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\DOI{10.5802/crchim.417}
\datereceived{2025-08-20}
\daterevised{2025-08-30}
\dateaccepted{2025-09-01}
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\dateposted{2025-10-21}
\begin{document}

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\CDRsetmeta{articletype}{review}

\title{High specificity is a key parameter for copper ligands as future
therapeutic agents for Alzheimer's, Wilson's, and cancer diseases}

\alttitle{Une haute sp\'{e}cificit\'{e} est d\'{e}terminante pour des
ch\'{e}lateurs du cuivre en tant que futurs agents th\'{e}rapeutiques
(maladies d'Alzheimer, de Wilson, et \`{a} cellules canc\'{e}reuses)} 


\dedicatory{This review is dedicated to Dr. Marguerite Piti\'{e},
${\dagger}$ 2011, {in memoriam}}

\author{\firstname{Anne} \lastname{Robert}\CDRorcid{0000-0002-9092-6776}\IsCorresp}
\address{Laboratoire de Chimie de Coordination du CNRS, LCC-CNRS, Inserm ERL 1289 MAAP, Universit\'{e} de Toulouse, 205 route de Narbonne, 31077 Toulouse cedex, France}
\email%[A. Robert]
{anne.robert@lcc-toulouse.fr}

\author{\firstname{Yan} \lastname{Liu}\CDRorcid{0000-0002-3864-1992}\IsCorresp}
\address{School of Chemical Engineering and Light Industry, Guangdong University of Technology, Higher Education Mega Center, Guangzhou 510006, PR China}
\email%[Y. Liu]
{yanliu@gdut.edu.cn}

\author{\firstname{Michel} \lastname{Nguyen}\CDRorcid{0000-0002-6496-0495}}
\addressSameAs{1}{Laboratoire de Chimie de Coordination du CNRS, LCC-CNRS, Inserm ERL 1289 MAAP, Universit\'{e} de Toulouse, 205 route de Narbonne, 31077 Toulouse cedex, France}
\email%[M. Nguyen]
{michel.nguyen@lcc-toulouse.fr}

\author{\firstname{Fran\c{c}oise} \lastname{Benoit-Vical}\CDRorcid{0000-0002-9468-4823}}
\addressSameAs{1}{Laboratoire de Chimie de Coordination du CNRS, LCC-CNRS, Inserm ERL 1289 MAAP, Universit\'{e} de Toulouse, 205 route de Narbonne, 31077 Toulouse cedex, France}
\email%[F. Benoit-Vical]
{francoise.benoit-vical@lcc-toulouse.fr}

\author{\firstname{Bernard}\nobreakauthor\lastname{Meunier}\CDRorcid{0000-0003-2200-7142}\IsCorresp}
\addressSameAs{1}{Laboratoire de Chimie de Coordination du CNRS, LCC-CNRS, Inserm ERL 1289 MAAP, Universit\'{e} de Toulouse, 205 route de Narbonne, 31077 Toulouse cedex, France}
\addressSameAs{2}{School of Chemical Engineering and Light Industry, Guangdong University of Technology, Higher Education Mega Center, Guangzhou 510006, PR China}
\email%[B. Meunier]
{bmeunier@lcc-toulouse.fr}

\shortrunauthors

\keywords{\kwd{Alzheimer's disease}
\kwd{Cancer}
\kwd{Chelator}
\kwd{Copper}
\kwd{Medicinal chemistry}
\kwd{Redox metal}
\kwd{Wilson's disease}}

\altkeywords{\kwd{Maladie d'Alzheimer}
\kwd{Cancer}
\kwd{Ch\'{e}lateur}
\kwd{Cuivre}
\kwd{Chimie th\'{e}rapeutique}
\kwd{M\'{e}tal \`{a} activit\'{e} r\'{e}dox}
\kwd{Maladie de Wilson}}

\thanks{CNRS, Inserm, GDUT}

\begin{abstract}
The loss of copper homeostasis is considered a characteristic feature
of several major human diseases such as Alzheimer's, Wilson's, or
cancer diseases. Consequently, the design of new efficient ligands
targeting copper regulation is a major concern of therapeutic
bioinorganic chemistry and should take in consideration the chelator
structures for being copper-specific and the redox properties of the
corresponding complexes to obtain suitable pharmacological activities.
As an improvement of previously developed amine ligands,
N4-tetradentate 8-aminoquinoline chelators, are able to inhibit memory
and behavior impairment in Alzheimer mice, by generating square planar
Cu(II) complexes suitable for the efficient extraction of copper ions
from metallated amyloid peptides, leading to a reduction of the
neurotoxic oxidative stress in the brain. These chelators also reverse
the hepatotoxic accumulation of copper in animals with Wilson's
disease and inhibit the proliferation and migration of human cancer
cell lines. This review summarizes the gradual and logical development
of twenty-five years of research in our group on copper-specific
chelators and their promising development as potential therapeutic
tools against diseases in which copper is involved. 
\end{abstract}

\begin{altabstract}
La perte de l'hom\'{e}ostasie du cuivre endog\`{e}ne est une
caract\'{e}ristique essentielle de certaines pathologies humaines comme
la maladie d'Alzheimer, la maladie de Wilson et le cancer. La mise au
point de ligands sp\'{e}cifiques du cuivre capables de r\'{e}guler
in vivo la r\'{e}partition de ce m\'{e}tal constitue donc
un probl\`{e}me de chimie bioinorganique et une voie majeure
d'avanc\'{e}es possibles en chimie th\'{e}rapeutique. La structure de
ces ligands et les propri\'{e}t\'{e}s oxydo-r\'{e}ductrices des
complexes qui en r\'{e}sultent sont d\'{e}cisives pour obtenir les
activit\'{e}s pharmacologiques d\'{e}sir\'{e}es. Mettant \`{a} profit
notre exp\'{e}rience des ligands amin\'{e}s, nous avons
d\'{e}velopp\'{e} des ch\'{e}lateurs N4-t\'{e}tradentates
comportant un motif 8-aminoquinol\'{e}ine et une chaine lat\'{e}rale
amin\'{e}e. Par l'extraction des ions cuivriques pi\'{e}g\'{e}s par
les peptides amylo\"{i}des et la r\'{e}duction du stress oxydant
neurotoxique dans le cerveau, ces ch\'{e}lateurs sont capables
d'inhiber le d\'{e}clin cognitif et comportemental de souris
mod\`{e}les de la maladie d'Alzheimer. Ces ch\'{e}lateurs
inhibent \'{e}galement l'accumulation h\'{e}patotoxique de cuivre
chez les souris atteintes de la maladie de Wilson, ainsi que la
prolif\'{e}ration et la migration de cellules canc\'{e}reuses
humaines. Cette revue r\'{e}sume le d\'{e}veloppement graduel et logique de
vingt-cinq ans de recherche dans notre groupe sur les
ch\'{e}lateurs sp\'{e}cifiques du cuivre, ainsi que leur
d\'{e}veloppement possible comme candidats-m\'{e}dicaments contre les
maladies dans lesquelles le cuivre est impliqu\'{e}.
\end{altabstract}

