Synthesis, structural characterization, DNA/Protein binding and in vitro cytotoxicity of isomeric ruthenium carbonyl complexes

Article abstract of DOI:10.1016/j.jorganchem.2014.04.003

The reaction of salicylaldehyde-4(N)-ethylthiosemicarbazone, [H ₂-(Sal-etsc)] with [RuHCl(CO)(PPh₃)₃] afforded two isomeric ruthenium(II) carbonyl complexes namely [Ru(H-Sal-etsc)(CO) Cl(PPh₃)₂] (1) a

Full text of DOI:10.1016/j.jorganchem.2014.04.003

Accepted Manuscript
Synthesis, structural characterization, DNA/Protein binding and in vitro cytotoxicity of
isomeric ruthenium carbonyl complexes
P. Kalaivani, R. Prabhakaran, F. Dallemer, E. Vaishnavi, P. Poornima, V. Vijaya
Padma, R. Renganathan, K. Natarajan
PII:
S0022-328X(14)00173-9
10.1016/j.jorganchem.2014.04.003
JOM 18538
DOI:
Reference:
To appear in: Journal of Organometallic Chemistry
Received Date: 19 February 2014
Revised Date: 28 March 2014
Accepted Date: 1 April 2014
Please cite this article as: P. Kalaivani, R. Prabhakaran, F. Dallemer, E. Vaishnavi, P. Poornima, V.
Vijaya Padma, R. Renganathan, K. Natarajan, Synthesis, structural characterization, DNA/Protein
binding and in vitro cytotoxicity of isomeric ruthenium carbonyl complexes, Journal of Organometallic
Chemistry (2014), doi: 10.1016/j.jorganchem.2014.04.003.
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ACCEPTED MANUSCRIPT
New structurally different isomeric ruthenium(II) complexes
were obtained from a single pot reaction of [RuHCl(CO)(PPh3)3]
and salicylaldehyde-4(N)-ethylthiosemicarbazone. The
complexes exhibited better cytotoxic profile against A549 and
HepG2 cell lines.

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Highlights
ꢀ Structurally different isomeric ruthenium(II) carbonyl complexes from one pot.
ꢀ The complexes bound electrostatically with CT-DNA
ꢀ Fluorophore- quencher mechanism in lysozyme binding was static quenching
ꢀ Relatively higher cytotoxicity then cisplatin and other Ru(II) analogues

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Synthesis, structural characterization, DNA/Protein binding and in vitro
cytotoxicity of isomeric ruthenium carbonyl complexes
a
a*
b
c
d
P. Kalaivani R. Prabhakaran , F. Dallemer , E. Vaishnavi , P. Poornima , V. Vijaya
,
Padma d, R. Renganathan c and K. Natarajan a*
a Department of Chemistry, Bharathiar University, Coimbatore 641 046, India
b
Laboratoire Madirel CNRS UMR7246, Aix-Marseille Université, Centre de Saint-Jérôme, bât.
MADIREL, 13397 Marseille Cedex 20
c Department of Chemistry, Bharathidasan University, Trichirappalli 620 015, India
d Department of Biotechnology, Bharathiar University, Coimbatore 641 046, India
ABSTRACT
The reaction of salicylaldehyde-4(N)-ethylthiosemicarbazone, [H2-(Sal-etsc)] with
[RuHCl(CO)(PPh3)3] afforded two isomeric ruthenium(II) carbonyl complexes namely [Ru(H-
Sal-etsc)(CO)Cl(PPh3)2] (1) and [Ru(Sal-etsc)(CO)(PPh3)2].Cl (2). The new complexes were
1
31
characterized by various spectroscopic techniques (IR, Electronic, H-NMR and P-NMR) and
X-ray crystallography. In this reaction, bidentate monobasic coordination with the formation of
more strain NS four member chelate in (1) and tridentate monobasic coordination with the
formation of ONS five and six member rings in (2) were observed. The binding interaction of
complexes (1 and 2) to calf thymus(CT) DNA /Lysozyme has been explored. The cytotoxic
nature of the complexes was evaluated on human lung/liver cancer cells, A549 and HepG2.
Keywords: Ruthenium(II) complexes; Salicylaldehyde-4(N)-ethylthiosemicarbazone ; CT-DNA
binding; Lysozyme protein binding; three-dimensional fluorescence spectroscopy; cytotoxicity.
Corresponding authors Tel.: +91-422-2428319; Fax: +91-422-2422387.
E-mail address: rpnchemist@gmail.com (R. Prabhakaran); k_natraj6@yahoo.com
(K. Natarajan)
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1. Introduction
Thiosemicarbazones are an important group of multidentate ligand coordinating
to the metal ion through the sulphur and one of the hydrazinic nitrogen atoms N2 or N1 [1-6].
The chemistry of thiosemicarbazone complexes of the transition metal ions has been receiving
significant current attention, mainly because of their structural and bioinorganic relevances [7-
12]. A large number of the thiosemicarbazone complexes have found wide range of medicinal
applications due to their potentially beneficial biological properties including antiproliferative
activity [13-19]. Studies have demonstrated that thiosemicarbazones are potent inhibitors of the
enzyme ribonucleotide reductase (RR) and are capable of interrupting DNA synthesis and repair
[20]. It is a known fact that some drugs have increased activity when administered as metal
complexes [21,22], and their interaction with biological molecules have been reported [23].
Cisplatin is one of the most widely used metallo drugs for solid tumors. Despite its success,
clinical use of cisplatin is limited due to acquired and intrinsic resistance of cancer cells to the
drug and its high toxicity to some normal cells. Hence, there exists an immense interest to
improve the design of metallo drugs having reduced toxicity and a high spectrum of activity,
especially drugs that would show activity against cell lines resistant to cisplatin. In the search for
anticancer agents containing metals other than platinum, ruthenium compounds turned out to be
the most promising ones [24]. The binding ability of ruthenium mimic to iron in certain
biological molecules which includes serum transferrin and albumin. These two proteins are used
by mammals to solubilise and exploiting the mechanisms that the body has evolved for non toxic
transport of iron, thereby reducing its toxicity. Lysozyme, an antimicrobial protein, is capable of
destroying the cell walls of certain bacteria. Besides its antimicrobial activity, it has many other
biological functions including anti-inflammatory, anti-viral, antihistaminic and anti-tumor
activities acting as mild antiseptic [25]. Lysozyme, belonging to Low molecular weight proteins,
it may be suitable carriers to target drugs to the kidney [26]. It is worthwhile to mention that
they are an excellent system for studying the protein surface recognition and it is an extensively
characterized protein with well-defined geometry [27,28]. Many researcher published large
amount of results explaining the interaction of drug with human serum albumin. Yet, few results
have been involved in drug binding to lysozyme. Therefore, studies on the interaction between
Lysozyme and drugs or biological activity substances are of importance in view of realizing
disposition, transportation and metabolism of drugs or biological activity substances as well as
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efficacy process. While considerable amount of work has been performed on the biological
activity of ruthenium complexes [29,30], the pursuit for the development of new bio-active
substances still persists. In continuation with our investigation [6] herein, we report the
DNA/Protein binding, cytotoxicity (MTT, LDH, and NO) and cellular uptake studies of two new
isomeric ruthenium(II) thiosemicarbazone complexes (1 and 2) which were obtained from single
pot.
