Discovery of a series of ruthenium(II) derivatives with α-dicarbonylmonoxime as novel inhibitors of cancer cells invasion and metastasis

Article abstract of DOI:10.1016/j.jorganchem.2017.05.024

A series of novel ruthenium(II)-cymene complexes (1–9) with substituted α-dicarbonylmonoximes of general formula [Ru(η⁶-cymene)(L)Cl] (L?=?N,O-chelating bidentate α-dicarbonylmonoxime derivatives) have been synthesized and characterized by elemental analysis, IR, ¹H NMR, ¹³C NMR spectroscopies, and in three cases by single crystal X-ray diffraction analysis. The most effective compound 9 displays remarkable anti-invasion and anti-metastasis properties without apparent cytotoxicity toward three different human cancer cell lines (MCF-7, Hela and HepG2). Further protein level studies suggest that the anti-metastasis activity of the complexes may result from the increasing expression of E-cadherin and reducing expression of Vimentin.

Full text of DOI:10.1016/j.jorganchem.2017.05.024

Journal of Organometallic Chemistry 842 (2017) 82e92
Contents lists available at ScienceDirect
Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
Discovery of a series of ruthenium(II) derivatives with a-
dicarbonylmonoxime as novel inhibitors of cancer cells invasion and
metastasis
Yihui He, Huiying Xue, Wendian Zhang, Li Wang, Guangya Xiang, Lei Li**, Xianmei Shang*
Tongji School of Pharmacy, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel ruthenium(II)-cymene complexes (1e9) with substituted a-dicarbonylmonoximes of
general formula [Ru(
h
6-cymene)(L)Cl] (L ¼ N,O-chelating bidentate
a-dicarbonylmonoxime derivatives)
Received 25 December 2016
Received in revised form
2 May 2017
Accepted 10 May 2017
Available online 11 May 2017
have been synthesized and characterized by elemental analysis, IR, 1H NMR, 13C NMR spectroscopies, and
in three cases by single crystal X-ray diffraction analysis. The most effective compound 9 displays
remarkable anti-invasion and anti-metastasis properties without apparent cytotoxicity toward three
different human cancer cell lines (MCF-7, Hela and HepG2). Further protein level studies suggest that the
anti-metastasis activity of the complexes may result from the increasing expression of E-cadherin and
reducing expression of Vimentin.
Keywords:
Ruthenium(II) complexes
Synthesis
© 2017 Elsevier B.V. All rights reserved.
Cytotoxicity
Anti-metastasis
1. Introduction
manner were synthesized and characterized [20], and all com-
plexes exhibited very strong in vitro protein tyrosine kinase
The discovery of the anticancer properties of cisplatin in the
1960s was a breakthrough event in metal-based drugs field [1,2].
Since then a tremendous number of novel metal complexes have
been synthesized and evaluated to find species with better anti-
cancer properties, lower toxic side effects, and less tumor resis-
tance to cisplatin [3e6]. Of the alternatives to platinum-based
anticancer drugs, most progress has been made with agents based
on ruthenium that tend to exhibit fewer side effects than platinum-
based complexes [7e13], and so far, three ruthenium(III) com-
pounds, NAMI-A [14], KP1019 [15] and KP1339 [16] have already
reached phase II clinical trials, by displaying antitumor activity
against primary tumors and metastasis, and showing low toxicity
and favorable clearance properties [17,18].
inhibitory activity with IC50 values in the range of 0.02e3.11 mM.
Tyrosine kinases are important cellular signaling proteins that have
a variety of biological activities including cell proliferation and
migration. Inhibition of angiogenic tyrosine kinases has been
developed as a systemic treatment strategy for cancer, and three
anti-angiogenic tyrosine kinase inhibitors (sunitinib, sorafenib and
pazopanib) have been approved for treatment of patients with
advanced cancer on account of excellent anti-metastasis effect [21].
The epithelial-mesenchymal transition (EMT) program broadly
regulates invasion and metastasis, and E-cadherin (a key cell-to-cell
adhesion molecule) and vimentin are important markers of EMT
[22], so the anti-metastasis action concerned relevant protein
expression level studies. As an extension of our previous research
on Ru(II)-p-cymene complexes with oximato ligands, with the re-
ports that some oxime organic derivatives have anticarcinogenic
activities and the amino-oxime Ru(II) complexes (having bidentate
N, N-donors) have shown to modulate both adhesion and migra-
tory capabilities of PC-3 cells [26], we are interested in the prop-
erties of the Ru(II)-p-cymene complexes with oxime ligands.
Because the binding mode of the oxime group depends to a great
extent on the presence of a neighboring donor group in the same
In addition to Ru(III) compounds, Ru(II)-arene complexes have
been demonstrated great therapeutic potential, although none of
these compounds has yet entered clinical trials [11,19]. In our pre-
vious studies a series of ruthenium(II)-p-cymene complexes con-
taining oxamato ligands coordinated in a bidentate O, O-donors
* Corresponding author.
** Corresponding author.
E-mail addresses: leileilesure@163.com (L. Li), shangxianmei@hust.edu.cn
(X. Shang).
ligand, and the
a-dicarbonylmonoximes have bidentate O, N-do-
nors and constitute very important of chelating agents with
http://dx.doi.org/10.1016/j.jorganchem.2017.05.024
0022-328X/© 2017 Elsevier B.V. All rights reserved.

Y. He et al. / Journal of Organometallic Chemistry 842 (2017) 82e92
83
bioactivity [23,24], we are wondering if the ruthenium complexes
with this special kind of monoximes show anti-invasion and anti-
metastasis activity.
In the 1H NMR spectra of 1e9, the expected resonances are
observed for the cymene and the substituted -diketone mon-
oxime. As a result of the coordination of the substituted bidentate
a
In the current work, we will combine ruthenium-p-cymene
chelate mode unit, downfield shifts (0.10e0.20 ppm) of the ligand
units with
a
-dicarbonylmonoxime ligands to generate a series of
protons are observed relative to the free
Similar downfield shifts were also detected for the coordinated p-
cymene in 1e9 as compared to the cymene ligand in [RuCl(
6-p-
cymene)( -Cl)]2. The spectra of the complexes 1e9 did not show
any signal for NeOH, indicating the deprotonation of oxime proton
leaving oxygen uncoordinated (crystal structures will be discussed
later). The 13C NMR spectra for 1e9 also show the expected reso-
nance signals.
a-diketone monoxime.
new organo-ruthenium(II) compounds, and the antitumor activity
of ruthenium(II)-cymene-dicarbonylmonoxime complexes will be
evaluated. The apoptosis and the cell cycle arrest will be investi-
gated by flow cytometry for the antiproliferative mechanism.
Scratch wound healing assay and transwell studies will also be
investigated on the migration and invasive of human cancer cells
for anti-metastasis effect. Further protein level research will be
carried out by western blot analysis for preliminary anti-invasion
and anti-metastasis mechanism.
h
m
2.3. X-ray diffraction analysis
2. Results and discussion
Single crystals were grown from the mixture of dichlor-
omethaneen-hexane by slow evaporation at room temperature or a
lower temperature for 1, 3 and 5. The crystal structures of 1, 3 and 5
were determined by X-ray crystallography (see Experimental sec-
tion for details of the data collections and structure refinements).
The molecular structures of 1, 3 and 5 are illustrated in Figs. 1e3,
respectively. The most relevant parameters of bond distances and
angles of the complexes are given in the legends of Figs. 1e3.
