Compounds as NLRP3 inhibitors and compositions and uses thereof

Abstract

NSIs with anti-inflammatory activity are provided, as are methods of using the NSIs to inhibit inflammation and prevent or treat 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, or an autoimmune or autoinflammatory disease.

Description

[0001] COMPOUNDS AS NLRP3 INHIBITORS AND COMPOSITIONS AND USES THEREOF

[0002] CROSS-REFERENCE TO REEATED APPLICATIONS

[0003] This application claims benefit of United States provisional patent application 63 / 664,426 filed June 26, 2024.

[0004] STATEMENT OF FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0005] This invention was made with government support under Grant Number: IU01AG076481 awarded by National Institutes of Health. The United States government has certain rights in the invention.

[0006] BACKGROUND OF THE INVENTION

[0007] Field, of the Invention

[0008] The invention generally relates to small molecule compounds that modulate the innate immune responses and methods of their use to inhibit inflammatory responses associated with innate immune dysregulation or over-activation. In particular, the invention provides small molecules which are NLRP3 inhibitors (NSIs), and methods of using these compounds and analogs thereof, e.g. to prevent or treat NRLP3 inflammasome dysregulation associated diseases and conditions, 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, CO VID-19, diabetes, macular degeneration, and autoimmune / autoinflammatory diseases.

[0009] Description of Related Art

[0010] 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)- ip 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.

[0011] 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.

[0012] 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 non- steroid 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 (MO As) holds great promise to provide effective treatments.

[0013] 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-ip 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.

[0014] 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 / PS1 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.

[0015] 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-ip 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-ip 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.

[0016] 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.

[0017] 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 COVID-19 exhibit mild-to-medium symptoms, however, 5-10% of COVID-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.

[0018] 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 CO VID- 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 COVID-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 COVID- 19.

[0019] SUMMARY OF THE INVENTION

[0020] The development of small molecule NSIs has attracted extensive attention as this signaling pathway has been indicated as having a critical role in many human diseases, such as AD, MS, AMI, TBI, PD, AMI, heart failure, diabetes, COVID-19, arthritis, macular degeneration, and autoinflammatory diseases. Therefore, small molecule NSIs can be effective therapeutic agents with broad indications. We have designed and developed new chemical entities as novel NSIs. Thus, these compounds, depicted in generic Formula I below, are NSIs and represent novel therapeutic agents for AD, MS, AMI, PD, TBI, CO VID-19, heart failure, arthritis, diabetes, macular degeneration, and gout.

[0021] Other features and advantages of the present invention will be set forth in the description of 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.

[0022] DETAILED DESCRIPTION

[0023] It is an object of this invention to provide a compound of Formula I:

[0024] Formula I wherein

[0025] A is: benzene, pyridine, pyrimidine, or 1,2-diazine;

[0026] R1is: unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted C1-C8 alkyl; unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted C1-C8 alkoxyl; amino; nitro; OH; or halogen;

[0027] R4is: halogen; amino; nitro; or cyano; cyclic or acyclic, substituted or unsubstituted C1-C8 alkyl.

[0028] R2, R3, and R5are the same or different and are independently selected from: 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; and cyano;

[0029] W is: unbranched, branched, saturated, unsaturated, substituted or unsubstituted Cl- C4 alkyl; NH; S; or SO2;

[0030] 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;

[0031] D is present or absent and when present is an aliphatic cyclic ring with an optional R6 substituent;

[0032] Y is present or absent and when present is N or C;

[0033] Z is present or absent and when present is N or C;

[0034] 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;

[0035] M is: CO; CO-N-RIO; SO2; or CS;

[0036] 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;

[0037] R6, R7a, R7b and R8 are the same or different, present or absent, and each independently represent: 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.

[0038] In a particular embodiment of the invention, there is provided a compound of formula IA:

[0039] Formula IA wherein

[0040] R9 is H; C1 -C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, or substituted or unsubstituted alkyl;

[0041] R4 is halogen; cyclic or acyclic, substituted or unsubstituted C1-C8 alkyl.

[0042] RIO is: where R1 1 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

[0043] L and E are covalently bonded (linked) together to form: where X is independently C, N, or O and forms an aromatic heterocyclic ring with the R7 substituent.

[0044] All other components are as described for Formula I.

[0045] In a particular embodiment of the invention, there is provided a compound of Formula IB:

[0046] Formula IB wherein

[0047] R9 is H; C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, or substituted or unsubstituted alkyl;

[0048] R4 is halogen; cyclic or acyclic, substituted or unsubstituted C1-C8 alkyl.

