Synthesis, interaction with DNA, cytotoxicity, cell cycle arrest and apoptotic inducing properties of ruthenium(II) molecular "light switch" complexes

Article abstract of DOI:10.1016/j.ejmech.2013.04.006

In an endeavor toward the development of metal-based anticancer drugs, we present here the design, synthesis and characterization of three ruthenium(II) functionalized phenanthroline complexes with extended π-conjugation. These complexes have been shown t

Full text of DOI:10.1016/j.ejmech.2013.04.006

European Journal of Medicinal Chemistry 64 (2013) 410e421
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, interaction with DNA, cytotoxicity, cell cycle arrest and
apoptotic inducing properties of ruthenium(II) molecular “light
switch” complexes
C. Shobha Devi a, D. Anil Kumar b, Surya S. Singh b, Nazar Gabra a, N. Deepika a,
,
Y. Praveen Kumar a, S. Satyanarayana a
*
a Department of Chemistry, Osmania University, Hyderabad 500007, Andhra Pradesh, India
b Department of Biochemistry, Osmania University, Hyderabad 500007, India
a r t i c l e i n f o
a b s t r a c t
Article history:
In an endeavor toward the development of metal-based anticancer drugs, we present here the design,
synthesis and characterization of three ruthenium(II) functionalized phenanthroline complexes with
extended p-conjugation. These complexes have been shown to act as promising CT-DNA intercalators as
Received 22 August 2012
Received in revised form
1 April 2013
Accepted 2 April 2013
Available online 15 April 2013
evidenced by UVevisible, luminescence, emission quenching by [Fe(CN)6]4ꢀ, DNA competitive binding
with ethidium bromide and salt dependent studies. All three complexes [Ru(Hdpa)2PPIP]2þ (1),
[Ru(Hdpa)2PIP]2þ (2), [Ru(Hdpa)24HEPIP]2þ (3) clearly demonstrated that they can bind to DNA through
the intercalation mode. Cell viability experiments indicated that all complexes showed significant dose
dependent cytotoxicity in selected cell lines. The apoptosis and cell cycle arrest were also investigated.
The complexes were docked into DNA-base-pairs using the ‘GOLD’ (Genetic Optimization for Ligand
Docking), docking program.
Keywords:
Ruthenium(II) complexes
Fluorescence
Cytotoxicity
Ó 2013 Elsevier Masson SAS. All rights reserved.
Flow cytometry
Docking
1. Introduction
for transferrin and by the necessity of its reductive activation in
cells [8,9]. These characters could be due to the potency of ruthe-
The field of metal-based chemotherapy has opened up after the
serendipitous discovery of cisplatin (a metal coordination com-
pound containing no organic units) by Barnett Rosenberg [1,2].
Cisplatin is highly effective against testicular and ovarian carci-
nomas as well as bladder, head and neck tumors [3,4]. However
platinum based drugs have limited clinical utility due to associated
toxic effects [5]. These limitations have prompted a search for more
effective and less toxic alternative metal-based antitumor agents. In
this respect ruthenium complexes have attracted a great deal of
attention due to their strong DNA-binding ability and remarkable
anticancer activity [6]. In particular ruthenium(II) polypyridyl
complexes acts as good noncovalent probes for DNA. In addition
these complexes are water soluble, coordinately saturated and inert
to substitution. Ruthenium complexes show lower general toxicity
than platinum compounds [7]. Ruthenium belongs to the same
group of elements as iron, which is reflected by its strong affinity
nium complexes to mimic the binding of iron to molecules of bio-
logical significance; this ability is an attractive feature of ruthenium
complexes [10]. Therefore these compounds are well suited for
medicinal applications as an alternative to platinum antitumor
drugs [11].
Two ruthenium-based anticancer drugs, NAMI-A (ImH[trans-
RuCl4(DMSO)(Im)]) and KP1019 (InH[trans-RuCl4In2]) [12,13] have
successfully completed phase I clinical trials for the treatment of
metastatic tumors and colon cancers. Though both of them have
their own limitations [14] in their ability as drugs, these limitations
paved the way for the development of new ruthenium(II) anti-
cancer complexes. A few studies have been carried out on the
anticancer activity of ruthenium(II) polypyridyl complexes [15e17],
but many more studies need to be undertaken to develop a novel
and interesting group of antitumor transition metal complexes.
The design, synthesis and reactivity of novel ruthenium complexes
have become the central focus of research in several laboratories,
including ours.
Literature reveals that the ruthenium(II) polypyridyl complexes
have a wide range of applications as anticancer and antibacterial
* Corresponding author. Tel.: þ91 40 2768233.
E-mail address: ssnsirasani@gmail.com (S. Satyanarayana).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.04.006

