Synthesis, characterization, DNA interaction and in vitro cytotoxicity activities of ruthenium(II) Schiff base complexes

Article abstract of DOI:10.1016/j.molstruc.2012.07.021

DNA binding, cleavage and cytotoxicity characteristics of a novel Schiff base ligand 3-(benzothiazol-2-yliminomethyl)-naphthalen-2-ol and ruthenium(II) complexes have been investigated. The DNA interaction properties of the complexes have been investigated using absorption spectra, as well as gel electrophoresis studies. Intrinsic binding constant (K_b) has been estimated under similar set of experimental conditions. Absorption spectral study indicate that the ligand and ruthenium(II) complexes has intrinsic binding constant in the range of 1.4-7.2 × 10⁴ M^-1. Ruthenium(II) complexes show more binding ability than the ligand. Further, in vitro cytotoxicity study of the ligand and the complexes exhibited antitumor activity against HeLa and HEp2 tumor cells.

Full text of DOI:10.1016/j.molstruc.2012.07.021

Journal of Molecular Structure 1030 (2012) 95–103
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Synthesis, characterization, DNA interaction and in vitro cytotoxicity activities
of ruthenium(II) Schiff base complexes
Subbaiyan Sathiyaraj a, Ray J. Butcher b, Chinnasamy Jayabalakrishnan a,
⇑
a Post Graduate and Research Department of Chemistry, Sri Ramakrishna Mission Vidyalaya College of Arts and Science, Coimbatore 641 020, Tamil Nadu, India
b Department of Chemistry, Howard University, Washington, DC 20059, USA
h i g h l i g h t s
"
"
"
"
"
Preparation of Schiff base ligand and its ruthenium(II) complexes.
Structure of ligand was confirmed by X-ray analysis.
DNA interaction and in vitro cytotoxicity activities were carried out.
Ruthenium(II) complexes show more binding ability than the ligand.
Ligand and the complexes showed better cytotoxicity against HeLa cancer cell line.
a r t i c l e i n f o
a b s t r a c t
Article history:
DNA binding, cleavage and cytotoxicity characteristics of a novel Schiff base ligand 3-(benzothiazol-2-
yliminomethyl)-naphthalen-2-ol and ruthenium(II) complexes have been investigated. The DNA interac-
tion properties of the complexes have been investigated using absorption spectra, as well as gel electro-
phoresis studies. Intrinsic binding constant (Kb) has been estimated under similar set of experimental
conditions. Absorption spectral study indicate that the ligand and ruthenium(II) complexes has intrinsic
binding constant in the range of 1.4–7.2 ꢀ 104 Mꢁ1. Ruthenium(II) complexes show more binding ability
than the ligand. Further, in vitro cytotoxicity study of the ligand and the complexes exhibited antitumor
activity against HeLa and HEp2 tumor cells.
Received 16 February 2012
Received in revised form 30 April 2012
Accepted 13 July 2012
Available online 26 July 2012
Keywords:
Ruthenium(II) complexes
Aminobenzothiazole
Crystal structure
DNA-interaction
Cytotoxicity
Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction
[6–8]. It has been reported that the intercalating ability of the com-
plex was involved in the planarity of ligands, the coordination
In recent years, many researches [1–3] have been focused on
interaction of small molecules with DNA. DNA is generally the pri-
mary intracellular target of anticancer drugs, so the interaction be-
tween small molecules and DNA can cause DNA damage in cancer
cells, blocking the division of cancer cells, and resulting in cell
death [4,5]. Small molecule can interact with DNA through the fol-
lowing three non-covalent modes: intercalation, groove binding
and external static electronic effects. Among these interactions,
intercalation is one of the most important DNA-binding modes,
which is related to the antitumor activity of the compound. Re-
cently, there is a great interest on the binding of transition metal
complexes with DNA, owing to their possible applications as new
cancer therapeutic agents and their photochemical properties that
make them potential probes of DNA structure and conformation
geometry, ligand donor atom type, the metal ion type [9–11]. So
the development of synthetic, sequence-selective DNA binding
and cleavage agents for DNA itself and for new potential DNA-tar-
geted antitumor drugs is essential for further expected applica-
tions in molecular biology, medicine, and related fields. Schiff
bases form an interesting class of ligands that has enjoyed popular
use in the coordination chemistry of transition, inner transition
and main group elements [12,13] Schiff bases have been reported
to show a variety of biological actions by virtue of the azomethine
linkage, which is responsible for various antibacterial, antifungal,
herbicidal and clinical activities [14–16]. Benzothiazole ring is
present in various marine or terrestrial natural compounds which
have useful biological activities [17]. Transition metal ions play a
vital role in a vast number of widely different biological processes.
In particular, ruthenium complexes are better suited for medical
applications because of their favorable rate of ligand exchange
and their ability to mimic iron binding to certain biomolecules
⇑
Corresponding author. Tel.: +91 422 2692461.
E-mail address: cjayabalakrishnan@gmail.com (C. Jayabalakrishnan).
0022-2860/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molstruc.2012.07.021

96
S. Sathiyaraj et al. / Journal of Molecular Structure 1030 (2012) 95–103
Table 1
[18]. Metal complexes of Schiff bases derived from heterocyclic
compounds containing nitrogen, sulfur and/or oxygen as ligand
atoms are of interest as simple structural models of more compli-
cated biological systems [19].
Herein, we report the synthesis, characterization, DNA binding,
cleavage and cytotoxic abilities of ruthenium(II) complexes con-
taining 3-(benzothiazol-2-yliminomethyl)-naphthalen-2-ol. Schiff
base derived from 2-aminobenzothiazole and 2-hydroxy-1-naph-
thaldehyde. Schiff base was structurally characterized by single
crystal X-ray crystallography.