\editornote{Article submitted by invitation}
%\alteditornote{Article soumis sur invitation}

\maketitle

%\vspace*{-6pt}

%\vspace*{6pt}

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\section{Introduction: copper in human health and disease}
\vspace*{-2pt}
\raggedbottom
After iron and zinc, copper is the third essential metal ion in living
systems \cite{1}. The highest concentrations of copper are found in
liver and brain. The copper concentration in human frontal lobe and
cerebellum is ranging from 60 to 110~$\upmu$M \cite{2}. In living
organisms, copper exists in the oxidized Cu(II) and reduced Cu(I)
forms. In biological conditions, the interconversion between these two
oxidation states is easy and facilitates the role of this metal as
catalytic co-factor in various copper enzymes involved in electron
transfers and oxidations.

Copper ions are found in many different 
\mbox{metalloenzymes}
(laccases, tyrosinase, superoxide 
dismutase, ascorbate oxidase,
cytochrome-c oxidase to cite a few ones) and metalloproteins
(hemocyanin, ceruloplasmin) \cite{3,4}. Among these copper proteins
involved in vital processes, cytochrome-c oxidase is a large
transmembrane terminal heme/copper oxidase of the respiratory electron
transport chain. Its redox centers involved in electron transport
consist of two heme moieties and two copper centers that catalyze the
reduction of dioxygen to water with the concomittant pumping of four
protons through the mitochondrial membrane \cite{5} (Equation~(\ref{eqn1})).
{\begin{eqnarray}
\mathrm{O}_2 \; \xrightarrow{4 \mathrm{e}^{-}, 4 \mathrm{H}^{+}} \;
2 \mathrm{H}_2 \mathrm{O}
\label{eqn1}
\end{eqnarray}}\unskip

Since the air-stable oxidation state ${+}$II of copper can be easily
reduced to the highly reactive state (${+}$I) by endogenous reducing
agents (e.g., ascorbate, glutathione, NADPH) \cite{6}, its acquisition,
distribution and regulation in living systems is strictly controlled by
copper carriers and chaperone proteins \cite{7}. As a low-valent
transition metal ion, Cu(I) behaves in the same way as Fe(II), being
able to induce the \textit{electron-by-electron} reduction of dioxygen
to water in all aerobic organisms (Scheme~\ref{sch1}). This catalytic
reaction is potentially deleterious because it produces superoxide
radical anion $\mathrm{O}_{2}^{\bullet -}$, then hydrogen peroxide
$\mathrm{H}_{2}\mathrm{O}_{2}$ and finally hydroxyl radical
$\mathrm{HO}^{\bullet}$, the most aggressive form among the
reactive/{reduced oxygen species} (ROS). 

\begin{scheme}
\includegraphics{sc01}
\vspace*{6pt}
\caption{\label{sch1}Step-by-step reduction of dioxygen catalyzed by
low-valent transition metals such as Cu(I) or Fe(II).}
\vspace*{-10pt}
\end{scheme}

Superoxide is a relatively mild reductant and hydrogen peroxide
is the required cofactor of the ubiquitous peroxidases. In healthy
organisms, the concentrations of superoxide and hydrogen peroxide are
strictly controlled by metallo-enzymes. The efficient superoxide
dismutases (SODs), Cu/Zn-superoxide dismutase (${=}$ SOD-1), to name one
of them, have a key role in the regulation of this metabolic pathway,
by converting two superoxide radical anions to molecular oxygen and
hydrogen peroxide through the redox activity of its copper centre,
while zinc plays a structural role (Equation~(\ref{eqn2})).

Cu/Zn-SOD (${=}$ SOD-1) is present in nearly all compartments of
human cells, nucleus, mitochondria, cytosol, and peroxisomes.
{\begin{eqnarray}
2 \mathrm{O}_2^{\bullet -}\; \xrightarrow{2 \mathrm{H}^{+}}\;
\mathrm{O}_2 + \mathrm{H}_2 \mathrm{O}_2
\label{eqn2}
\end{eqnarray}}\unskip

Hydrogen peroxide is used as cofactor by peroxidases, but any excess of
production of this peroxide is controlled by catalases that are highly
efficient at $\mathrm{H}_{2}\mathrm{O}_{2}$ disproportionation. In the
case of $\mathrm{H}_{2}\mathrm{O}_{2}$ overproduction, the
three-electron reduction product of dioxygen, hydroxyl radical
$\mathrm{HO}^{\bullet}$, is produced. This radical is the strongest
oxidizing agent (standard redox potential ${=}$ 2.8~V/NHE) after fluorine
gas (2.9~V/NHE) and, unlike $\mathrm{O}_{2}^{\bullet -}$ or
$\mathrm{H}_{2}\mathrm{O}_{2}$, no specific detoxifying enzymes exist
for $\mathrm{HO}^{\bullet}$. At diffusion-controlled rates,
$\mathrm{HO}^{\bullet}$ hydroxylates aliphatic C--H bonds, or performs
the one-electron oxidation of rather inert molecules, or modifies
unsaturated compounds by addition \cite{8}. All biomolecules of living
organisms can be quickly oxidized by $\mathrm{HO}^{\bullet}$, leading
to extensive damage up to cell death. The only way to prevent the
formation of this deleterious radical is to control the upstream
reduction of molecular oxygen. Involvement of an oxidative stress
induced by misregulated copper in the brain has been documented as a
key feature of Alzheimer's disease and proposed to be important for the
development of the brain pathology \cite{9,10,11}. It is also well
known that the deregulation of copper homeostasis is involved in other
inherited or sporadic neurodegenerative pathologies like Parkinson's
disease, amyotrophic lateral sclerosis that is caused by mutations of
SOD-1 \cite{12}, or prion-mediated encephalopathies \cite{13}. Copper
ions are also essential for the function of several brain
metalloenzymes like tyrosinase \cite{14} or dopamine
${\upbeta}$-hydroxylase \cite{15}.

In addition, any deficiency of a human copper carrier protein involved
in the regulation of copper uptake, may generate a pathology. This is
the case for Wilson's disease, a genetic condition caused by mutations
of the \textit{Atp7b} gene coding for the copper carrier ATP7B in
charge of excreting copper from liver to bile. Impairment of ATP7B
causes massive accumulation of copper in liver, brain, and other
organs, resulting in liver disease and neurological disorders
\cite{16,17,18}. Moreover, copper    is essential for efficient iron
and zinc trafficking in mammals. The major copper transporter
ceruloplasmin exhibits a ferroxidase activity that promotes the Fe(II)
oxidation to facilitate the iron loading into ferritin, and Zn(II) is a
substrate of ferroportin \cite{19}. So it should be noted that the
roles of these three vital metal ions, copper, zinc, and iron, are
often interconnected in different proteins or enzymes. Consequently,
any metal chelator, if considered as drug candidate, should be specific
for one of these metal ions to avoid as much as possible perturbations
of metalloproteins dependent on the two other metal ions. This is a
prerequisite point in the rational design of a copper ligand for being
beneficial in the therapy of a copper-related disease.

