Ruthenium(II) Complex Containing a Redox-Active Semiquinonate Ligand as a Potential Chemotherapeutic Agent: From Synthesis to in Vivo Studies

Article abstract of DOI:10.1021/acs.jmedchem.0c00431

Chemotherapy remains one of the dominant treatments to cure cancer. However, due to the many inherent drawbacks, there is a search for new chemotherapeutic drugs. Many classes of compounds have been investigated over the years to discover new targets and synergistic mechanisms of action including multicellular targets. In this work, we designed a new chemotherapeutic drug candidate against cancer, namely, [Ru(DIP)₂(sq)](PF₆) (Ru-sq) (DIP = 4,7-diphenyl-1,10-phenanthroline; sq = semiquinonate ligand). The aim was to combine the great potential expressed by Ru(II) polypyridyl complexes and the singular redox and biological properties associated with the catecholate moiety. Experimental evidence (e.g., X-ray crystallography, electron paramagnetic resonance, electrochemistry) demonstrates that the semiquinonate is the preferred oxidation state of the dioxo ligand in this complex. The biological activity of Ru-sq was then scrutinized in vitro and in vivo, and the results highlight the promising potential of this complex as a chemotherapeutic agent against cancer.

Full text of DOI:10.1021/acs.jmedchem.0c00431

Subscriber access provided by BIU Pharmacie | Faculté de Pharmacie, Université Paris V
Article
A Ruthenium(II) Complex Containing a Redox-Active Semiquinonate Ligand
as Potential Chemotherapeutic Agent: From Synthesis to In Vivo Studies
Anna Notaro, Angelo Frei, Riccardo Rubbiani, Marta Jakubaszek, Uttara Basu, Severin Koch, Cristina Mari,
Mazzarine Dotou, Olivier Blacque, Jeremie Gouyon, Fethi Bedioui, Nils Rotthowe, Rainer F. Winter, Bruno
Goud, Stefano Ferrari, Mickael Tharaud, Martina Rezácová, Jana Humajová, Pavel Tomšík, and Gilles Gasser
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00431 • Publication Date (Web): 22 Apr 2020
Downloaded from pubs.acs.org on April 22, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 96
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table of Content Graphic
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 96
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
A Ruthenium(II) Complex Containing
a Redox-Active Semiquinonate
Ligand as Potential Chemotherapeutic
Agent: From Synthesis to in vivo
Studies
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Anna Notaro,a,# Angelo Frei,b,# Riccardo Rubbiani,b,# Marta Jakubaszek,a, e
a
b
b
a
Uttara Basu, Severin Koch, Cristina Mari, Mazzarine Dotou, Olivier
b
c
c
d
d
Blacque, Jérémie Gouyon, Fethi Bedioui, Nils Rotthowe, Rainer F. Winter,
e
,f,g
h
i
Bruno Goud, Stefano Ferrari, Mickaël Tharaud, Martina Řezáčová, Jana
j
i
a,*
Humajová, Pavel Tomšík, and Gilles Gasser
a
Chimie ParisTech, PSL University, CNRS, Institute of Chemistry for Life and
Health Sciences, Laboratory for Inorganic Chemical Biology, F-75005 Paris,
France.
b
c
Department of Chemistry, University of Zurich, Winterthurerstrasse 190,
8057 Zurich, Switzerland.
Chimie ParisTech, PSL University, CNRS, Institute of Chemistry for Life and
Health Sciences, Team Synthèse, Electrochimie, Imagerie et Systèmes
Analytiques pour le Diagnostic, F-75005 Paris, France.
1
ACS Paragon Plus Environment

