Cancer-Specific, Intracellular, Reductive Activation of Anticancer PtIV Prodrugs

Article abstract of DOI:10.1002/chem.201701192

Because cellular uptake of anticancer Pt^II and Pt^IV drugs occurs by different mechanisms, the latter ones can exhibit substantial activity towards cells, which have either intrinsic or acquired resistance towards Pt^II drugs. However, this positive effect is diminished due to reductive activation of Pt^IV drugs in extracellular space that can be one of the reasons why they have not yet been approved for clinical use despite over 60 clinical trials conducted worldwide. Herein, we suggest a solution to this problem by achieving highly specific intracellular versus extracellular prodrug reduction. In particular, we prepared a hybrid Pt^IV prodrug containing two pro-reductants. This hybrid was uptaken by cells, the pro-reductants were activated in the cancer-specific microenvironment (high H₂O₂), and reduced Pt^IV by two one-electron transfers. The drug formed in this way induced cell death both in cisplatin-sensitive and resistant cell lines, but remained nontoxic to normal cells.

Full text of DOI:10.1002/chem.201701192

A Journal of
Accepted Article
Title: Cancer specific, intracellular, reductive activation of anticancer
Pt(IV)-prodrugs
Authors: Andriy Mokhir, Viktor Reshetnikov, and Steffen Daum
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To be cited as: Chem. Eur. J. 10.1002/chem.201701192
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Cancer specific, intracellular, reductive activation of anticancer Pt(IV)-
prodrugs
Viktor Reshetnikov,[a] Steffen Daum[a] and Andriy Mokhir*[a]
Friedrich-Alexander-Universität Erlangen-Nürnberg, Department Chemistry and Pharmacy,
Organic Chemistry II, Henkestr. 42, 91301 Erlangen, Germany,
Contact information of all authors: Viktor Reshetnikov, MSc, viktor.reshetnikov@fau.de;
Steffen Daum, MSc., steffen.daum@fau.de; Andriy Mokhir, Prof. Dr., Andriy.Mokhir@fau.de
Abstract: Since cellular uptake of anticancer PtII- and PtIV-drugs occurs via different
mechanisms, the latter ones can exhibit substantial activity towards cells, which have either
intrinsic or acquired resistance towards PtII-drugs. However, this positive effect is diminished
due to reductive activation of PtIV-drugs in extracellular space that can be one of the reasons
why they were not yet approved for clinical use despite over 60 clinical trials conducted
worldwide. Herein we suggest a solution to this problem by achieving highly specific
intracellular versus extracellular prodrug reduction. In particular, we prepared a hybrid PtIV-
prodrug containing two pro-reductants. This hybrid is uptaken by cells, the pro-reductants are
activated in the cancer specific microenvironment (high H2O2) and reduce PtIV via two one-
electron-transfers. The drug formed in this way induces cell death both in cisplatin sensitive
and resistant cell lines, but remains nontoxic to normal cells.
PtII complexes cisplatin, carboplatin and oxaliplatin are approved for clinical use and broadly
used anticancer drugs.[1] They act by the related mechanism including cellular uptake, loss of
leaving groups with formation of active [L2Pt(OH2)2]2+ complexes and tight binding to N7-
atoms in purine nucleobases within genomic DNA leading to initiation of cell death.[2]
However, despite their worldwide application for the treatment of testicular, ovarian, lung,
head and neck as well as colorectal cancer,[3] a number of disadvantages remains including
severe dose-limiting toxicity,[4] intrinsic resistance of some tumors as well as resistance
acquired in the result of multiple treatments.[5] The resistance is caused by many, often
simultaneously acting factors.[4,5a] However, reduced drug uptake plays an important role in
the majority of cases.[6] In an attempt to solve these problems PtIV-prodrugs were
introduced.[1,7] Their design relies on the hypothesis that they remain stable in the
extracellular space, are uptaken by cells and reduced by ascorbate (Asc), glutathione (GSH)
and macromolecular reductants[8] with formation of PtII-drugs, which further act as described
above. Whereas the classical PtII-drugs are uptaken both via passive diffusion and active
transport,[9] the uptake of PtIV-prodrugs is believed to involve only passive diffusion.[10]
Therefore, a general solution of the problem of the cancer resistance towards PtII-drugs
caused by the insufficient uptake can include the application of PtIV-prodrugs. Unfortunately,
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the expected positive effect is diminished due to substantial reduction of PtIV-prodrugs in the
extracellular space. For example, half-life of PtCl4(trans-1,2-diaminocyclohexane)
(ormaplatin) in blood plasma was found to be only 3 s at 37°C, whereas cis,trans,cis -
PtCl2(OAc)2(NH3)(cyclohexylamine) (satraplatin) is quickly converted under these conditions
to four metabolites, one of which is cis-PtCl2(NH3)(cyclohexylamine) (JM118).13 Another
clinically tested PtIV-complex cis,trans,cis-PtCl2(OH)2(iso-propylamine)2 (iproplatin) has the
lowest reduction potential in this series. However, though it is stable in the extracellular
space, its intracellular activation is weak and, correspondingly, its anticancer activity is low.
