Christian Vanasschen
email: c.vanasschen@fz-juelich.de

Enikő Molnár

Gyula Tircso
email: gyula.tircso@science.unideb.hu

Ferenc K Kálmán

Éva Tóth

Marie Brandt

Heinz H Coenen

Bernd Neumaier

Gyula Tircsó

Novel CDTA-based, Bifunctional Chelators for Stable and Inert Mn II Complexation: Synthesis and Physicochemical Characterization

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

In recent years, research on contrast agents (CAs) for magnetic resonance imaging (MRI) increasingly focused on paramagnetic Mn II complexes as alternatives to the well-established Gd III chelates routinely used in the clinical setting. [START_REF] Drahoš | Manganese(II) Complexes as Potential Contrast Agents for MRI[END_REF][START_REF] Molnár | Picolinate-Containing Macrocyclic Mn2+ Complexes as Potential MRI Contrast Agents[END_REF][START_REF] Forgács | Mono-, Bi-, and Trinuclear Bis-Hydrated Mn2+ Complexes as Potential MRI Contrast Agents[END_REF][START_REF] Forgács | A Bisamide Derivative of [Mn(1,4-DO2A)] -Solution Thermodynamic, Kinetic, and NMR Relaxometric Studies[END_REF] As a matter of fact, some Gd III CAs have been associated with tissue-deposition of toxic (free) gadolinium in patients suffering from chronic kidney disease (CKD), which causes nephrogenic systemic fibrosis (NSF), a potentially lethal fibrotic disease of the skin and internal organs. [START_REF] Grobner | Gadolinium -a specific trigger for the development of nephrogenic fibrosing dermopathy and nephrogenic systemic fibrosis?[END_REF][START_REF] Marckmann | Nephrogenic Systemic Fibrosis: Suspected Causative Role of Gadodiamide Used for Contrast-Enhanced Magnetic Resonance Imaging[END_REF] Very recently, a review by Kanal and Tweedle summarized several studies even highlighting the deposition of Gd III in the brain of non-renally impaired patients, further questioning the use of certain marketed Gd-based CAs. [START_REF] Kanal | Residual or Retained Gadolinium: Practical Implications for Radiologists and Our Patients[END_REF] Compared to Gd III , Mn II has the major advantage of being essential to the human body as trace amounts are involved in several enzymatic as well as metabolic processes in vivo. [START_REF] Roth | Metallomics and the Cell[END_REF] In addition, the Mn II ion possesses all the requisite physical properties for T1-weighted MR applications: fast water exchange kinetics, slow longitudinal electronic relaxation and a high spin quantum number (S = 5/2). [START_REF] Drahoš | Manganese(II) Complexes as Potential Contrast Agents for MRI[END_REF] Despite the biogenic nature of Mn II , the relatively high paramagnetic metal concentrations typically used in MRI (0.05 -0.30 mmol.kg -1 ) require the cation to be chelated within a thermodynamically stable and kinetically inert complex to be used as a contrast agent. However, to allow for efficient in vivo imaging, manganese chelates should not only fulfill thermodynamic and kinetic stability requirements but also exhibit high relaxivity values. Relaxivity (r1) is a measure of CA efficiency and is defined as the enhancement of the T1 relaxation rate of water protons induced by a millimolar CA concentration (r1 = Δ(1/T1)/[CA] in mM -1 s -1 ); the higher the relaxivity, the higher the positive contrast on T1-weighted MR images. Importantly, to attain elevated r1 values, the paramagnetic complex needs to possess at least one water molecule coordinated to the central metal ion (hydration number, q, should be ≥ 1). Taking into account the coordination number (CN) of Mn II (CN = 6-7), the chelator of choice should therefore contain a maximum of six donor groups in order to allow the Mn II ion to carry a water coligand. Unfortunately, recent literature has shown that applying this rationale does not guarantee the obtained chelates to fulfill both stability/inertness and relaxivity criteria (in this manuscript, the terms lability/inertness describe chelate dissociation kinetics rather than solvent exchange kinetics). For example, [Mn(NOTA)] -(NOTA = 1,4,7-triazacyclononane-1,4,7-triacetic acid, see Chart 1) was shown to be exceptionally stable thermodynamically as well as kinetically inert but the complex does not contain a coordinated water molecule (q = 0) despite the hexadentate nature of the chelator. [START_REF] Drahos | Dissociation kinetics of Mn2+ complexes of NOTA and DOTA[END_REF] On the other hand, open-chain hexacoordinate [Mn(EDTA)] 2-combines q = 1 and a good thermodynamic stability with extremely high kinetic lability. [START_REF] Rolla | 1H and 17O NMR Relaxometric and Computational Study on Macrocyclic Mn(II) Complexes[END_REF][START_REF] Kálmán | Kinetic Inertness of the Mn2+ Complexes Formed with AAZTA and Some Open-Chain EDTA Derivatives[END_REF] Notably, the case of the cyclen derivatives 1,4-DO2A and 1,7-DO2A (denticity = 6; cyclen = 1,4,7,10-tetraazacyclododecane; Chart 1) appears particularly remarkable. Although both isomers form Mn II complexes that display similarly high stability [START_REF] Rolla | 1H and 17O NMR Relaxometric and Computational Study on Macrocyclic Mn(II) Complexes[END_REF] and inertness, [START_REF] Garda | Physico-chemical properties of MnII complexes formed with cis-and trans-DO2A: thermodynamic, electrochemical and kinetic studies[END_REF] the position of the pendant acetate arms has a striking impact on the hydration number as q = 0 for [Mn(1,7-DO2A)] versus q = 0.9 for [Mn(1,4-DO2A)] (the latter exists as a mixture of two species; i.e. ≈ 87 % having q = 1 and ≈ 13 % q = 0). [START_REF] Rolla | 1H and 17O NMR Relaxometric and Computational Study on Macrocyclic Mn(II) Complexes[END_REF] Those three examples illustrate that the design of Mn II -based CAs suitable for in vivo application, i.e. associating sufficient thermodynamic stability with considerable kinetic inertness and good T1-contrast enhancement, is a highly challenging task.

In 2012, Kálmán and Tircsó assessed the kinetic inertness of several Mn II complexes of openchain EDTA derivatives and reported their dissociation half-life under physiological conditions, t1/2 (pH = 7.4). [START_REF] Kálmán | Kinetic Inertness of the Mn2+ Complexes Formed with AAZTA and Some Open-Chain EDTA Derivatives[END_REF][START_REF] Margerum | Differential kinetic analysis of the metallic elements[END_REF] The main finding of this study was that the rigidifying effect of the cyclohexyl bridge of CDTA (trans-1,2-diaminocyclohexane-N,N,N´,N´-tetraacetic acid, Chart 1) results in a marked increase in kinetic inertness of the corresponding Mn II chelate when compared to EDTA, having a flexible ethylene bridge (t1/2 = 12 h for [Mn(CDTA)] 2-versus ≈ 5 min for [Mn(EDTA)] 2-). Also taking into account the monohydrated nature (q = 1, [START_REF] Gale | Direct Measurement of the Mn(II) Hydration State in Metal Complexes and Metalloproteins through 17O NMR Line Widths[END_REF] which results in a relaxivity value of r1 = 3.65 mM -1 s -1 at 20 MHz and 25°C), as well as the good thermodynamic stability of [Mn(CDTA)] 2-(log KML = 14.32), it appears that this manganese complex displays all the required features for in vivo MR imaging. [START_REF] Kálmán | Kinetic Inertness of the Mn2+ Complexes Formed with AAZTA and Some Open-Chain EDTA Derivatives[END_REF] Our interest in Mn II -based CAs has been induced by the capability to produce the positronemitting radionuclide manganese-52g ( 52g Mn) which has promising decay properties for PET imaging (t1/2 = 5.6 d, β + -decay intensity: 29.6%, max. β + -energy: 575 keV). [START_REF] Topping | Manganese-52 positron emission tomography tracer characterization and initial results in phantoms and in vivo[END_REF][START_REF] Lewis | 52Mn Production for PET/MRI Tracking Of Human Stem Cells Expressing Divalent Metal Transporter 1 (DMT1)[END_REF] We have recently shown that radiolabeling CDTA with an isotopic 52g/ [START_REF] Swift | Erratum: NMR-Relaxation Mechanisms of 17O in Aqueous Solutions of Paramagnetic Cations and the Lifetime of Water Molecules in the First Coordination Sphere[END_REF] Mn mixture (or c.a. 52g Mn, c.a. = carrier-added) provides the corresponding bimodal PET/MR tracer with ease. [START_REF] Vanasschen | Radiolabelling with isotopic mixtures of 52g/55Mn(ii) as a straight route to stable manganese complexes for bimodal PET/MR imaging[END_REF] This technique allows overcoming the major hurdle to the design of PET/MR probes, i.e. the huge sensitivity difference between both techniques, which necessitates nanomolar versus millimolar concentration of PET and MR reporter molecules, respectively. Additionally, radiolabeling with c.a. 52g Mn guarantees that both reporter molecules are chemically identical, thereby ensuring equivalent in vivo behavior. Dual-modal PET/MR probes not only allow to combine the exquisite spatial resolution of MRI with the remarkably high sensitivity of PET, they also generate a significant contrast enhancement on the MR image, the latter of which can be made biomarker-responsive by appropriate chelate design. [START_REF] De Rosales | Potential clinical applications of bimodal PET-MRI or SPECT-MRI agents[END_REF][START_REF] Loving | Redox-Activated Manganese-Based MR Contrast Agent[END_REF][START_REF] Davies | Environmentally responsive MRI contrast agents[END_REF][START_REF] Frullano | Bimodal MR-PET Agent for Quantitative pH Imaging[END_REF] Importantly, such information cannot be obtained through PET/MR protocols performed by injecting a sole PET tracer.

In order to further benefit from the synergistic combination of PET and MR imaging, a bimodal manganese tracer should ideally possess at least one of the following features: (i) specificity to a tissue/disease of interest, (ii) marked MR contrast enhancement (i.e. high r1), or (iii) biomarker responsiveness (i.e. r1 varies with the occurrence of a biological process, which can hence be monitored). Achievement of any of those three properties, respectively, requires the manganese complex (i) to be derivatized with a targeting moiety, (ii) to be coupled to a multivalent, small and rigid molecular entity [START_REF] Zhu | Hexameric MnII Dendrimer as MRI Contrast Agent[END_REF][START_REF] Mastarone | A Modular System for the Synthesis of Multiplexed Magnetic Resonance Probes[END_REF][START_REF] Verwilst | A Tripodal Ruthenium-Gadolinium Metallostar as a Potential αvβ3 Integrin Specific Bimodal Imaging Contrast Agent[END_REF][START_REF] Song | Synthesis of Multimeric MR Contrast Agents for Cellular Imaging[END_REF] , or (iii) to be functionalized with a scaffold whose chemical modification through a biological event influences the CA relaxivity. [START_REF] Davies | Environmentally responsive MRI contrast agents[END_REF] A bifunctional manganese(II) chelator (BFMnC), i.e. a ligand bearing a functional group for further synthetic modification, is therefore needed to design such "optimized" PET/MR tracers.

Until now, reports describing tailor-made BFMnC for high stability/relaxivity CAs are rather scarce. Very recently, the Caravan group took advantage of the trans-1,2-diaminocyclohexane backbone to build the PyC3A chelator (Chart 2, A1), which forms a very stable and inert monoaquated Mn II complex, as well as the corresponding BFMnC, tBu-PyC3A-NHS (Chart 2, A2). [START_REF] Gale | A Manganese Alternative to Gadolinium for MRI Contrast[END_REF] After coupling four molecules of A2 to a fibrin-binding peptide and Mn II chelation, the obtained high relaxivity MR CA permitted efficient thrombus detection in a rat model combined with rapid elimination from the body without dechelation. Previously, the same research group synthesized a backbone-substituted EDTA precursor (Chart 2, B) and reported its 6-fold attachment to a central cyclotriphosphazene core. [START_REF] Zhu | Hexameric MnII Dendrimer as MRI Contrast Agent[END_REF] The corresponding hexameric Mn II dendrimer exhibited high relaxivity and was proposed as a non-Gd III CA ideally suited for CKD patients given its mixed renal and hepatobiliary clearance.

