The present disclosure relates to compounds that act as inhibitors of NLRP3 inflammasomes; pharmaceutical compositions comprising the compounds; and methods of treating cancer and disorders associated with inflammation and inflammaging.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing in XML file (Name: 762525_BIOT-007PC_ST26.xml; Size: 2,722 bytes; Date of Creation: Mar. 24, 2025) is incorporated herein by reference in its entirety.

BACKGROUND

Aging frailty poses a very concerning problem for the overall health and well-being of individuals and is characterized as a syndrome of multisystem physiological dysregulation. Aging frailty is a geriatric syndrome characterized by weakness, low physical activity, slowed motor performance, exhaustion, and unintentional weight loss (Yao, X. et al., Clinics in Geriatric Medicine 27(1): 79-87 (2011)). Furthermore, there are many studies showing a direct correlation between aging frailty and inflammation (Hubbard, R. E., et al., Biogerontology 11(5):635-641 (2010)). Immunosenescence is characterized by a low grade, chronic systemic inflammatory state known as inflammaging (Franceshi, C. et al., Annals of the New York Academy of Sciences 908:244-254 (2000)). This heightened inflammatory state or chronic inflammation found in aging and aging frailty leads to immune dysregulation and a complex remodeling of both innate and adaptive immunity.

Inhibiting the NLRP3 inflammasome, an oligomeric protein complex that includes ASC and caspase-1, mediates inflammation in an extensive number of preclinical models (Schwaid, A. G., J. Med. Chem. 2021, 64(1), 101-122). At the same time, the NLRP3 inflammasome is part of a larger pro-inflammatory pathway, whose modulation is also being explored. NLRP3 is an inflammasome sensor protein that has been well studied in a number of disease contexts. Many different indications are associated with the NLRP3 inflammasome including diseases related to aging, cryopyrin-associated periodic syndrome (CAPS), nonalcoholic steatohepatitis (NASH), gout, coronary artery disease, Crohn's disease, osteoarthritis, rheumatoid arthritis, Alzheimer's disease, Parkinson's disease, intestinal disorders, acute respiratory distress syndrome (ARDS), amyotrophic lateral sclerosis (ALS), cancer, and dermatological diseases.

NLRPs, including NLRP3, have been implicated in various eye diseases with not all being related to aging (Niu, L., et al., PLoS ONE 10(5): e0126277; and Mugisho, O., et al., Experimental Eye Research 215 (2022) 108911). For example, the NLRP3 inflammasome has been shown to play a role in both glaucoma, an ocular disease most commonly occurring in older adults, and dry eye, which can occur in people of any age.

The NLRP3 inflammasome is therefore a promising drug target. The breadth of the indications it is implicated in speak to the need for therapeutics that target the NLRP3 inflammasome.

SUMMARY

Provided here are compounds that inhibit the NLRP3 inflammasome. As such, these compounds are useful in the treatment of a variety of indications, including inflammaging and inflammation.

In particular, provided herein is a compound that binds to amino acids of Y143, R147, F257, Y258, H260, E263, V264, L272, L275, I276, C279, F299, G328, L331, L332, L335, and C514 of a NLRP3 amino acid sequence of SEQ ID NO: 1. Such a compound is useful for the treatment of a variety of conditions in a subject, such as cancer, inflammaging, inflammation, and age-related diseases.

In an aspect, provided herein is a compound of Formula I:

or a pharmaceutically acceptable salt thereof.

or a pharmaceutically acceptable salt thereof.

or a pharmaceutically acceptable salt thereof.

In yet another aspect, provided herein is a pharmaceutical composition comprising a compound described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

In yet another aspect, provided herein is a method of inhibiting NLRP3 inflammasome in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound described herein.

DETAILED DESCRIPTION

Provided here are compounds that inhibit the NLRP3 inflammasome. In a non-limiting embodiment, the compounds bind to amino acids of Y143, R147, F257, Y258, H260, E263, V264, L272, L275, 1276, C279, F299, G328, L331, L332, L335, and C514 of a NLRP3 amino acid sequence of SEQ ID NO: 1. Such compounds are useful for the treatment of a variety of conditions in a subject, such as cancer, inflammaging, inflammation, and age-related diseases. As such, these compounds, as well as pharmaceutical compositions that comprise these compounds, are useful in the treatment of a variety of indications, including cancer, inflammaging, inflammation, and age-related diseases.

These compounds are also useful for treating obesity and obesity-related disorders.

Definitions

Listed below are definitions of various terms used to describe the compounds and compositions disclosed herein. These definitions apply to the terms as they are used throughout this specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group.

Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and peptide chemistry are those well-known and commonly employed in the art.

As used herein, the articles “a” and “an” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.

As used herein, the term “about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. As used herein when referring to a measurable value such as an amount, a temporal duration, and the like, the term “about” is meant to encompass variations of ±10%, including ±5%, ±1%, and +0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

The term “administration” or the like as used herein refers to the providing a therapeutic agent to a subject. Multiple techniques of administering a therapeutic agent exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary, and topical administration.

As used herein, the term “contacting” refers to the bringing together of indicated moieties in an in vitro system or an in vivo system. For example, “contacting” a cell with a compound includes the administration of a compound of the present invention to an individual, subject, or patient, such as a human, as well as, for example, introducing a compound into a sample containing a purified preparation containing the cell.

The term “treat,” “treated,” “treating,” or “treatment” includes the diminishment or alleviation of at least one symptom associated or caused by the state, disorder or disease being treated. In certain embodiments, the treatment comprises alleviating the symptoms of inflammaging and age-related disorders.

As used herein, the term “prevent” or “prevention” means no disorder or disease development if none had occurred, or no further disorder or disease development if there had already been development of the disorder or disease. Also considered is the ability of one to prevent some or all of the symptoms associated with the disorder or disease.

As used herein, the term “patient,” “individual,” or “subject” refers to a human or a non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and marine mammals. Preferably, the patient, subject, or individual is human.

As used herein, the terms “effective amount,” “pharmaceutically effective amount,” and “therapeutically effective amount” refer to a nontoxic but sufficient amount of an agent to provide the desired biological result. That result may be reduction or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An appropriate therapeutic amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

As used herein, “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the present disclosure and are physiologically acceptable to the patient. Supplementary active compounds may also be incorporated into the compositions. The “pharmaceutically acceptable carrier” may further include a pharmaceutically acceptable salt of the compound disclosed herein. Other additional ingredients that may be included in the pharmaceutical compositions are known in the art and described, for example, in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

As used herein, an “epitope” refers to a surface or region on one or more entities (e.g., an NLRP3 polypeptide) that is capable of interacting with a binding molecule (e.g., the compounds of the disclosure). For example, a protein epitope may contain one or more amino acids and/or post-translational modifications (e.g., phosphorylated residues) which interact with the binding molecule. In some embodiments, an epitope may be a “conformational epitope,” which refers to an epitope involving a specific three-dimensional arrangement of the entity(ies) having or forming the epitope. For example, conformational epitopes of proteins may include combinations of amino acids and/or post-translational modifications from folded, non-linear stretches of amino acid chains.

As used herein, “inflammaging” is defined as chronic sterile inflammation that is associated with numerous age-related diseases.

As used herein, “age-related disorder” refers to disorders that are associated with the aging processStated alternatively, age-related disorders are diseases associated with the elderly. Non-limiting examples of age-related diseases include atherosclerosis and cardiovascular disease, cancer, arthritis, cataracts, osteoporosis, type 2 diabetes, hypertension, and Alzheimer's disease. The incidence of all of these diseases increases exponentially with age.

As used herein, “GLP-1” refers to glucagon-like peptide-1, which is a 30- or 31-amino-acid-long peptide hormone deriving from the tissue-specific posttranslational processing of the proglucagon peptide. It is produced and secreted by intestinal enteroendocrine L-cells and certain neurons within the nucleus of the solitary tract in the brainstem upon food consumption. Beside the insulinotropic effects, GLP-1 has been associated with numerous regulatory and protective effects. Glucagon-like peptide-1 receptor agonists have gained approval as drugs to treat diabetes and obesity.

As used herein, “comorbidity” refers to a disease or medical condition that is simultaneously present with another disease or medical condition in a patient.

At various places in the present specification, certain features of the compounds are disclosed in groups or in ranges. It is specifically intended that such a disclosure include each and every individual subcombination of the members of such groups and ranges. For example, the term “C1-6 alkyl” is specifically intended to individually disclose (without limitation) methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl and C6 alkyl.

The term “substituted” means that an atom or group of atoms formally replaces hydrogen as a “substituent” attached to another group. The term “substituted,” unless otherwise indicated, refers to any level of substitution, e.g., mono-, di-, tri-, tetra- or penta-substitution, where such substitution is permitted. The substituents are independently selected, and substitution may be at any chemically accessible position. It is to be understood that substitution at a given atom is limited by valency. It is to be understood that substitution at a given atom results in a chemically stable molecule. The phrase “optionally substituted” means unsubstituted or substituted. The term “substituted” means that a hydrogen atom is removed and replaced by a substituent. A single divalent substituent, e.g., oxo, can replace two hydrogen atoms.

The term “Cn-m” indicates a range which includes the endpoints, wherein n and m are integers and indicate the number of carbons. Examples include C1-4, C1-6 and the like.

As used herein, the term “alkyl,” by itself or as part of another substituent means, unless otherwise stated, a straight or branched chain hydrocarbon having the number of carbon atoms designated (i.e., C1-C6alkyl means an alkyl having one to six carbon atoms) and includes straight and branched chains. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert butyl, pentyl, neopentyl, and hexyl. Other examples of C1-C6alkyl include ethyl, methyl, isopropyl, isobutyl, n-pentyl, and n-hexyl.

The term “alkenyl” employed alone or in combination with other terms, refers to a straight-chain or branched hydrocarbon group corresponding to an alkyl group having one or more double carbon-carbon bonds. An alkenyl group formally corresponds to an alkene with one C—H bond replaced by the point of attachment of the alkenyl group to the remainder of the compound. The term “Cn-m alkenyl” refers to an alkenyl group having n to m carbons. In some embodiments, the alkenyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms. Example alkenyl groups include, but are not limited to, ethenyl, n-propenyl, isopropenyl, n-butenyl, sec-butenyl and the like.

The term “alkynyl” employed alone or in combination with other terms, refers to a straight-chain or branched hydrocarbon group corresponding to an alkyl group having one or more triple carbon-carbon bonds. An alkynyl group formally corresponds to an alkyne with one C—H bond replaced by the point of attachment of the alkyl group to the remainder of the compound. The term “Cn-m alkynyl” refers to an alkynyl group having n to m carbons. Example alkynyl groups include, but are not limited to, ethynyl, propyn-1-yl, propyn-2-yl and the like. In some embodiments, the alkynyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms.

The term “alkylene,” employed alone or in combination with other terms, refers to a divalent alkyl linking group. An alkylene group formally corresponds to an alkane with two C—H bond replaced by points of attachment of the alkylene group to the remainder of the compound. The term “Cn-m alkylene” refers to an alkylene group having n to m carbon atoms. Examples of alkylene groups include, but are not limited to, ethan-1,2-diyl, ethan-1,1-diyl, propan-1,3-diyl, propan-1,2-diyl, propan-1,1-diyl, butan-1,4-diyl, butan-1,3-diyl, butan-1,2-diyl, 2-methyl-propan-1,3-diyl and the like.

The term “alkoxy,” employed alone or in combination with other terms, refers to a group of formula —O-alkyl, wherein the alkyl group is as defined above. The term “Cn-m alkoxy” refers to an alkoxy group, the alkyl group of which has n to m carbons. Example alkoxy groups include methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), t-butoxy and the like. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, the term “haloalkyl” refers to an alkyl group, as defined above, substituted with one or more halo substituents, wherein alkyl and halo are as defined herein. Haloalkyl includes, by way of example, chloromethyl, trifluoromethyl, bromoethyl, chlorofluoroethyl, and the like.

As used herein, the term “halo” or “halogen” alone or as part of another substituent means, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom, preferably, fluorine, chlorine, or bromine, more preferably, fluorine or chlorine.

As used herein, the term “cycloalkyl” means a non-aromatic carbocyclic system that is fully saturated having 1, 2 or 3 rings wherein such rings may be fused. The term “fused” means that a second ring is present (i.e., attached or formed) by having two adjacent atoms in common (i.e., shared) with the first ring. Cycloalkyl also includes bicyclic structures that may be bridged or spirocyclic in nature with each individual ring within the bicycle varying from 3-8 atoms. The term “cycloalkyl” includes, but is not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[3.1.0]hexyl, spiro[3.3]heptanyl, and bicyclo[1.1.1]pentyl. In an embodiment, “cycloalkyl” is C3-10 cycloalkyl. In another embodiment, “cycloalkyl” is C3-6 cycloalkyl.

As used herein, the term “aromatic” refers to a carbocycle or heterocycle with one or more polyunsaturated rings and having aromatic character, i.e., having (4n+2) delocalized π (pi) electrons, where n is an integer.

As used herein, the term “aryl” means an aromatic carbocyclic system containing 1, 2 or 3 rings, wherein such rings may be fused, wherein fused is defined above. If the rings are fused, one of the rings must be fully unsaturated and the fused ring(s) may be fully saturated, partially unsaturated or fully unsaturated. The term “aryl” includes, but is not limited to, phenyl, naphthyl, indanyl, and 1,2,3,4-tetrahydronaphthalenyl. In some embodiments, aryl groups have 6 carbon atoms. In some embodiments, aryl groups have from six to ten carbon atoms. In some embodiments, aryl groups have from six to sixteen carbon atoms.

