Highly cytotoxic diruthenium trithiolato complexes of the type [(η6-p-MeC6H4Pri) 2Ru2(μ2-SR)3]+: Synthesis, characterization, molecular structure and in vitro anticancer activity

Article abstract of DOI:10.1039/c3nj00476g

A new series of cationic dinuclear p-cymene ruthenium complexes bridged by three thiophenolato ligands containing various substituents mainly in meta and ortho positions, [(η⁶-p-MeC₆H₄Pr ⁱ)₂Ru₂(μ₂-SR)₃] ⁺ (R = 3-C₆H₄Me: 1; R = 3-C₆H ₄OMe: 2; R = 3-C₆H₄OEt: 3; R = 3-C ₆H₄CF₃: 4; R = 3-C₆H ₄NH₂: 5; R = 3-C₆H₄Cl: 6; R = 2-C₆H₄Me: 7; R = 2-C₆H₄OMe: 8; R = 2-C₆H₄Prⁱ: 9; R = 2-C₆H ₄CF₃: 10; R = npt: 11 (npt = 2-naphthyl); R = mco: 12 (mco = 4-methylcoumarinyl); R = 3,5-C₆H₃Me₂: 13; R = 3,5-C₆H₃(CF₃)₂: 14; R = 3,5-C ₆H₃Cl₂: 15; R = 3,4-C₆H ₃(OMe)₂: 16), have been prepared from the reaction of the neutral p-cymene diruthenium dichloride dimer, [(η⁶-p-MeC ₆H₄Prⁱ)₂Ru₂Cl ₄], with the corresponding thiophenol RSH. All cationic complexes have been isolated as their chloride salts and fully characterized by spectroscopic and analytical methods. The molecular structures of 10 and 15 have been solved by a single-crystal X-ray structure analysis of [10]Cl and [15]Cl, which show that the two ruthenium atoms adopt a pseudo-octahedral geometry without a metal-metal bond in accordance with the noble gas rule. All complexes are highly cytotoxic towards human ovarian cancer cells, the IC₅₀ values being mostly in the nanomolar range. Complex 9 shows the highest cytotoxicity with an IC₅₀ value of 0.03 μM towards the A2780 cell line and the cisplatin-resistant mutant A2780cisR. The cytotoxicity of these complexes, which belong to the most active ruthenium anticancer compounds reported so far, can be correlated with the lipophilicity of the corresponding thiols. In comparison with the previous series, the results demonstrate that the positions of the substituents in the thiopenolato bridges are not as important as the nature of the substituents, alkyl substituents being the best ones in line with their lipophilic character.

Full text of DOI:10.1039/c3nj00476g

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Highly cytotoxic diruthenium trithiolato complexes of
the type [(g6-p-MeC6H4Pri)2Ru2(l2-SR)3]+: synthesis,
characterization, molecular structure and in vitro
anticancer activity†‡
Cite this: DOI: 10.1039/c3nj00476g
Federico Giannini,a Lydia E. H. Paul,a Julien Furrer,*a Bruno Therrienb and
Georg Suss-Fink*b
¨
A new series of cationic dinuclear p-cymene ruthenium complexes bridged by three thiophenolato ligands
containing various substituents mainly in meta and ortho positions, [(Z6-p-MeC6H4Pri)2Ru2(m2-SR)3]+ (R =
3-C6H4Me: 1; R = 3-C6H4OMe: 2; R = 3-C6H4OEt: 3; R = 3-C6H4CF3: 4; R = 3-C6H4NH2: 5; R = 3-C6H4Cl: 6;
R = 2-C6H4Me: 7; R = 2-C6H4OMe: 8; R = 2-C6H4Pri: 9; R = 2-C6H4CF3: 10; R = npt: 11 (npt = 2-naphthyl);
R = mco: 12 (mco = 4-methylcoumarinyl); R = 3,5-C6H3Me2: 13; R = 3,5-C6H3(CF3)2: 14; R = 3,5-C6H3Cl2:
15; R = 3,4-C6H3(OMe)2: 16), have been prepared from the reaction of the neutral p-cymene diruthenium
dichloride dimer, [(Z6-p-MeC6H4Pri)2Ru2Cl4], with the corresponding thiophenol RSH. All cationic complexes
have been isolated as their chloride salts and fully characterized by spectroscopic and analytical methods.
The molecular structures of 10 and 15 have been solved by a single-crystal X-ray structure analysis of [10]Cl
and [15]Cl, which show that the two ruthenium atoms adopt a pseudo-octahedral geometry without a
metal–metal bond in accordance with the noble gas rule. All complexes are highly cytotoxic towards human
ovarian cancer cells, the IC50 values being mostly in the nanomolar range. Complex 9 shows the highest
cytotoxicity with an IC50 value of 0.03 mM towards the A2780 cell line and the cisplatin-resistant mutant
A2780cisR. The cytotoxicity of these complexes, which belong to the most active ruthenium anticancer
compounds reported so far, can be correlated with the lipophilicity of the corresponding thiols. In
comparison with the previous series, the results demonstrate that the positions of the substituents in the
thiopenolato bridges are not as important as the nature of the substituents, alkyl substituents being the
best ones in line with their lipophilic character.
Received (in Victoria, Australia)
6th May 2013,
Accepted 8th July 2013
DOI: 10.1039/c3nj00476g
www.rsc.org/njc
In the search for anticancer agents containing metals other
than platinum, ruthenium compounds turned out to be the
1. Introduction
most promising ones.2 The ligand exchange kinetics of metal
complexes in aqueous solution, which seem to be crucial for
the anticancer activity, are very similar for platinum(II) and
ruthenium(II) complexes.3 Ruthenium complexes containing
imidazole (imi) and indazole (ind) introduced by B. K. Keppler4
and G. Sava5 were found to be active against a number of tumors;
after extensive preclinical tests, the compounds [indH][trans-
Ru(N-ind)2Cl4] (KP1019) and [imiH][trans-Ru(N-imi)(S-dmso)Cl4]
(NAMI-A) went into clinical trials.2
The field of arene ruthenium anticancer compounds was
initiated in 1992 by D. Tocher, who had observed a cytotoxicity
enhancement by coordinating the anticancer agent metronidazole
to a benzene ruthenium dichloro fragment.6 Later on, this field
was pioneered by P. J. Dyson1 and by P. J. Sadler,7 who reported
in 2001 the arene ruthenium complexes [(Z6-p-MeC6H4Pri)Ru-
Water-soluble arene ruthenium complexes are easily taken up
by living cells, because they seem to have the right balance
between hydrophilic and lipophilic properties. Combined
with the synthetic diversity and the fact that ruthenium is a
non-toxic metal, water-soluble arene complexes are ideally
suited for medicinal applications.1
a
¨
¨
Department fur Chemie und Biochemie, Universitat Bern, CH-3012 Bern,
Switzerland. E-mail: julien.furrer@dcb.unibe.ch; Fax: +41 (0) 31 631 4887;
Tel: +41 (0) 31 631 4383
b
´
ˆ
ˆ
Institut de Chimie, Universite de Neuchatel, CH-2000 Neuchatel, Switzerland.
E-mail: georg.suess-fink@unine.ch; Fax: +41 (0) 32 718 2511;
Tel: +41 (0) 32 718 2405
† Dedicated to Professor Bernard Meunier on the occasion of his retirement.
‡ Electronic supplementary information (ESI) available. CCDC 927646 and
927647. For ESI and crystallographic data in CIF or other electronic format see
DOI: 10.1039/c3nj00476g
(pta)Cl2] (pta
=
1,3,5-triaza-7-phospha-tricyclo-[3.3.1.1]decane),
c
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termed RAPTA-C8 and [(C6H5Ph)Ru(en)Cl]+ (en = 1,2-ethylenedi-
amine)9 to show antimetastatic or antitumoral properties; both
groups studied arene ruthenium complexes of these types exten-
sively.10 Of particular interest are dinuclear arene ruthenium
complexes, for which the cytotoxicity could be correlated with
lipophilicity and water solubility: C. G. Hartinger reported a series of
ruthenium complexes containing a pyridone-derived linker as an
O,O-O,O-doubly bridging ligand, (Z6-p-MeC6H4Pri)Ru(O2C6H5N-
(CH2)nNC6H5O2)Ru(Z6-p-MeC6H4Pri) (n = 2–12), which showed high
activity against various cancer cell lines with a pronounced influence
of the spacer length on the cytotoxicity.11
Scheme 1 Synthesis of complexes 1–16 as the chloride salts.
methods as orange to red air stable chloride salts in good to
excellent yields (Scheme 1). The analytical data are given in the
Experimental section.
