← Back
A Cancer Cell-Selective and Low-Toxic Bifunctional Heterodinuclear Pt(IV)-Ru(II) Anticancer Prodrug.
Article
Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
pubs.acs.org/IC
A Cancer Cell-Selective and Low-Toxic Bifunctional Heterodinuclear
Pt(IV)−Ru(II) Anticancer Prodrug
Lili Ma,†,§ Xudong Lin,‡ Cai Li,†,§ Zoufeng Xu,†,§ Chun-Yin Chan,† Man-Kit Tse,† Peng Shi,‡
and Guangyu Zhu*,†,§
†
Department of Chemistry, City University of Hong Kong, 83 Tat Chee Ave, Hong Kong SAR, People’s Republic of China
Department of Biomedical Engineering, City University of Hong Kong, 83 Tat Chee Ave, Hong Kong SAR, People’s Republic of
China
§
City University of Hong Kong Shenzhen Research Institute, Shenzhen 518057, People’s Republic of China
‡
S Supporting Information
*
ABSTRACT: Although different types of metal-based anticancer complexes have
been synthesized, novel complexes to reduce the serious side effect of cisplatin and
conquer cancer metastasis are still highly desired. Here, we report the synthesis,
characterization, and biological activity of a novel heterodinuclear Pt(IV)−Ru(II)
anticancer prodrug. The Pt(IV)−Ru(II) complex exhibits good stability in both
water and PBS solution. Biological evaluation revealed that this bifunctional
Pt(IV)−Ru(II) complex utilizes the advantages of two metal centers to have both
cytotoxicity and antimetastatic property as designed. Although the complex has
comparable cytotoxicities to cisplatin in tested cancer cell lines, this prodrug
selectively kills cancer but not normal cells, and the IC50 values of the Pt(IV)−
Ru(II) complex are 7−10 times higher than those of cisplatin toward normal cells.
The cancer cell selectivity is further demonstrated by a cancer−normal cell
coculture system. In addition, the antimetastatic properties of the heterodinuclear
complex are assessed by using highly metastatic human breast cancer cells, and the
results show that the migration and invasion of cancer cells are effectively restrained after the treatment. Moreover, the Pt(IV)−
Ru(II) complex displays lower toxicity than cisplatin in developing zebrafish embryos. We, therefore, report an example of
heterodinuclear Pt(IV)−Ru(II) complex not only to defeat both drug resistance and cancer metastasis but also having
significantly improved cancer cell selectivity and reduced in vivo toxicity than cisplatin.
■
INTRODUCTION
Pt(II)-based anticancer agents including cisplatin and its
analogues are among the most widely used and efficient
small-molecule drugs in clinical cancer chemotherapy.1 The
serious side effects and the incidence of drug resistance
promote the exploitation of new types of metal-based
anticancer drug candidates.2 Pt(IV) complexes represent a
promising family of nonconventional Pt-based anticancer
agents due to their inertness under physiological conditions,
easy modification through axial ligands, and the ability to be
activated to Pt(II) by reducing agents after entering cancer
cells.3−5 In the meantime, efforts have been devoted to
searching for novel pharmaceutical agents bearing other
metals.6 For example, different from cytotoxic Pt complexes,
Ru anticancer drug candidates including NAMI-A and RAPTAC are well-known for their impressive antimetastatic properties
with commonly moderate or low cytotoxicity.7,8
It is suggested that the incorporation of different metals into
one molecule may induce additive or even synergistic effects.
The toxicity of the resulted heteronuclear metal complexes,
however, is a great concern due to the presence of different
types of metals, and the cancer-cell selectivity of heteronuclear
© XXXX American Chemical Society
complexes is sometimes overlooked. Indeed, even for
mononuclear metal complexes in clinical settings, the doserelated toxicity is usually the cause for therapy discontinuation.9−11 On the other hand, although some heteronuclear
complexes containing Pt have been reported, most work mainly
focused on the study of metal−DNA interactions and/or the
ability to overcome cisplatin resistance.12−20 Only few cis/
transPt(II)−Ru(III) complexes were reported to be able to
effectively combat cancer cell motility.19 Novel heterodinuclear
Pt−Ru complexes with high cytotoxicity and antimetastatic
properties against cancer cells but with improved cancer cellselectivity and low toxicity have been rarely reported and are
highly desired.
Herein, we report the synthesis, characterization, and
biological evaluation of complex 5, a heterodinuclear Pt(IV)−
Ru(II) complex bearing both cisplatin and arene−Ru(II)
moieties. The Pt(IV) unit has advantages over its Pt(II)
congener, including but not limited to the kinetic inertness to
avoid undesirable side reactions to biomolecules and the
Received: January 7, 2018
A
DOI: 10.1021/acs.inorgchem.8b00053
Inorg. Chem. XXXX, XXX, XXX−XXX
Article
Inorganic Chemistry
Scheme 1. (A) Synthetic Route of Complex 5a and (B) Synthetic Route of Arene-Ru(II) Compound 6b
Reagents and conditions: (i) benzoic anhydride, 60 °C, 12 h in DMF; (ii) succinic anhydride, 60 °C, 12 h in DMF; (iii) EDCI/NHS, r.t., 12 h in
acetone; (iv) N-(3-aminopropyl)-imidazole, r.t., 12 h in acetone; (v) CymRu(II)(O^O)H2O, r.t., 12 h in methanol. bReagents and conditions: (vi)
succinic anhydride, r.t., 12 h in DMF; (vii) CymRu(II)(O^O)H2O, r.t., 4 h in DCM.
a
Table 1. Cytotoxicities of Complex 5 and Control Compounds by MTT Assay after 72 h Treatment. IC50 Values Are Expressed
As the Concentrations of Pt (μM)
cell lines
type
cisplatin
compound 6
cisplatin + compound 6b
complex 5
A2780
A2780cisR
RFa
A549
MDA-MB-231
MRC-5
WI38
Hs27
NIH3T3*
SIc
cancer
cancer
1.5 ± 0.3
9.7 ± 1.0
6.5
3.3 ± 2.5
14 ± 1
2.9 ± 0.5
3.2 ± 0.8
12 ± 1
4.4 ± 2.0
0.88
299 ± 96
447 ± 185
1.5
577 ± 78
1.4 ± 0.2
7.8 ± 2.3
5.6
1.7 ± 0.5
>100
1.6 ± 0.3
2.1 ± 0.1
6.9 ± 4.0
3.2
7.1 ± 3.0
30 ± 9
18 ± 2
24 ± 7
>60
46 ± 16
2.5
cancer
cancer
normal
normal
normal
normal
normal
0.98
a
RF (resistant factor): IC50 in A2780cisR/IC50 in A2780. bA mixture of cisplatin and an equal equivalent of arene−Ru(II) compound 6. cSI
(selectivity index) is defined as IC50 in MRC-5/IC50 in A549. *Mouse source.
