Novel tetranuclear ruthenium(II) arene complexes showing potent cytotoxic and antimetastatic activity as well as low toxicity in vivo

Article abstract of DOI:10.1016/j.ejmech.2019.06.061

Ruthenium complexes have attracted a surge of interest as anticancer drug candidates because of their low toxicity, diversity in mode-of-actions and non-cross drug resistance with conventional platinum-based agents. Despite remarkable advances, only a lim

Full text of DOI:10.1016/j.ejmech.2019.06.061

Accepted Manuscript
Novel tetranuclear ruthenium(II) arene complexes showing potent cytotoxic and
antimetastatic activity as well as low toxicity in vivo
Mohamed Kasim Mohamed Subarkhan, Lulu Ren, Binbin Xie, Chao Chen, Yuchen
Wang, Hangxiang Wang
PII:
S0223-5234(19)30588-4
DOI:
https://doi.org/10.1016/j.ejmech.2019.06.061
EJMECH 11464
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 8 March 2019
Revised Date: 19 June 2019
Accepted Date: 21 June 2019
Please cite this article as: M.K. Mohamed Subarkhan, L. Ren, B. Xie, C. Chen, Y. Wang, H. Wang,
Novel tetranuclear ruthenium(II) arene complexes showing potent cytotoxic and antimetastatic activity as
well as low toxicity in vivo, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.06.061.
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ACCEPTED MANUSCRIPT
Novel tetranuclear ruthenium(II) arene complexes
showing potent cytotoxic and antimetastatic activity as
well as low toxicity in vivo
Mohamed Kasim Mohamed Subarkhan,a Lulu Ren,b Binbin Xie,b Chao Chen,c Yuchen Wang,a
and Hangxiang Wang a,*
a The First Affiliated Hospital; Key Laboratory of Combined Multi-Organ Transplantation,
Ministry of Public Health, School of Medicine, Zhejiang University, Hangzhou, 310003, PR
China.
b Department of Medical Oncology; Sir Run Run Shaw Hospital; School of Medicine, Zhejiang
University, Hangzhou, 310016, PR China.
c College of Life Sciences, Huzhou University, Huzhou, 313000, PR China.
Corresponding Author: Hangxiang Wang, The First Affiliated Hospital, School of Medicine,
Zhejiang University; 79, Qingchun Road, Hangzhou, 310003, China. Phone: +86-571-
88208173; Fax: +86-571-88208173; E-mail: wanghx@zju.edu.cn
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ABSTRACT
Ruthenium complexes have attracted a surge of interest as anticancer drug candidates because of
their low toxicity, diversity in mode-of-actions and non-cross drug resistance with conventional
platinum-based agents. Despite remarkable advances, only a limited number of ruthenium
complexes have been demonstrated to kill cancer cells and suppress metastasis simultaneously.
Here, two organometallic tetranuclear Ru(II) arene complexes (Ru-1 and Ru-2) have been
synthesized and evaluated for their in vitro activity against a panel of human cancer cell lines,
including a cisplatin-resistant human lung cancer A549 cell line. A superior cytotoxic activity of
the ruthenium complexes compared to cisplatin across distinct cell lines was observed. Further
examination of the mechanism indicated that anticancer activity was accomplished by inducing
apoptosis in cancer cells. In addition, we found that such compounds exhibited promising
antimetastatic activity and reduced the invasiveness of cancer cells. Importantly, choosing Ru-1
as a target compound, a significantly enhanced safety profile relative to cisplatin in animals was
validated, suggesting that these complexes can be used as promising candidates for cancer
therapy and deserve further investigation.
KEYWORDS:
tetranuclear Ru(II) arene complexes, in vitro cytotoxicity, apoptosis, antimetastasis, drug toxicity
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1. INTRODUCTION
Since the first discovery of cisplatin in the 1960s [1], platinum agents have been extensively used
as standard-of-care drugs to clinically treat a variety of types of cancer [2]. However, severe side
effects (e.g., nephrotoxicity and myelosuppression) and intrinsic or acquired resistance of using
these platinum agents have significantly impeded the therapeutic benefits. Consequently, there is
a considerable incentive for the exploration of novel non-platinum anticancer agents over the
past decades [3-5]. In line with this consideration, a number of metallodrugs have been
synthesized and evaluated in preclinical animal models, and some of them have entered different
stages of clinical trials [6, 7]. However, despite remarkable advances, none of metallodrugs have
been developed as successfully as platinum drugs.
Ruthenium complexes have attracted considerable interest as alternative chemotherapeutics to
platinum-based agents. Currently, several ruthenium compounds, such as [Na]trans-[Ru(N-
ind)2Cl4] (NKP1339) and [imiH]trans-[Ru(N-imi)(S-dmso)Cl4] (NAMI-A), have entered phase II
clinical trials [8]. For instance, NAMI-A shows negligible cytotoxicity toward tumor cells but is
efficient against tumor metastasis and angiogenesis. On the other hand, during the last decade,
ruthenium(II) (Ru(II)) arene complexes have been particularly interesting as anticancer drug
candidates, presumably due to their chemical properties, air stability, aqueous solubility, and
structural diversity [9]. The arene ligands are able to coordinate with the Ru(II) metals strongly,
thereby making the overall complexes inert toward substitutions and stabilizing Ru(II) ions in a
low oxidation state [10-13]. Thus, arene coordination can confer complexes with enhanced
hydrophobicity compared with other types of Ru(II) complexes, thereby altering cellular uptake.
In addition, the remaining three ruthenium coordination sites can be occupied with other ligands,
readily forming a “piano-stool” geometry, which is typical for Ru(II) arene complexes [14-16].
