Synthesis, characterization, photocleavage, cytotoxicity in vitro, apoptosis, and cell cycle arrest of ruthenium(II) complexes

Article abstract of DOI:10.1080/00958972.2012.680592

Two new ruthenium(II) complexes, [Ru(dmp)₂(BHIP)]²⁺ (1) and [Ru(dmb)₂(BHIP)]²⁺ (2), were synthesized and characterized by elemental analysis, ESI-MS, and ¹H NMR. DNA-binding constants of these complex

Full text of DOI:10.1080/00958972.2012.680592

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Synthesis, characterization,
photocleavage, cytotoxicity in vitro,
apoptosis, and cell cycle arrest of
ruthenium(II) complexes
a
a
a
a
Qi-Feng Guo , Si-Hong Liu , Qing-Hua Liu , Hui-Hua Xu
,
a
a
a
a
Jian-Hua Zhao , Hai-Feng Wu , Xin-Yan Li & Jian-Wei Wang
a
Department of Orthopaedics , Guangzhou First Municipal
People's Hospital Affiliated to Guangzhou Medical College ,
Guangzhou 510180 , PR China
Published online: 03 May 2012.
To cite this article: Qi-Feng Guo , Si-Hong Liu , Qing-Hua Liu , Hui-Hua Xu , Jian-Hua Zhao ,
Hai-Feng Wu , Xin-Yan Li & Jian-Wei Wang (2012) Synthesis, characterization, photocleavage,
cytotoxicity in vitro, apoptosis, and cell cycle arrest of ruthenium(II) complexes, Journal of
Coordination Chemistry, 65:10, 1781-1791, DOI: 10.1080/00958972.2012.680592
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Journal of Coordination Chemistry
Vol. 65, No. 10, 20 May 2012, 1781–1791
Synthesis, characterization, photocleavage, cytotoxicity in vitro,
apoptosis, and cell cycle arrest of ruthenium(II) complexes
QI-FENG GUO*, SI-HONG LIU, QING-HUA LIU, HUI-HUA XU,
JIAN-HUA ZHAO, HAI-FENG WU, XIN-YAN LI and JIAN-WEI WANG
Department of Orthopaedics, Guangzhou First Municipal People’s Hospital Affiliated to
Guangzhou Medical College, Guangzhou 510180, PR China
(
Received 8 December 2011; in final form 28 February 2012)
2
þ 2þ
2 2
Two new ruthenium(II) complexes, [Ru(dmp) (BHIP)] (1) and [Ru(dmb) (BHIP)] (2), were
1
synthesized and characterized by elemental analysis, ESI-MS, and H NMR. DNA-binding
constants of these complexes with calf-thymus DNA (ct-DNA) were determined to be 2.09
)
4
ꢂ1 ꢂ1
5
ꢂ1 ꢂ1
(
ꢀ0.18) ꢁ 10 (mol L
)
(s ¼ 2.58) and 1.48 (ꢀ0.17) ꢁ 10 (mol L
(s ¼ 1.57), respectively.
Viscosity measurements show that 1 and 2 interact with ct-DNA by intercalation. Upon
irradiation at 365 nm, 1 and 2 induce cleavage of pBR322 DNA. The cytotoxicity of these
complexes was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay. The apoptosis induced by the complexes was studied by flow cytometry. The
results of the cell cycle arrest show that 2 can inhibit the proliferation of BEL-7402 cells in the
G0/G1 phase.
Keywords: Ruthenium(II) complexes; Photocleavage; Cytotoxicity; Apoptosis; Cell cycle arrest
1. Introduction
Interaction of ruthenium(II) complex with DNA has been extensively studied [1–10]. In
general, Ru(II) polypyridyl complexes bind DNA with non-covalent interactions such
as electrostatic binding, groove binding, and intercalation. Many ruthenium(II)
II
þ
complexes show unique properties, [Ru (tpy)(pic)(H O)] can induce DNA cleavage
2
2
in the presence of KHSO [11], [Ru(Melm)(iip)] [12] can induce uncoiling of calf-
2
thymus DNA (ct-DNA), [Ru(phen) (APIP)] [13], and [Ru(dmb) (pdpt)] [14] show
þ
5
þ
2þ
2
2
high antioxidant activity against hydroxyl radical. Studies on bioactivity of
þ
ruthenium(II) complexes have also been examined [15]. [(3-Py)Ru(phen) (tmopp)]
2
can effectively inhibit the proliferation of HepG-2 cells with a low IC value
5
0
ꢂ1
6
(
18.7 ꢀ 1.3 mg mL ) [16], [ꢀ -C Me RuCl(dppz)](CF SO ) shows high cytotoxicity to
6
6
3
3
ꢂ1 2þ
MCF-7 cells (IC ¼ 2.1 ꢀ 0.6 mmol L ) [17], [Ru(dip) (dcdppz)]
can induce the
apoptosis of BEL-7402 cells [18], and [Ru(bpy) (DNPIP)] suggests the antiprolifera-
5
0
2
2
þ
2
tive mechanism on HepG-2 was S-phase arrest [18]. In this article, a new ligand, BHIP,
was synthesized. This ligand has one more bromide than HPIP [19]. To evaluate the
*
Corresponding author. Email: qifengguoqfg@126.com
Journal of Coordination Chemistry
ISSN 0095-8972 print/ISSN 1029-0389 online ß 2012 Taylor & Francis
http://dx.doi.org/10.1080/00958972.2012.680592

