An unexpected in-solution instability of diiodido analogue of picoplatin complicates its biological characterization

Article abstract of DOI:10.1039/d1dt00740h

Complex cis-[PtI2(NH3)(pic)] (1; pic = 2-methylpyridine), a diiodido analogue of clinically studied picoplatin (2), is unstable in solution, which is intriguingly connected with the release of its pic ligand. This observation complicates the biological testing of e.g. cytotoxicity in human cancer cells for 1.

Full text of DOI:10.1039/d1dt00740h

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An unexpected in-solution instability of diiodido
analogue of picoplatin complicates its biological
characterization†
Cite this: Dalton Trans., 2021, 50,
6071
Received 4th March 2021,
Accepted 6th April 2021
Pavel Štarha, * Bohuslav Drahoš
and Radovan Herchel
DOI: 10.1039/d1dt00740h
rsc.li/dalton
Complex cis-[PtI2(NH3)(pic)] (1; pic = 2-methylpyridine), a diiodido dichlorido complexes (such as picoplatin) is based on the re-
analogue of clinically studied picoplatin (2), is unstable in solution, placement of both chlorido ligands by iodido ones.6 In this
which is intriguingly connected with the release of its pic ligand. manner, multiple Pt(II) iodido complexes have been recently
This observation complicates the biological testing of e.g. cyto- reported as having various pharmacological advantages over
toxicity in human cancer cells for 1.
their direct chlorido analogues as well as over conventional Pt
drugs. Complex cis-[PtI2(NH3)2], an iodido analogue of cispla-
tin, was more effective than cisplatin in various cancer cells
including the cells resistant towards Pt drugs.7 In contrast to
cisplatin, cis-[PtI2(NH3)2] kept both its iodido ligands, while
one of its NH3 ligand was replaced by the His15 imidazole of
hen egg white lysozyme (HEWL), used as the model protein.8
Similarly, cytotoxic cis-diiodido Pt(II) complexes released
N-donor aliphatic amines in the presence of various bio-
molecules.9 On the other hand, cis-[PtI2(naza)2] complexes,
containing 7-azaindole (naza) derivatives, did not release their
heterocyclic N-donor ligands and did not interact (in contrast
to their chlorido analogues) with GSH and DNA models.10
Complexes trans-[PtI2(am)(py)] (am = methanamine or propan-
2-amine) released pyridine (py) only when irradiated.11
Platinum-based drugs (e.g., cisplatin; Fig. 1) represent a clini-
cally successful group of complexes, because they have been
used worldwide for the treatment of cancer for more than 40
years.1 Among Pt(II) complexes, an infinite number of com-
pounds have been studied for their anticancer activity, follow-
ing multiple strategies developed over the years for the
improvement of potency and reducing the numerous side-
effects of cisplatin and its analogues.
One of the strategies is based on rational chemical modifi-
cation in order to reduce the interactions with various intra-
cellular biomolecules, such as reduced glutathione (GSH),
known to deactivate Pt(II) drugs. To date, the cis-ammine-
dichlorido-2-methylpyridineplatinum(II) complex (picoplatin,
JM473; Fig. 1) is the most successful of complexes following
this strategy.2 Picoplatin showed high anticancer activity
in vitro3 and in vivo2 as well as in various clinical trials in
patients suffering from e.g. small-cell lung cancer.4 The main
advantage of picoplatin is reported as lying in its efficacy
toward cancer cells with acquired resistance against other Pt-
based drugs.3,5 However, the in vitro cytotoxicity of picoplatin
is lower than that observed for cisplatin and oxaliplatin even
in the used cisplatin- (DMS53CisR) or oxaliplatin-
(HCT116oxalR) resistant cancer cells, respectively.
With respect to the formerly reported observations for pico-
platin and simple Pt(II) diiodido complexes, it was of interest
for us to investigate complex cis-[PtI2(NH3)(pic)] (1; Fig. 1).
