RU2848120C1 - Kinase nek7 inhibitors - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a compound of formula (I)

or a pharmaceutically acceptable salt thereof, wherein A is C₆ aryl, C₆ cycloalkyl or 6-membered monocyclic heteroaryl, having one nitrogen heteroatom, each of which is optionally substituted with one or two R⁵; X is N or CH; Y is NH; R¹ is H; R² is H, C₃-C₈ cycloalkyl, 4–6 membered heterocyclyl having one ring heteroatom selected from nitrogen, oxygen and sulphur, or 6-membered heteroaryl having one ring heteroatom selected from nitrogen, each of which is optionally substituted with another substituent selected from halogen, hydroxyl and C₁-C₆ alkyl; or R² is methyl, ethyl or 2-methylpropyl, each of which is optionally substituted with one more substituent selected from hydroxyl, C₂-C₆ alkenyl or C₁-C₆ alkoxy; R³ is a heteroaryl selected from isoxazolyl, 1,3,4-oxadiazolyl, 1,2,4-triazolyl, thiazolyl, isothiazolyl and 1,2,4-thiadiazolyl, each of which is optionally substituted with one substitute selected from the group consisting of C₁-C₆ alkyl, C₁-C₆ haloalkyl, C_1-2 haloalkylC₃ cycloalkyl and C_1-2 alkylC_3-4 cycloalkyl; R⁴ is H; and R⁵ in each case independently represents halogen, C₁-C₆ alkyl, C₁-C₆ hydroxylalkyl, pharmaceutical compositions containing such compounds, and a method for treating an NLRP3-mediated disorder.

EFFECT: novel compounds which can be used to treat an NLRP3-mediated disorder are obtained.

37 cl, 2 tbl, 73 ex

Description

State of the art

Field of technology

Embodiments of the present invention generally relate to compounds and methods for their preparation and use as therapeutic or prophylactic agents, such as for the treatment of inflammation.

Description of the prior art

Inflammasomes are multiprotein complexes whose activation plays a central role in innate immunity and inflammation. Four inflammasomes have been described to date: NLRP1, NLRC4, NLRP3, and AIM2. The NLRP3 inflammasome consists of NLRP3, ASC, and caspase-1. Its activation leads to the activation of caspase-1, which promotes the secretion of IL-1β and IL-18, cytokines that mediate inflammation in animal models of certain autoimmune diseases, myocardial infarction, metabolic syndromes, inflammatory bowel disease, and macrophage activation syndrome.

NEK7 is a member of the NIMA-related kinase (NEK) family, which acts as NLRP3-binding proteins, regulating its oligomerization and activation. NEK7 is a serine/threonine kinase required for mitotic entry, cell cycle progression, cell division, and mitotic progression. It is expressed in various tissues, including the brain, heart, lungs, liver, and spleen. Overexpression of NEK7 induces the production of abnormal cells, which is closely associated with tumors such as retinoblastoma, gallbladder cancer, and head and neck carcinoma.

A large number of inhibitors are widely used to disrupt effector signaling pathways involving IL-1β or IL-18 without abolishing the inflammatory response. Inhibitors of NLRP3 inflammasome activation, which block the NLRP3-NEK7 interaction, may have therapeutic or preventive activity in several human diseases, such as type 2 diabetes (T2D), atherosclerosis, gout, and neurodegenerative diseases. However, the precise mechanism of NLRP3-NEK7 interaction is poorly understood.

Accordingly, there is a need to develop inhibitors that directly target NEK7 to address the NLRP3 inflammasome-modulated inflammatory response in a range of pathological conditions, including gout, atherosclerosis, type 2 diabetes, metabolic syndrome, macular degeneration, Alzheimer's disease, multiple sclerosis, and inflammatory bowel disease. Embodiments of the present invention address this need and provide additional related advantages.

The essence of the invention

In brief, embodiments of the present invention provide compounds, including pharmaceutically acceptable salts, stereoisomers, and prodrugs thereof, that are capable of modulating the activity of the NLRP3 inflammasome.

In one aspect, the invention relates to compounds of structure (I):

their pharmaceutically acceptable salts, stereoisomers or prodrugs, wherein each of A, X, Y, R ¹ , R ² , R ³ and R ⁴ has the meanings specified below.

In another aspect, pharmaceutical compositions containing the compounds of the present invention and methods of using them for treating inflammation are also provided.

Detailed description

In the following description, certain specific details are set forth to provide a thorough understanding of various embodiments of the invention. However, one skilled in the art will appreciate that the disclosure may be practiced without these details.

Unless the context otherwise requires, in the present description and claims, the word "comprise" and its variations such as "comprises" and "comprising" should be interpreted in an open, inclusive sense, i.e., as "including, but not limited to."

In this specification, any concentration range, percentage range, ratio range, or integer range shall be understood to include the value of any integer within the stated range and, where appropriate, fractions thereof (e.g., one tenth and one hundredth of an integer), unless otherwise specified. As used herein, the terms "about" and "approximately" shall mean ±20%, ±10%, ±5%, or ±1% of the stated range, value, or structure, unless otherwise specified. The singular forms "a" and "b" as used herein shall be understood to refer to "one or more" of the listed components. The use of an alternative (e.g., "or") shall be understood to mean any one, both, or any combination of the alternatives.

Reference in this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. As used in the description and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

"Amino" refers to the radical -NH ₂ .

"Carboxy" or "carboxyl" refers to the radical _-CO2H .

"Cyano" refers to the radical -CN.

"Hydroxy" or "hydroxyl" refers to the radical -OH.

"Nitro" refers to the radical -NO ₂ .

"Oxo" refers to the =O substituent.

"Thiol" refers to the -SH substituent.

"Thioxo" refers to the =S substituent.

"Alkyl" refers to a saturated hydrocarbon radical, straight or branched chain, consisting solely of carbon and hydrogen atoms, having from one to twelve carbon atoms (C ₁ -C ₁₂ alkyl), from one to eight carbon atoms (C ₁ -C ₈ alkyl), or from one to six carbon atoms (C ₁ -C ₆ alkyl), or any value within these ranges, such as C ₄ -C ₆ alkyl, and the like, and which is attached to the rest of the molecule by a single bond, such as methyl, ethyl, n -propyl, 1-methylethyl (isopropyl), n -butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), 3-methylhexyl, 2-methylhexyl, and the like. The number of carbon atoms mentioned refers to the carbon skeleton and the branching of the carbon chain, but does not include carbon atoms belonging to any substituents. Unless otherwise specifically stated in the description, the alkyl group is optionally substituted.

"Alkenyl" refers to an unsaturated straight or branched chain hydrocarbon radical consisting solely of carbon and hydrogen atoms, which contains one or more carbon-carbon double bonds, having from two to twelve carbon atoms (C ₂ -C ₁₂ alkenyl), from two to eight carbon atoms (C ₂ -C ₈ alkenyl), or from two to six carbon atoms (C ₂ -C ₆ alkenyl), or any value within these ranges, and which is attached to the rest of the molecule by a single bond, for example, ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1,4-dienyl, and the like. The number of carbon atoms mentioned refers to the carbon skeleton and the branching of the carbon chain, but does not include the carbon atoms belonging to any substituents. Unless otherwise specifically stated in the description, an alkenyl group is optionally substituted.

The term "alkynyl" refers to an unsaturated straight or branched hydrocarbon radical having from 2 to 12 carbon atoms (C ₂ -C ₁₂ alkynyl), from two to nine carbon atoms (C ₂ -C ₉ alkynyl), or from two to six carbon atoms (C ₂ -C ₆ alkynyl), or any value in these ranges, and having at least one carbon-carbon triple bond. Examples of alkynyl groups can be selected from the group consisting of ethynyl, propargyl, but-1-ynyl, but-2-ynyl, and the like. The mentioned number of carbon atoms refers to the carbon skeleton and branching of the carbon chain, but does not include carbon atoms belonging to any substituents. Unless otherwise specifically stated in the description, the alkynyl group is optionally substituted.

"Alkoxy" refers to a radical of the formula -OR _a , where R _a is an alkyl radical, as defined above, containing from one to twelve carbon atoms (C ₁ -C ₁₂ alkoxy), from one to eight carbon atoms (C ₁ -C ₈ alkoxy), or from one to six carbon atoms (C ₁ -C ₆ alkoxy), or any value within these ranges. Unless otherwise specifically stated in the description, an alkoxy group is optionally substituted.

"Aminyl" refers to a radical of the formula -NR _a R _b , where R _a is H or C ₁ -C ₆ alkyl and R _b is C ₁ -C ₆ alkyl, as defined above. The C ₁ -C ₆ alkyl portion of the aminyl group is optionally substituted unless otherwise noted.

"Aminylalkylcycloalkyl" refers to a radical of the formula -R _a R _b NR _c R _d , wherein R _a is cycloalkyl as defined herein, R _b is C ₁ -C ₆ alkyl, R _c is H or C ₁ -C ₆ alkyl, and R _d is C ₁ -C ₆ alkyl as defined above. Cycloalkyl and each C ₁ -C ₆ alkyl portion of an aminylalkylcycloalkyl group are optionally substituted unless otherwise noted.

An "aromatic ring" refers to a cyclic planar molecule or molecular moiety (i.e., a radical) with a ring of resonant bonds that exhibits increased stability compared to other compounds with the same sets of atoms. Typically, aromatic rings contain a set of covalently bonded coplanar atoms and a number of π-electrons (e.g., alternating double and single bonds) that is even but not a multiple of 4 (i.e., 4n+2 π-electrons), where n=0, 1, 2, 3, etc.). Aromatic rings include, but are not limited to, phenyl, naphthenyl, imidazolyl, pyrrolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridonyl, pyridazinyl, and pyrimidonyl. Unless otherwise specifically stated in the description, an "aromatic ring" includes all radicals that are optionally substituted.

