Evaluation of DNA-binding, DNA cleavage, antioxidant and cytotoxic activity of mononuclear ruthenium(ii) carbonyl complexes of benzaldehyde 4-phenyl-3-thiosemicarbazones

Article abstract of DOI:10.1016/j.saa.2013.06.030

Two 4-phenyl-3-thiosemicarbazone ligands, (E)-2-(2-chlorobenzylidene)-N- phenylhydrazinecarbothioa-mide (HL¹) and (E)-2-(2-nitrobenzylidene)- N-phenylhydrazinecarbothioamide (HL²), and its ruthe-nium(ii) complexes were synthesized an

Full text of DOI:10.1016/j.saa.2013.06.030

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 115 (2013) 287–296
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Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Evaluation of DNA-binding, DNA cleavage, antioxidant and cytotoxic
activity of mononuclear ruthenium(II) carbonyl complexes
of benzaldehyde 4-phenyl-3-thiosemicarbazones
⇑
Krishnan Sampath, Subbaiyan Sathiyaraj, Chinnasamy Jayabalakrishnan
Post-Graduate and Research Department of Chemistry, Sri Ramakrishna Mission Vidyalaya College of Arts and Science, Coimbatore 641 020, Tamil Nadu, India
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ꢀ
ꢀ
ꢀ
ꢀ
Molecular structure of ligands was
elucidated by X-ray diffraction study.
The ligands and complexes interact
with CT-DNA, intercalatively.
The complexes can efficiently cleave
CT-DNA via oxidative cleavage.
The complexes possess significant
antioxidative property against DPPH
radical.
ꢀ
The complexes showed higher
cytotoxicity than the ligands against
tumor cells.
a r t i c l e i n f o
a b s t r a c t
Article history:
Two 4-phenyl-3-thiosemicarbazone ligands, (E)-2-(2-chlorobenzylidene)-N-phenylhydrazinecarbothioa-
mide (HL1) and (E)-2-(2-nitrobenzylidene)-N-phenylhydrazinecarbothioamide (HL2), and its ruthe-
nium(II) complexes were synthesized and characterized by physico-chemical and spectroscopic
methods. The Schiff bases act as bidentate, monobasic chelating ligands with S and N as the donor sites
and are preferably found in the thiol form in all the complexes studied. The molecular structure of HL1
and HL2 were determined by single crystal X-ray diffraction method. DNA binding of the compounds
was investigated by absorption spectroscopy which indicated that the compounds bind to DNA via inter-
calation. The oxidative cleavage of the complexes with CT-DNA inferred that the effects of cleavage are
dose dependent. Antioxidant study of the ligands and complexes showed significant antioxidant activity
against DPPH radical. In addition, the in vitro cytotoxicity of the ligands and complexes assayed against
HeLa and MCF-7 cell lines showed higher cytotoxic activity with the lower IC50 values indicating their
efficiency in killing the cancer cells even at low concentrations.
Received 27 April 2013
Accepted 5 June 2013
Available online 24 June 2013
Keywords:
Ruthenium(II) complexes
Thiosemicarbazones
DNA interaction
Antioxidant
Cytotoxicity
Ó 2013 Elsevier B.V. All rights reserved.
Introduction
therapeutic methods [1,2]. Despite many efforts in trying to unra-
vel the role of metal ions in biology, much remains to be learned
Biological inorganic chemistry is a discipline of increasing
importance in therapeutic as well as diagnostic medicine. It offers
the potential for design and preparation of novel complexes capa-
ble of treating diseases that are resistant to conventional
about how these metal cations are trafficked and stored prior to
their incorporation into different metalloproteins [3]. Metal chela-
tion is an excellent way to increase the lipophilic character of the
organic moiety. On coordination, ligands might improve their bio-
activity profiles, while some inactive ligands may acquire pharma-
cological properties and consequently they have become an
important class of structure – selective binding agents for nucleic
⇑
Corresponding author. Tel.: +91 9442001793.
E-mail address: drcjbstar@gmail.com (C. Jayabalakrishnan).
1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2013.06.030

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K. Sampath et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 115 (2013) 287–296
acids. Compounds that can selectively recognize a non-duplex
structure are involved in the control of gene expression. They have
considerable potential as chemotherapeutic agents for a variety of
diseases. Mechanism by which metal complexes interact with non-
duplex structure is similar to those seen in duplex DNA and RNA,
i.e. covalent binding, intercalation and groove binding [4,5]. Among
these DNA – targeted guest molecules, ruthenium based com-
pounds have attracted much attention because of low toxicity
and their promising anticancer activity [6,7]. Some chemical prop-
erties, such as rate of ligand exchange, range of accessible oxida-
tion states and ability to mimic iron in binding to certain
biological molecules make them fit for medicinal applications [8].
In particular, ruthenium(II) organometallics represents one of the
latest trends in metallodrug research [9].
Preparation of thiosemicarbazone ligands
(E)-2-(2-chlorobenzylidene)-N-phenylhydrazinecarbothioamide (HL1)
(1)
A methanol solution (50 mL) of 4-phenyl-3-thiosemicarbazide
(1.672 g, 0.01 mol) was added to a methanol solution (20 mL) con-
taining 2-chlorobenzaldehyde (1.12 mL, 0.01 mol). The mixture
was refluxed for an hour during which period a white color precip-
itate was formed. The reaction was cooled to room temperature
and the solid compound was filtered, washed and recrystallized
from methanol. White colored crystals, suitable for single crystal
X-ray diffraction analysis, were obtained by slow evaporation of
its methanolic solution. Yield: 86%; M.P: 159–160 °C. Anal. calcd.
