Chlorambucil conjugates of dinuclear p-cymene ruthenium trithiolato complexes: synthesis, characterization and cytotoxicity study in vitro and in vivo

Article abstract of DOI:10.1007/s00775-016-1353-z

Abstract: Four diruthenium trithiolato chlorambucil conjugates have been prepared via Steglich esterification from chlorambucil and the corresponding trithiolato precursors. All conjugates are highly cytotoxic towards human ovarian A2780 and A2780cisR cancer cell lines with IC₅₀ values in the nanomolar range. The conjugates exhibit selectivity towards A2780 cells as compared to non-cancerous HEK293 cells, while being only slightly selective for RF24 and A2780cisR cells. In vivo, the conjugate [10]BF₄ suppressed the growth of a solid Ehrlich tumor in immunocompetent NMRI mice but did not prolong their overall survival. The reactivity of the chlorambucil conjugates with glutathione, a potential target of the dinuclear ruthenium motive, and with the 2-deoxyguanosine 5′-monophosphate (dGMP—a model target of chlorambucil) was studied by mass spectrometry and NMR spectroscopy. The conjugates did not show catalytic activity for the oxidation of glutathione nor binding to nucleotides, indicating that glutathione oxidation and DNA alkylation are not key mechanisms of action. Graphical abstract: Four highly cytotoxic diruthenium trithiolato chlorambucil conjugates have been prepared. All conjugates exhibit selectivity towards A2780 cells as compared to HEK293 cells, while being only slightly active in RF24 and A2780cisR cells. In vivo, the best candidate suppressed the growth of a solid Ehrlich tumor in immunocompetent NMRI mice but did not prolong their overall survival.[Figure not available: see fulltext.]

Full text of DOI:10.1007/s00775-016-1353-z

J Biol Inorg Chem
DOI 10.1007/s00775-016-1353-z
ORIGINAL PAPER
Chlorambucil conjugates of dinuclear p‑cymene ruthenium
trithiolato complexes: synthesis, characterization and cytotoxicity
study in vitro and in vivo
David Stíbal1 · Bruno Therrien1 · Georg Süss‑Fink1 · Patrycja Nowak‑Sliwinska2 ·
Paul J. Dyson2 · Eva Čermáková4 · Martina Řezácˇová3 · Pavel Tomšík3
Received: 17 February 2016 / Accepted: 24 March 2016
© SBIC 2016
Abstract Four diruthenium trithiolato chlorambucil con-
jugates have been prepared via Steglich esterification from
chlorambucil and the corresponding trithiolato precursors.
All conjugates are highly cytotoxic towards human ovar-
ian A2780 and A2780cisR cancer cell lines with IC50 val-
ues in the nanomolar range. The conjugates exhibit selec-
tivity towards A2780 cells as compared to non-cancerous
HEK293 cells, while being only slightly selective for RF24
and A2780cisR cells. In vivo, the conjugate [10]BF4 sup-
pressed the growth of a solid Ehrlich tumor in immuno-
competent NMRI mice but did not prolong their overall
survival. The reactivity of the chlorambucil conjugates with
glutathione, a potential target of the dinuclear ruthenium
motive, and with the 2-deoxyguanosine 5′-monophos-
phate (dGMP—a model target of chlorambucil) was stud-
ied by mass spectrometry and NMR spectroscopy. The
conjugates did not show catalytic activity for the oxida-
tion of glutathione nor binding to nucleotides, indicating
that glutathione oxidation and DNA alkylation are not key
mechanisms of action.
Graphical abstract Four highly cytotoxic diruthenium
trithiolato chlorambucil conjugates have been prepared. All
conjugates exhibit selectivity towards A2780 cells as com-
pared to HEK293 cells, while being only slightly active
in RF24 and A2780cisR cells. In vivo, the best candidate
suppressed the growth of a solid Ehrlich tumor in immu-
nocompetent NMRI mice but did not prolong their overall
survival.
+
BF4
-
R = C6H5
CH2C6H5
C6H4-p-But
Ru
C6H4-p-OMe
R
S
O
O
S
S
R
Ru
Cl
N
* Bruno Therrien
bruno.therrien@unine.ch
Cl
* Pavel Tomšík
TomsikP@lfhk.cuni.cz
Keywords Arene ruthenium · Chlorambucil · Dinuclear
1
Institut de Chimie, Université de Neuchâtel, Neuchâtel,
Switzerland
complexes · Anticancer activity · In vivo study
2
Institut des Sciences et Ingénierie Chimiques, Ecole
Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
Abbreviations
cabCOOH Chlorambucil
Faculty of Medicine in Hradec Králové, Department
of Medical Biochemistry, Charles University in Prague,
Hradec Králové, Czech Republic
CTG
CellTiter-Glo® luminescent cell viability
assay
3
dGMP
dmso
2-deoxyguanosine 5′-monophosphate
Dimethylsulfoxide
4
Faculty of Medicine in Hradec Králové, Computer
Technology Center, Charles University in Prague, Hradec
Králové, Czech Republic
ECACC
European Centre of Cell Cultures
1 3

J Biol Inorg Chem
en
Ethylenediamine
cisplatin-resistant cancer cells as compared to chlorambu-
cil, to the parent RAPTA complex or to the mixture of both.
These conjugates are supposed to act by two distinct but
complementary modes of action, namely by protein ruthen-
ation and by DNA alkylation [19].
