Targeting the DNA-topoisomerase complex in a double-strike approach with a topoisomerase inhibiting moiety and covalent DNA binder

Article abstract of DOI:10.1039/c2cc31040f

Ru^II(arene)-flavonoids with high in vitro antitumour activity were synthesised. These compounds are capable of inhibiting human topoisomerase IIα and binding covalently to DNA.

Full text of DOI:10.1039/c2cc31040f

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Targeting the DNA-topoisomerase complex in a double-strike approach
with a topoisomerase inhibiting moiety and covalent DNA binderw
a
a
c
Andrea Kurzwernhart,ab Wolfgang Kandioller, Caroline Bartel, Simone Bachler,
¨
Robert Trondl,a Gerhard Muhlgassner,a Michael A. Jakupec,ab Vladimir B. Arion,ab
¨
Doris Marko,c Bernhard K. Kepplerab and Christian G. Hartinger*abd
Received 13th February 2012, Accepted 20th March 2012
DOI: 10.1039/c2cc31040f
RuII(arene)–flavonoids with high in vitro antitumour activity
were synthesised. These compounds are capable of inhibiting
human topoisomerase IIa and binding covalently to DNA.
Tumourigenic diseases are one of the major burdens of
mankind and many patients are still not treatable or do not
respond to standard drugs. This is often related to acquired or
intrinsic resistance which hampers the success of chemotherapy
with organic and inorganic anticancer agents, such as cisplatin.
In order to overcome this drawback, several approaches have
been used. The concept of multi-targeted anticancer agents
(Fig. 1), i.e., components of a molecule impact multiple separate
targets,1 has been shown to offer several advantages over
‘‘classic’’ chemotherapeutics, e.g., altered pharmacological
properties, metabolism and resistance development, tuneable
antitumour activity, ‘‘intramolecular’’ combination therapy,
and also selective targeted properties.1
Fig. 1 The concept of a multi-targeted small molecule. We aimed to
prepare a compound which is capable of binding via its ligand system
into the active site of a protein, whereas the metal fragment can form a
covalent bond to DNA.
advantageous toxicity profile.6 Flavonoids as secondary
metabolites of plants are such a compound class with a rich
variety of functions.7 Importantly, flavonoids are known to exhibit
properties such as antiradical and antioxidant, anti-inflammatory,
estrogenic, antimicrobial and also anticancer activity.8
We decided to link flavonoids to RuII(arene) moieties
(Scheme 1), since a few examples of metal–flavonoid com-
plexes are known to exhibit promising biological properties.9
However, these studies often did not aim to correlate the
biological activity with the inhibition of particular targets.10,11
Flavonoids act primarily through the inhibition of several
enzymes, and they have also been shown to interact with
human topoisomerases.7,12
Among the metal complexes developed as anticancer agents,
Ru(III) compounds are considered the most promising drug
candidates, and KP1019 and NAMI-A are currently under-
going clinical trials. More recently, RuII(arene) organometallics
have attracted considerable interest, and especially the RAPTA
family and ethylene-1,2-diamine complexes are at an advanced
preclinical development stage.2
One way to prepare biologically active molecules with multi-
targeted properties is to link metal fragments to bioactive
ligand systems. This strategy has already resulted in promising
approaches with compounds exhibiting novel modes of
action.3–5 Especially the use of ligand systems derived from
natural compounds appears attractive due to the often
The flavonol ligands 2a–d were prepared in two steps by a
Claisen–Schmidt condensation and subsequent Algar–Flynn–
Oyamada reaction (Scheme 1, ESIw).13–15 Ligands 2a–d were
converted in good yields into 3a–d with bis[dichlorido(Z6-p-cymene)-
ruthenium(II)] under alkaline conditions (Scheme 1).16 The
complexes only show minor signs of hydrolysis in aqueous
solution within 6 days.
a University of Vienna, Institute of Inorganic Chemistry,
Wa¨hringer Str. 42, 1090 Vienna, Austria.
E-mail: christian.hartinger@univie.ac.at; Fax: 43 1 4277 9526;
Tel: 43 1 4277 52609
b University of Vienna, Research Platform ‘‘Translational Cancer
Therapy Research’’, Wahringer Str. 42, A-1090 Vienna, Austria
¨
c University of Vienna, Institute of Food Chemistry and Toxicology,
In addition to characterisation by standard analytical methods
(Supporting Informationw), single crystals of 3bꢀCH3OH were
analysed by X-ray diffraction (Fig. 2).z 3b features a pseudo-
octahedral ‘‘piano-stool’’ configuration.17 The 3-hydroxyflavone
upon coordination to Ru acts as a bidentate ligand, forming
an envelope-like five-membered cycle, and the two Ru–O bonds
Wahringer Str. 38, 1090 Vienna, Austria
¨
d The University of Auckland, School of Chemical Sciences,
Private Bag 92019, Auckland 1142, New Zealand
w Electronic supplementary information (ESI) available: Materials and
methods, synthetic procedures, experimental setup. CCDC 826085.
For ESI and crystallographic data in CIF or other electronic format
see DOI: 10.1039/c2cc31040f
c
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Chem. Commun., 2012, 48, 4839–4841 4839

