Design of bifunctional pyridinophane ligands as Mn²⁺ MRI agents for diagnostic imaging of Alzheimer's disease

Abstract

Alzheimer's disease (AD) is a progressive neurodegenerative disorder whose prevalence is rising with the aging of the global population. Among the proposed pathological hallmarks, the beta-amyloid (Aβ) peptide aggregates and soluble Aβ oligomers are established biomarkers and remain valuable diagnostic targets. While positron emission tomography (PET) imaging agents dominate AD diagnostic imaging, there are no FDA-approved MRI agents for AD. Herein, we report five bifunctional chelators built on the 2,11-diaza[3.3](2,6)pyridinophane framework, and which were evaluated as chelators for Mn²⁺-based MRI contrast agents. Based on in vitro studies, including thermodynamic stability and kinetic inertness measurements, T₁ relaxivity and ¹⁷O transverse relaxivity measurements to extract hydration numbers and water-exchange parameters, we obtained a clear structure–activity correlation for the corresponding bifunctional chelators: anionic picolinate and acetate arms increase thermodynamic stability and kinetic inertness, while the benzothiazolyl-phenol arm accelerates water exchange. Importantly, a high hydration number alone is insufficient, as a rapid water exchange is also needed for an appreciable contrast. Moreover, we show both the bifunctional chelators and their Mn²⁺ complexes exhibit appreciable affinity for Aβ aggregates, both in vitro and in 5xFAD mouse brain sections. [Mn(TE-8)], the most kinetically inert complex with favorable relaxivity, log D, and Aβ affinity, was advanced to in vivo MRI studies. Unlike MnCl₂, which accumulates non-specifically, [Mn(TE-8)] cleared through renal and hepatobiliary routes and produced measurable brain contrast enhancement. Together, these results establish the diazapyridinophane scaffold as a viable first-generation platform for blood–brain barrier (BBB)–permeable Mn²⁺ MRI contrast agents.

Herein, five bifunctional pyridinophane chelators were employed to develop Mn(ii) MRI contrast agents for the detection of beta-amyloid peptide aggregates, one of the hallmarks of Alzheimer's disease.

Introduction

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that significantly impairs memory, cognition, and daily functioning.^1–3 AD is the leading cause of dementia and ranks as the fifth leading cause of death.⁴ In the United States, approximately 5.8 million individuals are currently living with AD dementia, a figure projected to increase to 13.8 million by 2050. Multiple mechanisms contribute to AD pathogenesis, including the accumulation of amyloid beta (Aβ) plaques, neurofibrillary tangles, oxidative stress, neuroinflammation, mitochondrial dysfunction, and synaptic impairment.^5–7 The amyloid hypothesis has been a predominant theory, positing that Aβ deposition is central to AD, followed by the formation of neurofibrillary tangles, neuronal death, and dementia.^8–10 Aβ peptide is generated by the abnormal cleavage of amyloid precursor protein (APP) by β- and γ-secretases, producing several forms, with Aβ₄₀ and Aβ₄₂ being the most prevalent.^11–13 Aβ₄₂ exhibits a greater tendency to aggregate, leading to the formation of Aβ plaques, which serve as a key biomarker for AD diagnosis.^5,14,15 Furthermore, both soluble and insoluble Aβ oligomers are considered highly neurotoxic.^16–19 Neuropathological studies and in vivo quantitative neuroimaging have demonstrated that cerebral Aβ accumulation is among the earliest preclinical biomarkers of AD.^20–22 Consequently, Aβ plaques and soluble oligomers represent promising targets for imaging agents aimed at the early diagnosis of the disease.

Currently, the diagnosis of AD employs a range of techniques, including behavioral and cognitive assessments, blood and cerebrospinal fluid (CSF) analyses, neurological evaluations, and brain imaging.^1,23–25 Due to the highly invasive nature of CSF collection, recent research has prioritized the identification of alternative non-invasive or minimally invasive biomarkers for early AD diagnosis. Positron emission tomography (PET) imaging agents targeting amyloid have been extensively investigated, with Pittsburgh compound B (PIB) recognized as a prominent agent that provides high image resolution and enables differentiation between AD and non-AD patients.^26–29 However, the use of PIB is constrained by its reliance on the short-lived isotope ¹¹C, which limits its commercial application. The Food and Drug Administration has subsequently approved PET imaging agents based on ¹⁸F, such as [¹⁸F]AV45, [¹⁸F]florbetaben, and [¹⁸F]flutemetamol (Fig. 1).^30–32 Although ¹⁸F offers a longer half-life than ¹¹C, its 110 minute half-life still presents logistical challenges. In contrast, radiometals such as ⁶⁴Cu are advantageous due to their extended half-lives. Importantly, our group has shown that these same frameworks tolerate conjugation to Aβ-binding fragments without loss of chelation, yielding functional bifunctional chelators (BFCs) for ⁶⁴Cu-based compounds that employ robust chelating frameworks such as 2,11-diaza[3.3](2,6)pyridinophane and 1,4,7-triazacyclononane (TACN).^33–38 The bifunctional chelators were further conjugated to small molecule ligands known to bind Aβ plaques, including Thioflavin T (ThT) and stilbene derivatives inspired by ¹⁸F-labeled FDA-approved compounds.^39–42

Fig. 1. (a) Representative small molecules commonly employed as positron emission tomography (PET) imaging agents for Alzheimer's disease, (b) design principles underlying the compounds investigated in this study, (c) previously reported simple pyridinophane ligands with potential for Mn²⁺ MRI applications,⁴³ and (d) bifunctional chelators examined in this study.

MRI is an appealing complement to PET, as it avoids ionizing radiation and offers higher spatial resolution, yet there are no FDA-approved MRI agents for AD diagnosis. Designing such an agent is difficult because the requirements pull in opposite directions: the physicochemical properties that make an effective MRI agent conflict with those needed for efficient blood–brain barrier (BBB) permeability. Successful MRI agent design must address criteria such as thermodynamic stability, kinetic inertness, and the presence of available coordination sites for water binding, which are essential for enhancing relaxivity and optimizing water exchange rates. To achieve brain penetration, MRI agents must also possess properties that facilitate passage through the BBB, a highly selective membrane that restricts central nervous system (CNS) access.^44,45 Key considerations include lipophilicity, molecular weight, topological polar surface area (TPSA), and the number of hydrogen bond donors and acceptors, all of which must remain within specific thresholds.^46–48 This opposite requirements between the hydrophilicity needed for relaxivity and lipophilicity needed for brain uptake is the central design challenge. Mn²⁺, administered as MnCl₂, has been utilized as contrast agents because Mn²⁺ can cross the BBB and accumulate in specific brain regions, such as the olfactory bulbs and hippocampal formation.^49–51 Mn²⁺-enhanced MRI (MEMRI) has been employed in numerous animal studies, including those focused on Alzheimer's disease, as Mn²⁺ can enter neurons via voltage-gated calcium ion channels due to the similar ionic radii of Mn²⁺ and Ca²⁺.^49,52–54 Recently, Brewster et al. reported one of the few examples of a Mn²⁺-based MRI agent for imaging amyloid-beta aggregates, utilizing the texaphyrin framework, which has demonstrated both diagnostic and therapeutic effects in Alzheimer's disease models.⁵⁵ Due to the limited literature on Mn²⁺-based MRI agents, further development of such agents is essential to facilitate early diagnosis of Alzheimer's disease.

