Tuning the hydrophobicity of ruthenium(II)-arene (RAPTA) drugs to modify uptake, biomolecular interactions and efficacy

Article abstract of DOI:10.1039/b705449a

The antitumour activity of the organometallic ruthenium(ii)-arene mixed phosphine complexes, [Ru(η⁶-p-cymene)Cl(PTA)(PPh ₃)]BF₄ 1b and [Ru(η⁶-C₆H ₅CH₂CH₂OH)Cl(PTA)(PPh₃)]BF ₄ 2b (PTA = 1,3,5-triaza-7-phosphaadamantane), have been evaluated in vitro and compared to their RAPTA analogues, [Ru(η⁶-p-cymene) Cl₂(PTA)] 1a and [Ru(η⁶-C₆H ₅CH₂CH₂OH)Cl₂(PTA)] 2a. The results show that the addition of the PPh₃ ligand to 2a increases the cytotoxicity towards the TS/A adenocarcinoma cancer cells, which correlates with increased uptake, but also increases cytotoxicity to non-tumourigenic HBL-100 cells, thus decreasing selectivity. The decrease in selectivity has been correlated to increased DNA interactions relative to proteins, demonstrated by reactivity of the compounds with a 14-mer oligonucleotide and the model proteins ubiquitin and cytochrome-c. This journal is The Royal Society of Chemistry.

Full text of DOI:10.1039/b705449a

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Tuning the hydrophobicity of ruthenium(II)–arene (RAPTA) drugs to modify
uptake, biomolecular interactions and efficacy†‡
Claudine Scolaro,a Adrian B. Chaplin,a Christian G. Hartinger,a Alberta Bergamo,b Moreno Cocchietto,b
Bernhard K. Keppler,d Gianni Savab,c and Paul J. Dyson*a
Received 12th April 2007, Accepted 22nd May 2007
First published as an Advance Article on the web 26th September 2007
DOI: 10.1039/b705449a
The antitumour activity of the organometallic ruthenium(II)–arene mixed phosphine complexes,
6
6
[Ru(g -p-cymene)Cl(PTA)(PPh3)]BF4 1b and [Ru(g -C6H5CH2CH2OH)Cl(PTA)(PPh3)]BF4 2b (PTA =
1,3,5-triaza-7-phosphaadamantane), have been evaluated in vitro and compared to their RAPTA
6
6
analogues, [Ru(g -p-cymene)Cl2(PTA)] 1a and [Ru(g -C6H5CH2CH2OH)Cl2(PTA)] 2a. The results show
that the addition of the PPh3 ligand to 2a increases the cytotoxicity towards the TS/A adenocarcinoma
cancer cells, which correlates with increased uptake, but also increases cytotoxicity to non-tumourigenic
HBL-100 cells, thus decreasing selectivity. The decrease in selectivity has been correlated to increased
DNA interactions relative to proteins, demonstrated by reactivity of the compounds with a 14-mer
oligonucleotide and the model proteins ubiquitin and cytochrome-c.
Introduction
Platinum-based drugs are among the most effective clinical agents
for the treatment of cancer with cisplatin, carboplatin, and
oxaliplatin in widespread use.1 These drugs exert their cytotoxicity
by binding to DNA and all three compounds are believed to
have similar molecular-level actions.2 However, they present some
clinical problems including acquired or intrinsic resistance that
limits the spectrum of cancers that can be treated.3 For most
forms of disseminated cancer, nevertheless, no curative therapy
is available, and the discovery and development of novel active
chemotherapeutic agents remains crucial.
A plethora of non-platinum metal complexes have also been
prepared and tested for anticancer activity.4 Notably, ruthenium
complexes have been found to be effective against cancers that
cannot be treated with platinum drugs and it was also found
that they exhibit a lower general toxicity compared to platinum
compounds.5,6 Two ruthenium compounds that have a different
spectrum of activity to platinum drugs7 are currently under-
going clinical evaluation.8 One, NAMI-A (see Chart 1), shows
high selectivity for solid tumour metastases and low toxicity at
pharmacologically active doses and has successfully completed
phase I clinical trials.9 Metastasis control is associated with a
series of biological activities that influence cell functions such
as adhesion, motility, and invasion of tumour cells10 involving
f-actin condensation and a reduction of gelatinolytic capacity.11
Chart 1 Clinically evaluated ruthenium-based anticancer drugs.
The other ruthenium compound, KP1019, was found to be active
against colorectal tumours12 and has successfully completed phase
I clinical trials.8a,13 Applying therapeutic doses of KP1019 neither
caused significant toxic side effects in test animals nor in patients
included in a clinical phase I study. Regarding the tumour-
inhibiting properties of KP1019, 5 out of 6 evaluable patients
treated with KP1019 showed disease stabilisation for up to 10
weeks.8a
More recently, increasing interest has focused on organometallic
compounds,14 specifically on ruthenium(II)–arene compounds
which show excellent antiproliferative properties in vitro and
in vivo.6,15 Our research has focused on compounds with
aInstitut des Sciences et Inge´nierie Chimiques, Ecole Polytechnique
Fe´de´rale de Lausanne (EPFL), 1015, Lausanne, Switzerland. E-mail: paul.
dyson@epfl.ch
6
the general formula [Ru(g -arene)Cl2(PTA)] (PTA = 1,3,5-
6
triaza-7-phosphaadamantane), the prototype being [Ru(g -p-
cymene)Cl2(PTA)], termed RAPTA-C. Several structurally di-
verse RAPTA derivatives have been studied and their in vitro
cytotoxicity has been evaluated.16,17 The in vivo effect on the
growth of lung metastases was also established for RAPTA-
bCallerio Foundation Onlus, Via A. Fleming 22–31, 34127, Trieste, Italy
cDipartimento di Scienze Biomediche, Universita` di Trieste, Via L. Giorgieri
7–9, 34127, Trieste, Italy
dInstitute of Inorganic Chemistry, University of Vienna, Wa¨hringer Straße
42, 1090, Vienna, Austria
6
C and RAPTA-B, i.e. [Ru(g -benzene)Cl2(PTA)].17 The PTA
† Based on the presentation given at Dalton Discussion No. 10, 3rd–5th
September 2007, University of Durham, Durham, UK.
‡ CCDC reference number 643753 for 1b. For crystallographic data in CIF
or other electronic format see DOI: 10.1039/b705449a
ligand provides a degree of water solubility (dependent on the
nature of the arene ligand) which facilitates administration and
transport in the body. Some related ruthenium cyclopentadienyl
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complexes containing the PTA ligand, viz. [RuCl(Cpꢀ)(PTA)2]
(Cpꢀ = C5H5 or C5Me5) that show modest biological activity have
been reported.18 It has also been shown that the aromatic ring in
RAPTA compounds may be replaced by the sulfur macrocycle
1,4,7-trithiacyclononane without changing the in vitro activity
too significantly.19 Moreover, some rhodium and osmium RAPTA
analogues have been evaluated in vitro in HT29 colon carcinoma,
A549 lung carcinoma and T47D breast carcinoma cell lines and
again, activities are not too dissimilar from the ruthenium(II)–
arene RAPTA complexes.20
Peruzzini et al.21 reported a series of water-soluble ruthenium
complexes of general formula [Ru(Cp)Cl(L)(Lꢀ)]n+ (L = PPh3, Lꢀ =
PTA, mPTA; L = Lꢀ = PTA, mPTA; mPTA = N-methyl-1,3,5-
triaza-7-phosphaadamantane). The interaction of the ruthenium
complexes with DNA was found to be strongly dependent on
the phosphine ligands present. Both methylation of the PTA or
substitution of PTA by PPh3 increases the reactivity with DNA.