[0049] D and E are covalently bonded directly to each other and form a two-ring system: All other components are as described for Formula I.

[0050] In a further embodiment, the present invention relates to the following compounds of formula (I):

[0051]

[0052] DISEASES AND CONDITIONS THAT ARE PREVENTED AND / OR TREATED

[0053] The NSIs disclosed herein are used to treat any disorder or condition associated with (e.g. caused by or related to or which exacerbates) unwanted NLRP3 inflammasome activation and / or consequences of such activation, e.g. unwanted production of pro- inflammatory cytokines pro-IL-ip 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 but not limited to multiple sclerosis, arthritis, type II diabetes mellitus, gout and ischemia.

[0054] 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 the 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.

[0055] Examples of particular auto-inflammatory diseases which may be prevented and / or treated by 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 (IE-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, Aphthous Stomatitis, 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.

[0056] In one aspect, the compounds are used to treat Multiple sclerosis (MS). MS refers to all types of MS including relapse-remitting, secondary progressive, and primary progressive MS. In one aspect, the compounds are used to treat neurodegenerative disorders including but not limited to AD, PD, ALS, and Huntington’s disease.

[0057] In one aspect, the compounds are used to prevent or treat ARDS / ALI and cytokine storm associated with COVID- 19.

[0058] COMPOSITIONS

[0059] The present invention provides compositions comprising one or more (at least one of the) compounds described herein, and / or pharmaceutically acceptable salts of the compounds. The compositions are generally for use in preventing or treating inflammation, e.g. inflammation caused by formation and activity of NLRP3 inflammasomes. The compositions include one or more substantially purified compounds as described herein, and a pharmacologically suitable (compatible, acceptable) carrier, which may be a physiologically acceptable carrier. The preparation of such compositions is known to those of skill in the art. Typically, such compositions are prepared either as liquid solutions or suspensions, however solid forms such as tablets, pills, powders and the like are also contemplated. Solid forms suitable for solution in, or suspension in, liquids prior to administration may also be prepared. The preparation may also be emulsified. The active ingredients may be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredients. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol and the like, or combinations thereof. In addition, the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and the like. In addition, the compositions may contain other agents with different but complementary activities, e.g. other antiinflammatory agents, analgesics, blood thinners, antihistamines, etc. If it is desired to administer an oral form of the compositions, various thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders and the like may be added. The compositions of the present invention may contain any such additional ingredients so as to provide the composition in a form suitable for administration. The final amount of compound in the formulations may vary. However, in general, the amount in the formulations will be from about 1-99%. Still other suitable formulations for use in the present invention can be found, for example in Remington's Pharmaceutical Sciences, Philadelphia, Pa., 19th ed. (1995).

[0060] As used herein, "pharmaceutically acceptable salts" refers to the relatively non-toxic, inorganic and organic acid addition salts, and base addition salts, of compounds of the present invention. These: salts can be prepared in situ during the final isolation and purification of the compounds. In particular, acid addition salts can be prepared by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Exemplary acid addition salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactiobionate, sulfamates, malonates, salicylates, propionates, methylene-bis-.beta.-hydroxynaphthoates, gentisates, isethionates, di-p-toluoyltartrates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, cyclohexylsulfamates and laurylsulfonate salts, and the like. See, for example S. M. Berge, et al., "Pharmaceutical Salts," J. Pharm. Sci., 66, 1- 19 (1977) which is incorporated herein by reference. Base addition salts can also be prepared by separately reacting the purified compound in its acid form with a suitable organic or inorganic base and isolating the salt thus formed. Base addition salts include pharmaceutically acceptable metal and amine salts. Suitable metal salts include the sodium, potassium, calcium, barium, zinc, magnesium, and aluminum salts. The sodium and potassium salts are preferred. Suitable inorganic base addition salts are prepared from metal bases which include sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide and the like. Suitable amine base addition salts are prepared from amines which have sufficient basicity to form a stable salt, and preferably include those amines which are frequently used in medicinal chemistry because of their low toxicity and acceptability for medical use. ammonia, ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N- benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, ephenamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids, e.g., lysine and arginine, and dicyclohexylamine, and the like.

[0061] Precursors (generally inactive precursors) of the compounds which are metabolized after administration to yield the compounds / active agents described herein in an active form are also encompassed.