These types of “piano-stool” anticancer complexes have been demonstrated to show enhanced
cellular uptake and interact with intracellular targets specifically [12]. Prior studies also indicated
that Ru(II) complexes bearing a π-conjugated arene ligand and various mono- or bidentate
ligands with distinct donor atoms, including N-S [17], N-O [18], N-N [19], N-P [20], O-O [21],
P-P [22] [21], and NHC[23], are regarded as cytotoxic drug candidates without exhibiting
antimetastatic activity. Therefore, ruthenium complexes that have the ability to simultaneously
kill cancer cells and suppress metastasis are promising drug candidates for the management of
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metastatic cancers from a clinical perspective.
In the continuing effort to develop Ru(II) complexes, we designed novel tetranuclear Ru(II)
arene complexes bearing aroylhydrazone ligands and explored them as efficaciously cytotoxic
and antimetastatic agents against cancer cell lines. Our molecular design is based on hydrazone
motifs. Hydrazone and its analogs are a class of the most vital natural products, showing
biological activities including anticancer properties [24]. Moreover, hydrazone exerts anticancer
activity via different modes of action, such as kinase inhibition, telomerase inhibition, and cell
cycle arrest [25, 26]. These unique functions make hydrazones attractive motifs for the creation
of therapeutic ruthenium complexes [27]. Ru(II) complexes have been broadly examined either
as single anticancer agents or in combination with other cytotoxic agents; therefore,
hybridization of arene Ru(II) with other bioactive pharmacophores is an effective strategy to
design novel anticancer agents.
The development of multinuclear metal complexes is also of considerable interest as drug
candidates for anticancer treatment [28]. For example, BBR3464 [29], a trinuclear cisplatin
compound, is 2-3 orders of magnitude more active than cisplatin in cisplatin-resistant cell lines.
However, this agent did not show significant clinical activity in phase II trials [30], suggesting
further structural optimization are needed. Despite these advancements, multinuclear Ru(II)
arene complexes are scarcely explored as anticancer agents to date [31]. Herein, we rationally
incorporated a hydrazone moiety into tetranuclear Ru(II) arene complexes to improve the
pharmacological activity. To test this rationale, two tetranuclear arene Ru(II) hydrazone
complexes (i.e., Ru-1 and Ru-2) were designed, and the anticancer activity was evaluated.
The in vitro cell-based results supported the high potency of using our Ru(II) complexes to
induce cell death against a small panel of cancer cell lines, including a cisplatin-resistant human
lung cancer A549 cell line. More interestingly, the Ru-1 complex was able to inhibit the
migration and invasion of cancer cells. Finally, the in vivo toxicity studies clearly demonstrated
that Ru-1 possessed higher safety margins in animals compared to the cisplatin agent, warranting
further investigation.
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2. RESULTS AND DISCUSSION
Synthesis and characterization of tetranuclear Ru(II) arene complexes
The hydrazone ligands were easily prepared in good yields by the condensation of benzil (L-1) and
oxalaldehyde (L-2) with isoniazid at a 1:2 molar ratio following a previously established protocol, as
shown in Scheme S1 [14]. These ligands were further allowed to react with the ruthenium(II) arene
precursor [(η6-p-cymene)RuCl2]2) in a 1:2 molar ratio in the presence of Et3N as the base at room
temperature. This synthetic scheme enables the final tetranuclear arene Ru(II) complexes (i.e., Ru-1
and Ru-2) to be obtained with the common formula [Ru4(η6-p-cymene)4 (L1-2)(Cl)6] in excellent
yields (Figure 1).
Figure 1. Structures of tetranuclear Ru(II) arene hydrazone complexes.
The Fourier-transform infrared (FT-IR) spectra of the free ligands displayed
νC=N and νC=O stretching vibrations in the region of 1548-1555 cm−1 and 1650-1656 cm−1, respectively
(Figure S1). This suggested that free hydrazone ligands were presented in the amide form in the solid
state. However, absorption peaks that are assigned to νN–H and νC-O stretching vibrations were not
observed in the complexes Ru-1 and Ru-2 (Figure S2). The coordination of the ligands to the Ru(II)
ion through an azomethine nitrogen is expected to reduce the electron density in the azomethine link,
which could induce spectral change. We indeed observed such a shift of the vibrational frequency to
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1487-1499 cm−1 upon ligand complexation, indicating the coordination of azomethine nitrogen to the
Ru(II) ion. On the other hand, the absorption bands in the spectral range of 1334-1336
cm−1 corresponded to the imidolate oxygen that was expected to coordinate to the Ru(II) metal.
Furthermore, the complexes showed the strong bands corresponding to νM-N in the region of 514-523
cm−1 (Figure S2). Therefore, the FTIR spectra confirmed the mode of coordination of the hydrazone
ligand to the ruthenium(II) ion via the azomethine nitrogen and imidolate oxygen [18, 32].
Accordingly, the ligands are coordinated to metal via imine nitrogen and imidolate oxygen
atoms.
Furthermore, we determined the absorption of Ru-1 and Ru-2 using a UV-vis spectrophotometer.
As depicted in Figure S3, Ru-1 and Ru-2 exhibited strong absorption at approximately 307-310 nm
and 255–257 nm, which correspond to highly intense π–π* and n–π* ligand-centered transitions,
respectively. Moreover, the relatively low absorption in the visible region from 479-484 nm could be
assigned to MLCT transitions [33]. We then carefully characterized both complexes using 1H and 13C
NMR spectra, and the characteristic signals from coordinated p-cymene appeared at the expected
shifts [34]. The 1H NMR spectra of both complexes were recorded in CDCl3 and are shown in Figure
S4-5. Multiplets observed in the region of δ 7.09-8.73 ppm were assigned to the aromatic protons of
hydrazone ligands in the complexes. In addition, the singlet peaks at 10.8 ppm are due to the presence
of the –NH proton in free ligands. The cymene protons appeared in the region of δ 5.83-3.67 ppm. In
addition, the methyl group of p-cymene appeared as a singlet at approximately δ 2.31–2.34 ppm.