The X-ray analysis clearly revealed complexes 1, 3 and 5 adopt
the similar three-legged piano-stool coordination geometry around
the ruthenium ion. Ru(II) ions are coordinated to carbonyl oxygen
2.1. Synthesis of the [Ru(h
6-p-cymene)(L)Cl] complexes
The synthesis of the title compounds (1e9) is as outlined in
Scheme 1. A series of new ruthenium(II) complexes [(
6-p-cymene)
Ru(L)Cl] (L ¼ N,O-chelating -dicarbonylmonoxime derivatives)
have been prepared by the reaction of [RuCl( -Cl)]2
6-p-cymene)(
with the appropriate -diketone monoxime in methanol at room
h
a
h
m
a
temperature. Compounds 1e9 are solids with intense colors, all of
which are stable when exposed to moist air. They are freely soluble
in DMSO, dichloromethane or chloroform and soluble in alcohols,
but insoluble in water.
and nitrogen of the
HL5) forming a five-member chelate ring with a mean bite angle of
77ꢁ (from 76.87(13)ꢁ to 77.13(7)ꢁ). The Ru- 6-arene centroid dis-
a
-diketone monoxime ligands (HL1, HL3 and
h
tances are 1.680 (1), 1.670 (3), 1.676 (5), suggesting that the
2.2. Spectral studies
ruthenium-cymene interaction is similar for the three ruthenium-
cymene complexes with different a-diketone monoxime ligands.
The elemental analysis data of the complexes are in good
agreement with the calculated values. In the IR spectra of the li-
gands, the stretching vibration bands of OeH appear at
3249e3444 cmꢀ1 respectively, and these bands disappear in the
spectra of 1e9, showing the deprotonation of eOH groups [25]. In
For 1, 3 and 5, the RueN, RueO1, RueCl and RueCcym distances
are all normal and they are similar to what have been observed in
structurally characterized Ru(II) complexes containing these bonds
[27e29]. It is important to note that the deprotonation of the oxime
(C]NeOH) and the coordination of the oxime nitrogen (not oxy-
gen), which results in the shortening of the NeO2
[1.256(2) ~ 1.282(5) Å] and C(O1)eC(N) [1.402(6) ~ 1.411(4) Å]
the spectra of complexes 1e9, the
n(C]O) band (at
1609e1639 cmꢀ1
)
is shifted to lower frequencies by about
24e70 cmꢀ1 compared to free ligands (nCO for substituted
a-dike-
tone monoxime are in the 1663e1726 cmꢀ1 range), indicating the
coordination of oxygen atom of the carbonyl moiety to Ru(II) ion
[25e27]. The striking feature common in all the spectra of the
distances and the lengthening of the CeN distance in the
ketoxime group. The facts suggest a delocalization of the electron
-ketoxime (O1eCeCeN1eO2)
a-
p
density on deprotonated side of the
moiety.
a
complexes are the noticeable shift of n(NeO) and n(C]N) stretching
vibrations relative to the free ligands. This suggests the coordina-
tion of the oxime group through nitrogen atom in all complexes.
2.4. Cytotoxicity studies
These phenomena indicate that the a-diketone monoxime coordi-
nated with central Ru atom in a N,O-chelating mode.
Eight synthesized ruthenium(II) compounds (1~5 and 7~9, 6 not
OH
N
R1
R1
R2
[Ru(η6-p-cymene)Cl2]2
O
O
NaOH
+
H2NOH HCl
Ru Cl
O
MeOH, r.t.
R2
O
N
R2
O
OH
R1
HL1: R1 = Me, R2 = Me;
HL2: R1 = H, R2 = Me;
HL3: R1 = Me, R2 = Et;
HL4: R1 = Et, R2 = Et;
HL5: R1 = Me, R2 = Ph;
HL6: R1 = Ph, R2 = Ph;
N
HL8:
[Ru(η6-p-cymene)(L)Cl]
O
O
N OH
1-9
HL9:
HL7: R1 = Me, R2
=
n-Pr;
Scheme 1. Synthesis of the [Ru(h
6-p-cymene)(L)Cl] complexes (1e9).

84
Y. He et al. / Journal of Organometallic Chemistry 842 (2017) 82e92
has been tested on various human tumor cell lines: human breast
cancer cell lines (MCF-7), human cervical cancer cell lines (Hela),
human hepatoma cell lines (HepG2).
As shown in Table 1, complexes 1e9 exhibit weak cytotoxicity
against three human cancer cell lines (MCF-7, Hela and HepG2)
with IC50 values above 57.9
mM, which are much higher than that of
cisplatin with IC50 values of 4.9e11.3
mM.
In comparison with the ligands (HL1 and HL9), almost all of
ruthenium(II)-cymene complexes show enhanced activities than
that of the ligands, confirming the beneficial effect of complexation
to arene ruthenium units.
Among 1, 2, 3, 4 and 7 with different straight-chain
ligands, 1 with the shortest length of carbon chain showed stronger
cytotoxicity with the IC50 values of 59.9e100.9 M (the order of
a-ketoximes
m
activities follows 1 > 2 > 7 > 3 > 4). For 5, 8 and 9 with different
aromatic rings ligands, they are weaker than 1 in anti-proliferative
activity, indicating the higher importance of alkyl substituent of
diketone monoxime ligands.
a-
In addition, for three human cancer cell lines treated in exper-
iment, the ruthenium(II)-cymene-ketoxime compounds are rela-
tively more sensitive to Hela cells.
2.5. Cell apoptosis analysis by flow cytometry
To gain more insights relative to the antitumor mechanism,
compounds 1, 2 and 5 with relative stronger activities and ligand
HL1 were chosen for further experiments. Since Hela cells appeared
to be the more sensitive than the other tested cells, we carried out
flow cytometry assays to determine the apoptosis level of Hela cells
exposed to 1, 2 and 5. The results of cell apoptosis analysis by flow
cytometry were shown in Table 2 and Fig. 4.
Fig. 1. Molecular structure of compound 1 showing the labeling scheme of the non-H
atoms. Selected bond lengths (Å) and angles (ꢁ): Ru(1)ꢀCl(1) 2.3970(6), Ru(1)ꢀO(1)
2.0694(14), Ru(1)ꢀN(1) 2.0414(17), N(1)ꢀO(2) 1.256(2), N(1)-C(3) 1.339(3), C(2)eC(3)
1.411(4), RuꢀCcym(max) 2.2207(19), RuꢀCcym(min) 2.1836(18), Cl(1)ꢀRu(1)ꢀO(1)
85.25(5), Cl(1)ꢀRu(1)ꢀN(1) 84.69(5), N(1)ꢀRu(1)ꢀO(1) 77.13(7), Cl(1)ꢀRu(1)ꢀcen-
troidcym 127.53, O(1)ꢀRu(1)ꢀcentroidcym 130.68, N(1)ꢀRu(1)ꢀcentroidcym 133.37.
We can see that the total apoptosis percentages of the positive
control are 14.14, 90.46 and 91.08% at the concentration of 2.0, 100
and 200 mM, respectively, and nearly equal to the negative control
(ca. 11.08%) at low concentrations, while the total apoptosis of
cisplatin increased obviously. This indicates cisplatin induced Hela
cells apoptosis in a dose-dependent manner.
For ligand HL1, the total apoptosis percentages are always below
10.01% at low (2 mM) or high (200 mM) concentrations. Compared
with the control group, these data show ligand HL1 could not
induce Hela cancer cell apoptosis, which correlate with its weak
cytotoxicity.