C. Shobha Devi et al. / European Journal of Medicinal Chemistry 64 (2013) 410e421
411
agents, which find their origin in intercalative interactions with
2.3.1. Synthesis of Ligand 2-(4-hydroxy,3-ethoxy-phenyl)-1H-
imidazo[4,5-f][1,10](phenanthro- line) (4HEPIP)
DNA. In recent years many authors have published their findings
about these complexes containing various intercalating ligands
[16,17]. The spectral characteristics and DNA binding properties of
the complexes are influenced by the planarity of the intercalating
ligand and nature of substituents on the ligand. In this paper we
report the synthesis and characterization of ruthenium(II) poly-
pyridyl complexes containing different intercalating ligands which
shows efficient DNA binding properties with the increase in
planarity of the ligands. These complexes have demonstrated DNA
binding ability hence we attempted to study the drug-DNA in-
teractions in detail. These complexes were evaluated for in-vitro
cytotoxicity on tumor cell lines such as A549 (Human alveolar
adenocarcinoma cell line), Du145 (Human Prostate cancer cell
lines), HeLa (Human cervical cancer cell line) and MCF-7 (Human
breast adenocarcinoma cell line). The complexes were docked into
DNA-base-pairs using the ‘GOLD’ docking program [18]. We found
that using such a computational method, specific DNA interaction
properties of the complexes can be identified. These observations
are in accordance with the experimental results.
Mixture of phen-dione (0.52 g, 2.50 mM), 4-hydroxy, 3-ethoxy-
benzaldehyde (0.40 g, 3.50 mM), ammonium acetate (3.88 g,
50 mM) and glacial acetic acid (15 mL) were refluxed together for
4 h as per Steck and Day [22], then cooled to room temperature and
diluted with water. Drop wise addition of ammonia gave a yellow
precipitate which was collected, washed with water, dried and
purified by recrystallization from pyridine-H2O (9:1, v/v); yield
0.71 g (70%). Analytical data: Elemental Analysis (Expt. and Calcd.
for C21H16N4): Expt. (%): C: 70.80; H: 4.60; N: 15.74; Calcd. C: 70.77;
H: 4.53; N: 15.72; ESI-MS (m/z): Calcd: 356, found: 357 [M þ H]þ. IR
(KBr, cmꢀ1): 3200e3015 (broad) (
1566 (
, C]C); 1H NMR 400 MHz (DMSO-d6, TMS
NH), 8.90 (d, 2H, H1), 7.90 (d, 2H, H3), 7.80 (d, 1H, H9), 7.70 (s, 1H,
12), 7.09 (d, 1H, H8), 6.90 (m, 2H, H2), 5.01 (s, 1H, OH), 4.15 (q, 2H, e
n
, NeH)(OeH), 1683 (
n, C]N),
n
d
): 9.65 (s, 1H,
H
OCH2) and 1.30 (t, 3H, CH3).
2.3.2. Synthesis of [Ru(Hdpa)2(PPIP)](ClO4)2$2H2O (1)
A mixture of cis-[Ru(Hdpa)2(Cl2)] (0.15 g, 0.26 mM) and PPIP
(0.10 g, 0.26 mM) was heated to reflux in water (30 mL) for 15 min
under N2 atmosphere. After this the solution was cooled; then the
reducing agent 30% H3PO3 neutralized with NaOH was added to the
reaction mixture and the reflux was continued for 3 h and treated
with an excess of NaClO4. The precipitated complex was dried,
dissolved in a small amount of acetonitrile and purified by chro-
matography over alumina using acetonitrileemethanol (3:1, v/v) as
an eluent. The red colored compound was obtained after drying in
vacuo. Yield, 0.16 g, 66%. Analytical data: Elemental Analysis (Expt.
and Calcd. for C45H38N10O11Cl2Ru): Expt. (%): C: 50.40; H: 3.50; N:
13.49%. Calcd. C: 50.65; H: 3.56; N: 13.17. ESI-MS (m/z): Calcd: 1066,
2. Experimental
2.1. Physical measurements
UVeVisible spectra were recorded with an Elico SL159 spec-
trophotometer. Fluorescence measurements were performed on
a Hitachi F-2500 spectrofluorimeter. IR spectra were recorded on
KBr disks on a PerkineElmer FT-IR-1605 spectrometer. 1H and 13
C
NMR spectra were recorded on a Bruker 400 MHz spectrometer
with DMSO-d6 as solvent at RT and tetramethylsilane (TMS) as
the internal standard. Microanalyses (C, H, N) were carried out
with a PerkineElmer 240 elemental analyzer. ESI-MS mass
spectra were recorded on ESI-MS Micro mass Quattro Lc triple
quadrupole mass spectrometer with Mass Lynx software (Man-
chester, UK) in m/z.
found: 1067 [M þ H]þ. IR (KBr, cmꢀ1): 3475e3315 (broad) (
n, NeH),
1662, 1615 (n, C]N), 1565, 1530 (n
, C]C), and 524 (RueN). 1H NMR
400 MHz (DMSO-d6, TMS d): 7.10 (t, 2H, H13), 7.20 (t, 1H, H14), 7.40
(t, 2H, H12), 7.77 (t, 2H, H2), 7.79 (d, 4H, H18), 7.80 (d, 2H, H8), 7.90 (d,
2H, H9), 8.20 (d, 4H, H19), 8.30 (d, 4H, H17), 8.90 (d, 2H, H3), 9.04 (d,
4H, H16), 9.80 (d, 2H, H1), 10.70 (s, 2H, NH). 13C[1H] NMR (DMSO-d6,
100 MHz): 115 (2C, C8), 117 (2C, C9), 119.10 (2C, C13), 119.60 (1C, C14),
2.2. Materials and methods
120.0 (2C, C10,11) 125.0 (2C, C6,7), 128.8 (2C, C2), 130.5 (4C, C17),
All reagents and solvents were purchased commercially and
were used as received unless otherwise noted. Ruthenium(III)
chloride trihydrate (RuCl3$3H2O), 1,10-phenanthroline mono-
hydrate and 2,20-dipyridylamine (Hdpa) were purchased from
Merck. CT (Calf Thymus) DNA was purchased from Aldrich. Super-
coiled pBR322 plasmid DNA (stored at ꢀ20 ꢁC) was obtained
from Fermentas life sciences, agarose (Genei), MTT (3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), DAPI
(40,6-diamidino-2-phenylindole, dihydrochloride) 0.1 mg/mL, and
cisplatin were purchased from Sigma Aldrich. Ultra-pure Milli-Q
138.90 (4C, C18), 142 (4C, C4,5)146 (2C, C15) 151.60 (2C, C12), 153.40
(2C, C3), 156.70 (4C, C16), 157.1 (4C, C20), 158.90 (4C, C19), 162.70 (2C,
C1).
2.3.3. Synthesis of [Ru(Hdpa)2(PIP)](ClO4)2$2H2O (2)
This complex was obtained by a procedure similar to that
described above; PIP (0.0769 g, 0.26 mM) was used in place of PPIP.
Yield: 65%. Analytical data: Elemental Analysis (Expt. and Calcd. for
C39H34N10O10Cl2Ru) Expt. (%): C: 48.40; H: 3.50; N: 14.40. Calcd. C:
48.04; H: 3.49; N: 14.37. ESI-MS (m/z): Calcd: 974, found: 975
water (18.2 m
U
) was used in all experiments. Double distilled water
[M þ H]þ. IR (KBr, cmꢀ1): 3324 (
n
, NeH), 1675, 1634 (
n
, C]N), 1582,
was used for preparing various buffers. All cell lines were obtained
from NCCS, Pune. Cells were routinely kept in DMEM or RPMI-1640
medium (Sigma Aldrich Ltd. supplemented with 10% (v/v) fetal
1532 (n d):
, C]C), 537 (RueN). 1H NMR 400 MHz (DMSO-d6, TMS
10.81 (s, 2H, NH), 9.05 (d, 2H, H1), 8.90 (d, 2H, H2), 8.30 (d, 2H, H3),
7.80 (d, 4H, H12), 7.70 (t, 2H, H9), 7.60 (t, 4H, H14), 7.55 (d, 4H, H15),
7.50 (d, 2H, H8), 7.10 (t, 4H, H13), 6.80 (t, 1H, H10). 13C[1H] NMR
(DMSO-d6, 100 MHz): 153.50 (4C, C15), 151.30 (2C, C1), 151.6 (4C,
bovine serum (FBS), 2 mM
L
-glutamine, 4.5 g Lꢀ1 glucose, 1ꢂ Non-
essential amino acids and 1ꢂ antibiotics consisting of penicillin/
streptomycin, gentamycin, amphotericin B, nystatin). Guava cell
cycle reagent kit, Guava Nexin reagent were provided by Millipore.
C16), 148.1 (2C, C11), 140.2 (2C, C4), 138.80 (4C, C12), 130.60 (2C, C3),
130.0 (2C, C8), 130.03 (2C, C5), 129.90 (2C, C2), 129.20 (4C, C13), 127.0
(2C, C6,7), 126.90 (1C, C10), 118.90 (4C, C14), 115.30 (2C, C9).
2.3. Synthesis and characterization of ligand and complexes
2.3.4. Synthesis of [Ru(Hdpa)2(4HEPIP)] (ClO4)2$2H2O (3)
The starting materials 1,10-phenanthroline-5,6-dione (phen-
dione) [19], (2-phenyl-1H-imidazo[4,5-f][1,10](phenanathroline))
(PIP), (2-(40-phenoxy-phenyl) imidazo[4,5-f][1,10]phenanthroline)
(PPIP) [20] and [Ru(Hdpa)2Cl2] [21] have been synthesized by
previously reported procedures.
This complex was obtained by a procedure similar to that
described above; 4-HEPIP (0.09 g, 0.26 mM) was used in place of
PPIP. Yield: 65%. Analytical data: Elemental Analysis (Expt. and
Calcd. for C41H38N10O12Cl2Ru) Expt. C: 47.40; H: 3.75; N: 13.20%.
Calcd. C: 47.59; H: 3.70; N: 13.54. ESI-MS (m/z): Calcd: 1034, found:

412
C. Shobha Devi et al. / European Journal of Medicinal Chemistry 64 (2013) 410e421
1035 [M þ H]þ. IR (KBr, cmꢀ1): 3418e3150 (broad) (
n
, OeH) (
n
, Ne
Fluorescence titration experiments were also performed by
adopting a procedure analogous to that used for the absorption
H), 1635 (n, C]N), 1581, 1526 (n
, C]C), and 538 (RueN). 1H NMR
400 MHz (DMSO-d6, TMS
d
): 1.43 (t, 3H, CH3), 4.10 (q, 2H, eOCH2),
titration experiment.
was employed (10
(10e150 M) was added. Emission quenching experiments were
A
fixed concentration of the complex
6.46 (t, 2H, H2), 6.80 (t, 4H, H15), 7.01 (s, 1H, H12), 7.05 (t, 4H, H16),
7.10 (d, 1H, H8), 7.20 (s, 1H, H9), 7.35 (d, 2H, H3), 7.70 (d, 1H, H1), 7.79
(s, 4H, H18), 8.01 (t, 4H, H17), 9.93 (s, 2H, NH) and 6.60 (s, 1H, OH).
13C[1H] NMR (DMSO-d6, 100 MHz): 14.70 (1C, CH3), 64.20 (1C, e
OCH2), 114.10 (1C, C9), 114.2 (1C, C10), 114.70 (1C, C8), 118.40 (1C,
mM) to which increments of DNA solution
m
carried out at room temperature by the addition of micro liter ali-
quots of 0.01 M K4[Fe(CN)6] to the 3 mL of complexes in TriseHCl
buffer. The quenching constant Ksv was determined by using Sterne
Volmer equation [25] I0/I ¼ 1 þ Ksv[Q] where I0 and I are the fluo-
rescence intensities in the absence and presence of the quencher
respectively, Q is the concentration of the quencher, Ksv is a linear
SterneVolmer quenching constant and is obtained from the slope.
Light switch on and off experiments were performed by using
equimolar concentrations (0.01 M) of CoCl2 (source of Co2þ ion) and
Na2EDTA (disodium ethylene diamine tetraacetate). These com-
C
12), 118.90 (2C, C2), 119.20 (2C, C3), 119.40 (1C, C11), 138.0 (4C, C15),
139.1 (4C, C4,5),149.0 (2C, C1),151.0 (2C, C13, 14),151.4 (2C, C6,7),152.0
(4C, C16), 152.70 (4C, C19) 153.0 (4C, C17), 153.90 (4C, C18). Structures
of these complexes are shown in Fig. 1.
2.4. DNA-Binding experiments
pounds were added in aliquots of micro liter into 10 mM of complex
Interaction of the complexes with CT-DNA was studied in Tris
buffer (5 mM TriseHCl, 50 mM NaCl, pH 7.2). The DNA had a ratio of
UV absorbance at 260 and 280 nm of about w1.9:1 indicating that
the DNA was sufficiently free of protein [23]. The concentration of
DNA in nucleotide i.e. phosphates or bases was determined spec-
trophotometrically by employing an extinction coefficient of
6600 Mꢀ1 cmꢀ1 at 260 nm [24]. Concentrated stock solutions of CT-
DNA were prepared in buffer; the concentration was determined by
UV absorbance at 260 nm. Stock solutions were stored at 4 ꢁC
and used within five days. Concentrated stock solutions of metal
complexes were prepared by dissolving in buffer at pH 7.1 in 5%
CH3OH 5 mM TriseHCl 50 mM NaCl and diluting suitably with
corresponding buffer to required concentrations for all the exper-
iments [15].
solutions in fluorescence titration. Salt dependent studies were
conducted in 5 mM TriseHCl and NaCl concentrations as indicated.
Viscosity experiments were carried out on Ostwald viscometer
immersed in thermostated water-bath maintained at 30 ꢃ 0.1 ꢁC
using BPE-buffer (6 mM Na2HPO4, 2 mM NaH2PO4, 1 mM Na2EDTA,
pH ¼ 7). Flow time was measured with a digital stopwatch and
every sample was measured at least thrice and was accepted if it
agreed within 0.1 s and an average flow time was calculated. Data
1/3
were presented as (
h
/ho)
vs the concentration of [Ru(II)]/[DNA],
where
h
is the viscosity of DNA in the presence of complexes and ho
is the viscosity of DNA alone.
2.5. Photocleavage experiments
Absorption titration experiment was performed at room tem-
perature to determine the binding affinity between the DNA and
the complexes. Constant concentration of the complex 3.0 mL
TAE-buffer (pH 8.0; 40 mM Tris-base, 20 mM acetic acid, 1 mM
EDTA) was used for gel-electrophoresis. TE-buffer (10 mM TriseHCl
and 1 mM Na2EDTA) used for dilution of pBR322 DNA. Supercoiled
(20
mM) was taken in a cuvette to which increments of the DNA
stock solution was added. DNA solution (concentration 0e100
mM)
pBR322 DNA (0.1 mg/mL) was treated with the complexes and the
was added to each cuvette reference and sample to eliminate the
absorbance of DNA itself. Solutions were mixed for 3 min and ab-
sorption spectra were recorded. Titrations were continued until
there was no further change noticed in the spectra.
mixtures were irradiated at 365 nm for 1 h. A loading buffer con-
taining 25% bromophenol blue, 0.25% xylene cyanol and 30% glyc-
erol (2
mL) was added. The samples were then analyzed by 0.8%
agarose gel electrophoresis at 50 V for 2 h. The gel was stained with
Fig. 1. Structures of 1, 2 and 3 complexes.

