Synthesis and characterization of Ru(II)-DMSO-Cl-chalcone complexes: DNA binding, nuclease, and topoisomerase II inhibitory activity

Article abstract of DOI:10.1021/ic202440r

The complexes of type cis-[Ru(S-DMSO)₃(R-CO-CH-CH-R′)Cl] (R = 2-hydroxyphenyl for all, R′ = phenyl 1, naphthyl 2, anthracenyl 3, thiophene 4, 3-methyl thiophene 5) are synthesized and characterized using spectroscopic (IR, ¹H and

Full text of DOI:10.1021/ic202440r

Article
pubs.acs.org/IC
Synthesis and Characterization of Ru(II)−DMSO−Cl−Chalcone
Complexes: DNA Binding, Nuclease, and Topoisomerase II Inhibitory
Activity
Ruchi Gaur and Lallan Mishra*
Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi-221005, India
S
* Supporting Information
ABSTRACT: The complexes of type cis-[Ru(S-DMSO)3(R
COCHCHR′)Cl] (R = 2-hydroxyphenyl for all, R′ =
phenyl 1, naphthyl 2, anthracenyl 3, thiophene 4, 3-methyl
thiophene 5) are synthesized and characterized using
1
spectroscopic (IR, H and 13C NMR, and UV−vis) and single
crystal X-ray diffraction techniques. Their crystal structures
show the formation of both intermolecular and intramolecular
H-bonding. The molecular assembly of complex 5 using
secondary interactions provides a butterfly structure. The
binding of complexes with calf thymus DNA is monitored
using UV−vis spectral titrations. The binding interaction of
complexes 1, 2, and 3 with DNA increases with increasing
conjugation of aromatic rings. However, complexes 4 and 5 interact with DNA strongly. The emission from ethidium bromide
(EB) bound DNA recorded in phosphate buffer solution (pH = 7.2) decreases by incremental addition of solution of the
complexes. The complexes 4 and 5 (100 μM) bind with the minor groove of DNA and cleave double-stranded pBR322 DNA
significantly even in the absence of an activator. In the presence of H2O2, they cleave supercoiled DNA via oxidative pathway
even at lower concentration (20 μM). Both complexes 4 and 5 inhibit topoisomerase II activity with IC50 values of 18 and 13.
These values suggest that 4 and 5 are potential topoisomerase II inhibitors as compared to some of known inhibitors like
novobiocin and etoposide.
substitution by DNA bases. The Ru(II) center bearing labile
ligands binds double-helical DNA forming monofunctional
adducts with DNA preferentially via N7 atom of its guanine
residues in a covalent binding mode.12 The noncovalent
interactions occur mainly through intercalation of aromatic
skeleton and its binding with minor groove of DNA. Thus,
DNA−drug intercalation could be stabilized significantly by π-
electron overlap and hydrophobic and polar interactions as well
as electrostatic forces of cationic intercalators with polyanionic
nucleic acid.13
Since tumors maintain a high level of topoisomerase II
(Topo II), inhibition of Topo II has also been considered as a
target for the design of several antitumor agents.14,15 The wide
variety of topoisomerase inhibitors including etoposide,
doxorubicin, mitoxantrone, amsacrine, and idarubicin kill cells
undergoing DNA replication, reading the DNA for protein
production or repair of DNA damage.16 According to previous
studies, Ru(II)(C6H6)(DMSO)Cl2 exhibits a strong DNA-
binding affinity and inhibits DNA relaxation activity of Topo
II.17 Thus, selection of synthetic precursor-like cis-Ru-
(DMSO)4Cl2 which contains DMSO and chloro groups as
labile ligands is justified. Additionally, chalcones being versatile
INTRODUCTION
■
The design and development of new anticancer drugs is an
active area of research in chemical science. After the discovery
of cisplatin by Rosenberg1 as an effective anticancer drug
having various side effects, the search for alternative metal
based drugs has been an important area of interest for
researchers. Among the several metals that are currently being
investigated for their anticancer activity, ruthenium occupies a
prominent position. In particular, cis-Ru(DMSO)4Cl2 is
targeted as an antitumor drug2 owing to its stabilization using
heteroaromatic ligands3 providing complexes like Na[trans-
RuCl4(S-DMSO)(Im)] (NAMI), [ImH][trans-RuCl4(DMSO-
S)Im] (NAMI-A), [IndH]trans-[Ru(N-Ind)2Cl4] (KP1019),4
and Ru(η6-p-cymene)Cl2(pta) (RAPTA).5 In this drug
discovery approach, DNA has been targeted extensively as
coordination of ruthenium atom to the nucleic bases is seen to
get enhanced through H-bonding interactions or weakened
because of steric interactions, suggesting the possibility to
design compounds to target specific nucleotides. Ruthenium
based antitumor complexes can interact with DNA by (1)
electrostatic interaction between a cationic metal complex with
DNA,6 (2) noncovalent hydrophobic surface binding, (3)
intercalation with DNA bases, and (4) covalent binding.7−11 It
is worth mentioning that labile ligands present in the
coordination sphere of a ruthenium ion facilitate their
Received: November 14, 2011
Published: February 21, 2012
© 2012 American Chemical Society
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Article
7.33 (m, 1H, Ar), 6.98 (d, J = 8.4 Hz, 1H, Ar), 6.59 (t, J = 7.5 Hz, 1H,
Ar), 3.62−3.15 (s and m, 18H, DMSO). 13C NMR (CDCl3, δ ppm):
188.53 (CO), 171.71 (Ar−CO), 140.48 (C-α), 121.66 (C-β),
136.66, 133.70, 132.38, 132.22, 131.48, 130.89, 128.95, 127.23, 126.66,
126.35, 125.62, 125.54, 125.23, 122.88, 115.30, (Ar), 47.40, 46.63,
45.95, 44.59, 44.19, 42.91 (CH3, DMSO). UV−vis (DMSO, 10−4 M):
λmax (nm) (εmax × 104 M−1 cm−1) 271 (1.10), 370 (0.996), 495 sh
(0.193).
precursor for the synthesis of biologically relevant flavones also
contributed to various biological properties.18−24 Therefore,
chalcones are considered as “privileged structure” by Evans et
al.25 as they bear bidentate (O,O) donor sites and could easily
chelate with Ru(II) center substituting labile ligands. The
chalcone bearing conjugated ligand frameworks may provide
additional opportunity for their interaction with a DNA helix.
Thus, presently synthesized and well characterized complexes
were allowed to interact with calf thymus (CT) DNA, and their
binding properties were monitored using UV−vis spectral
titrations and competitive binding experiments. The DNA
cleavage and topoisomerase inhibitory activities were moni-
tored using gel electrophoretic mobility assay technique.