As bioinorganic chemists initially involved in DNA cleavage by metal
complexes, our group became interested by using copper complexes as DNA
cleavers (Section~\ref{sec2} of the present review article), then some
copper ligands were designed for antitumor activity
(Sections~\ref{sec3} and \ref{sec6}). Different series of specific
copper chelators were then developed as potential drugs to treat
Alzheimer's disease (Section~\ref{sec4}) and Wilson's disease
(Section~\ref{sec5}). In relation to the state of the art, the present
review is therefore focused on the last twenty-five years of research
on copper ligands with potential therapeutic interest performed in our
research group, especially on the step-by-step rational design of
metal-selective and biologically efficient drugs.

\section{Copper complexes as DNA cleavers}\label{sec2}

Among the various metal complexes developed over the last three decades
as artificial nucleases, copper complexes of 1,10-phenanthroline (Phen)
have been widely used as DNA cleavers~\cite{20,21,22,23,24}. The
versatile Phen ligand is able to chelate copper ions as redox-active
Cu(Phen) and Cu(Phen)$_{2}$ complexes (for a general review on the
chemistry of phenanthrolines, see Ref.~\cite{24}). Interaction of the
redox-active Cu(I)/Cu(II)--phenanthroline complexes in the minor groove
of double-stranded DNA in the presence of
$\mathrm{H}_{2}\mathrm{O}_{2}$, triggers the oxidation of C1$^\prime$ and
C4$^{\prime}$ of 2-deoxyribose units, resulting in single-strand DNA
cleavage~\cite{20,21}. This cleavage can also be achieved by aerobic
oxidation in the presence of a reducing agent. Cu(Phen)$_{2}$ was found
significantly more active than Cu(Phen), but the association constant
for the second Phen ligand onto the copper ion is too low to enhance
DNA oxidation at submicromolar concentrations $(\log K_{\mathrm{app}} =
5.5)$~\cite{25}. Consequently, chelators named ``Clip-Phen'' were
prepared, containing two Phen residues linked through their C2 or C3
positions by an adjustable bridge, to favor the coordination of two
Phen units around a single copper ion (Figure~\ref{fig1})~\cite{26,27}.
The efficiency of oxidative DNA cleavage by Cu--Clip-Phen complexes,
under aerobic and physiological conditions in the presence of a
reductant, was evaluated by quantification of the cleavage of
supercoiled circular bacteriophage ${\Phi}$X174 DNA (form I) into
relaxed (form II) and circular (form III) forms. In these conditions,
Cu--Clip-Phen complexes exhibited a nuclease activity that was
dramatically higher than that of Cu--1,10-phenanthroline~\cite{26}.

\begin{figure}
\includegraphics{fig01}
\vspace*{-.2pc}
\caption{\label{fig1}Structures of the covalently linked
bis-phenanthroline ligands, 2-Clip-Phen and 3-Clip-Phen.}
\vspace*{-.8pc}
\end{figure}

The DNase activity of Clip-Phen was higher when the bridge was linked
at the C3 position of the Phen moiety (3-Clip-Phen series) compared to
the C2 position (2-Clip-Phen series) and when the bridge contained
three methylene units~\cite{27,28}. The most efficient chelator of this
series was 3-Clip-Phen (Figure~\ref{fig1}) with a serinol bridge at C3
position of the Phen rings, being 20--30 times more active as DNA
cleaver than its 2-Clip-Phen analog~\cite{27,28}. In fact, 2-Clip-Phen
in the presence of CuCl$_{2}$ (1:1 molar ratio) resulted in an
incomplete (70\%) cleavage of ${\Phi}$X174 form I into relaxed form
II (30\% of remaining form~I) whereas, in the same conditions,
3-Clip-Phen resulted in the complete cleavage of form I into form~II
(35\%) and circular form III (65\%), with an intense smear
corresponding to smaller DNA fragments.

In addition, the serinol bridge between the two Phen units allowed, via
its primary aliphatic amine, the vectorization and modulation of the
binding domain in these DNA cleavers. As expected, the attachment of
spermine, which has high affinity for the minor groove of double
helix DNA, to 2-Clip-Phen afforded a new
spermine--phenanthroline conjugate with an enhanced nuclease
efficacy~\cite{29}.

The electrochemical study of the 2- and 3-Clip-Phen series indicated
that the 2-Clip-Phen ligands are better to stabilize the tetrahedral
Cu(I) state of their complexes in comparison to the Cu(II) state. On
the other hand, 3-Clip-Phen derivatives facilitate the stabilization of
the corresponding Cu(II) complexes with a quasi-irreversible Cu(II)
${\rightarrow}$ Cu(I) reduction, suggesting that the reduced DNA
cleavage efficacy of Cu--2-Clip-Phen complexes might be correlated with
the higher stabilization of the cuprous oxidation state. However, the
absence of strict correlation between the redox properties of the
copper complexes and their DNA cleavage efficacy suggests that steric
or electrostatic parameters of the DNA interactions also take part in
the modulation of the nuclease activity of these copper complexes.

Noteworthy, several complexes of 2- or 3-Clip-Phen with Cu(II)
crystallized as a double-helical structure of stoichiometry
$\mathrm{Cu}_{2}\mathrm{L}_{2}$, while monomeric CuL species, with
various geometries, were predominant in solution~\cite{28}. The
structure and reactivity of these copper complexes appear to be
particularly versatile, a feature that opens the way for potential uses
as biological tools.


\section{Copper ligands/complexes with antitumor activity}\label{sec3}

Since the registration of cisplatin in the early 1980s, the role of
metal ligands and complexes in cancer research has been the subject of
many studies until now (for a recent review, see Ref.~\cite{30}). The
efficacy of the copper complexes of Clip-Phen as DNA cleavers suggested
that these chelators might also be considered as therapeutic agents
against cancer. In fact, research on the cytostatic and potential
antitumor activities of substituted Phen derivatives or related ligands
metalated with copper or other various metals has been investigated in
parallel to their DNase activity~\cite{31,32,33,34} and has remained an
active research field until recent years, with the aim of developing
novel metal-based anticancer drugs~\cite{35,36,37}.