Complexes 1, 2 and 5, at low concentrations (2.0 mM) do not
induce apoptosis in Hela cells. With the increase of the concen-
trations of 1, 2 and 5, the apoptosis rates increased obviously. The
total apoptosis percentages are up to 85.47 and 91.54% for 1, 81.51
and 94.55% for 2 and 83.10 and 88.24% for 5 at the concentrations of
100 and 200 mM, respectively, which are higher than the negative
control, but comparable with the positive control. Moreover, by
comparing the percentages of early apoptosis (Q4) and late
apoptosis (Q2) of cisplatin, we noticed that cisplatin can induce
both early apoptosis and late apoptosis of Hela cells, which is
different from the ruthenium-cymene-ketoxime complexes.
From the data of the apoptosis detection of 1, 2 and 5 (see
Table 2), we also can see that the percentages of early apoptosis
(Q4) are bigger than that of late apoptosis and necrotic cell (Q2).
Moreover, with the increasing concentrations of compounds, there
have been marked increases in the ratio of early apoptosis. The
results suggest that cell death might be mainly by means of early
apoptosis at high concentrations of the complexes, which is bene-
ficial for optimal antitumor agents.
Fig. 2. Molecular structure of compound 3 showing the labeling scheme of the non-H
atoms. Selected bond lengths (Å) and angles (ꢁ): Ru(1)ꢀCl(1) 2.3970(6), Ru(1)ꢀO(1)
2.071(2), Ru(1)ꢀN(1) 2.034(3), N(1)ꢀO(2) 1.282(5), N(1)-C(14) 1.332(5), C(13)eC(14)
1.402(6), RuꢀCcym(max) 2.205(4), RuꢀCcym(min) 2.149(4), Cl(1)ꢀRu(1)ꢀO(1) 86.08(7),
Cl(1)ꢀRu(1)ꢀN(1) 85.85(9), N(1)ꢀRu(1)ꢀO(1) 76.87(13), Cl(1)ꢀRu(1)ꢀcentroidcym
127.55, O(1)ꢀRu(1)ꢀcentroidcym 131.39, N(1)ꢀRu(1)ꢀcentroidcym 131.44.
determined owing to compound precipitation) and two ligands
(HL1 and HL9) were screened for preliminary in vitro antitumor
activity with cisplatin as a positive control. The in vitro cytotoxicity
2.6. Cell cycle analysis by flow cytometry
The cell cycle is a series of events leading to cell division and

Y. He et al. / Journal of Organometallic Chemistry 842 (2017) 82e92
85
Fig. 3. Molecular structure of compound 5 showing the labeling scheme of the non-H atoms. Selected bond lengths (Å) and angles (ꢁ): Ru(1)ꢀCl(1) 2.3984(6), Ru(1)ꢀO(1)
2.0739(15), Ru(1)ꢀN(1) 2.0261(16), N(1)ꢀO(2) 1.256(2), N(1)-C(18) 1.350(3), C(17)eC(18) 1.408(3), RuꢀCcym(max) 2.2415(18), RuꢀCcym(min) 2.171(2), Cl(1)ꢀRu(1)ꢀO(1) 84.67(5),
Cl(1)ꢀRu(1)ꢀN(1) 88.22(5), N(1)ꢀRu(1)ꢀO(1) 77.10(6), Cl(1)ꢀRu(1)ꢀcentroidcym 127.47, O(1)ꢀRu(1)ꢀcentroidcym 132.08, N(1)ꢀRu(1)ꢀcentroidcym 129.99.
Table 1
cell cycle progression in propidium-iodide-stained cells after Hela
cells were incubated with 100 M of complexes 1, 2 and 5 for 48 h.
The results of cell cycle analysis by flow cytometry are shown in
Fig. 5.
Cytotoxicity of eight ruthenium(II) compounds and two ligands (HL1 and HL9)
against three human cancer cell lines (MCF-7, Hela and HepG2). Data were presented
as mean S.D. of three independent experiments.
m
Compounds
IC50
(
m
M)
In the control, the percentage in the cells at G2/M phase is
10.68%, whereas the percentages in the cells at G2/M phase are
43.71, 37.79 and 30.43% after Hela cells were treated with com-
plexes 1, 2 and 5 for 48 h, respectively. Comparing with the control,
an enhancement of 33.03% for 1, 27.11% for 2 and 19.75% for 5 in the
cells at G2/M phase was found, which was accompanied a reduction
in the G0/G1 phase. Obviously, complexes 1, 2 and 5 showed similar
inhibitory effects in Hela cells on the cell cycle arrest. These data
suggest that complexes 1, 2 and 5 altered cell cycle progression
through inhibiting the cell growth of Hela cells at G2/M phase.
Cisplatin is a cell cycle non-specific drug. At the same concentra-
tion, cisplatin blocked Hela cells in S phase strongly, but blocked
same cells in G2-M phase slightly [31]. Therefore, the cycle arrest of
compound 1, 2 and 5 is different from cisplatin, but shows a similar
behavior with NAMI-A [36].
MCF-7
Hela
HepG2
1
2
3
4
5
7
8
90.0 8.8
59.9 5.1
57.9 6.9
171.9 14.7
341.9 24.2
78.8 5.5
159.2 16.8
99.7 18.8
122.4 8.7
>1000
100.9 14.7
130.8 83.9
337.2 28.0
500.1 60.3
76.8 6.2
327.2 25.1
283.9 33.2
132.8 12.8
>1000
119.4 24.5
264.0 15.9
359.3 30.4
126.8 11.7
263.3 20.2
282.5 34.0
285.2 34.0
>1000
9
HL1
HL9
436.3 53.4
11.3 1.9
162.5 19.5
4.9 0.6
190.9 24.6
3.1 0.4
cisplatin
replication [30]. In an attempt to study the mechanism of cell
apoptosis, flow cytometry analysis was performed to examine the
Table 2
Percentages of apoptosis of compounds (1, 2, 5 and HL1) against Hela cells in different concentrations with cisplatin as positive control.
Compd.
Conc.
M)
Q2 (late apoptosis and necrotic cell, %)
Q4 (early apoptosis, %)
Q2þQ4 (Total percentage)
(
m
Control
1
e
5.95
0.62
5.44
3.87
3.89
3.92
3.46
0.96
6.90
2.78
3.65
4.04
4.76
34.56
28.53
5.54
5.13
11.08
91.54
85.47
8.03
94.55
81.51
5.99
88.24
83.10
5.19
7.56
8.83
10.01
91.08
90.46
14.14
200
100
2
200
100
2
200
100
2
200
100
2
90.92
80.03
4.14
90.66
77.59
2.53
87.28
76.20
2.41
3.91
4.77
5.25
56.52
61.93
8.60
2
5
HL1
Cisplatin
200
100
2

86
Y. He et al. / Journal of Organometallic Chemistry 842 (2017) 82e92
2.7. Scratch wound healing assay
Cell has a natural tendency for migration that is a vital process in
the growth and safeguarding of tissue functions [32]. The cell
migration is very essential during embryogenesis, wound healing,
development of immune response, etc. It also takes place in several
diseases, especially in cancer, leading to invasion and metastasis
[33]. Therefore, we have investigated the effect of 1ꢀ9 on cancer
cell migration in highly metastatic (more migration rate) HepG2
cell line through scratch wound healing assay.
The experimental results show that compounds 2ꢀ8 seem no
obvious anti-migration effect. We observed the scratch assay im-
ages in HepG2 treated with 1 and 9 at 150 mM with time. The un-
treated control cells migrated rapidly in a time dependent fashion
due to metastatic property, while cells treated with 1 or 9 migrated
in a slow rate. The wound area almost remained unchanged in cells
incubated with 9, further indicating its good anti-migration effects.