Additionally, Wu et al. published a rigidified open-chain BFMnC (Chart 2, C) which was coupled to amphiphilic dextran micelles by CuAAC. [START_REF] Wu | Multivalent manganese complex decorated amphiphilic dextran micelles as sensitive MRI probes[END_REF] After Mn II loading, the increased T1-relaxivity of the obtained nanomicelles allowed for good MR signal enhancement of the vasculature. Although the kinetic inertness of the Mn II complexes of B and C has not been determined, data available in the literature indicate that both chelates are most certainly not as kinetically inert as the ones formed with more rigid hexadentate chelators like CDTA and PyC3A (t1/2 [Mn(EDTA)] 2-≈ 5 min., pH 7.4, 25 °C). [START_REF] Zhu | Hexameric MnII Dendrimer as MRI Contrast Agent[END_REF] Based on the favorable properties of [Mn(CDTA)] 2-as well as on prior experience with this ligand, [START_REF] Vanasschen | Radiolabelling with isotopic mixtures of 52g/55Mn(ii) as a straight route to stable manganese complexes for bimodal PET/MR imaging[END_REF] the synthesis of a CDTA-based BFMnC was undertaken.

N N tBuO 2 C CO 2 tBu tBuO 2 C CO 2 tBu OH N N N HO 2 C CO 2 H O B C N N CO 2 H CO 2 H HO 2 C N A1 N N CO 2 tBu CO 2 tBu tBuO 2 C N A2 O O N O O Chart 2.
Recently published BFMnCs (see text for references). It should be noted that the chelating motif of A2 and B was deprotected after coupling to a peptide/small multifunctional entity.

Herein, the rational design and synthesis of a CDTA-based BFMnC for stable Mn II complexation is described. A chemical handle for synthetic modification was implemented on the cyclohexyl ring of the chelator. The impact of this substituent on the characteristics of the corresponding Mn II complex was assessed in solution through thermodynamic, kinetic as well as NMR relaxometric studies performed on [Mn(4-HET-CDTA)] 2-as "model complex". The obtained results were compared to those reported for the parent [Mn(CDTA)] 2-and other relevant Mn II chelates.

Results and discussion

Design and synthesis of the bifunctional Mn II chelator (BFMnC)

At first glance, inserting the functional group for further derivatization on one of the pendant acetate substituents of CDTA appears as the shortest path to a bifunctional chelator. [START_REF] Gale | A Manganese Alternative to Gadolinium for MRI Contrast[END_REF] However, a chemical attachment point located that close to the future Mn II coordination cage may potentially alter the stability and/or the relaxivity of the final complex. In order to reduce this risk, it appeared safer to install the chemical handle on the cyclohexyl ring: for this reason, 3-cyclohexene-1-methanol was chosen as starting compound in our synthetic route.

The cyclohexene moiety of this inexpensive building block allows forming the trans-1,2diaminocyclohexane backbone, while its hydroxyl group can be converted into a highly reactive functional group for chemoselective coupling reactions. Among such transformations, the Cu Icatalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) [START_REF] Rostovtsev | A Stepwise Huisgen Cycloaddition Process: Copper(I)-Catalyzed Regioselective "Ligation" of Azides and Terminal Alkynes[END_REF] is a prominent member of the "click" reaction family [START_REF] Kolb | Click Chemistry: Diverse Chemical Function from a Few Good Reactions[END_REF] which has shown widespread use in the synthesis of targeted or other optimized imaging probes. [START_REF] Mastarone | A Modular System for the Synthesis of Multiplexed Magnetic Resonance Probes[END_REF][START_REF] Vanasschen | Gadolinium DOTA Chelates Featuring Alkyne Groups Directly Grafted on the Tetraaza Macrocyclic Ring: Synthesis, Relaxation Properties, "Click" Reaction, and High-Relaxivity Micelles[END_REF][START_REF] Baranyai | A shortcut to high-affinity Ga-68 and Cu-64 radiopharmaceuticals: one-pot click chemistry trimerisation on the TRAP platform[END_REF][START_REF] Mindt | Click Chemistry" Approach to the Efficient Synthesis of Multiple Imaging Probes Derived from a Single Precursor[END_REF][START_REF] Paulus | Development of a clickable bimodal fluorescent/PET probe for in vivo imaging[END_REF] CuAAC is of particular interest as the hydroxyl group of 3-cyclohexene-1-methanol enables an uncomplicated introduction of the required alkyne or azide functionalities, both of which are very inert to most chemical reagents and functional groups.

The here proposed target BFMnCs, i.e. CuAAC-reactive CDTA-based compounds 6a and 6b, are depicted in Scheme 1A. Strictly speaking, the latter are not bifunctional Mn II chelators per se, as tBu esters still have to be cleaved to unmask the CDTA chelating unit. However, deprotection should not be carried out before CuAAC coupling since CDTA forms very stable Cu II complexes (approx. 4-5 orders of magnitude more stable than [Mn(CDTA)] 2-) that might drastically complicate "click"catalyst removal. As the presence of copper likely precludes efficient radiolabeling of the final "click"conjugate, the selected approach towards bimodal PET/MR probes is based on the following reaction sequence: (i) CuAAC-coupling of protected derivative 6a or 6b to a molecule of interest, (ii) removing copper from the obtained conjugate, (iii) deprotecting the acetate pendant arms with TFA, (iv) final step radiolabeling with c.a. 52g Mn (Scheme 1B). It should be mentioned that due to the presence of excess copper and given the conditions required for tBu ester deprotection, the proposed approach is applicable for the coupling of relatively stable targeting vectors such as peptides rather than antibodies or other sensitive biomolecules. The synthesis of the BFMnCs started with the protection of the hydroxyl group of 3-cyclohexene-1-methanol as tert-butyldiphenylsilyl ether (Scheme 2). The second step already appeared as the most crucial transformation in the synthesis route as it is required to provide the trans-1,2diaminocyclohexane backbone needed to promote the kinetic inertness of the corresponding manganese chelate. [START_REF] Kálmán | Kinetic Inertness of the Mn2+ Complexes Formed with AAZTA and Some Open-Chain EDTA Derivatives[END_REF][START_REF] Gale | A Manganese Alternative to Gadolinium for MRI Contrast[END_REF] Initial measurements performed in our laboratory showed that the dissociation half-life (t1/2, pH 7.4, 25°C) is approx. 25-times higher for [Mn(trans-CDTA)] 2-than for [Mn(cis-CDTA)] 2-34 , further demonstrating that a suitable BFMnC has to be built on the trans-1,2diaminocyclohexane scaffold. The precursor of choice, trans-1,2-diazidocyclohexane 2, was obtained by 1,2-diazidonation of the alkene in the presence of NaN3 and Mn(OAc)3.2H2O. [START_REF] Snider | An Improved Procedure for the Conversion of Alkenes and Glycals to 1,2-Diazides Using Mn(OAc)3•2H2O in Acetonitrile Containing Trifluoroacetic Acid[END_REF] According to the literature, this reaction usually yields a mixture of cis-and trans-1,2-diazides when performed on cyclohexene derivatives, with the trans-isomer being formed predominantly. [START_REF] Snider | An Improved Procedure for the Conversion of Alkenes and Glycals to 1,2-Diazides Using Mn(OAc)3•2H2O in Acetonitrile Containing Trifluoroacetic Acid[END_REF][START_REF] Burns | New substituted 3amino-3,4-dihydro-2H-1,2-benzoxaborinine-8-carboxylic acid compounds are beta-lactamase inhibitors, useful for treating a subject having bacterial infection such as e.g. upper or lower respiratory tract infection[END_REF][START_REF] Minakawa | Imines that React with Phenols in Water over a Wide pH Range[END_REF][START_REF] Guo | Synthesis and evaluation of a cyclic imine derivative conjugated to a fluorescent molecule for labeling of proteins[END_REF] However, Galanski et al. as well as Habala et al. applied the same transformation to 4-methylcyclohexene and reported the stereospecific formation of the corresponding trans-1,2-diazide. [START_REF] Galanski | crystal structure and cytotoxicity of new oxaliplatin analogues indicating that improvement of anticancer activity is still possible[END_REF][START_REF] Habala | Methyl-substituted trans-1,2-cyclohexanediamines as new ligands for oxaliplatin-type complexes[END_REF] In accordance with the latter work, 2 was obtained stereospecifically as the trans-1,2-diazide, existing as two isomers which differ by the orientation of the protected hydroxymethyl substituent (axial/equatorial = 90:10 based on 1 H NMR; in both isomers, the two azide substituents are equatorial -see ESI for NMR spectra and a detailed discussion). To avoid complex 1 H and [START_REF] Margerum | Differential kinetic analysis of the metallic elements[END_REF] C NMR spectra and tedious peak assignment in the upcoming synthesis steps, it was decided to solely isolate the predominantly formed 4-axial isomer.

Removal of several by-products as well as the minor isomer (4-equatorial) was achieved by means of repeated column chromatography (petroleum ether/diethyl ether 99:1), yielding 62 % of isolated 4axial isomer. The quantitative reduction to trans-1,2-diamine 3 via hydrogenation over Pd/C catalyst was followed by full alkylation of the two primary amines with tert-butyl bromoacetate, producing tetraester 4 in 65% yield. In the next step, the 4-hydroxymethyl derivative 5 was obtained after deprotection using tetrabutylammonium fluoride (TBAF; 85% yield). MsCl, Et3N, CH2Cl2, 0°C → RT; 2. NaN3, DMF, 75°C, 70 % (2 steps).

OH OTBDPS OTBDPS N 3 N 3 OTBDPS NH 2 NH 2 N N O O O O O O O O O (i) (ii) (iii) (iv) (v)
Once the tBu ester-protected CDTA backbone was built, derivatization of the hydroxymethyl substituent of 5 with the alkyne/azide moiety needed for CuAAC coupling reactions was performed.

The triple bond was inserted via O-alkylation using propargyl bromide, cesium hydroxide monohydrate and tetrabutylammonium iodide (TBAI) in dichloromethane. This method proved to be extremely fast and efficient for alkylating two hydroxymethyl groups on a DOTA derivative (90 min reaction time, 90% yield). [START_REF] Vanasschen | Gadolinium DOTA Chelates Featuring Alkyne Groups Directly Grafted on the Tetraaza Macrocyclic Ring: Synthesis, Relaxation Properties, "Click" Reaction, and High-Relaxivity Micelles[END_REF] Carried out on 5, however, the reaction appeared significantly more sluggish, small amounts of starting material still being detected by TLC after 18 h stirring with 8 equivalents bromide and 4 equivalents base. When the reaction was performed in the presence of activated 4 Å powdered molecular sieve but without TBAI, [START_REF] Dueno | Cesium promoted O-alkylation of alcohols for the efficient ether synthesis[END_REF] the alcohol underwent almost full conversion within 18 hours at RT and 6a was obtained in 75% yield after column chromatography.

The azido-BFMnC 6b was synthesized by conversion of 5 to the corresponding mesylate followed by substitution of the latter with sodium azide in DMF. After removal of a low amount of elimination product which was formed during the first step, 4-A-CDTA(tBu)4 was isolated in 70% yield (over 2 steps). To the best of our knowledge, 6a and 6b are the first CDTA-based backbone-functionalized

BFMnCs published so far. The only other example of a bifunctional chelator specifically designed to yield Mn II -based CAs with elevated stability, inertness and relaxivity is tBu-PyC3A-NHS already mentioned in the introduction (structure A2 in Chart 2).

