Verdazil compounds as contrast agents for kidney magnetic resonance imaging

Description

1 / 116 VERDAZIL COMPOUNDS AS CONTRAST AGENTS FOR GENERATING KIDNEY FIELD MRI IMAGES

[001] The present description refers to metal-free contrast agents for magnetic resonance imaging, methods of manufacturing them and their uses. BASIS

[002] Chronic kidney disease (CKD) and acute kidney injury (AKI) represent a major health problem worldwide. CKD often develops slowly and without obvious symptoms in the early stages, but becomes progressively more debilitating in later stages, with limited chances of reversal. CKD outcomes are improved with early interventions, facilitated by early detection.

[003] The clinical gold standard for measuring renal function is the measurement of glomerular filtration rate (GFR). The clinical diagnosis of CKD in North America is defined as an estimated GFR (eGFR) < 60 mL / min / 1.73 m2 for more than 3 months, or a urinary albumin / creatinine ratio (ACR) > 30 mg / g for more than 3 months.

[004] However, the diagnostic values ​​for CKD were derived from large clinical studies in an ethnically limited population, significantly reducing the power. Petition 870250102279, dated 07 / 11 / 2025, page 10 / 144 2 / 116 diagnosis of these disease biomarkers. Furthermore, the underlying causes of CKD can vary among individuals, with some of the most common, including diabetes mellitus, cardiovascular disease, and kidney transplantation, potentially limiting the accuracy of eGFR measurements at the patient level. Nearly 30% of these patients may exhibit a 30% deviation from their actual eGFR. In addition, besides an individual's deviation from the derived population, the eGFR equation assumes steady-state creatinine levels and does not account for alterations or alternative pathways of creatinine production, leading to significant variability. Finally, a major limitation of eGFR measurement is that it does not provide physicians with spatial or structural information underlying renal dysfunction.

[005] There is a need for a more precise method of eGFR 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) can be a powerful tool for renal assessment in cases of suspected AKI and CKD. However, there is a strong negative bias against gadolinium-based contrast agents due to the possibility of inducing nephrogenic systemic fibrosis (NSF) in patients with limited renal function.

[006] There is a need for contrast agents that Petition 870250102279, dated 07 / 11 / 2025, page 11 / 144 3 / 116 can be used to provide a reliable, rapid, and quantitative clinical imaging approach for the early detection of CKD and other disorders. SUMMARY

[007] It is an object of the present invention to alleviate at least some of the deficiencies present in the prior art. Implementations of the present technology have been developed based on the inventors' understanding that there is a need for improved contrast agents for clinical purposes.

[008] The inventors have found that novel verdazil derivatives can provide metal-free contrast agents suitable for clinical use. The compounds provided in this document may enable reliable, rapid and / or quantitative clinical imaging, which may facilitate the diagnosis and early detection of various disorders. In particular, the compounds are advantageous for contrast-enhanced magnetic resonance imaging (CMR-MRI) and dynamic contrast-enhanced magnetic resonance imaging (DCI-MRI). In certain modalities, the compounds may demonstrate greater stability and / or reduced cytotoxicity compared to previous organic radical contrast agents.

[009] Swager et al. (U.S. Patent No. 8,715,621) described a variety of radicals that are useful as Petition 870250102279, dated 07 / 11 / 2025, page 12 / 144 4 / 116 polarizing compounds, but verdazil is used only as a radical initiator and not as an image-generating agent per se.

[0010] In one aspect, a compound represented by Structural Formula (I), or a pharmaceutically acceptable salt or ester thereof, is provided: in which: Ri and R-2 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.

[0011] In some embodiments, Ri and / or R2 are substituted or unsubstituted C1 to C2 alkyl groups. In some of these embodiments, Ri and / or R2 are substituted or unsubstituted C1 to C2 alkyl groups. In some of these embodiments, Ri and / or R2 are substituted or unsubstituted C4 to C2 cycloalkyl groups. In Petition 870250102279, dated 07 / 11 / 2025, p. 13 / 144 5 / 116 In some of these embodiments, Ri and / or R2 are substituted or unsubstituted C4 to Cs heterocycloalkyl groups. In some of these embodiments, Ri and / or R2 comprise a substituted or unsubstituted hydroxyl, amino, or thio group. In some embodiments, Ri and / or R2 are a substituted or unsubstituted benzaldehyde. In some embodiments, Ri and / or R2 comprise at least one heteroatom that is N, S, or O.

[0012] In certain embodiments of the compound of Formula (I), a compound represented by the Formula structural (I), or a salt or acceptable thereof: O 5N 6 Ni r2 where: Ri is selected from: ' Ίθ / / ^'cH3 R2 is selected from: pharmaceutically .Ri ester (I) Petition 870250102279, dated 07 / 11 / 2025, p. 14 / 144 6 / 116 OH OH OH OH OH NH2 in which: R is a monosaccharide; X is carbon (C) or oxygen (O); n is an integer from 1 to 5 (that is, 1, 2, 3, 4, or 5); Z is ether, ester, carbamate, thiocarbamate, urea, thiourea, hydrazone, amide, secondary amine, tertiary amine, disulfide, triazole, cyclooctyltriazolyl, cycloocta[d]pyridazyl or a group containing cyanobenzylthiazolyl; Y is a monosaccharide, a glycan, an amine containing a chemical moiety and a 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 R3 is selected from: Petition 870250102279, dated 07 / 11 / 2025, page 15 / 144 7 / 116 where Xi is any halogen.

[0013] In certain embodiments of the compound of Formula %R3 (I), R2 is n, where n and R3 are as described above.

[0014] In certain embodiments of the Formula compound X^y (I), R2 is n, where X, Z, Y and n are as described above.

[0015] In certain embodiments of the compound of Formula (I), a monosaccharide includes, but is not limited to, a 6-carbon sugar, for example, glucose, fructose, galactose or mannose.

[0016] 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 intended to be particularly limited; it may be a 6-carbon sugar (a hexose), a 5-carbon sugar (a pentose), a pyranose, a furanose, or another monosaccharide chemically suitable for binding to the compound.

[0017] In certain embodiments of the compound of Formula (I), the monosaccharide is a C3 to C7 carbohydrate, that is, possessing from 3 to 7 carbon atoms.

[0018] In certain embodiments of the compound of Formula (I), the monosaccharide is not glucose. Petition 870250102279, dated 07 / 11 / 2025, p. 16 / 144 8 / 116

[0019] 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: (II) where: Ri, Rs and en are as described above.

[0020] 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: (III) in which: Yi is selected from: Petition 870250102279, dated 07 / 11 / 2025, p. 17 / 144 9 / 116 X is either C or O; n is from 1 to 5; Z is ether, ester, carbamate, thiocarbamate, urea, thiourea, hydrazone, amide, secondary amine, tertiary amine, disulfide, triazole, cyclooctyltriazolyl, cycloocta[d]pyridazyl or a group containing cyanobenzyl thiazolyl; and Y2 is a monosaccharide, a glycan, an amine containing a chemical moiety and a 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.

[0021] In certain embodiments of the compound of Formula (III), a monosaccharide includes, but is not limited to, a 6-carbon sugar, for example, glucose, fructose, galactose or mannose.

[0022] 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 intended to be particularly limited; it may be a 6-carbon sugar, a 5-carbon sugar, or another monosaccharide chemically suitable for binding to the compound. In some embodiments, the monosaccharide is a C3 to C7 carbohydrate, that is, with 3 to 7 Petition 870250102279, dated 07 / 11 / 2025, p. 18 / 144 10 / 116 carbon atoms.

[0023] In certain embodiments of the compound of Formula (I), the compound is a compound shown in Table 1, or a pharmaceutically acceptable salt or ester thereof. The symbol is used to indicate a nitrogen radical in the compound.

[0024] In certain embodiments of the compound of Formula (I), the compound is not glucoverdazil. Table 1. Examples of Compounds of Formula (I), according to certain modalities Name Structure Glucoverdazil OH OH N HO, A- _N, / j YNY OH OH । Maltoverdazil OH HO^^Y°H <OHOH N-T° H0 .to ΑθΑγγ-N y OH OH । Celobioverdazil QH HO. .OH / OH ,I\L .0 *Y Y^ < OH N γ Ho\ ÁJwkAArNx / W Q YY N OH OH । Carboxiverdazil 1 0 ^ή'Ν^^οη o^n'N Petition 870250102279, dated 07 / 11 / 2025, page 19 / 144 11 / 116

[0025] It should be understood that all acidic, saline, basic, and other ionic and non-ionic forms of the compounds described in this document should be covered. For example, if a compound is shown as an acid in this Petition 870250102279, dated 07 / 11 / 2025, page 20 / 144 12 / 116 document, the salt forms of the compound will also be covered. Similarly, if a compound is shown as a salt, the acidic and / or basic forms will also be covered.

[0026] In certain modalities of the compound of Formula (I), the compound is suitable for use as a contrast agent. In some of these modalities, the compound is suitable for use in RMD-CE and / or RMD-DCE. In some of these modalities, the compound is suitable for biomedical imaging, such as, without limitation, for the diagnosis of renal dysfunction, for example, by determining and / or mapping the GFR.

[0027] In another aspect, a composition is provided comprising a compound of the description and a carrier.

[0028] In certain embodiments, the composition is a pharmaceutical composition comprising a compound of the description and a pharmaceutical carrier.

[0029] In certain modalities, the composition is suitable for use as a contrast agent.

[0030] In certain embodiments, the carrier is an aqueous solution. The carrier may be saline solution, water, phosphate-buffered saline (PBS), or 5% dextrose in water. Such compositions may be used for biomedical applications, such as imaging.

[0031] In a further aspect, a method is provided for the production of embodiments of the compound or composition, Petition 870250102279, dated 07 / 11 / 2025, page 21 / 144 13 / 116 as described in this document.

[0032] In another aspect, methods for generating biomedical images are provided, comprising administering a contrast agent to an individual and obtaining images of the contrast agent in the individual, wherein the contrast agent comprises the compound or composition as described in this document.

[0033] In certain modalities, biomedical imaging involves magnetic resonance imaging (MRI). MRI may involve contrast-enhanced magnetic resonance imaging (CEM-MRI) and / or dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI).

[0034] In certain modalities, a biomedical imaging method of the kidney is provided, comprising administering a contrast agent to an individual and obtaining images of the contrast agent in the individual, wherein the contrast agent comprises the compound or composition as described in this document. In some of these modalities, the method further comprises determining the individual's Glomerular Filtration Rate (GFR). In certain modalities, quantitative and / or qualitative information about renal function is obtained, such as, for example, the determination and / or spatial mapping of the individual's GFR. Such methods may be used, for example, to diagnose renal dysfunction and / or to monitor or evaluate the individual's renal function. Petition 870250102279, dated 07 / 11 / 2025, page 22 / 144 14 / 116

[0035] In certain modalities, a method is provided for diagnosing renal dysfunction, comprising administering a contrast agent to an individual and obtaining images of the contrast agent in the individual, wherein the contrast agent comprises the compound or composition as described in this document, and determining and / or mapping the individual's Glomerular Filtration Rate (GFR).

[0036] In certain modalities, a method is provided for monitoring, evaluating, or determining renal function, comprising administering a contrast agent to an individual and obtaining images of the contrast agent in the individual, wherein the contrast agent comprises the compound or composition as described in this document, and determining and / or mapping the individual's Glomerular Filtration Rate (GFR).

[0037] In certain modalities of the description methods, the individual has, is suspected of having, or is at risk of having renal dysfunction. The individual may have, be suspected of having, or be at risk of having chronic kidney disease (CKD), acute kidney injury (AKI), renal artery stenosis, urinary obstruction, and / or renal tumor or malignancy. In some modalities, the subject may be a candidate for kidney donation.

[0038] In certain embodiments of the description methods, the compound is glucoverdazil. Petition 870250102279, dated 07 / 11 / 2025, page 23 / 144 15 / 116

[0039] In a further aspect, the compound or composition as described in this document is provided for use in imaging, such as biomedical imaging. In certain embodiments, the compound or composition is for use in kidney imaging.

[0040] In another aspect, the compound or composition as described and / or claimed herein is provided for use as a contrast agent. The contrast agent can be used during imaging using modalities such as magnetic resonance imaging, MRI-C, MRI-D and the like.

[0041] In a further aspect, a metal-free contrast agent for biomedical imaging is provided comprising the compound or composition as described in this document.

[0042] In another aspect, a kit comprising the contrast compound, composition or agent as described in this document is provided. The kits may additionally comprise a buffer or excipient and / or instructions for use, for example, in biomedical imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The patent file or patent application contains at least one drawing executed in color. Copies of this patent publication or patent application with color drawing(s) will be provided by the Office upon request. Petition 870250102279, dated 07 / 11 / 2025, page 24 / 144 16 / 116 request and payment of the required fee.

[0044] For a better understanding of the invention and to show more clearly how it can be implemented, reference will now be made, by way of example, to the accompanying drawings, which illustrate aspects and features in accordance with embodiments of the present invention.

[0045] FIGS. 1A to 1F show paramagnetic characteristics and stability of glucoverdazil, according to certain embodiments of the present technology. FIG. 1A: Electron paramagnetic resonance (EPR) spectra of a 5 mM glucoverdazil solution in phosphate-buffered saline (PBS) acquired at room temperature; FIG. 1B: Ti- and T2-weighted magnetic resonance images of a 3 mM glucoverdazil solution in PBS acquired at 3 T; FIG. 1C: Longitudinal relaxivity of glucoverdazil at pH 7.4 in PBS at 3 T magnetic resonance. The stability of glucoverdazil (black) and TEMPO (pink) was determined by EPR during 2 hours of incubation in mouse serum (FIG. 1D) or in 4 mM sodium ascorbate buffer at pH 7.4 (FIG. 1E). FIG.1F: Storage stability of glucoverdazil 5 mM solutions left exposed to light at room temperature (pink) or left in the freezer (-20 °C) protected from light (black), as determined by EPR.

[0046] FIGS. 2A to 2C show the location and the Petition 870250102279, dated 07 / 11 / 2025, p. 25 / 144 17 / 116 In vivo clearance of glucoverdazil in healthy BALB / c mice. FIG. 2A: Magnetic resonance imaging of BALB / c mice was obtained both before injection and every 2.5 minutes after injection, following administration of glucoverdazil (3 mmol / kg). FIG. 2B: Regions of interest (ROIs) were selected and the mean intensity at each time point was obtained. Data are presented as means ± SEM for n = 9 mice. FIG. 2C: The semi-natural logarithmic transformation and the line of best fit (pink) are plotted from t = 2.5 min to t = 40 min of the renal 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. The renal decay time constant (k) and R2 are presented as means ± SD.

[0047] FIGS. 3A to 3D show RMD-DCE with glucoverdazil in a murine model of unilateral ureteral obstruction. FIG. 3A: Ti-weighted images of the kidneys at t = 2.5 min after injection (top) and RDTC maps (bottom) for the Placebo and UUO groups. FIG. 3B: RDTC values ​​for each kidney at each time point after injury. Data are presented as box-and-whisker plots of the single mean RDTC value of each kidney (ipsilateral or contralateral) individually, from each mouse (n = 5). FIG. 3C: Petition 870250102279, dated 07 / 11 / 2025, p. 26 / 144 18 / 116 Representative histology of paired kidneys from the placebo (left) and UUO (right) treatment groups stained with PAS. FIG. 3D: Serum creatinine levels of placebo (gray) and UUO (turquoise) mice on the two sampling days. Data are presented as individual SCr values. Statistical analysis was performed by two-way ANOVA for repeated measures, followed by a Tukey post-hoc test. In all graphs, * p < 0.05, ** p < 0.01 and **** p < 0.0001.

