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\DOI{10.5802/crchim.284}
\datereceived{2023-11-28}
\daterevised{2024-01-15}
\dateaccepted{2024-01-15}
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\dateposted{2024-02-28}
\begin{document}

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%\makeatletter
%\def\TITREspecial{\relax}
%\def\cdr@specialtitle@english{French Women in Chemistry in 2024}
%%\def\cdr@specialtitle@french{Les chimistes fran\c{c}aises en 2024}
%\makeatother

\title{Stable and inert manganese complexes for magnetic resonance
imaging}

\alttitle{Complexes de mangan\`{e}se stables et inertes pour l'imagerie
par r\'{e}sonance magn\'{e}tique}

\author{\firstname{Daouda} \lastname{Ndiaye}}
\address{Centre de Biophysique Mol\'{e}culaire, CNRS UPR4301,
Universit\'{e} d'Orl\'{e}ans, Rue~Charles Sadron, 45071 Orl\'{e}ans,
France}
\curraddr[D. Ndiaye]{Laboratoire Chimie et Biologie des M\'{e}taux, CEA-Grenoble, France}

\author{\firstname{\'{E}va} \lastname{T\'{o}th}\CDRorcid{0000-0002-3200-6752}\IsCorresp}
\addressSameAs{1}{Centre de Biophysique Mol\'{e}culaire, CNRS UPR4301,
Universit\'{e} d'Orl\'{e}ans, Rue~Charles Sadron, 45071 Orl\'{e}ans,
France}
\email[\'{E}. T\'{o}th]{eva.jakabtoth@cnrs-orleans.fr}

\keywords{\kwd{Manganese}
\kwd{MRI contrast agent}
\kwd{Relaxation agent}
\kwd{Bispidine}}

\altkeywords{\kwd{Mangan\`{e}se}
\kwd{Agent de contraste IRM}
\kwd{Agent de relaxation}
\kwd{Bispidine}}

\begin{abstract}
Paramagnetic Mn$^{2+}$ complexes are intensively
investigated as alternatives to replace the currently used, Gd-based
clinical contrast agents in Magnetic Resonance Imaging (MRI). Manganese
is an essential metal which alleviates its potential toxicity and the
environmental concerns related to the use of Gd$^{3+}$.
Thanks to its five unpaired electrons and slow electron spin
relaxation, Mn$^{2+}$ is a very efficient relaxation
agent. Given the high doses required for \textit{in vivo} MRI,
Mn$^{2+}$ needs to be chelated in thermodynamically stable
and kinetically inert complexes, despite its natural presence in the
body. We survey here the latest developments in the chemistry and the
preliminary \textit{in vivo} MRI evaluation of Mn$^{2+}$
complexes. We specifically focus on the molecular ligand design,
including linear, macrocyclic and bicyclic (bispidine) chelators that
allowed for substantially enhanced kinetic inertness of the complexes,
as well as for ligand selectivity for Mn$^{2+}$ versus the
main biological competitor Zn$^{2+}$. In addition to the
$+2$ form of manganese, Mn$^{3+}$ is also paramagnetic in
the high-spin state, with promising relaxation properties among
porphyrin complexes. Finally, examples will be presented to demonstrate
the first steps towards the development of redox sensors based on the
Mn$^{2+}$/Mn$^{3+}$ switch, detectable in
$^{1}$H or in $^{19}$F MRI. \vspace*{-1.5pt}
\end{abstract}

\begin{altabstract}
Les complexes paramagn\'{e}tiques de Mn$^{2+}$ font
l'objet de recherches intensives dans le but de remplacer les agents de
contraste \`{a} base de Gd$^{3+}$ actuellement
utilis\'{e}s dans l'imagerie m\'{e}dicale par r\'{e}sonance
magn\'{e}tique (IRM). Le mangan\`{e}se est un m\'{e}tal essentiel, ce
qui att\'{e}nue sa toxicit\'{e} potentielle et les pr\'{e}occupations
environnementales li\'{e}es \`{a} l'utilisation du
Gd$^{3+}$. Gr\^{a}ce \`{a} ses cinq \'{e}lectrons
non-appari\'{e}s et \`{a} sa relaxation \'{e}lectronique lente, le
Mn$^{2+}$ est un agent de relaxation efficace. \'{E}tant
donn\'{e} les doses \'{e}lev\'{e}es requises pour l'IRM \textit{in
vivo}, le Mn$^{2+}$ doit \^{e}tre ch\'{e}lat\'{e} dans des
complexes thermodynamiquement stables et cin\'{e}tiquement inertes,
malgr\'{e} sa pr\'{e}sence naturelle dans l'organisme. Nous
pr\'{e}senterons ici les d\'{e}veloppements chimiques r\'{e}cents et
l'\'{e}valuation IRM pr\'{e}clinique des complexes de
Mn$^{2+}$. Nous nous focaliseront particuli\`{e}rement sur
la conception mol\'{e}culaire des ligands dans les familles des
ch\'{e}lateurs lin\'{e}aires, macrocycliques ou encore bicycliques
(bispidines), qui ont permis une am\'{e}lioration spectaculaire de
l'inertie cin\'{e}tique des complexes, ainsi que la s\'{e}lectivit\'{e}
des ligands pour Mn$^{2+}$ par rapport au
Zn$^{2+}$, son principal comp\'{e}titeur biologique. En
plus de la forme +2 du mangan\`{e}se, Mn$^{3+}$ est
\'{e}galement paramagn\'{e}tique \`{a} l'\'{e}tat haut-spin, et
poss\`{e}de des propri\'{e}t\'{e}s de relaxation tr\`{e}s prometteuses
dans les complexes form\'{e}s avec des porphyrines. Enfin, nous
pr\'{e}senterons quelques exemples pour illustrer les premiers pas
effectu\'{e}s dans le d\'{e}veloppement de senseurs redox, bas\'{e}s
sur le couple Mn$^{2+}$/Mn$^{3+}$, avec une
d\'{e}tection potentielle en IRM du $^{1}$H ou du
$^{19}$F.
\end{altabstract}

\maketitle

\vspace*{3pt}

\twocolumngrid

\end{noXML}

\section{Introduction}

Starting with the discovery of X-ray at the end of the 19{th} century
and its immediate application to visualize the inside of the
body~\cite{1}, imaging technologies have revolutionized the way medical
doctors work. The imaging field witnessed a spectacular progress from
the 1970s, with the introduction of computerized image construction
procedures as well as novel technologies based on various physical
phenomena, including ultrasound, magnetic \mbox{resonance} or radioactive
emission. Imaging has also become a major investigation tool in
biological research to understand function and distribution of
biomolecules \textit{in vivo} or \textit{in cellulo}. Finally, the
pharmaceutical industry is also increasingly integrating imaging into
the drug development process, to follow drug biodistribution, metabolic
fate or therapeutic\break effect. 

Introduced in the early 1980s, Magnetic Resonance Imaging (MRI) has
become a widespread and powerful tool for establishing clinical
diagnosis and monitoring therapy. The use of paramagnetic metal
complexes as contrast-enhancing agents has greatly contributed to this
success~\cite{2}. MRI is based on the nuclear relaxation properties of
mostly water \mbox{protons.} Paramagnetic (or superparamagnetic) materials can
substantially reduce the relaxation rates of surrounding nuclei,
thereby improving image contrast between different tissues~\cite{3}. 