C. Shobha Devi et al. / European Journal of Medicinal Chemistry 64 (2013) 410e421
413
4
mL (from 1 mg/100
mL) ethidium bromide and photographed
2.10. Docking simulations
under UV light. The gels were viewed in a Gel doc system and
photographed using a CCD camera (Alpha InfoTech Corporation).
The molecular docking calculations on all three complexes were
done by using the 3.01 GOLD (Genetic Optimization for Ligand
Docking) program, which is based on Genetic Algorithms. This
method allows partial flexibility of the hydroxyl groups of the
respective DNA molecule and full flexibility of the ligand. The DNA
sequences used for the docking simulations were obtained from the
PDB protein data banks, and have double helices associated with
ligands. Using Discovery Studio 3.0, we built ((C-G-A-T-T-A-A-T-C-
G) and the PDB code: 1D49) initio double helix DNA decamer. In
order to evaluate the GOLD scoring function, all water molecules
were removed from the DNA molecules. The function fitted was
Gold Score:
2.6. Antimicrobial activity
The antimicrobial activity of the complexes was studied
against Escherichia coli and Neurospora crassa. Each of the ruth-
enium(II) complex was dissolved in DMSO at a concentration of
1 mg/mL. Paper discs of Whatman filter paper no. 1 were ster-
ilized in an autoclave. The paper disks were saturated with 10 mL
of the complex and were placed in the petri dishes containing LB
(Luria Bertini) agar media inoculated with E. coli and N. crassa
separately. The petri dishes were incubated at 37 ꢁC and the zone
inhibitions were recorded after 24 h. The results were also
compared with standard antibacterial drug Ampicillin at the
same concentration.
Fitness ¼ Sðhb ꢀ extÞ þ 1 : 3750 ꢂ Sðvdw ꢀ extÞ þ Sint
where S(hb ꢀ ext) is the DNA-ligand hydrogen bond score,
S(vdw ꢀ ext) is the DNA-ligand Vander Waals score, Sint is the
score from intramolecular ligand interactions [26].
2.7. Cell viability assay
To study the effects of the complexes on cell growth, selected
cell lines growing exponentially were added to 96-well plates
(Orange Scientific) at a density of 3 ꢂ 103 per well after counting on
Bright Line Heamocytometer (Sigma Ltd.). Stock solutions (10 mM)
of 1e3 were prepared by dissolving in DMSO and diluted with
RPMI-1640 or DMEM to the required concentrations prior to use
(final DMSO concentration, 0.1e1% v/v), which were proved to be
stable for at least 48 h at room temperature as monitored by UVe
3. Results and discussion
3.1. Synthesis and characterization
Ligands PPIP, PIP and 4HEPIP were prepared through conden-
sation of 1,10-phenanthroline-5,6-dione with respective aldehydes
using a method similar to that described by Steck and Day [22] in
70% yield. The corresponding ruthenium(II) complexes were syn-
thesized by direct reaction of [Ru(Hdpa)2Cl2] with the appropriate
precursor ligands. The desired complexes were isolated as their
perchlorate salts in 65% yield. All complexes were characterized by
elemental analyses, ESI-MS, UVeVIS, IR and NMR spectral tech-
niques. The C, H, N analyses of the complexes were consistent with
the formula of the complexes. In the electronspray ionization mass
spectrometry of complexes, the [M þ H]þ were observed, the ob-
tained molecular weights were consistent with the expected
values.
visible spectroscopy. Three complexes (1e100
added to the wells, ensuring an equal volume of 200
plates. Cell number/proliferation was measured after 48 h using a
standard MTT-based assay. 200 L of MTT [0.4 mg/mL in RPMI
m
M) were then
mL across the
m
medium without FCS] was added to each well and the plates were
incubated for 4 h at 37 ꢁC. After this, the medium was aspirated,
200 mL of DMSO (Sigma Ltd.) was then added to each well and the
plates were agitated gently for 5 min before measuring the optical
density at 620 nm in each well using Elisa reader (Thermo Scientific
Multi Skan EX). The IC50 values were calculated from the plotted
absorbance data for the doseeresponse curves. IC50 values (in
were expressed as the means ꢃ S.D.
mM)
The UVeVis absorption spectra of complexes showed bands in
UV region, 220e398 nm, characterized by intense
p / p* an intra-/
interligand transitions [27]. In addition to these bands absorptions
in visible region 450e470 nm can be attributed to metal-to-ligand
charge transfer (MLCT) Rudp / Lp* transitions. The infrared spectra
of 1e3 complexes showed broad band ranges from 3350 to
2.8. Cellular uptake. Flow cytometry
To study the cellular uptake of complexes, HeLa cells growing
exponentially were serum starved for 24 h in serum free media and
3050 cmꢀ1
1536 and 1474 (
(n
, NeH)(OeH), the bands at around 1625 (
n, C]N),
incubated with 50
were pelleted and washed with phosphate buffered saline. Com-
plexes at 20 M were added. One set of the cell suspension was
mM of complexes 1e3 for 4 h. After this, the cells
n
, C]C), which shifted to a lower frequency when
compared to the bands obtained for the free ligand indicates the
complexation. As reported [28] the metal-nitrogen stretching vi-
brations are expected to occur below 650 cmꢀl. The bands obtained
at 520e540 cmꢀ1 (RueN) for all complexes, further supported the
complex formation.
m
used for fluorescence intensity acquisition using the Express Pro
program in the flow cytometer (Guava). The rest of the cell sus-
pension was stained with Nexin and data acquired by flow cy-
tometer for apoptosis. In each case, 10,000 events were acquired.
In each of the complexes, the 1H NMR chemical shifts of protons
on the nitrogen atom of imidazole ring were not observed; since
the protons are very active and there is fast proton exchange be-
tween two imidazole N’s, therefore no peak was observed for the
NeH group of imidazole ring [29,30]. A characteristic peak for the
NeH group of the Hdpa ligand is observed at 11.00 to 9.91 for all
complexes. 1H NMR spectra of free ligand aromatic peaks appeared
2.9. Live cell confocal microscopy
The localization of complexes 1e3, was studied by confocal
microscopy. The HeLa cells were cultured on coverslips (corning
22 ꢂ 22 mm), incubated for 24 h until they reached 70% confluency.
These were then serum starved overnight, followed by incubation
at
shifted to down field and appears at
plexes. This down field shift may be due to complexation. 13C[1H]
NMR spectrum of ligand aromatic carbons is shown at 110e145.
The spectra of aromatic carbons of complexes resonated in the
range of 115e155 and shifted down field compared to the ligand.
d
6.9e8.9. After complex formation the aromatic peaks are
with 50
m
M of complexes 1e3, for varying amounts of time at 37 ꢁC.
d
7.3e9.3 in all three com-
Confocal microscopy imaging was carried out on one set without
any staining and one set was stained with DAPI (nuclear stain)
to identify localization of the complexes and any nuclear
disintegration.
d
d

