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Evaluation of heteroscorpionate ligands as scaffolds for the generation of Ruthenium(II) metallodrugs in breast cancer therapy.
Journal of Inorganic Biochemistry 253 (2024) 112486
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Evaluation of heteroscorpionate ligands as scaffolds for the generation of
Ruthenium(II) metallodrugs in breast cancer therapy
Elena Domínguez-Jurado a, b, Consuelo Ripoll a, c, Agustín Lara-Sánchez b, *, Alberto Ocaña d,
Iñigo J. Vitórica-Yrezábal e, Iván Bravo a, c, *, Carlos Alonso-Moreno a, b, *
a
Universidad de Castilla-La Mancha, Unidad nanoDrug, Facultad de Farmacia de Albacete, 02008 Albacete, Spain
Universidad de Castilla-La Mancha, Departamento de Química Inorgánica, Orgánica y Bioquímica, Facultad de Ciencias y Tecnologías Químicas-Centro de Innovación
en Química Avanzada (ORFEO-CINQA), Ciudad Real 13071, Spain
c
Universidad de Castilla-La Mancha, Departamento de Química Física. Facultad de Farmacia de Albacete, Albacete 02071, Spain
d
Experimental Therapeutics Unit, Hospital clínico San Carlos, IdISSC and CIBERONC, Madrid, Spain
e
Departamento de Química Inorgánica, Facultad de Ciencias, Universidad de Granada, Avda de Fuentenueva. s/n, 18071 Granada, Spain
b
A R T I C L E I N F O
A B S T R A C T
Keywords:
Heteroscorpionate ligands
TNBC
RAPTA derivatives
Preclinical evaluation
Uptake studies
Fluorescence lifetime imaging microscopy
The modular synthesis of the heteroscorpionate core is explored as a tool for the rapid development of
ruthenium-based therapeutic agents. Starting with a series of structurally diverse alcohol-NN ligands, a family of
heteroscorpionate-based ruthenium derivatives was synthesized, characterized, and evaluated as an alternative
to platinum therapy for breast cancer therapy. In vitro, the antitumoral activity of the novel derivatives was
assessed in a series of breast cancer cell lines using UNICAM-1 and cisplatin as metallodrug control. Through this
approach, a bimetallic heteroscorpionate-based metallodrug (RUSCO-2) was identified as the lead compound of
the series with an IC50 value range as low as 3–5 μM. Notably, RUSCO-2 was found to be highly cytotoxic in
TNBC cell lines, suggesting a mode of action independent of the receptor status of the cells. As a proof of concept
and taking advantage of the luminescent properties of one of the complexes obtained, uptake was monitored in
human breast cancer MCF7 cell lines by fluorescence lifetime imaging microscopy (FLIM) to reveal that the
compound is evenly distributed in the cytoplasm and that the incorporation of the heteroscorpionate ligand
protects it from aqueous processes, conversion in another entity, or the loss of the chloride group. Finally, ROS
studies were conducted, lipophilicity was estimated, the chloride/water exchange was studied, and stability
studies in simulated biological media were carried out to propose structure-activity relationships.
1. Introduction
Heteroscorpionate ligands are intriguing, versatile, and robust pol
ydentate compounds [1]. Due to their ease of structural modification,
multiple coordinating sites, and large steric bulk, this type of ligands has
been widely used as scaffolds to stabilize from early to late transition
metals [2,3]. In fact, we have witnessed the incorporation of different
types of anionic functional groups for many years, such as carboxylate,
alkoxide, thiolate, aryloxide, sulphonate, cyclopentadienyl, or acet
amidate in its structure, leading to novel features which broaden their
scope of application [4]. Many catalytic processes have been investi
gated within the corresponding inorganic entities [3]. Group 4 hetero
scorpionate catalysts have been mainly studied as catalyst precursors for
olefin polymerization [5]. Some ruthenium heteroscorpionate catalysts
were designed to achieve ring-closing metathesis [6]. Likewise, ruthe
nium carbonyl derivatives were successfully tested as catalysts in the
epoxidation of cyclohexene with different oxidizing agents [7]. Group 9
heteroscorpionate derivates provide an alternative to the Shilove
approach for the alkane C–H bond activation [8]. Heteroscorpionate
alkyl lanthanide catalysts displayed high catalytic activity in the
hydroamination processes [9], and magnesium, zinc, and aluminium
heteroscorpionate derivatives have been extensively described as very
active initiators for the ring-opening polymerization of lactones and
lactides [10–12]. Finally, aluminium heteroscorpionate complexes have
also been shown very active catalysts for cyclic carbonate synthesis [13].
Furthermore, it was noted that a large amount of research has been
devoted to the design and synthesis of metal complexes which structures
mimic the active sites of metalloenzymes [14,15].
* Corresponding author at: Universidad de Castilla-La Mancha, Unidad nanoDrug, Facultad de Farmacia de Albacete, 02008 Albacete, Spain.
E-mail address: carlos.amoreno@uclm.es (C. Alonso-Moreno).
https://doi.org/10.1016/j.jinorgbio.2024.112486
Received 23 September 2023; Received in revised form 14 January 2024; Accepted 15 January 2024
Available online 18 January 2024
0162-0134/© 2024 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/).
E. Domínguez-Jurado et al.
Journal of Inorganic Biochemistry 253 (2024) 112486
Despite these tridentate ligands represent a promising approach to
stabilize metals in their low oxidations state, and therefore improve
their adsorption and therapeutic activity, only a small number of studies
have been reported in the field of oncology [4,14,15]. Compared to the
platinum-based therapy in cancer: ruthenium complexes can be easily
tuned from a six-coordination octahedron mode with a wide range of
oxidation states leading to abundant metallodrug structures [16],
analogously to iron, they can be transported in the blood through
transferrin with faster absorption, metabolism and lower systemic
toxicity [17]; their different mechanism of action may overcome re
sistances generated after platinum therapy [18,19]. There are a few
ruthenium-based metallodrugs under clinical studies [20–22], and a
plethora of them in preclinical studies as therapeutic agents for the
treatment of breast cancer, ovarium cancer, and other common tumour
types [23–25]. At present, these ruthenium complexes include distinct
auxiliary ligands [26–28] and show different mechanisms of action,
such as tumour growth inhibition, cell cycle arrest, apoptosis, auto
phagy, and generation of reactive oxygen species (ROS) [29]. From the
pioneering work of Dyson et al. [30,31] in which RAPTA-C (see mo
lecular structure in Fig. 1) was presented to explore ruthenium(II) in the
search for other therapeutic alternatives to ruthenium(III) counterparts
that made progress in clinical phases, such as NAMI-A32 or KP1019 [33],
a plethora of derivatives of RAPTA-C26 [34–36] or the cyclopentadienylcounterparts [37–39] have been reported. Proposals of structur
al–activity relationships are not evident. In this sense, the best outcomes
were shown for those compounds maintaining the p-cymene moiety and
tunning or replacing the 1,3,5-triaza-7-phosphaadamantane (PTA)
auxiliary ligand. Thus, the water-soluble ruthenium(II) organometallic
compound UNICAM-1 based on the bis(3,5 dimethylpyrazol-1-yl)
methane ligand [40] (see molecular structure in Fig. 1), was evaluated
in vivo against triple-negative breast cancer (TNBC) to report low
toxicity and favourable clearance properties [41]. As far as we know,
only a few heteroscorpionate-based ruthenium metallodrugs have been
evaluated as an alternative to platinum therapy [42,43]. In this context,
the versatility of the coordination mode of the heteroscorpionate ligands
and their easily tunable core may provide exceptional and modulable
steric and electronic features required for the rational design of new
anticancer metallodrugs.
