Novel cyclometalated Ru(II) complexes containing isoquinoline ligands: Synthesis, characterization, cellular uptake and in vitro cytotoxicity

Article abstract of DOI:10.1016/j.ejmech.2020.112562

Two novel cyclometalated Ru(II) complexes containing isoquinoline ligand, [Ru(bpy)₂(1-Ph-IQ)](PF₆), (bpy = 2,2′-bipyridine; 1-Ph-IQ = 1-phenylisoquinoline; RuIQ-1) and [Ru(phen)₂(1-Ph-IQ)](PF₆) (phen = 1,10-phenanthroline; RuIQ-2) were found to show high cytotoxic activity against NCI–H460, A549, HeLa and MCF-7 cell lines. Notably, both of them exhibited IC₅₀ values that were an order of magnitude lower than those of clinical cisplatin and two structurally similar Ru(II)-isoquinoline complexes [Ru(bpy)₂(1-Py-IQ)](PF₆)₂ (Ru3) and [Ru(phen)₂(1-Py-IQ)](PF₆)₂ (Ru4) (1-Py-IQ = 1-pyridine-2-yl). The cellular uptake and intracellular localization displayed that the two cyclometalated Ru(II) complexes entered NCI–H460 cancer cells dominantly via endocytosis pathway, and preferentially distributed in the nucleus. Further investigations on the apoptosis-inducing mechanisms of RuIQ-1 and RuIQ-2 revealed that the two complexes could cause S, G2/M double-cycle arrest by regulating cell cycle related proteins. The two complexes also could reduce the mitochondrial membrane potential (MMP), promote the generation of intracellular ROS and trigger DNA damage, and then lead to apoptosis-mediated cell death. More importantly, RuIQ-2 exhibits low toxicity both towards normal HBE cells in vitro and zebrafish embryos in vivo. Accordingly, the developed complexes hold great potential to be developed as novel therapeutics for effective and low-toxic cancer treatment.

Full text of DOI:10.1016/j.ejmech.2020.112562

Journal Pre-proof
Novel cyclometalated Ru(II) complexes containing isoquinoline ligands: Synthesis,
characterization, cellular uptake and in vitro cytotoxicity
Jincan Chen, Jie Wang, Yuanyuan Deng, Baojun Li, Chengpeng Li, Yuxue Lin,
Dongbin Yang, Huanyun Zhang, Lanmei Chen, Tao Wang
PII:
S0223-5234(20)30534-1
DOI:
https://doi.org/10.1016/j.ejmech.2020.112562
EJMECH 112562
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 April 2020
Revised Date: 29 May 2020
Accepted Date: 10 June 2020
Please cite this article as: J. Chen, J. Wang, Y. Deng, B. Li, C. Li, Y. Lin, D. Yang, H. Zhang, L.
Chen, T. Wang, Novel cyclometalated Ru(II) complexes containing isoquinoline ligands: synthesis,
characterization, cellular uptake and in vitro cytotoxicity, European Journal of Medicinal Chemistry,
https://doi.org/10.1016/j.ejmech.2020.112562.
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Graphical Abstract
Novel cyclometalated Ru(II) complexes containing isoquinoline
ligands: synthesis, characterization, cellular uptake and in vitro
cytotoxicity
The cellular uptake, in vitro cytotoxicities, in vivo toxicity, cell cycle arrest and
apoptosis-inducing mechanism of two new cyclometalated ruthenium(II)-isoquinoline
complexes have been extensively explored by ICP-MS, MTT assay, flow cytometry,
zebrafish embryos model, inverted fluorescence microscope as well as western blot
experimental techniques.

Novel cyclometalated Ru(II) complexes containing isoquinoline
ligands: synthesis, characterization, cellular uptake and in vitro
cytotoxicity
a
a
a
a
b
a
Jincan Chen, Jie Wang, Yuanyuan Deng, Baojun Li, Chengpeng Li, Yuxue Lin,
c
c
a*
d,e*
Dongbin Yang, Huanyun Zhang, Lanmei Chen, Tao Wang,
a
Guangdong Key Laboratory for Research and Development of Nature Drugs, School of
Pharmacy, Guangdong Medical University, Zhanjiang, 524023, China
b
The Public Service Platform of South China Sea for R&D Marine Biomedicine Resources,
Marine Biomedical Research Institute, Guangdong Medical University, Zhanjiang, 524023, China
c
The Affiliated People’s Hospital of Hebi of Henan University, Hebi, 456030, China
d
The College of Nursing and Health, Zhengzhou University, Zhengzhou, 450001, China
e
Centre for Comparative Genomics, Murdoch University, Perth, WA 6150, Australia
*
Corresponding
author.
E-mail
addresses:
lanmeichen@126.com
(L.M.
Chen),
Tao.Wang@murdoch.edu.au (T. Wang)
Abstract
Two novel cyclometalated Ru(II) complexes containing isoquinoline ligand,
Ru(bpy) (1-Ph-IQ)](PF ), (bpy = 2,2’-bipyridine; 1-Ph-IQ= 1-phenylisoquinoline;
[
2
6
RuIQ-1) and [Ru(phen) (1-Ph-IQ)](PF ) (phen = 1,10-phenanthroline; RuIQ-2) were
2
6
found to show high cytotoxic activity against NCI-H460, A549, HeLa and MCF-7 cell
lines. Notably, both of them exhibited IC values that were an order of magnitude
5
0
lower than those of clinical cisplatin and two structurally similar Ru(II)
-isoquinoline
complexes [Ru(bpy) (1-Py-IQ)](PF ) (Ru3) and [Ru(phen) (1-Py-IQ)](PF ) (Ru4)
2
6 2
2
6 2
(1-Py-IQ=1-pyridine-2-yl). The cellular uptake and intracellular localization displayed
that the two cyclometalated Ru(II) complexes entered NCI-H460 cancer cells
dominantly via endocytosis pathway, and preferentially distributed in the nucleus.
1

Further investigations on the apoptosis-inducing mechanisms of RuIQ-1 and RuIQ-2
revealed that the two complexes could cause S, G2/M double-cycle arrest by
regulating cell cycle related proteins. The two complexes also could reduce the
mitochondrial membrane potential (MMP), promote the generation of intracellular
ROS and trigger DNA damage, and then lead to apoptosis-mediated cell death. More
importantly, RuIQ-2 exhibits low toxicity both towards normal HBE cells in vitro and
zebrafish embryos in vivo. Accordingly, the developed complexes hold great potential
to be developed as novel therapeutics for effective and low-toxic cancer treatment.
Keywords: Cyclometalated Ru(II) complexes, Isoquinoline, Apoptosis, DNA
damage, Low toxicity
1
. Introduction
In recent years, transition-metal complexes have gradually found their way in
various biomedical applications, ranging from antiangiogenic [1], antihypertension [2],
antibacterial [3], anti-inflammation [4], antimalarial [5], to anticancer [6-11]. Over the
past decades, platinum-based metal complexes have been intensively exploited in the
oncological field, given the successful introduction of cisplatin in the early 1980s [12,
1
3]. However, the clinical application of such platinum-based metal complex has been
heavily limited by issues including poor solubility, severe toxicity, and drug resistance
14, 15]. To circumvent these problems, considerable efforts have been made for the
[
development of alternative metal-based compounds. Among the various alternative
transition metals, organometallic ruthenium, featured by predictable geometry,
variable oxidation states and low toxicity to normal cells [16-18], has been
particularly emphasized.
By far, three Ru(III) drugs, namely NAMI-A [19], KP1019 [20], KP1339 [21],
and the Ru(II)-based therapeutic, TLD1433 [22], have progressed to different stages
in clinical trials. Although the further clinical investigations of NAMI-A [23] and
2

