Pyridinophane compounds and MRI contrast agents

Abstract

We report a chelator platform based on diazapyridinophane macrocycles as contrast agents to improve Magnetic Resonance Imaging. Lead compounds identified, [Mn(TE-1)]+ and [Mn(TE-4)], have good relaxivity and kinetic inertness in vivo. Spectrophotometric titrations and in vitro Zn challenge studies of [Mn(TE-4)] showed kinetic inertness with a rate of dissociation of 6.8 x 10-4 s-1, while [Mn(TE-1)]+ has a slower dissociation rate of 1.0 x 10-4 s-1 and a relaxivity of 3.6 mM -1 s-1 measured at 1.4 T magnetic field and 33 ºC. These Mn complexes were investigated by employing the 52Mn radioisotope, a positron emission tomography (PET) radiotracer, to probe kinetic inertness and stability in vivo. The Mn complexes of TE-1 and TE-4 exhibit different behavior in in vivo MRI studies given their different lipophilicity: [Mn(TE-4)] shows contrast enhancement in the kidney and bladder, indicating renal clearance, while the lipophilicity of [Mn(TE-1)]+ promotes a mixed biliary-renal excretion.

Description

[0001] PYRIDINOPHANE COMPOUNDS AND MRI CONTRAST AGENTS

[0002] RELATED APPLICATIONS

[0003] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent

[0004] Application No. 63 / 696,105, filed September 18, 2024, which is incorporated herein by reference.

[0005] GOVERNMENT SUPPORT

[0006] This invention was made with government support under AG083937 awarded by the National Institute of Health. The government has certain rights in the invention.

[0007] BACKGROUND OF THE INVENTION

[0008] Magnetic resonance imaging (MRI) is widely used to diagnose conditions affecting soft tissues such as tumors and joint injuries. Even though MRI provides high-resolution images, the inherent low sensitivity necessitates using contrast agents to improve the quality of MRI scans. For decades, gadolinium-based contrast agents with macrocyclic and acyclic ligands have been used extensively in pre-clinical and clinical applications due to their superb relaxation properties owing to the high spin of Gd3+(S = 7 / 2). However, several health effects, such as nephrogenic systemic fibrosis in patients with chronic kidney diseases and Gd leakage into the brain, have urged the community to explore Mn-based contrast agent alternatives. To replace Gd-based MRI agents, Mn- based agents should possess comparable relaxivities, which is challenging due to the lower spin of Mn2+(S = 5 / 2). Moreover, the Mn complexes should have high thermodynamic stability, good kinetic inertness to avoid decomplexation in vivo, and an inner sphere water molecule coordinated to the Mn center to enhance the relaxation properties of the contrast agent. Combining all these properties is challenging due to the low ligand field stabilization energy induced by the binding of chelators to the Mn center. MnDPDP, an FDA-approved Mn-based contrast agent later withdrawn from the market due to complex dissociation in vivo, highlights the importance of careful chelator design for Mn MRI agents. In addition to the stable55Mn isotope, the52Mn radioisotope (ty2= 5.56 days) provides a complementary radiotracer that can be used for positron emission tomography (PET) biodistribution studies. The longer half-life of this radioisotope allows for imaging over an extended time without the need for continuous injections of radioactivity. Surprisingly, the use of the52Mn radionuclide in PET imaging has been limited, despite its ease of production.

[0009] Many reported acyclic and macrocyclic chelators for Mn have shown favorable properties in recent years (Chart la). Generally, the Mn complexes of acyclic ligands are known to be kinetically labile, except for the Mn complex of PyC3A, a trans- 1,2-cyclohexane-based ligand that has shown great promise and is currently in clinical trials as an Mn MR contrast agent. More recently, Comba et al. reported a bispidine-type ligand showing good relaxivity and kinetic inertness. Macrocyclic

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[0011] UIUC2023-225-02(PCT) ligands generally suffer either from lower relaxivity due to their low hydration numbers q, such as for [Mn(DOTA)]2and [Mn(PCTA)], or limited kinetic inertness, such as for 15-membered rings. [Mn(D02A)] and [Mn(PC2A-EA)] are some of the limited examples that provide a compromise between relaxivity and kinetic inertness. More recently, MnCHXPYAN was reported as a promising52Mn PET imaging agent due to its kinetic inertness and in vivo stability. However, the relaxation properties of MnCHXPYAN are unsuitable for MRI applications, due to the lack of water molecule coordination to the metal center.

[0012] During the past decade, a large body of literature has explored potential chelators for Mn due to their favorable properties for MRI. However, macrocyclic chelators were mainly studied in vitro due to their lower relaxivity or poor kinetic inertness. Considering the limited number of Mn complexes that have shown a good compromise between thermodynamic stability, kinetic inertness, and good relaxivity, advances in the development of novel macrocyclic chelators that could satisfy all these properties are greatly needed.

[0013] SUMMARY

[0014] Based on precedents that [Mn(PCTA)] and related Mn complexes have higher thermodynamic stability and improved kinetic inertness compared to [Mn(D02A)] and [Mn(DOTA)], we envisioned modifying the ligand scaffold by replacing a tertiary amine with a pyridine (Chart 1c). This simple modification would allow us to rigidify the ligand and decrease its overall basicity, since pyridines have lower pXavalues than amines.

[0015] The more rigid pyridinophane ligands would potentially have higher relaxivity due to the reduced tumbling rate in the solution. Additionally, we hypothesized that the pyridinophane ligands and their chelating arms may favorably interact with albumin, increasing their MRI efficiency. Moreover, the lower overall basicity would potentially result in reduced proton competition for the bound metal complex, which is beneficial for thermodynamic stability and kinetic inertness considerations.

[0016] Herein, we report a series of pyridinophane ligands with different coordination numbers and pendant arms (i.e., picolinate, picolyl, and acetate), which are known to increase thermodynamic stability and kinetic inertness of the corresponding Mn complexes. Moreover, several novel chelators were synthesized to study the effects of symmetry, coordination number, and nature of pendant arms on the relaxation properties and MRI efficiency of those chelators. The complexes [Mn(TE-l)]+and [Mn(TE-4)] show an optimal combination of relaxivity, thermodynamic stability, and kinetic inertness (Chart lb). Surprisingly, both [Mn(TE-l)]+and [Mn(TE-4)] have a hydration number (i / ) value of 1.4, indicating an equilibrium between one and two water molecules bound to the Mn center. Importantly, having water molecules bound to the Mn center while having good kinetic inertness is rare. Since there are limited examples of Mn complexes that have been tested in vivo, we have employed [Mn(TE-l)]+and [Mn(TE-4)] in in vivo MRI imaging studies, as well as52Mn

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[0018] UIUC2023-225-02(PCT) biodistribution PET imaging studies. Overall, TE-1 and TE-4 have quite drastic differences in lipophilicity, leading to different clearance pathways in mice. In addition, the MRI studies were performed on a high-field 9.4 T MRI scanner, which provides greater resolution and a higher signal- to-noise ratio.

[0019] Chart 1. Structures of (a) reported and (b) new chelators, (c) The design principle of pyridinophane ligands.

[0020] (a) Reported ligands

[0021] 1,4 D02A 3,9 PC2A (R = H, R’ = CH2COO) BPPA

[0022] 3,6 PC2A (R = CH2COO, R’ = H,) PCTA (R = R’ = CH2COO)

[0023] (b) Ligands explored in this study

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[0025] UIUC2023-225-02(PCT)

[0026] (c) MRI Design Principles

[0027] Accordingly, this disclosure provides a compound of Formula I: or a salt and / or a hydrate thereof; wherein,

[0028] — represents one or more coordinate covalent bonds when NF is present in a metal complex of Formula I; or

[0029] — is absent when Mzis not present;

[0030] Mzis a metal or metal radioisotope, wherein z represents the oxidation state of the metal or metal radioisotope; or NF is absent;

[0031] G1is picolinyl, pyridinyl, carboxyl, or benzothiazolyl hydroxyphenyl;

[0032] G2is picolinyl, pyridinyl, carboxyl, or H; wherein picolinyl, pyridinyl, carboxyl, or benzothiazolyl hydroxyphenyl are substituted with R3; and

[0033] R1, R2, and R3are each independently H, halo, -(Ci-C6)alkyl, -O(Ci-C6)alkyl, or -N(R4)2 wherein each R4is independently H or -(Ci-Ce)alkyl; or the compound:

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[0035] UIUC2023-225-02(PCT)

[0036] (2-(3,7-diaza-l,5(2,6)-dipyridinacyclooctaphane-3-ylmethyl)-4-(benzo[r / / thiazol-2-yl)-6- methoxyphenol) or a metal complex thereof comprising NT, a salt, and / or a hydrate thereof.

[0037] This disclosure also provides a method for improving contrast of a target tissue in a subject imaged by magnetic resonance imaging (MRI) comprising administering a sufficient amount of a metal complex of one of the compounds described herein to the subject and imaging the subject by MRI, wherein the compound distributes to the target tissue and contrast of the target tissue is thereby improved.

[0038] This disclosure also provides a method for imaging a target tissue in a subject by positron emission tomography (PET) comprising administering a sufficient amount of a metal radioisotope complex of the one of the compounds described herein to the subject and imaging the subject by PET, wherein the compound distributes to the target tissue and the target tissue is thereby imaged.

[0039] The invention provides novel compounds of Formula, intermediates for the synthesis of compounds of Formula I, as well as methods of preparing compounds of Formula I. The invention also provides compounds of Formula I that are useful as intermediates for the synthesis of other useful compounds. The invention provides for the use of compounds of Formula I for the manufacture of contrast agenrt useful for imaging a mammal, such as a human.

[0040] The invention provides for the use of the compounds described herein for medical diagnosis. The diagnosis can be useful for the early detection of cognitive impairment, for example, Alzheimer’s disease. The invention also provides for the use of a compound as described herein for the manufacture of a contrast agent to aid in early detection of a disease in a mammal, for example, dementia in a human. The contrast agent can include a pharmaceutically acceptable diluent, excipient, or carrier.

[0041] BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The following drawings form part of the specification and are included to further demonstrate certain embodiments or various aspects of the invention. In some instances, embodiments of the invention can be best understood by referring to the accompanying drawings in combination with the detailed description presented herein. The description and accompanying drawings may highlight a certain specific example, or a certain aspect of the invention. However, one skilled in the art will understand that portions of the example or aspect may be used in combination with other examples or aspects of the invention.

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[0044] UIUC2023-225-02(PCT) Figure 1. ORTEPs (50% ellipsoid probability) of X-ray crystal structures of (a) [Mn(TE- 1)]+, (b) [Mn(TE-6)]+, (c) [Mn(TE-3)]2+, and (d) [Mn(TE-2)]+The structure of [Mn(TE-2)] is polymeric as crystalized and was truncated for clarity. Hydrogens were omitted for clarity, and the anions and solvent molecules are shown only for key interactions.

[0045] Figure 2. Speciation plot of TE-1 obtained from UV-VIS Spectrophotometric titrations with 60 mM of TE-1 ligand in the presence of 0.1 M KC1.

[0046] Figure 3. Speciation plot of TE-4 obtained from UV-vis spectrophotometric titrations with 60 mM of TE-4 ligand in the presence of 0.1 M KC1.

[0047] Figure 4. Speciation plot of [Mn(TE-l)]+obtained from HySS and using values in Table 2 and Table 3, corresponding to protonation and metal complex formation.

[0048] Figure 5. Speciation plot of [Mn(TE-4)] obtained from HySS and using values in Table 2 and Table 3 corresponding to protonation and metal complex formation.

[0049] Figure 6A-D. (a) HPLC traces corresponding to ImM [Mn(TE-l)]+in the presence of 25 equiv of ZnCh(b) Fitted 1storder decay kinetic data of [Mn(TE-l)]+with squares representing different time points obtained in the Zn challenge and the red line representing the best-fit line, (c) HPLC traces corresponding to ImM [Mn(TE-4)] in the presence of 25 equiv of ZnCb. (d) Fitted 1storder decay kinetic data with squares representing different time points obtained in the Zn challenge and the red line representing the best fit line.

[0050] Figure 7A-D. (a) Variable scan rate cyclic voltammograms corresponding to redox couple of Mnn / Mnniof 1 mM [Mn(TE-l)]+and 0.1 M NaCICh in 1 : 1 MeOH: H2O. The arrow represents the direction of the scan, (b) Variable scan rate cyclic voltammograms corresponding to redox couple of Mnn / Mnniof 1 mM [Mn(TE-4)] and 0.1 M NaCIOi H2O. (c) Differential pulse voltammetry corresponding to 1 mM [Mn(TE-l)]+. The two oxidative events are attributed to different coordination modes, (d) Differential pulse voltammetry corresponding to 1 mM [Mn(TE-4)].

[0051] Figure 8. (a) Relaxivity values of metal complex in 50 mM HEPES Buffer with 0.15 M NaCl. Error bars represent measurements of different batches of metal complex (b) Relaxivities of Mn complexes in the presence of 0.67 mM BSA. Relaxivities were obtained from the slope of concentration (mM) vs relaxation on a 1.4 T at 33 °C. Relaxivities of DOTA, PCTA, and 1,4-DO2A were only reported at 0.47 T magnetic field.

[0052] Figure 9A-D. (a) Plot to determine the number of BSA molecules binding to [Mn(TE-l)]. Using 440 mM of BSA and varying the concentration of [Mn(TE-l)] revealed n= 2, 2 molecules of [Mn(TE-l)] binding per BSA molecule, (b) Plot to determine the Kavalue of 0.5 mM [Mn(TE-l)] towards BSA. Kawas calculated using previously determined equations, (c) Plot to determine the number of BSA molecules binding to [Mn(TE-4)]. There was no clear inflection point and binding sites were assumed to be n=2. (d) Plot to determine the Kavalue of 0.5 mM [Mn(TE-4)] towards BSA.

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[0054] UIUC2023-225-02(PCT) Figure 10. Fitted17O transverse relaxivity plots of (a) [Mn( TE-1)]+and (b) [Mn(TE-4)]. The curve in Figure 10 was fitted with the Swift-Connick equation to obtain some of the physical parameters such as the enthalpy, entropy of activation of water binding, and the hyperfine coupling of the water molecule to the Mn center. The exchange rate can be calculated using the Eyring equation and setting the temperature to 310K.

[0055] Figure 11A-C. (a) RadioHPLC and the cold trace of [52Mn][Mn(TE-l)]+; the black trace represents the cold metal complex, the red trace represents the labeled [52Mn][Mn(TE-l)]+, and the blue trace represents the control of [52Mn]; (b) RadioHPLC and the cold trace of [52Mn][Mn(TE-4)]; (c) Apparent molar activity of [52Mn][Mn(TE-l)]+at 37 °C using two conditions for radiolabeling.

[0056] Figure 12. MR images of the abdominal area of mice after IV injection of 80 mmol / Kg of [Mn(TE-4)], showing contrast enhancement in the kidney and bladder and suggesting renal clearance.

[0057] Figure 13A-C. (a) Normalized signal intensity of kidneys for [Mn(TE-4)] was obtained by drawing ROIs on the kidneys of n=3 mice subtracting the Signal Intensity of muscle and dividing by the signal intensity of air (SI Kidney- SI muscle / SI air). Error bars represent the standard deviation for n=3 mice, (b) Normalized signal intensity of the liver was obtained by drawing ROIs on the liver of n=3 mice subtracting the signal intensity of muscle and dividing by the signal intensity of air (SI Liver- SI muscle / SI air). Error bars represent the standard deviation for n=3 mice, (c) Normalized signal intensity of the stomach was obtained by drawing ROIs on the stomach of n=3 mice subtracting the signal intensity of muscle and dividing by the signal intensity of air (SI Stomach - SI muscle / SI air). Error bars represent the standard deviation for n=3 mice.

[0058] Figure 14A-B. (a) PET images 45 min post injection of [52Mn][Mn(TE-l)]+and [52Mn][Mn(TE-4)]; (b) Biodistribution data in %ID / g in different organs 1 h post injection for [52Mn][Mn(acetate)], [52Mn][Mn(TE-l)]+and [52Mn][Mn(TE-4)]).

[0059] Figure 15. MR images of the abdominal area of mice after IP injection of 80 mmol / Kg of [Mn(TE-l)]+, showing contrast enhancement in the liver and kidney and suggesting a mixed hepatobiliary and renal clearance.