[0062] The therapeutic agents described herein are used alone or in combination with other suitable agents, e.g. other agents that prevent or treat inflammation (for example, by another mechanism), including but not limited to: IL-1R antagonists such as anakinra; monoclonal antibodies against interleukin ip such as canakinumab (Haris); various interleukin 1 binding proteins such as rilonacept; and the like. Accordingly, the compositions provided herein may include one or more of these additional agents.

[0063] The compositions (preparations) of the present disclosure may be administered by any of the many suitable means which are well known to those of skill in the art, including but not limited to: by injection (e.g. intravenous, intraperitoneal, intramuscular, subcutaneous, intra-aural, intraarticular, intramammary, and the like), by absorption through epithelial or mucocutaneous linings (e.g., nasal, oral, vaginal, rectal, gastrointestinal mucosal linings, and the like), by inhalation, orally, intranasally, by ingestion of a food or probiotic product containing the agent, topically (e.g. on areas such as eyes, skin, in ears or on inflamed areas), as eye drops, via sprays, incorporated into dressings or bandages (e.g. lyophilized forms may be included directly in the dressing), etc. Generally, the mode of administration is oral or by injection so as to effect systemic distribution of the agent, or locally by direct application, via an appropriate means, at or near a site of inflammation or a site where inflammation is likely to occur.

[0064] The amount of a compound that is administered varies depending on several factors, including the disease or condition being treated, the stage of the disease, the overall health of the subject, the subject’s age, gender and weight, etc. In general, the amount is in the range of from about 0.01 to about 100 mg / kg of body weight, and usually is in the range of from about 1 to about 20 mg / kg of body weight. The subjects (patients) that are treated as described herein are generally mammals, e.g. humans, but veterinary applications of this technology are also encompassed, e.g. for companion pets such as cats and dogs.

[0065] The compounds of the disclosure are utilized to prevent and / or to treat conditions and / or diseases associated with (e.g. caused by) NRLP3 inflammasome activity (i.e. to treat NRLP3 inflammasome- associated inflammation). By “prevent” we mean that the compounds are administered prophylactically to a subject who is likely to develop the disease or condition, but before symptoms or indications of disease develop, or early in development. For example, subjects who have experienced AMI may be treated as described herein in order to prevent subsequent adverse cardiac remodeling during the “second wave” of inflammation. Alternatively, or in addition, the compounds may be administered in order to treat conditions / diseases that have already developed (e.g. when symptoms are already being exhibited, or are observable or measurable). In this case, administration of the compounds ameliorates and may reverse the symptoms or at least arrest the disease (e.g. prevent further disease development or progress). Those of skill in the art will recognize that while a goal of prevention or treatment may be to completely prevent or alleviate disease symptoms, much benefit can also accrue if symptoms not fully eradicated but are lessened, decreased or their onset is slowed, even though a full-blown cure is not effected. The amount that is administered is a therapeutically effective amount, e.g. an amount that eradicates one or more symptoms of the disease or condition, or an amount that at least lessens one or more symptoms (at least one) of the disease or condition.

[0066] Methods of treating NRLP3 inflammasome-related diseases are provided. Such methods may include a step of identifying a subject in need of such treatment (e.g. a subject with one or more symptoms of an NRLP3 inflammasome-related disorder, or a subject who is likely to develop such a disorder). For example, patients who have or have had MS may be treated, as can patients for whom there is reason to suspect that a relapse of MS is likely to or might occur. The same is true for other conditions that are treated by the agents disclosed herein, i.e. a subject suitable for undergoing treatment may have one or more readily observable symptoms, or early symptoms, or a predisposition to development of the disease (e.g. genetically, due to life style, due to exposure to a substance that is known to cause inflammation, etc.) that is being prevented or treated; or may be experiencing or likely to experience a relapse.

[0067] As indicated above, the present invention inter alia provides the specified compounds for use in a method of preventing or treating NRLP3 inflammasome-associated inflammation, including but not limited to neuroinflammations associated with MS, AD, etc., as well as acute inflammation, or acute inflammatory response, which may occur in variety of illness in which an injury induces inflammation. Generally, at least one (one or more) of the compounds is administered. Further, the present invention may provide the specified compound as an active therapeutic ingredient in the specified method. The specified compound may be the only active agent that is administered, (or the only active agent in a composition), or may be administered with one or more other active agents (or present in a composition with one or more other active agents). Examples of other active agents include but are not limited to: at least one anti-inflammatory agent, many of which are known in the art; pain medication; nutraceuticals; steroids; NSAIDs; etc. “Administered with” refers to being included in a course of treatment with other agents over a period of hours, days, weeks, or longer; and / or being included in a single composition with (compounded with) other agents. Further, the present invention may provide the specified compound for use in a method of treatment of the human or animal body by therapy, the method comprising the specified method.

