R. J.; Fuller, R. J.; Drew,
S. C.; Gahan, L.; Hanson, J. R.; Moubaraki, B.; Murray, K. S.;
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