Compounds as NLRP3 pet radiotracers and compositions and uses thereof

Abstract

NLRP3 PET radiotracers are provided, as are methods of using the NLRP3 PET radiotracers to image and / or diagnose diseases and conditions associated with inflammation, such as multiple sclerosis (MS), Alzheimer's disease (AD), traumatic brain injury (TBI), Parkinson's disease (PD), acute myocardial infarction (AMI), heart failure, gout, rheumatoid arthritis, COVID- 19, diabetes, macular degeneration, and autoimmune / autoinflammatory diseases.

Description

[0001] COMPOUNDS AS NLRP3 PET RADIOTRACERS

[0002] AND COMPOSITIONS AND USES THEREOF

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims benefit of United States provisional patent application 63 / 664,422 filed June 26, 2024.

[0005] STATEMENT OF FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0006] This invention was made with government support under Grant Number: 1RF1AG076912-01A1 awarded by National Institutes of Health. The United States government has certain rights in the invention. x

[0007] BACKGROUND OF THE INVENTION

[0008] Field of the Invention

[0009] The invention generally relates to NLRP3 Positron Emission Tomography (PET) radiotracers that image neuroinflammation and monitor inflammatory responses and methods of their use as diagnostic and molecular imaging agents. In particular, the invention provides small molecule NLRP3 PET radiotracers, and methods of using the radiotracers to monitor the inflammatory status in or help diagnosis of diseases involving inflammation, such as multiple sclerosis (MS), Alzheimer’s disease (AD), traumatic brain injury (TBI), Parkinson’s disease (PD), acute myocardial infarction (AMI), heart failure, gout, rheumatoid arthritis, COVID-19, diabetes, macular degeneration, and autoimmune / autoinflammatory diseases.

[0010] Description of Related Art

[0011] Inflammasomes are cytosolic multiprotein complexes that play key roles in the innate immune responses in recognition of pathogen- and damage-associated molecular patterns (PAMPs and DAMPs). The canonical activation of inflammasomes leads to caspase- 1 activation and subsequent release of pro-inflammatory cytokines interleukin (IL)- 1 β and IL- 18. Consequently, an array of inflammatory responses and / or pyroptosis is initiated. To date, three types of inflammasomes assembled by sensor proteins have been extensively studied, and this includes the nucleotide-binding oligomerization domain [NOD] leucine rich repeat [LRR]- containing receptors (NLRs), protein absent in melanoma 2 (AIM2), and pyrin. Typically, inflammasomes are supramolecular assemblies composed of a sensor protein, an adaptor protein (apoptosis-associated speck-like protein containing a caspase recruitment domain - ASC), and an effector component, e.g., pro-caspase- 1. Among them, the NLRP3 recognize a plethora of signals, e.g., extracellular ATP, P-amyloid (AP), nigericin, and biologically relevant crystals including alum, calcium pyrophosphate dihydrate (CPPD), monosodium urate (MSU), silica, and asbestos, via mechanisms that are not yet understood. Cryopyrin-associated periodic syndrome (CAPS), a dominantly inherited auto-inflammatory disease, provides evidence to support the translational potential of targeting the NLRP3 inflammasome by its connection with gain-of- function mutations in NLRP3. Aberrant NLRP3 inflammasome activity is also thought to contribute to the pathogenesis of other complex diseases, notably metabolic disorders, obesity, atherosclerosis and neurodegenerative disorders such as Alzheimer’s disease (AD) and Parkinson’s diseases (PD). All these findings highlight the translational potential of the NLRP3 inflammasome as a promising drug target to develop novel treatments to achieve disease interventions.

[0012] Alzheimer’s disease (AD) is the most common type of dementia, and > 5 million Americans and up to 36 million individuals worldwide are currently affected by AD. In addition, > $260 billion is spent annually in the US alone on AD treatment and care, significantly exacerbating problems with the already overextended health care system. AD is projected to become a dominant health care expenditure over the next 3 decades. Unfortunately, existing treatments provide only temporary symptomatic relief. Pathologically, AD is uniquely characterized by the presence of extracellular senile plaques and intracellular neurofibrillary tangles, with P-amyloid (Ap) and hyper-phosphorylated tau being the ingredients, respectively. Potential disease-modifying therapeutics for AD have and are being tested, and most approaches under active pursuit are targeting Ap. However, successful results from the ongoing clinical trials have not been reported. Therefore, there is a desperate need to develop safe and effective AD treatments.

[0013] Among the indicated AD risk factors, neuroinflammation has been recognized as an essential player. Genetic, pathological, and epidemiological studies strongly support the essentiality of neuroinflammation in AD development and progression. Glial dysfunction due to inefficient phagocytosis or degeneration and elevated pro-inflammatory cytokines have been observed in preclinical AD models and in AD patients. Notably, no neuroinflammation was observed in the population with high Ap plaque but without dementia, thus suggesting its causative roles in cognitive impairment. Studies also found that chronic inflammation can induce AP and tau pathologies, instead of just being a passive response activated by plaques and tangles. Recent studies also showed neuroinflammation decades before cognitive impairments. Collectively, evidence strongly supports strategies targeting neuroinflammation for the prevention and treatment of AD. Although epidemiological studies touted the benefits of nonsteroid anti-inflammatory drugs (NSAIDs) in reducing the risk of AD, clinical studies found no efficacy of NSAIDs in improving cognitive functions in AD patients. Several explanations have been put forth to explain this discrepancy between epidemiological and clinical studies, e.g., treatment timing and duration, the specific drugs being evaluated, and trial design. A recent analysis of the ANDI dataset showed that one particular NSAID, diclofenac, is associated with reduced AD risk and slower cognition decline, but may have a cyclooxygenase independent mechanism. Thus, attenuation of neuroinflammation by novel mechanisms of action (MOAs) holds great promise to provide effective treatments.

[0014] Recently, emerging evidence has suggested a link between NLRP3 inflammasomes and AD development. NLRP3 can sense a plethora of exogenous and endogenous molecules including Ap and tau aggregates to activate the NLRP3 inflammasome. The levels of NLRP3, ASC, caspase- 1 , and down-stream effectors including IL- 1 P and IL- 18 were found to be upregulated in AD mouse models and AD patients. Recently, increased NLRP3 inflammasome activity, evidenced by active caspase- 1 and ASC levels, was also found in frontotemporal dementia (FTD) patients and in the tau22 FTD mouse model. Co-localization of NLRP3, ASC, and caspase- 1 was also seen in mouse AD models. Also, both IL-ip and IL- 18 have essential roles in AD pathologies, e.g., synaptic plasticity, Ap, and tau.