2. Results and discussion
2.1. Synthesis
The stoichiometric reaction of [H2-(Sal-etsc)] with [RuHCl(CO)(PPh3)3] afforded two
isomeric complexes having different structural features (Scheme 1.). The new complexes are
soluble in common organic solvents such as dichloromethane, chloroform, benzene, acetonitrile,
ethanol, methanol, dimethylsulfoxide and dimethylformamide. The analytical data of the
complexes agreed very well with the proposed molecular formulae.
O
H
CO
Ru
N
[RuHCl(CO)(PPh3)3]
OH
O
P
S
P
N
HN
S
N
N
+
N
P
C
H
.Cl
C
H
Benzene
5h
NH
N
N
Ru Cl
S
H
H
CO
P
P= PPh3
1
2
Scheme 1. Preparation of new ruthenium(II) complexes
2.2. Structural characterization
The IR spectra of the [H2-(Sal-etsc)] ligand and its complexes provide information about the
metal ligand bonding. A strong absorption at 1620 cm-1 in the ligand [H2-Sal-etsc] is assigned to
the presence of azomethine group ν(C=N). This band undergoes a negative shift of 15-24 cm-1 in
the complexes 1 and 2, which can be attributed to the donation of the lone pair of the azomethine
nitrogen to the metal ion during the coordination [31,32]. The ligand may exist in thione- thiol
tautomerization since it contains a thioamide (-NH-C=S) functional group. The absence of the
ν(S-H) stretch in the region 2500-2600 cm-1 and the presence of ν(N-H) stretch in the region 3131-
3312 cm-1 in the IR spectrum of the ligand indicate thione form in the solid state. This is further
inferred from the presence of a strong band in the region 817 cm-1 due to the ν(C=S) stretch which
completely disappeared in the complex 1 and a new band appeared at 748 cm-1 corresponding to
ν(C-S) indicating the enolisation of NH-C=S group and subsequent coordination through the
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sulphur atom [33,34]. But in complex 2, the absence of the ν(S-H) stretch and presence of ν(N-H)
stretch at 3053 cm-1 indicate the involvement of the thione sulphur in the coordination rather than
thiolate [6,35,36]. A broad band corresponding to ν(OH) appeared at 3400 cm-1 in the ligand has
been observed at 3371 cm-1 in the complex 1 indicating the non-participation of phenolic oxygen
in coordination [37]. But, this band disappeared completely in the complex 2, indicating the
involvement of phenolic oxygen in coordination. This was further supported by the increase in
the frequency of ν(C-O) absorption at 1312 cm-1 [31]. A sharp band at 1927 and 1969 cm-1 account
for the terminal carbonyl group in 1 and 2 respectively. In addition, vibrations corresponding to
the presence of triphenylphosphine also appeared in the expected region [38].
Electronic
spectra were recorded in dichloromethane solution. Both the complexes have been found to be
diamagnetic indicating the presence of ruthenium in +2 oxidation state. The ground state of
6
1
5
ruthenium(II) (t2g configuration) is A1g. The excited states corresponding to t2g
1
eg configuration are 3T1g, 3T2g, 1T1g and 1T2g in the order of increasing energy. Hence, four bands
1
3
1
3
1
1
are possible corresponding to the transitions A1g → T1g, A1g → T2g, A1g →1T1g and A1g →
1T2g, in the order of increasing energy. They showed intense absorption in the visible region (407
and 452 nm) that is probably due to the allowed MLCT transitions as is generally observed in
low spin d6 ruthenium complexes [39-41]. All other transitions in the UV region 266-268 nm and
361-379 nm were assigned to intra ligand and LMCT transitions respectively [42]. The 1H-NMR
spectra of the ligand and the complexes have been taken in DMSO. The spectra of the ligand and
new complexes showed a complex overlap of signals as a multiplet at δ 6.6-7.9 ppm range
corresponding to aromatic protons. The spectrum of [H2-(Sal-etsc)] showed a singlet at δ 9.65
ppm corresponding to (N(2)H-C=S) group [43], but in complex 1 there was no resonance
attributable to N(2)H, indicating coordination of the ligand in the anionic form after
deprotonation at N(2). However, in the complex 2, a singlet occurred at δ 9.3 ppm due to
(N(2)H-C=S-) group indicating that the ligand remains in its thionic form even after
complexation. This slight upfield shift showed that the thiosemicarbazone is coordinated to the
metal through thione sulphur atom rather than thiolate sulphur [6, 44]. Complex 1 exhibited a
singlet at δ 10.1 corresponding to phenolic -OH group [37]. In addition, there is a new sharp
singlet observed at δ 5.3 ppm in this complex which may be due to the presence of imine –NH
group that was formed from the intramolecular hydrogen bonding between phenolic -OH and N1
nitrogen atom [45]. Whereas the singlet corresponding to –OH group completely disappeared in
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complex 2 confirming the involvement of phenolic oxygen in coordination. Two singlets
appeared for complexes 1 and 2 at δ 8.42 and 9.3 ppm and at 8.0 and 9.6 ppm assigned to
azomethine proton and terminal -NH protons of the Schiff base respectively [33]. A quartet was
observed around δ 2.81-3.69pm due to the presence of methylene protons and methyl group
protons appeared as a triplet at δ 0.80-1.30 ppm in the ligand and complexes [46]. In order to
confirm the presence of triphenylphosphine, 31P NMR spectra were recorded. The singlet
observed at 28.12 and 29.64 ppm in complexes 1 and 2, respectively, suggested the presence of
two magnetically equivalent triphenylphosphines trans to each other.