414
C. Shobha Devi et al. / European Journal of Medicinal Chemistry 64 (2013) 410e421
Fig. 2. Absorption spectra of complexes 1, 2 and 3 in TriseHCl buffer upon addition of CT-DNA. [Ru] ¼ 20
mM. Arrow shows the absorbance change upon the increase of DNA
concentration. Inset: plots of [DNA]/(εa ꢀ εf) versus [DNA] for the titration of DNA with ruthenium(II) complexes.
3.2. Electronic absorption titration
where [DNA] is the concentration of DNA in base pairs, εa is the
apparent extinction coefficient obtained by calculating Aobsd
/
The absorption spectra of the complexes 1, 2 and 3 in the
absence and presence of calf thymus DNA are shown in Fig. 2. With
increasing concentration of DNA, the absorption spectra of the
complexes exhibited gradual decrease in absorption with a small
red shift. In addition, isosbestic points were observed at 447 nm for
1, at 361 nm for 2 and 274 nm for 3 respectively. These spectral
characteristics are consistent with such a mode of binding that
involves a strong stacking interaction between an aromatic chro-
mophore and the base pairs of DNA. It also suggested that com-
plexes 1e3 bind to DNA by intercalation in a manner similar to
known intercalators [Ru(L)2(dppz)] [31].
[complex], εf corresponds to the extinction coefficient of the com-
plex in its free form and εb refers to the extinction coefficient of the
complex in the fully bound form. Kb is the equilibrium binding
constant (in Mꢀ1) of the complex binding to DNA. When plotting
[DNA]/(εa ꢀ εf) vs [DNA], Kb is obtained by the ratio of the slope to
the intercept. The intrinsic binding constants Kb of complexes 1, 2
and 3 are given in Table 1. The DNA binding affinities of these
complexes follow the order 1 > 2 > 3. This data indicates that the
size and the shape of the intercalated ligand has a significant effect
on the strength of DNA binding and the most suitable intercalating
ligand leads to the highest affinity of complexes with DNA. As PPIP
In order to compare quantitatively the binding strength of the
complexes, the intrinsic binding constants Kb of the complexes with
DNA were obtained by monitoring the changes in absorbance at
398 nm for complex 1, 298 nm for complex 2 and 218 nm for
possesses a greater planar area and extended p system, it could
penetrate more deeply into the DNA base pairs. Therefore binding
constant for complex 1 is more than that of 2 and 3.
Due to the additional groups such as hydroxyl (eOH) and ethoxy
(eOEt) present in 4-HEPIP ligand exert some steric hindrance in
complex 3. These groups may come into close proximity of the base
pairs at the intercalation site therefore binding constant of complex
3 is less than 2. Hydroxyl (eOH) and ethoxy (eOEt) are electron
donor groups, this makes the intercalating ligand more electron
complex
3 with increasing concentration of DNA using the
following equation [32]:
À
Á
À
Á
À
À
Á
½DNAꢄ= εa ꢀ εf ¼ ½DNAꢄ= εb ꢀ εf þ 1= Kb εb ꢀ εf
Table 1
Absorption peaks, Absorption, Emission and quenching binding constants of ruthenium(II)complexes.
Complex
Absorption lmax(nm)
Hypochromism
Absorption binding constant Kb (Mꢀ1
)
Emission (K) binding constant
Ksv only comp
Comp þ DNA
1:50
1:200
1
2
3
228, 398, 457
218, 276, 470
298, 351, 464
20.1%
17.3%
13.2%
2.42 ꢂ 105
1.17 ꢂ 105
6.41 ꢂ 104
2.39 ꢂ 105
1.25 ꢂ 105
6.32 ꢂ 104
309
267
215
154
104
75
46
24
17

