Gold(Iii) to ruthenium(iii) metal exchange in dithiocarbamato complexes tunes their biological mode of action for cytotoxicity in cancer cells

Article abstract of DOI:10.3390/molecules26134073

Malignant tumors have affected the human being since the pharaoh period, but in the last century the incidence of this disease has increased due to a large number of risk factors, including deleterious lifestyle habits (i.e., smoking) and the higher longevity. Many efforts have been spent in the last decades on achieving an early stage diagnosis of cancer, and more effective cures, leading to a decline in age-standardized cancer mortality rates. In the last years, our research groups have developed new metal-based complexes, with the aim to obtain a better selectivity for cancer cells and less side effects than the clinically established reference drug cisplatin. This work is focused on four novel Au(III) and Ru(III) complexes that share the piperidine dithiocarbamato (pipe-DTC) as the ligand, in a different molar ratio. The compounds [AuCl2(pipeDTC)], [Au(pipeDTC)2]Cl, [Ru(pipeDTC)3] and β-[Ru2(pipeDTC)5] have been synthesized and fully characterized by several chemical analyses. We have then investigated their biological properties in two different cell lines, namely, AGS (gastric adenocarcinoma) and HCT116 (colon carcinomas), showing significant differences among the four compounds. First, the two gold-based compounds and β-[Ru2(pipeDTC)5] display IC50 in the μM range, significantly lower than cisplatin. Second, we showed that [AuCl2(pipeDTC)] and β-[Ru2(pipeDTC)5]Cl drive different molecular mechanisms. The first was able to induce the protein level of the DNA damage response factor p53 and the autophagy protein p62, in contrast to the second that induced the ATF4 protein level, but repressed p62 expression. This study highlights that the biological activity of different complexes bringing the same organic ligand depends on the electronic and structural properties of the metal, which are able to fine tune the biological properties, giving us precious information that can help to design more selective anticancer drugs.

Full text of DOI:10.3390/molecules26134073

molecules
Article
Gold(III) to Ruthenium(III) Metal Exchange in
Dithiocarbamato Complexes Tunes Their Biological Mode of
Action for Cytotoxicity in Cancer Cells
Maria Dalla Pozza 1, Christophe Orvain 2, Leonardo Brustolin 1, Nicolò Pettenuzzo 1, Chiara Nardon 1,
Christian Gaiddon 2,
*
and Dolores Fregona 1,
*
1
Department of Chemical Sciences, University of Padova, Via F. Marzolo 1, 35131 Padova, Italy;
maria.dalla-pozza@chimieparistech.psl.eu (M.D.P.); leo.brus90@gmail.com (L.B.);
nicolo.pettenuzzo.np@gmail.com (N.P.); chiara.nardon@unipd.it (C.N.)
Interface Recherche Fondamentale en Cancérologie, Université de Strasbourg, Inserm UMR_S 1113,
3 av. Molière, 67200 Strasbourg, France; orvain@unistra.fr
2
*
Correspondence: Gaiddon@unistra.fr (C.G.); dolores.fregona@unipd.it (D.F.);
Tel.: +33-38-827-7727 (C.G.); +39-(0)49-827-5159 (D.F.)
Abstract: Malignant tumors have affected the human being since the pharaoh period, but in the last
century the incidence of this disease has increased due to a large number of risk factors, including
deleterious lifestyle habits (i.e., smoking) and the higher longevity. Many efforts have been spent in
the last decades on achieving an early stage diagnosis of cancer, and more effective cures, leading to a
decline in age-standardized cancer mortality rates. In the last years, our research groups have devel-
oped new metal-based complexes, with the aim to obtain a better selectivity for cancer cells and less
side effects than the clinically established reference drug cisplatin. This work is focused on four novel
Au(III) and Ru(III) complexes that share the piperidine dithiocarbamato (pipe-DTC) as the ligand,
in a different molar ratio. The compounds [AuCl2(pipeDTC)], [Au(pipeDTC)2]Cl, [Ru(pipeDTC)3]
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Dalla Pozza, M.; Orvain, C.;
Brustolin, L.; Pettenuzzo, N.; Nardon,
C.; Gaiddon, C.; Fregona, D. Gold(III)
to Ruthenium(III) Metal Exchange in
Dithiocarbamato Complexes Tunes
Their Biological Mode of Action for
Cytotoxicity in Cancer Cells.
and β-[Ru2(pipeDTC)5] have been synthesized and fully characterized by several chemical analy-
ses. We have then investigated their biological properties in two different cell lines, namely, AGS
(gastric adenocarcinoma) and HCT116 (colon carcinomas), showing significant differences among
the four compounds. First, the two gold-based compounds and
the M range, significantly lower than cisplatin. Second, we showed that [AuCl2(pipeDTC)] and
-[Ru2(pipeDTC)5]Cl drive different molecular mechanisms. The first was able to induce the protein
β-[Ru2(pipeDTC)5] display IC50 in
Molecules 2021, 26, 4073. https://
doi.org/10.3390/molecules26134073
µ
β
level of the DNA damage response factor p53 and the autophagy protein p62, in contrast to the
second that induced the ATF4 protein level, but repressed p62 expression. This study highlights
that the biological activity of different complexes bringing the same organic ligand depends on the
electronic and structural properties of the metal, which are able to fine tune the biological properties,
giving us precious information that can help to design more selective anticancer drugs.
Academic Editor: Mauro Ravera
Received: 27 May 2021
Accepted: 29 June 2021
Published: 3 July 2021
Keywords: gastric cancer; metal complexes; chemotherapy; dithiocarbamates; drug mechanism of
action; ER stress; p53; autophagy
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
Nowadays, cancer represents the second most important cause of death and morbidity
Copyright:
© 2021 by the authors.
in Europe, after cardiovascular diseases [
small-molecule anticancer agent is cisplatin, which was serendipitously discovered back in
1965, by Barnett Rosenberg and Loretta Van Camp [ ]. Despite its efficacy against many
1]. In this context, the most used and effective
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This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
2
types of cancers, such as testicular, ovarian and bladder neoplasia, this anticancer agent
presents several severe side effects, including gastrointestinal symptoms, renal tubular
damage, and ototoxicity. Those side effects illustrate the lack of selectivity of cisplatin
for cancer cells, and cause a reduced therapeutic window, which represents the major
Molecules 2021, 26, 4073. https://doi.org/10.3390/molecules26134073
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 4073
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inconveniences of the applicability and efficacy of cisplatin [
3,4
]. These serious limitations,
along with the intrinsic or acquired resistance of some types of tumors to cisplatin [5], have
prompted researchers to design new platinum-based drugs with better pharmacological
efficacy and fewer side effects. Carboplatin and oxaliplatin are approved worldwide as the
second-generation platinum(II) drugs [6], and satraplatin was been the first platinum(IV)
complex to reach phase III clinical trials. Unfortunately, these compounds have also
displayed side effects and resistance mechanisms [7].
For these reasons, in the last decades researchers have focused on the design and
development of novel metal complexes using different transition metals, with the aim to
improve the effectiveness and diminish the side effects of platinum-based drugs. Among
the non-platinum antitumor agents, several gold(III) and ruthenium(III) complexes gained
particular interest because of their strong inhibitory activity on cancer cell growth [8–10].
In addition, the immunosuppressive and anti-inflammatory properties of the gold ion
itself [11] have made gold(III) complexes exciting for testing as anticancer compounds
since the immune tumoral microenvironment became a target of anticancer therapy. On
the other hand, ruthenium(III) complexes present several interesting aspects, such as (i) the
rate of ligand exchange comparable to that of Pt(II) compounds [12], (ii) multiple and
accessible oxidation states [13
environment [15 16], which make this metal particularly interesting in the development of
anticancer agents.
In this context, in the last decades, our research group has been designing some
gold(III)– and ruthenium(III) dithiocarbamato complexes [17 18]. Dithiocarbamates (DTC)
,14], and (iii) the ability to mimic iron in the physiological
,
,
have not accidentally been chosen as ligands; in fact, some dithiocarbamato salts have
displayed good efficacy as chemo-protectants during cisplatin treatment, decreasing its
nephrotoxicity without changing its antineoplastic activity [19]. Indeed, this class of
compounds could diminish the interactions between the metal center and the sulfur-
containing biomolecules, which are generally believed to have negative effects on the
therapeutic efficacy of platinum-based drugs [20,21]. Unfortunately, the applicability of
DTC salts as chemo-protectants is limited by the acute toxicity of free DTC in the body,
which biodegrade to different toxic metabolites, such as carbon disulfide, thiourea, and
alkyl amines [21]. Nevertheless, when the dithiocarbamates are coordinated to a metal
center, the resulting complex is expected to be stable because of the “chelate effect”. This
effect makes the complex decomposition unlikely to occur, excluding the possibility of
unwanted collateral reactions before reaching the cell. Therefore, our complexes have been
designed with the purpose of combining the anticancer activity of the metal center with
the protecting action of the sulfur chelating ligand [22].
In this work, we have focused our attention on two gold(III) and two ruthenium(III)
complexes with piperidine dithiocarbamate as the ligand, which show a satisfactory rate of
synthesis, purity and stability (Figure 1). We have synthesized the two gold derivatives,
although [AuCl2(pipeDTC)] has already been reported in the literature with a very different
synthetic way from our procedure, in order to compare the behavior of gold complexes
with that of ruthenium with the same ligand [23].
After the chemical characterization of the four compounds, special attention has
been addressed to the biological evaluation of their antiproliferative activity and their
mechanisms of action.