Crystal structures of complexes (1 and 2) along with their numbering schemes are given
in Fig. 1 and 2. Crystallographic data and bond parameters are given in table 1 and 2. Complexes
(1 and 2) crystallized in monoclinic space group C2/c and P21/n respectively. The ruthenium
atom in the complex 1 is in a distorted octahedral environment with two mutually trans
triphenylphosphine ligands (P1-Ru-P2 178.78(4)°). The chloride is in a trans position to the
sulphur atom (Cl1-Ru-S1 165.24(4)°) and the carbonyl group lies trans to the coordinated N2
atom with an angle of 166.6(1)°. The trans angles deviate from linearity and N2, S chelation
(four member ring) leads to small N2-Ru-S1 bite angle 65.23(8)°. The Ru-ligand distances,
namely Ru-C47 1.840(5) Å, Ru-P1 2.387(1) Å, Ru-P2 2.383(1) Å, Ru-S1 2.4102(8) Å and Ru-
Cl1 2.4302(8) Å are comparable with the distances found in other ruthenium complexes
containing triphenylphosphine in trans position [45,47-49]. Ru-N bond distance is longer due to
the trans effect of carbonyl group [49,50]. In the structure of 1, one weak intramolecular O(1)-
H(1)…N(1) hydrogen bond (D…A distance 2.72(8) Å and D-H…A 126.77°) can be found
(Table 3.). In 2, the ligand is coordinated to ruthenium by utilizing its phenolic oxygen, N1
nitrogen and thione sulphur atom and behaved as monobasic tridentate. The presence of a
chloride ion outside the coordination sphere compensated the charge of ruthenium as Ru2+. It
forms an intra molecular hydrogen bond; the hydrogen of N2 nitrogen is bonded to the chloride
ion which is in the outer sphere, with a N(2)-H(2N)…Cl(3) distance of 2.99(4) Å
[d (N(2)…Cl(3))=2.99(4) Å, N(2)-H(2N)…Cl(3)=167.07°] (Table 3). A molecule of
dichloromethane is present as solvent of crystallization in this crystal lattice. The P1-Ru-P2
[175.45(3)°], C47-Ru-N1 [176.3(1)°] and O1-Ru-S1 [171.08(7)°] trans angles deviate
significantly from the linearity and N1, S chelation (five member ring) leads to N(1)-Ru-S1 bite
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angle, 82.54(8)°. This has resulted in significant distortion in RuCONSP2 core from the ideal
octahedral geometry. All the Ru-ligand distances, namely Ru-C47 1.853(4) Å, Ru -P1 2.417(1)
Å, Ru-P2 2.406(1) Å, Ru-S1 2.360(1) Å, Ru-N1 2.077(3) Å and Ru-O 2.071(2) Å are
comparable with those reported earlier [51].
O
S
Ru
N
NH
N
H
VII
O
H
N
O
H
N
N
S
Ru
N
NH
OH
N
S
S
Ru
N
VIII
XI
N
NH
O
H
N
H
OH
I
H
N
SH
N
N
N
NH
SH
VI
II
O
H
H
O
H
H
N
N
SH
N
O
N
S
N
V
NH
H
O
H
N
H
N
N
H
N
N
H
N
S
SH
IV
III
O
H
H
N
N
NH
O
H
Ru
H
N
S
N
N
X
Ru
S
IX
Scheme 2. Possible mode of coordination of Salicylaldehyde-4(N)-ethylthiosemicarbazone
Salicylaldehyde-4(N)-ethylthiosemicarbazone is a multidentate fluxional molecule which
posses variable coordination modes (I-VI) as indicated in Scheme 2. Any two forms among them
can bring the isomerism upon coordination with a metal atom (VII-XI). Herein, salicylaldehyde-
4(N)-ethylthiosemicarbazone formed two structurally different ruthenium complexes from a
single reaction in which the ligand coordinated to ruthenium through its N2 nitrogen and thiolate
sulphur atoms in complex 1 (IX) and phenolic oxygen, N1 nitrogen and thione sulphur atoms in
complex 2 (VII). Though there are number of coordination modes are possible for
thiosemicarbazones, the most observed type is (VIII). However, there are few reports including
ours witnessed with the type (IX) [5d, 6, 45, 47-49]. The coordination mode in type (XI) (with
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five member ring) is common for thiosemicarbazones in the absence of third donor atom whereas
it is uncommon and scarcely found in the literature with presence of third donor atom [37].
Similarly, the coordination mode as indicated in VII also rarely found in the chemistry of
octahedral ruthenium complexes [5d]. However, the coordination mode observed in X has not
been found in ruthenium chemistry.
2.3. DNA binding studies
The DNA binding study is a simple mechanism of action responsible for cytotoxicity of
any new complex. Thus the interaction between DNA and metal complexes is of vital
importance in understanding the mechanism. Thus, the type of binding of the new ruthenium(II)
complexes 1and 2 with CT DNA were studied with the help of UV and fluorescence quenching
techniques.
2.3.1. Electronic absorption titration
Electronic absorption spectroscopy is an effective method to examine the binding modes and
magnitudes of metal complexes with DNA. The interaction of transition metal complexes with
DNA takes place via both covalent and/or non-covalent interactions [52]. In the case of covalent
binding, the labile ligand of the complexes is replaced by a nitrogen base of DNA such as
guanine N7 while the non- covalent DNA interactions include intercalative, electrostatic and
groove binding of metal complexes outside of DNA helix. Absorption titration experiment was
carried out to study the DNA binding property of the new Ru(II) complexes (1 and 2). The
absorption spectra of complexes at constant concentration (1 µM) in the presence of different
concentrations of CT-DNA (0.05-0.50 µM) are given in Fig. 3. The absorption spectra of
complex 1 showed intra ligand (IL) transition at 263 nm, as the DNA concentration is increased,
the hyperchromism (A = 0.0516 to 0.7636) with a blue shift of 7 nm was observed. The
absorption spectra of complex 2 exhibited hyperchromism at 265 nm (A = 0.1092 to 0.8295)
with 5 nm blue shift. The CT bands at 361 and 379 nm showed hypochromism without any shift
in the absorption maxima. The observed hyperchromic effect with blue shift in IL band
suggested that the new complexes bind to CT-DNA by external contact, possibly due to
electrostatic binding [53,54]. The intrinsic binding constant Kb is a useful tool to monitor the
magnitude of the binding strength of compounds with CT DNA (Table 4.). The intrinsic binding
constant Kb was determined by using Stern volmer equation (1).
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[DNA] ꢀ [εa-εf]) = [DNA] ꢀ [εb-εf]+1 ꢀ Kb[εb-εf]
(1)
The absorption coefficients εa, εf, and εb correspond to Aobsd/[DNA], the extinction
coefficient for the free complex and the extinction coefficient for the complex in the fully bound
form, respectively. The slope and the intercept of the linear fit of the plot of [DNA]/[εa - εf]
versus [DNA] give 1/[εa - εf] and 1/Kb[εb - εf], respectively. It can be determined by monitoring
the changes in the absorbance in the IL band at the corresponding λmax with increasing
concentration of DNA and is given by the ratio of of [DNA]/(εa- εf) versus [DNA] (Insets in Fig.
3). From the binding constant values, it is inferred that the complex 2 exhibited better binding
than 1.
2.3.2. Fluorescence titration
In the emission spectra, complex 1 had fluorescence at 351 nm (Fig. 4). If CT DNA
solution was added to this complex solution, the fluorescence intensity was increased (I = 59.44
to 67.09) with 1 nm blue shift in the absorption maxima. Similarly, the complex 2 showed
hyperchromism ( I= 30.83 - 78.24) at 352 nm with 2 nm blue shift. The enhanced fluorescence
intensity of the new complexes indicates the non intercalative binding mode of DNA.