Crystal and structure refinement data for Schiff base ligand (HL).
Empirical formula
C18 H12 N2 O S
Formula weight
Temperature
Wavelength
Crystal system
Space group
304.36
295(2) K
0.71073 Å
Monoclinic
P 21/n
Unit cell dimensions
a (Å)
b (Å)
c (Å)
9.7176(3)
15.0806(6).
9.7882(3)
a
(°)
b (°)
(°)
90
2. Experimental
101.713(3)
c
90
1404.56(8) Å3
1.439 Mg/m3
2.1. Materials and instrumentation
Volume
Density (calculated)
Crystal size
Theta range for data collection
Index ranges
0.47 ꢀ 0.38 ꢀ 0.24 mm3
Reagent grade chemicals were used without further purification
in all the synthetic work. All solvents were purified by standard
methods. 2-hydroxy-1-naphthaldehyde, 2-amino benzothiazole
were purchased from Sigma–Aldrich chemie. RuCl3ꢂ3H2O, tri-
phenyl phosphine/arsine were purchased from Himedia. Calf-thy-
mus (CT-DNA) was purchased from Bangalore Genei, Bangalore,
India. The Human Cervical cancer cell line HeLa and human laryn-
geal epithelial carcinoma cell line HEp2 were obtained from Na-
tional center for cell science (NCCS), pune, India.
5.13–34.98°
ꢁ15 < =h < =14
ꢁ19 < =k < =24
ꢁ15 < =l < =14
12583
5739 [R(int) = 0.0261]
Full-matrix least-squares on F2
5739/0/200
Reflections collected
Independent reflections
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
1.035
Final R indices [I > 2
R indices (all data)
Largest diff. peak and hole
r
(I)]
R1 = 0.0557, wR2 = 0.1303
R1 = 0.0956, wR2 = 0.1518
0.353 and ꢁ0.243 e.Åꢁ3
Infrared spectra were recorded on a FT-IR Perkin Elmer spectro-
photometer RXI model as KBR pellets in the range 4000–400 cmꢁ1
.
Elemental analyses were performed with a model Vario ELIII CHNS
at Sophisticated Test and Instrumentation Centre (STIC), Cochin
University, Kerala. Electronic spectra were recorded in DMSO solu-
tion in a Systronics 2202 Double beam spectrophotometer in 800–
200 nm range. 1H, 13C and 31P NMR spectra were recorded on Bru-
ker WM DCX 500 MHz instrument using TMS and ortho phosphoric
acid as an internal standard at SAIF, Indian Institute of Technology,
Chennai. Anti cancer studies was carried out at the Kovai Medical
Centre and Hospital Pharmacy College, Coimbatore, Tamil Nadu.
Table 2
Selected Bond lengths (Å) and angles (°) for Schiff base ligand (HL).
Bond lengths
Bond angles
S(1)AC(18)
S(1)AC(12)
O(1)AC(1)
O(1)AH(1A)
N(1)AC(11)
N(1)AC(12)
N(2)AC(12)
N(2)AC(13)
C(1)AC(10)
C(1)AC(2)
C(2)AC(3)
C(2)AH(2A)
C(3)AC(4)
C(3)AH(3A)
C(4)AC(5)
C(4)AC(9)
1.7332(16)
1.7462(14)
1.3348(18)
0.8200
1.3023(18)
1.3943(19)
1.2941(19)
1.3870(18)
1.406(2)
1.412(2)
1.353(2)
0.9300
C(18)AS(1)AC(12)
C(1)AO(1)AH(1A)
C(11)AN(1)AC(12)
C(12)AN(2)AC(13)
O(1)AC(1)AC(10)
O(1)AC(1)AC(2)
C(10)AC(1)AC(2)
C(3)AC(2)AC(1)
C(3)AC(2)AH(2A)
C(1)AC(2)AH(2A)
C(2)AC(3)AC(4)
C(2)AC(3)AH(3A)
C(4)AC(3)AH(3A)
C(5)AC(4)AC(9)
C(5)AC(4)AC(3)
88.78(7)
109.5
117.79(12)
110.50(12)
122.51(13)
116.80(13)
120.69(14)
120.06(15)
120.0
120.0
122.15(14)
118.9
2.2. Crystal structure determination
Single crystal of Schiff base ligand (HL) was grown by slow
evaporation of a solution of the ligand in chloroform. Selected crys-
tal data are given in Tables 1 and 2, Fig. 1. The X-ray diffraction
data for ligand (HL) were collected on a Bruker SMART CCD diffrac-
1.422(2)
0.9300
1.408(2)
1.418(2)
118.9
119.99(14)
121.16(14)
tion using graphite-monochromated Mo
K
a
radiation
(k = 0.71073 Å) by and scans. X-ray data reduction structure
u
x
solution and refinement were done using the SHEL XS-97 and
SHELXL-97 packages [20]. The structure was solved by direct
methods.