for C14H12ClN3S (%): C, 58.03; H, 4.17; N, 14.50; S, 11.07. Found
(%): C, 58.09; H, 4.10; N, 14.55; S, 11.11. IR (KBr, cmꢁ1): 1597
Thiosemicarbazones form an important class of compounds
because of their promising pharmacological properties, such as
trypanocidal activity, antitubercular activity, antioxidative activity
and antitumor activities [10–12]. Moreover, some thiosemicarba-
zones have shown to increase their biological activity by their abil-
ity to form chelates with specific metal ions [13]. On the other
hand, over production of activated oxygen species in the forms of
superoxide anion (HOꢀ) and hydroxyl radical (HOꢀ), generated by
normal metabolic process, is considered to be the main contributor
to oxidative damages to biomolecules such as DNA, lipids and pro-
teins, thus accelerating cancer, aging, inflammation, cardiovascular
and neurodegenerative diseases [14]. The potential value of antiox-
idants has prompted scientists to search for compounds for
improving antioxidant activity and cytotoxicity [15]. With this
background in mind, we herein report on the synthesis and charac-
terization of ruthenium(II) complexes of 2-chloro/nitro benzalde-
hyde 4-phenyl-3-thiosemicarbazones. The crystal structure of the
thiosemicarbazone ligands have been determined by X-ray crystal-
lography. The investigation of the biological properties of the li-
gands and ruthenium(II) complexes focused on the binding
properties with CT-DNA was performed by UV spectroscopy and
the cleavage properties of the complexes was performed by gel
electrophoresis with CT-DNA. Finally, we have studied their anti-
oxidative property against DPPH radical and in vitro antitumor
activity against HeLa and MCF-7 cancer cell lines.
m
(p ?
(C@N); 860
m(C@S). UV–vis (DMSO), kmax (nm): 315, 368
pꢂ, n ? pꢂ). 1H NMR (DMSO-d6): d 10.20 (s, 1H, hydrazine
NH); d 8.60 (s, 1H, phenyl NH); d 12.01 (s, 1H, HAC@N); d 7.21–
8.47 (m, 8H, aromatic). 13C NMR (DMSO-d6): d 139 (C@S); d 176
(C@N); d 125–139 (aromatic).
(E)-2-(2-nitrobenzylidene)-N-phenylhydrazinecarbothioamide (HL2)
(2)
It was prepared using the same procedure as described for HL1
with 4-phenyl-3-thiosemicarbazide (1.672 g, 0.01 mol) and 2-
nitrobenzaldehyde (1.511 g, 0.01 mol). A yellow product was
formed. The solid compound was filtered, washed and recrystal-
lized from methanol. Yellow colored crystals, suitable for single
crystal X-ray diffraction analysis, were obtained by slow evapora-
tion of its methanolic solution. Yield: 82%; M.P: 190–191 °C. Anal.
calcd. for C14H12N4O2S (%): C, 55.99; H, 4.03; N, 18.65; S, 10.68.
Found (%): C, 55.91; H, 4.08; N, 18.66; S, 10.63. IR (KBr, cmꢁ1):
1599
408 (
m
(C@N); 846
m(C@S). UV–vis (DMSO), kmax (nm): 312, 368,
p
?
pꢂ, n ? pꢂ). 1H NMR (DMSO-d6): d 10.20 (s, 1H, hydrazine
NH); d 8.59 (s, 1H, phenyl NH); d 12.12 (s, 1H, HAC@N); d 7.21–
8.06 (m, 8H, aromatic). 13C NMR (DMSO-d6): d 148 (C@S); d 176
(C@N); d 124–139 (aromatic).
Single crystal X-ray diffraction studies
Single crystal X-ray diffraction data of 1 and 2 were collected at
room temperature on a Bruker AXS KAPPA APEX2 CCD diffractom-
eter equipped with a fine focused sealed tube. The unit cell param-
eters were determined and the data collections of ligands 1 and 2
Experimental
Materials and methods
were performed using
a
graphite-mono chromate Mo
Ka
All the chemicals used were chemically pure and of AR grade.
Calf-thymus DNA (CT-DNA) was purchased from Bangalore Genei,
Bangalore, India. The Human Cervical (HeLa) and human breast
(MCF-7) cancer cell lines were obtained from National centre for
cell science (NCCS), Pune, India. Solvents were purified according
to the standard procedure [16]. The metal precursors
[RuHCl(CO)(PPh3)3] and [RuHCl(CO)(AsPh3)3] were prepared by lit-
erature methods [17,18]. Microanalyses (C, H, N and S) were per-
formed on a Vario EL III CHNS analyser at STIC, Cochin University
of Science and Technology, Kerala, India. IR spectra were recorded
as KBr pellets in the 400–4000 cmꢁ1 region using a Perkin Elmer
FT-IR 8000 spectrophotometer. Electronic spectra were recorded
in DMSO solution with a Systronics double beam UV–vis spectro-
photometer 2202 in the range 200–800 nm. 1H, 13C and 13P NMR
spectra were recorded on a Bruker AV III 500 MHZ instrument
using TMS and ortho phosphoric acid as an internal standard at
SAIF, Indian Institute of Technology Madras, Chennai, Tamil Nadu.
DNA cleavage studies were carried out using Gelstan-Gel docu-
mentation system. Antioxidant and anti cancer studies were car-
ried out at the Kovai Medical Centre and Hospital Pharmacy
College, Coimbatore, Tamil Nadu. Melting points were recorded
with Veego VMP-DS heating table.
(k = 0.71073 Å) radiation by
u
and scans. The data collected
x
were reduced SAINT program [19] and the empirical absorption
corrections were carried out using the SADABS program [20]. The
structure of the compounds 1 and 2 was solved by direct methods
[21] using SHELXS-97, which revealed the position of all non-
hydrogen atoms, and was refined by full-matrix least squares on
F2 (SHELXL-97) [22]. All non-hydrogen atoms were refined aniso-
tropically, while the hydrogen atoms were placed in calculated
positions and refined as riding atoms.