■
RESULTS AND DISCUSSION
Complex 5 was derived from the asymmetric substitution of
c,c,t-[Pt(NH3)2Cl2(OH)2] stepwise (Scheme 1). First, the
reaction of c,c,t-[Pt(NH3)2Cl2(OH)2] with two equivalents of
benzoic anhydride yielded c,c,t-[Pt(NH3)2Cl2(OH)(benzoate)]
(1). Compound 2 was obtained by the reaction of compound 1
with 6 equiv of succinic anhydride. The carboxylic group of 2
was activated by EDC/NHS chemistry to obtain an NHS ester,
followed by conjugation with N-(3-aminopropyl)-imidazole
through an amide bond to form compound 4. Finally, the
arene−Ru(II) moiety with an oxalate leaving group was applied
to 4 to produce the final product 5.21,22 Additionally, the
arene−Ru(II) compound 6 was synthesized as a control
(Scheme 1). The compounds were fully characterized by 1H,
13
C, 195Pt NMR spectroscopy, ESI-MS, and CHN elemental
analysis (Figures S1−S16). The purities of compounds 4 and 5
preferred reduction inside cancer cells. To obtain such a
bifunctional heterodinuclear anticancer agent, a cytotoxic
Pt(IV) unit is conjugated with an arene−Ru(II) center through
an imidazole linker. The contribution of Pt to cytotoxicity and
Ru to antimetastatic property was illustrated, and the cancer
cell-selectivity was scrutinized. The possible relationship
between cancer cell selectivity and cellular accumulation was
examined. Furthermore, zebrafish embryos were utilized to test
the in vivo toxicity of complex 5. We report a unique example of
a heterodinuclear Pt(IV)−Ru(II) complex combining the
cytotoxic and antimetastatic properties of both metal centers
with impressive cancer cell selectivity in vitro and low toxicity in
vivo.
B
DOI: 10.1021/acs.inorgchem.8b00053
Inorg. Chem. XXXX, XXX, XXX−XXX
Article
Inorganic Chemistry
Figure 1. Cellular accumulation of complex 5 and cisplatin in (A) A549 and (B) MRC-5. Cells were treated with 100 μM compounds for 1 h. Cell
numbers were recorded, and the Pt or Ru levels were determined by ICP-MS. Data were obtained from two independent experiments and expressed
as pmol Pt or Ru per 106 cells.
Serious side effects of cisplatin, a metal-based DNA damaging
agent without cancer cell selectivity, are always a concern in
clinical chemotherapy. The effect of complex 5 on the
proliferation of normal cells was subsequently tested. WI38
and MRC-5 normal human lung fibroblast, Hs27 human
foreskin fibroblast, and NIH3T3 mouse musculus fibroblast
cells were selected for the evaluation (Table 1). Intriguingly,
complex 5 shows dramatically decreased cytotoxicities in all the
normal cells tested. The IC50 values of complex 5 are 7- to 10fold higher than those of cisplatin in WI38, MRC-5, and
NIH3T3 cells. Compared to a mixture of cisplatin and
compound 6, the heterodinuclear complex 5 shows 11-times
increased IC50 values in MRC-5 cells. In addition, upon
treatment with 60 μM of complex 5 for 72 h, the viability of
Hs27 cells does not change significantly (Figure S22). The
selectivity index (SI), defined as the ratio of the IC50 value in
MRC-5 to that in A549 cells, is 2.5 for complex 5 but only 0.88
and 0.98 for cisplatin and the mixture, respectively. Taken
together, complex 5 shows identical cytotoxicities to those of
cisplatin or a mixture of cisplatin and compound 6 in difference
types of human cancer cells, but its cytotoxicities are much
lower than both in tested normal cells, indicating the
significantly improved cancer cell selectivity. The cancer cell
selectivity of complex 5 was further demonstrated by coculture
of MRC-5 and A549 cells, both of which are originated from
the lung and can be easily distinguished by their shapes.
Compared to the untreated or cisplatin-treated groups, upon
treatment with 50 μM complex 5 for 48 h, very few cancer cells
were left, and the normal cells were still alive (Figure S23). The
result confirms that the heterodinuclear complex 5 is able to kill
cancer cells effectively and selectively.27
To probe whether the origin of cancer cell selectivity of
complex 5 was from the difference in cellular accumulation,
A549 and MRC-5 cells were treated with 100 μM complex 5 or
cisplatin for 1 h, and the cellular levels of Pt and Ru were
determined by ICP-MS (Figure 1). In A549 and MRC-5 cells,
the accumulation levels of complex 5 are identical to those of
cisplatin (expressed as Pt levels). For instance, in A549 cells,
the cellular level of complex 5 is 69.2 pmol Pt/106 cells, and the
value for cisplatin is 57.8 pmol Pt/106 cells. Similarly, in MRC5 cells, the cellular uptake of complex 5 and cisplatin are 121.5
pmol Pt/106 cells and 97.6 pmol Pt/106 cells, respectively. It is
noteworthy that both complex 5 and cisplatin show stronger
abilities to internalize into MRC-5 cells than A549 cells.
Therefore, cellular accumulation may not contribute to the
cancer cell-selectivity of complex 5.
Unlike cisplatin and other types of cytotoxic Pt compounds,
the antimetastatic property of Ru complexes is commonly more
attractive than their cytotoxicity. Both Ru(III) (e.g., NAMI-A)
were further tested by HPLC (Figure S17), which are 95% and
96%, respectively.
The stability of the heterodinuclear complex 5 in water was
studied by 1H NMR. Mononuclear Pt(IV) compound 4 was
included as a control. No significant changes are observed in
the spectra of both compounds 4 and 5 after 24 h, indicating
their stability in water and excluding the possibility of the ester
bond hydrolysis in the Pt(IV) center (Figures S18 and S19).23
Subsequently, the stability test was carried out in PBS (pH =
7.4). Neither compound 4 nor 5 changes significantly after 24 h
under this condition (Figures S20 and S21). The great stability
of the compounds will benefit their further biological
applications.
Cytotoxicities of complex 5 were evaluated by MTT assay in
a panel of human cancer cell lines including A2780 ovarian,
A549 non-small-cell lung, and MDA-MB-231 breast cancer
cells, which are widely used for the assessment of metal-based
anticancer drugs.24,25 Cisplatin-resistant A2780cisR cells,
generated from their parental cells, were also employed.26
Cisplatin, arene−Ru(II) compound 6, and a mixture of cisplatin
and an equal equivalent of compound 6 were tested as controls.