1
782
Q.-F. Guo et al.
Scheme 1. The structures of 1 and 2.
effect of Br on bioactivity, two new ruthenium(II) polypyridyl complexes,
2
þ
[
bromo-4-hydroxyphenyl)imizado[4,5-f][1,10]phenanthroline) and [Ru(dmb) (BHIP)]
Ru(dmp) (BHIP)]
(1) (dmp ¼ 2,9-dimethyl-1,10-phenanthroline, BHIP ¼ 2-(3-
2
2
þ
2
0
0
(
2) (dmb ¼ 4,4 -dimethyl-2,2 -bipyridine, scheme 1), were synthesized and characterized
1
by elemental analysis, electrospray ionization mass spectrometry (ESI-MS), and H
NMR. Their DNA-binding behaviors were investigated by electronic absorption
titration, viscosity measurements, and photocleavage. The cytotoxicity of these
complexes against BEL-7402, Hela, MCF-7, and MG-63 cells were evaluated by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The apoptosis of
BEL-7402 cells induced by 1 and 2 was investigated by flow cytometry. The cell cycle
arrest was also studied.
2
2
. Materials and methods
.1. Synthesis of ligand and complexes
2.1.1. Synthesis of BHIP. A mixture of 1,10-phenanthroline-5,6-dione (0.315 g,
1
.5 mmol) [20], 3-bromo-4-hydroxyphenylaldehyde (0.302 g, 1.5 mmol), ammonium
acetate (2.31 g, 30 mmol), and glacial acetic acid (30 mL) was refluxed with stirring for
h. The cooled solution was diluted with water and neutralized with concentrated
2
aqueous ammonia. The precipitate was collected and purified by column chromatog-
raphy on silica gel (60–100 mesh) with ethanol as eluent to give the compound as yellow
powder. Yield: 80%. Anal. Calcd for C H N BrO: C, 58.33; H, 2.83; N, 14.32; Found
1
9
11
4
þ
1
(
%): C, 58.18; H, 2.95; N, 14.47. FAB-MS: m/z ¼ 392.3 [M þ 1] . H NMR (500 MHz,
DMSO-d ): 9.92 (d, 2 H, J ¼ 6.0 Hz), 8.89 (d, 2 H, H , J ¼ 8.0 Hz), 8.41 (d, 1H,
6
i
J ¼ 2.5 Hz), 7.22 (d, 1 H, J ¼ 6.5 Hz), 7.82 (dd, 2H, J ¼ 4.5, J ¼ 4.5 Hz), 7.15 (d, 1H,
J ¼ 8.0 Hz), 3.40 (s, 1H, H ).
O–H
þ2
2
.1.2. Synthesis of [Ru(dmp) (BHIP)] (1). A mixture of cis-[Ru(dmp) Cl ] ꢃ 2H O
2
2
2
2
(0.260 g, 0.5 mmol) [21] and BHIP (0.196 g, 0.5 mmol) in ethanol (30 mL) was refluxed
under argon for 8 h to give a clear red solution. Upon cooling, a red precipitate was