Preparation of ruthenium(II) complexes
Synthesis of [RuCl(CO)(PPh3)2L1] (3)
A methanolic solution (20 mL) containing HL1 (1) (0.145 g,
0.5 mmol) and triethylamine (0.07 mL, 0.5 mmol) were added to
[RuHCl(CO)(PPh3)3] (0.475 g, 0.5 mmol) in benzene (20 mL). The
resulting red color solution was refluxed for 8 h. The reaction mix-
ture was then cooled to room temperature, which results in the
formation of red color precipitate. It was filtered off and then sub-
jected to purification by TLC. This solid was recrystallized from
CH2Cl2/Hexane mixture. Our efforts to obtain single crystal of the
complexes were unsuccessful. Yield: 56%. M.P: 240–242 °C. Anal.

K. Sampath et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 115 (2013) 287–296
289
calcd. for C51H41Cl2N3OP2RuS (%): C, 62.64; H, 4.23; N, 4.30; S, 3.28.
Found (%): C, 62.67; H, 4.28; N, 4.39; S, 3.23. IR (KBr, cmꢁ1): 1482
m
(C@N); 742 m(CAS); 1911 m(C„O). UV–vis (DMSO), kmax (nm):
317, 368 (ILCT), 411 (MLCT). 1H NMR (DMSO-d6): d 8.66 (s, 1H,
phenyl NH); d 12.11 (s, 1H, HAC@N); d 6.89–7.79 (m, 39H, aro-
matic). 13C NMR (DMSO-d6): d 136 (CAS); d 182 (C@N); d 193
(C„O); d 123–134 (aromatic). 31P NMR (DMSO-d6): d 36 (PPh3).
Synthesis of [RuCl(CO)(AsPh3)2L1] (4)
It was prepared by using the same procedure as described for 3
with HL1 (1) (0.145 g, 0.5 mmol) and [RuHCl(CO)(AsPh3)3] (0.54 g,
0.5 mmol). Yellow colored powder obtained. Yield: 55%. M.P:
220–221 °C. Anal. calcd. for C51H41Cl2N3OAs2RuS (%): C, 57.47; H,
3.88; N, 3.94; S, 3.01. Found (%): C, 57.45; H, 3.82; N, 3.92; S,
Fig. 1. ORTEP diagram of 1 with thermal ellipsoid at 50% probability.
3.03. IR (KBr, cmꢁ1): 1474
m(C@N); 737 m(CAS); 1938 m(C„O).
UV–vis (DMSO), kmax (nm): 320, 370 (ILCT), 401, 480 (MLCT). 1H
NMR (DMSO-d6): d 8.63 (s, 1H, phenyl NH); d 12.08 (s, 1H,
HAC@N); d 6.80–7.91 (m, 39H, aromatic). 13C NMR (DMSO-d6): d
136 (CAS); d 183 (C@N); d 191 (C„O); d 123–135 (aromatic).
Synthesis of [RuCl(CO)(PPh3)2L2] (5)
It was prepared by using the same procedure as described for 3
with HL2 (2) (0.150 g, 0.5 mmol) and [RuHCl(CO)(PPh3)3] (0.475 g,
0.5 mmol). Brown colored powder obtained. Yield: 59%. M.P:
222–224 °C. Anal. calcd. for C51H41ClN4O3P2RuS (%): C, 61.97; H,
4.18; N, 5.67; S, 3.24. Found (%): C, 61.92; H, 4.20; N, 5.71; S,
3.22. IR (KBr, cmꢁ1): 1518
m(C@N); 743 m(CAS); 1934 m(C„O).
Fig. 2. ORTEP diagram of 2 with thermal ellipsoid at 50% probability.
UV–vis (DMSO), kmax (nm): 312, 368, 408 (ILCT), 428 (MLCT). 1H
NMR (DMSO-d6): d 8.59 (s, 1H, phenyl NH); d 12.25 (s, 1H, H–
C@N); d 6.85–7.87 (m, 39H, aromatic). 13C NMR (DMSO-d6): d
138 (C–S); d 181 (C@N); d 194 (C„O); d 123–134 (aromatic). 31P
NMR (DMSO-d6): d 36 (PPh3).
A solution of CT-DNA in the buffer, gave a ratio of UV absorbance
of about 1.8–1.9 at 260 and 280 nm, indicating that the DNA was
sufficiently free of protein. The CT-DNA concentration per nucleo-
tide was determined spectrophotometrically by employing an
extinction coefficient of 6600 Mꢁ1 cmꢁ1 at 260 nm. The compounds
were dissolved in a mixed solvent of 5% DMSO and 95% Tris–HCl
buffer. Stock solutions were stored at 4 °C and used within 4 days.
Absorption titration experiments were performed with fixed con-
Synthesis of [RuCl(CO)(AsPh3)2L2] (6)
It was prepared by using the same procedure as described for 3
with HL2 (2) (0.150 g, 0.5 mmol) and [RuHCl(CO)(AsPh3)3] (0.54 g,
0.5 mmol). Orange colored powder obtained. Yield: 56%. M.P:
263–265 °C. Anal. calcd. for C51H41ClN4O3As2RuS (%): C, 56.91; H,
3.84; N, 5.21; S, 2.98. Found (%): C, 56.98; H, 3.86; N, 5.25; S,
centrations of the complexes (25
lM) with varying concentration
of DNA (0–50 M). While measuring the absorption spectra, an
l
equal amount of DNA was added to both the test solution and the
reference solution to eliminate the absorbance of DNA itself.
2.93. IR (KBr, cmꢁ1): 1517
m(C@N); 739 m(CAS); 1933 m(C„O).
UV–vis (DMSO), kmax (nm): 317, 368, 408 (ILCT), 437, 473 (MLCT).
1H NMR (DMSO-d6): d 8.57 (s, 1H, phenyl NH); d 12.13 (s, 1H,
HAC@N); d 6.78–8.21 (m, 39H, aromatic). 13C NMR (DMSO-d6): d
139 (CAS); d 182 (C@N); d 192 (C„O); d 122–134 (aromatic).
