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Synthesis, Characterization, Cytotoxic Activity, and Metabolic Studies of Ruthenium(II) Polypyridyl Complexes Containing Flavonoid Ligands.
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Article
Synthesis, Characterization, Cytotoxic Activity, and Metabolic
Studies of Ruthenium(II) Polypyridyl Complexes Containing
Flavonoid Ligands
Alexandra-Cristina Munteanu,# Anna Notaro,# Marta Jakubaszek, Joseph Cowell, Mickaël Tharaud,
Bruno Goud, Valentina Uivarosi, and Gilles Gasser*
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ABSTRACT: Four novel monocationic Ru(II) polypyridyl
complexes were synthesized with the general formula [Ru(DIP)2flv]X, where DIP is 4,7-diphenyl-1,10-phenanthroline, flv
stands for the flavonoid ligand (5-hydroxyflavone in [Ru(DIP)2(5OHF)](PF6), genistein in [Ru(DIP)2(gen)](PF6), chrysin in
[Ru(DIP)2(chr)](OTf), and morin in [Ru(DIP)2(mor)](OTf)),
and X is the counterion, PF−6 , and OTf ̅ (triflate, CF3SO3̅),
respectively. Following the chemical characterization of the
complexes by 1H and 13C NMR, mass spectrometry, and elemental
analysis, their cytotoxicity was tested against several cancer cell
lines. The most promising complex, [Ru(DIP)2(gen)](PF6), was
further investigated for its biological activity. Metabolic studies
revealed that this complex severely impaired mitochondrial
respiration and glycolysis processes, contrary to its precursor, Ru(DIP)2Cl2, which showed a prominent effect only on the
mitochondrial respiration. In addition, its preferential accumulation in MDA-MB-435S cells (a human melanoma cell line previously
described as mammary gland/breast; derived from metastatic site: pleural effusion), which are used for the study of metastasis,
explained the better activity in this cell line compared to MCF-7 (human, ductal carcinoma).
■
INTRODUCTION
Cancer, listed as a chronic degenerative noncommunicable
disease by the World Health Organization (WHO), is a leading
cause of death worldwide.1 Despite the clinical success of
several platinum-based drugs (e.g., cisplatin, carboplatin, and
oxaliplatin),2 their efficacy is impeded by intrinsic and acquired
resistance and dose-limiting toxicity.3 Therefore, the search for
more effective therapeutic strategies has led to the development of other metal complexes with anticancer properties.4
Ruthenium (Ru)-based compounds have emerged as potential
anticancer drug candidates due to their unique physicochemical and biological properties,5−8 generally lower systemic
toxicity (in animal models), and higher cellular uptake
compared to platinum complexes.5 NAMI-A,9,10 KP1019,11,12
and its water-soluble sodium salt IT-139 (formerly KP1339)13
are Ru complexes that have been evaluated in clinical trials as
chemotherapeutic agents for the treatment of cancer. NAMI-A
is an antimetastatic drug candidate with diverse mechanisms of
action.14−17 Unfortunately, during a phase I/II study, its
clinical activity was found to be disappointing, which led to the
discontinuation of the trials. These poor results were mainly
attributed to dose-limiting adverse events associated with the
treatments.10
© XXXX American Chemical Society
Therefore, current trends in the development of novel Rubased anticancer drug candidates aim to meet the need for
more efficient treatments and improved toxicological profiles
for the emergent drugs. For instance, Ru(II) polypyridyl
complexes have shown great potential,18,19 finding applications
in tumor diagnosis,20 as antineoplastic agents,19,21 and as
photosensitizers for photodynamic therapy (PDT).22,23 The
most successful compound bearing a Ru(II) polypyridyl
scaffold, TLD-1433,24 has recently entered phase II clinical
studies as a photosensitizer for intravesical PDT against
bladder cancer.25,26
Moreover, very interesting results have been found for
heteroleptic complexes of Ru(II), bearing an O,O-chelating
ligand. For instance, RAPTA complexes with curcuminoid
ligands (IC50 values ≤1 μM) displayed novel binding modes
with biomolecular targets and high cancer cell selective
Received: December 6, 2019
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Figure 1. Chemical structures of flavonoids 5-hydroxyflavone, chrysin, genistein, and morin.
Scheme 1. Synthesis of Complexes of the Type [Ru(DIP)2(flv)]Xa
a
Where flv = flavonoid and X = counterion. (I) DIP, LiCl, DMF, reflux, 24 h, 78%. (II) (i) NaOH, 5-hydroxyflavone, ethanol, reflux, 2 h; (ii)
NH4PF6, ethanol/H2O (1:10), 25%. (III) (i) silver triflate, ethanol, RT, 1h. (IV) sodium ethoxide, genistein, ethanol, reflux, 2 h; (ii) NH4PF6,
ethanol/H2O (1:10), 13%. (V) sodium ethoxide, chrysin, ethanol, reflux, 2 h, 16%. (VI) NEt3, TMSBr, THF, RT, 1h. VII) sodium ethoxide,
Ru(DIP)2(OTf)2, ethanol, reflux, 2 h, 35%.
with the flavonoids shown in Figure 1 as O,O-chelating ligands.
Flavonoids are a naturally occurring subclass of polyphenols
with high structural versatility.29 They have been extensively
studied in the design of novel anticancer drug candidates. As a
result, two derivatives of the flavonoid chrysin (Figure 1),
namely flavopiridol and P276-00, have entered clinical
activity.27 In addition, RuII(η6-p-cymene) complexes with
flavonol-derived ligands were found to have potent cytotoxic
activity against several human cancer cell lines with IC50 values
in the low micromolar range.28
These recent discoveries have prompted us toward the study
of the therapeutic potential of Ru(II) polypyridyl complexes
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Table 1. IC50 Values for Flavonoid Ligands, Cisplatin, Doxorubicin, [Ru(DIP)2(5-OHF)](PF6), [Ru(DIP)2(gen)](PF6),
[Ru(DIP)2(chr)](OTf), [Ru(DIP)2(mor)](OTf), and Ru(DIP)2Cl2 in Different Cell Linesa
IC50 (μM)
compound
MCF-7
FaDU
MDA-MB-435S
U87
RPE-1
HEK293
5-hydroxyflavone
genistein
chrysin
morin
cisplatin
doxorubicin
Ru(DIP)2Cl2
[Ru(DIP)2(5-OHF)](PF6)
[Ru(DIP)2(gen)](PF6)
[Ru(DIP)2(chr)](OTf)
[Ru(DIP)2(mor)](OTf)
>100
>100
62.59 ± 3.23
>100
19.69 ± 1.63
9.39 ± 1.37
>50
>50
16.67 ± 3.93
>50
>50
>100
>100
95.06 ± 11.55
>100
5.17 ± 0.21
1.55 ± 0.18
>50
38.21 ± 5.22
5.21 ± 0.73
>50
>50
>100
>100
79.37 ± 8.13
>100
17.62 ± 0.54
5.55 ± 1.37
27.73 ± 5.33
24.48 ± 1.92
2.64 ± 0.43
27.73 ± 5.33
>50
>100
>100
91.14 ± 13.76
>100
6.94 ± 0.46
0.59 ± 0.03
25.59 ± 0.29
30.72 ± 1.48
5.21 ± 1.74
25.59 ± 0.29
>50
>100
>100
>100
>100
39.9 ± 9.14
14.9 ± 1.31
3.13 ± 0.28
19.72 ± 8.23
2.36 ± 0.77
23.21 ± 8.08
>50
>100
75.85 ± 0.84
26.80 ± 2.79
>100
2.27 ± 0.67
0.21 ± 0.03
12.11 ± 1.30
26.46 ± 3.20
0.72 ± 0.10
33.02 ± 3.25
>50
a
48 h treatment.
