DNA-binding and cleavage, cytotoxicity properties of Ru(II) complexes with 2-(4′-chloro-phenyl) imidazo[4,5-f][1,10]phenanthroline, ligand and their "light switch" on-off effect

Article abstract of DOI:10.1007/s00044-013-0617-1

Three new complexes of the type [Ru(phen)₂PIP-Cl](1) [Ru(bpy)₂PIP-Cl](2) and [Ru(dmp)₂PIP-Cl](3) (phen = 1,10-phenanthroline; bpy = 2,2′-bipyridine; dmb = 4,4-dimethyl-2,2′- bipyridine), PIP-Cl = 2-(4′-chloro-phenyl) imida

Full text of DOI:10.1007/s00044-013-0617-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0617-1
ORIGINAL RESEARCH
DNA-binding and cleavage, cytotoxicity properties
of Ru(II) complexes with 2-(40-chloro-phenyl)
imidazo[4,5-f][1,10]phenanthroline, ligand
and their ‘‘light switch’’ on–off effect
•
Nazar Mohammed Gabra Bakheit Mustafa
•
•
Yata Praveen Kumar C. Shobha Devi M. Shilpa
•
•
•
Kotha Laxma Reddy S. Satyanarayana
Received: 28 December 2012 / Accepted: 4 May 2013
Ó Springer Science+Business Media New York 2013
Abstract Three new complexes of the type [Ru(phen)2-
PIP-Cl](1) [Ru(bpy)2PIP-Cl](2) and [Ru(dmp)2PIP-Cl]
(3) (phen = 1,10-phenanthroline; bpy = 2,20-bipyridine;
dmb = 4,4-dimethyl-2,20-bipyridine), PIP-Cl = 2-(40-chloro-
phenyl) imidazo[4,5-f][1,10]phenanthroline) were synthesized
and characterized by using UV–VIS, IR and 1H-NMR,
13C-NMR spectral methods. Absorption spectroscopy, emis-
sion spectroscopy, viscosity measurements and DNA melting
techniques were used to investigate the binding of these
Ru(II) complexes with calf thymus DNA, and photocleavage
studies were used to investigate the binding of these
complexes with plasmid DNA. The spectroscopic studies
together with viscosity measurements and DNA melting
studies supported fact that Ru(II) complexes bind to
CT-DNA(calf thymus DNA) by an intercalation mode via
PIP-Cl into the base pairs of DNA. Upon irradiation, these
novel Ru(II) complexes cleave the plasmid pBR 322 DNA
from the supercoiled form I to the open circular form II.
Introduction
Considerable attention has been given to the design of small
molecules that bind to DNA with site selectivity so as to
develop new therapeutics and chemical probes for nucleic
acid sites and structure, as well as novel diagnostic agents
targeted to the double helical DNA (Jenkins et al., 1992; He
et al., 1998). Small molecule serves as analogs in studies of
protein-nucleic acid recognition, provides site-specific
reagents for molecular biology and yields rationales for new
drug design. The effect of size, shape, hydrophobicity and
charge on the binding of the complex to DNA has been ana-
lyzed by changing the type of heteroaromatic ligand or metal
center. In particular, ruthenium (II) complexes with polypyr-
idine ligands, due to a combination of easily constructed rigid
chiral structures spanning all three spatial dimensions and a
rich photophysical repertoire, have attracted considerable
attention. Three types of binding modes, that is, intercalation
mode, groove-binding mode and electrostatic-binding mode,
have been proposed and further developed to explain the
interaction mechanism between the complexes and DNA
(Lincoln and Norde’n, 1998; Choi et al., 1997; Xiong and Ji,
1999). Although a considerable number of complexes have
been reported (Hiort et al., 1993; Dupureur and Barton, 1997;
Lincolnetal.,1996),itisnotablethatmostofthemarefocused
on the prototypes [Ru(bpy)2L]2? or [Ru(phen)2L]2? with
symmetric intercalative aromatic ligand (L), such as dpq,
dppz, pip and their substitution derivates. Recently, some
studies on Ru(II) polypyridyl-type complexes with asym-
metric aromatic ligand including similar complexes with two
Ru(II) atoms have been reported (Zou et al., 1999; Deng et al.,
2003; Jiang, 2004; Jiang et al., 2003). However, most of these
complexes contain only planar aromatic ligands and investi-
gation of such complexes with ligands containing substituents
as DNA-binding reagent is relatively few. In fact, most of
Keywords Polypyridyl complex ꢀ Photocleavage ꢀ
In vitro cytotoxicity ꢀ Docking
N. M. Gabra ꢀ B. Mustafa ꢀ Y. P. Kumar ꢀ C. Shobha Devi ꢀ
M. Shilpa ꢀ K. L. Reddy ꢀ S. Satyanarayana (&)
Department of Chemistry, Osmania University,
Hyderabad 500007, Andhra Pradesh, India
e-mail: ssnsirasani@gmail.com
N. M. Gabra
Department of Chemistry, Red Sea University, P.O. Box 24,
Port Sudan, Sudan
e-mail: nazargabra@yahoo.com
123

Med Chem Res
these complexes exhibit interesting propertiesuponbinding to
DNA (Barton and Raphael, 1984; Pyle et al., 1989; Morgan
et al., 1991; Xiong et al., 1999).
ammonia gave a yellow precipitate which was collected
washed with water, purified and dried, yield (72 %),
Analytical data Anal. Calcd C19H11N4 for (%) C: 65.81;
In this paper, we report the synthesis of three complexes
of Ru(II) containing a ligand with phenolic and chloro
substituents at the 2- and 4-position of the phenyl group,
and their binding properties with calf thymus DNA using
absorption, fluorescence spectroscopy and viscosity mea-
surements. Their photocleavage behavior toward pBR-233
and the mechanism for cleavage were also investigated.
