Studies of ruthenium(ii)-2,2′-bisimidazole complexes on binding to G-quadruplex DNA and inducing apoptosis in HeLa cells

Article abstract of DOI:10.1039/c3nj00542a

Three ruthenium(ii) complexes [Ru(bpy)₂(biim)]²⁺ (1), [Ru(phen)₂(biim)]²⁺ (2) and [Ru(p-mopip) ₂-(biim)]²⁺ (3) (where bpy is 2,2′-bipyridine, phen is 1,10-phenanthroline, biim is 2,2′-bisimidazole and p-mopip is 2-(4-methoxyphenyl)-imidazo-[4,5f]phenanthroline), have been synthesized and characterized. The interactions of human telomeric DNA oligomers 5′-G ₃(T₂AG₃)₃-3′ (HTG21) with ruthenium(ii) complexes were investigated via UV-vis, fluorescence resonance energy transfer (FRET) melting assay, polymerase chain reaction (PCR) stop assay, and circular dichroism (CD) measurements. The results indicated that the three ruthenium(ii) complexes could stabilize the formation of human telomeric G-quadruplex DNA, and complex 2 was found to be the most efficient. In vitro cytotoxicity assay by MTT also showed that complex 2 was superior to complexes 1 and 3 in inhibiting the growth of cancer cells. Telomeric repeat amplification protocol (TRAP) showed that complexes 2 and 3 led to an inhibition of the telomerase activity, and complex 2 was the significantly better inhibitor. Flow cytometric analysis and evaluation of mitochondrial membrane potential demonstrated that complex 2 inhibited the growth of HeLa cells through induction of apoptotic cell death, as evidenced by the depletion of mitochondrial membrane potential in HeLa cells.

Full text of DOI:10.1039/c3nj00542a

NJC
View Article Online
View Journal
PAPER
0
Studies of ruthenium(II)-2,2 -bisimidazole complexes
on binding to G-quadruplex DNA and inducing
apoptosis in HeLa cells†
Cite this: DOI: 10.1039/c3nj00542a
Yu Xia, Qingchang Chen, Xiuying Qin, Dongdong Sun, Jingnan Zhang and Jie Liu*
2+
2+
2+
Three ruthenium(II) complexes [Ru(bpy)
2
(biim)] (1), [Ru(phen)
2
2
(biim)] (2) and [Ru(p-mopip) -(biim)]
0
0
(
3) (where bpy is 2,2 -bipyridine, phen is 1,10-phenanthroline, biim is 2,2 -bisimidazole and p-mopip is
2
-(4-methoxyphenyl)-imidazo-[4,5f]phenanthroline), have been synthesized and characterized. The
0
0
interactions of human telomeric DNA oligomers 5 -G (T AG ) -3 (HTG21) with ruthenium(II) complexes
3 2 3 3
were investigated via UV-vis, fluorescence resonance energy transfer (FRET) melting assay, polymerase
chain reaction (PCR) stop assay, and circular dichroism (CD) measurements. The results indicated that
the three ruthenium(II) complexes could stabilize the formation of human telomeric G-quadruplex DNA,
and complex 2 was found to be the most efficient. In vitro cytotoxicity assay by MTT also showed that
complex 2 was superior to complexes 1 and 3 in inhibiting the growth of cancer cells. Telomeric repeat
amplification protocol (TRAP) showed that complexes 2 and 3 led to an inhibition of the telomerase
activity, and complex 2 was the significantly better inhibitor. Flow cytometric analysis and evaluation of
mitochondrial membrane potential demonstrated that complex 2 inhibited the growth of HeLa cells
through induction of apoptotic cell death, as evidenced by the depletion of mitochondrial membrane
potential in HeLa cells.
Received (in Montpellier, France)
2
2nd May 2013,
Accepted 21st August 2013
DOI: 10.1039/c3nj00542a
www.rsc.org/njc
A number of small molecules have been reported to efficiently
stabilize G-quadruplex DNA, and recently some metal complexes
1
Introduction
The human telomeric DNA consists of the tandem repeat sequence as G-quadruplex DNA stabilizers were reported.10–14 The metal
[
TTAGGGCCCTAA]
n
. Several kilobases of this sequence are paired can play a major structural role in organizing the ligands into an
with a complementary strand to form duplex DNA, but about two optimal structure for G-quadruplex DNA interaction. It is likely
1
hundred bases are unpaired as a single-stranded overhang.
that such stabilization occurs due to p–p interactions between the
This single G-rich strand has the inclination to adopt higher- large aromatic ligands of the ruthenium(II) complexes and DNA
order and functionally useful four-stranded structures called guanine residues. Ruthenium(II) complexes have prominent DNA
2
15
G-quadruplexes. Recent studies suggest that the G-quadruplex binding properties.
For example, the typical ruthenium
3
2+
0
0
structures exist in vivo and such structures form in telomeric complex [Ru(bpy) (dppz)] (dppz = dipyrido[3,2-a:2 ,3 -c]phena-
DNA at specific times during the cell cycle. It has been shown zine) is known as DNA ‘‘light switch’’.
that the stabilization of G-quadruplexes can effectively inhibit tightly intercalate between the duplex DNA base pairs and
telomerase activity which is expressed in over 85% of tumor cell stabilize the DNA. Some of them have been studied as syn-
lines but in relatively few normal cell types and ultimately alter thetic restriction enzymes, nucleic acid probes, anticancer drugs,
2
4
16,17
Such complexes can
1
8
5
6
,7
19–21
telomere maintenance. Telomere maintenance is very signif- and DNA footprinting agents, etc.
icant for the unlimited proliferative potential of cancer cells.
Rickling et al. studied the
8
4+
2 2
action of the dinuclear [(tap) Ru(tpac)Ru(tap) ] complex (tapc =
Consequently, the inhibition of telomerase activity by inducing tetrapyridoacridine) with the [TTAGGG] sequence. They found
4
G-quadruplex formation is very important for developing new that the complex damaged the sequences by photobridging
9
anticancer drugs.
intramolecularly two or more G bases with the metallic [(tap)
2-
4
+
Ru(tpac)Ru(tap) ] . The photoinduced bridging process ‘‘froze’’
2
2
2
Department of Chemistry, Jinan University, Guangzhou 510632, P. R. China.
the folded G-quadruplex conformation. Thomas et al. reported
that the dinuclear tppz-based systems could bind with high
affinity to quadruplex DNA at high ionic strengths through
E-mail: tliuliu@jnu.edu.cn; Fax: +86-20-8522-1263
1
†
Electronic supplementary information (ESI) available: ESI-MS and H NMR
spectra of complexes 1, 2 and 3 (Fig. S1, S2 and S3). Absorbance spectra (lmax/nm)
2
3
6
ꢁ1
the 22-mer d(AG
3 2 3 3 3
[T AG ] )[G ] human telomeric sequence.
and DNA-binding constants K
0.1039/c3nj00542a
b
(ꢀ 10
M ) of complexes 1, 2 and 3. See DOI:
1
The international community has widely recognized that some
This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013
New J. Chem.