Complex [Ru(L3)(DMSO)3Cl] (3). Yield: 0.400 g (58%). Mp: >200
°C. Anal. Calcd for C29H33O5S3ClRu Found (Calcd) (%): C 50.14
(50.17); H 4.74 (4.79). ESI-MS: m/z: 694 [M]+, 659 [M − Cl]+, 581
[M − Cl − DMSO]+, 503 [M − Cl − 2DMSO]+, 425 [M − Cl −
3DMSO]+. IR (KBr pellet, cm−1): 3006(m) υ(C−H), 1612(s) υ(C
1
O), 1107(s) υ(SO), 426(m) υ(Ru−S). H NMR (CDCl3, δ ppm):
8.58 (m, 2H, Hα and Ar), 8.27 (d, J = 7.8 Hz, 1H, Ar), 8.05 (d, J = 7.2
Hz, 1H, Ar), 7.72 (m, 6H, Ar and Hβ), 7.33 (m, 1H, Ar), 7.02 (d, J =
8.7 Hz, 1H, Ar), 6.53 (t, J = 7.5 Hz, 1H, Ar), 3.64−3.19 (s and m,
18H, DMSO). 13C NMR (CDCl3, δ ppm): 188.29 (CO), 171.87
(Ar−CO), 140.41 (C-α), 121.55 (C-β), 136.75, 133.07, 132.33,
131.27, 129.90, 129.56, 129.00, 128.72, 126.76, 125.63, 125.49, 125.03,
115.35 (Ar), 47.54, 46.65, 46.35, 44.76, 44.12, 42.90 (CH3, DMSO).
UV−vis (DMSO, 10−4 M): λmax (nm) (εmax × 104 M−1 cm−1)
357(0.647), 444 (1.089), 516(0.037).
EXPERIMENTAL SECTION
■
Materials. The starting material cis,fac-[RuCl2(DMSO-S)3(DMSO-
O)] was prepared from RuCl3·3H2O using a reported procedure.26
However, different aldehydes, RuCl3·3H2O, 2-hydroxy acetophenone,
and agarose were purchased from Sigma Aldrich Chemical Co. Pvt.
Ltd., India. Solvents were purchased from E. Merck and used as
received. Calf thymus (CT) DNA and supercoiled (SC) plasmid DNA
pBR322 (as a solution in Tris buffer and cesium chloride purified),
with a length of 4361 base pairs, were purchased from Bangalore
Genei, India.
Complex [Ru(L4)(DMSO)3Cl] (4). Yield: 0.400 g (67%). Mp: >200
°C. Anal. Calcd for C19H27O5S4ClRu Found (Calcd) (%): 37.56
(38.02); H 4.74 (4.53). IR (KBr pellet, cm−1): 3010(m) υ(C−H),
1
1613(s) υ(CO), 1098(s) υ(SO), 427(m) υ(Ru−S). H NMR
Instrumental Methods. Infrared, UV−vis, and luminescence
spectra were recorded on VARIAN 3100 FTIR, Jasco UV-630
spectrophotometer and Perkin-Elmer LS-45 spectrophotometer,
respectively. Elemental analysis and mass measurements were carried
out using a Carbo-Erba elemental analyzer 1108 and JEOL SX-102
mass spectrometer, respectively. 1H NMR spectra were recorded using
JEOL AL 300 MHz spectrometer and TMS as internal reference.
Preparation of Chalcones. The chalcones, 1-(2-hydroxyphenyl)-
3-(1-phenyl)propenone (L1), 1-(2-hydroxyphenyl)-3-(1-naphathyl)-
propenone (L2), 1-(2-hydroxyphenyl)-3-(9-anthracyl)propenone
(L3), 1-(2-hydroxyphenyl)-3-(2-thienyl)propenone (L4), and 1-(2-
Hydroxyphenyl)-3-(3-methyl-2-thienyl)-2-propen-1-one (L5), were
synthesized and characterized using methods reported elsewhere.27,28
Synthesis of [Ru(L1)(DMSO)3Cl] (1). A solution of cis,fac-
[RuCl2(DMSO-S)3(DMSO-O)] (484 mg, 1 mmol) dissolved in
methanol (20 mL) was added dropwise to a methanolic solution
(15 mL) of L1 (224 mg, 1 mmol) containing an equimolar amount of
NEt3. After complete addition, the color of the resulting solution
changed from orange to red, and stirring was continued for 12 h at
room temperature. The red crystalline solid thus obtained was filtered
and washed with methanol followed by diethyl ether and then dried in
vacuo. Yield: 0.350 g (59%). Mp: >200 °C. The material is partially
soluble in H2O, ethanol, methanol, and tetrahydrofuran while it is
highly soluble in acetone, dichloromethane, acetonitrile, DMSO, and
chloroform. Anal. Calcd for C21H29O5S3ClRu Found (Calcd) (%): C
41.93 (42.45); H 4.69 (4.92). IR (KBr pellet, cm−1): 2924(m) υ(C
(CDCl3, δ ppm): 7.73 (m, 2H, thiophene and Hα), 7.45 (m, 2H, Ar
and thiophene), 7.30 (m, 2H, Hβ and thiophene), 7.1 (m, 1H, Ar),
6.94 (d, J = 8.7 Hz, 1H, Ar), 6.59 (t, J = 7.5 Hz, 1H, Ar), 3.55−3.09 (s
and m, 18H, DMSO). 13C NMR (CDCl3, δ ppm): 187.98 (CO),
171.57 (Ar−CO), 140.51 (C-α), 121.78 (C-β), 136.52, 128.44, 122.93
(thiophene), 136.11, 131.77, 129.14, 125.55, 115.28 (Ar), 42.95, 44.28,
44.65, 45.69, 46.58, 47.38 (CH3, DMSO). UV−vis (DMSO, 10−4 M):
λmax (nm) (εmax × 104 M−1 cm−1) 291 (0.49), 368 (1.22), 501 (0.23).
Complex [Ru(L5)(DMSO)3Cl] (5). Yield: 0.370 g (56%). Mp: >200
°C. Anal. Calcd for C20H29O5S4ClRu Found (Calcd) (%): C 39.26
(39.11); H 3.92 (4.76) %. IR (KBr pellet, cm−1): 3091(m) υ(C−H),
1
1612(s) υ(CO), 1102(s) υ(SO), 424(m) υ(Ru−S). H NMR
(CDCl3, δ ppm): 7.87 (d, J = 15 Hz, 1H, Hα), 7.73 (d, J = 8.4 Hz, 1H,
Ar), 7.38 (m, 3H, Ar, Hβ and thiophene), 6.94 (m, 2H, Ar and
thiophene), 6.59 (t, J = 7.5 Hz, 1H, Ar), 3.56−3.08 (s and m, 18H,
DMSO), 2.34 (s, 3H, CH3). 13C NMR (CDCl3, δ ppm): 187.98 (C
O), 171.50 (Ar−CO), 142.27 (C-α), 121.54 (C-β), 131.44, 131.82,
136.38 (thiophene), 134.92, 134.77, 127.88, 125.52, 115.26 (Ar),
47.36, 46.61, 44.55, 44.29, 43.06 (CH3, DMSO), 14.30 (CH3). UV−
vis (DMSO, 10−4 M): λmax (nm) (εmax × 104 M−1 cm−1) 292 (0.51),
374 (1.54), 504 (0.37).