2.3.3. Ethidium bromide displacement study
The competitive EB binding studies may be carried out to further examine the binding
mode of each complex with CT-DNA. Emission spectra of EB bound to CT- DNA in the
absence and presence of each complex have been recorded for [EB] = 1ꢁM, [DNA] = 1ꢁM for
increasing amounts of each complex. In general, the intrinsic fluorescence intensity of DNA is
very low, and that of EB in Tris buffer is also not high due to quenching by the solvent
molecules. However, the fluorescence intensity of EB will be developed by the addition of DNA
because of its intercalation into the DNA. In our experiment, as exemplified in Fig. 5, the
fluorescence intensities of EB bound to CT-DNA at 620 nm showed increasing trend with
increasing concentration of the complexes (1 and 2). This indicated that the fluorescence
intensity of EB cannot be quenched by the addition of metal complexes. This observation is often
considered as they can bind weakly to the DNA probably by electrostatic interaction.
2.4. Lysozyme binding studies
Lysozyme has many physiological and pharmaceutical functions, the effectiveness of
drugs depends on their binding ability. Therefore, it is valuable to investigate the binding
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properties of new Ru(II) complexes 1 and 2 with Lysozyme. UV–Vis absorption measurement is
a valuable method to explore the structural change and the complex formation. The UV spectrum
of Lysozyme with the addition of complexes 1 and 2 at 280 nm (Fig. 6) showed that the
absorbance of Lysozyme increased with the addition of complexes 1 and 2. This indicates that
there is an interaction between Lysozyme and complexes 1 and 2 and it involved the formation
of ground state complexes of the type Lysozyme–complex.
2.4.1. Fluorescence quenching studies
The influence of complex on the fluorescence intensity of Lysozyme is shown in Fig. 7. The
fluorescence of Lysozyme was quenched effectively with the blue shift from 343 nm to 335 in its
emission maximum and this may be due to the binding of complex on the active sites in protein
[55]. The strong quenching of the Lysozyme fluorescence suggests that the chromophore of
Lysozyme was positioned in a more hydrophobic environment after addition of complexes 1 and
2 [56]. Generally, quenching types often include static and dynamic quenching. Static quenching
refers to fluorophore–quencher complex formation and the dynamic quenching refers to a
process that the fluorophore and the quencher come into contact during the transient existence of
the excited state. In order to confirm the quenching mechanism, the fluorescence quenching data
were analyzed using the Stern–Volmer equation (2), the ratio of the fluorescence intensity in the
absence of (I0) and in the presence of (Icorr- corrected fluorescence intensity) the quencher is
related to the concentration of the quencher [Q] by a coefficient Ksv.
I0/Icorr = 1+ KSV [Q]
(2)
The linear curve is obtained within the concentration studied (Fig. 8) and Ksv can be derived
from the slope of the curve, since the lifetime of Lysozyme is in the order of 10-9
s, the calculated bimolecular quenching rate constant (kq) using Ksv= kqτ0 was found to be higher
than the maximum collisional quenching (kq) of various kind of quenchers (Table 5.) to
biopolymers (2.0 x 1010 M−1s−1). All the evidence suggests that the probable quenching
mechanism of Lysozyme -complex interaction was initiated by complex formation as static
quenching which was in accordance with the absorption spectra results.
2.4.2. Binding constants and the number of binding sites
From the fluorescence spectra, we could obtain some information on the binding of small
molecules to proteins such as the binding mechanism, binding constant and binding site. When
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small molecules bind independently to a set of equivalent sites on a macromolecule, the
equilibrium between free and bound molecules can be described by using Scatchard equation 3.
F0-F
log
= log K + n log[Q]
F
→
(3)
Where K is the binding constant of quencher with Lysozyme, n is the number of binding sites, F0
and F are the fluorescence intensity in the absence and presence of the quencher. The value of K
can be determined from the slope of log [(F0 − F)/F] versus log [Q] as shown in Fig. 9. The
calculated value of binding constant (K) and the number of binding sites (n) were listed in table
6. The binding behaviour of the complex follows order, 2 > 1.The values suggest that the binding
occurs between the complex and Lysozyme.
2.5. Conformational Investigation
2.5.1. Synchronous fluorescence spectra
Synchronous fluorescence spectroscopy (SFS) is a multidimensional fluorescence
technique that gives information about the conformational change of protein molecular
environment in a vicinity of the fluorophore functional groups [57]. The fluorescence of
Lysozyme is due to tryptophan and tyrosine residues. Among them tryptophan lies in the active
site of protein. When the D value (ꢂλ) between excitation and emission wavelength are
stabilized at 60 nm, the synchronous fluorescence gives the characteristic information of
tryptophan residues [58]. The variation in the tryptophan emission is the consequence of the
protein conformational changes. Therefore, to explore the structural change of Lysozyme,
Synchronous fluorescence spectra of complexes 1 and 2 with Lysozyme at ∆λ=15 nm and ∆λ=60
nm were recorded. Results showed that, both tryptophan and tyrosine show a decrease in
intensity but the tryptophan spectrum is accompanied with blue shift. It reveals that the binding
around Trp residues is strengthened. Synchronous spectra of Lysozyme in the absence and
presence of complex 2 was shown in fig. 10. Hence the results clearly indicate that the
complexes 1 and 2 bind to active site in the protein which makes them as potential molecules for
biological applications.
2.5.2. Three-dimensional fluorescence spectra
The three-dimensional fluorescence spectra have become a popular fluorescence analysis
technique in recent years [59,60]. It can comprehensively exhibit the fluorescence information of
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the sample, which makes the investigation of the characteristic conformational change of protein
to be more scientific and credible. If there is a shift at the excitation or emission wavelength
around the fluorescence peak, or appearance of a new peak or disappearance of existing peak, it
could be an important hint to suggest conformational changes to the protein. Fig. 11 presented
the three dimensional fluorescence spectrum and their characteristics of Lysozyme (A),
Lysozyme–1 (B) and Lysozyme –2 (C) and the corresponding parameters presented in Table 6.
In Fig. 11, peak 1 is the Rayleigh scattering peak (λex = λem), peak 2 is the second-ordered
scattering peak (λem= 2λex). Peak 1 mainly reveals the spectral behavior of Trp and Tyr residues.
The reason is that when Lysozyme is excited at 280 nm, it mainly reveals the intrinsic
fluorescence of Trp and Tyr residues. Beside peak 1, there is another strong fluorescence peak 2,
which mainly exhibit the fluorescence characteristic of polypeptide backbone structures [61].
