Anticancer Activity of Bifunctional Organometallic Ru(II) Arene Complexes Containing a 7-Hydroxycoumarin Group

Article abstract of DOI:10.1021/acs.organomet.7b00842

Three organometallic Ru(arene) compounds bearing 7-hydroxycoumarin have been designed and synthesized. Resulting Ru(II) complexes 2-4 showed potent cytotoxicity against the tested cancer cell lines, much higher than either the ligand L1 or unfunctionalized complex 1 alone. Further study indicated complexes 2-4 can activate the expression of Bax, induce cytochrome c release from the mitochondria, and finally activate caspase-3, hinting that these compounds probably induce cell apoptosis by mitochondrial pathway. Additional molecular docking study showed that complexes 2-4 have the potential to bind with the MEK1, and Western blot analysis confirmed that the Ru(II) complexes can prevent the phosphorylation of MEK1 and ERK1 in HCT116 cells, which helps us to further understand the anticancer mechanisms of the newly synthesized complexes. Overall, our research suggested that the ruthenium-coumarin complexes could induce cell apoptosis via inhibition of the mitochondrial and ERK signal pathways.

Full text of DOI:10.1021/acs.organomet.7b00842

Article
Cite This: Organometallics XXXX, XXX, XXX−XXX
Anticancer Activity of Bifunctional Organometallic Ru(II) Arene
Complexes Containing a 7‑Hydroxycoumarin Group
Jian Zhao,†,‡ Dingyi Zhang,† Wuyang Hua,† Wanchun Li,† Gang Xu,†,‡ and Shaohua Gou*,†,‡
†Pharmaceutical Research Center and School of Chemistry and Chemical Engineering and ‡Jiangsu Province Hi-Tech Key Laboratory
for Biomedical Research, Southeast University, Nanjing 211189, China
S
* Supporting Information
ABSTRACT: Three organometallic Ru(arene) compounds bearing 7-
hydroxycoumarin have been designed and synthesized. Resulting Ru(II)
complexes 2−4 showed potent cytotoxicity against the tested cancer cell
lines, much higher than either the ligand L1 or unfunctionalized complex
1 alone. Further study indicated complexes 2−4 can activate the
expression of Bax, induce cytochrome c release from the mitochondria,
and finally activate caspase-3, hinting that these compounds probably
induce cell apoptosis by mitochondrial pathway. Additional molecular
docking study showed that complexes 2−4 have the potential to bind with
the MEK1, and Western blot analysis confirmed that the Ru(II)
complexes can prevent the phosphorylation of MEK1 and ERK1 in
HCT116 cells, which helps us to further understand the anticancer
mechanisms of the newly synthesized complexes. Overall, our research
suggested that the ruthenium-coumarin complexes could induce cell
apoptosis via inhibition of the mitochondrial and ERK signal pathways.
arene complexes such as [(C6H5Ph)-Ru(en)Cl][PF6] (RM175,
en = ethylenediamine) and [(piPrC6H4Me)Ru(pta)Cl2]
(RAPTA-C, pta = 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]-
decane) are also showing great therapeutic potential (Figure
1).19−21 In general, arene Ru(II) complexes present a “piano
stool” geometry in which arene forms the seat and the other
ligands resemble the legs.22−24 The hydrophobic arene group
can influence the cellular uptake and affect the kinetic reactivity
of the ruthenium(II) complex,25,26 while the rest coordination
ligands may provide more opportunities to design anticancer
agents with different modes of action.27−31
The ERK/MAPK (RAS/RAF/MEK/ERK) pathway plays a
central role in regulating cellular growth, proliferation, and
survival.32 MEK is an intermediary in the ERK/MAPK
pathway, which can be activated by RAF, leading to the
subsequent phosphorylation of ERK1/2.33 Hence, MEK has
emerged as a promising molecular target for tumor therapy.34
Coumarins, a wide class of natural compounds, showed
versatile biological functions including anti-inflammatory,
antioxidant, antithrombotic, and antidepressant.35,36 Moreover,
coumarin as well as its derivatives is one of the most important
natural products presenting anticancer properties. Coumarins
exhibited anticancer activities through various mechanisms, for
example kinase inhibition, telomerase inhibition, heat shock
protein (HSP90) inhibition, and cell cycle arrest.37,38 SAR
(structure−activity relationship) study revealed that the
INTRODUCTION
■
Platinum-based anticancer drugs, such as cisplatin, carboplatin,
and oxaliplatin, are well-established treatment options for a
wide range of tumors, particularly in colorectal, testicular and
nonsmall cell lung cancers, but they are limited by severe side
effects and acquired drug resistance.1−6 Numerous researches
indicated that ruthenium-based compounds could be regarded
as the promising candidates for alternative agents to take the
place of platinum drugs in cancer therapy due to their lower
toxicity and high selectivity.7−14 So far, two ruthenium(III)
compounds, [ImH][trans-Ru(DMSO)(Im)Cl4] (NAMIA, Im =
imidazole) and [IndH][trans-Ru(Ind)2Cl4] (KP1019, Ind =
indazole), have entered clinical trials (Figure 1). However,
NAMI-A failed in phase II tests, while KP1019 and its sodium
salt KP1339 (sodium [trans-RuCl4(1H-indazole)2]) are still
under clinical trials.10,15−18 Meanwhile, organometallic Ru(II)
Received: November 22, 2017
Figure 1. Related anticancer ruthenium complexes in this paper.
