New ruthenium(II)-letrozole complexes as anticancer therapeutics

Article abstract of DOI:10.1021/jm301103y

Novel ruthenium-letrozole complexes have been prepared, and cell viability of two human cancer cell types (breast and glioblastoma) was determined. Some ruthenium compounds are known for their cytotoxicity to cancer cells, whereas letrozole is an aromatas

Full text of DOI:10.1021/jm301103y

Article
pubs.acs.org/jmc
New Ruthenium(II)−Letrozole Complexes as Anticancer Therapeutics
Annie Castonguay, Cedric Doucet, Michal Juhas, and Dusica Maysinger*
́
Department of Pharmacology and Therapeutics, McGill University, 3655 Promenade Sir-William-Osler, Montreal, Quebec H3G 1Y6,
Canada
S
* Supporting Information
ABSTRACT: Novel ruthenium−letrozole complexes have
been prepared, and cell viability of two human cancer cell
types (breast and glioblastoma) was determined. Some
ruthenium compounds are known for their cytotoxicity to
cancer cells, whereas letrozole is an aromatase inhibitor
administered after surgery to post-menopausal women with
hormonally responsive breast cancer. A significant in vitro
activity was established for complex 5·Let against breast cancer
MCF-7 cells and significantly lower activity against glioblasto-
ma U251N cells. The activity of 5·Let was even higher than
that of 4, a compound analogous to the well-known drug
RAPTA-C. Results from the combination of 5·Let (or 4) with 3-methyladenine (3-MA) or with curcumin, respectively, revealed
that the resultant cancer cell death likely involves 5·Let-induced autophagy.
to the different geometries that they adopt.4 Ruthenium-based
drugs are very appealing to study, not only to better understand
INTRODUCTION
■
Breast cancer is the most frequently diagnosed cancer and the
their modes of action but also because they were found to have
leading cause of cancer death among women, accounting for
certain advantages over platinum-based drugs: (i) they are
23% of total cancer cases in women, and 14% of cancer deaths.1
active against cisplatin resistant cell lines, notably the breast
The development of cancer therapies over the past few decades
T47D cells and the ovarian A2780cisR cells, and (ii) they have
has focused principally on chemotherapy combined with other
treatment approaches such as surgery, radiotherapy, and
targeted therapy. A major drawback of chemotherapy is that
a low occurrence of side effects due to their higher selectivity
for cancer cells compared with normal cells.4 Like cisplatin,
ruthenium complexes interact with DNA but, as anticipated,
the drugs used are also toxic to normal cells, leading to
they interact differently. Interestingly, some ruthenium
numerous undesirable side effects (bone and back pain, blood
compounds bind more strongly, leading to an adduct that is
clots, weakness, fatigue, stroke, hair loss, etc.). Moreover, the
more resistant to cell repair mechanisms.5 Ruthenium drugs are
use of a single agent often fails to achieve complete cancer
remission,2 largely due to the development of cancer cell
also believed to induce cancer cell death by other mechanisms;
for instance, they are believed to enter cancer cells by
resistance. An appealing strategy to increase the survival
mimicking iron, a ruthenium congener of the periodic table,
chances of breast cancer victims is to create multitasking
(i) by binding to human serum proteins found at the cells
drugs, able to promote cell death by simultaneously targeting
surface6a and (ii) by interacting with mitochondria.6b Another
selected signal transduction molecules. In addition to possible
synergistic effects resulting from the combined use of two drugs
(which can lead to shorter-term treatments), another great
mode of action for cancer therapeutics is the so-called
“activation by reduction” mechanism, which has been under
debate for decades but which only applies to Ru(III) drugs.7 It
advantage of this approach is the potential to gain some control
is noteworthy that two Ru(III) complexes have entered phase II
of the hydrophilicity and the size of the entire delivery system,
increasing drug efficiency and reducing side effects. We have
undertaken the preparation of ruthenium−letrozole multi-
tasking drugs; the selection of the two components is justified
clinical trials: KP1019 and NAMI-A (Figure 1a). KP1019 was
developed for solid tumors, whereas NAMI-A has been
prepared as an antimetastatic drug.4 Also, several Ru(II)
complexes bearing an arene ligand are under preclinical
evaluation, such as RAPTA-C and Ru-arene-en (Figure 1a).
below.
Cell Killing Agent: Ruthenium. A well-known example of
a cell killing drug is cisplatin, a transition metal-based complex
Cell Growth Inhibiting Agent: Letrozole. Estrogen
receptor positive (ER+) is a very common type of breast
that has been administered to cancer patients for decades and is
known to interact with DNA.3 During the last 20 years,
ruthenium complexes of various kinds have emerged in the
cancer and is diagnosed when estrogen receptors α (ERα) are
found in excess in the tumor cells of the victim. After surgical
literature, mainly designed to replace cisplatin; it was believed
that the interaction of ruthenium complexes with DNA would
Received: July 26, 2012
differ from that of cisplatin (or carboplatin and oxaliplatin), due
Published: September 19, 2012
© 2012 American Chemical Society
8799
dx.doi.org/10.1021/jm301103y | J. Med. Chem. 2012, 55, 8799−8806

Journal of Medicinal Chemistry
Article
catalytically converts androgens to estrogens, notably testoster-
one into estradiol, an estrogen responsible for the growth of
breast cancer tumors. In post-menopausal women, the
production of estrogens is no longer governed by the ovaries
and mainly relies on the catalytic action of aromatase. Letrozole
(Femara) (Figure 1b) is an example of drugs that can
coordinate to the aromatase iron center and deactivate it by
preventing androgens from approaching its catalytic site.
Letrozole was approved by the FDA a decade ago as a first-
line treatment due to its proven superiority to the widely used
prodrug tamoxifen9 and is commonly administered to post-
menopausal women suffering from an ER+ breast cancer.
