Mixed ligand ruthenium(III) complexes of benzaldehyde 4-methyl-3- thiosemicarbazones with triphenylphosphine/triphenylarsine co-ligands: Synthesis, DNA binding, DNA cleavage, antioxidative and cytotoxic activity

Article abstract of DOI:10.1016/j.molstruc.2013.04.051

The new ruthenium(III) complexes with 4-methyl-3-thiosemicarbazone ligands, (E)-2-(2-chlorobenzylidene)-N-methylhydrazinecarbothioamide (HL¹) and (E)-2-(2-nitrobenzylidene)-N-methylhydrazinecarbothioamide (HL²), were prepared and cha

Full text of DOI:10.1016/j.molstruc.2013.04.051

Journal of Molecular Structure 1046 (2013) 82–91
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Mixed ligand ruthenium(III) complexes of benzaldehyde
4-methyl-3-thiosemicarbazones with triphenylphosphine/triphenylarsine
co-ligands: Synthesis, DNA binding, DNA cleavage, antioxidative and
cytotoxic activity
⇑
K. Sampath, S. Sathiyaraj, G. Raja, C. Jayabalakrishnan
Post-Graduate and Research Department of Chemistry, Sri Ramakrishna Mission Vidyalaya College of Arts and Science, Coimbatore 641 020, Tamil Nadu, India
g r a p h i c a l a b s t r a c t
h i g h l i g h t s
ꢀ
ꢀ
ꢀ
ꢀ
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 complex 3 shows higher IC50
value than the other complexes
against tumor cells.
a r t i c l e i n f o
a b s t r a c t
Article history:
The new ruthenium(III) complexes with 4-methyl-3-thiosemicarbazone ligands, (E)-2-(2-
chlorobenzylidene)-N-methylhydrazinecarbothioamide (HL1) and (E)-2-(2-nitrobenzylidene)-N-meth-
ylhydrazinecarbothioamide (HL2), were prepared and characterized by various physico-chemical and
spectroscopic methods. The title compounds 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 ligands and complexes were investigated by absorption spectroscopy and IR spectroscopy. It reveals
that the compounds bind to nitrogenous bases of DNA via intercalation. The oxidative cleavage of the com-
plexes with CT-DNA inferred that the effects of cleavage are dose dependent. Antioxidant study of the
ligands and complexes showed the significant antioxidant activity against DPPH radical. In addition, the
in vitro cytotoxicity of the ligands and complexes against MCF-7 cell line was assayed which showed higher
cytotoxic activity with the lower IC50 values indicating their efficiency in killing the cancer cells even at low
concentrations.
Received 28 November 2012
Received in revised form 1 April 2013
Accepted 22 April 2013
Available online 28 April 2013
Keywords:
Ruthenium(III) complexes
4-Methyl-3-thiosemicarbazones
DNA binding
Oxidative cleavage
Antioxidant
Cytotoxicity
Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction
platinum compounds constitute a discrete class of inorganic
chemotherapeutics, which are widely used in the clinic as anti-
The great majority of antitumor drugs are either synthetic or
natural products that are based on organic compounds, but
tumor agents. Inspite of their important activity against various
human cancers, platinum compounds are not sufficiently effective
against some of most frequent human cancers, such as breast and
colon tumors. Moreover, patients treated with cisplatin often de-
velop acquired resistance and experience serious adverse effects
⇑
Corresponding author. Tel.: +91 422 2692461.
E-mail address: drcjbstar@gmail.com (C. Jayabalakrishnan).
0022-2860/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molstruc.2013.04.051

K. Sampath et al. / Journal of Molecular Structure 1046 (2013) 82–91
83
[1–3]. Thus, both the advantages and the drawbacks of platinum
chemotherapy have motivated scientists to search for other metal
based drugs with better therapeutic properties. At this juncture,
ruthenium, a race transition metal of the platinum group, has
emerged as an attractive alternative due to several favorable prop-
erties suited to rational anticancer drug design and biological
applications. Biologically compatible ligand – exchange kinetics
of ruthenium complexes similar to those of platinum complexes,
a higher coordination number that could potentially be used to fine
– tune the properties of the complexes, and low toxicity (than their
platinum counterparts) towards healthy tissues by mimicking iron
in binding to many biological molecules are the advantages of
using ruthenium complexes [4]. Moreover, the entrance of two
ruthenium(III) based drugs, NAMI-A [5] and KP109 [6] into clinical
trials for the treatment of metastatic tumors increased interest on
this metal.
Technology, Kerala, India. IR spectra were recorded as KBr pellets
in the 400–4000 cmꢁ1 region using a Perkin Elmer FT-IR 8000 spec-
trophotometer. Electronic spectra were recorded in DMSO solution
with a Systronics double beam UV–vis spectrophotometer 2202 in
the range 200–800 nm. 1H NMR and 13C NMR spectra were re-
corded on a Bruker AV III 500 MHZ instrument using TMS as an
internal reference. Magnetic susceptibility measurements of the
complexes were recorded using Guoy balance. EPR spectra were
recorded on a varian E-112 ESR spectrophotometer at X-band
microwave frequencies for powdered samples at room tempera-
ture. EI mass spectrum of the complex was recorded on a JEOL
GCMATE II mass spectrometer. Antioxidant and Anti cancer studies
were carried out at the Kovai Medical Centre and Hospital Phar-
macy College, Coimbatore, Tamil nadu. Melting points were re-
corded with Veego VMP-DS heating table.
Thiosemicarbazones form an interesting class of compounds
with a wide range of pharmacological applications [7]. The biolog-
ical activity of thiosemicarbazones is related to their chelating abil-
ity with transition metal ions. This chelating ability is a function of
the unique characteristics of the mixed hard – soft NS donor atoms
in the structure. The structure of thiosemicarbazones offers up a
variety of donor atom sets such that they are versatile ligands in
both neutral and anionic forms [8]. Incorporation of metals onto
these thiosemicarbazone ligands can result in alteration or
enhancement of their biological activity [9]. During the last two
or three decades, there has been an interesting attention on the
binding study of small molecules to DNA, since it is an important
genetic substance in organisms [10,11]. Errors in gene expression
can often cause disease and play a secondary role in the outcome
and severity of human diseases [12]. A complete understanding
of DNA – drug binding is significant in the rational design of
DNA structural probe, DNA foot printing, sequence – specific cleav-
ing agents and potential anti-cancer drugs [13]. Hence, it is essen-
tial to explore the interactions of metal complexes with DNA to
design effective chemotherapeutic agents and better anticancer
drugs.
2.2. Preparation of ligands
2.2.1. (E)-2-(2-chlorobenzylidene)-N-methylhydrazinecarbothioamide
(HL1) (1)
A methanolic solution (50 mL) of 4-methyl-3-thiosemicarba-
zide (1.051 g, 0.01 mol) was added to
a methanol solution
(50 mL) containing 2-chlorobenzaldehyde (1.12 mL, 0.01 mol).
The mixture was refluxed for an hour during which period a white
color precipitate 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: 89%; M.P:
200–201 °C. Anal. calcd. for C9H10ClN3S (%): C, 47.47; H, 4.43; N,
18.45; S, 14.08. Found (%): C, 47.40; H, 4.45; N, 18.43; S, 14.01.
IR (KBr, cmꢁ1): 1585
(nm): 305, 360 (
m
?