View Article Online
Paper
NJC
DNA oligomers HTG21 were purchased from Shanghai Sangon
Biological Engineering Technology & Services (Shanghai, China)
and used without further purification. The concentration of HTG21
was determined by measuring the absorbance at 260 nm after
melting. Single-strand molar absorptivities were calculated from
mononucleotide data using a nearest-neighbour approximation.
The formation of intramolecular G-quadruplexes was carried out as
follows: the oligonucleotide samples, dissolved in different buffers,
were heated to 90 1C for 5 min, gently cooled to room temperature,
and then incubated at 4 1C overnight. Buffer: 10 mM Tris-HCl,
pH = 7.4. Solutions of DNA in 5 mM Tris-HCl/50 mM NaCl
buffer in water gave a ratio of UV absorbance at 260 and
2
80 nm, A260/A280, of 1.9. The concentration of DNA in nucleo-
tide phosphate (NP) was determined by UV absorbance at
60 nm after 1 : 100 dilution. The molar absorptivity, e260, was
2
ꢁ1
ꢁ1
determined to be 6600 M cm . Stock solutions were stored
Scheme 1 Synthetic routes for the preparation of complexes 1, 2 and 3.
at 4 1C and used after no more than 4 days.
2.2 Physical measurements
ruthenium complexes exhibit great cytotoxicity toward cancer
cells but low toxicity to normal cells, they are easily absorbed by
Microanalysis (C, H and N) was carried out using a Perkin-
Elmer 240C elemental analyzer. Electrospray ionization mass
2
4,25
tumor tissue and rapidly excreted from the body.
It is very inspiring that the promising ruthenium anticancer
agents, [ImH][trans-RuCl (Im)(DMSO)] (NAMI-A, where DMSO =
dimethyl sulfoxide and Im = imidazole), [IndH][trans-RuCl (Ind) ]
spectrometry (ESI-MS) was performed on a LQC system (Finngan
1
MAT, USA) using CH
3
CN as the mobile phase. H NMR spectra were
4
recorded on a Varian Mercury-plus 300 NMR spectrometer with
d6-DMSO as the solvent and SiMe4 as an internal standard at
300 MHz at room temperature. All chemical shifts were given
relative to TMS (tetramethylsilane). UV-Vis spectra were measured
on a Perkin-Elmer Lambda-850 spectrophotometer. Circular
dichroism (CD) spectra were recorded on a Jasco J-810 spectro-
polarimeter.
4
2
(
(
KP1019, where Ind = indazole) and trans-[RuCl (Ind) ]IndH
4 2
2
6–28
NKP-1339),
have successfully entered clinical trials. These
reports make ruthenium complexes containing imidazole and
2
9,30
their derivative ligands draw much attention.
In the past
few years, our group has been committed to the research on
antitumor properties of ruthenium complexes including their
design, synthesis, structural modification, biological activity 2.3 Synthesis and characteristics
3
1
and mechanisms. In our previous studies, we found that
ruthenium complexes containing methylimidazole ligands,
Ruthenium(III) chloride hydrate (Alfa Aesar), ethanedial
Sigma), 1,10-phenanthroline (Sigma), and 4-methoxybenzalde-
hyde (Sigma) were used. The compounds 2,2 -bisimidazole,
(
2
+
such as [Ru(MeIm) (N–N)] [N–N = tip, iip, dppz, dpq], had
some antitumor activity. We designed three new complexes
(biim)] (1), [Ru(phen)
biim)] (3), all the three complexes have bisimidazole as the
0
4
1
2
,10-phenanthroline-5,6-dione, p-mopip and cis-[Ru(bpy) Cl ]ꢂ
2
2
2
+
2+
[
(
Ru(bpy)
2
+
2
(biim)] (2) and [Ru(p-mopip)
2
-
H O and cis-[Ru(phen) Cl ]ꢂ2H O were prepared and charac-
2
2
2
2
2
33–35
terized according to methods described in the literature.
.3.1 Synthesis of cis-[Ru(bpy) (biim)](PF . A mixture of
cis-[Ru(bpy) Cl O (0.26 g, 0.5 mmol), biim (0.067 g,
]ꢂ2H
.5 mmol), and ethylene glycol (20 mL) was refluxed for 6 h
main ligand (Scheme 1). It was well known that the H linking to
the N atom of bisimidazole may form hydrogen-bonding with
the functional groups located on the edges of DNA to favor
interaction of the main ligand, and thus such a ligand strength-
ens the DNA-binding affinity of the complex. The interaction
of human telomeric G-quadruplex DNA with the three com-
plexes has been studied. Importantly, complex 2 exhibited
potent antitumor activities and effectively inhibited telomerase
activity. The biological properties of complex 2, the most active
ruthenium complex among the three, were studied by showing
its apoptosis-inducing activities and related signaling pathways
in HeLa tumor cells.
2
2
6 2
)
2
2
2
0
under argon. The cooled reaction mixture was diluted with
water (50 mL). Saturated aqueous sodium hexafluorophosphate
solution was added under vigorous stirring, and filtered. The
dark red solid was collected and washed with small amounts of
water, and diethyl ether, then dried under vacuum, and purified
by column chromatography on alumina using acetonitrile–
toluene (12 : 1 v/v) as the eluant. The solvent was removed
3
2
under reduced pressure and red microcrystals were obtained
1
(
(
0.25 g, 60% yield). H NMR (300 MHz, [D
6
]DMSO, 25 1C): d 8.75
d, J = 7.8 Hz, 4H, bpy-H , H
0
), 8.11 (t, J = 7.5 Hz, J = 6.9 Hz,
6
6
1
2
4
(
H
H, bpy-H , H
0
), 7.84 (d, J = 6.5 Hz, 4H, bpy-H , H4 ), 7.46
0
3
3
4
2
2
Experimental
t, J = 6.5 Hz, 4H, bpy-H , H
0
), 7.56 (d, J = 6.2 Hz, 2H, biim-H ,
5
5
5
.1 Reagents and materials
All the reagents and solvents were purchased from commercial m/z = 547.1 [M] (see Fig. S1, ESI†). C26
sources and used without further purification. Human telomeric C 37.29, H 2.65, N 13.38%; found C 37.27, H 2.66, N 13.35%.
+
0
), 6.44 (d, J = 6.3 Hz, 2H, biim-H
, H
0
). MS (ESI , CH
H N Ru (547.58): calcd
22 8
CN):
5
4
4
3
+
New J. Chem.
