Monochalcoplatin: An Actively Transported, Quickly Reducible, and Highly Potent PtIV Anticancer Prodrug

Article abstract of DOI:10.1002/anie.201804314

Recently, Pt^IV prodrugs have attracted much attention as the next generation of platinum-based antineoplastic drug candidates. Here we report the discovery and evaluation of monochalcoplatin, a monocarboxylated Pt^IV prodrug that is among the most cytotoxic Pt^IV prodrugs to date. Compared with its dicarboxylated counterpart chalcoplatin, monochalcoplatin accumulates astonishingly effectively and rapidly in cancer cells, which is not ascribed to its lipophilicity. The prodrug is quickly reduced, causes DNA damage, and induces apoptosis, resulting in superior cytotoxicity with IC₅₀ values in the nanomolar range in both cisplatin-sensitive and -resistant cells; these IC₅₀ values are up to 422-fold higher than that of cisplatin. A detailed mechanistic study reveals that monochalcoplatin actively enters cells through a transporter-mediated process. Moreover, monochalcoplatin shows significant antitumor activity in an in vivo colorectal tumor model. Our study implies a practical strategy for the design of more effective Pt^IV prodrugs to conquer drug resistance by tuning both cellular uptake pathways and activation processes.

Full text of DOI:10.1002/anie.201804314

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Accepted Article
Title: Monochalcoplatin: an actively transported, quickly reducible, and
highly potent Pt(IV) anticancer prodrug
Authors: Lili Ma, Wang Na, Rong Ma, Cai Li, Man-Kit Tse, and
Guangyu Zhu
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201804314
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10.1002/anie.201804314
Angewandte Chemie International Edition
COMMUNICATION
Monochalcoplatin: an actively transported, quickly reducible, and
highly potent Pt(IV) anticancer prodrug
Lili Ma, Na Wang,‡ Rong Ma,‡ Cai Li, Zoufeng Xu, Man-Kit Tse, and Guangyu Zhu*
Abstract: Recently, Pt(IV) prodrugs have attracted much attention as
the next generation of platinum-based antineoplastic drug candidates.
Here we report the discovery and evaluation of monochalcoplatin, a
monocarboxylated Pt(IV) prodrug that is among the most cytotoxic
Pt(IV) prodrugs to date. Compared with its dicarboxylated counterpart
chalcoplatin, monochalcoplatin astonishingly effectively and rapidly
accumulates in cancer cells, which is not ascribed to its lipophilicity.
The prodrug is quickly reduced, causes DNA damage, and induces
apoptosis, resulting in superior cytotoxicity with IC50 values in the
nanomolar range in both cisplatin-sensitive and -resistant cells, which
is up to 422-fold higher than cisplatin. A detailed mechanistic study
pathways. The prodrug displays a distinctive mode of action,
further indicating the role of the p53 agonist.
reveals that monochalcoplatin actively enters cells through
a
transporter-mediated process. Moreover, monochalcoplatin shows
significant antitumor activity in an in vivo colorectal tumor model. Our
study implies a practical strategy for the design of more effective
Pt(IV) prodrugs to conquer drug resistance by tuning both cellular
uptake pathways and activation processes.
Figure 1. Chemical structures of compounds 1-6.
Among non-conventional platinum-based anticancer agents that
do not obey the original “structure-activity relationships” of
cisplatin, Pt(IV) prodrugs have recently shown significantly
promising antineoplastic activity.[1] The octahedral geometry of
the Pt(IV) center endows the metal with inertness before entering
cells, and the reducing environment within cancer but not normal
cells allows for the efficient activation of the prodrugs, releasing
the reduced Pt(II) moiety to induce DNA damage.[2] These
properties make these prodrugs able to have reduced side effects,
to achieve oral availability, and to overcome cisplatin resistance.