(C@N); 951
m(C@S). UV–vis (DMSO), kmax
p
p⁄, n ? p⁄). 1H NMR (DMSO-d6): d 8.60
(s, 1H, hydrazine NH); d 8.47 (s, 1H, methyl NH); d 11.67 (s, 1H,
HAC@N); d 3.03 (d, 3H, CH3); d 7.39–8.30 (m, 4H, aromatic).
13C NMR (DMSO-d6): d 142 (C@S); d 178 (C@N); d 31 (CH3); d
127–133 (aromatic).
In the case of pharmaceuticals, the binding capacity of thio-
semicarbazones is further increased by condensation of the thio-
semicarbazide with an aldehyde containing heteroatom [14,15].
This aroused our interest in the synthesis of ruthenium(III) thio-
semicarbazone complexes. Herein, we report the synthesis, charac-
terization, DNA binding, DNA cleavage, antioxidative and
cytotoxicity studies of ruthenium(III) complexes of 2-choloro/nitro
benzaldehyde 4-methyl-3-thiosemicarbazones. The crystal struc-
ture of the thiosemicarbazone ligands have been determined by
X-ray crystallography. The investigation of the biological proper-
ties of the ligands and ruthenium(III) complexes has been focused
on the binding properties with CT-DNA performed by UV spectros-
copy and cleavage properties of the complexes performed by gel
electrophoresis with CT-DNA. Finally, we have studied their anti-
oxidative property against DPPH radical and in vitro cytotoxicity
activity against MCF-7 cancer cell line.
2.2.3. (E)-2-(2-nitrobenzylidene)-N-methylhydrazinecarbothioamide
(HL2) (2)
It was prepared using the same procedure as described for HL1
with 4-methyl-3-thiosemicarbazide (1.051 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: 85%; M.P: 229–230 °C. Anal.
calcd. for C9H10N4O2S (%): C, 45.37; H, 4.23; N, 23.51; S, 13.46.
Found (%): C, 45.39; H, 4.29; N, 23.52; S, 13.42. IR (KBr, cmꢁ1):
1530
396 (
m
p
(C@N); 850
?
m(C@S). UV–vis (DMSO), kmax (nm): 305, 368,
p⁄, n ? p⁄). 1H NMR (DMSO-d6): d 8.64 (s, 1H, hydrazine
NH); d 8.48 (s, 1H, methyl NH); d 11.79 (s, 1H, HAC@N); d 3.04 (d,
3H, CH3); d 7.61–8.43 (m, 4H, aromatic). 13C NMR (DMSO-d6): d
148 (C@S); d 178 (C@N); d 31 (CH3); d 125–137 (aromatic).
2. Experimental
2.3. Single crystal X-ray diffraction studies
2.1. Materials and instrumentation
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
All the chemicals used were chemically pure and AR grade. Calf-
thymus DNA (CT-DNA) was purchased from Bangalore Genei, Ban-
galore, India. Solvents were purified and dried according to the
standard procedure [16]. The metal precursors [RuCl3(PPh3)3] and
[RuCl3(AsPh3)3] were prepared by literature methods [17,18].
Micro analyses (C, H, N and S) were performed on a Vario EL III
CHNS analyzer at STIC, Cochin University of Science and
were performed using
a
graphite-mono chromate Mo
Ka
(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

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K. Sampath et al. / Journal of Molecular Structure 1046 (2013) 82–91
[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.
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 com-
pounds were dissolved in a mixed solvent of 5% DMSO and 95%
Tris–HCl buffer. Stock solutions were stored at 4 °C and used with-
in 4 days. Absorption titration experiments were performed with
2.4. Preparation of ruthenium(III) complexes
fixed concentrations of the complexes (25
lM) with varying con-
centration of DNA (0–50 M). While measuring the absorption
l
2.4.1. Synthesis of [RuCl2(PPh3)2L1] (3)
spectra, an equal amount of DNA was added to both the test solu-
tion and the reference solution to eliminate the absorbance of DNA
itself.
A methanolic solution (20 mL) containing HL1 (1) (0.110 g,
0.5 mmol) and triethylamine (0.07 mL, 0.5 mmol) were added to
[RuCl3(PPh3)3] (0.452 g, 0.5 mmol) in benzene (20 mL). The result-
ing red color solution was refluxed for 8 h. The reaction mixture
was then cooled to room temperature, following which the result-
ing dark brown color precipitate was filtered off and then purity
was checked by TLC. This solid was crystallized from CH2Cl2/hex-
ane mixture. Our sincere effort to obtain single crystal of the com-
plexes went unsuccessful. Yield: 55%. M.P: 221–222 °C. Anal. calcd.
for C45H39Cl3N3P2RuS (%): C, 58.54; H, 4.26; N, 4.55; S, 3.47. Found
(%): C, 58.51; H, 4.29; N, 4.51; S, 3.40. EI-MS: Found m/z = 923 (M+)
2.5.2. Nuclease activity using gel electrophoresis
The DNA cleavage activity of the ruthenium(III) 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
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
(calculated m/z = 923.25 for M+). IR (KBr, cmꢁ1): 1580
(CAS); 541 (RuAN). UV–vis (DMSO), kmax (nm): 317, 365, 411
m
(C@N); 747
of CT-DNA, 30; 60; 90 lM of each complex (3–6) in 5% DMSO and
m
m
95% Tris–HCl buffer (5 mM, pH 7.2) with 50 mM NaCl and 60
lM of
(ILCT), 435 (LMCT). EPR (300 K, ‘g’ value): 1.95.
leff (300 K): 1.62 lB.
H2O2.
The samples with sufficient buffer were incubated for 2 h at
37 °C. After incubation, 1 L of loading buffer (0.25% bromophenol
2.4.2. Synthesis of [RuCl2(AsPh3)2L1] (4)
l
It was prepared using the same procedure as described for 3
with HL1 (1) (0.110 g, 0.5 mmol) and [RuCl3(AsPh3)3] (0.565 g,
0.5 mmol). Brown colored crystalline powder was obtained. Yield:
54%. M.P: 215–217 °C. Anal. calcd. for C45H39Cl3N3As2RuS (%): C,
53.45; H, 3.89; N, 4.16; S, 3.17. Found (%): C, 53.49; H, 3.91; N,
4.11; S, 3.12. EI-MS: Found m/z = 1011 (M+) (calculated
blue, 0.25% xylene cynol and 60% glycerol) was added to the
reaction mixture and loaded onto a 1% agarose gel containing
1.0 lg/mL of ethidium bromide. The electrophoresis was carried
out for 2 h at 50 V in Tris–acetic acid EDTA buffer. The bands were
visualized under UV light and photographed.
m/z = 1011.15 for M+). IR (KBr, cmꢁ1): 1579
522 (RuAN). UV–vis (DMSO), kmax (nm): 310, 368 (ILCT), 404
m(C@N); 742
m
(CAS);
2.5.3. Antioxidant activity
m
The 2,2-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging
activity of the compounds was measured according to the method
of Elizabeth and Rao [23]. The DPPH radical is a stable free radical
having a kmax at 517 nm. A fixed concentration of the experimental
(LMCT). EPR (300 K, ‘g’ value): 2.31. leff (300 K): 1.65 lB
.