[0060] Figure 16A-C. (a) Normalized signal intensity of kidneys for [Mn(TE-l)]+was obtained by drawing ROIs on the kidneys of n=3 mice subtracting the signal intensity of muscle and dividing by the signal intensity of air (SI Kidney- SI muscle / SI air). Error bars represent the standard deviation for n=3 mice, (b) Normalized signal intensity of the liver was obtained by drawing ROIs on the liver of n=3 mice subtracting the signal intensity of muscle and dividing by the signal intensity of air (SI Liver- SI muscle / SI air). Error bars represent the standard deviation for n=3 mice, (c) Normalized signal intensity of the stomach was obtained by drawing ROIs on the stomach of n=3 mice subtracting the signal intensity of muscle and dividing by the signal intensity of air (SI Stomach- SI muscle / SI air). Error bars represent the standard deviation for n=3 mice.

[0061] Figure 17. UV-VIS spectrum of the titration of TE-8 from pH 2.3 to 11.0 and corresponding speciation diagram.

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[0063] UIUC2023-225-02(PCT) Figure 18A-B. CVs of ImM of TE-2, TE-3, TE-5, and TE-6. CVs are done using Glassy carbon electrode and Ag / AgCl reference with 0.1M NaC104 as supporting electrolyte.

[0064] Figure 19A-C. CVs of ImM of TE-7, TE-8, TE-9, TE-10, and TE-11. CVs are done using Glassy carbon electrode and Ag / AgCl reference with 0.1M NaC104 as supporting electrolyte.

[0065] DETAILED DESCRIPTION

[0066] Magnetic Resonance Imaging (MRI) is a widely used diagnostic tool, yet it exhibits inherent low sensitivity that necessitates the use of contrast agents to improve the quality of MRI scans. Gadolinium-based contrast agents, and more recently manganese-based agents, have been used in pre-clinical and clinical applications due to their relaxation properties. The development of macrocyclic chelators for Mn2+has been limited due to low relaxivities or poor kinetic inertness of the corresponding Mn complexes, and thus the development of new chelators to support Mn contrast agents with high relaxivity and good kinetic inertness is consequential.

[0067] Herein, we report an attractive chelator platform based on the diazapyridinophane macrocycle. We have synthesized a library of chelators to study the effect of coordination number and choice of pendant arms on the efficiency of the corresponding Mn MRI agents, and we have identified two lead compounds, [Mn(TE-l)]+and [Mn(TE-4)], which have good relaxivity and kinetic inertness in vivo. Through spectrophotometric titrations and in vitro Zn challenge studies, [Mn(TE- 4)] showed an appreciable kinetic inertness with a rate of dissociation of 6.8 x 10'4s’1, while [Mn(TE- 1)]+has an even slower dissociation rate of 1.0 x 10’4s’1and an exceptional relaxivity of 3.6 mM s’1measured at 1.4 T magnetic field and 33 °C. These Mn complexes were further investigated by employing the52Mn radioisotope, a positron emission tomography (PET) radiotracer, which allowed us to probe their kinetic inertness and stability in vivo.

[0068] The Mn complexes of TE-1 and TE-4 exhibit drastically different behavior in in vivo MRI studies given their different lipophilicity: [Mn(TE-4)] shows significant contrast enhancement in the kidney and bladder, indicating primarily renal clearance, while the lipophilicity of [Mn(TE-l)]+promotes a mixed biliary-renal excretion. Overall, the performance of [Mn(TE-4)] both in vitro and in vivo lends promise to its potential development as an MRI contrast agent and also as a dual PET / MR imaging agent.

[0069] Additional information and data that can be used with aspects of the invention described herein is found in the following publication: El Sayed T, et al., Pyridinophane Ligands: An Attractive Chelator Platform for Mn-Based Imaging Agents. ChemRxiv. 2025; doi:10.26434 / chemrxiv-2025- snpm4, which is incorporated herein by reference.

[0070] Definitions.

[0071] The following definitions are included to provide a clear and consistent understanding of the specification and claims. As used herein, the recited terms have the following meanings. All other

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[0073] UIUC2023-225-02(PCT) terms and phrases used in this specification have their ordinary meanings as one of skill in the art would understand. Such ordinary meanings may be obtained by reference to technical dictionaries, such as Hawley ’s Condensed Chemical Dictionary 14thEdition, by R.J. Lewis, John Wiley & Sons, New York, N.Y., 2001.

[0074] References in the specification to "one embodiment", "an embodiment", etc., indicate that the embodiment described may include a particular aspect, feature, structure, moiety, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, moiety, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, moiety, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such aspect, feature, structure, moiety, or characteristic with other embodiments, whether or not explicitly described.

[0075] The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" includes a plurality of such compounds, so that a compound X includes a plurality of compounds X. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for the use of exclusive terminology, such as "solely," "only," and the like, in connection with any element described herein, and / or the recitation of claim elements or use of "negative" limitations.

[0076] The term "and / or" means any one of the items, any combination of the items, or all of the items with which this term is associated. The phrases "one or more" and "at least one" are readily understood by one of skill in the art, particularly when read in context of its usage. For example, the phrase can mean one, two, three, four, five, six, ten, 100, or any upper limit approximately 10, 100, or 1000 times higher than a recited lower limit. For example, one or more substituents on a phenyl ring refers to one to five, or one to four, for example if the phenyl ring is disubstituted.

[0077] As will be understood by the skilled artisan, all numbers, including those expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, are approximations and are understood as being optionally modified in all instances by the term "about." These values can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings of the descriptions herein. It is also understood that such values inherently contain variability resulting from the standard deviations found in their respective testing measurements. When values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value without the modifier "about" also forms a further aspect.

[0078] The terms "about" and "approximately" are used interchangeably. Both terms can refer to a variation of ± 5%, ± 10%, ± 20%, or ± 25% of the value specified. For example, "about 50" percent can in some embodiments carry a variation from 45 to 55 percent, or as otherwise defined by a particular claim. For integer ranges, the term "about" can include one or two integers greater than

[0079] 500.163W01 9

[0080] UIUC2023-225-02(PCT) and / or less than a recited integer at each end of the range. Unless indicated otherwise herein, the terms "about" and "approximately" are intended to include values, e.g., weight percentages, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, composition, or embodiment. The terms "about" and "approximately" can also modify the endpoints of a recited range as discussed above in this paragraph.

[0081] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges recited herein also encompass any and all possible subranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values. It is therefore understood that each unit between two particular units are also disclosed. For example, if 10 to 15 is disclosed, then 11, 12, 13, and 14 are also disclosed, individually, and as part of a range. A recited range (e.g., weight percentages or carbon groups) includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art, all language such as "up to", "at least", "greater than", "less than", "more than", "or more", and the like, include the number recited and such terms refer to ranges that can be subsequently broken down into sub-ranges as discussed above. In the same manner, all ratios recited herein also include all sub-ratios falling within the broader ratio. Accordingly, specific values recited for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for radicals and substituents. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0082] This disclosure provides ranges, limits, and deviations to variables such as volume, mass, percentages, ratios, etc. It is understood by an ordinary person skilled in the art that a range, such as “number 1” to “number ”, implies a continuous range of numbers that includes the whole numbers and fractional numbers. For example, 1 to 10 means 1, 2, 3, 4, 5, ... 9, 10. It also means 1.0, 1.1, 1.2. 1.3, ... , 9.8, 9.9, 10.0, and also means 1.01, 1.02, 1.03, and so on. If the variable disclosed is a number less than “number 10”, it implies a continuous range that includes whole numbers and fractional numbers less than number 10, as discussed above. Similarly, if the variable disclosed is a number greater than “number 10”, it implies a continuous range that includes whole numbers and fractional numbers greater than number 10. These ranges can be modified by the term “about”, whose meaning has been described above.

[0083] The recitation of a), b), c), ... or i), ii), iii), or the like in a list of components or steps do not confer any particular order unless explicitly stated.

[0084] One skilled in the art will also readily recognize that where members are grouped together in a common manner, such as in a Markush group, the invention encompasses not only the entire group

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[0086] UIUC2023-225-02(PCT) listed as a whole, but each member of the group individually and all possible subgroups of the main group. Additionally, for all purposes, the invention encompasses not only the main group, but also the main group absent one or more of the group members. The invention therefore envisages the explicit exclusion of any one or more of members of a recited group. Accordingly, provisos may apply to any of the disclosed categories or embodiments whereby any one or more of the recited elements, species, or embodiments, may be excluded from such categories or embodiments, for example, for use in an explicit negative limitation.

[0087] The term "contacting" refers to the act of touching, making contact, or of bringing to immediate or close proximity, including at the cellular or molecular level, for example, to bring about a physiological reaction, a chemical reaction, or a physical change, e.g., in a solution, in a reaction mixture, in vitro, or in vivo.

[0088] As used herein, "subject" or “patient” means an individual having symptoms of, or at risk for, a disease or other malignancy. A patient may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such a mouse model as described herein. Likewise, the patient may include either adults or juveniles (e.g., children). Moreover, patient may mean any living organism, preferably a mammal (e.g., human or non-human) that may benefit from the administration of compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish and the like. In one embodiment of the methods provided herein, the mammal is a human.

[0089] As used herein, the terms “providing”, “administering,” “introducing,” are used interchangeably herein and refer to the placement of a compound of the disclosure into a subject by a method or route that results in at least partial localization of the compound to a desired site. The compound can be administered by any appropriate route that results in delivery to a desired location in the subject.

[0090] The compound and compositions described herein may be administered with additional compositions to prolong stability and activity of the compositions, or in combination with other therapeutic drugs.

[0091] An "effective amount" refers to an amount effective to bring about a recited effect, such as an amount necessary to image tissues in an animal. Determination of an effective amount is typically within the capacity of persons skilled in the art, especially in light of the detailed disclosure provided herein. The term "effective amount" is intended to include an amount of a compound or reagent described herein, or an amount of a combination of compounds or reagents described herein, e.g., that

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[0093] UIUC2023-225-02(PCT) is effective to image tissues in an animal. Thus, an "effective amount" generally means an amount that provides the desired effect.

[0094] The term “substantially” as used herein, is a broad term and is used in its ordinary sense, including, without limitation, being largely but not necessarily wholly that which is specified. For example, the term could refer to a numerical value that may not be 100% the full numerical value. The full numerical value may be less by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, or about 20%.

[0095] Wherever the term “comprising” is used herein, options are contemplated wherein the terms “consisting of’ or “consisting essentially of’ are used instead. As used herein, “comprising” is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of' excludes any element, step, or ingredient not specified in the aspect element. As used herein, "consisting essentially of' does not exclude materials or steps that do not materially affect the basic and novel characteristics of the aspect. In each instance herein any of the terms "comprising", "consisting essentially of' and "consisting of' may be replaced with either of the other two terms. The disclosure illustratively described herein may be suitably practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.

[0096] This disclosure provides methods of making the compounds and compositions of the invention. The compounds and compositions can be prepared by any of the applicable techniques described herein, optionally in combination with standard techniques of organic synthesis. Many techniques such as etherification and esterification are well known in the art. However, many of these techniques are elaborated in Compendium of Organic Synthetic Methods (John Wiley & Sons, New York), Vol. 1, Ian T. Harrison and Shuyen Harrison, 1971; Vol. 2, Ian T. Harrison and Shuyen Harrison, 1974; Vol. 3, Louis S. Hegedus and Leroy Wade, 1977; Vol. 4, Leroy G. Wade, Jr., 1980; Vol. 5, Leroy G. Wade, Jr., 1984; and Vol. 6; as well as standard organic reference texts such as March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th Ed., by M. B. Smith and J. March (John Wiley & Sons, New York, 2001); Comprehensive Organic Synthesis. Selectivity, Strategy & Efficiency in Modem Organic Chemistry. In 9 Volumes, Barry M. Trost, Editor-in-Chief (Pergamon Press, New York, 1993 printing); Advanced Organic Chemistry, Part B: Reactions and Synthesis, Second Edition, Cary and Sundberg (1983); for heterocyclic synthesis see Hermanson, Greg T., Bioconjugate Techniques, Third Edition, Academic Press, 2013.

[0097] The formulas and compounds described herein can be modified using protecting groups. Suitable amino and carboxy protecting groups are known to those skilled in the art (see for example, Protecting Groups in Organic Synthesis, Second Edition, Greene, T. W., and Wuts, P. G. M., John Wiley & Sons, New York, and references cited therein; Philip J. Kocienski; Protecting Groups (Georg Thieme Verlag Stuttgart, New York, 1994), and references cited therein); and Comprehensive

[0098] 500.163W01 12

[0099] UIUC2023-225-02(PCT) Organic Transformations, Larock, R. C., Second Edition, John Wiley & Sons, New York (1999), and referenced cited therein.

[0100] The term "halo" or "halide" refers to fluoro, chloro, bromo, or iodo. Similarly, the term "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0101] The term "alkyl" refers to a branched or unbranched hydrocarbon having, for example, from 1-20 carbon atoms, and often 1-12, 1-10, 1-8, 1-6, or 1-4 carbon atoms; or for example, a range between 1-20 carbon atoms, such as 2-6, 3-6, 2-8, or 3-8 carbon atoms. As used herein, the term “alkyl” also encompasses a “cycloalkyl”, defined below. Examples include, but are not limited to, methyl, ethyl, 1 -propyl, 2-propyl (Ao-propyl), 1 -butyl, 2-methyl-l -propyl (isobutyl), 2-butyl (secbutyl), 2-methyl-2-propyl (Abutyl), 1 -pentyl, 2-pentyl, 3 -pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl,

[0102] 3 -methyl- 1 -butyl, 2-methyl-l -butyl, 1 -hexyl, 2-hexyl, 3 -hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl,

[0103] 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3 -pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, hexyl, octyl, decyl, dodecyl, and the like. The alkyl can be unsubstituted or substituted, for example, with a substituent described below or otherwise described herein. The alkyl can also be optionally partially or fully unsaturated. As such, the recitation of an alkyl group can include an alkenyl group or an alkynyl group. The alkyl can be a monovalent hydrocarbon radical, as described and exemplified above, or it can be a divalent hydrocarbon radical (i.e., an alkylene).

[0104] An alkylene is an alkyl group having two free valences at a carbon atom or two different carbon atoms of a carbon chain. Similarly, alkenylene and alkynylene are respectively an alkene and an alkyne having two free valences at two different carbon atoms, or an alkenylene can have the two free valences on the same carbon.

[0105] The term "cycloalkyl" refers to cyclic alkyl groups of, for example, from 3 to 10 carbon atoms having a single cyclic ring or multiple condensed rings. Cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantyl, and the like. The cycloalkyl can be unsubstituted or substituted. The cycloalkyl group can be monovalent or divalent and can be optionally substituted as described for alkyl groups. The cycloalkyl group can optionally include one or more cites of unsaturation, for example, the cycloalkyl group can include one or more carbon-carbon double bonds, such as, for example, 1 -cyclopent- 1-enyl, 1 -cyclopent-2-enyl, 1 -cyclopent-3 -enyl, cyclohexyl, 1- cyclohex-l-enyl, 1 -cyclohex-2-enyl, 1 -cyclohex-3 -enyl, and the like.

[0106] The term “heteroatom” refers to any atom in the periodic table that is not carbon or hydrogen. Typically, a heteroatom is O, S, N, P. The heteroatom may also be a halogen, metal or metalloid.

[0107] The term "heterocycloalkyl" or “heterocyclyl” refers to a saturated or partially saturated monocyclic, bicyclic, or polycyclic ring containing at least one heteroatom selected from nitrogen, sulfur, oxygen, preferably from 1 to 3 heteroatoms in at least one ring. Each ring is preferably from 3- to 10-membered, more preferably 4 to 7 membered. Examples of suitable heterocycloalkyl substituents include pyrrolidinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, piperidinyl, piperazinyl,

[0108] 500.163W01 13

[0109] UIUC2023-225-02(PCT) tetrahydropyranyl, morpholinyl, 1,3-diazapanyl, 1,4-diazapanyl, 1 ,4-oxazepanyl, and 1,4- oxathiapanyl. The group may be a terminal group or a bridging group.