Previous inspiring work on Pt(IV) prodrugs mainly focused on the
modification of axial ligands to tune lipophilicity or kinetics, the
application of drug carriers to target cancer cells, and the
generation of dual- or multi-targeting Pt(IV) complexes using
bioactive moieties to achieve enhanced pharmacological
effects.[2c, 3]
In the process of obtaining novel platinum anticancer agents,
we have synthesized and biologically evaluated chalcoplatin, a
“dual-targeting” Pt(IV) prodrug containing two moieties of
chalcone, which is an inhibitor of p53-MDM2 interaction, in the
axial position (Figure 1).[4] Compared to cisplatin, chalcoplatin
shows elevated cytotoxicity in the micromolar range in p53 wild-
type human cancer cells, and this Pt(IV) prodrug enters cells more
efficiently than cisplatin, likely due to its lipophilicity. Chalcoplatin
significantly stabilizes p53 and triggers downstream apoptotic
Although different Pt(IV) prodrugs with bioactive axial ligands
have been prepared, few efforts have been put forward to design
Pt(IV) prodrugs with transporter-mediated cellular uptake
mechanisms and instant activation processes. Here we report our
discovery that monochalcoplatin, a monocarboxylated Pt(IV)
prodrug, but not chalcoplatin or other analogues, is
actively/facilitated transported into cells and rapidly reduced,
which leads to significant cell death in a short time frame. Our
finding points to the importance of designing small-molecule non-
functionalized Pt(IV) prodrugs with unique structures that can be
actively transported into cells and subsequently quickly activated,
which lies in the first two steps of their mechanism of action. By
tackling these factors, downstream processes including DNA
damage and cellular responses are strengthened and the
anticancer efficacy especially in cisplatin-refractory tumor is
dramatically enhanced.
We first synthesized monochalcoplatin as well as several other
chalcoplatin analogues (Figures 1). As the building block of
chalcone, 4-formylphenoxyacetic acid was used as the axial
ligand as well. To synthesize monocarboxylated Pt(IV)
compounds 1, 3, and 5, the carboxyl groups of ligands were
activated by 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide
(EDCI)/N-hydroxysuccinimide (NHS) to yield NHS esters, which
subsequently
reacted
with
c,c,t-[Pt(NH3)2Cl2(OH)2]
or
[Pt(DACH)(ox)(OH)2] (Figures S1 and S2).[2b] The dicarboxylated
Pt(IV) compounds 4 and 6 were obtained as well (Figure S2).
These compounds were fully characterized by 1H, 13C, 195Pt NMR,
ESI-MS, and CHN elemental analysis (Figures S4-S22). The
stability of compounds 1–6 in PBS was confirmed by RP-HPLC
(Figure S23).
Cytotoxicities of these Pt(IV) compounds were first screened in
A2780 cisplatin-sensitive and A2780cisR cisplatin-resistant
ovarian human cancer cells (Table S1). Oxaliplatin-based Pt(IV)
compounds are significantly less active than their corresponding
[*]
Dr. L. Ma, N. Wang, R. Ma, C. Li, Z. Xu, Dr. M.-K. Tse, Prof. Dr. G
Zhu
Department of Chemistry, City University of Hong Kong
83 Tat Chee Ave, Kowloon Tong, Hong Kong SAR (P. R. China)
Email: guangzhu@cityu.edu.hk
Dr. L. Ma, N. Wang, C. Li, Z. Xu, Prof. Dr. G Zhu
City University of Hong Kong Shenzhen Research Institute,
Shenzhen (P.R. China)
‡ These authors contribute equally to this work
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201804314
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COMMUNICATION
cisplatin-based ones. For cisplatin-based scaffolds, the
dicarboxylated Pt(IV) complexes are less potent than their
monocarboxylated counterparts, no matter the axial ligand is
chalcone or 4-formylphenoxyacetic acid. Among these
compounds, monochalcoplatin displays the highest cytotoxicities.
We, therefore, focused on this monocarboxylated Pt(IV) complex
in the following tests.
A detailed cytotoxicity test of cisplatin, chalcoplatin, and
monochalcoplatin was performed. Besides A2780 and A2780cisR
cells, other p53 wild-type human cancer cells including A549,
MCF-7, HCT116, and HeLa were utilized. Cisplatin-resistant
A549cisR cells were also included. The half maximal inhibitory
concentration (IC50) values of cisplatin for 72 h treatment are in
the typical micromolar range in the cisplatin-sensitive cell lines,
and the drug is not significantly active in the resistant cell lines
(Table 1). Chalcoplatin is more active than cisplatin in most of the
cells tested, although its IC50 values are still in the micromolar
range. Monochalcoplatin is significantly more active than both
cisplatin and chalcoplatin, and the IC50 values of
monochalcoplatin for 1 h, the Pt levels in genomic DNA are 13 ±
3 and 19 ± 3 ng Pt/106 cells in A2780 and A2780cisR cells,
respectively. To further reveal whether monochalcoplatin was
able to cause DNA damage within a short time, the expression
levels of γH2A.X were examined by immunoblotting (Figure S27).
