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\DOI{10.5802/crchim.363}
\datereceived{2024-07-26}
\dateaccepted{2024-11-18}
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\begin{document}

\begin{noXML}

\CDRsetmeta{articletype}{clarification}

\title{Towards more efficient, more specific, and safer MRI contrast
agents: a Portuguese--French collaborative journey}

\alttitle{Vers des agents de contraste IRM plus efficaces, plus
sp\'{e}cifiques et plus s\^{u}rs : un parcours collaboratif
portugais-fran\c{c}ais}

\author{\firstname{Sara Martinho Almeida} \lastname{Pinto}\CDRorcid{0000-0003-3817-1182}}
\address{Coimbra Chemistry Centre - Institute of Molecular Sciences (CQC-IMS), 
Chemistry Department of the University of Coimbra, 
Portugal}

\author{\firstname{Sara} \lastname{Lacerda}\CDRorcid{0000-0003-3865-4379}}
\address{Centre de Biophysique Mol\'{e}culaire, CNRS - UPR 4301, 
Universit\'{e} d'Orl\'{e}ans, Orl\'{e}ans, 
France}

\author{\firstname{Jean-Fran\c{c}ois} \lastname{Morfin}}
\addressSameAs{2}{Centre de Biophysique Mol\'{e}culaire, CNRS - UPR 4301, 
Universit\'{e} d'Orl\'{e}ans, Orl\'{e}ans, 
France}

\author{\firstname{Rafael Tiago} \lastname{Aroso}\CDRorcid{0000-0002-2657-9212}}
\addressSameAs{1}{Coimbra Chemistry Centre - Institute of Molecular Sciences (CQC-IMS), 
Chemistry Department of the University of Coimbra, 
Portugal}

\author{\firstname{Daniela Sofia de Sousa} \lastname{~Teixeira}\CDRorcid{0000-0001-8302-9260}}
\addressSameAs{1}{Coimbra Chemistry Centre - Institute of Molecular Sciences (CQC-IMS), 
Chemistry Department of the University of Coimbra, 
Portugal}
\addressSameAs{2}{Centre de Biophysique Mol\'{e}culaire, CNRS - UPR 4301, 
Universit\'{e} d'Orl\'{e}ans, Orl\'{e}ans, 
France}

\author{\firstname{Zolt\'{a}n} \lastname{Garda}\CDRorcid{0000-0002-3904-2959}}
\addressSameAs{2}{Centre de Biophysique Mol\'{e}culaire, CNRS - UPR 4301, 
Universit\'{e} d'Orl\'{e}ans, Orl\'{e}ans, 
France}

\author{\firstname{Maria Miguens} \lastname{Pereira}\IsCorresp\CDRorcid{0000-0003-4958-7677}}
\addressSameAs{1}{Coimbra Chemistry Centre - Institute of Molecular Sciences (CQC-IMS), 
Chemistry Department of the University of Coimbra, 
Portugal}
\email[M. M. Pereira]{mmpereira@qui.uc.pt}

\author{\firstname{Carlos Frederico Gusm\~ao Campos} \lastname{~Geraldes}\IsCorresp\CDRorcid{0000-0002-0837-8329}}
\addressSameAs{1}{Coimbra Chemistry Centre - Institute of Molecular Sciences (CQC-IMS), 
Chemistry Department of the University of Coimbra, 
Portugal}
\email[C. F. G. C. Geraldes]{geraldes@ci.uc.pt}

\author{\firstname{\'{E}va} \lastname{T\'{o}th}\IsCorresp\CDRorcid{0000-0002-3200-6752}}
\addressSameAs{2}{Centre de Biophysique Mol\'{e}culaire, CNRS - UPR 4301, 
Universit\'{e} d'Orl\'{e}ans, Orl\'{e}ans, 
France}
\email[\'{E}. T\'{o}th]{eva.jakabtoth@cnrs.fr}

\shortrunauthors

\keywords{\kwd{Magnetic resonance imaging}
\kwd{Contrast agents}
\kwd{Lanthanide}
\kwd{Manganese}
\kwd{Porphyrins}}

\altkeywords{\kwd{Imagerie par r\'{e}sonance magn\'{e}tique}
\kwd{Agents de contraste}
\kwd{Lanthanide}
\kwd{Mangan\`{e}se}
\kwd{Porphyrines}}

\begin{abstract}
Although complexes of the paramagnetic Gd(III) ion have been used with
great success for the past 40  years to enhance image contrast in
magnetic resonance imaging (MRI), important challenges such as 
improving the relaxation efficacy, the specificity, the
safety, and the environmental impact of these agents remain in this field. Thanks to a
collaborative effort between the Centre of Molecular Biophysics in
Orl\'{e}ans, France and the Coimbra Chemistry Centre - Institute of
Molecular Sciences in Coimbra, Portugal, there have been important
advances in the chemistry of MRI probes. The authors have developed rigid,
medium-sized, and bishydrated Gd(III) complexes specifically optimized
for high-field applications with successful validation in
vivo. For the specific targeting of amyloid peptides implicated in
many pathologies such as Alzheimer's disease and diabetes, they have
designed benzothiazole-derivative metal chelates with detection
potential in MRI, optical, and nuclear imaging modalities. The authors have
gained valuable insights into the aggregation behavior of these
complexes in solution and how this affects peptide binding affinity.
More recently, manganese porphyrin complexes have been investigated with
the objective of visualizing the tissue redox state based on the
Mn(II)/Mn(III) redox switch, which can be followed not only in
classical \textsuperscript{1}H detection but also in \textsuperscript{19}F MRI.
\end{abstract}