[0110] The term "aryl" refers to an aromatic hydrocarbon group derived from the removal of at least one hydrogen atom from a single carbon atom of a parent aromatic ring system. The radical attachment site can be at a saturated or unsaturated carbon atom of the parent ring system. The aryl group can have from 6 to 30 carbon atoms, for example, about 6-10 carbon atoms. The aryl group can have a single ring (e.g., phenyl) or multiple condensed (fused) rings, wherein at least one ring is aromatic (e.g., naphthyl, dihydrophenanthrenyl, fluorenyl, or anthryl). Typical aryl groups include, but are not limited to, radicals derived from benzene, naphthalene, anthracene, biphenyl, and the like. The aryl can be unsubstituted or optionally substituted with a substituent described below. For example, a phenyl moiety or group may be substituted with one or more substituents Rxwhere Rxis at the ortho-, meta-, or / % / ra-position, and X is an integer variable of 1 to 5.

[0111] The term "heteroaryl" refers to a monocyclic, bicyclic, or tricyclic ring system containing one, two, or three aromatic rings and containing at least one nitrogen, oxygen, or sulfur atom in an aromatic ring. The heteroaryl can be unsubstituted or substituted, for example, with one or more, and in particular one to three, substituents, as described in the definition of "substituted". Typical heteroaryl groups contain 2-20 carbon atoms in the ring skeleton in addition to the one or more heteroatoms, wherein the ring skeleton comprises a 5-membered ring, a 6-membered ring, two 5- membered rings, two 6-membered rings, or a 5 -membered ring fused to a 6-membered ring. Examples of heteroaryl groups include, but are not limited to, 2H-pyrrolyl, 3H-indolyl, 4H- quinolizinyl, acridinyl, benzo[b]thienyl, benzothiazolyl, 0-carbolinyl, carbazolyl, chromenyl, cinnolinyl, dibenzo[b,d]furanyl, furazanyl, furyl, imidazolyl, imidizolyl, indazolyl, indolisinyl, indolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthyridinyl, oxazolyl, perimidinyl, phenanthridinyl, phenanthrolinyl, phenarsazinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, thiadiazolyl, thianthrenyl, thiazolyl, thienyl, triazolyl, tetrazolyl, and xanthenyl. In one embodiment the term "heteroaryl" denotes a monocyclic aromatic ring containing five or six ring atoms containing carbon and 1, 2, 3, or 4 heteroatoms independently selected from non-peroxide oxygen, sulfur, and N(Z) wherein Z is absent or is H, O, alkyl, aryl, or (Ci-C6)alkylaryl. In some embodiments, heteroaryl denotes an ortho-fused bicyclic heterocycle of about eight to ten ring atoms derived therefrom, particularly a benzo-derivative or one derived by fusing a propylene, trimethylene, or tetramethylene diradical thereto.

[0112] As used herein, the term "substituted" or “substituent” is intended to indicate that one or more (for example, in various embodiments, 1-10; in other embodiments, 1-6; in some embodiments 1, 2, 3, 4, or 5; in certain embodiments, 1, 2, or 3; and in other embodiments, 1 or 2) hydrogens on the group indicated in the expression using “substituted” (or “substituent”) is replaced with a selection

[0113] 500.163W01 14

[0114] UIUC2023-225-02(PCT) from the indicated group(s), or with a suitable group known to those of skill in the art, provided that the indicated atom’s normal valency is not exceeded, and that the substitution results in a stable compound. Suitable indicated groups include, e.g., alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, hydroxyalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, alkylamino, dialkylamino, carboxyalkyl, alkylthio, alkylsulfinyl, and alkylsulfonyl. Substituents of the indicated groups can be those recited in a specific list of substituents described herein, or as one of skill in the art would recognize, can be one or more substituents selected from alkyl, alkenyl, alkynyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, alkylamino, dialkylamino, trifluoromethylthio, difluoromethyl, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl, and cyano. Suitable substituents of indicated groups can be bonded to a substituted carbon atom include F, Cl, Br, I, OR', 0C(0)N(R')2, CN, CF3, OCF3, R', O, S, C(O), S(O), methylenedioxy, ethylenedioxy, N(R')2, SR', SOR', SO2R', SO2N(R')2, SO3R', C(O)R', C(O)C(O)R', C(O)CH2C(O)R', C(S)R', C(O)OR', OC(O)R', C(0)N(R')2, 0C(0)N(R')2, C(S)N(R')2, (CH2)O-2NHC(0)R', N(R')N(R')C(O)R', N(R')N(R')C(O)OR', N(R')N(R')C0N(R')2, N(R')SO2R', N(R')SO2N(R')2, N(R')C(O)OR', N(R')C(O)R', N(R')C(S)R', N(R')C(0)N(R')2, N(R')C(S)N(R')2, N(COR')COR', N(OR')R', C(=NH)N(R')2, C(O)N(OR')R', or C(=NOR')R' wherein R’ can be hydrogen or a carbon-based moiety (e.g., (Ci-Ce)alkyl), and wherein the carbon-based moiety can itself be further substituted. When a substituent is monovalent, such as, for example, F or Cl, it is bonded to the atom it is substituting by a single bond. When a substituent is divalent, such as O, it is bonded to the atom it is substituting by a double bond; for example, a carbon atom substituted with O forms a carbonyl group, C=O.

[0115] Embodiments of the Technology.

[0116] 1. A compound of Formula I: salt and / or a hydrate thereof; wherein,

[0117] — represents one or more coordinate covalent bonds when NF is present in a metal complex of Formula I; or

[0118] — is absent when Mzis not present;

[0119] Mzis a metal or metal radioisotope, wherein z represents the oxidation state of the metal or metal radioisotope; or

[0120] 500.163W01 15

[0121] UIUC2023-225-02(PCT) Mzis absent;

[0122] G1is picolinyl, pyridinyl, carboxyl, or benzothiazolyl hydroxyphenyl;

[0123] G2is picolinyl, pyridinyl, carboxyl, or H; wherein picolinyl, pyridinyl, carboxyl, or benzothiazolyl hydroxyphenyl are substituted with R3; and

[0124] R1, R2, and R3are each independently H, halo, -(Ci-C6)alkyl, -O(Ci-C6)alkyl, or -N(R4)2 wherein each R4is independently H or -(Ci-Ce)alkyl; or the compound 2-(3,7-diaza-l,5(2,6)-dipyridinacyclooctaphane-3-ylmethyl)-4- (benzo[c / / thiazol-2-yl)-6-methoxyphenol or a metal complex thereof comprising Mz, a salt, and / or a hydrate thereof.

[0125] 2. The compound of embodiment 1, wherein G1is carboxyl.

[0126] 3. The compound of embodiment 1, wherein G1is 2-picolynyl.

[0127] 4. The compound of any one of embodiments 1-3, wherein G2is carboxyl or picolinyl.

[0128] 5. The compound of any one of embodiments 1-3, wherein G2is benzothiazolyl hydroxyphenyl.

[0129] 6. The compound of embodiment 5, wherein R3is -O(Ci-C6)alkyl.

[0130] 7. The compound of any one of embodiments 1-3, wherein G2is:

[0131] 8. The compound of any one of embodiments 1-7, wherein R1and R2are H.

[0132] 9. The compound of any one of embodiments 1-8, wherein Mzis a transition metal or lanthanide.

[0133] 10. The compound of any one of embodiments 1-8, wherein Mzis manganese.

[0134] 11. The compound of any one of embodiments 1-10, wherein the oxidation state (z) of the metal is +2 or +3.

[0135] 12. The compound of any one of embodiments 1-8, wherein Mzis Mn2+.

[0136] 13. The compound of any one of embodiments 1-8, wherein Mzis absent.

[0137] 14. The compound of embodiment 1, wherein the compound is: or a radioisotope, a salt and / or a hydrate thereof.

[0138] 500.163W01 16

[0139] UIUC2023-225-02(PCT) 15. The compound of embodiment 1, wherein the compound is: or a metal complex comprising Mz, a salt, and / or a hydrate thereof.

[0140] 16. The compound of embodiment 1, wherein the compound is: or a metal complex comprising Mz, a salt, and / or a hydrate thereof. In some embodiments, the compound is a complex of Mn, wherein the compound is one of TE-2, TE-3, and TE-5 to TE-11:

[0141] 500.163W01 17

[0142] UIUC2023-225-02(PCT)

[0143] 17. The compound of any one of embodiments 1-16, wherein the compound is a hydrated compound comprising between 1 and 2 water molecules.

[0144] 18. A method for improving contrast of a target tissue in a subject imaged by magnetic resonance imaging (MRI) comprising administering a sufficient amount of a metal complex of the compound of any one of embodiments 1-17 to the subject and imaging the subject by MRI, wherein the compound distributes to the target tissue and contrast of the target tissue is thereby improved.

[0145] 19. A method for imaging a target tissue in a subject by positron emission tomography (PET) comprising administering a sufficient amount of a metal radioisotope complex of the compound of any one of embodiments 1-17 to the subject and imaging the subject by PET, wherein the compound distributes to the target tissue and the target tissue is thereby imaged.

[0146] 500.163W01

[0147] UIUC2023-225-02(PCT) 20. The method of embodiment 18 or 19, wherein the target tissue is kidney, liver, stomach, brain, a cancer or a combination thereof.

[0148] 21. A method for diagnosing Alzheimer’s disease in a subject comprising: a) administering a sufficient amount of a metal complex of the compound of any one of embodiments 1-17 to the subject; b) imaging the subject by magnetic resonance imaging (MRI); and c) detecting the presence or absence of Amyloid- / >eta plaques in the subject’s brain, wherein the compound binds to Amyloid- / >eta plaques if present; wherein a presence or absence of Alzheimer’s disease is thereby diagnosed.

[0149] Results and Discussion.

[0150] Design and synthesis of macrocyclic chelators. The pyridinophane ligand framework was selected since the rigidity of the ligand scaffold could enhance the relaxivity of the Mn complexes. The rigidity of the chelator favorably enhancing the relaxivity has been shown by Toth et al. when utilizing the bispidine ligand family, which seems to have similar rigidity and proton sponge behavior as the pyridinophane chelator family. Garai et al. recently reported [Mn (BPPA)] as a good chelator for52Mn / 55Mn. While the Gd complexes of TE-1 and TE-4 have been reported by Iglesias et al. and Sherry et al., respectively, they have never been employed in Mn complexes. In addition, our group has recently reported TE-3 as a dual Cu(II) / Cu(I) chelator. We wanted to repurpose these chelators for Mn as well as synthesize new chelators to study how the structure of these chelators affects their function as MRI contrast agents. Our approach was to vary the donor arms on the amine N- substituents to incorporate carboxylate, pyridyl, and picolinate arms that have been shown to enhance thermodynamic stability, kinetic inertness, and relaxivity. Varying the coordination number could indicate whether an optimal geometry would be a good promise between all these competing properties required for the design of good MRI contrast agents.

[0151] The synthesis of the symmetric ligands starts with the Richman- Atkins cyclization of precursors SI and S2 to affordTsN4 (S3, Scheme 1). This is subsequently deprotected with sulfuric acid to affordHN4, which is further reacted with various alkyl bromide substrates in an SN2 reaction. Hydrolysis of ester intermediates affords TE-1 and TE-4 in good yields. For TE-3, the synthesis utilizesHN4 which is later reacted with picoline bromide to afford TE-3 in good yields.

[0152] Scheme 1. Synthetic scheme of symmetric chelators.

[0153] 500.163W01 19

[0154] UIUC2023-225-02(PCT)

[0155] For the synthesis of unsymmetric ligands, the selective protection of one of the secondary amines was employed with 0.5 equiv of BOC2O to afford the desired monoprotected product, along with unreacted starting material. The monoprotected product was subjected to an SN2 reaction to yield S6 in decent yield (Scheme 2). The N-Boc group is hydrolyzed with 4 M HC1 in dioxane to afford S7 in good yields; S7 is a common intermediate for making all three novel unsymmetric ligands TE-2, TE-5, and TE-6. TE-2 was synthesized from S7 using the Eschweiler-Clarke synthesis in 72% yield. In addition, S7 was reacted with picoline bromide followed by the deprotection of the picolinate ester to afford TE-6 in 22% yield over two steps. Alternatively, S7 was reacted with tertbutyl bromoacetate to afford S8 which is then hydrolyzed to afford TE-5 in decent yield. Having a family of pyridinophane ligands with different chelating arms and denticities could indicate factors that are important to consider when designing chelators for Mn.

[0156] 500.163W01 20

[0157] UIUC2023-225-02(PCT) Scheme 2. Synthetic scheme of unsymmetric chelators.

[0158] S8 TE-6

[0159] Metal complex synthesis and characterization. The metal complexes were prepared anaerobically by mixing the corresponding ligand with Mn(C104)2 in the presence of a base. The metal complexes were isolated as a powder and vapor diffusion recrystallizations were set up. Gratifyingly, we were able to crystalize some of the complexes and evaluate their coordination geometry in the solid state (Figure 1). Crystals of [Mn(TE-l)]C104 were grown by vapor diffusion of Et2O into a mixture of MeOH and H2O. Surprisingly, TE-1 wraps around Mn and forms a distorted eight-coordinate square antiprismatic complex, with the tertiary amines having the longest bond lengths of 2.493 and 2.478 A (Table 1); one of the picolinate arms of TE-1 is protonated, resulting in the carbonyl group binding to the metal center, while the other picolinate arm is binding through the anionic oxygen.

[0160] 500.163W01 21

[0161] UIUC2023-225-02(PCT) Table 1. Key Mn-L bond lengths (in A) for the Mn complexes.

[0162] For TE-6, the corresponding Mn metal complex adopts a distorted seven-coordinate pentagonal bipyramidal geometry, with the Mn-amine bond lengths being elongated (Table 1). For [Mn(TE-2)]+, the structure was obtained from acetone diffusion in water to reveal a polymeric structure, with one of the oxygen atoms of the picolinate arm from another molecule binding to the Mn center, making it a seven-coordinate metal center. For both [Mn(TE-3)]2+and [Mn(TE-2)]+, even though there are only 6 donor atoms from the multidentate ligand, a seventh coordination site is occupied by a bridging ligand or a perchlorate anion. The high coordination environment adopted by the Mn complexes highlights the coordination preferences for Mn2+having a large ionic radius compared to other transition metals. Despite the crystal structures not showing water directly coordinated to the Mn center, in some structures water is hydrogen bonding in the secondary sphere with the picolinate arm or the perchlorate anion. It is important to note that the structures of these metal complexes in solution will differ significantly from the solid-state structure, due to the dynamic flexibility of the ligand framework. For all the metal complexes, the oxidation state of the Mn center is confirmed to be 2+ based on the number of counteranions and the protonation state of the ligand.

[0163] Thermodynamic stability & kinetic inertness of the Mn complexes. Because the metal-ligand reaction involves the competition of the protons for the basic donor atoms in the ligand and the metal ion, quantifying complex formation equilibria requires prior knowledge of ligand protonation constants. In aqueous solutions, UV-vis pH spectrophotometric titrations in the presence of 0.1 M KC1 were used to determine the protonation constants of the ligands. Pyridinophane ligands act as good proton sponges, making it difficult for them to be fully deprotonated except at basic pH. At physiological pH, the pyridinophane ligands exist as protonated species with the protons being on the basic amines / pyri dines of the macrocycle (Figure 2, Figure 3). The most basic sites were tentatively assigned to the tertiary nitrogens and pyridines. The symmetric ligands showed a variety of deprotonation events, with the picolinic acid moiety of TE-1 having pKa’s of 4.44 and 3.08, corresponding to the pyridine and the carboxylic acid moieties, respectively (Table 2). These values align with other reported ligands bearing those arms. These pKa’s are slightly different from the reported values by Iglesias et al. (Inorg. Chem. 2012, 51 (20), 10893) due to performing UV-vis

[0164] 500.163W01 22

[0165] UIUC2023-225-02(PCT) titrations rather than potentiometric titrations which typically utilize higher concentrations of ligand and metal complexes. It is challenging to assign the basic p a s, as sometimes pyridine groups in pyridinophane ligands tend to have higher p a s compared to tertiary amines. The most basic proton is thought to be enclosed in the macrocyclic ligand cavity, thus the proton sponge nature of these ligands. For TE-3, the pyridyl arm has a p.& of 3.16, similar to a cyclam ligand bearing a pyridyl arm. The pyridyl arm can be deprotonated at a wide range of p a s ranging from 2.45 to 3.91, depending on the pyridine substitution.