In addition to PET and MRI, fluorescent probes that generate detectable signals upon interaction with Aβ aggregates have been employed. Common design strategies for these probes include restriction of intramolecular rotation (RIR), modulation of intramolecular charge transfer (ICT), and adjustment of fluorescence in response to the local polarity and microenvironment of the fluorophore.⁵⁶ For RIR-based probes, binding to Aβ restricts molecular motion and suppresses nonradiative relaxation, which enhances fluorescence. This approach was recently demonstrated with ZY-12-OMe, a stilbene-based Aβ probe that exhibits enhanced, blue-shifted fluorescence upon Aβ binding due to restricted aromatic ring rotation and changes in the local hydrophobic environment.^42,57 In contrast, donor-π-acceptor fluorophores can undergo twisted intramolecular charge transfer (TICT) in aqueous solution, resulting in weak fluorescence. However, binding within the hydrophobic environment of Aβ aggregates can suppress TICT and promote a more emissive state.^58–60 Recent examples include BODIPY-based near-infrared probes such as BQ-1, which was specifically developed for Aβ42 aggregates. BQ-1 revealed a remarkable 189.5-fold increase in fluorescence upon binding, attributed to a TICT-based mechanism.⁶¹ Other fluorophore scaffolds, such as benzothiazole, cyanine, and distyrylbenzene derivatives have also been investigated to achieve Aβ-responsive fluorescence and enhance sensitivity for various Aβ species.

Recent studies by our group^43,62 and Toàn et al.⁶³ have demonstrated that the pyridinophane framework is well suited for Mn²⁺ due to its thermodynamic stability, kinetic inertness, and favorable relaxivity properties. In this study, the pyridinophane ligand framework was functionalized with a benzothiazole group, a small molecule structurally similar to ThT and PIB. This design strategy was previously applied to ⁶⁴Cu chelators, where the pyridinophane framework underwent a Mannich reaction to append the benzothiazole group to the secondary amine of the macrocycle. The remaining secondary amine was modified with various chelating arms, including picolyl, picolinate, and acetate groups, or was left unmodified or methylated. Some of these compounds had been previously reported and were repurposed for Mn²⁺, while others were newly synthesized for this investigation. To address the complex requirements for developing a Mn²⁺ MRI agent for Alzheimer's disease, a library of compounds was evaluated for their coordination abilities, specifically thermodynamic stability, kinetic inertness, and redox stability with Mn²⁺. Additional assessments included in vitro binding affinity to Aβ₄₂, topical staining of bifunctional compounds on mouse brain slices, log D evaluation, and cell toxicity to identify candidates for in vivo evaluation. In vivo MRI studies were performed on the most promising compounds. Results were compared to MnCl₂, a non-specific MRI agent, to assess each compound's effectiveness in enhancing contrast in murine models.

Results and discussion

Synthesis of organic ligands

The synthesis of the chelators involved a Richman-Atkins cyclization of S1 and S2 to produce ^TsN4. Subsequent deprotection of ^TsN4 with sulfuric acid yielded ^HN4, which served as a common precursor for the unsymmetric chelators examined in this study. The objective was to employ the benzothiazole fragment, known for its interaction with Aβ fibrils, and to systematically vary the substituent on the secondary amine to determine the optimal coordination number for Mn²⁺ complexes. Different synthetic routes were selected based on the chelating arm, as certain arms are acid sensitive and the benzothiazole moiety may be sensitive to either acid or base. For TE-7, synthesis commenced from ^HN4, followed by selective protection with di-tert-butyl dicarbonate (Boc₂O), yielding tert-butyl 3,7-diaza-1,5(2,6)-dipyridinacyclooctaphane-3-carboxylate (^HNBoc, S7) in 40% yield. An S_N2 reaction between ^HNBoc and 2-bromomethylpyridine·HBr in the presence of Na₂CO₃ produced a mixture of Boc-protected and Boc-deprotected products, likely due to the mildly acidic conditions of the HBr salt. The crude mixture underwent Boc deprotection with 4 M HCl in dioxane, affording S8 in 37% yield over two steps. Installation of the benzothiazole group via a Mannich reaction with paraformaldehyde yielded TE-7 after column purification. For TE-8, ^HNBoc (S7) underwent an S_N2 reaction with picolinate methyl bromide in the presence of Na₂CO₃, affording tert-butyl 7-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-3,7-diaza-1,5(2,6)-dipyridinacyclooctaphane-3-carboxylate (^BocN4picolinate) in 46% yield (Fig. 2). Subsequent Boc deprotection with 4 M HCl in dioxane, followed by a Mannich reaction to introduce the benzothiazole group, afforded the protected TE-8 ligand in 53% yield. Basic hydrolysis with LiOH was employed to hydrolyze the picolinate group, thereby minimizing product decomposition, as the bifunctional ligands may be sensitive to acidic conditions. For TE-9, a distinct synthetic route was utilized: ^HN4 was reacted with tert-butyl bromoacetate to yield both mono- and di-alkylated products, which were separated by column chromatography on basic alumina to isolate the desired mono-alkylated product in 35% yield. A subsequent Mannich reaction on the mono-alkylated product afforded protected TE-9 in 67% yield. The synthesis of TE-10 involved reacting ^HNBoc with the benzothiazolyl-phenol and paraformaldehyde via a Mannich reaction, affording the protected ligand in 58% yield. Deprotection of the Boc group with 4 M HCl in dioxane produced the final ligand in 76% yield. TE-11 was synthesized according to a previously reported procedure, with minor modifications to the purification process, utilizing a basic alumina column to achieve a 60% yield of the final ligand.

Fig. 2. Synthetic scheme corresponding to the synthesis of bifunctional chelators explored in this study.

Mn complex synthesis and X-ray characterization

Metal complexes were synthesized under anaerobic conditions by combining the appropriate ligands with Mn(ii) salts, including Mn(ClO₄)₂, Mn(OTf)₂, or MnCl₂, in the presence of a base. The resulting Mn²⁺ complexes were isolated as powders, and single crystals suitable for X-ray diffraction analysis were obtained through vapor diffusion or solvent-layering techniques. Crystals of [Mn(TE-7)]OTf were grown by layering a solution of the Mn complex in acetonitrile and methanol with diethyl ether. The complex crystallized in the centrosymmetric space group P1̄, with the Mn2 atom located on a crystallographic inversion center (Fig. 3a). The coordination environment of [Mn(TE-7)] is best described as a distorted pentagonal bipyramidal geometry. Notably, the axial amine donor atoms N3 and N4 exhibit elongated Mn–N bond distances of 2.407 and 2.415 Å (Table 1), consistent with relatively weak axial coordination.

Fig. 3. ORTEPs (50% ellipsoid probability) of X-ray crystal structures of (a) [Mn(TE-7)]⁺ and (b) [Mn(TE-11)]Cl². Most hydrogen atoms were omitted for clarity, and anions and solvent molecules are shown only for key interactions.

Table 1. Mn–ligand distances (Å) for Mn complexes.