[0048] FIGS. 4A to 4D show DCE-MRI with glucoverdazil of folic acid-induced nephropathy. FIG. 4A: Ti-weighted images of the kidneys at t = 2.5 min after injection (top) and RDTC maps (bottom). FIG. 4B: RDTC values ​​for the kidneys at each time point after injury. Data are presented as box-and-whisker plots of the single mean RDTC value of both kidneys from each mouse (n = 5). FIG. 4C: Representative histology of the kidneys (top) with magnifications of the cortical or medullary regions (bottom) stained with PAS. Black arrows indicate positive histological staining for fibrotic areas, represented by a light blue color. FIG. 4D: Serum creatinine levels of ANA mice at each post-injury time point (n = 5). Statistical analysis was performed using a two-way repeated measures ANOVA, followed by Tukey's posthoc test. In all graphs, * p < 0.05, ** p < 0.01 Petition 870250102279, dated 07 / 11 / 2025, p. 27 / 144 19 / 116 and *** p < 0.001.

[0049] FIGS. 5A to 5D show the determination of glomerular filtration rate (GFR) by transdermal fluorescence and dynamic contrast-enhanced magnetic resonance imaging (DCM-MRI) in mice with folic acid nephropathy (FA), according to certain modalities of the present technology. FIG. 5A: Normalized fluorescence intensity of transdermal fluorescence clearance of FITC-sinistrine in FA mice on days 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 intensity of DCM-MRI of glucoverdazil-enhanced examinations over time for FA mice on days 0, 15, and 30 (black, pink, and turquoise, respectively). The data are presented as the mean of each time point for each replicate ± SEM for n = 5 mice. FIG.Figure 5C: GFR values ​​for ANA mice determined by transdermal fluorescence (gray) or DCE-MRI (pink). Data are presented as box-and-whisker plots of GFR for each mouse. Statistical analysis was performed by two-way ANOVA for mixed measures, followed by a Tukey post-hoc test. The p-value for each test is indicated in the graph. FIG. 5D: Illustration of glucoverdazil clearance through the kidney by DCE-MRI to detail the differences between. Petition 870250102279, dated 07 / 11 / 2025, page 28 / 144 20 / 116 transdermal and MRI-DCE measurements.

[0050] FIG. 6 shows an illustration of the data acquisition and image mapping workflow of RMD-DCE, according to certain modalities of the present technology.

[0051] FIG. 7 shows the 1H NMR spectrum of compound 2 synthesized according to certain embodiments of the present technology.

[0052] FIG. 8 shows the 13C NMR spectrum of compound 2 synthesized according to certain embodiments of the present technology.

[0053] FIG. 9 shows the 1H NMR spectrum of the intermediate compound synthesized according to certain embodiments of the present technology.

[0054] FIG. 10 shows the 13C NMR spectrum of the intermediate compound synthesized according to certain embodiments of the present technology.

[0055] FIG. 11 shows the 1H NMR spectrum of compound 3 synthesized according to certain embodiments of the present technology.

[0056] FIG. 12 shows the 13C NMR spectrum of compound 3 synthesized according to certain embodiments of the present technology.

[0057] FIG. 13 shows high-performance liquid chromatography traces of compounds 3 and 4 synthesized for Petition 870250102279, dated 07 / 11 / 2025, p. 29 / 144 21 / 116 verify the radical activity of the compound after the radicalization step, according to certain embodiments of the present technology.

[0058] FIG. 14 shows (A) a comparison between the RDTC value of transdermal fluorescence 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 RMD-DCE with glucoverdazil. (B) A comparison between the RDTC value in healthy BALB / c mice using voxel-derived intensity measurements or the whole ROI of a slice at each time point (n = 14 mice for both). Data are presented as box-and-whisker plots of single RDTC (A, transdermal) or single mean RDTC (A, RM and B). Statistical analysis was performed by one-way ANOVA followed by a Tukey post-hoc test. In all plots, ns indicates not significant and **** indicates p < 0.0001.

[0059] FIG. 15 shows the cytotoxicity assessment of glucoverdazil at different concentrations in H460 after 4 h (A) and 24 h (B) incubations with the respective concentration. Data are represented as means ± SD of n = 3 replicates. Statistical analysis was performed by one-way ANOVA followed by a Tukey post-hoc test.

[0060] FIG. 16 shows the cytotoxicity assessment of glucoverdazil, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) Petition 870250102279, dated 07 / 11 / 2025, page 30 / 144 22 / 116 and 2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) in human renal proximal tubule cells at concentrations of 10 mM after 4 h (A) or 24 h (B) incubations under cell culture conditions. Data are represented as means ± SD of n = 3 replicates. Statistical analysis was performed by one-way ANOVA followed by a Tukey post-hoc test. * p < 0.05 of live and dead cell populations compared to populations from the other conditions.

[0061] FIG. 17 shows the uptake of glucoverdazil in human renal proximal tubule cells after 24 hours of incubation in 10 mM glucoverdazil under cell culture conditions. Uptake was measured by EPR activity compared to a known concentration and normalized for the number of cells loaded in the EPR tube. Data are represented as means ± SD of n = 3 replicates. Statistical analysis was performed by one-way ANOVA followed by a Tukey post-hoc test.

[0062] FIGS. 18A to F show RMD-DCE data with glucoverdazil in the unilateral ureteral obstruction (UUO) model. FIG. 18A: Normalized intensity curves over time of UUO mice on day 0. The curve is shown as normalized mean intensity ± SEM. FIG. 18B: Semi-natural logarithmic regression curve of FIG. 18A with the line of best fit from t = 0 min to at = 40 min. The data are Petition 870250102279, dated 07 / 11 / 2025, p. 31 / 144 23 / 116 shown as mean RDTC (k) and R2 ± SD value. RDTC values ​​for the regions of interest (ROIs) of the cortex and renal medulla and pelvis (MRP) for the placebo group (FIG. 18C) and the UUO group (FIG. 18D). Data are presented as box-and-whisker plots of the single mean RDTC value of each kidney (ipsilateral or contralateral) of each region, individually, of each mouse (n = 5). The same data and plots for AUC are shown in FIGS. 18E and 18F for the placebo and UUO groups, respectively. Statistical analysis was performed by two-way repeated measures ANOVA, followed by a Tukey post-hoc test. In all graphs, ns is not significant, * p < 0.05, *** p < 0.001 and **** p < 0.0001.

[0063] FIGS. 19A to 19C show RMD-DCE data with glucoverdazil in the folic acid nephropathy (FA) model. FIG. 19A: RDTC values ​​for the regions of interest (ROIs) of the cortex and renal medulla and pelvis (MRP). Data are presented as box-and-whisker plots of the single mean RDTC value for each mouse (n = 5). The same data and plots for AUC are shown in FIG. 19B. FIG. 19C: Time-normalized intensity curves for the whole-kidney FA model, as well as for the cortex and MRP regions for each of the time points after injury. Curves are presented as the mean normalized intensity at each time point ± SEM. Statistical analysis was Petition 870250102279, dated 07 / 11 / 2025, page 32 / 144 24 / 116 performed by two-way repeated measures ANOVA, followed by a Tukey post-hoc test. In all graphs, ns not significant, * p < 0.05, ** p < 0.01 and **** p < 0.001. DETAILED DESCRIPTION

[0064] Glomerular filtration rate (GFR) measurement is the clinical gold standard for measuring renal function. However, it relies on incorporating physiological, demographic, and blood metabolite levels into an equation derived from a limited and underrepresentative population, and does not provide spatial information about renal dysfunction. Dynamic contrast-enhanced magnetic resonance imaging (DCI-MRI) can be powerful for renal assessment in cases of suspected AKI and CKD; however, there is a strong negative bias against gadolinium-based contrast agents due to the possibility of inducing nephrogenic systemic fibrosis in patients with limited renal function.

[0065] To purposefully develop a contrast agent for RMD-DCE suitable for use as a diagnostic tool for renal pathology, a class of extremely stable nitrogen-centered organic radicals, known as verdazils, was evaluated. Verdazils provide excellent T1 to 3T shortening, derived from an extremely stable radical, without signal loss in highly reducing environments. This study demonstrated that a verdazil modified by Petition 870250102279, dated 07 / 11 / 2025, page 33 / 144 25 / 116 glucose, glucoverdazil, showed no extrarenal uptake in vitro and in vivo, with contrast imaging limited to the kidneys and bladder. Renal functional deficits were demonstrated over time in murine models of unilateral ureteral obstruction (UUO) and folic acid-induced nephropathy (ANA), with renal functional maps correlated with histological and blood biomarkers of renal dysfunction. Using the ANA model, glucoverdazil clearance rates were shown to be a reliable measure of GFR, as demonstrated by comparison with a validated transdermal fluorescence technique. The present description is based, at least in part, on the finding that glucoverdazil may be an extremely potent metal-free magnetic resonance (MRI) contrast agent.In certain modalities, the contrast agents described can provide reliable GFR measurements, free from error-prone population-derived equations; spatial or structural information underlying renal dysfunction; and / or enable a personalized, image-based medicine approach for nephrology or other areas. In certain modalities, the compounds of the metal-free contrast agents provided are stable and non-toxic.

[0066] In order to provide a clear and consistent understanding of the terms used in this report Petition 870250102279, dated 07 / 11 / 2025, page 34 / 144 26 / 116 descriptive, some definitions are provided below. Furthermore, unless defined otherwise, all technical and scientific terms used in this document have the same meaning commonly understood by someone skilled in the art to which this invention relates.

[0067] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and / or descriptive report may mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more of one." Similarly, the word "another" may mean "at least one second or more."

[0068] As used in this descriptive report and claims, the words comprising (and any form of comprising, such as comprising and comprises), having (and any form of having, such as having and has), including (and any form of including, such as including and includes) or containing (and any form of containing, such as containing and contains), are inclusive or open-ended and do not exclude additional elements or process steps not mentioned.

[0069] The term approximately is used to indicate that a value includes an inherent error variation for the device or method employed to determine the value.

[0070] The terms derivative and variant are used interchangeably in this document. Petition 870250102279, dated 07 / 11 / 2025, page 35 / 144 27 / 116

[0071] The term individual, as used in this document, includes eukaryotes, such as mammals, for example, humans, sheep, cattle, horses, pigs, dogs, cats, non-human primates, mice and rats. The terms individual and patient are used interchangeably in this document.

[0072] Definitions of specific functional groups and chemical terms are described in more detail below. For the purposes of this description, chemical elements are identified according to the Periodic Table of Elements, CAS version, Chemistry and Physics Handbook, 75th edition, back cover, and specific functional groups are generally defined as described therein. In addition, the general principles of organic chemistry, as well as specific functional fractions and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March's Advanced Organic Chemistry, 5th 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, 3rd edition, Cambridge University Press, Cambridge, 1987; the entire content of each of which is incorporated herein by reference.

[0073] It should be noted that a compound of the description, as described in this document, may be replaced by Petition 870250102279, dated 07 / 11 / 2025, page 36 / 144 28 / 116 any number of substituents or functional fractions. In general, the term substituted, preceded or not by the term optionally, and the substituents contained in the formulas of this invention, refer to the substitution of hydrogen radicals in a given structure by the radical of a specified substituent. When more than one position in any structure can be substituted by more than one substituent selected from a specified group, the substituent may be the same or different at each position. As used in this document, the term substituted is considered to include all permitted substituents of organic compounds. In a broad sense, permitted substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds.For the purposes of this description, heteroatoms such as nitrogen may have hydrogen substituents and / or any permitted substituents from organic compounds described in this document that satisfy the valences of the heteroatoms. Furthermore, this description is not intended to be limited in any way by the permitted substituents from organic compounds.

[0074] The term acyl, as used in this document, refers to a group with the general formula —C(=O)R°, where R° is a substituted or unsubstituted hydroxyl group, thiol. Petition 870250102279, dated 07 / 11 / 2025, p. 37 / 144 29 / 116 substituted or unsubstituted, substituted or unsubstituted amino, cyclic or acyclic aliphatic, cyclic or acyclic heteroaliphatic, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. Exemplary acyl groups include carboxylic acids (—CO2H), ketones (as an acetyl group [—(C=O)CHsl], esters, amides, carbonates, carbamates, and ureas. Acyl substituents include, but are not limited to, any of the substituents described in this document that result in the formation of a stable chemical moiety (e.g., 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).

[0075] The term aliphatic, as used in this document, includes saturated and unsaturated, non-aromatic, 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 understood by one skilled in the art, aliphatic is intended to include, but is not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl and cycloalkynyl groups. Therefore, as used in this document, the term alkyl includes Petition 870250102279, dated 07 / 11 / 2025, p. 38 / 144 30 / 116 linear, branched, and cyclic alkyl groups. A similar convention applies to other generic terms such as alkenyl, alkynyl, and the like. Furthermore, as used in this document, the terms alkyl, alkenyl, alkynyl, and the like encompass substituted and unsubstituted groups. In certain embodiments, as used in this document, aliphatic is used to indicate those aliphatic groups (cyclic, acyclic, substituted, unsubstituted, branched, or unbranched) that have from 1 to 6 carbon atoms, or from 2 to 6 carbon atoms. In certain embodiments, an aliphatic group has from 1 to 5 or 2 to 5 carbon atoms. In certain embodiments, an aliphatic group has from 1 to 4 or 2 to 4 carbon atoms. In certain embodiments, an aliphatic group has from 1 to 3 or 2 to 3 carbon atoms. In certain embodiments, an aliphatic group has 1 to 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 from 1 to 6 carbon atoms (C1-C2). Substituents of aliphatic groups include, but are not limited to, any of the substituents described in this document, which result in the formation of a stable chemical moiety (e.g., an aliphatic group substituted by one or more aliphatic, alkyl, alkenyl, alkynyl groups). Petition 870250102279, dated 07 / 11 / 2025, p. 39 / 144 31 / 116 heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, sulfinyl, sulfonyl, oxo, imino, thio-oxo, phosphine, cyano, amino, azido, nitro, hydroxyl, thio and / or halo).

[0076] The term alkyl, as used in this document, refers to saturated hydrocarbon radicals, linear or branched chain, derived from a hydrocarbon chemical moiety containing between one and twenty carbon atoms by the removal of a single hydrogen atom. In some embodiments, the alkyl group employed in the invention contains from 1 to 6 carbon atoms (C1-β). In another embodiment, the alkyl group employed contains from 1 to 5 carbon atoms. In other embodiments, the alkyl group contains from 1 to 4 carbon atoms. In another embodiment, the alkyl group contains from 1 to 3 carbons. In other embodiments, the alkyl group contains from 1 to 2 carbons. In other embodiments, the alkyl group contains 1 carbon atom. Examples of alkyl radicals include, among others, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, sec-pentyl, isopentyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, sec-hexyl and the like, which may contain one or more substituents.Alkyl group substituents include, but are not limited to, any of the substituents described in this document that result in the formation of a stable chemical moiety (e.g., an alkyl group substituted by one or more aliphatic, heteroaliphatic, heterocyclic groups). Petition 870250102279, dated 07 / 11 / 2025, p. 40 / 144 32 / 116 aryl, heteroaryl, acyl, sulfinyl, sulfonyl, oxo, imino, thio-oxo, cyano, amino, azido, nitro, hydroxyl, thio and / or halo).