Compared with untreated control, the wound closure of 1 was
much lower than that of 9 in 6 h of incubation. Whereas in 24 h,
compound 9 showed the lowest wound closure (Fig. 6) [34]. This
result indicates that these complexes may have great potential to be
anti-metastatic through the inhibition of cancer cells migration.
2.8. Invasion assay
Invasive ability was measured in a transwell cell culture
chamber according to the method of Albini [35], and the images of
invasion assay are shown in Fig. 7.
From the images of the transwell assay, we observed that there
were a lot of HepG2 cells in the blank group (control), while the
HepG2 cells treated with 1 or 9 were much less than that of control
group, indicating that 1 and 9 are able to inhibit the invasion of
HepG2 cells, and the anti-invasive ability of 9 is stronger than that
of 1.
2.9. Effects of expression of E-cadherin and Vimentin
Recent experimental evidence suggests when lack of in vitro
cytotoxicity is associated to inhibition of matrigel invasion, it may
predict in vivo-selective antimetastasis activity of ruthenium
complexes [36]. In order to understand the molecular targeting and
mechanism of action of ruthenium(II)-cymene compounds, ac-
cording to the invasion assay results, we choose compound 9, the
anti-metastasis compound with low cytotoxicity, to study the
preliminary anti-metastasis action by Western blotting.
The activation of anti-Bcl-2 E-cadherin, anti-Bcl-2 vimentin,
anti-cyclin-D1 and anti-cyclin-E1 antibody was assayed by western
blot analysis. As shown in Fig. 8, we can see the high expression of
both cyclin D1 and cyclin E1 in control group. After the treatment of
HepG2 cells with different concentrations of compound 9, the
expression levels of cyclin-D1 and cyclin-E1 decreased significantly,
indicating that compound 9 can block cells in the G1 phase, down-
regulate the expression of cyclin-D1 and cyclin-E1, and inhibit
cyclin-D1/cyclin-E1 in vitro. Thus the expression of both cyclin-D1
and cyclin-E1 is correlated with the antiproliferative effect of
compound 9.
E-cadherin is an important cell adhesion molecule cadherin
family member, which play an important role in the maintenance of
Fig. 4. Apoptosis detection Hela cells using FITC-PI assay after 48 h. The total per-
centage of apoptotic cells was considered as Q2 þ Q4. Plot presents the fluorescence
data of propidium iodide (PI) and Annexin V fluorescence in corresponding to (a) 1,
2
2
m
M, (b) 1, 100
m
M, (c) 1, 200 M, (d) 2, 2
m
mM, (e) 2, 100 mM, (f) 2, 200 mM, (g) 5,
mM (h) 5, 100
m
M, (i) 5, 200 mM, (j) control.

Y. He et al. / Journal of Organometallic Chemistry 842 (2017) 82e92
87
Fig. 5. Cell cycle distribution of Hela cells after the treatment with 100 mM of compounds (A). Data analysis of cell cycle arrest (B).
cell adhesion, cell polarity and cell communication [22]. Vimentin
is a key member of intermediate filament protein family, which is
known to play a significant role in the maintenance of cell integrity
and against external stress injury [37]. EMT is one of the main
mechanisms of promoting tumor invasion and metastasis [37,38].
E-cadherin and Vimentin are important markers of EMT. As shown
in Fig. 8, we can observe the expression of Vimentin decreased
slightly, whereas the expression of E-cadherin increased obviously.
The increased expression of E-cadherin and reduced expression of
Vimentin illustrate the potent ability of compound 9 in anti-tumor
invasion and metastasis. These observations also suggest that
cyclin-D1, cyclin-E1 and vimentin may be the cellular targets of
these ruthenium complexes with a-dicarbonylmonoximes.
3. Conclusions
In this work we have reported the synthesis of a series of new
ruthenium(II)-cymene complexes with substituted -dicarbo-
a
nylmonoxime ligands (1ꢀ9). All compounds were subjected to
characterization by elemental analysis, IR, 1H NMR, 13C NMR

88
Y. He et al. / Journal of Organometallic Chemistry 842 (2017) 82e92
Fig. 6. Images of scratch assay in HepG2 cells treated with 1 or 9 at 150 mM at 0 h, 6 h and 24 h (culture medium as control) (left). Wound closure (%) was quantified by calculating
the change of wound width (right). Data were presented as mean S.D. of three independent experiments. Statistical analyses were performed using Student's t-test. **P < 0.01 and
***P < 0.001.
Fig. 7. Images of transwell assay in HepG2 cells treated with 1 or 9 at 100 mM at 24 h (culture medium as control).
cytotoxicity against three different human cancer cells (MCF-7, Hela
and HepG2). Relative to cisplatin, these ruthenium(II)-cymene
complexes are found to be lack of in vitro cytotoxicity.
Apoptosis and cell cycle analysis showed that the selected three
ruthenium compounds induced a similar accumulation of Hela cells
in the G2-M phase, while cisplatin is known to induce initial S-
phase arrest as a DNA replication blockade. This shows that the
ruthenium(II) complexes can have an action mechanism different
from that of Pt drugs. For this type of relatively non-cytotoxic
compounds [Ru(
derivatives), the main targets should not be DNA.
h
6-cymene)(L)Cl] (L ¼
a-dicarbonylmonoxime
In addition, scratch wound healing assay and transwell test
showed that 1 and 9 could inhibit tumor cells migration and in-
vasion of matrigel, indicating that these complexes may have great
anti-metastatic potential. Protein level analysis shows that the anti-
metastasis action is associated with the increasing expression of E-
cadherin and reducing expression of Vimentin.
Fig. 8. The expression of anti-Bcl-2 E-cadherin, anti-Bcl-2 Vimentin, anti-Cyclin-D1
and anti-Cyclin-E1 antibody were assayed by western blot analysis, after the treat-
ment of HepG2 cells with compound 9 at 100 mM and 120 mM.
In summary, our study provides a novel type of ruthenium(II)
therapeutic agents that aim to block cancer cell migration and in-
vasion without exerting cell toxicity. Both 1 and 9 have shown to
have some similar antitumor effects to NAMI-A, namely low cyto-
toxicity in vitro, and remarkable anti-invasion and metastasis
properties. The preliminary anti-migration activity of these
spectroscopies, and in three cases by single crystal X-ray diffraction
analysis. The X-ray structure analyses revealed a pseudo-octahedral
'piano-stool' configuration for the metals with bidentate coordi-
nation through oxime-N and
a-ketone-O, forming a nearly planar
five-membered metallocycle. Eight complexes were tested for their

Y. He et al. / Journal of Organometallic Chemistry 842 (2017) 82e92
89
compounds may be a result of abnormal expression of E-cadherin
and vimentin. Further studies deserve to be undertaken which may
be helpful for the design of new ruthenium-based anticancer
agents.
CeCH3), 22.4, 23.2 (CH3C6H4CH(CH3)2), 30.9 (CH3C6H4CH(CH3)2),
83.0, 84.0, 84.8, 86.5, 100.9, 104.3 (CH3C6H4CH(CH3)2), 145.0
(OeN]CeH), 202.0 (O]CeCH3) ppm.