© XXXX American Chemical Society
A
DOI: 10.1021/acs.organomet.7b00842
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substitution on the coumarin ring can strongly affect the
anticancer activity of the resulting compounds.35,37 Among the
diverse substitutions on the coumarin framework, O-substituted
analogs of 7-hydroxycoumarins have shown desirable anticancer
activities and attracted extensive interest in recent years.35
Moreover, some studies recently revealed that 7-hydroxycou-
marin derivatives can inhibit the ERK/MAPK pathway via
MEK1 targeting.33,39 These characters make coumarin a
promising scaffold for anticancer agents.40−42
data are in good agreement with the corresponding structure of
complexes 2−4. Besides, the absorption and emission spectra
were studied (Figure 2). Notably, complexes 2−4 showed
similar absorption and emission spectra, but the fluorescence
intensity of ligand L1 was much higher than that of complexes
2−4.
Since Ru(II) complexes have been widely studied either as
single anticancer agents or in combination with other cytotoxic
agents, hybridization of Ru(II) complexes and other bioactive
pharmacophores is an effective strategy to design novel
anticancer agents.14 On the basis of the above study, it is
rational to introduce a 7-hydroxycoumarin moiety to Ru(II)
complexes to improve the pharmacological activity of the arene
Ru(II) complexes. According to our assumption, the newly
synthesized complexes could exhibit their anticancer activity
through multiple mechanisms. Herein, a series of ruthenium−
coumarin complexes were designed to evaluate the biological
activities as well as the possible underlying anticancer
mechanisms.
Figure 2. UV−visible absorbance (a) and emission spectra (b) of
ligand L1 and complexes 2−4 in H2O/MeOH (5:95) at 25 °C.
In Vitro Cytotoxicity. The cytotoxic activities of L1 and
complexes 1−4 against HCT-116 (colorectal cancer), HepG-2
(hepatocellular carcinoma), and A549 (nonsmall cell lung
cancer) cell lines were investigated by MTT assay with cisplatin
as a positive control. The IC50 values (dose required to inhibit
50% cellular growth) were determined from dose-survival
curves (Table 1). According to the IC50 values, both L1 and
RESULTS AND DISCUSSION
■
Synthesis and Characterization. Ligand L1 and com-
plexes 2−4 were prepared by following the procedure shown in
Scheme 1-2. 7-Hydroxycoumarin was first reacted with
Table 1. In Vitro Cytotoxicity (IC50 μM) of Ligand L1 and
Complexes 1−4 against Human Cancer Cell Lines
IC50 (μM)
a
Scheme 1. Preparation of Ligand L1 and Complex 2
a
b
c
compd
L1
HCT-116
>500
HepG2
>500
>500
A549
>500
>500
complex 1
complex 2
complex 3
complex 4
cisplatin
460.7 21.4
65.6 4.6
85.07 7.4
131.7 7.5
15.2 1.3
78.7 4.8
118.4 9.6
143.3 7.9
13.6 2.8
72.2 6.8
86.51 5.5
161.4 7.5
16.5 1.5
a
b
Human colorectal cancer cell line. Human hepatocellular carcinoma
c
cell line. Human nonsmall-cell lung cancer cell line.