KP1019 [20] have been limited by low therapeutic efficiency and poor water
solubility, respectively, with improved solubility and promising anticancer efficacy,
KP1339 is currently undergoing clinical trials [21,24]. TLD1433, the first
Ru(II)-based photosensitizer for PDT treatment of bladder cancer, recently entered
phase IB clinical trials [22, 24]. However, the mechanism of action of these Ru-based
drugs has not been fully understood yet.
Recently, the cyclometalated Ru(II) complexes, as a new type of non-platinum
metal-based compounds, have attracted worldwide attention due to their strong safety
profiles and potential anti-cancer effects, with many of them displayed different
mechanisms of cytotoxic action compared with the classical cisplatin [25-28]. The
cyclometalated Ru(II) compounds, in which one or more of the nitrogen donors of
compounds are replaced by carbon donor atoms, are relatively stable in structure since
the bond distances of Ru-C are shorter than that of Ru-N bond [29, 30]. Also, it has
been shown that the cyclometalation of Ru(II) compounds formed by substituting
Ru-N with Ru-C bond can improve the liposolubility of these compounds and result
in increased cellular uptake of tumour tissues, an essential factor for high-efficient
anti-cancer effect [27, 31, 32].
The development of Ru(II)-polypyridyl complexes with bioactive alkaloids as
ligands provides a new strategy for designing novel metal-based antitumor drugs with
improved activity [33-35]. Isoquinoline (IQ) and its derivatives are a class of nitrogen
heterocyclic alkaloids widely existing in nature and possessing a variety of important
biological functions, such as anticancer, analgesic, and anti-inflammatory effects [36].
It has been reported that isoquinoline alkaloids could inhibit the proliferation,
migration and invasion of tumour cells through multiple pathways, including cycle
arrest, apoptosis, inhibiting the activity of cyclooxygenase-2 (COX-2), topoisomerase
and angiogenesis, etc. [37]. Also, according to the recent study performed by Liang et
al., the biological activity of the isoquinoline alkaloids could be further improved via
2
+
2+
2+
coordination of isoquinoline derivatives with metals such as Zn , Ni , Cu [38].
The implication of these is that designing Ru(II) based cyclometalated isoquinoline
3

compounds, via employing synthetic diversity of the Ru(II) complexes, represents an
appealing strategy for new anticancer drug development.
Based on this hypothesis, in this work, with 1-phenylisoquinoline as a ligand,
+
two new cyclometalated Ru(II) complexes [Ru(bpy) (1-Ph-IQ)] (RuIQ-1) and
2
+
[
Ru(phen) (1-Ph-IQ)] (RuIQ-2) (1-Ph-IQ = 1-phenylisoquinoline) (Fig. 1) were
2
designed and synthesized. As experimental controls, the non-cyclometalated
ruthenium(II) isoquinoline complexes [Ru(bpy) (1-Py-IQ)](PF ) (Ru3) and
2
6 2
[
Ru(phen) (1-Py-IQ)](PF ) (Ru4) (1-Py-IQ=1-pyridine-2-yl), with a similar structure
2 6 2
to RuIQ-1 and RuIQ-2, were also prepared and characterized, and the corresponding
cytotoxicity of Ru3 and Ru4 was explored as well. Further mechanism studies
showed that the cyclometalated Ru(II) complexes, especially complex RuIQ-2, could
effectively constrain the viability of the NCI-H460 cells through inducing S, G2/M
double-cycle arrest, DNA damage, the generation of the reactive oxygen species
(ROS) and ROS-mediated mitochondrial dysfunction. In addition, when the toxicity
of complex RuIQ-2 to zebrafish embryos was investigated, it was confirmed that the
cyclometalated Ru(II) complexes containing isoquinoline ligands possessed superior
safety profiles and therefore held great potential to be further developed as a novel
chemotherapeutic agent for effective lung cancer treatment.
4

Fig. 1. The chemical structure of Ru(II) complexes RuIQ-1, RuIQ-2, Ru3, Ru4 and their ligands.
2
. Results and discussion
2
.1. Synthesis, characterization and UV-visible spectral studies
The synthetic routes of precursors cis-[Ru(L) Cl ] (L=bpy, phen), ligand 1-Ph-IQ
2
2
and the cyclometalated Ru(II) complexes RuIQ-1, RuIQ-2 are described in Scheme 1.
Firstly, the ligand 1-Ph-IQ was synthesized according to the previously reported
method [39]. Secondly, based on the reports [40, 41], reflowing RuCl
3
·nH O, LiCl
2
and bpy or phen in a solution of N,N-Dimethylformamide (DMF) at 140 °C overnight,
and the precursors cis-[Ru(L) Cl ]·2H O (L=bpy, phen) were obtained. Finally,
2
2
2
complexes RuIQ-1 and RuIQ-2 were obtained by reacting cis-[Ru(L) Cl ] (L = bpy,
2
2
phen) with 1-Ph-IQ in ethyl alcohol at 80 °C overnight under the protection of argon.
Ru(II) isoquinoline complexes Ru3 and Ru4 were synthesized and obtained
5

according to previously reported methods [42, 43]. Then, complexes RuIQ-1,
RuIQ-2, Ru3, Ru4 were characterized by elemental analysis, ESI-MS (Fig. S1-S4 in
1
supplementary materials), H NMR (Fig.S5-S8). The counter anion of all these
-
complexes is PF .
6
Scheme 1. The synthetic route of precursors cis-[Ru(bpy) Cl ], cis-[Ru(phen) Cl ], ligand
2
2
2
2
1
-Ph-IQ and RuIQ-1, RuIQ-2.
The UV/Vis absorption spectra of RuIQ-1 and RuIQ-2 in phosphate buffered
saline (PBS), saline solution (NaCl), ethanol (C H OH), and methyl cyanide (CH CN)
2
5
3
at 298 K are displayed in Fig. 2. As shown in Fig. 2, RuIQ-1 and RuIQ-2 showed
intense absorption bands that in the range of 250-300 nm, which were attributed
*
mainly to intra-ligand π-π transitions [42, 44, 45]. Differing from RuIQ-1, the
maximum absorption peak of RuIQ-2 was correspondingly blue-shifted from 270 nm
to 266 nm, the reason probably lies in the fact that the ancillary ligand phen in
RuIQ-2 has better π-π conjugated system. Besides, the relatively weak bands at the
*
range of 300–380 nm are most likely attributed to π–π transitions, the relatively
6

