PLATFORM FOR TARGETED MR IMAGING

RELATED APPLICATIONS

[0001] This application claims priority to, and benefit of, United Kingdom Patent Application No. 2216233.3, filed November 1, 2022, the content of which is incorporated by reference in its entirety.

FIELD

[0002] This disclosure relates to a manganese bifunctional chelating agent conjugation platform for targeted MR imaging. The use of a manganese 5-(4-isothiocyanatophenyl)- 10,15,20-(tri-4-sulfonatophenyl)porphyrin complex is ideal for the versatile design and synthesis of new MRI molecular agents. This platform affords several distinct advantages: (1) it is amenable to pre-metalated conjugation, (2) it is chemically inert and compatible with a much greater variety of synthesis conditions compared to current bifunctional chelating agents (BFCAs), (3) it allows direct conjugation to biomolecules in a single step and dispenses with intermediate deprotection steps, and (4) the stability of Mn chelation under all reaction conditions tested ensures improved product homogeneity. Specifically, Mn complexation by MnP-NCS drastically increases its reactivity with nucleophiles, enabling conjugation with even weak nucleophiles and without requiring a catalyst.

BACKGROUND

[0003] Current paramagnetic contrast agents provide largely non-specific contrast enhancement on magnetic resonance imaging (MRI). The majority of existing agents cannot achieve the specificity required for targeted imaging of molecular entities, as only minor tweaks in pharmacokinetics and bio-distribution are possible. Gadolinium (Gd)- based chelates have been utilized in diagnostic magnetic resonance imaging (MRI) for over three decades by virtue of its superior contrast enhancement of tissue abnormalities. In most patients, Gd is safely eliminated through the kidneys into urine. However, the current use of gadolinium (Gd) poses risks of toxicity to patients when it becomes decoupled and released in its ionic form. By 2006, a severe contraindication was discovered between Gd administration in patients with impaired renal function and high levels of Gd accumulation - this condition called nephrogenic systemic fibrosis (NSF) is one characterized by extensive systemic fibrosis that leads to severe pain, immobility, and even death. Over the past decade, the incidence of NSF has greatly declined due to several precautionary measures: Gd -based agents are not used in patients with renal insufficiency to ensure rapid elimination, high-dose applications have been curtailed, and less stable linear chelates have been replaced with macrocyclic structures. Despite these measures, new and more subtle complications are coming to light. Recent studies have begun to show anthropogenic Gd contamination of the environment from patient scans. While others are suggesting long-term Gd retention in patients with normal renal function with patients reporting milder NSF-like symptoms. The toxicity and safety issues associated with Gd-based agents have directed the focus of most MRI contrast agent development towards retaining the use of Gd but reducing risk by improving its safety profile.

[0004] The development and clinical translation of more specialized Gd-based contrast agents to improve the diagnostic capabilities of MRI is seemingly stalled. Use of such agents which possess specific functionalities for biological markers result in a prolonged elimination half-life. As elimination half-life increases, so does the risk of undesired free Gd release and deposition in the body. Any retention of Gd agents, even if retained from tissue targeting, thereby risks contraindications in patients with even normal renal function. Such a risk has limited the use of Gd Agents from a vast array of untapped applications that require prolonged retention of the contrast agent in the body. Without definitively addressing the issue of Gd toxicity, current designs of MRI contrast agents are constrained to a narrow scope of applications.

[0005] The last hurdle is particularly relevant and necessary to overcome if we wish to design a highly versatile MRI contrast agent platform. The use of pendant carboxylate groups for Gd coordination makes current MRI chelates susceptible to side reactions (at the time of functionalization and conjugation). To avert side reactions that reduce both yield and destabilize Gd coordination of the resulting complex, protective groups are sometimes added. However, adding protective groups introduces additional reaction steps and precludes pre-metalation of the chelate, all of which pose real problems to product consistency and purity when large molecules are involved, and completely exclude conjugation reactions involving solids. [0006] Thus, it would be very advantageous to provide a chelating agent conjugation platform for targeted MR imaging which avoids the above-mentioned problematic toxicity issues associated with gadolinium (Gd).

SUMMARY

[0007] The present disclosure provides a manganese bifunctional chelating agent conjugation platform for targeted MR imaging. The chelating agent platform disclosed herein provides chemistries and uses of the paramagnetic metal, manganese (Mn), for synthesizing targeted MRI contrast agents for high-specificity molecular imaging. The chelating agent uses porphyrin as a bifunctional chelating agent (BFCA), as this dual functionality provides both stable metal chelation and conjugation to biomolecules or drugs via a pendant electrophile or nucleophile.

[0008] A first aspect of the present disclosure relates to compounds of Formula (I), and pharmaceutically acceptable salts or tautomers thereof, wherein:

E is NH, O, or S;

Z is absent, CH2, NH, O, or S; and

R is a targeting group. [0009] In some embodiments, R comprises a protein, peptide, antibody, glycoprotein, glycosaminoglycan, polysaccharide, dendrimer, lipid, steroid, drug, polymer, acrylate, or small molecule moiety targeting group.

[0010] In some embodiments, Z is CH2, NH, O, or S. In some embodiments, Z is absent, NH, O, or S.

[0011] In some embodiments, E is S. In some embodiments, E is S; and Z is NH. In some embodiments, E is S; and Z is O. In some embodiments, E is S; and Z is S.

[0012] In some embodiments, E is O. In some embodiments, E is O; and Z is absent, CH2 or O. In some embodiments, E is O; and Z is absent or CH2. In some embodiments, E is O; and Z is O.

[0013] In some embodiments, E is NH. In some embodiments, E is NH; and Z is NH.

[0014] In some embodiments R is an antibody or glycoprotein with specificity for a biological antigen.

[0015] In some embodiments, R is albumin.

[0016] In some embodiments, R comprises a small molecule or a drug. In some embodiments, R comprises a 1,2-diamine-substituted aromatic compound, wherein the aromatic compound is optionally further substituted. In some embodiments, R further comprises a spacer moiety which is an optionally substituted alkylene or heteroalkylene having a chain length of 3 to 10 atoms.

[0017] In some embodiments, R is a 5 -hydroxyindole, 5-hydroxytryptamide, serotonin, serotonin hydrochloride, or 5-hydroxyindole-3 -acetic acid. In some embodiments, the compound is for imaging myeloperoxidase.

[0018] In some embodiments, R is 4,4-diphenylcyclohexan-l-ol. In some embodiments, R is cholic acid, deoxycholic acid glycocholic acid, glycolithocholic acid, glycochenodeoxycholic acid, lithocholic acid, chenodeoxycholic acid, taurochenodeoxycholic acid, taurocholic acid, or other bile acid derivative.

[0019] In some embodiments, R is a fatty acid. In some embodiments, the fatty acid is steric acid or lauric acid.

[0020] In some embodiments, R comprises an acrylate. [0021] In some embodiments, R comprises a metal-free porphyrin. In some embodiments, said metal free porphyrin is any one of 10,15,20-(tri-4- sulfonatophenyl)porphyrin, (di-4-sulfonatophenyl)porphyrin, (4- sulfonatophenyl)porphyrin, uroporphyrinogen I and III, hematoporphyrin, photofrin and Tetra(4-carboxyphenyl)porphyrin), benzoporphyrin, porphycene, corrin, corphin, sirohydrochlorin, chlorin, phthalocyanine, hydrazine formyl -hydrazine, l-(4-Amino-l- piperazine), or (R)-2-amino-4-phenylbutanehydrazide.

[0022] In some embodiments, R is a polysaccharide or a glycosaminoglycan. In some embodiments, the polysaccharide or glycosaminoglycan comprises free carboxylic acids. In some embodiments, the polysaccharide is a mannuronate and/or guluronate based polysaccharide. In some embodiments, the polysaccharide is alginate. In some embodiments, the glycosaminoglycan is hyaluronic acid, chondroitin sulfate, dermatan sulfate or heparin.

[0023] In some embodiments, R is a protein or a polysaccharide. In some embodiments, R is a protein and the protein is collagen, silk, elastin, or any other protein-based compound. In some embodiments R is a polysaccharide and the polysaccharide is chitosan. In some embodiments, R is a polysaccharide or a glycosaminoglycan. In some embodiments, R is a polysaccharide and the polysaccharide polyvinyl alcohol, cellulose, callose, chitin, dextran, cyclodextran, amylose, glucose, arabinose, ribose, threose, fructose, galactose, xylose, manose, maltose, lactose, talose, sucrose, pectin, amylopectin, carrageanan starch or glycogen. In some embodiments, R is a glycosaminoglycan and the glycosaminoglycan is heparin sulfate or keratan sulfate.

[0024] In another aspect, provided a compound of Formula (I), (la), (lb), or (Ic), or a pharmaceutically acceptable salt or tautomer thereof, for use as an MRI contrast agent. In some embodiments, provided is a compound of Formula (I), (la), (lb), or (Ic), or a pharmaceutically acceptable salt or tautomer thereof, for use as an MRI contrast agent in a subject in need thereof.

[0025] In another aspect, provided is a method of functionalizing a protein or antibody with a pre-metalated Mn ²⁺ porphyrin reagent. In one embodiment, the pre-metalated Mn ²⁺ porphyrin reagent is MnP-NCS. [0026] In another aspect, provided is a method of functionalizing alginate with a pre- metalated Mn ²⁺ porphyrin reagent. In one embodiment, the pre-metalated Mn ²⁺ porphyrin reagent is MnP-NCS.

[0027] In another aspect, provided is a one-step method of labeling a primary alcohol, a secondary alcohol, or an electron-deficient aryl amine with an Mn ²⁺ porphyrin reagent. In one embodiment, the Mn ²⁺ porphyrin reagent is MnP-NCS.

[0028] In another aspect, provided is a method of labeling a carboxylate with an Mn ²⁺ porphyrin reagent. In one embodiment, the Mn ²⁺ porphyrin reagent is MnP-NCS.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The following drawings are illustrative of the disclosure and are not to be construed as limiting the disclosure. The drawings are not necessarily to scale.

[0030] Figure 1. Reaction of manganese 5-(4-isothiocyanatophenyl)-10,15,20-(tri-4- sulfonatophenyl)porphyrin (MnP-NCS): (1) with primary or secondary alcohols results in either a thiocarbamate or a carbamate linkage; (2) with primary or secondary amines results in a thiourea, guanidine, or guanidinium linkage; (3) with primary or secondary carboxylic acids results in the formation of an amide linkage; and (4) with thiols results in a dithiocarbamate linkage.

[0031] Figure 2 is a molecular structure showing that aryl isothiocyanate substitution with electron-withdrawing groups destabilizes the isothiocyanate, leading to enhanced reactivity; electron-donating groups stabilizes the isothiocyanate, leading to resistance against reaction with nucleophiles.

[0032] Figure 3 shows a detailed breakdown of common post-metalation bioconjugate techniques used by BFCAs and the complexity compared to pre-labeling.

[0033] Figure 4 shows a library of exemplary MRI contrast agents synthesized from MnP-NCS. Some agents adopt pre-labeling methods of conjugation.

[0034] Figure 5 shows the general procedure for MnP-NCS labeling of protein. Subscript n indicates an integer value of MnP labels per protein. [0035] Figure 6 illustrates labeling of human serum albumin (HSA) with stoichiometric excess of MnP-NCS as described in Example 1. Molecular weights obtained by matrix- assisted laser desorption/ionization (MALDI); manganese atoms per HSA protein; tagging efficiency % based upon stoichiometric excess used. [0036] Figure 7 is a plot of T ₁ relaxation value (ms) versus ratio of MnP conjugated to HSA and shows T1 measured for HSA control and the following (MnP)n-HSA conjugates, wherein n represents the average number of MnP labels per HSA: (MnP) _3.5 -HSA, (MnP)6-HSA, (MnP)9-HSA, and (MnP)20-HSA. [0037] Figure 8 shows in-vivo T1-weighted spin-echo images of healthy rats injected with (MnP) _n -HSA agents with either 3.5 or 6 MnP bound to each HSA protein with associated tables containing tissue T1 and T2 relaxation times. [0038] Figure 9 shows the chemical reaction of conjugation of MnP-NCS with APO- NH ₂ to form the fibrosis agent MnP-APO. APO-NH ₂ is unmetallated MnP-NH _2. [0039] Figure 10 Mass Spectrum obtained for MnP-APO with an Agilent 6538 Q-TOF system in ESI MS Negative mode. MS (ESI) m/z calcd for [M-2H] ² C89H56MnN10O18S7 ^2- : 915.56. Found: 915.56. Calcd for [M-3H] ^3- C ₈₉ H ₅₄ MnN ₁₀ O ₁₈ S ₇ ^3- : 610.0363. Found: 610.0366. [0040] Figure 11 is a plot of porphyrin-bound collagen to collagen ratio versus the concentration of free porphyrin (uM) and shows the binding of MnP-APO (circle) and MnP-NH2 porphyrin precursor (triangle) to type I rat collagen at 37 ^o C, demonstrating specificity of fibrosis targeted MRI agent MnP-APO for collagen I. [0041] Figure 12 is a plot of T1 values (ms) versus contrast concentration (µM) obtained for collagen films incubated with MnP-APO (circle) or MnP-NH2 (square) solutions. [0042] Figure 13 shows T1-weighted SPGR images and T1 maps of mouse heart obtained post-injection of MnP-APO fibrosis agent in healthy and isoproterenol-treated mice with heart failure. [0043] Figure 14 depicts T1 (ms) values measured in mouse hearts obtained post- injection of MnP-APO fibrosis agent in healthy and isoproterenol-treated mice with heart failure. [0044] Figure 15 shows Mass Spectrum obtained for MnP-NO with an Agilent 6538 Q- TOF system in ESI MS Negative mode. MS (ESI) m/z calculated for [M-1H] ^1- C57H45MnN8O11S4-: 1200.15. Found 1200.15. MS (ESI) m/z calculated for [M2-6H] ⁴ ((C ₅₇ H ₄₃ MnN ₈ O ₁₁ S ₄ ) ₂ ) ^4- : 500.0663. Found: 599.0669. MS (ESI) m/z calculated for [M+ acetate- 2H] ^2- ((C59H47MnN8O13S4) ^2- : 629.08. Found: 629.08. Mass spectrum is consistent with dimers formed by sulfonatophenyl metalloporphyrins. [0045] Figure 16 shows T ₁ reduction of collagen gels due to binding with MnP-NO agent. Activation of MnP-NO is dependent on concentration of NO donor Diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate. [0046] Figure 17 shows specificity of MnP-NO agent to nitric oxide activation over other endogenous radical species. Post-filtration T1 is lowest in the presence of nitric oxide, indicating greatest binding to MnP-NO agent in the presence of nitric oxide. [0047] Figure 18 shows that the MnP-NO agent is activated by both (1) use of a nitric oxide donor Diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate and (2) macrophage-like cells stimulated with lipopolysaccharide (LPS). Bound proteins separated on SDS page gel due to binding to MnP-NO agent shows reduced T1 relaxation time. [0048] Figure 19 shows a representative T ₁ map demonstrating lower T ₁ (ms) in the inflamed myocardium of isoproterenol-treated mice after administration of MnP-NO agent. Reduced T ₁ is seen on Day 2; T1 returns to baseline at later imaging timepoints. [0049] Figure 20 shows that the MnP-NO agent provides significant T ₁ (ms) reduction in the inflamed myocardium of isoproterenol-treated mice compared to healthy control mice. [0050] Figure 21 Mass Spectrum obtained for MnP-S-BP with an Agilent 6538 Q-TOF system in ESI MS Negative mode. MS (ESI) m/z calculated for [M2-2Na] ⁴ ((C ₆₃ H ₄₄ MnN ₅ Na ₂ O ₁₀ S ₄ ) ₂ ) ^4- : 629.56. Found: 629.57. MS (ESI) m/z calculated for [M- 2H] ^2- C63H45MnN5O10S4-: 607.0720. Found 607.0718. MS (ESI) m/z calculated for [M- 3H] ^3- C ₆₃ H ₄₄ MnN ₅ O ₁₀ S ₄ -: 404.38. Found 404.38. MS (ESI) m/z calculated for [M ₂ -H] (C63H46MnN5Na2O10S4)-: 1215.15. Found:1215.15. [0051] Figure 22 Mass Spectrum obtained for MnP-O-BP with an Agilent 6538 Q-TOF ((C ₆₃ H ₄₄ MnN ₅ Na ₂ O ₁₁ S ₃ ) ₂ ) ^4- : 621.57. Found: 621.58. MS (ESI) m/z calculated for [M- 2H ] ^2- C ₆₃ H ₄₅ MnN ₅ O ₁₁ S ₃ -: 599.0826. Found 599.0835. MS (ESI) m/z calculated for [M- 3H] ^3- C ₆₃ H ₄₄ MnN ₅ O ₁₁ S ₃ -: 399.05. Found 399.05

[0052] Figure 23 shows Ti-weighted SPGR images and Ti Maps of mouse aorta (arrow) obtained post-injection of our blood-pool agent MnP-O-BP.