X-ray Crystallographic Studies. Crystals of complexes suitable
for X-ray diffraction were grown at room temperature in a mixture of
dichloromethane/petroleum ether (40−60 °C) solvent. The X-ray
crystallographic data were recorded by mounting single-crystal of the
complexes separately on a glass fiber. Oxford diffraction XCALIBUR-S
CCD area detector diffractometer was used for the determination of
cell and intensity data collection. Appropriate empirical absorption
corrections were applied using multiscan programs. Monochromated
Mo Kα radiation (λ = 0.710 73 Å) was used for the measurements.
The crystal structures were solved by direct methods and refined by
full-matrix least-squares SHELXL-97,29 and special computations were
carried out using PLATON.30
Electrochemical Studies. Cyclic voltammetry was performed on a
CHI 620c electrochemical analyzer. A glassy carbon working electrode,
platinum wire auxiliary electrode, and Ag/Ag+ reference electrode were
used in a standard three-electrode configuration. Tetrabutylammo-
nium perchlorate (TBAP) was used as a supporting electrolyte, and
the solution concentration was kept as 10−3 M.
1
H), 1628(s) υ(CO), 1099 υ(SO), 425(m) υ(RuS). H NMR
(CDCl3, δ ppm): 7.76 (d, J = 8.4 Hz, 1H, Ar), 7.61 (m, 4H, Ar and
Hα), 7.43 (m, 4H, Ar and Hβ), 7.32 (m, 1H, Ar), 6.96 (d, J = 8.4 Hz,
1H, Ar), 6.58 (t, J = 7.5 Hz, 1H, Ar) 3.56−3.09 (s and m, 18H,
DMSO). 13C NMR (CDCl3, δ ppm): 188.89 (CO), 171.64 (Ar
CO), 143.57 (C-α), 121.70 (C-β), 136.6, 134.91, 132.21, 130.58,
129.05, 128.24, 125.60, 124.31, 115.22 (Ar), 47.38, 46.61, 45.84, 44.64,
44.24, 42.87 (CH3, DMSO). UV−vis (DMF, 10−4 M): λmax (nm) (εmax
× 104 M−1 cm−1) 239 (1.51), 329(1.45), 494 sh (0.273).
Other complexes were also prepared adopting the procedure similar
to that used for the preparation of complex 1 using L2, L3, L4, L5
separately in place of L1.
Complex [Ru(L2)(DMSO)3Cl] (2). Yield: 0.388 g (60%). Mp: >200
°C. Anal. Calcd for C25H31O5S3ClRu (2) Found (Calcd) (%): C 46.41
(46.61); H 4.78 (4.85). IR (KBr pellet, cm−1): 2922(m) υ(CH),
Absorption Titration. The experiments of DNA binding with
complexes were carried out in Na-phosphate buffer solution (pH 7.2).
The absorption ratio of CT DNA solutions at λmax 260 and 280 nm
was found as 1.9:1. It showed that DNA is sufficiently free from
1
1616(s) υ(CO), 1100(s) υ(SO), 428(m) υ(RuS). H NMR
(CDCl3, δ ppm): 8.4 (d, J = 15 Hz, 1H, Hα), 8.093 (d, J = 7.8 Hz, 1H,
Ar), 7.89 (m, 4H, Ar), 7.74 (d, J = 15 Hz, 1H, Hβ), 7.59 (m, 3H, Ar),
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protein impurities. The concentration of DNA was determined using
UV−vis absorbance and the molar absorption coefficient (6600 M−1
cm−1) at 260 nm.31 The absorption titrations of the complexes (10 μM
in Na-phosphate buffer containing 0.01% DMSO) against CT DNA
were performed by monitoring their absorption spectra with
incremental addition of CT DNA within 1−10 μM concentration.
The spectra were recorded after equilibration for 3 min, allowing the
complexes to bind to the CT-DNA. The intrinsic binding constant
(Kb) was calculated from a plot of [DNA]/(εa − εf) versus [DNA]
using the following equation:
supercoiled DNA from being converted into relaxed DNA (IC50
values) was calculated from the midpoint concentration of the
complex-induced DNA unwinding. To check whether the order of
addition affected the results, experiments were also carried out in ice,
in which the whole reaction mixture was assembled.
RESULTS
■
Description of Molecular Structure. The complexes
(Scheme 1) were found to be air stable both in solid as well as
−1
Scheme 1. Schematic Representation of Synthetic Strategy
[DNA]/(ε − ε )=[DNA]/(ε − ε )+[K (ε − ε )]
(1)
a
f
b
f
b
b
f
[DNA] is the concentration of DNA in base pairs. The apparent
absorption coefficients εa, εf, and εb correspond to Aobsd/[Ru], the
extinction coefficient for free ruthenium(II) complexes and extinction
coefficient for the ruthenium(II) complex in fully bound form,
respectively.32 The value of Kb was calculated as the ratio of the slope
to the intercept.
Competitive Binding with Ethidium Bromide. Relative binding
of ruthenium complexes with CT DNA was studied by fluorescence
spectral method using bound CT DNA solution in Na phosphate
buffer solution (pH 7.2). In a typical experiment, 20 μL of CT-DNA
solution (A260 = 2.0) was added to 2 mL of ethidium bromide (EB) in
buffer solution, and the fluorescence intensity was measured at
excitation wavelength λ, 510 nm; maximum emission was observed at
λ, 600 nm. Aliquots of 0.1 mM solution of the complexes were then
added to DNA-EB, and resulting fluorescence spectra were recorded
after each addition until maximum reduction in the intensity of
fluorescence occurred. The Stern−Volmer quenching constant33 in
each complex was calculated using the equation given as
in solution. The molecular structures along with crystallo-
graphic numbering schemes of complexes are illustrated in
Figure 1a−e. Crystallographic data and selected bond distance/
bond angle data are shown in Table 1 and Supporting
Information, respectively. The complexes 1, 2, and 4 crystal-
lized as block-shaped crystals in a triclinic crystal system with
space group P1 while complexes 3 and 5 crystallized in
̅
I /I = 1 + K r
(2)
o
sv
monoclinic crystal system with space group P21/c and C2/c,
respectively. All the complexes exhibited distorted octahedral
geometry around the metal center as expected from low spin d6
Ru(II) ion. No solvent accessible voids could be seen from the
packing pattern of the complexes 1, 2, 3, and 4, while in
complex 5 dichloromethane and water molecules were present.
