Evaluation of arene ruthenium(ii) N-heterocyclic carbene complexes as organometallics interacting with thiol and selenol containing biomolecules

Article abstract of DOI:10.1039/c2dt32319b

Metal complexes with N-heterocyclic carbene (NHC) ligands have been widely used in catalytic chemistry and are now increasingly considered for the development of new chemical tools and metal based drugs. Ruthenium complexes of the type (p-cymene)(NHC)RuCl₂ interacted with biologically relevant thiols and selenols, which resulted in the inhibition of enzymes such as thioredoxin reductase or cathepsin B. Pronounced antiproliferative effects could be obtained provided that an appropriate cellular uptake was achieved. Inhibition of tumor cell growth was accompanied by a perturbation of metabolic parameters such as cellular respiration. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c2dt32319b

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Evaluation of arene ruthenium(II) N-heterocyclic
carbene complexes as organometallics interacting with
Cite this: Dalton Trans., 2013, 42, 1657
thiol and selenol containing biomolecules†
a
b
c
a
Luciano Oehninger, Maria Stefanopoulou, Hamed Alborzinia, Julia Schur,
a
d
e
Stephanie Ludewig, Kazuhiko Namikawa, Alvaro Muñoz-Castro,
d
a
c
b
Reinhard W. Köster, Knut Baumann, Stefan Wölfl, William S. Sheldrick and
a
Ingo Ott*
Metal complexes with N-heterocyclic carbene (NHC) ligands have been widely used in catalytic chemistry
and are now increasingly considered for the development of new chemical tools and metal based drugs.
2
Ruthenium complexes of the type (p-cymene)(NHC)RuCl interacted with biologically relevant thiols and
Received 2nd October 2012,
Accepted 26th October 2012
selenols, which resulted in the inhibition of enzymes such as thioredoxin reductase or cathepsin
B. Pronounced antiproliferative effects could be obtained provided that an appropriate cellular uptake
was achieved. Inhibition of tumor cell growth was accompanied by a perturbation of metabolic para-
meters such as cellular respiration.
DOI: 10.1039/c2dt32319b
www.rsc.org/dalton
moderate) could be confirmed for the prototypic ruthenium
NHC complexes of the Grubbs type.
Ruthenium complexes themselves have been playing a
Introduction
1
9
Metal complexes with N-heterocyclic carbene (NHC) ligands
have attracted an enormous degree of attention in the research sharply increasing role in inorganic medicinal chemistry and
2
0–22
community due to their excellent catalytic properties. Thus, lead compounds have been successfully established.
organometallic chemistry makes use of the fact that NHCs, as Accordingly, these transition metal complexes are now com-
persistent forms of divalent carbon, are able to form stable monly considered as the second most promising type of new
complexes with various transition metals that can for example anticancer metal species after the blockbuster platinum cyto-
be used as catalysts for hydroformylation and olefin statics. The possible modes of action of ruthenium complexes
1
–3
metathesis.
Besides their wide use in catalysis, an increas- are under intensive discussion but evidence suggests that for
ing number of reports describe metal NHC complexes with many derivatives the interaction with proteins plays an impor-
2
3–27
interesting biological properties and hence this type of coordi- tant role.
nation compound has been more frequently used in bioorgano-
Concerning the biological activity of ruthenium NHC
4
–6
metallic chemistry.
In particular gold(I) and silver(I) NHC complexes, the available literature is limited to a few
complexes have demonstrated considerable potential for the reports, which however indicate that pharmacologically
7
–18
development of new antitumor agents and anti-infectives.
active species can be achieved from this type of compound.
Not surprisingly, in a recent report biological activity (although This includes the preliminary biological effects of Grubbs
catalysts, toxicity studies in zebrafish or antimicrobial
1
9,28–33
properties.
a
Institute of Medicinal and Pharmaceutical Chemistry, Technische Universität
With the aim of designing ruthenium NHC complexes
with strong biological activity as prospective anticancer
drugs, we prepared a series of arene ruthenium(II) benzimid-
azolylidene carbene complexes and studied their behaviour
under a broad variety of relevant chemical and biological
assays. The choice of the benzimidazolylidene NHC ligand was
Braunschweig, Beethovenstr. 55, D-38106 Braunschweig, Germany.
E-mail: ingo.ott@tu-bs.de
b
Lehrstuhl für Analytische Chemie, Ruhr-Universität Bochum, D-44780 Bochum,
Germany
c
Institute of Pharmacy and Molecular Biotechnology, Ruprecht-Karls-Universität
Heidelberg, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany
d
9,10
Division of Cell Biology and Cell Physiology, Zoological Institute, Technische
motivated by our recent studies on gold(I) NHC complexes
whereas the (arene)RuCl fragment was selected as an organo-
2
Universität Braunschweig, Spielmannstr. 8, D-38106 Braunschweig, Germany
e
Department of Chemical Sciences, Universidad Andres Bello, Republica 275,
metallic pharmacophore, which has been intensively and
successfully studied in the field of bioorganometallic
Santiago, Chile
†
Electronic supplementary information (ESI)
0.1039/c2dt32319b
available. See DOI:
2
3–25,34–39
1
chemistry.
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Scheme 1 Synthesis procedure for 1a–4a. (a) Alkylhalide,
2 3 3
K CO , CH CN,
6
reflux, 6 h; (b) Ag
–8 h.