[0053] Figure 24 compares Tl relaxation times (ms) of mouse aorta obtained after i.v. injection of blood-pool agent MnP-O-BP or unfunctionalized MnTPPS4.

[0054] Figure 25 shows graph tracking Mn concentration in organs measured by ICP over 7 days after administration of MnP-O-BP

[0055] Figure 26 shows mass Spectrum of MnP conjugated to carboxylate of stearic acid obtained with an Agilent 6538 Q-TOF system in ESI MS Negative mode. MS (ESI) m/z calculated for [M2-6H] ^4- ((C ₆₂ H ₆₀ MnN ₅ O ₁₀ S ₃ ) ₂ ) ^4- : 592.6447. Found: 592.6464. Is consistent with dimers formed by sulfonatophenyl metalloporphyrins.

[0056] Figure 27 shows mass Spectrum of MnP conjugated to carboxylate of 3,5- Dihydroxycyclohexanecarboxylic acid obtained with an Agilent 6538 Q-TOF system in ESI MS Negative mode. MS (ESI) m/z calculated for [M-1H] ¹ " C ₅₁ H ₃₈ MnN ₅ O ₁₂ S ₃ - : 1063.11. Found 1063.11. MS (ESI) m/z calculated for [M2-6H] ^4- ((C ₅₁ H ₃₆ MnN ₅ O ₁₂ S ₃ ) ₂ ) ^4- : 530.5457. Found: 530.5451. MS (ESI) m/z calculated for [M+ acetate- 2H] ^2- (C ₅₃ H ₄₀ MnN ₅ O ₁₄ S ₃ ) ^2- : 560.56. Found: 560.56. Mass spectrum is consistent with dimers formed by sulfonatophenyl metalloporphyrins.

[0057] Figure 28 shows Mass Spectrum of MnP conjugated to carboxylate of deoxycholic acid obtained with an Agilent 6538 Q-TOF system in ESI MS Negative mode. MS (ESI) m/z calculated for [M - 2H]- C ₆₈ H ₆₇ MnN ₅ O ₁₂ S ₃ ^2- : 1296.33. found: 1296.32. Calculated for [M - 2H] ^2- C ₆₈ H ₆₆ MnN ₅ O ₁₂ S ₃ ^2- : 646.66. found: 646.65. Calculated for [M - 3H] ^3- C ₆₈ H ₆₄ MnN ₅ O ₁₂ S ₃ ^3- : 431.10. found: 431.10. With acetic acid C ₇₀ H ₆₈ MnN ₅ O ₁₄ S ₃ ^2- Calculated 1353.33 found 1353.33.

[0058] Figure 29 shows Ti-weighted SPGR images and Ti maps of mouse aorta obtained post-injection of our blood-pool agent MnP -Deox.

[0059] Figure 30 compares Tl relaxation times (ms) of mouse aorta obtained after i.v. injection of blood-pool agent MnP -Deox or unfunctionalized MnTPPS4. [0060] Figure 31 shows graph tracking Mn concentration in organs measured by ICP over 7 days post-MnP-Deox administration.

[0061] Figure 32 shows mass spectrum of thiocarbamate product from MnP conjugation to alcohol of 3, 5 -Dihydroxycyclohexanecarboxylic acid obtained by Waters ACQUITY H-class UHPLC system with PDA detector and single quadrupole MS detector in positive mode. MS (ESI) m/z calcd for [M] ⁺ C ₅₂ H ₃₉ MnN ₅ O ₁₃ S ₄ ⁺ : 1124.08. Found: 1124.47.

[0062] Figure 33 shows mass spectrum of carbamate product from MnP conjugation to alcohol of 3, 5 -Dihydroxy cyclohexanecarboxylic acid obtained by Waters ACQUITY IT- class UHPLC system with PDA detector and single quadrupole MS detector in positive mode. MS (ESI) m/z calcd for [M] ⁺ C ₅₂ H ₃₉ MnN ₅ O ₁₄ S ₃ ⁺ : 1108.10. Found: 1108.54.

[0063] Figure 34 shows the Mass spec ESI of MnP -Deox product conjugated via the alcohol obtained with an Agilent 6538 Q-TOF system in ESI MS Negative mode. MS (ESI) m/z calculated for [M2-6H] ^4- ((C ₆₉ H ₆₄ MnN ₅ O ₁₃ S ₄ )2) ^4- : 676.6387 Found: 676.6392. MS (ESI) m/z calculated for [M2-8H] ^4- ((C ₆ 9H ₆ 3MnN ₅ O ₁₃ S ₄ ) ₂ ) ^6- : 450.76. Found: 450.76. Mass spectrum is consistent with dimers formed by sulfonatophenyl metalloporphyrins.

[0064] Figure 35 graphs the percentage of porphyrin bound to collagen versus time to assess the leaching of different porphyrins from collagen gels after in-situ labeling.

[0065] Figure 36 shows Ti and T2 maps obtained for collagen gels labeled with MnP- Col and collagen controls at 5 and 60 days.

[0066] Figure 37 shows Ti and T2 values measured for collagen gels labeled with MnP- Col and collagen controls at 5 and 60 days.

[0067] Figure 38 shows the fabrication of MnP -labeled polyurethane hydrogel discs where MnP is bound to hydrogel by UV -catalyzed vinyl polymerization and hydration in saline.

[0068] Figure 39 graphs the percentage of porphyrin leached from labeled polyurethane hydrogel discs versus time. Stability of MnP labeling was measured by UV-vis spectroscopy, where non-covalently bound MnP (square) and covalently bound MnP- NCS (circle) leached out of hydrogel are compared after incubation in saline at 37°C, n=3. [0069] Figure 40 shows the T1 map of MnP-labeled polyurethan hydrogel discs in a 12- well plate phantom. Discs were labeled with either 2 or 4 ppm acrylate-functionalized manganese porphyrin.

[0070] Figure 41 shows in-vivo MRI of polyurethane hydrogel implants of MnP-labeled and unlabeled implants. Ti maps, Ti-weighted spoiled gradient echo (SPGR) with 10° flip angle, Ti-weighted spin echo (SE), and T2-weighted turbo spin echo (TSE) in the sagittal orientation are shown at 1 and 7 weeks post-implantation.

[0071] Figure 42 graphs the mean Ti (ms) and standard deviation of Ti (ms) versus time, demonstrating in-vivo MRI tracking of MnP-labeled polyurethane hydrogel degradation in rats

[0072] Figure 43 graphs Ti value (ms) versus time to demonstrate in-vitro stability of TI values in labeled alginate (N=3) in PBS at 37°C.

[0073] Figure 44 shows Ti -weighted SE and Ti maps obtained from longitudinal rat imaging over 30 days of implanted MnP-NCS labeled alginate/chitosan gel and unlabeled control. Ti-weighted SE images were used to localize implants while Ti Maps were used to calculate Ti reduction from MnP-NCS labeling.

[0074] Figure 45 graphs T i value (ms) versus time to assess stability of T i reduction over one month from MnP labeling in all labeled alginate/chitosan gel implants and controls. MnP-NCS labeled alginate (MnP-Alginate) produced significant contrast in comparison to control, which it maintained over 30 days.

[0075] Figure 46 summarizes the available chemistry for MnP-NCS and shows the chemical versatility of MnP-NCS platform.

DETAILED DESCRIPTION

[0076] Various embodiments and aspects of the disclosure will be described with reference to details discussed below. Numerous specific details are described to provide a thorough understanding of various embodiments of the present disclosure. However, in certain instances, well-known or conventional details are not described in order to provide a concise discussion of embodiments of the present disclosure. Definitions

[0077] As used herein, the terms, “comprises” and “comprising” are to be construed as being inclusive and open ended, and not exclusive. Specifically, when used in this specification including claims, the terms, “comprises” and “comprising” and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps, or components.

[0078] As used herein, the term “exemplary” means “serving as an example, instance, or illustration,” and should not be construed as preferred or advantageous over other configurations disclosed herein.

[0079] As used herein, the terms “about” and “approximately”, when used in conjunction with ranges of dimensions of particles, compositions of mixtures or other physical properties or characteristics, are meant to cover slight variations that may exist in the upper and lower limits of the ranges of dimensions so as to not exclude embodiments where on average most of the dimensions are satisfied but where statistically dimensions may exist outside this region. Unless otherwise specified, the terms “about” and “approximately” mean plus or minus 25 percent or less. It is not the intention to exclude embodiments such as these from the present disclosure.

[0080] It is to be understood that unless otherwise specified, any specified range or group is as a shorthand way of referring to each and every member of a range or group individually, as well as each and every possible sub-range or sub-group encompassed therein and similarly with respect to any sub-ranges or sub-groups therein. Unless otherwise specified, the present disclosure relates to and explicitly incorporates each and every specific member and combination of sub-ranges or sub-groups.

[0081] As used herein, the term "on the order of', when used in conjunction with a quantity or parameter, refers to a range spanning approximately one tenth to ten times the stated quantity or parameter.

[0082] As used herein, phrase “small molecule” refers to a water-soluble molecule which can ideally exit the vasculature and enter the extravascular space. Ideally, this molecular weight would be under 5 kDa, but could go up to 50 kDa and can include biomolecules which are not saccharides, proteins, or lipids.

[0083] As used herein, phrase “biological targets” refers to specific molecules whose presence is specifically associated and indicative an endogenous environment or process. This can include differential presence in tissues and their compartments, metabolic processes, pathologies, and cell types.

[0084] The following description of the embodiments of the disclosure has been presented to illustrate the principles of the disclosure and not to limit the disclosure to the particular embodiment illustrated. It is intended that the scope of the disclosure be defined by all of the embodiments encompassed within the following claims and their equivalents.

Compounds

[0085] The present disclosure provides an MRI contrast agent comprised of an MRI- active manganese porphyrin (MnP) connected to a linker and functionalized R group.

[0086] The compound disclosed herein and shown in Formula (I) is an MRI-active manganese porphyrin (MnP) connected to an R group via a linker (-NHC(=E)Z-). Specificity is achieved via the R group, which are molecules with specificity for different biological targets, including specific tissue or cell types, or biological processes. Targeting can be based on physical affinity or exclusive chemical activation by an endogenous substrate. Possible R groups include proteins, small peptides, antibodies, glycoproteins, glycosaminoglycans, polysaccharides, lipids steroids, drugs, and small molecules possessing at least one nucleophile, including primary and secondary amines, alcohols, thiols, or carboxylic acids. The target itself can also be a protein, small peptide, antibody, glycoprotein, glycosaminoglycan, polysaccharide, lipid, steroid, and metabolite. When MnP is conjugated to a specific R group, the conjugated Ti MRI contrast agent achieves similar affinity or chemical reactivity to these biological substrates. The R group -conjugated MnP is, thus, able to accumulate at a particular substrate, producing a Ti reduction and enabling qualitative and quantitative imaging of specific biomarkers associated with biological function and disease.

[0087] In addition to targeting biological substrates, materials can also be targeted. In this case, R groups are synthetic or natural materials, soluble polymers, dendrimers, nanoparticles, or reagents for synthesizing materials and nanoparticles. These materials can be used for implantable prosthetics, cellular and acellular scaffolds for tissue engineering, and materials for drug delivery, to name a few. Conjugating MnP to these R groups enables in-vivo MRI of materials - as the MnP -conjugated R group is an integral component of the material, MRI contrast will accurately reflect spatial location and extent, material dimensions, and material density.

[0088] Linker selection is dependent on the initial nucleophile (alcohol, amine, carboxylic acid, thiol) available for conjugation on the R group, and the reaction conditions, see Figure 1. The presence of a flexible linker offers several advantages over directly attaching the R group to the porphyrin. First, flexibility of the linker bridge prevents the rigid nature of the MnP porphyrin from interfering with the R group. By allowing the R group to freely rotate and bend, the bridge better conserves the free steric effects necessary for R group affinity to a substrate. This is also important when the R group is part of a material, as increased rigidity of the R group can interfere with the molecular structure of the material, resulting in undesirable mechanical alterations. A flexible linker minimizes these mechanical changes by enabling proper orientation of molecules and minimizing steric interference from MnP-R inflexibility.

[0089] Finally, for bulky R groups, a rigid MnP-R structure would impede its ability to reach molecular targets; a flexible linker is needed to minimize the molecular radius and allow the MnP-R to adopt multiple orientations, facilitating transport from blood into the extravascular space and then through the extracellular matrix to the intended target.

[0090] In a first aspect, the present disclosure provides a compound of Formula (I): or a pharmaceutically acceptable salt or a tautomer thereof, wherein: E is NH, O, or S;

Z is absent, CH2, NH, O, or S; and

R is a targeting group. In some embodiments, Z is CH2, NH, O, or S. Z is absent, NH, O, or S.

[0091] In some embodiments, E is S and the compound is a compound of Formula (la) or a pharmaceutically acceptable salt or a tautomer thereof. In some embodiments of the compounds of Formula (la), Z is NH, O, or S.