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Figure 1. Chemical structures of the ligand K pipeDTC and the gold and ruthenium piperidine dithiocarbamato derivatives.
The compounds’ names and their abbreviations are reported here.
2. Results and Discussion
2.1. Syntheses and Characterization
The four synthesized compounds were prepared, starting from the common ligand
piperidine dithiocarbamato, K pipeDTC. The synthesis of the piperidine dithiocarbamato
salt follows the general route, where the acid–base equilibrium, derived from nucleophilic
attack of the amine to the electrophilic carbon of CS2, is shifted to products by the addition
of one equivalent of base.
For the syntheses of the gold piperidine dithiocarbamates, we started by prepar-
ing the dinuclear precursor [Au(I)(pipeDTC)]2 by the reduction in H[AuCl4]
xH2O with
·
one equivalent of Na2SO3, followed by the addition of one equivalent of K pipeDTC
salt. Then, the compound [AuCl2(pipeDTC)] was synthesized by the oxidative addition
of Cl2 to a suspension of the previously obtained Au(I)-pipeDTC precursor, whereas
[Au(pipeDTC)2]Cl was obtained by adding one equivalent of K pipeDTC to a solution of
the neutral [AuCl2(pipeDTC)] compound.
Concerning ruthenium complexes, both [Ru(pipeDTC)3] and [Ru2(pipeDTC)5]Cl were
obtained from the reaction between the precursor RuCl3 and the three equivalents of the
dithiocarbamato salt K pipeDTC, followed by purification by column gravity chromatogra-
phy. This final step was crucial to separate the mononuclear paramagnetic [Ru(pipeDTC)3]
from the dinuclear diamagnetic [Ru2(pipeDTC)5]Cl.
The starting ligand and the four obtained complexes were characterized by means of
several techniques. The proposed stoichiometry of the four compounds was confirmed by
elemental analyses, indicating a good correlation between the calculated and found values.
FT-IR spectroscopy was useful to identify the synthesized complexes coordination
mode. The presence of a single band v(CSS) in the region 950–1050 cm−1 indicates a
symmetric bidentate coordination mode of the ligand, accordingly to the literature data [24].
Moreover, passing from the pipeDTC ligand to the complexes, a shift in the v(N-CSS) and
v(CSS) to lower wavenumbers is observed, due to the electron-withdrawing character
of the metal center, which is mainly evident for gold complexes (see Table 1 for the IR
vibrations list).

