Maleimide-functionalised platinum(IV) complexes as a synthetic platform for targeted drug delivery

Article abstract of DOI:10.1039/c3cc39258a

Maleimide-functionalised Pt(iv) complexes with highly selective binding properties to thiol groups were synthesised as precursors for binding of thiol-containing tumour-targeting molecules like human serum albumin.

Full text of DOI:10.1039/c3cc39258a

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Maleimide-functionalised platinum(IV) complexes as a
synthetic platform for targeted drug delivery†
Cite this: Chem. Commun., 2013,
49, 2249
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Verena Pichler,z Josef Mayr,z Petra Heffeter,*bc Orsolya Domotor, Eva A. Enyedy,
´
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¨ ¨
Received 28th December 2012,
Accepted 31st January 2013
Gerrit Hermann,e Diana Groza,c Gunda Kollensperger,e Markus Galanksi,a
¨
Walter Berger,bc Bernhard K. Kepplera and Christian R. Kowol*ab
DOI: 10.1039/c3cc39258a
www.rsc.org/chemcomm
Maleimide-functionalised Pt(IV) complexes with highly selective
To cope with the enhanced ROS stress, malignant tissues
binding properties to thiol groups were synthesised as precursors establish reductive conditions based on increased extra- and
for binding of thiol-containing tumour-targeting molecules like intracellular levels of several bio-reductants such as glutathione
human serum albumin.
or thioredoxin.10 This should initiate tumour-associated
platinum(IV) reduction with release of the axial moieties and
Use of metal-based drugs in cancer therapy is well established simultaneous generation of the respective platinum(II) drugs
due to the worldwide success of the platinum(II) drugs cisplatin, with enhanced reactivity (Fig. 1).1,11 However, there is increasing
carboplatin and oxaliplatin.1,2 Nevertheless, therapy with these evidence that also platinum(IV) compounds are not specifically
compounds is characterised by severe adverse effects as a result activated only in tumour tissue but also in healthy tissues, espe-
of unselective damage to healthy tissues and acquired or cially in the presence of hemeproteins.12–14 Thus, it is of interest to
intrinsic resistance.3 One approach to overcome these draw- further enhance the tumour-targeting of platinum(IV) drugs.
backs of platinum(II) chemotherapeutics is the development
A well-established targeting strategy is the use of albumin, the
of platinum(IV) complexes. This class of compounds is kineti- most abundant protein in human blood serum which is known to
cally more inert and it is widely accepted that they act as accumulate in malignant and inflammatory tissues due to the
prodrugs. Thus, platinum(IV) complexes open up the possi- EPR (enhanced permeability and retention) effect.15,16 Moreover,
bility to develop platinum drugs with reduced toxicity and albumin can also be actively incorporated into cancer cells by
enable potential oral application. The most prominent endocytosis.17 Human serum albumin (HSA) possesses a single
platinum(IV) drug is satraplatin which was investigated in free thiol group (cysteine-34), which enables selective and defined
advanced phase III clinical trials.2,4 The additional axial preparation of drug–HSA conjugates using maleimide as a
ligands of platinum(IV) complexes allow coupling of bioactive coupling moiety. The favourable properties of HSA-coupled
molecules like estrogen,5 ethacrynic acid,6 folic acid,7 carbon anticancer drugs were already clinically proven for doxorubicin
nanotubes8 or gold nanoparticles.9
(INNO206; aldoxorubicin), currently in phase II clinical
studies18 and a HSA-based nanoparticle encapsulating paclitaxel
a University of Vienna, Institute of Inorganic Chemistry, Waehringer Strasse 42,
A-1090, Vienna, Austria. E-mail: christian.kowol@univie.ac.at;
Fax: +43-1-4277-52680; Tel: +43-1-4277-52609
b Research Platform ‘‘Translational Cancer Therapy Research’’ University of Vienna,
Waehringer Strasse 42, A-1090, Vienna, Austria
c Institute of Cancer Research and Comprehensive Cancer Center,
Medical University of Vienna, Borschkegasse 8a, A-1090, Vienna, Austria.