4.2.3. Synthesis of [Ru(h
6-p-cymene)(L3)Cl] (3)
4. Experimental section
Compound 3 was prepared analogously by following the
method and conditions described for 1 but using HL3 (1.0 mmol)
4.1. Materials and physical measurements
RuCl3$3H2O and 1,2-indanedione (HL8) were purchased from
and [Ru(
obtained. Yield: 35.6%. M.p.: 107.8e109.4 ꢁC. Anal.Calcd. for
15H22ClNO2Ru: C 46.81, H 5.76, N 3.64. Found: C 47.26, H 5.53, N
3.69. IR (KBr): v ¼ 1629 (C]O/N]C), 1524, 1340, 994 (N]O/NeO),
519 (RueN), 465 (RueO) cmꢀ1 1H NMR (CDCl3):
h
6-p-cymene)Cl2]2 (0.5 mmol). Dark red crystals were
C
Aldrich. [Ru(
h
6-p-cymene)Cl2]2 [39] and the ligands 2,3-
pentanedione-2-oxime (HL3), 2,3-hexanedione-2-oxime (HL4),
a
-
.
d
¼ 1.20 (t, 3H,
benzil monoxime (HL6), 3,4-hexanedione-3-oxime (HL7) and ace-
naphthenequinone monooxime (HL9) were synthesized using
literature methods [40,41]. The ligands diacetylmonoxime (HL1),
(Z)-2-oxopropanal oxime (HL2), 1-phenyl-1,2-propanedione-2-
oxime (HL5) and all other chemicals were obtained from Sino-
pharm Chemical Reagent Co. Ltd., China and were used as received.
All biological materials were purchased from Wuhan Goodti
meBioeTechnology Co. Ltd., China. Elemental analyses were per-
formed with a Perkin-Elmer 2400 analyzer, series II. IR spectra were
recorded from 4000 to 400 cmꢀ1 with a Bruker Vertex 70 FTeIR
spectrophotometer on KBr pellets; only significant bands are cited
in the text. NMR spectra were recorded with a Bruker AM-400
spectrometer. 1H and 13C chemical shifts are reported relative to
tetramethylsilane.
CH3CH2, J ¼ 7.2 Hz), 1.24 (d, 3H, CH3C6H4CH(CH3)2, J ¼ 7.2 Hz), 1.26
(d, 6H, CH3C6H4CH(CH3)2, J ¼ 7.2 Hz), 1.90 (s, 3H, N]CeCH3), 2.27
(s, 3H, CH3C6H4CH(CH3)2), 2.60e2.67 (m, 2H, O]CeCH2CH3),
2.84e2.91 (m, 1H, CH3C6H4CH(CH3)2),
d
¼ 5.33 (m, 2H, -C6H4ꢀ),
5.47 (m, 1H, -C6H4-), 5.55 (m, 1H, -C6H4-) ppm. 13C NMR (CDCl3):
d
¼ 9.4 (CH3CH2C]O), 10.8 (CH3C]N), 18.5 (CH3C6H4CH(CH3)2),
22.0, 22.4 (CH3C6H4CH(CH3)2), 30.0 (O]CeCH2CH3), 30.9
(CH3C6H4CH(CH3)2), 83.7, 84.3, 84.8, 86.0, 99.5, 103.3
(CH3C6H4CH(CH3)2), 152.3 (OeN]C-CH3), 205.1 (O]C-CH2CH3)
ppm.
4.2.4. Synthesis of [Ru(h
6-p-cymene)(L4)Cl] (4)
Compound 4 was prepared analogously by following the
method and conditions described for 1 but using HL4 (1.0 mmol)
and [Ru(h
6-p-cymene)Cl2]2 (0.5 mmol). Red solids were obtained.
4.2. Synthesis of the [Ru(
h
6-p-cymene)(L)Cl] complexes (1e9)
Yield: 24.3%, M.p.: 77.7e79.3 ꢁC. Anal.Calcd. for C16H24ClNO2Ru: C
48.18, H 6.06, N 3.51. Found: C 48.58, H 5.73, N 3.59. IR (KBr):
v ¼ 1627 (C]O/N]C), 1524, 1350, 943 (N]O/NeO), 525 (RueN),
4.2.1. Synthesis of [Ru(h
6-p-cymene)(L1)Cl] (1)
244.8 mg (0.4 mmol) [Ru(
6-p-cymene)Cl2]2 was added to a
h
473 (RueO) cmꢀ1
J ¼ 7.6 Hz), 1.17e1.23 (m, 9H, CH3CH2C]O, CH3C6H4CH(CH3)2),
2.23e2.30 (m, 4H, N]CeCH3, CH3CH2C]N), 2.37e2.44 (m, 1H,
CH3CH2C]N), 2.56e2.62 (m, 2H, O]CeCH2CH3), 2.80e2.86 (m,
.
1H NMR (CDCl3):
d
¼ 0.92 (t, 3H, CH3CH2C]N,
methanol (15 mL) solution of 80.9 mg (0.8 mmol) diacetyl mon-
oxime (HL1), and the mixture was stirred at room temperature for
1.5 h. The solvent was removed under reduced pressure, and the
crude product was purified by silica gel column chromatography
(Vchloroform: Vacetone ¼ 10:1). The solid was then recrystallized from
dichloromethaneen-hexane. Purple crystals were obtained. Yield:
51.7%. M.p: 142.0e143.7 ꢁC. Anal.Calcd. for C14H20ClNO2Ru: C 45.34,
H 5.44, N 3.78. Found: C 45.71, H 5.24, N 3.85. IR (KBr): v ¼ 1637
(C]O/N]C), 1529, 1330, 977 (N]O/NeO), 519 (RueN), 478 (RueO)
1H, CH3C6H4CH(CH3)2),
d
¼ 5.26 (d, 1H, -C6H4-, J ¼ 6.0 Hz), 5.34 (d,
1H, -C6H4-, J ¼ 6.0 Hz), 5.50 (d, 1H, -C6H4ꢀ, J ¼ 6.0 Hz), 5.53 (d, 1H,
-C6H4-, J ¼ 6.0 Hz) ppm. 13C NMR (CDCl3):
d
¼ 9.7 (CH3CH2C]N),
11.0
(CH3CH3CH2C]O),
18.4
(CH3CH2C]N), 18.5
(CH3C6H4CH(CH3)2), 21.9, 22.4 (CH3C6H4CH(CH3)2), 30.0 (O]
CeCH2CH3), 30.9 (CH3C6H4CH(CH3)2), 83.1, 84.1, 84.8, 86.3, 100.4,
103.1 (CH3C6H4CH(CH3)2), 158.1 (OeN]CH2CH3), 205.1(O]
CH2CH3) ppm.
cmꢀ1
.
1H NMR (CDCl3):
d
¼ 1.24 (d, 3H, CH3C6H4CH(CH3)2,
J ¼ 6.8 Hz), 1.25 (d, 3H, CH3C6H4CH(CH3)2, J ¼ 6.8 Hz), 1.90 (s, 3H,
N]CeCH3), 2.26 (s, 3H, CH3C6H4CH(CH3)2), 2.35 (s, 3H, O]CeCH3),
2.85e2.92 (m, 1H, CH3C6H4CH(CH3)2),
d
¼ 5.30 (d, 1H, -C6H4-,
4.2.5. Synthesis of [Ru(h
6-p-cymene)(L5)Cl] (5)
J ¼ 6.0 Hz), 5.33 (d, 1H, -C6H4-, J ¼ 6.0 Hz), 5.49 (d, 1H, -C6H4-,
Compound 5 was prepared analogously by following the
J ¼ 6.0 Hz), 5.52 (d, 1H, -C6H4ꢀ, J ¼ 6.0 Hz) ppm. 13C NMR (CDCl3):
method and conditions described for 1 but using HL5 (1.0 mmol)
d
¼ 11.2 (N]CeCH3), 18.5 (CH3C6H4CH(CH3)2), 22.0 (O]CeCH3),
and [Ru(
tained. Yield: 27.0%. M.p.: 155.8e157.4 ꢁC. Anal.Calcd. for
19H22ClNO2Ru: C 52.71, H 5.12, N 3.24. Found: C 53.13, H 4.94, N
3.35. IR (KBr): v ¼ 1635 (C]O/N]C), 1521, 1341, 971 (N]O/NeO),
513 (RueN), 451 (RueO) cmꢀ1. 1H NMR (DMSO-d6):
h
6-p-cymene)Cl2]2 (0.5 mmol). Purple crystals were ob-
22.4, 24.2 (CH3C6H4CH(CH3)2), 30.8 (CH3C6H4CH(CH3)2), 83.2, 84.0,
84.5, 86.4, 100.3, 103.6 (CH3C6H4CH(CH3)2), 152.8 (N]CeCH3),
201.5 (O]CeCH3) ppm.
