Potent half-sandwich Ru(Ⅱ) N^N (aryl-BIAN) complexes: Lysosome-mediated apoptosis, in vitro and in vivo anticancer activities

Article abstract of DOI:10.1016/j.ejmech.2020.112763

Herein a new series of organometallic half-sandwich Ru(Ⅱ) complexes bearing aryl-BIAN chelating ligands with various electron-withdrawing and electron-donating substituents have been developed as theranostic agents. All the complexes display much higher anti-proliferative potency than the clinical chemotherapeutic drug cisplatin towards seven cancer cell lines. The anti-proliferative efficacy of these complexes is correlated to their electron-withdrawing ability. Interestingly, complex Ru1 also potently suppresses cancer cell migration in vitro and effectively inhibit tumor growth in vivo in a CT26 colon cancer mouse xenograft model. Mechanisms of action studies display that Ru1 can favorably accumulate in lysosome and exerts anti-cancer potency by inducing a series of events related to lysosomal dysfunction in CT26 cells. Interestingly, inhibition of lysosomal enzymes leads to suppression of cytotoxicity and apoptosis induced by Ru1. Our results elucidate that complex Ru1 can elicit cytotoxicity through lysosome-mediated apoptosis in vitro and suppress tumor growth in vivo.

Full text of DOI:10.1016/j.ejmech.2020.112763

European Journal of Medicinal Chemistry 207 (2020) 112763
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Potent half-sandwich Ru(II) N^N (aryl-BIAN) complexes: Lysosome-
mediated apoptosis, in vitro and in vivo anticancer activities
,
,
Zhishan Xu a b, Jie Huang c **, Deliang Kong b, Yuliang Yang b, Lihua Guo b, Xianglei Jia d,
Genshen Zhong e, Zhe Liu b
,
*
a College of Chemistry, Chemistry Engineering and Materials Science, Shandong Normal University, Jinan, 250014, China
b Institute of Anticancer Agents Development and Theranostic Application, The Key Laboratory of Life-Organic Analysis and Key Laboratory of
Pharmaceutical Intermediates and Analysis of Natural Medicine, Department of Chemistry and Chemical Engineering, Qufu Normal University, Qufu,
273165, China
c Qingdao University of Science and Technology, Qingdao, 266061, China
d Henan Key Laboratory of Neural Regeneration, The First Affiliated Hospital of Xinxiang Medical University, Weihui, 453100, China
e Henan Collaborative Innovation Center of Molecular Diagnosis and Laboratory Medicine, School of Laboratory Medicine, Xinxiang Medical University,
Xinxiang, 453003, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein a new series of organometallic half-sandwich Ru(II) complexes bearing aryl-BIAN chelating li-
gands with various electron-withdrawing and electron-donating substituents have been developed as
theranostic agents. All the complexes display much higher anti-proliferative potency than the clinical
chemotherapeutic drug cisplatin towards seven cancer cell lines. The anti-proliferative efficacy of these
complexes is correlated to their electron-withdrawing ability. Interestingly, complex Ru1 also potently
suppresses cancer cell migration in vitro and effectively inhibit tumor growth in vivo in a CT26 colon
cancer mouse xenograft model. Mechanisms of action studies display that Ru1 can favorably accumulate
in lysosome and exerts anti-cancer potency by inducing a series of events related to lysosomal
dysfunction in CT26 cells. Interestingly, inhibition of lysosomal enzymes leads to suppression of cyto-
toxicity and apoptosis induced by Ru1. Our results elucidate that complex Ru1 can elicit cytotoxicity
through lysosome-mediated apoptosis in vitro and suppress tumor growth in vivo.
Received 18 June 2020
Received in revised form
9 August 2020
Accepted 15 August 2020
Available online 22 August 2020
Keywords:
Ruthenium
Metallodrug
Anticancer
Lysosome-mediated apoptosis
In vivo
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
[7e9]. Consequently, it is desirable to develop other metal-based
anticancer agents with improved therapeutic efficacy.
Cancer has become the leading cause of fatal diseases and im-
poses huge serious economic burdens on cancer suffers all over the
world [1e3]. The standard methods of cancer treatment often
include surgery, radiation therapy, and chemotherapy or a combi-
nation of these methods [4e6]. Platinum-based anti-cancer drugs
are typical representatives of metal-based chemotherapeutic
agents, such as cisplatin, carboplatin and oxaliplatin. Cisplatin was
introduced into clinics in 1978 and achieved great success for many
types of cancer treatment. However, their severe unintended and
undesirable adverse effects and the progressive acquisition of drug
resistance have strongly limited their therapeutic effects in clinic
Quite recently, a large number of metal-based complexes, such
as palladium, gold, osmium, iridium, rhodium, and ruthenium
complexes, have shown better anti-cancer properties than
platinum-based drugs against many types of cancer cells [10e17].