Similar behaviour has also been observed for platinum
thiosalicylate complexes, [Pt(SC6H4CO2)(L)] (L = PTA or PPh3),22
where the PPh3 derivative exhibits the highest antitumour activity
toward leukemia P388 cells.23 Some complexes containing PPh3
such as cis-[PtCl(PPh3)2(8-MTT)], cis-[Pt(PPh3)2(8-MTT)2], and
cis-[Pt(PPh3)2(8-MTT)(8-TTH)] are stronger inhibitors of the
cisplatin-resistant SKOV3 cell line than analogous complexes
containing PTA (8-TTH2 = 8-thiotheophylline; 8-MTTH = 8-
methylthiotheophylline).24,25 The activity of the thiopurine plat-
inum complexes on cisplatin-sensitive T2 cells is also strongly
affected by the nature of the phosphine ligand, the complexes con-
taining PPh3 being significantly more active than those containing
PTA.
In this paper we use the strategy described by Peruzzini to
modify the hydrosolubility of the RAPTA compounds using
PPh3 in combination with PTA. In a previous study we at-
tached arene ligands with hydrogen bonding substituents to the
RAPTA framework as we hypothesised that it would increase
the cytotoxicity of the RAPTA compounds by increasing their
tendency to bind DNA/RNA via increased hydrogen bonding
interactions.16 While we were able to demonstrate increased
reactivity with oligonucleotides the increased hydrophilicity of
the compounds resulted in a dramatic decrease in uptake by the
cells and correspondingly their cytotoxicity decreased. Thus, by
incorporating a hydrophobic PPh3 into the RAPTA framework
we hope to reduce the hydrosolubility of the compounds with
hydrogen bonding substituents and increase their uptake, and
accordingly, their cytotoxicity.
Scheme 1 Synthesis of ruthenium(II)–arene mixed phosphine complexes
1b and 2b, and the structures of RAPTA-C 1a and RAPTA-OH 2a.
2JP-Ru-P coupling constant of 54 Hz, corroborating the coordination
of both phosphines to the ruthenium centre. Furthermore, the
electrospray ionisation mass spectra (ESI-MS) of 1b and 2b display
the desired parent ions with the expected isotopic abundances.
The structure of 1b has been established in the solid state by
single crystal X-ray diffraction and is depicted in Fig. 1. The
structure exhibits the typical ‘piano-stool’ geometry around the
ruthenium atom and has a close resemblance to that of the related
6
bisphosphine complex, [Ru(g -p-cymene)Cl(PPh3)2]BF4 [Ru–P =
26
2.3649(6), 2.4042(6) A], other bisphosphine complexes,27 and
˚
RAPTA-C 1a,28 although the Ru–PTA bond length is slightly
elongated in comparison [2.3201(14) A vs. 2.297(3) (average over
˚
two independent molecules) in RAPTA-C 1a].
Results and discussion
Fig. 1 ORTEP representation of 1b; thermal ellipsoids are drawn
at 50% probability level and the counter ion is omitted for clarity.
6
Substitution of the labile CH3CN ligand in [Ru(g -p-
cymene)Cl(CH3CN)(PPh3)]BF4 with PTA in CH2Cl2 at room
temperature affords the mixed phosphine complex, [Ru(g -p-
◦
˚
Key bond lengths (A) and angles ( ): Ru1–Cl1, 2.4069(15); Ru1–P1,
2.3201(14); Ru1–P2, 2.3590(14); Ru1–Cavg , 2.26(4); Cl1–Ru1–P1, 82.78(5);
Cl1–Ru1–P2, 90.35(5); P1–Ru1–P2, 95.32(5).
6
cymene)Cl(PTA)(PPh3)]BF4 1b, in good yield (Scheme 1). The cor-
responding RAPTA complex bearing a hydroxyl-functionalised
Cytotoxicity and uptake studies
6
g -arene, C6H5CH2CH2OH, is prepared by ligand substitution
6
of the chloride ligand in [Ru(g -C6H5CH2CH2OH)Cl2(PPh3)], to
The MTT test was carried out on compounds 1b and 2b using
the tumourigenic TS/A and non-tumourigenic HBL-100 cell
lines, previously used to evaluate the activity of other RAPTA
compounds.17 The effects of 1b and 2b on the growth of these cell
lines were evaluated after 24, 48 and 72 h and the corresponding
give 2b in high yield. Full synthetic, spectroscopic and analytical
details are provided in the Experimental section, but of note, the
31P NMR spectra of 1b and 2b exhibit a characteristic pair of
doublets at ca. 30 ppm (PPh3) and ca. −42 ppm (PTA), with a
5066 | Dalton Trans., 2007, 5065–5072
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Table 1 IC50 values on the TS/A and HBL-100 cell lines after 72 h
incubation
Compounds
IC50 (TS/A)/lM
IC50 (HBL-100)/lM
> 300
1a17
1b
> 300
> 300
> 300
124 13
37
> 300
82
3
2a17
2b
9
Table 2 Ruthenium uptake after treatment of TS/A cells with 100 lM of
drug for 24 h. Each number is the mean S.E. of an experiment made in
triplicate
Compounds
Intracellular uptake/
Intracellular uptake/
lg 10−6 cells
×10−4
M
1a17
1b
0.12 0.02
0.13 0.03
0.06 0.01
0.20 0.02
2.55 0.06
1.71 0.40
1.33 0.20
2.63 0.23
Fig. 2 Negative ion ESI mass spectrum (deconvoluted) of a 5 : 1
incubation mixture of 2b + 14-mer in water (37 ◦C, 72 h incubation).
2a17
2b
Table 3 Species observed after deconvolution of the ESI spectra from the
incubation of the compounds with the 14-mer 5ꢀ-ATACATGGTACATA-
3ꢀ (ratio 5 : 1)a
IC50 values resulting from an average of two experiments are listed
in Table 1.
Compounds Adducts
As expected, the cytotoxicity of 2b is greatest in the tumour
cells, the other three compounds not showing any significant
cytotoxicity up to 300 lM. The increased cytotoxicity of 2b, at
least relative to 2a, corresponds well with increased drug uptake
(Table 2), being ca. 3 fold higher. In contrast, the addition of the
PPh3 ligand to RAPTA-C has only a minor effect on cytotoxicity
probably due to the poor solubility of 1b in the medium.