Nuclease activity using gel electrophoresis
The DNA cleavage activity of the ruthenium(II) complexes was
monitored by agarose gel electrophoresis on CT-DNA. The tests
were performed in the absence and presence of activating agent,
H2O2 under aerobic condition. Hydrolytic cleavage was monitored
DNA binding – titration experiments
All the experiments involving the binding of compounds with
CT-DNA were carried out in a double distilled water buffer with
tris(hydroxymethyl)-aminomethane (Tris, 5 mM) and sodium
chloride (50 mM) and adjusted to pH 7.2 with hydrochloric acid.
by 30 lM of CT DNA and 40 lM of each complex (3–6) in 5% DMSO
and 95% Tris–HCl buffer (5 mM, pH 7.2) with 50 mM NaCl.
For oxidative cleavage, each reaction mixture contained 30
lM
of CT DNA, 30; 60; 90 lM of each complex (3–6) in 5% DMSO and
Scheme 1. Synthetic route of the ruthenium(II) thiosemicarbazone complexes, where E = P/As.

290
K. Sampath et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 115 (2013) 287–296
Table 1
2.7. In vitro anticancer activity assay
Experimental data for crystallographic analyses.
Cytotoxicity of the compounds was carried out on human cervi-
cal (HeLa) and human breast (MCF-7) cancer cell lines. Cell viability
was carried out using the MTT assay method [24]. The HeLa and
MCF-7 cells were grown in eagles minimum essential medium
(EMEM) containing 10% fetal bovine serum (FBS). For the screening
experiment, the cells were seeded onto 96-well plates at plating
density of 10,000 cells/ well and incubated to allow for cell attach-
ment at 37 °C, 5% CO2, 95% air and 100% relative humidity for 24 h
prior to the addition of the compounds. The compounds were dis-
solved in DMSO and diluted in the respective medium containing
1% FBS. After 24 h the medium was replaced with the respective
medium with 1% FBS containing the compounds at various concen-
trations and incubated at 37 °C under conditions of 5% CO2, 95% air
and 100% relative humidity for 48 h. Triplication was maintained
and the medium not containing the compounds served as control.
Compound
1
2
CCDC Number
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
Space group
Unit cell dimensions
a (Å)
911889
C
911890
C14H12N4O2S
300.34
14H12ClN3S
289.78
296(2)
0.71073
Triclinic
P-1
296(2)
0.71073
Triclinic
P-1
7.4469(15)
8.2008(15)
12.180(3)
93.317(6)
92.825(6)
110.689(6)
692.8(2)
2
1.389
0.415
300
0.30 ꢃ 0.25 ꢃ 0.20
7.3916(3)
8.4061(3)
12.2232(5)
90.383(2)
93.940(2)
110.124(2)
711.06(5)
2
1.403
0.237
b (Å)
c (Å)
a
(°)
b (°)
c
(°)
Volume (Å3)
Z
Density (cal.) (mg/m3)
Abs. coefficient (mmꢁ1
F (000)
After 48 h, 15 l
L of MTT (5 mg mLꢁ1) in phosphate buffered saline
)
(PBS) was added to each well and incubated at 37 °C for 4 h. The
medium with MTT was then removed and the formed formazan
crystals were dissolved in 100 lL of DMSO. The absorbance was
then measured at 570 nm using microplate reader. The % cell inhi-
bition was determined using the following formula and a graph was
plotted with the percentage of cell inhibition versus concentration.
From this, the IC50 was calculated:% cell Inhibition = [mean OD of
untreated cells (control)/mean OD of treated cells (control)] ꢃ 100.
312
Crystal size (mm3)
0.30 ꢃ 0.25 ꢃ 0.20
1.67–28.28
ꢁ9 6 h 6 h9,
ꢁ9 6 k 6 10,
ꢁ15 6 l 6 16
5966
h range for data collection (°) 1.68–27.92
Index ranges
ꢁ9 6 h 6 9,
ꢁ10 6 k 6 6,
ꢁ15 6 l 6 15
4908
Reflections collected
Independent reflections
3035
3373
[R(int) = 0.0247]
Semi-empirical from
equivalents
0.9216 and 0.8856
Full-matrix least-
squares on F2
3035/0/172
1.048
R1 = 0.0565,
wR2 = 0.1469
R1 = 0.0863,
wR2 = 0.1821
[R(int) = 0.0161]
Semi-empirical from
equivalents
0.9541 and 0.9322
Full-matrix least-
squares on F2
3373/0/194
0.971
R1 = 0.0415,
wR2 = 0.1246
R1 = 0.0522,
wR2 = 0.1373
Absorption correction
Max. and min. transmission
Refinement method
Results and discussion
Analytical and spectroscopic data for the ligands and its
complexes indicate a 1:1 metal–ligand stoichiometry for all the
complexes. The synthetic route of the complexes and the proposed
structure of the complexes are shown in Scheme 1. The complexes
are soluble in most common organic solvents like CH2Cl2, CHCl3,
DMF, DMSO, etc.
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2
r
(I)]
R indices (all data)
X-ray crystallography
95% Tris–HCl buffer (5 mM, pH 7.2) with 50 mM NaCl and 60
H2O2.
The samples with sufficient buffer were incubated for 2 h at
37 °C. After incubation, 1 L of loading buffer (0.25% bromophenol
blue, 0.25% xylene cynol and 60% glycerol) was added to the reac-
tion mixture and loaded onto a 1% agarose gel containing 1.0 g/
mL of ethidium bromide. The electrophoresis was carried out for
2 h at 50 V in Tris–acetic acid EDTA buffer. The bands were visual-
ized under UV light and photographed.
lM of
The ORTEP representation of ligands, 1 and 2 are shown in Figs.
1 and 2 respectively. Relevant data collection and details of the
structure refinement are summarized in Table 1. The selected bond
lengths and bond angles for compounds 1 and 2 are given in Tables
2 and 3, respectively. The ligands, 1 and 2 crystallizes in a triclinic
space group P-1 with two crystallographically independent
l
l
Table 2
Antioxidant activity
Selected bond lengths (Å).