All compounds have been fully characterized by spectro-
scopic and analytical methods, the 1H and 13C NMR data being
particularly meaningful. For instance, the 1H NMR spectrum of
complex 1 presents four resonances in the aromatic region
between 7.8 and 7.1 ppm: a singlet at 7.69 ppm being assigned
to the ortho proton in position 2 of the aromatic ring of the
thiophenolato ligands, the two doublets at 7.67 and 7.17 ppm
being attributed to the ortho and para protons, and a doublet of
doublets at 7.27 ppm related to the meta proton. The meta
methyl groups of the thiopenolato ligands give rise to a singlet
at 2.45 ppm. Characteristic for p-cymene ruthenium complexes
is the low field shift for the resonances of the aromatic p-cymene
protons, for which the coordination to the ruthenium center
causes a chemical shift of the four doublets to lower frequencies
between 5.5 and 5.0 ppm. The resonances in the low frequency
region are caused by the aliphatic protons of the p-cymene
ligands: a septuplet at 1.94 ppm and the two doublets at 0.91
and 0.81 ppm caused by the two isopropyl groups and the singlet
at 1.63 ppm caused by the two methyl substituents (Scheme 2).
As an example of the complexes with an ortho substituent on
the thiophenolato ligands, the 1H NMR spectrum of complex 8
is discussed, while the 1H NMR spectrum of complex 15 serves as an
example of complexes with two substituents on the thiophenolato
ligands; the spectrum of 8 reveals three characteristic resonances in
the aromatic region: a doublet at 7.85 ppm due to the ortho proton,
a triplet of doublets at 7.36 ppm being assigned to the para proton
and a multiplet at 7.00 ppm being attributed to the two meta
protons. The region between 5.6 and 4.90 ppm presents four
doublets caused by the aromatic protons of the two p-cymene rings.
The methoxy group gives rise to a strong singlet at 4.10 ppm, a
septuplet at 1.99 ppm and two doublets at 0.86 and 0.67 ppm are
assigned to the isopropyl protons, and a singlet at 1.64 ppm is
Recently, we found cationic dinuclear arene ruthenium
complexes of general formula [(Z6-arene)2Ru2(m2-SR)3]+ to be
highly cytotoxic as chloride salts towards human ovarian cancer
cells A2780 and their cisplatin-resistant mutant A2780cisR, the
IC50 values being mostly in the submicromolar range.12 Incu-
bation with possible biological targets such as nucleotides,
peptides and amino acids revealed interactions with only
cysteine and glutathione, causing oxidation to cystine and
oxidized glutathione (GSSG), respectively, as observed by
NMR spectroscopy. The complexes can be recovered intact after
oxidation, which prompted us to suggest a catalytic role of the
ruthenium complex in its biological mode of action,13,14
a
mechanism that had been postulated for the first time by
Sadler.15
A systematic study of dinuclear trithiophenolato compounds
of general formula [(Z6-p-MeC6H4Pri)2Ru2(m2-S-4-C6H4X)3]Cl (X being
different functional groups in the para position of the thiophenolato
substituents)16 and [(Z6-p-MeC6H4Pri)2Ru2(m2-SR1)2(m2-SR2)]Cl (with
different thiolato bridges)17 suggested the cytotoxic activity to be
influenced by the lipophilicity and the Hammett’s constants of the
corresponding thiols, although a direct correlation between cyto-
toxicity, catalytic oxidation activity and redox potentials could not be
established, which is perhaps not surprising because the biological
activity may be mainly determined by the different cellular uptake of
the compounds.
As an extension of this work, we herein report the synthesis
and molecular structure, the in vitro anticancer activity and the
catalytic glutathione oxidation activity of a new series of sixteen
trithiophenolato-bridged dinuclear p-cymene ruthenium complexes
of general formula [(Z6-MeC6H4Pri)2Ru2(m2-SR)3]+, R being aromatic
substituents with various functional groups mainly in the meta and
ortho positions.
1
assigned to the methyl group of the p-cymene rings. The H NMR
spectrum of complex 15 shows nearly the same signals in the low
frequency region, but in the aromatic region between 8 and 7 ppm
only two singlets at 7.79 and 7.41 ppm show up, assignable to the
two ortho protons and the para proton of the thiophenolato ligands.
Interestingly, only one doublet at 5.56 ppm for the aromatic protons
of the two p-cymene ligands is observed in this case, whereas the
remaining protons collapse in a tight multiplet.
2. Results and discussion
2.1. Synthesis and characterization
The p-cymene ruthenium complex [(Z6-p-MeC6H4Pri)2Ru2Cl4] reacts
in refluxing ethanol with an excess of the corresponding thiol
to give the trithiolato complexes [(Z6-p-MeC6H4Pri)2Ru2(m2-SR)3]+
(R = 3-C6H4Me: 1; R = 3-C6H4OMe: 2; R = 3-C6H4OEt: 3; R =
3-C6H4CF3: 4; R = 3-C6H4NH2: 5; R = 3-C6H4Cl: 6; R = 2-C6H4Me:
7; R = 2-C6H4OMe: 8; R = 2-C6H4Pri: 9; R = 2-C6H4CF3: 10; R = npt:
2.2. Molecular structures of 10 and 15
11 (npt = 2-naphthyl); R = mco: 12 (mco = 4-methylcoumarinyl); R = Suitable crystals for X-ray structure analysis were obtained for
3,5-C6H3Me2: 13; R = 3,5-C6H3(CF3)2: 14; R = 3,5-C6H3Cl2: 15; R = complexes [10]Cl (R = 2-C6H4CF3) and [15]Cl (R = 3,5-C6H3Cl2).
3,4-C6H3(OMe)2: 16), which are isolated by chromatographic Both salts co-crystallize with solvent molecules and the
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Scheme 2 Schematic representation of compounds [1]Cl–[16]Cl.
stoichiometry in the crystals is [10]ClÁCH2Cl2 and [15]ClÁ which diastereotopic protons for the p-cymene ligands are
2CHCl3, respectively. These solvent molecules not only fill the observed by NMR spectroscopy (see the Experimental part).
voids in the crystal packing, they also form a series of weak
C–HÁ Á ÁCl interactions with their neighboring cations ([10]+ or
2.3. Cytotoxicity studies
[15]+) and anions (ClÀ): the CÁ Á ÁCl distances ranging from 3.3 to Complexes 1–16 were evaluated as the chloride salts for their
3.5 Å in [10]ClÁCH2Cl2 and from 3.3 to 3.7 in [15]ClÁ2CHCl3. The in vitro anticancer activity towards the human ovarian cancer
Ortep drawings including the atom labeling scheme for 10 and cell line A2780 and its cisplatin-resistant mutant A2780cisR,
15 are shown in Fig. 1 and 2, together with selected bond using the cell counting kit 8 assay (Dojindo), which measures
lengths and angles.
mitochondrial dehydrogenase activity as an indication of cell
In both structures, the ruthenium atoms adopt a pseudo- viability. The IC50 values, which represent the concentration of
octahedral geometry with three sulfur atoms and the p-cymene the complex that is required for 50% in vitro inhibition, are
ligand that formally occupies three coordination sites. The reported in Table 1.
