# Mn 2+ Bispidine Complexes as Potential Magnetic Resonance Imaging Agents: Noncoordinating Peripheral Functional Groups Affect Structural, Kinetic, and Relaxation Properties

Maryame Sy, Daouda Ndiaye, Hervé Meudal, Loïc J Charbonnière, Aline M Nonat, Éva Tóth

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Maryame Sy, Daouda Ndiaye, Hervé Meudal, Loïc J Charbonnière, Aline M Nonat, et al. . Mn 2+ Bispidine Complexes as Potential Magnetic Resonance Imaging Agents: Noncoordinating Peripheral Functional Groups Affect Structural, Kinetic, and Relaxation Properties. ChemistryEurope, 2025, 10.1002/ceur.202500263 . hal-05348993

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# Mn²⁺ Bispidine Complexes as Potential Magnetic Resonance Imaging Agents : Noncoordinating Peripheral Functional Groups Affect Structural, Kinetic, and Relaxation Properties

Maryame Sy, Daouda Ndiaye, Hervé Meudal, Loïc J. Charbonnière,* Aline M. Nonat,* and Éva Tóth*

Mn²⁺ bispidine complexes combine unprecedented kinetic inertness with high relaxivity and are under scrutiny as potential magnetic resonance imaging (MRI) probes. This study demonstrates that, beyond the rigid, preorganized bispidine structure and the first coordination sphere of the Mn²⁺ ion, peripheral functional groups also play a role in enhancing those kinetic and relaxation properties. Three penta-dentate ligands have been designed, synthesized, and their Mn²⁺ complexes investigated in order to elucidate the effect of noncoordinating carboxylate and amine pendants on the bispidine scaffold. While thermodynamic stability is not influenced by these variations in the peripheral structure, kinetic inertness is increased two-threefold when the ligand bears both carboxylate and amine

functions. The synergistic effect of these groups could be related to the formation of a relatively stable dinuclear MnLZn complex, which can slow down transmetallation with Zn²⁺, the most important competitor of Mn²⁺ in vivo. Water exchange rate on these monohydrated chelates remains unaffected by variations in the complex charge. On the other hand, the simultaneous presence of deprotonated carboxylate and protonated amine functions enhances proton relaxivity, likely via reorganization of the second sphere water shell. These insights into structure-activity relationships help fine-tune the safety and the efficacy of MRI agents based on Mn²⁺-bispidines.

## 1. Introduction

In recent years, intensive research has been carried out to identify safer and ecologically more sustainable alternatives to Gd³⁺-based magnetic resonance imaging (MRI) contrast agents. This has been initially promoted by the emergence of nephrogenic systemic fibrosis in patients with renal failure[1] and by the observation of

Gd-deposition in certain organs,[2] particularly in the brain,[3] following repeated Gd-injections. Today, ecological considerations are also increasingly important, since gadolinium is now commonly detected in the environment at concentrations exceeding natural background levels.[4,5] Paramagnetic complexes of Mn²⁺ or Fe³⁺ endowed with high-spin (S = 5/2) and relatively long electron spin relaxation times (τ₅ ≈ 10⁻⁹ s) are the most obvious candidates to replace gadolinium-based relaxation probes in MRI.[6,7] Both Mn and Fe are essential metal ions,[8] furthermore, their abundance in the earth crust (second and third most abundant transition metals) makes them also economically attractive. Most attention has focused on Mn²⁺ chelates,[9,10] though an increasing number of reports concern Fe³⁺-based potential MRI probes.[11-13] Mn²⁺ is also a unique metal ion that offers detection in both MRI and positron emission tomography (PET)[14] as it possesses a β⁺ emitting isotope, ⁵²Mn²⁺, adapted to image slow biological processes (t₁/₂ = 5.6 d; β⁺-energy 0.6 MeV).[15] For in vivo imaging applications, Mn²⁺ must be complexed by a polydentate ligand that forms a stable and inert chelate to prevent free metal release in the body, which could be harmful at the elevated concentration needed for MRI (0.1 mmol/kg clinical dose).[16-19] Further requirements involve good relaxivity, thus the presence of at least one inner sphere water molecule, high aqueous solubility, and a favorable pharmacokinetic profile implying rapid and predominantly renal elimination. In the d⁶ electronic configuration of Mn²⁺, weak-field ligands are mandatory to provide complexes with the metal in high-spin state (5 unpaired electrons). In this

M. Sy, L. J. Charbonnière, A. M. Nonat
Equipe de Synthèse pour l'Analyse
Département des Sciences Analytiques
CNRS
IPHC UMR, 7178
Université de Strasbourg
27, Rue Becquerel, F-67 037 Strasbourg, France
E-mail: L.Charbonn@unistra.fr
Aline.Nonat@unistra.fr

D. Ndiaye,[+] H. Meudal, É. Tóth
Centre de Biophysique Moléculaire
CNRS UPR 4301
Université d'Orléans
Rue Charles Sadron, F-45071 Orléans, France
E-mail: eva.jakabtoth@cnrs-orleans.fr

[+]Present address: Institut de Chimie – UMR7177, Université de Strasbourg, F-67000 Strasbourg, France

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Supporting information for this article is available on the WWW under https://doi.org/10.1002/ceur.202500263

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© 2025 The Author(s). ChemistryEurope published by Chemistry Europe and Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
<figure><img src="image_5.png" /></figure>

case, the lack of crystal field stabilization energy makes it difficult to achieve high stability and kinetic inertness. Among the various ligand classes investigated for Mn²⁺ complexation, those based on the linear t-CDTA[20,21] (PyC3A[22–25]) and the macrocyclic DO2A-[20,26,27] or PC2A-type chelators[21,28,29] allowed combining the best Mn²⁺ complex stabilities and MRI properties

(Scheme 1). Today, MnPyC3A and a MnPC2A-derivative are in clinical trials.[30–32]

Ligands based on the bispidine (3,7-diazabicyclo[3.3.1]nonane) bicycle[33] represent a unique class for Mn²⁺ chelation, as they provide unprecedented kinetic and thermodynamic properties. We evidenced not only excellent relaxation efficacy

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

$R = H : H_2PC2A-EA$

$R = -CH_2-Py : H_2PC2A-DPA$

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

$H_2(1,4-DO2A)$

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

$R = H : H_3PyC3A$

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

<figure><img src="image_10.png" /></figure>

$R1 = CO_2H and R2 = NH_2 : H_3L^1$

$R1 = CO_2H and R2 = NH(COMe) : H_3L^2$

$R1 = CH_2OH and R2 = NH_2 : HL^3$

<figure><img src="image_11.png" /></figure>

$R1 = CO_2H and R2 = (CH_2)_2CO_2H : H_4L^4$

$R1 = CO_2H and R2 = H : H_3B^1$

$R1 = PO_3H_2 and R2 = H : H_4B^2$

<figure><img src="image_12.png" /></figure>

$R = CH_2CO_2H : HB^3$

$R = CH_2Py : B^4$

$R = CH_2Pa : HB^5$

<figure><img src="image_13.png" /></figure>

● = Pyridyl (Py)