Herein, we report the preparation of the first ruthenium−
letrozole complexes; the special feature is their potential to
simultaneously kill cancer cells (ruthenium) and inhibit the
growth of the surviving ones (letrozole). We also report our
results on their toxicity against human breast MCF-7 cancer
cells and compare the results with the ones obtained for human
glioblastoma brain U251N cancer cells, one of the deadliest
forms of cancer because of its highly invasive nature.10 We have
also investigated the combination of our most potent
ruthenium drugs with a well-known autophagy inhibitor, 3-
methyladenine (3-MA), and a drug that is known to mainly
induce cell death via autophagy, curcumin.
RESULTS AND DISCUSSION
■
Figure 1. (a) Ruthenium compounds for cancer therapy and (b)
inhibition of aromatase by letrozole.
Complex Synthesis and Characterization. Cationic
ruthenium−letrozole complex 2a, in which two letrozole
ligands are coordinated to the metal via only one nitrogen
atom, was obtained with 44% yield when dichloro(benzene)-
ruthenium(II) dimer 1 was heated with 4 equiv of letrozole
(Figure 2). Interestingly, when only 2 equiv of the aromatase
inhibitor was used instead, compound 2a was again obtained as
the major species. To our knowledge, this is the first example of
the coordination of letrozole to a group 8 metal, which is of
great importance to further understand how this drug might
also coordinate to iron, a congener of ruthenium in the periodic
table, which is the metal responsible for the catalytic activity of
the aromatase enzyme. Only two reports of letrozole−metal
removal of breast tumor(s), it is usual to administer a cell
growth inhibiting drug in order to avoid ER+ cancer recidivism.
A well-known example of this type of drug is tamoxifen
(prodrug), which acts as a selective estrogen receptor down-
regulator since its mode of action is to bind to the estrogen
receptors and consequently to prevent tumor growth (Figure
1b). Another way of achieving the same goal is simply by
blocking the action of an enzyme called aromatase, a member
of the cytochrome P450 family, consisting of an iron center
surrounded by a porphyrin and a cysteine unit.8 This enzyme
Figure 2. Synthesis of letrozole−ruthenium complexes.
8800
dx.doi.org/10.1021/jm301103y | J. Med. Chem. 2012, 55, 8799−8806

Journal of Medicinal Chemistry
Article
interactions were found in the literature; they showed that the
enzyme inhibitor can coordinate to Cu(I) in a tetradentate
fashion to afford a polymer, {[Cu(LTZ)](BF4)·CHCl3}n,11 or
can coordinate to Cu(II), Ni(II), or Co(II) by only one
nitrogen of the triazole ring,12 as observed in the present study.
The nature of 2a was unambiguously confirmed by high-
resolution (and high-accuracy) mass spectrometry, as well as by
elemental analysis. Characterization of the complex by NMR
spectroscopy was less straightforward since the letrozole ligands
in 2a were found to be relatively labile in solution. For instance,
when a 1H NMR spectrum of 2a was recorded in CD2Cl2, 50%
of the molecules were found to undergo replacement of one
letrozole ligand, most likely by the chloride counterion. As a
consequence an equimolar mixture of cationic complex 2a,
neutral complex 2b (the new species obtained from the
displacement of one letrozole unit), and letrozole was
produced. Markedly, when spectra of 2a were recorded in
coordinating solvents such as DMSO-d6 or methanol-d4, one
letrozole from all the molecules was found to readily leave the
coordination sphere of the ruthenium, leading to the exclusive
observation, by 1H NMR, of signals similar to the ones
previously attributed to 2b. It is noteworthy that in those
coordinating solvents, the complete displacement of both
letrozole ligands was also observed over time. The lability of the
aromatase inhibitor(s) in 2a was also observed in the solid state
by X-ray crystallography studies. When single crystals were
grown over 12 h by the diffusion of pentane into a freshly
prepared and concentrated solution of 2a in CDCl3, two types
of crystals were found to coexist, corresponding to 2a and 2b
(Figure 2). The ORTEP (Oak Ridge thermal-ellipsoid plot)
diagram for the solid-state structure analysis of complex 2b is
presented in Figure 3.13 Crystals of 2a were less easy to handle
than those of 2b, losing their crystalinity within a few minutes
when removed from the CDCl3/pentane solvent mixture at
room temperature. This observation is consistent with the high
content of residual electronic density found in the structure of
2a, which might be due to the presence of solvent molecules
within the crystal structure. For comparison purposes, the
synthesis of a cymene analogue was attempted using the same
procedure as the one used to prepare 2a, starting with
dichloro(p-cymene)ruthenium(II) dimer ([RuCl2(cymene)]2).
Unfortunately, the solid obtained was only soluble in DMSO-d6
1
and H NMR spectroscopy revealed the presence of two free
letrozole ligands per cymene unit, which might indicate that the
steric hindrance of the arene ring is a factor enhancing letrozole
ligand lability in solution.
