Synthesis, characterization and anticancer mechanism studies of fluorinated cyclometalated ruthenium(ii) complexes

Article abstract of DOI:10.1039/d0dt01412e

The drug-resistance of cancer cells has become a major obstacle to the development of clinical drugs for chemotherapy. In order to overcome cisplatin-resistance, seven cyclometalated ruthenium(ii) complexes were synthesized with a varying degree of fluorine substitution, for use as anticancer agents. A cytotoxicity assay testified that the complexes possessed a more cytotoxic effect than cisplatin towards the cisplatin-resistant cell line A549R. The number of fluorine atoms regulated the lipophilicity of the complexes, but the relationship was not linear.Ru1containing one fluorine atom had the highest lipophilicity and the best therapeutic effect. The complexes enter cells through an energy-dependent pathway and then localize in the nuclei and mitochondria. The complexes induced nuclear dysfunction by the inhibition of DNA replication as well as mitochondrial dysfunction by the loss of membrane potential. The damage to these vital organelles leads to cell apoptosisviathe caspase 3/7 pathway. Our results indicated that the modulation of the number of fluorine atoms in therapeutic agents can have a profound effect andRu1is a complex with a high potential as a drug for the treatment of cisplatin-resistant cancer.

Full text of DOI:10.1039/d0dt01412e

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Journal Name
ARTICLE
Synthesis, characterization and anticancer mechanism studies of
fluorinated cyclometalated ruthenium(II) complexes†
a
a
b
a
a
a
Ya Wen, Cheng Ouyang, Quanwen Li, Thomas W. Rees, Kangqiang Qiu,* Liangnian Ji and Hui
Chao*
Received 00th January 20xx,
Accepted 00th January 20xx
a,c
DOI: 10.1039/x0xx00000x
The drug-resistance of cancer cell has become a major obstacle to the development of clinical drugs for chemotherapy. In
order to overcome cisplatin-resistance, seven cyclometalated ruthenium(II) complexes were synthesized with a varying
degree of fluorine substitution, for use as anticancer agents. Cytotoxicity assay testified the complexes possessed a more
cytotoxic effect than cisplatin toward the cisplatin-resistant cell line A549R. The number of fluorine atoms regulates the
lipophilicity of the complexes, but the relationship is not linear. Ru1 containing one fluorine atom has the highest lipophilicity
and the best therapeutic effect. The complexes enter cells through an energy-dependent pathway and localize in nuclei and
mitochondria. The complexes induce nuclear dysfunction by the inhibition of DNA replication as well as mitochondrial
dysfunction by loss of membrane potential. The damage to these vital organelles leads to cell apoptosis via the caspase 3/7
pathway. Our results indicate modulation of the number of fluorine atom in therapeutic agents can have a profound effect
and Ru1 is a complex with a high potential as a drug for the treatment of cisplatin-resistant cancer.
www.rsc.org/
power house and their dysfunction inhibits energy-dependent
1
7
Introduction
cellular processes. Heavy-metal complexes, especially those
6
8
10
possessing d , d , and d electronic structures, can be detected
easily inside living cells allowing their intracellular activity and
location to be determined.
complexes have been designed and employed for
chemotherapy to solve the problems of drug resistance towards
metallodrugs such as cisplatin.
Ruthenium based coordination complexes have been shown
to be highly promising chemotherapeutic agents.2
ruthenium complexes have recently been in clinical trials.
Cancer is the second leading cause of death worldwide and was
responsible for approximately 9.6 million deaths in 2018. With
an aging and growing population, cancer incidence and
mortality increase year on year. There were ~18.1 million new
cancer cases in 2018 and it is predicted that the number of new
cancer patients will increase by ~27 million by 2030. To
address this global problem, many therapeutic technologies
and drugs have developed.
1
8-20
Organelle-targeting metal
1
2,14,21
1
,2
2,23
Three
3
-6
As a traditional clinical
2
4,25
antineoplastic method, chemotherapy has saved many lives and
still is an important tool in the fight against cancer. However,
with long-term treatment, cancer can become drug resistant,
Ruthenium complexes have also been investigated as
endoplasmic reticulum (ER), mitochondria, lysosome, and
nucleus-targeting drugs, and are highly effective against
7
-11
which lowering the treatment efficacy.
It is therefore
2
6-29
cisplatin resistant cells.
Aside from subcellular targeting,
necessary to develop novel effective anticancer strategies and
agents to overcome the challenge of drug-resistant cancer.
Recently, organelle-targeting chemotherapy has attracted
tremendous attention due to the significance of organelles in
cellular function. The damage of organelles causes dysfunction,
leading to cell death.
genetic material and nuclear dysfunction prevents DNA
replication and transcription. Mitochondria are the cells
therapeutic efficacy is highly dependent on cellular uptake. Our
group has developed cyclometalated nucleus-targeting
ruthenium polypyridyl complexes with increased cellular
uptake. By replacing one of the polypyridyl ligand coordination
sites nitrogen atoms with carbon, the overall charge of the
complex decreases from +2 to +1, resulting in increased
lipophilicity and high cellular uptake. Mitochondria-targeting
cycloruthenated polypyridyl complexes have also been
developed and shown to be highly effective for the treatment
of cisplatin-resistant cancer.
Due to its strong electron-withdrawing effect, fluorine is
often incorporated into compound in order to tune optical and
physical properties allowing fine control of their potency.
