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Conformational control of anticancer activity: the application of arene-linked dinuclear ruthenium(ii) organometallics
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Cite this: Chem. Sci., 2014, 5, 2536
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Conformational control of anticancer activity: the
application of arene-linked dinuclear ruthenium(II)
organometallics†
Benjamin S. Murray,* Laure Menin, Rosario Scopelliti and Paul J. Dyson*
Dinuclear metal complexes have emerged as a promising class of biologically active compounds which
possess unique anticancer activity. Here, we describe a novel series of arene-linked dinuclear
organometallic Ru(II) complexes, where the relative conformation of the ruthenium centres is controlled
by the stereochemical configuration of 1,2-diphenylethylenediamine linker moieties, as confirmed by Xray crystallography. The reactivity and cytotoxicity of these compounds is compared to flexible dinuclear
and mononuclear analogues, demonstrating in all cases the complexes can undergo aquation,
coordinate to typical biological donor ligands and importantly, in the case of dinuclear analogues,
crosslink oligonucleotide and peptide sequences. Differences in the conformation of the isomeric
dinuclear compounds lead to significantly different levels of cytotoxicity against A2780, A2780cisR and
HEK-293 cell lines; isomers with a closed conformation are significantly more cytotoxic than isomers
with a more open conformation and they are also significantly less susceptible to acquired resistance
mechanisms operating in the A2780cisR cell line. These rigid dinuclear compounds possess markedly
increased cytotoxicity relative to the non-cytotoxic mononuclear analogues that does not appear to be
related to differences in complex lipophilicity or cellular uptake, which, in general, remain similar in
Received 11th January 2014
Accepted 24th March 2014
magnitude across the series. Thus, the molecular conformation of such dinuclear species may be crucial
in determining the nature of the adducts formed on coordination to biological targets in a cellular
DOI: 10.1039/c4sc00116h
environment, and opens up a novel route toward the development of more active metal-based
www.rsc.org/chemicalscience
anticancer agents.
Introduction
Metal-based compounds offer considerable potential in
medicinal chemistry where the careful choice of metal may
afford compounds possessing geometrical, coordination and
potentially catalytic properties not accessible through purely
organic molecules. As a result, the judicious combination of a
metal ion and associated ligands may result in complexes
capable of unique biological activity. In the context of developing metal-based drugs for the treatment of cancer, considerable effort has been directed toward the development of novel
platinum complexes,1 stemming from the clinical success of
cisplatin (and subsequent derivatives).
Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de
Lausanne (EPFL), CH-1015, Lausanne, Switzerland. E-mail: paul.dyson@ep.ch;
Fax: +41 (0)21 693 97 80; Tel: +41 (0)21 693 98 54
† Electronic supplementary information (ESI) available: Experimental procedures
for all novel compounds and copies of nuclear magnetic resonance spectra, X-ray
diffraction parameters, HPLC chromatograms for arene ligands of 5a and 6a,
UV-vis spectra and selected mass spectra. CCDC 978401–978404. For ESI and
crystallographic data in CIF or other electronic format see DOI:
10.1039/c4sc00116h
2536 | Chem. Sci., 2014, 5, 2536–2545
Apart from the platinum family of metallodrugs several other
metals have been utilised in the development and identication
of further classes of compounds that exhibit favourable medicinal attributes. Of these, ruthenium-based organometallic
compounds constitute a rapidly developing eld that continues
to yield complexes exhibiting diverse biological activity.2 Prominent examples include the [Ru(h6-arene)(en)Cl]+ family of
organometallics that has yielded compounds with a comparable
cytotoxicity to that of cisplatin in certain cell lines.3 Structurally
related compounds based on the [Ru(h6-arene)(L)Cl]+ scaffold,
with organic ligands (L) chosen because of their various biological activities, also exhibited signicant antiproliferative
activity against a range of cancer cell lines.4–6 An alternative
strategy, based on the development of kinetically inert ruthenium half-sandwich complexes as potent inhibitors of protein
kinases, has also led to complexes exhibiting high cytotoxicity
against the HCT-116 cell line.7 Ru(II)–arene complexes have also
been incorporated into multinuclear systems and assessed for
their anticancer activity. These have included metalla-cycles,8–12
metalla-cages13–15 and dendrimer-based systems.16 Of particular
interest to us are the [Ru(h6-arene)Cl2(PTA)] (RAPTA) series of
organometallic ruthenium(II) compounds which display selective activity on metastatic tumours in vivo.17,18 Recent work
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observed that proteins are a major intracellular target of the
RAPTA compounds,19 including the histone proteins of the
nucleosome core particle. Structural studies also show that
RAPTA-C binds to the protein component of the nucleosome
core particle in preference to the nucleic acid component.20 In
addition, subcellular localisation of RAPTA-T in A2780 and
A2780cisR cells revealed higher metal to protein ratios in the
particulate fraction (including the mitochondria) relative to the
cytosol and nuclear fractions.19 Further accounts have described
selective binding of RAPTA-C within protein mixtures21 and also
potent enzyme inhibition by RAPTA compounds.22,23 These
studies offer an insight into the potential of the RAPTA
compounds to exert their biological activity through protein
interactions rather than DNA damaging mechanisms characteristic of the vast majority of platinum metallodrugs reported so
far. We reasoned that dinuclear analogues of the RAPTA series
may exhibit a different spectrum of biological activity compared
to mononuclear RAPTA derivatives, potentially retaining the
propensity of the mononuclear RAPTA compounds to bind
proteins while acting via crosslinking of target biomolecules
through long-range interactions rather than short range interactions typical of mononuclear species. In the eld of platinum
metallodrugs the formation of dinuclear analogues has been a
successful route toward the development of compounds capable
of unique binding modes and interactions with DNA.24 This
approach has led to compounds of increased potency compared
to established mononuclear platinum compounds which retain
high activity in cell lines resistant to cisplatin.25,26 In contrast,
dinuclear organometallic ruthenium(II) arene compounds are
underexplored.27 In the most comprehensive of these studies to
date a series of dinuclear ruthenium compounds linked via
maltol-derived ligands were investigated.28–30 The cytotoxicity of
these compounds was found to be tuned by modication of the
chain length of the alkyl component of the linker ligand, with
cytotoxicity correlating well to the experimentally determined
lipophilicity of the resulting complexes.28 Of notable interest is
the observation that not only could these complexes crosslink
two DNA duplexes but also that DNA–protein crosslinks could be
formed, potentially a novel mode of action for such complexes.30
We hypothesised that the use of linker ligands rigid enough
to x the relative orientation of the metal centres in a dinuclear
RAPTA complex would allow access to a series of isomeric
structures of differing conformation. Such complexes would
allow an assessment of the effect of conformation on biological
activity to be probed, including the manner in which isomeric
complexes of different conformation interact with potential
target biomolecules.