H2A.X plays a key role in DNA repair, and phosphorylation of Ser
139 will occur to form γH2A.X rapidly after the formation of DNA
double strand breaks (DSBs).[6] Compared to cisplatin,
monochalcoplatin evokes a remarkably increased level of γH2A.X
in A2780 cells after 4 h, and a similar tendency was observed after
6 h.
The cell-based assays mentioned above confirm that
monochalcoplatin quickly induces apoptosis and is significantly
cytotoxic but they do not reveal the reason. We next examined
reduction processes because such properties may significantly
influence the cytotoxicity of Pt(IV) prodrugs. Cyclic voltammetry
measurement shows that monochalcoplatin and chalcoplatin
have Ep values of -0.85 V and -0.73 V (vs. Fc+/Fc= +0.05 V),
respectively (Figure S28).[4] The similar and moderately low
monochalcoplatin are in the nanomolar range in all the cells tested. reduction potentials of monochalcoplatin and chalcoplatin imply
For example, the IC50 values of chalcoplatin in A2780 and A549
cells are 830 and 1900 nM, respectively, while those of
monochalcoplatin are as low as 10 and 80 nM, respectively.
Compared with cisplatin, monochalcoplatin displays up to a 422-
fold increase in cytotoxicity in the cells tested. Thus,
monochalcoplatin is among the most cytotoxic Pt(IV) prodrugs to
date. Furthermore, monochalcoplatin is able to overcome
cisplatin resistance, and the IC50 values are 70 and 140 nM in
A2780cisR and A549cisR cells, respectively. In A549 and
A549cisR cells, the resistant factors (RF), defined as the ratio of
the IC50 value in cisplatin-resistant cells to that in cisplatin-
sensitive cells, is 4.3 for cisplatin, and the value decreases to 1.8
for monochalcoplatin. A similar trend is also found in A2780 and
A2780cisR cells (Table 1). Although monochalcoplatin shows
striking cytotoxicities in cancer cells, the selectivity index (SI) of
monochalcoplatin is comparable to that of cisplatin. Additionally,
monochalcoplatin exhibits excellent cell-killing effect in a 3-D
spheroid model generated from MCF-7 cells (Figure S24).
that they are not easily reduced before their cellular entrance.
Although chalcoplatin and monochalcoplatin have similar
reduction potentials, their activation process may not be exactly
the same. Indeed, the reduction speed is also a factor mediating
the anticancer activity of Pt(IV) prodrugs.[7] We assume that
monochalcoplatin may be quickly reduced from Pt(IV) to Pt(II)
inside the cells to execute its DNA-damaging function. To
corroborate our hypothesis, the Pt(IV) prodrugs were incubated in
and without the presence of sodium ascorbate (NaAs), a cellular
reducing agent, and the fractions of intact Pt(IV) prodrug were
quantified by RP-HPLC (Figures 2 and S29). Both complexes are
stable without NaAs, and chalcoplatin is slowly reduced in the
presence of NaAs. In striking contrast, monochalcoplatin is
reduced very quickly under this condition. After only 2 h, the
percentage of Pt(IV) form remained drops to 71%, and the value
decreases to 50% after 6 h. This instant activation process may
contribute to monochalcoplatin’s quick mode of action, although
there is a risk of reduction in the blood stream.
The ability of monochalcoplatin to induce apoptosis and to bind
to nucleotides was subsequently examined. Monochalcoplatin,
but not cisplatin and chalcoplatin, was able to induce apoptosis
after 6 h (Figure S25).[5] After the treatment of 10 μM
Table 1. IC50 values (nM) of Pt compounds. Cells were treated for 72 h and
cell viability was determined by MTT assay.