Molecular modeling was carried out to further explore the
binding selectivity of duplex oligonucleotides. The result
should be of value in understanding the binding mode of
the complex to DNA, as well as laying the foundation for
the rational design of a DNA molecular light switch and
DNA-cleaving agents (Kratochwil et al., 1999).
H: 3.20; N: 16.16; found C: 65.73; H: 3.12; N: 15.98; LC-MS
1
in DMSO M/Z: 347.5 H-NMR (DMSO-d6, 400 MHZ
d
ppm): 9.10 (d, 2H), 8.80 (d, 2H), 8.25 (d, 2H), 7.70 (m, 2H),
7.53 (d, 2H), 7.25(s, 1H),7.10 (d,2H), 6.90 (s, 1H), 6.65 (d,
2H). 13C-NMR (200 MHz, DMSO-d6, 298 K, d ppm):
156.36 (C-k), 154.33 (C-i), 152.99 (C-a), 146.72 (C–c),
132.81 (C-e), 131.25 (C-m), 129.49 (C-o), 128.29 (C-d, C-n),
125.06 (C-g), 122.84 (C-b, C-f), 121.85 (C-j), 118.23 (C-l).
Synthesis of [Ru(phen) PIP-Cl](ClO4)2ꢀ2H2O
2
A
mixture of cis-[Ru(phen)2(Cl)2]ꢀ2H2O (0.10 g,
0.16 mmol) and Cl-PIP (0.079 g, 0.16 mmol) in ethanol
(30 ml) was refluxed under nitrogen for 8 h. After cooling,
the clear solution was filtered. The filtrate was treated with
a saturated solution of NaClO4, and a red precipitate was
obtained. The solid was collected washed with small
amounts of water, ethanol and diethyl ether and then dried
under vacuum, yield (69 %),
Experimental
Materials
Analytical data for RuC43H27N9Cl3O11 Calcd. (%) C:
63.83; H: 3.42; N: 13.75. Found: C: 63.90; H: 3.37; N:
13.86. LC-MS in DMSO M/Z: 1071.5. IR: 1484 (C=C),
1627 (C=N), 722 (Ru–N(L)), 627 cm-1 (Ru–N(PIP-Cl).
1H-NMR (DMSO-d6, 400 MHZ d ppm): 9.13 (d, 2H), 8.79
(d, 4H), 8.40 (s, 3H), 8.16 (d, 4H), 8.09 (d, 2H), 8.04 (d,
2H),7.70 (t,2H), 7.45 (d, 2H), 7.15 (d, 2H). 13C-NMR
(200 MHz, DMSO-d6, 298 K, d ppm): 156.51 (C-k),
153.28 (C-i), 151.21 (C-e) 150.86 (C-1, C-a), 147.85 (C-5),
146.17 (C-3,C–c), 137.27 (C-m, C-4), 131.61 (C-o), 131.00
(C-d), 128.35 (C-6, C-n), 127.72 (C-g), 126.65 (C-f),
123.64 (C-2, C-b), 119.58 (C-l, C-j).
RuCl3, 1,10-phenanthroline monohydrate and 2,20-bipyri-
dine were purchased from Merck (India). Calf thymus(CT)
DNA, 3,4-diaminobenzophenone, 4-chloro-2-(1H-imi-
dazo[4,5-f][1,10]phenanthroline-2-yl)phenol and 4,40-
dimethyl-2,20-bipyridine were obtained from Sigma. The
supercoiled pBR-322 DNA (Fermentas Life Science, India)
was used as received. All other common chemicals and
solvents were procured from locally available sources. All
the solvents were purified before use as per standard proce-
dures (Perrin et al., 1980). Deionized, double-distilled water
was used for preparing various buffers. Solutions of DNA in
Tris–HCl buffer (pH = 7.2), 50 mM NaCl gave a ratio of
UV absorbance at 260 and 280 nm of 1.8–1.9, indicating that
the DNA was sufficiently free of protein (Marmur, 1961).
The concentration of CT-DNA was determined spectro-
photometrically using the molar absorption coefficient
6,600 M-1 cm-1 (260 nm) (Rreichmann et al., 1954).
Synthesis of [Ru(bpy)2 PIP-Cl](ClO4)2ꢀ2H2O
This complex was synthesized using the same procedure
described for complex (1) yield (58 %).
Analytical data for RuC39H27N8Cl3O11: Calcd. (%) C:
63.90; H: 3.37; N: 13.86. Found: C: 63.96; H: 3.35; N: 13.90.
LCMS in DMSO M/Z: 1023.5. IR: 1466 (C=C), 1603 (C=N),
768 (Ru–N(L)), 627 cm-1 (Ru–N(PIP-Cl)). 1H-NMR
(DMSO-d6, 400 MHZ d ppm): 9.10(d, 2H), 8.90 (m, 2H), 8.25
(t, 3H), 8.66 (t, 3H), 8.12 (m, 4H), 8.08 (d, 2H), 7.93 (t,2H),
7.65 (t,2H), 7.42 (m, 2H), 7.13 (d, 1H). 13C-NMR (200 MHz,
DMSO-d6, 298 K): 157.38 (C-5), 157.16 (C-k), 156.44 (C-i),
151.96 (C-a, C-1), 145.81 (C-e), 138.45 (C-3, C–c), 131.75
(C-m), 128.16 (C-n, C-o C-d), 126.53 (C-g,C-4), 124.93 (C-b,
C-f), 123.69 (C-2), 119.64 (C-j), 115.61 (C-l).