[0015] In addition to the pathogenic effects of the NLRP3 inflammasome underlying AD, pharmacological and genetic downregulation of this complex in preclinical AD model suggests translational potential in developing AD therapeutics. Knockout of NLRP3 or caspase- 1 ameliorated Ap pathology and improved spatial memory functions in transgenic APP / PS 1 mice. Another study employing 5XFAD mice carrying the ASC+ / _genotype also supported this notion Furthermore, recent studies in Tau22 mice demonstrated that knockout of NLRP3 or ASC reduced tau phosphorylation and aggregation. Also, deletion of NLRP3 in aged mice showed protective activity from aging-related cognitive decline, suggesting its central role in the inflammatory responses of normal aging. Small molecule NLRP3 selective inhibitors (NSIs) also showed beneficial effects in improving cognitive functions in AD mouse models. Recent studies have also indicated that NLRP3 inflammasome dysregulation is a mechanism of connecting gut microbiota change to neuroinflammation. This is consistent with the observation of increased NLRP3 inflammasome activity of monocytes from AD patients, and is in line with early studies demonstrating infiltration of peripheral monocytes to the CNS in AD. Collectively, these results indicate essential and convergent roles of the NLRP3 inflammasome axis in AD development, thus strongly suggesting the NLRP3 inflammasome as a viable target to develop effective therapeutics for AD.

[0016] Multiple sclerosis (MS) is an immune-mediated and neurodegenerative disorder characterized by neuroinflammation and demyelination. Currently there is no cure for MS and current medications mainly speed up recovery, reduced relapse rates, or manage symptoms. The immunopathology of MS is characterized by the infiltration of myelin-reactive T cells into the central nervous system (CNS) and induction of demyelination which disrupts the communication of the nervous system. Although the exact etiology and pathogenesis of MS remain unknown, emerging evidence supports a critical role for NLRP3 inflammasomes and IL- 1 P in the pathogenesis of MS. Clinical studies showed that expression of caspase- 1, IL-ip, and IL- 18 was elevated in MS plaques and peripheral mononuclear cells of MS patients. Intriguingly, MS-like lesions were observed in a Muckle -Wells syndrome (MWS) patient who had a disease- susceptible mutation in the Nlrp3 gene. Absence of the inflammasome products caspase- 1, IL-ip and IL- 18 rendered mice resistance to experimental autoimmune encephalomyelitis (EAE), a mouse model that mimics human MS. Animal studies have shown that NLRP3 deficiency substantially delayed onset and reduced severity of EAE symptoms, decreased neuroinflammation, demyelination and oligodendrocyte loss progression. Recently, the effectiveness of IFN-P, a drug that has been used for more than 15 years as a first- line treatment for human MS, was found to depend on the NLRP3 inflammasome, suggesting that IFN-P may therapeutically target the NLRP3 inflammasome-IL- 1 P axis in MS. Given the fact that many MS patients fail to respond to currently available MS treatments, including IFN-P, development of novel small molecule inhibitors targeting the NLRP3 inflammasome pathway will provide new opportunities to disease intervention for therapeutic benefits in the clinic.

[0017] NLRP3 inflammasomes also play critical roles in the inflammatory responses to myocardial injury during AMI. In the early phases of AMI, the acute ischemic injury induces the expression of NLRP3 inflammasome components (priming), which concomitantly provides the stimuli leading to NLRP3 activation and formation of the macromolecular aggregate (trigger), leading to an active inflammasome. Caspase- 1 is detected in the heart starting 3 - 6 hours after ischemia and its activity peaks between 24 and 72 hours, while low grade activation persists for weeks after the initial insult. Reperfusion, while it effectively reduces infarct size, does not prevent activation of the NLRP3 inflammasome and leads to further injury through caspase- 1- dependent inflammatory cell death. To support this notion, studies demonstrated that mice with genetic deletion of NLRP3 or ASC exhibited smaller infarct size in experimental AMI model, and reduced tendency toward adverse remodeling and heart failure, consistent with previously reported central role of caspase- 1 in AMI. Transgenic mice expressing constitutively active caspase- 1 , on the other hand, developed adverse cardiac remodeling and heart failure.

[0018] The outbreak of the coronavirus disease 2019 (CO VID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has evolved into a global pandemic. Although the approval of COVID-19 vaccines has significantly protected the general population, the emergence of viral variants continues to represent a tremendous challenge and burden on our society. The majority of patients with CO VID- 19 exhibit mild-to-medium symptoms, however, 5-10% of CO VID- 19 patients become significantly ill suffering with excessive immune response dysregulation and high mortality. One of the clinical signs of critically ill COVID-19 patients is the resulting complications of acute respiratory distress syndrome (ARDS) and acute lung injury (ALI), which leads to respiratory and multi-organ failures, and ultimately patient death. In addition, ARDS by itself is a life-threatening condition of seriously ill patients associated many risk factors. Thus, effective treatments to mitigate ARDS / ALI are urgently needed.

[0019] Recent studies have emerged to suggest a critical role for the NLRP3 inflammasome in the observed cytokine storm and the development of ARDS / ALI in COVID- 19. Intriguingly, early studies have revealed the essential role of the NLRP3 inflammasome in the development of ARDS / ALI. Recent studies using animal models and CO VID- 19 patient samples also demonstrated NLRP3 activation by SARS-CoV-2 via multiple mechanisms. More importantly, NLRP3 activation is observed in COVID-19 patients and is associated with lesions of the nervous systems / lungs and disease severity. Furthermore, NLRP3 inflammasomes are over activated in elderly individuals and age is one of the strongest predictors of COVID-19 mortality with 80% of COVID- 19 deaths in the USA being in people of >65 old. In addition, many major risk factors associated with CO VID-19 contraction, e.g., diabetes and obesity, have demonstrated strong links with NLRP3 inflammasome dysregulation. Therefore, novel NSIs represent a promising approach to develop effective treatments to mitigate the complications of ARDS / ALI and inflammatory responses in CO VID-19. Besides its therapeutic potential, the NLRP3 inflammasome has recently been exploited as a potential biomarker for inflammatory conditions to assist diagnosis and clinical evaluation of new therapeutics, given its association with the innate immune responses and development of inflammation. The level of NLRP3 in cerebrospinal fluid (CSF) has been studied as a predictor of the severity of and to discriminate between community-acquired bacterial meningitis (CABM) and viral meningitis (VM) in patients. The changes of the CSF level of NLRP3 have also been tested as a potential biomarker for septic patients. Serum protein levels of ASC, caspase- 1, IL-1β and IL- 18 have been investigated as biomarkers for TBI, MS and stroke patients. Researchers also investigated NLRP3 as a predictive biomarker for glioma patients and cardiometabolic syndrome. Notably, a recent study suggested that activation of the NLRP3 inflammasome could serve as a predictor for COVID-19 patients.