a
complex 1 exhibited negligible cytotoxocity against the tested
cell lines, while complexes 2−4 showed modest antiproliferative
activity with the IC50 range from 65.6 to 161.4 μM. Obviously,
the cytotoxicity of complexes 2−4 is much greater than either
ligand L1 or unfunctionalized complex 1 alone, indicating that
both ruthenium moieties and coumarin analog played
important roles in generating cytotoxic activity. However, the
cytotoxicity of complexes 2−4 was much lower than that of
cisplatin. It is worth noting that the IC50 values of the resulting
Ru(II) arene complexes could be as low as submicromolar, but
often were >100 μM such as RAPTA-C with excellent in vivo
activity.43,44 To our knowledge, the relatively hydrophobic
cymene ligand of the arene Ru(II) could promote trans-
membrane transport, which may increase the cellular uptake of
the 7-hydroxycoumarin moiety, besides these components
could work synergistically toward different targets on the tumor
cells. Introduction of the 7-hydroxycoumarin moiety to the
Ru(II) complex is an effective way to improve the cytotoxic
activity of the Ru(II) arene complex.
Conditions: (a) K2CO3, KI, acetone, reflux, 8 h; (b) TBTU, Et3N,
DMF, 40 °C, 4 h; (c) CH2Cl2, 60 °C, 2 h.
a
Scheme 2. Preparation of Complexes 3 and 4
a
Conditions: (a) H2O, 50 °C, 24 h; (b) anhydrous methnol, 50 °C, 12
h.
bromoacetic acid and then with 4-pyridinemethanol to produce
ligand L1. Complex 2 was subsequently synthesized by treating
the dimer [Ru(η6-p-cymene)Cl2]2 and L1. For complexes 3 and
4, the dimer [Ru(η6-p-cymene)Cl2]2 was first treated with silver
carboxylate and then with L1 to give the target complexes. The
synthesized complexes were characterized by elemental
analysis, 1H and 13C NMR spectra (see Supporting
Information) along with ESI-MS spectrometry. The spectral
Cell Cycle. The perturbation effects of ligand L1 and
complexes 1−4 on cell cycle progression of HCT-116 cells
were analyzed by flow cytometry (Figure 3). The cell cycle data
clearly showed that complexes 2−4 blocked the cell cycle
mainly at G2/M phase with respect to the untreated control,
B
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Figure 3. Cell cycle distribution upon treatment with ligand L1 and
complexes 1−4 in HCT-116 cells.
while free ligand L1 induced the cell cycle at G0/G1 phase
under the test conditions, indicating that there may be a little
difference in the mechanisms of ligand L1 and complexes 2−4.
Besides, the percentage of cancer cells arrested at the G2/M
phase by complex 2 (46.00%) was higher than those of
complexes 3 (43.45%) and 4 (38.11%), demonstrating that
treatment with complex 2 affected the G2/M populations more
than treatment with complexes 3 and 4. However, complex 1
induced negligible changes on cell cycle distribution. This study
indicated that complexes 2−4 arrested the cell cycle in G2/M
phase in HCT-116 cells, which is similar to most of the
antineoplastic drugs which induce the cell cycle in the G2/M or
S phases.45,46
Figure 5. Flow cytometry analysis for apoptosis of HCT-116 cells
induced by complexes 1−4 at the same concentration of 150 μM for
24 h. Lower left, living cells; lower right, early apoptotic cells; upper
right, late apoptotic cells; upper left, necrotic cells. Inserted numbers in
the profiles indicate the percentage of the cells present in this area.
respectively, for 24 h at a concentration of 150 μM. The result
is shown in Figure 5. Obviously, treatment with complexes 2−4
increased the early apoptotic cell populations of the HCT-116
cells compared with the negative control. The relative order of
inducing apoptosis against HCT-116 cells is 2 (67.4%) > 3
(64.6%) > 4 (55.3%) > 1 (1.29%), which is consistent with the
result of cytotoxicity study to some extent. Overall, complexes
2−4 caused cells apoptosis rather than necrosis, indicating that
these three complexes produced cancer cell death through an
apoptotic pathway.
To further investigate the apoptosis pathway induced by
complexes 2−4, Western blot assay was applied to evaluate the
expression of the apoptosis-related proteins in HCT-116 cells.