Ru2S3 unit forms a trigonal-bipyramidal framework with no
All complexes except 12 are highly cytotoxic towards human
metal–metal bond; the RuÁ Á ÁRu distances being 3.3472(8) in 10 ovarian cancer cells, with IC50 values in the nanomolar range.
and 3.3518(5) Å in 15. The Ru–S bond distances in both cations All complexes except 16 exhibit similar activities against both
range from 2.3821(17) to 2.4239(15) Å and the Ru–S–Ru angles cell lines, A2780 and A2780cisR. Complex 9 (where R =
range from 87.85(4) to 88.82(5)1; these values are similar to 2-C6H4Pri) shows the highest cytotoxic effect for both ovarian
those found in the p-cymene derivatives [(Z6-p-MeC6H4- cancer cell lines, with an IC50 value of 0.03 mM towards A2780
Pri)2Ru2(m2-S-4-C6H4Br)3]+ and [(Z6-p-MeC6H4Pri)2Ru2(m2-S-4- and A2780cisR cell lines. Following the tendency we found for
C6H4Me)3]+.18,19 In both structures, the R groups of the thiolato the other classes of dinuclear trithiolato p-cymene ruthenium
bridges, R = 2-C6H4CF3 in 10 and 3,5-C6H3Cl2 in 15, are not in complexes,13,14,16,17 complexes 1–16 are amongst the most
the plane of the three sulfur atoms, thus generating a propeller active anticancer arene ruthenium compounds ever reported.
type chirality. The tilt of the R groups remains in solution, for For comparison, we also included cisplatin as a benchmark
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Table 1 Comparison of cytotoxicities of 1–16 with the calculated log P values
for the corresponding thiols
Complex
Log P (RSH)
IC50 [mM] A2780
IC50 [mM] A2780cisR
1
2
3
4
5
6
7
8
2.98 Æ 0.28
2.54 Æ 0.30
3.07 Æ 0.30
3.49 Æ 0.36
1.27 Æ 0.29
3.10 Æ 0.29
2.98 Æ 0.28
2.24 Æ 0.30
3.86 Æ 0.28
3.55 Æ 0.36
3.75 Æ 0.28
2.83 Æ 0.42
3.44 Æ 0.28
4.74 Æ 0.47
3.69 Æ 0.32
2.38 Æ 0.32
0.235 Æ 0.033
0.153 Æ 0.014
0.126 Æ 0.024
0.085 Æ 0.004
0.869 Æ 0.059
0.177 Æ 0.012
0.332 Æ 0.015
0.155 Æ 0.011
0.030 Æ 0.001
0.122 Æ 0.019
0.070 Æ 0.012
7.73 Æ 1.491
0.083 Æ 0.005
0.075 Æ 0.004
0.066 Æ 0.005
0.070 Æ 0.012
0.230 Æ 0.019
0.201 Æ 0.004
0.177 Æ 0.013
0.075 Æ 0.005
2.249 Æ 0.271
0.216 Æ 0.016
0.354 Æ 0.011
0.210 Æ 0.012
0.031 Æ 0.001
0.119 Æ 0.006
0.049 Æ 0.004
Z10
9
10
11
12
13
14
15
16
0.044 Æ 0.002
0.059 Æ 0.005
0.063 Æ 0.003
0.638 Æ 0.057
(R = 3,5-C6H3Me2) and 9 (R = 2-C6H4Pri), see Table 1. Ligands
containing alkoxyl substituents such as complex 3 (R =
3-C6H4OEt), 2 (R = 3-C6H4OMe) and 8 (R = 2-C6H4OMe) have
approximately the same cytotoxic values between 0.12 and
0.16 mM. For these complexes, as well as for those containing
only aliphatic substituents on the thiolato ligands, the disub-
stitution has a beneficial influence on the cytotoxicity: indeed,
complex 16 [R = 3,4-C6H3(OMe)2] has a lower IC50 value as
complex 2 (R = 3-C6H4OMe). The heteroatom-containing com-
plexes show almost the same cytotoxicity without correlation to
the position in the thiophenolato ligand, the IC50 values being
around 0.15 mM, with the exception of 4 (R = 2-C6H4CF3) with a
lower and 5 (R = 3-C6H4NH2) with a higher IC50 value.
Fig. 1 Ortep diagram of 10 with 35% probability level thermal ellipsoids, hydro-
gen atoms, chloride and dichloromethane being omitted for clarity. Selected bond
lengths (Å) and angles (1): Ru1–Ru2 3.3472(8), Ru1–S1 2.4033(15), Ru1–
S2 2.4028(15), Ru1–S3 2.3821(17), Ru2–S1 2.4093(15), Ru2–S2 2.3857(16), Ru2–
S3 2.4239(15); Ru1–S1–Ru2 88.26(5), Ru1–S2–Ru2 88.82(5), Ru1–S3–Ru2 88.41(5).
2.4. Correlation between cytotoxicity and lipophilicity
The in vitro activity of anticancer drugs can often be related in
part to their lipophilic character, the resulting hydrophobicity
may contribute to an increased uptake of the compound by the
cells, thereby enhancing the antiproliferative activity.20 In the
case of the complexes 1–16, the Ru2S3 core with two p-cymene
ligands remains the same for all complexes, the lipophilicity
should vary only as a function of the RSH log P parameters,
where the partition coefficient log P, calculated using the
ACD/ChemSketch software,21,22 reflects the lipophilicity of the
substituents.
Fig. 2 Ortep diagram of 15 with 35% probability level thermal ellipsoids,
hydrogen atoms, chloride and chloroform molecules being omitted for clarity.
Selected bond lengths (Å) and angles (1): Ru1–Ru2 3.3518(5), Ru1–S1 2.4193(12),
Ru1–S2 2.3952(12), Ru1–S3 2.4080(12), Ru2–S1 2.4126(11), Ru2–S2 2.4106(12),
Ru2–S3 2.3974(12); Ru1–S1–Ru2 87.85(4), Ru1–S2–Ru2 88.45(4), Ru1–S3–Ru2
88.46(4).
The partition coefficients of the thiols given in Table 1 are
plotted in Fig. S1 (ESI‡) against the IC50 values of the related
complexes 1–16. From these plots a correlation between lipo-
(IC50 2.94 Æ 0.17 mM for A2780 and 21.90 Æ 0.80 mM for philicity and cytotoxic activity is evidenced by the black regres-
A2780cisR). sion line; complexes derived from thiols with increasing log P
The character of the substituents in the thiolato ligands parameters up to the value 4 show a steady decrease of their
plays an important role in the differences in terms of cytotoxi- IC50 values, which seem to increase again after log P = 4.5, the
city observed for this series of complexes, whereas the position tendency being the same for both cell lines. From these plots it
of the substituents in the thiolato ligands seems to have little can be seen that complexes with ligands derived from thiols
influence on the in vitro anticancer activity. Whatever the substi- with log P values in the range between 3.5 and 4.0 show the
tuent position is, complexes with no heteroatom exhibit increasing highest cytotoxicities, whereas complexes with ligands derived
IC50 values that parallel the aliphatic character and lipophilicity from thiols with log P values less than 3.5 belong to the less
of the corresponding thiol, going from 1 (R = 3-C6H4Me) to 13 active one of the series, presumably due to an insufficient
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cellular uptake, since the less lipophilic compounds have more turnover frequencies for each complex as a catalyst at about 50%
difficulties to cross cell membranes.
conversion of GSH to GSSG, are reported in Table 2, in compar-
ison with the corresponding IC50 values. All the complexes
studied are completely intact after a catalytic run and active for
further runs. Since the [(Z6-p-MeC6H4Pri)2Ru2(m2-SR)3]+ com-
plexes are inert to substitution and stable towards oxygen and
water, we expect them to enter the cancer cells intact.
As expected, a direct correlation between cytotoxicity and cata-
lytic glutathione oxidation activity could not be established. One of
the most cytotoxic complexes of the series, 11 (with IC50 values of
0.07 mM for A2780 and 0.05 mM for A2780cisR), has a high catalytic
activity (TOF50 = 13.30 hÀ1) in line with its high cytotoxicity (low
IC50). Complex 12 (IC50 > 7 mM for both cell lines) shows a lower
TOF50 value of 5.59 hÀ1, as expected for lower cytotoxicity (high
IC50). However, the most cytotoxic complex 9 (IC50 = 0.03 mM for
both cell lines) has only a TOF50 of 5.39 hÀ1, in the same order of
magnitude as the two least cytotoxic complexes.
The lack of a direct correlation between the catalytic oxida-
tion activity and the cytotoxicity of these complexes is not really
surprising, since the biological activity of compounds depends
also on the cellular uptake and on the ability to penetrate
cancer cell membranes. This ability is mainly a function of
their lipophilicity and correlates therefore with the partition
coefficients of the compounds. Nevertheless, the catalytic
glutathione oxidation may play a role in the general mode of
action of these complexes.
2.5. Catalytic glutathione oxidation
The tripeptide glutathione, a thiol present in living cells with
concentrations ranging from 0.1 to 10 mM, is the major intra-
cellular reducing agent; it deactivates reactive oxygen species and
serves as a cofactor for redox modulating enzymes.23 Glutathione
is found in living cells either free or bound to proteins. Free
glutathione is present mainly in its reduced form (GSH), which
can be converted to the oxidized disulfide form (GSSG) during
oxidative stress and reverted to the reduced form by glutathione
reductase.24 Maintaining an optimal GSH : GSSG ratio in the
cells is critical for survival, and a deficiency of GSH can result in
oxidative damage. This ratio is superior to 100 in normal resting
cells, whereas in various situations of oxidative stress this ratio is
reported to decrease to values between 10 and 1.25 An imbalance
of GSH levels is observed in a wide range of pathologies,
including cancer;26 elevated GSH levels increase antioxidant
capacity and resistance to oxidative stress, and this is observed
in many types of cancer.26–28 A depletion of GSH could therefore
affect the ability of cancer cells to cope with oxidation damage.29
Recently, we found the series of highly cytotoxic complexes
of the general formula [(Z6-p-MeC6H4Pri)2Ru2(m2-SR1)2(m2-SR2)]+
(where R1 are aliphatic groups and R2 are p-substituted phenyl
groups) to catalyze efficiently the oxidation of GSH to GSSG.
