DNA interaction, antioxidant, and in vitro antitumor activity of binuclear ruthenium(III) complexes of benzothiazole-substituted ferrocenyl thiosemicarbazones

Article abstract of DOI:10.1007/s00044-013-0698-x

In our search for new anticancer drugs, a series of binuclear ruthenium(III) thiosemicarbazone complexes of the type [RuCl ₂(EPh₃)]₂L (where E = P/As; L = binucleating monobasic tridentate thiosemicarbazone ligand) have been synthesized. Structural features were determined by various physico-chemical and spectral techniques. The interactions of these complexes with CT-DNA were investigated by absorption spectral study, indicates that the binuclear ruthenium(III) complexes form adducts with DNA and has intrinsic binding constant in the range of 1.0 × 10⁴-7.9 × 10⁴ M^-1. The free radical scavenging activity of binuclear ruthenium(III) complexes have been determined by their interaction with the stable DPPH free radical. All the complexes exhibited significant antiproliferative activity against human breast cancer line, MCF-7. This research may provide knowledge that is an excellent backdrop for the rational design of promising drugs.

Full text of DOI:10.1007/s00044-013-0698-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0698-x
ORIGINAL RESEARCH
DNA interaction, antioxidant, and in vitro antitumor activity
of binuclear ruthenium(III) complexes of benzothiazole-
substituted ferrocenyl thiosemicarbazones
•
Krishnan Sampath Subbaiyan Sathiyaraj
•
Chinnasamy Jayabalakrishnan
Received: 14 December 2012 / Accepted: 8 August 2013
Ó Springer Science+Business Media New York 2013
Abstract In our search for new anticancer drugs, a series
of binuclear ruthenium(III) thiosemicarbazone complexes
of the type [RuCl2(EPh3)]2L (where E = P/As; L = binu-
cleating monobasic tridentate thiosemicarbazone ligand)
have been synthesized. Structural features were determined
by various physico-chemical and spectral techniques. The
interactions of these complexes with CT-DNA were inves-
tigated by absorption spectral study, indicates that the
binuclear ruthenium(III) complexes form adducts with DNA
and has intrinsic binding constant in the range of 1.0 9 104–
7.9 9 104 M-1. The free radical scavenging activity of
binuclear ruthenium(III) complexes have been determined
by their interaction with the stable DPPH free radical. All
the complexes exhibited significant antiproliferative activity
against human breast cancer line, MCF-7. This research may
provide knowledge that is an excellent backdrop for the
rational design of promising drugs.
the use of current platinum drugs, there are some major
problems associated with the side effects, such as nausea,
kidney failure etc., typical of heavy metal toxicity, in
addition to the major problem of resistance (Wang and
Lippard, 2005; Angeles-Boza et al., 2004). Hence, attempts
are being made to replace these drugs with suitable alter-
natives, and in this direction, a number of non platinum
complexes have been synthesized and screened for their
potential anticancer activity. In the field of non platinum
compounds exhibiting anticancer properties, ruthenium
complexes find very promising alternative to platinum,
showing activity on tumors which developed resistance to
cisplatin or in which cisplatin is inactive (Kostova, 2006);
thus indicating that it may be a strong candidate to form a
basis for rational anticancer drug design (Clarke et al.,
1999). The binuclear ruthenium complexes have been
found to have better biological activity than mononuclear
complexes (Manimaran and Jayabalakrishnan, 2010).
Moreover, bimetallic coordination complexes have
numerous applications, such as in the treatment of cancer
(Wang et al., 2001), as antiviral agents (Tarasconi et al.,
2000), and for other biological properties. Interestingly,
two ruthenium(III) complexes, NAMI-A and KP1019, have
successfully completed clinical trials for the treatment of
metastatic tumors increased interest in this metal (Bergamo
et al., 2002; Hartinger et al., 2006).
