Maleimide-functionalised organoruthenium anticancer agents and their binding to thiol-containing biomolecules

Article abstract of DOI:10.1039/c1cc14713g

Ru^II(arene) anticancer compounds with maleimide functionality were prepared to allow selective interaction with thiol-containing biomolecules and thereby enforcing the selective delivery of the compounds to the tumour.

Full text of DOI:10.1039/c1cc14713g

View Online / Journal Homepage / Table of Contents for this issue
Chemical Communications
www.rsc.org/chemcomm
Volume 48 | Number 10 | 1 February 2012 | Pages 1325–1608
ISSN 1359-7345
COMMUNICATION
Hartinger et al.
Maleimide-functionalised organoruthenium anticancer agents and their binding to thiol-containing biomolecules

Dynamic Article Links
ChemComm
View Online
Cite this: Chem. Commun., 2012, 48, 1475–1477
www.rsc.org/chemcomm
COMMUNICATION
Maleimide-functionalised organoruthenium anticancer agents and their
binding to thiol-containing biomoleculeswz
Muhammad Hanif,ab Alexey A. Nazarov,c Anton Legin,a Michael Groessl,c
Vladimir B. Arion,a Michael A. Jakupec,a Yury O. Tsybin,c Paul J. Dyson,c
Bernhard K. Kepplera and Christian G. Hartinger*ad
Received 31st July 2011, Accepted 27th September 2011
DOI: 10.1039/c1cc14713g
RuII(arene) anticancer compounds with maleimide functionality
were prepared to allow selective interaction with thiol-containing
biomolecules and thereby enforcing the selective delivery of the
compounds to the tumour.
Metal-based drugs are applied for the treatment of different
diseases and in particular platinum complexes are among the
most widely used anticancer chemotherapeutics.1 One of the
Fig. 1 Strategy for tagging bioactive Ru complexes to thiol-containing
major drawbacks of Pt-based drugs is their low degree of
selectivity for tumour tissue, which results in a number of
serious side effects during the medication.1 Several different
strategies have been proposed to overcome the limitations of
Pt complexes, and targeting via tethering Pt to macromolecular
drug carriers appears promising.2 Notably, Kratz and co-workers
reported on Pt complexes linked to maleimides,3 known
to react selectively with thiols of biomolecules4 for transport
of bioactive moieties by human serum albumin (HSA) in
the blood serum. Such macromolecules can extravasate into
the tumour tissue via leaky vessels exploiting the enhanced
permeability and retention (EPR) effect. The EPR effect is
characterised by increased permeability from the blood vessels
into tumours because of rapid and defective angiogenesis.
Furthermore, drug accumulation is improved by a dysfunctional
lymphatic drainage in tumours.5
biomolecules.
biological activity.6–8 We have developed a series of Ru(arene)
compounds in which the arene is functionalised with a maleimide
moiety (Fig. 1), following a recently reported strategy.9 In
addition, the Ru coordination sphere is completed with chlorido
leaving groups and P-based co-ligands, i.e., carbohydrate-
derived phosphites and 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]-
decane (pta) and for comparison the bulky and lipophilic
triphenylphosphine, resembling drug candidates with known
anticancer activity.6,10,11 Ru(arene)(pta) complexes have been
shown to inhibit tumours in vivo,12 in particular with respect to
metastases which are often difficult to treat.13 On the other
hand Ru(arene) moieties linked to sugar derivatives were
developed to target a cytotoxic moiety selectively into the
tumour by exploiting the high energy demand of tumours
which can only be satisfied by glycolysis due to insufficient
supply via the blood stream.11,14,15
More recently, bioorganometallics and in particular anti-
cancer Ru(arene) complexes were found to exhibit promising
Complexes 1–4 were prepared by reacting dimeric
[Ru(Z6-N-benzylmaleimide)Cl2]2 with the respective ligand in
CH2Cl2 or dimethylformamide (DMF; Scheme 1). The com-
pounds were characterised by NMR spectroscopy, electro-
spray ionisation mass spectrometry (ESI MS), and elemental
analysis (see Supporting information for detailsz). The
31P{1H} NMR spectra feature several signals which coalesce
at elevated temperature, indicating the presence of a single
species (see Fig. S1z). Such behaviour has neither been
observed for the parent compound [dichlorido(Z6-p-cymene)-
(1,3,5-triaza-7-phosphaadamantane)ruthenium(II)] (RAPTA-C)
nor for related sugar-phosphite complexes with their p-cymene
ligands.5,11 The bulky maleimide substituent causes hindered
rotation along the Ru-arenecentroid axis compared to methyl/
isopropyl residues.
a University of Vienna, Institute of Inorganic Chemistry,
Waehringer Str. 42, A-1090 Vienna, Austria.
E-mail: christian.hartinger@univie.ac.at; Fax: +43-1-4277-9526;
Tel: +43-1-4277-52609
b Department of Chemistry, COMSATS Institute of Information
Technology Abbottabad Campus, University Road,
Abbottabad-22060, Pakistan
c
´
Institut des Sciences et Ingenierie Chimiques, Ecole Polytechnique
´
´
Federale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland
d The University of Auckland, School of Chemical Sciences,
Private Bag 92019, Auckland 1142, New Zealand
w This article is part of the ChemComm ‘Emerging Investigators 2012’
themed issue.
z Electronic supplementary information (ESI) available: Materials
and Methods, Synthetic procedures, NMR, SEC and ESI-MS studies
on the binding to thiols, X-ray diffraction data. CCDC 837010. For
ESI and crystallographic data in CIF or other electronic format see
DOI: 10.1039/c1cc14713g
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 1475–1477 1475

