Synthesis, DNA-binding, photocleavage, cytotoxicity and antioxidant activity of ruthenium (II) polypyridyl complexes

Article abstract of DOI:10.1016/j.ejmech.2009.10.043

Two new ligands maip (1a), paip (1b) with their ruthenium (II) complexes [Ru(bpy)₂(maip)](ClO₄)₂ (2a) and [Ru(bpy)₂(paip)](ClO₄)₂ (2b) have been synthesized and characterized. The results s

Full text of DOI:10.1016/j.ejmech.2009.10.043

European Journal of Medicinal Chemistry 45 (2010) 564–571
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, DNA-binding, photocleavage, cytotoxicity and antioxidant
activity of ruthenium (II) polypyridyl complexes
,
a
b
,
a
c,
*
Yun-Jun Liu a , Cheng-Hui Zeng , Hong-Liang Huang , Li-Xin He , Fu-Hai Wu
*
*
a School of Pharmacy, Guangdong Pharmaceutical University, Guangdong, Guangzhou 510006, PR China
b School of Life Science and Biopharmaceuticals, Guangdong Pharmaceutical University, Guangdong, Guangzhou 510006, PR China
c School of Public Health, Guangdong Pharmaceutical University, Guangdong, Guangzhou 510006, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Two new ligands maip (1a), paip (1b) with their ruthenium (II) complexes [Ru(bpy)2(maip)](ClO4)2 (2a)
and [Ru(bpy)2(paip)](ClO4)2 (2b) have been synthesized and characterized. The results show that
complexes 2a and 2b interact with DNA through intercalative mode. The cytotoxicity of these
compounds has been evaluated by MTT assay. The experiments on antioxidant activity show that these
compounds exhibit good antioxidant activity against hydroxyl radical (OHꢀ).
Received 26 September 2009
Received in revised form
25 October 2009
Accepted 27 October 2009
Available online 4 November 2009
Crown Copyright Ó 2009 Published by Elsevier Masson SAS. All rights reserved.
Keywords:
Ruthenium complexes
DNA-binding
Cytotoxicity
Antioxidant activity
1. Introduction
binding and intercalation. In recent years, the antitumor activity
of Ru(II) complex has been investigated extensively [7,9]. The
Metal complexes binding nucleic acid are currently investi-
gated because of their utility as DNA structural probes, DNA foot
printing and sequence-specific cleavage agents and potential
anticancer drug [1–4]. Binding of small molecules with DNA has
been studied extensively since DNA is the material of inherence
and controls the structure and function of cells [5]. In this respect
ruthenium (II) complexes have attracted a great deal of attention
due to their strong DNA-binding and potential anticancer [6–12].
In fact, the activity of many anticancer, antimalarial and anti-
bacterical agents finds its origin in intercalative interactions with
DNA [13]. Many researchers have focused on the DNA-binding
mechanism of Ru(II) complexes with DNA. The previous studies
showed that Ru(II) complexes could bind DNA in a non-covalent
interactions fashion such as electrostatic binding, grooving
results demonstrated that some polypyridyl Ru(II) complexes
possess excellent antitumor activity toward selected tumor
cell lines and might be as potential candidate for drug. In
this study, two new ligands maip 1a (maip ¼ 2-(3-amino-
phenyl)imidazo[4,5-f][1,10]phenanthroline), paip 1b (paip ¼ 2-
(4-aminophenyl)imidazo[4,5-f][1,10]phenanthroline) and their
complexes [Ru(bpy)2(maip)](ClO4)2 2a (bpy ¼ 2,20-bipyridine)
and [Ru(bpy)2(paip)](ClO4)2 2b (Scheme 1) have been synthe-
sized and characterized by elemental analysis, FAB-MS, ES-MS
and 1H NMR. The DNA-binding behaviors were studied with
spectroscopic titration, viscosity measurements, thermal dena-
turation and photocleavage. The spectroscopic titration and
viscosity changes of calf thymus (CT-DNA) show that these
complexes interact with DNA through intercalative mode. The
studies on mechanism of photocleavage reveal that singlet
oxygen (1O2) and superoxide anion radical (O2 ꢁ) may play an
ꢀ
important role in the photocleavage. The cytotoxicity of ligands
1a, 1b and their complexes 2a and 2b have been evaluated
by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) assay. The experiments on antioxidant activity show
that ligands 1a, 1b and complexes 2a, 2b exhibit good antioxi-
dant activity against hydroxyl radical (OHꢀ).
Abbreviations: Maip, 2-(3-aminophenyl)imizado[4,5-f][1,10]phenanthroline;
paip, 2-(4-aminophenyl)imizado[4,5-f][1,10]phenanthroline; bpy, 2,20-bipyridine;
CT-DNA, calf thymus; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide; pip, 2-(20-phenyl)imizado[4,5-f][1,10]phenanthroline.
