Verdazil compounds as contrast agents in magnetic resonance imaging of the kidneys

Abstract

Verdazil derivative compounds and compositions thereof, useful as contrast agents for biomedical imaging, are provided. The compounds and compositions of this disclosure can be used, for example, for quantitative and / or qualitative assessment of glomerular filtration rate (GFR) in subjects, providing a reliable and rapid assessment of renal function.

Description

Technical Field

[0001] The present disclosure relates to a metal-free contrast agent for magnetic resonance imaging, a method for producing the same, and use thereof.

Background Art

[0002] Chronic kidney disease (CKD) and acute kidney injury (AKI) are major global health burdens. Chronic kidney disease (CKD) often progresses slowly in the early stages and shows no clear symptoms, but gradually becomes severe enough to cause debilitation in the later stages, and the prospects for recovery are limited. The prognosis of chronic kidney disease is improved by early intervention, which is facilitated by earlier detection.

[0003] The gold standard for measuring renal function clinically is the measurement of glomerular filtration rate (GFR). The clinical diagnosis of chronic kidney disease (CKD) in North America is defined by the state where the estimated glomerular filtration rate (eGFR) is less than 60 mL / min / 1.73m , , ,

[0004] , , 2 ,

[0005] persisting for more than 3 months, or the state where the urinary albumin-to-creatinine (ACR) exceeds 30 mg / g persisting for more than 3 months.

[0004] [[ID=2l]] However, the diagnostic values for chronic kidney disease (CKD) have been derived based on large-scale clinical studies targeting ethnically limited populations, and thus, the diagnostic ability of these disease biomarkers is significantly reduced. Furthermore, the causes underlying chronic kidney disease (CKD) vary among individual patients, and the most common ones include diabetes, cardiovascular disease, and kidney transplantation, which can reduce the accuracy of eGFR measurement in individual patients. In about 30% of these patients, a deviation of 30% from the true eGFR value may be observed. Furthermore, in addition to the deviation of subjects from the derivation population, the eGFR calculation formula assumes a steady state of serum creatinine levels and does not consider changes in creatinine production or alternative pathways, resulting in large variations. Finally, a major limitation of glomerular filtration rate (GFR) measurement is that it cannot provide physicians with spatial or structural information underlying renal dysfunction.

[0005] There is a need for a more accurate method of estimating glomerular filtration rate (GFR) that can provide spatial or structural information without using the patient's race or other demographic attributes. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) can be a powerful tool for evaluating renal function in cases suspected of acute kidney injury (AKI) and chronic kidney disease (CKD). However, there are strong negative views and prejudices against gadolinium-based contrast agents because of the potential to induce nephrogenic systemic fibrosis (NSF) in patients with reduced renal function.

[0006] There is a need for contrast agents that can be used to provide a reliable, rapid, and quantitative clinical imaging approach for the early detection of chronic kidney disease (CKD) and other diseases. SUMMARY OF THE INVENTION

[0007] An object of the present invention is to improve at least a part of the drawbacks found in the prior art. Embodiments of the present technology have been developed based on the inventors' recognition of the need for improved contrast agents in clinical applications.

[0008] The inventors have found that novel verdazyl derivatives can provide metal-free contrast agents suitable for clinical use. The compounds provided in the present disclosure are reliable, enable rapid and / or quantitative clinical imaging, and can facilitate the early diagnosis and detection of various diseases. In particular, the compounds are suitable for contrast-enhanced magnetic resonance imaging (CE-MRI) and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). In certain embodiments, the compounds may exhibit higher stability and / or reduced cytotoxicity compared to conventional organic radical contrast agents.

[0009] Swager et al. (U.S. Patent No. 8,715,621) describe various radicals useful as polarizing agents, but verdazyl is used only as a radical initiator and is not itself used as an imaging agent.

[0010] In one embodiment, a compound represented by structural formula (I), or a pharmaceutically acceptable salt or ester thereof, is provided. [ka] (I)

[0011] Here,

[0012] R1 and R2 are, independently, a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted cyclic alkyl group, a substituted or unsubstituted heterocyclic alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted arylalkyl group, or a substituted or unsubstituted heteroaryl group.

[0013] In some embodiments, R1 and / or R2 are substituted or unsubstituted C1-C5 alkyl groups. In some of such embodiments, R1 and / or R2 represent substituted or unsubstituted C1-C6 alkyl groups. In some of such embodiments, R1 and / or R2 are substituted or unsubstituted C4-C8 cyclic alkyl groups. In some of such embodiments, R1 and / or R2 are substituted or unsubstituted C4-C8 heterocyclic alkyl groups. In some of such embodiments, R1 and / or R2 include substituted or unsubstituted hydroxyl, amino, or thio groups. In some embodiments, R1 and / or R2 are substituted or unsubstituted benzaldehyde. In some embodiments, R1 and / or R2 include at least one heteroatom selected from nitrogen (N), sulfur (S), or oxygen (O).

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

[0015] Here,

[0016] R1 is selected from the following: [ka]

[0017] R2 is selected from the following: [ka]

[0018] Here,

[0019] R is a monosaccharide,

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

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

[0022] Z is an ether, ester, carbamate, thiocarbamate, urea, thiourea, hydrazone, amide, secondary amine, tertiary amine, disulfide, triazole, cyclooctyltriazolyl, cycloocta[d]pyridadyl, or cyanobenzylthiazolyl-containing group.

[0023] Y is a monosaccharide, sugar chain, substructure having an amine group separated by a substituted α-carbon and a carboxylate, peptide, nanoparticle, dendrimer, antibody, antibody fragment, nucleic acid, aptamer, organic target ligand, or R3.

[0024] R3 is selected from the following groups:

[0025] Here, X1 is any halogen.

[0026] In a particular embodiment of the compound of formula (I), R2 is [ka] Here, n and R3 are as described above.

[0027] In a particular embodiment of the compound of formula (I), R2 is [ka] Here, X, Z, Y, and n are as described above.

[0028] In some embodiments of the compound of formula (I), the monosaccharides include, but are not limited to, hexacarbon sugars (e.g., glucose, fructose, galactose, mannose).

[0029] In certain embodiments of the compounds of formula (I), the monosaccharide is glucose, fructose, tagatose, galactose, mannose, xylose, or ribose. It should be understood that the monosaccharide is not particularly limited and may be a six-carbon sugar (hexose), a five-carbon sugar (pentose), pyranose, furanose, or other monosaccharides suitable for chemical bonding to the compound.

[0030] In certain embodiments of the compounds of formula (I), the monosaccharide is a C3-C7 carbohydrate, i.e., having 3-7 carbon atoms.

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

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

[0033] Here, R1, R3, and n are as described above in this specification.

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

[0035] Here,

[0036] Y1 is selected from the following: [ka]

[0037] X is either C or O,

[0038] n is an integer from 1 to 5.

[0039] Z is an ether group, ester group, carbamate group, thiocarbamate group, urea group, thiourea group, hydrazone group, amide group, secondary amine group, tertiary amine group, disulfide group, triazole group, cyclooctyltriazolyl group, cycloocta[d]pyridazyl group, or cyanobenzylthiazolyl-containing group.

[0040] Y2 is a monosaccharide, sugar chain, substructure having an amine group and carboxylate separated by a substituted α-carbon, peptide, nanoparticle, dendrimer, antibody, antibody fragment, nucleic acid, aptamer, or organic target ligand.

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

[0042] In certain embodiments of the compounds of formula (III), the monosaccharide is glucose, fructose, tagatose, galactose, mannose, xylose, or ribose. It should be understood that the monosaccharide is not particularly limited and may be a C6 monosaccharide, a C5 monosaccharide, or any other monosaccharide that is chemically suitable for bonding to the compound. In some embodiments, the monosaccharide is a C3-C7 carbohydrate, i.e., having 3-7 carbon atoms.

[0043] In certain embodiments of the compound represented by formula (I), the compound is one of the compounds listed in Table 1 or a pharmaceutically acceptable salt or ester thereof. The "·" symbol is used to indicate a radical on the nitrogen atom in the compound.

[0044] In certain embodiments of the compound represented by formula (I), the compound is not glucoverdazil.

[0045] [Table 1] TIFF2026511165000011.tif134170

[0046] It should be understood that the compounds described herein encompass all forms, including acids, salts, bases, and other ionic and nonionic forms. For example, if a compound is described as an acid in this disclosure, its salt form is also included. Similarly, if a compound is described as a salt, the corresponding free acid and / or free base forms are also included.

[0047] In certain embodiments of the compound represented by formula (I), the compound is suitable for use as a contrast agent. In some such embodiments, the compound is suitable for use in contrast-enhanced magnetic resonance imaging (CE-MRI) and / or dynamic contrast-enhanced MRI (DCE-MRI). In some such embodiments, the compound is suitable for biomedical imaging, for example, but not limited to, diagnosing renal dysfunction by determining and / or mapping glomerular filtration rate (GFR).

[0048] In another embodiment, compositions comprising the compounds and carriers of the present disclosure are provided.

[0049] In certain embodiments, the composition is a pharmaceutical composition comprising the compounds and pharmaceutical carriers of the Disclosure.

[0050] In certain embodiments, the composition is suitable for use as a contrast agent.

[0051] In certain embodiments, the carrier is an aqueous solution. The carrier may be physiological saline, water, phosphate-buffered saline (PBS), or a 5% glucose aqueous solution. Such compositions can be used for biomedical applications, such as biomedical imaging.

[0052] Further embodiments provide methods for producing embodiments of the compounds or compositions described herein.

[0053] In another embodiment, a biomedical imaging method is provided, which includes administering a contrast agent to a subject and imaging the contrast agent within the subject, wherein the contrast agent includes the compounds or compositions described in this disclosure.

[0054] In certain embodiments, biomedical imaging includes magnetic resonance imaging (MRI). MRI may include contrast-enhanced magnetic resonance imaging (CE-MRI) and / or dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI).

[0055] In certain embodiments, a method for biomedical imaging of the kidney is provided, comprising administering a contrast agent to a subject and imaging the contrast agent within the subject, wherein the contrast agent comprises a compound or composition described in this disclosure. In some such embodiments, the method further includes determining the subject's glomerular filtration rate (GFR). In certain embodiments, quantitative and / or qualitative renal function information is obtained, which may include, for example, determining or spatially mapping the subject's glomerular filtration rate (GFR). Such methods can be used, for example, to diagnose renal impairment and / or to monitor or evaluate the subject's renal function.

[0056] In certain embodiments, a method is provided for diagnosing renal impairment, comprising administering a contrast agent to a subject and imaging the contrast agent within the subject, wherein the contrast agent comprises a compound or composition described herein, and for determining and / or mapping the glomerular filtration rate (GFR) of the subject.

[0057] In certain embodiments, a method is provided for monitoring, evaluating, or measuring renal function, the method comprising administering a contrast agent to a subject, imaging the contrast agent within the subject, and determining and / or mapping the subject's glomerular filtration rate (GFR), the contrast agent comprising a compound or composition described in this disclosure.

[0058] In certain embodiments of the methods of this disclosure, the subject has, is suspected of having, or is at risk of having renal impairment. The subject may 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 renal malignancy or renal tumor. In some embodiments, the subject may be a candidate kidney donor.

[0059] In certain embodiments of the methods disclosed herein, the compound is glucoverdazil.

[0060] In yet another embodiment, compounds or compositions described in this disclosure are provided for use in imaging (e.g., biomedical imaging). In a particular embodiment, the compounds or compositions are used for imaging of the kidney.

[0061] In further embodiments, compounds or compositions described in this disclosure and / or claims are provided for use as contrast agents. Contrast agents can be used in imaging modalities such as magnetic resonance imaging (MRI), contrast-enhanced magnetic resonance imaging (CE-MRI), and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI).

[0062] In yet another embodiment, a metal-free contrast agent for biomedical imaging is provided, comprising the compounds or compositions described herein.

[0063] In yet another embodiment, kits comprising the compounds, compositions, or contrast agents described herein are provided. The kits may further include buffers or excipients, and / or instructions for use relating to biomedical imaging, for example.

[0064] The patent or application file must include at least one color drawing. A copy of the published patent or patent application, including the color drawing, will be provided by the Japan Patent Office upon request and payment of the required fees.

[0065] To better understand the present invention and to more clearly demonstrate how it can be implemented, the accompanying drawings illustrating embodiments and features of the present invention are shown below as examples. [Brief explanation of the drawing]

[0066] [Figure 1A]Figures 1A-1F show the paramagnetic properties and stability of glucoverdazil according to specific embodiments of this disclosure. Figure 1A: EPR spectrum of a 5 mM glucoverdazil solution in phosphate-buffered saline (PBS) acquired at room temperature; Figure 1B: T1-weighted and T2-weighted MRI images of a 3 mM glucoverdazil solution in PBS acquired at 3T; Figure 1C: Longitudinal relaxation ability of glucoverdazil in PBS under pH 7.4 conditions, as shown in 3T MRI. The stability of glucoverdazil (black) and TEMPO (pink) was evaluated by EPR measurement during a 2-hour incubation in mouse serum (Figure 1D) or in 4 mM sodium ascorbate buffer at pH 7.4 (Figure 1E). Figure 1F: Storage stability of a 5 mM glucoverdazil solution at room temperature with light exposure (pink) or frozen storage at -20°C under light shielding (black), as measured by EPR. [Figure 1B] Refer to the explanation in Figure 1A. [Figure 1C] Refer to the explanation in Figure 1A. [Figure 1D] Refer to the explanation in Figure 1A. [Figure 1E] Refer to the explanation in Figure 1A. [Figure 1F] Refer to the explanation in Figure 1A. [Figure 2A] Figures 2A-2C show the localization and clearance of glucoverdazil in vivo in healthy BALB / c mice. Figure 2A: Magnetic resonance imaging (MRI) imaging of BALB / c mice was performed before and every 2.5 minutes after administration of glucoverdazil (3 mmol / kg). Figure 2B: A region of interest (ROI) was selected, and the mean intensity at each time point was obtained. Data are shown as mean ± SEM for n=9 mice. Figure 2C: Semi-logarithmic transformation using natural logarithms and linear regression lines (pink) are shown for the range of t=2.5 min to t=40 min in the renal clearance curve. Data are shown as mean ± standard error (SEM) for 9 mice (n=9) at each time point. The best-fit line was determined for each curve. Renal decay time constant (k) and R2 are shown as mean ± SD. [Figure 2B] Refer to the explanation in Figure 2A. [Figure 2C]Refer to the explanation in Figure 2A. [Figure 3A] Figures 3A-3D show DCE-MRI with glucoverdazil contrast in a mouse model of unilateral ureteral obstruction (UUO). Figure 3A: T1-weighted images (top) and renal decay time constant (RDTC) maps (bottom) of the kidneys at t=2.5 minutes post-administration in the Sham and UUO groups. Figure 3B: RDTC values ​​of each kidney at each time point after injury. Data are shown as box plots of a single mean RDTC value for each kidney (ipsilateral or contralateral) for each mouse (n=5). Figure 3C: Representative histological images of a pair of kidneys stained with PAS in the sham treatment group (left) and the unilateral ureteral obstruction (UUO) treatment group (right). Figure 3D: Serum creatinine values ​​on two measurement days in sham surgery mice (gray) and unilateral ureteral obstruction (UUO) treated mice (blue-green). Data are shown as individual serum creatinine (SCr) values. Statistical analysis was performed using repeated measures two-way ANOVA, followed by Tukey's post-hoc test. In all graphs, * indicates p<0.05, ** indicates p<0.01, and **** indicates p<0.0001. [Figure 3B] Refer to the explanation in Figure 3A. [Figure 3C] Refer to the explanation in Figure 3A. [Figure 3D] Refer to the explanation in Figure 3A. [Figure 4A] Figures 4A-4D show DCE-MRI with glucoverdazil enhancement in folate-induced nephropathy. Figure 4A: T1-weighted image (top) and RDTC map (bottom) of the kidney at t=2.5 minutes after injection. Figure 4B: RDTC values ​​of the kidney at each time point after injury. Data are shown as box plots of a single mean RDTC value obtained from both kidneys of each mouse (n=5). Figure 4C: Representative histological image of the kidney stained with PAS (top) and magnified image of the cortical or medullary region (bottom). Black arrows indicate histologically positive staining of fibrotic areas (shown in light blue). Figure 4D: Serum creatinine levels of FAN mice at each time point after injury (n=5). Statistical analysis was performed using repeated measures two-way ANOVA followed by Tukey's post-hoc test. In all graphs, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001. [Figure 4B] See the explanation in Figure 4A. [Figure 4C] See the explanation in Figure 4A. [Figure 4D] See the explanation in Figure 4A. [Figure 5A] Figures 5A-5D show the calculation of glomerular filtration rate (GFR) in folate-induced nephropathy (FAN) mice by transcutaneous fluorescence and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in a specific embodiment of this technology. Figure 5A shows the normalized fluorescence intensity of FITC-sinistrin transcutaneous fluorescence clearance in FAN mice (black, pink, and turquoise represent days 0, 15, and 30, respectively). Data are shown as the mean ± SEM for each time point in each replicate of mice (n=6). Figure 5B: Time course of normalized DCE-MRI intensity mediated by glucoverdazil in FAN mice (days 0, 15, and 30; black, pink, and turquoise, respectively). Data are shown as the mean ± standard error (SEM) for each time point in n=5 mice. Figure 5C: Glomerular filtration rate (GFR) values ​​in FAN mice determined by transcutaneous fluorescence (gray) or DCE-MRI (pink). The data shows the glomerular filtration rate (GFR) of each mouse in a box plot. For statistical analysis, a mixed-design two-way ANOVA was used, followed by Tukey's post-hoc test. The p-values ​​for each test are indicated in the graph. Figure 5D: A diagram showing glucoverdazil clearance via the kidney by DCE-MRI to illustrate the difference between transcutaneous measurement and DCE-MRI measurement. [Figure 5B] Refer to the explanation in Figure 5A. [Figure 5C] Refer to the explanation in Figure 5A. [Figure 5D] Refer to the explanation in Figure 5A. [Figure 6] Figure 6 is an explanatory diagram of the DCE-MRI data acquisition and image mapping workflow according to a specific embodiment of this technology. [Figure 7] Figure 7 shows the 1H NMR spectrum of synthetic compound 2 according to a specific embodiment of this technology. [Figure 8] Figure 8 shows the 13C NMR spectrum of synthetic compound 2 according to a specific embodiment of this technology. [Figure 9]Figure 9 shows the 1H NMR spectrum of a synthetic intermediate compound in a specific embodiment of this technology. [Figure 10] Figure 10 shows the 13C NMR spectrum of a synthetic intermediate compound according to a specific embodiment of this technology. [Figure 11] Figure 11 shows the 1H NMR spectrum of synthetic compound 3 according to a specific embodiment of this technology. [Figure 12] Figure 12 shows the 13C NMR spectrum of synthetic compound 3 according to a specific embodiment of this technology. [Figure 13] Figure 13 shows high-performance liquid chromatography (HPLC) chromatograms of synthetic compounds 3 and 4 obtained to verify the radical activity of the compounds after the radicalization step in a specific embodiment of this technology. [Figure 14A] Figures 14A and 14B show (A) a comparison of transcutaneous fluorescence RDTC values ​​in healthy BALB / c mice (n=6) calculated using MediBeacon software and RDTC values ​​in healthy BALB / c mice (n=14) calculated using glucoverdazil-enhanced DCE-MRI. (B) a comparison of RDTC values ​​in healthy BALB / c mice (both n=14) at each time point, using intensity measurements obtained at the voxel level or from the entire region of interest (ROI) of each slice. Data are shown as box plots of a single RDTC value in A (transcutaneous) or a single mean RDTC value in A (MRI) and B. Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. In all graphs, ns indicates that the graph is not statistically significant, and **** indicates a p-value < 0.0001. [Figure 14B] Refer to the explanation in Figure 14A. [Figure 15A] Figures 15A and 15B show the cytotoxicity assessment of H460 large cell lung cancer cells after incubation with various concentrations of glucoverdazil for 4 hours (A) and 24 hours (B). Data are presented as mean ± standard deviation (SD) of repeated measures for n=3. Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. [Figure 15B] Refer to the explanation in Figure 15A. [Figure 16A] Figures 16A and 16B show the cytotoxicity assessment of human renal proximal tubular cells after incubation for 4 hours (A) or 24 hours (B) at 10 mM concentrations of glucoverdadil, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), and 2,2,6,6-tetramethylpiperidine-1-yloxyl (TEMPO) under cell culture conditions. Data are presented as mean ± standard deviation (n=3). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. * indicates the proportion of live and dead cells that showed a statistically significant difference (p<0.05) compared to other conditions. [Figure 16B] Refer to the explanation in Figure 16A. [Figure 17] Figure 17 shows the uptake of glucoverdazil into human renal proximal tubular cells incubated in a 10 mM glucoverdazil solution for 24 hours under cell culture conditions. Uptake was measured based on comparison with EPR activity at known concentrations and normalized by the number of cells loaded into the EPR tubes. Data are expressed as the mean ± SD of repeated measures for n=3. Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. [Figure 18A]Figures 18A-18F show DCE-MRI data with glucoverdazil contrast in a unilateral ureteral obstruction (UUO) model. Figure 18A: Normalized intensity-versus-time curve in UUO-treated mice (day 0). Curves are shown as mean normalized intensity ± SEM. Figure 18B: Best-fitted line from t=0 to t=40 minutes on the semi-logarithmic (natural logarithmic) regression curve of Figure 18A. Data are shown as mean RDTC value (k) and R2 value (±SD). RDTC values ​​of regions of interest (ROI) in the cortex and medulla-renal pelvis (MRP) in the sham group (Figure 18C) and the UUO group (Figure 18D). Data are shown as box plots, showing a single mean RDTC value obtained for each region of each kidney (ipsilateral or contralateral) for each mouse (n=5). Similar data and plots regarding AUC are shown in Figures 18E and 18F for the sham surgery group and the UUO group, respectively. Statistical analysis was performed using repeated measures two-way ANOVA followed by Tukey's post-hoc test. In all graphs, ns indicates no significant difference, * indicates p<0.05, *** indicates p<0.001, and **** indicates p<0.0001. [Figure 18B] See the explanation in Figure 18A. [Figure 18C] See the explanation in Figure 18A. [Figure 18D] See the explanation in Figure 18A. [Figure 18E] See the explanation in Figure 18A. [Figure 18F] See the explanation in Figure 18A. [Figure 19A]Figures 19A-19C show DCE-MRI data with glucoverdazil contrast in a folate-induced nephropathy (FAN) model. Figure 19A: RDTC values ​​in regions of interest (ROI) of the cortex and medulla-pelvis (MRP). Data are shown as box plots of a single mean RDTC value obtained from each mouse (n=5). Figure 19B shows the same data and AUC plot. Figure 19C: Normalized intensity-versus-time curves at each time point after injury for the whole kidney, cortex, and MRP region in the FAN model. Curves are shown as mean normalized intensity (±SEM) at each time point. Statistical analysis was performed using repeated measures two-way ANOVA followed by Tukey's post-hoc multiple comparison test. In all graphs, ns indicates no significant difference, * indicates p<0.05, ** indicates p<0.01, and **** indicates p<0.001. [Figure 19B] See the explanation in Figure 19A. [Figure 19C] See the explanation in Figure 19A. [Modes for carrying out the invention]

[0067] Glomerular filtration rate (GFR) measurement is the gold standard for assessing renal function in clinical practice. However, this method relies on inputting physiological, demographic, and blood-derived metabolite levels into a formula derived from a limited, less representative population, and does not provide spatial information about renal impairment. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) can be useful for evaluating renal function in cases suspected of acute kidney injury (AKI) or chronic kidney disease (CKD), but there is a strong negative bias against the use of gadolinium-based contrast agents because they can induce nephrogenic systemic fibrosis in patients with impaired renal function.

[0068] With the aim of intentionally developing a contrast agent for DCE-MRI suitable for renal pathological diagnosis, a class of extremely stable nitrogen-centered organic radicals called verdazil was evaluated. Verdazil is derived from an extremely stable radical, exhibits excellent T1 shortening effect at 3T, and does not cause signal loss even in highly reducing environments. This disclosure shows that glucoverdazil, a glucose-modified form of verdazil, does not show extrarenal uptake in either in vitro or in vivo, and that image contrast is limited to the kidney and bladder. In mouse models of unilateral ureteral obstruction (UUO) and folate-induced nephropathy (FAN), renal function decline was shown over time, and renal function maps correlated with histological and serum biomarkers of renal injury. Using the FAN model, compared with validated transcutaneous fluorescence techniques, the clearance rate of glucoverdazil was shown to be a reliable indicator of GFR. This disclosure is based, at least in part, on the finding that glucoverdazil could be an extremely potent contrast agent for metal-free magnetic resonance imaging (MRI). In certain embodiments, the contrast agents of the Disclosure may provide reliable glomerular filtration rate (GFR) measurements that are independent of error-prone population-derived estimation formulas and provide underlying spatial or structural information for renal dysfunction, and / or enable image-based personalized medicine approaches in nephrology or other fields. In certain embodiments, the compounds of the Disclosure provide metal-free, stable, and non-toxic contrast agents.

[0069] To ensure a clear and consistent understanding of the terms used herein, several definitions are provided below. Furthermore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this invention pertains.

[0070] In the claims and / or specification, the "a" or "an" used with the term "comprising" may mean "one," but it can also mean "one or more," "at least one," and "one or more." Similarly, the word "another" can mean at least a second one, or even more.

[0071] As used herein and in the claims, the terms “comprising” (including its conjugations “comprise” and “comprises”), “having” (including “have” and “has”), “including” (including “include” and “includes”), or “containing” (including “contain” and “contains”) are open-ended and do not exclude additional undescribed elements or steps.

[0072] In this specification, the term "about" indicates that it includes an error range inherent to the apparatus or method used to determine the value.

[0073] In this specification, the terms “derivative” and “variant” are used synonymously.

[0074] As used herein, the term "subject" includes eukaryotes (e.g., mammals), with specific examples including humans, sheep, cattle, horses, pigs, dogs, cats, non-human primates, mice, and rats. In this specification, the terms "subject" and "patient" are used synonymously.

[0075] Definitions of specific functional groups and chemical terms are described in more detail below. In this disclosure, the notation of chemical elements follows the CAS periodic table of elements found on the inside cover of the 75th edition of the Handbook of Chemistry and Physics, and the definitions of specific functional groups generally follow those of the same book. Furthermore, general principles of organic chemistry, as well as specific functional groups and reactivity, are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987, the entire contents of each of these references are incorporated herein by reference.

[0076] As described herein, it will be understood that the compounds of this disclosure may be substituted with any number of substituents or functional groups. In general, the term “substituted” (whether prefixed with “arbitrarily” or not) and the substituents shown in the structural formulas of the present invention mean the substitution of a hydrogen radical in a given structure with a radical of a particular substituent. If multiple positions in any structure can be substituted with multiple substituents selected from a particular group, the substituents at each position may be identical or different from one another. In this specification, “substituted” encompasses substitution with all substituents permissible in organic compounds. In a broad sense, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents in organic compounds. In this disclosure, heteroatoms such as nitrogen may have hydrogen substituents and / or any substituents permissible in the organic compounds described herein that satisfy the valence of the heteroatom. Furthermore, this disclosure is not intended to be limited in any aspect by any substituents permissible in organic compounds.

[0077] In this specification, the term "acyl" refers to the general formula -C(=O)R o It means a base represented by R oThese are substituted or unsubstituted hydroxyl groups, substituted or unsubstituted thiol groups, substituted or unsubstituted amino groups, substituted or unsubstituted aliphatic groups (cyclic or acyclic), substituted or unsubstituted heteroaliphatic groups (cyclic or acyclic), substituted or unsubstituted aryl groups, or substituted or unsubstituted heteroaryl groups. Examples of acyl groups include carboxylic acids (-CO2H), ketones (e.g., acetyl group [-(C(=O)CH3)]), esters, amides, carbonate esters, carbamates, and urea. Acyl substituents are, but are not limited to, those substituents described herein that can form a stable site. Examples include aliphatic groups substituted with one or more aliphatic groups, alkyl groups, alkenyl groups, alkynyl groups, heteroaliphatic groups, heterocyclic groups, aryl groups, heteroaryl groups, acyl groups, sulfinyl groups, sulfonyl groups, oxo groups, imino groups, thiooxo groups, cyano groups, amino groups, azide groups, nitro groups, hydroxyl groups, thio groups, and / or halogen groups.

[0078] As used herein, “aliphatic” means saturated or unsaturated non-aromatic hydrocarbons, including linear (i.e., unbranched), branched, acyclic or cyclic (i.e., carbocyclic) hydrocarbons, or polycyclic hydrocarbons, which may optionally be substituted with one or more functional groups. As those skilled in the art will understand, as used herein, “aliphatic” means alkyl groups, alkenyl groups, alkynyl groups, cyclic alkyl groups, cyclic alkenyl groups, and cyclic alkynyl groups, but is not limited to these. Thus, as used herein, “alkyl group” includes linear alkyl groups, branched alkyl groups, and cyclic alkyl groups. A similar convention applies to other general terms such as “alkenyl” and “alkynyl.” Furthermore, as used herein, terms such as “alkyl group,” “alkenyl group,” and “alkynyl group” encompass both substituted and unsubstituted groups. In certain embodiments, as used herein, “aliphatic” means an aliphatic group (cyclic, acyclic, substituted, unsubstituted, branched or unbranched) having 1 to 6 or 2 to 6 carbon atoms. In certain embodiments, the aliphatic group has 1 to 5 or 2 to 5 carbon atoms. In certain embodiments, the aliphatic group has 1 to 4 or 2 to 4 carbon atoms. In certain embodiments, the aliphatic group has 1 to 3 or 2 to 3 carbon atoms. In certain embodiments, the aliphatic group has 1 to 2 carbon atoms. In certain embodiments, the aliphatic group has 1 carbon atom. In certain embodiments, the aliphatic group has 2 carbon atoms. In certain embodiments, the aliphatic group has 1 to 6 carbon atoms (C 1~6 The substituents of the aliphatic group include, but are not limited to, any of the substituents described herein that form a stable site (e.g., an aliphatic group substituted with one or more aliphatic groups, alkyl groups, alkenyl groups, alkynyl groups, heteroaliphatic groups, heterocyclic groups, aryl groups, heteroaryl groups, acyl groups, sulfinyl groups, sulfonyl groups, oxo groups, imino groups, thiooxo groups, phosphino groups, cyano groups, amino groups, azide groups, nitro groups, hydroxyl groups, thio groups, and / or halogen groups).

[0079] As used herein, "alkyl group" refers to a saturated linear or branched hydrocarbon group derived from a hydrocarbon substructure having 1 to 20 carbon atoms by removing a single hydrogen atom. In some embodiments, the alkyl groups used in the present invention have 1 to 6 carbon atoms (C 1~6 ). In other embodiments, the alkyl group used has 1 to 5 carbon atoms. In yet another embodiment, the alkyl group has 1 to 4 carbon atoms. In yet another embodiment, the alkyl group has 1 to 3 carbon atoms. In yet another embodiment, the alkyl group contains 1 to 2 carbon atoms. In yet another embodiment, the alkyl group has 1 carbon atom. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, sec-pentyl, isopentyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, sec-hexyl, etc., and may have one or more substituents. Examples of substituents on alkyl groups include, but are not limited to, those substituents described herein that form a stable site (for example, alkyl groups substituted with one or more aliphatic groups, heteroaliphatic groups, heterocyclic groups, aryl groups, heteroaryl groups, acyl groups, sulfinyl groups, sulfonyl groups, oxo groups, imino groups, thiooxo groups, cyano groups, amino groups, azide groups, nitro groups, hydroxyl groups, thio groups, and / or halogen groups).

[0080] As used herein, the term “alkenyl group” means a monovalent group obtained by removing a single hydrogen atom from a linear or branched hydrocarbon substructure having at least one carbon-carbon double bond. In certain embodiments, the alkenyl group used in the present invention has 2 to 6 carbon atoms. In some embodiments, the alkenyl group used in the present invention has 2 to 5 carbon atoms. In other embodiments, the alkenyl group used has 2 to 4 carbon atoms. In yet another embodiment, the alkenyl group has 2 to 3 carbon atoms. In yet another embodiment, the alkenyl group has 2 carbon atoms. Examples of alkenyl groups include ethenyl, propenyl, butenyl, and 1-methyl-2-buten-1-yl, which may have one or more substituents. The substituents of the alkenyl group include, but are not limited to, any of the substituents described in this disclosure that form a stable site (for example, an alkenyl group substituted with one or more aliphatic groups, heteroaliphatic groups, heterocyclic groups, aryl groups, heteroaryl groups, acyl groups, sulfinyl groups, sulfonyl groups, oxo groups, imino groups, thiooxo groups, cyano groups, amino groups, azide groups, nitro groups, hydroxyl groups, thio groups, and / or halogen groups).

[0081] As used herein, "alkynyl" refers to a monovalent group obtained by removing a single hydrogen atom from a straight-chain or branched-chain hydrocarbon having at least one carbon-carbon triple bond. In certain embodiments, the alkynyl group used in the present invention has 2 to 6 carbon atoms. In some embodiments, the alkynyl group used in the present invention has 2 to 5 carbon atoms. In other embodiments, the alkynyl group used has 2 to 4 carbon atoms. In yet another embodiment, the alkynyl group has 2 to 3 carbon atoms. In yet another embodiment, the alkynyl group has 2 carbon atoms. Typical alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), and 1-propynyl, and may have one or more substituents. Substituents for the alkynyl group include, but are not limited to, any of the substituents described herein, as long as they result in the formation of a stable site (for example, an alkynyl group substituted with one or more aliphatic groups, heteroaliphatic groups, heterocyclic groups, aryl groups, heteroaryl groups, acyl groups, sulfinyl groups, sulfonyl groups, oxo groups, imino groups, thiooxo groups, cyano groups, amino groups, azide groups, nitro groups, hydroxyl groups, thio groups, and / or halogen groups).

[0082] In this specification, "amino" refers to the group of formula (-NH2). "Substituting amino" refers to the group of formula (-NHR h ) or (-NR h 2) refers to the base, where R hThe substituents other than hydrogen that can form a stable site (for example, amino groups substituted with one or more aliphatic groups, alkyl groups, alkenyl groups, alkynyl groups, heteroaliphatic groups, heterocyclic groups, aryl groups, heteroaryl groups, acyl groups, sulfinyl groups, sulfonyl groups, amino groups, nitro groups, hydroxyl groups, and / or thio groups). The “suitable amino protecting groups” as used herein are well known to those skilled in the art and include those described in detail in *Protecting Groups in Organic Synthesis*, 3rd edition, by TW Greene and PGM Wuts (John Wiley & Sons, 1999), which are incorporated herein by reference. Suitable amino protecting groups include methyl carbamate, ethyl carbamate, 9-fluorenyl methyl carbamate (Fmoc), 9-(2-sulfo)fluorenyl methyl carbamate, 9-(2,7-dibromo)fluorenyl methyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxantyl)] methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (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-t-butylphenyl)-1-methylethylcarbamate (t-Bumeoc), 2-(2′-and 4′-pyridyl)ethylcarbamate (Pyoc), 2-(N,N-Dicyclohexylcarboxamide) ethylcarbamate, t-butylcarbamate (BOC), 1-adamantylcarbamate (Adoc), vinylcarbamate (Voc), allylcarbamate (Alloc), 1-isopropylallylcarbamate (Ipaoc), cinnamylcarbamate (Coc), 4-nitrocinnamylcarbamate (Noc), 8-quinolylcarbamate, N-hydroxypiperidinylcarbamate, alkyldithiocarbamate, benzylcarbamate (Cbz), p-methoxybenzylcarbamate (Moz), p- Trobenzylcarbamate, p-bromobenzylcarbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzylcarbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethylcarbamate, diphenylmethylcarbamate, 2-methylthioethylcarbamate, 2-methylsulfonylethylcarbamate, 2-(p-toluenesulfonyl)ethylcarbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenylcarbamate (Mtpc), 2,4-dimethylthio Phenylcarbamate (Bmpc), 2-phosphonioethylcarbamate (Peoc), 2-triphenylphosphonioisopropylcarbamate (Ppoc), 1,1-dimethyl-2-cyanoethylcarbamate, m-chloro-p-acyloxybenzylcarbamate, p-(dihydroxyboryl)benzylcarbamate, 5-benzisoxazolylmethylcarbamate, 2-(trifluoromethyl)-6-chromonylmethylcarbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzylcarbamate, o-nitrobenz Carbamate, 3,4-dimethoxy-6-nitrobenzylcarbamate, phenyl(o-nitrophenyl)methylcarbamate, phenothiazinyl-(10)-carbonyl derivative, N'-p-toluenesulfonylaminocarbonyl derivative, N'-phenylaminothiocarbonyl derivative, t-amylcarbamate, S-benzylthiocarbamate, p-cyanobenzylcarbamate, cyclobutylcarbamate, cyclohexylcarbamate, cyclopentylcarbamate, cyclopropylmethylcarbamate, p-decyloxybenzylcarbamate, 2,2-Dimethoxycarbonylvinylcarbamate, o-(N,N-dimethylcarboxamide)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamide)propylcarbamate, 1,1-dimethylpropynylcarbamate, di(2-pyridyl)methylcarbamate, 2-furylmethylcarbamate, 2-iodoethylcarbamate, isobornylcarbamate, isobutylcarbamate, isonicotinylcarbamate, p-(p'-methoxyphenylazo)benzylcarbamate, 1-methylcyclobutylcarbamate, 1-methyl Lucyclohexylcarbamate, 1-methyl-1-cyclopropylmethylcarbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethylcarbamate, 1-methyl-1-(p-phenylazophenyl)ethylcarbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethylcarbamate, phenylcarbamate, p-(phenylazo)benzylcarbamate, 2,4,6-tri-t-butylphenylcarbamate, 4-(trimethylammonium)benzylcarbamate, 2,4,6-trimethylbenzyl Lucarbamate, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivatives, benzamide, p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide, acetamide, (N'-dithiobenzyloxycarbonylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenoxy (L)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazofenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine derivative, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-Tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexane-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexane-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyrroline-3-yl)amine, Quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzosperylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2-picolylamino N'-oxide, N-1,1-dimethylthiomethyleneamine, N-benzylideneamine, Np-methyl Toxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N-(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine, Np-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine, N-borane derivatives, N-diphenyl Rubonic acid derivatives, N-[phenyl(pentacarbonylchromium or tungsten)carbonyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosamine, amine N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidate, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4-Dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys), p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, phenacylsulfonamide are available.,

[0083] The term "aryl" as used herein refers to a stable aromatic carbocyclic monocyclic or polycyclic ring system having 3 to 20 ring atoms, all of which are carbon atoms and may be substituted or unsubstituted. In certain embodiments of the present invention, "aryl" refers to an aromatic ring system of C4-C 20 which is monocyclic, bicyclic or tricyclic consisting of 1 to 3 aromatic rings, including but not limited to phenyl, biphenyl, naphthyl, etc. The aryl may optionally be substituted with one or more substituents. Aryl substituents include, but are not limited to, the substituents described in this disclosure that can form a stable site (e.g., an aryl group substituted with one or more of an aliphatic group, heteroaliphatic group, heterocyclic group, aryl group, heteroaryl group, acyl group, sulfinyl group, sulfonyl group, oxo group, imino group, thioxo group, cyano group, amino group, azide group, nitro group, hydroxy group, thio group, and / or halogen group).

[0084] The terms "direct bond" or "bond" refer to a single, double, or triple bond between two groups. In certain embodiments, "direct bond" refers to a single bond between two groups.

[0085] As used herein, the terms "halo" and "halogen" refer to atoms selected from fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br), and iodine (iod, -I).

[0086] The term “heteroaliphatic” as used herein means non-aromatic aliphatic compounds, including both saturated and unsaturated compounds, that can take the form of linear (i.e., unbranched), branched, acyclic, cyclic (i.e., heterocyclic) or polycyclic hydrocarbons, which are optionally substituted with one or more functional groups and, for example, contain one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of carbon atoms. As will be understood by those skilled in the art, “heteroaliphatic” as used herein includes, but is not limited to, heteroalkyl groups, heteroalkenyl groups, heteroalkynyl groups, and heterocyclic groups. Accordingly, “heteroalkyl” as used herein includes linear alkyl groups, branched alkyl groups, and cyclic alkyl groups as defined herein, which are optionally substituted with one or more functional groups and, for example, contain one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of carbon atoms. Similar conventions apply to other general terms such as “heteroalkenyl” and “heteroalkynyl.” Furthermore, as used herein, terms such as “heteroalkyl,” “heteroalkenyl,” and “heteroalkynyl” encompass both substituted and unsubstituted groups. In certain embodiments, “heteroaliphatic” as used herein refers to a heteroaliphatic group having 1 to 6 or 2 to 6 carbon atoms (cyclic or acyclic, substituted or unsubstituted, branched or unbranched). In certain embodiments, the heteroaliphatic group has 1 to 5 or 2 to 5 carbon atoms. In certain embodiments, the heteroaliphatic group has 1 to 4 or 2 to 4 carbon atoms. In certain embodiments, the heteroaliphatic group has 1 to 3 or 2 to 3 carbon atoms. In certain embodiments, the heteroaliphatic group has 1 to 2 carbon atoms. In certain embodiments, the heteroaliphatic group has 1 carbon atom. In certain embodiments, the heteroaliphatic group has 2 carbon atoms.Substituents of heteroaliphatic groups include, but are not limited to, any of the substituents described herein, insofar as they form a stable site (for example, heteroaliphatic groups substituted with one or more aliphatic groups, alkyl groups, alkenyl groups, alkynyl groups, heteroaliphatic groups, heterocyclic groups, aryl groups, heteroaryl groups, acyl groups, sulfinyl groups, sulfonyl groups, oxo groups, imino groups, thiooxo groups, phosphino groups, cyano groups, amino groups, azide groups, nitro groups, hydroxyl groups, thio groups, and / or halogen groups).

[0087] In this specification, "heteroaryl" means a stable aromatic monocyclic or polycyclic ring system having 3 to 20 ring atoms. One of these ring atoms is selected from S, O, or N, 0, 1, or 2 additional ring atoms are independently selected from S, O, or N as additional heteroatoms, and the remaining ring atoms are carbon, which are bonded to the rest of the molecule via one of the ring atoms. Representative heteroaryl groups include, but are not limited to, pyrrolyl, pyrazolyl, imidazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridadinyl, triazinyl, tetradinyl, pyrrolidinyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, indazolyl, quinolidinyl, sinnolinyl, quinazolinyl, phthalazinyl, naphthridinyl, quinoxalinyl, thiophenyl, thianaphthenyl, furyl, benzofuryl, benzothiazolyl, thiazolinyl, isothiazolyl, thiadiazolinyl, oxazolyl, isoxazolyl, and oxadiazolyl. These may be substituted with one or more substituents. The heteroaryl substituent may be any of the substituents described in this disclosure that form a stable site, but is not limited to these. Examples include heteroaryl groups substituted with one or more aliphatic groups, heteroaliphatic groups, heterocyclic groups, aryl groups, heteroaryl groups, acyl groups, sulfinyl groups, sulfonyl groups, oxo groups, imino groups, thiooxo groups, cyano groups, amino groups, azide groups, nitro groups, hydroxyl groups, thio groups, and / or halogen groups.

[0088] The terms “heterocyclic” or “heterocyclic group” refer to non-aromatic, partially unsaturated or fully saturated 3- to 10-membered ring systems, including 3- to 8-membered monocyclic rings, and bicyclic or tricyclic ring systems in which an aromatic 5- or 6-membered aryl or heteroaryl group may be fused to the non-aromatic ring. These heterocyclic rings include those having 1 to 3 heteroatoms independently selected from oxygen, sulfur, and nitrogen, where the sulfur and nitrogen heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. In certain embodiments, the term “heterocyclic” refers to a non-aromatic 5-membered, 6-membered, or 7-membered ring, or polycyclic group, where at least one of the ring atoms of the group is a heteroatom selected from O, S, and N (where the nitrogen and sulfur heteroatoms may optionally be oxidized), the remaining ring atoms are carbon atoms, and the group is bonded to the rest of the molecule via any of the ring atoms. Heterocyclic groups include, but are not limited to, bicyclic or tricyclic groups. These consist of fused 5-membered, 6-membered, or 7-membered rings having 1 to 3 heteroatoms independently selected from oxygen, sulfur, and nitrogen. Herein, (i) each 5-membered ring has 0 to 2 double bonds, each 6-membered ring has 0 to 2 double bonds, and each 7-membered ring has 0 to 3 double bonds; (ii) the nitrogen heteroatom and sulfur heteroatom may be optionally oxidized; (iii) the nitrogen heteroatom may be optionally quaternized; and (iv) any of the above heterocyclic rings may be fused with an aryl ring or a heteroaryl ring. Representative heterocycles include azilidinyl, azetidinyl, 1,3-diazetidinyl, piperidinyl, piperazinyl, azokanyl, thiranyl, thietanyl, tetrahydrothiophenyl, dithiolanyl, thiacyclohexanyl, oxylanyl, oxetanyl, tetrahydrofuryl, tetrahydropyranyl, dioxanyl, oxathiolanyl, morpholinyl, thioxanyl, and tetrahydronaphthyl, which may have one or more substituents.Substituents include, but are not limited to, the substituents described herein, and which result in the formation of a stable site (for example, heterocyclic groups substituted with one or more aliphatic groups, heteroaliphatic groups, heterocyclic groups, aryl groups, heteroaryl groups, acyl groups, sulfinyl groups, sulfonyl groups, oxo groups, imino groups, thiooxo groups, cyano groups, amino groups, azide groups, nitro groups, hydroxyl groups, thio groups, and / or halogen groups).

[0089] In this specification, the term "hydrogen" means all isotopes having atomic number 1. Generally, hydrogen is a stable isotope containing zero or one neutron, i.e. 1 H or 2 This refers to H (deuterium). In certain embodiments, hydrogen is present in its natural isotopic abundance. In other embodiments, it is specifically selected that a deuterium atom is present at at least one position.

[0090] In this specification, the terms “hydroxy” or “hydroxyl” refer to a group represented by the formula (-OH). A “substituted hydroxyl group” is a group represented by the formula (-ORi), where Ri is a substituent other than hydrogen that provides a stable site (for example, a hydroxyl group, aliphatic group, alkyl group, alkenyl group, alkynyl group, heteroaliphatic group, heterocyclic group, aryl group, heteroaryl group, acyl group, sulfinyl group, and / or sulfonyl group substituted with a suitable hydroxyl protecting group). The “suitable hydroxyl protecting groups” as used herein are well known to those skilled in the art and include those described in detail in *Protecting Groups in Organic Synthesis* by TW Greene and PGM Wuts (3rd edition, John Wiley & Sons, 1999). The entire contents of that book 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, and 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-methoxypiperidine-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuryl, tetrahydrothiofuryl, 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-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxide, diphenylmethyl, p,p'-dinitrobenzyldihydryl, 5-dibenzosbergine L, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl, 4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4′,4″-tris(levurinoyloxyphenyl)methyl, 4,4′,4″-tris(benzoyloxyphenyl)methyl, 3-(imidazole-1-yl)bis(4′,4″-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, 1,3-benzodithiolan-2-yl, benzoisothiazolyl S,S-dioxide, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethyltexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (rebrinate), 4,4-(ethylenedimethyl O) Pentanoate (levulinoyl dithioacetal), pivaloate, adamantanoate, crotonoate, 4-methoxycrotonoate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkylmethyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), alkylethyl 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-benzylthiocarbonate, 4-ethoxy-1-naphthyl carbonate, methyldithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2 ,6-Dichloro-4-methylphenoxyacetate, 2,6-Dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-Bis(1,1-dimethylpropyl)phenoxyacetate, Chlorodiphenylacetate, Isobutyrate, Monosuccinoate, (E)-2-methyl-2-butenoate, o-(methoxycarbonyl)benzoate, α-Naphthoate, Nitrate, Alkyl N,N,N′,N′-Tetramethylphosphodiamidate, Alkyl N-phenylcarbamate, Borate, Dimethylphosphinthioyl Protecting groups used to protect alkyl 2,4-dinitrophenyl sulfenates, sulfates, methanesulfonates (mesylates), benzylsulfonates, and tosylates (Ts), 1,2- or 1,3-diols include methylene acetals, ethylidene acetals, 1-t-butylethylidene ketals, 1-phenylethylidene ketals, (4-methoxyphenyl)ethylidene acetals, 2,2,2-trichloroethylidene acetals, acetonides, cyclopentylidene ketals, cyclohexylidene ketals, and cycloheptylides. Ketal, benzylidene acetal, p-methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal, 3,4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethylene orthoester, 1-methoxyethylidene orthoester, 1-ethoxyethylidene orthoester, 1,2-dimethoxyethylidene orthoester, α-methoxybenzylidene orthoester, 1-(N,N-dimethylamino)ethylidene derivative, α-(N,These include N'-dimethylamino)benzylidene derivatives, 2-oxacyclopentylidene orthoesters, di-t-butylsilylene groups (DTBS), 1,3-(1,1,3,3-tetraisopropyldisiloxanylidene) derivatives (TIPDS), tetra-t-butoxydisiloxane-1,3-diylidene derivatives (TBDS), cyclic carbonate esters, cyclic borate esters, ethylborate esters, and phenylborate esters.

[0091] In this specification, the term "imino" refers to the formula (=NR r ) refers to the base, where R r This means hydrogen, or any substituent described herein, that forms a stable site (for example, a suitable amino protecting group; a substituted or unsubstituted amino group; an acyl group; a cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkyl group; a cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkenyl group; a cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkynyl group; a cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkyl group; a cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkenyl group; a cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkynyl group; a substituted or unsubstituted aryl group; or a substituted or unsubstituted heteroaryl group).

[0092] In this specification, the term "isocyano" refers to the base of formula (-NC).

[0093] In this specification, the term "nitro" refers to the group represented by the formula (-NO2).

[0094] As used herein, the term "nitroxide" refers to a stable cyclic or acyclic nitroxide group. In certain embodiments, a stable nitroxide refers to a chemically stable nitroxide that can be obtained as a pure product and is suitable for storage and laboratory handling. In certain embodiments, a stable nitroxide refers to a cyclic or acyclic nitroxide having two groups that do not contain α-hydrogens. Exemplary cyclic and acyclic nitroxides are described in Keana, Chemical Reviews (1978) 78:37-64, the entire contents of which are incorporated herein by reference.

[0095] In this specification, "oxo" refers to the group represented by the formula (=O).

[0096] In this specification, the terms "thio" or "thiol" refer to the group of formula (-SH). A "substituted thiol" refers to the group of formula (-SR). r This refers to the group represented by ), where R r The substituent is a substituent other than hydrogen, which forms a stable site (for example, a thio group substituted with one or more aliphatic groups, alkyl groups, alkenyl groups, alkynyl groups, heteroaliphatic groups, heterocyclic groups, aryl groups, heteroaryl groups, acyl groups, sulfinyl groups and / or sulfonyl groups).

[0097] As used herein, the term "thiooxo" refers to the group represented by the formula (=S).

[0098] In this specification, "sulfinyl" refers to the formula R f This refers to the sulfinyl group represented by -S(=O)-, where R f The group may be an optionally substituted aliphatic group, heteroaliphatic group, aryl group, or heteroaryl group. The term "alkylsulfinyl" is used with respect to R f The term "arylsulfinyl" refers to a sulfinyl group which is an alkyl group which may be optionally substituted. f This refers to a sulfinyl group having an optionally substituted aryl or heteroaryl group.

[0099] In this specification, the term "sulfonyl" refers to the compound of formula R g This refers to the sulfonyl group represented by -S(=O)2-, where R gThis may be an aliphatic group, a heteroaliphatic group, an aryl group, or a heteroaryl group, which may be optionally substituted. The term "alkylsulfonyl" is R g This refers to a sulfonyl group which may be an optionally substituted alkyl group. The term "arylsulfonyl" is R g This refers to a sulfonyl group which may be an aryl or heteroaryl group to which the aryl group may be optionally substituted. Exemplary aryl or alkylsulfonyl groups include tosyl(toluenesulfonyl, CH3C6H4SO2-), mesyl(methylsulfonyl, CH3SO2-), and trifluoromethanesulfonyl (CF3SO2-).

[0100] As used herein, "stable part" preferably refers to a part that has sufficient stability to be manufactured (including in-situ manufacturing) and maintains its integrity for a period sufficient to serve the purposes detailed herein.

[0101] As used herein, "stable radical" means a free radical that is stable enough to be manufactured (including in-situ manufacturing) and that retains its integrity for a period sufficient to be useful for the purposes detailed herein.

[0102] In certain embodiments, one or more (or all) of the hydrogen atoms in the compound of structural formula (I), (II), or (III) are 2 It is H (deuterium).

[0103] Embodiments of this technology include compounds that are verdazil derivatives (for example, compounds of formula (I), formula (II), and formula (III), as well as the compounds shown in Table 1), as described herein. The compounds disclosed herein are metal-free contrast agents that exhibit superior stability and / or lower toxicity compared to conventional contrast agents. This compound is useful in clinical settings for biomedical imaging, including but not limited to contrast-enhanced magnetic resonance imaging (CE-MRI) and dynamic contrast-enhanced MRI (DCE-MRI), providing reliable, rapid, and / or quantitative clinical imaging that facilitates the early diagnosis and detection of various diseases. Each aspect of this technology includes compositions and pharmaceutical compositions containing the compound, methods for producing the compound, and uses thereof.

[0104] composition In certain embodiments, the compounds of this disclosure may be contained in a composition or pharmaceutical composition.

[0105] Pharmaceutical compositions are generally formulated to suit the intended method or route of administration. Examples of non-limiting routes of administration include oral or parenteral administration, such as intramuscular, intravenous, subcutaneous (e.g., injection or implant), intraperitoneal, intrathecal, or intra-articular administration. In some embodiments, pharmaceutical compositions are provided in single-use containers (e.g., single-use vials, ampoules, syringes, or auto-injectors). In other embodiments, pharmaceutical compositions are provided in multi-use containers (e.g., multi-use vials). The compounds and compositions described herein can be administered to a subject by any suitable method known in the art.

[0106] The term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, used to support or deliver any composition or component described herein. Each carrier must be “acceptable” in the sense that it is compatible with the composition and its components and is not harmful to the patient. Examples of materials that may function as pharmaceutically acceptable carriers include, but are not limited to, the following: (1) sugars, e.g., lactose, glucose, and sucrose; (2) starches, e.g., corn starch and potato starch; (3) cellulose and its derivatives, e.g., sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) tragacanth powder; (5) malt; (6) gelatin; (7) talc; (8) excipients, e.g., cocoa butter and suppository wax; (9) oils, e.g., peanut oil, cottonseed oil, safflower oil, sesame oil, and olive oil. , corn oil and soybean oil; (10) glycols, e.g., propylene glycol; (11) polyols, e.g., glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, e.g., ethyl oleate and ethyl laurate; (13) agar; (14) buffers, e.g., 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; and (21) other non-toxic suitable substances used in pharmaceutical formulations.

[0107] In certain embodiments, the compositions and pharmaceutical compositions of the present disclosure may exist in the form of micelles. In some micelles, the compositions of the present disclosure are mixed with nonpolar radicals (e.g., radicals containing perfluorinated substructures). The compositions or pharmaceutical compositions may also contain surfactants. A non-limiting example of a perfluorinated radical is the TEMPO group having a fluorinated chain (e.g., a perfluorinated chain) (described in Pozzi, Adv. Synth. Cat. 347:677 (2005)), which is incorporated herein by reference). An exemplary perfluorinated radical is C6-C at the 4-position of TEMPO via an amide or sulfonamide group. 20 (For example, C8~C 12 ) is TEMPO with a perfluoroalkyl group attached. Suitable surfactants include, but are not limited to, perfluorosulfone carboxylic acids, especially C4~C 12 Acids such as C6, C7, C8, C9, C 10 , C 11 and C 12 Examples of acids include, but are not limited to, ammonium perfluorooctanoate (FC143), perfluorooctanesulfonic acid (PFOS), and perfluorononanoic acid (PFNA).

[0108] The compositions and pharmaceutical compositions of this disclosure may also include additional components such as stabilizers, preservatives, and dispersants. The compositions and pharmaceutical compositions of this disclosure may also include additional components suitable for the intended purpose, such as excipients, dyes, etc., suitable for imaging and / or administration to a subject.

[0109] A “pharmaceutically acceptable salt” of a compound means a pharmaceutically acceptable salt of the compound. Preferred salts of the compound are those that maintain or enhance the biological efficacy and properties of the free acids and free bases of the parent compound as defined in this disclosure, or that utilize the basic, acidic, or charged functional groups inherent in the molecule, and that are not biologically or otherwise undesirable. Examples of pharmaceutically acceptable salts are, for example, described in Berge et al., “Pharmaceutical Salts” (J. Pharm. Sci. 66, 1-19 (1977)).Non-limiting examples of such salts include: (1) Acid addition salts: those formed by adding inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, nitric acid, phosphoric acid, carbonate-forming agents to a basic or positively charged functional group, or acetic acid, propionic acid, lactic acid, oxalic acid, glycolic acid, pivalic acid, t-butylacetic acid, β-hydroxybutyric acid, valeric acid, hexanoic acid, cyclopentanepropionic acid, pyruvic acid, malonic acid, succinic acid, malic acid, and maleic acid. Ionic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, cyclohexylaminosulfonic acid, benzenesulfonic acid, sulfanilic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 3-phenylpropionic acid, laurylsulfonic acid, lauryl sulfate (1) Base addition salts: These are formed when an acidic proton present in the parent compound is replaced by an alkali metal ion (e.g., lithium, sodium, potassium), an alkaline earth metal ion (e.g., magnesium, calcium, barium), or another metal ion such as aluminum, zinc, or iron, or when it is coordinated with an organic base such as ammonia, ethylamine, diethylamine, ethylenediamine, N,N'-dibenzylethylenediamine, ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, piperazine, chloroprocaine, procaine, choline, or lysine.

[0110] Pharmaceutically acceptable salts can be prepared from parent compounds having basic or acidic functional groups by known chemical methods. Generally, such salts are prepared by reacting the free acidic or free basic form of a compound with a stoichiometric amount of a suitable base or acid in water or an organic solvent, or a mixture thereof. The salt may be prepared in situ during the final isolation or purification of the compound, or it may be prepared by separately reacting the free acidic or free basic form of the compound with the desired corresponding base or acid and then isolating the salt thus formed. The term "chemically acceptable salt" also includes amphoteric compounds in which cationic and anionic groups are covalently bonded within the same molecule, as these are "internal salts."

[0111] use Dynamic contrast-enhanced MRI (DCE-MRI) analyzes the contrast enhancement pattern of tissue over time after administration of a paramagnetic contrast agent. This is achieved by acquiring a baseline image without contrast enhancement, and then acquiring a series of images over time during and after the arrival of the contrast agent in the target tissue. The acquired signals are used to generate an intensity-versus-time curve of the tissue, which reflects the tissue's response to the arrival of the contrast agent as an enhancement value. Dynamic contrast-enhanced MRI (DCE-MRI) is used in the study of a wide range of pathological conditions, including cardiac diseases (particularly myocardial infarction), stroke and other brain diseases, various neoplasms with an emphasis on anti-angiogenic therapy and early detection, and evaluation of the peripheral vascular and musculoskeletal systems.

[0112] Most commonly, gadolinium (Gd) chelates are used as contrast agents in dynamic contrast-enhanced MRI (DCE-MRI). However, Gd chelate contrast agents are not ideal for many applications. Free gadolinium (Gd 3+Gadolinium is known to be toxic and needs to be strongly complexed with a ligand for use in humans. There are concerns about the potential toxicity of gadolinium complexes in patients with renal failure, particularly the risk of inducing nephrogenic systemic fibrosis (NSF) in patients with impaired renal function. Therefore, there is a need for contrast agents with higher stability and / or lower toxicity for DCE-MRI use in clinical settings.

[0113] The applications of the compounds and compositions of this technology are not limited and can be used as contrast agents for imaging, for example, in contrast-enhanced magnetic resonance imaging (CE-MRI) and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Therefore, in certain embodiments, the compounds described herein are contrast agents used for imaging such as magnetic resonance imaging (MRI), contrast-enhanced magnetic resonance imaging (CE-MRI), and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). It is understood that any suitable magnetic resonance imaging (MRI) or other imaging technique may be used in combination with the compounds and compositions described herein (see, for example, Westerbrook et al., MRI in Practice Ed., Blackwell Publishing, Oxford, UK, 2005, the contents of which are incorporated herein by reference). Furthermore, this method can be carried out under any magnetic field strength. In some embodiments, the magnetic field strength may be about 0.1 T to about 30 T, for example, 3 T. In these magnetic fields, the frequency of the electromagnetic waves required to excite the electron spin transition of the unpaired electrons in the polarizer ranges from approximately 2.8 GHz to approximately 840 GHz. For example, the radiation may originate from a 140 GHz gyrotron.

[0114] Chronic kidney disease (CKD) continues to be a significant healthcare burden internationally. CKD often progresses slowly in its early stages without obvious symptoms, but in later stages, the condition becomes progressively more severe with limited reversal potential. While often attributed to long-term hypertension and diabetes, it can also occur after acute kidney injury (AKI), resulting from a rapid and severe decline in kidney function.

[0115] The outcomes of chronic kidney disease (CKD) are improved by earlier intervention made possible by earlier detection. In North America, the clinical diagnosis of chronic kidney disease (CKD) is defined as an estimated glomerular filtration rate (eGFR) of 60 mL / min / 1.73 m². 2 It is defined as a condition of less than 3 months persisting, or a condition of urinary albumin to creatinine (ACR) greater than 30 mg / g persisting for more than 3 months. Because these diagnostic values ​​are derived from large-scale clinical studies conducted on ethnically limited populations, the diagnostic power of these disease biomarkers is significantly diminished. Furthermore, the underlying causes of chronic kidney disease (CKD) vary among individuals, with the most common being diabetes, cardiovascular disease, and kidney transplantation, which can limit the accuracy of eGFR measurement at the patient level. This is because approximately 30% of these patients may show a 30% deviation from their true eGFR value. In addition to individual deviation from the derived population, such large variability arises because eGFR estimation formulas assume steady-state creatinine concentration and do not consider changes in creatinine production or alternative pathways. Developing more accurate glomerular filtration rate (GFR) estimation methods that are independent of race and other patient demographic characteristics is an urgent task. Most importantly, these values ​​fail to provide physicians with the spatial or structural information underlying renal dysfunction. Renal biopsy can provide histopathological data that can predict the outcome of chronic kidney disease, offering spatial data on specific renal lesions as well as overall renal function. However, biopsy is an invasive procedure with inherent risks and cannot be repeatedly used to spatiotemporally characterize kidney disease. Clinically, GFR (glomerular filtration rate) remains the gold standard indicator of renal function, and its measurement requires a reliable, rapid, and / or quantitative clinical imaging approach.

[0116] In certain embodiments, the compounds and compositions described herein provide metal-free contrast agents for biomedical imaging. In certain embodiments, the compounds and compositions described herein provide metal-free alternatives to Gd-based contrast agents to facilitate the implementation of CE-MRI and / or DCE-MRI. In certain embodiments, the compounds and compositions described herein are selectively taken up by the kidney and are therefore used for biomedical imaging of the kidney. However, it should be understood that the uses of the compounds and compositions described herein are not intended to be particularly limited. For example, the compounds and compositions may be used for imaging other tissues, depending on the uptake or distribution of the compound after administration to the subject, and other considerations that determine suitability for a particular use.

[0117] In certain embodiments of the imaging methods of this disclosure, imaging of the kidney is possible. Such methods may, but are not limited to, provide quantitative and / or qualitative renal function information, such as the determination and / or mapping of the glomerular filtration rate (GFR) in a subject. In certain embodiments, such imaging is free from toxicological concerns related to conventional contrast agents in patients with renal impairment and / or does not require the use of the patient's race or other demographic characteristics.

[0118] In certain embodiments, a biomedical imaging method is provided which includes administering a contrast agent to a subject and imaging the contrast agent within the subject, wherein the contrast agent includes compounds or compositions described herein. The biomedical imaging may be magnetic resonance imaging (MRI), such as, for example, contrast-enhanced magnetic resonance imaging (CE-MRI) or dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). In certain embodiments, biomedical imaging is used to image the kidney. In some such embodiments, imaging is used to monitor or evaluate the renal function of a subject. In some such embodiments, imaging is used to determine and / or map the glomerular filtration rate (GFR) in the subject. Thus, the method can provide quantitative and / or qualitative renal function information, including, for example, the glomerular filtration rate (GFR) of a subject.

[0119] In one embodiment, a method is provided for measuring the renal function of a subject. The method includes the steps of administering a contrast agent to a subject and imaging the contrast agent within the subject, wherein the contrast agent comprises a compound or pharmaceutical composition described in this disclosure, and further includes the steps of determining and / or mapping the glomerular filtration rate (GFR) of the subject.

[0120] In certain embodiments, a method is provided for diagnosing renal impairment in a subject, comprising administering a contrast agent containing a compound or composition described in the Disclosure to the subject, imaging the contrast agent within the subject, and determining and / or mapping the glomerular filtration rate (GFR) of the subject.

[0121] In certain embodiments, a method is provided for diagnosing chronic kidney disease (CKD), acute kidney injury (AKI), renal artery stenosis, urinary tract obstruction, and / or renal tumor or renal malignancy in a subject, the method comprising administering a contrast agent to the subject, imaging the contrast agent within the subject, and determining and / or mapping the glomerular filtration rate (GFR) of the subject, wherein the contrast agent includes compounds or compositions described herein.

[0122] In certain embodiments, a method for determining whether a subject meets the requirements for a kidney donor includes the steps of administering a contrast agent to the subject and imaging the contrast agent within the subject, wherein the contrast agent comprises a compound or composition described herein, and further includes the steps of determining and / or mapping the subject's glomerular filtration rate (GFR).

[0123] In certain embodiments of the methods described herein, the subject has, is suspected of having, or is at risk of having renal impairment. The subject may have, be suspected of having, or be at risk of having any of the following: chronic kidney disease (CKD), acute kidney injury (AKI), renal artery stenosis, urinary tract obstruction, renal tumor, or renal malignancy. The subject may be a kidney donor or a kidney donor candidate.

[0124] kit This disclosure also provides kits containing the compounds or compositions described herein. The kits generally take the form of physical structures housing various components and can be used, for example, in carrying out the methods described herein. For example, a kit may contain one or more compounds or compositions described herein (for example, provided in sterile containers), and such compounds or compositions may be in the form of pharmaceutical compositions suitable for administration to a subject. The compounds or compositions may be provided in a ready-to-use form, or in a form requiring reconstitution or dilution before administration (for example, a powder). If the compounds or compositions are in a form requiring reconstitution or dilution by the user, the kit may also include diluents (for example, sterile water), buffers, pharmaceutically acceptable excipients, etc., packaged together with or separately from the compounds or compositions. Each component of the kit may be housed in an individual container, and all of these containers may be housed in a single package. The kit of the present invention may be designed to meet the conditions necessary for properly preserving the components housed inside it (e.g., refrigeration or freezing).

[0125] The kit may further include a label or packaging leaflet containing identification information for the components included in the kit and instructions on how to use them. The label or packaging leaflet may include manufacturer information such as lot number and expiration date. The label or packaging leaflet may be, for example, integrated into the physical structure containing the components, housed separately within the physical structure, or affixed to the components of the kit (e.g., ampoules, tubes, or vials).

[0126] Examples The present invention will be more readily understood by referring to the following embodiments. These embodiments are provided for illustrative purposes and should not be construed as limiting the scope of the invention in any way.

[0127] Unless otherwise specified or unless the context clearly indicates otherwise, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. It should be understood that any methods and materials similar or equivalent to those described herein may be used in carrying out or testing the present invention.

[0128] Example 1. Synthesis of Modified Verdazyl Compounds Various compounds represented by structural formula (I) can be prepared according to Scheme 1 below, which shows a general verdazil synthesis scheme including modifications of R1 (bonded to the nitrogen atoms at positions 1 and 5 of the verdazil ring) and R2 (bonded to the carbon atom at position 3 of the same ring). In Scheme 1, the reaction conditions are as follows: (a) 15% phosgene solution in toluene, 0°C to 20°C, overnight; (b) HCl in a miscible solvent, reflux temperature, 2 hours; (c) R2 group with an aldehyde and a non-nucleophilic base (2 equivalents) in a miscible solvent, room temperature, overnight; (d) potassium ferricyanide and sodium bicarbonate in a polar solvent, until foaming stops. The synthesis procedure shown in Scheme 1, which produces intermediate 1 in the process leading to the synthesis of formula (I), (II), or (III), can be carried out using diphosgene, triphosgene, carbonyldiimidazole, disuccinimidyl carbonate, and bis(pentafluorophenyl) carbonate in addition to phosgene. [ka] Scheme 1. Synthesis of modified verdazyl compounds in specific embodiments

[0129] Various compounds of structural formula (I) can also be prepared according to Scheme 2 below, which shows a general scheme for verdazil synthesis, starting with compound 1 produced as described in Scheme 1, and modifying R1 (bonded to nitrogen at positions 1 and 5 of the ring) and R2 (bonded to carbon at position 3 of the ring). In summary, a large quantity of repeating carbon chains having alcohol groups protected with polyethylene glycol polymer or PMB of arbitrary length is synthesized, oxidized to an aldehyde, the verdazil ring is cyclized using the aldehyde, and then deprotected to obtain modified verdazil compounds having free alcohols. In the initial step, any group having an aldehyde group at one end and a protected functional group containing a heteroatom (e.g., a protected alcohol, a protected amine, a protected thiol, a masked acid, etc.) at the other end can be used. Next, the alcoholic hydroxyl group (OH) can be converted into any leaving group or reactive site that reacts with a nucleophile, thereby allowing any functional group having an N, S, or O nucleophile to be attached.

[0130] In Scheme 2, the reaction conditions are as follows: (a) H2O / acetonitrile, NaOAc, and the following formula: [ka] [ka] (or any group having an aldehyde at one end and a protected heteroatom at the other end may be used), (b) HCl in a miscible solvent, reflux temperature, for 2 hours, (c) appropriate solvent and conditions selected according to the particular reaction, (d) potassium ferricyanide and sodium bicarbonate in a polar solvent until foaming stops. [ka] Scheme 2. Synthesis of modified verdazyl compounds according to a specific embodiment.

[0131] Example 2. Optimized target-directed synthesis of glucoverdazil The synthesis of glucoverdazil is outlined in Scheme 3 below. By combining it with the previously reported 6-oxoverdazil synthesis method, we identified an optimized synthetic route for glucoverdazil that achieves the high molecular purity and scalability required for in vivo contrast agents. The hydrazine side chains reported in previous literature were usually limited to short carbon chains or aryl groups (Patra et al., 2016; Pare et al., 2005; Solea et al., 2018; Barclay et al., 2002; Calabretta et al., 1991; References 33, 35-38). The side chain was modified with an isopropyl group. The isopropyl group introduced into the side chain is bulky enough to protect delocalized radicals and also improves serum retention after injection. In previous synthesis, N-Boc isopropylhydrazine was obtained in large quantities from the N-Boc hydrazine precursor according to the synthesis method reported by Calabretta et al. (Kumar et al., 2018). However, in this case, commercially available isopropyl BOC hydrazine was used. Compound 2 has been synthesized by several reported methods, all of which involve the disubstitution reaction of COCl2 using phosgene solution or solid triphosgene (Solea et al., 2018; Le et al., 2016). Glucoverdazil was synthesized using both forms of phosgene, and similar results were obtained in both cases. Here, a 15% phosgene solution in toluene was selected. As first reported by Pare et al. (Solea et al., 2018), a synthetic route with excellent yield and purity was obtained by recrystallizing the crude product after the phosgene step with heptane. Boc deprotection was performed in ethanolic hydrochloric acid to form an intermediate compound, and then a non-radical tetradinanon ring (compound 3) containing D-glucose was generated in the same manner as first reported by Le et al. (2016). Finally, following Le et al., the oxidation of compound 3 was also carried out using potassium ferricyanide. Potassium ferricyanide is a far milder oxidizing agent than benzoquinone, which has been classically used in most existing Verdazil-related literature, and is also easier to purify.

[0132] Many of the previous studies reporting these reactions were insufficient in terms of characterization. Here, high purity 1 H and 13 In addition to the 1C spectrum, high-resolution mass spectrometry of the corresponding synthetic intermediates at each step is also reported (Figures 7-12). Purity measurements of compound 4 by EPR spectroscopy and analytical high-performance liquid chromatography demonstrated that this approach to glucoverdazil completely converted non-radical compound 3 to compound 4 (Figure 13). [ka] Scheme 3. Overview of the synthesis of Glucoverdazil 4

[0133] Example 3. Characterization of glucoverdazil as a contrast agent with MRI activity. Refer to Figures 1A to 1F to measure the paramagnetic properties and stability of glucoverdazil.

[0134] For stability testing of glucoverdazil, the EPR instrument was calibrated using samples of glucoverdazil or TEMPO in phosphate-buffered saline (PBS) prior to stability testing. After calibration, solutions of glucoverdazil or TEMPO were prepared (20 mM in mouse serum, or 5 mM in 4 mM sodium ascorbate buffer at pH 7.4). A single spectrum was obtained, and the height of the highest intensity peak was fixed for each compound. Subsequently, EPR measurements were performed every 5 seconds for 2 hours (mouse serum) or 1.5 hours (ascorbic acid) to measure the rate of change in activity. For stability testing of glucoverdazil in water, 5 mM samples were prepared and left standing in a fume hood exposed to light, or wrapped in aluminum foil and left standing in a dark refrigerator at 4°C. These solutions were sampled periodically, and after calibrating the EPR with a newly prepared 5 mM glucoverdazil sample, EPR measurements were performed.

[0135] The EPR spectrum of glucoverdazil was consistent with previous reports, and multiple EPR peaks characterized highly delocalized radicals within the tetradinanon ring (Figure 1A; Massolle et al., 2018; Tain et al., 2017). The identification of these free radicals suggested that glucoverdazil may exhibit contrast enhancement in MRI (Wahsner et al., 2019). Magnetic resonance imaging (MRI) of a glucoverdazil solution phantom in phosphate-buffered saline (PBS) showed a twofold increase in T1 contrast, while the T2 effect remained unchanged compared to water (Figure 1B). The relaxation ability of glucoverdazil was lower than expected compared to the values ​​reported for GBCA, but the contrast effect was comparable to other previously reported organic radical compounds, with a longitudinal relaxation ability (r1) of 0.30 mM. -1 s -1 ±0.3mM -1 s -1 (Figure 1C; Le et al., 2016; Matsumoto et al., 2022; Chevalier et al., 2009). Contrast enhancement was similar to that of TEMPO, but the tetradinanon radical was substantially more stable than its nitroxy ORCA counterpart (Figures 1D and 1E). Neither glucoverdazil nor TEMPO showed any change in radical activity in mouse serum (Figure 1D). On the other hand, in the presence of ascorbic acid, a mild biological reducing agent, no decrease in the glucoverdazil radical was observed, whereas the nitroxy radical of TEMPO was completely reduced (Figure 1E).

[0136] The time-dependent stability of the glucoverdazil radical in solution was evaluated by storing the solution under direct light at room temperature or in complete darkness at -20°C. Periodic EPR measurements were performed on these solutions. When stored on a benchtop, glucoverdazil maintained over 50% of its radical activity even after 4 months, and in frozen solutions, it maintained over 80% of its radical activity even after 1 year (Figure 1F). These results demonstrate not only the reduction resistance of delocalized glucoverdazil radicals under bioreducing conditions, but also the important properties of shelf life and storage stability for commonly used MRI contrast agents.

[0137] Prior to in vivo experiments, the cytocompatibility of glucoverdazil was evaluated in H460 lung cancer epithelial cells. The results showed no cytotoxicity compared to untreated cells at concentrations up to 10 mM (Figure 15).

[0138] To evaluate the cell viability of H460 cells, large cell lung cancer cells (H460) were cultured in RPMI-1640 (RPMI) medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P / S) until confluence reached 80%, at which point they were subcultured. After three subculturings, the cells were seeded in 6-well plates and cultured until 80% confluence was reached. For each condition, cells were seeded in three wells. Subsequently, the cells were cultured for 4 or 24 hours in standard medium supplemented with glucoverdadil concentrations of 0 mM, 2.5 mM, 5 mM, or 10 mM. At each time point, the medium was aspirated and the cells were washed three times with Dulbecco's phosphate-buffered saline (PBS) at 37°C. Subsequently, the cells were detached with trypsin-EDTA, centrifuged at 400×g (5 min, 4°C), and the supernatant was aspirated and removed. The cell pellet was then resuspended in 1 mL of phosphate-buffered saline (PBS) containing 0.2 μM calcein acetoxymethyl ester (calcein-AM, which fluorescently stains live cells green) and 16 μM ethidium homodimer-1 (which fluorescently stains dead cells red). By flow cytometry (Beckman Coulter Galios flow cytometer), using 488 nm excitation, the live and dead cell populations were counted using a 525 nm / 40 nm bandpass filter for calcein acetoxymethyl ester (live cells, green) and a 620 nm / 20 nm bandpass filter for ethidium homodimer-1 (dead cells, red). After the procedure, the viable cell population under each condition was determined by comparing the total number of single-stained calcein AM-positive cells with the total number of single-stained cells that were either live or dead.

[0139] The suitability of glucoverdazil as an in vivo contrast agent was evaluated after intravenous administration to nine BALB / c mice. The dose was set at 3 mmol / kg, based on the difference in r1 values ​​between glucoverdazil and gadovist® (approximately 10-fold) and the clinically recommended standard dose of gadovist® (0.1 mmol / kg). This dose of glucoverdazil was still significantly lower than the maximum concentration used to evaluate cytocompatibility. Baseline voxel intensity was set by pre-injection scans, and T1-weighted images were acquired every 3 minutes after injection. Contrast enhancement was limited in muscle and liver, and uptake and clearance were clearly limited to the urinary tract (Figure 2A). Overall, 5 minutes after administration, signal intensity changes of 127%±9% in muscle, 121%±10% in liver, and 184%±21% in kidney were observed compared to the pre-contrast scan (Figure 2B). The mean post-administration clearance time, determined by the return of the renal region of interest (ROI) to baseline intensity, was approximately 40 minutes, coinciding with the point at which the increase in bladder ROI signal plateaued. At this point, signal changes of 120%±8% were observed in muscle, 115%±7% in liver, 127%±8% in kidney, and 438%±48% in bladder. The clearance dynamics of glucoverdazil across the kidney were consistent with monophasic exponential decay, and the slope of the semi-logarithmic plot using natural logarithms indicated the renal decay time constant (RDTC, k, unit: min) for glucoverdazil clearance in renal tissue. -1 We were able to determine the (Figure 2C) value. Linear regression was performed consistently from the maximum contrast intensity at t=2.5 min to t=40 min, where the return to baseline was most consistent. The mean k value obtained from 9 healthy BALB / c mice was -0.124 min. -1 ±0.012 min -1 So, the average R 2 The value was 0.97 ± 0.05, demonstrating excellent reproducibility of this baseline measurement in healthy renal function.

[0140] Since it was observed that glucoverdazil is mainly taken up by the kidney, a cytocompatibility test using high concentrations of glucoverdazil was repeated in human renal proximal tubular cells (hRPT, Figure 16A, B).

[0141] To evaluate the viability of human renal proximal tubular (hRPT) cells, hRPT cells were cultured in epithelial cell medium (EpiMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (P / S), and epithelial cell proliferation supplement (EpiCGS) until confluence reached 80%, at which point they were subcultured. The cells were subcultured three times before seeding into 6-well plates until 80% confluence was reached. For each condition, cells were seeded into three wells. The cells were then incubated for 4 or 24 hours in standard medium, or in standard medium supplemented with 10 mM glucoverdazil, 10 mM 5,5-dimethyl-1-pyrroline-N-oxide (DMPO, nitron spin trap), or 10 mM 2,2,6,6-tetramethylpiperidine-1-yloxyl (TEMPO, nitroxy radical). The culture medium was aspirated and removed at each time point, and the cells were washed three times with Dulbecco's phosphate-buffered saline (PBS) at 37°C. Then, the cells were detached with trypsin-EDTA, centrifuged at 400×g (4°C for 5 minutes), and the supernatant was aspirated and removed. The cells were then resuspended in 1 mL of phosphate-buffered saline containing 0.2 μM calcein acetoxymethyl ester (fluoresces green on living cells) and 16 μM ethidium homodimer-1 (fluoresces red on dead cells).

[0142] Using flow cytometry (Beckman Coulter Galios flow cytometer), with excitation light at 488 nm, a 525 nm / 40 nm bandpass filter was used for calcein acetoxymethyl ester (live cells, green), and a 620 nm / 20 nm bandpass filter was used for ethidium homodimer-1 (dead cells, red) to count the populations of viable and dead cells. After the procedure, using Kaluza analysis software (Beckman Coulter), the viable cell population for each condition was determined by comparing the total number of single-stained calcein AM-positive cells with the total number of cells that were positive for single-staining as either live or dead cells.

[0143] Glucoverdazil did not show a significant increase in cell death compared to untreated cells even after 24 hours of culture, whereas in the presence of TEMPO, over 90% of hRPTs were killed after only 4 hours of culture. This clear difference in cytocompatibility highlights another important performance difference between TEMPO and the tetradinanon derivative ORCA.

[0144] Intracellular uptake of glucoverdazil by hRPT cells was evaluated by EPR spectroscopy. To evaluate glucoverdazil uptake in hRPT cells, hRPT cells were cultured in epithelial cell medium (EpiMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (P / S), and epithelial cell proliferation supplement (EpiCGS) until confluence reached 80%, at which point they were subcultured. After three subculturings, the cells were seeded in 6-well plates and cultured until confluence reached 80%. Cells were seeded in three wells (triplicates) for each condition. Subsequently, the cells were cultured for 24 hours in normal medium or medium supplemented with 10 mM glucoverdazil. The medium was aspirated and the cells were washed three times with Dulbecco's phosphate-buffered saline (PBS) at 37°C. Subsequently, the cells were detached with trypsin-EDTA, centrifuged at 400×g at 4°C for 5 minutes, and the supernatant was aspirated and removed. The cells were then resuspended in 100 μL of phosphate-buffered saline (PBS). The concentrated cell solution was transferred to an EPR tube. To determine the cell count in each solution, 1 μL aliquots were taken and diluted. The EPR instrument was adjusted to match a freshly prepared 5 mM solution of glucoverdazil in phosphate-buffered saline (PBS), and the samples were then measured by EPR spectroscopy. The concentration was measured based on a predetermined standard curve, and then normalized by a predetermined cell count to obtain the glucoverdazil (nM) per cell.

[0145] As a result, no detectable levels of glucoverdazil uptake were observed (Figure 17). The low uptake of glucoverdazil in extraurinal tissues, the good reproducibility of contrast agent excretion from the kidney to the bladder, the relatively short renal clearance time of glucoverdazil, and the demonstrated cytocompatibility suggest that glucoverdazil may be suitable for renal function assessment based on magnetic resonance imaging.

[0146] Example 4. DCE-MRI with glucoverdazil contrast for acute kidney injury due to unilateral ureteral obstruction (UUO). The efficacy of glucoverdazil as a DCE-MRI contrast agent for acute kidney injury (AKI) due to obstructive nephropathy was evaluated using a mouse model of unilateral ureteral obstruction (UUO). Surgical obstruction of the left ureter hinders urine excretion, causing hydronephrosis and significantly reducing the renal function of the ipsilateral kidney. Both a sham surgery group (only contact with surgical instruments was made with the left kidney) and a surgically unilateral ureteral obstruction group (UUO, ligation of the left kidney) were evaluated. Time-course changes in voxel intensity within the kidney were calculated for the entire left and right kidneys (Figures 3A-3D), as well as for the cortical and medullary / renal pelvis (MRP) regions of each kidney (Figures 18A-18F). Voxel-level mapping of the renal decay time constant (RDTC) was used to evaluate changes in renal function. In the sham group, no significant changes in kidney morphology were observed between days 0, 3, and 7. On the other hand, in the unilateral ureteral obstruction (UUO) model mouse group, the morphology of the kidney on the ureteral obstruction side (ipsilateral) changed significantly on days 3 and 7 compared to day 0. Hydronephrosis became evident on day 3 as the disappearance of the medullary region and worsened further on day 7, which is a characteristic finding of the UUO model (Xiong et al., 2021). No significant morphological changes were observed in the kidney contralateral to the ligated ureter in UUO-treated mice.

[0147] Prior to analyzing data from diseased mice, the RDTC value (renal decay time constant) was determined in preoperative BALB / c mice to define the optimal time interval for glucoverdazil clearance (Figures 18A-18F). In sham-treated mice, no significant changes were observed in RDTC values ​​on days 3 and 7 compared to day 0, either graphically (Figure 3A) or quantitatively (Figure 3B) across both kidneys. However, in UUO-treated mice, a significant change was observed in the ipsilateral kidney's RDTC value, with k = -0.135 min on day 0. -1 ±0.018 min -1 Therefore, on the third day, k = -0.028 min -1 ±0.014 min -1 On the 7th day, k = -0.013 min -1 ±0.032 min -1The value increased to [value]. RDTC levels in the contralateral kidney of UUO-treated mice showed no change between day 0 and day 3 (k = -0.133 min, respectively). -1 ±0.020 min -1 and k = -0.117 min -1 ±0.014 min -1 ) However, it increased significantly on day 7 (k = -0.097 min -1 ±0.010 min -1 ). Changes in the physiological function of the contralateral kidney after unilateral ureteral obstruction (UUO) have been predicted in rodent models, and induction of macrophage-to-myofibroblast transition (Figueroa et al., 2019), fibrosis (Xiong et al., 2021; Bianco et al., 2019), and changes in mitochondrial function in the cortex (Eddy et al., 2012) have already been reported. These data demonstrate that glucoverdadil-mediated DCE-MRI can detect renal dysfunction in the contralateral kidney early after ipsilateral ureteral obstruction (UUO).

[0148] Histological analysis and serum creatinine (SCr) analysis were performed on mice in both groups, and the pathological conditions observed in qualitative MRI images and renal decay time constant (RDTC) maps were confirmed. Renal function was also evaluated using the gold standard method. Morphology and fibrosis staining of the kidneys of the sham surgery group and the ipsilateral kidney of the UUO-treated mice showed no significant abnormalities. However, clear hydronephrosis was observed in the kidney ipsilateral to the ureteral obstruction (Figure 3C). No significant changes in serum creatinine levels were observed in sham surgery mice between 0 and 7 days postoperatively, but a significant increase was observed in unilateral ureteral obstruction (UUO)-treated mice (Figure 3D). These observed changes in serum creatinine (SCr) are in good agreement with findings previously reported in the UUO model and reproduce 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 observed in SCr supports the use of RDTC as an indicator of renal function in glucoverdazil-mediated DCE-MRI.

[0149] While ipsilateral kidney damage due to ureteral obstruction was evident on anatomical MRI, the UUO model, using standard glucoverdazil-mediated DCE-MRI techniques with simplified kinetic mapping, demonstrated the ability to show both local and structural abnormalities across both kidneys, suggesting functional changes occurring in the contralateral kidney even before fibrosis staining became positive. Region-specific analyses were performed to identify glucoverdazil clearance in the cortex, medulla, and renal pelvis (Figures 18A-18F). Here, impaired glucoverdazil clearance due to renal dysfunction was clearly identified, and the sites of pathological manifestation within the kidney were simultaneously mapped. This is extremely useful for evaluating acute kidney injury (AKI) (Matsumoto et al., 2022; Chevalier et al., 2009; Xiong et al., 2021).

[0150] Example 5. DCE-MRI with glucoverdadil enhancement of the transition from acute to chronic kidney injury mediated by folate-induced nephropathy (FAN). Next, we attempted to evaluate renal function in a more complex fibrosis-driven kidney disease model mediated by folate-induced nephropathy (FAN). In addition to RDTC values ​​and AUC, supporting histological evaluations and serum creatinine levels (SCr) were also assessed (Figures 4A-D). FAN is caused by folate crystals formed in the renal tubules after systemic administration of folate (Jiang et al., 2018). This crystallization leads to the initial phase of severe acute kidney injury (AKI), followed by renal scar formation with fibrosis, which causes a long-term progressive decline in renal function, leading to chronic kidney disease (CKD) approximately 3 weeks after folate injection. We conducted the folate-induced nephropathy (FAN) model using BALB / c mice, rather than the commonly used C57BL / 6 mice. This is because C57BL / 6 mice have an extremely high mortality rate during the acute kidney injury phase, while BALB / c mice have high resistance to this phase.

[0151] Anatomical imaging revealed a decrease in overall kidney size in both kidneys from day 0 to day 30. This finding has already been reported in a folate-induced nephropathy (FAN) model (Doi et al., 2006; Figure 4A). Following glucoverdazil-mediated DCE-MRI, RDTC levels were measured in both kidneys before folate administration and at 15 and 30 days after administration (shown as a graph in Figure 4A and quantitatively in Figure 4B). A significant increase in RDTC was observed on day 15 (k = -0.154 min on day 0). -1 ±0.025 min -1 On the 15th day, k = -0.082 min -1 ±0.008 min -1 ), and then, on day 30, RDTC returned to baseline values ​​(k = -0.139 min). -1 ±0.016 min -1 In the acute kidney injury (AKI) phase of folate-induced nephropathy (FAN), RDTC levels in the medulla and cortex were significantly increased, indicating both poor ureteral outflow and glomerular filtration. In the early stages of chronic kidney disease (CKD), recovery of RDTC levels is expected by day 30. This is because acute kidney injury (AKI) often presents with much more severe renal impairment than the early stages of CKD, and this finding is consistent with literature using the same post-injury time point (Doi et al., 2006). The RDTC map at day 30 showed localized striped areas with relatively high RDTC in the cortex (Figure 4A right, black arrow). This maps the striped structure of fibrosis in the kidney at day 30, as confirmed by histological evaluation (Figure 4C, light blue areas indicated by black arrows are indicators of fibrotic tissue). Therefore, by integrating spatial and dynamic information into a single renal map, as demonstrated in the RDTC image (Figure 4A), an improvement in diagnostic accuracy can be achieved that cannot be obtained with existing nephrological methods that are limited to spatial or dynamic information alone.

[0152] Kidneys were removed at 0, 15, and 30 days after folic acid injection, and histological evaluation was performed to confirm both acute kidney injury (AKI) and chronic kidney disease (CKD) (Figure 4C). In addition, blood samples were taken at the same time points (0, 15, and 30 days) to measure serum creatinine (SCr) (Figure 4D). Histological evaluation revealed that the fibrotic area in the kidney had significantly increased by 15 days, while its severity had decreased by 30 days (Figure 4C). Serum creatinine (SCr) showed a slight increase from 0 days to 15 days, which was expected as such, as a slight increase in SCr is usually seen in acute kidney injury (AKI) (Doi et al., 2006; Waikar et al., 2009). However, at day 30 in the early stages of chronic kidney disease (CKD), serum creatinine levels were significantly elevated compared to days 0 and 15, which is a strong indicator of severe renal dysfunction associated with folate-induced nephropathy (FAN) and the early stages of CKD (Aparicio-Trejo et al., 2020; Yam, 2021; Scarfe et al., 2018). Evaluation of the FAN model highlighted the importance of applying a renal assessment method that combines spatial and temporal evaluations. In severe acute kidney injury (AKI), a mild increase in serum creatinine levels (SCr) was observed, but the renal decay time constant (RDTC) value increased significantly, with differential effects across regions within the kidney (Figure 4A). The RDTC map has unique utility in showing discrete regions (i.e., cortical striae) with more severe renal impairment, which may correspond to fibrotic areas observed in histological evaluation (Figure 4C).

[0153] Example 6. Comparison of DCE-MRI via glucoverdazil with validated glomerular filtration rate (GFR) measurement methods. While measuring glomerular filtration rate (GFR) has extremely high clinical value, current measurement methods are limited to measuring dynamic indicators of renal function and rely on techniques prone to errors. To develop an image-based approach for assessing renal function, a comparison was attempted between glucoverdazil-mediated DCE-MRI and transcutaneous fluorescence monitoring, an established and validated GFR measurement method. Transcutaneous GFR measurement is based on intravenous administration of a fluorescent molecule (e.g., FITC-sinistrin) that is cleared only by filtration, and transcutaneous monitoring of fluorescence in the blood pool over time. In transdermal absorption technology, a monophase exponential decay model is applied to determine the RDTC value of the fluorescence intensity versus time curve, and then converted to glomerular filtration rate (GFR) using a predetermined correction factor (Schreiber et al., 2012; Friedemann et al., 2016; Shmarlouski et al., 2017; Scarfe et al., 2018; Schock-Kusch et al., 2013). Transdermal GFR measurements were used as a basis for deriving the glucoverdadil-specific correction factor.

[0154] In the second cohort of FAN mice, transdermal administration (Figure 5A) and DCE-MRI via glucoverdazil (Figure 5B) were performed on day 0 (black), day 15 (pink), and day 30 (cyan). Both methods showed characteristic monophase decay curves, and RDTC values ​​were calculated after quasi-logarithmic transformation based on natural logarithms. On day 0, the mean RDTC value measured by DCE-MRI via glucoverdazil was k = -0.135 min -1 ±0.022 min -1 ) is the RDTC value (k=-0.075min) measured by transcutaneous fluorescence. -1 ±0.011 min -1) was significantly different (Figure 14). These differences in RDTC values ​​were not surprising given the different data sampling locations. Transcutaneous measurement evaluates signals originating from the blood pool within a few millimeters of the skin surface, while dynamic contrast-enhanced MRI (DCE-MRI) evaluates signal clearance in the kidney tissue itself. With the transcutaneous absorption technique, the baseline glomerular filtration rate (GFR) was 1584 ± 238 μL / min / 100g body weight. The values ​​measured in BALB / c mice were consistent with reported literature values ​​(Yan et al., 2021). Baseline RDTC data obtained from BALB / c mice on day 0 were pooled, and a glucoverdadil-specific correction coefficient for converting RDTC to GFR was derived using the mean GFR value obtained by the transcutaneous absorption technique. Using this coefficient, GFR was calculated from the tRDTC value at each post-injury time point and compared with the GFR value obtained by the transcutaneous absorption technique (Figure 5C). No significant difference was found between the GFR values ​​obtained by the two methods.

[0155] In conclusion, the preparation of glucoverdazil, an ORCA derived from tetradinanone, was optimized and scaled up, demonstrating superior redox stability and cytocompatibility compared to conventionally used nitroxy radical contrast agents. Because glucoverdazil's specific uptake is limited to the kidney, ureter, and bladder, it is considered particularly useful for DCE-MRI in the kidney. Glucoverdazil was applied to imaging a UUO model of severe acute kidney injury and a FAN model progressing from acute kidney injury to chronic kidney disease, and local changes in renal function within the kidney were indicated as renal decay time constants (RDTC). Voxel-level mapping of AUC in both models did not correlate reliably with histological changes and changes in serum creatinine levels (SCr) as well as RDTC mapping, which fully supported renal injury. Benchmarking against validated transcutaneous fluorescence GFR measurement methods, glucoverdazil demonstrated reliable GFR determination by DCE-MRI. Importantly, this approach to GFR measurement not only adds spatial information to the gold standard nephrological assessment, but also allows for GFR determination without relying on patient demographic characteristics, which have been shown to be prone to error. Overall, glucoverdazil may enable safer MRI-based diagnosis in patients with known or suspected acute kidney injury (AKI) and / or chronic kidney disease (CKD). Given the molecular properties of this organic radical, particularly its non-intracellular uptake, excellent cytocompatibility and preliminary biocompatibility, renal localization, and rapid clearance, tetradinanone-based ORCA constitutes a novel and promising class of metal-free MRI contrast agents.

[0156] Experimental methods in Examples 1-6 Common reagents:All chemical reagents were purchased from Sigma-Aldrich and used as is unless otherwise noted. Only N'-(propan-2-yl)(tert-butoxy)carbohydrazide was purchased from AABlocks. All cell culture reagents and consumables were purchased from ThermoFisher. However, epithelial cell medium and epithelial cell growth supplements were purchased from ScienCell.

[0157] EPR spectrum: All EPR spectra were acquired at room temperature using a Bruker EMXplus EPR spectrometer. All NMR spectra were measured using a Bruker AVANCE II 400 or Bruker AVANCE III HD 600. All MRI imaging was performed using a 3T preclinical MRI system (MR Solutions, Ltd.). In the analysis of all MRI image data, only slices depicting the target tissue were included in the analysis (i.e., in kidney imaging, only slices depicting the kidney were used). All data processing, mapping, and calculation of various quantities were performed using programs written in MATLAB® 2020A. GraphPad Prism 9.5 was used to create all graphs and figures and to calculate statistical analysis results.

[0158] Animal studies using in vivo magnetic resonance imaging (MRI):All animal experiments using in vivo magnetic resonance imaging (MRI) were conducted in accordance with the animal use protocol HIe-3640-R1 approved by the IACUC at the University of Ottawa. Mice were anesthetized with isoflurane and placed on a heated cradle before being inserted into the MRI machine. T1-weighted RARE images were acquired before contrast agent administration, and then continuously acquired every 2.5 minutes for 60 minutes after administration. T1-weighted imaging: slice thickness 1 mm, imaging field of view (FOV) 50 × 50 mm, mean 3, matrix size 96 × 96, TE 11 ms, echo interval 7 ms, TR 720 ms, acquisition time 2 minutes 16 seconds. In all cases, 3 mmol / kg of contrast agent was administered intravenously via a tail vein catheter, and the catheter was flushed with saline to ensure that the entire volume of contrast agent was administered.

[0159] MRI data processing and analysis:MRI data processing and analysis were performed as follows: (1) Intensity vs. Time curves. Relevant slices were adjusted to remove the effects of the automatic gain function associated with magnetic resonance imaging, and all scans and slices were normalized to a water-filled fiducial marker placed beside the mouse during all scans. Using MATLAB® routines, the region of interest (ROI) for each slice was plotted at each scan time point, and voxel-level intensity vs. time data was created. The obtained data was normalized so that the minimum intensity voxel of the first scan was set to 100%, and presented as the average normalized intensity of the entire ROI at each time point. (2) RDTC values ​​and image maps. The natural logarithm of the intensity values ​​of each region of interest (ROI) in the voxel-level intensity vs. time curves was taken to create a semi-logarithmic curve. Next, linear regression was applied in the range from t=2.5 min to t=40 min, and k (renal decay time constant (RDTC), min-1) was calculated from the slope of this curve at each voxel. The RDTC values ​​shown in the graph are the average RDTC values ​​per voxel. The k map was superimposed on the image acquired at t=0 minutes. (3) Area under the curve and image map. Baseline correction was performed by subtracting the minimum voxel value of each curve from the intensity-time curve for each voxel, and the baseline was set to 0 at all time points. For each voxel, the curve was integrated using the trapezoid rule (trapezoid function) to calculate the AUC (area under the curve) value. The AUC value shown in the graph is the average AUC value per voxel. The AUC map was superimposed on the image acquired at t=0 minutes. A schematic diagram of the DCE-MRI data acquisition and image mapping workflow is shown in Figure 6.

[0160] Evaluation of glucoverdazil's tissue localization using DCE-MRI: Glucoverdazil-enhanced MRI scans were obtained from nine healthy BALB / c mice as described above, and regions of interest (ROIs) were defined in the kidney, liver, bladder, and muscle tissue, and normalized intensity-versus-time curves were obtained as described above.

[0161] Serum creatinine measurement:For all disease models, blood was collected sequentially from the saphenous vein on day 0 and at each point after injury, prior to the performance of glucoverdazil-enhanced MRI. The collected blood was centrifuged for 10 minutes (room temperature, 900 × g) and the serum fraction was recovered. The samples were stored at -80°C until use. Serum creatinine (SCr) was measured using a quantitative HPLC method with a creatinine standard curve, with some modifications to a previously reported method (Bello et al., 2019) (Agilent 1260 Infinity with diode array detector, Agilent Zorbax 300-SCX column with inner diameter 2.1 mm × length 50 mm, particle size 5 μm). In summary, creatinine was dissolved in the HPLC mobile phase (15 mM sodium acetate buffer at pH 4.2 containing 4% methanol and 1% acetonitrile (AcN)), serially diluted, and the HPLC peaks detected at 234 nm were integrated using a Bruker HyStar PP to create a standard curve of creatinine from 0 μM to 12.5 μM. Analysis of standards and samples was performed under isocratic conditions with the mobile phase at a flow rate of 0.5 mL / min. To thawed mouse serum samples, a solution of acetonitrile (AcN) containing 0.5% acetate was added to the serum in a 4:1 ratio to precipitate proteins, and creatinine was extracted. The samples were vortexed and then allowed to stand at -20°C for 30 minutes to obtain complete precipitation and sedimentation. The samples were then centrifuged at 12,000 × g (4°C for 10 minutes), and the supernatant was transferred to a new tube. To remove acidified acetonitrile (AcN), the tubes were dried by heating and vacuum centrifugation at 50°C for 45 minutes. The resulting pellet was resuspended in 60 μL of mobile phase, and the sample was subjected to HPLC analysis. Elution peaks corresponding to the retention times observed on the standard curve were integrated after detection at 234 nm.

[0162] Animal models of kidney disease: For all mice in the disease models of unilateral ureteral obstruction (UUO) and folate-induced nephropathy (FAN), glucoverdazil-enhanced MRI was obtained as described above, and a region of interest (ROI) was set in the renal tissue, and normalized intensity-versus-time curves were also obtained as described above.

[0163] Unilateral ureteral obstruction (UUO):A mouse model of unilateral ureteral obstruction (UUO) in acute kidney injury (AKI) was created using C57BL / 6 mice according to the literature (Vanholder et al., 2021). In essence, 10 female C57BL / 6 (8 weeks old) mice were randomly assigned to either a sham surgery group (5 mice) or a unilateral ureteral obstruction (UUO) treatment group (5 mice). The mice were imaged using glucoverdazil as a contrast agent immediately before surgery (day 0). The mice were anesthetized by continuous inhalation of isoflurane. The left kidney of the mice was accessed laparoscopically, and the left ureter was either lightly touched with surgical instruments (sham surgery group) or obstructed by ligation with sutures (unilateral ureteral obstruction (UUO) group). The wound was sutured closed, and the mice were imaged by glucoverdazil-enhanced MRI on days 3 and 7 post-injury. On day 7, the mice were euthanized by cervical dislocation. The kidney was removed, fixed with paraformaldehyde, then tissue sections were prepared, stained with PAS, and images were acquired using a slide scanner.

[0164] Folic acid-induced nephropathy (glucoverdil-enhanced MRI):A folate-induced nephropathy (FAN) model for the transition from acute kidney injury (AKI) to chronic kidney disease (CKD) was implemented in BALB / c mice, following a modified version of a common procedure reported in the literature (Van Buren et al., 2011; Chawla et al., 2014; Levin et al., 2011; Chen et al., 2019; Gama et al., 2021). In CD1 and C57 / Bl6 mice, a folate (FA) dose of 250 mg / kg resulted in extremely high mortality. BALB / c mice have been shown to exhibit higher resistance to obstruction-induced injury and to induce chronic kidney disease (CKD) with higher reproducibility (Luis-Lima et al., 2017; Niemantsversriet et al., 2021). Five BALB / c mice were imaged on day 0 using glucoverdazil-enhanced MRI. Immediately after scanning, mice were intraperitoneally injected with folic acid (FA) dissolved in 0.3 M sodium bicarbonate solution at a dose of 125 mg / kg. The first five days after folic acid injection are the period when the effects of folic acid are strongest, and daily subcutaneous fluid administration was required. By day 7, the condition of the mice was stable and they could be maintained under normal housing conditions without fluid replacement. Mice were re-imaged by glucoverdazil-enhanced MRI on days 15 and 30 after injury. On day 30, the mice were euthanized by cervical dislocation. The kidneys were removed, fixed with paraformaldehyde, sections were prepared, stained with PAS, and images were obtained using a slide scanner. A group of mice that underwent the same disease-inducing treatment without any glucoverdazil-enhanced MRI were euthanized on day 15 to obtain histological evaluation at this point and to maintain the continuity of longitudinal MRI data in this disease model.

[0165] Folate-induced nephropathy (transcutaneous fluorescence):In a parallel group of six mice, renal disease was induced using the same method as the contrast-enhanced magnetic resonance imaging (CMRI) group, except that transcutaneous fluorescence was measured instead of magnetic resonance imaging as described in previous literature (Levey et al., 2020; Romagnani et al., 2017; Srivastava et al., 2021; Nikken et al., 2007; Warwick et al., 2022). In general, prior to any data acquisition, the fur on the right dorsal lateral side of each mouse was removed. The following day, a transcutaneous fluorescence monitor (MediBeacon, Inc.) was fixed to the shaved area using a special adhesive patch with a window. The battery was connected to the transcutaneous monitor, and a 5-minute baseline was established. FITC-sinistrin solution (150 μL, 0.2 mg / kg) was administered intravenously via the tail vein, and data were acquired over 55 minutes. Data collection on day 0 was performed immediately before intraperitoneal injection of folic acid, and this was repeated on days 15 and 30 after injury. At each time point, the mice were euthanized by cervical dislocation.

[0166] Conversion from RDTC value to glomerular filtration rate:Data obtained by transcutaneous fluorescence were analyzed using proprietary software (MediBeacon, Inc.), and RDTC and glomerular filtration rate (GFR) values ​​were calculated based on fitting to a pharmacokinetic model. GFR and RDTC values ​​were calculated using this software by applying a monophase decay model to uncorrected raw data. MediBeacon uses conversion coefficients to directly convert RDTC values ​​to glomerular filtration rate (GFR), which were predetermined based on mouse data created by MediBeacon and compared with GFR measured by conventional methods (Levey et al., 2020; Romagnani et al., 2017; Srivastava et al., 2021; Nikken et al., 2007; Warwick et al., 2022). Based on the consistency of RDTC values ​​in 14 healthy BALB / c mice, the conversion coefficient from glucoverdazil RDTC to GFR was derived based on the average GFR value of 6 healthy BALB / c mice measured by transcutaneous fluorescence. The mean GFR value was normalized using the difference obtained by comparing the pooled RDTC value with the transdermal RDTC value, and a conversion coefficient was derived. Next, this conversion was applied to the mean RDTC value shown in the data, and the results of comparing GFR between the two methods were obtained.

[0167] Synthesis of N-({N'-[tert-butoxycarbonyl]-N-(propan-2-yl)hydrazinecarbonyl}(propan-2-yl)amino)(tert-butoxy)formamide (2):To toluene (50 mL), which had been pre-dried using a 4 Å molecular sieve, anhydrous Et3N (4 mL, 28.8 mmol) was added, followed by N'-(propan-2-yl)(tert-butoxy)carbohydrazide (4 g, 23 mmol). The solution was cooled to 0°C while stirring under an N2 atmosphere. A 15% phosgene solution (9 mL, 12.7 mmol) in toluene was added dropwise over approximately 1 minute (caution is required when adding phosgene as it is extremely toxic). The reaction mixture was stirred at 0°C for 1 hour, then heated to room temperature (rt) and stirred for a further 18 hours. The reaction was stopped by adding methanol (50 mL), and the reaction mixture was stirred at room temperature for 30 minutes, after which the solvent was removed under reduced pressure. The mixture was diluted with 10% NH4OH aqueous solution (75 mL) and then extracted with ethyl acetate (15 mL x 3). The combined organic layer was washed with saturated brine (40 mL), dried over anhydrous sodium sulfate (Na₂SO₄), filtered, and the solvent was removed by distillation to obtain a white powder. The powder was dissolved in 80 mL of heated dry heptane and allowed to stand at 4°C for 18 hours to induce crystallization of the product. The crystals were filtered off and washed with hexane. The product was dried under high vacuum (colorless crystals, compound 2, 2.55 g, 59%). 1 H NMR (400MHz, CDCl3) δ6.40 (s, DO exchange, 2H), 4.15 (s, 2H), 1.43 (s, 18H), 1.12 (s, 12H). 13 ¹³C NMR (150 MHz, CDCl3): δ = 155.9, 81.1, 52.6, 50.3, 28.2, 19.2 (broad signal). HRMS (ESI): Calculated value (¹³C 17 H 34 N4O5Na[M+Na]+): 397.2411, measured value: 397.2427.

[0168] Synthesis of 1,3-diamino-1,3-bis(propan-2-yl)urea:Compound 2 (2.55 g) was resuspended in ethanol (25 mL) in a 100 mL round-bottom flask, and the mixture was heated to 80°C with an air condenser attached. Concentrated hydrochloric acid (10 mL) was added dropwise, and the solution was stirred at 80°C for 30 minutes. The solution was cooled to room temperature, and the solvent was removed by distillation. The crude product was co-evaporated sequentially once each 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 obtain 1,3-diamino-1,3-bis(propan-2-yl)urea dihydrochloride as a colorless solid in quantitative yield. 1 ¹H NMR (400 MHz, deuterated methanol (MeOH-D4)): δ 4.23 (heptet, J=6.8 Hz, 2H), 1.35 (doublet, J=6.8 Hz, 12H). 13 C NMR (150MHz, deuterated methanol (MeOH-D4)) δ=162.3, 56.2, 18.9.HRMS (ESI): C7H 18 The calculated value of N4ONa[M+Na]+ is 197.1355, and the measured value is 197.1378.

[0169] Synthesis of 6-[(1S,2R,3R,4R)-1,2,3,4,5-pentahydroxypentyl]-2,4-bis(propan-2-yl)-1,2,4,5-tetradinan-3-one (3): 1,3-Diamino-1,3-bis(propan-2-yl)urea dihydrochloride (1.55 g, 6.3 mmol) was resuspended in H2O (10 mL) with stirring at room temperature. An aqueous solution (5 mL) containing D-glucose (1.2 g, 6.7 mmol) and sodium acetate (NaOAc, 1.1 g, 13.4 mmol) was added dropwise over 1 minute, and the mixture was then stirred at room temperature for 18 hours. The reaction mixture was extracted with n-butanol (10 mL x 6 times). The combined organic layer was dried over Na2SO4, filtered, and the solvent was removed by distillation. The resulting oily substance was sequentially co-evaporated with methanol, toluene, and petroleum ether (50 mL each). The resulting product was dried overnight under high vacuum to obtain pale yellow crystals (compound 3, 1.48 g, 70%). 1 H NMR (400MHz, deuterated methanol (MeOH-D4)) δ4.53(m,2H),4.06-3.95(m,2H),3.84-3.70(m,2H),3. 68-3.57(m,2H),3.53(d,J=2.7Hz,1H),1.13(dd,J=6.8,3.4Hz,6H),1.08(d,J=6.5Hz,6H).13 C NMR (150MHz, deuterated methanol (MeOH-d4)) δ=155.7,73.0,72.4,72.2,72.0,70.0,65.0,19.8,19.7,19.3,18.9.HRMS(ESI):C 13 H 28 Calculated value for N4O6Na[M+Na]+: 359.1910, measured value: 359.1907.

[0170] Synthesis of 3-oxo-6-[(1S,2R,3R,4R)-1,2,3,4,5-pentahydroxypentyl]-2,4-bis(propan-2-yl)-1,2,3,4-tetrahydro-1,2,4,5-tetrazin-1-yl(4): Compound 3 (1.48 g, 4.41 mmol) was resuspended in H2O (5 mL) at room temperature with stirring. In a separate container, potassium ferricyanide (4.44 g, 13.5 mmol) was mixed with 80 drops (approximately 4.5 mL) of NaHCO3 (2 M), followed by the addition of H2O (5 mL). This mixture was dissolved using an ultrasonic bath. The resulting solution was added dropwise to the original reaction mixture over 1 minute with stirring, and then stirred at room temperature for approximately 30 minutes or until foaming ceased. The mixture was extracted six times with n-butanol (10 mL). The combined organic layer was dried over Na2SO4, filtered, and concentrated. The resulting oil was first co-evaporated with methanol (50 mL), then co-evaporated three times with toluene (50 mL each), cooled to 0°C, and then co-evaporated with petroleum ether (50 mL). The obtained product was dried overnight under high vacuum to yield a bright yellow fine powder (glucoverdazil 4, 1.09 g, 74%). Since this compound was a radical species, structural analysis by NMR was not possible. In addition to changes in elution time in HPLC, HRMS and EPR spectroscopy were used to confirm the structure and purity. Figure 12 shows the HPLC chromatogram. HRMS(ESI):C 13 H 25 The calculated value of N4O6Na[M+Na]+ is 356.1676, and the measured value is 356.1672.

[0171] Phantom MRI and determination of the longitudinal relaxation ability of glucoverdazil:Contrast agent samples were prepared in 1× phosphate-buffered saline (PBS) and packed into standard NMR tubes. These were then inserted into 50 mL Falcon tubes containing ultrasound gel to form an MRI phantom. The MRI phantom was placed in a 38 mm diameter transmit / receive volume coil and inserted into the MRI machine. For phantom evaluation, a multislice RARE (Rapid Imaging with Refocused Echoes) pulse sequence was performed on T1-weighted images using the following parameters: slice thickness = 5 mm, imaging field of view (FOV) = 40 × 40 mm, mean = 3, matrix size = 96 × 96, TE = 11 ms, echo interval = 7 ms, TR = 720 ms, acquisition time = 2 minutes 16 seconds. For T2-weighted images, all parameters except TE = 68 ms and TR = 4800 ms were the same as for T1-weighted images, and the acquisition time was 7 minutes 28 seconds.

[0172] For relaxation measurements, the same imaging phantom was used, and the contrast agent concentration was set to 1-3 mM. These concentrations were verified by electron paramagnetic resonance (EPR) spectroscopy. To measure the longitudinal relaxation rate (R1), an inversion-recovery RARE sequence was performed with the following parameters: slice thickness 5 mm, imaging field of view (FOV) 50 × 50 mm, mean = 1, matrix size 96 × 96, TE = 17 ms, TR = 5000 ms, TI = 50, 75, 100, 150, 200, 250, 300, 400, 600, 800, 1200, 2400, 4800 ms, with an acquisition time of 2 minutes 30 seconds per TI. The longitudinal relaxation rate was calculated using the mapping2 MATLAB® routine, created by J. Barral, M. Etezadi-Amoli, E. Gudmundson, and N. Stikov (2009) and modified by J. Rioux (2022). The longitudinal relaxation ability (r1) was determined from the slope of the graph plotting 1 / T1 against contrast agent concentration.

[0173] Stability measurement of Glucoverdazil:Prior to stability measurements, the EPR instrument was calibrated to samples of glucoverdazil or TEMPO in phosphate-buffered saline (PBS). After calibration, solutions of glucoverdazil or TEMPO were prepared (20 mM in mouse serum, or 5 mM in 4 mM sodium ascorbate buffer at pH 7.4). A single spectrum was obtained, and the height of the highest intensity peak was fixed for both compounds. Subsequently, to measure the percentage change in activity, EPR measurements were performed every 5 seconds for 2 hours in mouse serum and for 1.5 hours in ascorbic acid. To measure the stability of glucoverdazil in water, a 5 mM sample was prepared and allowed to stand in a fume hood exposed to light, or wrapped in aluminum foil and left to stand in the dark at 4°C (in a refrigerator). Samples were periodically taken from these solutions, and after calibrating the EPR instrument with a freshly prepared 5 mM glucoverdazil sample, EPR measurements were performed.

[0174] Evaluation of cell viability of H460 cells: Large cell lung cancer cells (H460) were cultured in RPMI-1640 (RPMI) medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P / S), and subcultured when confluence reached 80%. After subculturing three times, the cells were seeded into 6-well plates and cultured until confluence reached 80%. Cells were seeded to form three wells for each condition. The cells were then cultured for 4 or 24 hours under conditions of 0 mM, 2.5 mM, 5 mM, or 10 mM glucoverdazil added to standard medium. At each time point, the medium was aspirated and the cells were washed three times with Dulbecco's phosphate-buffered saline (PBS) at 37°C. Subsequently, the cells were detached with trypsin-EDTA, centrifuged at 400×g (5 minutes, 4°C), the supernatant was aspirated and removed, and the cells were resuspended in 1 mL of phosphate-buffered saline (PBS) containing 0.2 μM calcein acetoxymethyl ester (fluoresces green on living cells) and 16 μM ethidium homodimer-1 (fluoresces red on dead cells).

[0175] The viable and dead cell populations were counted by flow cytometry (Beckman Coulter Galios flow cytometer). Excitation was set to 488 nm, with a 525 / 40 nm bandpass filter used for calcein acetoxymethyl ester (live cells, green) and a 620 / 20 nm bandpass filter used for ethidium homodimer-1 (dead cells, red). After the procedure, the viable cell population under each condition was determined using Kaluza analysis software (Beckman Coulter) by comparing the number of single-stained calcein AM-positive cells with the total number of single-stained cells that were either viable or dead.

[0176] Evaluation of human renal proximal tubular (hRPT) cell viability: The viable cell population under each condition was determined using Kaluza analysis software (Beckman Coulter) by comparing the number of calcein AM-positive cells with the total number of cells detected by single-staining of live or dead cells. hRPT cells were cultured in epithelial cell medium (EpiMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (P / S), and epithelial cell proliferation supplement (EpiCGS), and passaged when confluence reached 80%. After three passages, the cells were seeded in 6-well plates and cultured until confluence reached 80%. Cells were seeded in three wells for each condition. The cells were then incubated for 4 or 24 hours in standard medium, or in standard medium supplemented with 10 mM glucoverdazil, 10 mM 5,5-dimethyl-1-pyrroline-N-oxide (DMPO, nitron spin trap), or 10 mM (2,2,6,6-tetramethylpiperidine-1-yl)oxyl (TEMPO, nitroxy radical). At each time point, the medium was aspirated and the cells were washed three times with Dulbecco's phosphate-buffered saline (PBS) at 37°C. The cells were then detached with trypsin-EDTA, centrifuged at 400 × g (5 minutes, 4°C), the supernatant was aspirated, and the cells were resuspended in 1 mL of phosphate-buffered saline containing 0.2 μM calcein acetoxymethyl ester (fluorescing green into live cells) and 16 μM ethidium homodimer-1 (fluorescing red into dead cells).

[0177] Live and dead cell populations were counted by flow cytometry (Beckman Coulter Galios flow cytometer). Under 488 nm excitation, a 525 nm / 40 nm bandpass filter was used for calcein acetoxymethyl ester (live cells, green) and a 620 nm / 20 nm bandpass filter was used for ethidium homodimer-1 (dead cells, red). After the procedure, the viable cell population under each condition was determined using Beckman Coulter's Kaluza analysis software by comparing the total number of calcein AM-positive cells by single staining with the total number of cells that were single-stain-positive, either live or dead.

[0178] Evaluation of glucoverdazil uptake in hRPT cells: hRPT cells were cultured in epithelial cell medium (EpiMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (P / S), and epithelial cell proliferation supplement (EpiCGS), and subcultured when confluence reached 80%. After three subcultures, the cells were seeded in 6-well plates and cultured until confluence reached 80%. Cells were seeded in three wells for each condition. Cells were cultured for 24 hours in standard medium or medium supplemented with 10 mM glucoverdazil. The medium was aspirated and the cells were washed three times with Dulbecco's phosphate-buffered saline (PBS) at 37°C. Then, the cells were detached with trypsin-EDTA, centrifuged at 400×g (5 minutes, 4°C), the supernatant was aspirated and removed, and the cells were resuspended in 100 μL of phosphate-buffered saline (PBS). The concentrated cell solution was transferred to an EPR tube. One μL aliquot was taken and diluted to measure the number of cells in each solution.

[0179] The EPR instrument was adjusted to a freshly prepared 5 mM glucoverdazil solution in phosphate-buffered saline (PBS), and then the sample was measured using EPR. The concentration was measured by comparing it to a pre-established standard curve, and then normalized by a predetermined cell number to calculate the glucoverdazil (nM) per cell.

[0180] Chromatogram of high-performance liquid chromatography:To confirm the completion of radicalization from compound 3 to compound 4, HPLC chromatography, high-resolution mass spectrometry (HRMS), and EPR spectroscopy were used in combination. H2O containing 0.5% TFA and acetonitrile containing 0.5% TFA were used as mobile phases, and an HPLC gradient was employed in which the proportion of acetonitrile was increased from 1% to 100% over 20 minutes. The elution time for compound 3 was consistently 10.5 minutes, but increased to 11.8 minutes after radicalization and the elimination of two protons from the verdazil ring. Furthermore, radicalization resulted in an increased absorbance at 452 nm for compound 4, while no absorbance was observed at this wavelength for the non-radical compound 3. High-performance liquid chromatography (HPLC) chromatograms of compounds 3 and 4, obtained to verify the radical activity of the compounds after the radicalization step, are shown in Figure 13. References

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[0237] The present invention will be described in detail with reference to embodiments, but these embodiments are merely illustrative and not limiting.

[0238] Other embodiments are also possible that employ the principles of the present invention and fall within the spirit and scope of the invention as defined by the claims appended herein.

[0239] All the contents of all references and other materials cited herein are hereby incorporated herein by reference in their entirety.

Claims

1. A compound of formula (I), or a pharmaceutically acceptable salt or ester thereof, 【Chemistry 1】 (I) R １ The following can be selected: 【Chemistry 2】 R ２ The following can be selected: 【Transformation 3】 R is a monosaccharide; X is carbon (C) or oxygen (O); n is 1 to 5; Z is selected from ethers, esters, carbamates, thiocarbamates, urea, thiourea, hydrazones, amides, secondary amines, tertiary amines, disulfides, triazoles, cyclooctyltriazolyl, cycloocta[d]pyridadyl, and cyanobenzylthiazolyl-containing groups; Y is a monosaccharide, sugar chain, substructure having amine and carboxylate groups separated by a substituted α-carbon, peptide, nanoparticle, dendrimer, antibody, antibody fragment, nucleic acid, aptamer, organic target ligand, and R ３ Selected from, R ３ The following can be selected: 【Chemistry 4】 X １ is a compound that is any halogen.

2. R ２ teeth, 【Transformation 5】 The compound according to claim 1.

3. R ２ teeth, 【Transformation 6】 The compound according to claim 1.

4. The aforementioned compound is given by formula (II): 【Transformation 7】 (II) The compound according to claim 1, which is a compound of the same, or a pharmaceutically acceptable salt or ester thereof.

5. The aforementioned compound is of formula (III): 【Transformation 8】 (III) A compound of, or a pharmaceutically acceptable salt or ester thereof, Y １ The following can be selected: 【Chemistry 9】 X is either C or O, n is between 1 and 5. Z is selected from ether groups, ester groups, carbamate groups, thiocarbamate groups, urea groups, thiourea groups, hydrazone groups, amide groups, secondary amine groups, tertiary amine groups, disulfide groups, triazole groups, cyclooctyltriazolyl groups, cycloocta[d]pyridazyl groups, and cyanobenzylthiazolyl-containing groups. Y ２ is the compound according to claim 1, selected from monosaccharides, glycans, partial structures having an amine and a carboxyl group separated by a substituted α-carbon, peptides, nanoparticles, dendrimers, antibodies, antibody fragments, nucleic acids, aptamers, and other organic targeting ligands.

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

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

8. The aforementioned compound, 【Chemistry 10】 【Chemistry 11】 【Chemistry 12】 【Chemistry 13】 【Chemistry 14】 【Chemistry 15】 【Chemistry 16】 【Chemistry 17】 [Chemistry 18] The compound according to claim 1, or a pharmaceutically acceptable salt or ester thereof.

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

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

11. A compound according to any one of claims 1 to 8 or a composition according to claim 9 or 10 for use in biomedical imaging.

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

13. The compound or composition for use according to claim 11 or 12, wherein the compound is glucoverdadil.

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

15. The method according to claim 14, wherein the biomedical imaging includes magnetic resonance imaging (MRI).

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

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

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

19. The method according to any one of claims 14 to 18, wherein the imaging is used to evaluate renal function.

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

21. The method according to any one of claims 14 to 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 tract obstruction, renal tumor or renal malignancy, or is a kidney donor.

22. The method according to any one of claims 14 to 20, further comprising measuring the glomerular filtration rate (GFR) of the subject.

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

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

25. Administering a contrast agent to the subject, To image the subject with the contrast agent, A method for diagnosing renal dysfunction in a subject, comprising measuring and / or mapping the glomerular filtration rate (GFR) of the subject, wherein the contrast agent comprises a compound according to any one of claims 1 to 8 or a composition according to claim 9 or 10.

26. Administering a contrast agent to the subject, To image the subject with the contrast agent, A method for monitoring, evaluating, or measuring the renal function of a subject, comprising measuring and / or mapping the glomerular filtration rate (GFR) of the subject, wherein the contrast agent comprises a compound according to any one of claims 1 to 8 or a composition according to claim 9 or 10.

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

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

29. The compound is a compound according to any one of claims 1 to 8, other than glucoverdadil, or a composition according to claim 9 or 10.

30. Use of a compound according to any one of claims 1 to 8 or a composition according to 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 according to claim 30, wherein the biomedical imaging includes magnetic resonance imaging (MRI).

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

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

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

35. The use according to any one of claims 30 to 34, wherein the imaging is used to evaluate renal function.

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

37. The use according to 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, renal tumor or renal malignancy, or is a kidney donor.

38. The use according to any one of claims 30 to 36, further comprising measuring the glomerular filtration rate (GFR) of the subject.

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

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

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

42. The use according to claim 41, further comprising imaging the contrast agent in the subject and measuring and / or mapping the glomerular filtration rate (GFR) of the subject.

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

44. The use according to claim 43, further comprising imaging the contrast agent in the subject and measuring and / or mapping the glomerular filtration rate (GFR) of the subject.

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

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