This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013

View Article Online
NJC
Paper
2
.3.2 Synthesis of cis-[Ru(phen)
Cl
]ꢂ2H
.5 mmol), and ethylene glycol (20 mL) was refluxed for 6 h
2
(biim)](PF
6
)
2
. A mixture of concentration due to dilution at the end of each titration were
cis-[Ru(phen)
2
2
2
O (0.284 g, 0.5 mmol), biim (0.067 g, negligible.
0
under argon. The cooled reaction mixture was diluted with
water (50 mL). Saturated aqueous sodium hexafluorophosphate
solution was added under vigorous stirring, and filtered. The The fluorescent labeled oligonucleotide, F21T (5 -FAM-G
dark red solid was collected and washed with small amounts of TAMRA-3 , FAM:6-carboxyfluorescein, TAMRA:6-carboxytetra-
water, and diethyl ether, then dried under vacuum, and purified methylrhodamine), used as the FRET probe was diluted in
by column chromatography on alumina using acetonitrile– Tris-HCl buffer (10 mM, pH 7.4) containing KCl (60 mM) and
toluene (13 : 1 v/v) as the eluant. The solvent was removed then annealed by heating to 92 1C for 5 min, followed by slow
2
.5 Fluorescence resonance energy transfer (FRET)
0
[T
AG
]
-
3
2
3
3
0
under reduced pressure and red microcrystals were obtained cooling to room temperature, overnight. Fluorescence melting
1
(
0.22 g, 49% yield). H NMR (300 MHz, [D ]DMSO, 25 1C): d 8.79 curves were determined using a Bio-Rad iQ5 real time PCR
6
(
d, J = 7.7 Hz, 2H, phen-H ), 8.66 (d, J = 8.3 Hz, 2H, phen-H ), detection system, by using a total reaction volume of 20 mL,
2
9
8.35 (m, J
1
= 7.2 Hz, J
2
= 5.5 Hz, 6H, phen-H
4
, H
7
, H
5
), 8.11 with labeled oligonucleotide (1 mM) and different concentra-
(
7
d, J = 6.7 Hz, 2H, phen-H
.43 (d, J = 7.1 Hz, 2H, phen-H
biim-H , H ), 6.39 (d, J = 5.6 Hz, 2H, biim-H
CH CN): m/z = 595.2 [M] (see Fig. S2, ESI†). C30
6
), 8.01 (d, J = 7.9 Hz, 2H, phen-H
), tions of complexes in Tris-HCl buffer (10 mM, pH 7.4) contain-
8
), 7.68 (d, J = 6.2 Hz, 2H, ing KCl (60 mM). Fluorescence readings with an excitation wave
3
+
0
4 4
, H
0
). MS (ESI , at 470 nm and detection at 530 nm were taken at intervals of
5
5
+
Ru 1 1C over the range 30–95 1C, with a constant temperature being
3
H
22
N
8
(
595.62): calcd C 40.69, H 2.50, N 12.65%; found C 40.65, maintained for 30 s prior to each reading to ensure a stable
H 2.52, N 12.61%.
value. The melting of the G-quadruplex was monitored alone or
2
.3.3 Synthesis of cis-[Ru(p-mopip) (biim)](PF ) . A mix- in the presence of various concentrations of complexes. Final
2 6 2
ture of cis-[Ru(p-mopip)
(
2
Cl
2
]ꢂ2H
O (0.43 g, 0.5 mmol), biim analysis of the data was carried out by using Origin 6.0.
2
0.067 g, 0.5 mmol), and ethylene glycol (20 mL) was refluxed
for 6 h under argon. The cooled reaction mixture was diluted
with water (50 mL). Saturated aqueous sodium hexafluorophos-
phate solution was added under vigorous stirring, and filtered. The oligonucleotide HTG21 (5 -G [T AG ] -3 ) and the corre-
2.6 PCR stop assay
0
0
3 2 3 3
The dark red solid was collected and washed with small sponding complementary sequence (HTG21rev, ATCGCTTCTCG-
amounts of water, and diethyl ether, then dried under vacuum, TCCCTAACC) were used. The reactions were performed in 1 ꢀ
and purified by column chromatography on alumina using PCR buffer, containing each oligonucleotide (10 pmol each),
acetonitrile–ethanol (8 : 1 v/v) as the eluant. The solvent was dNTPs (0.16 mM), Taq polymerase (2.5 U), and different con-
removed under reduced pressure and red microcrystals were centrations of complexes. Reaction mixtures were incubated in a
1
obtained (0.32 g, 54% yield). H NMR (300 MHz, [D
6
]DMSO, thermocycler under the following cycling conditions: 94 1C for
2
5 1C): d 9.05 (d, J = 7.1 Hz, 4H, p-mopip), 8.91 (d, J = 7.8 Hz, 4H, 3 min, followed by 30 cycles of 94 1C for 30 s, 58 1C for 30 s,
p-mopip), 8.33 (m, J = 8.8 Hz, J
1
2
= 7.2 Hz, 4H, p-mopip), 8.00 and 72 1C for 30 s. PCR products were then analyzed on a
(
6
d, J = 8.5 Hz, 4H, p-mopip), 7.81 (d, J = 8.5 Hz, 4H, p-mopip), nondenaturing polyacrylamide gel (15%) in 1 ꢀ TBE and silver
+
.4–7.6 (d, J = 6.2 Hz, 4H, biim-H , H
0
, H , H
0
). MS (ESI , stained.
4
4
5
5
+
CH CN): m/z = 887.3 [M] (see Fig. S3, ESI†). C H N O Ru
3
46 34 12 2
(
887.91): calcd C 46.91, H 2.91, N 14.27%; found C 46.95,
2.7 Circular dichroism (CD)
H 2.94, N 14.22%.
CD experiments were performed on a JASCO-J810 circular
dichroism spectrophotometer. A quartz cuvette with 4 mm path
length was used for the spectra recorded over a wavelength
2.4 Absorbance spectra
Absorbance spectra titrations were carried out at room tem- range of 230–400 nm at 1 nm bandwidth, 1 nm step size, and
perature to determine the binding affinity between DNA and 0.5 s time per point. The oligomer HTG21 was diluted from
the complex. Initially, 3 mL water solutions of the blank buffer stock to the correct concentration (2 mM) in Tris-HCl buffer
and the ruthenium complex sample (10 mM) dissolved in (10 mM, pH 7.4) and then annealed by heating to 90 1C for
distilled water were placed in the reference and sample cuvettes 5 min, gradually cooled to room temperature, and incubated at
(
1.0 cm path length), respectively, and then the first spectrum 4 1C overnight. Then, CD titration was performed at a fixed
was recorded in the range of 200–600 nm. During the titration, HTG21 concentration (2 mM) with various concentrations
–10 mL aliquot of buffered DNA solution was added to each (2.0 mol equiv.) of the complexes in buffer at 25 1C. After each
1
cuvette to eliminate the absorbance of DNA, and the solutions addition of the complexes, the reaction was stirred and allowed
were mixed by repeated inversion. Complex–DNA solutions to equilibrate for at least 3 min until no elliptic changes were
were incubated for 5 min before absorption spectra were recorded. observed and a CD spectrum was collected. Buffer baseline
The titration processes were repeated until there was no change in was collected in the same cuvette and subtracted from the
the spectra for four titrations at least, indicating that binding sample spectra. Final analysis of the data was carried out using
saturation had been achieved. The changes in the metal complex Origin 6.0.