[0020] In the past decade, a number of small molecule NLRP3 inflammasome inhibitors have been reported to interfere with the inflammasome pathway via different mechanisms of action (MO As). Among them, some of those that bind directly to the NLRP3 protein showed promising effects in preclinical animal models. Structurally, most of them are either derived from a sulfonylurea chemical scaffold or contain an electrophilic moiety, i.e., a Michael acceptor, exemplified by MCC950, CY-09, Tranilast, and Oridonin. Interestingly, only MCC950 was tried as a positron emission tomography (PET) radiotracer to image the NLRP3 inflammasome activity. Unfortunately, the radiotracer, [nC]MCC950, showed poor brain uptake, thus limiting its further use as a radioligand for CNS applications. Therefore, CNS penetrant NLRP3 PET radiotracers are urgently needed, given the important roles of this inflammasome in neurodegenerative disorders. There is a need to explore their potential as tools to validate the NLRP3 inflammasome as a biomarker for neuroinflammation.

[0021] SUMMARY OF THE INVENTION

[0022] This disclosure describes the design and development of new chemical entities as novel NSIs for use, for example, as PET radiotracers. These PET radiotracers, which bind to the NLRP3 protein, are valuable tools for detecting and visualizing inflammasomes and thus to detect and / or diagnose diseases related to inflammation, such as AD, MS, AMI, TBI, PD, AMI, heart failure, diabetes, COVID- 19, arthritis, macular degeneration and autoinflammatory diseases. Significantly, the compounds penetrate the CNS. For example, they can cross the blood-brain barrier and so can be used to visualize inflammation in the brain. The compounds are depicted in generic Formula I below.

[0023] Other features and advantages of the present invention will be set forth in the description of the invention that follows, and in part will be apparent from the description or may be learned by practice of the invention. The invention will be realized and attained by the compositions and methods particularly pointed out in the written description and claims hereof.

[0024] BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Fig. 1A-D. In vitro autoradiography of [18F]YM-III-109 in mouse brain tissues. A) Representative autoradiographic images of mouse brains (sagittal).The baseline sections were treated with [18F]YM-III-109 only, and the blocking sections were co-treated with YM-III-109 at 10 pM with [18F]YM-III-109; B). Quantification of radioactivity in brain regions of A; (C) Representative autoradiographic images of mice brains (sagittal) from WT, nlrp3- / - and AD mice. (D) Relative radioactive uptake of [18F] YM-III-109. Data as mean ± SD (n=8). Data are expressed as the density light units per square millimeter (DEU / mm2).

[0026] Fig. 2. Biodistribution histogram of [18F]YM-III-109 in C57BE / 6 mice (n = 5). All data are the mean ± standard deviation. Data are expressed as the percentage of injected dose per cubic centimeter (% ID / cc).

[0027] Fig. 3. Representative sagittal PET / CT image of [18F] YM-III-109 in baseline (left) and blocking (right) experiments (summed 20-60 min).

[0028] Fig. 4A-C. A and B).TACs of whole brain uptake (n = 5 for Baseline, n = 4 for Blocking). means whole brain radioactivity normalized by maximal blood radioactivity; C) AUC for TACs of baseline and blocking (~46% reduction in AUC).

[0029] Fig. 5. PET / CT imaging blocking studies of [18F]YM-III-109 in AD (left) and WT (right) mice. Summed 0-60 min.

[0030] Fig. 6. TACs of whole brain uptake (n = 4 for AD mice, n = 4 for WT mice).

[0031] DETAILED DESCRIPTION

[0032] It is an object of this invention to provide new chemical entities that are small molecules that bind to the NERP3 protein in inflammasomes. In some aspects, the new chemicals are radiolabeled (e.g. with C-l 1 or F-18) and used as PET radiotracers. The compounds have a formula as depicted in Formula I:

[0033] Formula I wherein

[0034] A is a 6-membered ring selected from benzene, pyridine, pyrimidine, and 1,2-diazine;

[0035] R1 is: an unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted C1-C8 alkyl; an unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted C1-C8 alkoxy 1; amino; nitro; OH; or halogen;

[0036] R4 is: halogen, amino, nitro or cyano;

[0037] R2, R3, and R5 are the same or different and are independently: H; C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkyl; C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkoxyl; C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkylcarbonyl; halogen; hydroxyl; amino; nitro; or cyano;

[0038] W is: an unbranched, branched, saturated, unsaturated, substituted or unsubstituted C1-C4 alkyl; NH; S; or SO2;

[0039] B is: 1,2,3-traizole; 1,2,4 triazole; 1,2-diazole; 1,3-diazole; 1,2,4 oxadizole; isoxazole; or 1,3,4-oxadiazole;

[0040] D is present or absent and when present is an aliphatic cyclic ring with an optional R6 substituent;

[0041] Y is present or absent and when present is N or C;

[0042] Z is present or absent and when present is N or C;

[0043] E is: a 5C or 6C aromatic ring with an optional R7 substituent; or two 5C or 6C aromatic rings each with an optional R7 substituent;

[0044] M is: CO; CO-N-RIO; SO2; or CS;

[0045] L is: a linker sequence comprising an unbranched, branched, saturated, unsaturated, substituted or unsubstituted C3-C6 alkyl; cyclopropyl with an optional R8 substituc with an optional R8 substituent and n = 0-4;

[0046] R6 and R7 and R8 are the same or different, are optional, and are independently: H; Cl- C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkyl; C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkoxyl; C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkylcarbonyl, halogen, hydroxyl, amino, nitro, cyano, ester and carboxylic acid.

[0047] In some aspects, the compounds of Formula I are radiolabeled for use as PET radiotracers, e.g. with at least one C-l 1 and / or at least one F-18. When labeled, the label(s) is / are located within R1 or R2 or R3 or R4 or R5 or R6 or R7.

[0048] In some aspects of Formula I, the compounds are represented by Formula II: wherein

[0049] R9 is H; C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, or substituted or unsubstituted alkyl; R4 is halogen;

[0050] R10 is: where R11 is: H; C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkyl; C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkoxyl; Cl- C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkylcarbonyl, halogen, hydroxyl, amino, nitro or cyano; and

[0051] L and E are covalently bonded (linked) together to form: where

[0052] X is independently C, N, or O and forms an aromatic heterocyclic ring with the R7 substituent.