Rather than evaluating the Mn II chelates of deprotected 6a/6b in terms of equilibrium, kinetic and relaxometric properties, the "model chelator" 4-HET-CDTA was synthesized (8a, Scheme 3; the 4substituent is axial whereas the iminodiacetate groups are diequatorial -see ESI for NMR spectra and a detailed discussion). The latter was obtained by "clicking" alkyne 6a with 2-azidoethanol in the presence of copper(I) iodide [START_REF] Vanasschen | Gadolinium DOTA Chelates Featuring Alkyne Groups Directly Grafted on the Tetraaza Macrocyclic Ring: Synthesis, Relaxation Properties, "Click" Reaction, and High-Relaxivity Micelles[END_REF] followed by tBu ester cleavage of intermediate 7a using TFA. 4-HET-CDTA bears a 1,2,3-triazole moiety on its cyclohexyl ring substituent, as will be the case for the "click"-conjugates intended to be radiolabeled with 52g/55 Mn II (Scheme 1B). Therefore, the parameters obtained for [Mn(4-HET-CDTA)] 2-should be much more relevant than data generated with the chelates of deprotected 6a/6b. While the hydroxyethyl side-chain of 4-HET-CDTA is expected to havelittle to no effect on the thermodynamic properties of the final complex, the potential impact of the coordinating N-substituted 1,2,3-triazole moiety on the structural, dynamic and relaxometric properties of the chelate is of particular interest. 42,[START_REF] Stasiuk | Click chemistry with lanthanide complexes: a word of caution[END_REF] A "model chelator" obtained by "clicking" and deprotecting 6b would have a triazole moiety that is

In that context, as a "model chelator" derived from 6b would have a shorter and less flexible linker between the cyclohexane ring and the triazole than Synthesis of model compound 4-HET-CDTA (8a) by "click"-coupling between 6a and 2azidoethanol followed by deprotection of the acetate pendant arms. Reagents and conditions: (i) 2-azidoethanol, CuI, DIPEA, 2,6-lutidine, CH2Cl2, RT, 70%; (ii) TFA/CH2Cl2 1:1, RT, 76%.

Solution equilibrium studies

Determination of protonation constants

As a first step, the protonation constants of the 4-HET-CDTA chelator were measured by pHpotentiometric titration in 0.15 M NaCl, which is considered to better mimic the conditions present in biological fluids than other electrolyte solutions do. The ligand protonation constants are defined as shown in eq. 1:

] H ][ L H [ ] L H [ 1 i i H i + - = K (eq. 1)
where i = 1, 2 and L denotes the deprotonated ligand.

The protonation constants obtained for 4-HET-CDTA along with the data corresponding to the parent CDTA are listed and compared in Table 1. As can be seen from those values, the presence of the 1,2,3-triazole-bearing substituent on the cyclohexane ring of 4-HET-CDTA results in a minor decrease of the basicity of the amine nitrogen atoms (∆ logK1 H = 0.5, ∆ logK2 H = 0.2), most likely due to the slight electron-withdrawing effect of the triazole unit. This makes the 4-HET-CDTA ligand altogether marginally less basic than non-functionalized CDTA (Δ Σ log Ki H = 0.8). For comparison purposes, Table 1 also lists the data reported for EDTA and 1,4-DO2A (Chart 1), a macrocyclic chelator whose Mn II complex was shown to combine a reasonably high relaxivity with good thermodynamic stability and kinetic inertness. [START_REF] Rolla | 1H and 17O NMR Relaxometric and Computational Study on Macrocyclic Mn(II) Complexes[END_REF][START_REF] Garda | Physico-chemical properties of MnII complexes formed with cis-and trans-DO2A: thermodynamic, electrochemical and kinetic studies[END_REF] The 1,4-DO2A ligand is significantly more basic than 4-HET-CDTA, CDTA and EDTA (Δ Σ log Ki H = 4.4, 3.6 and 5.3 respectively). 4-HET-CDTA [a] CDTA [b] EDTA [b] 1,4-DO2A [c] log K1 H 8.99(2)

Table 1. Protonation constants of 4-HET

9.36 9.17

Stability of the complexes formed with Mn II and other essential metal ions

In order to obtain thermodynamic insights of the formation of 4-HET-CDTA complexes with Mn II as well as the essential metals Cu II , Mg II , Ca II and Zn II , their stability and protonation constants (KML and KMHiL, respectively) were determined by pH-potentiometric titration, excepted for Cu II . For the chelate formed with this ion, pH-potentiometry had to be complemented with UV-Vis spectrophotometric measurements. Although the titration data were fitted by considering the formation of mononuclear complexes (ML), the existence of protonated species at lower pH values had to be taken into account for Ca II and Mn II (MHL complex) as well as for Zn II and Cu II (MH2L complex). The formation of ternary hydroxydo complexes (ML(OH)), which usually appear at higher pH, was not confirmed for any of the studied metal ions up to pH = 11.80. The thermodynamic stability and protonation constants of the different metal chelates are expressed by eq. 2 and 3, respectively.

] L ][ M [ ] ML [ ML = K (eq. 2) ] H ][ L MH [ ] L MH [ 1 i i L MH i + - = K where i = 1, 2 (eq. 3)
For the Cu II system, it was not possible to obtain an acceptable fit of the experimental pHpotentiometric data (volume of NaOH added vs. pH) since the absence of free Cu II was observed even at the most acidic titration point (pH = 1.73). Therefore, the formation of this complex had to be studied under fairly acidic conditions using a batch method (samples prepared over the As expected, the highest stability constant was measured for the Cu II complex, the latter ion also ). In the presence of 1.0 M Na I ions, the ligand forms a weak sodium complex in alkaline solution which leads to a reduced basicity (a similar decrease was reported for the protonation constants of several polyaminocarboxylate ligands such as EDTA, CDTA and DOTA). [START_REF] Chen | Solution speciation in the aqueous Na(I)-EDTA and K(I)-EDTA systems[END_REF][START_REF] Carr | Complexometric titration for the determination of sodium ion[END_REF][START_REF] Delgado | Metal complexes of cyclic tetra-azatetra-acetic acids[END_REF] Focusing solely on the data for the Mn II complexes, one observes that the thermodynamic stability decreases in the order 1,4-DO2A > CDTA > 4-HET-CDTA > EDTA, which is in perfect agreement with the Σ log Ki H values discussed in the previous section. Interestingly, the pMn value for [Mn(1,4-DO2A)] is significantly lower than those calculated for the Mn II complexes of EDTA, 4-HET-CDTA and CDTA. This can be explained in terms of differences between the ligand protonation constants (e.g. the conditional stability of the given complex is the lowest among these chelates): while for 1,4-DO2A the first two protonation constants (log K1 H and log K2 H ) are greater than pH 7.4, only log K1 H is > 7.4 for EDTA, CDTA and 4-HET-CDTA. Consecutively, the proton competition at physiological pH is considerably greater for the 1,4-DO2A ligand than for the CDTA's, resulting in a lower conditional stability constant as well as a lower pMn value for its Mn II complex. The [Mn(4-HET-CDTA)] 2-complex was further studied by 1 H-relaxometry. In order to confirm the equilibrium model, the relaxivity versus pH profile was compared with the species distribution curves calculated by using the stability data determined through pH-potentiometry. Figure 2 shows that the relaxivity of [Mn(4-HET-CDTA)] 2-is nearly constant in the pH range 3.8 -12.20, suggesting that neither protonation nor deprotonation occurs in the quoted pH range (i.e. only the [Mn(4-HET-CDTA)] 2-chelate is present in solution in the given pH range). The significant increase in relaxivity below pH = 3.8 is due to the protonation of the Mn II complex followed by the progressive release of the Mn II ion as a result of the chelate dissociation at lower pH. Notably, the observed relaxivity increase at acidic pH coincides with the chelate dissociation suggested by the species distribution curves, thereby confirming the equilibrium model. 2.

The relaxivity of [Mn(4-HET-CDTA)] 2-was determined by titrating a solution of the ligand with Mn II and found to be r1 = 4.56 mM -1 s -1 (20 MHz, 25 o C, see Fig. S1 in ESI). This value is significantly higher than expected for a monoaquated Mn II complex of low molecular weight formed with a CDTAtype ligand (r1 = 3.65 mM -1 s -1 for [Mn(CDTA)] 2-under identical conditions). [START_REF] Kálmán | Kinetic Inertness of the Mn2+ Complexes Formed with AAZTA and Some Open-Chain EDTA Derivatives[END_REF] It is well-known that the relaxivity of paramagnetic metal chelates depends on many physico-chemical parameters, the most important ones being the number of coordinated water molecules (q), the water exchange rate (kex), the electron spin relaxation times (T1,2e) and the rotational correlation time (τR); the latter is directly related to the molecular weight. Even for ligands possessing identical donor atoms in nature and number, the corresponding Mn II complexes may have very discrepant relaxivity values (e.g. r1 = 1.5, 2.1 and 3.2 mM -1 s -1 at 20 MHz and 25 °C for the Mn II complexes of hexadentate chelators 1,7-DO2A, 1,4-DO2A and EDTA, respectively). In order to identify the parameters responsible for the increased r1 value of [Mn(4-HET-CDTA)] 2-, an in-depth evaluation of the chelate was carried out by performing NMRD (nuclear magnetic relaxation dispersion) and [START_REF] Vanasschen | Radiolabelling with isotopic mixtures of 52g/55Mn(ii) as a straight route to stable manganese complexes for bimodal PET/MR imaging[END_REF] O NMR experiments (vide infra).

Dissociation kinetics of the [Mn(4-HET-CDTA)] 2-complex

In contrast to Gd III , Mn II has an endogenous elimination pathway via the hepatobiliary system.

However, exposure to large doses of the free ion has acute toxic effects as Mn II accumulates in the brain, leading to symptoms of dementia known as "manganese-induced Parkinsonism" or "manganism". [START_REF] Cersosimo | The diagnosis of manganese-induced parkinsonism[END_REF] It is nowadays recognized that kinetic inertness, which assesses the inertness of a complex towards metal release, is more important than thermodynamic stability when in vivo application of MRI CAs is considered. [START_REF] Sherry | A primer on gadolinium chemistry[END_REF] In order to evaluate the inertness of [Mn(4-HET-CDTA)] 2- under physiological conditions, the rates of the transmetallation reactions between the complex and endogenous Cu II were studied (eq. 4) (the complex used in the studies was characterized by HPLC and MS methods, see Figure S2 and S3 in ESI). UV-Vis spectrophotometric monitoring of those reactions was performed in the presence of a high Cu II excess (10-40 fold) to ensure pseudo-first-order conditions (pH range 4.05 -5.03).

[Mn(4-HET-CDTA)] 2-+ Cu II [Cu(4-HET-CDTA)] 2-+ Mn II (eq. 4)

Since a pseudo-first-order process is considered, the rate of the dissociation reactions can be expressed as described in eq. 5, where kobs is a pseudo-first-order rate constant, and [MnL]t is the total concentration of the Mn II chelate:

t obs t ] MnL [ dt ] MnL [ d k = - (eq. 5)
Figure 3 shows the calculated kobs rate constants as a function of the acid concentration (see Fig. S4 in ESI for the cCuII-dependence of the pseudo-first order rate constants). As highlighted by Figure 3, the kobs values increase with increasing acid concentration (decreasing pH) as well as with decreasing concentration of the exchanging ion. The relation between the kobs values and pH may be explained by a rate-determining step in which the proton-assisted dissociation of the Mn II chelate is followed by a fast complexation of the Cu II ion by the free ligand. Since the dependence of kobs on [H + ] results in a straight line, the exchange reaction may occur via spontaneous as well as proton-assisted pathways. Additionally, the fact that kobs values are inversely proportional to the Cu II concentration indicates that a stable, catalytically inactive dinuclear intermediate, usually referred to as "dead-end" complex ([Mn(L)Cu]) is formed (its existence was confirmed by MS spectroscopy, see ESI Figure S5). [START_REF] Kasprzyk | Kinetics of interaction of metal ions with two tetraazatetraacetate macrocycles[END_REF] Altogether, these experimental observations illustrate that the spontaneous dissociation, the proton-assisted dissociation and the metal ion-catalyzed dissociation have to be taken into account as possible pathways for the decomplexation of [Mn(4-HET-CDTA)] 2- (see Fig. 4).