Despite showing effective anticancer properties, the bis-
tridentate cycloruthenates were rarely reported for organelle-
1
2-15
The nucleus, for example, contains
2
9
1
6
2
7
a. MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of
Chemistry, Sun Yat-Sen University, Guangzhou 510275, P. R. China. E-mail:
ceschh@mail.sysu.edu.cn, qiukq3@mail.sysu.edu.cn
b.
School of Materials Science and Engineering, Nankai University, Tianjin 300350, P.
30-33
R. China.
c.
MOE Key Laboratory of Theoretical Organic Chemistry and Functional Molecule,
School of Chemistry and Chemical Engineering, Hunan University of Science and
Technology, Xiangtan, 400201, P. R. China
3
4,35
targeting therapy.
ruthenium metallodrugs, bis-tridentate cycloruthenates with
To achieve better antineoplastic
†
1
Electronic supplementary information (ESI) available: additional data. See DOI:
0.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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Fig. 1 (A) The general chemical structure of Ru0-5. (B-H) Crystal structures of the complexes Ru0-5. For clarity, the solvent molecules, the hydrogen atoms,
and the counter anions are omitted.
varied fluorine atom substitution were studied for the cytotoxicity against non-tumor cells than towards cancer cell
treatment of cisplatin resistant cancer. Seven fluorinated lines. Interestingly, Ru0-5 exhibit a more cytotoxic effect
cycloruthenated complexes (Ru0-5, Fig. 1A) were designed and compared to cisplatin against the cisplatin resistant cell line
synthesized in this work. The complexes exhibit the ability to A549R. Ru1 has the highest cytotoxicity towards the resistant
against cisplatin-resistant cancer cells and were found to mainly cell line with an IC50 value of 1.44 μM, which is 79.3 times lower
accumulate in the nucleus and mitochondria, inducing nuclear than that of cisplatin. The IC50 values of Ru1-4 against A549R
and mitochondrial dysfunction, and resulting in cancer cell cells are more potent than that of the non-fluorinated Ru0,
death by apoptosis.
indicating the addition of fluorine is an effective strategy.
However, Ru5 which has the highest degree of fluorination is
the least effective of the Ru complexes studied. To investigate
the influence of fluorine substituents on the anticancer effects
of the complexes, the lipophilicity, cellular uptake, intracellular
localization and uptake mechanism of the complexes were
studied.
Results and discussion
Synthesis and characterization
The complexes were synthesized through the synthetic route
shown in Scheme S1. The fluorinated terpyridyl ligands (L0-5)
were obtained first. Ru0-5 were then synthesized by the
3 2
reaction of RuCl •6H O and the corresponding ligands via a Lipophilicity
two-step reaction. The Ru(II) complexes were purified by
Lipophilicity is well known to play a key role in cytotoxicity.29
A
column chromatography on alumina with acetonitrile and
1
19
toluene, and characterized by ES-MS, H NMR and F NMR (Fig.
S1-S20). The molecular structures of the complexes were also
confirmed by single-crystal X-ray diffraction (Fig. 1B-H). The
crystal data and structural refinements are shown in Table S1-
S4. Selected bond lengths and angles are presented in Table S5-
S11.
Table 1 IC50 (μM) values of Ru0-5 and cisplatin towards selected human
cancer and non-tumor cell lines. Values are the average of at least three
separate experiments.
Complexes
HeLa
A549
A549R
LO2
Ru0
Ru1
4.31 ± 0.22 4.71 ± 0.14 5.53 ± 0.20 7.72 ± 0.24
0.97 ± 0.11 1.25 ± 0.09 1.44 ± 0.14 4.26 ± 0.37
1.44 ± 0.14 2.20 ± 0.16 3.11 ± 0.41 3.91 ± 0.35
6.71 ± 0.56 4.12 ± 0.33 4.54 ± 0.22 10.7 ± 1.94
2.12 ± 0.08 2.22 ± 0.15 2.69 ± 0.09 4.45 ± 0.30
3.44 ± 0.41 3.87 ± 0.20 3.85 ± 0.36 5.12 ± 0.42
5.87 ± 0.36 6.12 ± 0.33 6.43 ± 0.45 9.27 ± 0.96
Cytotoxicity assay
In order to explore the antitumor potential of Ru0-5, the
complexes were assessed by MTT assay, against three human
cancer cell lines (HeLa, A549 and A549R) and a non-tumor
human cell line LO2. The cells were treated with various
concentrations of Ru0-5 (as well as cisplatin as a positive control)
for 48 h. The resulting IC50 values for the tested complexes are
shown in Table 1. All the ruthenium complexes displayed good
cytotoxicity activity towards the three selected cancer cell lines,
with IC50 values ranging from 0.97 to 6.43 μM and show a lower
Ru2
Ru3a
Ru3b
Ru4
Ru5
Cisplatin
8.93 ± 0.79 17.2 ± 1.30
114 ± 10.1
18.8 ± 1.20
2
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UV-vis absorption “shake flask” method was used to determine
the n-octanol/water partition coefficients (log PO/W) of the
complexes as 1.04 for Ru0, 2.14 for Ru1, 1.23 for Ru2, 0.41 for
Ru3a, 0.79 for Ru3b, 0.44 for Ru4 and 0.05 for Ru5. The log Po/w
values do not correlate to the number of fluorine atoms on the
complexes. Furthermore, the log Po/w values have a negative
correlation to the IC50 values of Ru0-5 for A549R cells. Ru1 with
the highest log Po/w value displays the best cytotoxic activity.