Synthesis of 2-(40-chloro-phenyl) imidazo[4,5-
f][1,10]phenanthroline (PIP-Cl)
A
solution of 1,10-phenanthroline-5,6-dione (0.53 g,
2.50 mmol), 5-chloro-2-hydroxybenzaldehyde (0.547 g,
3.50 mmol) and ammonium acetate in 10 ml glacial acetic
acid were refluxed together for 2 h as per Steck and Day
(Tan et al., 2005) and then cooled to room temperature and
diluted with water 25 cm3. Dropwise addition of liquor
123

Med Chem Res
Synthesis of [Ru(dmb)2 PIP-Cl](ClO4)2ꢀ2H2O
intensity at 561 nm of complexes in the absence and
presence of CT-DNA.
This complex was synthesized using the same procedure
described for complex (1) yield: 60 %.
Viscosity experiments were carried out using an Ost-
wald viscometer maintained at a constant temperature
30.0 0.1 °C in a thermostatic water bath. Calf thymus
DNA samples, approximately 200 base pairs in average
length, were prepared by sonicating in order to minimize
complexities arising from DNA flexibility (Satyanarayana
et al., 1993). Data were presented as (g/g0)1/3 versus the
concentration of Ru(II) complexes, where g is the viscosity
of DNA in the presence of complexes and g0 is the vis-
cosity of DNA alone. Viscosity values were calculated
from the observed flow time of DNA-containing solutions
(t [ 100 s) corrected for the flow time of buffer alone (t0)
(Moucheron et al., 1997; Ashwini Kumar et al., 2010).
The DNA melting experiments were carried out by
controlling the temperature of the sample cell with a Shi-
madzu circulating bath while monitoring the absorbance at
260 nm. For the gel electrophoresis experiments, super-
coiled pBR-322 DNA (100 lM) was treated with the Ru(II)
complexes in 50 mM Tris–HCl, 18 mM NaCl buffer pH
7.8, and the solutions were then irradiated at room tem-
perature with a UV lamp (365 nm, 10 W). The samples
were analyzed by electrophoresis for 1 h at 100 V on a
0.8 % agarose gel in Tris–acetic acid–EDTA buffer, pH
7.2. The gel was stained with l lg/mL ethidium bromide
and photographed under UV light.
Analytical data for RuC43H35N8Cl3O11: Calcd. (%) C:
63.47; H: 3.99; N: 13.30. Found: C: 63.45; H: 4.15; N:
13.25. LCMS in DMSO M/Z: 1083. IR: 1484 (C=C), 1619
(C=N), 737 (Ru–N(L)), 626 cm-1 (Ru–N(pip-Cl). 1H-
NMR (DMSO-d6, 400 MHZ d ppm): 9.12 (d, 2H), 8.93 (m,
4H), 8.83 (s, 1H), 8.13 (d, 1H), 8.07 (s, 1H), 7.87 (d, 2H),
7.67 (d, 2H), 7.32 (d,2H), 6.98 (m,4H), 2.55 (d, 6H), 2.45
(d,6H). 13C-NMR (200 MHz, DMSO-d6, 298 K): 156.91
(C-5), 156.50 (C-k), 154.53 (C-i), 151.13 (C-a, C-1),
149.59 (C-3), 148.63 (C-e), 145.86 (C–c), 131.20 (C-m),
128.82 (C-o), 126.51 (C-n, C-d), 125.65 (C-4), 124.20
(C-2), 123.77 (C-f), 123.53 (C-g), 123.43 (C-b), 119.47
(C-l), 114.77 (C-j), 21.05 (C-6).
Physical measurements
Microanalyses (C, H, N) were carried out on a Perkin-Elmer
240 elemental analyzer. NMR spectra were recorded on
Bruker 400 MHz spectrometer with DMSO-d6 as a solvent at
room temperature and tetramethylsilane (TMS) as the internal
standard. Mass spectra were recorded with LC-MS 2010 A
Shimadzu, Japan. IR spectra were recorded, in KBr phase on
Perkin-Elmer FTIR-1605 spectrophotometer. UV–Visible
spectra were recorded on Elico Bio-spectrophotometer model
BL198. Emission spectra were carried out with Elico Bio-
spectrofluorimeter mode SL174 luminescence spectrometer
at room temperature. Spectroscopic titrations were carried out
at room temperature to determine the binding affinity between
DNA and the complex. Initially, 3 mL solutions of the blank
buffer and the ruthenium complexes sample (20 lM) were
placed in the reference and sample cuvette (1 cm path length),
respectively, and then first spectrum was recorded in the range
of 200–600 nm. During the titration, aliquot (1–1 lL) of
buffered DNA solution (concentration of *5–10 mM in base
pairs)wasaddedtoeachcuvettetoeliminatetheabsorbanceof
DNA itself, and the solutions were mixed for *5 min; the
absorptionspectrawererecorded. Thetitrationprocesseswere
repeated until there was no change in the spectra for four
titrations at least; indicating binding saturation had been
achieved. The changes in the metal complex concentration
due to dilution at the end of each titration were negligible. The
above procedure was repeated minimum of three times (Na-
gababu et al., 2011).