[0068] Also provided are methods of preventing or decreasing NREP3 inflammasome activation in a cell, comprising contacting the cell with an amount of at least one compound as disclosed herein. The amount is sufficient to prevent, entirely or at least by about 50% of the level of activation that occurs in the absence of the at least one compound. The cell may be in vitro or in vivo, e.g. in a test tube, petri dish, culture flask, etc. in a laboratory setting, or in a living being (e.g. a mammal) or part thereof, e.g. in an organ, limb, etc.

[0069] Also provided are methods of preventing, inhibiting or decreasing activity of an NREP3 inflammasome or a plurality of NREP3 inflammasomes. The methods comprise contacting the NREP3 inflammasome(s) with at least one compound disclosed herein. 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, thereby preventing, inhibiting or decreasing the activity of an NREP3 inflammasome or the plurality of NREP3 inflammasomes.

[0070] METHODS OF MAKING THE COMPOUNDS

[0071] Also encompassed herein are methods of making the disclosed compounds.

[0072] In some aspects, the methods are as illustrated in Scheme 1:

[0073] Scheme 1 where D, E, R2, R3, R4, R5, R7, R9 and RIO are as defined as described for Formula 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 / HoO; 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.

[0074] Before exemplary embodiments of the present invention are described in greater detail, 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.

[0075] 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.

[0076] 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. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

[0077] 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 publication dates which may need to be independently confirmed.

[0078] 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 antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0079] 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.

[0080] EXAMPLES

[0081] Example 1. NSIs prevent the release of IL-1 f> in vitro to NLRP3 inflammasome pathway

[0082] Cultured mouse macrophages were treated with LPS followed by ATP to induce the formation of the NLRP3 inflammasomes and measure the release of mature IL-ip in the supernatant (Table 1).

[0083] The ability of the exemplary compounds listed in Table 1 to inhibit the release of IL- ip was measured.

[0084] Table 1

[0085] Legend : +- 1 4-+ IC50 <0.1 «M; +++ IC500.1< x < 1 z / M; ++ IC501 < x < 10 / / M, + IC5010 / / M < x.

Claims

CLAIMSWe claim:

1. A compound of Formula I:Formula I whereinA is: benzene, pyridine, pyrimidine, or 1,2-diazine;R1is: unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted C1-C8 alkyl; unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted C1-C8 alkoxyl; amino; nitro; OH; or halogen;R4is: halogen; amino; nitro; or cyano;R2, R3, and R5are the same or different and are independently selected from: 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; and cyano;W is: 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; or 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 substituc-C-(CH2)n- with an optional R8 substituent and n = 0-4;R6, R7a, R7b and R8 are the same or different, present or absent, and each independently represent:: 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 un substituted alkoxyl; C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkylcarbonyl, halogen, hydroxyl, amino, nitro, cyano, ester and carboxylic acid.

2. The compound of claim 1, having Formula IA:Formula IA whereinR9 is H; C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, or substituted or unsubstituted alkyl;R4 is halogen;RIO is:where Rl l is: H; C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or un substituted 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 the R7 substituent.

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

4. The compound of claim 1, wherein the compound is selected from the group consisting of:5. A method of preventing or treating a disease or condition caused by inflammation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one compound of any of claims 1-4.

6. The method of claim 5, wherein the disease or condition is multiple sclerosis (MS), Alzheimer’s disease (AD), traumatic brain injury (TBI), Parkinson’s disease (PD), acute myocardial infarction (AMI), heart failure, gout, rheumatoid arthritis, CO VID-19, diabetes, macular degeneration, or an autoimmune or autoinflammatory disease.

7. A method of preventing or decreasing NOD-like receptor protein 3 (NLRP3) inflammasome activation in a cell, comprising contacting the cell with an amount of a compound of any of claims 1-4, wherein the amount is sufficient to prevent or decrease NLRP3 inflammasome activation in the cell.

8. A method of inhibiting or decreasing activity of a NOD-like receptor protein 3 (NLRP3) inflammasome, comprising contacting the NLRP3 inflammasome with a compound of any of claims 1-4 under conditions that permit the compound to bind to and inhibit of decrease the activity of the NLRP3 inflammasome.

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