[0166] Table 2. Protonation constants of synthesized chelators TE-1, TE-4, TE-3, TE-2, TE-6, TE-5, and comparison to other Mn chelators. Titrations were performed from pH 2-11, in the presence of 0.1 M KC1. avalues outside the titration range.

[0167] For the unsymmetric ligands, there was a deprotonation event with a p a that is distinctly more basic highlighting the fact that pyridinophane ligands are proton sponges that are difficult to fully deprotonate. Comparing the deprotonation of BPPA with TE-2, which differ in their chemical structure by a methyl group on the amine group, TE-2 should be more difficult to deprotonate due to the more electron-rich nature of the ligand. This is highlighted in that the proton enclosed in the macrocyclic cavity of the ligand is deprotonated at logKuL12.66 for TE-2, while the highest pKa of BPPA is 8.65 (Table 2). Simple modifications of the chelating arms on the tertiary amines significantly affect the overall chelator's basicity. TE-5 has a simple replacement of a carboxylate group compared to a picolinate group in TE-1. The carboxylate group is likely hydrogen bonding with the picolinate group in solution, which would raise the overall basicity of the ligand. This is evident by the higher deprotonation constants for TE-5 compared to TE-1. For TE-6, replacing the carboxylate arm with a pyridyl arm lowers the basicity of the ligand in comparison to TE-5 due to the 500.163W01 23 UIUC2023-225-02(PCT) reduced hydrogen bonding capability of this chelator. Finally, the dianionic ligands TE-4 and TE-5 have similar basicity to 3,9-PC2A.

[0168] After obtaining the protonation constants, the metal complexes were prepared in situ in the presence of 0.1 M KC1, and the stability constants were determined. TE-4 had the highest logKxini. of 18.74 among this series of ligands, which was expected based on literature precedents showing that carboxylate arms endow the highest thermodynamic stability for Mn2+(Table 3). Moreover, the pMn value of 8.04 for TE-4 is comparable to that for PyC3A, a ligand being evaluated in clinical trials as a Mn MRI contrast agent. TE-2 had a dramatically lower logKxini. compared to BPPA, which differs only by a methyl group. This was not unexpected based on the higher basicity of TE-2 which is expected to form protonated metal complexes at physiological pH. Moreover, the steric hindrance introduced by the addition of the methyl group may result in less stable metal complexes. It is expected that picolinate arms endow high thermodynamic stability, but this was not observed in this ligand series. The stability constant logKxini. values are usually not good indicators for thermodynamic stability due to the different ligand denticities and basicities. The pM value established by Raymond et al. gives a more accurate trend for the metal scavenging ability for a particular chelator. The pM values were calculated with conditions proposed by Toth et al. (Inorg. Chem. 2010, 49 (7), 3224), to allow for direct comparison with chelators in the literature. The thermodynamic stability of the symmetric ligands was higher than that of the unsymmetric ligands. In general, the thermodynamic stability of the complexes was lower than PCTA and DOTA. We did not anticipate this based on Green’s report that pMn for pyridinophane ligands is generally higher than amine-based macrocyclic ligands such as DOTA or PCTA. A potential explanation for the lower- than-expected stability constants is the presence of the protonated M(LH) species at physiological pH (Figure 4, Figure 5). Overall, the Mn2+complexes of this ligand series exhibit significant thermodynamic stabilities to be further evaluated as potential Mn MRI contrast agents.

[0169] Table 3. Stability constants and pMn values for ligands TE-1, TE-2, TE-4, TE-3, TE-6 and TE-5 and related reported ligands with Mn2+.

[0170] 500.163W01 24

[0171] UIUC2023-225-02(PCT) apMn calculated as -log[Mn]free where [Mn2+] = 105M, [L] = 105M.

[0172] The kinetic inertness of MRI contrast agents is of utmost importance since there are many endogenous metal ions in vivo. Transmetallation experiments employing Zn2+are typically used to assess the kinetic inertness of Mn2+complexes. Usually, these measurements are done using T2 relaxometry, since free Mn2+should have higher relaxation than chelated Mn. For convenience, we opted to use high performance liquid chromatography (HPLC) to study the rate of transmetallation of our complexes with Zn2+. We synthesized both Mn and Zn complexes independently to obtain their retention times (Figure 6). We also decided to premix both Mn and Zn complexes to ensure that we could separate them. The standard conditions of challenging the Mn complex with 25 equiv of Zn2+were employed. Based on the rates of dissociation, the seven-coordinate complexes of this series were quite labile; hence, they are likely not suitable for use as MRI contrast agents.

[0173] The Mn complexes of TE-1 and TE-2 were the most kinetically inert among the series (Table 4). The kinetic inertness could be attributed to the picolinic acid moiety. For [Mn(TE-l)]+, the metal exchange rate is slower, likely due to the geometric reorganization from 8 to 6 coordinates upon Zn coordination. The rate of transmetallation of TE-2 with ZnCh is surprisingly slower than the newly developed [Mn(BPPA)]. The higher kinetic inertness of [Mn(TE-2)] compared to [Mn(BPPA)] could be attributed to the steric bulk of the methyl group slowing down the exchange of Zn2+with the Mn complex. For [Mn(TE-l)]+, the rate of dissociation corresponding to l.OxlO'4s'1is significantly slower than 3,9-PC2A and PyC3a. For [Mn(TE-4)], the dissociation rate is almost identical to PyC3A, which is currently in clinical trials.

[0174] Table 4. The rate of transmetallation of the complexes reported in this series. aValue for Kinetic inertness of TE-5 was obtained from challenge of 1 mM [Mn(TE-5)] with 1 mM of ZnCl2.

[0175] When comparing [Mn(TE-6)]+and [Mn(TE-l)]+, a simple replacement of a picolinic acid arm with a pyridyl arm accelerates the metal exchange, highlighting the importance of donor arm selection. Notably, the chelating arm may endow thermodynamic stability but may be detrimental to the kinetic inertness of the metal complex. This is evident for [Mn(TE-3)]2+, which has a decent thermodynamic stability but is kinetically labile, highlighting that pyridyl groups may not be suitable for Mn chelation. Based on the rates of dissociation of [Mn(TE-l)]+and [Mn(TE-4)], we anticipated that both complexes should be stable in vivo. It is worth noting that the conditions employed herein

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[0177] UIUC2023-225-02(PCT) for kinetic inertness are harsher than in vivo conditions, as the concentration of Zn2+in blood is expected to be around 10 mM, yet such harsh kinetic inertness conditions provide a good benchmark, similar to previous reports.

[0178] Electrochemical properties ofMn complexes. As Mn can take multiple oxidation states, it is important to probe the oxidation potentials since Mn3+is usually a less potent MRI agent. Therefore, we wanted to investigate the oxidation potentials of these Mn complexes to see whether Mn3+was accessible. Cyclic voltammetry (CV) measurements vs. Ag / AgCl as the reference electrode were employed to evaluate the redox potentials and E1 / 2 values of the metal complexes, while differential pulse voltammetry (DPV) was used for cases where the redox events were irreversible. For the Mn complexes evaluated in this study, the oxidation potentials were assigned to the oxidation of Mn2+to Mn3+based on the similarity of the oxidation potentials reported for other ligand systems (Table 5).

[0179] Table 5. Mnn / Mnniredox potentials obtained from differential pulse voltammetry (DPV).

[0180] The irreversible oxidation event for the [Mn(TE-l)]+complex is likely related to the eightcoordinate geometry of the Mn center, which requires an important reorganization of the square antiprismatic geometry to the octahedral coordination preferred by Mn3+(Figure 7). In addition, the presence of two oxidation potentials, as observed in DPV, is indicative of two conformations that exist in solution (Figure 7). One of these conformations was tentatively assigned to an aqua complex with one of the picolinate arms not binding to the Mn center. The conformational flexibility of pyridinophane ligands giving rise to distinct oxidative events has been previously reported by our group (Inorg. Chem. 2014, 53 (24), 13112). For [Mn(TE-4)], a quasi-reversible oxidation event is observed, which is in line with an octahedral geometry that does not undergo extensive structural reorganization upon oxidation to Mn3+.

[0181] The pyridinophane-based Mn complexes explored here are likely resistant to oxidation in biological media which is attributed to higher oxidation potentials when compared to the reduction of O2 to H2O (Table 5). The high oxidation potentials of [Mn(TE-3)]2+and [Mn(HTE-2)]2+could be explained based on the titration data, as the predominant species in solution is the dicationic metal complex of TE-3 and the protonated metal complex of TE-2. The 2+ charge explains the higher oxidation potential in comparison to the other Mn complexes: when comparing the oxidation potentials to other Mn complexes such as NOTA or 1,4- DO2A, the pyridinophane ligands possess higher oxidation potentials due to the lower basicity of the pyridines compared to tertiary amines as less electron-rich metal complexes are less prone to oxidation. However, when comparing our

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[0183] UIUC2023-225-02(PCT) systems to the 3,6-PC2A ligand containing a pyridyl group, the oxidation potential for [Mn(TE-4)] is similar to that of the Mn complex of 3,6-PC2A (Table 5).

[0184] Relaxivity properties ofMn complexes. The relaxivities of the Mn complexes were measured in 50 mM of N-(2 -hydroxyethyl) piperazine-JV-ethanesulfonic acid (HEPES) buffer in the presence of 0.15 M NaCl for ionic strength which are common conditions. Excitingly, the measured relaxivities for most of the Mn complexes investigated herein surpass the relaxivity of [Mn(PyC3a)]. For example, [Mn(TE-l)]+has a relaxivity of 3.6 mM'1s'1measured at 1.4 T magnetic field at 33 °C, comparable to the reported record relaxivity of 3.17 mM'1s'1for the Mn complex of a bispidine ligand. Moreover, the relaxivities of the Mn2+complexes of pyridinophane based ligands are higher than those of the Mn complexes of common macrocyclic chelators such as PCTA, DOTA, and 1,4- DO2A (Figure 8). Since these common macrocyclic ligands have only been measured at 0.47 T magnetic field, it is hard to make definitive comparisons. However, relaxivity values are expected to decrease with increasing field strength. Based on the obtained relaxivities, we hypothesize that the rigidity of the pyridinophane ligands makes them an exceptional chelating platform for Mn2+.

[0185] When the complexes are incubated with 0.67 mM of BSA, there is a noticeable relaxivity enhancement for the metal complexes suggesting the metal complexes have some affinity to BSA (Figure 8b). To better understand this behavior, we have measured the affinity of these Mn complexes towards BSA using relaxometry. The experiment involves titrating different concentrations of metal complexes while keeping the concentration of BSA constant at 440 mM. The inflection point in Figure 9 shows the number of metal complexes bound to BSA when the binding pockets of the protein are saturated with the metal complexes. When the binding pockets are saturated, relaxivity contributions from the unbound metal complexes are observed, which reduces the apparent relaxivity. To obtain the binding affinity constants (Ka), a concentration of 0.5 mM of each metal complex was used, while the concentration of BSA was varied. The Kavalues were obtained by fitting the data to reported equations to yield 7.43 x 103M'1for [Mn(TE-l)]+and 7.22 x 103M'1for [Mn(TE-4)] (Table 6). For [Mn(TE-3)]2+, the Kavalue of 2.02 x 104M'1is the highest among this series.

[0186] These affinity constants indicate strong interactions between the Mn complexes and BSA. By comparison, the previously reported Complex 1 supported by a chelator bearing the picolinic acid pendant arm has been shown to bind albumin with an affinity constant of 1.6 x 103M'1. The non- covalent and hydrogen bonding interactions between the metal complexes and BSA enhance the relaxivity of the metal complexes by reducing the tumbling rate in solution. Interestingly, the symmetric Mn complexes [Mn(TE-l)+, [Mn(TE-4)], and [Mn(TE-3)]2+, along with pseudo-symmetric complex [Mn(TE-6)]+, exhibit higher affinity towards BSA when compared to the unsymmetric complexes [Mn(TE-2)]+and [Mn(TE-5)]+. This trend could be due to the specificity of the BSA binding pocket towards symmetric complexes. Importantly, the affinities of the Mn complexes described herein are similar to MnLMe, a Mn blood pool MRI contrast agent.

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[0188] UIUC2023-225-02(PCT) Table 6. Determined BSA affinities for the Mn complexes.

[0189] To further understand the underlying physical properties such as hydration number (c / ) and water exchange rate (Kex) that distinguishes the pyridinophane ligand platform in terms of relaxivity from many macrocyclic ligands, we have employed17O transverse relaxivity measurements. The maximum17O l / Ta value allows for an estimation of the hydration number. The metal complex concentrations employed were 4-8 mM, which is the concentration typically used in literature. Both [Mn(TE-l)]+and [Mn(TE-4)] had a q value of 1.4, while the other Mn complexes exhibit lower q values.17O transverse relaxation measurements also provide several additional kinetic parameters, such as the water exchange rate, which correlates with the contrast enhancement of Mn complexes. The curve shape indicates whether the water exchange is slow, fast, or intermediate; based on the shapes of the17O graphs, [Mn(TE-l)]+is in the faster exchange regime when compared to [Mn(TE- 4)] (Figure 10). The additional kinetic parameters determined suggest that [Mn(TE-4)] and [Mn(TE- 1)]+undergo different mechanisms of water binding (Table 7). For [Mn(TE-4)], the water binding follows an associative mechanism, as suggested by the negative AS1value.

[0190] Table 7. Extracted parameters obtained from17O transverse relaxivity measurements.

[0191] By comparison, for [Mn(TE-l)]+the water molecule likely binds to the metal center and displaces the picolinate arm in an interchange mechanism, since the AS1value is close to 0. The interchange mechanism for the water coordination is also supported by the CV measurements showing two different conformations in solution (Figure 7). In the literature, eight-coordinate Mn complexes were presumed to be inadequate for MRI applications as they are coordinatively saturated and cannot accommodate water binding. However, based on the17O transverse relaxivity results, [Mn(TE-l)]+has a q value of 1.4, suggesting that eight-coordinate complexes could still allow for water exchange by a dissociative or interchange mechanism; a q value of 1.4 has been also observed for other macrocyclic ligands containing picolinate arms. Except for the Mn2+complexes of TE-2 and TE-3 having the slowest exchange rates for this series, the other Mn complexes have comparable exchange rates (Table 7).

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[0193] UIUC2023-225-02(PCT) The results obtained from17O transverse relaxivity measurements are in line with the observed speciation for these Mn complexes. For example, at physiological pH complex [Mn(TE- 3)]2+exists as two species in solution, corresponding to MnL and MnLOH. The presence of the latter hydroxo species in solution could explain the lower exchange rate compared to the other six- coordinate complexes. Hydroxide ions will slow the water exchange rate, as supported by the lower water exchange rate measured for [Mn(TE-3)]2+compared to other metal complexes. Generally, relaxivity should increase as the molecular weight of the metal complex increases, resulting in a slower tumbling rate that enhances relaxivity. Indeed, [Mn(TE-l)] has the highest molecular weight, which could explain the high relaxivity measured for this complex. Moreover, two picolinate arms enhance the water exchange rate as the carbonyl groups are likely hydrogen bonding with water, increasing the mean residence time of water molecules interacting with the Mn center.

[0194] Despite its lower molecular weight, the relaxivity of [Mn(TE-4)] is comparable to other Mn complexes in this series. The higher-than-expected relaxivity of this complex is possibly governed by the hydration number q of 1.4, as well as the appreciable water exchange rate of 3.5 x 107s'1that is likely enhances by the presence of the acetate arms. Overall, while the Mn complexes developed herein seem to have a slightly lower thermodynamic stability compared to other reported Mn complexes, the relaxivities of these Mn complexes surpass many reported Mn complexes.

[0195] 52Mn Radiolabeling Studies. We then employed the52Mn radioisotope to label the optimal chelators developed herein and used the radiolabeled complexes for in vivo biodistribution studies. The radiolabeling conditions using HEPES buffer at pH 7 were selected based on literature precedents showing that these were the optimal conditions for complexation. The radiolabelling was performed at 37 °C and 80 °C for 30 min and 1 h using 625 nmol / mCi specific activity of52Mn, and 0.1 M Na citrate was used as the mobile phase to obtain radiochemical yields. Aliquots of the radiochemical reactions were analyzed using radio-HPLC, which aligned well with the isolated cold complexes (Figure 11).

