A Dogma in Doubt: Hydrolysis of Equatorial Ligands of PtIV Complexes under Physiological Conditions

Article abstract of DOI:10.1002/anie.201900682

Due to their high kinetic inertness and consequently reduced side reactions with biomolecules, Pt^IV complexes are considered to define the future of anticancer platinum drugs. The aqueous stability of a series of biscarboxylato Pt^IV complexes was studied under physiologically relevant conditions. Unexpectedly and in contrast to the current chemical understanding, especially oxaliplatin and satraplatin complexes underwent fast hydrolysis in equatorial position (even in cell culture medium and serum). Notably, the resulting hydrolysis products strongly differ in their reduction kinetics, a crucial parameter for the activation of Pt^IV drugs, which also changes the anticancer potential of the compounds in cell culture. The discovery that intact Pt^IV complexes can hydrolyze at equatorial position contradicts the dogma on the general kinetic inertness of Pt^IV compounds and needs to be considered in the screening and design for novel platinum-based anticancer drugs.

Full text of DOI:10.1002/anie.201900682

A Journal of the Gesellschaft Deutscher Chemiker
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Accepted Article
Title: Doubt on a dogma: aquation of equatorial ligands of Pt(IV)
complexes under physiological conditions
Authors: Alexander Kastner, Isabella Poetsch, Josef Mayr, Jaroslav V.
Burda, Alexander Roller, Petra Heffeter, Bernhard K. Keppler,
and Christian R. Kowol
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201900682
Angew. Chem. 10.1002/ange.201900682
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10.1002/anie.201900682
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Doubt on a dogma: aquation of equatorial ligands of Pt(IV)
complexes under physiological conditions
Alexander Kastner, Isabella Poetsch, Josef Mayr, Jaroslav V. Burda, Alexander Roller, Petra Heffeter,
Bernhard K. Keppler and Christian R. Kowol*
Dedicated to Prof. Dr. Wolfgang Weigand on the occasion of his 60th birthday.
advantage of Pt(IV) complexes are the two additional axial
ligands, which can be used to optimize chemical and biological
properties, like lipophilicity and reduction potential. Furthermore,
bioactive substances or drug-targeting moieties can be used as
axial ligands.[8b, 12] Despite these benefits, no Pt(IV) drug has
been clinically approved so far.[2] Nevertheless, some of them
entered clinical trials. However, Tetraplatin, ctc-[Pt(DACH)Cl4]
Abstract Due to their higher kinetic inertness and consequently
reduced side reactions with biomolecules, Pt(IV) complexes are
considered as the future of anticancer platinum drugs. We
investigated the aqueous stability of a series of biscarboxylato
Pt(IV) complexes under physiologically relevant conditions.
Unexpectedly and in contrast to the current chemical
understanding, especially oxaliplatin and satraplatin complexes
revealed fast hydrolysis in equatorial position (even in cell
culture medium and serum). Notably, the formed hydrolysis
products strongly differ in their reduction kinetics, a crucial
parameter for activation of Pt(IV) drugs also changing the
anticancer potential of the compounds in cell culture. The
discovery that intact Pt(IV) complexes can hydrolyze at
equatorial position, contradicts the dogma on the general kinetic
inertness of Pt(IV) compounds and needs to be considered in
the screening and design for novel anticancer drugs.