Here we report the synthesis, binding studies with potential
biological targets and in vitro biological evaluation of a series of
dinuclear Ru(II)–arene compounds, where the conformation of
the RAPTA units relative to each other is controlled by the
stereochemical conguration of a 1,2-diphenylethylenediamine
(DPEN) linker molecule. Compounds with a linking group
possessing either an (R,R)- or (S,S)-conguration exhibit a more
‘closed’ conformation whereas those with a (R,S)-conguration
possess an open conformation not dissimilar to that of a
complex linked via a exible linker (Fig. 1). Such
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conformational control could signicantly inuence the interactions of each complex with biomolecular targets, ultimately
yielding a different range of adducts, and potentially controlling
the cytotoxicity and selectivity.
In addition to the rigid dinuclear complexes described above
a further dinuclear complex, linked together using the exible
ethylene diamine analogue, and a mononuclear analogue are
also reported. Our approach was to develop dinuclear Ru(II)–
arene compounds linked together through the arene ligand in
order to retain the core coordination environment around
ruthenium as in the original RAPTA series to avoid signicantly
perturbing the coordination mode of these dinuclear complexes
with potential biological targets.
Results and discussion
Synthesis and characterisation
The synthesis of the ruthenium dimers, and the mononuclear
analogue, proceeded via amide-forming reactions between the
common intermediate complex, 1, bearing a carboxylic acid
substituent on the arene ligand, and the selected amines
(Scheme 1). Complex 1 includes a single chelating oxalatomoiety as a protecting group in order to suppress undesired
reactions with the ruthenium centre during amide bond
formation. Reactions between 1 and the desired amines were
performed utilising the coupling agent O-(benzotriazol-1-yl)N,N,N0 ,N0 -tetramethyluronium tetrauoroborate (TBTU) with
N,N-diisopropylethylamine (DIPEA) in DMF, to either yield 3a
and 4a as DMF-insoluble yellow powders that were isolated by
ltration, or monomer analogue 2a and dinuclear compounds
5a and 6a as crude yellow powders following precipitation with
acetone. All compounds were further puried by recrystallisation until analytically pure (see ESI†). Each oxalato-protected
ruthenium complex was successfully converted to its chloridoanalogue by dissolution in an anhydrous solution of HCl in
methanol – the yellow solutions immediately turned red in
colour followed by precipitation of the desired dinuclear
compounds 3b–6b as their HCl salts, or in the case of monomer
analogue 2b the product was obtained by precipitation with
diethyl ether. All compounds (1–6b) are water soluble; the oxalato-protected compounds (2a–6a) possess low solubility in
DMSO and other organic solvents. All compounds were characterised by 1H, 31P and 13C NMR, high-resolution mass spectrometry and elemental analysis (see ESI†).
The molecular structures of 1, 2a, 4a, 5a and 6a (Fig. 2 and
S1†) were conrmed by single crystal X-ray crystallography on
Fig. 1 Newman projections of predicted conformations of dinuclear
complexes 4a–6a and 4b–6b based on ligand steric demand.
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Synthesis and numbering scheme of the mononuclear oxalato-complex 2a, dinuclear oxalato-complexes 3a–6a and chloridoanalogues 2b–6b.
Scheme 1
crystals grown via the slow diffusion of diethyl ether into a
methanolic solution of the complex (2a), or the slow diffusion of
acetone into a solution of the complex in H2O and acetone (1,
4a, 5a and 6a). In each case the coordination environment
around the central ruthenium ions is similar, adopting the
characteristic “piano-stool” conguration. Structural data
Fig. 2 Molecular structures of 1 (top left), 2a (top right), 4a (middle)
and 5a/6a (bottom). Hydrogen atoms have been omitted for clarity.
Atoms are colour coded: ruthenium – cyan, phosphorus – orange,
oxygen – red, nitrogen – purple, carbon – grey. See ESI† for figures of
the compounds including atom labels.
2538 | Chem. Sci., 2014, 5, 2536–2545
conrms the stereoisomeric complexes, 4a, 5a and 6a, occupy
conformations predicted by the Newman projections of the
DPEN linkers (Fig. 1). For the enantiomeric complexes 5a and 6a
linked by (1S,2S)-DPEN or (1R,2R)-DPEN respectively, the
structures are of a concave conformation with the trans-geometry of the phenyl rings of the linker backbone forcing the two
RAPTA units into positions adjacent to one another.‡ In
contrast, the conformation of the diastereomeric complex 4a
linked by (1R,2S)-DPEN is found to be more linear with the two
RAPTA substituents being related through a centre of inversion.