Page 3 of 96
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
d
Department of Chemistry, University of Konstanz, Universitätsstrasse 10, D-
78457 Konstanz, Germany.
e
f
Institut Curie, PSL University, CNRS UMR 144, F-75005, Paris, France.
Institute of Molecular Cancer Research, University of Zurich, CH-8057, Zurich,
Switzerland.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
g
h
Institute of Molecular Genetics of the Czech Academy of Sciences, Videnska 1083,
1
43 00 Prague, Czech Republic.
Université de Paris, Institut de physique du globe de Paris, CNRS, F-75005 Paris,
France.
i
j
Department of Medical Biochemistry, Faculty of Medicine in Hradec Kralove,
Charles University, Šimkova 870, 500 03 Hradec Kralove, Czech Republic.
Department of Medical Biochemistry, Faculty of Medicine in Prague, 150 06
Prague, Czech Republic.
#
these authors have contributed equally to the work
*
Corresponding author: E-mail: gilles.gasser@chimeparistech.psl.eu; WWW:
www.gassergroup.com; Phone: +33 1 44 27 56 02
ORCID Number
Anna Notaro: 0000-0003-0148-1160
Angelo Frei: 0000-0001-6169-2491
Marta Jakubaszek: 0000-0001-7590-2330
Uttara Basu: 0000-0002-0509-2421
Mazzarine Dotou: 0000-0001-8781-6763
Olivier Blacque: 0000-0001-9857-4042
2
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 96
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Fethi Bedioui: 0000-0002-0063-4412
Bruno Goud: 0000-0003-1227-4159
Stefano Ferrari: 0000-0002-6607-215X
Martina Řezáčová: 0000-0001-5370-2290
Jana Humajová (Mattová): 0000-0001-8099-6781
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Pavel Tomšík: 0000-0002-4366-075X
Gilles Gasser: 0000-0002-4244-5097
Keywords: Bioinorganic Chemistry, Cancer, DNA, Medicinal Inorganic
Chemistry, Ruthenium.
3
ACS Paragon Plus Environment

Page 5 of 96
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Abstract
Chemotherapy remains one of the dominant treatments to cure cancer.
However, due to the many inherent drawbacks, there is a surge for new
chemotherapeutic drugs. Many classes of compounds have been investigated
over the years in order to discover new targets and synergistic mechanisms of
action including multicellular targets. In this work, we designed a new
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
chemotherapeutic drug candidate against cancer, namely [Ru(DIP) (sq)]PF
2
6
(Ru-sq) (DIP = 4,7-diphenyl-1,10-phenanthroline; sq = semiquinonate ligand).
The aim was to combine the great potential expressed by Ru(II) polypyridyl
complexes and the singular redox and biological properties associated to the
catecholate moiety. Experimental evidences (e.g. X-ray crystallography,
electron paramagnetic resonance, electrochemistry) demonstrate that the
semiquinonate is the preferred oxidation state of the dioxo ligand in this
complex. The biological activity of Ru-sq was then scrutinised in vitro and in
vivo, and the results highlight the auspicious potential of this complex as a
chemotherapeutic agent against cancer.
4
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 96
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Introduction
In the last decades, the search for new chemotherapeutic agents against
cancer has challenged scientists worldwide. Chemotherapy, together with
surgery, radiotherapy and immunotherapy, is used in a combined modality
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
therapy to treat cancer. The goal of this combination is to overcome the
drawbacks of each singular treatment to afford the best chances of survival for
1
the patients. Cisplatin is one of the most common chemotherapeutic agents
utilized against cancer. However, its severe side effects are limiting its clinical
use.2–6 Therefore, many other platinum-based drug candidates have been
investigated over the last 40 years leading to the worldwide clinical approval of
carboplatin and oxaliplatin.7,8 On the basis of these ground-breaking
discoveries and the observed occurrence of resistance with platinum treatment,
a large number of metal complexes based on other metals than platinum have
been examined.9–18 In this field, ruthenium complexes play a central role due to
their inherent advantages (e.g., multiple stable oxidation states, well-
established chemistry, etc.).19–23 Interestingly, Ru(III) and Ru(II) complexes
19
usually have ligand exchange kinetics similar to those of Pt(II) complexes. In
5
ACS Paragon Plus Environment