GSH
GSSG
ind
Reduced glutathione
Oxidized glutathione
Indazole
imi
Imidazole
MTD
SEM
VUFB
Maximum tolerated dose
Standard error of the mean
Research Institute for Pharmacy and
Biochemistry
Complexes
of
the
general
formula
[(η6-
arene)2Ru2(μ-SR)3]+ are highly cytotoxic to human ovar-
ian cancer cells, they are in fact among the most active
arene ruthenium anti-cancer compounds reported so far,
the IC50 values of these compounds being in the nanomolar
range for A2780 human ovarian cancer cells and for the cis-
platin-resistant A2780cisR cells [23–25]. Like other arene
ruthenium complexes, thiolato-bridged arene ruthenium
complexes were recently coupled to oligopeptides, the
resulting complexes having significantly increased solubil-
ity in water while maintaining their high cytotoxicity [26].
These dinuclear arene ruthenium trithiolato complexes can
also be functionalized at only one of the thiolato ligands, in
particular since mixed complexes of the type [(η6-arene)2-
Ru2(SRal)2(SRar)]+ are easily accessible with aliphatic (al)
and aromatic (ar) substituents at the thiolato bridges [27].
We therefore prepared the neutral dithiolato precur-
sors [(η6-p-Me-C6H4-Pri)2-Ru2(SCH2R)2Cl2] (R = Ph: 1,
R = CH2Ph: 2, R = C6H4-p-But: 3, R = C6H4-p-OCH3:
4) from p-cymene ruthenium dichloride dimer and the cor-
responding thiol, as well as the cationic mixed trithiolato
complexes [(η6-p-Me-C6H4-Pri)2-Ru2(SCH2R)2-(SC6H4-
p-OH)]+ (Ph: 5, R = CH2Ph: 6, R = C6H4-p-But: 7) by
reaction with p-mercaptophenol according to published
methods [27–29]. The new derivative [(η6-p-Me-C6H4-
Pri)2-Ru2(SCH2C6H4-p-OMe)2(SC6H4-p-OH)]+ (8) was
synthesized in an analogous way. The phenolic group of
the mixed thiolato-bridged cationic complexes 5–8 can be
subsequently functionalized with chlorambucil. Herein we
report the synthesis, the characterization and the in vitro
activity of four thiolato-bridged arene ruthenium chlo-
rambucil conjugates and an in vivo evaluation of the most
promising derivative.
Introduction
Inspired by the discovery of the anticancer properties of
cis-Pt(NH3)2Cl2 (cisplatin) by Rosenberg in 1970 [1],
research on metal-based anticancer agents expanded to
metals other than platinum [2–4]. Already in 1980, Clarke
showed that certain ruthenium complexes are cytotoxic
to cancer cells [5]. Moreover, in vivo they often possess
a lower general toxicity than platinum complexes. Based
on these encouraging biological properties and the fact
that their physico-chemical properties can be easily tuned,
ruthenium complexes are among the most studied non-plat-
inum metal-based drugs [6–9].
Among ruthenium complexes, arene ruthenium deriva-
tives exhibit promising anti-cancer activity. A wide and
diverse range of arene ruthenium complexes has been
studied [10–12], including [(η6-arene)Ru(P-pta)Cl2]
(pta = 1,3,5-triaza-7-phospha-tricyclo-[3.3.1.13,7]-decane)
complexes termed RAPTA [13, 14] as well as [(η6-arene)
Ru(N,N-en)Cl]+ (en = 1,2-ethylenediamine) complexes
[15, 16]. Moreover, coupling of organic molecules to arene
ruthenium complexes can increase their solubility, facilitate
their transport into cells or inhibit unique cancer targets.
For instance, Hartinger’s group designed an arene ruthe-
nium peptide conjugate by a click reaction of the ruthenium
moiety containing a pyronato ligand with the neuropeptide
[Leu5]-enkephalin [17]. The resulting conjugate showed
antiproliferative activity in human ovarian carcinoma cells
with an IC50 value of 13 µM, whereas the peptide or the
ruthenium moiety alone were hardly cytotoxic. Similarly,
the ruthenium complex [(η6-C6H5CH2NH2)Ru(P-pta)Cl2]
was coupled to ethacrynic acid, a compound that inhibits
the enzyme glutathione-S-transferase. The resulting com-
plex was found to bind to glutathione-S-transferase, where-
upon the metal moiety was enzymatically cleaved and
released into the sensitized cancer cells [18]. Very recently,
RAPTA-type complexes were reported, in which the arene
ligand was functionalized by chlorambucil [19], an anti-
cancer drug routinely employed in the treatment of chronic
myeloid leukemia, myeloma and chronic lymphocytic leu-
kemia [20–22]. Some of these chlorambucil-functionalized
RAPTA complexes show superior anticancer activity to
Results and discussion
Synthesis and characterization
The three previously reported trithiolato complexes 5–7 were
synthesized according to published methods [27–29]. The
new complex 8 was synthesized in an analogous fashion,
isolated as a chloride salt and fully characterized. To allow
the conjugation of the cationic trithiolato complexes with
chlorambucil (cabCOOH), compounds [5–8]Cl were reacted
with sodium tetrafluoroborate to yield the correspond-
ing tetrafluoroborate salts, which were reacted with chlo-
rambucil under Steglich esterification reaction conditions
1 3

J Biol Inorg Chem
+
BF4
+
-
Cl-
Ru
Ru
Ru
Cl
R
R
R
S
R
S
S
S
O
O
S
S
OH
S
S
R
R
Cl
Ru
Ru
Ru
Cl
N
R = C6H5; 1
R = C6H5; [5]Cl
R = C6H5; [9]BF4
CH2C6H5; 2
CH2C6H5; [6]Cl
CH2C6H5; [10]BF4
C6H4-p-But; [11]BF4
C6H4-p-OMe; [12]BF4
C6H4-p-But; 3
C6H4-p-But; [7]Cl
C6H4-p-OMe; [8]Cl
Cl
C6H4-p-OMe; 4
Scheme 1 Synthesis of the precursor complexes [5–8]Cl from complexes 1–4, and the chlorambucil conjugates [9–12]BF4 (see “Experimental
part” for the conditions)
Table 1 IC50 values (nM) of complexes 1–4 and [5–8]Cl on human
ovarian carcinoma cells
Table 2 IC50 values (nM) of [9–12]BF4 against human carcinoma
cell lines A2780 and A2780cisR determined using the CTG assay
after 72 h
Compound
A2780
A2780cisR
Compound
A2780
A2780cisR
HEK293
RF24
1
2940 600
200 50
>5000
3600 800
310 80
>5000
[9]BF4
75 14
321 44
353 10
335 50
591 62
>10,000
335 26
448 40
152 15
325 45
280 36
770 100
N.D.