1
H-NMR spectra of the complexes in D2O shows that the
central linker signicantly inuences molecular conformation.
The spectrum of 3a (Fig. S30†), in which the RAPTA-units are
linked through a exible ethylene diamine moiety, is relatively
simple. Both sets of methylene protons and arene protons
manifest as a single set of resonances. In contrast, the spectra of
dinuclear complexes constructed with the stereoisomeric 1,2diphenylethylenediamine linking groups are more complex
(Fig. 3), the form of each being dependent on the stereochemical conguration of the linker used. The 1H NMR spectra of the
enantiomeric complexes 5a and 6a are identical and possess
four sets of Ru-coordinated arene resonances which are slightly
shielded relative to equivalent resonances observed in the
spectra of 1–3a. The methylene protons give rise to a complex
set of resonances between 2.4–2.6 ppm whereas resonances
corresponding to the aromatic protons of the linker consist of
two peaks between 7.1–7.3 ppm. For the diastereomeric
complex, 4a, two of the four Ru-coordinated arene resonances
overlap, with all arene resonances having a different chemical
shi compared to those of 5a and 6a. Interestingly, one of the
arene resonances of 4a (5.08 ppm) is signicantly more shielded
relative to the other arene resonances. In addition, the resonance set corresponding to the methylene protons is more
shielded and occurs between 2.16–2.33 ppm. These observations correlate well with the observation in the X-ray structure of
4a that the corresponding protons are shown to reside above the
plane of the linker phenyl groups – a magnetic environment
which would provide shielding from the applied magnetic eld.
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1
H NMR spectra of 4a (top) and 5a (bottom) (D2O, 400 MHz,
298 K, 10 mM). Resonances are identified with coloured circles: linker
phenyl – red, Ru arene – blue, CH – green, PTA – orange, CH2 – black
and CH3 – purple.
Fig. 3
Furthermore, in the spectrum of 4a the linker phenyl protons
give rise to a single resonance at 7.4 ppm. The increased
complexity in the spectra of 4a–6a relative to the spectrum of 3a
reects the increased central rigidity present in the former
complexes due to the DPEN linkers. In addition, the shielding
observed in the spectrum of 4a provides direct evidence that the
conformation observed in the solid state is also preserved in
aqueous solution.
Aquation and stability studies
Many metal complexes, including cisplatin and several families
of organometallic ruthenium complexes, are activated on
cellular internalization (low [Cl]) by aquation of labile metal–
chlorido bonds, the aquated species being signicantly more
labile toward donor groups of biological targets.31 Early work17
with a prototypical example of the RAPTA series, RAPTA-C,
revealed that upon dissolution in pure water or 4 mM NaCl
solution the complex rapidly undergoes exchange of both
chlorido ligands for aqua ligands. In 100 mM saline solution
this process is completely suppressed.
Oxalato- and chlorido-containing ruthenium compounds
synthesised in this work were assessed for their stability and
reactivity in phosphate buffer (10 mM) at pH 7.2 in the presence
of either 5 mM or 100 mM NaCl at 298 K. The UV-vis spectra of
the chlorido analogues 2b–5b immediately change upon
dissolution in phosphate buffer (10 mM) containing NaCl (5
mM), indicative of a change in coordination environment at the
Ru centre. The half-lifes of the chlorido complexes (2b–5b) were
estimated from the change in absorbance with time (see
Fig. S5–S9†). In phosphate buffer 3b possessed the shortest halflife (8.5 min) followed by 2b (10.5 min), 4b (13.6 min) then 5b
(17.3 min), the more closed structure of the latter compound
presumably slows the aquation process for this complex. For
comparison, under these conditions RAPTA-C has a half-life
shorter than all compounds described here (6.8 min). These
results indicate that although the different structures and
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conformations of compounds 2b–5b affect the kinetics of ligand
exchange at ruthenium, the ‘active’ aqua species of each
complex may be readily formed on dissolution in low [Cl]
aqueous solution. In the presence of elevated NaCl concentrations (100 mM) no aquation of these compounds was observed,
in accordance with previous results.17 The oxalato-protected
compounds 2a–5a were also examined by 31P NMR spectroscopy
in the presence of HEPES–phosphate buffer (5 mM or 100 mM
NaCl), and in the presence of BSA, CT-DNA and glutathione. In
each case the appearance of no new 31P-NMR signals were
observed over a period of 72 h at 310 K, demonstrating that the
oxalato complexes are inert towards aquation or ligand
exchange under ambient conditions on the timescale of the
experiment. In contrast, on incubation of 2a–5a in RPMI or
DMEM media used for cell culture it was found that a new
resonance at 32.4 ppm gradually appears in the 31P NMR
spectra over 48 h, with incubation of the complexes in DMEM
resulting in the largest transformation (Fig. S10–S16†). The new
resonance at 32.4 ppm may be attributed to the exchange of
the oxalato ligand for a carbonato ligand as sodium bicarbonate
is present in the RPMI and DMEM media used at concentrations of 23.8 mM and 44.0 mM respectively. Subsequent
experiments involving the incubation of 2a with NH4CO3H (23.8
mM) in phosphate buffer (pH 7.4, 50 mM) conrmed this
hypothesis, as the 31P NMR spectrum aer 24 h exhibits the
same transformation as the spectra recorded in RPMI/DMEM.
