From pyrone to thiopyrone ligands-rendering maltol-derived ruthenium(II)-arene complexes that are anticancer active in vitro

Article abstract of DOI:10.1021/om900483t

Ru(II)-arene complexes with pyrone-derived Iigands are rendered active against cancer cells by replacement of the coordinated 0,0 donor with an S,0 donor. The different stabilities of these systems may explain the observed influence of the donor atoms on

Full text of DOI:10.1021/om900483t

Organometallics 2009, 28, 4249–4251 4249
DOI: 10.1021/om900483t
From Pyrone to Thiopyrone Ligands-Rendering Maltol-Derived
Ruthenium(II)-Arene Complexes That Are Anticancer Active in Vitro
Wolfgang Kandioller,† Christian G. Hartinger,*,† Alexey A. Nazarov,*,†,‡
Maxim L. Kuznetsov,§ Roland O. John,† Caroline Bartel,† Michael A. Jakupec,†
Vladimir B. Arion,† and Bernhard K. Keppler†
†University of Vienna, Institute of Inorganic Chemistry, Waehringer Str. 42, A-1090 Vienna, Austria,
‡
ꢀ
ꢀ ꢀ
Institut des Sciences et Ingenierie Chimiques, Ecole Polytechnique Federale de Lausanne (EPFL), CH-1015
ꢀ
§
Lausanne, Switzerland, and Centro de Quımica Estrutural, Complexo I, Instituto Superior Tecnico,
´
Technical University of Lisbon, Av. Rovisco Pais, 1049-001 Lisbon, Portugal
Received June 8, 2009
Summary: Ru(II)-arene complexes with pyrone-derived li-
gands are rendered active against cancer cells by replacement
of the coordinated O,O donor with an S,O donor. The different
stabilities of these systems may explain the observed influence
of the donor atoms on the anticancer activity in vitro.
Scheme 1. Synthesis of the Complexes 2a-d
Metal complexes are playing an important role in the
treatment of cancer, and many promising compounds have
been developed in recent years.1-4 Ruthenium complexes have
been shown to be among the most promising candidates for
new metal-based anticancer drugs. Two of them, KP1019 and
NAMI-A, are currently undergoing clinical trials.2,5 Their low
general toxicity might be explained by their modes of action,
including protein binding and activation by reduction.5-7
More recently, bioorganometallic chemistry has emerged
as a new source of anticancer metallodrugs, with titanocene
dichloride being the prototype agent of this compound
class.4,8,9 Furthermore, organometallic Ru(II) compounds
that are stabilized in their þ2 oxidation state by coordination
of an arene ligand have been investigated for their anticancer
properties. These piano-stool complexes have been pio-
neered by the Dyson and Sadler groups,10,11 who developed
compounds with pta (1,3,5-triaza-7-phoshatricyclo[3.3.1.1]-
decane) and en (ethylenediamine) ligands, respectively.10 For
the [(η6-arene)RuII(X)(Y)] complexes, DNA base selectivity
strongly depends on the character of the chelating ligand Y-
exchange of the neutral ethylenediamine by anionic acetyla-
cetonate shifts the affinity from guanine to adenine.12 In
addition to en and pta complexes, maltol-derived mono- and
polynuclear ruthenium and osmium complexes have been
developed.13-15 The linking of two pyridone moieties
opened up new possibilities for tuning the in vitro anticancer
activity and lipophilicity, and compounds with interduplex
cross-linking capacity were obtained.14,16-18 In the case of
the mononuclear Ru(II) complexes, an increase in cytotoxic
activity was achieved by derivatization of the pyrone ring
with lipophilic aromatic substituents.13
In order to study the Ru-ligand interaction and its effect on
the in vitro anticancer activity, Ru(II)-cymene complexes
(Scheme 1) with pyrones and their corresponding, more
lipophilic thiopyrones as chelating agents were prepared.15,19
Such (thio)pyrone systems have already found application in
*To whom correspondence should be addressed. E-mail:
christian.hartinger@univie.ac.at (C.G.H.); alex.nazarov@univie.ac.at
(A.A.N.).