Keywords Binuclear ruthenium(III) complexes ꢀ
Benzothiazolyl thiosemicarbazide ꢀ DNA binding ꢀ
DPPH ꢀ MCF-7
Introduction
Though many cisplatin analogs have been screened as
potential antitumor agents, in addition to cisplatin, only
carboplatin and oxaliplatin have entered into clinical use
(Kelland et al., 1999). In spite of the achievements made in
Antioxidants have become one of the major areas of
research. Antioxidants are extensively studied for their
ability to protect organism and cell from damage that are
induced by free radicals. Thiosemicarbazones are versatile
ligands, bearing suitable donor atoms for coordination to
metals. The transition metal complexes of thiosemicarba-
zones are of the considerable pharmacological interest since
their derivatives have shown a broad spectrum of chemo-
therapeutic properties such as antitumor, antioxidative
K. Sampath ꢀ S. Sathiyaraj ꢀ C. Jayabalakrishnan (&)
Post-Graduate and Research Department of Chemistry, Sri
Ramakrishna Mission Vidyalaya College of Arts and Science,
Coimbatore 641 020, Tamil Nadu, India
e-mail: drcjbstar@gmail.com
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Med Chem Res
Synthesis of thiosemicarbazone ligands
activity, etc. (Singh et al., 2012; Prabhakaran et al., 2011a,
b). Benzothiazolyl thiosemicarbazones represent a very
interesting class of compounds because of their wide
applications in antibacterial, antifungal, and antimalarial
(Pandeya et al., 1999). On the other hand, the potential to
use ferrocenyl compounds as medicals and chemothera-
peutic agents for the treatment of cancer has attracted many
authors in the last two decades (Liu et al., 2003; Top et al.,
2001). Ferrocenyl thiosemicarbazone complexes were
found to be active against protozoa, tumors, and fungi
(Petering et al., 1964). Despite the wealth of information
available on this topic, only very few reports are available
on the related ruthenium complexes. Hence, in view of the
aforementioned facts, the present article embodies the
various spectroscopic characterization binuclear ruthe-
nium(III) complexes containing benzothiazole-substituted
ferrocenyl thiosemicarbazone ligands. Further, the biolog-
ical experiments on DNA binding, antioxidant, and cyto-
toxicity studies of all the synthesized complexes were also
carried out.
To a methanolic solution (30 mL) of 6-nitro/H-2-benzo-
thiazolyl thiosemicarbazide (20 mmol), diacetylferrocene
(10 mmol) in methanol (30 mL) was added and stirred
along with a few drops of glacial acetic acid. The mixture
was then refluxed for about 8 h. The resultant product was
washed with methanol, and the purity of the ligands was
checked by TLC. The outline of synthesis of thiosemicar-
bazone ligands is given in Scheme 1.
H2L1
Yield 82 %; color: dark brown; M.P.: 298 °C. Anal. calc
for C30H24N10S4FeO4: C, 46.63; H, 3.13; N, 18.13; S,
16.60. Found: C, 46.62; H, 3.10; N, 18.23; S, 16.69. IR
(cm-1): 1,649 (C=N), 826 (C=S), 1,511 (C=N thiazole
1
ring). UV–Vis (in DMSO); kmax, nm; 308, 368, 430. H
NMR (ppm); 7.39–8.23 (m, Ar–H), 2.28 (s, CH3), 8.67 (s,
NH), 11.3 (s, SH), 4.59–4.79 (m, Cp ring-H). 13C NMR
(ppm); 117–132 (Ar, C), 27 (CH3), 141 (C–S), 159 (C=N
imine), 165 (thiazole C=N), 71–81 (C, Cp ring).
Experimental
H2L2
Materials and methods
Yield 79 %; color: black; M.P.: 289 °C. Anal. calc for
C30H26N8S4Fe: C, 52.78; H, 3.84; N, 16.41; S, 18.79. Found:
All the chemicals used were chemically pure and AR
grade. Solvents were purified and dried according to the
standard procedure (Vogel, 1989). The metal precursors
[RuCl3(PPh3)3] and [RuCl3(AsPh3)3] were prepared by
literature methods (Chatt et al., 1968; Thangadurai and
Natarajan, 2001). Calf-thymus DNA (CT-DNA) was pur-
chased from Bangalore Genei, Bangalore, India. Microa-
nalyses (C, H, N, & S) were performed 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 400–4,000 cm-1 region using a Perkin
Elmer FT IR 8000 spectrophotometer. Electronic spectra
were recorded in DMSO solution with a Systronics double
beam UV–Vis spectrophotometer 2202 in the range
CH3
C
O
H
H
N
S
N
C
S
+
NH2
Fe
N
2
R
H3C
O
Reflux, 8 h
Methanol
H3C
C
N
R
SH
C
N
N
S
N
1
200–800 nm. H NMR and 13C NMR spectra were recor-
Fe
H
H
N
ded on a Bruker AV III 500 MHZ instrument using TMS as
an internal reference. Magnetic susceptibility measure-
ments of the complexes were recorded using Guoy balance.