As shown in Figure 6, complexes 2−4 upregulated the
Cellular Uptake. To gain insight into the cellular behavior
of the ruthenium-coumarin complexes, the intracellular uptake
of complex 2 was studied by laser scanning confocal
microscope (LSCM). As shown in Figure 4, complex 2 could
Figure 6. (a) HCT-116 cells treated with complexes 2−4 (20 μM) for
12 h were examined for the expression of apoptosis-regulated proteins
using Western blot analysis. The data are representative of three
independent experiments. (b) Densitometric analysis of the expression
of apoptosis-regulated proteins normalized with GAPDH. The relative
expression of each protein was represented by the density of the
protein band/density of GAPDH band.
Figure 4. Confocal microscopy images of HCT-116 cells incubated
with complex 2 for 3 h at 37 °C. Pictures show cells in green channel,
bright-field, and the corresponding merged images: (a) complex 2 at
10 μM; (b) complex 2 at 30 μM. Cell images were obtained at an
excitation wavelength of 405 nm with a (475−575 nm) emission filter.
enter into the HCT-116 cells causing intracellular fluorescence
after 3 h incubation. Moreover, it was apparent that the
intracellular fluorescence intensity of complex 2 at the
concentration of 30 μM was higher than that of the
concentration at 10 μM, indicating that the cellular uptake of
complex 2 occurred in a concentration-dependent manner.
Apoptosis Study. The determination of the cellular death
mechanism (necrosis or apoptosis) was performed by using an
Annexin V-FITC/propidium iodide (PI) assay (Figure 5).
Complexes 1−4 were incubated with HCT-116 cells,
expression levels of cytochrome c (cytosol) and Bax, cleaved
PARP and caspase-3, and downregulated the expression of
procaspased-3, demonstrating that these complexes could
activate the expression of Bax, induce cytochrome c release
from the mitochondria, and finally activate caspase-3. This
study indicated that complexes 2−4 probably induced cell
apoptosis by mitochondrial pathway.
Docking Study. It was reported that coumarins can dock
into the allosteric site of the MEK1 structure.39 To further
discover the anticancer mechanism of the newly synthesized
C
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ruthenium-coumarin complexes which shared the coumarin
scaffold, docking study with crystal structure of MEK1 (Protein
Databank (PDB) ID: 1S9J) was performed using Autodock 4.2
package.39,47 The structures of complexes 2−4 were optimized
at the M06-L/6-31G(d,p)//LanL2DZ level using Gaussian09
(Figure S9).48 The docking results are shown in Figure 7.
Figure 8. (a) HCT-116 cells treated with complexes 2 and 3 for 12 h
were examined for the expression of P-MEK and P-ERK proteins using
Western blot analysis. The data are representative of three
independent experiments. (b) Densitometric analysis of the expression
of P-MEK and P-ERK proteins normalized with β-actin. The relative
expression of each protein was represented by the density of the
protein band/density of β-actin band.
concentration-dependent manner in HCT116 cells, implying
the inhibition of the downstream molecules in MAPK signal
transduction.
CONCLUSION
■
In this study, three organometallic Ru(arene) compounds
bearing 7-hydroxycoumarin pharmacophores have been
synthesized and characterized. The newly synthesized ruthe-
nium-coumarin complexes showed improved cytotoxicity
against the tested cell lines, especially complex 2, which
exhibited the strongest cytotoxic activity. It was found that
neither the ligand L1 nor the unfunctionalized ruthenium
analogue complex 1 showed activity on their own, indicating
that the Ru(arene) compound and coumarin have a positive
cooperative effect on cancer cells. Flow cytometry studies
showed that complexes 2−4 can block the cell cycle in G2/M
phase and produce death of tumor cells through an apoptotic
pathway. Moreover, complexes 2−4 can activate the expression
of Bax, induce cytochrome c release from the mitochondria,
and finally activate caspase-3, hinting that these compounds
probably induce cell apoptosis by mitochondrial pathway.
Additional molecular docking study showed that complexes 2−
4 have the potential to bind with the MEK1, and Western blot
analysis confirmed that the resulting Ru(II) complexes can
prevent the phosphorylation of MEK1 and ERK1 in HCT116
cells. All these results suggested that the newly synthesized
ruthenium-coumarin complexes could induce cell apoptosis via
inhibition of the mitochondrial and ERK signal pathways. Since
cancer is a complicated disease, anticancer agents that act at
multiple targets can improve their efficacy, which are thought to
be effective way for anticancer drug design. Overall, our
research indicates that the coordination of the bioactive ligand
to metal fragments with established anticancer properties to
obtain a synergistic effect on targeting the tumor cells may be a
reasonable approach for antitumor drug development.