$R1 = CO_2^− and R2 = NH_3^+ : [MnHL^1]$

$R1 = CO_2^− and R2 = NH(COMe) : [MnL^2]$

$R1 = CH_2OH and R2 = NH_3^+ : [MnHL^3]$

<caption>**Scheme 1.** Ligands and MnL complexes are discussed in the text. Pa = Picolinic acid (6-methylene-2-carboxypyridine).</caption>
<figure><img src="image_14.png" /></figure>

for the monohydrated [MnHL¹] complex formed with the pentadentate L¹ chelator (Scheme 1), but also a complete lack of dissociation over a period of 130 d. at pH 6, 37 °C, in the presence of 50 equiv. of Zn²⁺, making it the most inert mono-hydrated Mn²⁺ complex known to date.[³⁴] Under similar conditions, the best macrocyclic chelates like MnPC2A-EA and MnPC2A-DPA have dissociation half-lives of 54.4 and 64.5 h,[²⁹] respectively. In [MnHL¹], the metal ion is coordinated by the two amines from the bicycle, two pyridine nitrogen atoms, and the carboxylate group of the lysine chain, in addition to one hydration water molecule. The exceptional resistance to dissociation was related to the highly preorganized and rigid structure of the bispidine skeleton, which constitutes a coordination cage perfectly adapted to Mn²⁺, preventing potential attacks by metal cations or protons, as in the case of cyclams[³⁵,³⁶] and cryptands.[³⁷,³⁸] We recently established that such excellent kinetic inertness is retained by Mn²⁺ bispidine complexes exclusively in chair-chair conformation, but not in boat-chair conformation.[³⁹]

Bispidine ligands of higher denticity, such as the heptadentate B³ and B⁴ and the octadentate B⁵ (Scheme 1), showed another remarkable coordination property. By offering different coordination modes to Mn²⁺ and Zn²⁺ (respectively forming octa- and hexacoordinate complexes), they provide the first examples of selectivity (of over ten orders of magnitude) for Mn²⁺ vs. Zn²⁺,[⁴⁰,⁴¹] in contrast to the general Irving–Williams rules.[⁴²] Although less inert than [MnHL¹], these chelates are also very resistant to dissociation.

Some of these bispidine complexes have been injected into healthy mice. The in vivo MRI experiments,[³⁴,⁴⁰] realized at 40–80% lower injected doses than those clinically used, still showed good signal enhancement. The complexes underwent fast renal clearance, and liver uptake was negligible. Ex vivo biodistribution by inductively coupled plasma optical emission spectroscopy (ICP-OES) Mn detection in various tissues confirmed full elimination of the probes from the animals.

While several of the advantageous properties are common to Mn²⁺ chelates with pentadentate bispidine ligands in chair–chair conformation,[³⁴,³⁹–⁴¹,⁴³] [MnHL¹] stands out with its superior kinetic inertness and relaxation efficacy. Indeed, for [MnB¹]⁻ bearing an acetate function on the N7 position, kinetic inertness was found to decrease even if it remained exceptionally good, with ≈46% dissociation in 120 d, at pH 6,[⁴³] while [MnHL¹] does not show any Mn²⁺ release (37 °C, in the presence of 50 equiv. of Zn). This suggests a potential role of the noncoordinating primary amine group of the lysine. We hypothesized that this amine and the noncoordinating carboxylate (Scheme 1) together can provide a binding pocket for Zn²⁺, which further slows down Zn²⁺ transmetalation and reinforces the kinetic inertness of [MnHL¹]. Furthermore, the relaxivity of [MnHL¹] is also remarkably high ($r_f = 4.28 \text{ mM}^{-1} \text{ s}^{-1}$, 25 °C, pH 7, 20 MHz) as compared not only to typical monohydrated Mn²⁺ chelates,[²²] but also to the other bispidine derivatives cited above (+9% in average). Explanations involve the presence of a second-sphere contribution and a slower rotational dynamics, with a potential impact of the noncoordinating carboxylates and the lysine side chain.

Given the growing interest in this family of bispidine chelators for the design of highly inert and efficient MRI agents, it is essential to elucidate the influence of noncoordinating peripheral functional groups on the structural, thermodynamic, and kinetic properties and on the relaxivity of their Mn²⁺ complexes. Toward this end, we report here the synthesis of three new ligands L², L³, and L⁴, and a comprehensive characterization of their Mn²⁺ complexes (Scheme 1). Ligands L² and L³ differ from L¹, respectively, by the substitution of the lysine amine with an acetamide function and by the replacement of the noncoordinating carboxylates with hydroxyl groups. These structures were designed to assess how the nature of the peripheral functional groups, which do not interfere with Mn²⁺ coordination, influences the physico-chemical properties of the complexes, and more particularly, if there is a specific interplay between the ε-NH₃⁺ of the L-lysine side chain and the carboxylate functions in [MnHL¹]. The potential role of the lateral amine was further assessed by including the glutamate analog L⁴ in the comparison. Finally, a variable pH NMR study was performed on [MnHL¹] and [MnHL¹]²⁺ to better understand their solution behavior. These insights will be valuable to derive bispidine structures with optimized properties for contrast agent development.

## 2. Results and Discussion

### 2.1. Synthesis and Complex Preparation

The model ligand L¹ was synthesized following the synthetic route developed by Roux et al.[⁴⁴] with several improvements in the purification steps in order to improve synthesis yield and reproducibility. First, the precipitation of bispidone 1 was optimized by switching from diethyl ether to acetonitrile, resulting in an off-white product with a 67% yield. The Boc deprotection was carried out directly on the crude product obtained from the reduction of compound 1 to 2, followed by the saponification of the methyl ester functions with LiOH, and L¹ was purified using only a single flash purification chromatography step on a reverse phase C18 column (H₂O/ACN, 0.1% TFA) at the end of the synthesis. This refined approach achieved an overall yield of 21%, slightly improving the original 18% yield while enhancing reproducibility. The reduction of the central ketone is the limiting step in the synthesis, and optimizations could be considered to improve its facial regioselectivity.