Importantly, the presence of the PPh3 ligand decreases the
selectivity of the compounds towards the tumour cells as evidenced
by the lower IC50 values of 1b and 2b in the non-tumourigenic
HBL-100 cells (used to model healthy cells) relative to 1a and 2a.
The higher hydrophobicity of the ruthenium(II)–arene compounds
with the PPh3 ligands would appear to have the effect of increasing
the general cytotoxicity of the complexes, which may also be
connected to differences in reactivity with various biomolecules
(see later).
6
[14-mer + Ru(PTA) + Ru(g -p-cymene)(PTA)]
1a
1b
2a
[14-mer + Ru(PTA)] (92%)
6
[14-mer + Ru(g -p-cymene) (PTA)] (22%)
14-mer (18%)
14-mer
[14-mer + 2Ru(PTA)]
[14-mer + 2Ru(C6H5CH2CH2OH)(PTA)] (80%)
[14-mer + 2Ru(PTA) + 2Ru(C6H5CH2CH2OH)(PTA) +
Cl−] (80%)
[14-mer + Ru(C6H5CH2CH2OH)(PTA) +
Ru(C6H5CH2CH2OH)] (55%)
[14-mer + 2Ru(PTA) + Ru(C6H5CH2CH2OH)(PTA)] (45%)
2b
[14-mer + Ru(PTA)(PPh3)]
a Mass of the 14-mer is 4271. The percentage in parentheses corresponds
to the relative intensity (first species at 100%).
Comparative interactions with model biomolecules
the high affinity of 2b towards nucleobase targets. In comparison,
1a and 2a are less reactive. It appears that as a first contact between
the ruthenium(II)–arene compound and the oligonucleotide, the
structure of the arene ligand is important to favour an interaction,
as demonstrated elsewhere.30 Most probably, the binding of
compound takes place initially via the rapid hydrolysis of the
chloride in solution, followed by substitution of the water ligands
for the 14-mer. Next, the arene ligand may be lost to give the
possibility to the oligonucleotide to occupy the surroundings of the
ruthenium centre. As postulated, these data indicate that hydrogen
bonding between the arene ligand in 2b and the DNA will favour
a first contact.
The traditional target for metal-based anticancer drugs is DNA,
and damage of DNA is implicated in the activity of several impor-
tant metal-based anticancer drugs.29 Accordingly, the reactivity
of 1b, 2b and their precursors RAPTA-C 1a and RAPTA-OH
2a with the 14-mer 5ꢀ-ATACATGGTACATA-3ꢀ, was studied by
electrospray ionisation mass spectrometry (ESI-MS). Essentially,
the compounds were incubated in water with the 14-mer at 37 ◦C
for 72 h in a 5 : 1 (ruthenium : 14-mer) ratio and the products
analysed by ESI-MS. Following incubation, the solutions were
diluted with DMBA (dimethylbenzylamine), and injected into
the mass spectrometer. The negative ion ESI deconvolution mass
spectrum for the incubation with 2b is shown in Fig. 2 as an
illustrative example, complete data are summarised in Table 3.
Inspection of these data reveals a number of trends. First, 2b is
able to bind to the oligonucleotide as shown by the peak at m/z
4790 (relative abundance 100%) which corresponds to [14-mer-
Ru(PTA)(PPh3)] (no interaction is detected with 1b, presumably
due to its insolubility in the incubation medium). It is noteworthy
that no unreacted 14-mer is present in the spectrum of 2b indicating
Ubiquitin (Ub) and cytochrome-c (Cyt-C) have been used as
model proteins to study the binding of metal complexes to proteins
using mass spectrometry with both clinically established and de-
velopmental drugs. It was reported that the reactivity of cisplatin,
transplatin, carboplatin, and oxaliplatin toward cytochrome-c
is quite similar.31 The reaction of the ruthenium–arene com-
6
plex, [(g -biphenyl)Ru(en)Cl][PF6], toward cytochrome-c has been
studied by mass spectrometric and NMR methods and it was
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6
found that when incubating Cyt-C with a ten-fold excess of [(g -
bip)Ru(en)Cl][PF6] in water and in TEAA buffer at pH 8.7 and
7.6, respectively, resulted in the formation of a monoruthenated
species.32 RAPTA compounds have also been shown to bind
to Cyt-C and lysozyme with His33 was proposed as a major
interaction site in the former.33 Reactions of ubiquitin with
platinum complexes including cisplatin, transplatin, and asym-
metric trans-Pt complexes have also been reported.34 Cisplatin
was found to form 1 : 1 and 1 : 2 adducts, while transplatin
forms mainly Pt(NH3)2ClUb. The asymmetric Pt(II) complexes
were reported to coordinate exclusively to Met1 of ubiquitin which
is also supposed to be the main target for cisplatin but not for
transplatin.
Similarly, complexes 1a, 1b, 2a and 2b were incubated with
Ub or Cyt-C at molar ratios of 5 : 1 and analysed by offline
nanoelectrospray ionisation-ion trap-mass spectroscopy (nESI-
IT-MS).
Fig. 3 Time course of the reaction of 2a with Ub followed by
nESI-QToF-MS; Ub and 2a were incubated at a molar ratio of 1 : 5
at 37 ◦C.
6
Ub reacts with 1a to form the monoruthenated adduct Ru(g -
p-cymene)–Ub with a mass of 8807 Da after 5 h incubation.
The corresponding peak in the deconvoluted mass spectrum was
the most abundant signal, followed by the signal of Ub (8575
Da, 52%). After incubation for 1 day the signal for Ub was no
longer present. As for the 14-mer, 1b did not react with Ub.
The most abundant peak in the mass spectrum corresponded to
Ub conjugates were found as in the IT mode but at a significantly
lower intensity.
The reaction of Cyt-C with the Ru complexes was followed by
nESI-IT-MS. The neutral complexes 1a and 2a were found to form
6
6
the complex [Ru(g -p-cymene)Cl(PPh3)(PTA)]+. In contrast to 1a,
with Cyt-C the same adducts as with Ub, i.e., Ru(g -arene)–Cyt-C,
but the reaction kinetics were markedly decreased. The complexes
with the PPh3 ligands did not form any detectable adducts even
after 6 days of incubation. These data are summarised in Table 4.
incubation of 2a with ubiquitin for 5 h affords a mass spectrum
in which the most abundant peak corresponds to Ub with peaks
of lower relative intensity at 8706 Da (54%) and 8785 Da (19%)
6
that correspond to a RuCl–Ub adduct and the Ru(g -arene)–
Table 4 Species observed from the incubation mixture of the ruthenium
Ub conjugate. The analysis after 1 day of incubation yielded the
compounds and Cyt-C in water after 5 h incubation at 37 ◦
C
6
trifunctional Ru(g -arene)–Ub species as the peak with the highest
relative intensity (a considerable amount of Ub was still detected,
ca. 52%). After 2 days, a spectrum with only a Ru(g -arene)–Ub
Compound
Adducts
6
6
1a
1b
2a
2b
[Cyt-C + Ru(g -p-cymene)]
None observed
[Cyt-C + Ru(g -C6H5CH2CH2OH)]
None observed
adduct was observed. The reaction of 2b was found to proceed
more slowly than the parent compound 2a, but under the applied
conditions forms the same adducts, although the Ub peak is still
the most abundant protein peak in the mass spectrum. Note that
even after 6 days of incubation, all the protein peaks are minor
6
The differences in reaction kinetics for the different proteins
might be explained by the availability of metal-binding sites on the
surface of proteins.35 Although Cyt-C contains twice the number
of methionines, three more histidines and two cysteine residues
than Ub, reactivity towards Cyt-C is slower than that of Ub.