Figure 7. Binding mode of complexes 2−4 within the MEK1 (1S9J)
allosteric catalytic site, yellow dashed lines indicate the H-bond
interaction between the complex and MEK1: (a) complex 2; (b)
complex 3; (c) complex 4.
Obviously, for complexes 2−4, the carbonyl oxygen from the
coumarin ring formed two critical hydrogen bonds with
residues Val211 and Ser212 in a similar manner to other
published MEK1 inhibitors,39,49 indicating that the introduction
of the ruthenium moieties did not affect the binding ability of
coumarin to MEK1. Moreover, the ester carbonyl group of
complexes 2−4 also made a hydrogen bond with the backbone
of Arg234. Overall, the docking experiments demonstrated that
complexes 2−4 have the potential to bind with the MEK1.
Effects of Complexes 2 and 3 on MEK/ERK Signaling.
To further determine the effect of these complexes on the
ERK/MAPK pathway signaling, complexes 2 and 3 were
selected to explore the effect on the protein expression of P-
MEK1 and P-ERK1 in HCT116 cells. As shown in Figure 8, the
expression of P-ERK1 was decreased after treatment with
complexes 2 and 3, reflecting the inhibition of the activity of
MEK1. Moreover, complexes 2 and 3 also showed significant
effect in suppressing MEK1 phosphorrylation, indicating that
complexes 2 and 3 could also block the MEK1 being
phosphorylated by upstream kinase. Thus, complexes 2 and 3
can prevent the phosphorylation of MEK1 and ERK1 in a
EXPERIMENTAL SECTION
■
Materials and Measurements. All chemicals and solvents were
of analytical reagent grade and used without further purification.
[Ru(η6-p-cymene)Cl2]2 were prepared according to previous
reports.50 1H and 13C NMR spectra were measured on Bruker
spectrometers. Mass spectra were measured by an Agilent 6224 ESI/
TOF MS instrument. Elemental analysis of C, H, and N used a Vario
MICRO CHNOS elemental analyzer (Elementar). Cancer cells were
obtained from Jiangsu KeyGEN BioTECH company (China). Cell
cycle and apoptosis experiments were measured on a BD Accuri C6
flow cytometer and analyzed by Cell Quest software. UV−visible
measurements were performed on a Shimadzu UV2600 instrument
equipped with a thermostatically controlled cell holder. Fluorescence
D
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measurements were performed using a FluoroMax-4 fluorometer.
Scans were run at room temperature with excitation and emission slit
widths of 2.5 nm. The concentration of ligand L1 and complexes 2−4
was 30 and 10 μM for absorption and emission spectra study,
respectively.
(1H, d, J = 8.4 Hz, OCCHCH(Ar)), 8.01−8.04 (1H, d, J = 9.6 Hz,
CHCHCO), 8.45−8.47 (2H, d, J = 6.0 Hz, NCH(Py)) ppm. 13C
NMR (75 MHz, DMSO-d6) δ 17.68, 22.59, 30.94, 64.22, 65.32, 80.50,
83.01, 97.32, 100.91, 102.12, 113.11, 113.46, 123.86, 130.06, 144.72,
148.60, 153.26, 155.66, 160.67, 161.04, 164.78, 168.47 ppm.
Synthesis of L1. 7-Hydroxycoumarin (1.62 g, 10 mmol),
potassium carbonate (6.91 g, 50 mmol), bromoacetic acid (6.95 g,
50 mmol), and acetone (250 mL) were added into a 500 mL round-
bottom flask. Then a small amount of potassium iodide was added and
the mixture was refluxed for 8 h. After reaction finished, water (200
mL) was added, and the pH was adjusted to ∼3 by 5% HCl aqueous.
Then acetone was removed under reduced pressure at room
temperature, and white deposits (a) were obtained by filtration.
Intermediate a (1.10 g, 5 mmol) and TBTU (1.61 g, 5 mmol) were
dissolved in 100 mL of DMF, and then 4-pyridinemethanol (0.55 g, 5
mmol) and TEA (0.51 g, 5 mmol) were added and the reaction stirred
at 40 °C for 4 h. The solvent was then removed by evaporation under
reduced pressure. Column chromatography (eluent 15:1 DCM/
methanol) gave L1 as white solid. Yield: 0.98 g (62.8%). ESI-MS: m/z
Complex 4. Yield: 0.19 g (69.7%). Light-orange powder. Anal.