EPR spectra were recorded on a varian E-112 ESR spec-
trophotometer at X-band microwave frequencies for pow-
dered samples at room temperature. EI mass spectrum of
the complex was recorded on a JEOL GCMATE II mass
spectrometer. Antioxidant and Anticancer studies were
carried out at the Kovai Medical Centre and Hospital
Pharmacy College, Coimbatore, Tamil Nadu. Melting
points were recorded with Veego VMP-DS heating table
and were uncorrected.
S
N
C
N
HS
N
C
R
CH3
Abbreviation
H2L1
R
NO2
H
H2L2
Scheme 1 The outline of synthesis of binucleating thiosemicarba-
zone ligands
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Med Chem Res
C, 52.70; H, 3.82; N, 16.42; S, 18.89. IR (cm-1): 1,631 (C=N),
S, 7.82. Found: C, 48.31; H, 3.25; N, 8.52; S, 7.90. EI-MS:
Found m/z = 1,639 (M?) (calculated m/z = 1,639.19 for
M?). IR (cm-1): 1,595 (C=N), 747 (C–S), 1,436 (C=N
thiazole ring), 527 (Ru–N), 424 (Ru–S). UV–Vis (in
DMSO); kmax, nm; 305, 368, 432, 471. EPR (300 K, ‘g’
value): 2.00. leff (300 K): 1.61 lB.
807 (C=S), 1,543 (C=N thiazole ring). UV–Vis (in DMSO);
1
max, nm; 305, 368, 428. H NMR (ppm); 7.41–8.23 (m, Ar–
k
H), 2.29 (s, CH3), 8.68 (s, NH), 11.9 (s, SH), 4.60–4.80 (m, Cp
ring-H). 13C NMR (ppm); 119–131 (Ar, C), 27 (CH3), 140 (C–
S), 157 (C=N imine), 165 (thiazole C=N), 71–81 (C, Cp ring).
Synthesis of binuclear ruthenium(III)
thiosemicarbazone complexes
{[RuCl2(AsPh3)]2L1}
Yield 64 %; color: black; M.P.: [300 °C. Anal. calc for
C66H52N10Ru2FeO4As2S4Cl4: C, 45.90; H, 3.03; N, 8.11;
S, 7.43. Found: C, 45.94; H, 3.09; N, 8.19; S, 7.47. EI-MS:
Found m/z = 1,727 (M?) (calculated m/z = 1,727.08 for
M?). IR (cm-1): 1,581 (C=N), 740 (C–S), 1,483 (C=N
thiazole ring), 559 (Ru–N), 474 (Ru–S). UV–Vis (in
DMSO); kmax, nm; 308, 368, 430, 471. EPR (300 K, ‘g’
value): 1.94. leff (300 K): 1.64 lB.
All the reactions were carried out under anhydrous condition.
The monobasic tridentate thiosemicarbazones (0.1 mmol)
were added to a solution of [RuCl3(EPh3)3] (0.1 mmol),
(E = P/As) in 1:2 molar ratio in benzene–methanol/tetra-
hydrofuran (50 mL) mixture, and then refluxed for 10 h. The
resulting solution was concentrated to about 3 mL and the
complexes were precipitated by the addition of small quan-
tity of petroleum ether (60–80 °C). The complexes were then
filtered off, washed with petroleum ether, and recrystallized
from tetrahydrofuran/CH2Cl2/petroleum ether and dried
under vacuum. Schematic route for synthesis of binuclear
ruthenium(III) thiosemicarbazone complexes is given in
Scheme 2.