However, it was not possible to establish a direct correlation
between their in vitro anticancer activity (IC50) and their catalytic
activity (TOF50).17
3. Conclusion
For the present series of complexes [(Z6-p-MeC6H4Pri)2-
Ru2(m2-SR)3]+ containing thiophenolato ligands with ortho and
meta substituents, the catalytic activity for the oxidation of GSH
to GSSG was also analyzed by NMR spectroscopy. Since the
glutathione autoxidation in the presence of O2 is less than 5%
in 24 h, we incubated the two most cytotoxic complexes 9 and
11 (lowest IC50) and the two least cytotoxic complexes 5 and
12 (highest IC50) with GSH in a 1 : 100 ratio, in a solution of
D2O/DMSO-d6 (99 : 1), at pD 7 and 37 1C and in an aerobic
atmosphere (see Table 2).
To the best of our knowledge, the new diruthenium trithiolato
complexes, all obtained in good to excellent yields, are among
the most cytotoxic arene ruthenium compounds ever reported.
Interestingly, all complexes show comparable effects on both,
cisplatin-sensitive and cisplatin-resistant human ovarian cancer
cells. Based on our results for the compounds [(Z6-p-MeC6H4Pri)2-
Ru2(m2-S-4-C6H4X)3]Cl with different functional groups X in the
para position of the thiophenolato substituents,16 it can be
assumed that, also for the new complexes with substituents
in meta and ortho positions, the catalytic oxidation of GSH
to GSSG plays a role in the biological activity of these com-
plexes, but other modes of action probably involving non-
covalent interactions with enzymes and/or DNA, may also be
involved.
From the results obtained, we can confirm that the lipophilicity
plays an important role for these complexes. A comparison with
the para-substituted analogues [(Z6-p-MeC6H4Pri)2Ru2(m2-S-4-
C6H4X)3]Cl16 shows that the position of the substituent in the
aromatic rings of the thiolato bridges is not so important
as the nature of the substituent. In the series of the mixed
thiolato derivatives [(Z6-p-MeC6H4Pri)2Ru2(m2-SR1)2(m2-SR2)]Cl,17
it turned out that with different thiolato bridges it is possible to
compensate the effect of one thiolato bridge by the other one.
Overall, it can be generalized that the cytotoxicity of this type of
complexes without heteroatoms in the thiolato bridges parallels
the aliphatic character and the lipophilicity of the corresponding
thiols.
The 1H-NMR spectra confirm the complete oxidation of GSH
to GSSG within 24 h in the four experiments, as evidenced by
the complete disappearance of the b-CH2 resonances of GSH at
d 3.01 ppm and the simultaneous appearance of two new
signals at d 3.06 ppm and d 3.34 ppm, and by the replacement
of the a-CH resonance of GSH at d 4.63 ppm with a new signal
at d 4.81 ppm. The TOF50 values, which correspond to the
Table 2 Comparison of cytotoxicity (IC50) with catalytic activity (TOF50) of the
most and the least cytotoxic complexes
IC50 [mM]
A2780
IC50 [mM]
A2780cisR
Complex
R
TOF50 (hÀ1
)
9
11
5
2-C6H4Pri
npt
3-C6H4NH2
mco
5.39
13.30
6.09
0.030 Æ 0.001 0.031 Æ 0.001
0.070 Æ 0.012 0.049 Æ 0.004
0.869 Æ 0.059 2.249 Æ 0.271
7.73 Æ 1.491 Z10
12
5.59
c
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4. Experimental
4.1. Materials and methods
139.2, 130.1, 125.1, 118.5, 113.4, 107.6, 99.9, 85.5, 85.1, 84.9,
83.8, 55.9, 30.6, 22.6, 22.0, 17.7 ppm.
4.2.3. Data for [3]Cl. Yield: 173.4 mg (90%).
The starting material [(Z6-p-MeC6H4Pri)2Ru2Cl4] was prepared
according to published methods.30 All other reagents were
commercially available and were used as received. NMR spectra
were recorded with Bruker 400 MHz and 500 MHz spectro-
meters. Electrospray mass spectra were obtained in positive- or
negative-ion mode with an LCQ Finningan mass spectrometer.
Microanalyses were performed by the Mikroelementaranaly-
tisches Laboratorium, ETH, Zu¨rich (Switzerland).
1
C
44H55ClO3Ru2S3Á2CH2Cl2 (1008.17): calcd C 53.02, H 5.60%;
found C 52.97, H 5.70%. ESI MS (MeOH): m/z = 930.7 [M]+.
1H NMR (400 MHz, CDCl3): d = 7.49 (d, 3J = 7.6 Hz, 3 H, SC6H4-
m-OCH2CH3), 7.44 (m, 3 H, SC6H4-m-OCH2CH3), 7.34 (t, 3J =
8.4 Hz, 3 H, SC6H4-m-OCH2CH3), 6.94 (d, 3J = 7.6 Hz, 3 H, SC6H4-
m-OCH2CH3), 5.37 (d, 3J = 4 Hz, 2 H, p-MeC6H4Pri), 5.23 (d, 3J =
4 Hz, 2 H, p-MeC6H4Pri), 5.13 (d, J = 4 Hz, 2 H, p-MeC6H4Pri),
3
5.10 (d, 3J = 4 Hz, 2 H, p-MeC6H4Pri), 4.13 (q, 3J = 6.8 Hz,
6 H, SC6H4-m-OCH2CH3), 1.97 [sept., 3J = 6.80 Hz, 2 H,
4.2. Synthesis of complexes [(g6-p-MeC6H4Pri)2Ru2(l2-SR)3]+
(1–16)
p-MeC6H4CH(CH3)2], 1.65 (s,
(t, J = 6.8 Hz, 9 H, SC6H4-m-OCH2CH3), 0.92 [d, J = 4 Hz, 6 H,
6
H, p-CH3C6H4Pri), 1.50
3
3
A solution of [(Z6-p-MeC6H4Pri)2Ru2Cl4] (122.4 mg, 0.2 mmol) p-MeC6H4CH(CH3)2], 0.80 [d, 3J = 8 Hz, 6 H, p-MeC6H4CH(CH3)2] ppm.
in technical grade EtOH (40 ml) was heated under reflux. As 13C{1H} NMR (100 MHz, CDCl3): d = 158.9, 138.9, 130.1, 124.8,
soon as the starting material was completely dissolved, a 119.0, 114.3, 107.5, 99.9, 85.5, 84.9, 84.8, 83.7, 63.9, 30.6, 22.7,
solution of 6 equivalents of the corresponding thiol in 5 ml 21.9, 17.7, 14.9 ppm.
EtOH was added dropwise (1,3-MeC6H4SH: 143 ml; 1,3-
4.2.4. Data for [4]Cl. Yield: 149.40 mg (72%). C41H40F9ClRu2S3Á
MeOC6H4SH: 148 ml; 1,3-EtOC6H4SH: 169 ml; 1,3-CF3C6H4SH: 12EtOH (1060.57): calcd C 48.69, H 4.37%; found C 48.85, H 4.58%.