2
O in CH
2
Cl
2
, 4 h; (c) bis[dichlorido(η -p-cymene)ruthenium(II)],
3
Chemistry
The target compounds were obtained in a three step procedure
Fig. 1 Optimized molecular geometries (hydrogen atoms are omitted for
4
0
(see Scheme 1). Initially the ligands were synthesized by
clarity).
refluxing benzimidazole with an excess of an alkylhalide in the
presence of K CO giving as a product the corresponding ben-
2
3
zimidazolium halide. The benzimidazol-2-ylidene ruthenium(II) details of DFT calculations). Expectedly, the chlorido ligands
complexes were then obtained in a one-pot reaction by were more polarized but had a higher BDE in the range of
treating the respective benzimidazolium cation with Ag O fol- 124.72 to 126.58 kcal mol− . As confirmed exemplarily by
1
2
6
lowed by reaction with bis[dichlorido(η -p-cymene)ruthenium(II)]. UV-Vis spectroscopy with 4a, the NHC ligand was stably coor-
In this step the treatment with Ag O resulted in the for- dinated to ruthenium in buffer solution over at least 10 hours
2
mation of a silver intermediate followed by conversion of the at 37 °C (see ESI†).
intermediate into the ruthenium NHC complex by means of a
transmetallation reaction. The target compounds were purified
Inhibition of disulfide reductases (TrxR and GR) and proteases
(
trypsin and catB)
as appropriate by filtration over celite, recrystallization and hot
filtration.
Proteins, and in particular cysteine and selenocysteine contain-
1
13
The complexes were characterized by H NMR, C NMR, ing enzymes, have been identified as critical targets for ruthe-
1
9,23–27
and mass spectroscopy as well as by elemental analysis. nium metallodrugs.
In this study, the inhibition of the
Although complexes 1a–4a are expected to have a formal sym- selenoenzyme TrxR and the cysteine protease cathepsin B
metry plane, for complex 2a the methylene protons of the NHC (catB) exposed for 75 minutes to the compounds was investi-
1
ligand are non-equivalent in H NMR spectra due to an ortho- gated. The disulfide reductase GR and the serine protease
gonal position adopted by the carbene to the ruthenium–arene trypsin were used as controls to check for the specificity of the
4
1,42
axis as has already been reported in the literature.
A
enzymatic inhibition. Besides the ruthenium(II) complexes 1a–
similar phenomenon was observed for complex 4a, for which 4a, the ruthenium dimer ([(p-cymene)RuCl ] ) and the benz-
2
2
the asymmetry is in the (N-CH
cases it can be concluded that there is a non-equivalent dispo-
2
-C
6
H
5
) benzyl protons. In both imidazolium derivative 2 were included in the study.
As expected, the ruthenium free compound 2 showed no
sition from the side chains in solution that leads to broaden- inhibitory effects on any of the studied enzymes (see Table 1).
1
ing of the respective signals in the H NMR spectra. For the Strong inhibitory properties were observed for the ruthenium
methyl groups of the isopropyl side chains of 3a two doublets complexes against TrxR (IC50 values between 0.30 and
at 1.77 and 1.70 ppm were observed demonstrating that these 3.74 μM). Their activity in this assay surpassed that of recently
signals are also not equivalent. The optimized molecular geo- biologically studied Grubbs type catalysts and was comparable
9
,10,19
metries of 1a–4a calculated by DFT denote a general trend in with that of gold(I) benzimidazolylidene derivatives.
the orientation of the p-cymene ligand with respect to the Effects against GR were in all cases weaker compared with
NHC moiety ensuring the lowest steric hindrance with the iso- TrxR (IC50 values between 1.84 to 75.29 μM). Moderate inhi-
propyl group of the arene ligand opposite to the substituted bition was noted with the ruthenium complexes 1a–4a against
NHC ligand (see Fig. 1). The distances between ruthenium and catB (IC50 values between 8.35 and 17.77 μM) but no inhibition
its ligands vary only marginally in the series 1a–4a (see of trypsin activity up to the highest concentration used
Table SI1 in the ESI†). The geometrically most favorable struc- (100 μM) was observed. The dinuclear complex ([(p-cymene)-
tures are consistent with the solution-state conformations RuCl ] ) was active against TrxR and GR, but did not reach an
2
2
1
according to the H NMR measurements concerning the non- IC50 value against catB or trypsin at the highest concentration
equivalency observed in the NHC side chains of 2a–4a. Com- applied (100 μM).
paring calculated bond dissociation energies (BDE) and
The inhibition of the enzymes by the ruthenium complexes
natural population analysis (NPA) showed that both organo- expectedly involves ligand exchange reactions between (seleno)-
metallic fragments had similar BDE (in the range of cysteines in the active sites and the ligands coordinated to
−
1
5
9.46–64.01 kcal mol ) as well as NPA values (see ESI† for ruthenium. This can lead to a time-dependent covalent
1
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Table 1 Inhibition of TrxR and GR; results are expressed as means (±error) of repeated experiments. “>100” indicates that no IC50 value was reached up to 100 μM.
In case there was a significant difference to the control at 100 μM (but above the 50% level) the respective value is given between brackets
IC50 TrxR (μM)
IC50 GR (μM)
IC50 catB (μM)
IC50 trypsin (μM)
2
>100
>100
26.24
3.00
75.29
2.12
1.84
>100
>100 (66%
15.69
8.35
10.24
17.77
>100
>100
>100
>100
>100
>100
±
0.05
±0.70
±1.3
[
(p-cym)RuCl
]
2 2
0.78
0.30
3.74
0.74
0.78
)
±
0.03
0.05
0.92
0.56
±0.02
±1.07
1
2
3
4
a
a
a
a
±
±7.38
±1.42
±
±0.27
±0.26
±
±0.85
±3.35
Fig. 2 Time dependent inhibition of complex 1a against catB (10 μM) and TrxR
0.5 μM).