The S(2)−O(3) bond distances [1.47(5), 1.48(18),
1.46(4),1.48(5), 1.49(7) Å] in complexes were found to be
comparable with that observed in free DMSO [1.49(1) Å].34
The chalcone exists in bis chelating coordination mode (η2),
and chelating angles were found as 88.20(15)°, 88.25(5)°,
88.72(14)°, 88.30(16)°, and 88.80(3)° in the molecular
structure of complexes 1, 2, 3, 4, and 5, respectively.
Here, Iο and I are the fluorescence intensity in the absence and
presence of complexes, respectively, Ksv is a linear Stern−Volmer
quenching constant, and r is the ratio of the total concentration of a
complex to that of DNA. The value of Ksv is given by the ratio of slope
to intercept in a plot of Iο/I versus [complex]/[DNA].
DNA Cleavage Studies Using Agarose Gel Electrophoresis.
In the gel electrophoresis experiments, supercoiled pBR322 DNA was
treated with metal complexes, and the mixture was incubated for 30
min at 37 °C. The samples were then analyzed by 1.5% agarose gel
electrophoresis [Tris−acetic acid−ethylenediaminetetraacetic acid
(EDTA) (TAE) buffer, pH 8.3] for 3 h at 50 mV. The gel was
stained with 0.5 μg mL−1 ethidium bromide, visualized by UV light,
and photographed for analysis. The extent of cleavage of the SC DNA
was determined by measuring the intensities of the bands using Alpha
Innotech gel documentation system (AlphaImager 2200)and Gen-
osens 1510 documentation and analysis system. The experiments were
also carried out in the presence of activators, viz., NaN3 (1O singlet
oxygen trapper), sodium formate (OH− radical scavanger), sodium
ascobate (reducing agent), and H2O2, which were added to SC DNA
prior to the addition of complexes 4 and 5 (acetonitrile 0.01%) only.
Topoisomerase Inhibition Assay. DNA topoisomerase II (Topo
II) from Escherichia coli was purchased from New England Biolabs, and
no further purification was performed. One unit (U) of the enzyme
was defined as the amount that completely relaxed 0.5 μg of negatively
charged supercoiled pBR322 plasmid DNA in 30 min at 30 °C under
standard assay conditions. The reaction mixture (20 μL) consists of 35
mM Tris−HCl (pH 7.5), 24 mM KCl, 4.0 mM MgCl2, 2 mM DTT
(dithiothreitol), 5 mM spermidine, 0.1 mg/mL BSA, 1.75 mM ATP,
6.5% glycerol, 0.30 μg of pBR322 DNA, and 1 U of Topo II together
with variable concentration of Ru(II) complexes (0−20 μM). The
corresponding reaction mixtures were incubated at 37 °C for 30 min,
and then they were terminated by the addition of 3 μL of 5× stop
solution dye consisting of 0.25% bromophenol blue, 4.5% sodium
dodecyl sulfate, and 45% glycerol. The electrophoresis of the samples
was carried out through 1% agarose in TAE buffer at 50 V for 2 h. The
gel was stained with 1 μg mL−1 EB and photographed under UV light.
The concentration of the inhibitor that prevented 50% of the
The bond lengths (Ru−O, Ru−S, and Ru−Cl) vary in the
order (Ru−Cl) > (Ru−S) > (Ru−O).27 The C1−O1−Ru1−
O2-C7 atoms lie in the same plane. The O(1)−Ru(1)−S(2)
angles in all complexes were found to be significantly smaller
than 180°; it may be due to steric repulsion of the methyl group
of DMSO and phenyl ring of the chalcone. The two oxygen
atoms of the ligand are bonded to the Ru(II) center, and the
Ru(1)−O(1) distance varies from 2.0514(17) to 2.091(4) Å
whereas the Ru(1)−O(2) distance varied from 2.055(4) to
2.0726(17) Å and was found to be consistent with the reported
values.35 Additionally, Ru−S distance varied from 2.246(10) to
2.266(9) Å, and Ru−Cl bond length was found from 2.407(3)
to 2.418 (15) Å. Probably this longer distance for Ru−Cl as
compared to the Ru−S bond made fast substitution of Cl by
nucleobases.
The molecular structures of the complexes obtained from
their X-ray diffraction studies were supported by their
spectroscopic measurements in the solution. The IR spectra
of the chalcones in general display characteristic peaks at
3446−3458 and 1638−1689 cm−1 assigned to υ(OH) and
υ(CO) vibration, respectively, and were found to be
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Figure 1. Molecular structure of (a) complex 1, (b) complex 2, (c) complex 3, (d) complex 4, (e) complex 5. Hydrogen atoms are omitted for
clarity.
consistent with the earlier report.27,28 As compared to the
spectra of free ligands, the IR spectra of their complexes
displayed peaks at 1628, 1615, 1612, 1617, and 1616 cm−1,
respectively, and supported the coordination of their υ(CO)
group. Since the υ(O−H) vibration of free ligands disappeared
in the spectra of their corresponding complexes, it is considered
that the deprotonated OH group has coordinated with the
metal ion. Additional peaks observed at 1100−1050 and 425−
430 cm−1 are assigned to υ(SO) and υ(Ru−S) vibrations.