"Aryl" refers to a carbocyclic ring system radical containing from 6 to 18 carbon atoms, such as from 6 to 10 carbon atoms ( _C6 - _C10 aryl) and at least one carbocyclic aromatic ring. For purposes of embodiments of the present invention, an aryl radical is a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused or bridged ring systems. Aryl radicals include, but are not limited to, aryl radicals derived from aceanthrilene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, ac-indacene, c-ndacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene. Unless otherwise specifically stated in the description, an aryl group is optionally substituted.

"Cyanoalkyl" refers to an alkyl group containing at least one cyano substituent. The -CN substituent may be on a primary, secondary, or tertiary carbon atom. Unless otherwise specifically stated in the description, a cyanoalkyl group is optionally substituted. "Carbocyclic" or "carbocycle" refers to a ring system in which each of the ring atoms is carbon.

"Cycloalkyl" refers to a non-aromatic monocyclic or polycyclic carbocyclic radical consisting solely of carbon and hydrogen atoms, which may include fused or bridged ring systems, having from three to fifteen ring carbon atoms ( _C3 - _C15 cycloalkyl), from three to ten ring carbon atoms ( _C3 - _C10 cycloalkyl), or from three to eight ring carbon atoms ( _C3 - _C8 cycloalkyl), or any value within these ranges, such as from three to four carbon atoms ( _C3 - _C4 cycloalkyl), and which is saturated or partially unsaturated and attached to the rest of the molecule by a single bond. Monocyclic radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic radicals include, for example, adamantyl, norbornyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like. Unless otherwise specifically stated in the description, the cycloalkyl group is optionally substituted.

"Alkylcycloalkyl" refers to a radical of the formula -R _a R _b , where R _a is a cycloalkyl group and R _b is an alkyl group as defined above. Unless otherwise specifically stated in the description, an alkylcycloalkyl group is optionally substituted.

"Fused" refers to any ring structure described herein that is fused to another ring structure.

"Halogen" refers to bromine, chlorine, fluorine or iodine.

"Haloalkyl" refers to an alkyl radical as defined above that is substituted by one or more halo radicals as defined above, such as trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl, and the like. Unless otherwise specifically stated in the description, a haloalkyl group is optionally substituted.

"Halocycloalkyl" refers to a cycloalkyl radical, as defined above, which is substituted by one or more halo radicals, as defined above, such as trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl, and the like. Unless otherwise specifically stated in the description, a halocycloalkyl group is optionally substituted.

"Haloalkylcycloalkyl" refers to a radical group of the formula -R _a R _b , where R _a is a cycloalkyl group and R _b is a haloalkyl group as defined above. Unless otherwise specifically stated in the description, a haloalkylcycloalkyl group is optionally substituted.

"Halocycloalkylalkyl" refers to a radical group of the formula -R _a R _b , where R _a is an alkyl group and R _b is a halocycloalkyl group as defined above. Unless otherwise specifically stated in the description, a halocycloalkylalkyl group is optionally substituted.

"Heterocyclylcycloalkyl" refers to a radical group of the formula -R _a R _b , where R _a is a cycloalkyl group and R _b is a heterocyclyl group, as defined herein. Unless otherwise specifically stated in the description, a heterocyclylcycloalkyl group is optionally substituted.

"Hydroxyalkyl" refers to an alkyl radical, as defined above, that is substituted by one or more hydroxyl radicals. The hydroxyalkyl radical is attached to the main chain through a carbon atom of the alkyl group. Unless otherwise specifically stated in the description, the hydroxyalkyl group is optionally substituted.

"Heterocyclyl" refers to a 3- to 18-membered, for example, 3- to 10-membered, or 3- to 8-membered, non-aromatic ring radical containing from one to ten ring carbon atoms (e.g., two to ten) and from one to six ring heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. Unless otherwise specifically stated in the description, a heterocyclyl radical is partially or fully saturated and represents a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused, spirocyclic, and/or bridged ring systems. The nitrogen, carbon, and sulfur atoms in a heterocyclyl radical are optionally oxidized, and the nitrogen atoms may be optionally quaternized. Examples of such heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, furanonyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, hexahydro-1H-pyrrolizine, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, oxiranyl, piperidinyl, piperazinyl, 4-piperidonyl, azetidinyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxothiomorpholinyl and 1,1-dioxothiomorpholinyl. Unless otherwise specifically stated in the description, the heterocyclyl group is optionally substituted.

"Halogenoheterocyclyl" refers to a heterocyclyl group containing at least one halogen substituent. The halogen substituent may be on a primary, secondary, or tertiary carbon atom. Unless otherwise specifically stated in the description, a halogenoheterocyclyl group is optionally substituted.

"Halogenoheterocyclylalkyl" refers to a radical group of the formula -R _a R _b , where R _a is an alkyl group and R _b is a haloheterocyclyl group, as defined herein. Unless otherwise specifically stated in the description, a haloheterocyclylalkyl group is optionally substituted.

"Heterocyclylalkyl" refers to a radical group of the formula -R _a R _b , where R _a is an alkyl group and R _b is a heterocyclyl group, as defined herein. Unless otherwise specifically stated in the description, a heterocyclylalkyl group is optionally substituted.

"Heteroaryl" refers to a 5- to 18-membered, for example 5- to 6-membered, ring system radical containing from one to thirteen ring carbon atoms, from one to six ring heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur, and at least one aromatic ring. Heteroaryl radicals may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen, carbon, or sulfur atoms in the heteroaryl radical may be optionally oxidized; the nitrogen atom may be optionally quaternized. Examples include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyridazinyl, 1-oxidopyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl and thiophenyl (i.e. thienyl). Unless otherwise specifically stated in the description, a heteroaryl group is optionally substituted.

Oxazolyl, isoxazolyl, 1, 2, 3-oxadiazolyl, 1, 2, 4-oxadiazolyl, 1, 2, 5-oxadiazolyl, 1, 3, 4-oxadiazolyl, 1, 2, 3-triazolyl, 1, 2, 4-triazolyl, thiazolyl, isothiazolyl, 1, 2, 3-thiadiazolyl, 1, 2, 4-thiadiazolyl, 1, 2, 5-thiadiazolyl, and 1, 3, 4-thiadiazolyl refer to the following structures, respectively:

wherein oxazolyl, isoxazolyl, 1, 2, 3-oxadiazolyl, 1, 2, 4-oxadiazolyl, 1, 2, 5-oxadiazolyl, 1, 3, 4-oxadiazolyl, 1, 2, 3-triazolyl, 1, 2, 4-triazolyl, thiazolyl, isothiazolyl, 1, 2, 3-thiadiazolyl, 1, 2, 4-thiadiazolyl, 1, 2, 5-thiadiazolyl and 1, 3, 4-thiadiazolyl are attached to the remainder of the molecule by a covalent bond to one of the carbon atoms in the oxazolyl, isoxazolyl, 1, 2, 3-oxadiazolyl, 1, 2, 4-oxadiazolyl ring, 1, 2, 5-oxadiazolyl, 1, 3, 4-oxadiazolyl, 1, 2, 3-triazolyl, 1, 2, 4-triazolyl, thiazolyl, isothiazolyl, 1, 2, 3-thiadiazolyl, 1, 2, 4-thiadiazolyl, 1, 2, 5-thiadiazolyl and 1, 3, 4-thiadiazolyl.

The term "substituted" as used herein means any of the above groups (e.g., alkyl, alkenyl, alkylene, alkylcarbonyl, alkoxy, alkoxyalkyl, aminylalkyl, aryl, cyanoalkyl, cycloalkyl, haloalkyl, heterocyclyl, heterocyclene, heterocyclylalkyl, heteroaryl, heteroarylalkyl and/or hydroxyalkyl) wherein at least one hydrogen atom (e.g., 1, 2, 3, or all hydrogen atoms) is replaced by a bond to a non-hydrogen substituent. Examples of non-hydrogen substituents include, but are not limited to: amino, carboxyl, cyano, hydroxyl, halogen, nitro, oxo, thiol, thioxo, alkyl, alkenyl, alkylcarbonyl, alkoxy, aryl, cyanoalkyl, cycloalkyl, haloalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl and/or hydroxylalkyl substituents, each of which may also be optionally substituted with one or more of the above substituents.

In some specific embodiments, the optional substituents are independently selected from the group consisting of halogen, hydroxyl, cyano, aminyl, _C1 - _C6 alkyl, _C2 - _C6 alkenyl, _C2 - _C6 alkynyl, _C1 - _C6 haloalkyl, _C3 - _C8 cycloalkyl, _C3 - _C8 halocycloalkyl, _C6 - _C10 aryl, 5- or 6-membered heteroaryl, _C1 - _C6 alkoxy, and 3-8 membered heterocyclyl.

The term "effective amount" or "therapeutically effective amount" refers to that amount of a compound described herein that is sufficient to accomplish the intended use, including, but not limited to, the treatment of a disease, as defined below. A therapeutically effective amount may vary depending on the intended therapeutic use ( in vivo ) or the subject and disease state being treated, such as the weight and age of the subject, the severity of the disease state, the route of administration, and the like, which can be readily determined by one of skill in the art. This term also applies to a dose that induces a specific response in target cells, such as a decrease in platelet adhesion and/or cell migration. The specific dose will vary depending on the particular compound selected, the administration regimen to be followed, whether it is administered in combination with other compounds, the timing of administration, the tissue into which it is administered, and the physical delivery system in which it is delivered.