(DACH
=
(1R,2R)-(−)-1,2-diaminocyclohexane),
was
discontinued because of its neurotoxicity[13], while Iproplatin, ctc-
[Pt(IPA)2(OH)2Cl2] (IPA = isopropylamine) did not show superior
anticancer activity.[14] The most prominent representative
Satraplatin,
ctc-[Pt(NH3)(CHA)(OAc)2Cl2]
(CHA
=
cyclohexylamine) ultimately failed to show improved overall
survival in a phase III study as well.[2] Nevertheless, current
research strongly focuses on Pt(IV) complexes, as they are
considered as the next generation of platinum-based anticancer
drugs.[7] Consequently, the underlying chemical properties and
reactivities are of high importance for the specific design of
novel drugs with optimized biological properties. As already
mentioned above, Pt(IV) complexes are considered as
kinetically inert. However, there are a few reports that the axial
ligands can be hydrolysed when electron-withdrawing ligands
like dichloroacetate are present.[15] Concerning the equatorial
ligands of Pt(IV) complexes, a study using plasma from patients
treated with satraplatin showed not only the parent Pt(II) drug
JM118, but unexpectedly also the mono- and di-hydrated Pt(IV)-
species, where one or two equatorial chlorido ligands were
exchanged with hydroxido ligands.[16] This is in strong contrast to
the “dogma” of kinetic inertness of Pt(IV) drugs. As such a
hydrolysis would strongly impact on the chemical characteristics
(e.g. reduction potential) and consequently also the biological
properties, in this study we investigated the hydrolysis of the
equatorial ligands for a panel of different Pt(IV) complexes in
comparison to satraplatin (Figure 1).
Pt(II) complexes still play a very important role in cancer
treatment[1], being part of about 50% of all chemotherapies.[2]
Cis-, carbo- and oxaliplatin, are widely used against various
forms of cancer.[3] Furthermore, very recent clinical data show
impressive synergistic effects of platinum drugs together with
checkpoint inhibitor immunotherapy.[4] Their mode of action
involves aquation, binding to DNA and thereby inducing
apoptosis.[5] However, Pt(II) complexes do not only bind to DNA
in the tumor tissue, but also in healthy cells, resulting in (severe)
side-effects like nephro-, neuro- and gastrointestinal-tract
toxicity.[6] To reduce these adverse effects, Pt(IV) complexes
attract increasing interest.[7] Such low-spin d6 octahedral
complexes are considered to be kinetically more inert[8] and
consequently far less reactive towards biomolecules.[9]
Activation by reduction to the active Pt(II) complexes can occur
via low-molecular weight compounds like ascorbate and
glutathione[10], and/or high-molecular weight proteins.[11] Another
[*]
MSc. A. Kastner, Dr. J. Mayr, Dipl.-Ing. A. Roller, Ass.-Prof. Dr. C.
R. Kowol, Prof. Dr. Dr. B. K. Keppler
University of Vienna, Faculty of Chemistry, Institute of Inorganic
Chemistry, Waehringer Strasse 42, A-1090, Vienna, Austria
E-mail: christian.kowol@univie.ac.at
MSc. I. Poetsch, Assoc.-Prof. Dr. P. Heffeter
Institute of Cancer Research and Comprehensive Cancer Center,
Medical University of Vienna, Borschkegasse 8a, A-1090, Vienna,
Austria.
Prof. Dr. J. V. Burda
Department of Chemical Physics and Optics, Faculty of
Mathematics and Physics, Charles University, Ke Karlovu 3, 121 16
Prague 2, Czech Republic
Assoc.-Prof. Dr. P. Heffeter, Prof. Dr. Dr. B. K. Keppler, Ass.-Prof.
Dr. C. R. Kowol
Research Cluster “Translational Cancer Therapy Research”,
Vienna, Austria
Figure 1. Investigated Pt(IV) compounds.
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201900682
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Figure 2. Time-dependent Hydrolysis of 3 (50 µM in PB) at 37°C and pH 7.4
with formation of the hydroxido (3a) and dihydroxido (3b) complex.
Table 1. Relative amount in % of the respective hydrolysis products of
complexes 1-4 determined by HPLC-MS after incubation of 50 µM compound
at 37°C for 24 h in PB at different pH values.
To get a first overview, we analyzed the hydrolytic stability of the
model complexes 1-3 and satraplatin 4 (Figure 1) under
physiologically relevant conditions. All complexes possess two
acetato ligands in axial position and a cisplatin (1), oxaliplatin (2),
carboplatin (3) or cis-amminedichlorido(cyclohexylamine) (4)
equatorial core. The complexes were incubated in phosphate
buffer (PB) at 37°C and pH 7.4 for 24 h and monitored using
HPLC-MS. Indeed, after 24 h the majority of satraplatin (72%
hydroxido and 3% dihydroxido species) and the oxaliplatin-
based complex 3 (43% hydroxido and 14% dihydroxido; Figure
2) were already hydrolyzed. In contrast, the cisplatin analogue 1
was widely stable (5% hydroxido species) and the carboplatin
derivative was completely unchanged (Figure S1). To exclude a
significant impact of the column on the hydrolysis process the
incubated solutions were also directly injected into the mass
spectrometer. This revealed the same hydrolyzed products as
observed with HPLC-MS (data not shown).