1H and 13C NMR spectral data of the complexes are
incorporated in the Experimental Section and Supporting
Information. The OH proton observed in the spectra of free
chalcones disappeared in the spectra of corresponding
complexes, again supporting the deprotonation of the OH
group. An appreciable downfield shift of ethylenic protons was
also observed as a consequence of the coordination of
neighboring CO group. Since there is no peak observed
for O-bonded DMSO at δ ∼2.72 ppm, all DMSO is assigned to
be S-coordinated to the metal center. In 13C NMR spectra, C
O carbon of the chalcones in the complexes appeared at δ
187.98−188.89 ppm while the O−C−Ar carbon atoms of
chalcones appeared at δ 171.49−171.90 ppm. The carbon
atoms of phenyl groups as well as methyl groups of DMSO
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a
Table 1. Selected Crystallographic Data of Complexes 1, 2, 3, 4, and 5
1
2
3
4
5
formula
C21H29O5S3ClRu
594.17
C25H31O5S3ClRu
644.20
C29H33O5S3ClRu
694.25
C19H27O5S4ClRu
600.17
C41H58O12S8Cl4Ru2
1343.37
M
cryst syst
triclinic
triclinic
monoclinic
150(2)
triclinic
monoclinic
150(2)
T (K)
150(2)
293(2)
293(2)
space group
P1
̅
P1
̅
P21/c
P1
̅
C2/c
a/Å
8.3200(4)
12.4615(9)
12.9858(10)
107.394(7)
103.270(5)
94.901(5)
1233.17(14)
2
8.3503(3)
12.0760(7)
13.9832(6)
73.436(4)
83.884(3)
88.764(4)
1343.78(11)
2
8.3752(3)
24.7738(9)
14.2933(6)
90
8.2466(4)
12.3466(7)
12.9838(7)
106.590(5)
101.295(4)
95.912(4)
1224.52(11)
2
23.2656(17)
11.7850(5)
23.5597(18)
90
b/Å
c/Å
α (deg)
β (deg)
95.429(4)
90
120.357(10)
90
γ (deg)
V/ Å3
2952.3(2)
4
5574.0(6)
4
Z
Dc/mg m−3
1.600
1.592
1.562
1.628
1.601
reflns collected/unique
7811/5293
5293/0/280
0.0331
8822/5709
5709/0/316
0.0189
22 306/5164
5164/0/358
0.0906
9084/5381
5381/0/128
0.0199
19 642/4897
4897/0/310
0.0738
data/restraints/params
R(int)
θ range for data collection (deg)
2.91−25.00
98
2.91−25.00
99.4
3.29−25.00
99.8
2.91−25.00
98
3.46- 25.00
99.8
completeness to θ = 25.00
wR2
0.1762
0.0734
0.0841
0.2190
0.2509
R1
0.0614
0.0295
0.0432
0.0680
0.0974
GOF
1.105
0.738
0.809
1.012
1.221
largest diff. peak and hole (e Å−3
)
2.277 and −2.320
1.100 and −0.347
1.004 and −0.375
2.888 and −2.563
4.354 and −1.592
a
Refinement method: full-matrix, least-squares on F2.
appeared in the ranges δ 115.22−136.75 and δ 40.98−47.66
ppm, respectively.
The ESI-MS of a representative complex 3 was also recorded
to support the stability of complexes in the solution. It showed
molecular ion peak at m/z 694 assigned to [M]+. However,
additional peaks observed at 659, 581, 503, and 425 were
assigned to [M − Cl]+, [M − Cl − DMSO]+, [M − Cl −
2DMSO]+, [M − Cl − 3DMSO]+, respectively.
In the electronic spectra of complexes, the intense bands
observed at higher energy showed greater dependence on the
nature of the O,O-ligands. The remaining less intense bands
were attributed to charge transfer transitions from chlorides to
Ru(II) ion. The low spin ruthenium(II) ion (d6 configuration)
provides filled metal orbitals of proper symmetry and interacts
with relatively low lying π* orbitals of the ligand. The broad
bands observed at λmax 494−516 nm arise from dπ(RuII)→
π*(L1−5) metal-to-ligand charge-transfer (MLCT) transitions.
It is reported that substitution by a more conjugated ligand
enhances π-delocalization and hence lowers the energy between
dπ(RuII)→π*(ligand) MLCT band.36 So an attempt was made
to correlate such variation in complexes 1, 2, and 3. The higher
energy bands observed at 230−375 nm in the spectra of the
complexes are assigned to intraligand and π−π* transitions.
These bands were red-shifted as compared to the band
observed from respective ligands. The spectral features of the
complexes are shown Figure 2.
Cyclic Voltammetry. Cyclic voltammetry of complexes was
carried out in dichloromethane at 298 K using ferrocene/
ferrocenium (Fc/Fc+) as an internal standard (0.10 V (80 mv)
vs Ag/Ag+). The mixed-ligand complexes showed irreversible
oxidation peak from 0.79 to 0.93 V assigned to RuIII/RuII redox
couple;37 the data are summarized in Table 3, and figures are
shown as Supporting Information. This irreversibility could be
considered in view of the transient lifetime of the reduced state
as reported earlier.38 However, the lower value of oxidation
Figure 2. Overlay UV−vis spectra of complexes 1−5 recorded in
dichloromethane (1 × 10−4 M). Inset: MLCT region of the complex 3.
potential for Ru(III)/Ru(II) couple in complex 1 could be
attributed to the stronger σ-donor and weaker π-acceptor
ability of the ligands, consequently stabilizing the higher
oxidation state of Ru(III).39 The oxidation potential data
suggested that metal based oxidation varied in order of E1/2(3)
< E1/2(5) < E1/2(4) < E1/2(2) < E1/2(1). This order goes with
the order of their MLCT transition energies. The lower value of
E1/2 for the oxidation of Ru(II) to Ru(III) found in complex 5
as compared to its value in complex 4 could be considered due
to the presence of electron repelling the methyl group in the
thiophene ring of ligand.40
DNA Interaction. Electronic absorption spectroscopy is
found to be an effective tool to examine the binding mode of
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Figure 3. UV−vis absorption spectra of (a) complex 1 (10 μM) in the absence and in the presence of increasing amounts of DNA = 0−100 μM and
(b) complex 5 (10 μM) in the absence and in the presence of increasing amounts of DNA = 0−100 μM. Arrow shows the absorbance changes upon
increasing DNA concentration.
Figure 4. Emission spectra from EB bound DNA in the absence (---) and in the presence of [complex 1 and 5] 0−4 μM concentration, [EB] 10 μM,
[DNA] 10 μM. Arrow shows changes in the emission intensity upon addition of increasing concentration of the complex. Inset: Plots of Io/I vs
[Ru]/[DNA] with experimental data points.
DNA with metal complexes.41 Thus, to explore the possibility
of binding of each complex to CT-DNA, spectroscopic
titrations of the solution of complexes separately with CT-
DNA were carried out. The ruthenium(II) complexes can bind
to double-stranded DNA in different binding modes on the
basis of their structure, charge, and type of ligands. In order to
compare the binding strength of the complexes with CT DNA,
the intrinsic binding constant Kb was calculated by monitoring
the changes in their absorbance with increasing concentration
of DNA (Figure 3). The value of Kb was obtained as a ratio of
slope to intercept obtained from the plot of [DNA]/(εa − εf)
versus [DNA]. The Kb values were obtained as 4.22 × 105, 5.29
× 105, 7.09 × 105, 4.9 × 106, and 5.7 × 106 for complexes 1, 2,
3, 4, and 5, respectively. The Kb values thus vary in the order Kb
(5) > Kb (4) > Kb (3) > Kb (2) > Kb (1).
