Phorbiplatin, a Highly Potent Pt(IV) Antitumor Prodrug That Can Be Controllably Activated by Red Light

Article abstract of DOI:10.1016/j.chempr.2019.08.021

Selective activation of prodrugs within a tumor is particularly attractive because of their low damage to normal tissue. Here, we report the design, photoactivation mechanism, and antitumor activity of a red-light-activatable Pt(IV) prodrug based on oxaliplatin, a first-line clinical antineoplastic. This small-molecule prodrug, designated as phorbiplatin, has controllable activation property: it is shown to be inert in the dark but under short-period irradiation with low intensity of red light (7 mW/cm²), without the need of any external catalyst, phorbiplatin is rapidly reduced to oxaliplatin. The prodrug displays photocytotoxicity that is up to 1,786 times greater than that of oxaliplatin in human carcinoma cells, and it is also significantly active in vivo. The controllable activation property and superior antitumor activity of phorbiplatin may suggest a novel strategy for the design of visible light-activatable platinum prodrugs to reduce the adverse effects and conquer drug resistance of traditional platinum chemotherapy. Currently, most of the small-molecule anticancer drugs used in clinics do not have controllable activation properties, leading to undesired side effects. Anticancer drugs with “on-site” activation properties are highly demanded. Here, we report the development of a small-molecule anticancer prodrug that can be controllably activated by a red light. The prodrug is stable in the dark even in a reducing environment and shows minimum dark toxicity to the cells. Under irradiation with low intensity of red light, the prodrug utilizes a unique photoactivation mechanism to be quickly and efficiently activated, releasing oxaliplatin, a widely used antineoplastic agent. The activated prodrug displays significantly increased cytotoxicity in human cancer cells compared with oxaliplatin, and it is able to kill tumor cells much more efficiently than oxaliplatin in a mouse tumor model. Our work significantly contributes to the development of photoactivatable anticancer prodrugs, especially by red light. We report the design, evaluation, and photoactivation mechanism of phorbiplatin, a platinum(IV) antitumor prodrug that can be controllably activated by red light. Phorbiplatin maintains its integrity without irradiation, but under irradiation with red light, the prodrug is quickly and efficiently activated, releasing oxaliplatin and PPA. The prodrug shows significant antitumor activity both in vitro and in vivo.

Full text of DOI:10.1016/j.chempr.2019.08.021

Article
Phorbiplatin, a Highly Potent Pt(IV) Antitumor
Prodrug That Can Be Controllably Activated
by Red Light
Zhigang Wang, Na Wang,
Shun-Cheung Cheng, ..., Hajime
Hirao, Chi-Chiu Ko, Guangyu
Zhu
wangzg@szu.edu.cn (Z.W.)
guangzhu@cityu.edu.hk (G.Z.)
HIGHLIGHTS
The first small-molecule Pt(IV)
prodrug that can be activated by
red light
Study on the unique
photoreduction mechanism of the
Pt(IV) prodrug
Significantly improved antitumor
activity both in vitro and in vivo
We report the design, evaluation, and photoactivation mechanism of phorbiplatin,
a platinum(IV) antitumor prodrug that can be controllably activated by red light.
Phorbiplatin maintains its integrity without irradiation, but under irradiation with
red light, the prodrug is quickly and efficiently activated, releasing oxaliplatin and
PPA. The prodrug shows significant antitumor activity both in vitro and in vivo.
Wang et al., Chem 5, 1–15
December 12, 2019 ª 2019 Elsevier Inc.
https://doi.org/10.1016/j.chempr.2019.08.021

Please cite this article in press as: Wang et al., Phorbiplatin, a Highly Potent Pt(IV) Antitumor Prodrug That Can Be Controllably Activated by Red
Light, Chem (2019), https://doi.org/10.1016/j.chempr.2019.08.021
Article
Phorbiplatin, a Highly Potent Pt(IV)
Antitumor Prodrug That Can
Be Controllably Activated by Red Light
Zhigang Wang,1,2,* Na Wang,1,4 Shun-Cheung Cheng, Kai Xu, Zhiqin Deng, Shu Chen,1,4
1
1
1,4
Zoufeng Xu,1,4 Kai Xie, Man-Kit Tse, Peng Shi, Hajime Hirao, Chi-Chiu Ko, and Guangyu Zhu1,4,5,*
3
1
3
1
1
SUMMARY
The Bigger Picture
Selective activation of prodrugs within a tumor is particularly attractive because
of their low damage to normal tissue. Here, we report the design, photoactiva-
tion mechanism, and antitumor activity of a red-light-activatable Pt(IV) prodrug
based on oxaliplatin, a first-line clinical antineoplastic. This small-molecule pro-
drug, designated as phorbiplatin, has controllable activation property: it is
shown to be inert in the dark but under short-period irradiation with low inten-
Currently, most of the small-
molecule anticancer drugs used in
clinics do not have controllable
activation properties, leading to
undesired side effects. Anticancer
drugs with ‘‘on-site’’ activation
properties are highly demanded.
Here, we report the development
of a small-molecule anticancer
prodrug that can be controllably
activated by a red light. The
prodrug is stable in the dark even
in a reducing environment and
shows minimum dark toxicity to
the cells. Under irradiation with
low intensity of red light, the
prodrug utilizes a unique
2
sity of red light (7 mW/cm ), without the need of any external catalyst, phorbi-
platin is rapidly reduced to oxaliplatin. The prodrug displays photocytotoxicity
that is up to 1,786 times greater than that of oxaliplatin in human carcinoma
cells, and it is also significantly active in vivo. The controllable activation prop-
erty and superior antitumor activity of phorbiplatin may suggest a novel strat-
egy for the design of visible light-activatable platinum prodrugs to reduce
the adverse effects and conquer drug resistance of traditional platinum
chemotherapy.
INTRODUCTION
Controllable activation of prodrugs at the tumor site has been widely explored in
cancer chemotherapy, aiming to increase the therapeutic window of conventional
photoactivation mechanism to be
quickly and efficiently activated,
releasing oxaliplatin, a widely
used antineoplastic agent. The
activated prodrug displays
1
,2
chemotherapeutics with limited tumor-targeting property. Both endogenous ac-
tivators within tumor microenvironments including overexpressed tumor-associated
3
4
5
6
enzymes, elevated levels of reactive oxygen species (ROS), hypoxia and low pH,
7
8
9
and exogenous activators including catalysts, light, and ultrasound have been uti-
lized to activate prodrugs. Among them, photoactivation is particularly attractive
because it can enable the activation of prodrugs in a high spatial and temporal
significantly increased cytotoxicity
in human cancer cells compared
with oxaliplatin, and it is able to
kill tumor cells much more
1
0
manner. In addition, modern lasers and fiber optics can deliver light to any tissue
1
1
in the body, making it a solid means for controllable and targeted cancer therapy.
efficiently than oxaliplatin in a
mouse tumor model. Our work
significantly contributes to the
development of photoactivatable
anticancer prodrugs, especially by
red light.
Despite the great success of platinum chemotherapy including cisplatin, carboplatin,
and oxaliplatin in clinics, their toxic side effects, due to non-specific activation of the
1
2–16
drugs, as well as drug resistance issues hinder their broader applications.
Devel-
opment of photoactivatable platinum-based anticancer prodrugs is highly
appealing. Efforts have been devoted to the design of Pt(IV)-based photoactivatable
1
7–20
complexes. Examples include diiodo and diazido Pt(IV) complexes.
However,
the in vivo application of these complexes is limited by slow photoreaction, low sta-
bility in physiological conditions, and/or limited activation wavelengths in the region
of UVA or blue light to efficiently activate the complexes, which has poor tissue pene-
tration depth. To extend the activation wavelength of the trans-diam(m)ine diazido
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1

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Light, Chem (2019), https://doi.org/10.1016/j.chempr.2019.08.021
Pt(IV) complexes, upconversion nanoparticles (UCNPs), which are able to convert
near-infrared (NIR) light to UV light, have been conjugated with the Pt(IV) complexes
2
1–23
to indirectly activate them.
However, the photoactivation efficiency via UCNPs is
low; the activation needs a high power laser and a long irradiation time. Small-mole-
cule platinum prodrugs that are stable in physiological conditions and can be quickly
activated by red or NIR light are highly desired.