In this context, the cytostatic properties of a series of 3-Clip-Phen
derivatives were evaluated on the classic L1210 murine leukemia cell
line. The concentration inhibiting the cell growth by 50\% after 48~h
(IC$_{50}$) of 3-Clip-Phen was close to that of Phen itself
(1.15 and $2.5~\upmu$M, respectively), although
Cu--3-Clip-Phen was a better DNA cleaver than
Cu(Phen)$_{2}$~\cite{33}. This result suggested that the
cytostatic activity was not strictly correlated to the DNase activity.

Conversely, 3-propyl-Clip-Phen, with a simple aliphatic linker
(Figure~\ref{fig2}), exhibited a significantly higher activity against
L1210 cells ($\mathrm{IC}_{50} = 0.25~\upmu\mathrm{M}$) compared to
3-Clip-Phen (Figure~\ref{fig1}). Analogs of 3-propyl-Clip-Phen
containing two to five methylene units in the linker (3-alkyl-Clip-phen
series) (Figure~\ref{fig2}), were also evaluated. Their activity was
significantly correlated to their number of methylene units in the
linker, itself correlated to hydrophobicity (Figure~\ref{fig3}). The
longer the linker, the more active the compound was, with an 
$\mathrm{IC}_{50}$
value of $0.13~\upmu\mathrm{M}$ for 3-pentyl-Clip-phen. In order to
investigate whether the hydrophilic primary amine of the linker of
3-Clip-phen $(\log P_{\mathrm{calc}} = 2.47$, $\mathrm{IC}_{50} =
1.15~\upmu\mathrm{M})$ could hamper its biological activity, this
ligand was compared to the slightly more hydrophobic 3-acetyl-Clip-Phen
$(\log P_{\mathrm{calc}} = 2.80)$. However, the antiproliferative
activity of 3-acetyl-Clip-Phen was lower at $5~\upmu\mathrm{M}$,
indicating that the correlation between biological activity and log
$P_{\mathrm{calc}}$ was limited to the 3-aliphatic-Clip-Phen
series~\cite{33}.


\begin{figure}
\includegraphics{fig02}
\vspace*{-.2pc}
\caption{\label{fig2}Structures of alkyl substituted 3-Clip-Phen
ligands, and 3,8-substituted-1,10-phenanthrolines.}
\vspace*{-.8pc}
\end{figure}


\begin{figure}
\includegraphics{fig03}
\caption{\label{fig3}Antiproliferative activity of 3-alkyl-Clip-Phen
derivatives measured as 1/IC$_{50}$ value with respect to
the number of methylene units in the linker ($n$, blue trace) or to the
hydrophobicity ($\log P_{\mathrm{calc}}$, red trace). The
dotted lines stand for polynomial trend curves of degree 2.}
\end{figure}

The putative role of the alkoxy substituent at the C3-position in the
phenanthroline of Clip-Phen derivatives was evaluated by comparing
their activities to that of monomeric Phen derivatives, 3-methoxy- and
3,8-dimethoxy-1,10-phenantholine. The $\mathrm{IC}_{50}$ values of
3-methoxy- and 3,8-dimethoxy-1,10-phenantholine were 
$1.5~\upmu\mathrm{M}$ and $1.4~\upmu\mathrm{M}$, respectively, in the
same range as for Phen itself ($2.5~\upmu\mathrm{M}$), suggesting that
the alkoxy substituents at the C3- and C8-position were not
responsible for the higher cytostatic activity of 3-alkyl-Clip-Phen. On the other hand, the attachment position of the linker on the
phenanthroline units is an important parameter, since
2-propyl-Clip-Phen exhibited a much poorer activity ($\mathrm{IC}_{50}$
value ${>}$~$100~\upmu\mathrm{M}$) than its regio-isomer
3-propyl-Clip-Phen ($1.15~\upmu\mathrm{M}$)~\cite{33}.

The conjugation of 3-Clip-Phen with DNA binders and/or cell penetration
agents such as spermine or polyarginine (Arg)$_{9}$ did not increase
the antiproliferative activity. The $\mathrm{IC}_{50}$ values of
3-Clip-Phen--spermine and 3-Clip-Phen--(Arg)$_{9}$ were 
$12~\upmu\mathrm{M}$ and $1.9~\upmu\mathrm{M}$, respectively, against
L1210 cells~\cite{33}, despite the fact that 3-Clip-Phen--spermine
exhibited a much higher nuclease activity than 3-Clip-Phen on
supercoiled ${\Phi}$X174 DNA~\cite{29}.

Importantly, Cu(II) complexes of Clip-Phen derivatives were prepared,
and their cytostatic activities were evaluated. While complexation of
copper failed to improve the activity of 3-Clip-Phen derivatives
regardless of the composition of the linker, the activity of
2-propyl-Clip-Phen dramatically increased with copper complexation
($\mathrm{IC}_{50} = 2~\upmu\mathrm{M}$, compared to $\mathrm{IC}_{50}>
100~\upmu\mathrm{M}$ for the free ligand). These results confirmed
that the attachment position of the linker induced large variations in
the biological properties of Clip-Phen derivatives~\cite{33}. 


\section{Copper chelators against Alzheimer's\newline disease}\label{sec4}

Among the various putative targets involved in Alzheimer's disease
(AD), the loss of metal ion homeostasis in AD brain is well
documented~\cite{38,39,40,41}. Post-mortem analyses of the brain of
patients with AD indicated that amyloid plaques contain an excess of
copper, iron, and zinc by a factor of 5.7, 2.8, and 3.1, respectively,
compared to the levels of normal brains~\cite{9}.
Copper--amyloid complexes, in the presence of endogenous
reductants, catalyze the reduction of dioxygen to generate reduced ROS
(Scheme~\ref{sch1})~\cite{40,42}. Production of ROS is the molecular
signature of chronic inflammation reported in AD brain and involved in
neuronal death~\cite{43}. In addition, copper ions sequestered in
amyloid plaques may be responsible for a deficit in copper for cerebral
copper-dependent enzymes, like superoxide dismutase-1,~\cite{44} or
copper oxidases involved in the biosynthesis of neurotransmitters.
Therefore, restoration of copper homeostasis in the brain is a
multifunction drug target that requires the design of specific
chelators~\cite{38,11,45,46}. Because of the deleterious
oxidative stress catalyzed by out-of-control redox-active metal ions,
this approach should be considered an essential strategy among all
various attempts in AD drug design.