C
d
¼ 1.19 (d, 3H,
4.2.2. Synthesis of [Ru(
h
6-p-cymene)(L2)Cl] (2)
CH3C6H4CH(CH3)2, J ¼ 6.8 Hz), 1.20 (d, 3H, CH3C6H4CH(CH3)2,
J ¼ 6.8 Hz), 1.90 (s, 3H, O]CeCH3), 2.17 (s, 3H, CH3C6H4CH(CH3)2),
2.75e2.80 (m, 1H, CH3C6H4CH(CH3)2), 5.52 (d, 1H, -C6H4-,
J ¼ 6.0 Hz), 5.56 (d, 1H, -C6H4-, J ¼ 6.0 Hz), 5.69 (d, 1H, -C6H4-,
J ¼ 6.0 Hz), 5.81 (d, 1H, -C6H4-, J ¼ 6.0 Hz), 7.51e7.64 (m, 5H, AreH)
Compound 2 was prepared analogously by following the
method and conditions described for 1 but using HL2 (1.0 mmol)
and [Ru(
obtained. Yield: 31.3%. M.p.: 134.1e135 ꢁC. Anal. Calcd. for
13H18ClNO2Ru: C 43.76, H 5.08, N 3.93. Found: C 44.11, H 4.92, N
4.06. IR (KBr): v ¼ 1619 (C]O/N]C), 1513, 1358, 981 (N]O/NeO),
483 (RueN), 450 (RueO) cmꢀ1 1H NMR (CDCl3):
h
6-p-cymene)Cl2]2 (0.5 mmol). Orange-red solids were
C
ppm. 13C NMR (DMSO-d6):
d
¼ 18.4 (CH3C6H4CH(CH3)2), 22.0 (O]
CeCH3), 22.1, 22.4 (CH3C6H4CH(CH3)2), 30.9 (CH3C6H4CH(CH3)2),
83.8, 84.7, 85.7, 87.2, 101.0, 103.2 (CH3C6H4CH(CH3)2), 126.5, 128.5,
129.0, 129.3, 132.3, 136.0 (C6H5eC]N), 152.1 (OeN]CeC6H5),
195.2 (O]CeCH3) ppm.
.
d
¼ 1.24 (d, 3H,
CH3C6H4CH(CH3)2, J ¼ 7.2 Hz), 1.26 (d, 3H, CH3C6H4CH(CH3)2,
J ¼ 7.2 Hz), 2.26 (s, 3H, O]CeCH3), 2.28 (s, 3H,CH3C6H4CH(CH3)2,
2.84e2.91 (m, 1H, CH3C6H4CH(CH3)2), 5.28 (d, 1H, -C6H4-,
J ¼ 6.0 Hz), 5.37 (d, 1H, -C6H4-, J ¼ 6.0 Hz), 5.50 (d, 1H, -C6H4-,
J ¼ 6.0 Hz), 5.53 (d, 1H, -C6H4ꢀ, J ¼ 6.0 Hz), 7.43 (s, 1H, OeN]CeH)
4.2.6. Synthesis of [Ru(h
6-p-cymene)(L6)Cl] (6)
Compound 6 was prepared analogously by following the
ppm. 13C NMR (CDCl3):
d
¼ 18.5 (CH3C6H4CH(CH3)2), 22.0 (O]
method and conditions described for 1 but using HL6 (1.0 mmol)

90
Y. He et al. / Journal of Organometallic Chemistry 842 (2017) 82e92
and [Ru(
h
6-p-cymene)Cl2]2 (0.5 mmol). Purple crystals were ob-
8.24 (d, 1H, AreH, J ¼ 7.2 Hz), 8.34 (d, 1H, AreH, J ¼ 8.0 Hz) ppm. 13
C
tained. Yield: 28.6%. M.p.: 192.6e194.2 ꢁC. Anal. Calcd. for
NMR (CDCl3):
d
¼
18.5 (CH3C6H4CH(CH3)2), 22.1, 22.4
C
24H24ClNO2Ru: C 58.24, H 4.89, N 2.83. Found: C 58.78, H 4.69, N
2.93. IR (KBr): v ¼ 1638 (C]O/N]C), 1512, 1373, 999 (N]O/NeO),
505 (RueN), 479 (RueO) cmꢀ1 1H NMR (CDCl3),
(CH3C6H4CH(CH3)2), 30.9 (CH3C6H4CH(CH3)2), 82.2, 83.4, 83.9, 85.7,
99.3, 103.2 (CH3C6H4CH(CH3)2), 120.9, 124.0, 125.7, 125.8, 127.5,
128.5, 128.6, 130.3, 133.2, 136.0 (AreH), 155.2 (N]C), 199.7 (O]C)
ppm.
.
d
¼ 1.32 (d, 3H,
CH3C6H4CH(CH3)2, J ¼ 6.8 Hz), 1.34 (d, 3H, CH3C6H4CH(CH3)2,
J ¼ 6.8 Hz), 2.36 (s, 3H, CH3C6H4CH(CH3)2), 2.94e3.02 (m, 1H,
CH3C6H4CH(CH3)2), 5.42 (d, 1H, -C6H4-, J ¼ 6.0 Hz), 5.47 (d, 1H,
-C6H4-, J ¼ 6.0 Hz), 5.57 (d, 1H, -C6H4-, J ¼ 6.0 Hz), 5.67 (d, 1H -C6H4-
4.3. X-ray measurements
, J ¼ 6.0 Hz),
d
¼ 7.06e7.38 (m, 10H, AreH) ppm. 13C NMR (DMSO-
Suitable single crystals of compounds 1, 3 and 5 were mounted
in glass capillaries for X-ray structural analysis. Diffraction data
were collected on a Bruker Smart APEX-II CCD diffractometer with
d6),
d
¼ 18.5 (CH3C6H4CH(CH3)2), 22.1, 22.4 (CH3C6H4CH(CH3)2),
40.0 (CH3C6H4CH(CH3)2), 83.9, 85.2, 85.9, 87.7, 102.0, 103.5
(CH3C6H4CH(CH3)2), 128.6, 128.76, 129.1, 130.6, 131.7, 132.3, 135.8
((eC6H5)2), 155.7 (N]CeC6H5), 193.7 (O]CeC6H5) ppm.
Mo Ka (
l
¼ 0.71076 Å) radiation at room temperature. During the
intensity data collection, no significant decay was observed. The
intensities were collected for Lorentz-polarization effects and
empirical absorption with the SADABS program. The structure was
solved by direct methods using the SHELXL-97 program. All non-
hydrogen atoms were found from the difference Fourier synthe-
ses. The H atoms were included in calculated positions with
isotropic thermal parameters related to those of the supporting
carbon atoms but were not included in the refinement. All calcu-
lations were performed using the Bruker Smart program [42].