In particular, ruthenium (Ru) complexes have attracted attention of
researchers because of beneficial biological properties, it has been
considered to be potential chemotherapeutic agents worthy of
further research [18e20]. Three Ru complexes, i.e., NAMI-A, KP1019
and NKP-1339/IT139, have entered clinical trials [21,22]. In recent
years, the exploration of half-sandwich Ru(II) complexes has
attracted much attention because of abundant modification po-
tentials and tunable chemical reactivity [23e25]. A wide range of
half-sandwich Ru(II) complexes bearing modified N, N-chelated
ligands, those based on the
to improve their anti-cancer activity [26e28]. The
were based on DAB and on BIAN backbones (DAB ¼ 1,4-diaza-1,3-
h
6-arene scaffold, have been developed
-diimines used
* Corresponding author.
** Corresponding author.
a
E-mail addresses: huangjiesci@163.com (J. Huang), liuzheqd@163.com (Z. Liu).
https://doi.org/10.1016/j.ejmech.2020.112763
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

2
Z. Xu et al. / European Journal of Medicinal Chemistry 207 (2020) 112763
butadiene; BIAN ¼ bis (imino) acenaphthene). Nitrogen-donor
a
-
were refluxed for 10 h. The more detailed procedures are provided
in ESIy. All half-sandwich Ru(II) complexes (Ru1eRu5) were iso-
lated as PF6ꢀ salts and were authenticated by 1H NMR spectroscopy,
(Figs. S1e5, ESIy), Mass spectrometry (Figs. S6e10, ESIy) and CHN
elemental analysis.
diimine, as the most robust and versatile ligands, has attracted
considerable attention because of finely electronic and tunable
steric properties in synthetic coordination chemistry [29,30]. We
have developed a series of half-sandwich Ru(II) complexes bearing
a
-diimine ligands that showed promising anti-proliferative po-
tency against cancer cells [31]. The aryl-BIANs proved to be
promising nitrogen-donor ligands for the formation of catalysts.
However, Ru(II) complexes bearing aryl-BIAN-chelated ligands
have not been reported, they also did not undergo systematic
biomedical evaluation in vitro and in vivo. Moreover, the electronic
effects play an important role in anti-cancer activity of metal-based
complexes [32]. In this regard, the exploration of new half-
sandwich Ru(II) complexes bearing modified aryl-BIAN-chelated
ligands with better biomedical properties is of particular
importance.
2.2. Inhibition of cell proliferation
To explore the anti-proliferative activity of complexes Ru1eRu5
in vitro, a panel of cancer cell lines, including A549, HepG2, HeLa,
HT29, HCT-116, SMMC-7721 and CT26 were treated for 24 h with
different concentrations of complexes Ru1eRu5 or cisplatin as a
positive control and then measured using the MTT-based assay. The
results of IC50 values are shown in Table 1. Viability of A549, HepG2,
HeLa, HT29, HCT116, SMMC-7721 and CT26 cells was significantly
inhibited by complexes Ru1eRu5, with IC50 values ranging from 2.3
Inspired by above findings, herein a series of new half-sandwich
Ru (II) complexes bearing aryl-BIAN-chelated ligands with different
electron-withdrawing and electron-donating groups substituents
were successfully designed and synthesized, and their anti-cancer
activities and mechanisms of action (MoAs) were studied in vitro
and in vivo. The results exhibited higher anti-proliferative activity
of half-sandwich Ru (II) complexes than that of cisplatin. Among
these complexes Ru1eRu5, Ru1, bearing the strong electron-
withdrawing fluorine (F) group, showed the highest potency
against these cancer cell lines tested, suggesting that the strategic
introduction of electron-withdrawing groups is effective. Ru1 was
used as a model complex for the investigation of potential anti-
cancer MoAs in CT26 cells. Finally, in vivo the anticancer efficacy of
the Ru1 was assessed in a CT26 colon cancer mouse xenograft
model. To our knowledge, complex Ru1 is the first half-sandwich
Ru (II) organometallic complex bearing aryl-BIAN-chelated li-
gands that has been reported to curb tumor growth in vivo. Our
work provides novel design strategy for half-sandwich Ru (II)
organometallic complexes to improve anti-cancer activity.
to 8.8 mM. Importantly, in comparison with cisplatin, complexes
Ru1eRu5 displayed much stronger anti-proliferative activity
against all these cancer cell lines screened. In particular,
Ru1(2.3 0.3
activity than cisplatin (25.6 3.4
(8.8 0.6 M) exhibited the lowest cytotoxicity in these complexes,
mM) displayed about 11-fold higher anti-proliferative
mM) in CT26 cells. Although Ru5
m
it was found to still display much higher anti-proliferative activity
in comparison with that of cisplatin against HepG2 cells, altogether
implying that complexes Ru1eRu5 could be more active over
cisplatin among the cancer therapy. We next tested the anti-
proliferative activities of complexes Ru1eRu5 against CT26 cells,
HepG2 cells and NIH3T3 cells (mouse fibroblasts normal cell) by
MTT assay after 72 h incubation, anti-proliferative activities of
complexes Ru1eRu5 for 72 h treatment were more significant
compared to corresponding that of 24 h treatment with Ru1eRu5,
and Ru1 (0.9 0.1
mM) still displayed about 10-fold higher anti-
proliferative activity than cisplatin (10.5
1.4 M) in CT26 cells
m
for 72 h treatment. Unfortunately, no selectivity was observed for
complexes Ru1eRu5 between cancer cells versus NIH3T3 cells (see
Table 2).