This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013
New J. Chem.

View Article Online
Paper
.8 Cell culture
NJC
2
2.11 Flow cytometric analysis
Cells were cultured in a RPMI 1640 medium supplemented with The cell cycle distribution was analyzed using flow cytometry as
heat inactivated fetal bovine serum (FBS, 10%), penicillin previously described. Cells exposed to the complexes were
ꢁ1
ꢁ1
(
100 mg mL ), and streptomycin (100 mg mL ). Cells were trypsinized and washed with PBS. After adding 70% ethanol
maintained at 37 1C in a 5% CO
was changed twice weekly.
2
incubator, and the medium and overnight fixation at ꢁ20 1C, the trypsinized cells were
stained with propidium iodide (PI) for 4 h in the dark. The DNA
content was measured using a Epics XL-MCL flow cytometer
(Beckman Coulter, Miami, FL) and the cell cycle distribution
was analyzed by MultiCycle software (Phoenix FlowSystems,
2.9 MTT assay
HeLa (human cervical cancer), A549 (human lung carcinoma)
and HepG2 (human hepatocellular liver carcinoma) cells were
grown in a RPMI 1640 medium supplemented with FBS (10%),
San Diego, CA). Apoptotic cells with hypodiploid DNA content
were measured by quantifying the sub-G1 peak in the cell cycle
pattern. Each experiment per sample was determined by recording
ꢁ
1
ꢁ1
penicillin (100 mg mL ) and streptomycin (100 mg mL ). They
were incubated at 37 1C in a humidified incubator with 5% CO
and 95% air. Cells at the exponential growth phase were diluted
10 000 events.
2
m
2.12 Evaluation of mitochondrial membrane potential (DW )
3
ꢁ1
to 2.5 ꢀ 10 cells mL with RPMI 1640, and then seeded in
The mitochondrial membrane potential was measured by the
9
6-well culture clusters (Costar) at a volume of 100 mL per well,
0
0
0
0
JC-1 (5,5 ,6,6 -tetrachloro-1,1 ,3,3 -tetraethylbenzimidazolylcarbo-
cyanine iodide) probe purchased from Beyotime Institute of
Biotechnology. Firstly, cells in 6-well plates were trypsinized and
and incubated for 24 h at 37 1C in 5% CO . Then the cells were
2
treated with various concentrations of the complex (5, 10, 25,
ꢁ
1
5
0, 75, 100, 150 and 200 mmol L ); the medium and drug-free
ꢁ
1
resuspended in 0.5 mL of PBS buffer containing 10 mg mL of
JC-1. After 10 min incubation at 37 1C in the dark, the supernatant
was immediately removed by centrifugation. After being washed
and resuspended in PBS, the stained cells were immediately
analyzed using flow cytometry. The percentage of the green
fluorescence from JC-1 monomers was used to demonstrate
control samples were prepared simultaneously. After incuba-
ꢁ
1
tion of the cells for up to 48 h, MTT (100 mL, 5 mg mL
solution was added to each well. After a further period of
incubation (4 h at 37 1C in 5% CO ) the cell lysate (100 mL)
was added to each well. After 12 h at 37 1C, the plates were
analyzed on a microplate reader at a wavelength of 570 nm
)
2
the cells that lost DCm.
(the absorbance of the complexes at this wavelength can be
neglected). The percent growth inhibitory rate of treated cells
was calculated as (Acontrol ꢁ Adrug/Acontrol ꢁ Acell-free) ꢀ 100%,
where A is the mean value calculated by using the data from
three replicate tests. The IC50 values were determined by
plotting the percentage viability versus concentration on a
logarithmic graph and reading the concentration at which 50%
of cells were viable relative to the control.
3
Results and discussion
3.1 DNA-binding studies by absorbance spectroscopy
Electronic absorbance spectroscopy is one of the most useful
ways to investigate the interactions of complexes with DNA.
Complex binding to DNA usually results in hypochromism and
red shift. The extent of the hypochromism and red shift
parallels the binding affinity. The absorbance spectra of
3
6
3
7
2.10 TRAP assay
complexes 1, 2 and 3 in the absence and presence of HTG21
The telomerase extract was prepared from HepG-2 cells. A
modified version of the TRAP assay was used in this experi-
ment. PCR was performed in a final 50 mL reaction volume
composed of a 45 mL reaction mix containing 20 mM Tris-HCl
(at a constant concentration of complexes, [Ru] = 10 mM) were
obtained. With the increase in concentration of HTG21, all the
absorbance bands of the complexes displayed clear hypochro-
mism. The hypochromism (H%), as defined by H% = 100%
(
6
3
2
pH 8.0), 50 mM deoxynucleotide triphosphates, 1.5 mM MgCl ,
ꢁ
1
(
Afree ꢁ Abound)/Afree, of MLCT bands at about 480 nm of 1, 2 and
3 mM KCl, 1 mM EDTA, 0.005% Tween 20, 20 mg mL BSA,
0
0
3 were 13%, 29% and 23% with slight red shifts, respectively
(Fig. 1 and eqn (1), ESI†). Hypochromism and red shifts
indicated strong interactions between the three complexes
.5 pmol of primer HTG21 (5 -G (T AG ) -3 ), 18 pmol of primer
3
2
0
3 3
0
TS (5 -AATCCGTCGAGCAGAGTT-3 ), 22.5 pmol of primer CXext
0
0
(5 -GTGCCCTTACCCTTACCCTTACCCTAA-3 ), 2.5 U of Taq DNA
and HTG21. The intrinsic binding constants K
b
7
of 1, 2 and 3
polymerase, and 100 ng of telomerase. Complexes or distilled
water was added at a volume of 5 mL. PCR was performed in
an Eppendorf Master cycler equipped with a hot lid and
incubated for 30 min at 30 1C, followed by 92 1C 30 s, 52 1C
6
ꢁ1
ꢁ1
were measured to be 1.15 ꢀ 10 M , 2.91 ꢀ 10 M and 1.5 ꢀ
7
ꢁ1
1
0 M , respectively, from the decay of the absorbance (see
Table S1 and Fig. S4, ESI†). The binding constant K of complex
is larger than that of complexes 1 and 3, which indicates that
complex 2 is bound to HTG21 more tightly than complexes
b
2
30 s, and 72 1C 30 s for 30 cycles. After amplification, 8 mL of
loading buffer (containing 5 ꢀ Tris-Borate-EDTA buffer (TBE
buffer), 0.2% bromophenol blue, and 0.2% xylene cyanol) was
added to the reaction. A 15 mL aliquot was loaded onto a 10%
non-denaturing acrylamide gel (19 : 1) in 1 ꢀ TBE buffer and
1
and 3.