The oxalato-protected complexes 2a–6a are stable toward
aquation and inert in the presence of typical biological ligands,
whereas media containing bicarbonate at concentration levels
typically found in biological systems (10–30 mM), results in the
gradual exchange of the oxalato ligand for a carbonato ligand,
providing a possible route toward the intracellular activation of
the oxalato-protected compounds.
Amino acid and nucleotide binding studies
In order to assess the ability of the complexes to coordinate to
and crosslink biological targets, binding studies were performed with guanosine 50 -monophosphate, L-histidine and a
range of model oligonucleotides and peptides.
To establish whether each ruthenium ion of the dinuclear
compounds 3b–6b could coordinate simultaneously to target
ligands, binding studies were performed initially with guanosine 50 -monophosphate and L-histidine – both being good
ligands for RAPTA-type compounds as constituents of DNA or
proteins, respectively.20,32 For each dinuclear complex 3b–6b,
following incubation in the presence of 2 equivalents of 50 -GMP
(72 h, 310 K, pH 4.5 unbuffered), electrospray ionization mass
spectrometric analysis revealed the presence of a range of 2 : 1
and 1 : 1 50 -GMP–metal complex adducts. Interestingly, for the
complexes with the closed conformation, 5b and 6b, and the
exible ethylene diamine linker, 3b, these adducts include
examples where all four labile chlorido ligands are lost and
replaced with a single 50 -GMP molecule – indicating both
ruthenium centres are bridged via coordination to the same 50 GMP ligand. 1H and 31P NMR spectroscopy indicated the most
likely coordination sites are N7 and phosphate oxygen (see
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below). For the more open complex, 4b, the conformation of
which is expected to be less favourable for intramolecular
bridging coordination to occur, the corresponding peaks in the
mass spectrum are of a relatively low intensity, the peaks corresponding to 2 : 1 50 -GMP–metal complex adducts are of
higher relative intensity (Fig. S17†). Studies with the monomer
analogue 2b reveal the formation of only 1 : 1 50 -GMP–metal
complex adducts, even in the presence of 2 equivalents 50 -GMP.
For incubations of dinuclear compounds 3b–5b with 2 equivalents of 50 -GMP at pH 7.5 (310 K, 60 mM ammonium acetate)
only 1 : 1 adducts were detected. Adducts include examples
where all four chlorido ligands are replaced by a single 50 -GMP
ligand, alongside similar adducts also containing a coordinated
acetate ligand. It is likely that at pH 7.5 the deprotonation of the
50 -GMP phosphate (pKa of 6.49 reported for PO3H of 50 -GMP33)
facilitates the formation of intramolecularly bridged 1 : 1
adducts in preference to the formation of 2 : 1 50 -GMP–metal
complex adducts. Complementary binding studies between 50 GMP and 3b were also monitored by 1H and 31P NMR spectroscopy over 24 h (pD ¼ 7.5, 200 mM HEPES buffer, 310 K)
(Fig. S18†). Spectra are complex due to the formation of diastereoisomeric adducts upon coordination with 50 -GMP.
Multiple new 50 -GMP H8 resonances in the region 8.00–8.85
ppm were identied by 1H NMR spectroscopy corresponding to
adducts involving coordination of N7 of 50 -GMP to the ruthenium ion. Concurrent analysis with 31P NMR spectroscopy
revealed the appearance of new 50 -GMP resonances (2.90 to
8.45 ppm) indicative of phosphate coordination to the ruthenium ion alongside N7 coordination. This data complements
the observation of intramolecular crosslinking of these dinuclear RAPTA analogues by 50 -GMP as observed in the mass
spectrometric studies. A further distinctive proton resonance
set, centred at 7.05 ppm, also gradually appeared in the
aromatic region of the 1H NMR spectra. This was deduced to be
due to formation of the free arene ligand, indicating that upon
coordination of 50 -GMP to 3b, potentially through both phosphate and N7 coordination, the arene ligand is lost from the
adduct. In addition, the colour of the solution changed from
yellow to black over the 24 h incubation period indicative of
complex decomposition. Similar 50 -GMP–Ru adduct(s) were not
identied during the mass spectrometric studies although the
free arene ligand was detected by ESI-MS in the incubations
performed at pH 7.5. The loss of the arene ligand is not
unprecedented with similar loss of the arene ligand being
reported previously in binding studies of RAPTA-C with a 14-mer
oligonucleotide.34
Binding experiments were also performed with 2 equivalents of L-histidine at pH 7.1 and monitored by 1H and 31P
NMR spectroscopy and ESI-MS. As with 50 -GMP, ESI-MS
revealed the formation of a range of 1 : 1 adducts between Lhistidine and dinuclear compounds 3b–6b. Bridging of Lhistidine between the two ruthenium ions is observed for all
the dinuclear compounds; characterised by peaks corresponding to the loss of all four chlorido ligands in exchange
for a single histidine, although the intensity of these peaks are
relatively low compared to the intensity of other 1 : 1 adduct
peaks (Fig. S19†). Peaks corresponding to 2 : 1 L-histidine–
2540 | Chem. Sci., 2014, 5, 2536–2545
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metal complex adducts are also observed in all the spectra of
the dinuclear compounds, but are of low relative intensity
(<15%). For the mononuclear analogue 2b, in the presence of 2
equivalents of L-histidine, only peaks corresponding to 1 : 1
adducts are observed. 1H NMR spectroscopy indicates that loss
of the arene does not take place during the 24 h incubation,
and, unlike the 50 -GMP incubation, the colour of the solution
remains yellow.
