Influence of carboxylic acid functionalities in ruthenium (II) polypyridyl complexes on DNA binding, cytotoxicity and antioxidant activity: Synthesis, structure and in vitro anticancer activity

Article abstract of DOI:10.1016/j.ejmech.2012.11.024

Four new Ru(II) complexes [RuHCl(bpy)(PPh₃)(CO)] (1), [RuHCl(bpy)(AsPh₃)(CO)] (2) (bpy = 2,2′-bipyridine), [RuCl(HL)(PPh₃)₂(CO)] (3) and [RuCl(HL)(AsPh ₃)₂(CO)] (4) (HL = 2,2′-bipyridine-4,4′- dicarboxylic acid) were synthesized and characterized. X-ray diffraction was used to characterize 3 in solid state. The interactions of these complexes with DNA were explored by different techniques which revealed that the complexes could bind to CT-DNA through non-intercalation. The in vitro cytotoxic and antioxidant activities of the complexes validated against a panel of cancer cell lines and free radicals showed that 3 and 4 possess quite high anticancer and antioxidant activities over 1, 2 and standard drugs. An apparent dependence of biological activities on incorporation of COOH in bipyridine moiety was noticed: Inclusion of COOH caused significant differences in DNA binding, cytotoxicity and antioxidant activity.

Full text of DOI:10.1016/j.ejmech.2012.11.024

European Journal of Medicinal Chemistry 59 (2013) 253e264
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Influence of carboxylic acid functionalities in ruthenium (II) polypyridyl
complexes on DNA binding, cytotoxicity and antioxidant activity: Synthesis,
structure and in vitro anticancer activity
Thangavel Sathiya Kamatchi a, Nataraj Chitrapriya b, Seog K. Kim b, Frank R. Fronczek c,
,
Karuppannan Natarajan a
*
a Department of Chemistry, Bharathiar University, Coimbatore 641046, Tamilnadu, India
b Department of Chemistry, Yeungnam University, Gyeongsan City, Gyeong-buk 712-749, Republic of Korea
c Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803, USA
a r t i c l e i n f o
a b s t r a c t
Four new Ru(II) complexes [RuHCl(bpy)(PPh3)(CO)] (1), [RuHCl(bpy)(AsPh3)(CO)] (2) (bpy ¼ 2,20-bipyr-
idine), [RuCl(HL)(PPh3)2(CO)] (3) and [RuCl(HL)(AsPh3)2(CO)] (4) (HL ¼ 2,20-bipyridine-4,40-dicarboxylic
acid) were synthesized and characterized. X-ray diffraction was used to characterize 3 in solid state. The
interactions of these complexes with DNA were explored by different techniques which revealed that the
complexes could bind to CT-DNA through non-intercalation. The in vitro cytotoxic and antioxidant
activities of the complexes validated against a panel of cancer cell lines and free radicals showed that 3
and 4 possess quite high anticancer and antioxidant activities over 1, 2 and standard drugs. An apparent
dependence of biological activities on incorporation of COOH in bipyridine moiety was noticed: Inclusion
of COOH caused significant differences in DNA binding, cytotoxicity and antioxidant activity.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Article history:
Received 11 August 2012
Received in revised form
22 October 2012
Accepted 17 November 2012
Available online 27 November 2012
Keywords:
Ruthenium
Bipyridine
Dicarboxylic acid
DNA interaction
Cytotoxicity
Antioxidant activity
1. Introduction
involved in clinical trials [9e11]. It is known that ruthenium
complexes are well suited for medical applications due to the fact
Though possible remedial measures are available at present to
tackle any disease, continuous search in trying to find better and
more effective drugs is on the increase. This is particularly true in
the case of cancer where cisplatin and its analogs such as carbo-
platin, and oxaliplatin during chemotherapy result in drawbacks,
including intrinsic or acquired resistance, and toxicity. So, the
efforts to mitigate the drawbacks have prompted chemists to
synthesize a variety of analogs, which is valuable not only for
providing better drugs but also for offering useful tools in the study
of the molecular mechanisms underlying tumor development. In
this connection, ruthenium based coordination and organometallic
complexes have shown enormous impact. Though a wide range of
ruthenium complexes have been described in the literature, only
a few of them show outstanding anticancer activity [1e8] and two
of them, for instance NAMI-A and KP1019 (Fig. 1.), are currently
that they have a very similar rates of ligand exchange to those of
platinum(II) antitumor drugs [12], a wide range of accessible
oxidation states under physiological environment and an ability of
the ruthenium to mimic iron in binding to certain molecules of
biological significance [1,2,9]. Under aqueous conditions, three
predominant oxidation states are known for Ru, namely, Ru(II),
Ru(III), and Ru(IV), all of them mostly presenting an octahedral
configuration. This octahedral geometry appears to be partially
responsible for the differences observed in the mechanism of action
of ruthenium complexes compared to cisplatin. It is well known
that the hypoxic environment of many tumors favor the reduction
of Ru(III) compounds (which are relatively slow in binding to most
biological substrates) to Ru(II) species, which bind more rapidly [2].
In addition, the redox potential exhibited between different
accessible oxidation states of ruthenium complexes enables the
body to catalyze oxidation and reduction reactions with relative
ease, depending on physiological environment. Moreover, the
biochemical changes that accompany cancer alter physiological
environment, enabling ruthenium complexes to be selectively
* Corresponding author. Tel.: þ91 422 2428319; fax: þ91 422 2422387.
E-mail address: k_natraj6@yahoo.com (K. Natarajan).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.11.024

254
T. Sathiya Kamatchi et al. / European Journal of Medicinal Chemistry 59 (2013) 253e264
modulate the solubility of the complex, cell transport, and biolog-
ical activity [32] and the ancillary ligand of polypyridyl ruth-
enium(II) complexes could play a key role in the spectral properties
and interactions with DNA [33e36]. This has inspired us to
synthesize a series of new ruthenium complexes containing
bipyridine and bipyridine dicarboxylic acids with PPh3/AsPh3 as
ancillary ligands.
Since DNA is considered to be one of the major pharmacological
targets of anticancer drugs, the objective of the present work is to
understand in detail the DNA binding mode of the new complexes
reported in this paper. Hence, the interactions of these complexes
with DNA were studied by electronic absorption, luminescence
quenching, thermal melting and viscosity measurements. It is also
known that the uncontrolled production of free radical reactive
oxygen species such as superoxide anion ðO2$ꢀÞ, hydroxyl radical
(OHꢁ) and hydrogen peroxide (H2O2) can induce DNA damage in
humans. Such a damage to DNA has been suggested to contribute to
aging and various diseases including cardiovascular, cancer and
chronic inflammation [37,38]. This has prompted us to test the
synthesized complexes as free radical scavengers against DPPH
radical, hydroxyl radical, nitric oxide radical, superoxide anion
radical, hydrogen peroxide and Fe2þ ion. Further, the in vitro
anticancer activities of the synthesized complexes were also
determined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
2H-tetrazolium bromide) assay, SRB ([9-(2-carboxyphenyl)-6-
diethylamino-3-xanthenylidene]-diethylammonium chloride) assay
and trypan blue dye ((3Z,30Z)-3,30-[(3,30-dimethylbiphenyl-4,40-
diyl)di(1Z)hydrazin-2-yl-1-ylidene]bis(5-amino-4-oxo-3,4-dihydr-
onaphthalene-2,7-disulfonic acid)) exclusion assay on a panel of
cancer cell lines.
Fig. 1. Chemical structures of promising ruthenium drugs under investigation: NAMI-
A and KP1019.
activated in cancer tissues. Since a wide range of ligand geometry
exists in their complexes, ruthenium compounds bind to DNA
affecting its conformation differently than cisplatin and its analogs.
Besides, non-nuclear targets, such as the mitochondrion and the
cell surface, have also been implicated in the antineoplastic activity
of some ruthenium complexes. Some other factors that might be
involved in the mechanism underlying the cytotoxic effects of
ruthenium complexes have also been proposed, such as inhibition
of metastases, DNA damage, production of reactive oxygen species,
interactions with proteins, activation of genes of the endoplasmic
reticulum stress pathway and triggering apoptosis [13e18].
Among ruthenium complexes studied for anticancer applica-
tion, the group of ruthenium complexes with pyridyl-based ligands
is of special interest, due to a combination of easily constructed
rigid chiral structures and useful photophysical properties. They
have been studied mainly because, the large, rigid, multidentate
polypyridyl ligands confer shape and chirality to the ruthenium
complexes that could be utilized to achieve customized DNA-
binding properties (enantioselective recognition of DNA, DNA
structure, DNA mediated electron transfer, DNA foot printing
agents and cleavage). This has provided the motivation to develop
polypyridyleRu complexes as DNA-targeting anticancer agents,
and a large number of such ruthenium complexes have been
screened for anticancer activity, a majority of these complexes
contain bidentate ligands with functional auxiliary ligands [19e31].
Moreover it has been proved that an appropriate attachment of the
carboxylic acid (COOH) group in coordination complexes could
2. Results and discussion
2.1. Synthesis and characterization of the complexes
Reactions of the bipyridine (bpy) and bipyridine dicarboxylic
acid (H2L) with [RuHCl(CO)(PPh3)3] and [RuHCl(CO)(AsPh3)3] gave
new complexes of the types [RuHCl(bpy)(EPh3)(CO)] and [RuCl(H-
L)(EPh3)2(CO)] where E ¼ P/As, as sketched out in Scheme 1. All the
complexes are air stable for extended periods and remarkably
Scheme 1. General scheme for the synthesis of new ruthenium complexes.