3.2 The studies of thermodynamic stabilization
electrophoresed at 200 V for 1 h. Gels were fixed and then To evaluate the stabilization of complexes 1, 2 and 3 for
0
0
stained with AgNO
3
.
G-quadruplex F21T (sequence: 5 -FAM-G [T AG ] -TAMRA-3 ,
3 2 3 3
New J. Chem.
This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013

View Article Online
NJC
Paper
Fig. 2 (a) Melting curves of G-quadruplex F21T (1 mM) in the absence of
complexes (black) and in the presence of complex 1 (red), 2 (blue) and 3 (green)
at a concentration of 1 mM in Tris-HCl buffer (10 mM, pH 7.4) with 60 mM KCl. (b)
DNA stabilization temperature versus the concentration of complexes 1, 2 and 3
binding to F21T.
stabilization effect of these three complexes on G-quadruplex
m
F21T. The DT value of complex 2 was the highest, indicating
that complex 2 possessed a stronger stabilizing ability to the
F21T G-quadruplex than complexes 1 and 3.
3.3 Inhibition of amplification of HTG21
In order to further evaluate the ability of complexes 1, 2 and 3 to
stabilize G-quadruplex DNA, polymerase chain reaction (PCR)
stop assay was carried out to ascertain whether complexes were
Fig. 1 Absorbance spectra of complexes 1 (a), 2 (b) and 3 (c) in buffer at 25 1C in
the presence of increasing amounts of HTG21. [Ru] = 10 mM, [DNA] = 0–10 mM
from top to bottom. Arrows indicate the change in absorbance upon increasing
the DNA concentration.
0
0
bound to the test oligomer (5 -G
G-quadruplex structure. In the presence of the complexes
3
(T
2
AG
3
)
3
-3 ) and stabilized the
39
0
0
tested, the template sequence 5 -G (T AG ) -3 was induced
3
2
3 3
into a G-quadruplex structure that blocked the hybridization
0
with a complementary primer sequence. In that case, the 5 to
0
mimicking the human telomeric repeat, where FAM is the 3 primer extension by DNA Taq polymerase was arrested and
donor fluorophore 6-carboxyfluorescein and TAMRA is the the final double-stranded DNA PCR product could not be
acceptor 6-carboxytetramethylrhodamine), FRET melting detected. The inhibitory effect of 1 and 3 was gradually
3
8
experiments were carried out. Fig. 2a shows the melting enhanced as the concentrations were increased from 2.5 to
curves of G-quadruplex F21T (0.2 mM) in the presence of 1, 2 15 mM with no PCR product detected at 17.5 mM (Fig. 3).
and 3. It was clear that 1, 2 and 3 at a concentration of 1 mM Meanwhile, 2 at a concentration of 10.0 mM could completely
could raise the melting temperature of the G-quadruplex by inhibit the formation of the PCR product. The IC50 values of 1, 2
about 8 1C, 13 1C and 11 1C, respectively. Fig. 2b shows that the and 3 were estimated to be 16.28 (ꢃ1.07), 7.45 (ꢃ0.63) and
stabilization of G-quadruplex DNA depended on the concen- 13.14 (ꢃ0.83) mM, respectively. The PCR assay further indicated
tration of complexes. The results demonstrated an obvious that complex 2 is a better G-quadruplex binder.
This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013
New J. Chem.

View Article Online
Paper
NJC
Fig. 3 Dose-dependent inhibition of HTG21 PCR amplification by complexes
1
, 2 and 3.
3.4 Circular dichroism (CD) spectra
Circular dichroism (CD) spectroscopy can be used to monitor
4
0
the formation of the G-quadruplex structure. Fig. 4 displays
the CD spectra for the titration of HTG21 oligonucleotide with
increasing amounts of complexes. In the absence of salt, the CD
spectrum of the HTG21 was found to have a major positive
band at 253 nm and a positive band near 295 nm (Fig. 4a, black
line), which indicates the coexistence of a single strand, parallel
4
1
and antiparallel G-quadruplex. Upon titration with complexes
, 2 and 3 into HTG21 oligonucleotide, dramatic changes in the
1
CD spectra were observed.
As shown in Fig. 4a, the maximum at 253 nm was gradually
suppressed and shifted toward 245 nm, while the band cen-
tered at about 293 nm increased dramatically with an increase
in the concentration of 1. Meanwhile, a major negative band at
about 261 nm started to appear. The CD spectrum of this new
DNA conformation was virtually consistent with the CD spectra
of antiparallel G-quadruplexes described in previous studies,
where the major positive band was usually observed around
2
90 nm with a negative band at 265 nm and a smaller positive
4
2
band at 246 nm. As the concentration of 1 further increased
to 7 mM, a strong shoulder located near 275 nm as the spectral
characteristic of a parallel G-quadruplex appeared. These
results indicated that 1 could induce the guanine-rich DNA to
form the mixed parallel–antiparallel G-quadruplex structure. At
the same time, a strong and positive induced CD signal was
observed between 320 and 380 nm. This induced CD signal was
additional evidence for the interaction between the G-quadruplex
43
and complex 1.
The CD spectra of complex 2 titrated into the HTG21
oligonucleotide in the absence of salt was similar to, but not
identical, that of complex 1 in the wavelength region below
300 nm. In Fig. 4b, it can be seen that titration of 2 into this
DNA solution resulted in significant changes in the CD spec-
trum, including the observation of enhancement of the maxi-
mum at 290 nm and suppression of the band at 252 nm,
shifting to 260 nm, and the emergence of a strong shoulder
at 275 nm and a negative peak near 240 nm. The presence of
these signals suggested that 2 can also induce the HTG21 to
form the mixed parallel–antiparallel G-quadruplex structure.
It is worth noting that the shoulder near 275 nm induced by 2 is
much more dramatical than that induced by 1. The reason
4
4
Fig. 4 CD spectra of the HTG21 in the presence of increasing amounts of
complexes 1 (a), 2 (b) and 3 (c). [HTG21] = 2 mM, in 10 mM Tris-HCl, pH = 7.4,
the complex concentration was 0–10 mM. (d) Illustration of how three ruthenium
complexes induce single-stranded human telomeric DNA to form a mixed
seems to be that the planar arrangement of the phen rings and G-quadruplex.