[0196] TE-1 and TE-5 achieved quantitative radiolabeling in 60 min at 37 °C, while TE-3 and TE-4 required heating to 80 °C to achieve near quantitative radiolabeling (Table 8). Because bifunctional chelators containing antibodies and small molecules may be sensitive to high temperatures, the mild radiolabeling conditions of TE-1 are advantageous. Because [52Mn][Mn(TE-l)] showed mild radiolabeling conditions, we performed an apparent molar activity (AMA) study where we varied the concentration of the ligand. The apparent molar activity was found to be 4259 mCi / pmol, which is higher than the apparent molar activity for [52Mn][Mn(DOTA)]. Based on these findings, we suggest that pyridinophane ligands with picolinic acid arms exhibit favorable chelation properties for generating radiolabeled Mn complexes.

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[0198] UIUC2023-225-02(PCT) Table 8. Radiochemical yields of52Mn complexes, as determined by RadioTLC (ND: not determined).

[0199] We also performed logD measurements using octanol / PBS to evaluate the lipophilicity of the52Mn complexes reported herein. Based on our findings, [52Mn][Mn(TE-l)] was the most lipophilic compound with a logD value of 0.04 ± 0.06, followed by [52Mn][Mn(TE-5)] with a value of -1.39 ± 0.09, and then [52Mn][Mn(TE-3)] with a value of -2.09 ± 0.05 (Table 8). The lipophilicity of [Mn(TE-l)]+could be attributed to having a mono-cationic complex with two picolinic acid arms. The replacement of one picolinic acid arm with an acetate makes the corresponding metal complex more hydrophilic. For TE-3, the ligand is neutral and the corresponding Mn2+complex is dicationic, which could explain the hydrophilic nature of this metal complex.

[0200] In vivo MR Imaging Studies. With a scarcity of Mn complexes tested in vivo, SNQ SNQ Q encouraged to test our lead compounds to evaluate their performance. Prior to in vivo experiments, a cell toxicity assay was performed with lead compounds. Based on thermodynamic stability, kinetic inertness, and relaxivity measurements, we chose the [Mn(TE-l)]+and [Mn(TE-4)] complexes to evaluate in vivo. Despite the good kinetic inertness of good relaxivity of [Mn(TE-2)], its lower thermodynamic stability prevented us from testing this compound in vivo.

[0201] The instability of seven-coordinate complexes towards transmetallation with ZnCh makes them ineffective MRI contrast agents. Given that the concentration of Zn in the blood is roughly 10 mM, the Zn challenge with 25 mM of ZnCb is performed under harsh conditions. For [Mn(TE-l)]+, the compound was formulated in HEPES buffer with 8% MeOH due to solubility issues, while for [Mn(TE-4)] the compound was formulated with Chelex-treated Millipore water. Three mice were administered 80 mmol / kg [Mn(TE-4)] intravascularly. For [Mn(TE-l)]+, we performed the injections intraperitoneally due to the solubility constraints of the compound. For [Mn(TE-4)], a more hydrophilic compound, contrast enhancement was observed in the kidneys and bladder, and the intensity of the signal in the organs gradually increased for 35 minutes (Figure 12).

[0202] The contrast enhancement persists for a longer period compared to other recently developed Mn MRI contrast agents, which could be attributed to the affinity of the pyridinophane ligands towards BSA. For example, MS325, a Gd agent known to bind plasma proteins, has a slow renal excretion due to high protein binding. Therefore, we speculate that the interaction of these pyridinophane ligands with albumin protein in the blood could lead to a longer circulation time of the MRI contrast agent in the body. Based on the normalized signal intensities of the organs, [Mn(TE-4)] is excreted primarily through the kidneys, along with a smaller uptake of the compound and excretion

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[0204] UIUC2023-225-02(PCT) through the liver. The signal intensity curves in different organs resemble the behavior of MnLMe, a recently reported Mn-based blood pool agent (Figure 13). Dramatic enhancement in T1 -weighted images in comparison to many reported Mn MRI contrast agents is attributed to the binding of [Mn(TE-4)] to murine serum albumin in vivo. The prolonged circulation time of the MRI contrast agent could be beneficial for a longer acquisition time, circumventing the need for continuous infusion of the agents. There was no significant uptake of [Mn(TE-4)] in the stomach or liver, which indicates that the compound is primarily excreted renally and similar to other small contrast agents. Overall, taking the in vitro data into consideration along with the in vivo data makes this macrocyclic Mn complex attractive for further pre-clinical development.

[0205] 52Mn Biodistribution Studies. [52Mn] [Mn(TE-4)] was then used to study the biodistribution of the Mn complex in vivo and evaluate the integrity of the Mn complex upon excretion. After the radiolabeling of the compound, a Sep-Pak column was used to purify the compound before injection in mice. The compound showed good stability in the formulation. The PET imaging experiment and the biodistribution data corroborated well with the MRI data, showing mainly renal excretion of the compound (Figure 14a). Additionally, the biodistribution data of [52Mn] [Mn(TE-4)] collected 1 h post-injection shows that the compound was mostly eliminated from the body, given the low ID / g observed in the different organs (Figure 14b).

[0206] The excretion of the radioactive [52Mn] [Mn(TE-4)] is faster than the excretion of the MRI formulation, likely due to the differences in concentrations of compound used in PET and MR imaging, respectively. The biodistribution of free Mn2+that we obtained resembles the reported ones in the literature, (PLoS One 2017, 12 (3)) as free Mn2+mostly accumulates in the heart, pancreas, spleen, kidney, and liver; since free Mn2+mimics Ca2+, it is usually taken up by voltage-dependent channels in cardiomyocytes and neurons. Slow elimination of radioactivity from organs is indicative of unstable radiocomplexes, whereas stable radiocomplexes are often eliminated rapidly through the hepatobiliary or digestive systems. The biodistribution of free52Mn that is distinct from [52Mn][Mn(TE-4)], along with the rapid clearance of the52Mn complex from organs indicate that the complex is stable in vivo. Moreover, the urine metabolite analysis showed that the compound was >93% intact, suggesting that the stability of [52Mn] [Mn(TE-4)] could be attributed to the rigidity of the pyridinophane ligand. Recently, Iglesias et al. reported Mn-CHXPYAN as a promising52Mn PET tracer showing good stability in vivo, which was also attributed to the increased rigidity of the complex. Their findings corroborate well with our results, and suggests that the rigidity of the chelator could be crucial when designing new Mn chelators for MR / PET imaging applications.

[0207] For [Mn(TE-l)]+, a more lipophilic compound, contrast enhancement was observed in the kidney, liver, and stomach (Figure 16). Similarly to TE-4, the normalized signal intensity of the liver and kidney increases over time, but some clearance is observed throughout imaging. Given the solubility limitations of the compound, an intraperitoneal injection was used which could explain the clearance of the compound. Since this type of injection bypasses the circulatory system, there will be

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[0209] UIUC2023-225-02(PCT) minimal albumin binding, hence the compound is expected to be cleared faster. Contrast enhancement in the liver and the kidney suggests a mixed hepatobiliary and renal excretion of the compound. The uptake in the liver could be due to the lipophilic nature of the Mn complex.

[0210] [52Mn][Mn(TE-l)]+was then used to study its biodistribution in vivo. The PET images obtained at 45 min post-injection show a significant uptake of [52Mn][Mn(TE-l)]+in the gallbladder and small intestines (Figure 14a). The biodistribution data confirms that the lipophilic nature of [52Mn][Mn(TE-l)]+is influencing the elimination pathway of the compound. It is difficult to compare the MRI and PET data since the mice injections were different. At the lower concentrations used in PET, we did not observe any solubility problems of the Mn complex; hence, an intravascular injection was used for PET imaging and [52Mn][Mn(TE-l)] biodistribution. Even though the injection route of the MRI and PET formulations of [Mn(TE-l)] was different, both experiments corroborate the lipophilic nature of the complex, given the fate of the metal complex in vivo. It is worth noting that liver uptake could be attributed to the lipophilicity of the compound or the release of free Mn2+that is rapidly uptaken by hepatocytes. Given the urine metabolite stability showing over 99% stability of52Mn [Mn(TE-l)]+, we are inclined to believe that hepatic uptake is due to the compound's lipophilicity. For biodistribution studies, [52Mn] [Mn(acetate)] was used as a comparison to both [52Mn][Mn(TE-4)] and [52Mn][Mn(TE-l)]+, showing the organ uptake of the synthesized complexes is different from free52Mn2+. Overall, for55 / 52Mn[Mn(TE-l)]+to be clinically useful, more studies focused on studying the pharmacokinetics of elimination are needed to show whether the compound is excreted within 24 h.

[0211] Conclusion. We have synthesized several new chelators based on the pyridinophane macrocycle and containing various chelating arms, and then studied their Mn chelating properties in detail. The Mn complexes of TE-1 and TE-4 were found to have optimal in vitro properties such as good relaxivity, decent thermodynamic stability, and good kinetic inertness. The extensive coordination study revealed that seven-coordinate complexes [Mn(TE-5)]+and [Mn(TE-6)]+were kinetically labile and were not suitable for further development. The relaxivity of [Mn(TE-l)]+surpasses [Mn(PyC3a)] and is comparable to [Mn(Bispidine)]. Despite TE-1 and TE-4 having lower thermodynamic stability than other chelators, the compromise between their kinetic inertness and relaxation properties is promising for in vivo studies. Given the good relaxation properties and kinetic inertness, we decided to test these compounds in murine models using a 9.4 T MRI scanner. The contrast enhancement observed for [Mn(TE-4)] primarily in the kidney and bladder indicates renal excretion, which is typical for small molecules. For [Mn(TE-l)]+, the contrast enhancement was observed in the kidney, liver, and stomach, consistent with the lipophilicity of this compound, as measured using52Mn radiolabeling.

[0212] The biodistribution and PET imaging studies corroborated well with the MRI experiments. The slow excretion of the MRI agents was attributed to albumin affinity, which prolongs the blood circulation time of the MRI contrast agents. Albumin affinity is typically advantageous for cancer

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[0214] UIUC2023-225-02(PCT) imaging since compounds having albumin affinity are uptaken by cancer cells and are not cleared rapidly from the body. For [Mn(TE-l)]+, PET imaging studies showed significant uptake in the gallbladder and small intestines. Overall, [Mn(TE-4)] shows promise in both in vitro and in vivo experiments, encouraging us to pursue its potential development as an MRI contrast agent and also as a dual PET / MR imaging agent.

[0215] II. Pyridinophane contrast agents for Alzheimer’s disease diagnosis by magnetic resonance imaging.

[0216] The invention includes a series of novel compounds based on pyridinophane ligands and demonstrates their potential as manganese-based Magnetic Resonance Imaging (MRI) agents. Traditionally, most contrast agents that are approved by the FDA for human use are gadolinium- based agents. Despite the strong paramagnetic properties of Gd, several reports of Nephrogenic Systemic Fibrosis (NSF) in patients with compromised renal function have raised concerns. Gd agents present huge challenges for coordination chemists since chelator design plays a crucial role in avoiding the release of free Gd ions in vivo. To address the toxicity issues, essential metal ions such as Fe(III) and Mn(II) have been explored. Telescan which was the only Mn-approved MRI agent has been discontinued due to the release of Mn(II) ions from the chelator. The free Mn is taken up by hepatocytes, providing contrast between healthy and abnormal liver cells. Even though Mn is biogenic, developing chelators with high thermodynamic stability and kinetic inertness is necessary.

[0217] MnPyC3a is the only general Mn contrast agent currently in clinical trials. Its higher thermodynamic stability, kinetic inertness, and comparable contrast ability to commercially used Gd agents make it a good alternative. However, developing liver-specific contrast agents is of great interest since they could help in diagnosing hepatic tumors. Moreover, liver-specific agents are beneficial for patients with renal compromise since the contrast agents are partially eliminated through the liver. Developing more specific Mn MRI agents such as liver-specific is limited especially at higher field scanners (9.4T). The higher field scanners provide higher-resolution images and shorter acquisition times. Developing contrast agents for higher field scanners is challenging due to the strict influence of higher magnetic fields on molecular parameters used to optimize newly developed agents. Due to the difficulty of developing MRI agents at higher fields, there have been very limited examples of using 9.4 T for in vivo imaging in mice specifically utilizing Gd probes. To date, there have not been any Mn MRI agents tested in vivo at these higher field scanners.

[0218] On the other hand, to the best of our knowledge, no Mn MRI agents for diagnosing Alzheimer’s disease (AD) have been developed to date. AD is a common neurodegenerative disease characterized by cognitive decline. The etiology of the disease is not well understood. However, there are several biomarkers observed throughout the progression of the disease such as the aggregation of AP into soluble oligomers, plaques, and neurofibrillary tangles (NFTs). These biomarkers can serve as handles for diagnosing the disease early providing better life quality for patients.

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[0220] UIUC2023-225-02(PCT) The compounds explored in this series are classified into different categories. The first set of compounds is classified as extracellular or liver-specific MRI agents. The Mirica group has reported the pyridinophane framework (Chart 2a) as a strong metal chelator for 1strow transition metals such as Cu. Moreover, the Toth group has shown that pyridinophane derived ligands can also stabilize Mn and have favorable properties such as high thermodynamic stability, kinetic inertness and good relaxivity. Our series varies the donor groups (Chart 2a) on the tertiary amines to explore the effect of coordination number, charge, and lipophilicity on the contrast ability of the Mn complexes. In general, the symmetric ligands had a higher thermodynamic stability and relaxivity compared to the asymmetric ones. The relaxivity was evaluated on a 1.4 T tabletop NMR in PBS and incubated with 0.67mM Bovine Serum Albumin (BSA). Several compounds showed enhanced relaxivity in the presence of BSA indicating their high affinity to one of the most abundant proteins in the blood pool. The number of waters coordinated to the metal center dictates the efficiency of MRI agents. This property has been evaluated using17O transverse relaxivity. The lead compounds were evaluated on a 9.4 T MRI scanner in vitro. Preliminary mice studies showed good contrast enhancement in the kidneys and liver using a dose of 80 pmol / Kg which is lower than the commercial dose used for Gd agents.

[0221] Chart 2. Structure of synthesized chelators. b. Synthesized chelators as Alzheimer’s disease MRI agents.

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[0223] UIUC2023-225-02(PCT)

[0224] The second set of compounds is designed for diagnosing Alzheimer’s disease. MRI agents specifically for Alzheimer’s disease are scarce. There have been very few examples of Gd-based agents reported in the literature. There are stringent requirements for MRI diagnostic agents for Alzheimer’s disease which include good water solubility, lipophilicity, and good relaxivity. Combining all these properties is difficult, causing no advancements of any candidates to clinical trials. The Mirica group has shown that phenyl benzothiazole (BTZ) (Chart 2b) attached to the pyridinophane framework has a high affinity to A0 plaques. We sought this framework has various advantages. First, the second tertiary amine could be functionalized with various chelating arms that would enhance the thermodynamic stability of the metal complexes. Second, the BTZ groups would act as a strong chelating group and A0 binding. Upon binding to A0, there should be an enhancement in relaxivity due to the slow tumbling rate of the metal complex in solution.

[0225] Even though there has been a lot of research to replace Gd agents with biogenic metals, there has not been any liver-specific Mn agents approved for human use. Moreover, there has not been any Mn agent that could be used at higher scanner fields. The 9.4 T MRI scanners could provide higher resolution images and could reduce the acquisition time of scans. The lead compounds have shown promise in mice at 9.4 T and could be developed further in clinical trials. The compounds tested herein showed some contrast in the liver indicating partial clearance of the contrast agent through the liver. These compounds developed are attractive since they show some partial liver clearance despite lacking traditional liver targeting groups that require lengthy synthetic steps. The lead compounds in this series shown in Chart 2a are accessible synthetically without the need for lengthy synthetic steps. Moreover, these compounds could be radiolabeled with52Mn along with55Mn and be used as a dual PET -MR imaging agent. Dual imaging modality can be powerful in diagnosing diseases as PET is a very sensitive imaging technique while MRI provides higher tissue resolution images.

[0226] The second suite of compounds developed as Mn MRI agents for Alzheimer’s disease would be one of the first Mn MRI agents suitable for Alzheimer’s disease. There are very limited examples of Gd-based agents that have been reported in the literature. Most of these compounds had poor water solubility or poor blood-brain barrier (BBB) permeability. Wong et.al reported a dual Gd MRI-NIR agent that has shown promise in vivo in terms of BBB permeability and relaxivity. However, there have been no reports of Mn agents designed for Alzheimer’s disease. Developing Mn agents for Alzheimer’s disease could help improve patients’ lives through early diagnosis of the disease. Currently, clinical diagnostic tests that could be used to detect the onset of the disease are lacking.