Ruthenium(II) complexes
1783
obtained by dropwise addition of saturated aqueous NaClO solution. The crude
4
product was purified by column chromatography on neutral alumina oxide with a
mixture of CH CN–toluene (3 : 1, v/v) as eluent. The red band was collected. The
3
solvent was removed under reduced pressure and a red powder was obtained. Yield:
1%. Anal. Calcd for C H BrCl N O Ru: C, 50.96; H, 3.18; N, 10.12. Found (%):
47 35 2 8 9
7
C, 50.55; H, 2.94; N, 10.51. ESI-MS [CH CN, m/z]: 907.6 ([M–2ClO –H] ), 454.5
þ
3
4
2
þ
1
(
[M–2ClO4] ). H NMR (500 MHz, DMSO-d ): ꢁ 8.89 (d, 2H, J ¼ 8.4 Hz), 8.82 (d, 2H,
6
J ¼ 8.3 Hz), 8.40 (t, 4H, J ¼ 8.0 Hz), 8.36 (d, 1H, J ¼ 2.1 Hz), 8.22 (d, 2H, J ¼ 8.8 Hz),
.08 (d, 1H, J ¼ 2.1 Hz), 7.96 (d, 2H, J ¼ 8.4 Hz), 7.43 (dd, 2H, J ¼ 5.5, J ¼ 5.5 Hz), 7.35
d, 2H, J ¼ 8.4 Hz), 7.26 (d, 2H, J ¼ 5.1 Hz), 7.08 (d, 1 H, J ¼ 8.6 Hz), 3.35 (s, 1H,
), 2.06 (s, 6H), 1.93 (s, 6H).
8
(
H
O–H
2
þ
2
.1.3. Synthesis of [Ru(dmb) (BHIP)]
2
(2). This complex was synthesized in a
manner identical to that described for 1, with cis-[Ru(dmb) Cl ] ꢃ 2H O (0.280 g,
2
2
2
0
.5 mmol) [22] in place of cis-[Ru(dmp) Cl ] ꢃ 2H O. Yield: 72%. Anal. Calcd for
2
2
2
C H BrCl N O Ru: C, 48.74; H, 3.33; N, 10.57. Found (%): C, 48.55; H, 3.44; N,
4
3
35
2
8
9
þ 2þ
0.72. ESI-MS [CH CN, m/z]: 859.3 ([M–2ClO –H] ), 430.5 ([M–2ClO ] ). H NMR
3 4 4
1
1
(
500 MHz, DMSO-d ): ꢁ 9.03 (d, 2H, J ¼ 7.6 Hz), 8.69 (d, 4H, J ¼ 8.5 Hz), 8.45 (d, 1H,
6
J ¼ 2.0 Hz), 8.16 (d, 1H, J ¼ 2.1 Hz), 8.01 (d, 2H, J ¼ 5.5 Hz), 7.87 (dd, 2H, J ¼ 5.3,
J ¼ 5.3 Hz), 7.38 (dd, 4H, J ¼ 6.4, J ¼ 5.9 Hz), 7.14 (d, 2H, J ¼ 7.0 Hz), 6.99 (d, 1H,
J ¼ 8.6 Hz), 3.35 (s, 1H, H
), 2.12 (s, 6H), 2.07 (s, 6H).
O–H
Caution: Perchlorate salts of metal compounds with organic ligands are potentially
explosive, and only small amounts of the material should be prepared and handled with
great care.
2.2. Physical measurements
ct-DNA was obtained from the Sino-American Biotechnology Company. pBR322
DNA was obtained from Shanghai Sangon Biological Engineering & Services Co., Ltd.
Dimethyl sulfoxide (DMSO) and RPMI 1640 were purchased from Sigma. Cell lines of
hepatocellular origin (BEL-7402), human epithelial carcinoma (Hela), breast cancer
(MCF-7), and human osteosarcoma (MG-63) were purchased from American Type
Culture Collection, agarose and ethidium bromide were obtained from Aldrich.
RuCl ꢃ ꢂH O was purchased from Kunming Institution of Precious Metals.
3
2
1,10-Phenanthroline was obtained from Guangzhou Chemical Reagent Factory.
Doubly-distilled water was used to prepare buffers (5 mmol L ) tris(hydroxymethy-
ꢂ1
ꢂ1
l)aminomethane-HCl (Tris-HCl), 50 mmol L NaCl, pH ¼ 7.2). A solution of ct-DNA
in the buffer gave a ratio of UV absorbance at 260 nm and 280 nm of ca 1.8ꢂ1.9 : 1,
indicating that the DNA was sufficiently free of protein [23]. The DNA concentration
per nucleotide was determined by absorption spectroscopy using the molar absorption
ꢂ1 ꢂ1 ꢂ1
coefficient (6600 (mol L
)
cm ) at 260 nm [24].
Microanalysis (C, H, and N) was carried out with a Perkin-Elmer 240Q elemental
analyzer. Fast atom bombardment (FAB) mass spectra were recorded on a VG ZAB-
HS spectrometer in a 3-nitrobenzyl alcohol matrix. ESI-MS were recorded on a LCQ
system (Finnigan MAT, USA) using methanol as mobile phase. The spray voltage, tube
lens offset, capillary voltage, and capillary temperature were set at 4.50 kV, 30.00 V,