[0077] The term alkenyl, as used in this document, denotes a monovalent group derived from a linear or branched hydrocarbon chemical moiety with 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 from 2 to 6 carbon atoms. In some embodiments, the alkenyl group employed in the invention contains from 2 to 5 carbon atoms. In another embodiment, the alkenyl group employed contains from 2 to 4 carbon atoms. In other embodiments, the alkenyl group contains from 2 to 3 carbon atoms. In another embodiment, the alkenyl group contains 2 carbons. Alkenyl groups include, for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl and the like, which may contain one or more substituents.Alkenyl group substituents include, among others, any of the substituents described in this document that result in the formation of a stable chemical moiety (e.g., an alkenyl group substituted by one or more aliphatic, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, sulfinyl, sulfonyl, oxo, imino, thio-oxo, cyano, amino, azido, nitro, hydroxyl, thio and / or halo groups). Petition 870250102279, dated 07 / 11 / 2025, p. 41 / 144 33 / 116

[0078] The term alkynyl, as used in this document, refers to a monovalent group derived from a linear or branched chain hydrocarbon with 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 from 2 to 6 carbon atoms. In some embodiments, the alkynyl group employed in the invention contains from 2 to 5 carbon atoms. In another embodiment, the alkynyl group employed contains from 2 to 4 carbon atoms. In still other embodiments, the alkynyl group contains from 2 to 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 contain one or more substituents.Alkynyl group substituents include, but are not limited to, any of the substituents described in this document that result in the formation of a stable chemical moiety (e.g., an alkynyl group substituted by one or more aliphatic, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, sulfinyl, sulfonyl, oxo, imino, thioxo, cyano, amino, azido, nitro, hydroxyl, thio, and / or halo groups).

[0079] The term amino, as used in this document, refers to a group of the formula (—NH2). A substituted amino refers to a group of the formulas (—NHRh) or (— Petition 870250102279, dated 07 / 11 / 2025, p. 42 / 144 34 / 116 NRh2), where Rh can be any substituent except hydrogen, resulting in the formation of a stable chemical moiety (e.g., an amino group substituted by one or more aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, sulfinyl, sulfonyl, amino, nitro, hydroxyl, and / or thio groups). A suitable “protecting amino group,” as used in this document, is well known in the art and includes 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 carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxantyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethyl carbamate (Adpoc), carbamate of 1,1-dimethyl2-haloethyl, 1,1-dimethyl-2,2-dibromoethyl carbamate (DBt-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-t-butylphenyl)-1-methylethyl (t. Petition 870250102279, dated 07 / 11 / 2025, page 43 / 144 35 / 116 Bumeoc), 2-(2'- and 4'-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclo-hexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), aliphatic carbamate (Alloc), 1-isopropylaliphatic carbamate (Ipaoc), cinamila carbamate (Coc), 4-nitrocinamila carbamate (Noc), 8-quinolyl carbamate, Nhidroxypiperidinila carbamate, alquilditio carbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl, 2,4-dichlorobenzyl carbamate, 4-methylsulfonylbenzyl carbamate (Msz), 9antrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenosulfonyl)ethyl carbamate, de [2-(1,3ditianyl)]methyl (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc),2triphenylphosphonioisopropyl carbamate (Ppoc), 1,1-dimethyl2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6chromonylmethyl carbamate (Tcroc), m-nitrophenyl, 3,5-dimethoxybenzyl carbamate, onitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, Petition 870250102279, dated 07 / 11 / 2025, page 44 / 144 36 / 116 Phenyl(o-nitrophenyl)methyl carbamate, phenothiazinyl-(10)-carbonyl derivative, N'-ptoluenosulfonilaminocarbonyl derivative, N'phenylaminothiocarbonyl derivative, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzil carbamate, cyclobutyl carbamate, Cyclo-hexila carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxycarbonylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborin carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p'-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methylcyclohexyla carbamate, 1-methyl-1-cyclopropylmethyl carbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl1-(p-phenylazophenyl)ethyl carbamate,1-methyl-1-phenylethyl carbamate, 1-methyl-1-(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-pyridylcarboxamide, Petition 870250102279, dated 07 / 11 / 2025, page 45 / 144 37 / 116 derivative of N-benzoylphenylalanine, benzamide, p-phenylbenzamide, o-nitrophenylacetamide, onitrophenoxyacetamide, acetoacetamide, (N'-dithiobenzyloxycarbonylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide, 2-methyl-2-(onitrophenoxy)propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinamide, derivative of N-acetylmethionine, onitrobenzamide, o-(benzoyloxymethyl)benzamide, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N1,1,4,4-tetramethyldisylalazacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3pyrolin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,N-5dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4methoxyphenyl)diphenylmethyl]amine (MMTr), N-9phenyfluorenylamine (PhF), N-2,7-dichloro-9fluorenylmethylenoamine, N-ferroxydomylmethylamine, N-2ó2ó(Fc N-1,1-dimethylthiomethylenoamine, Nbenzylidenoamine, Np-methoxybenzylidenoamine, NPetition 870250102279, dated 07 / 11 / 2025, p. 46 / 144, 38 / 116 diphenylmethyleneamine, N-[(2-pyridyl)mesethyl]methyleneamine, N-(N',N'-dimethylaminomethylene)amine, N,N'-isopropylidenediamine, Np-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-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, N-amine oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, onitrobenzenesulfenamide (Nps), 2,4dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys), p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6,-trimetil-4-methoxybenzenossulfonamida (Mtr), 2,4,6trimethoxybenzenossulfonamida (Mtb), 2,6-dimethyl-4methoxybenzenossulfonamida (Pme), 2,3,5,6-tetramethyl-4methoxybenzenossulfonamida (Mte), 4methoxybenzenossulfonamida (Mbs), 2,4,6trimetilbenzenossulfonamida (Mts), 2,6-dimethoxy-4metilbenzenossulfonamida (iMds), 2,2,5,7,8, Petition 870250102279, de 07 / 11 / 2025, pág. 47 / 144 39 / 116 pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4',8'-dimethoxynaphthylmethyl) benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide and phenacylsulfonamide.

[0080] The term aryl, as used in this document, refers to a stable mono- or polycyclic aromatic ring system with 3 to 20 ring atoms, all of which ring atoms are carbon, and which may be substituted or unsubstituted. In certain embodiments of the present invention, aryl refers to a C4-C20 mono-, bi- or tricyclic aromatic ring system with one, two or three aromatic rings which include, but are not limited to, phenyl, biphenyl, naphthyl and the like, which may contain one or more substituents. Aryl substituents include, but are not limited to, any of the substituents described in this document that result in the formation of a stable chemical moiety (e.g., an aryl group substituted by one or more aliphatic, heteroaliphatic, heterocyclic, aryl, heteroaryl; acyl, sulfinyl, sulfonyl, oxo, imino, thio-oxo, cyano, amino, azido, nitro, hydroxyl, thio and / or halo groups).

[0081] The term direct link or link refers to a single, double, or triple link between two groups. In certain modalities, a direct link refers to a Petition 870250102279, dated 07 / 11 / 2025, p. 48 / 144 40 / 116 simple link between two groups.

[0082] The terms halo and halogen, as used in this document, refer to an atom selected from fluorine (fluoro, —F), chlorine (chlorine, —Cl), bromine (bromine, —Br), and iodine (iodine, —I).

[0083] The term heteroaliphatic, as used in this document, includes saturated and unsaturated, non-aromatic hydrocarbons, linear (i.e., unbranched), branched, acyclic, cyclic (i.e., heterocyclic) or polycyclic, which are optionally substituted by one or more functional groups and which contain one or more oxygen, sulfur, nitrogen, phosphorus or silicon atoms, for example, in place of carbon atoms. As will be understood by one skilled in the art, heteroaliphatic is intended to include, but is not limited to, heteroalkyl, heteroalkenyl, heteroalkynyl and heterocyclyl groups. Thus, as used in this document, the term heteroalkyl includes linear, branched and cyclic alkyl groups, as defined in this document, which are optionally substituted by one or more functional groups and which contain one or more oxygen, sulfur, nitrogen, phosphorus or silicon atoms, for example, in place of carbon atoms.A similar convention applies to other generic terms, such as heteroalkenyl, heteroalkynyl, and the like. Furthermore, accordingly. Petition 870250102279, dated 07 / 11 / 2025, p. 49 / 144 41 / 116 used in this document, the terms heteroalkyl, heteroalkenyl, heteroalkynyl and the like encompass substituted and unsubstituted groups. In certain embodiments, as used in this document, heteroaliphatic is used to indicate those heteroaliphatic groups (cyclic, acyclic, substituted, unsubstituted, branched or unbranched) that have from 1 to 6 carbon atoms or from 2 to 6 carbon atoms. In certain embodiments, a heteroaliphatic group has from 1 to 5 or from 2 to 5 carbon atoms. In certain embodiments, a heteroaliphatic group has from 1 to 4 or from 2 to 4 carbon atoms. In certain embodiments, a heteroaliphatic group has from 1 to 3 or from 2 to 3 carbon atoms. In certain embodiments, a heteroaliphatic group has from 1 to 2 carbon atoms. In certain embodiments, a heteroaliphatic group has 1 carbon atom. In certain forms, a heteroaliphatic group has 2 carbon atoms.Heteroaliphatic group substituents include, but are not limited to, any of the substituents described in this document that result in the formation of a stable chemical moiety (e.g., a heteroaliphatic group substituted with one or more aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, sulfinyl, sulfonyl, oxo, imino, thiooxo, phosphine, cyano, amino, azido, nitro, hydroxyl, thio and / or halo groups). Petition 870250102279, dated 07 / 11 / 2025, p. 50 / 144 42 / 116

[0084] The term heteroaryl, as used in this document, refers to a stable mono- or polycyclic aromatic ring system with 3 to 20 ring atoms, of which one ring atom is selected from S, O, and N; zero, one, or two ring atoms are additional heteroatoms selected independently from S, O, and N; and the remaining ring atoms are carbon, the radical being attached 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, pyrrolizinyl, indolyl, quinolinyl, isoquinolinyl, benzoimidazolyl, indazolyl, quinolinyl, isoquinolinyl, quinolizinyl, cinolinyl, quinazolinyl, phthalaziline, naphtridinyl, quinoxalinyl, thiophenyl, tianaftenyl, furanyl, benzofuranyl, benzothiazolyl, thiazolinyl, isothiazolyl, thiadiazolinyl, oxazolyl, isoxazolyl, oxadiaziolyl, oxadiaziolyl and the like, which may contain one or more substituents. Heteroaryl substituents include, but are not limited to, any of the substituents described in this document that result in the formation of a stable chemical moiety (e.g., a heteroaryl group substituted by one or more aliphatic, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, sulfinyl, sulfonyl, oxo, imino, thiooxo, cyano, amino groups). Petition 870250102279, dated 07 / 11 / 2025, p. 51 / 144 43 / 116 azido, nitro, hydroxyl, thio and / or halo).

[0085] The term heterocyclic or heterocyclyl, as used in this document, refers to a non-aromatic, partially unsaturated or fully saturated ring system of 3 to 10 members, which includes single rings of 3 to 8 atoms in size, and bietricyclic ring systems that may include five- or six-membered aromatic aryl or heteroaryl groups fused to a non-aromatic ring. These heterocyclic rings include those with one to three heteroatoms independently selected from oxygen, sulfur, and nitrogen, in which the nitrogen and sulfur heteroatoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized.In certain embodiments, the term heterocyclic refers to a non-aromatic polycyclic ring or group of 5, 6, or 7 members, in which 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 linked to the rest of the molecule through any of the ring atoms. Heterocyclic groups include, but are not limited to, a bi- or tricyclic group, comprising fused rings of five, six, or seven members having between one and three heteroatoms independently selected from oxygen, sulfur, and nitrogen. Petition 870250102279, dated 07 / 11 / 2025, page 52 / 144 44 / 116 that (i) each 5-membered ring has from 0 to 2 double bonds, each 6-membered ring has from 0 to 2 double bonds and each 7-membered ring has from 0 to 3 double bonds, (ii) the nitrogen and sulfur heteroatoms may be optionally oxidized, (iii) the nitrogen heteroatom may be optionally quaternized and (iv) any of the above heterocyclic rings may be fused to an aryl or heteroaryl ring. Exemplary heterocycles include azacyclopropanil, azacyclobutanil, 1,3-diazatidinyl, piperidinyl, piperazinyl, azocanil, tiaranil, thietanyl, tetrahydrothiophenyl, dithiolanil, thiacyclohexanil, oxanil, oxetanil, tetrahydrofuranil, tetrahydropuranil, dioxanil, oxathiolanil, morpholinyl, thioxanil, tetrahydronaphthyl and the like, which may contain one or more substituents.Substituents include, but are not limited to, any of the substituents described in this document that result in the formation of a stable chemical moiety (e.g., a heterocyclic group substituted by one or more aliphatic, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, sulfinyl, sulfonyl, oxo, imino, thio-oxo, cyano, amino, azido, nitro, hydroxyl, thio and / or halo groups).

[0086] The term hydrogen, as used in this document, refers to any isotope with atomic number 1. Typically, hydrogen refers to stable isotopes. Petition 870250102279, dated 07 / 11 / 2025, p. 53 / 144 45 / 116 containing zero or one neutron (i.e., XH 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 deuterium present.

[0087] The term hydroxyl or hydroxyl group, as used in this document, refers to a group of the formula (—OH). A substituted hydroxyl group refers to a group of the formula (—ORi), where Ri can be any substituent except hydrogen, resulting in a chemically stable moiety (e.g., a hydroxyl group substituted by 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 in this document, is well known in the art and includes 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. Petition 870250102279, dated 07 / 11 / 2025, page 54 / 144 46 / 116 pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, S,S-dioxide of 4methoxytetrahydrothiopyranyl, 1-[(2-chloro-4-methyl)phenyl]-4methoxypiperidin-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7aocta-hydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, pmethoxyphenyl, 2,4-dinitrophenyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, phalobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, pphenylbenzyl, 2-picolyl, 4-picolyl,3-Methyl-2picolyl N-oxide, diphenylmethyl, p,p'-dinitrobenzhydryl, 5dibenzosuberyl, triphenylmethyl, a-naphthyldiphenylmethyl, pmethoxyphenyldiphenylmethyl, 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',4tris(benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)bis(4',4, Petition 870250102279, dated 07 / 11 / 2025, page 55 / 144 47 / 116 dimethoxyphenyl)methyla, 1,1-bis(4-methoxiphenyl)-1'pyrenylmethyla, 9-anthryla, 9-(9-phenyl)xanthenyla, 9-(9-phenyl10-oxo)anthryla, 1,3-benzodithiolan-2-yl, S,S-dióxido de benzisothiazolila, trimethylsily (TMS), triethylsily (TES), tri-isopropylsily (TIPS), dimethylisopropylsily (IPDMS), diethylisopropylsily (DEIPS), dimethyltexylsily, t-butyldimethylsily (TBDMS), t-butyldiphenylsily (TBDPS), tribenzilsily, tri-p-xylylsily, triphenylsily, diphenylmethylsily (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, pchlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinate), pivaloate, adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), methyl alcohol carbonate, 9-fluorenylmethyl carbonate (Fmoc), ethyl alcohol carbonate,alkyl carbonate 2,2,2-trichloroethyl (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2(phenylsulfonyl)ethyl carbonate (Psec), 2(triphenylphosphonium)ethyl carbonate (Peoc), alkyl isobutyl carbonate, vinyl alkyl carbonate, alkyl carbonate allyl, p-nitrophenyl alkyl carbonate, alkyl benzyl carbonate, p-methoxybenzyl alkyl carbonate, Petition 870250102279, of 07 / 11 / 2025, p. 56 / 144 48 / 116 alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-1naphthyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4azidobutyrate, 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(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate, o(methoxycarbonyl)benzoate, α-naphthoate, nitrate, alkyl N,N,N',N'tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfonate, sulfate, methanesulfonate (mesylate), benzylsulfonate and tosylate (Ts).To protect 1,2- or 1,3-diols, protecting groups include methylene acetal, ethylidene acetal, 1-t-butylethylidene acetal, 1-phenylethylidene acetal, (4-methoxyphenyl)ethylidene acetal, 2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene acetal, cyclohexylidene acetal, cycloheptylidene acetal, benzylidene acetal, p-methoxybenzylidene acetal, 2,4-dimethoxybenzylidene acetal, 3,4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethylene acetal, ortho dimethoxymethylene ester, ortho. Petition 870250102279, dated 07 / 11 / 2025, page 57 / 144 49 / 116 1-methoxyethylidene ester, 1-ethoxyethylidine ortho ester, 1,2-dimethoxyethylidene ortho ester, amethoxybenzylidene ortho ester, 1-(N,N-dimethylamino) ethylidene derivative, a-(N,N'-dimethylamino)benzylidene derivative, 2-oxacyclopentylidene ortho ester, di-tbutylsilylene group (DTBS), 1,3-(1,1,3,3tetraisopropyldisiloxanylidene) derivative (TIPDS), tetrabutoxydisiloxane-1,3-di-ylidene derivative (TBDS), cyclic carbonates, cyclic boronates, ethyl boronate and phenyl boronate.