[0053] All other components are as described for Formula I. In some aspects, the compounds of Formula II are radiolabeled for use as PET radiotracers, e.g. with at least one C-l 1 and / or at least one F-18. When labeled, the label(s) is / are located within R2 or R3 or R4 or R5 or R7 or R9 and / or RIO. In some aspects of Formula I, the compounds are represented by Formula III: wherein

[0054] R9 is H; C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, or substituted or unsubstituted alkyl;

[0055] R4 is halogen;

[0056] D and E are covalently bonded directly to each other and form a two-ring system:

[0057] All other components are as described for Formula I. In some aspects, the compounds of Formula III are radiolabeled for use as PET radiotracers, e.g. with at least one C-11 and / or at least one F-11. When labeled, the label(s) is / are located within R2 or R3 or R4 or R5 or R6 or R7 or R9.

[0058] In some aspects, the present disclosure provides the labeled PET radiotracer of Formula IV :

[0059] Also encompassed are salts (e.g. CT, SO4 , etc.), hydrates and stereoisomers of each of the compounds and radiolabeled compounds disclosed herein.

[0060] As used herein, “F-18” refers to [18 FJFluorodeoxyglucose (INN), or fluorodeoxy glucose F 18 (US AN and USP), also commonly called fluorodeoxyglucose and abbreviated [18 FJFDG, 2-[18 FjFDG or FDG, is a radiopharmaceutical, specifically a radiotracer, used in the medical imaging modality positron emission tomography (PET). Chemically, it is 2-deoxy-2-[18 F]fluoro-D-glucose, a glucose analog, with the positron- emitting radionuclide fluorine- 18 substituted for the normal hydroxyl group at the C-2 position in the glucose molecule.

[0061] As used herein, “C-11” refers to Carbon- 11 choline, which is used as the basis of some medical imaging technologies. Because of its involvement in biologic processes, choline is related to diseases, leading to the development of medical imaging techniques to monitor its concentration. When radiolabeled withnCH3, choline is used as a tracer in PET imaging. By monitoring the gamma radiation resulting from the decay of carbon-11, the uptake, distribution, and retention of carbon- 11 choline can be monitored.

[0062] Methods of labeling compounds with C-11 and / or F-18 are known in the art. Briefly, [HCJCCh was obtained via the 14N (p, a) 11C reaction on nitrogen with 2.5% oxygen, with 11 MeV protons (Siemens Eclipse cyclotron), and trapped on molecular sieves in a TRACERlab FX-Mel synthesizer (GE Healthcare). [ 11CJCH4 was obtained by the reduction of [11CJCO2 in the presence of Ni / hydrogen at 350 °C. The resulting [11CJCH4 passed through an oven containing 12 to produce [11CJCH3I via a radical reaction.

[0063] The prepared [11CJCH3I was trapped in anhydrous DMF (300 pL) containing precursor (1.0 mg) and K2CO3 (5.0 mg). The reaction vessel was heated at 80 °C and kept there for 3 min. The radioactive mixture containing target radiotracer was quenched by addition of an HPLC mobile phase (0.7 mL) and then applied to a reverse phase semipreparative HPLC (Phenomenex Gemini-NX 5u C18 110A, 250 x 10 mm, 5.0 mL / min, a gradient of 10-90 % CH3CN in H2O). A radioactive fraction having a retention time of 15.2 min was collected in a flask, and diluted in water (30 mL). The final product was reformulated by loading onto a solid-phase exchange (SPE) C-18 cartridge (Waters WAT020515 Sep-Pak Plus Short Cl 8), rinsing with water (4 x 5 mL), eluting with EtOH (0.3 mL), and diluting with saline (2.7 mL). The chemical and radiochemical purity of the final product was tested by analytical HPLC (V ARIAN Puruit XRs 5 Cl 8, 150 x 4.6 mm), eluting with a gradient of 10-90 % CH3CN in H2O of 0.1% TFA, at a flow rate of 2 mL / min. Confirmation of the identity of radiotracer was achieved by co-injection with parent compound as reference standard. For the determination of molar radioactivity, mass (pmol) of radiotracer with a known radioactivity was determined by HPLC comparison of UV absorbance at 254 nm with those of known concentrations of non-radioactive parent compound.

[0064] A ruthenium-mediated radiofluorination method was employed to afford [18FJ- radiotracer by using air-stable ruthenium complex CpRu(COD)Cl, chloroimidazolium chloride, and [18F]fluoride. Specifically, precursors were first activated by CpRu(COD)Cl to form an T|67t- coordinated ruthenium-phenol complex, which was used to elute [18F]fluoride off an anion exchange cartridge by the subsequent addition of chloroimidazolium chloride. The elution efficiency of [18F]fluoride achieved 90%. After adding solvent (DMSO / acetonitrile (v / v) = 1:1), the reaction mixture was heated to 125 °C for 30 min, followed by high performance liquid chromatography (HPLC) purification and reformulation to afford radiotracer, respectively, in sterile saline containing <10% (v / v) ethanol. The overall yield for the radiolabeling chemistry was 25% (non-decay corrected) in a total synthesis time of 120 min from end of cyclotron bombardment. The specific activity was around 5.1 Ci / pmol, and the radiochemical purity was confirmed being greater than 98% at time of injection

[0065] DISEASES AND CONDITIONS THAT ARE DETECTED AND / OR DIAGNOSED, USING THE COMPOUNDS DESCRIBED HEREIN

[0066] By binding to the NLRP3 protein of inflammasomes, the compounds disclosed herein can be used to detect (visualize, image, etc.) and / or diagnose disorders and conditions associated with (e.g. caused by or related to or exacerbated by) unwanted activation of NLRP3 containing inflammasomes, and / or consequences of such activation such as the unwanted production of pro-inflammatory cytokines pro-IL-lB and pro-IL-18. Such diseases / conditions may be caused by so-called sterile inflammation (e.g. various inflammatory diseases, second wave inflammation after heart attack, stroke or other ischemic or traumatic injury), or by inflammation that is caused by an infection (e.g. by an infectious organism such as a bacterium or virus). Such diseases and conditions result from a wide array of stimuli. For example, numerous microbes including various bacteria, viruses, fungi, and protozoan parasites can activate the NLRP3 inflammasome, e.g., the bacterial toxin nigericin has also been reported to induce the activation of NLRP3 by causing potassium efflux in a pannexin-1 -dependent manner. In addition to microbial activators, endogenous “danger” signals such as ATP, monosodium urate (MSU) activate the NLRP3 inflammasome, as do various other types of cellular damage resulting e.g. from metabolic stress, ischemia and trauma. The NLRP3 inflammasome is implicated in metabolic disorders and sterile inflammatory responses including multiple sclerosis, arthritis, type II diabetes mellitus, gout and ischemia. A number of endogenous and exogenous crystalline molecules activate the NLRP3 inflammasome, e.g. uric acid crystals and calcium pyrophosphate dihydrate, the causative agents of gout and pseudogout respectively. Silica and asbestos particles, which cause fibrotic lung disorders silicosis and asbestosis respectively, also activate the NLRP3 inflammasome. Release of ATP from necrotic cells is a danger signal that activates the innate or sterile inflammatory immune response. Inhibiting NLRP3 inflammasome activation has beneficial effects in preventing the damage mediated by the sterile inflammatory response in diseases such as renal-, cardiac-, and cerebral-ischemia. In addition, necrosis-induced sterile inflammation in trauma and secondary to infections and sepsis are modulated by the inhibitors of the NLRP3 pathway described herein. The NLRP3 inflammasome can also be activated by molecules associated with stress or danger, including crystalline and particulate substances.