trials.30,31 Although not yet fully understood, the cytotoxic
activity of flavonoids is believed to rely upon the modulation of
cellular processes that include proliferation, differentiation,
apoptosis, metastasis, and oxidative stress.29,32,33 Moreover,
naturally occurring flavonoid aglycons display exceptionally
low, if any, systemic toxicity. It should be noted, however, that
the absence of acute toxic effects is related to their low water
solubility and bioavailability.34,35
The present work focuses on the synthesis of four novel
monocationic Ru(II) polypyridyl complexes with the general
formula [Ru(DIP)2flv]X, where DIP is 4,7-diphenyl-1,10phenanthroline, flv stands for the flavonoid ligand (5hydroxyflavone in [Ru(DIP)2(5-OHF)](PF6), genistein in
[Ru(DIP)2(gen)](PF6), chrysin in [Ru(DIP)2(chr)](OTf),
and morin in [Ru(DIP)2(mor)](OTf)), and X is the
counterion (PF6̅ or OTf ̅ (triflate)). Following the successful
synthesis and characterization, the antiproliferative activity of
the complexes was tested against different cell lines. For the
most potent compound of the series, metabolic studies were
performed and compared with the Ru(DIP)2Cl2 precursor.
complexes of 5-OHF, genistein, and chrysin, where the
flavonoids coordinate via the 4,5-O,O site, the selective
protection of the oxygen atoms at the 3, 7, 2′, and 4′ positions
was necessary.
Therefore, the synthesis of [Ru(DIP)2(mor)](OTf) involved an additional protection step shown in Scheme 1.
Following a similar procedure to Qi et al.,42 the selective
protection at the 2′, 4′, 3, and 7 positions with trimethylsilyl
(TMS) protecting group was achieved. The protection step
was performed in the presence of triethylamine and TMS-Br in
THF and, following an aqueous workup, the protected morin
was used in the complexation step without any further
purification. The complexation reaction was performed as
described above. Interestingly, during the course of the
complexation reaction, the TMS protecting groups were
hydrolyzed, negating the need for a deprotection step.
Following the successful synthesis of [Ru(DIP)2(mor)](OTf),
coordination at the 4, 5-O,O site was confirmed by 1D and 2D
NMR studies. It was noticed during the course of the NMR
experiments that [Ru(DIP)2(mor)](OTf) exists as a mixture
of two isomers in solution. The second isomer is presumed to
be the result of the morin binding via the 3,4-O,O site. The rate
of isomerization between the two isomers, however, is slow,
with approximately 25% of the 3,4-O,O complex being visible
by 1H NMR after 5 days in solution (Figure S5). It should be
noted that [Ru(DIP)2(mor)](OTf) is stable for over 6 months
if stored as a powder at −20 °C.
The identity of the compounds was confirmed by ESI-MS
and NMR spectroscopy (Figures S1−S9), and their purity was
confirmed by microanalysis. All complexes are chiral and were
isolated as a racemic mixture of Δ and Λ enantiomers. No
attempt to obtain enantiopure complexes was made in this
work. All four complexes are stable in the solid state and
soluble in methanol, DCM, DMSO, and DMF and moderately
soluble in acetone and acetonitrile. Because the stability and
aggregation of metal-based drug candidates is an important
parameter, stability studies were undertaken.43−45 Preliminary
studies (Figures S10−S13) showed that [Ru(DIP)2(5-OHF)](PF6), [Ru(DIP)2(gen)](PF6), and [Ru(DIP)2(chr)](OTf)
are stable in DMSO over 5 days. The stability of [Ru(DIP)2(mor)](OTf), on the other hand, was tested in DMF
due to the slower isomerization rate when compared to that in
DMSO. Taking this into account, NMR analysis in DMF over
5 days shows no degradation of the product (Figure S13).
■
RESULTS AND DISCUSSION
Synthesis and Characterization of the Ru(II) Complexes. The synthesis of the Ru(II) complexes was achieved in
a 2-step process for [Ru(DIP)2(5-OHF)](PF6), a 3-step
process for [Ru(DIP)2(gen)](PF6) and [Ru(DIP)2(chr)](OTf), and a 4-step process for [Ru(DIP)2(mor)](OTf)
(Scheme 1). Briefly, RuCl2(dmso)4,36 DIP, and LiCl were
refluxed in DMF to afford Ru(DIP)2Cl2 in a 72% yield after
precipitation with acetone.37 Ru(DIP)2Cl2 was then refluxed in
a nitrogen atmosphere for 1.5−2 h with the appropriate
flavonoid in the presence of sodium ethoxide in dry ethanol.
Complexes [Ru(DIP)2(5-OHF)](PF6) and [Ru(DIP)2(gen)](PF6) (25 and 13%, respectively) were obtained after
precipitation with a large excess of NH4PF6 and further
purification. Complexes [Ru(DIP)2(chr)](OTf) and [Ru(DIP)2(mor)](OTf) (16 and 35%, respectively) were obtained
via a ruthenium triflate intermediate. Briefly, Ru(DIP)2Cl2 and
silver triflate were stirred to afford [Ru(DIP)2(OTf)2], and the
appropriate flavonoid was added after filtration of AgCl in the
presence of sodium ethoxide.