Fig. 3 (right) Binding of 1a–4a to albumin. Values are represented as percen-
(
tage of ruthenium bound to BSA as means (±error) of repeated experiments.
inhibition of the enzymes. Accordingly, an enzymatic inhi-
bition assay for TrxR and catB was performed exemplarily
with 1a at a fixed concentration close to the respective IC50
value (see Fig. 2) over a period of 180 minutes. Whereas TrxR
inhibition was almost complete at the beginning of the experi-
ment, the inhibition of catB activity showed a clear depen-
dency on the exposure time with a progressive decrease of the
enzymatic activity. Thus, during the first 20 minutes of incu-
bation with 10 μM of 1a no inhibition of catB activity could be
noted, but after 75 minutes a considerable inhibition (67% of
the untreated control) was observed. After 3 hours of exposure
the enzymatic activity of catB decreased to 15%.
bound ruthenium, for 4a equilibrium values were reached
after 1–2 hours and remained around 70%. Generally, the
amount of ruthenium attached to albumin increased with the
size and lipophilicity of the side chains of the NHC ligands
(
values after 24 h: 1a: 19%, 2a: 27%, 3a: 42%, 4a: 68%).
In order to characterize the peptide binding site of the sub-
stances 1a and 4a the capabilities of electrospray ionization
tandem mass spectrometry were employed. For this purpose
1a and 4a were incubated with both a cysteine- and a seleno-
cysteine-containing peptide (AGCVGAGLIK and AGUVGAGLIK
respectively) in an approach to mimic their interaction
with the active center of enzymes. Subsequent MS analysis
revealed molecular ions corresponding to the masses of the
peptide modified by the addition of the mass of the appropri-
ate substance after loss of one or both chlorides or after loss
of further ligands. Both cysteine and selenocysteine could be
characterized as the preferred binding site by means of
tandem MS analysis as shown in Fig. 4.
Overall the results in summary indicate a preference for
inhibition of the selenoenzyme TrxR, which is faster and more
efficiently inhibited compared with the enzymes with relevant
cysteines in their active sites (here: catB).
Interaction with non-enzymatic thiol and selenol containing
biomolecules
Compound 1a was incubated with N-acetylcysteine in
1
In order to study the reactivity towards (seleno)cysteine con- DMSO-d
over a period of 24 hours and H NMR spectra were
6
taining biomolecules in more detail, the interaction with taken. Several changes in the spectra could be observed over
albumin, (seleno)cysteine-containing peptides and N-acetyl- time, which indicated a reaction between N-acetylcysteine and
cysteine was evaluated. A time dependent binding study to the ruthenium complex. As shown in Fig. 5 a signal at
2
albumin, which contains an accessible cysteine binding site, 9.62 ppm corresponding to the protonated C of benzimid-
was performed for 1a–4a, denoting substantial differences azole-2-ylidene was formed over time, which confirmed the
between the individual complexes (see Fig. 3). While for 1a–3a loss of the NHC ligand. From the NH of N-acetylcysteine
binding was effectively complete at the beginning of the (a doublet at 8.24 ppm) two further signals emerged at lower
measurements and ranged roughly in the range of 10–50% of fields (doublets at 8.34 ppm and 8.66 ppm), which can be
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interpreted as the result of coordination between ruthenium Cl /H
Dalton Transactions
−
2
O exchange reactions are typical for arene ruthenium
and N-acetylcysteine. Other spectral changes included the complexes (see also the theoretical considerations in the
3
4,39
shifting of the aromatic signals of the p-cymene ligand and ESI†).
further signals emerging from N-acetylcysteine. Altogether
the experiments indicated effects on both the NHC and the
p-cymene ligands as well as the formation of chelate complexes
between N-acetylcysteine and ruthenium (see the ESI† for
more details).
It can be speculated that initially the chlorido ligands are
replaced by the respective selenol or thiol, which may lead to a
labilisation of the coordinated arene and NHC ligands. Con-
cerning the reactions in an aqueous environment intermediate
formation of aqua complexes must be taken into account since
Binding to DNA
In order to study if binding to different biomolecules is also of
importance for ruthenium(II) NHC complexes, DNA was
exposed to 1a and 4a at a nucleotide/ruthenium ratio of 100/1
for 2 hours. Afterwards the DNA was precipitated, the bound
ruthenium was quantified by high-resolution continuum
source atomic absorption spectroscopy and the amount of pre-
cipitated DNA by UV/Vis spectroscopy. Both complexes bound
to DNA with low nucleotide/ruthenium ratios of 73 796-
(
±15 578)/1 for 1a and 17 869(±2723)/1 for 4a, respectively.
Calculated as percentage the obtained data showed that for 1a
approximately 14% and for 4a approximately 47% of the total
ruthenium were attached to DNA. Overall this indicated the
same trend as observed with binding to albumin. However, the
low nucleotide/ruthenium ratios are not indicative of a strong
interaction with DNA (platinum complexes such as cisplatin or
carboplatin reach much higher ratios under similar experi-
4
3
mental conditions ).
Effects on tumor cell proliferation, cellular uptake and toxicity
in zebrafish embryos
Potential antiproliferative effects of the compounds were inves-
tigated in two tumor cell lines (namely, MCF-7 human breast
adenocarcinoma and HT-29 colon carcinoma). Pronounced
activity (IC50 values in the low micromolar range) was observed
with 4a in both cancer cell lines. In contrast, 2 as well as the
ruthenium complexes [(p-cymene)RuCl ] did not trigger any
2+
Fig. 4 MS/MS spectrum of the molecular ion [peptide + Ru] at m/z 497
after a 1 h incubation at 37 °C of a 1 : 1 mixture of complex 4a with the peptide
H-AGCVGAGLIK-OH. The asterisk (*) represents the ruthenium atom. Spectra
2
2
antiproliferative effects up to a concentration of 100 μM (see
obtained with both peptides after 1 and 4 hours as well as with 1a are provided Table 2). For 1a and 2a in both cell lines and for 3a in HT-29
in the ESI.†
no IC50 values could be calculated as proliferation at 100 μM
1
Fig. 5 H NMR spectra of a mixture 1 : 2 of complex 1a and N-acetylcysteine in DMSO-d
6
monitored over time.