broad and weak absorption at 470-570 nm are contributed by the metal-to-ligand
charge transfer (MLCT) absorption.
Fig. 2. UV-Vis spectra of Ru(II) complexes in different solvents at 298 K. (A) RuIQ-1 (20 μM).
(B) RuIQ-2 (20 μM).
2
.2. Properties of the cyclometalated Ru(II) complexes in solution
The stability of drugs plays a vital role in cellular internalization and target
binding. Thus, we analyzed the aqueous stability of RuIQ-1 and RuIQ-2 by UV/Vis
1
absorption spectroscopy and H NMR. First of all, we observed that there was no
apparent change in the UV-Vis absorption of Ru(II) complexes in different solvents
(
PBS, saline solution, C H OH, and CH CN) at 298 K (Fig. 2A and 2B). Then, we
2 5 3
analyzed the time-dependent absorption spectra of RuIQ-1 and RuIQ-2 in PBS at
98 K (Fig. S9). Similarly, the absorption spectra of RuIQ-1 and RuIQ-2 did not
2
change significantly within 12 h. Finally, long-term stability monitoring was assessed
1
by H NMR to simulate the physiological conditions. As demonstrated in Fig. S10
and Fig. S11, after one week incubating RuIQ-1 and RuIQ-2 in DMSO-d /D O (v/v
6
2
1
=
3:1), the H NMR spectra did not show any significant changes compared with the
original samples.
It must be noted that the Ru(II) complexes may precipitate or bind with certain
biological molecules such as various plasma proteins after systematic administration,
which prevents them from reaching biological targets with high concentration. To test
the potential binding of Ru (II) complexes to such internal molecules, the absorption
spectra of RuIQ-1 and RuIQ-2 incubated in aqueous solutions containing bovine
serum albumin (BSA) was monitored by UV-Vis spectra at 298 K. From Fig. S12A
7

and Fig. S12B, it was clear that there was no obvious absorbance change in UV-Vis
spectra within 24 h, suggesting no prominent binding of Ru(II) complexes with
plasma proteins. Besides, the ESI-MS spectra of Ru(II) complexes showed abundant
peaks of molecular ions coinciding with the molecular weights of the compounds (Fig.
S12C and Fig. S12D), indicating that the precipitation or forming into other
compounds did not occur under the tested conditions. The results of all these
suggested that RuIQ-1 and RuIQ-2 were quite stable in aqueous solution.
2
.3. Cytotoxicity assay in vitro
After confirming the stability of the synthesized RuIQ-1, RuIQ-2, Ru3, Ru4,
the in vitro cytotoxicity of them, together with precursors and ligand, were assessed
on NCI-H460 (human lung cancer), A549 (human non-small cell lung cancer), HeLa
(
human
-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, with the
clinical cisplatin as control. The IC values of the tested reagents to the illustrated
cervical
cancer)
and
MCF-7
(breast
cancer)
cells
via
3
5
0
cancer cells were shown in Table 1. As displayed, after the treatment of test cancer
cells for 48 h, the IC50 values of the ligand 1-Ph-IQ, precursors cis-[Ru(L) Cl ]
2
2
(
L=bpy, phen) recorded a high value (approached or exceeded 200 μM) against all the
tested cancer cells, and thus deemed as inactive. On the other hand, while complexes
Ru3 and Ru4 showed moderate activity towards the tested cancer cells (with IC50
values ranging from 69.1 to 88.7 μM), the new cyclometalated complexes RuIQ-1
and RuIQ-2 displayed higher cytotoxicity effects toward all the test cancer cells, with
IC50 values ranging from 1.8 to 7.2 μM. As observed on the human lung cancer
NCI-H460 cells, RuIQ-1 and RuIQ-2 showed IC50 values (2.1 μM and 1.8 μM,
respectively) that are over 32- and 38-fold lower than that of Ru3 (69.1 μM), and over
3
6- and 43-fold lower than that of complex Ru4 (77.6 μM). Moreover, the activities
of RuIQ-1 and RuIQ-2 were about 12-fold and 14-fold higher than that of the clinical
cisplatin against NCI-H460 cells. Further investigation illustrated that RuIQ-1 and
RuIQ-2 inhibited the viability of NCI-H460 cells in
a
time- and
8

concentration-dependent manner (Fig.S13). Therefore, our results provided
substantial evidence that the integration of the ligand 1-Ph-IQ to organometallic Ru(II)
center represents a rational strategy for the development of novel chemical
compounds with enhanced anti-cancer effects.
To evaluate the potential side effects, additional cytotoxicity tests were
conducted using complexes RuIQ-1, RuIQ-2, Ru3, Ru4 to against the normal HBE
(human bronchial epithelial) cells (Table 1). Compared with Ru3, Ru4 and cisplatin
control, which displayed comparable toxicity to cancer cells and normal HBE cells,
complexes RuIQ-1 and RuIQ-2 exhibited much lower toxicity to the normal HBE
cells. These results suggested that RuIQ-1 and RuIQ-2 possess a preferable
therapeutic profile against cancer, especially lung cancer cells. This observation was
further supported by the selectivity index (SI) assay (Table 1), where a clear tread was
recorded as RuIQ-2 (11.1) > RuIQ-1 (10.5) > Ru3 (1.4) > Ru4 (1.3) > cisplatin (0.6).
Hence, RuIQ-2 demonstrated the highest safety profile for potential efficient cancer
treatment. The above analysis also confirms that in vitro cytotoxicity of complexes
RuIQ-1 and RuIQ-2 agrees with those of other cyclometalated Ru(II) complexes [26,
4
2]. This may attribute to the high hydrophobicity and cellular uptake caused by the
unique structural characteristics of the cyclometalated ligands.
Since NCI-H460 cells were especially susceptible to RuIQ-1 and RuIQ-2, with
over 12- and 14-fold lower IC50 values than that of cisplatin under the identical
conditions, this cell line was then selected as a cell model for further exploration of
the potential mechanisms accounting for the growth inhibition effect of
cyclometalated Ru(II)-isoquinoline compounds.
*
Table 1. Cytotoxicity assay in vitro (IC50 ) and Log Po/w values.
Complexes
IC50 (μM)
Log Po/w
c
NCI-H460
A549
MCF-7
HeLa
HBE
SI
-
b
b
cis-[Ru(bpy)
2
Cl
2
]
194.2±6.51
>200
>200
>200
189.2±4.31
>200
>200
197.5±6.12
>200
>200
>200
>200
-
b
cis-[Ru(phen)
2
Cl
2
]
-
b
ab
1
-Ph-IQ
172.0±3.9
2.1±0.2
>200
194.7±6.1
7.2±0.4
-
a
RuIQ-1
3.2±0.1
4.3±0.3
22.0±0.8
10.5
1.02±0.10
9