In vitro cytotoxicity assay
Cytotoxicity of complexes 1, 2 and 3 was evaluated by
using standard MTT (3-(4,5-dimethylthiazole)-2,5-diphe-
nyltetrazolium bromide) assay (Pyle, 1990). Cells were
placed in 96-well microassay culture plates (8 9 103 cells
per well) and grown overnight at 37 °C in a 5 % CO2
incubator. The cytotoxicity was evaluated against a panel
of four different human cancer cell lines, namely HeLa
(ATCC No. CCL-2) derived from human cervical cancer
cells, A549 (ATCC No. CCL-185) derived from human
alveolar adenocarcinoma epithelial cells, MDA-MB-231
(ATCC No. HTB-26) and MCF7 (ATCC No. HTB-22)
derived from human breast adenocarcinoma cells.
Docking simulations
Molecular docking of synthetic compounds into the three-
dimensional X-ray structure of DNA (PDB code: 1Y9H)
was carried out using the GOLD (Genetic Optimization for
Ligand Docking) software package (version 3.0.1) as
implemented through the graphical user interface Silver
Descriptor 1.1
Fluorescence emission titration experiments were per-
formed at a fixed metal complex concentration (10 lM) to
which DNA (1–160 lM) was added in a volume of
(10–100 lL). Excitation wavelength was kept constant,
and emission was recorded. Fluorescence emission
enhancement was based on comparison of emission
The 3D structures of DNA molecules were built and
saved in Mol2 format with the aid of the molecular
123

Med Chem Res
modeling program Discovery Studio 3.0. These partial
charges of Mol2 files were further modified by using the
Mercury (version 2.2), so that the charges of the nonpolar
hydrogens atoms assigned to the atom to which the
hydrogen is attached. The resulting files were saved as pdb
files. GOLD DOCK 3.0.1 was employed for all docking
calculations. The Silver Descriptor interface program was
used to generate the docking input files. Ten runs were
generated by using genetic algorithm searches. Automatic
was prepared by a method similar to that described by
Steck and Day [50]. Refluxing of 1,10-phenanthroline-5,
6-dione with the appropriate, mole ratio of 2-(4-chloro-
phenyl) imidazo[4,5-f][1,10]phenanthroline and ammo-
nium acetate in glacial acetic acid for 2 h produced the
desired ligand in high yields. The complexes 1, 2 and 3
were prepared by direct reaction of ligand with appropriate
mole ratios of the precursor complexes in ethanol. The
desired Ru(II) complexes were isolated as their perchlo-
rates and were purified by column chromatography. Three
˚
settings were used, and the results differing within 1.5 A in
1
positional root-mean-square deviation (RMSD) were clus-
tered together, and the results of the most favorable free
energy of binding were selected as the resultant complex
structures.
complexes and ligand PIP-Cl were characterized by H,
13C-NMR, IR spectra mass spectroscopy and elemental
analysis. In the IR spectrum, two peaks appeared in the
region 722 and 627 cm-1 which corresponds to M–N
stretching of metal ancillary of ligand and metal PIP-Cl
ligand.
Results and discussion
Absorption spectral studies
Synthesis and characterization
The application of electronic absorption spectroscopy in
DNA-binding studies is one of the most useful techniques.
Complex binding with DNA through intercalation usually
The synthetic routes to the ligand and its Ru(II) complexes
1, 2 and 3 are summarized in Fig. 1. The ligand, PIP-Cl,
2+
HO
H
N
N
N
N
N
N
[Ru(phen)2Cl2]
Ru
N
N
Cl
5
1
2
1
4
OH
Cl
6
3
2+
OHC
O
b
c
d
HO
a
N
h
H
N
k
l
N
N
N
m
n
H
HO
f
[Ru(bpy)2Cl2]
N
i
j
e
N
N
N
Ru
N
1.NH4OAc
2.AcOH(glacial)
3.Reflux
N
O
g
o
Cl
N
PIP-Cl
N
N
Cl
5
1
1,10-phenanthroline
5,6-dione
4
2
2
3
2+
N
H
HO
N
[Ru(dmb)2Cl2]
N
N
Ru
N
N
N
N
Cl
5
1
4
2
3
3
6
Fig. 1 Synthesis of the complexes [Ru(phen)2PIP-Cl]2? (1), [Ru(bpy)2PIP-Cl]2? (2) and [Ru(dmb)2PIP-Cl]2? (3)
123

Med Chem Res
where [DNA] is the concentration of DNA in base pairs,
the apparent absorption coefficients ea, ef and eb correspond
to Aobsd/[Ru(II)], the extinction coefficients for the free
Ru(II) complex, extinction coefficients of complex in the
presence of DNA and the extinction coefficients for the
Ru(II) complex in the fully bound form, respectively. In
plots [DNA]/(ea - ef) versus [DNA], K is given by the
ratio of slope to intercept. Intrinsic binding constants Kb of
[Ru(phen)2PIP-Cl]2?(1), [Ru(bpy) PIP-Cl]2?(2) and
[Ru(dmb)2 PIP-Cl]2?(3) were obtained about 6.4 9 105,
4.2 9 105 and 2.1 9 105, respectively (Table 1). The val-
ues are comparable with those of some known DNA in-
tercalators, such as [Ru(phen)2 fyip]2?, [Ru(bpy)2 fyip]2?
(Shilpa et al., 2011;Devi et al., 2012;Yata et al., 2012).