[0092] In some embodiments, E is O and the compound is a compound of Formula (lb)

or a pharmaceutically acceptable salt or a tautomer thereof. In some embodiments of the compounds of Formula (lb), Z is absent, CH2 or O.In some embodiments of the compounds of Formula (lb), Z is CH2 or O. In some embodiments of the compounds of Formula (lb), Z is absent or O.

[0093] In some embodiments, E is NH, and the compound is a compound of Formula (Ic) or a pharmaceutically acceptable salt or a tautomer thereof. For example, in some embodiments of Formula (Ic), E is protonated and is in the form of NH2 ⁺ . In some embodiments of the compounds of Formula (Ic), Z is NH.

[0094] In some embodiments, R is or comprises a protein, small peptide, antibody, glycoprotein, glycosaminoglycan, polysaccharide, dendrimer, lipid, steroid, drug, polymer, acrylate, or small molecule.

[0095] In some embodiments, the compound of Formula (I) is obtained from the reaction between MnP-NCS and R’, wherein R’ is a protein, small peptide, antibody, glycoprotein, glycosaminoglycan, polysaccharide, dendrimer, lipid, steroid, drug, polymer, acrylate, or small molecule possessing at least one nucleophile. In some embodiments, the at least one nucleophile of R’ may include at least one group selected from primary amines, secondary amines, aromatic amines, primary alcohols, secondary alcohols, aromatic alcohols, thiols and carboxylic acids, or a combination thereof. In some embodiments, R’ is a carboxylic acid which is acrylate, and R is an alkene. In some embodiments, the at least one nucleophile of R’ may include at least one group selected from primary amines, secondary amines, aromatic amines, primary alcohols, secondary alcohols, aromatic alcohols, and thiols, or a combination thereof, wherein after reaction with MnP-NCS, the amine nitrogen, alcohol oxygen, or thiol sulfur atom of the nucleophile of R’ corresponds to Z of the linker of the compound of Formula (I), (la), (lb), or (Ic), and the remainder of R’ corresponds to the structure of R.

[0096] When R is described as a particular structural moiety, such as a protein, small peptide, antibody, glycoprotein, glycosaminoglycan, polysaccharide, dendrimer, lipid, steroid, drug, polymer, acrylate, or small molecule which is conjugated to MnP-NCS via an amine, alcohol, or thiol, it is to be understood that an amine nitrogen, alcohol oxygen, or thiol sulfur atom of the moiety corresponds to Z of the linker, and the remainder of the moiety corresponds to R. For example, it is to be understood that “R is 4,4- diphenylcyclohexan-l-ol” is interpreted to mean that Z of Formula (I) is O, and R is [0097] When R is described as a particular structural moiety, such as a protein, small peptide, antibody, glycoprotein, glycosaminoglycan, polysaccharide, dendrimer, lipid, steroid, drug, polymer, acrylate, or small molecule which is conjugated to MnP-NCS via a carboxylate, it is to be understood that the CH ₂ adjacent to the carboxylate may correspond to Z, and the remainder of the moiety may correspond to R. For example, it (which has the formula CH ₃ (CH ₂ ) ₁₆ CO ₂ H) may be interpreted as Z of Formula (I) is CH2, and R is the aliphatic chain of stearic acid less one methylene group (i.e., CH ₃ (CH ₂ ) ₁₅ ). [0098] When R is described as a particular structural moiety, such as a protein, small peptide, antibody, glycoprotein, glycosaminoglycan, polysaccharide, dendrimer, lipid, steroid, drug, polymer, acrylate, or small molecule which is conjugated to MnP-NCS via a carboxylate, it is to be understood that Z may be absent, and the remainder of the moiety excluding the carboxylate may correspond to R. For example, it is to be understood that (which has the formula CH3(CH2)16CO2H) may be interpreted as Z of Formula (I) is absent, and R is the aliphatic chain of stearic acid (i.e., CH ₃ (CH ₂ ) ₁₆ ). [0099] In some embodiments, R is or comprises an antibody/glycoprotein with specificity for a biological antigen, and the linker is a thiourea linker wherein E is S and Z is NH, or the linker is guanidine wherein E is NH, or the linker is guanidinium wherein E is NH2 ⁺ . [0100] In some embodiments, R is albumin, and the linker is a thiourea linker, wherein E is S and Z is NH. [0101] In some embodiments, R comprises a small molecule or a drug. [0102] In some embodiments, R comprises a metal-free porphyrin. In some embodiments, the metal free porphyrin may be any one of 10,15,20-(tri-4- sulfonatophenyl)porphyrin, (di-4-sulfonatophenyl)porphyrin, (4- sulfonatophenyl)porphyrin, uroporphyrinogen I and III, hematoporphyrin, photofrin and Tetra(4-carboxyphenyl)porphyrin), benzoporphyrin, porphycene, corrin, corphin, sirohydrochlorin, chlorin, phthalocyanine, hydrazine formyl-hydrazine, 1-(4-Amino-1- piperazine), or (R)-2-amino-4-phenylbutanehydrazide. [0103] In some embodiments, R comprises a small molecule. In some embodiments, the small molecule comprises a 1,2-diamine-substituted aromatic compound. In some embodiments, the 1,2-diamine-substituted aromatic compound is optionally further substituted. In some embodiments, the 1,2-diamine-substituted aromatic compound is capable of benzotriazole formation in the presence of nitric oxide. In some embodiments, the small molecule comprises 1,2-diaminobenzene. In some embodiments, the small molecule comprises optionally substituted 1,2-diaminobenzene. In some embodiments, the small molecule is 1,2-diaminobenzene, optionally substituted by methyl. In some embodiments, the small molecule is 1,2-diaminobenzene optionally substituted at the 4- position (e.g., 4-methyl- 1,2-diaminobenzene). In some embodiments, R further comprises a spacer moiety between the linker and the small molecule. In some embodiments, the spacer moiety comprises an optionally substituted alkylene or heteroalkylene. In some embodiments, the spacer moiety comprises a heteroalkylene optionally substituted by oxo. In some embodiments, the spacer moiety comprises an optionally substituted alkylene or heteroalkylene having a linear chain length of 3 to 10 atoms. In some embodiments, the spacer moiety comprises an optionally substituted alkylene or heteroalkylene having a linear chain length of 6 atoms. For example, in one embodiment,

[0104] In some embodiments, R is a polysaccharide or a glycosaminoglycan, and the linker is thiocarbamate (where E is S and Z is O) or carbamate (where E is O and Z is O). In some embodiments, carboxylic acids of R remain free carboxylic acids, i.e. do not participate in the conjugation reaction (e.g. with the porphyrin). In some embodiments, the polysaccharide is a mannuronate and/or guluronate-based polysaccharide. In some embodiments, the polysaccharide is alginate. In some embodiments, the glycosaminoglycan is hyaluronic acid, chondroitin sulfate, dermatan sulfate, or heparin.

[0105] In some embodiments, R is a polysaccharide or a glycosaminoglycan, and of the linker is an amide linker where E is O and Z is CH2. In some embodiments, R is a polysaccharide or a glycosaminoglycan, and of the linker is an amide linker where E is O and Z is absent. In some embodiments, the polysaccharide is a mannuronate and/or guluronate based polysaccharide. In some embodiments, the polysaccharide is alginate. In some embodiments, the glycosaminoglycan is hyaluronic acid, chondroitin sulfate, dermatan sulfate or heparin. [0106] In some embodiments, R is or comprises 4,4-diphenylcyclohexan-l-ol, and the linker is thiocarbamate (where E is S and Z is O) or carbamate (where E is O and Z is O).

In some embodiments,

[0107] In some embodiments, R is comprises a steroid. In some embodiments, R is cholic acid, deoxycholic acid, glycocholic acid, glycolithocholic acid, glycochenodeoxycholic acid, lithocholic acid, chenodeoxycholic acid, taurochenodeoxycholic acid, taurocholic acid, or other bile acid derivative; and the linker is an amide linker where E is O and Z is absent.

[0108] In some embodiments, R is a protein or a polysaccharide, and the linker is a thiourea linker where E is S and Z is NH. In some embodiments, the protein is selected from the group consisting of collagen, silk, elastin, and any other protein-based compound. In some embodiments, the polysaccharide is chitosan.

[0109] In some embodiments, R is a polysaccharide or a glycosaminoglycan, and the linker is thiocarbamate (wherein E is S and Z is O) or carbamate (wherein E is O and Z is O). In some embodiments, the polysaccharide is any one of polyvinyl alcohol, cellulose, callose, chitin, dextran, cyclodextran, amylose, glucose, arabinose, ribose, threose, fructose, galactose, xylose, manose, maltose, lactose, talose, sucrose, pectin, amylopectin, carrageanan starch, and glycogen. In some embodiments, the glycosaminoglycan is heparin sulfate or keratan sulfate.

[0110] In some embodiments, R is a fatty acid, and the linker is an amide linker wherein E is O and Z is CH2. In some embodiments, the fatty acid is stearic acid or lauric acid.

[OlH] In some embodiments, R is a fatty acid, and the linker is an amide linker wherein E is O and Z is absent. In some embodiments, Z is absent and R is the aliphatic chain of stearic acid or lauric acid.

[0112] In some embodiments, R comprises an acrylate.

[0113] In some embodiments, R’ is an acrylate and R is an alkene. [0114] In some embodiments, R is 5 -hydroxyindole, 5-hydroxytryptamide, serotonin, serotonin hydrochloride, or 5-hydroxyindole-3 -acetic acid. In some embodiments, the compound of Formula (I) is for imaging myeloperoxidase activity.

[0115] In some embodiments, the compound of Formula (I) is selected from the compounds provided in Table 1.

Table 1

Functionalized Manganese Porphyrin Compounds (MnPs)

[0116] The present disclosure also provides a functionalized MRI-active manganese porphyrin (MnP) useful for preparing an MRI contrast agent disclosed herein. In some embodiments, the functionalized MnP is manganese (III) 5-(4-isothiocyanatophenyl)- 10,15,20-(tri-4-sulfonatophenyl)porphyrin (MnP-NCS) which has the following structure:

In some embodiments, provided is a manganese porphyrin complex selected from the group consisting of:

Mn(III) 5-(4-aminophenyl)-10, 15,20-(tri-4-sulfonatophenyl)porphyrin (MnP-NH ₂ )

Mn(III) 5,10,15,20-(tetra-4-sulfonatophenyl)porphyrin (Mn-TPPS4)

-(4-aminophenyl)-10,15,20-(tri-4-sulfonatophenyl)porphyrin (APO-NH2) 5-(4-isothiocyanatophenyl)-10,15,20-(tri-4-sulfonatophenyl)p orphyrin (APO-NCS)

Use as an MRI contrast agent

[0117] In another aspect of the invention, provided is use of the compound of Formula (I) as a manganese (Mn) based MRI contrast agent. In some embodiments, the use of some of the compounds may be as a contrast agent in specific MR imaging of selected antigen. In some embodiments, the use of some of the compounds may be as an intravascular contrast agent in MR angiography.

[0118] In some embodiments, the use of some of the compounds may be as a contrast agent in MR imaging of collagenous, elastic, and fibrotic tissue, wherein the MR imaging of collagenous, elastic, and fibrotic tissue is used to the diagnosis or study of associated pathologies including heart disease, kidney disease, liver disease, bowel disease, vascular disease, brain disease and injury, and cancer.

[0119] In some embodiments, the use of some of the compounds may be as a contrast agent in MR imaging of nitric oxide production, wherein the nitric oxide production is produced from iNOS, eNOS, and nNOS. In some embodiments, the MR imaging of nitric oxide production is used for the diagnosis or study of inflammatory associated with infection, heart disease, kidney disease, liver disease, bowel disease, arthritis and cancer. [0120] In some embodiments, the use of some of the compounds may be as a contrast agent in MR imaging in the fabrication of MR trackable implants, hydrogels, nanoparticles and polymers.

[0121] In some embodiments, the use of some of the compounds may be as a contrast agent in MR imaging for MR trackable drug delivery. The compound may be used for the fabrication of MR active nanoparticles for intravascular imaging.

[0122] In some embodiments, the use some of the compounds may be as MR active micelles for MR angiography with longer blood-pool retention.

[0123] In some embodiments, the use of some of the compounds may be for the preparation of MR labeled polymers produced from free-radical polymerization.

[0124] In some embodiments, the use of some of the compounds may be as a contrast agent in MR imaging for the study and diagnosis of inflammatory associated with infection, heart disease, kidney disease, liver disease, bowel disease, arthritis, and cancer.

Method of synthesis of compounds of Formula (I)

[0125] In some embodiments, the structures are synthesized as follows.

When E = S and Z = NH, O or S (Compound of Formula (la)

[0126] Conjugation of MnP-NCS to amines (primary, secondary, aromatic) results in a thiourea product where Z = NH; conjugation of MnP-NCS to thiols results in a dithiocarbamate product where Z = S; and conjugation of MnP-NCS to alcohols (primary or secondary) results in a thiocarbamate where Z = O. When the R-Group possesses only a single one of these nucleophiles for conjugation with MnP-NCS, either reagent may be used at a stoichiometric excess, e.g., under 10 times excess. In some embodiments, if each individual R-group possesses multiple potential sites of conjugation, a stoichiometric excess of MnP-NCS may be used. Selection of the stoichiometric excess depends on both the number of relevant nucleophilic sites available on the R-Group and the desired degree of conjugation ratio of MnP to R-Group. For all conditions, MnP-NCS and the R-group reagents may be dissolved in any order under vigorous stirring and left until the reaction is judged to be complete with monitoring by chromatographic means.

[0127] An aqueous route of synthesis may be used in the cases where the R-group is only soluble or stable in water. In these cases, an inorganic base like sodium hydroxide, calcium hydroxide, or a carbonate buffer is added to the stirred reaction solution to increase alkalinity of solution. In some embodiments, the inorganic base is a carbonate. In some embodiments, the final pH < 9.5. In some embodiments, the pH can be lowered to 8, e.g., to prevent denaturation of more sensitive R-Group (proteins). In some embodiments, reactions can be conducted on ice. In some embodiments, reactions can be performed at room temperature. Reaction times can also range from 15 minutes to no more than 24 hours. When products are small molecular mass (<I0 kDa) they can be purified by affinity chromatography. When products have an intermediate or large molecular mass (>5000 kDa) they may be purified by methods of size exclusion including chromatography, spin filtration or dialysis.

[0128] In some embodiments, non-aqueous means of conjugation are used, e.g., to minimize reaction with water and take advantage of the versatility of MnP-NCS solubility. In some embodiments, the synthesis is performed in dimethylacetamide. In some embodiments, the synthesis can include other polar aprotic solvents e.g. dimethylformamide, dimethyl sulfoxide and acetonitrile. In some embodiments, prior to reaction MnP-NCS is dried under vacuum at an elevated temperature <100°C for at least an hour to remove coordinated water. In some embodiments, the R-groups and solvents are dried prior to use. In some embodiments, reactions are performed under a dry inert atmosphere, e.g. Nitrogen or Argon at room temperature.