Complex	TE-7	TE-11
Mn1–N1	2.294(3)	2.2545(12)
Mn1–N2	2.290(3)	2.2390(12)
Mn1–N3	2.407(3)	2.4091(12)
Mn1–N4	2.415(4)	2.3665(12)
Mn1–N5	2.297(4)
Mn1–O1	2.240(3)
Mn1–O2	2.275(3)
Mn1–Cl1		2.4288(4)
Mn1–Cl2		2.4177(4)

The benzothiazolyl moiety contains a μ-oxygen bridge derived from the deprotonated phenol group, linking two Mn²⁺ centers. One Mn²⁺ ion is coordinated by the pyridinophane ligand framework, while the second Mn²⁺ ion is preferentially bound by the O atoms from the benzothiazolyl fragment, including both the methoxy and the hydroxyl groups. The weakly coordinating triflate anion also participates in coordination to both Mn centers. This type of μ-phenoxo-bridged Mn²⁺ coordination environment is commonly observed in manganese-containing enzymes, such as catalases.⁶⁴ The formation of such structures is facilitated by the relatively large ionic radius, high-spin d⁵ electronic configuration, and kinetic lability of Mn²⁺. Consequently, Mn²⁺ readily accommodates additional weak donor ligands, including oxygen-containing groups and weakly coordinating anions, which can contribute to stabilization of the metal center. Although direct correlations between solid-state and solution-phase structures should be made cautiously, it is expected that the weakly bound triflate ligands dissociate in solution and are replaced by solvent molecules, particularly water. Such water-accessible coordination sites are advantageous for MRI applications, as they can enhance water exchange and improve relaxivity, thereby increasing imaging efficiency.

For [Mn(TE-11)], MnCl₂ was used as the Mn salt, yielding a (TE-11) Mn–dichloride complex with an octahedral geometry. In this structure, the benzothiazolyl group does not coordinate to the Mn center, and the longest bonds are Mn–Cl1 and Mn–Cl2 (Fig. 3b). These chloride ligands are expected to dissociate readily in solution, allowing water molecules to bind to the Mn²⁺ center. This hypothesis is supported by ¹⁷O transverse relaxivity measurements. Attempts to crystallize the other three complexes were unsuccessful.

Determination of protonation constants and stability constants of Mn²⁺ complexes

Spectrophotometric titrations were used to determine the protonation constants of ligands containing functional groups such as pyridines, picolinates, and benzothiazoles. These groups undergo protonation and deprotonation, which are accompanied by changes in their UV-vis absorption spectra. The phenol group within the benzothiazole moiety serves as an effective spectroscopic probe because its deprotonation induces a bathochromic shift in the absorption spectrum. This shift is attributed to increased electron density and enhanced delocalization of the negative charge from the phenolate oxygen into the aromatic ring, which reduces the HOMO–LUMO energy gap and results in a red shift in absorption (Fig. 4). The absorbance changes were monitored by tracking the λ_max of the absorption bands corresponding to the different protonation states of the phenol group.

Fig. 4. (a) UV-Vis absorption spectra of TE-8 and (c) TE-9 (4 × 10⁻⁵ M, I = 0.1 M KCl, 25 °C) were recorded at various pH values. (b and d) Speciation plots show the distribution of TE-8 and TE-9 species, respectively. Dotted blue triangles indicate spectral changes at λ_max 329 nm (TE-8) and 326 nm (TE-9), with best fit lines in red. Dotted yellow triangles indicate changes at λ_max 367 nm (TE-8) and 356 nm (TE-9), with best fit lines in black. The bathochromic shifts at 367 nm (TE-8) and 356 nm (TE-9) result from deprotonation of the phenol group in the benzothiazolyl moiety. Ligands were prepared in DMSO and diluted to micromolar concentrations, with final DMSO content below 2% (v/v). (e and g) UV-Vis spectra of [Mn(TE-8)] and [Mn(TE-9)] (4 × 10⁻⁵ M, I = 0.1 M KCl, 25 °C) were measured at varying pHs. (f and h) Speciation plots show species distribution. Blue and yellow triangles represent changes at λ_max values of 327/324 nm and 367/359 nm, with the best-fit lines shown. Bathochromic shifts at 367 nm (TE-8) and 359 nm (TE-9) result from phenol deprotonation. Ligands were prepared in DMSO, while the metal salt was prepared in water.

Potentiometric titrations could not be performed due to the limited aqueous solubility of these compounds. Therefore, the ligands were prepared as DMSO stock solutions and diluted to micromolar concentrations, ensuring that the final DMSO content in the titration vessel remained below 2% (v/v). The pH-dependent changes in UV-vis were sufficiently pronounced to enable reliable determination of pK_a values. Boros et al.⁶⁵ have indicated that absorbance changes greater than 0.2 are desirable for spectrophotometric titrations. In the present study, the observed absorbance changes met this criterion, supporting the use of spectrophotometric titration to determine protonation constants. All titrations were conducted in the presence of 0.1 M KCl to maintain constant ionic strength. Pyridinophane ligands act as effective proton sponges, impeding complete deprotonation except under basic pH conditions.^66,67 A comparison of the most basic proton, attributed to the tertiary amine pK_a of TE-7 and TE-9, indicates that substituting a pyridyl arm with an acetate arm slightly reduces the overall basicity of the ligands. The most basic pK_a of TE-7 is 10.56, whereas that of TE-9 is 10.06 (Table 2). The most acidic pK_a of TE-7, with a value of 1.83, likely corresponds to deprotonation of the pyridyl arm, which aligns with previous reports on related macrocyclic ligands of cyclam containing a pyridyl arm (Table 2).⁶⁸ For TE-9, the most acidic pK_a likely corresponds to deprotonation of an acetate group, consistent with prior work on analogous chelators bearing acetate arms. A comparison of TE-10 and TE-11, which differ by a methyl group, demonstrates that TE-11 is more basic than TE-10. These differences were expected, as N-methylation increases the amine's electron-donating ability through hyperconjugation from the methyl group.

Table 2. Protonation constants of synthesized chelators TE-8, TE-8, TE-9, TE-10, and TE-11. Titrations were performed in the presence of 0.1 M KCl. Ligands were prepared in DMSO and diluted to micromolar concentrations, with final DMSO content below 2% (v/v).

	log KHL	log KH2L	log KH3L	log KH4L
TE-1a	9.84(3)	5.36(4)	3.56(5)
TE-4a	9.25(3)	5.99(4)	3.09(7)	2.84(6)
TE-7	10.56(6)	7.99(6)	5.27(6)	1.83(7)
TE-8	11.54(3)	8.52(3)	6.43(3)	2.75(4)
TE-9	10.06(1)	7.23(5)	5.47(5)	3.29(2)
TE-10	8.72(1)	5.59(3)	5.20(7)	2.85(3)
TE-11	10.25(7)	7.85(7)	4.43(9)	4.01(8)
BPPAb	8.65(1)	6.26(1)	2.92(1)
1,4-Et4DO2Ac	11.5(1)	11.1(3)	4.4(2)	3.6(2)

^aPreviously reported pyridinophane-based ligands using potentiometric titrations in 0.16 M NaCl.⁴³

^bPyridinophane-based ligand reported by Toàn et al.,⁶³ using potentiometric titrations in 0.16 M NaCl.

^cDO2A-type ligands reported by Xu et al.,⁶⁹ using potentiometric titrations in 0.1 M KCl.

