# Towards more efficient, more specific, and safer MRI contrast agents: a Portuguese-French collaborative journey

Sara Martinho Almeida Pinto, Sara Lacerda, Jean-François Morfin, Rafael Tiago Aroso, Daniela Sofia de Sousa Teixeira, Zoltán Garda, Maria Miguens Pereira, Carlos Frederico Gusmão Campos Geraldes, Éva Tóth

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Sara Martinho Almeida Pinto, Sara Lacerda, Jean-François Morfin, Rafael Tiago Aroso, Daniela Sofia de Sousa Teixeira, et al. Towards more efficient, more specific, and safer MRI contrast agents: a Portuguese-French collaborative journey. Comptes Rendus. Chimie, 2025, 28 (G1), pp.95-110. 10.5802/crchim.363. hal-05184946

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Comptes Rendus
Chimie

Sara Martinho Almeida Pinto, Sara Lacerda, Jean-François Morfin, Rafael Tiago Aroso, Daniela Sofia de Sousa Teixeira, Zoltán Garda, Maria Miguens Pereira, Carlos Frederico Gusmão Campos Geraldes and Éva Tóth

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

Volume 28 (2025), p. 95-110

Online since: 20 January 2025

https://doi.org/10.5802/crchim.363

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# Towards more efficient, more specific, and safer MRI contrast agents: a Portuguese-French collaborative journey

Sara Martinho Almeida Pinto<sup>®,a</sup>, Sara Lacerda<sup>®,b</sup>, Jean-François Morfin<sup>b</sup>, Rafael Tiago Aroso<sup>®,a</sup>, Daniela Sofia de Sousa Teixeira<sup>®,a,b</sup>, Zoltán Garda<sup>®,b</sup>, Maria Miguens Pereira<sup>*,®,a</sup>, Carlos Frederico Gusmão Campos Geraldes<sup>*,®,a</sup> and Éva Tóth<sup>*,®,b</sup>

<sup>a</sup> Coimbra Chemistry Centre - Institute of Molecular Sciences (CQC-IMS), Chemistry Department of the University of Coimbra, Portugal

<sup>b</sup> Centre de Biophysique Moléculaire, CNRS - UPR 4301, Université d'Orléans, Orléans, France

E-mails: mmpereira@qui.uc.pt (M. M. Pereira), geraldes@ci.uc.pt (C. F. G. C. Geraldes), eva.jakabtoth@cnrs.fr (É. Tóth)

**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 Orléans, 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 ¹H detection but also in ¹⁹F MRI.

**Keywords.** Magnetic resonance imaging, Contrast agents, Lanthanide, Manganese, Porphyrins.

Manuscript received 26 July 2024, accepted 18 November 2024.

## 1. Introduction

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 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 [1-3]. 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 (T¹) and transverse (T²) relaxation properties of

<footnote>* Corresponding authors</footnote>
these protons are exploited to produce detailed im-
ages of internal structures [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 inten-
sities. However, the inherent contrast is often in-
sufficient for accurate diagnosis, and therefore con-
trast enhancing agents are needed. These param-
agnetic or superparamagnetic drugs can reduce the
longitudinal and transverse relaxation times of the
surrounding water protons, thereby considerably im-
proving the diagnostic quality of the images [4–6].

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 morphologi-
cal and functional abnormalities, enhance blood ves-
sels in MR angiography, and to assess blood-brain
barrier degradation [7,8].

In recent years, with the overall aims of improv-
ing sensitivity, specificity, and safety of MRI con-
trast agents to promote earlier and more reliable
detection of diseases such as cancer and neurode-
generative disorders, research has been pursued in
several domains: (a) increasing the relaxation effi-
ciency of the complexes [6]; (b) addressing contrast
agents to specific biological targets [3,9]; (c) detecting
variations in tissue physical-chemical properties like
pH, temperature, cation concentration, and redox
state with smart probes [10–13]; and (d) substitut-
ing Gd(III) with more biocompatible paramagnetic
metal ions such as Mn(II), Mn(III), and Fe(III) [14–
18]. Coordination chemistry is fundamental to ad-
dressing these challenges. Within this general con-
text, this review aims to focus on the collaborative
achievements between research groups in the Cen-
tre of Molecular Biophysics, CNRS, Orléans and in
the Coimbra Chemistry Centre - Institute of Molec-
ular 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

extended to the design of MRI agents with increased
efficiency or various targeting capabilities. In partic-
ular, a lot of effort has been dedicated to the visu-
alization 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 fo-
cus. Finally, detection has been also extended to ¹⁹F
MRI, which offers complementary advantages over
classical ¹H MRI. The excellence of this collaborative
research, leading to 36 joint publications altogether,
was recognized by the Portuguese and French Acad-
emies of Sciences with the attribution of the Mariano
Gago Prize in 2022. This minireview illustrates, with-
out the intention to be exhaustive, some of the main
achievements of the Coimbra-Orléans collaboration
via a selected number of representative examples.