[0129] The reaction begins once MnP-NCS and the R-group are mixed in any order under stirring and may include the addition of a catalyst depending on nucleophile for conjugation on R-Group. In the case of an R-group possessing a thiol, alcohol (primary) or amine (primary, secondary, or aromatic), no additional base or catalyst is required when the R-group is used at a stoichiometric excess of 5 to 10 times.

[0130] In some embodiments, if the stoichiometric excess of the R-group is under 5 times, or the amine is in the form of a protonated salt (like hydrochloride), a non- nucleophilic base is added. In some embodiments, the non-nucleophilic base is Triethylamine or N,N-Diisopropylethylamine. In some embodiments, the non- nucleophilic base is a stronger base, e.g. l,8-Diazabicyclo[5.4.0]undec-7-ene. In some embodiments, bases are added in excess, e.g. at a 4x stoichiometric excess to MnP-NCS. Reaction times are most commonly between 15 to 60 minutes but may approach but do not generally exceed 24 hours. In most cases, the first step of purification from nonaqueous synthesis involves precipitating the porphyrin product into a solvent, e.g. ether, acetone or ethyl acetate. If significant porphyrin remains dissolved in organic phase, acetic acid can be incrementally added until all of the porphyrin product is precipitated. Following this, products can undergo previously mentioned methods of purification.

When E = S and Z = O

[0131] In some embodiments, when conjugation to the R-group is done via an alcohol, e.g. a secondary alcohol, the synthesis includes use of l,8-Diazabicyclo[5.4.0]undec-7- ene as a base. In some embodiments, the l,8-Diazabicyclo[5.4.0]undec-7-ene is used at an equivalent or small molar excess relative to MnP-NCS. In some embodiments, reactions are performed at about 60°C or lower. In some embodiments, reactions are performed at about 50°C. In some embodiments, reactions are performed at about 50°C and do not exceed about 8 hours.

When E = S and Z = O and R-Group contains 1 < carboxylic acids

[0132] In some embodiments, conjugation to the R-group is done via alcohol in the presence of carboxylic acids, and no base is added. In some embodiments, a strong protonated acid is added, e.g. to block reaction with carboxylates. In some embodiments, MnP-NCS itself provides the source of sulfonic acid. In some embodiments, MnP-NCS is provided as a blend of MnP-NCS as a sulfonic acid and sodium salt. In some embodiments, MnP-NCS is provided as a salt with the conjugate acid of non-nucleophilic amine bases, e.g. I,8-Diazabicyclo[5.4.0]undec-7-ene. In some embodiments, the reaction is performed at elevated temperatures of about 80°C. In some embodiments, reaction times are between about 6 and about 24 hours.

Method of synthesis of compounds of Formula (lb)

E = O and Z = O or absent

[0133] Similarly, to methods of synthesis for Formula (I), stoichiometries are determined based upon the nature of the R-group having either a single point for conjugation, or multiple points of conjugation. In some embodiments, synthesis is non-aqueous, e.g., with use of dimethylacetamide or DMSO, or other polar aprotic solvents like dimethylformamide or acetonitrile. In some embodiments, prior to reaction MnP-NCS is dried under vacuum at an elevated temperature, e.g., <100°C for at least an hour to remove coordinated water. In some embodiments, R-groups and solvents are dried prior to use and reactions are performed under a dry inert atmosphere, e.g. Nitrogen or Argon at room temperature. MnP-NCS and the R-group reagents are first mixed in any order under stirring with the reaction starting upon the addition of a catalyst. Purification procedures are identical to those outlined for the synthesis of Formula (I).

When E = O and Z = O

[0134] A carbamate linkage where Z = O can be produced directly in one step from the reaction of MnP-NCS with an R-group possessing an alcohol. Synthesis methods are nearly identical to the non-aqueous conjugation to alcohols outlined above for Formula (I). After the MnP-NCS and R-group reagents are stirring in reaction solution, catalytic quantities of dibutyltin dilaurate and a large excess non-nucleophilic amine base such as Triethylamine or N,N-Diisopropylethylamine are added at about 93°C and the reaction is left for at least 12 hours.

[0135] Alternatively, this product can be produced by the conversion of a thiocarbamate- linked MnP conjugate whose synthesis is describe above for Formula (I). This method requires two steps and provides greater yields and for when carboxylic acids are present on the R group. The first step follows the method described above for Formula (I) for conjugation to alcohols in non-aqueous conditions. Following this, conversion is effectively accomplished within 12 hours after increasing temperature above 80°C and adding excess non-nucleophilic amine base such as Triethylamine or N,N- Diisopropylethylamine or l,8-Diazabicyclo[5.4.0]undec-7-ene.

When E = O and Z = absent

[0136] An amide linkage where Z = CH3 is produced from the reaction of MnP-NCS with an R-group possessing a carboxylic acid or carboxylate salt. MnP-NCS and the R- Group reagent containing a carboxylic acid are first mixed together before an amine base is added like l,8-Diazabicyclo[5.4.0]undec-7-ene, TEA, DIPEA or other. If in the form of a carboxylate salt, no base is needed. Reaction time should exceed 1 hour but should not surpass 6 hours. Compatible temperatures range from about 0°C to about 93°C but are highly dependent upon the solubility behavior of the carboxylate R-Group. Ideally, reactions are performed at about 60°C for about 1 hour. Alternatively, MnP-NFE may be coupled with carboxylates in aqueous 2 -(N-morpholino)ethane sulfonic acid buffer with use of a coupling agent like l-Ethyl-3-(3dimethylaminopropyl)carbodiimide.

Methods of synthesis of Formula (Ic)

When E = NH and Z = NH [0137] The guanidine linkage where Z= NH is produced by the conversion of a thiourea linked MnP conjugate whose synthesis is described above for Formula (I). This conversion is simply achieved by stirring a thiourea linked MnP conjugate in a solution of concentrated ammonium acetate at 95°C.

[0138] The compound of Formula (I) is an MRI-active manganese porphyrin (MnP) connected an R group via a linker (-NHC(=E)Z-). Specificity is achieved via the R group, which are molecules with specificity for different biological targets, including specific tissue or cell types, or biological processes. Targeting can be based on physical affinity or exclusive chemical activation by an endogenous substrate. Possible R groups include proteins, small peptides, antibodies, glycoproteins, glycosaminoglycans, polysaccharides, lipids steroids, drugs, and small molecules possessing at least one nucleophile, including primary and secondary amines, alcohols, thiols, or carboxylic acids.

[0139] The target itself, to which the R-group binds, can also be a protein, small peptide, antibody, glycoprotein, glycosaminoglycan, polysaccharide, lipid, steroid, and metabolite. When MnP is conjugated to a specific R group, the conjugated Ti MRI contrast agent achieves similar affinity or chemical reactivity to these biological substrates. The R group -conjugated MnP is, thus, able to accumulate at a particular substrate, producing a Ti reduction and enabling qualitative and quantitative imaging of specific biomarkers associated with biological function and disease.

[0140] In addition to targeting biological substrates, materials can also be targeted. In this case, R groups are synthetic or natural materials, nanoparticles, or reagents for synthesizing materials and nanoparticles. These materials can be used for implantable prosthetics, cellular and acellular scaffolds for tissue engineering, and materials for drug delivery, to name a few. Conjugating MnP to these R groups enables in-vivo MRI of materials - as the MnP-conjugated R group is an integral component of the material, MRI contrast will accurately reflect spatial location and extent, material dimensions, and material density.

[0141] The linker selection is dependent on the initial nucleophile (alcohol, amine, carboxylic acid, thiol) available for conjugation on the R group, and the reaction conditions as illustrated in Figure 1. The presence of a flexible linker offers several advantages over directly attaching the R group to the porphyrin. First, flexibility of the linker bridge prevents the rigid nature of the MnP porphyrin from interfering with the R group. By allowing the R group to freely rotate and bend, the bridge better conserves the free steric effects necessary for R group affinity to a substrate. This is also important when the R group is part of a material, as increased rigidity of the R group can interfere with the molecular structure of the material, resulting in undesirable mechanical alterations. A flexible linker minimizes these mechanical changes by enabling proper orientation of molecules and minimizing steric interference from MnP-R inflexibility. Finally, for bulky R groups, a rigid MnP-R structure would impede its reaching molecular targets; a flexible linker is needed to minimize the molecular radius and allow the MnP- R to adopt multiple orientations, facilitating transport from blood into the extravascular space and then through the extracellular matrix to the intended target.

[0142] Functionalization of porphyrins and photodynamic therapy agents involve functionalizing apo-porphyrins with electrophilic groups for conjugation to biomolecules (Pathak et al., 2021). These porphyrins are metal-free, and functionalization of nucleophiles into electrophiles is easily achieved. Analogous conversions were observed to be unsuccessful on a porphyrin with a manganese core. It is understood that metal- mediated withdrawal of electron density would subsequently deactivate peripheral nucleophiles, making them less amendable to conversion and activation to electrophiles. For example, when a nucleophilic amine of MnP-NFF was activated to an electrophilic succinimidyl ester, unsuccessful results were obtained. Also unsuccessful were mild conditions and use of 1 , 1 '-thiocarbonyldi2( lH)-pyridone for converting MnP-NFF amine to an isothiocyanate, strategies used for un-metalated porphyrins. Much harsher conditions and a large excess of thiophosgene were used to convert MnP-NFF amines to isothiocyanates. Importantly, Mn insertion must precede conjugating the porphyrin to the R group, because the harsh reaction required will cleave linkages and many potential R groups.

[0143] It is noteworthy that despite scarce effort on conjugating metalated porphyrins (particularly Mn) to R groups with affinity to biological targets, in the work that has been published (Giuntini et al., 2011), the focus was not on MRI but on a superoxide dismutase mimic or mutagenic drug. Importantly, the porphyrins functionalized for use in photodynamic therapy, superoxide dismutase mimicry, or as mutagenic drugs are designed to be cell permeable. The conjugative methods used for these compounds are not suitable for the sulfonated porphyrins of the present disclosure, because sulfonates eliminate cell permeability, handicap radical generation, and possess electrostatics ill- suited to superoxide dismutase mimics.

[0144] The present disclosure further introduces the sulfonated MnP as a conjugation platform for synthesizing targeted MRI probes. Its resistance to oxidation/reduction ensures the Mn(III) ion remains in the appropriate oxidation state for coordination stability and stable Ti relaxivity. Furthermore, the high polarity -3 charge on sulfonates ensures that MnP-R is water soluble and does not bio-accumulate or enter cells. This property prevents MnP-R from undergoing phase 1 metabolism that could hydrolyze linkages and potentially subject MnP-R to oxidation or reduction. Any free MnP-R is also excreted immediately by the kidneys and liver without modification. Table 2 lists without limitation various R groups suitable for the present MnP MRI agents

Table 2:

[0145] Amide, carbamate, and guanidine linkages are not traditionally considered a product of isothiocyanate conjugation. For example, carboxylic acids are not generally targeted because they are known to interfere with isothiocyanate conjugation. However, the present disclosure establishes that conjugating MnP-NCS with carboxylic acids produces amide linkages, as well as conjugating MnP-NCS with alcohols or amines will produce a carbamate or guanidine linkage, respectively, under certain conditions. Thiocarbamate linkages are uncommon, and despite a few examples in FITC, have not been reported in MRI The reaction conditions required to obtain these thiocarbamate linkages are simply incompatible with existing MRI platforms. Even more challenging is producing disubstituted thiocarbamates from NCS and a secondary alcohol. NCS reagents are known to be unreactive, even with use of catalysts. However, the present disclosure describes conjugating NCS to weaker nucleophiles. Furthermore, aryl isothiocyanates para-substituted with an electron-donating group (examples shown in Figure 2) to which all existing isothiocyanate bifunctional chelating agents (manganese or gadolinium) belong, are even less reactive than most isothiocyanates, because according to the Hammett equation, the electron donation and resonance stabilization stabilizes the isothiocyanate against nucleophilic attack. Due to this incompatibility, MRI metal chelates are not customarily conjugated to an alcohol by an isothiocyanate, nor do they usually possess a thiocarbamate linkage. Additionally, the pendant bases, like carboxylates, used by current bifunctional chelation methods for metal stabilization, interfere with conjugation to weaker nucleophiles like carboxylates and alcohols. However, in the present disclosure, are described conjugations for isothiocyanate- functionalized chelates. [0146] In a further embodiment, the present disclosure relates to the reactivity of the isothiocyanate on MnP-NCS. Un-metalated APO-NCS, despite possessing 3 electronwithdrawing sulfonates, is inferior to MnP-NCS for conjugating alcohols. This unexpected reactivity of the isothiocyanate on MnP-NCS is understood to be derived from metalating APO-NCS. Furthermore, linkages previously reported from alcohol conjugation by isothiocyanates in general may be inaccurate (Giuntini et al., 2011). It was observed that a carbamate linkage was the main product, not a thiocarbamate linkage as is previously reported. Lack of characterization underlies this inaccuracy in previous reports, as linker characterization is challenging on large macromolecules which are conjugated by isothiocyanate based fluorescence conjugated products like fluorescein isothiocyanate (FITC) conjugated dextran. Accordingly, the present disclosure is further directed to linkers resulting from MnP-NCS conjugation to alcohol.

[0147] Conjugating isothiocyanate with carboxylic acids has traditionally been difficult. This is because unprotected carboxylic acids do interfere with NCS conjugation. This interference also makes selective conjugation to alcohol in the presence of carboxylic acid challenging. Examples of NCS conjugation to an R group through anything except an amine when carboxylic acids are present are non-existent, if not rare, because the latter is known to cause interference. In fact, all current clinically used chelates use polyaminocarboxylate cores, and established NCS chemistry is restricted to the most reactive nucleophilic amines of lysine. The use of any weaker nucleophiles leads to significant competition from free carboxylates and reactions both intramolecularly and between NCS-functionalized chelates. The present disclosure demonstrates that the weakest nucleophiles (i.e. secondary alcohol) can be selectively targeted by NCS for conjugation over carboxylic acids without use of protective groups.

[0148] In a further embodiment of the present disclosure are described, nitric oxide (NO) agent and fibrosis agent. The NO agents of the present disclosure may possess an R group described herein for use in medical imaging. The fibrosis agents of the present disclosure also may contain R groups described herein conjugated to a probe for medical imaging or suggested for targeting collagen or fibrosis (although apo -porphyrins have been used as fluorescent stains in histology). In a further embodiment of the disclosure, a method is described for reducing Mn-sulfonated porphyrins’ affinity to collagen or tumors by inserting Mn into sulfonated porphyrins.

Approach to Conjugation [0149] Bioconjugation refers to the tagging of drugs or biomolecules such as polysaccharides, proteins, and antibodies, and is performed to enable specific detection, tracking, and even quantitation of biological targets. To date, only fluorescent imaging boasts an extensive toolkit of specialized agents and conjugated biomolecules that include proteins, carbohydrates, lipids, steroids, nucleotides, drugs, and both synthetic and natural biomaterials. No other imaging modality matches this massive library of imaging probes, because the extensive selection of conjugates and conjugative techniques available to fluorescent imaging is not available to radiochemistry and MRI contrast agents. This disparity is attributed to distinct platform -related limitations and to fundamental differences in the way each field approaches bioconjugation, which is outlined in Figure 3.