2. Results and discussion
Within this class, with regard to the effect of substitutions on
chelating ligand (aryl-BIAN) on anti-proliferative potency, overall,
complexes containing electron-donating groups (Me and OMe) on
chelating ligand (aryl-BIAN) exhibited less potency in comparison
with those complexes with electron-withdrawing groups (F, Cl and
Br). The complex Ru1 containing the strong electron-withdrawing F
2.1. Synthesis and characterization
The synthetic route to new half-sandwich Ru(II) complexes
(Ru1eRu5) are shown in Scheme 1. The aryl-BIAN ligands were
synthesized according to literature methods reported previously
[33,34]. The five half-sandwich Ru(II) complexes (Ru1eRu5) were
prepared with moderate yields by reacting corresponding aryl-
group exhibited the highest potency (2.3 0.3
Ru5 with the electron-donating OMe groups showed the worst
anticancer potency (8.8 0.6 M). The IC50 value showed the
following trends in anti-proliferative potency of complexes
mM), while complex
m
BIAN ligands and the dimer [(h
6-p-cymene)RuCl2]2 in toluene
Scheme 1. Synthetic route to half-sandwich Ru(II) complexes.

Z. Xu et al. / European Journal of Medicinal Chemistry 207 (2020) 112763
3
Table 1
In vitro cytotoxic IC50 values (m
M) of RuIIcomplexes against cancerous cell lines for 24 h.
Complex
A549
CT26
HCT116
HT29
HeLa
HepG2
SMMC-7721
Ru1
Ru2
Ru3
Ru4
Ru5
Cisplatin
2.9 0.4
3.9 0.3
4.3 0.4
5.2 0.6
6.8 0.8
22.1 2.9
2.3 0.3
3.5 0.4
4.2 0.5
4.9 0.4
6.4 0.7
25.6 3.4
2.7 0.4
3.8 0.4
4.8 0.4
5.5 0.7
6.7 0.8
42.8 5.9
3.2 0.5
4.1 0.5
5.4 0.6
5.8 0.6
7.9 0.9
18.9 1.9
2.9 0.4
4.0 0.6
5.1 0.5
5.1 0.7
6.9 0.4
10.2 1.8
3.2 0.3
4.1 0.6
4.7 0.7
6.0 0.7
8.8 0.6
20.9 2.5
3.1 0.5
4.2 0.5
4.4 0.6
5.9 0.9
7.7 0.8
19.5 2.1
experiments, the 1H NMR experiment results showed that complex
Ru1 was stable in 75% DMSO‑d6/25% D2O at 310 K for at least 72 h
(Fig. S11, ESIy). Finally, the cellular uptake levels of complexes
Ru1eRu5 were quantitatively determined by Atomic absorption
spectrometry. After incubation with 10 mM complexes Ru1eRu5
for 1 h, the cellular accumulation amounts for complexes Ru1eRu5
were 92.6, 80.8, 81.2, 59.5 and 53.7 (ppb/106 cells), respectively
(Table S1, ESIy), most likely due to the complexes containing
electron-donating groups (Me and OMe) on chelating ligand (aryl-
BIAN) exhibited less cellular accumulation amounts in comparison
with those complexes with electron-withdrawing groups. The
anticancer potency of complexes Ru1eRu5 was correlated with
their cellular uptake level.
Table 2
In vitro cytotoxic IC50 values (m
M) of RuIIcomplexes against different cell lines for
72 h.
Complex
CT26
HepG2
NIH3T3
Ru1
Ru2
Ru3
Ru4
Ru5
Cisplatin
0.9 0.1
1.2 0.1
1.6 0.2
2.1 0.2
2.9 0.4
10.5 1.4
1.7 0.2
2.8 0.3
3.0 0.4
3.9 0.5
4.7 0.4
13.6 2.2
2.8 0.2
3.4 0.5
2.8 0.3
4.1 0.5
4.8 0.6
15.1 2.3
Ru1eRu5: F > Cl > Br > Me > OMe. Further analysis demonstrated
that with the increase of electron-withdrawing ability, the anti-
cancer potency of these complexes was found to be enhanced,
which is be consistent with the reported N, N-chelated half-
sandwich Iridium (III) complexes [35]. In all these cancer cell
lines tested, because of the complex Ru1 exhibited strong potency
in complexes Ru1eRu5, the mechanism of action and its anticancer
potency were further investigated in vitro and in vivo.