Furthermore, the two isopropyl methyl protons of p-cymene appeared as two doublets in the region
of δ 0.55-1.23 ppm, and the methine protons are present in the region of δ 2.34-2.87 ppm as a septet.
Furthermore, the 13C NMR spectra of the complexes are shown in Figure S6-7; the signals of the
aromatic carbons showed singlet resonances at approximately 112.4-136.4 ppm. The resonance
due to C=N and C–O was observed at approximately 165.0 ppm and 174.0 ppm, respectively.
The signals observed at 78.6-85.6 ppm and 7.9–102.0 ppm confirmed the presence of the p-
cymene ligand in the complexes [25]. ESI-MS analysis was performed to validate the final
adducts, showing that the peaks at m/z 1460.6 and 1305.6 can be assigned to the tetranuclear
form of the Ru-1 and Ru-2 complexes, respectively (Figure S8-9).
After systemic injection, noncovalent interactions between ruthenium complexes with
abundant serum proteins will occur, which may cause loss-of-function of the administered drugs.
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The formation of new noncovalent species could manifest as a gradual shift in the UV-vis profile
[35]. Therefore, to examine this possibility, we monitored the UV-vis spectra of the complexes
in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and
in phosphate-buffered saline (PBS) containing 10% DMSO [36]. In DMEM with 10% FBS, both
the Ru-1 and Ru-2 complexes exhibited minimal changes in the UV-vis profiles, indicating
negligible associations with serum proteins [37]. In addition, the two complexes remained intact
toward hydrolysis in the mixture of DMSO:PBS, manifesting high stability over 72 h at 8 h
intervals (Figure S10).
In vitro cytotoxicity against human cancer cells
Next, we assessed the in vitro cytotoxicity of the Ru-1 and Ru-2 complexes against various
human cancer cell lines (i.e., A549, A549cisR, MCF-7, LoVo, and HuH-7). After a 72-h incubation of
the compounds, the cell viability was determined by the conventional MTT assay. In this experimental
setting, cisplatin was also included as a reference. We summarized the values of the half maximal
inhibitory concentration (IC50) in Table 1. Undoubtedly, we observed the superior activity of using
Ru-1 and Ru-2 relative to cisplatin in all tested cancer cell lines (Figure 2). More interestingly, both
complexes Ru-1 and Ru-2 showed much lower IC50 values in cisplatin-resistant A549cisR cells. For
instance, cisplatin displayed an IC50 value of 17.24 ± 1.5 µM in A549cisR cells; however, Ru-1
and Ru-2 significantly decreased these values to 3.39 ± 0.5 and 5.70 ± 0.3 µM in A549cisR cells,
respectively. Several cellular processes can contribute the resistance of cells toward cisplatin,
including the decrease in intracellular drug uptake, the enhanced level of thiol-rich proteins (e.g.,
metallothionein and glutathione), the increased DNA repair, and the tolerance of cell-death
pathways [38]. However, we observed potent cytotoxicity of Ru-1 and Ru-2 in cisplatin-resistant
A549 cells, as evidenced by the drug resistance factor (Table 1). Therefore, the complexes
presented here hold the potential to overcome drug resistance induced by cisplatin.
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Figure 2. In vitro cytotoxicity in (a) A549 cells, (b) A549cisR cells, (c) MCF-7 cells, (d) LoVo cells and (e)
HuH-7 cells after 72-h treatment with Ru-1, Ru-2 and cisplatin.
Table 1. Antitumor potential of Ru(II) arene complexes after 72 h of incubation (expressed as IC50±SD in
µM). [a]
Cell line
A549
Type of cancer cell line
Lung carcinoma
Ru-1 (FI)[b]
cisplatin
Ru-2 (FI)
1.39 ± 0.15 (1.9)
3.39 ± 0.47 (5.1)
2.43
1.41 ± 0.23 (1.9)
5.70 ± 0.33 (3.0)
4.04
2.68 ± 0.16
17.24 ± 1.52
6.43
A549cisR Lung carcinoma
[c]RF
MCF-7
LoVo
Breast adenocarcinoma
2.63 ± 0.30 (1.6)
2.04 ± 0.10 (1.7)
1.71 ± 0.20 (1.8)
2.91 ± 0.35 (1.4)
2.09 ± 0.77 (1.6)
1.98 ± 0.30 (1.5)
4.24 ± 0.28
3.45 ± 0.27
3.04 ± 0.29
Colon adenocarcinoma
HuH-7
Hepato cellular carcinoma
RAW 264.7 Murine macrophage cells
10.47 ± 1.18 (0.70) 7.91 ± 1.50 (0.92) 7.30 ± 1.20
Human umbilical vein
HUVEC
6.16 ± 0.26 (0.80) 5.89 ± 0.18 (0.83) 4.90 ± 0.4.3
endothelial cells
[d] log Po/w
-0.68
0.53
--
[a] Determined by MTT assay. IC50 values indicate the molar concentrations of whole Ru(II) complexes
Ru-1 and Ru-2, and cisplatin required to inhibit 50% of cell growth with respect to control groups. The
data obtained are based on the average of three independent experiments, and the reported errors are the
corresponding standard deviations.
[b] FI (fold increase) is defined as IC50 (cisplatin)/IC50 (Ru-1 or Ru-2).
[c] RF (resistant factor) is defined as IC50 in A549cisR/IC50 in A549.
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[d] The log Po/w values determined via the shake-flask method against n-octanol/water (1:1, v/v) partition.