These spectral characteristics of the large hypochromism
and clear red shift as well as the large Kb value observed
suggest that complexes most likely intercalatively bind to
DNA, involving a strong stacking interaction between the
aromatic chromophore and the base pairs of DNA. The
difference in binding strength of complexes 1 and 2 is
probably caused by the different ancillary ligands. The four
additional methyl groups in complex 3 relative to complex
2 exert some steric hindrance. Therefore, complex 1 is
probably more deeply interacted and more tightly bound to
the adjacent DNA base pairs than complex 2. Similarly, the
difference in binding strength of complexes 1 and 3 is due
to the difference in the ancillary ligands. On going from
phen to dmb, the planarity area and hydrophobicity
decrease, leading to a lower binding affinity. The DNA-
binding affinities of these complexes follow the order:
[Ru(phen)2PIP-Cl]2?(1) [ [Ru(bpy)2PIP-Cl]2?(2) [ [Ru
(dmb)2PIP-Cl]2?(3).
4.50E-03
4.00E-03
3.50E-03
3.00E-03
2.50E-03
2.00E-03
1.50E-03
1.00E-03
5.00E-04
0.00E+00
0
0.002
0.004
0.006
[DNA]
Wavelength(Nanometer)
Fig. 2 Absorption spectra of [Ru(bpy)2PIP-Cl]2?in the absence (top)
and presence (lower) of DNA in Tris–HCl buffer. The absorbance
changes upon increasing the CT-DNA concentration (10, 20, 30,
40 lL of DNA addition), [Ru] = 10 lM, [DNA] = 0–126 lM. The
arrow shows the intensity change upon increasing the DNA
P
P
concentration. Inset plots of [DNA]/(
titration of DNA with the Ru(II)
–
a
f) versus [DNA] for the
results in hypochromism and bathochromism, because of
the intercalative mode involving a stacking interaction
between an aromatic chromophore and the base pair of
DNA. The extent of the hypochromism commonly parallels
the intercalative binding strength. The absorption spectra of
complex 2 in the absence and presence of CT-DNA are
given in Fig. 2. As increasing the concentration of CT-
DNA, the MLCT transition bands of complexes 1, 2 and 3
at 453, 445 and 422 nm exhibit hypochromism of about,
18.5 14.2 and 11.5 % as well as an insignificant batho-
chromism about 7.2, 6.1 and 5.3 nm, respectively. These
results are similar to those reported earlier for various
metallointercalators (Pyle, 1990; Wolfe et al., 1987). Based
on these observations, we presume that there are some
interactions between the complexes and the base pairs of
DNA. In order to compare quantitatively the binding
strength of the three complexes, the intrinsic binding
constants Kb of the three complexes with CT-DNA were
obtained by monitoring the changes of absorbance in the
MLCT band with increasing concentration of DNA using
the following equation (Zhen et al., 1999) through a plot of
[DNA]/[ea - ef] versus [DNA].
Fluorescence spectroscopic studies
Luminescence spectroscopy is one of the most common
and at the same time most sensitive ways to analyze drug–
DNA interactions. Support for the above intercalative
binding mode also comes from the emission measurement
of the complexes. In the absence of DNA, complexes 1, 2
and 3 can emit luminescence in Tris buffer at ambient
temperature with maxima at 560 nm. Binding of the three
complexes to DNA was found to increase the fluorescence
intensity. The results of emission titration for the complex
1 in the absence and presence of CT-DNA are shown in
½DNAꢁ=ðea ꢂ efÞ ¼ ½DNAꢁ=ðeb ꢂ efÞ þ 1=Kbðeb ꢂ efÞ
Table 1 Results of DNA-
binding data of Ru(II)
complexes
Complex
Tm Hypochromicity (%) Absorption kmax (nm) Dk (nm) Kb
free bound
[Ru(phen)2PIP-Cl]2? (1) 70
14.2
18.5
11.5
416–422
447–453
438–445
6
7
7
6.4 9 105
4.2 9 105
2.1 9 105
[Ru(bpy)2PIP-Cl]2? (2)
[Ru(dmb)2PIP-Cl]2? (3)
64
68
CT-DNA Tm = 60 °C
123

Med Chem Res
Table 2 Emission data of complexes
820
720
620
520
420
320
220
120
20
Complexes
Ex peak Em peak Fluorescence
binding constant
[Ru(phen)2PIP-Cl]2? (1) 468
610
691
470
6.9 9 105
5.4 9 105
1.4 9 105
[Ru(bpy)2PIP-Cl]2? (2)
[Ru(dmb)2PIP-Cl]2? (3)
468
467
that of in the absence of DNA for complex 2 and 1.79 times
larger than that of in the absence of DNA for complex 3,
respectively. This implies that these complexes strongly
interact with DNA and can be efficiently protected by DNA
(Table 2), since the hydrophobic environment inside the
DNA helix reduces the accessibility of solvent water. In
this way, the mobility of the complexes is restricted at the
binding site, leading to a decrease in the vibrational modes
of relaxation.