Today, clinically approved MRI contrast agents are complexes of the
rare earth metal ion gadolinium (Gd$^{3+}$). With its seven unpaired
electrons ($S = 7/2$) and exceptionally slow electron spin
relaxation due to a symmetric electron configuration, Gd$^{3+}$ is the
most paramagnetic metal ion in the periodic table and a very efficient
relaxation agent. Gd$^{3+}$ complexes were considered for
a long time as safe drugs which are fully eliminated from the body
shortly after their injection. However, since 2006, several
Gd$^{3+}$-related toxicity cases were reported, mostly in
kidney-impaired patients presenting slow renal excretion~\cite{4}. In
this novel, occasionally fatal disease called nephrogenic systemic
fibrosis, Gd deposition was detected in the body, originating from the
dissociation of the injected agent releasing free, non-complexed
Gd$^{3+}$. This could be directly related to the low kinetic inertness,
thus low resistance of the chelates to dissociation. The drug safety
agencies in the USA and Europe rapidly responded by the withdrawal of
several, less inert chelates and by restricting the use of any
Gd$^{3+}$-based agent in kidney-impaired patients. Long-term
accumulation of very small, but detectable amounts of Gd$^{3+}$ has
been also observed in various organs of patients, without toxic
effects~\cite{5}. Lately, the presence of Gd in river and coastal
waters due to contrast agent applications is arising as an important
environmental pollution issue~\cite{6,7}. Altogether, these problems
have promoted intensive research in the community of coordination
chemists to identify safer alternatives to Gd$^{3+}$\break complexes. 

Among potential metal ions, Mn$^{2+}$ is the most obvious candidate to
replace Gd$^{3+}$ in MRI contrast agent applications~\cite{8,9}. In its
high-spin state, Mn$^{2+}$ has five unpaired electrons ($S =
5/2$) and slow electron spin relaxation, which are the most critical
requirements for a significant relaxation effect. The first relaxation
agent used in MRI by Lauterbur was MnCl$_{2}$~\cite{10}, and
Mn$^{2+}$ continues to be applied in specific animal experiments to
track excitable cells like neurons and cardiac cells where it enters as
a Ca$^{2+}$ surrogate (Manganese-Enhanced MRI $=$ MEMRI)~\cite{11}. 

Manganese is an essential metal, having multiple biological roles.
Despite the natural presence of manganese in the body, injecting free
Mn$^{2+}$ at the typically high MRI contrast agent doses
(0.05--0.30 mmol per kg body weight) could be problematic,
potentially causing neurotoxicity and Parkinson-like
symptoms~\cite{12}. Therefore, Mn$^{2+}$ needs to be chelated in stable
and inert complexes for \textit{in vivo} applications. 


{In this context, a great number of novel ligands have been proposed
for Mn}$^{2+}$ {chelation during the last decade}~{\cite{8, 13}. Stable
and inert complexation of Mn}$^{2+}$ {is, however, a coordination
chemistry challenge. Indeed, in the high spin d}$^{5}$ {electron
configuration, Mn}$^{2+}$ {has no ligand-field stabilization energy,
thus its complexes are less stable than those} {formed} {with} {other
divalent transition metal analogues, including} {the most relevant
biological competitor Zn}$^{2+}$. The thermodynamic stability of the
metal chelates is expressed by the stability constant,
$\log K_{\mathrm{ML}}$ ($K_{\mathrm{ML}} = [\mathrm{ML}]/
[\mathrm{M}][\mathrm{L}]$, where the
values in brackets correspond to equilibrium concentrations of the
complex ML, the metal M and the fully deprotonated ligand L,
respectively). Conditional stabilities are often compared for different
complexes in order to get rid of the difference in ligand protonation
constants. For potential Mn$^{2+}${-based contrast agents, conditional
stability is most commonly expressed with the 
$\mathrm{pMn} = -\log[\mathrm{Mn}]$
value, where {[}Mn{]} is the equilibrium concentration of free
Mn}$^{2+}${, calculated for 10}$^{-5}$ {M total concentrations of the
metal and the ligand each, at pH 7.4~\cite{14}. In addition to the
thermodynamic stability, the kinetic inertness, i.e. resistance of the
chelate to dissociation, which would lead to the release of free metal
ion in biological media, is another important requirement for}
{\textit{in vivo}} {use. Dissociation of metal complexes typically
occurs by acid- and metal-mediated processes. Concerning the
metal-catalysis, endogenous metal ions, such as Cu}$^{2+}$ {or
Zn}$^{2+}$ {are the most important to promote transmetallation, thus
Mn}$^{2+}$ {release}~{\cite{15}. Kinetic inertness is often regarded as
even more relevant than high thermodynamic stability, because once the
complex is injected into the body, it is not in thermodynamic
equilibrium; yet it should stay intact all along its way through the
body until full excretion. Mn}$^{2+}$ {complexes are typically labile,
and until very recently, it seemed extremely challenging to attain
substantial increase in their kinetic inertness.} \looseness=-1

{For good MRI efficiency, Mn}$^{2+}$ {complexes need to contain at
least one water molecule in their inner coordination sphere which makes
high thermodynamic stability and kinetic inertness even more difficult
to achieve. The MRI efficiency of a contrast agent is given by its
relaxivity} ($r_{1}$), {which is defined as the paramagnetic
enhancement of the longitudinal water proton relaxation rate induced by
a unity (millimolar) concentration of the probe (the metal
ion)}~\cite{3}. The relaxation effect of the paramagnetic metal
complex on the surrounding water protons is governed by two major
contributions, called outer and inner sphere relaxivity, both linearly
dependent on $S(S + 1)$. This implies that relaxivities will be lower
for Mn$^{2+}$ ($S = 5/2$) than for Gd$^{3+}$ ($S = 7/2$) complexes
provided the other determining parameters are similar~\cite{16}, thus
higher doses might be required for Mn$^{2+}${-based MRI agents to
achieve a similar contrast enhancement effect.}

{Outer sphere relaxivity arises from dipole--dipole interactions between
the electron spin of the metal and the nuclear spin of the water
protons diffusing around the complex. The inner-sphere term, which is
more interesting since it can be optimized by rational molecular
design, is related to water (or proton) exchange between the inner
sphere of the metal ion and bulk water. As dipolar interactions are
strongly dependent on the distance between the electron and nuclear
spins, inner sphere protons experience a much stronger paramagnetic
relaxation effect, and this is then transferred to the surrounding
water by chemical exchange of the inner sphere water. Inner sphere
relaxivity is thus dependent on different structural and dynamic
parameters of the complex, such as the number of water molecules
directly coordinated to the metal ion (hydration number,} $q$), {the
rate of exchange of these water molecules with bulk water}
($k_{\mathrm{ex}}$), {the rotational motion of the complex
characterized by the rotational correlation time} ($\tau_{R}$), {and
the relaxation times of the electron spin of the metal}
($T_{{1\mathrm{e}}}$ {and} $T_{{2\mathrm{e}}}$). {Inner sphere
relaxivity is linearly proportional to the hydration number, however,
given the relatively low stability of Mn}$^{2+}$ {complexes, it seems
difficult to increase relaxivity by designing bishydrated chelates.} 


{One Mn}$^{2+}$ {complex was previously approved for clinical contrast
agent use} (Teslascan$^{\text{\textregistered}}$), {but later
withdrawn. This non-hydrated MnDPDP chelate (DPDP $=$
N,N$'$-dipyridoxylethylenediamine-N,N$'$-diacetate-5,5$'$-bis(phosphate);
Scheme~\ref{sch1}) is not stable} {\textit{in vivo}}{, but releases free
Mn}$^{2+}$ {which is mostly responsible for its relaxation effect.
Although no toxicity problems were associated to the use of MnDPDP, it
is obvious that today no Mn}$^{2+}$ {complex with such limited
stability could be accepted for human applications.}

Beside the $2+$ oxidation state, manganese can also exist as a
Mn$^{3+}$ cation in aqueous solution, including under biological
conditions. High spin Mn$^{3+}$ is also paramagnetic. Although
Mn$^{3+}$ has lower electron spin ($S = 4/2$) and typically
faster electron spin relaxation than Mn$^{2+}$, several examples, in
particular in the family of Mn$^{3+}$ porphyrin complexes, evidence
that this higher oxidation state can also have interesting relaxation
properties with potential applications in MRI~\cite{17}. 