## 2. Strategies to increase the efficiency of MRI agents

The MRI efficiency of a paramagnetic metal chelate
is expressed by its proton relaxivity, r₁, defined as the
paramagnetic enhancement of the longitudinal wa-
ter proton relaxation rate referred to 1 mM concen-
tration. Proton relaxivity is the sum of inner- and
outer-sphere contributions originating from dipolar
interactions between the electron spin of the para-
magnetic 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 deter-
mined 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
(kex), the rotational correlation time of the chelate
(τR), and the electron spin relaxation times of the
metal ion (T₁₂e) [20]. For small molecular weight
Gd(III) complexes, such as clinical agents, relaxiv-
ity is limited by fast rotation. Therefore, many dif-
ferent approaches have been explored in the past in
order to slow down the motional dynamics of the
chelates and increase relaxivity. Slow rotation pro-
moted by macromolecular systems can indeed lead
to a remarkable (5–10 fold) relaxivity improvement
between 20 and 60 MHz. However, this effect van-
ishes at higher frequencies. Given the great increase
<figure><img src="image_3.png" /><figcaption>**Figure 1.** (a) Structure of the metallostar and of the Gd₃L1 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 Gd₃L1 injections at 8 mmol·Gd·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 Gd₃L1 due to its threefold higher relaxivity (adapted with permission from *Contrast Media Mol. Imag.* [19]).</figcaption></figure>

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 [21,22] or a benzene-core trimer [19,23] (Figure 1), 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 1). This excellent relaxation efficiency is retained in vivo as evidenced by MRI experiments on healthy mice [19,21]. For both compounds, the MRI data have been further complemented by biodistribution studies performed in γ-scintigraphy imaging in rats using the ¹⁵³Sm analogue. Both MRI and γ-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 ef-
forts have been also dedicated to the optimization
of the water exchange rate. While for typical DOTA-
or DTPA-type small molecular weight Gd(III) com-
plexes, 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 limit-
ing parameter [24]. Consequently, it is important
to design chelates with accelerated water exchange.
It has been shown that nine-coordinate, monohy-
drated Gd(III) complexes undergo dissociative wa-
ter exchange [20], implying that the rate-determining
step is the dissociation of the leaving water mole-
cule 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 el-
ement determining the exchange rate. The steric
crowding could be increased by replacing an ethy-
lene by a propylene bridge in the DTPA backbone
(EPTPA) [25,26] or an acetate with a propionate pend-
dant arm in DOTA (DO3A-prop) [27], leading to two
and one orders of magnitude increase in the water
exchange rate, respectively (Scheme 1). Both of these
approaches have been further combined with simul-
taneous optimization of the rotational motion of the
chelates. For instance, a long hydrophobic chain has
been attached to the ligand (EPTPAC16; Scheme 1).
This promotes the formation of micelles in aqueous
solution of the Gd(III) chelate, endowed with slow
rotation and consequently improved proton relaxiv-
ities [26,28]. Indeed, the relaxivity of Gd(EPTPAC16)
amounts to 9.1 $mM^{-1} \cdot s^{-1}$ in the monomeric and to
$20.6 \ mM^{-1} \cdot s^{-1}$ in the micellar form (measured at
0.2 mM and 2.0 mM, below and above the critical
micellar concentration [cmc], respectively) as com-
pared to $3.3 \ mM^{-1} \cdot s^{-1}$ for GdDTPA (20 MHz, 25 °C).
Some of the complexes have been also successfully
evaluated in vivo in MRI experiments performed on
rats [29].