It is noteworthy that morin bears three possible coordination sites (Figure 1), and literature data suggest that the
preferred binding site of metal ions to morin is the 3,4-O,O
site.38−41 Therefore, to allow for comparison to the Ru(II)
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Figure 2. ICP-MS data of cellular uptake of tested compounds in MDA-MB-435S and MCF-7 cell lines. (a) Total cellular accumulation (2 h
treatment, 5 μM). (b) Time-dependent cellular accumulation in MDA-MB-435S cell line. (c) Time-dependent cellular accumulation in the MCF-7
cell line. (d) Mechanism of cellular uptake of Ru(DIP)2Cl2 in tested cell lines (2 h treatment, 5 μM). (e) Mechanism of cellular uptake of
[Ru(DIP)2(gen)](PF6) in tested cell lines (2 h treatment, 5 μM). Data of (a), (d), and (e) are presented as the mean ± SD of at least 3 technical
replicates. Data of (b) and (c) are presented as the mean ± SD of at least 3 biological replicates.
Cytotoxicity, Cellular Uptake, and Metabolic Studies.
The biological activity of the complexes was tested on MDAMB-435S (human, melanoma), FaDU (human, pharynx
carcinoma), MCF-7 (human, ductal carcinoma), U87
(human, glioblastoma), RPE-1 (human, normal retinal
pigmented epithelium), and HEK 293 (human embryonic
kidney) cell lines using a fluorometric cell viability assay.46
Cisplatin and doxorubicin were tested in the same conditions
as positive controls.47,48 Ru(DIP)2Cl2 as well as the flavonoids
5-hydroxyflavone, genistein, chrysin, and morin were used as
additional controls. The IC50 (half maximal inhibitory
concentration) values obtained in this study are reported in
Table 1 (all cytotoxicity graphs are available in Figure S14).
The literature cites good to excellent cytotoxic activity for
other 5-hydroxyflavone, chrysin, and morin metal complexes,41,49−52 results that prompted us to the design of
these compounds. It is noteworthy that complexes of morin
(bound via the 3,4-O,O site) and chrysin bearing a Ru(II)
polypyridyl scaffold have been previously reported. Their
cytotoxic activity was studied on HeLa (cervical carcinoma),
SW620 (colorectal adenocarcinoma, metastatic), HepG2
(hepatocellular carcinoma), and MCF-7 cell lines with IC50
values ranging from 7.64 to >100 μM.41 [Ru(DIP)2(mor)](OTf), however, was found to be essentially nontoxic, with
IC50 values above 50 μM in all cell lines tested, while
[Ru(DIP)2(5-OHF)](PF6) and [Ru(DIP)2(chr)](OTf) exerted moderate toxicity toward some of the cell lines tested.
Interestingly, the most promising complex identified in this
study is the complex bearing the flavonoid genistein,
([Ru(DIP)2(gen)](PF6)), with IC50 values comparable to
those of both cisplatin and doxorubicin. Genistein is
considered a suitable lead for anticancer drug development,
and derivatives have been synthesized to enhance its cytotoxic
activity.53−57 It should be stated that among all chemical
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Figure 3. (a) Mito stress test profile in MDA-MB-435S cells after 24 h treatment. Oxygen consumption rate changes after treatment with specific
electron transport chain inhibitors. Oligomycin (inhibitor of ATP synthase (complex V)), FCCP (uncoupling agent), antimycin-A (complex III
inhibitor), and rotenone (complex I inhibitor). (b) Glycolysis stress test profile in MDA-MB-435S cells after 24 h treatment. Extracellular
acidification rate that corresponds to the glycolysis process changes after treatment with glucose (basal level of glycolysis in cells), oligomycin
(inhibitor of ATP synthase (complex V), mitochondria inhibition), 2-deoxyglucose (analogue of glucose that inhibits glycolytic pathway).
cellular accumulation in the two cell lines tested. The obtained
results confirm previous conclusions that all tested compounds
accumulate more in the MDA-MB-435S cell line than in MCF7 cells. After 24 h incubation time, a similar uptake of
Ru(DIP)2Cl2 and [Ru(DIP)2(gen)](PF6) was found in MDAMB-435S (∼30 ng of metal in 106 cells) in comparison with
cisplatin (∼4 ng of metal in 106 cells). On the other hand,
[Ru(DIP)2(gen)](PF6) accumulates much more in MCF-7
cells than the two other compounds after 24 h (∼2 ng of metal
in 106 cells as compared to ∼1 ng) and 48 h (∼5 ng of metal in
106 cells compared to ∼1 ng). Notably, there is a discrepancy
between the amount of metal detected in the total
accumulation and the time dependent accumulation experiments in both cell lines at the 2 h time point (shown in Figures
2a−c). This can be explained by the different mechanisms of
uptake of the Ru complexes (see below) and the availability of
the complexes in cellular media (5 times lower concentration
of the compounds in the time dependent experiments).
To understand the nature of the mechanism of uptake
(passive or active) of the tested complexes, cells were
pretreated with various inhibitors or kept at different
temperatures. A temperature of 4 °C was used to slow passive
diffusion, as well as active transportation. To block cellular
metabolism, pretreatments with ATP production inhibitors 2deoxy-D-glucose and oligomycin were performed. Chloroquine
or ammonium chloride (NH4Cl) impede endocytic pathways,
and tetraethylammonium chloride stops the cation transporters. Following pretreatments, cells were incubated with
[Ru(DIP)2(gen)](PF6) or Ru(DIP)2Cl2 (2 h, 5 μM) and
subsequently analyzed via ICP-MS (Figures 2d and 2e).
Inhibition of active uptake mechanisms did not significantly
perturb accumulation of [Ru(DIP)2(gen)](PF6) in both cell
lines tested, demonstrating that the mechanism responsible for
its accumulation is energy independent (passive). On the other
hand, Ru(DIP)2Cl2 is taken up via a passive mechanism by the
MCF-7 cell line and an active mechanism by the MDA-MB435S cell line. As shown for other similar ruthenium
complexes, this observation indicates that slight changes in
lipophilic properties and structure play a decisive role in the
cellular uptake of Ru(II) polypyridyl complexes.61−63
To better understand the effect of the flavonoid complex of
interest on the cellular metabolism of MDA-MB-435S cells, a
Seahorse XF Analyzer was used. This device allows for the real
time measurement of the oxygen consumption rate (OCR)
and extracellular acidification rate (ECAR) in cells. First, the
derivatives of genistein, scarce data exist regarding its metal
complexes. For instance, a homoleptic copper(II) genistein
complex was reported to enhance the cytotoxic activity of the
ligand against four cancer cell lines, including 518A2
melanoma and MCF-7/Topo breast carcinoma cell lines.52
Unfortunately, [Ru(DIP)2(gen)](PF6) exerted no selectivity
between cancerous and noncancerous cell lines with
comparable IC50 values. However, this drawback is commonly
faced in medicinal chemistry and could be improved by the
introduction of a targeting moiety.