163 ml; 1,3-NH2C6H4SH: 128 ml; 1,3-ClC6H4SH: 140 ml; 1,2- ESI MS (MeOH): m/z = 1003.02 [M + H]+. 1H NMR (400 MHz,
MeC6H4SH: 142 ml; 1,2-MeOC6H4SH: 146 ml; 1,2-PriC6H4SH: CDCl3): d = 8.50 (d, J = 7.4 Hz, 3 H, SC6H4-m-CF3), 8.05 (s, 3 H,
3
182 ml; 1,2-CF3C6H4SH: 159 ml; npt-SH: 192 mg; mco-SH: 230 mg; SC6H4-m-CF3), 7.72 (t, 3J = 6.82 Hz, 3 H, SC6H4-m-CF3), 7.64 (d, 3J =
1,3,5-Me2C6H3SH: 164 ml; 1,3,5-(CF3)2C6H3SH: 203 ml; 1,3,5- 6.82 Hz, 3 H, SC6H4-m-CF3), 5.68 (m, 2 H, p-MeC6H4Pri), 5.34 (m,
Cl2C6H3SH: 215 mg; 3,4-(MeO)2C6H3-1-SH: 174 ml). The resulting 4 H, p-MeC6H4Pri), 5.26 (m, 2 H, p-MeC6H4Pri), 1.89 [sept., 3J =
solution was refluxed for 18 h. Then, the solvent was evaporated, 6.90 Hz, 2 H, p-MeC6H4CH(CH3)2], 1.62 (s, 6 H, p-CH3C6H4Pri), 0.88
and the residue was purified by column chromatography on [d, 3J = 6.90 Hz, 6 H, p-MeC6H4CH(CH3)2], 0.79 [d, 3J = 6.90 Hz, 6 H,
silica gel using dichloromethane–ethanol (5: 1) as an eluent. The p-MeC6H4CH(CH3)2] ppm. 13C{1H} NMR (100 MHz, CDCl3): d =
yellow to reddish products were isolated as chloride salts and 138.9, 137.3, 133.1, 130.7, 128.8, 125.1, 125.0, 108.1, 100.5, 85.9,
dried under vacuum.
85.6, 85.3, 84.1, 30.7, 22.4, 21.8, 17.6 ppm.
4.2.1. Data for [1]Cl. Yield: 145.0 mg (83%). C41H49ClRu2S3Á
4.2.5. Data for [5]Cl. Yield: 141.09 mg (80%).
38H46N3ClRu2S3Á2CH2Cl2 (921.05): calcd C 50.21, H 5.14%;
1
14CH2Cl2 (896.86): calcd C 55.24, H 5.56%; found C 55.07,
C
H 6.06%. ESI MS (MeOH): m/z = 841.11 [M + H]+. 1H NMR found C 50.13, H 5.60%. ESI MS (MeOH): m/z = 844.09 [M +
(400 MHz, CDCl3): d = 7.69 (s, 3 H, SC6H4-m-CH3), 7.67 H]+. 1H NMR (400 MHz, CDCl3): d = 7.55 (s, 3 H, SC6H4-m-NH2),
3
(d, J = 7.42 Hz, 3 H, SC6H4-m-CH3), 7.27 (m, 3 H, SC6H4-m-CH3), 7.11 (d, 3J = 7.90 Hz, 3 H, SC6H4-m-NH2), 7.03 (t, 3J = 7.90 Hz, 3
3
3
7.17 (d, J = 7.42 Hz, 3 H, SC6H4-m-CH3), 5.35 (d, J = 5.39 Hz, H, SC6H4-m-NH2), 6.61 (d, 3J = 7.90 Hz, 3 H, SC6H4-m-NH2), 5.57
2 H, p-MeC6H4Pri), 5.21 (d, 3J = 5.39 Hz, 2 H, p-MeC6H4Pri), 5.11 (d, J = 5.75 Hz, 2 H, p-MeC6H4Pri), 5.32 (d, J = 5.75 Hz, 2 H,
3
3
(d, 3J = 5.39 Hz, 2 H, p-MeC6H4Pri), 5.08 (d, 3J = 5.39 Hz, p-MeC6H4Pri), 5.25 (d, 3J = 5.75 Hz, 2 H, p-MeC6H4Pri), 5.19
2 H, p-MeC6H4Pri), 2.45 (s, 9 H, SC6H4-m-CH3), 1.94 [sept., J = (d, 3J = 5.75 Hz, 2 H, p-MeC6H4Pri), 2.06 [sept., 3J = 6.95 Hz, 2 H,
3
6.95 Hz, 2 H, p-MeC6H4CH(CH3)2], 1.63 (s, 6 H, p-CH3C6H4Pri), p-MeC6H4CH(CH3)2], 1.66 (s, 6 H, p-CH3C6H4Pri), 0.94 [d, J =
3
0.91 [d, 3J = 6.83 Hz, 6 H, p-MeC6H4CH(CH3)2], 0.81 [d, 3J = 6.83 Hz, 6.90 Hz, 6 H, p-MeC6H4CH(CH3)2], 0.83 [d, J = 6.90 Hz, 6 H,
3
6 H, p-MeC6H4CH(CH3)2] ppm. 13C{1H} NMR (100 MHz, CDCl3): p-MeC6H4CH(CH3)2] ppm. 13C{1H} NMR (100 MHz, CDCl3): d =
d = 138.9, 137.6, 133.2, 129.5, 129.3, 129.1, 107.4, 99.8, 85.5, 147.4, 138.9, 129.1, 121.7, 119.5, 114.8, 107.1, 99.7, 85.5, 85.3,
84.8, 83.55, 82.3, 30.6, 22.0, 21.6, 21.4, 17.7 ppm.
84.6, 84.3, 30.6, 22.8, 22.0, 17.6 ppm.
4.2.2. Data for [2]Cl. Yield: 162.0 mg (88%). C41H49O3Cl-
Ru2S3 (923.61): calcd C 53.32, H 5.35%; found C 53.42, H 5.95%.
4.2.6. Data for [6]Cl. Yield: 110.5 mg (59%).
4
C
38H40Cl4Ru2S3Á5 EtOH (973.73): calcd C 49.33, H 4.76%; found
ESI MS (MeOH): m/z = 889.09 [M + H]+. 1H NMR (400 MHz, C 49.20, H 4.94%. ESI MS (MeOH): m/z = 902.94 [M + H]+.
CDCl3): d = 7.48 (m, 3 H, SC6H4-m-OCH3), 7.43 (m, 3 H, SC6H4- 1H NMR (400 MHz, CDCl3): d = 8.01 (d, 3J = 7.80 Hz, 3 H, SC6H4-
m-OCH3), 7.32 (t, 3J = 8.06 Hz, 3 H, SC6H4-m-OCH3), 6.90 m-Cl), 1 27.80 (m, 3 H, SC6H4-m-Cl), 7.46 (t, J = 7.85 Hz, 3 H,
3
3 3
3
(m, 3 H, SC6H4-m-OCH3), 5.42 (d, J = 5.80 Hz, 2 H, p-MeC6H4- SC6H4-m-Cl), 7.36 (d, 3J = 7.85 Hz, 3 H, SC6H4-m-Cl), 5.57 (d, 3J =
3
3
Pri), 5.26 (d, J = 5.80 Hz, 2 H, p-MeC6H4Pri), 5.17 (d, J = 5.80 5.95 Hz, 2 H, p-MeC6H4Pri), 5.33 (d, 3J = 5.95 Hz, 2 H,
Hz, 2 H, p-MeC6H4Pri), 5.12 (d, 3J = 5.80 Hz, 2 H, p-MeC6H4Pri), p-MeC6H4Pri), 5.28 (d, 3J = 5.95 Hz, 2 H, p-MeC6H4Pri), 5.25
3.93 (s, 9 H, SC6H4-m-OCH3), 1.98 [sept., 3J = 6.95 Hz, 2 H, (d, 3J = 5.95 Hz, 2 H, p-MeC6H4Pri), 1.99 [sept., 3J = 6.85 Hz, 2 H,
p-MeC6H4CH(CH3)2], 1.66 (s, 6 H, p-CH3C6H4Pri), 0.93 [d, J = p-MeC6H4CH(CH3)2], 1.67 (s, 6 H, p-CH3C6H4Pri), 0.95 [d, J =
6.90 Hz, 6 H, p-MeC6H4CH(CH3)2], 0.82 [d, J = 6.90 Hz, 6 H, 6.90 Hz, 6 H, p-MeC6H4CH(CH3)2], 0.85 [d, J = 6.90 Hz, 6 H,
p-MeC6H4CH(CH3)2] ppm. 13C{1H} NMR (100 MHz, CDCl3): d = 159.6, p-MeC6H4CH(CH3)2] ppm. 13C{1H} NMR (100 MHz, CDCl3): d = 139.6,
3
3
3
3
c
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134.2, 131.9, 131.6, 131.1, 128.7, 107.9, 100.4, 85.8, 85.3, 85.2,
83.9, 30.8, 22.5, 22.0, 17.8 ppm.