### 3. Design of lanthanide-based bimodal MRI and optical imaging probes

In biomedical imaging, there has been a continu-
ous interest in combining two (or more) detection
modalities in bimodal (or multimodal) approaches
in order to ascertain the results [30]. Ideally, a bi-
modal imaging probe should incorporate, within the

same molecular entity, detection capabilities in both
modalities. Since lanthanide ions have very simi-
lar coordination chemistry but highly versatile mag-
netic and optical properties, lanthanide complexes
provide an ideal platform for the development of bi-
modal optical and MRI agents [31–38]. Indeed, sev-
eral 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 gen-
erate characteristic lanthanide luminescence [39].
The very sharp emission bands that are independent
of experimental conditions, the long emission life-
times, or the high resistance to photo-bleaching are
all advantageous features of luminescent lanthanide
probes with respect to organic fluorophores [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 biodis-
tribution, thereby allowing for straightforward over-
lapping 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 con-
sequences for the optical properties of the lumines-
cent analogue given the non-radiative energy dissi-
pation due to O–H resonances. This has to be there-
fore compensated by efficient sensitization of the lu-
minescence using well-adapted chromophore anten-
nas. The situation is even more challenging with
bishydrated complexes, which provide increased re-
 laxation efficacy for the Gd(III) analogues or for lan-
 thanide 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 de-
rivative of the DO3A-prop ligand (DO3A-prop-pyr,
Scheme 1) has been complexed to Gd(III) as well
as to the near-infrared emitting Nd(III) and Yb(III)
ions [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 $mM^{-1} \cdot s^{-1}$, 20 MHz, 25 °C) resulting from the si-
multaneous optimization of the rotational dynamics
and of the water exchange.
<figure><img src="image_4.png" /></figure>

$$ \begin{gathered} [\text{Gd(DOTA)(H}_2\text{O})]^- \\ k_{\text{ex}}^{298} = 4.1 \times 10^6 \text{ s}^{-1} \end{gathered} $$

<figure><img src="image_5.png" /></figure>

$$ \begin{gathered} R = H \quad [\text{Gd(DO3A-prop)(H}_2\text{O})]^- \\ k_{\text{ex}}^{298} = 61 \times 10^6 \text{ s}^{-1} \end{gathered} $$

$$ \begin{gathered} R = \text{NH}_2 \quad [\text{Gd(DO3A-propNH}_2\text{)(H}_2\text{O})]^- \\ k_{\text{ex}}^{298} = 51 \times 10^6 \text{ s}^{-1} \end{gathered} $$

<figure><img src="image_6.png" /></figure>

$$ \begin{gathered} R = \text{O-CH}_2\text{CH}_2\text{NH}^- \quad [\text{Gd(DO3A-amide-prop)(H}_2\text{O})]^- \\ k_{\text{ex}}^{298} = 40 \times 10^6 \text{ s}^{-1} \end{gathered} $$

<figure><img src="image_7.png" /></figure>

$$ \begin{gathered} R = \text{O-CH}_2\text{CH}_2\text{NH-CH}_2\text{CH}_2\text{CH}_2\text{NH}^- \quad [\text{Gd(DO3A-prop-pyr)(H}_2\text{O})]^- \\ k_{\text{ex}}^{298} = 62 \times 10^6 \text{ s}^{-1} \end{gathered} $$

<figure><img src="image_8.png" /></figure>

$$ \begin{gathered} [\text{Gd(DTPA)(H}_2\text{O})]^{2-} \\ k_{\text{ex}} = 4.1 \times 10^6 \text{ s}^{-1} \end{gathered} $$

<figure><img src="image_9.png" /></figure>

$$ \begin{gathered} R = H \quad [\text{Gd(EPTPA)(H}_2\text{O})]^{2-} \\ k_{\text{ex}} = 330 \times 10^6 \text{ s}^{-1} \end{gathered} $$

$$ \begin{gathered} R = \text{CH}_2\text{-OH} \quad [\text{Gd(EPTPACH}_2\text{OH)(H}_2\text{O})]^{2-} \\ k_{\text{ex}} = 88 \times 10^6 \text{ s}^{-1} \end{gathered} $$

$$ \begin{gathered} R = \text{CH}_2\text{-O-CO-C}_{15}\text{H}_{31} \quad [\text{Gd(EPTPAC16)(H}_2\text{O})]^{2-} \\ k_{\text{ex}} = 170 \times 10^6 \text{ s}^{-1} \end{gathered} $$

