DNA interaction and cytotoxicity studies of ruthenium(III) complexes containing 3-(benzothiazol-2-yliminomethyl)-naphthalen-2-ol ligand

Article abstract of DOI:10.1080/00958972.2013.764995

A new Schiff base, 3-(benzothiazol-2-yliminomethyl)-naphthalen-2-ol, has been synthesized and characterized by elemental analysis, Fourier transform infrared spectroscopy (FT-IR), UV-vis, nuclear magnetic resonance, and single-crystal X-ray diffraction. Ruthenium(III) complexes of the Schiff base were synthesized and characterized by analytical and spectroscopic (FT-IR, UV-vis, and electron paramagnetic resonance) data as well as magnetic susceptibility measurements. DNA-binding properties of the ligand and its ruthenium(III) complexes have been investigated by electronic absorption spectroscopy. The three ruthenium(III) complexes were tested for DNA cleavage. Further in vitro study of the cytotoxity of the ligand and the complexes on human cervical cancer cell line and human laryngeal epithelial carcinoma cell line were carried out.

Full text of DOI:10.1080/00958972.2013.764995

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DNA interaction and cytotoxicity
studies of ruthenium(III) complexes
containing 3-(benzothiazol-2-
yliminomethyl)-naphthalen-2-ol ligand
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 , India
b Department of Chemistry , Howard University , Washington ,
DC , USA
Accepted author version posted online: 17 Jan 2013.Published
online: 25 Feb 2013.
To cite this article: Subbaiyan Sathiyaraj , Ray J. Butcher & Chinnasamy Jayabalakrishnan (2013)
DNA interaction and cytotoxicity studies of ruthenium(III) complexes containing 3-(benzothiazol-2-
yliminomethyl)-naphthalen-2-ol ligand, Journal of Coordination Chemistry, 66:4, 580-591, DOI:
10.1080/00958972.2013.764995
To link to this article: http://dx.doi.org/10.1080/00958972.2013.764995
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Journal of Coordination Chemistry
Vol. 66, No. 4, 20 February 2013, 580–591
DNA interaction and cytotoxicity studies of ruthenium(III)
complexes containing 3-(benzothiazol-2-yliminomethyl)-
naphthalen-2-ol ligand
SUBBAIYAN SATHIYARAJ†, RAY J. BUTCHER‡ and CHINNASAMY
JAYABALAKRISHNAN*†
†Post Graduate and Research Department of Chemistry, Sri Ramakrishna Mission
Vidyalaya College of Arts and Science, Coimbatore, India
‡Department of Chemistry, Howard University, Washington, DC, USA
(Received 8 June 2012; in final form 11 October 2012)
A new Schiff base, 3-(benzothiazol-2-yliminomethyl)-naphthalen-2-ol, has been synthesized and
characterized by elemental analysis, Fourier transform infrared spectroscopy (FT-IR), UV–vis,
nuclear magnetic resonance, and single-crystal X-ray diffraction. Ruthenium(III) complexes of the
Schiff base were synthesized and characterized by analytical and spectroscopic (FT-IR, UV–vis,
and electron paramagnetic resonance) data as well as magnetic susceptibility measurements. DNA-
binding properties of the ligand and its ruthenium(III) complexes have been investigated by elec-
tronic absorption spectroscopy. The three ruthenium(III) complexes were tested for DNA cleavage.
Further in vitro study of the cytotoxity of the ligand and the complexes on human cervical cancer
cell line and human laryngeal epithelial carcinoma cell line were carried out.
Keywords: Ruthenium(III) complexes; Crystal structure; DNA-interaction; Cytotoxicity
1. Introduction
Metal complexes that can interact with DNA have been studied as DNA footprint, novel
chemotherapeutics, and highly sensitive diagnostic agents [1–6]. Transition metals play a
very important role in organisms, and their complexes can interact non-covalently with
nucleic acid by intercalation, groove-binding or external electrostatic binding [3,7–11].