Calcd (%) for C30H29NO9Ru: C 55.55, H 4.51, N 2.16. Found: C
1
55.72, H 4.54, N 2.03. ESI-MS: m/z [M + H]+ = 650.1 (50%). H
NMR (400 MHz, CDCl3) δ 1.28−1.30 (6H, d, J = 6.8 Hz, CH−
(CH3)2), 2.17 (3H, s, CH3), 2.76−2.85 (1H, m, overlapped, J = 6.8
Hz, CH(CH3)2), 2.82−2.86 (1H, d, J = 15.6 Hz, OOCCH2COO),
3.43−3.47 (1H, d, J = 15.6 Hz, OOCCH2COO), 4.85 (2H, s, O
CCH2O), 5.28 (2H, s, Py−CH2O), 5.35−5.37 (2H, d, J = 6.0 Hz,
CH3CCH(Ar)), 5.50−5.52 (2H, d, J = 6.0 Hz, (CH3)2CHCCH(Ar)),
6.28−6.31 (1H, d, J = 9.2 Hz, CHCHCO), 6.80 (1H, s,
OCCHC(Ar)), 6.89−6.91 (1H, d, J = 8.4 Hz, OCCHCH(Ar)),
7.28−7.29 (2H, d, J = 6.0 Hz, CCH(Py)), 7.43−7.45 (1H, d, J = 8.4
Hz, OCCHCH(Ar)), 7.69−7.72 (1H, d, J = 9.6 Hz, CHCHCO),
8.66−8.68 (2H, d, J = 6.4 Hz, NCH(Py)) ppm. 13C NMR (100 MHz,
DMSO-d6) δ 17.88, 22.37, 30.66, 46.73, 64.07, 65.17, 81.42, 82.00,
97.27, 101.69, 102.58, 112.86, 113.57, 113.87, 123.40, 129.34, 143.48,
147.20, 152.89, 155.58, 160.50, 160.94, 167.64, 174.56 ppm.
Docking Studies. Docking studies were carried out using
Autodock Dock 4.2. The crystal data of MEK1 was obtained from
the Protein Databank (PDB ID: 1S9J). Small molecules in 1S9J were
removed prior to the docking by software PyMOL. The docking
simulation was performed with the Lamarckian genetic algorithm for
as much as 150 docking runs. Each run of the docking operation was
terminated after a maximum of 2 500 000 energy evaluations. During
docking studies, the protein structure was kept rigid. Rotation in
complexes 2−4 was permitted about all single bonds.
Cell Culture. Three human tumor cell lines, HCT-116 (human
colorectal cancer), HepG2 (human hepatocellular carcinoma), and
A549 (human non-small-cell lung cancer) were maintained in a
humidified atmosphere of 5% CO2 and 95% air at 37 °C. They were
cultured in RPMI-1640 medium supplemented with 10% fetal bovine
serum (FBS), 100 μg/mL penicillin, and 100 μg/mL streptomycin.
MTT Assay. The IC50 values of all compounds were determined by
means of the colorimetric assay MTT assay. This assay is based on the
cleavage of the yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl tetrazolium bromide; MTT, Sigma), forming purple
formazan crystals by viable cells. The cultured cells were plated in 96-
well culture plates at a density of 5000 cells per well and incubated for
24 h at 37 °C in a 5% CO2 incubator. The compounds were dissolved
in DMF and then diluted to the required concentration with culture
medium (the final concentration of DMF was less than 0.4%). The
diluted complexes were then added to the wells, and cells were
incubated at 37 °C for 72 h. Afterward, the cells were treated with 20
μL MTT dye solution (5 mg/mL) for 4 h cultivation. The media with
MTT were removed and replaced with DMSO (150 μL). The UV
absorption intensity was detected with an ELISA reader at 490 nm.
The IC50 values were calculated by SPSS software after three parallel
experiments.