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[0228] UIUC2023-225-02(PCT) Early diagnosis and detection of this disease could minimize cognitive damage and delay the progression of the disease. We therefore developed Mn MRI agents that have good relaxivity and good affinity to A[3 fibrils. A|3 oligomers and plaques are considered one of the earliest biomarkers throughout the progression of the disease. MRI is an indispensable technique that could monitor the disease progression and evaluate the responsiveness of patients to potential drugs. Data for compounds shown in Chart 2 are shown in Figure 17-19 and Table 9-11 below.

[0229] Table 9. Formation constants of Mn metal complexes for Alzheimer’s disease and their thermodynamic stability expressed as pM value.

[0230] Table 10. Relaxivities of compounds containing A[3 binding moiety with 0.67mM BSA.

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[0232] UIUC2023-225-02(PCT) Table 11. Extracted parameters from 170 studies of all Mn complexes.

[0233] Pharmaceutical Formulations.

[0234] The compounds described herein can be used to prepare therapeutic pharmaceutical compositions, for example, by combining the compounds with a pharmaceutically acceptable diluent, excipient, or carrier. The compounds may be added to a carrier in the form of a salt or solvate. In cases where compounds are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compounds as salts may be appropriate. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids that form a physiologically acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate, a-ketoglutarate, and -glycerophosphate. Suitable inorganic salts may also be formed, including hydrochloride, halide, sulfate, nitrate, bicarbonate, and carbonate salts.

[0235] Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid to provide a physiologically acceptable ionic compound. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example, calcium) salts of carboxylic acids can also be prepared by analogous methods.

[0236] The compounds of the formulas described herein can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient, in a variety of forms. The forms can be specifically adapted to a chosen route of administration, e.g., oral or parenteral administration, by intravenous, intramuscular, topical or subcutaneous routes.

[0237] The compounds described herein may be systemically administered in combination with a pharmaceutically acceptable vehicle, such as an inert diluent or an assimilable edible carrier. For oral administration, compounds can be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into the food of a patient's diet. Compounds may also be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations

[0238] 500.163W01 37

[0239] UIUC2023-225-02(PCT) typically contain at least 0.1% of active compound. The percentage of the compositions and preparations can vary and may conveniently be from about 0.5% to about 60%, about 1% to about 25%, or about 2% to about 10%, of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions can be such that an effective dosage level can be obtained.

[0240] The tablets, troches, pills, capsules, and the like may also contain one or more of the following: binders such as gum tragacanth, acacia, com starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as com starch, potato starch, alginic acid and the like; and a lubricant such as magnesium stearate. A sweetening agent such as sucrose, fructose, lactose or aspartame; or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring, may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propyl parabens as preservatives, a dye and flavoring such as cherry or orange flavor. Any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and devices.

[0241] The active compound may be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can be prepared in glycerol, liquid polyethylene glycols, triacetin, or mixtures thereof, or in a pharmaceutically acceptable oil. Under ordinary conditions of storage and use, preparations may contain a preservative to prevent the growth of microorganisms.

[0242] Pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions, dispersions, or sterile powders comprising the active ingredient adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. The ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions, or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and / or antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example,

[0243] 500.163W01 38

[0244] UIUC2023-225-02(PCT) sugars, buffers, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by agents delaying absorption, for example, aluminum monostearate and / or gelatin.

[0245] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, optionally followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation can include vacuum drying and freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the solution.

[0246] For topical administration, compounds may be applied in pure form, e.g., when they are liquids. However, it will generally be desirable to administer the active agent to the skin as a composition or formulation, for example, in combination with a dermatologically acceptable carrier, which may be a solid, a liquid, a gel, or the like.

[0247] Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina, and the like. Useful liquid carriers include water, dimethyl sulfoxide (DMSO), alcohols, glycols, or water-alcohol / glycol blends, in which a compound can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using a pump-type or aerosol sprayer.

[0248] Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses, or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

[0249] Examples of dermatological compositions for delivering active agents to the skin are known to the art; for example, see U.S. Patent Nos. 4,992,478 (Geria), 4,820,508 (Wortzman), 4,608,392 (Jacquet et al.), and 4,559,157 (Smith et al.). Such dermatological compositions can be used in combinations with the compounds described herein where an ingredient of such compositions can optionally be replaced by a compound described herein, or a compound described herein can be added to the composition.

[0250] Useful dosages of the compounds described herein can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Patent No. 4,938,949 (Borch et al.). The amount of a compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular compound or salt selected but also with the route of administration, the nature of the condition being treated, and the age and condition of the patient, and will be ultimately at the discretion of an attendant physician or clinician.

[0251] In general, however, a suitable dose will be in the range of from about 0.5 to about 100 mg / kg, e.g., from about 10 to about 75 mg / kg of body weight per day, such as 3 to about 50 mg per

[0252] 500.163W01 39

[0253] UIUC2023-225-02(PCT) kilogram body weight of the recipient per day, preferably in the range of 6 to 90 mg / kg / day, most preferably in the range of 15 to 60 mg / kg / day.

[0254] The compound is conveniently formulated in unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of active ingredient per unit dosage form. In one embodiment, the invention provides a composition comprising a compound of the invention formulated in such a unit dosage form.

[0255] The compound can be conveniently administered in a unit dosage form, for example, containing 5 to 1000 mg / m2, conveniently 10 to 750 mg / m2, most conveniently, 50 to 500 mg / m2of active ingredient per unit dosage form. The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations.

[0256] The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.

[0257] The following Examples are intended to illustrate the above invention and should not be construed as to narrow its scope. One skilled in the art will readily recognize that the Examples suggest many other ways in which the invention could be practiced. It should be understood that numerous variations and modifications may be made while remaining within the scope of the invention.

[0258] EXAMPLES

[0259] Example 1. Synthetic methods.

[0260] General Procedures: All reagents used as purchased from commercial sources unless stated otherwise. Pyridine ditosyl (SI) and sodium amide salt (S2) were synthesized from previously reported procedures. TE-4 final ligand and precursor were synthesized from H2N4 from previously reported procedures. S12 and TE-8 were synthesized using previously published procedures. All solutions were prepared using Millipore metal-free water. UV-visible spectra were recorded on a Varian Cary 50 Bio spectrophotometer and reported as / .max, nm (s, M-Icm' '). Fluorescence data was recorded on a SpectraMax M2e plate reader (Molecular Devices).JH (300 MHz) NMR and13C (126 MHz) NMR were recorded on a VARIAN VXR 500 with UNITY INOVA Console spectrometer. Chemical shifts were reported in parts per million referenced to residual solvent peaks. ESLMS experiments were performed on a Waters Q-TOF Ultima ESI mass spectrometer with electron spray ionization source by the Mass Spectrometry Lab at UIUC.

[0261] 500.163W01 40

[0262] UIUC2023-225-02(PCT)

[0263] To a three-neck flask, S2 (18.071g, 0.0202 mol, 2 equiv) is added and dissolved in 500 mL

[0264] MeCN.S2 is heated to 70 °C prior to the addition of SI. SI is weighed and sonicated in 500mL of

[0265] MeCN then added dropwise using an addition funnel over 8 hours. After the addition is complete, the reaction is stirred under refluxing conditions for 48 h. After 48 h, the MeCN evaporates, yielding a white / beige solid. The solid is redissolved in 300 ml MeOH and stirred for 1 hour then vacuum filtered yielding a mixture of S3 and undesired trimer. A crude NMR is obtained to determine the dimer / trimer ratio (usually 7:1). The TsN4 dimer / trimer mixture is dissolved in 300ml of MeOH and depending on the dimer / trimer ratio (12M HCL is added to selectively protonate the dimer). The dimer / trimer mix is stirred overnight and the following day, the mixture is vacuum filtered. The solid is TsN4 trimer and the solution is TsN4 dimer. The methanol is evaporated yielding an off- white solid (5.74 g of dimer obtained, 47% yield). ' H NMR (500 MHz, CDCL) 8 7.97 (d, J = 7.7 Hz, 2H), 7.74 (d, J= 7.9 Hz, 4H), 7.49 (d, J= 7.6 Hz, 4H), 7.39 (d, J= 8.0 Hz, 4H), 5.06 (s, 8H), 2.44 (s, 6H). HRMS-ESI (m / z): [M +H]+ calcd for C28H29N4O4S2, 549.1550; found 549.1650.

[0266] TSN4-HC1 (6.33g, 10.92 mol) was dissolved in 64 mL 90% sulfuric acid. This mixture was stirred and refluxed at 110 °C for 3 hours. After cooling, the solution was diluted with 700 mL of water. In an ice bath the solution was treated with sodium hydroxide to make the pH around 14. The aqueous layer was separated over two batches and the resulting solution was extracted with DCM (3 X 350 mL). The combined organic layers were dried over anhydrous K2CO3, and filtered. The filtrate was concentrated to dryness to give a white solid, HN4 (2.43g, 93% yield).1H NMR (500 MHz, CDCh) 8 7.11 (t, J = 7.5 Hz, 2H), 6.53 (d, J = 7.5 Hz, 4H), 3.99 (s, 8H).

[0267] S13

[0268] 500.163W01 41

[0269] UIUC2023-225-02(PCT) NaBH4 (0.983 g, 26.0 mmol) was added slowly to a stirred solution of dimethyl pyridine-2,6- dicarboxylate (5.04 g, 26.0 mmol) in a 3:7 mixture of DCM / MeOH (175 mL) at 0 °C, over 30 min. The solution was allowed to warm to RT and stirred for a further 3 h. The reaction mixture was quenched by the addition of aqueous saturated NH4CI solution (50 mL), the methanol was removed under reduced pressure, and the remaining residue was extracted with DCM (3x 75 mL). The organic phases combined were dried over Na2SO4 and evaporated under reduced pressure to yield the crude product as a colorless oil. The crude product was purified by silica gel chromatography (Combiflash automated purification system; A: DCM, B: MeOH; 0%B to 15% B) to yield the product as a white crystalline solid (1.55g, 37%). Alternatively, the product can be recrystallized from a mixture of 1:1 DCM: Hexanes. 'H NMR (500 MHz, CDC13) 8 8.02 (d, J = 7.7 Hz, 1H), 7.84 (td, J = 7.7, 1.1 Hz, 1H), 7.54 (d, J= 7.8 Hz, 1H), 4.86 (s, 2H), 3.99 (d, J= 1.2 Hz, 3H), 3.50 (d, J= 15.0 Hz, 1H).

[0270] Phosphorus tribromide (1.2 mL, 11.12 mmol, 1.2 equiv.) was added slowly using a syringe to a stirred solution of methyl 6-(hydroxymethyl)picolinate (1.55 g, 9.27 mmol) in MeCN (25 mL) at 0 °C. The reaction mixture was stirred for 2.5 h at RT, and the resulting yellow solution quenched by addition of saturated NaHCCh solution (50 mL). The product was extracted with DCM (3 x 50mL), the combined organic phase dried over Na2SO4, and the solvent removed under reduced pressure to yield the product as a white crystalline solid (1.72 g, 79%). 'H NMR (499 MHz, CDCh) 8 8.06 (dt, J = 7.8, 1.1 Hz, 1H), 7.87 (td, J= 7.7, 1.1 Hz, 1H), 7.69 (dt, J= 7.8, 1.1 Hz, 1H), 4.64 (d, J= l.l Hz,

[0271] 2H), 4.01 (d, J = 1.1 Hz, 3H).

[0272] To a round bottom flask, S4 (0.133g, 0.55 mmol) was added along with picolinate bromide (0.254g, 1.11 mmol, 2equiv.) and K2CO3 (0.306g, 2.22 mmol, 4equiv.). The reaction was stirred in dry MeCN under reflux for 48 h. The potassium carbonate was fdtered off and the MeCN was evaporated yielding a fluffy white compound. The crude product was purified by flash column chromatography on silica gel (A: DCM and B: MeOH 0% B to 10% B with 1% tri ethyl amine). The compound is obtained as a white solid (0.222 g, 70% yield). 'H NMR (500 MHz, CDCh) 8 8.08 -

[0273] 500.163W01 42

[0274] UIUC2023-225-02(PCT) 7.93 (m, 4H), 7.71 (dd, J = 6.1, 2.7 Hz, 2H), 7.15 (t, J = 7.6 Hz, 2H), 6.63 (d, J = 7.6 Hz, 4H), 4.55 (s, 4H), 4.17 (d, J = 14.0 Hz, 4H), 3.85 (s, 6H), 3.74 (d, J = 14.1 Hz, 4H).

[0275] To a round bottom flask, S9 (0.185g,0.343mmol) was added followed by the addition of 6M

[0276] HC1. The reaction was refluxed for 48 h then taken off the heat. The reaction mixture was fdtered hot, and the filtrate was evaporated yielding a sticky white solid. The solid was redissolved 2x5mL of DI water and evaporated yielding an off-white solid (0.187g, 80% yield).1H NMR (500 MHz, D2O) 8 8.40 (t, J= 7.9 Hz, 2H), 8.07 (d, J= 7.9 Hz, 2H), 7.90 (d, J= 7.9 Hz, 2H), 7.59 (t, J= 7.8 Hz, 2H),

[0277] 7.00 (d, J = 7.8 Hz, 4H), 4.37 - 4.08 (m, 8H).

[0278] S4 S5

[0279] To a round bottom flask, S4 (0.750g, 3.12 mmol) was added and dissolved in dichloromethane. Triethylamine was added to the solution using a syringe (0.948g, 1.31ml, 9.36 mmol, 3equiv.). The solution was stirred at 0°C using an ice bath for 15 min before adding BOC2O. A solution of BOC2O is prepared by dissolving BOC2O (0.340g, 1.502 mmol, 0.5 equiv.) in 15ml of di chloromethane and added dropwise over 30 minutes. The reaction is stirred for 4 hours then the reaction is washed with Sat’d K2CO3 and the aqueous layer is extracted 3x with di chloromethane. The organic layer is then dried over anhydrous K2CO3 and evaporated. The residue was purified by flash column chromatography on silica gel (A: DCM and B: MeOH 0% B to 10% B with 1% tri ethyl amine). The compound is obtained as a white solid (0.467 g, 44% yield). 'H NMR (500 MHz, CDCI3) 8 7.28 (dt, J = 11.4, 7.6 Hz, 2H), 7.05 (d, J = 7.8 Hz, 1H), 6.94 (d, J = 7.6 Hz, 1H), 6.84 - 6.74 (m, 2H), 4.78 (s, 2H), 4.73 (s, 2H), 4.22 (d, J = 3.2 Hz, 4H), 1.55 (s, 10H).13C NMR (126 MHz, CDCI3) 8 158.05, 157.36, 157.14, 155.08, 154.17, 153.81, 137.24, 137.09, 122.44, 121.62, 120.92, 120.89, 80.70, 55.42, 54.92, 54.56, 53.45, 28.58.

[0280] 500.163W01 43

[0281] UIUC2023-225-02(PCT)

[0282] To a round bottom flask was added S5 (0.261 g, 0.765mmol) along with Na2CO3(0.244g, 2.30mmol, 3equiv.) and MeCN (20ml). The reaction mixture is stirred for 10 minutes at room temperature prior to the addition of bromopicolinate (0.175g, 0.765 mmol) as a solid. The reaction was stirred at a refluxing temperature overnight. The following day, Na2COs is filtered off and the MeCN is evaporated. The residue is redissolved in DCM and washed with sat. K2CO3. The aqueous layer is extracted 3x with DCM. The combined organic layer is dried over anhydrous K2CO3. The drying agent is filtered off and the organic layer is concentrated down yielding an off-white powder (0.210g, 56% yield).1H NMR (500 MHz, CDCI3) 8 8.10 (dd, J= 13.0, 7.6 Hz, 2H), 8.03 - 7.93 (m, 1H), 7.25 - 7.17 (m, 2H), 6.96 - 6.86 (m, 4H), 4.82 (s, 2H), 4.78 (s, 4H), 4.33 (s, 2H), 4.05 (d, J = 4.5 Hz, 4H), 4.01 (s, 3H), 1.61 (s, 9H).