Here, we report the design, mechanistic investigation, and antitumor activity of a
small-molecule Pt(IV) prodrug that can be activated by red light in a controllable
fashion. We have designed a photoactivatable oxaliplatin-based Pt(IV) prodrug,
taking advantage of the fact that Pt(IV) complexes have an octahedral geometry
in which two axial ligands can tune the properties of the complexes and the
2
4–26
saturated coordination sphere gives low reactivity and side effects.
This
prodrug is kinetically stable in the presence of cellular reducing agents but can
be quickly activated under low-power red-light irradiation. This prodrug, proposed
to function in a unique photoactivation mechanism, shows significant antitumor
activity both in vitro and in vivo. We believe this strategy will open a window for
the development of Pt(IV) prodrugs with controllable activation properties and
low side effects.
RESULTS AND DISCUSSION
Rational Design of a Red Light Activatable Pt(IV) Prodrug
To build up a Pt(IV) prodrug that can be efficiently activated by visible light,
especially red light, a photo-absorber with strong absorption in the red-light
region is needed to collect the energy from light, and the photo-absorber may
transfer energy to the Pt center to facilitate the reduction. Indeed, the excited
state of photo-absorbers has been shown to regulate redox reactions through
2
7,28
energy or electron transfer.
Bearing this in mind, we rationally designed a
red-light activatable Pt(IV) prodrug, phorbiplatin {[Pt(DACH)(PPA)(OH)(ox)],
DACH = (1R,2R)-1,2-diaminocyclohexane, ox = oxalate}, in which the pyropheo-
phorbide a (PPA) ligand is located at the axial position of the oxaliplatin-
based Pt(IV) prodrug (Figure 1A). PPA has the advantage of high absorbance
2
9,30
around 650 nm, with a high quantum yield of singlet oxygen.
PPA and its
derivatives have been used as photosensitizers in photodynamic therapy and
photo-catalysts in several redox reactions through electron transfer under
3
1–35
irradiation.
drugs to elicit promising anticancer effects.
chosen based on the findings that tetracarboxylato Pt(IV) compounds are
In addition, PPA derivatives have been conjugated to platinum(II)
3
6–39
The oxaliplatin scaffold was
4
0–42
generally stable toward reduction.
This prodrug supposedly exists in its
intact Pt(IV) form in the dark but is activated to release oxaliplatin and PPA under
irradiation of red light. Such a stable Pt(IV) prodrug holds promise for minimizing
the side effects that originate from non-specific activation of the prodrug by
cellular reductants.
1Department of Chemistry, City University of
Hong Kong, Hong Kong SAR, P. R. China
2
School of Pharmaceutical Sciences, Health
Science Center, Shenzhen University, Shenzhen
18060, P. R. China
5
Preparation and Characterization of Phorbiplatin
3
Department of Biomedical Engineering, City
University of Hong Kong, Hong Kong SAR, P. R.
China
The oxaliplatin-based Pt(IV) scaffold [Pt(DACH)(OH)
2
(ox)] was prepared as
previously described, and the N-hydroxysuccinimide (NHS) ester of PPA was
4City University of Hong Kong Shenzhen Research
Institute, Shenzhen 518057, P. R. China
4
3
2
obtained. The ester subsequently reacted with [Pt(DACH)(OH) (ox)] to yield
phorbiplatin (Figure S1). The purity of the prodrug is 96%, as determined by
analytical reversed-phase HPLC (RP-HPLC, Figure S2). The prodrug was
5Lead Contact
*Correspondence: wangzg@szu.edu.cn (Z.W.),
1
13
195
characterized spectroscopically by ESI-HRMS, H, C, and
and fluorescence spectroscopy (Figures S3–S8).
Pt NMR, UV-Vis,
guangzhu@cityu.edu.hk (G.Z.)
https://doi.org/10.1016/j.chempr.2019.08.021
2
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Figure 1. Chemical Structure, Stability, Activation Property, and DNA Binding Ability of Phorbiplatin
(
(
(
(
A) Chemical structure of phorbiplatin.
B) RP-HPLC (254 nm) chromatograms of cell lysate of A2780 cells treated with phorbiplatin (5 mM) for 8 h.
C) RP-HPLC (254 nm) chromatograms of oxaliplatin (50 mM); PPA (50 mM); phorbiplatin (50 mM) in the dark; phorbiplatin with 1 equiv. sodium ascorbate
2
NaAs) (50 mM) in the dark; and phorbiplatin with 1 equiv NaAs (50 mM) under irradiation (650 nm, 7 mW/cm ) for 10 min. The compounds were incubated
1
2
3
at room temperature in a PBS buffer (pH 7.4) containing 5% acetonitrile and 0.5% DMF. Peak of oxaliplatin; peak of phorbiplatin; peak of PPA.
(
D) ESI-MS (positive mode) results of phorbiplatin in the presence of NaAs under irradiation at the retention time of 1.8 min (indicated as 1 in Figure C) in
+
+
the m/z range of 390–430, corresponding to [oxaliplatin+H] , calculated at 398.06, found 398 and [oxaliplatin+Na] , calculated at 420.05, found 420.
2
(
E) Time-dependent reduction of phorbiplatin (10 mM in a PBS buffer containing 1 mM ascorbate) under irradiation (650 nm, 7 mW/cm ) or kept in the
dark. Also, see Figure S21.
F) Percentage of Pt bound to ct-DNA. Phorbiplatin or oxaliplatin (10 mM) was incubated with ct-DNA (150 mg), the mixture was kept in the dark or
(
2
ꢀ
irradiated (650 nm, 7 mW/cm , 30 min), and then the system was further incubated in the dark at 37 C for 24 h (mean G SD; n = 2).
Stability of Phorbiplatin
We first examined the stability of phorbiplatin in phosphate buffered saline (PBS,
pH = 7.4) in the dark by RP-HPLC. As shown in Figure S9, after incubation for 24 h
ꢀ
at 37 C, 96% of phorbiplatin still remained, indicating the high stability of the
compound. In the presence of 1 mM ascorbate (100 equiv.), 89% of phorbiplatin
remained after 24 h, indicating that the prodrug is quite stable in the dark, even in
the presence of the reducing agent (Figure S10). We also prepared the di-carboxy-
lato derivative of phorbiplatin for comparison. The reaction of phorbiplatin with
acetic anhydride yields acetylated phorbiplatin (Ac-phorbiplatin) (Figure S11),
1
13
195
the target compound, which was characterized by ESI-HRMS, H, C, and
Pt
NMR (Figures S12–S16). Ac-phorbiplatin was stable in PBS buffer after incubation
for 24 h (Figure S17). In the presence of ascorbate, however, 55% of Ac-phorbiplatin
was reduced after incubation for 24 h (Figure S18). Therefore, Ac-phorbiplatin was
not considered any further. The stability of phorbiplatin in the presence of another
reducing agent, 2-(N-morpholino)ethanesulfonic acid (MES) was tested, and
ꢀ
phorbiplatin also remained in its intact form after 24 h at 37 C (Figure S19). We
further tested the stability of phorbiplatin in cells. Cell lysate of A2780 human
ovarian carcinoma cells treated with phorbiplatin for 8 h was analyzed by RP-HPLC
(
Figure 1B). The ratio of the peaks representing phorbiplatin and PPA in the lysate
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is 4:1, indicating that 80% of phorbiplatin is kept intact in cells, although there is a
possibility that other forms of phorbiplatin metabolite in cells are undetectable
from HPLC.
Photoactivation Properties of Phorbiplatin
The photoactivation property of phorbiplatin was subsequently investigated.
When a solution of phorbiplatin containing ascorbate was irradiated by red light
2
at 650 nm at a low power density of 7 mW/cm , the height of the peak of phorbiplatin
in the HPLC chromatogram quickly decreased, and the peak heights for PPA and
oxaliplatin increased simultaneously, indicating the reduction of Pt(IV) to Pt(II)
along with the dissociation of the axial PPA ligand (Figure 1C). The formation of
oxaliplatin and oxidized ascorbate was further confirmed by ESI-MS (Figures 1D
and S20). After irradiation for 10 min, 81% of phorbiplatin was reduced, and most
of the complexes were reduced within 30 min (Figures 1E and S21). Irradiation of
phorbiplatin in the absence of ascorbate also resulted in the release of PPA and
oxaliplatin, although the reaction speed was much slower (Figures S22 and S23).