Many transition metal complexes have been investigated as probes of DNA structure, agents
for mediation of strand scission of duplex DNA and chemotherapeutic agents [12–14]. Some
Pt(II) complexes (cisplatin and carboplatin) have found their way into pharmaceutical use as
clinical antitumor drugs [15]. Schiff bases show a variety of biological actions by virtue of
the azomethine linkage, which is responsible for various antibacterial, antifungal, herbicidal,
and clinical activities [16–18]. Polyfunctional ligands based on benzazole derivatives, such
as 2-aminobenzothiazole, 2-aminothiazole, and 2-aminobenzoxazole have biological activity
as fungicides, antibiotics, pesticides, and neuroprotectors [19–21]. Thiazole derivatives
are widely used in the synthesis of medical products such as sulfathiazole [22].
*Corresponding author. Email: srkvmca@md4.vsnl.net.in
Journal of Coordination Chemistry
ISSN 0095-8972 print/ISSN 1029-0389 online Ó 2013 Taylor & Francis
http://dx.doi.org/10.1080/00958972.2013.764995

Ruthenium(III) complexes
581
A large number of ruthenium Schiff base complexes have been reported and their cata-
lytic and biological properties studied [23–29]. Intercalation, hydrogen bonding, and π–π
interactions are implicated in the mode of action of ruthenium complexes as antitumor and
antimetastatic agents. Ruthenium–halogen bonds in several ruthenium complexes exhibit-
ing anticancer activity suggest that these bonds may also play an important role. Herein,
we report the synthesis and characterization of a new Schiff base derived from the conden-
sation of 2-aminobenzothiazole with 2-hydroxy-1-naphthaldehyde and its ruthenium(III)
complexes. DNA binding and cleavage behaviors were examined using UV spectroscopic
and gel electrophoresis techniques, respectively, and cytotoxic abilities of ruthenium(III)
complexes were examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro-
mide (MTT) assay. The Schiff base was structurally characterized by single-crystal X-ray
diffraction.
2. Experimental
2.1. Materials and instrumentation
Reagent grade chemicals were used without purification in all the synthetic work. All sol-
vents were purified by standard methods. 2-Hydroxy-1-naphthaldehyde and 2-amino ben-
zothiazole were purchased from Sigma-Aldrich. RuCl3·3H2O, triphenylphosphine/arsine
was purchased from Himedia. [RuCl3(PPh3)3] [30], [RuCl3(AsPh3)3] [31], and
[RuBr3(PPh3)3] [32] were prepared by reported methods. Calf-thymus DNA (CT-DNA)
was purchased from Bangalore Genei, Bangalore, India. The human cervical cancer cell
line (HeLa) and human laryngeal epithelial carcinoma cell line (HEp2) were obtained from
the NCCS, Pune, India.
Infrared spectra were recorded on a FT-IR Perkin Elmer spectrophotometer RXI model
as KBr pellets from 4000 to 400 cmꢀ1. Elemental analyzes were performed with a Vario
ELIII CHNS at the Sophisticated Test and Instrumentation Center (STIC), Cochin Univer-
sity, Kerala. Electronic spectra were recorded in dimethylsulfoxide (DMSO) solution in a
1
Systronics 2202 double-beam spectrophotometer from 800 to 200 nm. H and 13C spectra
were recorded on a Bruker WM DCX 500 MHz instrument using TMS as an internal stan-
dard at SAIF, Indian Institute of Technology, Chennai. Magnetic susceptibility measure-
ments of the complexes were recorded using a Guoy balance at room temperature. X-band
electron paramagnetic resonance spectra of the complexes were recorded on a Varian E-
112 spectrometer using TCNE as the standard at the SAIF, Indian Institute of Technology
Bombay, Mumbai, India. DNA cleavage studies were carried out using Gelstan, Gel docu-
mentation system. Anticancer studies were carried out at the Kovai Medical Center and
Hospital Pharmacy College, Coimbatore, Tamilnadu. Melting points were recorded with a
Veego DS model apparatus and are uncorrected.