It is to be understood that if an aryl, heteroaryl, cycloalkyl, or heterocyclyl moiety may be bonded or otherwise attached to a designated moiety through differing ring atoms (i.e., shown or described without denotation of a specific point of attachment), then all possible points are intended, whether through a carbon atom or, for example, a trivalent nitrogen atom. For example, the term “pyridinyl” means 2-, 3- or 4-pyridinyl, the term “thienyl” means 2- or 3-thienyl, and so forth.

As used herein, the term “Nucleotide-binding oligomerization domain, Leucine rich Repeat and Pyrin domain containing 3” or “NOD-, LRR-, and pyrin domain-containing protein 3” or “NLRP3” refers to UNIPROT reference number Q96P20 and the amino acid sequence of SEQ ID NO: 1, shown below.

NLRP3 is a member of the Nod-like receptor (NLR) family of proteins. NLRP3 is an intracellular sensor that detects a broad range of danger signals and environmental insults resulting in a protective pro-inflammatory response designed to impair pathogens and repair tissue damage via the formation and activation of the NLRP3 inflammasome (Coll, R. C., et al., Trends Pharmacol. Sci., 2022, 43(8), 653-668). NLRP3 is highly expressed in subsets of peripheral leukocytes and microglia of the central nervous system.

NLRP3 displays a tripartite structure consisting of a Pyrin Domain (PYD), a central nucleotide binding and oligomerization domain of the NACHT subfamily of NTPases, and a Leucine Rich Repeat domain (LRR). The NACHT domain has ATPase activity that is required to induce an inactive ADP-bound decameric assembly in cells (Hochheiser, I., et al., Nature, 2022, 604, 184-189), (Brinkschulte, R., et al., Commun. Biol., 2022, 5, 1176).

Assembly of the NLRP3 inflammasome leads to caspase 1-dependent secretory release of the pro-inflammatory cytokines IL-1β and IL-18 as well as to gasdermin D-mediated pyroptotic cell death.

In an aspect, provided herein is a compound that binds to one or more amino acids of Y143, R147, F257, Y258, H260, E263, V264, L272, L275, I276, C279, F299, G328, L331, L332, L335, and C514 of a NLRP3 amino acid sequence of SEQ ID NO: 1.

In an embodiment, the compound binds to at least amino acids of L275 and C279 of a NLRP3 amino acid sequence of SEQ ID NO: 1. In another embodiment, the compound binds to amino acids of Y143, R147, F257, Y258, H260, E263, V264, L272, L275, 1276, C279, F299, G328, L331, L332, L335, and C514 of a NLRP3 amino acid sequence of SEQ ID NO: 1.

Compounds

Provided herein are compounds that are inhibitors of the NLRP3 inflammasome and are thus useful in the treatment of inflammatory disorders, including cancer and other proliferation diseases.

In an aspect, provided herein is a compound of Formula I:

In an embodiment,

In an embodiment,

In another embodiment,

In yet another embodiment,

In another embodiment,

or a pharmaceutically acceptable salt thereof.

In still another embodiment, Ring A is phenyl.

In an embodiment, Ring B is selected from the group consisting of C6-10 aryl, 5-6 membered heteroaryl, C3-6 cycloalkyl, and 3-6 membered heterocycloalkyl. In another embodiment, Ring B is phenyl. In yet another embodiment, Ring B and R5 are absent.

In another embodiment, R2 is H.

In an embodiment, R3 is C1-6 alkyl substituted by OH or C1-3 alkoxy. In still another embodiment, R3 is C1-6 alkyl substituted by OH. In an embodiment, R3 is C1-6 alkyl substituted by C1-3alkoxy. In an embodiment, R3 is selected from the group consisting of C1-6 alkyl, C3-6 cycloalkyl, and 3-6 membered heterocycloalkyl, all of which are substituted by OH, C1-6 alkoxy, NH2, NH(C1-6 alkyl), N(C1-6 alkyl)2, halo, SO2R6, or C0-3 alkyene-5-10 membered heteroaryl. In another embodiment, R3 is C1-6 alkyl substituted by 3-6 membered heterocycloalkyl optionally substituted with one or two halo or OH. In yet another embodiment, R3 is 3-6 membered heterocycloalkyl is optionally substituted by C1-6alkyl. In an embodiment, R3 is C1-6 alkyl substituted by N(Ra)2. In another embodiment, each Ra is independently H or C1-6 alkyl.

In yet another embodiment, R3 is C1-6 alkyl substituted by OH or C1-3 alkoxy. In still another embodiment, R3 is C1-6 alkyl substituted by OH. In an embodiment, R3 is C1-6 alkyl substituted by C1-3alkoxy. In an embodiment, R3 is selected from the group consisting of C1-6 alkyl, C3-6 cycloalkyl, and 3-6 membered heterocycloalkyl, all of which are substituted by OH, C1-6 alkoxy, NH2, NH(C1-6 alkyl), N(C1-6 alkyl)2, halo, SO2R6, or C0-3 alkyene-5-10 membered heteroaryl. In another embodiment, R3 is C1-6 alkyl substituted by 3-6 membered heterocycloalkyl optionally substituted with one or two halo. In yet another embodiment, R3 is 3-6 membered heterocycloalkyl is optionally substituted by C1-6 alkyl. In an embodiment, R3 is C1-6 alkyl substituted by N(Ra)2. In another embodiment, each Ra is independently H or C1-6 alkyl.

In another embodiment, R2 and R3, together with the atom to which they are attached, form a ring selected from the group consisting of C3-6 cycloalkyl and 3-6 membered heterocycloalkyl, both of which is substituted by OH. In yet another embodiment, R2 and R3, together with the atom to which they are attached, form C3-6 cycloalkyl substituted by OH. In still another embodiment, R2 and R3, together with the atom to which they are attached, form 3-6 membered heterocycloalkyl substituted by OH.

In an embodiment, each R4 is independently selected from the group consisting of halo, C1-6alkoxy, OC3-6 cycloalkyl, and C1-6 haloalkyl. In another embodiment, each R4 is independently selected from the group consisting of halo, C1-3 alkoxy, O-cyclopropyl, and C1-3 haloalkyl. In an embodiment, R4 is C1-6 alkoxy.

In yet another embodiment, each R5 is independently selected from the group consisting of C1-6 alkyl, halo, CN, and C(O)NH(C1-6 alkyl). In still another embodiment, each R5 is independently selected from the group consisting of C1-3 alkyl, halo, CN, and C(O)NH(C1-3 alkyl). In another embodiment, R5 is selected from the group consisting of C1-6 alkyl, halo, OH, C1-6 alkoxy, CN, and C1-6 haloalkyl.

In an embodiment, each R9 is independently selected from the group consisting of C1-3 alkyl, C1-6 alkoxy, C1-3 alkyl-OH, and halo.

In another embodiment, m is 0, 1, or 2. In yet another embodiment, m is 0. In still another embodiment, m is 1. In an embodiment, m is 2.

In another embodiment, n is 1. In yet another embodiment, n is 0. In still another embodiment, n is 2.

In another embodiment, p is 0, 1, or 2. In yet another embodiment, p is 0. In still another embodiment, p is 1. In an embodiment, p is 2.

In another embodiment, the compound of Formula I is selected from the group consisting of a compound in Table 1.

or a pharmaceutically acceptable salt thereof.

In another embodiment, the compound of Formula I is selected from the group consisting of a compound in Table 1A.

or a pharmaceutically acceptable salt thereof.

In another embodiment, the compound of Formula I is selected from the group consisting of a compound in Table 1B.

In another embodiment, the compound of Formula I is selected from the group consisting of a compound in Table 1C.

In an embodiment,

In another embodiment,

or a pharmaceutically acceptable salt thereof.

In still another embodiment, Ring A is phenyl.

In an embodiment, Ring B is selected from the group consisting of phenyl, 5-6 membered heteroaryl, C3-6 cycloalkyl, and 3-6 membered heterocycloalkyl. In another embodiment, Ring B is phenyl. In yet another embodiment, Ring B and R5 are absent.

In an embodiment, each R4 is independently selected from the group consisting of C1-6 alkoxy and OC3-6 cycloalkyl. In another embodiment, each R4 is independently selected from the group consisting of C1-3 alkoxy and O-cyclopropyl. In yet another embodiment, R4 is C1-3 alkoxy. In still another embodiment, R4 is O-cyclopropyl. In an embodiment, R4 is O-cyclobutyl.

In another embodiment, each R5 is independently selected from the group consisting of halo, CN, CONH2, and CONH(C1-6 alkyl). In yet another embodiment, R5 is halo. In still another embodiment, R5 is CN. In an embodiment, R5 is CONH2. In another embodiment, CONH(C1-3 alkyl).

In yet another embodiment, each R9 is independently selected from the group consisting of C1-6 alkyl-OH, halo, OH, NH2, NH(C1-6 alkyl), and N(C1-6 alkyl)2. In still another embodiment, R9 is C1-6alkyl-OH. In an embodiment, R9 is OH. In another embodiment, R9 is halo. In yet another embodiment, R9 is NH2. In still another embodiment, R9 is NH(C1-3 alkyl). In an embodiment, R9 is N(C1-3 alkyl)2.

In another embodiment, m is 0, 1, or 2. In yet another embodiment, m is 0. In still another embodiment, m is 1. In an embodiment, m is 2.

In another embodiment, n is 0, 1, or 2. In yet another embodiment, n is 0. In still another embodiment, n is 1. In an embodiment, n is 2.

In another embodiment, p is 0, 1, or 2. In yet another embodiment, p is 0. In still another embodiment, p is 1. In an embodiment, p is 2.

In another embodiment, the compound of Formula II is selected from the group consisting of a compound in Table 2.

or a pharmaceutically acceptable salt thereof.

In another embodiment, the compound of Formula II is selected from the group consisting of a compound in Table 2A.

Ex No.
Structure

or a pharmaceutically acceptable salt thereof.

In another embodiment, the compound of Formula II is selected from the group consisting of a compound in Table 2B.

Ex No.
Structure

In an aspect, provided herein is a compound of Formula III:

In an embodiment,

In another embodiment,

In another embodiment,

In another embodiment, the compound of Formula III is a compound of Formula IIIa:

or a pharmaceutically acceptable salt thereof.

In still another embodiment, Ring A is phenyl.

In an embodiment, Ring B is selected from the group consisting of C6-10 aryl, 5-6 membered heteroaryl, C3-6 cycloalkyl, and 3-6 membered heterocycloalkyl. In another embodiment, Ring B is phenyl. In yet another embodiment, Ring B and R5 are absent.

In another embodiment, R2 is H.

In yet another embodiment, R3 is C1-6 alkyl substituted by OH or C1-3 alkoxy. In still another embodiment, R3 is C1-6 alkyl substituted by OH. In an embodiment, R3 is C1-6 alkyl substituted by C1-3alkoxy. In an embodiment, R3 is selected from the group consisting of C1-6 alkyl, C3-6 cycloalkyl, and 3-6 membered heterocycloalkyl, all of which are substituted by OH, C1-6 alkoxy, NH2, NH(C1-6 alkyl), N(C1-6 alkyl)2, halo, SO2R6, or C0-3 alkyene-5-10 membered heteroaryl. In another embodiment, R3 is C1-6 alkyl substituted by 3-6 membered heterocycloalkyl optionally substituted with one or two halo or OH. In another embodiment, R3 is C1-6 alkyl substituted by 3-6 membered heterocycloalkyl optionally substituted with one or two halo. In yet another embodiment, R3 is 3-6 membered heterocycloalkyl is optionally substituted by C1-6 alkyl. In an embodiment, R3 is C1-6 alkyl substituted by N(Ra)2. In another embodiment, each Ra is independently H or C1-6 alkyl.

In another embodiment, R2 and R3, together with the atom to which they are attached, form a ring selected from the group consisting of C3-6 cycloalkyl and 3-6 membered heterocycloalkyl, both of which is substituted by OH. In yet another embodiment, R2 and R3, together with the atom to which they are attached, form C3-6 cycloalkyl substituted by OH. In still another embodiment, R2 and R3, together with the atom to which they are attached, form 3-6 membered heterocycloalkyl substituted by OH.

In an embodiment, each R4 is independently selected from the group consisting of halo, C1-6alkoxy, OC3-6 cycloalkyl, and C1-6 haloalkyl. In another embodiment, each R4 is independently selected from the group consisting of halo, C1-3 alkoxy, O-cyclopropyl, and C1-3 haloalkyl. In an embodiment, R4 is C1-6 alkoxy.

In yet another embodiment, each R5 is independently selected from the group consisting of C1-6 alkyl, halo, CN, and C(O)NH(C1-6 alkyl). In still another embodiment, each R5 is independently selected from the group consisting of C1-3 alkyl, halo, CN, and C(O)NH(C1-3 alkyl). In another embodiment, R5 is selected from the group consisting of C1-6 alkyl, halo, OH, C1-6 alkoxy, CN, and C1-6haloalkyl.

In an embodiment, each R9 is independently selected from the group consisting of C1-3 alkyl, C1-6 alkoxy, C1-3 alkyl-OH, and halo.

In another embodiment, m is 0, 1, or 2. In yet another embodiment, m is 0. In still another embodiment, m is 1. In an embodiment, m is 2.

In another embodiment, n is 1. In yet another embodiment, n is 0. In still another embodiment, n is 2.

In another embodiment, p is 0, 1, or 2. In yet another embodiment, p is 0. In still another embodiment, p is 1. In an embodiment, p is 2.

In another embodiment, the compound of Formula I is selected from the group consisting of a compound in Table 3.

In another embodiment, the compound of Formula I is selected from the group consisting of a compound in Table 3A.

