EP4688758A1

EP4688758A1 - Verdazyl compounds as contrast agents for magnetic resonance imaging of kidneys

Info

Publication number: EP4688758A1
Application number: EP24773734.9A
Authority: EP
Inventors: Nicholas David CALVERT; Alexia Margarite KIRBY; Aidan Alexander TORRENS; Adam Jason SHUHENDLER
Original assignee: Yellowbird Diagnostics Inc
Current assignee: Yellowbird Diagnostics Inc
Priority date: 2023-03-23
Filing date: 2024-03-22
Publication date: 2026-02-11
Also published as: CN121263409A; JP2026511165A; AU2024238841A1; KR20260021591A; WO2024192530A1; IL323529A

Abstract

There are provided verdazyl derivative compounds and compositions thereof useful as contrast agents for biomedical imaging. Compounds and compositions of the disclosure may be used for example for quantitative and/or qualitative assessments of glomerular filtration rate (GFR) in a subject, providing reliable and rapid assessment of kidney function.

Description

VERDAZYL COMPOUNDS AS CONTRAST AGENTS FOR MAGNETIC RESONANCE IMAGING OF KIDNEYS

FIELD

[0001] The present disclosure relates to metal-free contrast agents for magnetic resonance imaging, their methods of manufacture and their uses.

BACKGROUND

[0002] Chronic kidney disease (CKD) and acute kidney injury (AKI) are a major health burden globally. CKD often develops slowly and without obvious symptoms in the early stages but becomes progressively more debilitating in later stages with limited chances for reversal. CKD outcomes are improved with early interventions facilitated by earlier detection.

[0003] The gold standard clinically for measuring kidney function is glomerular filtration rate (GFR) measurement. Clinical diagnosis of CKD in North America is defined as an estimated GFR (eGFR) of < 60 mL/min/1.73 m² for more than 3 months, or a urine albumin-to-creatinine (ACR) > 30 mg/g for more than 3 months.

[0004] However, the CKD diagnostic values have been derived from large clinical studies in an ethnically limited population, significantly reducing the diagnostic power of these biomarkers of disease. Additionally, underlying causes of CKD can vary between individuals, where some of the most common, including diabetes mellitus, cardiovascular disease, and kidney transplant, can limit the accuracy of eGFR measurements at the patient level. Almost 30% of these patients can present with a 30% deviation from their true eGFR. Furthermore, on top of the deviation of an individual from the derivative population, the eGFR equation assumes steady-state creatinine levels and does not account for alterations in or alternate routes of creatinine production, leading to large variability. Finally, a major limitation of GFR measurement is that is does not provide physicians with spatial or structural information underlying the renal dysfunction.

[0005] There is a need for a more accurate method of GFR estimation that is free from the use of race or other demographic characteristics of the patient and that can provide spatial or structural information. Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) could be a powerful tool for renal assessment in cases of suspected AKI and CKD, however a strong negative bias against gadolinium-based contrast agents exists due to the possibility of inducing nephrogenic systemic fibrosis (NSF) in patients with limited renal function.

[0006] There is a need for contrast agents that can be used to provide a reliable, rapid, and quantitative clinical imaging approach for early detection of CKD and other disorders.

SUMMARY

[0007] It is an object of the present invention to ameliorate at least some of the deficiencies present in the prior art. Embodiments of the present technology have been developed based on the inventors’ appreciation that there is a need for improved contrast agents for clinical purposes.

[0008] Inventors have discovered that novel verdazyl derivatives can provide metal- free contrast agents suitable for use clinically. Compounds provided herein can allow reliable, rapid, and/or quantitative clinical imaging, which can facilitate early diagnosis and detection of various disorders. In particular, compounds are advantageous for contrast enhanced magnetic resonance imaging (CE-MRI) and dynamic contrast enhanced magnetic resonance imaging (DCE-MRI). In certain embodiments, compounds may demonstrate greater stability and/or reduced cytotoxicity compared to previous organic radical contrast agents.

[0009] Swager et al. (U.S. Patent No. 8,715,621) described a variety of radicals that are useful as polarizing compounds, but verdazyl is used only as a radical initiator and not as an imaging agent per se.

[0010] From one aspect, there is provided a compound represented by structural Formula (I), or a pharmaceutically acceptable salt or ester thereof:

[0011] wherein:

[0012] Ri and R2 are independently substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, or substituted or unsubstituted heteroaryl.

[0013] In some embodiments, Ri and/or R2 is substituted or unsubstituted Ci to C5 alkyl. In some such embodiments, Ri and/or R2 is substituted or unsubstituted Ci to Ce alkyl. In some such embodiments, Ri and/or R2 is substituted or unsubstituted C4 to Cs cycloalkyl. In some such embodiments, Ri and/or R2 is substituted or unsubstituted C4 to Cs heterocycloalkyl. In some such embodiments, Ri and/or R2 comprises a substituted or unsubstituted hydroxyl, amino or thio group. In some embodiments, Ri and/or R2 is a substituted or unsubstituted benzaldehyde. In some embodiments, Ri and/or R2 comprises at least one heteroatom which is N, S, or O.

[0014] In certain embodiments of the compound of Formula (I), there is provided a compound represented by structural Formula (I), or a pharmaceutically acceptable salt or ester thereof:

[0015] wherein:

[0016] Ri is selected from:

[0017] R2 is selected from:

[0018] wherein:

[0019] R is a monosaccharide;

[0020] X is carbon (C) or oxygen (O);

[0021] n is an integer from 1 to 5 (i.e., 1, 2, 3, 4, or 5);

[0022] Z is ether, ester, carbamate, thiocarbamate, urea, thiourea, hydrazone, amide, secondary amine, tertiary amine, disulfide, triazole, cyclooctyltriazolyl, cycloocta[ ]pyridazyl, or a cyanobenzyl thiazolyl-containing group;

[0023] Y is a monosaccharide, a glycan, a moiety bearing amine and carboxylate separated by a substituted alpha carbon, a peptide, a nanoparticle, a dendrimer, an antibody, an antibody fragment, a nucleic acid, an aptamer, an organic targeting ligand, or R3; and

[0024] R3 is selected from: [0025] wherein Xi is any halogen.

[0026] In certain embodiments of the compound of Formula (I), R2 is , where n and R3 are as described above.

[0027] In certain embodiments of the compound of Formula (I), R2 is

, where X, Z, Y, and n are as described above.

[0028] In certain embodiments of the compound of Formula (I), a monosaccharide includes but is not limited to a 6-carbon sugar, e.g., glucose, fructose, galactose, or mannose.

[0029] In certain embodiments of the compound of Formula (I), the monosaccharide is glucose, fructose, tagatose, galactose, mannose, xylose, or ribose. It should be understood that the monosaccharide is not meant to be particularly limited; it may be a 6- carbon sugar (a hexose), a 5-carbon sugar (a pentose), a pyranose, a furanose, or other monosaccharide suitable chemically for attaching to the compound.

[0030] In certain embodiments of the compound of Formula (I), the monosaccharide is a C3 to C7 carbohydrate, i.e., having from 3 to 7 carbon atoms.

[0031] In certain embodiments of the compound of Formula (I), the monosaccharide is not glucose.

[0032] In certain embodiments of the compound of Formula (I), the compound is represented by structural Formula (II), or a pharmaceutically acceptable salt or ester thereof:

[0033] wherein: Ri, R3 and n are as described hereinabove.

[0034] In certain embodiments of the compound of Formula (I), the compound is represented by structural Formula (III), or a pharmaceutically acceptable salt or ester thereof:

[0035] wherein:

[0037] X is C or O;

[0038] n is from 1 to 5;

[0039] Z is ether, ester, carbamate, thiocarbamate, urea, thiourea, hydrazone, amide, secondary amine, tertiary amine, disulfide, triazole, cyclooctyltriazolyl, cycloocta[ ]pyridazyl, or a cyanobenzyl thiazolyl-containing group; and

[0040] Y2 is a monosaccharide, a glycan, a moiety bearing amine and carboxylate separated by a substituted alpha carbon, a peptide, a nanoparticle, a dendrimer, an antibody, an antibody fragment, a nucleic acid, an aptamer, or an organic targeting ligand.

[0041] In certain embodiments of the compound of Formula (III), a monosaccharide includes but is not limited to a 6-carbon sugar, e.g., glucose, fructose, galactose, or mannose.

[0042] In certain embodiments of the compound of Formula (III), the monosaccharide is glucose, fructose, tagatose, galactose, mannose, xylose, or ribose. It should be understood that the monosaccharide is not meant to be particularly limited; it may be a 6- carbon sugar, a 5-carbon sugar, or other monosaccharide suitable chemically for attaching to the compound. In some embodiments, the monosaccharide is a C3 to C7 carbohydrate, i.e., having from 3 to 7 carbon atoms.

[0043] In certain embodiments of the compound of Formula (I), the compound is a compound set forth in Table 1, or a pharmaceutically acceptable salt or ester thereof. The symbol is used to indicate a nitrogen radical in the compound.

[0044] In certain embodiments of the compound of Formula (I), the compound is not glucoverdazyl.

Table 1. Examples of Compounds of Formula (I), in accordance with certain embodiments.

[0045] It should be understood that all acid, salt, base, and other ionic and non-ionic forms of compounds described herein are intended to be encompassed. For example, if a compound is shown as an acid herein, the salt forms of the compound are also encompassed. Likewise, if a compound is shown as a salt, the acid and/or basic forms are also encompassed.

[0046] In certain embodiments of the compound of Formula (I), the compound is suitable for use as a contrast agent. In some such embodiments, the compound is suitable for use for CE-MRI and/or DCE-MRI. In some such embodiments, the compound is suitable for biomedical imaging, such as without limitation for diagnosis of renal dysfunction, e.g., by determining and/or mapping GFR.

[0047] From another aspect, there is provided a composition comprising a compound of the disclosure and a carrier. [0048] In certain embodiments, the composition is a pharmaceutical composition comprising a compound of the disclosure and a pharmaceutical carrier.

[0049] In certain embodiments, the composition is suitable for use as a contrast agent.

[0050] In certain embodiments, the carrier is an aqueous solution. The carrier may be saline, water, phosphate-buffered saline (PBS), or dextrose 5% in water. Such compositions may be used for biomedical applications such as imaging.

[0051] From a yet further aspect, there is provided a method of making embodiments of the compound or composition as described herein.

[0052] From another aspect, there are provided methods of biomedical imaging, comprising administering a contrast agent to a subject and imaging the contrast agent in the subject, wherein the contrast agent comprises the compound or composition as described herein.

[0053] In certain embodiments, the biomedical imaging comprises magnetic resonance imaging (MRI). The MRI may comprise contrast enhanced magnetic resonance imaging (CE-MRI) and/or dynamic contrast enhanced magnetic resonance imaging (DCE- MRI).

[0054] In certain embodiments, there is provided a method of biomedical imaging of the kidney, comprising administering a contrast agent to a subject and imaging the contrast agent in the subject, wherein the contrast agent comprises the compound or composition as described herein. In some such embodiments, the method further comprises determining the subject’s Glomerular Filtration Rate (GFR). In certain embodiments, quantitative and/or qualitative kidney functional information is obtained, such as without limitation determining and/or mapping spatially the subject’s GFR. Such methods may be used for example to diagnose kidney dysfunction and/or to monitor or assess kidney function in the subject.

[0055] In certain embodiments, there is provided a method of diagnosing kidney dysfunction, comprising administering a contrast agent to a subject and imaging the contrast agent in the subject, wherein the contrast agent comprises the compound or composition as described herein, and determining and/or mapping the subject’s Glomerular Filtration Rate (GFR).

[0056] In certain embodiments, there is provided a method of monitoring, assessing or determining kidney function, comprising administering a contrast agent to a subject and imaging the contrast agent in the subject, wherein the contrast agent comprises the compound or composition as described herein, and determining and/or mapping the subject’s Glomerular Filtration Rate (GFR).

[0057] In certain embodiments of methods of the disclosure, the subject has, is suspected of having, or is at risk of renal dysfunction. The subject may have, be suspected of having, or be at risk of chronic kidney disease (CKD), acute kidney injury (AKI), renal artery stenosis, urinary obstruction, and/or a renal tumor or malignancy. In some embodiments, the subject may be a candidate kidney donor.

[0058] In certain embodiments of methods of the disclosure, the compound is glucoverdazyl.

[0059] From a yet further aspect, there is provided the compound or composition as described herein for use in imaging, such as biomedical imaging. In certain embodiments, the compound or composition is for use in imaging the kidney.

[0060] From a yet other aspect, there is provided the compound or composition as described and/or claimed herein for use as a contrast agent. The contrast agent may be used during imaging using modalities such as MRI, CE-MRI, DCE-MRI, and the like.

[0061] From a further aspect, there is provided a metal-free contrast agent for biomedical imaging comprising the compound or composition as described herein.

[0062] From yet another aspect, there is provided a kit comprising the compound, composition or contrast agent as described herein Kits may further comprise a buffer or excipient, and/or instructions for use, e.g., in biomedical imaging.

BRIEF DESCRIPTION OF THE DRAWINGS

[0063] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0064] For a better understanding of the invention and to show more clearly how it may be carried into effect, reference will now be made by way of example to the accompanying drawings, which illustrate aspects and features according to embodiments of the present invention, and in which:

[0065] FIGs. 1A-1F show paramagnetic characteristics and stability of glucoverdazyl, according to certain embodiments of the present technology. FIG. 1A: Electron paramagnetic resonance (EPR) spectra of a 5 mM glucoverdazyl solution in phosphate buffered saline (PBS) acquired at room temperature; FIG. IB: Ti and T2 weighted MRI images of 3 mM glucoverdazyl solution in PBS acquired at 3 T; FIG. 1C: Longitudinal relaxivity of glucoverdazyl at pH 7.4 in PBS at 3 T MRI. The stability of glucoverdazyl (black) and TEMPO (pink) was determined by EPR during 2 hr incubation in mouse serum (FIG. ID) or 4 mM sodium ascorbate buffer at pH 7.4 (FIG. IE). FIG. IF: The storage stability of 5 mM glucoverdazyl solutions left exposed to light at room temperature (pink) or left in the freezer (-20°C) protected from light (black) as determined by EPR.

[0066] FIGs. 2A-2C show glucoverdazyl localization and clearance in vivo in healthy BALB/c mice. FIG. 2A: MRI scans of BALB/c mice were acquired both preinjection and every 2.5 min post-injection following the administration of glucoverdazyl (3 mmol/kg). FIG. 2B: Regions-of-interest (ROIs) were selected and the average intensity at each timepoint was acquired. Data are presented as means ± SEM for n = 9 mice. FIG. 2C: The semi-natural logarithmic transformation and line of best fit (pink) is drawn from t = 2.5min to t = 40 min of the kidney clearance curve. Data are presented as means of each time point ± SEM for n = 9 mice. The line of best fit was determined for individual curves. Renal decay time constant (k) and R² are shown as means ± SD.

[0067] FIGs. 3A-3D show glucoverdazyl-enhanced DCE-MRI in a mouse model of unilateral ureteral obstruction. FIG. 3A: Ti-weighted images of kidneys att = 2.5 min postinjection (top) and RDTC maps (bottom) for Sham and UUO groups. FIG. 3B: RDTC values for each kidney at each post-injury time-point. Data are presented as box-and- whisker plots of the single average RDTC value from each kidney (ipsi- or contralateral) individually, from each mouse (n = 5). FIG. 3C: Representative histology of paired kidneys from sham (left) and UUO (right) treatment groups stained by PAS. FIG. 3D: Serum creatinine levels of sham (grey) and UUO (turquoise) mice on the two sampled days. Data are presented as individual values of SCr. Statistical analysis was done by repeated measures two-way ANOVA followed by a Tukey post-hoc test. In all graphs, * /?<0.05, **/?<0.01, and ****/?<0.0001.

[0068] FIGs. 4A-4D show glucoverdazyl-enhanced DCE-MRI of folic acid-induced nephropathy. FIG. 4A: Ti-weighted images of kidneys at t = 2.5 min post-injection (top) and RDTC maps (bottom). FIG. 4B: RDTC values for kidneys at each post-injury time point. Data are presented as box-and-whisker plots of the single average RDTC value from both kidneys of each mouse (n = 5). FIG. 4C: Representative histology of kidneys (top) with enlargements of the cortical or medullary regions (bottom) stained by PAS. Black arrows indicate positive histological staining for fibrotic areas, represented by a light blue color. FIG. 4D: Serum creatinine levels of FAN mice at each post-injury time point (n = 5). Statistical analysis was done by repeated measures two-way ANOVA followed by a Tukey post-hoc test. In all graphs, */?<0.05, **/?<0.01, and ***/?<0.001.