This effect might be because the PPA ligand itself acts as a weak reductant through
decomposition under irradiation, as the degradation products were observed in the
HPLC chromatogram as small peaks at the retention time of 4 min and 12.5 min
4
4
(
Figure S23).
Photoreduction Mechanism of Phorbiplatin
We further studied the mechanism of the photoreduction. Since no spectral overlap
was observed between the emission band of PPA (em = 671 nm) and the absorption
2
of the dihydroxido Pt(IV) compound [Pt(DACH)(OH) (ox)] (Figure S24), direct energy
transfer is not likely to occur between PPA and the Pt(IV) center. As PPA is able to
regulate redox reactions through photo-induced electron transfer (PET), conjugation
of PPA at the axial position of Pt(IV) may result in a direct electron transfer from the
excited state of PPA to the Pt(IV) center under irradiation. Using PPA in
45
dimethylformamide (DMF; F
F
= 0.31) as a reference, the quantum yield of
phorbiplatin is determined to be 0.32 in DMF, which is similar to that of PPA. In
addition, the fluorescence lifetimes of phorbiplatin and PPA in PBS buffer
containing DMF are 7.09 ns and 7.08 ns, respectively (Figure S25). The similar quantum
yield and emission lifetime suggest that the singlet excited state of PPA is not directly
involved in the reduction process, excluding the possibility of a direct electron transfer
from the singlet excited state of PPA to the Pt(IV) center under irradiation.
Given the observations that the photoreduction process of phorbiplatin is much
more efficient in the presence of ascorbate (Figures S21 and S22) and that
ascorbate is able to reduce PPA analogs to its reduced forms including p radical
4
6–50
anion under irradiation through PET,
we hypothesized that ascorbate might
first reduce the PPA moiety within phorbiplatin, and then the reduced PPA
further reduces the Pt(IV) center. To corroborate this hypothesis, we first investi-
gated the photo-induced reduction of PPA in the presence of ascorbic acid.
Degassed solutions were used to prevent the auto-oxidation of reduced
products by oxygen. Pyridine containing water was chosen as the solvent due to
4
6,51
the observation that the reduced form of PPA analogs has a long life in it.
As shown in Figure S26A, the spectra of free PPA did not change significantly
upon irradiation. Notably, in the presence of ascorbic acid, the spectra of PPA
changed rapidly under irradiation, indicating the formation of reduced products.
The differential absorbance of irradiated PPA in the presence of ascorbic acid
(
Figure S26C) is similar to that of mono-reduced PPA p radical anion obtained
from electrochemical reduction (Figure S27B), implying that mono-reduced PPA
4
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p radical anion is the main reduced product. Return to darkness led to the recovery
of the spectral changes, which is due to the back-reaction of reduced PPA with the
oxidized ascorbic acid (Figure S26D). We then studied the cyclic voltammetry of
PPA and phorbiplatin in DMF, and the redox potentials are reported versus the
+
E
1/2 of Fc /Fc. As shown in Figure 2A, the reduction potentials (E1/2) of free PPA
d
to PPA p radical anion (PPA ) and PPA p radical dianion (PPA
ꢁ
d
2ꢁ
) are ꢁ1.51
and ꢁ1.81 V, respectively. For phorbiplatin, the respective potentials associated
with the PPA moiety are slightly higher at E1/2 = ꢁ1.47 and ꢁ1.79 V, respectively.
A new irreversible reduction peak at E
p
= ꢁ1.32 V represents the reduction of
Pt(IV) (Figure 2B), which is comparable to the values reported for other mono-car-
4
0
boxylato oxaliplatin-based Pt(IV) prodrugs. By employing these data, we esti-
mated the thermodynamic driving force, DG 0P ET, for PET between the ascorbate
and the excited PPA using the well-known Rehm-Weller equation (see the electro-
chemistry studies and energetics considerations in the Supplemental Information).
The estimated DG0 values for the reactions of ascorbate with the singlet excited
PET
d
state and the triplet excited state of PPA to yield PPA are ꢁ0.64 and ꢁ0.16 eV,
ꢁ
d
respectively, indicating the PET reduction of PPA to PPA by ascorbate is thermo-
ꢁ
dynamically favorable in both its singlet and triplet excited states. The DGET of
d
ꢁ
electron transfer process from PPA
to Pt(IV) was estimated to be ꢁ0.26 eV,
indicating that the process is thermodynamically favorable as well.
We further elucidate the mechanism by using emission quenching study and
transient absorption spectroscopy. We measured the emission intensity and decay
of both PPA and phorbiplatin in the presence of ascorbate. As shown in Figure S28,
upon addition of an increased concentration of ascorbate, the emission intensity of
neither PPA nor phorbiplatin changed significantly. The emission lifetimes of PPA
and phorbiplatin in the presence of ascorbate are identical to those of free PPA
and phorbiplatin (Figure S29), respectively. These results indicate that ascorbate
does not react with the singlet excited PPA under irradiation. Thus, ascorbate
might reduce the triplet excited state of PPA under irradiation. Indeed, the reduc-
tion of porphyrin by ascorbate through its triplet excited state has been reported
5
1,52
previously.
We further measured the nanosecond transient absorption
(
ns-TA) spectra of PPA and phorbiplatin in degassed solutions. The ns-TA spectra
of PPA show ground-state bleaching at 412 nm and excited-state absorption over
the range of 440–550 nm (Figure 2C). The spectral features are consistent with the
5
3
reported triplet state absorption of PPA derivative. The decay time of excited-
state absorption at 450 nm of PPA is 6.6 ms from the single exponential fitting
(
Figure S30A). The ns-TA spectra and decay time at 450 nm of phorbiplatin are
similar to those of PPA (Figures 2D and S30B) and could account for the important
role of the PPA moiety within phorbiplatin. The ns-TA spectral features of PPA in
the presence of ascorbate are similar to those of free PPA (Figures 2E and S31).
In stark contrast, compared with free PPA and phorbiplatin, the decay time of ab-
sorption of PPA at 450 nm in the presence of ascorbate increases dramatically to
1
11 ms (Figure S30C). This long half-life species is reasonably identified as the
d
reduced form of PPA (PPA ) based on our result mentioned above that PPA
ꢁ
d
ꢁ
was formed in the presence of ascorbic acid under irradiation as well as in view
of previous reports on the spectral features of reduced porphyrins under irradiation
5
0,54,55
and the porphyrin p radical anion has prolonged decay time.
The difference
d
between PPA (Figure 2E) and the triplet excited PPA (Figure 2C) is difficult to be
d
observed in the ns-TA spectra due to the spectral overlap of PPA with the PPA
ꢁ
ꢁ
5
6
triplet absorption. The ns-TA spectrum of phorbiplatin in the presence of ascor-
bate (Figure 2F) is similar to that of PPA with ascorbate (Figure 2E), due to the pres-
ence of PPA moiety within phorbiplatin. The decay time of phorbiplatin in the
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Figure 2. Cyclic Voltammetry, Transient Absorption Spectra, and Proposed Reduction Mechanism of Phorbiplatin
(
(
(
A) Cyclic voltammogram of PPA in DMF.
+
B) Cyclic voltammogram of phorbiplatin in DMF; the data are reported versus the E1/2 of Fc /Fc.
C–F) Transient absorption spectra of 10 mM PPA, 10 mM phorbiplatin, 10 mM PPA with 1 mM ascorbate, and 10 mM phorbiplatin with 1 mM ascorbate in
PBS buffer containing 50% ACN. Also, see Figure S30. The high decreased absorption around 670 nm at time 0 is due to emission disturbance.
G) Proposed mechanism of photo-induced reduction of phorbiplatin by ascorbate. Abbreviation is as follows: dehydroascorbic acid, DHA.
(
6
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presence of ascorbate at 450 nm, however, decreased significantly to 3.0 ms
d
Figure S30D), indicating a fast reaction of the formed PPA within phorbiplatin.
ꢁ
(
d
ꢁ
We speculate that this reaction is described as an electron transfer from PPA
to the Pt(IV) center, which results in the formation of PPA and Pt(III).