As used herein, "therapy" or "treatment" refers to an approach for obtaining beneficial or desired results with respect to a disease, disorder, or medical condition, including, but not limited to, a therapeutic effect and/or a prophylactic effect. A therapeutic effect is understood to mean the elimination or alleviation of the underlying disease being treated. Furthermore, a therapeutic benefit is achieved by eliminating or improving one or more physiological symptoms associated with the underlying disease, such that the subject experiences an improvement, despite the fact that the subject may still suffer from the underlying disease. A prophylactic effect includes delaying or eliminating the manifestation of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, stopping, or reversing the progression of a disease or condition, or any combination thereof. In some embodiments, for prophylactic purposes, compositions are administered to a subject at risk of developing a particular disease or to a subject reporting one or more physiological symptoms of a disease, even if a diagnosis of this disease may not be made.

The terms "co-administration," "administration in combination with," and their grammatical equivalents, as used herein, encompass the administration of two or more agents to an animal, including humans, such that both agents and/or their metabolites are present in the subject at the same time. Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which both agents are present.

"Pharmaceutically acceptable salt" refers to acid addition salts and base addition salts.

"Pharmaceutically acceptable acid addition salt" refers to those salts that retain the biological effectiveness of the free bases, are biologically tolerable, or are otherwise biologically suitable for administration to a subject. See, generally, S. M. Berge, et al. , "Pharmaceutical Salts," J. Pharm. Sci., 1977, 66:1-19, and Handbook of Pharmaceutical Salts, Properties, Selection, and Use, Stahl and Wermuth, Eds., Wiley-VCH and VHCA, Zurich, 2002. Preferred pharmaceutically acceptable acid addition salts are those that are pharmacologically effective and suitable for contact with patient tissues without undue toxicity, irritation, or allergic reaction. Pharmaceutically acceptable acid addition salts which are formed with inorganic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxoglutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucinous acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, etc.

"Pharmaceutically acceptable base addition salt" refers to those salts that retain the biological effectiveness of the free acids, are biologically tolerable, or otherwise biologically suitable for administration to a subject. See , generally, S. M. Berge, et al. , "Pharmaceutical Salts," J. Pharm. Sci., 1977, 66:1-19, and Handbook of Pharmaceutical Salts, Properties, Selection, and Use, Stahl and Wermuth, Eds., Wiley-VCH and VHCA, Zurich, 2002. Preferred pharmaceutically acceptable base addition salts are those that are pharmacologically effective and suitable for contact with patient tissues without undue toxicity, irritation, or allergic reaction. Pharmaceutically acceptable base addition salts are prepared by adding an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines and basic ion exchange resins such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dinol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benetamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

In some embodiments, pharmaceutically acceptable salts include quaternary ammonium salts, such as alkyl halide quaternary amine salts (e.g., methyl bromide).

The terms "antagonist" and "inhibitor" are used interchangeably and refer to a compound that has the ability to inhibit the biological function of a target protein, whether by inhibiting the activity or expression of a protein such as the NLRP3 or NEK7 inflammasome or the NLRP3-NEK7 inflammasome association. Accordingly, the terms "antagonist" and "inhibitors" are defined in the context of the biological role of the target protein. Although the preferred antagonists disclosed in this application specifically interact with (e.g., bind to) the target, compounds that inhibit the biological activity of the target protein through interaction with other participants in the signal transduction pathway of which the target protein is a member are also specifically included within the scope of this definition. The preferred biological activity inhibited by the antagonist is related to the development, growth, or spread of a tumor.

The term "agonist," as used herein, refers to a compound that has the ability to initiate or enhance the biological function of a target protein by inhibiting the activity or expression of the target protein. Accordingly, the term "agonist" is defined in the context of the biological role of the target polypeptide. Although the preferred agonists described in this application specifically interact with (e.g., bind to) the target, compounds that initiate or enhance the biological activity of the target polypeptide through interaction with other participants in the signal transduction pathway of which the target polypeptide is a member are also specifically included within the scope of this definition.

Signaling is the process by which stimulatory or inhibitory signals are transmitted into and within a cell to elicit an intracellular response.

The term "selective inhibition" or "selectively inhibits" refers to a biologically active agent, which refers to the ability of the agent to preferentially reduce target signaling activity over non-target signaling activity through direct or indirect interaction with the target.

"Subject" refers to an animal, such as a mammal, such as a human. The methods described herein may be useful in both human and veterinary medicine. In some embodiments, the subject is a mammal, and in some embodiments, the subject is a human.

"Mammals" include humans and both domestic animals, such as laboratory animals and pets (e.g., cats, dogs, pigs, cattle, sheep, goats, horses, rabbits), and non-domestic animals, such as wild animals and the like.

"Prodrug" means a compound that can be converted under physiological conditions or by solvolysis into a biologically active compound described herein (e.g., compounds of structure (I)). Thus, the term "prodrug" refers to a pharmaceutically acceptable precursor of a biologically active compound. In some aspects, a prodrug is inactive when administered to a subject but is converted in vivo into the active compound, for example, by hydrolysis. A compound that is a prodrug often offers advantages in solubility, histocompatibility, or sustained release in a mammalian subject (see, e.g. , Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier, Amsterdam). A discussion of prodrugs is provided in Higuchi, T., et al., "Pro-drugs as Novel Delivery Systems," ACS Symposium Series, Vol. 14, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference in their entireties. The term "prodrug" also includes any covalently linked carriers that release the active compound in vivo when such prodrug is administered to a mammalian subject. Prodrugs of the active compound, as described herein Prodrugs, as described in the document, are typically prepared by modifying functional groups present in the active compound so that these modified compounds are cleaved either by routine manipulation or in vivo to yield the parent active compound. Prodrugs include compounds wherein a hydroxy, amino, or thiol group is bonded to any group that, when the prodrug of the active compound is administered to a mammalian subject, cleaves to form a free hydroxy, free amino, or free mercapto group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives of the hydroxyl functional group or acetamide, formamide, and benzamide derivatives of the amine functional group in the active compound, and the like.

The term " in vivo " refers to an event that occurs within the subject's body.

The embodiments described herein also encompass all pharmaceutically acceptable compounds of structure (I).

Some embodiments also encompass in vivo metabolic products of the disclosed compounds. Such products may be formed, for example, as a result of oxidation, reduction, hydrolysis, amidation, esterification, and the like of the administered compound, primarily through enzymatic processes. Accordingly, embodiments include compounds produced by a method comprising administering a compound of the present invention to a mammal for a period of time sufficient to form its metabolic product. Such products are typically identified by administering a radiolabeled compound of the present invention in a detectable dose to an animal, such as a rat, mouse, guinea pig, monkey, or human, allowing sufficient time for the metabolic process to occur and isolating its metabolites from urine, blood, or other biological samples.

"Stable compound" and "stable structure" are intended to mean a compound that is sufficiently robust to withstand isolation to an acceptable degree of purity from a reaction mixture and formulation into an effective therapeutic agent.

Solvates of the compounds described herein are often formed during crystallization. As used herein, the term "solvate" refers to a complex that includes one or more compounds of the present invention with one or more solvent molecules. In some embodiments, the solvent is water, in which case the solvate is a hydrate. Alternatively, in other embodiments, the solvent is an organic solvent. Thus, the compounds of the present invention can exist as hydrates, including monohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate, and the like, as well as the corresponding solvated forms. In some aspects, the compounds of the present invention are true solvates, while in other cases, the compounds of the present invention simply retain extraneous water or are a mixture of water and some extraneous solvent.

"Optional" or "optionally" means that the subsequently described circumstantial event may or may not occur, and that the description includes instances where the stated event or circumstance occurs and instances where it does not. For example, "optionally substituted aryl" means that the aryl radical may or may not be substituted, and that the description includes both substituted aryl radicals and unsubstituted aryl radicals.

"Pharmaceutical composition" refers to a composition of the compounds of the present invention and a vehicle conventional in the art for delivering the compounds of the present invention to mammals, such as humans. Such a vehicle includes all pharmaceutically acceptable carriers, diluents, or excipients.

"Pharmaceutically acceptable carrier, diluent, or excipient" includes, without limitation, any adjuvant, vehicle, excipient, lubricant, sweetener, diluent, preservative, dye/coloring agent, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solubilizer, or emulsifier.

"Stereoisomer" refers to a compound composed of the same atoms linked by the same bonds but having different three-dimensional structures that are not interchangeable. The present invention contemplates various stereoisomers and mixtures thereof and includes "enantiomers," which refers to two stereoisomers whose molecules are incompatible when superimposed as mirror images of each other.

The compounds of the present invention (i.e., the compounds of structure (I)) or pharmaceutically acceptable salts thereof may contain one or more centers of geometric asymmetry and thus may form stereoisomers, such as enantiomers, diastereomers, and other stereoisomeric forms that are defined in terms of absolute stereochemistry as (R)- or (S)- or as (D)- or (L)- for amino acids. Thus, embodiments include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)- isomers can be prepared using chiral synthons or chiral reagents or separated using conventional techniques, such as chromatography and fractional crystallization. Conventional methods for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or racemate of a salt or derivative) using, for example, chiral high-pressure liquid chromatography (HPLC). When compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless otherwise noted, the compounds are intended to include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.