The results are summarized in Table 1. Cisplatin shows
cytotoxicity in both cancer and normal cells, and the IC50 values
were in the low micromolar range. As expected, the cytotoxicity
of arene−Ru(II) compound 6 is low, indicated by the high IC50
values (>100 μM). In addition, we examined the cytotoxicity of
a mixture of cisplatin and an equal equivalent of compound 6.
The mixture shows slightly improved cytotoxicities than
cisplatin in the tested cancer cells, including A2780,
A2780cisR, and A549 cells. Moreover, in A549 cells, the
combination index (CI) tested by Chou-Talalay assay was 0.54,
indicating a synergistic effect for the cotreatment of cisplatin
and compound 6. However, the mixture also exhibits a higher
cytotoxicity than cisplatin in MRC-5 human normal lung
fibroblasts. Cotreatment of cisplatin and compound 6 results in
enhanced cytotoxicity but not cancer cell selectivity. We
subsequently examined the cytotoxicity of heterodinuclear
complex 5. Complex 5 displays low micromolar IC50 values in
the tested cancer cells, which are slightly higher than or
identical to those of cisplatin and a mixture of cisplatin and
compound 6. For example, the IC50 values of cisplatin and the
mixture are 1.5 μM and 1.4 μM in A2780 cells, respectively, and
that of complex 5 is 2.1 μM. In cisplatin-resistant A2780cisR
cells, complex 5 shows a lower IC50 value (6.9 μM) than
cisplatin (9.7 μM) as well as the mixture (7.8 μM), indicating
the effectiveness of complex 5 in the resistant cells. The IC50
values of complex 5 are 7.1 and 30 μM in A549 and MDA-MB231 cells, respectively.
C
DOI: 10.1021/acs.inorgchem.8b00053
Inorg. Chem. XXXX, XXX, XXX−XXX
Article
Inorganic Chemistry
Figure 2. Migration inhibition of MDA-MB-231 cells by wound healing assay. Cells were treated with different concentrations of complex 5. After
calcein-AM staining, images were taken at 0, 8, and 24 h by SPE confocal (10×). Scale bar, 250 μm. The area of the wounds was measured by the
ImageJ software. The wound closure ratios were calculated from three to four replicates.
Figure 3. As expected, a large number of cells in the control
group invades to the lower side of the insets, and the invasion
and Ru(II) complexes (e.g., RAPTA-C) have shown their
ability to actively suppress cancer metastasis, which is a
complicated process from a series of actions.28−30 To
investigate whether the incorporation of arene−Ru(II) moiety
into complex 5 is able to functionalize the heterodinuclear
complex with antimetastatic ability, we investigated two
important steps of metastasis, namely cancer cell migration
and invasion.31 The ability of complex 5 to modulate cancer cell
metastasis was measured by using highly metastatic MDA-MB231 human breast cancer cells. First, the migration inhibition
effect of complex 5 was assessed by a wound healing assay
(Figure 2).32 After the formation of a cell monolayer, “wounds”
with similar sizes were created, and the initial areas were
recorded. Data are listed in Table S1. Subtoxic concentrations
of complex 5 were used to minimize its cell-killing effect, and
the cell viability was above 75% at 175 μM (Figure S24). In the
untreated group, 31% of the scratched area is filled with cells
after 8 h. In the treated groups, wound closure ratios range
from 15% to 8% with increasing concentrations of complex 5
(from 75 μM to 175 μM), indicating that cell migration is
effectively diminished by the complex. A similar trend is
observed after 24 h. A total of 76% of the wounds are healed in
the untreated group. In contrast, the wound closure ratio is
only 25% for the cells treated with 175 μM complex 5. Even at
the lowest concentration used, the wound closure ratio is 43%,
which is much lower than the group without treatment. These
results show that the heterodinuclear Pt(IV)−Ru(II) complex
is able to restrain cancer cell migration in a time- and
concentration-dependent manner.
Next, the potential of complex 5 to overcome cancer cell
invasion was tested by using a transwell invasion assay.32,33 The
microporous insert wells were precoated with a proper
concentration of matrigel to mimic the extracellular matrix. A
DMEM medium with 10% FBS was added to the receiver wells
as a chemoattractant (Figure S25). MDA-MB-231 cells were
treated with 100 μM complex 5 for 24 h, and the cells without
treatment were set as a control. After invading to the lower side
of the inset wells, cells were stained with crystal violet, and the
invasion values were calculated by measuring the UV
absorbance at 590 nm. Representative images are shown in
Figure 3. A transwell invasion assay showing the invasion inhibition of
MDA-MB-231 by complex 5. Cells were treated with 100 μM complex
5 for 24 h. Images were taken by a Leica DMI3000 B inverted
microscope (20×). Scale bar, 75 μm. Cell invasion ratios were
measured by crystal violet assay. The invasion ratio of cells without
drug treatment was set as 100%. Data were calculated from two to
three replicates.
ratio is defined as 100%. Notably, with the treatment of
complex 5, the invasion ratio reduces to 63.6%. These results
demonstrate that complex 5 is able to efficiently inhibit the
migration and halt the invasion of MDA-MB-231 cells.
Finally, the in vivo toxicity assessment of cisplatin and
complex 5 was conducted using zebrafish embryos.34 With the
high degree of homology to mammals, rapid postfertilization
development, small size, and optical transparency, zebrafish
embryos have become a widely used model organism for drug
discovery and toxicology evaluation.35,36 The embryos were
treated with increasing concentrations of cisplatin or complex 5.
The cumulative survival and hatching status of the embryos
were recorded and evaluated every 24 h (Figure 4 and Table
S2). The mortality and hatching of the embryos are dependent
on the concentration of cisplatin and complex 5. Without
treatment, almost all of the zebrafish embryos survive, and they
finally develop into juvenile zebrafish. After 96 h of treatment,
only when the concentration of cisplatin is 30 μM or lower, the
survival rate can maintain over 90%, and the value drops to 75%
D
DOI: 10.1021/acs.inorgchem.8b00053
Inorg. Chem. XXXX, XXX, XXX−XXX
Inorganic Chemistry
Article
■
CONCLUSIONS
In conclusion, we report the first example of a heterodinuclear
Pt(IV)−Ru(II) complex not only to conquer both drug
resistance and cancer metastasis but also having significantly
improved cancer cell selectivity and reduced in vivo toxicity
than cisplatin. The complex utilizes the unique properties of
both Pt and Ru metals as designed, and its cytotoxic and
antimetastatic properties are confirmed. Although the complex
has improved cytotoxicity compared with cisplatin in the
resistant cells, its IC50 values in several normal cells increase by
an order of magnitude, and the complex shows great cancer cell
selectivity in a coculture system in vitro. The cancer cell
selectivity is not due to the cellular accumulation, and we
assume that the complex may have selective activation in cancer
cells, leading to cancer cell-selectivity. The low toxicity profile
of complex 5 is further illustrated by an in vivo toxicity
assessment using zebrafish embryos. This cancer cell selective
and low-toxic bifunctional heterodinuclear compound distinguishes itself from many other types of multinuclear metalbased complexes including the ones made from our own group
because their cancer cell selectivity and toxicity are greatly
concerned.10,21 The detailed intracellular fate of complex 5 as
well as the profound mechanism of superior cancer cell
selectivity are still under investigation.