Pre-labeled conjugation

[0150] Fluorophores (e.g. fluorescein, rhodamine) are inherently fluorescent. For this reason, their functionalization and subsequent coupling can be considered a pre-labelled method of conjugation, as the inherently strong absorption and emission of fluorophores is transferred to downstream products and conjugates, thus eliminating the need for postconjugate activation. Purification of intermediate products is straightforward, owing to easy visualization of fluorophores on chromatography. Most importantly, fluorophores are compatible with virtually all conjugation reactions due to their remarkable inertness. The result is an extremely broad platform that is compatible with many functionalization techniques and reaction conditions without the need for protective groups. Dispensing with protecting group chemistry, fluorescent conjugation can be achieved by directly modifying the functional groups of biomolecules in their native forms.

[0151] Post-labeled conjugation

[0152] In contrast to fluorescent imaging agents, radiochemistry and MRI contrast agent chemistry traditionally use post-labeling methods to functionalize biomolecules. Bifunctional chelating agents (BFCA) are used to conjugate to biomolecules, analogous to conjugation of fluorophores to biomolecules. Unlike fluorophores, however, BFCAs do not have inherent radiological activity (for nuclear medicine) or paramagnetism (for MRI). Contrast generation is conferred to BFCAs only upon metalation, which is accomplished post-conjugation. The chelation of radiometals or paramagnetic metals serves another critical purpose: it “stabilizes” metals that are highly toxic in their free ionic form. In radiochemistry, where biomolecules undergo fluorination or become isotopic derivatives, the need for post-conjugation is obvious. Radioactive half-lives are so short that metalation must be performed right before their use in nuclear medicine imaging. MRI contrast agents are not limited by these constraints and, theoretically, can be pre-metalated before conjugation. However, because much of the chemistry for synthesizing and conjugating MRI contrast agents was adopted directly from radiochemistry as a consequence of sharing the same BFCA platforms, the benefits of pre-metalated conjugation have not been fully explored. Another culprit is the core polyaminocarboxylate structure used in BFCAs: it favors post-conjugation metalation.

[0153] The overall ramification on Gd chelate design is a greater emphasis on ease of metalation, reaction speed, and mild conditions - stability and chemical inertness are relegated. It is noteworthy that because BFCAs do not possess the chemical inertness of fluorescent platforms (they are extremely sensitive to chelating contaminant metals and are best used in as few reaction steps as possible), the onus of achieving chemical versatility and compatibility is placed, instead, on the target biomolecule. A suitable target must be modifiable and able to undergo multiple reaction steps before conjugation to BFCAs, a process that can substantially modify the biomolecule and its functional groups from their native form. Consequently, reaction procedures for BFCA conjugation and subsequent post-labelling are comparatively complex and tedious compared to prelabeled conjugation as depicted in Figure 3. Post-labeling of BFCA also leads to product heterogeneity and some toxicity risks. These issues arise from the conjugated R group interfering with the metalation process through steric blockage and competitive coordination of metals. This leads to quality control issues of batch variation and release of weakly coordinated metals posing issues of toxicity. Despite the benefits of the pre- metalated approach, it has been rarely used for BFCA due to chemical restrictions. All current BFCAs utilize pendant polyaminocarboxylates for metal coordination. Without the use of protective groups used during post-metalation conjugation, these pendant carboxylates act as competitive nucleophiles during conjugation to an R-group. This can lead to poor yields, mixed products, and destabilized chelates that pose risks of toxicity.

Gd chelation stability and Gd toxicity

[0154] While free Gd is highly toxic, the use of some non-specific Gd agents is still recognized as safe for use in healthy patients. Current Gd complexes are believed to have sufficient stability when the half-life of elimination is approximately 1.5 hours or less. However, renal impairment can lead to prolonged circulation and tissue retention of Gd contrast agents, extending elimination half-life to the range of 4 to 36 hours. By 2006, use of Gd-based contrast agents become contraindicated in patients with kidney disease due to nephrogenic systemic fibrosis (NSF). Consequently, use of most Gd agents that posed the highest risk of NSF were suspended at the recommendation of regulatory agencies in the United States and Europe. Those Gd based agents still acceptable for use must possess high stability and rapid elimination from the body. As a result, clinical translation of Gd-based targeted agents is currently hindered, because their mechanism of accumulation prolongs incubation time similarly to renal deficiency. For example, before the risks of NSF were known, the albumin -binding Ablavar was a targeted agent that was successfully adopted into the clinic. While there is still insufficient data on the risks of NSF posed by Ablavar, its parent DTPA later became suspended from use as an MRI agent due to risks of NSF. With the functionalization of MnDTPA for albumin binding, Ablavar extended its elimination half-life from 1.6 to 18.5 hours, surpassing the 11 -hour elimination half-life of MnDTPA in contraindicated patients with severe renal deficiency. With the risks associated with such prolonged incubation of Gd agents, targeted contrast agents must be both stable but remain non-toxic upon metal release.

New approach to MRI contrast agent design

[0155] The present disclosure is directed to manganese 5-(4-isothiocyanatophenyl)- 10,15,20-(tri-4-sulfonatophenyl)porphyrin as a superior electrophilic platform to replace the current polyaminocarboxylate-based BFCAs for the design and synthesis of new specialized T1 MRI contrast agents. This platform both resolves toxicity issues while enabling new chemistry not previously possible with current polyaminocarboxylate - based BFCAs. An exemplary suite of MRI contrast agents enabled by our new manganese porphyrin (MnP) platform is presented in Figure 4. The use of manganese (Mn) in place of Gd reduces all risks of toxicity associated with free metal release. This is because Mn is both an essential dietary mineral and is currently approved for intravenous use in its free ionic form as MnCh (LumenHance). Further, it does not pose as much of a risk as an environmental pollutant as Gd given its natural abundance in plants and animals. The use of a manganese 5-(4-isothiocyanatophenyl)-10,15,20-(tri-4- sulfonatophenyl)porphyrin (MnP-NCS) complex is ideal for the versatile design and synthesis of new MRI molecular agents. This platform affords several distinct advantages: (1) it is amenable to pre-metalated conjugation, (2) it is chemically inert and compatible with a much greater variety of synthesis conditions compared to current BFCAs, (3) it allows direct conjugation to biomolecules in a single step and dispenses with intermediate deprotection steps, and (4) the stability of Mn chelation under all reaction conditions tested ensures improved product homogeneity. In the present disclosure, it is understood that Mn complexation by MnP-NCS drastically increases the reactivity of the isothiocyanate with nucleophiles, enabling conjugation with even weak nucleophiles and without requiring a catalyst. This allowed the production of a variety of MRI contrast agents depicted in Figure 4. This disclosure further describes a MnP platform which exhibits unmatched and unexpected stability against the displacement of manganese under extreme acid and zinc stress testing (Vollett et al., 2023).

Conjugation to lysine for albumin labeling

[0156] Protein and peptide conjugation is the most ubiquitous use of Gd BFCAs. However, labeling of these large macromolecules has suffered from poor reaction efficiency. Further, the molecular complexity of these targets results in direct interference of the metalation process by blocking and competing for Gd coordination, leading to quality control and toxicity issues.

[0157] The present disclosure, however, provides a different outcome. In the present disclosure MnP-NCS is both capable and highly efficient for pre-metalated conjugation to HSA with higher conjugation efficiency than any previous platform using the reaction scheme depicted in Figure 5.

Conjugation to aryl amine for fibrosis targeting

[0158] Fibrosis is a pathology common to a myriad of conditions involving chronic inflammation and/or repeated tissue injury. It can arise in almost any organ and involves the replacement of healthy tissue with permanent scar made of connective tissue, primarily collagen I. Fibrotic changes are a hallmark of pulmonary fibrosis, liver cirrhosis, kidney disease, inflammatory bowel syndrome, vascular disease, and heart failure. Advanced fibrosis can lead to organ failure and even death. Early detection is, therefore, key to effective intervention. Non-invasive methods for detecting fibrosis are non-specific. Biopsy remains the gold standard and can differentiate among healthy tissue, old scar, and fibrogenesis. However, it is non-ideal and a non-invasive version of biopsy is the only solution to tracking disease longitudinally. Towards this goal, some collagen-specific gadolinium MRI contrast agents have been designed and evaluated in animals (Desogere et al., 2019). Yet, their indiscriminate binding to all types of collagen falls short of the desired ability to diagnose scar versus ongoing fibrotic disease.

Nitric Oxide Agent

[0159] Nitric oxide (NO) plays a critical role in the healthy function of organs but is also a marker of inflammation and can even provide a target for treating inflammatory disease . The far-ranging utility of NO imaging is seen in its diverse applications: neurodegenerative conditions such as Parkinson’s and ischemic brain injury, cardiovascular diseases like atherosclerosis, and both non-ischemic and ischemic heart failure, and others ranging from arthritis, gastrointestinal, lung, kidney, and liver disease. To date, NO imaging has been imaged almost exclusively with fluorescent dyes, which provide very limited penetration depths. To probe deep into the body, MRI probes sensitive to NO have also been reported, but their inherently low sensitivity, and requirement that they be delivered at the site of inflammation, precludes broad utility (Sharma et al., 2014). In the present disclosure is described a NO-responsive MRI agent that accumulates via covalent bonding with interstitial proteins in the presence of NO, thereby generating an MRI signal proportional to the local NO concentration. The present disclosure further describes the synthesis and in-vitro MRI demonstration of said MRI contrast agent (MnP-NO) for the molecular imaging of NO. The disclosure further shows that functionalization of MnP with a (2-amino-4-methylphenyl)carbamate enables in- situ, NO-activated conjugation of MnP to matrix proteins. The disclosure further describes a NO-responsive MRI agent which enables quantitative measurement of NO concentration in vivo.

Reaction with Sterically Hindered Alcohol for Reversible Albumin Binding

[0160] Water is the solvent of life and primary component of all living organisms. As most biomolecules are either product or participants in the predominantly aqueous environment, they must be sufficiently soluble to interface with water. For this reason, alcohol moieties (with their hydrophilic hydroxyl group) are ubiquitous in biology, with over 65% of biomolecules and 40% of current drugs possessing one or more hydroxyl groups. This makes establishing a method for one-step conjugation to alcohols by an electrophilic BFCA very desirable to minimize alterations to biomolecules and simplify synthesis. [0161] For the first test and demonstration of our MnP-NCS platform’s reactivity to secondary alcohols, MRI blood-pool agent is synthesized via conjugation with 4,4- diphenylcyclohexan-l-ol (BP) in a single step depicted in Figure 22 and 24. This albumin-binding moiety BP is currently used in the clinically approved gadolinium blood-pool agent Gadofosveset, which utilizes a diethylenetriaminepentaacetic acid chelate (Gd-DTPA). However, synthesis of Gadofosveset is laborious, requiring 5 to 6 steps to functionalize BP with the gadolinium chelate (McMurry, 1996). In addition to its laborious synthesis, Gadofosveset was pulled from the market a year after its core chelate structure, Gadopentetic acid, was recommended for suspension by the European Medicines Agency in 2017. The synthesis of a manganese porphyrin analogue using facile one-step conjugation shows the effectiveness of the method of preparation described herein. The resulting MnP-BP product has the benefit of easier synthesis, while the use of manganese for Ti relaxation helps reduce potential toxicity associated with Gd agents. This further acts as a model demonstration of how the facile conjugation of MnP- NCS can improve the MRI functionalization of currently utilized molecules possessing alcohols.

[0162] The synthesis of a blood-pool agent (MnP-BP) by conjugating MnP-NCS with the sterically hindered alcohol of 4,4-diphenylcyclohexan-l-ol without prior activation is described herein. In the present disclosure, it is also understood that chemical species of the linkage may be controlled with reaction conditions towards a thiocarbamate or carbamate.

Carboxylic acid conjugation

[0163] Carboxylic acids usually require activation before conjugation. This can be done in two ways. The first is a one-step procedure with the use of activating agents like carbodiimides, which enable its coupling with nucleophiles like amines. The second way is to convert carboxyl groups to electrophiles by functionalizing them into activated esters, like succinimidyl, or by their substitution with a good leaving group. Current BFCAs based upon polyaminocarboxylates cannot be pre-metalated during coupling, because the pendant carboxylates must be fully protected to prevent their unintentional coupling to nucleophiles and intramolecular cyclization. Thus, de-protection and metalation must always follow this form of conjugation. Additionally, this reaction is often extremely slow, a recent example being an amine-fimctionalized DOTA platform requiring several days.

[0164] While carboxylates are known to interfere with isothiocyanate conjugation, they are not usually targeted for conjugation by isothiocyanates. The disclosure describes a facile single-step method for direct conjugation to carboxylates without the need for activating agents or isocyanates. The reaction is extremely fast and efficient, achieving yields of 100% within just 1 hour to form an amide bond from MnP-NCS reaction with carboxylates Figure 29. While it is possible that this reaction can be utilized by current isothiocyanate BFCA platforms, it would still require deprotection and metalation steps to prevent loss of the pendant carboxylates to side reactions.

Conjugation to alcohols over carboxylates

[0165] Given the known interference of carboxylates with isothiocyanates and other electrophilic conjugation, specific conditions that favor conjugation to secondary alcohols over carboxylates without the need for additional steps to protect carboxylate groups are needed. The exclusive conjugation of MnP-NCS to alcohols over carboxylic acids could be achieved in the absence of base outlined in Figure 36 and Figure 37 to achieve either the thiocarbamate or carbamate product, respectively. Conjugation of MnP-NCS to alcohols in the presence of unprotected carboxylates is demonstrated for 3, 5 -Dihydroxycyclohexanecarboxylic acid and deoxycholic acid with resulting mass spectrums provided in Figure 38 and Figure 39.

Material Labeling

[0166] The study of biomaterials is fundamental to the field of tissue engineering and regenerative medicine. Advancement in this field has the potential of providing patients with an alternative to transplantation, one that uses healthy cells and structural matrix materials to regenerate new, healthy tissue. One of the greatest challenges facing this field, however, is the complex nuances tied to understanding how the body responds to these implants. The materials used must initially approximate the structural and mechanical properties of native tissue, but then dynamically adapt to in-vivo regeneration. Thus, longitudinal tracking of the implant is necessary for monitoring its dynamic response in the body. Response parameters to monitor include: material migration, structural changes, and material degradation (that will be accelerated by an inflammatory response to foreign material). The inflammatory response is what directs regeneration, and controlling this response is a necessity for successful scaffold integration. This response, however, is highly material-specific and can be modulated by many factors, such as the material chemical structure, its surface properties, and supplementation with drugs or cells. For example, the vast expanse in structural properties attempted by researchers - by changing scaffold pore size and shape, fiber diameter, and fiber orientation - also alter implant degradation, and it is impossible to predict its effect on in-vivo inflammatory response without a non-invasive method for material monitoring. Non-invasive methods for in-vivo material tracking are scarce but much needed. We propose that T1 -enhanced contrast-enhanced Tl-weighted MRI is ideal for non-invasive imaging of biomaterial implants.