Embodiments of the present invention include all possible rotamers and conformationally constrained states of the compounds of the invention. Also included are atropisomers, which are stereoisomers arising from hindered rotation about a single bond, where energy differences due to steric strain or other factors create a rotational barrier high enough to isolate individual conformers. For example, some compounds of the present invention may exist as mixtures of atropisomers or purified or enriched for the presence of a single atropisomer.

In some embodiments, the compounds of structure (I) are a mixture of enantiomers or diastereomers. In other embodiments, the compounds of structure (I) are substantially a single enantiomer or diastereomer.

"Tautomer" refers to a proton shift from one atom of a molecule to another atom of the same molecule. Thus, embodiments include tautomers of the compounds of the present invention.

The chemical names and structure diagrams used in this document are a modified form of the IUPAC nomenclature system, generated using ACD/Name Version 9.07 and/or ChemDraw Professional Version 17.0.0.206 naming program (CambridgeSoft). For complex chemical names used in this document, the substituent group is typically listed before the group to which it is attached. For example, cyclopropylethyl contains an ethyl skeleton with a cyclopropyl substituent. Except as noted below, all bonds are labeled in the chemical structure diagrams in this document, excluding all bonds on some carbon atoms that are assumed to be bonded to enough hydrogen atoms to complete the valence.

Connections

The invention relates to compounds, including their pharmaceutically acceptable salts, stereoisomers and prodrugs, which are capable of modulating the activity of the NLRP3 inflammasome.

Embodiments of the present invention provide a compound having the following structure (I):

or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof, wherein:

A is _C6 - _C10 aryl, _C3 - _C10 cycloalkyl, 3-10 membered heterocyclyl, or 5-6 membered monocyclic heteroaryl, each of which is optionally substituted with one or more ^R5 ;

X represents N or CH;

Y represents CHOH or NH;

R ¹ is H or C ₁ -C ₆ alkyl;

R ² is C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₈ cycloalkyl, 3-8 membered heterocyclyl or 5- or 6-membered heteroaryl, each of which is optionally substituted with another substituent selected from halogen, hydroxyl, cyano, aminyl, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ alkoxy and 3-8 membered heterocyclyl;

R ³ is heteroaryl selected from oxazolyl, isoxazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,3-triazolyl, 1, 2, 4-triazolyl, thiazolyl, isothiazolyl, 1, 2, 3-thiadiazolyl, 1, 2, 4-thiadiazolyl, 1, 2, 5-thiadiazolyl and 1, 3, 4-thiadiazolyl, each of which is optionally substituted with another substituent selected from amino, halogen, cyano, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, _C1 - _C6 haloalkyl, _C3 - _C8 cycloalkyl, _C3 - _C8 alkylcycloalkyl, _C3 - _C8 haloalkylcycloalkyl, _C3 - _C8 aminylalkylcycloalkyl, _C1 - _C6 cyanoalkyl _C1 - _C6 aminyl, _C1 - _C6 hydroxylalkyl, 3-8-membered heterocyclyl, 3-8-membered heterocyclylalkyl, 3-8-membered heterocyclylcycloalkyl, 3-8-membered haloheterocyclyl, 3-8-membered haloheterocyclylalkyl, _C3 - _C8 halocycloalkyl and _C3 - _C8 halocycloalkylalkyl, and combinations thereof;

R ⁴ is H, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₈ cycloalkyl, 3-8-membered heterocyclyl, C ₆ -C ₁₀ aryl, or 5- or 6-membered heteroaryl, each of which is optionally substituted with another substituent selected from halogen, hydroxyl, cyano, aminyl, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, and C ₁ -C ₆ alkoxy; and

R ⁵ in each occurrence is independently halogen, cyano, C ₁ -C ₆ alkyl, C ₁ -C ₆ hydroxylalkyl or C ₁ -C ₆ haloalkyl.

In some embodiments of structure (I), A is _C6 - _C10 aryl, _C3 - _C10 cycloalkyl, 3-10 membered heterocyclyl, or 5-6 membered monocyclic heteroaryl, each of which is optionally substituted with one or more ^R5 ;

X represents N or CH;

Y represents CHOH or NH;

R ¹ is H or C ₁ -C ₆ alkyl;

R ² is C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₈ cycloalkyl, 3-8-membered heterocyclyl or 5- or 6-membered heteroaryl, each of which is optionally substituted with another substituent selected from halogen, hydroxyl, cyano, aminyl, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ alkoxy and 3-8-membered heterocyclyl;

R ³ is heteroaryl selected from oxazolyl, isoxazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,3-triazolyl, 1, 2, 4-triazolyl, thiazolyl, isothiazolyl, 1, 2, 3-thiadiazolyl, 1, 2, 4-thiadiazolyl, 1, 2, 5-thiadiazolyl and 1, 3, 4-thiadiazolyl, each of which is optionally substituted with a further substituent selected from halogen, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ haloalkyl, _C3 - _C8 cycloalkyl and _C3 - _C8 halocycloalkyl;

R ⁴ is H, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₈ cycloalkyl, 3-8-membered heterocyclyl, C ₆ -C ₁₀ aryl, or 5- or 6-membered heteroaryl, each of which is optionally substituted with another substituent selected from halogen, hydroxyl, cyano, aminyl, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, and C ₁ -C ₆ alkoxy; and

R ⁵ in each occurrence is independently halogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkyl, or C ₁ -C ₆ haloalkyl.

In one embodiment, there is provided a compound of structure (I) or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof, wherein:

A is _C6 - _C10 aryl, _C3 - _C10 cycloalkyl, 3-10 membered heterocyclyl, or 5-6 membered monocyclic heteroaryl, each of which is optionally substituted with one or more ^R5 ;

X represents N or CH;

Y represents CHOH or NH;

R ¹ is H or C ₁ -C ₆ alkyl;

R ² is H, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₈ cycloalkyl, 3-8-membered heterocyclyl or 5- or 6-membered heteroaryl, each of which is optionally substituted with another substituent selected from halogen, hydroxyl, cyano, aminyl, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ alkoxy and 3-8 membered heterocyclyl;

R ³ is heteroaryl selected from oxazolyl, isoxazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,3-triazolyl, 1, 2, 4-triazolyl, thiazolyl, isothiazolyl, 1, 2, 3-thiadiazolyl, 1, 2, 4-thiadiazolyl, 1, 2, 5-thiadiazolyl and 1, 3, 4-thiadiazolyl, each of which is optionally substituted with another substituent selected from amino, halogen, cyano, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, _C1 - _C6 haloalkyl, _C3 - _C8 cycloalkyl, _C3 - _C8 alkylcycloalkyl, _C3 - _C8 haloalkylcycloalkyl, _C3 - _C8 aminylalkylcycloalkyl, _C1 - _C6 cyanoalkyl, _C1 -C6 aminyl, _C1 - _C6 hydroxylalkyl, _3-8 -membered heterocyclyl, 3-8-membered heterocyclylalkyl, 3-8-membered heterocyclylcycloalkyl, 3-8-membered haloheterocyclyl, 3-8-membered haloheterocyclylalkyl, _C3 - _C8 halocycloalkyl and _C3 - _C8 halocycloalkylalkyl, and combinations thereof;

R ⁴ is H, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₈ cycloalkyl, 3-8-membered heterocyclyl, C ₆ -C ₁₀ aryl, or 5- or 6-membered heteroaryl, each of which is optionally substituted with another substituent selected from halogen, hydroxyl, cyano, aminyl, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, and C ₁ -C ₆ alkoxy; and

R ⁵ in each occurrence is independently halogen, cyano, C ₁ -C ₆ alkyl, C ₁ -C ₆ hydroxylalkyl, C ₁ -C ₆ alkoxy, or C ₁ -C ₆ haloalkyl.

In a certain embodiment, R ¹ is H. In other embodiments, R ¹ is C ₁ -C ₆ alkyl, such as methyl.

In one embodiment, compounds of structure (I) are provided, wherein R ² is branched C ₄ -C ₆ alkyl, C ₃ -C ₄ cycloalkyl, C ₃ -C ₈ heterocyclyl, or 5- or 6-membered heteroaryl, each of which is optionally substituted with another substituent selected from halogen, hydroxyl, cyano, aminyl, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ alkoxy, and 3-8-membered heterocyclyl.

In another embodiment, compounds of structure (I) are provided, wherein R ² is branched C ₄ -C ₆ alkyl, C ₃ -C ₄ cycloalkyl, or C ₃ -C ₈ heterocyclyl, each of which is optionally substituted with another substituent selected from halogen, hydroxyl, cyano, aminyl, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ alkoxy, and 3-8-membered heterocyclyl.

In certain embodiments, R ² is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, each of which is optionally substituted with another substituent selected from halogen, hydroxyl, cyano, aminyl, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ alkoxy, and 3-8-membered heterocyclyl.

In other embodiments, R ² is methyl, isopropyl, 2-methylpropyl, or allyl, each of which is optionally substituted with another substituent selected from halogen, hydroxyl, cyano, aminyl, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ alkoxy, and 3-8-membered heterocyclyl.

In other embodiments, R ² is methyl, ethyl, isopropyl, 2-methylpropyl, or allyl, each of which is optionally substituted with another substituent selected from halogen, hydroxyl, cyano, aminyl, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ alkoxy, and 3-8-membered heterocyclyl.

In other embodiments, R ² is oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, or dioxidotetrahydrothiophenyl, each of which is optionally substituted with another substituent selected from halogen, hydroxyl, cyano, aminyl, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ alkoxy, and 3-8-membered heterocyclyl.

In other embodiments, R ² is oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, azetidinyl, or dioxidotetrahydrothiophenyl, each of which is optionally substituted with another substituent selected from halogen, hydroxyl, cyano, aminyl, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ alkoxy, and 3-8-membered heterocyclyl.