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Table 1. List of the main IR vibrations of the metal complexes.
v(N-CSS)
v(CSS)
Compound
−1
−1
(cm
)
(cm
)
K pipeDTC
1417
1560
1582
965
[Au(pipeDTC)2]Cl
[AuCl2(pipeDTC)]
[Ru(pipeDTC)3]
[Ru2(pipeDTC)5]Cl
1003
1006
1002
1001
1488, 1455, 1440
1505, 1441
The 1H-NMR spectra were recorded in deuterated acetonitrile CD3CN (400.13 MHz,
298 K) to assess the completeness of the dithiocarbamato salt formation. On passing from
the free amine to the corresponding DTC ligand, the signal ascribed to the N–H piperidine
proton disappears and the C–H signals closest to the nitrogen atom (
lower fields (from 2.68 to 4.34 ppm). Indeed, the electron-withdrawing character of the
NCSS moiety of pipeDTC influences the resonance of the protons, making them resonate
at a lower field. On the other hand, the pipeDTC and protons are less affected by
α protons) shift to
α
β
γ
the deshielding effect of the nitrogen atom, thus resonating at higher fields compared
1
to the
α
protons (Table 2). The H-NMR spectra are influenced by the diamagnetic or
paramagnetic character of the metal center. In fact, the spectra of the gold complexes
present peaks in the normal range 0–12 ppm, indicating the diamagnetic character of the
Au(III) center. On the contrary, the spectrum of the [Ru(pipeDTC)3] complex shows a shift
in the
α protons to lower fields, due to the paramagnetic character of the Ru(III) metal
center (Figure S1). On the other hand, the dinuclear complex [Ru2(pipeDTC)5]Cl spectrum
does not present any signal outside the 0–12 ppm range, pointing out the diamagnetic
nature of this derivative (Table 2). This is ascribable to the presence of antiferromagnetic
coupling of the two Ru(III) ions.
Table 2. List of the proton chemical shifts (ppm) of the N–H piperidine, and the DTC derivatives.
α-CH2
(ppm)
β-CH2
(ppm)
γ-CH2
(ppm)
Compound
piperidine
K pipeDTC
2.68
4.34
3.75
3.80
1.40
1.50
1.78
1.79
1.40
1.60
1.78
1.79
[AuCl2(pipeDTC)]
[Au(pipeDTC)2]Cl
26.02
21.95
[Ru(pipeDTC)3]
1.01
1.55
3.95
1.55
[Ru2(pipeDTC)5]Cl
3.88
All the spectra were recorded with a 400.13 MHz spectrometer at 298 K in CD3CN.
As reported in Figure S2, the complexity of the [Ru2(pipeDTC)5]Cl spectrum is caused
by the different coordination mode adopted by the ligands [25], which led them to be
non-magnetically equivalent.
The interaction of Au 1:1, Au 1:2 and Ru 2:5 (see Figure 1 for the structures) in in vitro
active complexes with some model molecules were carried out by 1H-NMR spectroscopy.
The biologically relevant biomolecules, such as the sulfur-containing amino acids cysteine
and methionine, and the histidine, were mimicked by 1-propanethiol (PrSH), dimethyl
sulfide (DMS) and 1-methylimidazole (1-MeIm), respectively. The analyses were carried out
over 24 h at 310 K, in acetonitrile, as it is the most suitable solvent in terms of volatility and
solubility of both the model molecules and the complexes. In fact, the [AuCl2(pipeDTC)]
complex was poorly soluble in DMSO, while chloroform was not chosen because its
volatility did not allow analyses at 37◦C. The molar ratio between the complex and the
-

Molecules 2021, 26, 4073
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model molecule was 1:1 for DMS, and 1:2 for PrSH and 1-MeIm [26]. This study highlighted
that all the three compounds are inert toward dimethyl sulfide and 1-methylimidazole, but
underwent reduction in the presence of the thiol moiety (Figures S3–S11).
2.2. Biological Activity
As a preliminary screening, the in vitro cytotoxicity of the four complexes was investi-
gated toward the adenocarcinoma gastric cell line (AGS) and the human colon carcinoma
cell line (HCT116). These cell lines were chosen since colon cancer is one of the most fre-
quent tumors in industrialized countries, and gastric cancer is one of the most aggressive.
For instance, the overall survival rate at 5 years for a gastric cancer patient is below 25%,
and the median survival is below 12 months [27,28]. First, the inhibition of cell proliferation
(IC50) in two-dimensionally (2D) cultured cells was evaluated over 48 h and 72 h, by means
of the MTT assay.
The results were compared to the reference drug cisplatin. The exposure of both
the cell lines to increasing concentrations of Au 1:1, Au 1:2 and Ru 2:5 led to a notable
inhibition of cell survival, with IC50 values below 1
than those of cisplatin (12–21 M, respectively). Similar results were obtained for 72 h
of treatment. Conversely, Ru 1:3 does not display interesting cytotoxic activity, with IC50
values higher than 70 M. Concerning the HCT116 cell line, the two gold compounds show
µM over 48 h, which are much lower
µ
µ
strong cytotoxicity after 48 h, but this activity diminishes after 72 h (Table 3).
Table 3. IC50 values (µM) for the different compounds (Figure 1), calculated after 48 h and 72 h.
48 h
72 h
Compound
HCT116
AGS
HCT116
AGS
Au 1:1
Au 1:2
Ru 2:5
cis-Pt
0.3 ± 0.2
0.7 ± 0.5
0.28 ± 0.07
12 ± 2
0.9 ± 0.2
0.3 ± 0.2
0.65 ± 0.08
21 ± 2
1.1 ± 0.7
1.0 ± 0.2
0.25 ± 0.09
5.6 ± 0.6
>70
0.4 ± 0.2
0.4 ± 0.2
0.5 ± 0.1
8 ± 2
Ru 1:3
>70
>70
>70
The error was calculated as standard deviation of the average IC50 values using three or more indepen-
dent experiments.
The screening of anticancer drugs on a platform, using 2D-cultured cell lines, does not
have the ability to reproduce all the in vivo conditions of the tumor environment [29–31]
.
Based on this observation, we chose to confirm the cytotoxic properties of the most active
compounds using HCT116 cultured in 3D, which reproduces the physiological conditions
more closely (cells in aggregates presenting different levels of contact with the surround-
ings). The effect of the drugs was measured by their impact on the size of the sphere, and
more quantitatively by assessing cell survival via the resazurin assay. Interestingly, the two
gold DTC derivatives and the ruthenium DTC derivatives showed different behaviors in 3D
culture compared to the 2D cultures. Unfortunately, both of the gold(III) DTC derivatives
did not show any cytotoxic effect in a 3D cell culture, contrary to what was observed in the
2D model.
This inefficacy in the 3D model may suggest a short-term cytostatic effect for these
complexes. In this regard, a previous study carried out on gold DTC derivatives of the
type [AuIIICl2(dmdt)] and [AuIIIBr2(esdt)] (dmdt = dimethyldithiocarbamato; esdt = ethyl-
sarcosinedithiocarbamate), by real-time xCELLigence analysis, confirmed that they in-
duce a strong cell growth inhibition after 24 h, but the cells restore viability afterwards
(26–90 h) [32]. The precise molecular mechanisms that are responsible for the short-term
effect or the lack of activity on cell aggregates are unclear and need further investigation.
For instance, a drugs stability inside the cells, or the difficulty of the drugs to penetrate
within the cell aggregates may play a role.
On the contrary, [Ru2(pipeDTC)5]Cl displayed a promising cytotoxic effect in the
3D system (IC50
5
±
2
µM, Figure 2a), much higher than that of cisplatin (63
±
25 µM).