4.2.11. Data for [11]Cl. Yield: 137.7 mg (70%).
50H49ClRu2S3ÁEtOH (1029.78): calcd C 60.65, H 5.38%; found
C
4.2.7. Data for [7]Cl. Yield: 112.95 mg (65%). C 60.35, H 5.50%. ESI MS (MeOH): m/z = 949.3 [M + H]+.
C
41H49ClRu2S3Á CH2Cl2 (896.86): calcd C 55.24, H 5.56%; found 1H NMR (400 MHz, CDCl3): d = 8.52 (s, 3 H, SC10H7), 8.04
1
4
C 55.13, H 6.04%. ESI MS (MeOH): m/z = 841.11 [M + H]+. (m, 6 H, SC10H7), 7.92 (m, 6 H, SC10H7), 7.57 (m, 6 H, SC10H7),
1H NMR (400 MHz, CDCl3): d = 7.96 (d, 3J = 7.85 Hz, 3 H, SC6H4- 5.51 (d, 3J = 5.5 Hz, 2 H, p-MeC6H4Pri), 5.31 (d, 3J = 5.5 Hz, 2 H,
o-CH3), 7.29 (m, 3 H, SC6H4-o-CH3), 7.27 (m, 3 H, SC6H4-o-CH3), p-MeC6H4Pri), 5.28 (d, 3J = 5.5 Hz, 2 H, p-MeC6H4Pri), 5.24
7.23 (m, 3 H, SC6H4-o-CH3), 5.34 (d, 3J = 5.75 Hz, 2 H, (d, 3J = 5.5 Hz, 2 H, p-MeC6H4Pri), 1.96 [sept., 3J = 6.40 Hz, 2 H,
p-MeC6H4Pri), 5.19 (d, 3J = 5.75 Hz, 2 H, p-MeC6H4Pri), 5.13 p-MeC6H4CH(CH3)2], 1.58 (s, 6 H, p-CH3C6H4Pri), 0.81 [d, J =
3
3
3
3
(d, J = 5.75 Hz, 2 H, p-MeC6H4Pri), 5.09 (d, J = 5.75 Hz, 2 H, 6.80 Hz, 6 H, p-MeC6H4CH(CH3)2], 0.71 [d, J = 6.80 Hz, 6 H,
p-MeC6H4Pri), 2.86 (s, 9 H, SC6H4-o-CH3), 1.95 [sept., 3J = p-MeC6H4CH(CH3)2] ppm. 13C{1H} NMR (100 MHz, CDCl3): d =
6.95 Hz, 2 H, p-MeC6H4CH(CH3)2], 1.58 (s, 6 H, p-CH3C6H4Pri), 135.4, 133.4, 133.0, 132.2, 129.8, 129.1, 128.0, 127.9, 127.3,
0.84 [d, 3J = 6.90 Hz, 6 H, p-MeC6H4CH(CH3)2], 0.70 [d, 3J = 127.0, 108.0, 99.7, 85.9, 85.3, 84.6, 83.7, 30.8, 22.7, 21.9,
6.90 Hz, 6 H, p-MeC6H4CH(CH3)2] ppm. 13C{1H} NMR (100 MHz, 17.8 ppm.
CDCl3): d = 140.7, 137.0, 133.0, 130.7, 128.7, 127.2, 107.2, 99.9,
84.7, 83.8, 83.4, 83.0, 30.7, 22.6, 22.0, 21.3, 17.7 ppm.
4.2.12. Data for [12]Cl. Yield: 183.5 mg (85%).
50H49O6ClRu2S3ÁEtOHÁCH2Cl2 (1210.71): calcd C 52.58, H
C
4.2.8. Data for [8]Cl. Yield: 109.9 mg (60%). 4.75%; found C 52.84, H 5.00%. ESI MS (MeOH): m/z = 1045.3
C
41H49O3ClRu2S3Á CH2Cl2 (987.33): calcd C 51.09, H 5.20%; [M + H]+. H NMR (400 MHz, CDCl3): d = 8.33 (d, J = 8.4 Hz, 3
1
3
3
4
found C 50.95, H 5.75%. ESI MS (MeOH): m/z = 888.1 [M]+. H, SC6H3C3HO2-7-CH3), 7.85 (d, 3J = 8.4 Hz, 3 H, SC6H3C3HO2-7-
1H NMR (400 MHz, CDCl3): d = 7.85 (d, 3J = 7.80 Hz, 3 H, SC6H4- CH3), 7.73 (s,
3 H, SC6H3C3HO2-7-CH3), 6.36 (s, 3 H,
o-OCH3), 7.36 (t, 3J = 7.80 Hz, 3 H, SC6H4-o-OCH3), 7.00 (m, 6 H, SC6H3C3HO2-7-CH3), 5.79 (d, 3J = 6.0 Hz, 2 H, p-MeC6H4Pri),
3
SC6H4-o-OCH3), 5.43 (d, J = 5.60 Hz, 2 H, p-MeC6H4Pri), 5.33 5.42 (d, 3J = 6.0 Hz, 2 H, p-MeC6H4Pri), 5.38 (d, 3J = 6.0 Hz, 2 H,
3
3
(d, J = 5.60 Hz, 2 H, p-MeC6H4Pri), 5.18 (d, J = 5.60 Hz, 2 H, p-MeC6H4Pri), 5.31 (d, 3J = 6.0 Hz, 2 H, p-MeC6H4Pri), 2.53
p-MeC6H4Pri), 5.02 (d, 3J = 5.60 Hz, 2 H, p-MeC6H4Pri), 4.10 (s, 9 H, SC6H3C3HO2-7-CH3), 1.98 [sept., 3J = 6.90 Hz, 2 H,
(s, 9 H, SC6H4-o-OCH3), 1.99 [sept., 3J = 6.80 Hz, 2 H, p-MeC6H4CH(CH3)2], 1.67 (s, 6 H, p-CH3C6H4Pri), 0.91 [d, J =
3
3
3
p-MeC6H4CH(CH3)2], 1.64 (s, 6 H, p-CH3C6H4Pri), 0.86 [d, J = 6.80 Hz, 6 H, p-MeC6H4CH(CH3)2], 0.76 [d, J = 6.80 Hz, 6 H,
3
6.90 Hz, 6 H, p-MeC6H4CH(CH3)2], 0.67 [d, J = 6.90 Hz, 6 H, p-MeC6H4CH(CH3)2] ppm. 13C{1H} NMR (100 MHz, CDCl3): d =
p-MeC6H4CH(CH3)2] ppm. 13C{1H} NMR (100 MHz, CDCl3): d = 160.6, 153.2, 152.7, 143.0, 130.1, 126.2, 120.1, 119.9, 115.3,
160.0, 134.5, 129.7, 125.7, 121.6, 111.5, 107.3, 100.4, 84.5, 83.9, 108.3, 100.8, 85.7, 85.6, 85.4, 84.3, 30.5, 22.9, 21.9, 19.0,
83.8, 83.6, 56.4, 30.6, 22.8, 21.3, 17.8 ppm.
18.1 ppm.
4.2.13. Data for [13]Cl. Yield: 119.3 mg (65%).
4.2.9. Data for [9]Cl. Yield: 111.9 mg (58%). C47H61ClRu2S3Á
12CH2Cl2 (1002.25): calcd C 56.92, H 6.24%; found C 56.66,
C
44H55ClRu2S3ÁEtOH (963.78): calcd C 57.32, H 6.38%; found
H 6.63%. ESI MS (MeOH): m/z = 925.2 [M + H]+. 1H NMR C 56.99, H 6.64%. ESI MS (MeOH): m/z = 883.16 [M + H]+.