Page 7 of 96
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
addition, ruthenium complexes have found applications in different fields of
medicinal chemistry against cancer since they behave differently depending on
the biological settings, therefore displaying different biological targets.24–29 KP-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1019, IT-139 (formerly NKP-1339) and NAMI-A are, to date, the only three Ru
complexes to have reached clinical trial as anticancer agents. Their mechanism
of action involves ligand exchange, resembling therefore the one of cisplatin.30–
35
Of note, TLD-1433, a substitutionally inert Ru(II) polypyridyl complex, has
recently entered phase II clinical trial as a photosensitizer for photodynamic
therapy (PDT).36,37 Another very promising class of ruthenium complexes are
the coordinatively saturated and substitutionally inert ruthenium polypyridyl
complexes. These compounds have been intensely investigated over the last
years and several applications as potential chemotherapeutic agents have
24
been unearthed. At first, most of the bio-activity of these compounds was
associated with interactions with DNA.38–41 However, over the years, many
other modes of action were identified, such as the trigger of mitochondrial
dysfunction,42–44 Topoisomerases I and II inhibition,45,46 modification of cell
47
24
membranes and others. Due to the great opportunities offered by this class
6
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 96
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
of Ru compounds, in this work, we designed a new Ru polypyridyl complex,
namely [Ru(DIP) (sq)](PF ) (Ru-sq, Scheme 1a) where DIP is 4,7-diphenyl-
2
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1,10-phenanthroline and sq is a semiquinonate ligand, which was found to be
a very interesting anticancer drug candidate. Semiquinonate is a so-called ‘non-
innocent’ ligand as its electrochemical properties strongly resemble that of the
48
metal center. Semiquinonate is the oxidised form of catechol, a well-known
dioxo ligand, which can exist in three redox forms, namely catecholate (cat),
semiquinonate (sq) and quinone (q) (Scheme 1b).49 Catecholate and its
oxidation products have already been intensively investigated as ligands.50,51
However, the focus of these studies has mostly been on the unique
electronic/redox properties of metal complexes containing such ‘non-innocent’
dioxo ligands.52–55 Catechols are also known as pan-assay interference
compounds (PAINS) due to their redox and chelating properties.56
Nevertheless, catecholate and its derivatives have also shown potential in
different fields of biological interest,57–61 such as cancer chemoprevention,59
60
61
antifungal activity and the inhibition of the spontaneous Aβ fibril formation,
which is a key target for the treatment of Alzheimer´s disease. Worthy of note,
7
ACS Paragon Plus Environment

Page 9 of 96
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
vanadium compounds carrying catechol-like ligands have been investigated by
Crans and co-workers.62,63 During these studies, particularly potent cytotoxic
vanadium (V) catecholate complexes toward bone cancer cells were unveiled.62
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
III
+
The cytotoxicity on glial cells of [Ru (NH ) (catecholate)] was also investigated
3
4
58
in 2007 by Almeida and co-workers. In this case, the catechol was found to
be more cytotoxic than the Ru(III) complex itself with an EC of 0.342 mM
50
against rat astrocytes and 0.568 mM against human glioblastoma GL-15 cell
III
+
line, while the [Ru (NH ) (catecholate)] complex had EC = 1.380 mM and
3
4
50
58
EC = 2.6 mM against rat astrocytes and human glioblastoma, respectively.
50
Further studies suggested that depletion of glutathione and induction of
apoptosis were possible explanations for the cytotoxicity observed for catechol
towards mouse neuroblastoma N2a cell line.57 These preliminary studies
rationalize our choice to integrate catechol and its oxidation products into a
Ru(II) polypyridyl complex. To the best of our knowledge, [Ru(DIP) (sq)](PF )
2
6
is the first Ru(II) polypyridyl complex containing a catechol moiety to be deeply
investigated from both a physico-chemical and biological point of view. The
complex was isolated as a racemic mixture of ∆ and Λ enantiomers. No effort
8
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 96
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
was made in this work to isolate pure enantiomers. As described below, in vitro
and in vivo studies demonstrate a significant potential of this compound as a
chemotherapeutic agent against cancer.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a)
+
PF6-
N
N
N
N
N
N
O
N
N
I
II
Ru(DMSO)4(Cl)2
Ru
Ru
Cl
Cl
O
b)
O
O
O
O
-e-
-e-
O
O
+e-
+e-
cat
sq
q
Scheme 1. a) Synthesis of [Ru(DIP) (sq)](PF ). I) DIP, LiCl, DMF, reflux, 24 h,
2
6
78%; II) (i) NaOH, catechol 2-propanol, reflux, 24h; (ii) air, 2 h; (iii) NH PF , 2-
4
6
propanol/H O (1:8), 19%. b) Catecholate (cat) and its oxidised forms, semiquinonate
2
(sq) and quinone (q).
Results and Discussion
Synthesis and characterization of [Ru(DIP) (sq)](PF )
2
6
9
ACS Paragon Plus Environment