Cell lines
cisplatin
chalcoplatin
mono-
chalcoplatin
10 ± 3
70 ± 10
7.0
80 ± 10
140 ± 10
1.8
240 ± 8
23 ± 6
FI[a]
A2780
A2780cisR
RF[b]
A549
A549cisR
RF
1320 ± 490
17425 ± 4375
12
1380 ± 160
5880 ± 420
4.3
830 ± 570
1590 ± 250
1.9
132
249
----
17
42
----
75
422
50
23
64
----
1900 ± 700[d]
1600 ± 500[d]
0.84
Figure 2. RP-HPLC analysis of remaining Pt(IV) complexes. 50 μM Pt(IV)
complexes were incubated in and without the presence of 2 mM sodium
ascorbate in a solution of PBS/MeOH (9/1, V/V, pH 7.4) at 37 °C in dark.
MCF-7
HCT116
HeLa
18020 ± 400
9703 ± 793
9080 ± 3200
2760 ± 420
3220 ± 800
2
4800 ± 1000[d]
800 ± 400[d]
1500 ± 800[d]
9000 ± 400[d]
620 ± 200
4.7
180 ± 10
120 ± 20
50 ± 20
1.5
MRC-5
WI38
Another common reason for improved cytotoxicity of a Pt drug
is increased cellular uptake. The LogPo/w values of chalcoplatin
and monochalcoplatin are determined to be 1.39 ± 0.06 and 1.29
± 0.03, respectively. The monocarboxylated Pt(IV) prodrug is
slightly less lipophilic than its dicarboxylated counterpart due to
SI[c]
[a] FI (fold increase) is defined as IC50(cisplatin)/IC50(monochalcoplatin)
[b] RF (resistant factor) is defined as IC50 in A2780CisR/IC50 in A2780 or
as IC50 in A549cisR/IC50 in A549
[c] SI (selectivity index) is defined as IC50 in MRC-5/IC50 in A549
[d] Data were cited from Chem. Commun., 2015, 51, 6301
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10.1002/anie.201804314
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COMMUNICATION
monosubstitution and the remaining hydroxyl group on the Pt(IV)
moiety,[8] indicating that the cellular entrance of monochalcoplatin
by just passive diffusion may not be as efficient as that of
chalcoplatin. Next, Pt accumulation in A2780 and A2780cisR cells
upon treatment with 10 µM Pt compounds for 6 h was studied
(Figure 3A, B). Cisplatin treatment results in 5.2 and 2.1 ng Pt/106
cells in A2780 and A2780cisR cells, respectively, and the
numbers increase to 18.1 and 9.9 ng Pt/106 cells, respectively, for
chalcoplatin-treated cells. These results are consistent with our
previous work under the same condition.[4] In stark contrast,
monochalcoplatin accumulates much more efficiently. The levels
of Pt are 189 and 229 ng Pt/106 cells in A2780 and A2780cisR
cells, respectively, showing a 36 and 111-fold increase over
cisplatin. A similar trend is also observed in A549 cells (Figure
S30), and the concentration of Pt inside the cells is estimated to
be 2.4 mM based on the cell volume,[9] which is much higher than
the treatment concentration, indicating an active transport
process. These levels are among the highest for currently known
Pt(IV) prodrugs in the same cell lines in a similar time frame.[2c, 10]
This greatly enhanced accumulation of monochalcoplatin might
be partially related to the rapid reduction and subsequent
aquation processes that lead to Pt trapping. This significantly
elevated cellular levels of Pt are believed to be one of the major
reasons for monochalcoplatin’s remarkable cytotoxicity.
Figure 3. Cellular accumulation of cDDP, chalcoplatin, and monochalcoplatin.
(A) A2780 cells, 6 h treatment; (B) A2780cisR cells, 6 h treatment; (C) A2780
cells, 1 h treatment; (D) A2780cisR cells, 1 h treatment. **, p< 0.01; ***, p< 0.001.
Further investigation was undertaken to reveal the possible
cellular entrance pathways of monochalcoplatin. The compound
shares similar lipophilicity with chalcoplatin but the accumulation
is astonishingly higher, showing that monochalcoplatin may enter
cells via energy-dependent pathways including active transport
and endocytosis. To investigate these possibilities, A2780 and
A2780cisR cells were treated with 10 µM chalcoplatin or
monochalcoplatin for 1 h at either 37 °C or 4 °C and Pt levels were
assessed. Statistically significantly decreased Pt levels were
found at 4 °C compared to 37 °C, indicating the role of energy-
dependent transport.[11] For example, the Pt levels in
monochalcoplatin-treated A2780cisR cells are 86 ± 23 and 5.6 ±
1.0 ng Pt/106 cells at 37 °C and 4 °C, respectively (Figure 3C, D).