Drug selectivity in killing cancer cells over noncancerous cells
One of the major limitations of existing antitumor drugs is their poor selectivity for killing
cancer cells over noncancerous cells, which usually causes side effects and impairs the dose
intensification of drugs in clinic. To assess whether Ru-1 and Ru-2 exert the activity as cancer-
selective agents, we additionally tested the cytotoxicity in noncancerous cell lines including
murine macrophage RAW 264.7 and human umbilical vein endothelial HUVEC cells. Cisplatin
exhibited the high cytotoxicity in both cells; the IC50 values in noncancerous cells are
comparable with those in cancer cells (Table 1 and Figure S11). Interestingly, the complexes
Ru-1 and Ru-2 were less toxic than cisplatin in both tested RAW 264.7 and HUVEC cells.
The selectivity index (SI) for each complex can be defined as the ratio of the IC50 value in
noncancerous cells to the IC50 value in cancer cells and is summarized in Figure 3. The
cytotoxicity difference between cancer cells over noncancerous cells was higher of using the
Ru(II) complexes than that of using cisplatin. In particular, Ru-1 demonstrated the superior
selectivity for killing cancer cells over noncancerous cells (Figure 3). These results thus
evidenced the advantages of the complexes Ru-1 and Ru-2 as cancer-selective agents, and they
should have the potential to reduce the toxicity to healthy cells and organs when considering the
in vivo use.
Figure 3. The selectivity index of Ru(II) complexes and cisplatin. The selectivity index is defined as the ratio
of the IC50 value in RAW 264.7 or HUVEC cells to the IC50 value in cancer cells.
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The EdU incorporation assay was further performed to evaluate the antiproliferation activity
of the Ru-1 and Ru-2 complexes in A549 cells after 24 h of treatment (Figure 4a). We chose the
IC20 concentration for this in vitro experiment. The results clearly showed that the cisplatin
treatment only produced a negligible effect on the proliferation of A549 cells, whereas Ru-1 and
Ru-2 significantly reduced their activity. The induction of A549 cell apoptosis by Ru-1, Ru-2
and cisplatin was investigated by apoptosis assays using AO/EB staining and fluorescence
microscopy. AO is a vital dye that can stain both live and dead cells and shows green
fluorescence. EB only stains cells that have lost their membrane integrity and exhibits red
fluorescence. Necrotic cells are stained in red but have nuclear morphologies that resemble those
of viable cells. Apoptotic cells appear green and exhibit morphological changes, such as cell
blebbing and the formation of apoptotic bodies [39]. As shown in Figure 4c, untreated A549
cells showed consistently green fluorescence with normal morphologies; however, the A549 cells
treated with Ru-1, Ru-2 and cisplatin showed red orange fluorescence with fragmented
chromatin and apoptotic bodies under fluorescence microscope observation, suggesting that low
concentrations of Ru-1, Ru-2 and cisplatin predominantly induced apoptosis in A549 cells [40].
Lipophilicity plays a vital role in influencing the antitumor activity of a given Ru(II) arene
compound. We thus determined the distribution coefficients (log P) by employing the “shake-
flask” method to correlate the cytotoxicity with this factor. As illustrated in Figure S12 and
Table 1, Ru-1 and Ru-2 exhibited log Po/w values of -0.68 and 0.53, respectively. The Ru-1
complex, showing high lipophilicity, may more easily penetrate cell membranes to enhance
apoptosis than the Ru-2 complex [41].
The superior cytotoxic activity of Ru-1 and Ru-2 may also be partially attributed to the
extended π-π* conjugation of phenyl ring resulting from ligand coordination of the arene Ru(II)
ion [42]. Numerous Ru(II) arene complexes have been assessed for their antiproliferative activity
[14, 43]. However, complexes bearing hydrazone units with multiple Ru(II) metal centers have
not been explored thus far. This lack of study prompted us to further develop novel multinuclear
Ru(II) candidates and explore the efficacies for cancer therapy [44]. Gratifyingly, through
rationally tailoring four metal active sites, the in vitro activities could be further optimized and
improved on the basis of the tetranuclear Ru(II) arene scaffold. Moreover, the increased activity
and selectivity of these tetrametallic complexes might be attributed to synergistic cooperation
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between the four metals and the hydrazone ligands, thereby supporting our design rationale for
generating multinuclear Ru(II) candidates. Further, attempts could be made to create new Ru(II)
complexes as potential anticancer candidates by incorporating different spacers or altering
substituents on the arene moiety.
Figure 4. a) A Click-iT EdU assay for determining the proliferation of A549 cells. The cells were treated
with Ru-1 (0.4 µM), Ru-2 (0.4 µM) and cisplatin (0.9 µM) for 24 h. b) Quantification of cell
proliferation. The data are presented as the means ± s.d.; n≥5 regions with a total of 1500-2000 cells
analyzed; **p<0.01. c) Dual AO/EB fluorescent staining of A549 cells after treatment with Ru-1 (0.4
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µM), Ru-2 (0.4 µM) and cisplatin (0.9 µM) for 24 h. d) Cell death rates in AO/EB staining were
quantified. The data are presented as the means ± s.d.; n≥5 regions with a total of 1500-2000 cells
analyzed; **p<0.01.