E-mail: petra.heffeter@meduniwien.ac.at; Fax: +43-1-40160-957555;
Tel: +43-1-40160-57557
d Department of Inorganic and Analytical Chemistry, University of Szeged,
´
´
Dom ter 7., H-6720 Szeged, Hungary
e Division of Analytical Chemistry, Department of Chemistry, University of Natural
Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria
† Electronic supplementary information (ESI) available: Materials and methods,
Synthetic procedures, pre-investigations for HSA binding, X-ray diffraction data,
RP-HPLC studies with cysteine, SEC-ICP-MS data. CCDC 916782. For ESI and
crystallographic data in CIF or other electronic format see DOI: 10.1039/c3cc39258a
‡ These authors contributed equally to this work.
Fig. 1 The concept of maleimide-functionalised platinum(IV) prodrugs.
Chem. Commun., 2013, 49, 2249--2251 2249
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The stability of the novel complexes in aqueous solution was
measured with RP-HPLC over a period of 24 h. For both
maleimide derivatives 3a and 3b slow hydrolysis could be
observed (o1% per hour, Fig. S1, ESI†). In comparison, the
recorded chromatograms of succinimide derivative 4b showed
no change within 24 h suggesting that the changes in the case
of 3a and 3b originate from slow hydrolysis of the maleimide
moiety. Subsequently, the complexes were incubated with an
excess of cysteine to analyse the reactivity of the thiol group
with the maleimide moiety (Fig. S2, ESI†). The results showed
an immediate and complete reaction of cysteine with com-
plexes 3a and 3b (o2 min). In contrast, the succinimide
derivative 4b showed no reaction during the same time (1.5 h,
Fig. S3, ESI†), which proves selective binding of cysteine to the
maleimide moiety without any reaction with the platinum
centre. The binding rate of 3a and 3b to HSA was measured
in aqueous solution in a 4 : 1 albumin-to-Pt ratio. The analyses
indicated rapid albumin linkage with a halftime of B1 h for
both maleimide complexes (Fig. 3). To further investigate
albumin binding in detail, 3a, 3b, and 4b were incubated with
HSA for 4 h at various concentrations in phosphate buffer,
followed by addition of excess 2,20-dithiodipyridine, which
reacts with the remaining free thiol groups of cysteine-34 and
results in generation of 2-thiopyridone, which can be quantified
by UV-vis spectroscopy (see ESI†). In the case of 4b, the thiol
groups of HSA remain unreacted at all tested concentrations,
whereas an exponential decrease in free SH was found for the
maleimide-containing complexes 3a and 3b (Fig. 4). These
data reveal that around 80% of the maleimide-containing
platinum(IV) compound is bound to HSA after 4 h incubation
at a concentration of >30 mM. The original concentration of free
SH groups from HSA was B25 mM (see ESI†) and a binding
saturation was reached at ca. 25–30 mM of the Pt-complex,
which suggests a 1 : 1 Pt–HSA adduct possibly due to steric
hindrance of the second maleimide group.
Scheme 1 Synthesis of novel platinum(IV) complexes ((a): N-N = 1,2-ethane-
1,2-diamine, Y = Cl; (b): N-N = 1R,2R-cyclohexanediamine, Y2 = oxalate).
(Abraxanes) approved for treatment of breast cancer and non-
small cell lung cancer (NSCLC).19
Herein, we report on the synthesis of the first platinum(IV)
complexes containing a maleimide moiety at the axial position,
which enables simple and fast coupling of thiol-containing
targeting molecules. This was confirmed by investigations with
cysteine and HSA and their anticancer activity was tested in vivo.
The starting compounds 1a and 1b were synthesised using
standard literature procedures, followed by oxidation with H2O2
(2a and 2b, Scheme 1).20 The maleimide-containing linker was
generated by conversion of maleimidopropanoic acid to iso-
cyanate, which was subsequently coupled with the platinum
centre by reaction with the hydroxido groups of the corresponding
platinum(IV) precursor in DMF yielding 3a (67%) and 3b (44%).