2.3. Synthesis of the Schiff base ligand
The Schiff base ligand 3-(benzothiazol-2-yliminomethyl)-naph-
thalen-2-ol (HL) was isolated from the condensation reaction of 2-
hydroxy-1-naphthaldehyde (1.20 g. 10 mmol) and 2-amino benzo-
thiazole (1.5 g, 10 mmol) in ethanol under reflux for 6 h. After cool-
ing the reaction mixture to room temperature, the solid product
formed was filtered, washed with ethanol and dried in vacuo
(Scheme 1). Yield 72%; Color: Orange; M.P: 148 °C. This solid was
recrystallized from chloroform, yielding orange crystals suitable
for X-ray diffraction analysis. Anal. Calc for C18H12N2OS: C, 70.02;
H, 4.61; N, 9.15; S, 10.46. Found: C, 70.30; H, 4.33; N, 8.92; S,
10.12. IR (cmꢁ1): 3396 (ph-OH), 1622 (C@N), 1599 (C@N thiazole
ring), 1311 (ph-CO). UV–vis (in DMSO); kmax, nm; 276, 365, 445,
476. 1H NMR (ppm); 8.72 (1H, s, HC@N), 10.12 (1H, s, ArAOH),
7.24–8.27 (m, ArAH). 13C NMR (ppm); 152 (C@N imine), 166
(C@N thiazole), 117–136 (ArAC).
Fig. 1. ORTEP for HL.
2.4. Synthesis of [RuCl(CO)(PPh3)L] (1)
To a solution of Schiff base ligand (HL) (0.060 g, 0.2 mmol) in
methanol (20 cm3) was added triethylamine (0.020 g, 0.2 mmol)

S. Sathiyaraj et al. / Journal of Molecular Structure 1030 (2012) 95–103
97
OH
511. 1H NMR (ppm); 8.93(1H, s, HC@N), 7.26–7.70 (m, ArAH).
13C NMR (ppm); 160 (C@N imine), 171 (C@N thiazole), 192
(C„O), 127–133 (ArAC).
HO
OHC
+
N
S
N
S
Ethanol
6 h
N
C
H
NH2
Scheme 1. Formation of Schiff base ligand PPh3 (Triphenyl phosphine); AsPh3
(Triphenyl arsine); py (pyridine).
2.7. DNA-binding and cleavage assay
2.7.1. Electronic absorption spectroscopy
Electronic absorption titration of the ligand and its complexes
were carried out in aqueous buffer solution (50 mm NaCl, 5 mM
Tris–HCl, pH 7.1) at fixed complex concentration (10 lM) while
gradually increasing the concentration of CT-DNA. The absorption
data were analyzed to evaluate the intrinsic binding constant Kb,
which can be determined from the following equation, [24]
followed by [RuHCl(CO)(PPh3)3] [21] (1.91 g, 0.2 mmol) and the
mixture was refluxed for 6 h to afford a reddish orange solution.
The solvent was then evaporated under reduced pressure and the
solid mass, thus obtained, was subjected to purification by TLC.
This solid was recrystallized from CH2Cl2/Hexane mixture. Our sin-
cere effort to obtain single crystal of the complexes went unsuc-
cessful. Yield: 58%; Color: reddish brown; M.P: 155 °C. Anal. Calc
for C37H26ClN2O2PRuS: C, 60.86; H, 3.59; N, 3.84; S, 4.39. Found:
C, 60.65; H, 3.25; N, 3.52; S, 4.12. IR (cmꢁ1); 1618 (C@N), 1573
(C@N thiazole ring), 1350 (ph-CO), 1925 (C„O). UV–vis (in
DMSO); kmax, nm; 252, 296, 365, 416, 524. 1H NMR (ppm); 9.12
(1H, s, HC@N), 6.26–7.83 (m, ArAH). 13C NMR (ppm); 162 (C@N
imine), 170 (C@N thiazole), 194 (C„O), 128–138 (ArAC).
½DNAꢃ=ðea
ꢁ
ef Þ ¼ ½DNAꢃ=ðeb
ꢁ
ef Þ þ 1=kbðeb
ꢁ
ef
Þ
where [DNA] is the concentration of DNA in base pairs, the
apparent absorption coefficient ea ef and eb correspond to Aobsd/
,
[complex], the extinction coefficient of the free compound and
the extinction coefficient of the compound when fully bound to
DNA respectively. In plots of [DNA]/(ea ef) vs [DNA], Kb is given
ꢁ
by the ratio of slope to the intercept.
2.5. Synthesis of [RuCl(CO)(AsPh3)L] (2)
2.7.2. DNA cleavage
The DNA cleavage activity of the Schiff base metal complexes
was monitored by agarose gel electrophoresis on CT DNA. The tests
were performed under aerobic condition with H2O2 as an oxidant.
Complex 2 was synthesized as described for (1) by utilizing
[RuHCl(CO)(AsPh3)3] [22] (2.168 g; 0.2 mmol) and the Schiff base
ligand (HL) (0.060 g; 0.2 mmol) as in Scheme 2. Yield: 63%; Color:
orange; M.P: 190 °C. Anal. Calc for C37H26ClN2O2AsRuS: C, 57.41; H,
3.39; N, 3.62; S, 4.14. Found: C, 57.30; H, 3.15; N, 3.47; S, 4.08. IR
(cmꢁ1): 1598 (C@N), 1578 (C@N thiazole ring), 1324 (ph-CO),
1965 (C„O). UV–vis (in DMSO); kmax, nm; 252, 296, 361, 445,
531. 1H NMR (ppm); 9.02 (1H, s, HC@N), 7.08–8.12 (m, ArAH).
13C NMR (ppm); 159 (C@N imine), 169 (C@N thiazole), 191
(C„O), 114–134 (ArAC).
Each reaction mixture contained 30
of each complex in DMSO and 60
l
M of CT DNA, 30 and 60
lM
lM of hydrogen peroxide in
50 mM Tris–HCl, (pH 7.1). The reaction was incubated at 37 °C
for 2 h. After incubation, 1 l of loading buffer (0.25% bromophenol
blue, 0.25% xylene cynol and 60% glycerol) was added to the reac-
tion mixture and loaded onto a 1% agarose gel containing 1.0 g/ml
l
l
of ethidium bromide. The electrophoresis was carried out for 2 h at
50 V in Tris–acetic acid EDTA buffer. The bands were visualized un-
der UV light and photographed.