[0067] Examples of auto-inflammatory’ diseases which may be detected, e.g., imaged and / or diagnosed by PET scans using the agents described herein include but are not limited to: i) joint, bone and muscle diseases such as rheumatoid arthritis, psoriatic arthritis, osteoarthritis, ankylosing spondylitis, erosive osteoarthritis of the hand, recurrent multifocal osteomyelitis, traumatic knee injury; relapsing polychondritis, etc.; ii) hereditary systemic autoinflammatory diseases such as familial Mediterranean fever (FMF), cryopyrin-associated periodic syndrome (CAPS); Muckle-Wells Syndrome, TNF receptor-associated periodic syndrome (TRAPS), hyper-IgD syndrome (HIDS), periodic fever, aphthous stomatitis, pharyngitis and adenitis (PFAPA), deficiency of interleukin- 1 (IL- 1) receptor antagonist (DIRA), etc.; iii) systemic inflammatory diseases such as systemic juvenile idiopathic arthritis, adult-onset Still’s disease, Schnitzler syndrome, Behcet’s disease, PFAPA (Periodic Fever, Apthous Sstomatitis, Pharyngitis, Adenitis), SAPHO (synovitis, acne, pustulosis, hyperostosis, osteitis) syndrome, macrophage activation syndrome, etc.; and iv) common inflammatory diseases such as gout, Type 1 diabetes, Type 2 diabetes, metabolic syndrome, insulin resistance, stroke, heart attack, myocarditis, cardiac toxicity due to drug or radiation, ischemic heart disease, cardiomyopathy on a familial or genetic basis, heart failure, cardiac arrest and anoxic brain injury, acute and chronic lung injury due to infection, ischemia, toxin, trauma; dry eye syndrome, pustular psoriasis; neutrophilic dermatoses; acute or chronic hepatitis due a virus, toxin, ischemia or drug; acute or chronic renal injury due to ischemia, hypertension, diabetes, toxin or drugs; sepsis, septic shock; etc.

[0068] In one aspect, the PET radiotracers are used to image and / or diagnose multiple sclerosis (MS). MS refers to all types of MS including relapse-remitting, secondary' progressive, and primary progressive MS.

[0069] In one aspect, the PET radiotracers are used to image and / or diagnose neurodegenerative disorders that refer to AD, PD, ALS, and Huntington’s disease.

[0070] In one aspect, the PET radiotracers are used to image and / or diagnose ARDS / ALI and cytokine storm associated with COVID-19.

[0071] IMAGING AND DIAGNOSING

[0072] In some aspects, the compounds of the disclosure are utilized in vivo as radioactive agents to label and then image (e.g. visualize, locate, etc.) NRLP3 inflammasomes. The agents thus detect conditions and / or diseases associated with (e.g. caused by) unwanted increases in the number of NRLP3 inflammasomes in an area of interest as an indicator of an increase in unwanted inflammasome activity in the area, e.g. to identify inflammasome- associated inflammation. In some aspects, the compounds of the disclosure are utilized as markers (e.g. diagnostic markers) of the conditions / diseases. Accordingly, methods of imaging NRLP3 inflammasome-related diseases in subject in need thereof are provided. Such methods may include a step of identifying a subject in need of such imaging (e.g. a subject with one or more symptoms of an NRLP3 inflammasome-related disorder, or a subject who is likely to develop or at risk of developing such a disorder) and then conducting the imaging. Imaging may be performed e.g. as a PET scan. For example, patients who have or who have had MS are imaged, as are patients for whom there is reason to suspect that the onset or a relapse of MS is likely to occur.

[0073] A subject suitable for undergoing imaging may have one or more readily observable symptoms of, or early symptoms of, or a predisposition to development of the disease (e.g. genetically, due to lifestyle, due to exposure to a substance that is known to cause inflammation, surgery, injury, etc.) that is being imaged. Since the compounds are capable of entering the CNS and can penetrate the blood brain barrier, the present invention inter alia provides the specified compounds for use in methods of imaging NRLP3 inflammasome- associated neuroinflammation associated with MS, AD, etc.. However, the agents are also used to image acute inflammation, or acute inflammatory responses, which may occur in variety of illnesses or injuries in which inflammation is induced.

[0074] Methods of imaging such as PET scans are known in the art. Briefly, a PET (positron emission tomography) radioactive tracer is injected into the body, which then moves through the body and concentrates in specific organs or tissues. The PET scanner detects the radioactive emissions from the tracer, and a computer converts this data into images. As preparation, the patient may be asked to fast or avoid certain medications before the scan, depending on the specific scan and the patient's condition. A small amount of radioactive tracer is injected into a vein in the arm or hand and the patient rests for about 30 to 60 minutes while the tracer is absorbed by the body and distributed to the target area(s). The exact volume that is injected varies depending on the activity (radioactivity) of the tracer, but a common example is 400 MBq of agent in a 5 mL saline solution. This is generally followed by a saline flush of about 35 mL

[0075] For scanning, the patient lies on a narrow table that slides into a large, doughnutshaped PET scanner. The scanner has detectors that surround the body and detect the signals from the tracer. The PET scanner sends the signals of radioactivity to a computer, which analyzes the signals and creates 3D images of the organ or tissue being scanned. The images are displayed on a monitor, and a radiologist or nuclear medicine specialist interprets them to identify any abnormalities or changes and makes a diagnosis based on that information.

[0076] In some aspects, the compounds disclosed herein are used in combined PET / CT scans, e.g., PET scans performed in conjunction with a computed tomography (CT) scan, which provides anatomical images along with the functional information from the PET scan. This combination helps to pinpoint the location of abnormalities more accurately.