a
a
RuIQ-2
Ru3
1.8±0.3
69.1±3.4
77.6±4.1
25.9±1.5
2.5±0.5
74.5±3.7
79.3±3.8
27.3±1.8
7.0±0.3
4.7±0.4
80.2±4.1
82.2±3.5
20.0±0.3
11.1
1.4
1.3
0.6
1.12±0.09
-1.65±0.12
-1.07±0.08
ND
b
b
b
b
b
b
b
b
b
a,b
a,b
a,b
72.0±3.4
88.7±4.7
21.8±2.2
96.1±3.4
97.6±4.2
16.4±1.9
b
b
Ru4
a,b
Cisplatin
15.0±2.0
*
is the illustrated cells were treated for 48 h, and the data is expressed as mean ± standard
a
deviation (Mean ± SD). indicates the same complex, different cell groups and NCI-H460 cell
b
group bilateral t-test, P <0.05; indicates bilateral t-test between the same cell line, different
c
complex treatment groups and complex RuIQ-2 treatment group, P <0.05. SI (Selectivity Index)
=
IC (HBE)/IC (NCI-H460).
50 50
2
.4. Lipophilicity and cellular uptake of Ru(II) complexes
It is generally acknowledged that the lipophilicity of the metal-based agents plays
a vital influence on their cellular uptake, cytotoxicity, and subsequent cancer
therapeutic [26, 45-47]. In this study, the lipophilicity, expressed as oil-water partition
coefficient (logP ), is evaluated by the “shake-flask” method using inductively
o/w
coupled plasma mass spectrometry (ICP-MS). As shown in Fig. 3A, the
cyclometalated complexes (RuIQ-1 and RuIQ-2) are more lipophilic than the
non-cyclometalated complexes (Ru3 and Ru4), with the order of the logPo/w values
being recorded: RuIQ-2 (1.12) > RuIQ-1 (1.02) > Ru4 (-1.07) > Ru3 (-1.65).
Complex RuIQ-2 displayed the highest logPo/w value, suggesting that this complex is
the most hydrophobic, which may contribute to its highest cytotoxicity. By contrast,
complexes Ru3 and Ru4 exhibited negative logPo/w values, indicating that they are
hydrophilic, that is why they gave moderately cytotoxicities. It is very interesting to
note that the only difference among complexes RuIQ-1, RuIQ-2, Ru3 and Ru4 is the
molecular structure, but they display obvious differences in their logPo/w values, and
then results in different cytotoxicities.
To investigate the relationship between the cellular uptake rate and the
cytotoxicity of RuIQ-1 and RuIQ-2, ICP-MS was performed to quantitative
determine of the ruthenium level inside of the NCI-H460 cells, with the results being
6
represented as ng of ruthenium per 10 cells. Our results showed that a noticeable
time- and concentration-dependent increase in the uptake of RuIQ-1 and RuIQ-2 by
NCI-H460 cells (Fig. 3B). As expected, complex RuIQ-2 exhibited higher cellular
1
0

uptake than that of RuIQ-1 in all the test time points and drug concentrations, which
due to its highest hydrophobicity. The studies of Barton and Chao et al. have
illustrated that the most liposoluble Ru(II) complexes presented the greatest uptake
[
48-50].
When the cellular distribution of complexes RuIQ-1 and RuIQ-2 was measured in
NCI-H460 cells by ICP-MS, it was evident that, after 6 h incubation using 2 μM of
the chemicals, RuIQ-1 and RuIQ-2 mainly accumulated in the nucleus and
mitochondria, with only a small fraction of them detectable in the cytoplasm. Since
the cell nucleus contains DNA etc. most of the cellular genetic material, the
accumulation of RuIQ-1 and RuIQ-2 in the nucleus may cause suppression of
cellular DNA transcription and replication, and exhibit their biological activities.
These results also indicated that RuIQ-1 and RuIQ-2 may mainly target nucleus and
mitochondria, similar with the previously reported cyclometalated Ru(II) complexes
such as [Ru(bpy)(phpy)(dppz)](ClO ) [26] and [Ru(L) (1-Ph-βC)](PF ) (L=dmb, bpy)
4
2
6
[
42].
1
1

Fig. 3. Lipophilicity and cellular uptake of Ru(II) complexes. (A) LogPo/w values of Ru(II)
complexes. (B) Cellular ruthenium concentrations determined in NCI-H460 cells after 1, 3, and 6
h incubated with RuIQ-1 and RuIQ-2 at 1, 2, 4 μM, respectively. (C) Subcellular distribution of
Ru(II) complexes in NCI-H460 cells after incubated with RuIQ-1 (2 μM) or RuIQ-2 (2 μM) for
different period. (D) Intracellular uptake of Ru(II) complexes in NCI-H460 cells under different
*
endocytosis-inhibited conditions. Data were collected from three individual experiments ( p <
**
***
0
.05, p < 0.01, and p < 0.001).
2
.5. The mechanism of cellular uptake
The ways of drug entering into the cells have an essential impact on their biological
activity. Generally, the drug molecules enter cancer cells via active and passive
pathways. Active transport is energy dependent, on the contrary, passive transport is a
non-energy-dependent way [51]. To gain more insight into the detailed routes of
complexes RuIQ-1 and RuIQ-2 into cells, we treated NCI-H460 cells with Ru(II)
complexes at either low temperature (4 °C) or NaN in combination with DOG, a
3
commonly used strategy to block adenosine 5′-triphosphate (ATP) dependent active
mechanisms. The results showed that both low temperature and DOG+NaN could
3
strongly block the cellular uptake of RuIQ-1 and RuIQ-2 (Fig. 3D), denoting that
1
2

both RuIQ-1 and RuIQ-2 are taken up by NCI-H460 cells via active transport
pathway. Endocytosis is the most common active transport, and engaged in the
cellular uptake of a wide variety of exogenous molecules. In order to further
investigate the internalization pathway of RuIQ-1 and RuIQ-2, we used two
endocytosis inhibitors, i.e. nystatin (clathrin-mediated endocytosis) and sucrose (lipid
raft-mediated endocytosis) [51]. In our study, it was found that the cellular uptake
levels of both RuIQ-1 and RuIQ-2 were significantly reduced in the sucrose group,
with only a moderate decrease in the nystatin group. These results manifest that the
cross-membrane transportation of RuIQ-1 and RuIQ-2 mainly via the
clathrin-mediated endocytosis pathway.
2
.6. Cyclometalated Ru(II) complexes induce apoptosis in NCI-H460 cells
Apoptosis has been observed to engage in the anti-cancer activity of many of the
previously reported drugs [52-54]. Next, the Hoechst 33342 staining technique,
accompanied by fluorescence microscopy assay were performed to determine whether
such a mechanism involved in the potential anti-cancer effect of the complexes
RuIQ-1 and RuIQ-2. As shown in Fig. 4A and 4B, after RuIQ-2 treatment, the
NCI-H460 cells showed noticeable apoptotic morphology, including nuclear
shrinkage and chromatin condensation, in a time- and concentration-dependent
manner. Similar apoptotic features were also observed after RuIQ-1 treatment (Fig.
S14A). These results strongly suggested that the involvement of apoptosis in the Ru(II)
complexes-mediated cell death.
To provide further information about Ru(II) complexes-mediated cell death,
Annexin V/PI staining was performed, and the results were analyzed by Flow
Cytometry. We can clearly observe that the Ru(II) complexes induced apoptosis of the
NCI-H460 cells in a concentration-dependent manner (Fig. 4C and Fig. S14B).
Differing from the control group, where the percentage of the early apoptotic cells
recorded 2.24% and 1.73%, after the cells were incubated with RuIQ-1 and RuIQ-2
at 4 μM for 24 h, the percentage of early apoptotic cells increased to 42.96% for
1
3