2.6. Synthesis of [RuCl(CO)(py)L] (3)
Complex 3 was synthesized as described for (1) by utilizing
[RuHCl(CO)py(PPh3)2] [23] (1.538 g; 0.2 mmol) and the Schiff base
ligand (HL) (0.060 g; 0.2 mmol) as in Scheme 2. Yield: 54%; Color:
brown; M.P: 182 °C. Anal. Calc for C24H16ClN3O2RuS: C, 52.70; H,
2.95; N, 7.68; S, 5.58. Found: C, 52.95; H, 2.77; N, 7.61; S, 5.68. IR
(cmꢁ1): 1604 (C@N), 1575 (C@N thiazole ring), 1338 (ph-CO)
1938 (C„O). UV–vis (in DMSO); kmax, nm; 255, 294, 347, 432,
2.8. Cytotoxicity assay
The human cervical cancer cell line (HeLa) and human laryngeal
epithelial carcinoma cell line (HEp2) was obtained from National
Centre for Cell Science (NCCS), Pune, and grown in Eagles Mini-
mum Essential Medium containing 10% fetal bovine serum (FBS).
All cells were maintained at 37 °C, 5% CO2, 95% air and 100% rela-
tive humidity. Maintenance cultures were passaged weekly, and
the culture medium was changed twice a week.
The monolayer cells were detached with trypsin–ethylenedi-
aminetetraacetic acid (EDTA) to make single cell suspensions and
viable cells were counted using a hemocytometer and diluted with
medium with 5% FBS to give final density of 1 ꢀ 105 cells/ml. one
hundred microlitres per well of cell suspension were seeded into
96-well plates at plating density of 10,000 cells/well and incubated
to allow for cell attachment at 37 °C, 5% CO2, 95% air and 100% rel-
ative humidity. After 24 h the cells were treated with serial con-
centrations of the extracts and fractions. They were initially
dissolved in neat dimethylsulfoxide (DMSO) and further diluted
in serum free medium to produce various concentrations. One
hundred microlitres per well of each concentration was added to
plates to obtain final concentrations of 1.0, 0.5, 0.25, 0.125, and
HO
[RuHCl(CO)(B)(EPh3)2]
N
S
+
N
H
Methanol
Reflux 6 h
B
OC
Cl
Ru
N
O
N
S
0.063 lg/ml. The final volume in each well was 200 ll and the
H
plates were incubated at 37 °C, 5% CO2, 95% air and 100% relative
humidity for 48 h. The medium containing without samples were
served as control. Triplicate was maintained for all concentrations.
MTT is a yellow water soluble tetrazolium salt. A mitochondrial
enzyme in living cells, succinate-dehydrogenase, cleaves the tetra-
zolium ring, converting the MTT to an insoluble purple formazan.
where, B=PPh3/AsPh3/py; E=PPh3/AsPh3
PPh3 (Triphenyl phosphine); AsPh3 (Triphenyl arsine); py (pyridine)
Scheme 2. Formation of ruthenium(II) Schiff base complexes.

98
S. Sathiyaraj et al. / Journal of Molecular Structure 1030 (2012) 95–103
Therefore, the amount of formazan produced is directly propor-
tional to the number of viable cells.
reader. The % cell inhibition was determined using the following
formula.
After 48 h of incubation, 15 ll of MTT (5 mg/ml) in phosphate
buffered saline (PBS) was added to each well and incubated at
37 °C for 4 h. The medium with MTT was then flicked off and the
formed formazan crystals were solubilized in 100 ll of DMSO
%Growth Inhibition ¼ 100 ꢁ AbsðsampleÞ=AbsðcontrolÞ ꢀ 100:
Nonlinear regression graph was plotted between % cell inhibi-
tion and Log10 concentration and IC50 was determined using
GraphPad Prism software [25,26].
and then measured the absorbance at 570 nm using micro plate
HL
[RuCl(CO)(py)L]
Fig. 2. IR Spectra of ligand HL and Complex [RuCl(CO)(py)L].

S. Sathiyaraj et al. / Journal of Molecular Structure 1030 (2012) 95–103
99
3. Results and discussion
3.1. Crystal structure of 3-(benzothiazol-2-yliminomethyl)-
naphthalen-2-ol (HL)
The molecular structure of HL, along with the atom numbering
scheme is given in Fig. 1.
The crystal data and structural refinement parameters are given
in Table 1 and selected bond lengths and angles are given in Table
2. The compound crystallized into a monoclinic lattice with space
group P 21/n. The azomethine bond, C12AN1 1.3943(19) Å is in
conformity with a formal C@N double bond and the C1AO1 bond
distance of 1.3348(18) Å, slightly shorter than the normal CAO sin-
gle bond distance. The bond distance for C12AN2 1.2941(19) Å and
C12AS1 1.7462(14) Å for thiazole group are closer to CAN and the
CAS single bond, respectively. The single crystal X-ray diffraction
study on his compound exits in imine-ol form without any
ambiguity.