2.4.3. Synthesis of [RuCl2(PPh3)2L2] (5)
It was prepared using the same procedure as described for 3
with HL2 (2) (0.138 g, 0.5 mmol) and [RuCl3(PPh3)3] (0.452 g,
0.5 mmol). Brown colored crystalline powder was obtained. Yield:
56%. M.P: 291–293 °C. Anal. calcd. for C45H39Cl2N4O2P2RuS (%): C,
57.88; H, 4.21; N, 6.00; S, 3.43. Found (%): C, 57.91; H, 4.29; N,
6.05; S, 3.48. EI-MS: Found m/z = 933 (M+) (calculated
compound (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
m/z = 933.81 for M+). IR (KBr, cmꢁ1): 1524
522 (RuAN). UV–vis (DMSO), kmax (nm): 320, 368, 406 (ILCT),
m
(C@N); 742
m(CAS);
run in triplicate, and various concentrations (20–100
the compounds used to fix concentration at which the
compounds showed 50% of activity. In addition, the percentage of
activity was calculated using the formula, of suppression
lg/mL) of
m
a
435 (LMCT). EPR (300 K, ‘g’ value): 2.60. leff (300 K): 1.69 lB
.
%
2.4.4. Synthesis of [RuCl2(AsPh3)2L2](6)
ratio = [(A0 ꢁ AC)/A0] ꢂ 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.
It was prepared using the same procedure as described for 3
with HL2 (2) (0.138 g, 0.5 mmol) and [RuCl3(AsPh3)3] (0.565 g,
0.5 mmol). Dark red colored crystalline powder was obtained.
Yield: 54%. M.P: 278–280 °C. Anal. calcd. for C45H39Cl2N4O2As2RuS
(%): C, 52.90; H, 3.85; N, 5.48; S, 3.14. Found (%): C, 52.99; H, 3.84;
N, 5.51; S, 3.19. EI-MS: Found m/z = 1021 (M+) (calculated
2.5.4. In vitro cytotoxic activity assay
Cytotoxicity of the compounds were carried out on human
breast cancer cell line (MCF-7) was obtained from National Centre
for Cell Science (NCCS), Pune, India. Cell viability was carried out
using the MTT assay method [24]. MCF-7 cell was 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 attachment at 37 °C, 5% CO2, 95% air
and 100% relative humidity for 24 h prior to the addition of the
compounds. The compounds were dissolved 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 concentrations and
incubated at 37 °C under conditions of 5% CO2, 95% air and 100%
m/z = 1021.70 for M+). IR (KBr, cmꢁ1): 1523
522 (RuAN). UV–vis (DMSO), kmax (nm): 317, 368, 406 (ILCT),
m(C@N); 740 m(CAS);
m
430 (LMCT). EPR (300 K, ‘g’ value): 2.21. leff (300 K): 1.62 lB
.
2.5. Pharmacological properties
2.5.1. DNA binding experiments
All the experiments involving the binding of compounds with
CT-DNA were carried out in a doubly distilled water buffer with
tris(hydroxymethyl)-aminomethane (Tris, 5 mM) and sodium
chloride (50 mM) and adjusted to pH 7.2 with hydrochloric acid.
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

K. Sampath et al. / Journal of Molecular Structure 1046 (2013) 82–91
85
relative humidity for 48 h. Triplication was maintained and the
medium not containing the compounds served as control. After
48 h, 15
L of MTT (5 mg mLꢁ1) in phosphate buffered saline
l
(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 micro plate reader. The % cell inhi-
bition was determined using the following formula and a graph
was plotted with the percentage of cell inhibition versus concen-
tration. From this, the IC50 was calculated: % cell Inhibition = [mean
OD of untreated cells (control)/mean OD of treated cells
(control)] ꢂ 100.
3. Results and discussion
Fig. 1. ORTEP diagram of 1 with thermal ellipsoid at 50% probability.
Analytical and spectroscopic data for the ligands and its com-
plexes indicate a 1:1 metal–ligand stoichiometry for all the com-
plexes. 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, and DMSO.
3.1. X-ray crystallography
The ORTEP representation of compounds 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, bond angles and torsion angles for compounds 1
and 2 are given in Tables 2–4 respectively.
The ligand 1 crystallizes in a monoclinic space group C2/c with
eight crystallographically independent molecules in the unit cell.
Its existence in the thione form is confirmed by the C@S bond
length of 1.6884(14) Å. The molecule exists in the E conformation
about N2AN3 bond as evidenced by the C2AN2AN3AC3 torsion
angle of 175.6(1)°. The azomethine bond length, C3AN3, 1.276(2)
Å is in conformity with a formed C@N double bond length,
1.28 Å. The ligand 2 crystallizes in a triclinic space group P-1 with
two crystallographically independent molecules in the unit cell.
The azomethine bond length, C7AN2, 1.276(1) Å is in conformity
with a formal C@N double bond length, 1.28 Å and C8AS1 bond
distance of 1.683(1) Å is very close to a formal C@S bond length,
1.60 Å. It confirms the existence of the thiosemicarbazone in the
thione form in the solid state. The thione sulfur atom S(1) is trans
to the azomethine nitrogen atom N(2) which is confirmed by a tor-
sion angle of 179.68(8) for S(1)AC(8)AN(3)AN(2) bond.
Fig. 2. ORTEP diagram of 2 with thermal ellipsoid at 50% probability.
frequency of the free ligands 1 and 2 respectively was shifted to
lower frequency in the spectra of the complexes at 1523–
1580 cmꢁ1 indicating that the coordination through N atom [25].
A band appeared at 951 cmꢁ1 for 1 and 850 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 740–747 cmꢁ1
indicating that the other coordination is through thiolate sulfur
after enolization followed by deprotonation on sulfur [26]. In all
the complexes, the medium intensity band in the region 522–
541 cmꢁ1 is attributed to RuAN [27]. Overall, the complexes con-
tain 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.
3.2. Infrared spectra
The IR spectra of the free ligands were compared with those of
the metal complexes in order to study the binding mode(s) of the
thiosemicarbazone ligand to metal and the IR spectrum of the
ligands 1 and 2 is shown in Figs. S1 and S2. A medium sharp band
at 1530 and 1585 cmꢁ1 due to the azomethine C@N stretching
3.3. Electronic spectra
The electronic spectra of the ligands and its complexes were re-
corded in DMSO. The spectra of the free ligands showed bands at
Scheme 1. Synthetic route of the ruthenium(III) thiosemicarbazone complexes, where E = P/As.

86
K. Sampath et al. / Journal of Molecular Structure 1046 (2013) 82–91
Fig. S3. The 1H NMR spectra of the free ligands showed a singlet in
Table 1
Experimental data for crystallographic analyses.
the region d 8.60–8.64 for the hydrazine NH proton [33] whereas
triplet in the region d 8.47–8.48 for the methyl NH proton. The
ACH@N proton displayed a sharp singlet at d 11.67–11.79 in the
spectra of the ligands. The aromatic protons observed in the region
d 7.39–8.43. For ligands, the chemical shift at d 3.03–3.04 is due to
methyl proton. In the 13C NMR spectra of the ligands, the signal at d
142–148 corresponds to the thioamide carbon (C@S) and the signal
at d 178 is due to the azomethine carbon atom. The aromatic car-
bons of the free ligands show signal in the region d 125–138. The
signal due to methyl carbon of ligands appears at d 31.