RuIQ-1 and 57.31% for RuIQ-2. Comparing the apoptotic effect, it was clear that the
complex RuIQ-2 displayed more effective apoptotic activity than RuIQ-1, with the
percentage of total apoptotic cells recorded 51.23% for RuIQ-1 and 68.05% for
RuIQ-2. This observation is consistent with their cytotoxic activity. Since complex
RuIQ-2 presents more effective growth inhibition and apoptosis induction effects in
the NCI-H460 cells than that of RuIQ-1, the apoptosis-induced mechanism of it was
further investigated.
Apoptotic pathways can be divided into the intrinsic and extrinsic ways
according to which caspases are involved [55]. As an essential regulatory protein in
the downstream pathway of apoptosis, caspase plays a vital role in the initiation and
performance of apoptosis. Caspase, especially caspase-3 activation, subsequently can
lead to the cleavage of poly ADP-ribose polymerase (PARP), serving as a biochemical
marker of cells apoptosis [56]. To investigate the molecular events initiated by
RuIQ-2, after treatment for 24 h, the activities of caspase-3, -8 and -9 were analyzed
by western blot assay. According to Fig. 4D, co-incubation with 1, 2, and 4 μM of
RuIQ-2 for 24 h caused a significant in the protein expression levels of the
cleaved-PARP, cleaved caspase-3, -8 and -9. Meanwhile, with the shearing of
caspase-8, caspase-9, caspase-3 and PARP, the total expression levels of caspase-3
and PARP experienced a significant declined. All these findings demonstrated that
RuIQ-2-induced apoptosis in NCI-H460 cells through caspase-dependent extrinsic
and intrinsic pathways.
1
4

Fig. 4. RuIQ-2 induced apoptosis in NCI-H460 cells. (A) NCI-H460 cells were stained with
Hoechst 33342 after co-incubation with 1, 2, and 4 μM of RuIQ-2 for 12 h. (B) NCI-H460 cells
were stained with Hoechst 33342 after co-incubation with RuIQ-2 at 2 μM for different time. (C)
NCI-H460 cells apoptosis was detected by Annexin V/PI assay after treatment with 1, 2, and 4 μM
of RuIQ-2 for 24 h. (D) The expression of Cleaved caspase-9, -8, -3, caspase-3, PARP, Cleaved
PARP were evaluated by western blot analysis in a concentration-dependent manner with RuIQ-2
treatment for 24 h.
2
.7. Cyclometalated Ru(II) complexes induce mitochondrial dysfunction
Since mitochondrion can release pro-apoptotic factors such as cytochrome c and
other apoptosis-inducing factors, hence, it plays a vital role in the apoptotic process
52-54, 57]. To investigate the effects of Ru(II) complexes on mitochondrial
[
membrane potential (MMP), decline of which is a marker of mitochondrial
dysfunction, the changes in MMP were detected by the fluorescent probe
5
,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidalylcarbo cyanine iodide (JC-1).
When MMP is low, JC-1 monomer emits green fluorescence signal, while when MMP
is high, JC-1 aggregate displays red fluorescence signal. In our study, after treatment
1
5

with 1, 2, and 4 μM of RuIQ-1 or RuIQ-2 for 12 h, the red fluorescence saw
prominent decrease while significant increase in the green fluorescence occurred (Fig.
5
A and Fig. S15A). Similar results were recorded when treatment with 2 μM of
RuIQ-1 or RuIQ-2 for a series of different time points (Fig. 5B and Fig. S15B). All
these findings indicate the decline of MMP. In addition, for RuIQ-2, the changes of
MMP were detected quantitatively by using flow cytometry (Fig. 5C, D). As
presented in Fig. 5C, after treatment with RuIQ-2 for 12 h, the green fluorescence of
the JC-1 monomers increased from 16.0% (1 μM) to 56.7% (4 μM). The decline of
MMP is also a marker of apoptosis. To further confirm the mitochondrial pathways in
RuIQ-2-induced apoptosis, we detected the expression of Bcl-2, Bax, Bcl-xl, and Bad
in mitochondrial membrane and cytochrome c protein in cytoplasm using western blot
analysis. As shown in Fig. 5E, with the increase of RuIQ-2 concentration, the
expression of Bcl-2 and Bcl-xl (anti-apoptotic proteins) showed notable attenuation.
At the same time, the expression of Bax and Bad (pro-apoptotic proteins), as well as
the content of cytochrome c protein in the cytoplasm experienced a significant
increase. These results collectively indicated that RuIQ-2 induced apoptosis in
NCI-H460 cells through the mitochondrial apoptotic pathway.
1
6

Fig. 5. RuIQ-2 induced mitochondrial dysfunction in NCI-H460 cells. (A) Fluorescence
microscopy analysis of cellular MMP level by JC-1 staining after treatment with RuIQ-2 at 1, 2
and 4 μM for 12 h. (B) Fluorescence microscopy analysis of cellular MMP level by JC-1 staining
after 2 μM of RuIQ-2 treatment for different time. (C) Flow cytometry analysis of cellular MMP
level after treatment with RuIQ-2 at 1, 2 and 4 μM for 12 h. (D) Flow cytometry analysis the ratio
of red/green fluorescent intensity after treatment with RuIQ-2 at 1, 2 and 4 μM for 12 h. Data
*
**
***
were collected from three individual experiments ( p <0.05, p < 0.01, and p < 0.001). (E)
Western blot analysis of the expression levels of Bcl-2, Bax, Bcl-xl, Bad in mitochondrial
membrane and cytochrome c protein in cytoplasm after RuIQ-2 (2 μM ) treatment for 24 h.
2
.8. Cyclometalated Ru(II) complexes stimulate the generation of intracellular ROS
It has been demonstrated that ROS could induce chain of
mitochondria-associated events, such as the decline of MMP and apoptosis [52-54,
a
1
7