Competitive Binding of Complexes with Ethidium
Bromide. Ethidium bromide (EB) is a standard intercalating
agent of DNA. Hence, a competitive binding study using
ethidium bromide (EB) bound to DNA was carried out by
successive addition of 0−0.25 μM of each complex to 10 μM
DNA solutions containing 10 μM solution of EB in Na-
phosphate buffer (pH 7.2). The emission spectra of EB-DNA
system in the presence and absence of ruthenium complexes 1
and 5 are shown in Figure 4. The plots of Iο/I versus
[complex]/[DNA] are shown in Figure 4. They support the
linear Stern−Volmer equation. The Stern−Volmer constant
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Figure 5. (a) Ethidium bromide stained agarose gel (1%) of pBR322 plasmid DNA (300 ng μL−1) in the presence of complex 4 after 1 h of
incubation: lane 1, DNA control; lane 2, pBR322 + 25 μM; lane 3, pBR322 + 50 μM; lane 4, pBR322 + 75 μM; lane 5, pBR322 + 100 μM. (b)
Cleavage of supercoiled pBR322 DNA showing the decrease in SC DNA and the formation of NC DNA with increasing concentration of complex 4.
Figure 6. (a) Ethidium bromide stained agarose gel (1%) of pBR322 plasmid DNA (300 ng μL−1) in the presence of complex 5 after 1 h of
incubation: lane 1, DNA control; lane 2, pBR322 + 10 μM; lane 3, pBR322 + 25 μM; lane 4, pBR322 + 50 μM; lane 5, pBR322 + 75 μM; lane 6,
pBR322 + 100 μM. (b) Cleavage of supercoiled pBR322 DNA showing the decrease in SC DNA and the formation of NC DNA with increasing
concentration of complex 5.
Figure 7. Ethidium bromide stained agarose gel (1%) of pBR322 plasmid DNA (300 ng μL−1) in the presence of 20 μM complexes after 1 h of
incubation: lane 1, DNA control; lane 2, pBR322 + 4 + DAPI; lane 3, pBR322 + 4 + MG; lane 4, pBR322 + 5 + DAPI; lane 5, pBR322 + 5 + MG.
(Ksv) is evaluated as 5.3, 4.2, 3.8, 1.8, and 1.2 for complexes 5,
4, 3, 2, and 1, respectively, which were in parity with the extent
of displacement of ethidium bromide by the complexes. The
hydrogen bond may be formed between the sulfur of the
thiophene group, present in the ligand of the complexes 4 and
5, and the complementary functional group present on the edge
of the DNA.42
Nuclease Activity of the Complexes in the Absence of
Activators. To assess the DNA cleavage ability of the
complexes, supercoiled pBR322 DNA (300 ng μL−1) was
incubated separately with 100 μM of all complexes in 5 mM
Tris-HCl/50 mM NaCl buffer at pH 7.2 for 1 h without
addition of any activator as depicted in the Supporting
Information. Control experiment showed that SC DNA
(form I) was cleaved only by complexes 4 and 5, and formed
NC DNA (form II). However, a preliminary experiment
showed that L1−L5 separately did not cause any cleavage
(Supporting Information) of DNA at 100 μM concentration.
In order to study DNA cleavage pattern of complexes 4 and
5, different concentrations (0−100 μM) of 4 and 5 were
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Figure 8. (a) Ethidium bromide stained agarose gel (1.5%) of pBR322 plasmid DNA (300 ng μL−1) in the presence of complex 4 (20 μM) after 1 h
of incubation: lane 1, DNA control; lane 2, DNA + NaN3 (50 μM); lane 3, DNA + NaN3 (50 μM) + 4; lane 4, DNA + sodium formate (50 μM);
lane 5, DNA + sodium formate (50 μM) + 4; lane 6, DNA + sodium ascorbate; lane 7, DNA + sodium ascorbate + 4; lane 8, DNA + H2O2 (50 μM);
lane 9, DNA + H2O2 (50 μM) + 4. (b) Ethidium bromide stained agarose gel (1.5%) of pBR322 plasmid DNA (300 ng μL−1) in the presence of
complex 5 (20 μM) after 1 h of incubation: lane 1, DNA control; lane 2, DNA + NaN3 (50 μM); lane 3, DNA + NaN3 (50 μM) + 5; lane 4, DNA +
sodium formate (50 μM); lane 5, DNA + sodium formate (50 μM) + 5; lane 6, DNA + H2O2 (50 μM); lane 7, DNA + H2O2 (50 μM) + 5.
incubated with pBR322 DNA for 1 h separately in 15 μL of the
two separate reaction mixtures. Both complexes caused a linear
increase in the intensity of NC DNA band accompanied with
the similar decline in SC DNA in a concentration dependent
manner in aqueous buffer solution. Complexes 4 and 5 both
converted more than 90% of SC form into NC form at a
concentration of 100 μM. Complex 4 was generated into both
nicked circular and linear forms from its SC form at a
concentration of 100 μM as depicted in Figure 5 while 100 μM
of 5 completely converted into nicked circular form as shown in
Figure 6. DNA cleavage by complex 5 is significantly higher
than that observed with complex 4, which could be related to
the presence of the electron releasing methyl group as
substituent.
Figure 9. Ethidium bromide stained agarose gel (1%) of pBR322
plasmid DNA (300 ng μL−1) in the presence of complex 5 (20 μM)
after 1 h of incubation: lane 1, DNA control; lane 2, DNA + 5; lane 3,
DNA + H2O2 (50 μM) + 5; lane 4, DNA + H2O2 (50 μM) + sodium
azide (50 μM); lane 5, DNA + H2O2 (50 μM) + 3-mercaptopropionic
acid (50 μM) + 5.
DNA Cleavage in Presence of Minor and Major
Groove Binding Agents. The potential interacting site of
the complex with supercoiled plasmid pBR322 DNA was
performed by recognition elements, minor groove binding
agent 4′,6-diamidino-2-phenylindole (DAPI) and major groove
binding agent, methyl green (MG).43 The supercoiled DNA
was treated separately with DAPI and MG prior to the addition
of the complexes 4 and 5. The degree of binding of the
complex with pBR322 DNA in the presence of DAPI and MG
is found as 79% and 21% in complex 4 and 86% and 24% in
complex 5, respectively (Figure 7).
Investigation of DNA Cleavage in Presence of
Activator and Radical Scavengers. The involvement of
reactive oxygen species (hydroxyl, singlet oxygen, and hydrogen
peroxide) in the nuclease mechanism could be inferred by
monitoring the quenching of DNA cleavage in the presence of
radical scavengers44 in the solution. Complexes 1, 2, and 3 did
not cleave DNA in the presence of scavengers like NaN3 (1O
singlet oxygen trapper), sodium formate (OH• radical
scavenger), and H2O2 (both oxidizing agent as well as reducing
agent). However, complexes 4 and 5 in the presence of H2O2
converted from I form to II form of DNA even at lower
concentration (20 μM) as depicted in Figure 8a,b. It is further
supported by the experiment using a representative complex 5
as depicted in Figure 9 (lane 4) that H2O2 in the presence of
NaN3 is unable to assist the cleavage of DNA. It is perhaps due
to trapping of O2 (H2O2 → H2O + O2) by NaN3, hence
inhibiting the oxidation of DNA bases, especially guanine.