\begin{altabstract}
Alors que les complexes de l'ion paramagn\'{e}tique Gd(III) sont
utilis\'{e}s avec beaucoup de succ\`{e}s depuis pr\`{e}s de quarante
ans pour am\'{e}liorer le contraste des images en imagerie par
r\'{e}sonance magn\'{e}tique, d'importants d\'{e}fis restent \`{a}
relever dans ce domaine afin d'am\'{e}liorer l'efficacit\'{e} de
relaxation, la sp\'{e}cificit\'{e} ou la biocompatibilit\'{e} des
agents, et de diminuer leur impact environnemental. Gr\^{a}ce \`{a} un
effort de collaboration entre le Centre de Biophysique Mol\'{e}culaire
d'Orl\'{e}ans, en France et le Centre de Chimie de Coimbra - Institut
des Sciences Mol\'{e}culaires de Coimbra, au Portugal, d'importants
progr\`{e}s ont \'{e}t\'{e} r\'{e}alis\'{e}s dans la chimie des sondes
IRM. Nous avons d\'{e}velopp\'{e} des complexes de Gd(III)
bishydrat\'{e}s, rigides et de taille moyenne, sp\'{e}cifiquement
optimis\'{e}s pour les applications \`{a} haut champ, qui ont
\'{e}t\'{e} valid\'{e}s avec succ\`{e}s in vivo. Pour le ciblage
sp\'{e}cifique des peptides amylo\"{i}des, impliqu\'{e}s dans de
nombreuses pathologies telles que la maladie d'Alzheimer ou le
diab\`{e}te, nous avons con\c{c}u des complexes portant un groupement
benzothiazole, avec un potentiel de d\'{e}tection en IRM, en imagerie
optique et nucl\'{e}aire. Nous avons montr\'{e} que leur agr\'{e}gation
en solution a un impact fort sur la reconnaissance des peptides. Plus
r\'{e}cemment, des complexes de porphyrine de mangan\`{e}se ont
\'{e}t\'{e} \'{e}tudi\'{e}s dans le but de visualiser l'\'{e}tat
r\'{e}dox des tissus sur la base d'un changement r\'{e}dox
Mn(II)/Mn(III), qui peut \^{e}tre suivi non seulement par une
d\'{e}tection IRM de \textsuperscript{1}H classique, mais \'{e}galement en
IRM de \textsuperscript{19}F.
\end{altabstract}

\maketitle

\twocolumngrid

\end{noXML}

\let\rmmu\upmu

\section{Introduction}\label{sec1} 
Magnetic resonance imaging (MRI) is currently one of the most widely
used and powerful diagnostic imaging modalities in clinical practice,
and it has also become an important tool in biomedical research. 
MRI offers high spatial and temporal 
\mbox{resolution} and is capable of
visualizing soft tissue alterations in order to detect, for instance,
chronic inflammation, tumors, tissue perfusion, and other abnormalities,
making it an invaluable tool for diagnosing various
diseases~\cite{1,2,3}. This non-invasive technique is based on applying
an external magnetic field to align the spin moments of protons in the
water molecules within the tissues or organs of interest. After
low-energy radio frequency irradiation, the longitudinal $(T^{1})$ and
transverse $(T^{2})$ relaxation properties of these protons are
exploited to produce detailed images of internal 
structures~\cite{2}.

Thus, the images generated by MRI result from differences in the
density (i.e., concentration) and the nuclear relaxation properties of
protons, mainly of water, which produce variations in signal
intensities. However, the inherent contrast is often insufficient for
accurate diagnosis, and therefore contrast enhancing agents are needed.
These paramagnetic or superparamagnetic drugs can reduce the
longitudinal and transverse relaxation times of the surrounding water
protons, thereby considerably improving the diagnostic quality of the
images~\cite{4,5,6}.  

All commercial MRI contrast agents as well as the overwhelming
majority of potential probes reported in the literature are
gadolinium(III) (Gd) chelates, which involve the use of
polyamino-polycarboxylate ligands. Gd(III) complexes formed with
macrocyclic (gadoteric acid, gadobutrol, gadopiclenol) or linear
(gadopentetic acid) structures have been thus far the golden standard
in clinical practice. They have been efficiently used for instance to
highlight morphological and functional abnormalities, enhance blood
vessels in MR angiography, and to assess blood--brain barrier 
degradation~\cite{7,8}.

In recent years, with the overall aims of improving sensitivity,
specificity, and safety of MRI contrast agents to promote earlier and
more reliable detection of diseases such as cancer and
neurodegenerative disorders, research has been pursued in several
domains:  (a)~increasing the relaxation efficiency of the 
complexes~\cite{6}; (b)~addressing contrast agents to specific
biological  targets~\cite{3,9};  (c)~detecting variations in tissue
physical--chemical properties like pH, temperature, cation
concentration, and redox state with smart probes~\cite{10,11,12,13}; and
(d)~substituting Gd(III) with more biocompatible paramagnetic metal
ions such as Mn(II), Mn(III), and Fe(III)~\cite{14,15,16,17,18}.
Coordination chemistry is fundamental to addressing these challenges.
Within this general context, this review aims to focus on the
collaborative achievements between research groups in the Centre of
Molecular Biophysics, CNRS, Orl\'{e}ans and in the Coimbra Chemistry
Centre - Institute of Molecular Sciences (CQC-IMS), Chemistry
Department of the University of Coimbra, Portugal over the past two
decades. This highly fruitful partnership began with the investigation
of basic coordination properties of macrocyclic lanthanide chelates,
and has been later 
\mbox{extended} to the design of MRI agents with increased
efficiency or various targeting capabilities. In particular, a lot of
effort has been dedicated to the visualization of amyloid peptides,
mostly in relation to Alzheimer's disease. More recently, the
monitoring of the tissue redox environment, using Mn-complexes of
tetrapyrrolic macrocycles, has become a major focus. Finally, detection
has been also extended to  $\mbox{}^{19}$F MRI, which offers
complementary advantages over classical  $\mbox{}^{1}$H MRI. The
excellence of this collaborative research, leading to 36 joint
publications altogether, was recognized by the Portuguese and French
Academies of Sciences with the attribution of the Mariano Gago Prize in
2022. This minireview  illustrates, without the intention to be
exhaustive, some of the main achievements of the Coimbra--Orl\'{e}ans
collaboration via a selected number of representative
examples.