5
7]. Hence, ROS is considered to be a mediator of apoptosis [58, 59], and exploring
the cellular ROS levels is of great importance to elucidate the underlying mechanism
of Ru(II) complexes-induced apoptosis. To investigate whether Ru(II) complexes
stimulate ROS generation and accumulation, 2,7-dichloride-hydrofluorescein
diacetate (DCFH-DA) fluorescent probe-based imaging assay was performed. We
observed that the fluorescence intensity in NCI-H460 cells both increased in a time-
and concentration-dependent manner after RuIQ-1 or RuIQ-2 treatment (Fig. S16
and S17), suggesting the generation and accumulation of ROS. Moreover, the cellular
ROS level was further measured by flow cytometry after RuIQ-2 treatment. As
shown in Fig. 6A and Fig. S18, after 12 h of co-incubation with 1, 2, and 4 μM of
RuIQ-2, a noticeable enhanced in the mean fluorescent intensity (M) was observed,
with the DCF fluorescence increased from 1.31 to 6.51, which is approximately 5-
fold higher than that of the control group. So we came to the conclusion that the
cyclometalated Ru(II) complexes could induce the generation of ROS in NCI-H460
cells.
As reported, the ROS can be generated either endogenously during the process of
mitochondrial oxidative phosphorylation, or produced from the interaction between
exogenous sources such as xenobiotic compounds [60]. So, mitochondria are both
source and target of ROS [61]. The cyclometalated complexes studied in this work
can induce the decline of mitochondrial membrane potential (MMP) and results in
mitochondrial dysfunction, which causes the damage of respiratory chain, and
2
−
generates radical and non-radical species such as superoxide anion (O ) and
hydrogen peroxide (H O ). Based on the above information, these cyclometalated
2
2
Ru(II) complexes may stimulate the mitochondria based ROS generation. This
speculation correlates with our previous publication [58], the Ru(II)
complex-triggered ROS generation could be effectively blocked by using
cyclosporine A (CsA), a confirmed mitochondrial permeability transition pore (MPTP)
opening inhibitor.
To further explore the effect of ROS activation induced by complex RuIQ-2 on
1
8

mitochondria and cell apoptosis, two ROS inhibitors, i.e. glutathione (GSH) and
N-acetyl-l-cysteine (NAC), were used in our study. After the addition of either NAC
or GSH, the fluorescence intensity of DCF displayed significant reduction (Fig.S19),
indicating the inhibition of ROS generation in NCI-H460 cells. Next, NCI-H460 cells
were treated with 2 μM of RuIQ-2 combined with/without GSH (5 mM) or NAC (10
mM) for 12 h. After stained with Annexin V-FITC/PI, the percentage of apoptotic
cells was measured using flow cytometry. From Fig. 6B and Fig. 6C, it was evident
that both NAC and GSH could block RuIQ-2-induced apoptosis in NCI-H460,
denoting that ROS generation played an essential role in RuIQ-2-induced apoptosis
in the tested NCI-H460 cells. This mechanism was further demonstrated by
subsequent western blot assay. The results in Fig. 6D manifested that pretreatment of
NAC or GSH inhibited cytochrome c release in the cytoplasm and efficiently reversed
the RuIQ-2-induced PARP and caspase-3 phosphorylation. The implication of all
these is that RuIQ-2-induced apoptosis in NCI-H460 cells is triggered by
ROS-mediated mitochondrial dysfunction.
1
9

Fig. 6. RuIQ-2 stimulated generation of intracellular ROS. (A) Flow cytometry analysis of
cellular ROS level by DCFH-DA staining in a concentration-dependent manner upon RuIQ-2
treatment for 12 h. (B) The percentage of apoptotic cells were detected by Annexin V-FITC/PI
double staining assay using flow cytometry after 2 μM RuIQ-2 treatment with or without
antioxidants NAC (10 mM) or GSH (5 mM). (C) Cell viability was assessed by MTT assay after
1
2 h of RuIQ-2 treatment with or without NAC (10 mM) or GSH (5 mM). (D) Western Blot
analysis of the protein expression levels of PARP, cytochrome c and cleaved caspase-3 after
RuIQ-2 (2 μM) treatment for 24 h with or without NAC (10 mM) or GSH (5 mM).
2
.9. Cyclometalated Ru(II) complexes trigger DNA damage and S, G2/M double-cycle
arrest
DNA damage, such as the change of cellular DNA structure, can lead to inhibition
of DNA replication. When DNA damage cannot be repaired, it may cause cell cancer.
So, DNA damage is considered as a mark of apoptosis [62-65]. We have reported that
many Ru(II) complexes could promote the generation of cellular ROS levels, which in
turn attack DNA and results in apoptosis [42, 51, 66, 67]. To determine whether or not
these complexes can induce DNA damage, the single-cell gel electrophoresis assay
(comet assay) was conducted in our study. As shown in Fig. S20A and Fig. 7A, in the
control group, NCI-H460 cells failed to show a comet-like appearance. However,
after RuIQ-1 (1 μM) or RuIQ-2 (1 μM) treatment for 12 h, NCI-H460 cells exhibited
well-formed comet tails, indicating the existence of severe DNA damage. When the
treatment concentration was increased to 4 μM, significantly prolonged comet tails
appeared, denoting that more DNA damage occurred with the increase of complexes
concentration. Furthermore, it was also found that NCI-H460 cells presented
well-formed comet tails in a time-dependent manner after 2 μM of RuIQ-2 treatment
(Fig. 7B). To provide solid evidence about the RuIQ-2-induced DNA damage,
western blot analysis was scheduled (Fig. 7C and 7D). Coincident with our previous
reports, RuIQ-2 treatment resulted in significantly up-regulated DNA damage
markers, including the phosphorylation levels of ATM (Ser1984), Histone H2A.X
(Ser139), ATR (Ser 428) and p53 (Ser15) [42, 51]. Meanwhile, the phosphorylation
levels of Chk1 and Chk2 were also up-regulated. Moreover, several cell cycle
arrest-related key downstream proteins (e.g. p21, p27) were simultaneously activated
2
0

(Fig.7D).
The comet tail observed does not mean that the ruthenium complexes directly
damage DNA. It has been reported that excess intracellular ROS could attack DNA,
resulting in DNA damage [68, 69]. So, the observed DNA damage may results from
the increased intracellular ROS levels. This has been confirmed by our previous study
(the cyclometalated Ru(II) β-carboline complexes could induce DNA damage through
ROS overproduction [42]. Many Ru(II) complexes can bind to DNA in a noncovalent
interaction such as electrostatic binding, groove binding, or intercalation [70-73]. We
didn't do DNA-binding experiments in this work. However, according to our previous
studies [42, 66, 74, 75], we speculate that the binding affinity of the title complexes
towards DNA is not very strong because they don’t have a large planar aromatic
ligand (intercalative), such as dppz, dpq and pip [66, 74, 75], so the interaction
between them and DNA is not supposed to be the primary reason of apoptosis.
From the biology aspects, DNA is the key information carrier, so it is highly stable
over the evolution, except for the DNA enzyme mediated degradation. Differing from
necrosis mediated cell death, which is caused by external factors such as trauma or
infection, the apoptosis, featured by fragmentation of nuclei, is a secondary response
to DNA damage, with the biological goal of protecting a multicellular organism
against a damaged cell. As a result, the apoptosis mediated cell death has been
commonly measured using the comet assay to detect DNA damage of cells after
treatment with complexes. Comets with almost all DNA in the tail are often referred
to as ‘hedgehog’ comets and are widely assumed to represent apoptotic cells. In
summary, the title complexes could induce mitochondrial dysfunction and the
generation of intracellular ROS, which may indirectly lead to DNA damage. Also, the
comet assay (DNA fragments appearance) could serve as a mark for apoptosis
assessment.
2
1