1
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Table 2 Antiproliferative effects in MCF-7 and HT-29 cells, results are expressed
as IC50 values as means (±error) of repeated experiments. “>100” indicates that
no IC50 value was reached up to 100 μM (the percentage of remaining cell
biomass at 100 μM is given between brackets in this case)
The influence on the cellular metabolism was monitored
online by the use of a metabolic sensor chip analysis system
(
(
BIONAS) with 1a (inactive against cancer cell growth) and 4a
active against cancer cell growth) as examples for complexes
IC50 MCF-7 (μM)
IC50 HT-29 (μM) with differing biological properties. The used biosensor chip
system allows to record changes of the cell layer (reflecting
morphological changes), the respiration rate (cellular oxygen
2
>100 (100%)
>100 (100%)
>100 (100%)
[
(p-cym)RuCl
]
2 2
>100 (100%)
±5.84
±7.44
consumption) as well as the extracellular acidification rate
of living cells over an extended time span (see Fig. 7). For
complex 1a at a concentration of 10 μM no relevant effects on
MCF-7 cell metabolism were noted with only a slight decrease
of impedance after longer exposure. However, at 100 μM the
impedance kept steadily decreasing after approx. 13 hours
of the treatment and the respiration rate was immediately
lowered after the compound was administered. This shows
that at higher concentrations tumor cell integrity can be
clearly affected by 1a, although an IC50 value in conventional
proliferation experiments is not reached. As expected 4a was
clearly more effective and showed dose-dependent effects on
all measured metabolic parameters reflecting the onset of cell
death. Of particular interest is the increase in extracellular
acidification at 10 μM of 4a. This is related to an increased
glycolysis rate as a compensatory effect of the cells towards the
action of the compound. This phenomenon has also been
1
2
3
4
a
a
a
a
>100 (63%
>100 (73%
)
)
>100 (70%
>100 (68%
>100 (61%
)
)
)
±3.62
±5.01
±5.26
±
1.64
51.67
±0.93
±0.97
2.07
2.40
1
0,19,44
observed with various other metal based drugs.
Next we studied the toxicity of 1a (inactive against cancer
cell growth) and 4a (active against cancer cell growth) in zebra-
fish embryos, which can be used as a model to study effects of
general toxicity in an organism more complex than cultured
Fig. 6 Cellular uptake of 10 μM of 4a in cell culture media with and without
fetal calf serum (FCS) into MCF-7 cells. Values are represented as μmol of ruthe-
nium per mg protein as means (±error) of repeated experiments. No cellular cells.
ruthenium was detected in the case of 1a–3a.
3
0,45
The embryos were exposed to different concen-
trations of the respective complex for 96 hours and living
embryos were counted (Fig. 8). In good agreement with the cell
culture experiments complex 1a did not trigger substantial
was inhibited less than 50%. However, at this concentration lethality up to the highest concentration used (100 μM). With
proliferation was decreased roughly by 30–40%.
4a concentrations higher than 15 μM in combination with an
Similar to the results of the albumin binding study, these exposure of at least 48 hours were lethal for more than 50% of
data indicated an influence of lipophilicity and cellular uptake the zebrafish embryos, a dosage of 40 μM was lethal for more
on the antiproliferative potential. In view of this fact, the cellu- than 50% of the embryos within 24 hours. Concentrations of
lar ruthenium accumulation of the complexes 1a–4a in MCF-7 4a around its IC50 value in cancer cells did not lead to signifi-
cells was quantified by high-resolution continuum source cant lethality for zebrafish embryos. Low toxicity for ruthe-
atomic absorption spectroscopy. Taking the results of the nium carbene complexes in zebrafish embryos had been
albumin binding studies into account, these cellular uptake reported previously, although in rats some toxic effects had
experiments were performed in a comparative manner using been observed.3
0,32,33
For obvious reasons a direct comparison
serum supplemented and serum-free cell culture media. between results from cultured human cancer cells and zebra-
Serum is routinely added as a nutrient to standard cell culture fish embryos is difficult, nevertheless, the results indicate that
media and contains high levels of albumin and other proteins. it might be possible to identify an appropriate effective but
No cellular ruthenium could be detected for compounds non-toxic dose range in future in vivo studies on ruthenium
1
a–3a in the presence or absence of serum, respectively. In NHC derivatives.
contrast for 4a a time dependent increase in cellular ruthe-
nium was measured after 6 and 24 hours, which is in good
agreement with its enhanced cytotoxicity. The intracellular
ruthenium levels after exposure for 24 hours with a serum-con-
Conclusions
taining medium were lower than those in a serum-free In conclusion the obtained results suggested that complexes of
medium, indicating that protein binding negatively influenced the type (arene)(NHC)RuCl display potent enzyme inhibitory
2
the uptake of this compound upon longer exposure (see effects, which were translated into strong cytotoxic effects in
Fig. 6).
case the cellular uptake of the complexes was strong enough
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Fig. 7 Real-time response of MCF-7 cell metabolism in response to 1a and 4a. Treatment (exposure) started after 5 h of equilibration and was continued for 24 h.
Cell impedance (a), standard respiration rate (b) and acidification rate (c); RM = “running medium” (without compound).
Fig. 8 Survival of zebrafish embryos exposed for 6, 24, 48, 72 and 96 hours to different concentrations of 1a (left) and 4a (right). Results are expressed as means
(±error) of two independent experiments. 50 embryos were exposed to each concentration per experiment.