Based on the evidence described above, we propose a photoactivation mechanism of
phorbiplatin, which is illustrated in Figure 2G. First, phorbiplatin (1) is excited to its
singlet excited state (2) under irradiation, and the singlet excited phorbiplatin (2)
goes to its triplet excited state (3) through intersystem crossing (ISC). The energies
of 2 and 3 with respect to 1 were estimated by time-dependent density functional the-
ory (TDDFT) calculations as 47.6 and 29.9 kcal/mol, respectively. The triplet excited
PPA moiety within phorbiplatin (3) can accept an electron from an electron donor
(
e.g., ascorbate) to generate a Pt(IV) complex containing ground state PPA p radical
d
ꢁ
d
ꢁ
anion (PPA ) (4). Then, PPA transfers one electron to the Pt(IV) center to yield a Pt(III)
complex (5) or (6) along with the disassociation of the corresponding axial ligands. The
57
formed Pt(III) complex will then be easily and rapidly reduced to a Pt(II) complex. The
proposed mechanism of action was also probed by density functional theory (DFT) cal-
culations. The relative potential energies of a few species in the reaction processes
were calculated. DE is ꢁ0.8 kcal/mol for the conversion of the Pt(IV) complex 1 to
the Pt(III) complex 5, whereas it is ꢁ3.4 kcal/mol for the reaction from 5 to the final
products. These values suggest that the reactions are thermodynamically favorable.
Nevertheless, Pt(III) will not be thermally accessible from Pt(IV), and light is needed
here. DFT calculations also suggest that the pathway via 5 is more stable than that
via 6. A di-carboxylato Pt(IV) complex is generally thought to have a higher reduction
24,58
potential (easier to be reduced) than its mono-carboxylato derivative.
Therefore,
according to our proposed mechanism, Ac-phorbiplatin, a di-carboxylato Pt(IV) com-
plex, might undergo faster photo-induced reduction than phorbiplatin through inner
sphere electron transfer. Indeed, the photoreduction rate of Ac-phorbiplatin is signif-
icantly faster than that of phorbiplatin. For example, 92% of Ac-phorbiplatin is reduced
after irradiation for only 1 min, while the value is as low as 17% for phorbiplatin (Fig-
ure S32). This information further supports the validity of our proposed mechanism
of the photoreduction process.
Photoreduction of Phorbiplatin by Other Biomolecules
Given the high oxidation ability of excited PPA, many reductants will be able to reduce
5
9
PPA under irradiation. Thus, we tested the ability of 2-(N-morpholino)ethanesulfonic
acid (MES) and glutathione (GSH) as reductants in the photoreduction reaction of phor-
biplatin. As shown in Figure S33, MES and GSH promote the reduction of phorbiplatin
0
under irradiation as expected. It has been reported that guanosine 5 -monophosphate
0
(
GMP) and adenosine 5 -monophosphate (AMP) in DNA can transfer an electron to
6
0
PPA under irradiation as well. We, therefore, co-incubated phorbiplatin with calf
thymus (ct)-DNA without other common reductants to investigate the reduction and
the subsequent binding to DNA. Theoretically, phorbiplatin is not able to bind cova-
lently to ct-DNA in the dark, and only when it is reduced to oxaliplatin, can the reduced
product bind to ct-DNA. As shown in Figure 1F, a trace amount of Pt is detected on
DNA in the dark. By contrast, under irradiation, the level of Pt on DNA is similar to
that of oxaliplatin, indicating that phorbiplatin is reduced to oxaliplatin. Therefore,
DNA serves as a reductant in the system, phorbiplatin is efficiently reduced to oxalipla-
tin, and the resultant oxaliplatin binds to DNA.
Cellular Accumulation of Phorbiplatin
The biological activity of phorbiplatin was subsequently evaluated. We first tested
the cellular accumulation of phorbiplatin by ICP-MS. Platinum accumulation in
Chem 5, 1–15, December 12, 2019
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Light, Chem (2019), https://doi.org/10.1016/j.chempr.2019.08.021
Figure 3. Cellular Accumulation, Genomic DNA Binding Property, and ROS Generation Ability of
Phorbiplatin
(
(
(
A) Time-dependent cellular accumulation of Pt in A2780 cells treated with 0.5 mM phorbiplatin
mean G SD; n = 3).
B) Confocal images of A2780 cells treated with 1 mM complexes for 8 h. The blue color corresponds
to nuclei stained by 0.2 mM hoechst 33342 (ex = 405 nm, em = 420–480 nm), and the red color is the
fluorescence of PPA or phorbiplatin (ex = 405 nm, em = 650–700 nm) (scale bar,10 mm).
(
C) Pt levels in genomic DNA of A2780 cells. The cells were treated with phorbiplatin (0.5 mM) for 8 h,
2
followed by irradiation at 650 nm (7 mW/cm ) for 15 min or in the dark, and further incubated for 8 h
before the measurement of Pt in DNA (mean G SD; n = 3).
(
(
(
2
D) Fluorescent images of H DCFDA-stained A2780 cells treated with PPA (5 mM) or phorbiplatin
2
5 mM) for 8 h followed by irradiation at 650 nm (7 mW/cm ) for 5 min (ex = 488 nm, em = 517–527 nm)
scale bar, 50 mm). Also, see Figure S35.
A2780 cells increases dramatically over incubation time before 8 h and remains
steady after 8 h (Figure 3A). Thus, an incubation time of 8 h was selected for the
following biological assays. The cellular distribution of phorbiplatin was also exam-
ined. After treatment of A2780 cells with phorbiplatin at a concentration of 1 mM for 8
h, most of the compound stays in the cytoplasm (Figure 3B).
Cytotoxicity of Phorbiplatin
We subsequently examined the cytotoxicity of phorbiplatin together with
oxaliplatin and PPA in platinum-sensitive (A2780) and platinum-resistant
(
A2780cisR) human ovarian cancer cells. Human breast cancer cells MCF-7 were
6
1
also used because of their poor response to platinum drugs. These types of cells
have been extensively applied in the evaluation of novel metal-based anticancer
6
2–64
agents.
Cells were treated with the compounds for 8 h, followed by irradiation
or incubation in the dark, and the cell viability was measured by MTT assay after 40 h.
As shown in Table 1, oxaliplatin shows IC50 values in the typical micromolar range
and is not active in A2780cisR cells. Under irradiation, the IC50 values of oxaliplatin
do not change significantly from those in the dark, indicating that irradiation has
8
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Table 1. Cytotoxicity of Oxaliplatin, PPA, and Phorbiplatin
a
Cell Line
Irradiation
IC50 (mM)
Oxaliplatin
76 G 4
FI
PPA
Phorbiplatin
>10
A2780
in the dark
>10
under irradiation
in the dark
68 G 9
0.34 G 0.05
>10
0.13 G 0.01
>10
523
974
1786
58
A2780cisR
MCF-7
162 G 9
under irradiation
in the dark
185 G 8
0.23 G 0.01
>10
0.19 G 0.01
>10
110 G 4.3
78.6 G 8.7
8.7 G 0.9
7.6 G 1.3
122 G 5.2
under irradiation
in the dark
0.20 G 0.02
>10
0.044 G 0.004
>10
4
T1
under irradiation
in the dark
0.16 G 0.02
>10
0.13 G 0.004
MRC-5
>10
2
Cells were treated with complexes for 8 h, followed by irradiation at 650 nm (7 mW/cm ) for 15 min or in
the dark. Cell viability was determined by MTT assay at 40 h after irradiation (mean G SD; n = 3).
a
FI is defined as IC50 of oxaliplatin/IC50 of phorbiplatin.
a negligible effect on the cytotoxicity of oxaliplatin. PPA shows low cytotoxicity to
the tested cells in the dark and is active under irradiation, behaving as a typical
6
5,66
photosensitizer as previously reported.
Phorbiplatin is non-toxic to the tested
cells in the dark, and the IC50 value is higher than 10 mM, suggesting the inert
properties of this Pt(IV) prodrug before activation. By contrast, under irradiation,
phorbiplatin is very active in the tested cells with IC50 values in the nanomolar range.