T. Sathiya Kamatchi et al. / European Journal of Medicinal Chemistry 59 (2013) 253e264
255
soluble in methanol, ethanol, chloroform, dichloromethane, DMF
and DMSO. The synthesized complexes were then characterized by
elemental analysis, IR, and 1H NMR spectroscopic techniques. The
analytical data of the new complexes agree well with the proposed
at 95 K. The crystal was a solvate containing disordered methanol
molecule. The solvent contribution to the structure factors was
removed using the SQUEEZE procedure [39], amounting to 3.75
methanol molecules per Ru complex. The Ru(II) ion exhibits a hexa
coordination with an octahedral geometry, where equatorial
coordination comes from two ring nitrogens of bidentate chelating
ligand HL, a chloride and a carbonyl carbon. A pair of triphenyl-
phosphines completes the axial coordination. The complex lies on
a two-fold rotation axis in the crystal. The two-fold rotation axis
causes superposition of the CO and Cl ligands and also the COOH
and COOꢀ groups. The þ2 oxidation state of the complex is
compensated by a chloride ion and a deprotonated carboxylate
anion. The coordination environment around the Ru(II) ion can be
molecular formulas. The IR peak shift of yC
] vibrations of the
N
ligands gave an idea about the involvement of the ligands in the
coordination with ruthenium ion. The retention of the IR band
around 3373e3414 cmꢀ1 in 3 and 4 confirms the non participation
of COOH group of bipyridine dicarboxylic acid in coordination,
which is consistent with the results of NMR and X-ray analysis. It
has been observed that a molecule of bipyridine replaced a pair of
a PPh3/AsPh3 in the case of [RuHCl(bpy)(EPh3)(CO)] complexes and
one molecule of bipyridine dicarboxylic acid replaced, a hydride ion
and a PPh3/AsPh3 in the case of [RuCl(HL)(EPh3)2(CO)] complexes.
The solid state structure of the complex 3 was determined by single
crystal X-ray crystallographic studies. It revealed that bipyridine
dicarboxylic acid is coordinated to the metal ion as neutral biden-
tate NN donor. However one of the carboxylic acid groups has been
found to be deprotonated.
considered as being
Though the PPh3 ligands usually prefer to occupy mutually cis
position for better -interaction [40] in this complex the presence
of CO, a stronger -acidic ligand, might have forced the bulky PPh3
a distorted octahedron {RuN2P2(CO)Cl}.
p
p
ligands to take trans position for steric reasons. The N(1)eRu(1)e
Cl(1) bond angle is 170.0(1)ꢃ showing that Cl atom lies trans to
ring nitrogen. The RueN1, RueN’1, RueC and RueCl bond lengths
found in the complex agree well with that reported for similar
other ruthenium complexes [41e43]. The two RueP bond lengths
are identical by symmetry and are comparable to those reported for
other Ru(II) complexes containing coordinated PPh3 [44,45].
2.2. X-ray structural characterization
2.2.1. Crystal structure of 3
The structure of 3 has been established by single crystal X-ray
analysis. The ORTEP drawing is displayed in Fig. 2. The details
concerning the data collection and structure refinement of the
complex are summarized in Table 1. Selected geometrical param-
eters (inter atomic distances and angles) and hydrogen bond
distances are given in Table 2. Single crystals of 3 were obtained
from slow evaporation of the mixture of chloroform and methanol
(1:1) at room temperature as rectangular prism. An orange single
crystal of approximate unit cell dimension 0.23 ꢂ 0.18 ꢂ 0.15 was
isolated and the single crystal X-ray diffraction data were collected
2.3. DNA binding properties
2.3.1. Electronic absorption titration
The mode and strength of binding of the complexes to calf
thymus (CT) DNA have been usually studied by different spectro-
scopic techniques. We have carried out electronic absorption
spectral measurements to determine the equilibrium binding
constant (Kb) of the complexes to CT DNA by monitoring the change
Fig. 2. X-ray crystal structure and atom numbering scheme for 3 as displacement ellipsoids at 50% probability level. The hydrogen atoms (except one carboxylic acid proton) have
been omitted for clarity, and the disorder is not illustrated.

256
T. Sathiya Kamatchi et al. / European Journal of Medicinal Chemistry 59 (2013) 253e264
Table 1
Table 2
Crystal data and details of data collection for 3.
Selected bond lengths (Å) and angles (ꢃ) for 3.
3
Complex 3
ꢀ
CCDC deposit number
Empirical formula
Formula weight
843879
Interatomic distances (A)
Ru1eC7
Ru1eCl1
Ru1eN1
Ru1eN’1
Ru1eP1
Ru1eP’1
C7eO3
C49H37ClN2O5P2Ru$3.75(CH3OH)
1052.42
95
0.71073
Orthorhombic
C2221
1.819(14)
2.424(3)
2.105(3)
2.105(3)
2.386(1)
2.386(1)
1.31(2)
Temperature (K)
ꢀ
Wavelength (A)
Crystal system
Space group
Unit cell dimensions
ꢀ
a(A)
35.965(4)
11.049(2)
14.482(2)
5754.8(15)
4
1.215
0.424
2174
2.6e26.3ꢃ
ꢀ44 ꢅ h ꢅ 44
ꢀ13 ꢅ k ꢅ 13
ꢀ17 ꢅ l ꢅ 17
9123/5651, 0.055
0.972
N1eC5
N’1eC5
1.354(5)
1.354(5)
ꢀ
b(A)
c(A)
Volume (A )
Bond angles(ꢃ)
C7eRu1eN1
C7eRu1eN’1
C7eRu1eCl1
C7eRu1eP1
C7eRu1eP’1
N1eRu1eCl1
N1eRu1eP1
N1eRu1eP’1
N1eRu1eN’1
N’1eRu1eCl1
N’1eRu1eP1
N’1eRu1eP’1
Cl1eRu1eP1
Cl1eRu1eP’1
P1eRu1eP’1
ꢀ
3
ꢀ
93.70(6)
171.00(6)
95.50(6)
90. 30(6)
89.90 (6)
170.00(1)
88.29(8)
91.52(8)
77.60 (1)
93.30(1)
91.52(8)
88.29(8)
87.95(7)
92.22(7)
179.75(4)
Z
Density (calculated, g cmꢀ3
)
Absorption coefficient (mmꢀ1
F(000)
)
q
range for data collection
Index ranges
Reflections collected/unique, Rint
Completeness to qmax
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices
Full-matrix least-squares on F2
5651/0/280
1.019
R1 ¼ 0.0462 [I > 2
s(I)]
ꢃ
ꢀ
wR2 ¼ 0.1087 (all data)
0.44; ꢀ0.38
Hydrogen-bonding distances (A) and angles ( ) for 3
ꢀꢀ3
Largest diff. peak and hole/e A
Complex
3
DeH/A
DeH
0.951(4)
H/A
1.632(4)
D/A
D-H/A
O1eH1/O1i
2.491(6)
148.0(3)
Symmetry code (i) ꢀx, y, 3/2 ꢀ z
in the absorption intensity of the spectral band at w310 nm for all
the complexes. In general, a hypochromism and a red shift are
associated with the binding of the complex to the helix because of
the interaction between the aromatic chromophore of the complex
and the base pairs of the DNA. The magnitude of the hypochromism
and red shift depends on the strength of interaction between DNA
and the complex. In our study, the electronic absorption spectral
bands of the complexes were recorded over the range 200e800 nm
in DMSO and buffer (3:97). The complexes have been found to be
efficient light absorbers throughout the UV and visible regions. The
complexes exhibited MLCT bands around 400e420 nm and
a shoulder at 330 nm. Absorption titration experiment was per-
formed by maintaining the metal complex concentration constant
than 4, in accord with the extent of hypochromism and red shift in
the * absorption band (w310 nm). Since the primary ligand of
pep
all the complexes is the same, the differences in the DNA-binding
affinity can only be attributed to the presence of ancillary ligands.
Given the close structural similarity between the complexes, we are
not sure as to why this should be. These spectral features suggested
that 3 and 4 interact with DNA through a mode that is similar to
that of 1 and 2, but with small binding affinity as compared to 1 and
2. It may probably be due to the influence of substitution on
bipyridine moiety and ancillary ligands. In order to compare
quantitatively the binding strength of the complexes, the intrinsic
binding constants Kb of the complexes with DNA were obtained by
monitoring the changes in absorbance at 310 nm with increasing
concentration of DNA using the following equation
(10
mM) and varying the concentration of nucleic acid (from 0 to
100
mM). The spectral changes of 1e4 upon addition of CT-DNA are
shown in Fig. 3.
The absorption spectra of 1 on the addition of DNA showed
a 36% hypochromism with considerable red shift in the
pep*
ꢀ
ꢁ
À
Á
À
Á
À
Á
ligand-centered (LC) band at the ratio of 0.11, whereas 2 showed
only a 22% of hypochromicity with substantial red shift in IL band at
0.14 ratio. The results suggested an intimate association of these
complexes with DNA and also it was likely that these complexes
may bind to the helix by intercalation or groove binding mode. The
extent of hypochromism and red shift is more in 1 than 2, indicating
strong binding affinity of 1 to CT-DNA. Isobestic points have been
clearly identified at 290 nm for 1, 340 nm for 2, and at 295 nm for 3
and 4. The presence of the isosbestic points in these titrations
suggested that chemical equilibria exist between the bound and the
free metal complexes. For 3, the hypochromism observed in IL
bands is 16% at the 0.11 ratio whereas for 4 (hypo 8%) were observed
in the presence of DNA. It implies that a relatively weak association
of 4 to the helix. It can be seen from Fig. 3 that as the DNA
concentration is increased, there is hypochromism of the main
absorption band though no obvious red shift of the same band was
observed in the case of 3 and 4. Hence, 3 and 4 clearly bind to DNA
by non-intercalative mode. However, 3 is having a higher affinity
½DNAꢄ= εa ꢀ εf ¼ ½DNAꢄ= εb ꢀ εf þ 1= Kb εb ꢀ εf
where in, [DNA] is the concentration of DNA in base pairs, εa, εf, and
εb are the apparent-, free- and bound-metal-complex extinction
coefficients respectively. Kb is the equilibrium binding constant of
complex binding to DNA. Each set of data, when fitted to the above
equation, gave a straight line with a slope of 1/(εb ꢀ εf) and a y-
intercept of 1/Kb (εb ꢀ εf) and Kb was determined from the ratio of
the slope to intercept (Fig. 4).
The binding constants for the complexes were found to be
4.1 ꢂ 103 Mꢀ1, 6.5 ꢂ 102, 2.6 ꢂ 102 Mꢀ1 and 1.1 ꢂ 102 Mꢀ1 for 1, 2, 3
and 4 respectively. Compared [46] with those of the so-called DNA-
classical intercalators (ethidium bromide, Kb, 1.8 ꢂ 106 Mꢀ1 in
25 mM TriseHCl/40 mM NaCl buffer, pH 7.9) and partial inter-
calating metal complexes ([Ru(phen)2(dppz)]2þ, Kb > 106 Mꢀ1), the
observed binding constants indicate that these Ru(II) complexes
interact with DNA with less affinity and may be by groove of DNA.