Breast cancer (BC) remains a worldwide health problem with >3.5
million cases expected in 2050. Therapies to treat this disease include
the use of chemotherapy and targeted therapies, although the efficacy of
many of them in practice is very limited due to the generation of resis
tance and the presence of adverse effects on the patient. Currently, there
is no effective treatment for some subtypes of breast cancer, such as
TNBC or HER2+ in advanced stages. Therefore, there is an urgent need
to develop new effective therapeutic agents. In this regard, novel ther
apeutic alternatives based on ruthenium metallodrugs are actively being
explored for the treatment of TNBC [37,44–47].
In this work, alcohol-based NN heteroscorpionate ligands have been
assessed as auxiliary scaffolds for the generation of heteroscorpionatebased ruthenium derivatives. Due to the chemical robustness shown in
previously reported organometallic analogues with this family of het
eroscorpionate ligands [13,48], and the excellent antimetastatic prop
erties showed by RAPTA derivatives in vivo [26], several RAPTA/
heteroscorpionate hybrid ruthenium derivatives were designed, syn
thesized, and fully characterized by analytical, spectroscopic methods
and X-ray crystallography studies. The novel complexes were tested
against a panel of breast cancer tumoral cells and IC50 was calculated
finding the leading agent of the series. The uptake was studied by timeresolved fluorescence microscopy of a fluorescent derivative.
2. Results and discussion
2.1. Synthesis and structural characterization of ruthenium compounds
Heteroscorpionate ligands L1-L5 were synthesized following the
protocol previously reported for the synthesis of L1 and L2 [13]. After
deprotonation at the methylene group of bis(3,5-dimethylpyrazol-1-yl)
methane with BunLi, an addition reaction with the corresponding
aldehyde, the lithium salts were obtained as powered solids. The hy
drolysis of the lithium salts with a saturated solution of NH4Cl afforded
the ligands L1-L5 as white or brown solids in good yields (ca. 80%)
(Fig. 2). As expected, 1H and 13C{1H} NMR spectra of L1-L5, exhibited
two sets of resonances for the protons and carbons from the pyrazole
rings, indicating they were not equivalent. These data were attributed to
the presence of a chiral center in the Ca carbon of the ligand L1-L5
precursors (see Fig. S1-S6 in the Supporting Information). It should be
noted that the L3 ligand incorporates an anthracene group that will
provide optical properties to the ruthenium counterpart to facilitate
uptake studies.
The proposed cationic ruthenium complexes (RUSCO-1-RUSCO-5)
were obtained after stirring a mixture of the corresponding ligand and
the ruthenium precursor [RuCl2(p-cym)]2 in MeOH/THF 3:1 (Fig. 3).
The complex formation was monitored by 1H NMR to be completed
within a 4-h reaction time in all cases. RUSCO-1-RUSCO-5 were further
purified by crystallization and were isolated in good yields (ca. 75%).
These ruthenium complexes were soluble in DMSO and CH2Cl2 and
showed minor solubility in water. Complexes were characterized spec
troscopically (see Experimental Section and Fig. S7-S16 in the Sup
porting Information). The 1H NMR and 13C{1H}-NMR spectra of these
complexes showed two different sets of resonances for protons and
carbons of both pyrazole rings, indicating they were not equivalent, the
resonances for the different groups from the alcohol moiety, two
different resonances for the methyl groups from the isopropyl moiety
and the corresponding resonances for the p-cymene ring (Fig. 4). 1H–1H
COSY experiments and 1H–13C heteronuclear correlation (g-HSQC)
ascertained the assignment of most of the resonances (see Experimental
Section and Fig. S17 in the Supporting Information). Based on spectro
scopic data, ionic compounds are obtained with a chloride anion as a
counterion and with a distorted-octahedral environment around the
ruthenium centre in which the heteroscorpionate ligand is coordinated
to the metal centre in a neutral κ2-NN coordination mode (Fig. 3).
Single crystal X-ray diffraction studies for RUSCO-2 and RUSCO-3
confirmed the solid-state structure of ruthenium compounds. The cor
responding ORTEP views are represented in Fig. 5. The crystallographic
data and selected bond distances and angles are collected in Tables S1
and S2 in the Supporting Information, respectively. The molecular
structures revealed a formally six-coordinated Ru(II) cation unit in
which the ruthenium atom shows the typical “piano-stool” structure
[49] with the metal centre coordinated to the heteroscorpionate ligand
in a κ2-NN coordination mode, a chloride atom, and the p-cymene group
in a η6-hapticity coordination mode (Fig. 5). The second chlorine atom
appears as a counterion forming a hydrogen bond with the hydroxyl
moiety of the heteroscorpionate ligand [O(1)-H(1)⋅⋅⋅Cl(2) 2.297 Å for
Fig. 1. Chemical structure of RAPTA-C and UNICAM-1 metallodrugs.
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Journal of Inorganic Biochemistry 253 (2024) 112486
Fig. 2. Synthesis of precursors L1-L5.
Fig. 3. Synthesis of ruthenium-based agents (1–5). Yields ~80%.
Fig. 4. 1HNMR spectrum of RUSCO-2 in CDCl3.
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Journal of Inorganic Biochemistry 253 (2024) 112486
saturated solution of the compounds. Then, according to the Interna
tional Pharmacopeia [50,51], RUSCO-1 and RUSCO-4 are classified as
soluble (35.06 mg/mL and 79.60 mg/mL, respectively), RUSCO-2 and
RUSCO-3 are slightly soluble (6.84 mg/mL and 1.04 mg/mL, respec
tively) and RUSCO-5 is sparingly soluble (10.06 mg/mL). According to
NMR experiments, ruthenium compounds degraded in chloroform after
72 h, generating a precipitate at the bottom of the NMR tube and
affording a mixture of aromatic species from which the free p-cymene
group was identified (see Fig. S18-S22 for RUSCO-1-RUSCO-5 in the
Supporting Information). Metallodrugs are commonly dissolved in a
mixture of DMSO:H2O to perform biological assays which in any case
did not exceed 0.25% v/v of DMSO. RAPTA complexes are prone to
hydrolysis which is problematic for clinical evaluation trials. The sta
bility of RUSCO-1-RUSCO-5, in DMSO‑d6:D2O, CDCl3 and simulated
biological medium (DMSO‑d6:D2O after adding NaCl), was monitored
by NMR together with RAPTA-C as a control (see Fig. S18-S27 for
RUSCO-1-RUSCO-5 and RAPTA-C stability in Fig. S29 of the Supporting
Information). The set of signals belonging to RUSCO compounds per
sisted throughout stability experiments in contrast to RAPTA-C. The
existence of only one pattern for the ruthenium derivatives discarded the
fast chloride dissociation from RUSCO-1-RUSCO-5, contrary to that
found for RAPTA-C. This was demonstrated by comparing the spectra
before and after the addition of silver nitrate to the solution (see Fig. S28
for RUSCO-1, RUSCO-4, and RUSCO-5, as representative spectra)
[52,53]. The silver nitrate forces the exchanged of the chloride moiety
for one molecule of water. This generates the appearance of a precipitate
(AgCl) and in turn, produces changes in the NMR spectrum. Therefore,
the chelating ligand improves the stability of the ruthenium derivatives,
while the arene group facilitates the uptake and interaction with po
tential targets. The loss of the arene group gives rise to ruthenium(III)
derivatives and, therefore, causes an in-drop of antitumoral activity of
the ruthenium metallodrugs. Tunning both building blocks may allow an
improvement in the pharmacological profile of such a family of metal
lodrugs. Heteroscorpionate ligands are very well-known entities to sta
bilize early to late transition metals. From a chemical point of view, the
robust nature of the heteroscorpionate ligand presents an ideal template
for both high-throughput and rational metallodrug design. As it hap
pens, the incorporation of such a robust ligand allowed an improvement
in the stability of the ruthenium compounds.