[0167] The present approach as disclosed herein to functionalizing material with MRI contrast exploits bright (i.e., Ti) contrast, as Ti relaxation circumvents limitations of dark T2 contrast (including obliterating signal from surrounding anatomy, non-specific distinction amongst multiple endogenous sources of dark contrast). Therefore, a bright- contrast approach is the only one that supports high, accurate definition of material boundaries.

[0168] Another feature of the approach disclosed herein is to employ covalent conjugation, as this is necessary for achieving quantification of material degradation (and not to agent leaching from material). Currently, no Ti platform exists for material functionalization that is safe to use for long-term tracking. In fact, no known Ti conjugate has the chemical versatility to label most biomaterials. Many materials require conjugation through means that are incompatible with the current chemical capabilities of polyaminocarboxylate-based BFCAs. This incompatibility arises out of the necessity that BFCAs be pre-metalated before conjugation to materials. However, most of these reactions require protection of the pendant carboxylic acids until the final step, at which point metalation cannot be done.

Collagen Labeling

[0169] Collagen is the primary component of extra cellular matrix providing the structural element of most tissues and organs. Consequently, it is an important biomaterial of research for use in tissue implants and cellular scaffolds intended for regeneration. While FITC conjugated collagen I is commercially available for use in fluorescent tracking of collagen implants, it cannot provide adequate tracking in deep tissues due to the limited penetration depth of fluorescent imaging. The covalent labeling of collagen, like most proteins, is done through lysine coupling with an electrophile. While this is compatible with current Gd BFCAs, it is irreconcilable with post- conjugative metalation. The neutral conditions necessary for the metalation of BFCAs with GdC? would cause crosslinking and precipitation of acid-soluble collagen. Taking the approach of using a pre-metalated BFCA functionalized with an activated ester or isothiocyanate would bypass this problem and likely allow similar conjugation to collagen using similar established protocols for fluorescent agents. However, the final preparation of soluble labeled collagen requires extended time in acetic acid solution. These conditions are associated with the release of free Gd from chelates. Additionally, the prolonged incubation time expected for the degradation and clearance of collagen would likely extend the half-life of the Gd chelate well beyond what is acceptable.

[0170] The present disclosure describes a MnP-NCS platform that is compatible with existing collagen labeling protocols established for previous isothiocyanate functionalized fluorophores. Furthermore, the resulting NCS bond is stable at physiological conditions for a month.

Labeling during radical polymerization

[0171] In the instant disclosure, a Ti contrast agent is designed for labeling polymers that form via free radical polymerization (e.g. polyHEMA, poly(vinyl alcohol), poly(ethylene glycol), polyethylene, Teflon, and approximately half of polymers or synthetic rubbers). The distinctive feature of our new MnP is the ability to covalently label a growing polymer chain during polymer propagation to produce stable binding and a homogenous distribution. As proof-of-concept, a fast-degradable polyurethane hydrogel ionomer was chosen as a polymer. This hydrogel has been tailored to fully erode in physiological conditions within three to six months, the approximate interval to achieve functionally mature blood vessels. Two main degradation processes affect the volume and density of the hydrogel: volumetric swelling and resorption. Volumetric swelling results from hydrolysis of crosslinker chains and the network polymer mesh becoming looser. Hydrolysis of the crosslinker chains also leads to the dissolution of degradation products from the hydrogel that are resorbed by cells, leading to a decrease in both volume and density. The potential of in-vivo tracking of polymer implants on MRI using present MnP contrast agent. [0172] The MnP-vinyl MRI agent developed herein also demonstrates the ability to accurately locate labeled soft tissue implants in vivo. By measuring differences in signal intensity unique to the labeling agent, the labeled implant can distinguish physiological processes occurring at the implant site. The volume of the implant can be measured over time, enabling accurate tracking of the implant biodegradation. With this innovative polymer labeling MRI platform, physicians now have access to key insights to help them make proper assessments of the performance of medical devices inserted in patients.

Alginate labeling

[0173] Alginate has long been the most popular biomaterial due it its availability, ease of preparation, biocompatibility, biodegradability, and nontoxicity. It is soluble at physiological pH and has excellent crosslinking capabilities. Many alginate applications have been translated into the clinic, as for wound dressing and in controlled drug delivery. Its versatility as a drug carrier is seen in the example of drug release: as insoluble physical barrier to protect drugs from stomach acid and intravenous uses as dendrimers, nanocrystals, emulsions, liposomes, micelles, and lipid or polymeric nanoparticles. Newer forms of alginate drug delivery fall in the domain of regenerative medicine: injectable alginate hydrogels or drug -embedded wound dressings allow localized delayed drug release. The disclosure herein also describes a method to track alginate on MRI, with the goal of advancing progress towards the effective use of alginate in regenerative medicine and drug delivery. MRI-active alginate would allow time-course monitoring of alginate delivery and migration, thereby opening the door to improvements in delivery vehicle or scaffold design. Current methods for labeling alginate and many other materials for MRI are lacking as they rely on temporary absorption of a Ti reducing agent into a solid. Covalent conjugation is required for extended tracking in-vivo. Current MRI contrast agents are incapable of covalently labeling alginate given that metalation post conjugation is impossible given that alginate is insoluble in the presence of free multivalent Gd or Mn. Furthermore, conjugation to the carboxylates or alcohols of alginate with a metalated complex requires a uniquely stable chelate without pendant coordinated bases in need of protection. The MnP-NCS platform described herein is uniquely suited for this task. Chemistry enabled by MnP-NCS

[0174] Manganese porphyrin isothiocyante (MnP-NCS) enables new chemistry that would otherwise be impossible or very difficult with current Gd or Mn chelates. We adopt a pre-metalation approach, as conventional post-metalation designs suffer from nonspecific metal absorption and retention. However, Mn ²⁺ chelates have been excluded from pre-metalation chemistry, since they do not have sufficient stability in acid to enable an equivalent DOTA-based BFCA. To date, no pre-metalated Mn ²⁺ complex is capable of functionalization. MnP-NCS is the first Mn chelate capable of pre-metalated functionalization for conjugation to proteins and antibodies.

Agents for which MnP-NCS is the only possible conjugation method

[0175] . However, post-conjugation metalation is incompatible with alginate conjugation, as adding a multivalent Gd ³⁺ into alginate solution will cause instantaneous precipitation.

Agents for which MnP-NCS is the only one-step conjugation method

[0176] This includes all reactions with secondary alcohols and other small molecules, including polysaccharides with primary and secondary alcohols. New agents in this category are a blood-pool agent (MnP-BP) and a nitric-oxide specific agent (MnP-NO). Conjugation of secondary alcohols is normally very slow, as they are weak nucleophiles. In contrast, our MnP-NCS platform is unmatched in its activation of electrophiles by electron withdrawal due to it possessing 3 sulfonates and a positively charged metal. In the specific case of MnP-NO, the succinimidyl reaction is incompatible with unprotected polyaminocarboxylate (de-protection is incompatible with the product due to hydrolysis).

Agents for which MnP-NCS is the only practical solution

[0177] This includes reactions with weak nucleophiles stronger than secondary alcohols (e.g., primary alcohols, electron-deficient aryl amines). New agents in this category include anew collagen-targeted fibrosis agent (MnP-APO). These weak nucleophiles will see slow, inefficient reactions with current DOTA-based BFCAs that provide pre- metalated functionalization. Furthermore, current BFCA is susceptible to Gd release during the long incubation times in acidic solution and in the presence of complexing amino acids. Agents for which MnP-NCS simplifies synthesis via one-step conjugation or improves efficiency

[0178] All agents in this category, except those conjugated to lysine (albumin and collagen), cannot be done in a single step by any current BFCA. However, even for lysine, MnP-NCS improves conjugation efficiency.

The chemistries discovered in the MnP-NCS platform disclosed herein are described below may extend to current BFCAs.

Reaction between NCS and secondary alcohol

[0179] In the present disclosure is provided a DBU catalysis of the reaction between NCS and secondary alcohols. DBU is used also outside of polymer chemistry for NCS conjugation. It is noteworthy that previous efforts to react secondary alcohols with NCS via DBU, failed due to the use of aliphatic NCS molecules. It is possible that the benzyl- NCS BFCAs are just reactive enough that DBU could enable their conjugation with secondary alcohols. However, the BFCA would have to be protected to prevent conjugation to its own pendant coordinating bases.

Reaction between NCS and carboxylate

[0180] The present disclosure further describes the use of NCS for conjugation to carboxylates.

MnP-NCS lipophilic salts

[0181] The lipophilic salts simplify or improve some reactions by eliminating the need to add base. This is particularly useful in reactions sensitive to base (e.g. conjugation to alcohols, conjugation to acrylate).

Conversion of thiourea linkages to guanidinium

[0182] Thiourea linkages of MnP conjugates can be converted to guanidinium with use of a saturated solution of ammonium acetate, and heating overnight at 90°C.

MnP-R purification

[0183] The unique ability of MnP-R products to form large aggregates at low pH (<3) enables easy purification and quantitative recovery from several organic solvents with the addition of an organic acid. Scheme 1: General methods for conjugation of MnP-NCS to alcohols (including secondary alcohols) to form thiocarbamate linkers

Scheme 2: General methods for conjugation of MnP-NCS to alcohols (including secondary alcohols) to form carbamate linkers

Scheme 3: General methods for conjugation of MnP-NCS to carboxylates to form amide linkers

Scheme 4: General methods for conjugation of MnP-NCS to alcohols in the presence of free carboxylates to form thiocarbamate linkers

B = H ⁺ , Na ⁺ , TBAH ⁺ , HDBU ⁺

Scheme 5: Conjugation of MnP-NCS to alcohols in the presence of free carboxylates to form carbamate linkers

References

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Desogere, P., Montesi, S. B., & Caravan, P. (2019). Molecular Probes for Imaging Fibrosis and Fibrogenesis. Chemistry (Weinheim an Der Bergstrasse, Germany), 25(5), 1128-1141. https://doi.org/10.1002/chem.201801578

Giuntini, F., Alonso, C. M. A., & Boyle, R. W. (2011). Synthetic approaches for the conjugation of porphyrins and related macrocycles to peptides and proteins. Photochemical & Photobiological Sciences : Official Journal of the European Photochemistry Association and the European Society for Photohiology, 10(5), 759-791. https://doi.org/10.1039/c0pp00366b

McMurry, T. J. ; S. H. ; S. D. M. ; L. R. B. (1996). Diagnostic imaging contrast agents with extended blood retention.

Pathak, P., Zarandi, M. A., Zhou, X., & Jayawickramarajah, J. (2021). Synthesis and Applications of Porphyrin-Biomacromolecule Conjugates. Frontiers in Chemistry, 9(November), 1-30. https://doi.org/10.3389/fchem.2021.764137

Sharma, R., Seo, J.-W., & Kwon, S. (2014). In Vivo Imaging ofNitric Oxide by Magnetic Resonance Imaging Techniques. Journal of Nanomaterials, 2014, 1—13. https://doi.org/10.1155/2014/523646

Vollett, K. D. W., Szulc, D. A., & Cheng, H.-L. M. (2023). A Manganese Porphyrin Platform for the Design and Synthesis of Molecular and Targeted MRI Contrast Agents. International Journal of Molecular Sciences, 24(11). https://doi.org/10.3390/ijms24119532 ENUMERATED EMBODIMENTS

Embodiment 1. A compound of Formula (I): wherein:

E is NH, O, or S;

Z is absent, CEE, NH, O, or S; and

R comprises a protein, peptide, antibody, glycoprotein, glycosaminoglycan, polysaccharide, dendrimer, lipid, steroid, drug, polymer, acrylate, or small molecule moiety targeting group; or a pharmaceutically acceptable salt or tautomer thereof.

Embodiment 2: The compound of Embodiments 1, wherein E is S, or a pharmaceutically acceptable salt or tautomer thereof.

Embodiment 3: The compound of Embodiment 1 or 2, wherein E is S; and Z is

NH, O, or S; or a pharmaceutically acceptable salt or tautomer thereof.

Embodiment 4: The compound of any one of Embodiments 1-3, wherein E is S; and Z is NH; or a pharmaceutically acceptable salt or tautomer thereof.

Embodiment 5: The compound of any one of Embodiments 1-3, wherein E is S; and Z is O; or a pharmaceutically acceptable salt or tautomer thereof. Embodiment 6: The compound of any one of Embodiments 1-3, wherein E is S; and Z is S; or a pharmaceutically acceptable salt or tautomer thereof. Embodiment 7: The compound of Embodiment 1, wherein E is O, or a pharmaceutically acceptable salt or tautomer thereof Embodiment 8: The compound of Embodiment 1 or 7, wherein E is O; and Z is absent, CH2 or O; or a pharmaceutically acceptable salt or tautomer thereof. Embodiment 9: The compound of Embodiments 1, 7, or 8, wherein E is O; and Z is absent orCH ₂ ; or a pharmaceutically acceptable salt or tautomer thereof. Embodiment 10: The compound of Embodiments 1, 7, or 8, wherein E is O; and Z is O; or a pharmaceutically acceptable salt or tautomer thereof. Embodiment 11: The compound of Embodiment 1, or a pharmaceutically acceptable salt or tautomer thereof, wherein E is NH. Embodiment 12: The compound of Embodiments 1 or 11, wherein E is NH; and Z is NH, or a pharmaceutically acceptable salt or tautomer thereof. Embodiment 13: The compound of Embodiments 1, 4, or 12, or a pharmaceutically acceptable salt or tautomer thereof, wherein R is an antibody or glycoprotein with specificity for a biological antigen. Embodiment 14: The compound of Embodiment 1 or 4, or a pharmaceutically acceptable salt or tautomer thereof, wherein R is albumin. Embodiment 15: The compound of any one of Embodiments 1-8, or a pharmaceutically acceptable salt or tautomer thereof, wherein R comprises a small molecule or a drug. Embodiment 16: The compound of any one of Embodiments 1-8, or a pharmaceutically acceptable salt or tautomer thereof, wherein R comprises a metal-free porphyrin. Embodiment 17: The compound of Embodiment 1 or 16, wherein said metal free porphyrin is any one of 10,15,20-(tri-4-sulfonatophenyl)porphyrin, (di-4- sulfonatophenyl)porphyrin, (4-sulfonatophenyl)porphyrin, uroporphyrinogen I and III, hematoporphyrin, photofrin and Tetra(4-carboxyphenyl)porphyrin), benzoporphyrin, porphycene, corrin, corphin, sirohydrochlorin, chlorin, phthalocyanine, hydrazine formyl-hydrazine, l-(4-Amino-l-piperazine), or (R)-2-amino-4- phenylbutanehydrazide .