2.2. Crystal structure determination
Single crystal of the Schiff base (HL) was obtained by slow solvent evaporation of the
ligand in chloroform. Selected crystal data are given in tables 2 and 3. The X-ray diffrac-
tion data for HL were collected on a Bruker SMART CCD diffractometer using graphite-
monochromated Mo Kα radiation (λ = 0.71073 Å) by / and ω scans. X-ray data reduction

582
S. Sathiyaraj et al.
Figure 1. Labeled ORTEP diagram of HL with thermal ellipsoid at 50% probaility.
HO
HO
N
S
N
S
Ethanol
6 h
N
C
H
+
OHC
NH2
Scheme 1. Formation of Schiff base ligand.
structure solution and refinement were done using the SHELXS-97 and SHELXL-97 pack-
ages [33]. The structure (figure 1) was solved by direct methods.
2.3. Synthesis of the Schiff base ligand
3-(Benzothiazol-2-yliminomethyl)-naphthalen-2-ol (HL) was isolated from condensation of
2-hydroxy-1-naphthaldehyde (1.20 g, 10 mmol) and 2-amino benzothiazole (1.5 g,
10 mmol) in ethanol under reflux for 6 h. After cooling the reaction mixture to room tem-
perature, the solid product formed was filtered, washed with ethanol and dried in vacuo
(scheme 1). Purity was checked by TLC. This solid was recrystallized from chloroform,
yielding orange crystals suitable for X-ray diffraction analysis.
2.4. Synthesis of ruthenium(III) Schiff base complexes
The new ruthenium complexes were prepared by the following general procedure (scheme
2). To a solution of [RuX3(EPh3)3] (0.1 mmol) in benzene (X = Cl/Br, E = PPh3/AsPh3) HL
(0.1 mmol) in chloroform was added and the mixture was refluxed for 6 h. The solvent
was then evaporated under reduced pressure and the solid mass filtered and washed with
petroleum ether. The purity was checked by TLC and subjected to purification by column

Ruthenium(III) complexes
583
chromatography. This solid was recrystallized from CH2Cl2/n-hexane mixture. Our efforts
to obtain single crystals of the complexes were not successful.
2.5. DNA-binding and cleavage assay
2.5.1. 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 μM) while gradually increasing the con-
centration of CT-DNA. Absorption data were analyzed to evaluate the intrinsic binding
constant Kb, which can be determined from the following equation [34]:
½DNAꢁ=ðea ꢀ ef Þ ¼ ½DNAꢁ=ðeb ꢀ ef Þ þ 1=Kbðeb ꢀ ef Þ
where [DNA] is the concentration of DNA in base pairs, the apparent absorption
coefficient ɛa, ɛf and ɛb 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] / (ɛa ꢀ ɛf) versus [DNA], Kb is given by the ratio of slope
to intercept.
2.5.2. DNA cleavage studies. Cleavage experiments of supercoiled pBR322 DNA (1 μg)
in 5 mM Tris-HCl, 50 mM NaCl, buffer at pH 7.1, were carried out using agarose gel
electrophoresis. The samples were incubated for 2 h at 37 °C. A loading buffer containing
25% bromophenol blue, 0.25% xylene cyanol, and 60% glycerol was added and electro-
phoresis was carried out at 50 V for 2 h in tris-acetic acid/ethylenediaminetetraacetic acid
(EDTA) buffer using 1% agarose gel containing 1.0 μg/mL ethidium bromide. The bands
were visualized under UV light and photographed. The cleavage efficiency was measured
by determining the ability of the complex to convert supercoiled DNA (SC) to nicked
circular form (NC) and linear form (LC).