Thus, optimization of the reduction potential of PtIV-complexes does not allow eliminating
their extracellular reduction without affecting the intracellular activation. The outlined intrinsic
problem can arguably be one of the reasons why over 60 clinical trials of PtIV-complexes did
not yet lead to a single approval.[2,7]
Cancer cells and tumors are substantially different from their parent healthy cells and tissues.
For example, it is known that tumor microenvironment contains high concentration of reactive
-
oxygen species (ROS= H2O2, O2 , HO, 1O2).[11] The latter property is believed to be crucially
important for the cancer phenotype.[12] Therefore, ROS-activated prodrugs can potentially be
designed for many different cancer types that makes them especially attractive alternatives
to currently used chemotherapeutics with narrow modes of action. For example, we have
recently developed prodrugs (e.g. 1, Figure 1), which are activated by ROS within cancer
cells with the release of N-alkylaminoferrocenes (AmFc, e.g. 2).[13] The latter products are
strong one-e--reducing agents as evidenced by their low oxidation potential (-0.37 V versus
ferrocene[13a,14]). In the preliminary experiment we observed that 2 is oxidized by PtIV-prodrug
5 with formation of ion 2+ as indicated by the appearance of the characteristic broad
absorbance band at ~900 nm in the UV-visible spectrum of a mixture of 5 and 2 (2 eq)
(supporting information (SI), Figure S11). As expected, parent prodrug 1 was found to be
unable to reduce the PtIV complex at the same conditions. Based on these data we
envisioned that combination of AmFc-based prodrugs and PtIV-complexes stable in the
extracellular space could provide hybrids, which will be activated exclusively in cancer cells,
but remain stable and inactive both in the extracellular space and in normal cells. We
expected that these prodrugs will exhibit lower toxicity and higher anticancer activity towards
cisplatin-resistant cells than previously known PtIV prodrugs.
To prove this idea, we prepared prodrug 8 containing two pro-reductant AmFc moieties
covalently linked to the PtIV-complex by a “click” reaction between substrate 6 and complex 7
(Figure 1, SI). Identity of 8 and its purity were confirmed by ESI-TOF mass spectrometry
1
13
(Figure 2A), H and C NMR spectroscopy (SI) and elemental analysis (SI). The suggested
mechanism of ROS-induced activation of 8 is outlined in Figure 1. Under cancer specific
conditions (high concentration of ROS) C-B bonds of 8 will be first oxidized in the presence
of ROS that will trigger formation of intermediate 10 containing two AmFc fragments (e--
donors).[13] Since these two e--donors are positioned in close proximity to an e--acceptor PtIV,
we expected that an e--transfer between them will be a quick and efficient process leading to
formation of cisplatin. A competing direct oxidation of the AmFc’s by endogenous ROS is a
bimolecular process, which at low concentrations of the reagents is expected to be much
slower than the desired intramolecular process.