MnL

k MnL Mn 2+ + H n L K MnHL MnHL k H K MnLCu Mn(L)Cu k Cu Mn 2+ + CuL
] [Cu ] [H 1 ] [Cu ] [H MnLCu MnHL I 3 1 MnL obs II I K K k k k k + + + + = + + (eq. 7)
As a result of the fitting of equation 7, the rate constant characterizing the spontaneous dissociation (kMnL) was found to be very small and affected by a large error, while k3, describing the metal-assisted dissociation, was negative. This indicates that the spontaneous and metal-assisted pathways contribute only negligibly to the dissociation of [Mn(4-HET-CDTA)] 2-, justifying to fix the values of the corresponding rate constants to zero in equation 7. In addition, the protonation constant of the complex determined by pH-potentiometric titration is much lower than the pH range evaluated in the kinetic study. Thus, the simplified equation 8 was applied for the fitting of the experimental kobs

values. ] [Cu 1 ] [H ] [Cu ] [H 1 ] [H MnLCu 1 MnLCu MnHL 1 obs II II K k K K k k + = + + = + + + (eq. 8)
The results of this kinetic data refinement are summarized in Table 3 along with the values for the Mn II complexes of CDTA and 1,4-DO2A. The comparison of the k1 values for [Mn(4-HET-CDTA)] 2- and [Mn(CDTA)] 2-indicates that the presence of the ring substituent in 4-HET-CDTA has a beneficial, although minor, effect on the rate of acid catalyzed dissociation, this being mainly due to the lower ligand basicity. The most relevant comparison of the kinetic inertness of both chelates is based on their half-lives of dissociation, t1/2, calculated near physiological conditions (pH = 7.4, cCuII = 1×10 -5 M). Based on the t1/2 values, the Mn II complex of 4-HET-CDTA appears to be ≈ 35% more inert than its non-substituted analogue (t1/2 = 16.2 h versus 12.1 h, respectively). This result may be rationalized in terms of rigidity as the substituent located in 4-position of the cyclohexane ring renders the whole ligand scaffold less flexible. The positive impact of an increased ligand rigidity on the kinetic inertness has already been demonstrated for both Mn II and Gd III chelates. [START_REF] Kálmán | Kinetic Inertness of the Mn2+ Complexes Formed with AAZTA and Some Open-Chain EDTA Derivatives[END_REF][START_REF] Tircsó | Approaching the Kinetic Inertness of Macrocyclic Gadolinium(III)-Based MRI Contrast Agents with Highly Rigid Open-Chain Derivatives[END_REF][START_REF] Bonnet | A Pyridine-Based Ligand with Two Hydrazine Functions for Lanthanide Chelation: Remarkable Kinetic Inertness for a Linear, Bishydrated Complex[END_REF][START_REF] Gale | Gd(CyPic3A)(H2O)2]-: a stable, bis(aquated) and highrelaxivity Gd(iii) complex[END_REF] The macrocyclic complex [Mn(1,4-DO2A)], which also dissociates mainly via the proton-assisted pathway, [START_REF] Garda | Physico-chemical properties of MnII complexes formed with cis-and trans-DO2A: thermodynamic, electrochemical and kinetic studies[END_REF] is characterized by an approx. 3-fold lower k1 value than [Mn(4-HET-CDTA)] 2-. As a direct consequence, the calculated half-life of dissociation under physiological conditions, t1/2, is substantially higher for [Mn(1,4-DO2A)] compared to [Mn(4-HET-CDTA)] 2-. Interestingly, 1,4-DO2AM (bisamide derivative of 1,4-DO2A; see Chart 1) forms an even more inert Mn II complex (k1 = 8.7 M -1 s -1 ; t1/2 = 556 h) thanks to its two non-protonable amide groups which prevent the formation of the monoprotonated intermediate species. [START_REF] Forgács | A Bisamide Derivative of [Mn(1,4-DO2A)] -Solution Thermodynamic, Kinetic, and NMR Relaxometric Studies[END_REF] Those results are not unexpected as Mn II complexes formed with linear ligands are known to be less inert than their macrocyclic analogues of same denticity.

Strictly speaking, CDTA and PyC3A were the only open-chain ligands capable of forming Mn II chelates with acceptable kinetic inertness reported so far; 4-HET-CDTA can thus be considered as a novel addition to this rather limited group.

Table 3.

Constants and calculated half-lives (t1/2 = ln 2/kobs, pH = 7.4, [Cu II ] = 1×10 -5 M) characterizing the dissociation of the Mn II complexes formed with 4-HET-CDTA, CDTA and 1,4-DO2A (25 °C).

4-HET-CDTA [a] CDTA [b] 1,4-DO2A [c] k1 (M -1 s -1 ) 297( 8) 400 100 k2 (M -2 s -1 ) [d] -- In order to characterize the relaxation properties of the [Mn(4-HET-CDTA)] 2-complex and to explain its high relaxivity (r1 = 4.56 mM -1 s -1 at 20 MHz and 25 o C), nuclear magnetic relaxation dispersion (NMRD) profiles (r1 vs. magnetic field strength) were recorded in the frequency range of 10 kHz -400 MHz at 298, 310 and 323 K (Fig. 5). As mentioned above, the relaxivity of paramagnetic metal complexes is related to several physico-chemical parameters (e.g. q, kex, τR, T1,2e).

The independent assessment of some of these is a key issue, therefore NMRD data are often completed with variable-temperature [START_REF] Vanasschen | Radiolabelling with isotopic mixtures of 52g/55Mn(ii) as a straight route to stable manganese complexes for bimodal PET/MR imaging[END_REF] O NMR measurements. While the transverse (1/T2) and the longitudinal (1/T1) [START_REF] Vanasschen | Radiolabelling with isotopic mixtures of 52g/55Mn(ii) as a straight route to stable manganese complexes for bimodal PET/MR imaging[END_REF] O relaxation rates give access to the water exchange rate (kex) and to the rotational correlation time (τR), respectively, the [START_REF] Vanasschen | Radiolabelling with isotopic mixtures of 52g/55Mn(ii) as a straight route to stable manganese complexes for bimodal PET/MR imaging[END_REF] O chemical shift is related to the number of water molecules directly coordinated to the paramagnetic metal center (q). As shown in Fig. 5, all three NMRD curves have the typical shape of rapidly tumbling, low molecular weight complexes with a single dispersion at 1-10 MHz. The relaxivity of [Mn(4-HET-CDTA)] 2-decreases with increasing temperature over the entire frequency range, evidencing that the relaxivity is limited by fast rotation, as was expected. Information about the water-exchange kinetics was obtained by measuring the [START_REF] Vanasschen | Radiolabelling with isotopic mixtures of 52g/55Mn(ii) as a straight route to stable manganese complexes for bimodal PET/MR imaging[END_REF] O relaxation rates for an aqueous [Mn(4-HET-CDTA)] 2-solution and a diamagnetic reference at different temperatures (9.4 T, see Fig. 6). As [START_REF] Vanasschen | Radiolabelling with isotopic mixtures of 52g/55Mn(ii) as a straight route to stable manganese complexes for bimodal PET/MR imaging[END_REF] O T1 values and the chemical shifts showed negligible differences between the Mn II complex and the reference, they are not reported. The 17 O 1/T2r data have been analyzed using the Swift-Connick equations, assuming a simple exponential behavior of the electron spin relaxation (see ESI for detailed equations). [START_REF] Swift | NMR-Relaxation Mechanisms of O17 in Aqueous Solutions of Paramagnetic Cations and the Lifetime of Water Molecules in the First Coordination Sphere[END_REF][START_REF] Swift | Erratum: NMR-Relaxation Mechanisms of 17O in Aqueous Solutions of Paramagnetic Cations and the Lifetime of Water Molecules in the First Coordination Sphere[END_REF] Based on the analogy with [Mn(EDTA)] 2-, the hydration number was fixed to q = 1, a value which has also been confirmed experimentally for [Mn(CDTA)] 2-. [START_REF] Gale | Direct Measurement of the Mn(II) Hydration State in Metal Complexes and Metalloproteins through 17O NMR Line Widths[END_REF] We would like to mention that we obtain q = 0.81 using Gale's method to assess q from the maximum value of the transverse [START_REF] Vanasschen | Radiolabelling with isotopic mixtures of 52g/55Mn(ii) as a straight route to stable manganese complexes for bimodal PET/MR imaging[END_REF] O relaxation rate. [START_REF] Gale | Direct Measurement of the Mn(II) Hydration State in Metal Complexes and Metalloproteins through 17O NMR Line Widths[END_REF] The fitting procedure allowed the calculation of the water exchange rate, kex 298 , its activation enthalpy, ΔH ‡ , and the electronic relaxation rate of the Mn II ion, 1/T1e 298 , which are given in Table 4. The scalar coupling constant, AO/ħ, was set to 40×10 6 rad s -1 , a value typically obtained experimentally but also by DFT calculations. [START_REF] Rolla | 1H and 17O NMR Relaxometric and Computational Study on Macrocyclic Mn(II) Complexes[END_REF] . From Fig. 6 ER is the activation energy for the rotational motion of the complex.

To describe the rotational dynamics of the complex, the 1 H NMRD data were fitted according to the Solomon-Bloembergen-Morgan (SBM) [START_REF] Bloembergen | Proton Relaxation Times in Paramagnetic Solutions. Effects of Electron Spin Relaxation[END_REF] and Freed models [START_REF] Freed | Dynamic effects of pair correlation functions on spin relaxation by translational diffusion in liquids. II. Finite jumps and independent T1 processes[END_REF] 4. Although the quality of the fit is slightly worse for the 25°C data at high field than for the lower temperatures, it remains acceptable. The rotational correlation time (τR 298 ) of [Mn(4-HET-CDTA)] 2-is longer than those of typical low-molecular-weight complexes without appended groups (e.g. ≈ 84% increase compared to [Mn(EDTA)] 2-, ≈ 98% increase versus [Mn(1,4-DO2A)]), [START_REF] Rolla | 1H and 17O NMR Relaxometric and Computational Study on Macrocyclic Mn(II) Complexes[END_REF] which is in agreement with the higher molecular mass of the chelate. This increased τR 298 value is thus responsible for the remarkably high relaxivity of [Mn(4-HET-CDTA)] 2-at 20 MHz (25 °C). Mn II -based imaging probes should ideally possess all of the following three characteristics to allow for efficient in vivo MR or PET/MR imaging: an elevated T1-relaxivity for good contrast enhancement, a high thermodynamic stability to ensure that the chelator strongly binds Mn II and, especially, a sufficient kinetic inertness to avoid, or at least minimize, complex dissociation prior to excretion from the body. In view of the currently available literature and the data obtained in the present study, 4-HET-CDTA (and most likely all "click"-conjugates derived from 6a and 6b) is among the few Mn II chelators that satisfy all of the above requirements. The most relevant data for comparing those few complexes are summarized in Table 5.

Table 5. Relevant relaxometric, thermodynamic and kinetic parameters (25 °C) for comparing Mn II complexes of 4-HET-CDTA and other hexadentate chelators reported in the literature.

Parameter 4-HET-CDTA [a] CDTA PyC3A [b] 1,4-DO2A [c] 1,4-DO2AM [d] r1 (mM the "click"-conjugates derived from 6a/6b, relaxivities are expected to be higher than for [Mn(CDTA)] 2-(r1 > 3.65 mM -1 s -1 due to an increase in τR); [g] ref. [START_REF] Rolla | 1H and 17O NMR Relaxometric and Computational Study on Macrocyclic Mn(II) Complexes[END_REF] ; [h] pMn values were calculated by using the approach of É.

Tóth et al. (25 o C, pH = 7.4 and cMnII = cL = 1×10 -5 M 44 ); [i] [Mn(PyC3A)] -is ≈ 1.6-fold more inert to Zn IIinduced transmetallation than [Mn(CDTA)] 2-at pH 6.0 and 37 °C (no data reported for pH 7.4, 25 °C). [START_REF] Gale | A Manganese Alternative to Gadolinium for MRI Contrast[END_REF] Albeit [Mn(1,4-DO2A)] and, especially, [Mn(1,4-DO2AM)] 2+ show very promising inertness towards dissociation, they suffer from (i) a reduced hydration number (q = 0.87) which negatively impacts the relaxivity, (ii) slower complexation kinetics than open-chain CDTA derivatives (due to the macrocyclic nature), which might be an issue in radiolabeling experiments, (iii) the absence of reported bifunctional derivatives. It should also be mentioned that a group of extremely inert CDTAbisamide-based Mn II chelates has been patented recently. [START_REF] Baranyai | Ethylenediaminetetraacetic acid bis(amide) derivatives and their respective complexes with Mn(II) ion for use as MRI contrast agent[END_REF] The here described 4-P-CDTA(tBu)4 and 4-A-CDTA(tBu)4 are, along with tBu-PyC3A-NHS reported by Caravan et al., [START_REF] Gale | A Manganese Alternative to Gadolinium for MRI Contrast[END_REF] the only rationally designed BFMnCs enabling the in vivo evaluation of Mn II -based PET/MR tracers.