\begin{figure*}
\includegraphics{fig01}
\vspace*{2pt}
\caption{\label{fig1}(a)~Structure of the metallostar and of the
Gd$_{3}$L1 trimer having optimized relaxivity for high-field MRI
applications.  (b)~Relaxivities at different frequencies and
temperatures compared to the clinical agents GdDOTA and GdDTPA. 
(c)~Dynamic contrast enhanced magnetic resonance images acquired
at 9.4~T (400~MHz proton Larmor frequency) after GdDOTA or Gd$_{3}$L1
injections at 
$8~\mathrm{mmol}{\cdot}\mathrm{Gd}{\cdot}\mathrm{kg}^{-1}$ dose. For
both complexes, marked signal intensity increase is observed in the
vascular system, renal cortex, and medulla; the signal enhancement is
considerably higher with Gd$_{3}$L1 due to its threefold higher
relaxivity (adapted with permission from \textit{Contrast Media Mol.\ 
Imag.}~\cite{23}).}
\vspace*{4pt}
\end{figure*}

\section{Strategies to increase the efficiency of MRI
agents}\label{sec2} 
The MRI efficiency of a paramagnetic metal chelate is expressed by its
proton relaxivity,  $r_{1}$, defined as the paramagnetic enhancement of
the longitudinal water proton relaxation rate referred to 1 mM
concentration. Proton relaxivity is the sum of inner- and outer-sphere
contributions originating from dipolar interactions between the
electron spin of the paramagnetic metal ion and the nuclear spins of
the water protons in the inner coordination sphere or of those
diffusing in the proximity of the complex (outer sphere), respectively.
The inner-sphere term can be modulated by chemical design, as it is
determined by structural and dynamic properties of the metal complex.
Among these, the most important are the number of inner-sphere
coordinated water molecules  $(q)$, their exchange rate with bulk water
$(k_{\mathrm{ex}})$, the rotational correlation time of the chelate 
$(\tau_{\mathrm{R}})$, and the electron spin relaxation times of the metal ion
$(T_{1,2e})$~\cite{19}. For small molecular weight Gd(III) complexes,
such as clinical agents, relaxivity is limited by fast rotation.
Therefore, many different approaches have been explored in the past in
order to slow down the motional dynamics of the chelates and increase
relaxivity. Slow rotation promoted by macromolecular systems can indeed
lead to a remarkable (5--10 fold) relaxivity improvement between
20 and 60~MHz. However, this effect vanishes at higher frequencies. Given
the great increase in detection sensitivity (and consequently of image
resolution) with magnetic field, there is a clear interest in
performing MRI at high fields (4.7--9.4~T, corresponding to
200--400~MHz proton Larmor frequency) especially for preclinical
studies, where the small animal size calls for high sensitivity. The
relaxivity optimization at these magnetic fields requires a different
strategy, in particular, appropriate fine-tuning of the rotational
motion by using medium-sized complexes instead of macromolecules. In
addition, the rigidity of the molecules is another key element in order to
exclude internal flexibility, which could be detrimental to
relaxivity gain. We have achieved this with various molecular design
approaches, such as the creation of a metallostar~\cite{20,21} or a
benzene-core  trimer~\cite{23,22}  (Figure~\ref{fig1}), both bearing
bis(hydrated) Gd(III) chelates to maximize the relaxivity. Indeed,
inner-sphere relaxivity is linearly proportional to the hydration
number; thus increasing $q$ has a direct effect, which is independent
of the magnetic field. Thanks to the combined effects of the optimal
molecular size, the rigidity, and the presence of two inner-sphere water
molecules, these complexes are endowed with remarkably high proton
relaxivities at high magnetic fields as compared to clinical agents 
(Figure~\ref{fig1}). This excellent relaxation efficiency is retained
in vivo as evidenced by MRI experiments on healthy
mice~\cite{23,20}. For both compounds, the MRI data have been further
complemented by biodistribution studies performed in
$\rmgamma$-scintigraphy imaging in rats using the $\mbox{}^{153}$Sm
analogue. Both MRI and $\rmgamma$-scintigraphy confirmed fast renal
elimination of the two compounds, leakage to the extracellular space in
the muscle tissue, and no permeation to the brain with intact
blood--brain barrier, all important for the development of
general-purpose MRI agents. 

In the search for higher MRI efficiency, many efforts have been also
dedicated to the optimization of the water exchange rate. While for
typical DOTA- or DTPA-type small molecular weight Gd(III) complexes, the
water exchange process is fast enough not to be limiting for proton
relaxivity, for slowly rotating macromolecular systems with optimized
rotational dynamics, water exchange can become the limiting 
parameter~\cite{24}. Consequently, it is important to design chelates
with accelerated water exchange. It has been shown that
nine-coordinate, monohydrated Gd(III) complexes undergo dissociative
water  exchange~\cite{19}, implying that the rate-determining step is
the dissociation of the leaving water molecule before the entry of 
the incoming water. For such water exchange processes, steric crowding
around the water binding site was found to be the key element 
determining the exchange rate. The steric crowding could be increased by
replacing an ethylene by a propylene bridge in the DTPA backbone 
(EPTPA)~\cite{25,26} or an acetate with a propionate pendant arm in
DOTA  (DO3A-prop)~\cite{27}, leading to two and one orders of magnitude
increase in the water exchange rate, respectively  (Scheme~\ref{sch1}).
Both of these approaches have been further combined with simultaneous
optimization of the rotational motion of the chelates. For instance, a
long hydrophobic chain has been attached to the ligand (EPTPAC16; 
Scheme~\ref{sch1}). This promotes the formation of micelles in aqueous
solution of the Gd(III) chelate, endowed with slow rotation and
consequently improved proton relaxivities~\cite{26,28}. Indeed, the
relaxivity of Gd(EPTPAC16) amounts to 
$9.1~\mathrm{mM}^{-1}{\cdot}\mathrm{s}^{-1}$ in the monomeric and to 
$20.6~\mathrm{mM}^{-1}{\cdot}\mathrm{s}^{-1}$ in the micellar form
(measured at 0.2~mM and 2.0~mM, below and above the critical micellar
concentration [cmc], respectively) as compared to 
$3.3~\mathrm{mM}^{-1}{\cdot}\mathrm{s}^{-1}$ for GdDTPA (20~MHz,
25~\textdegree C). Some of the complexes have been also successfully
evaluated in vivo in MRI experiments performed on 
rats~\cite{29}. 