For the synthesis of ligand L², the bispidol intermediate 2 was prepared according to the procedure described by Roux et al.[⁴⁴] The synthesis began with the removal of the Boc protecting group, yielding compound 3 with a 51% yield. Subsequently, the primary amine of L-lysine underwent peptide coupling with N-succinimidyl acetate in the presence of N-methylmorpholine in DMF, affording compound 4 with a 59% yield. Finally, hydrolysis of the methyl esters produced ligand L² with an overall yield of 20% in four steps from bispidone 1. The ¹H NMR spectrum confirms successful coupling, highlighted by the acetate methyl signal at 1.96 ppm (Figure S1, Supporting Information).
<figure><img src="image_15.png" /></figure>

Ligand L³ was obtained from bispidol 5 by first reducing the methyl ester functions in the presence of lithium tetrahydroborate in THF at room temperature. This was followed by the deprotection of the Boc protecting group, resulting in ligand L³ as a TFA salt (L³·3TFA) with a 68% yield. As shown in **Figure 1**, the ¹H NMR spectrum of L³ is quite typical. In addition to the usual AB systems of protons 6 and 8 in the azacylohexane ring, the two CH₂ groups resulting from the reduction of the ester functions also form AB spin systems. Peak assignments were made by analyzing nuclear overhauser effect spectroscopy (NOESY), correlation spectroscopy (COSY), and hetereonuclear single quantum coherence (HSQC) spectra. Due to the asymmetric carbon of L-lysine, the molecule does not possess a plane of symmetry. The numbering

for the annotation of the NMR spectrum has been simplified for clarity, and this same convention is used consistently throughout the study (**Scheme 2**).

The coupling between the apical proton 9 and the axial protons 6ax and 8ax is clearly observed in the NOESY spectrum of ligand L³, indicative of the bispidine ring adopting a chair-chair conformation. Notably, this coupling is absent in the NOESY spectrum of intermediate 5, implying that the bicyclic structure is in a boat-chair conformation or an intermediate state prior to deprotection. Upon deprotection under acidic conditions, it returns to the typical chair-chair conformation. This pH-induced conformational change has been previously reported, demonstrating that varying pH allows for a reversible switch between conformations.[45]

<figure><img src="image_16.png" /><figcaption>**Figure 1.** ¹H NMR spectrum of L³ (500 MHz, MeOD, 25 °C).</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>**Scheme 2.** Synthesis of ligands and proton numbering.</figcaption></figure>
<figure><img src="image_18.png" /></figure>

Ligand L⁴ was synthesized following an adaptation of the pro-
tocol developed for L¹. Bispidone 6 was obtained through a double
Mannich reaction between the piperidinone P₁, L-glutamic acid,
and formaldehyde. The main challenge in this reaction was purifi-
cation, as compound 6 was difficult to precipitate, yielding only
34% through precipitation in acetonitrile. The second step involved
reducing the central ketone to yield bispidol 7. As before, this inter-
mediate was not isolated, but directly underwent saponification of
the methyl ester groups with LiOH, producing ligand L⁴ with an
overall yield of 20% for the two steps. 1D and 2D ¹H NMR spectra
(Figure S1–10, Supporting Information) reveal the distinct signals
of a bispidol in chair–chair conformation with the tertiary alcohol
group oriented toward N3, demonstrating the same facial regiose-
lectivity observed in the NaBH₄ reduction of L-lysine derivatives,
where hydride addition occurs on the N7 face.

For the MnL (or ZnL) samples used in kinetic, NMR, and relaxo-
metric studies, stock solutions of the complexes were prepared by
mixing aqueous MnCl₂ (or ZnCl₂) and ligand solutions of known
concentrations, in equimolar quantities. The pH was set to
≈6.5 with a NaOH solution and controlled regularly over time
to ensure full complexation (especially for MnL complexes, which
form slowly). Full complex formation could be verified by the sta-
bilization of the relaxivity values (for MnL) over time; ZnL formation
is instantaneous. The lack of free Mn was checked by the xylenol-
orange test, and the Mn concentration was systematically con-
trolled by ICP-OES or bulk magnetic susceptibility measurements.

## 2.2. Thermodynamic Stability and Kinetic Inertness of MnL Complexes

The thermodynamic stability of the complexes is dependent on
ligand basicity, thus first pH-potentiometric titrations (Figure S11,
Supporting Information) have been carried out on L², L³, and L⁴
ligand solutions to assess the protonation constants. Their analy-
sis yielded 3, 4, and 5 logK<sub>H</sub> values, respectively, for L², L³, and L⁴

(Table 1). In the basic pH region, we observed a single (L², L⁴) or
two (L³) protonation steps. By analogy to L¹ and other bispidine
ligands,[40,41,43] we attributed the first protonation step to one of
the tertiary amine groups of the bispidine bicycle; and for L³, as in
the case of L¹, the second protonation constant corresponds to
the lysine ε-amino group. The protonation scheme of the bicycle
is intimately linked to its conformation; simultaneous protonation
of N3 and N7 sites is impossible in the chair–chair conforma-
tion.[39,45,46] Based on the protonation constants, one can con-
clude that all three ligands adopt a chair–chair conformation,
which prevents the protonation of the second bicycle amine
and which is known to offer the most favorable metal coordina-
tion environment. The subsequent protonation constants fall into
the pH region 2–5 and correspond to carboxylate functions, with
the exception of L³, where the last protonation step occurs on a
pyridine. Pyridine protonation was also detected for other bispi-
dine derivatives.[41]

Stability constants have been determined for Mn²⁺ and Zn²⁺ complexes by pH-potentiometric titration in the 2–11 pH range (Table 1, Figure S11, Supporting Information). Due to the slow formation of the Mn²⁺ complexes with all ligands studied, batch samples were prepared between pH 2 and 7, while direct titrations were performed in the basic pH range, as well as for Zn-L systems. As for MnL¹, the stability constants determined for MnL², MnL³, and MnL⁴ are relatively low compared to the best Mn²⁺ chelating ligands described in the literature.[6] The pMn values are also considerably lower than those of MnPC2A-EA, for instance (pMn = 9.27).[28] The stability of ZnL complexes is higher compared to their Mn²⁺ analogs, obeying the Irving–Williams series.[42] We should note that even higher stability constants can be expected with Cu²⁺; logK<sub>CuL</sub> = 19.2 was reported, for instance, for the CuB1 analog.[47] For ML³ (M = Mn²⁺ or Zn²⁺) complexes, in the basic pH region, we observed a protonation constant practically identical to the ligand logK<sub>H2r</sub>, corresponding to the protonation of the noncoordinating lysine amine. For the