The fluorescence intensity of peak 2 decreased a lot after interacting with new ruthenium(II)
complexes (1 and 2), which implied that the peptide strand structure of Lysozyme was changed.
The reduction in the peak intensities of 1 and 2 indicate the interaction between Lysozyme and
new ruthenium(II) complexes. The binding induced a slight change in the folding of the
polypeptides of Lysozyme, which resulted in a conformational changes by increasing the
exposure of hydrophobic regions which had been obscured [62]. All these phenomena and
analyses show that the binding of new ruthenium(II) complexes (1 and 2), to Lysozyme induced
secondary structure changes in Lysozyme.
2.6. Cytotoxic studies
Cell proliferation assay using MTT reveals that the complexes could reduce the cell
viability of lung (A549) and liver (HepG2) cancer cells in a dose dependent manner (Fig. 12).
The cells were treated with different concentrations (1.0, 10.0, 25.0, 50.0ꢁM) of complexes and
the cell viability was measured as percentage by comparing with control. Cisplatin was used as
positive control. While comparing the IC50 values of the complexes, it has been observed that the
complexes could induce cell death more significantly than cisplatin in A549 cells and not in the
HepG2 cells (Table 7). By comparing the cell viability of complexes and ligands, there was a
significant reduction in the viability of complexes than the ligand.
Cell membrane integrity test was done by measuring LDH release in the cells into the
culture medium to assess the cytotoxic nature of a compound. LDH is a cytosolic enzyme
released into cell culture supernatant due to compromised membrane integrity, which is
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associated with cell death. Table 8 shows the percentage of LDH released from the A549 and
HepG2 cells after 48 hours of treatment with each complex. The significant increase of LDH
level in the culture supernatant of the complex treated cells confirmed the cytotoxic effect of
complexes on lung and liver cancer cells. Complexes induced cytotoxicity can be demonstrated
by the increase in LDH leakage and associated loss of cell viability due to the destabilization of
membrane phospholipids.
The nitrite is the stable product of the nitric oxide released in response to oxidative stress,
the amount of nitrite in the culture medium corresponds to the level of nitric oxide. Hence the
level of nitrite is estimated to measure the NO produced after complex treatment. Treatment of
A549 and HepG2 cells with the complexes resulted in enhanced NO release than the untreated
cells (Table 9). NO is a gaseous signaling molecule and a well-known, short-lived free radical
which is produced non-enzymatically by iNOS and causes damage to most biomolecules,
including DNA and protein. Two major NOS-isoenzymes are present in cells: endothelial,
membrane-bound, constitutively active NOS (eNOS), and cytoplasmic inducible NOS (iNOS)
[63]. Previous studies showed that increased ROS generation lead to the activation of iNOS
which in turn releases NO. RNS are by-products of NO production in living cells. Up-regulated
RNS production can cause cell damage or death, through nitration of biological target molecules
such as DNA, lipids, and proteins [64]. Inducible NOS was positively correlated with tumor
growth in various tumors, indicating that the two major determinants for the effect of NO appear
to be its production and tumor type. Excessive NO production results in limited angiogenesis
and, in some tumor cells, increased apoptosis [65,66]. Hence from the present study it is obvious
that the complexes could induce the NO production in A549 and HepG2 cells which may result
in apoptotic execution.
Information regarding the accumulation of the complexes in intracellular compartments
of the cells is paramount importance. The cells were treated with the complexes and intracellular
accumulation was assessed as described in materials and methods. It has been observed that
more than 20% of each complex was internalized at the treated concentration (Fig. 13; Table 10).
In addition, as it is observed in the results of the cell proliferation assay, the complex 2 showed
less IC50 value than complex 1 in both the cell lines tested, the intracellular accumulation of the
complex 2 was significantly higher than the complex 1. This may be due to the increased
electronic delocalization through six and five member rings and subsequent increase in the
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positive charge on the metal atom which leads to the enhanced permeability of the complexes in
to the cancer cells. While comparing the activities of the newly synthesized complexes with the
previously reported complexes, the new complexes showed higher activity in terms of reduction
in cell viability and cytotoxicity [6]. Though the intercellular uptake of the new complexes in
terms of the percentage (%) accumulation inside the cells was less than the previously reported
complexes, the new complexes exhibited better activity. Further, this proves that the cytotoxicity
was not disproportionately influenced by the different level of cellular uptake of the complexes.
In addition, the intracellular accumulation of the complexes by A549 cells were also
photographed using a fluorescent microscope (Fig. 14) and cellular uptake of the complexes by
A549 cells has been clearly confirmed.
Since the complexes exhibit lower IC50 value than cisplatin in A549 cells, the role of
ROS on the complex induced cytotoxicity was investigated in the presence of glutathione; a
strong antioxidant in cell viability assay. It has been observed that the presence of glutathione
significantly increased the cell viability of A549 cells while treating with the complexes (Fig.
15). These results indicate that the glutathione could neutralize the complex induced oxidative
stress by ROS hypergeneration and thus the cell viability is significantly increased. From this, it
is concluded that the cytotoxic activity of the complexes may be due to ROS hypergeneration.
3. Conclusions
The reaction of salicylaldehyde-4(N)-ethylthiosemicarbazone [H2-(Sal-etsc)] with
[RuHCl(CO)(PPh3)3] have been carried out. The stoichiometric reaction afforded two isomeric
products having different structural features in each case and they were characterized by the
analytical, IR, electronic, NMR spectroscopy and X-ray crystallography. In the complex 1,
thiosemicarbazone coordinated through N(2) nitrogen and thiolate sulphur by forming more
strained four member ring. However, monobasic tridentate coordination behaviour of the ligand
was observed in 2 by utilizing N1 hydrazinic nitrogen, thione sulphur and phenolic oxygen. Both
the complexes (1 and 2) containing biologically active thiosemicarbazone moiety with different
coordination mode tempted us to study their biological properties and hence, an attempt was
made to compare their chelation with their biological properties. DNA and protein are the main
cellular targets for the biological activity thus the new complexes have been subjected to study
their binding properties. Results obtained from the electronic absorption spectroscopy,
fluorescence spectroscopy and ethidium bromide displacement studies revealed that the
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complexes could interact with CT-DNA through non-intercalative mode. The interaction with the
protein Lysozyme, the complexes exhibited good binding affinity in the order of 2 > 1. There is a
single class of binding site in the surrounding of tryptophan base pair at the interface of
Lysozyme; the results of synchronous fluorescence spectroscopy and three-dimensional
fluorescence spectra indicated that the structure of these tryptophan residues was significantly
altered through binding. The IC50 value of the complexes in A549 cell line was relatively high as
compared with the standard cisplatin. The LDH and NO release studies confirmed that the
complex 2 was more active than complex 1. In addition, intracellular uptake of the complex 2
was significantly more active than the complex 1. The complexes exhibited their cytotoxicity
through the induction of oxidative stress.