New J. Chem.
This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013

View Article Online
NJC
Paper
Table 1 IC50 values (mM) of the complexes 1, 2 and 3 after 48 h treatment in
three selected cell lines
IC50 (mM)
Complexes
HeLa
A549
HepG2
1
2
3
89.4 ꢃ 2.3
13.5 ꢃ 0.5
67.2 ꢃ 1.5
7.6 ꢃ 0.4
127 ꢃ 1.5
19.2 ꢃ 0.7
32.4 ꢃ 0.9
13.6 ꢃ 0.3
103 ꢃ 1.8
29.7 ꢃ 0.6
59.1 ꢃ 0.8
26.8 ꢃ 0.5
CDDP
their appropriate spacing make 2 ideal to be stacked on top of
the guanine tetrads.
In Fig. 4c, it can be seen that the bands at 252 and 292 nm
greatly weaken with the addition of complex 3 into the same
DNA solution, without the appearance of any new bands. The
extraordinary result showed that complex 3 had no ability to
induce the formation of mixed G-quadruplex structures. The Fig. 5 Complex 2 induced apoptosis in HeLa cells. (A) The change of cell
spectral changes indicate that 1 and 2 can induce human morphology observed using an inverted microscope (ꢀ200). Cells were treated
with different concentrations of complex 2 for 48 h. (B) Complex 2 induced
telomeric DNA to form a mixed/hybrid type G-quadruplex
apoptotic morphological changes of HeLa cells. HeLa cells were treated with
structure (Fig. 4d).
complex 2 for 48 h, stained with Hoechst 33342 and photographed using
fluorescence microscopy (ꢀ400). The red arrows indicate the condensed or
3.5 In vitro cytotoxicity assay
fragmented nucleus and multi-blebbing cells, respectively.
In vitro cytotoxicity assay of complexes 1, 2 and 3 was evaluated
by means of MTT assay against three human cancer cell lines
fluorescence microscopy. Apoptotic and necrotic cells can be
distinguished from one another in fluorescence microscopy.
(using cisplatin as the positive control) including HeLa (human
4
6
cervical cancer), A549 (human lung carcinoma) and HepG2
4
5
After treatment of HeLa cells with 2 for a period of 48 h,
apoptotic bodies displaying different size and irregular morphology
were observed as shown in Fig. 5B. However, staining bright,
condensed chromatin and fragmented nuclei did not appear in
the normal control group. The cells treated with 2 did exhibit
morphologic features of apoptosis, and further quantitative analy-
sis of changes in apoptosis is necessary.
(
human hepatocellular liver carcinoma). After the cancer cells
were incubated at various concentrations of tested complexes
for 48 h, each complex exhibited different antitumor activities.
The IC50 values of three complexes and cisplatin are shown
in Table 1. The results indicated that the cancer cells tested are
susceptible to the complexes. As shown in Table 1, IC50 values
of 1 on three cell lines range from 89.4 mM to 127 mM, which
exhibited moderate cytotoxic activity. Compared to 1, the IC50
value of 3 is lower, which shows that 3 is more active in the cell
lines tested than 1. It is interesting that complex 2 exhibited the
highest antiproliferative activities in three cancer cell lines
among the three complexes, as evidenced by the lowest IC50
values. Notably, 2 exhibited a broad spectrum of inhibition
on human cancer cells, with IC50 values ranging from 13.5 to
3.6 TRAP assay
In order to examine the ability of the complexes to inhibit the
telomerase activity, the telomeric repeat amplification protocol
4
7
(
TRAP) assay was performed. Complexes 2 and 3 were tested
in this experiment. Fig. 6 shows the in vitro inhibitory effect of
and 3. The process of inhibition of telomerase activity was
2
29.7 mM, indicating the high cytotoxic effects of 2 on cancer
investigated in a dose-dependent manner, and the number of
bands obviously decreased with respect to the control, in the
drug concentration range of 1–20 mM. The two complexes tested
cells. It is also worth noting that 2 shows a distinct preference
for HeLa cells, thus HeLa cells were chosen as a cell model for
further investigation of the mechanisms underlying the anti-
proliferative action of 2.
To clarify how complex 2 affected HeLa cell growth, the
changes in cell morphology were examined using an inverted
microscope (Fig. 5A). We found that HeLa cells treated with
complex 2 for 48 h exhibited marked morphologic signs of
apoptosis in a dose-dependent manner, where lots of cells
became round, detached cells increased and adherent cells
gradually decreased, then a large number of suspended
cells appeared, accompanied with cell debris, apoptotic bodies
and other characteristics. To observe the morphologic charac-
teristics of apoptotic nuclei, HeLa cells were stained with
Hoechst 33342 after exposure to 2 (10 mM) and detected using Fig. 6 The influence of complexes 2 and 3 on the telomere activity of HeLa cells.
This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013
New J. Chem.

View Article Online
Paper
NJC
Fig. 8 Complex 2 induced the depletion of mitochondrial membrane potential
(
DC
analyzed using JC-1 flow cytometry. The number in each dot plot represents the
percentage of cells that lost DC
m
) in HeLa cells. Cells treated with different concentrations of complex 2 were
Fig. 7 Quantitative analysis of complex 2 induced apoptotic cell death (48 h) in
HeLa cells using flow cytometry. The cells treated with different concentrations of
complex 2 for 48 h were collected and stained with PI after fixation.
m
.
pathways associated with the initiation of apoptotic cascades.
Therefore, mitochondrial dysfunction was investigated by mea-
suring changes in the mitochondrial membrane potential (MMP,
led to an inhibition of the telomerase activity, but there were
differences in the extent of inhibition. Fig. 6 clearly revealed
that 2 exhibited greater telomerase inhibitory activity than
complex 3. A number of methods have been previously used
to confirm that complex 2 is a more effective stabilizer of
HTG21, and shows stronger inhibition activity against cancer
cells. According to all the above results, we can infer that
telomerase is one of the targets for inhibiting tumors induced
by the ruthenium complexes.
m
DC ) using flow cytometry after staining live cells with the
0
0
0
0
cationic dye JC-1 (5,5 ,6,6 -tetrachloro-1,1 ,3,3 -tetraethylbenzimi-
dazolylcarbocyanine iodide). JC-1 exhibits potential-dependent
accumulation in mitochondria, indicated by a fluorescence emis-
sion shift from red to green. As depicted in Fig. 8, complex 2
significantly induced a dose-dependent decline of DCm in HeLa
cells. The cell ratio of mitochondrial depolarization increased
roughly by nearly 20 times, changing from 1.1% (control) to 2.9%
(2.5 mM), then 6.9% (5 mM), finally up to 18.7% (10 mM). Its
3.7 Cell cycle arrest and induction of apoptosis
m
accumulation in HeLa cells then induced the loss of DC , led
The inhibition of cancer cell proliferation, the cessation of cell-
cycle progression and the induction of apoptosis have all been
targeted in chemotherapeutic strategies for the treatment of
cancer. We therefore evaluated whether complex 2 altered the
cell cycle of HeLa cells and induced apoptosis of HeLa cells
using flow cytometric analyses. The cell-cycle phase distribu-
tion in HeLa cells after 48 h exposure to different concentra-
tions of 2 was analysed.
to the release of mitochondrial contents and apoptosis-related
factors such as cytochrome c and other apoptosis-inducing fac-
tors, then DNA damage occurred and morphological changes
appeared. The decline of DCm further confirmed that 2 induced
apoptosis in HeLa cells through the endogenous mitochondria-
mediated pathway.