2.4. Suppression of tumor growth in a mouse CT26 colon cancer
cells xenograft tumor model
Given that potent anti-proliferative effect exhibited by these
complexes Ru in vitro, we assessed the biological efficacy of Ru1 on
transplanted tumor in mouse xenograft model in vivo. CT26 colon
cancer cells were implanted to BALB/c mice to generate tumor
xenograft models. On day 3 after inoculation of CT26 cancer cells,
mice were treated with 7 repeated doses for 7 days with intrave-
nous injection of Ru1 (5 mg/kg and 8 mg/kg dissolved in a vehicle
solution) or with vehicle (3% DMSO and 97% of a saline solution (v/
v)). On day 24, mice bearing xenografts of CT26 colon cancer cells
were sacrificed and the size of the tumor was determined. It can be
seen from Fig. 2 that the Ru1 treatment group was found to
2.3. Water solubility, stability and cellular uptake
The water solubility and stability of complexes were investi-
gated by UVeVis spectroscopy and 1H NMR experiments, respec-
tively. Complexes Ru1eRu5 had solubilities of 105.66 mg/L,
102.22 mg/L, 135.98 mg/L, 137.36 mg/L and 105.80 mg/L, respec-
tively (Fig. 1). Overall, Complexes Ru1eRu5 showed good solubility.
The stability of complex Ru1 was further investigated by 1H NMR
Fig. 1. Calibration of UV-spectroscopic measurement for solubility of complex Ru1 (A), Ru2 (B), Ru3 (C), Ru4 (D) and Ru5 (E) by the UV-spectroscopic assay at 293 nm.

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Z. Xu et al. / European Journal of Medicinal Chemistry 207 (2020) 112763
Fig. 2. Ru1 suppressed tumor growth in a mouse CT26 colon cancer cells xenograft tumor model in vivo. Mice harboring CT26 tumors were injected with vehicle or with Ru1 (5 mg/
kg and 8 mg/kg daily for 7 days, n ¼ 6). (A) Average tumor volumes. (B) Changes of body weight. (C) Average tumor weight. (D) Tumors separated from CT26 tumor xenograft-
bearing mice. (*P < 0.05 vs Control; **P < 0.01 vs Control).
suppress the rate of tumor growth by 34.6 2.4% (5 mg/kg) and
46.3 2.9% (8 mg/kg), respectively, in comparison with a vehicle
control. Parallelly, complex Ru1 decreased the tumor weight by
33.2 2.9% (5 mg/kg) (P < 0.05) and 45.7 3.3% (8 mg/kg) (P < 0.01)
in comparison with a vehicle control, respectively (Fig. 2C and
Table S2, ESIy). Importantly, there were no outward signs of Ru1
(5 mg/kg and 8 mg/kg) toxicity or obvious body weight loss was
found over the course of the experiment (Fig. 2B), these data
indicated that Ru1 was well tolerated in vivo.
2.6. Cellular localization
Recently, numerous organometallic anticancer complexes have
been documented to localize in different subcellular organelles and
cause cell damage [10,40,41]. To further confirm the intracellular
distribution of Ru1, localizations of complex Ru1 with the
organelle-specific commercial stain Lyso-tracker Deep Red (LTDR)
and Mito-Tracker Deep Red FM (MTDR) in CT26 cells were deter-
mined using confocal microscopy, respectively. After treatment for
30 min, as shown in Fig. 4, Ru1 can effectively penetrate into
CT26 cells, and specifically localized in the lysosome. Pearson’s
colocalization coefficient of complex Ru1 with LTDR was deter-
mined to be 0.86. Meanwhile, minimal colocalization coefficient
(0.13) for Ru1 and MTDR was observed. These results indicated that
complex Ru1 could possess lysosomal specificity.
2.5. Inhibition of cell migration
Malignant cells with metastatic ability can escape from the
primary tumor site and migrate to distant organs and further adapt
to the new tumor microenvironment, it aids in the survival and
induces tumorigenesis [36,37]. To investigate whether Ru1 inhibits
cells migration, After Ru1 treatment for 24 h, we evaluated the
inhibition effect of Ru1 on CT26 cells migration using wound
healing assay. As shown in Fig. 3A, Ru1 (0.5 ꢁ IC50) impeded
2.7. Elevation of intracellular ROS levels
It has been documented that metal-based complexes can
directly induce lysosomal dysfunction, causing ROS overproduction
and cell death [42,43]. Since Ru1 showed potent cytotoxicity to
CT26 cells and lysosomal specificity as discussed above, we further
investigate the effect of Ru1 on intracellular ROS expression, CT26
cancer cells were treated with indicated concentrations of Ru1 for
24 h and the changes of ROS levels were determined by a DCFH-DA
assay using flow cytometry. As can be seen from Fig. 5 and Table S3,
CT26 cells migration at a wound closure ratio of 8.4
1.3% in
comparison with control group of 37.1 4.2%, indicating that Ru1
could inhibit dramatically CT26 cells migration. Cancer cells
migration, invasion and metastasis mainly depend on the degra-
dation of various cell adhesion molecules and extracellular matrix.
It is mediated in part by extracellular matrix metalloproteinases
(MMPs) in these processes [38]. In addition, a strong correlation
between matrix metalloproteinase-2 (MMP-2) and matrix
metalloproteinase-9 (MMP-9) has been reported in these processes
of cancer metastasis [39]. Given that Ru1 inhibited cell migration,
we wondered whether Ru1 would have effect on expression levels
of cell migration-related proteins in CT26 cells. After CT26 cells
were treated with Ru1 for 24 h, the expression levels of cell
migration-related proteins were analyzed by flow cytometry. Ru1
treatment clearly decreased the expression of MMP-2 and MMP-9.