<table><caption>**Table 1.** Ligand protonation constants, stability constants of ML complexes, and pMn values. Values in parentheses correspond to one standard deviation. *I* = 0.15 M NaCl, 25 °C.</caption><thead><tr><th></th><th>L<sup>1[34]</sup></th><th>L<sup>2</sup></th><th>L<sup>3</sup></th><th>L<sup>4</sup></th><th>B<sup>1[43]</sup></th><th>PC2A-EA<sup>[28]</sup></th></tr></thead><tbody><tr><td>logK<sub>H1</sub></td><td>11.44</td><td>11.71(2)</td><td>11.90(7)</td><td>9.52(4)</td><td>9.54</td><td>11.34</td></tr><tr><td>logK<sub>H2</sub></td><td>10.31</td><td>4.93(3)</td><td>10.47(5)</td><td>5.06(7)</td><td>5.11</td><td>8.93</td></tr><tr><td>logK<sub>H3</sub></td><td>4.71</td><td>2.89(3)</td><td>2.93(6)</td><td>4.22(6)</td><td>2.99</td><td>6.91</td></tr><tr><td>logK<sub>H4</sub></td><td>2.76</td><td>–</td><td>2.17(5)</td><td>2.67(7)</td><td>1.80</td><td>1.97</td></tr><tr><td>logK<sub>H5</sub></td><td>2.22</td><td>–</td><td>–</td><td>2.31(4)</td><td>–</td><td>–</td></tr><tr><td>logK<sub>ZnL</sub></td><td>15.59</td><td>14.84(3)</td><td>15.76(3)</td><td>14.22(6)</td><td>13.72</td><td>–</td></tr><tr><td>logK<sub>ZnHL</sub></td><td>10.33</td><td>3.77(1)</td><td>10.28(3)</td><td>4.67(6)</td><td>3.26</td><td>–</td></tr><tr><td>logK<sub>ZnH2L</sub></td><td>3.28</td><td>–</td><td>3.05(5)</td><td>3.27(5)</td><td>–</td><td>–</td></tr><tr><td>logK<sub>MnL</sub></td><td>12.21</td><td>12.85(9)</td><td>12.71(5)</td><td>12.80(1)</td><td>11.26</td><td>19.01</td></tr><tr><td>logK<sub>MnHL</sub></td><td>10.42</td><td>4.43(5)</td><td>10.40(4)</td><td>4.89(9)</td><td>3.20</td><td>6.88</td></tr><tr><td>logK<sub>MnH2L</sub></td><td>3.87</td><td>–</td><td>3.43(2)</td><td>3.28(9)</td><td>–</td><td>2.50</td></tr><tr><td>pMn<sup>a)</sup></td><td>6.65</td><td>6.77</td><td>6.56</td><td>7.83</td><td>7.06</td><td>9.27</td></tr><tr><td>pZn<sup>a)</sup></td><td>8.29</td><td>7.77</td><td>8.01</td><td>8.55</td><td>8.29</td><td>–</td></tr></tbody></table>

<footnote><sup>a)</sup> C<sub>M2+</sub> = c<sub>L</sub> = 10<sup>-5</sup> M, pH 7.4.</footnote>
<figure><img src="image_19.png" /></figure>

two other complexes, there is no protonation step that could correspond to a tertiary amine of the bicycle, which is in accordance with the chair-chair conformation of the bispidine. Indeed, such protonation has been observed in the basic region exclusively for complexes in boat-chair conformation.[39] Overall, and as expected, variations in the ligand structure outside the metal coordination sphere have little influence on the thermodynamic stability of the ML complexes.

Species distribution curves have been calculated by using the stability and protonation constants obtained (Figure 2). For MnL² and MnL³, they were further confirmed with the pH-dependency of the water proton relaxation rates. They show full complexation above pH 6, and in the case of MnL³, the presence of the mono-protonated [MnHL³]²⁺ species at physiological pH, as for [MnHL¹].[34] These pH-dependent R₁ₚ values have also been fitted by fixing the stability and protonation constants obtained from potentiometry (Table 1) and by assuming that they are the sum of contributions from all species present at a given pH. This allowed us to estimate relaxivity values of 4.6 ± 0.1 and 4.0 ± 0.2 mM⁻¹ s⁻¹ for the protonated [MnHL²] and [MnH₂L³]³⁺ species, respectively, slightly higher than those of [MnL²]⁻ (and [MnHL³]²⁺.

High resistance of MnL complexes to dissociation under non-equilibrium conditions is requisite for safe in vivo use. In the objective of evaluating the contribution of the uncoordinating carboxylate and amine groups to the exceptional kinetic

inertness of [MnHL¹], we have assessed the kinetic inertness for [MnL²]⁻ and [MnHL³]²⁺ first in highly acidic solutions where dissociation is faster (0.01–1 M HCl, 25 °C, Figure 3a). The observed rate constants show a second-order dependence on proton concentration and were fitted to Equation (1):

$$k_{\text{obs}} = k_0 + k_1 [H^+] + k_2 [H^+]^2 \quad (1)$$

The k₁ and k₂ values obtained from the fit are reported in Table 2, while k₀ was fixed to 0 as commonly done,[34] otherwise the fit yielded negative values and large errors. The results confirm, first of all, the exceptional kinetic inertness of Mn²⁺ complexes formed with these pentadentate bispidine ligands, which is again superior for [MnHL¹] than for the two other systems [MnL²]⁻ and [MnHL³]²⁺. While the exact mechanism of the acid-catalyzed dissociation has not been investigated for bispidine complexes, we can draw a reasonable parallel with typical azamacrocyclic metal chelates where mechanistic studies have been done.[48] Thus, we can assume that in the proton-assisted dissociation route, the proton is attached first to a carboxylate function, and in this step, the metal remains coordinated inside the bispidine cage. The rate-determining step is expected to be the transfer of the proton from the carboxylate to one of the bicyclic amine nitrogens, which will lead to the metal release. This proton transfer is known to be slowed down by ligand rigidity.

<figure><img src="image_20.png" /><figcaption>Figure 2. Species distribution curves calculated for Mn-L² a) and Mn-L³ systems (1 mM) b) and pH-dependent variation of the paramagnetic enhancement of the water proton relaxation rates, R₁ₚ (▲) measured for cₘᵢ; nL = 1 mM at 25 °C, 60 MHz; the gray line represents the fit of the relaxation rates as described in the text. l = 0.15 M NaCl.</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>Figure 3. a) Pseudo-first-order dissociation rate constants determined in acidic solutions (l = 0.1 M NaCl + HCl). Curves represent the fit of the experimental points. b) Time-dependent variation of transverse, r₂ ([MnL²]⁻ and [MnHL³]²⁺) or longitudinal relaxivity, r₁ ([MnHL¹]) in a 1 mM MnL solution at pH 6, 37 °C, and 60 MHz, in the presence of 50 equiv. of Zn²⁺ (l = 0.1 M NaCl).</figcaption></figure>
<figure><img src="image_22.png" /><figcaption>**Table 2.** Dissociation percentages, half-lives ($t_{1/2}$) at 37 °C, and rate constants characterizing the proton-assisted dissociation of $Mn^{2+}$ complexes ($k_1$ and $k_2$) at 25 °C. *I* = 0.1 M NaCl.</figcaption></figure>