* Corresponding authors. Tel.: þ86 20 39352122; fax: þ86 20 39352128.
E-mail addresses: lyjche@163.com (Y.-J. Liu), hhongliang2004@yahoo.com.cn
(H.-L. Huang), fuhaiwu@163.com (F.-H. Wu).
0223-5234/$ – see front matter Crown Copyright Ó 2009 Published by Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.10.043

Y.-J. Liu et al. / European Journal of Medicinal Chemistry 45 (2010) 564–571
565
Scheme 1. The structure of complexes [Ru(bpy)2(maip)]2þ and [Ru(bpy)2(paip)]2þ
.
2. Results and discussion
at 460 nm for 2a and 462 nm for 2b are assigned to the metal-to-
ligand charge transfer (MLCT) transition, the bands at the range of
2.1. Synthesis and characterization
300–400 nm are attributed to
below 300 nm are attributed intraligand (IL)
p–
p* transitions, and the bands
p–
p* transitions.
Complexes 2a and 2b were prepared by heating of ligand maip
or paip with cis-[Ru(bpy)2Cl2] [14]. The ligands maip and paip were
prepared with a method similar to that described by Steck and Day
[15]. The structures of ligands and their complexes were confirmed
by elemental analysis, FAB-MS, ES-MS, and 1H NMR. For each of the
two complexes, two sets of NMR signal were observed. One set
corresponds to the ancillary ligand (bpy), and the other set corre-
sponds to the intercalative ligand (maip or paip). For maip or paip
and complexes 2a and 2b, the chemical shifts of protons on the
nitrogen atom of imidazole ring were not observed, probably
because the protons are very active and easy to be exchanged
quickly between the two nitrogens of the imidazole ring in
solution.
For metallo-intercalators, DNA-binding is associated with
hypochromism and a red shift in the MLCT and ligand bands [16].
Fig. 1 shows the absorption spectra of complexes 2a and 2b in the
presence of increasing concentration of DNA. As increasing the
concentration of CT-DNA, the MLCT transition band of complexes
2a at 460 nm and 2b at 462 nm exhibits hypochromism of 26.3%
and 30.8%, and bathochromism of 2 and 3 nm, respectively. These
spectral characteristics obviously suggest that complexes 2a and 2b
interact with DNA most likely through a mode that involves
a stacking interaction between the aromatic chromophore and the
base pairs of DNA.
In order to further elucidate the binding strength of the
complexes, the intrinsic constants Kb were determined by moni-
toring the changes of absorbance in the MLCT band with increasing
concentration of CT-DNA. The values of Kb are 2.06 ꢂ 0.22 ꢃ105
(s ¼ 2.24) and 3.12 ꢂ 0.15 ꢃ105 (s ¼ 1.76) Mꢁ1 for 2a and 2b,
respectively. These values are comparable to that of complexes
The complexes were also characterized by electrospray mass
spectrometry (ES-MS). In the ES-MS spectra for the complexes 2a
and 2b, as expected, the intense signals for [M-ClO4]þ, [M-2ClO4-
H]þ and [M-2ClO4]2þ were observed, the obtained molecular
weights are consistent with the expected values.
[Ru(bpy)2(pip)]2þ (4.70 ꢂ 0.20 ꢃ 105 Mꢁ1
)
[17], [Ru(bpy)2(m-
npip)]2þ
(2.40 ꢂ 0.30 ꢃ 105 Mꢁ1
)
and
[Ru(bpy)2(p-npip)]2þ
(8.20 ꢂ 0.50 ꢃ 105 Mꢁ1) [18], but smaller than that of complexes
[Ru(bpy)2(dppz)]2þ (4.90 ꢃ 106 Mꢁ1) [19] and [Ru(bpy)2(HBT)]2þ
(5.71 ꢂ0.20 ꢃ 107 Mꢁ1) [6] (Fig. 1).
2.2. DNA-binding studies
2.2.1. Electronic absorption spectra
The absorption spectra of the two Ru(II) complexes are charac-
terized by intense p–
p* ligand transitions in the UV and metal-to-
2.2.2. Viscosity measurements
Optical photophysical probes provide necessary but not suffi-
cient clues to support a binding mode and viscosity experiment is
considered as one of the least ambiguous and the most critical tests
ligand charge transfer (MLCT) transition in the visible region. The
absorption spectra of complexes 2a and 2b mainly consist of three
resolved bands in the range 200–600 nm. The lowest energy bands
Fig. 1. Absorption spectra of complex 2a (a) and 2b (b) in Tris–HCl buffer upon addition of CT-DNA. [Ru] ¼ 20
concentration. Plots of (3a 3f)/(3b 3f) versus [DNA] for the titration of DNA with Ru(II) complexes.
mM. Arrow shows the absorbance change upon the increase of DNA
ꢁ
ꢁ

566
Y.-J. Liu et al. / European Journal of Medicinal Chemistry 45 (2010) 564–571
intensities of complexes 2a and 2b grow to about 1.70 and 2.72
times larger than those in the absence of DNA, respectively. The
enhancement of emission intensity is indicative of binding of the
complexes to the hydrophobic pocket of DNA, since the hydro-
phobic environment inside the DNA helix reduces the accessibility
of water molecules to the complex and the complex mobility is
restricted at the binding site, leading to decrease of the vibrational
modes of relaxation.