On the other hand, differences in pharmacological profiles can be
attributed to the ability of this family of compounds to penetrate bio
logical membranes, which in turn is mainly dependent on lipophilicity
factors. Calculated logarithmic octanol/water partition coefficients
(clog P) [54] for RUSCO-1-RUSCO-5 were obtained using the software
Molinsipiration (see Table S3 and Fig. S30 in the Supporting Informa
tion) [55]. The increasing size of the substituents on the hetero
scorpionate scaffold might explain the observed lipophilicity pattern.
Fig. 5. ORTEP plot in OLEX2 for the structure of RUSCO-2 (A) and RUSCO-3
(B). Thermal ellipsoids are given at the 30% probability level. All Hydrogen
atoms have been omitted except for the OH for clarity. Ruthenium atoms are
shown in dark blue, iron in orange, nitrogen in light blue, oxygen in red,
chloride in green, and carbon and hydrogen in white. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web
version of this article.)
RUSCO-2 and O(1 A)-H(1 A)⋅⋅⋅Cl(04) 2.266 Å for RUSCO-3]. RUSCO-2
crystallizes as an enantiopure complex in the chiral space group P212121
and only shows the S enantiomer in the unit cell with a Flack parameter
of − 0.01(3), while RUSCO-3 crystallizes as a racemic mixture of both
enantiomers in the asymmetric unit. The average Ru–N and Ru–Cl
bond distances for both complexes are 2.119(9) Å and 2.410(2) Å,
respectively, which are in good agreement with the literature data
[32,33]. Bond angles confirmed the proposed “piano-stool” structure
(See Table S2 in the Supporting Information).
2.3. Antitumoral properties of ruthenium metallodrugs
The antitumoral properties of ruthenium complexes were evaluated
in a panel of representative breast tumour cell lines including triple
negative (BT549 and MDA-MB-231), HER2+ (SKBR3), and ER+/PR+
(MCF7) using UNICAM-1 and cisplatin as reference for metallodrugs. As
can be seen in Fig. 6, most ruthenium compounds at 4 μM showed better
antiproliferative effects than the reference ruthenium metallodrugs
UNICAM-1, used as controls in all the cancer cell lines evaluated.
RUSCO-2 was the most active ruthenium metallodrug of the series (see
IC50 values in Table 1 and dose-response curve for RUSCO-1, RUSCO-2,
and RUSCO-3 in Fig. S31 in the Supporting Information). Ligands L1-L5
did not show significant cytotoxicity.
RUSCO-2 displayed higher cytotoxicity than cisplatin at low con
centrations whereas increasing the doses makes both equipotent. These
results indicate that tunning the core of the heteroscorpionate ligand led
to a significant increase in the cytotoxicity of the resulting complexes.
Notably, RUSCO-2 was found to be highly cytotoxic in TNBC cell lines,
2.2. Study of the stability of the ruthenium derivatives in aqueous media
The precursors L1-L5 were not soluble in water but the cationic
complexes RUSCO-1-RUSCO-5 were air-stable, very soluble in chlori
nated solvents and other polar solvents such as methanol and ethanol
and have different solubility in water. Solubility in water has been
measured by UV–vis spectroscopy. A calibration curve has been made
for each compound and the solubility has been calculated using a
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Journal of Inorganic Biochemistry 253 (2024) 112486
Fig. 6. Impact on cell viability of RUSCO-1-RUSCO-5, UNICAM-1, and cisplatin at 4 μM. (A) Screening of several ruthenium compounds by exploring cell viability in
BT549, SKBR3, MDA-MB-231, and MCF7 breast cancer cell lines for 72 h evaluated using MTTs. Data are the average +/− standard deviation (SD) of three in
dependent experiments performed in triplicate. To determine significant statistical differences, a Student’s t-test was used. The values for the statistical analyses are *
p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.
Table 1
IC50 values for ruthenium complexes in a panel of breast cancer tumour cells.a
Cell Line
BT549
MDA-MB-231
SKBR3
MCF7
HaCaT
HEK-293
a
IC50 values (μM)
RUSCO-1
RUSCO-2
RUSCO-3
RUSCO-4
RUSCO-5
UNICAM-1
Cisplatin
14.20 ± 1.39
12.79 ± 0.64
8.50 ± 0.50
13.63 ± 0.57
–
–
3.45 ± 0.45
3.19 ± 0.76
4.07 ± 2.10
5.10 ± 0.20
3.36 ± 0.20
5.40 ± 1.64
13.93 ± 0.77
12.85 ± 1.68
9.57 ± 1.24
12.93 ± 1.89
–
–
>20
>20
>20
18.24 ± 0.87
–
–
>20
14.53 ± 2.49
16.30 ± 1.43
15.68 ± 0.17
–
–
>20
>20
>20
>20
>20
>20
5.50 ± 0.28
11.14 ± 1.55
6.69 ± 0.65
10.12 ± 2.10
2.01 ± 0.04
11.86 ± 2.29
Values are means ± SDs obtained by the MTT assay (exposure time: 72 h).
suggesting a mode of action independent of the receptor status of the
cells. TNBC lacks effective treatments, with a five-year survival rate of
20–90% depending on its stage [56]. The clinical relevance of TNBC
entails the need to explore other alternatives to address this disease:
improve existing treatments or generate new drugs [28].
The toxicity in non-transformed cells was also evaluated in vitro
(Table 1). To do so, the non-transformed but immortalized keratinocyte
cell line HaCaT and the immortalized human embryonic kidney cell line
HEK 293, as a proof of concept, were treated with only the lead com
pound of the series and using cisplatin as the reference metallodrug.
RUSCO-2, as the lead compound of the series, did not reduce the toxicity
of cisplatin showed in non-tumoral cells (see Fig. 7 and Table 1).
2.4. Reactive Oxygen Species (ROS) generation by RUSCO compounds
Metal complexes can directly or indirectly alter the cellular redox
balance through redox pathways. Consequently, studies on reactive
oxygen species (ROS) generation have been carried out to ascertain
whether the observed antitumor activity could be attributed to redox
reactions [15]. The assessment of ROS generation by RUSCO-1-RUSCO5 was conducted in the MCF7 breast cancer cell line, using H2O2 as a
positive control. All the complexes were tested at a concentration of 3
μM. As depicted in Fig. 8, none of the compounds generated ROS
compared to the negative control. Therefore, the superior activity of
compound RUSCO-2 is not attributed to ROS generation.
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Journal of Inorganic Biochemistry 253 (2024) 112486
Fig. 7. Impact on cell viability of RUSCO-2 and cisplatin in non-transformed HaCaT cells (A) and in an immortalized human embryonic kidney cell line, HEK 293 (B),
for 72 h evaluated using MTTs. Data are the average +/− standard deviation (SD) of three independent experiments performed in triplicate.