Met1 and His68 of Ub are easier to access than the potential
metal-binding sites in Cyt-C as they are more exposed on the
protein surface (Fig. 4). What is clear, however, is that the more
6
compared to the signal of [Ru(g -arene)Cl(PPh3)(PTA)]+.
For comparison purposes, analysis of the incubation solutions
was also conducted using nanoelectrospray ionisation-quadrupole
time of flight-mass spectrometry (nESI-QToF-MS). The reaction
mixtures were pre-concentrated on a C18 column and, by reversing
the flow, the samples were introduced into the mass spectrometer.
However, in comparison to the analyses performed on the ion
trap system, the formation of a lower number of adducts with
lower intensities were observed. Ub (8562 Da) was found to
6
form with 1a and 2a trifunctional adducts of the type Ru(g -
arene)–Ub (8795 and 8783 Da for 1a and 2a, respectively) and
6
to a minor degree of bifunctional Ru(g -arene)(PTA)–Ub (for 2a
the time course of the reaction is presented in Fig. 3; a similar
reactivity was found for 1a). In contrast, the compounds 1b and
2b behaved quite differently: while 1b did not show any reaction
with ubiquitin; 2b reacts with Ub to form Ru(PPh3)(PTA)–Ub
(9080 Da, 5%) after 20 h of incubation. The peaks of the complex
cations [Ru(g -arene)Cl(PPh3)(PTA)]+ (m/z 689.8 and 677.8 for 1b
and 2b, respectively) were the most abundant, and the binding of 2b
6
Fig.
4
Molecular structures of (left) ubiquitin36 and (right)
cytochrome-c37 with the His, Met and Cys residues highlighted as stick
graphics (PyMOL v0.98, (c) DeLano Scientific LLC).
toward Ub was found to be slower than of the neutral compound
6
2a. After incubation for 20 h, for 1a and 2a the same Ru(g -arene)–
5068 | Dalton Trans., 2007, 5065–5072
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hydrophobic (and sterically demanding) complexes 1b and 2b are
less reactive towards both proteins than 1a and 2a. Moreover,
selective (preferential) interactions are also observed.
(660.89 g mol−1)·0.6(CH3CH2CH2CH2CH3): C, 56.29; H, 5.61; N,
1.99. Found: C, 56.64; H, 5.28; N, 2.31.
[Ru(g6-p-cymene)Cl(PTA)(PPh3)]BF4 1b
Concluding remarks
From an overview of the data presented herein, it would appear
that as the structure of RAPTA compounds are modified in such a
way that DNA binding interactions are favoured over interactions
with proteins, the general toxicity of the compounds increases. It is
possible that this trend could be a general phenomenon although
further experiments of different classes of anticancer compounds
would be required to substantiate this view. It would certainly
be interesting to evaluate the platinum compounds described
in the Introduction that were modified in a similar manner on
non-tumourigenic cells. Nevertheless, this work demonstrates the
importance of finding targets other than DNA for the design of
new putative anticancer reagents that will exhibit high selectivity
and thus reduced general side-effects.
6
A solution of [Ru(g -p-cymene)Cl(CH3CN)(PPh3)]BF4 (0.50 g,
0.76 mmol) and PTA (0.15 g, 0.99 mmol) in CH2Cl2 (25 ml)
was stirred at RT for 90 min. The solution was then washed
with water (2 × 50 ml), dried with Na2SO4 and the product was
precipitated by the addition of pentane. Yield: 0.44 g (75%) as a
1
yellow microcrystalline powder. H NMR (CDCl3): d 7.48–7.70
(m, 15H, PPh3), 6.69 (dd, JHH = 6.2, JPH = 5, 1H, H6), 5.94
3
3
3
3
Experimental
(d, JHH = 5.8, 1H, H5), 5.80 (d, JHH = 6.1, 1H, H3), 5.13 (dd,
3JHH = 6.4, JPH = 5, 1H, H2), 4.45 (d, JHH = 13.1, 3H, H12),
3
2
ꢀ
Reactions were carried out under a nitrogen atmosphere and
solvents were purged with nitrogen before use. 1,3,5-Triaza-7-
4.36 (d, JHH = 13.2, 3H, H12 ), 4.26 (d, JHH = 14.3, 3H, H11),
2
2
ꢀ
3.83 (d, JHH = 14.2, 3H, H11 ), 2.71 (sept, JHH = 6.9, 1H, H8),
2
3
6
phosphaadamantane (PTA),38 [Ru(g -p-cymene)Cl2(PPh3)],39 and
1.29 (d, JHH = 7, 3H, H9), 1.27 (d, JHH = 7, 3H, H10). 13C{ H}
NMR (CDCl3): d 134.4 (d, 2JPC = 10, PPh3), 133.1 (d, 1JPC = 48,
PPh3), 131.6 (d, 4JPC = 2, PPh3), 128.9 (d, 3JPC = 10, PPh3), 125.2
3
3
1
6
40
[Ru(g -C6H5CH2CH2OH)Cl2]2 were prepared as described else-
where. All other chemicals are commercial products and were
used as received. NMR spectra were recorded with a Bruker
Avance 400 spectrometer at room temperature, unless otherwise
stated. Chemical shifts are given in ppm and coupling constants
(J) in Hz. ESI mass spectra for characterisation of synthetic
compounds were recorded on a Thermo Finnigan LCQ DECA
XPPlus according to a literature procedure41 and microanalyses
were performed at the EPFL.
(d, JPC = 4, C4), 101.7 (s, C1), 97.9 (d, JPC = 4, C2), 93.8 (d,
2
2
2JPC = 8, C3), 91.0 (d, JPC = 2, C6), 88.2 (d, JPC = 8, C5), 72.5
2
2
(d, JPC = 8, C12), 53.7 (d, JPC = 15, C11), 31.0 (s, C8), 22.0 (s,
3
1
C9/10), 21.5 (s, C10/9), 17.7 (s, C7). 31P{ H} NMR (CDCl3) d 29.9 (d,
1
2JPP = 54, 1P, RuPPh3), −42.8 (d, 2JPP = 54, 1P, RuPTA). 11B{1H}
1
NMR (CDCl3) d −0.7 (pent, JBF = 1, BF4). ESI–MS (CH2Cl2)
positive ion: m/z, 690 [M]+; negative ion: m/z, 87 [BF4]−. Calcd
for C34H41BClF4N3P2Ru (776.99 g mol−1): C, 52.56; H, 5.32; N,
5.41. Found: C, 52.31; H, 5.36; N, 5.33.
[Ru(g6-p-cymene)Cl(CH3CN)(PPh3)]BF4
[Ru(g6-C6H5CH2CH2OH)Cl2(PPh3)]
6
A suspension of [Ru(g -p-cymene)Cl2(PPh3)] (5.22 g, 9.18 mmol)
and NH4BF4 (1.25 g, 11.04 mmol) in CH3CN (180 ml) was
stirred at RT for 4 days. The solvent was then removed and
the residue extracted with CH2Cl2 through Celite. The product
was precipitated following concentration and addition of pentane.