According to UV-visible spectroscopy the solution of 8 (25 µM) in the mixture of phosphate-
buffered saline (PBS, 10 mM phosphate, 150 NaCl, pH 7) and N,N-dimethylformamide
(DMF, 1 %, v/v, SI) containing GSH (5 mM) and Asc (1 mM) is rather stable. In particular, its
absorbance at 347 nm (A347nm) is decreased down to 87 % of the initial value during its
incubation for 12 h at 37°C (Figure 2B). GSH and Asc were added to simulate conditions of
the intracellular space.[15] Since concentrations of these reducing agents in the blood plasma
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are substantially lower (GSH: 2-20 µM; Asc: ~50 µM) than those, which we used, it is
expected that the stability of 8 outside of cells will be even higher. In contrast, A347nm was
found to be continuously decreased down to 44 % of the initial value during the incubation of
8 in the presence of H2O2 (0.2 mM) indicating transformation of prodrug 8. The product
released in the latter reaction was identified by 195Pt-NMR spectroscopy and ESI-TOF mass
spectrometry (MS) (Figure 2C, D). In particular, in the NMR spectra of prodrug 8 and control
5 signals at = +1142 and +1170 ppm respectively, which are characteristic for PtIV, were
observed. Upon prolonged incubation of 8 with H2O2 this signal was shifted to -2100 ppm,
which is in agreement with cisplatin formation (SI). Furthermore, in the mass spectrum of
solution of 8 (5 mM) incubated with excess H2O2 (20 mM) for 12 h at 37 °C followed by the
addition of AgNO3 (9.4 mM) and 15N-labeled cisplatin 13 (internal standard, [13+Ag+]+, m/z
412.8498) a peak corresponding to Ag+ adduct with cisplatin [M+Ag]+ (m/z 405.8984) was
observed, whereas it was practically absent in the spectrum of control mixture lacking H2O2.
To obtain quantitative information on the ROS-induced activation of 8, we studied this and a
series of control reactions by thin layer chromatography (TLC, SI). In particular, we confirmed
that in a mixture of PtIV-prodrug 5 (0.25 mM) and pro-reductant 3 (2 eq) in the presence of
H2O2 a substantial portion of 5 (43 %) is converted to cisplatin. However, at more
physiologically relevant low concentrations (prodrug 5: 25 µM; pro-reductant 3: 2 eq) the
reaction does not occur, which is an expected result for a bimolecular process. In contrast,
prodrug 8 (25 µM) is activated efficiently even at low concentrations (74 % conversion to
cisplatin), since in this case the reduction is an intramolecular process, which is not
concentration dependent. Also in this experiment we observed that 8 is stable in the absence
of H2O2 (2.7 % conversion to cisplatin), which is in agreement with both UV-visible and MS
data discussed above.
Encouraged by these results we studied effects of hybrid 8 and controls on human ovarian
carcinoma cell line A2780, which is known to be sensitive to cisplatin.[8c] First, we
investigated the efficiency of prodrug uptake by A2780 cells by using the chromogenic assay
for intracellular boron as previously described.[13] In particular, we found that 8 is uptaken by
A2780 cells slightly less efficiently (p<0.001, N=5) than control 3 (0.33 versus 0.45; the
permeability was set to “1” for reference 1) that was in agreement with lower lipophilicity of 8
(logP= 0.3 + 0.1 versus 0.6 + 0.1 for 3, p<0.05, N=3). Furthermore, we observed that the
mixture of 3 and 5 exhibits the same effect on the cell viability as PtIV-prodrug 5 alone: IC50=
9 + 1 µM (Table 1). Since the AmFc-based prodrug 3 is substantially less active (IC50= 39 + 2
µM, p<0.001, N=3), we concluded that the antiproliferative effect of the mixture is defined
mainly by the PtIV component. In contrast, the activity of hybrid 8 was found to be
substantially stronger: IC50= 2.5 + 0.5 µM, p<0.001, N=3. The latter value coincides with that
obtained for cisplatin (IC50= 2.1 + 0.5 µM) that may indicate quantitative reduction of PtIV in 8
within cancer cells. Importantly, this process is cancer-cell specific, since prodrug 8 was
found to be non-toxic towards normal fibroblast (HDFa) cells up to its solubility limit. Next, we
evaluated the effect of 8 towards cisplatin-resistant cell line A2780cis, where the resistance
is caused by the reduced drug uptake.[16] We were pleased to observe, that 8 exhibits high
activity towards these cells (IC50= 6 + 1 µM, resistance factor, RF= 2.4), whereas cisplatin
was substantially less active (IC50= 13 + 1 µM, resistance factor, RF= 6.2, p<0.01, N=3).