1
784
Q.-F. Guo et al.
ꢄ
2
3.00 V, and 200 C, respectively, and the quoted m/z values are for the major peaks in
1
the isotope distribution. H NMR spectra were recorded on a Varian-500 spectrometer.
All chemical shifts were given relative to tetramethylsilane. UV-Vis spectra were
recorded on a Shimadzu UV-3101PC spectrophotometer at room temperature.
2
.3. DNA-binding and photoactivated cleavage
The DNA-binding and photoactivated cleavage experiments were performed at room
ꢂ1
ꢂ1
temperature. Buffer A (5 mmol L Tris hydrochloride, 50 mmol L NaCl, pH 7.0) was
used for absorption titration, luminescence titration, and viscosity measurements.
ꢂ1
ꢂ1
Buffer B (50 mmol L Tris-HCl, 18 mmol L NaCl, pH 7.2) was used for DNA
photocleavage experiments.
The absorption titrations of the complex in buffer were performed using a fixed
ꢂ1
concentration (20 mmol L ) for complex to which increments of the DNA stock
solution were added. Ru-DNA solutions were allowed to incubate for 5 min before
absorption spectra were recorded. The intrinsic binding constants K, based on the
absorption titration, were measured by monitoring changes in absorption at the metal-
to-ligand charge transfer (MLCT) band with increasing concentration of DNA
according to the literature [25].
Viscosity measurements were carried out using an Ubbelodhe viscometer maintained
ꢄ
at 25.0 (ꢀ0.1) C in a thermostatic bath. DNA samples approximately 200 base pairs in
average length were prepared by sonication to minimize complexities arising from DNA
flexibility [26]. The relative viscosities were measured [27, 28] and the data were treated
with the same methods reported by Cohen [29].
For the gel electrophoresis experiment, supercoiled pBR322 DNA (0.1 mg) was
treated with the Ru(II) complexes in buffer B, and the solution was then irradiated at
room temperature with a UV lamp (365 nm, 10 W) for 45 min. The samples were
analyzed by electrophoresis for 1.5 h at 80 V on a 0.8% agarose gel in TBE
ꢂ1
ꢂ1
(
89 mmol L Tris-borate acid, 2 mmol L EDTA, pH ¼ 8.3). The gel was stained
ꢂ1
with 1 mg mL ethidium bromide and photographed on an Alpha Innotech IS-5500
fluorescence chemiluminescence and visible imaging system.
2
.4. Cytotoxicity assay
Standard MTT assay procedures were used [30]. Cells were placed in 96-well microassay
3
ꢄ
culture plates (8 ꢁ 10 cells per well) and grown overnight at 37 C in a 5% CO
2
incubator. The complexes tested were dissolved in DMSO and diluted with RPMI 1640
and then added to the wells to achieve final concentrations ranging from 10 to
1 mol L . Control wells were prepared by addition of culture medium (100 mL).
Wells containing culture medium without cells were used as blanks. The plates were
incubated at 37 C in a 5% CO incubator for 48 h. Upon completion of the incubation,
2
stock MTT dye solution (20 mL, 5 mg mL ) was added to each well. After 4 h
ꢂ6
ꢂ4
ꢂ1
0
ꢄ
ꢂ1
incubation, buffer (100 mL) containing DMF (50%) and sodium dodecyl sulfate (20%)
was added to solubilize the MTT formazan. The optical density of each well was then
measured on a microplate spectrophotometer at a wavelength of 490 nm. The IC50
values were determined by plotting the percentage of viability versus concentration on a
logarithmic graph and reading off the concentration at which 50% of the cells remain