Complex 1, an diiodido analogue of picoplatin, is known in
the literature as an intermediate in the synthesis of picoplatin
(used in this work for comparative purposes; 2, Fig. 1).12
Complex 1 was identified using elemental analysis, NMR, ESI+
mass spectrometry (MS) and FT-IR spectroscopy. All H and C
1
atoms of 1 were detected in the H and 13C NMR spectra and
assigned using 2D experiments (ESI, Fig. S1–S4†).
The mentioned low cytotoxicity of picoplatin indicates that
there is room for improvement of its activity. One of the suc-
cessful strategies for the improvement of potency of Pt(II)
Department of Inorganic Chemistry, Faculty of Science, Palacký University in
Olomouc, 17. listopadu 1192/12, 771 46 Olomouc, Czech Republic.
E-mail: pavel.starha@upol.cz
†Electronic supplementary information (ESI) available: Experimental section,
discussion of NMR results, Fig. S1–S10 and Tables S1 and S2. See DOI: 10.1039/
d1dt00740h
Fig. 1 Structural formulas of cisplatin, picoplatin (2) and complex cis-
[PtI2(NH3)(pic)] (1); pic = 2-methylpyridine.
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1
Being aware of a contradiction between the results reported ESI, Fig. S5†). The last set of H NMR signals of 1 observed in
for Pt(II) diiodido complexes with NH3 or aliphatic amines 50% DMF-d7/50% D2O solution, represented by the resonances
(release of N-donor ligand(s) in the presence of at 7.92 ppm and 7.42 ppm, most likely belongs to the trans-
biomolecules),8,9 as compared with complexes containing [PtI2(NH3)(pic)] species (1t). This is indirectly evidenced by the
heterocyclic ligands (no release of N-donor ligands),10c and detection of the cis-to-trans isomerization (together with the
with an intention to investigate the cytotoxicity of 1, it was release of the pic ligand) in DMF-d7 (without D2O).
1
imperative for us to pay special attention to its solution behav- Specifically, three sets of H NMR signals were detected in the
iour studies (1H NMR, HPLC, UV-Vis), since speciation under DMF-d7 solution of 1, represented by the characteristic C6–H
(pseudo)physiological conditions is generally accepted as a and C7–H3 signals at 8.98/9.04/8.48 ppm, and 3.17/3.14/
crucial factor for new metal complexes developed for biological 2.48 ppm, respectively, for 1, 1t and free pic (ESI, Fig. S6 and
testing.13 It is worth noting that DMF was used for solution S7†). Furthermore, the release of the NH3 ligand can be ruled
behaviour studies, because 1 was not sufficiently soluble in the out because the total integral intensity of the broad multiplet
1
mixtures of other organic solvents (e.g., methanol, ethanol or centred at 4.40 ppm in the H NMR spectrum of 1 in DMF-d7
acetone) with water. DMSO was excluded from the in-solution correlated well with the total integral intensity of three
stability studies in this phase, because its negative effect on detected C7–H3 resonances belonging to 1, 1t and free pic
the stability of various metallodrugs, including cisplatin, was (ESI, Fig. S7†).
reported in the literature.14
In total for the 1H NMR studies, although the extent of
We started with studies of the hydrolysis of picoplatin (2) by hydrolysis of 1 (ca. 25% at t = 96 h) is comparable with that of
1H NMR (50% DMF-d7/50% D2O), clearly detecting one new set picoplatin (2), only ca. 10% of the initial complex 1 was
of signals in the 1H NMR spectra of 2 (e.g., doublet at detected in the 1H NMR spectrum after 96 h of standing at
7.54 ppm; Fig. 2). An equilibrium was reached after 72 h with 37 °C, which is caused by the pic release from 1 (ca. 13% at t =
ca. 22% of
(according to the previously determined pKa values15); the After 6 h of standing, ca. 95% of 1 was still present in 50%
sample was incubated at 37 °C between the measurements. DMF-d7/50% D2O, while only ca. 55% of 1 remained intact
2
hydrolysed to cis-[Pt(H2O)(NH3)(OH)(pic)]+ 96 h) and by the trans-isomerization of 1 (ca. 50% at t = 96 h).