Coordination of phosphorus ligands to transition metals
favors hydrophilicity and promotes interactions with DNA,14
leading to enhancement of the complex cytotoxicity. For
example, the introduction of 1, 3, 5-triaza-7-
phosphatricyclo[3.3.1.1]decane (PTA) into organometallic
Ru(II)−arene systems resulted not only in formation of
water-soluble complexes but also in selective antimetastatic
activity. Also, it has been shown that when modified with a
triphenylphoshine ligand, the RAPTA structure forms a
complex with enhanced cytotoxicity and cellular uptake, and
with higher affinity toward DNA.15 We reasoned that it would
be of interest to study the effect of the substitution of one of
the letrozole ligands in 2a by a triphenylphosphine. When the
reaction of complex 2a with 1 equiv of triphenylphosphine was
followed by 31P{1H} NMR spectroscopy, it was noted that the
singlet resonance corresponding to the free triphenylphosphine
disappeared and that a new singlet resonance at 23 ppm
emerged, which was found to correspond to the neutral 4
complex (Figure 2). The expected monosubstituted cationic
complex might have formed but probably underwent a
substitution reaction of the remaining letrozole ligand by the
chloride counterion. The identity of the complex was confirmed
by comparing its NMR spectra with the ones displayed by the
product obtained from the well-known reaction of 1 with 2
equiv of triphenylphosphine. Since the presence of the chloride
counterion in 2a was most likely the main factor preventing us
from preparing a complex bearing both a letrozole and a
triphenylphosphine, we attempted the preparation of a new
cationic letrozole−ruthenium complex, taking advantage of the
less coordinating nature of the tetraphenylborate or tetra-
fluoroborate counterions. Complex 2a reacted sluggishly with
sodium tetraphenylborate or sodium tetrafluoroborate under
the conditions used but the desired analogue of 2a, complex 3,
was successfully prepared with 73% yield when 1 was heated
with 4 equiv of letrozole and an excess of ammonium
tetrafluoroborate for 1 h (Figure 2). As expected, the letrozole
ligands in that complex were less labile in solution than the
ones in 2a. Fortunately, it was possible to achieve the synthesis
of our target 18-electron compound 5 (Figure 2), by
substituting only one letrozole ligand from 3, by a
triphenylphosphine. Complexes 3 and 5 were characterized
by NMR spectroscopy, high-resolution (and high-accuracy)
mass spectrometry, and by elemental analysis.16 Their nature
was unambiguously confirmed by X-ray crystallography, and the
ORTEP diagram for the state structure analysis of complex 3 is
presented in Figure 3.13
Cytotoxicity Studies. The in vitro toxicity of letrozole
complexes 2a, 3, and 5·Let and the letrozole free complex 4 (25
μM) was assessed against the human breast cancer MCF-7 cells
(after 24 h), by the MTT assay and cell counting (Figure 4a,
Figure S1, Supporting Information). The two analogues 2a and
3, each bearing two letrozole ligands, did not significantly
reduce mitochondrial metabolic activity (2a, 82% 3%; 3, 79%
3%; p < 0.001), compared with the effect of equimolar or
Figure 3. ORTEP diagrams for complexes 2b and 3. Thermal
ellipsoids are shown at the 50% probability level.
8801
dx.doi.org/10.1021/jm301103y | J. Med. Chem. 2012, 55, 8799−8806

Journal of Medicinal Chemistry
Article
(Figure 4a, 61%
6%, p < 0.001).This cytotoxicity is even
higher than that observed for 4, a compound analogous to
RAPTA-C, which is a well-known antimetastatic ruthenium
drug under preclinical evaluation (Figure 4a, 71% 2%, p <
0.001). Among all the drugs tested under estrogen-deprived
conditions, 5·Let remained the most potent drug for which the
mitochondrial metabolic activity decreased dramatically (Figure
4a, 27% 4%, p < 0.001). This result might indicate a killing
effect from the ruthenium moiety, the compound being 2 times
more cytotoxic than one would have expected from the additive
effect of the ligands tested alone, under those conditions.
Moreover, this result demonstrates the importance of fine-
tuning the drug solubility and size to favor the most appropriate
interactions of the metal with biological molecules for
optimizing a killing effect. The IC50 value for 5·Let in MCF-7
cells is 36 6 μM (for 24 h), which is in the range of the IC50
values reported for other Ru(II) complexes against human
cancer cells. For instance, an IC50 value of 19.58 2.37 μM (72
h) was reported for complex [Ru(η6-p-cymene)(curcuminato)-
Cl],17a an IC50 value of 11.3 1.4 μM (72 h) was reported for
complex [RuCl(Tp)(PTA)(PPh3)] (Tp = trispyrazolylbora-
te),17b and IC50 values in the range of 1.8 0.5 to 115.0 0.6
μM (96 h) were reported for complexes [Ru(η6-p-cymene)-
Cl2{Ph2P(CH2)nS(O)xPh-κ-P}] (x = 0−2; n = 1−3),17c against
MCF-7 breast cancer cells.
Compounds tested in MCF-7 cells were also tested in
glioblastoma brain U251N cells (after 24 h exposure, Figure
4b). None of the compounds were cytotoxic, except letrozole
(50 μM) and 5·Let (25 μM), which displayed a small decrease
in mitochondrial metabolic activity. These in vitro results,
however, cannot be directly translated to the effectiveness in
animals with glioblastoma xenographs or ectopic tumors. For
example, ruthenium complex NAMI-A (Figure 1a) was poorly
effective in vitro, but turned out to be remarkably effective in
mice, in vivo.18
Figure 4. Mitochondrial metabolic activity determined by MTT assay
in human breast cancer cells (MCF-7) (a) and glioblastoma cancer
cells (U251N) (b), treated with 2a (25 μM), 3 (25 μM), 4 (25 μM),
5·Let (25 μM), triphenylphosphine (25 μM), and letrozole (50 μM).
Results from estrogen-deprived MCF-7 cells are depicted with open
bars, and those from MCF-7 cells grown in the presence of serum are
depicted as filled bars. All values are expressed as means (from at least
three independent experiments) SEM relative to untreated controls
(100%). Significant differences: *p < 0.05; ***p < 0.001.
Combination Studies. Cell death occurs as a consequence
of three different processes: apoptosis, autophagy, and
necrosis.19 It is important to learn more about the
mechanism(s) by which individual anticancer drugs operate
to create powerful anticancer treatments, especially ones using
specific drug combinations. Numerous anticancer drugs are
known to induce cell death by apoptosis, while some others are
known to induce cell death via an autophagy pathway,20 which
is a self-cannibalization process that also can contribute to
tumor resiliency.21 Since inhibition of autophagy might prevent
the growth of established tumors, the study of autophagy
inhibitors used in combination with drugs that trigger
“cytoprotective” autophagy holds promise for improved
treatment of cancer. We have investigated whether an
autophagy process might be triggered by our two most potent
ruthenium drug candidates, 4 and 5·Let. To do so, we assessed
their in vitro toxicity in combination with a well-known
autophagy inhibitor, 3-methyladenine (3-MA), against the
previously investigated cells, breast MCF-7 (Figure 5a) and
glioblastoma brain U251N (Figure 5b,c), using the MTT assay.