Embodiment 18: The compound of any one of Embodiments 1-8, wherein R comprises a 1,2-diamine-substituted aromatic compound, wherein the aromatic compound is optionally further substituted.

Embodiment 19: The compound of Embodiment 1 or 18, wherein R further comprises a spacer moiety which is an optionally substituted alkylene or heteroalkylene having a chain length of 3 to 10 atoms.

Embodiment 20: The compound according to any one of Embodiments 1, 5, 9, or

10, wherein R is a polysaccharide or a glycosaminoglycan.

Embodiment 21 : The compound of Embodiments 1 or 20, wherein the polysaccharide or glycosaminoglycan comprises free carboxylic acids.

Embodiment 22: The compound of Embodiment 1, 20 or 21, wherein the polysaccharide is a mannuronate and/or guluronate based polysaccharide.

Embodiment 23: The compound of Embodiments 1, 20 or 21, wherein R is a polysaccharide and the polysaccharide is alginate.

Embodiment 24: The compound of Embodiment 1, 20 or 21, wherein R is a glycosaminoglycan and the glycosaminoglycan is hyaluronic acid, chondroitin sulfate, dermatan sulfate or heparin.

Embodiment 25: The compound of Embodiment 1, 5, or 10, wherein R is 4,4- diphenylcyclohexan- 1 -ol .

Embodiment 26: The compound of Embodiment 1 or 9, wherein R is cholic acid, deoxycholic acid glycocholic acid, glycolithocholic acid, glycochenodeoxycholic acid, lithocholic acid, chenodeoxycholic acid, taurochenodeoxycholic acid, taurocholic acid, or other bile acid derivative.

Embodiment 27 : The compound of Embodiment 1 or 4, wherein R is a protein or a polysaccharide.

Embodiment 28: The compound of Embodiment 1 or 27, wherein R is a protein and the protein is collagen, silk, elastin, or any other protein-based compound. Embodiment 29: The compound of Embodiment 1 or 27, wherein R is a polysaccharide and the polysaccharide is chitosan.

Embodiment 30: The compound of Embodiment 1, 5 or 10, wherein R is a polysaccharide or a glycosaminoglycan.

Embodiment 31 : The compound of Embodiment 1 or 30, wherein R is a polysaccharide and the polysaccharide polyvinyl alcohol, cellulose, callose, chitin, dextran, cyclodextran, amylose, glucose, arabinose, ribose, threose, fructose, galactose, xylose, manose, maltose, lactose, talose, sucrose, pectin, amylopectin, carrageanan starch or glycogen.

Embodiment 32: The compound of Embodiment 1 or 30, wherein R is a glycosaminoglycan and the glycosaminoglycan is heparin sulfate or keratan sulfate.

Embodiment 33: The compound of Embodiment 1 or 9, wherein R is a fatty acid.

Embodiment 33: The compound of Embodiment 1 or 33, wherein the fatty acid is stearic acid or lauric acid.

Embodiment 34: The compound of any one of Embodiments 1-4, wherein R comprises an acrylate.

Embodiment 35: The compound of Embodiment 1, wherein said R group is a 5- hydroxyindole, 5-hydroxytryptamide, serotonin, serotonin hydrochloride, 5- hydroxyindole-3 -acetic acid for imaging myeloperoxidase.

Embodiment 36: The use of the compound according to any one of Embodiments

1-35 as an MRI contrast agent.

EXAMPLES

Example 1: Preparation of MnP-labeled human serum albumin (MnPn-HSA)

[0184] Manganese 5-(4-isothiocyanatophenyl)-10,15,20-(tri-4- sulfonatophenyl)porphyrin (MnP-NCS) was added at different stoichiometric excess (Ox, 5x, lOx, 20x and 40x) to aqueous human serum albumin (has) solutions under vigorous stirring. Sodium carbonate buffer (0.1 M) solution was added to adjust the pH of the reaction to pH 9, and the reaction mixture was left for 24 hours. Purification of MnPn-HSA was performed with 50 mL Amicon ultra spin filters with cut-off of 50 kDa to remove unbound porphyrin. Reaction solutions were repeatedly spin filtered with PBS (9x) well past the point that the resulting filtrate was transparent.3x more filtrations were performed with deionized water to remove PBS salt. Reaction solutions were then lyophilized, producing a red solid which could be dissolved and used directly as intravascular contrast agent. [0185] Matrix-assisted laser desorption/ionization (MALDI) was used to determine MnP-NCS tagging efficiency by change in average molecular weight of HSA. The stoichiometric conditions used in the reaction and resulting tagging efficiency is depicted in Figure 6. High tagging efficiencies of 70% and to 48% were achieved, far exceeding any previous tagging efficiency reported for albumin labeling by either fluorescent agents for BFCA used for MRI or radiopharmaceuticals (Vollett et al., 2023). [0186] Phantom MRI scans testing of the albumin bound HSA (MnP-Alb) for T1 relaxation is depicted in Figure 7 where resulting T ₁ relaxation increased with greater degree of labeling. We named each based upon the measured ratio of MnP to HSA on mass spec (MnP-3.5Alb, MnP-6Alb, MnP-9Alb, MnP-19Alb). Given that both MnP- 3.5Alb or MnP-6Alb products possessed sufficient T1 relaxivity and had the highest labeling efficiencies, we used them for testing specificity of MnP labelled HSA for the intravascular compartment in-vivo. Results from imaging a rat after injection of either MnP- _3.5 Alb or MnP- ₆ Alb is included in Figure 8. These agents were found to provide stable T1 relaxation in blood for at least 40 minutes, demonstrating that MnP-NCS conjugated to HSA is an effective manganese blood-pool agent. This supports the effectiveness and functionality of MnP-NCS labeling of proteins for MRI imaging.

Example 2: Preparation of MnP-labeled APO porphyrin (MnP-APO) [0187] To produce an MRI contrast agent for imaging collagen, MnP-NCS was conjugated to APO-NH ₂ with the reaction scheme depicted in Figure 9. First, 10 mg of MnP-NCS (10 µmol) and 10 mg of APO-NH ₂ (10 µmol) were dissolved in 300 uL of DMSO with stir bar. Next, 9.5 µL N, N-diisopropylethylamine (DIPEA) was added to the reaction solution under vigorous stirring and left for 6 hr at room temperature. The reaction was monitored by TLC with a mobile phase of CHCl ₃ :MeOH:H ₂ O 6:3:1 with a couple drops of dilute HCl. The reaction solution was then purified by precipitating in 10 mL ethyl ether. The resulting brown precipitate was rinsed 3 times with 5 mL ethyl acetate. The resulting solid was purified by flash chromatography with C18 cartridge and was eluted with a gradient of aqueous 0.1M ammonium acetate and 1:4 acetonitrile:methanol. The pH of mobile phase was adjusted to just under a pH of 8.5 by ammonium hydroxide to reduce porphyrin aggregation. Fractions characterized by relevant peaks were lyophilized to remove volatile buffer. Confirmation of successful conjugation of the fibrosis agent (MnP-APO) was performed with mass spec ESI as shown in Figure 10. Example 3: MnP-APO exhibits improved in vitro binding to collagen relative to MnP-NH2 [0188] Porphyrin stock solutions were prepared by weighing out lyophilized products and dissolving in PBS to a concentration of approximately 500 uM. This stock solution was dispensed into each well. [0189] Lyophilized rat tail collagen was dissolved overnight at 4°C in 0.02 M acetic acid at a concentration of 5mg/mL. Each well was filled with 25 uL of collagen solution before 10, 25, 50, 100 and 250 uM) was dispensed into wells with lyophilized collagen (N=2) before plates were shaken and then placed into an incubator at 37°C overnight, allowing collagen to gel with tape covering plates to prevent evaporation. The next day, the solution was removed from each well for weighing and measurement by ICP-OES. The results were interpreted using Eq.1, which is commonly used for plotting enzyme kinetics. Equation 1. where [bound] is p p y bound to collagen, [collagen] is the concentration of collagen, N is number of binding sites per collagen chain, [free] is concentration of free porphyrin, and Kd is the dissociation constant or concentration when binding sites are half saturated. [0190] The data was measured from IPC and a Scatchard model was for measuring affinity as depicted in Figure 11 It was determined that MnP-APO had N = 19.3 ± 0.98 binding sites and a dissociation constant of Kd = 4.73 ± 1.7 µM. These values indicated at least two orders of magnitude improved binding affinity than that of MnP-NH ₂ (K _d = >100 µM), confirming that the presence of the APO functional group significantly improved binding to collagen. [0191] Next, to verify that MnP-APO provided a high T ₁ relaxivity for contrast enhancement on MRI, MnP-APO was measured against the clinically used agent Gadovist at 3T. A relaxivity of 15.15 mM ^-1 s ^-1 was demonstrated for the MnP-APO, and a relaxivity of 4.4 mM ^-1 s ^-1 was demonstrated for Gadovist. The collagen gels previously used for measuring binding affinity of MnP-APO were then used as phantoms to test the ability of MnP-APO for reducing Ti contrast of collagen. Gels were washed with PBS to remove unbound porphyrin and then placed in an incubator at 38°C, with PBS solution replacement three times daily. After three days, T i was measured for these collagen gels at 3T. Results from phantom scans are plotted in Figure 12. The conjugation of MnP- NCS to the APO-NH2 restored significant affinity to acid-soluble collagen while also increasing Ti relaxivity in comparison to the unfiinctionalized MnP-NH ₂ .

Example 4: MnP-APO 3T MRI imaging in a mouse model of fibrosis

[0192] In-vivo study on the effectiveness of MnP-APO was performed as a blood-pool agent in mice. In brief, the fibrosis detecting agent MnP-APO was administered through intravenous tail injection to either healthy mice or those with isoproterenol -induced heart failure. Imaging was performed on a preclinical 3T scanner. The agent provided significant contrast to the cardiac tissue of isoproterenol-treated mice compared to healthy hearts; this is demonstrated in images provided in Figure 13 with comparative T1 values graphed in Figure 14. This demonstrated that MnP-APO is an effective contrast agent for the detection of heart disease associated with fibrosis.

Example 5: Synthesis of a nitric oxide imaging agent (MnP-NO) Ar, 24hr

[0193] Synthesis of the NO agent (MnP-NO) was performed in three steps depicted above. First 4.4 mg of 4-amino-l-butanol (40 pmol) was dissolved in 100 uL of DMAc with stir bar. Ten mg of MnP-NCS (10 pmol) was dissolved in 500 uL of DMAc; the solution was then slowly added dropwise to a 4-amino-l-butanol solution mixture under vigorous stirring and then left for 30 min at room temperature. Upon completion of reaction, the product was crashed out in 10 mL ethyl acetate and centrifuged. The resulting green precipitate was rinsed with ethyl acetate and re-dissolved in 500 uL of DMAc, with the precipitation procedure repeated. The solid product was dissolved in deionized water and run through a pre-loaded amberlite IR120 ion exchange resin to exchange excess amine reagent with sodium. The resulting solution was lyophilized to produce a green powder before storage in the freezer at -20 ^o C. [0194] The second step was performed with 10 mg of MnP-butanol (10 µmol) dissolved in 500 uL anhydrous DMAc with stirring under argon at room temperature. To the - Disuccinimidyl carbonate (30 µmol). The reaction was left under vigorous stirring for 5 h at room temperature, with monitoring using TLC plates with a CHCl3:Methanol:H2O 6:3:1 mobile phase with a couple drops of dilute HCl. The reaction solution was then purified by precipitating product (MnP-Succ) in 40 mL acetone. The resulting green precipitate was rinsed 3 times with 10 mL THF. The resulting solid was lyophilized overnight to yield a green powder. [0195] The third and final step was performed by dissolving 10 mg of MnP-Succ (10 µmol) in 500 uL anhydrous DMAc with stirring under argon at room temperature. To the reaction mixture we added 4.2 uL triethylamine (TEA, 30 µmol) and 3.7 mg of 3,4- diaminotoluene (30 µmol). The reaction was left under vigorous stirring for 5 h at room temperature, with monitoring using TLC plates with a CHCl ₃ :Methanol:H ₂ O 6:3:1 mobile phase with a couple drops of dilute HCl. The reaction solution was then purified by precipitating out the product in 10 mL ether. The resulting red precipitate was rinsed 3 times with 5 mL ethyl acetate. The resulting solid was purified by flash chromatography with C18 cartridge and was eluted with a gradient of aqueous (0.5M ammonium acetate) and acetonitrile. Fractions from relevant peaks were lyophilized, followed by vacuum drying at 50 ^o C to remove volatile buffer. Confirmation of product was performed by mass to charge determined by ESI in Figure 15. MnP-NO was also determined to have a higher relaxivity than clinical Gd agent Gadovist with 12.1 mM ^-1 s ^-1 and 4.4 mM ^-1 s ^-1 , respectively. Example 6: MnP-NO detects nitric oxide in vitro and in an in vivo mouse model of inflammation [0196] To confirm that MnP-NO is capable of providing Ti contrast enhancement of collagen dependent on the concentration of NO, 200 uL of 5 mg/mL collagen solution was dispensed into 96 well plates and lyophilized overnight. Dried collagen samples were transferred to 48 well plates with 3 collagen pieces per plate. 500 uL of an approximately 160 uM solution of NO agent dissolved in PBS was added to each plate and left for 1 hour to soak with light mixing. A 100 mM NO donor methanol solution and 10 mM NO donor DMSO solution were prepared and then dispensed into each collagen/NO agent well to make the following: (1) NO donor concentrations of 10, 5, and 2.5 mM prepared by lOOmM NO donor methanol solution, and (2) 1, 0.75, 0.5, 0.25, 0.1, 0.05, 0.025 and 0 mM prepared with 10 mM NO donor (Diethylammonium (Z)-1-(N,N- diethylamino)diazen-l-ium-l,2-diolate) DMSO solution. The experiment was left for 2 hours at room temperature with light shaking before collagen gels were removed and rinsed with PBS; gels were then placed in 10 mL of PBS and left in an incubator for a week with one change in PBS solution after 3 days. Collagen samples were then sonicated for 2 hours at 45°C to remove unbound collagen before transfer into 384 plates for MRI.

[0197] Results are depicted in Figure 16 indicating that Ti of collagen gels decreased with increasing concentrations of NO donor. This suggests the possibility that MnP-NO may enable the quantitative determination of NO concentration in-vivo. Selectivity of MnP-NO for nitric oxide over other radical species was also confirmed by testing relative activation and resulting reduction in Ti which is depicted in Figure 17.

[0198] MnP-NO was also demonstrated to be activated in-vitro by nitric oxide generated by macrophage -like cells stimulated with lipopolysaccharide in Figure 18. This supports that MnP-NO is just as effective at detecting nitric oxide generated endogenously as it by an NO donor.

[0199] MnP-NO was also demonstrated to successfully detect NO generated in an isoproterenol-induced cardiac inflammation model. Inflammation was detected by MnP- NO in the myocardium of isoproterenol -treated mice with a significant reduction in Ti as observed on Ti maps included in Figure 19 with averages plotted in Figure 20.