3.2. Spectroscopic data
3.2.1. Infrared spectra
The important IR spectral data of the ligand were compared
with those of the ruthenium(II) complexes in order to confirm
the binding mode of the shiff base ligand to ruthenium ion in the
complexes were shown Fig. 2. IR spectra of the Schiff base (HL)
has the most characteristic bands appeared at 3396 cmꢁ1
Fig. 3. UV–visible spectra of ligand HL and Complex [RuCl(CO)(PPh3)L].
m
(OAH), 1622 cmꢁ1
m
m
m
(C@N azomethine), 1599 cmꢁ1
(ph-CO), and 744 cmꢁ1
(CASAC). The band
(C@N) azomethine group of the Schiff base
m(C@N thia-
zole ring), 1311 cmꢁ1
at 1622 cmꢁ1 due to
m
served in all the complexes due to M ? L transitions are possible
in the visible region in the range of 416–531 nm [32–34]. The other
bands in the region 252–365 nm region were assignable to ligand
underwent a shift to lower frequency (1618–1598 cmꢁ1) after
complexation, indicating the bonding of unsaturated nitrogen of
the azomethine group to the metal ion [27]. The phenolic CAO
stretching vibrations that appeared at 1311 cmꢁ1 in the Schiff base
[28] underwent a shift towards higher frequencies in the com-
plexes. This shift confirms the participation of oxygen in the
CAOAM bond. In the low frequency region 430–470 cmꢁ1 is attrib-
uted to (MAO) and the region 560–580 cmꢁ1 to (MAN) [29]. IR
spectrum of the ligand of revealed a medium band at 1599 cmꢁ1
(C@N) thiazole ring, which is shifted to lower frequency at about
(1578–1573 cmꢁ1) after complexation, which also indicates that
it has been affected upon coordination to ruthenium metal ions.
The unchanged band after complexation at 748 cmꢁ1 in the free li-
gand suggests non-involvement of the coordination which was as-
centered (LC) transitions and have been designated as
p–
pꢄ and
n–pꢄ transition. The pattern of the electronic spectra of all the com-
plexes indicated the presence of an octahedral environment
around the ruthenium(II) ion, similar to that of other ruthenium(II)
octahedral complexes [35].
3.2.3. NMR spectra
The 1H NMR spectra of ligand and complexes were recorded in
DMSO-d6 solution for confirming the binding mode of the Schiff
base to ruthenium ion shown in Figs. 4–6. The aromatic protons
for the ligand appeared as a multiplet at 7.24–8.27 ppm. On com-
plexation, the protons on the phenyl ring remain more or less un-
changed in the complexes, even if there are slight variation in their
resonances due to the delocalization of electron density in the sys-
tem [36] and these signals in the complexes cannot be distin-
guished from the aromatic signals of PPh3/AsPh3 due to their
extensive overlap appeared at 7.26–8.12 ppm [37]. The proton of
the hydroxyl group appears as broad singlet at 10.12 (Ph-OH)
ppm for free Schiff base ligand. In the spectra of the complexes,
the resonance arising from the hydroxyl (Ph-OH) proton is not ob-
served, indicating the coordination of the hydroxyl oxygen to the
metal ion [8]. The signal due to the azomethine proton (-HC@N)
is found to be considerably deshielded at 8.93–9.12 ppm relatively
to that of the free Schiff base ligand 8.72 ppm as a consequence of
electron donation to the metal center.
signed to m(CASAC). In all the complexes, the strong band in the
region 1963–1925 cmꢁ1 is due to terminally coordinated carbonyl
group. For the complex [RuCl(CO)(py)L)], the IR spectrum showed a
medium intensity band at 1119 cmꢁ1 which is characteristics of
coordinated nitrogen base. Characteristic bands for triphenyl phos-
phine/arsine are also present in the expected region [30].
3.2.2. Electronic spectra
The electronic spectra of the Schiff base HL and its complexes
were taken in DMSO and shown in Fig. 3. Two very strong bands
at 365 and 276 nm were observed in the spectra of the ligand,
which is attributed to n–pꢄ and
p–
pꢄ transitions in the aromatic
ring and C@N chromophore [31]. The electronic spectra of all com-
The 13C NMR data were recorded in DMSO-d6 solution and the
assignment of ligand and the complexes are given. 13C NMR spec-
tra of all Schiff base ligand displayed a single resonance at 152 ppm
[38] showed that the azomethine carbon atoms were equivalent,
which also confirm the structure of the ligand. The signal at
166 ppm corresponds to thiazolic C@N carbon [39]. The downfield
shift of these two signals at 162–159 and 169–171 ppm clearly
indicates that both the C@N carbons were affected by coordination
[40]. The aromatic carbons of free ligand and the corresponding
plexes showed five bands in the region 252–531 nm. All the ruthe-
1
nium(II) complexes in an octahedral environment is A1g arising
from the t62g configuration and the excited states corresponding
to the t52g eg1 configuration are 3T1g, 3T2g, 1T1g and 1T2g. Hence, four
bands corresponding to the transition 1A1g ? 3T1g 1A1g ? 3T2g,
,
1A1g ? 1T1g and 1A1g ? 1T2g are possible in order of increasing en-
ergy. The low energy bands in the visible region are assigned to the
charge transfer (CT) transitions. The charge transfer bands ob-

100
S. Sathiyaraj et al. / Journal of Molecular Structure 1030 (2012) 95–103
Fig. 4. 13C NMR spectrum of HL.
Fig. 5. 1H NMR spectrum of [RuCl(CO)(PPh3)L].
Fig. 6. 31P NMR spectrum of [RuCl(CO)(PPh3)L].

S. Sathiyaraj et al. / Journal of Molecular Structure 1030 (2012) 95–103
101
complexes show signals in the region 114–138 ppm. For all the
complexes, the terminal carbonyl group appeared in the range
191–194 ppm [41].