1
2
The 2,2-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging
activity of the compounds was measured according to the method
of Elizabeth [23]. The DPPH radical is a stable free radical having a
kmax at 517 nm. A fixed concentration of the experimental com-
C(1)AC(6)
C(1)AC(2)
C(1)AH(1)
C(2)AC(3)
C(5)AC(6)
C(5)AH(5)
C(6)AN(3)
C(7)AN(3)
C(7)AN(2)
C(7)AS(1)
C(8)AN(1)
C(8)AC(9)
C(8)AH(8)
C(9)AC(14)
C(9)AC(10)
C(14)ACl(1)
N(1)AN(2)
N(2)AH(2N2)
N(3)AH(3N3)
1.374(5)
1.386(4)
0.9300
1.380(5)
1.379(4)
0.9300
1.429(4)
1.339(4)
1.350(3)
1.679(3)
1.282(4)
1.454(4)
0.9300
1.401(4)
1.404(4)
1.736(3)
1.379(3)
0.8600
S(1)AC(7)
1.6787(15)
1.2766(19)
1.3724(18)
1.203(2)
1.329(2)
1.4295(19)
0.8600
1.3500(19)
0.87(2)
1.2040(18)
1.470(2)
1.466(2)
0.9300
N(3)AC(8)
N(3)AN(2)
O(2)AN(4)
N(1)AC(7)
N(1)AC(6)
N(1)AH(1N1)
N(2)AC(7)
N(2)AH(2N2)
N(4)AO(1)
N(4)AC(10)
C(8)AC(9)
C(8)AH(8)
C(9)AC(14)
C(9)AC(10)
C(10)AC(11)
C(4)AC(5)
C(4)AH(4)
C(5)AH(5)
pound (100 lL) was added to a solution of DPPH in methanol
(0.3 mM, 1 mL) and the final volume was made up to 4 mL with
double distilled water. DPPH solution with methanol was used as
a positive control and methanol alone acted as a blank. The solu-
tion was incubated at 37 °C for 30 min in dark. The decrease in
absorbance of DPPH was measured at 517 nm. The tests were
run in triplicate, and various concentrations (20–100 lg/mL) of
the compounds used to fix a concentration at which the com-
pounds showed 50% of activity. In addition, the percentage of activ-
ity was calculated using the formula, % of suppression ratio = [(A0–
Ac)/A0] ꢃ 100. A0 and Ac are the absorbance in the absence and
presence of the tested compounds respectively. The 50% activity
(IC50) can be calculated using the percentage of activity.
1.397(2)
1.400(2)
1.381(2)
1.383(2)
0.9300
0.8600
0.9300

K. Sampath et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 115 (2013) 287–296
291
molecules in the unit cell. The ligands existence in the thione form
is confirmed by the C@S bond length, 1.679(3) Å for 1 and
1.678(15) Å for 2. The azomethine bond length, C8AN1
(1.282(4) Å) for 1 and C8AN3 (1.2766(19) Å) for 2 is in conformity
with a formed C@N double bond length (1.28 Å). The single crystal
X-ray diffraction study confirms the existence of the thiosemicar-
bazone in the thione form without any ambiguity.
d 6.78–7.87, due to the delocalisation of electron density in the sys-
tem [29] and these signals in the complexes cannot be distin-
guished from the aromatic signals of PPh3/ AsPh3 due to their
extensive overlap [30]. For ligands and complexes, the chemical
shift at d 8.57–8.66 is due to phenyl NH proton.
In the 13C NMR spectra of the ligands, the signal at d 139–148
corresponds to the thioamide carbon (C@S), which disappears in
the spectra of the complexes and a new signal, (CAS) at d 136–
139 indicates the coordination of sulphur via deprotonation [31].
The spectra of the ligands displayed a single resonance at d 176
due to the azomethine carbon atom, downfield shift of this signal
at d 181–183 clearly indicates the coordination of C@N carbon
atom. The aromatic carbons of the free ligands and complexes
show signal in the region d 122–139. For all the complexes, the ter-
minal carbonyl group C„O appeared at d 191–194 [32].
Infrared spectra
The IR bands for the metal complexes derived from the benzal-
dehyde thiosemicarbazone ligands are most useful in attempting
to determine the mode of coordination. A medium sharp band at
1597 and 1599 cmꢁ1 due to the azomethine C@N stretching fre-
quency of the free ligands 1 and 2 respectively was shifted to lower
frequency in the spectra of the complexes at 1474–1518 cmꢁ1 indi-
cating that the coordination through N atom [25]. A band appeared
at 860 cmꢁ1 for 1 and 846 cmꢁ1 for 2 due to vibration of the C@S
double bond which disappeared in the spectra of the complexes
and a new band, CAS appeared at 737–743 cmꢁ1 indicating that
the other coordination is through thiolate sulphur after enolization
followed by deprotonation on sulphur [26]. Further, the replace-
ment of the hydride ion in the metal precursors by the ligands
has been confirmed by the absence of a band around 2020 cmꢁ1
in all the complexes [27]. In all the complexes, the strong band
in the region 1911–1938 cmꢁ1 is due to terminally coordinated
carbonyl group. Overall, the complexes contain monobasic NS
coordinated thiosemicarbazones. In addition, the characteristic
absorption bands due to triphenylphosphine and triphenylarsine
were also observed for all the complexes in their expected regions.
In order to confirm the presence of triphenylphosphine group,
31P NMR spectra were recorded for the complexes, 3 and 5. A sharp
singlet observed at d 36 for the complexes, 3 and 5 was due to the
presence of magnetically equivalent phosphorous atoms and thus
suggesting the presence of two triphenylphosphine groups in a po-
sition trans to each other [33].