View Online
Scheme 1 Synthesis of RuII(Z6-N-benzylmaleimide) anticancer
compounds.
Fig. 3 Time course of the decrease of maleimide CH signals due to
reaction with Cys (k = 0.07 minꢀ1 [0.988]), GSH (k = 0.13 minꢀ1
[0.985]) and NAC (k = 0.09 minꢀ1 [0.998]) in D2O as compared to the
reaction of prehydrolysed 1 with Cys (k = 0.08 minꢀ1 [0.995]). The
data was normalised to the signal of d7-DMF as internal standard.
Fig. 2 1H NMR spectra of the reaction of 1 with Cys (1 : 1.2) in
D2O/100 mM NaCl. d7-DMF was used as internal standard (7.8 ppm).
Single crystals of 1 suitable for X-ray diffraction were grown
by slow diffusion of diethyl ether into a DMF/methanol
solution (the molecular structure of 1 is shown in Fig. S2z).
The ruthenium–centroidarene distance and the Ru-donor atom
bond lengths are similar to those for RAPTA-C for which two
crystallographically independent molecules were found in the
unit cell (see Table S2z).5 However, the P–Ru–Cl and the
Cl–Ru–Cl angles are very different which is due to the different
substituents at the arene moiety.
Fig. 4 MSn study to characterise the formed adduct in the reaction of
1 with Cys in aqueous solution; top: full scan mass spectrum, middle:
MS2 of parent ion at m/z 566, bottom: MS3 of parent ion at m/z 408.
with thiols. Compound 1 was allowed to hydrolyse for 24 h in
D2O (indicated by a shift in the 31P{1H} NMR spectra from
approximately ꢀ32 to ꢀ28 ppm) and afterwards Cys was
added. Similar kinetics were observed indicating high selectivity
for the nucleophilic addition of the thiol to the maleimide
moiety, especially under physiological conditions (phosphate
buffer pH 7.4, 100 mM NaCl; data not shown).
In order to determine the selectivity of thiols for the
maleimide functionality of 1 but not for its Ru centre, the
compound was reacted with the biomolecules Cys, glutathione
(GSH), N-acetylcysteine (NAC) and N-acetylcysteine methyl
ester (N-AcCysMe) in a molar ratio of 1 : 1.2 (complex : Cys) in
D2O/100 mM NaCl (pH 5.5) at room temperature. NaCl was
added to suppress aquation of the Ru centre.11,16 The 1H NMR
spectra show that the maleimide CH protons at ca. 6.8 ppm
disappear rapidly relative to the d7-DMF internal standard
assuming pseudo-first order kinetics (Fig. 2 and 3), accompanied
by labilisation of the arene–Ru bond and resulting in additional
signals at approximately 7.2 ppm (within 1 day, see Fig. S3z).
In the case of Cys, GSH and NAC the reactions proceeded
fairly quickly and within approximately 60 min, 1 was quanti-
tatively converted into the respective adduct (Fig. 3). The
reaction with N-AcCysMe in D2O/100 mM NaCl was too
rapid to be followed by NMR spectroscopy, which is related to
the pH in the incubation solution.
In addition to the NMR studies, ESI-Fourier transform ion
cyclotron resonance (FT-ICR)-mass spectra were recorded in
order to unambiguously characterise the formed species in the
reaction of 1 with the biological thiols. The reaction between
1 and Cys in a molar ratio of 1 : 1 yielded a species at
m/z 566.05635 (Fig. 4 and S4z), which can be assigned to the
[1 + Cys ꢀ 2Cl ꢀ H]+ ion, accompanied by a decrease of the
signal at m/z 463.04862 [1 ꢀ 2Cl + OH]+.
Fragmentation of the adduct ion at m/z 566 by collision-
induced dissociation resulted in the formation of two main
main product ions at m/z 408.97846 and 479.02212 (Fig. 4),
which were identified as fragments with the pta ligand
and with a part of the Cys residue cleaved, respectively. An
MS3 experiment with m/z 408 as precursor ion revealed the
sequential cleavage of the Cys backbone by decarboxylation
Furthermore, the influence of aquation of 1 was studied in
order to address the role of the metal centre on the reaction
c
1476 Chem. Commun., 2012, 48, 1475–1477
This journal is The Royal Society of Chemistry 2012