Knowledge of protonation constants is required before conducting metal complex titrations because ligand protonation and metal complex formation compete in solution. To ensure the accuracy of the stability constants, ligand protonation constants are entered into HypSpec software together with metal formation constants. This approach allows for differentiation between simple ligand protonation and complex formation. Stability constants of the Mn²⁺ complexes were determined using spectrophotometric titrations. In this method, the ligand and a source of Mn²⁺ salt were premixed to form the complex in situ. The resulting complex was then dissolved in 0.1 M KCl to maintain ionic strength, and the pH was adjusted using 0.15 M KOH. Similar to the free ligands, the Mn²⁺ complexes exhibited a bathochromic shift upon phenol deprotonation, resulting in measurable changes in the UV-Vis spectra (Fig. 4). Among the Mn²⁺ complexes in this series, [Mn(TE-8)] exhibited the highest log K_MnL value of 17.13. This observation aligns with the finding that chelators with higher denticity, such as those with seven coordinating atoms, generally form the most thermodynamically stable complexes, as reported by Uzal-Varela et al.⁷⁰ The second highest log K_MnL value was observed for [Mn(TE-9)] at 15.75. Previous studies indicate that picolinate and acetate ligands, classified as borderline bases according to Pearson's hard and soft acid–base theory, are well matched to the Mn²⁺ ion, which is a borderline acid. These ligand arms typically confer significant thermodynamic stability. While log K_MnL values represent the thermodynamic stability of Mn complexes with specific protonation states, calculated pMn values more accurately reflect the stability of Mn complexes at defined pH values. Within this series, [Mn(TE-9)] displayed the highest pMn value of 9.04, followed by [Mn(TE-8)] with a pMn value of 8.52. Overall, both TE-8 and TE-9 ligands demonstrated strong affinity for Mn²⁺ ions, making them promising candidates for further evaluation. A comparison of the stability constants for Mn²⁺ complexes of the bifunctional ligands described herein and the previously reported Mn²⁺ complexes of TE-1, TE-4, and BPPA ligands indicates that the stability constants and pMn values are similar to those obtained by potentiometric titrations (Table 3). In certain cases, such as the substitution of a carboxylate arm of TE-4 with a benzothiazolylphenolate arm in TE-9, a slight reduction in log K_MnL and pMn was observed, likely due to increased steric hindrance around the metal center. The lower log K_MnL values for more sterically hindered ligands can be partially explained by steric interactions within the metal-binding cavity, which may impede optimal Mn²⁺ coordination. This trend has also been reported by Uzal-Varela et al.,⁷⁰ who attributed the decreased stability constants of Mn²⁺ complexes to the presence of bulkier substituents on the ligands.

Table 3. Stability constants of synthesized chelators TE-8, TE-8, TE-9, TE-10, and TE-11 with Mn²⁺. Titrations were performed in the presence of 0.1 M KCl. Ligands were prepared in DMSO (final DMSO <2%), metal salt in water.

	log KMnLH2	log KMnLH	log KMnL	log KMnLOH	pMna
[Mn(TE-1)]b			13.20(4)	9.63(4)	7.94
[Mn(TE-4)]b			16.76(5)	10.18(5)	9.98
[Mn(TE-7)]+		6.05(3)	13.75(3)	5.98(4)	7.53
[Mn(TE-8)]	5.4(1)	6.68(8)	17.13(9)	7.55(8)	8.52
[Mn(TE-9)]		5.49(1)	15.75(1)	7.55(1)	9.04
[Mn(TE-10)]2+		5.46(6)	12.72(6)	5.76(6)	7.51
[Mn(TE-11)]2+		5.15(1)	14.45(1)	9.48(1)	8.01
[Mn(BPPA)]+c		1.69(3)	16.45(1)	12.19(2)	10.08
[Mn(1,4-Et4DO2A)]d		4.5(1)	17.9(1)		7.5

^a pMn calculated as −log[Mn]_free where [Mn²⁺] = 10⁻⁵ M, [L] = 10⁻⁵ M, pH = 7.4.

^bReported by our group for related Mn²⁺ complexes using potentiometric titrations in 0.16 M NaCl.⁴³

^cReported by Toàn et al.⁶³ for an Mn²⁺ complex using potentiometric titrations in 0.16 M NaCl.

^dXu et al.⁶⁹ Reported DO2A-type Mn²⁺ complexes characterized using potentiometric titrations.

Evaluation of kinetic inertness and electrochemical properties

Mn²⁺ complexes are intrinsically labile due to the negligible ligand-field stabilization of the d⁵ configuration, making resistance to transmetalation a key design criterion. In line with the Irving-Williams series, Zn²⁺ readily outcompetes Mn²⁺ for ligand binding, necessitating a stringent evaluation of kinetic inertness. Kinetic inertness was assessed by exposing Mn²⁺ complexes to 25 equivalents of ZnCl₂ and monitoring the decrease in Mn²⁺ complexes alongside the corresponding increase in Zn²⁺ complexes using high-performance liquid chromatography (HPLC), as the two complexes exhibit distinct retention times. The challenge conditions of 25 equiv. of ZnCl₂ were selected based on other studies, as they provide a good comparison with other complexes in literature.^63,71 Certain Mn²⁺ complexes, including [Mn(TE-9)] and [Mn(TE-10)]²⁺, were unstable under Zn²⁺ challenge. Specifically, [Mn(TE-9)] dissociated immediately under these conditions. For [Mn(TE-10)]²⁺, a color change from yellow to brown was observed during the experiment, suggesting that the Mn²⁺ complex may undergo oxidation over time. The oxidation of Mn²⁺ phenolate compounds has been previously reported, as phenolates can function as redox-non-innocent ligands, rendering them redox-unstable or highly reactive.⁷² In contrast, Mn²⁺ complexes of TE-7, TE-8, and TE-11 displayed dissociation half-lives of 37, 120, and 15 min, respectively (Fig. 5a, b, Fig. S41, and Table 4). Increased coordination number correlated with enhanced resistance to Zn²⁺-mediated transmetallation, as observed for [Mn(TE-7)]⁺ and [Mn(TE-8)]. Ligand design exerts a dominant influence on kinetic inertness. For example, picolinate donor arms outperformed picolyl arms in terms of kinetic inertness. Additionally, N-methylation of amines enhanced the kinetic inertness of the corresponding metal complex, as demonstrated for [Mn(TE-11)]²⁺. Notably, [Mn(TE-8)] exhibited the longest half-life, establishing it as the most kinetically inert complex in this series and a leading candidate for in vivo application.

Fig. 5. (a) HPLC traces of [Mn(TE-8)] over the course of the challenge experiment with 25 equiv. ZnCl₂. (b) % remaining [Mn(TE-8)] as a function of time based on the integration area of peak. (c) Variable scan rate cyclic voltammograms of [Mn(TE-8)] in 1 : 1 (MeCN : H₂O) with 100 mM NaClO₄ as supporting electrolyte. (d) Differential Pulse Voltammogram of [Mn(TE-8)]. (e) Variable scan rate cyclic voltammograms of [Mn(TE-9)] in 1 : 1 (MeCN : H₂O) with 100 mM NaClO₄ as supporting electrolyte. (f) Differential Pulse Voltammogram of [Mn(TE-9)].