2
[10]BF4
[11]BF4
[12]BF4
Cisplatina
55
6
3
183 25
600 72
860 60
451 65
4
235 50
47.8 3.0
74.4 2.8
163 8.0
320 80
860 60
>5000
1580 300
4400 400
[5]Cl
[6]Cl
[7]Cl
[8]Cl
Cisplatina
42.9 1.0
49.9 1.9
59.5 1.3
109 30
>10,000
Values are given as the means the standard error of the mean
a
Taken from Ref. [35]
Values are given as the means the standard error of the mean
values are compared with values for the neutral dithiolato
complexes 1–4 and cisplatin (Table 1). In general, the neu-
tral dithiolato complexes are less cytotoxic than the cati-
onic trithiolato complexes; the new derivative 8 confirms
this tendency. The chloride salts of the cationic trithiolato
complexes [5–8]Cl exhibit IC50 values in the nanomolar
range.
Chlorambucil, N,N-bis(2-chloroethyl)-p-aminophenylb-
utyric acid, is a drug routinely employed in the treatment of
chronic myeloid leukemia, myeloma and chronic lympho-
cytic leukemia [20, 21]. Its anticancer properties involve
direct binding to DNA, specifically to the N7 of guanines.
Chlorambucil can form either monofunctional nucleotide
adducts, or interact with both of its electrophilic sites at
once, forming either intrastrand or interstrand DNA cross-
links [22].
a
Taken from Ref. [35]
to give the chlorambucil conjugates [(η6-p-MeC6H4-
Pri)2-Ru2(SCH2R)2(SC6H4-p-OOCcab)]+ R = C6H5: 9,
R = CH2C6H5: 10, R = C6H4-p-But: 11, R = C6H4-p-OMe:
12) (Scheme 1). The cationic complexes are isolated as the
tetrafluoroborate salts in the form of orange crystalline pow-
ders. Compounds [9–12]BF4 are well soluble in chlorinated
solvents, alcohols, acetonitrile, THF and DMSO. All com-
1
plexes have been fully characterized by H and 13C NMR
spectroscopy, mass spectrometry and elemental analysis, the
analytical data are given in the Experimental Part.
Cytotoxicity studies
The in vitro anticancer activity of the chlorambucil con-
jugates [9–12]BF4 was evaluated against the human ovar-
ian cancer cell lines A2780 and A2780cisR with acquired
resistance to cisplatin using the CellTiter-Glo® lumines-
cent cell viability assay (CTG) that determines the number
of viable cells in culture based on quantitation of the ATP
present, an indicator of metabolically active cells. The IC50
The cytotoxicity of chlorambucil conjugates [9–12]
BF4 was studied on four cell lines: non-cancerous human
embryonic kidney cells (HEK293), immortalized human
endothelial cells (RF24) and the human ovarian cancer
A2780 and A2780cisR cell lines. The IC50 values for the
two ovarian cancer cell lines are in the nanomolar range
(Table 2), albeit higher than those of the chloride salts of
1 3

J Biol Inorg Chem
Fig. 1 Dose-dependent cell viability after administration of [9–12]BF4. Values are presented as the means the standard error of the mean
the corresponding trithiolato complexes [5–8]Cl that are
not conjugated to chlorambucil (Table 1).
glutathione is reported to be at least partially responsible
for the high in vitro cytotoxicity of trithiolato dinuclear
arene ruthenium complexes [31]. On the other hand, the
mode of action of chlorambucil is its covalent binding to
DNA nucleotides, namely to the N7 position of guanine.
Both of these targets were investigated in order to see if
the chlorambucil conjugates retain the mode of action of
its respective parts.
The cell viability experiments conducted in the four
cell lines (Fig. 1) show that the chlorambucil conjugates
[10]BF4 and [12]BF4 exhibit selectivity for the A2780
cell line at the dose range 50–300 nM (complex 10) or
10–100 nM (complex 12) as compared to HEK293 cells.