[0088] The term imino, as used in this document, refers to a group of the formula (=NRr), wherein Rr corresponds to hydrogen or any substituent as described in this document, which results in the formation of a stable chemical moiety (e.g., a suitable amino protecting group; substituted or unsubstituted amino; acyl; cyclic or acyl alkyl, branched or unbranched, substituted or unsubstituted; cyclic or acyl alkenyl, branched or unbranched, substituted or unsubstituted; cyclic or acyl alkynyl, branched or unbranched, substituted or unsubstituted; cyclic or acyl heteroalkyl, branched or unbranched, substituted or unsubstituted; Petition 870250102279, dated 07 / 11 / 2025, p. 58 / 144 50 / 116 substituted; cyclic or acylic heteroalkynyl, branched or unbranched, substituted or unsubstituted; aryl substituted or unsubstituted; or unsubstituted heteroaryl).

[0089] The term isocyan, as used in this document, refers to a group of the formula (—NC).

[0090] The term nitro, as used in this document, refers to a group of the formula (—NO2).

[0091] The term nitroxide, as used in this document, refers to a stable nitroxide group that may be cyclic or acyclic. In certain embodiments, a stable nitroxide refers to a chemically stable nitroxide that can be obtained in pure form, stored, and handled in a laboratory. In certain embodiments, a stable nitroxide refers to a cyclic or acyclic nitroxide containing two groups that do not contain alpha hydrogens. Exemplary cyclic or acyclic nitroxides are provided in Keana, Chemical Reviews (1978) 78:37 to 64, the entirety of which is incorporated herein by reference.

[0092] The term oxo, as used in this document, refers to a group in the formula (=O).

[0093] The term thio or thiol, as used in this document, refers to a group of the formula (—SH). A substituted thiol refers to a group of the formula (—SRr), where Rr is any substituent except hydrogen, which Petition 870250102279, dated 07 / 11 / 2025, p. 59 / 144 51 / 116 results in the formation of a stable chemical moiety (e.g., a thio group substituted by one or more aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, sulfinyl and / or sulfonyl groups).

[0094] The term thio-oxo, as used in this document, refers to a group of the formula (=S).

[0095] The term sulfinyl, as used in this document, refers to a group of the formula Rf—S(=O)—, where Rf may be an aliphatic, heteroaliphatic, aryl, or optionally substituted heteroaryl group. The term alkylsulfinyl refers to a sulfinyl group, where Rf may be an optionally substituted alkyl group. The term arylsulfinyl refers to a sulfinyl group, where Rf may be an optionally substituted aryl or heteroaryl group.

[0096] The term sulfonyl, as used in this document, refers to the group of formula Rg—S(=O)2—, where Rg may be an aliphatic, heteroaliphatic, aryl, or optionally substituted heteroaryl group. The term alkylsulfonyl refers to a sulfonyl group, where Rg may be an optionally substituted alkyl group. The term arylsulfonyl refers to a sulfonyl group, where Rg may be an optionally substituted aryl or heteroaryl group. Examples of aryl or alkylsulfonyl groups include tosyl Petition 870250102279, dated 07 / 11 / 2025, p. 60 / 144 52 / 116 (toluenesulfonyl, CH3C6H4SO2—), mesyl (methylsulfonyl, CH3SO2—) and trifluoromethanesulfonyl (CF3SO2—).

[0097] The term “stable chemical moiety,” as used in this document, preferably refers to a chemical moiety that has sufficient stability to permit manufacturing (including in situ manufacturing) and that maintains its integrity for a period of time sufficient to be useful for the purposes detailed in this document.

[0098] The term “stable radical,” as used in this document, refers to a free radical that has sufficient stability to permit fabrication (including in situ fabrication) and that maintains its integrity for a period of time sufficient to be useful for the purposes detailed in this document.

[0099] In certain embodiments, one or more (or all) of the hydrogen atoms present in a compound of Structural Formulas (I), (II) or (III) are 2H.

[00100] Aspects of the present technology comprise compounds that are derived from verdazil, as described in this document (e.g., compounds of Formula (I), Formula (II), Formula (III), compounds shown in Table 1). The compounds provide metal-free contrast agents that are more stable and / or less toxic than previous contrast agents. Compounds are clinically useful for medical imaging, such as, Petition 870250102279, dated 07 / 11 / 2025, p. 61 / 144 53 / 116 among others, RMD-CE and RMD-DCE, and can provide reliable, rapid and / or quantitative clinical images to facilitate the diagnosis and early detection of various disorders. Aspects of the present technology comprise pharmaceutical compositions and compounds, including the compounds, methods of production of such compounds and uses thereof. Compositions

[00101] In certain embodiments, the compounds of the present description may be present in a composition or in a pharmaceutical composition.

[00102] Pharmaceutical compositions are generally formulated to be compatible with the intended method or route of administration; exemplary routes of administration include, but are not limited to, oral or parenteral, for example, intramuscular, intravenous, subcutaneous (e.g., injection or implant), intraperitoneal, intrathecal, or intra-articular. In some embodiments, the pharmaceutical composition is provided in a single-use container (e.g., a single-use vial, syringe, or autoinjector), while in other embodiments, a multi-use container (e.g., a multi-use vial) is provided. The compounds and compositions provided in this document may be administered to an individual in any appropriate manner known in the art. Petition 870250102279, dated 07 / 11 / 2025, page 62 / 144 54 / 116

[00103] 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 the transport of any composition in question or component thereof. Each carrier must be “acceptable” in the sense of being compatible with the composition in question and its components and not harmful to the patient.Some examples of materials that can 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 cellulose derivatives, such as sodium carboxymethylcellulose, ethylcellulose, 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 solution; (18) Ringer's solution; (19) ethyl alcohol; (20). Petition 870250102279, dated 07 / 11 / 2025, page 63 / 144 55 / 116 phosphate buffer solutions; and (21) other compatible non-toxic substances used in pharmaceutical formulations.

[00104] In certain embodiments, the compositions and pharmaceutical compositions of the description are present in the form of micelles. For some micelles, a composition of the description is mixed with a nonpolar radical, for example, a radical containing a perfluorinated chemical moiety. A composition or pharmaceutical composition may also include a surfactant. A non-limiting type of perfluorinated radical is a TEMPO group with a fluorinated tail, for example, 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 linked to a C6-C20 perfluoroalkyl group (such as C8-C12) 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 C1, C7, C8, C9, C10, C11 and C12 acids.Exemplary surfactants include, without limitation, ammonium perfluorooctanoate (FC143), perfluorooctanesulfonic acid (PFOS), and perfluorononanoic acid (PFNA).

[00105] The pharmaceutical compositions and compounds described may also include additional components such as stabilizers, preservatives, dispersants and the like. Petition 870250102279, dated 07 / 11 / 2025, page 64 / 144 56 / 116 The pharmaceutical compositions and compounds described may also include additional components suitable for the intended purpose, for example, suitable for obtaining images and / or administering to an individual, such as excipients, colorants and the like.

[00106] A “pharmaceutically acceptable salt” of a compound means a salt of a compound that is pharmaceutically acceptable. Desirable salts of a compound are those that retain or enhance the biological efficacy and free acid and base properties of the original compound, as defined in this document, or that take advantage of an intrinsically basic, acidic, or charged functionality in 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. 66, 1 in 19 (1977).Non-limiting 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 acid, glycolic acid, pivalic acid, t-butylacetic acid, β-acid. Petition 870250102279, dated 07 / 11 / 2025, page 65 / 144 57 / 116 hydroxybutyric acid, valeric acid, hexanoic acid, cyclopentanopropionic 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-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 3-phenylpropionic acid, laurylsulfonic acid, laurylsulfuric 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, hydroxynaphthoic acid, salicylic acid, ascorbic acid,Stearic acid, muconic acid and the like; (2) base addition salts, formed when an acidic proton present in the original compound 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, Petition 870250102279, dated 07 / 11 / 2025, page 66 / 144 58 / 116 ethylamine, diethylamine, ethylenediamine, N,N'dibenzylethylenediamine, ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, piperazine, chloroprocaine, procaine, choline, lysine and the like.

[00107] Pharmaceutically acceptable salts can be synthesized from an original compound containing a basic or acidic chemical moiety by conventional chemical methods. Generally, such salts are prepared by reacting the free acidic or basic forms of the compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent, or a mixture of both. Salts can be prepared in situ, during the isolation or final purification of a compound, or by the separate reaction of a compound in its free acidic or basic form with the desired corresponding base or acid, and isolating the salt thus formed. The term “pharmaceutically acceptable salts” also includes zwitterionic compounds containing a cationic group covalently bonded to an anionic group, as these are “internal salts”. Uses

[00108] Dynamic contrast-enhanced MRI (DCE-MRI) analyzes the temporal enhancement pattern of a tissue after the introduction of a paramagnetic contrast agent. This is achieved by acquiring baseline images without contrast enhancement, followed by a series of images acquired over time during Petition 870250102279, dated 07 / 11 / 2025, page 67 / 144 59 / 116 and after the arrival of the contrast agent in the tissue of interest. The acquired signal is used to generate a temporal intensity curve for the tissue, which reflects the tissue's response to the arrival of the contrast agent in enhancement values. DMR-DCE has been used to study a wide variety of conditions, including cardiac pathologies, notably infarction, stroke and other brain disorders, a wide range of neoplasms with emphasis on antiangiogenic treatment and early detection, as well as investigations of the peripheral vascular and musculoskeletal systems.

[00109] Most often, gadolinium (Gd) chelates are used as contrast agents for MRI-DCE. However, Gd chelate contrast agents are not ideal for many applications. Free gadolinium (Gd3+) is known to be toxic and must be strongly complexed by a ligand to be used in humans. There are concerns regarding the potential toxicity of gadolinium complexes in patients with renal impairment, particularly regarding 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 MRI-DCE for clinical use.

[00110] The uses of the compounds and compositions of the present technology are not limited and may include agents Petition 870250102279, dated 07 / 11 / 2025, page 68 / 144 60 / 116 contrast for image generation, for example, for contrast-enhanced MRI (CE-MRI), dynamic contrast-enhanced MRI (DCE-MRI), and similar modalities. Thus, in certain modalities, the compounds described in this document are contrast agents used for image generation, for example, MRI, CE-MRI, DCE-MRI, and so on. It should be understood that any suitable magnetic resonance or other imaging technique can be used in conjunction with the compounds and compositions described in this document (for example, see RMD in Practice Ed., by Westerbrook et al., Blackwell Publishing, Oxford, UK, 2005, the contents of which are incorporated herein by reference). Furthermore, the methods can be performed under any magnetic field intensity. In some modalities, the field may have an intensity in the range of about 0.1 T to about 30 T, for example, at 3 T.The radiation to excite the electron spin transitions in the unpaired electron(s) of the polarizing agent in these fields will be in the range of about 2.8 GHz to about 840 GHz. For example, the radiation could be from a 140 GHz gyrotron.

[00111] Chronic kidney disease (CKD) remains a major international health problem. CKD usually develops slowly and without obvious symptoms in the early stages, but becomes progressively more debilitating in later stages, with limited chances of reversal. Petition 870250102279, dated 07 / 11 / 2025, page 69 / 144 61 / 116 This disease is often attributed to hypertension and long-standing diabetes, but it is also a possible consequence of acute kidney injury (AKI), resulting from a sudden and drastic decline in kidney function.

[00112] CKD outcomes are improved with early interventions facilitated by early detection. The 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 urinary albumin / creatinine ratio (ACR) > 30 mg / g for more than 3 months. These diagnostic values ​​were derived from large clinical studies in an ethnically limited population, significantly reducing the diagnostic power of these disease biomarkers. Furthermore, the underlying causes of CKD can vary among individuals, where some of the most common, including diabetes mellitus, cardiovascular disease, and kidney transplantation, can limit the accuracy of eGFR measurements at the patient level, as nearly 30% of these patients may present with a 30% deviation from their actual eGFR.Furthermore, in addition to an individual's deviation from the derived population, the eGFR equation assumes steady-state creatinine levels and does not consider alterations or alternative pathways of creatinine production, leading to this large variability. The development of more precise methods for estimation has become imperative. Petition 870250102279, dated 07 / 11 / 2025, page 70 / 144 62 / 116 of the GFR, free from the use of race or other patient demographic characteristics. More importantly, these values ​​do not provide physicians with spatial or structural information underlying renal dysfunction. Renal biopsy can provide predictive histopathological data for CKD outcomes, providing spatial data on specific renal lesions and not just on overall renal function. However, biopsies are invasive procedures with their own inherent risk, preventing their repeated use to characterize renal disease spatiotemporally. Clinically, GFR remains the gold standard as an indicator of renal function, and there is a need for reliable, rapid, and / or quantitative clinical imaging approaches for its measurement.

[00113] In certain modalities, the compounds and compositions described in this document provide metal-free contrast agents for medical imaging. In certain modalities, the compounds and compositions described in this document provide metal-free alternatives to Gd-based contrast agents to facilitate MRI-CE and / or MRI-DCE. In certain modalities, the compounds and compositions described in this document are selectively absorbed in the kidney and are therefore used for medical imaging of the kidney. It should be understood, however, that the use of the compounds and compositions described in this document Petition 870250102279, dated 07 / 11 / 2025, p. 71 / 144 63 / 116 is not intended to be particularly limited; for example, compounds and compositions may be used for imaging other tissues, depending on the uptake / distribution of the compound after administration to an individual and other considerations that determine suitability for a specific use.

[00114] In certain modalities of the imaging methods described, images of the kidney are provided. Such methods can provide quantitative and / or qualitative renal functional information, such as, without limitation, the determination and / or mapping of the GFR in an individual. In certain modalities, this imaging is free from toxicological concerns of prior contrast agents in patients with renal dysfunction and / or free from the use of race or other demographic characteristics of the patient.