Table 4. Rates of transmetallation of the Mn complexes for the ligands reported in this series and relevant analogs, measured in presence of 25 equiv. Zn²⁺.

Metal complex	Rate (s−1)	t 1/2 (min)
[Mn(TE-1)]a	1.0 × 10−4	116
[Mn(TE-4)]a	6.8 × 10−4	17
[Mn(TE-7)]+	3.0 × 10−4	37
[Mn(TE-8)]	9.7 × 10−5	120
[Mn(TE-11)]2+	8.0 × 10−4	15
PyC3Ab	6.76 × 10−4	17
BPPAc	8.29 × 10−5	140

^aDissociation kinetics were conducted in water at 25 °C, which differs from the conditions used for other complexes, where experiments were performed at 37 °C in buffered solutions. Previously reported Mn²⁺ complexes by our group.⁴³

^bRef. 71.

^cRef. 63.

In addition to evaluating the kinetic inertness of the metal complexes, redox stability was assessed using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The experimental setup consisted of a glassy carbon working electrode, an Ag/AgCl reference electrode, and a platinum wire auxiliary electrode. When the oxidation potential was irreversible, DPV was employed to estimate the oxidation potential. Measurements were conducted in 1 : 1 MeCN : H₂O with 100 mM NaClO₄ as the supporting electrolyte. Since Mn³⁺ is generally a less effective relaxation agent than Mn²⁺, due to fewer unpaired electrons and a reduced impact on the dipolar moments of water protons, it was necessary to determine the oxidation potential of the Mn²⁺ complexes. The overall formal charges of the complexes in solution were assigned from the ligand speciation and the number of deprotonated anionic donor arms at physiological pH: [Mn(TE-7)]⁺, [Mn(TE-10)]²⁺, and [Mn(TE-11)]²⁺ were considered positively charged, while [Mn(TE-8)] and [Mn(TE-9)] were considered neutral. Analysis of the cyclic voltammograms and differential pulse voltammograms for [Mn(TE-7)]⁺, [Mn(TE-10)]²⁺, and [Mn(TE-11)]²⁺ revealed multiple oxidation features, indicating the presence of several species in solution (Fig. S41). Complete assignment of each species is challenging, as Mn complexes with oxygen-containing ligands often dimerize, frequently bridged by oxo (μ-O), hydroxo (μ-OH), or carboxylato (μ-OAc) groups.^64,73,74 For [Mn(TE-7)]⁺, crystallization of the complex demonstrated bridging ligands in the solid state. The solid-state structure may reflect one of the solution-state structures, potentially explaining the multiple oxidative features of Mn(ii)/Mn(iii) observed in Fig. S72. For [Mn(TE-8)] and [Mn(TE-9)], the oxidation potentials were less positive than those of the other complexes, consistent with their neutral character, as both possess two anionic donor arms coordinating to the Mn²⁺ center (Fig. 5c–f).

Evaluation of relaxation properties

The relaxivities of the Mn²⁺ complexes were evaluated under conditions consistent with previously reported studies involving pyridinophane ligands.^43,62,63 Specifically, relaxivities were measured in 50 mM HEPES buffer at pH 7.4. Among the compounds tested, [Mn(TE-7)]⁺, [Mn(TE-8)], and [Mn(TE-10)]²⁺ exhibited the highest r₁ relaxivity values of 3.38, 3.03, and 3.22 mM⁻¹ s⁻¹, respectively (Fig. 6). Comparison with non-targeted, simple Mn²⁺ complexes such as [Mn(TE-1)] and [Mn(TE-4)] revealed that the bifunctional chelators exhibited relaxivity values in a similar range.⁴³ This finding suggests that the benzothiazole moiety does not adversely affect the relaxivity of these bifunctional chelators relative to the non-targeted simple complexes, including [Mn(TE-1)], [Mn(TE-4)], and [Mn(BPPA)], as previously explored by Toàn et al.⁶³ and our research group.⁴³ Additionally, [Mn(TE-9)], which contains an acetate chelating arm, demonstrated lower relaxivity compared to [Mn(TE-8)], which features a picolinate chelating arm. This trend was also observed for the simple Mn²⁺ complexes. A comparison of the relaxivities of [Mn(TE-2)]⁺⁶² and [Mn(TE-8)], which differ in their N-amine substituents, shows that [Mn(TE-8)] exhibits slightly higher relaxivity. While [Mn(TE-2)]⁺ contains an N-methyl group,⁶² [Mn(TE-8)] incorporates a benzothiazolyl moiety. The enhanced relaxivity of [Mn(TE-8)] may be attributed to its greater molecular weight, which could contribute to slower molecular tumbling and consequently improved relaxation efficiency.

Fig. 6. Relaxivity values of the metal complex were measured in 50 mM HEPES buffer containing 0.15 M NaCl. Relaxivities were determined from the slope of the relaxation rate versus concentration using a 1.4 T NMR spectrometer at 33 °C. The relaxivity values for [Mn(TE-1)] and [Mn(TE-4)] were reported in a previous study.⁴³.

To elucidate the origin of the relaxivity enhancement observed in these bifunctional Mn²⁺ complexes, transverse ¹⁷O relaxivity experiments were conducted. Multiple factors influence the relaxation properties of contrast agents, including rotational correlation time (τ_r), which depends on the molecular weight of the agent, hydration number (q), water exchange rate (k_ex), ionizable proton exchange rate, the choice of buffer or solvent, and the field strength of the NMR instrument. The hydration number is typically determined using a method reported by Gale et al., which involves dividing the maximum ¹⁷O transverse relaxivity value by 510 mM⁻¹ s⁻¹, corresponding to q = 1.⁷⁵ However, this approach is limited when the water exchange rate is either too rapid or too slow, as observed for the compounds investigated in this study. In such cases, the maximum transverse relaxivity is not observed for all Mn²⁺ complexes because the presence of water in the samples causes freezing below 0 °C (273 K), thereby limiting further temperature reduction. Xu et al. recently demonstrated that their Mn²⁺ complexes with DO2A-derived ligands were in the fast exchange regime.⁶⁹ Consequently, they performed a global fit of either the hydration number (q) or the hyperfine coupling (A₀/h), holding the other parameter constant due to their interdependence. To determine the q values for these bifunctional complexes, a global fit of the Swift–Connick equation was conducted, following the approach of Xu et al.,⁶⁹ while maintaining a constant hyperfine coupling. The resulting q values were fractional, indicating a coordination equilibrium between hydrated (q = 1) and non-hydrated (q = 0) forms, rather than a single, well-defined inner-sphere water molecule.