C. Shobha Devi et al. / European Journal of Medicinal Chemistry 64 (2013) 410e421
415
Fig. 3. Emission spectra of complexes 1, 2 and 3 in TriseHCl buffer at 25 ꢁC upon addition of CT-DNA. Arrow shows the intensity change upon increasing CT-DNA concentrations.
denser, hence interaction between DNA and the complex decreases.
These results are in agreement with the reported complexes
[33,34].
468 nm and emission peaks at 606, 608 and 611 nm for 1, 2 and 3
complexes respectively. Binding data were cast into the Scatchard
plot of r/Cf vs r, where r is the binding ratio (Cb/[DNA]) and Cf is the
free ligand concentration. Nonlinear least square fitting of the data
yield K. As previously reported [35], the emission binding constant
values of the three complexes are in agreement with the value
obtained from the absorption titration experiment. The K (emission
binding constant) values are given in Table 1. Emission quenching
experiments using [Fe(CN)6]4ꢀ as quencher further supported the
above proposal (Fig. 4), absence of DNA, complexes were efficiently
quenched by quencher, while presence of DNA the quenching was
small, because the highly negatively charged [Fe(CN)6]4ꢀ would be
repelled by the negative charge of the DNA phosphate backbone
which would hinder the quenching of the emission of the bound
complexes. All complexes showed linear SterneVolmer plots, Ksv
values are given in Table 1.
The Kb values of the complexes studied are higher than that of
analogous ruthenium(II) complexes reported [Ru(bpy)2PPIP]
(4.3 ꢂ 104) [20]. This may also be attributed to the ancillary ligand, i.e.
Hdpa compared to bipyridine. The only structural difference be-
tween [Ru(Hdpa)2(PPIP)]2þ and [Ru(bpy)2(PPIP)]2þ is the amine e
NHe between two pyridine moieties its incorporation significantly
increased the binding constant of the complex. Thus it may be the e
NHe group in the Hdpa ligand which would make significant con-
tributions to the binding of the complex into the DNA.
3.3. Emission studies
As shown in Fig. 3, upon addition of CT-DNA, the fluorescence
emission intensities of the complexes increases. This implies that
complexes can strongly interact with DNA and be protected by the
DNA efficiently. Since the hydrophobic environments inside the
DNA helix reduces the accessibility of solvent water molecules to
the complex and the complex mobility is restricted at the binding
site, it leads to decrease of relaxation and hence fluorescence in-
tensity increases. The excitation peaks appeared at 460, 475 and
Upon addition of DNA to the complex, its emission intensity was
increased (light switch on), this is because complex binds to DNA to
form DNA-[Ru(Hdpa)2(PIP)]2þ. The emission of this bound complex
was quenched by Cobalt(II) ion, thus turning the light switch off
[36]. The addition of Co2þ to complex bound to DNA resulted in the
loss of luminescence due to the formation of Co2þ
e
[Ru(Hdpa)2(PIP)]2þ heterometallic complex. Addition of EDTA into
the buffer system containing of Co2þe[Ru(Hdpa)2(PIP)]2þ the
emission intensity of the complex was recovered again (light
switch on). This indicated that heterometallic complex became free
again due to EDTAeCo2þ complex formation. Similar observations
were obtained for all the three complexes. The emission of DNA-
intercalated complex (light switch on) can be quenched by transi-
tion metal ions, thus turning the light switch off.
Fig. 4. Emission quenching of complexes 1, 2 and 3 [Ru] ¼ 20
m
M, with K4[Fe(CN)6] in
Fig. 5. Effect of increasing amount of ethidium bromide (EtBr), complexes 1, 2 and 3 on
relative viscosity of CT-DNA at 25 (ꢃ0.1) ꢁC. [DNA] ¼ 0.25 mM.
the absence (a), presence (b) and excess of DNA (c).

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C. Shobha Devi et al. / European Journal of Medicinal Chemistry 64 (2013) 410e421
Table 2
Antibacterial activity of ruthenium(II) complexes at 20 mg/mL.
Complex
Bacterial species
Fungal species
E. coli
N. crassa
DMSO
Nil
Nil
1
10
7
2
8
6
3
7
5
Ampicillin
18e22
13e15
Values of Zone of inhibition (mm, including the diameter of the disc).
of the complexes decreased proportionately to that of proven
intercalators [39] like ethidium bromide.
3.6. Photocleavage
It is reported that many antitumor agents can interact with DNA
and cause DNA strand scission [40] and also the photocleavage
activity of various Ru(II) and Co(III) complexes have been reported
[41e43] for this reason we have also studied the capacity of the
present complexes to cleave pBR322 DNA by gel electrophoresis as
shown in Fig. 7. Gel electrophoresis separation of pBR322 DNA was
studied after incubation with complexes and irradiation at 365 nm.
No DNA cleavage was observed for the control in which the com-
plex was absent. When this circular DNA was subjected to elec-
trophoresis, relatively fast migration was observed for the
supercoiled form (form I). As scission occurred on one strand, the
supercoiled DNA generated electrophoretically slower-moving
open circular form (form II). When both strands were cleaved, a
linear form (III) was generated that migrated between forms I and II
[44]. The delivery of high concentration metal ion to the helix, may
generate singlet oxygen (1O2) or hydroxyl radicals (*OH) which
yields an efficient DNA cleavage reaction. To identify the nature of
the reactive species responsible for photoactivated cleavage of
plasmid DNA, we have further investigated the DNA cleavage
experiment in the presence of the singlet oxygen (1O2) scavenger
histidine. The DNA cleavage of the plasmid by complexes 1e3 was
inhibited in the presence of histidine (lanes d, h and l) (Fig. 7.),
suggesting that 1O2 is likely to be the reactive species responsible
for the cleavage reaction. Further, the control (in which the complex
was absent) confirmed that the complexes did cause photo-
sensitized cleavage.
Fig. 6. NaCl concentration dependency of the equilibrium binding constants for DNA
binding ligands. Data are given for complexes 1, 2 and 3.
3.4. Viscosity measurements
The measurement of viscosity of DNA is regarded as the least
ambiguous and the most critical test of a DNA binding model in
solution in the absence of X-ray crystallographic structural data
[37]. Intercalation mode is expected to lengthen the DNA helix as
the base pairs are pushed apart to accommodate the bound ligand,
leading to an increase in viscosity [37]. The effects of complexes 1, 2,
3 and ethidium bromide (EtBr) on the viscosity of rod-like DNA, are
shown in Fig. 5. As expected, EtBr increases the relative specific
viscosity for the lengthening of the DNA double helix resulting from
intercalation. The increase in the degree of viscosity, may depend
on the binding affinity of DNA. These results also suggested that all
complexes intercalate between the base pairs of DNA and parallel
the result obtained by absorption, fluorescence and quenching
measurements.
3.5. Salt dependent studies
The salt dependence studies of complexes binding to DNA were
determined. The clear dependence of the binding constants for
these complexes upon Naþ binding to DNA was analyzed by Record
theory [38]. From this theory, the slope of the lines provide an es-
timate of Z
j
, where Z is the charge on the complex and
j
is the
3.7. Antimicrobial activity
fraction of counter ions associated with each DNA phosphate
(
j
¼ 0.88 for double-stranded B-form DNA). The data in Fig. 6
The antibacterial and antifungal activity result indicates that the
complexes showed good activity against E. coli and N. crassa were
given in Table 2. Complex 1 showed slightly more activity than 2
and 3. The results were expressed as zone inhibition diameter
(in mm). DMSO control showed a negligible activity as compared
with the complexes. The antimicrobial activity increased as the
concentration of the complexes increased [45].
shows that the slopes of the lines are greater than 1, being 1.35,
1.30 and 1.23 for 1, 2 and 3 complexes respectively. These values are
less than the theoretically expected value of Z
Such lower values could arise from coupled anion release (from the
ligand) or from changes in ligand or DNA hydration upon binding.
By increasing the Naþ concentrations, the relative binding affinities
j
¼ 2 ꢂ 0.88 ¼ 1.76.
Fig. 7. Photoactivated cleavage of pBR322 DNA in the presence of different concentrations of 1, 2 and 3 complexes after irradiation at 365 nm for 30 min. Lanes d, g and l are
complexes in presence of histidine (2 mM).