<table><thead><tr><th></th><th>[MnHL<sup>1</sup>]<sup>b)</sup></th><th>[MnL<sup>2</sup>]<sup>-</sup></th><th>[MnHL<sup>3</sup>]<sup>2+</sup></th><th>[MnB<sup>1</sup>]<sup>-c)</sup></th></tr></thead><tbody><tr><td>Dissociation %</td><td>No dissociation</td><td>≈18%<sup>d)</sup></td><td>≈33%<sup>d)</sup></td><td>≈46%<sup>d)</sup></td></tr><tr><td>(pH 6)<sup>a)</sup></td><td></td><td>(110 d)</td><td>(106 d)</td><td>(120 d)</td></tr><tr><td>t<sub>1/2</sub> (pH 4)<sup>a)</sup></td><td>33 d</td><td>10 d</td><td>12 d</td><td>4.5 d</td></tr><tr><td>k<sub>1</sub> × 10<sup>-3</sup> (s<sup>-1</sup> M<sup>-1</sup>)</td><td>1.6</td><td>4.3 ± 0.1</td><td>2.5 ± 0.1</td><td>17.9</td></tr><tr><td>k<sub>2</sub> × 10<sup>-4</sup> (s<sup>-1</sup> M<sup>-2</sup>)</td><td>5.0</td><td>13 ± 2</td><td>7.4 ± 0.9</td><td>—</td></tr></tbody></table>

In such a scenario, it can be reasonably assumed that in [MnHL¹] the strong hydrogen binding between the lysine ε-amine and a noncoordinating carboxylate further rigidifies the complex structure (see below), which could likely explain its slightly higher resistance to acid-catalyzed dissociation.

The inertness was further investigated via transmetallation reactions in the presence of 50 equiv. $Zn^{2+}$ at different pH values, at 37 °C (Figure S12, Supporting Information). For direct comparison, we represent the evolution of the relaxivity as a function of time at pH 6 (Figure 3b).

In contrast to [MnHL¹], which did not show any dissociation over a period of 130 d at pH 6, [MnL²] and [MnHL³]²⁺ undergo partial dissociation (≈18% in 110 d and ≈33% in 106 d, respectively). At pH 4, the estimated dissociation half-life of [MnHL¹] (33 d) is three times superior to that of [MnL²]⁻ (10 d) and [MnHL³]²⁺ (12 d). These results reveal a synergistic contribution of the uncoordinated carboxylate and lysine ε-amine groups to the high kinetic inertness of [MnHL¹].

We hypothesized that the presence of these functional groups in the ligand, which are not coordinated to $Mn^{2+}$, could increase the kinetic inertness by coordinating and thus "scavenging" the transmetalating $Zn^{2+}$ ion. To investigate such potential $Zn^{2+}$ complexation, we exploited the fact that the lysine ε-amino group is protonated below pH 9, allowing $Zn^{2+}$ complexation to be monitored by pH-potentiometry. Hence, we carried out a potentiometric titration of a [MnHL¹] solution prepared at pH 7.3 to which one equivalent of $Zn^{2+}$ was added (Figure S11a, Supporting Information). After $Zn^{2+}$ addition, the pH drops from 7.3 to 3.4, confirming the release of the amine proton upon coordination to the $Zn^{2+}$ ion. The pH-potentiometric titration of this solution with NaOH indeed indicated the consumption of one equivalent of base between pH 3.4 and 6, suggesting the formation of a $MnL¹:Zn^{2+}$ adduct with equimolar stoichiometry. Given the very slow dissociation of [MnHL¹], the titration curve could be analyzed to estimate a stability constant of $logK_{MnLZn} = 9.88$ for the heteronuclear adduct ($K_{MnLZn} = [MnL¹Zn]/[MnL¹][Zn]$. Above pH 7, the formation of hydroxocomplexes is detected, and from pH 9.5, precipitation is observed. The species distribution curves were calculated, indicating the formation of the dinuclear species in the pH 3.4–6 region (Figure 4). This could also be corroborated with the pH-dependent relaxivities measured in an aqueous solution containing [MnHL¹] and 1 equiv. $Zn^{2+}$. The formation of dimer/oligomers could be excluded by the lack of any change in DOSY spectra between solutions containing L¹ and

<figure><img src="image_23.png" /><figcaption>**Figure 4.** Species distribution curves calculated for [MnHL¹] in the presence of 1 equiv. of $Zn^{2+}$, and pH-dependent relaxivities (Δ) measured at 25 °C, 60 MHz. *I* = 0.15 M NaCl. (Δ) and (Δ) represent the situation (pH and $r_i$) before and after addition of $Zn^{2+}$, respectively.</figcaption></figure>

increasing equivalents of $Zn^{2+}$ (up to 2, Figure S13a, Supporting Information). We should note that the pH-dependent relaxivity curve in Figure 4 seems slightly shifted to higher pH with respect to the formation process of the dinuclear complex. This can be likely related to the simultaneous deprotonation process of [MnH₂L¹]⁺ to [MnHL¹], characterized by slightly lower relaxivity.

Notably, a small instantaneous increase (≈20%) of the longitudinal relaxivity is observed upon adding $Zn^{2+}$ to the [MnHL¹] solution at pH 6, while the relaxivity no longer changes afterward for months (Figure 3b). This intriguing phenomenon, detected to a much lower extent (≈6–8%) for the other complexes, is fully reproducible. The relaxivity increase diminishes with decreasing pH and disappears below pH 4. The instantaneous formation of the dinuclear ([MnL¹Zn]⁺) complex will likely induce modification in the second sphere relaxivity of the complex, which was previously found important, as well as in the rotational correlation time ($τ_{rh}$), explaining the observed relaxivity increase. Notably, MnPC2A-DPA[29] and MnPyc3A-BPEN[24] (Scheme 1) bearing a Zn-binding unit, which were developed as Zn-sensors acting in the presence of HSA, also had enhanced kinetic inertness as compared to the parent MnPC2A-EA and MnPyc3A complexes, and their complexation with $Zn^{2+}$ also yielded a small relaxivity increase (≈10%, without HSA). Overall, these data indicate that the presence of additional chemical functions in the $Mn^{2+}$ complex, which are available for weak $Zn^{2+}$ coordination, are interesting design elements to improve kinetic inertness.