685
687
689
691
693
695
697
699
Wavelength (nm)
Fig. 3 Emission spectra of complexes of [Ru(phen)2PIP-Cl]2? in
Tris–HCl buffer at 25 °C upon addition of CT-DNA, [Ru] = 20 lM,
[DNA] = 0–120 lM. The arrow shows the increase in intensity upon
increasing CT-DNA concentrations
Fig. 3. Upon addition of CT-DNA, the emission intensity
increases steadily and reaches 1.91 times larger than that in
the absence of DNA for complex 1, 1.30 times larger than
Differential luminescence quenching was also utilized in
monitoring DNA binding. A highly negatively charged
1
7
A
6
5
4
3
2
1
0
B
C
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
[Fe(CN)6]4-mM
2
37
8
7
6
5
4
3
2
1
0
A
A
6
5
4
3
2
1
0
B
B
C
C
0
0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08
0
0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08
[Fe(CN)6]4-mM
[Fe(CN)6]4-mM
Fig. 4 Emission quenching curves of complexes 1, 2 and 3. Quenching in the absence of DNA (a), quenching in the presence of DNA (b) (1:20).
Excess of DNA (1:200 lM) (c)
123

Med Chem Res
Viscosity measurements
quencher is expected to be repelled by the negatively
charged phosphate backbone, and therefore, a DNA-bound
cationic molecule should be readily quenched (Lakowicz
and Webber, 1973). Figure 4 shows the steady-state emis-
sion quenching experiments using [Fe(CN)6]4- as quencher.
In the absence of DNA, all three complexes were efficiently
quenched by [Fe(CN)6]4-, but when bound to DNA the
complexes were protected from the quencher. This may be
explained by repulsion between the highly negatively
charged [Fe(CN)6]4- and the DNA polyanion backbone
which hinders access of [Fe(CN)6]4- to the DNA-bound
complexes. The quenching studies indicate that the DNA-
binding abilities of the complexes follow the order: 1, 3 and
2. Steady-state emission quenching experiments using
[Fe(CN)6]4- as quencher were also used to observe the
binding of the complexes with CT-DNA. The Stern–Volmer
quenching constant (Ksv) can be determined by using Stern–
Volmer equation (Satyanarayana et al., 1992).
Measurement of DNA viscosity is regarded as the least
ambiguous and the most essential analysis of the binding
mode of DNA in solution, and particularly affords stronger
evidence for an intercalative DNA-binding mode. The
viscosity changes are the consequence of a change in
length of the DNA. A classical intercalator like ethidium
bromide reveals a significant increase in the viscosity of the
DNA solution, due to an increase in the separation of base
pairs at the intercalation sites and hence an enhancement in
overall DNA length (Satyanarayana et al., 1993; Lecomte
et al., 1992) In contrast, partial or nonintercalation binding
modes, such as electrostatic or covalent binding, typically
reduce or make no change in the DNA solution viscosity
(Arounaguiri and Maiya, 1996). The effects of complexes
1–3 on the viscosity of DNA are shown in Fig. 5. The
viscosity of DNA is increased with the increment of each
one of complexes 1–3, and it is similar to the behavior of
well-known DNA intercalator ([Ru(bpy)2(dppz)]2?) (Gra-
I0=I ¼ 1 þ Ksv½Qꢁ
ham et al., 1980), on the other hand, for [Ru(bpy)3]2?
,
where I0 and I are the intensities of the fluorophore in the
absence and presence of quencher, respectively, Q is the
concentration of the quencher and Ksv is a linear Stern–
Volmer quenching constant. In the quenching plot of
I0/I versus [Q], Ksv is given by the slope. Figure 4 shows
the Stern–Volmer plots for both the free complex in solu-
tion and the complex in the presence of increasing amounts
of DNA. All the complexes show linear Stern–Volmer
plots. The Ksv values for the complexes in the absence of
DNA were 482, 319 and 272 nm, for complexes (1), (2)
and (3), respectively. In the presence of DNA, the Ksv
values were 261, 190 and 163 for complexes (1), (2) and
(3), respectively. Hence, the Ksv values are smaller in the
presence of DNA. At high concentration of DNA (1:200;
Ru2?: DNA), the plots have essentially zero slope, indi-
cating that the bound species is inaccessible to quencher.
which has also been well known to bind to DNA in an
electrostatic mode. There is no effect on the relative vis-
cosity of DNA solution. The increase in viscosity for
complexes 1, 2 and 3 was less compared to the ethidium
bromide. The experimental results further support that all
the three complexes bind to DNA in an intercalation mode.
Thermal denaturation study
The melting of DNA can be used to distinguish between
those molecules which bind via intercalation and those
which bind externally. The melting temperature Tm, at
which 50 % of the DNA has become single-stranded, can
be determined from the thermal denaturation curves of
DNA. In the absence of any added complex, the melting
transition of CT-DNA was sharp. Intercalation of organic
dyes or metallointercalators generally results in a consid-
erable stabilization of the DNA duplex with a corre-
sponding large increase in melting temperature (Tm). In the
presence of intercalators, the Tm rises sharply until all the
intercalating sites are saturated, after which the stabiliza-
tion was due to electrostatic binding, and Tm increases less
steeply (Haq et al., 1995). Thermal denaturation experi-
ments carried out on CT-DNA in the absence of any added
complex revealed that the Tm for the duplex was 60.5 °C
under our experimental conditions.
12
a
10
b
8
c
6
4
2
d
0
0.25
0.45
0.65
0.85
1.05
1.25
1.45
The observed melting temperature in the presence of
complexes 1, 2 and 3 was 72.4 0.3, 68.2 0.3,
64.8 0.3, respectively. The experimental results indicate
that the complex (1) exhibits larger DNA-binding affinity
than complex 3.
[Complex]/[DNA]
Fig. 5 Effect of increasing amount of complexes on the relative
viscosities of CT-DNA at 25 0.1 oC EB (a), [Ru(phen)2PIP-Cl]2?