Thus, the dynamic behavior of RUSCO-3 shows a monoexponentially
decay upon excitation at 368 nm leading to a fluorescence lifetime of
9.76 ns, see Fig. 9B, which make it easy to monitor by FLIM. Such mi
croscopy is an excellent tool to monitor both cellular uptake and intra
cellular diffusion of fluorescent drugs because of the high sensitivity of
fluorescence lifetimes to the local environment, as well as to possible
chemical transformations that drugs or pre-drugs may undergo. Being
very useful to follow action mechanisms and intracellular localization in
compartments and/or organelles. The FLIM images of MCF7 cell line
following at 1, 2, 4, 8, 24 and 48-h incubation (Fig. 9C) demonstrate the
rapid internalization and excellent stability in the biological environ
ment of RUSCO-3. The compound is observed exclusively in the cyto
plasm from the first hour of incubation and is not present in the nucleus
at longer times. However, in the images, a certain accumulation is
observed in very specific areas within the cytoplasm suggesting two
different populations of RUSCO-3 coexisting.
To begin with the histogram analysis, the overall average lifetime
distribution for all the cell lines is narrow and spans from 5 to 12.5 ns.
No differences are observed in the histograms at different incubation
times, all being centered around 8.3 ns. Detailed analysis of the lifetime
distribution histograms requires two Gaussian functions for an accurate
fit, which suggests that RUSCO-3 is present in different forms in the cell
environment (Fig. 9D and S32 in the Supporting Information). The
deconvolution shows that the dominant contribution arises from a
population of RUSCO-3 molecules with lifetimes of 5–10 ns, with an
average lifetime centered at 8.1 ns (55%) and appears homogeneously
distributed within the cytoplasm. On the other hand, the maximum
value of the lifetime for the second population is significantly different:
~9 ns centered and shifted up to 12.5 ns (45%). This shift to longer
lifetimes suggests a more hydrophobic environment for this second
population [59], which are represented as sub-micron aggregates in the
images, probably due to accumulation in lipid droplets, endoplasmic
reticulum via interactions with macromolecules, or mitochondria.
To clarify the nature of these accumulations of RUSCO-3 with a
hydrophobic environment inside, cell colocalization experiments were
performed. MCF7 cells were treated with a specific mitochondrial dye
(mitotracker) capable of exclusively staining the mitochondria. Thus, we
can excite both RUSCO-3 and the mitotracker to obtain simultaneous
colocalization. Fig. 9E shows the images when the mitrotracker and
RUSCO-3 are excited separately and when both merges. The results
show that mitochondria are not the preferred site of accumulation.
Finally, further analysis in the nucleus and cytoplasm as a region of
interest shows that lifetime distribution histogram shifts to lower
average lifetime values (7.7 ns) in the nucleus when compared to the
cytoplasm (8.3 ns), and besides that, RUSCO-3 population is practically
Fig. 8. Mean Fluorescence Intensity as a quantification of ROS generation by
RUSCO-1-RUSCO-5 and comparing it to H2O2 as a positive control. Data are the
average +/− standard deviation (SD) of three independent experiments per
formed in triplicate. To determine significant statistical differences, a Student’s
t-test was used. The values for the statistical analyses are * p ≤ 0.05; ** p ≤
0.01; *** p ≤ 0.001; **** p ≤ 0.0001.
2.5. Uptake studies for RUSCO compounds
Taking advantage of the fact that RUSCO-3 displays luminescent
properties, this compound was monitored in human breast cancer MCF7
cell lines by fluorescence lifetime imaging microscopy (FLIM) as a proof
of concept to gain insight into their mechanism of action (Fig. 9). First,
the photophysical behavior of L3 and RUSCO-3 was studied. Fig. 9A and
S34 shows the absorbance and fluorescence spectra of this complex and
its ligand in pH 7.4 PBS buffer. The electronic absorption spectrum
shows high energy contributions within 200–300 nm with a welldefined intense band centred at 257 nm that can be assigned to intra
ligand π → π* and n → π* transitions from heteroscorpionate and pcymene ligands [57]. The typical spectrum of anthracene with vibronic
structure can be observed at 370 nm, whereas no MLCT transitions at
low energies are observed [58]. In such a way that the fluorescence
spectrum corresponds exclusively to the emission of the anthracene
ligand as an independent entity: a mirror image emission spectrum
within 370–600 nm where the vibronic structure is easily appreciated.
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Journal of Inorganic Biochemistry 253 (2024) 112486
Fig. 9. A) Absorption and emission spectra of RUSCO-3 pH 7.4 PBS buffer. B) Fluorescent decay profile of RUSCO-3 in pH 7.4 PBS buffer. C) FLIM images of MCF7
cell lines after treatment with RUSCO-3 at different times (1 h - 48 h). D) Overall histogram and deconvolution of the average emission lifetimes (1 h – 48 h) collected
as average of each FLIM images. E) MCF7 cell treated with mitotracker, RUSCO-3, and merged.
negligible in the nucleus when compared to the cytoplasm (7% vs. 93%)
according to the areas of the respective histograms (see Fig. S33).
Madrid, Spain). The UV–vis absorption spectra were recorded at room
temperature using a Cary 100 spectrophotometer (Agilent, Madrid,
Spain) using a slit width of 0.4 nm and a scan rate of 600 nm/min.
Microanalyses were performed with a PerkinElmer 2400 CHN analyzer
3. Experimental section
3.1. Synthesis and characterization
3.1.2. Synthesis and characterization of ligands L1-L5
To a solution of bis(3,5-dimethyl-1H-pyrazol-1-yl)methane (2.00 g,
9.77 mmol) in THF (50 mL) at − 78 ◦ C was added dropwise n-butyl
lithium 1.6 M (6.11 mL, 9.77 mmol). After 1 h stirring time under N2
atmosphere, the solution was transferred via cannula to another Schlenk
containing the corresponding aldehyde (9.79 mmol), and the mixture
was allowed to stir for 1 h at room temperature. An aqueous saturated
NH4Cl solution (50 mL) was added to protonate the ligand. After
extracting the organic phase and removing the solvent under vacuum,
the resultant solid was washed with n-hexane. Filtration of the precipi
tate afforded a yellowish solid. L1 and L2 were synthesized following the
procedure described in the literature.13
3.1.1. General procedure
Synthesis reactions were performed using standard Schlenk and
glove-box techniques under an atmosphere of dry nitrogen. THF and
hexane were pre-dried over sodium wire and distilled under nitrogen.
CDCl3, DMSO‑d6, and D2O were stored over activated 4 Å molecular
sieves and degassed by several freeze-thaw cycles. All NMR experiments
were conducted in deuterated solvents at 297 K in a Varian FT-400
spectrometer (VARIAN Inc., Palo Alto city, California, USA) equipped
with a 4 nucleus ASW PFG 1H/19F/13C/{15N–31P}. The 1H π/2 pulse
length was adjusted for each sample. 1H- and 13C{1H}-NMR chemical
shifts (δ) are given in ppm relative to TMS. Coupling constants (J) are
documented in Hz. The solvent signals were used as references and
chemical shifts were converted to the TMS scale. IR experiments were
conducted on an FT/IR-4000 series Jasco instruments (Jasco analytics,
3.1.3. 1-(anthracen-9-yl)-2,2-bis(3,5-dimethyl-1H-pyrazol-1-yl)ethan-1ol (bpzanteH; L3)
Yield: 3.26 g, 7,86 mmol, 80%. 1H NMR (400 MHz, CDCl3) δ:
7
E. Domínguez-Jurado et al.
Journal of Inorganic Biochemistry 253 (2024) 112486
(CH, iPr-p-cym), 23.3 (Me, iPr-p-cym), 22.7 (Me, iPr-p-cym), 21.1 (Me, pcym), 18.8 (Me3), 17.4 (Me3’), 17.1 (Me, p-tolyl), 12.3 (Me5), 10.9
(Me5’). UV–vis: maximum absorbance at 207 nm. Elemental analysis
calculated (%) for C29H38Cl2N4ORu: C, 55.23; H, 6.07; N, 8.88 found C,
55.30; H, 6.26; N, 8.71.