{[RuCl2(PPh3)]2L2}
Yield 59 %; color: brown; M.P.: [ 300 °C. Anal. calc for
C66H54N8Ru2FeP2S4Cl4: C, 51.17; H, 3.51; N, 7.23; S,
8.28. Found: C, 51.18; H, 3.55; N, 7.25; S, 8.21. EI-MS:
Found m/z = 1,549 (M?) (calculated m/z = 1,549.19 for
M?). IR (cm-1): 1,613 (C=N), 749 (C–S), 1,471 (C=N
thiazole ring), 540 (Ru–N), 424 (Ru–S). UV–Vis (in
DMSO); kmax, nm; 308, 366, 430, 470. EPR (300 K, ‘g’
value): 1.95. leff (300 K): 1.65 lB.
{[RuCl2(PPh3)]2L1}
Yield 60 %; color: dark brown; M.P.:[300 °C. Anal. calc
for C66H52N10Ru2FeO4P2S4Cl4: C, 48.36; H, 3.20; N, 8.54;
{[RuCl2(AsPh3)]2L2}
H3C
C
R
N
SH
C
N
Yield 62 %; color: dark brown; M.P.:[300 °C. Anal. calc
for C66H54N8Ru2FeAs2S4Cl4: C, 48.42; H, 3.32; N, 6.84; S,
7.83. Found: C, 48.45; H, 3.33; N, 6.84; S, 7.83. EI-MS:
Found m/z = 1,637 (M?) (calculated m/z = 1,637.09 for
M?). IR (cm-1): 1,616 (C=N), 741 (C–S), 1,457 (C=N
thiazole ring), 544 (Ru–N), 424 (Ru–S). UV–Vis (in
DMSO); kmax, nm; 305, 368, 435, 466. EPR (300 K, ‘g’
value): 2.11. leff (300 K): 1.62 lB.
N
S
N
Fe
H
H
N
S
N
C
N
HS
N
C
R
CH3
Benzene-Methanol/THF
reflux, 10 h
2 [RuCl3(EPh3)3]
EPh3
DNA binding: titration experiments
Cl
Cl
Ru
H3C
C
All the experiments involving the binding of complexes
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 sufficiently
free of protein. The CT-DNA concentration per nucleotide
was determined spectrophotometrically by employing an
extinction coefficient of 6,600 M-1 cm-1 at 260 nm. The
complexes were dissolved in a mixed solvent of 5 %
R
N
S
C
N
N
S
N
Fe
H
H
N
S
N
C
S
N
N
C
R
CH3
Ru
Cl
Cl
EPh3
Scheme 2 Schematic route for synthesis of binuclear ruthenium(III)
thiosemicarbazone complexes
123

Med Chem Res
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 concentrations of
the complexes (25 lM) with varying concentration of
DNA (0–40 lM). While measuring the absorption spectra,
an equal amount of DNA was added to both the test
solution and the reference solution to eliminate the absor-
bance of DNA itself.
buffered saline 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 inhibition was deter-
mined using the following formula and a graph was plotted
with the percentage of cell inhibition versus concentration.
From this, the IC50 was calculated: % cell inhibi-
tion = [mean OD of untreated cells (control)/mean OD of
treated cells (control)] 9 100.
Antioxidant activity
The 2,2-diphenyl-2-picryl-hydrazyl (DPPH) radical scav-
enging activity of the complexes was measured according
to the literature method (Elizabeth and Rao, 1990). The
DPPH radical is a stable free radical having a kmax at
517 nm. A fixed concentration of the experimental com-
plex (100 lL) was added to a solution of DPPH in meth-
anol (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 solution was incubated at 37 °C
for 30 min in the 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 complexes used to fix a concentration at which the
compounds showed 50 % of activity. In addition, the per-
centage of activity was calculated using the formula, % of
suppression ratio = [(A0 - AC)/A0] 9 100. A0 and AC are
the absorbances in the absence and presence of the tested
complexes, respectively. The 50 % activity (IC50) can be
calculated using the percentage of activity.