[0200] The present disclosure is directed to a new MRI contrast agent for the MRI detection of NO. The in-vitro data demonstrates that MnP-NO may enable, for the first time, significant Ti contrast enhancement from NO generation associated with local inflammation in tissue. Such an agent has a potential to detect inflammatory diseases at their earliest stages and monitor progression and response to treatment.

Example 7: Synthesis and Evaluation of a MnP conjugated thiocarbamate blood pool agent (MnP-S-BP)

[0201] The MnP-NCS conjugated blood pool product with thiocarbamate linkage (MnP- S-BP) was produced in the ways depicted above. In brief 5 mg of MnP-NCS (5.77 pmol) and 7.28 mg of 4,4-diphenylcyclohexan-l-ol (11.54 pmol) was added to a reaction flask and dried under vacuum at 100°C for 1 hour. The reaction flask was then purged with argon before 500 pL of anhydrous DMAc was added and then vigorously stirred at room temperature. Then 29 pmol of DBU was added to reaction before heated with stirring to 50°C, and monitored for completion with TLC using ACN: H ₂ O:acetic acid (80:20: 1) as mobile phase. Reaction solution was then purified by precipitating in 10 mL ethyl acetate.

[0202] The resulting brown precipitate was rinsed 3 times with 5 mL ethyl acetate. Solid product was then dissolved in 1 mL H2O and washed 3 times with 5 mL ethyl acetate. This product was then purified by reverse phase chromatography using a octylsilane column and a H2O: acetonitrile mobile phase, with the aqueous phase composed of 0.5M ammonium acetate buffer. The ratio of H2O in the mobile phase was increased until MnP- S-BP eluted. This solution was then lyophilized overnight yielding MnP-S-BP as a green solid. Identity of products was confirmed by their mass to charge ratios obtained by mass spectroscopy in Figure 21.

[0203] Alternatively, this synthesis was performed without the use of base as either the regular MnP-NCS with sodium cations, or as a Tetrabutylammonium salt (TBA-MnP- NCS) or DBU salt (DBUH-MnP-NCS) at 80°C. Reaction times were extended with sodium MnP-NCS requiring at least 48 hours while the lipophilic salts requiring 2 to 24 hours.

[0204] MnP-NCS was an ideal Ti conjugating platform for conjugation to alcohols, as it achieves better reaction efficiencies and shorter reaction times for conjugations to a sterically hindered alcohol than what has been previously reported for conjugation to primary alcohols (Bernhard et al., 2012).

Example 8: Synthesis of MnP labeled carbamate blood pool agent (MnP-O-BP)

[0205] The MnP-NCS conjugated BP with carbamate linkage (MnP-O-BP) was produced in the way depicted above. In brief, 5 mg of MnP-NCS (5.77 umol) and 7.28 mg of 4,4-diphenylcyclohexan-l-ol (11.54 umol) was added to a reaction flask and dried under vacuum at 100°C for 1 hour. The reaction flask was then purged with argon before 500 pL of anhydrous DMSO was added and then vigorously stirred at room temperature before a drop of dibutyltin(IV) dilaurate and N,N-diisopropylethylamine (DIPEA) was added. Reaction solutions were then heated with stirring to 96°C, and monitored for completion with TLC using can :H2O: acetic acid (80:20: 1) as mobile phase. Reaction solution was then purified by precipitating in 10 mL ethyl acetate. The resulting brown precipitate was rinsed 3 times with 5 mL ethyl acetate. Solid product was then dissolved in 1 mL H2O and washed 3 times with 5 mL ethyl acetate. This product was then purified by reverse phase chromatography using a octyl silane column and a H2O: acetonitrile mobile phase, with the aqueous phase composed of 0.5M ammonium acetate buffer. This solution was then lyophilized overnight yielding MnP-O-BP as a green solid. Identity of products was confirmed by their mass to charge ratios obtained by mass spectroscopy in Figure 22. Much higher yields were obtained by following the synthesis steps for MnP- S-BP using DBU, but extending reaction times to 12 or 24 hours. Alternatively, the base free synthesis method was used, but the use of DMSO as solvent resulted in the MnP-0- BP product.

Example 9: Evaluation of MnP-O-BP as a blood pool agent in vivo in mice

[0206] Viability of MnP-O-BP as a blood-pool agent was tested in vivo in mice. In brief, MnP-BP was administered in a mouse through intravenous tail injection. Imaging was performed on a preclinical 3T scanner. The agent remained within the intravascular space and provided detailed imaging of the vasculature depicted in the T1 maps and SPGR images included in Figure 23, showing stable contrast over an hour. The measured T1 values of MnP-O-BP within the aorta overtime are compared to the MnTPPS4 porphyrin control in Figure 24. Manganese concentration was observed to decrease significantly in organs after 7 days according to Figure 25, indicating excretion from the body.

[0207] The in-vitro testing showed MnP-O-BP bound to albumin, and in-vivo results confirmed a significant and stable reduction of Ti in the vasculature of a mouse. The synthesis was of MnP-S-BP and MnP-O-BP using DBU as a probe conjugating catalyst to react MnP-NCS to a sterically hindered alcohol.

Example 10: Synthesis of MnP-carboxylic acid conjugates including MnP-Deox [0208] MnP-NCS reacts quantitatively with the carboxylates of stearic acid, 3,5- Dihydroxycyclohexanecarboxylic acid, and deoxycholic acid when mixed in DMSO or DMAc as sodium salts, or with their carboxylic acidic derivatives in the presence of base as outlined above. Conjugation of MnP-NCS to carboxylates is achieved with several small molecules. The mass spec mass spec results provided for stearic acid in Figure 26, 3,5-Dihydroxycyclohexanecarboxylic acid in Figure 27 and deoxycholic acid (MnP- Deox) in Figure 28. Example 11: Evaluation of MnP-Deox as a blood pool agent in mice [0209] An in-vivo study of the effectiveness of MnP-Deox as a blood-pool agent was performed in mice. In brief, the blood-pool agent MnP-Deox was administered in a mouse through intravenous tail injection. Imaging was performed on a preclinical 3T scanner. The agent remained within the intravascular space and provided detailed imaging of the vasculature providing prolonged stable contrast enhancement depicted in the T1 maps and SPGR images included in Figure 29 showing stable contrast over an hour. The measured T1 values of MnP-Deox within the aorta over time are compared to the unfunctionalized MnTPPS4 in Figure 30. Manganese concentrations were observed to decrease significantly in organs after 7 days according to Figure 31, indicating excretion from the body. [0210] In-vitro testing showed it bound to albumin, and in-vivo results confirmed a significant and stable reduction of T1 in the vasculature of a mouse. [0211] Example 12: Labeling of collagen gels with MnP-NCS [0212] Labeling of collagen gels in situ was performed similarly to proteins outlined in Figure 5. In brief dispensing 1mL of either 10 mg/mL or 3mg/mL bovine type I collagen solutions (advanced biomatrix) into 14 mL round bottom falcon tubes on ice. Next, 0.25 mL of cold stock solutions of either MnP-NCS, APO-NH2, MnTPPS4 or MnP-NH2 were dispensed into tubes with collagen gel (n=2) and mixed thoroughly. Next, 0.25 mL of cold, freshly prepared 0.5M Na2CO3 was added and mixed with collagen gels. Gels were then left in the fridge for 24 hours at 4 ^o C to solidify before being transferred to 50mL falcon tubes filled with PBS and left in an incubator at 37 ^o C for 24 hours. Example 13: Preparation of MnP labeled acid soluble collagen

[0213] Preparation of MnP labeled acid soluble collagen is similar to the previously described reaction for conjugation to collagen as described in Example 12; however, instead of allowing collagen gels to form, MnP-NCS porphyrin was added to collagen solution and vortexed followed by the slow dropwise addition of 0.5 M Na2CCh with intermediate shaking until PH ~ 9. This was left for 1 hour before reaction was stopped by adding a 3x volume of PBS to falcon tubes and centrifuging at 16’000 rpm to remove unreacted porphyrin. This rinsing procedure was repeated three times before gels were transferred to a beaker and a 5x volume of 0.5 M acetic acid was added to dissolve collagen with stirring overnight. Salt and excess acetic acid was then removed by dialyzing collagen solution in 50 kDa Mw cut off tubing against 50 mM acetic acid in deionized water. Dialysis was performed for a week with dialysate replaced at least 3 times a day. The resulting desalted collagen solution was then centrifuged at 16’000 rpm for 2 hours to remove any remaining insoluble collagen before supernatant was collected, lyophilized, and then stored at -80°C.

[0214] Labeling of collagen with MnP-NCS was confirmed by retention in the collagen gels relative to MnP-NH2 and MnTPPS4 porphyrin used as controls, depicted in Figure 35. It was observed that over 90% of the porphyrin controls had leached out. In contrast, a significant quantity of MnP-NCS remained in gels, with 31.4 ± 1.8% and 56.0 ± 1.7% remaining in 3mg/mL and 10 mg/mL collagen gels, respectively, with only a minor decrease observed over 556 days. These results demonstrate that MnP-NCS was capable of labeling collagen and prevented the rapid leaching of the metalated MnP porphyrin.

[0215] To test effectiveness of collagen labeling for Ti relaxation, MnP labeled acid soluble collagen (MnP-Col) was blended with regular acid soluble collagen at 25 % (w/w) to form 10 mg/mL collagen gels. Resulting Ti maps were depicted in Figure 36 with values tabulated in Figure 37. No significant leaching of MnP was observed on day 5 or 60. We also obtained MRI phantoms of MnP-Col labelled gels. The blending of MnP- Col with acid-soluble collagen resulted in significant Ti reduction in collagen scaffolds, a reduction that remained for 2 months. Example 14: Synthesis of methacrylate functionalized MnP (MnP-vinyl) [0216] Synthesis of methacrylate functionalized MnP is depicted above. In brief MnP- NCS was conjugated to excess 2-aminoethyl methacrylate under basic conditions with 30 equivalencies of DIPEA in DMAc at room temperature for 1 hour to form a stable thiourea linkage between the porphyrin and the vinyl moiety to yield MnP-vinyl. MS (ESI) m/z Calculated for [M - 2H] ^2- C ₅₁ H ₃₅ MnN ₆ O ₁₁ S ₄ ^2- : 545.03. found: 545.03. Calculated for [M - H]- C51H37MnN6O11S4-: 1092.08 found: 1092.07. Example 15: MnP labeling of hydrogel [0217] MnP labeling of polyurethane diacrylate-co-poly(acrylic acid) hydrogel is depicted in Figure 38 and was prepared as polyurethane diacrylate and acrylic acid in a 50:50 weight percent; these were dissolved overnight with 0.3 % weight of 2-hydroxy-2- methylpropiophenone photo-initiator (Sigma-Aldrich). MnP-vinyl agent dissolved in ultrapure water at concentrations ranging from 0 5 mg/mL was added to the acrylate solution for a 20% volume of aqueous solution to weight of resin. The solution was mixed for one hour and sterile-filtered through a 0.22 m filter (MilliPore). The solution was pipetted into a polypropylene 96-well plate (Greiner) and irradiated with UV light ( max = 365 nm, Phoseon Technology FireEdge 300) for 1 minute to initiate free radical polymerization of the acrylate groups. The crosslinked discs were collected from the 96- well plate. The extent of reacted vinyl groups in the discs was determined by FTIR. The buffered saline (DPBS -/-, Sigma-Aldrich) for seven days until equilibrium water uptake is achieved with DPBS replacement on each day. [0218] The stability of bound MnP in the hydrogels was assessed and compared to unbound Mn-TPPS4 loaded hydrogels in buffered saline at physiological temperature. Measurement of free MnP from labeled hydrogels revealed minimal MnP release, less than 2% over 28 days depicted in Figure 39. Unbound commercially available Mn-TPPS4 was quickly released from the hydrogel, over 40% within the first 4 days. The thiourea and vinyl bonds that conjugate the MnP to the hydrogel were stable, enabling MnP to be released with the degrading hydrogel. An MRI phantom of MnP-labeled hydrogels is depicted in Figure 40. showing substantial T1 reduction for sensitive hydrogel detection on MRI Labeling with 4 ppm MnP produced a significant T1 reduction (T1 = 517±36 ms versus 879±147 ms unlabeled).

Example 16: Studying the efficiency of the contrast agent in vivo for allowing implant tracking.

[0219] Unlabeled hydrogel and 4 ppm MnP-labeled hydrogel discs implanted subcutaneously at random locations in the dorsum of the same animals were tracked for 7 weeks and explanted for histological and manganese content analyses. Labeled implants were substantially brighter on Tlw SPGR and Tlw SE images compared to unlabeled controls depicted in Figure 41. The signal enhancement was reproducible between different animals and between different implant sites within the same animal. Quantitative T1 mapping revealed a significant T1 reduction of 41% 1 week postimplantation and a sustained, significant difference between labeled and unlabeled implants over the entire 7-week interval p < 0.01, depicted in Figure 42.

Example 17: Synthesis and evaluation of MnP-alginate gels

[0220] MnP labeled alginate was prepared as depicted above, where 40 mg of DBU- MnP-NCS (46.12 pmol) and tetrabutylammonium alginate (500 mg) was mixed with 20mL DMSO at 60°C for 2 hours. The reaction was stopped by adding the reaction mixture to a stirring solution of acidic isopropyl alcohol (0.6M HC1) where it precipitated out as green fibers. The solution was decanted and then washed three times with a 1: 1 ethanol:water solution (100 mL). These green fibers were added to a stirring solution of MilliQ water (50 mL) and titrated with a 1 M Na2CCf solution until fibers completely dissolved and continued until the pH was ~7. The resulting solution was then precipitated as a green fiber in 300 mL methanol to remove remaining unbound porphyrin resulting in MnP -labeled alginate (MnP -alginate).

[0221] To demonstrate long-term stability and MRI tracking of MnP -alginate gels, 4% MnP -alginate was blended with 4% chitosan gel before crosslinking at a pH of 1 . The resulting gels were imaged demonstrating stability of labeling over 184 days on MRI with results plotted in Figure 43.

Example 18: In vivo tracking of MnP-alginate implants

[0222] In-vivo tracking of labeled implants in rats was conducted to verify that the implants could be tracked. The MnP-labeled gels provided significant and stable Ti reduction in labeled gels over the course of 30 days, with representative Ti map depicted in Figure 44 and the average Ti values plotted in Figure 45. Most implanted alginate/chitosan gels remained stable in vivo for nearly a month.

Equivalents

[0223] The details of one or more embodiments of the disclosure are set forth in the accompanying description above. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the methods and materials are now described. Other features, objects, and advantages of the disclosure will be apparent from the description and from the claims. In the specification and the appended claims, the singular forms include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents and publications cited in this specification are incorporated by reference.

The foregoing description has been presented only for the purposes of illustration and is not intended to limit the disclosure to the precise form disclosed, but by the claims appended hereto.