[Ru(DIP)2(gen)](PF6) showed good activity toward the
MDA-MB-435S cell line, with an IC50 of 2.64 μM. Currently,
this cell line is identified as a melanoma cell line, which derives
from the pleural effusion of a 31-year-old female with
metastatic, ductal adenocarcinoma of the breast and considered still valuable for the study of metastasis.58,59 The lower
activity expressed by the complex toward the MCF-7 cell line
(IC50 = 16.67 μM) led us to study the cellular uptake and
mechanism of uptake of this complex in two different cell lines
derived from breast tissue. In these experiments, cells were
treated with 5 μM of [Ru(DIP)2(gen)](PF6) for 2 h, and the
metal content was analyzed via inductively coupled plasma
mass spectrometry (ICP-MS). Cisplatin and Ru(DIP)2Cl2
were tested in the same conditions as controls. The viability
of the cells after the 2 h treatment was additionally tested,
confirming that the acquired results were obtained from living
cells (Figure S14). Figure 2a shows that the cellular uptake is
much lower for the MCF-7 cell line when compared to MDAMB-435S for all the tested compounds. Interestingly, Ru(DIP)2Cl2 accumulates more in MDA-MB-435S compared to
[Ru(DIP)2(gen)](PF6), in the same cell line, but shows lower
cytotoxicity than the flavonoid complex. This observation can
be rationalized by the explanation provided by Policar et al. in
2014 where they state that IC50 is a resultant value of cellular
uptake, interaction with cellular target, and its intrinsic
toxicity.60 Therefore, one could argue that the higher activity
expressed by [Ru(DIP)2(gen)](PF6) toward MDA-MB-435S
when compared to MCF-7 cells comes as a consequence of its
higher cellular uptake. To understand the kinetics of the tested
compounds in the chosen cell lines, we performed timedependent accumulation experiments. Ruthenium and platinum contents in treated cells were measured by ICP-MS after
2, 12, 24, and 48 h. In this analysis, the concentration of the
tested compounds was decreased to 1 μM to reduce cell loss
during the experiment. Figures 2b and 2c show the changes in
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studies, which will aim to identify the cellular targets of the
complex and possible interactions with protein transporters.
Because the current treatment of advanced melanoma provides
modest results, this work may open new opportunities in the
search for chemopreventive and/or chemotherapeutic agents
for human cancers, especially melanoma.
influence on the oxidative phosphorylation was measured. As
shown in Figures 3a and S15, 24 h treatment with flavonoid
complex [Ru(DIP)2(gen)](PF6) and its precursor Ru(DIP)2Cl2 strongly inhibit mitochondrial respiration. Cells
do not respond to the oligomycin injection, which inhibits
ATP synthase,64 nor to the FCCP, which will interfere with the
mitochondrial membrane proton gradient.65 ATP production,
as well as spare respiratory capacity (calculated as the
difference between maximal and basal respiration), are
extremely low, further confirming nonfunctioning mitochondria in treated MDA-MB-435S cells.
Next, the effect on the glycolysis process was investigated.
Figures 2b and S16 show interesting differences between the
modes of action of [Ru(DIP)2(gen)](PF6) and Ru(DIP)2Cl2.
During the glycolysis stress test, the first injection is made with
a saturated solution of glucose. This treatment should trigger
the glycolysis process in cells and consequently lead to higher
ECAR. Surprisingly, MDA-MB-435S cells treated with [Ru(DIP)2(gen)](PF6) showed no increase in ECAR values
following injection of the saturated glucose solution. This
observation is a clear indication of the impaired glycolytic
process. On the other hand, cells treated with Ru(DIP)2Cl2
showed similar glycolysis levels when compared to those of the
untreated cells. This suggests that the cytosolic process of ATP
production is impaired in [Ru(DIP)2(gen)](PF6) treated cells
but not in those treated with Ru(DIP)2Cl2. Furthermore, the
lack of response to the oligomycin injection in cells treated
with both complexes agrees with the results obtained via the
mito stress test, which suggests nonfunctioning mitochondria
after both treatments. Interestingly, the complexes [Ru(DIP) 2 (sq)](PF 6 ), [Ru(DIP) 2 (mal)](PF 6 ), and [Ru(DIP)2(3-methoxysq)](PF6), recently reported by our group,
also showed impaired mitochondrial function but did not show
any effect on the glycolysis process.66−68 This illustrates how
subtle structural changes in the complexes bearing the same
Ru(DIP)2 core but different dioxo ligands can result in
significantly different behavior of the complexes in living cells.
■
EXPERIMENTAL SECTION
Materials. All chemicals were either of reagent or analytical grade
and used as purchased from commercial sources without additional
purification. Ruthenium trichloride hydrate was provided by I2CNS,
and 4,7-diphenyl-1,10-phenanthroline, lithium chloride (anhydrous,
99%), the flavonoids, and tetrabutylammonium hexafluorophosphate
were provided by Sigma-Aldrich. All solvents were purchased of
analytical or HPLC grade. When necessary, solvents were degassed by
purging with dry, oxygen-free nitrogen for at least 30 min before use.
Preparative thin layer chromatography (TLC) glass plates were used
(Analtech, Sigma-Aldrich, Steinheim, Germany, 20 × 20 cm; 1500 μm
thickness).
Instrumentation and Methods. Amber glass or clear glassware
wrapped in tin foil were used when protection from the light was
necessary. Schlenk glassware and a vacuum line were employed when
reactions sensitive to moisture/oxygen had to be performed under a
nitrogen atmosphere. Thin layer chromatography (TLC) was
performed using silica gel 60 F-254 (Merck) plates with detection
of spots being achieved by exposure to UV light. Eluent mixtures are
expressed as volume to volume (v/v) ratios. 1H and 13C NMR spectra
were measured on Bruker Avance III HD 400 MHz or Bruker Avance
Neo 500 MHz spectrometers using the signal of the deuterated
solvent as an internal standard.69 The chemical shifts δ are reported in
ppm (parts per million) relative to tetramethylsilane (TMS) or signals
from the residual protons of deuterated solvents. The following
abbreviations were used to designate multiplicities: s = singlet, d =
doublet, app t = apparent triplet, m = multiplet, dd = double−doublet,
br = broad. Chemical shifts were expressed in ppm. ESI experiments
were carried out using a 6470 Triple Quad (Agilent Technologies).