[0069] FIGs. 5A-5D show determination of glomerular filtration rate (GFR) by transdermal fluorescence and dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) in folic acid nephropathic (FAN) mice, according to certain embodiments of the present technology. FIG. 5A: Normalized fluorescence intensity of the transdermal fluorescence clearance of FITC-sinistrin in FAN mice on day 0, 15, and 30 (black, pink, and turquoise, respectively). Data are presented as the mean of each time point for each replicate ± SEM for n = 6 mice. FIG. 5B: Normalized DCE-MRI intensity of glucoverdazyl-enhanced scans over time for FAN mice on day 0, 15, and 30 (black, pink, and turquoise, respectively). Data are presented as the mean of each time point for each replicate ± SEM for n = 5 mice. FIG. 5C: GFR values for FAN mice determined by either transdermal fluorescence (grey) or DCE-MRI (pink). Data are presented as box-and- whisker plots of the GFR for each mouse. Statistical analysis was done by mixed measures two-way ANOVA followed by a Tukey post-hoc test. The p value for each test is labelled on the graph. FIG. 5D: Illustration of glucoverdazyl clearance through the kidney by DCE- MRI to elaborate on differences between transdermal and DCE-MRI measurements.

[0070] FIG. 6 shows an illustration of the DCE-MRI data acquisition and image mapping workflow, according to certain embodiments of the present technology.

[0071] FIG. 7 shows the ^JH NMR spectrum of synthesized compound 2, according to certain embodiments of the present technology. [0072] FIG. 8 shows the ¹³C NMR spectrum of synthesized compound 2, according to certain embodiments of the present technology.

[0073] FIG. 9 shows the ^JH NMR spectrum of synthesized intermediate compound, according to certain embodiments of the present technology.

[0074] FIG. 10 shows the ¹³C NMR spectrum of synthesized intermediate compound, according to certain embodiments of the present technology.

[0075] FIG. 11 shows the ^JH NMR spectrum of synthesized compound 3, according to certain embodiments of the present technology.

[0076] FIG. 12 shows the ¹³C NMR spectrum of synthesized compound 3, according to certain embodiments of the present technology.

[0077] FIG. 13 shows high performance liquid chromatography traces of synthesized compounds 3 and 4 to verify radical activity of the compound after radicalization step, according to certain embodiments of the present technology.

[0078] FIG. 14 shows (A) comparison between the transdermal fluorescence RDTC value in healthy BALB/c mice (n = 6) as determined by MediBeacon software and the RDTC value in healthy BALB/c mice (n = 14) as determined by glucoverdazyl DCE-MRI. (B) A comparison between RDTC value in healthy BALB/c mice by using intensity measurements derived per voxel or from the entire ROI of a slice at each timepoint (n = 14 mice for both). Data are presented as box-and-whisker plots of the single RDTC (A, transdermal) or single average RDTC (A, MRI and B). Statistical analysis was done by one-way ANOVA followed by a Tukey post-hoc test. In all graphs, ns indicates nonsignificant and **** indicates /?<0.0001. [0079] FIG. 15 shows cytotoxicity evaluation of glucoverdazyl at different concentrations in H460 after 4 hr (A) and 24 hr (B) incubations with the respective concentration. Data are represented as means ± SD of n = 3 replicates. Statistical analysis was done by one-way ANOVA followed by a Tukey post-hoc test.

[0080] FIG. 16 shows cytotoxicity evaluation of glucoverdazyl, 5,5-dimethyl-l- pyrroline N-oxide (DMPO), and 2,2,6,6-Tetramethylpiperidin-l-yl)oxyl (TEMPO) in human renal proximal tubule cells at 10 mM concentrations after 4 hr (A) or 24 hr (B) incubations under cell culture conditions. Data are represented as means ± SD of n = 3 replicates. Statistical analysis was done by one-way ANOVA followed by a Tukey post- hoc test. */?<0.05 of the live and dead cell populations compared to the populations of the other conditions.

[0081] FIG. 17 shows glucoverdazyl uptake in human renal proximal tubules cells after 24 hr incubation in 10 mM glucoverdazyl under cell culture conditions. Uptake was measured by EPR activity compared to a known concentration and normalized to number of cells loaded into the EPR tube. Data are represented as means ± SD of n = 3 replicates. Statistical analysis was done by one-way ANOVA followed by a Tukey post-hoc test.

[0082] FIGs. 18A-F show glucoverdazyl -enhanced DCE-MRI data in the unilateral ureter obstruction (UUO) model. FIG. 18A: Normalized intensity-over-time curves of UUO mice on day 0. Curve is presented as mean normalized intensity ± SEM FIG. 18B: The semi-natural-log regression curve of FIG. 18A with the line of best fit from t = 0 min to t = 40 min. Data are presented as mean RDTC (k) and R² value ± SD. RDTC values for the cortex and medulla & renal pelvis (MRP) regions-of-interest (ROIs) for the sham group (FIG. 18C) and UUO group (FIG. 18D). Data are presented as box-and-whisker plots of the single average RDTC value from each kidney (ipsi- or contralateral) from each region, individually, from each mouse (n = 5). The same data and plots for AUC are shown in FIGs. 18E and 18F for the sham and UUO groups, respectively. Statistical analysis was done by repeated measures two-way ANOVA followed by a Tukey post-hoc test. In all graphs, ns non-significant, * p < 0.05, *** p < 0.001, and **** p < 0.0001.

[0083] FIGs. 19A-19C show glucoverdazyl -enhanced DCE-MRI data in the folic acid nephropathy (FAN) model. FIG. 19A: RDTC values for the cortex and medulla & renal pelvis (MRP) regions-of-interest (ROIs). Data are presented as box-and- whisker plots of the single average RDTC value from each mouse (n = 5). The same data and plots for AUC are shown in FIG. 19B. FIG. 19C: The normalized intensity-over-time curves for the FAN model over the entire kidney, as well as for the cortex and MRP regions for each of the post injury-time points. Curves are presented as the mean normalized intensity at each time point ± SEM. Statistical analysis was done by repeated measures two-way ANOVA followed by a Tukey post-hoc test. In all graphs, ns non-significant, * p < 0.05, ** p < 0.01, and **** p < 0.001.

DETAILED DESCRIPTION

[0084] Glomerular filtration rate (GFR) measurement is the gold standard clinically for measuring kidney function. However, it relies on entering physiological, demographic, and blood-based metabolite levels into an equation derived from a limited, underrepresentative population, and does not provide spatial information about kidney dysfunction. Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) could be powerful for renal assessment in cases of suspected AKI and CKD, however a strong negative bias against gadolinium-based contrast agents exists due to the possibility of inducing nephrogenic systemic fibrosis in patients with limited renal function.

[0085] To purposefully develop a contrast agent for DCE-MRI suitable for use a as renal pathology diagnostic, a class of extremely stable nitrogen-centered organic radicals known as verdazyls was evaluated. Verdazyls provide excellent T1 -shortening at 3 T derived from an extremely stable radical, with no loss of signal in highly reducing environments. It was demonstrate herein that a glucose-modified verdazyl, glucoverdazyl, showed no extrarenal uptake in vitro and in vivo, with image contrast being limited to kidney and bladder. Renal functional deficits were demonstrated over time in mouse models of unilateral ureter obstruction (UUO) and folic acid-induced nephropathy (FAN), with functional kidney maps correlating with histological and blood biomarkers of kidney dysfunction. Using the FAN model, it was shown that glucoverdazyl clearance rates were a reliable measure of GFR, as demonstrated by comparison to a validated transdermal fluorescence technique. The present disclosure is based, at least in part, on the finding that glucoverdazyl can be an extremely powerful metal-free MRI contrast agent. In certain embodiments, contrast agents of the disclosure can provide reliable GFR measurements free from error-prone, population-derived equations; spatial or structural information underlying renal dysfunction; and/or enable an imaging-based personalized medicine approach to nephrology or other areas. In certain embodiments, compounds of the provide metal-free contrast agents which are stable and non-toxic.

[0086] In order to provide a clear and consistent understanding of the terms used in the present specification, a number of definitions are provided below. Moreover, unless defined otherwise, all technical and scientific terms as used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention pertains.

[0087] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one”, but it is also consistent with the meaning of “one or more”, “at least one”, and “one or more than one”. Similarly, the word “another” may mean at least a second or more.

[0088] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “include” and “includes”) or “containing” (and any form of containing, such as “contain” and “contains”), are inclusive or open-ended and do not exclude additional, unrecited elements or process steps.

[0089] The term “about” is used to indicate that a value includes an inherent variation of error for the device or the method being employed to determine the value.

[0090] The terms “derivative” and “variant” are used interchangeably herein.

[0091] The term “subject,” as used herein, include eukaryotes, such as mammals, e.g., humans, ovines, bovines, equines, porcines, canines, felines, non-human primates, mice, and rats. The terms “subject” and “patient” are used interchangeably herein.

[0092] Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75^th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March Mar ch's Advanced Organic Chemistry, 5^th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3^rd Edition, Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference.

[0093] It will be appreciated that a compound of the disclosure, as described herein, may be substituted with any number of substituents or functional moieties. In general, the term “substituted” whether preceded by the term “optionally” or not, and substituents contained in formulas of this invention, refer to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent. When more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. For purposes of this disclosure, heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms. Furthermore, this disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds.

[0094] The term “acyl,” as used herein, refers to a group having the general formula — C(=O)R°, where R° is substituted or unsubstituted hydroxyl, substituted or unsubstituted thiol, substituted or unsubstituted amino, substituted or unsubstituted, cyclic or acyclic aliphatic, substituted or unsubstituted, cyclic or acyclic heteroaliphatic, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. Exemplary acyl groups include carboxylic acids ( — CO2H), ketones (such as an acetyl group [ — (C=O)CH3], esters, amides, carbonates, carbamates, and ureas. Acyl substituents include, but are not limited to, any of the substituents described herein, that result in the formation of a stable moiety (for example, an aliphatic group substituted with one or more aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, sulfinyl, sulfonyl, oxo, imino, thiooxo, cyano, amino, azido, nitro, hydroxy, thio, and/or halo groups).

[0095] The term “aliphatic,” as used herein, includes both saturated and unsaturated, nonaromatic, straight chain (i.e., unbranched), branched, acyclic, cyclic (i.e., carbocyclic), or polycyclic hydrocarbons, which are optionally substituted with one or more functional groups. As will be appreciated by one of ordinary skill in the art, “aliphatic” is intended herein to include, but is not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties. Thus, as used herein, the term “alkyl” includes straight, branched and cyclic alkyl groups. An analogous convention applies to other generic terms such as “alkenyl”, “alkynyl”, and the like. Furthermore, as used herein, the terms “alkyl”, “alkenyl”, “alkynyl”, and the like encompass both substituted and unsubstituted groups. In certain embodiments, as used herein, “aliphatic” is used to indicate those aliphatic groups (cyclic, acyclic, substituted, unsubstituted, branched or unbranched) having 1-6 carbon atoms, or 2-6 carbon atoms. In certain embodiments, an aliphatic group has 1-5 or 2-5 carbon atoms. In certain embodiments, an aliphatic group has 1-4 or 2-4 carbon atoms. In certain embodiments, an aliphatic group has 1-3 or 2-3 carbon atoms. In certain embodiments, an aliphatic group has 1-2 carbon atoms. In certain embodiments, an aliphatic group has 1 carbon atom. In certain embodiments, an aliphatic group has 2 carbon atoms. In certain embodiments, an aliphatic group has 1-6 carbon atoms (Ci-e). Aliphatic group substituents include, but are not limited to, any of the substituents described herein, that result in the formation of a stable moiety (for example, an aliphatic group substituted with one or more aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, sulfinyl, sulfonyl, oxo, imino, thiooxo, phosphino, cyano, amino, azido, nitro, hydroxy, thio, and/or halo groups).

[0096] The term “alkyl,” as used herein, refers to saturated, straight- or branched- chain hydrocarbon radicals derived from a hydrocarbon moiety containing between one and twenty carbon atoms by removal of a single hydrogen atom. In some embodiments, the alkyl group employed in the invention contains 1-6 carbon atoms (Ci-e). In another embodiment, the alkyl group employed contains 1-5 carbon atoms. In still other embodiments, the alkyl group contains 1-4 carbon atoms. In yet another embodiment, the alkyl group contains 1-3 carbons. In yet other embodiments, the alkyl group contains 1-2 carbons. In yet other embodiments, the alkyl group contains 1 carbon atom. Examples of alkyl radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, sec-pentyl, iso-pentyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, sechexyl, and the like, which may bear one or more substituents. Alkyl group substituents include, but are not limited to, any of the substituents described herein, that result in the formation of a stable moiety (for example, an alkyl group substituted with one or more aliphatic, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, sulfinyl, sulfonyl, oxo, imino, thiooxo, cyano, amino, azido, nitro, hydroxy, thio, and/or halo groups).

[0097] The term “alkenyl,” as used herein, denotes a monovalent group derived from a straight- or branched-chain hydrocarbon moiety having at least one carbon-carbon double bond by the removal of a single hydrogen atom. In certain embodiments, the alkenyl group employed in the invention contains 2-6 carbon atoms. In some embodiments, the alkenyl group employed in the invention contains 2-5 carbon atoms. In another embodiment, the alkenyl group employed contains 2-4 carbon atoms. In still other embodiments, the alkenyl group contains 2-3 carbon atoms. In yet another embodiment, the alkenyl group contains 2 carbons. Alkenyl groups include, for example, ethenyl, propenyl, butenyl, l-methyl-2- buten-l-yl, and the like, which may bear one or more substituents. Alkenyl group substituents include, but are not limited to, any of the substituents described herein, that result in the formation of a stable moiety (for example, an alkenyl group substituted with one or more aliphatic, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, sulfinyl, sulfonyl, oxo, imino, thiooxo, cyano, amino, azido, nitro, hydroxy, thio, and/or halo groups).

[0098] The term “alkynyl,” as used herein, refers to a monovalent group derived from a straight- or branched-chain hydrocarbon having at least one carbon-carbon triple bond by the removal of a single hydrogen atom. In certain embodiments, the alkynyl group employed in the invention contains 2-6 carbon atoms. In some embodiments, the alkynyl group employed in the invention contains 2-5 carbon atoms. In another embodiment, the alkynyl group employed contains 2-4 carbon atoms. In still other embodiments, the alkynyl group contains 2-3 carbon atoms. In still other embodiments, the alkynyl group contains 2 carbon atoms. Representative alkynyl groups include, but are not limited to, ethynyl, 2- propynyl(propargyl), 1-propynyl, and the like, which may bear one or more substituents. Alkynyl group substituents include, but are not limited to, any of the substituents described herein, that result in the formation of a stable moiety (for example, an alkynyl group substituted with one or more aliphatic, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, sulfinyl, sulfonyl, oxo, imino, thiooxo, cyano, amino, azido, nitro, hydroxy, thio, and/or halo groups).