In other embodiments, R ² is oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, azetidinyl pyrrolidinyl or dioxidotetrahydrothiophenyl, each of which is optionally substituted with another substituent selected from halogen, hydroxyl, cyano, aminyl, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ alkoxy and 3-8-membered heterocyclyl.

In yet another embodiment, R ² is pyridinyl optionally substituted with another substituent selected from halogen, hydroxyl, cyano, aminyl, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ alkoxy, and 3-8-membered heterocyclyl.

In any of the above embodiments, R ² is unsubstituted. In other of the previous embodiments, R ² is substituted with one or more of hydroxyl and fluoro.

In any of the above embodiments, R ² is unsubstituted. In other of the previous embodiments, R ² is substituted with one or more of hydroxyl, methyl, methoxy, and fluoro.

In more specific embodiments, R ² has one of the following structures:

In other specific embodiments, R ² has one of the following structures:

In other specific embodiments, R ² has one of the following structures:

In some embodiments, R ² is optionally substituted with one or more substituents selected from the group consisting of halogen, hydroxyl, cyano, aminyl, C ₁ -C ₆ alkyl, and 3-8-membered heterocyclyl.

In some embodiments, R ² does not have the following structures:

.

In any of the above embodiments, R ³ is oxadyl, isoxazolyl, 1, 2, 3-oxadiazolyl or 1, 3, 4-oxadiazolyl, each of which is optionally substituted with another substituent selected from halogen, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ haloalkyl, C ₃ -C ₈ cycloalkyl and C ₃ -C ₈ halocycloalkyl. For example, in some embodiments, R ³ is isoxazolyl optionally substituted with another substituent selected from halogen, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ haloalkyl, C ₃ -C ₈ cycloalkyl, and C ₃ -C ₈ halocycloalkyl. In other specific embodiments, R ³ is substituted with C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, C ₃ -C ₈ cycloalkyl, or C ₃ -C ₈ halocycloalkyl.

In other embodiments, R ³ is oxazolyl, isoxazolyl, 1, 2, 3-oxadiazolyl, 1, 3, 4-oxadiazolyl, thiazolyl, isothiazolyl, 1, 2, 4-thiadiazolyl, 1, 3, 4-thiadiazolyl, or 1, 2, 4-triazolyl, each of which is optionally substituted with another substituent selected from halogen, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ haloalkyl, C ₃ -C ₈ cycloalkyl, cyano, C ₁ -C ₆ aminyl, C ₁ -C ₆ hydroxylalkyl, 3-8 membered heterocyclyl, and C ₃ -C ₈ halocycloalkyl, or combinations thereof.

In some embodiments, R ³ is isoxazolyl optionally substituted with another substituent selected from halogen, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ haloalkyl, C ₃ -C ₈ cycloalkyl, cyano, C ₁ -C ₆ aminyl, C ₁ -C ₆ hydroxylalkyl, 3-8-membered heterocyclyl and C ₃ -C ₈ halocycloalkyl, or combinations thereof.

In some embodiments, R ³ is thiazolyl optionally substituted with another substituent selected from halogen, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ haloalkyl, C ₃ -C ₈ cycloalkyl, cyano, C ₁ -C ₆ aminyl, C ₁ -C ₆ hydroxylalkyl, 3-8 membered heterocyclyl and C ₃ -C ₈ halocycloalkyl, or combinations thereof.

In some embodiments, R ³ is isothiazolyl optionally substituted with another substituent selected from halogen, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ haloalkyl, C ₃ -C ₈ cycloalkyl, cyano, C ₁ -C ₆ aminyl, C ₁ -C ₆ hydroxylalkyl, 3-8-membered heterocyclyl and C ₃ -C ₈ halocycloalkyl, or combinations thereof.

In some embodiments, R ³ is 1,2,4-thiadiazolyl optionally substituted with another substituent selected from halogen, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ haloalkyl, C ₃ -C ₈ cycloalkyl, cyano, C ₁ -C ₆ aminyl, C ₁ -C ₆ hydroxylalkyl, 3-8-membered heterocyclyl and C ₃ -C ₈ halocycloalkyl, or combinations thereof.

In some embodiments, R ³ is 1,3,4-thiadiazolyl optionally substituted with another substituent selected from halogen, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ haloalkyl, C ₃ -C ₈ cycloalkyl, cyano, C ₁ -C ₆ aminyl, C ₁ -C ₆ hydroxylalkyl, 3-8-membered heterocyclyl and C ₃ -C ₈ halocycloalkyl, or combinations thereof.

In some embodiments, R ³ is 1,3,4-oxadiazolyl optionally substituted with another substituent selected from halogen, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ haloalkyl, C ₃ -C ₈ cycloalkyl, cyano, C ₁ -C ₆ aminyl, C ₁ -C ₆ hydroxylalkyl, 3-8 membered heterocyclyl and C ₃ -C ₈ halocycloalkyl, or combinations thereof.

In some embodiments, R ³ is 1,2,4-triazolyl optionally substituted with another substituent selected from halogen, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ haloalkyl, C ₃ -C ₈ cycloalkyl, cyano, C ₁ -C ₆ aminyl, C ₁ -C ₆ hydroxylalkyl, 3-8-membered heterocyclyl and C ₃ -C ₈ halocycloalkyl, or combinations thereof.

In other embodiments, R ³ is substituted with C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, C ₃ -C ₈ cycloalkyl, cyano, C ₁ -C ₆ aminyl, C ₁ -C ₆ hydroxylalkyl, 3-8-membered heterocyclyl, or C ₃ -C ₈ halocycloalkyl, or a combination thereof.

In various embodiments, R ³ has one of the following structures:

In other embodiments, R ³ has one of the following structures:

In other embodiments, R ³ has one of the following structures:

In other embodiments, R ⁴ is H. In other embodiments, R ⁴ C ₁ -C ₆ is alkyl, such as methyl.

In some embodiments, Y is CHOH. In other embodiments, Y is NH.

In other embodiments, X is N. In other embodiments, X is CH.

In various embodiments, A is _C6 - _C10 aryl, _C3 - _C10 cycloalkyl, or 5-6-membered monocyclic heteroaryl, each of which is optionally substituted with one or more ^R6 . A is intended to be a divalent radical.

In some embodiments, A is a divalent optionally substituted _{C 6-10 aryl} . In some embodiments, A is a divalent optionally substituted 3-8-membered saturated or partially unsaturated carbocyclic ring. In some embodiments, A is a divalent optionally substituted 3-10-membered heterocyclic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, A is a divalent optionally substituted 5-6-membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In some embodiments, A is a divalent group selected from phenyl, pyridinyl, cyclohexyl, and cyclohexenyl; each of which is optionally substituted.

In other embodiments, A is phenyl. In other embodiments, A is saturated or unsaturated cyclohexyl. In other embodiments, A is pyridinyl.

In other embodiments, A is pyrimidinyl, which is optionally substituted.

In any of the above embodiments, A is unsubstituted. In various of the above embodiments, A is substituted with one or more R ⁵ . For example, in some embodiments, R ⁵ is halogen. In other embodiments, R ⁵ is fluoro. In various other embodiments, R ⁵ is chloro.

In some embodiments, R ⁵ is cyano. In some embodiments, R ⁵ is C ₁ -C ₆ alkyl. In some embodiments, R ⁵ is methyl. In some embodiments, R ⁵ is C ₁ -C ₆ haloalkyl. In some embodiments, R ⁵ is difluoromethyl. In other embodiments, R ⁵ is C ₁ -C ₆ hydroxylalkyl. In some embodiments, R ⁵ is -CH ₂ OH.

In some embodiments, A is a divalent group selected from phenyl, naphthyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, adamantyl, cyclooctyl, [3.3.0]bicyclooctanyl, [4.3.0]bicyclononanyl, [4.4.0]bicyclodecanyl, [2.2.2]bicyclooctanyl, fluorenyl, indanyl, tetrahydronaphthyl, acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazoline, carbazolyl, NH-carbazolyl, carbolinyl, chromanyl, chromenil, cinnolinyl, decahydroquinolinyl, dithiazinyl, tetrahydrofuranyl, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3-indolyl, isoindolinyl, isoindolenyl, isobenzofuranyl, isochromanil, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl;- 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxazolidinyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, thiadiazinyl, 1,2,3-thiadiazolayl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl, oxetanyl, azetidinyl and xanthenyl; each of which is optionally substituted.

In particular embodiments, A has one of the following structures:

In other specific embodiments, A has one of the following structures:

In a particular embodiment, the compound of structure (I) is an NLRP3 inflammasome modulator.

In a specific embodiment, the compound of structure (I) is an inhibitor of NEK7 in a patient or in a biological sample.

In various embodiments, the compound has one of the structures presented in Table 1 below, or a pharmaceutically acceptable salt, stereoisomer, or prodrug thereof. The compounds in Table 1 were prepared as described in the examples or by methods known in the art and analyzed using mass spectrometry and/or ^1H NMR.