The possibility of endocytosis and macropinocytosis was
subsequently determined, as these processes are responsible for
the uptake of certain types of metal complexes.[12] Pretreatment
with wortmannin (inhibition of endocytosis and macropinocytosis
by blocking phosphoinositide 3-kinase)[13] does not significantly
alter the level of Pt accumulation for chalcoplatin or
monochalcoplatin. These results indicate that active transport but
not passive diffusion or endocytosis/macropinocytosis is the
primary uptake route for monochalcoplatin. Our initial experiment
using copper transporter 1 (Ctr1)-knockout cells has shown that
Ctr1 is not responsible for the uptake of monochalcoplatin (Figure
S31), although it plays roles in the cellular accumulation of Pt in
cisplatin.[14] We also measured the cellular accumulation of
oxaliplatin-based Pt(IV) compounds bearing one or two chalcone
axial ligands (compounds 7 and 8, Figure S3). These mono- and
di-carboxylated Pt(IV) compounds have similar levels of Pt
accumulation in A2780 cells, which are significantly lower than
that of monochalcoplatin (Figure S32). Thus, the remarkable
cellular accumulation of monochalcoplatin is more likely due the
unique structure of the entire molecule.
Figure 4. The growth of HCT116 tumor in xenograft model with different
treatment. The xenograft tumor model was established in BALB/c nude mice by
subcutaneous injecting HCT116 colon carcinoma cells. The indicated
compounds were injected intravenously (i.v.) every two days for 4 times totally,
including monochalcoplatin (0.72 mg-Pt/kg), cisplatin (0.95 mg-Pt/kg), and
vehicle (1% DMF and 1% Tween-80 in 0.9% NaCl) as control. Mean ± SD, n=
5. **, p< 0.01. Student’s t-Test.
The antitumor efficacy of monochalcoplatin in vivo was further
demonstrated. The in vitro results show that monochalcoplatin
has significantly higher cytotoxicity than cisplatin in HCT116 cells.
Therefore, a xenograft tumor model was established in BALB/c
nude mice by subcutaneous injecting HCT116 colon carcinoma
cells. Each of five HCT116-transplanted mice received vehicle,
cisplatin (0.95 mg-Pt/kg), or monochalcoplatin (0.72 mg-Pt/kg)
intravenously (i.v.) every two days for total four treatments.
Compared to the control group, cisplatin-treated group has no
significant difference in tumor volume. On day 27, the average
tumor volume is 993.9 ± 367.6 mm3 upon the treatment of cisplatin,
and the value is 1617.0 ± 596.7 mm3 for the control group.
Remarkably, monochalcoplatin effectively suppresses the tumor
growth. The average tumor volume in monochalcoplatin-treated
group after 27 days is 320.2 ± 91.7 mm3, which is 32% of that in
cisplatin-treated group (p< 0.01), and 20% of that in the control
group (p< 0.01) (Figure 4). After termination of the experiments,
the tumor of each animal was collected and the weight was
recorded (Figure S33). Upon the treatment of monochalcoplatin,
the tumor weight decreased significantly, which is 33% and 25%
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Scheme 1. Proposed cellular entrance pathway and reduction process of
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Acknowledgements
We thank the National Natural Science Foundation of China
(Grant No. 21371145) and the City University of Hong Kong
(Projects 9667148, 9667131) for funding support.
Keywords: Pt(IV) prodrugs • cisplatin • active transport •
antitumor activity • prodrug activation
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10.1002/anie.201804314
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COMMUNICATION
A monocarboxylated
Pt(IV) prodrug is actively
transported into cells and
reduced promptly,
Lili Ma, Na Wang,‡ Rong Ma,‡
Cai Li, Zoufeng Xu, Man-Kit Tse,
and Guangyu Zhu*
Page No. – Page No.
resulting in nanomolar
range IC50 values in vitro
and effective tumor
Monochalcoplatin: an actively
transported, quickly reducible,
and highly potent Pt(IV)
anticancer prodrug
growth inhibition in vivo.
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