Notably, repetition of these experiments at pH values of 8.0 and
9.0 even resulted in distinctly accelerated hydrolysis (Table 1).
At pH 9.0 the parental oxaliplatin derivative 3 completely
disappeared with exclusive formation of the dihydroxido species.
Also satraplatin was fully converted into its hydrolyzed species
(25% hydroxido/75% dihydroxido). In contrast, in case of the
cisplatin analogue 1 still ~70% of intact complex could be
observed together with ~20% hydroxido and 5 % dihydroxido
species. Carboplatin was again completely unaffected. These
findings indicated a distinct pH-dependency of the reaction,
which could be exemplarily proven for 3 revealing a perfect
linear correlation between pH and the reaction rate (Figure S2).
To rule out the involvement of phosphate in the hydrolysis, other
buffer systems i.e. HEPES and ammonium carbonate were
investigated for 3 yielding comparable results (Figure S3). Next,
3 was investigated in cell culture medium (RPMI-1640), which
showed similar ratios of hydroxido/dihydroxido complex
formation as in pure PB solution (Figure S3). Notably, the pH
value of fresh cell culture medium was about 7.4 but raised up to
pH 8–9 within 24 h.
Compound
pH
Species
1
5
2
/
3
43
14
32
53
/
4
% Hydroxido
% Dihydroxido
% Hydroxido
% Dihydroxido
% Hydroxido
% Dihydroxido
72
3
7.4
/
/
10
2
/
76
19
25
75
8.0
9.0
/
22
5
/
/
100
Consequently, the cell culture medium was phosphate-buffered
(150 mM), generating stable pH values over >24 h. Finally, the
hydrolysis of 3 was also investigated in mouse serum (also
buffered with 150 mM phosphate), again yielding similar results
(Figure S3). This clearly indicates that all the components of cell
culture medium or serum (amino acids, proteins, inorganic salts
and vitamins) have no influence on the hydrolysis process of the
Pt(IV) complex. Instead this reaction is solely dependent on the
presence of water as a solvent and the appropriate pH value.
Notably, the hydrolysis of oxaliplatin with ~10%[17] is distinctly
slower compared to 3 with ~50% after 24 h at pH 7.4. A likely
explanation is, that the Pt(II) complex with a monodentate
oxalate ligand possesses a pKa of 7.23[18] and therefore an aqua
ligand is partially present. This enables a fast ring-closing
reverse reaction to the bidentate-bound oxalate and reformation
of oxaliplatin. Consequently, the release of oxalate is
suppressed. In contrast, the pKa of the Pt(IV) complex 3a is
much lower at 3.5 (see below). Therefore a hydroxido ligand is
still present at pH 7.4 and the reverse reaction back to 3 is
hindered. This results in a shift of the equilibrium towards the
dihydroxido species and as a consequence in faster hydrolysis
than in case of Pt(II).
To investigate if the hydrolysis of 3 takes place via an attack on
the electrophilic carboxylic group of the oxalate ligand or the
platinum itself, we analyzed the hydrolysis process in H218O
(buffered with 50 mM phosphate). This measurement resulted in
an
exact
mass
of
m/z
=
488
for
ctc-
[Pt(DACH)(OAc)2(18OH)2] + Na+, which proves that the platinum
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core is directly attacked and not the oxalato ligand (Figure S4).