\begin{scheme*}
\includegraphics{sc01}
\vspace*{.5pc}
\caption{\label{sch1}Accelerating water exchange for DOTA- and
DTPA-type Gd(III) complexes by increased steric crowding around the
water binding site. The water exchange rates, $k_{\mathrm{ex}}$,
indicated refer to 298~K.}
\end{scheme*}

\section{Design of lanthanide-based bimodal MRI and optical imaging
probes}\label{sec3} 
In biomedical imaging, there has been a continuous interest in
combining two (or more) detection modalities in bimodal (or multimodal)
approaches in order to ascertain the results~\cite{30}. Ideally, a
bimodal imaging probe should incorporate, within the same molecular
entity, detection capabilities in both modalities. Since lanthanide
ions have very similar coordination chemistry but highly versatile
magnetic and optical properties, lanthanide complexes provide an ideal
platform for the development of bimodal optical and MRI 
agents~\cite{31,32,33,34,35,36,37,38}. Indeed, several lanthanide ions
can have luminescence emission in the visible region or in the near-infrared
region (NIR), provided they are sensitized via an appropriate chromophore
that is able to absorb the exciting light and transfer the energy to
the lanthanide ion to generate characteristic lanthanide
luminescence~\cite{39}. The very sharp emission bands that are
independent of experimental conditions, the long emission lifetimes, or
the high resistance to photo-bleaching are all advantageous features of
luminescent lanthanide probes with respect to organic
fluorophores~\cite{40}. The same ligand can be applied to complex
Gd(III), adapted to MRI detection, and another luminescent lanthanide
ion for optical detection, and the two complexes are expected to have
an identical biodistribution, thereby allowing for straightforward
overlapping of the results obtained with the two different imaging
modalities. However, while the coordination of one inner-sphere water
molecule in the Gd(III) chelate is indispensable to MRI, it has
negative consequences for the optical properties of the luminescent
analogue given the non-radiative energy dissipation due to O--H
resonances. This has to be therefore compensated by efficient
sensitization of the luminescence using well-adapted chromophore
antennas. The situation is even more challenging with bishydrated
complexes, which provide increased relaxation efficacy for the Gd(III)
analogues or for lanthanide ions with near-infrared emission. 

We have investigated different ligand systems in the context of
developing lanthanide-based bimodal, optical, and MRI agents. For
instance, a pyrene derivative of the DO3A-prop ligand (DO3A-prop-pyr, 
Scheme~\ref{sch1}) has been complexed to Gd(III) as well as to the
near-infrared emitting Nd(III) and Yb(III) ions~\cite{28}. Here the
pyrene itself can be used as a fluorophore, but it was also
demonstrated that it can also sensitize the near-infrared luminescence
of Nd(III) and Yb(III). The aggregated micellar form of the Gd(III)
complex displays an impressive relaxivity 
($32~\mathrm{mM}^{-1}{\cdot}\mathrm{s}^{-1}$,  20~MHz, 25~\textdegree
C) resulting from the simultaneous optimization of the rotational
dynamics and of the water exchange. 

In another approach, the pyridine N-oxide coordinating function was
used to replace two acetate arms in \textit{trans} position of the DOTA
to obtain the DO2A-trans-(py$^{\mathrm{NO}}$)$^{2}$ ligand 
(Scheme~\ref{sch2}), and its Ln(III) complexes were investigated as
potential bimodal probes. Interestingly, the water exchange rate on the
Gd(III) analogue is only 50\% higher than that on GdDOTA despite the
increased steric hindrance induced by the two six-membered chelate
rings. This limited increase was attributed to the positive charge of
the chelate, which is known to slow down the dissociation of the leaving
water molecule at the rate-determining step. The luminescence
properties have been assessed for several lanthanide complexes (Eu(III)
and Tb(III) emitting in the visible, and Yb(III), Nd(III), Ho(III), and
Pr(III) emitting in the NIR region). Remarkably, the pyridine N-oxide was
found to be a very efficient chromophore antenna for all these metal
ions in aqueous solution, with a particularly interesting quantum yield
for Yb(III) (0.73\%  in H$_{2}$O)~\cite{41}. 

\begin{scheme}
\includegraphics{sc02}
\vspace*{.5pc}
\caption{\label{sch2}Chemical structure of the
DO2A-trans-(py$^{\mathrm{NO}})_{2}$ ligand.}
\vspace*{-.5pc}
\end{scheme}

\section{Metal complexes targeted at amyloid\newline peptides}\label{sec4}
Even though the ability to form amyloid fibrils is nowadays considered a
natural property of polypeptide chains, it has been recognized early on
that amyloid forms of certain proteins are characteristic of various
pathologies and can be considered relevant biomarkers for their
imaging detection. The most well-known examples are A$\rmbeta$ in the
context of Alzheimer's disease, amylin in type 2 diabetes mellitus, and
$\rmalpha$ synuclein in Parkinson's disease, just to name a few.
Amyloidogenesis involves all different processes corresponding to the
unfolding and misfolding of proteins that are not corrected by protein
quality control and degradation mechanisms, and thus lead to
misassembled, soluble or insoluble, toxic, and oligomeric or polymeric
fibrillary aggregates. The presence of these protein forms affects
cell or organ function, potentially leading to cell death.
Amyloidogenesis often occurs well before the clinical onset of 
diseases. Therefore, the detection of misassembled protein forms
represents an important goal for molecular imaging diagnosis of 
amyloid-related pathologies. 