Molecules 2021, 26, 4073
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Moreover, the cytotoxic effect was correlated with the drug concentration-dependent
decrease in spheroids’ diameter after the treatment (Figure 2b), thus indicating a progressive
decrease in cell–cell interaction, which is associated with cell death.
Figure 2.
calculated from three independent experiments; Cells were cultured for 7 days and then treated for
7 days. ( ) The 3D spheroids after treatment with different concentrations (0 M (NT)–10 M) of
(a) IC50 values of the 3D-cultured HCT116 cell line. The average of IC50 (red line) was
b
µ
µ
[Ru2(pipeDTC)5]Cl.
2.3. Investigation of the Mechanism of Action
DNA has been described to be a target for different types of metal-based complexes,
especially ruthenium complexes [33]. In order to understand if DNA was a possible target
for the complexes, an EMSA (electrophoretic mobility shift assay) [34] was carried out.
This qualitative assay showed that the three active compounds did not interact with DNA
as much as cisplatin. In fact, no shift in the bottom band (supercoiled form of plasmid)
was observed for the samples incubated with Au 1:1, Au 1:2, and Ru 2:5, when compared
with the control (namely, the plasmid without a complex). On the contrary, a shift in the
supercoiled plasmid band was observable in the cisplatin-treated samples (Figure 3), as
expected, since DNA is its major target [3]. From this preliminary observation, we can

Molecules 2021, 26, 4073
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conclude that the tested compounds do not interact with DNA when compared to a smaller
concentration of CisPt. Anyway, more in-depth studies are needed to confirm this result.
Figure 3. Electrophoretic mobility of pJoJo plasmid DNA in presence of the different DTC derivatives and cisplatin. The
plasmid was incubated with the complexes for 24 h at room temperature. For the gold and ruthenium compounds the
following concentrations were realized: column 1–4: 15 µM, 30 µM, 45 µM, 60 µM, respectively. For cisplatin: column 1–4:
1 µM, 5 µM, 10 µM, 15 µM, respectively.
It has been elucidated, from previous studies, that ruthenium derivatives are located
in various cellular compartments (i.e., ER, mitochondria, nucleus for Ru derivatives, and
cytoplasmic area for Au(III) derivatives) [35,36]. In this work, we focused our attention on
the possible pathways implicated in causing cell death. To understand the molecular mech-
anisms involved in the cytotoxicity of the Au 1:2 and Ru 2:5 compounds, we investigated
the activation of pro-cell death mechanisms. We first analyzed the protein level of p53, a
transcription factor induced by several cellular stresses, which can, in response, induce
different types of cell deaths as apoptosis. In addition, it has been demonstrated that over
50% of human tumors are characterized by mutations of p53, leading to the enhanced sur-
vivability of tumor cells [37–39]. The role of p53 is particularly important in gastric cancer,
where the inactivation of p53 correlates with the response to chemotherapy [40]. Western
blot was used to measure the p53 protein levels [41]. AGS and HCT116 cells express a
wild-type p53. AGS cells were treated over 24 h and 48 h, with both of the two complexes,
while HCT116 cells were treated only with the most promising [Ru2(pipeDTC)5]Cl species,
always using cisplatin as a reference drug. The protein levels for p53 were quantified and
standardized with the protein levels for actin, to be indicated as a percentage relative to the
positive control cisplatin. As expected, cisplatin significantly induced p53 protein levels
at 24 h. Different results were obtained for the Au 1:2 and Ru2:5 compounds, as follows:
(i) The AGS cells treated with [Au(pipeDTC)2]Cl showed a dose-dependent increase in p53
protein levels, starting after 24 h and lasting up to 48 h (Figure 4); (ii) Ru 2:5 did not induce
p53 protein levels in both of the cell lines (Figure 4). The fact that this compound did not
bind DNA and did not induce p53 expression suggests that this complex may act indepen-
dently of p53, in contrast to some ruthenium complexes previously described [42], and may
resemble more in their mode of action to ruthenium complexes such as RDC11 [43]. This
observation is very encouraging, since the ability of compounds that require p53 to induce
cell death is rather debilitated in cancer cells with the p53 mutation, which occurs in about
50% of cases [44]. Therefore, antitumor agents triggering cell death independently of p53
pathways could be a promising strategy in the treatment of p53-mutated tumors [42,45].

Molecules 2021, 26, 4073
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Figure 4. Expression of cleaved caspase-3, p53 and p62. Cisplatin was used as positive control (IC50). IC25, IC50 and IC75
were utilized to treat the cells. This image is representative of three independent experiments. The expression of proteins in
AGS cells for [Au(pipeDTC)2]Cl and in HCT116 cells for [Ru2(pipeDTC)3] is reported.
We then evaluated the cleavage of caspase-3 to estimate if the activity of these com-
pounds is associated with cell apoptosis activated by caspase-3 cleavage [46]. This protein
belongs to the family of cysteine proteases, largely known for their role in controlling cell
death and inflammation. Caspase-3 is the most widely studied among the effector caspases,
since it plays a key role in both the extrinsic and intrinsic pathway of apoptosis [46,47]. For
both of the tested compounds in both cell lines, no cleavage of caspase-3 was observed
under our conditions, in contrast to the treatment with cisplatin (Figure 4 and Figure S12).
This suggested that these complexes probably induce cell death via other mechanisms.
Hence, we focused our attention on the possible activation of autophagy. Autophagy is a
cellular degradation pathway for the clearance of damaged or superfluous proteins and
organelles, which maintain cell homeostasis and viability [48,49]. In this scenario, p62 is
a stress-inducible cellular protein that plays a key role as a selective autophagy marker.
This protein has a pivotal role, as it is of the main importance in promoting viability, but,
on the other hand, a higher expression of p62 is also associated with cell death induction.
This last effect in the case of metabolic stress, hypoxia, nutrition deficiency or therapeutic
treatment, leads to a non-apoptotic cell death (autophagic cell death, ACD) [49]. Hence, we
examined the levels of the p62 protein in cells treated with our complexes. Intriguingly,
Au 1:2 induces a dose-response increase in p62 levels, while an opposite behavior is ob-
served in the Ru 2:5-treated cells, which display a dose-dependent decrease in p62 protein
levels (Figure 4).
As Au 1:2 induced p53 protein levels, we investigated whether p53 was playing a role
in the p62 increase. To do so, the cells were co-treated over 48 h with pifithrin-
is a known inhibitor of p53 function [51]. Interestingly, in the presence of the p53 inhibitor
pifithrin- , the complex Au 1:2 decreased the expression levels of p62, whereas pifithrin did
α [50], which
α
not change the activity of the ruthenium derivative (Figure 5). From the collected results,
we can propose that the Au 1:2 mechanism of action might be p53-dependent, and that it