Multiparameter fitting of the temperature dependence of the ¹⁷O T₂ value yields the water exchange rate (k_ex), along with the associated activation enthalpy and entropy for the water exchange process. Analysis of ln(1/T_2r) versus temperature, combined with curve fitting using the Swift-Connick equation, enables classification of the complexes into fast, intermediate, or slow exchange regimes. Most bifunctional Mn²⁺ complexes, specifically [Mn(TE-7)]⁺ through [Mn(TE-10)]²⁺, exhibit fast exchange rates (Fig. S44) compared to previously reported parent compounds^43,62 that lack the benzothiazolyl group. The only exception is [Mn(TE-11)]²⁺, which exhibits a significantly reduced exchange rate, as indicated by the curve in Fig. S45 and the calculated value in Table 5. The observed enhancement in exchange rates is attributed to the presence of the phenol group. Previous structure–activity relationship studies on Gd cyclen complexes have demonstrated that replacing a carboxylate arm in DOTA-type compounds with phenols and phosphonates substantially increases the exchange rates of the corresponding Gd³⁺ complexes.⁷⁶ In this series, the benzothiazolyl group functions as a binder to Aβ plaques and substantially increases the exchange rate with water molecules. For [Mn(TE-11)]²⁺, the markedly reduced exchange rate is likely due to the presence of the N–Me group. The absence of chelating groups that could serve as hydrogen bond acceptors, or the presence of a pyridyl group that could dissociate to allow water binding, may explain why [Mn(TE-11)]²⁺ has the slowest exchange rate. For [Mn(TE-7)]⁺, the high molecular weight of the Mn²⁺ complex, which slows the tumbling rate in solution, as well as steric compression around the Mn²⁺ center, are proposed as the primary factors contributing to the enhanced relaxivity of this compound. For [Mn(TE-8)], the appreciable relaxivity of 3.03 mM⁻¹ s⁻¹ is attributed to the slower tumbling rate in solution or the enhanced exchange rate, which may result from the additive effect of the phenol group and the picolinate arm that could enhance water exchange through hydrogen bonding. The observed large k_ex and small ΔH values indicate that the transition state associated with the association or dissociation of water molecules has a low energy barrier.⁶⁹ Additionally, the 6- and 7-coordinate states exhibit comparable energies. Overall, based on its in vitro relaxation properties, [Mn(TE-8)] emerges as a promising candidate for further in vivo evaluation.

Table 5. Parameters obtained from ¹⁷O transverse relaxivity measurements; values are shown as ranges based on error of fit.

Complex	q	ΔH‡ (kJ mol−1)	A 0/h (107 rad s−1)	T 1e (ns)	k ex (107 s−1)	τ m (ns)
[Mn(TE-7)]+	0.4 ± 0.1	22.4	4.0a	0.0081	594	0.17
[Mn(TE-8)]	0.5 ± 0.3	15.6	3.70a	0.005	240	0.42
[Mn(TE-9)]	0.7 ± 0.1	15.4	3.98a	3.1	461	0.22
[Mn(TE-10)]2+	0.5 ± 0.1	16.3	3.81a	5.1	133	0.75
[Mn(TE-11)]2+	0.6 ± 0.3	20.0	4.28a	0.03	8.06	12.4

^aThese values were held constant during the fitting.

Evaluation of Aβ-binding properties

Before in vivo evaluation, interactions between bifunctional chelators and amyloid-beta (Aβ) aggregates are typically assessed using titration-based fluorescence enhancement assays or competitive binding assays.⁷⁷ Fluorescence binding assays depend on the activation of intrinsic fluorescence when these molecules interact with Aβ₄₂ aggregates or fibrils. Titration with varying amounts of fluorescent probe increases fluorescence until a saturation curve is achieved. In this study, the molecules did not exhibit fluorescence activation; therefore, a competitive binding assay was performed using the established fluorescence reporter Thioflavin-T (ThT). ThT binds to Aβ₄₂ by targeting cross-β-sheet structures.⁷⁸ Upon binding to the rigid amyloid structure, ThT rotation is restricted, resulting in fluorescence activation. Several molecules, including Pittsburgh compound B (PIB) and other PET imaging agents such as ¹⁸F-flutemetamol, share structural similarities with ThT. The newly designed and repurposed bifunctional chelators containing the benzothiazole group also possess these structural features and were therefore expected to interact with Aβ₄₂ binding pockets also targeted by ThT. Sequential titration of increasing concentrations of the chelators displaces ThT from its binding pocket, leading to a decrease in fluorescence. The resulting competitive binding curves were fitted using a one-site competition model to determine K_i values. The K_i values for the higher denticity ligands TE-7, TE-8, and TE-9 were 0.66 μM, 1.01 μM, and 1.28 μM, respectively, whereas TE-10 and TE-11 exhibited binding affinities of 3.0 μM and 2.0 μM against Aβ₄₂ (Fig. 7 and Fig. S46). Notably, the bulkier ligands TE-7, TE-8, and TE-9 showed higher affinity for Aβ₄₂ than the unsubstituted ligands. This observation may be attributed to the fact that benzothiazole derivatives are known to have defined binding pockets near the C-terminal and central regions of Aβ, with key interacting amino acid residues including phenylalanine (Phe), glutamate (Glu), aspartate (Asp), glycine (Gly), isoleucine (Ile), methionine (Met), valine (Val), and alanine (Ala).^78,79 These residues interact with benzothiazole compounds through π–π stacking, hydrophobic interactions, and hydrogen bonding. The presence of picolyl, picolinate, and acetate groups was hypothesized to enhance these interactions, which could explain the lower affinity of TE-10 and TE-11 compared to TE-7, TE-8, and TE-9.

Fig. 7. Thioflavin T (ThT) fluorescence competition assays of (a) TE-8 (b) TE-9 (c) [Mn(TE-8)] (d) [Mn(TE-9)] with ThT-bound Aβ₄₂ fibrils ([Aβ] = 5 μM, [ThT] = 2 μM, λ_ex = 435 nm, λ_em = 485 nm). (e) Bar graph comparing the Ki values of ligands explored in this study and their corresponding Mn²⁺ complexes.

Compared with other fluorescent probes, such as CRANAD-based or BODIPY-based compounds that exhibit nanomolar affinities towards Aβ aggregates,^58,59 the bifunctional compounds examined in this study exhibit moderate affinities in the high nanomolar to low micromolar range. However, since these compounds are intended for use as MRI contrast agents that are typically administered at millimolar concentrations, their affinities towards Aβ aggregates is considered to be adequate, and it is anticipated that these probes will effectively saturate the binding sites of Aβ plaques in vivo.

The binding affinity of the metal complexes was evaluated using the same methodology as for the simple ligands, as MRI contrast agents are administered as metal complexes in the bloodstream. Therefore, the binding affinity of the metal complexes is more relevant for biological applications. Notably, the Mn²⁺ complexes [Mn(TE-8)] and [Mn(TE-9)] did not show reduced affinity towards amyloid beta (Aβ). In contrast, the other three complexes, [Mn(TE-7)]⁺, [Mn(TE-10)]²⁺, and [Mn(TE-11)]²⁺, exhibited lower affinity compared to their corresponding ligands. The observation that [Mn(TE-8)] maintained similar affinity to its ligand TE-8 is promising, as Mn²⁺ complexes are the biologically relevant species for MRI applications; these pre-formed complexes are formulated and administered intravenously in mice. Consistent with their speciation, both [Mn(TE-8)] and [Mn(TE-9)] are neutral at physiological pH, each bearing two anionic donor arms. Their higher affinity, relative to the other complexes, may be attributed to the ability of neutral molecules to interact more effectively with the hydrophobic pockets of amyloid beta due to reduced solvation. In general, charged molecules solvate water molecules more efficiently, which can hinder their binding to hydrophobic pockets. The energetic barrier for binding these neutral complexes to amyloid beta fibrils may be lower than that for charged Mn²⁺ complexes, potentially explaining the observed differences in affinity towards Aβ fibrils.