Metal-based imaging agents capable of visualizing amyloid peptides have
been the focus of very intensive research for many years now, with
the most attention being directed at Alzheimer's disease by using nuclear
imaging techniques~\cite{42,43,44}. We have been long engaged in this
research via the development of potential imaging agents for
A$\rmbeta$ and amylin. Our probes are constructed in a modular way,
consisting of a macrocyclic, DOTA-type metal chelate, an amyloid
recognition moiety based on the benzothiazole derivative Pittsburg
compound B (PiB), and a linker between the two. The modularity allows
for a better understanding and control of the role of each element in
amyloid binding properties. The DOTA-type chelator makes the probes
easily adaptable to various imaging modalities, as it can efficiently
complex various metal ions suitable for MRI (Gd(III)), for optical
detection (luminescent lanthanides), and for nuclear imaging
(e.g.,  $\mbox{}^{111}$In for single-photon emission computed tomography
and  $\mbox{}^{68}$Ga for positron emission tomography [PET]). 

\begin{scheme*}
\vspace*{-.2pc}
\includegraphics{sc03}
\vspace*{.5pc}
\caption{\label{sch3}Amyloid-targeted PiB-derivative ligands.}
\vspace*{-.5pc}
\end{scheme*}

We have investigated the influence of different structural variations
on  peptide binding properties: the length and the chemical nature
of the linker, the orientation of the benzothiazole targeting unit, and
the charge of the metal complex. The first generation of compounds
(L1--L4,  Scheme~\ref{sch3})~\cite{42,45,46,48,49,50,51} contained a
short spacer between the metal chelate and the PiB, and their Gd(III)
complexes displayed dissociation constants ($K_{\mathrm{d}}$) in the range of
67--194~$\rmmu\mathrm{M}$ determined by surface plasmon
resonance (SPR) for the binding with $\mathrm{A}\rmbeta_{1\ndash 40}$
aggregates. Neutral complexes were found to have higher affinity than
negatively charged complexes. Overall, the decrease in affinity  was
around five orders of magnitude for these complexes as compared to the
PiB itself. 
\mbox{Proton} relaxivity measurements also evidenced binding to
amyloid  $\mathrm{A}\rmbeta_{1\ndash 40}$ as well as to human serum albumin
(HSA), yielding, respectively, two- and fourfold relaxivity increase
upon binding of GdL1 for instance. Ex vivo
immunohistochemistry revealed selective targeting of A$\rmbeta$ plaques
in human brain tissue. Octanol/water partition coefficients varied
between  ${-}$0.15 (for GdL1) and ${+}$0.32 (for GdL4), and such relatively
hydrophilic molecules do not readily cross the blood--brain barrier,
which would be important in the context of Alzheimer's disease. In vivo
brain uptake was assessed for the L1 ligand labeled with
$\mbox{}^{111}$In in wild-type mice and showed 0.36 and 0.5\%ID/g in
the cortex and in the cerebellum (at 2~min), respectively, and rapid clearance, which
might be sufficient for nuclear imaging if the probe is retained in the
Alzheimer brain. In vivo PET imaging using the
$\mbox{}^{68}$Ga-labeled analogues showed somewhat higher brain uptake
in transgenic Alzheimer mice than in control animals with
$\mbox{}^{68}$GaL2 but not with  $\mbox{}^{68}$GaL1. However,
autoradiography on human Alzheimer brain tissues failed to detect
amyloid deposits~\cite{48}. 

With the objective of increasing lipophilicity, a second family was
later designed by changing the orientation of the PiB and using longer
spacers (L5 and L6, Scheme~\ref{sch3}). Despite its higher
lipophilicity (log  $P_{\mathrm{oct}/\mathrm{water}}= 0.63)$,
$\mbox{}^{111}$InL5 did not have much enhanced brain uptake (0.12\%ID/g
at 30~min post-injection vs 0.07\%ID/g for $\mbox{}^{111}$InL2), which
was explained by its higher molecular weight~\cite{52}. 

Amylin fibrils, accumulated in pancreatic islets in the case of type 2
diabetes, represent a biomarker that is more accessible for the
imaging agent than A$\rmbeta$ in the brain. We have therefore assessed
the binding properties of GdL1, GdL2, GdL5, and GdL6 to amyloid amylin
as well, in comparison to  $\mathrm{A}\rmbeta_{1\ndash 40}$. These studies
have been performed by SPR at different peptide immobilization levels
on the SPR chips, standard  ${\sim}$4000 RU and very high ${\sim}$9000
RU, and the data were analyzed by fitting a 1:1 Langmuir isotherm. We
could show that the binding affinity increases when the metal complex
is placed further away from the peptide recognition unit
(Table~\ref{tab1}). In particular, in the case of GdL6 containing the
longest C10 spacer and when using high peptide immobilization levels
on the SPR chips, the data revealed nanomolar affinity for both
peptides  ($K_{\mathrm{d}}= 4.4$~nM and 4.5~nM for  $\mathrm{A}\rmbeta_{1\ndash 40}$ and
amylin, respectively), similar to that of the PiB alone. This was the
first time that such high affinity was reported for a metal chelate. On
the other hand, none of these systems have shown any notable
selectivity for one peptide over the other. 

\begin{table*}
\caption{\label{tab1}Dissociation constant ($K_{\mathrm{d}}$) determined by
fitting the SPR data to 1:1 Langmuir isotherms~\cite{52}\vspace*{-3pt}}
\begin{tabular}{cccc} 
\thead
$K_{\mathrm{d}}$ & $\mathrm{A}\rmbeta_{1\ndash 40}$ & Amylin & HSA \\
\endthead
GdL1 & 170~$\rmmu$M$^{\mathrm{c}}$ &
154~$\rmmu$M & 1100~$\rmmu$M$^{\mathrm{d}}$ \\
GdL2 & 71~$\rmmu$M  & 
8.3~$\rmmu$M & 1700~$\rmmu$M \\
GdL5 & 16~$\rmmu$M & 
17.2~$\rmmu$M & 300~$\rmmu$M \\ 
GdL6$^{\mathrm{a}}$ & 5~$\rmmu$M &
3~$\rmmu$M & 36~$\rmmu$M \\
GdL6$^{\mathrm{b}}$ & 
\parbox[t]{8.2pc}{\centering 4.4~nM \textit{(from data between 0 and 125~nM)}} & 
\parbox[t]{8.2pc}{\centering 4.5~nM \textit{(from data between 0 and 62.5~nM)}} & -
\vspace*{2pt}
\botline
\end{tabular}
\tabnote{${}^{\mathrm{a}}$Immobilization:  ${\sim}$4000 RU;
${}^{\mathrm{b}}$Immobilization: ${\sim}$9000 RU;
${}^{\mathrm{c}}$Measured for LaL1;
${}^{\mathrm{d}}$Measured by relaxometric titration.\vspace*{-3pt}}
\end{table*}