4
8
As shown in Fig. 7, treatment with 2 led to a marked dose-
dependent increase in the proportion of cells in the G0/G1
phase. There were 77.1% and 86.1% cells in G0/G1 phase after
4
Conclusions
0
In summary, three new ruthenium(II)-2,2 -bisimidazole com-
plexes have been synthesized and made to interact with human
telomeric G-quadruplex DNA. The experimental results implied
that the three complexes could bind tightly to the human
telomeric DNA. Complex 2 could significantly stabilize the
G-quadruplex structure and exhibited more efficiency in indu-
cing the formation of mixed/hybrid type G-quadruplexes com-
pared to complexes 1 and 3. This result might be explained by
the fact that its auxiliary phen ligands are available for the p–p
interactions between the aromatic phen ligands and DNA
guanine residues, meanwhile, the great steric hindrance of
the p-mopip ligands of complex 3 make them difficult to
externally bind to the stacks of guanine quartets within a
quadruplex or intercalating between the stacks. In vitro cyto-
48 h treatment with 2 at concentrations of 5 and 10 mM,
respectively, compared with 75.6% in untreated cultures; at
the same time, the proportion of cells in the G2/M phase
decreased significantly from 4.2% to 1.2%. This confirmed that
the 2-treated cells were blocked in the G0/G1-phase. The
alteration of cells in the S phase after 48 h treatment with
different concentrations of 2 was not regular. Furthermore, a
population of sub-G1-phase cells, a characteristic of apoptosis,
was increased dramatically with an increase in the concen-
tration of 2. These results indicated that cell death induced by
complex 2 is mainly caused by induction of apoptosis.
3.8 Induction of mitochondrial dysfunction
Mitochondria control the life and death of a cell, deciding the toxicity assay data show that complex 2 exhibited more potent
4
9
fate of a cell by controlling the process of apoptosis. Mito- antitumor activities against cancer cell lines, compared to com-
chondria act as a point of integration for apoptotic signals plexes 1 and 3. This result showed that the antitumor activity is
originating from both the extrinsic and intrinsic apoptotic closely related to their ability to interact with G-quadruplex DNA,
5
0
pathways. Mitochondrial dysfunction and the release of apop- and complex 2 could effectively inhibit tumor cell growth. TRAP
togenic factors are critical keys in triggering various apoptotic assay demonstrated that complex 2 exhibited stronger inhibitory
New J. Chem.
This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013

View Article Online
NJC
Paper
activity towards telomerase. Thus, the inhibition of telomerase 12 J. E. Reed, A. A. Arnal, S. Neidle and R. Vilar, J. Am. Chem.
activity by inducing G-quadruplex formation is very important
Soc., 2006, 128, 5992–5993.
for developing anticancer drugs. Complex 2 induced HeLa cells 13 N. V. Anantha, M. Azam and R. D. Sheardy, Biochemistry,
to generate a significant peak of apoptosis in the sub-G1 phase,
1998, 37, 2709–2714.
indicating that complex 2 inhibits HeLa cells proliferation by 14 H. Yu, X. Wang, M. Fu, J. Ren and X. Qu, Nucleic Acids Res.,
induction of apoptosis. Further investigation showed that the
2008, 36, 5695–5703.
treatment of the complex in HeLa cells could result in significant 15 B. M. Zeglis, V. C. Pierre and J. K. Barton, Chem. Commun.,
depletion of DC in a dose-dependent manner, and it is a
2007, 4565–4579.
relatively early event in complex 2-induced apoptosis. Taken 16 M. R. Gill and J. A. Thomas, Chem. Soc. Rev., 2012, 41,
together, these results demonstrated that complex 2 could effec-
3179–3192.
tively promote the apoptosis of tumor cells by acting on mito- 17 H. Song, J. T. Kaiser and J. K. Barton, Nat. Chem., 2012, 4,
chondrial apoptotic pathways. The present results should be of
615–620.
value in further understanding the interaction of G-quadruplexes 18 G. N. Parkinson, M. P. Lee and S. Neidle, Nature, 2002, 417,
m
with metal complexes, as well as offer valuable information for
designing new antitumor therapeutics.
876–880.
19 K. E. Erkkila, D. T. Odom and J. K. Barton, Chem. Rev., 1999,
9
9, 2777–2796.
0 C. Metcalfe and J. A. Thomas, Chem. Soc. Rev., 2003, 32,
15–224.
1 U. Schatzschneider, J. Niesel, I. Ott, R. Gust, H. Alborzinia
and S. W o¨ lfl, ChemMedChem, 2008, 3, 1104–1109.
2 S. Rickling, L. Ghisdavu, F. Pierard, P. Gerbaux, M. Surin,
P. Murat, E. Defrancq, C. Moucheron and A. Kirsch-De
Mesmaeker, Chem.–Eur. J., 2010, 16, 3951–3961.
2
2
2
Acknowledgements
2
This work was supported by the National Natural Science
Foundation of China (20871056, 21171070, 21371075), the
Planned Item of Science and Technology of Guangdong Province
(c1011220800060, c1211220800571), the Natural Science Foun-
dation of Guangdong Province and the Fundamental Research
Funds for the Central Universities.
2
2
2
3 C. Rajput, R. Rutkaite, L. Swanson, I. Haq and J. A. Thomas,
Chem.–Eur. J., 2006, 12, 4611–4619.
4 F. Linares, M. A. Galindo, S. Galli, M. A. Romero, J. A.
Navarro and E. Barea, Inorg. Chem., 2009, 48, 7413–7420.
5 O. Zava, S. M. Zakeeruddin, C. Danelon, H. Vogel, M. Gratzel
and P. J. Dyson, ChemBioChem, 2009, 10, 1796–1800.
Notes and references
1
2
3
N. W. Kim, M. A. Piatyszek, K. R. Prowse, C. B. Harley, M. D.
West, P. L. Ho, G. M. Coviello, W. E. Wright, S. L. Weinrich 26 J. M. Rademaker-Lakhai, D. van den Bongard, D. Pluim,
and J. W. Shay, Science, 1994, 266, 2011–2015.