\subsection{Clioquinol (CQ) and PBT2}
The 8-hydroxyquinoline derivatives clioquinol (CQ) and PBT2
(Figure~\ref{fig4}) have been the first copper/zinc ligands considered
as potential anti-AD agents to limit the interactions between metal
ions and amyloid peptide (A${\upbeta}$). Clioquinol was able to
decrease A${\upbeta}$ deposits in brain and to improve learning and
memory abilities of transgenic AD mice~\cite{47,48}. Unfortunately,
this simple chelator, previously used as antifungal and antiprotozoal
drug, was withdrawn from the market in 1983, due to its neurotoxicity
attributed to zinc chelation~\cite{15,50}. In fact, the
8-hydroxyquinolines are non-specific metal chelators~\cite{51,52}, and
the affinity constant of CQ for Cu(II) is only one order of magnitude
higher than that for Zn(II) ($\log K = 10$ and 9,
respectively)~\cite{38,53}. In this series, PBT2~\cite{38} was less
toxic~\cite{48}. However, phase II clinical trial of PBT2 as anti-AD
drug was stopped due to its lack of efficacy~\cite{54}.


\begin{figure}
\includegraphics{fig04}
\vspace*{-2pt}
\caption{\label{fig4}Structures of copper chelators developed as drugs
against Alzheimer's disease. Z~stands for an adjustable linker.}
\vspace*{-6pt}
\end{figure}


In fact, CQ and PBT2 are bi- or tridentate ligands. Consequently, their
corresponding Cu(II) complexes have a 1:2 metal/ligand
stoichiometry~\cite{38,55}. Moreover, addition of stoichiometric
amounts of CQ or PBT2 to Cu(II)--A${\upbeta}$ in vitro does not
efficiently remove Cu from Cu(II)--A${\upbeta}$, but generates stable
ternary CQ-- or PBT2-- Cu(II)--A${\upbeta}$
complexes~\cite{55,56}. Since \mbox{8-hydroxyquinolines}
are unable to extract copper(II) from soluble amyloids, these ligands
should not be considered as regulators of the copper homeostasis in AD
brains. Moreover, their flexible coordination properties allow them to
accommodate either Cu(II) or Cu(I), reducing their capacity to inhibit
the oxidative stress induced by Cu--A${\upbeta}$~\cite{57}.

Additionnally, copper is a required cofactor for several essential
metalloenzymes, for example, for the tyrosinase that produces L-DOPA
from tyrosine and catecholamine neurotransmitters~\cite{14}.
Circadian rhythm dysfunction occurs in AD, and copper was recently
reported to modulate rest--activity cycles via norepinephrine
produced by copper enzyme dopamine ${\upbeta}$-hydroxylase~\cite{15}.
These data indicate that the chance to get an efficient copper ligand
from the shelves to treat copper-related disease are limited, due to
the necessity of understanding the whole picture of the various
parameters in copper coordination chemistry. Specific copper chelators
should result from a rational design in order to create drugs as
specific as possible. These ligands should not disrupt the regular
activity of copper enzymes or proteins and must have suitable
pharmacological and safety profiles. Moreover, they should be able to
cross the blood--brain barrier after an oral administration, to
avoid any painful mode of administration. The following sections are
reporting our successive works to reach this goal.


\subsection{Bis(8-hydroxyquinolines)}
In line with our expertise on Clip-Phen as copper chelators endowed
with nuclease and cytostatic 
\mbox{activities} (see above), we prepared a
series of chelators using two 8-hydroxyquinoline motifs covalently
linked at their C2 position. Contrary to mono(8-hydroxy)quinolines,
bis(8-hydroxy)quinolines (Figure~\ref{fig3}) offer tetradentate 
N$_2$O$_2$ binding
sites to form copper complexes with a ligand/metal ratio ${=}$ 1:1. The
affinity of bis(8-hydroxyquinolines) for Cu(II) is higher than that of
mono(8-hydroxy)quinolines by 4 to 6 orders of magnitude, with $\log
K_{\mathrm{app}}$ of 15.5--16.6 at physiological pH~\cite{60,61}.
However, their selectivity for copper with respect to zinc is only 100
to 1000 [$\log K_{\mathrm{app}}$ for Zn(II) ${=}$ 12.5--14.2]. These
ligands are efficient at solubilizing A${\upbeta}$ peptides and are
able to inhibit $\mathrm{H}_{2}\mathrm{O}_{2}$ production by the
Cu--A${\upbeta}$/ascorbic acid system. These results were superior to
those obtained with the corresponding 8-hydroxyquinoline monomers, thus
validating the strategy of tetradentate chelators. However, their
affinity for Zn(II) is still too high to consider these ligands as
biologically pertinent copper regulators~\cite{62}. 


\subsection{Bis(8-aminoquinolines)}
To proceed to the transfer of copper from A${\upbeta}$ to glutathione
(an efficient copper carrier) and, therefore, to restore the copper
homeostasis in AD brains, we designed a series of tetradentate ligands L
based on a bis(8-aminoquinoline) scaffold, which are specific for
copper coordination (Figure~\ref{fig4}, PA1637, 1, 2)~\cite{11}. These
ligands have a high affinity for Cu(II) ions,   with log
$K_{\mathrm{app}}$ values ranging from 14 to 16 at pH 7.4, and a low
affinity for Zn(II), resulting in very high selectivity for Cu(II) with
respect to Zn(II), with a  log
[$K_{\mathrm{app}}$(Cu--L)/$K_{\mathrm{app}}$(Zn--L)] value over 12
\cite{63}. In vitro, at micromolar concentrations, they efficiently
extract Cu(II) from Cu(II)--A${\upbeta}$, to provide the Cu(II)--L
complex (Scheme~\ref{sch2})~\cite{55}. The presence of an amino
function at the C8-position of the quinoline skeleton is required for
the specific chelation of copper.


\begin{scheme}
\includegraphics{sc02}
\vspace*{6pt}
\caption{\label{sch2}Transfer of copper from Cu--A${\upbeta}$ to
Cu-proteins, assisted by a specific Cu(II) chelator. L stands for
TDMQ20; GSH stands for glutathione. {Reprinted with permission
from Ref.~\cite{41}. Copyright 2019 American Chemical Society}.}
\vspace*{-1.2pc}
\end{scheme}


Importantly, this series of chelators offers a well-defined square
planar coordination suitable for Cu(II) but not for Cu(I). So, in the
presence of glutathione (GSH), a reducing agent and a competitive
ligand for copper, the Cu(II)--L complex readily releases
its copper ion to glutathione to generate a Cu--GSH complex
that will be able to transfer copper to regular metal carriers 
(Scheme~\ref{sch2})~\cite{64}.

In such conditions, the bis(8-AQ) ligand is released and should
be able to act as a catalyst (Scheme~\ref{sch2}). Bis(8-AQ) ligands may
therefore be considered as suitable copper regulators of the copper
homeostasis. Conversely, it was also shown that the copper complex of
the mono(8-hydroxyquinoline) derivative PBT2 was unable to release
copper in the presence of GSH. 