Cell Cycle Measurement. HCT-116 cells were transferred into 6-
well plates and cultured overnight at 37 °C. Then, ligand L1 (30 μM)
and complexes 1−4 (30 μM) were incubated with cells for 24 h. All
adherent and floating cells were collected and washed with PBS and
fixed with 70% ethanol at 4 °C for 24 h. After being centrifuged, cells
were stained with 50 μg/mL propidium iodide solution containing 100
μg/mL RNase for 0.5 h at 37 °C. The samples were measured by flow
cytometry (FAC Scan, Becton Dickenson) using Cell Quest software
and recording propidium iodide (PI) in the FL2 channel.
1
[M + H]+ = 312.1. H NMR (400 MHz, CDCl3) δ 4.81 (2H, s, O
CCH2O), 5.27 (2H, s, Py-CH2O), 6.29−6.32 (1H, d, J = 9.2 Hz,
CHCHCO), 6.80−6.81 (1H, d, J = 2.4 Hz, OCCHC(Ar)), 6.89−
6.92 (1H, d-d, J = 8.8, 2.4 Hz, OCCHCH(Ar)), 7.25−7.26 (2H, d, J =
5.6 Hz, CCH(Py)), 7.41−7.43 (1H, d, J = 8.4 Hz, OCCHCH(Ar)),
7.65−7.68 (1H, d, J = 9.2 Hz, CHCHCO), 8.62−8.63 (2H, d, J =
6.0 Hz, NCH(Py)) ppm. 13C NMR (100 MHz, DMSO-d6) δ 65.21,
65.26, 101.75, 112.74, 113.51, 113.99, 122.12, 129.10, 143.18, 143.64,
150.20, 155.64, 160.54, 160.82, 167.63 ppm.
Synthesis of Complex 2. A solution of L1 (0.16 g, 0.5 mmol) in
dichloromethane (10 mL) was added to a suspension of [Ru(η6-p-
cymene)Cl2]2 (0.13 g, 0.21 mmol) in dichloromethane (15 mL)
dropwise. The mixture was stirred at 60 °C for 2 h. Then, the reaction
mixture was concentrated to 5 mL and cooling to room temperature.
The crude product was separated, washed with with Et2O (3 × 10 mL)
and hexane (20 mL) (3 × 10 mL), and dried in a vacuum-dryer. Yield:
0.14 g (54.0%). Light-orange powder. Anal. Calcd (%) for
C27H27Cl2NO5Ru: C 52.52, H 4.41, N 2.27. Found: C 52.38, H
1
4.58, N 2.26. ESI-MS: m/z [M − Cl]+ = 582.1. H NMR (600 MHz,
CDCl3) δ 1.31−1.32 (6H, d, J = 6.6 Hz, CH−(CH3)2), 2.11 (3H, s,
CH3), 2.97−3.01 (1H, m, J = 6.6 Hz, CH(CH3)2), 4.82 (2H, s, O
CCH2O), 5.25−5.26 (2H, d, J = 4.8 Hz, CH3CCH(Ar)), 5.27 (2H, s,
Py−CH2O), 5.46−5.47 (2H, d, J = 4.8 Hz, (CH3)2CHCCH(Ar)),
6.29−6.30 (1H, d, J = 9.6 Hz, CHCHCO), 6.81 (1H, s,
OCCHC(Ar)), 6.87−6.88 (1H, d, J = 8.4 Hz, OCCHCH(Ar)), 7.22
(2H, m, CCH(Py)), 7.42−7.44 (1H, d, J = 8.4 Hz, OCCHCH(Ar)),
7.68−7.70 (1H, d, J = 9.6 Hz, CHCHCO), 9.01 (2H, m, NCH(Py))
ppm. 13C NMR (150 MHz, DMSO-d6) δ 18.29, 22.29, 30.70, 64.33,
65.28, 82.25, 82.81, 97.19, 101.76, 103.65, 112.76, 113.60, 113.97,
122.67, 129.34, 143.36, 146.07, 155.05, 155.64, 160.49, 160.85, 167.58
ppm.
General Procedure for Synthesis of Complexes 3 and 4.
[Ru(η6-p-cymene)Cl2]2 (0.13 g, 0.21 mmol) and silver carboxylate
(0.5 mmol) were stirred in water at 50 °C for 24 h. The mixture was
filtered to remove the AgCl precipitate. The solvent was removed
under vacuum, and the residue was redissolved in methanol (20 mL).