Meanwhile, Ru1 treatment also reduced the expression of tran-
scription nuclear factor (NF)-kB p65, which regulates the expres-
sion and activities of MMP-2 and MMP-9, as summarized in Fig. 3B
and Fig. S12, ESIy. The results indicated that Ru1 could impede
ESIy, the intracellular the fluorescence signal elevated in
a
concentration-dependent manner after Ru1 treatment compared
with control group. These results confirmed that ROS over-
production could act as a player in Ru1-induced cell death.
2.8. Induction of lysosomal dysfunction
As demonstrated above, complex Ru1 possesses lysosomal
specificity. Emerging evidence supports that lysosome dysfunction
is considered to be one of the main toxicity paradigms of metal-
based compounds, which may be characterized by abnormal acti-
vation of lysosomal enzyme, the change of lysosome-associated
membrane proteins (LAMPs) expression, and permeability of ly-
sosomes [44]. In order to measure the effect of Ru1 on lysosome
effectively cell migration through downregulation of NF-
kB p65,
MMP-2 and MMP-9 expression in CT26 cells.

Z. Xu et al. / European Journal of Medicinal Chemistry 207 (2020) 112763
5
Fig. 3. CT26 cells treated with complex Ru1 for 24 h at 0.5 ꢁ IC50 concentration. (A) Wound-healing assay, typical images were taken at 0 and 24 h (*P < 0.01 vs Control). (B) Levels
of NF- B p65, MMP-2 and MMP-9 expression were measured using flow cytometry. (*P < 0.05 vs Control, **P < 0.01 vs Control).
k
stability, acridine orange (AO), a fluorescent indicator of lysosome,
was utilized for measuring the permeability of the lysosome. AO in
nuclei and cytosol emits green fluorescence, while displays red
fluorescence in lysosomes. When the rupture of lysosomes
happened, the red fluorescence gradually weakens and only green
fluorescence from AO can be observed. After treatment for 24 h
followed by confocal microscopy analysis, Ru1 visibly decreased the
red fluorescence in a dose-dependent manner in CT26 cells, which
implied that Ru1 could increase permeability of lysosomes (Fig. 6A).
The rupture of lysosomes can evoke lysosomal hydrolases release,
such as cathepsin B, into the cytosol to sensitize cells to the
lysosome-induced cell death [45]. To explore the effect of Ru1 on
cathepsin B release, after treatment with Ru1 for 24 h, the Magic
Red MR-(RR)2, as fluorogenic cathepsin B substrate, was utilized to
investigate the activity of cathepsin B by confocal microscopy
analysis. As shown in Fig. 6B, the red fluorescent product mainly
aggregated in the lysosomes in CT26 cells control group, while with
the increased concentration of Ru1, the red fluorescent product
diffused into other organelles areas. which indicated that Ru1 could
increase cathepsin B release from lysosomal lumen to the cytosol.
For comparison, we also performed the effect of Ru1
anticancer activity. Additionally, lysosome-associated membrane
proteins (LAMPs) expression is an indicator of lysosome function
and the study that metal-based complex-induced down-regulation
of LAMP1 expression accompanies lysosomal dysfunction has been
reported [46]. To investigate the effect of Ru1 on the expression of
LAMP1, CT26 cells were treated with Ru1 for 24 h. As shown in
Fig. 6C, we found that the levels of LAMP1 expression were
increased with the increased concentration of Ru1 compared with
control group in CT26 cells. Collectively, these results indicated that
Ru1 can induce lysosomal dysfunction by down-regulation of
LAMP1 expression, following the increase of cathepsin B release
from lysosomal lumen to the cytosol in CT26 cells.
2.9. Effects of leupeptin on cell viability and sphere formation in
Ru1-treated CT26 cells
Given that Ru1 can accumulate in subcellular organelle lyso-
some and induce release of lysosomal content from lysosomal
lumen to the cytosol, we supposed that the lysosome could play an
important role in Ru1-induced cytotoxicity. To further determine
the role of lysosomes in cytotoxicity induced by Ru1, we used
leupeptin, a lysosomal inhibitor, the cell proliferation and cells
sphere formation assays were determined after lysosome inhibi-
tion. It was well documented that leupeptin curbed the activity of
lysosomal hydrolases, such as cysteine, threonine, serine proteases,
which are considered to be the basic lysosomal enzymes [47,48].