The IC50 value for the chlorambucil conjugate [10]BF4 is
55
6 nM for A2780 cells, similar to that of its precur-
Interestingly, [10]BF4 does not oxidize glutathione,
although its precursor [6]Cl has a reported turnover fre-
quency of 7.02 h−1 for the catalytic glutathione oxidation
[27]. After 24 h of incubation at 37 °C, the NMR spectrum
shows only the peaks of the reduced form of glutathione,
no formation of the disulfide being detected.
The interaction of [10]BF4 with 2-deoxyguanosine
5′-monophosphate (dGMP) was studied using mass spec-
trometry at two different pH values, at pH = 3.5 without
the addition of any buffer or base, and after the addition of
0.1 M NaOH, which raised the pH to 8. According to bio-
logical studies of chlorambucil [32], the alkylation of gua-
nines proceeds much faster at elevated pH. However, in our
case the MS spectra did not show any adducts of 2-deoxy-
guanosine 5′-monophosphate and [10]BF4 even at pH = 8.
The only detected products aside from the original complex
were the two hydrolysis products of the chlorambucil moi-
ety of the conjugate, as shown in Fig. 2. When the same
sor [6]Cl (74.4 2.8 nM), while for chlorambucil alone it
is approximately 22-fold higher (1200 nM) [30]. Complex
[11]BF4, at low doses (up to 70 nM), is the most selective
one towards A2780cisR cells, which is not observed in the
higher dose range. In general, the four conjugates are less
cytotoxic to the RF24 and A2780cisR cells at lower doses,
while at higher doses the difference in cytotoxic activity is
not retained.
Interactions with 2‑deoxyguanosine 5′‑monophosphate
and glutathione
In order to study the mechanism of action of the new
chlorambucil conjugates, the conjugate with the high-
est activity in vitro, [10]BF4, was incubated with the
tripeptide glutathione and with the nucleotide 2-deoxy-
guanosine 5′-monophosphate. The catalytic oxidation of
1 3

J Biol Inorg Chem
Fig. 2 Hydrolysis products of compound [10]BF4 after 24 h incubation at 37 °C with four equivalents of 2-deoxyguanosine 5′monophosphate
1
experiment was followed by H NMR spectroscopy, no
adduct formation was observed (data not shown).
These results, although preliminary, suggest that the
conjugation of the thiolato-bridged arene ruthenium com-
plex to chlorambucil hampers the chlorambucil moiety to
interact with nucleotides. Conversely, the conjugation also
blocks the catalytic activity of the dinuclear trithiolato
unit and the mode of action of this compound is therefore
unclear.
In vivo study
To investigate the effects of [10]BF4 on tumor develop-
ment and survival of adult female immunocompetent
NMRI mice, we used a solid Ehrlich tumor implanted
Fig. 3 Weight of the solid Ehrlich tumor (in grams) on day 11 of
mice injected on days 1, 4 and 8 i.p. with [10]BF4 in doses of 10, 15,
or 20 mg/kg, or chlorambucil 5 mg/kg. Values are the means
the
subcutaneously as an experimental model for breast can-
cer. The compound doses were selected according to the
dose-finding study, which had revealed a maximum tol-
erated dose (MTD) of 20 mg/kg. For the MTD assess-
ment, two or three healthy mice per group were observed
for weight loss (the limit was 10 %) over 14 days after
treatment. Figure 3 shows the weight of resected tumors
after treatment with saline in propane-1,2-diol, [10]
BF4 administered at 20, 15 or 10 mg/kg, or chlorambucil
(5 mg/kg), respectively, measured on day 11. [10]BF4 at
the dose of 15 mg/kg had a significant inhibitory effect
on tumor growth (TGI = 44.4 %, P = 0.0474). That was
standard error of the mean (SEM) (n = 7 in each group). Values that
are significantly different from the control experiment are labeled by
* (*P < 0.05, ***P < 0.001)
similar to the activity of the positive control, chlorambucil
(TGI = 47.4 %, P = 0.0268).
The mean overall survival of sham-treated control
tumor-bearing mice was 21.6 days. [10]BF4 at both 10 and
15 mg/kg showed a tendency to prolong the survival albeit
the effect was not significant (P = 0.458 and 0.693, respec-
tively). The group receiving [10]BF4 at 20 mg/kg could not
1 3

J Biol Inorg Chem
Fig. 4 Kaplan–Meier analysis
of survival. Only the adminis-
tration of chlorambucil at 5 mg/
kg significantly prolonged the
survival of tumor-bearing mice
compared to tumor-bearing
controls treated with saline
i.p. (control). Neither 10 mg
nor 15 mg of [10]BF4/kg body
weight had any statistically
significant effect. The com-
pounds were administered i.p.
on days 1, 4 and 8 after tumor
inoculation
be evaluated because of their bad general condition. Unlike
the healthy mice in the MTD finding study, the tumor-
bearing mice treated with 20 mg/kg of [10]BF4 displayed
severe symptoms of intoxication and had to be euthanized.
Only chlorambucil prolonged the mean survival time sig-
nificantly when compared with sham-treated tumor-bearing
control mice (P = 0.0401). Chlorambucil was also more
effective when compared with its conjugate, [10]BF4, at
both evaluated doses (P < 0.01). A Kaplan–Meier analysis
of survival can be seen in Fig. 4.