Elemental analysis was performed at Science Centre, London
Metropolitan University using Thermo Fisher (Carlo Erba) Flash
2000 Elemental Analyzer, configured for %CHN. IR spectra were
recorded with a SpectrumTwo FTIR Spectrometer (Perkin−Elmer)
equipped with a Specac Golden GateTM ATR (attenuated total
reflection) accessory; applied as neat samples; 1/λ in cm−1.
Synthesis and Characterization. RuCl2(dmso)4. RuCl2(dmso)4
was synthesized following an adapted literature procedure.36
Spectroscopic data were in agreement with the literature.36
Ru(DIP)2Cl2. Ru(DIP)2Cl2 was synthesized following an adapted
literature procedure.36 Spectroscopic data were in agreement with the
literature.37,66
[Ru(DIP)2(5-OHF)](PF6). Ru(DIP)2Cl2 (0.20 g, 0.24 mmol) and aq.
NaOH (0.38 mL, 1 M) were dissolved in ethanol (20 mL). The
solution was degassed for 20 min, and 5-hydroxyflavone (0.09 g, 0.38
mmol) was added. The resulting mixture was heated to reflux for 1.5 h
under a N2 atmosphere and protected from light. The mixture was
cooled to RT while still protected from light, and the solvent was
removed under vacuum. The residual solid was redissolved in ethanol
(10 mL), and H2O (100 mL) and NH4PF6 (1.00 g, 6.13 mmol) were
added. The precipitate formed was filtered, washed with H2O (3 × 50
mL) and Et2O (3 × 50 mL), and collected. The solid with Et2O (10
mL) and then heptane (10 mL) was sonicated for 10 min and then
centrifuged. This procedure was repeated three times for each solvent.
The solid was collected with DCM and dried under vacuum to deliver
[Ru(DIP)2(5-OHF)](PF6) (0.07 g, 0.061 mmol, 25% yield) as a
purple solid. 1H NMR (400 MHz, CD2Cl2): δ/ppm = 9.54 (d, J = 5.5
Hz, 1H), 9.38 (d, J = 5.5 Hz, 1H), 8.27 (d, J = 8.7 Hz, 2H), 8.21−
8.16 (m, 3H), 8.11 (d, J = 5.5 Hz, 1H), 7.96 (dd, J = 9.4, 5.5 Hz, 2H),
7.92−7.89 (m, 2H), 7.78−7.50 (m, 23H), 7.42 (dd, J = 10.5, 5.5 Hz,
2H), 7.35 (app t, J = 8.3 Hz, 1H), 6.74 (s, 1H), 6.65 (dd, J = 11.6, 8.3
Hz, 2H); 13C NMR (125 MHz, CD2Cl2): δ/ppm = 179.9, 168.1,
160.0, 158.1, 153.5, 153.1, 151.6, 151.1, 151.0, 150.2, 149.8, 149.6,
■
CONCLUSIONS
Briefly, four monocationic Ru(II) polypyridyl complexes with
the general formula [Ru(DIP)2flv]X were synthesized. The
cytotoxicity of these complexes was tested against different
cancerous and healthy cell lines, and the most promising
compound identified was [Ru(DIP)2(gen)](PF6) with cytotoxicity comparable to that of cisplatin and doxorubicin. The
complex displayed good activity toward the MDA-MB-435S
cell line (IC50 = 2.64 μM), a melanoma cell line derived from
the pleural effusion of a female with metastatic breast
adenocarcinoma, used for the study of metastasis. Interestingly,
genistein was not cytotoxic (IC50 > 100 μM), and the
precursor, Ru(DIP)2Cl2, was only moderately active (IC50 =
27.73 μM). [Ru(DIP)2(gen)](PF6) was found to be taken up
more efficiently by MDA-MB-435S cell lines than MCF-7, a
commonly used breast cancer cell line, in both cases via a
passive transportation mechanism. Further metabolic studies in
the MDA-MB-435S cell line revealed that [Ru(DIP)2(gen)](PF6) not only inhibits mitochondrial respiration but also
interferes with the cytosolic glycolysis process in comparison to
Ru(DIP)2Cl2. This result suggests that addition of the
flavonoid moiety changes the behavior of the complex in
living cells and allows for a more complex mode of action,
leading to cell death. Therefore, we consider [Ru(DIP)2(gen)](PF6) to be a suitable candidate for further
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148.0, 147.7, 146.3, 146.2, 136.2, 136.2, 136.0, 136.0, 134.3, 131.8,
131.0, 129.9, 129.9, 129.7, 129.7, 129.6, 129.5, 129.5, 129.4, 129.2,
129.1, 128.6, 128.6, 128.4, 126.0, 125.9, 125.8, 125.7, 125.6, 125.4,
124.7, 124.5, 118.3, 113.0, 105.9, 100.3. MS (ESI+): m/z 1003.22
[M]+. Elemental Analysis: calcd for C63H41F6N4O3PRu = C, 65.91; H,
3.60; N, 4.88. Found = C, 65.70; H, 3.58; N, 4.55.