(1) Guo, Z.; Sadler, P. J. Adv. Inorg. Chem. 2000, 49, 183–306.
(2) Alessio, E.; Mestroni, G.; Bergamo, A.; Sava, G. Curr. Top. Med.
Chem. 2004, 4, 1525–35.
(3) Jakupec, M. A.; Galanski, M.; Arion, V. B.; Hartinger, C. G.;
Keppler, B. K. Dalton Trans. 2008, 183–194.
(4) Strohfeldt, K.; Tacke, M. Chem. Soc. Rev. 2008, 37, 1174–1187.
(5) Hartinger, C. G.; Zorbas-Seifried, S.; Jakupec, M. A.; Kynast, B.;
Zorbas, H.; Keppler, B. K. J. Inorg. Biochem. 2006, 100, 891–904.
(6) Groessl, M.; Reisner, E.; Hartinger, C. G.; Eichinger, R.; Semenova,
O.; Timerbaev, A. R.; Jakupec, M. A.; Arion, V. B.; Keppler, B. K. J. Med.
Chem. 2007, 50, 2185–2193.
(7) Dyson, P. J.; Sava, G. Dalton Trans. 2006, 1929–1933.
(8) Hartinger, C. G.; Dyson, P. J. Chem. Soc. Rev. 2009, 38, 391–401.
(9) Jaouen, G., Bioorganometallics; Wiley-VCH: Weinheim, Germany,
2006; p 444.
(13) Peacock, A. F. A.; Melchart, M.; Deeth, R. J.; Habtemariam, A.;
Parsons, S.; Sadler, P. J. Chem. Eur. J. 2007, 13, 2601–2613.
(14) Mendoza-Ferri, M. G.; Hartinger, C. G.; Eichinger, R. E.;
Stolyarova, N.; Jakupec, M. A.; Nazarov, A. A.; Severin, K.; Keppler,
B. K. Organometallics 2008, 27, 2405–2407.
(15) Kandioller, W.; Hartinger, C. G.; Nazarov, A. A.; Kasser, J.;
John, R.; Jakupec, M. A.; Arion, V. B.; Dyson, P. J.; Keppler, B. K.
J. Organomet. Chem. 2009, 694, 922–929.
(16) Mendoza-Ferri, M. G.; Hartinger, C. G.; Nazarov, A. A.;
Kandioller, W.; Severin, K.; Keppler, B. K. Appl. Organomet. Chem.
2008, 22, 326–332.
(17) Mendoza-Ferri, M. G.; Hartinger, C. G.; Mendoza, M. A.;
Groessl, M.; Egger, A.; Eichinger, R. E.; Mangrum, J. B.; Farrell, N. P.;
Maruszak, M.; Bednarski, P. J.; Klein, F.; Jakupec, M. A.; Nazarov, A. A.;
Severin, K.; Keppler, B. K. J. Med. Chem. 2009, 52, 916–925.
(10) Ang, W. H.; Dyson, P. J. Eur. J. Inorg. Chem. 2006, 4003–4018.
(11) Yan, Y. K.; Melchart, M.; Habtemariam, A.; Sadler, P. J. Chem.
Commun. 2005, 4764–4776.
(12) Fernandez, R.; Melchart, M.; Habtemariam, A.; Parsons, S.;
Sadler, P. J. Chem. Eur. J. 2004, 10, 5173–5179.
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(18) Novakova, O.; Nazarov, A. A.; Hartinger, C. G.; Keppler, B. K.;
Brabec, V. Biochem. Pharmacol. 2009, 77, 364–374.
(19) Lewis, J. A.; Puerta, D. T.; Cohen, S. M. Inorg. Chem. 2003, 42,
7455–7459.
r
2009 American Chemical Society
Published on Web 07/17/2009
pubs.acs.org/Organometallics

4250 Organometallics, Vol. 28, No. 15, 2009
Kandioller et al.