Results and discussion
Structure of the complexes
A new series of binuclear ruthenium(III) thiosemicarba-
zone complexes of the type {[RuCl2(EPh3)]2L} (E = P/As;
L = binucleating monobasic tridentate thiosemicarbazone
ligands) were synthesized by reacting ruthenium(III) pre-
cursors [RuCl3(EPh3)3] with binucleating monobasic tri-
dentate thiosemicarbazone ligands in 2:1 molar ratio,
respectively, in benzene–methanol/tetrahydrofuran mix-
ture. The synthesized binuclear ruthenium(III) thiosemi-
carbazone complexes are stable in air at room temperature,
non-hygroscopic in nature, and soluble in common sol-
vents, such as dichloromethane, dimethylformamide,
dimethylsulphoxide, etc. The analytical data of the com-
plexes are in good agreement with the calculated values,
thus confirming the proposed hetero bimetallic composi-
tion for all the complexes.
In vitro anticancer activity assay
IR spectra
Cytotoxicity of the complexes 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 (Mossman,
1983). MCF-7 cell was grown in Eagles minimum essential
medium 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 com-
pounds at various concentrations 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.
After 48 h, 15 lL of MTT (5 mg mL-1) in phosphate
The IR bands for the metal complexes derived from the
binucleating ferrocenyl thiosemicarbazone ligands are
most useful in attempting to determine the mode of coor-
dination. The free ligands display m(C=S) absorptions in the
region 807–826 cm-1, shifts to 740–749 cm-1 in the
spectra of the complexes. This observation may be attrib-
uted to the enolization of –NH–C=S and subsequent
coordination through the deprotonated sulfur (Thangadurai
and Natarajan, 2001). A strong band in the range
1,631–1,649 cm-1 due to the azomethine group m(C=N) of
free ligands was shifted to lower frequency in the spectra of
complexes in the range 1,581–1,616 cm-1 indicating that
the other coordination is through azomethine nitrogen atom
(Bechford et al., 2009). IR spectrum of the ligands revealed
a medium intensity band in the region 1,511–1,543 cm-1
m(C=N) thiazole ring, which is shifted to lower frequency in
the range 1,436–1,483 cm-1 after complexation, which
also indicates that it has been affected upon coordination
to the metal ion (Prasad et al., 2010). The m(C–S–C) at
123

Med Chem Res
617–621 cm-1 of the thiazole ring remains unchanged
which demonstrated that the thiazole group of sulfur does
not coordinate to the ruthenium metal (Alam et al., 2008).
Absorptions at 527–559 and 424–474 cm-1 in the spectra
of the complexes are attributed to the m(Ru–N) and m(Ru–S)
(Manivannan et al., 2007). In addition, other characteristic
bands due to triphenylphosphine/triphenylarsine are also
present in the expected region.
group. The aromatic protons for all the ligands appeared as
multiplets at 7.39–7.83 ppm.
13C NMR spectra of all the ligands displayed a single
resonance at 157–159 ppm due to the azomethine carbon
atoms, which also confirms the structure of the ligands (Pal
et al., 2000). The signal at 165 ppm corresponds to thiaz-
olic C=N carbon (Desai et al., 2008). The thiolic carbon of
the thiosemicarbazones appeared at 140–141 ppm. The
signal due to the methyl carbon of the ligands appears at
27 ppm. The aromatic carbons of free ligands show signal
in the region 117–132 ppm. For ligands, the signal at
71–81 ppm is assigned to cyclopentadienyl ring carbons
(Casas et al., 2004).
Electronic spectra
The electronic spectra of the ligands and its complexes
were taken in DMSO. The spectra of the free ligands
showed bands at 305–308 and 368 nm are attributed to p–
p* and n–p* transitions in the aromatic ring, thioamide,
and imine chromophore. For all the ligands and complexes,
the shoulder in the region 428–430 nm is attributed to the
transition of 3d electron on iron to either non-bonding or
antibonding orbitals of the cyclopentadienyl ring (Pra-
bhakaran et al., 2011a, b). These peaks have been shifted in
the spectra of the complexes, reveals that the ligands are
involved in the coordination with the ruthenium metal ion.
The electronic spectra of all the complexes showed four
bands in the region 305–471 nm. The ground state of
Magnetic moment and EPR spectra
The room temperature magnetic susceptibility measure-
ments of the binuclear ruthenium(III) complexes showed
that they are paramagnetic (lB = 1.61–1.65), corresponds
to single unpaired electron in a low-spin 4d5 configuration,
and confirms that ruthenium is in ?3 oxidation state in all
the complexes. It has been observed that the lB values
indicate a single unpaired electron and are consistent with
noninteracting d5 centers or the absence of any strong
magnetic interactions between the two ruthenium centers
of the molecule (Manimaran and Jayabalakrishnan, 2010).