Page 11 of 96
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
The synthesis of the target compound [Ru(DIP) (sq)](PF ) was achieved in a 2-
2
6
64
step synthesis (Scheme 1a). Briefly, the known Ru(DMSO) Cl , DIP and LiCl
2
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
were refluxed in DMF to afford Ru(DIP) Cl in 72% yield after precipitation with
2
2
acetone.65 The compound was then refluxed in a nitrogen atmosphere
overnight with catechol in the presence of NaOH in 2-propanol. The oxidation
step of the catecholate to the semiquinonate was performed by exposing the
solution of the Ru complex in 2-propanol to air for 2 h. [Ru(DIP) (sq)](PF ) was
2
6
obtained in 19% yield after precipitation with a large excess of NH PF and
4
6
purification via silica gel chromatography. The identity of the product was
1
confirmed by HR-MS and NMR spectroscopy. H-NMR spectra showed a
characteristic peak broadening in the aromatic region between 7–9 ppm due to
the paramagnetism of the complex. In the 13C NMR and 2D H- C HSQC
spectra (Figure S1) ten inequivalent CH carbons were observed. The purity of
the product was confirmed by microanalysis.
1
13
X-ray Crystallography of [Ru(DIP) (sq)](Cl)
2
10
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 96
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The crystal structure of [Ru(DIP) (sq)](Cl) was determined by a single crystal
2
X-ray diffraction study. Suitable single crystals were grown from slow diffusion
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
of diethylether into a solution of the product prior to precipitation with NH PF6
4
in MeCN. The crystal structure revealed two independent Ru molecules (Ru-1
and Ru-2 in Figure S2), two chloride counter ions (from LiCl) and three water
molecules in the asymmetric unit (monoclinic P2 /c space group). Both Cl
1
atoms are disordered over two sets of sites with site-occupancy ratios of
0.299/0.701(3) and 0.244/0.756(5). The H atoms of the isolated water
molecules could be introduced in the final refinements, but their positions were
kept fixed to satisfy reliable hydrogen bonding. The molecular structure of one
of the independent Ru molecules is shown in Figure 1 and a selection of the
most relevant bond lengths and angles are provided in Tables S1 and S2
(additional crystallographic information can be found in the supporting
information). The X-ray crystal structure determination also provided evidence
for the nature of the dioxo ligand, as it can exist as catecholate, semiquinonate
and quinone.49,53,66 The typical range for the C-O bond length of such a ligand
coordinated to a metal is 1.34–1.47 Å for the catecholate form, 1.27–1.31 Å for
11
ACS Paragon Plus Environment

Page 13 of 96
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
the semiquinonate form and around 1.23 Å for the quinone.49,66 The C-O bond
distances of the dioxo ligand in [Ru(DIP) (sq)](Cl) are 1.309(4), 1.314(4),
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1.315(4) and 1.319(4) Å, which suggest that it is present in its semiquinonate
form.49
Figure 1. Molecular structure of [Ru(DIP) (sq)](Cl). The asymmetric unit
2
contains two crystallographically independent Ru cations, only one of which is
-
presented. The Cl counter ions, H atoms and solvent molecules are omitted for
clarity. The thermal ellipsoids are shown at the 30% probability level.
Electrochemistry
12
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 96
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The electrochemistry of [Ru(DIP) (sq)](PF ) (abbreviated as Ru-sq) was
2
6
investigated using cyclic voltammetry (CV) and rotating disc electrode
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
voltammetry
(RDE)
in
DMF
containing
tetrabutylammonium
hexafluorophosphate 0.1 M. The RDE voltammogram shown in Fig. S3 exhibits
four well-defined, reversible waves, in addition to that of decamethylferrocene,
which was used as internal reference with a half-wave potential of 0.030 V vs
the Saturated Calomel Electrode (SCE). The four features related to the Ru-sq
have the same intensity, which attests that the related redox processes involve
the same number of exchanged electrons. By comparison with the data
reported in the literature for closely related complexes under similar
conditions,52,67,68 the underlying redox processes were assigned as shown in
Table S3. The oxidation located at + 0.647 V vs SCE can be attributed to the
Ru(II)/Ru(III) redox couple while the sq/cat redox couple can be associated to
the first reduction process at -0.249 V vs SCE. The following two processes, at
more negative potentials, can be assigned to the sequential reductions of the
0/-
ancillary ligands (DIP ). Of note, the latter are separate couples with quite
some substantial redox splitting.52,67 These data clearly show how the presence
13
ACS Paragon Plus Environment