For example, in A2780 cells, the IC50 of phorbiplatin under irradiation is as low
as 0.13 mM. Compared with oxaliplatin, the photocytotoxicity of phorbiplatin in-
creases by a factor of 523. Phorbiplatin is also active in platinum-resistant
A2780cisR and MCF-7 cells with IC50 values of 0.19 and 0.044 mM, respectively,
and the photocytotoxicity of phorbiplatin increases by factors of 974 and 1786
compared with oxaliplatin, respectively. In addition, phorbiplatin is non-toxic to
the tested normal human lung fibroblasts MRC-5 in the dark, indicating its low
toxicity to normal cells. We also tested the photocytotoxicity of phorbiplatin in
4
T1 murine cancer cell line. The tumor growth of 4T1 cells in BALB/c mice closely
mimics stage IV human breast cancer, and this cell line has been widely used in
6
7–69
antitumor studies.
Phorbiplatin shows 58-fold increased photocytotoxicity
compared with oxaliplatin in 4T1 cells, indicating its significant activity against late
stage breast cancer cells.
Action Mechanism of Phorbiplatin in Cells
More biological assays were carried out to study the fate of phorbiplatin in cells.
First, the cellular accumulation levels of Pt in A2780, A2780cisR, and MCF-7
cells treated with phorbiplatin and oxaliplatin for 8 h were measured, and the results
are shown in Figure S34. Compared with oxaliplatin, phorbiplatin accumulates more
efficiently in the cells. For example, the cellular accumulation levels for phorbiplatin
6
and oxaliplatin in A2780 cells are 6.4 G 1.2 and 0.9 G 0.1 ng Pt/10 cells, respec-
tively. This high level of accumulation may be ascribed to the increased lipophilicity
7
0
of phorbiplatin (LogP = 1.25) compared with that of oxaliplatin (LogP = ꢁ1.76).
To confirm whether phorbiplatin can be reduced to oxaliplatin and subsequently bind
to genomic DNA in cells, we measured platinum levels in the genomic DNA of A2780
cells treated with phorbiplatin. Under irradiation, the platinum level in DNA is
1
10 G 9.7 pg Pt per mg DNA, whereas the value is only 28 G 5.5 pg Pt per mg DNA
in the dark (Figure 3C). This result suggests that a small portion of phorbiplatin may
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be reduced in cells in the dark after 16 h, but under irradiation, phorbiplatin is rapidly
reduced, resulting in a high level of Pt in genomic DNA.
PPA reportedly acts as a photosensitizer to kill cells by inducing ROS under
7
1
irradiation. We measured the ROS levels of A2780 cells treated with phorbiplatin
to examine its ability to induce ROS using H DCFDA as a fluorescent probe. As shown
2
in Figures 3D and S35, compared with untreated cells and cells treated with phorbipla-
tin or PPA in the dark, cells treated with phorbiplatin or PPA under irradiation show
significantly increased fluorescence intensity, suggesting elevated levels of cellular
ROS. Therefore, PPA, especially after the dissociation with Pt in cancer cells after
irradiation, may also contribute to the cytotoxicity of phorbiplatin. The ability of
phorbiplatin to generate ROS was further proved by an in vitro cell-free assay based
on a chemical probe, 1,3-diphenylisobenzofuran (DPBF) (Figure S36), and the results
suggest that phorbiplatin efficiently induces ROS under irradiation.
To further elucidate the contribution of different ROS species in the photocytotox-
icity of phorbiplatin, we measured the photocytotoxicity of the compounds in the
presence of singlet oxygen inhibitor L-histidine and hydroxyl radical inhibitor
D-mannitol. As shown in Table S1, these inhibitors did not change the cytotoxicity
of oxaliplatin significantly. In the presence of L-histidine, the photocytotoxicity of
PPA decreased, indicating that singlet oxygen plays some roles. In the presence
of D-mannitol, however, the photocytotoxicity of PPA decreased more significantly,
suggesting that the photocytotoxicity of PPA mainly relies on the generation of
hydroxyl radicals. This result is consistent with other reports that a PPA derivative
5
3
generates more hydroxyl radicals than singlet oxygen. For phorbiplatin, in the
presence of L-histidine, the photocytotoxicity slightly decreased, and this phenom-
enon was also observed in the presence of D-mannitol. Therefore, both single oxy-
gen and hydroxyl radicals contribute to the photocytotoxicity of phorbiplatin.
We further tested the ability of phorbiplatin to induce apoptosis using an Annexin
V/7-aminoactinomycin d (7-AAD) double staining assay, and the result is shown in
Figure S37. A2780 cells treated with phorbiplatin under irradiation show high frac-
tions of apoptotic (29.1%) and dead (68.0%) cells compared with all the other groups
including untreated cells, cells treated with oxaliplatin, and cells treated with PPA
under irradiation. Therefore, phorbiplatin induces cell death through apoptotic
pathways.
In vivo Antitumor Activity of Phorbiplatin
Based on the in vitro results, we argue that the photocytotoxicity of phorbiplatin is
due to its high accumulation in cancer cells followed by the generation of active
Pt(II) species together with ROS including singlet oxygen and hydroxyl radicals un-
der irradiation. This multi-action property might result in high antitumor activity
in vivo. The lack of oxygen in the hypoxic tumor microenvironment will reduce
the generation of ROS and the photocytotoxicity of PPA. Our hypothesis is that
although the generation of ROS from phorbiplatin may be limited under hypoxia,
this prodrug can still generate Pt(II) to exhibit cell-killing effects, which may lead
to enhanced antitumor activity compared with oxaliplatin and PPA, or even a
mixture.
To corroborate this hypothesis, we further studied the antitumor activity of phorbi-
platin in vivo using a murine mammary adenocarcinoma 4T1 xenograft model.
BALB/c mice bearing the 4T1 tumor model were treated with PPA or phorbiplatin,
and the fluorescence of the complex was monitored. As shown in Figure S38, the
10
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Figure 4. Antitumor Activity of Phorbiplatin In vivo
(
A) The growth of 4T1 tumors in a xenograft model treated with various complexes and the relative
tumor volumes are presented. Arrows represent the time of complex administration and
irradiation. Also, see Figure S39.
(
(
(
B) Excised tumor weight at the endpoint.
C) Photographs of excised tumors at the endpoint.
D) The bodyweight of the mice. The complexes were i.v. injected 5 times in total at a dose of
3
6
.5 mmol-Pt/kg. 4 h after i.v. injection, the tumor sites of irradiation group were irradiated with
2
60 nm light at a power density of 100 mW/cm for 10 min. The tumor volume of each group was
measured every two days for 12 days. (Statistical analysis was carried out using Student’s t test.
*p < 0.01, ***p < 0.001, mean G SD; n = 5).
*
mice showed strong fluorescence near tumors 4 h post-injection of PPA or phorbi-
platin. The fluorescence at the tumor site kept decreasing and disappeared after
2
4 h. Thus, we chose 4 h after injection as the time point for irradiation. BALB/c
3
mice bearing approximately 40 mm 4T1 tumors were intravenously injected with
different complexes at a dose of 3.5 mmol-Pt/kg every two days for a total of five
2
treatments. After 4 h, tumors were irradiated with red light (660 nm, 100 mW/cm )
for 10 min. As indicated in Figures 4A and S39, oxaliplatin at this dose exhibited
limited effects on the tumor growth, and a mixture of PPA and oxaliplatin with irra-
diation weakly inhibited the tumor growth. Phorbiplatin in the dark also weakly in-
hibited the tumor growth, which might be due to the slow reduction of phorbiplatin
and the following release of oxaliplatin after a long time in vivo. Remarkably, phor-
biplatin with irradiation significantly inhibited the tumor growth, with 67% reduction
in tumor volume and 62% reduction in tumor weight compared with the control
group (Figures S39, 4B, and 4C). These results suggest significantly improved in vivo
antitumor effects of phorbiplatin compared with oxaliplatin or even a mixture of PPA
and oxaliplatin. In addition, the bodyweight of the mice treated with phorbiplatin
did not change significantly, highlighting the safety of phorbiplatin (Figure 4D).
Moreover, histological analysis (H&E staining) shows that tumor damage occurs
more significantly in the phorbiplatin-treated group under irradiation than other
groups, suggesting the superior antitumor activity of phorbiplatin (Figure S40). Be-
sides, the results of H&E staining in Figure S40 show that there is liver damage in the
mice treated with the mixture of oxaliplatin and PPA under irradiation, raising a
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safety concern of the mixture. By contrast, no liver damage was found in the mice
treated with phorbiplatin under irradiation, and the other organs are also in good
conditions. These results further confirmed the safety of phorbiplatin.