4. Experimental Work
4.1. Materials
The ligand [H2-(Sal-etsc)] and the ruthenium precursor [RuHCl(CO)(PPh3)3] were
synthesized according to standard literature procedures [67-69]. All the reagents used in this
study were analar grade and the solvents were purified and dried according to the standard
procedure [70].
4.2. Preparation of new ruthenium (II) complexes
The Schiff base [H2-(Sal-etsc)] (0.024 g; 0.105 mmol) was added to a solution of
[RuHCl(CO)(PPh3)3] (0.100 g; 0.105 mmol) in dry benzene (20 cm3). The mixture was heated
under reflux for 5 h and was subjected to thin layer chromatography where two spots were
identified. They were isolated by silica gel column chromatography. The first compound was
isolated from a greenish yellow band by using 1:19 ethyl acetate: petroleum ether as eluent.
Followed by this, a dark yellow band containing the second complex was eluted by using 2: 18
methanol: benzene as eluents. Both the complexes were recrystallized in
dichloromethane/acetonitrile mixture which afforded yellow (1) and orange (2) crystals.
[Ru(H-Sal-etsc)(CO)Cl(PPh3)2] (1): Yield: 42 % , M.p. 160°C.
Anal. Calcd. for
C47H42N3SO2ClRuP2 : C, 61.94; H, 4.64; N, 4.60, S, 3.51.Found:C, 61.76; H, 4.53; N, 4.56; S,
3.42 %. FT-IR (cm-1) in KBr: 3371 (νO-H), 1605 (νC=N), 1253 (νC-O), 748 (νC-S), 1927 (νC≡O) 1432,
1088, 694 cm-1 (for PPh3) ; UV- vis (CH2Cl2), λmax: 263 (35,440) nm (dm3mol-1cm-1) (intra-
ligand transition); 357 (4,640), 378 (4,419) nm (dm3mol-1cm-1) (LMCT s→d); 452 (3,160) nm
(dm3mol-1cm-1) MLCT transition ; 1H NMR (DMSO-d6, ppm): δ 10.1 (s, 1H, -O-H), 8.42 (s, 1H,
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-CH=N), 5.3 (s, imine - NH), 9.3 (s, terminal -NH), 3.1 (q, -CH2-), 0.80 (t, -CH3), 6.71–7.81 (m,
aromatic). 31P NMR (DMSO-d6, ppm): 28.12 ppm.
[Ru(Sal-etsc)(CO)(PPh3)2].Cl (2): Yield : 48%, M.p. 231°C. Anal. Calcd. for
C47H42N3SO2ClRuP2 : C, 61.94; H, 4.64; N, 4.60, S, 3.51.Found:C, 61.79; H, 4.52; N, 4.54; S,
3.46 %. FT-IR (cm-1) in KBr: 1596 (νC=N), 1312 (νC-O), 795 (νC-S), 1969 (νC≡O) 1437, 1091, 697
cm-1 (for PPh3) ; UV- vis (CH2Cl2), λmax: 265(56,000) nm (dm3mol-1cm-1) (intra-ligand
transition); 361 (5,950), 379 (4,330) (dm3mol-1cm-1) (LMCT s→d); 407 (2,820) nm (dm3mol-
1cm-1) MLCT transition ; 1H NMR (DMSO-d6, ppm): δ 8.0 (s, 1H, -CH=N), 9.3 (s, -HN-C=S),
-
9.6 (s, terminal -NH), 2.81 (q, -CH2 ), 1.2 (t,-CH3), 6.20–7.60 (m, aromatic). 31P NMR (DMSO-
d6, ppm): 29.64 ppm.
4.3. Measurements
Infrared spectra were measured as KBr pellets on a Nicolet Avatar Model FT-IR
spectrophotometer in the 400- 4000 cm-1 range. Elemental analyses of carbon, hydrogen,
nitrogen, and sulfur were determined using Vario EL III CHNS at the Department of Chemistry,
Bharathiar University, Coimbatore, India. The electronic spectra of the complexes were recorded
in dichloromethane using a JASCO V-630 spectrophotometer in the 200–800-nm range.
1
31
Emission spectra were recorded using a JASCO FP-6600 spectrofluorometer. H NMR and P
NMR spectra were taken in DMSO at room temperature with a Bruker 400 MHz instrument with
chemical shifts relative to tetramethylsilane and orthophosphoric acid respectively. Melting
points were measured in a Lab India apparatus. Single-crystal data collections for the new Ru(II)
complexes were performed at 293 K with a Bruker SMART 1000 CCD diffractometer using
monochromatic Mo Kα (λ = 0.71073 Å ) radiation. The data were collected and processed using
the software program SAINT and the software package SHELXTL used for space group
determination, structure solution, and refinement. The structures were solved by direct methods,
completed with difference Fourier syntheses, and refined with full-matrix least-squares using
SHELXL-97. All non-hydrogen atoms were refined with anisotropic displacement parameters.
All scattering factors and anomalous dispersions factors are contained in the SHELXTL [71,72].
4.4. DNA binding study
DNA binding studies have been done according to the method described in the earlier
reports [3,4,6].
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4.5. Lysozyme binding studies
Lysozyme was purchased from Hi Media, India. Lysozyme was prepared in phosphate
buffer of pH 7.6 and stored in the dark at 4° C for use. The concentration of Lysozyme was
determined spectrophotometrically by using ε280 (Lysozyme) = 37646 M−1 cm−1 [73].
Concentrated stock solutions of complexes were prepared by dissolving the complex in
phosphate buffer: DMSO (100:1) and diluted suitably with phosphate buffer to required
concentrations for all the experiments. The steady-state fluorescence spectra of Lysozyme were
recorded under an excitation wavelength of 280 nm and the emission was monitored at 342 nm.
The excitation, emission slit widths (each 5 nm) were maintained constant for all the experiment.
A scan rate of 500nm/min was used. A 3 ml solution, containing appropriate concentration of
Lysozyme (1 x 10−6 M), was titrated by successive additions of the complexes. In order to
correct the inner filter effect the following equation 4, is used
Fcorr= Fobs*10 Aexc+Aem
2
(4)
Where Fcorr is the corrected fluorescence value, Fobs the measured fluorescence value,
Aexc the absorption value at the excitation wavelength, and Aem the absorption value at the
emission wavelength [74]. Conformational changes were studied using synchronous fluorescence
measurement. The spectra were measured at two different ∆λ (difference between the excitation
and emission wavelengths of Lysozyme) values such as 15 and 60 nm. The three-dimensional
fluorescence spectrum was performed under the following conditions: the emission wavelengths
at 290–450 nm, the excitation at 280 nm and other parameters are similar to quenching
experiments.