In another aspect, provided herein is a pharmaceutical composition comprising a compound described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

The compounds disclosed herein may exist as tautomers and optical isomers (e.g., enantiomers, diastereomers, diastereomeric mixtures, racemic mixtures, and the like).

It is generally well known in the art that any compound that will be converted in vivo to provide a compound disclosed herein is a prodrug within the scope of the present disclosure.

Compounds provided herein can also include all isotopes of atoms occurring in the intermediates or final compounds. Isotopes include those atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium. One or more constituent atoms of the compounds of the invention can be replaced or substituted with isotopes of the atoms in natural or non-natural abundance. In some embodiments, the compound includes at least one deuterium atom. For example, one or more hydrogen atoms in a compound of the present disclosure can be replaced or substituted by deuterium. In some embodiments, the compound includes two or more deuterium atoms. In some embodiments, the compound includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 deuterium atoms. Synthetic methods for including isotopes into organic compounds are known in the art (Deuterium Labeling in Organic Chemistry by Alan F. Thomas (New York, N.Y., Appleton-Century-Crofts, 1971; The Renaissance of H/D Exchange by Jens Atzrodt, Volker Derdau, Thorsten Fey and Jochen Zimmermann, Angew. Chem. Int. Ed. 2007, 7744-7765; The Organic Chemistry of Isotopic Labelling by James R. Hanson, Royal Society of Chemistry, 2011). Isotopically labeled compounds can used in various studies such as NMR spectroscopy, metabolism experiments, and/or assays.

In embodiments, the compounds provided herein have an isotopic enrichment factor for each designated deuterium atom of at least 3500 (52.5% deuterium incorporation at each designated deuterium atom), at least 4000 (60% deuterium incorporation), at least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium), at least 5500 (82.5% deuterium incorporation), at least 6000 (90% deuterium incorporation), at least 6333.3 (95% deuterium incorporation), at least 6466.7 (97% deuterium incorporation), at least 6600 (99% deuterium incorporation), or at least 6633.3 (99.5% deuterium incorporation).

Methods of Treatment

In an aspect, provided herein is a method of inhibiting NLRP3 inflammasome in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound disclosed herein.

In another aspect, provided herein is a method of modulating GLP-1 in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound disclosed herein.

In another aspect, provided herein is a method of treating a disease or disorder associated with GLP-1 activity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound disclosed herein.

In an embodiment, the disease or disorder associated with GLP-1 activity is obesity. In an embodiment, the obesity is dietary-induced obesity.

In another aspect, provided herein is a method of treating obesity-related comorbidity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound disclosed herein.

In an embodiment, the obesity-related comorbidity is selected from the group consisting of diabetes, heart failure, metabolic dysfunction-associated steatohepatitis (MASH), and a renal disease.

In another aspect, provided herein is a method of treating obesity-related disease, disorder, or comorbidity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound disclosed herein and a GLP-1 modulator.

In an embodiment, the GLP-1 modulator is selected from the group consisting of semaglutide, dulaglutide, exenatide, liraglutide, and lixisenatide.

In another aspect, provided herein is a method of treating inflammation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound disclosed herein.

In another aspect, provided herein is a method of treating inflammaging in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound disclosed herein.

In another aspect, provided herein is a method of treating cryopyrin-associated periodic syndrome (CAPS) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound disclosed herein.

In an embodiment, the CAPS is selected from the group consisting of familial cold autoinflammatory syndrome, Muckle-Wells syndrome, and neonatal-onset multisystem inflammatory disease.

In another aspect, provided herein is a method of treating a dermatologic disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound disclosed herein.

In an embodiment, the dermatologic disease is selected from the group consisting of psoriasis, urticaria, skin photoaging, and eczema.

Also provided herein is a method of using the compounds provided herein for treatment or amelioration of aging or an aging-related condition negatively impacting longevity or quality of life, wherein the aging-related condition negatively impacting longevity or quality of life is selected from the group consisting of inflammation, anemia, hyperglycemia, dyslipidemia, hyperinsulinemia, insulin resistance, immunosuppression, liver disease, iron overload, hypertrigliceridemia, impaired skin integrity, wound healing, scarring, pain, allergies, sleep disorders and problems, gastrointestinal disorders and problems, Th1-type inflammation, Th2-type inflammation, an inflammatory disease involving T-cell dependent B cell proliferation, T-cell dependent B cell proliferation, allergy, asthma, atherosclerosis, autoimmunity, hypercholesterolemia, chronic inflammation, chronic obstructive pulmonary disease (COPD), Crohn's disease, cutaneous responses to tissue damage, fibrosis, hematological oncology, metabolic diseases, cardiovascular disease, organ transplantation, psoriasis, liver fibrosis, dermatitis, pulmonary fibrosis, pulmonary responses to respiratory infections, restenosis, rheumatoid arthritis, sarcoidosis, stromal biology in tumors, systemic lupus erythematosus (SLE), ulcerative colitis, vascular inflammation, and diseases that are driven or exacerbated by one or more factors selected from the group consisting of alpha smooth muscle actin (αSMA), CD40, CD69, collagen I, collagen III, decorin, e-selectin, eotaxin 3 (CCL26), fibroblast proliferation, human leukocyte antigen-DR isotype (HLA-DR), immunoglobulin G, interferon gamma-induced protein 10 (IP-10/CXCL10), interferon-inducible T cell alpha chemoattractant (I-TAC/CXCL11), interleukin (IL)-1, IL-1.alpha., IL-2, IL-6, IL-8 (CXCL8), IL-10, IL-17A, IL-17F, keratin 8/81, macrophage colony-stimulating factor (M-CSF), matrix metalloproteinase (MMP)-1, MMP-9, monocyte chemoattractant protein 1 (MCP-1), monokine induced by gamma interferon (MIG/CXCL9), plasminogen activation inhibitor 1 (PAI-1), prostaglandin E2 (PGE2), serum amyloid A, T or B cell proliferation, tissue plasminogen activator (tPA), tumor necrosis factor alpha (TNF.alpha.), vascular cell adhesion molecule (VCAM-1), and vascular endothelial growth factor 2 (VEGFR2), comprising: administering to a subject in need thereof a compound provided herein.

In an aspect, provided herein is a method of reversing a normal aging process in subject comprising administering to the subject a therapeutically effective amount of a compound provided herein or a pharmaceutically acceptable salt thereof.

In another aspect, provided herein is a method of reversing a normal aging process in subject comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a compound provided herein or a pharmaceutically acceptable salt thereof.

In yet another aspect, provided herein is a method of extending lifespan of a subject comprising administering to the subject a therapeutically effective amount of a compound provided herein or a pharmaceutically acceptable salt thereof.

In still another aspect, provided herein is a method of extending lifespan of a subject comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a compound provided herein or a pharmaceutically acceptable salt thereof.

In another aspect, provided herein is a method to slow down and mitigate the aging process in a subject comprising administering to the subject a therapeutically effective amount of a compound provided herein or a pharmaceutically acceptable salt thereof.

In another aspect, provided herein is a method of inhibiting or modulating the pro-inflammatory pathway in a cell comprising contacting the cell with a compound provided herein, or a pharmaceutically acceptable salt thereof. In yet another aspect, provided herein is a method of inhibiting or modulating NLRP3 in a cell comprising contacting the cell with a compound provided herein, or a pharmaceutically acceptable salt thereof.

Treatment of a cell (in vitro or in vivo) that expresses a NLRP3 inflammasome with a compound provided herein can result in inhibiting the pro-inflammatory pathway and inhibiting downstream events related to the signaling pathway such as inflammation or inflammaging.

In another aspect, provided herein is a method of treating a neurosensory disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound disclosed herein.

In an embodiment, the neurosensory disease is selected from the group consisting of hearing loss, hearing injury, and ocular disease.

In another aspect, provided herein is a method of treating an ocular disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound disclosed herein.

In an embodiment, the ocular disease is an age-related eye disease (ARED). In an embodiment, the ocular disease is retinal and optic nerve injury. In another embodiment, the ocular disease is age-related macular degeneration (AMD). In an embodiment, the age-related macular degeneration is non-neovascular geographic atrophic (“dry”) AMD. In an embodiment, the age-related macular degeneration is neovascular exudative (“wet”) AMD.

In another embodiment, the ocular disease is diabetic retinopathy. In another embodiment, the ocular disease is diabetic macular edema (DME). In another embodiment, the ocular disease is geographic atrophy. In another embodiment, the geographic atrophy is in the back of the eye. In an embodiment, the ocular disease is retinal disease.

In an embodiment, the ocular disease is dry eye. In an embodiment, the ocular disease is severe dry eye. In an embodiment, the ocular disease is Sjogren's syndrome dry eye (SSDE). In an embodiment, the ocular disease is non-Sjogren's syndrome dry eye (NSSDE).

In an embodiment, the ocular disease is glaucoma. In an embodiment, the ocular disease is cataracts.

In an embodiment, the ocular disease is not an age-related eye disease. In an embodiment, the ocular disease is selected from a corneal ulcer, a non-healing ocular burn, uveitis, a persistent corneal defect, inflammatory keratitis, retinitis pigmentosa, and retinopathy of prematurity.

In yet another aspect, provided herein is a method of treating an inflammatory disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound disclosed herein.

In an embodiment, the inflammatory disorder is selected from the group consisting of allergy, asthma, atopic dermatitis, atherosclerosis, autoimmune diseases, coeliac disease, chronic inflammation, glomerulonephritis, hepatitis, inflammatory bowel disease, preperfusion injury, SARS-CoV-2 infection, transplant rejection, heart disease, diabetes, arthritis, Crohn's disease, ulcerative colitis, non-alcoholic steatohepatitis (NASH), gout, coronary artery disease, rheumatoid arthritis, intestinal disorders, and acute respiratory distress syndrome (ARDS). In an embodiment, the inflammatory disorder is diabetes-associated atherosclerosis. In another embodiment, the inflammatory disorder is kidney injury in diabetic nephropathy. In an embodiment, the inflammatory disorder is low-grade inflammation. In an embodiment, the inflammatory disorder is graft versus host disease (GvHD).

In another embodiment, the inflammatory disorder is a neuroinflammatory disease. In yet another embodiment, the inflammatory disorder is inner ear inflammation. In another embodiment, the inflammatory disorder is an ocular disease.

In an embodiment, a chronic inflammation comprises a tissue inflammation. Tissue inflammation is a chronic inflammation that is confined to a particular tissue or organ. In an embodiment, a tissue inflammation comprises, e.g., a skin inflammation, ocular inflammation, a muscle inflammation, a tendon inflammation, a ligament inflammation, a bone inflammation, a cartilage inflammation, a lung inflammation, a heart inflammation, a liver inflammation, a pancreatic inflammation, a kidney inflammation, a bladder inflammation, a stomach inflammation, an intestinal inflammation, a neuron inflammation, and a brain inflammation.

In another embodiment, a chronic inflammation comprises a systemic inflammation. Although the processes involved are identical to tissue inflammation, systemic inflammation is not confined to a particular tissue but in fact overwhelms the body, involving the endothelium and other organ systems. When it is due to infection, the term sepsis applied, with the term bacteremia being applied specifically for bacterial sepsis and viremia specifically to viral sepsis. Vasodilation and organ dysfunction are serious problems associated with widespread infection that may lead to septic shock and death.

In yet another embodiment, a chronic inflammation comprises an arthritis. Arthritis includes a group of conditions involving damage to the joints of the body due to the inflammation of the synovium including, without limitation osteoarthritis, rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthropathies like ankylosing spondylitis, reactive arthritis (Reiter's syndrome), psoriatic arthritis, enteropathic arthritis associated with inflammatory bowel disease, Whipple disease and Behcet disease, septic arthritis, gout (also known as gouty arthritis, crystal synovitis, metabolic arthritis), pseudogout (calcium pyrophosphate deposition disease), and Still's disease. Arthritis can affect a single joint (monoarthritis), two to four joints (oligoarthritis) or five or more joints (polyarthritis) and can be either an auto-immune disease or a non-autoimmune disease.

In still another aspect, provided herein is a method of treating an age-related disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound disclosed herein.

In another aspect, provided herein is a method of treating obesity-related comorbidity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound disclosed herein.

In an embodiment, the obesity-related comorbidity is selected from the group consisting of diabetes, heart failure, metabolic dysfunction-associated steatohepatitis (MASH), and a renal disease.

In another aspect, provided herein is a method of treating a metabolic condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound disclosed herein.

In an embodiment, the metabolic condition is selected from the group consisting of diabetes, obesity, cystic fibrosis, and hyperthyroidism. In an embodiment, the metabolic condition is insulin resistance. In an embodiment, the metabolic condition is an ischemic stroke concomitant with diabetes.

In yet another aspect, provided herein is a method of treating a neurodegenerative disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound disclosed herein.

In an embodiment, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), and Batten disease.

In an aspect, provided herein is a method of treating a disease or disorder of the inner ear in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound disclosed herein.

In an embodiment, the disease or disorder of the inner ear is selected from the group consisting of hearing loss, hearing impairment, vertigo, Meniere's disease, and tinnitus. In another embodiment, the disease of the inner ear is hearing loss. In yet another embodiment, the disease of the inner ear is hearing impairment.

In another embodiment, the hearing loss is age-related, noise-induced, or the result of a viral infection. In yet another embodiment, the viral infection is Zika virus or coronavirus.

In another aspect, provided herein is a method of treating a neurosensory disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound of the present disclosure.