Compound
1
2
CCDC Number
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
Space group
Unit cell dimensions
a (Å)
893840
C9H10ClN3S
227.71
893999
C9H10N4O2S
238.27
296(2)
0.71073
Triclinic
P-1
296(2)
0.71073
Monoclinic
C2/c
14.3409(11)
8.0487(7)
18.6839(14)
90
90.868(2)
90
7.3403(4)
8.0726(4)
10.1536(5)
87.194(2)
70.544(2)
72.861(2)
541.30(5)
2
b (Å)
c (Å)
3.5. Magnetic moment and EPR spectra
a
(°)
b (°)
c
(°)
The room temperature magnetic susceptibility measurements
of the ruthenium(III) complexes shows that they are paramagnetic.
The magnetic moment value, lB = 1.62–1.69 corresponds to single
Volume (Å3)
2156.4(3)
8
Z
Density (cal.) (mg/m3) 1.403
1.462
unpaired electron in a low-spin 4d5 configuration and confirms
that ruthenium is in +3 oxidation state in all the complexes [27].
All the complexes are uniformly paramagnetic with magnetic
moments corresponding to one unpaired electron at room temper-
ature (low-spin Ru(III), t52g). The X-band EPR spectra of powdered
samples of the complexes were recorded at room temperature
and the EPR spectrum of the complex 4 is shown in Fig. 3. The nat-
ure of the spectra revealed the absence of any hyperfine splitting
due to interactions with any other nuclei present in the complexes.
All the complexes exhibit a single isotropic resonance with g values
in the range 1.95–2.60. Although the complexes have some distor-
tion in their octahedral geometries, the observation of isotropic
lines in the EPR spectra may be due to the occupancy of the un-
paired electron in a degenerate orbital [34]. The nature of spectra
obtained is in good agreement with that of previously reported
ruthenium(III) octahedral complexes [26,24].
Abs. coefficient
(mmꢁ1
F(000)
0.511
0.290
)
944
248
Crystal size (mm3)
h Range for data
collection (°)
0.40 ꢂ 0.35 ꢂ 0.30
0.40 ꢂ 0.30 ꢂ 0.20
2.18–28.29
2.64–28.16
Index ranges
ꢁ18 6 h 6 17
ꢁ10 6 k 6 10
ꢁ24 6 l 6 24
9667
ꢁ9 6 h 6 9,
ꢁ10 6 k 6 10,
ꢁ13 6 l 6 13
8930
Reflections collected
Independent
reflections
2652 [R(int) = 0.0315]
2585 [R(int) = 0.0196]
Absorption correction
Semi-empirical from
equivalents
0.8617 and 0.8216
Semi-empirical from
equivalents
0.9443 and 0.8928
Max. and min.
transmission
Refinement method
Full-matrix least-
squares on F2
2652/0/129
Full-matrix least-
squares on F2
2585/0/146
Data/restraints/
parameters
Goodness-of-fit on F2
Final R indices
1.130
1.028
3.6. Mass spectral analysis
R1 = 0.0333,
wR2 = 0.1010
R1 = 0.0400,
wR2 = 0.1164
R1 = 0.0337,
wR2 = 0.0947
R1 = 0.0377,
wR2 = 0.0992
[I > 2r(I)]
The mass spectrum of the ruthenium(III) complexes is in good
agreement with the proposed molecular structure and the mass
spectrum of the complex 6 is shown in Fig. S4. The molecular ion
peak, [M+] appears at m/z = 923, 1011, 933 and 1021 confirms
the stoichiometry of the complexes 3, 4, 5, and 6, respectively.
R indices (all data)
305 and 360–396 nm are attributed to
p
ꢁ
p⁄ and n ꢁ p⁄ transi-
tions in the aromatic ring, thioamide and imine chromophore
[28]. These peaks have been shifted in the spectra of the complexes
which reveal that the ligands are involved in the coordination with
ruthenium metal ion. The electronic spectra of all the complexes
show three to four bands in the region 310–435 nm. The ground
state of ruthenium(III) (t25g configuration) is 2T2g, while the first ex-
3.7. Pharmacological properties
3.7.1. DNA binding – absorption titration experiments
It is a well-known fact that DNA is the primary pharmacological
target of many antitumor compounds, and hence, the interaction
between DNA and metal complexes are of paramount importance
in understanding the mechanism. The interaction of transition me-
tal complexes with DNA takes place via both covalent and/or non-
covalent interactions. In the case of covalent binding, the labile li-
gand of the complexes is replaced by a nitrogen base of DNA such
as guanine N7 while the non-covalent DNA interactions include
intercalative, electrostatic and groove binding of metal complexes
outside of a DNA helix [35].
Electronic absorption spectroscopy is an effective method to
examine the binding modes of metal complexes with DNA. Com-
pounds binding through intercalation usually results in hypochro-
mism with or without small red or blue shift, since the
intercalative mode involves a strong interaction between the pla-
nar aromatic chromophore and the base pairs of DNA [36]. The re-
sults of absorption spectra of the compounds in the absence and
presence of CT-DNA are given in Fig. 4. Upon increasing the con-
centration of DNA to the test compounds, the absorption bands
of the ligands 1 and 2 exhibits hypochromism of 5.65% and
17.34% with blue shifts of 2 nm at 310 and 280 nm, whereas the
2
cited doublet levels in the order of increasing energy are A2g and
2T1g, which arise from t24ge1g configuration [29]. The lower wave-
length bands are characterized as ligand centered transitions
occurring within the ligand orbitals. Since in a d5 system, and espe-
cially in ruthenium(III) that has relatively high oxidizing proper-
ties, the charge transfer bands of the type
L y ? t2g are
p
prominent in the low energy region obscuring the weaker bands
due to d–d transition. Therefore, it becomes difficult to assign con-
clusively the bands of ruthenium(III) complexes appearing in the
visible region. Hence, all the bands that appear in this region
(404–473 nm) have been assigned to charge transfer transitions,
which are in conformity with the assignments made for similar
ruthenium(III) octahedral complexes [30–32].
3.4. NMR spectra
The NMR spectra of the ligands were recorded in DMSO-d6 solu-
tion and the 1H and 13C NMR spectra of the ligand (2) are shown in

K. Sampath et al. / Journal of Molecular Structure 1046 (2013) 82–91
87
Table 2
Table 4
Selected bond lengths (Å).
Selected torsion angles [°].