The conjugate [10]BF4 at any dose tested did not have
a statistically significant therapeutic effect in comparison
to chlorambucil alone. When the dose of 20 mg/kg was
applied, which is stoichiometrically almost equivalent to
5 mg/kg chlorambucil, [10]BF4 turned out to be more sys-
temically toxic and less potent than chlorambucil itself. It
is quite possible that, even if the conjugate was completely
hydrolyzed into the putatively more active components,
the summation of their toxicity might hinder the body’s
own defense mechanisms such as the anticancer immune
surveillance. Studies in more cancer models are needed
to determine whether or not the conjugation in question
increases the anticancer potential of the components.
difference in the cytotoxicity towards the cisplatin-resistant
cell line A2780cisR shows the clear influence of the chlo-
rambucil moiety. The cisplatin-resistant cancer cells are
known to be cross-resistant to chlorambucil [33], presum-
ably due to the similar modes of action of the two agents.
The difference in the spectrum of anticancer activity of the
chlorambucil conjugates compared to their trithiolato pre-
cursors suggests that chlorambucil is cleaved after uptake
into cells with both parts of the conjugate subsequently
exhibiting the cytotoxic effects by their respective modes
of action.
In a breast cancer model in immunocompetent mice,
[10]BF4 at the dose of 15 mg/kg shows a relevant effect on
the inhibition of tumor growth. However, it did not show
a statistically significant effect on the survival of tumor-
bearing mice, possibly due to the high systemic toxicity of
[10]BF4. In conclusion, the studies show that conjugation
of chlorambucil with dinuclear p-cymene ruthenium trithi-
olato complexes did not result in the intended synergistic
enhancement of the anticancer properties of both com-
ponents. The different modes of action do not seem to be
cooperative.
Experimental part
Conclusions
Materials and methods
Four chlorambucil conjugates of trithiolato arene ruthenium
complexes were synthesized, fully characterized and stud-
ied for their anticancer properties. In vitro studies showed
the complexes to be highly cytotoxic, their IC50 values
being comparable to those of the trithiolato precursors. The
The dithiolato and trithiolato complexes 1–4 and [5–7]Cl
were prepared according to published methods [27–29]. All
other reagents were commercially available and were used
without further purification. NMR spectra were recorded
1 3

J Biol Inorg Chem
with a Bruker 400 MHz spectrometer. Electrospray mass
spectra were obtained in positive mode with an LCQ Finni-
gan mass spectrometer. The purity of the compounds was
established by elemental analysis and was above 95 % for
all compounds. The synthesis of chlorambucil conjugates
[9–12]BF4 was carried out using standard Schlenk tech-
niques whereas all the other reactions were performed in
air.
another Schlenk tube containing a solution of chlorambu-
cil (2.0 equivalents) and 4-(dimethyl-amino)pyridine (1.0
equivalent) in CH2Cl2 (5 mL). A solution of dicyclohexyl-
carbodiimide (2.0 equivalents) in CH2Cl2 (5 mL) was
added dropwise to this mixture. The reaction was stirred at
room temperature under nitrogen atmosphere for 16 h. The
solvent was then removed under reduced pressure, the resi-
due dissolved in cold acetonitrile, filtered through a syringe
filter, evaporated to dryness and chromatographed on silica