<caption>**Scheme 1.** Accelerating water exchange for DOTA- and DTPA-type Gd(III) complexes by increased steric crowding around the water binding site. The water exchange rates, $k_{\text{ex}}$, indicated refer to 298 K.</caption>
<figure><img src="image_10.png" /></figure>

**DO2A-*trans*-(*py*NO)₂**

**Scheme 2.** Chemical structure of the DO2A-*trans*-(*py*NO)₂ ligand.

In another approach, the pyridine N-oxide co-
ordinating function was used to replace two ac-
etate arms in *trans* position of the DOTA to obtain
the DO2A-*trans*-(*py*NO)² ligand (Scheme 2), and its
Ln(III) complexes were investigated as potential bi-
modal 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 re-
gion). Remarkably, the pyridine N-oxide was found
to be a very efficient chromophore antenna for all
these metal ions in aqueous solution, with a partic-
ularly interesting quantum yield for Yb(III) (0.73% in
H₂O) [41].

**4. Metal complexes targeted at amyloid peptides**

Even though the ability to form amyloid fibrils is
nowadays considered a natural property of polypep-
tide chains, it has been recognized early on that
amyloid forms of certain proteins are characteristic
of various pathologies and can be considered rele-
vant biomarkers for their imaging detection. The
most well-known examples are Aβ in the context of
Alzheimer's disease, amylin in type 2 diabetes mel-
litus, and α synuclein in Parkinson's disease, just to

name a few. Amyloidogenesis involves all different
processes corresponding to the unfolding and mis-
folding of proteins that are not corrected by pro-
tein quality control and degradation mechanisms,
and thus lead to misassembled, soluble or insoluble,
toxic, and oligomeric or polymeric fibrillary aggre-
gates. 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 misas-
sembled 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 inten-
sive research for many years now, with the most at-
tention being directed at Alzheimer's disease by using
nuclear imaging techniques [42–44]. We have been
long engaged in this research via the development
of potential imaging agents for Aβ and amylin. Our
probes are constructed in a modular way, consisting
of a macrocyclic, DOTA-type metal chelate, an amy-
loid recognition moiety based on the benzothiazole
derivative Pittsburg compound B (PiB), and a linker
between the two. The modularity allows for a bet-
ter understanding and control of the role of each ele-
ment in amyloid binding properties. The DOTA-type
chelator makes the probes easily adaptable to vari-
ous imaging modalities, as it can efficiently complex
various metal ions suitable for MRI (Gd(III)), for opti-
cal detection (luminescent lanthanides), and for nu-
clear imaging (e.g., ¹¹¹In for single-photon emission
computed tomography and ⁶⁸Ga for positron emis-
sion tomography [PET]).

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 gener-
ation of compounds (L1–L4, Scheme 3) [42,45–50]
contained a short spacer between the metal chelate
and the PiB, and their Gd(III) complexes displayed
dissociation constants (K_d) in the range of 67–194 µM
determined by surface plasmon resonance (SPR) for
the binding with Aβ₁⁻₄₀ aggregates. Neutral com-
plexes were found to have higher affinity than neg-
atively charged complexes. Overall, the decrease in
affinity was around five orders of magnitude for these
complexes as compared to the PiB itself. Proton
<figure><img src="image_11.png" /><figcaption>**Scheme 3.** Amyloid-targeted PiB-derivative ligands.</figcaption></figure>

relaxivity measurements also evidenced binding to amyloid Aβ₁-40 as well as to human serum albumin (HSA), yielding, respectively, two- and four-fold relaxivity increase upon binding of GdL1 for instance. Ex vivo immunohistochemistry revealed selective targeting of Aβ 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 ¹¹¹In 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 ⁶⁸Ga-labeled analogues showed somewhat higher brain uptake in transgenic Alzheimer mice than in control animals with ⁶⁸GaL2 but not with ⁶⁸GaL1. However, autoradiography on human Alzheimer brain tissues failed to detect amyloid deposits [47].

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 3). Despite its higher lipophilicity (log P<sub>oct/water</sub> = 0.63), ¹¹¹InL5 did not have much enhanced brain uptake (0.12%ID/g at 30 min post-injection vs 0.07%ID/g for ¹¹¹InL2), which was explained by its higher molecular weight [51].