View Online
Table 1 In vitro anticancer activity (mean IC50 values ꢁ standard
deviation) of 1–4 in human ovarian (CH1), colon (SW480) and non-
small cell lung cancer (A549) cells (exposure time 96 h)
IC50 values/mM
CH1
SW480
A549
Compound
1
2
3
26 ꢁ 1
14 ꢁ 1
15 ꢁ 2
15 ꢁ 3
65 ꢁ 15
191 ꢁ 49
13 ꢁ 1
4640
63 ꢁ 4
92 ꢁ 31
116 ꢁ 5
4640
13 ꢁ 1
4
RAPTA-C
22 ꢁ 4
170 ꢁ 60
Cys residue (Cys34). These data indicate that Cys containing
biomolecules could act as carriers for such a compound class
as evidenced by their cytotoxicity toward cancer cells. Indeed,
we can speculate that HSA can deliver and ultimately release
the Ru(arene) fragments although further work must be
carried out to confirm this hypothesis.
Fig. 5 SEC–ICP-MS chromatograms for the reaction with human
serum albumin and human serum (1 : 1).
with the S remaining bound to the maleimide residue (Fig. 4).
These data indicate that the maleimide group preferentially
reacts with Cys and that the metal centre is not directly
involved in the adduct formation. Similar observations were
made for the reactions with GSH and NAC. In the reaction
of 1 with NAC the most abundant peak was assigned to
[1 ꢀ 2 Cl + NAC ꢀ H]+ (m/z 608.06700) and with GSH to
[1 ꢀ 2 Cl + GSH ꢀ H]+ (m/z 752.12089).
This work was supported by the Higher Education
Commission of Pakistan, the Austrian Exchange Service
¨
(OAD), the Hochschuljubilaumsstiftung Vienna, COST D39
¨
and CM0902 Fund. We gratefully acknowledge Alexander
Roller for collecting the X-ray diffraction data, Michaela Hejl
for performing in vitro anticancer assays and Prof. Markus
Galanski for recording the 2D NMR spectra.
In an attempt to characterise the reaction of 1 with HSA,
1H NMR spectroscopy and size exclusion chromatography
hyphenated to inductively coupled plasma mass spectrometry
(SEC–ICP-MS) studies were performed on reaction mixtures
containing 1 and HSA in molar ratios from 1 : 1 to 2 : 1
(protein : 1; see Fig. 5 and S5z). The maleimide CH protons are
overlapped by proton signals of aromatic amino acids of HSA,
however, the peaks assignable to d7-DMF and the maleimide
moiety were clearly distinguishable. The NMR spectra indicate a
significantly lower reactivity to HSA compared to the small
molecules. A similar picture was seen in the SEC–ICP-MS in
which only a small amount of Ru was detected bound to HSA
after about 3 h. However, after 72 h in serum the majority of
Ru was found in the 60–80 kDa fraction containing HSA
(Fig. 5 and Fig. S6z for SEC–UV/vis chromatograms).
Notes and references
1 M. A. Jakupec, M. Galanski, V. B. Arion, C. G. Hartinger and
B. K. Keppler, Dalton Trans., 2008, 183–194.