Yield: 4.82 g (80%) as a yellow powder. NMR data are in agreement
with the analogous PF6 complex.42 1H NMR (CDCl3): d 7.46–
6
A
suspension of [Ru(g -C6H5CH2CH2OH)Cl2]2 (0.80 g,
1.36 mmol) and PPh3 (0.89, 3.39 mmol) in 2 : 1 CH2Cl2–
MeOH (30 ml) was stirred at RT for 90 min forming a dark
homogeneous phase. The solution was then slowly concentrated
in vacuo resulting in precipitation of the product as an orange–red
solid. Yield: 1.07 g (71%). 1H NMR (CDCl3): d 7.68–7.83 (m, 6H,
3
7.68 (m, 15H, PPh3), 6.33 (d, JHH = 6.0, 1H, H5), 5.92 (d,
3
3
3JHH = 5.9, 1H, H3), 5.38 (d, JHH = 5.9, 1H, H2), 4.70 (d,
ortho-PPh3), 7.32–7.50 (m, 9H, PPh3), 5.64 (d, JHH = 5.8, 2H,
3
3
3
3
3JHH = 6.2, 1H, H6), 3.11 (sept, JHH = 6.8, 1H, H8), 1.99 (s,
H3), 5.05 (dd, JHH = 5, JHH = 5, 2H, H2), 3.88 (dt, JHH = 5,
3
3H, H12), 1.74 (s, 3H, H7), 1.38 (d, JHH = 7, 3H, H9), 1.36 (d,
3JPH = 3, 1H, H1), 4.14 (t, 3JHH = 5.4, 2H, H6), 2.92 (t, 3JHH = 5.1,
1
1
3JHH = 7, 3H, H10). 31P{ H} NMR (CDCl3) d 35.7 (s, RuPPh3).
2H, H5), 2.86 (br, 1H, OH). 13C{ H} NMR (CDCl3): d 134.2 (d,
1
4
3
11B{1H} NMR (CDCl3) d −0.8 (pent, JBF = 1, BF4). ESI-MS
2JPC = 10, PPh3), 130.5 (d, JPC = 2, PPh3), 128.2 (d, JPC = 10,
(CH2Cl2) positive ion: m/z, 533 (23%) [M − CH3CN]+, 574
[M]+; negative ion: m/z, 87 [BF4]−. Calcd for C30H32BClF4NPRu
PPh3), 108.7 (d, 2JPC = 9, C4), 91.7 (d, 2JPC = 5, C3), 86.0 (br, C2),
82.8 (s, C1), 60.7 (s, C6), 35.4 (s, C5). 31P{ H} NMR (CDCl3) d 27.8
1
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(s, RuPPh3). Calcd for C26H25Cl2OPRu (556.43 g mol−1): C, 56.12;
Table 5 Crystallographic data for 1b
H, 4.53. Found: C, 55.76; H, 4.57.
Formula
M
T/K
C34H41BClF4N3P2Ru
776.97
140(2)
[Ru(g6-(C6H5CH2CH2OH)Cl(PTA)(PPh3)]BF4 2b
Cryst. syst.
Space group
Monoclinic
P2(1)/c
13.6698(10)
14.4658(9)
17.1533(6)
˚
a/A
˚
b/A
˚
c/A
a/◦
b/◦
d/◦
99.576(5)
3
˚
V/A
3344.7(3)
4
1.543
Z
Density/g cm−3
l/mm−1
0.697
6
A
solution of [Ru(g -C6H5CH2CH2OH)Cl2(PPh3)] (0.20 g,
h range/◦
2.98 ≤ h ≤ 25.02
0.36 mmol), PTA (0.085 g, 0.54 mmol) and NH4BF4 (0.057 g,
0.54 mmol) in 1 : 1 CH2Cl2–MeOH (20 ml) was stirred at 40 ◦C for
1 h. The solvent was removed in vacuo and the residue suspended in
CH2Cl2 (50 ml), then washed with water (2 × 50 ml). The aqueous
phase was extracted with CH2Cl2 (3 × 50 ml) and the combined
organic phases dried with Na2SO4. Concentration and addition of
excess hexane gave the product as a yellow powder. Yield: 0.24 g
(87%). 1H NMR (CDCl3): d 7.45–7.60 (m, 15H, PPh3), 6.44–6.57
(m, 1H, H2), 6.22 (d, 3JHH = 5.5, 1H, H3), 5.05 (d, 3JHH = 6.2, 1H,
H5), 5.41–5.51 (m, 1H, H6), 4.39–4.48 (m, 1H, H1), 4.31 (s, 6H,
Measured reflns
Unique reflns
No. data/restr./param.
R1, wR2 [I > 2r(I)]a
GoFb
19880
5666 [Rint = 0.0773]
5666/0/418
0.0384, 0.0571
0.774
2
1/2
a R1 = RꢁFo | − |Fc ꢁ/R|Fo |, wR2 = {R[w(Fo − Fc2)2]/R[w(Fo2)2]}
.
b GoF = {R[w(Fo − Fc2)2]/(n − p)} where n is the number of data and
p is the number of parameters refined.
2
1/2
cultured according to a standard procedure,47 and maintained in
RPMI-1640 medium (EuroClone, Wetherby, UK) supplemented
with 10% Fetal Bovine Serum (FBS, Invitrogen, Milano, Italy),
2 mM L-glutamine (EuroClone, Wetherby, UK) and 50 lg ml−1
gentamycin sulfate solution (EuroClone, Wetherby, UK). The cell
line was kept in an incubator with 5% CO2 and 100% relative
humidity at 37 ◦C. Cells from a confluent monolayer were removed
from flasks by a trypsin–EDTA solution (EuroClone, Wetherby,
UK).
HBL-100 non-tumorigenic human breast cells, obtained from
ATCC (American Type Culture Collection), were maintained in
McCoy’s 5A medium (Sigma, St. Louis, MO, USA) supplemented
with 10% FBS, 2 mM L-glutamine, 100 UI ml−1 penicillin, and
100 lg ml−1 streptomycin (EuroClone, Whetherby, UK) in a
humidified atmosphere with 5% CO2 at 37 ◦C.