Molecules 2021, 26, 4073
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could act by inducing an enforced autophagy that leads to cell death via a non-apoptotic
pathway. Differently, the Ru 2:5 complex seems to not involve autophagy in the cell death
pathway, and intriguingly the p53-independent mechanism of action of this compound is
also confirmed by the presence of the p53 inhibitor agent.
Figure 5. Expression of the autophagy biomarker p62 in HCT116 cells with and without co-treatment with pifithrin-
α over
48 h. Cisplatin was used as positive control (IC50). This image is representative of three independent experiments.
As the ruthenium-based complex has sidestepped all the planned “check points”,
in terms of unravelling its mechanism of action, the endoplasmic reticulum (ER)–stress
pathway was then considered. Indeed, we previously showed that several ruthenium
complexes can induce several markers of the ER stress pathway [35,43,52,53]. Alterations
in the folding capacity of the ER, caused by a variety of endogenous and exogenous stimuli,
prompt a cellular stress condition known as ER stress. In turn, ER stress activates an
intracellular signal transduction pathway, called the unfolded protein response (UPR),
which is essential to re-establish ER homeostasis [54]. One of the key regulators of the
UPR is the activating transcription factor 4 (ATF4), which monitors the transcription of
the essential genes for adaptive functions. Intriguingly, ATF4 can be involved in both a
pro-survival and pro-apoptotic role during ER stress conditions, depending on the duration
as well as the severity of the ER stress [55]. In fact, under ER stress conditions, an increased
translation of selected mRNAs occurs, including ATF4, which monitors the transcription
of essential genes for adaptive functions. Focusing on cancer cells, increased levels of
ATF4 compared to healthy cells are usually observed, leading to stress adaptation and,
consequently, to cancer cell survival. Indeed, the inhibition of ATF4, or a decrease in its
levels, results in increased stress, causing cell death [56]. Remarkably, in other cases, the
long-term activation of the UPR axis may evoke a paradoxical response via the initiation
of apoptotic cell death, highlighting the dual role of this protein in the fate of cancerous
cells [55]. Based on this information, we investigated whether our complexes might
regulate the protein level of ATF4, as a marker of the induction of the ER stress pathway.
The ruthenium complex was able to significantly induce the protein level of ATF4 in cancer
cells, in contrast to cisplatin (Figure 6). Interestingly, the response was dose- and time-
dependent. At 24 h of treatment, induction was present for a low concentration of the drug,
while a high concentration reduced the ATF4 protein level. However, at 48 h of treatment,
all of the concentrations were able to induce the ATF4 protein levels. Interestingly, the
inhibition of p53 by pifithrin significantly increased the ATF4 protein level, but did not
significantly affect the ability of Ru 2:5 to induce ATF4 levels. Hence, this result indicated
that the Ru 2:5 complex can induce the expression of at least one marker of the ER stress
pathway, suggesting that part of its mode of action may trigger this pathway independently
of p53. Additional functional studies will be required to further establish the role of the ER
stress pathways in Ru 2:5 cytotoxicity.

Molecules 2021, 26, 4073
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Figure 6. Expression of the ER stress biomarker ATF4 after treatment with Ru 2:5 in HCT116 cell lines. Cisplatin was used
as positive control (IC50). This image is representative of three independent experiments.
3. Materials and Methods
Chemicals and biological materials. The following chemicals were purchased and used
as provided by suppliers: tetrachloroauric acid trihydrate (99%), ruthenium(III) chloride
trihydrate ( 99%), potassium tetrachloroplatinate(II) ( 99%), manganese dioxide (99%),
silver nitrate ( 99%), potassium iodide (99%), sodium sulfite ( 98%), sodium chloride
(99%), piperidine (99%), hydrogen chloride (37%), carbon disulfide anhydrous ( 99%)
≥
≥
≥
≥
≥
(Sigma-Aldrich). Potassium hydroxide (99%) (VWR). Solvents: dichloromethane, diethyl
ether, chloroform, ethanol, ammonium hydroxide (33%) (Sigma-Aldrich). The 1H-NMR
chemical shifts (δ) of the signals are given in ppm and referenced to residual protons in
deuterated solvent acetonitrile-d (CD3CN 1.94 ppm) (VWR). DMSO (>99.9%, for biolog-
ical treatments), methanol (99.8%), Tris-glycine buffer 10x, Dulbecco modified Eagle’s
medium (D-MEM), RPMI medium, L-glutamine, penicillin, streptomycin, fetal bovine
serum (FBS), trypsin (0.05%, EDTA 0.02% in PBS), in vitro toxicology assay kit (MTT-
and resazurin-based), dithiothreitol (DTT) (
≥
99.0%), Laemmli buffer, sodium dodecyl
sulfate (SDS) ( 99.0%), Luminata Forte substrate, Luminata Crescendo substrate, polyacry-
≥
lamide solution 50 wt.% in H2O, N,N,N0,N0-tetramethylethylenediamine (Sigma-Aldrich).
Ammonium persulfate (APS) (90%), nitrocellulose membranes, Ready Gel® precast poly-
acrylamide gels, TEMED, glycerol (BIO RAD). AGS cells, HCT116 cells (American Type
Culture Collection, ATCC); pJoJo plasmid was provided by P.Collombat (Université Côte
d’Azur, Inserm). Antibodies (Table 1).
Synthesis of the K PipeDTC ligand. KOH (2.272 g, 40 mmol) was dissolved in 100 mL of
ethanol and 4 mL (40.5 mmol) of piperidine was added with the solution mixture set at
◦
0 C. Successively, an excess of carbon disulfide (CS2) was added, leading to a yellow
mixture, which was kept under vigorous stirring for 1 h. Afterwards, the solvent volume
was reduced and the product precipitated by adding cold diethyl ether, filtered and washed
with cold ethanol, before being dried under vacuum in the presence of P2O5. Piperidine
dithiocarbamate potassium salt: aspect: white solid. Yield: 95%. Anal. Calc. C6H10KNS2
(MW = 199.38 g/mol): C, 36.14; H, 5.06; N, 7.03; S, 32.16. Found: C, 35.84; H, 5.02; N, 7.05;
1
S, 32.22. H-NMR (CD3CN, 300.13 MHz):
δ (ppm): 1.50 (m, 4H); 1.60 (m, 2H); 4.34 (t, 4H).
Medium FT-IR (KBr): (cm−1) = 1417.12 (v N-CSS), 965.00 (va CSS).
Synthesis of the precursor [AuI(pipeDTC)]2. H[AuCl4]
dissolved in a saturated saline solution at 0 C, followed by reduction Au(III)
1 equivalent of Na2SO3 under stirring. The bright yellow solution turns colorless, and
immediately 355.1 mg (1.78 mmol) of K PipeDTC was added, leading to the precipitation
of an orange solid. The mixture was left under vigorous stirring for 5 min, and then the
precipitate was centrifuged, washed three times with 5 mL of water and dried under
·
xH2O (714.7 mg, 1.78 mmol) was
Au(I) with
◦
→