2.6. Cytotoxicity assay
The human cervical cancer cell line (HeLa) and human laryngeal epithelial carcinoma cell
line (HEp2) were obtained from the National Center for Cell Science (NCCS), Pune and
grown in Eagles Minimum Essential Medium containing 10% fetal bovine serum (FBS).
All cells were maintained at 37 °C, 5% CO2, 95% air, and 100% relative humidity. Mainte-
nance cultures were passaged weekly, and the culture medium was changed twice a week.
The monolayer cells were detached with trypsin-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 ꢂ105 cells/mL. One hundred microliters 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% relative
humidity. After 24 h, the cells were treated with serial concentrations of the extracts and
fractions. They were initially dissolved in neat DMSO and further diluted in serum-free
medium to produce various concentrations. One hundred microliters per well of each
concentration was added to plates to obtain final concentrations of 1.0, 0.5, 0.25, 0.125,

584
S. Sathiyaraj et al.
and 0.063 μg/mL. The final volume in each well was 200 μL, and the plates were incu-
bated at 37 °C, 5% CO2, 95% air, and 100% relative humidity for 48 h. The medium with-
out samples served as control. Triplicates were maintained for all concentrations.
MTT is a yellow water-soluble tetrazolium salt. A mitochondrial enzyme in living cells,
succinate-dehydrogenase, cleaves the tetrazolium ring, converting the MTT to an insoluble
purple formazan. Therefore, the amount of formazan produced is directly proportional to
the number of viable cells.
After 48 h of incubation, 15 μL of MTT (5 mg/mL) in phosphate-buffered saline 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 μL of DMSO; the
absorbance at 570 nm was measured using a microplate reader. The % cell inhibition was
determined using the following formula.
% Growth inhibition ¼ 100 ꢀ Abs ðsampleÞ=Abs ðcontrolÞ ꢂ 100
A non-linear regression graph was plotted between % cell inhibition and log10 concen-
tration, and IC50 was determined using Graph Pad Prism software [35,36].
3. Results and discussion
Three air stable, mononuclear octahedral ruthenium(III) Schiff base complexes, [RuX2(EPh3)L]
(where X = Cl or Br; E = P or As; L= monobasic tridentate Schiff base), have been prepared by
reaction of [RuX3(EPh3)3] with HL in 1 : 1 M ratio in chloroform–benzene mixture. The analyt-
ical data (table 1) for the new complexes agree well with the proposed molecular formulas
(scheme 2).
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 figure 1.
The crystal data and structural refinement parameters are given in table 2 and selected bond
lengths and angles are given in table 3. The compound crystallized into a monoclinic lattice
with space group P 21/n. The azomethine bond, C12–N1 1.3943(19) Å, is in conformity
with a formal C=N double bond, and the C1–O1 bond distance of 1.3348(18) Å is slightly
shorter than the normal C–O single-bond distance. The bond distance for C12–N2 1.2941
(19) Å and C12–S1 1.7462(14) Å for thiazole are close to C–N and C–S single bonds. This
compound exits in imine-ol form without any ambiguity.
Table 1. Analytical data of ligand and ruthenium(III) complexes.
Elemental analysis calculated (found)
Ligand and
complexes
Yield Melting
Color
(%)
point (°C)
C%
H%
N%
S%
HL
Orange
Brown
72
62
67
64
148
170
158
145
70.02 (70.30) 4.61 (4.33) 9.15 (8.92) 10.46 (10.12)
[RuCl2(PPh3)L]
[RuCl2(AsPh3)L] Brown
[RuBr2(PPh3)L] Dark brown
58.62 (58.37) 3.55 (3.69) 3.80 (3.75)
55.32 (55.09) 3.35 (3.61) 3.58 (3.89)
52.31 (52.62) 3.17 (3.38) 3.39 (3.11)
4.35 (4.09)
4.10 (3.89)
3.38 (3.53)

Ruthenium(III) complexes
585
HO
[RuX3(EPh3)3]
N
S
+
N
H
chloroform-benzene
Reflux 6 h
X
X
N
S
EPh3
O
Ru
N
H
where X=Cl or Br; E=P or As
Scheme 2. Formation of ruthenium(III) Schiff base complexes.