Conclusions

In view of designing Mn II -based MR and PET/MR imaging agents combining the optimal balance of thermodynamic stability, kinetic inertness and elevated relaxivity with targeting or bioresponsive properties, two CuAAC-reactive BFMnCs have been prepared. 4-P-CDTA(tBu)4 (6a) and 4-A-CDTA(tBu)4 (6b), bearing an alkyne/azide substituent on the cyclohexane ring, were obtained via a 6step synthesis involving the build-up of a functionalized trans-1,2-diaminocyclohexane core. To evaluate the potential impact of a triazole-bearing substituent on the physico-chemical properties of the corresponding Mn II complex, the model chelator 4-HET-CDTA (8a) was synthesized from 6a. The protonation constants revealed that 4-HET-CDTA is slightly less basic than parent CDTA, most likely due to the weak electron-withdrawing effect of the triazole moiety. As a direct consequence, the thermodynamic stabilitiy of the complexes formed with Mn II as well as with essential metal ions (Cu II , Zn II , Ca II and Mg II ) are marginally lower for 4-HET-CDTA compared to CDTA (log KMnL = 13.80 versus 14.32, respectively). Nonetheless, the conditional stability of [Mn(4-HET-CDTA)] 2-is almost identical to that of [Mn(CDTA)] 2-as demonstrated by the calculated pMn values (pMn = 8.62 versus

8.68, respectively). A kinetic investigation of the transmetallation reaction between [Mn(4-HET-

CDTA)] 2-and Cu II ions has shown that complex dissociation mainly occurs via a proton-assisted mechanism: here, the reduced basicity of 4-HET-CDTA has a positive impact on the proton-assisted dissociation which is slightly slower for [Mn(4-HET-CDTA)] 2-than for [Mn(CDTA)] 2-(k1 = 297 vs. 400 M -1 s -1 , respectively). Most importantly, the dissociation half-life calculated near physiological conditions, which is the best parameter for evaluating the kinetic inertness of a complex, is ≈ 35%

higher for [Mn(4-HET-CDTA)] 2-with t1/2 = 16.2 h compared to 12.1 h for [Mn(CDTA)] 2-(pH 7.4, 25 °C). This may be explained by the combined effects of reduced basicity and increased rigidity of the ligand. As demonstrated by the fitting of [START_REF] Vanasschen | Radiolabelling with isotopic mixtures of 52g/55Mn(ii) as a straight route to stable manganese complexes for bimodal PET/MR imaging[END_REF] O NMR data and 1 H NMRD profiles, the remarkably high relaxivity of [Mn(4-HET-CDTA)] 2-(r1 = 4.56 mM -1 s -1 at 20 MHz, 25 °C), which evidences the presence of an inner-sphere water (q = 1), is due to an increased rotational correlation time (τR 298 = 105 ps). Additionally, the fast water-exchange is in good agreement with the kex value reported for [Mn(CDTA)] 2-(kex 298 = 17.6 x 10 7 vs. 14.0 x 10 7 s -1 , respectively) given their identical coordination cage.

Compound 6a is currently employed for the synthesis of a target-specific 52g/55 Mn-conjugate to be evaluated in hybrid imaging experiments.

Experimental section

Synthesis

All commercially purchased reagents and solvents were used as received and without further purification. NMR spectra were acquired on a Bruker Avance 600 (600 MHz for 1 H) spectrometer and processed/analyzed using MestreNova 8.1 software (Mestrelab Research). High resolution mass spectrometry (HRMS) was performed using a hybrid linear ion trap FT-ICR mass spectrometer LTQ-FT Ultra (Thermo Fisher Scientific, Bremen, Germany) equipped with a 7 T supra-conducting magnet.

The electrospray ionisation (ESI) source was operated in negative or positive mode. Infrared (IR) spectra were measured on a Shimadzu FTIR-8400 Fourier Transform Infrared spectrophotometer (confirmation of the presence of azide functionalities in compounds 2 and 6b).

4-(((tert-Butyldiphenylsilyl)oxy)methyl)-cyclohexene (1)

By use of an addition funnel, tert-butyldiphenylsilyl chloride (4.343 g, 15.49 mmol) in 7 mL CH2Cl2

was added dropwise to a solution of 3-cyclohexene-1-methanol (1.688 g, 14.75 mmol) and imidazole (2.109 g, 30.97 mmol) in 20 mL CH2Cl2, resulting in the formation of a precipitate. After stirring this suspension for 18 h at room temperature (RT), CH2Cl2 was added (30 mL) and the reaction quenched with saturated NH4Cl (30 mL). The layers were separated and the aqueous phase was extracted with CH2Cl2 (2 x 30 mL). The combined organic extracts were washed with brine (30 mL), dried over MgSO4, filtered and concentrated under reduced pressure. The obtained oily residue was purified by silica gel column chromatography (petroleum ether/EtOAc 99:1, Rf = 0.84) to yield 1 as a colorless oil (5.015 g, 97 % yield). Note: 1 could not be ionized in ESI-MS and thus, not be identified.

4-(((tert-Butyldiphenylsilyl)oxy)methyl)-trans-1,2-diazidocyclohexane (2)

In a 250 mL round-bottomed flask, a mixture of alkene 1 (3.323 g, 9.479 mmol), NaN3 (3.081 g, 47.393 mmol) and Mn(OAc)3.2H2O (7.859 g, 28,437 mmol) was put under argon atmosphere, suspended in a mixture of dry CH3CN/CH2Cl2 (85:15, 100 mL) and cooled to -20°C. After addition of 10 mL TFA, the reaction was vigorously stirred for 4 h at -15 to -20°C. The reaction was then quenched by adding NaHSO3 (10%, 75 mL) and the aqueous phase was extracted with EtOAc (3 x 75 mL). The combined organic fractions were successively washed with a saturated Na2CO3 solution (2 x 70 mL) and brine (2 x 70 mL), dried over MgSO4 and evaporated in vacuo. [START_REF] Drahoš | Manganese(II) Complexes as Potential Contrast Agents for MRI[END_REF] H and [START_REF] Margerum | Differential kinetic analysis of the metallic elements[END_REF] C NMR analysis of the crude oil indicated that the expected trans-diazide, bearing the tertbutyl(methoxy)diphenylsilane group in an axial position, had been formed preferentially (axial/equatorial ≈ 90:10). The latter was isolated from several impurities as well as from the equatorially-substituted isomer by careful and repeated silica gel column chromatography (petroleum ether/Et2O 99:1). Individual column fractions were tested for purity by TLC (petroleum ether/EtOAc 97:3, Rf = 0.61) and combined accordingly. The pure 4-axial trans-diazide 2 was obtained as a yellowish oil (2.254 g, 62 % yield).

Caution: Particular care should be taken when performing this reaction. Handling large amounts of sodium azide may be associated with the exposure to highly toxic HN3, therefore all manipulations should be carried out in a well-ventilated fume hood. Regarding the potential explosion hazard of organic azides, we did not experience any problems with compound 2, neither during the extraction step, nor during evaporation under vacuum at 40°C. IR: νN3 = 2097.66 cm -1

Note: 2 could not be ionized in ESI-MS and thus, not be identified. Elemental analysis was not performed due to potential explosion hazard of organic azides.

4-(((tert-Butyldiphenylsilyl)oxy)methyl)-trans-1,2-diaminocyclohexane (3)

Diazide 2 (1.526 g, 3.51 mmol) and 481 mg 10% Pd/C were placed in a 100 mL round-bottomed flask, suspended in dry EtOAc/MeOH (75:25, 76 mL) and flushed with argon for 10 minutes. The reaction mixture was then hydrogenated at atmospheric pressure and RT for 4 h. After removal of the catalyst by filtration over Celite, the filtrate was evaporated to dryness to yield diamine 3 as a slightly yellowish oil (1.344 g ≈ quantitative yield). The obtained diamine was used in the following synthesis step without further purification.

Tetra-tert-butyl 4-(((tert-butyldiphenylsilyl)oxy)methyl)-trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetate (4)

Diamine 3 (1.344 g, 3.51 mmol) and K2CO3 (2.911 g, 21.07 mmol) were introduced into a 250 mL round-bottomed flask and placed under argon atmosphere. After addition of dry DMF (90 mL) and tert-butyl bromoacetate (2.644 mL, 17.56 mmol), the suspension was heated to 50°C for approximately 6 h. The reaction mixture was then diluted with EtOAc (50 mL), the remaining K2CO3 removed by centrifugation and the filtrate diluted with additional 400 mL of EtOAc. The obtained organic fraction was then washed with water (100 mL) and with brine (2 x 100 mL). The organic layer was dried over MgSO4 and the solvents evaporated under reduced pressure. Purification of the crude oil was carried out by silica gel column chromatography, eluting first with a CH2Cl2/EtOAc 95:5 mixture containing 1% Et3N, then slowly increasing the EtOAc content up to 15%. Individual column fractions were tested for purity by TLC (petroleum ether/EtOAc 80:20 + 1% aq. NH3, Rf = 0.69) and combined accordingly. Compound 4 was recovered as a yellow-orange oil (1.900 mg, 65% yield).

Equilibrium studies

The chemicals (MCl2 salts) used in the studies were of the highest analytical grade obtained form Sigma-Aldrich and Strem Chemicals Inc. companies. The concentration of the stock solutions was determined by complexometric titration using a standardized Na2H2EDTA solution and appropriate indicators (Patton & Reeder (CaCl2), Eriochrome Black T (MgCl2 and MnCl2), xylenol orange (ZnCl2), murexid (CuCl2)). [START_REF] Schwarzenbach | Complexometric titrations[END_REF] The pH-potentiometric titrations were carried out with a Methrohm 888 Titrando titration workstation, using a Metrohm-6.0233.100 combined electrode. The titrated solutions (6.00 mL) were thermostated at 25 °C, and samples were stirred and kept under inert gas atmosphere (N2) to avoid the effect of CO2. The concentration of the 4-HET-CDTA ligand was determined by pH-potentiometric titration, comparing the titration curves obtained in the presence and absence of moderate Mn 2+ excess. The protonation constants of 4-HET-CDTA as well as the stability and protonation constants of the complexes formed with Mg II , Ca II , Mn II , Cu II and Zn II were also determined by pH-potentiometric titration. The metal-to-ligand concentration ratios were 1:1 and 2:1 (the concentration of the ligand was generally 2.50 -3.00 mM). The pH-potentiometric titrations curves were measured in the pH range 1.8 -11.8 and 86 -278 mL NaOH-pH data pairs were recorded and fitted simultaneously.

The stability constant and solution speciation of the [Mn(4-HET-CDTA)] 2-complex was confirmed using 1 H relaxometry in order to support the value obtained by pH-potentiometry (for details about T1 measurements, see the "Relaxivity determination" section below). 5.00 mL of a 1. complex was determined at 21 wavelengths (800-580 nm range) by recording the spectra of 1.70×10 - 3 , 2.57×10 -3 and 3.42×10 -3 M complex solutions, while for the Cu II ion, previously published molar absorptivity values (determined under identical conditions) [START_REF] Molnár | Picolinate-Containing Macrocyclic Mn2+ Complexes as Potential MRI Contrast Agents[END_REF] were used for the data fitting. The molar absorption coefficients of diprotonated [CuH2(4-HET-CDTA)] were calculated during the simultaneous data refinement (UV-visible, and pH-potentiometric titration curves obtained at various metal to ligand concentrations). The fitting parameters for the absorbance data are as follows 4.34×10 - 3 , 3.65×10 -3 , 3.29×10 -3 , 3.18×10 -3 , 3.26×10 -3 , 2.63×10 -3 , 2.48×10 -3 , 2.24×10 -3 , 2.10×10 -3 , 1.78×10 -3 , 1.91×10 -3 , 1.79×10 -3 , 2.25×10 -3 , 2.49×10 -3 , 2.75×10 -3 , 2.74×10 -3 , 2.89×10 -3 , 2.75×10 -3 , 2.79×10 -3 , 2.76×10 -3 , 2.58×10 -3 , which indicate that the error of the fitting is being kept below 1% for all wavelengths. The protonation (ligand and complexes) and stability constants (complexes) were calculated from the titration data with the PSEQUAD program. 64

Kinetic studies

The dissociation rates of the [Mn(4-HET-CDTA)] where At, A0 and Ae are the absorbance at time t, at the start and at equilibrium of the reaction, respectively. The calculations were performed with the computer program Micromath Scientist, version 2.0 (Salt Lake City, UT, USA) by using a standard least-squares procedure.