DNA binding – titration experiments
Monitoring the effect of adding increasing amounts of DNA on
the absorption spectrum of a metal complex is one of the most
widely used method for determining overall binding constants. A
complex bound to DNA through intercalation is characterized by
the change in absorbance (hypochromism) and red shift in wave-
length, due to the intercalative mode involving a stacking interac-
tion between the aromatic chromophore and the DNA base pairs
[34]. The results of absorption spectra of the compounds in the ab-
sence and presence of CT-DNA are given in Fig. 4. Upon increasing
the concentration of DNA to the test compounds, the absorption
bands of the ligands 1 and 2 exhibited hypochromism of 5.65%
Electronic spectra
The electronic spectra of the ligands show bands at 312–315
and 368–408 nm. The first band at 312–315 nm, is assigned to a li-
gand
p ?
pꢂ transition, while the band at 368–408 nm is assigned
to an n ? pꢂ transition associated with the imine and thioamide
function of the thiosemicarbazones. The spectra of the complexes
showed three to five bands in the region 312–480 nm. The lower
wavelength bands (312–320 and 368–370 nm) are characterized
as ligand centered transitions occurring within the ligand orbitals.
These bands are shifted when compared to ligands indicating the
involvement of imine nitrogen and thionyl sulphur in coordination
with ruthenium atom [26]. The bands appearing in the region 401–
432 nm are assigned to charge transfer transitions arising from the
excitation of an electron from metal t2g level to an unfilled molec-
ular orbital derived from the pꢂ level of the ligands [28]. The pat-
tern of the electronic spectra for the complexes are similar to
other ruthenium(II) octahedral complexes [26,27].
Table 3
Selected bond angles (°).
1
2
C(6)AC(1)AC(2)
119.6(3)
120.2
119.7
119.1(3)
120.5
120.5
116.3(3)
124.6(2)
119.1(2)
120.5(3)
119.8
C(8)AN(3)AN(2)
C(7)AN(1)AC(6)
C(7)AN(1)AH(1N1)
C(6)AN(1)AH(1N1)
C(7)AN(2)AN(3)
C(7)AN(2)AH(2N2)
N(3)AN(2)AH(2N2)
O(2)AN(4)AO(1)
O(2)AN(4)AC(10)
O(1)AN(4)AC(10)
N(1)AC(7)AN(2)
N(1)AC(7)AS(1)
114.99(13)
124.91(13)
117.5
C(6)AC(1)AH(1)
C(5)AC(4)AH(4)
C(6)AC(5)AC(4)
117.5
C(6)AC(5)AH(5)
C(4)AC(5)AH(5)
N(3)AC(7)AN(2)
N(3)AC(7)AS(1)
N(2)AC(7)AS(1)
N(1)AC(8)AC(9)
N(1)AC(8)AH(8)
C(9)AC(8)AH(8)
C(14)AC(9)AC(10)
C(14)AC(9)AC(8)
C(10)AC(9)AC(8)
C(11)AC(10)AC(9)
C(11)AC(10)AH(10)
C(9)AC(10)AH(10)
C(13)AC(12)AH(12)
C(11)AC(12)AH(12)
C(13)AC(14)AC(9)
C(13)AC(14)ACl(1)
C(9)AC(14)ACl(1)
C(8)AN(1)AN(2)
C(7)AN(2)AN(1)
C(7)AN(2)AH(2N2)
N(1)AN(2)AH(2N2)
C(7)AN(3)AC(6)
C(7)AN(3)AH(3N3)
C(6)AN(3)A H(3N3)
120.62(13)
118.2(14)
121.1(14)
122.22(17)
119.82(14)
117.96(15)
116.44(14)
124.33(12)
119.22(12)
119.53(14)
120.2
120.2
121.44(14)
120.3
120.42(17)
119.8
119.8
119.09(17)
120.5
120.5
121.43(17)
119.3
119.3
120.56(18)
119.7
119.7
119.8
NMR spectra
117.4(3)
121.6(3)
121.0(3)
121.3(3)
119.3
119.3
119.9
119.9
121.4(3)
118.7(2)
119.9(2)
114.8(2)
120.8(2)
119.6
119.6
124.8(2)
117.6
N(2)AC(7)AS(1)
N(3)AC(8)AC(9)
N(3)AC(8)AH(8)
C(9)AC(8)AH(8)
C(9)AC(10)AN(4)
C(10)AC(11)AH(11)
C(3)AC(4)AC(5)
C(3)AC(4)AH(4)
C(5)AC(4)AH(4)
C(6)AC(5)AC(4)
C(6)AC(5)AH(5)
C(4)AC(5)AH(5)
C(13)AC(14)AC(9)
C(13)AC(14)A H(14)
C(9)AC(14)A H(14)
C(12)AC(13)A C(14)
C(12)AC(13)AH(13)
C(14)AC(13)AH(13)
The NMR spectra of the compounds were recorded in DMSO-d6
solution for confirming the binding mode of the ligands to the
ruthenium atom and the 1H, 13C and 31P NMR spectra of the com-
plex 3 are shown in Fig. 3. The 1H NMR spectra of the free ligands
showed a singlet at d 10.20 for the hydrazine NH proton, which is
absent in the spectra of the complexes indicating the enolization
and deprotonation of the ANHAC@S group prior to coordination
of ligand to metal through thiolate sulphur. The ACH@N proton
observed at d 12.01–12.12 in the spectra of the ligands, is shifted
slightly downfield in the spectra of the complexes in the region d
12.08–12.25. This observation supports the involvement of AC@N
chromophore in coordination. The aromatic protons, observed as
multiplets in the region d 7.21–8.47 in the spectra of the ligands,
are remain more or less unchanged in the complexes in the region
117.6

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K. Sampath et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 115 (2013) 287–296
Fig. 3. 1H (A), 13C (B) and 31P NMR spectrum of the complex 3.
and 25.42% with red shifts of 2 nm at 310 and 352 nm, respec-
tively, whereas the absorption bands of complexes 3 and 4 exhib-
ited a hypochromism of about 17.30% at 360 nm and 40.64% at
353 nm with red shifts of 6 nm, respectively. However, complexes
5 at 352 nm and 6 at 352 nm exhibited a hypochromism of about
9.74% and 6.05% with red shifts of 3 and 2 nm, respectively. These
results suggested an intimate association of the test compounds
with CT-DNA, and it is also likely that these compounds bind to
the DNA helix via intercalation. After the compounds intercalate
to the base pairs of DNA, the pꢂ orbital of the intercalated com-
pounds could couple with
p orbitals of the base pairs, thus decreas-
ing the
p
?