Table 2. Crystal and structure refinement data for HL.
Empirical formula
Formula weight
Temperature
Wavelength
Crystal system
Space group
Unit cell dimensions
a (Å)
C18H12N2OS
304.36
295(2) K
0.71073 Å
Monoclinic
P 21/n
9.7176(3)
b (Å)
c (Å)
15.0806(6)
9.7882(3)
α (°)
90
β (°)
γ (°)
Volume
Z
101.713(3)
90
1404.56(8) Å3
4
Density (calculated)
Crystal size
Theta range for data collection
Index ranges
1.439 Mg/m3
0.47 ꢂ 0.38 ꢂ 0.24 mm3
5.13–34.98°
ꢀ15 6 h 6 14,
ꢀ19 6 k 6 24,
ꢀ15 6 l 6 14
12,583
Reflections collected
Independent reflections
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2σ(I)]
R indices (all data)
5739 [R(int) = 0.0261]
Full-matrix least-squares on F2
5739/0/200
1.035
R1 = 0.0557, wR2 = 0.1303
R1 = 0.0956, wR2 = 0.1518
0.353 and -0.243 e Åꢀ3
Largest diff. peak and hole

586
S. Sathiyaraj et al.
Table 3. Selected bond lengths (Å) and angles (°) for HL.
Bond lengths
Bond angles
S(1)–C(18)
S(1)–C(12)
O(1)–C(1)
O(1)–H(1A)
N(1)–C(11)
N(1)–C(12)
N(2)–C(12)
N(2)–C(13)
C(1)–C(10)
C(1)–C(2)
C(2)–C(3)
C(2)–H(2A)
C(3)–C(4)
C(3)–H(3A)
C(4)–C(5)
C(4)–C(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)–S(1)–C(12)
C(1)–O(1)–H(1A)
C(11)–N(1)–C(12)
C(12)–N(2)–C(13)
O(1)–C(1)–C(10)
O(1)–C(1)–C(2)
C(10)–C(1)–C(2)
C(3)–C(2)–C(1)
C(3)–C(2)–H(2A)
C(1)–C(2)–H(2A)
C(2)–C(3)–C(4)
C(2)–C(3)–H(3A)
C(4)–C(3)–H(3A)
C(5)–C(4)–C(9)
C(5)–C(4)–C(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
1.422(2)
0.9300
1.408(2)
1.418(2)
118.9
119.99(14)
121.16(14)
3.2. Spectral characterization
3.2.1. Infrared spectra. IR spectral data of the ligand were compared with those of the
ruthenium(III) complexes to confirm the binding mode of HL to ruthenium and values are
listed in table 4. HL has the most characteristic bands at 3396 cmꢀ1 ν(O–H), 1622 cmꢀ1
ν(C=N azomethine), 1599 cmꢀ1 ν(C=N thiazole ring), 1311 cmꢀ1 ν(Ph–CO), and 744 cmꢀ1
ν(C–S–C). The band at 1622 cmꢀ1 due to ν(C=N) underwent a shift to lower frequency
(1601–1598 cmꢀ1) after complexation, indicating bonding of unsaturated nitrogen of the
azomethine to ruthenium [37]. The phenolic C–O stretch that appeared at 1311 cmꢀ1 in
the ligand [38] shifts to higher frequencies (1340–1338 cmꢀ1) in the complexes. This shift
confirms participation of oxygen in C–O–M bond. The low frequency at 430–470 cmꢀ1 is
attributed to (M–O) and 560–580 cmꢀ1 to (M–N) [39]. IR spectrum of the ligand revealed
a medium band at 1599 cmꢀ1 for C=N of the thiazole ring, which shifts to lower fre-
quency at 1586–1580 cmꢀ1 after complexation, also indicating coordination to ruthenium.