Crystallographic details are reported in Table 3.
4.2.7. Synthesis of [Ru(h
6-p-cymene)(L7)Cl] (7)
Compound 7 was prepared analogously by following the
method and conditions described for 1 but using HL7 (1.0 mmol)
and [Ru(h
6-p-cymene)Cl2]2 (0.5 mmol). Purple solids were ob-
tained. Yield: 47.4%. Anal.Calcd. for C16H24ClNO2Ru: C 48.18, H 5.76,
N 3.51. Found: C 48.32, H 5.69, N 3.67. IR (KBr): v ¼ 1622 (C]O/N]
C), 1525, 1337, 973 (N]O/NeO), 522 (RueN), 455 (RueO) cmꢀ1. 1H
NMR (CDCl3):
d
¼ 0.97 (t, 3H, CH3CH2CH2, J ¼ 7.6 Hz), 1.24 (d, 3H,
CH3C6H4CH(CH3)2, J ¼ 7.2 Hz), 1.26 (d, 3H, CH3C6H4CH(CH3)2,
J ¼ 7.2 Hz),1.69 (m, 2H, CH3CH2CH2),1.90 (s, 3H, N]CeCH3), 2.26 (s,
3H, CH3C6H4CH(CH3)2), 2.57e2.60 (t, 2H, O]CeCH2CH2),
4.4. Cytotoxic activity in vitro
2.83e2.90 (m, 1H,CH3C6H4CH(CH3)2),
d
¼ 5.33 (t-like, 2H, -C6H4-),
The following cell lines were used for biological assays: human
breast carcinoma cell line (MCF-7), cervical carcinoma cell line
(Hela) and human liver hepatocellular carcinoma cell lines
(HepG2). They were cultured with RPMI-1640 medium (Solarbio)
containing 10% fetal bovine serum (Zhejiang Tianhang Biological
Technology Co. Ltd., China), and placed at 37 ꢁC in humidified in-
cubators in an atmosphere of 5% CO2.
5.47 (d, 1H, -C6H4-), 5.54 (d, 1H, -C6H4-) ppm. 13C NMR (CDCl3):
d
¼
11.0
(CH3CH2CH2C]O),
13.9
(CH3C]N),
21.9,
18.4
22.4
(CH3C6H4CH(CH3)2),
19.0 (CH3CH2CH2C]O),
(CH3C6H4CH(CH3)2), 30.9 (CH3C6H4CH(CH3)2), 38.9 (O]C-
CH2CH2CH3), 83.6, 84.2, 84.6, 86.2, 99.8, 103.3 (CH3C6H4CH(CH3)2),
152.8 (OeN]C-CH3), 204.8 (O]CeCH2CH3) ppm.
The complexes were dissolved in DMSO at a concentration of
20 mM as stock solution, and diluted in culture medium at con-
4.2.8. Synthesis of [Ru(h
6-p-cymene)(L8)Cl] (8)
Compound 8 was prepared analogously by following the
centrations of 1000, 400, 160, 64 and 25.6 mM as working solution.
To avoid DMSO toxicity, the concentration of DMSO was less than
0.2% (v/v) in all experiments [43].
The cancer cells in exponential phase were digested with
trypsin, and seeded into a 96-well plate at density of 1 ꢂ 105.
Twelve hours later, new culture medium was added to replace the
previous one, then the compounds were added to the wells to
achieve final concentrations. Upon completion of the incubation for
method and conditions described for 1 but using HL8 (1.0 mmol)
and [Ru(
tained. Yield: 22.6%. M.p.:183.9e186.0 ꢁC. Anal.Calcd. for
19H20ClNO2Ru: C 52.96, H 4.68, N 3.25. Found: C 53.39, H 4.48, N
3.36. IR (KBr): v ¼ 1609 (C]O/N]C), 1568, 1333, 967 (N]O/NeO),
513 (RueN), 449 (RueO) cmꢀ1 1H NMR (CDCl3):
h
6-p-cymene)Cl2]2 (0.5 mmol). Black crystals were ob-
C
.
d
¼ 1.17 (d, 6H,
CH3C6H4CH(CH3)2, J ¼ 6.8 Hz), 2.25 (s, 3H, CH3C6H4CH(CH3)2),
2.79e2.86 (m, 1H, CH3C6H4CH(CH3)2), 3.58 (dd, CH2eC]N,
J ¼ 21.6 Hz), 5.41, 5.43 (d, 1H, -C6H4-, J ¼ 6.0 Hz), 7.06e7.12 (m, 4H,
-C6H4-), 7.52 (d, 1H, AreH, J ¼ 7.2 Hz), 7.53 (t-like, 1H, AreH, J ¼ 7.2,
7.6 Hz), 7.61 (d, 1H, AreH, J ¼ 7.6 Hz), 7.70 (t-like, 1H, AreH, J ¼ 6.4,
7.6 Hz), 7.90 (d, 1H, AreH, J ¼ 7.6 Hz) ppm. 13C NMR (DMSO-d6):
48 h, MTT dye solution (20
After 4 h incubation, all the solution inside each well was sucked
out carefully and then added DMSO (150 L) to solubilize the MTT
mL, 5 mg/mL) was added to each well.
m
formazan. The OD of each well was measured on a microplate
spectrophotometer at a wavelength of 492 nm. The dose causing
50% inhibition of cell growth (IC50) was determined from the curve
of inhibiting percentage versus dose.
d
¼ 21.0 (CH3C6H4CH(CH3)2), 24.4 (CH3C6H4CH(CH3)2), 27.1 (CH2-
C]N), 33.4 (CH3C6H4CH(CH3)2), 126.5, 129.3, 135.0, 135.1
(CH3C6H4CH(CH3)2), 123.9, 127.9, 128.3, 135.3, 145.8, 148.9 (AreH),
158.2 (N]CeAr), 197.3 (O]CeAr) ppm.
4.5. Cell apoptosis assay
4.2.9. Synthesis of [Ru(
h
6-p-cymene)(L9)Cl] (9)
The cervical carcinoma cells (Hela) in exponential phase were
digested with trypsin, then seeded into a 12-well plate at density of
3 ꢂ 105, and grown in an atmosphere of 5% CO2 at 37 ꢁC. Twelve
hours later, new culture medium was added to replace the previous
one, and then the compounds were added to the wells at the
concentrations of 2, 100 and 200 mM, respectively. After 48 h of
culture, cells were collected (including cells in culture medium and
cells attached to the bottom of the bottle wall), then centrifuged
Compound 9 was prepared analogously by following the
method and conditions described for 1 but using HL9 (1.0 mmol)
and [Ru(h
6-p-cymene)Cl2]2 (0.5 mmol). Purple crystals were ob-
tained. Yield: 28.6%. M.p. > 300 ꢁC. Anal.Calcd. for C22H20ClNO2Ru:
C 56.59, H 4.32, N 3.00. Found C 56.79, H 4.15, N 3.10. IR (KBr):
v ¼ 1613 (C]O), 1570 (C]N), 1374, 952 (N]O/NeO), 509 (RueN),
443 (RueO) cmꢀ1
.