C9H10ClN3S (1)
C9H10N4O2S (2)
C9H10ClN3S (1)
C9H10N4O2S (2)
Cl(1)AC(5)
S(1)AC(2)
N(1)AC(2)
N(1)AC(1)
N(1)AH(1)
N(2)AC(2)
N(2)AN(3)
N(2)AH(2)
N(3)AC(3)
C(1)AH(1A)
C(1)AH(1B)
C(1)AH(1C)
C(3)AC(4)
C(3)AH(3)
C(4)AC(5)
C(4)AC(9)
C(5)AC(6)
C(6)AC(7)
C(6)AH(6)
C(7)AC(8)
C(7)AH(7)
C(8)AC(9)
C(8)AH(8)
C(9)AH(9)
1.7357(17)
C(1)AC(2)
C(1)AC(6)
C(1)AH(1)
C(2)AC(3)
C(2)AH(2)
C(3)AC(4)
C(3)AH(3)
C(4)AC(5)
C(4)AH(4)
C(5)AC(6)
C(5)AN(1)
C(6)AC(7)
C(7)AN(2)
C(7)AH(7)
C(8)AN(4)
C(8)AN(3)
C(8)AS(1)
C(9)AN(4)
C(9)AH(9A)
C(9)AH(9B)
C(9)AH(9C)
N(1)AO(1)
N(1)AO(2)
N(2)AN(3)
N(3)AH(3N3)
N(4)AH(4N4)
1.3783(18)
1.3960(17)
0.9300
1.384(2)
0.9300
1.377(2)
0.9300
1.3893(17)
0.9300
1.3999(15)
1.4656(16)
1.4700(15)
1.2761(15)
0.9300
1.3224(16)
1.3572(15)
1.6834(11)
1.4522(16)
0.9600
H(1)AN(1)AC(1)AH(1A) 125.8(2)
C(6)AC(1)AC(2)AC(3)
ꢁ2.0(2)
1.5(2)
0.7(2)
1.6884(14)
1.321(2)
1.4474(19)
0.8600
1.3540(17)
1.3696(17)
0.8600
1.2764(18)
0.9600
0.9600
0.9600
1.4616(19)
0.9300
1.3951(19)
1.396(2)
1.381(2)
1.373(3)
0.9300
H(1)AN(1)AC(1)AH(1C) ꢁ114.1(2) C(1)AC(2)AC(3)AC(4)
C(2)AN(1)AC(1)AH(1B) ꢁ174.2(2) C(2)AC(3)AC(4)AC(5)
H(1)AN(1)AC(2)AS(1)
C(1)AN(1)AC(2)AN(2)
C(2)AN(2)AN(3)AC(3)
H(2)AN(2)AC(2)AN(1)
N(3)AN(2)AC(2)AS(1)
N(2)AN(3)AC(3)AC(4)
N(3)AC(3)AC(4)AC(5)
N(3)AC(3)AC(4)AC(9)
H(3)AC(3)AC(4)AC(5)
H(3)AC(3)AC(4)AC(9)
C(3)AC(4)AC(5)AC(l1)
C(3)AC(4)AC(5)AC(6)
C(9)AC(4)AC(5)AC(l1)
C(3)AC(4)AC(9)AC(8)
C(5)AC(4)AC(9)AH(9)
C(l1)AC(5)AC(6)AC(7)
C(4)AC(5)AC(6)AH(6)
C(5)AC(6)AC(7)AH(7)
H(6)AC(6)AC(7)AC(8)
C(6)AC(7)AC(8)AH(8)
H(7)AC(7)AC(8)AC(9)
C(7)AC(8)AC(9)AH(9)
H(8)AC(8)AC(9)AC(4)
ꢁ177.5(1) C(3)AC(4)AC(5)AC(6)
ꢁ177.4(2) C(3)AC(4)AC(5)AN(1)
ꢁ2.5(2)
174.98(12)
0.33(18)
176.07(12)
1.94(18)
ꢁ175.39(11)
ꢁ173.54(11)
9.13(18)
175.6(1)
179.5(1)
179.6(1)
179.0(1)
171.9(1)
ꢁ8.9(2)
ꢁ8.1(2)
171.1(1)
ꢁ3.3(2)
177.1(1)
177.5(1)
C(2)AC(1)AC(6)AC(5)
C(2)AC(1)AC(6)AC(7)
C(4)AC(5)AC(6)AC(1)
N(1)AC(5)AC(6)AC(1)
C(4)AC(5)AC(6)AC(7)
N(1)AC(5)AC(6)AC(7)
C(1)AC(6)AC(7)AN(2)
C(5)AC(6)AC(7)AN(2)
15.00(17)
ꢁ169.65(11)
C(4)AC(5)AN(1)AO(1) ꢁ148.74(13)
C(6)AC(5)AN(1)AO(1) 28.77(17)
C(4)AC(5)AN(1)AO(2) 28.70(16)
ꢁ176.9(2) C(6)AC(5)AN(1)AO(2) ꢁ153.79(12)
ꢁ177.8(1) C(6)AC(7)AN(2)AN(3) ꢁ176.86(10)
ꢁ179.2(1) N(4)AC(8)AN(3)AN(2) 1.15(17)
1.382(3)
0.9300
1.380(2)
0.9300
0.9600
0.9600
1.2232(16)
1.2256(15)
1.3664(13)
0.8600
ꢁ179.7(2) S(1)AC(8)AN(3)AN(2)
179.68(8)
ꢁ178.8(2) C(7)AN(2)AN(3)AC(8) 178.73(11)
ꢁ178.8(2) N(3)AC(8)AN(4)AC(9) 176.90(12)
179.0(2)
179.0(2)
179.2(2)
179.2(2)
S(1)AC(8)AN(4)AC(9)
ꢁ1.55(19)
0.9300
0.8600
absorption bands of complexes 3 and 4 exhibits a hypochromism
of about 6.23% at 347 nm and 9.20% at 360 nm with blue shifts
of 3 nm, respectively. However, complexes 5 at 359 nm and 6 at
363 nm exhibits a hypochromism of about 6.28% and 5.02% with
red shifts of 4 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 inter-
calation. After the compounds intercalate to the base pairs of DNA,
Table 3
Selected angles (°).
C9H10ClN3S (1)
C9H10N4O2S (2)
C(2)AN(1)AC(1)
C(2)AN(1)AH(1)
C(1)AN(1)AH(1)
C(2)AN(2)AN(3)
C(2)AN(2)AH(2)
N(3)AN(2)AH(2)
C(3)AN(3)AN(2)
N(1)AC(1)AH(1A)
N(1)AC(1)AH(1B)
H(1A)AC(1)AH(1B)
N(1)AC(1)AH(1C)
H(1A)AC(1)AH(1C)
H(1B)AC(1)AH(1C)
N(1)AC(2)AN(2)
N(1)AC(2)AS(1)
N(2)AC(2)AS(1)
N(3)AC(3)AC(4)
N(3)AC(3)AH(3)
C(4)AC(3)AH(3)
C(5)AC(4)AC(9)
C(5)AC(4)AC(3)
C(9)AC(4)AC(3)
C(6)AC(5)AC(4)
C(6)AC(5)ACl(1)
C(4)AC(5)ACl(1)
C(7)AC(6)AC(5)
C(7)AC(6)AH(6)
C(5)AC(6)AH(6)
C(6)AC(7)AC(8)
C(6)AC(7)AH(7)
C(8)AC(7)AH(7)
C(9)AC(8)AC(7)
C(9)AC(8)AH(8)
C(7)AC(8)AH(8)
C(8)AC(9)AC(4)
C(8)AC(9)AH(9)
C(4)AC(9)AH(9)
124.55(14)
117.7
117.7
119.25(12)
120.4
120.4
115.70(12)
109.5
109.5
109.5
109.5
109.5
109.5
116.62(13)
124.28(11)
119.11(11)
120.10(13)
119.9
C(2)AC(1)AC(6)
C(2)AC(1)AH(1)
C(6)AC(1)AH(1)
C(1)AC(2)AC(3)
C(1)AC(2)AH(2)
C(3)AC(2)AH(2)
C(4)AC(3)AC(2)
C(4)AC(3)AH(3)
C(2)AC(3)AH(3)
C(3)AC(4)AC(5)
C(3)AC(4)AH(4)
C(5)AC(4)AH(4)
C(4)AC(5)AC(6)
C(4)AC(5)AN(1)
C(6)AC(5)AN(1)
C(1)AC(6)AC(5)
C(1)AC(6)AC(7)
C(5)AC(6)AC(7)
N(2)AC(7)AC(6)
N(2)AC(7)AH(7)
C(6)AC(7)AH(7)
N(4)AC(8)AN(3)
N(4)AC(8)AS(1)
N(3)AC(8)AS(1)
N(4)AC(9)AH(9A)
N(4)AC(9)AH(9B)
H(9A)AC(9)AH(9B)
N(4)AC(9)AH(9C)
H(9A)AC(9)AH(9C)
H(9B)AC(9)AH(9C)
O(1)AN(1)AO(2)
O(1)AN(1)AC(5)
O(2)AN(1)AC(5)
C(7)AN(2)AN(3)
C(8)AN(3)AN(2)
C(8)AN(3)AH(3N3)
N(2)AN(3)AH(3N3)
C(8)AN(4)AC(9)
C(8)AN(4)AH(4N4)
C(9)AN(4)AH(4N4)
121.93(11)
119.0
119.0
120.31(12)
119.8
119.8
119.88(12)
120.1
120.1
119.00(12)
120.5
120.5
122.81(11)
115.88(10)
121.27(10)
116.01(11)
119.20(10)
124.64(11)
118.24(10)
120.9
the p⁄ orbital of the intercalated compounds could couple with
p
orbitals of the base pairs, thus decreasing the
p
?