In an embodiment, the neurosensory disease is selected from the group consisting of amyotrophic lateral sclerosis (ALS), traumatic brain injury, Parkinson's disease, and Alzheimer's disease. In another embodiment, the neurosensory disease is ALS. In yet another embodiment, the neurosensory disease is traumatic brain injury. In still another embodiment, the neurosensory disease is Parkinson's disease. In an embodiment, the neurosensory disease is Alzheimer's disease.

In yet another aspect, provided herein is a method of treating, preventing, or mitigating an adverse effect related to the administration of a T cell engaging agent in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound disclosed herein.

In an embodiment, the adverse effect is cytokine release syndrome (CRS). In another embodiment, the adverse effect is fever, hypotension and/or hypoxia.

In yet another embodiment, the adverse effect is an elevated serum level of one of more cytokine(s), particularly one or more cytokine(s) selected from the group consisting of IL-1 b, IL-6 and IL-8. In another embodiment, the adverse effect is an elevated serum level of one of more cytokine(s), particularly one or more cytokine(s) selected from the group consisting of IL-1b, IL-6, IL-18, and TNF-α. In another embodiment, the adverse effect is an elevated serum level of one of more cytokine(s), particularly one or more cytokine(s) selected from the group consisting of IL-1 b, IL-6, and IL-18.

In an embodiment of the methods, the subject is a human.

In another aspect, the disclosure provides a compound disclosed herein, or a pharmaceutically acceptable salt thereof, for use in the manufacture of a medicament for treating or preventing a disease in which NLRP3 inflammasome plays a role.

In an aspect, provided herein is a method of treating a condition selected from the group consisting of autoimmune diseases, inflammatory diseases, proliferative and hyperproliferative diseases, immunologically-mediated diseases, bone diseases, metabolic diseases, neurological and neurodegenerative diseases, cardiovascular diseases, hormone related diseases, allergies, asthma, and Alzheimer's disease. In other embodiments, said condition is selected from a proliferative disorder and a neurodegenerative disorder.

One aspect of this disclosure provides compounds that are useful for the treatment of diseases, disorders, and conditions characterized by excessive or abnormal cell proliferation. Such diseases include, but are not limited to, a proliferative or hyperproliferative disease, and a neurodegenerative disease. Examples of proliferative and hyperproliferative diseases include, without limitation, cancer.

Therefore, in an aspect, provided herein is a method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound disclosed herein, or a pharmaceutically acceptable salt thereof.

In an embodiment, the cancer is a CD20-expressing cancer. In another embodiment, the cancer is a B-cell cancer.

In yet another embodiment, the cancer is selected from the group consisting of Non-Hodgkin lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), high grade B cell lymphoma (HGBCL), primary mediastinal large B-cell lymphoma (PMBCL), follicular lymphoma (FL), mantle-cell lymphoma (MCL) and marginal zone lymphoma (MZL).

In another embodiment, the cancer is selected from the group consisting of myeloma, lymphoma, or a cancer selected from gastric, renal, head and neck, oropharangeal, non-small cell lung cancer (NSCLC), endometrial, hepatocarcinoma, non-Hodgkin's lymphoma, and pulmonary.

In an embodiment, the cancer is selected from the group consisting of prostate cancer, colon cancer, lung cancer, squamous cell cancer of the head and neck, esophageal cancer, hepatocellular carcinoma, melanoma, sarcoma, gastric cancer, pancreatic cancer, ovarian cancer, breast cancer.

In an embodiment, the cancer is selected from the group consisting of tumors, neoplasms, carcinomas, sarcomas, leukemias, lymphomas and the like. For example, cancers include, but are not limited to, mesothelioma, leukemias and lymphomas such as cutaneous T-cell lymphomas (CTCL), noncutaneous peripheral T-cell lymphomas, lymphomas associated with human T-cell lymphotrophic virus (HTLV) such as adult T-cell leukemia/lymphoma (ATLL), B-cell lymphoma, acute nonlymphocytic leukemias, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, lymphomas, and multiple myeloma, non-Hodgkin lymphoma, acute lymphatic leukemia (ALL), chronic lymphatic leukemia (CLL), Hodgkin's lymphoma, Burkitt lymphoma, adult T-cell leukemia lymphoma, acute-myeloid leukemia (AML), chronic myeloid leukemia (CML), or hepatocellular carcinoma. Further examples include myelodysplastic syndrome, childhood solid tumors such as brain tumors, neuroblastoma, retinoblastoma, Wilms' tumor, bone tumors, and soft-tissue sarcomas, common solid tumors of adults such as head and neck cancers (e.g., oral, laryngeal, nasopharyngeal and esophageal), genitourinary cancers (e.g., prostate, bladder, renal, uterine, ovarian, testicular), lung cancer (e.g., small-cell and non-small cell), breast cancer, pancreatic cancer, melanoma and other skin cancers, stomach cancer, brain tumors, tumors related to Gorlin syndrome (e.g., medulloblastoma, meningioma, etc.), and liver cancer. Additional exemplary forms of cancer which may be treated by the subject compounds include, but are not limited to, cancer of skeletal or smooth muscle, stomach cancer, cancer of the small intestine, rectum carcinoma, cancer of the salivary gland, endometrial cancer, adrenal cancer, anal cancer, rectal cancer, parathyroid cancer, and pituitary cancer.

In another aspect, provided herein is the use of one or more compounds of the disclosure in the manufacture of a medicament for the treatment of cancer, including without limitation the various types of cancer disclosed herein.

In some embodiments, the compounds of this disclosure are useful for treating cancer, such as colorectal, thyroid, breast, and lung cancer; and myeloproliferative disorders, such as polycythemia vera, thrombocythemia, myeloid metaplasia with myelofibrosis, chronic myelogenous leukemia, chronic myelomonocytic leukemia, hypereosinophilic syndrome, juvenile myelomonocytic leukemia, and systemic mast cell disease. In some embodiments, the compounds of this disclosure are useful for treating hematopoietic disorders, in particular, acute-myelogenous leukemia (AML), chronic-myelogenous leukemia (CML), acute-promyelocytic leukemia, and acute lymphocytic leukemia (ALL).

In another aspect, provided herein is a method of treating obesity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound disclosed herein.

In an embodiment, the obesity is dietary-induced obesity.

In another aspect, provided herein is a method of treating obesity-mediated metabolic disorders in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound disclosed herein.

In an embodiment, the obesity-mediated metabolic disorder is non-alcoholic fatty liver disease (NAFLD). In another embodiment, the obesity-mediated metabolic disorder is insulin resistance. In yet another embodiment, the obesity-mediated metabolic disorder is obesity-induced inflammation.

In yet another aspect, provided herein is a method of improving lipid metabolism in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound disclosed herein.

In still another aspect, provided herein is a method of reducing hypothalamic gliosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound disclosed herein.

In another aspect, provided herein is a method of reducing leptin levels in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound disclosed herein.

In another aspect, provided herein is a method of reducing low-density lipoprotein (LDL) cholesterol in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound disclosed herein.

In an embodiment of the methods, the method treats obesity in the subject, e.g., reduces obesity in the subject.

Compounds of the present disclosure can be administered intratympanically, wherein a long, narrow, bore needle is passed through the ear canal and through the eardrum to administer medications into the middle ear space where they are absorbed by the inner ear.

According to the methods of treatment of the present disclosure, disorders are treated or prevented in a subject, such as a human or other animal, by administering to the subject a therapeutically effective amount of a compound of the disclosure, in such amounts and for such time as is necessary to achieve the desired result. The term “therapeutically effective amount” of a compound of the disclosure, as used herein, means a sufficient amount of the compound so as to decrease the symptoms of a disorder in a subject. As is well understood in the medical arts a therapeutically effective amount of a compound of this disclosure will be at a reasonable benefit/risk ratio applicable to any medical treatment.

In general, compounds of the disclosure will be administered in therapeutically effective amounts via any of the usual and acceptable modes known in the art, either singly or in combination with one or more therapeutic agents. A therapeutically effective amount may vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used and other factors. In general, satisfactory results are indicated to be obtained systemically at daily dosages of from about 0.03 to 2.5 mg/kg per body weight. An indicated daily dosage in the larger mammal, e.g., humans, is in the range from about 0.5 mg to about 100 mg, conveniently administered, e.g., in divided doses up to four times a day or in retard form. Suitable unit dosage forms for oral administration comprise from ca. 1 to 50 mg active ingredient.

In certain embodiments, a therapeutic amount or dose of the compounds of the present disclosure may range from about 0.1 mg/Kg to about 500 mg/Kg, alternatively from about 1 to about 50 mg/Kg. In general, treatment regimens according to the present disclosure comprise administration to a patient in need of such treatment from about 10 mg to about 1000 mg of the compound(s) of this disclosure per day in single or multiple doses. Therapeutic amounts or doses will also vary depending on route of administration, as well as the possibility of co-usage with other agents.

Upon improvement of a subject's condition, a maintenance dose of a compound, composition or combination of this disclosure may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained; when the symptoms have been alleviated to the desired level, treatment should cease. The subject may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.

It will be understood, however, that the total daily usage of the compounds and compositions of the present disclosure will be decided by the attending physician within the scope of sound medical judgment. The specific inhibitory dose for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.

The disclosure also provides for a pharmaceutical combination, e.g., a kit, comprising a) a first agent which is a compound of the disclosure as disclosed herein, in free form or in pharmaceutically acceptable salt form, and b) at least one co-agent. The kit can comprise instructions for its administration.

The protein kinase inhibitors or pharmaceutical salts thereof may be formulated into pharmaceutical compositions for administration to animals or humans. These pharmaceutical compositions, which comprise an amount of the protein inhibitor effective to treat or prevent a protein kinase-mediated condition and a pharmaceutically acceptable carrier, are other embodiments of the present disclosure.

In an aspect, provided herein is a kit comprising a compound capable of inhibiting NLRP3 inflammasome activity selected from one or more compounds of disclosed herein, or pharmaceutically acceptable salts thereof, and instructions for use in treating a disorder associated with NLRP3 inflammasomes.

In another aspect, the disclosure provides a kit comprising a compound capable of inhibiting NLRP3 inflammasome activity selected from a compound disclosed herein, or a pharmaceutically acceptable salt thereof.

In yet another aspect, provided herein is a kit comprising a compound disclosed herein, or a pharmaceutically acceptable salt thereof for the treatment of any of the indications disclosed herein.

The following examples further illustrate aspects of the present disclosure. However, they are in no way a limitation of the teachings of the present disclosure as set forth.

EXAMPLES

The compounds and methods disclosed herein are further illustrated by the following examples, which should not be construed as further limiting. The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of organic synthesis, cell biology, cell culture, and molecular biology, which are within the skill of the art.

Abbreviations

Example 1: Synthetic Procedures

Synthesis of Common Intermediates

To a solution of starting material 4 (9 g, 74.26 mmol, 1 eq) in DCM (800 mL) was added MgSO4 (178.77 g, 1.49 mol, 20 eq) and the corresponding aldehyde (25.89 g, 148.51 mmol, 28.29 mL, 2 eq), then the mixture was stirred at 25° C. for 12 h. LCMS showed the starting material was consumed completely and desired mass was detected. The crude mixture was filtered, and the filtrate was concentrated under reduced pressure.

To a solution of starting material 5 (9 g, 74.26 mmol, 1 eq) in DCM (800 mL) was added MgSO4 (178.77 g, 1.49 mol, 20 eq) and the corresponding aldehyde (25.89 g, 148.51 mmol, 28.29 mL, 2 eq), then the mixture was stirred at 25° C. for 12 h. LCMS showed the starting material was consumed completely and desired mass was detected. The crude mixture was filtered, and the filtrate was concentrated under reduced pressure.

General Procedure 1 (GP1): Michael Addition to Imine

To a solution of starting material 1 (1 g, 4.54 mmol, 1 eq) in THF (20 mL) was added LDA (2 M, 1.5 eq) at −70° C. under N2, then stirred at −70° C. for 0.5 h. Then the corresponding sulfinamide (1.5 eq) in THF (10 mL) was added dropwise and the reaction was stirred for 1 h under N2. The reaction progress was monitored by LCMS. Upon completion, the reaction mixture was poured into a sat. aq. NH4Cl solution (150 mL), the mixture was extracted with EtOAc (3×100 mL), the combined organic phases were washed with brine (100 mL), dried over anhydrous Na2SO4, and concentrated to give a crude product. The crude material was purified by flash silica gel chromatography.

To a solution of compound 5 (730 mg, 1.47 mmol, 1 eq) in THF (10 mL) was added NaH (88.02 mg, 2.20 mmol, 60% purity, 1.5 eq) in ten batches at 0° C. under N2, and stirred for 30 min at 0° C. Then the reaction was added CH3I (416.50 mg, 2.93 mmol, 182.68 μL, 2 eq) at 0° C. and stirred for 1 h at 25° C. under N2. LCMS showed starting material was remained and desire product was detected. The mixture was poured into sat. aq. NH4Cl solution (50 mL) at 0° C. and the mixture was extracted with EtOAc (3×50 mL), then washed with brine (2×50 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to afford a residue. The residue was purified by column chromatography on silica and concentrated under reduced pressure.

General Procedure 3 (GP3): Cleavage of Ellman Auxiliary

To a solution of the corresponding sulfinamide (6.1 g, 11.92 mmol, 1 eq) in THF (0.2 M) and H2O (0.2 M) was added 12 (0.2 eq). The reaction mixture was stirred at 50° C. for 12 h. LCMS showed the starting material was consumed completely. The mixture was poured in sat. aq. Na2SO3 and extracted with EtOAc (3×100 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to give a residue. The residue was purified by silica column chromatography.