O NMR and 1 H NMRD measurements

Longitudinal (1/T1) and transverse (1/T2) [START_REF] Vanasschen | Radiolabelling with isotopic mixtures of 52g/55Mn(ii) as a straight route to stable manganese complexes for bimodal PET/MR imaging[END_REF] O relaxation rates and chemical shifts of an aqueous solution of [Mn(4-HET-CDTA)] 2-(pH = 7.3, 3.0 mM) and a diamagnetic reference (HClO4 acidified water, pH = 3.3) were measured in the temperature range 273 -348 K using a Bruker Avance 400 (9.4 T, 54.2 MHz) spectrometer. The temperature was determined according to previous calibration by means of ethylene glycol and methanol as standards. [START_REF] Raiford | Calibration of methanol and ethylene glycol nuclear magnetic resonance thermometers[END_REF] 1/T1 and 1/T2 values were determined by the inversion-recovery and the Carr-Purcell-Meiboom-Gill spin-echo technique, respectively. [START_REF] Meiboom | Modified Spin-Echo Method for Measuring Nuclear Relaxation Times[END_REF] The technique used for the [START_REF] Vanasschen | Radiolabelling with isotopic mixtures of 52g/55Mn(ii) as a straight route to stable manganese complexes for bimodal PET/MR imaging[END_REF] O NMR measurements has been described previously. [START_REF] Micskei | Oxygen-17 NMR study of water exchange on gadolinium polyaminopolyacetates [Gd(DTPA)(H2O)]2-and [Gd(DOTA)(H2O)]-related to NMR imaging[END_REF] Susceptibility corrections of the chemical shifts were avoided by placing the sample in a glass sphere fitted into a 10 mm NMR tube. [START_REF] Vanasschen | Radiolabelling with isotopic mixtures of 52g/55Mn(ii) as a straight route to stable manganese complexes for bimodal PET/MR imaging[END_REF]

Relaxivity determination

The longitudinal water proton relaxation rate (r1 = 1/T1 -1/Tw) was measured at 20 MHz with a Bruker Minispec MQ-20 relaxometer (Bruker Biospin, Germany). Samples were thermostated by using a circulating water bath at 25.0 ± 0.2 o C. The longitudinal relaxation times (T1) were measured by the inversion-recovery method (180 o ─ τ ─ 90 o ), averaging 5-6 data points for each concentration point obtained from 14 different τ values (τ values ranging between 0 to at least 6 times the expected T1).

The relaxivity of the complex was determined by titrating a nearly 1.0 mM ligand solution with a Mn II stock solution at pH=7.32 (50 mM HEPES buffer, I = 0.15 M NaCl, 25 o C). Under these conditions, only one Mn II ion containing species is present in solution (i.e. the [Mn(4-HET-CDTA)] 2-complex, as supported by the r1 vs. pH profile). Plotting 1/T1 values as a function of [Mn II ] for the samples with [Ligand] > [Mn II ] gives a straight line, the slope of which is equal to the relaxivity of the complex.

For Table of Contents Only

Synopsis

The synthesis of two CDTA-based bifunctional Mn II chelators (BFMnCs) reactive in "click" chemistry is reported. A "click"-conjugate of one BFMnC was used to perform a detailed thermodynamic, kinetic and relaxometric assessment of the corresponding Mn II complex. As demonstrated by the exquisite balance between thermodynamic stability, kinetic inertness and relaxivity of the chelate, the reported BFMnCs are promising precursors for Mn II -based PET/MR tracers ( 52g/55 Mn II ) as well as MR contrast agents (Mn II ) of the next-generation.

Scheme 1 .

 1 Scheme 1. (A) Structure of the desired BFMnCs 6a (or 4-P-CDTA(tBu)4) and 6b (or 4-A-CDTA(tBu)4). (B) Synthesis strategy for a targeted bimodal PET/MR probe starting from 6a.

Scheme 2 .

 2 Scheme 2. Synthetic scheme for the preparation of 4-P-CDTA(tBu)4 (6a) and 4-A-CDTA(tBu)4 (6b). Reagents and conditions: (i) TBDPSCl, imidazole, CH2Cl2, RT, 97 %. (ii) NaN3, Mn(OAc)3.2H2O, TFA, CH3CN/CH2Cl2, -20°C, 62 %. (iii) H2, Pd/C, EtOAc/MeOH, RT, quantitative. (iv) tert-butyl bromoacetate, K2CO3, DMF, 50°C, 65 %. (v) TBAF, THF, 0°C → RT, 85 %. (a) propargyl bromide, CsOH.H2O, CH2Cl2, 0°C → RT, 75 %. (b) 1.

  Scheme 3.Synthesis of model compound 4-HET-CDTA (8a) by "click"-coupling between 6a and 2-

  -CDTA, CDTA, EDTA and 1,4-DO2A (I = 0.15 M NaCl, T = 25 o C).

  [H + ] range of 0.02 -1.0 M; [H + ] + [Na + ] = 1.0 M). The corresponding absorption spectra recorded for the Cu II : 4-HET-CDTA : H + system between 400 -875 nm are shown in Figure 1. These data were fitted simultaneously with the ones obtained by pH-potentiometry (titration at 1:1 and 2:1 metal-to-ligand ratio in the pH range 1.73 -11.80 and 1.70 -5.00, respectively). The thermodynamic stability constants obtained for the different 4-HET-CDTA complexes are presented in Table 2 and compared with those of their EDTA, CDTA and 1,4-DO2A analogues. The values highlight that 4-HET-CDTA forms chelates of slightly lower stabilitiy than CDTA (∆log KML = 0.4 -0.7) which is the consequence of the lower ligand basicity. In the case of the Mn II complexes, the calculated pMn value (which can be considered as a conditional stability constant; pMn = -log [Mn II ]free at 25 o C, pH = 7.4 and cMnII = cL = 1×10 -5 M) 44 is almost identical for both complexes (pMn = 8.62 vs. 8.68, respectively).

Figure 1 .

 1 Figure 1. Absorption spectra of the [Cu(4-HET-CDTA)] 2-system as a function of cH+. Conditions: cL = 3.475 mM, cCuII = 3.422 mM, 25 o C, 1.0 M (Na + +H + )Cl -, cH+ in the samples was 18.8; 34.1; 49.7; 99.6; 149; 200; 250; 349; 450 and 999 mM (from top to bottom).

  leading to the largest difference in thermodynamic stability between 4-HET-CDTA and CDTA complexes (Δ log KCuL = 1.5). This may be attributed to the lower ligand basicity as the stability constant of [Cu(4-HET-CDTA)] 2-had to be determined in 1.0 M instead of 0.15 M NaCl (log K1 H = 8.78(2), log K2 H = 5.68(3), logK3 H = 3.59(3), logK4 H = 2.59(3) and logK5 H 1.24(3) making Σ log Κi Η = 21.88 in 1.0 M NaCl and 25 o C

  [a] The data in brackets are the standard deviations; [b] data taken from the Diploma work of Veronika Józsa, 2015, University of Debrecen, Debrecen, Hungary; [c] ref. 4 ; [d] pMn values were calculated by using the approach of É. Tóth et al. (25 o C, pH = 7.4 and cMnII = cL = 1×10 -5 M) 44 ; [e] ref. 11 ; [f] ref. 12 ; [g] determined by UV-Vis spectrophotometry.

Figure 2 .

 2 Figure 2. Relaxivity of [Mn(4-HET-CDTA)] 2-as a function of pH (blue dots; 20 MHz, 25 o C; pH range 1.75 -12.20) and the species distribution curves (solid lines) calculated by using the stability constants presented inTable 2.

Figure 3 .

 3 Figure 3. cH+ dependence of the pseudo-first-order rate constants (kobs) for the [Mn(4-HET-CDTA)] 2- complex (0.4 mM) at different exchanging metal ion concentrations. cCuII = 4.28 mM (10-fold excess; blue triangles), 8.26 mM (20-fold excess; red diamonds), 12.8 mM (30-fold excess; purple squares) and 17.1 mM (40fold excess; green dots). The corresponding pH values were the following: 5.03. 4.79, 4.56, 4.26 and 4.05 (from left to right).

Figure 4 .

 4 Figure 4. Possible reaction pathways for the dissociation of the [Mn(4-HET-CDTA)] 2-complex. The different kx and Kx constants are defined in the text.

Figure 5 . 1 H

 51 Figure 5. 1 H NMRD profiles for [Mn(4-HET-CDTA)] 2-(pH = 7.4) recorded at 25 °C (triangles), 37 °C (squares) and 50 o C (diamonds).

  it appears that the reduced transverse[START_REF] Vanasschen | Radiolabelling with isotopic mixtures of 52g/55Mn(ii) as a straight route to stable manganese complexes for bimodal PET/MR imaging[END_REF] O relaxation rates (1/T2r) obtained for [Mn(4-HET-CDTA)] 2-are in the fast exchange region above 285K, while they turn to the intermediate exchange regime below this temperature. The analysis of the[START_REF] Vanasschen | Radiolabelling with isotopic mixtures of 52g/55Mn(ii) as a straight route to stable manganese complexes for bimodal PET/MR imaging[END_REF] O NMR data showed that the contribution of the electron spin relaxation term in the overall correlation time (1/τc = kex + 1/T1e) governing the transverse relaxation rate becomes negligible at high temperatures (approx. 3 %), which ensures the correct determination of the water exchange rate. The maximum contribution of 1/T1e amounts to 30 % at the lowest temperatures and the 1/T2r data cannot be fitted by neglecting the contribution of T1e. The kex 298 value obtained for [Mn(4-HET-CDTA)] 2-is very close to that of [Mn(CDTA)] 2-, which is expected considering their identical coordination cage. On the other hand, the water exchange is approximately three times slower than for [Mn(EDTA)] 2-(kex 298 = 47.1×10 7 s -1 ), most likely due to the more flexible EDTA skeleton which facilitates the rearrangement of the coordination environment during the water exchange process, and about 6-times slower than for the macrocyclic chelate [Mn(1,4-DO2A)].[START_REF] Rolla | 1H and 17O NMR Relaxometric and Computational Study on Macrocyclic Mn(II) Complexes[END_REF] Moreover, the positive value of the activation entropy suggests a dissociatively activated water exchange mechanism. Most importantly, the water exchange of [Mn(4-HET-CDTA)] 2-remains fast enough to consider BFMnC 6a or 6b for the design of multimeric CAs with optimized relaxivity.[START_REF] Zhu | Hexameric MnII Dendrimer as MRI Contrast Agent[END_REF]

  characterizing the inner-(IS) and outer-sphere (OS) relaxation mechanisms, respectively, and the Swift-Connick[START_REF] Swift | NMR-Relaxation Mechanisms of O17 in Aqueous Solutions of Paramagnetic Cations and the Lifetime of Water Molecules in the First Coordination Sphere[END_REF][START_REF] Swift | Erratum: NMR-Relaxation Mechanisms of 17O in Aqueous Solutions of Paramagnetic Cations and the Lifetime of Water Molecules in the First Coordination Sphere[END_REF] equations describing solvent exchange (see ESI for detailed equations). The water exchange parameters have been set to the values determined by the fitting of the[START_REF] Vanasschen | Radiolabelling with isotopic mixtures of 52g/55Mn(ii) as a straight route to stable manganese complexes for bimodal PET/MR imaging[END_REF] O NMR data, while the following parameters were fixed to reasonable estimates: the distance between the metal ion and the inner and outer sphere water protons were set to rMnH = 2.83 Å and aMnH = 3.6 Å, respectively.[START_REF] Rolla | 1H and 17O NMR Relaxometric and Computational Study on Macrocyclic Mn(II) Complexes[END_REF] ; the diffusion coefficient and its activation energy were assigned to DMnH = 26×10 -10 m 2 s -1 and EMnH = 18 kJ mol -1 ; Ev was set to 1 kJ mol -1 . The fit of the NMRD data yielded τv 298 = 12.2 ps for the correlation time describing the modulation of the ZFS, and ∆ 2 = 2.3×10 20 s -2 for the trace of the square of the ZFS tensor. The other best-fit parameters obtained are given in Table

Figure 6 .