As these small-molecule binding experiments conrm all
dinuclear complexes 3b–6b readily coordinate to appropriate
donor ligands simultaneously through both ruthenium
atoms, binding studies were expanded to include a short
model peptide sequence and a single-stranded 13-mer
oligonucleotide.
Oligonucleotide and peptide binding studies
Oligonucleotide binding studies were performed on the
13-mer sequence 50 -ATACATCGTACAT-30 in unbuffered
aqueous solutions (72 h, 310 K, pH ¼ 4.5, 0.2 mM complex and
2 eq. oligonucleotide). The reaction mixture was then diluted
and directly analysed using ESI-MS in negative ionization
mode. In higher mass regions of the spectra peaks
attributable to 1 : 1 oligonucleotide–metal complex adducts
are observed in which the metal complexes have lost their
chlorido ligands. No higher order adducts were detected.
However, the predominant species in these spectra correspond to oligonucleotide–metal complex adducts where the
arene ligand is lost leaving the Ru–PTA fragment coordinated
to the oligonucleotide. These results correlate with the NMR
binding studies of 50 -GMP with 3b where loss of the arene
ligand was observed.
Peptide binding studies utilised a fragment of amyloid bprotein (residues 1–16, H-Asp-Ala-Glu-Phe-Arg-His-Asp-SerGly-Tyr-Glu-Val-His-His-Gln-Lys-OH). This 16-mer contains
three histidine residues as well as one lysine and two glutamate residues – all of which have been observed to coordinate
to RAPTA compounds in crystallographic studies.20 Incubations were performed in unbuffered aqueous solutions (72 h,
310 K) in a 1 : 1 peptide–metal complex ratio. Using ESI-MS in
all cases a range of 1 : 1 adducts were observed with no 2 : 1
or higher order complex–peptide or peptide–complex adducts
detected. As with the small-molecule binding studies, 1 : 1
dinuclear metal complex–peptide adducts are detected where
all four labile chlorido ligands are lost (and, in some cases,
loss of PTA ligands is also observed) and substituted by a
single peptide. The loss of two or three ligands at each Ru
centre implies that each metal of the dinuclear compounds
must be coordinated to one or more amino acid residues of
the peptide. It is likely that crosslinked species where
each metal centre is bound to a different amino acid residue
form a proportion of these 1 : 1 adducts. ESI mass spectra of
peptide adducts of 4b, 5b and 6b reveals a similar
peak distribution (Fig. S20†). To probe the nature of the
binding within these 1 : 1 adducts electron-transfer dissociation (ETD) fragmentation studies were performed on
selected ions.
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Electron-transfer dissociation (ETD) fragmentation and ion
mobility-mass spectrometry (IM-MS) studies
ETD fragmentation of peptides is an established technique
used to randomly fragment the N–Ca bonds of a peptide backbone. This technique oen preserves post-translational modications of the peptide side chains, such as phosphorylation
and glycosylation, allowing their identication and localisation
in a peptide sequence to be determined.35 More recently, this
technique has been used to identify drug metallation sites in
peptide36 and protein samples.37 We used ETD to probe the
binding of 2b–6b to the 16-mer peptide H-Asp-Ala-Glu-Phe-ArgHis-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-OH (see ESI† for a
description of data analysis, see Table 1 for adducts analysed
and location of metallated residues). Analysis of the ETD
spectra of the 1 : 1 [peptide + 2b + 2H 2Cl]4+ and [peptide + 2b
+ 2H 2Cl PTA]4+ adducts revealed complex binding at the
His6, His13 and His14 sites. These observations could correspond to crosslinking of sites His6, His13 or His14 through
metallation by a single Ru complex where crosslinking then
breaks apart upon ETD fragmentation, or could correspond to a
population of three metallated peptides within the sample,
where the site of modication is solely at His6, His13 or His14.
For dinuclear complexes 3b–6b, ETD spectra provided direct
evidence that each of the complexes was able to crosslink the
peptide through simultaneous coordination of each ruthenium
centre to one or more different histidine residues at His6, His13
and His14 (Fig. 4). Further peptide adducts of each metal
complex were analysed where either a single metallated histidine site was identied or no metallated amino acid sites were
clearly identied (although metallation at the terminal amino
acid residues was discounted). These spectra provide only a
partial picture of the sites of metallation in these adducts but,
given the 1 : 1 peptide–complex stoichiometries of the adducts,
they also provide evidence towards crosslinking of the peptide
through the histidine residues. It was noticeable that ETD
spectra with 4b–6b are very similar for each particular adduct,
exhibiting virtually identical fragment distributions in each
case (Fig. S21 and S22†). These results suggest that although 4b
and 5b/6b possess different conformations there is sufficient
exibility in these peptide adducts to allow crosslinking
through the same histidine positions. To probe these adducts
further complementary binding studies were performed using
Table 1 Peptide–complex adducts subjected to electron-transfer
dissociation fragmentation and identified sites of metallation
Adduct
Identied site of metallation
[Peptide + 2b + 2H 2Cl]4+
[Peptide + 2b + 2H 2Cl PTA]4+
[Peptide + 3b + H 4Cl]5+
[Peptide + 3b PTA 4Cl]4+
[Peptide + 3b 4Cl]4+
[Peptide + X + H 4Cl PTA]5+
(X ¼ 4b, 5b or 6b)
[Peptide + X + H 4Cl]5+ and
[peptide + X 4Cl PTA]4+
(X ¼ 4b, 5b or 6b)
His6, His13 and His14
His6, His13 and His14
His6 and His13
His6, other sites unidentied
His6, other sites unidentied
His6 and His13 or His14
Central amino acid residues
(no metallation observed at
terminal residues)