p⁄ transition
energies, hence resulting in hypochromism [37]. In order to com-
pare quantitatively the binding strength of the compounds, the
intrinsic binding constants (Kb) of them with CT-DNA were deter-
mined from the following equation.
½DNAꢃ=ðea
ꢁ
ef Þ ¼ ½DNAꢃ=ðeb
ꢁ
ef Þ þ 1=Kbðeb
ꢁ
ef
Þ
where [DNA] is the concentration of DNA in base pairs and the appar-
ent absorption coefficient ea ef and eb correspond to Aobs/[com-
,
pound], the extinction coefficient of the free compound and the
extinction coefficient of the compound when fully bound to DNA,
119.9
117.43(13)
121.13(13)
121.44(13)
121.83(15)
117.85(12)
120.32(12)
119.39(16)
120.3
120.3
120.28(16)
119.9
119.9
120.15(18)
119.9
120.9
116.84(10)
123.79(9)
119.36(9)
109.5
109.5
109.5
109.5
109.5
109.5
123.29(12)
118.67(11)
117.98(12)
116.73(10)
119.28(10)
120.4
120.4
123.48(11)
118.3
119.9
120.88(16)
119.6
119.6
118.3
Fig. 3. EPR spectrum of the complex 4.

88
K. Sampath et al. / Journal of Molecular Structure 1046 (2013) 82–91
respectively. The plot of [DNA]/(ea
and the intercept which are equal to 1/(eb
respectively; Kb is the ratio of the slope to the intercept. The magni-
ꢁ
e
f) versus [DNA] gave a slope
stretching), PO2 stretching vibrations and deoxyribose stretching of
DNA backbone are confined in the spectral region 1800–700 cmꢁ1
ꢁ
e
f) and 1/Kb
(eb
ꢁ
ef),
.
Therefore, this particular region is of interest here. The vibrational
bands of DNA at 1711, 1660, 1611 and 1494 cmꢁ1 are assigned to
guanine (G), thymine (T), adenine (A) and cytosine (C) nitrogenous
bases, respectively [38,39]. Bands at 1225 and 1084 cmꢁ1 denote
phosphate stretching vibrations. These are the prominent bands
of pure DNA, which are monitored during compound–DNA interac-
tudes of intrinsic binding constants (Kb) were calculated to be
1.7( 0.04) ꢂ 104 Mꢁ1, 4.6( 0.08) ꢂ 103 Mꢁ1, 1.2( 0.14) ꢂ 106 Mꢁ1
,
5.6( 0.09) ꢂ 105 Mꢁ1
,
3.9( 0.12) ꢂ 104 Mꢁ1 and 1.6( 0.11)
ꢂ 104 Mꢁ1 for compounds 1, 2, 3, 4, 5 and 6 respectively. The ob-
served values of Kb reveals that the ruthenium(III) complexes (3–6)
bind strongly than the respective ligands (1 and 2) to DNA via inter-
calative mode. From the results obtained, it has been found that com-
plex 3 strongly bound with CT-DNA relative to that with 4, 5 and 6,
and the order of binding affinity is 3 > 4 > 5 > 6 > 1 > 2.
tion ([Compound] = 25 lM; [DNA] = 30 lM) in this study. Changes
in these bands (shifting and intensity) of free DNA (A), upon inter-
action with ligand 1 (B) and complex 4 (C) are shown in Fig. 5. After
compounds, 1–6, addition to DNA solution, guanine band at 1711
shifts to 1712–1715 cmꢁ1
, thymine band at 1660 shifts to
1665 cmꢁ1 and adenine band at 1611 shifts towards lower wave
number 1608–10 cmꢁ1. These shifting can be attributed to direct
binding to guanine (N7), thymine (O2) and adenine (N7) of DNA
bases. No major shifting is observed for phosphate asymmetric
and symmetric vibrations. Hence, it is inferred that the compounds
3.7.2. DNA binding – FTIR analysis
It is well known in the literature that the study of the binding
between DNA and various compounds is studied with IR spectros-
copy in order to confirm where the ligand binds (DNA bases, phos-
phate backbone). Ring vibrations of nitrogenous bases (C@O, C@N
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 lM, [DNA] = 0–50 lM. Arrow
shows the absorption intensities decrease upon increasing DNA concentrations (inset: plot between [DNA] and [DNA]/[ea
ꢁ
e
f] ꢂ 10ꢁ8).

K. Sampath et al. / Journal of Molecular Structure 1046 (2013) 82–91
89
are bind to CT-DNA through nitrogenous bases rather than phos-
phate backbone [40].
3.7.3. DNA cleavage study by gel electrophoresis
Suitably designed metal complexes, after binding to DNA, can
induce several changes in the DNA conformation, such as bending,
local denaturation, intercalation, micro loop formation and subse-
quent DNA shorting lead to decrease in molecular weight of DNA
[41]. 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. 6 and 7 respectively. Fig. 6 shows the hydrolytic cleavage
Fig. 6. Gel electrophoresis diagram showing the hydrolytic cleavage of CT-DNA
(30 lM) incubated at 37 °C for a period of 2 h at a concentration of 40 lM of
complex 3–6 in 5% DMSO and 95% 5 mM Tris–HCl/50 mM NaCl buffer at pH 7.2.
activity of the complexes, 3–6 at 40 lM. A control experiment
using DNA alone does not show any significant cleavage of DNA
(Lane 1). Further, when DNA is allowed to interact with the com-
plexes, no considerable difference in the intensity of the bands
for metal bound DNA as compared to control DNA was observed.