All the complexes are uniformly paramagnetic with
magnetic moments, correspond to one unpaired electron at
room temperature (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, {[RuCl2(PPh3)]2L2} is shown in Fig. 2. The
nature 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 1.94–2.11. Although
the complexes have some distortion in their octahedral
geometries, the observation of isotropic lines in the EPR
spectra may be due to the occupancy of the unpaired
electron in a degenerate orbital (Khan et al., 1990). The
nature of spectra obtained is in good agreement with that of
previously reported binuclear ruthenium(III) complexes
(Manimaran and Jayabalakrishnan, 2010; Samanta et al.,
2001).
2
ruthenium(III) (t52g configuration) is T2g, while the first
excited doublet levels in the order of increasing energy are
2A2g and 2T1g, which arise from t24g e1g configuration
(Ballhausen, 1962). The lower wavelength bands are
characterized as ligand-centered transitions occurring
within the ligand orbitals. In a d5 system, and especially in
ruthenium(III) that has relatively high oxidising properties,
the charge transfer bands of the type Lpy ? t2g are
prominent in the low-energy region obscuring the weaker
bands due to d–d transition. Therefore, it becomes difficult
to assign conclusively the bands, 466–471 nm of ruthe-
nium(III) complexes appearing in the visible region.
Hence, all the bands that appear in this region have been
assigned to charge transfer transitions, which are in con-
formity with the assignments made for similar binuclear
ruthenium(III) complexes (Manimaran and Jayabalakrish-
nan, 2010; Samanta et al., 2001).
NMR spectra
The formations of ligands were conveniently monitored by
Mass spectral analysis
peak ratios in the 1H and 13C NMR spectra. The 1H and 13
C
NMR spectra of H2L2 are given in Fig. 1. The signal due to
–SH and –NH protons in the ligands appears at
11.3–11.9 ppm and 8.67–8.68 ppm (West et al., 1998). For
ligands, the chemical shift at 4.59–4.80 ppm is due to
cyclopentadienyl ring protons (Sing et al., 2002). The
ligands displayed a signal at 2.28–2.35 ppm due to methyl
The mass spectrum of all the binuclear ruthenium(III)
complexes is in good agreement with the proposed
molecular structure and the mass spectrum of the com-
plexes, {[RuCl2(AsPh3)]2L1} and {[RuCl2(PPh3)]2L2}, is
shown in Fig. 3. The molecular ion peak, [M?] appeared at
m/z = 1,639, 1,727, 1,549 and 1,637 confirms the
123

Med Chem Res
Fig. 1 1H (a) and 13C (b) NMR
spectrum of the ligand, H2L2
stoichiometry of the complexes, {[RuCl2(PPh3)]2L1},
{[RuCl2(AsPh3)]2L1}, {[RuCl2(PPh3)]2L2}, and {[RuCl2
(AsPh3)]2L2}, respectively.
DNA binding–absorption titration experiments
A majority of reports indicate that the presence of labile
ligand units play a vital role in the DNA-binding strategy of
metal complexes. Effectiveness of the DNA binding
depends on the nucleophilic nature of the labile ligand
attached to the metal center and the nature of metal ion itself
(Kamath et al., 2012). In the present complexes, the
architecture of ligand is such that it provides at least one
labile ligand viz., thiosemicarbazone group, which is
Fig. 2 EPR spectrum of the complex, {[RuCl2(PPh3)]2L2}
123

Med Chem Res
Fig. 3 EI-mass spectra of the
complexes,
{[RuCl2(AsPh3)]2L1} and
{[RuCl2(PPh3)]2L2}
responsible for the interaction of the compounds with N-7
of nucleotide, which leads to the perturbation of DNA
structure, and hence inhibiting the replication. In the present
investigation by following the changes in absorbance and
shift in the wavelength as a function of added concentration
of DNA, the binding of metal complexes (25 lM) to CT-
DNA in different concentrations (0–40 lM) has been
characterized classically. The electronic spectra of the
complexes upon the addition of DNA are shown in Fig. 4.