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Cellular uptake and localization
It has been reported that the cellular uptake of organometallic
3
6
drugs dramatically influences their cytotoxicity.
The
differences in vitro cytotoxic activity for Ru0-5 indicate the
cellular uptake of the complexes might be different. The
intracellular distribution of the complexes is another key factor
which cytotoxic activity can depend. Therefore, the uptake
content and distribution of the complexes in A549R cells (drug-
resistant cells) and LO2 cells (non-tumor cells) was quantified by
inductively coupled plasma mass spectrometry (ICP-MS). As
shown in Fig. 2, the complexes display a much higher cellular
uptake level in A549R cells compared to non-tumor LO2 cells,
demonstrating the selectivity of the complexes between cancer
cells and non-tumor cells. The complexes were found to mainly
localize in mitochondria and nuclei in both cell lines. Due to its
low lipophilicity and therefore poor ability to penetrate the cell
membrane, Ru5 has a lowest uptake level. The cellular uptake
level of Ru0 was found to be very high despite its low
cytotoxicity. This demonstrates the enhanced toxicity of Ru1 is
not due to its uptake alone.
Fig. 3 ICP-MS data for Ru0, Ru1 and Ru5 (1.0 μM) incubated with A549R cells
for over time.
The cellular uptake levels in A549R cells over time of
complexes Ru0 (un-fluorinated), Ru1 (most cytotoxic) and Ru5
(lowest lipophilicity) were studied by ICP-MS (Fig. 3). The
cellular uptake of the complexes was found to depend on
treatment time and reached saturation within 2 h. In addition,
the Ru complexes were not expelled from the cells within 8 h,
indicating that the uptake mechanism of the Ru complexes is
different from cisplatin.
In order to determine the energy-dependency of Ru
complexes cell entry mechanism, A549R cells were incubated
with the complexes at 4 °C and pre-treated with various
condition: metabolic inhibitors 2-deoxy-D-glucose and
3
7,38
oligomycin,
4
the endocytosis inhibitors NH Cl and
3
9
chloroquine to examine the endocytic pathway, and a high
potassium environment (170 mM K ) to reduce the cell
+
3
8
membrane potential. In Fig. S21, the results demonstrate that
there was no significant change in the high potassium
environment. In contrast, the metabolic inhibitors and
endocytosis inhibitors both reduced Ru complexes uptake. The
results indicate that the Ru complexes are uptaken via an
energy-dependent pathway, such as endocytosis and active
transport pathways.
Nuclear dysfunction
5
-Ethynyl-2'-deoxyuridine (EdU) is a thymidine analogue that
can incorporated into DNA replication, serving as a fluorescent
4
0,41
marker of active proliferating cells.
The nucleus is a main
target of the Ru complexes, so an EdU assay was used to detect
the effect of the complexes on DNA replication (Fig. 4). In the
control group, a large amount of newly replicated DNA was
detected in the cell nucleus. With 50 μM cisplatin treatment, a
small inhibitory effect was found in A549R cells, while the DNA
replication of most cells was found to stop with 100 μM cisplatin.
In the case of the ruthenium complexes, the inhibitory effect
increases significantly with concentration of complexes
Fig. 2 ICP-MS data for A549R cells (A) and LO2 cells (B) incubated with 1.0
μM of Ru0-5 for 9 h. Cytoplasm here refers specifically to cytoplasm without increasing. As in the cytotoxicity assay, Ru1 was found to have
the mitochondria.
the best inhibitory effect.
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Fig. 4 The antiproliferative effects on A549R cells by different concentrations of ruthenium complexes and cisplatin for 24 h. Scale bar: 100 μm.
Mitochondrial dysfunction
Cell death pathway
Mitochondria are another cellular target of the complexes. There are three classical types of cell death, including apoptosis,
Mitochondrial dysfunction induced by Ru0, Ru1 and Ru5 was necrosis and autophagy. Many chemotherapy drugs induce cell
4
7,48
determined by measuring changes in the mitochondrial death by apoptosis.
To further verify the mechanism of cell
4
2,43
membrane potential (MMP).
a hallmark event in the early stages of apoptosis.
The depolarization of MMP is death induced by the complexes, the related apoptosis assays
4
4,45
JC-1 is a were carried out. After MMP loss we would expect to observe
cationic dye that is widely used for detecting MMP. The the activation of the caspase family, especially caspase 3/7,
decrease of MMP can be easily detected by a transformation of which plays an important role in the initiation of apoptosis. The
JC-1 from red fluorescence to green fluorescence, and the activity of caspase 3/7 was assessed through the Caspase-Glo
4
6
transformation is an early indicator of apoptosis. In Fig. 5, 3/7 assay. As shown in Fig. 6, a low concentration of the
when A549R cells were exposed to an increasing concentration complexes caused a small increase of caspase 3/7 expression in
of the complexes, the fluorescence of JC-1 changes from red to A549R cells, while a high concentration caused a distinct
green, indicating MMP decrease. For Ru0 and Ru5, the MMP of increase in caspase 3/7 expression. At 1 μM and 2 μM
A549R cells is basically the same as the control at 0.5 μM and concentrations, Ru1 induced much higher expression of caspase
red fluorescence can still be found at 2 μM. However, for Ru1, 3/7 than the other complexes and cisplatin. The results indicate
most red fluorescence disappeared at 1 μM, indicating that the ruthenium complexes induce cancer cell death via an
significant MMP decrease. This experiment shows that all three apoptosis pathway.
compounds induce MMP loss while Ru1 is the most effective.