[Ru(DIP)2(gen)](PF6). Ru(DIP)2Cl2 (0.20 g, 0.24 mmol) was
dissolved in ethanol (20 mL). The solution was degassed for 20
min, and silver triflate (0.13 g, 0.52 mmol) was added. The mixture
was stirred at RT for 1 h, protected from light, under a N2
atmosphere. The crude reaction mixture was filtered, and the filtrate
was degassed for 20 min. To the degassed solution, genistein (0.10 g,
0.38 mmol) and an ethanolic solution of sodium ethoxide (21%, 285
μL) were added. The mixture was heated to reflux for 2 h under N2
atmosphere while protected from light. The mixture was cooled to
RT, and the solvent was removed under vacuum. The residual solid
was dissolved in ethanol (10 mL), and H2O (100 mL) and NH4PF6
(1.00 g, 6.13 mmol) were added. The precipitate which formed was
filtered and washed with H2O (3 × 50 mL), heptane (3 × 50 mL),
and Et2O (2 × 50 mL). The solid was collected with DCM and dried
under vacuum to deliver the crude product. Purification was achieved
via preparative TLC (DCM/ethyl acetate/methanol 79/20/1). The
product was collected from the prep TLC with methanol, and the
solvent was subsequently removed under reduced pressure. The solid
with Et2O (10 mL) and then heptane (10 mL) was sonicated for 10
min and then centrifuged. This procedure was repeated three times
for each solvent. The solid was collected with DCM and dried under
vacuum to deliver [Ru(DIP)2(gen)](PF6) (0.04 g, 0.033 mmol, 14%)
as a deep purple solid. 1H NMR (400 MHz, CD3OD): δ/ppm = 9.59
(d, J = 5.5 Hz, 1H), 9.21 (d, J = 5.5 Hz, 1H), 8.42 (d, J = 5.5 Hz, 1H),
8.28 (dd, J = 9.4, 1.4 Hz, 2H), 8.20 (dd, J = 9.4, 3.7 Hz, 2H), 8.10
(dd, J = 5.5, 2.3 Hz, 2H), 8.00 (d, J = 5.5 Hz, 1H), 7.82−7.73 (m,
5H), 7.72−7.53 (m, 18H), 7.50 (d, J = 5.5 Hz, 1H), 7.38 (d, J = 5.5
Hz, 1H), 6.50 (d, J = 8.7 Hz, 2H), 6.26 (d, J = 8.7 Hz, 2H), 6.10 (s,
1H); 13C NMR (125 MHz, CD3OD): δ/ppm = 178.2, 169.5, 165.5,
160.9, 158.1, 155.2, 155.1, 153.0, 152.7, 152.6, 152.1, 151.2, 150.9,
150.9, 149.6, 149.1, 147.8, 147.5, 137.7, 137.6, 137.6, 137.5 131.1,
131.1, 131.0, 130.8, 130.5, 130.4, 130.3, 130.2, 130.1, 130.1, 130.1,
129.7, 129.7, 129.6, 129.5, 126.9, 126.8, 126.7, 126.7, 126.6 125.9,
125.8, 124.2, 123.6, 115.3, 109.3, 92.4, 58.3. MS (ESI+): m/z 1035.5
[M]+. Elemental Analysis: calcd for C63H41F6N4O5PRu = C, 64.12; H,
3.50; N, 4.75. Found = C, 64.51; H, 3.45; N, 4.48.
[Ru(DIP)2(chr)](OTf)·4H2O. Ru(DIP)2Cl2 (0.50 g, 0.60 mmol) was
dissolved in ethanol (30 mL). The solution was degassed for 20 min
and silver triflate (0.34 g, 1.32 mmol) was added. The mixture was
stirred at RT for 1 h protected from light, under a N2 atmosphere.
The crude reaction mixture was filtered and the filtrate was degassed
for 20 min before chrysin (0.24 g, 0.96 mmol) and an ethanolic
solution of sodium ethoxide (21%, 717 μL) were added. The mixture
was heated to reflux for 2 h under N2 atmosphere and protected from
light. The mixture was cooled to RT while still protected from light,
and the solvent was removed under vacuum. The residual solid was
collected in DCM (20 mL) and filtered through Celite. The solvent
was removed under vacuum to deliver the crude product. Purification
was achieved via preparative TLC (DCM/ethyl acetate/methanol 79/
20/1). The product was collected from the prep TLC with methanol,
and the solvent was subsequently removed under reduced pressure.
The solid with Et2O (10 mL) and then heptane (10 mL), was
sonicated for 10 min and then centrifuged. This procedure was
repeated three times for each solvent. The solid was collected with
DCM and dried under vacuum to afford [Ru(DIP)2(chr)](OTf)
(0.12 g, 0.09 mmol, 16% yield) as a deep purple solid. 1H NMR (400
MHz, CD2Cl2-d2): δ/ppm = 9.56 (d, J = 5.5 Hz, 1H), 9.32 (d, J = 5.5
Hz, 1H), 8.20−8.09 (m, 4H), 8.09−7.99 (m, 2H), 7.84−7.80 (m,
2H), 7.76 (d, J = 7.3 Hz, 2H), 7.69−7.36 (m, 24H), 7.34 (d, J = 5.5
Hz, 1H), 7.28 (d, J = 5.5 Hz, 1H), 6.48 (s, 1H), 6.17 (br d, J = 2.2 Hz,
1H), 6.04 (br d, J = 2.2 Hz, 1H). 13C NMR (125 MHz, CD2Cl2): δ/
ppm = 178.2, 169.1, 160.0, 159.4, 153.7, 153.4, 152.3, 152.0, 151.6,
150.7, 150.2, 150.2, 147.9, 147.7, 146.3, 146.2, 136.9, 136.8, 136.7,
136.6, 131.8, 131.7, 130.4, 130.4, 130.2, 130.1, 129.9, 129.8, 129.7,
Article
129.6, 129.6, 129.5, 129.0, 129.0, 128.8, 126.3, 126.2, 126.1, 125.8,
125.1, 107.7, 105.5, 104.6, 92.3. MS (ESI+): m/z 1019.6 [M]+, (ESI): m/z 149.2 [OTf]̅. Elemental Analysis: calcd for C64H49F3N4O11RuS
= C, 61.97; H, 3.99; N, 4.51. Found = C, 62.09; H, 3.93; N, 4.28.
[Ru(DIP)2(mor)](OTf). A: Morin (0.56 g, 1.85 mmol) was
suspended in dry tetrahydrofuran (50 mL) and triethylamine (1.55
mL, 11.1 mmol) was added. The mixture was stirred at RT under a N2
atmosphere for 15 min before TMS-Br (1.47 mL, 11.1 mmol) was
added. The mixture was stirred at RT under a N2 atmosphere for 2.5 h
before being added to a separating funnel. H2O (50 mL) was added,
and the product was extracted in DCM and dried on Na2SO4. The
solvent was removed under vacuum to yield the crude product A.