[00115] In certain modalities, biomedical imaging methods are provided, comprising administering a contrast agent to an individual and obtaining images of the contrast agent in the individual, wherein the contrast agent comprises a compound or composition as described in this document. Medical imaging may be, for example, and without limitation, magnetic resonance imaging (MRI), such as MRI-CE or MRI-DCE. In certain modalities, biomedical imaging is used to obtain images of the kidney. In some of these Petition 870250102279, dated 07 / 11 / 2025, page 72 / 144 In 64 / 116 modalities, image generation is used to monitor or assess renal function in an individual. In some of these modalities, image generation is used to determine and / or map the GFR in an individual. The methods can therefore provide quantitative and / or qualitative renal functional information, such as, without limitation, the individual's GFR.

[00116] In certain embodiments, methods are provided for measuring renal function in an individual, comprising administering a contrast agent to an individual and obtaining images of the contrast agent in the individual, wherein the contrast agent comprises a compound or composition as described in this document, and determining and / or mapping the individual's GFR.

[00117] In certain modalities, methods are provided for diagnosing renal dysfunction in an individual, comprising administering a contrast agent to the individual and obtaining images of the contrast agent in the individual, wherein the contrast agent comprises a compound or composition as described in this document, and determining and / or mapping the individual's GFR.

[00118] In certain modalities, methods are provided to diagnose chronic kidney disease (CKD), acute kidney injury (AKI), renal artery stenosis, urinary obstruction, and / or renal tumor or malignancy in an individual. Petition 870250102279, dated 07 / 11 / 2025, page 73 / 144 65 / 116 comprising administering a contrast agent to the individual and obtaining images of the contrast agent in the individual, wherein the contrast agent comprises a compound or composition as described in this document, and determining and / or mapping the individual's GFR.

[00119] In certain embodiments, methods are provided to determine whether an individual meets the requirements for kidney donation, comprising administering a contrast agent to the individual and obtaining images of the contrast agent in the individual, wherein the contrast agent comprises a compound or composition as described in this document, and determining and / or mapping the individual's GFR.

[00120] In certain modalities of the methods provided in this document, the individual has, is suspected of having, or is at risk of having renal dysfunction. An individual may have, be suspected of having, or be at risk of having chronic kidney disease (CKD), acute kidney injury (AKI), renal artery stenosis, urinary obstruction, or renal tumor or malignancy. An individual may be a kidney donor or a candidate for kidney donation. Kits

[00121] Kits comprising a compound or composition as described herein are also provided in this document. The kits generally take the form of a physical structure housing several components and may be Petition 870250102279, dated 07 / 11 / 2025, p. 74 / 144 66 / 116 used, for example, in the practice of the methods provided in this document. For example, a kit may include one or more compounds or compositions described in this document (provided, for example, in a sterile container), which may be in the form of a pharmaceutical composition suitable for administration to an individual. The compound or composition may be provided in a ready-to-use form or in a form that requires, for example, reconstitution or dilution (e.g., a powder) before 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 packaged in an individual container, and all containers may be in a single package.A kit of the present invention can be designed for the conditions necessary to properly maintain the components housed therein (e.g., refrigeration or freezing).

[00122] A kit may also contain a label or leaflet, including identifying information about the components contained therein and instructions for use. Labels or leaflets may include manufacturer information, such as batch numbers and expiration dates. The label or leaflet may be, for example, Petition 870250102279, dated 07 / 11 / 2025, page 75 / 144 67 / 116 example, integrated into the physical structure that houses the components, contained separately within the physical structure, or attached to a component of the kit (e.g., an ampoule, tube, or vial). EXAMPLES

[00123] The present invention will be more easily understood with reference to the following examples, which are provided to illustrate the invention and should not be construed as limiting its scope in any way.

[00124] Unless defined otherwise or the context clearly indicates otherwise, all technical and scientific terms used in this document have the same meaning commonly understood by one skilled in the art to which this invention pertains. It should be understood that any methods and materials similar or equivalent to those described in this document may be used in the practice or testing of the invention. Example 1. Synthesis of modified verdazil compounds

[00125] A variety of compounds of Structural Formula (I) can be prepared according to Scheme 1 below, which shows a general scheme for the synthesis of verdazil with modification at R1 (bonded to nitrogen at positions 1 and 5 of the ring) and R2 (bonded to carbon at position 3 of the ring). In Scheme 1, the reaction conditions are as follows: Petition 870250102279, dated 07 / 11 / 2025, p. 76 / 144 68 / 116 following: (a) 15% Phosgene in Toluene, 0 °C to 20 °C, overnight; (b) HCl in miscible solvent, reflux temperature, 2 hours; (c) R2 group containing aldehyde 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 described in Scheme 1 that generates intermediate 1 in the pathway to the synthesis of formulas (I), (II) or (III) can be carried out, in addition to phosgene, using diphosgene, triphosgene, diimidazole carbonyl, disuccinimidyl carbonate, bis-pentafluorophenyl carbonate. Scheme 1. Synthesis of modified verdazil compounds according to certain embodiments.

[00126] A variety of compounds of Structural Formula (I) can also be prepared according to Scheme 2 below, which shows a general scheme for the synthesis of verdazil with modification at R1 (linked to nitrogen at positions 1 and 5 of the ring) and R2 (linked to carbon at position 3 of the ring), starting with compound 1 generated as described in Scheme 1. In summary, large quantities of polyethylene glycol polymer of any length or repeating order of Petition 870250102279, dated 07 / 11 / 2025, page 77 / 144 69 / 116 carbon with a PMB-protected alcohol can be produced, then oxidized to an aldehyde, followed by closure of the verdazil ring with it and deprotection to give a verdazil compound modified 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 into any leaving group or reactive loop that interacts with nucleophiles, allowing the binding of any functional group to an N, S, or O nucleophilic site.

[00127] In Scheme 2, the reaction conditions are (a) HyO / acetonitrile NaOAc and or following (or any group having an aldehyde at one end and a protected heteroatom at the other end may be used); (b) HCl in miscible solvent, reflux temperature, 2 hours; (c) Appropriate solvent and conditions selected for the specific reaction; (d) Potassium ferricyanide and sodium bicarbonate in polar solvent, until effervescence ceases. Petition 870250102279, dated 07 / 11 / 2025, p. 78 / 144 70 / 116 Scheme 2. Synthesis of modified verdazil compounds according to certain embodiments. Example 2. Optimized targeted synthesis of glucoverdazil

[00128] An overview of the glucoverdazil synthesis is shown in Scheme 3 below. A combination of previously reported 6-oxoverdazil syntheses was used to identify an optimized route for glucoverdazil, producing the high level of molecular purity and scalability required for an in vivo contrast agent. The hydrazine side chains previously reported in the literature were generally 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). The side chains were functionalized with isopropyl groups, which are bulky enough side chains to help protect the delocalized radical while improving serum retention after injection. In previous syntheses, isopropyl hydrazine N-boc was generated from the hydrazine N-boc precursor, following Petition 870250102279, dated 07 / 11 / 2025, p. 79 / 14471 / 116 a synthesis previously reported by Calabretta et al. in large quantities (Kumar et al., 2018). However, commercially available isopropyl boc hydrazine was used in this case. Compound 2 was synthesized in several reported ways, all of which are COCle disubstitutions, either as a phosgene solution or solid triphosgene (Solea et al., 2018; Le et al., 2016). Glucordasil was synthesized with both forms of phosgene with similar results. In this paper, a 15% phosgene solution in toluene was chosen. A route with superior yield and purity was achieved by recrystallization with heptane of the crude product after the phosgene step, as first reported by Paré et al. (Solea et al., 2018). After deprotection of boc 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 first reported by Le et al.Finally, the oxidation of compound 3 was also carried out, 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 most of the available literature on verdazil.

[00129] Much of the previous literature that reported these reactions was incomplete with regard to Petition 870250102279, dated 07 / 11 / 2025, page 80 / 144 72 / 116 characterization. In this document, high-purity XH and 13C spectra are reported, as well as high-resolution mass spectrometry for each step and associated intermediates (FIGs. 7 to 12). The purity of compound 4 was determined by EPR spectroscopy and analytical high-performance liquid chromatography, demonstrating that this approach for glucoverdazil produced the fully converted compound 4 from the non-radical 3 (FIG. 13). COCíj (15% toluene) Et3N O'C ambient temperature, during the night i)HCI / EtOH (80°C. 1 h ) --li) O-Glucose NaOAc H2O (room temperature, overnight) 70% yield 74% yield Scheme 3. Overview of glucoverdazil synthesis 4. Example 3. Characterization of glucoverdazil as an active contrast agent in magnetic resonance imaging.

[00130] Referring to FIGS. IA to IF, the paramagnetic characteristics and stability of glucoverdazil were determined.

[00131] For glucoverdazil stability measurements, the EPR was adjusted for a glucoverdazil or TEMPO sample in PBS before any stability measurements. Once adjusted, solutions of glucoverdazil or TEMPO were prepared (20 mM in mouse serum or 5 mM in 4 mM sodium ascorbate buffer pH 7.4). A single spectrum Petition 870250102279, dated 07 / 11 / 2025, page 81 / 144 73 / 116 was acquired and the height of the most intense peak for each compound was recorded. EPR scans were then acquired every 5 s for 2 h (mouse serum) or 1.5 h (ascorbate) to measure the percent change in activity. For stability measurements of glucoverdazil in water, a 5 mM sample was prepared and left in a fume hood exposed to light, or wrapped in aluminum foil and left in a dark refrigerator at 4 °C. Periodically, these solutions were sampled and measured by EPR after adjustment, using a freshly prepared 5 mM glucoverdazil sample.

[00132] The RPE spectrum for glucoverdazil agreed with those previously reported, with the multiple RPE peaks being characteristic of the high degree of radical delocalization within the tetrazinanone ring (Fig. 1A; Massolle et al., 2018; Tain et al., 2017). Confirmation of this free radical indicated that glucoverdazil could exhibit contrast enhancement on magnetic resonance imaging (Wahsner et al., 2019). Magnetic resonance imaging of glucoverdazil solution phantoms in PBS showed a two-fold increase in contrast at Ti, but no change in T2 effects compared to water (FIG. 1B). The relaxivity of glucoverdazil was, as expected, lower than that reported for GBCAs, but the contrast effects were of similar magnitude to those of other Petition 870250102279, dated 07 / 11 / 2025, page 82 / 144 74 / 116 previously reported organic radical compounds, with a longitudinal relaxivity (r1) of 0.30 mM-1s-1 ± 0.3 mM1s-1 (Fig. 1C; Le et al., 2016; Matsumoto et al., 2022; Chevalier et al., 2009). Although contrast enhancement was similar to that of TEMPO, the tetrazinanone radical was substantially more stable than the nitroxy ORCA equivalent (FIG. 1D, E). Neither glucoverdazil nor TEMPO showed any alteration in radical activity in mouse serum (FIG. 1D); however, in the presence of ascorbate, a mild biological reductant, there was no loss of the glucoverdazil radical, but rather complete reduction of the nitroxy radical of TEMPO (FIG. 1E).

[00133] The stability of the glucoverdazil radical in solution over time was evaluated by storing the solutions at room temperature under direct light or at -20 °C in complete darkness. Periodic EPR sampling of these solutions showed that glucoverdazil retained more than 50% of its radical activity after 4 months of benchtop storage, while the frozen solution retained more than 80% of its radical activity after 1 year (FIG. 1F). This result demonstrated the resilience of the delocalized glucoverdazil radical under bioreducing conditions, as well as its shelf life and storage capacity, both critical characteristics of commonly used magnetic resonance contrast agents. Petition 870250102279, dated 07 / 11 / 2025, page 83 / 144 75 / 116

[00134] Prior to in vivo experimentation, cytocompatibility with glucoverdazil was evaluated in H460 lung carcinoma epithelial cells and did not demonstrate cytotoxicity compared to untreated cells at concentrations up to 10 mM (FIG. 15).

[00135] To evaluate cell viability in H460 cells, large cell lung cancer (H460) cells were cultured in RPMI-1640 medium (RPMI) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P / S) to 80% confluence, at which point they were subcultured. Cells were subcultured three times before being seeded in a 6-well plate and cultured to 80% confluence. Cells were seeded to obtain 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 glucoverdazil for 4 or 24 hours. At the respective time points, the medium was aspirated and the cells were washed three times with Dulbecco's phosphate-buffered saline (PBS) at 37 °C.Next, the cells were diluted with trypsin-EDTA, centrifuged at 400 xg (5 minutes, 4 °C), aspirated, and resuspended in 1 mL of PBS solution containing 0.2 pM calcein-acetoxymethyl ester (fluorescent green staining for live cells) and 16 pM ethidium-1 homodimer (fluorescent staining of...). Petition 870250102279, dated 07 / 11 / 2025, p. 84 / 144 76 / 116 red for dead cells). Live and dead cell populations were counted by flow cytometry (Gallis Beckman-Coulter Flow Cytometer) using an excitation of 488 nm 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 homodimer1 (dead cells, red). After completion, the viable cell population for each condition was determined by comparing the total number of calcein-AM positive cells, individually stained, with the combined total of cells that were individually stained as positive for live or dead, using the Kaluza analysis software (Beckman-Coulter).

[00136] The suitability of glucordazil to the contrast medium was evaluated in vivo after intravenous injections in 9 BALB / c mice. An administered dose of 3 mmol / kg was chosen based on the difference in r1 between glucordazil and Gadovist® (~10 times), and the clinically recommended standard dose of Gadovist® of 0.1 mmol / kg. This dose of glucordazil was still well below the maximum concentration evaluated for cytocompatibility. After injection, Ti-weighted images were acquired every 3 minutes after a pre-injection scan, which was used to establish the baseline voxel intensity. Contrast-limited enhancement was observed in muscle and liver, with uptake and Petition 870250102279, dated 07 / 11 / 2025, page 85 / 144 77 / 116 clearances were clearly isolated for the urinary system (FIG. 2A). Overall, signal changes relative to pre-contrast scans of 127% ± 9% were observed in muscle, 121% ± 10% in liver, and 184% ± 21% in kidneys 5 minutes after injection (FIG. 2B). The mean clearance time after injection, determined by the return of renal ROI to baseline intensity, was approximately 40 minutes, coinciding with the plateau of bladder ROI signal increase. At this time, signal changes of 120% ± 8% were observed in muscle, 115% ± 7% in liver, 127% ± 8% in kidneys, and 438% ± 48% in bladder. The kinetics of glucoverdazil clearance by the kidneys corresponded to that of monophasic degradation, allowing the determination of the renal degradation time constant (RDTC, k in min-1) for glucoverdazil clearance in renal tissue from the slope of the semi-natural logarithmic graph of the data (FIG. 2C).Linear regression was consistently performed from the maximum contrast intensity at t = 2.5 minutes to the most consistent return-to-baseline time of t = 40 minutes. The mean k value determined in the 9 healthy BALB / c mice was -0.124 min-1 ± 0.012 min-1, with a mean R² of 0.97 ± 0.05, demonstrating excellent reproducibility of this baseline measure of healthy renal function.

[00137] With the observation that glucoverdazil was being absorbed primarily by the kidneys, studies of Petition 870250102279, dated 07 / 11 / 2025, page 86 / 144 78 / 116 cytocompatibility tests were repeated with high concentrations of glucoverdazil in human renal proximal tubule cells (hRPT; FIG. 16).