Ru(II) arene complexes induce cancer cell apoptosis
Apoptosis and cell cycle arrest in the cells are the main reasons accounting for the inhibition of
cell growth [45]. To examine whether the inhibition of cancer cell growth was a consequence of
apoptosis induced by the complexes, we performed an Alexa Fluor 488 Annexin V/propidium
iodide (PI) double-staining assay in A549 cells. During apoptosis, phosphatidylserine will be
exposed on the outer leaflet of the cell membrane, which can be specifically detected by the
binding of fluorescently labeled annexin V [46]. After treatment of the cells (1 µM, Ru-1, Ru-2
and cisplatin) for 24 h, fluorescence-activated cell sorting (FACS) was conducted to analyze the
number of apoptotic cells. As shown in Figure 5a, the incubation of cells with the complexes
induced a high level of apoptosis, and mainly, a late apoptotic event was observed. In addition,
compared with cisplatin, Ru-1 and Ru-2 were more potent in inducing apoptosis in A549 cells,
which was consistent with the MTT assay results [18]. Deregulated cell-cycle control is a
fundamental aspect of cancer, and the process is related to the proliferation and death of cancer
cells [37]. Therefore, the e ect of complexes on cell cycle progression was investigated using
FACS. As shown in Figure 5c, upon exposure of A549 cells to drugs at a 1 µM concentration
for 24 h, the percentage of cells in the G0/G1 phase decreased from 67.13% to 28.3%, 24.18%,
and 30.5% for Ru-1, Ru-2 and cisplatin, respectively. Notably, Ru-1 and Ru-2 elicited a strong
S phase arrest in cells, accounting for 68.2% and 73.10% of the cell population, respectively
(untreated cells, 21.30%). Thus, the enrichment of the S phase of cancer cells may result in
apoptosis by disrupting the cell cycle [47].
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Figure 5. Ru(II) complexes significantly induce apoptosis in A549 cells. The concentrations of Ru-1,
Ru-2 and cisplatin used in these studies were 1 µM. (a-b) Apoptosis of A549 cells after treatment with
compounds was determined by an Alexa Fluor 488 Annexin V/propidium iodide (PI) double-staining
assay. The apoptotic ratio is shown in the upper panel, and the quantitative results are shown in the lower
panel. (c-d) A549 cells were treated with drugs for 24 h, and the cell cycle was analyzed by FACS. The
histogram shows the distribution of the cell cycle, indicating that Ru(II) complexes mainly arrested cells
at the S phase arrest.
Ru(II) arene complexes inhibit cell invasion and migration
Metastasis and invasion are important incidences in the later period of cancer progression.
Therefore, the inhibition of metastasis and invasion is critical for efficient cancer treatment [48].
Cell migration occurs during physiological processes, which play a crucial role in the
progression of various diseases, including cancer. In vitro migration assays are essential to
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understand the mechanism of cell migration and to identify the inhibitory ability of the
complexes. The migration is measured by determining the space in the wound closure that is
occupied by cells 24 h after treatment (Figure 6a and b). In A549 cells, migration was
significantly reduced after treatment with Ru-1 and Ru-2 at 0.4 µM, while cisplatin (0.9 µM)
was clearly less active. For example, the ratios of wound closure were 18.8%, 23.3%, and 42.3%
for Ru-1, Ru-2, and cisplatin, respectively [49]. In addition, during the progression of cancer,
tumor cells acquire the ability to penetrate the surrounding tissues in a process called invasion.
These metastatic cells enter lymphatic or vascular circulation and move through the circulatory
system and attach to a new distant location, producing secondary tumors. An effective anticancer
drug should be able to impair the movement of cancer cells from the primary sites to other
organs in cancer patients. Therefore, the invasiveness of cancer cells was examined by Transwell
assay [50]. The invasive A549 cells were seeded on a Matrigel-coated membrane and treated
with Ru-1, Ru-2 and cisplatin (0.4, 0.4 and 0.9 µM, respectively) for 24 h. Upon the treatment
with the Ru-1 and Ru-2 complexes, the numbers of invaded cells were significantly reduced
compared to those of the untreated cells (Figure 6c and d) [51]. Taken together, these results
provide evidence that in addition to cytotoxic activity, these tetranuclear Ru(II) complexes have
the ability to suppress metastasis and invasion of cancer cells.
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Figure 6. a) The migration of A549 cells was observed by a wound-healing assay. The cells were treated
with Ru-1, Ru-2 and cisplatin for 24 h. b) Quantification analysis showed that Ru-1, Ru-2 and cisplatin
reduced A549 migration. c) Invasion of A549 cells was observed by Transwell assay. A549 cells were
treated with drugs for 24 h, and the invading cells in the lower chamber were visualized by microscopy.
d) The number of invading cells upon treatment with drugs was significantly reduced compared with that
of the untreated cells.
In vivo systemic toxicity
Finally, to explore the systemic toxicity, a series of animal experiments were carefully
conducted. Because of the superior cytotoxicity and antimetastatic activity of Ru-1 observed in
the cell-based results, we chose Ru-1 as a model compound to evaluate the safety profile in
healthy ICR mice. The mice (n = 9, four females and five males in each group) were
intraperitoneally injected with Ru-1 (dissolved in DMSO, 3, 6, 12, 18, and 25 mg/kg) five
successive times every other day. For comparisons, saline, the vehicle DMSO and cisplatin
administered in its clinical formulation (3, 6, and 12 mg/kg) were injected. The body weights and
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survival of the mice were recorded during the period of the study. Unfortunately, all of the mice
receiving cisplatin at a dose of 6 mg/kg died, whereas the survival rate in Ru-1-treated mice (6
mg/kg) was 100% (Figure 7), and the body weight remained stable (Figure S13). We further
intensified the doses of the Ru-1 complex in animals and found that seven out of nine mice could
tolerate the dose of 12 mg/kg. The LD50 values were estimated on the basis of the threshold at
which the body weight loss exceeded 20%. Impressively, the LD50 value of Ru-1 (18.1 mg/kg,
95% LD50, 13.9-25.8 mg/kg) was at least 6-fold higher than that of cisplatin (less than 3 mg/kg).
Figure 7. Survival of healthy ICR mice following intraperitoneal injection of saline, DMSO, cisplatin and
Ru-1 for five successive times every other day. The mice were defined as dead when the body weight loss
exceeded 20%. Arrows indicate the intraperitoneal injections.