[0099] The term “amino,” as used herein, refers to a group of the formula ( — NH2). A “substituted amino” refers to a group of the formulae ( — NHR¹¹) or ( — NR¹¹2), wherein R^h can be any substituted except hydrogen which result in the formation of a stable moiety (for example, an amino group substituted with one or more aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, sulfinyl, sulfonyl, amino, nitro, hydroxy, and/or thio groups). A “suitable amino-protecting group,” as used herein, is well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. Suitable amino-protecting groups include methyl carbamate, ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7- di -t-buty 1 - [9-( 10,10-dioxo- 10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD- Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2- trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), l-(l-adamantyl)-l- methylethyl carbamate (Adpoc), l,l-dimethyl-2-haloethyl carbamate, l,l-dimethyl-2,2- dibromoethyl carbamate (DB-t-BOC), l,l-dimethyl-2, 2, 2 -tri chloroethyl carbamate (TCBOC), 1 -methyl- l-(4-biphenylyl)ethyl carbamate (Bpoc), l-(3,5-di-t-butylphenyl)-l- methylethyl carbamate (t-Bumeoc), 2-(2'- and 4'-pyridyl)ethyl carbamate (Pyoc), 2-(N,N- dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1 -isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8- quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithio carbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitobenzyl carbamate, p- bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4- methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p- toluenesulfonyl)ethyl carbamate, [2-(l,3-dithianyl)]methyl carbamate (Dmoc), 4- methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2- phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc), l,l-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p- (dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate, 2- (trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5- dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, phenothiazinyl-(10)-carbonyl derivative, N'-p-toluenesulfonylaminocarbonyl derivative, N'-phenylaminothiocarbonyl derivative, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxycarbonylvinyl carbamate, o-(N,N- dimethylcarboxamido)benzyl carbamate, l,l-dimethyl-3-(N,N- dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di (2- pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p'-methoxyphenylazo)benzyl carbamate, 1 -methylcyclobutyl carbamate, 1 -methylcyclohexyl carbamate, 1 -methyl- 1- cyclopropylmethyl carbamate, l-methyl-l-(3,5-dimethoxyphenyl)ethyl carbamate, 1- methyl-l-(p-phenylazophenyl)ethyl carbamate, 1 -methyl- 1 -phenylethyl carbamate, 1- methyl-l-(4-pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, 2,4,6- trimethylbenzyl carbamate, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3- pyridyl carb oxami de, N-benzoylphenylalanyl derivative, benzamide, p-phenylbenzamide, o-nitophenyl acetamide, o-nitrophenoxyacetamide, acetoacetamide, (N'- dithiobenzyloxycarbonylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o- nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o- phenylazophenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o- nitrocinnamide, N-acetylmethionine derivative, o-nitrobenzamide, o- (benzoyloxymethyl)benzamide, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N- dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-l, 1,4,4- tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted l,3-dimethyl-l,3,5- triazacyclohexan-2-one, 5-substituted 1,3 -dibenzyl- 1, 3, 5-triazacy cl ohexan-2-one, 1- substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2- (trimethylsilyl)ethoxy]methylamine (SEM), N-3 -acetoxypropylamine, N-(l-isopropyl-4- nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4- methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fem), N-2- picolylamino N'-oxide, N-l, 1 -dimethylthiomethyleneamine, N-benzylideneamine, N-p- methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2- pyridyl)mesityl]methyleneamine, N — (N',N'-dimethylaminomethylene)amine, N,N'- isopropylidenediamine, N-p-nitrobenzylideneamine, N-salicylideneamine, N-5- chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N- cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-l-cyclohexenyl)amine, N-borane derivative, N-diphenylborinic acid derivative, N-[phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N- nitrosoamine, amine N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, o- nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys), p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6- trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4- methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6- dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6- sulfonamide (Pmc), methanesulfonamide (Ms), P-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4',8'-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacyl sulfonamide.

[00100] The term “aryl,” as used herein, refer to stable aromatic mono- or polycyclic ring system having 3-20 ring atoms, of which all the ring atoms are carbon, and which may be substituted or unsubstituted. In certain embodiments of the present invention, “aryl” refers to a mono, bi, or tricyclic C4-C20 aromatic ring system having one, two, or three aromatic rings which include, but not limited to, phenyl, biphenyl, naphthyl, and the like, which may bear one or more substituents. Aryl substituents include, but are not limited to, any of the substituents described herein, that result in the formation of a stable moiety (for example, an aryl group substituted with one or more aliphatic, heteroaliphatic, heterocyclic, aryl, heteroaryl; acyl, sulfinyl, sulfonyl, oxo, imino, thiooxo, cyano, amino, azido, nitro, hydroxy, thio, and/or halo groups).

[00101] The term “direct bond” or “bond” refers to a single, double or triple bond between two groups. In certain embodiments, a “direct bond” refers to a single bond between two groups.

[00102] The terms “halo” and “halogen” as used herein refer to an atom selected from fluorine (fluoro, — F), chlorine (chloro, — Cl), bromine (bromo, — Br), and iodine (iodo, -I). [00103] The term “heteroaliphatic,” as used herein, includes both saturated and unsaturated, nonaromatic, straight chain (i.e., unbranched), branched, acyclic, cyclic (i.e., heterocyclic), or polycyclic hydrocarbons, which are optionally substituted with one or more functional groups, and that contain one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms, e.g., in place of carbon atoms. As will be appreciated by one of ordinary skill in the art, “heteroaliphatic” is intended herein to include, but is not limited to, heteroalkyl, heteroalkenyl, heteroalkynyl, and heterocyclyl moieties. Thus, as used herein, the term “heteroalkyl” includes straight, branched and cyclic alkyl groups, as defined herein, which are optionally substituted with one or more functional groups, and that contain one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms, e.g., in place of carbon atoms. An analogous convention applies to other generic terms such as “heteroalkenyl”, “heteroalkynyl”, and the like. Furthermore, as used herein, the terms “heteroalkyl”, “heteroalkenyl”, “heteroalkynyl”, and the like encompass both substituted and unsubstituted groups. In certain embodiments, as used herein, “heteroaliphatic” is used to indicate those heteroaliphatic groups (cyclic, acyclic, substituted, unsubstituted, branched or unbranched) having 1-6 carbon atoms, or 2-6 carbon atoms. In certain embodiments, a heteroaliphatic group has 1-5 or 2-5 carbon atoms. In certain embodiments, a heteroaliphatic group has 1-4 or 2-4 carbon atoms. In certain embodiments, a heteroaliphatic group has 1-3 or 2-3 carbon atoms. In certain embodiments, a heteroaliphatic group has 1-2 carbon atoms. In certain embodiments, an heteroaliphatic group has 1 carbon atom. In certain embodiments, a heteroaliphatic group has 2 carbon atoms. Heteroaliphatic group substituents include, but are not limited to, any of the substituents described herein, that result in the formation of a stable moiety (for example, a heteroaliphatic group substituted with one or more aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, sulfinyl, sulfonyl, oxo, imino, thiooxo, phosphino, cyano, amino, azido, nitro, hydroxy, thio, and/or halo groups). [00104] The term “heteroaryl,” as used herein, refer to stable aromatic mono- or polycyclic ring system having 3-20 ring atoms, of which one ring atom is selected from S, O, and N; zero, one, or two ring atoms are additional heteroatoms independently selected from S, O, and N; and the remaining ring atoms are carbon, the radical being joined to the rest of the molecule via any of the ring atoms. Exemplary heteroaryls include, but are not limited to pyrrolyl, pyrazolyl, imidazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, tetrazinyl, pyyrolizinyl, indolyl, quinolinyl, isoquinolinyl, benzoimidazolyl, indazolyl, quinolinyl, isoquinolinyl, quinolizinyl, cinnolinyl, quinazolynyl, phthalazinyl, naphthridinyl, quinoxalinyl, thiophenyl, thianaphthenyl, furanyl, benzofuranyl, benzothiazolyl, thiazolynyl, isothiazolyl, thiadi azolynyl, oxazolyl, isoxazolyl, oxadiaziolyl, oxadiaziolyl, and the like, which may bear one or more substituents. Heteroaryl substituents include, but are not limited to, any of the substituents described herein, that result in the formation of a stable moiety (for example, a heteroaryl group substituted with one or more aliphatic, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, sulfinyl, sulfonyl, oxo, imino, thiooxo, cyano, amino, azido, nitro, hydroxy, thio, and/or halo groups).

[00105] The term “heterocyclic,” or “heterocyclyl,” as used herein, refers to an nonaromatic, partially unsaturated or fully saturated, 3- to 10-membered ring system, which includes single rings of 3 to 8 atoms in size, and bi- and tri-cyclic ring systems which may include aromatic five- or six -membered aryl or heteroaryl groups fused to a non-aromatic ring. These heterocyclic rings include those having from one to three heteroatoms independently selected from oxygen, sulfur, and nitrogen, in which the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. In certain embodiments, the term heterocylic refers to a non-aromatic 5-, 6-, or 7-membered ring or polycyclic group wherein at least one ring atom is a heteroatom selected from O, S, and N (wherein the nitrogen and sulfur heteroatoms may be optionally oxidized), and the remaining ring atoms are carbon, the radical being joined to the rest of the molecule via any of the ring atoms. Heterocyclyl groups include, but are not limited to, a bi- or tri-cyclic group, comprising fused five, six, or seven-membered rings having between one and three heteroatoms independently selected from the oxygen, sulfur, and nitrogen, wherein (i) each 5-membered ring has 0 to 2 double bonds, each 6-membered ring has 0 to 2 double bonds, and each 7-membered ring has 0 to 3 double bonds, (ii) the nitrogen and sulfur heteroatoms may be optionally oxidized, (iii) the nitrogen heteroatom may optionally be quaternized, and (iv) any of the above heterocyclic rings may be fused to an aryl or heteroaryl ring. Exemplary heterocycles include azacyclopropanyl, azacyclobutanyl, 1,3-diazatidinyl, piperidinyl, piperazinyl, azocanyl, thiaranyl, thietanyl, tetrahydrothiophenyl, dithiolanyl, thiacyclohexanyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropuranyl, dioxanyl, oxathiolanyl, morpholinyl, thioxanyl, tetrahydronaphthyl, and the like, which may bear one or more substituents. Substituents include, but are not limited to, any of the substituents described herein, that result in the formation of a stable moiety (for example, a heterocyclic group substituted with one or more aliphatic, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, sulfinyl, sulfonyl, oxo, imino, thiooxo, cyano, amino, azido, nitro, hydroxy, thio, and/or halo groups).

[00106] The term “hydrogen,” as used herein, refers to any isotope having an atomic number of 1. Typically, hydrogen refers to stable isotopes containing either zero or one neutrons (i.e., ^JH or ²H, also known as deuterium). In certain embodiments, hydrogen is present in its normal isotopic abundance. In other embodiments, at least one position is specifically selected to have a deuterium present.

[00107] The term “hydroxy,” or “hydroxyl,” as used herein, refers to a group of the formula ( — OH). A “substituted hydroxyl” refers to a group of the formula ( — ORi), wherein Ri can be any substitutent except hydrogen which results in a stable moiety (for example, a hydroxy group substituted with a suitable hydroxyl protecting group, an aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, sulfinyl, and/or sulfonyl group). A “suitable hydroxyl protecting group” as used herein, is well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. Suitable hydroxyl protecting groups include methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p- methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2- methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3- bromotetrahydropyranyl, tetrahydrothiopyranyl, 1 -methoxy cyclohexyl, 4- methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4- methoxytetrahydrothiopyranyl S,S-dioxide, l-[(2-chloro-4-methyl)phenyl]-4- methoxypiperidin-4-yl (CTMP), l,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1 -ethoxy ethyl, l-(2-chloroethoxy)ethyl, 1 -methyl- 1 -methoxy ethyl, 1 -methyl- 1 -benzyloxy ethyl, 1- methyl-l-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2- (phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p- halobenzyl, 2, 6-di chlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3- methyl-2-picolyl N-oxido, diphenylmethyl, p,p'-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, a-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p- methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4'- bromophenacyloxyphenyl)diphenylmethyl, 4,4',4'-tris(4,5- dichlorophthalimidophenyl)methyl, 4,4',4"-tris(levulinoyloxyphenyl)methyl, 4,4',4"- tris(benzoyloxyphenyl)methyl, 3-(imidazol-l-yl)bis(4',4"-dimethoxyphenyl)methyl, 1,1- bis(4-methoxyphenyl)-l'-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl- 10-oxo)anthryl, l,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethyl silyl (TMS), triethylsilyl (TES), triisopropyl silyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t- butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethyl silyl (DPMS), t-butylmethoxyphenyl silyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, di chloroacetate, tri chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3- phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate, adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate, 9- fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-l-napththyl carbonate, methyl dithiocarb onate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2- (methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-

(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6- dichloro-4-(l,l,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(l, 1- dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)- 2-methyl-2-butenoate, o-(methoxycarbonyl)benzoate, a-naphthoate, nitrate, alkyl N,N,N',N'-tetramethylphosphorodiamidate, alkyl N-phenyl carbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts). For protecting 1,2- or 1,3-diols, the protecting groups include methylene acetal, ethylidene acetal, 1-t-butylethylidene ketal, 1- phenylethylidene ketal, (4-methoxyphenyl)ethylidene acetal, 2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene ketal, cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal, p-methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal, 3,4- dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethylene ortho ester, 1-m ethoxy ethylidene ortho ester, 1 -ethoxy ethylidine ortho ester, 1,2-dimethoxy ethylidene ortho ester, a- methoxybenzylidene ortho ester, l-(N,N-dimethylamino)ethylidene derivative, a-(N,N'- dimethylamino)benzylidene derivative, 2-oxacyclopentylidene ortho ester, di-t- butylsilylene group (DTBS), l,3-(l,l,3,3-tetraisopropyldisiloxanylidene) derivative (TIPDS), tetra-t-butoxydisiloxane-l,3-diylidene derivative (TBDS), cyclic carbonates, cyclic boronates, ethyl boronate, and phenyl boronate.

[00108] The term “imino,” as used herein, refers to a group of the formula (=NR5), wherein R^r corresponds to hydrogen or any substitutent as described herein, that results in the formation of a stable moiety (for example, a suitable amino protecting group; substituted or unsubstituted amino; acyl; cyclic or acylic, branched or unbranched, substituted or unsubstituted alkyl; cyclic or acylic, branched or unbranched, substituted or unsubstituted alkenyl; cyclic or acylic, branched or unbranched, substituted or unsubstituted alkynyl; cyclic or acylic, branched or unbranched, substituted or unsubstituted heteroalkyl; cyclic or acylic, branched or unbranched, substituted or unsubstituted heteroalkenyl; cyclic or acylic, branched or unbranched, substituted or unsubstituted heteroalkynyl; substituted or unsubstituted aryl; or substituted or unsubstituted heteroaryl).

[00109] The term “isocyano,” as used herein, refers to a group of the formula ( — NC).

[00110] The term “nitro,” as used herein, refers to a group of the formula ( — NO2).

[00111] The term “nitroxide,” as used herein, refers to a stable nitroxide group which may be cyclic or acyclic. In certain embodiments, a stable nitroxide refers to a chemically stable nitroxide which may be obtained in pure form, stored, and handled in the laboratory. In certain embodiments, a stable nitroxide refers to a cyclic or acyclic nitroxide which contains two groups which do not contain alpha hydrogens. Exemplary cyclic or acyclic nitroxides are provided in Keana, Chemical Reviews (1978) 78:37-64, the entirety of which is incorporated herein by reference.

[00112] The term “oxo,” as used herein, refers to a group of the formula (=0).

[00113] The term “thio,” or “thiol,” as used herein, refers to a group of the formula

( — SH). A “substituted thiol” refers to a group of the formula ( — SR^r), wherein R^r is any substitutent, except hydrogen, which results in the formation of a stable moiety (for example, a thio group substituted with one or more aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, sulfinyl, and/or sulfonyl).

[00114] The term “thiooxo,” as used herein, refers to a group of the formula (=S).

[00115] The term “sulfinyl,” as used herein, refers to a group of the formula R^f — S(=O) — wherein R^fmay be an optionally substituted aliphatic, heteroaliphatic, aryl, or heteroaryl. The term “alkyl sulfinyl” refers to a sulfinyl group where R^fmay be an optionally substituted alkyl group. The term “aryl sulfinyl” refers to a sulfinyl group where R^f may be an optionally substituted aryl or heteroaryl group.

[00116] The term “sulfonyl,” as used herein, refers the group of the formula R^g — S(=O)2 — , wherein R^gmay be an optionally substituted aliphatic, heteroaliphatic, aryl, or heteroaryl. The term “alkyl sulfonyl” refers to a sulfonyl group where R^gmay be an optionally substituted alkyl group. The term “aryl sulfonyl” refers to a sulfonyl group where R^gmay be an optionally substituted aryl or heteroaryl. Exemplary aryl or alkyl sulfonyl groups include tosyl (toluene sulfonyl, CEECeEUSCh — ), mesyl (methyl sulfonyl, CH3SO2 — ), and trifluoromethanesulfonyl (CF3SO2 — ). [00117] The term “stable moiety,” as used herein, preferably refers to a moiety which possess stability sufficient to allow manufacture (including manufacture in situ)., and which maintains its integrity for a sufficient period of time to be useful for the purposes detailed herein.

[00118] The term “stable radical,” as used herein, refers to a free radical that possesses stability sufficient to allow manufacture (including manufacture in situ)., and which maintains its integrity for a sufficient period of time to be useful for the purposes detailed herein.

[00119] In certain embodiments, one or more (or all) of the hydrogen atoms present in a compound of Structural Formulae (I), (II) or (III) are ²H.

[00120] Aspects of the present technology comprise compounds which are verdazyl derivatives, as described herein (e.g., compounds of Formula (I), Formula (II), Formula (III), compounds shown in Table 1). The compounds provide metal-free contrast agents which are more stable and/or less toxic than previous contrast agents. The compounds are useful clinically for medical imaging, such as without limitation CE-MRI and DCE-MRI, and can provide reliable, rapid, and/or quantitative clinical imaging to facilitate early diagnosis and detection of various disorders. Aspects of the present technology comprise compositions and pharmaceutical compositions including the compounds, methods of making such compounds, and uses thereof.