In conclusion, to the best of our knowledge, we developed the first small-molecule
Pt(IV) prodrug that can be efficiently and controllably activated by red light in a
spatial and temporal fashion. This prodrug is stable in the dark, even in the presence
of reductants. Upon red light irradiation, the prodrug is reduced rapidly to oxalipla-
tin and PPA. Studies on the photoactivation process indicate that the prodrug uti-
lizes a unique mechanism to be photo-reduced: after photoactivation to the triplet
excited state, PPA is reduced under irradiation in the presence of ascorbate, and
the reduced PPA further reduces the Pt(IV) center to initiate the photoreduction pro-
cess. The PPA axial ligand acts as a ‘‘photo-induced redox relay’’ to transfer electrons
to the Pt(IV) center. To the best of our knowledge, this is also the first example of a
small-molecule Pt(IV) prodrug containing a functionalized axial ligand that can facil-
itate the electron transfer process from a reducing agent to the Pt center under irra-
diation. The prodrug shows negligible toxicity to cells in the dark, while upon irradi-
ation, it exhibits a remarkable ability to kill cancer cells with up to a 1,786-fold
increase in photocytotoxicity compared with oxaliplatin. Mechanistic investigations
indicate that oxaliplatin and PPA resulting from the prodrug activation induce DNA
damage and ROS species, respectively, to effectively kill cancer cells in a combina-
torial mode. The prodrug is also much more photoactive in vivo against the growth
of breast tumor in BALB/c mice compared with oxaliplatin, PPA, and even a mixture
of oxaliplatin and PPA. These results confirm that phorbiplatin is an effective red
light-activatable anticancer prodrug, which is able to overcome drug resistance is-
sues of traditional platinum drugs. Compared with the recently reported photoacti-
vation of Pt(IV) prodrugs by adding a photocatalyst externally with the irradiation of
blue light, phorbiplatin, as a single agent, does not require the delivery of an
external catalyst into the tumor region and is applicable in vivo due to the utilization
7
2,73
of red light.
This novel strategy of building photoactivatable platinum prodrugs
opens up a new direction for the design of Pt(IV) prodrugs with controllable activa-
tion properties. Our current efforts aim to apply this strategy to other photo-ab-
sorbers to obtain more potent Pt(IV) anticancer prodrugs.
EXPERIMENTAL PROCEDURES
The experimental procedures are included in the Supplemental Information.
SUPPLEMENTAL INFORMATION
Supplemental Information can be found online at https://doi.org/10.1016/j.chempr.
2
019.08.021.
ACKNOWLEDGMENTS
We thank the Hong Kong Research Grants Council (CityU 11304318 and 11307419),
National Natural Science Foundation of China (21877092), City University of Hong
Kong (projects 9610369 to H.H. and 9667148 to G.Z.), the Science Technology
and Innovation Committee of Shenzhen Municipality (JCYJ20170307091106444),
and Shenzhen University (project 2018022) for funding support.
AUTHOR CONTRIBUTIONS
G.Z. and Z.W. conceived the project. Z.W. and G.Z. designed the experiments and
wrote the manuscript. Z.W. designed and conducted the chemical synthesis, charac-
terization, reduction, and in vitro biological assays of phorbiplatin. N.W. carried out
12
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the in vivo animal assays. Z.W., S.-C.C., and C.-C.K. performed the transient absorp-
tion measurement. K.Xu and H.H. carried out the DFT calculations. Z.X. carried out
1
the H NMR experiments. Z.W. and Z.D. carried out the synthesis and characteriza-
tion of Ac-phorbiplatin. S.C. measured the fluorescence properties of the com-
pounds. Z.W., N.W., K.Xie, and P.S. carried out the animal imaging assays. M.-K.T
1
95
conducted the
manuscript.
Pt NMR experiments. All authors edited and approved the final
DECLARATION OF INTERESTS
G.Z., Z.W., and Z.X. are listed as inventors on a patent application by City University
of Hong Kong describing the preparation and therapeutic use of phorbiplatin. The
patent is pending in the USA (application number 15/917,966). The other authors
declare no competing financial interests.
Received: August 12, 2018
Revised: April 29, 2019
Accepted: August 22, 2019
Published: September 23, 2019
REFERENCES
1
. Lakshmanan, S., Gupta, G.K., Avci, P.,
Chandran, R., Sadasivam, M., Jorge, A.E., and
Hamblin, M.R. (2014). Physical energy for drug
delivery; poration, concentration and
9. Tung, C.H., Han, M.S., Kim, Y., Qi, J., and
O’Neill, B.E. (2017). Tumor ablation using low-
intensity ultrasound and sound excitable drug.
J. Control. Release 258, 67–72.
platinum(IV)–azide complexes. Angew. Chem.
Int. Ed. 42, 335–339.
1
9. Mackay, F.S., Woods, J.A., Heringov a´ , P.,
Kasp a´ rkov a´ , J., Pizarro, A.M., Moggach, S.A.,
Parsons, S., Brabec, V., and Sadler, P.J. (2007).
A potent cytotoxic photoactivated platinum
complex. Proc. Natl. Acad. Sci. USA 104,
20743–20748.
activation. Adv. Drug Deliv. Rev. 71, 98–114.
1
0. Yang, Y., Mu, J., and Xing, B. (2017).
Photoactivated drug delivery and bioimaging.
Wiley Interdiscip. Rev. Nanomed.
Nanobiotechnol. 9.
2
. Du, B.J., Li, D., Wang, J., and Wang, E.K. (2017).
Designing metal-contained enzyme mimics for
prodrug activation. Adv. Drug Deliv. Rev. 118,
7
8–93.
1
1. Yun, S.H., and Kwok, S.J.J. (2017). Light in
diagnosis, therapy and surgery. Nat. Biomed.
Eng. 1.
20. Farrer, N.J., Woods, J.A., Salassa, L., Zhao, Y.,
Robinson, K.S., Clarkson, G., Mackay, F.S., and
Sadler, P.J. (2010). A potent trans-diimine
platinum anticancer complex photoactivated
by visible light. Angew. Chem. Int. Ed. 49,
8905–8908.
3
. Zhu, L., Wang, T., Perche, F., Taigind, A., and
Torchilin, V.P. (2013). Enhanced anticancer
activity of nanopreparation containing an
MMP2-sensitive PEG-drug conjugate and cell-
penetrating moiety. Proc. Natl. Acad. Sci. USA
12. Wang, D., and Lippard, S.J. (2005). Cellular
processing of platinum anticancer drugs. Nat.
Rev. Drug Discov. 4, 307–320.
110, 17047–17052.
21. Min, Y., Li, J., Liu, F., Yeow, E.K., and Xing, B.
4
. Kim, E.J., Bhuniya, S., Lee, H., Kim, H.M.,
Cheong, C., Maiti, S., Hong, K.S., and Kim, J.S.
13. Kelland, L. (2007). The resurgence of platinum-
based cancer chemotherapy. Nat. Rev. Cancer
7, 573–584.
(2014). Near-infrared light-mediated
photoactivation of a platinum antitumor
prodrug and simultaneous cellular apoptosis
imaging by upconversion-luminescent
(2014). An activatable prodrug for the
treatment of metastatic tumors. J. Am. Chem.
Soc. 136, 13888–13894.
1
4. Rabik, C.A., and Dolan, M.E. (2007). Molecular
mechanisms of resistance and toxicity
associated with platinating agents. Cancer
Treat. Rev. 33, 9–23.
nanoparticles. Angew. Chem. Int. Ed. 53, 1012–
1016.
5
6
. Hunter, F.W., Wouters, B.G., and Wilson, W.R.
(2016). Hypoxia-activated prodrugs: paths
22. Dai, Y., Xiao, H., Liu, J., Yuan, Q., Ma, P., Yang,
D., Li, C., Cheng, Z., Hou, Z., Yang, P., et al.
(2013). In vivo multimodality imaging and
cancer therapy by near-infrared light-triggered
trans-platinum prodrug-conjugated
forward in the era of personalised medicine. Br.
J. Cancer 114, 1071–1077.
15. Galluzzi, L., Senovilla, L., Vitale, I., Michels, J.,
Martins, I., Kepp, O., Castedo, M., and
. Noh, J., Kwon, B., Han, E., Park, M., Yang, W.,
Cho, W., Yoo, W., Khang, G., and Lee, D.