2
H10), 4.18 (d, JHH = 14.5, 3H, H9), 3.80–4.04 (m, 2H, H8), 3.92
(d, 2JHH = 13.2, 3H, H9ꢀ ), 2.67 (t, 3JHH = 5, 2H, H7), 2.29 (br, 1H,
1
OH). 13C{ H} NMR (CDCl3): d 134.3 (d, 2JPC = 10, PPh3), 133.3
(d, 1JPC = 49, PPh3), 131.7 (d, 4JPC = 2, PPh3), 129.0 (d, 3JPC = 10,
PPh3), 118.2 (d, 2JPC = 5, C4), 97.8 (d, 2JPC = 8, C5), 97.5 (d, 2JPC
=
=
4, C6), 91.7 (d, 2JPC = 9, C3), 89.6 (br, C2), 85.0 (s, C1), 72.4 (d, 3JPC
1
8, C10), 60.1 (s, C8), 53.6 (d, 1JPC = 15.4, C9), 35.6 (s, C7). 31P{ H}
NMR (CDCl3) d 32.0 (d, 2JPP = 53, 1P, RuPPh3), −42.0 (d, 2JPP
=
=
1
54, 1P, RuPTA). 11B{1H} NMR (CDCl3) d −0.8 (pent, JBF
1, BF4). ESI–MS (CH2Cl2) positive ion: m/z, 678 [M]+; negative
ion: m/z, 87 [BF4]−. Calcd for C32H37BClF4N3OP2Ru (764.94 g
mol−1)·1/2(H2O): C, 49.66; H, 4.95; N, 5.43. Found: C, 49.55; H,
5.14; N, 5.25.
Crystallography†
Cell viability was determined by the trypan blue dye exclusion
test. For experimental purposes, the cells were sown in multiwell
cell culture plastic plates (Corning Costar Italia, Milano, Italy).
Cell growth was determined by the MTT viability test.48 Cells
were sown on 96-well plates and after 24 h were incubated with
the appropriate compound, prepared by dissolving in a medium
containing 5% of serum for 24, 48 and 72 h. Solutions of the
substances were prepared by diluting a freshly prepared stock
solution of compound 2b in water (10−2 M) and compound 1b
in DMSO (10−2 M). Maximum DMSO concentration in the
cells was 1% v/v. Analysis was performed at the end of the
incubation time. Briefly, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide] dissolved in PBS (5 mg ml−1) was
added (10 ll per 100 ll of medium) to all wells and the plates
were then incubated at 37 ◦C with 5% CO2 and 100% relative
humidity for 4 h. After this time, the medium was discarded and
100 ll of DMSO (Sigma, St. Louis, MO, USA) were added to each
well according to the method of Alley et al.49 Optical density was
measured at 570 nm on a SpectraCount Packard (Meriden, CT)
instrument.
Crystals suitable for X-ray diffraction were obtained from slow
diffusion of pentane into a CHCl3 solution of 1b. Relevant details
about the structure refinements are given in Table 5, and selected
geometrical parameters are included in the captions of Fig. 1. Data
collection was performed on a KUMA CCD diffractometer sys-
˚
tem using graphite monochromated Mo Ka radiation (0.71073 A)
and a low-temperature device. Data reduction was performed
using CrysAlis RED.43 Structures were solved using SIR97,44 and
refined (full-matrix least squares on F2) using SHELXTL.45 All
non-hydrogen atoms were refined anisotropically, with hydrogen
atoms placed in calculated positions using the riding model. The
graphical representation was made with ORTEP3.46
Cell growth inhibition effects on TS/A adenocarcinoma cells
TS/A murine adenocarcinoma cell line, initially obtained from
Dr G. Forni (CNR, Centro di Immunogenetica ed Oncologia
Sperimentale, Torino, Italy) belong to the tumour cell lines panel of
the Callerio Foundation and is stored in liquid nitrogen. Cells were
5070 | Dalton Trans., 2007, 5065–5072
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Determination of intracellular ruthenium
Protein binding studies
Ruthenium cell uptake was determined by atomic absorption
spectroscopy (AAS) on samples processed using the procedure
of Tamura and Arai with slight modifications.50 For each complex
tested, a 6-well plate was prepared by seeding 1.25 × 105 TS/A cells
in 3 ml of complete medium with 5% FBS to each experimental
and control well. The plate was incubated for 24 h at 37 ◦C. The
wells were then washed three times with PBS. Control wells were
filled with 3 ml of complete medium and experimental wells with
3 ml of a 100 lM solution of ruthenium compounds prepared
Ubiquitin (from bovine red blood cells, min. 90%) and
cytochrome-c (from horse heart, 96%) were purchased from
Sigma (Steinheim, Germany). Formic acid (98–100%), acetonitrile
(HPLC gradient grade), and water (HPLC gradient grade) came
from Merck (Darmstadt, Germany), Fisher Scientific (Loughbor-
ough, UK), and Acros Organics (Geel, Belgium), respectively.
Complexes (1a, 1b, 2a and 2b) were incubated at molar ratios
of 5 : 1 with the proteins in aqueous solution at 37 ◦C yielding a
concentration of 10 lM protein. In order to dissolve the compound
1b, DMSO was added with a final DMSO concentration of 1% in
H2O. Before nESI-IT-MS analysis, the incubation solutions were
mixed at a ratio of 1 : 3 with ACN : H2O : formic acid (68 : 32 : 0.7)
and the samples were analysed immediately after mixing (after 5 h,
1 day, 2 days, 3 days, and 6 days incubation).
◦
in complete medium. The plate was incubated for 24 h at 37 C
before the cells were collected and counted with the trypan blue
exclusion test and the intracellular concentration of ruthenium
was determined. After this treatment, the cells were dried in
R
Nalgeneꢀ cryogenic vials (a first drying step was performed
overnight at 80 ◦C and a second step at 105 ◦C until the samples
reached a constant weight). The dried cells were decomposed by
the addition of an aliquot of tetramethylammonium hydroxide
(25% in water) (Aldrich Chimica, Gallarate, Milano, Italy) and
of milliQ water at a ratio of 1 : 1 directly in each vial at
room temperature under shaking. Final volumes were adjusted
to 1 ml with milliQ water. The concentration of ruthenium in
TS/A tumour cells was measured in triplicate by flameless atomic
absorption spectroscopy (AAS) using a Zeeman graphite tube
atomizer, model SpectrAA-300, supplied with a specific ruthenium
emission lamp (hollow cathode lamp P/N 56–101447-00; Varian,
Mulgrave, Victoria, Australia). Quantification of ruthenium was
carried out in 10 ll samples at 349.9 nm with an atomising
temperature of 2500 ◦C, using argon as carrier gas at a flow rate of
3.0 l min−1 (for further details concerning the furnace parameter
settings, see ref. 51). Before each analysis, a five-point calibration
curve was obtained to check the range of linearity using ruthe-
nium custom-grade standard 998 mg ml−1 (Inorganic Ventures,
Lakewood, N.J.).
nESI-IT-MS. Analyses were performed on a Thermo Finni-
gan LCQ Deca XP Plus quadrupole ion-trap instrument in positive
ion mode. The capillary temperature was set at 180 ◦C and
the source voltage to 1.51 kV, with a mass range from 300 to
2000. The acquisition was performed with Tune Plus 1.3 SR1
program (Thermo Finnigan) and the deconvolution of data were
performed with Bioworks Browser 3.0 (Thermo Finnigan) on a
Windows 2000 Prof. SR4 PC system using Biomass Calculation
and Deconvolution software.
nESI-QToF-MS. The samples were introduced into the mass
spectrometer by means of a Waters modular CapLC system.