(see results with complex 4a). For complexes with insufficient action of the complexes is based on the interaction with bio-
cellular uptake (1a–3a) some efficacy against tumor cell growth logically relevant thiols and selenols, such as the enzymes
was observed only at the highest studied concentration thioredoxin reductase or catB. The lipophilicity of the side
(100 μM). The inhibitory effects on tumor cell growth were chains of the NHC ligand influenced the binding to albumin
accompanied by an appropriate decrease in cell impedance and the cellular ruthenium uptake. Ruthenium could also be
and cellular respiration. Toxic effects in zebrafish embryos bound to purified DNA, however, to a lower extent than
were noted in concentrations much higher than those in the observed with albumin. The relevance of this interaction with
cell culture experiments. There is evidence that the mode of DNA is not clear so far.
1
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6
In consequence, ruthenium NHC complexes can be used to
DICHLORIDO[1,3-DIETHYLBENZIMIDAZOL-2-YLIDENE](η -P-CYMENE)RUTHENIUM(II)
design new tools for chemical biology as well as to develop (2A). Yield: 0.107
g
(0.22 mmol, 72%) orange powder.
1
metal based drugs. Further variation of the coordinated NHC
ligand is expected to lead to complexes with a distinct biologi- (m, 2H, ArH4/7), 5.50 (2H, MeC
H NMR (CD Cl ): (ppm) 7.54–7.46 (m, 2H, ArH5/6), 7.36–7.25
2 2
3
6
H
4
–, JHH = 6.0 Hz), 5.09 (2H,
3
cal activity profile.
MeC H –, J
6 4 HH
= 6.0 Hz), 4.99 (m, broad, 2H, –NCH Me), 4.47
2
3
(
m, broad, 2H, –NCH
2
Me), 3.00 (hept, 1H, CHMe
2
,
J
HH
=
=
3
7
7
.0 Hz), 1.89 (s, 3H, C
.0 Hz), 1.27 (d, 6H, (CH ) CHC H –, J = 7.0 Hz). C NMR
HH
6
H
4
CH ), 1.56 (t, 6H, –NCH CH , JHH
3
2
3
13
3
Experimental section
General
3 2
6
4
(
CD
2
Cl
2
): (ppm)16.26 (–CH(CH
3
)
2
), 18.68 (–NCH
), 45.17 (–NCH
2.51 and 87.62 (CH from p-cymene), 99.38 and 110.68 (qua-
).
), 188.90 (NHC); MS(EI): 480.1
M + H ). Elemental analysis for C H N Cl Ru (% calcd/
2
CH
3
), 22.48
(
C
6
H
4
CH
3
), 31.01 (p-cymene –CH(Me)
2
2
CH ),
3
All reagents and the solvents were used as received from
Sigma, Aldrich, or Fluka. Rat liver TrxR, baker’s yeast GR and
trypsin from bovine pancreas were obtained from Sigma, catB
8
ternary C from p-cymene), 111.09 and 122.99 (CH from C
35.59 (quaternary C from C
6 4
H
1
(
6 4
H
1
+
was obtained from Calbiochem. H NMR were recorded on a
2
1
28
2
2
1
3
Bruker DRX-400 AS NMR System and C NMR were recorded
on a Bruker AV II-600 AS NMR System, MS spectra were
recorded on a FinniganMAT4515. The purity of the target com-
pounds (>95%) was confirmed by elemental analysis (Flash
EA112, Thermo Quest Italia). For all compounds undergoing
biological evaluation, the experimental values differed less
than 0.5% from the calculated ones. Bis[dichlorido(η -p-
cymene)ruthenium(II)] and the benzimidazolium cations 1–4
were synthesized as described in the literature.
found): C (52.50/52.64), H (5.87/5.72), N (5.83/5.65).
6
DICHLORIDO[1,3-DIISOPROPYLBENZIMIDAZOL-2-YLIDENE](η -P-CYMENE)-
RUTHENIUM(II) (3A). Yield: 0.087 g (0.17 mmol, 55%) red powder.
1
3
H NMR (CDCl ): (ppm) 7.71–7.64 (m, 2H, ArH5/6), 7.24–7.17
3
(
2
m, 2H, ArH4/7), 5.81 (hept, 2H, –NCHMe , JHH = 7.0 Hz),
3
3
5
6
C
.62 2H, MeC H –, J
= 6.0 Hz), 5.23 (2H, MeC H –, J
=
6
4
HH
6
4
HH
6
3
.0 Hz), 2.99 (hept, 1H, CHMe
CH ), 1.77 (d, 6H, NCH(CH
NCH(CH ) , J = 7.2 Hz), 1.39 (d, 6H, (CH ) CHC H –, J
2
, JHH = 6.8 Hz), 2.13 (s, 3H,
3
6
H
4
3
3 2
) , JHH = 6.8 Hz), 1.70 (d, 6H,
9
,10,46
3
3
=
3
1
2
3
HH
3 2
6
4
HH
7
.0 Hz). C NMR (CDCl
3
): (ppm) 18.66 (cymene –CH(CH
3
)
2
),
General procedure for synthesis of the ruthenium NHC
complexes
20.00, 22.29, 22.64 and 22.92 (s, –NCH(CH
3
)
2
), 31.01
(
p-cymene –CH(Me) ) 53.74 (–NCH(Me) ), 83.52 and 86.58
2 2
The respective benzimidazolium cation (0.32 mmol) and Ag
2
O
(CH from p-cymene), 97.41 and 106.78 (quaternary C from
). 134.17 (quater-
(0.0371 g, 0.16 mmol) were added to a dried Schlenk tube. The p-cymene), 112.93 and 121.82 (CH from C
6 4
H
+
mixture was flushed three times with N and then dry CH Cl nary C from C H ), 187.48 (NHC); MS(EI): 508.1 (M + H ).