B: Ru(DIP)2Cl2 (0.83 g, 1.00 mmol) was dissolved in ethanol (50
mL). The solution was degassed for 20 min, and silver triflate (0.56 g,
2.20 mmol) was added. The mixture was stirred at RT for 1 h
protected from light, under a N2 atmosphere. The crude reaction
mixture was filtered, and the filtrate was degassed for 20 min before
product A and an ethanolic solution of sodium ethoxide (21%, 750
μL) were added. The mixture was heated to reflux for 2 h under N2
atmosphere and protected from light. The mixture was cooled to RT
while still protected from light, and the solvent was removed under
vacuum. The residual solid was collected in DCM (20 mL) and
filtered through Celite. The solvent was removed under vacuum to
deliver the crude product. Purification was achieved via preparative
TLC (DCM/ethyl acetate/methanol 79/20/1). The product was
collected from the prep TLC with methanol, and the solvent was
subsequently removed under reduced pressure. The solid with Et2O
(10 mL) and then heptane (10 mL) was sonicated for 10 min and
then centrifuged. This procedure was repeated three times for each
solvent. The solid was collected with DCM and dried under vacuum
to afford [Ru(DIP)2(mor)](OTf) (0.42 g, 0.35 mmol, 35% yield) as a
deep purple solid. 1H NMR (400 MHz, DMF-d7): δ/ppm = 11.85 (s,
1H), 9.73 (dd, J = 10.1, 5.5 Hz, 2H), 8.53 (d, J = 5.5 Hz, 1H), 8.45
(d, J = 5.5 Hz, 1H), 8.42−8.20 (m, 7H), 7.93−7.49 (m, 25H), 6.45
(dd, J = 8.7, 2.4 Hz, 1H), 6.06 (d, J = 2.4 Hz, 1H), 5.99 (s, 1H), 5.76
(s, 1H). 13C NMR (125 MHz, DMF-d7): δ/ppm = 158.9, 158.0,
155.0, 154.7, 151.9, 151.8, 151.8, 151.5, 149.7, 149.6, 147.3, 147.0,
145.7, 145.5, 143.3, 136.4, 136.1, 136.0, 130.3, 130.2, 130.0, 129.4,
129.3, 129.2, 129.1, 128.8, 128.2, 128.0, 126.4, 126.3, 125.9, 125.9,
125.8, 125.7, 125.1, 125.0, 112.5, 108.0, 104.9, 95.7. MS (ESI+): m/z
1067.9 [M]+, (ESI-): m/z 149.3 [OTf]̅. Elemental Analysis: calcd for
C64H41F3N4O10RuS = C, 63.20; H, 3.40; N, 4.60. Found = C, 62.77;
H, 3.33; N, 4.45.
Stability Studies. The stability in DMSO-d6 or DMF-d7 at room
temperature was assessed by 1H NMR over 96 h.
Cytotoxicity Assay Using a 2D Cellular Model. Cytotoxicity of
[Ru(DIP) 2 (5-OHF)](PF 6 ), [Ru(DIP) 2 (gen)](PF 6 ), [Ru(DIP)2(chr)](OTf), [Ru(DIP)2(mor)](OTf), Ru(DIP)2Cl2, cisplatin, and doxorubicin was assessed by a fluorometric cell viability assay
using Resazurin (ACROS Organics). Briefly, cells were seeded in
triplicate in 96-well plates at a density of 4 × 103 cells/well in 100 μL.
After 24 h, cells were treated with increasing concentrations of the
ruthenium complexes. Dilutions were prepared as follows: 0.250 mM
stock in DMSO ([Ru(DIP)2(5-OHF)](PF6), [Ru(DIP)2(gen)](PF6),
and [Ru(DIP)2(chr)](OTf)) or DMF ([Ru(DIP)2(mor)](OTf) and
Ru(DIP)2Cl2), which were further diluted to 100 μM in cell media.
After 48 h of incubation, the medium was removed, and 100 μL of
complete medium containing resazurin (0.2 mg/mL final concentration) was added. After 4 h of incubation at 37 °C, the fluorescence
signal of resorufin product was read (ex: 540 nm em: 590 nm) in a
SpectraMax M5 microplate Reader. IC50 values were then calculated
using GraphPad Prism software.
GraphPad Prism Calculations of IC50 Values. XY analysis with
three replicate values in side by side subcolumns was chosen. Inserted
raw data obtained from SpectraMax M5 microplate reader was treated
as follows: X values were transformed to be logarithmic; data were
normalized to the lowest Y value. Data were then analyzed with XY
analysis “Nonlinear regression (curve fit)” then “log(inhibitor) vs.
normalized response”.
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Cytotoxicity Assay Using a 2D Cellular Model F (2 h
Incubation). Cytotoxicity of [Ru(DIP)2(gen)](PF6) and cisplatin
was assessed by a fluorometric cell viability assay using Resazurin
(ACROS Organics). Briefly, cells were seeded in triplicate in 96-well
plates at a density of 4 × 103 cells/well in 100 μL. After 24 h, cells
were treated with increasing concentrations of the complexes.
Dilutions were prepared as described in the section titled Cytotoxicity
Assay Using a 2D Cellular Model. After 2 h incubation, the medium
was removed, and 100 μL of complete medium containing resazurin
(0.2 mg/mL final concentration) was added. After 4 h of incubation
at 37 °C, the fluorescence signal of resorufin product was read (ex:
540 nm em: 590 nm) in a SpectraMax M5 microplate Reader. IC50
values were then calculated using GraphPad Prism software as stated
before.
Sample Preparation for Cellular Uptake. MDA-MB-435S and
MCF-7 cells were seeded at a density of 2 × 106 in 10 cm plates. The
next day, cells were treated with 5 μM concentration of [Ru(DIP)2(gen)](PF6), Ru(DIP)2Cl2, or cisplatin. Dilutions were
prepared as described in the section titled Cytotoxicity Assay Using
a 2D Cellular Model. After 2 h, cells were washed, collected, counted,
and snap frozen in liquid nitrogen and stored at −20 °C. ICP-MS
samples were prepared as follows: samples were digested using 70%
nitric acid (1 mL, 60 °C, overnight). Samples were then further
diluted 1:100 (1% HCl solution in MQ water) and analyzed using
ICP-MS.
Sample Preparation for Studies on the Mechanism of
Cellular Uptake. Samples were prepared as previously reported.66
Briefly, MDA-MB-435S and MCF-7 cells were seeded at a density of 2
× 106 in 10 cm dishes and were pretreated the following day with the
corresponding inhibitors or kept at a specific temperature for 1 h.
Next, cells were washed with PBS and incubated with 5 μM of
[Ru(DIP)2(gen)](PF6) or Ru(DIP)2Cl2 for 2 h (low temperature
samples were still kept at 4 °C). Dilutions were prepared as described
in the section titled Cytotoxicity Assay Using a 2D Cellular Model.
Subsequently, cells were washed with PBS, collected, counted, and
snap frozen in liquid nitrogen. Pellets were stored at −20 °C. ICP-MS
samples were prepared as follows: samples were digested using 70%
nitric acid (1 mL, 60 °C, overnight), further diluted 1:100 (1% HCl
solution in MQ water), and analyzed using ICP-MS.