9.50–7.24 (set of bs, 9H, anthracene group, 7.46 (d, JHH = 8.9 Hz, 1H,
CHa), 6.79 (d, JHH = 8.9 Hz, 1H, CH-OH), 5.94 (s, 1H, H4), 5.47 (s, 1H,
OH), 5.01 (s, 1H, H4’), 2.38 (s, 3H, Me3), 1.96 (s, 3H, Me3’), 1.95 (s, 3H,
Me5), 0.89 (s, 3H, Me5’). 13C{1H}- NMR (101 MHz, CDCl3) δ:
149.1–120.0 (C3,3′, C5,5′ and carbon from anthracene group), 106.8 (C4),
104.9 (C4’), 70.8 (CH-OH), 70.4 (Ca), 14.0 (Me3), 13.8 (Me3’), 10.8
(Me5), 9.5 (Me5’). UV–vis: two maximum absorbances at 255 and 207
nm. Elemental analysis calculated (%) for C26H26N4O: C, 76.07; H, 6.38;
N, 13.65; found: C, 76.16; H, 6.41; N, 13.46.
3.1.8. [RuCl(κ2-NN-bpzFerrH)(p-cym)][Cl] (RUSCO-2)
Yield: 0.28 g, 0.38 mmol, 79%. 1H NMR (400 MHz, CDCl3) δ: 7.10 (d,
JHH = 7.0 Hz, 1H, OH), 6.75 (brs, 1H, Ar-p-cym), 6.50 (brs, 1H, Ar-pcym), 6.08 (s, 1H, H4), 6.01 (brs, 1H, Ar-p-cym), 5.89 (s, 1H, H4’), 5.85
(brs, 1H, Ar-p-cym), 5.60 (d, JHH = 9.5 Hz, 1H, CHa), 5.49 (m, 1H, CHOH), 4.66 (s, 1H, C5H4), 4.31 (s, 5H, C5H5), 4.17 (s, 1H, C5H4), 3.99 (s,
1H, C5H4), 3.31 (s, 1H, C5H4), 3.12 (m, 1H, iPr-p-cym), 2.60 (s, 3H, Me3),
2.55 (s, 3H, Me3’), 2.47 (s, 3H, Me-p-cym), 2.40 (s, 3H, Me5), 1.55 (s, 3H,
Me5’), 1.42 (d, JHH = 6.7 Hz, 3H, iPr-p-cym), 1.31 (d, JHH = 6.7 Hz, 3H,
i
Pr-p-cym). 13C{1H}-NMR (101 MHz, CDCl3) δ: 156.5–125.8 (C3,3′, C5,5′
and quaternary carbons of p-cym), 110.0, 109.5 (C4,4′), 110.8–80.0 (Arp-cym), 72.9 (Ca), 69.2 (C5H5 and CH-OH), 68.3–65.8 (C5H4), 31.3 (CH,
i
Pr-p-cym), 23,4, 23.1 (Me, iPr-p-cym), 18.9 (Me, p-cym), 17.4 (Me3),
17.2 (Me3’), 12.5 (Me5), 11.6 (Me5’). UV–vis: maximum absorbance at
207 nm. Elemental analysis calcd (%) for C32H40Cl2FeN4ORu: C, 55.78;
H, 5.85; N, 8.13; found C, 55.84; H, 5.90; N, 8.04.
3.1.4. 2,2-bis(3,5-dimethyl-1H-pyrazol-1-yl)-1-(pyridin-2-yl)ethan-1-ol
(bpzpyeH; L4)
Yield: 2.47 g, 7.92 mmol, 81%. 1H NMR (400 MHz, CDCl3) δ 8.56 (d,
JHH = 4.8 Hz, 1H, CH6 pyridine), 7.51 (m, JHH = 7.7 Hz, 1H, CH4 pyr
idine), 7.16 (m, 1H, CH5 pyridine), 7.07 (d, J = 7.8 Hz, 1H, CH3 pyri
dine), 6.44 (d, J = 6.6 Hz, 1H, CHa), 5.93 (d, J = 6.6 Hz, 1H, CH-OH),
5.77 (s, 1H, H4), 5.63 (s, 1H, H4’), 5.36 (s, 1H, OH), 2.20 (s, 3H, Me3),
2.17 (s, 3H, Me3’), 2.07 (s, 3H, Me5), 1.93 (s, 3H, Me5’). 13C{1H}- NMR
(101 MHz, CDCl3) δ 157.7 (C2 pyridine), 148.7 (C6 pyridine), 148.6,
148.3, 140.5, 139.7(C3,3′, C5,5′), 136.5 (C4 pyridine), 123.0 (C5 pyri
dine), 122.5 (C3 pyridine), 106.4 (C4), 105.9 (C4’), 74.9 (Ca), 72.4 (CHOH), 13.7 (Me3), 13.7 (Me3’), 10.9 (Me5), 10.6 (Me5’). UV–vis:
maximum absorbance at 207 nm. Elemental analysis calculated (%)
C17H21N5O: C, 65.57; H, 6.80; N, 22.49; found: C, 65.61; H, 6.83; N,
22.11.
3.1.9. [RuCl(κ2-NN-bpzanteH)(p-cym)][Cl] (RUSCO-3)
Yield: 0.29 g, 0.41 mmol, 82%. 1H NMR (400 MHz, CDCl3) δ:
9.61–6,49 (m, 9H, anthracene group), 7.96 (d, JHH = 8.4 Hz, 1H, CHOH), 7.44 (d, JHH = 8.0 Hz, 1H, CHa), 6.98 (brs, 1H, Ar-p-cym), 6.34 (d,
JHH = 5.7 Hz, 1H, Ar-p-cym), 6.22 (s, 1H, H4), 6.05 (d, JHH = 5.1 Hz, 1H,
Ar-p-cym), 5.96 (d, JHH = 5.5 Hz, 1H, Ar-p-cym), 5.00 (s, 1H, H4’), 3.27
(m, 1H, iPr-p-cym), 2.70 (s, 3H, Me3), 2.64 (s, 3H, Me3’), 2.30 (s, 3H, Mep-cym), 2.27 (s, 3H, Me5), 1.46 (d, JHH = 6.6 Hz, 3H, iPr-p-cym), 1.20 (d,
JHH = 6.9 Hz, 3H, iPr-p-cym), 0.38 (s, 3H, Me5’). 13C{1H}-NMR (101
MHz, CDCl3) δ: 158.3, 156.5, 147.3, 145.4 (C3,3′, C5,5′), 131.7–125.2
(carbons from anthracene group and quaternary carbons of p-cym),
110.8–80.0 (Ar-p-cym), 110.6 (C4), 108.6 (C4’), 71.2 (CH-OH), 70.7
(Ca), 32.0 (CH, iPr-p-cym), 24.2, 21.8 (Me, iPr-p-cym), 18.5 (Me, p-cym),
17.4 (Me3), 16.9 (Me3’), 13.3 (Me5), 9.8 (Me5’). UV–vis: maximum
absorbance at 207 nm. Elemental analysis calculated (%) for
C36H40Cl2N4ORu: C, 63.47; H, 5.92; N, 8.22; found C, 63.52; H, 6.08; N,
8.19.