 6 Figure 6. Variable-temperature reduced transverse 17 O relaxation rates (1/T2r) recorded for [Mn(4-HET-CDTA)] 2-at 9.4 T. The solid lines correspond to the best fit of the data.

1 H

 1 NMR (600 MHz, CDCl3) δ 7.58 (dt, J = 7.9, 1.5 Hz, 4H), 7.37 -7.25 (m, 6H), 3.47 -3.29 (m, 10H, 4 x CH2CO2 t Bu + CH2OTBDPS), 2.88 (m, 2H, 2 x CH-N), 1.94 -1.78 (m, 2H, chx ring protons), 1.70 (m, 1H, chx ring proton), 1.36 (m, 40H, 4 x C(CH3)3 + 4 chx ring protons), 0.96 (s, 9H, C(CH3)3).

  The calibration of the electrode was performed by two point calibration (KH-phthalate (pH = 4.005) and borax (pH = 9.177) buffers). The calculation of [H + ] from the measured pH values was performed with the use of the method proposed by Irving et al.[START_REF] Irving | A study of some problems in determining the stoicheiometric proton dissociation constants of complexes by potentiometric titrations using a glass electrode[END_REF] by titrating a 0.01 M HCl solution (I = 0.15 M NaCl) with a standardized NaOH solution. The differences between the measured (pHread) and calculated pH (-log [H + ]) values were used to obtain the equilibrium H + concentrations from the pHdata obtained in the titrations. The ion product of water was determined from the same experiment in the pH range 11.4 -12.0.

  02 mM [Mn(4-HET-CDTA)] 2-sample were titrated with solid NaOH or gaseous HCl (in order to avoid sample dilution) to adjust the pH in the range 1.75 -12.20, followed by recording and averaging of 4-6 T1 values for each pH data point. Due to the high conditional stability of [Cu(4-HET-CDTA)] 2-, the formation of the complex was complete (nearly 100%) even at pH = 1.73 (starting point of the pH-potentiometric titration). For this reason, 10 out-of-cell (batch) samples containing a slight excess of ligand and the Cu II ion were prepared ([Lig] = 3.475 mM, [Cu 2+ ] = 3.422 mM, 25 o C, 1.0 M (Na + +H + )Cl -). The samples, whose acidity was varied in the concentration range of 18.8 -999 mM, were equilibrated for 1 day before recording the absorption spectra at 25 o C in Peltier thermostated semi-micro 1 cm Hellma® cells using a Varian CARY 1E, UV-Vis spectrophotometer. The molar absorptivity of the [Cu(4-HET-CDTA)] 2-

  2-chelate were studied at 25 o C (Peltier thermostated) and 0.15 M NaCl ionic strength by monitoring the formation of the Cu II complex at 290 nm via UV-Vis spectrophotometry (Varian CARY 1E instrument) in 1 cm quartz cuvettes. All dissociation reactions were performed under pseudo-first order conditions where the exchanging metal ion (Cu II ) was in 10 -40-fold excess relative to the complex ([complex] = 4.0×10 -4 M, pH range 4.05 -5.03). The kinetic studies were carried out in a non-coordinating buffer to maintain the pH in the samples constant (0.05 M N,N'-dimethylpiperazine (dmp, logK2 H = 4.19)). The pseudo-first-order rate constants (kobs) were calculated by fitting the absorbance-time data pairs to equation 9:

  O enriched water (10% H2 17 O, CortecNet) was added to the solutions to reach about 1% enrichment for improving sensitivity. Proton NMRD profiles of the [Mn(4-HET-CDTA)] 2- complex (1.00 mM, pH = 7.4) were recorded in aqueous solution 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. Temperature monitoring was performed with a VTC91 temperature control unit and maintained by a gas flow. The temperature was determined according to previous calibration with a Pt resistance temperature probe. The least-squares fit of the 17 O NMR and of the NMRD data was performed using Visualiseur/Optimiseur (F. Yerly, VISUALISEUR 3.3.7, Lausanne, Switzerland, 2006; F. Yerly, OPTIMISEUR 3.3.7. Lausanne, Switzerland, 2006) running on a MATLAB 8.3.0 (R2014a) platform.

Table 2 .

 2 Stability constants (log KML), protonation constants (log KMHiL) and pMn values for metal complexes formed with 4-HET-CDTA, CDTA, EDTA and 1,4-DO2A (I = 0.15 M NaCl, T = 25 o C).

			4-HET-CDTA [a]	CDTA [b]	EDTA [b]	1,4-DO2A [c]
	Mg II	log KML log KMHL	8.69(1) -	9.14 3.53	7.61 -	--
	Ca II	log KML log KMHL	9.86(1) 3.50(2)	10.23 3.58	9.53 2.92	8.62 -
		log KMnL	13.80(3)	14.32 [e]	12.46 [e]	15.68 [f]
	Mn II	log KMnHL log KMnH2L	2.56(5) -	2.90 [e] 1.89 [e]	2.95 [e] -[e]	4.15 -
		pMn	8.62 [d]	8.68 [d]	7.83 [d]	7.27 [d, f]
		log KML	16.08(1)	16.75	15.92	18.03
	Zn II	log KMHL	2.58(1)	2.57	3.23	3.58
		log KMH2L	1.63(3)	1.58	1.50	1.65
		log KML	18.30(2) [g]	19.78 [g]	19.02 [g]	24.43 [g]
	Cu II	log KMHL	3.02(2)	2.91	3.15	2.95
		log KMH2L	1.46(2)	1.10	2.04	-

Table 4 .

 4 Best-fit parameters obtained from the analysis of[START_REF] Vanasschen | Radiolabelling with isotopic mixtures of 52g/55Mn(ii) as a straight route to stable manganese complexes for bimodal PET/MR imaging[END_REF] O NMR and 1 H NMRD data for the Mn II

	complexes of 4-HET-CDTA, CDTA, EDTA and 1,4-DO2A.
	Parameter	4-HET-CDTA	CDTA [d]	EDTA [e]	1,4-DO2A [f]
	kex 298 (×10 7 s -1 ) [a]	17.6 ± 4.4	14.0	47.1	113.4
	∆H ‡ (kJ mol -1 ) [a]	36.2 ± 2.8	42.5	33.5	29.4
	∆S ‡ (J K -1 mol -1 ) [a]	+34 ± 7	-	-	-
	AO/ (x10 6 rad s -1 ) [a]	40 [c]	4.2	-40.5	43
	1/T1e 298 (×10 7 s -1 ) [a]	7 ± 2.4	0.65	-	-
	ER (kJ mol -1 ) [b],[h]	26.2 ± 1.4	-	21.8	19.1
	τR 298 (ps) [b]	104.9 ± 5.0	-	57	46

  This work; [b] ref. 26 ; [c] ref. 12 ; [d] ref. 4 ; [e] 20 MHz; [f] given the superior molecular weight of 4-HET-CDTA, this value cannot be strictly compared with the r1 data of other chelates in this table. Nonetheless, for all

	t1/2 (h)	16.2	12.1	-[i]	48.3	556
	BFMnC	6a/6b	6a/6b	tBu-PyC3A-NHS	-	-
	[a]
	-1 s -1 ) 25/37 o C [e]	4.56 [f] /3.43	3.65/2.75	3.3/2.5	2.1 [g] / -	2.5/ -
	q	1	1	1	0.87 [g]	0.87
	log KMnL	13.80	14.32	14.14	15.68	12.64
	log KMnL pH=7.4	-	12.34 [b]	11.34	10.10 [b]	-
	pMn [h]	8.62	8.68	8.17	7.27	6.98

  H NMR (600 MHz, CDCl3) δ 7.75 (dt, J = 7.9, 1.6 Hz, 4H), 7.51 -7.41 (m, 6H), 5.74 (d, J = 2.3 Hz, 2H), 3.63 (d, J = 6.1 Hz, 2H), 2.24 -2.04 (m, 3H), 1.99 -1.77 (m, 3H), 1.43 -1.30 (m, 1H), 1.13 (s,

	9H).
	13 C NMR (151 MHz, CDCl3) δ 135.66 (CH Ar), 134.10 (C Ar), 129.55 (CH Ar), 127.63 (CH Ar),
	127.11 & 126.34 (C=C), 68.63 (CH2-OTBDPS), 36.41, 28.33, 26.94 (C(CH3)3), 25.43, 24.88, 19.40
	(C(CH3)3).

[START_REF] Drahoš | Manganese(II) Complexes as Potential Contrast Agents for MRI[END_REF]

  exchange resin (Cl -form, 100-200 mesh; before sample loading, the resin was converted to the OH - form by washing with large volumes of aq. 1M NaOH followed by water rinses until neutral pH of the eluate). After washing of the column with decarbonated water (200 mL), ligand 8a was eluted using a