Compositions

[00121] In certain embodiments, the compounds of the present disclosure can be present in a composition or a pharmaceutical composition. [00122] Pharmaceutical compositions are generally formulated to be compatible with the intended method or route of administration; exemplary routes of administration include without limitation oral or parenteral, e.g., intramuscular, intravenous, subcutaneous (e.g., injection or implant), intraperitoneal, intrathecal, or intraarticular. In some embodiments, the pharmaceutical composition is provided in a single-use container (e.g., a single-use vial, ampoule, syringe, or autoinjector, whereas a multi-use container (e.g., a multi-use vial) is provided in other embodiments. Compounds and compositions provided herein may be administered to a subject in any appropriate manner known in the art.

[00123] The term "pharmaceutically acceptable carrier" refers to a pharmaceutically- acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any subject composition or component thereof. Each carrier must be "acceptable" in the sense of being compatible with the subject composition and its components and not injurious to the patient. Some examples of materials which may serve as pharmaceutically acceptable carriers include, without limitation: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[00124] In certain embodiments, compositions and pharmaceutical compositions of the disclosure are present in the form of micelles. For some micelles, a composition of the disclosure is mixed with a nonpolar radical, e.g., a radical containing a perfluorinated moiety. A composition or pharmaceutical composition may also include a surfactant. One non-limiting type of a perfluorinated radical is a TEMPO group with a fluorinated tail, e.g. a perfluorinated tail (described in Pozzi, Adv. Synth. Cat. 347:677 (2005), the contents of which are incorporated by reference). An exemplary perfluorinated radical is TEMPO attached to a C6-C20 (such as Cs -C12) perfluoroalkyl group via an amide or sulfonamide group at the 4-position of TEMPO. Suitable surfactants include, without limitation, perfluorinated sulfonic carboxylic acids, particularly C4 -C12 acids such as Ce, C7, Cs, C9, C10, C11 and C12 acids. Exemplary surfactants include without limitation ammonium perfluorooctanoate (FC 143), perfluorooctanesulfonic acid (PFOS) and perfluorononanoic acid (PFNA).

[00125] Compositions and pharmaceutical compositions of the disclosure may also include additional components such as stabilizers, preservatives, dispersants, and the like. Compositions and pharmaceutical compositions of the disclosure may also include additional components suitable for the intended purpose, e.g., suitable for imaging and/or administration to a subject, such as excipients, dyes, and the like.

[00126] A "pharmaceutically acceptable salt" of a compound means a salt of a compound that is pharmaceutically acceptable. Desirable are salts of a compound that retain or improve the biological effectiveness and properties of the free acids and bases of the parent compound as defined herein or that take advantage of an intrinsically basic, acidic or charged functionality on the molecule and that are not biologically or otherwise undesirable. Examples of pharmaceutically acceptable salts are also described, for example, in Berge et al., "Pharmaceutical Salts", J. Pharm. Sci. 66, 1-19 (1977). Nonlimiting examples of such salts include: (1) acid addition salts, formed on a basic or positively charged functionality, by the addition of inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, nitric acid, phosphoric acid, carbonate forming agents, and the like; or formed with organic acids such as acetic acid, propionic acid, lactic acid, oxalic, glycolic acid, pivalic acid, t-butylacetic acid, P-hydroxybutyric acid, valeric acid, hexanoic acid, cyclopentanepropionic acid, pyruvic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2- hydroxyethanesulfonic acid, cyclohexylaminosulfonic acid, benzenesulfonic acid, sulfanilic acid, 4-chlorobenzenesulfonic acid, 2-napthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 3 -phenyl propionic acid, lauryl sulphonic acid, lauryl sulfuric acid, oleic acid, palmitic acid, stearic acid, lauric acid, embonic (pamoic) acid, palmoic acid, pantothenic acid, lactobionic acid, alginic acid, galactaric acid, galacturonic acid, gluconic acid, glucoheptonic acid, glutamic acid, naphthoic acid, hydroxynapthoic acid, salicylic acid, ascorbic acid, stearic acid, muconic acid, and the like; (2) base addition salts, formed when an acidic proton present in the parent compound either is replaced by a metal ion, including, an alkali metal ion (e.g., lithium, sodium, potassium), an alkaline earth ion (e.g., magnesium, calcium, barium), or other metal ions such as aluminum, zinc, iron and the like; or coordinates with an organic base such as ammonia, ethylamine, diethylamine, ethylenediamine, N,N'-dibenzylethylenediamine, ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, piperazine, chloroprocain, procain, choline, lysine and the like.

[00127] Pharmaceutically acceptable salts may be synthesized from a parent compound that contains a basic or acidic moiety, by conventional chemical methods. Generally, such salts are prepared by reacting the free acid or base forms of compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two. Salts may be prepared in situ, during the final isolation or purification of a compound or by separately reacting a compound in its free acid or base form with the desired corresponding base or acid, and isolating the salt thus formed. The term "pharmaceutically acceptable salts" also include zwitterionic compounds containing a cationic group covalently bonded to an anionic group, as they are "internal salts".

Uses

[00128] Dynamic contrast-enhanced MRI (DCE-MRI) analyzes the temporal enhancement pattern of a tissue following the introduction of a paramagnetic contrast agent. This is achieved by the acquisition of baseline images without contrast enhancement, followed by a series of images acquired overtime during and after the arrival of the contrast agent in the tissue of interest. The acquired signal is used to generate a time intensity curve for the tissue, which mirrors the tissue’s response to the arrival of contrast agent in enhancement values. DCE-MRI has been used to study a wide variety of conditions including pathologies of the heart, notably infarction, stroke and further cerebral afflictions, a wide range of neoplasms with an emphasis on anti angiogenic treatment and early detection, as well as investigations of the peripheral vascular and musculoskeletal systems.

[00129] Most often gadolinium (Gd) chelates are used as contrast agents for DCE- MRI. However, Gd-chelate contrast agents are not ideal for many applications. Free gadolinium (Gd³⁺) is known to be toxic and must be tightly complexed by a ligand to be used in humans. There has been concern about the possible toxicity of gadolinium complexes in patients with renal insufficiency, particularly the risk of inducing nephrogenic systemic fibrosis (NSF) in patients with limited renal function. Therefore, there is a need for more stable and/or less toxic contrast agents for DCE-MRI for clinical use.

[00130] Uses of compounds and compositions of the present technology are not limited and may include as contrast agents for imaging, e.g., for contrast-enhanced MRI (CE-MRI), dynamic contrast-enhanced MRI (DCE-MRI), and the like. Thus in certain embodiments, compounds described herein are contrast agents used for imaging, e.g., MRI, CE-MRI, DCE-MRI, and so on. It is to be understood that any suitable MRI or other imaging technique may be used in conjunction with compounds and compositions described herein (e.g., see MRI in Practice Ed. by Westerbrook et al., Blackwell Publishing, Oxford, UK, 2005, the contents of which are incorporated herein by reference). Further, methods may be performed under any magnetic field strength. In some embodiments, the field may have a strength in the range of about 0.1 T to about 30 T, e.g, at 3 T. The radiation for exciting electron spin transitions in the unpaired electron(s) of the polarizing agent at these fields will be in the range of about 2.8 GHz to about 840 GHz. For example, the radiation may be from a 140 GHz gyrotron.

[00131] Chronic kidney disease (CKD) continues to be a major international healthcare burden. CKD often develops slowly and without obvious symptoms in the early stages but becomes progressively more debilitating in later stages with limited chances for reversal. This disease is often attributed to long-term hypertension and diabetes but is also a potential outcome following an acute kidney injury (AKI), the result of a sudden and dramatic decline in kidney function.

[00132] CKD outcomes are improved with early interventions facilitated by earlier detection. Clinical diagnosis of CKD in North America is defined as an estimated glomerular filtration rate (eGFR) of < 60 mL/min/1.73 m² for more than 3 months, or a urine albumin-to-creatinine (ACR) > 30 mg/g for more than 3 months. These diagnostic values have been derived from large clinical studies in an ethnically limited population, significantly reducing the diagnostic power of these biomarkers of disease. Additionally, underlying causes of CKD can vary between individuals, where some of the most common, including diabetes mellitus, cardiovascular disease, and kidney transplant, can limit the accuracy of eGFR measurements at the patient level, as almost 30% of these patients can present with a 30% deviation from their true eGFR. Furthermore, on top of the deviation of an individual from the derivative population, the eGFR equation assumes steady-state creatinine levels and does not account for alterations in or alternate routes of creatinine production, leading to this large variability. It has become imperative that more accurate methods for GFR estimation be developed that are free from the use of race or other demographic characteristics of the patient. Most importantly, these values fail to provide physicians with spatial or structural information underlying the renal dysfunction. Kidney biopsy can provide histopathological data predictive of CKD outcomes, delivering spatial data about specific kidney lesions and not just overall kidney function. However, biopsies are invasive procedures with their own inherent risk, precluding their repeated use to spatiotemporally characterize kidney disease. Clinically, GFR remains the gold standard as an indicator for kidney function, and there is a need for reliable, rapid, and/or quantitative clinical imaging approaches for its measurement.

[00133] In certain embodiments, compounds and compositions described herein provide metal-free contrast agents for medical imaging. In certain embodiments, compounds and compositions described herein provide metal-free alternatives to Gd-based contrast agents for facilitating CE-MRI and/or DCE-MRI. In certain embodiments, the compounds and compositions described herein are taken up selectively in the kidney and are therefore used for medical imaging of the kidney. It should be understood however that the use of compounds and compositions described herein is not meant to be particularly limited; for example, compounds and compositions may be used for imaging other tissues, depending on the compound’s uptake/distribution after administration to a subject and other considerations that determine suitability for a particular use.

[00134] In certain embodiments of imaging methods of the disclosure, imaging of the kidney is provided. Such methods may provide quantitative and/or qualitative kidney functional information, such as without limitation determination and/or mapping of GFR in a subject. In certain embodiments, such imaging is free of toxicological concerns of previous contrast agents in patients with renal dysfunction, and/or free from the use of race or other demographic characteristics of the patient. [00135] In certain embodiments, there are provided methods of biomedical imaging, comprising administering a contrast agent to a subject and imaging the contrast agent in the subject, wherein the contrast agent comprises a compound or composition as described herein. The medical imaging may be, for example and without limitation, magnetic resonance imaging (MRI) such as CE-MRI or DCE-MRI. In certain embodiments, the biomedical imaging is used to image the kidney. In some such embodiments, the imaging is used to monitor or assess kidney function in the subject. In some such embodiments, the imaging is used to determine and/or map GFR in a subject. Methods may therefore provide quantitative and/or qualitative kidney functional information such as without limitation the subject’s GFR.

[00136] In certain embodiments, there are provided methods of measuring kidney function in a subject, comprising administering a contrast agent to a subject and imaging the contrast agent in the subject, wherein the contrast agent comprises a compound or composition as described herein, and determining and/or mapping the subject’s GFR.

[00137] In certain embodiments, there are provided methods of diagnosing kidney dysfunction in a subject, comprising administering a contrast agent to the subject and imaging the contrast agent in the subject, wherein the contrast agent comprises a compound or composition as described herein, and determining and/or mapping the subject’s GFR.

[00138] In certain embodiments, there are provided methods of diagnosing chronic kidney disease (CKD), acute kidney injury (AKI), renal artery stenosis, urinary obstruction, and/or a renal tumor or malignancy in a subject, comprising administering a contrast agent to the subject and imaging the contrast agent in the subject, wherein the contrast agent comprises a compound or composition as described herein, and determining and/or mapping the subject’s GFR. [00139] In certain embodiments, there are provided methods of determining if a subject meets requirements for kidney donation, comprising administering a contrast agent to the subject and imaging the contrast agent in the subject, wherein the contrast agent comprises a compound or composition as described herein, and determining and/or mapping the subject’s GFR.

[00140] In certain embodiments of methods provided herein, the subject has, is suspected of having, or is at risk of renal dysfunction. A subject may have, be suspected of having, or be at risk of chronic kidney disease (CKD), acute kidney injury (AKI), renal artery stenosis, urinary obstruction, or a renal tumor or malignancy. A subject may be a kidney donor or a candidate kidney donor.

Kits

[00141] There are also provided herein kits comprising a compound or composition as described herein. Kits are generally in the form of a physical structure housing various components and may be used, for example, in practicing the methods provided herein. For example, a kit may include one or more compound or composition disclosed herein (provided in, e.g., a sterile container), which may be in the form of a pharmaceutical composition suitable for administration to a subject. The compound or composition can be provided in a form that is ready for use or in a form requiring, for example, reconstitution or dilution (e.g., a powder) prior to administration. When the compounds or compositions are in a form that needs to be reconstituted or diluted by a user, the kit may also include diluents (e.g., sterile water), buffers, pharmaceutically acceptable excipients, and the like, packaged with or separately from the compounds or compositions. Each component of the kit may be enclosed within an individual container, and all of the various containers may be within a single package. A kit of the present invention may be designed for conditions necessary to properly maintain the components housed therein (e.g., refrigeration or freezing). [00142] A kit may also contain a label or packaging insert including identifying information for the components therein and instructions for their use. Labels or inserts can include manufacturer information such as lot numbers and expiration dates. The label or packaging insert may be, e.g., integrated into the physical structure housing the components, contained separately within the physical structure, or affixed to a component of the kit (e.g., an ampoule, tube or vial).

EXAMPLES

[00143] The present invention will be more readily understood by referring to the following examples, which are provided to illustrate the invention and are not to be construed as limiting the scope thereof in any manner.

[00144] Unless defined otherwise or the context clearly dictates 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 invention belongs. It should be understood that any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention.

Example 1. Synthesis of modified verdazyl compounds.

[00145] A variety of compounds of Structural Formula (I) can be prepared according to Scheme 1 below, which shows a general scheme for verdazyl synthesis with modification to Ri (attached to nitrogen at positions 1, 5 in the ring) and R2 (attached to carbon at position 3 in the ring). In Scheme 1, reaction conditions are as follows: (a) 15% Phosgene in Toluene, 0°C - 20°C, overnight; (b) HC1 in miscible solvent, reflux temperature, 2 hrs; (c) Aldehyde-bearing R2 group in miscible solvent with 2 equivalents of non-nucleophilic base, room temperature, overnight; and (d) Potassium ferricyanide and sodium bicarbonate in polar solvent, until effervescence ceases. The synthetic procedure outlined in Scheme 1 that generates intermediate 1 on the way to synthesis of formulas (I), (II), or (III) can be performed, in addition to phosgene, using diphosgene, triphosgene, carbonyl diimidazole, disuccinimidyl carbonate, bis-pentafluoro phenyl carbonate.

Scheme 1. Synthesis of modified verdazyl compounds in accordance with certain embodiments.

[00146] A variety of compounds of Structural Formula (I) can also be prepared according to Scheme 2 below, which shows a general scheme for verdazyl synthesis with modification to Ri (attached to nitrogen at positions 1, 5 in the ring) and R2 (attached to carbon at position 3 in the ring), starting with compound 1 generated as described in Scheme 1. In brief, large quantities of either any length polyethylene glycol polymer or carbon repeat with a PMB protected alcohol can be made, then oxidized to an aldehyde, followed by closing the verdazyl ring with it and deprotecting to give a modified verdazyl compound with a free alcohol. It should be noted that, for the first steps, any group having an aldehyde at one end and a protected heteroatom at the other end (e.g., a protected alcohol, a protected amine, a protected thiol, a masked acid, etc.) can be used. The alcohol (OH) can then be functionalized to any leaving group or reactive handle that will interact with nucleophiles, allowing attachment of any functional group with an N, S, or O nucleophilic site.

[00147] In Scheme 2, reaction conditions are as follows: (a) H2O/ Acetonitrile, NaOAc, and either (or any group having an aldehyde at one end and a protected heteroatom at the other end can be used); (b) HC1 in miscible solvent, reflux temperature, 2 hrs; (c) Appropriate solvent and conditions selected for the specific reaction; (d) potassium ferricyanide and sodium bicarbonate in polar solvent, until effervescence ceases.

Scheme 2. Synthesis of modified verdazyl compounds in accordance with certain embodiments.

Example 2. Optimized Targeted Synthesis of Glucoverdazyl.