3.1.5. 2,2-bis(3,5-dimethyl-1H-pyrazol-1-yl)-1-(quinolin-2-yl)ethan-1-ol
(bpzqeH; L5)
Yield: 2.53 g, 7.01 mmol, 72%. 1H NMR (400 MHz, CDCl3) δ 8.09 (d,
J = 8.5 Hz, 1H, CH8 quinoline), 8.00 (d, JHH = 8.5 Hz, 1H, CH3 quino
line), 7.78 (d, JHH = 8.1 Hz, 1H, CH5 quinoline), 7.71 (m, 1H, CH7
quinoline), 7.53 (m, 1H, CH6 quinoline), 7.18 (d, JHH = 8.5 Hz, 1H, CH4
quinoline), 6.59 (d, JHH = 6.8 Hz, 1H, CHa), 6.12 (s, 1H, CH-OH), 5.78 (s,
1H, H4), 5.64 (s, 1H, H4’), 5.60 (s, 1H, OH), 2.22 (s, 3H, Me3), 2.20 (s,
3H, Me3’), 2.15 (s, 3H, Me5), 1.92 (s, 3H, Me5’). 13C{1H}-NMR (101
MHz, CDCl3) δ 159.1–110.0 (C3,3′, C5,5′ and carbon from quinoline
group), 106.5 (C4), 106.1 (C4’), 75.0 (CH-OH), 72.6 (Ca), 13.8 (Me3,3′),
11.1 (Me5), 10.7 (Me5,5′). UV–vis: maximum absorbance at 207 nm.
Elemental analysis calculated (%) for C21H23N5O: C, 69.78; H, 6.41; N,
19.38; found C, 69.88; H, 6.66; N, 19.23.
3.1.10. [RuCl(κ2-NN-bpzpyeH)(p-cym)][Cl] (RUSCO-4)
Yield: 0.25 mg, 0.40 mmol, 80%. 1H NMR (400 MHz, CDCl3) δ: 8.27
(d, JHH = 4.0 Hz, 1H, pyridine group), 8.04 (d, JHH = 7.9 Hz, 1H, pyr
idine group), 7.74 (m, 1H, pyridine group), 7.29 (d, JHH = 5.9 Hz, 1H,
CHa), 7.15 (dd, JHH = 7.4, 4.9 Hz, 1H, pyridine group), 6.46 (d, JHH =
6.2 Hz, 1H, Ar-p-cym), 6.29 (d, JHH = 6.2 Hz, 1H, Ar-p-cym), 6.10 (m,
4H, Ar-p-cyme, H4, CHOH), 5.85 (s, 1H, H4’), 3.13 (m, 1H, iPr-p-cym),
2.60 (s, 3H, Me3), 2.59 (s, 3H, Me3’), 2.46 (s, 3H, Me-p-cym), 2.38 (s, 3H,
Me5), 1.62 (s, 3H, Me5’), 1.23 (d, JHH = 6.8 Hz, 3H, iPr-p-cym), 1.16 (d,
JHH = 6.8 Hz, 3H, iPr-p-cym). 13C{1H}-NMR (101 MHz, CDCl3) δ:
158.8–122.8 (C3,3’,C5,5′, pyridine group and quaternary carbons of pcym), 109.7 (C4), 109.3 (C4), 110.5–80.0 (Ar-p-cym), 71.6 (CH-OH),
70.9 (Ca), 31.3 (CH, iPr-p-cym), 24.1, 23.2 (Me, iPr-p-cym), 18.6 (Me, pcym), 17.2 (Me3), (Me3’), 12.4 (Me5), 11.2 (Me5’). UV–vis: maximum
absorbance at 207 nm. Elemental analysis calculated (%) for
C27H35Cl2N5ORu: C, 52.51; H, 5.71; N,11.34; found C, 52.67; H, 5.82; N,
11.22.
3.1.6. Synthesis and characterization of ruthenium compounds RUSCO-1RUSCO-5
The ruthenium dimer [RuCl2(p-cym)]2 (0.25 mmol, 0.15 g) was
dissolved in MeOH/THF 3:1 and stirred for 30 min. The corresponding
ligand (0.50 mmol) was added to the red solution and the mixture was
allowed to stir for 4 h at room temperature and under N2 atmosphere.
The colour of the solution shifted from red to orange. The solvent was
removed under vacuum and the residual power was washed with
diethylether (5 mL) and then with n-hexane (10 mL). The solid was dried
under vacuum providing the ruthenium complexes as orange solids.
UNICAM-1 was synthesized following the procedure described in the
literature [40].
3.1.7. [RuCl(κ2-NN-bpzteH)(p-cym)][Cl] (RUSCO-1)
Yield: 0.253 g, 0.40 mmol, 80%. 1H NMR (400 MHz, CDCl3) δ 7.02
(d, JHH = 7.7 Hz, 2H, p-tolyl), 6.95 (d, JHH = 8.0 Hz, 2H, p-tolyl), 6.50
(brs, 1H, CHa), 6.23 (d, JHH = 5.8 Hz, 1H, Ar-p-cym), 6.12 (s, 1H, H4),
6.02 (d, JHH = 5.9 Hz, 1H, Ar-p-cym), 5.90 (brs, 2H, H4’ and 1H Ar-pcym), 5.82 (brs, 1H, Ar-p-cym), 5.69 (d, JHH = 9.6 Hz, 1H, CH-OH), 3.16
(m, 1H, iPr-p-cym), 2.65 (s, 3H, Me3), 2.58 (s, 3H, Me3’), 2.44 (s, 3H,
Me5), 2.37 (s, 3H, Me-p-cym), 2.28 (s, 3H, Me-p-tolyl), 1.34 (s, 3H,
Me5’), 1.32 (d, JHH = 2.7 Hz, 3H, iPr-p-cym), 1.30 (d, JHH = 2.7 Hz, 3H,
i
Pr-p-cym). 13C{1H}-NMR (101 MHz, CDCl3) δ: 156.9–125.0 (C3,3′, C5,5′,
quaternary carbons of p-cym and carbons of Ar-p-tolyl), 110.0, 109.4
(C4,4′), 100.3, 86.4, 85.7, 81.1 (Ar-p-cym), 72.7 (Ca), 70.8 (CH-OH), 31.3
3.1.11. [RuCl(κ2-NN-bpzqeH)(p-cym)][Cl] (RUSCO-5)
Yield: 0.29 g, 0.43 mmol, 86%. 1H NMR (400 MHz, CDCl3) δ:
8.22–7.52 (m, 6H, quinoline group), 6.62 (d, JHH = 5.1 Hz, 1H, Ar-pcym), 6.35 (d, JHH = 5.6 Hz, 1H, Ar-p-cym), 6.28 (brs 2H, CH-OH and
CHa), 6.13 (s, 1H, H4), 6.10 (d, JHH = 5.7 Hz, 1H, Ar-p-cym), 6.07 (d,
JHH = 5.2 Hz, 1H, Ar-p-cym), 5.87 (s, 1H, H4’), 3.14 (m, 1H, iPr-p-cym),
2.64 (s, 3H, Me3), 2.62 (s, 3H, Me3’), 2.50 (s, 3H, Me5), 2.41 (s, 3H, Me8
E. Domínguez-Jurado et al.
Journal of Inorganic Biochemistry 253 (2024) 112486
p-cym), 1.68 (s, 3H, Me5’), 1.24 (d, JHH = 6.7 Hz, 3H, iPr-p-cym), 1.10 (d,
JHH = 6.7 Hz, 3H, iPr-p-cym). 13C{1H}-NMR (101 MHz, CDCl3) δ:
159.3–126.3 (C3,3’,C5,5′, quinoline group and quaternary carbons of pcym), 109.8, 109.5 (C4,4′), 110.8–80.1 (Ar-p-cym), 70.4, 70.3 (Ca and
CH-OH), 31.4 (CH, iPr-p-cym), 23,6, 22.8 (Me, iPr-p-cym), 18.5 (Me, pcym), 18.7 (Me3), 17.5 (Me3’), 17.3 (Me5), 11.7 (Me5). UV–vis:
maximum absorbance at 207 nm. Elemental analysis calculated (%) for
C31H37Cl2N5ORu: C, 55.77; H, 5.59; N,10.49; found C, 55.90; H, 5.70; N,
10.48.