gel (solvent system CH2Cl2/acetone 7:1). The first orange
band was collected and evaporated to give the products as
orange powders.
Synthesis and data for [(η6‑p‑MeC6H4Pri)2Ru2(SCH2C6
H4‑p‑OMe)2(SC6H4‑p‑OH)] [8]Cl
[(η6-p-MeC6H4Pri)2Ru2(SCH2C6H4-p-OMe)2Cl2] (100 mg,
0.118 mmol) was dissolved in EtOH (50 mL). Then
46 mg (0.354 mmol) of 4-hydroxythiophenol, dissolved
in 10 mL of EtOH, were added to this solution drop-
wise and the solution was refluxed over-night. The solu-
tion was concentrated to dryness, the residue dissolved
in CH2Cl2 (1 mL) and subjected to column chromatogra-
phy on silica gel (solvents CH2Cl2/EtOH 7:1). The red-
dish band was collected and evaporated to dryness to
give the product as an orange powder. Yield: 110.6 mg
(89 %). C42H51ClO3Ru2S3: calcd. C, 53.80; H 5.48;
found C, 53.42; H, 5.34. ESI MS: (MeOH + CH2Cl2):
m/z = 903.1 [M]+. 1H NMR (400 MHz, CDCl3): δ = 10.30
Data for [9]BF4
Yield: 29.7 mg (84 %). C54H64BCl2F4NO2Ru2S3·0.75CH2
Cl2: calcd. C, 51.42; H, 5.16; N, 1.10; found C, 51.64; H,
5.18; N, 1.15. ESI MS: (MeOH + CH2Cl2): m/z = 1128.4
[M]+. 1H NMR (400 MHz, CDCl3): δ = 7.82 (d, 3J = 8 Hz,
2H, SC6H4-p-O), 7.59–7.35 (m, 10H, SCH2C6H5), 7.15
[d, 3J = 8 Hz, 2H, OCC3H6-p-C6H4N(C2H4Cl)2], 7.05
3
3
(d, 2H, J = 8 Hz, p-SC6H4O), 6.76 [d, J = 8 Hz, 2H,
3
OCC3H6-p-C6H4N(C2H4Cl)2], 5.15 [d, J = 5 Hz, 2H, p-
3
CH3C6H4CH(CH3)2], 4.99 [d, J = 5 Hz, 2H, p-CH3C6
3
H4CH(CH3)2], 4.86 [d, J = 5 Hz, 2H, p-CH3C6H4CH
3
3
(s, 1H, SC6H4-p-OH), 7.48 (d, 2H, J = 8 Hz, SC6H4-
(CH3)2], 4.71 [d, J = 5 Hz, 2H, p-CH3C6H4CH(CH3)2],
p-OH), 7.42 (t, 4H, SCH2C6H4-p-OCH3), 7.22 (d, 2H,
3J = 8 Hz, SC6H4-p-OH), 6.98 (t, 4H, SCH2C6H4-p-
3.75 [m, 4H, OCC3H6-p-C6H4N(C2H4Cl)2], 3.66 [m,
4H, OCC3H6-p-C6H4N(C2H4Cl)2, 2H, SCH2C6H5], 3.46
3
3
OCH3), 5.03 [d, 2H, J = 6 Hz, p-CH3C6H4-CH-(CH3)2],
(s, 2H, SCH2C6H5), 2.68 [t, J = 7 Hz, 2H, OCC3H6-
3
3
4.91 [d, 2H, J = 6 Hz, p-CH3C6H4-CH-(CH3)2], 4.71 [t,
p-C6H4N(C2H4Cl)2], 2.59 [t, J = 7 Hz, 2H, OCC3H6-
4H, p-CH3C6H4-CH-(CH3)2], 3.88 (d, 6H, 3J = 6 Hz,
p-C6H4N(C2H4Cl)2], 2.05 [quint, 3J
=
7 Hz, 2H,
3
SCH2C6H4-p-OCH3), 3.53 (s, 2H, SCH2C6H4-p-OCH3),
OCC3H6-p-C6H4N(C2H4Cl)2], 1.92 [sept, J = 7 Hz, 2H,
3
3.34 (s, 2H, SCH2C6H4-p-OCH3), 2.02 [sept, J = 7 Hz,
p-CH3C6H4-CH-(CH3)2], 1.76 [s, 6H, p-CH3C6H4-CH-
3
2H, p-CH3C6H4-CH-(CH3)2], 1.69 [s, 6H, p-CH3C6H4-CH-
(CH3)2], 0.99 [d, J = 7 Hz, 6H, p-CH3C6H4-CH-(CH3)2],
3
3
(CH3)2], 1.03 [d, J = 7 Hz, 6H, p-CH3C6H4-CH-(CH3)2],
0.92 [d, J = 7 Hz, 6H, p-CH3C6H4-CH-(CH3)2] ppm. 13C
3
0.97 [d, J = 7 Hz, 6H, p-CH3C6H4-CH-(CH3)2] ppm. 13C
NMR (100 MHz, CDCl3): δ = 150.96, 139.73, 133.59,
129.93, 129.53, 129.35, 128.77, 128.61, 128.15, 122.30,
107.06, 100.27, 84.11, 83.62, 82.46, 40.09, 39.91, 30.88,
23.20, 22.36, 18.00 ppm.
NMR (100 MHz, CDCl3): δ = 159.44, 159.32, 157.81,
133.88, 131.69, 130.6, 130.40, 115.38, 114.00, 113.85,
106.95, 99.97, 83.72, 83.59, 82.28, 55.99, 55.46, 39.51,
39.27, 30.87, 23.15, 22.42, 17.99 ppm.
Data for [10]BF4
General procedure for the synthesis of chlorambucil
conjugates [9–12]BF4
Yield: 31.5 mg (81 %). C56H68BCl2F4NO2Ru2S3: calcd.
C, 54.10; H, 5.51; N, 1.13; found C, 53.71; H, 5.53; N,
1.23. ESI MS: (MeOH/CH2Cl2): m/z = 1158.0 [M]+.
In a Schlenk tube, 30 mg of [(η6-p-MeC6H4Pri)2Ru2
(SCH2R)2(SC6H4-p-OH)]Cl (R = C6H5 0.032 mmol,
R = CH2C6H5 0.032 mmol, R = C6H4-p-But 0.029 mmol,
R = C6H4-p-OMe 0.030 mmol) were dissolved in distilled
dichloromethane (10 mL), ten equivalents of NaBF4 were