2.2.4. Thermal denaturation
Thermal behaviors of DNA in the presence of complexes can give
an insight into their conformational changes when temperature is
raised, and offer information about the interaction strength of
complexes with DNA. The melting temperature Tm, which is
defined as the temperature where half of the total base pairs is
unbonded, is usually introduced. It is well known that when the
temperature in solution increases, the double-stranded DNA
gradually dissociates to single strands, and generates a hyper-
chromic effect on the absorption spectra of DNA base pairs
Fig. 2. Effect of increasing amounts of complexes 2a (-) and 2b (C) and [Ru(bpy)3]2þ
(:) on the relative viscosity of calf thymus DNA at 25 (ꢂ0.1) ꢄC. [DNA] ¼ 0.30 mM.
(l
¼ 260 nm). The intercalation of natural or synthesized organic-
and metallo-intercalators generally results in considerable
a
increase in the melting temperature (Tm). A large change in the Tm
of DNA is indicative of a strong interaction between the complexes
and DNA. The effect of complexes [Ru(bpy)2(maip)]2þ and
[Ru(bpy)2(paip)]2þ on the melting temperature of CT-DNA in buffer
is shown in Fig. 4. In the absence of any added complexes, the
thermal denaturation carried out for DNA gave a Tm of 68.9 ꢂ 0.2 ꢄC
under our experimental conditions. The observed melting
temperatures in the presence of complexes 2a and 2b were
77.6 ꢂ 0.2 ꢄC and 80.3 ꢂ 0.2 ꢄC, respectively. The large increases in
of the binding modes of complexes to DNA. The viscosity of a DNA
solution is sensitive to the addition of organic drugs and metal
complexes bound by intercalation. In general, a classical interca-
lation mode results in lengthening the DNA helix, as base pairs are
separated to accommodate the binding ligand, leading to increase
of DNA viscosity; a partial and/or nonclassical intercalation of
ligand could bend (or kink) the DNA helix, reduces its effective
length and, concomitantly, its viscosity [20,21]. The effects of
complexes 2a and 2b, together with [Ru(bpy)3]2þ on the relative
viscosity of rod-like DNA are shown in Fig. 2. [Ru(bpy)3]2þ has been
known to bind to DNA in electrostatic mode, it exerts essentially no
effect on DNA viscosity. On increasing the concentration of
complexes 2a and 2b, the relative viscosity of the DNA increased
steadily, this is the result of an increase in length of the DNA duplex
following intercalation. The results suggest that complexes 2a and
2b intercalate between the base pairs of CT-DNA (Fig. 2).
Tm of the two Ru(II) complexes (the D
Tm is 8.9 and 11.4 ꢄC for 2a and
2b) are comparable to that observed for Ru(II) complexes [10,22,23]
and lend strong support for intercalation into the helix of DNA. The
experimental results also indicate that complex 2b exhibits larger
DNA-binding affinity than complex 2a does.
The binding constants of complexes 2a and 2b to CT-DNA at Tm
were determined by McGee’s equation [24].
1
1
Tm0
1
Tm
R
Hm
n
ꢁ
¼
lnð1 þ KLÞ
(1)
D
2.2.3. Luminescence spectroscopic studies
where T0m and Tm are the melting temperature of CT-DNA alone and
in the presence of complex, respectively. Hm is the enthalpy of
DNA melting (per bp), R is the gas constant, K is the DNA-binding
constant at Tm, L is the free Ru(II) complex concentration, and n is
the binding site size.
The luminescence titration experiment was carried out in
aqueous solution. As shown in Fig. 3, an obvious increase in emis-
sion intensity was observed for the two complexes. As increasing
the concentration of CT-DNA, when the ratio of [DNA]/[Ru] of 9:1
for 2a and 8:1 for 2b reached a saturating value, the emission
D
Fig. 3. Emission spectra of complex 2a (a) and 2b (b) in Tris–HCl buffer in the absence and presence of CT-DNA. Arrow shows the intensity change upon increasing DNA
concentrations.