Samples were loaded onto a C18 cartridge (Symmetry 300TM, C18,
5 lm NanoEaseTM) and desalted with 0.1% HCOOH for 3 min at a
flow rate of 30 ll min−1. The 10-port valve was then switched such
that the samples were sprayed directly into the mass spectrometer.
The electrospray potential (ca. 2 kV) was applied to the liquid via
a low dead volume stainless steel union. A small amount (ca. 2.5
psi) of nebulising gas was introduced around the spray tip to aid
the electrospray process. Data were acquired using a Micromass
Q-Tof Ultima mass spectrometer, equipped with Z-spray nanoflow
electrospray ion source. The mass spectrometer was operated in
the positive-ion mode with a source temperature of 80 ◦C and
a counter current gas flow rate of 40 l h−1. The instrument was
calibrated with phosphoric acid in the range of 100–2000 m/z. All
data were processed using MassLynx version 4.0 software using
Max Ent Electrospray software algorithm.
Oligonucleotide binding
The 14-mer oligonucleotide (5ꢀ-ATACATGGTACATA-3ꢀ) was
obtained from MWG biotech AG (Ebersberg, Germany) and
the concentration was taken to be 100 lM as specified by the
supplier. The samples were prepared in a ratio of 1 : 5 by
mixing the 14-mer (50 lM) with an aqueous solution of the
ruthenium complex (250 lM). In order to dissolve the compound
1b, DMSO was added with a final DMSO concentration of
1% in H2O. The samples were maintained at 37 ◦C for 72 h
with vigorous shaking. The ESI measurements were performed
on a Micromass Q-Tof Ultima. When necessary the samples
Acknowledgements
We thank the Swiss National Science Foundation, the EPFL, the
Austrian Science Fund (C. G. H.; Schro¨dinger Fellowship J2613-
N19) and COST (Switzerland) for financial support. The present
work was carried out with contributions from the Laboratory for
the Identification of New Antimetastasis Drugs and developed
under the COST D20 action.
R
were desalted through a Microconꢀ centrifugal filter device
(Millipore) directly after the incubation. Then the solutions were
diluted twice with DMBA (25 lM; dimethylbenzylamine) and
injected into the mass spectrometer. The spectra were recorded
in negative mode and before every series of measurements the
spectrometer was calibrated with H3PO4. The source temperature
was set at 373 K and the cone voltage to 35 keV, with a mass
range from 400 to 2000. The acquisition and the deconvolution
of data were performed on a Windows XP PC system using
Mass Lynx (version 4.0) and the Max Ent Electrospray software
algorithm.
References
1 (a) T. Boulikas and M. Vougiouka, Oncol. Rep., 2003, 10, 1663–1682;
(b) A. Pasini and F. Zunino, Angew. Chem., Int. Ed. Engl., 1987, 26,
615–624.
This journal is
The Royal Society of Chemistry 2007
Dalton Trans., 2007, 5065–5072 | 5071
©

View Article Online
2 (a) B. Lippert, Cisplatin, Chemistry and Biochemistry of a Leading
Anticancer Drug, Wiley-VCH, Weinheim, 1999; (b) J. Reedijk, Chem.
Commun., 1996, 801–806; (c) J. D. Roberts, J. Peroutka and N. J. Farrell,
Inorg. Biochem., 1999, 77, 51–57.
3 (a) E. Wong and C. M. Giandomenico, Chem. Rev., 1999, 99, 2451–
2466; (b) M. Galanski, M. A. Jakupec and B. K. Keppler, Curr. Med.
Chem., 2005, 12, 2075–2094.
22 L. J. McCaffrey, W. Henderson, B. K. Nicholson, J. E. Mackay and
M. B. Dinger, J. Chem. Soc., Dalton Trans., 1997, 2577–2586.
23 W. Henderson, L. J. McCaffrey and B. K. Nicholson, J. Chem. Soc.,
Dalton Trans., 2000, 2753–2760.
24 A. Romerosa, P. Bergamini, V. Bertolasi, A. Canella, M. Cattabriga, R.
Gavioli, S. Man˜as, N. Mantovani and L. Pellacani, Inorg. Chem., 2004,
43, 905–913.
4 (a) M. D. Hall, R. C. Dolman and T. W. Hambley, in Metal Ions in
Biological Systems, ed. A. Sigel and H. Sigel, Dekker, New York, 2004,
vol. 42, pp. 297–322; (b) K. R. Barnes and S. J. Lippard, in Metal Ions
in Biological Systems, ed. A. Sigel and H. Sigel, Dekker, New York,
2004, vol. 42, pp. 143–177.
5 (a) I. Kostova, Curr. Med. Chem., 2006, 13, 1085–1107; (b) M. Galanski,
V. B. Arion, M. A. Jakupec and B. K. Keppler, Curr. Pharm. Des., 2003,
9, 2078–2089.
25 A. Romerosa, C. Lo´pez-Magan˜a, M. Saoud, S. Man˜as, E. Colacio and
J. Sua´rez-Varela, Inorg. Chim. Acta, 2000, 307, 125–130.
26 R. Lalrempuia, P. J. Carroll and M. R. Kollipara, J. Coord. Chem.,
2003, 56, 1499–1504.
27 A. B. Chaplin and P. J. Dyson, Organometallics, 2007, 26, 2447.
28 C. S. Allardyce, P. J. Dyson, D. J. Ellis and S. L. Heath, Chem. Commun.,
2001, 15, 1396–1397.
29 O. A. Olivero, C. Semino, A. Kassim, D. M. Lopez-Larraza and M. C.
Poirier, Mutat. Res., 1995, 346, 221–230.
30 H. Chen, J. A. Parkinson, S. Parsons, R. A. Coxall, R. O. Gould and
P. J. Sadler, J. Am. Chem. Soc., 2002, 124, 3064.
6 P. J. Dyson and G. Sava, Dalton Trans., 2006, 1929–1933.
7 E. Alessio, G. Mestroni, A. Bergamo and G. Sava, Met. Ions Biol. Syst.,
2004, 42, 323–351.
8 (a) C. G. Hartinger, S. Zorbas-Seifried, M. A. Jakupec, B. Kynast, H.
Zorbas and B. K. Keppler, J. Inorg. Biochem., 2006, 100, 891–904; (b) E.
Alessio, G. Mestroni, A. Bergamo and G. Sava, Curr. Top. Med. Chem.,
2004, 4, 1525–1535.
9 J. M. Rademaker-Lakhai, D. Bongard Van den, D. Pluim, J. H. Beijnen
and J. H. Schellens, Clin. Cancer Res., 2004, 10, 3717–3727.
10 (a) G. Sava, F. Frausin, M. Cocchietto, F. Vita, E. Podda, P. Spessotto,
A. Furlani, V. Scarcia and G. Zabucchi, Eur. J. Cancer, 2004, 40, 1383–
1396; (b) S. Zorzet, A. Bergamo, M. Cocchietto, A. Sorc, B. Gava, E.