<footnote><sup>a)</sup>50 equiv. $Zn^{2+}$, 37 °C; <sup>b)</sup>ref. [34]; <sup>c)</sup>ref. [43]; <sup>d)</sup>d = days. Values in parentheses represent the experimental times.</footnote>
<figure><img src="image_24.png" /></figure>

## 2.3. ¹H/¹³C NMR Study and Relaxation Properties of MnL Complexes

In order to gain further insight into the solution behavior and the arrangement of the non-coordinating carboxylates and the lysine ε-amino group in [MnHL¹], 1 and 2 D NMR ¹H total correlation spectroscopy (TOCSY) and HSQC) spectra have been recorded for L¹ and [MnHL¹] at different pHs (Figure 5 and 6), and also for [MnHL³]²⁺ at pH 7 for comparison, at 700 MHz (Figure 7). As compared to free L¹, the ¹H NMR spectrum of the paramagnetic [MnHL¹] complex shows signal broadening for protons H11, H12, H13, H14, and H15, while the signals for protons H2/4, H6/8, H9, and H10 tend to disappear (Figure 5) because of stronger electron-nuclear dipolar interactions, in accordance with the 1/r⁶ dependence of the Mn²⁺-induced paramagnetic relaxation enhancement effect (r is the Mn²⁺-¹H distance).[49] We also note that the ¹H signals of the two pyridines are nonequivalent in [MnHL¹] and that protons Hd and Hd' disappear.

Mn²⁺ complexation is also confirmed by HSQC and TOCSY spectra (Figure 6a,b and S13b, Supporting Information) (5.61 ≤ pD ≤ 9.51), as indicated by the absence of correlations for protons Hd and Hd' in the aromatic region as well as protons from the bispidine scaffold (H2/4, H9, H10, H6/H8). In parallel, new correlations appear in the 1.5–3.5 ppm ¹H chemical shift range. At pD 5.61, a single and distinct correlation is observed for the methyl protons (H15, ¹H/¹³C: 1.7 ppm / 42.1 ppm, Figure 6c, green) along with additional correlations attributed to lysine side chain protons. Unlike in the free ligand (blue), in the Mn²⁺ complex, the lysine protons (H12/H12', H13/H13', and H14/H14') become split, suggesting conformational restriction of the lysine moiety. The most plausible cause of this rigidity is the intramolecular hydrogen bonding between the lysine amine and a noncoordinating carboxylate (Figure 6d). [MnHL³]²⁺ lacks such conformational

restriction, as evidenced by the HSQC spectrum recorded under comparable conditions (Figure 7). This highlights the role of the carboxylate group in stabilizing strong intramolecular hydrogen-bonding interactions, which further rigidify the structure.

Surprisingly, this correlation pattern of [MnHL¹] changes with pD (Figure S14, Supporting Information). In contrast to the sole species present at pD 5.61 with the conformationally locked lysine side chain, at pD 7.51 an additional species emerges in a ≈1:1 ratio and becomes predominant at pD 9.51 (95% of the population, estimated from correlation integrals; Figure 6c). In this second species, H12/H12', H13/H13', and H14/H14' protons are no longer split, indicating increased conformational flexibility. Potentiometric data evidence the same, single species, [MnHL¹], at pD 5.61 and 7.41, and even at pD 9.51, only 5% of the complex is deprotonated ([MnL¹]⁻).[34] On the other hand, one can consider that proton exchange on the lysine ε-amino group is a primarily base-catalyzed process,[50] implying increasingly faster proton exchange upon increasing pH. Faster proton exchange can lead to progressive weakening of the hydrogen bond between the amine and the carboxylate, resulting in free motion of the lysine chain at higher basicity. Interestingly, this pH-dependent flexibility of the lysine sidechain does not have detectable consequences on the relaxivity of [MnHL¹] in the pH range 5.6–9.5.[34] This means that the rotational correlation time of the Mn-water proton vector is little affected by the side chain flexibility, or the effect is compensated by variations in the second sphere relaxivity due to rearrangement of second sphere water molecules (see below).

## 2.4. Relaxation Properties

The functional groups of the ligand that do not participate in Mn²⁺ coordination also affect the relaxivity of these bispidine complexes.

<figure><img src="image_25.png" /><figcaption>**Figure 5.** ¹H NMR spectra of H₂L¹⁻ (—) and [MnHL¹] (—) in D₂O at pH 7.1 (pD 7.51), 25 °C, cH₂L₁ = cMnHL₁ = 2.5 mM, 700 MHz (equ stands for equatorial and ax for axial).</figcaption></figure>
<figure><img src="image_26.png" /></figure>

<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /><figcaption>**Figure 6.** a) Overlay of HSQC spectra of L¹ at pD 7.51 (blue) and [MnHL¹] at pD 7.4 (red), and b) enlargement corresponding to 0.8 ppm < δ < 3.5 ppm (cL = cMnHL = 2.5 mM, 25 °C, 700 MHz). c) Zoom (1.1 ppm < δ < 3.5 ppm) of HSQC spectra of L¹ at pD 7.51 (blue) and [MnHL¹] at pD 7.4 (red), pD 5.61 (green), and pD 9.41 (black) (cL = cMnHL = 2.5 mM, 25 °C, 700 MHz; relaxation delay 1.3 s). d) Schematic representation of [MnHL¹] with proton numbering.</figcaption></figure>

**Table 3** illustrates the comparison of the r₁ values at 20 and 60 MHz (25 °C) for a selection of MnL chelates. They all have a similar metal coordination sphere with one water molecule and similar molecular weight, thus the relaxivity differences can be reasonably attributed to differences in the second sphere relaxivity contribution. Indeed, the relaxation enhancement is a sum of contributions from inner, second, and outer sphere mechanisms, originating respectively from the exchange of inner or second sphere water molecules with bulk, or from the diffusion of outer sphere water around the complex. The comparison in Table 3 indicates that the presence of a higher number of charged functions (carboxylate and amine) has a non-negligible effect to enhance relaxivity. In particular, the influence of the noncoordinating carboxylates seems important, as demonstrated by the 10% higher relaxivities for [MnHL¹] vs. [MnHL³]²⁺, likely related to the charged carboxylate functions which maintain more second sphere water molecules in the proximity of the paramagnetic center. In overall, these r₁ values are higher than relaxivities of typical monohydrated Mn²⁺ complexes reported in the literature (for instance, 3.3 mM⁻¹s⁻¹[²²] for [Mn(PCy3A)]⁻ or 3.52 mM⁻¹s⁻¹[²⁸] for [Mn(PC2A-EA)]⁺ at 60 MHz, 25 °C), and they

are comparable to those of clinical Gd³⁺-based agents, despite the lower electron spin of Mn²⁺.