In addition, the transformation between the Mn$^{3+}$ and Mn$^{2+}$
forms mediated by biological redox active species can be exploited for
the design of redox responsive MRI probes. As the Mn$^{3+}$ and
Mn$^{2+}$ states are characterized by different relaxation properties,
a redox change will be detectable on the Magnetic Resonance images, and
this can report on the redox state of the biological
environment~\cite{18}.

{We should also mention that Mn has a positron-emitting radionuclide,}
$^{52}$Mn, {with interesting decay features for application in Positron
Emission Tomography (PET)}
($t_{1/2} = 5.6$ d, ${\upbeta}^{+}${-decay intensity: 29.6\%,
max.} ${\upbeta}^{+}${-energy: 575 keV).} {The relatively
long half-life makes} $^{52}${Mn suitable for the imaging of the slow
biodistribution of macromolecules (such as monoclonal antibodies), over
periods of days. For PET applications, highly stable and inert Mn
chelation is very important to prevent off-target signals originating
from released free} $^{52}${Mn.} 


{The increasing interest in} $^{52}$Mn {is also related to the
important advances in bimodal PET/MRI}. Mn$^{2+}$ {is the only example
where a} {\textit{unique}} {metal ion offers detection capability in
both MRI and PET, which obviously alleviates the chemical design of
bimodal PET/MRI agents} {\cite{19}. Instead of conjugating individual
PET and MRI probes, an appropriate} {isotopic}
$^{52/\mathrm{nat}}$Mn mixture provides the bimodal PET/MR tracer
with ease, ensures that PET and MR reporter complexes are present in a
concentration adapted for each imaging modality (PET is
${>}$10$^{6}$ {times more sensitive than MRI) and have
strictly identical} {\textit{in vivo}} {biodistribution.}

In overall, for any bioimaging application, Mn$^{2+}$ needs to be
chelated in complexes of high thermodynamic stability and kinetic
inertness. When MRI applications are concerned, such high stability and
inertness of the complex need to be maintained while preserving one
inner sphere water molecule to ensure relaxation efficiency. Another
feature to consider is the rich redox chemistry of Mn, which implies
that the ligand should stabilize the required redox state under
biological conditions. To meet all these requirements together, novel
ligands have to be\break designed. 

Here, we give an overview of the recent advances in the development of
Mn-based potential MR imaging agents. In parallel to the on-going
progress in the chemistry of imaging agents, the first steps have also
been made towards clinical translation, as two small molecular weight
Mn$^{2+}$ chelates passed to Phase~1 human trials as potential
extracellular, ``general-purpose'' MRI probes to replace the current
Gd$^{3+}$-based agents~\cite{20,21}. Our objective is not to be
exhaustive in this review. We will mainly focus on the chemical design
of the ligand in order to tackle the most challenging questions: how to
improve the kinetic inertness for the Mn$^{2+}$ complexes and how to
achieve selectivity for Mn$^{2+}$ over the main biological competitor
Zn$^{2+}$. While we will select examples from different ligand families
including also the design of smart imaging agents, derived from the
best chelators, we will survey more closely the latest results obtained
with bispidine-type complexes, which brought spectacular progress in
these respects. Finally, concerning Mn$^{3+}$ chelates, there is much
less data available in the literature, but some recent examples
indicate that novel opportunities might be opened in this field. 

\section{Linear chelators for Mn$^{2+}$ complexation}

Mn$^{2+}$ complexes of ethylenediamine-tetraacetate (EDTA) and its
derivatives have been known for more than 50 years. These chelates are
typically seven-coordinated with one inner sphere water molecule, which
made them also interesting early on for paramagnetic relaxation
studies~\cite{22}. In the context of MRI contrast agent development,
derivatization of MnEDTA with hydrophobic moieties such as an
ethoxybenzyl (EOB), a benzothiazole-aniline (BTA) or a benzyloxymethyl
(BOM) has been more recently reported to target the liver
(Mn(EOB-EDTA), Mn(BTA-EDTA))~\cite{23,24} or to induce non-covalent
binding to human serum albumin (Mn(EDTA-BOM))~\cite{25} for
angiographic imaging (Scheme~\ref{sch1}). These modifications influence the
biological fate of the complexes, but have limited impact on their
thermodynamic stability or kinetic inertness, and in particular, this
latter remains relatively low. The relaxivity is slightly increased for
Mn(EDTA-BOM) or Mn(EDTA-BOM$_{2}$) complexes (3.60 and 4.30
mM$^{-1}{\cdot}$s$^{-1}$, 20 MHz, 25~\textdegree C)~\cite{25} versus MnEDTA
(3.00 mM$^{-1}{\cdot}$s$^{-1}$)~\cite{25}, in accordance with the increased
molecular size. 

\begin{scheme*}[t!]
\includegraphics{sc01}
\vspace*{9pt}
\caption{\label{sch1}Mn$^{2+}$ complexes formed with
linear ligands.}
\end{scheme*}

The most important developments in this family concern the
rigidification of the ligand skeleton in the objective of improving
kinetic inertness. Several design elements have been tested in this
direction. First of all, the ethylene backbone was replaced with
considerably less flexible moieties such as a cyclohexane, a benzene or
a pyridine ring, and this led to improved resistance to complex
dissociation. The effect of rigidity was first demonstrated by
comparing the kinetic inertness of MnEDTA and Mn(\textit{t-}CDTA)
($t$-CDTA~$=$~\textit{trans}-1,2-diaminocyclohexane-N,N,N$'$,N$'$-tetraacetate;
Scheme~\ref{sch1}) in Cu$^{2+}$ exchange reactions~\cite{26}. These
evidenced a two orders of magnitude slower proton-assisted dissociation
for Mn(\textit{t}-CDTA), resulting in a dissociation half-life of
$t_{1/2} = 12$ h estimated for physiological conditions, versus 0.076 h
for MnEDTA (pH 7.4 and
10$^{-5}$ M Cu$^{2+}$ concentration). The two stereoisomer
forms of the cyclohexane-based CDTA ligand, the \textit{trans}
\textit{t}-CDTA and the \textit{cis} \textit{c}-CDTA provide a
different coordination cage for metal complexation; it is considerably
larger for \textit{t}-CDTA with a maximum distance of 4 \AA{} between
the two nitrogen atoms, as compared to 3.1 \AA{} for
\textit{c}-CDTA~\cite{27}. Yet, DFT calculations indicate only minor
differences in the metal coordination environments in the solution
structures, with bond distances differing in less than 0.035 \AA{}. The
DFT results suggest remarkably higher stability for the \textit{trans}
than for the \textit{cis} analogue, with an energy difference of 35.4
kJ${\cdot}$mol$^{-1}$. This was linked to the more strained
geometry of the ligand in the \textit{cis}- than in the
\textit{trans}-stereoisomer in accommodating the relatively large
Mn$^{2+}$ ion~\cite{28}. Experimental data confirm these findings.
Although the thermodynamic stability constants for\break Mn(\textit{c}-CDTA)
and Mn(\textit{t}-CDTA) are similar (log$K_{\mathrm{MnL}}$
values are 14.19 and 14.32, respectively), the conditional stability as
expressed by the pMn value is higher for Mn(\textit{t}-CDTA) (8.68
versus 7.82; $c_{\mathrm{L}} = c_{\mathrm{Mn}} = 10^{-5}$ M, 
pH 7.4). The most striking difference is in
the kinetic inertness of the complexes, the \textit{trans}-isomer being
250 times more inert than the \textit{cis}-analogue, as it was assessed
in Cu$^{2+}$ transmetalation reactions~\cite{28}. The \textit{t}-CDTA
was then used for the development of a bifunctional chelator, and the
Mn$^{2+}$ complex was evaluated with respect to its MRI
properties~\cite{29}. 