As a reference complex, which is unreactive towards thiols, the
succinimide functionalised compound 4b was synthesised using
an analogous procedure in good yield (53%). All complexes were
characterised by one- and two-dimensional NMR spectroscopy,
ESI-mass spectrometry and elemental analysis (see ESI†).
The results of an X-ray crystallographic study of § 3a are
shown in Fig. 2. The complex crystallised in the monoclinic space
group C2/c, with octahedral coordination geometry. In the equa-
torial plane the bidentate ethane-1,2-diamine and two chlorido
ligands are coordinated to the platinum(IV) core, whereas at the
axial position the two 2-maleimideethylcarbamoyl moieties are
bound. The bond lengths are in good accordance with those of
comparable compounds in the literature20,21 with Pt–Cl at around
2.3 Å and both Pt–O and Pt–N close to 2.0 Å (for bond lengths and
angles see Tables S1 and S2, ESI†).
To investigate the drug–protein binding properties of 3b in
fetal calf serum (FCS), SEC-ICP-MS measurements of sulphur
and platinum were performed (Fig. 5). The majority (>70%) of
platinum was detected in the frame of 8–9 min, which corre-
sponds to the retention time of albumin (see ESI†). Only small
amounts of 3b were found in the low molecular weight fraction,
while 4b was exclusively detected in this fraction.
Fig. 2 ORTEP plot of 3a with atom labelling scheme, the thermal ellipsoids have
been drawn at the 50% probability level.
Fig. 3 Binding studies of 3b with albumin using RP-HPLC.
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resulting in significantly reduced tumour growth in the case of
4b and even disease stabilisation in the case of the maleimide-
containing 3b. Thus, 3b had a significantly higher anticancer
activity (p o 0.01 at day 15) against CT-26 tumours than 4b
resulting in a distinctly lower mean tumour burden (0.38 g vs.
0.18 g for 4b and 3b, respectively, at day 15). Together, this
suggests that the albumin binding of 3b leads either to pro-
longed plasma half-life of the drug and/or a more selective
accumulation in the malignant tissue due to the EPR effect.
Although the exact nature of the involved bio-reductants and
the (sub)cellular localisation of drug activation is speculative so
far, our data prove that maleimide-mediated binding to serum
albumin is a feasible strategy to enhance anticancer activity of
platinum(IV) drugs in vivo.
Fig. 4 Free SH content of HSA at various platinum complex–HSA ratios after 4 h
incubation, 3a (J), 3b (m), 4b (&) [cHSA = 99.2 mM; pH = 7.40, 20 mM phosphate
puffer + 0.10 M NaCl, 2% (m/m) DMSO].
This work was supported by the ‘‘Fonds der Stadt Wien fu¨r
¨
innovative interdisziplinare Krebsforschung’’ (to P.H. and
´
C.R.K.), the OTKA project 103905 (to E.A.E.), the Austrian
´
Science Fund FWF (project L568 to W.B.) and E.A. Enyedy
gratefully acknowledges the financial support of J. Bolyai
research fellowship. We are thankful to V. Arion for collecting
X-ray diffraction data and G. Zeitler for animal care.
Notes and references
§ Crystallographic details: 3a: C16H22Cl2N6O8Pt, Mr = 692.39, 0.12 ꢀ
0.10 ꢀ 0.02 mm, monoclinic, C2/c, a = 26.9636(16) Å, b = 7.7770(4) Å, c =
11.3395(6) Å, b = 106.782(2)1, V = 2276.6(2) Å3, Z = 4, rcalcd = 2.020 g cmꢁ3
,
T = 150(2) K, l = 0.71073 Å, m = 6.454 mmꢁ1, R1 = 0.0181, wR2 = 0.0438,
GOF = 1.007; for description of data collection and refinement see ESI.
Fig. 5 Size-exclusion chromatography (SEC)-ICP-MS determination of 3b (50 mM)
in fetal calf serum after 2 h of incubation.
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c
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