[00138] To assess the viability of human renal proximal tubule (hRPT) cells, hRPT cells were cultured in epithelial cell culture medium (EpiMEM) supplemented with 10% FBS, 1% P / S, and epithelial cell growth supplement (EpiCGS) to 80% confluence, at which point they were passaged. The cells were passaged three times before being seeded in a 6-well plate and cultured to 80% confluence. Cells were seeded to obtain triplicate wells of each condition. The cells were then incubated in their regular media supplemented with regular media, 10 mM glucoverdazil, 10 mM 5,5-dimethyl-1-pyrroline N-oxide (DMPO, a nitrone spin trap) or 10 mM (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO, a nitroxy radical), for 4 or 24 hours.At the respective times, the medium was aspirated and the cells were washed three times with Dulbecco's phosphate-buffered saline (PBS) at 37 °C. Then, the cells were harvested with trypsin-EDTA, centrifuged at 400 x g (5 minutes, 4 °C), aspirated, and resuspended in 1 mL of PBS solution containing 0.2 pM calcein-acetoxymethyl ester (fluorescent green staining for live cells) and 16 pM of... Petition 870250102279, dated 07 / 11 / 2025, page 87 / 144 79 / 116 ethidium homodimer-1 (fluorescent red staining for dead cells).

[00139] Live and dead cell populations were counted by flow cytometry (Beckman-Coulter Gallios flow cytometer) using an excitation of 488 nm 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 completion, the viable cell population for each condition was determined by comparing the total number of calcein-AM positive cells, individually stained, with the combined total of cells that were individually stained as positive for live or dead, using the Kaluza analysis software (Beckman-Coulter).

[00140] Although glucoverdazil did not result in a significant increase in cell death compared to untreated cells after 24 h of incubation, > 90% of hRPT were dead after only 4 h of incubation with TEMPO. This striking difference in cytocompatibility highlights a fundamental additional performance difference between TEMPO and tetrazinanone-derived ORCAs.

[00141] The cellular uptake of glucoverdazil by hRPT cells was evaluated by EPR spectroscopy. To evaluate the uptake of glucoverdazil in hRPT cells, hRPT cells were cultured in cell culture medium. Petition 870250102279, dated 07 / 11 / 2025, page 88 / 144 80 / 116 epithelial cells (EpiMEM) supplemented with 10% FBS, 1% P / S, and epithelial cell growth supplement (EpiCGS) were subcultured to 80% confluence, at which point they were subcultured. Cells were subcultured three times before being seeded into 6-well plates and cultured to 80% confluence. Cells were seeded to obtain triplicate wells of each condition. Cells were then incubated in their regular media supplemented with regular medium or 10 mM glucoverdazil and incubated for 24 hours. The medium was aspirated, and the cells were washed three times with Dulbecco's phosphate-buffered saline (PBS) at 37 °C. Next, the cells were harvested with trypsin-EDTA, centrifuged at 400 xg (5 minutes, 4 °C), aspirated, and resuspended in 100 μl of PBS. The concentrated cell solutions were transferred to EPR tubes. A 1 μl aliquot was retained and diluted to obtain the number of cells in each solution.The EPR was adjusted for a freshly prepared 5 mM glucoverdazil solution in PBS, and then the samples were measured by EPR. The concentration was measured relative to a previously determined standard curve and then normalized to the previously determined number of cells to obtain nM of glucoverdazil per cell.

[00142] The results indicated that glucoverdazil was not absorbed detectably (FIG. 17). The smallest Petition 870250102279, dated 07 / 11 / 2025, page 89 / 144 81 / 116 uptake of glucoverdazil in extraurinary tissues, reproducible efflux of renal contrast to the bladder, relatively rapid renal clearance time of glucoverdazil, and its demonstrated cytocompatibility suggest that glucoverdazil may be suitable for evaluating renal function by magnetic resonance imaging. Example 4. MRI-DCE with Glucoverdazil of Acute Kidney Injury due to Unilateral Ureteral Obstruction.

[00143] A murine model of unilateral ureteral obstruction (UUO) was used to determine the efficacy of glucoverdazil as a DCE-RMD agent for acute kidney injury (AKI) caused by obstructive nephropathy. Surgical obstruction of the left ureter prevents fluid clearance, leading to hydronephrosis and a drastic reduction in renal function of the ipsilateral kidney. Murine groups were evaluated: placebo (left kidney touched by surgical instrument) and surgical UUO (left kidney ligated). The change in voxel-by-voxel intensity over time within the kidneys was determined for the entire left and right kidney (FIGs. 3A to 3D), as well as for their cortex and medullary / renal pelvis (MRP) regions (FIGs. 18A to F). Voxel-by-voxel mapping of RDTC was used to assess changes in renal function. There was no perceptible change in renal morphology between days 0, 3, or 7 in the placebo group of mice; However, the morphology of the kidney ipsilateral to the obstruction of Petition 870250102279, dated 07 / 11 / 2025, pp. 90 / 144 82 / 116 ureter changed drastically on days 3 and 7 compared to day 0 in the UUO mouse group. Hydronephropathy was evident by ablation of the medullary region on day 3, which continued to worsen on day 7, a characteristic of the UUO model (Xiong et al., 2021). The kidney contralateral to the ligated ureter in UUO mice showed no obvious morphological changes.

[00144] RDTC was determined in BALB / c mice prior to surgery in order to define the optimal time interval for glucoverdazil clearance before analysis of data from diseased mice (FIGs. 18A to F). In placebo-treated mice, no significant change in RDTC was observed graphically (FIG. 3A) and quantitatively (FIG. 3B) on days 3 or 7 relative to day 0 in either kidney. In mice treated with UUO, however, a significant change in ipsilateral kidney RDTC was observed, increasing from k = -0.135 min-1 ± 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. Contralateral kidney RDTC in UUO mice remained unchanged between days 0 and 3 (k = -0.133 min-1 ± 0.020 min-1 versus k = 0.117 min-1 ± 0.014 min-1, respectively), but showed a significant increase in RDTC on day 7 (k = -0.097 min-1 ± 0.010 min-1).Changes in contralateral kidney physiology after UUO are expected in rodent models with induction. Petition 870250102279, dated 07 / 11 / 2025, pp. 91 / 144 83 / 116 of the macrophage-myofibroblast transition (Figueroa et al., 2019), fibrosis (Xiong et al., 2021; Bianco et al., 2019), and alteration of cortical mitochondrial function (Eddy et al., 2012) previously reported. These data demonstrated that glucoverdazil-mediated RMDCE was able to detect contralateral kidney functional impairment early after ipsilateral ureteral obstruction.

[00145] Histological and serum creatinine (SCr) analyses were performed on both groups of mice to confirm the pathology observed through qualitative magnetic resonance imaging and CTRD mapping, as well as for renal function using a gold-standard technique. Both the morphology and fibrosis staining of placebo kidneys and the ipsilateral kidney in UUO-treated mice showed no significant alterations, while the kidney ipsilateral to the ureteral obstruction clearly showed hydronephropathy (FIG. 3C). No significant changes in Scr were detected in placebo mice between days 0 and 7 after surgery, while a significant increase was observed in UUO mice (FIG. 3D). These changes observed in CrS align well with those previously reported in UUO models and recapitulate the changes observed in RDTC (Vielhauer et al., 2001; Martinez-Klimova et al., 2019; Fink et al., 1987).The fact that AUC measurements do not parallel the change measured in CrS corroborates this. Petition 870250102279, dated 07 / 11 / 2025, page 92 / 144 84 / 116 the use of RDTC as a measure of renal function by glucoverdazil-mediated MRI-DCE.

[00146] Although damage to the ipsilateral kidney due to ureteral obstruction was evident even on anatomical magnetic resonance imaging, the UUO model demonstrated that standard glucoverdazil-mediated MRI-DCE techniques with simple kinetic mapping can be used to show regional and structural defects in both kidneys, alerting to functional changes arising in the contralateral kidney even before positive staining for fibrosis. A regional analysis of the data discriminating glucoverdazil clearance from the cortex versus medulla and renal pelvis was performed (FIGs. 18A to F). In this paper, an impairment in glucoverdazil clearance caused by a decrease in renal function was clearly discerned, while also mapping where the pathology was occurring within the kidney, which is extremely valuable for the assessment of AKI (Matsumoto et al., 2022; Chevalier et al., 2009; Xiong et al., 2021). Example 5. RMD-DCE with Glucoverdazil for Acute to Chronic Kidney Injury due to Folic Acid-Induced Nephropathy (ANA).

[00147] Next, renal function was evaluated in a more complex model of kidney disease, driven by fibrosis, mediated by acid-induced nephropathy. Petition 870250102279, dated 07 / 11 / 2025, pp. 93 / 144 85 / 116 folic acid (ANF). Both RDTC and AUC were evaluated, as well as supporting histology and CrS (FIG. 4). ANF is the result of the formation of tubular folic acid crystals after systemic administration of folic acid (Jiang et al., 2018). This crystallization causes an initial phase of severe AKI, followed by fibrotic renal scarring that leads to a progressive and long-term decline in renal function, resulting in CKD approximately 3 weeks after folate injection. The ANF model was implemented in BALB / c mice instead of the more commonly used C57Bl / 6 strain, since BALB / c mice were more resistant to the AKI phase, which presented an extremely high mortality rate in C57Bl / 6 mice.

[00148] Through anatomical imaging, a reduction in overall kidney size was observed from day 0 to day 30 in both kidneys, which had been previously reported for the ANA model (Doi et al., 2006; FIG. 4A). After glucoverdazil-mediated RMD-DCE, RDTC was determined for both kidneys before folate administration and 15 and 30 days after administration (FIG. 4A graphically and FIG. 4B quantitatively). A significant increase in RDTC was observed on 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 on day 30 (k = -0.139 min-1 ± 0.016 min-1). The AKI phase of FAN resulted Petition 870250102279, dated 07 / 11 / 2025, pp. 94 / 144 86 / 116 in a significant increase in medullary and cortical RDTC, indicative of impaired ureteral drainage and impaired glomerular filtration. Recovery of RDTC on day 30 is anticipated in the early stages of CKD, as AKI often presents with a much more severe level of renal dysfunction than the early stages of CKD, which is consistent with the literature that uses the same temporal points after injury (Doi et al., 2006). RDTC maps on day 30 indicated the presence of striated regions located within the cortex, showing relatively higher RDTC (FIG. 4A right, black arrows), which map the fibrotic striations in the kidneys on day 30, assessed by histology (FIG. 4C, light blue regions are indicators of fibrotic tissue highlighted by black arrows). Therefore, the combination of spatial and temporal information in maps of individual kidneys, as was done in the case of CT scans (FIG.4A) provides enhanced diagnostic power not offered by existing nephrological techniques, which are limited to spatial or kinetic information only.

[00149] Kidneys were collected on days 0, 15, and 30 after folate injection and evaluated histologically to confirm both AKI and CKD (FIG. 4C), and blood was collected at the same time intervals for SR determination (FIG. 4D). Histological evaluation Petition 870250102279, dated 07 / 11 / 2025, pp. 95 / 144 87 / 116 revealed substantially increased fibrotic regions in the kidney on day 15, but which decreased in severity on day 30 (FIG. 4C). Systemic creatinine (Scr) showed a slight elevation on day 15 compared to day 0, which was expected, as acute renal failure (ARF) generally shows only small elevations in Scr (Doi et al., 2006; Waikar et al., 2009). However, on day 30, during the early stages of chronic kidney disease (CKD), Scr increased significantly compared to day 0 and day 15, which is a strong indicator of a severe decrease in renal function associated with antinuclear antibody (ANA) and the early stages of CKD (Aparicio-Trejo et al., 2020; Yam, 2021; Scarfe et al., 2018). The importance of applying a method for renal assessment combining spatial and temporal evaluation was highlighted by the ANA model assessment. During severe AKI, slight elevations in CrS were observed, but a much larger increase in RDTC was obtained with differential regional effects throughout the kidney (FIG. 4A).Of singular value, the RDTC maps indicated discrete regions of greater renal dysfunction (i.e., cortical striae), which may correspond to areas of fibrosis observed in histological evaluations (FIG. 4C). Example 6. Comparison of glycoderdazil-mediated DCE-RM with a validated measure of GFR

[00150] Although GFR measurement is of immense clinical value, current methods rely on error-prone techniques, limited to kinetic measurements of renal function. Petition 870250102279, dated 07 / 11 / 2025, pp. 96 / 144 88 / 116 Aiming at the development of an image-based approach for the assessment of renal function, this study sought to compare glucoverdazil-mediated RMD-DCE with an established and validated measure of GFR, namely, transdermal fluorescence monitoring. The transdermal GFR technique is based on the intravenous injection of a fluorescent molecule (e.g., FITC-sinistrine), which is cleared exclusively by filtration, and transdermal monitoring of blood pool fluorescence over time. The transdermal technique applies a monophasic decay model to determine the RDTC of the fluorescence intensity versus time curve, which is then corrected for 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).Transdermal GFR measurements were used as a reference for deriving the specific correction factor for glucoverdazil.

[00151] In a second cohort of ANA-induced mice, both transdermal measurements (FIG. 5A) and glucoverdazil-mediated RMD-DCE measurements (FIG. 5B) were performed on day 0 (black), day 15 (pink), and day 30 (cyan). Both techniques resulted in the characteristic monophasic decay curve, yielding RDTC values ​​after seminatural logarithmic transformations. On day 0, the RDTC Petition 870250102279, dated 07 / 11 / 2025, page 97 / 144 The mean 89 / 116 determined by glucoverdazil-mediated RMD-DCE (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 not surprising, given the different data collection sites. While transdermal measurements assess the signal from blood accumulation within the first few millimeters of the skin surface, the RMD-DCE technique assesses signal clearance from within the renal tissue itself. With the transdermal technique, the baseline GFR = 1584 ± 238 pI / min / 100 g body weight was determined for BALB / c mice, which is in agreement with values ​​reported in the literature (Yan et al., 2021). By pooling baseline RDTC data obtained for BALB / c mice on day 0 and using the mean GFR measurement from the transdermal technique, a specific correction factor for glucoverdazil was derived for the conversion of RDTC to GFR.With this factor, the GFR was calculated from the t-RDTC values ​​for each time point after the injury and compared with the GFR values ​​determined by the transdermal technique (FIG. 5C), demonstrating that there was no significant difference in the GFR determined by the two methods.

[00152] In conclusion, a tetrazinanone-derived ORCA preparation, glucoverdazil, was optimized and scaled up and demonstrated superior redox stability and cytocompatibility compared to other agents. Petition 870250102279, dated 07 / 11 / 2025, pp. 98 / 144 90 / 116 nitroxy radical contrast agents previously used. Glucoverdazil appears to be especially useful for renal RMD-DCE due to its specific uptake limited to the kidneys, ureter, and bladder. Glucoverdazil has been applied to image the UUO model of severe AKI and the ANA model of AKI progression to CKD, showing regional functional changes in the kidneys in the form of RDTC. Voxel-wise AUC mapping for both models did not correlate with histological and CrS changes as reliably as RDTC mapping, which fully corroborated renal dysfunction. By comparison with a validated transdermal fluorescence recording method for measuring GFR, glucoverdazil has been shown to provide reliable GFR determination by RMD-DCE.It is important to emphasize that this approach to GFR measurement not only adds a spatial component to the gold-standard nephrological assessment, but also determines GFR without relying on the patient's demographic characteristics, which have proven prone to errors. In general, glucoverdazil can provide more reliable diagnoses by magnetic resonance imaging in patients with known or suspected AKI and / or CKD. Given the molecular properties of this organic radical, especially its inability to penetrate the cell, excellent cytocompatibility and preliminary biocompatibility, localization in the kidneys, and rapid clearance, ORCAs... Petition 870250102279, dated 07 / 11 / 2025, pp. 99 / 144 91 / 116 tetrazinanone-based contrast agents represent a promising new class of metal-free magnetic resonance contrast agents. Experimental Procedures for Examples 1 through 6.