Ruthenium(II) complexes
1785
viable relative to the control. Each experiment was repeated at least three times to
get the mean values. Four different tumor cell lines were the subjects of this study:
BEL-7402, Hela, MCF-7, and MG-63 (purchased from American Type Culture
Collection).
2.5. Apoptotic assay by flow cytometry
6
After chemical treatment, 1 ꢁ 10 cells were harvested, washed with phosphate buffer
ꢄ
saline (PBS), then fixed with 70% ethanol, and finally maintained at 4 C for at least
1
5
1
2 h. Then the pellets were stained with the fluorescent probe solution containing
ꢂ1
ꢂ1
0 mg mL propidium iodide (PI) and 1 mg mL annexin in PBS on ice in the dark for
5 min. Then the fluorescence emission was measured at 530 nm and 575 nm (or
equivalent) using 488 nm excitation by a FACS Calibur flow cytometer (Beckman
Dickinson & Co., Franklin Lakes, NJ). A minimum of 10,000 cells were analyzed per
sample.
2
.6. Cell cycle arrest
BEL-7402 cells were seeded into six-well plates (Costar, Corning Corp, New York) at a
5
density of 2 ꢁ 10 cells per well and incubated for 24 h. The cells were cultured in RPMI
ꢄ
1640 supplemented with 10% of fetal bovine serum and incubated at 37 C and 5%
CO . The medium was removed and replaced with medium (final DMSO concentration,
2
ꢂ1
1
% v/v) containing the complex (25 mmol L ). After incubation for 24 h, the cell layer
was trypsinized and washed with cold PBS and fixed with 70% ethanol. Twenty mL of
ꢂ1
ꢂ1
RNAse (0.2 mg mL ) and 20 mL of PI (0.02 mg mL ) were added to the cell
ꢄ
suspensions and incubated at 37 C for 30 min. Then the samples were analyzed by a
FACS Calibur flow cytometer (Beckman Dickinson & Co., Franklin Lakes, NJ). The
4
number of cells analyzed for each sample was more than 10 [31].
3
3
. Results and discussion
.1. Synthesis and characterization
BHIP was synthesized with a method similar to that described by Steck and Day [32].
Complexes 1 and 2 were prepared by refluxing BHIP with cis-[Ru(dmp) Cl ] ꢃ 2H O or
2
2
2
cis-[Ru(dmb) Cl ] ꢃ 2H O in ethanol. The structures of the ligand and its complexes
2
2
2
1
were confirmed by elemental analysis, FAB-MS, ES-MS, and H NMR. The chemical
shifts of protons on nitrogen of imidazole were not observed, probably because the
protons are very active and easily exchanged between the two nitrogen atoms of
imidazole in solution. In the ESI-MS spectra for 1 and 2, as expected, intense signals for
þ 2þ
M–2ClO –H] and [M–2ClO ] were observed; the obtained molecular weights were
4 4
[
consistent with the expected values.