Table 1. Representative compounds of structure (I) No. Structure Name 1 1-(4-(4-amino-1-isopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)phenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea 2 1-(4-(4-amino-1-(oxetan-3-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)phenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea 3 1-(4-(4-amino-1-(oxetan-3-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)phenyl)-3-(5-( tert- butyl)isoxazol-3-yl)urea 4 1-(4-(4-amino-1-(tetrahydro-2 H -pyran-4-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)phenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea 5 1-(4-(4-amino-1-(tetrahydrofuran-3-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)phenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea 6 1-(4-(4-amino-1-isopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-chlorophenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea 7 1-(4-(4-amino-1-cyclobutyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)phenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea 8 1-(4-(4-amino-1-cyclobutyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea 9 1-(4-(4-amino-1-(2-hydroxy-2-methylpropyl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)phenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea 10 1-(4-(4-amino-1-(2-hydroxy-2-methylpropyl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea 11 1-(4-(4-amino-1-isopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea 12 1-(4-(4-amino-1-(oxetan-3-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea 13 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea 14 1-(4-(4-amino-1-(1-(oxetan-3-yl)piperidin-4-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea 15 1-(4-(4-amino-1-(4-hydroxycyclohexyl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea 16 1-(4-(4-amino-1-(3,3-difluorocyclobutyl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea 17 1-(4-(4-amino-1-(pyridin-4-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea 18 1-(4-(4-amino-1-(1,1-dioxidotetrahydrothiophen-3-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea 19 1-(4-(4-amino-1-(tetrahydro-2 H -pyran-3-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea 20 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)phenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea 21 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea 22 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(1,1,1-trifluoro-2-methylpropan-2-yl)isoxazol-5-yl)urea 23 1-(4-(4-amino-1-(oxetan-3-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(5-( tert- butyl)isoxazol-3-yl)urea 24 1-(4-(4-amino-1-(tetrahydrofuran-3-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea 25 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)urea 26 1-(4-(1-allyl-4-amino-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(5-( tert- butyl)isoxazol-3-yl)urea 27 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(2-fluoropropan-2-yl)isoxazol-5-yl)urea 28 1-(4-(4-amino-1-methyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-chlorophenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea 29 1-(4-(4-amino-1-methyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea 30 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[4,3- c ]pyridin-3-yl)-2-fluorophenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea 31 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[4,3- c ]pyridin-3-yl)phenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea 32 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(1-methylcyclopropyl)isoxazol-5-yl)urea 33 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(5-( tert- butyl)-1,3,4-thiadiazol-2-yl)urea 34 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(4-( tert- butyl)thiazol-2-yl)urea 35 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-( tert- butyl)isothiazol-5-yl)urea 36 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(5-( tert- butyl)-1,3,4-oxadiazol-2-yl)urea 37 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-( tert- butyl)-1,2,4-thiadiazol-5-yl)urea 38 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(1-( tert- butyl)-1 H -1,2,4-triazol-3-yl)urea 39 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(trifluoromethyl)isoxazol-5-yl)urea 40 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(pentan-3-yl)isoxazol-5-yl)urea 41 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-isopropylisoxazol-5-yl)urea 42 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-ethylisoxazol-5-yl)urea 43 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-( sec- butyl)isoxazol-5-yl)urea 44 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(1-methylcyclobutyl)isoxazol-5-yl)urea 45 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[4,3- c ]pyridin-3-yl)-2-fluorophenyl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea 46 1-(4-(4-amino-1-(3-hydroxycyclobutyl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea 47 1-(5-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)pyridin-2-yl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea 48 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-methylphenyl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea 49 1-(4-(4-amino-1-(1-methylazetidin-3-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea 50 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2,5-difluorophenyl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea 51 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-(hydroxymethyl)phenyl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea 52 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(2-cyanopropan-2-yl)isoxazol-5-yl)urea 53 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(hydroxymethyl)isoxazol-5-yl)urea 54 1-(5-(4-amino-1-cyclopropyl-1 H -pyrazolo[4,3- c ]pyridin-3-yl)pyridin-2-yl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea 55 1-(4-(4-amino-1-(2-hydroxyethyl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea 56 1-(4-(4-amino-1-(2-hydroxyethyl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)urea 57 1-(4-(4-amino-1-(2-methoxyethyl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea 58 1-(4-(4-amino-1-(2-methoxyethyl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)urea 59 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-methylphenyl)-3-(5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)urea 60 1-(4-(4-amino-1-(3-hydroxycyclobutyl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)urea 61 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[4,3- c ]pyridin-3-yl)-2-fluorophenyl)-3-(5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)urea 62 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2,5-difluorophenyl)-3-(5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)urea 63 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-(hydroxymethyl)phenyl)-3-(5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)urea 64 1-(5-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)pyridin-2-yl)-3-(5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)urea 65 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(tert-butyl)-4-methylisoxazol-5-yl)urea 66 1-(4-(4-amino-1-(2-hydroxy-2-methylpropyl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea 67 1-(4-(4-amino-1-(2-hydroxy-2-methylpropyl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)urea 68 1-(4-(4-amino-1-(1-methylazetidin-3-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)urea 69 1-(4-(4-amino-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea 70 1-(4-(4-amino-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)urea 71 1-(4-(4-amino-1-(1-methylpiperidin-4-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea 72 1-(4-(4-amino-1-(1-methylpiperidin-4-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)urea 73 1-(4-(4-amino-1-(1-methylpyrrolidin-3-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea 74 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-(difluoromethyl)phenyl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea 75 1-(5-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-3-fluoropyridin-2-yl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea 76 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(1-((dimethylamino)methyl)cyclopropyl)isoxazol-5-yl)urea 77 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-((3,3-difluoroazetidin-1-yl)methyl)isoxazol-5-yl)urea 78 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(1-(4-methylpiperazin-1-yl)cyclopropyl)isoxazol-5-yl)urea 79 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(2-(4-methylpiperazin-1-yl)propan-2-yl)isoxazol-5-yl)urea 80 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(1-hydroxy-2-methylpropan-2-yl)isoxazol-5-yl)urea

It is understood that in the present description, combinations of substituents and/or variables of the depicted formulas are permissible only if such combinations result in stable compounds.

In a further embodiment, various compounds of the present invention that exist in free base or acid form can be converted into their pharmaceutically acceptable salts by treatment with an appropriate inorganic or organic base or acid using methods known to those skilled in the art. Salts of the compounds of the present invention can be converted into their free base or acid form using standard methods.

Methods for preparing the compounds described in this document are provided below. In general, the starting components can be obtained from sources such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, etc., or synthesized according to sources known to those skilled in the art (see, for example, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition (Wiley, December 2000)) or prepared as described herein.

The following general reaction schemes illustrate typical methods for preparing compounds of structure (I):

or pharmaceutically acceptable salts, stereoisomers or prodrugs thereof, wherein each of A, X, Y, R ¹ , R ² , R ³ and R ⁴ have the meanings specified herein.

General reaction scheme 1

The following general reaction scheme, where X ¹ is a halogen and X, R ¹ , R ² and A have the meanings described herein, illustrates exemplary methods for preparing intermediate amines B:

As shown in the general reaction scheme 1, functionalization of pyrazolopyrimidine with an alkyl- or aryl-boronic acid or a suitable electrophile in the presence of a base yields intermediate A, which can then be subjected to palladium-catalyzed arylation (followed by a reduction step if necessary) to form intermediate amine B.

General reaction scheme 2

The following general reaction scheme illustrates examples of methods for preparing carbamate intermediates C:

As shown in general reaction scheme 2, intermediates C can be prepared in the presence of base by reacting phenyl chloroformates and the indicated heteroarylamine (an amino-substituted analog of R ³ ). General reaction scheme 2 shows the preparation of compounds where R ⁴ is H; however, compounds where R ⁴ is other than H can be prepared by analogous methods by installing R ⁴ after the preparation of intermediate I or by using an appropriately substituted heteroarylamine.

General reaction scheme 3

The following general reaction scheme illustrates examples of methods for preparing compounds of structure (I):

Intermediates B and intermediates C are treated with a base such as trimethylamine in THF to give compounds of structure (I).

General reaction scheme 4

The following general reaction scheme illustrates examples of methods for preparing compounds of structure (I):

Intermediate B was reacted with phenyl chloroformate under appropriate conditions to give intermediate C. Intermediate C was then coupled with an amine using a suitable base (e.g., trimethylamine, DIPEA, DMAP, etc.) in THF to give compounds of structure (I).

Any of the above reaction schemes can be modified at any stage to add and/or modify a substituent or change the order of steps, as appropriate, at any stage of the overall synthesis of the desired compounds. For example, one skilled in the art will readily appreciate that the carbamate analog of intermediate B can alternatively be prepared and reacted with the amine analog R ³ to yield compounds of structure (I).

Those skilled in the art will also appreciate that in the processes for preparing the compounds described herein, functional groups of the intermediates may require protection with suitable protecting groups. Such functional groups include, but are not limited to, hydroxy, amino, mercapto, and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl (e.g., tert-butyldimethylsilyl, tert-butyldiphenylsilyl, or trimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, amidino, and guanidino include tert-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protecting groups for mercapto include -C(O)-R" (where R" is alkyl, aryl, or arylalkyl), p-methoxybenzyl, trityl, and the like. Suitable protecting groups for carboxylic acids include alkyl, aryl, or arylalkyl esters. Protecting groups are optionally added or removed according to standard methods known to those skilled in the art and described herein. The use of protecting groups is described in detail in Green, T. W. and P. G. M. Wutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley. Those skilled in the art will appreciate that the protecting group can also be a polymeric resin, such as Wang resin, Rink resin, or 2-chlorotrityl chloride resin.

Those skilled in the art will also appreciate that, although such protected derivatives of the compounds of the present invention may not possess pharmacological activity per se, they can be administered to a mammal and then metabolized in the body to form the pharmacologically active compounds of the present invention. Such derivatives may therefore be described as "prodrugs." Prodrugs of the compounds of the present invention are included within the scope of embodiments of the invention.