2 S. van Zutphen and J. Reedijk, Coord. Chem. Rev., 2005, 249,
2845–2853.
3 A. Warnecke, I. Fichtner, D. Garmann, U. Jaehde and F. Kratz,
Bioconjugate Chem., 2004, 15, 1349–1359.
4 P. Haquette, M. Salmain, K. Svedlung, A. Martel, B. Rudolf,
J. Zakrzewski, S. Cordier, T. Roisnel, C. Fosse and G. Jaouen,
ChemBioChem, 2007, 8, 224–231.
5 C. S. Allardyce, P. J. Dyson, D. J. Ellis and S. L. Heath, Chem.
Commun., 2001, 1396–1397.
6 C. G. Hartinger and P. J. Dyson, Chem. Soc. Rev., 2009, 38, 391–401.
7 A. Bergamo and G. Sava, Dalton Trans., 2011, 40, 7817–7823.
8 G. Gasser, I. Ott and N. Metzler-Nolte, J. Med. Chem., 2011, 54,
3–25.
9 P. Haquette, B. Talbi, S. Canaguier, S. Dagorne, C. Fosse,
A. Martel, G. Jaouen and M. Salmain, Tetrahedron Lett., 2008,
49, 4670–4673.
The in vitro activity of 1–4 was established in human ovarian
(CH1), colon (SW480) and non-small cell lung cancer (A549)
cells and compared to RAPTA-C (Table 1). In the chemosensitive
cell line CH1, all maleimide-functionalised compounds are
more active than the parent compound RAPTA-C. The role
of the phosphorus-containing ligand on the anticancer activity
becomes evident when comparing the data for SW480 and
A549 cells, in which the compounds bearing more liphophilic
carbohydrate-derived or triphenylphospine ligands are signifi-
cantly more active than the pta analogue and RAPTA-C.
In conclusion, we have functionalised anticancer RuII(arene)
compounds with maleimide moieties that undergo selective
reaction with thiol-containing biological nucleophiles via the
maleimide residue. This behaviour was observed in presence
of amino acids as well as of HSA, having an accessible
10 P. J. Dyson, Chimia, 2007, 61, 698–703.
11 I. Berger, M. Hanif, A. A. Nazarov, C. G. Hartinger, R. O. John,
M. L. Kuznetsov, M. Groessl, F. Schmitt, O. Zava, F. Biba,
V. B. Arion, M. Galanski, M. A. Jakupec, L. Juillerat-Jeanneret,
P. J. Dyson and B. K. Keppler, Chem.–Eur. J., 2008, 14, 9046–9057.
12 S. Chatterjee, S. Kundu, A. Bhattacharyya, C. G. Hartinger and
P. J. Dyson, JBIC, J. Biol. Inorg. Chem., 2008, 13, 1149–1155.
13 C. Scolaro, A. Bergamo, L. Brescacin, R. Delfino, M. Cocchietto,
G. Laurenczy, T. J. Geldbach, G. Sava and P. J. Dyson, J. Med.
Chem., 2005, 48, 4161–4171.
14 I. Berger, A. A. Nazarov, C. G. Hartinger, M. Groessl,
S.-M. Valiahdi, M. A. Jakupec and B. K. Keppler, ChemMedChem,
2007, 2, 505–514.
15 C. G. Hartinger, A. A. Nazarov, S. M. Ashraf, P. J. Dyson and
B. K. Keppler, Curr. Med. Chem., 2008, 15, 2574–2591.
16 C. Scolaro, C. G. Hartinger, C. S. Allardyce, B. K. Keppler and
P. J. Dyson, J. Inorg. Biochem., 2008, 102, 1743–1748.
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 1475–1477 1477