Prior studies showed that cisplatin could lead to serious symptoms such as hyperemia, edema,
and inflammatory infiltration of the kidney, accompanied by hydropic or ballooning
degeneration of proximal tubular epithelial cells, cytoplasmic relaxation, and exudates from
glomerular capsules [52, 53]. Thus, we performed histological analyses to examine the damage
of the drugs to major organs. Representative images using hematoxylin-eosin (H&E) staining are
shown in Figure 8 and Figure S10. The results revealed a mass of vacuolization (i.e.,
accumulation of white vesicles) in the cell cytoplasm of renal tubules in the cisplatin-treated
mice, which indicated high cisplatin-induced renal damage. Fortunately, no damage was
observed in major organs, including the kidney, in the mice receiving Ru-1 (6 mg/kg), showing
similar histological characteristics as those of saline-treated mice. More encouragingly,
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administration of Ru-1 at high doses of up to 12 mg/kg also did not cause obvious damage to
these organs (Figure S10).
Figure 8. H&E stained tissue slices of the organs (heart, kidney, liver, lung, and spleen) excised from the mice
treated with saline, cisplatin (3 mg/kg) and Ru-1 (6 mg/kg). The images on the right are the enlargement of the
region in the white rectangle.
To further validate the damage of cisplatin and Ru-1 toward the kidney, we conducted the
TUNEL assay. As shown in Figure 9, intraperitoneal injection of cisplatin resulted in extensive
apoptosis in kidney sections, which was well correlated with the H&E results. Conversely,
negligible nephrotoxicity in the mice administered Ru-1 at a dose of 6 mg/kg was observed.
Taken together, all results suggest that the compound Ru-1 exhibits low systemic toxicity and is
potentially more tolerated by animals than cisplatin.
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Figure 9. Representative TUNEL analysis of the excised kidneys from the mice treated with saline,
cisplatin and Ru-1 complexes. The images on the second line are the enlargement of the region in the
white rectangle. Obviously, potent induction of apoptosis was observed in the kidneys of the mice
receiving cisplatin (3 mg/kg) but not in the kidneys of the mice receiving Ru-1 (6 mg/kg).
3. CONCLUSION
In conclusion, two novel tetranuclear Ru(II) arene complexes (Ru-1 and Ru-2) have been
synthesized and examined for their in vitro activity against human cancer cell lines. The
complexes, especially Ru-1, exhibited higher cytotoxic potency relative to cisplatin by
efficaciously inducing cell apoptosis. In addition, the complexes showed antimetastatic activity,
reducing the invasiveness of cancer cells. Finally, in vivo toxicity studies suggested a remarkable
alleviation of systemic toxicity using the Ru-1 complex compared with cisplatin. Given the
intrinsic feature of Ru-1 in circumventing cisplatin resistance and in showing negligible
nephrotoxicity, these Ru(II) arene complexes should find potential use in patients with impaired
renal function and deserve further investigation.
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4. Experimental section
Materials and methods for the synthesis of tetranuclear Ru(II) arene complexes
6-p-cym)RuCl2]2 was purchased from Sigma-Aldrich (Shanghai, China). Benzil, oxalaldehyde
[(η
and isoniazid were purchased from TCI (Shanghai, China). All other compounds and solvents
were purchased from J&K Chemical (Shanghai, China). All reactions were performed in a dry
atmosphere. Thin-layer chromatography (TLC) was performed on silica gel 60 F254 precoated
aluminum sheets (Merck) and visualized by fluorescence quenching. Chromatographic
purification was accomplished using flash column chromatography on silica gel (neutral,
Qingdao Haiyang Chemical Co., Ltd). The Fourier-transform infrared (FT-IR) spectra of the
samples were recorded on an Avatar370 IR spectrophotometer (Thermo Nicolet, USA) using
pressed KBr discs. 1H and 13C NMR spectra were recorded in DMSO or CDCl3 on a Bruker 400
spectrometer and calibrated to the residual solvent peak or tetramethylsilane (= 0 ppm).
Multiplicities are abbreviated as follows: s = singlet, d = doublet, m = multiplet. Mass
spectrometry ESI-MS was recorded on an AB Triple TOF 5600+System (AB SCIEX,
Framingham, USA). The theoretical calculations were performed using IsoPro software [54].
Reverse-phase high-performance liquid chromatography (RP-HPLC) measurements were carried
out on a Shimadzu LC 20 system.
Preparation of benzil isoniazid ligands
A mixture of isoniazid (10 mmol) and benzil and oxalaldehyde (5 mmol) in ethanol (20 mL)
containing a drop of conc. HCl was refluxed for 30 min [55, 56]. The white solid formed was
collected by filtration, washed with ethanol and diethyl ether and dried in vacuo. Yield: 75–90%.
NMR data are shown in Figure S14-15.
Synthesis of tetranuclear Ru(II) arene isoniazid complexes
A mixture containing [(η
6-p-cym)RuCl2]2 (10 mmol), isoniazid ligands (10 mmol) and Et3N (0.2 ml)
in 1:1 equivalent of benzene (20 ml) was stirred at room temperature for 6 h. The orange precipitate
obtained was filtered, washed with hexane and dried in vacuo. The progress of the reaction was
monitored via TLC.