Page 15 of 96
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
of the semiquinonate ligand influences the redox properties of the metal centre,
causing a shift to lower potential. The couple of Ru (III)-quinone ligand is not
observed in these conditions since they are possibly located outside the anodic
limit of a DMF-based electrolyte. Moreover, the CV experiment (Figure S3)
indicates the reversibility of the redox processes, at least on the voltammetric
timescale.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Electron Paramagnetic Resonance
Ru-sq in its native state is Electron Paramagnetic Resonance (EPR) active in
DCM due to the presence of an unpaired spin as already confirmed by X-ray
crystallography (Figure S4a). At room temperature, a rather broad isotropic
signal was observed. Its g-value of 2.0244 is in line with a ligand-centred spin
density and deviates only slightly from the free electron value g of 2.0023. This
e
behaviour is in strong contrast to a metal-centred spin of a Ru(III) complex,
which would only become observable at low temperatures due to rapid
relaxation and display a broad, axial or rhombic signal with large anisotropy.53,69
The reduced form Ru-cat (Scheme 2) was generated by the reaction of Ru-sq
14
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 96
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
with equimolar amounts of cobaltocene (Cp Co, E = -0.880 V vs SCE in
2
1/2
70
DMF/0.1 M NBu PF ) (Figure S4b). Owing to the presence of a low-spin Ru(II)
4
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ion and a closed-shell catecholate ligand this species is EPR silent. The same
+
holds also true for oxidized, dicationic Ru -sq (Scheme 2), which was prepared
by treatment of Ru-sq with an excess of 1,1’-diacetylferrocenium
hexafluoroantimonate (Ac FcSbF , E = 0.940 V vs SCE in DMF/0.1 M
2
6
1/2
70,71
NBu PF ) (Figure S4c).
The absence of an EPR signal indicates that the
4
6
+
unpaired spins at the Ru(III) ion (Ru ) and the sq ligand are
antiferromagnetically coupled.
+
2+
N
e-
N
-e-
N
-
N
O
N
N
N
O
N
N
N
O
N
N
Ru
Ru
Ru
+ -
+e-
e
O
O
O
+
Ru-cat
Ru-sq
Ru -sq
+
Scheme 2. Structures of Ru-cat, Ru-sq, and Ru -sq, carrying a catecholate or a
+
semiquinonate ligand and Ru in oxidation state +II (Ru) or +III (Ru ), respectively.
Stability in DMSO and human plasma
15
ACS Paragon Plus Environment

Page 17 of 96
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
The stability of a compound plays an important role in its biological activity and
6
viability. Therefore, the integrity of Ru-sq was first assessed in DMSO-d using
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1H NMR spectroscopy. Ru-sq was found to be stable in DMSO over 8 days. No
change in the NMR spectra of the complex was observed over 8 days (Figure
S5). Next, to obtain a preliminary insight into the behaviour of Ru-sq under
physiological conditions, the stability of Ru-sq in human plasma was
investigated by UPLC following a procedure already established by our group.43
Ru-sq was incubated in human plasma at 37°C for 0 h, 4 h, 6 h, 12 h, 20 h and 24
h using diazepam as an internal standard. The UV traces of the UPLC analysis are
shown in Figure S6a. The concentration of Ru-sq was normalized with respect to the
internal standard and plotted against time. The linear trend shown in Figure S6b clearly
demonstrates that between 6 and 20 h, a decomposition of 50% of the
compound was observed, to reach a total degradation of the compound after
24 h.
Cytotoxicity Studies
16
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 96
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
After a full characterisation of Ru-sq, its potential activity as a chemotherapeutic
agent was investigated starting from the biological evaluation of its behaviour
against cancer cells in monolayer cell cultures. The cytotoxicity of Ru-sq
towards HeLa (human cervical adenocarcinoma), A2780 (human ovarian
carcinoma), A2780 cis (human cisplatin resistant ovarian carcinoma), A2780
ADR (human doxorubicin resistant ovarian carcinoma), CT-26 (mouse colon
adenocarcinoma), CT-26 LUC (mouse colon adenocarcinoma stably
expressing luciferase), RPE-1 (human normal retina pigmented epithelial) and
MRC-5 (human normal lung fibroblast) cell lines was therefore investigated
using a fluorometric cell viability assay (single graphs available in Figures S7).72
Cytotoxicity of cisplatin and doxorubicin was determined in the same cell lines
as positive controls and, as additional controls, Ru(DIP) Cl and catechol were
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
also tested.73,74 As shown in Table 1 where IC (the half maximal inhibitory
50
concentration) values are reported, Ru-sq displayed IC values between the
50
high nanomolar and low micromolar range on the cell lines investigated in this
study, while the Ru(DIP) Cl precursor and the catechol ligand itself showed
2
2
much lower cytotoxicity. Very impressively, Ru-sq exerted an activity 40 times
17
ACS Paragon Plus Environment