Fig. 5 The fluorescence images of A549R cells incubated with different concentrations of ruthenium complexes for 12 h before staining with JC-1 to reveal
changes in MMP. Scale bar: 20 μm.
4
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apoptotic/necrotic cells. The signals can be divided into three
classes: viable cells (annexin V-/PI-, red), early apoptotic cells
(
annexin V+/PI-, green), and late apoptotic/necrotic (annexin
4
9,50
V+/PI+, blue).
In Fig. 7, under the same treatment conditions,
Ru1 showed the most annexin V+/PI+ cells with the highest
expression of annexin V. The number of annexin V+/PI+ cells
was shown to increase with increase in complex concentration,
indicating that the complexes induce apoptosis in A549R cells.
Conclusions
In conclusion, seven ruthenium complexes were synthesized
and characterized as anticancer agents containing a varied
number of fluorine atoms. The fluorinated cyclometalated
ruthenium complexes were found to localize in both the
mitochondria and the nucleus of A549R cells, inhibiting DNA
replication, and inducing loss of MMP leading to cell apoptosis
Fig. 6 Caspase 3/7 activity measurements. A549R cells were incubated with by the caspase 3/7 pathway. The results show that the fluorine
different concentrations of Ru complex or cisplatin for 12 h.
atom can regulate the lipophilicity of the complexes and Ru1
with one fluorine atom possesses the best anticancer activity.
Ru1 accumulates in A549R cells and showed a more than 70-
To quantify the cells in the different stages of apoptosis, flow fold increased cytotoxic effect compared to cisplatin. In contrast
cytometry analysis of living cells double labelled with annexin V- to Ru0, the addition of a fluorine atom in Ru1 leads to greatly
FITC (annexin V) and propidium iodide (PI) was performed. In improved efficacy. Ru5, which has five fluorine atoms, is the
early apoptosis, the membrane phosphatidylserine (PS) least effective drug due to its poor lipophilicity and cell uptake.
translocates from the inner plasma membrane to the outer The results indicate the degree of fluorination can be used to
leaflet. Annexin V is a specific fluorescence probe to detect PS tune the anticancer properties of the complexes and Ru1 is a
on the plasma membrane surface. PI can pass through the complex with great potential as a therapeutic agent with
plasma membrane of dead cells to stain the nucleus, which selectivity towards cancer cell lines and high efficacy against
allows the further distinction of early apoptotic and late cisplatin-resistant cell line A549R.
Fig. 7 Different concentration of ruthenium complexes induced apoptotic A549R cell death as detected by the annexin V-FITC/PI assay (12 h).
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After cooling down to the room temperature, 40 mL water was
poured down, and then filtered. The filtrate was collected and
Experimental section
General information
NH
ether. The crude product was obtained after dried and purified
through neutral Al column (100-200 mesh) with
4 6
PF was added. The residue was filtered and washed by
a
2 3
O
The reagents and solvents for chemical synthesis were of
analytical grade, and of biological grade for cell experiments. All
of them were obtained from commercial sources and used as
received unless specified. Ruthenium chloride hydrate,
toluene/MeCN (v/v, 1:1) as the eluent. The solvent was
removed via rotary evaporator, and then the purified Ru0 was
obtained.
Except the synthesis of Ru5, the synthesis methods of Ru1-4
were similar as that of Ru0.
The method for L5 was a little different from above way with
some little changes. Potassium t-butoxide (3.36 g, 30.0 mmol, 3
eq.) and 2-acetylpyridine (2.20 mL, 20.0 mmol, 2 eq.) was added
to dry DMF (32 mL), and creamy yellow suspension was
obtained. Then, 2,3,4,5,6-pentafluorobenzaldehyde (2.00 g,
5
,5',6,6'-tetrachloro-1,1'-3,3'-tretraethyl-benzimidazolylcarbo-
cyanine iodide (JC-1), cisplatin and 3-(4,5-dimethylthiazol-2-yl)-
,5-diphenyltetrazolium bromide (MTT) were obtained from
2
Sigma-Aldrich.
2-phenylpyridine,
2-acetylpyridine,
4-
2,4,6-
fluorobenzaldehyde,
3,4-difluorobenzaldehyde,
trifluorobenzaldehyde,
2,3,5,6-tetrafluorobenzaldehyde,
2
,3,4,5,6-pentafluorobenzaldehyde and potassium t-butoxide
1
0.0 mmol, 1 eq.) was quickly poured into above mixture. After
(KTB), acetic acid, ammonium acetate, triethylamine and
that, the red mixture was stirred at room temperature for 12 h
under Ar atmosphere. Then, a solution of ammonium acetate
ethylene glycol were obtained from Energy-Chemical (China).
Dulbecco's modified Eagle medium (DMEM) and fetal bovine
serum (FBS) were obtained from Gibco. Ultrapure Milli-Q water
was used in all experiments. The ligand 6-phenyl-2,2'-bipyridine
(15.8 g) in ethanol and acetic acid (2:1, 190 mL) was added, and
the mixture was refluxed for another 5 h by strong stirred. After
the solution cooled down to the room temperature, a lot of
yellow microcrystal was obtained. The microcrystal was not
needed to be purified further before used.