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 Aβ in the brain. We have therefore assessed the binding

properties of GdL1, GdL2, GdL5, and GdL6 to amyloid amylin as well, in comparison to Aβ₁-40. These studies have been performed by SPR at different peptide immobilization levels on the SPR chips, standard ~4000 RU and very high ~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 1). 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<sub>d</sub> = 4.4 nM and 4.5 nM for Aβ₁-40 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.

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 carrying out a Scatchard linearization analysis of the SPR data, we could evidence different affinity regimes for each
<table><caption>**Table 1.** Dissociation constant ($K_d$) determined by fitting the SPR data to 1:1 Langmuir isotherms [51]</caption><thead><tr><th>K<sub>d</sub></th><th>Aβ<sub>1–40</sub></th><th>Amylin</th><th>HSA</th></tr></thead><tbody><tr><td>GdL1</td><td>170 μM<sup>c</sup></td><td>154 μM</td><td>1100 μM<sup>d</sup></td></tr><tr><td>GdL2</td><td>71 μM</td><td>8.3 μM</td><td>1700 μM</td></tr><tr><td>GdL5</td><td>16 μM</td><td>17.2 μM</td><td>300 μM</td></tr><tr><td>GdL6<sup>a</sup></td><td>5 μM</td><td>3 μM</td><td>36 μM</td></tr><tr><td>GdL6<sup>b</sup></td><td>4.4 nM (from data<br>between 0 and 125 nM)</td><td>4.5 nM (from data<br>between 0 and 62.5 nM)</td><td>-</td></tr></tbody></table>

<sup>a</sup>Immobilization: ~4000 RU; <sup>b</sup>Immobilization: ~9000 RU; <sup>c</sup>Measured for LaL1; <sup>d</sup>Measured by relaxometric titration.

of the complexes as indicated by clear breakpoints on the linearized Scatchard plots (Figure 2). 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 Aβ<sub>1–40</sub> or amylin (Table 1).

The presence of complexes also influences the aggregation process of the peptide as has been shown for Aβ<sub>1–40</sub>. 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 Aβ<sub>1–40</sub> [50]. 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 β-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 [51].

Finally, the biodistribution of radiolabeled <sup>111</sup>InL5 and <sup>111</sup>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 <sup>111</sup>InL5 and <sup>111</sup>InL6, respectively), which is very promising for potential amylin imaging in diabetic pancreas [51].

**5. Manganese complexes of tetrapyrrolic macrocycles as potential contrast agents**

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 [53]. 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 Gd-enhanced MRI, though without toxic effects [54,55]. Furthermore, recent years have witnessed increasing awareness about the presence of gadolinium not only in wastewater but also in natural water [56]. 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. Although Mn is more paramagnetic in the +2 redox
<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /><figcaption>**Figure 2.** Top: SPR binding plots fitted as 1:1 Langmuir isotherm functions for the interaction of GdL6 with Aβ₁-40 (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 *Sens. Diagn.* [43]. Bottom: Sketch of the aggregation of amyloid-targeted, PiB-derivative probes and its influence on peptide binding affinities. Reproduced with permission from *Chem. Eur. J.* [52].</figcaption></figure>

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
<figure><img src="image_14.png" /><figcaption>**Scheme 4.** Synthesis of Mn(III) phthalocyanines.</figcaption></figure>

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 [57]. 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 [58]. The synthetic strategy is depicted in Scheme 4. 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 ultra-

sound as an alternative heating source, yielding **1a** and **b** [59,60]. The condensation of **1a** or **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 °C, respectively, yielding the desired biocompatible phthalocyanines **2a** and **2b** in ≈60% yield. Aiming at the preparation of manganese(III) phthalocyanines, the demetalation of **3a** and **3b** was pursued in the reflux of pyridine and pyridinium HCl salt. The final step was the complexation of free-base phthalocyanines **3a** and **3b**, with Mn(OAc)₂ in DMF as the solvent, under air atmosphere. Mn(III)-phthalocyanines **4a** and **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 **4b** water soluble, alkylation with iodomethane was additionally performed. Both phthalocyanines presented relatively low r₁ relaxivities (4.0–5.7 mM⁻¹·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 [61–63] and on responsive MRI probes in Orléans [13, 64, 65], we have designed a family of porphyrins with the objective of creating Mn-based redox MRI probes [66].

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 [67].