The 1H NMR studies indicated that a hydrolysis, which is after 24 h (Fig. 2). This is important for the upcoming studies
usually involved in the mechanism of action of cytotoxic Pt(II) of the cellular accumulation by human cancer cells, which
complexes (including picoplatin, 2),15 occurred also for the should shed light on whether 1 could be subjected to the
iodido complex 1 (Fig. 2), because the mentioned doublet in vitro cytotoxicity testing. In other words, we aim to investi-
appeared at 7.54 ppm also for 1. Remarkably for 1 and in gate more deeply, whether the herein reported instability of 1
sharp contrast to 2, two other sets of pic signals were detected means just its undesirable extracellular deactivation14,16 or
1
in the H NMR spectra of 1 along with the mentioned signals there is a possibility of intracellular activation, depending on
of 1 and its hydrolysate. This implied that two other processes the cellular accumulation rate of 1. A similar effect has recently
are in progress for 1 under the used conditions. In particular, been investigated for the unstable, highly cytotoxic VV
a metabolism of 1 is unambiguously connected with the complex, which is supposed to be beneficial for a decrease of
release of the pic ligand, as proved by the presence of the C6– toxicity in the treated patients.17
1
H and C7–H3 resonances of free pic detected in the H NMR
Also of interest, the formerly reported Pt(II) diiodido com-
spectrum of 1 at 8.49 and 2.58 ppm, respectively (Fig. 2 and plexes were more stable in solution than their chlorido
analogues,7,9,18 which was not the case of the herein studied
picoplatin (2) and its markedly less stable diiodido analogue 1.
It should be noted that the N-donor ligand release, as observed
for some Pt diiodido complexes in the presence of various
biomolecules8,9 or when irradiated11 (see above), was also
reported for some Pt dichlorido complexes studied in the cell
culture medium19 or blood serum supplemented with meth-
ionine or N-acetylcysteine,20 but, to the best of our knowledge,
it has never been observed directly in solution (i.e., without
any biomolecules), as described in this work for 1.
The HPLC chromatogram of the freshly prepared solution
of picoplatin (2) in 50% MeCN/50% H2O (v/v) contained two
dominant peaks (tR = 2.25 and 3.90 min; Fig. 3). Two new
peaks were observed in the HPLC chromatogram of 2 at tR
=
5.23 and 6.12 min during its incubation up to 96 h of standing
at 37 °C (Fig. 3 and ESI, Fig. S8†). For two reasons, these new
signals are connected with the hydrolysis of 2. At first, their
coupled ESI+ MS showed the {[Pt(NH3)(pic)] – H}+ (304.1 m/z;
calc. 304.0 m/z), {[Pt(pic)] + MeCN – H}+ (328.0 m/z; calc. 328.0
Fig. 2 The results of the 1H NMR solution behaviour studies of 1
(black), given together with picoplatin (2; red) and free 2-methylpyridine
(pic; green); the samples were prepared in 50% DMF-d7/50% D2O and
incubated at 37 °C between the measurements.
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Fig. 4 The UV-Vis spectroscopy results as obtained at different time-
points for 1 (grayscale; 50% DMF/50% H2O, 37 °C). The spectrum of the
cis-[Pt(H2O)(NH3)(OH)(pic)]+ hydrolysate (red) is given for comparative
purposes.
Fig. 3 The results of the HPLC/ESI+ MS experiments performed for 1
1 and the spectrum of the co-studied cis-[Pt(H2O)(NH3)(OH)
(pic)]+ (measured for comparative purposes) suggested that the
mentioned hydrolysis is not the only process for 1 under the
used experimental conditions and that its solution behaviour
has to be understood as more complex than for the co-studied
picoplatin. This assumption of the presence of another Pt-
(black),
2 (picoplatin; red) and free 2-methylpyridine (pic; green).
Compounds were dissolved in 50% MeCN/50% H2O, and 1 and 2 were
recorded at multiple time-points up to 96 h of standing at 37 °C.