5
1
(pbpy) was prepared according to the reported method.
Before the experiments, DMSO was used to dissolve the
complexes. During the experiments with cells, the
concentration of DMSO was less than 1% (v/v).
Secondly, RuCl
mg, 1.00 mmol, 1 eq.) were refluxed with EtOH (125 mL). After
h, the mixture was cooled, and the formed precipitate was
3 2
•6H O (262 mg, 1.00 mmol, 1 eq.) and L5 (399
Thermo LTQ XL mass spectrometer was used for measuring
electrospray ionization mass spectrum (ESI-MS). Nuclear
magnetic resonance spectrometer (Bruker AVANCE AV 500) was
used for measuring NMR spectra and tetramethylsilane (TMS)
was used as a standard. UV-vis spectra were recorded on a
Perkin-Elmer Lambda 850 spectrophotometer. ICP-MS was
conducted on iCAP-RQ inductively coupled plasma mass
spectrometer (ICP-MS Thermo Elemental Co., Ltd). Rigaku R-
AXIS SPIDER Image Plate diffractometer, with Mo Kα radiation
3
collected. The precipitate was washed by EtOH and ether
several times, and dried under vacuum. The intermediate (140
mg, 230 µmol, 1 eq.) was further reacted with pbpy (53.6 mg,
2
30 µmol, 1 eq.) and triethylamine (1 mL) in EtOH (10 mL) under
Ar atmosphere at 78 °C for 8 h. After cooling down to the room
temperature, 40 mL water was poured down, and then filtered.
The filtrate was collected and NH
was filtered and washed by ether. The crude product was
obtained after dried and purified through a neutral Al
4 6
PF was added. The residue
(λ = 0.71073 Å), was used for X-ray diffraction measurements.
2 3
O
column (100-200 mesh) with toluene/MeCN (v/v, 1:1) as the
eluent. The solvent was removed via rotary evaporator, and
then the purified Ru5 was obtained.
Synthesis of the ruthenium complexes
The complexes were synthesized by similar methods according
to previous studies.27 Firstly, for the synthesis of the ligand 4'-
phenyl-2,2':6',2''-terpyridine (L0), a mixture of EtOH (25 mL) ,
KOH (560 mg, 10 mmol, 1 eq), 2-acetylpyridine (1.21 g, 10 mmol,
[
RuL0(pbpy)](PF
purple crystal. ES-MS (MeOH): m/z = 642.2 [M – PF
500 MHz, d -DMSO): δ = 9.29 (s, 2H), 8.95 (d, J = 8.1 Hz, 2H),
.74 (d, J = 8.2 Hz, 1H), 8.69 (d, J = 8.0 Hz, 1H), 8.39 (t, J = 8.6 Hz,
6
) (Ru0). Yield: 138 mg, 175 µmol, 76%. Deep
-
+ 1
6
] . H NMR
(
6
8
3
4
2
eq.), benzaldehyde (530 mg, 5 mmol, 1 eq.) and NH
3 2
•H O
H), 8.15 (t, J = 8.0 Hz, 1H), 7.97-7.93 (m, 1H), 7.87 (dd, J = 11.2,
.4 Hz, 3H), 7.70 (t, J = 7.7 Hz, 2H), 7.58 (t, J = 7.4 Hz, 1H), 7.54
(14.5 mL) were added accordingly into a 100 mL round bottom
flask and stirred at room temperature for 24 h. Then the cream
white precipitate was filtered and washed with EtOH, water and
diethyl-ether three times. The ligand isolated as a white solid
was dried and saved for subsequent use.
(
d, J = 5.3 Hz, 1H), 7.36 (d, J = 5.5 Hz, 2H), 7.16(dt, J = 12.9, 6.2
Hz, 3H), 6.68 (dd, J = 11.0, 4.0 Hz, 1H), 6.46 (dd, J = 11.0, 4.4 Hz,
H), 5.66 (d, J = 7.5 Hz, 1H).
RuL1(pbpy)](PF ) (Ru1). Yield: 133 mg, 166 µmol, 72%. Deep
purple crystal. ES-MS (MeOH): m/z = 660.2 [M – PF
500 MHz, d -DMSO): δ = 9.28 (s, 2H), 8.94 (d, J = 8.1 Hz, 2H),
.71 (dd, J = 26.4, 8.1 Hz, 2H), 8.43 (dd, J = 27.2, 6.6 Hz, 3H), 8.15
t, J = 8.0 Hz, 1H), 7.94 (t, J = 7.8 Hz, 1H), 7.87 (t, J = 7.3 Hz, 3H),
1
[
6
Secondly, RuCl
mg, 1.00 mmol, 1 eq.) were refluxed with EtOH (125 mL). After
h, the mixture was cooled, and the formed precipitate was
3 2
•6H O (262 mg, 1.00 mmol, 1 eq.) and L0 (309
-
+ 1
6
] . H NMR
(
8
(
6
3
collected. The precipitate was washed by EtOH and ether
several times, and dried under vacuum. The intermediate (119
mg, 230 µmol, 1 eq.) was further reacted with 6-phenyl-2,2'-
bipyridine (pbpy, 53.6 mg, 230 µmol, 1 eq.) and triethylamine (1
mL) in glycol (10 mL) under Ar atmosphere at 120 °C for 8 h.