[00148] An overview of the synthesis of glucoverdazyl is shown in Scheme 3 below. A combination of previously reported 6-oxoverdazyl syntheses were used to identify an optimized route to glucoverdazyl yielding the high level of molecular purity and scalability that is required for an in vivo contrast agent. The previously reported hydrazine side chains in the literature were usually limited to short carbon chains or aryl groups (Patra et al., 2016; Pare et al., 2005; Solea et al., 2018; Barclay et al., 2002; Calabretta et al., 1991)ref 33, 35-38). Side chains were functionalized with isopropyl groups which are bulky enough side chains to help protect the delocalized radical while also improving serum retention after injection. In earlier syntheses, A-boc isopropyl hydrazine were generated from the N- boc hydrazine precursor following a previously reported synthesis by Calabretta et al. in large quantities (Kumar et al., 2018). However, commercially available isopropyl boc hydrazine was used in this instance. Compound 2 has been synthesized in a number of reported ways, all of which are di-substitutions of COCh, either as a phosgene solution or solid triphosgene (Solea et al., 2018; Le et al., 2016)). Glucoverdazyl was synthesized with both forms of phosgene with similar results. Here, the 15% phosgene in toluene solution was chosen. A route with superior yield and purity was achieved through heptane recrystallization of the crude product after the phosgene step, as first reported by Pare et al. (Solea et al., 2018). Following hoc deprotection in ethanolic hydrochloric acid to form an intermediate compound, the non-radical tetrazinanone ring (compound 3) with D- glucose was generated in the same manner as was first reported by Le et a/., 2016. Finally, oxidation of compound 3 was also performed as reported by Le et al. using potassium ferricyanide, a much milder oxidant with an easier purification process compared to the more classically used benzoquinone seen in the majority of the available verdazyl literature.

[00149] Much of the past literature that has reported on these reactions has been incomplete with respect to characterization. Here high purity 'H and ¹³C spectra are reported, as well as high-resolution mass spectrometry for each step and associated intermediates (FIGs. 7-12). The purity of compound 4 was determined by EPR spectroscopy and analytical high-performance liquid chromatography, demonstrating that this approach to glucoverdazyl yielded compound 4 fully converted from non-radical 3 (FIG. 13)

Scheme 3. Overview of the synthesis of glucoverdazyl 4.

Example 3. Characterization of Glucoverdazyl as an MRI-Active Contrast Agent.

[00150] Referring to FIGs. 1A-1F, the paramagnetic characteristics and stability of glucoverdazyl were determined. [00151] For glucoverdazyl stability measurements, the EPR was tuned to a sample of glucoverdazyl or TEMPO in PBS prior to any stability measurements. Once tuned, solutions of glucoverdazyl or TEMPO were prepared (20 mM in mouse serum or 5 mM in a 4 mM sodium ascorbate buffer pH 7.4). A single spectrum was acquired and the peak height of the most intense peak for either compound was locked. EPR scans were then acquired every 5 s for 2 hr (mouse serum) or 1.5 hr (ascorbate) to measure percent change in the activity. For stability measurements of glucoverdazyl in water, a 5 mM sample was prepared and left in a fume hood exposed to light, or wrapped in tinfoil and left in a dark fridge at 4°C. Periodically, these solutions were sampled and measured by the EPR after it was tuned using a freshly prepared 5 mM sample of glucoverdazyl.

[00152] The EPR spectrum for glucoverdazyl agreed with those previously reported, with the multiple EPR peaks being characteristic of the high degree of radical delocalization within the tetrazinanone ring (Fig. 1A; Massolle et al., 2018; Tain et al., 2017). The confirmation of this free radical indicated that glucoverdazyl could present MRI contrast enhancement (Wahsner et al., 2019). MR imaging of solution phantoms of glucoverdazyl in PBS showed a two-fold increase in Ti contrast, but no change in T2 effects relative to water (Fig. IB). The relaxivity of glucoverdazyl was expectedly less than that reported for GBCAs, but the contrast effects were of similar magnitude as other previously reported organic radical compounds, with a longitudinal relaxivity (n) of 0.30 mM'¹s'¹ ± 0.3 mM'¹s'¹ (Fig. 1C; Le et al., 2016; Matsumoto et al., 2022; Chevalier et al., 2009). While contrast enhancement was similar to TEMPO, the tetrazinanone radical was substantially more stable than the nitroxy ORCA counterpart (Fig. ID, E). Neither glucoverdazyl nor TEMPO showed any change in radical activity in mouse serum (Fig. ID), however in the presence of ascorbate, a mild biological reductant, there was no loss of the glucoverdazyl radical but complete reduction of the TEMPO nitroxy radical (Fig. IE).

[00153] The stability of the glucoverdazyl radical in solution over time was evaluated by storing solutions at room temperature in direct light or at -20°C in complete darkness. Periodic EPR sampling of these solutions showed that glucoverdazyl maintained over 50% of its radical activity after 4 months while stored on the benchtop, while the frozen solution maintained over 80% of its radical activity after 1 year (Fig. IF). This result demonstrated the resiliency of the glucoverdazyl delocalized radical in bioreductive conditions, as well as shelf-life and storability, both of which are critical features of commonly used MRI contrast agents.

[00154] Prior to in vivo experimentation, glucoverdazyl cytocompatibility was evaluated in H460 lung carcinoma epithelial cells and demonstrated no cytotoxicity compared to untreated cells in concentrations up to 10 mM (FIG. 15).

[00155] To assess cell viability in H460 cells, large-cell lung cancer cells (H460) were grown in RPMI-1640 (RPMI) media supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S) until 80% confluent, at which point they were passaged. Cells were passaged three times before being seeded in to a 6-well plate and grown until 80% confluent. Cells were seeded to have triplicate wells of each condition. Cells were then incubated in their regular media supplemented with 0 mM, 2.5 mM, 5 mM, or 10 mM glucoverdazyl, for either 4 hrs or 24 hrs. At the respective time points, the media was aspirated and cells were washed three times with 37°C Dulbecco’s phosphate-buffered saline (PBS). Afterwards, cells were lifted with trypsin-EDTA, centrifuged at 400 xg (5 min, 4°C), aspirated, then resuspended in a 1 mL PBS solution containing 0.2 pM calcein- acetoxymethyl ester (fluorescently staining live cells green) and 16 pM ethidium homodimer-1 (fluorescently staining dead cells red). Live and dead cell populations were counted by flow cytometry (Beckman-Coulter Gallios Flow Cytometer) using a 488 nm excitation with a 525 nm / 40 nm bandpass filter for calcein-acetoxymethyl ester (live cells, green) and a 620 nm / 20 nm bandpass filter for ethidium homodimer-1 (dead cells, red). After performing, the viable cell population for each condition was determined by comparing the total number of singly-stained, calcein-AM-positive cell counts to the combined total of cells that were singly-stained as positive for live or dead using Kaluza analysis software (Beckman-Coulter).

[00156] Glucoverdazyl was evaluated for contrast media suitability in vivo following intravenous injections to 9 BALB/c mice. An administered dose of 3 mmol/kg was chosen based on the difference in n between glucoverdazyl and Gadovist® (~10-fold), and the standard clinically recommended Gadovist® dose of 0.1 mmol/kg. This dose of glucoverdazyl was still well below the maximum concentration evaluated for cytocompatibility. Following injection, Ti-weighted images were acquired every 3 min following a pre-inj ection scan, which was used to establish baseline voxel intensity. Limited contrast enhancement was observed in the muscle and liver, with uptake and clearance clearly isolated to the urinary system (Fig. 2A). Overall, signal changes relative to pre-contrast scans by 127% ± 9% in the muscle, 121% ± 10% in the liver, and 184% ± 21% in the kidneys 5 min post injection were observed (Fig. 2B). The average clearance time after injection, determined by the return of the kidney ROI to baseline intensity, was approximately 40 min, coinciding with the plateau of the bladder ROI signal increase. At this timepoint, signal changes by 120% ± 8% in the muscle, 115% ± 7% in the liver, 127% ± 8% in the kidneys, and 438% ± 48% in the bladder were observed. The clearance kinetics of glucoverdazyl through the kidneys matched that of one-phase decay, affording the determination of the renal decay time constant (RDTC, k in min'¹) for glucoverdazyl clearance kidney tissue from the slope of the semi-natural log plot of the data (Fig. 2C). The linear regression was consistently performed from the contrast intensity maximum at t = 2.5 min to the most consistent return to baseline time of t = 40 min. The average k determined from the 9 healthy BALB/c mice was -0.124 min'¹ ± 0.012 min'¹, with an average R² of 0.97 ± 0.05, demonstrating excellent reproducibility of this baseline measure of healthy kidney function. [00157] With the observation that glucoverdazyl was being primarily taken up within the kidneys, cytocompatibility studies were repeated with high glucoverdazyl concentrations in human renal proximal tubule cells (hRPT; FIG. 16).

[00158] For assessment of viability of human renal proximal tubule (hRPT) cells, hRPT cells were grown in Epithelial Cell Media (EpiMEM) media supplemented with 10% FBS, 1% P/S, and epithelial cell growth supplement (EpiCGS) until 80% confluent, at which point they were passaged. Cells were passaged three times before being seeded in to a 6-well plate and grown until 80% confluent. Cells were seeded to have triplicate wells of each condition. Cells were then incubated in their regular media supplemented with regular media, 10 mM glucoverdazyl, 10 mM 5,5-dimethyl-l-pyrroline N-oxide (DMPO, a nitrone spin trap), or 10 mM (2,2,6,6-Tetramethylpiperidin-l-yl)oxyl (TEMPO, a nitroxy radical), for either 4 hrs or 24 hrs. At the respective time points, the media was aspirated and cells were washed three times with 37°C Dulbecco’s phosphate-buffered saline (PBS). Afterwards, cells were lifted with trypsin-EDTA, centrifuged at 400 xg (5 min, 4°C), aspirated, then resuspended in a 1 mL PBS solution containing 0.2 pM calcein- acetoxymethyl ester (fluorescently staining live cells green) and 16 pM ethidium homodimer-1 (fluorescently staining dead cells red).

[00159] Live and dead cell populations were counted through flow cytometry (Beckman-Coulter Gallios Flow Cytometer) using a 488 nm excitation with a 525 nm / 40 nm bandpass filter for calcein-acetoxymethyl ester (live cells, green) and a 620 nm / 20 nm bandpass filter for ethidium homodimer- 1 (dead cells, red). After performing, the viable cell population for each condition was determined by comparing the total number of singly- stained, calcein-AM-positive cell counts to the combined total of cells that were singly- stained as positive for live or dead using Kaluza analysis software (Beckman-Coulter).

[00160] While glucoverdazyl resulted in no significant increase in cell death compared to untreated cells after a 24 h incubation, >90% of the hRPT were dead after only a 4 h incubation with TEMPO. This stark difference in cytocompatibility highlights an additional key performance difference between TEMPO and tetrazinanone-derived ORCAs.

[00161] The cell uptake of glucoverdazyl by hRPT cells was evaluated by EPR spectroscopy. For evaluation of glucoverdazyl uptake in hRPT cells, hRPT cells were grown in Epithelial Cell Media (EpiMEM) media supplemented with 10% FBS, 1% P/S, and epithelial cell growth supplement (EpiCGS) until 80% confluent, at which point they were passaged. Cells were passaged three times before being seeded in to 6-well plates and grown until 80% confluent. Cells were seeded to have triplicate wells of each condition. Cells were then incubated in their regular media supplemented with regular media or 10 mM glucoverdazyl, and incubated for 24 hrs. The media was aspirated and cells were washed three times with 37°C Dulbecco’s phosphate-buffered saline (PBS). Afterwards, cells were lifted with trypsin-EDTA, centrifuged at 400 xg (5 min, 4°C), aspirated, then resuspended in 100 pl of PBS. The concentrated cell solutions were transferred to EPR tubes. A 1 pl aliquot was retained and diluted to obtain the number of cells in each solution. The EPR was tuned to a 5 mM solution of freshly prepared glucoverdazyl in PBS, then samples were measured by the EPR. Concentration was measured against a previously determined standard curve, then normalized to the number of cells previously determined to obtain nM glucoverdazyl per cell.

[00162] The results indicated that glucoverdazyl was not taken up in any detectable manner (FIG. 17). The lower glucoverdazyl uptake in extraurinary tissues, the reproducible kidney contrast efflux to the bladder, the relatively fast renal clearance time of glucoverdazyl, and its demonstrated cytocompatibility suggested that glucoverdazyl may be well suited to MRI-based evaluation of kidney function.

Example 4. Glucoverdazyl-DCE-MRI of Acute Kidney Injury through Unilateral Ureter obstruction. [00163] A unilateral ureter obstruction (UUO) mouse model was used to determine the effectiveness of glucoverdazyl as a DCE-MRI agent for an acute kidney injury (AKI) caused by obstructive nephropathy. The surgical obstruction of the left ureter prevents fluid clearance, leading to hydronephrosis and dramatically reduced kidney function of the ipsilateral kidney. Both sham (left kidney was touched by a surgical instrument) and surgical UUO (left kidney was ligated) mouse groups were evaluated. The change in voxelwise intensity over time within the kidneys was determined for the entire left and right kidney (FIGs. 3A-3D), as well as their cortex and medullary/renal pelvis (MRP) regions (FIGs. 18A-F). Voxel-wise mapping of the RDTC was used to evaluate changes in kidney function. There was no discernible change in kidney morphology between days 0, 3, or 7 in the sham group of mice, however the morphology of the kidney ipsilateral to the ureter obstruction changed dramatically on days 3 and 7 relative to day 0 in the UUO mouse group. Hydronephropathy was evident by the ablation of the medullary region on day 3, which continued to worsen at day 7, a hallmark of the UUO model (Xiong et al., 2021). The kidney contralateral to the ligated ureter in the UUO mice did not show any obvious morphological changes.

[00164] The RDTC was determined in BALB/c mice prior to surgery in order to define the optimal time interval for glucoverdazyl clearance prior to the analysis of data from diseased mice (FIGs. 18A-F). In the sham-treated mice, no significant changes in RDTC were noted graphically (FIG. 3A) and quantitatively (FIG. 3B) on days 3 or 7 relative to day 0 across both kidneys. In UUO-treated mice, however, a significant change in RDTC of the ipsilateral kidney was observed, increasing from k = -0.135 min ⁴ ± 0.018 min'¹ on day 0 to k = -0.028 min'¹ ± 0.014 min'¹ on day 3, and k = -0.013 min'¹ ± 0.032 min'¹ on day 7. The RDTC of the contralateral kidney in UUO mice was unchanged between days 0 and 3 (k = -0.133 min'¹ ± 0.020 min'¹ versus k = -0.117 min'¹ ± 0.014 min' respectively), but showed a significant increase in RDTC by day 7 (k = -0.097 min'¹ ± 0.010 min'¹). Altered physiology of the contralateral kidney following UUO is expected in rodent models, with the induction of macrophage-to-myofibroblast transition (Figueroa et al., 2019), fibrosis (Xiong et al., 2021; Bianco et al., 2019), and altered cortical mitochondrial function (Eddy et al., 2012) previously reported. This data demonstrated that glucoverdazyl-mediated DCE-MRI was able to detect the contralateral kidney functional impairment early after ipsilateral ureter obstruction.

[00165] Histological and serum creatinine (SCr) analysis was performed on both groups of mice to confirm the pathology observed through qualitative MRI images and RDTC maps, as well as to kidney function using a gold standard technique. Both the morphology and fibrosis staining of the sham kidneys and the ipsilateral kidney in UUO- treated mice were unremarkable, while the kidney ipsilateral to ureter obstruction clearly showed hydronephropathy (FIG. 3C). No significant change in SCr was detected in the sham mice between days 0 and 7 post-surgery, while a significant increase was observed in the UUO mice (FIG. 3D) These observed changes in SCr align well with those previously reported in UUO models and recapitulate the observed alterations in RDTC (Vielhauer et al., 2001; Martinez-Klimova et al., 2019; Fink et al., 1987). The fact that AUC measurements did not parallel the measured alteration in SCr supports the use of RDTC as a measure of kidney function by glucoverdazyl-mediated DCE-MRI.

[00166] While the damage caused to the kidney ipsilateral to the ureteral obstruction was evident even on anatomical MRI, the UUO model demonstrated that standard glucoverdazyl-mediated DCE-MRI techniques with simple kinetic mapping can be used to show regional and structural defects across both kidneys, alerting to the functional changes arising in the contralateral kidney even before positive fibrosis staining. A regional analysis of the data discriminating glucoverdazyl clearance from the cortex versus medulla & renal pelvis was performed (FIGs. 18A-F). Here, an impairment in glucoverdazyl clearance caused by a decrease in kidney function was clearly discerned, while also mapping where the pathology was occurring within the kidney, which is extremely valuable towards the evaluation of AKI (Matsumoto et al., 2022; Chevalier et al., 2009; Xiong et al., 2021). Example 5. Glucoverdazyl-DCE-MRI of Acute-to-Chronic Kidney Injury through Folic Acid-Induced Nephropathy (FAN).