[0283] To a round bottom flask was added S5 (0.135 g, 0.765mmol) along with 4M HCL in dioxane (3.0ml). The reaction was stirred at room temperature for 3h. As the reaction progressed, the product began precipitating out. The excess dioxane was evaporated and the solid was basified using saturated K2CO3. The basic layer was extracted 3x15mL of DCM. The organic layer was dried with anhydrous K2CO3. The drying agent is filtered off and the DCM evaporated yielding an off-white powder (0.0679 g, % yield). 'H NMR (500 MHz, CDC13) 8 8.12 (d, J = 7.8 Hz, 1H), 8.09 (dd, J = 7.7, 1.1 Hz, 1H), 7.95 (t, J = 7.7 Hz, 1H), 7.11 (t, J = 7.6 Hz, 2H), 6.73 (dd, J = 7.7, 0.9 Hz, 2H), 6.52 (dd, J = 7.6, 1.0 Hz, 2H), 4.33 (s, 2H), 4.03 (s, 3H), 4.00 (s, 4H), 3.94 (s, 4H).13C NMR (126 MHz, CDCI3) 8 165.91, 160.73, 159.47, 158.75, 157.91, 147.54, 137.58, 135.58, 126.56, 123.95, 121.96, 120.32, 119.77, 65.06, 64.06, 55.89, 53.44, 53.02.

[0284] 500.163W01

[0285] UIUC2023-225-02(PCT)

[0286] To a round bottom flask, S7 (0.144g, 0.369 mmol) was added followed by the addition of ImL of formaldehyde and 10 ml of formic acid. The reaction mixture was refluxed for 15h. The following day, the reaction mixture was cooled down and about 5mL of concentrated HC1 were added to the reaction vessel. The solvent was then evaporated yielding an oil. About 15mL of NaOH were used to basify the product which was then extracted with 3x20mL of DCM. The organic layer with anhydrous K2CO3. The drying agent is filtered off and the DCM evaporated yielding an off- white powder (0.106g, 74% yield). 'HNMR (500 MHz, CDC13) 8 8.17 (d, J = 7.7 Hz, 1H), 7.67 (t, J = 7.6 Hz, 1H), 7.13 (d, J = 7.5 Hz, 1H), 7.05 (t, J = 7.6 Hz, 2H), 6.46 (dd, J = 7.7, 3.1 Hz, 4H), 4.17 (d, J = 14.2 Hz, 2H), 4.06 (d, J = 14.0 Hz, 2H), 4.03 (s, 2H), 3.46 (d, J = 14.0 Hz, 2H), 3.25 (d, J = 14.2 Hz, 2H), 2.91 (s, 3H).13C NMR (126 MHz, CDCk) 8 169.00, 156.82, 156.50, 156.09, 155.68, 154.57, 136.65, 135.59, 122.53, 121.90, 120.26, 120.12, 76.22, 64.73, 63.42, 62.59, 55.46, 47.85, 28.69.

[0287] To a round bottom flask, S7 (0.120g, 0.308mmol) was added along with Na2C03(0.098g, 0.925mmol, 3equiv.) and MeCN (20ml). The reaction mixture is stirred for 10 minutes at room temperature prior to the addition of bromopicoline (0.078g, 0.308 mmol) as a solid. The reaction initially turns pink then slowly starts turning orange. The reaction was stirred under refluxing conditions for 24 hours. The following day, the reaction was dark red. The Na2COs was filtered off and the MeCN was evaporated yielding a red oil that was basified with saturated K2CO3. The basic layer was extracted 3x25mL of DCM. The organic layer was dried with anhydrous K2CO3. The drying agent is filtered off and the DCM evaporated yielding brown oil. This residue was subjected to the following reaction without any further purification. The residue was dissolved in lOmL of 6M HC1 and the reaction was stirred under refluxing conditions for 24h. The solvent was then evaporated, and the mixture was purified on a reverse phase column (5 % A to 100% A A: H2O with 0 .1%TFA

[0288] 500.163W01 45

[0289] UIUC2023-225-02(PCT) and B: MeCN with 0.1% TFA). The collected fractions were evaporated, and the obtained oil was washed 3x5mL of IM HC1 to exchange TFA with HC1. The product was obtained as a yellow powder (0.022g, 15% yield over two steps).JH NMR (500 MHz, D2O) 8 8.37 (d, J= 5.8 Hz, 1H), 8.33 (td, J = 7.9, 1.5 Hz, 1H), 8.28 (t, J= 7.8 Hz, 1H), 7.99 (dd, J= 11.0, 7.8 Hz, 3H), 7.72 (t, J= 7.7 Hz, 3H), 7.12 (t, J= 8.5 Hz, 4H), 4.57 (s, 2H), 4.48 (s, 2H), 4.34 (d, J= 16.3 Hz, 8H).13C NMR (126 MHz, D2O) 8 162.95, 155.59, 155.39, 153.83, 152.21, 147.21, 146.02, 143.93, 142.51, 141.21, 129.54,

[0290] 127.97, 126.47, 125.77, 122.47, 122.44, 58.85, 57.78, 57.44, 57.19.

[0291] To a round bottom flask, S7 (0.064g, 0.165mmol) was added along with Na2C03(0.053g, 0.495mmol, 3equiv.) and MeCN (10ml). The reaction mixture is stirred for 10 minutes at room temperature prior to the addition of tertbutylbromoacetate (0.032g, 240ml, 0.165 mmol) using a micropipette. The reaction was then stirred under refluxing conditions overnight. The following day, Na2COs was filtered off and the MeCN was evaporated yielding an oil that was basified with saturated K2CO3. The basic layer was extracted 3x15mL of DCM. The organic layer was dried with anhydrous K2CO3. The drying agent is filtered off and the DCM evaporated yielding an off-white powder (0.050g, 60%yield).1H NMR (500 MHz, CDCI3) 8 8.04 d, 2H), 7.92 (t, 1H), 7.20 - 7.06 (m, 2H), 6.85 (d, 4H), 4.27 (s, 2H), 3.99 (q, J= 9.0 Hz, 11H), 3.61 (s, 2H), 1.51 (s, 9H).

[0292] To a round bottom flask, S8 (0.050g, 0.369 mmol) was added followed by the addition of lOmL of 6M HC1. The reaction mixture was stirred under refluxing conditions overnight. The following day, the solvent was evaporated yielding a white powder (0.020g, 47% yield). 'H NMR (500 MHz, D2O) 8 8.12 (t, J= 7.8 Hz, 1H), 8.03 (d, J= 8.0 Hz, 1H), 7.84 (d, J= 7.8 Hz, 1H), 7.61 (t, J= 7.8 Hz, 2H), 7.14 (d, J= 7.8 Hz, 2H), 7.07 (d, J= 7.8 Hz, 2H), 4.78 (s, 2H), 4.51 (d, J= 10.2 Hz, 8H), 4.12 (s, 2H).

[0293] 500.163W01

[0294] UIUC2023-225-02(PCT)

[0295] To a round bottom flask, S5 (0.14g, 0.41mmol) was added along with Na2COs (0.13g, 1.23 mmol, 3 equiv.) and picoline bromide (0.103g, 0.41 mmol) in dry MeCN. The color of the reaction turns pink right after the addition of picoline bromide. The reaction is refluxed overnight and as the reaction progresses, it turns to light brown. The following day, the Na2COs is filtered off and the MeCN is evaporated yielding oil. The oil is basified with saturated K2CO3, and the basic layer is extracted with 3x15ml of DCM. The organic layer is dried with anhydrous K2CO3 then filtered and evaporated. A mass spectrum indicated that there is a mixture of desired product boc protected product and deprotected product. The crude reaction mixture was subjected to boc deprotection. 4M HC1 in dioxane was added to the reaction mixture and the reaction was stirred for 3h. After 3 hours, the excess dioxane is evaporated yielding a light brown precipitate. The precipitate is basified with saturated K2CO3, and the basic layer is extracted with 3x15ml of DCM. The organic layer is dried with anhydrous K2CO3 then filtered and evaporated yielding the desired product (0.050 g, 37% yield over two steps). 'H NMR (500 MHz, CDCI3) 8 8.65 (d, J = 5.0 Hz, 1H), 7.84 - 7.79 (m, 2H), 7.28 - 7.21 (m, 1H), 7.13 (t, J= 7.6 Hz, 2H), 6.77 (d, J= 7.7 Hz, 2H), 6.54 (d, J= 7.5 Hz, 2H), 4.25 (s, 2H), 4.03 (s, 4H), 3.97 (s, 4H).

[0296] To a 3-neck flask was added S9 (0.050g, 0.151mmol) along with paraformaldehyde (0.024g, 0.831 mmol, 5equiv.) and suspended in lOmL of dry MeCN. The reaction was heated under refluxing conditions for one hour. Benzothiazole (0.058g, 0.227mmol, 1.5equiv) was dissolved in lOmL of hot MeCN and added dropwise to the reaction over 30 min. After the addition of benzothiazole, the reaction was stirred under refluxing conditions for 24h. The crude reaction mixture was purified by flash column chromatography on a basic alumina column (A: DCM and B: MeOH 0% B to 10% B). The product was obtained as a yellow oil (0.016g, 18% yield). 'H NMR (500 MHz, CDCI3) 8 8.63 (d, 7 = 4.9 Hz, 1H), 8.05 (d, 7= 8.1 Hz, 1H), 7.91 (d, 7= 8.0 Hz, 1H), 7.83 - 7.74 (m, 2H), 7.68 (d, J = 2.0 Hz, 1H), 7.52 (d, J = 2.0 Hz, 1H), 7.49 (d, J= 7.7 Hz, 1H), 7.38 (t, J= 7.6 Hz, 1H), 7.25 (d, J = 5.4 Hz, 1H), 7.20 (t, J= 7.6 Hz, 2H), 6.86 (dd, J= 12.1, 7.6 Hz, 4H), 4.29 (s, 2H), 4.21 (s, 2H), 4.12

[0297] 500.163W01

[0298] UIUC2023-225-02(PCT) (s, 4H), 4.10 (s, 3H), 4.03 (s, 4H).13C NMR (126 MHz, CDCh) 5 171.44, 167.08, 158.53, 156.78, 154.33, 153.14, 149.17, 148.26, 147.77, 135.60, 135.20, 125.24, 124.09, 123.80, 122.35, 122.19, 121.73, 121.61, 121.32, 120.51, 119.76, 109.21, 64.83, 62.76, 62.72, 62.33, 55.24, 52.41. HRMS- ESI (m / z)-. [M +H]+ calcd for C35H33N6O2S; 601.2310 found; 601.239.

[0299] To a 3-neck flask was added S7 (0.100g, 0.256 mmol) along with paraformaldehyde (0.038g, 1.283 mmol, 5equiv.) and suspended in 20mL of dry MeCN. The reaction was heated under refluxing conditions for one hour. Benzothiazole (0.131g, 0.513mmol, 2 equiv.) was dissolved in lOmL of hot MeCN and added drop wise to the reaction over 30 min. After the addition of benzothiazole, the reaction was stirred under refluxing conditions for 24h. The crude reaction mixture was purified by flash column chromatography on a basic alumina column (A: DCM and B: MeOH 0% B to 10% B). The product was obtained as a light-yellow powder (0.089g, 53% yield). 'H NMR (500 MHz, CDCh) 8 8.01 (t, J= 8.7 Hz, 2H), 7.96 (d, J= 8.2 Hz, 1H), 7.87 (t, J= 7.7 Hz, 1H), 7.81 (d, J= 7.9 Hz, 1H), 7.58 (d, J= 2.1 Hz, 1H), 7.43 (d, J = 2.0 Hz, 1H), 7.40 (t, J= 7.7 Hz, 1H), 7.28 (t, J= 7.6 Hz, 1H), 7.11 (t, J= 7.6 Hz, 2H), 6.77 (dd, J= 9.9, 7.6 Hz, 4H), 4.23 (s, 2H), 4.19 (s, 2H), 4.02 (s, 3H), 4.00 (s, 4H), 3.95 (s, 3H), 3.94 (s, 4H).13C NMR (126 MHz, CDCh) 8 167.04, 164.84, 159.55, 156.58, 154.43, 153.13, 149.15, 147.76, 146.51, 136.59, 135.20, 133.77, 125.44, 125.23, 124.08, 123.79, 122.94, 122.07, 121.68, 120.50, 119.74, 64.45, 62.83 ,62.30, 55.23, 52.42, 51.98. HRMS-ESI (m / z): [M +H]+calcd for C37H35N6O4S; 659.236 found; 659.2436.

[0300] To a round bottom flask, S10 (0.020g, 0.030mmol) was added along with Li OH (0.004g, 0.089mmol, 3equiv.). The starting material was dissolved in 6 mL of a 1:1 mixture of MeOH and DI H2O. The reaction mixture was stirred at room temperature for 6 h. The solvents were evaporated, and the residue was redissolved in MeOH and fdtered through a celite plug then the MeOH was evaporated yielding a yellow powder as a lithium salt (0.013g, 67% yield). 'H NMR (500 MHz, CD3OD) 8 8.13 (d, J= 7.7 Hz, 1H), 7.95 (t, J= 7.7 Hz, 1H), 7.87 (dd, J= 14.2, 8.0 Hz, 2H), 7.64 (d, J= 7.6 Hz, 1H), 7.61 - 7.57 (m, 1H), 7.45 (s, 1H), 7.43 (d, J= 8.0 Hz, 1H), 7.31 (t, J= 7.4 Hz, 1H),

[0301] 500.163W01 48

[0302] UIUC2023-225-02(PCT) 7.22 (t, J= 7.7 Hz, 2H), 6.67 (dd, J= 25.4, 7.7 Hz, 4H), 4.36 (d, J= 14.7 Hz, 2H), 4.29 - 4.21 (m, 4H), 4.07 (s, 3H), 4.00 (s, 1H), 3.52 (d, J= 14.5 Hz, 2H), 3.42 (t, J= 16.2 Hz, 2H).

[0303] To a 3-neck flask was added S5 (0.141g, 0.414 mmol) along with paraformaldehyde (0.12g, 4.14mmol, lOequiv.) and suspended in 15mL of dry MeCN. The reaction was heated under refluxing conditions for one hour. Benzothiazole (0.160g, 0.621 mmol, 1.5 quiv.) was dissolved in lOmL of hot MeCN and added dropwise to the reaction over 30 min. After the addition of benzothiazole, the reaction was stirred under refluxing conditions for 24h. The crude reaction mixture was purified by flash column chromatography on a silica column (A: DCM and B: MeOH 0% B to 10% B with 1% tri ethyl amine). The product was obtained as a light-yellow powder (0.147g, 58% yield).1H NMR (500 MHz, CDCh) 8 8.04 (d, J= 8.1 Hz, 1H), 7.89 (d, J= 7.9 Hz, 1H), 7.66 (d, J= 2.1 Hz, 1H), 7.52 (d, J= 2.0 Hz, 1H), 7.48 (t, J= 7.6 Hz, 1H), 7.36 (t, J= 7.6 Hz, 1H), 7.26 - 7.17 (m, 2H), 6.96 (d, J = 7.7 Hz, 1H), 6.88 (q, J= 7.6 Hz, 3H), 4.82 (s, 2H), 4.78 (s, 3H), 4.28 (s, 2H), 4.08 (s, 6H), 1.60 (s, 9H).

[0304] To a round bottom flask was added SI 1 (0.035 g, 0.057 mmol) along with 4M HCL in dioxane (2.0ml). The reaction was stirred at room temperature for 3h. As the reaction progressed, the product began precipitating out. The excess dioxane was evaporated and the solid was basified using saturated K2CO3. The basic layer was extracted 3x15mL of DCM. The organic layer was dried with anhydrous K2CO3. The drying agent is fdtered off and the DCM evaporated yielding an off-yellow powder (0.022g, 76% yield). ' H NMR (500 MHz, CDCI3) 8 8.02 (d, J = 8.1 Hz, 1H), 7.87 (d, J = 7.9 Hz, 1H), 7.63 (s, 1H), 7.52 (s, 1H), 7.46 (t, J= 7.6 Hz, 1H), 7.34 (t, J= 7.6 Hz, 1H), 7.16 (t, J= 7.6 Hz, 2H), 6.74 (d, J= 7.7 Hz, 2H), 6.64 (d, J= 7.5 Hz, 2H), 4.29 (s, 2H), 4.16 (s, 4H), 4.06 (d, J= 8.8 Hz, 7H).