1
786
Q.-F. Guo et al.
Figure 1. Absorption spectra of complexes in Tris-HCl buffer upon addition of ct-DNA in the presence of
ꢂ1
(
a) 1 and (b) 2. [Ru] ¼ 20 mmol L . Arrows show the absorbance change upon increase of DNA
a f b f
concentration. Plots of (" –" )/(" –" ) vs. [DNA] for titration of DNA with Ru(II) complexes.
3.2. Electronic absorption titration
In the presence of increasing amounts of ct-DNA, absorption spectra of 1 and 2 are
shown in figure 1. The hypochromism of the MLCT band of 1 at 468 nm and 2 at 467 nm
upon binding to DNA are 26.78% and 28.19% with 2 and 3 nm red shifts, respectively.
These spectral characteristics suggest that these complexes interact with DNA most
likely through a mode that involves a stacking interaction between the aromatic
chromophore and the base pairs of DNA. DNA-binding constants were obtained by
monitoring the changes in absorbance at the MLCT band with increasing concentration
4
of DNA. The intrinsic binding constants were determined to be 2.09 (ꢀ0.18) ꢁ 10
ꢂ1 ꢂ1
5
ꢂ1 ꢂ1
)
(
values are smaller than those of [Ru(bpy) (dppz)]
mol L
)
(s ¼ 2.58) and 1.48 (ꢀ0.17) ꢁ 10 (mol L
(s ¼ 1.57), respectively. These
ꢂ1 ꢂ1
2
þ
6
(410 (mol L
)
) [33], but
2
2
þ
comparable to those of DNA intercalators [Ru(dmp) (HAPIP)]
hydroxyl-5-aminophenyl)imidazo[4,5-f][1,10]phenanthroline, 3.30 ꢁ 10 (mol L
(HAPIP ¼ 2-(2-
2
4
ꢂ1 ꢂ1
) )
2
þ
[
34], [Ru(dmb) (BFIP)]
2
(BFIP ¼ 2-benzo[b]furan-2-yl-1H-imidazo[4,5-f][1,10]phe-
4
ꢂ1 ꢂ1 2þ
nanthroline, 3.20 ꢁ 10 (mol L
)
)
[35], and [Ru(dmb) (ITAP)]
(ITAP ¼
ꢂ1 ꢂ1
) ) [36]. The difference
2
4
Isatino[1,2-b]-1,4,8,9-tetraazatriphenylene, 4.50 ꢁ 10 (mol L
between the two intrinsic constants is caused by different ancillary ligands. Complex 1
shows less binding strength to ct-DNA. Substitution on 2- and 9-positions of the
ancillary phen ligands must cause severe steric constraints near the core of Ru(II) when
the complex intercalates into the DNA base pairs. The methyl groups may come into
close proximity of base pairs at the intercalative site. These steric clashes prevent the
complex from intercalating effectively, decreasing the intrinsic constant. Such steric
0
interactions would not be present with substitution on the 4- and 4 -positions of ancillary
bpy ligands [37].
3
.3. Viscosity measurements
Changes in relative viscosity of DNA solutions have proven useful for assignment of
mode of binding to DNA. The relative change in viscosity was measured using ct-DNA

Ruthenium(II) complexes
1787
ꢄ
Figure 2. Effect of increasing amounts of 1 (ꢅ) and 2 (g) on the relative viscosity of ct-DNA at 25 (ꢀ0.1) C.
ꢂ1
[
DNA] ¼ 0.25 mmol L
.
Figure 3. Photoactivated cleavage of pBR322 DNA in the presence of different complexes upon irradiation
at 365 nm for 45 min.
with increasing concentrations of 1 and 2. The effect of 1 and 2 on the relative viscosity
of rod-like DNA are shown in figure 2. On increasing concentrations of 1 and 2, the
relative viscosity of DNA solution increased steadily. These results suggest that 1 and 2
intercalate between the base pairs of ct-DNA. Enhancements of the relative viscosity of
DNA follow the order 2 4 1, consistent with the DNA-binding affinities.
3
.4. Photocleavage of pBR322 DNA
When circular plasmid DNA is subjected to electrophoresis, relatively fast migration
will be observed for the intact supercoil form (Form I); if scission occurs on one strand
(
(
nicking), the supercoil will relax to generate a slower-moving open circular form
Form II) [38]. The gel electrophoresis separation of pBR322 DNA after incubation
with the Ru(II) complexes and irradiation at 365 nm for 45 min is shown in figure 3. No
obvious DNA cleavage was observed for controls in which complexes were absent or
incubation of the plasmid with the Ru(II) complex in dark. Both complexes exhibited
concentration-dependent, single-strand cleavage of supercoiled Form I to nicked Form
II. At increasing concentration of the Ru(II) complexes, the amount of Form I of
pBR322 DNA diminishes gradually, whereas that of Form II increases. Comparing the