T. Sathiya Kamatchi et al. / European Journal of Medicinal Chemistry 59 (2013) 253e264
257
0.8
0.4
(1)
0.0
0.6
(2)
0.3
0.0
0.8
(3)
(4)
0.4
0.0
0.6
0.3
0.0
300
400
500
Wavelength (nm)
Fig. 4. Plot of (εa ꢀ εf)/(εb ꢀ εf) vs [DNA] for the titration of DNA with complexes 1e4
and solid line is linear fitting of the data.
Fig. 3. UV spectra of 1e4 in the absence (- - -) and in the presence of CT-DNA in
increasing amounts, [complex] ¼ 10
mM, [DNA] ¼ 0e100
mM.
EB with dodecamer DNA have been previously reported [51]. The
titration of 4 with DNA-bound EB (from 0 to 25
mM) was also per-
2.3.2. EB competition assay
formed, and the results clearly indicate that the addition of the 4 did
not affect the emission spectrum of EB (data not shown), indicating
their poor DNA binding ability. Hence, it is concluded that the Ru(II)
complexes with bipyridine ligands competitively replaced interca-
lated EB markedly and more effectively than that of the complexes
containing bipyridine dicarboxylic acid ligands. Among 1e3, the
ability of 1 to displace DNA intercalator EB from CT DNA was greater
than that of the other complexes.
It has been shown that the competitive binding experiments
with a well-established quenching assay based on the displace-
ment of the intercalating drug ethidium bromide (EB) from CT-DNA
give better information about the DNA binding properties of
complexes [47]. If a complex can displace EB from DNA-bound EB,
the fluorescence of EB will be sharply decreased due to the fact that
the free EB molecules are much less fluorescent than the DNA
bound EB molecules because the surrounding water molecules
quench the fluorescence of free EB [48]. However, not only the DNA
intercalators but also groove DNA binders could cause the reduc-
tion in the emission intensities of DNA bound EB [49], but only
moderately for the latter case. For example, the non-intercalating
DNA groove binding agents of berenil, bisamidines, spermine, and
spermidine were reported [50] to be capable of displacing EB from
DNA. In order to get a clear picture on the nature of binding of the
new complexes to DNA, emission spectra of EB bound to DNA in the
absence and in the presence of complexes were recorded. Changes
in the emission spectra of the DNA bound EB upon increasing the
concentration of 1, 2 and 3 are illustrated in Fig. 5. The results
showed that the reduction in emission of DNA-bound EB was slow
and only 36,16 and 12% at very high concentration ratio (0.1) for 1, 2
and 3 respectively.
The quenching plot (Fig. 6.) illustrates that the removal of EB
bound to DNA by 1, 2 and 3 are in good agreement with the linear
SterneVolmer equation [52]
I0=I ¼ 1 þ Ksq
r
where I0 and I represent the fluorescence intensities in the absence
and presence of the complex, respectively, and r is the concentra-
tion ratio of the complex to DNA. Ksq is a linear SterneVolmer
quenching constant dependent on the ratio of the bound concen-
tration of EB to the concentration of DNA. The Ksq value is 6.2 ꢆ 0.2,
2.04 ꢆ 0.4 and 1.80 ꢆ 0.4 for 1, 2 and 3 respectively. This result also
proves that complexes with bipyridine ligands are strongly bound
with DNA than that of other complexes.
It is seen from Fig. 5 that the reduction in emission intensity is
weak, in contrast to the proven DNA intercalators. The slow emis-
sion reduction and partial displacement of EB from the DNA-EB
adduct observed on the additions of 1, 2 and 3 have been attrib-
uted to competition of complexes with EB for binding to the grooves
of DNA. A simultaneous binding mode of a groove and intercalator
2.3.3. DNA melting experiments
It has been observed that the thermal behaviors of DNA in the
presence of complexes give insight into their conformational
changes when temperature is raised, and binding of metal
complexes to the double stranded DNA usually stabilizes the duplex
structure to some extent depending on the strength of interaction

258
T. Sathiya Kamatchi et al. / European Journal of Medicinal Chemistry 59 (2013) 253e264
(1)
(1)
30
20
10
1.5
1.2
0.9
0
(2)
(2)
30
1.12
20
10
0.98
1.20
0
(3)
30
(3)
1.05
0.90
20
10
0
0.00
0.02
0.04
0.06
0.08
0.10
[Complex]/[DNA]
550
600
650
700
Fig. 6. SterneVolmer quenching plot of EB bound to CT-DNA by ruthenium complexes
1e3.
Wavelength (nm)
Fig. 5. Emission spectra of EB bound to DNA in the absence (- - -) and in the presence
of 1e3.
usually increases the relative specific viscosity due the lengthening
of the DNA double helix which results from intercalation. Among
the four Ru(II) complexes 1, 2 and 3 decreases the viscosity of rod
like DNA. This result suggested that 1, 2 and 3 are non-intercalators,
and possibly groove binders. The groove binder does not lengthen
the DNA molecules and does not increase the viscosity of DNA
solutions [57]. On increasing the concentration of 4, the relative
viscosity of DNA did not show much alteration, which is consistent
with the well known [Ru(bpy)3]3þ. Such a trend is typical of elec-
trostatic mode [58]. This result suggested that the complexes
with nucleic acid [53]. Hence, melting of CT DNA in the presence of
added complexes was studied. As shown in Fig. 7, the melting
temperature of calf-thymus DNA in the absence of complexes was
measured as 60.30 ꢆ 0.5 ꢃC. The binding metal complexes should
lead to an increase in the melting temperature (Tm) of DNA as
compared to DNA itself. In our case, only an insignificant shift in Tm
was observed in the presence of 4, indicating mainly surface
aggregation or electrostatic interaction of the complexes to the CT-
DNA. In contrast, the addition of 1, 2 and 3 to the DNA solution
resulted in slight increase of Tm (the D
Tm is 3 ꢃC for 1, 2 ꢃC for 2 and
0.9
0.8
0.7
3) value of the DNA. This is comparable to that observed for DNA
groove binders [54]. Since none of the six Ru (II) complexes
exhibited large Tm values, intercalative DNA binding mode could
unequivocally be excluded and the small shift in Tm values suggest
only an unfavored interaction with CT-DNA. The experimental
results also confirm that complexes with bipyridine moiety
exhibited larger DNA-binding affinity than that of the complexes
containing bipyridine carboxylic acid.
2.3.4. Viscometric studies
The viscosity measurement is important in the investigation of
binding mode of complexes with DNA in solution in the absence of
crystallographic structural data [55] and it is sensitive to change in
length of DNA. The effect of increasing amounts of complexes and
EB on the relative viscosities of CT-DNA is shown in Fig. 8. The
effects of the complexes on the viscosities of rod-like DNA together
with [Ru(bpy)3]2þ and ethidium bromide [56] are also shown in
Fig. 8 for comparison purpose. Under appropriate conditions, EB
30
40
50
60
70
80
90
100
Temperature oC
Fig. 7. Thermal denaturation plots of 100
mM CT-DNA alone and in the presence of
complexes at R ¼ [Ru]/[DNA] ¼ 0.1. DNA (-), 1(C), 2(:), 3 (+), 4(A).