7
6
.55 (t, J = 8.5 Hz, 3H), 7.37 (d, J = 5.2 Hz, 2H), 7.20-7.13 (m, 3H),
1
9
.68 (t, J = 7.4 Hz, 1H), 6.46 (t, J = 7.3 Hz, 1H), 5.66 (s, 1H).
F
6
NMR (470 MHz, d -DMSO) δ -111.13, -111.15.
6
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
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Page 7 of 10
Pleas De ad l to o nn oT tr aa nd sj au cs t ti omn as rgins
Journal Name
ARTICLE
[
RuL2(pbpy)](PF
purple crystal. ES-MS (MeOH): m/z = 678.2 [M – PF
500 MHz, d -DMSO): δ = 9.31 (s, 2H), 8.95 (d, J = 8.1 Hz, 2H), collection and refinement and the crystal parameters are shown
.74 (d, J = 8.1Hz, 1H), 8.69 (d, J = 8.0 Hz, 1H), 8.56 (ddd, J = in Table S1-S4. In Table S5-S11, bond length (Å) and selected
6
View Article Online
) (Ru2). Yield: 163 mg, 198 µmol, 86%. Deep thermal parameters and isotropic thermal parameters were
-
+
1
DOI: 10.1039/D0DT01412E
6
] . H NMR applied to refine all non-hydrogen atoms. The details of the data
(
6
8
1
1
7
=
o
2.3, 7.7, 2.2 Hz, 1H), 8.40 (d, J = 8.1 Hz, 1H), 8.31 (d, J = 8.4Hz, bond angles ( ) are presented. The supplementary
H), 8.16 (t, J = 8.0Hz, 1H), 7.97-7.93 (m, 1H), 7.92-7.85 (m, 3H), crystallographic data (CCDC 1611114 for Ru0, CCDC 1610273 for
.79 (dd, J = 19.1, 8.6 Hz, 1H), 7.53 (d, J = 5.2 Hz, 1H), 7.37 (d, J Ru1, CCDC 1610282 for Ru2, CCDC 1611011 for Ru3a, CCDC
5.5 Hz, 2H), 7.20-7.13 (m, 3H), 6.69 (t, J = 7.0 Hz, 1H), 6.45 (t, 1611142 for Ru3b, CCDC 1610281 for Ru4 and CCDC 1611141
J = 7.3 Hz, 1H), 5.63 (d, J = 7.5 Hz, 1H). F NMR (470 MHz, d
DMSO) δ -135.98, -136.03, -136.46, -136.51.
1
9
6
-
for Ru5) can be obtained free of charge from the Cambridge
Crystallographic Data Centre via
[
RuL3a(pbpy)](PF
Deep purple crystal. ES-MS (MeOH): m/z = 696.2 [M – PF
NMR (500 MHz, d -DMSO): δ = 9.33 (s, 2H), 8.95 (d, J = 8.1 Hz,
H), 8.75 (d, J = 8.2 Hz, 1H), 8.70 (d, J = 8.0 Hz, 1H), 8.50 (dd, J
9.6, 6.7 Hz, 2H), 8.41 (d, J = 8.0 Hz, 1H), 8.17 (t, J = 8.0 Hz, 1H),
.99-7.85 (m, 4H), 7.52 (d, J = 5.3 Hz, 1H), 7.39(d, J = 5.5 Hz, 2H), method was used to determine the n-octanol/water (log Po/w
.23-7.18 (m, 2H), 7.16-7.12 (m, 1H), 6.68 (dd, J = 10.8, 4.1 Hz, partition coefficients of the ruthenium complexes. First, a
6
) (Ru3a). Yield: 130 mg, 154 µmol, 67%. https://summary.ccdc.cam.ac.uk/structure-summary-form.
-
+ 1
6
] . H
6
Log P measurement
shake-flask ultraviolet absorptive spectrophotometry
2
=
7
7
1
A
2
9
)
1
9
H), 6.45 (t, J = 7.3 Hz, 1H), 5.60 (d, J = 7.5 Hz, 1H). F NMR (470 mixture of 50 mL n-octanol and 50 mL aqueous NaCl (0.9%, w/v)
-DMSO) δ -134.31, -134.39, -160.65, -160.73, -160.81. was left shaking at 37 °C for 48 h. The ruthenium complex
RuL3b(pbpy)](PF ) (Ru3b). Yield: 151 mg, 179 µmol, 78%. standard solutions were prepared using the water phase or
Deep purple crystal. ES-MS (MeOH): m/z = 696.2 [M – PF
NMR (500 MHz, d -DMSO): δ = 9.06 (s, 2H), 8.76 (t, J = 8.2 Hz, solutions (10 μM) were prepared with mixed solvent containing
H), 8.71 (d, J = 8.0 Hz, 1H), 8.41 (d, J = 8.1 Hz, 1H), 8.18 (t, J = 25 mL of n-octanol and 25 mL of aqueous NaCl (0.9%, w/v), and
.0 Hz, 1H), 7.98-7.94 (m, 1H), 7.89-7.83 (m, 3H), 7.61 (t, J = 8.6 shaken for 48 h. Third, the concentrations of the ruthenium
) and organic phase (C ) of the
m, 3H), 6.69 (t, J = 7.1 Hz, 1H), 6.48 (t, J = 7.3 Hz, 1H), 5.66 (d, J detecting solution were determined using ultraviolet
MHz, d
[
6
6
-
+ 1
6
] . H organic phase. Secondly, the ruthenium complex detection
6
3
8
Hz, 2H), 7.55 (d, J = 5.2 Hz, 1H), 7.41 (d, J = 5.5 Hz, 2H), 7.21-7.14 complexes in the water phase (C
w
o
(
=
-
7.4 Hz, 1H). 19F NMR (470 MHz, d
104.71, -108.61, -108.62.