This result suggests that the nuclease activity of the complexes
does not involve by hydrolytic pathway. These experimental facts
demonstrated that a combination of both the ruthenium com-
plexes and activating agent, H2O2 are required to show effective
cleavage of CT-DNA. Fig. 7 reveals that the oxidative cleavage of
CT-DNA induced by complexes, 3–6 in the presence of H2O2. Con-
trol experiment using DNA alone (Lanes 1 and 9) and DNA with
H2O2 (Lanes 2 and 10) does not show any significant cleavage of
CT-DNA even on longer exposure time. When the DNA is allowed
to interact with the complexes (3–6) at various concentrations
Lane 1: control, Lanes 2–5: DNA + complexes 3–6, respectively.
complex, 3 shows potential nuclease activity against DNA at low
concentration, 30 lM itself, indicates a superior DNA cleavage than
the other complexes. The cleavage activity may be due to the inter-
calative interaction of the complexes with the DNA strands in the
presence of H2O2 induces oxidative DNA cleavage [42].
3.7.4. Antioxidant activity
Since the DNA interaction experiments conducted so far reveals
that the compounds exhibit good DNA binding affinity and good
cleavage activity, it is considered worthwhile to study the antiox-
idant activity of these compounds. Hence, we carried out experi-
ments to explore the free radical scavenging activity of the
compounds, with the hope of developing potential antioxidants
and therapeutic reagents for respiratory diseases, such as asthma,
emphysema and asbestosis [43]. 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
(30, 60 and 90 lM), a substantial decrease in the intensities of
the bands for the metal bound DNA as compared to untreated con-
trol DNA was observed, which suggests the cleavage of DNA by me-
tal complexes [42]. As can be seen from Lanes 5, 8, 13 and 16 of
complexes 3, 4, 5 and 6 respectively, the bands were completely
disappeared, indicating the sufficient cleavage of DNA by ruthe-
nium(III) complexes at 90 lM concentration. Interestingly, the
Fig. 7. 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. Lanes 1 and 9: DNA. Lanes 2 and 10: DNA + H2O2. Lanes 3–5: 3 (30, 60
lM) 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
Fig. 5. Stacked view of FT-IR spectra of DNA, free CT-DNA (A) and upon interaction
with ligand (B) and complex (C), respectively, ([Compound] = 25 M;
[DNA] = 30
M) in the region of 1800–700 cmꢁ1
and 90
11–13:
90 lM) + DNA + H2O2.
l
5
M) + DNA + H2O2. Lanes 6–8: 4 (30, 60 and 90
l
M) + DNA + H2O2. Lanes
(30, 60 and
1
4
l
(30, 60 and 90 M) + DNA + H2O2. Lanes 14–16: 6
l
l
.

90
K. Sampath et al. / Journal of Molecular Structure 1046 (2013) 82–91
becomes paired off in the presence of a free radical scavenger, this
absorption vanishes, and the resulting decolorization is stoichiom-
etric with respect to the number of electrons taken up. The DPPH
assay of the tested compounds is shown in Fig. 8, it is seen from
the results that all the complexes exhibited moderate activity com-
pared to the standard Ascorbic acid (Aca). The IC50 values indicated
that the compounds showed antioxidant activity in the order of
3 > 4 > 5 ꢄ 6 > 1 > 2. Complex 3 showed a higher antioxidant activ-
ity followed by 4, whereas 5 and 6 exhibited similar antioxidant
activity. From the above results, the scavenging effect of the free
ligands is significantly less when compared to that of their corre-
sponding ruthenium(III) complexes. This might be due to the d5
low spin electronic configuration and the availability of an odd
electron in ruthenium(III) complexes, which increases the capacity
to stabilize the unpaired electrons and thereby scavenge the free
radicals [43].
Fig. 9. % Growth inhibition of MCF-7 cell line as a function of concentration of the
compounds 1–6.
3.7.5. In vitro anticancer activity evaluation
The effect of ligands and ruthenium(III) complexes was exam-
ined on human breast cancer cell line, MCF-7 using MTT assay, a
colorimetric determination of cell viability during in vitro treat-
ment with drug. The assay, developed as an initial stage of drug
screening, measures the amount of MTT reduction by mitochon-
drial dehydrogenase and assumes that cell viability (corresponding
to the reductive activity) is proportional to the production of pur-
ple formazan that is measured spectrophotometrically [44]. A low
IC50 is desired and implies cytotoxicity or proliferation at low drug
concentration. The activity that corresponds to the inhibition of
cancer cell growth at a maximum level is shown in Fig. 9 and Table
5. Upon increasing the concentration of complexes, the results of
MTT assays reveal that the complex 3 showed a higher cytotoxic
effect followed by 4. Complexes, 5 and 6 showed significant IC50
values but the cytotoxic effect of these complexes are less when
compared to the other two complexes 3 and 4. It is to be noted that
Table 5
Cytotoxic activity of the ligands and ruthenium(III)
complexes against the selected cancer line, MCF-7.
Compounds
IC50 (lM)
1
2
3
4
5
6
>100
>100
9.50
18.54
75.64
85.50
bation period (48 h) and hence the data are highly significant. The
findings of the in vitro cytotoxic activities confirm the binding of
the complexes to DNA, which consequently leads to cell death.
the ligands does not show any significant activity (IC50 > 100 lM),
which confirmed that the chelation of the ligands with the ruthe-
nium(III) ion is the only responsible factor for the observed cyto-
toxic property of the complexes. Comparison of the anticancer
activity of the ruthenium(III) complexes reveal that upon changing
the thiosemicarbazone moieties and ancillary ligands the activity
of the complexes gets varied. The complexes of chlorobenzalde-
hyde thiosemicarbazone moiety exhibited higher cytotoxic effects
than the complexes of nitrobenzaldehyde thiosemicarbazone.
Moreover, the complexes containing triphenylphosphine as a co-li-
gand showed better cytotoxic effects than the complexes contain-
ing triphenylarsine. For clinical purposes, the compounds that
show cytotoxicity in shorter time periods are preferred. The data
obtained for our compounds showed cytotoxicity with short incu-
4. Conclusion
Four ruthenium(III) complexes bearing NS-thiosemicarbazone
ligands and the PPh3/AsPh3 co-ligands are synthesized and charac-
terized by various spectroscopic techniques. Thiosemicarbazone li-
gands have also been characterized by crystallographically. An
octahedral geometry has been tentatively assigned for all the com-
plexes. All the newly synthesized compounds have been subjected
to examine their biological property like DNA binding, DNA cleav-
age, antioxidant and cytotoxicity under in vitro experimental condi-
tions. The DNA binding ability of the ligands and complexes
assessed by absorption spectra suggests an intercalative mode of
binding with binding constants ranging from 103 to 106 Mꢁ1. The
DNA cleavage capabilities of complexes in the presence of H2O2 re-
veal their potential nuclease activity to cleave CT-DNA. The com-
plex 3 shows superior cleavage activity at low concentration than
the other complexes. The antioxidant activity shows that all the
ruthenium(III) complexes can serve as potential antioxidants than
the ligands against DPPH radical in the order 3 > 4 > 5 ꢄ 6 > 1 > 2.
In addition, the in vitro cytotoxicity of the complexes possesses sig-
nificant activity against MCF-7 cell line. Complex 3 exhibits good
cytotoxic effect than the other complexes and ligands. At this junc-
ture, it is notable to mention that the major chemical and biological
findings of this study throw some light on the potential of these
complexes in a reasonable range of concentrations under in vitro
conditions. The results of biological studies revealed that the elec-
tron donating ability of the substituent, chlorine on the thiosemi-
carbazone ligands in the complexes increases the biological
activity. In addition, the liphophilic effect of triphenylphosphine
in the complexes could enhance the biological activity.