Page 19 of 96
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
higher than cisplatin against a cisplatin resistant cell line. On the other hand,
the cytotoxicity of doxorubicin and Ru-sq against a doxorubicin resistant cell
line appeared to be in the same order of magnitude. Overall, complex Ru-sq
displays a cytotoxicity, which is comparable to doxorubicin and much higher
than the one of cisplatin. However, no selectivity was observed between
cancerous and non-cancerous cell lines. This shortcoming is often faced in
medicinal chemistry and could be improved by the introduction of a targeting
moiety or by nanoformulation.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 1. IC values of Ru-sq, the Ru(DIP) Cl precursor and the catechol ligand
50
2
2
in tested cell lines; cisplatin and doxorubicin were used as positive controls.
A2780 A2780
ADR cis
CT-26
LUC
MRC-
5
IC (μM)
HeLa A2780
CT-26
RPE-1
5
0
9
.28 ± 4.00 ± 8.32 ± 18.33 2.60 ± 2.42 ± 30.24
11.20
Cisplatin
0
.20 0.76 0.71 ± 2.92 0.18 0.23
± 5.11 ± 2.32
0
.082
Doxorubic 0.34 ± 0.19 ± 5.94 ± 0.54 ±
0.18 ± 0.89 ± 3.37 ±
0.006 0.17 1.24
±
in
0.02
0.03
0.58
0.04
0.003
0
.50 ± 0.67 ± 4.13 ± 0.45 ± 1.00 ± 1.51 ± 0.90 ± 0.95 ±
0.01 0.04 0.2 0.03 0.03 0.14 0.04 0.09
5.03 4.69 ± 78.27 6.36 ± 9.20 ± 6.65 ± 3.13 ± 5.54 ±
Ru-sq
1
Ru(DIP)2
Cl2
± 0.4
0.14
± 4.9
0.57
1.22
0.5
0.07
0.39
18
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 96
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
4.55
±
2
2.80
5.96
16 ±
4.14
11.56
>100
>100
>100
>100
Catechol
±
± 0.40
11.30
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
We then decided to pursue the investigation of Ru-sq behaviour in a Multi
75
Cellular Tumour Spheroids (MCTS) model. It was previously shown that such
a model mimics the in vivo microenvironment and tumour metabolism.76,77
Moreover, large MCTS develop a central necrosis core similar to that found in
78
the inner core of tumours. These unique features give a better representation
of a cancer model compared to a 2D model, lowering the disparity between in
vitro and in vivo models.78 Table 2 shows the IC50 values obtained via a
luminescent cell viability assay for compounds that were administered to HeLa
MCTS for 48 h (single graphs are availabe in Figure S8). The Ru(DIP) Cl
2
2
precursor and the catechol ligand were tested as additional controls and
exhibited lower cytotoxicity than Ru-sq. Catechol showed no toxicity towards
the cell line tested (IC > 100 μM) while the precursor displayed a cytotoxicity
50
comparable to cisplatin. Cisplatin was used as a positive control and the results
19
ACS Paragon Plus Environment