The kinetic inertness is further improved with the PhDTA ligand, where
an aromatic benzene ring rigidifies the molecular skeleton (PhDTA $=$
\textit{o}-phenylenediamine-N,N,N$'$,N$'$-tetraacetate;
Scheme~\ref{sch1}). The dissociation half-life of MnPhDTA is estimated to be
$t_{1/2} = 19$ h at pH 7.4~\cite{30}. On the other
hand, the conditional stability constant (pMn $=$ 8.16) and the
relaxivity (3.72 mM$^{-1}{\cdot}$s$^{-1}$; 20 MHz,
25~\textdegree C) remain similar to those of Mn(\textit{t}-CDTA). Other
ligands containing a pyridine in their backbone were also investigated,
however, these ligands did not lead to improved inertness of the
Mn$^{2+}$ complexes~\cite{31}.

Amphiphilic anionic \textit{t}-CDTA derivatives have been recently
reported for specific uptake by hepatocytes through one or two
bi-directional transmembrane Organic Anion Transporting Polypeptide
(OATP) transporters. The complexes have been characterized through
relaxivity measurements, \textit{in vivo} liver imaging in mice, and
quantitation of \textit{in vitro} cell uptake through ectopically
expressed human OATP1 transporters~\cite{32}. 

The group of Caravan developed the
N-picolyl-N,N$'$,N$'$-trans-1,2-cyclohexylenediaminetriacetate (PyC3A;
Scheme~\ref{sch1}) ligand by further rigidifying \textit{t}-CDTA by the
substitution of one carboxylate with a pyridine. MnPyC3A was proposed
in 2015 as an extracellular contrast agent alternative to
Gd$^{3+}$-based contrast agents~\cite{33}, and it reached Phase 1 human
studies in 2022~\cite{20}. MnPyC3A is endowed with good thermodynamic
and conditional stability (log$K_{\mathrm{MnL}} = 14.14$ and
pMn $=$ 8.17), and its kinetic inertness exceeds that of the clinical
contrast agent GdDTPA; in the presence of 25-fold excess of Zn$^{2+}$,
pH 6, 37~\textdegree C, the dissociation is 20-times faster for GdDTPA
than for MnPyC3A. The relaxivity of MnPyC3A is 3.3
mM$^{-1}{\cdot}$s$^{-1}$ (20 MHz, 25~\textdegree C),
in coherence with its molecular size and monohydrated nature, and it is
slightly increased in serum due to moderate interaction with serum
proteins.

Thanks to its slightly lipophilic structure, MnPyC3A undergoes combined
hepatobiliary and renal excretion, and no \textit{in vivo} degradation
was detected for the complex. MR Imaging evaluation of this potential
contrast agent has been realized in various animal models with
side-by-side comparison to clinical agents. In a nonhuman primate
model, MnPyC3A provided signal enhancement in arteries versus muscle
which was comparable to GdDTPA in a standard dynamic MR angiography
protocol~\cite{34}. Pharmacokinetics was also found comparable to that
of clinically used extracellular Gd-based agents~\cite{34}. In rat
models of breast cancer and metastatic liver disease, it was found to
be equally efficient to delineate tumors as the clinical
Gd$^{3+}$ probes~\cite{35}. MnPyC3A was recently tested in
pigs for tissue characterization of acute myocardial infarction, and it
appeared to be equally effective as gadolinium-based agents for the
assessment of myocardial infarction location and size~\cite{36}.

Several liver-specific probes have been also derived from MnPyC3A. In a
family of nine complexes, a lead compound substituted with a
benzyloxy-moiety on the pyridine was retained based on its high
relaxivity, rapid blood clearance and avid hepatocellular uptake, the
two latter properties directly correlated with the log\textit{P}
values~\cite{37}. Injection of this lead Mn(PyC3A-3-OBn) complex
(Scheme~\ref{sch1}) into a mouse model with colorectal liver metastasis resulted
in excellent tumor visualization as hypointense\break spots. 

MnPyC3A was conjugated to bis-pyridyl-ethylamine (BPEN), the well-known
Zn$^{2+}$-binding moiety, to derive a responsive agent for the
\textit{in vivo} detection of Zn$^{2+}$released in different mouse
tissues, in particular to image ${\upbeta}$-cell function in the
pancreas and glucose-stimulated zinc secretion in the
prostate~\cite{38}. Mn(PyC3A-BPEN) (Scheme~\ref{sch1}), like previously reported
Gd-based zinc-responsive agents based on BPEN, shows only a modest
$r_{1}$ increase in the presence of Zn$^{2+}$
alone, but when Human Serum Albumin (HSA) is also present, a ternary
Mn(PyC3A-BPEN)-Zn-HSA complex forms with significantly enhanced
relaxivity, allowing for Zn$^{2+}$ detection. Interestingly, the
presence of the BPEN unit was found to improve the kinetic inertness of
the complex toward transmetalation by Zn$^{2+}$ and transchelation by
HSA. Biodistribution and excretory characterization studies indicated
that Mn(PyC3A-BPEN) is eliminated from mice without dissociation, by
combined renal and hepatobiliary\break pathways.

Stasiuk and collaborators reported a pentadentate Schiff-base type
Mn$^{2+}$ complex, MnL$^{\mathrm{Me}}$ which was obtained in a
single-pot template reaction~\cite{39} (Scheme~\ref{sch1}). In solution, this
chelate has two inner sphere water molecules, and correspondingly
elevated proton relaxivity which further increases in the presence of
serum albumin ($r_{1} = 5.7$
mM$^{-1}{\cdot}$s$^{-1}$ and 21.1
mM$^{-1}{\cdot}$s$^{-1}$, respectively, 298 K, 20
MHz). The inertness of this complex might be insufficient for further
development. In the presence of 25-fold excess of Zn$^{2+}$ at pH 6.4,
MnL$^{\mathrm{Me}}$ was found to fully dissociate in
${\sim}$10 min. After injection of MnL$^{\mathrm{Me}}$ in
a clinical dose (0.1 mmol${\cdot}$kg$^{-1}$) in a healthy mouse,
urine samples were collected and analyzed by LC-MS, showing that at 40
min post injection, 24\% of the applied MnL$^{\mathrm{Me}}$ was
excreted into the bladder in an intact\break form. 