	13 C NMR (151 MHz, CDCl3) δ 170.51, 170.49 (C=O), 134.54 (CH Ar), 132.89, 132.86 (C Ar), EtOAc (70 mL) and washed with brine (2 x 30 mL). Drying over MgSO4 and evaporation yielded a IR: νN3 = 2097.66 cm -1
	128.48, 128.47 (CH Ar), 126.58 (CH Ar), 79.45 & 79.43 (CO2C(CH3)3), 66.49 (CH2-OTBDPS), 58.05 golden-brown oil which was chromatographed over a silica gel column using an elution gradient
	& 56.50 (2 x CH-N), 53.06 & 52.87 (CH2CO2 t Bu), 34.01, 27.12 (CO2C(CH3)3), 27.06, 25.88 (CH2Cl2/MeOH 99:1 + 0.5% aq. NH3 to CH2Cl2/MeOH 95:5 + 0.5% aq. NH3); individual column Tetra-tert-butyl 4-((1-(2-hydroxyethyl)-1H[1,2,3]triazol-4-yl)methoxy)methyl-trans-1,2-
	(C(CH3)3), 23.57, 22.38, 18.21 (C(CH3)3). fractions were tested for purity by TLC (100% EtOAc, Rf = 0.79; CH2Cl2/MeOH 97:3 + 0.5% aq. diaminocyclohexane-N,N,N',N'-tetraacetate, 4-HET-CDTA(tBu)4 (7a) formic acid gradient (0.005, 0.01, 0.02, 0.04, 0.08, 0.15, 0.3 and 1 M aq. HCO2H). Freeze-drying of
	HRMS (FT-ICR, positive), m/z: 839.52353 ([M+H] + ; calculated for C47H75N2O9Si + : 839.52363), m/z: NH3, Rf = 0.24) and combined accordingly. Pure 6a was obtained as a golden-yellow oil (767 mg, 2-Azidoethanol (107.3 mg, 1.232 mmol; synthesized as reported by Lu et al.) 61 , DIPEA (358 μL, the fractions containing the desired product (0.15, 0.30 and 1 M aq. HCO2H) yielded 4-HET-CDTA as
	861.50456 ([M+Na] + ; calculated for C47H74N2NaO9Si + : 861.50558). 75% yield). 2.054 mmol), 2,6-lutidine (244 μL, 2.054 mmol) and CuI (219.5 mg, 1.130 mmol) were added an off-white solid (191 mg, 76% yield).
	1 H NMR (600 MHz, CDCl3) δ 4.11 (d, J = 2.4 Hz, 2H, CH2-C≡CH), 3.48 (d, J = 3.7 Hz, 8H, 4 x successively to a 50 mL round-bottomed flask containing alkyne 6a (656 mg, 1.027 mmol). The flask 1 H NMR (600 MHz, D2O) δ 8.05 (s, 1H, triazole H), 4.58 (m, 2H, HC=C-CH2-O), 4.50 (t, J = 5.0 Hz,
	Tetra-tert-butyl 4-(hydroxymethyl)-trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetate (5) CH2CO2 t Bu), 3.42 -3.30 (m, 2H, CH2-O), 2.98 (m, 1H, CH-N), 2.87 (m, 1H, CH-N), 2.40 (t, J = 2.3 was put under argon atmosphere, and the reagents were suspended in anhydrous CH2Cl2 (25 mL). 2H, N-CH2-CH2-OH), 4.09 -3.36 (m, 12H, 4 x CH2CO2 t Bu + HC=C-CH2-O-CH2 + CH2-OH), 3.24
	Tetrabutylammonium fluoride (5.66 mL of a 1.0 M solution in THF, 5.66 mmol) was slowly added Hz, 1H, C≡CH), 2.02 -1.90 (m, 2H, chx ring protons), 1.90 -1.81 (m, 1H, chx ring protons), 1.45 (s, After 2 h of vigorous stirring at RT, the reaction mix was diluted with CH2Cl2 (25 mL) and washed (td, J = 11.5, 3.6 Hz, 1H, CH(2)N3), 3.09 (td, J = 11.9, 3.5 Hz, 1H, CH(1)N3), 2.17 (m, 1H, chx ring
	under argon atmosphere to an ice-cooled solution of compound 4 (1.900 g, 2.264 mmol) in dry THF 40H, 4 x C(CH3)3 + 4 chx ring protons). twice with aq. 10% CuSO4.5H2O (2 x 10 mL) to get rid of 2,6-lutidine. In order to remove the copper proton), 2.07 (m, 1H, chx ring proton), 1.96 -1.85 (m, 1H, chx ring proton), 1.76 (m, 1H, chx ring
	(25 mL). After 24 h stirring at RT, the reaction was quenched by addition of a saturated NH4Cl 13 C NMR (151 MHz, CDCl3) δ 171.57 & 171.53 (C=O), 80.53 & 80.52 (CO2C(CH3)3), 80.12 (C≡CH), ions chelated by the clicked product, a 0.4 M aqueous DTPA solution (pH 7.1, 25 mL) was added to proton), 1.63 -1.30 (m, 3H, chx ring protons).
	solution (30 mL). EtOAc was then added (150 mL), the phases separated and the aqueous layer 74.07 (C≡CH), 73.48 (CH2-O), 59.87 (CH-N), 58.16 (CH2-C≡CH), 57.73 (CH-N), 54.03 & 53.87 the organic fraction and the heterogeneous mixture was stirred vigorously for 1-2 h. Once the intensely 13 C NMR (151 MHz, D2O) δ 172.25 & 170.89 (C=O), 143.46 (HC=C-CH2-O), 125.68 (HC=C-CH2-
	extracted with EtOAc (3 x 30 mL). The combined organic fractions were washed with NaHCO3 sat. (CH2CO2 t Bu), 32.60, 28.66, 28.15 (CO2C(CH3)3), 25.31, 23.73. blue aqueous phase was discarded, a fresh 0.4 M DTPA solution (25 mL) was added to the O), 71.06 (HC=C-CH2-O-CH2), 63.42 (CH-N), 62.44 (HC=C-CH2-O-CH2), 59.91 (CH2-OH), 58.43
	(50 mL), dried over MgSO4 and evaporated to dryness. Purification of the crude oil was performed by HRMS (FT-ICR, positive), m/z: 639.42166 ([M+H] + ; calculated for C34H59N2O9 + : 639.42151), m/z: dichloromethane fraction and the resulting mixture was stirred overnight. The organic phase was then (CH-N), 53.85 (CH2CO2H, low intensity, broad) 52.85 (N-CH2-CH2-OH), 49.88 (CH2CO2H, low
	silica gel column chromatography (gradient: CH2Cl2/EtOAc 95:5 + 1% Et3N to CH2Cl2/EtOAc 85:15 661.40408 ([M+Na] + ; calculated for C34H58N2NaO9 + : 661.40345). dried over MgSO4 and evaporated under vacuum. Purification of the crude oil was performed by silica intensity, broad), 31.73, 24.72, 24.63, 19.46.
	+ 1% Et3N); individual column fractions were tested for purity by TLC (100% EtOAc, Rf = 0.36) and gel column chromatography (CH2Cl2/MeOH 97:3 + 1% NH3); individual column fractions were tested
	combined accordingly. Pure alcohol 5 was recovered as a yellow-orange oil (1.160 g, 85% yield). Tetra-tert-butyl 4-(azidomethyl)-trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetate, for purity by TLC (CH2Cl2/MeOH 97:3 + 1% NEt3, Rf = 0.30). 4-HET-CDTA(tBu)4 (7a) was obtained
	1 H NMR (600 MHz, CDCl3) δ 3.54 -3.40 (m, 10H, 4 x CH2CO2 t Bu + CH2OH), 2.98 (m, 1H, CH-N), 4-A-CDTA(tBu)4 (6b) as a yellow-orange oil (521 mg, 70% yield).
	2.85 (m, 1H, CH-N), 2.54 (s, broad, 1H, OH), 2.00 -1.80 (m, 3H, chx ring protons), 1.46 (m, 40H, 4 Alcohol 5 (190 mg, 0.316 mmol) and triethylamine (110.2 μL, 0.79 mmol) in anhydrous CH2Cl2 (12 CH2-
	x C(CH3)3 + 4 chx ring protons). mL) were placed under argon atmosphere and cooled to 0°C. Methanesulfonyl chloride (48.9 μL, CH2-OH), 4.04 (t, J = 5.0 Hz, 2H, CH2-OH), 3.69 -3.19 (m, 10H, 4 x CH2CO2 t Bu + HC=C-CH2-O-
	13 C NMR (151 MHz, CDCl3) δ 171.62 & 171.57 (C=O), 80.60 & 80.52 (CO2C(CH3)3), 65.64 0.632 mmol) was slowly added to this solution. After 2-3 h at 0°C, the reaction mixture was diluted CH2), 2.93 (m, 1H, CH-N), 2.84 (m, 1H, CH-N), 2.09 -1.68 (m, 3H, chx ring protons), 1.45 (s, 40H, 4
	(CH2OH), 60.30 & 57.64 (2 x CH-N), 54.07 & 54.03 (CH2CO2 t Bu), 35.23, 28.37, 28.13 with diethyl ether (40 mL), washed with water (20 mL) and brine (20 mL) and dried over MgSO4. x C(CH3)3 + 4 chx ring protons).
	(CO2C(CH3)3), 25.10, 23.89. Evaporation in vacuo yielded the crude mesylate (199 mg, ≈ 0.293 mmol) which was further reacted 13 C NMR (151 MHz, CDCl3) δ 171.68 (C=O), 145.46 (HC=C-CH2-O), 123.68 (HC=C-CH2-O), 80.66
	HRMS (FT-ICR, positive), m/z: 601.40583 ([M+H] + ; calculated for C31H57N2O9 + : 601.40586), m/z: by dissolution in anhydrous DMF (10 mL), addition of NaN3 (57.1 mg, 0.879 mmol) and stirring at (CO2C(CH3)3), 73.93 (HC=C-CH2-O-CH2), 64.68 (HC=C-CH2-O-CH2), 61.06 (CH2-OH), 59.91 &
	623.38780 ([M+Na] + ; calculated for C31H56N2NaO9 + : 623.38780). 75°C for 24 h. Once the solution was allowed to cool down, diethyl ether was added (50 mL), the 57.90 (CH-N), 54.02 (CH2CO2 t Bu), 52.58 (N-CH2-CH2-OH), 32.78, 28.77, 28.14 (CO2C(CH3)3),
	reaction mixture was washed with water (25 mL) and brine (25 mL) and dried over MgSO4. Crude 25.38, 23.80.
	Tetra-tert-butyl 4-(prop-2-ynyloxymethyl)-trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetate, azide was purified by silica gel column chromatography (CH2Cl2/EtOAc 95:5 + 1% Et3N); individual HRMS (FT-ICR, positive), m/z: 748.44578 ([M+Na] + ; calculated for C36H63N5NaO10 + : 748.44671).
	4-P-CDTA(tBu)4 (6a) column fractions were tested for purity by TLC (petroleum ether/EtOAc 80:20 + 1% aq. NH3, Rf =
	Alcohol 5 (962 mg, 1.601 mmol) and activated powdered 4 Å molecular sieves (1.5 g) were suspended 0.48). Finally, 6b was recovered as a yellowish oil (139 mg, 70 % over 2 steps). 4-((1-(2-Hydroxyethyl)-1H[1,2,3]triazol-4-yl)methoxy)methyl-trans-1,2-diaminocyclohexane-
	in 15 mL anhydrous CH2Cl2, placed under argon atmosphere and cooled to 0°C. To this mixture, a 1 H NMR (600 MHz, CDCl3) δ 3.64 -3.29 (m, 8H, 4 x CH2CO2 t Bu), 3.25 -3.09 (m, 2H, CH2N3), 3.04 N,N,N',N'-tetraacetic acid, 4-HET-CDTA (8a)
	solution of propargyl bromide (80% in toluene, 518 μL, 4.803 mmol) and cesium hydroxide (m, 1H, CH-N), 2.94 (m, 1H, CH-N), 2.03 -1.79 (m, 3H, chx ring protons), 1.46 (m, 40H, 4 x 7a (363 mg, 0.500 mmol) was dissolved in a 1:1 TFA/CH2Cl2 mixture (10 mL) and stirred at RT for
	monohydrate (806.6 mg, 4.803 mmol) were successively added and the suspension was vigorously C(CH3)3 + 4 chx ring protons). 18 h. Evaporation of the solvents under reduced pressure yielded an orange oil which was dissolved in
	stirred. After 30 min, the ice bath was removed and the reaction was left at RT for approximately 18 h. 13 C NMR (151 MHz, CDCl3) δ 171.47 & 171.39 (C=O), 80.68 & 80.67 (CO2C(CH3)3), 58.77 & 57.22 10 -2 M aq. HCl (1.5 mL, pH ≈ 2) and loaded onto a Dowex 50WX4 (H+ form, 100-200 mesh) cation-
	Full consumption of the adduct was verified by TLC (100% EtOAc). If needed, 2 equivalents of (2 x CH-N), 56.13 (CH2N3), 54.15 & 53.98 (CH2CO2 t Bu), 32.72, 28.77, 28.15 (CO2C(CH3)3), 25.67, exchange resin. The latter was washed with water (200 mL) and the ammonium salt of 8a was
	propargyl bromide and CsOH.H2O were added and reacted for a few additional hours. The reaction 23.28.
	mixture was then diluted with EtOAc (15 mL), centrifuged to remove insolubilities, re-diluted with HRMS (FT-ICR, positive), m/z: 626.41240 ([M+H] + ; calculated for C31H56N5O8 + : 626.41234).

1 H NMR (600 MHz, CDCl3) δ 7.76 (s, 1H, triazole H), 4.72 -4.41 (m, 4H, HC=C-CH2-O + Nrecovered by elution with 0.5 M aq. NH3. The yellow-orange residue obtained after evaporation of solvents was dissolved in 1M aq. NH3 (1.2 mL, pH ≈ 10) and transferred onto a Dowex 1X8 anion-HRMS (FT-ICR, positive), m/z: 502.21430 ([M+H] + ; calculated for C20H32N5O10 + : 502.21437).

O NMR and 1 H NMRD evaluation of the [Mn(4-HET-CDTA)] 2-complex

Acknowledgements C.V., H.H.C and B.N. thank the Helmholtz Association for funding of this work through Helmholtz-Portfolio Topic "Technology and Medicine". Part of this research was funded by the Hungarian Scientific Research Fund (OTKA K-120224 project), Le Studium, Loire Valley Institute for Advanced Studies (Gy.T. and F.K.K.) and the János Bolyai Research Scholarship (Gy.T. and F.K.K.) of the Hungarian Academy of Sciences. E.T. acknowledges support of the Ligue contre le cancer (France).

The research was also supported by the EU and co-financed by the European Regional Development Fund under the project GINOP-2.3.2-15-2016-00008. The authors are further indebted to Katalin Takács for her help with the pH-potentiometric data collection and Dr. Dezső Szikra for his help with the MS studies.