Pharmaceutical compositions

Other embodiments relate to pharmaceutical compositions. The pharmaceutical composition comprises any one (or more) of the above compounds and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is formulated for oral administration. In other embodiments, the pharmaceutical composition is formulated for injection. In yet another embodiment, the pharmaceutical compositions comprise a compound described herein and an additional therapeutic agent (e.g., an anticancer agent). Non-limiting examples of such therapeutic agents are described below.

Suitable routes of administration include, but are not limited to, oral, intravenous, rectal, aerosol, parenteral, ophthalmic, pulmonary, transmucosal, transdermal, vaginal, aural, nasal, and topical administration. Additionally, by way of example only, parenteral administration includes intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intralymphatic, and intranasal injections.

In some embodiments, a compound as described herein is administered locally rather than systemically, such as by injecting the compound directly into an organ, often as a depot or sustained-release preparation. In specific embodiments, long-acting preparations are administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Furthermore, in other embodiments, the compound is delivered in a targeted drug delivery system, such as a liposome coated with an organ-specific antibody. In such embodiments, the liposomes are targeted to and selectively taken up by the organ. In yet other embodiments, a compound as described herein is provided in the form of a quick-release preparation, an extended-release preparation, or an intermediate-release preparation. In yet other embodiments, the compound described herein is administered locally.

In methods of treatment according to embodiments of the invention, an effective amount of at least one compound of structure (I) is administered to a subject suffering from, or diagnosed with, a disease, disorder, or medical condition. Effective amounts or doses can be determined using methods such as modeling, dose escalation studies, or clinical trials, for example, the route or mode of administration or delivery of the drug, the pharmacokinetics of the drug, the severity and course of the disease, disorder, or condition, the subject's previous or current therapy, the subject's health status and response to drugs, and the judgment of the treating physician.

The compounds of the invention are effective over a wide dosage range. For example, when treating adults, doses of 10 to 5000 mg, 100 to 5000 mg, 1000 to 4000 mg per day, and 1000 to 3000 mg per day are examples of dosages used in some embodiments. The exact dosage will depend on the route of administration, the form in which the compound is administered, the subject being treated, the body weight of the subject being treated, and the preference and experience of the treating physician.

In some embodiments, the compounds of the present invention are administered as a single dose. Typically, such administration will be by injection, such as intravenous injection, for rapid delivery. However, other routes are also used if necessary. A single dose of a compound of the invention can also be used to treat an acute condition.

In some embodiments, the compounds of the present invention are administered in multiple doses. In some embodiments, dosing is about once, twice, three times, four times, five times, six times, or more than six times per day. In other embodiments, dosing is about once a month, once every two weeks, once a week, or once every other day. In another embodiment, the compounds of the present invention and another agent (e.g., an anticancer agent) are administered together from about once a day to about 6 times per day. In another embodiment, administration of the compounds of the present invention and the agent lasts less than about 7 days. In another embodiment, administration lasts more than about 6, 10, 14, 28 days, two months, six months, or one year. In some cases, continuous administration is achieved and maintained for as long as necessary.

Administration of the compounds of the present invention can be continued for as long as necessary. In some embodiments, the compounds of the present invention are administered for more than 1, 2, 3, 4, 5, 6, 7, 14, or 28 days. In some embodiments, the compounds of the present invention are administered for less than 28, 14, 7, 6, 5, 4, 3, 2, or 1 day. In some embodiments, the compounds of the present invention are administered on a long-term, chronic basis, such as to treat chronic effects.

In some embodiments, the compounds of the present invention are administered in separate dosage forms. It is known in the art that due to intersubject variability in compound pharmacokinetics, individualization of the dosing regimen is necessary for optimal therapy.

In some embodiments, the compounds described herein are formulated into pharmaceutical compositions. In specific embodiments, the pharmaceutical compositions are formulated in a conventional manner using one or more physiologically acceptable carriers containing excipients and auxiliaries that facilitate processing of the compounds of the invention into preparations that can be used pharmaceutically. The proper formulation depends on the chosen route of administration. Any pharmaceutically acceptable methods, carriers, and excipients are used as appropriate for preparing the pharmaceutical compositions described herein: Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins1999).

The present document provides pharmaceutical compositions comprising one or more compounds of structure (I) and a pharmaceutically acceptable carrier.

Disclosed herein are pharmaceutical compositions comprising one or more compounds selected from compounds of structure (I) and a pharmaceutically acceptable diluent(s), excipient(s), and carrier(s). In some embodiments, the disclosed compounds are administered as pharmaceutical compositions in which one or more compounds selected from compounds of structure (I) are mixed with other active ingredients, as in a combination therapy. All combinations of active substances described in the "Combination Therapies" section below and throughout this disclosure are encompassed herein. In specific embodiments, the pharmaceutical compositions include one or more compounds of structure (I).

In a particular embodiment, the pharmaceutical compositions of the compounds of structure (I) are NLRP3 inflammasome modulators.

In a specific embodiment, pharmaceutical compositions of compounds of structure (I) inhibit NEK7 when administered to a patient or biological sample.

The pharmaceutical composition used in the present application refers to a mixture of one or more compounds selected from the compounds of structure (I) with other chemical components such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents and/or excipients. In some embodiments, the pharmaceutical composition facilitates the administration of the compound to the body. In some embodiments, therapeutically effective amounts of one or more compounds selected from the compounds of structure (I) provided herein are administered in a pharmaceutical composition to a mammal suffering from the disease, disorder or medical condition being treated. In specific embodiments, the mammal is a human. In some embodiments, therapeutically effective amounts vary depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used and other factors. The compounds described herein are used alone or in combination with one or more therapeutic agents as components of mixtures.

In one embodiment, one or more compounds selected from the compounds of structure (I) are prepared as aqueous solutions. In specific embodiments, the aqueous solution is selected from, by way of example only, a physiologically compatible buffer, such as Hanks' solution, Ringer's solution, or physiological saline buffer. In other embodiments, one or more compounds selected from the compounds of structure (I) are prepared for transmucosal administration. In specific embodiments, transmucosal formulations include penetrants that are suitable for permeating a barrier. In other embodiments, in which the compounds described herein are prepared for other parenteral injections, suitable dosage forms include aqueous or non-aqueous solutions. In specific embodiments, such solutions include physiologically compatible buffers and/or excipients.

In another embodiment, the compounds described herein are prepared for oral administration. The compounds described herein are prepared by combining the active compounds, for example, with pharmaceutically acceptable carriers or excipients. In various embodiments, the compounds described herein are prepared in oral dosage forms, which include, by way of example only, tablets, powders, pills, dragees, capsules, liquids, gels, syrups, elixirs, slurries, suspensions, and the like.

In some embodiments, pharmaceutical preparations for oral use are prepared by mixing one or more solid excipients with one or more compounds described herein, optionally milling the resulting mixture, and processing the mixture of granules after adding suitable auxiliary substances, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations such as: for example, corn starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, microcrystalline cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose; or others, such as: polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. In specific embodiments, disintegrants are optionally added. Disintegrating agents include, by way of example only, cross-linked croscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

In one embodiment, dosage forms such as dragee cores and tablets are provided with one or more suitable coatings. In specific embodiments, concentrated sugar solutions are used to coat the dosage form. The sugar solutions optionally contain additional components, such as, for example, gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyes and/or pigments are also optionally added to the coatings for identification. Furthermore, dyes and/or pigments are optionally used to characterize various dose combinations of the active compounds.

In some embodiments, therapeutically effective amounts of at least one of the compounds described herein are incorporated into other oral dosage forms. Oral dosage forms include hard capsules made from gelatin, as well as soft, sealed capsules made from gelatin and a plasticizer, such as glycerin or sorbitol. In specific embodiments, hard capsules comprise the active ingredients in admixture with one or more fillers. Fillers include, by way of example only, lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In other embodiments, soft capsules comprise one or more active compounds dissolved or suspended in a suitable liquid. Suitable liquids include, by way of example only, one or more fatty oils, liquid paraffin, or liquid polyethylene glycol. In addition, stabilizers are optionally added.

In other embodiments, the compounds described herein are prepared for parenteral injection, including compositions suitable for bolus injection or continuous infusion. In specific embodiments, injection dosage forms are presented in unit dosage form (e.g., in ampoules) or in multi-dose containers. Preservatives are optionally added to injection dosage forms. In other embodiments, the pharmaceutical compositions are prepared in a form suitable for parenteral administration as sterile suspensions, solutions, or emulsions in oily or aqueous vehicles. Compositions for parenteral injection optionally contain excipients such as suspending, stabilizing, and/or dispersing agents. In specific embodiments, parenteral dosage forms include aqueous solutions of the active compounds in water-soluble form. In further embodiments, suspensions of one or more compounds selected from the compounds of structure (I) are prepared as suitable oily injection suspensions. Suitable lipophilic solvents or carriers for use in the pharmaceutical compositions described herein include, by way of example only, fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. In some specific embodiments, aqueous injection suspensions contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension contains suitable stabilizers or agents that increase the solubility of the compounds, allowing for the preparation of highly concentrated solutions. Alternatively, in other embodiments, the active ingredient is in powder form for constitution with a suitable vehicle, such as sterile, pyrogen-free water, before use.