T. Sathiya Kamatchi et al. / European Journal of Medicinal Chemistry 59 (2013) 253e264
259
favored and hence, relatively small binding affinity is only observed
for 3 and 4, when compared to that of 1 and 2.
1.15
1.10
1.05
1.00
2.4. Cytotoxic activity
The cytotoxic activity of the new complexes was evaluated using
different assays namely MTT, SRB and trypan blue dye exclusion
assay on a panel of cancer cell lines. The MTT assay is based on the
mitochondrial reduction of the tetrazolium salt by actively growing
cells to produce blue insoluble formazan crystals i.e. only live cells
reduce yellow MTT to blue formazan products. The SRB assay is
based on binding of the dye to basic amino acids of cellular
proteins, and colorimetric evaluation provides an estimate of total
protein mass, which is related to cell number. The dye exclusion
test is based on the ability of viable cells to be impermeable to
trypan blue. When membrane integrity of the cells is compromised,
there is an uptake of the dye into the cells so that viable cells, which
are unstained, appear clear with a refractile ring around them and
nonviable cells appear as dark blue colored with no refractile ring
around them. HeLa (human cervical cancer cells), HEp-2 (human
laryngeal epithelial carcinoma cells) and Hep G2 (human liver
carcinoma cells) were utilized for MTT assay, SKOV3 (Human
Ovarian adeno carcinoma), SKMel2 (Human Skin Melanoma) and
HCT-15 (Human Colon tumor) cell lines used for SRB assay and
dalton lymphoma ascites (DLA) and ehrlich ascites carcinoma cells
(EAC) used for trypan blue dye exclusion method. In all the assays,
cisplatin, a clinically used antitumor agent was also evaluated for
positive control. Appropriate ruthenium precursors and ligands
used in the synthesis of the complexes were also tested for cyto-
toxicity. Reproducible IC50 values were obtained for all the
complexes and are given in Table 3. The effects of 1e4, precursors,
ligands and cisplatin on the viability of these cells were evaluated
after an exposure period of 48 h in MTT and SRB assays. Though we
have used three different assays (MTT, SRB, and trypan blue) having
different working principles to study the cytotoxic activity of
complexes on different cell lines, correlated anti-tumor activity
results were obtained for all the complexes in all the three assays.
The ligands and ruthenium precursors did not show any
0.00
0.02
0.04
0.06
0.08
0.10
[Complex]/[DNA]
Fig. 8. Effect of increasing amounts of complexes and EB on the relative viscosities of
;
CT-DNA in 5 mM TriseHCl buffer (pH 7.0). EB (+), [Ru(bpy)3]2þ(-), 1(C), 2(:), 3 ( ),
4(=).
containing non planar ligand moiety (bipyridine) bind to DNA
strongly compared to complexes with carboxylic acid bipyridine
moiety. Such behavior is very similar to that of the groove-binding.
2.3.5. Discussion
The above spectral results together with binding constant
revealed that complex 1 bind to DNA through groove binding mode
with higher affinity than the other Ru(II) complexes (2, 3 and 4).
The difference in DNA-binding affinity observed for 1 and 2 may be
due to the presence of different ancillary ligands. Complex 2 which
contain AsPh3 can hinder the strength of binding of the chelating
ligand into DNA when compared to PPh3. The effective binding
strength of 1 could be explained by relatively small steric hindrance
of PPh3. Furthermore, complexes containing bipyridine (1, 2)
showed better binding affinity compared to the complexes con-
taining bipyridine dicarboxylic acids (3 and 4). This might be due to
the substitution of carboxyl acid groups in bipyridine leading to
differences in space configuration and the electron density distri-
bution affecting the effective binding to DNA. Moreover, since all
the octahedral complexes bind to DNA in three dimensions, the
presence of ancillary ligands may also have an effect on the binding
property. This has been reflected in exhibiting better binding
affinity for 1 and 2 which contained only one PPh3 or AsPh3
compared to 3 and 4 which contain two PPh3 or AsPh3 exerting
more steric hindrance. Therefore, 1 and 2 are probably more tightly
bound to DNA helix than 3 and 4. In addition to this, one of the
carboxylic acid groups which exist in anionic form in 3 and 4 will
electrostatically repel with negatively charged phosphate ion.
Hence, the strong DNA interaction would not be particularly
significant activity even up to 1000
mM of concentration on all
cancer cells tested, which confirmed that the chelation of the ligand
with the Ru(II) ion being the only responsible factor for the
observed cytotoxic properties of the new complexes. As shown in
Table 3, the tested complexes showed moderate to potent anti-
cancer activities against the tested cells, with IC50 values ranging
from 5.1 to 560 mg/mL. In general 3 and 4 are most promising and
exhibited comparable cytotoxic properties. The activity of 3 is
better than that of 4 and outperforming cisplatin. When the IC50
value of 3 is compared with that of cisplatin against DLA, it is found
that the cytotoxic activity of 3 is about ten times higher than that of
cisplatin. Complex 3 showed almost equipotent activity with
cisplatin (IC50, 16.2 ꢆ 0.7) against HeLa cell line (IC50, 16.8 ꢆ 5.9)
Table 3
IC50 (mg/mL) of ruthenium complexes and cisplatin against various cancer cell lines.
Complexes
IC50(m
g/mL)a
MTT assay
SRB assay
Trypan blue dye exclusion assay
HeLa
Hep-G2
HEp-2
SKOV3
SKMel2
HCT-15
DLA
EAC
1
2
3
4
48.8 ꢆ 2.3
59.3 ꢆ 0.4
16.8 ꢆ 5.9
18.5 ꢆ 6.3
16.2 ꢆ 0.7
57.2 ꢆ 3.7
64.8 ꢆ 0.9
20.4 ꢆ 1.8
27.2 ꢆ 0.4
12.5 ꢆ 2.5
400 ꢆ 3.0
560 ꢆ 6.0
187 ꢆ 2.0
219 ꢆ 3.0
8.3 ꢆ 0.2
118.7 ꢆ 9.8
138.41 ꢆ 2.1
11.50 ꢆ 0.7
15.88 ꢆ 0.0
13.65 ꢆ 1.4
147.72 ꢆ 6.7
159.97 ꢆ 0.8
9.06 ꢆ 0.1
130.38 ꢆ 3.9
178.60 ꢆ 2.7
8.50 ꢆ 0.1
100 ꢆ 4.0
240 ꢆ 1.0
120 ꢆ 3.0
5.1 ꢆ 0.2
300 ꢆ 5.0
12.4 ꢆ 0.1
31.94 ꢆ 2.3
32.5 ꢆ 0.2
14.87 ꢆ 0.1
9.77 ꢆ 0.3
10.62 ꢆ 0.2
7.66 ꢆ 0.5
9.23 ꢆ 2.5
49.56 ꢆ 3.5
Cisplatin
a
Concentration of the drug that produces 50% inhibition of the cancer cells.

260
T. Sathiya Kamatchi et al. / European Journal of Medicinal Chemistry 59 (2013) 253e264
Table 4
Antioxidant activity of the ligands, precursors, new complexes, Vitamin C and BHT against various radicals.
Compounds
IC50(mM)
DPPHꢁ
OHꢁ
93.7 ꢆ 1.7
NOꢁ
O2
H2O2
Metal chelating
ꢀ$
Bpy
H2L
88.6 ꢆ 0.4
101.4 ꢆ 1.3
199.7 ꢆ 3.4
243.5 ꢆ 4.5
72.3 ꢆ 2.4
79.8 ꢆ 3.7
45.9 ꢆ 0.2
56.7 ꢆ 1.1
147.6 ꢆ 4.2
86.2 ꢆ 1.8
103.5 ꢆ 2.5
97.4 ꢆ 1.8
212.2 ꢆ 4.8
256.3 ꢆ 5.9
76.2 ꢆ 2.4
81.1 ꢆ 1.8
55.1 ꢆ 2.6
65.9 ꢆ 2.1
215.8 ꢆ 2.7
154.3 ꢆ 2.4
93.9 ꢆ 4.7
109.3 ꢆ 1.1
267.4 ꢆ 3.6
294.2 ꢆ 4.8
83.5 ꢆ 2.6
96.8 ꢆ 0.9
61.5 ꢆ 1.7
73.6 ꢆ 2.2
221.4 ꢆ 1.2
131.6 ꢆ 1.5
114.4 ꢆ 2.9
105.8 ꢆ 2.5
288.5 ꢆ 3.2
302.2 ꢆ 2.7
81.7 ꢆ 2.7
90.9 ꢆ 0.4
56.5 ꢆ 2.9
68.1 ꢆ 1.6
238.5 ꢆ 3.6
149.8 ꢆ 4.3
50.8 ꢆ 0.9
66.2 ꢆ 1.2
387.9 ꢆ 1.8
413.1 ꢆ 2.3
>500
91.7 ꢆ 0.3
234.9 ꢆ 1.5
289.8 ꢆ 5.8
60.1 ꢆ 0.5
69.4 ꢆ 1.3
41.4 ꢆ 0.7
54.6 ꢆ 2.8
232.8 ꢆ 1.9
163.4 ꢆ 0.7
[RuHCl(CO)(PPh3)3]
[RuHCl(CO)(AsPh3)3]
1
2
3
4
>500
59.8 ꢆ 4.0
67.3 ꢆ 2.9
e
Vitamin C
BHT
e
whereas 4 showed IC50 of 31.94 ꢆ 2.87 against EAC cells compared
to cisplatin (IC50, 32.5 ꢆ 0.20). The IC50 values suggest a variation of
cell sensitivity in the cell lines studied and was in the sequence of
3e4 > 1e2, which is inconsistent with the trends of their DNA-
binding abilities. Another generalization is that there is no
substantial variation in the cytotoxic activities when triphenyl-
phosphine is replaced by triphenylarsine in the complexes.
Considering the specific structures of 1, 2 and 3, 4, it appears that
the carboxyl moiety of 3 and 4 may play a critical role for the
significant cytotoxic activity on the tested cancer cell lines. The
COOH group of 3 and 4 could readily bind to certain biomolecules
over expressed on surfaces of the rapidly growing cancer cells. The
efficacy of 3 and 4 in inhibiting the growth of HeLa, Hep G2 and
SKOV3 tumor cell lines are much better than that reported for other
similar ruthenium complexes [59e61]. Cytotoxic effects of 1 and 2
are low to moderate on all the tested cell lines and 2 did not show
3e4 > 1e2 > Ligands > Precursors:
The appropriate attachment of COOH group is only the
responsible factor, which formulates 3 and 4 superior to 1 and 2 in
all the radical scavenging assays. The hydroxyl radical scavenging
power of the tested complexes was the most (41.4 ꢆ 0.7
the superoxide anion radical scavenging ability was the least
(73.6 ꢆ 2.2 M). It is observed that 1 and 2 did not produce any
noteworthy metal chelating activity even after 500 M of concen-
mM), and
m
m
tration on Fe2þ ions implying that these complexes are virtually
devoid of metal chelating properties where as 3 and 4 showed
moderate to high metal chelating activity which might be due to
the chelation of Fe2þ ion by the electron rich anion COOꢀ and
uncoordinated COOH group. It is to be noted that no significant
radical scavenging activities were observed in all the experiments
carried out with ruthenium precursor complexes and ligands under
the same experimental conditions. From the above results, it can be
concluded that the scavenging effects of the precursor complexes
and free ligands is significantly less when compared to their cor-
responding Ru(II) complexes, which is mainly due to the chelation
of the organic ligand with the Ru(II) ion. Although the radical
scavenging mechanism of the complexes under study remains
unclear, the experimental results are helpful in designing more
effective antioxidant agents against free radicals.
any significant activity even up to 500 mM concentration on HEp-2
tumor cells.
2.5. Antioxidant activity
Free radicals that are generated in many bioorganic redox
processes may induce oxidative damage in various components
of the body (lipids, proteins and DNA) and have been implicated
in aging and a number of life-limiting chronic diseases such as
cancer, hypertension, cardiac infarction, atherosclerosis, rheuma-
tism, cataracts and others [62,63]. Efforts to counteract the
damage caused by these species are gaining acceptance as a basis
for novel therapeutic approaches and the field of preventive
medicine is experiencing an upsurge of interest in medically
useful antioxidants. We carried out experiments to explore the
free radical scavenging ability of the new complexes, with a hope
to develop potential antioxidants and therapeutic reagents. The
IC50 values of all the complexes (Table 4) obtained from different
type of assay experiments strongly support that the complexes
presented in this work possess excellent antioxidant activities,
which are better than that of the standard antioxidants including
natural antioxidant vitamin C and synthetic antioxidant BHT.
The reactivity of free radicals can be neutralized by the donation
of an electron or hydrogen. Lower the IC50, greater the hydrogen
donating ability and thus the antioxidant activity of the free radical
scavengers. Consistent with the above fact, 3 and 4 showed better
radical scavenging activity than 1 and 2. This might be due to the
presence of carboxylic acid in 3 and 4 thus making those complexes
efficient hydrogen donors to stabilize the unpaired electrons and
thereby scavenging the free radicals. Despite the complexes 1e2
having RueH bond, the observed decreasing rank order of radical
scavenging activity is
3. Conclusions
The present contribution describes the synthesis of four new
ruthenium complexes comprising versatile polypyridyl ligands.
Chelating ligands employed include 2,20-bipyridine and 2,20-
bipyridine-4,40-dicarboxylic acid. All the complexes have been
extensively characterized. The structure of complex 3 was estab-
lished by X-ray crystallography. Single crystal XRD upshots of
complexes revealed an octahedral geometry around the ruthenium
ion with bipyridine dicarboxylic acid as a neutral bidentate donor
with the involvement of both the nitrogen atoms of bipyridine
ring. The incorporation of simple modifications in ancillary ligand
sphere and COOH group in the bipyridine ring resulted in signifi-
cant differences in DNA binding behaviors for complexes. More-
over, the fine points obtained from cytotoxic studies and various
antioxidant assays showed that the Ru(II) complexes enclosing
2,20-bipyridine-4,40-dicarboxylic acid showed good signs of
significant cytotoxic activity and excellent radical scavenging
ability. On the whole, the introduction of COOH group in the
bipyridine ring markedly increases the antitumor and antioxidant
efficiency of the synthesized complexes. With careful selection of
the substituents on the ligands, the anticancer properties of the
complexes can be improved, and efficient multifunctional mole-
cules can be designed. Work is currently in progress to explore