3
(400 MHz, CDCl3): d = 7.95 (d, J = 7.90 Hz, 3 H, SC6H4-o-Pri), 1H NMR (400 MHz, CDCl3): d = 7.45 (s, 6 H, SC6H3-3,5-CH3),
7.36 (m, 6 H, SC6H4-o-Pri), 7.20 (m, 3 H, SC6H4-o-Pri), 5.28 6.98 (s, 3 H, SC6H3-3,5-CH3), 5.27 (d, 3J = 5.60 Hz, 2 H,
3
3
(d, J = 5.70 Hz, 2 H, p-MeC6H4Pri), 5.18 (d, J = 5.70 Hz, 2 H, p-MeC6H4Pri), 5.17 (d, 3J = 5.60 Hz, 2 H, p-MeC6H4Pri), 5.07
p-MeC6H4Pri), 5.05 (d, 3J = 5.70 Hz, 2 H, p-MeC6H4Pri), 4.99 (d, J = 5.60 Hz, 2 H, p-MeC6H4Pri), 5.03 (d, J = 5.60 Hz, 2 H,
3
3
(d, 3J = 5.70 Hz, 2 H, p-MeC6H4Pri), 4.19 [sept., 3J = 6.80 Hz, 3 H, p-MeC6H4Pri), 2.41 [s, 18 H, SC6H3-3,5-CH3], 1.96 [sept., 3J =
SC6H4-o-CH(CH3)2], 1.97 [sept., 3J
=
6.80 Hz,
p-MeC6H4CH(CH3)2], 1.60 (s, 6 H, p-CH3C6H4Pri), 1.50 [d, J = 0.93 [d, 3J = 6.90 Hz, 6 H, p-MeC6H4CH(CH3)2], 0.85 [d, 3J =
6.90 Hz, 18 H, SC6H4-o-CH(CH3)2], 0.80 [d, 3J = 6.90 Hz, 6 H, 6.90 Hz, H, p-MeC6H4CH(CH3)2] ppm. 13C{1H} NMR
p-MeC6H4CH(CH3)2], 0.68 [d, 3J
6.90 Hz, H, (100 MHz, CDCl3): d = 138.6, 137.4, 130.3, 130.2, 107.5, 99.6,
p-MeC6H4CH(CH3)2] ppm. 13C{1H} NMR (100 MHz, CDCl3): 85.5, 84.7, 84.6, 83.4, 30.5, 22.5, 22.0, 21.5, 17.7 ppm.
2
H, 6.80 Hz, 2 H, p-MeC6H4CH(CH3)2], 1.65 (s, 6 H, p-CH3C6H4Pri),
3
6
=
6
d = 151.3, 135.3, 133.3, 129.3, 126.8, 125.7, 106.8, 100.0, 84.6,
83.7, 83.6, 83.5, 31.0, 30.5, 23.9, 22.5, 21.2, 17.8 ppm.
4.2.14. Data for [14]Cl. Yield: 134.1 mg (54%).
44H37F18ClRu2S3Á3 EtOH (1287.61): calcd C 43.53, H 4.02%;
C
4.2.10. Data for [10]Cl. Yield: 151.2 mg (73%). found C 43.75, H 3.77%. ESI MS (MeOH): m/z = 1206.99 [M +
41H40F9ClRu2S3 (1037.54): calcd C 47.46, H 3.89%; found C H]+. 1H NMR (400 MHz, CDCl3): d = 8.35 (s, 6 H, SC6H3-3,5-CF3),
C
47.17, H 4.07%. ESI MS (MeOH): m/z = 1003.0 [M + H]+. 1H NMR 7.94 (s, 3 H, SC6H3-3,5-CF3), 5.62 (m, 2 H, p-MeC6H4Pri), 5.35
3
(400 MHz, CDCl3): d = 8.31 (d, J = 7.2 Hz, 3 H, SC6H4-o-CF3), (m, 6 H, p-MeC6H4Pri), 1.89 [sept., 3J = 6.90 Hz, 2 H,
3
7.78 (d, J = 7.2 Hz, 3 H, SC6H4-o-CF3), 7.61 (m, 6 H, SC6H4-o- p-MeC6H4CH(CH3)2], 1.71 (s, 6 H, p-CH3C6H4Pri), 0.90 [m, 12 H,
3
CF3), 5.18 (m, 8 H, p-MeC6H4Pri), 2.01 [sept., J = 6.80 Hz, 2 H, p-MeC6H4CH(CH3)2] ppm. 13C{1H} NMR (100 MHz, CDCl3):
3
p-MeC6H4CH(CH3)2], 1.57 (s, 6 H, p-CH3C6H4Pri), 0.83 [d, J = d = 140.5, 132.5, 124.0, 122.5, 121.3, 109.2, 101.0, 86.8, 86.0,
3
6.90 Hz, 6 H, p-MeC6H4CH(CH3)2], 0.71 [d, J = 6.90 Hz, 6 H, 84.9, 84.0, 31.0, 22.3, 21.6, 17.9 ppm.
p-MeC6H4CH(CH3)2] ppm. 13C{1H} NMR (100 MHz, CDCl3): d =
137.2, 135.5, 133.4, 133.0, 129.3, 127.1, 122.7, 108.0, 100.7, 84.9,
84.5, 84.4, 83.6, 30.5, 22.7, 20.9, 17.6 ppm.
4.2.15. Data for [15]Cl. Yield: 141.4 mg (68%).
C
38H37Cl7Ru2S3 (1040.22): calcd C 43.88, H 3.59%; found C
1
43.87, H 3.73%. ESI MS (MeOH): m/z = 1004.9 [M]+. H NMR
c
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(400 MHz, CDCl3): d = 7.79 (s, 6 H, SC6H3-3,5-Cl), 7.41 (s, 3 H, refinement and all further calculations were carried out using
SC6H3-3,5-Cl), 5.56 (d, 3J = 5.6 Hz, 2 H, p-MeC6H4Pri), 5.32 SHELXL-97.31 The H-atoms were included in calculated posi-
(m, 6 H, p-MeC6H4Pri), 2.06 [sept., 3J = 6.80 Hz, 2 H, tions and treated as riding atoms using the SHELXL default
3
p-MeC6H4CH(CH3)2], 1.64 (s, 6 H, p-CH3C6H4Pri), 1.02 [d, J = parameters. The non-H atoms were refined anisotropically,
6.8 Hz, 6 H, p-MeC6H4CH(CH3)2], 0.95 [d, 3J = 6.8 Hz, 6 H, using weighted full-matrix least-square on F2 (Table 3). Fig. 1
p-MeC6H4CH(CH3)2] ppm. 13C{1H} NMR (100 MHz, CDCl3): d = and 2 were drawn with ORTEP.32
140.6, 135.5, 130.6, 129.1, 108.7, 100.8, 86.1, 85.4, 85.1, 83.9,
31.0, 22.5, 22.0, 18.1 ppm.
The files CCDC 927646 [10]ClÁCH2Cl2 and 927647 [15]ClÁ2CHCl3
contain the supplementary crystallographic data for this paper.
4.2.16. Data for [16]Cl. Yield: 166 mg (82%).
1
4.4. Cell culture and inhibition of cell growth
C
44H55ClO6Ru2S3Á8CH2Cl2 (1024.32): calcd C 51.35, H 5.41%;
found C 51.65, H 5.60%. ESI MS (MeOH): m/z = 979.1 [M + H]+.
1H NMR (400 MHz, CDCl3): d = 7.55 (d, 3J = 8.4 Hz, 3 H, SC6H3-
Human A2780 and A2780cisR ovarian carcinoma cells were
obtained from the European Centre of Cell Cultures (ECACC,
Salisbury, UK) and maintained in culture as described by the
provider. The cells were routinely grown in RPM1 1640 medium
which contained fetal calf serum (FCS) (10%), 2 mM Gln and
1% antibiotics (penicillin/streptomycin) at 37 1C and CO2 (5%).
Cytotoxicity was determined using the cell counting kit 8
(Dojindo). Therefore, the cells were seeded in 96-well plates
as monolayers with 100 mL of cell solution (approximately
10 000 cells) per well. Compounds were dissolved in DMSO,
then dissolved in the culture medium and serially diluted to the
appropriate concentration, to give a final DMSO concentration
of 1%. 100 mL of drug solution was added to each well and the
plates were incubated for 96 h. After incubation the culture
medium was removed completely and subsequently, 10 mL kit
solution and 100 mL fresh medium were added to the cells. The
plates were incubated for a further 90 minutes. The optical
density, directly proportional to the number of surviving cells,
was quantified at 450 nm using a multiwall plate reader and the
fraction of surviving cells was calculated from the absorbance
of untreated control cells. Evaluation is based on means from
four independent experiments, each comprising four micro-
cultures per concentration level.
3
3,4-OCH3), 7.50 (s, 3 H, SC6H3-3,4-OCH3), 6.95 (d, J = 8.4 Hz,
3 H, SC6H3-3,4-OCH3), 5.47 (d, 3J = 5.6 Hz, 2 H, p-MeC6H4Pri), 5.30
(d, 3J = 5.6 Hz, 2 H, p-MeC6H4Pri), 5.23 (d, 3J = 5.6 Hz, 2 H,
p-MeC6H4Pri), 5.17 (d, 3J = 5.6 Hz, 2 H, p-MeC6H4Pri), 4.07 (s, 9 H,
SC6H3-3,4-OCH3), 3.94 (s, 9 H, SC6H3-3,4-OCH3), 1.98 [sept., 3J =
6.80 Hz, 2 H, p-MeC6H4CH(CH3)2], 1.63 (s, 6 H, p-CH3C6H4Pri),
0.95 [d, 3J = 7.2 Hz, 6 H, p-MeC6H4CH(CH3)2], 0.87 [d, 3J =
6.8 Hz, 6 H, p-MeC6H4CH(CH3)2] ppm. 13C{1H} NMR (100 MHz,
CDCl3): d = 149.6, 148.7, 128.9, 125.7, 115.7, 111.9, 107.3, 99.3,
85.8, 85.2, 84.6, 83.6, 56.9, 56.2, 30.7, 22.8, 22.1, 17.7 ppm.