1
788
Q.-F. Guo et al.
Figure 4. Cell viability of 1 and 2 on BEL-7402 (a), Hela (b), MCF-7 (c), and MG-63 (d) cell proliferation
in vitro. Each point is obtained from three independent experiments with the mean ꢀ standard error.
effect on cleavage of pBR322 DNA, 2 exhibits more effective DNA cleavage than 1.
This may be attributed to the different DNA-binding affinity of two Ru(II) complexes.
3
.5. Cytotoxicity in vitro
The cytotoxicity in vitro of Ru(II) complexes was determined against BEL-7402, Hela,
MCF-7, and MG-63 cell lines using the MTT assay. Due to low aqueous solubility,
1
and 2 were dissolved in DMSO and blank samples containing the same amount of
DMSO were used as controls. The cell viability is depicted in figure 4. The cytotoxicity
of complexes was concentration-dependent. The cell viability decreased with increasing
concentrations of 1 and 2. The IC50 values were calculated (table 1). Both complexes
demonstrate high in vitro cytotoxicity against selected tumor cell lines. Comparing IC50
values of 1 and 2, 2 appeared to be more active than 1 against all the cell lines,
consistent with the DNA-binding affinities of 1 and 2. Comparing the IC values of
5
0
2
þ
ꢂ1
these complexes with that of [Ru(phen) (HPIP)] (4100 mmol L ) on BEL-7402 cells
2
[
19], owing to the Br in HPIP, the cytotoxicities of 1 and 2 were largely enhanced.
3
.6. Apoptosis assay by flow cytometer
In order to gain insight into the type of cell death induced by 1 and 2, apoptotic assays
on BEL-7402 cells were investigated by cell apoptosis analyses and the percentage of

Ruthenium(II) complexes
1789
Table 1. The IC50 values of 1 and 2 against selected cell lines.
ꢂ1
IC50 (mmol L
Hela
)
Complex
BEL-7402
MCF-7
MG-63
1
2
25.5
16.9
27.8
13.2
39.4
16.5
19.4
17.1
Figure 5. Cell cycle distribution of BEL-7402 cells as analyzed by FACS calibur flow cytometry.
Control (a), exposed to 2 (25 mmol L , (b)) for 24 h.
ꢂ1
apoptotic and necrotic cells determined by flow cytometry. In the control (a), the
percentage of apoptotic (A) and necrotic (N) cells were 0.20% and 0.09%, respectively.
In the presence of 1 (b) and 2 (c) (Supplementary material), the percentage of apoptosis
(
0
A) and necrotosis (N) of BEL-7402 cells was 37.64% and 7.33% for 1 and 16.52% and
ꢂ1
.46% for 2. Cell apoptosis increased by 37.44% and 16.32% at 25 mmol L . However,
the ratios of apoptosis versus necrotosis are 5.14 and 35.91 for 1 and 2, respectively.
Comparing the ratios under identical conditions, 2 shows higher apoptotic effect than 1.
These results are consistent with those obtained from the IC50 values.
3
.7. Cell cycle arrest
Studies on ruthenium complex-induced anti-proliferative action on cancer cells have
attracted much attention [39–41]. Complex 2 was chosen for investigating cell cycle
arrest as it showed higher cytotoxicity than 1 towards all cell lines. Induction of
apoptosis of cells can inhibit cancer cell proliferation. After treatment of BEL-7402 cells
with 2 for 24 h, the cell cycle of BEL-7402 cells was investigated by flow cytometry in
PI-stained cell method. Figure 5 shows that treatment of BEL-7402 cells with 2 caused
an increase at G0/G1 phase, accompanied by corresponding reduction in the percentage
of cells in S phase. The percentage of enhancement in G0/G1 phase was 7.32%. In
addition, the percentage of apoptosis (sub-G1) also shows an enhancement of 21.33%.

1
790
Q.-F. Guo et al.
These data suggest that the antiproliferative mechanism on BEL-7402 cells was a G0/G1
phase arrest and apoptosis.
4
. Conclusions
2þ
Two new ruthenium(II) complexes, [Ru(dmp) (BHIP)] (1) and [Ru(dmb) (BHIP)]
2
þ
2
2
(
2), were synthesized and characterized. Viscosity measurements suggest that 1 and 2
interact with ct-DNA by intercalation. Under identical conditions, 2 showed more
effective DNA cleavage than 1. The results from cytotoxicity assay show that 2 exhibits
higher cytotoxic activity than 1 against selected tumor cell lines. The apoptotic assay
demonstrates that 2 showed more effective apoptosis of BEL-7402 cells than 1. For 1
and 2, bioactivities are consistent with the DNA-binding affinities. Additionally, 2 can
inhibit the proliferation of BEL-7402 cells in the G0/G1 phase.
Acknowledgments
This work was supported by the Science and Technology Foundation of Guangdong
Province (No. 2010B31500005), Science and Technology Planning Project Pillar
Program of Guangzhou Municipality (No. 2010 J-E021-1), and Medical Scientific
Research Foundation of Guangzhou Municipality (201102A212029) of China.
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