Y.-J. Liu et al. / European Journal of Medicinal Chemistry 45 (2010) 564–571
567
Fig. 4. Thermal denaturation of CT-DNA in the absence (-) and presence of complexes
2a (C) and 2b (:). [Ru] ¼ 32 M, [DNA] ¼ 80 M.
m
m
The value of
D
Hm ¼ 6.9 kcal molꢁ1 was determined by different
scanning calorimetry [20]. On the basis of the absorption spectra
titration experiment and the neighbor-exclusion principle, the
values of n for complexes 2a and 2b were 2.24 and 1.76 bp (base
pairs). The binding constants K for complexes 2a and 2b at Tm were
calculated to be 2.34 ꢃ103 and 7.54 ꢃ103 Mꢁ1
,
respectively.
According to the van’t Hoffs equations (2)–(4) [25]
ꢀ
ꢁ
ꢀ
ꢁ
K2
K1
D
H0
R
1
1
ln
¼
ꢁ
(2)
T1 T2
D
G0T ¼ ꢁRT ln K
(3)
(4)
D
GT0
¼
D
H ꢁ T
D
S0
where K1 and K2 are the DNA-binding constants of the complex at the
temperature of T1 and T2, respectively.
changes of standard enthalpy, standard free energy and standard
entropy of binding of the complex to CT-DNA. The values
D
H0, G0T and S0 are the
D
D
Fig. 5. (a) Photoactivated cleavage of pBR 322 DNA in the presence of different
concentrations of Ru(II) complexes after irradiation at 365 nm for 30 min. (b) Photo-
activated cleavage of supercoiled pBR 322 DNA by complexes 2a (20 mM) and 2b
(20 mM) in the absence and presence of different inhibitors [100 mM mannitol,
of
D
G0T,
D
H0, and
D
S0 were ꢁ73.94 kJ molꢁ1
,
ꢁ30.31 kJ molꢁ1
200 mM dimethylsulfoxide (DMSO), 1000 U mlꢁ1 superoxide dismutase (SOD), 1.2 mM
distidine] after irradiation at 365 nm for 30 min. (c) Bar diagram representation of the
effect of inhibitors on the photoactivated cleavage of complexes 2a and 2b.
and ꢁ146.4 J molꢁ1 Kꢁ1 for complex 2a, and ꢁ58.92 kJ molꢁ1
,
ꢁ31.34 kJ molꢁ1 and ꢁ92.55 J molꢁ1 Kꢁ1 for complex 2b. The nega-
tive binding free energies suggest that the sum of the free energies of
the free complexes 2a and 2b to DNA are higher than that of adduct. It
is obvious that the binding of complexes to CT-DNA is energetically
highly favorable at room temperature and the binding reaction was
enthalpically driven. The negative entropy values indicate that the
degree of freedom of the Ru(II) complexes is decreased after the
binding, and that the DNA conformational freedom is also reduced
upon complex-DNA-binding.
irradiation at 365 nm for 30 min. No obvious DNA cleavage was
observed for controls in which complex was absent, or incubation
of the plasmid with the Ru(II) complex in dark (data not presented).
With increasing concentration of the Ru(II) complexes, the amount
of Form I of pBR 322 DNA diminishes gradually, whereas that of
2.2.5. Photoactivated cleavage of pBR 322 DNA
Table 1
The cleavage reaction on plasmid DNA by Ru(II) complexes can
be monitored by agarose gel electrophoresis, when circular plasmid
DNA is subject 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). If both strands are cleaved,
a linear form (Form III) that migrates between Form I and Form II
will be generated [26]. Fig. 5a shows gel electrophoresis separation
of pBR 322 DNA after incubation with the Ru(II) complexes and
The IC50 values of 1a, 2a, 1b and 2b against selected cell lines.
Compound
IC50 (mM)
BEL-7402
C-6
hepG-2
MCF-7
1a
2a
1b
2b
83.79 ꢂ 2.96
87.00 ꢂ 3.68
35.07 ꢂ 3.70
95.33 ꢂ 2.91
20.12 ꢂ 2.35
29.12 ꢂ 3.58
31.31 ꢂ 4.33
36.22 ꢂ 2.74
22.77 ꢂ 3.10
10.26 ꢂ 2.78
40.12 ꢂ 3.46
41.06 ꢂ 2.98
36.72 ꢂ 3.63
102.01 ꢂ 4.55
26.25 ꢂ 3.12
20.78 ꢂ 2.95
28.60 ꢂ 2.34
32.76 ꢂ 3.24
23.00 ꢂ 3.54
11.34 ꢂ 2.38
cisplatin

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Y.-J. Liu et al. / European Journal of Medicinal Chemistry 45 (2010) 564–571
Fig. 6. (a) Cell viability of complex 2a (a) and 2b (b) on tumor (BEL-7402, C-6, hepG-2 and MCF-7) cell proliferation in vitro. Each data point is the mean ꢂ standard error obtained
from at least three independent experiments. (b) Light microscopy of BEL-7402 cell line after treated for 48 h in the absence I (control) and presence of different concentrations
complex 2b: II: [Ru] ¼ 6.25
m
M; III: [Ru] ¼ 12.5
mM; IV: [Ru] ¼ 25
mM; V: [Ru] ¼ 50
mM; VI: [Ru] ¼ 100
mM; VII: [Ru] ¼ 200
mM. Cell was observed using an inverted microscope and
photographed by a digital camera.