C. Shobha Devi et al. / European Journal of Medicinal Chemistry 64 (2013) 410e421
417
A549, Du145, HeLa and MCF-7 to the synthesized complexes at
increasing concentrations for 48 h. 1% DMSO concentration was
maintained for both, cells treated with the complexes and un-
treated cells (which were taken as negative control). Cisplatin was
used as the positive control. All three complexes exhibited in-vitro
cytotoxicity against the selected cell lines. These complexes also
exhibited dose-dependent growth inhibitory effect against the
tested cell lines. It clearly demonstrates that upon increasing the
concentration of complexes an obvious decrease was observed in
cell viability (Fig. 8). Among all the complexes, complex 1 exhibited
promising growth inhibitory effect against HeLa cell line. Further-
more, complex
1 displayed slightly higher cytotoxicity than
cisplatin to HeLa cells. These results are in good agreement with the
previously reported ruthenium polypyridyl complexes [15,16,47]
(Table 3).
3.9. Cell cycle arrest by flow cytometry
The cell-membrane is the structure that protects living cells
from the surrounding environment, only allowing the movement of
compounds generally with small molecular size across this barrier
into the cell. As the complexes are stable in aqueous solution and
luminescence intensity increase upon binding with DNA, the
cellular uptake properties of ruthenium complexes can be studied
by using flow cytometry and confocal microscopy [48]. Cells not
treated with complexes exhibited neglectable luminescence. In-
Fig. 8. In-vitro cytotoxicity of complexes 1, 2 and 3 on HeLa tumor cells. Cytotoxicity
was measured by MTT reduction assay after 48 h. Untreated cells are used as the
control.
Table 3
IC50
(mM) values of the ruthenium(II) complexes and cisplatin against selected cell
lines.
cubation with 50
mM complexes for 4 h caused changes in the
Complex
A549
Du145
HeLa
MCF-7
fluorescence profiles in the following order 1 > 2 > 3 (Figs. 9 and
10). Median red fluorescence values of 1e3 and untreated cells
(control), are 106.33, 44.11, 12.28 and 2.1 respectively.
The effect of all three complexes on the cell cycle of HeLa cells
was studied by flow cytometry in propidium iodide(PI) stained cells
after treatment with complexes for 12 h. The representative DNA
distribution Histograms of control and HeLa cells in the presence of
all three ruthenium complexes are shown in Fig. 11. On treating
1
2
3
15 ꢃ 1.5
47 ꢃ 1.1
68 ꢃ 1.9
12 ꢃ 1.0
23 ꢃ 0.9
26 ꢃ 1.9
52 ꢃ 0.5
6 ꢃ 1.5
18 ꢃ 1.1
31 ꢃ 0.9
45 ꢃ 0.5
19 ꢃ 1.2
37 ꢃ 1.1
41 ꢃ 1.2
62 ꢃ 1.9
21 ꢃ 1.2
Cisplatin
IC50 values are given in mM, cisplatin is included for comparison. Data are presented
as mean values ꢃ standard deviations and cell viability assessed after 48 h of
incubation.
complexes (50 mM) the percentage of HeLa cells increased in the
3.8. In vitro cytotoxicity studies
G0/G1 phase (control 68.8%, (1) 79.3%, (2) 76.9% and (3) 72.4%)
reflecting a significant inhibition of cell cycle progression. These
results indicated that antiproliferative mechanism induced by all
three complexes on HeLa cells is in the G0/G1 phase.
Cytotoxicity tests were performed using the MTT assay [46],
following exposure to the selected human tumor cell lines such as
Control
Control
Control
Complex 1
Complex 2
Complex 3
10e0
10e1
10e2
10e3
10e4
10e0
10e1
10e2
10e3
10e4
10e0
10e1
10e2
10e3
10e4
Red Fluorescence (RED-HLog)
Red Fluorescence (RED-HLog)
Red Fluorescence (RED-HLog)
Fig. 9. Red fluorescence analyzed by flow cytometry. Representative histograms indicates cultures treated with untreated cells (control), 50
mM of complexes 1, 2 and 3 (for 4 h).

418
C. Shobha Devi et al. / European Journal of Medicinal Chemistry 64 (2013) 410e421
A
Control & Complexes 3, 2, 1
10e0
10e1
Red Fluorescence (RED-HLog)
10e2
10e3
10e4
B
6 h
15min
3 h
Complex 1
50 µM
Fig. 10. Flow cytometric results of HeLa cells incubated with blank medium and complexes 1 (20 mM), 2 (20 mM), and 3 (20 m
M) at 37 ꢁC for 2 h. 10,000 events were collected in the
FL2 channel (excitation, 488 nm; emission, 585e620 nm) (A). Confocal images of HeLa cells treated with 1. Cells were incubated with 50 mM complex 1 for different time intervals at
37 ꢁC and observed by confocal microscopy (B).
Fig. 11. Effects of control, complexes 1, 2 and 3 on HeLa tumor cells after 12 h, distribution among cell cycle phases.