Kroemer, G. (2012). Molecular mechanisms of
cisplatin resistance. Oncogene 31, 1869–1883.
upconverison nanoparticles. J. Am. Chem. Soc.
(2015). Amplification of oxidative stress by a
135, 18920–18929.
dual stimuli-responsive hybrid drug enhances
cancer cell death. Nat. Commun. 6, 6907.
16. Dasari, S., and Tchounwou, P.B. (2014).
Cisplatin in cancer therapy: molecular
mechanisms of action. Eur. J. Pharmacol. 740,
364–378.
23. Xu, S., Zhu, X., Zhang, C., Huang, W., Zhou, Y.,
and Yan, D. (2018). Oxygen and Pt(II) self-
generating conjugate for synergistic photo-
chemo therapy of hypoxic tumor. Nat.
Commun. 9, 2053.
7
. Weiss, J.T., Dawson, J.C., Macleod, K.G.,
Rybski, W., Fraser, C., Torres-S a´ nchez, C.,
Patton, E.E., Bradley, M., Carragher, N.O., and
Unciti-Broceta, A. (2014). Extracellular
17. Kratochwil, N.A., Zabel, M., Range, K.J., and
Bednarski, P.J. (1996). Synthesis and X-ray
palladium-catalysed dealkylation of 5-fluoro-1-
propargyl-uracil as a bioorthogonally activated
prodrug approach. Nat. Commun. 5, 3277.
crystal structure of trans,cis-[Pt(OAc)
2
I
2
(en)]: a
24. Hall, M.D., Mellor, H.R., Callaghan, R., and
Hambley, T.W. (2007). Basis for design and
development of platinum(IV) anticancer
complexes. J. Med. Chem. 50, 3403–3411.
novel type of cisplatin analog that can be
photolyzed by visible light to DNA-binding and
cytotoxic species in vitro. J. Med. Chem. 39,
2499–2507.
8
. Lameijer, L.N., Ernst, D., Hopkins, S.L., Meijer,
M.S., Askes, S.H.C., Le D e´ v e´ dec, S.E., and
Bonnet, S. (2017). A red-light-activated
ruthenium-caged NAMPT inhibitor remains
phototoxic in hypoxic cancer cells. Angew.
Chem. Int. Ed. 56, 11549–11553.
25. Johnstone, T.C., Suntharalingam, K., and
Lippard, S.J. (2016). The next generation of
platinum drugs: targeted Pt(II) agents,
nanoparticle delivery, and Pt(IV) prodrugs.
Chem. Rev. 116, 3436–3486.
18. M u¨ ller, P., Schr o¨ der, B., Parkinson, J.A.,
Kratochwil, N.A., Coxall, R.A., Parkin, A.,
Parsons, S., and Sadler, P.J. (2003). Nucleotide
cross-linking induced by photoreactions of
Chem 5, 1–15, December 12, 2019
13

Please cite this article in press as: Wang et al., Phorbiplatin, a Highly Potent Pt(IV) Antitumor Prodrug That Can Be Controllably Activated by Red
Light, Chem (2019), https://doi.org/10.1016/j.chempr.2019.08.021
2
2
2
6. Wang, X., and Guo, Z. (2013). Targeting and
delivery of platinum-based anticancer drugs.
Chem. Soc. Rev. 42, 202–224.
39. Brunner, H., and Gruber, N. (2004). Carboplatin-
containing porphyrin–platinum complexes as
cytotoxic and phototoxic antitumor agents.
Inorg. Chim. Acta 357, 4423–4451.
a-phosphatidylcholine vesicles. Photochem.
Photobiol. Sci. 5, 317–325.
54. Neta, P., Scherz, A., and Levanon, H. (1979).
Electron transfer reactions involving porphyrins
and chlorophyll a. J. Am. Chem. Soc. 101,
3624–3629.
7. Romero, N.A., and Nicewicz, D.A. (2016).
Organic photoredox catalysis. Chem. Rev. 116, 40. Zhang, J.Z., Wexselblatt, E., Hambley, T.W.,
10075–10166.
and Gibson, D. (2012). Pt( iv ) Analogs of
oxaliplatin that do not follow the expected
correlation between electrochemical reduction 55. Neta, P. (1981). One-electron transfer reactions
potential and rate of reduction by ascorbate.
8. Rybicka-Jasi nꢀ ska, K., Shan, W., Zawada, K.,
Kadish, K.M., and Gryko, D. (2016). Porphyrins
as photoredox catalysts: experimental and
theoretical studies. J. Am. Chem. Soc. 138,
involving zinc and cobalt porphyrins in aqueous
solutions. J. Phys. Chem. 85, 3678–3684.
Chem. Commun. 48, 847–849.
4
4
4
1. Thiabaud, G., McCall, R., He, G., Arambula,
J.F., Siddik, Z.H., and Sessler, J.L. (2016).
Activation of platinum(IV) prodrugs by
motexafin gadolinium as a redox mediator.
Angew. Chem. Int. Ed. 55, 12626–12631.
1
5451–15458.
56. Whitten, D.G., Yau, J.C.N., and Carroll, F.A.
(
1971). Photochemistry and oxidation-
2
9. Feng, X., Jiang, D., Kang, T., Yao, J., Jing, Y.,
Jiang, T., Feng, J., Zhu, Q., Song, Q., Dong, N.,
et al. (2016). Tumor-homing and penetrating
peptide-functionalized photosensitizer-
conjugated PEG-PLA nanoparticles for chemo-
photodynamic combination therapy of drug-
resistant cancer. ACS Appl. Mater. Interfaces 8,
reduction reactions of tin porphyrins. J. Am.
Chem. Soc. 93, 2291–2296.
57. McCormick, M.C., Keijzer, K., Polavarapu, A.,
2. Varbanov, H.P., Valiahdi, S.M., Kowol, C.R.,
Jakupec, M.A., Galanski, M., and Keppler, B.K.
Schultz, F.A., and Baik, M.H. (2014).
Understanding intrinsically irreversible, non-
Nernstian, two-electron redox processes: a
combined experimental and computational
study of the electrochemical activation of
platinum(IV) antitumor prodrugs. J. Am. Chem.
Soc. 136, 8992–9000.
(2012). Novel tetracarboxylatoplatinum(IV)
complexes as carboplatin prodrugs. Dalton
Trans. 41, 14404–14415.
17817–17832.
3
3
0. Stamati, I., Kuimova, M.K., Lion, M., Yahioglu,
G., Phillips, D., and Deonarain, M.P. (2010).
Novel photosensitisers derived from
pyropheophorbide-a: uptake by cells and
photodynamic efficiency in vitro. Photochem.
Photobiol. Sci. 9, 1033–1041.
3. Zhang, J.Z., Bonnitcha, P., Wexselblatt, E.,
Klein, A.V., Najajreh, Y., Gibson, D., and
Hambley, T.W. (2013). Facile preparation of
mono-, di- and mixed-carboxylato platinum(IV)
complexes for versatile anticancer prodrug
design. Chem. Eur. J. 19, 1672–1676.
58. Wilson, J.J., and Lippard, S.J. (2011). Synthesis,
characterization, and cytotoxicity of
platinum(IV) carbamate complexes. Inorg.
Chem. 50, 3103–3115.
1. Duan, X., Chan, C., Guo, N., Han, W.,
Weichselbaum, R.R., and Lin, W. (2016).
Photodynamic therapy mediated by nontoxic
core-shell nanoparticles synergize with
immune checkpoint blockade to elicit
antitumor immunity and antimetastatic effect
on breast cancer. J. Am. Chem. Soc. 138,
4
4. Bonnett, R., and Mart ı´ nez-Botella, G. (2001).
Photobleaching of sensitisers used in
photodynamic therapy. Tetrahedron 40, 9513–
59. Mauzerall, D. (1960). The photoreduction of
porphyrins and the oxidation of amines by
photo-excited dyes. J. Am. Chem. Soc. 82,
9547.
1832–1833.
45. Al-Omari, S., and Ali, A. (2009). Photodynamic
activity of pyropheophorbide methyl ester and
pyropheophorbide a in dimethylformamide
solution. Gen. Physiol. Biophys. 28, 70–77.
