PEOPLE
MARIE CURIE ACTIONS
Intra-European Fellowships (IEF)
DINURU
273658
FINAL REPORT
Period: 01.07.2012 to 30.06.2013
July 2013
1.
Publishable summary report
The continuing search for drugs that possess greater efficacy, are more selective, suffer
from lower incidences of resistance and exhibit lower general toxicity is essential against the
backdrop of figures showing that the number of people worldwide who die from cancer is
expected to increase over the next 25 years and that one in three Europeans will develop
cancer during their lifetime.
In the context of developing metal-based drugs for the treatment of cancer,
platinum complexes have received great attention – stemming from the success of cisplatin
(and derivatives). However, the use of cisplatin is limited by its associated side effects and
drug resistance. To circumvent these problems, and in efforts to identify more selective
compounds of increased activity, other metals have been utilized in the development of
novel drug candidates. Ruthenium-based organometallic compounds are amongst the most
promising alternatives and several families of such compounds have been evaluated and
have shown diverse biological activity profiles. One of these, the RAPTA series of
organometallic ruthenium(II) compounds, display selective activity on metastatic tumours in
vivo and experimental evidence has shown proteins are major intracellular targets of these
compounds, in contrast to the DNA damaging mechanism of action exerted by current
platinum-based metallodrugs.
Taking advantage of previous research on the RAPTA series of compounds and current
understanding of their mechanism of action, the main objectives of this project were:
The development of dinuclear analogues of the RAPTA series of compounds, including
conformationally rigid examples, to probe the influence of relative metal conformation
on mode of action and biological efficacy.

Screening of the compounds in appropriate cell lines using MTT assays to enable an
initial qualitative structure-activity analysis to be made.

Identification of molecular targets and characterization of complex-target interactions.
Work performed within the scope of this project took place in the Laboratory of
Organometallic and Medicinal Chemistry (Prof. Paul J. Dyson) at EPFL. The results and
progress achieved for each of the objectives is given below.
1. Synthesis of dinuclear organometallic Ru(II) compounds
In comparison to dinuclear platinum compounds there have been relatively few examples of
dinuclear ruthenium compounds synthesised in the context of anticancer drug
development, with no examples based on the RAPTA series being reported. However, from
previously published work on mononuclear RAPTA compounds it was clear that the arene
and PTA ligands may be further derivatized, thus providing a route toward the linking of two
structures through covalent tethering of two arene or two PTA ligands.
Various synthetic routes were explored, ultimately allowing access to dinuclear
organometallic ruthenium(II) compounds, tethered through the PTA or the arene ligands.
For each family of dinuclear compounds, chiral linkers based on 1,2diphenylethylenediamine (1,2-DPEN) were used that forced the RAPTA units into different
relative conformations. In addition, analogous flexible
linkers were also utilised. For the arene linked-compounds,
crystallization conditions were found that provide crystals
suitable for X-ray diffraction of dinuclear complexes with
chiral 1,2-DPEN linkers (see inset), confirming the relative
conformation of the two RAPTA units in these molecules
were as predicted.
2. In vitro evaluation of cellular cytotoxicity.
All compounds were screened for cytotoxic activity toward several tumorigenic and nontumorigenic cell lines using the MTT assay. For dinuclear compounds linked via their PTA
ligands no relationship between linker structure and cytotoxicity was observed with all
compounds exhibiting similar levels of moderate cytotoxicity toward all cell lines, indicative
of no cellular selectivity. In contrast, for the arene-linked compounds, cytotoxic activity was
observed to be dependent on the structure of the organic linker, with structures exhibiting a
more closed conformation being significantly more active than those with a more open
linear conformation. Furthermore, complexes with the closed conformation retained their
high level of cytotoxicity in the cisplatin resistant A2780cisR cell line whilst complexes with
the open conformation exhibited reduced cytotoxicity.
3. Identification of molecular targets and characterization of complex-target interactions.
The origins of the different cytotoxicities of the arene-linked dinuclear complexes was
probed via coordination studies with various biological targets, including amino acids,
nucleotides, peptides and oligonucleotides. In small molecule binding studies a clear
relationship was found between complex conformation and the nature of adducts formed.
Compounds with the closed conformation exhibited a higher proportion of 1:1 crosslinked
adducts with 5'-GMP and L-histidine whilst for the more open structures a higher proportion
of 2:1 ligand:complex adducts was
observed. Binding studies with model
peptides or oligonucleotides revealed
all dinuclear complexes were able to
crosslink these sequences through
simultaneous binding of both
ruthenium
centres
to
amino
acid/nucleotide residues.
Further
evaluation of peptide (H-Asp-Ala-GluPhe-Arg-His-Asp-Ser-Gly-Tyr-Glu-ValHis-His-Gln-Lys-OH) crosslinking was
achieved through electron-transfer
dissociation (ETD) fragmentation of
selected adducts (see inset). This study revealed coordination of the metal complexes to
the histidine residues of the peptide in all cases. Given the dramatically different
conformations observed for dinuclear compounds constructed with 1,2-DPEN ligands, it is
reasonable to conclude that the tertiary structure of such peptide adducts must be different
in each case.
Perspectives
Results from the research carried out within the scope of this Marie Curie fellowship have
provided an intriguing insight into how the biological activity of dinuclear metal complexes
may be influenced through control of complex conformation. The development of novel
metallodrugs is a promising approach in the search for compounds with anticancer activity.
This work feeds into this area by demonstrating that control of molecular conformation can
modify complex-ligand interactions, ultimately leading to increased activity, and also drug
resistance mechanisms operating in tumorigenic cells may be overcome.
2.
Use and dissemination of foreground

Invited keynote presentation at the International Symposium on Metal Complexes,
Burgos, Spain, 16th-20th June 2013.

Oral presentation and poster at the 20th EuCheMS conference on Organometallic
Chemistry, St Andrews, Scotland, 30th June – 4th July 2013.

Poster at the Fall meeting of the Swiss Chemical Society, Lausanne, Switzerland, 6 th
September 2013.
Manuscripts are currently in preparation to report the findings of this project and are
planned to be published over the next three months.