Form II increases, when concentration reached 50
m
M for 2a and 2b,
dimethylsulfoxide (DMSO), superoxide dismutase (SOD) enzyme
and histidine (Fig. 5b). The cleavage of the plasmid was not inhibited
in the presence of hydroxyl radical (OHꢀ) scavengers such as
mannitol [27] and dimethylsulfoxide [28], which indicated that
hydroxyl radical was not likely to be the cleaving agent. In the
presence of superoxide dismutase (SOD), a facile superoxide anion
radical (Oꢀ2ꢁ) quencher, the cleavage was obviously improved, which
indicated that Oꢀ2ꢁ might be an inhibitor in the photoactivated
cleavage of the plasmid and reducing the amount of Oꢀ2ꢁ can improve
DNA was completely converted from Form I to Form II. Under the
same experimental conditions, complex 2b exhibits more effective
DNA cleavage activity than complex 2a. The different cleaving
efficiency may be ascribed to the different DNA-binding affinity of
two Ru(II) complexes.
In order to observe the reactive species that are responsible for
the photoactivated cleavage of the plasmid DNA, the DNA cleavage
experiment has been carried out in the presence of the D-mannitol,

Y.-J. Liu et al. / European Journal of Medicinal Chemistry 45 (2010) 564–571
569
2.2.7. Antioxidant activity
Since complexes exhibit good DNA-binding affinity, it is
considered worthwhile to study other potential aspects of these
complexes, such as antioxidant and antiradical activity. It has been
reported that the free radical-scavenging ability of chromones is
excellently directed toward hydroxyl radical (OHꢀ) [30]. In this
paper, the antioxidant activity of the ligands 1a, 1b and complexes
2a and 2b against hydroxyl radical (OHꢀ) was investigated. As
shown in Fig. 7, the inhibitory effect of the ligands 1a, 1b and their
Ru(II) complexes 2a, 2b on OHꢀ was concentration related and the
suppression ratio increased with increasing of sample concentra-
tion in the range of 0.2–1.4
tration, the antioxidant activity of complexes 2a and 2b is higher
than that of ligands 1a and 1b, at the high concentration (1.4 M),
mM. Fig. 7 shows at the low concen-
m
the hydroxyl radical-scavenging activity of 2a and 2b is slightly
weaker than those of 1a and 1b. The antioxidant activity against
hydroxyl radical of complexes 2a and 2b is comparable under the
same experimental condition. It was believed that the information
obtained from the present work would ultimately be helpful to
develop new potential antioxidants and new therapeutic reagents
for some diseases.
Fig. 7. Scanvenging effect of the ligands and complexes on hydroxyl radicals.
(-) maip, (C) paip, (:) [Ru(bpy)2(maip)]2þ, (;) [Ru(bpy)2(paip)]2þ. Experiments
were performed in triplicate.
3. Conclusion
the cleavage effect. Superoxide dismutase strongly enhancing the
yield of cleavage has also been observed in the photoactivated
cleavage of [Ru(bpy)2(Fpp)]2þ (Fpp ¼ 2-(3,30-difluoro-3,4-methyl-
enedioxyphenyl)imidazo[4,5-f][1,10]phenanthroline) [7]. In the
presence of the singlet oxygen (1O2) scavenger histidine [29], the
DNA cleavage of the plasmid by complexes 2a and 2b was inhibited,
suggesting that 1O2 is likely to be the reactive species responsible for
the cleavage reaction. Fig. 5c shows the bar diagram representation
of the percentage of cleavage (C) for complexes 2a and 2b.
Two new Ru(II) complexes of [Ru(bpy)2(maip)]2þ and
[Ru(bpy)2(paip)]2þ have been synthesized and characterized. The
DNA-binding behaviors of two Ru(II) complexes have also been
examined by spectroscopic methods, viscosity measurement and
thermal denaturation. The results indicate that the two complexes
can intercalate into DNA base pairs via intercalative ligands. When
irradiated at 365 nm, complexes 2a and 2b can efficiently cleave the
plasmid pBR 322 DNA. The studies on mechanism of photocleavage
reveal that singlet oxygen (1O2) and superoxide anion radical (O2ꢀꢁ
)
may play an important role in the photocleavage. Cytotoxicity assay
shows that the ligands and their complexes all displayed antitumor
activity against tumor cells tested. Antioxidant activity experiments
show that ligands and their Ru(II) complexes exhibit good antiox-
idant activity against hydroxyl radical (OHꢀ).