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ion mobility-mass spectrometry (IM-MS). IM-MS has been
recently used to probe conformational changes in the protein
ubiquitin on platination with cisplatin38 and also in combination with fragmentation techniques to identify drug metallation
sites on peptides.36
We used IM-MS to probe the different complex–peptide
adducts analysed by ETD fragmentation and comparisons of
their arrival time distributions (ATDs) have been made (Table 2,
Fig. 5, 6 and S23–S26†). For the mononuclear complex-peptide
adduct formed through loss of all chlorido and a PTA ligand
[peptide + 2b + 2H 2Cl PTA]4+ a single peak was observed in
the ATD indicating this adduct exists as a single isomer in the
gas phase. For the equivalent adducts of the dinuclear
complexes ([peptide + X 4Cl PTA]4+ (X ¼ 3b–6b)) the ATDs
are more complex and exhibit two peaks (Fig. 5) indicating these
adducts all exist as two isomeric species in the gas phase. For
1 : 1 peptide–complex adducts, formed through loss of all
chlorido ligands from Ru whilst retaining all their PTA ligands
([peptide + X + nH nCl]4+ (X ¼ 2b–6b)), the ATDs in each case
consist of two peaks. These results show each adduct in this
series also exists as two isomeric species in the gas phase. A
comparison of the dri times at which each peak in the ATDs is
centred revealed that peaks for the [peptide + X + nH nCl]4+
adducts are all centred at equal or longer dri times than their
corresponding [peptide + X + nH nCl PTA]4+ adducts (X ¼
2b–6b). This reects the larger size of the former adducts due to
their retention of the PTA ligand, and highlights the important
role played by the ligand set around the Ru ion in determining
the shape of the adduct formed. The split distributions
observed in the ATDs of all the dinuclear Ru compound–peptide
adducts are likely due to crosslinking of the peptide by the
dinuclear compounds between His6–His13 and His6–His14 to
yield two adducts of different size. In contrast, the single peak
observed in the ATD of the adduct [peptide + 2b + 2H 2Cl
PTA]4+ may be due to the metallation at the three histidine sites,
as observed in the ETD analysis, yielding three adducts of
identical size. An alternative interpretation is that the peptide is
crosslinked by a single metal complex through sites His6, His13
and His14 to yield a single adduct; this interpretation correlates
with the metal fragment of this adduct having lost two chlorido
and one PTA ligand. Similar to the ETD analysis of the peptide
adducts of the isomeric complexes 4b–6b no differences are
observed in the ATDs of their peptide adducts, or between the
ATDs of the [X 4Cl + 2OH + 2H2O]2+ (X ¼ 4b–6b, Fig. S25†)
ions themselves. Combined, these data indicate that despite the
different conformations of the three complexes, as conrmed
by crystallographic and 1H NMR analysis, they have an identical
size, at least in the gas phase. In addition, these data show that
adducts formed between the peptide and the metal complexes
are similar in terms of their size and in terms of their preferential binding sites.
Evaluation of in vitro anticancer activity
The cytotoxicity of 2a–6a and 2b–6b was assessed in human
ovarian carcinoma (A2780), human ovarian carcinoma with
acquired resistance to cisplatin (A2780cisR) and human
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Fig. 4 ETD LTQ Orbitrap FTMS of the [H-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-OH + 3b + H 4Cl]5+ adduct
(top) and the [H-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-OH + 5b + H 4Cl PTA]5+ adduct (bottom) after a 100
ms interaction period with fluoroanthene radical anions. Fragments corresponding to metallation at His6 are labelled in black whereas fragments
labelled in red are those corresponding to metallation at His13. Fragments labelled with * correspond to a metallated fragment.
Data from IM-MS studies showing ion mobility drift times of peak maxima, measured monoisotopic masses of selected peptide adducts
or complex ions and the corresponding theoretical values
Table 2
Peptide adduct/ion
Ion mobility dri
time(s) (ms)
Measured monoisotopic
mass (m/z)
Theoretical monoisotopic
mass (m/z)
[Peptide + 2b + 2H 2Cl PTA]4+ (C97H140N28O29Ru)
[Peptide + 3b 4Cl PTA]4+ (C112H159N32O30PRu2)
[Peptide + 4b 4Cl PTA]4+ (C124H167N32O30PRu2)
[Peptide + 5b 4Cl PTA]4+ (C124H167N32O30PRu2)
[Peptide + 6b 4Cl PTA]4+ (C124H167N32O30PRu2)
[Peptide + 2b + 2H 2Cl]4+ (C103H152N31O29PRu)
[Peptide + 3b 4Cl]4+ (C118H171N35O30P2Ru2)
[Peptide + 4b 4Cl]4+ (C130H179N35O30P2Ru2)
[Peptide + 5b 4Cl]4+ (C130H179N35O30P2Ru2)
[Peptide + 6b 4Cl]4+ (C130H179N35O30P2Ru2)
[4b + 2OH + 2H2O 4Cl]2+ (C46H66N8O6P2Ru2)
[5b + 2OH + 2H2O 4Cl]2+ (C46H66N8O6P2Ru2)
[6b + 2OH + 2H2O 4Cl]2+ (C46H66N8O6P2Ru2)
[Peptide + 4H]4+ (C84H123N27O28)
2.93
2.88, 3.15
2.98, 3.42
2.98, 3.42
2.98, 3.42
2.98, 3.09
2.93, 3.42
3.04, 3.53
2.98, 3.53
2.98, 3.58
2.66, 2.88
2.66, 2.88
2.66, 2.88
2.66
565.77
666.78
704.80
704.80
704.80
605.04
706.06
744.07
744.08
744.07
546.12
546.12
546.12
489.51
565.73
666.74
704.76
704.76
704.76
605.00
706.01
744.03
744.03
744.03
546.13
546.13
546.13
489.48
embryonic kidney (HEK 293) cell lines using the MTT assay
(Table 3). As discussed earlier, the cytotoxicity of the original
RAPTA series toward a range of cancer cell lines is low (IC50
2542 | Chem. Sci., 2014, 5, 2536–2545
oen >300 mM) and these high IC50 values are mirrored by the
values determined for mononuclear compounds 2a and 2b,
which are >300 mM in the three cell lines. All dinuclear
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Fig. 5 Comparison of the ATDs of the [peptide + 2b 2Cl PTA +
2H]4+ (bottom), [peptide + 3b 4Cl PTA]4+ (middle) and [peptide +
4b 4Cl PTA]4+ (top) adducts. Inset shows the isotope profile of the
corresponding mass peaks in the mass spectrum.