[00167] It was next sought to evaluate renal function in a more complex, fibrosis- driven model of kidney disease mediated through folic acid-induced nephropathy (FAN). Both RDTC and AUC, as well as supporting histology and SCr were evaluated (FIG. 4). FAN is the result of tubular folic acid crystal formation following systemic administration of folic acid (Jiang et al., 2018). This crystallization causes an initial phase of severe AKI, which is followed by fibrotic renal scarring that leads to a long term, progressive decline in kidney function, resulting in CKD around 3-weeks post-folate injection. The FAN model was implemented in BALB/c mice rather than the more commonly used C57B1/6 strain, as BALB/c mice were more resistant to the AKI phase, which had an extremely high mortality rate in C57B1/6 mice.

[00168] Through anatomical imaging, a reduction in overall kidney size from day 0 to day 30 in both kidneys was noted, which has previously been reported for the FAN model (Doi et al., 2006; FIG. 4A). Following glucoverdazyl-mediated DCE-MRI, the RDTC was determined for both kidneys prior to the administration of folate, and 15- and 30-days following administration (FIG. 4A graphically, and FIG. 4B quantitatively . A significant increase in RDTC was observed at day 15 (k = -0.154 min'¹ ± 0.025 min'¹ on day 0 and k = -0.082 min'¹ ± -0.008 min'¹ on day 15), followed by a return to baseline RDTC at day 30 (k = -0.139 min'¹ ± 0.016 min'¹). The AKI phase of FAN resulted in a significant increase in medullary and cortical RDTC, indicative of both poor drainage into the ureter and poor glomerular filtration. The recovery of RDTC by day 30 is anticipated in the early stages of the CKD phase of the disease, since AKI often presents with a much more severe level of kidney dysfunction than early stages of CKD, which agreed with literature using the same post-injury time points (Doi et al., 2006). The RDTC maps at day 30 indicated the presence of localized, striated regions within the cortex presenting relatively higher RDTC (FIG. 4A right, black arrows), which map the fibrotic striations at day 30 kidneys evaluated by histology (FIG. 4C, light blue regions are indicators of fibrotic tissue highlighted by black arrows). Therefore, the combination of spatial and temporal information into single kidney maps, as was done in the case of RDTC images (FIG. 4A), afford an increased diagnostic power not afforded by existing nephrological techniques limited either to spatial or kinetic information alone.

[00169] Kidneys were harvested at days 0, 15, and 30 post-folate injection and evaluated by histology to confirm both AKI and CKD (FIG. 4C), and blood sampling was performed over the same time intervals for SCr determination (FIG. 4D). Histological evaluation revealed substantially increased fibrotic regions in the kidney at day 15, but which decreased in severity at day 30 (FIG. 4C). SCr showed a slight elevation on day 15 relative to day 0, which was expected as AKI usually shows only small elevations in SCr (Doi et al., 2006; Waikar et al., 2009). However, by day 30 during early stages of CKD, SCr was significantly increased compared to both day 0 and day 15, which is a strong indicator of a severe decrease in kidney function associated with FAN and early stages of CKD (Aparicio-Trejo et al., 2020; Yam, 2021; Scarfe et al., 2018). The importance of applying a method for kidney evaluation combining spatial and temporal evaluation was highlighted by the assessment of the FAN model. During severe AKI, minor elevations in SCr were seen, but a much larger increase in RDTC was obtained with differential regional effects across the kidney (FIG. 4A). Of unique value, RDTC maps indicated discrete regions of greater kidney dysfunction (i.e., cortical striations), which may correspond to areas of fibrosis observed in the histological evaluations (FIG. 4C).

Example 6. Comparison of Glucoverdazyl-mediated DCE-MRI to a Validated Measure of GFR.

[00170] While the measurement of GFR is of immense clinical value, current methods rely on error-prone techniques limited to kinetic measures of kidney function. Towards the development of an imaging-based approach to renal functional evaluation, a comparison of glucoverdazyl-mediated DCE-MRI to an established and validated measure of GFR was sought, namely transdermal fluorescence monitoring. The transdermal GFR technique relies on the intravenous injection of a fluorescent molecule (e.g., FITC-sinistrin) which is cleared solely by filtration, and the transdermal monitoring of blood-pool fluorescence over time. The transdermal technique applies a one-phase decay model to determine the RDTC of the fluorescence intensity versus time curve, which is then corrected to GFR by a previously determined correction factor (Schreiber et al., 2012; Friedemann et al., 2016; Shmarlouski et al., 2017; Scarfe et al., 2018; Schock-Kusch et al., 2013). The transdermal GFR measurements were used as a benchmark against which the glucoverdazyl-specific correction factor could be derived.

[00171] In a second cohort of FAN-induced mice, both transdermal (FIG. 5A) and glucoverdazyl-mediated DCE-MRI (FIG. 5B) measurements were performed on day 0 (black), day 15 (pink) and day 30 (cyan). Both techniques resulted in the hallmark one- phase decay curve, yielding RDTC values after semi-natural log transformations. On day 0, the mean RDTC determined by glucoverdazyl-mediated DCE-MRI (k =-0.135 min'¹ ± 0.022 min'¹) was significantly different from the RDTC determined by transdermal fluorescence (k = -0.075 min'¹ ± 0.011 min'¹) (FIG. 14). The difference in these RDTCs was unsurprising given the different locations for data sampling. Whereas transdermal measurements evaluate the signal from blood pool within the first few millimeters of the surface of the skin, the DCE-MRI technique evaluates signal clearance from within the kidney tissue itself. With the transdermal technique, baseline GFR = 1584 ± 238 pl/min/lOOg b.w. was determined for BALB/c mice, which is in line with literature values reported (Yan et al., 2021). By pooling the baseline RDTC data obtained for day 0 BALB/c mice, and by using the average GFR measurement from the transdermal technique, a glucoverdazyl-specific correction factor for the conversion of RDTC to GFR was derived. With this factor, GFR was calculated from t RDTC values for each post-injury time point and compared them to the GFR values determined by the transdermal technique (FIG. 5C), demonstrating that there was no significant difference in GFR determined by the two methods.

[00172] In conclusion, a preparation of a tetrazinanone-derived ORCA, glucoverdazyl, has been optimized and scaled and demonstrated its superior redox stability and cytocompatibility relative to previously used nitroxy-radical contrast agents. Glucoverdazyl appears especially useful for renal DCE-MRI due to its specific uptake limited to the kidneys, ureter, and bladder. Glucoverdazyl has been applied to imaging the UUO model of severe AKI, and FAN model of AKI-to-CKD progression, showing regional functional changes within the kidneys in the form of RDTC. The voxel-wise mapping of AUC for both models did not correlate with histological and SCr changes as reliably as did mapping RDTC, which fully corroborated kidney dysfunction. Through benchmarking to a validated transdermal fluorescent recording method of measuring GFR, it was demonstrated that glucoverdazyl affords the reliable determination of GFR by DCE- MRI. Importantly, this approach to GFR measurement not only adds a spatial component to the gold standard nephrology assessment, but also determines GFR free from the reliance upon patient demographic characteristics, which has been shown to be error-prone. Overall, glucoverdazyl may provide safer MRI-based diagnoses in patients with known or suspected AKI and/or CKD. Given the molecular properties of this organic radical, especially its inability to enter the cell, excellent cytocompatibility and preliminary biocompatibility, localization to the kidneys and quick clearance, tetrazinanone-based ORCAs represent a novel and promising class of metal-free MRI contrast agent.

Experimental Procedures for Examples 1 to 6.

[00173] General Reagents. All chemical reagents were purchased from Sigma- Aldrich and used as is unless otherwise reported, with the exception of N'-(propan-2- yl)(tert-butoxy)carbohydrazide, which was purchased from AABlocks. All cell culture reagents and consumables were purchased from ThermoFisher, with the exception of the Epithelial Cell Media and Epithelial Cell Growth Supplement, which were purchased from ScienCell.

[00174] EPR spectra. All EPR spectra were acquired on a Bruker EMX plus EPR at room temperature. All NMR spectra were acquired on a Bruker AVANCE II 400 or a Bruker Avance III HD 600. All MRI acquisitions were performed on a 3 T pre-clinical MRI (MR Solutions, Ltd.). For all MRI image data analysis, only relevant slices of the tissue of interest were included in analysis (i.e., scans for kidney region only used slices with the kidney visible). All data processing, mapping, and quantity generation was done using a program written in MATLAB 2020A®. GraphPad Prism 9.5 was used to generate all graphs, graphical figures, and statistical results.

[00175] In vivo MRI Animal Studies. All in vivo MRI animal studies were conducted under Animal Use Protocol HIe-3640-Rl approved by the IACUC at the University of Ottawa. Mice were anesthetized with isoflurane, placed on a heated cradle and inserted into the MRI. Consecutive Tl-weighted RARE images were acquired prior to and every 2.5 min thereafter for 60 minutes after contrast agent injection. Tl-weighted Imaging: slice thickness of 1 mm, FOV of 50x50 mm, averages = 3, matrix size = 96x96, TE = 11 ms, echo spacing = 7 ms, TR = 720 ms, and acquisition time of 2 minutes 16 seconds. In all cases 3 mmol/kg of contrast agent was injected intravenously through a tail vein catheter, which was flushed with saline to ensure full dose of contrast agent was received.

[00176] MRI data processing and analysis. MRI data processing and analysis was conducted as follows: (1) Intensity-Over-Time Curves. Relevant slices were adjusted to remove any automatic gain functions-associated with the MRI, and all scans and slices were normalized to water-filled fiducial marker placed alongside the mice during all scans. A MatLab routine was used to draw slicewise regions-of-interest (ROIs) at each scan time point to generate voxelwise intensity over time data, presented as the mean intensity of the total ROI for each timepoint normalized to 100% for the lowest intensity voxel for the first scan. (2) Renal Decay Time Constant Values and Image Maps. The natural logarithm of each ROI intensity value in the voxel wise intensity-over-time curve was taken to generate a semi-log curve. A linear regression was then applied from t = 2.5 min to t = 40 min, and the voxelwise slope of this curve yielded k (renal decay time constant (RDTC), min-1). RDTC values shown in graphs are the mean RDTC value per voxel. Maps of k were overlaid on top of the image acquired at t = 0 min. (3) Area-Under-the-Curve Values and Image Maps. A baseline correction was applied to each of the voxelwise intensity-overtime curves by subtracting the lowest voxel value, setting the baseline to 0 across all time points. The integral of this curve for each voxel was acquired using the trapezoids function to generate an area-under-the-curve (AUC) value. AUC values shown in graphs are the mean AUC value per voxel. Maps of AUC were overlaid on top of the image acquired at t = 0 min. An illustration of DCE-MRI data acquisition and image mapping workflow is shown in FIG. 6.

[00177] Evaluation of Glucoverdazyl Tissue Localization by DCE-MRI. Glucoverdazyl contrast-enhanced scans acquired from 9 healthy BALB/c mice were acquired as described above, and normalized intensity-over-time curves were acquired as described above, with ROIs drawn for kidney, liver, bladder, and muscle tissues.

[00178] Serum Creatinine Measurements . For all disease models, blood was drawn serially from the saphenous vein at day 0 and at each post-injury time point prior to glucoverdazyl-enhanced MRI. The blood was centrifuged for 10 min (room temperature, 900 xg) and the serum was collected from the fractionated sample. Samples were stored at -80oC until use. Serum creatinine (SCr) was determined by quantitative HPLC (Agilent 1260 Infinity with diode array equipped with a 2.1 mm x 50 mm, 5 pm particle size Agilent Zorbax 300-SCX column) against a creatinine standard curve by modifying a previously reported method (Bello et al., 2019). Briefly, creatinine was dissolved in HPLC mobile phase (15 mM sodium acetate buffer at pH 4.2 with 4% methanol and 1% acetonitrile (AcN)) and serially diluted to create a creatinine standard curve from 0 pM to 12.5 pM through integration of the produced HPLC peak at 234 nm (Bruker HyStar PP). Standards and samples were acquired at a flow rate of 0.5 mL/min in an isocratic concentration of mobile phase. Creatinine was extracted from mouse serum samples after thawing by precipitating proteins by adding a 4: 1 ratio of AcN solution containing 0.5% acetic acid to serum. Samples were vortexed, then left at -20°C for 30 min to allow complete precipitation and settling. Samples were then centrifuged at 12,000 xg (10 min, 4°C), and the supernatant transferred to a new tube. Tubes were dried to remove acidified AcN through heated vacuum centrifugation for 45 min at 50°C. The resulting pellet was resuspended in 60 pl of mobile phase and samples were subjected to HPLC with integration of the matched-time elution peak seen in the standard curve at 234 nm.

[00179] Animal Models of Kidney disease . Glucoverdazyl contrast-enhanced scans for all mice in both UUO and FAN disease models were acquired as described above, and normalized intensity-over-time curves were acquired as described above, with ROIs drawn for kidney tissue.

[00180] Unilateral Ureter Obstruction. The unilateral ureter obstruction (UUO) mouse model of acute kidney injury (AKI) was performed in C57/B16 mice as was previously reported in the literature (Vanholder et al., 2021). Briefly, 10 female C57/B16 (8 weeks old) mice were sorted into groups of 5 for either the sham procedure or UUO procedure. Mice were imaged with glucoverdazyl contrast immediately prior to surgery (day 0). Mice were anesthetized by constant isoflurane inhalation. The left kidney of the mouse was accessed laparoscopically and the left ureter was either touched gently with a surgical instrument (sham group) or tied shut with a suture (UUO group). The wound was sutured closed and mice were imaged by glucoverdazyl contrast MRI 3-days and 7-days post injury. On day 7, mice were sacrificed by cervical dislocation. The kidneys were removed and fixed in paraformaldehyde, after which they were sectioned and stained with PAS, with images acquired using a slide scanner. [00181] Folic Acid-Induced Nephropathy (Glucoverdazyl Contrast MRI). The folic acid-induced nephropathy (FAN) model of AKI-to-chronic kidney disease (CKD) injury was performed in BALB/c mice following a modification to general procedures previously reported in the literature (Van Buren et al., 2011; Chawla et al., 2014; Levin et al., 2011; Chen et al., 2019; Gama et al., 2021). An extremely high mortality rate at doses of 250 mg/kg of folic acid (FA) administered to CD1 and C57/B16 mice was noticed. BALB/c mice have been shown to be more resistant to obstruction-mediated injuries and to more reliably generate CKD (Luis-Lima et al., 2017; Niemantsversriet et al., 2021). Five BALB/c mice were imaged by glucoverdazyl-enhanced MRI at day 0. Immediately following scans, mice were injected intraperitoneally with 125 mg/kg FA in a 0.3 M sodium bicarbonate solution. Daily subcutaneous fluid support was necessary for the first 5 days following FA injection, where the effects of the FA are most severe. By day 7, mice were stabilized and were housed normally without any fluid assistance. Mice were reimaged by glucoverdazyl-enhanced MRI 15-days and 30-days post injury. On day 30, mice were sacrificed by cervical dislocation. The kidneys were removed and fixed in paraformaldehyde, after which they were sectioned and stained with PAS, with images acquired using a slide scanner. A group of mice underwent the same disease induction without any glucoverdazyl-enhanced MRI and were sacrificed at day 15 to obtain histology for this time point and to maintain uninterrupted longitudinal MRI data for this disease model.

[00182] Folic Acid-Induced Nephropathy (Transdermal Fluorescence). A parallel group of 6 mice had kidney disease induced identically to those in the contrast MRI group, except that transdermal fluorescence measurements, as previously described in the literature, were performed instead of MRI (Levey et al., 2020; Romagnani et al., 2017; Srivastava et al., 2021; Nikken et al., 2007; Warwick et al., 2022). Briefly, prior to any data acquisition, hair was removed from the right dorsolateral aspect of the mice. The next day, a transdermal fluorescence monitor (MediBeacon, Inc.) was affixed to the shaved area by a proprietary windowed adhesive patch. The battery was connected to the transdermal monitor and a 5 min baseline was established. A solution of FITC-sinistrin (150 pl, 0.2 mg/kg) was injected intravenously through the tail vein, and data was collected for 55 min. Day 0 data collection immediately preceded intraperitoneal FA injection and was repeated 15-days and 30-days post injury, at which point mice were sacrificed by cervical dislocation.