[0077] Methods of conducting PET scans and of obtaining PET scan images are known in the art, for example, those described in US patent application 20230346324, and issued US patents 10688200, 11576628, and 11627920, 11844636 and 12032107, the complete contents of each of which is hereby incorporated by reference in entirety. Methods of interpreting PET scan images, such as by using computer readable media that is programmed with instructions to process and output the PET images, are also encompassed herein and are known and described, for example, in the US patents and patent applications listed above.

[0078] The agents are used to bind to and image inflammasomes (thereby detecting inflammation) in organs, tissues and cells, including but not limited to: the central nervous system components (e.g. brain, nerves, spinal cord, etc.), liver, kidney, blood, heart, lungs, skin, pancreas, intestines, ovaries, glands, muscles, fatty tissue, _and the like.

[0079] The product of at least one PET scan of an organ, tissue, etc. of a subject that is obtained is a PET image or scan of the area that was radio-labeled and scanned. Such images are interpreted (“read”) by a person skilled in the art such as a physician, a radiologist, a neuroradiologist, etc. and the information obtained by the specialist is used e.g. to diagnose a disease and / or to confirm a diagnosis. Exemplary diseases that can be diagnosed, or for which a diagnosis can be confirmed, include but are not limited to MS, AD and others disclosed elsewhere herein.

[0080] In addition, this technology is used to monitor the progression of a disease, e.g. with or without treatment. Monitoring involves periodic PET scans of a subject in need thereof at spaced apart time points. For example, if the subject is on medication, images are obtained before medication is taken and after it has been taken and the images are compared to assess if the medication is efficacious, e.g. to assess whether or not inflammation has been decreased. Based on this information, the same treatment regimen may be continued, or a more rigorous regime may be undertaken (higher doses, change to a stronger medication, etc.), or treatment may be stopped or doses decreased to a maintenance dose, etc. depending on the analysis of professionals and the subject’s wishes.

[0081] The agents may be used to monitor the progress of a disease, whether or not medical intervention occurs. This can facilitate the choice of options for a physician, a patient or the family of a patient as to what treatment options are available and when and whether treatment should be started or stopped.

[0082] Also provided are methods of visualizing an NRLP3 inflammasome or a plurality of NRLP3 inflammasomes. The methods comprise contacting the NRLP3 inflammasome(s) with at least one compound disclosed herein, the at least one compound being labeled with C- 11 and / or F-l 1. The step of contacting is performed under physiological conditions that permit the at least one compound to bind to the NLRP3 protein of the NLRP3 inflammasome(s), e.g. in buffer or saline at a pH of from about 7.0-7.5, such as about 7.35- 7.45. The NLRP3 inflammasome can then be visualized by detecting radiation emitted from C-l 1 and / or F-l 1 of labeled compounds that are bound to the NRLP3 inflammasome(s), e.g. by PET imaging.

[0083] COMPOSITIONS

[0084] This disclosure also provides compositions comprising at least one (e.g. one or more) of the agents described herein. The compositions comprising the agents are introduced into an in vivo subject. Thus, the compositions must be physiologically compatible (pharmaceutically acceptable) and generally comprise at least one agent as disclosed herein and a pharmaceutically acceptable carrier. The in vivo subject is usually a mammal such as a human. However, their use in non-human animals is also encompassed, e.g. for veterinary’ purposes.

[0085] As used herein, the "pharmaceutically acceptable carrier" is any of the known physiologically compatible carriers, such as solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption retarders, etc. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epithelial administration (e.g., by injection or infusion). The compositions are generally maintained at or near physiological pH, e.g. in the range of from about 7.35 to about 7.45. The pharmaceutically acceptable compounds described herein may comprise one or more pharmaceutically acceptable salts. "Pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the original compound and does not give any undesired toxic effects. Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitrate, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphite, and aliphatic mono- and dicarboxylic acids, and phenyl substitutions. Examples include those derived from non-toxic organic acids such as alkanoic acid, hydroxyalkanoic acid, aromatic acid, aliphatic and aromatic sulfonic acid. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium and calcium, as well as N, N'-dibenzylethylenediamine, N-methylglucamine, chloroprocine, choline, diethanolamine, ethylenediamine and prokine. Examples thereof include those derived from non-toxic organic amines.

[0086] The pharmaceutical compositions described herein may also contain pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium hydrogen sulfate, sodium pyrosulfate, sodium sulfate; (2) fat-soluble antioxidants such as ascorbic palmitate, butylated hydroxyanisole (BHA), butylated hydroxy toluene (B HT), lecithin, propyl gallate, alpha-tocopherol; and / or (3) citric acid, ethylenediamine tetraacetic acid (EDTA). Metal chelating agents such as sorbitol, tartrate acid, and phosphoric acid may also be included.

[0087] Examples of suitable aqueous and non-aqueous carriers that are used in the pharmaceutical compositions described herein are water, ethanol, polyols (glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, and olive oil. Vegetable oils may be used, as well as injectable organic esters such as ethyl oleate. Appropriate fluidity can be maintained, for example, by using a coating material such as reticin, by maintaining the required particle size in the case of dispersion, and / or by using a surfactant.

[0088] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers and dispersants. Prevention of microbial survival can be ensured both by sterilization and / or by containing various antibacterial and antifungal agents such as parabens, chlorobutanols, phenol sorbates and the like. Further, in some aspects, the compositions contain a tonicity agent such as sugar and sodium chloride. In addition, the absorption delay of the injectable dosage form can be brought about by the inclusion of agents that delay the absorption, such as aluminum monostearate and gelatin.

[0089] Pharmaceutically acceptable carriers and preparations include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of injectable sterile solutions or dispersions. The use of such solvents and agents for pharmaceutically active substances is known in the art. Further other active compounds may also be included in the compositions, such as various drugs, analgesics to reduce pain at injection sites, anti- inflammation agents, relaxation agents to calm the patient, etc.

[0090] Pharmaceutical compositions should generally be sterile and stable in manufacturing and storage conditions. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentration and sterilized e.g. by microfiltration, heat, etc. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and / or a suitable mixture thereof. Appropriate fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by using surfactants. In many cases, it is preferable to include, for example, sugar, a polyalcohol such as mannitol or sorbitol, or an isotonic agent such as sodium chloride in the composition. The absorption retardation of the injectable composition can be brought about by including, for example, an absorption retarder such as monostearate and gelatin in the composition.

[0091] For sterile powders for preparing injectable sterile solutions, production methods include but are not limited to vacuum drying and freeze-drying.

[0092] The amount of agent that is combined with a carrier material to produce a single dosage form, and the volume of a dosage form, can vary depending on the subject being imaged and the method of administration. Generally, the amount is that for which a detectable signal is obtained. Generally, in percentage, this amount is about 0.01% to about 99%' of the agent in a composition, or from about 0.1% to about 70%, or from about 1 to 10, 20, 30, 40 or 50%.