Alessio, E. Iengo and G. Sava, J. Pharmacol. Exp. Ther., 2000, 295,
927–933.
11 (a) B. Gava, S. Zorzet, P. Spessotto, M. Cocchietto and G. Sava,
J. Pharmacol. Exp. Ther., 2006, 317, 284–291; (b) G. Sava, S. Zorzet, C.
Turrin, F. Vita, M. Soranzo, G. Zabucchi, M. Cocchietto, A. Bergamo,
S. DiGiovine, G. Pezzoni, L. Sartor and S. Garbisa, Clin. Cancer Res.,
2003, 9, 1898–1905; (c) A. Vacca, M. Bruno, A. Boccarelli, M. Coluccia,
D. Ribatti, A. Bergamo, S. Garbisa, L. Sartor and G. Sava, Br. J. Cancer,
2002, 86, 993–998.
31 A. Casini, C. Gabbiani, G. Mastrobuoni, L. Messori, G. Moneti and
G. Pieraccini, ChemMedChem, 2006, 1(4), 413–417.
32 F. Wang, J. Bella, J. A. Parkinson and P. J. Sadler, JBIC, J. Biol. Inorg.
Chem., 2005, 10(2), 147–155.
33 A. Casini, G. Mastrobuoni, W. H. Ang, C. Gabbiani, G. Pieraccini,
G. Moneti, P. J. Dyson and L. Messori, ChemMedChem, 2007, 2, 631–
635.
34 (a) D. Gibson and C. E. Costello, Eur. J. Mass Spectrom., 1999, 5(6),
501–510; (b) T. Peleg-Shulman, Y. Najajreh and D. J. Gibson, Inorg.
Biochem., 2002, 91(1), 306–311; (c) L. Balter and D. Gibson, Rapid
Commun. Mass Spectrom., 2005, 19(24), 3666–3672; (d) Y. Najajreh,
T. Peleg-Shulman, O. Moshel, N. Farrell and D. J. Gibson, Biol. Inorg.
Chem., 2003, 8(1–2), 167–175; (e) C. G. Hartinger, W. H. Ang, A. Casini,
L. Messori, B. K. Keppler and P. J. Dyson, J. Anal. At. Spectrom., 2007,
DOI: 10.1039/b703350h.
35 A. R. Timerbaev, C. G. Hartinger, S. S. Aleksenko and B. K. Keppler,
Chem. Rev., 2006, 106, 2224–2248.
36 S. Vijay-Kumar, C. E. Bugg and W. J. Cook, J. Mol. Biol., 1987, 194,
12 M. H. Seelig, M. R. Berger and B. K. Keppler, J. Cancer Res. Clin.
Oncol., 1992, 118, 195–200.
13 M. A. Jakupec, V. B. Arion, S. Kapitza, E. Reisner, A. Eichinger, M.
Pongratz, B. Marian, N. Graf von Keyserlingk and B. K. Keppler,
Int. J. Clin. Pharmacol. Ther., 2005, 43, 595–596.
531–544.
37 G. W. Bushnell, G. V. Louie and G. D. Brayer, J. Mol. Biol., 1990, 214,
585–595.
38 D. J. Daigle, A. B. Pepperman and S. L. Vail, J. Heterocycl. Chem.,
1974, 11, 407–408.
14 (a) C. S. Allardyce, A. Dorcier, C. Scolaro and P. J. Dyson, Appl.
Organomet. Chem., 2005, 19, 1–10; (b) A. Vessieres, S. Top, W. Beck, E.
Hillard and G. Jaouen, Dalton Trans., 2006, 529–541.
15 M. Melchart and P. J. Sadler, in Bioorganometallics, ed. G. Jaouen,
Wiley-VCH, Weinheim, 2006, pp. 39–62.
16 C. Scolaro, T. J. Geldbach, S. Rochat, A. Dorcier, C. Gossens, A
Bergamo, M. Cocchietto, I. Tavernelli, G. Sava, U. Rothlisberger and
P. J. Dyson, Organometallics, 2006, 25, 756–765.
17 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.
39 M. A. Bennett and A. K. Smith, J. Chem. Soc., Dalton Trans., 1974,
233–241.
40 T. Onishi, Y. Miyaki, H. Asano and H. Kurosawa, Chem. Lett., 1999,
28, 809.
41 P. J. Dyson and J. S. McIndoe, Inorg. Chim. Acta, 2003, 354, 68–74.
42 A. B. Chaplin, C. Fellay, G. Laurenczy and P. J. Dyson, Organometallics,
2007, 26, 586.
43 Oxford Diffraction Ltd., Abingdon, Oxfordshire, 2003.
44 A. Altomare, M. C. Burla, M. Camalli, G. L. Cascarano, C. Giacov-
azzo, A. Guagliardi, A. G. G. Moliterni, G. Polidori and R. Spagna,
J. Appl. Crystallogr., 1999, 32, 115–119.
18 D. N. Akbayeva, L. Gonsalvi, W. Oberhauser, M. Peruzzini, F. Vizza, P.
Bru¨ggeller, A. Romerosa, G. Sava and A. Bergamo, Chem. Commun.,
2003, 264–265.
19 B. Serli, E. Zangrando, T. Gianferrara, C. Scolaro, P. J. Dyson, A.
Bergamo and E. Alessio, Eur. J. Inorg. Chem., 2005, 3423–3434.
20 A. Dorcier, W. H. Ang, S. Bolan˜o, L. Gonsalvi, L. Juillerat-Jeannerat,
G. Laurenczy, M. Peruzzini, A. D. Phillips, F. Zanobini and P. J. Dyson,
Organometallics, 2006, 25, 4090–4096.
45 (a) Bruker AXS, Inc., Madison, WI, 1997; (b) G. M. Sheldrick,
SHELXTL, University of Go¨ttingen, Germany, 1997.
46 L. J. Farrugia, J. Appl. Crystallogr., 1997, 30, 565.
47 P. Nanni, C. De Giovanni, P. L. Lollini, G. Nicoletti and G. Prodi, Clin.
Exp. Metastasis, 1983, 1, 373–385.
48 T. Mosmann, J. Immunol. Methods, 1983, 65, 55–63.
49 M. C. Alley, D. A. Scudiero, A. Monks, M. L. Hursey, M. J. Czerwinski,
D. L. Fine, B. J. Abbott, J. G. Mayo, R. H. Shoemaker and M. R. Boyd,
Cancer Res., 1988, 48, 589–601.
21 A. Romerosa, T. Campos-Malpartida, C. Lidrissi, M. Saoud, M.
Serrano-Ruiz, M. Peruzzini, J. A. Garrido-Ca´rdenas and F. Garca´a-
Maroto, Inorg. Chem., 2006, 45, 1289–1298.
50 H. Tamura and T. Arai, Bunseki Kagaku, 1992, 41, 13–17.
51 M. Cocchietto and G. Sava, Pharmacol. Toxicol., 2000, 87, 193–197.
5072 | Dalton Trans., 2007, 5065–5072
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