This is in line with H218O studies of the hydrolysis of the axial
dichloroacetate ligands of mitaplatin.[19]
To gain more insights on the exact impact of the different
equatorial ligands, the additional Pt(IV) model complexes 5-7
(Figure 1) were synthesized. Table 2 presents an overview on
the speed of hydrolysis of all complexes 1-7 after incubation in
PB at pH 7.4 and 37°C for 24 h.
quantum-chemical data was calculated. The BE obtained from
energy decomposition of the individual reactants (Table S1) are
in good agreement with the trends mentioned above. For
example: comparison of the BE of the leaving chlorido ligands
resulted also in the order 5~4>6>1 (in case of 4 the weaker-
bound chlorido ligand was considered). Furthermore, BE of the
ammine ligands in complex 1, 2 and 7 were in line with the
experimental data, although the BE of the acetato ligands are
quite different. These results were also verified by extended-
transition state natural orbitals for chemical valence (ETS-
NOCV)[20] analysis and average ionization local potential (ALIP)
Table 2. Overview on the impact of equatorial ligands on the rate of hydrolysis
in % of complexes 1-7 determined by HPLC-MS of 50 µM compound after 24
h incubation in PB at 37°C.
maps[21]
.
Additionally, the heights of activation barrier
%
%
Stable
calculations of the hydrolysis (Scheme S1) of the equatorial
ligands correlate very well with the HPLC-MS experiments
(Table 1). As summarized in Table 3, the activation energies for
the hydrolysis in case of 3 and 4 are distinctly lower compared to
1 and 2. This confirms that the higher σ-donor properties of a
secondary amine compared to simple NH3 ligands are important
for an accelerated reaction, which explains the differences
observed in the HPLC-MS experiments.[22] Furthermore, also the
second hydrolysis step ΔE(TS2) of 3, where the monodentate-
bound oxalate ligand is finally released, possesses a distinctly
lower activation barrier compared to 4 with two chlorido ligands
in exact agreement with the HPLC-MS studies (Table 1). The
calculations in case of 4 further revealed a higher trans-effect of
the CHA ligand compared to NH3 explaining the two peaks
observed with HPLC-MS. This is in accordance with a report on
the respective Pt(II) complex JM118.[23]
Complex
Hydroxido
Dihydroxido
ligandb
Labile ligand
1
2
3
4
5
6
5
0
0
0
NH3
NH3
Cl
CBDCA
oxalate
Cl
41
72a
81
42
0
13
3
DACH
NH3/CHA
DACH
en
18
0
Cl
Cl
7
0
NH3
oxalate
a
two different mono-hydroxido species can be observed with a ratio of ~
b
1:6 DACH = (1R,2R)-(−)-1,2-diaminocyclohexane; CHA = cyclohexylamine;
en = ethylenediamine; CBDCA = 1,1-cyclobutanedicarboxylic acid.
The following trends could be observed: 1) when comparing the
derivatives with
a chlorido ligand as leaving group, the
oxaliplatin-like complex (DACH; 5) has the fastest hydrolysis
with complete disappearance of the parental compound after 24
h, followed by satraplatin (NH3/CHA; 4), the ethylenediamine
complex (en; 6) and the cisplatin derivative (NH3; 1) with just 5%
hydrolysis. 2) Comparison of the complexes with two NH3
equatorial ligands shows generally very slow hydrolysis rates
with the cisplatin derivative 1 as the fastest (5% hydrolysis),
followed by the completely stable oxalate-bearing complex 7 and
the carboplatin analogue 2. 3) When comparing complexes with
the same amine ligand but chlorido vs. oxalato leaving groups
(e.g. 5 vs 3), the complex with chlorido ligands hydrolyses faster
(81% hydroxido for 5) than the one with the bidentate oxalate
ligand (41% for 3). However, when exchanging the DACH ligand
in complexes 5 and 3 for two NH3 moieties, the hydrolytic
stability dramatically increases with only 5% hydroxide species
for 1 and no hydrolysis for 7. This impressively shows that the
influence of the leaving group (chlorido vs. oxalate) has much
less impact on the hydrolysis compared to the stable amine
ligand (DACH vs. NH3). Notably, incubation of 1-7 in cell culture
medium (RPMI-1640) resulted in similar amounts of hydrolysis
products. As RPMI-1640 contains ~100 mM NaCl, this also
confirms that the presence of chloride ions cannot significantly
change the rate of hydrolysis (this could be also supported by
comparison of 4, 5 and 6 in 50 mM PB vs. 50 mM PBS with 150
mM NaCl).