T. Sathiya Kamatchi et al. / European Journal of Medicinal Chemistry 59 (2013) 253e264
261
these and related systems in detail to derive structure activity
relationships.
H2O)/nm 266 (ε/dm3 molꢀ1 cmꢀ1 24,257), 311 (17,590) and 404
(3060); IR (KBr disks, nmax/cmꢀ1) 1472 (C]N); 1941 (C^O); 330
(RueCl); 1H NMR; dH(500 MHz; DMSO-d6; 25 ꢃC, TMS): 9.12 (1H,
d, J ¼ 9.0 Hz, ]CH6), 8.56 (1H, d, J ¼ 4.5 Hz, ]CH6’), 7.99 (1H, t,
J ¼ 7.5 Hz, ]CH5), 7.79 (1H, t, J ¼ 7.5 Hz, ]CH5’), 7.22 (1H, t,
J ¼ 6.5 Hz, ]CH4), 6.54 (1H, t, J ¼ 6.5 Hz, ]CH4’),7.39 (2H, d,
J ¼ 5.0 Hz, ]CH3, ]CH3’), 7.14e7.39 (15 H, m, Ph), ꢀ11.47 (1H, s,
RueH).
4. Experimental section
4.1. General procedures
All chemicals were reagent grade and were used as received from
commercial suppliers unless otherwise stated. All the solvents were
degassed and distilled according to standard procedures [64].
Commercially available RuCl3$3H2O (Himedia) was used without
further purification. The starting complexes [RuHCl(CO)(PPh3)3]
[65], [RuHCl(CO)(AsPh3)3] [66]were prepared as reported earlier.
The ligand 2,20-bipyridine was purchased from Merck chemicals.
2,20-bipyridine-4,40-dicarboxylic acid was purchased from Sigmae
Aldrich. Melting points were determined with Lab India instru-
ment. Elemental analyses (C, H, N, S) were performed on Vario EL III
Elementar elemental analyzer. Electronic absorption spectra were
recorded using JASCO 600 spectrophotometer. Nicolet Avatar Model
FT-IR spectrophotometer was used to record the IR spectra (4000e
400 cmꢀ1) of the free ligands and the complexes. 1H NMR spectra
were recorded on Bruker AMX 500 at 500 MHz using tetrame-
thylsilane as an internal standard. Calf thymus DNA (CT-DNA) was
purchased from Sigma and dissolved in 5 mM Tris HCl buffer (pH 7.2)
containing 100 mM NaCl and 1 mM EDTA. It was dialyzed several
times against 5 mM Tris HCl buffer. All experiments involving
interactions of complexes with CT-DNA were carried out in Tris HCl
buffer (pH 7.0). The concentrations of DNA, complexes 1, 2, 3 and 4
were determined spectrophotometrically using their extinction
4.2.3. Synthesis of [RuCl(HL)(PPh3)2(CO)] (3)
An ethanolic (20 mL) solution of 2,20-bipyridine-4,40-dicarbox-
ylic acid (H2L) (0.026 g, 0.106 mmol) was added to a refluxing
solution of [RuHCl(CO)(PPh3)3], (0.101 g, 0.106 mmol) in ethanol
(20 mL). The mixture was heated under reflux for 2 h. The solution
was filtered while hot, reduced to half of its volume and left for
slow evaporation. The semi-crystalline product that separated out
was filtered off, washed with ethanol and dried under vacuum. X-
ray quality crystals of the product were grown by recrystallization
from dichloromthane/methanol (1:1) mixture.
Yield ¼ 0.075 g, 77%; m.p. 158 ꢃC; elemental analysis calcd. (%)
for C49H37ClN2O5P2Ru: C, 63.13; H, 4.00; N, 3.01%. Found: C, 63.21;
H, 4.01; N, 3.01%; UVevisible: lmax(solvent : 5% DMSO/H2O)/nm
266 (ε/dm3 molꢀ1 cmꢀ1 34,840), 315 (25,870) and 404 (3090); IR
(KBr disks, nmax/cmꢀ1) 3414 (OH); 1714 (C]O); 1635 (COO,
asymm); 1433 (COO, symm); 1484 (C]N); 1230 (CeO); 1941
(C^O); 326 (RueCl); 1H NMR; dH(500 MHz; DMSO-d6; 25 ꢃC,
TMS): 13.62 (1H, s, OH), 9.31 (1H, d, J ¼ 5.5 Hz, ]CH6), 7.89 (1H, d,
J ¼ 5.5 Hz, ]CH6’), 7.69 (1H, dd, Jo ¼ 5.5, Jm ¼ 1 Hz, ]CH5), 6.90
(1H, dd, Jo ¼ 5.5, Jm ¼ 1 Hz, ]CH5’), 8.51 (1H, s, ]CH3), 8.47 (1H, s,
]CH3’),7.22e7.40 (30H, m, Ph).
coefficients ε310nm ¼ 18,600 Mꢀ1 cmꢀ1, ε311nm ¼ 17,590 Mꢀ1 cmꢀ1
,
ε315nm ¼ 25,870 Mꢀ1 cmꢀ1, ε310nm ¼ 23,130 Mꢀ1 cmꢀ1 respectively.
Fluorescence spectra were measured on a Perkin Elmer spectroflu-
orimeter. Antioxidant activity measurements were done using UV
spectrophotometer (UV-1800, Shimadzu).
4.2.4. Synthesis of [RuCl(HL)(AsPh3)2(CO)] (4)
It was synthesized using the same procedure as described for 3
with the ligand (H2L) (0.026 g, 0.106 mmol) and precursor complex
[RuHCl(CO)(AsPh3)3] (0.115 g, 0.106 mmol).
4.2. Synthesis of complexes
Yield ¼ 0.092 g, 86%; m.p. 162 ꢃC; elemental analysis calcd. (%)
for C49H37ClN2O5As2Ru: C, 57.69; H, 3.65; N, 2.75%. Found: C, 57.82;
H, 3.66; N, 2.74%; UVevisible: lmax(solvent: 5% DMSO/H2O)/nm 260
(ε/dm3 molꢀ1 cmꢀ1 28,355), 310 (23,130) and 405 (2310); IR (KBr
disks, nmax/cmꢀ1) 3399 (OH); 1719 (C]O); 1625 (COO, asymm);
1434 (COO, symm); 1482 (C]N); 1230 (CeO); 1942 (C^O); 329
(RueCl); 1H NMR; dH(500 MHz; DMSO-d6; 25 ꢃC, TMS): 13.47 (1H, s,
OH), 9.41 (1H, d, J ¼ 5.5 Hz, ]CH6), 7.92 (1H, d, J ¼ 5.5 Hz, ]CH6’),
7.76 (1H, dd, Jo ¼ 5.5, Jm ¼ 1 Hz, ]CH5), 6.83 (1H, dd, Jo ¼ 5.5,
Jm ¼ 1 Hz, ]CH5’), 8.55 (1H, s, ]CH3), 8.73 (1H, s, ]CH3’),7.27e
7.46 (30 H, m, Ph).
4.2.1. Synthesis of [RuHCl(bpy)(PPh3)(CO)] (1)
[RuHCl(CO)(PPh3)3] (0.101 g, 0.106 mmol) and the ligand 2,20-
bipyridine (0.017 g, 0.106 mmol) were suspended in distilled
ethanol (20 mL). Half an hour at reflux was needed to solubilize
both the metal and chelating partner. The resulting yellow solution
was maintained at reflux for 2 h. This solution was allowed to stand
at room temperature for 3 days. The resulting yellow precipitate
was filtered off, washed with ethanol, diethyl ether and dried under
vacuum to afford complex 1.
Yield ¼ 0.056 g, 92%; m.p. 143 ꢃC; elemental analysis calcd. (%)
for C29H24ClN2OPRu: C, 59.64; H, 4.14; N, 4.80%. Found: C, 59.58; H,
4.13; N, 4.81%; UVevisible: lmax(solvent: 5% DMSO/H2O)/nm 266
(ε/dm3 molꢀ1 cmꢀ1 25,358), 310 (18,600) and 408 (2640); IR (KBr
disks, nmax/cmꢀ1) 1479 (C]N); 1936 (C^O); 331 (RueCl); 1H NMR;
dH(500 MHz; DMSO-d6; 25 ꢃC, TMS): 9.00 (1H, d, J ¼ 9.0 Hz, ]CH6),
8.43 (1H, d, J ¼ 4.5 Hz, ]CH6’), 7.97 (1H, t, J ¼ 7.5 Hz, ]CH5), 7.85
(1H, t, J ¼ 7.5 Hz, ]CH5’), 7.02 (1H, t, J ¼ 6.5 Hz, ]CH4), 6.33 (1H, t,
J ¼ 6.5 Hz, ]CH4’),7.41 (2H, d, J ¼ 5.0 Hz, ]CH3, ]CH3’), 7.17e7.33
(15H, m, Ph), ꢀ11.34 (1H, s, RueH).
4.3. X-ray crystallography
X-ray diffraction measurements were performed at low
temperature on a Nonius Kappa CCD diffractometer equipped with
graphite monochromated Mo K
a radiation and an Oxford Cryo-
stream cryostat. The structure of the complex was solved by direct
methods and refinements were carried out by full matrix least-
square techniques. The hydrogen atoms were generally visible in
difference maps, and were placed in idealized positions and treated
as riding in the refinements. Disorder was present in the structure,
including disordered solvent. The Ru complex lies on a twofold axis,
thus the carbonyl and chloro ligand are superimposed, as are the
COOꢀ and COOH. Cl1, C7, O3 and the H atom on O1 were assigned
half populations, the displacement parameters of C7 and O3 were
constrained to be identical, and no further restraints were applied.
Contribution to the structure factors from disordered solvent was
removed for refinement using the SQUEEZE procedure. The
following computer programs were used: structure solution SIR-97
4.2.2. Synthesis of [RuHCl(bpy)(AsPh3)(CO)] (2)
The complex was prepared as a crystalline yellow solid by the
same procedure as described above replacing [RuHCl(CO)(PPh3)3]
by [RuHCl(CO)(AsPh3)3] (Ru, 0.115 g, 0.106 mmol; bpy, 0.017 g,
0.106 mmol).
Yield ¼ 0.059 g, 89%; m.p. 151 ꢃC; elemental analysis calcd. (%)
for C29H24ClN2OAsRu: C, 55.47; H, 3.85; N, 4.46%. Found: C,
55.56; H, 3.86; N, 4.45%; UVevisible: lmax(solvent: 5% DMSO/