[00153] General Reagents. All chemical reagents were purchased from Sigma-Aldrich and used as is, unless otherwise indicated, with the exception of N'-(propan-2-yl)(tert-butoxy)carbohydrazide, which was purchased from AABlocks. All reagents and consumables for cell culture were purchased from ThermoFisher, with the exception of Epithelial Cell Medium and Epithelial Cell Growth Supplement, which were purchased from ScienCell.

[00154] 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 NMR acquisitions were performed on a 3T preclinical MRI scanner (MR Solutions, Ltd.). For all MRI image data analyses, only relevant slices of the tissue of interest were included in the analysis (i.e., scans of the renal region used only slices with the kidney visible). All processing, mapping, and generation of data quantities were performed using a program written in MATLAB 2020A®. GraphPad Prism 9.5 was used to generate all graphs, graphic figures, and Petition 870250102279, dated 07 / 11 / 2025, pp. 100 / 144 92 / 116 statistical results.

[00155] In vivo MRI studies in animals. All in vivo MRI studies in animals were conducted under the Animal Use Protocol HIe-3640-R1, approved by the IACUC of the University of Ottawa. Mice were anesthetized with isoflurane, placed in a heated cradle, and inserted into the magnetic resonance imaging system. Consecutive Ti-weighted RARE images were acquired before and every 2.5 minutes for 60 minutes after contrast agent injection. Ti-weighted image: slice thickness 1 mm, field of view 50 x 50 mm, mean = 3, matrix size = 96 x 96, TE = 11 ms, echo spacing = 7 ms, TR = 720 ms, and acquisition time 2 minutes and 16 seconds. In all cases, 3 mmol / kg of contrast agent was injected intravenously through a catheter in the caudal vein, which was irrigated with saline solution to ensure delivery of the full dose of contrast agent.

[00156] Magnetic resonance data processing and analysis. Magnetic resonance data processing and analysis were conducted as follows: (1) Intensity-Over-Time Curves. Relevant slices were fitted to remove any automatic gain functions associated with magnetic resonance, and all scans and slices were normalized to a water-filled fiducial marker placed next to the mice. Petition 870250102279, dated 07 / 11 / 2025, pp. 101 / 144 93 / 116 during all scans. A MatLab routine was used to draw regions of interest (ROIs) in slices at each scan time point to generate intensity data over time in voxels, presented as the average total ROI intensity for each time point normalized to 100% for the lowest intensity voxel for the first scan. (2) Renal deterioration time constant values ​​and image maps. The natural logarithm of each ROI intensity value on the intensity curve over time in voxels was used to generate a semi-log curve. A linear regression was then applied from t = 2.5 minutes to at = 40 minutes, and the slope in voxels of this curve yielded k (renal deterioration time constant (RDTC), min-1). The RDTC values ​​shown in the graphs are the average RDTC value per voxel. Maps of k were overlaid on the image acquired at t = 0 minutes. (3) Area Under the Curve Values ​​and Image Maps.A baseline correction was applied to each of the intensity curves over time, voxel by voxel, subtracting the smallest voxel value, setting the baseline to 0 at all time points. The integral of this curve for each voxel was acquired using the trapezoid function to generate an area under the curve (AUC) value. The AUC values ​​shown in the graphs are the average AUC value per voxel. The AUC maps were overlaid on the image acquired at t = 0 minute. Petition 870250102279, dated 07 / 11 / 2025, pages 102 / 144 94 / 116 An illustration of the data acquisition and image mapping workflow using RMD-DCE is shown in FIG. 6.

[00157] Evaluation of Glucoverdazil Tissue Localization by MDRC-DCE. Glucoverdazil contrast 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 renal, hepatic, bladder, and muscle tissues.

[00158] Serum Creatinine Measurements. For all disease models, blood was serially collected from the saphenous vein on day 0 and at each time point after injury, prior to MRI with glucoverdazil. Blood was centrifuged for 10 minutes (room temperature, 900 xg) and serum was collected from the fractionated sample. Samples were stored at -80 °C until use. Serum creatinine (CrS) was determined by quantitative HPLC (Agilent 1260 Infinity diode array equipped with a 2.1 mm x 50 mm, 5 µm particle size Agilent Zorbax 300-SCX column) relative to a creatinine standard curve, modifying a previously reported method (Bello et al., 2019). In summary, creatinine was dissolved in the 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. Petition 870250102279, dated 07 / 11 / 2025, pp. 103 / 144 95 / 116 by integrating the HPLC peak produced at 234 nm (Bruker HyStar PP). Standards and samples were acquired at a flow rate of 0.5 mL / min at an isocratic mobile phase concentration. Creatinine was extracted from mouse serum samples after thawing by protein precipitation by adding a 4:1 ratio of 0.5% acetic acid-containing NaCl solution to the serum. Samples were vortexed and left at -20 °C for 30 minutes to allow complete precipitation and sedimentation. Samples were then centrifuged at 12,000 x g (10 min, 4 °C) and the supernatant transferred to a new tube. Tubes were dried to remove acidified NaCl by heated vacuum centrifugation for 45 minutes at 50 °C. The resulting granule was resuspended in 60 μl of mobile phase and the samples were subjected to HPLC with integration of the elution peak at a corresponding time observed on the standard curve at 234 nm.

[00159] Animal Models of Renal Disease. Contrast-enhanced images with glucordazil for all mice in the UUO and ANA disease models were acquired as described above, and normalized intensity-over-time curves were acquired as described above, with ROIs drawn for renal tissue.

[00160] Unilateral Ureteral Obstruction. The murine model of acute kidney injury (AKI) with ureteral obstruction. Petition 870250102279, dated 07 / 11 / 2025, pages 104 / 144 A unilateral 96 / 116 (UUO) procedure was performed in C57 / B16 mice, as previously reported in the literature (Vanholder et al., 2021). Briefly, 10 female C57 / B16 mice (8 weeks old) were divided into groups of 5 for either the placebo procedure or the UUO procedure. The mice underwent glucoverdazil contrast imaging immediately before surgery (day 0). The mice were anesthetized by continuous isoflurane inhalation. The left kidney of the mouse was accessed laparoscopically, and the left ureter was gently tapped with a surgical instrument (placebo group) or closed with a suture (UUO group). The wound was closed with a suture, and the mice underwent glucoverdazil contrast-enhanced magnetic resonance imaging 3 and 7 days post-injury. On day 7, the 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 by a slide scanner.

[00161] Folic Acid-Induced Nephropathy (Glucoverdazil Contrast-Induced Nephropathy). The folic acid-induced nephropathy (AAN) model of acute kidney injury (AKI) for chronic kidney disease (CKD) was performed in BALB / c mice following a modification of the 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., 2011; Chen et al., 2019; Gama et al., 2011; Chen et al., 2019; Gama et al. Petition 870250102279, dated 07 / 11 / 2025, pp. 105 / 144 (97 / 116 al., 2021). An extremely high mortality rate was observed with doses of 250 mg / kg of folic acid (FA) administered to CD1 and C57 / Bl6 mice. BALB / c mice have been shown to be more resistant to obstruction-mediated lesions and to generate CKD more reliably (Luis-Lima et al., 2017; Niemantsversriet et al., 2021). Five BALB / c mice underwent magnetic resonance imaging with glucoverdazil on day 0. Immediately after the examinations, the mice were injected intraperitoneally with 125 mg / kg of FA in a 0.3 M sodium bicarbonate solution. Daily subcutaneous fluid support was required during the first 5 days after FA injection, where the effects of FA are most severe. By day 7, the mice were stabilized and housed normally without any fluid assistance. The mice underwent magnetic resonance imaging again with glucoverdazil 15 and 30 days after the injury.On day 30, the 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 MRI with glucoverdazil and were sacrificed on day 15 to obtain histology to that point in time and to maintain uninterrupted longitudinal MRI data. Petition 870250102279, dated 07 / 11 / 2025, pp. 106 / 144 98 / 116 this disease model.

[00162] Folic Acid-Induced Nephropathy (Transdermal Fluorescence). A parallel group of 6 mice had kidney disease induced identically to those in the contrast-enhanced 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, before any data acquisition, hair was removed from the right dorsolateral aspect of the mice. The following day, a transdermal fluorescence monitor (MediBeacon, Inc.) was attached to the shaved area using a patented adhesive with a window. The battery was connected to the transdermal monitor and a 5-minute baseline was established. A solution of FITC-sinistrina (150 pL, 0.2 mg / kg) was injected intravenously via the caudal vein, and data were collected for 55 minutes.Data collection on day 0 immediately preceded intraperitoneal injection of FA and was repeated 15 and 30 days after injury, at which time mice were sacrificed by cervical dislocation.

[00163] Conversion of RDTC to Glomerular Filtration Rates. Data acquired by transdermal fluorescence were analyzed using proprietary software (MediBeacon, Inc.) to generate RDTC and rate values. Petition 870250102279, dated 07 / 11 / 2025, pp. 107 / 144 99 / 116 glomerular filtration rate (GFR) based on pharmacokinetic model adjustment. GFR and RDTC data were generated using this software with a monophasic decay model of the raw data, without corrections. MediBeacon uses a previously determined factor that can directly convert RDTC to GFR, based on mouse data generated by them and compared to GFR measured by 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 RDTCs in the 14 healthy BALB / c mice measured, a conversion factor for RDTC with glucoverdazil to GFR was derived based on the mean GFR value of the 6 healthy BALB / c mice measured by transdermal fluorescence. By comparing the difference between combined RDTC and transdermal derived RDTC to normalize the mean GFR value, the conversion factor was derived.This conversion was then applied to the average RDTC value shown in the data to generate a comparison of the GFR between the two methods.

[00164] Synthesis of N-({N'-[(tert-butoxy)carbonyl]-N(propan-2-yl)hydrazinecarbonyl}(propan-2-yl)amino)(tert-butoxy)formamide (2). Dry EtaN (4 mL, 28.8 mmol) was added to a toluene solution (50 mL, pre-dried with 4 Å molecular sieves) followed by the addition of 4 g of N'-(propan-2-yl)(tert-butoxy)carbohydrazide (4 g, 23 mmol). The solution was cooled to 0 °C while being stirred in Petition 870250102279, dated 07 / 11 / 2025, pp. 108 / 144 100 / 116 atmosphere of N2. A 15% phosgene solution in toluene (9 mL, 12.7 mmol) was added dropwise over approximately 1 minute (phosgene is highly toxic; care must be taken when adding it), and the reaction mixture was stirred for 1 hour at 0 °C, then heated to room temperature (rt) and stirred for a further 18 h. The reaction was quenched by the addition of MeOH (50 mL), stirred for 30 min at room temperature, and evaporated. The mixture was diluted with 10% NH4OH solution (75 mL), followed by extraction with EtOAc (3 x 15 mL). The combined organic was washed with brine (40 mL), dried with Na2SO4, filtered, and evaporated, resulting in a white powder. The powder was dissolved in 80 mL of hot, dry heptane and left aside for 18 h at 4 °C to induce crystallization of the product. The crystals were filtered and washed with hexane. The product was dried under high vacuum (colorless crystals, compound 2, 2.55 g, 59%).1H NMR (400 MHz, CDCla) δ 6.40 (s, D2O exchange, 2H), 4.15 (s, 2H), 1.43 (s, 18H), 1.12 (s, 12H). 13C NMR (150 MHz, CDCla) δ = 155, 9, 81, 1, 52, 6, 50.3, 28.2, 19.2 (wide signal) HRMS (ESI): Calculated for Ci7H34N4OsNa [M+Na] + : 397.2411, found 397.2427.

[00165] Synthesis of 1,3-diamino-1,3-bis(propan-2-yl)urea. Compound 2 (2.55 g) was resuspended in EtOH (25 mL) in a 100 mL round-bottom flask and heated to 80 °C (air condenser). Concentrated HCl (10 mL) was added. Petition 870250102279, dated 07 / 11 / 2025, pp. 109 / 144 101 / 116 drop by drop and the solution was stirred for 30 minutes at 80 °C. The solution was cooled to room temperature and the solvent was evaporated. The crude product was co-evaporated consecutively once with methanol, toluene, and petroleum ether (50 mL for each). The crude product with sufficient purity for the subsequent step was dried under high vacuum, resulting in 1,3-diamino-1,3-bis(propan-2-yl)urea dihydrochloride as a colorless solid in quantitative yield. 1H NMR (400 MHz, MeOH-D4) δ 4.23 (heptide, J = 6.8 Hz, 2H), 1.35 (d, J = 6.8 Hz, 12H). 13C NMR (150 MHz, MeOH-D4) δ = 162.3, 56.2, 18.9. HRMS (ESI): Calculated for CvHis^ONa [M+Na] + : 197.1355, found 197.1378.

[00166] Synthesis of 6-[(1S,2R,3R,4R)-1,2,3,4,5-pentahydroxypentyl]-2,4-bis(propan-2-yl)-1,2,4,5-tetrazinan-3one (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 room temperature. A solution of 5 mL of D-glucose (1.2 g, 6.7 mmol) and NaOAc (1.1 g, 13.4 mmol) in water was added dropwise over 1 minute, followed by stirring at room temperature for 18 h. The reaction mixture was extracted with n-butanol (6 x 10 mL). The combined organic extract was dried with Na2SO4, filtered, and evaporated. The resulting oil was consecutively co-evaporated with methanol, toluene, and petroleum ether (50 mL of each). The product Petition 870250102279, dated 07 / 11 / 2025, pp. 110 / 144 The resulting 102 / 116 was dried in high vacuum overnight, producing light yellow crystals (compound 3, 1.48 g, 70%). 1H NMR (400 MHz, MeOH-D4) δ 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) δ = 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 Ci3H2sN4OeNa [M+Na] + : calculated 359, 1910, found 359, 1907.

[00167] Synthesis of 3-oxo-6-[(1S,2R,3R,4R)-1,2,3,4,5-pentahydroxypentyl]-2,4-bis(propan-2-yl)-1,2,3,4-tetrahydro-1,2,4,5-tetrazin-1-yl (4). Compound 3 (1.48 g, 4.41 mmol) was resuspended in H2O (5 mL with stirring (room temperature)). In a separate container, potassium ferricyanide (4.44 g, 13.5 mmol) was mixed with 80 drops (~4.5 mL) of NaHCO3 solution (2 M), followed by the addition of water (5 mL); the mixture was solubilized using an ultrasonic bath. The resulting solution was added dropwise over 1 minute to the original reaction mixture, with stirring followed by stirring (at room temperature) for about 30 minutes or until effervescence ceased. The mixture was extracted with n-butanol (6 x 10 mL). The combined organic mixture was dried with Na2SO4, filtered, and evaporated. The resulting oil was co-evaporated consecutively with methanol (50 Petition 870250102279, dated 07 / 11 / 2025, pp. 111 / 144 The compound was eluted three times with toluene (50 mL each time), cooled to 0 °C, followed by coevaporation with petroleum ether (50 mL). The resulting product was dried under high vacuum overnight to yield a bright yellow fine powder (glucoverdazil 4, 1.09 g, 74%). Given that the compound was radical in nature, it could not be distinguished by NMR. Changes in elution time by HPLC, as well as HRMS and EPR, were used to confirm the structure and purity. HPLC traces can be seen in FIG. 12. HRMS (ESI): Calculated for CH iO / N / ; [M+Na]+: calculated 356.1676, found 356.1672.

[00168] Magnetic resonance imaging (MRI) with phantom and determination of the longitudinal relaxivity of glucoverdazil. Contrast samples were prepared in 1x PBS in standard MRI tubes, which were then inserted into a 50 mL Falcon tube containing ultrasound gel, containing the MRI phantom. The MRI phantom was placed in a 38 mm diameter send-receive volume coil and inserted into the MRI. A Rapid Imaging with Refocused Echoes (RARE) multidetector pulse sequence was implemented for phantom evaluation using the following parameters for Ti-weighted images: slice thickness of 5 mm, field of view of 40 x 40 mm, averages = 3, matrix size = 96 x 96, TE = 11 ms, echo spacing = 7 ms, TR = 720 ms and acquisition time of 2 minutes and 16 seconds. For T2-weighted images, all parameters were the same as for images. Petition 870250102279, dated 07 / 11 / 2025, pp. 112 / 144 104 / 116 weighted in Ti, except TE = 68 ms and TR = 4800 ms, and the acquisition time was 7 minutes and 28 seconds.

[00169] For relaxivity measurements, the same image generation phantom was used with contrast concentrations of 1 to 3 mM, which were verified by electron paramagnetic spectroscopy. To measure the longitudinal relaxation rate (r1), an inversion recovery RARE sequence was implemented with the following parameters: slice thickness of 5 mm, field of view of 50 x 50 mm, mean = 1, matrix size = 96 x 96, 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 and 30 seconds per TI. Longitudinal relaxation rates were extracted using the MATLAB mapping2 routine, written by J. Barral, M. Etezadi-Amoli, E. Gudmundson, and N. Stikov (2009) and modified by J. Rioux (2022). Longitudinal relaxivity (r1) was extracted from the slope of the graph of 1 / Ti versus contrast agent concentration.

[00170] Stability measurements of glucoverdazil. The EPR was adjusted for a sample of glucoverdazil or TEMPO in PBS before any stability measurements. Once adjusted, solutions of glucoverdazil or TEMPO were prepared (20 mM in mouse serum or 5 mM in 4 mM sodium ascorbate buffer pH 7.4). A single spectrum was Petition 870250102279, dated 07 / 11 / 2025, pp. 113 / 144 105 / 116 acquired and the height of the most intense peak for each compound was recorded. EPR scans were then acquired every 5 s for 2 h (mouse serum) or 1.5 h (ascorbate) to measure the percent change in activity. For stability measurements of glucoverdazil in water, a 5 mM sample was prepared and left in a fume hood exposed to light, or wrapped in aluminum foil and left in a dark refrigerator at 4 °C. Periodically, these solutions were sampled and measured by EPR after adjustment, using a freshly prepared 5 mM glucoverdazil sample.

[00171] Evaluation of cell viability in H460 cells. Large cell lung cancer (H460) cells were cultured in RPMI-1640 medium (RPMI) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P / S) to 80% confluence, at which point they were re-passed. Cells were re-passed three times before being seeded in a 6-well plate and cultured to 80% confluence. Cells were seeded to obtain 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 glucoverdazil for 4 or 24 hours. At the respective time points, the medium was aspirated and the cells were washed three times with Dulbecco's phosphate-buffered saline (PBS) at 37 °C. Next, the cells were harvested with trypsin-EDTA, centrifuged at 400 xg (5 Petition 870250102279, dated 07 / 11 / 2025, pages 114 / 144 106 / 116 minutes, 4 °C), aspirated and resuspended in 1 mL of PBS solution containing 0.2 pM calcein-acetoxymethyl ester (fluorescent green for live cells) and 16 pM ethidium homodimer-1 (fluorescent red for dead cells).

[00172] Live and dead cell populations were counted by flow cytometry (Beckman-Coulter Gallios flow cytometer) using an excitation of 488 nm 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 completion, the viable cell population for each condition was determined by comparing the total number of calcein-AM positive cells, individually stained, with the combined total of cells that were individually stained as positive for live or dead cells, using the Kaluza analysis software (Beckman-Coulter).

[00173] Evaluation of the viability of human renal proximal tubule (hRPT) cells. hRPT cells were cultured in epithelial cell culture medium (EpiMEM) supplemented with 10% FBS, 1% P / S and epithelial cell growth supplement (EpiCGS) until they reached 80% confluence, at which point they were subjected to passage. The cells were passed three times before Petition 870250102279, dated 07 / 11 / 2025, pages 115 / 144 107 / 116 cells were seeded in a 6-well plate and cultured until they reached 80% confluence. Cells were seeded to obtain triplicate wells of each condition. The cells were then incubated in their regular media supplemented with regular media, 10 mM glucoverdazil, 10 mM 5,5-dimethyl-1-pyrroline N-oxide (DMPO, a nitrone spin trap), or 10 mM (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO, a nitroxy radical), for 4 or 24 hours. At the respective times, the medium was aspirated and the cells were washed three times with Dulbecco's phosphate-buffered saline (PBS) at 37 °C. Next, the cells were harvested with trypsin-EDTA, centrifuged at 400 xg (5 minutes, 4 °C), aspirated, and resuspended in 1 mL of PBS solution containing 0.2 pM calcein-acetoxymethyl ester (fluorescent green staining for live cells) and 16 pM ethidium homodimer-1 (fluorescent red staining for dead cells).

[00174] Live and dead cell populations were counted by flow cytometry (BeckmanCoulter Gallios flow cytometer) using an excitation of 488 nm 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 procedure, the cell population Petition 870250102279, dated 07 / 11 / 2025, pages 116 / 144 The 108 / 116 viable cells for each condition were determined by comparing the total number of individually stained calceinAM-positive cell counts with the combined total of cells that were individually stained as positive for live or dead cells using Kaluza analysis software (BeckmanCoulter).

[00175] Evaluation of glucoverdazil uptake in hRPT cells. hRPT cells were cultured in epithelial cell culture medium (EpiMEM) supplemented with 10% FBS, 1% P / S, and epithelial cell growth supplement (EpiCGS) to 80% confluence, at which point they were passed through. Cells were passed through three times before being seeded into 6-well plates and cultured to 80% confluence. Cells were seeded to obtain triplicate wells of each condition. Cells were then incubated in their regular media supplemented with regular medium or 10 mM glucoverdazil and incubated for 24 hours. The medium was aspirated, and the cells were washed three times with Dulbecco's phosphate-buffered saline (PBS) at 37 °C. Next, the cells were diluted with trypsin-EDTA, centrifuged at 400 xg (5 minutes, 4 °C), aspirated, and resuspended in 100 μl of PBS. The concentrated cell solutions were transferred to EPR tubes.A 1 μl aliquot was retained and diluted to obtain the number. Petition 870250102279, dated 07 / 11 / 2025, pp. 117 / 144 109 / 116 cells in each solution.

[00176] The EPR was adjusted for a freshly prepared 5 mM glucoverdazil solution in PBS, and then the samples were measured by EPR. The concentration was measured relative to a previously determined standard curve and then normalized to the previously determined number of cells to obtain nM of glucoverdazil per cell.

[00177] High-performance liquid chromatography traces. To verify the completion of the radicalization of compound 3 to 4, HPLC traces were used in conjunction with HRMS and EPR. A 20-minute HPLC gradient was used with 0.5% TFA H2O, increasing from 1% to 100% over 20 minutes with 0.5% TFA acetonitrile. The elution time of compound 3 was consistently 10.5 minutes, while after radicalization and the loss of two protons from the verdazyl ring, this time increased to 11.8 minutes. Furthermore, radicalization induced higher absorbance at 452 nm for compound 4, while no activity at this wavelength was observed in the non-radical compound 3. High-performance liquid chromatography traces of compounds 3 and 4 to verify the radical activity of the compound after the radicalization step are shown in Figure 13. REFERENCES

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[00179] Although this invention is described in detail with reference to its embodiments, these embodiments are offered to illustrate, but not to limit, the invention. It is possible to create other embodiments that employ the principles of the invention and that fall within its essence and scope, as defined by Petition 870250102279, dated 07 / 11 / 2025, pp. 124 / 144 116 / 116 attached claims.

[00180] The content of all documents and references cited in this document are incorporated herein by reference in their entirety. Petition 870250102279, dated 07 / 11 / 2025, pages 125 / 144

Claims

1 / 9 CLAIMS 1. Compound of Formula (I), or a pharmaceutically acceptable salt or ester thereof, for use as a contrast agent in magnetic resonance imaging: Rix JL .Ri δN�^NΓ 1 4NAL· N2 r2 (I) characterized in that: Ri is selected from: wherein R is a monosaccharide or CH2OH; Petition 870250085850, dated 23 / 09 / 2025, p. 47 / 56 2 / 9 Rs is selected from: °~ò O oA NO2 -Xi '0 Xi where Xi is any halogen. Compound according to the claim characterized in that Rs 3. Compound agreement characterized by the fact that Formula (II), or one of the same:

4. Compound claims 1 a monosaccharide is a 5. Compound claims 1 to 3 with claim 3 is a pharmaceutically effective salt or ester compound according to any 3, characterized by the fact that one of the monosaccharides is glucose, fructose, galactose or mannose. Petition 870250085850, dated 23 / 09 / 2025, p. 48 / 56 3 / 9 6. Compound according to claim 1, characterized in that the compound is: V OH OH OH OH OH OH HO. .OH ,OH Ύ < OH HO. / 0 HO V ΌH ÕH or a pharmaceutically acceptable salt or ester thereof.

7. Composition, characterized in that it comprises the compound, as defined in any one of claims 1 to 6, and a carrier.

8. Composition according to claim 7, the composition being characterized in that it is a pharmaceutical composition and the carrier is a pharmaceutically acceptable carrier.

9. Compound, according to any one of claims 1 to 6, or composition, according to any Petition 870250085850, dated 09 / 23 / 2025, page 49 / 56 4 / 9 one of claims 7 or 8, characterized in that the compound is glucoverdazil.

10. A method for generating biomedical images, characterized by comprising administering a contrast agent to an individual and obtaining images of the contrast agent in the individual, wherein the contrast agent comprises the compound as defined in any one of claims 1 to 6, or the composition as defined in any one of claims 7 or 8.

11. Method according to claim 10, characterized in that the generation of biomedical images comprises magnetic resonance imaging (MRI).

12. Method, according to claim 11, characterized in that the MRI is a dynamic contrast-enhanced magnetic resonance imaging (DCI-MRI).

13. Method, according to claim 11, characterized in that the MRI is a contrast-enhanced magnetic resonance imaging (CMR).

14. Method, according to any one of claims 10 to 13, characterized in that the image generation is a kidney image generation.

15. Method, according to any one of claims 10 to 14, characterized in that image generation is used to assess renal function. Petition 870250085850, dated 09 / 23 / 2025, pp. 50 / 56 5 / 9 16. A method, according to any one of claims 10 to 15, characterized in that the individual has, is suspected of having, or is at risk of having renal dysfunction.

17. A method, according to any one of claims 10 to 15, characterized in that the individual has, is suspected of having, or is at risk of having chronic kidney disease (CKD), acute kidney injury (AKI), renal artery stenosis, urinary obstruction, or renal tumor or malignancy, or is a kidney donor.

18. A method, according to any one of claims 10 to 16, characterized in that it further comprises determining the Glomerular Filtration Rate (GFR) of an individual.

19. A method, according to any one of claims 10 to 18, characterized in that quantitative and / or qualitative information about renal function is obtained.

20. A method, according to any one of claims 10 to 19, characterized in that it further comprises mapping the Glomerular Filtration Rate (GFR) of the individual.

21. Method for diagnosing renal dysfunction in an individual, characterized by comprising administering a contrast agent to the individual, obtaining images of the contrast agent in the individual and determining and / or mapping the Glomerular Filtration Rate (GFR) of the individual, wherein the contrast agent comprises the compound as defined in any one of claims 1 to 6, or the composition as defined in any one of claims 7 or 8.

22. A method for monitoring, evaluating, or determining renal function in an individual, characterized by comprising administering a contrast agent to the individual, obtaining images of the contrast agent in the individual, and determining and / or mapping the individual's Glomerular Filtration Rate (GFR), wherein the contrast agent comprises the compound as defined in any one of claims 1 to 6, or the composition as defined in any one of claims 7 or 8.

23. Method, according to any one of claims 10 to 22, characterized in that the compound is glucoverdazil.

24. Contrast agent for biomedical imaging, characterized in that it comprises the compound as defined in any one of claims 1 to 6, or the composition as defined in any one of claims 7 or 8.

25. Use of a compound, as defined in any one of claims 1 to 6, or of the composition, as defined in any one of claims 7 or 8, characterized in that it is a contrast agent for biomedical imaging, wherein the contrast agent is formulated for administration to an individual.

26. Use, according to claim 25, characterized in that the generation of biomedical images comprises magnetic resonance imaging (MRI).

27. Use, according to claim 26, characterized in that the MRI is a dynamic contrast-enhanced magnetic resonance imaging (DCI-MRI).

28. Use, according to claim 26, characterized in that the MRI is a contrast-enhanced magnetic resonance imaging (CMR).

29. Use, according to any one of claims 25 to 28, characterized in that the image generation is a kidney image generation.

30. Use, according to any one of claims 25 to 29, characterized in that image generation is used to assess renal function.

31. Use, according to any of claims 25 to 30, characterized by the fact that the individual has, is suspected of having, or is at risk of having renal dysfunction.

32. Use, according to any of claims 25 to 31, characterized by the fact that the individual has, is suspected of having, or is at risk of having chronic kidney disease (CKD), acute kidney injury (AKI), renal artery stenosis, urinary obstruction, or renal tumor or malignancy, or is a kidney donor.

33. Use, according to any one of claims 25 to 31, characterized in that the use further comprises the determination of the individual's Glomerular Filtration Rate (GFR).

34. Use, according to any of claims 25 to 32, characterized in that quantitative and / or qualitative information about renal function is obtained.

35. Use, according to any one of claims 25 to 34, characterized in that the use further comprises mapping the individual's Glomerular Filtration Rate (GFR).

36. Use of a compound, as defined in any one of claims 1 to 6, or of the composition, as defined in any one of claims 7 or 8, characterized in that it acts as a contrast agent for the diagnosis of renal dysfunction in an individual, wherein the contrast agent is formulated for administration to the individual.

37. Use, according to claim 36, characterized in that the use further comprises obtaining images of the contrast agent Petition 870250085850, dated 09 / 23 / 2025, pp. 54 / 56 9 / 9 in the individual and determining and / or mapping the individual's Glomerular Filtration Rate (GFR).

38. Use of the compound, as defined in any one of claims 1 to 6, or of the composition, as defined in any one of claims 7 or 8, characterized in that it is used as a contrast agent to monitor, evaluate or determine renal function in an individual, wherein the contrast agent is formulated for administration to the individual.

39. Use according to claim 38, the use being characterized in that it further comprises obtaining images of the contrast agent in the individual and determining and / or mapping the individual's Glomerular Filtration Rate (GFR).

40. Use, according to any one of claims 25 to 39, characterized in that the compound is glucoverdazil. Petition 870250085850, dated 09 / 23 / 2025, pp. 55 / 56

Images