2.2.6. In vitro cytotoxic assays
The cytotoxic activity of all these complexes and the corre-
sponding ligands against cell lines of BEL-7402 (hepatocellular), C-6
(Rat glioma), hepG-2 (hepatocellular) and MCF-7 (breast cancer)
was evaluated by MTT assay. The concentrations of them ranged
from 6.25
mM to 400
mM. The IC50 values obtained of ligands and
4. Experimental
their complexes against selected four tumor cell lines are shown in
Table 1. The resulting concentration–effect curves obtained with
continuous exposure for 48 h are depicted in Fig. 6a. After the
tumor cells were incubated in the presence of 1a, 2a, 1b and 2b for
48 h, the IC50 values for all of ligands and complexes ranged from
4.1. Material
Calf thymus DNA (CT-DNA) was obtained from the Sino-Amer-
ican Biotechnology Company. Doubly distilled water was used to
prepare buffers (5 mM Tris(hydroxymethylaminomethane)–HCl,
50 mM NaCl, pH ¼ 7.2). A solution of calf thymus DNA in the buffer
gave a ratio of UV absorbance at 260 and 280 nm of ca. 1.8–1.9:1,
indicating that the DNA was sufficiently free of protein [31]. The
DNA concentration per nucleotide was determined by absorption
spectroscopy using the molar absorption coefficient (6600
20.78 to 102.01 mM, suggesting that ligands and complexes
exhibited antitumor activity against all of these cell lines in
different degree. The IC50 values for complex 2a against cell lines of
BEL-7402, C-6, hepG-2 and MCF-7 were 87.00, 31.31, 41.06 and
28.60
mM, respectively, and the IC50 values for complex 2b were
95.33, 22.77, 102.01 and 23.00
mM. With comparison of complexes
2a and 2b, 2b appeared to be much cytotoxic against the cell lines of
C-6 and MCF-7. From IC50 values, we can conclude that when
ligands 1a and 1b bond with the Ru(II) metal center to form
complexes 2a and 2b, the antitumor activity of them has been
weakened. These compounds all exhibit relatively lower in vitro
cytotoxicity against the selected cell lines than cisplatin. Fig. 6a was
the cell viability for complexes 2a and 2b on the tumor cell lines
(BEL-7402, C-6, hepG-2 and MCF-7) in vitro. The toxicity of 2a and
2b was found to be concentration dependent, the cell viability
decreased with increasing the concentration of complexes. Fig. 6b
was the antiproliferative activity for complex 2b against tumor cells
of BEL-7402, with increasing the concentration of complex 2b (from
II to VII), the proliferation of tumor cells of BEL-7404 was effectively
inhibited.
M
ꢁ1 cmꢁ1) at 260 nm [32].
4.2. Physical measurements
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-nitro-
benzyl alcohol matrix. Electrospray mass spectra (ES-MS) were
recorded on an 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, 23.00 V and
200 ꢄC, respectively, and the quoted m/z values are for the major
peaks in the isotope distribution. 1H NMR spectra were recorded on
a Varian-500 spectrometer. All chemical shifts were given relative

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Y.-J. Liu et al. / European Journal of Medicinal Chemistry 45 (2010) 564–571
to tetramethylsilane (TMS). UV/vis spectra were recorded on
a Shimadzu UV-3101PC spectrophotometer at room temperature.
software. The percentage of cleavage (C) was calculated according to
Equation, where DI, DII and DIII are the IDVs of Form I (supercoil
form), Form II (nicking form) and Form III (linear form), respectively.
4.3. DNA-binding and photocleavage
DII þ 2DIII
C ¼
(6)
DI þ DII þ DIII
The DNA-binding and photoactivated cleavage experiments
were performed at room temperature. Buffer A [5 mM tris(hy-
droxymethyl)aminomethane (Tris)–hydrochloride, 50 mM NaCl,
pH 7.0] was used for absorption titration, luminescence titration
and viscosity measurements. Buffer B (50 mM Tris–HCl, 18 mM
NaCl, pH 7.2) was used for DNA photocleavage experiments. Buffer
C (1.5 mM Na2HPO4, 0.5 mM NaH2PO4, 0.25 mM Na2EDTA, pH 7.0)
was used for thermal DNA denaturation experiments.