2
2
2
6
4
was added (15 mL). The flask was closed and the solution Elemental analysis for
C
23
H
32
N
2
Cl
stirred for 4 h in the dark. A solution of bis[dichlorido(η -p- C (54.33/54.14), H (6.34/6.33), N (5.51/5.23).
cymene)ruthenium(II)] (0.0979 g, 0.16 mmol) in CH Cl was
2
Ru (% calcd/found):
6
6
[1,3-DIBENZYLBENZIMIDAZOL-2-YLIDENE]DICHLORIDO(η -P-CYMENE)-
2
2
added (10 mL) and the resulting solution was stirred for RUTHENIUM(II) (4A). Yield: 0.070 g (0.12 mmol, 35%) brown
1
3
–8 hours. The obtained suspension was filtered over celite powder.
H
3
NMR (CDCl ): (ppm) 7.37–7.35 (m, 4H,
(
281 nm) and concentrated in a vacuum. The orange residue –NCH ArH), 7.31–7.29 (m, 2H, –NCH ArH), 7.13–7.12 (m, 4H,
2
2
was recrystallized from CH
2
Cl
2
–n-hexane (10 mL/40 mL) at –NCH
2
ArH), 7.10–7.06 (m, 2H, ArH5/6), 7.05–7.01 (m, 2H,
2
4
°C. After filtration, an orange solid was obtained. The ArH4/7), 6.55 (d, 2H, NCH
2
Ar, JHH = 17.0 Hz) and 5.84 (d, 2H,
2
3
product was first washed with hot toluene, then with diethyl NCH Ar, J
ether and dried under vacuum. A second recrystallization from 5.06 (2H, MeC
= 17.0 Hz), 5.35 (2H, MeC H –, J
= 6.1 Hz),
2
HH
6
4
HH
3
6
H
4
–,
J
HH = 6.1 Hz), 2.73 (hept, 1H, CHMe
2
,
3
CH
2
Cl
2
–n-hexane (10 mL/40 mL) at 4 °C was necessary for
J
HH
=
6.9 Hz), 1.89 (s, 3H,
C
6
H
4
CH ), 1.17 (d, 6H,
3
3
13
complexes 3a and 4a. The synthesis of 1a and 2a by a different (CH ) CHC H –, J = 7.0 Hz). C NMR (CDCl ): (ppm) 18.28
method was reported before.
DICHLORIDO[1,3-DIMETHYLBENZIMIDAZOL-2-YLIDENE](η -P-CYMENE)- 52.97 (–NCH
3
2
6
4
HH
3
4
2
(–CH(CH
3
)
2
), 22.47 (C
6
H
4
CH
3
2
), 30.61 (p-cymene –CH(Me) ),
6
2
Ar), 84.62 and 85.44 (CH from p-cymene), 97.25
RUTHENIUM(II) (1A). Yield: 0.101 g (0.22 mmol, 72%) orange and 107.97 (quaternary C from p-cymene), 111.64, 123.13,
1
powder. H NMR (CD
2
Cl
2
): (ppm) 7.45–7.38 (m, 2H, ArH5/6), 127.80, 125.93, 127.48, 128.92, 135.54, 137.64 (18C, ArC),
3
+
7
5
(
.37–7.30 (m, 2H, ArH4/7), 5.51 (2H, MeC
.14 (2H, MeC H –, J
hept, 1H, CHMe
d, 6H, (CH CHC
ppm) 18.90 (–CH(CH ) ), 22.47 (C H CH ), 31.20 (p-cymene –
6
H
4
–, JHH = 6.0 Hz), 191.66 (NHC). MS(EI): 604.6 (M + H ). Elemental analysis for
3
= 6.0 Hz), 4.17 (s, 6H, –NCH ), 2.99 C H N Cl Ru (% calcd/found): C (61.59/61.26), H (5.33/5.21),
6
4
HH
3
21 32
2
2
3
2
, JHH = 7.0 Hz), 1.99 (s, 3H, C
6
H
4
CH
3
), 1.27 N (4.63/4.44).
3
13
(
3
)
2
6
H
4
–,
J
HH = 7.0 Hz). C NMR (CD
2
Cl ):
2
Theoretical calculations
(
3
2
6
4
3
CH(Me)
2
), 36.69 (–NCH
3
), 82.79 and 87.06 (CH from Relativistic density functional theory calculations were done by
4
7
p-cymene), 99.29 and 111.11 (quaternary C from p-cymene), using the ADF 2012 code, incorporating scalar corrections
1
10.23 and 123.38 (CH from C H ). 136.20 (quaternary C via the ZORA Hamiltonian. Triple-x ξ basis set plus two polariz-
6
4
+
from C
6
H
4
), 190.11 (NHC); MS(EI): 452.1 (M + H ). Elemental ation functions (STO-TZ2P) for valence electrons were
analysis for C19
H
24
N
2
2
Cl Ru (% calcd/found): C (50.44/49.85), employed within the generalized gradient approximation
H (6.19/5.58), N (5.35/5.25).
(GGA) according to the Perdew–Burke–Ernzerhof (PBE)
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Dalton Trans., 2013, 42, 1657–1666 | 1663

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exchange–correlation functional, due to its performance on determination by atomic absorption spectroscopy were stabil-
the calculations of inorganic and organometallic compounds. ized by addition of 30 μL of Triton X-100 (1%) and 60 μL of
2
10
Frozen core approximation was applied to the [1s –3d ] core 1 M HCl. The amount of ruthenium bound to DNA was
2
for Ru and [1s ] for C, N, and Cl, leaving the remaining elec- expressed as a nucleotide/ruthenium ratio and as percentage
trons explicitly treated variationally. Geometry optimizations of total ruthenium. Results were calculated as means of three
were done without any symmetry restraint, via the analytical independent experiments, which were performed with two
energy gradient method implemented by Verluis and Ziegler. replicates.