Thus, H2O2 induced DNA cleavage in the presence of
complexes 4 and 5 following an oxidative pathway.
Topoisomerase Inhibition Assay by the Ruthenium
Complexes. Topoisomerase inhibitory reactions were per-
formed by complexes 4 and 5. Complex 5 completely inhibited
Topo II without promoting the formation of linear DNA
products, and similar results were also observed with complex 4
as depicted in Figure 10. Both Ru(II) complexes inhibited the
activity of Topo II at a low concentration (IC50 < 20 μM),
comparable to some classical Topo II inhibitors as shown in
Table 2. Similar to that described before for Topo II, the DNA
strand passage assay was also used to distinguish the effects of
Ru(II) complexes on Topo II function from their effects on
DNA topology. The religation rate of the relaxed plasmid in the
presence of Ru(II) complex 4 is slower than that of complex 5
(Supporting Information). Both complexes 4 and 5 bind to
DNA with almost the same affinity and Topo II inhibitory
activity, indicating a similar mechanism of molecular action.
DISCUSSION
■
The chalcones can coordinate with cis-Ru(DMSO)4Cl2, either
by replacing two S-bonded DMSO ligands, or one S-bonded
and one O-bonded DMSO ligands, or one DMSO and one
chloro ligand. However, owing to the facial arrangement of a
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Figure 10. Effects of different concentrations of 4 and 5 on the activity of DNA topoisomerase II.
Table 2. Inhibitory Effects of Complexes 4 and 5 on
Activities of Topoisomerase II
actions are responsible for the construction of a linear chain in
1 and 2 at a distance of 2.897 and 2.868 Å, respectively
(Supporting Information). Additionally, the crystal structures of
1 and 4 also showed extensive intermolecular C−H···Cl
interactions as depicted in the Supporting Information.
Complexes 4 and 5 showed intramolecular C−H···S
interactions at 2.85 and 2.77 Å distances, respectively
(Supporting Information). The packing diagram of complex 5
along the a-axis showed formation of a double helical
arrangement which looked like a butterfly structure (Support-
ing Information) supported by cocrystallized water and
dichloromethane molecules. The shortest interlayer distance
(distance between adjacent Ru···Ru) is observed at 10.5, 8.41,
8.56, 6.84, and 8.35 Å in complexes 1, 2, 3, 4, and 5,
respectively. These distances are found to be shorter than the
distance 12.32 Å, bringing Ru···Ru interactions. The KPI
(Kitaigorodskii packing index) values47 69.1% with 98 grid
points, 69.5% without grid point, 68.8% again without grid
points, 68.1% with 60 grid points, and 68.1% with 32 grid
points in the complexes 1, 2, 3, 4, and 5, respectively, showed
compact packing in their crystal lattice.
topoisomerase II Inhibitory activity (IC50)
ref
camptothecin
doxorubicin
novobiocin
etoposide
>100
1
60
60
60
60
32
35
18
13
complex 4
complex 5
present work
present work
chloro and O-bonded DMSO groups, they were substituted by
the bidentate deprotonated chalcones chelated as η2-L (O,O).
Two DMSO-S ligands occupy the basal plane, and one chlorine
atom and another DMSO-S were bound at axial sites and
displayed facial geometry. The unit cell of the complexes
contains two discrete molecules, arranged in a head to tail
fashion, extending along the axis. In complexes, the chelating
chalcones adopt a flattened boat conformation with the H atom
on the C1 in axial position that points toward the S-coordinated
DMSO. A computational analysis using PLATON showed that
extensive intermolecular C−H···π interactions were displayed
from the crystal structure of the complexes (Supporting
Information). A weaker molecular force, the CH/π interaction
brings the hydrogen bond between soft acids and soft bases, is
recognized to play a substantial role in a variety of chemical and
biological phenomena.45 These interactions played a significant
role in the building of a supramolecular structure (Supporting
Information).46 Intermolecular C−H(CH3)···π(phenyl) inter-
The most important features of the 1H and 13C NMR spectra
of complexes are the following: (i) the coupling constants JHα−
Hβ = ∼15 Hz in complexes indicating the presence of a trans
configuration of ethylinic double bond, while coupling
constants of aromatic protons were found in the range J =
∼7−8 Hz; (ii) the C-β carbon atom resonance (δ = ∼140.41−
143.57 ppm) appeared downfield of those of the C-α atoms (δ
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∼ 121.78 ppm), because of the mesomeric deshielding effect of
the carbonyl group.
with the Ru(II) complexes bearing a complicated skeleton of
porphyrin ligand framework.54 However, potential binding of
complex 5 with DNA could be considered in view of the
presence of an electron releasing methyl group attached to
thiophene ring in its ligand structures which probably enhances
the electron density on the surface of chalcone and
concomitantly bring its stronger overlap with symmetrical
orbital of DNA bases. As compared to a furan ring containing
system, the increase in size of the central atom of the
heterocyclic ring increases hydrophobicity and hydrogen
binding affinity of DNA together with groove binding affinity,
especially at the A−T sequence of DNA.55 Thus, as expected
the role of the thiophene ring is important in bringing the
potential candidature of both complexes 4 and 5.
The emission intensity from EB is used as a spectral probe, as
it showed enhanced emission intensity when bound to the
hydrophobic part of DNA.56 The binding of complexes to DNA
could result in the displacement of the bound EB and could
cause a decrease in emission intensity. The Ksv values obtained
using emission data were found to be similar to that obtained
from the absorption titration measurements. Complexes 4 and
5 showed the highest Ksv values. It appears that the DNA helix
simultaneously accommodates both the complex and EB in the
grooves and enhances the hydrophobicity of the thiophene ring
in the DNA-bound complex, hence perturbing the DNA helix
strongly and displacing the bound EB more efficiently than that
of other complexes.
Spectroelectrochemical Correlation. The UV−vis ab-
sorption spectroscopy and cyclic voltammetry can be
complementary probes of charge-transfer processes within
metal complexes. The MLCT bands in the UV−vis spectrum of
metal complexes arise from an electronic excitation which is
equivalent to oxidation of the metal ion and reduction of the
ligand. The energy of the MLCT transition should equal the
absolute difference in potential between corresponding
oxidation and reduction processes, as a first approximation.
Lever and co-workers48,49 have demonstrated excellent linear
correlations between the MLCT energies. The energy of the
MLCT transition could be predicted with the help of observed
electrochemical data by considering eqs 3 and 4.50
ν
= 8065(ΔE°) + 3000
(3)
MLCT
III
II
ΔE° = E° (Ru − Ru ) − E° (L)
(4)
298 298
νMLCT is the frequency of the lowest energy MLCT transition
(in cm−1). The factor 8065 in eq 3 is used to convert potential
difference, ΔE, from volt to cm−1, and the term 3000 cm−1 is of
empirical origin whereas E°298(RuIII−RuII) and E°298 are the
formal potentials (in V) of the ruthenium(III)−ruthenium(II)
couple as well as the ligand reduction potential, respectively.