(b), [Ru(bpy)2PIP-Cl]2? (c) and [Ru(dmb)2PIP-Cl]2? (d)
123

Med Chem Res
Fig. 6 Salt dependence of the
equilibrium binding constants
for DNA binding of complexes
[Ru(phen)2PIP-Cl]2? (1),
1.4
1.2
1
[Ru(bpy)2PIP-Cl]2? (2) and
[Ru(dmb)2PIP-Cl]2? (3). The
lines indicate the slope of the
linear square fit to the data as
a -2.1, b –1.86, c -1.37
0.8
0.6
0.4
0.2
1
2
3
0
0.00E+00
1.00E-01
2.00E-01
3.00E-01
4.00E-01
Log(Na+)
estimate of SK = (d log[Kb]/d log[Na?]) = Zw, where Z is
the charge of the metal complex and w is 0.88 for
DNA[37,39]. Figure 6 shows the decrease of Kb of 1, 2 and
3 as the concentration of Na? is increased. As expected, the
plot becomes nonlinear at ionic strengths greater than
0.1 M (Record et al., 1978; Mudasir et al., 2006). The
slopes of the lines in Fig. 6 are being -2.1, -1.86 and
Salt titration
Reverse salt titrations of 1, 2 and 3 bound to DNA were
performed, and the results are shown in Fig. 6. The binding
constant at each titration point was then calculated, and a
plot of log[Kb] versus log[Na?] was constructed. From
polyelectrolyte theory, the slope of this graph provides an
Fig. 7 a,
b
[Ru(phen)2PIP-Cl]2? in Tris buffer (1), complex ? DNA (2), complex ? DNA ? Co2? (3) and com-
plex ? DNA ? Co2? ? EDTA (4)
123

Med Chem Res
-1.37 for 1, 2 and 3 complexes, respectively. The values of
complexes 1 and 2 are more than the theoretically expected
values of Zw (2 9 0.88 = 1.76) but complex 3 shows less
value. Such lower values could arise from coupled anion
release or from change in complex or DNA hydration upon
binding (4). The knowledge of Zw allows for a quantitative
estimation of the nonelectrostatic contribution to the DNA-
binding constant for these complexes.
circular plasmid DNA was subjected to electrophoresis,
relatively fast migration will be observed for the super-
coiled form (Form I). If scission occurs on one strand
(nicking), the supercoils will relax to generate a slower-
moving open circular form (Form II) (Marmur, 1961). If
both strands are cleaved, a linear form (Form III) will be
generated that migrates between Forms I and II. Figure 8
shows the gel electrophoretic separations of plasmid
pBR322 DNA after incubation with Ru(II) complexes and
irradiation at 365 nm. Fig. 8 reveals the conversion of
varying concentrations of [Ru(phen)2PIP-Cl]2?, [Ru(b-
py)2PIP-Cl]2?and [Ru(dmb)2PIP-Cl]2?. It can be seen that
with increasing the concentration of [Ru(phen)2PIP-Cl]2?
and [Ru(bpy)2PIP-Cl]2?, Form II increases gradually,
while Form I diminishes gradually. With increasing irra-
diation time, Form I of pBR322 DNA diminishes gradu-
ally, whereas the amount of Form II increases. This is the
result of single-stranded cleavage of pBR 322 DNA. It can
also be seen in Fig. 6c that neither irradiation of DNA at
Light switch on–off effect
Ruthenium(II) complexes bind to DNA through the inter-
calation of the PIP-Cl ligand between the bases of the
duplex, resulting in an increase in the luminescence
intensity, thus making it a ‘‘DNA light switch’’(Liu et al.,
2004). The [Ru(bpy)2(Cl-PIP)]2? DNA light switch can be
turned off in the presence of Co2?, Ni2? and Zn2?, and the
emission can be fully restored by the addition of EDTA.
The quenched change in the luminescence intensity of
DNA-bound Ru(II) complexes due to the interaction of
Co2? with DNA is shown in Fig 7a, b. Cycling of the DNA
light switch OFF and ON can be accomplished through the
successive introduction of Co2? and EDTA, respectively,
to solutions of DNA bound. The luminescence intensities
of DNA-bound Ru(II) in the presence of Co2? and EDTA
reveal the modulation of Co2? and EDTA to luminescence
intensities of DNA-bound Ru(II).
80
Complex 1
Complex 2
70
Complex 3
60
50
40
30
20
10
0
Photoactivated cleavage of pBR 322 DNA
There has been considerable interest in DNA endonucleo-
lytic cleavage reactions that are activated by metal ions
(Liu et al., 2005; Barton and Raphael, 1984). The delivery
of high concentrations of metal ion to the helix, in locally
generating oxygen or hydroxide radicals, yields an efficient
DNA cleavage reaction. DNA cleavage was monitored by
relation of supercoiled circular pBR 322 (Form I) into
nicked circular (Form II) and linear (Form III). When
A549
MCF-7
HELA
MDA-MB-231
Fig. 9 Percentage cell viability of complexes [Ru(phen)2PIP-Cl]2?
(1), [Ru(bpy)2PIP-Cl]2? (2) and [Ru(dmb)2PIP-Cl]2? (3), with
different cell lines. Complex concentrations were 25 lM, and cells
were incubated for 24 h
Fig. 8 Photo-activated
cleavage of pBR 322 DNA in
the presence of [Ru(phen)2PIP-
Cl]2? (1), [Ru(bpy)2PIP-Cl]2?