RuL4(pbpy)](PF
purple crystal. ES-MS (MeOH): m/z = 714.2 [M – PF
500 MHz, d -DMSO): δ = 9.14 (s, 2H), 8.76 (dd, J = 8.0, 5.1 Hz,
H), 8.72 (d, J = 8.0 Hz, 1H), 8.42 (d, J = 8.1 Hz, 1H), 8.20 (dd, J =
6
-DMSO) δ -104.70, -104.71, spectrophotometry. The n-octanol/water partition coefficient
(log P), is calculated by the following equation (1):
[
6
) (Ru4). Yield: 136 mg, 159 µmol, 69%. Deep
log P = lg (C
o
/C
w
)
(1)
-
+ 1
6
] . H NMR
(
6
Cell culture
3
1
3
4.0, 5.9 Hz, 2H), 8.00-7.95 (m, 1H), 7.87 (dd, J = 11.2, 4.4 Hz, The human cervix carcinoma cell line HeLa, the lung carcinoma
H), 7.57 (d, J = 5.2 Hz, 1H), 7.43 (d, J = 5.4 Hz, 2H), 7.25-7.19 cell lines A549 and A549R (cisplatin-resistant cells), and the
(
1
m, 2H), 7.16 (dd, J = 9.4, 3.5 Hz, 1H), 6.69 (dd, J = 10.9, 3.9 Hz, human non-tumor hepatocyte cell line LO2 were selected for
H), 6.48 (t, J = 7.3 Hz, 1H), 5.64 (d, J = 7.4 Hz, 1H). F NMR (470 this work. The cell lines were acquired from the Experimental
1
9
6
MHz, d -DMSO) δ -136.94, -136.97, -136.99, -137.02, -140.57, - Animal Centre of Sun Yat-Sen University (Guangzhou, China). All
1
40.60, -140.62, -140.65.
cells were maintained as monolayer cultures and incubated
[
RuL5(pbpy)](PF
6
) (Ru5). Yield: 88.8 mg, 101 µmol, 44%. with DMEM containing 10% fetal bovine serum (FBS) at 37 °C
-
+
Modena crystal. ES-MS (MeOH): m/z = 732.1 [M – PF
NMR (500 MHz, d -DMSO): δ 9.12 (s, 2H), 8.78 (t, J = 8.9 Hz, 3H),
.73 (d, J = 8.0 Hz, 1H), 8.44 (d, J = 8.0 Hz, 1H), 8.22 (t, J = 8.0 Hz,
H), 8.01-7.96 (m, 1H), 7.89 (dd, J = 10.7, 4.5 Hz, 3H), 7.59 (d, J
5.2 Hz, 1H), 7.46 (d, J = 5.5 Hz, 2H), 7.23 (dd, J = 9.6, 3.5 Hz, MTT assay was used for measuring the toxicity of the ruthenium
H), 7.20-7.15 (m, 1H), 6.71 (t, J = 7.4 Hz, 1H), 6.50 (t, J = 7.3 Hz, complexes towards cells. Approximately 1 × 10 cells were
6 2
] . 1H under 5% CO atmosphere.
6
8
1
=
2
1
1
1
Cytotoxicity tests
4
1
9
H), 5.56 (d, J = 7.5 Hz, 1H). F NMR (470 MHz, d
41.61, -141.63, -141.66, -141.68, -153.53, -153.57, -153.62, - and before culture medium was removed. The ruthenium
61.82, -161.83, -161.87, -161.88, -161.92, -161.93. complexes (or cisplatin) were added at different concentrations
diluted by DMEM with a final DMSO concentration of 1%, v/v)
and then were incubated for another 48 h. After that, 15 μL MTT
5 mg/mL) were added to each well and the cells incubated for
6
-DMSO) δ - seeded in every well in 96-well plates and incubated for 24 h,
(
X-ray crystallography
(
Rigaku R-AXIS SPIDER Image Plate diffractometer, with Mo Kα a further 4 h at 37 °C. Finally, the media was removed and
radiation (λ = 0.71073 Å) at 150 K, was used for X-ray diffraction replaced with 150 μL of DMSO in each well. After shaking for 5
measurements. The SHELXL 2014/7 and the SHELXL 2014/7 min, the OD590 values were measured with a Tecan Infinite
program packages were used to perform the full-matrix least- M200 monochromator-based multifunction microplate reader
2
squares refinement and structure solution based on F for the (Biorad, USA).