Table 3. Energy of activation barriers (in kcal/mol) for replacement of the first
(ΔE(TS1)) and the second (ΔE(TS2)) equatorial-leaving group.
Complex
ΔE(TS1)
ΔE(TS2)
1
2
3
4a
4b
31.1
27.3
31.6
30.4
27.0
22.6
25.7
24.8
26.1
25.0
a (trans to CHA); b (trans to NH3)
To test how the hydrolysis alters the chemical and biological
properties the two derivatives of 3 were synthesized. This was
achieved through incubation of 3 at pH 8–9 and 37°C and
subsequent purification via preparative HPLC. The hydroxido
1
(3a) and the dihydroxido (3b) species was characterized by H
and 13C-NMR, mass spectrometry and elemental analysis.
Furthermore, the pKa values of 3a and 3b were determined via
1H-NMR and found to be 3.5 and 4.0, respectively (Figure 3).
This is in line with data of 3a after ~3 h of incubation at pH of 2.5,
which resulted in a complete transformation back to its parental
complex 3. According to the pKa value the hydroxido ligand gets
protonated at such low pH values and the thereby generated
aqua ligand can be released under re-formation of the bidentate
oxalato complex 3. In contrast, 3a is stable at pH 5.5, which
again fits to the pKa value of 3.5.
In order to obtain deeper insight into the underlying binding
energies (BE) and heights of activation barriers, a set of
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Figure 3. Determination of pKa values via correlation between ppm-shift of the
acetato ligand and pD for 3a (left) and 3b (right).
As a next step, the thermodynamic reduction properties of 3, 3a
and 3b were compared using cyclic voltammetry. All three
complexes showed irreversible reduction peaks with decreasing
potentials the more hydroxido ligands are present in the
molecule (3: –630 mV vs. NHE; 3a: –670 mV vs. NHE; 3b: –920
mV vs. NHE). This trend is in line with data from similar Pt(IV)
complexes, however with one or two axial hydroxido groups.[25]
The kinetic reduction rates of 3, 3a, and 3b were investigated by
HPLC after incubation with 10 eq. of L-ascorbic acid at 20°C.
While 3 was completely stable within 6 h, 3a and 3b were
reduced much faster and fully converted to the respective Pt(II)
species already after 3-4 h (Figure 5). Consequently, these
hydroxide species are even faster reduced than the cisplatin
complex 1 which is well-known to be much more sensitive than
oxaliplatin or carboplatin derivatives.[26] Thus, although the
thermodynamic reduction potential decreases with increasing
number of OH groups, the reduction rate extremely accelerates.
Although this seems to be unexpected, these data are in line
with a study of Gibson et al.[25] using axial mono- and
dihydroxido derivatives of complex 3 and support the importance
of the Pt(IV) reduction kinetics.
After incubation of 3b in MeOH/Et2O a few single crystals could
be obtained and analyzed by X-ray diffraction. The structure
reveals an octahedral geometry with two axial acetato ligands
and the equatorial DACH moiety. However, the two hydroxido
groups were exchanged by methoxido ligands (Figure 4; for
bond lengths and angles see Table S5/S6) Notably, the crystals
contain both the R,R and S,S isomers as a racemic mixture.
This can be explained by the ~2% S,S isomer present in the
commercially available DACH compound and the often observed
preference of compounds to crystallize as a racemate and not
as the pure isomers.[24]
Figure 5. Reduction rate of 1 mM compounds 1-3, 3a and 3b at 20°C with 10
eq. L-ascorbic acid in 250 mM phosphate buffer at pH 7.4 monitored by HPLC.
Figure 4. X-ray crystal structure of 3b incubated in MeOH/Et2O (the disorder
of the DACH ligand is omitted).