For further assessment, nuclear magnetic relaxation dispersion (NMRD) profiles have been recorded for [MnHL³]²⁺ and [MnL⁴]²⁻ at 25 and 37 °C, in water (pH 7) in the proton Larmor frequency range of 0.01–80 MHz (Figure 8). We also carried out a variable temperature ¹⁷O NMR study to confirm the hydration number *q* and to determine the parameters that govern water exchange (Figure 8). By using Gale's approach,[⁵¹] the temperature-dependent ¹⁷O transverse relaxation rates (1/T₂) allowed to estimation of hydration numbers *q* = 0.9 and 0.7 for [MnHL³]²⁺ and [MnL⁴]²⁻, respectively, which, considering the error of this method, supports monohydration of the chelates (Figure S15, Supporting Information). We have then analyzed the ¹⁷O relaxation rates and the ¹H NMRD profiles of [MnHL³]²⁺ and [MnL⁴]²⁻ by using the common Solomon–Bloembergen–Morgan theory of paramagnetic relaxation and calculated the parameters describing water exchange and rotational dynamics (Table 3; see Table S1, Supporting Information for the full set of fitted parameters and Supporting Information for equations).
<figure><img src="image_29.png" /></figure>

<figure><img src="image_30.png" /><figcaption>**Figure 7.** HSQC spectrum of [MnHL<sup>3</sup>]<sup>2+</sup>. c<sub>MnHL</sub> = 2.5 mM, 25 °C, pH 7; 700 MHz; relaxation delay 1.3 s.</figcaption></figure>

<table><caption>**Table 3.** Proton relaxivities and parameters obtained from the fit of <sup>17</sup>O NMR and NMRD data. All complexes have q = 1.</caption><thead><tr><th></th><th>[MnHL<sup>1</sup>]<sup>d)</sup></th><th>[MnL<sup>2</sup>]<sup>-</sup></th><th>[MnHL<sup>3</sup>]<sup>2+</sup></th><th>[MnL<sup>4</sup>]<sup>2-</sup></th><th>[MnB<sup>1</sup>]</th><th>[MnB<sup>2</sup>]<sup>2-e</sup></th></tr></thead><tbody><tr><td>r<sub>1</sub>/mM<sup>-1</sup> s<sup>-1</sup></td><td>4.28<sup>a)</sup></td><td></td><td>3.91<sup>a)</sup></td><td>4.10<sup>a)</sup></td><td>3.64<sup>a)</sup></td><td>4.31<sup>a)</sup></td></tr><tr><td>k<sub>ex</sub><sup>298</sup>/10<sup>7</sup> s<sup>-1</sup></td><td>5.1</td><td>4.03<sup>b)</sup></td><td>3.71<sup>b)</sup></td><td>3.81<sup>b)</sup></td><td></td><td></td></tr><tr><td>ΔH<sup>‡</sup>/kJ mol<sup>-1</sup></td><td>10.6</td><td></td><td>7.4 ± 0.2</td><td>6.3 ± 0.1</td><td>5.5</td><td>0.12</td></tr><tr><td>ΔS<sup>‡</sup>/kJ mol<sup>-1</sup></td><td>-62</td><td></td><td>13.5 ± 0.4</td><td>7.9 ± 0.4</td><td>14.9</td><td>25.7</td></tr><tr><td>E<sub>rH</sub>/kJ mol<sup>-1</sup></td><td>22</td><td></td><td>-49</td><td>-83</td><td>-47</td><td>-42</td></tr><tr><td>τ<sub>rH</sub><sup>298</sup>/ps</td><td>100</td><td></td><td>30 ± 2</td><td>24 ± 2</td><td>20</td><td>19</td></tr><tr><td></td><td>72<sup>c)</sup></td><td></td><td>93 ± 2</td><td>94 ± 3</td><td>89</td><td>92<sup>c)</sup></td></tr><tr><td></td><td></td><td></td><td>78 ± 3<sup>c)</sup></td><td>79 ± 1<sup>c)</sup></td><td>65<sup>c)</sup></td><td></td></tr></tbody></table>

The rate ($k_{ex}^{298}$), the activation enthalpy ($\Delta H^{‡}$), and entropy ($\Delta S^{‡}$) of the water exchange, determined from the <sup>17</sup>O relaxation rates, are very similar for all the complexes, with the exception of the phosphonate derivative [MnB<sup>2</sup>]<sup>2-</sup>, indicating that these parameters are determined essentially by the inner coordination sphere. The strong negative activation entropies suggest an associatively activated mechanism for the water exchange, which implies the arrival of the incoming water molecule as the rate-determining step. This also explains the lower exchange rate of the phosphonate derivative [MnB<sup>2</sup>]<sup>2-</sup>, for which the increased negative charge around the water binding site is unfavorable to the approach of the incoming water.

The analysis of the NMRD data has been performed both without and with the assumption of a second sphere contribution to relaxivity. Without including a second sphere contribution in the analysis of the relaxivities, the fit yields rotational correlation times that are likely overestimated based on the molecular weight of the complexes. This was also the case for [MnHL<sup>1</sup>].

($\tau_{rH}^{298}$ = 100 ps).<sup>[34]</sup> For the glutamate derivative [MnL<sup>4</sup>]<sup>2-</sup>, we considered thus a second sphere relaxivity contribution by including two second sphere water molecules in the analysis with an estimated Mn-H proton distance of 3.2 Å, 50 ps residence time and an activation enthalpy of 10 kJ/mol, based on previous simulations on Gd<sup>3+</sup> complexes,<sup>[52]</sup> and similarly to the analysis of [MnHL<sup>1</sup>].<sup>[34]</sup> For [MnHL<sup>3</sup>]<sup>2+</sup>, however, a satisfactory fit could be obtained when a single second sphere water molecule was considered (with the same residence time and distance as above), and the rotational correlation time obtained in this analysis was very similar to $\tau_{rH}^{298}$ of [MnL<sup>4</sup>]<sup>2-</sup> and [MnHL<sup>1</sup>] calculated assuming two second sphere water molecules. This indicates a more important second sphere contribution for these latter two systems. One should be aware, however, that the second sphere contribution can only be estimated using assumptions regarding the number and the distance of the second sphere water molecules, and it is not straightforward to reliably assess those. Therefore, it is probably the most reasonable to simply

<footnote><sup>a)</sup>20 MHz and 25 °C; <sup>b)</sup>60 MHz and 25 °C; <sup>c)</sup>considering second sphere relaxivity contribution; <sup>d)</sup>Ref. [34]; <sup>e)</sup>Ref. [43].</footnote>
<figure><img src="image_31.png" /></figure>

<figure><img src="image_32.png" /><figcaption>**Figure 8.** NMRD profiles in water at 37 °C (▲) and 25 °C (♦) for [MnHL<sup>3</sup>]<sup>2+</sup> a) and [MnL<sup>4</sup>]<sup>2-</sup> b); reduced transverse <sup>17</sup>O relaxation rates (9.4 T) for [MnHL<sup>3</sup>]<sup>2+</sup> c) and [MnL<sup>4</sup>]<sup>2-</sup> d).</figcaption></figure>

compare the experimental relaxivities to conclude that while a second sphere relaxation contribution exists for all [MnHL¹], [MnL²]⁻, [MnHL³]²⁺ and [MnL⁴]²⁻ complexes, it is the most important for [MnHL¹].