4.6. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) assay
Effect of the ligand and complexes (1 and 2) on the viability of human lung cancer cells
(A549) and liver cancer cells (HepG2) were assayed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazoliumbromide (MTT) assay [75]. The cells were seeded at a density of 10,000
cells per well, in 200 µl RPMI 1640 medium and were allowed to attach overnight in a CO2
incubator and then the complexes dissolved in DMSO were added to the cells at a final
concentration of 1, 10, 25 and 50 µM in the cell culture media. After 48 hours, the wells were
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treated with 20 µl MTT (5 mg/ml PBS) and incubated at 37 °C for 4 hours. The purple formazan
crystals formed were dissolved in 200 µl DMSO and read at 570 nm in a micro plate reader.
In addition, the cell membrane integrity (LDH leakage), Nitric oxide scavenging and
cellular uptake studies were done according to the standard literature methods [76-78].
Supporting Information
Crystallographic data for [Ru(H-Sal-etsc)(CO)Cl(PPh3)2] (1) and [Ru(Sal-
etsc)(CO)(PPh3)2].Cl (CH2Cl2) (2) have been deposited at the Cambridge Crystallographic Data
Centre as supplementary publication (CCDC No. 856737 and 856736). The data can be obtained
free of charge at www.ccdc.cam.ac.uk/conts/retrieving.html or from the Cambridge
Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK [fax: + 44-1223/336-
033; e-mail: deposit@ccdc.cam.ac.uk].
ACKNOWLEDGMENT
The authors gratefully acknowledged Council of Science and Industrial Research, New
Delhi, India and Department of Science and Technology, New Delhi, India for their financial
assistance.
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Table 1 Crystal data and structure refinement of new Ru(II) complexes 1 and 2.
Table 2 Selected bond lengths (Å) and angles (°) of new Ru(II) thiosemicarbazone
complexes 1 and 2.
Table 3 Hydrogen bonding for complex 1 and 2 [Å and °].
Table 4 Binding constant for interaction of complexes with CT-DNA.
Table 5 Stern–Volmer constants (Ksv), quenching rate constants (kq), binding constants (K)
and number of binding sites calculated from the fluorescence data of interaction of lysozyme
with complex.
Table 6 Three dimensional fluorescence spectral characteristics of (A) Lysozyme (B)
Lysozyme+1 (C) Lysozyme+2.
Table 7 In vitro cytotoxicity of ligand, complexes 1 and 2 for human tumour cell lines A549
and HepG2.
Table 8 Percentage of lactate dehydrogenase released by the human cancer cell lines A549
and HepG2 after an incubation period of 48 hours with the ligand (23ꢀM for A549 and 20
ꢀM for HepG2)complexes 1(20ꢀM for A549 and 19 ꢀM for HepG2) and 2 (17ꢀM for A549
and 15 ꢀM for HepG2).
Table 9 nmoles of nitrite released by the human cancer cell lines A549 and HepG2 after an
incubation period of 48 hours with the ligand (23ꢀM for A549 and 20 ꢀM for
HepG2)complexes 1(20ꢀM for A549 and 19 ꢀM for HepG2) and 2 (17ꢀM for A549 and 15
ꢀM for HepG2).
Table 10 Percentage of cellular uptake of the complexes 1 and 2 by human cancer cell lines
A549 and HepG2 after an incubation period of 2 hours with the complexes 1(20ꢀM for A549
and 19 ꢀM for HepG2) and 2 (17ꢀM for A549 and 15 ꢀM for HepG2).
Fig.1. ORTEP diagram of 1 with hydrogen bonding (Thermal ellipsoids are shown at 50%
probability).
Fig. 2. ORTEP diagram of 2 with hydrogen bonding, solvent molecule removed for clarity
(Thermal ellipsoids are shown at 50% probability).
Fig. 3. Absorption titration spectra of 1 and 2 with increasing concentrations (0.05-0.5 ꢀM)
of CT-DNA (tris HCl buffer, pH 7); the inset shows binding isotherms with CT-DNA.
Fig. 4. Changes in the emission spectra of 1 and 2 with increasing concentrations
(0.05-0.5 ꢀM) of CT-DNA (tris HCl buffer, pH 7).

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Fig. 5. The emission spectra of the DNA–EB system (λexc = 515 nm, λem = 530–750 nm), in
the presence of complex 1 and 2. [DNA] = 1 µM, [EB] = 1 µM. The arrow shows the
emission intensity changes upon increasing the amount of complex.
Fig. 6. Absorption spectra of lysozyme (1×10−5M) in the absence and presence of complexes
1 and 2 (1 x 10-5M).
Fig. 7. Fluorescence quenching of lysozyme (1×10−5M) by complex 1 (A) and 2(B) (0-5 x
10-5 M).
Fig. 8. Plot of I0/I vs log [Q].
Fig. 9. Plot of log [(F0-F)/F] vs log [Q].
Fig. 10. Synchronous spectra of lysozyme (1 x 10-6 M) in the absence and presence of
complex 2 (0-5 x 10-5 M) in the wavelength difference of a) ∆λ =15 nm and b) ∆λ = 60 nm.
Fig. 11. Three dimensional fluorescence spectra for (A) Lysozyme (B) Lyso -1 (C) Lyso- 2,
λex/λem 280/340 nm.
Fig. 12. The cell viability of the lung (A549) and liver (HepG2) cancer cells after exposing to
ligand and complexes 1 and 2 along with Cisplatin for 48 h was depicted. Results shown are
mean±SEM, which are three separate experiments performed in triplicate.
Fig. 13. Cells were exposed to the complexes 1 (20ꢀM) and 2 (17ꢀM) for 48 h in the
presence and absence of glutathione and cell viability was assessed using MTT assay. Results
shown are mean±SEM, which are three separate experiments performed in triplicate.
Fig.14. Percentage of intracellular uptake of the complexes 1 and 2 by human cancer cell
lines A549 and HepG2 after an incubation period of 2 hours. Error bars represent the standard
error of mean (n=6).
Fig. 15. Intracellular fluorescence of the complexes 1 (20ꢀM) and 2 (17ꢀM) by A549 after an
incubation period of 2 hours were photographed. The figures are one of the representatives of
three independent experiments with similar results.

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Table 1 Crystal data and structure refinement of new Ru(II) complexes 1 and 2.