Firstly, after CT26 cells were incubated with Ru1 (1 ꢁ IC50) with or
(1 ꢁ IC50 ¼ 2.3
shown in Fig. S13, ESI.y, complex Ru1 (1 ꢁ IC50 ¼ 2.3
cantly increased the cathepsin B release compared to that of Ru5
(2.3 M) treated group in CT26 cells. The results showed that Ru1
mM) and Ru5 (2.3
mM) on cathepsin B release, as
mM) signifi-
m
exerted much stronger effects than Ru5 under the same concen-
tration used on CT26 cancer cells and therefore displayed the best

6
Z. Xu et al. / European Journal of Medicinal Chemistry 207 (2020) 112763
Fig. 4. Colocalization of complex Ru1 and mitochondria and lysosome in CT26 cells was observed using confocal microscopy, respectively. Confocal microscopy images of CT26 cells
co-labeled with complex Ru1 (5 M, 30 min) and MTDR (100 nM, 30 min) and LTDR (75 nM, 30 min). Complex Ru1 were collected at 520 20 nm (excitation: 488 nm), LTDR was
collected at 630 30 nm (excitation: 594 nm) and MTDR was collected at 710 20 nm (excitation: 633 nm), scale bars: 20 m.
m
m
Fig. 5. ROS level was determined using flow cytometry after CT26 cells were exposed to Ru1 for 24 h.
without leupeptin (10
mM) for 24 h, we performed the cell viability
inhibition of cells growth.
assay, As shown in Fig. 7A, Ru1 alone treatment significantly
decreased the viability of CT26 cells, importantly, the viability of
CT26 cells along with the lysosomal inhibition with leupeptin was
dramatically rescued. In addition, the long-term effect of coincu-
2.10. Effects of leupeptin on apoptosis in Ru1-treated CT26 cells
As demonstrated by the literature, lysosomes are associated
with a programmed cell death, called lysosomal cell death (LCD), in
which lysosome disruption or leakage of lysosomal enzymes from
lysosomal lumen to the cytosol leads to cell death [49,50]. Since the
lysosome could play a key player in Ru1-induced cytotoxicity as
discussed above, we further explored the involvement of LCD in
bation with Ru1 (0.25 ꢁ IC50) and leupeptin (2
mM) on CT26 cell
growth was determined by a cancer cells’ sphere formation assay.
As seen in Fig. 7B, the size of the cells sphere formation of CT26 cells
was increased more significantly in the coincubation group of Ru1
with leupeptin compared to Ru1 alone after 10 days of treatment.
These results implied that leupeptin pre-treatment can remarkably
decrease the inhibition of cells growth induced by Ru1, which
indicated that the lysosome could play a key player in Ru1-induced
Ru1-induced apoptosis. CT26 cells were treated by Ru1 (1 ꢁ IC50
)
for 24 h with or without lysosomal inhibitor leupeptin pre-
treatment for 2 h following flow cytometry analysis. As shown in

Z. Xu et al. / European Journal of Medicinal Chemistry 207 (2020) 112763
7
Fig. 6. CT26 cells were treated by Ru1 for 24 h with indicated concentrations. (A) AO (5
m
M) staining, lex ¼ 488 nm, lem ¼ 520 20 nm (green) and 630 20 nm (red). Scale bars:
10
20
m
m
m. (B) Observation of cathepsin B release from lysosomal lumen to the cytosol in CT26 cells with Magic Red MR-(RR)2 staining. lex ¼ 594 nm, lem ¼ 620 20 nm. Scale bars:
m. (C) Level of LAMP1 expression was determined by flow cytometry (*P < 0.01 vs Control, **P < 0.01 vs Control). (For interpretation of the references to colour in this figure
legend, the reader is referred to the Web version of this article).
Fig. 7. CT26 cells were treated with Ru1 by for 24 h in the absence or presence of leupeptin (10 mM) pretreatment for 1 h. (A) Lysosomal inhibitors leupeptin decreased cytotoxicity
of Ru1 (**P < 0.01 vs Control). (B) Leupeptin attenuated the inhibition of cell sphere formation in Ru1-treated CT26 cells.

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Z. Xu et al. / European Journal of Medicinal Chemistry 207 (2020) 112763
Fig. 8 and Table S4, ESIy, Ru1 alone treatment significantly
enhanced the Ru1-induced percentage of apoptosis. Notably, in line
with the notion of Ru1-induced cytotoxicity, along with the coin-
cubation with Ru1 and leupeptin in CT26 cells, the percentage of
apoptosis was completely reversed. To this end, these observations
confirmed that lysosome could play a key player in Ru1-induced
apoptosis.
in toluene (20 mL) were refluxed for 10 h in a Schlenk bottle under a
nitrogen atmosphere. After cooling to room temperature, solid
NH4PF6 (0.60 mmol) was added to the solution with stirring for 4 h.
After NH4PF6 was filtered off, the solvent
was removed under reduced pressure. The crude products were
purified by recrystallization using CH2Cl2/diethyl ether or CH2Cl2/
hexane solution, mixture was concentrated in a vacuum and loaded
on the column.