4.4. Cytotoxicity assay
Standard 3-(4,5-dimethylthiazole)-2,5-diphenyltetraazolium
bromide (MTT) assay procedures were used [36]. Cells were seeded
in 96-well microassay culture plates (2 ꢃ 104 cells per well) and
incubated overnight at 37 ꢄC in a 5% CO2 incubator. Test compounds
were then added to the wells to achieve final concentrations
ranging from 10ꢁ6 to 10ꢁ4 M. Control wells were prepared by
addition of culture medium (100 mL). Wells containing culture
medium without cells were used as blanks. The plates were incu-
bated at 37 ꢄC in a 5% CO2 incubator for 48 h. Upon completion of
The absorption titrations of the complex in buffer were per-
formed using a fixed concentration (20 mM) for complex to which
increments of the DNA stock solution were added. Ru-DNA solu-
tions were allowed to incubate for 5 min before the absorption
spectra were recorded. The intrinsic binding constants K, based on
the absorption titration, were measured by monitoring the changes
of absorption in the MLCT band with increasing concentration of
DNA using the following equation [33].
the incubation, stock MTT dye solution (20
added to each well. After 4 h incubation, buffer (100
m
L, 5 mg mLꢁ1) was
L) containing
m
N,N-dimethylformamide (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 viability versus concentration on a loga-
rithmic graph and reading off the concentration at which 50% of
cells remain 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 (hepa-
tocellular), C-6 (Rat glioma), hepG-2 (hepatocellular) and MCF-7
(breast cancer) (purchased from American Type Culture Collection).
rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ꢂ
ꢃ
b ꢁ b2 ꢁ 2K2Ct½DNAꢅ=s
3a
3b
ꢁ
ꢁ
3f
3f
¼
(5a)
(5b)
2KCt
b ¼ 1 þ KCt þ K½DNAꢅ=2s
where [DNA] is the concentration of CT-DNA in base pairs, the
apparent absorption coefficients 3a 3f and 3b correspond to Aobsed
,
/
[Ru], the absorbance for the free ruthenium complex, and the
absorbance for the ruthenium complex in fully bound form,
respectively. K is the equilibrium binding constant, Ct is the total
metal complex concentration in nucleotides and s is the binding
site size.
Thermal denaturation studies were carried out with a Perkin–
Elmer Lambda 35 spectrophotometer equipped with a Peltier
temperature-controlling programmer (ꢂ0.1 ꢄC). The melting
temperature (Tm) was taken as the mid-point of the hyperchromic
transition. The melting curves were obtained by measuring the
absorbance at 260 nm for solutions of CT-DNA (80
absence and presence of the Ru(II) complex (20 M) as a function of
the temperature. The temperature was scanned from 50 to 90 ꢄC at
a speed of 1 ꢄC minꢁ1. The data were presented as (A ꢁ A0)/(Af ꢁ A0)
versus temperature, where A, A0, and Af are the observed, the initial,
and the final absorbance at 260 nm, respectively.
4.5. Scavenger measurements of hydroxyl radical (OHꢀ)
The hydroxyl radical (OHꢀ) in aqueous media was generated by
the Fenton system [37]. The solution of the tested complexes was
prepared with DMF (N,N-dimethylformamide). The 5 ml assay
mixture contained following reagents: safranin (28.5
Fe(II) (100 M), H2O2 (44.0 M), the tested compounds (0.2–
1.4
mM), EDTA-
m
m
m
M) and a phosphate buffer (67 mM, pH ¼ 7.4). The assay
m
M) in the
mixtures were incubated at 37 ꢄC for 30 min in a water bath. After
which, the absorbance was measured at 520 nm. All the tests were
run in triplicate and expressed as the mean. Ai was the absorbance
in the presence of the tested compound; A0 was the absorbance in
the absence of tested compounds; Ac was the absorbance in the
absence of tested compound, EDTA-Fe(II), H2O2. The suppression
ratio (ha) was calculated on the basis of (Ai ꢁ A0)/(Ac ꢁ A0) ꢃ 100%.
m
Viscosity measurements were carried out using an Ubbelodhe
viscometer maintained at a constant temperature at 25.0 (ꢂ0.1) ꢄC
in a thermostatic bath. DNA samples approximately 200 base pairs
in average length were prepared by sonicating in order to minimize
complexities arising from DNA flexibility [34]. Flow time was
measured with a digital stopwatch, and each sample was measured
4.6. Synthesis of ligands and complexes
4.6.1. Synthesis of 2-(3-aminophenyl)imidazo[4,5-f][1,10]phenanth-
roline (maip, 1a)
three times, and an average flow time was calculated. Data were
A mixture of 2-(3-nitrophenyl)imidazo[4,5-f][1,10]phenanthro-
line (0.205 g, 0.5 mmol) [38] was completely dissolved in ethanol
(50 cm3) with stirring for 1 h. Then the Pd/C (0.20 g, 10% Pd) and
NH2NH2$H2O (6 ml) were added in the above solution and refluxed
for 3 h. The hot solution was filtered and evaporated to remove the
solvent under reduced pressure. The red compound obtained was
washed with cool ethanol and dried at 50 ꢄC in vaccuo. Yield: 75%.