After 24 h, the combination of complex 5·Let (0−100 μM)
with 3-MA (1 mM) did not lead to any enhancement of
cytotoxicity against breast MCF-7 cells (Figure 5a). This
implies that complex 5·Let does not induce a “cytoprotective”
autophagy process, as was reported for other Ru(II) drugs,
which used in combination with autophagy inhibitors enhanced
their cytotoxicity effect.22 The fact that no additive effect is
noted indicates that an autophagy killing process might have
even double (50 μM) letrozole concentrations. Similar results
were obtained for 4 and PPh3. In contrast, 5·Let significantly
reduced mitochondrial metabolic activity (62%
5%, p <
0.001), suggesting an additive toxic effect between triphenyl-
phosphine and two letrozole ligands, under comparable testing
conditions. Interestingly, when estrogen-deprived conditions
were used, the cytotoxicity of complexes 2a and 3 (as well as
the one corresponding to twice the concentration of letrozole
tested alone) was found to be approximately equal or even
slightly lower compared with the previous conditions used
(Figure 4a). A plausible explanation for this behavior might be
the low hydrophilicity of these compounds under the
conditions used or simply that the cells developed a letrozole
resistance. It should be stressed that the mitochondrial
metabolic activity decreased significantly for the other
compounds tested under estrogen-deprived conditions. This
observation highlights the role played by estrogens in the
growth of MCF-7 hormonally responsive cancer cells but also
the role played by the triphenylphosphine moiety, which is
present in the other compounds tested. This ligand is known to
enhance the anticancer properties of metallic complexes but
also proved to be surprisingly cytotoxic when tested alone
8802
dx.doi.org/10.1021/jm301103y | J. Med. Chem. 2012, 55, 8799−8806

Journal of Medicinal Chemistry
Article
complex 4 showed a considerable additive effect when
combined with the same autophagy inhibitor, over the same
range of concentrations (Figure 5c). This result suggests that 4
itself most likely does not induce autophagy, while 5·Let might
act by inducing cell killing autophagy (Figure 6). To clarify
these findings, we investigated the effect of the combination of
the same two ruthenium complexes with curcumin in
glioblastoma cells (U251N), a compound which is known to
promote cell death through induction of autophagy in glioma.23
The combination of complex 5·Let with curcumin (25 μM)
showed a large synergistic effect at 25 μM (Figure 5b) whereas
the combination of 4 (25 μM) with curcumin (Figure 5c) did
not lead to a comparable effect. These findings suggest different
modes of cell death induced by the two ruthenium complexes,
and further studies should elucidate signal transduction
pathways involved in these modes of cell death.
EXPERIMENTAL SECTION
■
Complex Synthesis and Characterization. All reagents were
used as received from commercial vendors. Letrozole was purchased
from AK Scientific, while RuCl3·xH2O, 1,4-cyclohexadiene, ammo-
nium tetrafluoroborate, and triphenylphosphine were acquired from
Sigma Aldrich. Experiments were performed under an argon
atmosphere, and solvents were dried on activated molecular sieves
columns using a solvent purification system. All NMR spectra were
recorded at room temperature on a 200−500 MHz Varian
spectrometer. 1H and 13C{1H} NMR spectra were referenced to
solvent resonances, whereas 31P{1H} NMR spectra were referenced to
an external 85% H3PO4 sample (0 ppm). All chemical shifts and
coupling constants are expressed in ppm and Hz, respectively. The
elemental analyses were performed by the Laboratoire d’Analyse
́
́ ́ ́ ́
Elementaire (Departement de Chimie, Universite de Montreal). The
high-resolution and high-accuracy mass spectra (ESI-MS) were
obtained using an Exactive Orbitrap spectrometer from ThermoFisher
Scientific (Department of Chemistry, McGill University). High-
performance liquid chromatography (HPLC, Waters Instrument,
reversed phase Symmetry C18 column 5 μm, 4.6 mm × 150 mm,
detection 254 nm, mobile phase ethanol/methanol/2-propanol, flow
rate 1.0 mL/min, retention time 9−11 min) was used to confirm the
purity (≥95%) of the ruthenium complexes.
[Ru(C6H6)(η1-Let)2Cl]Cl, 2. Letrozole (0.228 g, 0.80 mmol) was
added to a degassed suspension of 1 (0.100 g, 0.20 mmol) in ethanol
(35 mL), and the mixture was heated under reflux. After 1 h, the
mixture was allowed to cool to room temperature and was filtered on a
glass frit. The yellow-greenish precipitate that was collected was
dissolved in a minimum amount of methanol, and the solution was
filtered on a filter paper. When hexanes was added to the concentrated
filtrate, the desired product precipitated. Compound 2 was obtained as
a dark yellow powder (0.144 g, 44%) after it was collected on a filter
paper and washed with hexanes.24 1H NMR (CD2Cl2, 500 MHz) δ
5.70 (s, C6H6, 6H, 2b), 5.88 (s, C6H6, 6H, 2a), 7.03 (s, CH, 1H, 2b),
7.30−7.42 (m, ArH, 8H, 1a or 1b), 7.37 (s, CH, 2H, 2a), 7.48−7.58
(m, ArH, 8H, 2a or 2b), 7.64−7.73 (m, ArH, 8H, 2a or 2b), 8.35 (s,
Htriazole, 2H, 2a), 8.40 (s, Htriazole, 1H, 2b), 8.88 (s, Htriazole, 1H, 2b),
10.47 (s, Htriazole, 2H, 2a). 13C{1H} NMR (CD2Cl2, 125 MHz, 2a and
2b): δ 66.5−66.6 (CH), 83.3−85.8 (C6H6), 113.1−113.3 (CArCN),
118.2−118.3 (CN), 129.1−129.8 (CAr), 132.7−133.1 (CAr), 141.0−
144.2 (CHCAr), 151.9−154.0 (Ctriazole). Found (%): C, 53.09; H, 3.44;
N, 14.83. C40H28Cl2N10Ru1·5H2O requires C, 52.70; H, 4.17; N, 15.37.