added, and the mixture was stirred at room temperature
for 12 h. Then the solution was filtered through a syringe
filter (0.22 µm), passed through a small plug of silica gel
(solvent system CH2Cl2/acetone 7:1) and transferred into
3
1H NMR (400 MHz, CDCl3): δ = 7.73 (d, J = 8 Hz,
2H, SC6H4-p-O), 7.45–7.27 (m, 10H, SC2H4C6H5), 7.13
[d, 3J = 8 Hz, 2H, OCC3H6-p-C6H4N(C2H4Cl)2], 7.01
3
3
(d, 2H, J = 8 Hz, SC6H4-p-O), 6.68 [d, J = 8 Hz, 2H,
3
OCC3H6-p-C6H4N(C2H4Cl)2], 5.23 [d, J = 5 Hz, 2H, p-
3
CH3C6H4CH(CH3)2], 5.19 [d, J = 5 Hz, 2H, p-CH3C6
3
H4CH(CH3)2], 5.15 [d, J = 5 Hz, 2H, p-CH3C6H4CH
3
(CH3)2], 5.10 [d, J = 5 Hz, 2H, p-CH3C6H4CH(CH3)2],
1 3

J Biol Inorg Chem
3
3.72 [m, 4H, OCC3H6-p-C6H4N(C2H4Cl)2], 3.65 [m, 4H,
OCC3H6-p-C6H4N(C2H4Cl)2, 2H, SCH2C6H5], 3.08 (m,
4H, SC2H4C6H5), 2.88 (m, 2H, SC2H4C6H5), 2.66, [m,
p-CH3C6H4CH(CH3)2], 5.00 [d, J = 5 Hz, 2H, p-CH3
3
C6H4CH(CH3)2], 4.87 [d, J = 5 Hz, 2H, p-CH3C6H4CH
3
(CH3)2], 4.77 [d, J = 5 Hz, 2H, p-CH3C6H4CH(CH3)2],
3
3
4H, OCC3H6-p-C6H4N(C2H4Cl)2], 2.59 (t, J = 8 Hz, 2H,
3.86 (d, J = 11 Hz, 6H, SCH2C6H4-p-OCH3), 3.72 [m,
SC2H4C6H5), 2.06 [m, 2H, OCC3H6-p-C6H4N(C2H4Cl)2,
2H, p-CH3C6H4-CH-(CH3)2], 1.83 [s, 6H, p-CH3C6H4-CH-
4H, OCC3H6-p-C6H4N(C2H4Cl)2], 3.65 [m, 4H, OCC3H6-
p-C6H4N(C2H4Cl)2], 3.59 (s, 2H, SCH23C6H4-p-OCH3),
3.39 (s, 2H, SCH2C6H4-p-OCH3), 2.67 [t, J = 7 Hz, 2H,
OCC3H6-p-C6H4N(C2H4Cl)2], 2.58 [t, 3J = 7 Hz, 2H,
OCC3H6-p-C6H4N(C2H4Cl)2], 2.05 [m, 2H, OCC3H6-p-
C6H4N(C2H4Cl)2], 1.92 [m, 2H, p-CH3C6H4-CH-(CH3)2],
3
(CH3)2], 1.05 [t, J = 7 Hz, 12H, p-CH3C6H4-CH-(CH3)2]
ppm. 13C NMR (100 MHz, CDCl3): δ = 171.72, 150.92,
139.85, 133.53, 129.75, 128.85, 128.77, 128.72, 126.82,
122.15, 112.49, 107.03, 100.37, 84.18, 83.95, 83.19, 41.17,
40.41, 40.19, 38.67, 38.55, 33.87, 33.54, 30.88, 26.51,
23.21, 22.38, 17.80 ppm.
3
1.76 [s, 6H, p-CH3C6H4-CH-(CH3)2], 0.99 [d, J = 6 Hz,
6H, p-CH3C6H4-CH-(CH3)2], 0.93 [d, 3J = 6 Hz, 6H,
p-CH3C6H4-CH-(CH3)2] ppm. 13C NMR (100 MHz,
CDCl3): δ = 204.31, 171.29, 159.36, 150.94, 144.45,
134.95, 133.56, 131.64, 130.61, 130.40, 130.18, 123.70,
122.19, 114.05, 113.88, 112.24, 109.93, 107.10, 100.09,
83.87, 83.65, 82.40, 55.50, 55.44, 53.59, 40.55, 33.86,
33.55, 30.87, 26.53, 23.16, 22.34, 17.98 ppm.
Data for [11]BF4
Yield: 28.0 mg (79 %). C62H80BCl2F4NO2Ru2S3: calcd.
C, 56.10; H, 6.07; N, 1.06; found C, 56.19; H, 6.17; N,
1.05. ESI MS: (MeOH + CH2Cl2): m/z = 1240.6 [M]+.
3
1H NMR (400 MHz, CDCl3): δ = 7.80 (d, J = 8 Hz, 2H,
SC6H4-p-O), 7.45 [m, 8H, SCH2C6H4-p-C(CH3)3], 7.14
[d, 3J = 8 Hz, 2H, OCC3H6-p-C6H4N(C2H4Cl)2], 7.04
Cells and cell viability assays
3
3
(d, J = 8 Hz, 2H, SC6H4-p-O), 6.73 [d, J = 8 Hz, 2H,
Human A2780 and A2780cisR ovarian carcinoma cells
were obtained from the European Centre of Cell Cultures
(ECACC, UK). Non-cancerous human embryonic kidney
HEK293 cells were provided by the Institute of Pathol-
ogy, CHUV, Lausanne, Switzerland. Immortalized human
endothelial cells RF24 were kindly provided by the Angio-
genesis Laboratory of VU Medical Center, Amsterdam,
The Netherlands. A2780 and A2780cisR cells were rou-
tinely grown in RPMI 1640 medium supplemented with
GlutaMAX (Gibco), 10 % fetal calf serum and 1 % anti-
biotics (Penicillin/Streptomycin, Sigma), while HEK293
cells were grown in DMEM medium, both containing heat-
inactivated fetal calf serum (FCS, Sigma, USA) (10 %) and
1 % antibiotics at 37 °C and CO2 (5 %). RF24 cells were
maintained in RPMI 1640/DMEM (1:1) supplemented as
above.