The titrations result clearly shows that with increasing
concentration of DNA added to the complexes, significant
hyperchromism with red shift was observed. This can be
attributed to a strong interaction between DNA and com-
plexes. Based on the results obtained from the UV–Vis
titration, it is inferred that the complexes underwent a non-
intercalative mode of binding with DNA. Generally, hyp-
ochromism and hyperchromism are the two spectral fea-
tures which are closely connected with the double helix
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Fig. 4 Electronic spectra of complexes, {[RuCl2(PPh3)]2L1}, {[RuCl2(AsPh3)]2L1}, {[RuCl2(PPh3)]2L2}, and {[RuCl2(AsPh3)]2L2} in Tris–HCl
buffer upon addition of CT-DNA. [Complex] = 25 lM, [DNA] = 0–40 lM
structure of DNA. The observation of hypochromism is
indicative of intercalative mode of binding of DNA to the
complexes along with the stabilization of the DNA double
helix structure. On the other hand, the observation of hy-
perchromism is indicative of the break age of the secondary
structure of DNA. Hence, the observation of hyperchro-
mism with red shift for our complexes showed that the new
complexes interact with the secondary structure of CT-
DNA by breaking its double helix structure. In order to
compare quantitatively the binding strength of the com-
plexes, the intrinsic binding constants (Kb) of them with
CT-DNA were determined from the following equation.
½DNAꢁ=ðea ꢂ efÞ ¼ ½DNAꢁ=ðeb ꢂ efÞ þ 1=Kbðeb ꢂ efÞ
where [DNA] is the concentration of DNA in base pairs and
Fig. 5 Plots of [DNA]/[ea - ef] 9 10-8 versus [DNA] for the ruthe-
the apparent absorption coefficients ea, ef, and eb correspond
nium(III) complexes, {[RuCl2(PPh3)]2L1} (a), {[RuCl2(AsPh3)]2L1}
to Aobs/[compound], the extinction coefficient of the free
(b), {[RuCl2(PPh3)]2L2} (c), and {[RuCl2(AsPh3)]2L2} (d) with CT-
DNA
compound, and the extinction coefficient of the compound
when fully bound to DNA, respectively. The plot of [DNA]/
(ea - ef) versus [DNA] (Fig. 5) gave a slope and the inter-
cept which are equal to 1/(eb - ef) and 1/Kb (eb - ef),
respectively; Kb is the ratio of the slope to the intercept. The
magnitudes of intrinsic binding constants (Kb) were calcu-
lated to 7.9 9 104 M-1 {[RuCl2(PPh3)]2L1}, 3.8 9 104 M-1
{[RuCl2(AsPh3)]2L1}, 2.6 9 104 M-1 {[RuCl2(PPh3)]2L2},
and 1.0 9 104 M-1 {[RuCl2(AsPh3)]2L2}, respectively. The
significant difference in DNA-binding affinity of the
binuclear ruthenium(III) complexes can be understood as a
result of the fact that the complex with different ligands
shows stronger binding affinity with DNA. On comparing the
results, {[RuCl2(PPh3)]2L1} shows more binding affinity
than the other complexes. Interestingly, the Kb values
obtained for the above binuclear ruthenium(III) complexes
are comparable than those for the other known polypyridyl
ruthenium complexes (Liu et al., 2005; Zao et al., 2001).
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Med Chem Res
Fig. 6 Antioxidant activity of complexes, {[RuCl2(PPh3)]2L1} (a),
{[RuCl2(AsPh3)]2L1} (b), {[RuCl2(PPh3)]2L2} (c), {[RuCl2(AsPh3)]2L2}
(d) and standard ascorbic acid (Aca) against DPPH radical
Fig. 7 % Growth inhibition of MCF-7 cell line as a function of
concentration of the complexes, {[RuCl2(PPh3)]2L1} (a), {[RuCl2
(AsPh3)]2L1} (b), {[RuCl2(PPh3)]2L2} (c), and {[RuCl2(AsPh3)]2L2}
(d)
Radical scavenging activity
assay method that measures mitochondrial dehydrogenase
activity as an indication of cell viability. 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 Fig. 7.