Fig. 8. Antioxidant activity of compounds.

K. Sampath et al. / Journal of Molecular Structure 1046 (2013) 82–91
91
[14] R.N. Prabhu, D. Pandiarajan, R. Ramesh, J. Organomet. Chem. 694 (2009) 4170.
[15] J.S. Casas, M.S. Garcia-Tasende, J. Sordo, Coord. Chem. Rev. 209 (2000) 197.
[16] A.I. Vogel, Textbook of Practical Organic Chemistry, Longman, London, 1989.
[17] J. Chatt, G.J. Leigh, D.M.P. Mingos, R.J. Paske, J. Chem. Soc. A (1968) 2636.
[18] P. Viswanathamurthi, K. Natarajan, Ind. J. Chem. 38A (1999) 797.
[19] Siemens, SMART, SAINT, Area Detector Control and Integration software,
Siemens Analytical X-ray Instruments Inc., Madison, WI, USA, 1996.
[20] G.M. Sheldrick, SADABS, Program for Empirical Absorption Correction of Area
Detector Data, University of Gottingen, Germany, 1997.
Acknowledgement
Financial assistance received from the Council of Scientific and
Industrial Research, New Delhi, India [Grant No. 01(2308)/09/EMR-
II] is gratefully acknowledged.
Appendix A. Supplementary material
[21] G.M. Sheldrick, SHELXS-97, Program for the Solution of Crystal Structures,
University of Gottingen, Gottingen, Germany, 1997.
CCDC 893840 and 893999 contains the crystallographic data for
compounds 1 and 2 respectively. These data can be obtained free of
charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html, or
from the Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336 033; or e-mail: depos-
it@ccdc.cam.ac.uk. Supplementary data associated with this article
can be found, in the online version, at http://dx.doi.org/10.1016/
j.molstruc.2013.04.051.
[22] G.M. Sheldrick, Acta Crystallogr. A 46 (1990) 467.
[23] K. Elizabeth, M.N.A. Rao, Int. J. Pharmaceut. 58 (1990) 237.
[24] T. Mossman, J. Immnol. Methods 65 (1983) 55.
[25] N. Raja, R. Ramesh, Spectrochem. Acta A 75 (2010) 713.
[26] T.D. Thangadurai, K. Natarajan, Trans. Met. Chem. 26 (2001) 717.
[27] S. Manivannan, R. Prabhakaran, K.P. Balasubramanian, V. Dhanabal, R.
Karvembu, V. Chinnusamy, K. Natarajan, Appl. Organometal. Chem. 21
(2007) 952.
[28] R.K. Sharma, R.V. Singh, J.P.J. Tandon, Inorg. Nucl. Chem. 42 (1980) 1382.
[29] C.J. Ballhausen, Ligand Field Theory, McGraw Hill, New York, 1962.
[30] T.D. Thangadurai, K. Natarajan, Ind. J. Chem. 40A (2001) 573.
[31] C. Jayabalakrishnan, R. Karvembu, K. Natarajan, Synth. React. Inorg. Met. Org.
Chem. 32 (2002) 1101.
References
[32] A.K. Das, S.-M. Perg, S. Bhattarcharya, J. Chem. Soc. Dalton Trans. (2000) 181.
[33] P. Sengupta, R. Dinda, S. Ghosh, W.S. Sheldrick, Polyhedron 22 (2003) 447.
[34] M.M.T. Khan, D. Srinivas, R.I. Kureshy, N.H. Khan, Inorg. Chem. 29 (1990) 2320.
[35] Q.L. Zhang, J.G. Liu, H. Chao, G.Q. Xue, L.N. Ji, J. Inorg. Biochem. 83 (2001) 49.
[36] Z.-C. Liu, B.-D. Wang, B. Li, Q. Wang, Z.-Y. Yang, T.-R. Li, Y. Li, Eur. J. Med. Chem.
45 (2010) 5353.
[37] D.S. Raja, N.S.P. Bhuvanesh, K. Natarajan, Eur. J. Med. Chem. 46 (2011) 4584.
[38] A. Ahmed Ouameur, H.A. Tajmir-Riahi, J. Biol. Chem. 279 (2004) 42041.
[39] V.V. Andrushchenko, Z. Leonenko, H. Van de Sande, H. Wieser, Biopolymers 61
(2002) 243.
[40] D.K. Jangir, G. Tyagi, R. Mehrotra, S. Kundu, J. Mol. Struct. 969 (2010) 126.
[41] N.V. Kulkarni, A. Kamath, S. Budagumpi, V.K. Revankar, J. Mol. Struct. 1006
(2011) 580.
[42] A.H. Pathan, R.P. Bakale, G.N. Naik, C.S. Frampton, K.B. Gudasi, Polyhedron 34
(2012) 149.
[1] L.R. Kelland, Drugs 4 (2000) 1.
[2] M. Kartalou, J.M. Essigmann, Mutat. Res. 478 (2001) 23.
[3] Z.H. Siddik, Oncogene 22 (2003) 7265.
[4] P.C. Bruijninex, P.J. Sadler, Curr. Opin. Chem. Biol. 12 (2008) 197.
[5] G. Sava, I. Capozzi, A. Bergamo, R. Gagliardi, M. Cocchietto, L. Masiero, M.
Onisto, E. Alessio, G. Mestroni, S. Garbisa, Int. J. Cancer 68 (1996) 60.
[6] M. Galanski, V.B. Arion, M.A. Jakupec, B.K. Keppler, Curr. Pharm. Des. 9 (2003)
2078.
[7] H. Beraldo, D. Gambino, Med. Chem. 4 (2004) 31.
[8] F.A. Beckford, J. Thessing, A. Stott, A.A. Holder, O.G. Poluektov, L. Li, N.P.
Seeram, Inorg. Chem. Commun. 15 (2012) 225.
[9] D.K. Dermertzi, M.A. Demertzis, J.R. Miller, C.S. Frampton, J.P. Jasinski, D.X.
West, J. Inorg. Biochem. 92 (2002) 137.
[10] K.E. Erkkila, D.T. Odon, J.K. Barton, Chem. Rev. 99 (1999) 2777.
[11] A. Chouai, S.E. Wicke, C. Turro, J. Bacsa, K.R. Dunbar, D. Wang, R.P. Thummel,
Inorg. Chem. 44 (2005) 5996.
[43] T.S. Kamatchi, N. Chitrapriya, H. Lee, C.F. Fronczek, F.R. Fronczek, K. Natarajan,
Dalton Trans. 41 (2012) 2066.
[44] V. Moreno, M.F. Bardia, T. Calvet, J. Lorenzo, F.X. Aviles, M.H. Garcia, T.S.
Morais, A. Valente, M.P. Robalo, J. Inorg. Biochem. 105 (2011) 241.
[12] J. Hooda, D. Bednar ski, L. Irish, S.M. Firestine, Bioorg. Med. Chem. 14 (2006)
1902.
[13] V. Uma, V.G. Vaidyanathan, B.U. Nair, Bull. Chem. Soc. Jpn. 78 (2005) 845.