Figure 1. ORTEP plot of the molecular structure of 2b.
medicinal chemistry.20,21 The Ru(II)-cymene complexes were
obtained in good yields (64-85%) by deprotonation of the
ligand with sodium methoxide and reaction with bis[dichlorido-
(η6-p-cymene)ruthenium(II)]. The reaction was performed with
a slight excess of ligand to facilitate the purification. All
complexes have been fully characterized by 1D and 2D NMR
spectroscopy, mass spectrometry, and elemental analysis.
Single crystals of 2b were obtained from ethyl acetate, and
the molecular structure was determined by X-ray diffraction
analysis. The ruthenium center was found to adopt a piano-
stool configuration (Figure 1 and the Supporting Information).
Due to the larger sulfur atom in 2b, as compared to the oxygen
˚
in maltol-derived ligands, the Ru-S bond (2.3730(3) A) is signi-
˚
ficantly longer than the Ru-O bond (2.0808(10) A). This leads
to a strong distortion of the five-membered chelate ring of the
˚
complex. The length of the Ru-Cl bond in 2b (2.4331(4) A) is
comparable to the corresponding bond lengths of pyrone and
pyridone Ru(II)-arene complexes.14,15
The antiproliferative activities of 2a-d were investigated
in the human tumor cell lines SW480 (colon carcinoma) and
CH1 (ovarian carcinoma) by using the colorimetric MTT
assay (Figure 2). The IC50 values are presented in Table 1. As
reported earlier,14 the maltol complex 2a shows limited
cytotoxic activity, and the allomaltol derivative 2c has IC50
values >200 μM. In contrast, the thiopyrone complexes 2b,d
are at least by an order of magnitude (in IC50) more active
than their pyrone analogues. For complexes 2b,d, an inverted
sensitivity of SW480 cells and CH1 cells was observed, which
is in contrast with the case for a broad spectrum of other
compounds but parallels that observed with Ru(II)-arene
complexes containing an 8-quinolinolato ligand.22 The sub-
stitution pattern influences the activity, as inferred from the
2.7-3.9 times higher activity of 2b as compared to 2d. These
compounds are less cytotoxic than other ruthenium com-
plexes23 but are still in a reasonable range of activity in vitro
in comparison to other Ru-arene species.22 However, the in
vitro effects are only a first indication for potential activity,
which needs to be verified in vivo.
Figure 2. Hydrolysis of Ru-(thio)pyronato complexes (X=O, S),
yielding in the case of the pyrone ligands the dimeric [Ru2-
(cym)2(OH)3]þ species (as demonstrated by ESI-MS; top left).
When the aqua complexes 3a,c were reacted with imidazole, no
dimerization was observed (top right), as was the case for the
thiopyrone compounds 3b,d, which show significant cytotoxic
activity in human cancer cell lines (bottom).
Accordingly, hydrolysis and stability in water were investi-
gated by NMR spectroscopy in D2O or 10% DMSO-d6/90%
D2O solutions for the maltol and thiomaltol compounds,
respectively. The complexes 2a-d hydrolyze within seconds
to charged complexes by exchange of the chlorido ligand
with a water molecule (Figure 2). No coordination of
DMSO-d6 was observed, and identical NMR spectra
were obtained by activation of 2a,c with an equimolar
amount of AgNO3, which confirms the obtained results.
Similarly to the case of 2a,13 the formation of a dimeric
species was observed for 2c (Figure 2, top left), as demon-
strated by ESI-MS with a sample of 25 μM; this concentra-
tion was chosen in order to obtain a mass spectrum
containing both the intact complex and the dimeric species.
The amount of side product formed depends on the concen-
tration, time, and pH value of the solution, and its formation
can be prevented by addition of an equimolar amount of
imidazole, as also demonstrated by means of ESI-MS
(Figure 2, top right).
The in vitro inactivity of the maltolato complexes has been
attributed to the formation of dimers in aqueous solution.13
(20) Lewis, J. A.; Mongan, J.; McCammon, J. A.; Cohen, S. M.
ChemMedChem 2006, 1, 694–697.
(21) Thompson, K. H.; Barta, C. A.; Orvig, C. Chem. Soc. Rev. 2006,
35, 545–556.
(22) Schuecker, R.; John, R. O.; Jakupec, M. A.; Arion, V. B.;
Keppler, B. K. Organometallics 2008, 27, 6587–6595.