[0093] Alternatively, one or more different known imaging agents may also be utilized together with (in combination with) one or more labeled compounds as disclosed herein. KITS

[0094] Kits comprising the agents disclosed herein are also provided. Generally, such kits include a “dose” or a plurality of doses of a composition as described herein. The kit generally includes a packaged combination of a predetermined amount of reagents and instructions and a label indicating the intended use of the contents of the kit. The agents may be in the form of a sterile composition comprising at least one agent, either in a concentrated for ready for dilution in a sterile carrier, which may also be included in the kit; or in a dilute ready-to-use form; or as a sterile powder ready to mix with a sterile carrier, which may also be included in the kit. A sterile saline wash solution may also be included.

[0095] METHODS OF MAKING THE COMPOUNDS Also encompassed herein are methods of making the disclosed compounds.

[0096] In some aspects, the methods are as illustrated in Scheme 1: where D, E, R2, R3, R4, R5, R7, R9 and RIO are as defined as described for Formula I.

[0097] With respect to Scheme 1, the compounds of Ml, M2, M3, M4, and M6 are commercially available, are known in the literature or are obtained either by analogy with the processes described herein, or by conventional synthetic procedures, in accordance with standard techniques, from available starting materials using appropriate reagents and reaction conditions. M3 was prepared under click reaction conditions (e.g. CuSCU, sodium ascorbate, EtOH / khO; CuT, DMSO), followed by coupling with M4 and M6 to achieve. The coupling agent is preferably hexafluorophosphate azabenzotriazole tetramethyl uronium (HATU) and trimethylamine (Et3N) as a base.

[0098] It is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0099] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0100] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Representative illustrative methods and materials are herein described; methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.

[0101] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual dates of public availability and may need to be independently confirmed.

[0102] It is noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as support for the recitation in the claims of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitations, such as "wherein [a particular feature or element] is absent", or "except for [a particular feature or element]", or "wherein [a particular feature or element] is not present (included, etc.)...".

[0103] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0104] The invention is further described by the following non-limiting examples which further illustrate the invention, and are not intended, nor should they be interpreted to, limit the scope of the invention. EXAMPLES

[0105] EXAMPLE 1.

[0106] Our recent efforts identified a potent NLRP3 inhibitor, YM-III-109 (Formula IV), with an IC50 of 70 nM and a binding afinity Kd of 100 nM for inflammasomes. In vitro Absorption, Distribution, Metabolism, Excretion (ADME) studies revealed that YM-III-109 exhibits a half-life (Tl / 2) of 29 and 28 minutes in human and mouse hepatocytes, respectively. In vivo pharmacokinetic (PK) studies in mice demonstrated rapid and good brain penetration of this compound (B / P ratio of 0.47 at 30 minutes). Oral bioavailability was F% of 0.53) was observed as was high clearance after both IV and PO administration, leading to a Tl / 2 of 2.28 h and 2.76 h, respectively. The in vitro ADME and in vivo PK properties of YM-III-109 are summarized in Table 1. Although the PK properties of YN-III-109 in mice do not support further development of this compound as a therapeutic agent, the observed high clearance, short Tl / 2 along with the facts that it is a brain penetrant and has reasonable ADME properties in intact hepatocytes shows that this compound is a promising PET radiotracer.

[0107] Table 1. In vitro ADME and in vivo PK properties of YM-III-109

[0108] We successfully radiolabeled YM-III-109 with F-18 and obtained [18F]-YM-III-109 in high yield and purity (40% and >99%, respectively). Next, we performed autoradiography studies using WT and nlrp3- / - mouse brain tissues to establish the binding specificity of [18F]YM-III-109. As shown in Fig. 1A and IB, the results demonstrated strong baseline labeling of the brain tissues from WT C57BL / 6 mice by [18F]YM-III-109. The results also suggest the presence of NLRP3 in the brain tissues of WT mice. The binding of [18F] YM-III- 109 was significantly blocked by the parent compound YM-III-109, thus showing its binding specificity to the NLRP3 protein and supporting the use of [18F] YM-III-109 as a general tool to develop PET tracers. Notably, the labeling differences observed from the brain tissues of nlrp3- / - mice and WT mice (Fig. 1C and ID) clearly confirmed the specific labeling of NLRP3 by this radiotracer. Notably, it was also observed that the binding of [18F]YM-III- 109 is significantly higher in the 5xFAD mouse brain tissues than that in healthy control mice, and this is consistent with the literature that NLRP3 is overly activated in AD mouse models. Following the confirmation of specific labeling, we proceeded to conduct PET / CT imaging studies using the tracer [18F]YM-III-109 in C57BL / 6 mice (25-30 g, n = 5). The primary aim of these PET studies was to evaluate the in vivo properties of the tracer, including its distribution, brain penetration, and binding specificity. [18F]YM-III-109 was administered via tail vein injection, and PET scans were performed at multiple time points post-injection (2, 10, 30, and 60 minutes) to assess the biodistribution. The results, expressed as the percentage of injected dose per cubic centimeter (% ID / cc), are detailed in Fig.2. Quick initial uptake was observed in several organs: the brain, blood, liver, heart, lung, and kidney with recorded uptakes of 3.90% ID / cc, 12.86% ID / cc, 27.87% ID / cc, 12.24% ID / cc, 7.63% ID / cc, and 20.22% ID / cc respectively, at the 2-minute mark. Notably, the radioactivity in the liver and kidney demonstrated slower clearance rates, with levels at 16.06 % ID / cc and 11.08% ID / cc at 60 minutes post- injection, suggesting potential hepatobiliary and urinary pathways for the elimination of [18F]YM-III-109. Conversely, brain, blood, heart, and lung uptakes showed a fast wash out during the scanning period, reaching 0.93% ID / cc, 1.70% ID / cc, 1.66% ID / cc, and 1.77% ID / cc, respectively at the final time point. Muscle tissue displayed the lowest uptake, starting at 1.21% ID / cc at 2 minutes and slightly increasing to 1.33% ID / cc by 60 minutes post-injection. These findings provide crucial insights into the in vivo dynamics and elimination pathways of the tracer [18F]YM-III-109 in male C57BL / 6 mice.