General Procedure 4 (GP4): Silyl Protection of Hydroxyl Group

To a solution of the starting alcohol (1 eq) in DCM (0.1 M) was added imidazole (3 eq) and tert-Butyldimethylsilyl chloride (2 eq), the mixture was stirred at 20° C. for 12 h. The reaction was monitored by LCMS. Upon completion, the mixture was filtered, and the filtrate was concentrated to give a crude product. The crude product was purified by silica gel chromatography to give desire product.

General Procedure 5 (GP5): Secondary Amine Coupling

To a solution of the secondary amine (1 eq), carboxylic acid (1.1 eq), and EDCl (1.5 eq) in DCM (0.1 M) was added DMAP (0.1 eq), then stirred at 20° C. for 2 h. The reaction was monitored by LCMS. After completion, the reaction was concentrated under reduced to give a crude product. The crude product was purified by preparative-TLC.

Optionally, the following procedure can be utilized:

To a solution of the secondary amine (1.0 eq) and the carboxylic acid (1.0 eq) in DMF (0.25 M) was added CMPI (1.5 eq) (or other coupling agent such as HCTU, HATU or the like) and DIPEA (3 eq). The mixture was stirred at 50° C. for 12 h. After desired completion as tracked by LCMS. The mixture was poured into H2O and extracted with EtOAc. The combined organic layers were washed by brine, dried over Na2SO4, and concentrated under reduced pressure to give a crude residue.

To a solution of silyl protected alcohol (100 mg, 153.93 μmol, 1 eq) in THF (0.03 M; or other applicable solvent) was added HCl (1-150 eq). Additionally, other acids can be used in conjunction with HCl, on their own. Examples of other acids include, but are not limited to, TsOH, MsOH, H2SO4, HNO3, or similar (0.01-20 eq). The reaction mixture was stirred in the range of 0-90° C. for 1-24 h. The reaction mixture was adjusted to pH=7 with sat. aq. Na2CO3 and extracted with EtOAc (3×50 mL). The combined organic layers were dried over Na2SO4, then concentrated under reduced pressure to give a crude residue.

To a solution of the corresponding aldehyde (1 eq) in dioxane (0.03 M) was added hydrazine hydrate (98% purity, 2 eq), the mixture was stirred at 20° C. for 0.5 h, then stirred at 70° C. for 2 h. The reaction mixture was added into water (10 mL) at 20° C. and extracted with EtOAc (3×10 mL). The combined organic phases were dried over Na2SO4 and concentrated under reduce pressure to afford a residue. The crude product was purified by prep-TLC.

To a solution of the corresponding 4-chloro-pyrazolopyridine (1 eq), DIPEA (1 eq) in MeOH (0.06 M) was added Pd/C (10% purity, 0.0441 eq). The reaction mixture was stirred at 30° C. for 1 h under H2 (15 psi). The reaction mixture was filtered with diatomite and the filtrate was concentrated to afford the crude residue.

Optional GP8 when starting materials contain other aryl chloride moieties:

To a solution of the corresponding 4-chloro-pyrazolopyridine (1 eq) in THF (0.25 M) was added Pd(dppf)Cl2 (0.1 eq), TMEDA (1.7 eq), and NaBH4 (1.7 eq) under N2 atmosphere. The mixture was stirred at 20° C. for 2 h. After completion, as judged by LCMS, the mixture was poured into NH4Cl, the aqueous layer was extracted with EtOAc (×3). The combined organic layers were washed with brine (×5) dried over Na2SO4 (or similar drying agent). The solvent was removed in vacuo to afford the crude residue.

To a solution of the silyl protected compound (1 eq) in a solvent (solvent options: MeCN, Et2O, dioxane, MeOH, EtOH, i-PrOH, THF, DCM, DMF, or similar) at a concentration of 0.01-0.5 M, was added a fluoride ion source (1-20 eq, examples: TBAF, NH4F, HF, pyridine-HF, CsF, LiBF4, or similar). The reaction was stirred at 20-80° C., for 1-20 h, until starting material was consumed. A standard aqueous work-up was performed. The solvent was removed in vacuo to afford the crude product. The crude product was purified by column chromatography on silica gel eluted with petroleum ether/ethyl acetate to give the desired product.

General Procedure 10 (GP10): Ether Synthesis

To a solution of the hydroxy containing compound (1 eq) in a solvent (solvent options: MeCN, Et2O, dioxane, MeOH, EtOH, i-PrOH, THF, DCM, DMF, or similar) was added a base (1-5 eq, base options: MeLi, t-BuLi, PhLi, NaOMe, LiOMe, NaOEt, NaOt-Bu, LiOt-Bu, NaH, or similar) at −78-25° C.. The reaction was stirred until consumption of starting material, and then quenched by pouring into ice sat. aq. NH4Cl. The mixture was extracted with organic solvent (×3) and the combined organic phases were washed with brine. The organic phase was dried over sodium sulfate (or similar drying agent) and concentrated under reduced pressure to remove solvent. The resulting crude compound was purified by silica gel column chromatography (PE:EA, or similar applicable solvent system).

To a solution of the starting material (1 eq) in DMF (or other applicable solvent) was added a base (1.5 eq; base options: t-BuOLi, MeLi, t-BuLi, PhLi, NaOMe, LiOMe, NaOEt, NaOt-Bu, NaH, or similar) at 0° C. under N2 atmosphere. The reaction was stirred at 0° C. for 0.5-2 h. Then SEM-CI (or other protection reagent such as: MEM chloride, MOM chloride, BOM chloride, or similar) was added to the reaction and the resulting mixture was stirred at 20-30° C. for 1-6 h. The reaction mixture was quenched with NH4Cl under N2 and mixture was extracted with ethyl acetate (×3), the organic phases were combined and washed with brine (×3), then dried with anhydrous Na2SO4, filtered, and concentrated under reduced pressure to afford a crude product. The crude product was purified by flash silica gel chromatography.

To a solution of the starting material (1 eq) and trimethylboroxine (5 eq) in 1,4-dioxane (˜0.1 M; or other applicable solvent) was added Pd(dppf)Cl2 (0.1 eq) (or other palladium catalyst such as: Pd(dppf)Cl2·CH2Cl2, RuPhos Pd G3, Pd/C, or similar) and K2CO3 (3 eq), the mixture was stirred at 40-120° C. for 1-24 h. The reaction is monitored by LCMS, tracking consumption of starting material. After the reaction mixture was cooled to room temperature, the mixture was filtered through celite®, and the filtrate was concentrated to give a crude product. The crude product was purified by flash silica gel chromatography.

To a solution of the starting material (1 eq) and ethylboronic acid (3 eq) in 1,4-dioxane (˜0.1 M; or other applicable solvent) was added Pd(dppf)Cl2 (0.1 eq) (or other palladium catalyst such as: Pd(dppf)Cl2·CH2Cl2, RuPhos Pd G3, Pd/C, or similar) and K2CO3 (3 eq), the mixture was stirred at 40-120° C. for 1-24 h. The reaction is monitored by LCMS, tracking consumption of starting material. After the reaction mixture was cooled to room temperature, the mixture was filtered through celite®*, and the filtrate was concentrated to give a crude product. The crude product was purified by flash silica gel chromatography.

To a solution of the starting compound (1 eq) in DCM (or other applicable solvent) was added TFA (50-150 eq). The reaction mixture was stirred at 20-100° C. for 2-24 h. The reaction mixture was concentrated to give a crude residue. The residue was dissolved in a protic solvent and treated with Et3N (NH3—H2O, or similar base) to adjust to pH=8. The resulting mixture was quenched by pouring into H2O. The mixture was extracted with EtOAc (×3), the combined organic phases were washed with brine and dried over anhydrous sodium sulfate (or another drying agent). The solution was concentrated under reduced pressure to remove the solvent. The crude material can be purified by prep-TLC, column chromatography, or prep-HPLC before an optional following step.

To a solution of the starting compound (1 eq) in DCM (or other applicable solvent) was added BC13 (3 eq) at 0° C. The reaction mixture was stirred at 0-20° C. for 2-24 h under N2 atmosphere. The reaction was quenched with sat. aq. NaHCO3, the resulting mixture was extracted with DCM (×2) and the organic phases were washed with brine, then dried over anhydrous Na2SO4, filtered, and concentrated to give a residue. The crude material can be purified by prep-TLC, column chromatography, or prep-HPLC before an optional following step.

General Procedure 15 (GP15): Reduction

To a solution of the starting compound eq in EtOH (0.1 M) was added LiBH4 or a H4) in THF (2 M, 5 eq) at 0° C. The reaction mixture was stirred at 20-30° C. for 1-6 hr under N2 atmosphere. Upon desired completion the reaction mixture was poured into water and extracted with EA (×3). The combined organic phases were dried with Na2SO4 and concentrated to give the crude product.

To a solution of int-26 (1 eq) and the corresponding amine (1.5 eq) in DMF or other applicable solvent (0.2 M) was added K2CO3 or another similar base (2 eq). The mixture was stirred at 20-50° C. for 2-20 h. The reaction was tracked by LCMS. After the reaction was completed, the reaction mixture was poured into H2O solution at 0° C., and extracted with EtOAc (×2), the combined organic phase was washed with brine (×3), dried over anhydrous Na2SO4, and concentrated to give a crude product. The residue was purified by silica gel chromatography and concentrated.

To a solution of int-28 (1 eq) in MeOH (0.2 M) was added the corresponding amine (2 eq) and AcOH (0.1 eq). The reaction was stirred at 25° C. for 1.5 h. Then NaBH4 (3 eq) was added at 0° C. The reaction was stirred at 25° C. for 0.5 h under N2. Upon completion the mixture was poured into sat. aq. NH4Cl at 20° C. and extracted with DCM (×3). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to give a residue. The crude product was purified by flash silica gel chromatography.

Synthesis of Compounds 001-040 (C-Substituted)

Synthesis of Compound 001

Synthesis of Intermediate 16

Intermediate 14 was synthesized, starting from int-1, via GP1 through GP3

Intermediate 15 is commercially available.

Synthesis of Intermediate 17

Synthesis of Intermediate 18

Synthesis of Compound 001

Synthesis of Compound 002

Synthesis of Intermediate 20

Following a similar scheme to the synthesis of Compound 001, intermediate 20 was synthesized via GP1-GP5. The residue was purified by silica gel column chromatography (PE:EA=3:1). Compound 8 (550 mg, 617.85 μmol, 83.90% yield, 75% purity) was obtained as yellow oil. LCMS: tR=0.731 min; m/z=667.2[M+H]+.

Synthesis of Compound 002

Synthesis of Compound 003

Synthesis of Compound 004

Following a similar procedure to the synthesis of Compound 002, starting from int-14, compound 004 was synthesized via GP5-GP8. The residue was purified by two separate methods:

Synthesis of Compound 005

Synthesis of Compound 006

Following a similar procedure to the synthesis of Compound 002, starting from int-14, compound 006 was synthesized via GP5-GP7. The final step in the synthesis of compound 006 is as follows:

Synthesis of Compound 007

Synthesis of Compound 008

Intermediate 23 was synthesized, starting from int-1, via GP1 through GP3

Intermediate 24 is commercially available.

Synthesis of Compound 009

Synthesis of Compound 010

Synthesis of Compound 011

Synthesis of Compound 012

Following a similar procedure as compound 011, GP2-8 were utilized in the synthesis of compound 012.

Synthesis of Compound 013

Following a similar procedure to compound 002, compound 013 was synthesized starting from int-10 utilizing the following steps, in order: GP2-5, GP9-10, GP6-GP8; to afford the residue of crude compound 013.

Synthesis of Compound 014

Following a similar procedure to compound 002, compound 014 was synthesized starting from int-10 utilizing the following steps, in order: GP2-5, GP9-10, GP6-GP8; to afford the residue of crude compound 014.

Synthesis of Compound 015

Synthesis of Compound 016

Synthesis of Compound 017

Synthesis of Compound 018

Synthesis of Compound 019

Synthesis of Compound 020

Synthesis of Compound 021

Synthesis of Compound 022

Synthesis of Compound 023

Synthesis of Compound 024

Synthesis of Compound 025

Synthesis of Compound 026

Synthesis of Compound 027

Synthesis of Compound 028

Synthesis of Compound 029

Synthesis of Compound 030

Synthesis of Compound 031

Synthesis of Compound 032

Synthesis of Compound 033

Synthesis of Compound 034

Synthesis of Compound 035

Synthesis of Compound 036

Starting from int-11, the material was subjected to GP2-8, followed by oxidation with DMP (1.5 eq) in DCM (0.25 M) at 0° C. and stirred for 2 hr. The reaction was filtered, and the filtrate was concentrated to give the crude material which was purified by prep-TLC (SiO2, DCM:MeOH=10:1, Rf=0.7). Int-24 (25 mg, 50.40 μmol, 64.58% yield, 90% purity) was obtained as a colorless oil. LCMS: Rt=0.457 min, m/z=447.2 (M+H+).

Synthesis of Compound 037

Synthesis of Compound 038

To a solution of int-25 (2 g, 6.86 mmol, 1 eq), int-4 (914.97 mg, 7.55 mmol, 1.1 eq) in DCM (30 mL) was added Cs2CO3 (2.68 g, 8.24 mmol, 1.2 eq), the reaction was stirred at 20° C. for 12 h. TLC (PE:EtOAc=0:1; UV&DNP) showed starting material was consumed up and new spot was formed. The reaction was filtered and concentrated to give a crude product. The crude product was purified by silica gel chromatography (50% EtOAc in PE, Rf=0.60).