The unchanged band after complexation at 748 cmꢀ1 for ν(C–S–C) in the free ligand sug-
gests non-involvement of sulfur in coordination. Other characteristic bands due to triphen-
ylphosphine/arsine were also present in the expected region [40].
Table 4. IR and electronic spectroscopic data of ligand and ruthenium(III) complexes.
FT-IR cmꢀ1
UV–Vis
Ligand and
complexes
ν(C=N)
ν(C=N) ν(Ph-OH) ν(Ph-CO) thiazole
λ )
max, nm (ɛ, dm3 molꢀ1 cmꢀ1
HL
1622
1601
3396
–
1311
1338
1598
1586
276(1301), 365(1516), 445(2046), 476(2117)
256(1302), 297(1259), 364(1362), 409(1469),
438(1417), 520(701)
[RuCl2(PPh3)L]
[RuCl2(AsPh3)L]
[RuBr2(PPh3)L]
1598
1597
–
–
1340
1338
1583
1581
256(1300), 301(1274), 363(1361), 405(1306),
432(1412), 524(693)
255(1280), 289(1226), 368(1469), 416(1900),
526(899)

Ruthenium(III) complexes
587
3.2.2. Electronic spectra. Electronic spectra of HL and its complexes were recorded in
DMSO (table 4) and are given in Supplementary material. Two very strong bands at 365
and 276 nm were observed in the spectrum of the ligand, attributed to n–π⁄ and π–π⁄ tran-
sitions in the aromatic ring and C=N [41]. These peaks shift in spectra of the complexes.
Electronic spectra of all the complexes show four to five bands at 263–526 nm and their
5
assignments are given in table 4. The ground state of ruthenium(III) (t2g configuration) is
2
2T2g, while the first excited doublet levels in the order of increasing energy are A2g and
2T1g, arising from the t2g eg configuration [42]. In most ruthenium(III) complexes, elec-
tronic spectra show only charge transfer bands [43]. In a d5 system, and especially in
ruthenium(III) that has relatively high-oxidizing properties, charge transfer bands Lπy→t2g
are prominent in the low-energy region obscuring weaker d–d transitions. Therefore, it
becomes difficult to assign conclusively the bands of ruthenium(III) complexes appearing
in the visible region. Hence, all bands in this region have been assigned to charge transfer
transitions, in conformity with assignments made for similar ruthenium(III) complexes
[44–46].
4
1
3.2.3. 1H and 13C NMR spectra of the Schiff base. The formation of HL was moni-
1
tored by peak ratios in the H and 13C NMR spectra recorded in DMSO-d6 (Supplemen-
tary material). The aromatic proton for the ligand is a multiplet at 7.24–8.27 ppm. The
proton of the hydroxyl group is a broad singlet at 10.12 (Ph–OH) ppm for free HL. The
signal due to azomethine (–HC=N) is a singlet at 8.72 ppm. 13C NMR spectrum of
the Schiff base displays a single resonance at 152 ppm [47] which shows that the azome-
thine carbons are equivalent and also confirms the structure of the ligand. The signal at
166 ppm corresponds to the thiazolic C=N [48]. The resonance observed at 119–138 ppm
is assigned to phenyl.
3.2.4. Magnetic moment and EPR spectra. The room temperature magnetic suscepti-
bility measurements of the ruthenium(III) complexes showed that they are paramagnetic
(μeff = 1.85–1.96 BM) with a single unpaired electron in a low-spin 4d5 configuration and
confirms that ruthenium is in the +3 oxidation state.