3
OCC3H6-p-C6H4N(C2H4Cl)2], 5.11 [d, J = 6 Hz, 2H,, p-
CH3C6H4CH(CH3)2], 4.97 [d, 3J = 6 Hz, 2H, p-CH3C6H4C
3
H(CH3)2], 4.89 [d, J = 6 Hz, 2H, p-CH3C6H4CH(CH3)2],
3
4.60 [d, J = 6 Hz, 2H, p-CH3C6H4CH(CH3)2], 3.74 [m,
4H, OCC3H6-p-C6H4N(C2H4Cl)2], 3.66 [m, 4H, OCC3H6-
p-C6H4N(C2H4Cl)2], 3.61 [s, 2H, SCH2C6H4-p-C(CH3)3],
3
3.41 [s, 2H, SCH2C6H4-p-C(CH3)3], 2.67 [t, J = 7 Hz,
3
2H, OCC3H6-p-C6H4N(C2H4Cl)2], 2.58 [t, J = 7 Hz, 2H,
OCC3H6-p-C6H4N(C2H4Cl)2], 2.05 [quint, 3J = 7 Hz,
3
2H, OCC3H6-p-C6H4N(C2H4Cl)2], 1.89 [sept, J = 7 Hz,
2H, p-CH3C6H4-CH-(CH3)2], 1.76 [s, 6H, p-CH3C6H4-
CH-(CH3)2], 1.37 [s, 9H, SCH2C6H4-p-C(CH3)3], 1.33
[s, 9H, SCH2C6H4-p-C(CH3)3], 0.94 [d, 3J = 7 Hz,
6H, p-CH3C6H4-CH-(CH3)2], 0.89 [d, 3J = 7 Hz, 6H,
p-CH3C6H4-CH-(CH3)2] ppm. 13C NMR (100 MHz,
CDCl3): δ = 171.66, 151.57, 150.87, 136.68, 133.53,
129.88, 129.06, 125.51, 125.34, 122.25, 106.89, 100.54,
84.17, 83.47, 82.46, 39.94, 39.38, 34.74, 33.88, 33.45,
31.34, 30.79, 26.36, 23.05, 22.52, 18.07 ppm.
105 cells/well were seeded in 96-well cell culture plates
as described previously [34]. Briefly, 24 h after seeding,
culture medium with or without compounds was added
and cells were grown for an additional 72 h. Cell viability
was assessed using the CellTiter-Glo®. Luminescent Cell
Viability Assay (Promega, Madison, WI, USA) according
to the manufacturer’s instructions. Evaluation is based on
means from two independent experiments, each comprising
three microcultures per concentration level. 0.1 % DMSO
in 0.9 % NaCl was used as the control.
Data for [12]BF4
Yield: 21.7 mg (53 %). C56H68BCl2F4NO4Ru2S3: calcd.
C, 52.75; H, 5.37; N, 1.10; found C, 52.61; H, 5.40; N,
1.11. ESI MS: (MeOH + CH2Cl2): m/z = 1189.6 [M]+.
3
1H NMR (400 MHz, CDCl3): δ = 7.80 (d, J = 8 Hz,
2H, SC6H4-p-O), 7.44 (m, 4H, SCH2C6H4-p-OCH3), 7.12
[d, 3J = 8 Hz, 2H, OCC3H6-p-C6H4N(C2H4Cl)2], 7.02
(m, 2H, SC6H4-p-O, 2H, SCH2C6H4-p-OCH33), 6.95 (d,
3J = 8 Hz, 2H, SCH2C6H4-p-OCH3), 6.67 [d, J = 8 Hz,
Catalytic oxidation of glutathione
To evaluate the catalytic performance of the complexes for
the oxidation of the reduced form of GSH to the disulfide
form (GSSG), [10]BF4 (1 mg) was dissolved in 0.3 mL
3
2H, OCC3H6-p-C6H4N(C2H4Cl)2], 5.14 [d, J = 5 Hz, 2H,
1 3

J Biol Inorg Chem
of D2O and 0.3 mL of CD3CN, and 100 equiv. of GSH
(24.7 mg) were added to the solution. The sample was
subsequently analyzed by H NMR spectroscopy. The H
NMR spectra were recorded immediately after sample
preparation, and then every 30 min over the period of 24 h.
NCSS software were used for the calculations and statis-
tical evaluations. To calculate the tumor growth inhibition
(TGI), the following formula was used: THI (%) = [(mean
tumor weight in sham-treated controls − mean tumor
weight in treated mice)/mean tumor weight in sham-treated
controls] × 100 %.
1
1
Interaction with 2‑deoxyguanosine 5′‑monophosphate
Acknowledgments This work was financially supported by the
Swiss National Science Foundation (Projects 200020-143254 and
200020-131844), the Secrétariat d’Etat à l’éducation et à la recherche
(to PNS), and the PRVOUK P37/01 program initiated by Charles Uni-
versity in Prague. The authors also thank Ms. Petra Kazimirova and
Ms. Maria Perwein for their technical assistance. We also thank the
group of Dr. Stefan Schürch at the University of Bern for the micro-
analyses of the new compounds.
1 mg of [10]BF4 was dissolved in 0.3 mL of MeCN-d3 and
a solution of 1.1 mg of 2-deoxyguanosine 5′-monophos-
phate (4 equivalents) in 3 mL of D2O was added. The solu-
tion was incubated at 37 °C. NMR and MS spectra were
recorded immediately after mixing and after 24 h.
Animals and tumor model
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Due to the poor solubility of [10]BF4 in water, propane-
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saline, for which reason the i.p. route of administration
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were used for this study, they were obtained from Masaryk
University (Brno, Czech Republic). Animal care was con-
form to EU recommendations and in accordance with the
European convention for the protection of vertebrate ani-
mals used for experimental and other scientific purposes;
it was approved by the Ethical Commission of the Medi-
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