ruthenium complexes. Based on the parent atoms, anisotropic
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
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Pleas De ad l to o nn oT tr aa nd sj au cs t ti omn as rgins
Page 8 of 10
ARTICLE
Journal Name
Cellular uptake and localization
Analysis of MMP
View Article Online
DOI: 10.1039/D0DT01412E
5
A549R cells and LO2 cells were cultured in a 100 mm plate with A549R cells were cultured at a density of 1 × 10 cells per mL in
1
0 mL DMEM medium containing 10% FBS for 24 h. After 3.5 cm glass bottom dishes (Corning) for 48 h and then the cells
treatment with the ruthenium complexes (1.0 μM) for 9 h, the were incubated with different concentrations of ruthenium
cells were collected and counted. The cells were divided into complexes for 12 h. After the cells were washed twice with PBS,
portions and organelles extracted with the commercially the cells were incubated with pre-warmed staining working
obtained nucleus extraction kit or mitochondrial extraction kit solution containing JC-1 (5 μg/mL) for another 15 min. The cells
(Shanghai Sangon Biological Engineering Technology & Services were then washed twice with PBS and imaged by confocal
Co. Ltd.). The cytoplasm with mitochondria was obtained from luminescence microscopy (Zeiss LSM 710 laser microscopy
the nucleus extraction experiment. The samples were digested system)
with 60% HNO
3
at rt for one day, before dilution with Milli-Q
solutions. Inductively coupled plasma
2
H O to obtain 2% HNO
3
Caspase-3/7 activation detection assay
mass spectrometry (ICP-MS Thermo Elemental Co., Ltd) was
used to measure the ruthenium content of the samples.
A549R cells were seeded in white-walled solid-bottomed 96-
4
well plates at 1× 10 cells per well and incubated for 24 h. The
cells were then treated with different concentrations of
ruthenium complexes or cisplatin. After 12 h, the expression
Cellular uptake mechanism
The cellular uptake mechanism was performed according to Ref. index of caspase 3/7 in the A549R cells was investigated with a
5-37. For metabolic inhibition, the A549R cells were pre- Caspase-Glo 3/7 assay kit (Promega, USA) according to the
3
treated with inhibitors (50 mM 2-deoxy-D-glucose, 5 μM manufacturer's instructions and measured by a microplate
+
oligomycin, 50 mM NH
4
Cl or 100 μM chloroquine) or high K - reader (InfiniteM200 Pro, Tecan, Männedorf, Switzerland).
+
HBSS (containing 170 mM K ) for 1 h and then incubated with
the complexes. Subsequently, the culture medium was
removed and the cells were collected. After digestion with 60%
Annexin V/PI double staining
3
HNO at rt for one day, the samples were diluted with Milli-Q Annexin V and propidium iodide (PI) co-staining of the apoptotic
H
2
O to obtain 2% HNO
3
solutions. ICP-MS was used to measure membranes was performed by using the Annexin V/PI apoptosis
Kit following the manufacturer's protocol (MultiSciences
the ruthenium content.
(
Lianke) Biotech Co., Ltd.). A549R cells were cultured in a 6-well
5
plate at 5 × 10 cells per well for 48 h. Different concentrations
of ruthenium complexes or cisplatin were incubated with the
EdU assays
EdU (5-Ethynyl-2'-deoxyuridine) is
a
kind of thymidine cells for 12 h. The cells were collected, washed twice with PBS
nucleoside analogue, which can replace thymine (T) during cell and resuspended. The cells were then stained with Annexin V-
proliferation in synthetic DNA molecules. After a cell cycle, the FITC and PI for 15 min in the dark at room temperature. The
4
0,41
cells incubated with Edu are labeled.
Then, Apollo® samples were quantified by flow cytometry on a FACS Canto II
fluorescent dye can interact with EdU labelled cells to indicate (BD Biosciences, USA).
the replication-competence of DNA using a detection kit
(
Ribobio, Guangzhou, China). A549R cells were cultured in 96-
Conflicts of interest
There are no conflicts to declare.
well plates for 48 h. Different concentrations of ruthenium
complexes or cisplatin were the cells in logarithmic phase for 24
h. The cells were washed twice with PBS, and EdU was then
added to each well. The cells were incubated with EdU solution
(100 μL, 10 μM) for another 24 h. After washing with PBS twice, Acknowledgements
the cells were fixed with 4% paraformaldehyde (pH 7.4) for 30
min, and then the solution removed. Glycine (50 μL, 2 mg/mL)
solution was then added to interact with excess aldehyde
groups for 5 min. The solution was removed and the PBS was
added for 5 min, before removal and the addition of 100 μL PBS
with 0.5% Triton X-100 and 10 min incubation. The cells were
washed again with PBS. Each well was incubated with Apollo®
working solution in a dark environment for 30 min, and 100 μL
PBS with 0.5% Triton X-100 was then added to wash the cells 3
times. Finally, 100 μL Hoechst 33342 working solution was
added to each well and the cells were incubated for 30 min. The
cells were washed with PBS twice and 100 μL PBS was added to
each well. The cells were finally observed under an inverted
fluorescence microscope (Zeiss Axio Observer Z1, Germany).
This work was supported by the National Science Foundation of
China (Nos. 21525105, 21778079 and 21904144), and China
Postdoctoral Science Foundation (No. 2018M630988).
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Dalton Transactions
Page 10 of 10
View Article Online
DOI: 10.1039/D0DT01412E
Graphical Abstract
Fluorinated cyclometalated ruthenium(II) complexes cause cisplatin-resistant cancer
cells apoptosis by mitochondria and nuclear-targeting damage.