J. Szolom ´a jer, G. Paragi, G. Batta, C. F. Guerra, F. M.
J. H. Beijnen and J. H. Schellens, Clin. Cancer Res., 2004, 10,
3717–3727.
Bickelhaupt, Z. Kele, P. P ´a d ´a r, Z. Kupih ´a r and L. Kov ´a cs, 27 F. Barragan, D. Carrion-Salip, I. Gomez-Pinto, A. Gonzalez-
New J. Chem., 2011, 35, 476–482.
C. C. Chang, I. C. Kuo, I. F. Ling, C. T. Chen, H. C. Chen,
P. J. Lou, J. J. Lin and T. C. Chang, Anal. Chem., 2004, 76,
Canto, P. J. Sadler, R. de Llorens, V. Moreno, C. Gonzalez,
A. Massaguer and V. Marchan, Bioconjugate Chem., 2012, 23,
1838–1855.
4
490–4494.
28 V. Pierroz, T. Joshi, A. Leonidova, C. Mari, J. Schur, I. Ott,
L. Spiccia, S. Ferrari and G. Gasser, J. Am. Chem. Soc., 2012,
134, 20376–20387.
4
5
6
K. Paeschke, S. Juranek, D. Rhodes and H. J. Lipps, Chromo-
some Res., 2008, 16, 721–728.
A. Paul, B. Maji, S. K. Misra, A. K. Jain, K. Muniyappa and 29 I. Ascone, L. Messori, A. Casini, C. Gabbiani, A. Balerna,
S. Bhattacharya, J. Med. Chem., 2012, 55, 7460–7471.
A. M. Burger, F. Dai, C. M. Schultes, A. P. Reszka, M. J.
F. Dell’Unto and A. C. Castellano, Inorg. Chem., 2008, 47,
8629–8634.
Moore, J. A. Double and S. Neidle, Cancer Res., 2005, 65, 30 M. Groessl, E. Reisner, C. G. Hartinger, R. Eichinger,
1
489–1496.
O. Semenova, A. R. Timerbaev, M. A. Jakupec, V. B. Arion
and B. K. Keppler, J. Med. Chem., 2007, 50, 2185–2193.
31 D. Sun, Y. Liu, D. Liu, R. Zhang, X. Yang and J. Liu,
Chem.–Eur. J., 2012, 18, 4285–4295.
7
8
Y.-P. Huang, D.-M. Kong, Q.-M. Chen, H.-X. Shen and
H.-F. Mi, New J. Chem., 2004, 28, 1488–1493.
K. Masutomi, E. Y. Yu, S. Khurts, I. Ben-Porath, J. L. Currier,
G. B. Metz, M. W. Brooks, S. Kaneko, S. Murakami, J. A. 32 S. Shi, J. Liu, J. Li, K. C. Zheng, X. M. Huang, C. P. Tan,
DeCaprio, R. A. Weinberg, S. A. Stewart and W. C. Hahn,
Cell, 2003, 114, 241–253.
C. Zhao, J. Geng, L. Feng, J. Ren and X. Qu, Chem.–Eur. J.,
L. M. Chen and L. N. Ji, J. Inorg. Biochem., 2006, 100, 385–395.
33 J. Bolger, A. Gourdon, E. Ishow and J. P. Launay, Inorg.
Chem., 1996, 35, 2937–2944.
9
2011, 17, 8209–8215.
34 W. Paw and R. Eisenberg, Inorg. Chem., 1997, 36, 2287–2293.
1
1
0 S. N. Georgiades, N. H. Abd Karim, K. Suntharalingam and 35 J. C. Xiao and J. M. Shreeve, J. Org. Chem., 2005, 70,
R. Vilar, Angew. Chem., Int. Ed., 2010, 49, 4020–4034.
3072–3078.
1 J. E. Reed, S. Neidle and R. Vilar, Chem. Commun., 2007, 36 Z.-C. Liu, B.-D. Wang, Z.-Y. Yang, Y. Li, D.-D. Qin and
366–4368.
T.-R. Li, Eur. J. Med. Chem., 2009, 44, 4477–4484.
4
This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013
New J. Chem.

View Article Online
Paper
NJC
3
3
3
4
7 A. Tarushi, J. Kljun, I. Turel, A. A. Pantazaki, G. Psomas and 44 Y. Xu, Y. Noguchi and H. Sugiyama, Bioorg. Med. Chem.,
D. P. Kessissoglou, New J. Chem., 2013, 37, 342–355. 2006, 14, 5584–5591.
8 S. Cogoi, M. Paramasivam, B. Spolaore and L. E. Xodo, 45 X. Zhang, X. Li, S. Ye, Y. Zhang, L. Tao, Y. Gao, D. Gong,
Nucleic Acids Res., 2008, 36, 3765–8010.
9 D. Sun, R. Zhang, F. Yuan, D. Liu, Y. Zhou and J. Liu, Dalton
Trans., 2012, 41, 1734–1741.
M. Xi, H. Meng, M. Zhang, W. Gao, X. Xu, Q. Guo and
Q. You, Med. Chem., 2012, 8, 1012–1025.
46 M. Takaya, M. Nomura, T. Takahashi, Y. Kondo,
K. T. Lee and S. Kobayashi, Anticancer Res., 2009, 29,
995–1000.
0 E. M. Rezler, J. Seenisamy, S. Bashyam, M. Y. Kim, E. White,
W. D. Wilson and L. H. Hurley, J. Am. Chem. Soc., 2005, 127,
9
439–9447.
47 X. Chen, J. H. Wu, Y. W. Lai, R. Zhao, H. Chao and L. N. Ji,
Dalton Trans., 2013, 42, 4386–4397.
4
1 R. Rodriguez, G. D. Pantos, D. P. Goncalves, J. K. Sanders
and S. Balasubramanian, Angew. Chem., Int. Ed., 2007, 46, 48 Y. Jin, J. Liu, W.-T. Huang, S.-W. Chen and L. Hui, Eur. J.
405–5407. Med. Chem., 2011, 46, 4056–4061.
2 J. L. Mergny and J. C. Maurizot, ChemBioChem, 2001, 2, 124–132. 49 X. Yang, L. Chen, Y. Liu, Y. Yang, T. Chen, W. Zheng, J. Liu
3 E. W. White, F. Tanious, M. A. Ismail, A. P. Reszka, and Q. Y. He, Biochimie, 2012, 94, 345–353.
S. Neidle, D. W. Boykin and W. D. Wilson, Biophys. Chem., 50 C. A. Puckett, R. J. Ernst and J. K. Barton, Dalton Trans.,
5
4
4
2007, 126, 140–153.
2010, 39, 1159–1170.
New J. Chem.
This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013