■
EXPRIMENTAL SECTION
Materials and Instruments. Cisplatin was purchased from
Shandong Boyuan Pharmaceutical Co., Ltd., China. RuCl3·nH2O was
bought from J & K scientific. N-[3-(Dimethylamino)propyl]-N′ethylcarbodiimide hydrochloride (EDCI) and succinic anhydride were
purchased from Meryer. N-Hydroxysuccinimide (NHS) and N-(3aminopropyl)-imidazole were purchased from Sigma-Aldrich. N,N′dicyclohexylcarbodiimide (DCC) was ordered from International
Laboratory USA. Benzoic anhydride was bought from Energy
Chemical. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Life Technologies. Transwell
insert wells (Corning #3422, 6.5 mm diameter inserts, and 8.0 μm
pore size) and Matrigel (Corning #356237) were ordered from
Corning. Calcein-AM and crystal violet were purchased from SigmaAldrich. All agents and solvents were used as received without
additional drying or purification except otherwise indicated. All
reactions were carried out under atmosphere unless further
notifications. Bruker Ultrashield NMR spectrometers (300, 400, or
600 MHz) were used to detect 1H, 13C, and 195Pt NMR spectra at
room temperature. All NMR chemical shifts (δ) are reported in parts
per million (ppm) and referenced as described below. 1H and 13C
NMR spectra were referenced internally to residual solvent peaks
(DMSO-d6: 1H, δ 2.50; 13C, δ 39.5; D2O: 1H, δ 4.71). The 195Pt NMR
spectrum was referenced externally using standards of K2PtCl4 in D2O
(δ = −1628 ppm). Stability tests were carried out by 1H NMR with
water suppression, and D2O was used as an external standard. ESI-MS
was carried out on an Agilent API-2000 mass spectrometer (methanol
as solvent). Elemental analysis was performed by using a Vario Micro
elemental analyzer. Analytical reversed-phase HPLC was carried out by
using a Shimadzu Prominence System equipped with a DGU-20ASR
Degasser, two LC-20AT Liquid Chromatography Pumps, a SPD-20A
UV/vis detector, and a C18 column (Phenomenex, Gemini, 5 μm, 110
Å, 250 × 4.6 mm). An inductively coupled plasma-optical emission
spectrometer (ICP-OES, Optima 2100DV, PerkinElmer, USA) or
inductively coupled plasma-mass spectrometer (ICP-MS, NEXION
2000, PerkinElmer, USA) was applied to determine platinum and
ruthenium levels. Confocal images of the coculture assay and wound
healing assay were taken by a Leica SPE confocal microscope. Photos
of the transwell invasion assay were taken with a Leica DMI3000 B
inverted microscope. Images for in vivo toxicity test by zebrafish
Figure 4. Toxicity assessments of cisplatin and complex 5 using
zebrafish embryos. Survival rates of zebrafish embryos in the presence
of (a) cisplatin and (b) complex 5 are shown, together with hatching
rates of zebrafish embryos after the exposure to (c) cisplatin and (d)
complex 5. (e) Representative ecotoxicology images of zebrafish
embryos with treatment of complex 5 at different concentrations over
96 h. Data are collected from four replicates of two independent
experiments, and the mean values are presented. The data are also
listed in Table S2.
when 90 μM cisplatin is used (Figure 4a). In comparison to
cisplatin, the complex-5-treated group shows higher survival
rates at all the concentrations. The survival rate remains above
90% in the presence of 60 μM complex 5, and more than 83%
of zebrafish embryos survive when the concentration increases
to 90 μM (Figure 4b). The hatching rate upon treatment was
subsequently measured. In general, the embryos become
abnormal and are difficult to develop to juvenile zebrafish
with 45 μM or higher concentration of cisplatin. The hatching
rate is only 45% upon treatment with 45 μM cisplatin for 96 h.
When the concentration increases to 90 μM, only 10% of
embryos are able to develop to juvenile zebrafish (Figure 4c).
In contrast, the hatching rate upon treatment with even the
highest concentration of complex 5 still maintains at 50% after
96 h (Figure 4d), and the representative ecotoxicology images
of zebrafish embryos with the treatment of complex 5 are
shown in Figure 4e. Collectively, these results suggest that
complex 5 has a lower toxicity to zebrafish embryos than
cisplatin.
E
DOI: 10.1021/acs.inorgchem.8b00053
Inorg. Chem. XXXX, XXX, XXX−XXX
Article
Inorganic Chemistry
2H), 7.92−7.86 (m, 2H), 7.59−7.47 (m, 1H), 7.48−7.34 (m, 3H),
6.93−6.42 (m, 7H), 5.75 (d, J = 6.0 Hz, 2H), 5.52 (d, J = 6.0 Hz, 2H),
4.04 (t, J = 6.9 Hz, 2H), 3.01 (q, J = 5.7 Hz, 2H), 2.76−2.64 (m, 1H),
2.51 (d, J = 7.2 Hz, 2H), 2.32 (d, J = 7.2 Hz, 2H), 2.03 (s, 3H), 1.85
(m, 2H), 1.20 (d, J = 6.9 Hz, 6H). 13C NMR (150 MHz, DMSO-d6): δ
180.7, 173.8, 172.2, 165.3, 140.1, 133.7, 132.1, 129.8, 129.3, 128.3,
121.1, 100.0, 97.3, 83.0, 79.5, 45.3, 40.7, 35.6, 32.0, 30.8, 22.6, 17.8.
195
Pt NMR (128 MHz, DMSO-d6): δ 1205. ESI-MS (negative mode,
methanol) C29H38Cl2N5O9PtRu, [M − H]−, cald (m/z): 968.1.
Found: 968.3. Anal. Calcd for C29H39Cl2N5O9PtRu·2H2O: C, 34.67;
H, 4.31; N, 6.97. Found: C, 34.49; H, 4.486; N, 6.87. Pt/Ru ratio is
1:1.03 by ICP-OES, and the data are an average from four
independent experiments.