Page 21 of 96
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
7
9
are in line with literature data. The cytotoxicity of Ru-sq in HeLa MCTS was
impressively high after 48 h treatment, with IC ~ 14 μM, which is 3 times lower
50
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
than cisplatin or doxorubicin (IC ~ 47 µM or 39 µM, respectively). Noteworthy,
5
0
the cytotoxicity of Ru-sq was comparable to the one of doxorubicin after 72 h
80
treatment (IC ~ 11 μM). These studies clearly demonstrate the high potential
50
of Ru-sq as an anticancer drug candidate. The impressive bioactivity,
comparable to doxorubicin in monolayer cell culture, was also confirmed in the
3D tumour model – HeLa MCTS.
Table 2. IC values for Ru-sq, Ru(DIP) Cl precursor and the catechol ligand in
50
2
2
multicellular HeLa cancer cell spheroids (approximately 400 μm in diameter);
cisplatin and doxorubicin were used as positive controls.
Doxorubici
n
Ru(DIP) Cl
2
IC (μM)
Cisplatin
Ru-sq
Catechol
5
0
2
HeLa MCTS 46.49 ± 4.18 38.59 ± 0.43 14.11 ± 0.09 59.84 ± 3.05
>100
Spheroid integrity and growth upon treatment are very useful tools to determine
78
a potential drug activity. In this study MCTS were monitored over 13 days
after treatment with different concentrations of Ru-sq (Figure 2). Every 3 days,
20
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 96
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the spheroids were washed to remove dead cells and their diameters were
measured (Figure 2). It is important to note that at each washing step, half of
the media was removed and replaced with fresh one, diluting twice the quantity
of the compound in each well. The effect of Ru-sq on growth inhibition is dose-
dependent and already visible after 3 days. Low concentrations treatments (1
µM and 2.5 µM) led to regrowth of the spheroids after the first 72 h, while for 5
µM and 10 µM treatments, the regrowth is visible after 6 and 9 days,
respectively. Ru-sq treatment with concentrations higher than IC (20 µM and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
50
25 µM) completely inhibits the spheroids growth after 13 days of treatment.
Overall, we can conclude that Ru-sq treatment at 20 µM and 25 µM
concentrations, severely affect the size and the integrity of the spheroids after
13 days of treatment.
21
ACS Paragon Plus Environment

Page 23 of 96
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Growth kinetics of HeLa MCTS upon treatment with different
concentrations of Ru-sq (1, 2.5, 5, 10 and 20 µM). a) Images collected at day 0
(before treatment) and at day 3, 6, 9 and 13. b) MCTS diameter calculated at
different time points. Blue dotted line indicates day of seeding, red dashed line
indicates day of treatment, green dotted lines indicate days of washing.
Cell Death Mechanism
The excellent activity displayed by Ru-sq in HeLa MCTS encouraged us to perform
further experiments in order to obtain more insights into its in vitro behaviour. The first
step was the evaluation of the type of cell death occurring when cancer cells were
treated with Ru-sq. For this experiment, HeLa cells were analysed via flow
cytometry using the Annexin V and PI (propidium iodide) staining method.81
Staurosporine, a known inducer of apoptosis, was employed as a positive
22
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 96
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
control.82 As shown in Figure 3 and Figure S9 (dot plots), Ru-sq induced
significant apoptosis as early as 30 min treatment with progression from early
to late apoptosis at 4 h. The level of apoptosis induction by the complex after 4
h was comparable to that caused by 24 h staurosporine treatment. These data
clearly demonstrate that Ru-sq induces apoptosis as the only type of cell death
in HeLa cells.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Induction of apoptosis/necrosis in HeLa cells upon treatment with Ru-sq (10
μM) and staurosporine (1 μM) at different time frames. The values are expressed as a
mean ± S.D. (standard deviation) of three biological repeats.
23
ACS Paragon Plus Environment

Page 25 of 96
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Cellular uptake, intracellular distribution and DNA metalation studies
Next, the cellular uptake of Ru-sq and cisplatin was investigated in HeLa cells.
The amount of ruthenium accumulated was detected by Inductively Coupled
Plasma Mass Spectrometry (ICP-MS). Working concentrations and incubation
times were chosen to avoid extended cell mass loss due to the high cytotoxicity
of the complexes but considering a ruthenium final amount that could afford
determination of the metal content. After 2 h treatment (5 µM), Ru-sq
internalises slightly better than the drug cisplatin (Figure 4a). The low
accumulation of cisplatin in these working conditions is in agreement with the
literature data.83,84 To have more insights about uptake mechanisms of Ru-sq
into the HeLa cells, additional experiments were performed. Cells were pre-
treated with different inhibitors of uptake pathways or kept at different
temperatures to assess the energy dependence of the uptake mechanism(s)
(Figure S10). More specifically, low temperature (4ºC), pre-treatment with
metabolic inhibitors (which decreases ATP production), pre-treatment with
chloroquine or ammonium chloride (which mostly impede endocytic pathways),
or pre-treatment with tetraethylammonium chloride (mostly inhibiting cation
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
24
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 96
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
transporters) decreased Ru-sq accumulation in cells by half when compared to
37 ºC condition (see Figure S10). This outcome indicates that Ru-sq might be
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
transported into the HeLa cells by both energy dependent (active) and energy
independent (passive) pathways. Further cellular fractionation experiment
showed preferential accumulation of Ru-sq inside the nucleus (Figure 4b).
These findings suggest that the mode of action could be related to the damage
caused to DNA and/or to prevention of replication as well as transcription.85,86
In order to identify DNA as a potential target for the complex, the genetic
material was extracted from HeLa cells after 2 h treatment with Ru-sq or
cisplatin and the amount of metal analysed by ICP-MS. The DNA of cells
treated with Ru-sq displayed a metal content much higher when compared to
cisplatin. Taken together, these findings strongly suggest direct interaction with
DNA as a possible mechanism of action exerted by Ru-sq.
25
ACS Paragon Plus Environment