4.3. Single-crystal X-ray structure analyses
Crystals of compounds [10]ClÁCH2Cl2 and [15]ClÁ2CHCl3,
obtained by the slow evaporation of a dichloromethane
solution of [10]Cl and [15]Cl, respectively, were mounted on a
Stoe Image Plate Diffraction System equipped with a f circle
goniometer, using Mo-Ka graphite monochromated radiation
(l = 0.71073 Å) with f range 0–2001. The structures were solved
by direct methods using the program SHELXS-97, while the
Table 3 Crystallographic and structure refinement parameters for [10]ClÁCH2Cl2
and [15]ClÁ2CHCl3
Acknowledgements
[10]ClÁCH2Cl2
[15]ClÁ2CHCl3
This work was financially supported by the Swiss Science
National Foundation (projects no 200020-131844, 206021-
139078 and 200020-143254).
Chemical formula
Formula weight
C42H42Cl3F9Ru2S3
1122.43
C40H39Cl13Ru2S3
1278.88
Crystal system
Space group
Crystal colour and shape
Crystal size
a (Å)
b (Å)
c (Å)
Monoclinic
P21/c (no. 14)
Red block
0.19 Â 0.15 Â 0.15
13.2365(13)
19.4764(19)
17.2337(15)
94.607(11)
4428.5(7)
Monoclinic
P21/n (no. 14)
Orange block
0.20 Â 0.19 Â 0.15
13.2082(5)
13.1769(4)
28.5766(9)
92.029(3)
References
1 P. J. Dyson, Chimia, 2007, 61, 698.
2 I. Bratsos, St. Jedner, T. Gianferrara and E. Alessio, Chimia,
2007, 61, 692.
b (1)
V (Å3)
Z
4970.4(3)
4
4
3 J. Reedijk, Platinum Met. Rev., 2008, 52, 2.
T (K)
173(2)
1.683
1.073
173(2)
1.709
1.463
Dc (g cmÀ3
)
4 H. Keller and B. K. Keppler, US Pat., 4843069, 1989; cf.
B. K. Keppler, K.-G. Lipponer, B. Stenzel and F. Kratzin, in
Metal Complexes in Cancer Chemotherapy, ed. B. K. Keppler,
VCH Weinheim, 1993, p. 187.
5 G. Mestroni, E. Alessio and G. Sava, Int. Pat., PCT C 07F 15/00,
A61K 31/28, WO 98/00431, 1998; cf. G. Mestroni, E. Alessio,
G. Sava, S. Pacor and M. Coluccia, in Metal Complexes in Cancer
Chemotherapy, ed. B. K. Keppler, VCH Weinheim, 1993, p. 157.
6 L. D. Dale, J. H. Tocher, T. M. Dyson, D. I. Edwards and
D. A. Tocher, Anti-Cancer Drug Design, 1992, 7, 3.
m (mmÀ1
)
Scan range (1)
2.28 o y o 26.09
1.68 o y o 29.26
Unique reflections
Observed refls [I > 2s(I)]
Rint
8248
13 452
4556
0.0699
0.0472, wR2 0.1129
0.0911, wR2 0.1255
0.844
0.740, À0.855
9346
0.1486
0.0699, wR2 0.0898
0.1153, wR2 0.0991
1.096
1.374, À1.214
Final R indices [I > 2s(I)]a
R indices (all data)
Goodness-of-fit
Max, min Dr/e (ÅÀ3
)
Structures were refined on Fo2: wR2 = [S[w(Fo À Fc2)2]/Sw(Fo2)2]1/2
,
a
2
where wÀ1 = [S(Fo2) + (aP)2 + bP] and P = [max(Fo2, 0) + 2Fc2]/3.
c
New J. Chem.
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013

View Article Online
NJC
7 S. J. Dougan and P. J. Sadler, Chimia, 2007, 61, 704.
Paper
´
19 F. Cherioux, B. Therrien and G. Su¨ss-Fink, Eur. J. Inorg.
8 C. S. Allardyce, P. J. Dyson, D. J. Ellis and S. L. Heath, Chem.
Commun., 2001, 1396.
9 R. E. Morris, R. E. Aird, P. d. S. Murdoch, H. Chen,
J. Cummings, N. D. Hughes, S. Pearsons, A. Parkin, G. Boyd,
D. I. Jodrell and P. J. Sadler, J. Med. Chem., 2001, 44, 3616.
10 G. Su¨ss-Fink, Dalton Trans., 2010, 39, 1673.
Chem., 2003, 1043.
20 M. G. Mendoza-Ferri, C. G. Hartinger, M. A. Mendoza,
M. Groessl, A. E. Egger, R. E. Eichinger, J. B. Mangrum,
N. P. Farrell, M. Maruszak, P. J. Bednarski, F. Klein,
M. A. Jakupec, A. A. Nazarov, K. Severin and B. K. Keppler,
J. Med. Chem., 2009, 52, 916.
11 M. G. Mendoza-Ferri, C. G. Hartinger, R. E. Eichinger, 21 C. Hansch, A. Leo and R. W. Taft, Chem. Rev., 1991, 91, 165.
N. Stolyarova, K. Severin, M. A. Jakupec, A. A. Nazarov and 22 Advanced Chemistry Development, ACD/ChemSketch,
B. K. Keppler, Organometallics, 2008, 27, 2405; M. G. Mendoza-
Ferri, C. G. Hartinger, A. A. Nazarov, W. Kandioller, K. Severin
and B. K. Keppler, Appl. Organomet. Chem., 2008, 22, 326.
version 12.0, Advanced Chemistry Development, Toronto,
2012.
23 A. Meister, J. Biol. Chem., 1988, 263, 17205.
12 M. Gras, B. Therrien, G. Su¨ss-Fink, O. Zava and P. J. Dyson, 24 B. N. Ames, M. K. Shigenaga and T. M. Hagen, Proc. Natl.
Dalton Trans., 2010, 39, 10305. Acad. Sci. U. S. A., 1993, 90, 7915.
13 F. Giannini, G. Su¨ss-Fink and J. Furrer, Inorg. Chem., 2011, 25 H. M. Hassan and I. Fridovich, J. Bacteriol., 1980, 141, 156.
50, 10552. 26 D. M. Townsend and K. D. Tew, Oncogene, 2003, 22, 7369.
14 F. Giannini, L. E. H. Paul and J. Furrer, Chimia, 2012, 66, 775. 27 K. G. Balendiran, R. Dabur and D. Fraser, Cell Biochem.
15 S. J. Dougan, A. Habtemariam, S. E. McHale, S. Parsons and Funct., 2004, 22, 343.
P. J. Sadler, Proc. Natl. Acad. Sci. U. S. A., 2008, 105, 11628. 28 J. D. Hayes, J. U. Flanagan and I. R. Jowsey, Annu. Rev.
16 F. Giannini, J. Furrer, A.-F. Ibao, G. Su¨ss-Fink, B. Therrien, Pharmacol. Toxicol., 2005, 45, 51.
O. Zava, M. Baquie, P. J. Dyson and P. Stepnicka, JBIC, J. 29 P. K. Sasmal, C. N. Streu and E. Meggers, Chem. Commun.,
Biol. Inorg. Chem., 2012, 17, 951. 2013, 49, 1581.
17 F. Giannini, J. Furrer, G. Su¨ss-Fink, C. Clavel and P. J. Dyson, 30 M. A. Bennett, T.-N. Huang, T. W. Matheson and
J. Organomet. Chem., DOI: 10.1016/j.jorganchem.2013.04.049. A. K. Smith, Inorg. Synth., 1982, 21, 74.
18 F. Cherioux, B. Therrien and G. Su¨ss-Fink, J. Organomet. 31 G. M. Sheldrick, Acta Crystallogr., 2008, A64, 112.
ˇ
ˇ
ˇ
´
Chem., 2004, 357, 834.
32 L. J. Farrugia, J. Appl. Crystallogr., 1997, 30, 565.
c
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013
New J. Chem.