L1 (0.16 g, 0.5 mmol) was added, and the mixture was stirred at 50 °C
for 12 h. The solvent was reduced to 5 mL, and diethyl ether (25 mL)
was added to precipitate the product. The precipitate was filtered,
washed with Et2O (3 × 10 mL) and hexane (20 mL) (3 × 10 mL),
and dried in a vacuum-dryer.
Complex 3. Yield: 0.16 g (60.0%). Light-orange powder. Anal.
Calcd (%) for C29H27NO9Ru: C 54.89, H 4.29, N 2.21. Found: C
1
54.73, H 4.44, N 2.06. ESI-MS: m/z [M + Na]+ = 658.1. H NMR
Cellular Uptake. HCT116 cells were cultured in a 6-well plate
containing a coverslip. Then the cells were incubated with complex 1
at the concentrations of 10 and 30 μM for 3 h. After that, the cells
were washed with PBS for three times to wipe out the free drug. Then,
the coverslip was taken out and put on a glass slid. The glass slid was
detected by LSCM operating at a 405 nm excitation wavelength.
Apoptosis Study. Specific operations were as follows: After
induced apoptosis of HCT-116 cells by the addition of ligand L1 (30
(400 MHz, CDCl3) δ 1.24−1.26 (6H, d, J = 6.8 Hz, CH−(CH3)2),
2.03 (3H, s, CH3), 2.72−2.79 (1H, m, J = 6.8 Hz, CH(CH3)2), 5.12
(2H, s, OCCH2O), 5.36 (2H, s, Py−CH2O), 5.56−5.58 (2H, d, J =
6.0 Hz, CH3CCH(Ar)), 5.80−5.82 (2H, d, J = 6.0 Hz,
(CH3)2CHCCH(Ar)), 6.32−6.35 (1H, d, J = 9.2 Hz, CHCHCO),
7.00−7.02 (1H, d, J = 8.4 Hz, OCCHCH(Ar)), 7.09 (1H, s,
OCCHC(Ar)), 7.58−7.59 (2H, d, J = 6.0 Hz, CCH(Py)), 7.64−7.67
E
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μM) and complexes 1−4 (30 μM) for 24 h, cells were digested with
trypsin and washed twice with cold PBS and collected by
centrifugation (5 min, 25 °C, 2000 rpm). After that, cells were
resuspended in binding buffer (10 mM Hepes, 140 mM NaCl, 2.5 mM
CaCl2, pH 7.4) and incubated with annexin V-FITC (100 ng/mL) and
then with propidium iodide (2 μg/mL) for 15 min at room
temperature. The fluorescence was measured by an annexin V-FITC
apoptosis detection kit (Roche) according to the manufacturer’s
protocol, and cells were analyzed by a computer station running Cell
Quest software.
Western Blot Analysis. HCT-116 cells were cultured until the cell
density reached 80%. Then, the compounds with ideal concentrations
were added, and the cells were cultured for 12 h at 37 °C. Proteins
were extracted by lysis buffer. The concentration of protein was
measured by the BCA (bicinchoninic acid) protein assay with a
Varioskan multimode microplate spectrophotometer (Thermo,
Waltham, MA). Equivalent samples (20 mg/lane) were separated by
8−12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF)
Immobilon-P membrane (Bio-Rad) with a transblot apparatus (Bio-
Rad). The blots were blocked with 5% nonfat milk in TBST (Tris-
buffered saline plus 0.1% Tween 20) for 1 h, and then the membranes
were incubated with the indicated primary antibodies at 4 °C
overnight. After that, the membrane was washed with PBST three
times and subsequently probed by the appropriate secondary
antibodies conjugated to horseradish peroxidase for 1 h. Detection
was performed via Odyssey scanning system (Li-COR, Lincoln,
Nebraska). GAPDH or β-actin was used as loading control.
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ASSOCIATED CONTENT
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AUTHOR INFORMATION
Corresponding Author
*E-mail: sgou@seu.edu.cn.
■
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ORCID
Shaohua Gou: 0000-0003-0284-5480
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to the National Natural Science Foundation of
China (Grant No. 21601034) and Jiangsu Province Natural
Science Foundation (Grant No. BK20160664) for financial aid
to this work. We also thank the Fundamental Research Funds
for the Central Universities (Project 2242016K30020) for
supplying basic facilities to our key laboratory. We also express
our gratitude to the Priority Academic Program Development
of Jiangsu Higher Education Institutions for the construction of
fundamental facilities (Project 1107047002).
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