Fig. 7. RuIQ-2 induced DNA damage in NCI-H460 cells. (A) DNA fragmentation was examined
by comet assay. NCI-H460 cells were treated with RuIQ-2 at 1, 2 and 4 μM for 12 h. (B) DNA
fragmentation of NCI-H460 cells was examined, after treatment with RuIQ-2 (2 μM) for different
time period (3, 6, 12 h). (C) The level of DNA damage proteins (p-Histone H2A.X, p-ATM,
p-ATR, p-Chk1, p-Chk2) were assessed by western blot analysis, after treatment with RuIQ-2 at 1,
2
and 4 μM for 24 h. (D) Western blot analysis of the expression level of p-p53, p27, p21, after
treatment with various concentration of complex RuIQ-2 for 24 h.
DNA damage can directly affect DNA transcription, replication and cell cycle
arrest. To investigate whether the DNA damage caused by studied complexes on
NCI-H460 cells could affect cell cycle distribution of NCI-H460 cells, the cell cycle
ratio was detected by flow cytometry. The results in Fig. 8A and Fig. 8B displayed
that, after treating with RuIQ-2 at 4 μM for 24 h, compared with the control group,
2
2

the cell proportion of S phase nearly doubled, increased from 18.22% to 33.83%. A
similar trend was observed for the G2/M phase, which increased from 11.44% to
2
6.29%. These data suggested that RuIQ-2 could induce cell cycle arrest at S and
G /M phases in NCI-H460 cells. Similar S, G2/M double-cycle arrest results were
2
also observed in NCI-H460 cells after RuIQ-1 treatment (Fig. S20B).
As we all know, cell-cycle progression is regulated by forming a series of
specialized cyclin-dependent kinase (CDK)-cyclin complexes. To explore the
mechanism of RuIQ-2-induced cell cycle arrest, western blot analysis was employed
to examine the effect of RuIQ-2 treatment on the expression of regulatory proteins,
such as CDK2/Cyclin A, CDK1/Cyclin B. Our results showed that, after RuIQ-2
treatment, the expression of CDK1 and Cyclin B (key factors of G2/M phase arrest)
[
76, 77] significantly down-regulated (Fig. 8C). Although no apparent change of
CDK2 was observed, the expression of Cyclin A saw a prominent decrease. Since the
formation of CDK2-Cyclin A complex has been confirmed to be closely related to
phase S arrest [78], the reduction of Cyclin A expression definitely contributes to the
effect of RuIQ-2. Moreover, as shown in Fig.7D, both p21 and p27, two of the
well-known CDK inhibitors contributing to the G1-S transition blockage, also
displayed up-regulation [78, 79]. All of these findings demonstrated that both RuIQ-1
and RuIQ-2 could trigger DNA damage and S, G2/M double-cycle arrest via
suppressing the expression of regulatory proteins CDK1/Cyclin B and Cyclin A.
2
3

Fig. 8. RuIQ-2 induced cycle arrest in NCI-H460 cells. (A) Cell cycle distribution was performed
by PI staining after co-incubated with RuIQ-2 for 24 h. (B) Histograms show cell cycle
*
distribution of the NCI-H460 cells. Data were collected from three individual experiments ( p <
**
***
0
.05, p < 0.01, and p < 0.001). (C) Western blot analysis of expression of CDK1, CDK2,
Cyclin A, and Cyclin B. NCI-H460 cells were treated with RuIQ-2 at 1, 2 and 4 μM for 24 h.
2
.10. Complex RuIQ-2 shows low toxicity to developing zebrafish embryos
Compared with athymic nude mice models, zebrafish has potential advantages,
such as high reproductive rate and short growth period, etc. Recently, zebrafish has
rapidly developed to be a promising model for evaluating the toxicity of drugs [80-82].
In this work, the in vivo toxicity of complex RuIQ-2 was assessed on developing
zebrafish embryos.
As displayed in Fig. 9A, after exposure to RuIQ-2 at the expected treatment
concentrations (less than 16 μM), only minor toxicity was observed, with most of the
zebrafish embryos developing into juvenile zebrafish (more than 80% cumulative
hatch rate after 72 h) (Fig. 9B). And under such a concentration, the lethality rate was
lower than 40% even after prolonged 96 h incubation (Fig. 9C). When the
concentration of RuIQ-2 increased to 16 μM for 96 h, we observed that some
zebrafish embryos developed abnormally with a marked spinal curvature (Fig. 9A).
2
4

Moreover, when RuIQ-2 concentration was increased to 32 μM, a cumulative hatch
rate of less than 50% was recorded after 48 h incubation, and all the zebrafish
embryos died at the time point of 96 h. Therefore, it looks like that under the effective
concentration range (less than 16 μM), RuIQ-2 was generally safe for zebrafish
embryos. Since hypotoxicity to normal cells or organs is vital for developing novel
anticancer agents, complex RuIQ-2 is expected to be developed as a low-toxicity
agent against lung cancer cells.
Fig. 9. Toxicity assessment of RuIQ-2 in developing zebrafish embryos. (A) Toxicity of RuIQ-2
to zebrafish embryos at various concentrations within 96 h on a 4× objective lens in the
microscope. (B) Cumulative hatch rate of zebrafish embryos in the presence/absence of RuIQ-2 at
2
5

various concentrations every 24 h. (C) Lethality rate of zebrafish embryos in the presence/absence
of RuIQ-2 at different concentrations every 24 h.
3
. Conclusions
+
Two new cyclometalated Ru(II) complexes, i.e. [Ru(bpy) (1-Ph-IQ)] (RuIQ-1)
2
+
and [Ru(phen) (1-Ph-IQ)] (RuIQ-2), have been designed and synthesized. The
2
significant difference in the cytotoxicity between the cyclometalated Ru(II)
complexes RuIQ-1, RuIQ-2 and the non-cyclometalated complexes Ru3, Ru4 is due
to the obvious difference of lipophilicity caused by the small difference of their
molecular structure, i.e., substituting Ru-N with Ru-C bond. Our results suggested
that the coordination of the cyclometalated ligand 1-Ph-IQ to polypyridyl-Ru(II)
centers can effectively enhance cytotoxicity, and therefore providing a guideline for
future anti-cancer complexes exploration.
Our results indicated that RuIQ-2 could effectively induce S, G2/M double-cycle
arrest via suppressing the expression of regulatory proteins CDK1/Cyclin B and
Cyclin A. The mechanism studies showed that RuIQ-2-induced apoptosis is triggered
by ROS-mediated mitochondrial dysfunction. In a word, the Ru (II) complexes can
induce apoptosis of NCI-H460 cells by increasing intracellular ROS level, reducing
MMP and triggering DNA damage. More importantly, RuIQ-2 exhibited low toxicity
both toward normal HBE cells in vitro and zebrafish embryos in vivo. Although this
work is an in vitro assay, further in vivo assay using tumour bearing animal models
has been scheduled. Based on the current results, the cyclometalated Ru(II)
complexes developed in this work has great potential to be developed as novel lung
cancer therapeutic agents with fewer side effects.
4
. Experimental section
4
.1. Chemicals and reagents
Ultrapure MilliQ water was used in all experiments. DMSO, MTT, PBS, JC-1,
DCFH-DA, PI, Hoechst 33342, NAC, GSH, Annexin V-FITC Apoptosis Detection
2
6