ESI-MS m/z (+): 785.1 M+ (or [Ru(C6H6)(η1-Let)2Cl]+), 1607.2 [M+
+ M+ + Cl−]+.
Figure 5. Mitochondrial metabolic activity determined by MTT assay,
in human breast cancer cells (MCF-7) (a) and glioblastoma cancer
cells (U251N) (b, c), treated with complex 4 or 5·Let cotreated with
3-methyladenine (1 mM) and curcumin (25 μM), respectively. The
insets display the results obtained upon cell exposure to 3-
methyladenine (3-MA) and curcumin without any Ru complex
■
under comparable conditions. Ru complex ( ), Ru complex + 3-
▲
●
methyladenine (1 mM) ( ), Ru complex + curcumin (25 μM) ( ).
Results for estrogen-deprived MCF-7 cells are plotted with open
symbols, and those for MCF-7 cells grown in the presence of serum
are plotted with filled symbols. All values are expressed as means (from
representative experiments)
SEM relative to control (100%).
Significant differences in the insets: ***p < 0.001 (compared with
#
untreated control) and p < 0.001 (compared with 3-MA treatment).
For complete statistical details, see Supporting Information (statistical
analysis section).
[Ru(C6H6)(η1-Let)2Cl]BF4, 3. Letrozole (0.114 g, 0.40 mmol) was
added to a degassed suspension of 1 (0.050 g, 0.10 mmol) and
NH4BF4 (0.052 g, 0.50 mmol) in ethanol (10 mL), and the mixture
was refluxed for 1 h. The mixture was allowed to cool to room
temperature and was then filtered. The precipitate was washed with
methanol, followed by hexanes. A minimum of dichloromethane was
added on the filter paper to dissolve the precipitate, and the filtrate was
been triggered by 5·Let. The same result was also noted for the
combination of complex 4 with 3-MA, against the same cell line
(results not shown). In the case of glioblastoma U251N cells,
the combination of complex 5·Let (0−100 μM) and 3-MA (1
mM) did not display any additive effect (Figure 5b), while
8803
dx.doi.org/10.1021/jm301103y | J. Med. Chem. 2012, 55, 8799−8806

Journal of Medicinal Chemistry
Article
Figure 6. Different pathways by which complexes 4 and 5·Let might induce cancer cell death.
collected in a separate flask. A dark yellow powder was obtained after
the product was precipitated with hexanes and collected on a filter
paper. Compound 3 was obtained as pale yellow crystals (0.130 g,
73%) from the diffusion of pentane into a concentrated dichloro-
methane solution of the powder. 1H NMR (CDCl3, 500 MHz): δ 5.91
(s, C6H6, 6H), 7.03 (s, CH, 2H), 7.23 (d, J = 8, ArH, 4H), 7.43 (d, J =
8, ArH, 4H), 7.52 (d, J = 8, ArH, 4H), 7.67 (d, J = 8, ArH, 4H), 8.22
(s, Htriazole, 2H), 9.37 (s, Htriazole, 2H). 13C{1H} NMR (CDCl3, 125
MHz): δ 66.9 (CH, 2C), 85.6 (C6H6, 6C), 113.5 (CArCN, 2C), 113.6
(CArCN, 2C), 117.8 (CN, 2C), 118.0 (CN, 2C), 129.1 (CAr, 4C),
129.2 (CAr, 4C), 132.9 (CAr, 4C), 133.1 (CAr, 4C), 140.8 (CHCAr, 4C),
147.6 (Ctriazole, 2C), 152.0 (Ctriazole, 2C). Found (%): C, 53.79; H, 2.98;
N, 14.98. C40H28B1F4Cl1N10Ru1·H2O requires C, 53.92; H, 3.37; N,
15.72. ESI-MS m/z (+): 785.1 M+ (or [Ru(C6H6)(η1-Let)2Cl]+),
ides from Escherichia coli O111:B4 (LPS), curcumin, antimycin A, and
3-methyladenine were purchased from Sigma-Aldrich. Estrogen
receptor positive MCF-7 cell line was donated by Dr. Ursula Stochaj
(Department of Physiology, McGill University, Montreal, Canada),
and U251N glioblastoma cell line was donated by Dr. Josephine
Nalbantoglu (Departments of Neurology & Neurosurgery and
Medicine, McGill University, Montreal, Canada).
Cell Culture and Media. U251N glioblastoma and MCF-7 breast
adenocarcinoma were routinely cultured in Dulbecco’s modified Eagle
medium (DMEM) (Gibco), supplemented with 10% fetal bovine
serum (FBS) (Gibco) and 1% penicillin/streptomycin (Gibco). In
addition, MCF-7 were supplemented with 1% sodium pyruvate
(Gibco). MCF-7 cells that were treated under estrogen-deprived
conditions were washed three times with 1× phosphate buffered saline
(PBS) and prestarved for 3 h in serum-free, phenol-red-free medium.
Their treatment was performed using the same medium. For all
experiments, cells were seeded in 24-well plate (Corning) or 96-well
plate (Sarstedt) at a density of 3.5 × 104 cells/well (U251N) or 5.0 ×
104 cells/well (MCF-7) and maintained at 37 °C, 5% CO2 in a
humidified atmosphere and were grown in serum containing media for
24 h before cell treatments to attain confluency.
−
1657.2 [M+ + M+ + BF4 ]+.