Synthesis of Imidazole Ligand. N-(3-Aminopropylaminopropyl)-imidazole (715 μL, 6.0 mmol. 1.2 equiv) and succinic anhydride
(500 mg, 5.0 mmol. 1.0 equiv) were stirred in 0.7 mL of DMF for 12 h
at room temperature. Then, 50 mL of Et2O was added to the reaction
solution to get a white precipitate. The product was collected by
filtration and washed with Et2O (Scheme 1B). White powder, 1.0 g,
88.8%. 1H NMR (300 MHz, DMSO-d6): δ 11.93 (s, 1H), 7.92 (t, J =
6.0 Hz, 1H), 7.61 (s, 1H), 7.17 (s, 1H), 6.88 (s, 1H), 3.95 (t, J = 6.0
Hz, 2H), 3.00 (q, J = 6.0 Hz, 2H), 2.44 (t, J = 6.0, 2H), 2.31 (t, J = 6.0,
2H), 1.89−1.74 (m, 2H).
Synthesis of Arene−Ru(II) Compound 6. Compound 6 was
synthesized by adding the DCM solution of imidazole ligand (135 mg,
0.6 mmol, 1.0 equiv) dropwise to the DCM solution of CymRu(II)(O^O)H2O (202.0 mg, 0.6 mmol, 1.0 equiv). The mixture was reacted
at room temperature for 4 h in the dark. Then, DCM was removed by
rotary evaporator. Orange oil, 312.7 mg, 95%. 1H NMR (300 MHz,
CDCl3): δ 7.63 (s, 1H), 7.48 (t, J = 6.0 Hz, 1H), 6.88 (s, 2H), 5.64 (d,
J = 6.0 Hz, 2H), 5.42 (d, J = 6.0 Hz, 2H), 3.72 (t, J = 6.0 Hz, 2H),
3.20−3.08 (m, 2H), 2.819−2.75 (m, 1H), 2.72−2.68 (m, 2H), 2.56−
2.45 (m, 2H), 2.13 (s, 3H), 1.90−1.76 (m, 2H), 1.29 (d, J = 6.0 Hz,
6H).
Cell Lines and Cell Culture Conditions. A2780 and A2780cisR
cells were cultured in RPMI 1640 with 10% FBS and 100 units of
penicillin/streptomycin. A549, MDA-MB-231, NIH3T3, and Hs27
cells were cultured in DMEM containing 10% FBS and 100 units of
penicillin/streptomycin. WI38 and MRC-5 cells were cultured in
MEM with 10% FBS, 1% L-glutamine, 1% nonessential amino acids,
and 1% sodium pyruvate. Cisplatin-resistant cells A2780cisR were
generated from their parental A2780 cells. Briefly, A2780 cells were
cultured in complete medium containing 0.5 μg/mL cisplatin at the
beginning for the first screening, and the remaining cells were cultured
in complete medium containing 1.0 μg/mL cisplatin for at least 4
weeks until the resistance was obtained.26 All cells were incubated at
37 °C in 5% CO2.
Cytotoxicity Test. An MTT assay was used to evaluate the in vitro
cytotoxicity of the compounds. Cells were seeded in 96-well plates at a
density of 1500 to 2500 cells per well until the cell confluency reached
about 30%. Then, the medium was replaced by a drug-containing
medium. DMF was used as a supporting solvent, and its final
concentration was 0.5%. Cells incubated with a medium containing
0.5% DMF were set as controls. After 72 or 24 h of drug treatment, the
drug-containing medium was removed by FBS-free medium
containing 1 mg/mL MTT (0.2 mL per well). The medium was
replaced by DMSO after 2 to 4 h of staining (0.2 mL per well). The
absorbance was measured at 570 and 730 nm.
Coculture of MRC-5 and A549 Cells. Long-shape MRC-5 cells
(15 000 cells/well) and round-shape A549 cells (15 000 cells/well)
were seeded in a 24-well plate and were cultured in MEM with 10%
FBS, 1% L-glutamine, 1% nonessential amino acids, and 1% sodium
pyruvate. Sixteen hours later, the medium was replaced by a medium
containing 50 μM cisplatin or complex 5. Next, 0.5% DMF was used as
a supporting solvent, and cells treated with 0.5% DMF were set as a
control. After 48 h of treatment, the drug-containing medium was
removed, and cells were washed with PBS three times. Then, cells
were stained with calcein-AM and washed with PBS three times.
Calcein-AM was prepared to 1 mM stock solution in DMSO and was
diluted 2000 times with PBS before use. Images were taken with a laser
embryo were taken with an inverted microscope (Olympus IX81)
equipped with a cooled sCMOS camera (Neo, ANDOR).
HPLC Analysis of Compounds Purity. Phase A: milli-Q H2O
with 0.02% TFA. Phase B: acetonitrile (ACN) with 0.02% TFA.
Program: 0−12 min, 80% to 30% phase A; 12−14 min, 30% phase A;
14−15 min, 30% to 80% phase A; 16 min, stop. Flow rate: 1.0 mL/
min. Injection volume: 20 μL, @ 254 and 365 nm. For analysis of
compounds’ purity, a certain amount of compound 4 or 5 was
dissolved in Milli-Q-H2O and injected for a purity test. The integral
was calculated according to absorbance at 254 nm.
Stability Test by 1H NMR. The powder of compound 4 or 5 was
dissolved in Milli-Q-H2O or PBS with 1.6 or 2 mM as the final
concentration just before the test. 1H NMR spectra were acquired on a
Bruker Ultrashield 600 MHz NMR spectrometer with water
suppression.
Synthesis of Compound 1. The c,c,t-[Pt(NH3)2Cl2(OH)2]
(200.0 mg, 0.60 mmol, 1.0 equiv) and benzoic anhydride (270.0 mg,
1.20 mmol, 2.0 equiv) were heated to 60 °C in 2 mL of DMF for 16 h
in the dark. The suspension changed from yellow into white gradually.
Then, the product was collected by centrifugation and washed by
acetone and diethyl ether. White powder, 215.0 mg, 82.1%. 1H NMR
(300 MHz, DMSO-d6): δ 7.93−7.84 (m, 2H), 7.58−7.31 (m, 3H),
6.47−5.70 (m, 6H), 1.15 (t, J = 11.4 H, 1H). 13C NMR (100 MHz,
DMSO-d6): δ 173.5, 134.8, 131.1, 129.3, 127.7.
Synthesis of Compound 2. Compound 1 (50.0 mg, 0.11 mmol,
1.0 equiv) and succinic anhydride (70.0 mg, 0.70 mmol, 6.1 equiv)
were stirred in 1 mL of DMF at 60 °C overnight. The suspension
changed into a yellow solution gradually. A large amount of diethyl
ether was added to get a white precipitate. The product was washed
with diethyl ether. Light yellow powder, 20.0 mg, 33.8%. 1H NMR
(400 MHz, DMSO-d6): δ 12.11 (s, 1H), 7.88 (d, J = 7.6 Hz, 2H), 7.52
(t, J = 7.2 Hz, 1H), 7.43 (t, J = 7.6 Hz, 2H), 7.06−6.17 (m, 6H), 2.54
(t, J = 7.2 Hz, 2H), 2.40 (t, J = 7.2 Hz, 2H). 13C NMR (100 MHz,
DMSO-d6): δ 179.6, 173.8, 173.3, 133.1, 131.6, 129.4, 127.9, 30.4,
29.8.