[00183] Conversion from RDTC to Glomerular Filtration Rates. Data acquired from transdermal fluorescence was analyzed by proprietary software (MediBeacon, Inc.) to generate RDTC and glomerular filtration rate (GFR) values based pharmacokinetic model fitting. GFR and RDTC data were generated using this software with a one-phase decay model of the raw data with no corrections. MediBeacon uses a previously-determined factor that can directly convert RDTC to GFR, based on mouse data they generated and compared to measured GFR through traditional methods (Levey et al., 2020; Romagnani et al., 2017; Srivastava et al., 2021; Nikken et al., 2007; Warwick et al., 2022). Given the consistency of the RDTCs in the 14 measured healthy BALB/c mice, a conversion factor for glucoverdazyl RDTC to GFR was derived based on the average GFR value of the 6 healthy BALB/c mice measured through transdermal fluorescence. By comparing the difference between the pooled RDTC compared to the transdermal-derived RDTC to normalize the average GFR value, the conversion factor was derived. This conversion was then applied to the mean RDTC value presented in the data to generate a GFR comparison between the two methods.

[00184] Synthesis of N-({N'-[(tert-butoxy)carbonyl]-N-(propan-2- yl)hydrazinecarbonyl}(propan-2-yl)amino)(tert-butoxy)formamide (2). Dry EtiN (4 ml, 28.8 mmol) was added to a solution of toluene (50 mL, pre-died with 4 A molecular sieves) followed by the addition of 4 g of A" -(propan-2 -yl)(tert-butoxy)carbohydrazide (4 g, 23 mmol). The solution was cooled to 0°C while being stirred in the atmosphere of N2. A 15% phosgene solution in toluene (9 mL, 12.7 mmol) was added dropwise for ~1 min (phosgene is highly toxic, caution is needed when performing the addition), and the reaction mixture was stirred for 1 hr at 0°C, then warmed to room temperature (rt) and stirred for additional 18 h. The reaction was quenched by adding MeOH (50 mL), was stirred for 30 min at rt and was evaporated. The mixture was diluted with 10% solution of NH4OH (75 mL), followed by extraction with EtOAc (3 x 15 mL). The combined organic was washed with brine (40 mL), dried with Na2SC>4, filtered, and evaporated, resulting in a white powder. The powder was dissolved in 80 mL of hot, dry heptane and set aside for 18 hrs at 4°C to induce crystallization of the product. The crystals were filtered off and were washed with hexanes. The product was dried on the high vacuum (colourless crystals, compound 2, 2.55 g, 59%). 'H NMR (400 MHz, CDCh) 6 6.40 (s, D2O exch., 2H ), 4.15 (s, 2H), 1.43 (s, 18H), 1.12 (s, 12H).¹³C NMR (150 MHz, CDCh) 6 = 155.9, 81.1, 52.6, 50.3, 28.2, 19.2 (broad signal) HRMS (ESI): Calculated for Ci7H34N40sNa [M+Na]+: 397.2411, found 397.2427

[00185] Synthesis of l,3-diamino-l,3-bis(propan-2-yl)urea. Compound 2 (2.55g) was resuspended in EtOH (25 mL) in a 100 mL round bottom flask and heated to 80°C (air condenser). Concentrated HC1 (10 mL) was added dropwise and the solution was left stirring for 30 min at 80 °C. The solution was cooled to rt and the solvent was evaporated. The crude product was consecutively co-evaporated once with methanol, toluene, and petroleum ether (50 mL for each). The crude product of sufficient purity for the subsequent step was dried on the high vacuum giving l,3-diamino-l,3-bis(propan-2-yl)urea dihydrochloride as colourless solid in quantitative yield. ¹ H NMR (400 MHz, MeOHJZ ) 8 4.23 (heptet, J = 6.8 Hz, 2H), 1.35 (d, J= 6.8 Hz, 12H). ¹³C NMR (150 MHz, MeOH- D₄) 6 = 162.3, 56.2, 18.9. HRMS (ESI): Calculated for C7HisN₄ONa [M+Na]+: 197.1355, found 197.1378

[00186] Synthesis of 6- [(1 S,2R,3R,4R)- 1 ,2 ,3 ,4,5-pentahydroxypentyl]-2 ,4-bis(propan- 2-yl)-l,2,4,5-tetrazinan-3-one (3). 1,3 -Diamino- 1, 3 -bis(propan-2-yl)urea dihydrochloride, (1.55 g, 6.3 mmol) was resuspended in H2O (10 mL) with stirring at rt. A 5 mL solution of D-glucose (1.2 g, 6.7 mmol) and NaOAc, 1.1 g, 13.4 mmol) in water was added dropwise over 1 min, followed by stirring at rt for 18 hrs. The reaction mixture was extracted with //-butanol (6 ^ 10 mL). The combined organic extract was dried with Na2SO₄, was filtered, and was evaporated. The resulting oil was consecutively co-evaporated with methanol, toluene, and petroleum ether (50 mL of each). The resulting product was dried on the high vacuum overnight and giving pale yellow crystals (compound 3,1.48 g, 70%). ^JH NMR (400 MHz, MeOH-D₄) 6 4.53 (m, 2H), 4.06 - 3.95 (m, 2H), 3.84 - 3.70 (m, 2H), 3.68 - 3.57 (m, 2H), 3.53 (d, J= 2.7 Hz, 1H), 1.13 (dd, J= 6.8, 3.4 Hz, 6H), 1.08 (d, J= 6.5 Hz, 6H). ¹³C NMR (150 MHz, MeOH-D₄ 6 = 155.7, 73.0, 72.4, 72.2, 72.0, 70.0, 65.0, 19.8, 19.7, 19.3, 18.9. HRMS (ESI): Calculated for Ci3H₂sN₄O6Na [M+Na]+: calculated 359.1910, found 359.1907.

[00187] Synthesis of 3-oxo-6-[(lS,2R,3R,4R)-l,2,3,4,5-pentahydroxypentyl]-2,4- bis(propan-2-yl)-l,2,3,4-tetrahydro-l,2,4,5-tetrazin-l-yl (4). Compound 3 (1.48 g, 4.41 mmol) was resuspended in H2O (5 mL with stirring (rt)). In a separate vessel, potassium ferricyanide (4.44 g, 13.5 mmol) was mixed with 80 drops (~ 4.5 mL) of NaHCCh solution (2 M), followed by the addition of water (5mL); the mixture was solubilized using an ultrasound bath. Resulting solution was added dropwise over 1 min to the original reaction mixture with stirring followed by stirring (rt) for about 30 min or until effervescence stopped. The mixture was extracted with //-butanol (6 x 10 mL). The combined organic was dried with Na2SO₄, was filtered, and was evaporated. The resulting oil was consecutively co-evaporated with methanol (50 mL), three times with toluene (50 mL each time), was cooled to 0°C, followed by co-evaporation with petroleum ether (50 mL). The resulting product was dried on the high vacuum overnight to give a bright yellow fine powder (glucoverdazyl 4, 1.09 g, 74%). Given the compound was radical in nature it could not be characterized by NMR, changes in HPLC elution time, as well as HRMS and EPR were used to confirm structure and purity. HPLC traces can be seen in FIG. 12. HRMS (ESI): Calculated for Ci3H₂₅N₄O6Na [M+Na]+: calculated 356.1676, found 356.1672. [00188] Phantom MRI and determination of glucoverdazyl longitudinal relaxivity. Contrast agent samples were prepared in 1 x PBS in standard NMR tubes, which were then inserted into a 50 mL Falcon tube containing ultrasound gel, comprising the MRI phantom. The MRI phantom was placed into a 38-mm-diameter send and receive volume coil and inserted into the MRI. A multislice Rapid Imaging with Refocused Echoes (RARE) pulse sequence was implemented for evaluation of phantoms using the following parameters for '/'/-weighted imaging: slice thickness of 5 mm, FOV of 40x40 mm, averages = 3, matrix size = 96x96, TE = 11 ms, echo spacing = 7 ms, TR = 720 ms, and acquisition time of 2 minutes 16 seconds. For 7 - weighted imaging, all parameters were the same as for Ti- weighted images, except TE = 68 ms and TR = 4800 ms, and acquisition time was 7 minutes 28 seconds.

[00189] For relaxivity measurements, the same imaging phantom was used with contrast agent concentrations of 1 to 3 mM, which were verified by electron paramagnetic spectroscopy. To measure the longitudinal relaxation rate (Ri), an inversion recovery RARE sequence was implemented with the following parameters: Slice thickness of 5 mm, FOV of 50x50 mm, average = 1, matrix size = 96x96, TE = 17 ms, TR = 5000 ms, TI = 50, 75, 100, 150, 200, 250, 300, 400, 600, 800, 1200, 2400, and 4800 ms, and acquisition time of 2 minutes 30 seconds per TI. Longitudinal relaxation rates were extracted using the mapping2 MATLAB routine written by I. Barral, M. Etezadi-Amoli, E. Gudmundson, and N. Stikov (2009), and modified by I. Rioux (2022). The longitudinal relaxivity (n) was extracted from the slope of the plot of 1/Ti vs. contrast agent concentration.

[00190] Glucoverdazyl stability measurements. The EPR was tuned to a sample of glucoverdazyl or TEMPO in PBS prior to any stability measurements. Once tuned, solutions of glucoverdazyl or TEMPO were prepared (20 mM in mouse serum or 5 mM in a 4 mM sodium ascorbate buffer pH 7.4). A single spectrum was acquired and the peak height of the most intense peak for either compound was locked. EPR scans were then acquired every 5 s for 2 hr (mouse serum) or 1.5 hr (ascorbate) to measure percent change in the activity. For stability measurements of glucoverdazyl in water, a 5 mM sample was prepared and left in a fume hood exposed to light, or wrapped in tinfoil and left in a dark fridge at 4°C. Periodically, these solutions were sampled and measured by the EPR after it was tuned using a freshly prepared 5 mM sample of glucoverdazyl.

[00191] Assessment of cell viability in H460 cells. Large-cell lung cancer cells (H460) were grown in RPMI-1640 (RPMI) media supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S) until 80% confluent, at which point they were passaged. Cells were passaged three times before being seeded in to a 6-well plate and grown until 80% confluent. Cells were seeded to have triplicate wells of each condition. Cells were then incubated in their regular media supplemented with 0 mM, 2.5 mM, 5 mM, or 10 mM glucoverdazyl, for either 4 hrs or 24 hrs. At the respective time points, the media was aspirated and cells were washed three times with 37°C Dulbecco’s phosphate-buffered saline (PBS). Afterwards, cells were lifted with trypsin-EDTA, centrifuged at 400 xg (5 min, 4°C), aspirated, then resuspended in a 1 mL PBS solution containing 0.2 pM calcein- acetoxymethyl ester (fluorescently staining live cells green) and 16 pM ethidium homodimer-1 (fluorescently staining dead cells red).

[00192] Live and dead cell populations were counted by flow cytometry (Beckman- Coulter Gallios Flow Cytometer) using a 488 nm excitation with a 525 nm / 40 nm bandpass filter for calcein-acetoxymethyl ester (live cells, green) and a 620 nm / 20 nm bandpass filter for ethidium homodimer- 1 (dead cells, red). After performing, the viable cell population for each condition was determined by comparing the total number of singly- stained, calcein-AM-positive cell counts to the combined total of cells that were singly- stained as positive for live or dead using Kaluza analysis software (Beckman-Coulter).

[00193] Assessment of viability of human renal proximal tubule (hRPT) cells. hRPT cells were grown in Epithelial Cell Media (EpiMEM) media supplemented with 10% FBS, 1% P/S, and epithelial cell growth supplement (EpiCGS) until 80% confluent, at which point they were passaged. Cells were passaged three times before being seeded in to a 6- well plate and grown until 80% confluent. Cells were seeded to have triplicate wells of each condition. Cells were then incubated in their regular media supplemented with regular media, 10 mM glucoverdazyl, 10 mM 5,5-dimethyl-l-pyrroline N-oxide (DMPO, a nitrone spin trap), or 10 mM (2,2,6,6-Tetramethylpiperidin-l-yl)oxyl (TEMPO, a nitroxy radical), for either 4 hrs or 24 hrs. At the respective time points, the media was aspirated and cells were washed three times with 37°C Dulbecco’s phosphate-buffered saline (PBS). Afterwards, cells were lifted with trypsin-EDTA, centrifuged at 400 xg (5 min, 4°C), aspirated, then resuspended in a 1 mL PBS solution containing 0.2 pM calcein- acetoxymethyl ester (fluorescently staining live cells green) and 16 pM ethidium homodimer-1 (fluorescently staining dead cells red).

[00194] Live and dead cell populations were counted through flow cytometry (Beckman-Coulter Gallios Flow Cytometer) using a 488 nm excitation with a 525 nm / 40 nm bandpass filter for calcein-acetoxymethyl ester (live cells, green) and a 620 nm / 20 nm bandpass filter for ethidium homodimer- 1 (dead cells, red). After performing, the viable cell population for each condition was determined by comparing the total number of singly- stained, calcein-AM-positive cell counts to the combined total of cells that were singly- stained as positive for live or dead using Kaluza analysis software (Beckman-Coulter).

[00195] Evaluation of glucoverdazyl uptake in hRPT cells. hRPT cells were grown in Epithelial Cell Media (EpiMEM) media supplemented with 10% FBS, 1% P/S, and epithelial cell growth supplement (EpiCGS) until 80% confluent, at which point they were passaged. Cells were passaged three times before being seeded in to 6-well plates and grown until 80% confluent. Cells were seeded to have triplicate wells of each condition. Cells were then incubated in their regular media supplemented with regular media or 10 mM glucoverdazyl, and incubated for 24 hrs. The media was aspirated and cells were washed three times with 37°C Dulbecco’s phosphate-buffered saline (PBS). Afterwards, cells were lifted with trypsin-EDTA, centrifuged at 400 xg (5 min, 4°C), aspirated, then resuspended in 100 pl of PBS. The concentrated cell solutions were transferred to EPR tubes. A 1 pl aliquot was retained and diluted to obtain the number of cells in each solution.

[00196] The EPR was tuned to a 5 mM solution of freshly prepared glucoverdazyl in PBS, then samples were measured by the EPR. Concentration was measured against a previously determined standard curve, then normalized to the number of cells previously determined to obtain nM glucoverdazyl per cell.

[00197] High performance liquid chromatography traces. In order to verify the completion of radicalization from compound 3 to 4, HPLC traces were used in conjunction with HRMS and EPR. A 20 min HPLC gradient with 0.5% TFA H2O increasing 1% to 100% over 20 min with 0.5% TFA acetonitrile was used. Elution time of compound 3 was consistently 10.5 min, while after radicalization and loss of two protons from the verdazyl ring, this increased to 11.8 min. As well, radicalization induced greater absorbance at 452 nm for compound 4, while no activity at this wavelength is observed in the non-radical compound 3. High performance liquid chromatography traces of compounds 3 and 4 to verify radical activity of the compound after radicalization step are shown in FIG. 13.

REFERENCES

[00198] P. S. T. Yuen, S. R. Dunn, T. Miyaji, H. Yasuda, K. Sharma, R. A. Star, Am J Physiol Renal Physiol 2004, 286, 1116-1119.

[00199] T. N. Le, H. Grewal, V. Changoco, V. Truong, D. J. R. Brook, Tetrahedron 2016, 72, 6368-6374

[00200] A. Massolle, T. Dresselhaus, S. Eusterwiemann, C. Doerenkamp, H. Eckert, A.

Studer, J. Neugebauer, Physical Chemistry Chemical Physics 2018, 20, 7661-7675. [00201] R. W. Tain, A. M. Scotti, W. Li, X. J. Zhou, K. Cai, Adv Exp Med Biol 2017, 977, 73-79.

[00202] J. Wahsner, E. M. Gale, A. Rodriguez-Rodriguez, P. Caravan, Chem Rev 2019, 119, 957.

[00203] K. I. Matsumoto, I. Nakanishi, Z. Zhelev, R. Bakalova, I. Aoki, Antioxid Redox Signal 2022, 36, 95-121.

[00204] R. L. Chevalier, M. S. Forbes, B. A. Thornhill, Kidney Ini 2009, 75, 1145— 1152.

[00205] Y. Xiong, Y. Chang, J. Hao, C. Zhang, F. Yang, Z. Wang, Y. Liu, X. Wang, S. Mu, Q. Xu, Front Pharmacol 2021, 12, 83.

[00206] S. M. Figueroa, M. Lozano, C. Lobos, M. T. Hennrikus, A. A. Gonzalez, C. A. Amador, Front Pharmacol 2019, 10, 1314.

[00207] Y. Xiong, Y. Chang, J. Hao, C. Zhang, F. Yang, Z. Wang, Y. Liu, X. Wang, S. Mu, Q. Xu, Front Pharmacol 2021, 12, 83.

[00208] M. Bianco, J. A. Lopes, H. J. V. Beiral, J. D. D. Filho, S. P. Frankenfeld, R. S. Fortunato, C. R. Gattass, A. Vieyra, C. M. Takiya, PLoS One 2019, 14, DOI 10.1371/JOURNAL.PONE.0218986.