pꢂ transition energies, hence resulting in
hypochromism [35]. In order to compare quantitatively the bind-
ing strength of the compounds, the intrinsic binding constants

K. Sampath et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 115 (2013) 287–296
293
104 Mꢁ1 for compounds 1, 2, 3, 4, 5 and 6 respectively. The observed
values of Kb revealed that the ruthenium(II) complexes (3–6) bind
strongly than the respective ligands (1 and 2) to DNA via intercala-
tive mode. From the results obtained, it has been found that com-
plex 3 strongly binds with CT-DNA in comparison to that with 4,
5 and 6, and binding affinity was in the order 3 > 4 > 5 > 6 > 1 > 2.
The Kb values obtained for the above ruthenium(II) complexes are
comparable to those of the other known ruthenium(II) complexes
[36,37].
(Kb) of them with CT-DNA were determined from the following
equation.
½DNAꢄ=ðea
ꢁ
ef Þ ¼ ½DNAꢄ=ðeb
ꢁ
ef Þ þ 1=Kbðeb
ꢁ
ef
Þ
where [DNA] is the concentration of DNA in base pairs and the
apparent absorption coefficient ea ef and eb correspond to Aobs/
,
[compound], the extinction coefficient of the free compound and
the extinction coefficient of the compound when fully bound to
DNA, respectively. The plot of [DNA]/(ea
slope and the intercept which are equal to 1/(eb
f), respectively; Kb is the ratio of the slope to the intercept.
–e
f) versus [DNA] gave a
–ef) and 1/Kb
(eb
–e
DNA cleavage study by gel electrophoresis
The magnitudes of intrinsic binding constants (Kb) were calculated
to be 6.7( 0.04) ꢃ 103 Mꢁ1, 4.7( 0.09) ꢃ 103 Mꢁ1, 1.1( 0.12) ꢃ 105
Mꢁ1, 1.0( 0.09) ꢃ 105 Mꢁ1, 2.0( 0.13) ꢃ 104 Mꢁ1 and 1.5( 0.14) ꢃ
After binding to DNA, the metal complexes could induce several
changes in the DNA conformation, leading to decrease in molecular
Fig. 4. Electronic spectra of compounds 1 (A), 2 (B), 3 (C), 4 (D), 5 (E) and 6 (F) in Tris–HCl buffer upon addition of CT-DNA. [Complex] = 25
shows the absorption intensities decrease upon increasing DNA concentrations (Inset: Plot between [DNA] and [DNA]/[ea
f] X 10ꢁ8).
lM, [DNA] = 0–50 lM. Arrow
–e

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K. Sampath et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 115 (2013) 287–296
Fig. 5. Gel electrophoresis diagram showing the hydrolytic cleavage of CT-DNA
(30 M) incubated at 37 °C for a period of 2 h at a concentration of 40 M of
l
l
complex 3–6 in 5% DMSO and 95% 5 mM Tris–HCl/50 mM NaCl buffer at pH 7.2.
Fig. 7. Antioxidant activity of compounds, 1–6 with standard, ascorbic acid, Aca.
Lane 1: Control, Lanes 2–5: DNA + complexes 3–6, respectively.
Fig. 6. Gel electrophoresis diagram showing the oxidative cleavage of CT-DNA
(30
at pH 7.2 in the presence of H2O2 (60
period of 2 h. Lane 1 and 9: DNA. Lane 2 and 10: DNA + H2O2. Lane 3–5: 3 (30, 60
and 90 M) + DNA + H2O2. Lane 6–8: 4 (30, 60 and 90 M) + DNA + H2O2. Lane 11–
13: (30, 60 and 90 M) + DNA + H2O2. Lane 14–16: (30, 60 and
90 M) + DNA + H2O2.
l
M) by complexes 3–6 in 5% DMSO and 95% 5 mM Tris–HCl/50 mM NaCl buffer
lM) as a co-oxidant incubated at 37 °C for a
l
l
5
l
6
Fig. 8. Growth inhibition of MCF-7 cell line as a function of concentration of the
compounds 1–6.
l
longer exposure time. When the DNA is allowed to interact with
the complexes (3–6) at various concentrations (30, 60 and
90 lM), a substantial decrease in the intensities of the bands for
the metal bound DNA as compared to untreated control DNA was
observed, which suggests the cleavage of DNA by metal complexes
[39]. As can be seen from Lanes 5, 8, 13 and 16 of complexes 3, 4, 5
and 6 respectively, the bands have completely disappeared, indi-
weight of DNA [38]. The ability of metal complexes to perform DNA
cleavage is generally monitored by agarose gel electrophoresis and
in the present work CT-DNA was chosen to investigate its cleavage.
The cleavage experiments were carried out in the absence and
presence of activating agent, H2O2 under aerobic conditions and
are shown in Figs. 5 and 6 respectively. Fig. 5, shows the hydrolytic
cleavage activity of the complexes, 3–6 at 40 lM. A control exper-
cating cleavage of DNA by ruthenium(II) complexes at 90
centration. Interestingly, the complex, 3 shows potential nuclease
activity against DNA at low concentration, 30 M itself, indicates
lM con-
iment using DNA alone does not show any significant cleavage of
DNA (Lane 1). Further, when DNA is allowed to interact with the
complexes, no considerable difference between the intensity of
bands for metal bound DNA and control DNA was observed. This
suggests that the nuclease activity of the complexes does not in-
volve by hydrolytic pathway. These experimental facts demon-
strated that a combination of both the ruthenium complexes and
activating agent, H2O2 are required to show effective cleavage of
CT-DNA. Fig. 6 reveals that the oxidative cleavage of CT-DNA in-
duced by complexes, 3–6 in the presence of H2O2. Control experi-
ment using DNA alone (Lane 1 and 9) and DNA with H2O2 (Lane 2
and 10) does not show any significant cleavage of CT-DNA even on
l
a superior DNA cleavage than the other complexes. The cleavage
activity may be due to the intercalative interaction of the com-
plexes with the DNA strands in the presence of H2O2 induces oxi-
dative DNA cleavage [39].