262
T. Sathiya Kamatchi et al. / European Journal of Medicinal Chemistry 59 (2013) 253e264
[67], refinement SHELXL-97 [68], molecular diagrams, ORTEP-3 for
4.5.2. In vitro anticancer activity evaluation by SRB assay
Windows [69] and SQUEEZE [39].
Cells were plated in 96-multiwell plate (104 cells/well) for 24 h.
The complexes to be tested were dissolved in dimethyl sulphoxide.
Solutions of different concentration of the complexes and cisplatin
under test were added to the cell monolayer. Triplicate wells were
prepared for each individual concentration. The cytotoxicity assay
was done against SKMel2 (1 ꢂ 105 cells/mL), SKOV3 (1 ꢂ 105 cells/
mL), and HCT-15 (1 ꢂ 105 cells/mL) cell lines using a colorimetric
SRB assay method [71]. Exponentially growing cells were harvested
4.4. DNA binding experiments
The experiments were carried out in 5 mM TriseHCl buffer (pH
7.0) at ambient temperature and the complexes were dissolved in
5 mM TriseHCl buffer containing 5% DMSO. Changes in the fluo-
rescence emission spectrum of the ethidium bromide (referred to
as EB)eDNA complex were recorded under various complex
concentrations. The fluorescence spectra in the fluorimeter were
obtained at an excitation wavelength of 522 nm and an emission
wavelength of 584 nm. Melting profiles were measured at 260 nm
by a Cary 300 spectrophotometer. Readings were recorded for
every 1.5 ꢃC raise in temperature per minute. The viscosity
measurement was carried out using an Ubbelohde viscometer
immersed in a thermostatic water bath maintained at 25(ꢆ0.l) ꢃC.
DNA samples with approximately 200 base pairs in length were
prepared by sonication in order to minimize complexities arising
from DNA flexibility. Flow times were measured with a digital
stopwatch; each sample was measured three times, and an average
flow time was calculated. Relative viscosities for CT-DNA in the
presence and absence of the complex were calculated from the
and suspended in the culture media (100 mL, RPMI-1640) in a 96-
well plate. After 24 h of incubation at 37 ꢃC in humidified 5% CO2,
the cells were treated with varying concentrations of test
complexes (100 mL) and incubated further for 48 h under the same
conditions. The cells were fixed with 50% trichloroacetic acid and
stained for 30 min with SRB. The unbound dye was removed by 1%
acetic acid, and the protein-bound dye was extracted with 10 mM
tris base (pH 10.5) for 5 min. The optical density was measured at
520 nm in a micro plate reader to determine cell growth inhibition.
The results were expressed as the concentration at which there was
50% inhibition (IC50).
4.5.3. In vitro anticancer activity evaluation by trypan blue
exclusion method
The tumor cells (DLA and EAC) were aspirated from the perito-
neal cavity of tumor bearing mice and they were washed thrice with
normal saline and checked for viability using trypan blue dye
exclusion method. The viable cell suspension (1 ꢂ106 cells in 0.1 mL)
was added to tubes containing various concentrations of the test
complexes and the volume was made up to 1 mL using phosphate
buffered saline (PBS). Control tube contained only cell suspension.
These assay mixtures were incubated for 3 h at 37 ꢃC. The cell
suspension was further mixed with 0.1 mL of 1% trypan blue and
kept for 2e3 min and loaded on a hemocytometer. Dead cells take up
the blue color of trypan blue while live cells do not take up the dye.
The number of stained and unstained cells was counted separately.
relation
containing solution and t0 is the flow time of TriseHCl buffer
alone. Data are presented as ( h0 h
1/3 versus binding ratio, where
is the viscosity of CT-DNA in the presence of complex and h0 is the
viscosity of CT-DNA alone.
h
¼ (t ꢀ t0)/t0, where t is the observed flow time of DNA-
h/
)
4.5. In vitro anticancer activity evaluation
4.5.1. In vitro anticancer activity evaluation by MTT assay
Cytotoxicity studies of the complexes and cisplatin (positive
control) were carried out on human cervical cancer cells (HeLa),
human laryngeal epithelial carcinoma cells (HEp-2) and human
liver carcinoma cells (Hep G2), all of which were obtained from
National Centre for Cell Science (NCCS), Pune, India. Cell viability
was carried out using the MTT assay method [70]. The HeLa, Hep
G2 and HEp-2 cells were grown in Eagles minimum essential
medium containing 10% fetal bovine serum (FBS). For screening
%of cytotoxicity ¼ ½numberof deadcells=ðnumberof viablecells
þnumberof deadcellsÞꢄꢂ100
4.6. Antioxidant assays
experiments, the cells were seeded into 96-well plates in 100 mL
of respective medium containing 10% FBS, at plating density of
10,000 cells/well and incubated at 37 ꢃC, 5% CO2, 95% air and
100% relative humidity for 24 h prior to addition of complexes.
The complexes were dissolved in DMSO and diluted in respective
medium containing 1% FBS. After 24 h, the medium was replaced
with respective medium with 1% FBS containing the complexes
at various concentration and incubated at 37 ꢃC, 5% CO2, 95% air
and 100% relative humidity for 48 h. Triplicate was maintained
and the medium without the complexes and cisplatin were
The ability of ruthenium complexes to act as hydrogen donors or
free radical scavengers was tested by conducting a series of in vitro
antioxidant assays involving DPPH radical, hydroxyl radical, nitric
oxide radical, hydrogen peroxide, superoxide anion radical, metal
chelating assay and the results were compared with that of stan-
dard antioxidants including natural antioxidant Vitamin C and
synthetic antioxidant BHT (Butylated Hydroxy Toluene).
4.6.1. DPPHꢁ scavenging assay
served as negative control. After 48 h, 10 mL of MTT (5 mg/mL) in
The DPPH radical scavenging activity of the compounds was
measured according to the method of Blios [72]. The DPPH radical
is a stable free radical and due to the presence of an odd electron,
it shows a strong absorption band at 517 nm in visible spectrum. If
this electron becomes paired off in the presence of a free radical
scavenger, this absorption vanishes resulting in decolorization
stoichiometrically with respect to the number of electrons taken
up. Various concentrations of the experimental complexes were
phosphate buffered saline (PBS) was added to each well and
incubated at 37 ꢃC for 4 h. The medium with MTT was then
flicked off and the formed formazan crystals were dissolved in
100 mL of DMSO and then measured the absorbance at 570 nm
using micro plate reader. The % cell inhibition was determined
using the following formula, and a graph was plotted between %
of cell inhibition and concentration. From this plot, the IC50 value
was calculated.
taken and the volumes were adjusted to 100 mL with methanol.
About 5 mL of 0.1 mM methanolic solution of DPPH was added to
the aliquots of samples and standards (BHT and Vitamin C) and
shaken vigorously. Negative control was prepared by adding
% inhibition ¼ ½mean OD of untreated cellsðcontrolÞ=mean
OD of treated cellsðcontrolÞꢄ ꢂ 100
100
mL of methanol in 5 mL of 0.1 mM methanolic solution DPPH.
The tubes were allowed to stand for 20 min at 27 ꢃC. The