Complex Ru1: 1H NMR (500 MHz, CDCl3)
d 8.20e8.12 (m, 2H),
3. Conclusions
7.99 (d, J ¼ 8.3 Hz, 2H), 7.85e7.78 (m, 2H), 7.50e7.39 (m, 4H), 7.32
(td, J ¼ 8.4, 2.5 Hz, 2H), 6.88 (d, J ¼ 7.3 Hz, 2H), 5.50 (d, J ¼ 5.5 Hz,
2H), 5.42 (d, J ¼ 6.0 Hz, 2H), 2.46e2.35 (m, 1H), 1.93 (s, 3H), 1.10 (d,
J ¼ 6.9 Hz, 6H). ESI-MS (m/z): calcd for C34H28RuClN2F2: 639.0953,
found: 639.106. Elemental analysis calcd (%) for C34H28RuClN2PF8:
C, 52.08; H, 3.60; N, 3.57. Found: C, 52.12; H, 3.71; N, 3.46.
In conclusion, five novel organometallic half-sandwich Ru(II)
complexes Ru1eRu5 bearing aryl-BIAN-chelated ligands with
different electron-withdrawing and electron-donating groups
substituents have been synthesized and authenticated. All of these
newly prepared complexes display higher anti-proliferative po-
tency than the clinical chemotherapeutic drug cisplatin towards
seven cancer cell lines. The anti-proliferative efficacy of these
complexes is correlated to their electron-withdrawing ability.
Complex Ru1 is 11-fold more potency than cisplatin in CT26 cells.
Complex Ru1 can elevate intracellular ROS level and curb cancer
cell migration in vitro. In addition, in vivo complex Ru1 is found to
potently inhibit tumor growth in a CT26 colon cancer mouse
xenograft model. The further studies for anti-cancer mechanisms of
action display that Ru1 can specifically localize in lysosome and
exerts anti-cancer potency by inducing a cascade of events asso-
ciated with lysosomal dysfunction in CT26 cells including lyso-
somal damage, cathepsin B release, inhibition of LAMP1 expression
and apoptosis. Importantly, inhibition of lysosomal enzymes can
suppress effectively cytotoxicity and apoptosis induced by complex
Ru1. Our results indicate complex Ru1 can elicit cytotoxicity
through lysosome-mediated apoptosis in vitro and suppressing
tumor growth in vivo. In all, the understanding of mechanism for
the half-sandwich Ru(II) complexes-induced cell death would be
helpful to focus on the optimization of structure-activity on novel
Ru(II) complexes to achieve better anti-cancer efficacy.
Complex Ru2: 1H NMR (500 MHz, CDCl3)
d
8.10 (d, J ¼ 7.9 Hz,
2H), 8.00 (d, J ¼ 8.3 Hz, 2H), 7.77 (dd, J ¼ 8.4, 2.0 Hz, 2H), 7.70 (dd,
J ¼ 8.8, 1.4 Hz, 2H), 7.59 (dd, J ¼ 8.4, 2.0 Hz, 2H), 7.47 (t, J ¼ 7.8 Hz,
2H), 6.92 (d, J ¼ 7.3 Hz, 2H), 5.48 (d, J ¼ 6.0 Hz, 2H), 5.38 (d,
J ¼ 6.1 Hz, 2H), 2.49e2.44 (m, 1H), 1.97 (s, 3H), 1.11 (d, J ¼ 6.9 Hz,
6H). ESI-MS (m/z): calcd for C34H28RuCl3N2: 671.0362, found:
671.051. Elemental analysis calcd (%) for C34H28RuCl3N2PF6: C,
49.98; H, 3.45; N, 3.43. Found: C, 49.90; H, 3.53; N, 3.41.
Complex Ru3: 1H NMR (500 MHz, CDCl3)
d
8.05 (d, J ¼ 6.2 Hz,
2H), 7.99 (d, J ¼ 8.2 Hz, 2H), 7.86 (s, 2H), 7.80e7.68 (m, 4H), 7.47 (t,
J ¼ 7.4 Hz, 2H), 6.93 (d, J ¼ 6.9 Hz, 2H), 5.50 (d, J ¼ 3.3 Hz, 2H), 5.39
(d, J ¼ 1.7 Hz, 2H), 2.52e2.46 (m, 1H), 2.01 (s, 3H), 1.12 (d, J ¼ 6.6 Hz,
6H). ESI-MS (m/z): calcd for C34H28RuClBr2N2: 760.9331, found:
760.958. Elemental analysis calcd (%) for C34H28RuClBr2N2PF6: C,
45.08; H, 3.12; N, 3.09. Found: C, 45.14; H, 3.16; N, 3.13.
Complex Ru4: 1H NMR (500 MHz, CDCl3)
d
7.95 (d, J ¼ 8.3 Hz,
2H), 7.91 (d, J ¼ 7.7 Hz, 2H), 7.71 (d, J ¼ 7.8 Hz, 2H), 7.51 (d, J ¼ 6.6 Hz,
2H), 7.42 (t, J ¼ 7.8 Hz, 4H), 6.96 (d, J ¼ 7.3 Hz, 2H), 5.53 (d,
J ¼ 3.9 Hz, 2H), 5.39 (d, J ¼ 3.4 Hz, 2H), 2.59e2.46 (m, 1H), 2.55 (s,
6H), 2.07 (s, 3H), 1.12 (d, J ¼ 6.9 Hz, 6H). ESI-MS (m/z): calcd for
C
36H34RuClN2: 631.1454, found: 631.168. Elemental analysis calcd
(%) for C36H34RuClN2PF6: C, 55.71; H, 4.42; N, 3.61. Found: C, 55.82;
H, 4.35; N, 3.56.