The X-band EPR spectra of powdered samples of the complexes were recorded at room
temperature and the “g” values are listed in table 5. The EPR experiments provide only
the absolute “g” values and so neither their signs nor the correspondence of g1, g2 or g3 to
gx, gy or gz are known. All the complexes exhibited g? at 2.49, 2.37, and 2.40 and g|| at
2.24, 2.23 and 2.23 (table 5 and Supplementary material). The two different “g” values
(gx = gy≠gz) indicate a tetragonal distortion in these octahedral complexes. The presence of
Table 5. EPR and magnetic moment data of ruthenium(III) complexes.
Complexes
gx
gy
gz
hgi⁄
μeff (BM)
[RuCl2(PPh3)L]
[RuCl2(AsPh3)L]
[RuBr2(PPh3)L]
2.49
2.37
2.40
2.49
2.37
2.40
2.24
2.23
2.23
2.41
2.32
2.34
1.85
1.88
1.96
1=2
hgi⁄ = ½1=3gx2 þ 1=3gy2 þ 1=3gz2ꢁ
.

588
S. Sathiyaraj et al.
two “g” values also indicates axial symmetry. The nature and position of the lines in the
spectra of the complexes are similar to those of octahedral complexes [53].
3.3. DNA binding and cleavage
3.3.1. Electronic absorption spectroscopy. Transition metal centers are particularly
attractive moieties for the development of effective chemotherapeutic agents since they
exhibit well-defined coordination geometries and also often possess distinctive electro-
chemical or photo-physical properties, thus enhancing the functionality of the binding
agent [3]. The Schiff base and its complexes in DMSO-buffer mixture exhibit an intense
transition at 252–260 nm, attributed to the π–π⁄ transition that is unique to this Schiff base.
The binding of HL and complexes to the DNA helix have been characterized through
absorption spectral titrations by following changes in absorbance and shift in wavelength.
Figure 2 shows absorption spectra of ligand and ruthenium(III) complexes in the absence
and presence of CT-DNA. Addition of increasing amounts of CT-DNA result in hyperchro-
mism and blue shift. As the DNA-double helix possesses many hydrogen-bonding sites, it
is likely that amine groups of the amino acid chain form hydrogen bonds with DNA,
which may contribute to the hyperchromism observed. A similar hyperchromism has been
observed also for a ruthenium(III) complex bearing a Schiff base ligand [54].
Intrinsic binding constants (Kb) were calculated for HL and ruthenium(III) complexes,
1.4 ꢂ 104 Mꢀ1 [HL], 2.9 ꢂ 104 Mꢀ1 [RuCl2(PPh3)L], 1.9 ꢂ 104 Mꢀ1 [RuCl2(AsPh3)L],
2.2 ꢂ 104 Mꢀ1 [RuBr2(PPh3)L]. The different DNA-binding affinity of the three ruthenium
(III) complexes results from different co-ligands affecting binding affinity with DNA. Kb
Figure 2. Absorption spectral traces of HL, [RuCl2(PPh3)L], [RuCl2(AsPh3)L] and [RuBr2(PPh3)L] with
increasing concentration of CT-DNA in a Tris HCl-NaCl buffer (pH 7.1).

Ruthenium(III) complexes
589
Figure 3. Gel electrophoresis showing the chemical nuclease activity of pBR322 plasmid DNA (1 μg) with
different concentrations of the complexes; Lane 0: DNA control; Lane 1: 30 μM [RuCl2(PPh3)L] + DNA; Lane 2:
60 μM [RuCl2(PPh3)L] + DNA; Lane 3: 30 μM [RuCl2(AsPh3)L] + DNA; Lane 4: 60 μM [RuCl2(AsPh3)L] +
DNA; Lane 5: 30 μM [RuBr2(PPh3)L] + DNA; Lane 6: 60 μM [RuBr2(PPh3)L] + DNA.
values obtained for the above ruthenium(III) complexes are comparable with those
for [Ru(dmp)2(APIP)]2+ (APIP= 2-(2-aminophenyl)imidazo[4,5-f][1,10]phenanthroline),
2.3–3.3 ꢂ 104 Mꢀ1 [55].