T. Sathiya Kamatchi et al. / European Journal of Medicinal Chemistry 59 (2013) 253e264
263
absorbance of the sample was measured at 517 nm against the
4.6.6. Metal chelating activity
blank (methanol).
The chelation with ferrous ions by the experimental complexes
was estimated by the method of Dinis et al. [77]. Initially, about
4.6.2. OHꢁ scavenging assay
100
mL the samples and the standards were added to 50 mL solution
The hydroxyl radical scavenging activity of the complex has
been investigated by using the Nash method [73]. In vitro hydroxyl
radicals were generated by Fe3þ/ascorbic acid system. The detec-
tion of hydroxyl radicals was carried out by measuring the amount
of formaldehyde formed from the oxidation reaction with DMSO.
The formaldehyde produced was detected spectrophotometrically
at 412 nm. A mixture of 1.0 mL of iron-EDTA solution (0.13%
ferrous ammonium sulfate and 0.26% EDTA), 0.5 mL of EDTA
solution (0.018%), and 1.0 mL of DMSO (0.85% DMSO (v/v) in 0.1 M
phosphate buffer, pH 7.4) were sequentially added in the test
tubes. The reaction was initiated by adding 0.5 mL of ascorbic acid
(0.22%) and was incubated at 80e90 ꢃC for 15 min in a water bath.
After incubation, the reaction was terminated by the addition of
1.0 mL of ice-cold trichloroacetic acid (17.5% w/v). Subsequently,
3.0 mL of Nash reagent was added to each tube and left at room
temperature for 15 min. The intensity of the color formed was
measured spectrophotometrically at 412 nm against the reagent
blank.
of 2 mM FeCl2. The reaction was initiated by the addition of 200 mL
of 5 mM ferrozine and the mixture was shaken vigorously and left
standing at room temperature for 10 min. Absorbance of the
solution was then measured spectrophotometrically at 562 nm
against the blank (deionized water).
For the above six assays, all the tests were run in triplicate and
the percentage activity was calculated using the following equation
Scavenging activity ð%Þ ¼ ½ðA0 ꢀ A1Þ=A0ꢄ ꢂ 100
where, A0 is the absorbance of the control and
A1 is the absorbance of the complex/standard.
When the inhibition of the tested compounds is 50%, the tested
compound concentration is IC50
.
Acknowledgments
4.6.3. NOꢁ scavenging assay
Department of Science and Technology, New Delhi, India [Grant
No. SR/SI/IC-43/2007] for funds for the research and the University
Grants Commission, New Delhi, for the award of a Fellowship in
Science for Meritorious Students (RFSMS) to T. Sathiya Kamatchi
under the UGC-SAP-DRS programme are gratefully acknowledged.
We would like to thank Mr. V.S. Jamal Ahamed and Dr. Surk Sik
Moon for their help in cytotoxic studies (Department of Chemistry,
Kongju National University, Kongju 314-701, Republic of Korea).
Acknowledgment is also made to Mr. T. Sajeesh and Dr. T. Par-
imelazhagan, Department of Botany, Bharathiar University, India
for their help in radical scavenging assays.
Assay of nitric oxide (NOꢁ) scavenging activity is based on
the method [74], where sodium nitroprusside in aqueous
solution at physiological pH spontaneously generates nitric
oxide, which interacts with oxygen to produce nitrite ions. This
can be estimated using Greiss reagent. Scavengers of nitric
oxide compete with oxygen leading to reduced production of
nitrite ions. For the experiment, sodium nitroprusside (10 mM)
in phosphate buffered saline was mixed with a fixed concen-
tration of the complex, standards and incubated at room
temperature for 150 min. After the incubation period, 0.5 mL of
Greiss reagent containing 1% sulfanilamide, 2% H3PO4 and 0.1%
N-(1-naphthyl) ethylenediamine dihydrochloride was added.
The absorbance of the chromophore formed was measured at
546 nm.
Appendix A. Supplementary information
Supplementary information related to this article can be found
at http://dx.doi.org/10.1016/j.ejmech.2012.11.024.
4.6.4. H2O2 scavenging assay
The ability of the complexes to scavenge hydrogen peroxide was
determined using the method of Ruch et al. [75]. A solution of
hydrogen peroxide (2.0 mM) was prepared in phosphate buffer
(0.2 M, pH-7.4) and its concentration was determined spectro-
photometrically from absorption at 230 nm with molar absorptivity
References
[1] C.S. Allardyce, P.J. Dyson, Platinum Met. Rev. 45 (2001) 62e69.
[2] M.J. Clarke, Coord. Chem. Rev. 236 (2003) 209e233.
[3] C.X. Zhang, S.J. Lippard, Curr. Opin. Chem. Biol. 7 (2003) 481e489.
[4] W.H. Ang, P.J. Dyson, Eur. J. Inorg. Chem. (2006) 4003e4018.
[5] Y.K. Yan, M. Melchart, A. Habtemariam, P.J. Sadler, Chem. Commun. (2005)
4764e4776.
[6] L. Ronconi, P.J. Sadler, Coord. Chem. Rev. 251 (2007) 1633e1648.
[7] A.C.G. Hotze, M. Bacac, A.H. Velders, B.A.J. Jansen, H. Kooijman, A.L. Spek,
J.G. Haasnoot, J. Reedijk, J. Med. Chem. 46 (2003) 1743e1750.
[8] A.C.G. Hotze, A.H. Velders, F. Ugozzoli, M. Biagini Cingi, A.M. Manotti Lanfredi,
J.G. Haasnoot, J. Reedijk, Inorg. Chem. 39 (2000) 3838e3844.
[9] I. Kostova, Curr. Med. Chem. 13 (2006) 1085e1107.
[10] J.M. Rademaker-Lakhai, D. Van Den Bongard, D. Pluim, J.H. Beijnen,
J.H.M. Schellens, Clin. Cancer Res. 10 (2004) 3717e3727.
[11] C.G. Hartinger, S. Zorbas-Seifried, M.A. Jakupec, B. Kynast, H. Zorbas,
B.K. Keppler, J. Inorg. Biochem. 100 (2006) 891e904.
[12] J. Reedijk, Platinum Met. Rev. 52 (2008) 2e11.
[13] A. Bergamo, B. Gava, E. Alessio, G. Mestroni, B. Serli, M. Cocchietto, S. Zorzet,
G. Sava, Int. J. Oncol. 21 (2002) 1331e1338.
81 Mꢀ1 cmꢀ1. The complexes (100
(100
mG/mL), BHT and Vitamin C
mg/mL) were added to 3.4 mL of phosphate buffer together
with hydrogen peroxide solution (0.6 mL). An identical reaction
mixture without the sample was taken as negative control.
Absorbance of hydrogen peroxide at 230 nm was determined after
10 min against the blank (phosphate buffer).
ꢀ$
4.6.5. O2 scavenging assay
ꢀ$
The superoxide anion radical ðO2 Þ scavenging assay is based
on the capacity of the complexes to inhibit formazan formation
by scavenging the superoxide radicals generated in riboflavin-
light-NBT system [76]. Each 3 mL reaction mixture contained
50 mM sodium phosphate buffer (pH 7.6), 20
12 mM EDTA, 0.1 mg NBT and 1 mL complex solution (20e
100 g/mL). Reaction was started by illuminating the reaction
mg riboflavin,
[14] L.M. Wilhelmsson, F. Westerlund, P. Lincoln, B. Norden, J. Am. Chem. Soc. 124
(2002) 12092e12093.
[15] M.A. Jakupec, E. Reisner, A. Eichinger, M. Pongratz, V.B. Arion, M. Galanski,
C.G. Hartinger, B.K. Keppler, J. Med. Chem. 48 (2005) 31e37.
[16] K.S. Smalley, R. Contractor, N.K. Haass, A.N. Kulp, G.E. Atilla-Gokcumen,
D.S. Williams, H. Bregman, K.T. Flaherty, M.S. Soengas, E. Meggers, M. Herlyn,
Cancer Res. 67 (2007) 209e217.
[17] X. Meng, M.L. Leyva, M. Jenny, I. Gross, S. Benosman, B. Fricker, S. Harlepp,
P. Hebraud, A. Boos, P. Wlosik, P. Bischoff, C. Sirlin, M. Pfeffer, J.P. Loeffler,
C. Gaiddon, Cancer Res. 69 (2009) 5458e5466.
m
mixture with different concentrations of complex for 90 s.
Immediately after illumination, the absorbance was measured at
590 nm. The entire reaction assembly was enclosed in a box lined
with aluminum foil. Identical tubes with reaction mixture kept in
dark served as blanks.