4. Experimental section
Complex Ru5: 1H NMR (500 MHz, CDCl3)
d
7.96 (t, J ¼ 8.2 Hz,
4.1. Synthesis of complexes
4H), 7.75 (d, J ¼ 7.6 Hz, 2H), 7.47e7.42 (m, 2H), 7.22 (d, J ¼ 7.9 Hz,
2H), 7.12 (d, J ¼ 6.3 Hz, 2H), 7.03 (d, J ¼ 7.3 Hz, 2H), 5.42 (d,
J ¼ 6.3 Hz, 2H), 5.34 (d, J ¼ 6.2 Hz, 2H), 3.98 (s, 6H), 2.59e2.54 (m,
1H), 2.02 (s, 3H), 1.13 (d, J ¼ 6.9 Hz, 6H). ESI-MS (m/z): calcd for
General Method. Dimeric
ene)RuCl2]2 formed upon conversion of RuCl3 with
under refluxing in methanol [51]. Dimer [(
6-p-cymene)RuCl2]2
(0.05 mmol) and the corresponding aryl-BIAN ligands (0.10 mmol)
m
-chloro-bridged dimer [(
h
6-p-cym-
a
-terpinene
h
C36H34O2RuClN2: 663.1352, found: 663.154. Elemental analysis
Fig. 8. CT26 cells were treated with Ru1 by for 24 h in the absence or presence of leupeptin (10 mM) pretreatment for 1 h. Lysosomal inhibitors leupeptin attenuated the percentage
of apoptosis in Ru1-treated CT26 cells.

Z. Xu et al. / European Journal of Medicinal Chemistry 207 (2020) 112763
9
calcd (%) for C36H34O2RuClN2PF6: C, 53.50; H, 4.24; N, 3.47. Found:
4.6. Apoptosis assay
C, 53.45; H, 4.21; N, 3.52.
After exposing CT26 cells to complex Ru1 (1 ꢁ IC50) for 24 h with
or without lysosomal inhibitor leupeptin (10 M) pre-treatment for
2 h, the analysis of different stages of apoptosis populations of the
cells were analyzed using Annexin V-PE/7-AAD apoptosis staining
kit by flow cytometry.
m
4.2. Cell culture and reagents
The cancer cell lines A549, HepG2, HeLa, HT29, HCT-116, SMMC-
7721 and CT26 were preserved by our laboratory. All cancer cell
lines were grown in Dubelco’s Modified Eagle Medium (DMEM)
4.7. Solubility assay
supplemented with 10% fetal bovine serum (FBS) and 100 mg/mL
streptomycin þ100 U/mL penicillin solution. All Cell lines were
grown in a CO2 incubator at 37 ꢂC with a humidified atmosphere of
5% CO2 and 95% air. Phosphate-buffered saline (PBS), MTT, Annexin
V/7-AAD apoptosis detection Kit and DCFH-DA were purchased
from Keygen Biotech. These antibodies of anti-LAMP1, anti-MMP-2,
and anti-MMP-9 were purchased from Millipore.
The water solubility of the Ru1eRu5 was measured by UV-
spectroscopy [52,53]. A solution of the complexes Ru1eRu5 was
stocked in DMSO with a concentration of 10 mM. The stock solu-
tions were diluted in concentrations ranging from 10 mM to 200 mM
in 80% HEPES (pH ¼ 7.2e7.4) and 20% acetonitrile. The saturated
complex solutions were monitored by UV-spectroscopy and the
spectra were collected from 200 to 700 nm by TU-1901 UV
spectrophotometer.
4.3. MTT cell viability assay
The cancer cells A549, HepG2, HeLa, HT29, HCT-116, SMMC-
7721, CT26 and mouse fibroblasts normal cells NIH3T3 were plated
5000 cells per well and were pre-incubated at a 37 ꢂC in incubator
containing 5% CO2 overnight. The following day, all cell lines were
treated with compounds (Ru1eRu5) and cisplatin as indicated for
24 h or 72 h. After that, MTT was added into each well for further
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
incubation for 4 h. After adding 100 mL per well of DMSO solution
Acknowledgements
into 96-well plates, the plates were shaken for 0.5 h to dissolve fully
the purple formazan. The absorbance for the plate was determined
at 570 nm by a microplate reader. IC50 values quoted are
mean standard deviation.
This work was supported by National Natural Science Founda-
tion of China (Grant No. 21671118) and the Taishan Scholars
Program.
4.4. Tumor suppressive experiments in vivo
Appendix A. Supplementary data
7-8-week-old female BALB/c mice with the body weight
(18e22 g) were used in CT26 cells xenograft tumor animal models.
Animal experiments were approved and performed in accordance
with standard procedures and principles stated in the Guide for the
Care and Use of Laboratory Animals. Every effort was made to
reduce animal suffering among the experiments. After 5 days of
acclimation for mice, CT26 cells (1.0 ꢁ 106 cells) suspended in
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112763.
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