[Ru(C6H6)(η1-Let)2(PPh3)](BF4)(Cl), 5. Triphenylphosphine (0.040 g,
0.15 mmol) was added to a solution of 3 (0.100 g, 0.11 mmol) in
dichloromethane (6 mL), and the mixture was stirred at ambient
temperature for 48 h. The solvent was then concentrated, and the
product was purified by column chromatography (silica gel) using
dichloromethane, followed by acetone. Compound 5 (along with one
molecule of letrozole)16 was obtained as a light yellow powder (0.098
g, 75%). 1H NMR (CDCl3, 500 MHz): δ 5.79 (s, C6H6, 6H), 6.84 (s,
CH, 1H), 7.16 (s, CH, 1H), 7.22−7.38 (m, ArH, 21H), 7.57−7.73 (m,
ArH, 10H), 8.07 (br s, Htriazole, 1H), 8.18 (br s, Htriazole, 1H), 8.38 (s,
Htriazole, 1H), 9.30 (s, Htriazole, 1H). 13C{1H} NMR (CDCl3, 125 MHz):
δ 65.7 (s, CH, 1C), 66.6 (s, CH, 1C), 90.7 (d, J = 3, C6H6, 6C), 112.8
(s, CArCN, 1C), 113.1 (s, CArCN, 1C), 113.4 (s, CArCN, 2C), 118.0 (s,
Ruthenium Drug Solutions. Ruthenium drug 2a, 3, 4 and 5·Let
stock solutions (1 mM) were prepared using dimethylsulfoxide
(DMSO). Each stock solution was diluted in culture medium to
obtain working concentrations. The final (DMSO) concentration
never exceeded 0.5%, which was not toxic to the cells under the
conditions used.
Determination of Mitochondrial Metabolic Activity (MTT
Assay). Mitochondrial metabolic activity of cells was measured using
MTT assay. Cells were preliminarily treated with ruthenium
complexes 2a, 3, 4, and 5·Let solutions (in culture medium) at
concentrations between 0.01 and 100 μM. Based on preliminary
results, further experiments were performed at 10, 25, 50, 75, and 100
μM. Experiments were performed 24, 48, or 72 h after each treatment.
After the treatments were performed, medium was removed and
replaced with medium containing 500 μg/mL MTT. The cells were
then incubated for 30−90 min at 37 °C in order for the formazan salts
to form. Medium was removed, and cells were lysed using 500 μL of
DMSO and mixed gently for 5 min. The dissolved formazan crystals
were added in triplicate in a clear bottom 96-well plate (Sarstedt) and
quantified by measuring the absorbance of the solution at 595 nm
using Benchmark microplate reader (Bio-Rad). The extent of
formazan conversion is expressed in percentage relative to the
CN, 2C), 118.2 (s, CN, 1C), 118.3 (s, CN, 1C), 129.0 (d, J = 10, CPPh3
,
6C), 129.1 (s, CAr, 4C), 129.1 (s, CAr, 8C), 129.9 (s, CAr, 4C), 131.6 (d,
J = 2, CPPh3, 3C), 132.7 (d, J = 14, CPPh3, 3C), 133.1 (s, CAr, 8C), 133.8
(d, J = 10, CPPh3, 6C), 141.3 (s, CHCAr, 1C), 141.7 (s, CHCAr, 1C),
141.9 (s, CHCAr, 2C), 149.3 (s, Ctriazole, 2C), 152.1 (s, Ctriazole, 2C);
31P{1H} NMR (CDCl3, 200 MHz): δ 35.0; Found (%): C, 58.77; H,
3.54; N, 11.95. C58H43B1F4Cl1P1N10Ru1·3H2O requires C, 58.56; H,
4.12; N, 11.78. ESI-MS m/z (+): 762.1 M+ (or [Ru(C6H6)(η1-
−
Let)(PPh3)Cl]+), 1611.2 [M+ + M+ + BF4 ]+.
X-ray Diffraction Studies. Suitable crystals for X-ray diffraction data
were obtained by slow diffusion of pentane into concentrate
dichloromethane/chloroform-d solutions of 2a, 3, and 5·Let,
respectively. Data were collected at 150 K using a Bruker SMART
APEX II CCD X-ray diffractometer. Structure resolution and
refinement were performed with SHELXTL. H atoms were calculated
and constrained as riding on their bound atoms. CCDC 890790 and
890791 (2b and 3) contain the supplementary crystallographic data for
this paper. These data can be obtained free of charge via www.ccdc.
cam.ac.uk/data_request/cif or by emailing data_request@ccdc.cam.ac.
uk or by contacting The Cambridge Crystallographic Data Centre, 12,
Union Road, Cambridge CB2 1EZ, UK; fax +44 1223 336033.
Materials and Methods for Biology Experiments. 1-(4,5-
Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), lipopolysacchar-
untreated control. Results are expressed as mean
SEM obtained
from at least three independent experiments performed in triplicate.
Determination of Cell Death by Counting. Cell viability was
confirmed by cell counting using Hoechst 33258 (Sigma) Nuclear
Staining. Following treatments in 24-well plates, cells were fixed and
labeled with the fluorescent dye. Five images were taken per well using
Leica Application Suite with Leica DFC350FX monochrome digital
camera on Leica DM 4000B inverted fluorescent microscope with
8804
dx.doi.org/10.1021/jm301103y | J. Med. Chem. 2012, 55, 8799−8806

Journal of Medicinal Chemistry
Article
DAPI-1160A filter and 20× objective. The cells were subsequently
counted using ImageJ with Cell Counter plugin.
(4) Bergamo, A.; Sava, G. Ruthenium Anticancer Compounds: Myths
and Realities of the Emerging Metal-Based Drugs. Dalton Trans. 2011,
40, 7817−7823.
Statistical Analysis. All data are presented as the means
standard errors of the mean (SEM). A Student unpaired t test or
ANOVA coupled to Bonferroni correction were used for analyzing the
significance of the difference between the means. A p-value <0.05 (*)
was considered statistically significant. All experiments were performed
at least two or three times.