Finally, we studied the mechanism of antiproliferative activity of prodrug 8. During the
intracellular activation of 8, ferrocenium 11 is formed as a side product (Figure 1). In cells it
can be reduced with formation of the corresponding ferrocene. The latter compound can
-
potentially catalyze the conversion of O2 and H2O2 to highly reactive O2 and HO that should
enhance the intracellular oxidative stress.[13] We observed that in cell free settings controls
cisplatin and PtIV-prodrug 5 generate ROS weakly, as evidenced by only 1.6-1.7 fold
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enhancement of 2′,7′-dichlorofluorescin (DCFH) oxidation. In contrast, control 3 enhances
DCFH oxidation by 22 + 4 fold, whereas prodrug 8 is even a more efficient ROS inducer: 43
+ 10 fold enhancement, p<0.05, N=3. Related effects were obtained in the cellular assay. In
particular, both cisplatin and drug 5 were found to practically not affect intracellular ROS
level. Furthermore, control 3 and a mixture of 5 and 3 (2 eq) enhanced the oxidative stress
by 3.1-3.3 fold with respect to the non-treated cells, whereas hybrid prodrug 8 exhibited the
slightly stronger ROS-enhancing effect: 3.8-fold, p<0.05, N=6 (Figure 3). Based on these
data we concluded that intracellular ROS enhancement by prodrug 8 can contribute to its
antiproliferative properties. However, this cannot be the major factor, since control 3
releasing practically the same amount of ROS in cells as 8, exhibits relatively low
antiproliferative activity: IC50= 39 + 2 µM. Apart from ferrocenium 11, hybrid 8 releases
cisplatin upon its activation. The latter drug causes the characteristic effect on cell cycle
distribution in A2780 cells including the decrease of the G1/G0 cell population and the
increase of the S cell population (Figure 3): 67 + 3 % (G1/G0), 15 + 2 % (S), 18 + 4 % (G2) for
non-treated cells versus 44 + 2 % (G1/G0), 38.9 + 0.9 % (S), 17 + 3 % (G2) for the cisplatin-
treated cells. In contrast, control 3 practically does not affect the cell cycle. We observed that
hybrid 8-treated cells exhibit cell cycle distribution similar to that of cisplatin-treated ones: 57
+ 1 % (G1/G0), 29 + 2 % (S), 14 + 2 % (G2). These data are in agreement with the
intracellular release of active drug cisplatin from 8. It should be mentioned that the recently
published approach for reduction of PtIV prodrugs mediated by motexafin gadolinium relies
on their activation exclusively outside of cells.[17] Therefore, in contrast to our hybrid, these
PtIV complexes are expected to exhibit low activity towards cisplatin-resistant cells
analogously to the parent PtII-drugs.
In summary, we prepared PtIV-prodrug 8, which is activated exclusively in cancer cells
releasing toxic cisplatin and ferrocenium ions species. Its activity is retained both in cisplatin
sensitive and resistant cell lines, but it does not affect normal cells. The antiproliferative
mechanism relies mainly on the intracellular release of cisplatin, whereas ferrocene-
dependent enhancement of the oxidative stress supports this effect.
Acknowledgement:
We thank German Research Council (DFG, grant MO1418/7-1), German Academic
Exchange Service (DAAD, fellowship for VR) and Friedrich-Alexander-University of
Erlangen-Nürnberg for funding this project. We thank Dr. C. Färber and Prof. Dr. S. Harder
(Inorganic Chemistry, Friedrich-Alexander-University of Erlangen-Nürnberg) for acquisition of
195Pt-NMR spectra. Funding obtained within the program “major research instrumentation”
from German Research Council (400 and 600 MHz NMR spectrometers at the FAU) is
acknowledged.