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[Ru4(η
6-p-cymene)4 (L-1)(Cl)6] (Ru-1)
FT-IR (KBr):
ṽ
= 1499.1, 1334.3, 523.0 cm-1. UV-Vis (CH3CN, λmax/nm) (εmax/dm3 mol−1 cm−1):
484 (1581), 310 (4982), 258 (8768). ESI-MS (+ve mode): m/z 1460.6 [M + H - 4Cl]+
1
(calcd m/z 1459.1). H NMR (400 MHz, DMSO)
δ
(ppm): 7.09–8.72 (m, 18H, Ar), 5.78-5.83 (m,
4H, p-cym-H), 5.01-5.66 (m, 8H, p-cym-H), 3.67-4.02 (m, 4H, p-cym-H), 2.80-2.87 (m, 2H, p-cym
CH(CH3)2), 2.39-2.46 (m, 2H, p-cym CH(CH3)2), 1.99-2.09 (s, 12H, p-cym CCH3), 1.16-1.23 (m,
13
6H, p-cym CH(CH3)2), 0.55-1.23 (m, 6H, p-cym CH(CH3)2). C{1H} NMR (100 MHz, CDCl3,
δ,
ppm): 8.8, 18.5, 18.9, 21.5, 22.2, 22.6, 29.6, 30.8, 30.9, 79.6, 80.5, 80.7, 81.5, 82.2, 83.0, 85.6, 99.3,
100.6, 101.3, 102.0, 127.8, 128.1, 128.4, 128.5, 128.9, 129.0, 129.8, 129.8, 130.0, 130.1, 130.3, 130.5,
131.7, 133.9, 134.6, 135.8, 136.3, 136.4, 165.1, 166.4, 173.1, 174.0, 193.5. Anal. Calc. for
C66H74Cl6N6O2Ru4: C, 49.53; H, 4.66; N, 5.25%. Found: C, 50.06; H, 4.47; N, 5.13%. Orange
solid. Yield = 0.427 g (83%). HPLC (1:9 ACN/ H2O as mobile phase) (% purity):
nm; RT - 3.24 min.
≥95 % at 220
[Ru4(η
6-p-cymene)4 (L-2)(Cl)6] (Ru-2)
= 1487.8, 1336.8, 514.7 cm-1. UV-Vis (CH3CN, λmax/nm) (εmax/dm3 mol−1 cm−1):
479 (1212), 362 (2966), 307 (3133), 257 (5536). ESI-MS (-ve mode): m/z 1305.6 [M - H -
4Cl]+ (calcd m/z 1306.3). 1H NMR (400 MHz, DMSO)
(ppm): 7.49–8.73 (m, 8H, Ar), 5.83-5.77 (m,
FT-IR (KBr):
ṽ
δ
8H, p-cym-H), 5.14-5.32 (d, 4H, p-cym-H), 3.67-4.60 (d, 4H, p-cym-H), 3.02 (s, 6H, CH3), 2.79-2.86
(m, 2H, p-cym CH(CH3)2), 2.34-2.50 (m, 2H, p-cym CH(CH3)2), 2.08 (s, 6H, p-cym CCH3), 2.0 (s,
6H, p-cym CCH3), 1.18-1.20 (d, 12H, p-cym CH(CH3)2), 0.98-1.02 (d, 12H, p-cym
CH(CH3)2). 13C{1H} NMR (100 MHz, CDCl3,
δ, ppm): 7.9, 17.81, 20.9, 21.5, 45.2, 54.6, 78.8, 81.6,
82.7, 84.3, 98.1, 100.5, 112.4, 125.1, 127.7, 129.3, 129.7, 135.5, 160.6, 165.1, 174.1. Anal. Calc. for
C54H66Cl6N6O2Ru4: C, 44.71; H, 4.59; N, 5.80%. Found: C, 45.01; H, 4.77; N, 5.99%. Red-
orange solid. Yield = 0.303 g (81%). HPLC (1:9 ACN/ H2O as mobile phase) (% purity):
220 nm; RT - 3.24 min.
≥95 % at
The assessment of stability using UV–Vis spectroscopy
The complexes Ru-1 and Ru-2 were dissolved in phosphate-buffered saline (PBS) containing
10% DMSO and DMEM containing 10% FBS (without phenol red) at a final concentration of 50
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ACCEPTED MANUSCRIPT
µ
M. The absorption of the samples was monitored by UV–Vis spectroscopy over 24 h at 4 h
intervals.
Determination of Log P
Log Po/w values of Ru-1 and Ru-2 were determined using the shake-flask method. The
complexes were dissolved in water that was presaturated with n-octanol (for 24 h and left to
stand until phase separation occurred). The UV–Vis spectrum for each sample was obtained, and
the absorbances at the λmax of each compound were determined. Equal volumes of n-octanol
were added to each sample solution, and the heterogeneous mixtures were shaken for 2 h before
centrifuging at 4000 rpm for 1 min to achieve phase separation. The final absorbance of the
aqueous phase at the λmax of each compound was determined, and their water–octanol partition
coefficient was calculated. All experiments were performed in triplicate.
Cell culture
A549, A549cisR, MCF-7, LoVo, and HuH-7 cells were purchased from the cell bank of the
Chinese Academy of Sciences (Shanghai, China). A549, A549cisR and LoVo cells were cultured
in RPMI-1640 (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco). MCF-7 and
HuH-7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10%
FBS and 1% nonessential amino acids. All cells were maintained at 37 °C in 5% CO2.
Cell proliferation study by the EdU test
A549 cells were seeded into flat-bottomed 48-well plates with 2 × 104 cells per well and
incubated at 37 °C under a 5% CO2 atmosphere for 24 h. Ru-1, Ru-2 and cisplatin (0.4, 0.4 and
0.9 µM, respectively) were then added to the cells and incubated for an additional 24 h at 37 °C.
DNA synthesis was quantified at the end of the drug treatment using a Click-iT EdU Alexa Fluor
488 Assay Kit (Invitrogen) according to the manufacturer’s protocol. Briefly, EdU (5-ethynyl-2′-
deoxyuridine) was first added to each well and incubated for 2 h at 37 °C. Then, the cells were
fixed for 15 min at room temperature by adding 4% formaldehyde. Next, 0.5% Triton X-100 was
added to the cells and incubated for 10 min. Subsequently, azide-labeled Alexa Fluor 488 was
added to the cells, and they were incubated for 30 min in the dark. After staining the nuclei with
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Hoechst 33342 (Invitrogen) for 15 min, the cells were imaged using fluorescence microscopy
(Olympus, IX71).