Due to their amphiphilic nature, all these LnL complexes themselves undergo
 important aggregation in aqueous solution. These
``micellization'' processes have been investigated over a large
concentration range, from micro- to millimolar, of amphiphilic LnL
chelates. For these studies, different techniques have been applied,
each of them adapted to a different concentration range: fluorescence,
UV--vis, and relaxometry. The results when combined revealed more than one
cmc value, implying that different
aggregation regimes exist over the entire concentration range. The
existence of different aggregation states of the LnL complexes proved
to be very important for their peptide binding affinity. Indeed, by
\mbox{carrying} out a Scatchard linearization analysis of the SPR data, we
could evidence different affinity regimes for each of the complexes as
indicated by clear breakpoints on the linearized Scatchard plots 
(Figure~\ref{fig2}). These regimes are characteristic of the complex
while they remain independent of the peptide. Most importantly, the
breakpoints correspond to the cmc values previously determined. These
findings evidence for the first time that the aggregation of the probe
itself is important for its binding interaction with the peptide. Our
data indicate that the monomer form has the highest affinity. The
interaction of the probes with HSA has been also
investigated by SPR. While all complexes bind HSA, the affinities are
one to two orders of magnitude lower towards $\mathrm{A}\rmbeta_{1\ndash
40}$ or amylin  (Table~\ref{tab1}). 

\begin{figure*}
\includegraphics{fig02}
\vspace*{-1pt}
\caption{\label{fig2}Top: SPR binding plots fitted as 1:1 Langmuir
isotherm functions for the interaction of GdL6 with
$\mathrm{A}\rmbeta_{1\ndash 40}$ (A, C) and amylin (B, D). Peptide
immobilization was 4000 RU (A, B) and 9000 RU (C, D). For C and D,
points recorded only at low concentrations are represented. E and F are
respectively the Scatchard linearization plots of A and B. Reproduced
with permission from \textit{Sens.\ Diagn.}~\cite{43}. Bottom: Sketch
of the aggregation of amyloid-targeted, PiB-derivative
probes and its influence on peptide binding affinities. Reproduced with
permission from \textit{Chem.\ Eur.\ J.}~\cite{53}.}
\vspace*{-1pt}
\end{figure*}

The presence of  complexes also influences the aggregation process
of the peptide as has been shown for $\mathrm{A}\rmbeta_{1\ndash
40}$. In the case of LnL1--L3, combined UV--vis spectroscopy, circular
dichroism, dynamic light scattering, and transmission electron
spectroscopy data indicated that the small differences between these
structurally similar molecules can promote different behavior as LnL1
and LnL3 accelerated while LnL2 inhibited the aggregation of 
$\mathrm{A}\rmbeta_{1\ndash 40}$~\cite{51}. Fluorescent assays using
thioflavin T (ThT) were later applied to compare the effects of GdL2,
GdL5, and GdL6. The intercalation of this fluorescent dye into the
$\rmbeta$-sheets of amyloid fibrils yields a fluorescence change that
can be used to follow the aggregation process. Aggregation was slower
in the presence of GdL5 or GdL6, and these studies also showed
competition of these complexes with ThT for the same binding sites on
the peptide fibrils~\cite{52}. 

Finally, the biodistribution of radiolabeled  ${}^{111}$InL5 and 
${}^{111}$InL6 has been assessed in wild-type mice. As for the previous
complexes, brain uptake was low. There was no specific organ retention
in these healthy mice. The probes distributed to the pancreas at an
interesting level (2.9\%ID/g and 3.7\%ID/g for ${}^{111}$InL5 and 
${}^{111}$InL6, respectively), which is very promising for potential
amylin imaging in diabetic pancreas~\cite{52}. 

\begin{scheme*}
\includegraphics{sc04}
\vspace*{.5pc}
\caption{\label{sch4}Synthesis of Mn(III) phthalocyanines.}
\vspace*{-.6pc}
\end{scheme*}

\section{Manganese complexes of tetrapyrrolic macrocycles as potential
contrast agents}\label{sec5} 
Although the use of Gd(III)-based MRI agents to improve image quality has
been a key element in the clinical success of MRI,
lately there have been concerns about the safety of these
Gd(III) complexes. First, a new and potentially lethal disease,
nephrogenic systemic fibrosis (NSF) was discovered mostly in kidney
impaired patients and linked to Gd injections~\cite{54}. It has been
rapidly understood that in the case of slow kidney elimination,
open-chain Gd(III) complexes endowed with lower kinetic inertness might
undergo partial dissociation in vivo, and the free Gd(III) ions
released are the cause of NSF disease. This has led to the
withdrawal of several linear Gd(III) chelates from clinical practice
and warnings issued by drug safety authorities concerning MRI
examinations in kidney impaired patients. In parallel, Gd deposits have
been also detected in human bones and brains following 
\mbox{Gd-enhanced} MRI,
though without toxic effects~\cite{55,56}. Furthermore, recent years have
witnessed increasing awareness about the presence of gadolinium not
only in wastewater but also in natural water~\cite{57}. All these
concerns promote important research towards the replacement of Gd by
more biocompatible paramagnetic metal ions. Most of the research in
this field has been directed towards Mn(II) complexes. 
\mbox{Although} Mn is more
paramagnetic in the ${+}$2 redox state $(S = 5/2)$, the high spin state
of the ${+}$3 redox form is also a good relaxation agent  $(S = 2)$.
Manganese is an essential metal ion that clearly alleviates toxicity
problems. Even if in a non-complexed form, Mn(II) or Mn(III) would be
also toxic at the doses required for MRI, implying that it has to be
chelated in thermodynamically stable and kinetically inert complexes.
Mn(III) is very well stabilized by tetrapyrrolic macrocycles, such as
porphyrins and phthalocyanines. Indeed, Mn(III) porphyrin complexes have
been explored early on as potential MRI agents, however, without
further clinical development~\cite{58}. It was demonstrated that they
can have interestingly high proton relaxivities, presenting a different
magnetic field dependence from that of small molecular weight Gd(III)
or Mn(II) chelates. Nevertheless, low water solubility and aggregation
in aqueous solution can often be a problem. 