Sample Preparation for Time-Dependent Cellular Accumulation. MDA-MB-435S and MCF-7 cells were seeded at a density of
3 × 106 in 10 cm plates. The next day, cells were treated with 1 μM
concentration of [Ru(DIP)2(gen)](PF6), Ru(DIP)2Cl2, or cisplatin.
Dilutions were prepared as described in the section titled Cytotoxicity
Assay Using a 2D Cellular Model. After 2, 12, 24, and 48 h,
respectively, the cells were washed, collected, counted, and snap
frozen in liquid nitrogen and stored until further use at −20 °C. ICPMS samples were prepared as follows: samples were digested using
70% nitric acid (0.5 mL for the 2 and 12 h samples; 1 mL for the 24
and 48 h samples, 65 °C, overnight). The samples were further
diluted 1:50 (2 h samples) or 1:100 (12, 24, 48 h samples) in 1% HCl
solution in MQ water and analyzed using ICP-MS.
ICP-MS Studies. All ICP-MS measurements were performed on a
high resolution ICP-MS instrument (Element II, ThermoScientific)
located at the Institut de Physique du Globe de Paris (France). The
monitored isotopes were 101Ru and 195Pt. Daily, prior to the analytical
sequence, the instrument was first tuned to produce maximum
sensitivity and stability while also maintaining low uranium oxide
formation (UO/U ≤ 5%). The data were treated as follows:
intensities were converted into concentrations using uFREASI
(user-FRiendly Elemental dAta proceSsIng).70 This software,
developed for the HR-ICP-MS users community, is free and available
on http://www.ipgp.fr/~tharaud/uFREASI.
ICP-MS Data Analysis. Cellular Uptake Studies. The amount of
metal detected in the cell samples was transformed from parts per
billion into micrograms of metal. Data were subsequently normalized
to the number of cells and expressed as nanograms of metal/amount
of cells.
Mechanism of Uptake. The amount of ruthenium detected in cell
samples was transformed from parts per billion into micrograms of
Article
ruthenium, and values obtained were normalized to the number of
cells used for specific treatment. The value for the ruthenium found in
the 37 °C sample was used as a 100%.
Metabolic Studies. HeLa cells were seeded in Seahorse XFe96
well plates at a density of 10 × 103 cells/well in 80 μL. After 24 h, the
medium was replaced with fresh medium and cisplatin (1 μM),
genistein (1 μM), Ru(DIP)2Cl2 (1 μM), or [Ru(DIP)2(gen)](PF6)
(1 μM) were added. Dilutions were prepared as described in the
section titled Cytotoxicity Assay Using a 2D Cellular Model. After 24
h of incubation, the regular medium was removed, cells were washed
thrice using Seahorse Base Media and incubated in a non-CO2
incubator at 37 °C for 1 h.
Mito Stress Test. Mito stress assay was run using 1 μM oligomycin,
1 μM FCCP, and mixture of 1 μM antimycin-A/rotenone each in
ports A, B, and C, respectively, using the Seahorse XFe96 Extracellular
Flux Analyzer.
Glycolysis Stress Test. The glycolytic stress test was run using
glucose (10 mM), oligomycin (1 μM), and 2-deoxyglucose (50 mM)
in ports A, B, and C, respectively, using the Seahorse XFe96
Extracellular Flux Analyzer.
■
ASSOCIATED CONTENT
sı Supporting Information
*
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.inorgchem.9b03562.
H NMR spectra, 13C NMR spectra, fluorometric cell
viability assay, oxygen consumption rates, and extracellular acidification rates (PDF)
1
■
AUTHOR INFORMATION
Corresponding Author
Gilles Gasser − Chimie ParisTech, PSL University, CNRS,
Institute of Chemistry for Life and Health Sciences, Laboratory
for Inorganic Chemical Biology, F-75005 Paris, France;
orcid.org/0000-0002-4244-5097; Phone: +33 1 44 27 56
02; Email: gilles.gasser@chimeparistech.psl.eu;
www.gassergroup.com
Authors
Alexandra-Cristina Munteanu − Department of General and
Inorganic Chemistry, Faculty of Pharmacy, “Carol Davila”
University of Medicine and Pharmacy, 020956 Bucharest,
Romania
Anna Notaro − Chimie ParisTech, PSL University, CNRS,
Institute of Chemistry for Life and Health Sciences, Laboratory
for Inorganic Chemical Biology, F-75005 Paris, France
Marta Jakubaszek − Chimie ParisTech, PSL University, CNRS,
Institute of Chemistry for Life and Health Sciences, Laboratory
for Inorganic Chemical Biology, F-75005 Paris, France; Institut
Curie, PSL University, CNRS UMR 144, Paris, France
Joseph Cowell − Chimie ParisTech, PSL University, CNRS,
Institute of Chemistry for Life and Health Sciences, Laboratory
for Inorganic Chemical Biology, F-75005 Paris, France
Mickaël Tharaud − Université de Paris, Institut de Physique du
Globe de Paris, CNRS, F-75005 Paris, France
Bruno Goud − Institut Curie, PSL University, CNRS UMR 144,
Paris, France
Valentina Uivarosi − Department of General and Inorganic
Chemistry, Faculty of Pharmacy, “Carol Davila” University of
Medicine and Pharmacy, 020956 Bucharest, Romania
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.inorgchem.9b03562
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Author Contributions
#
A.-C.M. and A.N. contributed equally to this work.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This work was financially supported by an ERC Consolidator
Grant PhotoMedMet to G.G. (GA 681679) and has received
support under the program Investissements d’Avenir launched
by the French Government and implemented by the ANR with
the reference ANR-10-IDEX-0001-02 PSL (G.G.). Ile de
France Region is gratefully acknowledged for financial support
of the 500 MHz NMR spectrometer of Chimie ParisTech in
the framework of the SESAME equipment project. We
acknowledge the loan of Agilent’s equipment to Chimie
ParisTech. Part of this work was supported by IPGP
̂
multidisciplinary program PARI and by Region Ile-de-France
SESAME Grant 12015908. This project was also financially
supported by “Carol Davila” University of Medicine and
Pharmacy through Contract 23PFE/17.10.2018 funded by the
Ministry of Research and Innovation within PNCDI III,
Program 1, Development of the National RD system,
Subprogram 1.2, Institutional Performance, RDI excellence
funding projects.
■
Article
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