\section{Macrocyclic chelators for Mn$^{2+}$ complexation}

A great number of macrocycles of different sizes and bearing various
coordinating functions have been investigated for Mn$^{2+}$
complexation in the last two decades. The most important studies
involve 9-, 12- and 15-membered azamacrocycles and aza- or azaoxa crown
ethers, many of these have been recently reviewed~\cite{13}. We will
only focus on 12-membered rings, based either on the cyclen or on the
pyridine-containing pyclen macrocycle. 


MnDOTA (DOTA $=$ 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate) is
known for a long time to be very stable and highly inert~\cite{15, 40},
however, with eight coordinating functions from the ligand, there is no
space for inner sphere water in this complex, which limits its
relaxation efficacy to an outer sphere effect. One has to remove at
least two carboxylates from DOTA to allow for inner sphere water
coordination in the Mn$^{2+}$ complexes. Interestingly, for MnDO2A
chelates, the hydration state depends on the position of the two
carboxylate functions. A combined $^{17}$O NMR, NMRD (Nuclear Magnetic
Relaxation Dispersion) and DFT study indicated a hydration equilibrium
between nonhydrated ($q = 0$) and monohydrated ($q = 1$)
species in solution, with clear prevalence of the monohydrated form for
Mn(1,4-DO2A) (average \textit{q} is 0.9), while Mn(1,7-DO2A) was
essentially non-hydrated (1,4-DO2A $=$
1,4,7,10-tetraazacyclododecane-1,4-diacetate; 1,7-DO2A $=$
1,4,7,10-tetraazacyclododecane-1,7-diacetate; Scheme~\ref{sch2})~\cite{41}. In
accordance with this, proton relaxivities amount to 2.1
mM$^{-1}{\cdot}$s$^{-1}$ for Mn(1,4-DO2A) and to 1.5
mM$^{-1}{\cdot}$s$^{-1}$ for Mn(1,7-DO2A) (25~\textdegree C, 
30~MHz). It is surprising that despite this difference in the coordination
mode, the thermodynamic stabilities and the kinetic inertness of the
two isomers are comparable~\cite{42}. A detailed transmetalation study
of the complexes with Zn$^{2+}$ or Cu$^{2+}$ ions revealed that the
dissociation proceeds primarily by acid catalysis, and metal-assisted
pathways are negligible. The complexes are about 20 times more labile
than MnDOTA, with dissociation half-lives of 48.3~h (Mn(1,4-DO2A)) and
56.8 h (Mn(1,7-DO2A)) estimated for pH 7.4, to be compared to 1061 h
for MnDOTA. 1,4-DO2A was further functionalized with long aliphatic C12
and C16 chains on the secondary amines to promote micellization in
solution~\cite{43}. For the corresponding Mn$^{2+}$ chelates, this
leads to important decrease in the rotational motion, with a
concomitant enhancement of the proton relaxivity, up to seven-fold at
30 MHz, where rotation is an important factor for relaxivity [($r_{1} =
15.3$ mM$^{-1}{\cdot}$s$^{-1}$ for the Mn(1,4-DO2A)-(C16)$_{2}$ derivative
with two C16 chains, 298 K (Scheme~\ref{sch2})]. \looseness=-1

\begin{scheme*}[t!]
\vspace*{-2pt}
\includegraphics{sc02}
\vspace*{6pt}
\caption{\label{sch2}Mn$^{2+}$ complexes formed with macrocyclic ligands.}
\vspace*{-3pt}
\end{scheme*}


The incorporation of a pyridine into the 12-membered cyclen macrocycle
also rigidifies the molecules, as in the case the linear chelators.
Monofunctionalization of this pyclen cycle with one acetate (PC1A $=$
6-carboxymethyl-3,6,9,15-tetraazabicyclo{[}9.3.1{]}
\mbox{pentadeca-1(15),11,13-triene)} or one methylenephosphonate group
(PC1P $=$\break
6-dihydroxyphosphorylmethyl-3,6,9,15-tetraazabicy{\ubreak}clo{[}9.3.1{]}
pentadeca-1(15),11,13-triene) 
results in Mn$^{2+}$ complexes which are six-coordinate with one inner
sphere water (Scheme~\ref{sch2}), but they do not have sufficient thermodynamic
and redox stability, nor kinetic inertness~\cite{44}. Two acetate
functions were introduced on the pyclen either at the 3,6 or at the 3,9
positions (3,6-PC2A and 3,9-PC2A, respectively, Scheme~\ref{sch2}), and the
corresponding Mn$^{2+}$complexes were compared in a detailed
physical-chemical study~\cite{45}. In contrast to the DO2A analogues,
there is no difference in the hydration state of the Mn(3,6-PC2A) and
Mn(3,9-PC2A) chelates, which are both monohydrated, as evidenced by
$^{17}$O chemical shifts and relaxation rates. The relaxivities are
also similar, and typical of small monohydrated chelates (2.72 and 2.91
mM$^{-1}{\cdot}$s$^{-1}$, for Mn(3,6-PC2A) and Mn(3,9-PC2A), respectively, 
298~K, 0.49 T). The regioisomer Mn(3,9-PC2A) with the two acetate groups in
\textit{trans} position is endowed with a slightly higher thermodynamic
stability constant than Mn(3,6-PC2A) (log$K_{\mathrm{MnL}}
= 17.09$ versus 15.53, respectively) and conditional stability (pM $=$
8.64 versus 8.03; $c_{\mathrm{L}} = c_{\mathrm{Mn}} =
10^{-5}$ M, pH 7.4), while kinetic inertness is better
for Mn(3,6-PC2A), as shown by the longer dissociation half-life
calculated ($t_{1/2} = 63.2$ h versus 21.0 h for
Mn(3,6-PC2A) and Mn(3,9-PC2A), respectively; pH 7.4). Interestingly,
DFT calculations showed important differences in some nitrogen-Mn
distances between the two isomers, indicating more steric strain for
Mn(3,6-PC2A), which might be the reason for its lower stability. In
overall, both chelates satisfy the requirements of good stability and
inertness.\looseness=-1

The symmetrical 3,9-PC2A has been chosen for the design of various
molecular imaging agents. The pH-responsive 
probe Mn(3,9-PC2A-EA)
(Scheme~\ref{sch2})~\cite{46} undergoes protonation on the ethyleneamine
side-chain with log$K_{\mathrm{a}}$ of 6.88 which can thus
coordinate/decoordinate to/from the metal ion as a function of its
protonation state. As a consequence, the hydration number, thus the
relaxivity of Mn(3,9-PC2A-EA) increases from $q = 0$ and
$r_{1} = 2.04$ mM$^{-1}{\cdot}$s$^{-1}$, respectively, at pH 8.4 to
$q = 1$ and $r_{1} = 3.54$ mM$^{-1}{\cdot}$s$^{-1}$ at pH 6.0
(0.49 T, 25~\textdegree C). By attaching the BPEN Zn$^{2+}$ sensitive
unit to 3,9-PC2A, the Zn$^{2+}$-responsive probe Mn(PC2A-BPEN) was
prepared (Scheme~\ref{sch2})~\cite{47}. This probe functions according to a
similar mechanism described above for Mn(PyC3A-BPEN) by forming a
ternary complex with Zn$^{2+}$ and HSA, accompanied by a relaxivity
increase. Mn(PC2A-BPEN) could be used to visualize glucose-stimulated
Zn$^{2+}$ secretion in the prostate of a healthy mouse. 