(23) Jakupec, M. A.; Reisner, E.; Eichinger, A.; Pongratz, M.; Arion,
V. B.; Galanski, M.; Hartinger, C. G.; Keppler, B. K. J. Med. Chem.
2005, 48, 2831–2837.

Communication
Organometallics, Vol. 28, No. 15, 2009 4251
Table 1. Antiproliferative Activity, ΔG of Hydrolysis, and Bond Energies (kcal/mol) of 2a-d (20a-d)
IC50 (μM)
2a
2b
2c
2d
CH1
SW480
>100
>100
13 ( 4
5.1 ( 0.5
239 ( 22
359 ( 119
35 ( 8
20 ( 7
20a
20b
20c
20d
ΔGhydr
E(Ru-Cl)
E(Ru-O/S)O/SdC
E(Ru-O)O-C
4.2
3.3
7.1
34.6
35.7
4.9
3.6
7.9
34.4
36.2
10.6
26.8
36.9
11.1
26.4
36.6
Hydrolysis of 2b,d in 10% DMSO-d6/D2O results in the
rapid formation of aqua species, which are stable for more
than 24 h; in contrast to the case for the maltolato complexes,
no dimers have been observed. To explain these observa-
tions, theoretical DFT calculations (B3LYP, Gaussian 03
program package;24 see the Supporting Information for
computational details) of the metal-ligand bond energies
have been performed. They reveal that the Ru-SSdC bond
energies in the model complexes 20b,d (bearing benzene
instead of cymene in 2b,d) are higher by 7.8-8.0 kcal/mol
than the Ru-OO=C bond energies in 20a,c, respectively.
In contrast, the Ru-OO-C bond energies in thiopyrone
complexes are lower than those in pyrone species, but
only by 0.4-1.2 kcal/mol. The stronger binding of thiopyr-
ones to Ru as compared to pyrones explains why thiopyrone
complexes are stable in aqueous solution while pyrone
complexes undergo decomposition with release of the li-
gands 1a,c.
Theoretical calculations of 20a-d and 30a-d show that ΔG
values for the substitution of chloride with H2O are only
slightly positive (3.3-4.9 kcal/mol). Taking into account the
large excess of water, complexes 3a-d are the predominant
species in aqueous solutions. Lower Ru-Cl bond energies in
20b,d as compared to 20a,c, respectively, correlate with ΔG
values of hydrolysis (Table 1).
thiopyrone compounds behave quite differently in water
(dimer formation). These features have important implica-
tions for the mode of action of the compounds and result
in considerably active thiopyrone vs minimally active
pyrone complexes. Furthermore, complexes 2b,d are slightly
more active in SW480 cells, though CH1 cells are in our
experience more chemosensitive to the vast majority of
metal-based and other tumor-inhibiting compounds tested
so far.
€
Acknowledgment. We thank the Hochschuljubilaums-
stiftung Vienna, the Theodor-Korner-Fonds, COST
€
€
D39, and the Austrian Science Fund (Schrodinger Fel-
lowship J2613-N19 (C.G.H.) and project P18123-N11)
for financial support and the computer center of the
University of Vienna for computer time at the Linux-
PC cluster Schroedinger III. This research was supported
by a Marie Curie Intra European Fellowship within the
7th European Community Framework Programme pro-
ject 220890-SuRuCo (A.A.N.). We gratefully acknowl-
edge Alexander Roller for collecting the X-ray diffraction
data.
Supporting Information Available: Text, tables, figures, and a
CIF file giving the general procedure for the synthesis of the
complexes, characterization, elemental analysis data, X-ray
diffraction data, and computational details. This material is
available free of charge via the Internet at http://pubs.acs.org.
Crystallographic data for this paper are also available from the
Cambridge Crystallographic Database (CCDC 720741).
In summary, the modification of the first ligand sphere
and therewith of the stability of the complexes influences
significantly the in vitro anticancer activity. Pyrone and
(24) Gaussian 03, Revision D.01; Gaussian, Inc., Wallingford, CT, 2004.