Anal. Calcd. for C19H13N5: C, 73.30; H, 4.21; N, 22.49; Found: C,
73.28; H, 4.20; N, 22.47%. FAB-MS: m/z ¼ 412 [M þ 1]þ. 1H NMR
(500 MHz, DMSO-d6): 9.04 (d, 2H, Hc, J ¼ 8.5 Hz), 9.01 (d, 2H, Ha,
J ¼ 8.4 Hz), 7.83–7.85 (m, 2H, Hb), 7.62 (s, 1H, Hg), 7.51 (d, 1H, Hd,
J ¼ 8.3 Hz), 7.25 (t, 1H, He, J ¼ 7.6 Hz), 6.75 (d, 1H, Hf, J ¼ 8.6 Hz), 4.92
(s, 2H, HNH2).
1/3
presented as (
h
/h0
)
versus binding ratio [35], where h is the
viscosity of DNA in the presence of complexes and h0 is the viscosity
of DNA alone.
For the gel electrophoresis experiment, supercoiled pBR 322
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). The samples were analyzed by electropho-
resis for 1.5 h at 80 V on a 0.8% agarose gel inTBE (89 mM Tris-borate
acid, 2 mM EDTA, pH ¼ 8.3). The gel was stained with 1
mg/ml
ethidium bromide and photographed on an Alpha Innotech IS-5500
fluorescence chemiluminescence and visible imaging system. The
integrated density values (IDV) were given by FluorChem 5500

Y.-J. Liu et al. / European Journal of Medicinal Chemistry 45 (2010) 564–571
571
4.6.2. Synthesis of 2-(4-aminophenyl)imidazo[4,5-f][1,10]phenan-
throline (paip 1b)
Foundation of Guangdong Province (2009B030803057) and
Guangdong Pharmaceutical University for financial support.
This compound was synthesized in an identical manner to that
described for 1a, with 2-(4-nitrophenyl)imidazo[4,5-f][1,10]phe-
nanthroline in place of 2-(3-nitrophenyl)imidazo[4,5-f][1,10]phe-
nanthroline. Yield: 76%. Anal. Calcd. for C19H13N5: C, 73.30; H, 4.21;
N, 22.49; Found: C, 73.27; H, 4.22; N, 22.48%. FAB-MS: m/z ¼ 412
[M þ 1]þ. 1H NMR (500 MHz, DMSO-d6): 9.02 (d, 2H, Hc, J ¼ 8.4 Hz),
8.90 (d, 2H, Ha, J ¼ 8.6 Hz), 7.98 (d, 2H, Hd, J ¼ 8.4 Hz), 7.80–7.83 (m,
2H, Hb), 6.74 (d, 2H, He, J ¼ 8.2 Hz), 5.64 (s, 2H, HNH2).
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A mixture of cis-[Ru(bpy)2Cl2]$2H2O (0.260 g, 0.5 mmol) and
maip (0.156 g, 0.5 mmol) in ethanol (30 cm3) was refluxed under
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d
9.10 (d, 2H, Hc, J ¼ 8.5 Hz), 8.87 (d, 4H, H3,3 , J ¼ 8.5 Hz), 8.23
0
0
,
(t, 2H, Ha, J ¼ 7.8 Hz), 8.12 (t, 4H, H4,4 , J ¼ 7.6 Hz), 8.06 (d, 4H, H6,6
J ¼ 8.0 Hz), 7.93 (t, 2H, Hb, J ¼ 7.5 Hz), 7.86 (d, 1H, Hd, J ¼ 8.4 Hz),
7.59–7.62 (m, 1H, He), 7.56 (s, 1H, Hg), 7.35 (t, 2H, H5, J ¼ 7.5 Hz), 7.29
0
(t, 2H, H5 , J ¼ 7.6 Hz), 6.77 (d, 1H, Hf, J ¼ 8.4 Hz), 5.46 (s, 2H, HNH2).
4.6.4. Synthesis of [Ru(bpy)2(paip)](ClO4)2 2b
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724.5 ([M-2ClO4-H]þ), 362.4 ([M-2ClO4]2þ). 1H NMR (500 MHz,
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DMSO-d6):
d
9.06 (d, 2H, Hc, J ¼ 8.6 Hz), 8.89 (d, 4H, H3,3
,
0
J ¼ 8.5 Hz), 8.24 (t, 2H, Ha, J ¼ 7.7 Hz), 8.12 (t, 4H, H4,4 , J ¼ 7.6 Hz),
0
8.00 (d, 4H, H6,6 , J ¼ 8.2 Hz), 7.94 (d, 2H, Hd, J ¼ 8.0 Hz), 7.59–7.64
0
(m, 2H, Hb), 7.36 (t, 4H, H5,5 , J ¼ 7.6 Hz), 6.78 (d, 2H, He, J ¼ 8.2 Hz),
5.78 (s, 2H, HNH2).
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.
Acknowledgements
This work is supported by the National Nature Science
Foundation of China (30800227), the Science and Technology