The same 3,9-PC2A unit was also derivatized with a biphenyl moiety
which induces HSA-binding (Scheme~\ref{sch2})~\cite{48}. This leads to a
prolonged residential time of Mn(3,9-PC2A)-BP in the blood which makes
it interesting for angiographic imaging. Indeed, it could be used to
highlight microvasculature of the mouse brain. 


\section{Bispidine chelators}

All data on linear or macrocyclic Mn$^{2+}$ chelates indicate that
rigidity and preorganization of the ligand are key design elements to
promote kinetic inertness. This made us explore bispidine
(3,7-diazabicyclo{[}3.3.1{]}nonane) derivatives for Mn$^{2+}$
complexation. Bispidine-type chelators constitute a versatile family in
coordination chemistry with a highly preorganized and rigid
structure~\cite{49,50}. The bicyclic amine core can be appended with
various coordinating functions and in different positions in order to
match the coordination requirements of a large variety of metal ions of
various sizes, coordination numbers or coordination geometries. As a
function of the size and/or the position of the substituents, the
bicyclic skeleton can adopt different conformations and configurations,
among which the chair--chair conformer, with a ``bicyclic coordination
cage'' is the best suited for metal complexation (Scheme~\ref{sch3}a).
Bispidines were extensively studied for the complexation of various
transition metal and lanthanide ions, and many recent reports were
dedicated to the development of bispidine complexes as nuclear imaging
agents~\cite{51}. Interestingly, Mn$^{2+}$ was rarely in the scope of
such studies, and all previous Mn$^{2+}$-related data were restricted
to the solid state or to non-aqueous solvents. 

\begin{scheme*}
\includegraphics{sc03}
\vspace*{8pt}
\caption{\label{sch3}(a) Bispidine conformations, (b) bispidine ligands
for Mn$^{2+}$ complexation and (c) the putative structure of MnHBisp1.} 
\end{scheme*}

We have first investigated the 2,4-pyridyl-disubstituted bispidol
derivative H$_{4}$Bisp1 bearing one carboxylate (from a lysine) for
Mn$^{2+}$ complexation~\cite{52}. This ligand can coordinate the metal
in a five-dentate manner, involving the nitrogens from the two
pyridines, the heterobicycle (N3 and N7) as well as the methylene
carboxylate (Scheme~\ref{sch3}), leaving one coordination site for a water
molecule in the coordination sphere of the metal ion, important for
good relaxivity in MRI application. The thermodynamic stability of
MnBisp1, determined by pH-potentiometry, is relatively modest
(log$K_{\mathrm{MnL}} = 12.21$, pMn $=$ 6.65). However, once
the complex is formed, it is extremely resistant to dissociation. In a
standard transmetalation test carried out in the presence of 10 or 50
equiv. of Zn$^{2+}$ at pH 6 and 37~\textdegree C, no dissociation at
all was observed for 140 days, while the most inert non-bispidine
chelate, Mn(3,9-PC2A-BPEN) has a dissociation half-life of 64.5 h under
similar conditions (37~\textdegree C, pH 6.0, 25 Zn$^{2+}$
equivalents)~\cite{47}. Such high kinetic inertness is unprecedented
for a Mn$^{2+}$ chelate. MnHBisp1 (protonated on the lysine amine) has
also excellent relaxation properties, comparable to those of
Gd$^{3+}$-based agents, despite the difference in the electron spin of
the metals. The two non-coordinating carboxylate groups create an
additional second sphere contribution to relaxivity which, together
with the slightly higher molecular weight with respect to typical small
molecular weight contrast agents, is responsible for the unusually high
relaxivity ($r_{1} = 4.28$ mM$^{-1}{\cdot}$s$^{-1}$ and 3.37
mM$^{-1}{\cdot}$s$^{-1}$ at 25~\textdegree C and 37~\textdegree
C, respectively, 20~MHz, pH 7 in water). The \textit{in vivo} imaging
potential of MnHBisp1 was evaluated in a preliminary MRI experiment in
healthy mice at 7 T. Following intravenous injection of MnHBisp1 at
0.06 mmol/kg dose, the signal intensity was monitored over time in
various organs, which showed renal clearance and practically no liver
uptake (Figure~\ref{fig1}). This was further confirmed by \textit{ex vivo} ICP-OES
biodistribution measurements of Mn in various tissues. Moreover, Bisp1
was successfully labeled with $^{52}$Mn$^{2+}$, and the radiocomplex
proved to be stable in biological media. In addition to the MRI
potential of MnHBisp1, this opens interesting perspectives also for
immunoPET applications, after further conjugation of the\break bifunctional
ligand Bisp1 to biomolecules, such as antibodies.

\begin{figure*}[t!]
\includegraphics{fig01}
\caption{\label{fig1}MRI of healthy mice at 7 T following intravenous
injection of MnBisp1 at 0.06 mmol/kg dose. (a) Normalized signal
intensity in the kidney, muscle, liver and aorta plotted as a function
of time. Measurements were performed every 2 min for 4 mice. (b)
Sagittal T1-weighted MR images prior to injection and at 4, 15 min and
1 h post intravenous injection of the complex. Reproduced with
permission from reference~\cite{52}.}
\end{figure*}

Analogous bispidine ligands bearing methylenephosphonate (Bisp2) and
methylenecarboxylate (Bisp3) substituents instead of the lysine have
been also synthesized, and their Mn$^{2+}$ complexes have shown
similarly remarkable inertness and good proton relaxivities~\cite{53}.
$^{52}$Mn$^{2+}$ radiolabeling of the phosphonate derivative was
realized with excellent yields and the radiocomplex showed high
stability. 

In a recent comprehensive study combining structural NMR,
potentiometric, relaxometric and kinetic experiments with DFT
calculations, we have addressed how the substituents on the two
nitrogens of the heterobicycle (N3 and N7) affect the conformation of
the Mn$^{2+}$ complexes~\cite{54}. In these studies, we have included a
series of ligands Bisp3, Bisp4 and Bisp5 that possess
methylenecarboxylate substituents at the N7 or N3 positions or both,
respectively (Scheme~\ref{sch3}b). We could conclude that in this family the
conformation of the bispidine is determined by the nature of the N3
substituent (methyl or methylenecarboxylate), with very important
consequences on the thermodynamic stability, kinetic inertness,
hydration state and relaxation properties of the complexes. Only
ligands without N3-appended methylenecarboxylate adopt the chair--chair
conformation. Dissociation (as well as complex formation) is several
orders of magnitude slower for the Mn$^{2+}$ chelates in chair--chair
conformation. The hydration number and the relaxation properties are
also dependent on the ligand conformation: Mn$^{2+}$ chelates in
boat-chair conformation have less than one inner sphere water molecule,
as shown by $^{17}$O NMR data, which is unfavorable as it results in
lower relaxivities. In overall, we could evidence that chair--chair
conformation is required for the favorable properties of Mn$^{2+}$
bispidines. Another ligand, Bisp6 (Scheme~\ref{sch3}b) was also investigated to
address how steric crowding introduced on the ${\upalpha}$ carbon of
the N7 methylenecarboxylate substituent affects the kinetic inertness
of the Mn$^{2+}$ complex. As it was previously found in the family
Ln$^{3+}$-tetraazamacrocycles~\cite{55}, this modification on the
methylenecarboxylate arm further increases the resistance of the
Mn$^{2+}$ chelate to dissociation, leading to a dissociation half-life
which is roughly doubled for MnBisp6 with respect to the MnBisp3
analogue. 