264
T. Sathiya Kamatchi et al. / European Journal of Medicinal Chemistry 59 (2013) 253e264
[18] A. Greguric, I.D. Greguric, T.W. Hambley, J.R. Aldrich-Wright, J.G. Collins,
J. Chem. Soc. Dalton Trans. (2002) 849e855.
[47] D.L. Boger, B.E. Fink, S.R. Brunette, W.C. Tse, M.P. Hedric, J. Am. Chem. Soc. 123
(2001) 5878e5891.
[19] C.W. Jiang, H. Chao, X.L. Hong, H. Li, W.J. Mei, L.N. Ji, Inorg. Chem. Commun. 6
(2003) 773e775.
[48] D.L. Boger, B.E. Fink, M.P. Hedrick, J. Am. Chem. Soc. 122 (2000) 6382.
[49] D.J. Patel, Acc. Chem. Res. 12 (1979) 118e125.
[20] J.G. Liu, B.H. Ye, Q.L. Zhang, X.H. Zou, Q.X. Zhen, X. Tian, L.N. Ji, J. Biol. Inorg.
Chem. 5 (2000) 119e128.
[21] H.-L. Huang, Z.-Z. Li, Z.-H. Liang,Y.-J. Liu,Eur.J.Inorg.Chem. 36(2011)5538e5547.
[22] L. Salassa, T. Ruiu, C. Garino, A.M. Pizarro, F. Bardelli, D. Gianolio,
A. Westendorf, P.J. Bednarski, C. Lamberti, R. Gobetto, P.J. Sadler, Organome-
tallics 29 (2010) 6703e6710.
[23] Y. Xiong, X.F. He, X.H. Zou, J.Z. Wu, X.M. Chen, L.N. Ji, R.H. Li, J.Y. Zhou, K.B. Yu,
J. Chem. Soc. Dalton Trans. (1999) 19e23.
[24] Q.X. Zhen, B.H. Ye, Q.L. Zhang, J.G. Liu, H. Li, L.N. Ji, L. Wang, J. Inorg. Biochem.
76 (1999) 47e53.
[50] B.F. Cain, B.C. Baguley, W.A. Denny, J. Med. Chem. 21 (1978) 658e668.
[51] D. Banerjee, S.K. Pal, J. Phys. Chem. B 111 (2007) 5047e5052.
[52] J.R. Lakowicz, G. Webber, Biochemistry 12 (1973) 4161e4170.
[53] (a) A. Ambroise, B.G. Maiya, Inorg. Chem. 39 (2000) 4256e4263;
(b) P.U. Maheswari, M. Palaniandavar, J. Inorg. Biochem. 98 (2004) 219e230;
(c) F. Gao, H. Chao, F. Zhou, Y.X. Yuan, B. Peng, L.N. Ji, J. Inorg. Biochem. 100
(2006) 1487e1494;
(d) R.B. Nair, E.S. Teng, S.L. Kirkland, C.J. Murphy, Inorg. Chem. 37 (1998) 39e141.
[54] (a) N. Chitrapriya, Y.J. Jang, S.K. Kim, H. Lee, J. Inorg. Biochem. 105 (2011)
1569e1575;
[25] H.-L. Huang, Z.-Z. Li, Z.-H. Liang, J.-H. Yao, Y.-J. Liu, Eur. J. Med. Chem. 46
(2011) 3282e3290.
[26] S.S. Bhat, A.S. Kumbhar, P. Lonnecke, E.H. Hawkins, Inorg. Chem. 49 (2010)
4843e4853.
[27] V. Brabec, O. Novakova, Drug Resist. Updates 9 (2006) 111e122.
[28] K.E. Erkkila, D.T. Odom, J.K. Barton, Chem. Rev. 99 (1999) 2777e2795.
[29] Y.-J. Liu, C.-H. Zeng, H.-L. Huang, L.-X. He, F.-H. Wu, Eur. J. Med. Chem. 45
(2010) 564e571.
(b) Y. Fei, G. Lu, G. Fan, Y. Wu, Anal. Sci. 25 (2009) 1333e1338.
[55] J.B. Chaires, N. Dattagupta, D.M. Crothers, Biochemistry 21 (1982) 3933e
3940.
[56] E.J. Gabbay, R.E. Scofield, C.S. Baxter, J. Am. Chem. Soc. 95 (1973) 7850e7857.
[57] D. Suh, J.B. Chaires, Bioorg. Med. Chem. 3 (1995) 723e728.
[58] L. Lerman, J. Mol. Biol. 3 (1961) 18e30.
[59] F. Gao, X. Chen, J.-Q. Wang, Y. Chen, H. Chao, L.-N. Ji, Inorg. Chem. 48 (2009)
5599e5601.
[30] P.J. Dandlier, R.E. Holmlin, J.K. Barton, Science 274 (1997) 1465e1468.
[31] X. Chen, F. Gao, W.-Y. Yang, J. Sun, Z.-X. Zhou, L.-N. Ji, Inorg. Chim. Acta 378
(2011) 140e147.
[32] (a) C.M. Giandomenico, M.J. Abrams, B.A. Murrer, J.F. Vollano, M.I. Rheinheimer,
S.B. Wyer, G.E. Bossard, J.D. Higgins, Inorg. Chem. 34 (1995) 1015e1021;
(b) M. Galanski, B.K. Keppler, Inorg. Chem. 35 (1996) 1709e1711.
[33] Y. Xiong, L.N. Ji, Coord. Chem. Rev. 185 (1999) 711e733.
[34] J.W. Mei, J. Liu, K.C. Zheng, J. Li, Lin, H. Chao, X.An. Li, C.F. Yun, L.N. Ji, Dalton
Trans. 27 (2003) 1352e1359.
[60] L. Hajji, C. Saraiba-Bello, A. Romerosa, G. Segovia-Torrente, M. Serrano-Ruiz,
P. Bergamini, A. Canella, Inorg. Chem. 50 (2011) 873e882.
[61] D.-L. Ma, C.-M. Che, F.-M. Siu, M. Yang, K.-Y. Wong, Inorg. Chem. 46 (2007)
740e749.
[62] C. Soler Rivas, J.C. Espin, H. Wichers, Phytochem. Anal. 11 (2000) 330e338.
[63] K. Tsai, T.G. Hsu, K.M. Hsu, H. Cheng, T.Y. Liu, C.F. Hsu, C.W. Kong, Free Radical.
Biol. Med. 31 (2001) 1465e1472.
[64] A.I. Vogel, Textbook of Practical Organic Chemistry, fifth ed., Longman, London,
1989.
[35] J.G. Liu, Q.L. Zhang, X.F. Shi, L.N. Ji, Inorg. Chem. 40 (2001) 5045e5050.
[36] M. Mariappan, B.G. Maiya, Eur. J. Inorg. Chem. (2005) 2164e2173.
[37] K.B. Beckman, B.N. Ames, Phys. Rev. 78 (1998) 447e453.
[38] B. Halliwel, J.M.C. Gutteridge, Clarendon Press, Oxford, 1989, 494.
[39] Spek, Acta Cryst. D65 (2009) 148e155.
[40] F. Basuli, S.M. Peng, S. Bhattacharya, Inorg. Chem. 40 (2001) 1126e1133.
[41] E. Eskelinen, S. Luukkanen, M. Haukka, M. Ahlgren, T.A. Pakkanen, J. Chem.
Soc. Dalton Trans. (2000) 2745e2752.
[65] N. Ahmad, J.J. Levison, S.D. Robinson, M.F. Uttley, Inorg. Synth. 15 (1974) 48.
[66] R.A.S. Pelgado, W.Y. Lee, S.R. Choi, Y. Cho, M.J. Jun, Trans. Met. Chem. 16 (1991)
241e244.
[67] M.C. Altomare, M. Burla, G. Camalli, C. Cascarano, A. Giacovazzo, A.G.G. Guagliardi,
G. Moliterni, R. Polidori, J. Appl. Crystallogr. 32 (1999) 115e122.
[68] G.M. Sheldrick, Acta Cryst. A64 (2008) 112e122.
[69] L.J. Farrugia, J. Appl. Crystallogr. 30 (1997) 565e566.
[70] M. Blagosklonny, W.S. EI-diery, Int. J. Cancer 67 (1996) 386e392.
[71] M.A.A. Rahman, S.C. Cho, J. Song, H.T. Mun, S.S. Moon, Planta Med. 73 (2007)
1089e1094.
[42] A.I. Philippopoulos, A. Terzis, C.P. Raptopoulou, V.J. Catalano, P. Falaras, Eur. J.
Inorg. Chem. (2007) 5633e5644.
[43] M.I. Frazão Barbosa, E.M.A. Valle, S.L. Queiroz, J. Ellena, E.E. Castellano,
V.R.S. Malta, J.R. de Sousa, O. Piro, M.P. de Araujo, A.A. Batista, Polyhedron 29
(2010) 2297e2303.
[44] P. Sengupta, R. Dinda, S. Ghosh, W.S. Sheldrick, Polyhedron 20 (2001) 3349e3354.
[45] T. Sathiya Kamatchi, N. Chitrapriya, H. Lee, C.F. Fronczek, F.R. Fronczek,
K. Natarajan, Dalton Trans. 41 (2012) 2066e2077.
[72] M.S. Blois, Nature 29 (1958) 1199e1200.
[73] T. Nash, Biochem. J. 55 (1953) 416e421.
[74] L.C. Green, D.A. Wagner, J. Glogowski, P.L. Skipper, J.S. Wishnok,
S.R. Tannenbaum, Anal. Biochem. 126 (1982) 131e138.
[75] R.J. Ruch, S.J. Cheng, J.E. Klaunig, Carcinogenesis 10 (1989) 1003e1008.
[76] C. Beauchamp, I. Fridovich, Anal. Biochem. 44 (1971) 276e287.
[77] T.C.P. Dinis, V.M.C. Madeira, L.M. Almeida, Arch. Biochem. Biophys. 315 (1994)
161e169.
[46] M. Cory, D.D. Mckee, J. Kagan, D.W. Henry, J.A. Miller, J. Am. Chem. Soc. 107
(1985) 2528e2536.