Hydrolysate = cis-[Pt(H2O)(NH3)(OH)(pic)]+.
m/z) and {[Pt(NH3)(pic)] + H2O + OH + MeCN}+ (380.9 m/z; based pic-containing species in the used mixture of solvents is
1
calc. 381.1 m/z) species, which were not found in the ESI+ consistent with the conclusions gained from the H NMR and
mass spectrum of the fresh solution. Secondly, both peaks (tR HPLC experiments (vide supra). Also of importance, no UV-Vis
= 5.23 and 6.12 min) were also detected in the HPLC chroma- spectra band was detected at ca. 400 nm, as reported pre-
togram of the synthetically dechlorinated picoplatin.
viously for cis-[PtI2(H2O)2],7 indicating that the solution behav-
Concerning the diiodido complex 1, its freshly prepared iour of 1 is not connected with the formation of the mentioned
solution (50% MeCN/50% H2O, v/v) analysed by HPLC/ESI+ bis-aqua entity, resulting from the release of both the N-donor
MS showed the dominant peak at tR = 6.55 min (Fig. 3), ligands (i.e., NH3 and pic) of 1.
belonging to the initial complex ([PtI(NH3)(pic)]+; 431.8 m/z;
The chemical reactivity toward the solvolysis of complex 1
calc. 432.0 m/z). With the incubation of 1 at 37 °C, several new was also studied by theoretical methods employing the ORCA
HPLC peaks were observed in the obtained chromatograms. 4.2 computational package and using density functional
The peak centred at tR = 5.28 min is consistent with the hydro- theory (DFT) with the PBE0 hybrid functional.21 Generally,
lysis of 1, as observed also for picoplatin (2; see above). such solvolysis proceeds by the associative mechanisms in
Another HPLC peak (tR = 2.23 min), although at the same posi- square planar Pt(II) complexes through the five-coordinated
tion as detected for 2, had a different ESI+ mass spectrum bipyramidal transition state, in which incoming and leaving
(e.g., the dominant peak at 346.1 m/z; calc. 346.1 m/z for {[Pt ligands are weakly bound to platinum.22 Firstly, we have
(pic)] + OH + MeCN}+) than 2 with no chlorine-containing studied the first step in the solvolysis of both 1 and picoplatin
peaks detected. Most importantly, the peak at tR = 8.89 min (2) and considered not only solvolysis by water, but also by
was clearly detected in the HPLC chromatograms of 1 after 6 h DMF, which was used during the experimental studies. As
of incubation at 37 °C (Fig. 3 and ESI, Fig. S9†) and this peak expected, the halogenido ligand in the trans-position to pic is
unambiguously belongs to the released pic ligand (94.2 m/z; the most prone to solvolysis by both solvents, having the
calc. 94.1 m/z), which is very well consistent with the above-dis- lowest energy of the transition state (TS), albeit the second
cussed 1H NMR results.
halogenido ligand reached only slightly higher values of ΔG‡
The results of the time-dependent UV-Vis studies of 1 (up (ESI, Fig. S10†). The Gibbs free energies and selected structural
to 96 h of standing at 37 °C; 50% DMF/50% H2O, v/v) showed parameters are listed in ESI, Table S1.† Interestingly, the ener-
that there is a gradual change of its composition (Fig. 4). In gies of TS related to the solvolysis of N-donor ligands (pic and
particular, a decrease of intensity of the maximum detected at NH3) are lowered for 1 in comparison with 2.