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Table 1 In vitro anticancer activitya (IC50 values in mM) of 2a–d and
3a–d in ovarian (CH1), colon (SW480) and non-small cell lung
carcinoma (A549) compared to cisplatin and topoisomerase IIa
inhibition of 2a–d and 3a–db
IC50/mM
Topoisomerase
inhibitionb
CH1
SW480
A549
2a
3a
2b
3b
2c
3c
+
++
+
++
+
++
++
+++
—
1.9 ꢁ 0.2
2.1 ꢁ 0.2
11 ꢁ 3
25 ꢁ 10
20 ꢁ 2
9.6 ꢁ 1.5
6.3 ꢁ 1.1
7.2 ꢁ 0.5
7.0 ꢁ 0.9
7.9 ꢁ 2.1
3.7 ꢁ 0.4
3.8 ꢁ 0.5
3.3 ꢁ 0.4
1.1 ꢁ 0.1
81 ꢁ 9
1.8 ꢁ 0.2
17 ꢁ 2
1.56 ꢁ 0.04
1.7 ꢁ 0.4
37 ꢁ 10
18 ꢁ 1
Scheme 1 Synthesis of ligands (2a–d) and RuII(Z6-p-cymene) complexes
(3a–d): (a) NaOH; (b) H2O2; (c) NaOMe; (d) [Ru(Z6-p-cymene)Cl2]2.
2a/3a: R = H, 2b/3b: R = CH3, 2c/3c: R = F, 2d/3d: R = Cl.
2d
3d
0.60 ꢁ 0.10
0.86 ꢁ 0.06
0.14 ꢁ 0.03
7.9 ꢁ 1.2
9.5 ꢁ 0.5
1.3 ꢁ 0.4
cisplatinc
a
b
96 h exposure. Estimated 50% inhibitory activity:+ 440 mM,
++ E 20–40 mM, +++ o20 mM inhibitor. Taken from ref. 25.
c
activity have been reported. The major part of them are
polypyridyl- and related RuII complexes with DNA intercalating
ligands (for a review see ref. 22) and only a few RuII(arene)
complexes are known which inhibit topoisomerases.23,24 In this
study, human topoisomerase IIa catalytic activity was deter-
mined by means of the decatenation assay (Fig. 3; Supporting
informationw).
Catenated kinetoplast DNA (kDNA) was incubated with
topoisomerase IIa in the presence of different concentrations of
the flavone complexes 3a–d and their ligands 2a–d. Depending
on the substituent in para position of the phenyl ring, differing
potential to inhibit topoisomerase IIa was observed. The
chloro compound 3d is the most potent inhibitor, and in
general the extent of inhibition correlates well with the
in vitro anticancer activity (Table 1). The complexes were
generally more active than the ligands, which could however
at least be related to a partial release of the ligand. The role of
the metal centre in topoisomerase inhibition was recently
shown for Cu-thiosemicarbazonato complexes, where the
Cu compounds were about an order of magnitude more
potent than the respective ligands.26 However, RuII(arene)
complexes per se inhibit the enzyme only to a minor extent,
as demonstrated for [Ru(Z6-benzene)(DMSO)Cl2].27 To the best
of our knowledge, this is the first example of metal compounds
that show topoisomerase inhibitory potency correlating to their
antiproliferative activity.
Fig. 2 Molecular structure of the RuII(Z6-p-cymene) complex
3bꢀCH3OH.
are slightly different with 2.076(2) and 2.099(3) A, as observed
in structurally related compounds.17 The phenyl substituent of
the ligand is twisted with a torsion angle of 15.111.
The stability of the complexes in aqueous solution has been
1
studied by H NMR spectroscopy.
The in vitro anticancer activity of ligands 2a–d and complexes
3a–d was determined in the human cancer cell lines CH1
(ovarian carcinoma), SW480 (colon carcinoma) and A549
(non-small cell lung carcinoma) by means of the colorimetric
MTT assay (Table 1). Notably, the IC50 values of 3a–d were
found to be in the low mM range, and only a few examples with
similar in vitro anticancer activity have been reported.1,18–20
The substituent in para position of the phenyl ring has a
significant influence on the in vitro activity, with IC50 values
of the unsubstituted compound 3a being 2–3 times higher than
those of the most active chloro derivative 3d. Compared to
cisplatin, 3a–c are only 2–3 times less active and 3d even
exhibits the same activity in the SW480 cell line. In the CH1
and A549 cell lines 3a–d are about one order of magnitude less
active than cisplatin (Table 1), but significantly more active
than the majority of known tumour-inhibiting organoruthenium
compounds.
The altered topoisomerase IIa inhibitory activity of the