As a next step, the reactivity of 3b with different organic solvents
was investigated. In contrast to aqueous cell culture medium or
serum, incubation of 3b with e.g. DMSO, acetonitrile, MeOH or
EtOH for 1 h resulted in the exchange of one hydroxido ligand,
which could be proven by mass spectrometry and an altered
HPLC retention time (Figure S5). This indicates that at very high
excess, the hydroxido ligands indeed can be substituted, which
could also be used as a new synthetic pathway of introducing
equatorial ligands into already existing Pt(IV) complexes.
Table 4. IC50 values of 3, 3a and 3b against cancer cells after 72 h exposure.
Values represent mean ±SD from 3-4 biologically independent experiments
performed in triplicates.
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the equatorial amine ligands have the major impact on the rate
of hydrolysis. In addition, it is important to mention that (cancer)
cells possess different pH values in their organelles, e.g. up to
pH 8 in mitochondria.[27] Consequently, even higher levels of
hydrolysis can be expected, as this reaction is further
accelerated at more alkaline pH values. Under such conditions
also cisplatin derivatives start to hydrolyze to a significant
degree.
Cell line
3
3a
3b
IC50
±SD
IC50
±SD
IC50
±SD
HCT116
RKO
16.2
12.5
18.7
±2.0
±2.8
±2.2
16.6
15.6
13.7
±2.4
±4.9
±1.8
11.8
9.4
±2.1
±2.9
±2.1
CT-26
8.2
Thus, it is essential to carefully select the equatorial core of
Pt(IV) prodrugs not only according to the biological activity of the
active Pt(II) analogue, but also to the hydrolytic stability of the
Pt(IV) prodrugs (satraplatin<oxaliplatin<<cisplatin<carboplatin)
To evaluate, whether the changed chemical properties of the
hydrolysis products result in differences in biological activity, the
anticancer activity of 3, 3a and 3b against three cancer cell lines
(HCT116, RKO and CT-26) was evaluated. These experiments
revealed that 3b had a significantly lower IC50 value (up to 2-fold
more active) compared to the parental species 3 or the
monohydroxido species 3a (Figure 6, Figure S6 and Table 4).
An explanation for this could be that after reduction of 3a, the
hydroxido group in the respective Pt(II) complex is protonated
(pKa = 7.23)[18]. This aqua ligand represents a good leaving
group and facilitates ring-closure to the bidentate-bound oxalate
and reformation of oxaliplatin. Consequently, a very similar
cytotoxic activity 3 and 3a can be expected. In contrast, in case
of 3b, oxaliplatin cannot be regenerated, and instead the Pt(II)
complex [Pt(DACH)(H2O)OH]+ is formed, able to directly interact
with biological targets. In addition, cellular uptake of 3, 3a and
3b was studied on HCT-116 and CT-26 cells after 3 h incubation.
Notably, no significant differences in the uptake could be
observed, with the parental complex 3 showing the highest
cellular platinum levels (Figure S7).
and
the
rate
of
reduction
these
(satraplatin>cisplatin>>oxaliplatin~carboplatin).
All
parameters influence the biological properties and have to be
considered in the future design of novel Pt(IV) anticancer
prodrugs.
Acknowledgements
We are thankful to Dipl.-Ing. Dr. Wolfgang Kandoller for helpful
discussions and Martin Schaier and Sophie Neumayer for ICP-
MS measurements. JVB is grateful to the Grant Agency of the
Czech Republic project No 16-06240S for supporting this study.
Conflict of interest
The authors declare no conflict of interest.
Keywords: Platinum(IV) complexes • Anticancer • Hydrolysis •
Prodrug • Reduction
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Angewandte Chemie International Edition
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Less inert than assumed: The
hydrolytic stability of platinum(IV)
complexes was investigated and
substantial hydrolysis of equatorial
ligands could be observed for
various platinum(IV) compounds
under physiological relevant
conditions.
A. Kastner, I. Poetsch, J. Mayr, J. V.
Burda, A. Roller, P. Heffeter, B. K.
Keppler, C. R. Kowol
Page No. – Page No.
Doubt on a dogma: aquation of
equatorial ligands of Pt(IV)
complexes under physiological
conditions
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