In order to confirm the presence of triphenylphosphine group
and to determine the geometry of the complexes 31P NMR spectra
were recorded. 31P NMR spectra of the complex [Ru(CO)Cl(PPh3)L]
and [Ru(CO)Cl(py)L] were recorded in DMSO-d6 solution. The
observation of a sharp singlet at 28.2 ppm for the complex [Ru(-
CO)Cl(PPh3)L] confirms the presence of only one triphenylphos-
phine group. No signals were observed for complex
[Ru(CO)Cl(py)HL] confirms the absence of phosphine group.
Fig. 8. Electronic absorption spectra of the complex 1 in the absence and presence
of increasing amount of CT-DNA.
3.3. DNA binding and cleavage
3.3.1. Electronic absorption spectroscopy
Electronic absorption spectroscopy is one of the most useful
techniques for DNA-binding studies of metal complexes. The inter-
action of ruthenium complexes with CT-DNA were investigated by
UV absorption titrations. The binding of the ruthenium complexes
to DNA helices was characterized by following the changes in the
absorbance and shift in wavelength on each addition of DNA solu-
tion to the complex. The ruthenium(II) complexes in DMSO–buffer
mixture exhibit an intense transition in the region around 348–
252 nm, which is attributed to a
p–
pꢄ intraligand transition that
unique to this Schiff base. On the titration of CT-DNA with the
Schiff base ligand and complexes, a considerable increase or de-
crease in the absorption along with small blue shift was observed.
With increasing concentration of DNA, the absorption bands of the
ligand and complexes were affected to a considerable extent. For
ligand and complexes, the absorption spectra show clearly that
the addition of DNA to the complexes yields hyperchromism and
a blue shift to the ratio of [DNA]/[Ru]. Obviously, these spectral
characteristics suggest that all the complexes interact with DNA
most likely through a mode that involves a stacking interaction be-
tween the aromatic chromophore and the base pairs of DNA. Addi-
tion of increasing amounts of DNA resulted in hypsochromism of
the peak maxima in the UV–visible spectra of the ligand and com-
plexes Figs. 7–10. Complex interaction occurs with exterior phos-
phates of DNA via., electrostatic attraction [42]. In the plot of
Fig. 9. Electronic absorption spectra of the complex 2 in the absence and presence
of increasing amount of CT-DNA.
[DNA]/(ea ef) vs [DNA], the binding constant Kb is given by the ra-
ꢁ
tio of the slope to the y intercept. The intrinsic binding constant
(Kb) of ligand, (1) and (3) are of 1.4 ꢀ 104 Mꢁ1, 4.9 ꢀ 104 Mꢁ1
,
7.2 ꢀ 104 Mꢁ1. The DNA-binding constant of the title complexes
are comparable to those of some polypyridyl Ru(II) complexes
1.0–4.8 ꢀ 104 Mꢁ1 [43,44].
As the concentration of the DNA was increased, the absorption
bands of [RuCl(CO)(AsPh3)(HL)] (2) showed hyperchromism ini-
tially and on further increment, hypochromism of with a blue shift
(5 nm) was observed. An isosbestic point was observed at 265 nm.
Fig. 10. Electronic absorption spectra of the complex 3 in the absence and presence
of increasing amount of CT-DNA.
This behavior reveals an electrostatic association of the complex
with the helix surface [45] Hyperchromicity and hypochromicity
is the spectral feature of DNA concerning its double helix structure
[46]. Hyperchromic effect reflects the corresponding changes of
DNA in its conformation and structure after the complexes are
bound to DNA. Hypochromism results from contraction of DNA
in the helix axis while hyperchromism results from the damage
of DNA helix structure. The binding constant of the complex 2
could not be evaluated due to the random changes in the absorp-
tion on the addition of DNA.
3.3.2. DNA cleavage activity
The cleavage efficiency of the complexes compared with that of
the control is due to their efficient DNA-binding ability. The metal
complexes were able to convert super coiled DNA into open circu-
lar DNA. The general oxidative mechanisms proposed account for
Fig. 7. Electronic absorption spectra of the Schiff base ligand (HL) in the absence
and presence of increasing amount of CT-DNA.

102
S. Sathiyaraj et al. / Journal of Molecular Structure 1030 (2012) 95–103
than the ligand. However, these values are lower than the standard
anticancer drug cisplatin [48].
4. Conclusion
Three ruthenium(II) complexes containing 3-(benzothiazol-2-
yliminomethyl)-naphthalen-2-ol ligand, were synthesized and
characterized by various spectroscopic techniques. Schiff base li-
gand has also been characterized crystalographically. All the newly
synthesized complexes and ligand were evaluated for DNA-bind-
ing, DNA cleavage and cytotoxicity studies. DNA-binding behaviors
were examined by absorption spectroscopy. The results indicate
that intrinsic binding constant of complex 1, 3 and ligand varies
in the range of 1.4–7.2 ꢀ 104 Mꢁ1. Complex 2 on the other hand
shows dual mode of binding to DNA. The complexes and ligand be-
have as a DNA groove binder. DNA cleavage studies revealed that
all the three complexes have the ability to cleave nucleic acids
and the extent of the cleavage was found to be dose dependent.
Cytotoxicity evaluation in vitro shows that complex 3 displayed
better antitumor activity against selected cell lines. The ligand
and complexes 1 and 2 shows moderate antitumor activity.