As the first approach, we have reported the synthesis of biocompatible
manganese(III) phthalocyanines and their evaluation as potential
contrast agents~\cite{59}. The synthetic strategy is depicted in 
Scheme~\ref{sch4}. It starts with the functionalization of a
phthalonitrile with polyethylene glycol (PEG) or dimethylaminoethoxy
moieties by performing a nucleophilic substitution reaction between
these nucleophiles and 4-nitrophthalonitrile using ultrasound as an
alternative heating source, yielding \textbf{1a} and
\textbf{b}~\cite{60,61}. The condensation of \textbf{1a} or \textbf{1b}
phthalonitriles was performed using a mixture of DMAE/n-butanol as the
solvent and Zn(OAc)$_{2}$ as the template at room temperature and
100~\textdegree C, respectively, yielding the desired biocompatible
phthalocyanines \textbf{2a} and \textbf{2b} in ${\approx}$60\% yield.
Aiming at the preparation of manganese(III) phthalocyanines, the
demetalation of \textbf{3a} and \textbf{3b} was pursued in the reflux 
of pyridine and pyridinium HCl salt. The final step was the 
\mbox{complexation}
of free-base phthalocyanines \textbf{3a} and \textbf{3b}, with
Mn(OAc)$_{2}$ in DMF as the solvent, under air atmosphere.
Mn(III)-phthalocyanines \textbf{4a} and \textbf{4b} were obtained
following spontaneous oxidation of Mn(II) to Mn(III) by air oxygen in
36\% and 61\% overall yields. To turn the phthalocyanine \textbf{4b}
water soluble, alkylation with iodomethane was \mbox{additionally} performed.
Both phthalocyanines presented relatively low $r_{1}$ relaxivities 
($4.0\ndash5.7~\mathrm{mM}^{-1}{\cdot}\mathrm{s}^{-1}$ at 20~MHz) due
to aggregation in aqueous solution. No significant cytotoxicity to HeLa
cell cultures was observed after 2~h incubation and up to 2~mM
concentration. The reduction of Mn(III) to Mn(II) was also investigated
in both phthalocyanines, but the Mn(III) oxidation state proved to be
quite stable.

Building on the extensive experience on porphyrins in 
Coimbra~\cite{62,63,64} and on responsive MRI probes in 
Orl\'{e}ans~\cite{13,65,66}, we have designed a family of porphyrins with
the objective of creating Mn-based redox MRI probes~\cite{67}. 

\begin{figure}
\includegraphics{fig03}
\caption{\label{fig3}(a)~UV--vis reduction titration of 0.041~mM
Mn(III)-\textbf{7} with ascorbic acid, recorded in PBS (25~\textdegree
C, pH 7.4). Number of equivalents of ascorbic acid added: black, 0;  
red, 0.28; light blue, 0.57; light green, 0.85; pink, 6.4; 
dark  green, 11.4; dark blue, 25.3. (b)~UV--vis spectra of the reoxidation of
Mn(II)-\textbf{7} upon air 
exposure: black, Mn(II)-\textbf{7}  before exposure;  red, after 10 min
exposure; blue, after 4 h exposure; green, complete conversion to
Mn(III)-\textbf{7} (after 24~h exposure). (c)~Proton NMRD profiles
recorded at 298 K for Mn(III)-\textbf{7} (red) and Mn(II)-\textbf{7}
(blue). Adapted with permission from \textit{Dalton Trans.}\ 
(Ref.~\cite{67}).} 
\end{figure}

In living systems, the redox environments, both extra- and
intracellular are tightly regulated via the presence of different
biological redox couples, among which glutathione is a major actor
representing the highest concentration (1--11~mM in the intracellular
space). Many pathological states, including chronic inflammation,
ischemia, cancer, and so on, are associated with redox imbalance, which makes
the redox state an important biomarker for imaging detection~\cite{68}.

The switch of the Mn(III)/Mn(II) couple might offer an efficient way to
report on the tissue redox state~\cite{69,70,71,72,73}. The two forms have
different relaxation mechanisms obeying different magnetic field 
dependencies~\cite{72,74,75},  which can provide substantial relaxivity
differences with detectable signal change on MR images. Such a
redox MRI probe has to fulfill several requirements, such as rapid redox
transformation with a biologically relevant redox potential
accompanied by a significant relaxivity variation and stable
complexation of both Mn(II) and Mn(III) states. With respect to the
latter, porphyrins are quite interesting since they are among the very
few ligand systems capable of forming highly stable complexes with both
Mn(II) and Mn(III) ions. 

\begin{scheme*}
\includegraphics{sc05}
\vspace*{.5pc}
\caption{\label{sch5}Synthesis of PEGylated Mn(III) porphyrin.}
\vspace*{-.3pc}
\end{scheme*}