3.3.2. Cleavage of pBR322 DNA by ruthenium(III) complexes. The ability of ruthe-
nium(III) complexes to perform DNA cleavage is monitored by agarose gel electrophoresis
and the pBR322 plasmid DNA is involved. Agarose gel electrophoresis is shown in figure 3.
No DNA cleavage was observed for the control in which the metal complex was absent (lane
0), The relatively fast migration is the intact supercoil form (Form I) and the slower moving
migration is the open circular form (Form II), which was generated from supercoiled when
scission occurred on one strand [56,57]. With increasing concentration of the ruthenium(III)
complexes (lanes 1 and 2 for [RuCl2(PPh3)L], 3 and 4 for [RuCl2(AsPh3)L] and 5 and 6 for
[RuBr2(PPh3)L]), the amount of Form I of pBR322 DNA diminished gradually and the
amount of the nicked circular DNA (Form II) increased remarkably. When the concentration
increased to 60 μM for all the complexes, DNA was completely converted from Form I to
Form II. [RuCl2(PPh3)L] exhibits greater cleavage efficiency than other complexes which
can be attributed to the longer metal to ligand charge transfer excited state lifetime of
[RuCl2(PPh3)L].
3.4. Cytotoxic activity evaluation
To test the cytotoxicity of Schiff base ligand and ruthenium(III) complexes, HeLa and
HEp2 were cultured in the presence of varying concentrations of ligand and the corre-
sponding ruthenium(III) complexes for 48 h. The inhibitory concentration 50 (IC50) is
defined as the concentration required to reduce the size of the cell population by 50%. The
IC50 values of the ligand (IC50 = 175 μM) exhibit enhanced activity against HeLa cell line
and low activity (IC50 = 389 μM) against HEp2. The IC50 values for the complex are 105,
212 μM for [RuCl2(PPh3)L], 153, 275 μM for [RuCl2(AsPh3)L] and 123, 323 μM for
[RuBr2(PPh3)L] against HeLa and HEp2 cell lines, respectively. All ruthenium(III)
complexes show moderate activity against the two tumor cell lines. Thus, the IC50 value

590
S. Sathiyaraj et al.
for the ligand decreases with the coordination of it to ruthenium metal, which shows the
higher toxicity of the complex than the ligand. Even though they possess better cytotoxic-
ity, they could not reach the effectiveness of the standard drug cisplatin [58].
4. Conclusion
Three mononuclear ruthenium(III) Schiff base complexes were synthesized and character-
ized by various physico-chemical and spectroscopic methods, showing octahedral geome-
try. The binding of the ligand and the complexes with CT-DNA were tested using
absorption titration studies with binding affinity in the order [RuCl2(PPh3)L] >
[RuBr2(PPh3)L] > [RuCl2(AsPh3)L] > HL. Kb values obtained for the above ruthenium(III)
complexes are comparable than those for [Ru(dmp)2(APIP)]2+ (APIP= 2-(2-aminophenyl)
imidazo[4,5-f][1,10]phenanthroline), 2.3–3.3 ꢂ 104 Mꢀ1 [55]. The DNA cleavage results
showed that [RuCl2(PPh3)L] has more cleavage activity than the other complexes. In vitro
study of the cytotoxicity of the ligand and complexes on HeLa and HEp2 show moderate
antitumor activity against selected cell lines.
Supplementary material
Crystallographic data for the structural analysis have been deposited with the Cambridge
Crystallographic Data Center, 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 36033: Email: deposit@ccdc.cam.ac.uk or www.ccdc.cam.ac.uk/deposit).
Acknowledgements
We sincerely thank University Grants Commission (UGC), New Delhi for financial
support [Scheme No. 38-222/2009 (SR)]. One of the authors, RJB, wishes to acknowledge
the NSF-MRI program (Grant No. CHE-0619278) for providing funds to purchase the
diffractometer.
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