Molecules 2021, 26, 4073
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vacuum in the presence of P2O5. Bis(piperidine dithiocarbamate)digold(I): aspect: green–
yellow solid. Yield: 98%. Anal. Calc. C12H20Au2N2S4 (MW = 714.49 g/mol): C, 20.17;
H, 2.82; N, 3.92; S, 17.95. Found: C, 20.20; H, 2.82; N, 3.99; S, 17.89. Medium FT-IR (KBr):
(cm−1) = 1427.69 (v N-CSS), 967.97 (va CSS).
Synthesis of [AuCl2(pipeDTC)]. [AuI(pipeDTC)]2] (0.5 mmol) was suspended in 50 mL of
dichloromethane and the mixture was refluxed under stirring. After 10 min, an excess of
Cl2 (generated in a separate flask by mixing 300 mg of MnO2 with 5 mL of concentrated
HCl) was gurgled in the Au(I) suspension for 1 h. It was possible to observe that, after
some minutes, the solution turned brown with the dissolution of the Au(I) precursor, and
then became yellow. Then, the solution was cooled to room temperature and the solvent
half-reduced. The compound was precipitated with diethyl ether, washed with 2
×
4 mL
of diethyl ether and 2 5 mL of deionized water, and the orange product was dried under
×
vacuum in the presence of P2O5. Dichloro(piperidine dithiocarbamate)gold(III): aspect:
dark-yellow solid. Yield: 88%. Anal. Calc. C6H10AuCl2NS2 (MW = 428.15 g/mol): C, 16.83;
1
H, 2.35; N, 3.27; S, 14.98. Found: C, 17.02; H, 2.40; N, 3.14; S, 15.02. H-NMR (CD3CN,
400.13 MHz):
δ
(ppm): 1.78 (s, 6H); 3.75 (s, 4H). Medium FT-IR (KBr): (cm−1) = 1582
(v N-CSS), 1006 (va CSS).
Synthesis of [Au(pipeDTC)2]Cl. [AuIIICl2(pipeDTC)](123.1 mg, 0.288 mmol) was dissolved
in CHCl3 (60 mL), and, in a separate flask, the ligand K pipeDTC (57.3 mg, 0.288 mmol)
was dissolved in ethanol (1 mL). The ethanolic solution of the ligand was added dropwise
to the Au(III) precursor, and the solution turned from yellow to light orange. After 10 min,
the solvent was removed under reduced pressure. The residual solid was taken up with
CHCl3, and KCl was removed by filtration. The final product was collected by precipi-
tation with n-pentane and dried under vacuum in the presence of P2O5. Bis(piperidine
dithiocarbamate)gold(III) chloride: aspect: yellow–orange solid. Yield: 82%. Anal. Calc.
C12H20AuN2S4Cl (MW = 552.98 g/mol): C, 26.04; H, 3.61; N, 5.06; S, 23.19. Found: C,
1
25.86; H, 3.93; N, 4.83; S, 23,32. H-NMR (CD3CN, 400.13 MHz):
δ (ppm): 1.79 (m12H);
3.80 (m, 8H). Medium FT-IR (KBr): (cm−1) = 1560 (v N-CSS), 1003 ( va CSS).
Syntheses of [Ru2(pipeDTC)5]Cl and [Ru(pipeDTC)3]. Ruthenium(III)chloride trihydrate
(818.9 mg, 3.13 mmol) and K pipeDTC (187.3 mg, 9.39 mmol) were separately dissolved
in 5 mL of deionized water. The solution of ligand was added to Ru(III), and stirred at
room temperature for 1 h, yielding a brown precipitate that was centrifuged, washed with
2
×
10 mL of deionized water and dried under vacuum in the presence of P2O5. After
24 h, the isolated product was re-dissolved in 20 mL of CH2Cl2 and the solution filtered to
remove Ru oxides by-products. The obtained solution was purified via silica gel chromatog-
raphy. A gradient from CH2Cl2 100% to CH2Cl2/MeOH 90%:10% was used to first elute the
dark-green mononuclear complex [Ru(pipeDTC)3] and then the brown dinuclear derivative
[Ru2(pipeDTC)5]Cl as a mixture of
[Ru2(pipeDTC)5]Cl was isomerized to the thermodynamically stable
by reflux in methanol for 8 h. Both the mononuclear and the -dinuclear complexes were
re-precipitated from CH2Cl2 diethyl ether, washed with n-pentane, and dried in vacuum
in the presence of P2O5. -pentakis(piperidine dithiocarbamate)diruthenium(III)chloride:
α
and
β
isomers. Successively, the mixture of
α, β-
β-[Ru2(pipeDTC)5]Cl
β
β
aspect: brown solid. Yield: 30%. Anal. Calc. C30H50Ru2ClN5S10 (MW = 1039.00 g/mol): C,
1
34.64; H, 4.81; N, 6.73; S, 30.85. Found: C, 33.01; H, 4.73; N, 6.50; S, 30.37. H-NMR (CD3CN,
400.13 MHz):
δ
(ppm): 1.75 (m, 10H); 3.88 (m, 6H). Medium FT-IR (KBr): (cm−1) = 1505,
1441 (v N-CSS), 1001 (va CSS). Tris(piperidine dirhiocarbamate)ruthenium(III): aspect:
dark-green solid. Yield: 34%. Anal. Calc. C18H30RuN3S6 (MW = 581.91 g/mol): C, 37.13;
H, 5.16; N,7.22; S, 33.07. Found: C, 37.19; H, 5.01; N, 7.00; S, 33.97.1H-NMR (CD3CN, 400.13
MHz):
δ (ppm): 26.02 (m, 6H); 21.95 (m, 6H); 1.01 (m, 12H); 3.95 (m, 6H). Medium FT-IR
(KBr): (cm−1) 1488, 1455, 1440 (v N-CSS), 1002 (va CSS).