Brain section staining and log D measurements

The interactions between bifunctional chelators, their Mn²⁺ complexes, and native Aβ were assessed using histological staining of brain sections. Sections from 11-month-old 5xFAD mice were stained with the bifunctional chelators, followed by co-staining with the HJ3.4 antibody, which binds all forms of Aβ, including monomeric, soluble aggregates, fibrillar, and plaque-bound species.⁸⁰ Due to its broad binding properties, HJ3.4 is commonly used to quantify or visualize Aβ pathology by conjugation to a dye with a defined excitation and emission window, selected based on the fluorescence characteristics of the compounds under study.⁸⁰ In this experiment, AF594 was selected because the compounds exhibited fluorescence in the blue (DAPI) or green (GFP) channels, while AF594 emits in the red fluorescence window. The presence of various forms of amyloid beta was confirmed by staining with HJ3.4-AF594, with fluorescence observed in the red (Texas Red) channel. Immunostaining of TE-7, TE-10, and TE-11 demonstrated a correlation with in vitro Ki experiments. Pearson's R correlation values, calculated from the overlap of red fluorescence in the Texas Red channel (indicating the HJ3.4-AF594 labeling of Aβ plaques) and blue/green fluorescence in the DAPI/GFP channels (reflecting intrinsic compound fluorescence), were 0.61, 0.38, and 0.55 for TE-7, TE-10, and TE-11, respectively. Higher R values indicated greater colocalization between the compounds and Aβ plaques. The K_i affinity assays showed a similar trend, with TE-7 exhibiting a K_i of 0.66 μM, TE-10 a K_i of 3.0 μM, and TE-11 a K_i of 2.0 μM. The corresponding Mn²⁺ complexes were stained by preparing the Mn²⁺ complexes in situ before co-incubating the compounds with brain slices and the HJ3.4 antibody. These Mn²⁺ complexes generally demonstrated reduced affinity for Aβ, as indicated by lower Pearson's correlation values compared to the ligands. This observation is further supported by the affinity values derived from the K_i measurements.

For TE-8, the Pearson's R correlation value was the lowest among all evaluated chelators. The fluorescence spots corresponding to amyloid-beta plaques appeared more diffuse than the compound's fluorescence (Fig. 8). The inconsistency between the immunostaining and K_i experiments may be explained by two hypotheses. First, brain staining experiments require compounds with intrinsic fluorescence to visualize colocalization with amyloid-beta using fluorescence microscopy. TE-8 did not exhibit sufficient fluorescence, which may account for the lower Pearson's R value relative to the other compounds. Alternatively, TE-8 may not bind the same amino acid sequence as the HJ3.4 antibody. The HJ3.4 antibody recognizes a specific epitope in the N-terminal region (amino acid residues 1–13).⁸¹ Small-molecule dyes such as Congo red are known to bind to cross-β sheets.⁸² If TE-8 binds to a different amino acid residue, it may not demonstrate strong colocalization with the antibody. The cause of the disagreement between the two experiments cannot be definitively assigned. Despite the differences observed in the brain staining experiments for the TE-8 ligand, the corresponding Mn²⁺ complex demonstrated a similar R correlation value to the ligand, consistent with the K_i experiments. Given that [Mn(TE-8)] exhibited favorable K_i binding properties and an adequate brain staining profile, further in vivo evaluation was justified.

Fig. 8. Histological staining of 11-month-old female 5xFAD mice with TE-8 and TE-9 and their corresponding Mn²⁺ complexes (shown in blue and green channels, respectively) and costaining with AF594 fluorescently labeled antibody HJ3.4 (red channel). [Compound] = 25 μM; [HJ3.4-AF594] = 1 μg mL⁻¹. Scale bar: 125 μm.

In addition to in vitro K_i affinity assays and brain staining experiments, which can assess the affinity of newly developed bifunctional chelators for both fibrillar and native Aβ, several other properties are critical in determining whether compounds can penetrate the BBB, a highly restrictive interface that generally prevents the entry of compounds to protect the brain from neurotoxicity. Lipinski's Rule of 5, as well as other parameters such as topological surface area, plasma protein binding, and P-glycoprotein (P-gp) efflux susceptibility, often influence brain permeability.⁴⁷ Key criteria from Lipinski's Rule of 5 include a molecular weight less than 500 Da and log D values between 1 and 3, both of which are considered desirable for brain penetration.⁴⁷ To obtain log D, endpoint fluorescence readings were used as a readout in octanol-PBS partition experiments. Specifically, bifunctional chelators TE-7 through TE-11 were dissolved in a mixture of octanol and PBS, the tubes were vortexed and centrifuged, and the octanol layer was aliquoted into a 96-well plate. The remaining PBS layer was then back-extracted with octanol, and the logarithmic ratio of fluorescence between the two layers was calculated as the log D value. The log D values obtained for TE-7, TE-8, and TE-11 were promising (Table 6). However, when considering all other in vitro properties, TE-8 appears to be the most suitable candidate for in vivo evaluation.

Table 6. Summary of the molecular weights of all compounds evaluated in this study, as well as their log D values.

Compound	Molecular weight (g mol−1)	log D
TE-7	600.7	0.99 ± 0.06
TE-8	644.2	0.86 ± 0.02
TE-9	567.2	0.70 ± 0.03
TE-10	509.2	0.55 ± 0.03
TE-11	523.2	1.55 ± 0.02

In vivo MRI imaging

Prior to in vivo experimentation, cell-based assays were conducted to confirm the absence of toxicity of the compounds in murine models. Control experiments using MnCl₂ were performed in wild-type (WT) mice before evaluating the bifunctional compounds, and all in vivo MRI studies were performed on a 9.4 T scanner. Previous studies have demonstrated that MnCl₂ typically enters the brain through the BBB or the blood-cerebrospinal fluid (CSF) barrier.^83,84 Owing to the similar ionic radii of Mn²⁺ and Ca²⁺, Mn²⁺ generally enters neurons via voltage-gated calcium channels and accumulates in specific brain regions, such as the hippocampus and olfactory bulb.^49,85 The present control studies corroborated these findings, as Mn²⁺ accumulation was observed in these regions, evidenced by contrast enhancement in the Cornu Ammonis (CA) of the hippocampal region (Fig. 9). Whole-body MRI scans revealed that intravenous injection of MnCl₂ resulted in Mn²⁺ retention in multiple organs, including the heart, kidneys, liver, and intestines (Fig. S53). Analysis of normalized signal intensity graphs indicated a substantial increase in the signal-to-noise ratio (SNR) of MnCl₂ uptake, with a fourfold increase in the kidney and a fivefold increase in the liver (Fig. S53). The high signal-to-noise ratio is likely attributable to MnCl₂'s ability to bind multiple water molecules, which enhances relaxation properties and increases MRI contrast. However, MnCl₂ exhibits non-specific retention, limiting its suitability as an MRI agent due to a lack of specificity in excretion pathways. Previous studies examining the biodistribution of natural Mn²⁺ or ⁵²Mn support these observations, indicating that Mn²⁺ accumulates in various organs and displays an unfavorable biodistribution profile. The development of MRI agents for brain imaging remains challenging because compounds must be sufficiently lipophilic to cross the BBB, yet greater lipophilicity often leads to partial excretion via the hepatobiliary route.