TM
TM
Kit, QuantiPro BCA Assay Kit, ECL Start Western Blotting Detection Reagent
and endocytosis inhibitors including NaN3, DOG, Sucrose and Nystatin were
purchased from Sigma-Aldrich (St. Louis, MO, USA). NCI-H460, A549, HeLa,
MCF-7 and HBE cells were purchased from American Type Culture Collection
(ATCC, Manassas, VA). Cisplatin was purchased from Acros. Ruthenium standard
solution was purchased from Aladdin Chemistry Co. (Shanghai, China). Cell
Mitochondria Isolation Kit was purchased from Beyotime (Shanghai, China).
Antibodies were purchased from Cell Signaling Technology Company. Comet assay
reagent kit was purchased from Trevigen (Gaithersburg, MD, USA).
4
.2. Synthesis and characteristics
-Ph-IQ [39] and cis-[Ru(L) Cl ]·2H O (L=bpy, phen) [40, 41] were prepared
1
2
2
2
according to the methods in the literature.
Microanalyses were carried out with a Perkin-Elmer 240Q elemental analyzer.
Electrospray ionization mass spectrometry (ESI-MS) was recorded on Agilent
1
LC-MS6430B Spectrometer. H NMR spectra were recorded on a Bruker AVANCE
AV 400 NMR spectrometer using (CD ) SO as solvent at room temperature and TMS
3
2
as the internal standard. UV-Vis spectra were recorded on a Perkin–Elmer
Lambda-850 spectrophotometer (PerkinElmer, USA).
4
.2.1. Synthesis of [Ru(bpy) (1-Ph-IQ)](PF ) (RuIQ-1)
2 6
A mixture of cis-[Ru(bpy) Cl ]·2H O (0.24 g, 0.5 mM), 1-Ph-IQ (0.10 g, 0.5
2
2
2
mM), Ag(CF SO ) (0.26 g, 1 mM) and tetramethylammonium hydroxide (0.05 mL,
3
3
0
.5 mM) in anhydrous ethanol (10 mL) was refluxed under argon at 78 °C for 12 h to
produce a clear red solution. After the reaction, the red precipitate was obtained by a
dropwise addition of saturated aqueous KPF solution. Finally, the red precipitate was
6
dried under vacuum and purified by column chromatography on neutral alumina with
a mixture of CH CN-toluene (3:1, v/v) as eluent. Yield: 76%. Anal. Calc for
3
C H F N PRu: C 55.12%, H 3.44%, N 9.18%; Found: C 55.23%, H 3.41%, N
35
26
6
5
2
7

+
1
9
.20%. ESI-MS (MeCN): m/z = 617.79 ([M-PF ] ). H NMR (400 MHz, DMSO-d ) δ
6 6
8
.94 (dd, J = 7.3, 2.3 Hz, 1H), 8.79 (dt, J = 8.4, 1.1 Hz, 1H), 8.78 – 8.60 (m, 3H), 8.36
(
d, J = 7.9 Hz, 1H), 8.21 – 8.07 (m, 1H), 8.00 – 7.72 (m, 8H), 7.64 (ddd, J = 6.3, 2.2,
.1 Hz, 2H), 7.59 (ddd, J = 7.5, 5.4, 1.2 Hz, 1H), 7.47 (d, J = 1.0 Hz, 2H), 7.44 – 7.27
m, 3H), 6.96 (ddd, J = 8.1, 7.1, 1.4 Hz, 1H), 6.85 (td, J = 7.2, 1.2 Hz, 1H), 6.53 (dd, J
1
(
-1
=
7.5, 1.3 Hz, 1H). UV-Vis (λ/nm, ε/M )(PBS): 297(49200), 505(9550).
4
.2.2. Synthesis of [Ru(phen) (1-Ph-IQ)](PF ) (RuIQ-2)
2 6
Complex RuIQ-2 was synthesized in a manner identical to that described for
RuIQ-1, with [Ru(phen) Cl ]·2H O in place of [Ru(bpy) Cl ]·2H O. Yield: 71%.
2
2
2
2
2
2
Anal. Calc for C H F N PRu: C 57.78%, H 3.23%, N 8.64%; Found: C 57.70%, H
3
9
26
6
5
+
1
3
.25%, N 8.60%. ESI-MS (CH CN): m/z = 665.81 ([M-PF ] ). H NMR (400 MHz,
3 6
DMSO-d ) δ 8.96 (d, J = 8.3 Hz, 1H), 8.69 (dd, J = 8.2, 1.4 Hz, 1H), 8.63 – 8.45 (m,
6
3H), 8.39 (d, J = 8.0 Hz, 1H), 8.35 – 8.20 (m, 5H), 8.10 (ddd, J = 5.4, 3.9, 1.3 Hz, 2H),
7.93 (dd, J = 7.6, 1.9 Hz, 1H), 7.89 – 7.64 (m, 6H), 7.60 (dd, J = 8.1, 5.3 Hz, 1H),
7.52 (d, J = 6.3 Hz, 1H), 7.40 – 7.34 (m, 1H), 6.95 (ddd, J = 8.1, 7.1, 1.4 Hz, 1H),
6.76 (td, J = 7.3, 1.2 Hz, 1H), 6.43 (dd, J = 7.6, 1.3 Hz, 1H). UV-Vis (λ/nm,
-1
ε/M )(PBS): 267(54800), 496(11800).
4
.2.3. Synthesis of [Ru(bpy) (1-Py-IQ)](PF ) (Ru3) and [Ru(phen) (1-Py-IQ)](PF )
2 6 2 2 6 2
(Ru4)
N-Ts-phenethylamine and 2-pyridinecarboxaldehyde are dissolved in toluene,
boron trifluoride etherate is added dropwise in an ice bath, refluxed at 60 °C under
argon atmosphere, 30% NaOH, dehydrogenated at 120 °C to obtain 1-(2-pyridine
Base)-isoquinoline. Reflowing 1-(2-pyridyl)-isoquinoline and cis-[Ru(bpy) Cl ]·H O
2
2
2
or cis-[Ru(phen) Cl ]·H O in a solution of ethanol: water (1:1) overnight, and Ru3
2
2
2
and Ru4 were obtained finally.
1
Complex Ru3 was a brown solid weigh 0.076 g, and its yield was 82%. H NMR
(
400 MHz, DMSO-d ) δ 8.99 (d, J =8.3 Hz, 1H), 8.96 – 8.90 (m, 1H), 8.90 – 8.79 (m,
6
2
8