Acridine orange-ethidium bromide (AO-EB) staining
Dual AO-EB fluorescent staining was used to evaluate cell apoptosis in A549 cells upon
treatment with Ru-1, Ru-2 and cisplatin. Briefly, cells were seeded in 24-well plates at a density
of 5000 cells/well and incubated at 37 °C for 24 h. Ru-1, Ru-2 and cisplatin (0.4, 0.4 and 0.9
µM, respectively) were incubated with cells. After 24 h of incubation, the staining solution (10
µL) containing AO (100 µg/mL) and EB (100 µg/mL) was added to each well (500 µL).
Immediately, the cells were visualized using a fluorescence microscope (Olympus, BX-60,
Japan), and the percentage of dead cells was quantified in at least three random microscopic
fields.
Flow cytometry to determine cell cycle distribution and apoptosis
A549 cells were seeded into 6-well plates, incubated at 37 °C, and allowed to attach for 24 h.
Then, fresh media containing 1 µM Ru-1, Ru-2 or cisplatin were added and further incubated for
another 24 h. The untreated cells were included as the control. After drug treatment, the cells
were centrifuged at 1000 RPM for 5 min and washed with cold PBS. The cells were fixed with
75% ethanol at 4 °C overnight. The cells were then collected and washed twice with PBS.
Thereafter, the cells were stained with a solution containing propidium iodide (PI) (50 µg/mL)
and incubated in the dark for 30 min. Cell cycle distribution was then analyzed with a BD
FACSCantoTM II flow cytometer.
The cell apoptotic rate was determined by flow cytometry analysis with the fluorescein
isothiocyanate (FITC) Annexin V Apoptosis Detection Kit (Multi Sciences, China). A549
cells were collected by trypsinization, washed twice and resuspended in 500
µL 1 × binding
buffer with 5 L of FITC Annexin V and 10 L of PI. After incubation for 15 min, the
µ
µ
samples were subjected to analysis by flow cytometry. The results were analyzed with the BD
FACS Calibur™ system.
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Wound healing assay
A549 cells were grown to 90% confluence in a six-well plate after 24 h at 37 °C. A scratch
across the cell monolayer was produced using a sterile 10 L pipet tip. Following the treatment
µ
of cells with Ru-1, Ru-2 and cisplatin (0.4, 0.4 and 0.9 µM, respectively) or 0.1% DMSO as a
control, images of wounds were acquired by optical microscopy at time 0 after scratching and at
the end of a 24 h incubation period. To quantify the migration rate, the distance of the initial
wound was compared with the distance of the healing wound at 24 h after the scratch by the
pipet tip.
Cell invasion assay
Matrigel matrix (BD Biosciences) was placed into Transwell filters (30
Millipore) and allowed to complete gelation for 1 h at 37 °C. A total of 200
medium containing 4 × 104 A549 cells was added into the top chambers, and 700
µ
L/well, 8.0
L of RPMI-1640
L of RPMI-
µm PET,
µ
µ
1640 medium supplemented with 10% FBS was placed in the bottom chambers. Subsequently,
the wells in the top chambers were treated with Ru-1, Ru-2 and cisplatin (0.4, 0.4 and 0.9 µM,
respectively) for 24 h at 37 °C. After 24 h of incubation, cotton swabs were used to remove the
Matrigel and cells that remained in the top chambers. Next, A549 cells on the bottom surface of
the membrane were fixed with methanol for 10 min and stained with 0.5% crystal violet for 15
min. The invading cells on the membrane were washed with distilled water and photographed
under an optical microscope. The cells were counted in at least three random microscopic fields
(magnification, ×200). The experiments were repeated three times.
Animal experiments
All animal studies were conducted in accordance with the National Institute Guide for the Care
and Use of Laboratory Animals. The experimental protocols were approved by the Ethics
Committee of the First Affiliated Hospital, Zhejiang University School of Medicine.
In vivo toxicity
Healthy ICR mice (4-5 weeks old) were randomized into 9 groups (n = 9, four females and five
males in each group) and intraperitoneally injected with different doses of Ru-1 solution in
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DMSO (50
µ
L) every three days five times. Saline, DMSO, and cisplatin solution (Hospira
Australia Pty Ltd.) were used as controls. Cisplatin was administered at doses of 3, 6, and 12
mg/kg. Ru-1 was administered at doses of 3, 6, 12, 18, and 25 mg/kg. The body weight changes
of mice were monitored.
After receiving two injections of saline, DMSO, cisplatin and Ru-1, two mice in each group
were randomly selected and sacrificed by CO2 inhalation. The major organs, such as kidneys,
livers, lungs and spleens, were collected and fixed with 4% formaldehyde. Then, the tissues were
embedded in paraffin and sectioned into 5-µm-thick slices. These slices were stained with
hematoxylin and eosin (H&E, Sigma). For the terminal deoxynucleotidyl transferase-
mediated dUTP nick-end labeling (TUNEL) assay, the dewaxed and rehydrated kidney
sections were incubated with proteinase K for 15 min at 37 °C, rinsed with PBS twice, and
rinsed with the TUNEL In Situ Cell Death Detection Kit according to the manufacturer's
protocol (Sigma-Aldrich). The TUNEL-stained cells were counterstained with DAB (DAKO)
and visualized by optical microscopy in 10 random fields for each group.
Acknowledgement
This work was supported by the Zhejiang province fund (No. 519000-X81801), the National
Natural Science Foundation of China (Nos. 81571799 and 81773193), and the Zhejiang Province
Preeminence Youth Fund (LR19H160002).
Conflict of interest
The authors declare no potential conflicts of interest.
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