cultured (5 × 104 cells per plate) at 37 ◦ C in a 5% CO2 humidified at
mosphere with DMEM medium without phenol red. After 24 h RUSCO-3
(10 μM) was added to the cell and incubated for 1 and 8 h.
For colocalization experiments, cells were incubated with DMEM
medium without phenol red with RUSCO-3 (10 μM) for 24 h. The me
dium is then removed, three washes are carried out with PBS and DMEM
medium without phenol red and then mitotracker deep red FM (Ther
mofisher) (1 nM) is added and incubated for 10 min. Finally, the me
dium is removed and washed again with PBS and the images are
recorded in FLIM.
3.2. X-ray crystallography characterization
3.3.3. Fluorescence Lifetime Imaging of cells
Fluorescence lifetime images were recorded with a MicroTime 200
microscope (PicoQuant) equipped with a TCSPC card and two TAUSPAD-100 avalanche photodiode detectors. A 375 nm and 637 nm
pulsed diode lasers (LDH-D-C-375, 637 PicoQuant) were used as exci
tation source at 10 MHz repetition rate and a power of ~0.4 μW. The
emission was recorded with long-pass filter (− 519/19 LP). 80 × 80 μm
regions were scanned with 156 nm/pixel spatial resolution and 2 ms of
dwell time. FLIM images were processed using SymphoTime64 software
(PicoQuant). The lifetime distribution histograms were obtained from
FLIM images and were fitted to Gaussian curve. The FLIM images were
smoothed over 200 nm for clarity of presentation. The emission spectra
and the histograms were averaged over 3 independent measurements.
For colocalization experiments, firstly, the signal emitted by the mito
tracker upon excitation at 637 nm is recorded using the specific condi
tions of the compound (λem = 650–725 nm, 690/70 bandpass filter), and
then an additional image is recorded with the emission of RUSCO-3
upon excitation at 375 nm under its specific conditions (λem >405
nm, 405 bandpass filter). Two images were taken from each group of
cells (one corresponding to the mitotracker and the other to RUSCO-3),
using three different cell groups as samples (6 photos in total).
Prismatic crystals for RUSCO-2 and RUSCO-3 were mounted on a
mitogen mount and used for data collection on a Bruker D8 Venture with
a Photon II detector equipped with graphite monochromated MoKα ra
diation (λ = 0.71073 Å) (Bruker Apex2, Bruker AXS Inc., Madison, WI,
USA, 2004). The data reduction was performed with the APEX2 software
and corrected for absorption using SADABS [60]. Crystal structures were
solved by SHELXT program [61] and refined by full-matrix least-squares
on F2 including all reflections using anisotropic displacement parame
ters by means of the OLEX2 and SHELX crystallographic package
[61,62]. Final R(F), wR(F2), and goodness of fit agreement factors, de
tails on the data collection and analysis can be found in Table S1 of the
Supporting Information.
3.3. Uptake studies
3.3.1. Spectral equipment and measurements
Steady-state fluorescence (SSF) spectra were recorded on an FLS920
spectrofluorometer (Edinburgh Instruments) equipped with an MCPPMT (microchannel plate-photomultiplier tube) detector (R3809
model) and a TCSPC (time-correlated single photon counting) data
acquisition card (TCC900 model). A Xe lamp of 450 W was used as the
light source for SSF spectra, and a sub-nanosecond pulsed Light-Emitting
Diode, EPLED-360 (Edinburgh Photonics), was employed as a light
source for Time-resolved fluorescence decays (TRF). A TLC 50
temperature-controlled cuvette holder (Quantum Northwest) was used
to keep the temperature at 25 ◦ C during the spectra acquisition. Exci
tation and emission slits were both fixed at 4 nm, the step 1 nm and the
dwell time was 0.1 s. The excitation wavelength (λex) was 368 nm,
emission wavelength (λem) was 520 nm and the Δλem was 10 nm. All
measurements were performed using a 10 mm quartz cuvette (Hellma
Analytics).
Solutions of RUSCO-3 (10 μM) were prepared in pH 7.4 PBS solvent
from stock solution (1 mM) of RUSCO-3 in DMSO solvent.
The fluorescence intensity decay, I(t), was fitted to the following
multiexponential function using an iterative least-squares fit method.
n
∑
I(t) =
αi exp( − t/τi )
3.4. Biological assays
The compounds were dissolved in dimethyl sulfoxide (DMSO) before
performing each experiment. The maximal concentration used was 20
μM, due to limited water solubility; cisplatin was tested up to 14 μM. The
same volume of solvent was added to control conditions and did not
exceed 0.25% v/v.
3.4.1. Cell culture studies
The cell lines MCF7, SKBR3, BT549 and MDA-MB-231, the immor
talized non-transformed keratinocyte cell line HaCaT and the immor
talized human embryonic kidney cell line HEK 293 were acquired in
ATCC. All lines were grown in DMEM containing 10% fetal bovine
serum (FBS) and were supplemented with 100 U/mL penicillin, 5 mM Lglutamine, 100 μg/mL streptomycin at 37 ◦ C and 5% CO2 (SigmaAldrich, St. Louis, MO, USA). Cisplatin was purchased from Accord
Healthcare (the United Kingdom. MA).
(1)
i=1
3.4.2. MTT metabolization assays
For viability assessment of L1-L5 and RUSCO-1-RUSCO-5, cell pro
liferation was assayed by MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5
diphenyltetrazolium bromide) (Sigma Aldrich). Cell lines were plated at
5.000 cells per well in 96-multiwell plates. 24 h later, the cells were
treated at correspondent doses of the drugs for 72 h. After that, the
medium was aspirated and phenol red-free DMEM with MTT 0.5 mg/mL
was added for 15 min in growth conditions. The medium was removed
and MTT crystals were solubilized with 0.1 mL of dimethyl sulfoxide
(DMSO) (Sigma-Aldrich) and evaluated at an absorbance of 555 nm in a
multiwell plate reader.