́ ́
(5) (a) Brabec, V.; Novakova, O. DNA Binding Mode of Ruthenium
Complexes and Relationship to Tumor Cell Toxicity. Drug Resist.
Updates 2006, 9, 111−122. (b) Brabec, V. DNA Modifications by
Antitumor Platinum and Ruthenium Compounds: Their Recognition
and Repair. Prog. Nucleic Acid Res. Mol. Biol. 2002, 71, 1−68.
(6) (a) Pongratz, M.; Schluga, P.; Jakupec, M. A.; Arion, V. B.;
Hartinger, C. G.; Allmaier, G.; Keppler, B. K. Transferrin Binding and
Transferrin-Mediated Cellular Uptake of the Ruthenium Coordination
Compound KP1019, Studied by Means of AAS, ESI-MS and CD
Spectroscopy. J. Anal. At. Spectrom. 2004, 19, 46−51. (b) Kapitza, S.;
Pongratz, M.; Jakupec, M. A.; Heffeter, P.; Berger, W.; Lackinger, L.;
Keppler, B. K.; Marian, B. Heterocyclic Complexes of Ruthenium(III)
Induce Apoptosis in Colorectal Carcinoma Cells. J. Cancer Res. Clin.
2005, 131, 101−110.
(7) (a) Clarke, M. J. Ruthenium Metallopharmaceuticals. Coord.
Chem. Rev. 2003, 236, 209−233. (b) Clarke, M. J.; Bitler, S.; Rennert,
D.; Buchbinder, M.; Kelman, A. D. Reduction and Subsequent Binding
of Ruthenium Ions Catalyzed by Subcellular Components. J. Inorg.
Biochem. 1980, 12, 79−87.
(8) (a) Ghosh, D.; Griswold, J.; Erman, M.; Pangborn, W. X-ray
Structure of Human Aromatase Reveals an Androgen-Specific Active
Site. J. Steroid Biochem. 2010, 118, 197−202. (b) Ghosh, D.; Griswold,
J.; Erman, M.; Pangborn, W. Structural Basis for Androgen Specificity
and Oestrogen Synthesis in Human Aromatase. Nature 2009, 457,
219−223.
CONCLUSIONS
■
This study provides novel letrozole−ruthenium complexes as
chemotherapeutic agents. Letrozole coordinates to ruthenium-
(II) in a monodentate fashion via one nitrogen atom of the
triazole ring, similar to the coordination mode observed for
copper(II), nickel(II), or cobalt(II). However, this coordination
mode is different than the one noted for copper(I), where
letrozole binds to the metal by its four nitrogen atoms.
Letrozole ligand lability was found to be enhanced by the
coordinative nature of the complex counterion and by the
presence of alkyl substituents on the arene ring. Biological
experiments show that 5·Let is an effective cytotoxic agent
against the human breast cancer cells (MCF-7) and also,
although to a much smaller extent, against human glioblastoma
cells (U251N). The activity of 5·Let was significantly higher
than that of 4, a compound analogous to RAPTA-C, a
ruthenium drug presently in preclinical studies. Results from
the combined exposure of cancer cells to 5·Let (or 4) and 3-
methyladenine (3-MA) or curcumin point toward the critical
role of autophagy contributing to the 5·Let-induced cell death.
Taken together, these studies show the effectiveness of novel
Ru−letrozole complexes in significantly reducing viability of
different human cancer cell types.
(9) Buzdar, A. U. Phase III Study of Letrozole versus Tamoxifen as
First-Line Therapy of Advanced Breast Cancer in Postmenopausal
Women: Analysis of Survival and Update of Efficacy from the
International Letrozole Breast Cancer Group. J. Clin. Oncol. 2004, 22,
3199−3200.
(10) Wen, P. Y.; Kesari, S. Malignant Gliomas in Adults. New Engl. J.
Med. 2008, 359, 492−507.
(11) Yuan, R.-X.; Xiong, R.-G.; Abrahams, B. F.; Lee, G.-H.; Peng, S.-
M.; Che, C.-M.; You, X.-Z. A Cu(I) Coordination Polymer Employing
a Nonsteroidal Aromatase Inhibitor Letrozole as a Building Block. J.
Chem. Soc., Dalton Trans. 2001, 2071−2073.
(12) Tang, Y.-Z.; Zhou, M.; Huang, J.; Cao, Z.; Qi, T.-T.; Huang, G.-
H.; Wen, H.-R. Synthesis, Crystal Structure, and Characterization of
three New Letrozole Complexes. Z. Anorg. Allg. Chem. 2012, 638,
372−376.
ASSOCIATED CONTENT
■
S
* Supporting Information
Supplementary experimental details, Figure S1 showing results
of MTT assay for MCF-7 cells, and statistical details for Figure
5. This material is available free of charge via the Internet at
http://pubs.acs.org.
(13) X-ray diffraction studies carried out on crystals of 2a and 5 have
confirmed the empirical formulas and the connectivities in these
compounds, but the poor quality of the data obtained does not allow
firm commentary on the structural details.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: dusica.maysinger@mcgill.ca. Phone: 1-514-398-1264.
(14) Nazarov, A. A., Dyson, P. J. Phosphorus Compounds: Advanced
Tools in Catalysis and Material Sciences; Peruzzini, M., Gonsalvi, L,
Eds.; Catalysis by Metal Complexes series, Vol. 37; Springer: London,
2011.
(15) (a) Romerosa, A.; Campos-Malpartida, T.; Lidrissi, C.; Saoud,
́
M.; Serrano-Ruiz, M.; Peruzzini, M.; Garrido-Cardenas, J. A.; García-
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by NSERC (Natural Sciences and
Engineering Research Counsil) and CIHR (Canadian Institutes
of Health Research, Grants MOP 89995 and MOP 119425).