On the basis of these in vitro studies, the pharmacological activity of
PA1637 (Figure~\ref{fig4}) was evaluated on a murine model of AD. The
preclinical evaluation of drug candidates against AD has usually been
carried out on transgenic mice~\cite{48,65,66}. However, AD is not a
condition determined by a single gene mutation but a multi-parameter
pathology in which a large number of age-related genes are involved.
The fact that these transgenic mouse models do not accurately reflect
human pathogenesis is probably one of the reasons for the failures
during preclinical selection of drug candidates~\cite{67,68}. To
enlarge the panel of predictable mice models, we developed a
non-transgenic mouse model of AD. In regular mice (much cheaper than
transgenic ones), a memory impairment similar to the early stage of AD
is triggered by a single injection of 
$\mathrm{A}\upbeta_{1\ndash 42}$ oligomers
in the mouse cerebral lateral ventricles (intracerebroventricular [icv]
model)~\cite{69}. The animal model is validated by the fact that
injection of the control antisense peptide 
$\mathrm{A}\upbeta_{42\ndash 1}$ has
no effect on mouse memory and behavior. The ability of PA1637 to
inhibit the episodic memory loss was evaluated on this non-transgenic
murine model. After a short oral treatment with PA1637 (8 doses of
PA1637, 25~mg/kg each, over a 3-week period), the episodic memory of AD
mice was completely restored, similar to that of healthy animals, while
that of untreated AD mice was significantly impaired~\cite{69}. These
results clearly validated the oral administration of PA1637 to fully
inhibit the cognitive impairment induced by icv injection of amyloid
oligomers, a way to mimic the early stage of AD. At the moment, there
is no treatment able to regenerate functional neurons in the late
stages of AD. An efficient drug able to stop the development of AD in
the early stages will be welcome.


\subsection{Tetradentate mono-8-aminoquinolines\newline 
(TDMQ) against Alzheimer's disease}
In spite of the capacity of PA1637 to extract copper from
copper--amyloid in vitro and to inhibit the catalytic production of
$\mathrm{H}_{2}\mathrm{O}_{2}$ by Cu(II)--amyloid, its development was
stopped due to its rather low bioavailability. To enhance the
efficiency and druggability of such copper chelators, a new series of
tetradentate amine ligands (named TDMQs) was designed. TDMQs were based
on a mono(8-amino)quinoline motif substituted at the C2 position by a
nitrogen-containing side chain, able to offer a N4-tetradentate square
planar coordination site for Cu(II) complexes, similar to that of
bis(8-AQ) (Figure~\ref{fig4})~\cite{70,71}. Structural modulation of
the polyamine chain allowed tuning the geometry of the coordination
site and, consequently, the copper selectivity and the ability to
inhibit oxidative stress.

In this TDMQ series, the ligands having a side chain with $n = m = 2$
($2 + 2$ methylene groups), exhibited the highest affinity values for
Cu(II), with log $K_{\mathrm{app}}$~[Cu--L] values in the range 15--17,
and also the highest selectivity for Cu(II) with respect to Zn(II)
(ratio ${>}$ 11 log units)~\cite{71}. These chelators were
also able to transfer copper from Cu--A${\upbeta}$ to glutathione, and
to inhibit the aerobic production of ROS induced by Cu--A${\upbeta}$ in
the presence of a reductant~\cite{57}. These features are clearly
related to the capacity of these N4-tetradentate ligands to offer a
tetradentate square planar coordination sphere around the copper(II)
ion, generating copper complexes with four nitrogen ligands in a N4
equatorial plane coordination sphere~\cite{72}. Noteworthily, bis(8-AQ)
derivatives (structures depicted in Figure~\ref{fig4}) offered the same
coordination, resulting in similar correlations between the structures
of the Cu(II)--L complexes and their physicochemical properties such as
high affinity for Cu(II) and ability to inhibit ROS
production~\cite{63}.

Based on these results, TDMQ20 (Figure~\ref{fig4}) was selected for
evaluation of its anti-AD activity on three different mouse models. An
oral treatment with TDMQ20 was used to check the possibility of
reversing the cognitive and behavior impairments in two non-transgenic
models mimicking the early stage of AD and in a transgenic one modeling
a more advanced stage of AD. In the non-transgenic mice, memory
deficits were triggered by a single injection of the copper complex of
amyloid $\mathrm{A}\upbeta_{1\ndash 42}$ in the lateral ventricles
(icv-CuA${\upbeta}$ model) or hippocampus (hippo-CuA${\upbeta}$ model)
of regular mice. The third model was the classical transgenic mouse
5XFAD model~\cite{73,74}.

A short oral treatment with TDMQ20 of icv-CuA${\upbeta}$ or
hippo-CuA${\upbeta}$ mice (10~mg/kg, 8 doses over 16 days) fully
restored the cognitive status evaluated by the short-term novel object
recognition (NOR) assay~\cite{75}, in comparison to untreated AD
mice (Figure~\ref{fig5})~\cite{76}.


\begin{figure*}
\includegraphics{fig05}
\caption{\label{fig5}Declarative memory evaluated by the short-term
novel object recognition task (NOR). The recognition index for short
term memory in the icv-CuA${\upbeta}$ and the hippo-CuA${\upbeta}$
non-transgenic mouse models are reported in panels A and B,
respectively. In each case, the recognition index of AD mice which
received no drug-treatment (untreated group) was compared to that of
healthy mice (control group), and to the recognition index of AD mice
treated with TDMQ20 or clioquinol (CQ) ($8 \times 10$~mg/kg in 16
days). Each mark represents the result obtained with a single mouse;
horizontal lines represent mean values ${\pm}$ SEM. 
***$p < 0.001$, **$p < 0.01$, 
*$p < 0.05$ vs.\ the untreated AD group (ANOVA). {Adapted with
permission from Figure~2 of Ref.~\cite{76}. Copyright 2020 American
Chemical Society}.}
\end{figure*}

By comparison, the 8-hydroxyquinoline derivative clioquinol (CQ) did
not significantly improved the short-term memory of icv-CuA${\upbeta}$-
and hippo-CuA${\upbeta}$ mice. The level of malondialdehyde, the
signature of an oxidative stress, in the cortex of icv-CuA${\upbeta}$
was also reduced by TDMQ20. The three-month oral treatment of
transgenic 5XFAD mice with TDMQ20 also resulted in behavioral
improvements. Pharmacokinetic studies in rats indicated that TDMQ20 has
a good bioavailability and efficiently crosses the
blood--brain barrier after oral administration~\cite{77}.
Noteworthily, TDMQ20 did not exhibit significant acute or chronic
toxicity~\cite{76}.


In addition, a three-month oral treatment of 5XFAD mice with
TDMQ20 remarkably reduced the plaque loading in the cortex by about
70\%, suggesting a beneficial role of TDMQ20 in clearing pathological
A${\upbeta}$ deposits in AD mice. The treatment also significantly
increased the expression of the ChAT enzyme and the CHRM4 receptor, two
proteins involved in the cholinergic system~\cite{78}. All these
results strongly suggest that TDMQ20 is acting on several pathways of
this multifactorial disease.