Comparison of the ATDs of the [peptide + 2b 2Cl + 2H]4+
(bottom), [peptide + 3b 4Cl]4+ (middle) and [peptide + 4b 4Cl]4+
(top) adducts. Inset shows the isotope profile of the corresponding
mass peaks in the mass spectrum.
Fig. 6
compounds 3a–6a and 3b–6b are signicantly more cytotoxic
against the A2780 cell line than the mononuclear analogues. Of
these, the most cytotoxic compounds are those with the closed
conformation, i.e. 5a, 6a, 5b and 6b, with the chloridoanalogues (5b and 6b) being slightly more cytotoxic than the
oxalato-analogues (5a and 6a). Interestingly, the diastereoisomeric complexes 4a and 4b are signicantly less active, with
IC50 values increasing over 5-fold in the case of the chloridoanalogue and over 2-fold for the oxalato-analogue. The exible
dinuclear oxalato-complex, 3a, has a comparable cytotoxicity to
that of 4a whereas the IC50 value of the chlorido analogue, 3b, is
signicantly greater than that of 4b.
In the A2780cisR cell line the IC50 values of complexes 5a, 6a,
5b and 6b remain low, albeit, for complexes 6a and 6b, with the
linker of (R,R)-conguration, with a slight increase in IC50
values relative to those values obtained for the A2780 cell line.
The IC50 values of complexes 5a and 5b, with the linker of (S,S)-
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Chemical Science
conguration, remain essentially unchanged. In contrast, the
IC50 values for 3a and 3b (linked by the exible ethylenediamine
moiety) and 4a and 4b (with the linker of (R,S)-conguration)
increase dramatically. For example, 4a and 4b possess IC50
values 7 and 21 times, respectively, greater than those of their
most active diastereoisomers (5a and 5b) whereas 3a is 9 fold
less cytotoxic than the most active oxalato-compound 5a, and 3b
is essentially inactive in the A2780cisR cell line (IC50 > 300 mM).
In addition, compounds 5b and 6b are signicantly more active
than cisplatin in this cell line (over 3 fold in the case of 5b).
In the HEK-293 cell line, used as a model for non-tumorigenic cells, the activity of the compounds follows the pattern of
activity of the compounds observed in the A2780 cell line. The
cytotoxicity of 5a, 6a, 5b and 6b towards this cell line remains
higher than that of 3a, 3b, 4a and 4b with no signicant
differences in IC50 values between chlorido- and oxalatoanalogues observed for each case. One exception is that in this
cell line the activity of the chlorido complex 3b is greater than
that observed in the A2780 and A2780cisR cell lines and
comparable to the value of its oxolato analogue 3a, which in
other cell lines is more active.
The differences in activity between diastereoisomeric
complexes 4a–6a and 4b–6b are signicant, with an apparent
conformational dependence observed for both cytotoxic activity
and also for susceptibility to resistance in the A2780cisR cell
line. The complexes with the “closed” conformation consistently show high cytotoxicity against all cell lines examined, and
in the case of 5a and 5b, with the linker of (S,S)-conguration,
virtually no loss of activity against the A2780cisR cell line. In
contrast, the compounds with the more open structure (4a and
4b incorporating the linker of (R,S)-conguration), or those with
a exible ethylenediamine linker that may also be expected to
occupy an open conformation (3a and 3b), are signicantly less
cytotoxic and also show a dramatic decrease in cytotoxicity
against the A2780cisR cell line. To probe whether the origin of
these observations were due to the differential intracellular
uptake of these complexes, A2780 cells were incubated for 5 h
with 2a–6a (300 mM) and the intracellular ruthenium content
was determined using ICP-MS. It was found that the level of
internalised 4a (391 52 pmol Ru per 106 cells) is comparable
to that observed for 5a (332 25 pmol Ru per 106 cells).