The switch of the Mn(III)/Mn(II) couple might offer an efficient way to report on the tissue redox state [68–72]. The two forms have different relaxation mechanisms obeying different magnetic field dependencies [71, 73, 74], 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.

The first porphyrin that we developed as a potential redox-responsive MRI contrast agent was compound **7** (Scheme 5) [66]. Its synthesis started by PEGylation of free-base porphyrin **5** through nucleophilic aromatic substitution using NaH as the base and DMF as the solvent, yielding **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 °C. The final Mn(III) porphyrin **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

<figure><img src="image_15.png" /></figure>

**Figure 3.** (a) UV-vis reduction titration of 0.041 mM Mn(III)-7 with ascorbic acid, recorded in PBS (25 °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)-7 upon air exposure: black, Mn(II)-7 before exposure; red, after 10 min exposure; blue, after 4 h exposure; green, complete conversion to Mn(III)-7 (after 24 h exposure). (c) Proton NMRD profiles recorded at 298 K for Mn(III)-7 (red) and Mn(II)-7 (blue). Adapted with permission from *Dalton Trans.* (Ref. [66]).
<figure><img src="image_16.png" /><figcaption>**Scheme 5.** Synthesis of PEGylated Mn(III) porphyrin.</figcaption></figure>

Mn(II) oxidation states, and in aqueous solution, Mn(III)/Mn(II)-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 biological reductant, a rate constant $k_2$ of 46.1 $M^{-1}·s^{-1}$ was obtained, which corresponds to a half-life of few minutes in blood plasma, where ascorbic acid concentration reaches 20–40 µM. 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 3).

The presence of ¹⁹F atoms on Mn porphyrins, which is important to modulate their redox potential allowing reduction by ascorbate, opens further opportunities in MRI detection. ¹⁹F MRI has complementary advantages to classical ¹H MRI, including the lack of any background signal in biological
<figure><img src="image_17.png" /><figcaption>**Scheme 6.** Synthesis of fluorinated Mn(III) porphyrin.</figcaption></figure>

systems (hotspot imaging) or a wide range of acces-
sible ¹⁹F chemical shifts allowing for multiplex de-
tection. In the case of responsive probes, monitoring
biomarker-dependent variations in two indepen-
dent imaging modalities can further ascertain the
findings.

With this objective, we have recently reported
a novel fluorinated manganese porphyrin capable
of providing dual ¹H relaxivity and ¹⁹F MRI re-
sponse to redox changes [75]. The synthesis of
this porphyrin started by the condensation of 4-
(trifluoromethyl)benzaldehyde 8 with 1*H*-pyrrole 9
in a mixture of acetic acid/nitrobenzene, using NaY
zeolite as the Lewis acid (Scheme 6). Porphyrin 10

was obtained in 20% isolated yield. Complexation
with manganese was performed using the strategy
described above, giving **11** in 85% yield with an HPLC
purity of 98%. By using experimental methods and
density functional theory (DFT) calculations, por-
phyrin **11** was completely characterized and pre-
sented properties that made it a good starting point
for the future development of ¹⁹F redox contrast
agents. Despite its low solubility in aqueous medium,
this fluorinated Mn porphyrin evidenced ¹H relax-
ometric and ¹⁹F NMR properties in DMSO-d₆/H₂O,
which are interesting for differential redox MRI de-
tection. DFT calculations and experimental ¹⁹F re-
laxation data allowed us to conclude that an Mn–F
<figure><img src="image_18.png" /><figcaption>**Figure 4.** (a) Most stable structure obtained through DFT optimization at the B3LYP/6-31G(d,p) level of Mn(III)-11. (b) $^{19}$F MRI phantom images acquired at 1.1 mM Mn(III)/(II)-11 concentration in DMSO. (c) Monitoring reoxidation of Mn(II)-11 ($c_{MnL}$ = 1.6 mM) by air oxygen; signal-to-noise ratio (SNR; with respect to the Mn(III)-11 peak) as a function of oxidized Mn(III)-11 content (%). B: 7 T; TR: 60 ms; TE: 1.3 ms; FA: 90°; NA: 256; acquisition time: 8 min 11 s. Reproduced with permission from Ref. [75].</figcaption></figure>

distance of 9.7–10 Å, as in Mn(III)-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 7, porphyrin 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 4).

**6. Conclusion**

The collaborative work between the research groups at the Centre of Molecular Biophysics, CNRS, Orléans 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.

**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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