Synthesis of Compound 3. Compound 2 (160.0 mg, 0.30 mmol,
1.0 equiv), N-hydroxysuccinimide (NHS, 48.0 mg, 0.42 mmol, 1.4
equiv), and DCC (67.5 mg, 1.1 equiv) were stirred in 5 mL of acetone
at room temperature for 12 h in the dark. The suspension changed
into a yellow solution with white precipitate dicyclohexylurea (DCU).
The byproduct DCU was removed by filtration, and the yellow
solution was concentrated by rotary evaporation. Yellow powder,
180.2 mg, 94.5%. 1H NMR (400 MHz, DMSO-d6): δ 7.89 (d, J = 8.0
Hz, 2H), 7.53 (t, J = 7.3 Hz, 1H), 7.43 (t, J = 7.6 Hz, 1H), 7.05−6.27
(m, 6H), 2.88−2.79 (m, 6H), 2.68 (t, J = 6.8 Hz, 2H). 13C NMR (100
MHz, DMSO-d6): δ 178.2, 173.3, 170.2, 168.4, 133.0, 131.7, 129.4,
127.9, 33.4, 30.8, 29.6, 26.7.
Synthesis of Compound 4. Compound 3 (150.0 mg, 0.24 mmol,
1.0 equiv) was dissolved in 5 mL of acetone. A solution of N-(3aminopropyl)-imidazole (31.0 μL, 0.26 mmol. 1.1 equiv) in acetone
was added dropwise to compound 3. Yellow precipitate formed upon
the adding of N-(3-aminopropyl)-imidazole. After 12 h, the product
was collected by centrifugation and washed with diethyl ether. Light
yellow powder, 100.3 mg. 65.8%. 1H NMR (400 MHz, DMSO-d6): δ
7.92 (t, J = 5.2 Hz, 1H), 7.89 (d, J = 8.0 Hz, 2H), 7.63 (s, 1H), 7.53 (t,
J = 7.2 Hz, 1H), 7.43 (t, J = 7.6 Hz, 2H), 7.18 (s, 1H), 6.89 (s, 1H),
6.97−6.28 (m, 6H), 3.97 (t, J = 6.8 Hz, 2H), 3.06−2.96 (m, 2H), 2.31
(t, J = 7.6 Hz, 2H), 1.89−1.75 (m, 2H). 13C NMR (100 MHz, DMSOd6): δ 180.5, 173.7, 172.0, 137.8, 133.6, 132.1, 129.8, 128.8, 128.3,
119.8, 44.1, 36.2, 31.9, 31.7, 31.2, 31.2. 195Pt NMR (128 MHz, DMSOd6): δ 1202. ESI-MS (negative mod, methanol) C17H25Cl2N5O5Pt, [M
− H]−, cald (m/z): 644.1. Found: 644.2.
Synthesis of Complex 5. CymRu(II)(O^O)H2O was synthesized
by following the previous procedure.22 The solution of compound 4
(20.0 mg, 0.03 mmol, 1.0 equiv) in methanol was added dropwise into
a solution of CymRu(II)(O^O)H2O in methanol (10.1 mg, 0.03
mmol, 1.0 equiv). The mixture was stirred at room temperature in the
dark for 12 h. After removing solvent by rotary evaporation, the orange
product was washed with diethyl ether several times. Orange powder,
20.0 mg, 68.7%. 1H NMR (300 MHz, DMSO-d6): δ 7.99−7.91 (m,
F
DOI: 10.1021/acs.inorgchem.8b00053
Inorg. Chem. XXXX, XXX, XXX−XXX
Article
Inorganic Chemistry
confocal microscope (Leica SPE) with 10× magnification, and the
scale bar is 250 μm. The experiment was repeated with four replicates.
Cellular Accumulation. A549 cells or MRC-5 cells were seeded in
a six-well plate, and 100 μM of complex 5 or cisplatin was added when
the confluency of cells reached around 80%. Cells were incubated with
100 μM compound in complete medium (DMEM for A549 cell, and
MEM for MRC-5 cells) for 1 h. Then, cells were collected by
trypsinization, and the cell numbers were recorded. The levels of Pt
and Ru were determined by ICP-MS after digestion with concentrated
HNO3 at 65 °C overnight. Next, 0.5% DMF was used as a supporting
solvent in this experiment. Data were collected from two independent
experiments and expressed as pmol Pt or Ru/106 cells.
Migration Inhibition by Wound Healing Assay. MDA-MB-231
cells were suspended in DMEM containing 12% FBS and seeded in
24-well plates with a density of 280 000 cells per well. Cells were
allowed to attach and grow to form a confluent monolayer. Each well
of the plates was marked with a horizontal line passing through the
center of the bottom in advance. Wounds were created perpendicular
to the lines by tips, and unattached cells were removed by PBS
washing (pH 7.4). After calcein-AM staining, cells were washed with
PBS three times. Then, cells were incubated in DMEM with 1% FBS
containing different concentrations of complex 5 at 37 °C under 5%
CO2. Then, 1% FBS was used to suppress cell proliferation. DMF was
used as a supporting solvent, and the final concentration was 1%. Cells
with 1% DMF were set as a control. Images were captured at t = 0, 8,
and 24 h at the same position of each well. Photos were taken with a
laser confocal microscope (Leica SPE) with 10× magnification. Images
were generated by using the LAS AF Lite software, and the scale bar is
250 μm. The area of the wounds was measured by using the ImageJ
software based on bright field at each time point. In general, the
thresholds were adjusted, and the background was set to black first.
Then, the filled cells were highlighted from the images by intensity
through setting the filters. Finally, the outlines were drawn manually,
and the areas of wounds were measured. Wound closure ratio (%) =
[(original wound area − wound area at t)/original wound area] ×
100%. Data were calculated from three to four replicates.
Invasion Inhibition by Transwell Invasion Assay. Assay
preparation: MDA-MD-231 cells were starved in a serum-free medium
for 12−16 h before use. Transwell insert wells (Corning #3422, 6.5
mm diameter inserts, and 8.0 μm pore size) were precoated with
Matrigel 200−300 μg/mL and 100 μL/insert. After coating with
Matrigel, the inset wells were hydrated with 0.1 mL of serum-free
medium for 1 h. After harvesting by trypsinization, cells were washed
twice with serum-free medium and suspended in serum-free medium
(control group) or serum-free medium containing complex 5. DMF
was used as a supporting solvent, and the final concentration was 0.5%.