The first porphyrin that we developed as a potential
redox-responsive MRI contrast agent was compound \textbf{7} 
(Scheme~\ref{sch5})~\cite{67}. Its synthesis started by 
PEGylation of free-base porphyrin \textbf{5} through \mbox{nucleophilic}
aromatic substitution using NaH as the base and DMF as the solvent, yielding
\textbf{6} with 65\% isolated yield. The complexation reaction was
performed in sodium acetate/acetic acid buffer with Mn(OAc)$_{2}$ as the
metal salt at  120~\textdegree C. The final Mn(III) porphyrin
\textbf{7} was purified using extraction processes and was isolated
with 82\% yield. This new biocompatible fluorinated manganese porphyrin
is capable of stabilizing both Mn(III) and Mn(II) oxidation states, and
in aqueous solution, Mn(III)/Mn(II)-\textbf{7} is able to undergo
reversible reduction/oxidation reactions using ascorbic acid for the
reduction and O$_{2}$ for the oxidation. With
ascorbic acid as the 
\mbox{biological} reductant, a rate constant k$_{2}$ of 
$46.1~\mathrm{M}^1{\cdot}\mathrm{s}^{-1}$ was obtained, which
corresponds to a half-life of few minutes in blood plasma, where
ascorbic acid concentration reaches  20--40~$\rmmu$M. In addition, it
was also observed that with glutathione, it was not possible to achieve the
reduction of Mn(III) since glutathione carboxylate groups coordinate
to the metal in the axial position as was demonstrated by 
nuclear magnetic resonance (NMR) data.
Upon reduction of Mn(III) to Mn(II), a large increase in  proton
relaxivity was observed at low and medium magnetic fields
(${<}$60~MHz). Although the difference vanishes at higher fields with a
reversion of the order for very high fields, even at 80 MHz, the
relaxivity of the reduced form remains 30\% higher than that of the
oxidized form, which would correspond in MR images to remarkable
``turn-on'' response to a reducing environment  (Figure~\ref{fig3}).

\begin{scheme*}
\includegraphics{sc06}
\vspace*{.5pc}
\caption{\label{sch6}Synthesis of fluorinated Mn(III) porphyrin.}
\vspace*{-2pt}
\end{scheme*}

\begin{figure*}
\includegraphics{fig04}
\vspace*{2pt}
\caption{\label{fig4}(a)~Most stable structure obtained through DFT
optimization at the B3LYP/6-31G(d,p) level of Mn(III)-\textbf{11}.
(b)~$\mbox{}^{19}$F MRI phantom images acquired at 1.1~mM
Mn(III)/(II)-\textbf{11} concentration in DMSO.  (c)~Monitoring
reoxidation of Mn(II)-\textbf{11}  $(\mathrm{c}_{\mathrm{MnL}}=
1.6~\mathrm{mM})$ by air oxygen; signal-to-noise ratio (SNR; with
respect to the Mn(III)-\textbf{11} peak) as a function of oxidized
Mn(III)-\textbf{11} content (\%). B: 7~T;   TR: 60~ms; TE: 1.3~ms;
FA: 90\textdegree; NA: 256; acquisition time: 8~min 11~s.
Reproduced with permission from Ref.~\cite{76}.}
\end{figure*}

The presence of  ${}^{19}$F atoms on Mn porphyrins, which is
important to modulate their redox potential allowing reduction by
ascorbate, opens further opportunities in MRI detection. ${}^{19}$F MRI
has complementary advantages to classical ${}^{1}$H MRI, including the
lack of any background signal in biological systems (hotspot imaging)
or a wide range of accessible ${}^{19}$F chemical shifts allowing for
multiplex detection. In the case of responsive probes, monitoring
biomarker-dependent variations in two independent imaging modalities
can further ascertain the findings. 
\looseness=-1

With this objective, we have recently reported a novel fluorinated
manganese porphyrin capable of providing dual  ${}^{1}$H relaxivity and
${}^{19}$F MRI response to redox  changes~\cite{76}. The synthesis of
this porphyrin started by  the condensation of
4-(trifluoromethyl)benzaldehyde \textbf{8} with $1H$-pyrrole
\textbf{9} in a mixture of acetic acid/nitrobenzene, using NaY zeolite
as the Lewis acid  (Scheme~\ref{sch6}). Porphyrin \textbf{10} was obtained
in 20\% isolated yield. Complexation with manganese was performed using
the strategy described above, giving \textbf{11} in 85\% yield with an HPLC
purity of 98\%. By using experimental methods and 
density functional theory (DFT)
calculations, porphyrin \textbf{11} was completely characterized and
presented properties  that made it a good starting point for the
future development of ${}^{19}$F redox contrast agents. Despite its low
solubility in aqueous medium, this fluorinated Mn porphyrin evidenced 
${}^{1}$H relaxometric and ${}^{19}$F NMR properties in
DMSO-d$_{6}$/H$_{2}$O, which are interesting for differential redox MRI
detection. DFT calculations and experimental ${}^{19}$F relaxation data
allowed us to conclude that an Mn--F distance of 9.7--10~\AA{}, as in
Mn(III)-\textbf{11}, enables adequate paramagnetic effect of Mn(III) on
 ${}^{19}$F $T_{1}$ and  $T_{2}$ relaxation times, providing
sufficiently fast longitudinal relaxation without excessive
line broadening. Similarly to the previous porphyrin \textbf{7},
porphyrin \textbf{11} was also readily reduced in the presence of
ascorbic acid, evidenced by the broadening of the ${}^{19}$F NMR signal
for the Mn(II) form. By using phantom ${}^{19}$F MR images, a clear
decrease in MRI signal intensity was observed upon reduction from
Mn(III) to Mn(II), which was subsequently regenerated upon reoxidation
in air within 24~h  (Figure~\ref{fig4}).

\section{Conclusion}\label{sec6}
The collaborative work between the research groups at the Centre of
Molecular Biophysics, CNRS, Orl\'{e}ans in France and the 
CQC-IMS, Department
of Chemistry, University of Coimbra in Portugal has successfully
contributed to addressing major challenges in the development of MRI
agents over the past 20~years. Among the most significant recent
achievements, a large family of imaging agents targeted at amyloid
peptides have been designed and their in vitro characterization and in
vivo preclinical imaging investigation have yielded novel insights into the
understanding of amyloid binding properties of metal complexes. More
recently, redox-responsive probes based on Mn(III)/Mn(II) porphyrin
complexes have been developed with detection potential of the tissue redox
state in both  ${}^{1}$H and ${}^{19}$F MRI. The complementary
expertise of the two groups in the physical chemistry of MRI agents and
in the synthetic chemistry of porphyrins is a major asset for the
success of future work. 

\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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