1H NMR (DMSO-d6):
d
¼
1.17 (d, 6H,
CH3C6H4CH(CH3)2, J ¼ 6.8 Hz), 2.25 (s, 3H, CH3C6H4CH(CH3)2),
and washed twice with PBS. Cells were re-suspended in 300
mL of
2.79e2.86 (m, 1H, CH3C6H4CH(CH3)2), 7.06e7.12 (m,
binding buffer, and added 5 L of FITC and 10 L of PI to cells, and
m
m
4H,CH3C6H4CH(CH3)2), 7.64e7.67 (dd, 1H, AreH, J ¼ 7.2, 8.4 Hz),
7.84e7.88 (dd, 1H, AreH, J ¼ 7.2, 8.0 Hz), 7.54e7.99 (m, 2H, AreH),
vortexed for a while. The cell suspension was incubated in the dark
at 5e15 min at room temperature and then detected by flow

Y. He et al. / Journal of Organometallic Chemistry 842 (2017) 82e92
91
Table 3
Crystal data and structure refinement for compounds 1, 3 and 5.
Crystal data
Formula
1
3
5
C14 H20ClNO2Ru
370.83
C15H21ClNO2Ru
383.85
C19 H22ClNO2Ru
432.90
M(g molꢀ1
)
Crystal system
Space group
a (Å)
b (Å)
c (Å)
Monoclinic
P2(1)/n
8.5551(7)
12.5497(10)
14.6059(11)
90
101.0520(10)
90
1539.1(2)
4
Monoclinic
P2(1)/n
9.4075(10)
13.0758(14)
13.5095(14)
90
97.910(2)
90
1646.0(3)
4
Monoclinic
P2(1)/c
12.9955(11)
7.4206(7)
19.7038(17)
90
105.1830(10)
90
1833.8(3)
4
a
b
g
(deg)
(deg)
(deg)
Volume (Å3)
Z
Density (Mg/m3)
F000
1.600
752
1.549
780
1.568
880
Crystal size(mm3)
Index ranges
0.20 ꢂ 0.10 ꢂ 0.10
ꢀ12 ꢃ h ꢃ 11
ꢀ18 ꢃ k ꢃ 18
ꢀ21 ꢃ l ꢃ 21
2.16 to 31.50
98.0%
5029(0.0983)
16854
1.189
0.20 ꢂ 0.20 ꢂ 0.10
ꢀ13 ꢃ h ꢃ 13
ꢀ18 ꢃ k ꢃ 14
ꢀ19 ꢃ l ꢃ 19
2.18 to 30.50
99.9%
5014(0.0903)
17252
1.115
0.20 ꢂ 0.10 ꢂ 0.10
ꢀ18 ꢃ h ꢃ 18
ꢀ10 ꢃ k ꢃ 10
ꢀ26 ꢃ l ꢃ 28
1.62 to 31.00
99.4%
5815(0.1035)
19347
1.011
q
range(deg)
Completeness
N (Rint
)
Reflections collected
Absorption coefficient(mmꢀ1
Max. and min
)
0.8903 and 0.7969
0.982
0.8967 and 0.8078
1.026
0.9057 and 0.8234
1.089
GOF on F2
R1, wR2[I > 2
s
(I)]
R1 ¼ 0.0339
wR2 ¼ 0.0669
R1 ¼ 0.0475
wR2 ¼ 0.0736
R1 ¼ 0.0455
wR2 ¼ 0.1119
R1 ¼ 0.0626
wR2 ¼ 0.1211
R1 ¼ 0.0339
wR2 ¼ 0.0823
R1 ¼ 0.0424
wR2 ¼ 0.0937
R indices (all data)
cytometry (Beckman coulter flow cytometry).
phase were digested with trypsin and the medium was discarded
by centrifugation, then washed 1e2 times with PBS and re-
suspended with serum-free medium. Adjusting the cell density
was adjusted into 8 ꢂ 104/mL. Cell suspension with compounds
4.6. Cell cycle analysis
(200
150
containing 10% fetal bovine serum (500
mL, not containing fetal bovine serum) at a concentration of
The cervical carcinoma cells (Hela) were digested in exponential
phase with trypsin, then seeded into a 6-well plate at density of
3 ꢂ 106, and grown in an atmosphere of 5% CO2 at 37 ꢁC. Twelve
hours later, new culture medium was added to replace the previous
one and then the compounds were added to the wells at the con-
centration of 100 mM. After 48 h of culture, the culture medium was
discarded, and cells were collected by centrifugation. The cells was
washed twice with PBS, and centrifuged. The PBS was discarded,
mM was added to transwell chamber (Costar USA). The medium
mL) was added in 24-well
plate, then incubated at 37 ꢁC, saturated humidity and 5% CO2 for
24 h. Cells were fixed 15 min with 4% paraformaldehyde, then
wiped with a cotton swab on matrigel and transwell chamber. Then
the cells in 24-well plate were stained with 0.3% crystal violet
10 min, then washed with saline after dyeing and photographed
under a microscope.
and 300 mL of DNA staining solution and 10 mL of permeabilization
solution were added it, then the cell suspension was vortexed and
incubated for 30 min at 37 ꢁC, then detected by flow cytometry.
4.9. Western blot analysis
4.7. Scratch wound healing assay
HepG2 cells were harvested and lysed with RIPA lysis buffer by
incubation on ice for 10 min. After centrifugation at 8000 rpm for
5 min, supernatants were collected and concentrations of proteins
were measured using BCA reagent (purchased from Bio-Rad labo-
ratories, USA). The protein samples were denatured by boiling at
95 ꢁC for 5 min and loaded onto SDS-PAGE gel for electrophoresis.
The proteins were transferred onto PVDF membranes and the
membranes then incubated in the blocking solution (5% non-fat
dried milk) at room temperature for 0.5 h and were then incu-
bated with primary antibodies at 4 ꢁC overnight. The membranes
were subsequently incubated with secondary antibodies for 1 h.
Protein expression was normalized against tubulin expression.
Blotting images were acquired with the Odyssey infrared imaging
system (Li-COR Biosciences, USA) and analyzed by the software
provided by the manufacturer. Primary antibodies anti-Bcl-2 E-
cadherin (proteintech, USA) anti-Bcl-2 Vimentin (proteintech,
USA), anti-Cyclin-D1 (CST, USA) and anti-Cyclin-E1 antibody (Bos-
ter, Wuhan, China) were all used at a concentration of 1: 1000.
A vertical line with a marker pen was first drawn on the back of a
24-well plate for retained. Human hepatocellular carcinoma cells
(HepG2) in exponential phase were digested with trypsin, then
seeded into a 24-well plate (5 ꢂ 104 cells/well), and grown in 5%
CO2 at 37 ꢁC for 12 h. Cells mono-layers were wounded with a
sterile 100-mL pipette tip and washed with growth medium to
remove detached cells from the plates and immediately photo-
graphed under a microscope. Cells were exposed to compounds-
containing medium (not containing fetal bovine serum) at a con-
centration of 100 m
M and incubated at 37 ꢁC, saturated humidity, 5%
CO2 incubator, respectively, and the cells were photographed in 6 h
and 24 h using an Olympus BX41 microscope (Tokyo, Japan) and a
digital camera.
4.8. Transwell migration assay
Human hepatocellular carcinoma cells (HepG2) in exponential

92
Y. He et al. / Journal of Organometallic Chemistry 842 (2017) 82e92
B.K. Keppler, Dalton Trans. 44 (2015) 659e668.
Supplementary materials
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CCDCe1480620 (1), 1480631 (3), and 1480632 (5) contain the
supplementary crystallographic data for this paper. These data can
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Acknowledgments
This work has been supported by the National Natural Science
Foundation of China (No: 81102311), the Natural Science Founda-
tion of Hubei Province of China (No: 2016CFB433) and the Funda-
mental Research Funds for the Central Universities of China (No.
2015TS131).
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