[0305] 500.163W01

[0306] UIUC2023-225-02(PCT)

[0307] S12

[0308] To a 3-neck flask was added S12 (0.060g, 0.235 mmol) along with paraformaldehyde (0.035g, 1.16mmol, 5equiv.) and suspended in 15mL of dry MeCN. The reaction was heated under refluxing conditions for one hour. Benzothiazole (0.091g, 0.353 mmol, 1.5 quiv.) was dissolved in lOmL of hot MeCN and added drop wise to the reaction over 30 min. After the addition of benzothiazole, the reaction was stirred under refluxing conditions for 24h. The crude reaction mixture was purified by flash column chromatography on a basic alumina column (A: DCM and B: MeOH 0% B to 10% B). The product was obtained as a dark yellow powder (0.034, 28% yield). 1H NMR (500 MHz, CDC13) 8 8.06 (d, J= 8.2 Hz, 1H), 7.92 (d, J= 7.9 Hz, 1H), 7.69 (d, J= 2.0 Hz, 1H), 7.54 (s, 1H), 7.53 - 7.48 (m, 1H), 7.39 (t, J= 7.5 Hz, 1H), 7.20 (t, J= 7.6 Hz, 2H), 6.87 (dd, J= 16.4, 7.7 Hz, 4H), 4.31 (d, J = 6.9 Hz, 2H), 4.15 (s, 4H), 4.12 (d, J = 3.1 Hz, 3H), 3.89 (s, 4H), 2.77 (s, 3H).

[0309] Metal complex synthesis.

[0310] TE-1 Mn-TE-1

[0311] To a stirring solution of TE-1 (70 mg, 0.128 mmol) in MeCN was added Mnn(C104)2 6H2O (46 mg, 0.128 mmol) in MeCN. The cloudy white solution was stirred inside the glovebox overnight and the following day, the metal complex is precipitated using diethyl ether. The precipitate is collected and washed further with diethyl ether yielding the desired compound (53mg, 58% yield). X- ray crystals were obtained by dissolving the metal complex in methanol and adding a drop of water. This solution was set-up for diethyl ether vapor diffusion. ESI-MS (m / z): 282.6 [(TE- 1) Mn]2+; 564.1

[0312] 500.163W01 50

[0313] UIUC2023-225-02(PCT) To a stirring solution of TE-3 (30 mg, 0.07 mmol) in MeCN was added Mnn(C104)2 6H2O ( 25.7 mg, 0.07 mmol) in MeCN. The light brown solution was stirred inside the glovebox overnight and the following day, the metal complex is precipitated using diethyl ether. The precipitate is collected and washed with diethyl ether yielding the desired compound (32mg, 78% yield). ESI-MS (m / z): 238.6 [(TE-3) Mn]2+; 476.2 [(TE-3)Mn ]+; 522.2[(TE- Mn-TE-4

[0314] To a stirring solution of TE-4 (50 mg, 0.140 mmol) and CS2CO3 (59 mg, 0.168 mmol, 1.2 equiv.) in MeOH was added Mnn(C104)2 6H2O (59mg, 0.140 mmol) in MeOH. The off-white solution was stirred inside the glovebox overnight and the following day, the metal complex is precipitated using diethyl ether. The precipitate is collected and washed further with diethyl ether yielding the desired compound (66mg, 90% yield). ESI-MS (m / z): 410.1 [(TE-4) Mn+1]+; 205.5 [(TE- 4) Mn+2]+2.

[0315] To a stirring solution of TE-2 (35.0 mg, 0.090 mmol) in MeOH was added Mnn(C104)2 6H2O (32.5 mg, 0.090 mmol) in MeOH. The light-yellow solution was stirred inside the glovebox overnight and the following day, the metal complex is precipitated using diethyl ether. The precipitate is collected and washed further with diethyl ether yielding the desired compound (37mg, 76% yield). X- ray crystals were obtained by dissolving the metal complex in methanol and adding a drop of water. This solution was set-up for diethyl ether vapor diffusion. ESI-MS (m / z): 443.1 [(TE-2) Mn]+; 222.1

[0316] 500.163W01 51

[0317] UIUC2023-225-02(PCT) To a stirring solution of TE-5 (22.6 mg, 0.052 mmol) and CS2CO3 (0.034 mg, 0.1043 mmol, 2 equiv.) in MeOH was added Mnn(C104)2 6H2O (18.9mg, 0.052 mmol) in MeOH. The off-white solution was stirred inside the glovebox overnight and the following day, the metal complex is precipitated using diethyl ether. The precipitate is collected and washed further with diethyl ether yielding the desired compound (29mg, 94% yield).

[0318] To a stirring solution of TE-7 (29.3 mg, 0.063 mmol) and CS2CO3 (27 mg, 0.075 mmol, 1.2 equiv.) in MeOH was added Mnn(C104)2 6H2O (22.7 mg, 0.063 mmol) in MeOH. The light-yellow solution was stirred inside the glovebox overnight and the following day, the metal complex is precipitated using diethyl ether. The precipitate is collected and washed further with diethyl ether yielding the desired compound (25mg, 64% yield). X-ray crystals were obtained by dissolving the metal complex in methanol and adding a drop of water. This solution was set-up for diethyl ether vapor diffusion. ESI-MS (m / z): 520.1 [(TE-7)-Mn]+.

[0319] To a stirring solution of TE-3 (13.2 mg, 0.022 mmol) and K2CO3 (3.6 mg, 0.026 mmol, 1.2 equiv.) in MeOH was added Mnn(C104)2 6H2O (8.0 mg, 0.022 mmol) in MeOH. The light-yellow solution was stirred inside the glovebox overnight and the following day, the metal complex is precipitated using diethyl ether. The precipitate is collected and washed further with diethyl ether yielding the desired compound (25mg, 64% yield).

[0320] 500.163W01 52

[0321] UIUC2023-225-02(PCT) To a stirring solution of TE-6 (11.6 mg, 0.018 mmol) in DCM was added MnCh (2.3 mg, 0.018 mmol) in DCM. The yellow solution was stirred inside the glovebox overnight and the following day, the metal complex is precipitated using diethyl ether. The precipitate is collected and washed further with diethyl ether yielding the desired compound (8.5mg, 65% yield). ESI-MS (m / z):

[0322] To a stirring solution of TE-8 (8.7 mg, 0.015 mmol) and K2CO3 (2.3 mg, 0.016 mmol, 1.1 equiv.) in MeOH was added Mnn(OTf)2 (5.4 mg, 0.015 mmol) in MeOH. The light-yellow solution was stirred inside the glovebox overnight and the following day, the metal complex is precipitated using diethyl ether. The precipitate is collected and washed further with diethyl ether yielding the desired compound (4.5mg, 47% yield). ESI-MS (m / z):621.2 [(TE-8)-Mn+lH]+; 311.1 [(TE-8)-

[0323] To a stirring solution of TE-9 (12.1 mg, 0.023 mmol) and K2CO3 (3.8 mg, 0.028 mmol, 1.2 equiv.) in MeOH was added Mnn(C104)2 6H2O (8.4mg, 0.023 mmol) in MeOH. The light-yellow solution was stirred inside the glovebox overnight and the following day, the metal complex is precipitated using diethyl ether. The precipitate is collected and washed further with diethyl ether yielding the desired compound (8.5mg, 65 % yield).

[0324] 500.163W01 53

[0325] UIUC2023-225-02(PCT) To a stirring solution of TE-10 (14.6 mg, 0.029 mmol) and K2CO3 (3.1 mg, 0.034 mmol, 1.2 equiv.) in MeOH was added Mnn(C104)2 6H2O (10.4mg, 0.034 mmol) in MeOH. The light-yellow solution was stirred inside the glovebox overnight and the following day, the metal complex is precipitated using diethyl ether. The precipitate is collected and washed further with diethyl ether yielding the desired compound (11.6mg, 61 % yield).

[0326] Example 2. Pharmaceutical Dosage Forms.

[0327] The following formulations illustrate representative pharmaceutical dosage forms that may be used for the therapeutic or prophylactic administration of a compound of a formula described herein, a compound specifically disclosed herein, or a pharmaceutically acceptable salt or solvate thereof (hereinafter referred to as 'Compound X'):

[0328] (i) Tablet 1 mg / tablet

[0329] 'Compound X' 100.0

[0330] Lactose 77.5

[0331] Povidone 15.0

[0332] Croscarmellose sodium 12.0

[0333] Microcrystalline cellulose 92.5

[0334] Magnesium stearate 3,0

[0335] 300.0

[0336] (ii) Tablet 2 mg / tablet

[0337] 'Compound X' 20.0

[0338] Microcrystalline cellulose 410.0

[0339] Starch 50.0

[0340] Sodium starch glycolate 15.0

[0341] Magnesium stearate 5,0

[0342] 500.0

[0343] (iii) Capsule mg / capsule

[0344] 'Compound X' 10.0

[0345] Colloidal silicon dioxide 1.5

[0346] Lactose 465.5

[0347] Pregelatinized starch 120.0

[0348] Magnesium stearate 3,0

[0349] 600.0 fiv) Injection 1 (1 mg / mL) mg / mL

[0350] 'Compound X' (free acid form) 1.0

[0351] Dibasic sodium phosphate 12.0

[0352] Monobasic sodium phosphate 0.7

[0353] Sodium chloride 4.5

[0354] LO N Sodium hydroxide solution q.s.

[0355] (pH adjustment to 7.0-7.5)

[0356] Water for injection q.s. ad 1 mL

[0357] 500.163W01 54

[0358] UIUC2023-225-02(PCT) (v) Injection 2 (10 mg / mL) mg / mL

[0359] 'Compound X' (free acid form) 0.0

[0360] Monobasic sodium phosphate 0.3

[0361] Dibasic sodium phosphate 1.1

[0362] Polyethylene glycol 400 200.0

[0363] 0.1 N Sodium hydroxide solution q.s.

[0364] (pH adjustment to 7.0-7.5)

[0365] Water for injection q.s. ad 1 mL

[0366] (vi) Aerosol mg / can

[0367] 'Compound X' 20

[0368] Oleic acid 10

[0369] Trichloromonofluoromethane 5,000

[0370] Dichlorodifluoromethane 10,000

[0371] Dichlorotetrafluoroethane 5,000

[0372] (vii) Topical Gel 1 wt.%

[0373] 'Composition X' 5% Carbomer 934 1.25% Triethanolamine q.s. (pH adjustment to 5-7) Methyl paraben 0.2% Purified water q.s. to 100g

[0374] (viii) Topical Gel 2 wt.%

[0375] 'Composition X' 5% Methylcellulose 2% Methyl paraben 0.2% Propyl paraben 0.02% Purified water q.s. to 100g

[0376] (ix) Topical Ointment wt.%

[0377] 'Composition X' 5%

[0378] Propylene glycol 1%

[0379] Anhydrous ointment base 40%

[0380] Polysorbate 80 2%

[0381] Methyl paraben 0.2%

[0382] Purified water q.s. to 100g

[0383] (x) Topical Cream 1 wt.%

[0384] 'Composition X' 5% White bees wax 10% Liquid paraffin 30% Benzyl alcohol 5% Purified water q.s. to 100g

[0385] (xi) Topical Cream 2 wt.%

[0386] 'Composition X' 5% Stearic acid 10%

[0387] Glyceryl monostearate 3%

[0388] 500.163W01

[0389] UIUC2023-225-02(PCT) Polyoxyethylene stearyl ether 3% Sorbitol 5%

[0390] Isopropyl palmitate 2 %

[0391] Methyl Paraben 0.2%

[0392] Purified water q.s. to 100g

[0393] These formulations may be prepared by conventional procedures well known in the pharmaceutical art. It will be appreciated that the above pharmaceutical compositions may be varied according to well-known pharmaceutical techniques to accommodate differing amounts and types of active ingredient 'Compound X'. Aerosol formulation (vi) may be used in conjunction with a standard, metered dose aerosol dispenser. Additionally, the specific ingredients and proportions are for illustrative purposes. Ingredients may be exchanged for suitable equivalents and proportions may be varied, according to the desired properties of the dosage form of interest.

[0394] All publications, patents, and patent documents cited herein are incorporated by reference as though individually incorporated by reference. No limitations inconsistent with this disclosure are to be understood therefrom. The invention has been described with reference to various specific and preferred embodiments and techniques. However, many variations and modifications may be made while remaining within the spirit and scope of the invention.

[0395] While specific embodiments have been described above with reference to the disclosed embodiments and examples, such embodiments are only illustrative and do not limit the scope of the invention. Changes and modifications can be made in accordance with ordinary skill in the art without departing from the invention in its broader aspects as defined in the following claims.

[0396] 500.163W01 56

[0397] UIUC2023-225-02(PCT)

Claims

What is claimed is:

1. A compound of Formula I:or a salt and / or a hydrate thereof; wherein,— represents one or more coordinate covalent bonds when NF is present in a metal complex of Formula I; or— is absent when Mzis not present;Mzis a metal or metal radioisotope, wherein z represents the oxidation state of the metal or metal radioisotope; orMzis absent;G1is picolinyl, pyridinyl, carboxyl, or benzothiazolyl hydroxyphenyl;G2is picolinyl, pyridinyl, carboxyl, or H; wherein picolinyl, pyridinyl, carboxyl, or benzothiazolyl hydroxyphenyl are substituted with R3; andR1, R2, and R3are each independently H, halo, -(Ci-C6)alkyl, -O(Ci-C6)alkyl, or -N(R4)2 wherein each R4is independently H or -(Ci-Ce)alkyl; or the compound 2-(3,7-diaza-l,5(2,6)-dipyridinacyclooctaphane-3-ylmethyl)-4- (benzo[c / / thiazol-2-yl)-6-methoxyphenol or a metal complex thereof comprising Mz, a salt, and / or a hydrate thereof.

2. The compound of claim 1, wherein G1is carboxyl.

3. The compound of claim 1, wherein G1is 2-picolynyl.

4. The compound of claim 1, wherein G2is carboxyl or picolinyl.

5. The compound of claim 1, wherein G2is benzothiazolyl hydroxyphenyl.

6. The compound of claim 5, wherein R3is -O(Ci-C6)alkyl.500.163W01 57UIUC2023-225-02(PCT)7. The compound of claim 1, wherein G2is:

8. The compound of claim 1, wherein R1and R2are H.

9. The compound of claim 1, wherein Mzis a transition metal or lanthanide.

10. The compound of claim 1, wherein Mzis manganese.

11. The compound of claim 1, wherein the oxidation state (z) of the metal is +2 or +3.

12. The compound of claim 1, wherein Mzis Mn2+.

13. The compound of claim 1 , wherein Mzis absent.

14. The compound of claim 1, wherein the compound is:or a radioisotope, a salt and / or a hydrate thereof.500.163W01 58UIUC2023-225-02(PCT)15. The compound of claim 1, wherein the compound is:or a metal complex thereof comprising Mz, a salt, and / or a hydrate thereof.

16. The compound of claim 1, wherein the compound is:or a metal complex thereof comprising Mz, a salt, and / or a hydrate thereof.500.163W01UIUC2023-225-02(PCT)17. The compound of claim 1, wherein the compound is a hydrated compound comprising between 1 and 2 water molecules.

18. A method for improving contrast of a target tissue in a subject imaged by magnetic resonance imaging (MRI) comprising administering a sufficient amount of a metal complex of the compound of claim 1 to the subject and imaging the subject by MRI, wherein the compound distributes to the target tissue and contrast of the target tissue is thereby improved.

19. A method for imaging a target tissue in a subject by positron emission tomography (PET) comprising administering a sufficient amount of a metal radioisotope complex of the compound of claim 1 to the subject and imaging the subject by PET, wherein the compound distributes to the target tissue and the target tissue is thereby imaged.

20. The method of claim 18 or 19, wherein the target tissue is kidney, liver, stomach, brain, a cancer or a combination thereof.

21. A method for diagnosing Alzheimer’s disease in a subject comprising: a) administering a sufficient amount of a metal complex of the compound of any one of claims 1-17 to the subject; b) imaging the subject by magnetic resonance imaging (MRI); and c) detecting the presence or absence of Amyloid- / >eta plaques in the subject’s brain, wherein the compound binds to Amyloid- / >eta plaques if present; wherein a presence or absence of Alzheimer’s disease is thereby diagnosed.500.163W01 60UIUC2023-225-02(PCT)

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