To a solution of 2-bromoprop-1-ene (827.75 mg, 6.84 mmol, 607.74 μL, 1.2 eq) in THF (20 mL) was added n-BuLi (2.5 M, 5.47 mL, 2.4 eq) at −70° C. under N2 protected, then stirred at −70° C. for 0.5 h, then a solution of the purified compound from the prior step (2.5 g, 5.70 mmol, 1 eq) in THF (5 mL) was added dropwise to this reaction, the reaction was stirred at −70° C. for 1 h. LCMS showed starting material was consumed up and desire product was detected. The solution was slowly poured into saturated aqueous NH4Cl (300 mL) at 0° C. under N2. The layers were separated, the aqueous layer was extracted in EtOAc (100 mL×3), the combined organics were dried over Na2SO4 and filtered, and the solvent was evaporated in vacuo to give the crude residue. The crude product was purified by silica gel chromatography (7% EtOAc in MeOH, Rf=0.10) to give a product.

The product was subjected to GP2, GP3, and GP5 to give the intermediate amide product. The product (140 mg, 231.31 μmol, 1 eq) was dissolved in DCM (3 mL) and MeOH (3 mL) was treated with ozone (11.10 mg, 231.31 μmol, 1 eq) at −70° C. for 0.5 h, then the mixture was purged with nitrogen 3 times, PPh3 (121.34 mg, 462.63 μmol, 2 eq) was dissolved in DCM (1 mL) was added dropwise to the mixture at −70° C.. The mixture was warmed to 20° C. and stirred at 20° C. for 1 h. TLC (PE:EtOAc=0:1; UV) showed starting material was consumed up and new spots were formed. The reaction was concentrated to give a crude product. The crude product was purified by silica gel chromatography (35% EtOAc in PE, Rf=0.80) to give a product. This product was subjected to GP15 followed by GP13 and the resulting crude mixture was purified by prep-HPLC (column: Waters Xbridge 150×25 mm, 5 um; mobile phase: [water (NH4HCO3)-ACN]; gradient: 32%-62% B over 15 min) and lyophilized.

Synthesis of Compound 163

Synthesis of Compound 164

Synthesis of Compound 165

Synthesis of Compound 166

Synthesis of Compounds 041-062 (N-Substituted)

Synthesis of Compound 041

Synthesis of Compound 042

Synthesis of Compound 043

Synthesis of Compound 044

Synthesis of Compound 045

Synthesis of Compound 046

Synthesis of Compound 047

Synthesis of Compound 048

Synthesis of Compound 049

Synthesis of Compound 050

Synthesis of Compound 051

Synthesis of Compound 052

Compound 052 was synthesized by following a similar procedure to compound 041.

Synthesis of Compound 053

Synthesis of Compound 054

Synthesis of Compound 055

Compounds 056 and 057 were synthesized by following a similar procedure to compound 054. Intermediate 28 was subjected to GP17, GP5, and finally GP13 resulting in the crude mixture of 056 and 057. The product was purified by SFC (column: DAICEL CHIRALPAK AD (250 mm×50 mm, 10 um); mobile phase: [CO2-i-PrOH (0.1% NH3H2O)]; B %: 32%, isocratic elution mode). After SFC, the eluents were concentrated to remove organic solvents and the residual aqueous solutions were lyophilized.

Compound 058-061 were synthesized using the synthetic procedure shown above. Int-27 (1 eq) in THF (0.2M) was treated with MeMgBr (3 M, 3 eq) at −70° C. and stirred for 2 h. The resulting crude product was purified by silica gel chromatography. The purified product was dissolved in DCM and treated with Et3N (2 eq) and then SOBr2 (2 eq) at 0° C. The reaction was stirred at 20° C. for 12 h under N2 atmosphere. The crude product was purified by silica gel chromatography. The isolated compound was then subject to GP16, GP5, GP4, then GP12 to afford int-29. Intermediate 29 was then subject to GP13 followed by purification by SFC to afford the 4 corresponding products.

Synthesis of Compound 062

Compound 062 was synthesized starting from int-29 (above). Intermediate 29 was dissolved in DCM (0.1 M) and treated with DMP (3 eq) at 0° C.. The reaction was stirred for 1.5 h. The reaction was worked-up and the crude product was purified by prep-HPLC. The purified product was then dissolved in THF (0.15 M) and treated with MeMgBr (3 M, 5 eq) at 0° C. The mixture was stirred at 20° C. for 2 h. The reaction was quenched with sat. NH4Cl (50 mL) at 0° C. and extracted with EA (×3). The organic phases were washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated to give a residue. The residue was purified by preparative HPLC (column: Waters Xbridge 150×25 mm, Sum; mobile phase: [water (NH4HCO3)-ACN]; gradient: 36%-66% B over 9 min), LCMS showed the product was not clean and therefore was re-purified by prep-HPLC (column: Phenomenex luna C18 150×25 mm, 10 um; mobile phase: [water (FA)-ACN]; gradient: 15%-45% B over 9 min). Compound 062 (1.62 mg, 3.30 μmol, 2.09% yield, 100% purity) was obtained as white solid.

Synthesis of Compound 063

Synthesis of Compound 064

Synthesis of Compound 066

Synthesis of Compound 069

Synthesis of Compound 070

Synthesis of Compound 073

Synthesis of Compound 074

Synthesis of Compound 075

Synthesis of Compound 040

Compound 040 was prepared following the steps of GP4 and GP11-13, followed by oxidation with DMP in DCM or IBX in DMSO. The crude was treated and purified in the same manner as Compound 036. Compound 040 was then prepared according to the following scheme (see also synthesis of Compound 186 below):

Synthesis of Compound 224

Synthesis of Compound 039

Synthesis of Compound 067

Synthesis of Compound 136

Synthesis of Compound 137

Synthesis of Compound 138

Synthesis of Compound 139

Synthesis of Compound 155

Synthesis of Compound 184

Synthesis of Compound 068

Synthesis of Compound 096

Synthesis of Compound 135

Synthesis of Compound 146

Synthesis of Compound 149

Synthesis of Compound 156

Synthesis of Compound 158

Synthesis of Compound 161

Synthesis of Compound 162

Synthesis of Compound 167

Synthesis of Compound 180

Synthesis of Compound 189

Synthesis of Compound 227

Synthesis of Compound 193

Synthesis of Compound 194

Synthesis of Compound 195

Synthesis of Compound 196

Synthesis of Compound 197

Synthesis of Compound 198

Synthesis of Compound 199

Synthesis of Compounds 200 and 201

Synthesis of Compounds 202 and 203

Synthesis of Compounds 204 and 205

Synthesis of Compounds 208 and 209

Synthesis of Compound 211

Synthesis of Compound 212

Synthesis of Compound 213

Synthesis of Compound 206

Synthesis of Compound 207

Synthesis of Compound 214

Synthesis of Compound 215

Synthesis of Compound 216

Synthesis of Compound 219

Synthesis of Compounds 220 and 221

Synthesis of Compound 226

Synthesis of Compound 228

Synthesis of Compound 229

Synthesis of Compound 230

Synthesis of Compound 231

Synthesis of Compound 232

Synthesis of Compound 233

Synthesis of Compound 254

Synthesis of Compound 255

Synthesis of Compounds 256 and 257

Synthesis of Compounds 258 and 259

Synthesis of Compounds 260 and 261

Synthesis of Compound 262

Synthesis of Compound 279

Synthesis of Compound 280

Synthesis of Compound 077

Compound 077 was prepared by the following scheme.

Synthesis of Compounds 123 and 124

Synthesis of Compounds 129 and 130

Compounds 129 and 130 were prepared in a similar manner to compound 077.

Synthesis of Compound 187

Synthesis of Compound 217

Synthesis of Compound 218

Synthesis of Compounds 222 and 223

Synthesis of Compound 226

Synthesis of Compound 246

Synthesis of Compound 247

Synthesis of Compound 264

Synthesis of Compound 281

Synthesis of Compound 080

Compound 080 was prepared according to the following scheme:

Synthesis of Compound 083

Synthesis of Compounds 085 and 087

Compounds 085 and 087 were prepared by the steps of GP1-3 and GP5-8, where the product of GP6 was Boc-protected and the product on GP8 was deprotected under acidic conditions and then purified by SFC column: DAICEL CHIRALPAK AD (250 mm*30 mm, 10 um); mobile phase: [CO2-EtOH (0.1% NH3H2O)]; B %: 62.5%, isocratic elution mode to give two peaks.

Synthesis of Compounds 088 and 089

Compounds 088 and 089 were prepared by the steps of Scheme 3, GP1-3 and GP5-8, where the product of GP6 was Boc-protected and the product on GP8 was deprotected under acidic conditions and then purified by SFC (column: DAICEL CHIRALPAK AD (250 mm*30 mm, 10 um); mobile phase: [CO2-ACN/i-PrOH (0.1% NH3H2O)]; B %: 55%, isocratic elution mode) and concentrated and lyophilized to get the peak 1. Compound 089 (90 mg, 187.78 μmol, 55.58% yield, 98.8% purity) as an off-white solid.

Synthesis of Compound 081

Synthesis of Compound 082

Synthesis of Compound 090

Synthesis of Compound 091

Synthesis of Compounds 092 and 097

Compounds 092 and 097 were synthesized following the steps of GP1-3 and GP5-9, then GP11-13. The crude compounds 092 and 097 was purified by SFC (column: DAICEL CHIRALPAK AD (250 mm×50 mm, 10 um); mobile phase: [C02:i-PrOH (0.1% NH3H2O)]; B %: 30%, isocratic elution mode). The eluent was concentrated to remove organic solvents and the residual aqueous solution was lyophilized to afford the desired products.

Synthesis of Compound 095

Synthesis of Compound 079

Synthesis of Compound 210

Synthesis of Compound 105

Synthesis of Compound 106

Synthesis of Compound 107

Synthesis of Compound 108

Synthesis of Compound 109

Synthesis of Compound 110

Compound 110 was prepared in a similar manner to Compound 067 using 4-fluoropiperidine and was isolated as a light yellow oil (117 mg, 180.04 μmol, 50.66% yield). LCMS: Rt=0.502 min, m/z=650.2[M+H]+

Synthesis of Compound 111

Synthesis of Compound 112

Synthesis of Compound 113

Synthesis of Compound 114

Synthesis of Compound 115

Compound 115 was prepared in a similar manner to Compound 117. The amine starting material was prepared according to the following scheme:

Synthesis of Compound 116

Synthesis of Compound 117

Synthesis of Compound 118

Synthesis of Compound 119

Synthesis of Compound 120

Synthesis of Compounds 121 and 122

To a solution of compound 6 (2.31 g, 4.75 mmol, 1 eq) in DMF (25 mL) was added K2CO3 (1.31 g, 9.49 mmol, 2 eq) and MeNH2 (2 M, 11.87 mL, 5 eq), the mixture was stirred at 30° C. for 12 h. The reaction mixture was poured in water (30 mL), and extracted with EA (50 mL×3). The combined organic layers were washed by brine (50 mL×3) and dried over Na2SO4 and concentrated under reduced pressure to give a residue. Compound 7 (2.2 g, crude) was obtained as a yellow oil. LCMS: Rt=0.509 min, m/z=437.3 [M+H]+

To a mixture of compound 12 (90 mg, 152.08 μmol, 1 eq) in DCM (1 mL) was added TFA (1.54 g, 13.46 mmol, 1 mL, 88.52 eq) at 0° C., then the mixture was stirred at 25° C. for 3 h. The reaction mixture was poured into saturated NaHCO3 (20 mL) and extracted with DCM (30 mL×3). The combined organic layers were washed by brine (50 mL) and dried over Na2SO4 and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge 150×25 mm×5 um; mobile phase: [water (NH4HCO3)-ACN]; gradient: 35%-65% B over 15 min), the purified solution was lyophilized to give a white solid. Compound 13 (45 mg, 97.50 μmol, 64.11% yield) was obtained as a white solid. LCMS: Rt=0.423 min, m/z=462.2 [M+H]+

Synthesis of Compounds 125 and 126

Compounds 125 and 126 were prepared in a similar manner to compounds 121 and 122.

Synthesis of Compounds 127 and 128

Compounds 127 and 128 were prepared in a similar manner to compounds 121 and 122.

Synthesis of Compound 131

Synthesis of Compounds 132 and 133

Compounds 132 and 133 were prepared in a similar manner to Compound 131.

Synthesis of Compound 134

Synthesis of Compounds 141-144

Compounds 141-144 were prepared in a similar manner to Compounds 008 and 131.

Synthesis of Compound 148

Synthesis of Compounds 150 and 151

Compounds 150 and 151 were prepared in a similar manner to Compound 131 using TFA instead of zinc bromide.

Synthesis of Compounds 154-156

Compounds 152-154 were prepared in a similar manner to Compounds 008 and 131.

Synthesis of Compounds 159 and 160

Compounds 159 and 160 were prepared in a similar manner to Compounds 152 and 154.

Synthesis of Compounds 168-171

Compounds 168-171 were prepared in a similar manner to Compound 131.

Synthesis of Compound 172

Synthesis of Compounds 176 and 177

Compounds 173 and 174 were prepared in a similar manner to Compound 131.

Synthesis of Compounds 175 and 176

Compounds 175 and 176 were prepared in a similar manner to Compound 131.

Synthesis of Compound 177

Synthesis of Compounds 178 and 179

Compound 178 was prepared in a similar manner to Compound 131 using Compound 151 as starting material.

Synthesis of Compounds 181 and 182

Compounds 181 and 182 were prepared in a similar manner to the first step shown for Compound 131.

Synthesis of Compound 183

Synthesis of Compounds 065 and 071

Compounds 065 and 071 were prepared in a similar manner to Compound 131.