Antioxidant activity
Since the DNA interaction experiments have shown that the
compounds exhibit good DNA binding and cleavage activity, it is

K. Sampath et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 115 (2013) 287–296
295
Cytotoxic activity evaluation
The results of biological studies on DNA binding, DNA cleavage
and antioxidative studies for compounds, 1, 2, 3, 4, 5, and 6, have
encouraged us to test their cytotoxicity against a pair of selected
human cancer cell lines, human cervical (HeLa) and human breast
(MCF-7) cancer cells. Compounds were dissolved in DMSO, and
blank samples containing same volume of DMSO were taken as
controls to identify the activity of the solvent in this cytotoxicity
experiment. The results were analyzed by means of cell viability
curves and expressed with IC50 values in the studied concentration
range from 0.1 to 100 lM. The activity that corresponds to the
inhibition of cancer cell growth at a maximum level is shown in
Figs. 8 and 9, Table 4. All the complexes show good cytotoxic pro-
files, with low IC50 values in micromolar concentrations. The high-
est activity was noted for the complex 3 towards both the cancer
cell lines whereas the complex 6 shows, least activity towards
MCF-7 cancer cell line but failed to arrest the growth of HeLa can-
cer cells (up to a concentration of 100 lM). However, Complexes 4
and 5 showed significant IC50 values for HeLa and MCF-7 cancer
cell lines, the cytotoxic effect of these complexes are less when
compared to the complex 3. However, the ligands does not show
any significant activity (IC50 > 100 lM) against both the cell lines,
thus confirming that chelation of the ligands with the ruthe-
nium(II) ion is responsible for the observed cytotoxic property of
the complexes. Among the two different cell lines used in this
study, the proliferation of MCF-7 was arrested to a greater extent
than HeLa cells by the complexes except complex 3. Though the
complexes were active against tumor cell lines under in vitro cyto-
toxicity experiments, none of them could attain the effectiveness
of standard drug cisplatin [40]. Comparison of anticancer activity
of ruthenium(II) complexes reveals that the complexes of
chlorobenzaldehyde thiosemicarbazone moiety exhibited higher
cytotoxic effects than the complexes of nitrobenzaldehyde
thiosemicarbazone. Moreover, the complexes with triphenylphos-
phine as co-ligand showed better cytotoxic effects than the
complexes containing triphenylarsine. This might be due to the
lipophilic effect of triphenylphosphine in the ruthenium(II) com-
plexes which helps to cross the cytoplasmic membrane [41].
Hence, these complexes are active against the tumor cell lines un-
der in vitro conditions, and the results are comparable to those ob-
tained for other ruthenium(II) complexes [42].
Fig. 9. Growth inhibition of HeLa cell line as a function of concentration of the
compounds 1–6.
Table 4
Cytotoxic activity of the ligands and ruthenium(II) complexes against the MCF-7 and
HeLa cancer cell lines.
Compounds
IC50 values (
MCF-7
lM)
HeLa
1
2
3
4
5
6
>100
>100
18.60
19.52
27.58
41.16
12.75
>100
>100
13.93
24.16
57.26
>100
12.52
Reported value (Cisplatin)
Conclusion
considered worthwhile to study the antioxidant activity of these
compounds. 2,2-Diphenyl-2-picryl-hydrazyl (DPPH) assay is
widely used for assessing the ability of radical scavenging activity
and it is measured in terms of IC50 values. Because of the presence
of odd electron, DPPH shows a strong absorption band at 517 nm in
the visible spectrum. As this electron gets paired off in the pres-
ence of a free radical scavenger, absorption vanishes, and the
resulting decolourization is stoichiometric with respect to the
number of electrons taken up. The DPPH assay of the tested com-
pounds shown in Fig. 7, indicates that the all the complexes exhib-
ited higher antioxidant activity compared to the standard, ascorbic
acid (Aca). The IC50 values indicated that the compounds showed
antioxidant activity in the order of 3 > 4 > 5 > 6 > 1 > 2. Complex 3
showed a higher antioxidant activity compared to other three com-
plexes. From the above results, the scavenging effect of the free li-
gands is significantly less when compared to their corresponding
ruthenium(II) complexes which is due to the chelation of the or-
ganic ligands with the ruthenium(II) ion and the antioxidant activ-
ities of the complexes are in agreement with the reported
ruthenium(II) complexes [28].
In this work, a systematic approach to the synthesis of mononu-
clear ruthenium(II) carbonyl complexes containing benzaldehyde
4-phenyl-3-thiosemicarbazones were carried out and, were char-
acterized by various spectroscopic techniques. The structure and
geometry of the thiosemicarbazone ligands were analyzed by sin-
gle crystal X-ray diffraction. The DNA binding ability of the ligands
and complexes were assessed by absorption spectra which inferred
an intercalative mode of binding with binding constants ranging
from 103 to 105 Mꢁ1. The experimental results suggested that the
complex 3 can bind to DNA more strongly than the other three
complexes and free ligands. The DNA cleavage capabilities of com-
plexes in the presence of H2O2 revealed their potential nuclease
activity to cleave CT-DNA. The complex 3 showed superior cleav-
age activity at low concentration than the other complexes. The
antioxidant activity showed that all the ruthenium(II) complexes
can serve as potential antioxidants than the ligands against DPPH
radical in the order 3 > 4 > 5 > 6 > 1 > 2. Interestingly, all the com-
plexes showed higher scavenging activity than the standard, ascor-
bic acid. Moreover, the complexes exhibited good cytotoxic activity
against HeLa and MCF-7 cancer cell lines. The IC50 value indicates

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