6
0. D ı´ az-Espinosa, Y., Crespo-Hern a´ ndez, C.E.,
Alegr ´ı a, A.E., Garc ı´ a, C., and Arce, R. (2011).
Quenching enhancement of the singlet excited
state of pheophorbide-a by DNA in the
presence of the quinone carboquone.
Photochem. Photobiol. 87, 275–283.
1
6686–16695.
3
2. Ethirajan, M., Chen, Y., Joshi, P., and Pandey,
R.K. (2011). The role of porphyrin chemistry in
tumor imaging and photodynamic therapy.
Chem. Soc. Rev. 40, 340–362.
46. Bannister, T.T. (1959). Photoreduction of
chlorophyll a in the presence of ascorbic acid in
pyridine solutions. Plant Physiol. 34, 246–254.
6
1. Eckstein, N. (2011). Platinum resistance in
breast and ovarian cancer cell lines. J. Exp. Clin.
Cancer Res. 30, 91.
3
3. He, C., Duan, X., Guo, N., Chan, C., Poon, C.,
Weichselbaum, R.R., and Lin, W. (2016). Core-
shell nanoscale coordination polymers
combine chemotherapy and photodynamic
therapy to potentiate checkpoint blockade
cancer immunotherapy. Nat. Commun. 7,
47. Chibisov, A.K. (1969). A flash photolysis study of
intermediates in photochemical reactions of
chlorophyll. Photochem. Photobiol. 10,
331–347.
6
2. Knopf, K.M., Murphy, B.L., MacMillan, S.N.,
Baskin, J.M., Barr, M.P., Boros, E., and Wilson,
J.J. (2017). In vitro anticancer activity and in vivo
biodistribution of rhenium(I) Tricarbonyl aqua
complexes. J. Am. Chem. Soc. 139, 14302–
4
4
5
8. Suboch, V.P., Losev, A.P., and Gurinovitch, G.P.
1974). Photoreduction of protochlorophyll and
its derivatives. Photochem. Photobiol. 20,
83–190.
(
12499.
1
3
4. Shanmugam, S., Xu, J., and Boyer, C. (2015).
Utilizing the electron transfer mechanism of
chlorophyll a under light for controlled radical
polymerization. Chem. Sci. 6, 1341–1349.
14314.
9. Felix, C.C., Reszka, K., and Sealy, R.C. (1983).
Free radicals from photoreduction of
hematoporphyrin in aqueous solution.
Photochem. Photobiol. 37, 141–147.
63. Qin, H., Zhao, C., Sun, Y., Ren, J., and Qu, X.
(2017). Metallo-supramolecular complexes
enantioselectively eradicate cancer stem cells
in vivo. J. Am. Chem. Soc. 139, 16201–16209.
3
5. Xu, J., Shanmugam, S., Fu, C., Aguey-Zinsou,
K.F., and Boyer, C. (2016). Selective
photoactivation: From a single unit monomer
insertion reaction to controlled polymer
architectures. J. Am. Chem. Soc. 138, 3094–
0. Richoux, M.C., Neta, P., Harriman, A., Baral, S.,
and Habright, P. (1986). One- and two-electron
reduction of metalloporphyrins. Radiation
chemical, photochemical, and electrochemical
studies. Kinetics of the decay of p-radical
anions. J. Phys. Chem. 90, 2462–2468.
6
4. Venkatesh, V., Mishra, N.K., Romero-Canel o´ n,
I., Vernooij, R.R., Shi, H., Coverdale, J.P.C.,
Habtemariam, A., Verma, S., and Sadler, P.J.
3106.
(
2017). Supramolecular photoactivatable
3
6. Brunner, H., and Obermeier, H. (1994).
Platinum(II) complexes with porphyrin
anticancer hydrogels. J. Am. Chem. Soc. 139,
5656–5659.
5
1. Livingston, R., and McCartin, P.J. (1963). Some
observations related to the photoreduction of
chlorophyll. J. Am. Chem. Soc. 85, 1571–1573.
ligands—additive cytotoxic and photodynamic
effect. Angew. Chem. Int. Ed. 33, 2214–2215.
6
5. Savellano, M.D., Pogue, B.W., Hoopes, P.J.,
Vitetta, E.S., and Paulsen, K.D. (2005).
3
7. Lottner, C., Bart, K.C., Bernhardt, G., and
Brunner, H. (2002). Soluble
tetraarylporphyrinꢁplatinum conjugates as
cytotoxic and phototoxic antitumor agents.
J. Med. Chem. 45, 2079–2089.
52. Livingston, R., and Pugh, A.C.P. (1960). Role of
the triplet state in the photoreduction of
chlorophyll. Nature 186, 969–970.
Multiepitope HER2 targeting enhances
photoimmunotherapy of HER2-overexpressing
cancer cells with pyropheophorbide-a
immunoconjugates. Cancer Res. 65, 6371–6379.
53. Delanaye, L., Bahri, M.A., Tfibel, F., Fontaine-
Aupart, M.P., Mouithys-Mickalad, A., Heine, B.,
Piette, J., and Hoebeke, M. (2006). Physical
and chemical properties of
66. Wang, J., Liu, Q., Zhang, Y., Shi, H., Liu, H.,
Guo, W., Ma, Y., Huang, W., and Hong, Z.
(2017). Folic acid-conjugated
3
8. Lottner, C., Bart, K.C., Bernhardt, G., and
Brunner, H. (2002). Hematoporphyrin-derived
soluble porphyrinꢁplatinum conjugates with
combined cytotoxic and phototoxic antitumor
activity. J. Med. Chem. 45, 2064–2078.
pyropheophorbide-a methyl ester in ethanol,
phosphate buffer and aqueous dispersion of
small unilamellar dimyristoyl-l-
pyropheophorbide a as the photosensitizer
tested for in vivo targeted photodynamic
therapy. J. Pharm. Sci. 106, 1482–1489.
14
Chem 5, 1–15, December 12, 2019

Please cite this article in press as: Wang et al., Phorbiplatin, a Highly Potent Pt(IV) Antitumor Prodrug That Can Be Controllably Activated by Red
Light, Chem (2019), https://doi.org/10.1016/j.chempr.2019.08.021
6
6
7. Pulaski, B.A., and Ostrand-Rosenberg, S.
2001). Mouse 4T1 breast tumor model. Curr.
Protoc. Immunol. 2.
70. Rappel, C., Galanski, M., Yasemi, A., Habala, L.,
and Keppler, B.K. (2005). Analysis of
anticancer platinum(II)-complexes
by microemulsion electrokinetic
chromatography: separation of
72. Alonso-de Castro, S., Cortajarena, A.L.,
L o´ pez-Gallego, F., and Salassa, L. (2018).
Bioorthogonal catalytic activation
of platinum and ruthenium
(
8. Qi, J., Chen, C., Zhang, X., Hu, X., Ji, S., Kwok,
R.T.K., Lam, J.W.Y., Ding, D., and Tang, B.Z.
anticancer complexes by FAD
and flavoproteins. Angew. Chem. Int. Ed. 57,
3143–3147.
diastereomers and estimation of octanol–
water partition coefficients. Electrophoresis 26,
878–884.
(2018). Light-driven transformable optical agent
with adaptive functions for boosting cancer
surgery outcomes. Nat. Commun. 9, 1848.
73. Alonso-de Castro, S., Ruggiero, E., Ruiz-de-
Angulo, A., Rezabal, E., Mareque-Rivas, J.C.,
Lopez, X., L o´ pez-Gallego, F., and Salassa, L.
(2017). Riboflavin as a
7
1. Zhou, A., Wei, Y., Wu, B., Chen, Q., and Xing, D.
(2012). Pyropheophorbide a and c(RGDyK)
comodified chitosan-wrapped upconversion
nanoparticle for targeted near-infrared
photodynamic therapy. Mol. Pharmaceutics 9,
1580–1589.
6
9. Wang, T., Wang, D., Yu, H., Feng, B., Zhou, F.,
Zhang, H., Zhou, L., Jiao, S., and Li, Y. (2018). A
cancer vaccine-mediated postoperative
immunotherapy for recurrent and metastatic
tumors. Nat. Commun. 9, 1532.
bioorthogonal photocatalyst for
IV
the activation of a Pt prodrug. Chem. Sci. 8,
4619–4625.
Chem 5, 1–15, December 12, 2019
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