complexes as compared to the ligands may be explained by
the multi-targeted character of the complexes. In order to
demonstrate the potential of the compounds to interact covalently
with DNA as the second target molecule, the reactions of 3a–d
In order to demonstrate the multi-targeted character of
RuII(flavone) complexes, we studied the inhibition of human
topoisomerase IIa activity and the binding ability to DNA
models. Topoisomerase IIa is over-expressed in many types of
cancer and inhibitors, such as doxorubicin, etoposide and
mitoxantrone, are routinely used in the clinic,21 but only a
small number of Ru complexes with topoisomerase inhibitory
Fig. 3 Effect of complex 3b and ligand 2b on the catalytic activity of
topoisomerase IIa, as determined by the decatenation assay.
c
4840 Chem. Commun., 2012, 48, 4839–4841
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CCDC 826085 contains the supplementary crystallographic data
for this paper (The Cambridge Crystallographic Data Centre,
www.ccdc.cam.ac.uk/data_request/cif).
´
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with the DNA model compound 50-GMP were studied by
1H NMR spectroscopy. Complexes 3a–d reacted quickly with
the N7 atom of 50-GMP (H8 shift from d = 8.1 to approxi-
mately 7.6 ppm). Notably, the flavone ligand remains attached
to the Ru centre to interact with topoisomerase IIa. However,
the simultaneous interaction of the compounds with DNA
and the protein is difficult to prove and will be subject of a
separate study.
The flavonoids and their RuII(Z6-p-cymene) complexes
are fluorescent, with an emission maximum at ca. 520 nm
(3c; lex = 458 nm). This intrinsic property was used to localise
3c in SW480 cells in co-staining experiments with fluorescence
confocal laser scanning microscopy (Fig. 4). 3c and the
endoplasmic reticulum (ER) marker ER-TrackerTM Red
(lex = 587 nm, lem = 615 nm) give largely overlapping
signals, and therefore we conclude that the ER is the primarily
targeted organelle. This observation is common for lipophilic
compounds,28 and the ER might act as a reservoir for the
cytotoxic species.
In conclusion, the RuII(arene)-flavonoid system offers access
to multi-targeted anticancer drugs consisting of a DNA binding
metal centre and a biologically active ligand system inhibiting
topoisomerase IIa. With the accumulation in the endoplasmic
reticulum as a reservoir for the anticancer active moiety and the
covalent binding to DNA accompanied by increased topo-
isomerase IIa inhibitory activity as compared to its ligand 2d
and the high in vitro antitumour activity, the RuII(Z6-p-cymene)
complex 3d is a promising development candidate for an
anticancer drug following a double-strike approach.
´
We thank the University of Vienna, the Austrian Science
Fund (FWF), the Johanna Mahlke geb. Obermann Founda-
tion, and COST D39 for financial support. We gratefully
acknowledge Alexander Roller for collecting the X-ray
diffraction data.
References
z Crystallographic details: 3bꢀCH3OH: C27H29ClO4Ru, Mr = 554.02,
%
0.12 ꢂ 0.05 ꢂ 0.01 mm, triclinic, P1, a = 7.8882(5) A, b = 11.9690(9) A,
c = 13.5794(11) A, a = 74.879(5)1, b = 73.366(4)1, g = 89.101(4)1,
26 B. M. Zeglis, V. Divilov and J. S. Lewis, J. Med. Chem., 2011, 54, 2391.
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1999, 38, 4382.
28 D. Marko, K.-H. Merz, C. Kunz, A. Muller, N. Tarasova and
¨
G. Eisenbrand, Biochem. Pharmacol., 2002, 63, 669.
V = 1183.49(15) A3, Z = 2, rcalcd = 1.555 mg mꢃ3, m = 0.807 mmꢃ1
,
Mo-Ka, l = 0.71073 A, T = 100(2)K, 2ymax = 27.501, 5420 measured
independent reflections, Rint = 0.0972, R1 = 0.0466, wR2 = 0.1020;
description of data collection and refinement see supporting information;
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 4839–4841 4841