Unexpectedly, under identical incubation conditions the level of
6a (901 97 pmol Ru per 106 cells) is signicantly higher than
that observed for 5a despite it having a very similar cytotoxicity
prole. At present, the origins of this differential uptake are
unknown, though a recent report has shown the chirality of
substituents on the ruthenium arene ligand can have a significant inuence on biological activity.39 Compound 3a exhibits
slightly higher cellular uptake levels (474 21 pmol Ru per 106
cells) than 4a and 5a whereas the mononuclear analogue 2a
(459 89 pmol Ru per 106 cells) displays the highest level of
internalised complex of all the compounds. These results
indicate that while the levels of ruthenium cellular uptake do
not correlate with the cytotoxicity proles of the compounds at
72 h, all compounds, under the experimental conditions,
readily internalise in A2780 cells and at comparable levels
observed for RAPTA-T.19 In addition to these uptake studies
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Table 3 In vitro anticancer activity of compounds 2a–6b in human ovarian carcinoma (A2780), human ovarian carcinoma cisplatin resistant
(A2780cisR) and human embryonic kidney 293 (HEK-293) cell lines after 72 h exposure, octanol–water partition coefficients for compounds 2a–
6a and cellular (A2780) uptake of ruthenium after exposure to 2a–6a for 5 h exposure at 300 mM
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IC50 (mM)
Compound
A2780
A2780cisR
HEK-293
log P
Cellular uptake
(pmol ruthenium per 106 cells)
2a
3a
4a
5a
6a
2b
3b
4b
5b
6b
Cisplatin
>400
23 0.5
25 2.5
10 0.5
8.5 0.5
>300
64 9.5
20 0.5
3.7 0.6
3.7 0.6
1.5 0.2
>400
88 10
74 6
10 1
15 0.5
>300
>300
95 6.5
4.5 0.2
7.0 0.5
14.5 2
>400
29 2.5
19 4
61
6 0.5
>300
35 3
24 3.5
8 0.5
5.9 0.2
7.5 1
1.57
1.26
1.38
1.41
1.36
—
—
—
—
—
—
459 89
474 21
391 52
332 25
901 97
—
—
—
—
—
—
the log P values for complexes 2a, 3a, 4a, 5a and 6a were
determined using the shake-ask method.40 These experiments reveal the hydrophilic nature of all complexes with little
variation in log P within the series. Therefore it is reasonable
to conclude that the differences in the cytotoxicity, at least in
the A2780 cell line, must be due to cellular events associated
with the complexes and their ability to crosslink target
biomolecules as demonstrated in binding studies, and is not
due to signicant differences in complex hydrophilicity and
cellular uptake.
Conclusions
We have developed a new route to arene-functionalised organometallic Ru(II) compounds, allowing access to exible and
conformationally rigid dinuclear organometallic Ru(II)
complexes. Crystallographic studies and NMR spectroscopy
revealed that the stereochemical conguration of the 1,2diphenylethylenediamine (DPEN) linker controls whether the
dinuclear complexes have an open or closed conformation. The
range of adducts formed between these rigid dinuclear
complexes, 4b–6b and the small molecules 50 -GMP and L-histidine appears to be governed by the conformation of the
complex, and all dinuclear complexes 3b–6b are capable of
crosslinking model peptide/oligonucleotide sequences. The
mononuclear complexes, as observed for the original RAPTA
series, are non-cytotoxic whereas dinuclear complexes are
signicantly more cytotoxic. Although the different conformations of 4b vs. 5b/6b resulted in no differences in type and size
of adducts formed in model peptide binding studies, an
apparent conformational dependence on the cytotoxicity of
these dinuclear complexes was observed. Those with the more
closed conformation (5a–6b) are signicantly more cytotoxic
than those with the more open conformation (4a and 4b) and
are unaffected by resistance mechanisms operating in the
A2780cisR cell line. In contrast, 4a and 4b were signicantly less
cytotoxic toward this cell line than the A2780 cell line. However,
for all dinuclear complexes, cytotoxicity toward the cancerous
2544 | Chem. Sci., 2014, 5, 2536–2545
A2780 cell line and the non-cancerous HEK-293 cell line varied
little. Further studies revealed the hydrophilicity of the
compounds, as assessed by measurement of log P values, was
similar within the series 2a–6a, and uptake experiments with
A2780 cells revealed an appreciable level of complex association
in each case. Combined, these observations lead to the
conclusions that the different cytotoxicities of the mononuclear
and dinuclear compounds is linked to the ability of the complex
to crosslink biomolecular targets and that the cytotoxicity of the
dinuclear compounds is signicantly linked to their
conformation.
Acknowledgements
This work was supported by a Marie Curie Intra-European
Fellowship within the 7th European Community Framework
Programme (Project 273658-DINURU to B.S.M). We thank the
Nestle Institute of Health Sciences for access to the Synapt G2-S
quadrupole-time-of-ight HDMS mass spectrometer and Julien
Bourquin for assistance in data collection and analysis. We
thank Dr Luc Patiny for developing calculation tools for interpretation of ETD fragmentation spectra. We thank Baihua Ye
for chiral HPLC analysis and Prof. Nicolai Cramer for access to
these facilities.
Notes and references
‡ Structural data obtained from single crystals of samples of 5a and 6a is identical,
with each crystal found to contain a 50 : 50 mixture of 5a and 6a resulting in the
solved structures exhibiting disorder in the region of the chiral centres of the
DPEN linker molecule. As the (1S,2S)-DPEN and (1R,2R)-DPEN linkers utilised in
the synthesis of 5a and 6a respectively were commercial samples with a stated
enantiomeric excess of 98% and 99% respectively, we postulate the racemic
composition of crystals of 5a and 6a are not reective of the bulk sample, but are
most likely a result of the co-crystallization of the desired and undesired enantiomers present in the samples. Chiral HPLC was used to determine the
percentage of each enantiomer in bulk samples of 5a and 6a by analysis of the
arene ligand following cleavage from the Ru ions (see Experimental section and
Fig. S2–S4† for details). This analysis showed that 5a was obtained with an
enantiomeric excess of 99.4% and 6a was obtained with an enantiomeric excess of
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99.8%. These results conrm that the bulk samples of 5a and 6a are of a high level
of enantiomeric purity and indicate that co-crystallization of the enantiomeric
impurity in each sample with the bulk product is the source of the racemic crystals
obtained.
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