Cells with 0.5% DMF were set as a control. A 100 μL cell suspension
containing 50 000 cells was added to each insert. A total of 600 μL of
medium with 10% FBS as chemoattractant was added to the receiver
wells. The inserts were placed into the receiver wells gently without
making bubbles (Figure S25). Then, cells were incubated at 37 °C
under 5% CO2 for 24 h. After removing the medium, the inset wells
were removed and generally washed with PBS twice. Cells on the
upside of the insets were removed by cotton swabs and the insets
washed with PBS twice. Then, cells on the lower side of the inset wells
were fixed with 1.1% (w/v) glutaraldehyde for 15 min at room
temperature, followed by PBS washing two times and air-drying.
Subsequently, cells were stained with 0.1% crystal violet in 200 mM
boric acid solution for 20 min. After staining, cells were washed with
PBS twice and air-dried. Photos were taken by a Leica DMI3000 B
inverted microscope with 20× magnification, and the scale bar is 75
μm. Finally, the crystal violet was dissolved in 10% acetic acid (600
μL/well, 15 min with gently shaking at r.t.). Cell invasion ratios were
calculated according to the absorbance at 590 nm. Data were
calculated from two to three replicates.
In Vivo Toxicity Test by Using Zebrafish Embryos. The
embryos were obtained by random pairwise mating of wild-type adult
zebrafish (Danio rerio), which were maintained in aquaria under
standard laboratory conditions (at 28 ± 1 °C under a cycle of 14 h
light, 10 h dark). After collection, the eggs would be transferred to 9
cm Petri dishes containing 0.1 Hanks’ Balanced Salt Solution 30 (0.1
HBSS) at pH 7.46 (egg water) according to standard practices.
Zebrafish embryos were incubated in 24-well plates with 1 mL
solutions containing different concentrations (0, 15, 30, 45, 60, 75, and
90 μM) of cisplatin or complex 5 in egg water at 28 ± 1 °C. No
supporting solvent was used in this experiment.15 Embryos were used
per concentration, and four replicates from two independent
experiments were carried out. The hatching and growth of the
zebrafish embryos without and with cisplatin and complex 5 were
monitored every 24 h with an inverted microscope (Olympus IX81)
equipped with a cooled sCMOS camera (Neo, ANDOR). All animal
work was carried out with prior approval from the animal ethical
committee of City University of Hong Kong and was in accordance
with local animal care guidelines.
■
ASSOCIATED CONTENT
* Supporting Information
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorgchem.8b00053.
Figures S1−S25, Tables S1 and S2 (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: guangzhu@cityu.edu.hk.
ORCID
Peng Shi: 0000-0003-0629-4161
Guangyu Zhu: 0000-0002-4710-7070
Author Contributions
The manuscript was written through contributions of L.M.,
X.L., and G.Z. All authors have given approval to the final
version of the manuscript.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
We thank the National Natural Science Foundation of China
(Grant No. 21371145) and the City University of Hong Kong
(Projects 7004656, 9667131, and 9667148) for funding
support.
■
REFERENCES
(1) Kelland, L. The resurgence of platinum-based cancer chemotherapy. Nat. Rev. Cancer 2007, 7, 573−584.
(2) Burger, H.; Loos, W. J.; Eechoute, K.; Verweij, J.; Mathijssen, R.
H. J.; Wiemer, E. A. C. Drug transporters of platinum-based anticancer
agents and their clinical significance. Drug Resist. Updates 2011, 14,
22−34.
(3) Johnstone, T. C.; Suntharalingam, K.; Lippard, S. J. The Next
Generation of Platinum Drugs: Targeted Pt(II) Agents, Nanoparticle
Delivery, and Pt(IV) Prodrugs. Chem. Rev. 2016, 116, 3436−3486.
(4) Wilson, J. J.; Lippard, S. J. Synthetic methods for the preparation
of platinum anticancer complexes. Chem. Rev. 2014, 114, 4470−4495.
(5) Basu, U.; Banik, B.; Wen, R.; Pathak, R. K.; Dhar, S. The platin-X
series: activation, targeting, and delivery. Dalton Trans. 2016, 45,
12992−13004.
(6) Muhammad, N.; Guo, Z. Metal-based anticancer chemotherapeutic agents. Curr. Opin. Chem. Biol. 2014, 19, 144−153.
(7) Zhang, P.; Sadler, P. J. Redox-active metal complexes for
anticancer therapy. Eur. J. Inorg. Chem. 2017, 2017, 1541−1548.
(8) Alessio, E. Thirty years of the drug candidate NAMI-A and the
myths in the field of ruthenium anticancer compounds: a personal
perspective. Eur. J. Inorg. Chem. 2017, 2017, 1549−1560.
(9) Leijen, S.; Burgers, S. A.; Baas, P.; Pluim, D.; Tibben, M.; van
Werkhoven, E.; Alessio, E.; Sava, G.; Beijnen, J. H.; Schellens, J. H. M.
G
DOI: 10.1021/acs.inorgchem.8b00053
Inorg. Chem. XXXX, XXX, XXX−XXX
Article
Inorganic Chemistry
Phase I/II study with ruthenium compound NAMI-A and gemcitabine
in patients with non-small cell lung cancer after first line therapy.
Invest. New Drugs 2015, 33, 201−214.
(10) Bergamo, A.; Sava, G. Linking the future of anticancer metalcomplexes to the therapy of tumour metastases. Chem. Soc. Rev. 2015,
44, 8818−8835.
(11) Galanski, M.; Jakupec, M. A.; Keppler, B. K. Update of the
preclinical situation of anticancer platinum complexes: novel design
strategies and innovative analytical approaches. Curr. Med. Chem.
2005, 12, 2075−2094.
(12) Jovanovic, S.; Obrencevic, K.; Bugarcic, Z. D.; Popovic, I.;
Zakula, J.; Petrovic, B. New bimetallic palladium(II) and platinum(II)
complexes: studies of the nucleophilic substitution reactions,
interactions with CT-DNA, bovine serum albumin and cytotoxic
activity. Dalton Trans. 2016, 45, 12444−12457.
(13) Ramu, V.; Gill, M. R.; Jarman, P. J.; Turton, D.; Thomas, J. A.;
Das, A.; Smythe, C. A cytostatic ruthenium(II)−platinum(II)
bis(terpyridyl) anticancer complex that blocks entry into Sphase by
up-regulating p27KIP1. Chem. - Eur. J. 2015, 21, 9185−9197.
(14) van der Schilden, K.; Garcìa, F.; Kooijman, H.; Spek, A. L.;
Haasnoot, J. G.; Reedijk, J. A highly flexible dinuclear ruthenium(II)−
platinum(II) complex: Crystal structure and binding to 9-ethylguanine.
Angew. Chem., Int. Ed. 2004, 43, 5668−5670.