Synthesis of Compound 190

Synthesis of Compound 241

Synthesis of Compounds 282 and 283

Synthesis of Compound 296

Compound 296 was prepared in a similar manner to Compound 131. using formaldehyde instead of acetaldehyde and NaBH2CN instead of zinc bromide. Compound 296 (200 mg, 415.62 μmol, 27.22% yield, 98.2% purity) was obtained as a white solid. LCMS: RT=0.542 min, m/z=473.1 [M+H]+.

Synthesis of Compounds 297 and 298

Synthesis of Compound 140

Synthesis of Compound 147

Synthesis of Compound 157

To a solution of compound 4 (10.6 g, 40.41 mmol, 1 eq) in the THF (35 mL), MeOH (35 mL) and H2O (35 mL) was added LiOH·H2O (5.09 g, 121.22 mmol, 3 eq), then the mixture was stirred at 25° C. for 12 h. The mixture was adjusted to pH=2 with 1 N HCl solution. The reaction mixture was poured into H2O (200 mL) and extracted by EtOAc (200 mL×3), the organic phase was combined and washed with brine (200 mL), dried over Na2SO4 and concentrated to give a crude product. Compound 5 (9.6 g, crude) was obtained as white solid which was used directly for the next step. LCMS: Rt=0.352 min, m/z=233.3 (M+H+)

To a solution of the compound 7 (3.50 g, 5.49 mmol, 1 eq) in the THF (20 mL) was added HCl (2 M, 19.94 mL, 7.27 eq), then the mixture was stirred at 40° C. for 12 h. The mixture was adjusted pH to 9-10 by NaHCO3 and extracted by EtOAc (50 mL×2), then the organic phase was washed by brine (50 mL), dried over Na2SO4 and concentrated to give a residue. Compound 8 (3.3 g, crude) was obtained as yellow oil. The crude product was used directly for the next step. LCMS: Rt=0.599 min, m/z=479.2 (M+H+)

Synthesis of Compound 210

Synthesis of Compound 185

Synthesis of Compound 186

To a solution of compound 3 (90 mg, 130.05 μmol, 1 eq) in MeOH (1 mL) was added NH4F (48.17 mg, 1.30 mmol, 10 eq), the mixture was stirred at 60° C. for 2 h. The mixture was stirred at 40° C. for 5 h. The reaction mixture was poured into H2O (10 mL), and extracted with EA (10 mL×3). The combined organic phase was dried with Na2SO4 and concentrated to give the crude product. Compound 4 (76 mg, crude) as a light yellow oil. The crude product was used directly for the next step. LCMS: RT=0.638 min, m/z=578.4 [M+H]+

To a solution of compound 4 (100 mg, 173.08 μmol, 1 eq) in DMSO (2 mL) was added IBX (96.93 mg, 346.16 μmol, 2 eq). The reaction mixture was stirred at 25° C. for 3 h. The reaction mixture was poured into H2O (10 mL) under N2 and extracted by DCM (10 mL×3), the organic phase was combined and washed with brine (10 mL×3), dried with anhydrous Na2SO4, filtered and concentrated to give a crude product in vacuum. The crude product was purified by prep-TLC (PE:EA=0:1) and concentracted to give the product (Rf=0.8) compound 7 (84 mg, 81.70 μmol, 47.20% yield, 56% purity) was obtained as light-yellow oil. LCMS: RT=0.676 min, m/z=576.3 [M+H]+

Synthesis of Compound 188

Compound 188 was prepared following Scheme 4, GP1, GP5, GP13, and G16 using nitromethane and reducing with iron and ammonium chloride. The remaining of the synthesis shown below.

To a solution of Compound 12 and compound 12a (384.54 mg, 1.20 mmol, 3 eq) in dioxane (3 mL) was added Xphos Pd G4 (34.35 mg, 39.92 μmol, 0.1 eq). The reaction mixture was stirred at 80° C. for 16 hr. After the reaction was cooled into 20° C. and being filtered at 20° C. under N2, the filtrate was extracted with ethyl acetate (20 mL×2). The combined organic phase was evaporated under reduced pressure. The combined filter cake was quenched with 1 N HCl aqueous at 0° C. under N2. The residue was purified by column chromatography (SiO2, Commercial hexanes: Ethyl acetate=10/1 to 0/1) and by prep-TLC (SiO2, DCM:MeOH=10:1). Compound 13 (80 mg, 128.66 μmol, 32.23% yield, N/A purity) as yellow solid. LCMS: RT=0.853 min, m/z=622.4 [M+H]+

Compound 234 was prepared in accordance with the following scheme

To a 35 ml hydrogenation flask with a magnetic stir bar was added Pd/C (34.59 mg, 32.50 μmol, 10% purity, 0.15 eq) and followed by the addition of MeOH (4 mL) under argon, then added compound 5 (160 mg, 216.70 μmol, 1 eq) in MeOH (4 mL) and DIEA (28.01 mg, 216.70 μmol, 37.74 μL, 1 eq) under N2. The reaction mixture was stirred at 25° C. for 3 h under H2 (15 psi). The reaction mixture was filtered under N2 with diatomite and the filter cake was recycling, filter liquor was concentrated to give the crude. Compound 6 was obtained as a colorless oil. LCMS: RT=0.617 min, m/z=704.4 [M+H]+

Synthesis of Compound 244

Synthesis of Compound 235

Synthesis of Compound 240

Synthesis of Compound 236

Synthesis of Compound 242

Synthesis of Compound 237

Synthesis of Compound 243

Synthesis of Compound 238

Synthesis of Compound 239

Synthesis of Compound 245

Synthesis of Compound 248

Synthesis of Compound 249

Synthesis of Compound 250

Synthesis of Compound 251

Synthesis of Compound 252

Synthesis of Compound 253

Synthesis of Compound 265

Synthesis of Compound 266

Synthesis of Compound 277

Synthesis of Compound 278

Synthesis of Compound 267

Compound 267 was prepared following the procedures of GP2, GP3, and GP5 with (R)-2-methyl-N-((S)-(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-c]pyridin-7-yl)(1-trityl-1H-imidazol-4-yl)methyl)propane-2-sulfinamide as starting material and MsOH/NH2OH—HCl are reagents in GP3. Compound 267 was obtained as a white solid (45 mg, 94.71 μmol, 3.86% yield, 99.02% purity).

Synthesis of Compound 284

Synthesis of Compound 285

Synthesis of Compound 286

Synthesis of Compound 287

Synthesis of Compound 288

Synthesis of Compound 289

Synthesis of Compound 290

Synthesis of Compound 291

Synthesis of Compound 292

Synthesis of Compound 293

Synthesis of Compound 294

Synthesis of Compound 295

Synthesis of Compound 301

Synthesis of Compound 302

Synthesis of Compound 303

Synthesis of Compound 304

THP-1 cells are cultured in complete media (CM) until they reach logarithmic growth and achieve a viability >90%. CM is composed of RPMI-1640 (+Glutamax)/10% fetal bovine serum/55 μM β-mercaptoethanol/pen/strep. Cells are spun down and resuspended to 1,000,000 cells/mL in CM containing either 20 nM or 500 nM PMA. 150,000 cells (150 μL) are then added to each well of a 96-well TC plate and incubated for either 24 hr or 3 hr, respectively, in a standard cell culture incubator (37° C.; 5% CO2). After this incubation, the plate is tilted and media carefully removed. 200 μL of CM containing 100 ng/mL LPS is then added to the wells and the cells incubated an additional 3 hrs. The media is again removed and replaced with Opti-Mem medium containing pre-determined dilutions of test compounds in replicate wells. After a 30 min incubation, 10 μM nigericin (final concentration) in Opti-Mem medium with the corresponding concentration of compound is added to the wells for an additional 1 hr. Positive control wells contain 10 μM nigericin in Opti-Mem in the absence of test compound, while negative control wells contain Opti-Mem only. Supernatants are then transferred to a fresh 96-well plate for storage and assayed for IL-1β (human; DuoSet; R&D) and for TNFα (human; DuoSet; R&D) levels and relative pyroptosis using a CytoTox 96 Kit (do not freeze prior to testing; Promega). Once supernatants are removed, the relative viability of adherent cells in the 96-well TC plate are determined using a CellTiter-Glo® luminescent cell viability assay (Promega).

Table 4 below provides IC50 data for the compounds disclosed herein where “A” is indicative of an IC50 value of <100 nM, “B” is indicative of an IC50 value between 100 nM and 500 nM, “C” is indicative of an IC50 value between 500 nM and 1 uM, “D” is indicative of an IC50 value between 1 uM and 15 uM, and “E” is indicative of an IC50 value >15 uM.

001
A

002
A

003
A

004
A

005
A

006
A

007
A

009
A

013
A

014
A

015
A

016
E

017
A

018
A

019
A

022
A

023
A

024
A

025
A

026
A

027
A

028
E

029
A

030
E

031
E

033
A

034
E

035
A

036
A

037
A

038
A

040
A

041
A

042
A

045
A

046
A

048
A

049
A

051
A

052
E

053
A

054
E

055
A

056
A

058
A

060
E

061
E

062
A

063
A

064
A

065
A

066
A

067
A

068
A

070
A

071
A

072
A

073
A

074
A

075
A

076
A

077
A

078
A

079
A

080
A

081
A

082
A

083
A

084
A

085
A

086
A

087
A

089
A

090
A

091
A

093
A

094
A

095
A

096
A

097
A

098
A

099
A

100
A

101
A

102
A

103
A

104
A

106
A

107
A

108
A

109
A

110
A

111
A

112
A

113
A

114
A

115
A

116
A

117
A

119
A

120
A

121
A

122
C

123
C

125
A

126
A

127
A

128
A

129
C

130
A

131
A

133
A

135
A

136
A

137
A

138
A

139
A

140
A

141
A

143
A

144
A

147
A

148
A

149
A

151
A

152
C

153
A

154
A

155
A

156
A

157
A

159
A

160
A

161
A

162
A

163
A

164
A

165
A

166
A

167
A

168
A

169
A

170
A

171
A

172
A

173
A

174
A

175
A

177
A

178
A

179
A

180
C

181
A

182
A

183
A

184
A

186
A

187
A

188
A

189
A

190
A

193
A

194
A

195
A

196
A

197
A

198
A

199
A

202
A

203
A

205
A

206
A

207
A

209
A

211
A

212
A

213
A

214
A

215
A

216
A

217
A

218
A

219
A

221
A

222
C

223
A

224
A

225
A

227
A

228
A

229
A

230
A

231
A

232
A

233
A

236
A

238
A

239
A

240
C

241
A

244
A

245
A

246
A

247
A

248
A

249
A

250
A

251
A

252
A

253
A

254
A

255
A

257
A

259
A

261
A

262
A

263
A

264
A

265
A

266
A

277
A

278
A

279
A

280
A

282
A

283
A

284
A

285
A

286
A

287
C

289
A

290
A

291
A

292
A

293
A

294
C

298
A

NLRP3 belongs to the family of NOD-like receptors (NLRs), which are classified as AAA+ ATPases (ATPases associated with diverse cellular activities). AAA+ ATPases are triphosphate-nucleotide binding domains whose central β-sheet is defined by a characteristic parallel β2-β3-β4-β1-β5 topology, where the P-loop (or Walker A motif) is directly following the first β-strand (β1) and the Walker B motif is located on the third β-strand (β3) (Hochheiser and Geyer, 2023). The structure of the P-loop domain is characterized by a three-layered αβα sandwich of repeating β-loop-α units, also known as Rossmann-fold, where the β-strands are arranged in parallel orientation and surrounded by α-helices. NLRs constitute the subgroup of STAND ATPases within the AAA+ superfamily, which contain a C-terminal helical domain (HD1) followed by a winged helix domain (WHD) relative to the ATPase activity containing nucleotide-binding domain (NBD).

Compound 007 of the disclosure was used as an exemplary compound for determining the epitope to NLRP3, using cryogenic electron microscopy (cryo-EM).

The results of the cryo-EM assay found that Compound 007 binds in a pocket on the surface of the NACHT domain of NLRP3 (FIG. 1). The specific binding site is mostly composed of residues from the nucleotide-binding domain (NBD) but opposite to the ATP-binding site from the perspective of the central β-sheet (FIG. 2). The binding site is consistent with the finding that the 5-azaindazole-containing compounds either do not inhibit, or are very weak inhibitors of the intrinsic ATP-hydrolysis activity of NLRP3 (FIG. 6).

The defining feature of the interaction between the 5-azaindazole-containing compounds (Hartman et al., Bioorg. Med. Chem. Lett. 102: 129675. 2024) and NLRP3 is the formation of three hydrogen bonds to the backbone atoms of the terminal β-strand β2 in the NBD of NLRP3 (FIG. 3). The two nitrogen atoms (N32 and N31) form two donor-acceptor pairs with the backbone NH and CO groups of Tyr258 at a distance of 3.1 and 2.9 Angstroem, respectively, in perfect geometry (FIG. 3). This specific interaction is complemented by a third hydrogen bond between the CO moiety (O23) of the central peptide bond unit to the backbone NH group of His260 at 3.1 Angstroem (FIG. 3). The following biphenyl unit binds into a hydrophobic crevice formed by helices α2 and α4 of the NBD, where it interacts with the side chains of residues Leu275, Leu272, Leu335, Leu331, Leu332, Val264, the β-methylene group of Glu263, and Gly328 in a counterclockwise listing (FIG. 4). The ethoxy moiety in the meta-position of the first benzene ring of Compound 007 reaches out into a hydrophobic crevice formed by the side chains of Phe299, Leu272, Ile276 and Phe257 of NLRP3 contributing to the specificity of the interaction. The fluorine atom of the second benzene ring is embedded between the side chains of Leu331, Leu332 and L335 of NBD helix α4. The two benzene rings of the biphenyl unit are twisted relative to each other by 450 (FIG. 4).