The IC50 values were found to be 52.12 lM for
{[RuCl2(PPh3)]2L1}, 75.64 lM for {[RuCl2(AsPh3)]2L1},
85.16 lM for {[RuCl2(PPh3)]2L2}, and 85.80 lM for
{[RuCl2(AsPh3)]2L2}. Upon increasing the concentration of
complexes, the results of MTT assays reveal that the com-
plex {[RuCl2(PPh3)]2L1} shows a higher cytotoxic effect
followed by {[RuCl2(AsPh3)]2L1}, {[RuCl2(PPh3)]2L2},
and {[RuCl2(AsPh3)]2L2}. The findings of the in vitro
antitumor activities confirm the binding of the complexes to
DNA, which consequently leads to cell death.
Free radicals play an important role in the inflammatory
process. Many thiosemicarbazone complexes with a broad
spectrum of effects have been reported to act either as
inhibitors of free radical production or as radical scavengers
(Prabhakaran et al., 2011a, b). The 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
becomes paired off in the presence of a free radical scaven-
ger, this absorption vanishes, and the resulting decolouri-
zation is stoichiometric with respect to the number of
electrons taken up. The DPPH assay of the tested complexes
is shown in Fig. 6, it is seen from the results that all the
complexes exhibited moderate activity compared to the
standard Ascorbic acid (Aca). The IC50 values indicated
that the complexes showed antioxidant activity in the order
of {[RuCl2(PPh3)]2L1} [ {[RuCl2(AsPh3)]2L1} [ {[RuCl2
(PPh3)]2L2} [ {[RuCl2(AsPh3)]2L2}. Complex {[RuCl2
(PPh3)]2L1} showed a higher antioxidant activity than the
other complexes. The scavenging activity of the complexes
might be due to the d5 low-spin electronic configuration and
the availability of an odd electron in ruthenium(III) com-
plexes, which increases the capacity to stabilize the unpaired
electrons, and thereby scavenge the free radicals (Kamatchi
et al., 2012).
Conclusion
In this work, a systematic approach to the synthesis of four
new binuclear ruthenium(III) thiosemicarbazone com-
plexes and were characterized by various spectroscopic
techniques. An octahedral geometry has been tentatively
assigned for all the complexes. The DNA-binding study
suggests that all the complexes interact with DNA most
likely through a mode that involves a stacking interaction
between the aromatic chromophore and the base pairs of
DNA via electrostatically. The magnitudes of intrinsic
binding constant, Kb of the binuclear ruthenium(III) com-
plexes have been estimated in the range from
7.9 9 104 M-1 to 1.0 9 104 M-1. In addition, all the
complexes exhibit significant antioxidant activity and are
in the order of {[RuCl2(PPh3)]2L1} [ {[RuCl2(AsPh3)]2
In vitro anticancer activity evaluation
The positive results obtained from DNA binding and anti-
oxidative studies encouraged us to test its cytotoxic activity.
The complexes were evaluated for their cytotoxicity against
human breast cancer cell line (MCF-7) by means of MTT
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Med Chem Res
L1} [ {[RuCl2(PPh3)]2L2} [ {[RuCl2(AsPh3)]2L2}. More-
over, the complexes have potential in vitro antitumor
activity against MCF-7 cancer cell line. The IC50 value
indicates that the cytotoxic activity of {[RuCl2(PPh3)]2L1}
is higher than that of other three complexes. Animal model
studies are to be carried out with these complexes in order
to establish their potential as clinical candidates.
characterization, antibacterial activity, and DNA binding/cleav-
age studies. Med Chem Res 22:1948–1956
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Acknowledgments Financial assistance received from the Council
of Scientific and Industrial Research, New Delhi, India [Grant No.
01(2308)/09/EMR-II] is gratefully acknowledged.
Liu XW, Li J, Deng H, Zheng KC, Mao ZW, Ji LN (2005)
Experimental and DFT studies on the DNA-binding trend and
spectral properties of complexes [Ru(bpy)2L]2? (L = dmdpq,
dpq and dcdpq). Inorg Chim Acta 358:3311–3319
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