Finally, ¹H NMRD curves and transverse ¹⁷O relaxation rates have also been recorded in a solution containing [MnHL¹] and 1 equiv. of Zn²⁺ (Figure S17, Supporting Information). At all frequencies and both 25 and 37 °C, the relaxivities are slightly higher in the presence of Zn²⁺, as already observed above in the transmetalation study, as a consequence of the Zn²⁺ binding to the carboxylate and amine functions that do not participate in Mn²⁺ coordination. The ¹⁷O T₂ data evidence that the hydration number q = 1 is retained in the presence of Zn²⁺. The analysis of the data with the SBM theory indicates a slight increase in the water exchange rate (k<sub>ex</sub><sup>298</sup> = (8.2 ± 0.2) × 10<sup>7</sup> s<sup>-1</sup>) as well as in the rotational correlation time (τ<sub>rh</sub><sup>298</sup> = 124 ± 3 ps) as compared to that determined in the absence of Zn.

### 3. Conclusion

Overall, this study demonstrates that noncoordinating peripheral functional groups in bispidine ligands affect the structural,

kinetic, and relaxivity properties of their Mn²⁺ complexes, and therefore should be considered as relevant elements in the design of efficient and safe MRI probes. Three novel bispidine chelators (L2, L3, and L4) and their respective Mn²⁺ complexes were synthesized and characterized, highlighting the impact of the overall charge, the presence of potential metal coordinating functions on the periphery, and intramolecular hydrogen binding. While these changes do not affect the Mn²⁺ coordination mode and thermodynamic stability, they remarkably influence the kinetic inertness in the presence of the Zn²⁺ competitor, as well as the relaxivity of the complexes. Within this family, [MnHL¹] exhibits the highest kinetic inertness and relaxivity. The exceptional resistance of [MnHL¹] to transmetallation could be related, in addition to the highly rigid and preorganized bispidine structure, to possible Zn²⁺ complexation by the pending carboxylates and the lysine ε-amine to form a dinuclear [MnL¹Zn] species. A combined NMRD and ¹⁷O NMR study evidenced that the hydration number remains one for each of these complexes. The enhanced relaxivity of [MnHL¹] originates from optimized second-sphere effects, superior to that for the corresponding glutamate derivative [MnL⁴]²⁻. NMR studies revealed interactions between the ε-amino group of the lysine and carboxylate groups through hydrogen bonding, leading to rigidification of the lysine
<figure><img src="image_33.png" /></figure>

chain. Altogether, these findings provide valuable structural and mechanistic insights to help the design of Mn²⁺-based MRI probes with enhanced contrast agent efficacy and safety. Beyond MRI, optimization of the kinetic inertness is also useful in the development of bispidine complexes for nuclear imaging modalities, using Mn-52 as well as other radiometals.

## 4. Experimental Section

Synthetic details to obtain the ligands are provided in the Supporting Information. Potentiometric titrations were performed in 0.15 M aqueous NaCl under a nitrogen atmosphere, and the temperature was controlled at 25 ± 0.1 °C. For the determination of stability constants of MnL², MnHL³, and MnL⁴, batch samples (0.5 mL) were prepared between pH = 2.5 and 7.0 at a 1:1 Mn:L² ratio ($c_{Mn} = c_{L} = 2$ mM, 0.15 M NaCl). The samples were kept at 25 °C until the equilibrium state was reached (1 d at pH ≈ 2.5 and ≈2 h at pH ≈ 6). This was verified by monitoring the stabilization of the relativity and the pH values in the samples over time. Kinetic inertness of the complexes was assessed at 37 °C and in 0.1 M NaCl, via transmetallation studies of the MnL complex (1 mM) with Zn²⁺ at pH = 6 (0.030 M MES buffer), pH = 3.1 (0.050 M KH-phthalate buffer), pH = 3.7 and pH = 4.1 (0.03 M dimethylpiperazine), and pH = 5 (0.030 M N-methylpiperazine), in the presence of 50-fold excess of Zn²⁺, in pseudo-first order conditions. For the duration of the experiments (up to 5 months), the samples were stored in a thermostat at 37 °C, and the water proton relaxation rates at 60 MHz were monitored over time using a Bruker–Minispec relaxometer. The pH was controlled for each sample at the end of the kinetic measurements to confirm that it remained stable during the experiment. The dissociation was also followed at higher acidities (0.01–1 M), without Zn²⁺ (25 °C, I = 1 M NaCl + HCl).

Proton NMRD profiles of MnHL³ and MnL⁴ complexes were recorded in an aqueous solution ($c_{MnL} = 1$ mM, pH = 7) on a Stelar SMARTracer Fast Field Cycling relaxometer (0.01–10 MHz) and a Bruker WP80 NMR electromagnet adapted to variable field measurements (20–80 MHz) and controlled by a SMARTracer PC-NMR console. The temperature was monitored by a VTC91 temperature control unit and maintained by a gas flow. The temperature was determined by previous calibration with a Pt resistance temperature probe. Variable temperature ¹⁷O transverse ($T_2$) relaxation times and chemical shifts of aqueous solution of manganese complexes (for MnHL³: $c_{MnL} = 3.35$ mmol/kg, pH = 7.4; for MnL⁴: $c_{MnL} = 3.60$ mmol/kg, pH = 7.3) were measured using a Bruker ARX 400 spectrometer (9.4 T). To improve sensitivity in ¹⁷O NMR, ¹⁷O-enriched water (11.10% H₂¹⁷O, Cortecnet) was added to the solutions to yield ≈1% ¹⁷O enrichment. An acidified water solution (HClO₄, pH 3) was used as an external reference.[5,27,34,43,44,53–63]

### Supporting Information

The authors have cited additional references within the Supporting Information.[5,34,43,44,53-65]

### Acknowledgements

M.S. and D.N. contributed equally to this work. The authors thank the French Centre National de la Recherche Scientifique (CNRS) and the French National Research Agency (grant ANR-18-CE18-0008)

for funding. The authors thank the Mo2ving platform of CBM for NMR measurements.

### Conflict of Interest

The authors declare no conflict of interest.

### Data Availability Statement

The data that support the findings of this study are available in the supplementary material of this article.

**Keywords:** bispidine · kinetic inertness · manganese · magnetic resonance imaging contrast agent · relaxivity

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Manuscript received: July 9, 2025

Revised manuscript received: August 20, 2025

Version of record online: