VERDAZYL compounds as contrast agents for kidney magnetic resonance imaging

Abstract

Verazyl derivative compounds and compositions thereof for use as contrast agents for biomedical imaging are provided. The compounds and compositions of the present disclosure are useful, for example, for quantitative and / or qualitative assessment of glomerular filtration rate (GFR) in a subject, providing reliable and rapid assessment of renal function.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to a non-metallic contrast agent for magnetic resonance imaging, methods of its preparation and uses thereof. BACKGROUND

[0002] Chronic kidney disease (CKD) and acute kidney injury (AKI) are major global health burdens. CKD often develops slowly and has no noticeable symptoms in the early stages, but becomes progressively more debilitating later on with limited opportunities for reversal. CKD outcomes can be improved with early detection enabled by early intervention.

[0003] The gold standard for measuring kidney function in the clinic is glomerular filtration rate (GFR) measurement. In North America, the clinical diagnosis of CKD is defined as an estimated GFR (eGFR) < 60 mL / min / 1.73 m 2 and for more than 3 months, or urine albumin / creatinine ratio (ACR) > 30 mg / g and for more than 3 months.

[0004] However, the diagnostic values for CKD were derived from large clinical studies performed on a limited ethnic population, significantly reducing the diagnostic power of these disease biomarkers. Additionally, the underlying etiology of CKD varies among individuals, with some of the most common etiologies (including diabetes, cardiovascular disease, and kidney transplant) limiting the accuracy of eGFR measurements at the patient level. Nearly 30% of these patients have a 30% bias from their true eGFR. Furthermore, in addition to the bias of individuals from the source population, the eGFR equation assumes steady state creatinine levels and does not account for changes in creatinine production or alternative pathways, leading to large fluctuations. Finally, the main limitation of GFR measurement is the inability to provide physicians with spatial or structural information about the cause of kidney dysfunction.

[0005] There is a need for a more accurate method of GFR estimation that does not require the use of a patient's ethnicity or other demographic characteristics and that provides spatial or structural information. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) can be a powerful tool for kidney assessment in suspected AKI and CKD cases, but there is a strong negative bias against its use due to the potential for gadolinium-based contrast agents to induce nephrogenic systemic fibrosis in patients with limited kidney function.

[0006] There is a need for a contrast agent that can be used to provide a reliable, fast, and quantitative clinical imaging solution for early detection of CKD and other diseases. SUMMARY

[0007] It is an object of the present invention to improve at least some of the deficiencies existing in the prior art. The embodiments of the present technology have been developed based on the inventors' realizations that there is a need for improved contrast agents for clinical purposes.

[0008] The inventors have discovered that novel verdazyl derivatives can provide metal-free contrast agents suitable for clinical use. The compounds provided herein can enable reliable, fast, and / or quantitative clinical imaging, which can enable early diagnosis and detection of a variety of diseases. In particular, the compounds are advantageous for contrast-enhanced magnetic resonance imaging (CE-MRI) and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). In certain embodiments, the compounds can exhibit greater stability and / or reduced cytotoxicity compared to prior organic free radical contrast agents.

[0009] Swager et al. (U.S. Patent No. 8,715,621) describes a variety of radicals that can be used as polarizing compounds, but verdazyls are used only as radical initiators, not as imaging agents themselves.

[0010] In one aspect, provided is a compound represented by structural formula (I), or a pharmaceutically acceptable salt or ester thereof: , wherein: R1and R2are 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, R1and / or R2is substituted or unsubstituted C1to C5alkyl. In some such embodiments, R1and / or R2is substituted or unsubstituted C1to C6alkyl. In some such embodiments, R1and / or R2is substituted or unsubstituted C4to C8cycloalkyl. In some such embodiments, R1and / or R2is substituted or unsubstituted C4to C8heterocycloalkyl. In some such embodiments, R1and / or R2comprises a substituted or unsubstituted hydroxyl, amino, or sulfido group. In some embodiments, R1and / or R2is substituted or unsubstituted benzaldehyde. In some embodiments, R1and / or R2comprises at least one heteroatom, which is N, S, or O.

[0012] In certain embodiments of the compound of formula (I), provided is a compound represented by structural formula (I), or a pharmaceutically acceptable salt or ester thereof: , wherein: R1is selected from: , R2is selected from: , wherein: R is a monosaccharide; X is carbon (C) or oxygen (O); n is an integer from 1 to 5 (i.e., 1, 2, 3, 4, or 5); Z is an ether, ester, carbamate, thiocarbamate, urea, thiourea, hydrazone, amide, secondary amine, tertiary amine, disulfide, triazole, cyclooctyltriazolyl, cycloocta[ d ]pyridazinyl, or a cyano-benzyl-thiazolyl-containing group; Y is a monosaccharide, a polysaccharide, a moiety bearing an amine 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: , wherein X1 is any halogen.

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

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

[0015] In certain embodiments of the compound of formula (I), the monosaccharide includes, but is not limited to, a 6-carbon sugar, such as 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 can be a 6-carbon sugar (hexose), a 5-carbon sugar (pentose), a pyranose, a furanose, or other monosaccharide that is chemically suitable for attachment to the compound.

[0017] In certain embodiments of the compound of formula (I), the monosaccharide is a C3 to C7 carbohydrate, i.e., having 3 to 7 carbon atoms.

[0018] In certain embodiments of the compound of formula (I), the monosaccharide is not glucose.

[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: , wherein: R1, R3, and n 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: , wherein: Y1is selected from: , X is C or O; n is 1 to 5; Z is an ether, ester, carbamate, thiocarbamate, urea, thiourea, hydrazone, amide, secondary amine, tertiary amine, disulfide, triazole, cyclooctyltriazolyl, cycloocta[ d ]pyridazinyl, or a cyano-benzyl-thiazolyl-containing group; and Y2is a monosaccharide, a polysaccharide, a moiety bearing an amine 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), the monosaccharide includes, but is not limited to, a 6-carbon sugar, such as 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 can be a 6-carbon sugar, a 5-carbon sugar, or other monosaccharide that is chemically suitable for attachment to the compound. In certain embodiments, the monosaccharide is a C3 to C7 carbohydrate, i.e., having 3 to 7 carbon atoms.

[0023] In certain embodiments of the compound of formula (I), the compound is a compound listed 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 a glucoverdazyl.

[0025] Table 1. Examples of compounds of formula (I) according to certain embodiments.

[0026]

[0027]

[0028] It is understood that all acid, salt, base, and other ionic and non-ionic forms of the compounds described herein are intended to be encompassed. For example, if a compound is shown herein as an acid, salt forms of the compound are also intended to be encompassed. Likewise, if a compound is shown as a salt, acid and / or base forms are also encompassed.

[0029] In certain embodiments of the compounds of Formula (I), the compounds are suitable for use as contrast agents. In some such embodiments, the compounds are suitable for use in CE-MRI and / or DCE-MRI. In some such embodiments, the compounds are suitable for use in biomedical imaging, for example, but not limited to, the diagnosis of renal dysfunction, such as by determining and / or mapping GFR.

[0030] In another aspect, there is provided a composition comprising a compound of the disclosure and a carrier.

[0031] In certain embodiments, the composition is a pharmaceutical composition comprising a compound of the disclosure and a pharmaceutical carrier.

[0032] In certain embodiments, the composition is suitable for use as a contrast agent.

[0033] In certain embodiments, the carrier is an aqueous solution. The carrier can be saline, water, phosphate buffered saline (PBS), or a 5% dextrose in water solution. Such compositions can be used in biomedical applications such as imaging.

[0034] In a further aspect, there is provided a method of preparing an embodiment of a compound or composition described herein.

[0035] In another aspect, there is provided a method of biomedical imaging, comprising administering a contrast agent to a subject and imaging the contrast agent in the subject, wherein the contrast agent comprises a compound or composition described herein.

[0036] In certain embodiments, the biomedical imaging comprises magnetic resonance imaging (MRI). The MRI can comprise contrast-enhanced magnetic resonance imaging (CE-MRI) and / or dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI).

[0037] In certain embodiments, there is provided a method of renal biomedical imaging, comprising administering a contrast agent to a subject and imaging the contrast agent in the subject, wherein the contrast agent comprises a compound or composition described herein. In some such embodiments, the method further comprises determining the glomerular filtration rate (GFR) of the subject. In certain embodiments, quantitative and / or qualitative renal function information is obtained, for example, but not limited to, determining and / or spatially mapping the GFR of the subject. Such methods can be used, for example, to diagnose renal dysfunction in a subject and / or to monitor or assess renal function in a subject.

[0038] In certain embodiments, methods of diagnosing renal dysfunction are provided, comprising administering a contrast agent to a subject, and imaging the contrast agent in the subject, wherein the contrast agent comprises a compound or composition described herein, and determining and / or mapping the glomerular filtration rate (GFR) of the subject.

[0039] In certain embodiments, methods of monitoring, assessing, or determining renal function are provided, comprising administering a contrast agent to a subject, and imaging the contrast agent in the subject, wherein the contrast agent comprises a compound or composition described herein, and determining and / or mapping the glomerular filtration rate (GFR) of the subject.

[0040] In certain embodiments of the methods of the disclosure, the subject has, is suspected of having, or is at risk of having renal dysfunction. The subject can have, be suspected of having, or be at risk of having chronic kidney disease (CKD), acute kidney injury (AKI), renal artery stenosis, urinary tract obstruction, and / or a renal tumor or renal malignancy. In some embodiments, the subject can be a candidate kidney donor.

[0041] In certain embodiments of the methods of the disclosure, the compound is a glucosyl-verdazyl.

[0042] In still further aspects, uses of the compounds or compositions described herein for imaging, e.g., biomedical imaging, are provided. In certain embodiments, uses of the compounds or compositions for renal imaging are provided.

[0043] In another aspect, uses of the compounds or compositions described and / or claimed herein as contrast agents are provided. The contrast agents can be used during imaging using modalities such as MRI, CE-MRI, DCE-MRI, and the like.

[0044] In further aspects, metal-free contrast agents for biomedical imaging comprising the compounds or compositions described herein are provided.

[0045] In yet another aspect, kits comprising the compounds, compositions, or contrast agents described herein are provided. The kits can further comprise buffers or excipients, and / or instructions for use, e.g., in biomedical imaging. BRIEF DESCRIPTION OF DRAWINGS

[0046] The patent or application file contains at least one drawing. Copies of this patent or patent application publication with drawings will be provided by the Office upon request and payment of the necessary fee.

[0047] To better understand the invention and to more clearly illustrate how the invention can be practiced, the accompanying drawings are now illustrated by way of example, which show various aspects and features of embodiments according to the invention, and wherein: Figures 1A-1F The paramagnetic properties and stability of glucosyl-verdazyl according to certain embodiments of the present technology are shown. Figure 1A: Electron paramagnetic resonance (EPR) spectra of a solution of 5 mM glucosyl-verdazyl in phosphate-buffered saline (PBS) obtained at room temperature; Figure 1B: T1 and T2 weighted MRI images of a solution of 3 mM glucosyl-verdazyl in PBS obtained at 3 T; Figure 1C: Longitudinal relaxation rate of glucosyl-verdazyl in PBS at pH 7.4 under 3 T MRI. The EPR values ​​of glucosyl-verdazyl (Figure 1D) during 2 hours of incubation in mouse serum (Figure 1E) or 4 mM sodium ascorbate buffer at pH 7.4 (Figure 1D) are also shown. Black ) and TEMPO ( Pink The stability of ). Figure 1F: 5 mM glucosyl-verdazyl solution exposed to light at room temperature ( Pink Or place in a light-proof refrigerator (-20℃) Black Storage stability as determined by EPR in ( ).

[0048] Figures 2A-2C This shows the glucosyl-verdazyl in healthy BALB / c mice. In vivo Localization and clearance. Figure 2A: MRI scans of BALB / c mice obtained before injection and every 2.5 min after administration of glucosyl-verdazyl (3 mmol / kg). Figure 2B: Region of interest (ROI) selected and mean intensity obtained at each time point. Data are presented as mean ± SEM of 9 mice. Figure 2C: Semi-natural logarithmic transformation of the renal clearance curves from t = 2.5 min to t = 40 min and best-fit line (pink). Data are presented as mean ± SEM of n = 9 mice at each time point. Determination of the best-fit line for individual curves. Renal decay time constant (k) and R 2 Presented as mean ± SD.

[0049] Figures 3A-3D Figure 3A shows a glucose-verdazyl-enhanced DCE-MRI in a mouse model of unilateral ureteral obstruction. The image shows T1-weighted images of the kidneys in the sham-operated (Sham) group and the UUO group at t = 2.5 min after injection. Up ) and RDTC mapping ( DownFigure 3B: RDTC values ​​for each kidney at each time point after injury. Data are presented as box plots of individual mean RDTC values ​​for each kidney (ipsilateral or contralateral) from each mouse (n = 5). Figure 3C: Sham surgery stained with PAS ( Left ) and UUO ( Right Representative histology of paired kidneys in the treatment group. Figure 3D: Sham surgery ( Gray ) and UUO ( Turquoise Serum creatinine levels in mice on two sampling days. Data are presented as individual SCr values. Statistical analysis was performed using two-way ANOVA followed by repeated measures with Tukey post-hoc test. In all figures, *p<0.05, **p<0.01, and ****p<0.0001.

[0050] Figures 4A-4D Figure 4A shows a glucosyl-verdazyl-enhanced DCE-MRI of the kidney in folic acid-induced nephropathy. (Figure 4A: T1-weighted image of the kidney at t = 2.5 min post-injection) Up ) and RDTC mapping ( Down Figure 4B: RDTC values ​​of the kidneys at each time point after injury. Data are presented as box plots of individual mean RDTC values ​​from two kidneys of each mouse (n=5). Figure 4C: Kidneys stained with PAS ( Up Representative histology of ) with enlarged cortical or medullary regions ( Down Black arrows indicate positive histological staining in fibrotic areas, which are represented by light blue. Figure 4D: Serum creatinine levels in FAN mice at each time point after injury (n = 5). Statistical analysis was performed using two-way ANOVA followed by repeated measures with Tukey's post-hoc test. In all figures, *p < 0.05, **p < 0.01, and ***p < 0.001.

[0051] Figures 5A-5DDetermination of glomerular filtration rate (GFR) in folate nephropathy (FAN) mice by transdermal fluorescence and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) is shown in accordance with certain embodiments of the present technology. FIG. 5A: Normalized fluorescence intensity of FITC-Phloxine clearance at days 0, 15, and 30 (black, pink, and turquoise, respectively) in FAN mice. Data presented as mean ± SEM of n = 6 mice per time point per replicate. FIG. 5B: Normalized DCE-MRI intensity of Glucosyl-verdazyl-enhanced scans over time at days 0, 15, and 30 (black, pink, and turquoise, respectively) in FAN mice. Data presented as mean ± SEM of 5 mice per time point per replicate. FIG. 5C: GFR values determined by transdermal fluorescence (gray) or DCE-MRI (pink) in FAN mice. Data presented as boxplots of GFR per mouse. Statistical analysis was done by mixed-measures two-way ANOVA followed by Tukey’s post-hoc test. Values for each test are noted on the graph. FIG. 5D: Schematic of Glucosyl-verdazyl clearance through the kidney obtained by DCE-MRI to illustrate the difference between transdermal and DCE-MRI measurements. p

[0052] Figure 6 A schematic of the DCE-MRI data acquisition and image mapping workflow is shown in accordance with certain embodiments of the present technology.

[0053] Figure 7 A1H NMR spectrum of synthesized compound 2 is shown in accordance with certain embodiments of the present technology. 1

[0054] Figure 8 A1C NMR spectrum of synthesized compound 2 is shown in accordance with certain embodiments of the present technology. 13

[0055] Figure 9 A1H NMR spectrum of synthesized intermediate compound is shown in accordance with certain embodiments of the present technology. 1

[0056] Figure 10 A1C NMR spectrum of synthesized intermediate compound is shown in accordance with certain embodiments of the present technology. 13

[0057] Figure 11 A1H NMR spectrum of synthesized compound 3 is shown in accordance with certain embodiments of the present technology. 1

[0058] Figure 12 A1C NMR spectrum of synthesized compound 3 is shown in accordance with certain embodiments of the present technology. 13 ​​​​​​C NMR spectra.

[0059] Figure 13 High performance liquid chromatography traces of synthesized compounds 3 and 4 are shown to verify the radical activity of the compounds after the radicalization step, in accordance with certain embodiments of the present technology.

[0060] Figure 14 Transcutaneous fluorescence RDTC values in healthy BALB / c mice (n = 6) determined by MediBeacon software are shown in comparison to RDTC values in healthy BALB / c mice (n = 14) determined by glucose-based-verdazyl DCE-MRI (A). Comparison of RDTC values at each time point in healthy BALB / c mice by using intensity measurements derived per-voxel or whole-ROI from the slices (both n = 14 mice) (B). Data presented as boxplots of individual RDTC (A, transcutaneous) or individual mean RDTC (A, MRI and B). Statistical analysis was done by one-way ANOVA followed by Tukey’s post-hoc test. In all figures, ns indicates no significant difference and **** p < 0.0001.

[0061] Figure 15 Cell toxicity evaluation of glucose-based-verdazyl at different concentrations in H460 after incubation for 4 hours (A) and 24 hours (B) with the corresponding concentrations. Data presented as mean ± SD of n = 3 replicates. Statistical analysis was done by one-way ANOVA followed by Tukey’s post-hoc test.

[0062] Figure 16 Cell toxicity evaluation of glucose-based-verdazyl, 5,5-dimethyl-l-pyrroline-N-oxide (DMPO) and 2,2,6,6-tetramethylpiperidin-l-yl)oxy (TEMPO) in human renal proximal tubular cells after incubation for 4 hours (A) or 24 hours (B) at 10 mM concentration under cell culture conditions. Data presented as mean ± SD of n = 3 replicates. Statistical analysis was done by one-way ANOVA followed by Tukey’s post-hoc test. *p < 0.05 for live and dead cell populations compared to the number of other group conditions.

[0063] Figure 17 Glucose-based-verdazyl uptake in human renal proximal tubular cells after incubation for 24 h at 10 mM glucose-based-verdazyl under cell culture conditions. Uptake was measured by EPR activity in comparison to known concentrations and normalized to the number of cells loaded into the EPR tube. Data presented as mean ± SD of n = 3 replicates. Statistical analysis was done by one-way ANOVA followed by Tukey’s post-hoc test.

[0064] Figure 18 AF shows glucose-verdazyl-enhanced DCE-MRI data in a unilateral ureteral obstruction (UUO) model. Figure 18 A: Curve showing the change in normalized intensity over time in UUO mice on day 0. The curve is presented as mean normalized intensity ± SEM. Figure 18 B: Has the best-fit line from t = 0 min to t = 40 min Figure 18 The semi-natural logarithmic regression curve of A. Data are expressed as mean RDTC(k) and R0. 2 Values ​​are presented as ±SD. Sham surgery group ( Figure 18 C) and UUO group ( Figure 18 D) RDTC values ​​of the region of interest (ROI) in the midcortex and medulla & renal pelvis (MRP). Data are presented as box plots of individual mean RDTC values ​​for each kidney (ipsilateral or contralateral) in each region from each mouse (n = 5). Identical AUC data and plots for the sham-operated and UUO groups are shown below. Figure 18 Figures E and 18F are shown. Statistical analysis was performed by two-way ANOVA followed by Tukey post-hoc test. In all figures, ns: no significant difference, *p<0.05, ***p<0.001, and ****p<0.0001.

[0065] Figure 19 AC shows glucosyl-verdazyl-enhanced DCE-MRI data in a folate nephropathy (FAN) model. Figure 19 A: RDTC values ​​for the cortical and medullary & renal pelvis (MRP) regions of interest (ROIs). Data are presented as box plots of individual mean RDTC values ​​from each mouse (n = 5). The same AUC data and figures are shown below. Figure 19 As shown in B. Figure 19 C: Curves showing the normalized intensity of the entire kidney, cortex, and MRP region in the FAN model as a function of time at each post-injury time point. Curves are presented as mean normalized intensity ± SEM at each time point. Statistical analysis was performed by repeated measures two-way ANOVA followed by Tukey post-hoc test. In all figures, ns indicates no significant difference, *p<0.05, **p<0.01, and ****p<0.001. Detailed Implementation

[0066] Glomerular filtration rate (GFR) measurement is the clinical gold standard for measuring kidney function. However, it relies on the substitution of physiologic, demographic, and blood-based metabolite levels into an equation derived from a limited, under-representative population and does not provide spatial information about kidney dysfunction. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) can be a powerful tool for kidney assessment in suspected AKI and CKD cases, but there is strong negative bias against it due to the potential for gadolinium-based contrast agents to induce nephrogenic systemic fibrosis in patients with limited kidney function.

[0067] To develop a targeted contrast agent for DCE-MRI suitable for kidney pathology diagnosis, a class of extremely stable, nitrogen-centered organic radicals called verdazyls were evaluated. Verdazyls, based on extremely stable radicals, can provide excellent Tl shortening effects at 3 T with no signal loss in highly reducing environments. It is demonstrated herein that a glucose-modified verdazyl (glucosyl-verdazyl) provides In vitro and In vivo show no extrarenal uptake, with image contrast confined to the kidneys and bladder. In unilateral ureteral obstruction (UUO) and folate-induced nephropathy (FAN) mouse models, which show kidney function deficits over time, functional kidney maps correlate with histological and blood biomarkers of kidney dysfunction. Using the FAN model, it is shown that glucosyl-verdazyl clearance is a reliable measure of GFR, as demonstrated by comparison with a validated percutaneous fluorescence technique. The present disclosure is based (at least in part) on the discovery that glucosyl-verdazyl can be an extremely powerful metal-free MRI contrast agent. In certain embodiments, the present disclosure contrast agent can provide reliable GFR measurements without the error-prone population-derived equations, spatial or structural information about kidney dysfunction, and / or imaging-based personalized medicine approaches for nephrology or other fields. In certain embodiments, the present compounds provide stable and non-toxic metal-free contrast agents.

[0068] To provide a clear and consistent understanding of the specification, definitions for a number of terms used in the specification are provided below. Additionally, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0069] In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "providing," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0070] As used in the specification and claims, the word "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "include" and "includes") or "containing" (and any form of containing, such as "contain" and "contains") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0071] The term "about" is used to indicate that a value includes the inherent variations of error for the device or method being employed to determine the value.

[0072] The terms "derivative" and "variant" are used interchangeably herein.

[0073] The term "subject" as used herein includes eukaryotes, for example, mammals such as humans, sheep, cattle, horses, pigs, dogs, cats, non-human primates, mice, and rats. The term "subject" is used interchangeably with "patient" herein.

[0074] Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th Ed., inside cover, and specific functional groups are generally defined by the description therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Smith, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5thEd., John Wiley & Sons, New York, NY, 2001; and Greene and Wuts, Protective Groups in Organic Synthesis, 3rdEd., John Wiley & Sons, New York, NY, 1999. Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March's Advanced Organic Chemistry, 5 th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations , VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3 rd Edition, Cambridge University Press, Cambridge, 1987; the entire contents of each of the above documents are incorporated herein by reference.

[0075] It should be appreciated that the compounds of the present disclosure, as described herein, can be substituted with any number or combination of substituents or functional groups. Generally, the term“substituted”, whether preceded by the term“optionally” or not, and substituents contained in formulas of this application refer to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent. When more than one position in any given structure can be substituted with more than one substituent selected from a specified group, the substituents can be either the same or different at every position. As used herein, the term“substituted” is contemplated to include all allowed substituents of an organic compound. In a broad aspect, the allowed substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. For the purposes of this disclosure, heteroatoms such as nitrogen can have hydrogen substituents and / or the allowed substituents described herein for any organic compound that satisfies the valence of the heteroatom. Furthermore, the present disclosure is not intended to be limited in any way by the allowed substituents of an organic compound.

[0076] As used herein, the term "acyl" refers to a radical of the formula— C(=0)R°, where R° can be substituted or unsubstituted hydroxyl, substituted or unsubstituted thiol, substituted or unsubstituted amino, substituted or unsubstituted cyclic or acyclic aliphatic, substituted or unsubstituted cyclic or acyclic heteroaliphatic, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. Exemplary acyl groups include carboxylic acids (— C02H), ketones (e.g., an acetyl group [— (C=0)CH3]), esters, amides, carbonates, urethanes, and ureas. Acyl substituents include, but are not limited to, any substituents described herein that can form stable moieties (e.g., an aliphatic group substituted with one or more aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclyl, aryl, heteroaryl, acyl, sulfinyl, sulfonyl, oxo, imino, thia, cyano, amino, azido, nitro, hydroxyl, thiol, and / or halo groups).

[0077] As used herein, the term "aliphatic" includes saturated and unsaturated, non-aromatic, straight-chain (i.e., unbranched), branched, acyclic, cyclic (i.e., carbocyclic), or polycyclic hydrocarbons, which are optionally substituted by one or more functional groups. As will be appreciated by those skilled in the art, "aliphatic" is intended herein to include, but not be limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties. Thus, as used herein, the term "alkyl" includes straight-chain, branched-chain, and cyclic alkyl groups. Similar conventions apply to other generic terms such as "alkenyl," "alkynyl," and the like. Furthermore, as used herein, the terms "alkyl," "alkenyl," "alkynyl," and the like encompass both substituted and unsubstituted groups. In certain embodiments, as used herein, "aliphatic" is used to denote those aliphatic groups having from 1 to 6 carbon atoms or from 2 to 6 carbon atoms. In certain embodiments, the aliphatic groups have from 1 to 5 or from 2 to 5 carbon atoms. In certain embodiments, the aliphatic groups have from 1 to 4 or from 2 to 4 carbon atoms. In certain embodiments, the aliphatic groups have from 1 to 3 or from 2 to 3 carbon atoms. In certain embodiments, the aliphatic groups have from 1 to 2 carbon atoms. In certain embodiments, the aliphatic groups have 1 carbon atom. In certain embodiments, the aliphatic groups have 2 carbon atoms. In certain embodiments, the aliphatic groups have from 1 to 6 carbon atoms (C 1-6 ). Aliphatic group substituents include, but are not limited to, any substituents described herein that can form stable moieties (e.g., an aliphatic group substituted with one or more aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclyl, aryl, heteroaryl, acyl, sulfinyl, sulfonyl, oxo, imino, thia, cyano, amino, azido, nitro, hydroxyl, thiol, and / or halo groups).

[0078] As used herein, the term "alkyl" refers to a saturated straight-chain or branched-chain hydrocarbon radical derived by removal of a single hydrogen atom from a hydrocarbon moiety containing from 1 to 20 carbon atoms. In some embodiments, the alkyl groups employed by the present application contain 1-6 carbon atoms (C1-C6). In another embodiment, the alkyl groups employed contain 1-5 carbon atoms. In other embodiments, the alkyl groups contain 1-4 carbon atoms. In yet another embodiment, the alkyl groups contain 1-3 carbon atoms. In other embodiments, the alkyl groups contain 1-2 carbon atoms. In still other embodiments, the alkyl groups contain 1 carbon atom. Examples of alkyl radicals include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, s-pentyl, i-pentyl, t-pentyl, n-hexyl, s-hexyl, and the like, which can bear one or more substituents. Alkyl group substituents include, but are not limited to, any of the substituents described herein that are capable of forming stable moieties (e.g., alkyl groups substituted with one or more aliphatic, heteroaliphatic, heterocyclyl, aryl, heteroaryl, acyl, sulfinyl, sulfonyl, oxo, imino, thia, cyano, amino, azido, nitro, hydroxyl, thiol, and / or halo groups). 1-6 ). In another embodiment, the alkyl groups employed contain 1-5 carbon atoms. In other embodiments, the alkyl groups contain 1-4 carbon atoms. In yet another embodiment, the alkyl groups contain 1-3 carbon atoms. In other embodiments, the alkyl groups contain 1-2 carbon atoms. In still other embodiments, the alkyl groups contain 1 carbon atom. Examples of alkyl radicals include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, s-pentyl, i-pentyl, t-pentyl, n-hexyl, s-hexyl, and the like, which can bear one or more substituents. Alkyl group substituents include, but are not limited to, any of the substituents described herein that are capable of forming stable moieties (e.g., alkyl groups substituted with one or more aliphatic, heteroaliphatic, heterocyclyl, aryl, heteroaryl, acyl, sulfinyl, sulfonyl, oxo, imino, thia, cyano, amino, azido, nitro, hydroxyl, thiol, and / or halo groups).

[0079] As used herein, the term "alkenyl" refers to a monovalent radical derived from a straight-chain or branched-chain hydrocarbon moiety having at least one carbon-carbon double bond by removal of a single hydrogen atom. In certain embodiments, the alkenyl groups employed by the present application contain 2-6 carbon atoms. In some embodiments, the alkenyl groups employed by the present application contain 2-5 carbon atoms. In another embodiment, the alkenyl groups employed contain 2-4 carbon atoms. In other embodiments, the alkenyl groups contain 2-3 carbon atoms. In yet another embodiment, the alkenyl groups contain 2 carbon atoms. Alkenyl groups include, for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like, which can bear one or more substituents. Alkenyl group substituents include, but are not limited to, any of the substituents described herein that are capable of forming stable moieties (e.g., alkenyl groups substituted with one or more aliphatic, heteroaliphatic, heterocyclyl, aryl, heteroaryl, acyl, sulfinyl, sulfonyl, oxo, imino, thia, cyano, amino, azido, nitro, hydroxyl, thiol, and / or halo groups).

[0080] As used herein, the term "alkynyl" refers to monovalent groups derived from a straight or branched chain hydrocarbon by removal of one hydrogen atom, having at least one carbon-carbon triple bond. In certain embodiments, the alkynyl groups employed by the present application contain 2 to 6 carbon atoms. In some embodiments, the alkynyl groups employed by the present application contain 2 to 5 carbon atoms. In another embodiment, the alkynyl groups employed contain 2 to 4 carbon atoms. In other embodiments, the alkynyl groups contain 2 to 3 carbon atoms. In still other embodiments, the alkynyl groups contain 2 carbon atoms. Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1 -propynyl, and the like, which can bear one or more substituent groups. Alkynyl group substituents include, but are not limited to, any of the substituents described herein that are capable of forming stable moieties (e.g., alkynyl groups substituted with one or more aliphatic, heteroaliphatic, heterocyclyl, aryl, heteroaryl, acyl, sulfinyl, sulfonyl, oxo, imino, thia, cyano, amino, azido, nitro, hydroxyl, thiol, and / or halo groups).

[0081] As used herein, the term "amino" refers to a group of the formula (—NH2). "Substituted amino" refers to a group of the formula (—NHR h ) or (—NR h 2), where R hmay be any substituent other than hydrogen that is capable of forming a stable moiety (e.g., an amino group substituted with one or more aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclyl, aryl, heteroaryl, acyl, sulfinyl, sulfonyl, amino, nitro, hydroxyl, and / or thiol groups). As used herein, "suitable amino protecting groups" are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999, the entire contents of which are incorporated herein by reference. Suitable amino protecting groups include: methylcarbamate, ethylcarbamate, 9-fluorenylmethylcarbamate (Fmoc), 9-(2-sulfonato)fluorenylmethylcarbamate, 9-(2,7-dibromo)fluorenylmethylcarbamate, 2,7-di-tert-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 4-methoxybenzoylmethylcarbamate (Phenoc), 2,2,2-trichloroethylcarbamate (Troc), 2-trimethylsilylethylcarbamate (Teoc), 2-phenylethylcarbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethylcarbamate, 1,1-dimethyl-2,2-dibromoethylcarbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethylcarbamate (TCBOC), 1-methyl-1-(4-biphenylyl)ethylcarbamate (Bpoc), 1-(3,5-di-tert-butylphenyl)-1-methylethylcarbamate (t-Bumeoc), 2-(2'- and 4'-pyridyl)ethylcarbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamidyl)ethylcarbamate, tert-butylcarbamate (BOC), 1-adamantylcarbamate (Adoc), vinylcarbamate (Voc), allylcarbamate (Alloc), 1-isopropylallylcarbamate (Ipaoc), cinnamylcarbamate (Coc), 4-nitrocinnamylcarbamate (Noc), 8-quinolylcarbamate, N-hydroxypiperidinylcarbamate, alkyl dithio carbamate, benzylcarbamate (Cbz), p-methoxybenzylcarbamate (Moz), p-nitrobenzylcarbamate, p-bromobenzylcarbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzylcarbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethylcarbamate, benzhydrylcarbamate, 2-methylsulfenylethylcarbamate, 2-methylsulfonyl ethylcarbamate, 2-(p-toluenesulfonyl)ethylcarbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylsulfenylphenyloxy carbamate (Mtpc), 2,4-dimethylsulfenylphenylcarbamate (Bmpc), 2-phosphorylethylcarbamate (Peoc), 2-triphenylphosphorylisopropylcarbamate (Ppoc), 1,1-dimethyl-2-cyanoethylcarbamate, m-chloro-p-acetoxybenzylcarbamate, p-(dihydroxyboryl)benzylcarbamate, 5-benzisoxazolylmethylcarbamate, 2-(trifluoromethyl)-6-chromonylmethylcarbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzylcarbamate, o-nitrobenzylcarbamate, 3,4-dimethoxy-6-nitrobenzylcarbamate, phenyl(o-nitrophenyl)methylcarbamate, phenothiazinyl-(10)-carbonyl derivatives, N'-p-toluenesulfonylaminocarbonyl derivatives, N'-phenylaminothiocarbonyl derivatives, t-amylcarbamate, S-benzylsulfenylcarbamate, p-cyanobenzylcarbamate, cyclobutylcarbamate, cyclohexylcarbamate, cyclopentylcarbamate, cyclopropylmethylcarbamate, p-decyloxybenzylcarbamate, 2,2-dimethoxyvinylcarbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propylcarbamate, 1,1-dimethylpropynylcarbamate, di(2-pyridyl)methylcarbamate, 2-furylmethylcarbamate, 2-iodoethylcarbamate, isoborynlcarbamate, isobutylcarbamate, isonicotinylcarbamate, p-(p'-methoxyphenylazo)benzylcarbamate, 1-methylcyclobutylcarbamate, 1-methylcyclohexylcarbamate, 1-methyl-1-cyclopropylmethylcarbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethylcarbamate, 1-methyl-1-(p-phenylazophenyl)ethylcarbamate, 1-methyl-1-phenethylcarbamate, 1-methyl-1-(4-pyridyl)ethylcarbamate, phenylcarbamate, p-(phenylazo)benzylcarbamate, 2,4,6-tri-t-butylphenylcarbamate, 4-(trimethylammonio)benzylcarbamate, 2,4,6-trimethylbenzylcarbamate, formamido, acetylamino, chloroacetylamino, trichloroacetylamino, trifluoroacetylamino, phenylacetylamino, 3-phenylpropionylamino, picolinoylamino, 3-pyridinecarbonylamino, N-benzoylphenylalanyl derivative, benzoylamino, p-phenylbenzoylamino, o-nitrophenylacetylamino, o-nitrophenoxyacetylamino, acetoacetylamino, (N'-dithiobenzyloxycarbonylamino)acetylamino, 3-(p-hydroxyphenyl)propionylamino, 3-(o-nitrophenyl)propionylamino, 2-methyl-2-(o-nitrophenoxy)propionylamino, 2-methyl-2-(o-phenylazo-phenoxy)propionylamino, 4-chlorobutyrylamino, 3-methyl-3-nitrobutyrylamino, o-nitrocinnamoylamino, N-acetylmethionine derivative, o-nitrobenzoylamino, o-(benzoyloxymethyl)benzoylamino, 4,5-diphenyl-3-oxazolin-3-one, N-phthalimido, N-dithiobisuccinimido (Dts), N-2,3-diphenylmaleimido, N-2,5-dimethylpyrrolyl, N-1,1,4,4-tetramethyldisilylazide (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexyl-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexyl-2-one, 1-substituted 3,5-dinitro-4-pyridonyl, N-methylamino, N-allylamino, N-[2-(trimethylsilyl)ethoxy]methylamino (SEM), N-3-acetyloxypropylamino, N-(1-isopropyl-4-nitro-2-oxo-3-pyrrolin-3-yl)amino, quaternary ammonium salts, N-benzylamino, N-di(4-methoxyphenyl)methylamino, N-5-dibenzo-cycloheptylamino, N-tritylamino (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amino (MMTr), N-9-phenylfluorenylamino (PhF), N-2,7-dichloro-9-fluorenylmethylimino, N-ferrocenemethylamino (Fcm), N-2-picolylamino N-oxide, N-1,1-dimethylthiomethylimino, N-benzylideneamino, N-p-methoxybenzylideneamino, N-benzhydrylideneamino, N-[(2-pyridyl)mesitylidene]methylimino, N-(N',N'-dimethylaminomethylene)amino, N,N'-isopropylidenediamino, N-p-nitrobenzylideneamino, N-salicylideneamino, N-5-chlorosalicylidenamino, N-(5-chloro-2-hydroxyphenyl)benzaldehydeimino, N-cyclohexylmethyleneamino, N-(5,5-Dimethyl-3-oxo-1-cyclohexenyl)amino, N-borane derivatives, N-diphenylboronic acid derivatives, N-[phenyl(pentacarbonylchromium or tungsten)carbonyl]amino, N-copper chelate group, N-zinc chelate group, N-nitramino, N-nitrosoamino, amino N-oxide, diphenylphosphamide (Dpp), dimethylthiophosphamide (Mpt), diphenylthiophosphamide (Ppt), dialkylphosphamide ester group, dibenzylphosphamide ester group, diphenylphosphamide ester group, benzylsulfinamide group, o-nitrobenzenesulfinamide group (Nps), 2,4-dinitrobenzenesulfinamide group, pentachlorobenzenesulfinamide group, 2-nitro-4-methoxybenzenesulfinamide group, triphenylmethylsulfinamide group, 3-nitropyridinesulfinamide group (Npys), p-toluenesulfonamide group (Ts), benzenesulfonamide group, 2,3 The following are listed: 6-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylsomn-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilanesulfonamide (SES), 9-anthracitesulfonamide, 4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and benzoylmethylsulfonamide.

[0082] As used herein, the term "aryl" refers to a stable aromatic monocyclic or polycyclic ring system having 3 to 20 ring atoms, wherein all ring atoms are carbon and may or may not be substituted. In some embodiments of the invention, "aryl" refers to a monocyclic, bicyclic, or tricyclic C4-C ring system having one, two, or three aromatic rings. 20 Aromatic ring systems, wherein the aromatic ring includes, but is not limited to, phenyl, biphenyl, naphthyl, etc., and these groups may have one or more substituents. Aryl substituents include, but are not limited to, any substituents described herein that are capable of forming a stable moiety (e.g., an aryl group substituted with one or more aliphatic, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, sulfinyl, sulfonyl, oxo, imino, thiooxo, cyano, amino, azide, nitro, hydroxyl, thio, and / or halogenated groups).

[0083] The term "direct bond" or "bond" refers to a single, double, or triple bond between two groups. In some embodiments, "direct bond" refers to a single bond between two groups.

[0084] As used herein, the terms "halo" and "halogen" refer to an atom selected from fluorine (fluoro,—F), chlorine (chloro,—Cl), bromine (bromo,—Br), and iodine (iodo,—I).

[0085] As used herein, the term "heteroaliphatic" includes saturated and unsaturated, non-aromatic, straight-chain (i.e., unbranched), branched, acyclic, cyclic (i.e., heterocyclyl), or polycyclic hydrocarbons, which are optionally substituted with one or more functional groups, and contain one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms, for example, replacing carbon atoms. As will be appreciated by those of ordinary skill in the art, "heteroaliphatic" is intended to encompass, but not be limited to, heteroalkyl, heteroalkenyl, heteroalkynyl, and heterocyclyl moieties. Thus, as used herein, the term "heteroalkyl" includes straight chain, branched chain, and cyclic alkyl groups, as defined herein, which are optionally substituted with one or more functional groups, and contain one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms, for example, replacing carbon atoms. Similar conventions apply to other generic terms such as "heteroalkenyl," "heteroalkynyl," and the like. Furthermore, as used herein, the terms "heteroalkyl," "heteroalkenyl," "heteroalkynyl," and the like encompass both substituted and unsubstituted groups. In certain embodiments, as used herein, "heteroaliphatic" is used to denote those heteroaliphatic groups having 1-6 carbon atoms or 2-6 carbon atoms (cyclic, acyclic, substituted, unsubstituted, branched or unbranched). In certain embodiments, the heteroaliphatic group has 1-5 or 2-5 carbon atoms. In certain embodiments, the heteroaliphatic group has 1-4 or 2-4 carbon atoms. In certain embodiments, the heteroaliphatic group has 1-3 or 2-3 carbon atoms. In certain embodiments, the heteroaliphatic group has 1-2 carbon atoms. In certain embodiments, the heteroaliphatic group has 1 carbon atom. In certain embodiments, the heteroaliphatic group has 2 carbon atoms. Heteroaliphatic group substituents include, but are not limited to, any of the substituents described herein, which are capable of forming stable moieties (e.g., heteroaliphatic groups substituted with one or more aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclyl, aryl, heteroaryl, acyl, sulfinyl, sulfonyl, oxo, imino, thia, phosphino, cyano, amino, azido, nitro, hydroxy, thio, and / or halo groups).

[0086] As used herein, the term "heteroaryl" refers to a stable aromatic monocyclic or polycyclic ring system having 3-20 ring atoms, wherein one ring atom is selected from S, O, and N; zero, one, or two ring atoms are additional heteroatoms independently selected from S, O, and N; and the remaining ring atoms are carbon atoms, the radical being attached to the remainder of the molecule through any ring atom. Exemplary heteroaryl groups include, but are not limited to, pyrrolyl, pyrazolyl, imidazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, tetrazinyl, pyyrolizinyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, indazolyl, quinolinyl, isoquinolinyl, quinolizinyl, cinnolinyl, quinazolinyl, phthalazinyl, naphthyridinyl, quinoxalinyl, thienyl, sulfolyl, furanyl, benzofuranyl, benzothiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, oxadiazolyl, and the like, which can bear one or more substituents. Heteroaryl substituents include, but are not limited to, any of the substituents described herein that are capable of forming a stable moiety (e.g., a heteroaryl group substituted with one or more aliphatic, heteroaliphatic, heterocyclyl, aryl, heteroaryl, acyl, sulfinyl, sulfonyl, oxo, imino, thioxo, cyano, amino, azido, nitro, hydroxyl, thiol, and / or halo groups).

[0087] As used herein, the term "heterocyclyl" or "heterocyclyl group" refers to a non-aromatic, partially unsaturated or fully saturated 3- to 10-membered ring system, including monocyclic rings of 3 to 8 atoms in size, as well as bicyclic and tricyclic ring systems that can include an aromatic five- or six-membered aryl or heteroaryl group fused to a non-aromatic ring. These heterocyclic rings include those having 1 to 3 heteroatoms independently selected from oxygen, sulfur and nitrogen, wherein the nitrogen and sulfur heteroatoms can optionally be oxidized and the nitrogen heteroatoms can optionally be quaternized. In certain embodiments, the term heterocyclyl refers to a non-aromatic 5-, 6-, or 7-membered ring or polycyclic group in which at least one ring atom is a heteroatom selected from O, S and N (wherein the nitrogen and sulfur heteroatoms can optionally be oxidized), and the remaining ring atoms are carbon atoms, the radical being attached to the remainder of the molecule through any ring atom. Heterocyclyl groups include, but are not limited to, bicyclic or tricyclic groups comprising fused five-, six-, or seven-membered rings having one to three heteroatoms independently selected from oxygen, sulfur and nitrogen, wherein (i) each 5-membered ring has zero to two double bonds, each 6-membered ring has zero to two double bonds, each 7-membered ring has zero to three double bonds, (ii) the nitrogen and sulfur heteroatoms can optionally be oxidized, (iii) the nitrogen heteroatoms can optionally be quaternized, and (iv) any of the above heterocyclic rings can be fused to an aryl ring or a heteroaryl ring. Exemplary heterocyclyl groups include aziridinyl, azetidinyl, 1,3-diazetidinyl, piperidinyl, piperazinyl, azocanyl, thiaranyl, thietanyl, tetrahydrothiophenyl, dithiolanyl, thiacyclohexanyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, dioxanyl, oxathiolanyl, morpholinyl, thioxanyl, tetrahydronaphthyl, and the like, which can bear one or more substituents. Substituents include, but are not limited to, any of the substituents described herein that are capable of forming a stable moiety (e.g., a heterocyclyl group substituted with one or more aliphatic, heteroaliphatic, heterocyclyl, aryl, heteroaryl, acyl, sulfinyl, sulfonyl, oxo, imino, thioxo, cyano, amino, azido, nitro, hydroxyl, thiol, and / or halo groups).

[0088] As used herein, the term "hydrogen" refers to any isotope of atomic number 1. Typically, hydrogen refers to the stable isotope containing zero or one neutrons (i.e. 1 H or 2 H (also known as deuterium)). In certain embodiments, hydrogen is present at its normal isotopic abundance. In other embodiments, at least one position is specifically selected to have deuterium present.

[0089] As used herein, the term "hydroxy" or "hydroxyl" refers to a group of the formula (—OH). "Substituted hydroxy" refers to a group of the formula (—ORi), where R1can be any substituent other than hydrogen, which is capable of forming a stable moiety (e.g., a hydroxyl group substituted with a suitable hydroxyl protecting group, aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclyl, aryl, heteroaryl, acyl, sulfinyl, and / or sulfonyl group). "Suitable hydroxyl protecting groups" as used herein are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P.G.M. Wuts, 3rd edition, John Wiley & Sons, 1999, the entire contents of which are incorporated herein by reference. Suitable hydroxyl protecting groups include: methyl, methoxymethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, 1-[ (2-chloro-4-methyl)phenyl]-4-methoxy-piperidin-4-yl (CTMP), 1,4-dioxanyl-2-yl, tetrahydrofuranyl, tetrahydrothienyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-pyridinio, 4-pyridinio, 3-methyl-2-pyridinio N-oxide, benzhydryl, p,p-nitrobenzyi, 5-dibenzosuberyl, trityl, a-napthylbenzyl, p-methoxybenzyl, di(p-methoxyphenyl)benzyl, tri(p-methoxyphenyl)methyl, 4-(4'-bromophenoxyphenyl)benzyl, 4,4',4'-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4',4"-tris(levulinoyloxyphenyl)methyl, 4,4',4"-tris(benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)bis(4',4"-dimethoxyphenyl)methyl, 1,1-di(4-methoxyphenyl)-1'-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, 1,3-benzodithiolyl-2-yl, benzoisothiazolyl S,S-dioxide, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylhexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-cymylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butytmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, tritylacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinoyl), 4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate, adamantanoate, crotonoate, 4-methoxycrotonoate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphonio)ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate, alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzylthio carbonate, 4-ethoxy-l-naphthyl carbonate, methyldithio carbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylvalerate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethoxy, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethyloxy)methylbenzoate, 2,6-dichloro-4-methylphenoxyacetic acid, 2,6-dichloro-4-(l,l,3,3-tetramethylbutyl)phenoxyacetic acid, 2,4-di(l,l-dimethylpropyl)phenoxyacetic acid, chlorodiphenylacetic acid, isobutyric acid, monosuccinate, (E)-2-methyl-2-butenoic acid, o-(methoxycarbonyl)benzoic acid, a-naphthoic acid, nitric acid, alkyl N,N,N',N'-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, boronic acid, dimethylthiophosphonothioate, alkyl 2,4-dinitrophenylsulfinate, sulfate, methanesulfonate (mesylate), benzyl sulfonate, and p-toluenesulfonate (Ts). For protecting 1,2- or 1,3-diols, protecting groups include methylene acetal, ethylene acetal, 1-tert-butylethylene acetal, 1-phenylethylene acetal, (4-methoxyphenyl)ethylene acetal, 2,2,2-trichloroethylene acetal, acetonide, cyclopentylidene ketal, cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal, p-methoxybenzylidene acetal, 2,4-dimethoxybenzylidene acetal, 3,4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal, methoxybenzylidene acetal, ethoxybenzylidene acetal, dimethoxymethylene orthoate, 1-methoxyethylene orthoate, 1-ethoxyethylene orthoate, 1,2-dimethoxyethylene orthoate, a-methoxybenzylidene orthoate, 1-(N,N-dimethylamino)ethylene derivative, a-(N,N'-dimethylamino)benzylidene derivative, 2-oxacyclopentylidene orthoate, di-tert-butylsilylene group (DTBS), 1,3-(l,l,3,3-tetraisopropyldisiloxanylidene) derivative (TIPDS), tetra-tert-butoxydisiloxane-l,3-diylidene derivative (TBDS), cyclic carbonate, cyclic boronate, ethyl boronate, and phenyl boronate.

[0090] As used herein, the term "imino" refers to a radical of the formula (=NR r , wherein R rcorresponding hydrogen or any substituent described herein, which is capable of forming a stable moiety (e.g., a suitable amino protecting group; a substituted or unsubstituted amino; an acyl; a cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkyl; a cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkenyl; a cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkynyl; a cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkyl; a cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkenyl; a cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkynyl; a substituted or unsubstituted aryl; or a substituted or unsubstituted heteroaryl).

[0091] As used herein, the term "isocyano" refers to a group of formula (—NC).

[0092] As used herein, the term "nitro" refers to a group of formula (—NO2).

[0093] As used herein, the term "nitroxide" refers to a stable nitroxide radical, which can be cyclic or acyclic. In certain embodiments, a stable nitroxide refers to a chemically stable nitroxide radical, which can be obtained in pure form, stored and handled in the laboratory. In certain embodiments, a stable nitroxide refers to a cyclic or acyclic nitroxide radical containing two groups that do not contain an alpha hydrogen. Exemplary cyclic or acyclic nitroxides are provided in Keana, Chemical Reviews (1978) 78:37-64, which is incorporated herein by reference in its entirety.

[0094] As used herein, the term "oxo" refers to a group of formula (═O).

[0095] As used herein, the term "thio" or "thiol" refers to a group of formula (—SH). "Substituted thiol" refers to a group of formula (—SR r , wherein R r is any substituent other than hydrogen, which is capable of forming a stable moiety (e.g., a mercapto group substituted with one or more aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclyl, aryl, heteroaryl, acyl, sulfinyl, and / or sulfonyl groups).

[0096] As used herein, the term "sulfoxy" refers to a group of formula (═S).

[0097] As used herein, the term "sulfinyl" refers to a group of formula R f —S(═O)—, wherein R f may be an optionally substituted aliphatic, heteroaliphatic, aryl, or heteroaryl group. The term "alkylsulfinyl" refers to a group of formula R fSulfinyl which can be an optionally substituted alkyl group. The term "aryl sulfinyl" refers to R f Sulfinyl which can be an optionally substituted aryl or heteroaryl group.

[0098] As used herein, the term "sulfonyl" refers to a group of the formula R g —S(=0)2— group, wherein R g Sulfonyl which can be an optionally substituted aliphatic, heteroaliphatic, aryl, or heteroaryl group. The term "alkyl sulfonyl" refers to R g Sulfonyl which can be an optionally substituted alkyl group. The term "aryl sulfonyl" refers to R g Sulfonyl which can be an optionally substituted aryl or heteroaryl group. Exemplary aryl or alkyl sulfonyl groups include p-toluenesulfonyl (tosyl) (CH3C6H4SO2—), methanesulfonyl (methylsulfonyl, CH3SO2—), and trifluoromethanesulfonyl (CF3SO2—).

[0099] As used herein, the term "stable moiety" preferably refers to a moiety that has sufficient stability to be able to be prepared (including In situ prepared) and retain its integrity for a sufficient period of time for the purposes detailed herein.

[0100] As used herein, the term "stable radical" refers to a free radical that has sufficient stability to be able to be prepared (including In situ prepared) and retain its integrity for a sufficient period of time for the purposes detailed herein.

[0101] In certain embodiments, one or more (or all) of the hydrogen atoms present in the compounds of structural formula (I), (II), or (III) are 2 H.

[0102] As described herein, aspects of the technology include compounds that are verdazyl derivatives (e.g., compounds of formula (I), (II), (III), the compounds shown in Table 1). The compounds provide metal-free contrast agents that are more stable and / or less toxic than previous contrast agents. The compounds are clinically useful for medical imaging, such as, but not limited to, CE-MRI and DCE-MRI, and can provide reliable, fast, and / or quantitative clinical imaging to enable early diagnosis and detection of a variety of diseases. Aspects of the technology include compositions and pharmaceutical compositions containing the compounds, methods of making such compounds, and uses thereof.

[0103] Compositions In certain embodiments, the compounds of the present disclosure can be present in a composition or a pharmaceutical composition.

[0104] Pharmaceutical compositions are typically formulated to be compatible with their intended method or route of administration; exemplary routes of administration include, but are not limited to, oral or parenteral, such as intramuscular, intravenous, subcutaneous (e.g., injection or implantation), intraperitoneal, intrathecal, or intraarticular. In some embodiments, the pharmaceutical compositions are provided in single-use containers (e.g., single-use vials, ampules, syringes, or autoinjectors), while in other embodiments they are provided in multi-use containers (e.g., multi-use vials). The compounds and compositions provided herein can be administered to a subject by any appropriate means known in the art.

[0105] The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any subject composition or component thereof. Each carrier must be "acceptable" in the sense of being compatible with the subject composition and its components and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances used in pharmaceutical formulations.

[0106] In certain embodiments, the compositions and pharmaceutical compositions of the present disclosure are present in micellar form. For certain micelles, the compositions of the present disclosure are mixed with non-polar radicals, such as radicals containing perfluorinated moieties. The compositions or pharmaceutical compositions can also include a surfactant. One non-limiting type of perfluorinated radical is a TEMPO group with a fluorinated tail, such as a perfluorinated tail (described in Pozzi, Adv. Synth. Cat. 347:677 (2005), the contents of which are incorporated by reference). Exemplary perfluorinated radicals are attached to C6-C 20 (e.g., C8-C 12) perfluoroalkyl groups. Suitable surfactants include, but are not limited to, perfluoro sulfonate-based carboxylic acids, particularly C4-C 12 acids such as C6, C7, C8, C 10 , C 11 , and C 12 acids. Exemplary surfactants include, but are not limited to, ammonium perfluorooctanoate (FC 143), perfluorooctane sulfonic acid (PFOS), and perfluorononanoic acid (PFNA).

[0107] The compositions and pharmaceutical compositions of the present disclosure can also include additional components such as stabilizers, preservatives, dispersants, and the like. The compositions and pharmaceutical compositions of the present disclosure can also include additional components such as excipients, dyes, and the like that are suitable for the intended purpose, such as suitable for imaging and / or administration to a subject.

[0108] "Pharmaceutically acceptable salts" of a compound refer to salts of compounds that are pharmaceutically acceptable. Salts of the compounds of interest are those salts that retain or improve upon the biological effectiveness and properties of the free acids and free bases of the parent compounds defined herein, or that take advantage of the basic, acidic, or charged functionality inherent in the molecule, and are not biologically or otherwise undesirable. Examples of pharmaceutically acceptable salts are also described in, for example, Berge et al., "Pharmaceutical Salts", J. Pharm. Sci. 66, 1-19 (1977). Non-limiting examples of such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, nitric acid, phosphoric acid, carbonic acid, and the like; or with organic acids such as acetic acid, propionic acid, lactic acid, oxalic acid, glycolic acid, newt-butylacetic acid, beta-hydroxybutyric acid, valeric acid, hexanoic acid, cyclopentanepropionic acid, pyruvic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, cyclohexylsulfamic acid, benzenesulfonic acid, sulfanilic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 3-phenylpropionic acid, lauryl sulfonic acid, lauryl sulfuric acid, oleic acid, palmitic acid, stearic acid, lauric acid, embonic acid (pamoic acid), palmoic acid, pantothenic acid, lactobionic acid, alginic acid, galactaric acid, galacturonic acid, gluconic acid, glucoheptonic acid, glutamic acid, naphthalene-1 -carboxylic acid, hydroxynaphthoic acid, salicylic acid, ascorbic acid, stearic acid, muconic acid, and the like; (2) base addition salts formed when acidic protons present in the parent compound are replaced by metal ions, including alkali metal ions (e.g., lithium, sodium, potassium), alkaline earth ions (e.g., magnesium, calcium, barium), or other metal ions (e.g., aluminum, zinc, iron, and the like); or when acidic protons present in the parent compound are coordinated with organic bases (e.g., ammonia, ethylamine, diethylamine, ethylenediamine, N,N'-dibenzylethylenediamine, ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, piperazine, chloroprocaine, procaine, choline, lysine, and the like).

[0109] Pharmaceutically acceptable salts can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods. Generally, such salts are prepared by reacting the free acid or base forms of the compound with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of both. The salts can be prepared in situ during the final isolation or purification of the compounds. Salts can be prepared from the free acid or base forms of the compound by reaction of the free base or acid forms of the compound with a stoichiometric amount of the desired related base or acid, as appropriate, and separation of the resulting salt. The term "pharmaceutically acceptable salt" also includes zwitterionic compounds, which contain both cationic and anionic groups, and thus are "inner salts". These salts can be prepared in situ during the final isolation or purification of the compounds.

[0110] Use Dynamic contrast-enhanced MRI (DCE-MRI) analyzes the time-enhancement pattern of a tissue after the introduction of a paramagnetic contrast agent. This is achieved by acquiring a baseline image without contrast enhancement, and subsequently acquiring a series of images over time during and after the contrast agent reaches the target tissue. The acquired signal is used to generate a time-intensity curve for the tissue, which reflects the response of the tissue to the arrival of the contrast agent in terms of enhancement values. DCE-MRI has been used to study a variety of diseases, including cardiac conditions (especially infarction), stroke and other brain diseases, a variety of tumors (with emphasis on anti-angiogenic therapy and early detection), and investigation of the peripheral vasculature and musculoskeletal system.

[0111] The most common gadolinium (Gd) chelates are used as contrast agents for DCE-MRI. However, Gd chelate contrast agents are not ideal in many applications. Free gadolinium (Gd) 3+ ) is known to be toxic and must be tightly complexed by a ligand for use in humans. There has been concern about the possible toxicity of gadolinium complexes in patients with renal insufficiency, especially the risk of inducing nephrogenic systemic fibrosis (NSF) in patients with limited renal function. Therefore, there is a need for more stable and / or less toxic contrast agents for DCE-MRI for clinical applications.

[0112] The use of the compounds and compositions of the present technology is not limited and can include as contrast agents for imaging (e.g., for contrast-enhanced MRI (CE-MRI), dynamic contrast-enhanced MRI (DCE-MRI), etc.). Thus, in certain embodiments, the compounds described herein can be used as contrast agents for imaging (e.g., MRI, CE-MRI, DCE-MRI, etc.). It will be appreciated that any suitable MRI or other imaging technique can be used in conjunction with the compounds and compositions of the present technology (see, e.g., MRI in Practice Ed. by Westerbrook et al., Blackwell Publishing, Oxford, UK, 2005, the contents of which are incorporated by reference). Moreover, the methods can be performed at any magnetic field strength. In some embodiments, the field can have a strength ranging from about 0.1 T to 30 T (e.g., at 3 T). The range of radiation that excites electron spin transitions in the unpaired electrons of the polarizing agent at these fields would range from about 2.8 GHz to about 840 GHz. For example, the radiation can be from a 140 GHz gyrotron.

[0113] Chronic kidney disease (CKD) continues to be a major international medical burden. CKD often develops slowly and has no obvious symptoms in the early stages, but becomes progressively more debilitating later on, with limited opportunities for reversal. This disease is often attributed to long-term high blood pressure and diabetes, but is also a potential outcome following acute kidney injury (AKI), which is the result of a sudden and acute decline in kidney function.

[0114] CKD outcomes are improved with early intervention enabled by earlier detection. In North America, the clinical diagnosis of CKD is defined as an estimated glomerular filtration rate (eGFR) < 60 mL / min / 1.73 m 2and persisting for more than 3 months, or a urine albumin / creatinine ratio (ACR) > 30 mg / g and persisting for more than 3 months. These diagnostic values are derived from large clinical studies performed on ethnically limited populations, significantly reducing the diagnostic power of these disease biomarkers. Furthermore, the underlying etiology of CKD varies among individuals, with some of the most common etiologies (including diabetes, cardiovascular disease, and kidney transplant) limiting the accuracy of eGFR measurements at the patient level, as nearly 30% of these patients can have a 30% bias from their true eGFR. Additionally, beyond individual and source personnel bias, the eGFR equation assumes a steady state creatinine level and does not account for changes or alternative pathways of creatinine production, leading to such large fluctuations. It has become extremely important to develop more accurate GFR estimation methods that do not require the use of a patient's ethnicity or other demographic characteristics. Most importantly, these values do not provide physicians with spatial or structural information that leads to kidney dysfunction. Kidney biopsies can provide histopathological data that predicts the outcome of CKD, presenting spatial data of specific kidney lesions rather than just overall kidney function. However, biopsies are invasive procedures with their own inherent risks, hindering their repeated use to spatially and temporally characterize kidney disease. Clinically, GFR remains the gold standard as an indicator of kidney function, and a reliable, fast, and / or quantitative clinical imaging method is needed for its measurement.

[0115] In certain embodiments, the compounds and compositions described herein provide a metal-free contrast agent for medical imaging. In certain embodiments, the compounds and compositions described herein provide a metal-free alternative to gadolinium-based contrast agents for enabling CE-MRI and / or DCE-MRI. In certain embodiments, the compounds and compositions described herein are selectively taken up in the kidney and thus are used for kidney medical imaging. However, it should be understood that the use of the compounds and compositions described herein is not intended to be particularly limited; for example, the compounds and compositions can be used for imaging other tissues, depending on the uptake / distribution of the compounds upon administration to a subject and other considerations that determine suitability for a particular use.

[0116] In certain embodiments of the imaging methods of the present disclosure, imaging of the kidney is provided. Such methods can provide quantitative and / or qualitative kidney function information, such as but not limited to determining and / or mapping GFR in a subject. In certain embodiments, such imaging is free from the toxicological concerns of prior contrast agents in patients with kidney dysfunction, and / or does not require the use of a patient's ethnicity or other demographic characteristics.

[0117] In certain embodiments, methods of biomedical imaging are provided, comprising administering a contrast agent to a subject and imaging the contrast agent in the subject, wherein the contrast agent comprises a compound or composition as described herein. The medical imaging can be, for example, but is not limited to, magnetic resonance imaging (MRI), for example CE-MRI or DCE-MRI. In certain embodiments, the biomedical imaging is used to image the kidneys. In some such embodiments, the imaging is used to monitor or assess kidney function in a subject. In some such embodiments, the imaging is used to determine and / or map GFR in a subject. The methods can thus provide quantitative and / or qualitative kidney function information, for example, but not limited to, GFR in a subject.

[0118] In certain embodiments, methods of measuring kidney function in a subject are provided, comprising administering a contrast agent to a subject and imaging the contrast agent in the subject, wherein the contrast agent comprises a compound or composition as described herein, and determining and / or mapping GFR in the subject.

[0119] In certain embodiments, methods of diagnosing kidney dysfunction in a subject are provided, comprising administering a contrast agent to a subject and imaging the contrast agent in the subject, wherein the contrast agent comprises a compound or composition as described herein, and determining and / or mapping GFR in the subject.

[0120] In certain embodiments, methods of diagnosing chronic kidney disease (CKD), acute kidney injury (AKI), renal artery stenosis, urinary tract obstruction, and / or a renal tumor or malignant lesion in a subject are provided, comprising administering a contrast agent to a subject and imaging the contrast agent in the subject, wherein the contrast agent comprises a compound or composition as described herein, and determining and / or mapping GFR in the subject.

[0121] In certain embodiments, methods of determining whether a subject is eligible for kidney donation are provided, comprising administering a contrast agent to a subject and imaging the contrast agent in the subject, wherein the contrast agent comprises a compound or composition as described herein, and determining and / or mapping GFR in the subject.

[0122] In certain embodiments of the methods provided herein, the subject has, is suspected of having, or is at risk for having kidney dysfunction. The subject can have, be suspected of having, or be at risk for having chronic kidney disease (CKD), acute kidney injury (AKI), renal artery stenosis, urinary tract obstruction, or a renal tumor or malignant lesion. The subject can be a kidney donor or a candidate kidney donor.

[0123] Kits Also provided herein are kits comprising a compound or composition as described herein. A kit is generally in the form of a physical structure housing multiple components and can be used, for example, to carry out a method provided herein. For example, a kit can include one or more compounds or compositions disclosed herein (provided in, for example, a sterile container), which can be in the form of a pharmaceutical composition suitable for administration to a subject. The compound or composition can be provided in ready-to-use form, or in a form that requires reconstitution or dilution prior to administration (e.g., a powder). When the compound or composition is in a form that requires reconstitution or dilution by the user, the kit can also include a diluent (e.g., sterile water), buffer, pharmaceutically acceptable excipient, etc., packaged either with the compound or composition or separately. Each component of the kit can be sealed in an individual container, and multiple containers can all be within a single package. Kits of the application can be designed to maintain the components contained therein under appropriate conditions (e.g., refrigerated or frozen).

[0124] A kit can also include a label or package insert including instructions for use of the components thereof and information about them. The label or insert can include manufacturer's information, such as lot number and expiration date. The label or package insert can be integrated into the physical structure housing the components, included separately therein, or affixed to an element of the kit (e.g., an ampule, tube or vial).

[0125] Examples The present application will be more readily understood by reference to the following examples, which are provided to illustrate the present application and are not to be construed as limiting the scope thereof.

[0126] Unless otherwise defined or indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. It should be understood that any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application.

[0127] Example 1. Synthesis of Modified Verdazyl Compounds A variety of compounds of structural formula (I) can be prepared according to the following Scheme 1, which illustrates a general scheme for verdazyl synthesis with modifications to R1 (attached to the 1, 5 position nitrogens in the ring) and R2 (attached to the 3 position carbon in the ring). In Scheme 1, the reaction conditions are as follows: (a) 15% phosgene in toluene, 0°C - 20°C, overnight; (b) HCl in miscible solvent, reflux temperature, 2 hours; (c) R2 group with aldehyde in miscible solvent, add 2 equivalents of non-nucleophilic base, room temperature, overnight; and (d) potassium ferricyanide and sodium bicarbonate in polar solvent until bubbling stops. In addition to phosgene, the synthesis steps outlined in Scheme 1 can be carried out using diphosgene, triphosgene, carbonyldiimidazole, disuccinimidyl carbonate, bis-pentafluorophenyl carbonate, which generate intermediate 1 in the synthesis of formula (I), (II), or (III).

[0128]

[0129] Scheme 1. Synthesis of modified verdazyl compounds according to certain embodiments.

[0130] A variety of compounds of structural formula (I) can also be prepared according to the following Scheme 2, which illustrates a general scheme for verdazyl synthesis with modifications to R1 (attached to the 1, 5 position nitrogens in the ring) and R2 (attached to the 3 position carbon in the ring), starting with compound 1 generated as described in Scheme 1. Briefly, a large number of polyethylene glycol polymers of any length or carbon repeat units with PMB protected alcohols can be prepared and subsequently oxidized to aldehydes, followed by closing the verdazyl ring with aldehydes and deprotection to give modified verdazyl compounds with free alcohols. It should be noted that for the first step, any group with an aldehyde on one end and a protected heteroatom on the other end (such as a protected alcohol, a protected amine, a protected thiol, a masked acid, etc.) can be used. The alcohol (OH) functionality can then be functionalized to any leaving group or reaction handle that will interact with a nucleophile, enabling attachment of any functional group with N, S, or O nucleophilic sites.

[0131] In Scheme 2, the reaction conditions are as follows: (a) H2O / acetonitrile, NaOAc, and or (or any group with an aldehyde on one end and a protected heteroatom on the other end) in miscible solvent, reflux temperature, 2 hours; (c) appropriate solvent and conditions chosen for the specific reaction; (d) potassium ferricyanide and sodium bicarbonate in polar solvent until no more bubbling.

[0132]

[0133] Scheme 2. Synthesis of modified verdazyl compounds according to certain embodiments.

[0134] Example 2. Optimized targeted synthesis of glucosyl-verdazyl The synthesis of glucosyl-verdazyl is outlined in Scheme 3 below. The optimized route for glucosyl-verdazyl was determined using previously reported 6-oxoverdazyl syntheses, which produced In vivo high levels of molecular purity and quantifiability required for contrast agents. Hydrazine side chains previously reported in the literature are typically 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 chain was functionalized with isopropyl groups, which are bulky enough to help protect the delocalized radical while also improving serum retention after injection. In earlier syntheses, N The -boc isopropyl hydrazine was prepared in large quantities by reference to the previously reported synthesis by Calabretta et al. N The -boc hydrazine precursor was prepared in large quantities (Kumar et al., 2018). However, in this case, commercially available isopropyl boc hydrazine was used. Compound 2 has been synthesized by multiple reported methods, all of which displace COCl2(as a solution of phosgene or solid triphosgene) (Solea et al., 2018; Le et al., 2016). Both forms of phosgene were used to synthesize glucosyl-verdazyl with similar results. A 15% solution of phosgene in toluene was chosen for this study. The route with better yield and purity was achieved by heptane recrystallization of the crude product after the phosgene step as first reported by Paré et al. (Solea et al., 2018). After boc deprotection to form the intermediate compound in hydrochloric acid in ethanol, the non-radical tetrazinanone ring with D-glucose (compound 3) was generated in the same manner as first reported by Le et al. (2016). Finally, the oxidation of compound 3 was also performed using potassium ferricyanide as reported by Le et al., which is a much milder oxidizing agent compared to the more commonly used benzoquinone seen in most available verdazyl literature, and the purification process was easier.

[0135] Many past reports of these reactions are incomplete in terms of characterization. High purity 1 H and 13C-spectrum, and high-resolution mass spectra of each step and related intermediates ( Figures 7-12 The purity of compound 4 was determined by EPR spectroscopy and analytical high-performance liquid chromatography, indicating that this method of glucosyl-verdazyl produced compound 4 completely converted from non-radical 3. Figure 13 ).

[0136]

[0137] Scheme 3. Overview of the synthesis of glucosyl-verdazyl4.

[0138] Example 3. Characterization of glucosyl-verdazyl as an active contrast agent for MRI Reference Figures 1A-1F The paramagnetic properties and stability of glucosyl-verdazyl were determined.

[0139] For stability measurements of glucosyl-verdazyl, EPR was tuned to a glucosyl-verdazyl or TEMPO sample in PBS prior to any stability measurement. Once tuned, solutions of glucosyl-verdazyl or TEMPO were prepared (20 mM in mouse serum or 5 mM in 4 mM sodium ascorbate buffer at pH 7.4). Single spectra were obtained, and the peak height of the strongest peak for each compound was locked. Subsequently, EPR scans were obtained every 5 seconds for 2 hours (mouse serum) or 1.5 hours (ascorbate) to measure the percentage change in activity. For stability measurements of glucosyl-verdazyl in water, 5 mM samples were prepared and exposed to light in a fume hood or wrapped in foil and placed in a dark refrigerator at 4°C. These solutions were periodically sampled and measured by EPR after tuning with freshly prepared 5 mM glucosyl-verdazyl samples.

[0140] The EPR spectrum of glucosyl-verdazyl was consistent with previously reported ones, exhibiting multiple EPR peaks characteristic of high-degree radical delocalization within the tetrazinone ring (Fig. 1A; Massolle et al., 2018; Tain et al., 2017). This radical confirmation indicates that glucosyl-verdazyl can exhibit MRI contrast enhancement (Wahsner et al., 2019). MR imaging of the glucosyl-verdazyl solution phantom in PBS showed a two-fold increase in T1 contrast, but no change in T2 effect relative to water (Fig. 1B). The relaxation rate of glucosyl-verdazyl was expected to be lower than that of previously reported GBCAs, but the contrast effect was on the same order of magnitude as other previously reported organic radical compounds, with a longitudinal relaxation rate (r1) of 0.30 mM. -1 s -1 ± 0.3 mM -1 s -1 (Fig. 1C; Le et al., 2016; Matsumoto et al., 2022; Chevalier et al., 2009). Although the contrast enhancement was similar to that of TEMPO, the tetrazinone radical was significantly more stable than its nitroxy ORCA counterpart (Fig. 1D, Fig. 1E). Neither glucosyl-verdazyl nor TEMPO showed any change in radical activity in mouse serum (Fig. 1D); however, in the presence of the mild bioreducing agent ascorbate, glucosyl-verdazyl radicals were not lost, but TEMPO nitroxy radicals were completely reduced (Fig. 1E).

[0141] The stability of glucosyl-verdazyl in solution over time was assessed 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 glucosyl-verdazyl retained over 50% of its radical activity after 4 months of stage storage, while the frozen solution retained over 80% of its radical activity after 1 year (Figure 1F). This result demonstrates the resiliency of glucosyl-verdazyl delocalized radicals under bioreducing conditions, as well as its shelf life and storability—key characteristics of commonly used MRI contrast agents.

[0142] exist In vivo Prior to the experiment, the cytocompatibility of glucosyl-verdazyl was evaluated in H460 lung cancer epithelial cells, and it was shown that at concentrations up to 10 mM, it had no cytotoxicity compared to untreated cells. Figure 15 ).

[0143] To assess cell viability of H460 cells, large cell lung carcinoma cells (H460) were grown in RPMI-1640 (RPMI) medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P / S) until 80% confluency, at which point they were passaged. Cells were passaged three times, seeded in 6-well plates and grown until 80% confluency. Cells were seeded in triplicate wells for each condition. Cells were then incubated in their regular culture medium supplemented with 0 mM, 2.5 mM, 5 mM or 10 mM Glucosyl-verdazyl for 4 hours or 24 hours. At the respective time points, the culture medium was aspirated and cells were washed three times with 37°C Dulbecco's Phosphate Buffered Saline (PBS). Afterwards, cells were lifted with trypsin-EDTA, centrifuged (5 min, 4°C) at 400 x g, aspirated and then resuspended in 1 mL PBS solution containing 0.2 µM Calcein-acetoxymethyl ester (live cells stained green with fluorescence) and 16 µM Ethidium homodimer-1 (dead cells stained red with fluorescence). Live and dead cell populations were counted by flow cytometry (Beckman-Coulter Gallios Flow Cytometer) using 488 nm excitation, 525 nm / 40 nm bandpass filter for Calcein-AM (live cells, green) and 620 nm / 20 nm bandpass filter for Ethidium homodimer-1 (dead cells, red). After completion, the total number of single-stained Calcein-AM positive cells was compared to the combined total cell number of single-stained positive live or dead cells using Kaluza analysis software (Beckman-Coulter) to determine the live cell population in each condition.

[0144] Nine BALB / c mice Intravenous After injection, Glucosyl-verdazyl was evaluated for its ability to In vivo as a contrast agent. Based on the difference in r1 (about 10-fold) and the standard clinical recommendation of Gadovist ® on r1 (about 10-fold) and the standard clinical recommendation of Gadovist ®Dose (0.1 mmol / kg), an administration dose of 3 mmol / kg was chosen. This dose of glucosyl-verdazyl is still well below the maximum concentration used for cell compatibility assessment. After injection, T1 -weighted images were acquired every 3 min after the pre-injection scan used to establish baseline voxel intensity. Limited contrast enhancement was observed in muscle and liver, with uptake and washout clearly isolated to the urinary system (Fig. 2A). Overall, the observed signal change at 5 min post-injection relative to the pre-contrast scan was 127% ± 9% in muscle, 121% ± 10% in liver, and 184% ± 21% in kidney (Fig. 2B). The average washout time post-injection, determined by the return of the kidney ROI to baseline intensity, was approximately 40 min, coinciding with the plateau in bladder ROI signal growth. At this time point, the observed signal change was 120% ± 8% in muscle, 115% ± 7% in liver, 127% ± 8% in kidney, and 438% ± 48% in bladder. The clearance kinetics of glucosyl-verdazyl via the kidneys was consistent with a single-phase decay, with the renal decay time constant (RDTC, k, in min -1 ) determined from the slope of the semi-natural log plot of the data (Fig. 2C). Linear regression was always performed from the maximum contrast intensity at t = 2.5 min to the most consistent baseline recovery time at t = 40 min. The average k determined from 9 healthy BALB / c mice was -0.124 min -1 ± 0.012 min -1 , with an average R 2 of 0.97 ± 0.05, indicating excellent reproducibility for this baseline measurement of healthy kidney function.

[0145] Cell compatibility studies were repeated in human renal proximal tubule cells (hRPT) using high concentrations of glucosyl-verdazyl, observing that glucosyl-verdazyl was taken up primarily within the kidney. Figure 16

[0146] ​To assess human renal proximal tubule (hRPT) cell viability, hRPT cells were grown in epithelial cell growth medium (EpiMEM) supplemented with 10% FBS, 1% P / S, and epithelial cell growth supplement (EpiCGS) until 80% confluency, at which point they were passaged. Cells were passaged three times, seeded in 6-well plates, and grown until 80% confluency. Cells were seeded in triplicate wells per condition. Cells were then incubated in their regular culture medium supplemented with regular culture medium, 10 mM glucose-based verdazyl, 10 mM 5,5-dimethyl-1-pyrroline-N-oxide (DMPO, a nitrone spin-trap agent), or 10 mM (2,2,6,6-tetramethylpiperidin-1-yl)oxy (TEMPO, a nitroxide radical) for 4 hours or 24 hours. At the respective time points, the culture medium was aspirated and cells were washed three times with 37 °C Dulbecco’s phosphate-buffered saline (PBS). Afterwards, cells were lifted with trypsin-EDTA, centrifuged (5 min, 4 °C) at 400 x g, aspirated, and then resuspended in 1 mL of PBS solution containing 0.2 µM calcein-acetoxymethyl ester (live cells stained green with fluorescence) and 16 µM ethidium homodimer-1 (dead cells stained red with fluorescence).

[0147] Live and dead cell populations were counted by flow cytometry (Beckman-Coulter Gallios Flow Cytometer) using 488 nm excitation, with a 525 nm / 40 nm bandpass filter for calcein-acetoxymethyl ester (live cells, green) and a 620 nm / 20 nm bandpass filter for ethidium homodimer-1 (dead cells, red). Upon completion, the total number of single-stained calcein-AM positive cells was compared to the combined total cell number of live or dead cells that were single-stained positive using Kaluza analysis software (Beckman-Coulter) to determine the live cell population under each condition.

[0148] While glucose-based verdazyl did not result in a significant increase in cell death compared to untreated cells after 24 hours of incubation, >90% of hRPT were dead after only 4 hours of incubation with TEMPO. This significant difference in cell compatibility highlights an additional key performance difference between TEMPO and the tetrazonyl-derived ORCAs.

[0149] Cellular uptake of glucosyl-verdazyl by hRPT cells was assessed by EPR spectroscopy. For the assessment of glucosyl-verdazyl uptake in hRPT cells, hRPT cells were grown in epithelial cell medium (EpiMEM) supplemented with 10% FBS, 1% P / S, and epithelial cell growth supplement (EpiCGS) until 80% confluency at which point they were passaged. After three cell passages, cells were seeded in 6-well plates and grown until 80% confluency. Cells were seeded in triplicate wells for each condition. Cells were then incubated in their regular medium supplemented with regular medium or 10 mM glucosyl-verdazyl and incubated for 24 hours. The medium was aspirated and cells were washed three times with 37°C Dulbecco's phosphate-buffered saline (PBS). Afterwards, cells were lifted with trypsin-EDTA, centrifuged at 400 x g (5 min, 4°C), aspirated, and then resuspended in 100 μΐ of PBS. The concentrated cell solution was transferred to an EPR tube. A 1 μΐ aliquot was retained and diluted to obtain the cell number in each solution. The EPR was tuned to a 5 mM freshly prepared solution of glucosyl-verdazyl in PBS, and then the sample was measured by EPR. The concentration was determined by a pre-determined standard curve, then normalized to the pre-determined cell number to obtain the nanomoles (nM) of glucosyl-verdazyl per cell.

[0150] The results show that glucosyl-verdazyl is not taken up in any detectable way by the Figure 17 lower uptake of glucosyl-verdazyl in tissues outside the urinary system, reproducible excretion of the kidney contrast to the bladder, relatively fast kidney clearance time of glucosyl-verdazyl, and its proven cell compatibility, indicate that glucosyl-verdazyl can be very suitable for MRI-based kidney function assessment.

[0151] Example 4. Glucosyl-verdazyl-DCE-MRI of acute kidney injury caused by unilateral ureteral obstruction The effectiveness of glucosyl-verdazyl as a DCE-MRI agent for acute kidney injury (AKI) caused by obstructive nephropathy was determined using a unilateral ureteral obstruction (UUO) mouse model. Surgical obstruction of the left ureter prevents fluid clearance, leading to hydronephrosis and significantly reduced kidney function in the ipsilateral kidney. Groups of mice were evaluated for sham operation (left kidney touched by surgical instruments) and surgical UUO (left kidney ligated). For whole left and right kidneys ( Figures 3A-3D ) and their cortical and medullar / renal pelvis (MRP) regions ( Figure 18A-F), the voxel-wise intensity changes in the kidney were determined over time. The voxel-wise mapping using RDTC was used to assess the changes in kidney function. No significant changes in kidney morphology were observed between day 0, day 3 or day 7 in sham-operated mice, however, in the UUO mice group, the morphology of the kidney ipsilateral to the ureteral obstruction changed significantly at day 3 and day 7 relative to day 0. Hydronephrosis was evident at day 3 due to the ablation of the medullary region, which continued to worsen at day 7, which is a hallmark of the UUO model (Xiong et al., 2021). The contralateral kidney in UUO mice did not show any significant morphological changes.

[0152] Before analyzing the data from the diseased mice, to define the optimal time interval for the glucose-based verdazyl clearance, the RDTC of BALB / c mice was determined pre-surgery Figure 18 A-F). In sham-operated mice, no significant changes in the RDTC of both kidneys were observed at day 3 or day 7 relative to day 0 in the graph (Figure 3A) and quantification (Figure 3B). However, in UUO-treated mice, a significant change in the RDTC of the ipsilateral kidney was observed, increasing from k = -0.135 min -1 ± 0.018 min -1 at day 0 to k = -0.028 min -1 ± 0.014 min -1 at day 3 and k = -0.013 min -1 ± 0.032 min -1 at day 7. The RDTC of the contralateral kidney in UUO mice did not change between day 0 and day 3 (k = -0.133 min -1 ± 0.020 min -1 and k = -0.117 min -1 ± 0.014 min -1 ), but showed a significant increase at day 7 (k = -0.097 min -1 ± 0.010 min -1 ). In rodent models, physiological changes in the contralateral kidney are expected after UUO, which have been previously reported to induce macrophage-to-myofibroblast transition (Figueroa et al., 2019), fibrosis (Xiong et al., 2021; Bianco et al., 2019) and alterations in cortical mitochondrial function (Eddy et al., 2012). This data indicates that glucose-based verdazyl-mediated DCE-MRI is able to detect early contralateral kidney function impairment after ipsilateral ureteral obstruction.

[0153] Histology and serum creatinine (SCr) analysis were performed on both groups of mice to confirm the pathology observed by qualitative MRI images and RDTC maps, as well as to assess kidney function using gold standard techniques. Morphology and fibrosis staining of the sham kidneys and the contralateral kidneys of UUO-treated mice were unremarkable, however the ipsilateral kidneys of ureteral obstruction showed clear signs of hydronephrosis (Figure 3C). No significant changes in SCr were detected in sham mice at day 0 and day 7 post-surgery, however a significant increase was observed in UUO mice (Figure 3D). These changes observed in SCr are consistent with those previously reported in the UUO model and reproduced the changes observed in RDTC (Vielhauer et al., 2001; Martinez-Klimova et al., 2019; Fink et al., 1987). The fact that AUC measurements did not parallel the changes in SCr measured supports the use of RDTC as a measure of kidney function in glucose-based-verdazyl-mediated DCE-MRI.

[0154] While the damage caused by ureteral obstruction to the ipsilateral kidney was evident, even on anatomical MRI, the UUO model demonstrated that standard glucose-based-verdazyl-mediated DCE-MRI techniques utilizing simple kinetic mapping can be used to show regional and structural defects in both kidneys, even prewarning of functional changes occurring in the contralateral kidney before positive fibrosis staining. Regional analysis of glucose-based-verdazyl clearance data to distinguish cortical With medullary & renal pelvis was performed Figure 18 A-F). In this study, the impairment of glucose-based-verdazyl clearance resulting from reduced kidney function was clearly identified, while also mapping the location of the pathology occurring in the kidney, which is extremely valuable for the assessment of AKI (Matsumoto et al., 2022; Chevalier et al., 2009; Xiong et al., 2021).

[0155] Example 5. Glucose-based-verdazyl-DCE-MRI in acute to chronic kidney injury resulting from folate-induced nephropathy (FAN) Next, renal function was assessed in a more complex fibrosis-driven kidney disease model mediated by folate-induced nephropathy (FAN). RDTC and AUC were assessed along with supportive histology and SCr (Figure 4). FAN is the result of intratubular folate crystal formation following systemic administration of folate (Jiang et al., 2018). This crystallization leads to an initial period of severe AKI followed by a fibrotic renal scar that leads to a long-term progressive decline in kidney function, resulting in CKD approximately 3 weeks after injection of folate. The FAN model was established in BALB / c mice rather than the more commonly used C57B1 / 6 strain because BALB / c mice are more resistant to the AKI period, with C57B1 / 6 mice having a very high mortality rate during the AKI period.

[0156] By anatomical imaging, it was noted that the entire kidney size of both kidneys decreased from day 0 to day 30, which has been reported in previous FAN models (Doi et al., 2006; Figure 4A). RDTC of both kidneys was determined following glucose-based-verdazyl-mediated DCE-MRI prior to folate administration and 15 and 30 days post-administration (Figure 4A, in chart form, and Figure 4B, quantification). A significant increase in RDTC was observed at day 15 (day 0 k = -0.154 min -1 ± 0.025 min -1 and day 15 k = -0.082 min -1 ± 0.008 min -1 ), followed by a return to baseline RDTC at day 30 (k = -0.139 min -1 ± 0.016 min -1 ). The AKI period of FAN resulted in a significant increase in medullary and cortical RDTC, indicating poor drainage to the ureter and poor glomerular filtration. The RDTC recovery at day 30 is expected at an early stage of the disease CKD period, as AKI typically presents much more severe renal dysfunction than the early stage of CKD, which is consistent with literature using the same post-injury time point (Doi et al., 2006). The RDTC map at day 30 indicated the presence of localized striated regions within the cortex, presenting a relatively higher RDTC (Figure 4A right, black arrows), which mapped to the fibrotic striations of the kidney at day 30 assessed by histology (Figure 4C, light blue regions are the identification of fibrotic tissue highlighted by black arrows). Thus, the combination of spatial and temporal information into a single kidney map, as done in the example of RDTC images (Figure 4A), provides enhanced diagnostic capabilities that existing nephrology techniques, which are limited to either spatial information or kinetic information alone, cannot provide.

[0157] Kidneys were harvested at day 0, day 15, and day 30 post-folic acid injection and histological assessment was performed to confirm both AKI and CKD (Figure 4C), and blood was taken for SCr determination at the same time intervals (Figure 4D). Histological assessment showed a significant increase in the area of kidney fibrosis at day 15, but a decrease in severity at day 30 (Figure 4C). SCr showed a slight increase at day 15 relative to day 0, which is expected because AKI typically shows only a small SCr increase (Doi et al., 2006; Waikar et al., 2009). However, at day 30, during the early stage of CKD, SCr increased significantly relative to day 0 and day 15, which is a strong indicator of a severe decrease in kidney function associated with FAN and the early stage of CKD (Aparicio-Trejo et al., 2020; Yam, 2021; Scarfe et al., 2018). The evaluation by the FAN model highlights the importance of applying a kidney assessment method that combines spatial and temporal assessments. During severe AKI, a small increase in SCr was seen, but a much larger increase in RDTC occurred with a differential regional effect throughout the kidney (Figure 4A). The RDTC map has unique value, which indicates discrete regions of stronger kidney dysfunction (i.e., cortical stripes), which can correspond to the fibrotic regions observed by histological assessment (Figure 4C).

[0158] Example 6. Glucose-based verdazyl-mediated DCE-MRI comparison to validated GFR measurement Although the measurement of GFR has great clinical value, existing methods rely on error-prone techniques that are limited to the measurement of kidney function kinetics. To develop an imaging-based method to assess kidney function, glucose-based verdazyl-mediated DCE-MRI was compared to an established and validated GFR measurement, namely percutaneous fluorescence monitoring. The percutaneous GFR technique relies on the intravenous injection of a fluorescent molecule that is only cleared by filtration, such as FITC-inulin Intravenous and the percutaneous monitoring of blood-pool fluorescence over time. The percutaneous technique applies a single-phase decay model to determine the RDTC of the fluorescence intensity - time curve, which is subsequently corrected to GFR by a previously determined correction factor (Schreiber et al., 2012; Friedemann et al., 2016; Shmarlouski et al., 2017; Scarfe et al., 2018; Schock-Kusch et al., 2013). Using the percutaneous GFR measurement as a benchmark, it was then possible to derive a glucoverdazyl-specific correction factor.

[0159] In the second batch of FAN-induced mice, transdermal (Figure 5A) and glucose- based verdazyl-mediated DCE-MRI (Figure 5B) measurements were performed at day 0 (black), day 15 (pink) and day 30 (cyan). Both techniques yielded a characteristic mono-exponential decay curve, which after semi-natural logarithmic transformation yielded the RDTC values. At day 0, the mean RDTC (k = -0.135 min -1 ± 0.022 min -1 ) determined by glucose-based verdazyl-mediated DCE-MRI was significantly different ( Figure 14 ) than the RDTC (k = -0.075 min -1 ± 0.011 min -1 ) determined by transdermal fluorescence. Considering the different locations where the data were obtained, this difference in RDTCs is not surprising. But while the transdermal measurement assesses the signal from blood pools within the first few millimeters from the skin surface, the DCE-MRI technique assesses the signal clearance from within the kidney tissue itself. The baseline GFR of BALB / c mice measured with the transdermal technique was GFR = 1584 ± 238 µl / min / 100g b.w., which is in agreement with reported literature values (Yan et al., 2021). By integrating the baseline RDTC data obtained for BALB / c mice at day 0 and using the average GFR measurement from the transdermal technique, a glucose-based verdazyl-specific correction factor for the conversion of RDTC to GFR was derived. Using this factor, the GFR was calculated from the tRDTC values at each post-injury time point and compared to the GFR values determined by the transdermal technique (Figure 5C), showing that there was no significant difference in the GFR measured by the two methods.

[0160] In summary, the preparation of tetrazinone-derived ORCA (glucosyl-verdazyl) was optimized and scaled up, and shown to have superior redox stability and cell compatibility relative to previously used nitroxide contrast agents. Due to the specific uptake of glucosyl-verdazyl in the kidney, ureter, and bladder, it is particularly suitable for renal DCE-MRI. Glucosyl-verdazyl has been applied to UUO model of severe AKI and FAN model of AKI-to-CKD progression imaging, showing regional functional changes within the kidney in the form of RDTCs. The AUC voxel-wise maps of both models were less reliable in correlating with histology and SCr changes than mapping RDTCs, which adequately confirmed renal dysfunction. By benchmarking against the validated percutaneous fluorescence recording method of measuring GFR, glucosyl-verdazyl was shown to enable reliable GFR determination by DCE-MRI. Importantly, this method of GFR measurement not only adds a spatial component to the gold standard kidney assessment, but also does not rely on patient demographic determinants of GFR that have been shown to be error-prone. Overall, glucosyl-verdazyl can provide a safer MRI-based diagnosis for patients with known or suspected AKI and / or CKD. Considering the molecular properties of this organic radical, especially its inability to enter cells, excellent cell compatibility and preliminary biocompatibility, localization in the kidney and rapid clearance, tetrazinone-based ORCAs represent a new and promising class of metal-free MRI contrast agents.

[0161] Experimental procedures for examples 1 to 6 General reagents. All chemicals were purchased from Sigma-Aldrich and used directly unless otherwise reported, except N'-(propan-2-yl)(tert-butoxy)carbohydrazide was purchased from AABlocks. All cell culture reagents and consumables were purchased from ThermoFisher, except epithelial cell medium and epithelial cell growth supplement were purchased from ScienCell.

[0162] EPR spectra.All EPR spectra were obtained at room temperature conditions by Bruker EMX plus EPR. All NMR spectra were obtained by Bruker AVANCE II 400 or Bruker Avance III HD 600. All MRI acquisitions were performed on a 3 T preclinical MRI (MR Solutions, Ltd.). For all MRI image data analysis, only relevant slices of the tissue of interest were included (i.e. scans of the kidney region only used slices where the kidneys were visible). All data processing, mapping, and quantification generation were done using programs written in MATLAB 2020A®. All graphs, figures, and statistical results were generated using GraphPad Prism 9.5.

[0163] In vivo MRI animal Study 。 All In vivo MRI animal studies were performed under the University of Ottawa’s IACUC approved Animal Use Protocol Hie -3 640-R1. Mice were anesthetized with isoflurane, placed on a heated cradle and inserted into the MRI. Continuous T1 -weighted RARE images were acquired before and every 2.5 min after contrast injection for 60 min. T1 -weighted imaging: slice thickness of 1 mm, FOV of 50 x 50 mm, average = 3, matrix size = 96 x 96, TE = 11 ms, echo spacing = 7 ms, TR = 720 ms and acquisition time of 2 min 16 s. In all cases, 3 mmol / kg of contrast was injected intravenously through a tail vein catheter and flushed with saline to ensure the full dose of contrast was received.

[0164] MRI data processing and analysis. MRI data processing and analysis was performed as follows: (1) Plot of intensity versus time. (2) Image map of renal attenuation time constant values. The relevant slices were adjusted to remove any MRI-related automatic gain function and all scans and slices were normalized to a fiducial marker of filled water placed next to the mice during all scans. Using a MatLab program, region of interest (ROI) was drawn at the slice level at each scan time point to generate voxel-level intensity versus time data, presented as the average intensity of the total ROI at each time point, normalized to 100% with the lowest intensity voxel of the first scan as the baseline. (3) Image map of area under the curve values. Figure 6 The natural log of each ROI intensity value in the voxel-level intensity versus time curve was taken to generate a semi-log curve. Linear regression was then applied from t = 2.5 min to t = 40 min and the voxel-level slope of this curve is k (the renal decay time constant (RDTC, min-1 ). The RDTC values shown in the graph are the average RDTC values for each voxel. The map of k is overlaid on the image obtained at t = 0 min. Evaluation of tissue localization by DCE-MRI. . A baseline correction is applied to the curve of intensity per voxel level over time by subtracting the lowest voxel value to set the baseline to 0 for all time points. This curve of intensity per voxel over time is integrated using the trapezoidal function to generate an area under the curve (AUC) value. The AUC values shown in the graph are the average AUC values for each voxel. The map of AUC is overlaid on the image obtained at t = 0 min. A schematic of the image mapping workflow for DCE-MRI data acquisition is shown in Serum creatinine measurements.

[0165] Animal models of kidney disease. Glucosyl-verdazyl Unilateral ureteral obstruction. Glucosyl-verdazyl contrast-enhanced scans were obtained for 9 healthy BALB / c mice as described above and the normalized intensity over time curves were obtained as described above, and ROIs were drawn for the kidney, liver, bladder, and muscle tissue.

[0166] Folic acid-induced nephropathy (glucosyl-verdazyl contrast MRI). ​For all disease models, blood was continuously sampled from the saphenous vein on day 0 and at each post-injury time point prior to glucose-based verdazyl-enhanced MRI. Blood was centrifuged for 10 min (room temperature, 900 x g) and serum was collected from the fractionated sample. Samples were stored at -80 °C until use. Serum creatinine (SCr) was determined by quantitative HPLC (Agilent 1260 Infinity equipped with a 2.1 mm x 50 mm, 5 pm particle size Agilent Zorbax 300-SCX column with diode array) referencing a creatinine standard curve by a modified previously reported method (Bello et al., 2019). Briefly, creatinine was dissolved in HPLC mobile phase (15 mM sodium acetate buffer with 4% methanol and 1% acetonitrile (AcN) at pH 4.2) and serially diluted to yield a creatinine standard curve from 0 µM to 12.5 µM by integration of the HPLC peak (Bruker HyStar PP) produced at 234 nm. Standards and samples were obtained in an isocratic mobile phase at a flow rate of 0.5 mL / min. After thawing, creatinine was extracted from mouse serum samples by adding an AcN solution containing 0.5% acetic acid to the serum in a 4: 1 ratio. Samples were vortexed and then placed 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 was transferred to a new tube. The tubes were dried by heating at 50 °C for 45 minutes under vacuum to remove the acidified AcN. The resulting pellets were re-suspended in 60 µl of mobile phase and the samples were subjected to HPLC, integrating the elution peak at 234 nm for the matching time seen in the standard curve.

[0167] Folic acid-induced nephropathy (percutaneous fluorescence). Glucose-based verdazyl contrast-enhanced scans were obtained for all mice in both UUO and FAN disease models as described above and normalized intensity over time curves were obtained as described above, and ROIs of kidney tissue were plotted.

[0168] RDTC conversion to glomerular filtration rate.The unilateral ureteral obstruction (UUO) mouse model of acute kidney injury (AKI) was performed in C57 / Bl6 mice as reported in previous literature (Vanholder et al., 2021). Briefly, 10 female C57 / Bl6 mice (8 weeks old) were divided into 5 for either sham procedure or UUO procedure. Mice were imaged immediately before surgery (day 0) with the glucose-based verdazyl contrast agent. Mice were anesthetized by continuous isoflurane inhalation. The left kidney of the mice was exposed laparoscopically and the left ureter was gently touched with surgical instruments (sham group) or closed with a suture ligation (UUO group). The wound was sutured and mice were imaged by glucose-based verdazyl contrast MRI at day 3 and day 7 post-injury. Mice were sacrificed by cervical dislocation at day 7. Kidneys were removed and fixed in paraformaldehyde, after which they were sectioned and stained with PAS, images were obtained using a slide scanner.

[0169] Synthesis of N'-({N'-[(tert-butoxy)carbonyl]-N-(propan-2-yl)hydrazinylcarbonyl}(propan-2-yl)amino)(tert-butoxy)formamide (2). The folate-induced nephropathy (FAN) model of AKI to chronic kidney disease (CKD) injury was performed in BALB / c mice with modifications to the general procedure reported in previous literature (Van Buren et al., 2011; Chawla et al., 2014; Levin et al., 2011; Chen et al., 2019; Gama et al., 2021). CD1 and C57 / Bl6 mice were observed to have a very high mortality rate when administered a dose of 250 mg / kg of folate (FA). BALB / c mice have been shown to be more resistant to obstruction-mediated injury and more reliably develop CKD (Luis-Lima et al., 2017; Niemantsversriet et al., 2021). Five BALB / c mice were imaged by glucose-based verdazyl-enhanced MRI at day 0. Immediately after scanning, mice were injected intraperitoneally with 125 mg / kg FA in a 0.3 M sodium bicarbonate solution. Daily subcutaneous fluid support was required for the first 5 days after FA injection, as this is the period where the effects of FA are most severe. Mice became stable at day 7 and were normally housed without any fluid assistance. Mice were imaged again by glucose-based verdazyl-enhanced MRI at day 15 and day 30 post-injury. Mice were sacrificed by cervical dislocation at day 30. Kidneys were removed and fixed in paraformaldehyde, after which they were sectioned and stained with PAS, images were obtained using a slide scanner. One group of mice was subjected to the same disease induction without any glucose-based verdazyl-enhanced MRI, and was sacrificed at day 15 to obtain histological samples at this time point, and to keep longitudinal MRI data uninterrupted for this disease model.

[0170] Synthesis of 1,3-diamino-1,3-di(propan-2-yl)urea. 6 mice per group had the same induced kidney disease as the mice in the comparative MRI group, except that instead of MRI, percutaneous fluorescence measurements were performed as previously described (Levey et al., 2020; Romagnani et al., 2017; Srivastava et al., 2021; Nikken et al., 2007; Warwick et al., 2022). Briefly, prior to obtaining any data, the hair was removed from the right dorsolateral region of the mice. The next day, a percutaneous fluorescence monitor (MediBeacon, Inc.) was affixed to the shaved area with an adhesive patch through a dedicated window. The battery was connected to the percutaneous monitor and a 5 min baseline was established. A FITC-Phyco-Bloomsol solution (150 μΐ, 0.2 mg / kg) was injected intravenously through the tail vein and data were collected for 55 min. Day 0 data were collected immediately prior to intraperitoneal injection of folate and repeated on days 15 and 30 post-injury, at which time the mice were sacrificed by cervical dislocation.

[0171] Synthesis of 6-[(1S,2R,3R,4R)-1,2,3,4,5-pentahydroxypentyl]-2,4-di(propan-2-yl)-1,2,4,5-tetrazin-3-one (3). Data obtained from percutaneous fluorescence were analyzed by dedicated software (MediBeacon, Inc.) to generate RDTC and glomerular filtration rate (GFR) values based on pharmacokinetic model fitting. GFR and RDTC data were generated using this software employing a single-phase decay model on raw data and without correction. MediBeacon uses a predetermined coefficient that directly converts 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 RDTC in 14 measured healthy BALB / c mice, the conversion coefficient of glucose-based verdazyl RDTC to GFR was derived from the average GFR value measured by percutaneous fluorescence in 6 healthy BALB / c mice. The conversion coefficient was derived by comparing the difference between integrated RDTC and percutaneously derived RDTC normalized to the average GFR value. This conversion was subsequently applied to the average RDTC values presented in the data to generate a GFR comparison between the two methods.

[0172] Synthesis of 3-oxo-6-[(1S,2R,3R,4R)-1,2,3,4,5-pentahydroxypentyl]-2,4-di(propan-2-yl)-1,2,3,4-tetrahydro-1,2,4,5-tetrazin-1-yl (4). Figure 12To a toluene solution (50 mL, pre-dried over 4A molecular sieves) was added anhydrous Et3N (4 mL, 28.8 mmol) followed by 4 g of N'-(propan-2-yl)(tert- butoxy)carbohydrazide (4 g, 23 mmol). The solution was stirred under N2atmosphere and cooled to 0 °C. A 15% solution of phosgene in toluene (9 mL, 12.7 mmol) was added dropwise over ca. 1 min (phosgene is highly toxic and care needs to be taken while adding) and the reaction mixture was stirred at 0 °C for 1 h, then warmed to room temperature (rt) and stirred for an additional 18 h. The reaction was quenched by the addition of MeOH (50 mL), stirred at rt for 30 min and evaporated. The mixture was diluted with a 10% NH4OH solution (75 mL) followed by extraction with EtOAc (3 x 15 mL). The combined organic phases were washed with brine (40 mL), dried over Na2S04, filtered and evaporated to give a white powder. This powder was dissolved in 80 mL of hot anhydrous heptane and left to stand at 4 °C for 18 h to induce product crystallization. The crystals were filtered and washed with hexane. The product was dried under high vacuum (colorless crystals, compound 2, 2.55 g, 59%). .

[0173] Phantom MRI and In a 100 mL round bottom flask, compound 2 (2.55 g) was re-suspended in EtOH (25 mL) and heated to 80 °C (air condenser). Concentrated HC1 (10 mL) was added dropwise and the solution was stirred at 80 °C for 30 min. The solution was cooled to rt and the solvent evaporated. The crude product was co-evaporated successively with methanol, toluene and petroleum ether (50 mL each). The crude product of sufficient purity for the next step was dried under high vacuum to give 1,3-diamino-1,3-di(propan-2-yl)urea dihydrochloride as a colorless solid in quantitative yield. .

[0174] Determination of longitudinal relaxivity. Glucosyl-verdazyl stability measurements.D-glucose (1.2 g, 6.7 mmol) and NaOAc (1.1 g, 13.4 mmol) in water were added dropwise over 1 min, followed by stirring at rt for 18 h. The reaction mixture was extracted with n-butanol (6 x 10 mL). The combined organic extracts were dried over Na2SO4, filtered and evaporated. The resulting oil was co-evaporated successively with methanol, toluene and petroleum ether (50 mL each). The resulting product was dried under high vacuum overnight and gave light yellow crystals (Compound 3, 1.48 g, 70%). .

[0175] Cell viability assessment of H460 cells. Cell viability assessment of human renal proximal tubule (hRPT) cells. Compound 3 (1.48 g, 4.41 mmol) was re-suspended in H2O (5 mL with stirring (rt)). In a separate vessel, potassium ferricyanide (4.44 g, 13.5 mmol) was mixed with 80 drops (ca. 4.5 mL) of a NaHCO3solution (2 M), followed by water (5 mL); the mixture was dissolved using an ultrasonic bath. The resulting solution was added dropwise to the original reaction mixture with stirring over 1 min, followed by stirring (rt) for ca. 30 min or until bubbling ceased. The mixture was extracted with n-butanol (6 x 10 mL). The combined organic phases were dried over Na2SO4, filtered and evaporated. The resulting oil was co-evaporated successively with methanol (50 mL), three times with toluene (50 mL each), cooled to 0 °C and then co-evaporated with petroleum ether (50 mL). The resulting product was dried under high vacuum overnight to give a bright yellow fine powder (glucosyl-verdazyl 4, 1.09 g, 74%). Considering the radical nature of this compound, it could not be characterised by NMR, the structure and purity were confirmed using HPLC elution time shift and HRMS and EPR. See HPLC trace in Figure 2. HRMS (ESI): calculated for C Evaluation of glucosyl-verdazyl uptake in hRPT cells. . HRMS (ESI): calculated for C 13 H 25 N4O6Na [M+Na]+, calculated: 356.1676, found: 356.1672.

[0176] High-performance liquid chromatography traces. glucosyl-verdazyl Figure 13 rate Am J Physiol Renal PhysiolContrast agent samples were prepared in 1 x PBS in standard NMR tubes, which were then inserted into a 50 mL Falcon tube containing the ultrasound gel, constituting an MRI phantom. The MRI phantom was placed in a 38 mm diameter transmit and receive volume coil and inserted into the MRI. A multi-slice rapid acquisition with relaxation enhancement (RARE) pulse sequence was used for phantom evaluation, using the following parameters for Tl-weighted imaging: slice thickness of 5 mm, FOV of 40 x 40 mm, averages = 3, matrix size = 96 x 96, TR = 11 ms, echo interval = 7 ms, TR = 720 ms, and an acquisition time of 2 minutes 16 seconds. For T2-weighted imaging, all parameters were the same as for Tl-weighted imaging except TE = 68 ms and TR = 4800 ms, and an acquisition time of 7 minutes 28 seconds.

[0177] For relaxation rate measurements, the same imaging phantom was used, with a contrast agent concentration of 1 to 3 mM, which was verified by electron paramagnetic spectroscopy. To measure the longitudinal relaxation rate (Rl), an inversion recovery RARE sequence was used, with the following parameters: slice thickness of 5 mm, FOV of 50 x 50 mm, averages = 1, matrix size = 96 x 96, TE = 17 ms, TR = 5000 ms, Tl = 50, 75, 100, 150, 200, 250, 300, 400, 600, 800, 1200, 2400, and 4800 ms, and an acquisition time of 2 minutes 30 seconds for each Tl. The longitudinal relaxation rate was extracted using the mapping2 MATLAB program written by J. Barral, M. Etezadi-Amoli, E. Gudmundson, and N. Stikov (2009) and modified by J. Rioux (2022). The longitudinal relaxation rate (rl) was extracted from the slope of the plot of 1 / Tl versus contrast agent concentration.

[0178] TetrahedronPrior to any stability measurements, the EPR was tuned to either the glucose- based verdazyl or TEMPO sample in PBS. Once tuned, a solution of glucose-based verdazyl or TEMPO was prepared (20 mM in mouse serum, or 5 mM in pH 7.4 sodium ascorbate buffer). A single spectrum was obtained, and the peak height of the strongest peak of each compound was locked. EPR scan spectra were then obtained every 5 seconds for 2 hours (mouse serum) or 1.5 hours (ascorbate) to measure the percent change in activity. For the stability measurements of glucose-based verdazyl in water, a 5 mM sample was prepared and either exposed to light in a fume hood, or wrapped in tin foil and placed in a 4 °C dark refrigerator. Periodically, these solutions were sampled and measured by EPR after tuning with a freshly prepared 5 mM glucose-based verdazyl sample.

[0179] Physical Chemistry Chemical Physics Large cell lung carcinoma cells (H460) were grown in RPMI-1640 (RPMI) medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P / S) until 80% confluency, at which point they were passaged. Cells were passaged three times, seeded in a 6-well plate and grown until 80% confluency. Cells were seeded in triplicate wells for each condition. Cells were then incubated in their regular medium supplemented with 0 mM, 2.5 mM, 5 mM, or 10 mM glucose-based verdazyl for 4 hours or 24 hours. At the respective time points, the medium was aspirated and cells were washed three times with 37 °C Dulbecco’s phosphate-buffered saline (PBS). Afterwards, cells were lifted with trypsin-EDTA, centrifuged at 400 x g (5 min, 4 °C), aspirated, and then resuspended in 1 mL of PBS solution containing 0.2 µM calcein-acetoxymethyl ester (live cells stained green) and 16 µM ethidium homodimer-1 (dead cells stained red).

[0180] Live and dead cell populations were counted by flow cytometry (Beckman-Coulter Gallios Flow Cytometer) using 488 nm excitation, with a 525 nm / 40 nm bandpass filter for calcein-acetoxymethyl ester (live cells, green) and a 620 nm / 20 nm bandpass filter for ethidium homodimer-1 (dead cells, red). Once complete, the total number of singly stained calcein-AM positive cells was compared to the combined total cell number of live or dead cells that were singly stained positive using Kaluza analysis software (Beckman-Coulter) to determine the live cell population under each condition.

[0181] Adv Exp Med hRPT cells were grown in epithelial cell growth medium (EpiMEM) supplemented with 10% FBS, 1% P / S and epithelial cell growth supplement (EpiCGS) until 80% confluency, at which point they were passaged. Cells were passaged three times before seeding in 6-well plates and grown until 80% confluency. Cells were seeded in triplicate wells per condition. Cells were then incubated in their regular medium supplemented with regular medium, 10 mM glucose-based verdazyl, 10 mM 5,5-dimethyl-1-pyrroline-N-oxide (DMPO, a nitrone spin-trap agent) or 10 mM (2,2,6,6-tetramethylpiperidin-1-yl)oxy (TEMPO, a nitroxide radical) for 4 hours or 24 hours. At the respective time points, the medium was aspirated and cells were washed three times with 37°C Dulbecco’s phosphate-buffered saline (PBS). Afterwards, cells were lifted with trypsin-EDTA, centrifuged (5 min, 4°C) at 400 x g, aspirated and then resuspended in 1 mL PBS solution containing 0.2 µM calcein-acetoxymethyl ester (live cells stained green) and 16 µM ethidium homodimer-1 (dead cells stained red).

[0182] Live and dead cell populations were counted by flow cytometry (Beckman-Coulter Gallios Flow Cytometer) using 488 nm excitation, 525 nm / 40 nm bandpass filter for calcein-acetoxymethyl ester (live cells, green) and 620 nm / 20 nm bandpass filter for ethidium homodimer-1 (dead cells, red). Upon completion, the total number of singly stained calcein-AM positive cells was compared to the combined total cell number of live or dead cells singly stained positive using Kaluza analysis software (Beckman-Coulter) to determine the number of viable cells per condition.

[0183] BiolhRPT cells were grown in epithelial cell growth medium (EpiMEM) supplemented with 10% FBS, 1% P / S and epithelial cell growth supplement (EpiCGS) until 80% confluency at which point they were passaged. Cells were passaged three times before seeding in 6 well plates and grown until 80% confluency. Cells were seeded in triplicate wells for each condition. Cells were then incubated in their regular medium supplemented with regular medium or 10 mM glucosyl-verdazyl and incubated for 24 hours. The medium was aspirated and cells were washed three times with 37 °C Dulbecco's phosphate buffered saline (PBS). Afterwards, cells were lifted with trypsin-EDTA, centrifuged at 400 x g (5 min, 4 °C), aspirated and then resuspended in 100 μΐ of PBS. The concentrated cell solution was transferred to an EPR tube. A 1 μΐ aliquot was retained and diluted to obtain the number of cells in each solution.

[0184] The EPR was tuned to a 5 mM freshly prepared solution of glucosyl-verdazyl in PBS and then the sample was measured by EPR. The concentration was determined by a pre-determined standard curve and then normalized to the pre-determined number of cells to obtain the number of nanomoles (nM) of glucosyl-verdazyl per cell.

[0185] Chem To verify the completion of the radicalization of compounds 3 to 4, HPLC traces were combined with HRMS and EPR. A 20 minute HPLC gradient was performed with 0.5% TFA H20 (increasing from 1% to 100% over 20 min) with 0.5% TFA acetonitrile. The elution time of compound 3 was constant at 10.5 min, however after radicalization of the verdazyl ring and loss of two protons, the elution time increased to 11.8 min. At the same time, radicalization induced a larger absorbance at 452 nm for compound 4, however no activity at this wavelength was observed for the non-radicalized compound 3. The high performance liquid chromatography traces of compounds 3 and 4 verifying the radicalization step of the compound radical activity are shown in Rev .

[0186] References P. S. T. Yuen, S. R. Dunn, T. Miyaji, H. Yasuda, K. Sharma, R. A. Star, Antioxid Redox Signal 2004, 286 , 1116-1119. T. N. Le, H. Grewal, V. Changoco, V. Truong, D. J. R. Brook,Kidney Int 2016, 72 , 6368-6374 A. Massolle, T. Dresselhaus, S. Eusterwiemann, C. Doerenkamp, H. Eckert, A. Studer, J. Neugebauer, Front Pharmacol 2018, 20 ,7661-7675. R. W. Tain, A. M. Scotti, W. Li, X. J. Zhou, K. Cai, Front Pharmacol ​ 2017, 977 , 73-79. J. Wahsner, E. M. Gale, A. Rodríguez-Rodríguez, P. Caravan, ​ ​ 2019, 119 , 957. K. I. Matsumoto, I. Nakanishi, Z. Zhelev, R. Bakalova, I. Aoki, ​ 2022, 36 , 95-121. R. L. Chevalier, M. S. Forbes, B. A. Thornhill, ​ 2009, 75 ,1145-1152. Y. Xiong, Y. Chang, J. Hao, C. Zhang, F. Yang, Z. Wang, Y. Liu, X. Wang, S. Mu, Q. Xu, ​ 2021, 12 , 83. S. M. Figueroa, M. Lozano, C. Lobos, M. T. Hennrikus, A. A. Gonzalez, C. A. Amador, ​ 2019, 10 , 1314. Y. Xiong, Y. Chang, J. Hao, C. Zhang, F. Yang, Z. Wang, Y. Liu, X. Wang, S. Mu, Q. Xu, Front Pharmacol 2021, 12 , 83. M. Bianco, J. A. Lopes, H. J. V. Beiral, J. D. D. Filho, S. P. Frankenfeld, R. S. Fortunato, C. R. Gattass, A. Vieyra, C. M. Takiya, PLoS One 2019, 14 , DOI 10.1371 / JOURNAL.PONE.0218986. A. A. Eddy, J. M. Lopez-Guisa, D. M. Okamura, I. Yamaguchi, Pediatr Nephrol 2012, 27 , 1233. M. Patra, S. G. Awuah, S. J. Lippard, J Am Chem Soc 2016, 138, 12541. E. C. Pare, D. J. R. Brook, A. Brieger, M. Badik, M. Schinke, Org Biomol Chem 2005, 3, 4258. A. B. Solea, T. Wohlhauser, P. Abbasi, Y. Mongbanziama, A. Crochet, K. M. Fromm, G. Novitchi, C. Train, M. Pilkington, O. Mamula, Dalton Transactions 2018, 47, 4785-4789. T. M. Barclay, R. G. Hicks, M. T. Lemaire, L. K. Thompson, Z. Xu, Chem. Commun. 2002, 1688-1689. R. Calabretta, C. Gallina, C. Giordano, Synthesis (Stuttg) 1991, 536-539. V. Kumar, S. Shova, V. Maurel, G. Novitchi, C. Train, Eur J Inorg Chem 2018, 2018, 517-524. T.-N. Le, H. Grewal, V. Changoco, V. Truong, D. J. R. Brook, Tetrahedron 2016, 72, 6368-6374. V. Vielhauer, H. J. Anders, M. Mack, J. Cihak, F. Strutz, M. Stangassinger, B. Luckow, H. J. Grone, D. Schlondorff, Journal of the American Society of Nephrology 2001, 12 , 1173-1187. E. Martinez-Klimova, O. E. Aparicio-Trejo, E. Tapia, J. Pedraza-Chaverri, Biomolecules 2019, 9 , DOI 10.3390 / BIOM9040141. M. Fink, M. Henry, J. D. Tange, Pathology 1987, 19 , 143-149. K. Jiang, T. A. Ponzo, H. Tang, P. K. Mishra, S. I. Macura, L. O. Lerman, Am J Physiol Renal Physiol 2018, 315 , F1252. K. Doi, K. Okamoto, K. Negishi, Y. Suzuki, A. Nakao, T. Fujita, A. Toda, T. Yokomizo, Y. Kita, Y. Kihara, S. Ishii, T. Shimizu, E. Noiri, Am J Pathol2006, 168 , 1413. S. S. Waikar, J. v. Bonventre, Journal of the American Society of Nephrology 2009, 20 , 672-679. O. E. Aparicio-Trejo, S. H. Avila-Rojas, E. Tapia, P. Rojas-Morales, J. C. León-Contreras, E. Martínez-Klimova, R. Hernández-Pando, L. G. Sánchez-Lozada, J. Pedraza-Chaverri, Free Radic Biol Med 2020, 154 , 18-32. L. J. Yan, Animal Model Exp Med 2021, 4 , 329. L. Scarfe, D. Schock-Kusch, L. Ressel, J. Friedemann, Y. Shulhevich, P. Murray, B. Wilm, M. de Caestecker, J Vis Exp 2018, 2018 , DOI 10.3791 / 58520. W. Chaabane, F. Praddaude, M. Buleon, A. Jaafar, M. Vallet, P. Rischmann, C. I. Galarreta, R. L. Chevalier, I. Tack, Am J Physiol Renal Physiol 2013, 304 , 432-439. J. Friedemann, R. Heinrich, Y. Shulhevich, M. Raedle, L. William-Olsson, J. Pill, D. Schock-Kusch, Kidney Int 2016, 90 , 1377-1385. L. Scarfe, D. Schock-Kusch, L. Ressel, J. Friedemann, Y. Shulhevich, P. Murray, B. Wilm, M. de Caestecker, J Vis Exp 2018, 2018 , 58520. A. Shmarlouski, D. Schock-Kusch, Y. Shulhevich, V. Buschmann, T. Rohlicke, D. Herdt, M. Radle, J. Hesser, D. Stsepankou, IEEE Trans Biomed Eng 2016, 63 , 1742-1750. D. Schock-Kusch, S. Geraci, E. Ermeling, Y. Shulhevich, C. Sticht, J. Hesser, D. Stsepankou, S. Neudecker, J. Pill, R. Schmitt, A. Melk, PLoS One 2013, 8 , e71519. A. Schreiber, Y. Shulhevich, S. Geraci, J. Hesser, D. Stsepankou, S. Neudecker, S. Koenig, R. Heinrich, F. Hoecklin, J. Pill, J. Friedemann, F. Schweda, N. Gretz, D. Schock-Kusch, Am J Physiol Renal Physiol 2012, 303 , DOI 10.1152 / AJPRENAL.00279.2012. A. A. Eddy, J. M. López-Guisa, D. M. Okamura, I. Yamaguchi, Pediatr Nephrol 2012, 27 , 1233. M. Rabe, F. Schaefer, Nephron 2016, 133 , 53-61. H. M. Kang, S. H. Ahn, P. Choi, Y. A. Ko, S. H. Han, F. Chinga, A. S. D. Park, J. Tao, K. Sharma, J. Pullman, E. P. Bottinger, I. J. Goldberg, K. Susztak, Nature Medicine 2014 21:1 2014, 21 , 37-46. Q. Yuan, Y. Lv, H. Ding, Q. Ke, C. Shi, J. Luo, L. Jiang, J. Yang, Y. Zhou, Cell Death&Disease 2021 12:8 2021, 12 , 1-12. L. Marquez-Exposito, L. Tejedor-Santamaria, L. Santos-Sanchez, F. A. Valentijn, E. Cantero-Navarro, S. Rayego-Mateos, R. R. Rodrigues-Diez, A. Tejera-Munoz, V. Marchant, A. B. Sanz, A. Ortiz, R. Goldschmeding, M. Ruiz-Ortega, Front Pharmacol 2021, 12 , 1510. M. Parchure, R. Y. Ambaye, V. S. Lalitha, S. v. Gokhale, Experientia 1985, 41 , 72-73. E. Knock, L. Deng, Q. Wu, A. K. Lawrance, X. L. Wang, R. Rozen, J Nutr 2008, 138 , 653-658. A. K. Bello, P. E. Ronksley, N. Tangri, J. Kurzawa, M. A. Osman, A. Singer, A. K. Grill, D. Nitsch, J. A. Queenan, J. Wick, C. Lindeman, B. Soos, D. S. Tuot, S. Shojai, K. S. Brimble, D. Mangin, N. Drummond, JAMA Netw Open 2019, 2, e1910704. R. Vanholder, L. Annemans, A. K. Bello, B. Bikbov, D. Gallego, R. T. Gansevoort, N. Lameire, V. A. Luyckx, E. Noruisiene, T. Oostrom, C. Wanner, F. Wieringa, Clin Kidney J 2021, 14, 1719-1730. P. N. van Buren, R. Toto, Adv Chronic Kidney Dis 2011, 18 , 28-41. L. S. Chawla, P. W. Eggers, R. A. Star, P. L. Kimmel, New England Journal of Medicine 2014, 371 , 58-66. A. Levin, P. E. Stevens, Nature Reviews Nephrology 2011 7:8 2011, 7 ,446-457. T. K. Chen, D. H. Knicely, M. E. Grams, JAMA 2019, 322 , 1294. R. M. Gama, A. Clery, K. Griffiths, N. Heraghty, A. M. Peters, K. Palmer, H. Kibble, R. P. Vincent, C. C. Sharpe, H. Cairns, K. Bramham, PLoS One 2021,16 , e0255869. S. Luis-Lima, E. Porrini, Nephron 2017, 136 , 287-291. M. S. A. Niemantsverdriet, T. T. Pieters, I. E. Hoefer, M. C. Verhaar, J. A. Joles, W. W. van Solinge, W. M. Tiel Groenestege, S. Haitjema, M. B. Rookmaaker, PLoS One 2021, 16 , DOI 10.1371 / JOURNAL.PONE.0261977. A. S. Levey, S. M. Titan, N. R. Powe, J. Coresh, L. A. Inker, Clin J Am Soc Nephrol 2020, 15 , 1203-1212. P. Romagnani, G. Remuzzi, R. Glassock, A. Levin, K. J. Jager, M. Tonelli, Z. Massy, C. Wanner, H. J. Anders, Nat Rev Dis Primers 2017, 3 , DOI10.1038 / NRDP.2017.88. A. Srivastava, I. M. Schmidt, R. Palsson, A. Weins, J. v. Bonventre, V. Sabbisetti, I. E. Stillman, H. G. Rennke, S. S. Waikar, Kidney Int Rep 2021, 6 , 685-694. J. J. Nikken, G. P. Krestin, Eur Radiol 2007, 17 , 2780. J. Warwick, J. Holness, Semin Nucl Med 2022, 52 , 453-466. L. J. Yan,Animal Model Exp Med 2021, 4 , 329. While the application has been described in detail with respect to its embodiments, these embodiments are provided for illustrative purposes only and are not intended to limit the scope of the application. Other embodiments employing the principles of the application and falling within the spirit and scope of the appended claims are possible.

[0187] The contents of all files and references cited herein are hereby incorporated by reference in their entirety.

Claims

1. A compound of Formula (I) or a pharmaceutically acceptable salt or ester thereof: wherein: R1 is selected from: R2 is selected from: wherein X1 is any halogen. ， 4. The compound of claim 1, wherein the compound is a compound of Formula (II) or a pharmaceutically acceptable salt or ester thereof:

5. The compound of claim 1, wherein the compound is a compound of Formula (III), or a pharmaceutically acceptable salt or ester thereof: wherein: Y1 is selected from: X is C or O; n is 1-5; Y2 is selected from a monosaccharide, a polysaccharide, a moiety bearing an amine 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, and other organic targeting ligands. ， 6. The compound of any one of claims 1-5, wherein the monosaccharide is a 6-carbon sugar. ， wherein R is a monosaccharide; X is carbon (C) or oxygen (O); n is 1-5; Z is selected from the group consisting of ethers, esters, carbamates, thiocarbamates, ureas, thioureas, hydrazones, amides, secondary amines, tertiary amines, disulfides, triazoles, cyclooctyltriazolyl, cycloocta d ]pyridazinyl, and cyanobenzylthiazolyl-containing groups; Y is selected from the group consisting of monosaccharides, polysaccharides, moieties bearing amines and carboxylates separated by a substituted alpha carbon, peptides, nanoparticles, dendrimers, antibodies, antibody fragments, nucleic acids, aptamers, organic targeting ligands, and R3; and R3is selected from the group consisting of: ， 7. The compound of any one of claims 1-5, wherein the monosaccharide is glucose, fructose, galactose, or mannose.

2. The compound of claim 1, wherein R2 is .

3. The compound of claim 1, wherein R2 is .

8. The compound of claim 1, wherein the compound is: or a pharmaceutically acceptable salt or ester thereof. 。 9. A composition comprising the compound of any one of claims 1-8 and a carrier. ， 10. The composition of claim 9, wherein the composition is a pharmaceutical composition and the carrier is a pharmaceutically acceptable carrier.

11. The compound of any one of claims 1-8 or the composition of claim 9 or 10 for use in biomedical imaging. ， 12. The compound of any one of claims 1-8 or the composition of claim 9 or 10 for use as a contrast agent.

13. The compound or composition for use of claim 11 or 12, wherein the compound is glucoverdazyl. Z is selected from the group consisting of ethers, esters, carbamates, thiocarbamates, ureas, thioureas, hydrazones, amides, secondary amines, tertiary amines, disulfides, triazoles, cyclooctyltriazolyl, cycloocta d ]pyridazinyl, and cyanobenzylthiazolyl-containing groups; and 14. A method of biomedical imaging, comprising administering a contrast agent to a subject and imaging the contrast agent in the subject, wherein the contrast agent comprises the compound of any one of claims 1-8 or the composition of claim 9 or 10.

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

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

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

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

19. The method of any one of claims 14-18, wherein the imaging is for assessing kidney function.

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

21. The method of any one of claims 14-20, wherein the subject 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 a kidney tumor or a kidney malignancy, or the subject is a kidney donor. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 22. The method of any one of claims 14 to 20, further comprising determining the glomerular filtration rate (GFR) of the subject.

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

24. The method of any one of claims 14 to 23, further comprising mapping the glomerular filtration rate (GFR) of the subject.

25. A method of diagnosing renal dysfunction in a subject comprising: administering a contrast agent to the subject, imaging the contrast agent in the subject, and determining and / or mapping the glomerular filtration rate (GFR) of the subject, wherein the contrast agent comprises the compound of any one of claims 1 to 8 or the composition of claim 9 or 10.

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

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

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

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

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

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

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

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

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

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

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

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

38. The use of any one of claims 30 to 36, wherein the use further comprises determining the glomerular filtration rate (GFR) of the subject.

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

40. The use of any one of claims 30 to 39, wherein the use further comprises mapping glomerular filtration rate (GFR) of the subject.

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

42. The use of claim 41, wherein the use further comprises: The contrast agent is imaged in the subject, and glomerular filtration rate (GFR) of the subject is determined and / or mapped.

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

44. The use of claim 43, wherein the use further comprises: The contrast agent is imaged in the subject, and glomerular filtration rate (GFR) of the subject is determined and / or mapped.

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

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