[00209] A. A. Eddy, J. M. Lopez-Guisa, D. M. Okamura, I. Yamaguchi, Pediatr Nephrol 2012, 27, 1233.

[00210] M. Patra, S. G. Awuah, S. J. Lippard, J Am Chem Soc 2016, 138, 12541. [00211] E. C. Pare, D. J. R. Brook, A. Brieger, M. Badik, M. Schinke, Org Biomol Chem 2005, 3, 4258.

[00212] A. B. Solea, T. Wohlhauser, P. Abbasi, Y. Mongbanziama, A. Crochet, K. M. Fromm, G. Novitchi, C. Train, M. Pilkington, O. Mamula, Dalton Transactions 2018, 47, 4785-4789.

[00213] T. M. Barclay, R. G. Hicks, M. T. Lemaire, L. K. Thompson, Z. Xu, Chem. Commun. 2002, 1688-1689.

[00214] R. Calabretta, C. Gallina, C. Giordano, Synthesis (Stuttg) 1991, 536-539.

[00215] V. Kumar, S. Shova, V. Maurel, G. Novitchi, C. Train, EurJInorg Chem 2018, 2018, 517-524.

[00216] T. -N. Le, H. Grewal, V. Changoco, V. Truong, D. J. R. Brook, Tetrahedron

2016, 72, 6368-6374.

[00217] V. Vielhauer, H. J. Anders, M. Mack, J. Cihak, F. Strutz, M. Stangassinger, B. Luckow, H. J. Grone, D. Schlondorff, Journal of the American Society of Nephrology 2001, 12, 1173-1187.

[00218] E. Martinez-Klimova, O. E. Aparicio-Trejo, E. Tapia, J. Pedraza-Chaverri, Biomolecules 2019, 9, DOI 10.3390/BIOM9040141.

[00219] M. Fink, M. Henry, J. D. Tange, Pathology 1987, 19, 143-149.

[00220] K. Jiang, T. A. Ponzo, H. Tang, P. K. Mishra, S. I. Macura, L. O. Lerman, Am

J Physiol Renal Physiol 2018, 315, F1252. [00221] K. Doi, K. Okamoto, K. Negishi, Y. Suzuki, A. Nakao, T. Fujita, A. Toda, T. Yokomizo, Y. Kita, Y. Kihara, S. Ishii, T. Shimizu, E. Noiri, Am J Pathol 2006, 168, 1413.

[00222] S. S. Waikar, J. v. Bonventre, Journal of the American Society of Nephrology 2009, 20, 672-679.

[00223] O. E. Aparicio-Trejo, S. H. Avila-Rojas, E. Tapia, P. Rojas-Morales, J. C. Leon-Contreras, E. Martinez-Klimova, R. Hernandez-Pando, L. G. Sanchez- Lozada, J. Pedraza-Chaverri, Free Radic Biol Med 2020, 154, 18-32.

[00224] L. J. Yan, Animal Model Exp Med 2021, 4, 329.

[00225] L. Scarfe, D. Schock-Kusch, L. Ressel, J. Friedemann, Y. Shulhevich, P. Murray, B. Wilm, M. de Caestecker, J Vis Exp 2018, 2018, DOI 10.3791/58520.

[00226] W. Chaabane, F. Praddaude, M. Buleon, A. Jaafar, M. Vallet, P. Rischmann, C. I. Galarreta, R. L. Chevalier, I. Tack, Am J Physiol Renal Physiol 2013, 304, 432-439.

[00227] J. Friedemann, R. Heinrich, Y. Shulhevich, M. Raedle, L. William-Olsson, J. Pill, D. Schock-Kusch, Kidney Int 2016, 90, 1377-1385.

[00228] L. Scarfe, D. Schock-Kusch, L. Ressel, J. Friedemann, Y. Shulhevich, P. Murray, B. Wilm, M. de Caestecker, J Vis Exp 2018, 2018, 58520.

[00229] A. Shmarlouski, D. Schock-Kusch, Y. Shulhevich, V. Buschmann, T. Rohlicke, D. Herdt, M. Radle, J. Hesser, D. Stsepankou, IEEE Trans Biomed Eng 2016, 63, 1742-1750.

[00230] D. Schock-Kusch, S. Geraci, E. Ermeling, Y. Shulhevich, C. Sticht, J. Hesser, D. Stsepankou, S. Neudecker, J. Pill, R. Schmitt, A. Melk, PLoS One 2013, 8, e71519. [00231] A. Schreiber, Y. Shulhevich, S. Geraci, J. Hesser, D. Stsepankou, S. Neudecker, S. Koenig, R. Heinrich, F. Hoecklin, J. Pill, J. Friedemann, F. Schweda, N. Gretz, D. Schock-Kusch, Am J Physiol Renal Physiol 2012, 303, DOI 10.1152/ AJPRENAL.00279.2012.

[00232] A. A. Eddy, J. M. Lopez-Guisa, D. M. Okamura, I. Yamaguchi, Pediatr Nephrol 2Q12, 27, 1233.

[00233] M. Rabe, F. Schaefer, Nephron 2016, 133, 53-61.

[00234] H. M. Kang, S. H. Ahn, P. Choi, Y. A. Ko, S. H. Han, F. Chinga, A. S. D. Park, J. Tao, K. Sharma, J. Pullman, E. P. Bottinger, I. J. Goldberg, K. Susztak, Nature Medicine 201421:1 2014, 21, 37-46.

[00235] Q. Yuan, Y. Lv, H. Ding, Q. Ke, C. Shi, J. Luo, L. Jiang, J. Yang, Y. Zhou, Cell Death & Disease 2021 12:82021, 12, 1-12.

[00236] L. Marquez-Exposito, L. Tejedor-Santamaria, L. Santos-Sanchez, F. A. Valentijn, E. Cantero-Navarro, S. Rayego-Mateos, R. R. Rodrigues-Diez, A. Tejera- Munoz, V. Marchant, A. B. Sanz, A. Ortiz, R. Goldschmeding, M. Ruiz-Ortega, Front Pharmacol 2021, 12, 1510.

[00237] M. Parchure, R. Y. Ambaye, V. S. Lalitha, S. v. Gokhale, Experientia 1985, 41, 72-73.

[00238] E. Knock, L. Deng, Q. Wu, A. K. Lawrance, X. L. Wang, R. Rozen, J Nutr 2008, 138, 653-658. [00239] A. K. Bello, P. E. Ronksley, N. Tangri, J. Kurzawa, M. A. Osman, A. Singer, A. K. Grill, D. Nitsch, J. A. Queenan, J. Wick, C. Lindeman, B. Soos, D. S. Tuot, S. Shojai, K. S. Brimble, D. Mangin, N. Drummond, JAMA Netw Open 2019, 2, el910704.

[00240] R. Vanholder, L. Annemans, A. K. Bello, B. Bikbov, D. Gallego, R. T. Gansevoort, N. Lameire, V. A. Luyckx, E. Noruisiene, T. Oostrom, C. Wanner, F. Wieringa, Clin Kidney J 2021, 14, 1719-1730.

[00241] P. N. van Buren, R. Toto, Adv Chronic Kidney Dis 2011, 7S, 28-41.

[00242] L. S. Chawla, P. W. Eggers, R. A. Star, P. L. Kimmel, New England Journal of Medicine 2014, 371, 58-66.

[00243] A. Levin, P. E. Stevens, Nature Reviews Nephrology 2011 7:8 2011, 7, 446- 457.

[00244] T. K. Chen, D. H. Knicely, M. E. Grams, JAMA 2019, 322, 1294.

[00245] R. M. Gama, A. Clery, K. Griffiths, N. Heraghty, A. M. Peters, K. Palmer, H. Kibble, R. P. Vincent, C. C. Sharpe, H. Cairns, K. Bramham, PLoS One 2021, 16, e0255869.

[00246] S. Luis-Lima, E. Porrini, Nephron 2017, 136, 287-291.

[00247] M. S. A. Niemantsverdriet, T. T. Pieters, I. E. Hoefer, M. C. Verhaar, J. A. Joies, W. W. van Solinge, W. M. Tiel Groenestege, S. Haitjema, M. B. Rookmaaker, PLoS One 2021, 16, DOI 10.1371/JOURNAL.PONE.0261977.

[00248] A. S. Levey, S. M. Titan, N. R. Powe, J. Coresh, L. A. Inker, Clin J Am Soc Nephrol 2020, 15, 1203-1212. [00249] P. Romagnani, G. Remuzzi, R. Glassock, A. Levin, K. J. Jager, M. Tonelli, Z. Massy, C. Wanner, H. J. Anders, Nat Rev Dis Primers 2017, 3, DOI 10.1038/NRDP.2017.88.

[00250] A. Srivastava, I. M. Schmidt, R. Palsson, A. Weins, J. v. Bonventre, V. Sabbisetti, I. E. Stillman, H. G. Rennke, S. S. Waikar, Kidney Int Rep 2021, 6, 685-694.

[00251] J. J. Nikken, G. P. Krestin, Eur Radiol 2007, 17, 2780.

[00252] J. Warwick, J. Holness, Semin Nucl Med 2022, 52, 453-466.

[00253] L. J. Yan, Animal Model Exp Med 2021, 4, 329.

[00254] Although this invention is described in detail with reference to embodiments thereof, these embodiments are offered to illustrate but not to limit the invention. It is possible to make other embodiments that employ the principles of the invention and that fall within its spirit and scope as defined by the claims appended hereto.

[00255] The contents of all documents and references cited herein are hereby incorporated by reference in their entirety.

Claims

CLAIMS What is claimed is:

1. A compound of Formula (I), or a pharmaceutically acceptable salt or ester thereof: wherein:

Ri is selected from: wherein R is a monosaccharide; X is carbon (C) or oxygen (O); n is 1-5; Z is selected from ether, ester, carbamate, thiocarbamate, urea, thiourea, hydrazone, amide, secondary amine, tertiary amine, disulfide, triazole, cyclooctyltriazolyl, cycloocta[ ]pyridazyl, and cyanobenzyl thiazolyl-containing groups; Y is selected from a monosaccharide, a glycan, a moiety bearing amine and carboxylate separated by a substituted alpha carbon, a peptide, a nanoparticle, a dendrimer, an antibody, an antibody fragment, a nucleic acid, an aptamer, an organic targeting ligand, and R3; and R3 is selected from: wherein Xi is any halogen.

2. The compound of claim 1, wherein R2 is

3. The compound of claim 1, wherein R2 is

4. The compound of claim 1, wherein the compound is a compound of Formula (II), or a pharmaceutically acceptable salt or ester thereof:

5. The compound of claim 1, wherein the compound is a compound of Formula (III), or a pharmaceutically acceptable salt or ester thereof: wherein:

Yi is selected from:

X is C or O; n is 1-5;

Z is selected from ether, ester, carbamate, thiocarbamate, urea, thiourea, hydrazone, amide, secondary amine, tertiary amine, disulfide, triazole, cyclooctyltriazolyl, cycloocta[ ]pyridazyl, and cyanobenzyl thiazolyl-containing groups; and Y2 is selected from a monosaccharide, a glycan, a moiety bearing amine and carboxylate separated by a substituted alpha carbon, a peptide, a nanoparticle, a dendrimer, an antibody, an antibody fragment, a nucleic acid, an aptamer, and another organic targeting ligand.

6. The compound of any one of claims 1 to 5, wherein the monosaccharide is a 6-carbon sugar.

7. The compound of any one of claims 1 to 5, wherein the monosaccharide is glucose, fructose, galactose, or mannose.

8. The compound of claim 1, wherein the compound is:

or a pharmaceutically acceptable salt or ester thereof.

9. A composition comprising the compound of any one of claims 1 to 8 and a carrier.

10. The composition of claim 9, wherein the composition is a pharmaceutical composition, and the carrier is a pharmaceutically acceptable carrier.

11. The compound of any one of claims 1 to 8 or the composition of claim 9 or 10, for use in biomedical imaging.

12. The compound of any one of claims 1 to 8 or the composition of claim 9 or 10, for use as a contrast agent.

13. The compound or the composition for use of claim 11 or 12, wherein the compound is glucoverdazyl.

14. A method of biomedical imaging, comprising administering a contrast agent to a subject and imaging the contrast agent in the subject, wherein the contrast agent comprises the compound of any one of claims 1 to 8 or the composition of claim 9 or 10.

15. The method of claim 14, wherein the biomedical imaging comprises magnetic resonance imaging (MRI).

16. The method of claim 15, wherein the MRI is dynamic contrast enhanced magnetic resonance imaging (DCE-MRI).

17. The method of claim 15, wherein the MRI is contrast enhanced magnetic resonance imaging (CE-MRI).

18. The method of any one of claims 14 to 17, wherein the imaging is imaging of the kidney.

19. The method of any one of claims 14 to 18, wherein the imaging is used to assess kidney function.

20. The method of any one of claims 14 to 19, wherein the subject has, is suspected of having, or is at risk of renal dysfunction.

21. The method of any one of claims 14 to 20, wherein the subject has, is suspected of having, or is at risk of chronic kidney disease (CKD), acute kidney injury (AKI), renal artery stenosis, urinary obstruction, or a renal tumor or malignancy, or is a kidney donor.

22. The method of any one of claims 14 to 20, further comprising determining the subject’s Glomerular Filtration Rate (GFR).

23. The method of any one of claims 14 to 22, wherein quantitative and/or qualitative kidney functional information is obtained.

24. The method of any one of claims 14 to 23, further comprising mapping the subject’s Glomerular Filtration Rate (GFR).

25. A method of diagnosing kidney dysfunction in a subject, comprising administering a contrast agent to the subject, imaging the contrast agent in the subject, and determining and/or mapping the subject’s Glomerular Filtration Rate (GFR), wherein the contrast agent comprises the compound of any one of claims 1 to 8 or the composition of claim 9 or 10.

26. A method of monitoring, assessing or determining kidney function in a subject, comprising administering a contrast agent to the subject, imaging the contrast agent in the subject, and determining and/or mapping the subject’s Glomerular Filtration Rate (GFR), wherein the contrast agent comprises the compound of any one of claims 1 to 8 or the composition of claim 9 or 10.

27. The method of any one of claims 14 to 26, wherein the compound is glucoverdazyl.

28. A contrast agent for biomedical imaging comprising the compound of any one of claims 1 to 8 or the composition of claim 9 or 10.

29. The compound of any one of claims 1 to 8 or the composition of claim 9 or 10, provided that the compound is not glucoverdazyl.

30.Use of a compound of any one of claims 1 to 8 or the composition of claim 9 or 10 as a contrast agent for biomedical imaging, wherein the contrast agent is formulated for administration to a subject.

31. The use of claim 30, wherein the biomedical imaging comprises magnetic resonance imaging (MRI).

32. The use of claim 31, wherein the MRI is dynamic contrast enhanced magnetic resonance imaging (DCE-MRI).

33. The use of claim 31, wherein the MRI is contrast enhanced magnetic resonance imaging (CE-MRI).

34. The use of any one of claims 30 to 33, wherein the imaging is imaging of the kidney.

35. The use of any one of claims 30 to 34, wherein the imaging is used to assess kidney function.

36. The use of any one of claims 30 to 35, wherein the subject has, is suspected of having, or is at risk of renal dysfunction.

37. The use of any one of claims 30 to 36, wherein the subject has, is suspected of having, or is at risk of chronic kidney disease (CKD), acute kidney injury (AKI), renal artery stenosis, urinary obstruction, or a renal tumor or malignancy, or is a kidney donor.

38. The use of any one of claims 30 to 36, wherein the use further comprises determination of the subject’s Glomerular Filtration Rate (GFR).

39. The use of any one of claims 30 to 38, wherein quantitative and/or qualitative kidney functional information is obtained.

40. The use of any one of claims 30 to 39, wherein the use further comprises mapping the subject’s Glomerular Filtration Rate (GFR).

41. Use of a compound of any one of claims 1 to 8 or the composition of claim 9 or 10 as a contrast agent for diagnosing kidney dysfunction in a subject, wherein the contrast agent is formulated for administration to the subject.

42. The use of claim 41, wherein the use further comprises imaging the contrast agent in the subject, and determining and/or mapping the subject’s Glomerular Filtration Rate (GFR).

43. Use of compound of any one of claims 1 to 8 or the composition of claim 9 or 10 as a contrast agent for monitoring, assessing or determining kidney function in a subject, wherein the contrast agent is formulated for administration to the subject.

44. The use of claim 43, wherein the use further comprises imaging the contrast agent in the subject, and determining and/or mapping the subject’s Glomerular Filtration Rate (GFR).

45. The use of any one of claims 30 to 44, wherein the compound is glucoverdazyl.