Molecules 2021, 26, 4073
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3.1. Cell Lines and Culture Conditions
The 2D cell culture. Gastric adenocarcinoma AGS cells were maintained in RPMI
medium, while the colorectal cancer cells HCT116 were maintained in DMEM high-glucose
medium, both with 10% of FBS and incubated in presence of 5% CO2 at 37 ◦C.
To transfer cells from the Petri cell plate to the test plates (polystyrene, 96-well), the
following procedure was used: After removal of the medium, cells were washed with 5 mL
of EDTA and thus 5 mL of trypsin 0.5% was added. The Petri cell plate was then incubated
for 3–5 min to detach cells from the plate surface. After that, 10 mL of culture medium
was added to stop the trypsin reaction and then the suspension was transferred to a falcon
and centrifuged for 5 min at 800 rpm, to collect the cells on the bottom. Subsequently,
the supernatant was carefully removed, and 20 mL of new medium was added. The
cell suspension was diluted at a final concentration of 105 cells/mL and 100
µL of the
◦
cellular suspension was seeded in 96-well microplates and incubated at 37 C in a 5% CO2
atmosphere until reaching 70/80% confluence. The day after seeding, cells were treated
with vehicle (control; namely, DMSO or saline solution) or each compound (dissolved
in the vehicle DMSO/saline solution, 0.8% v/v) in fresh culture medium at the defined
concentrations (i.e., 10
72 h. Cell viability was evaluated via MTT assay, according to standard procedures [57
Cells were treated for 48 h and 72 h and then the medium was completely removed and
replaced with 100 L of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
µM, 5
µM, 2
µM, 1
µM, 0.5
µM and in some cases lower) for 48 or
,58].
µ
(10% MTT solution in PBS, phosphate-buffered saline solution, pH 7.4), and incubated
for 2 h at 37 ◦C. After removing incubation medium, formazan crystals were dissolved in
100 µL solution of DMSO, and MTT reduction was quantified by measuring the absorbance
at 570 nm. The percentage of surviving cells was calculated from the ratio of absorbance
between treated and untreated cells. Each treatment was performed in triplicate.
Cytotoxicity data were expressed as IC50 values, i.e., the concentration of the test
complex inducing 50% reduction in cell numbers compared with control cultures.
Cisplatin (in saline solution, NaCl 0.9% w/v) was used as reference drug.
The 3D cell culture. HCT116 cells were seeded at a density of 300 cells/well in ad hoc
conceived 96-well plates. In particular, 50
was dispensed in each well. Therefore, cells were incubated in a 5% CO2 incubator at 37 C
µ
L of cell suspension in DMEM (6000 cells/mL)
◦
for seven days and then 50 µL of medium drug solution at different concentrations was
added, without removing the previous medium. After that, cells were incubated again for
seven days and then incubated overnight with resazurin 10%. The optical density of each
well was quantified at 590 nm, using a multi-well plate reader (TriStar2 Berthold).
EMSA. Samples of plasmid DNA pJoJo (kindly provided by Prof. P. Collombat,
Université Côte d’Azur, Inserm) were incubated overnight with Ru 2:5, Au 1:2, Au 1:1 in a
20 mM NaCl solution (final volume 20 µL) at room temperature in the dark. The samples
were subjected to electrophoresis on 0.7% agarose gels, running at 25 ◦C with TAE buffer
with a voltage set at 50 V. The gels were then stained with EtBr, followed by detection at
302 nm. The following four different concentrations of complexes were used: (i) 15, 30, 45,
60 µM for Au 1:1, Au 1:2 and Ru 2:5, and (ii) 1, 5, 10, 15 µM for cisplatin.
Western Blot. Cells were seeded in 6-well plates at a density of 5
105 cells/well and
·
grown in a 5% CO2 incubator at 37 ◦C. In order to avoid cell scratching, cells were then
treated with a lysis buffer (Laemmli 2x buffer [59]) with the addition of DTT, and the protein
◦
suspension was denatured at 100 C for 5 min. Next, proteins were solved on a SDS-PAGE
gel and transferred to BIORAD nitrocellulose membrane. Membranes were then treated
with 20% methanol/10% triglycine solution, blocked with 5% milk powder in PBS + 0.05%
Tween 20 for 30 min,◦and subsequently incubated overnight with primary antibody in
blocking solution at 4 C. Following washes with 5% milk PBS, membranes were incubated
with 1:5000 HRP-conjugated secondary antibodies for 2 h at room temperature and washed
with PBS + 0.05% Tween 20. HRP reactivity was revealed using Luminata Crescendo or
Luminata Forte (Syngene).
β-actin was used as a loading control because of its ubiquitarian
expression across all eukaryotic cell types [60].