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Scheme and Figure legends:
Figure 1. Synthesis of hybrid prodrug 8 by coupling of alkyne 6 with azide 7 via Cu(I)-
catalyzed “click” reaction and the mechanism of activation of 8 at the cancer specific
conditions (high [ROS]). Structures of control compounds are given in the inset.
Figure 2. A: ESI-TOF mass spectrum of 8 (obs) and calculated isotopic distribution for
[8+H]+ (calcd): C66H86B2Cl2Fe2N10O12Pt. B: Changes of absorbance at 347 nm in UV-visible
spectra of the solution of cisplatin (25 µM) in DMF/phosphate buffer (1/99, v/v, pH 7) (black
triangles) and solutions of 8 (25 µM) containing either GSH (5 mM)/Asc (1 mM)/N,N,N’,N’-
ethylenediamine tetracetic acid (EDTA, 1 mM) mixture (black squares) or H2O2 (0.2 mM)
(open diamonds). C: An outline of H2O2-induced conversion of 8 to 12; 12 is detectable by
ESI-TOF MS as [12+Ag+]+ ion; 13 (2.4 mM) was added as a reference. D: ESI-TOF MS of (a)
8 (5 mM) treated with H2O2 (20 mM) for 12 h at 37 °C (upper spectrum), (b) the same as (a),
but H2O was added in place of H2O2 (middle spectrum), (c) reference compound 13 (bottom
spectrum). Green arrows label peaks of 13; a blue arrow labels a peak of 12. Integrals of
these peaks are shown below them (red colored); the integral of the peak at 405.8984 was
set to 1.0.
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Figure 3. Left plot: mean fluorescence (λex = 488 nm, λem = 530 nm) of A2870 cells loaded
with DCFH (10 µM) and either non-treated (labeled “cells”) or treated with prodrugs 3 and 8
as well as cisplatin (labeled “12”). Right plot: cell cycle distribution of A2870 cells, which were
either non-treated or treated with prodrugs 3 and 8 as well as cisplatin.
Text for the Table of Contents:
Reductive activation under oxidative conditions! A prodrug consisting of one Pt(IV)
center and two protected aminoferrocene-based pro-reductants was prepared. It is uptaken
by cells, activated in the cancer specific microenvironment (high H2O2) with reduction of
Pt(IV) via two intramolecular one-electron-transfers. The drug formed in this way induces cell
death both in cisplatin sensitive and resistant cell lines.
Tables:
Table 1. Antiproliferative activity of prodrugs and
control compounds towards cancer and normal cells.
Prodrugs/
drugs
Anticancer activity, IC50 (µM)[a]
A2780[b]
9 + 1
A2780cis[c] HDFa[d]
17.5 + 2.5 >50
5
3
39 + 2
9 + 1
-
-
>50
5(1 eq)/3(2 eq)
>50
8
2.5 + 0.5 6 + 1
2.1 + 0.3 13 + 1
>25
Cisplatin (12)
41 + 4
[a]
A2780 and A2780cis cells were incubated with
prodrugs/drugs for 96 h, whereas HDFa cells were
incubated for 48 h.
[b] A2870: human ovarian carcinoma cell line.
[c]
A2870cis: cisplatin resistant human ovarian
carcinoma cell line
[d]
HDFa: Human Dermal Fibroblasts adult cell line.
Other experimental details are described in the SI.
Keywords: Platinum(IV) prodrugs, Reactive Oxygen Species • Aminoferrocene • Cancer •
Electron transfer
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Figures
Figure 1.
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10.1002/chem.201701192
Chemistry - A European Journal
B
A
0,2
0,1
0
5 + 2
2+
5
500
750
1000
Wavelength [nm]
D
C
0,02
0,015
0,01
0,06
0,03
+ prodrug 5
[2]/[5]= 1.9
no prodrug
90
0,005
0
0
180
0
1
2
3
Time [min]
[2]/[5]
Figure 2.
Figure 3.
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10.1002/chem.201701192
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Table-of-contents figure
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