Fig. 9. (a) Axial MRI images of the mouse brain demonstrate contrast differences before and after intravenous MnCl₂ administration, with enhanced contrast observed in the hippocampus. (b) Normalized brain signal intensity following intravenous MnCl₂ injection was determined by delineating regions of interest (ROIs) in the brains of n = 3 mice, subtracting muscle signal intensity, and dividing by air signal intensity: (SI brain – SI muscle)/SI air. Error bars represent the standard deviation for n = 3 mice.

Based on the promising in vitro properties of [Mn(TE-8)], including high thermodynamic stability, robust kinetic inertness, appreciable relaxivity, and strong binding affinity toward Aβ₄₂ fibrils, as well as the non-specific biodistribution of MnCl₂, an in vivo evaluation of [Mn(TE-8)] was conducted. The Mn complex was formulated in 85% phosphate-buffered saline (PBS), 10% cremophor, and 5% dimethyl sulfoxide (DMSO), and administered intravenously to cohorts of wild-type and AD mice to assess brain uptake. Whole-body scans following [Mn(TE-8)] injection in wild-type mice demonstrated both renal and hepatobiliary excretion, as indicated by contrast enhancement in the kidneys and liver (Fig. 10). Normalized signal intensity plots showed that the signal-to-noise ratio (SNR) in the kidney and liver was lower than that observed with MnCl₂, consistent with the reduced water binding capacity of chelated Mn due to fewer available coordination sites (Fig. 10). Despite this, contrast enhancement remained significant, and rapid clearance from the heart was observed (Fig. 10). This rapid cardiac clearance is advantageous because it suggests that the complex remains intact in murine models. Additionally, strong SNR uptake in the bladder indicates partial renal excretion of [Mn(TE-8)], which is favorable since renally excreted compounds are eliminated efficiently. Injection of [Mn(TE-8)] into a 5xFAD mouse resulted in contrast enhancement in the kidney and liver, similar to observations in WT mice, as demonstrated in the imaging data (Fig. S67). The normalized signal intensity graphs show elimination from the heart, indicating that [Mn(TE-8)] remains intact (Fig. S69).

Fig. 10. Axial MRI images of the mouse abdominal region following administration of 80 μmol kg⁻¹ [Mn(TE-8)] demonstrate contrast enhancement in the liver and kidney, indicating mixed hepatobiliary and renal clearance. (a) Normalized signal intensity of the kidneys for [Mn(TE-8)] was calculated by drawing regions of interest (ROIs) on the kidneys of n = 3 wild-type (WT) mice, subtracting muscle signal intensity, and dividing by air signal intensity: (SI kidney – SI muscle)/SI air. Error bars indicate standard deviation for n = 3 mice. (b) Normalized signal intensity of the liver was calculated by drawing ROIs on the liver of n = 3 mice, subtracting muscle signal intensity, and dividing by air signal intensity: (SI liver – SI muscle)/SI air. Error bars indicate standard deviation for n = 3 mice. (c) Normalized signal intensity of the heart was calculated by drawing ROIs on the heart of n = 3 mice, subtracting muscle signal intensity, and dividing by air signal intensity: (SI heart – SI muscle)/SI air. (d) Normalized signal intensity of the bladder was calculated by drawing ROIs on the bladder of n = 3 mice, subtracting muscle signal intensity, and dividing by air signal intensity: (SI bladder – SI muscle)/SI air. Coronal MRI images of the mouse brain demonstrate contrast differences before and after intravenous [Mn(TE-8)] administration, with enhanced contrast observed in the brain. (e) Normalized brain signal intensity following intravenous [Mn(TE-8)] injection was determined by delineating regions of interest (ROIs) in the brains of n = 3 mice, subtracting muscle signal intensity, and dividing by air signal intensity: (SI brain – SI muscle)/SI air. Error bars represent the standard deviation for n = 3 mice.

Coronal MRI images of the brain following intravenous injection of [Mn(TE-8)] in n = 3 wild-type mice demonstrated brain uptake, as indicated by contrast enhancement (Fig. 10). Administration of [Mn(TE-8)] in a 5xFAD mouse produced comparable contrast enhancement, as demonstrated by MRI images and normalized brain signal intensity. Quantitative analysis within a delineated region of interest confirmed brain uptake following compound administration. This finding is promising, as this Mn²⁺ complex represents one of the first-generation compounds evaluated for this application.

Conclusions

In summary, we have designed and characterized a series of five bifunctional chelators that append a benzothiazolyl-phenolate amyloid-binding group to the diazapyridinophane macrocycle while retaining a second secondary amine for a tunable chelating arm. Rather than a single lead compound, the series yields a coherent set of structure–property relationships for the design of Mn²⁺ MRI contrast agents. Kinetic inertness toward Zn²⁺ tracked with denticity and with the nature of the donor arms, with the picolinate arms outperforming the picolyl arm, while N-methylation further improved inertness, such that [Mn(TE-8)] bearing a picolinate arm was the most inert complex in the series. Anionic donor arms rendered [Mn(TE-8)] and [Mn(TE-9)] neutral at physiological pH, which correlated with better retention of Aβ affinity upon metalation, consistent with reduced solvation favoring engagement of hydrophobic amyloid pockets. The benzothiazolyl–phenolate arm accelerated water exchange across the series, whereas [Mn(TE-11)] exhibited a slower exchange rate than the other compounds, which may be attributed to the presence of the methyl group. Guided by this profile, the [Mn(TE-8)] compound, which combines high Mn²⁺ affinity, the best kinetic inertness in the series, an appropriate log D and appreciable Aβ affinity, was employed in in vivo MRI studies at 9.4 T. Importantly, its behavior differs markedly from MnCl₂: instead of non-specific organ accumulation, [Mn(TE-8)] shows mixed renal and hepatobiliary clearance and rapid cardiac washout, indicating the complex remains intact in circulation, and it produces measurable brain contrast enhancement. While brain enhancement did not differ significantly between wild-type and AD mice, we note that the relaxivity was measured at 1.4 T, while MRI studies were performed at 9.4 T, so the in vitro values should guide rather than quantitatively predict contrast at the in vivo imaging field. These results thus suggest [Mn(TE-8)] is a first-generation, BBB-permeable Mn²⁺ contrast agent that establishes the diazapyridinophane scaffold as a viable platform for amyloid-targeted MRI. The most promising next step is to increase amyloid affinity and inner-sphere water access simultaneously, for example by strengthening the Aβ-binding motif and adjusting the chelating arm to raise q without slowing the water exchange rate, to achieve an in vivo Aβ-specific enhancement that is directly relevant to AD diagnosis.

Conflicts of interest

A patent (PCT Patent Application No. PCT/US2025/046362, entitled “Pyridinophane Compounds and MRI Contrast Agents”, Inventors: Liviu M. Mirica and Tarek El Sayed, filing date: September 15, 2025) has been filed on the synthesis and MRI applications of the pyridinophane chelators and their Mn complexes. All authors declare no other competing interests.

Data availability

Supplementary information (SI): synthetic procedures, spectroscopic studies, binding affinity studies, imaging studies, cytotoxicity studies, and crystallographic details. See DOI: https://doi.org/10.1039/d6qi01745b.

CCDC 2565696 and 2565697 contain the supplementary crystallographic data for this paper.^86a,b