[0109] We then further evaluated [18F]YM-III-109 for its suitability as a CNS PET radiotracer by examining its brain uptake. Detailed analyses were conducted using PET / CT imaging (sagittal, summed images from 20-60 minutes) and time-activity curves (TACs), both depicted in Fig. 3 and Fig. 4. Initial baseline studies indicated that [18F]YM-III-109 efficiently crossed the blood-brain barrier (BBB), achieving homogeneous distribution in the brain within 5 minutes post- injection. The tracer’s signal showed a gradual decline throughout the duration of the scanning. Notably, [18F]YM-III-109 demonstrated a significant increase in maximum brain uptake, reaching 4.17% ID / cc, which is substantially higher than the uptake values observed with our previously studied NLRP3 PET radiotracers. Then, we conducted self-blocking experiments using the parent YM-III-109 in C57BL / 6 mice (25-30 g, n = 4), as illustrated in Fig. 3 and Fig. 4. Contrary to expectations, brain uptake in self-blocking mice was significantly higher than that in baseline mice. This unexpected increase in uptake could partly be attributed to elevated levels of free [18F]YM-III-109 in the blood following pretreatment with YM-III-109. Due to this, the data on radioactivity accumulation was normalized against the maximal blood radioactivity to provide a clearer analysis of brain- specific activity. Pretreatment with YM-III-109 at a dosage of 0.3 mg / kg resulted in a notable reduction in brain radioactivity, decreasing by approximately 46% based on the area under the curve (AUC) analysis from 20 to 60 minutes post-injection.

[0110] Interestingly, during the PET scan, same elimination rate of [18F]YM-III-109 was observed under the self-blocking condition, suggesting reversible binding of the radiotracer in the mouse brain.

[0111] Next, we compared the brain uptake of [18F] YM-III-109 in WT mice and 5XFAD mice, a widely used preclinical AD mouse model. Detailed analyses were conducted using PET / CT imaging (sagittal, summed images from 0-60 minutes) and time-activity curves (TACs), both depicted in Fig. 5 and Fig. 6. Consistently, rapid brain uptake was observed for [18F]YM-III-109 by both WT and AD mice, achieving homogeneous distribution in the brain within 5 minutes post- injection. The tracer’s signal showed a gradual decline throughout the duration of the scanning. Notably, [18F] YM-III-109 demonstrated a significant increase in maximum brain uptake in AD mice when compared to the WT mice, which is consistent with the results from the autoradiography studies.

[0112] In summary, an NERP3 PET radiotracer, [18F]YM-III-109, was successfully obtained and was characterized in WT and AD mice. Autoradiography studies in WT, nlrp3- / - and AD mouse brain tissues clearly demonstrated its specific labeling and showed higher expression of NERP3 in AD mice. PET / CT studies of this radiotracer in WT and AD mice demonstrated rapid and improved brain uptake compared to previous PET tracers. Notably, self-blocking studies in WT mice supported selective labeling by this radiotracer in vivo. The results also revealed increased radioactivity in AD mice, suggesting increased expression of NLRP3 proteins, consistent with literature results. Taken together, [18F] YM-III-109 was successfully used as a CNS PET radiotracer.

[0113] While the invention has been described in terms of its several exemplary embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims. Accordingly, the present invention should not be limited to the embodiments as described above but should further include all modifications and equivalents thereof within the spirit and scope of the description provided herein.

Claims

CLAIMSWe claim:

1. A compound of Formula I:A is a 6-membered ring selected from benzene, pyridine, pyrimidine, and 1,2-diazine;R1 is: an unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted C1-C8 alkyl; an unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted C1-C8 alkoxyl; amino; nitro; OH; or halogen;R4 is: halogen, amino, nitro or cyano;R2, R3, and R5 are the same or different and are independently: H; C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkyl; C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkoxyl; C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkylcarbonyl; halogen; hydroxyl; amino; nitro; or cyano;W is: an unbranched, branched, saturated, unsaturated, substituted or unsubstituted Cl- C4 alkyl; NH; S; or SO2;B is: 1,2,3-traizole; 1,2,4 triazole; 1,2-diazole; 1,3-diazole; 1,2,4 oxadizole; isoxazole; or 1,3,4-oxadiazole;D is present or absent and when present is an aliphatic cyclic ring with an optional R6 substituent;Y is present or absent and when present is N or C;Z is present or absent and when present is N or C;E is: a 5C or 6C aromatic ring with an optional R7 substituent; or two 5C or 6C aromatic rings each with an optional R7 substituent;M is: CO; CO-N-RIO; SO2; or CS;L is: a linker sequence comprising an unbranched, branched, saturated, unsaturated,substituted or unsubstituted C3-C6 alkyl; cyclopropyl with an optional R8 substituent; N; or with an optional R8 substituent and n = 0-4;R6, R7 and R8 are the same or different, are optional, and are independently: H; C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkyl; C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkoxyl; C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkylcarbonyl, halogen, hydroxyl, amino, nitro, cyano, ester and carboxylic acid, and salts, hydrates and stereoisomers thereof.

2. The compound according to claim 1, having a Formula II:Formula II whereinR9 is H; C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, or substituted or unsubstituted alkyl;R4 is halogen;RIO is:where Rll is: H; C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkyl; C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkoxyl; Cl- C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkylcarbonyl, halogen, hydroxyl, amino, nitro or cyano; andL and E are covalently bonded (linked) together to form:where X is independently C, N, or O and forms an aromatic heterocyclic ring with theR7 substituent.

3. The compound according to claim 1, having a Formula III:Formula III whereinR9 is H; C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, or substituted or unsubstituted alkyl;R4 is halogen; and D and E are covalently bonded directly to each other and form a two-ring system:

4. A PET radiotracer comprising a compound of any of claims 1-3 labeled with C-11 or F-11.

5. The PET radiotracer, of claim 4, having Formula IV:

6. A pharmaceutical composition comprising at least one compound of claim 4 or 5, and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition of claim 6, wherein the at least one compound is the compound of Formula IV:

8. A method of imaging NLRP3 inflammasome-associated inflammation in a subject in need thereof, comprising administering to said subject a pharmaceutical composition of claim 6, and detecting radiation emitted from C-11 and / or F-11 labeled compounds within the subject.

9. The method of claim 8, wherein the step of detecting is performed by PET imaging or PET-CT imaging.

10. The method of claim 8, wherein said NRLP3 inflammasome-associated inflammation is selected from the group consisting of AD, PD, MS, TBI, AMI, heart failure, arthritis, diabetes, gout, COVID-19, and an autoinflammatory condition.

11. A method of visualizing an NRLP3 inflammasome, comprising contacting the NRLP3 inflammasome with at least one compound of any of claims 1-3 that is labeled with C-11 and / or F-11, wherein the step of contacting is performed under physiological conditions that permit the at least one compound to bind to the NLRP3 protein of the NLRP3 inflammasome, and visualizing the NRLP3 inflammasome by detecting C-11 and / or F-11 radiation emitted from the at least one compound bound to the NRLP3 inflammasome.

Images