Due to their reliability and easy use, the icv-CuA${\upbeta}$
and hippo-CuA${\upbeta}$ mice should be considered as robust
non-transgenic models to evaluate the activity of potential drugs in
the early stages of memory deficits. Moreover, among other possible
assays~\cite{79,80}, the short-term NOR test was found particularly
robust to evaluate the impairment of declarative memory in mice, making
it easy to select molecules for the treatment of the early stages of
AD. 

One should keep in mind that the few current AD therapies are
not curative and provide at best a short-term improvement in symptoms,
with potentially serious side effects. Moreover, their efficiency/cost
ratios are questionable. Despite intensive efforts on AD over the last
two decades in genetics, biochemistry, and cell biology, the pipeline
of new drugs is rather unproductive. As a reference point, lecanemab,
approved in the United States for the treatment of AD, is an
anti-amyloid monoclonal antibody administered intravenously every other
week with a post-infusion observation period with monitoring of the
effect by magnetic resonance imaging. In addition to these rather
strong constraints, treatment with lecanemab results in frequent and
potentially severe anticoagulation side effects, and this treatment is
not recommended for patients with a wide range of comorbidities, a very
frequent condition in aging patients~\cite{81}. In addition, the
European health agency (EMA) initially refused this treatment
(July 2024)~\cite{82}. Donanemab, another antibody targeting amyloids
recently approved by the FDA (June 2024), also induces fatal brain
bleeding in a few patients~\cite{83}. These facts strongly suggest
that efforts should be made to support the discovery of new ``small
molecules'', namely chemical agents, designed for an easy crossing of
the blood--brain barrier in order to produce an efficient
pharmacological effect on the early stages of AD, without strong
constraints or potentially severe adverse effects.

So the efficacy of TDMQ20 by oral administration at low doses
on three different AD mouse models with a very low toxicity, is an
encouragement for a future development of this specific copper
chelator, maybe in parallel with research of other molecules targeting
different mechanisms. However, considering that the introduction of a
new drug candidate in the pipeline of AD treatments is rather
challenging, we decided to investigate the correlation between the
copper chelating activity of TDMQ20 and its therapeutic interest in a
well-known copper-related disease, namely the Wilson's disease.


\section{Tetradentate mono-8-aminoquinoline\newline TDMQ20 and Wilson's
disease} \label{sec5}

Wilson's disease (WD) is a genetic disease caused
by mutations on the \textit{Atp7b} gene coding for the
copper carrier ATP7B, an ATPase in charge of incorporating copper in
apoceruloplasmin in the liver, leading to the elimination of this metal
ion in bile and then in feces. Deficiency in this copper carrier causes
accumulation of copper in the liver, generating acute or chronic
hepatitis and liver cirrhosis, potentially leading to a fulminant
hepatic failure. Copper is partially released in the bloodstream and
slowly eliminated in urine. In advanced stages of the disease,
neurological symptoms such as seizures or Parkinsonism appear, along
with psychiatric disorders. Accumulation of copper in various organs,
resulting especially in renal and cardiac pathologies as well as
hypoparathyroidism and osteoarticular damages, are correlated with a
fatal prognosis~\cite{17,18}. Lifelong treatment is therefore
necessary, using copper chelators to facilitate the excretion of
copper. The first-line drugs for WD treatment via oral administration
are currently D-penicillamine (DPA) and, to a lesser extent, trientine,
a non-selective polyamine chelator (Figure~\ref{fig6})~\cite{84}.


\begin{figure}
\includegraphics{fig06}
\caption{\label{fig6}Structures of copper chelators currently used in
the treatment of Wilson's disease (DPA and trientine), or in
development (TTM). $^{\mathrm{a}}$ Daily oral dosage for an adult~\cite{86}.}
\end{figure}


These two drugs are given at very high doses (1--2~g per day for an
adult) and exhibit adverse effects that may be serious enough to
require discontinuation of the treatment (in approximately 30\% of
patients for DPA)~\cite{85,86}. The efficacy of DPA in neurologic WD is
only moderate (55\% of improvement rate), and both DPA and trientine
induce a severe and irreversible neurological worsening in 10--50\% of
patients with previous neurological symptoms~\cite{87}. So there is a
real medical need for a specific copper chelator able to efficiently
regulate the copper excess, at lower doses than those used for DPA or
trientine and with lower side effects in the long term. Ammonium
tetrathiomolybdate has been proposed for more than 30 years~\cite{88},
recently replaced by bis-choline tetrathiomolybdate (TTM,
Figure~\ref{fig6})~\cite{89}. The $\mathrm{MoS}_{4}^{2-}$ ion chelates
copper to form very stable and highly insoluble \mbox{sulfur-bridged} Mo--Cu
\mbox{clusters~\cite{90,91}} \mbox{detected} in the liver of WD rat models~\cite{90},
resulting in accumulation of molybdenum in major organs (spleen, liver,
adrenal glands, kidney, and brain) following administration of
TTM~\cite{92,93}. The high affinity of TTM for copper is also
responsible for inhibition of major copper enzymes such as
ceruloplasmin, ascorbate oxidase, cytochrome oxidase, Cu/Zn-superoxide
dismutase and tyrosinase~\cite{94}.\looseness=1


The main drawback of DPA and trientine as ligands is their lack of
selectivity in metal chelation. In fact, both drugs coordinate with
high affinities a wide variety of metal ions and oxidation
states~\cite{95} (see 
footnote\footnote{{\vspace*{-22pt}\begin{eqnarray*}
&\displaystyle
\mathrm{M} + \mathrm{L} 
\mathop{\rightleftharpoons}\limits_{K_1}
\mathrm{ML}
\mathop{\rightleftharpoons}\limits_{K_2}^{\mathrm{L}} 
\mathrm{ML}_2 \\\\
&\displaystyle
K_1=\frac{[\mathrm{ML}]}{[\mathrm{M}][\mathrm{L}]} \quad
K_2=\frac{[\mathrm{ML}_2]}{[\mathrm{ML}][\mathrm{L}]}\quad
\beta_{1,2}=\frac{[\mathrm{ML}_2]}{[\mathrm{M}][\mathrm{L}]^2}
=K_1 K_2 
\\&\displaystyle \qquad\qquad\qquad\qquad\qquad
\qquad
(\mbox{usually noted } \beta_{2})
\end{eqnarray*}}\unskip}). 
For 
example, DPA coordinates $\mathrm{Cu}^{2+}$ with $\log K_{1} =
16.5$, but also $\mathrm{Cu}^{+}$\ $(\log K_{1} = 19.5)$,
$\mathrm{Zn}^{2+}$ ($\log K_{1} = 9.6$ and $\log \upbeta_{2} =
19.6$)~\cite{95}, as well as $\mathrm{Fe}^{2+}$, $\mathrm{Fe}^{3+}$,
$\mathrm{Co}^{2+}$, and $\mathrm{Co}^{3+}$~\cite{96}. In addition, the
structures of DPA--metal complexes can be diverse, including ternary
complexes involving another amino acid such as histidine or
methionine~\cite{97,98}, or mixed valence Cu(I)/Cu(II) cluster
complexes~\cite{99}. Moreover, the ability of DPA to act as a reductant
due to its thiol functionality, in addition to its capacity to
coordinate both Cu(II) and Cu(I) [or Fe(III) and Fe(II)], may confer to
DPA complexes the ability to trigger deleterious Fenton-like reaction
damages.