Fig. 11. Gel electrophoresis showing the chemical nuclease activity of the CT-DNA
incubated at 37 °C for 2 h with different concentration of complex 1, 2 and 3 in the
presence of H2O2 as an oxidant
(60 M) + complex 1 (30 M); lane 3, DNA + H2O2 (60
lane 4, DNA + H2O2 (60 M) + complex (30 M); lane 5, DNA + H2O2
(60 M) + complex 2 (60 M); lane 6, DNA + H2O2 (60
lane 7, DNA + H2O2 (60 M) + complex 3 (60 M); lane 8, DNA + H2O2 (60
;
lane 1, DNA control; lane 2, DNA + H2O2
l
l
l
M) + complex 1 (60 M);
l
l
2
l
l
l
lM) + complex 3 (30
lM);
l
l
l
M).
DNA cleavage by hydroxyl radicals via., abstraction of a hydrogen
atom from sugar units and predict the release of specific residues
arising from transformed sugars, depending on the position from
which the hydrogen atom is removed [47].
In the present study, the CT-DNA gel electrophoresis experi-
ment was conducted at 35 °C using our synthesized ruthenium(II)
complexes in the presence of H2O2 as an oxidant. It was found that,
at very low concentrations, few complexes exhibit nuclease activ-
ity in the presence of H2O2. Control experiment using DNA alone
does not show any significant cleavage of CT-DNA even on longer
exposure time. Hence, we conclude that the ruthenium(II) complex
cleaves DNA at different concentrations as compared with control
DNA Fig. 10. The amount of Form I diminished gradually, partly
converted to Form II, where as the intensity of the Form II band in-
creased as the concentration of the ruthenium(II) complexes as in-
creased, showing the potential chemical nuclease activity of the
Supplementary material
Crystallographic data for the structural analysis have been
deposited with the Cambridge Crystallographic Data Centre, CCDC
814145. Copy of this information may be obtained free of charge
from the Director, CCDC, 12 Union Road, Cambridge, CB21 EZ, UK
(fax: +44 1223 336033: e-mail: deposit@ccdc.cam.ac.uk or
www.ccdc.cam.ac.uk/deposi).
complexes. When the concentration increased to 60 lM for all
Acknowledgements
the complexes, DNA was completely converted from Form I to
Form II. The result showed that complex 1 has more cleavage activ-
ity than other complexes (see Fig. 11).
We sincerely thank University Grants Commission (UGC), New
Delhi for financial support [Scheme. No. 38-222/2009 (SR)]. One
of the author RJB wishes to acknowledge the NSF-MRI program
(grant No. CHE-0619278) for providing funds to purchase the
diffractometer.
3.4. Cytotoxic activity evaluation
References
To test the cytotoxicity of Schiff base ligand and ruthenium(II)
complexes, human cervical cancer cell line (HeLa) and the human
laryngeal epithelial carcinoma cell line (HEp2) were cultured in
the presence of varying concentrations of ligand and corresponding
ruthenium(II) complexes for 48 h. The inhibitory concentration 50
(IC50), defined as the concentration required to reduce the size of
the cell population by 50%. The IC50 values obtained of ligand
and its complexes against selected two tumor cell lines are given
[1] Y.-M. Song, Q. Wu, P.-J. Yang, N.-N. Luan, L.-F. Wang, Y.-M. Liu, J. Inorg.
Biochem. 100 (2006) 1685.
[2] M. Kozurkova, D. Sabolova, L. Janovec, J. Mikes, J. Koval, J. Ungvarsky, M.
Stefanisinova, P. Fedorocko, P. Kristian, J. Imrich, Bioorg. Med. Chem. 16 (2008)
3976.
[3] C.-P. Tan, J. Liu, L.-M. Chen, S. Shi, L.-N. Ji, J. Inorg. Biochem. 102 (2008) 1644.
[4] G. Zuber, J.C. Quada Jr, S.M.J. Hecht, Am. Chem. Soc. 120 (1998) 9368.
[5] S.M.J. Hecht, Nat. Prod. 63 (2000) 158.
[6] C. Metcalfe, J.A. Thomas, Chem. Soc. Rev. 32 (2003) 215.
[7] A. Silvestri, G. Barone, G. Ruisi, M.T. Lo Giudice, S.J. Tumminello, Inorg.
Biochem. 98 (2004) 589.
[8] M. Navarro, E.J. Cisneros-Fajardo, A. Sierralta, M. Fernández-Mestre, P. Silva, D.
Arrieche, E. Marchán, J. Biol. Inorg. Chem. 8 (2003) 401.
[9] H. Xu, K.-C. Zheng, H. Deng, L.-J. Lin, Q.-L. Zhang, L.-N. Ji, Dalton. Trans. 3 (2003)
2260.
[10] H. Xu, K.-C. Zheng, H. Deng, L.-J. Lin, Q.-L. Zhang, L.-N. Ji, New J. Chem. 27
(2003) 1255.
[11] M. Asadi, E. Safaei, B. Ranjbar, L. Hasani, New J. Chem. 28 (2004) 1227.
[12] S.R. Collinson, D.E. Fenton, Coord. Chem. Rev. 148 (1996) 19.
[13] A.K. Sharma, S. Chandra, Spectrochim. Acta A. 78 (2011) 337.
[14] H.F. Abd El-halim, M.M. Omar, G.G. Mohamed, Spectrochim. Acta A 78 (2011)
36.
[15] T.B.S.A. Ravoof, K.A. Crouse, M.I.M. Tahir, F.N.F. How, R. Rosli, D.J. Watkins,
Trans. Met. Chem. 35 (2010) 871.
[16] N.P. Priya, S. Arunachalam, A. Manimaran, D. Muthupriya, C. Jayabalakrishnan,
Spectrochim. Acta A 72 (2009) 6706.