The benzylic Ca position (C04) position of the 5-azaindiazole is chiral and in the (R)-configuration. The hydroxy group of the 2-hydroxyethyl moiety forms a hydrogen bond to the carboxyl side chain group of Glu263 (FIG. 3). Additional interactions are made to residue Cys514 at the tip of the β-sandwich loop in the winged-helix-domain (WHD) of NLRP3 at a distance of 3.3 Angstroem between the oxygen (001) and the sulfur atoms (FIG. 4). The methyl group at the NC position of the central peptide bond (C24) is in proximity (4.1 Angstroem) to the sidechain of Cys279 (FIG. 5).

The binding of Compound 007 to NLRP3 is complemented by an interaction of the nitrogen atom (N27) of the 5-azaindazole, which is in 3.4 and 4.6 Angstroem distance to the side chains of Arg147 and Tyr143 of the FISNA domain, respectively, coordinating the interaction of the inhibitor to the first subdomain of the NACHT (FIG. 4). Overall, the inhibitor Compound 007 performs interactions with 25 residues of the three subdomains NBD, FISNA and WHD, with Leu275 and Cys279 making the largest contribution to the buried surface area (BSA) of NLRP3 with 56 and 42 Å2, respectively.

Cloning, Expression and Purification of Human NLRP3

Full length, human NLRP3 (3-1036, UniProt accession code Q96P20), codon-optimized for Spodoptera frugiperda, was cloned in an in-house modified pACE-Bac1 vector containing an N-terminal MBP-tag, followed by a Tobacco etch virus (TEV) protease cleavage site. For recombinant protein expression of MBP-NLRP3, 1 L of Sf9 insect cells were infected with 3% v/v viral stock of the second virus passage. The expression culture was incubated for 72 h at 27° C. and 80 rpm, and was subsequently harvested by centrifugation at 2000 rpm for 20 min. Cell pellets were washed with PBS and subsequently used for protein purification or flash-frozen in liquid nitrogen and stored at −80° C. For protein purification, a 1 L cell pellet was solubilized in lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM TCEP, 1 mM ADP, 10 mM MgCl2) to which 1 μM Compound 007 from a 10 mM stock solution in DMSO was added. The lysis buffer was supplemented with 1 mM phenylmethylsulfonyl-fluoride (PMSF), followed by sonication (10 sec on; 5 sec off for 4 min at 40% intensity) on ice. The cell lysate was centrifuged at 25,000 rpm for 1 h and the supernatant was subsequently filtered with a 0.45 μm syringe filter, before it was applied onto a lysis buffer equilibrated 5 ml MBP-trap column (GE Healthcare) connected to an ÅKTA-Start FPLC system. The column was subsequently washed with 10 column volumes (CVs) of lysis buffer and the protein was eluted with 5 CVs of lysis buffer supplemented with 15 mM maltose. The NLRP3·Compound 007 complex was further purified using a Superose 6 increase 10/300 GL column (GE Healthcare) equilibrated with lysis buffer. Elution fractions were analyzed by SDS-PAGE and negative stain electron microscopy (EM), before they were plunge frozen for cryo-EM analysis.

A NACHT-transition LRR construct of human NLRP3 (aa 131-694) was subcloned into an in-house modified pACEBac1 vector containing an N-terminal MBP-tag, followed by a Tobacco etch virus (TEV) protease cleavage site. The Bac-to-Bac system was used to generate baculovirus infected Sf9 insect cells. For the expression of recombinant human NLRP3, insect cells were infected with 3% v/v of a viral stock of the second passage. The expression culture was incubated for 72 h at 27° C. and 80 rpm, and subsequently harvested by centrifugation at 2000 rpm for 20 min. The cell pellet was washed with PBS and either used for subsequent protein purification or frozen in liquid nitrogen and stored at −80° C. till further use. For protein purification, cells were resuspended in buffer A (20 mM Tris-HCl pH 7.8, 150 mM NaCl, 5 mM β-mercaptoethanol, 10 mM MgCl2, 1 mM ADP) supplemented with 1 mM PMSF and 1 μg/ml DNAse I, and lysed by sonication (6 sec on; 5 sec off for 4 min at 40% intensity on ice). The lysate was cleared from cell debris by centrifugation at 70,000×g for >30 minutes. The protein containing an N-terminal MBP tag was captured using a MBPTrap column (GE Healthcare) and the unbound fraction was washed off with buffer A. After elution with buffer A containing 10 mM maltose, TEV protease (1:50 w/w) was added for cleavage of the MBP-tag and the protein was dialyzed against buffer B (20 mM HEPES pH 7.8, 150 mM NaCl, 10 mM MgCl2, 1 mM ADP, 1 mM TCEP, 500 mM L-Arginine); overnight at 4° C. using the snake skin dialysis tubing with 3.5 kDa MWCO (Thermo Scientific). Protein was further purified using a Superdex 75 gel filtration column 16/600 (GE Healthcare) that was connected to a MBPTrap column for prolonged retention of the cleaved MBP tag and pre-equilibrated with buffer C (20 mM HEPES pH 7.8, 150 mM NaCl, 10 mM MgCl2, 1 mM ADP, 1 mM TCEP, 150 mM L-Arginine). Peak fractions containing monomeric NLRP3 were pooled, concentrated to 9.0 mg/ml (Amicon 10 kDa MWCO), snap frozen in liquid nitrogen, and stored at −80° C. For biochemical analysis, the protein was purified in absence of ADP and Compound 007. Due to lower solubility of the protein, the concentration was adjusted to 1 mg/ml.

Cryo-EM Grid Preparation, Data Collection and Processing

The purified NLRP3·Compound 007 complex was applied onto glow-discharged EM-grids (Quantifoil Cu300 R2/1+2 nm carbon support film), incubated for 30 s at 100% humidity and 4° C., before it was blotted for 5 s with blot force 5 and plunge frozen into liquid ethane using a Vitrobot mark IV plunge freezing device (Thermo Fisher Scientific). For dataset acquisition using the SerialEM version 4 automation software, 4,720 electron-event representation (EER) movies each consisting of 1122 raw frames were recorded on a Cs-corrected Krios Titan microscope operated at 300 kV and equipped with a Falcon4i camera at a total dose of 66.22 e−/A2 and a defocus range of −0.80 to −1.80 μm. For Cryo-EM data processing in cryoSPARC, the raw EER data were fractioned onto 40 movie frames with a unsampling factor of 2. Resulting movie stacks were aligned using the patch motion correction and patch CTF correction jobs. Using the blob picker, 3,114,152 particles were picked and detected of which 2,058,399 were extracted (search particle diameter 280 nm, 588 box size), of which 559,748 particles remained after multiple rounds of 2D classification. From these particles, three ab-initio models were generated, which were further subjected to heterogenous refinement. One model (468,974 particles) was further subjected to a non-uniform (NU)-refinement, followed by 3D classification allowing for ten 3D-classes. One class, comprising 30,200 particles, was further refined by another NU-refinement yielding a final reconstruction of the NLRP3·Compound 007 complex at 3.00 Å resolution (0.143 FSC). In this reconstruction, Compound 007 is clearly visible in the cryo-EM density map at a surface accessible binding pocket composed of the nucleotide-binding domain.

Thermal Shift Assay

Nano-differential scanning fluorimetry (nanoDSF) measurements were performed on a Prometheus NT.48 (NanoTemper) device to determine the thermal stability of the protein-ligand samples. For stability characterizations, NLRP3 was mixed with buffer or ligand in the presence of 2% DMSO. The sample was incubated for 30 minutes on ice before loading into capillaries (NanoTemper). The measurement was setup with a temperature ramp ranging from 15-95° C., a slope of 1.5° C./min, and 90% laser intensity.

For the analysis of the intrinsic ATP hydrolysis, 3 μM full length, wild type NLRP3 (peak 1) was incubated for 30 minutes on ice in the presence of 2% DMSO or 100 μM BAL-0028 or 100 μM Compound 007. After incubation, 100 μM of ATP was added and the reaction was incubated for 60 minutes at 25° C. Every 10 minutes a 10 μl sample was injected onto a 1260 Infinity II LC system connected to a reversed phase C18-silica column (Chromolith Performance, Merck) that was pre-equilibrated with buffer D (30 mM K2HPO4, 70 mM KH2PO4, 10 mM TBA-Br, 4% v/v acetonitrile; pH 6.5). The detector was setup to measure at 259 nm wavelength. Detected peaks for ADP and ATP were integrated and the ratio was used to calculate the molar concentrations of educt and product.

Surface Plasmon Resonance Spectroscopy

Surface plasmon resonance (SPR) spectroscopy experiments were performed on a Biacore 8K (GE Healthcare) device. The system was flushed with running buffer (10 mM HEPES pH 7.4, 200 mM NaCl, 0.5 mM ADP, 0.5 mM tris(2-carboxyethyl)phosphine (TCEP), 2 mM MgCl2, 1 g/L carboxymethyl dextran (CMD), 0.05% Tween20, 2% DMSO) at 25° C. A streptavidin functionalized sensor chip (Series S Sensor Chip SA, Cytiva) was conditioned with three consecutive injections of 1 M NaCl in 50 mM NaOH (10 μL/min) for 1 min. After purification of biotinylated NLRP3 (131-694) that was expressed as wildtype or mutants in the FreeStyle™ 293-F expression system, the protein was immobilized onto the sensor chip at 2 μL/min for 3000 s. The flow system was washed using 50% isopropanol in 1 M NaCl and 50 mM NaOH. Free streptavidin binding sites were blocked by four consecutive injections of Biotin-PEG (1000 nM, Mn 2,300 Da) for 2 min at 10 μL/min. For binding measurements in the single cycle mode, increasing concentrations of 2.3 to 600 nM BAL compounds were injected at 30 μL/min (association 240 s, dissociation 60/360 s). Data were collected at a rate of 10 Hz. The binding data were double referenced by blank cycle and reference flow cell subtraction. Data were corrected by a 4-point solvent correction. For determination of dissociation constants, processed data were fitted to a 1:1 interaction model using the Biacore Insight Evaluation Software (version 3.0.12.15655). For improved comparability of the NLRP3 variants and to account for their different immobilization levels, the measurements were normalized to the theoretical Rmax resulting in the bound fraction.

Compound 040 (“Cmpd 40”) was used to evaluate weight loss promotion in a diet-induced obesity (DIO) mouse model. The treatment groups were as follows:

Compound 040 was administered orally (PO) in a 50 mM citrate buffer (pH 4). Semaglutide was administered orally in a 20 mM citrate buffer (pH 7). The PO volume was 5 ul/gBW; and the subcutaneous (SC) volume was 4 ul/gBW. Mice were evaluated daily for body weight, food consumption, and water consumption. The mice were also evaluated weekly for body composition. Baseline and endpoint A1C and fasting glucose, endpoint HOMA-IR were also measured. The DIO mice were randomized for the drug treatment on the basis of equivalent body weight, body composition (fat mass, lean mass), 6 h fasting blood glucose, baseline blood A1C level, water consumption and food consumption as shown in FIG. 7.

The body weight change and body weight percentage changes are shown in FIG. 8 for mice receiving Compound 40 compared to a vehicle or semaglutide. Compound 40 showed a dose-dependent effect on reducing body weight of DIO mice. Table 5 summarizes the data shown in FIG. 8

Groups
baseline
DIO/VEH

The changes in food consumption in mice receiving no treatment (VEH), Compound 40, and semaglutide are shown in FIG. 9. Compound 40 showed a dose-dependent effect on reducing food consumption.

The changes in fat and lean percentages in mice receiving no treatment (VEH), Compound 40, and semaglutide are shown in FIG. 10. Compound 40 showed a dose-dependent effect on reducing fat mass to body weight percentage and maintaining lean mass to body weight percentage.

Compound 40, at doses of 50 mpk or higher, significantly reduced fasting glucose and improved insulin sensitivity in DIO mice as shown in FIG. 11.

FIG. 12. depicts a weight loss comparison between DIO mice receiving vehicle, semaglutide, NT-0796, WTX3232, or Compound 40

Compound 40, at doses of 50 mpk or higher, significantly reduced liver weight and inguinal fat weight. The inguinal fat to body weight percentage or muscle tissues to body weight percentage of Compound 40-treated DIO mice had no significant difference from Semaglutide treated mice as shown in FIG. 13.

The body weight change and body weight percentage changes are shown in FIGS. 14A and 14B for mice receiving Compound 096 or Compound 211 compared to a vehicle or semaglutide. Compounds 096 and 211 showed a dose-dependent effect on reducing body weight of DIO mice. Table 6 summarizes the data shown in FIG. 14

Groups
baseline

The changes in food consumption in mice receiving no treatment (VEH), Compound 096, Compound 211, and semaglutide are shown in FIG. 14. Compounds 096 and 211 showed a dose-dependent effect on reducing food consumption, which also caused a change in fat mass as shown in FIG. 15.

As can be seen in the above example and corresponding Figures, Compound 40, Compound 096, and Compound 211 significantly reduced body weight and food consumption of high fat DIO mice, which is comparable to the effect of Semaglutide.

Example 5: Foot Gout Model

Compound 096 and Compound 040 reduce swelling in a foot gout mouse model in a dose dependent manner as shown in FIG. 16 and FIG. 17. Compound 040 can be used as both a preventative and therapeutic treatment.

The disclosed subject matter is not to be limited in scope by the specific embodiments and examples described herein. Indeed, various modifications of the disclosure in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

All references (e.g., publications or patents or patent applications) cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Other embodiments are within the following claims.