[Ru(H-Sal-tsc)(CO)Cl(PPh3)2] [Ru(Sal-etsc)(CO)(PPh3)2].Cl
(CH2Cl2) (2)
Empirical formula
C47H42ClN3O2P2RuS
911.36
C48H44Cl3N3O2P2 RuS
996.28
Formula weight
Temperature
293 K
293 K
Wavelength
0.71073 Å
Monoclinic
C2/c
0.71073 Å
Monoclinic
P21/n
Crystal system
Space group
Unit cell dimensions
a
23.3204(3) Å
23.9347(4) Å
20.6176(2) Å
90º
13.646(5)Å
18.250(5)Å
20.6176(2) Å
90º
b
c
alpha
beta
124.016(1)º
92.36(2)º
gamma
90º
90º
Volume
9538.82 Å3
8
4612.03Å3
4
Z
Density (calculated)
Absorption coefficient
F(000)
1.269 Mg/m3
0.533 mm-1
3744
1.435Mg/m3
0.670 mm-1
2040
Theta range for data
collection
1.35 to 28.22º
2.47 to 28.24º
-30<=h<=31,-31<=k<=29,
-26<=l<=23
-18<=h<=17,-24=k<=22,
-24<=l<=22
Index ranges
Reflections
unique
collected
/ 44685/6650
0.0983]
[R(int)
=
39961/5423[R(int) = 0.1109]
28.24 º
Completeness to theta =
28.22 º
Full-matrix least-squares on
F2
Refinement method
Full-matrix least-squares on F2
Data
parameters
Goodness-of-fit on F^2
Final indices
/
restraints
/
11440 / 0 /515
0.886
11231 / 0/514
0.926
R
R1=0.0965, wR2 = 0.1227
R1=0.1655,wR2=0.1129
[I>2sigma(I)]
R indices (all data)
R1= 0.0502, wR2 = 0.1122
0.709 and -0.752 e.A-3
R1=0.0552,wR2=0.0897
0.534 and -0.941 e.A-3
Largest diff. peak and hole

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Table 2 Selected bond lengths (Å) and angles (°) of new Ru(II) thiosemicarbazone
complexes 1 and 2.
[Ru(H-Sal-etsc)(CO)Cl(PPh3)2] (1) [Ru(Sal-etsc)(CO)(PPh3)2].Cl (CH2Cl2) (2)
Bond lengths
Ru(1)-N(2)
Ru(1)-P(1)
Ru(1)-P(2)
Ru(1)-S(1)
Ru(1)-Cl(1)
Ru(1)-C(47)
Bond angles
2.199(4)
2.387(1)
2.383(1)
2.4102(8)
2.4302(8)
1.840(5)
Ru(1)-N(1)
Ru(1)-P(1)
Ru(1)-P(2)
Ru(1)-S(1)
Ru(1)-O(1)
Ru(1)-C(47)
2.077(3)
2.417(1)
2.406(1)
2.360(1)
2.071(2)
1.853(4)
C(47)-Ru(1)-N(2) 166.6(1)
C(47)-Ru(1)-)O(1)
C(47)-Ru(1)-P(2)
N(1)-Ru(1)-P(2)
C(47)-Ru(1)-P(1)
N(1)-Ru(1)-P(1)
P(2)-Ru(1)-P(1)
C(47)-Ru(1)-S(1)
N(1)-Ru(1)-S(1)
P(2)-Ru(1)-S(1)
P(1)-Ru(1)-S(1)
C(47)-Ru(1)-N(1)
N(1)-Ru(1)-O(1)
P(2)-Ru(1)-O(1)
P(1)-Ru(1)-O(1)
S(1)-Ru(1)-O(1)
94.3(1)
C(47)-Ru(1)-P(2)
N(2)-Ru(1)-P(2)
C(47)-Ru(1)-P(1)
N(2)-Ru(1)-P(1)
P(2)-Ru(1)-P(1)
C(47)-Ru(1)-S(1)
N(2)-Ru(1)-S(1)
P(2)-Ru(1)-S(1)
P(1)-Ru(1)-S(1)
90.7(1)
89.8(1)
89.52(8)
90.3(1)
92.28(8)
87.4(1)
89.32(8)
178.78(4)
101.3(1)
65.23(8)
88.27(4)
90.91(4)
90.70(8)
175.45(3)
94.5(1)
82.54(8)
89.47(3)
94.32(3)
176.3(1)
88.7(1)
C(47)-Ru(1)-Cl(1) 93.4(1)
N(2)-Ru(1)-Cl(1)
P(2)-Ru(1)-Cl(1)
P(1)-Ru(1)-Cl(1)
S(1)-Ru(1)-Cl(1)
100.08(8)
90.23(4)
90.33(4)
165. 24(4)
89.11(7)
87.53(7)
171.08(7)

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Table 3 Hydrogen bonding for complex 1 and 2 [Å and °].
D-H…A
d(D-H) d(H...A) d(D...A) <(DHA)
Complex 1
Complex 2
O(1)-H(1)...N(1)
N(2)-H(2A)...Cl(3)
1.08(5) 1.93(7)
0.999(0) 2.01(2)
2.72(8)
2.99(4)
126.77
167.07
Symmetry operation: (x, y, z) (1); Symmetry operation: (x, y, z) (2)
Table 4 Binding constant for interaction of complexes with CT-DNA.
System
Kb (x 105 M-1)
2.22±0.24
CT-DNA + 1
CT-DNA + 2
3.46±0.45
Table 5 Stern–Volmer constants (Ksv), quenching rate constants (kq), binding constants (K)
and number of binding sites calculated from the fluorescence data of interaction of lysozyme
with complex.
Complex
KSV x 104 M-1
kq x1013 M-1 s-1
K x103 M-1
n
1
2
3.16
5.42
2.11
5.18
6.48
6.78
0.871
0.830
Table 6 Three dimensional fluorescence spectral characteristics of (A) Lysozyme (B)
Lysozyme+1 (C) Lysozyme+2.
System
Peak 2 λex/ λem
∆λ
Intensity
Lysozyme (A)
Lysozyme+1 (B)
Lysozyme+2 (C)
280/340
280/335
280/338
60
55
58
961
814
823

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Table 7 In vitro cytotoxicity of ligand, complexes 1 and 2 for human tumour cell lines A549
and HepG2.
IC50 values (µM)
Complexes
A549
HepG2
20±0.98
19±0.87
15±1.01
9±0.85
[H2-(Sal-etsc)]
23±1.09
20±1.10
17±0.93
25±1.11
1
2
Cisplatin
Cells were exposed to different concentrations of ligand and complexes for 48 h and cell
viability was assessed using MTT assay. The IC50 values were calculated and tabulated.
Results shown are mean±SEM, which are three separate experiments performed in triplicate.
Table 8 Percentage of lactate dehydrogenase released by the human cancer cell lines A549
and HepG2 after an incubation period of 48 hours with the ligand (23ꢀM for A549 and 20
ꢀM for HepG2)complexes 1(20ꢀM for A549 and 19 ꢀM for HepG2) and 2 (17ꢀM for A549
and 15 ꢀM for HepG2).
LDH release (%)
Complexes
A549
HepG2
Control
0
0
[H2-(Sal-etsc)]
15.97±0.79
49.3±2.49
61.47±2.71
14.92±0.75
58.79±3.01
73.8±1.85
1
2
Results shown are mean±SEM, which are three separate experiments performed in triplicate.