We would like to thank Dr. A. Moores for providing laboratory
facilities, and Dr. J. Diamond for editorial assistance.
Maroto, F. Synthesis, Characterization, and DNA Binding of New
Water-Soluble Cyclopentadienyl Ruthenium(II) Complexes Incorpo-
rating Phosphines. Inorg. Chem. 2006, 45, 1289−1298. (b) Scolaro, C.;
Chaplin, A. B.; Hartinger, C. G.; Bergamo, A.; Cocchietto, M.;
Keppler, B. K.; Sava, G.; Dyson, P. J. Tuning the Hydrophobicity of
Ruthenium(II)-Arene (RAPTA) Drugs to Modify Uptake, Biomo-
lecular Interactions and Efficacy. Dalton Trans. 2007, 5065−5072.
(16) The free letrozole drug was not separated from the newly
formed complex 5, and this is the reason why the 5·Let notation is
used throughout the text. A small difference in the chemical shifts of
REFERENCES
■
(1) (a) Jemal, A.; Siegel, R.; Ward, E.; Hao, Y.; Xu, J.; Murray, T.;
Thun, M. J. Cancer Statistics, 2008. CaCancer J. Clin. 2008, 58, 71−
96.
1
peaks corresponding to the free letrozole drug was noted in the H
(2) Szakacs, G.; Paterson, J. K.; Ludwig, J. A.; Booth-Genthe, C.;
Gottesman, M. M. Targeting Multidrug Resistance in Cancer. Nat. Rev.
Drug Discovery 2006, 5, 219−234.
(3) Alderden, R. A.; Hall, M. D.; Hambley, T. W. The Discovery and
Development of Cisplatin. J. Chem. Educ. 2006, 83, 728−734.
NMR spectrum of the complex 5·Let mixture, and this small
ruthenium−letrozole interaction might explain why the free drug
remained with the complex in solution, even after extensive washings
of the complex with solvents in which letrozole is soluble and after
8805
dx.doi.org/10.1021/jm301103y | J. Med. Chem. 2012, 55, 8799−8806

Journal of Medicinal Chemistry
Article
column chromatography. The NMR data reported for complex 5 in
the experimental section includes the extra letrozole molecule.
(17) (a) Caruso, F.; Rossi, M.; Benson, A.; Opazo, C.; Freedman, D.;
Monti, E.; Gariboldi, M. B.; Shaulky, J.; Marchetti, F.; Pettinari, R.;
Pettinari, C. Ruthenium−Arene Complexes of Curcumin: X-Ray and
Density Functional Theory Structure, Synthesis, and Spectroscopic
Characterization, in Vitro Antitumor Activity, and DNA Docking
Studies of (p-Cymene)Ru(curcuminato)chloro. J. Med. Chem. 2012,
55, 1072−1081. (b) Garcia-Fernandez, A.; Diez, J.; Manteca, A.;
Sanchez, J.; Garcia-Navas, R.; Sierra, B. G.; Mollinedo, F.; Gamasa, M.
P.; Lastra, E. Antitumor Activity of New Hydridotris(pyrazolyl)borate
Ruthenium(II) Complexes Containing the Phosphanes PTA and 1-
CH3-PTA. Dalton Trans 2010, 39, 10186−10196. (c) Ludwig, G.;
́
Kaluđerovic, G. N.; Bette, M.; Block, M.; Paschke, R.; Steinborn, D.
Highly Active Neutral Ruthenium(II) Arene Complexes: Synthesis,
Characterization, And Investigation of Their Anticancer Properties. J.
Inorg. Biochem. 2012, 113, 77−82.
(18) Bergamo, A.; Zorzet, S.; Gava, B.; Sorc, A.; Alessio, E.; Iengo, E.;
Sava, G. Effects of NAMI-A and Some Related Ruthenium Complexes
on Cell Viability after Short Exposure of Tumor Cells. Anti-Cancer
Drug. 2000, 11, 665−672.
(19) Lockshin, R. A.; Zakeri, Z. Apoptosis, Autophagy, and More. Int.
J. Biochem. Cell Biol. 2004, 36, 2405−2419.
(20) Maiuri, M. C.; Zalckvar, E.; Kimchi, A.; Kroemer, G. Self-Eating
and Self-Killing: Crosstalk between Autophagy and Apoptosis. Nat.
Rev. Mol. Cell Biol. 2007, 8, 741−752.
(21) (a) Rodriguez-Rocha, H.; Garcia-Garcia, A.; Panayiotidis, M. I.;
Franco, R. DNA Damage and Autophagy. Mutat. Res., Fundam. Mol.
Mech. Mutagen. 2011, 711, 158−166. (b) Rabinowitz, J. D.; White, E.
Autophagy and Metabolism. Science 2010, 330, 1344−1348.
(22) Tan, C.; Lai, S.; Wu, S.; Hu, S.; Zhou, L.; Chen, Y.; Wang, M.;
Zhu, Y.; Lian, W.; Peng, W.; et al. Nuclear Permeable Ruthenium(II)
β-Carboline Complexes Induce Autophagy To Antagonize Mitochon-
drial-Mediated Apoptosis. J. Med. Chem. 2010, 53, 7613−7624.
(23) Aoki, H.; Takada, Y.; Kondo, S.; Sawaya, R.; Aggarwal, B. B.;
Kondo, Y. Evidence That Curcumin Suppresses the Growth of
Malignant Gliomas in Vitro and in Vivo through Induction of
Autophagy: Role of Akt and Extracellular Signal-Regulated Kinase
Signaling Pathways. Mol. Pharmacol. 2007, 72, 29−39.
(24) The resonances for complex 2a and 2b are both reported here
(equimolar mixture), and the spectra also showed the presence of an
equivalent of free letrozole, which is not reported (for letrozole NMR
data, see Supporting Information).
8806
dx.doi.org/10.1021/jm301103y | J. Med. Chem. 2012, 55, 8799−8806