{Bispidine ligands of higher denticity have been also investigated for
Mn}$^{2+}$ {complexation and found to exhibit other extraordinary
coordination\break chemistry features. While high kinetic inertness, as well
as relatively high thermodynamic stability could be now achieved for
Mn}$^{2+}$ {chelates, none of them so far provided selectivity for
Mn}$^{2+}$ {over other transition metal ions, in particular
Zn}$^{2+}${, its major biological competitor. This directly derives
from the large ionic radius of Mn}$^{2+}$ {and the spherical
distribution of its} {\textit{d}} {electrons. The Irving--Williams
series describes the order of complex stabilities with a particular
ligand in the first row of divalent transition metal cations, and
Mn}$^{2+}$ {complexes are usually the least stable:} Mn$^{2+}$
{${<}$} Fe$^{2+}$ {${<}$} Co$^{2+}$ {${<}$} Ni$^{2+}$
{${<}$} Cu$^{2+}$ {${>}$} Zn$^{2+}$. We hypothesized that
the rigid and preorganized nature of bispidines make them appropriate
to design ligands with high denticity and a perfectly adapted
coordination cavity for the ${\sim}$10\% larger size of Mn$^{2+}$
versus Zn$^{2+}$. This cavity would be too large for Zn$^{2+}$, which
could not accommodate all of the donor groups in its\break coordination
sphere. The higher number of coordinating functions would then lead to
a higher stability for Mn$^{2+}$ as compared to Zn$^{2+}$. Some
previous observations were already in support of preferential
coordination of larger metal ions by the rigid bispidine cavity,
resulting in divergence from the Irving--Williams series~\cite{56,57}.

Hepta- and octadentate bispidine ligands Bisp7, Bisp8 and Bisp9 have
been thus synthesized (Scheme~\ref{sch3})~\cite{58,59}. The Mn$^{2+}$ and
Zn$^{2+}$ complexes have been subject of solid-state X-ray and
computational studies to address their structure, and
pH-potentiometric, relaxometric and kinetic investigations have been
conducted to characterize their stability and inertness in aqueous
solution. The solid-state structures show octadentate coordination for
the Mn$^{2+}$, and hexadentate coordination for the Zn$^{2+}$
complexes, evidencing that the coordination cavity provided by these
ligands is well-suited for Mn$^{2+}$ but too large for Zn$^{2+}$. The
good match for Mn$^{2+}$ and the misfit for Zn$^{2+}$ was further
demonstrated by empirical force field calculations which indicated that
Mn$^{2+}$ has an optimum size, while Zn$^{2+}$ coordination induces a
ligand-based strain of ${\sim}$50 kJ/mol. Thermodynamic stability
constants determined by pH-potentiometric (Zn$^{2+}$) or relaxometric
(Mn$^{2+}$) titrations, further corroborated with $^{1}$H NMR and
UV--Vis data, confirmed the expectations from the structural and
computational studies. There is a spectacular, 5--10 orders of magnitude
difference in stability in favor of the Mn$^{2+}$ complexes over the
Zn$^{2+}$ analogues (log$K_{\mathrm{MnL}} = 24.2$, 24.7 and
19.47 for MnBisp7, MnBisp8 and MnBisp9, versus
log$K_{\mathrm{ZnL}} = 14.30$, 14.70 and 15.04 for ZnBisp7,
ZnBisp8 and ZnBisp9, respectively). These complexes represent the first
examples of real Mn$^{2+}$ selectivity in aqueous solution. The kinetic
inertness of these chelates also remains excellent, though slightly
lower than that of MnHBisp1. 

MnBisp9 has been fully characterized for its relaxation
properties~\cite{59}. Its relaxivity (4.44 mM$^{-1}{\cdot}$s$^{-1}$ at 20 MHz,
25~\textdegree C) is slightly superior to that of MnBisp1 which can be
accounted for the 17\% higher molecular weight, thus slower rotation.
This overbalances the lack of second-sphere contribution to relaxivity
which operates for MnBisp1 thanks to the non-coordinating carboxylate
groups, but which is not present for MnBisp9 where the carboxylates are
replaced by esters. A preliminary \textit{in vivo} MRI study has been
carried out with MnBisp9 in mice at 7 T. Even at an unusually low
injected dose (0.02~mmol/kg, 80\% below typical doses), good contrast
is observed in the kidneys. The signal variation over time indicates
rapid renal clearance and no significant liver uptake. \textit{Ex vivo}
ICP-MS data point to full elimination of the complex from the mice
after 24 h. 

\section{Mn$^{3+}$ complexes and redox-responsive MRI agents based on
the  Mn$^{2+}$/Mn$^{3+}$ switch}

\begin{scheme*}[t!]
\includegraphics{sc04}
\vspace*{6pt}
\caption{\label{sch4}Porphyrin Mn$^{3+}$ complexes.}
\vspace*{-2pt}
\end{scheme*}

The stabilization of the Mn$^{3+}$ state in aqueous solution and
physiological pH is by far not obvious, due to the strong tendency of
this cation to hydrolysis as well as to dismutation yielding Mn$^{2+}$
and Mn$^{4+}$. The need for an inner sphere water in MRI
applications makes stabilization even more difficult, and indeed, only
few types of Mn$^{3+}$ complexes are known with coordinated water
molecule at pH 7. Among these, Mn$^{3+}$ porphyrins represent a
specific class endowed with high stability, and they were indeed
considered in MRI and studied for their relaxation properties a long
time ago. Mn$^{3+}$ porphyrins have two water molecules coordinated to
the metal ion, above and below the porphyrin plane. The relaxivities
measured for some water soluble Mn$^{3+}$ porphyrins such as the
(5,10,15,20-tetrakis-(\textit{p}-sulfonatophenyl) porphinate)
derivative Mn(tpps) (Scheme~\ref{sch4}) were considered as anomalously high for
the given molecular size and electron spin, and this was tentatively
explained by the anisotropy of the electron density at the metal center
resulting in a closer approach of the Mn$^{3+}$ spin density to the
coordinated water protons~\cite{60,61}. More recently, there has been a
renewed interest in Mn$^{3+}$ porphyrins. Zhang and collaborators added
carboxylates on the porphyrin periphery to increase hydrophilicity and
facilitate \textit{in vivo} clearance of the Mntcp complex 
(Scheme~\ref{sch4})~\cite{17, 62}. The relaxivity, as well as the biodistribution have
been modulated by further structural modifications of this carboxylated
derivative, dimers endowed with HSA-binding capability have been
developed for blood pool imaging~\cite{63}, and specific liver
targeting was generated by introducing a \mbox{4-ethoxyphenyl} substituent on
the \mbox{porphyrin~\cite{64}.} Cell-penetrating Mn$^{3+}$ porphyrin
derivatives containing a Zn$^{2+}$-binding unit have been also
developed and successfully used for Zn$^{2+}$ detection~\cite{65,66}.
Phtalocyanine complexes of Mn$^{3+}$ have been also
investigated~\cite{67}, but like for porphyrins, water solubility
remains often limited. 