(2) and [Ru(dmb)2PIP-Cl]2? (3)
complexes, after irradiation at
365 nm. Lane 0 controls
plasmid DNA (untreated pBR
322), lanes 1–3, addition of
complexes 20, 40 and 60 M-1
123

Med Chem Res
Docking studies
Table 3 Percentage cell viability of different cell lines with ruthe-
nium complexes
For molecular docking of the complex with DNA sequence
d(CCATCGCTACC) after satisfactory spectroscopic mea-
surement of DNA-binding study of the complex, molecular
docking study was performed to understand the preferred
orientation of sterically acceptable complex using Cl-PIP
ligand with the DNA sequence. According to this docking
experiment, complex reasonably binds with DNA sequence
d(CCATCGCTACC). The docking result (Table 4) sug-
gested the best possible conformation of the complexes
interaction mainly through phenyl ring inside the DNA
major groove. It has been observed that the complex is
stabilized by electrostatic hydrogen bonding with DNA
bases, particularly involving O atoms on the phosphate of
DNA base pairs and N3 of guanine, in addition to van der
Waals and stacking bond interactions between electron
deficient chloro substituents phenyl ring system and pur-
ine–pyrimidine bases coordinated to Ru(II) ion, partici-
pates in hydrogen bonding, whereas the side chains
attached to Ru(II) complex enabled the molecules to stay in
the groove with the help of van der Waals forces. The
hydrogen bonding interactions involving the energy-mini-
mized docked poses of d(CCATCGCTACC) with com-
plexes are shown in Table 4. Thus, molecular docking
study together with spectroscopic studies indicated that
complex 1 interacts with the DNA through both covalent
and noncovalent interactions which perhaps owe to its
stronger bonding with DNA.
Complex
[Ru(phen)2PIP-Cl]2? (1) 25.03 14.23
[Ru(bpy)2PIP-Cl]2? (2)
22.37 54.3
[Ru(dmb)2PIP-Cl]2? (3) 28.29 71.78
A549 MCF-7 HELA MDA-MB-231
21.75 23.88
31.74 30.55
40.21 71.23
365 nm without Ru(II) nor incubation with Ru(II) without
light yields significant strand scission. It was likely that the
reduction of Ru(II) is the important step leading to DNA
cleavage.
In vitro cytotoxic assay
Cytotoxicity was tested for Ru(II) complexes by MTT
assay against several selected cell lines depicted in Fig. 9.
The following human cancer cell lines were used for the
experiment: human cervical cancer (HeLa), human breast
adenocarcinoma cell line (MDA-MB-231, MCF-7) and
human alveolar adenocarcinoma (A549), and the results
were compared to see the differences that might arise from
their structural differences. All compounds showed good
activity in the A549 cell line and moderate activity in
MDA-MB-231 cell lines Fig. 9, suggesting the different
ancillary ligands have little effect on their activity
(Table 3).
A minor difference was also noted for complexes 1 and
2 in HELA cell lines. The complex 3 having dmb ancillary
ligand showed very low or no activity at all in the cell lines
tested (Table 3), indicating that the methyl group is not
important for activity.
Antimicrobial activity
From the obtained data, it was clear that the complex 1
was more sensitive against the selected tumor cell lines
than complex 2, but these complexes all exhibited rela-
tively lower in vitro cytotoxicity against the selected cell
lines than cisplatin.
All the complexes exhibited varying antimicrobial activity
toward most of the microbes selected (Table 5) and were
found to be dose dependent. Most of the complexes
inhibited moderate growth of gram-negative bacteria
Escherichia coli (MTCC 443) and gram-positive
Table 4 The H-Bond, van der
Complex
H-bond
donor–acceptor length (A) (complex DNA)
Bond
van der Waals interactions Bond
Docking
Waals interaction and scores for
binding of Ru(II) and Co(III)
complexes to (1y9 h) DNA
decamer containing CG bases
using docking calculation
˚
˚
length (A) score
[Ru(phen)2PIP-Cl] O54-G13:H21
O54-G13:H22
2.413
2.658
2.555
2.161
1.857
2.0854
2.645
H78-DC17:H5
H66-DG16:H5
C35-DC10:OP1
C9-DC10:C5
1.852
1.365
2.456
2.885
2.722
2.456
1.645
1.810
2.530
2.702
1.497
1.759
39.656
22.458
18.422
H81-DG13:N2
H81-DC10:O2
[Ru(bpy)2PIP-Cl]
H77-DA9:OP1
H76-DA9:OP2
H76-DA9:OP5
C49-DG12:N7
C49-DG12:C8
H72-DT8:H71
H71-DT8:H2
[Ru(dmb)2PIP-Cl] H76-AD15:N3
H77-C10:O2
2.741
1.983
2.410
2.048
C14-DC10:OP1
C53-DT4:O4
H77-DG13:N3
H77-DG13:H21
O50-DG13:O2
O50-G13:H22
123

Med Chem Res
supercoiled Form I to the open circular Form II upon irradi-
ation, which may be taken as the potential DNA cleavage
reagent.
Table 5 Antibacterial activity of Ru(II) complexes at 20 lg/mL
Complex
Bacterial species
E. coli
S. aureus
Acknowledgments We are grateful to the Indian Council for Cul-
tural Relations (ICCR), Hyderabad, for financial support.
DMSO
Nil
15
24
11
22
18
Nil
13
21
10
14
18
[Ru(phen)2PIP-Cl]2? (1)
[Ru(bpy)2PIP-Cl]2? (2)
[Ru(dmb)2PIP-Cl]2? (3)
Septomycine
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