where αi and τi are the amplitude and lifetime for each ith term. The
mean lifetime of the decay was then calculated as:
n
∑
τ
i=1
m = n
∑
αi τ2i
αi τ i
(2)
i=1
3.3.2. Cell cultures for FLIM microscopy
Breast cancer cells MCF7 were grown in Dulbecco’s modified Eagle
medium (DMEM) without phenol red. Each medium was supplemented
with 10% inactivated fetal bovine serum, 1% L-glutamine, 1% peni
cillin/streptomycin. Cell cultures were incubated at 37 ◦ C in a saturated
humidity atmosphere with 5% of CO2. For FLIM experiments, cells were
seeded onto 20 mm square glass cover slides into 6-well plate and
3.4.3. Generation of ROS species
The fluorescence intensity related to the generation of ROS was
measured with a FLUOstar OPTIMA microplate reader. 2′,7′9
E. Domínguez-Jurado et al.
Journal of Inorganic Biochemistry 253 (2024) 112486
dichlorodihydrofluorescein diacetate (H2DCFDA) from Sigma Aldrich
was used as the ROS indicator. MCF7 cell line was plated at 2000 cells
per well in 96-multiwell plates and incubated for 24 h at 37 ◦ C and 5% of
CO2. After that, the medium was aspirated, and the cells were treated at
3 μM of each RUSCO compound and at 8 μM of H2O2 as the positive
control. 24 h later, the medium was removed, and the cells were washed
with PBS two times. 2′,7′-dichlorodihydrofluorescein diacetate was then
added at a final concentration of 10 μM in PBS. The plate was incubated
30 min and the fluorescence intensity was recorded at 520 nm.
are observed in the cytoplasm, one probably due to the free compound
and the other due to its accumulation in the hydrophobic zones of the
endoplasmic reticulum by interaction with macromolecules or inside
lipid droplets. Finally, RUSCO-3 scarcely reach the cell nucleus, ruling
out a mechanism of action based on DNA targeting in any way, as it has
been broadly reported for other metallodrugs [63].
While further rational modification of the ligand structure is required
together with further biological studies to ascertain the mechanism of
action, this work shows the potential of heteroscorpionate ligands in
both high throughputs testing and the rational design of new anticancer
metallodrugs as plausible therapeutic options for breast cancer therapy.
3.4.4. Statistical analysis
The in vitro experiments data are the average of three independent
experiments performed in triplicate, with error bars showing the stan
dard deviation of the triplicates. To determine if there are statistical
differences, a Student’s t-test was performed comparing the mean
cytotoxicity of each RUSCO at 4 μM with UNICAM-1 per cell line. The
values for the statistical analyses are: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤
0.001.
Funding sources
We gratefully acknowledge the financial support from the Ministerio
de Ciencia e Innovación y Agencia Estatal de la Investigación, Spain
(grants CPP2021-008597, PID2020-117788RB-I00 and RED2022134287-T funded by MCIN/AEI/10.13039/501100011033), grants
SBPLY/21/180501/000050 and SBPLY/21/180501/000132 funded by
JCCM and by EU through Fondo Europeo de Desarrollo Regional, grant
2021-GRIN-31240 funded by Universidad de Castilla-La Mancha and
Instituto de Salud Carlos III (grant number PI16/01121). Alberto
Ocaña’s lab is supported by the Instituto de Salud Carlos III (ISCIII,
PI19/00808).
4. Conclusions
Metallodrugs are a well-established group of chemotherapeutic
agents for the treatment of cancer. Most combination therapies include
platinum-based agents. However, platinum-based therapies often
develop resistance and display severe side effects. In recent years, the
biological properties and anticancer potential of ruthenium compounds
have been intensively investigated, nevertheless metallodrug discovery
still relies heavily on the screening of compound libraries. Rational
design of the ruthenium metallodrugs structure and elucidation of
structure-activity relations is fundamental to improving the pharmaco
logical profile of the therapeutic agents. In this sense, heteroscorpionate
ligands are prone to stabilize from early to late translation metals, being
potential candidates as building blocks for the next generation of novel
metallodrugs. To do so, several ruthenium-based metallodrugs were
obtained, fully characterized, and assessed as antitumoral agents against
a panel of representative breast cancer cells. In particular, the derivative
RUSCO-2 showed promising selectivity, as it preserved higher cytotox
icity than cisplatin against TNBC cells, being less toxic in nontransformed cells.
RUSCO-2 exhibited higher cytotoxicity within a panel of breast
cancer cells, representing the different subtypes of breast cancer. In
vitro, RUSCO-2 demonstrated a better antitumor profile compared to the
reference drug cisplatin and UNICAM-1. However, nonspecific cyto
toxicity effects on non-tumoral cells were also observed, similar to those
seen with cisplatin therapy. Despite attempts to establish a relationship
between structure and activity, such efforts were unsuccessful. After
conducting stability studies, ROS generation studies, and the estimation
of the lipophilicity values, the higher cytotoxicity of RUSCO-2 could not
be attributed. RUSCO-2 is not the most lipophilic compound of the se
ries, does not exhibit ROS generation, and shows similar solubility in
water as well as stability in organic solvents or simulated biological
media compared to its counterparts. Identifying a direct relationship
between cytotoxic activity and the chemical structure of a drug is
challenging. Numerous factors including physical and chemical prop
erties of the drugs, affinity for the drug target, or potential interactions
with other biomacromolecules could be involved in the antitumor ac
tivity of these compounds.
Results from uptake studies carried out on RUSCO-3, as a proof of
concept, may reveal several major phenomena for this family of de
rivatives. First, the uptake of RUSCO-3 is rapid at short incubation times
and is evenly distributed in the cytoplasm. Second, no significant
changes in the overall average lifetime distribution histograms are
observed over time in the different images, indicating that is not a
prodrug and that the incorporation of the heteroscorpionate ligand
protects it from aqueous processes, conversion in another entity, or the
loss of the chloride group. Third, two different populations of RUSCO-3
CRediT authorship contribution statement
Elena Domínguez-Jurado: Methodology, Investigation. Consuelo
Ripoll: Investigation, Formal analysis, Data curation. Agustín LaraSánchez: Writing – review & editing, Supervision, Resources. Alberto
Ocaña: Writing – review & editing, Resources. Iñigo J. VitóricaYrezábal: Investigation, Formal analysis. Iván Bravo: Writing – orig
inal draft, Supervision, Software, Methodology, Data curation,
Conceptualization. Carlos Alonso-Moreno: Writing – review & editing,
Writing – original draft, Validation, Supervision, Resources, Project
administration, Investigation, Funding acquisition, Conceptualization.
Declaration of competing interest
Carlos Alonso Moreno reports financial support was provided by
Spain Ministry of Science and Innovation. Carlos Alonso Moreno reports
financial support was provided by Junta de Comunidades de Castilla-La
Mancha Consejería de Educación Cultura y Deportes. Alberto reports
financial support was provided by Carlos III Health Institute.
Data availability
No data was used for the research described in the article.
Acknowledgements
The authors gratefully acknowledge financial support from ACEPAIN
foundation and AFANION. Consuelo Ripoll thanks the Junta de Comu
nidades de Castilla-La Mancha for her Postdoctoral fellowship (2018/
15132) and Elena Domínguez-Jurado to University of Castilla-La Man
cha for her Predoctoral fellowship (2020-PREDUCLM-16603).
Appendix A. Supplementary data
Crystallographic data table, NMR solution characterization, clogP
estimation, stability studies of compound RUSCO-1-RUSCO-5, and doseresponse curves for RUSCO-1-RUSCO-5 in a panel of breast cancer
tumour cells are depicted in the Supporting Information. CCDC
2277792-2277793 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
10
E. Domínguez-Jurado et al.
Journal of Inorganic Biochemistry 253 (2024) 112486
by contacting The Cambridge Crystallographic Data Centre, 12 Union
Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033. See doi:https
://doi.org/10.1039/x0xx00000x Supplementary data to this article
can be found online at https://doi.org/10.1016/j.jinorgbio.20
24.112486.
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