Mn porphyrins have been further explored in the objective of developing
redox sensitive agents. The redox state is an important characteristic
of tissues, and redox imbalance is associated to many pathologies which
can induce oxidative stress, generate reactive oxygen species, etc. The
redox homeostasis in cells and tissues is governed by different
biological molecules, such as cysteine, glutathione, hydrogen peroxide,
nicotinamide adenine dinucleotide (NADH), etc., and their imaging
detection has great interest. Given the different relaxation properties
of Mn$^{2+}$ and Mn$^{3+}$, this redox pair is very promising for the
design of redox sensitive MRI probes (Figure~\ref{fig2})~\cite{18}. For this,
the system needs to satisfy several requirements, including (a) stable
and inert complexation of both redox forms of the metal to prevent free
metal release, (b) a redox switch of the probe which is rapid and
occurs at a biologically relevant potential, and (c) a significant
relaxivity change between the two redox states allowing for MRI\break
monitoring.

\begin{figure*}
\includegraphics{fig02}
\caption{\label{fig2}The redox switch between Mn$^{3+}$ and Mn$^{2+}$
states is accompanied by a change in the relaxivity of the complexes,
as a consequence of variation in the electron spin (\textit{S}),
electron spin relaxation times ($T_{1,2\mathrm{e}}$), hydration number
(\textit{q}), or water exchange rate ($k_{\mathrm{ex}}$).}
{\vspace*{6pt}}
\end{figure*}

Following the pioneering work by Aime and collaborators on
Mn$^{2+}$/Mn$^{3+}$(tpps)~\cite{68}, as well as other examples of redox
responsive Mn-probes based on linear chelators such as the Janus ligand
JED (Scheme~\ref{sch5})~\cite{69,70,71,72}, we have been recently exploring a
PEGylated Mn-porphyrin derivative, Mn(pfpp(PEG)$_{4}$) 
(Scheme~\ref{sch5})~\cite{73}. The PEG chains have been introduced on the molecule to
increase water solubility, and additionally, fluorine atoms have been
added to modulate the redox potential. Both Mn$^{2+}$ and Mn$^{3+}$
forms are stably complexed in aqueous solution, with a reversible
switch between them. In the presence of ascorbic acid or
${\upbeta}$-mercaptoethanol, the Mn$^{3+}$ form undergoes reduction,
which is slowly but fully reversed in the presence of air oxygen. This
is accompanied by a ${\sim}$150\% relaxivity increase from the oxidized
to the reduced form at 20 MHz. No reduction was observed with
glutathione or cysteine, which can be explained by the coordination of
the glutathione carboxylate to the metal ion by replacing one
coordinated water molecule as evidenced by NMR data. Also,
Mn(pfpp(PEG)$_{4}$) had no detectable \textit{in vitro} cytotoxicity in
the typical MRI concentration range.

\begin{scheme*}[t!]
\includegraphics{sc05}
\vspace*{4pt}
\caption{\label{sch5}Mn$^{2+}$/Mn$^{3+}$ complexes investigated as
redox responsive probes.}
\vspace*{-2pt}
\end{scheme*}

With such fluorinated Mn-porphyrins, the redox detection can be
extended to $^{19}$F MRI in addition to $^{1}$H MRI, as we demonstrated
with another derivative, Mn(TPP-\textit{p}-CF$_{3}$) 
(Scheme~\ref{sch5})~\cite{74}. The two Mn redox states induce very 
different $^{1}$H and
$^{19}$F relaxometric properties which allow for differential redox MRI
detection. The $^{19}$F relaxation rates in these paramagnetic systems
are strongly dependent on the Mn$^{3+}$--F distance. In
Mn$^{3+}$(TPP-\textit{p}-CF$_{3}$), it is 9.7--10~\AA{}, as determined
from DFT calculations. This distance seems well tailored to allow for
adequate paramagnetic effect of Mn$^{3+}$ on $^{19}$F relaxation without
excessive line-broadening, which would be detrimental for $^{19}$F MRI
detection. Reduction of Mn$^{3+}$(TPP-\textit{p}-CF$_{3}$) with
ascorbic acid and its re-oxidation with air oxygen can be monitored by
UV--Vis spectrometry, $^{19}$F NMR and $^{1}$H relaxivity measurements.
Upon reduction, the $^{1}$H and $^{19}$F NMR signals of
Mn$^{3+}$(TPP-\textit{p}-CF$_{3}$) largely broaden and disappear. 
The~\mbox{reduction/oxidation} process can be readily visualized on phantom
$^{19}$F images acquired in DMSO/water mixture (Figure~\ref{fig3}). 

\begin{figure*}
\vspace*{-3pt}
\includegraphics{fig03}
\vspace*{-3pt}
\caption{\label{fig3}Monitoring oxidation of
{Mn}$^{2+}${(TPP-}{\textit{p}}{-CF}$_{3}$) ($c_{\mathrm{MnL}} = 1.6~\mathrm{mM}$
{in DMSO}) {by air oxygen. Variation of the Signal to Noise Ratio (SNR)
in MRI phantoms as a function of the oxidized
Mn}$^{3+}${(TPP-}{\textit{p}}{-CF}$_{3}${) content (\%). The} $^{19}${F
MRI phantom images for each point are represented above. $B = 7$ T;}
{TR: 60 ms; TE: 1.3 ms; FA: 90\textdegree; NA: 256; acquisition time 8
min 11 s.} {The signal of the Mn}$^{2+}$ {complex was saturated with a
selective pulse.} {Adapted with permission from ref.}~\cite{74}.}
\vspace*{-3pt}
\end{figure*}

$^{19}$F MRI detection of Mn$^{2+}$/Mn$^{3+}$ redox changes was also
realized using the HTFBED ligand (Scheme~\ref{sch5})~\cite{72}. The complexes
respond to different redox active species of the biological environment
including ROS, ascorbic acid or glutathione with concomitant changes in
the $^{19}$F as well as in the $^{1}$H MRI signal intensity. In
particular, an eightfold increase of the $^{19}$F signal was detected
upon\break oxidation from Mn$^{2+}$ to Mn$^{3+}$ form, and the \mbox{responsive}
properties of these probes could be also validated at the cellular
level in HepG2 cells.

\section{Conclusion}

Mn$^{2+}$ complexes represent the most viable alternatives to replace
Gd$^{3+}$-based MRI contrast agents in the clinical practice. In this
context, many chelators, including open-chain, macrocyclic and bicyclic
structures, have been explored in the last years for Mn$^{2+}$
complexation, and recently there has been important progress in
substantially improving the thermodynamic stability, the selectivity
and the kinetic inertness of Mn$^{2+}$ chelates. Endowed with a highly
preorganized and rigid skeleton, bispidine ligands proved to provide
unprecedented properties in Mn$^{2+}$ coordination chemistry, such as
extreme kinetic inertness, complex stability and Mn$^{2+}$ selectivity,
which can be further combined with excellent relaxation properties. An
increasing number of Mn$^{2+}$ complexes are successfully characterized
in \textit{in vivo} preclinical imaging, and some of them have reached
human clinical trials as potential MRI agents. The Mn$^{3+}$ state is
also increasingly explored, mainly in porphyrin complexes. Finally,
efforts are still being made in order to develop Mn-based redox
responsive agents. 

\section*{Declaration of interests}

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.

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