[17] V. Beneteau, T. Besson, J. Guillard, S. Leonce, B. Pfeiffer, Eur. J. Med. Chem. 34
(1999) 1053.
[18] G. Sathyaraj, T. Weyhermuller, B.U. Nair, Eur. J. Med. Chem. 45 (2010) 284.
[19] I. Sakyan, E. Logoglu, S. Arslan, N. Sari, N. Sakiyan, Bio Metals. 17 (2004) 115.
[20] G.M. Sheldrick, SHELXS -97 SHELXL -97 Fortran Programs for Crystal Structure
Solution and Refinement, University of Gottingen, Germany, 1997.
in Table 3. The IC50 values of the ligand (IC50 = 175
enhanced activity against HeLa cell line and low activity (IC50
389 M) against HEp2. The IC50 values are 149, 244 M for com-
plex 1. 123, 212 M for complex 2 and 87, 149 M for complex 3
lM) exhibit
=
l
l
l
l
against HeLa and HEp2 cell lines. All the ruthenium(II) complexes
show significant activity against the two tumor cell lines. Thus, the
IC50 value for the ligand decreases with the coordination of it to
ruthenium metal which shows the more toxicity of the complexes
Table 3
The IC50 values for Schiff base ligand and ruthenium(II) complexes against selected
cell lines.
Compound
HeLa
HEp2
>300
HL
175
149
123
l
l
l
M
M
M
lM
Complex 1
Complex 2
Complex 3
244
212
149
l
l
l
M
M
M
87 lM

S. Sathiyaraj et al. / Journal of Molecular Structure 1030 (2012) 95–103
103
[21] N. Ahmed, J.J. Levison, S.D. Robinson, M.F. Uttley, Inorg. Synth. 15 (1974) 48.
[22] R.A. Sanchez-Delgade, W.Y. Lee, S.R. Choi, Y.M.J. Cho, Y. Jun, Trans. Met. Chem.
16 (1991) 41.
[23] S. Gopinathan, I.R. Unny, S.S. Deshpande, C. Gopinathan, Ind. J. Chem. A 25
(1986) 1015.
[24] A. Wolf, G.H. Shimer, T. Meehan, Biochemistry 26 (1987) 6392.
[25] T. Mosmann, J. Immunol. Methods 65 (1983) 55.
[26] A. Monks, J. Natl Cancer Inst. 83 (1991) 757.
[27] R. Ramesh, M. Sivagamasundari, Synth. React. Inorg. Met-Org. Chem. 33 (2003)
899.
[28] R.C. Maurya, P. Patel, S. Rajput, Synth. React. Inorg. Met-org. Chem. 23 (2003)
817.
[29] K. Nakamoto, Infrared and Raman spectra of Inorganic and Co-ordination
compounds, Wiley Interscience, New York, 1971.
[30] A.K. Das, S.M. Peng, S.J. Bhattacharya, Chem. Soc. Jpn. 49 (1976) 287.
[31] R.K. Sharma, R.V. Singh, J.P.J. Tandon, Inorg. Nucl. Chem. 42 (1980) 1382.
[32] K. Natarajan, R.K. Poddar, C.J. Agarwala, Inorg. Nucl. Chem. 39 (1977) 431.
[33] A.B.P. Lever, Inorganic Electronic Spectroscopy, 2nd ed., Elsevier, New York,
1984.
[36] M. Maji, S. Ghosh, S.K. Chattopaghyay, T.C.W. Mak, Inorg. Chem. 36 (1997)
2938.
[37] R.V. Singh, S.C. Joshi, A. Gajraj, P. Nagpal, Appl. Organomet. Chem. 16 (2002)
713.
[38] J.T. Desai, C.K. Desai, K.R.I. Desai, Iran. Chem. Soc. 5 (2008) 67.
[39] S. Perz, C. Lopez, A. Caubet, X. Solans, M.F. Bardia, M. Gich, E. Molins, J.
Organomet. Chem. 692 (2007) 2402.
[40] K. Shankera, R. Rohinia, V. Ravindera, P.M. Reddy, Y.-P. Ho, Spectrochim. Acta A
73 (2009) 205.
[41] M.H. Desbosis, D. Astruc, Organometallics 8 (1989) 1841.
[42] M. Chauhan, F.J. Arjmand, Organomet. Chem. 692 (2007) 5156.
[43] X.W. Liu, J. Li, H. Deng, K.C. Zheng, Z.W. Mao, L.N. Ji, Inorg. Chim. Acta. 358
(2005) 3311.
[44] X.H. Zao, B.H. Ye, H. Li, Q.L. Zhang, H. Chao, J.G. Liu, L.N. Ji, X.Y. Li, J. Biol. Inorg.
Chem. 6 (2001) 143.
[45] D. Herebian, W.S.J. Sheldrick, Chem. Soc. Dalton Trans. (2002) 966.
[46] Q.S. Li, P. Yang, H.F. Wang, M.L. Guo, J. Inorg. Biochem. 64 (1996) 181.
[47] G. Prativel, M. Pitie, J. Bernadou, B. Meunier, Angew. Chem. Int. Ed. Eng. 30
(1991) 702.
[34] K. Chichak, U. Jacquenard, N.R. Branda, Eur. J. Inorg. Chem. (2002) 357.
[35] P. Sengupta, R. Dinda, S. Ghosh, Trans. Met. Chem. 27 (2002) 665.
[48] Y.-J. Liu, C.-H. Zeng, Z.-H. Liang, J.-H. Yao, H.-L. Huang, Z.-Z. Li, F.-H. Wu, Eur. J.
Med. Chem. 45 (2010) 3087.