C. Shobha Devi et al. / European Journal of Medicinal Chemistry 64 (2013) 410e421
419
Fig. 12. Nexin V staining shows induction of apoptosis of HeLa cells treated with complexes 1, 2 and 3 for 4 h. The percents of apoptotic cells were detected by analyzing Nexin V and
PI binding with the help of flow cytometry and EXPO32 MultiComp software. Viable cells did not bind to Nexin V or PI (lower left quadrant D3), early apoptotic cells bound to Nexin
V but excluded PI (lower right quadrant D4), and necrotic or late apoptotic cells were both Nexin V- and PI-positive (upper right quadrant D2), the upper left quadrant D1 contains
the nuclear debris or dead cells.
3.10. Apoptotic inducing activity
Representative images of the cells treated with vehicle (1% DMSO)
and all three complexes (50 M), bright field and DAPI stained
m
The apoptotic studies were carried out using flow cytometer and
the Nexin guava reagent. In the early stages of apoptosis, the cell
membrane can expose phosphatidylserine (PS) which is annexin
V-positive [49]. The measurement of annexin V binding to the cell
surface as an indicator for apoptosis has to be performed along with
a dye exclusion test, to establish the integrity of the cell membrane.
The percentage of apoptotic cells and changes in DNA content
distribution in HeLa cells treated by 1e3 were detected by
analyzing with Nexin 7-AAD, and Annexin V binding with the help
of flow cytometry. Viable cells did not bind to annexin V or PI (lower
left quadrant D3), early apoptotic cells bound to annexin V but
excluded PI (lower right quadrant D4) and necrotic or late apoptotic
cells were both annexin V and PI positive (upper right quadrant
D2). The upper left quadrant D1 contains dead cells. The percentage
of apoptotic cells in HeLa cells treated by complexes 1, 2 and 3 of
images are shown in Fig. 13. Control cells exhibit homogeneous
nuclear staining, and apoptotic cells display typical apoptotic
changes (e.g., staining bright, fragmented nuclei, cell shrinkage and
blebbing).
3.11. Molecular docking studies
Molecular docking studies were carried out to determine the
binding mode between Ru(II) complexes and the B-DNA. This was
used as templates for the docking studies. The aim was to study the
nucleobase sequence selectivity by computer simulation. The
relative orientation, interaction, Hydrogen bond and Vander Waals
in the docked complex determined from the results of the docking
study were analyzed using the graphical program Silver Visualizer
1.1. The views in Fig. 14, shows the different modes in which the
complex 1 intercalated into the B-DNA sequences. Docking of the
ruthenium-complexes onto B-DNA decamer (C-G-A-T-T-A-A-T-C-
G), RueN (guanine) interactions were also detected.
50 mM for 4 h increased in late apoptotic quadrant (upper right
quadrant D2). The results are depicted in Fig. 12. These results
indicated that the complexes can induce apoptosis of HeLa cell line.
The Confocal images were acquired after treating HeLa cells
Although the experimental results have shown that complexes
1, 2 and 3 can bind to DNA through intercalative mode and that the
with 50
mM of complexes 1e3 and counterstaining with DAPI.

420
C. Shobha Devi et al. / European Journal of Medicinal Chemistry 64 (2013) 410e421
Fig. 13. Confocal images of HeLa cells stained with three complexes. Control (untreated cells) with DAPI stained and bright filed images (A). Cells were incubated with 50 mM
complexes 1, 2 and 3 and observed by confocal microscopy (excitation, 488 nm; emission, 600e630 nm)(B), cells were incubated with 50 mM complexes 1-3 and stained with DAPI
(C). Blebbing of the nuclei is clearly observed.
DNA-binding affinity of complex 1 is stronger than that of complex
2 and 3, our calculational results further showed that the inter-
calative ligand of complex 1 interacts into DNA-base pairs through
major groove, whereas the intercalative ligand of complex 2 and 3
does so through the minor groove. Moreover, many binding details
in docking models are obtained. From Table 4 we can see the
following: (a) For complex 1, there is a strong hydrogen bond be-
tween H atom on the intercalative ligand and one O atom on a
Guanine of DNA base-pairs (N40-DG2:O6). The length of this
two O atoms of DNA-base-pairs, moreover, the lengths of these two
hydrogen bonds (H/O) (N40-DA5:O3 and N40-DT8:O4) are
ꢀ
ꢀ
2.642 A and 2.067 A, respectively. (c) For complex 3 there is one
strong hydrogen bond between one H atoms on the ligand and one
O atom on the phosphate of DNA-base-pairs (H73:DT8:O4), the
ꢀ
length of this hydrogen bond (H/O) is 3.047 A.
From the above analysis, we can clearly see that complexes 2
and 3 prefer AT e binding sites than GC e or mixed sequence
binding sites. Some [Ru(Hdpa)2(PIP/4HEPIP)](ClO4)2 (Hdpa ¼ 2,20-
dipyridylamine) complexes showed preferential binding to AT than
GC or mixed sequence but the origin of this selectivity was not
ꢀ
hydrogen bond (H/O) is 2.528 A. (b) For complex 2, there are two
strong hydrogen bonds between two H atoms on (PIP) ligand and
Fig. 14. Interaction between complex 1 and B-DNA. Complex is shown in Green color (A). The minimized model of the complex between complex 1 and B-DNA (B). (For inter-
pretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

C. Shobha Devi et al. / European Journal of Medicinal Chemistry 64 (2013) 410e421
421
Table 4
Acknowledgments
Hydrogen bonding interactions, Bond lengths and Dock scores of B-DNA with
ruthenium(II) complexes.
Council of Scientific and Industrial Research (CSIR, New Delhi,
India) is gratefully acknowledged for the financial support in the
form of Junior Research Fellow to C. Shobha Devi. Authors are also
thankful to Department of Biotechnology (DBT) ISLARE, Osmania
University.
Complex H eBond
donore
Bond
length (A) interactions
(Complex e DNA)
Vander Waals
Bond
length (A) score
Docking
ꢀ
ꢀ
acceptor
1
N40-DG2:O6 2.528
N40-DG22:C4
C6- DT4:C7
2.540
2.799
2.577
2.643
2.557
2.827
2.619
1.161
2.390
2.642
2.766
2.762
2.554
88.66
C25-DT4:O4
C51-DC9:OP1
N1-DA3:OP1
C1-DA3:OP1
C12- DA3:OP1
H75-DA7:H62
C11-DG2:C6
C2-DC1:OP1
C41-DT4:H73
C42- DT4:OP1
C13-DG2:OP1
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4. Conclusions
Three new ruthenium(II) polypyridyl complexes have been
synthesized and characterized. Binding of these complexes to CT-
DNA has been investigated in detail by electronic absorption
titration, steady state emission, viscosity and salt dependent
studies. The results suggest that all complexes bind to DNA in an
intercalative mode. When irradiated at 365 nm they were efficient
photocleavers. Antiproliferative effects on tumor cells exerted by
above complexes are consistent with their intracellular uptake
properties, which are significantly enhanced from 3e1. Further all
three complexes show effective cell growth inhibition by triggering
G0/G1 phase arrest and inducing apoptosis. Our data here suggests
that the complexes could be good antitumor drugs and a practical
probe of DNA sequence. The complexes are projected to be po-
tential anticancer molecules that may interfere with DNA replica-
tion where the intercalation is the preferred binding mode.
Additionally, the experimental results confirm the recognition of
DNA sites, which can help to promote novel rational design of drugs
for chemotherapy.
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