Due to the specificity of TDMQ20 for chelation of Cu(II) and the oral
bioavailability of this ligand , we decided to evaluate its activity
for the treatment of WD. In TX mice, a genetic model of WD~\cite{100},
the overload of copper in the liver was reduced in a dose-dependent
manner by a short oral treatment with TDMQ20 at doses ranging from 12.5
to 50~mg/kg/day (Figure~\ref{fig7}a). This decrease in hepatic copper
was correlated to an increase in fecal copper excretion
(Figure~\ref{fig7}b)~\cite{101}. These studies indicate that such low
doses of TDMQ20 are more efficient at improving the physiological
excretion pathway of copper in TX mice than DPA at 200~mg/kg/day.
TDMQ20 also increases the serum concentration of ceruloplasmin
(Figure~\ref{fig7}c). Such an effect is particularly important since
this concentration is a biomarker clinically used to evaluate the
efficency of chelation therapy in patients with WD.


\begin{figure*}
\vspace*{2pt}
\includegraphics{fig07}
\vspace*{2pt}
\caption{\label{fig7}Concentration of copper in liver (a) and feces (b)
in mg/kg, and of ceruloplasmin in serum (c) of TX mice (WD) after oral
treatment with TDMQ20 at 12.5~mg/kg/d (TDMQ20-L), 25~mg/kg/d
(TDMQ20-M), or 50~mg/kg/d (TDMQ20-H). WD mice orally treated by DPA at
200~mg/kg/d are given for comparison. Control mice are healthy C57BL/6
mice bearing no mutation on ATP7B. *$p < 0.05$,  **$p < 0.01$, ***$p <
0.001$ and  ****$p < 0.0001$. {Adapted from Ref.~\cite{101}}.}
\vspace*{2pt}
\end{figure*}


In addition, TDMQ20 up to $300~\upmu\mathrm{M}$ does not disturb the
activity of Cu/Zn-SOD, in contrast with DPA, which inhibits Cu/Zn-SOD
at a $50~\upmu\mathrm{M}$ concentration. In addition, the DPA--copper
complex produces damaging ROS in vitro in the presence of a reducing
agent, which is not the case with TDMQ20. In fact, DPA inhibits
catalase in vitro and could be responsible for chronic inflammation
through chronic disruption of $\mathrm{H}_{2}\mathrm{O}_{2}$ redox
homeostasis in vivo~\cite{102}. In conclusion, TDMQ20 should be
considered as a first-in-class drug candidate able to challenge DPA in
the treatment of WD.


\section{TDMQ20 copper chelator as anticancer agent}\label{sec6}

The possibility of using metal ligands in anticancer chemotherapy to
regulate metal homeostasis has not been extensively explored, although
it is known that the metal ion content is significantly higher in
cancer cells compared to normal ones~\cite{103}. In particular, copper
ions are involved in angiogenesis~\cite{104,105}, which is essential
for tumor growth and dissemination of cancer metastases~\cite{106,107}.
It has been evidenced that copper depletion inhibits angiogenesis in
cancer cells~\cite{108}. A few clinical trials have been reported with
copper chelators such as DPA, trientine, or TTM, three ligands that
have been documented for WD (see above, Figure~\ref{fig6}). However,
these attempts have not been successful, probably due to their lack of
metal selectivity and/or potential toxicity~\cite{109}. So having
TDMQ20 at hand as specific copper chelator, preliminary evaluation of
its activity against different cancer cell lines was carried out.
TDMQ20 was found cytotoxic against non-small cell lung carcinoma
(A549), cervix cancer HeLa and hepatocarcinoma HepG2 cells, with
$\mathrm{IC}_{50}$ values ranging from 14 to $16~\upmu\mathrm{M}$ in
vitro, lower than the reference drug 5-Fluorouracil (5-FU), and the
selectivity index of TDMQ20 was higher than that of 5-FU when using
non-cancer human cells as comparators. TDMQ20 also exhibited a
significant anti-migration activity on HeLa cells in vitro. Mechanistic
studies indicated that the activity of TDMQ20 probably involves the
overproduction of ROS, with the collapse of the \mbox{inner} transmembrane
potential in mitochondria and the induction of apoptosis~\cite{110}.


\section{Conclusion}

Since copper plays essential functions in human body, this metal ion
can be considered as a promising therapeutic target in several
diseases. A twenty-five-year review of the state of the art in rational
chelator design has led us from phenanthroline derivatives to the
N4-tetradentate mono-8-aminoquinoline series, especially the TDMQ20 as
a suitable copper(II) chelator for therapeutic use. Due to its
optimized coordination properties, this specific Cu(II) ligand exhibits
a promising activity on murine models of Alzheimer's and Wilson's
diseases, two diseases involving copper dyshomeostasis and copper
accumulation, respectively. Clinical development of TDMQ20 as a
therapeutic alternative to DPA for normalizing copper levels in WD
patients is currently under discussion. In addition, this chelator is
more active than the reference drug 5-FU on several human cancer cell
lines. Future clinical developments will be necessary to evidence which
therapeutic domain will be best tackled by TDMQ20. 

As a concluding remark, the specificity for copper chelation and the
structure and properties of the corresponding copper complex are
prerequesite parameters in the design of pharmacological ligands
suitable for targeting diseases triggered by the pathological
disruption of copper homeostasis. 

\vspace*{2pt}

\section*{Data availability}

\vspace*{2pt}

All data presented in this review article have been reported in the
cited articles of our group and in the associated supporting
information. 

\vspace*{1pt}

\section*{Acknowledgments}

\vspace*{1pt}

Authors gratefully acknowledge all colleagues, postdoctoral fellows,
PhD and master students, and collaborators who have been involved in
the research of specific metal regulators as potential drugs. 

\vspace*{1pt}

\section*{Declaration of interests}

\vspace*{1pt}

The authors do not work for, advise, own shares in, or receive funds
from any organization that could benefit from this article, and have
declared no affiliations other than their research organizations.

\vspace*{1pt}

\section*{Funding}

\vspace*{1pt}

Financial support was received from the CNRS, Inserm, and GDUT.

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