341 nm (t = 0 h) continued up to 72 h of standing, when an
Next, both steps of the solvolysis of halogenido ligands
equilibrium was reached with no further changes observed. were computed and relative Gibbs energies are depicted in
This is consistent with the observations reported for com- Fig. 5 and selected structural parameters are listed in ESI,
plexes cis-[PtI2(NH3)2]7 and [PtI2(dach)],18c both showing a Table S1.† Obviously, replacing chlorido ligands by iodido
gradual decrease of the band detected at ca. 350 nm, assigned ones led to lower energies of TS states in both steps of hydro-
to the hydrolytic replacement of the iodido ligands. Thus, the lysis thus speeding up the kinetics, which is in accordance
hydrolysis of the Pt–I bonds of 1 was also proved by UV-Vis with our experimental observations. Furthermore, we may
spectroscopy. Importantly, a difference between the spectra of notice that these reactions are less endergonic (ΔrG is lower)
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Fig. 5 Top: the molecular geometries obtained by DFT calculations for the hydrolysis of cis-[PtI2(NH3)(pic)] (1). Bottom: relative Gibbs free energies
of reactants (R), transition states (TS) and products (P) for solvolysis of cis-[PtI2(NH3)(pic)] (1) and cis-[PtCl2(NH3)(pic)] (picoplatin; 2) by water (left)
and DMF (right).
for 1. Similar results were found for solvolysis by DMF (Fig. 5),
but in this case, the energies of TS are rather higher than for
the hydrolysis, which means that DMF-solvolysis would be
slower. This nicely agrees with the NMR studies, in which the
hydrolysis was not observed in DMF unless it was mixed with
water. In all geometries of TS we observed the elongation of
Pt–X (X = I and Cl) and Pt–O bonds, e.g., the Pt–I bond varied
between 2.998 and 3.091 Å and Pt–O was close to 2.4 Å (ESI,
Table S1†). The <(X–Pt–O) angles, where X is the leaving halo-
Fig. 6 The Pt-based species detected in the water-containing solution
of 1.
genido ligand and O is the donor atom of incoming aqua or
the DMF ligand, are ca. 70–80°, and this angle is increasing in
testing of in vitro cytotoxicity in human cancer cells. Despite
the order Cl < I and H2O < DMF, which can be ascribed to
DMSO being reported as an unsuitable solvent for anticancer
steric effects (ESI, Table S1†).
Pt(II) complexes (as discussed above),14b we plan to investigate
Finally, thermodynamic data were computed for the cis–
the solution behaviour of 1 and 2 in this solvent as well as in
trans isomerization reactions of 1 and picoplatin (2) (ESI,
its mixture with water in the following study. In conclusion,
the results of the performed solution behaviour studies clearly
Table S2†). The Gibbs free energy of this reaction is almost
three-times higher for 1 showing deep impact of iodido
proved that the Pt(II) diiodido complex 1 undergoes several in-
ligands on the chemical properties of these complexes. Faster
solution processes (Fig. 6), which is in contrast to its chlorido
isomerization of 1 is also supported by the herein performed
analogue, i.e., clinically studied Pt(II) dichlorido complex pico-
platin (2).
experimental study.
In particular, besides the expected hydrolysis of the Pt–I
bonds of 1, we observed for the first time the release of the
N-donor ligand (heterocyclic pic in the case of this work). To
the best of our knowledge, the N-donor ligand release was
Conflicts of interest
observed for similar Pt(II) diiodido complexes only in the pres-
ence of relevant biomolecules8,9 or when irradiated.11 Since
the trans-effect of ligands of 1 follows an order I > py > NH3,
the NH3 release should predominate over the pic release from
the structure of 1. However, since the NH3 release was dis-
There are no conflicts to declare.
Acknowledgements
proved by the obtained results and direct evidence was The authors acknowledges the financial support from the pro-
obtained for the pic release, a contribution of the steric effects jects LO1305 and CZ.1.05/2.1.00/19.0377 and from the insti-
has to be taken into account as well for pic. Importantly, the tutional sources of the Department of Inorganic Chemistry,
instability of 1 under the aqueous conditions indicated that Palacký University in Olomouc, and supply of computational
further in-solution studies (e.g., interaction with intracellular resources by the project “e-Infrastruktura CZ” (e-INFRA
biomolecules, stability in the cell culture medium) are LM2018140) provided within the program Projects of Large
required to determine whether 1 could be submitted for the Research, Development and Innovations Infrastructures. We
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also thank Ms Natálie Macháňová for her help with syntheses,
Mrs Pavla Richterová for performing elemental analysis and
Dr Alena Šimová for recording FTIR spectra.
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