Molecules 2021, 26, 4073
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In Table 4 all the primary and secondary antibodies used to detect the proteins in this
work and their dilutions are reported.
Table 4. Detected proteins, primary and secondary antibodies, relative dilutions and substrates used in this work.
◦
◦
Protein
1 Antibody
Dilution
2 Antibody
Dilution
Substrate
polyclonal rabbit
(Cell Signaling)
anti-rabbit (goat)
(GE HealthCare)
caspase-3
1:1000
1:5000
Luminata Forte
monoclonal mouse
(Santa Cruz)
anti-mouse (goat)
(GE HealthCare)
Luminata
Crescendo
p62
p53
1:1000
1:1000
1:500
1:5000
1:5000
polyclonal rabbit
(Santa Cruz)
anti-rabbit (goat)
(GE HealthCare)
Luminata Forte
Luminata Forte
Polyclonal rat
(BioLegend)
anti-rat (rabbit)
(GE HealthCare)
ATF4
actin-β
1:10,000
1:10,000
Monoclonal mouse
(Chemicon)
anti-mouse (goat)
(GE HealthCare)
Luminata
Crescendo
1:1000
3.2. Instrumentation
Elemental analyses of carbon, nitrogen and hydrogen were carried out at the Micro-
analysis Laboratory (Chemistry Dept., University of Padova) with a Carlo Erba mod. 1108
CHNS-O microanalyzer.
Near-FT-IR spectra (400–4000 cm−1) were registered at room temperature (32 scans,
resolution 2 cm−1) by Nicolet Nexus 5SXC spectrophotometer. KBr pellets of samples were
prepared according to standard procedures. Spectra were processed with OMNIC 5.2 (Nico-
1
let Instrument Corporation). H-NMR spectra were recorded at 298 K on a Bruker Advance
DEX400 spectrophotometer equipped with a BBI-5 mm z-field gradient probe-head, and
a Silicon Graphics O2 workstation, operating in Fourier transform. Typical acquisition
parameters are as follows (1H: 400.13 MHz): 128 transients spectral width 7.5 kHz, 2 k
data points and a delay time of 1.0 s. Spectra were processed by using sine-square weight-
ing with a resolution of 1.0/3.0 Hz and a line-broadening threshold of 0.3/1.0 Hz. Data
processing was carried out by means of MestReNova version 6.2.0 (Mestrelab Research
S.L.). Peak multiplicity has been described as follows: s (singlet), t (triplet), q (quartet), sext.
(sextet) and m (multiplet). The 1H-NMR study of the interaction between selected models
of biomolecules and the metal–dithiocarbamato complexes was carried out dissolving
the metal–dithiocarbamato derivative (i.e., [AuCl2(pipeDTC)], [Au(pipeDTC)2]Cl, and
β-[Ru2(pipeDTC)5]Cl) in deuterated acetonitrile CD3CN (0.75 mL) to form 18–19 mM
solutions. The sample was put into an NMR tube and the biomolecule was added at the
right ratio (1:1 complex:model molecule for DMS and 1-MeIm, and 1:2 for PrSH).
Cell cultures were incubated at 37 ◦C in a 5% CO2-controlled atmosphere in Heracell
150i CO2 incubator (Thermo Scientific). Cellular viability was determined by absorbance
measurements at 570 nm (MTT) and 595 nm (resazurin) using a plate reader TriStar2 S LB
942 modular monochromator multimode reader (Berthold Technologies).
Data were processed by GraphPad Prism version 6.01 software.
Cells were controlled daily to check the confluence with the inverse microscope
Motic AT31E.
To assess the Western blot the membranes were detected with multi-fluorescence and
chemiluminescence imaging system G:BOX mini Pixi4 (Syngene).
The detection of agarose gels was carried out by AlphaImager mini spectrophotometer.
4. Conclusions
Among the various strategies to fight cancer, the one focused on metal-based chemother-
apy could be a winning approach. Our twenty years of studies, focused on the design of
new drugs with a lower toxicological impact than cisplatin, have highlighted compounds

Molecules 2021, 26, 4073
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based on gold and ruthenium, with truly interesting antineoplastic characteristics. In
this work, the anticancer activity of four gold and ruthenium complexes, with the ligand
piperidine dithiocarbamate, was assessed. Two frequent and aggressive types of cancer,
human gastric adenocarcinoma (AGS) and colorectal carcinoma (HCT116), were tested in
2D cell cultures, and IC50 values lower than or close to 1
of treatment, for three of the tested compounds.
µM were found after 24 h or 72 h
To get closer to the tridimensional aspect of a tumor, we subsequently referred to a 3D
model of HCT116 cells. These 3D experiments pointed out a very different behavior of the
two class of compounds, namely, the gold and the ruthenium derivatives. The results for
the gold-based complexes, seen in light of previous published data, suggest a short-term
cytostatic effect. The exact causes of this lack of efficacy in 3D cultures are unclear and need
further investigation. One explanation might be a reduced ability of the gold compound to
penetrate within the cell aggregates.
On the contrary, Ru 2:5 displayed a promising cytotoxic effect of one order of magni-
tude greater than cisplatin. The interaction NMR experiments of the different complexes
with model molecules, mimicking the major biological competitor of the ligands, pointed
out their general stability towards histidine and methionine, but also showed that they
undergo reduction in the presence of the model molecule mimicking cysteine. This result,
together with the fact that the EMSA assay showed that they do not strongly bind DNA as
cisplatin does, could be the key to start understanding the real mechanism of action.
While having the same ligands, gold and ruthenium derivatives have been shown to
exploit different pathways in preventing cancer cell growth. In particular, Ru 2:5 has been
shown to not involve neither the apoptotic pathway nor the autophagic one, displaying a
very promising p53-independent mechanism of action. This is a very important feature,
since many tumors have p53 as a mutated gene, leading to a decreased efficacy of many
drugs, including cisplatin, which we consider as a reference in our comparative studies.
The ability of this compound to sidestep the p53 pathway could be of strategic importance
in the incessant designing of more effective anticancer drugs. This striking experimental
evidence prompted us to deepen the study of this promising [Ru2(pipeDTC)5]Cl compound,
investigating the ER stress pathway. The results pointed out a general downregulation of
the ATF4 protein, a biomarker of ER stress with a key role in cell recovery, which in this
case could stimulate onco-suppressor action.
Even if the understanding of the type of induced cell death is troublesome, since some
regulatory mechanisms are shared between the different types of cell death, the results
collected in this work represent a promising starting point for already planned future
studies on these complexes with very interesting, but still unclear, mechanisms of action.
1
Supplementary Materials: The following are available online. H-NMR spectra of the ruthenium
compounds (Figures S1 and S2), 1H-NMR spectra of the interaction between complexes and model
molecules (Figures S3–S11), synthesis of cisplatin.
Author Contributions: Conceptualization, C.G., D.F.; methodology, L.B., C.O., N.P.; formal analysis,
C.O., C.G., D.F.; investigation, M.D.P., L.B., N.P.; resources, C.G., D.F.; data curation, N.P., L.B., C.N.;
writing—original draft preparation, M.D.P.; writing—review and editing, D.F., C.G; visualization,
M.D.P.; supervision, C.G., D.F.; project administration, D.F.; funding acquisition, C.G. All authors
have read and agreed to the published version of the manuscript.
Funding: This research was funded by Ligue contre le Cancer, IDEX from Strasbourg University,
Interdisciplinary Thematic Institute InnoVec.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The 1H-NMR spectra presented in this study are available in Supple-
mentary material.

Molecules 2021, 26, 4073
15 of 17
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are not available from the authors.
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