Bonding dissociation energies, corrected for the basis set
superposition error (BSSE), were obtained by using the frag-
ment analysis feature of the ADF code, where defined frag-
Antiproliferative effects, binding to albumin, cellular uptake
and AAS measurements
ments are employed as basis functions for the overall Experiments were performed according to recently described
1
9
calculations allowing us to determine specifically the bonding procedures. Compound 2a was used to calibrate the AAS
scheme acting in the interaction. Population analysis was spectrometer. The detection limit for Ru was 1.6 μM.
carried out on the basis of the natural population analysis
4
8
Effects on cell metabolism
(
NPA) scheme by using the NBO5 stand alone suite.
Online measurement of cell metabolism and morphological
changes was done using a Bionas2500 sensor chip system
1
Time dependent UV-vis and H NMR study
UV-vis absorption spectra were recorded using quartz cells (Bionas, Rostock, Germany). The metabolic sensor chips
0.2 cm path length) on a SPECORD 40 spectrometer (Analytik (SC 1000) include ion-sensitive field effect transistors to record
(
Jena) connected to an external computer. The data were pH changes, clark-type electrode to measure oxygen consump-
analyzed with the software WinASPECT Version 2.3.1.0 from tion, and interdigitated electrode structures for impedance
5
Analytik Jena. The measurements were performed at a concen- measurement under the cell layer. Approximately 1.5 × 10 of
tration of 250 μM in a mixture of 0.5% of acetonitrile in water MCF-7 cells were seeded directly onto each sensor chip in
1
at room temperature. H NMR spectra were recorded on a 450 μL of DMEM (PAA, E15-883) with penicillin/streptomycin
2
Bruker AVII-300 spectrometer at a concentration of 30 mM of and 10% (v/v) FCS (PAA) and incubated at 37 °C, 5% CO , and
compound and 60 mM of N-acetylcysteine using DMSO-d as a 95% humidity for about 24 h. The cell number used resulted
6
solvent. For both studies the probes were incubated between in approximately 80–90% confluence of the cells on the chip
measurements at 37 °C on an INCU-Line L10 incubator (VWR). surface which was the starting condition for online monitor-
ing. Sensor chips with cells were then transferred to the
Enzymatic inhibition assays
Bionas2500 analyzer in which the medium is continuously
To determine the inhibition of the enzymes TrxR, GR catB and exchanged in 8 min cycles (4 min exchange of medium and
trypsin established microplate reader based assays were per- 4 min without flow), during which the parameters were
1
9
formed. Commercially available isolated enzymes were used measured. The running medium used during analysis was
in all cases. The non-interference of the test compounds with DMEM (PAN, P03-0010) without carbonate buffer and only
the assay components was confirmed by control experiments weakly buffered with 1 mM Hepes and reduced FCS (0.1%).
using the respective enzyme free assay solutions.
For drug activity testing, the following steps were included: (1)
h equilibration with running medium (RM), (2) drug incu-
bation with substances freshly dissolved in medium at indi-
5
Binding to DNA
Experiments were performed according to
a
previously cated concentrations and indicated incubation time, (3) a drug
4
9,50
described method.
Salmon testes DNA (Sigma) was dis- free step in which cells are again fed with the running
solved in PBS pH 7.4 and drugs were added as stock solutions medium without substances, and (4) at the end of each experi-
in DMF. The final solutions contained 8.0 μM 1a or 4a, ment, the cell layer was removed by addition of 0.2% Triton
50 μg mL− salmon testes DNA and 0.1% v/v DMF. After vor- X-100 to obtain a basic signal without living cells on the
1
2
texing, the solutions were incubated at 37 °C for 2 h. Aliquots sensor surface as a negative control.
of 200 μL were mixed with 100 μL of 0.9 M sodium acetate and
Binding studies with cysteine- and selenocysteine-containing
peptides
9
3
1
00 μL of ice cold ethanol. Samples were stored at −20 °C for
0 min. The pellets were isolated by centrifugation (2390 g,
0 min, 4 °C) and resuspended in 300 μL of 0.3 M sodium The substances were dissolved in 40% acetonitrile at a concen-
acetate. 900 μL of ice cold ethanol were added, and the precipi- tration in the 40–59 mM range and incubated at 37 °C for 1
tate was collected after centrifugation (2390 g, 10 min, 4 °C). and 4 hours with a cysteine-containing decamer (AGCVGA-
Samples were washed twice with ice cold ethanol and were GLIK) and the analogous selenium-containing decamer
stored at −20 °C. The pellets were dissolved in 500 μL of water (AGUVGAGLIK) at a complex-to-peptide molar ratio of 1 : 1 and
(twice distilled), and the DNA content was determined by 5 : 1. The peptide was previously treated at a 1 : 5 ratio with
absorption reading at 260 nm in a microplate reader (Perkin- dithiothreitol for 1 h. Full-MS and MS/MS spectra were
Elmer Victor X4). Salmon testes DNA dissolved in water was recorded with a Finnigan LTQ XL mass spectrometer (Thermo
used for calibration purposes. Samples (240 μL) for ruthenium Electron Corp., San Jose, CA, USA), which was operated in the
1
664 | Dalton Trans., 2013, 42, 1657–1666
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Dalton Transactions
Paper
positive ion mode with a capillary temperature of 200 °C and a
spray voltage of 1.8 kV. Direct injection of samples into the
mass spectrometer was performed at a flow rate of 1.0 μL
min . The relative collision energy for collision-induced dis-
sociation was set at 35%.