The calculated and experimentally observed νMLCT transition
frequencies for the complexes are listed in Table 3. The
Cleavage Studies. There is substantial and continuous
interest in DNA endonucleolytic cleavage reactions that are
activated by metal ions. In general, the relaxed form of plasmid
DNA is generated due to the cleavage of one of the DNA
strands, known as nicking of DNA; the resultant opened
circular DNA is known as the nicked circular (form II) form
which migrated more slowly in agarose gel. If both strands are
cleaved, a linear (form III) form is generated which migrated
between I and II forms of DNA.57 Complexes 4 and 5 exhibited
good cleavage activity at 100 μM. However, in the presence of
minor groove binding agent DAPI, complexes 4 and 5 showed
intense cleavage of DNA even at 20 μM concentration. It
suggested a minor groove binding propensity. Additionally,
experiments also showed H2O2 induced DNA cleavage by
complexes 4 and 5 indicating that they followed an oxidative
pathway for the cleavage of plasmid DNA.
Table 3. Spectroelectrochemical Correlation Data for
Complexes 1−5
E298/V (ΔEp/mV)
νMLCT/cm−1
complex RuIII−RuII ligand reduction ΔEc/V
obsd
calcd
1
2
3
4
5
0.93
0.91
0.79
0.86
0.82
−1.20
−1.28
−1.24
−1.29
−1.32
2.13
2.19
2.03
2.15
2.14
20 243
20 202
19 350
19 841
19 960
20 174
20 660
19 368
20 335
20 254
calculated values lie within 900 cm−1 of the experimentally
observed energies, which were in very good agreement with the
previously observed correlation in other ruthenium com-
plexes.51
DNA Binding Studies. Generally, ruthenium complexes
form an adduct with DNA which contributes to their cytostatic
effect and brings changes in photophysical properties of
complexes which in turn have been exploited for monitoring
DNA binding properties of the complexes. Significant
hypochromicity along with a minor bathochromic shift of
MLCT band is observed, suggesting a primarily groove binding
nature of the complexes to CT DNA in buffer medium.52 Small
molecules that are known to display π stacking interaction
between two DNA base pairs are DNA intercalators which
show a much larger bathochromic shift and hypochromic effect
of the spectral bands.53 The more conjugated anthracene ring
of the ligand appended in complex 3 provides larger surface
area for the interaction with DNA, seems to facilitate partial
intercalation with base pair through DNA groove, and results in
a higher binding strength than that of complex 1 and 2. On the
other hand, complexes 4 and 5, bearing thiophene group in
their ligand skeleton, bind strongly with DNA molecule. Thus,
the DNA binding potential of these complexes containing
simple chalcone ligand framework is found to be comparable
Under physiological conditions, DNA replication, repair, and
transcription processes are significantly controlled by Topo II.58
The enzyme assists in these functions by altering the
topological properties of DNA, and it catalyzes by creating
the transient double strand breaks, transporting an intact
segment of DNA through the gap, and finally religating the
cleaved strands.59 Two distinct classes of Topo-II inhibitors
exist: (1) those that bind to and stabilize the DNA−Topo-II
cleavage complex, ultimately promoting the formation of
extremely double strand breaks (e.g., etoposide),60 and (2)
those, commonly referred as catalytic inhibitors, that antagonize
the ability of the enzyme to perform catalysis (e.g.,
merbarone).61 At low complex/DNA ratios, DNA only forms
loose aggregates (toroid with gaps), and there are still a number
of DNA binding sites in these loose structures accessible for
enzymes. However, at high complex/DNA ratios, DNA forms
compact aggregates (solid toroid or spherical aggregate), which
makes the binding site inaccessible and protects DNA from the
digestion of enzymes.62 The inhibition of topoisomerase largely
depends on the nature of the complexes and ligands, and the
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presence of uncoordinated sites in the skeleton of coordinated
ligands. Both complexes 4 and 5 bind separately to DNA with
almost similar affinity and Topo II inhibitory activity, indicating
a similar mechanism of molecular action. The precise molecular
mechanism of inhibition by these ruthenium complexes
remains unknown. It has been well-established that the redox
cycling of Fe(thiosemicarbazonato) complexes played a
significant role in their ribonucleotide reductase inhibition
and cytotoxicity.63 Indeed, similar results have recently been
reported for Cu(thiosemicarbazonato) complexes64 and for
Ru(II)(C6H6)(DMSO)Cl2 complex.17 They inhibit DNA
relaxation activity of Topo II by trapping it into a ternary
complex with DNA and cross-linking with Topo II. Ruthenium
complexes bound covalently to DNA could break the strand in
DNA by the generation of free radicals and other oxidation
products.65
(Grant 01 (2322)/ 09/EMR-II), is gratefully acknowledged and
help rendered by Prof. D. S. Pandey, Department of chemistry,
BHU Varanasi, for recording the CV.
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CONCLUSIONS
■
The present Article embodies the synthesis and characterization
of five new complexes of type cis-[Ru(DMSO)3L1−5Cl]
containing different chalcone derivatives (L1−5). The octahedral
Ru(II) center prefers the chelating (η2) binding mode of
chalcones. Cyclic voltammetry of the complexes shows that
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effect of ring conjugation was quite evident in the extent of
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the conjugation in the skeleton of corresponding ligand (3 > 2
> 1). However, complexes 4 and 5, bearing thiophene
derivatives in their structural framework, turned out to be the
most potential candidates and cleaved supercoiled pBR322
plasmid DNA efficiently following the oxidative pathway. Both
complexes prefer minor groove binding as evident from
potential cleavage of DNA in the presence of DAPI (DNA
minor groove binding agent). Both complexes 4 and 5 inhibited
Topo II activity more efficiently than those of many reported
inhibitors.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Crystallographic data in CIF format. Additional details, tables,
and figures. This material is available free of charge via the
Internet at http://pubs.acs.org. CCDC reference numbers
755185, 755186, 834036, 750139, and 78975 contain the
supplementary crystallographic data for 1, 2, 3, 4, and 5,
respectively. These data can be obtained free of charge via
http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the
Cambridge Crystallographic Data Centre, 12 Union Road,
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AUTHOR INFORMATION
Corresponding Author
*E-mail: lmishrabhu@yahoo.co.in. Phone: +91-542-6702449.
Fax: +91-542-2368127.
■
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ACKNOWLEDGMENTS
Financial support received from DBT New Delhi, India (Grant
BT/PR5910/BRB/10/406/2005), and CSIR New Delhi, India
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