(15) Miao, R.; Mongelli, M. T.; Zigler, D. F.; Winkel, B. S. J.; Brewer,
K. J. A multifunctional tetrametallic Ru−Pt supramolecular complex
exhibiting both DNA binding and photocleavage. Inorg. Chem. 2006,
45, 10413−10415.
(16) Prussin, A. J., II; Zhao, S.; Jain, A.; Winkel, B. S. J.; Brewer, K. J.
DNA interaction studies of tridentate bridged Ru(II)−Pt(II) mixedmetal supramolecules. J. Inorg. Biochem. 2009, 103, 427−431.
(17) Higgins, S. L. H.; White, T. A.; Winkel, B. S. J.; Brewer, K. J.
Redox, spectroscopic, and photophysical properties of Ru−Pt mixedmetal complexes incorporating 4,7-diphenyl-1,10-phenanthroline as
efficient DNA binding and photocleaving agents. Inorg. Chem. 2011,
50, 463−470.
(18) Jain, A.; Winkel, B. S. J.; Brewer, K. J. In vivo inhibition of E. coli
growth by a Ru(II)/Pt(II) supramolecule [(tpy)RuCl(dpp)PtCl2](PF6). J. Inorg. Biochem. 2007, 101, 1525−1528.
(19) Herman, A.; Tanski, J. M.; Tibbetts, M. F.; Anderson, C. M.
Synthesis, characterization, and in vitro evaluation of a potentially
selective anticancer, mixed-metal [ruthenium(III)−platinum(II)]
trinuclear complex. Inorg. Chem. 2008, 47, 274−280.
(20) Anderson, C. M.; Taylor, I. R.; Tibbetts, M. F.; Philpott, J.; Hu,
Y.; Tanski, J. M. Hetero-multinuclear ruthenium(III)/platinum(II)
complexes that potentially exhibit both antimetastatic and antineoplastic properties. Inorg. Chem. 2012, 51, 12917−12924.
(21) Ma, L.; Ma, R.; Wang, Z.; Yiu, S.-M.; Zhu, G. Heterodinuclear
Pt(IV)-Ru(II) anticancer prodrugs to combat both drug resistance and
tumor metastasis. Chem. Commun. 2016, 52, 10735−10738.
(22) Ang, W. H.; De Luca, A.; Chapuis-Bernasconi, C.; JuilleratJeanneret, L.; Lo Bello, M.; Dyson, P. J. Organometallic ruthenium
inhibitors of glutathione-S-transferase P1−1 as anticancer drugs.
ChemMedChem 2007, 2, 1799−1806.
(23) Wexselblatt, E.; Raveendran, R.; Salameh, S.; Friedman-Ezra, A.;
Yavin, E.; Gibson, D. On the stability of PtIV pro-drugs with
haloacetato ligands in the axial positions. Chem. - Eur. J. 2015, 21,
3108−3114.
(24) Wang, Z.; Xu, Z.; Zhu, G. A platinum(IV) anticancer prodrug
targeting nucleotide excision repair to overcome cisplatin resistance.
Angew. Chem., Int. Ed. 2016, 55, 15564−15568.
(25) Raveendran, R.; Braude, J. P.; Wexselblatt, E.; Novohradsky, V.;
Stuchlikova, O.; Brabec, V.; Gandin, V.; Gibson, D. Pt(IV) derivatives
of cisplatin and oxaliplatin with phenylbutyrate axial ligands are potent
cytotoxic agents that act by several mechanisms of action. Chem. Sci.
2016, 7, 2381−2391.
(26) Liang, X.-J.; Meng, H.; Wang, Y.; He, H.; Meng, J.; Lu, J.; Wang,
P. C.; Zhao, Y.; Gao, X.; Sun, B.; Chen, C.; Xing, G.; Shen, D.;
Gottesman, M. M.; Wu, Y.; Yin, J.-j.; Jia, L. Metallofullerene
nanoparticles circumvent tumor resistance to cisplatin by reactivating
endocytosis. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 7449−7454.
(27) Dhar, S.; Lippard, S. J. Mitaplatin, a potent fusion of cisplatin
and the orphan drug dichloroacetate. Proc. Natl. Acad. Sci. U. S. A.
2009, 106, 22199−22204.
(28) Dyson, P. J.; Sava, G. Metal-based antitumour drugs in the post
genomic era. Dalton Trans. 2006, 1929−1933.
(29) Gupta, G. P.; Massagué, J. Cancer metastasis: building a
framework. Cell 2006, 127, 679−695.
(30) Muller, A.; Homey, B.; Soto, H.; Ge, N.; Catron, D.; Buchanan,
M. E.; McClanahan, T.; Murphy, E.; Yuan, W.; Wagner, S. N.; Barrera,
J. L.; Mohar, A.; Verastegui, E.; Zlotnik, A. Involvement of chemokine
receptors in breast cancer metastasis. Nature 2001, 410, 50−56.
(31) Wan, L.; Pantel, K.; Kang, Y. Tumor metastasis: moving new
biological insights into the clinic. Nat. Med. 2013, 19, 1450−1464.
(32) Valster, A.; Tran, N. L.; Nakada, M.; Berens, M. E.; Chan, A. Y.;
Symons, M. Cell migration and invasion assays. Methods 2005, 37,
208−215.
(33) Albini, A.; Iwamoto, Y.; Kleinman, H. K.; Martin, G. R.;
Aaronson, S. A.; Kozlowski, J. M.; McEwan, R. N. A rapid in vitro assay
for quantitating the invasive potential of tumor cells. Cancer Res. 1987,
47, 3239−3245.
(34) Zon, L. I.; Peterson, R. T. In vivo drug discovery in the
zebrafish. Nat. Rev. Drug Discovery 2005, 4, 35−44.
(35) Lenis-Rojas, O. A.; Fernandes, A. R.; Roma-Rodrigues, C.;
Baptista, P. V.; Marques, F.; Perez-Fernandez, D.; Guerra-Varela, J.;
Sanchez, L.; Vazquez-Garcia, D.; Torres, M. L.; Fernandez, A.;
Fernandez, J. J. Heteroleptic mononuclear compounds of ruthenium(II): synthesis, structural analyses, in vitro antitumor activity and in
vivo toxicity on zebrafish embryos. Dalton Trans. 2016, 45, 19127−
19140.
(36) Wu, Q.; Zheng, K.; Liao, S.; Ding, Y.; Li, Y.; Mei, W. Arene
ruthenium(II) complexes as low-toxicity inhibitor against the
proliferation, migration, and invasion of MDA-MB-231 cells through
binding and stabilizing c-myc G-quadruplex DNA. Organometallics
2016, 35, 317−326.
H
DOI: 10.1021/acs.inorgchem.8b00053
Inorg. Chem. XXXX, XXX, XXX−XXX