6 G. Gasser, I. Ott and N. Metzler-Nolte, J. Med. Chem., 2011,
54, 3–25.
7 J. L. Hickey, R. A. Ruhayel, P. J. Barnard, M. V. Baker,
S. J. Berners-Price and A. Filipovska, J. Am. Chem. Soc.,
2008, 130, 12570–12571.
−
1
8
P. J. Barnard, M. V. Baker, S. J. Berners-Price and D. A. Day,
J. Inorg. Biochem., 2004, 98, 1642–1647.
Zebrafish embryo collection
9
R. Rubbiani, I. Kitanovic, H. Alborzinia, S. Can,
A. Kitanovic, L. A. Onambele, M. Stefanopoulou,
Y. Geldmacher, W. S. Sheldrick, G. Wolber, A. Prokop,
S. Wölfl and I. Ott, J. Med. Chem., 2010, 53, 8608–8618.
Zebrafish maintenance, breeding and raising of embryos were
5
1
performed as described. Shortly, fertilized eggs were col-
lected and raised at 28 °C in Petri dishes initially in embryo
water (0.06 g Sea Salt in 1 L distilled water). This medium was
replaced after about 10 hours by 30% Danieau (58 mM NaCl,
1
0 R. Rubbiani, S. Can, I. Kitanovic, H. Alborzinia,
M. Stefanopoulou, M. Kokoschka, S. Mönchgesang,
W. S. Sheldrick, S. Wölfl and I. Ott, J. Med. Chem., 2011, 54,
0
.7 mM KCl, 0.4 mM MgSO
HEPES buffer pH 7.2) containing 0.2 mM 1-phenyl-2-thiourea
PTU) for inhibiting pigment formation. At 24 hours post ferti-
lization (hpf) zebrafish embryos were dechorionated by enzy-
4 3 2
, 0.6 mM Ca(NO ) and 5 mM
8646–8657.
(
11 W. Liu, K. Bensdorf, M. Proetto, A. Hagenbach, U. Abram
and R. Gust, J. Med. Chem., 2011, 55, 3713–3727.
−
1
matic digestion with 2 mg ml protease for 20 min at room
temperature and washed thrice with a Danieau medium with
1
2 T. J. Siciliano, M. C. Deblock, K. M. Hindi, S. Durmus,
M. J. Panzner, C. A. Tessier and W. J. Youngs, J. Organomet.
Chem., 2011, 696, 1066–1071.
0
.2 mM PTU.
1
3 L. Eloy, A.-S. Jarrousse, M.-L. Teyssot, A. Gautier, L. Morel,
C. Jolivalt, T. Cresteil and S. Roland, ChemMedChem, 2012,
Zebrafish embryo toxicity
Substances were administered once to dechorionated 24 hpf
zebrafish embryos. For this purpose, 50 embryos per well were
placed in a 6-well-plate with different concentrations of sub-
stance in each well. Ruthenium complexes 1a and 4a were dis-
solved in DMF and the stock solutions were diluted (1 : 1000)
with 30% Danieau/0.2 mM PTU, thereby achieving final com-
pound concentrations of 0.2–100 μM. The control solution
contained the same concentration of DMF (0.1%). The develop-
ment of the embryos was monitored at 0, 6, 24, 48, 72 and
7
, 805–814.
1
1
4 S. Patil, A. Deally, B. Gleeson, H. Müller-Bunz, F. Paradisi
and M. Tacke, Metallomics, 2011, 3, 74–88.
5 S. Patil, A. Deally, B. Gleeson, F. Hackenberg, H. Müller-
Bunz, F. Paradisi and M. Tacke, Z. Anorg. Allg. Chem., 2011,
6
37, 386–396.
1
1
6 J. Lemke, A. Pinto, P. Niehoff, V. Vasylyeva and N. Metzler-
Nolte, Dalton Trans., 2009, 7063–7070.
7 S. D. Köster, H. Alborzinia, S. Can, I. Kitanovic, S. Wölfl,
R. Rubbiani, I. Ott, P. Riesterer, A. Prokop, K. Merz and
N. Metzler-Nolte, Chem. Sci., 2011, 3, 2062–2072.
9
6 hours after compound addition, which means that embryos
were screened for proper development and survival at 24, 30,
8, 72, 96 and 120 hpf embryos. Dead embryos were removed
4
1
1
2
8 E. Schuh, C. Pflüger, A. Citta, A. Folda, M. P. Rigobello,
A. Bindoli, A. Casini and F. Mohr, J. Med. Chem., 2012, 55,
from the well to prevent bacterial contamination; each experi-
ment was performed in duplicate.
5518–5528.
9 L. Oehninger, H. Alborzinia, S. Ludewig, K. Baumann,
S. Wölfl and I. Ott, ChemMedChem, 2011, 6, 2142–
2145.
Acknowledgements
0 A. Bergamo and G. Sava, Dalton Trans., 2011, 40, 7817–
Financial support by Deutsche Forschungsgemeinschaft (DFG,
7823.
project FOR630) and Fonds der Chemischen Industrie (FCI) is 21 W. H. Ang, A. Casini, G. Sava and P. J. Dyson, J. Organomet.
gratefully acknowledged.
Chem., 2011, 696, 989–998.
2
2 A. L. Noffke, A. Habtemariam, A. M. Pizarro and
P. J. Sadler, Chem. Commun., 2012, 48, 5219–5246.
3 J. Will, A. Kyas, W. S. Sheldrick and D. Wolters, J. Biol.
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