Page 27 of 96
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Cellular uptake (a), cellular fractionation (b) and DNA metalation (c) of HeLa
cells after treatment with tested compounds (5 µM, 2 h). Data are presented as the mean
± SD of at least 3 biological replicates.
JC-1 Mitochondrial Membrane Potential Test and Metabolic Studies
The search for additional mechanisms of action associated to the treatment of
Ru-sq led us to the investigation of the mitochondrial metabolism impairment.87
Firstly, the mitochondrial membrane potential was studied with the use of a
JC-1 (a membrane-permeant dye).88,89 At high
largely used indicator
potentials, the dye forms red emitting aggregates in the mitochondria
membrane, whereas at low potentials, it stays as a green emitting
monomer.88,89 The membrane potential is directly connected to oxidative
90
phosphorylation (the main mitochondrial function). HeLa cells were treated for
26
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 96
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
4 h with increasing concentrations of Ru-sq (from 0.2 µM to 0.6 µM). Figure
5a shows a slight decrease of the red fluorescence signal with increasing
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
concentrations of Ru-sq and a significant drop in the signal around the IC50
concentration (0.5 µM, marked in red). However, the dramatic collapse of
mitochondrial membrane potential could also be caused by ongoing apoptosis.91
Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), an uncoupling agent
9
2
92
that impairs the membrane potential was used as positive control. Comparison of
the results obtained with Ru-sq (0.5 µM) and FCCP treatment showed that the same
loss in potential was detected. These findings strongly suggest a contribution of the
membrane potential impairment to the cell death mechanism.
Figure 5. a) Fluorescence signal of JC-1 dye detected in HeLa cells treated for 24 h
with different concentrations of Ru-sq (from 0.2 µM to 0.6 µM). Bar marked in red
indicates the IC concentration (0.5 µM). FCCP is used as positive control, cisplatin
5
0
and DMSO (1%) are used as negative controls. b) Mito Stress Test profile in HeLa cells
27
ACS Paragon Plus Environment

Page 29 of 96
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
after 24 h treatment; oxygen consumption rate changes after treatment with specific
electron transport chain inhibitors. Oligomycin (inhibitor of ATP synthase (complex
V)), FCCP (uncoupling agent), Antimycin-A (complex III inhibitor) and Rotenone
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(complex I inhibitor).
Inspired by these findings, further studies on the metabolic pathways that could
be affected by the complex were performed. For this purpose, Seahorse XF
Analyzer was used to measure, in real time, the oxygen consumption rate
(OCR) and extracellular acidification rate (ECAR) of treated cells. Firstly, the
effect of Ru-sq on the oxidative phosphorylation in the HeLa cell line was
investigated. Mitochondrial respiration was found to be severely impaired in
cells treated with Ru-sq as opposed to the precursor Ru(DIP) Cl . This was
2
2
evident from the low basal respiration and the inhibition of ATP production
compared to untreated cells. The mitochondrial membrane of the cells treated
with Ru-sq, lost the capacity to restore the proton balance when treated with an
uncoupling agent (FCCP). The maximal respiration (the OCR value when the
mitochondrial membrane is uncoupled) and spare respiratory capacity
(difference in the OCR values between maximal respiration and basal
respiration) of the cells was reduced compared to untreated cells (Figure 5b
28
ACS Paragon Plus Environment