The pharmaceutical compositions comprise at least one pharmaceutically acceptable carrier, diluent, or excipient and one or more compounds selected from the compounds of structure (I) described herein as an active ingredient. The active ingredient is in the form of a free acid or free base, or in the form of a pharmaceutically acceptable salt. Furthermore, the methods and pharmaceutical compositions described herein include the use of N-oxides, crystalline forms (also known as polymorphs), as well as active metabolites of these compounds possessing a similar type of activity. All tautomers of the compounds described herein are included within the scope of the compounds provided herein. Furthermore, the compounds described herein include unsolvated forms, as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. Solvated forms of the compounds provided herein are also considered to be disclosed herein. In addition, the pharmaceutical compositions optionally include other drugs or pharmaceuticals, carriers, adjuvants such as preservatives, stabilizing, wetting or emulsifying agents, dissolution promoters, salts for regulating osmotic pressure, buffers and/or other therapeutically useful substances.

Methods for preparing compositions containing the compounds described herein include formulating the compounds with one or more inert, pharmaceutically acceptable excipients or carriers to form a solid, semi-solid, or liquid substance. Solid compositions include, but are not limited to, powders, tablets, dispersible granules, capsules, wafers, and suppositories. Liquid compositions include solutions in which the compound is dissolved, emulsions containing the compound, or a solution containing liposomes, micelles, or nanoparticles containing the compound as disclosed herein. Semi-solid compositions include, but are not limited to, gels, suspensions, and creams. The form of the pharmaceutical compositions described herein includes liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to use, or as emulsions. These compositions also optionally contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and the like.

In some embodiments, pharmaceutical compositions comprising one or more compounds selected from compounds of structure (I) are, for example, in liquid form, wherein the agents are present in solution, suspension, or both. Typically, when the composition is administered as a suspension, the first portion of the agent is in solution and the second portion of the agent is in particulate form suspended in a liquid matrix. In some embodiments, the liquid composition comprises a gel dosage form. In other embodiments, the liquid composition is aqueous.

In some embodiments, aqueous suspensions contain one or more polymers as suspending agents. Polymers include water-soluble polymers, such as cellulosic polymers, for example, hydroxypropyl methylcellulose, and water-insoluble polymers, such as cross-linked carboxy-containing polymers. Certain pharmaceutical compositions described herein include a mucoadhesive polymer selected from, for example, carboxymethylcellulose, carbomer (an acrylic acid polymer), poly(methyl methacrylate), polyacrylamide, polycarbophil, acrylic acid/butyl acrylate copolymer, sodium alginate, and dextran.

Pharmaceutical compositions also optionally include solubilizing agents to enhance the solubility of one or more compounds selected from compounds of structure (I). The term "solubilizing agent" generally includes agents that result in the formation of a micellar solution or a true solution of the agent. Certain suitable nonionic surfactants, such as polysorbate 80, can be used as solubilizing agents, as can ophthalmologically acceptable glycols, polyglycols such as polyethyleneglycol 400, and glycol ethers.

In addition, the pharmaceutical compositions optionally include one or more pH adjusting agents or buffering agents, including acids such as acetic, boric, citric, lactic, phosphoric, and hydrochloric acids; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and tris-hydroxymethylaminomethane; and buffers such as citrate/dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in an amount necessary to maintain the pH of the composition within an acceptable range.

The compositions also optionally include one or more salts in an amount necessary to adjust the osmolality of the composition to an acceptable range. Such salts include salts containing sodium, potassium, or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate, or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

Other pharmaceutical compositions optionally include one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances such as merphen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide, and cetylpyridinium chloride.

The compositions may include one or more surfactants to enhance physical stability or for other purposes. Suitable nonionic surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, such as polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkyl ethers and alkyl phenyl ethers, such as octoxynol 10 and octoxynol 40.

The compositions may include one or more antioxidants to enhance chemical stability where necessary. Suitable antioxidants include, by way of example only, ascorbic acid and sodium metabisulfite.

In some embodiments, aqueous suspension compositions are packaged in single-dose, non-reclosable containers. Alternatively, multi-dose, reclosable containers are used, in which case a preservative is typically included in the composition.

Alternative embodiments utilize other delivery systems for hydrophobic pharmaceutical compounds. Liposomes and emulsions are examples of delivery vehicles or carriers utilized herein. Some embodiments also utilize organic solvents, such as N-methylpyrrolidone. In other embodiments, the compounds described herein are delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various sustained-release materials can be utilized here. In some embodiments, sustained-release capsules release the compounds over periods ranging from several weeks to over 100 days. Depending on the chemical nature and biological stability of the therapeutic agent, additional protein stabilization strategies are utilized.

In some embodiments, the formulations described herein include one or more antioxidants, metal chelating agents, thiol-containing compounds, and/or other general-purpose stabilizers. Examples of such stabilizers include, but are not limited to: (a) from about 0.5% to about 2% w/v glycerol, (b) from about 0.1% to about 1% w/v methionine, (c) from about 0.1% to about 2% w/v monothioglycerol, (d) from about 1 mM to about 10 mM EDTA, (e) from about 0.01% to about 2% w/v ascorbic acid, (f) 0.003% to about 0.02% w/v polysorbate 80, (g) 0.001% to about 0.05% w/v polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrins, (l) pentosan polysulfate and other heparinoids, (m) divalent cations such as magnesium and zinc; or (n) combinations thereof.

In some embodiments, the concentration of one or more compounds selected from the compounds of structure (I) provided in the pharmaceutical compositions of the present invention is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25% 19%, 18.75%, 18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%, 16.75%, 16.50%, 16.25% 16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%, 13.75%, 13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25% 11%, 10.75%, 10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%, 8.25% 8%, 7.75%, 7.50%, 7.25% 7%, 6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%, 5.25% 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v.

In some embodiments, the concentration of one or more compounds selected from the compounds of structure (I) provided in the pharmaceutical compositions of the present invention is in the range of from about 0.0001% to about 50%, from about 0.001% to about 40%, from about 0.01% to about 30%, from about 0.02% to about 29%, from about 0.03% to about 28%, from about 0.04% to about 27%, from about 0.05% to about 26%, from about 0.06% to about 25%, from about 0.07% to about 24%, from about 0.08% to about 23%, from about 0.09% to about 22%, from about 0.1% to about 21%, from about 0.2% to about 20%, from about 0.3% to about 19%, from about 0.4% to about 18%, from about 0.5% to about 17%, from about 0.6% to about 16%, from about 0.7% to about 15%, from about 0.8% to about 14%, from about 0.9% to about 12%, from about 1% to about 10% w/w, w/v, or v/v.

In some embodiments, the amount of one or more compounds selected from the compounds of structure (I) present in the pharmaceutical compositions of the present invention is equal to or less than 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g, 0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007 g, 0.006 g, or 0.0001 g

In some embodiments, the amount of one or more compounds selected from the compounds of structure (I) present in the pharmaceutical compositions of the present invention is in the range of 0.0001-10 g, 0.0005-9 g, 0.001-8 g, 0.005-7 g, 0.01-6 g, 0.05-5 g, 0.1-4 g, 0.5-4 g, or 1-3 g.

Packaging materials for use in packaging the pharmaceutical compositions described herein include those described, for example, in U.S. Patent Nos. 5,323,907, 5,052,558, and 5,033,252. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, vials, tubes, inhalers, pumps, bags, bottles, containers, syringes, bottles, and any packaging material suitable for the selected dosage form and the intended route of administration and treatment. For example, the container(s) include one or more compounds described herein, optionally in a composition or in combination with another agent as described herein. The container(s) optionally have a sterile access port (e.g., the container is a bag for an intravenous solution or a vial with a stopper pierced by a hypodermic needle). Such kits optionally contain a compound with an identifying description or label or instructions relating to its use in the methods described herein.

For example, a kit typically includes one or more additional containers, each containing one or more different materials (such as reagents, optionally in concentrated form, and/or devices) desirable from a commercial and user perspective for using the compound described herein. Non-limiting examples of such materials include, but are not limited to, buffers, diluents, filters, needles, syringes; labels on the carrier, package, container, vial, and/or tube indicating the contents and/or instructions for use; and package inserts containing instructions for use. A set of instructions is also typically included. A label is not necessarily located on or associated with the container. For example, a label is located on the container where the letters, numbers, or other symbols that form the label are affixed, molded, or engraved on the container itself; a label is associated with the container if it is located within a vessel or carrier that also contains the container, for example, in the form of a package insert. Additionally, the label is used to indicate that the contents are to be used for a specific therapeutic application. Furthermore, the label includes instructions for using the contents, such as in the methods described herein. In some embodiments, the pharmaceutical compositions are provided in a package or dispenser device that contains one or more unit dosage forms containing the compound provided herein. For example, the package may comprise a metal or plastic foil, such as a blister pack. Alternatively, the package or dispenser may be accompanied by instructions for administration. Alternatively, the package or dispenser may be accompanied by a notice associated with the container, in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals, where the notice reflects the agency's approval of the human drug form or veterinary use. Such a notice, for example, is a label approved by the U.S. Food and Drug Administration for prescription drugs or an approved product insert. In some embodiments, compositions comprising a compound provided herein in a compatible pharmaceutical carrier are prepared, placed in an appropriate container, and labeled for treating the indicated condition.

Methods

Embodiments of the present invention are useful as modulators of the NLRP3 inflammasome by inhibiting NEK7 in host species. Therefore, compounds of structure (I) are also useful in the treatment of conditions mediated by effector signaling molecules such as IL-1β and IL-18.

The host or patient may belong to any mammalian species, such as primates, particularly humans; rodents, including mice, rats, and hamsters; rabbits; horses, cows, dogs, cats, etc. Animal models are of interest for experimental research, providing a model for the treatment of human diseases.

In one embodiment, the present invention is used as an inhibitor of the NLRP3 inflammasome activation mechanism. Therefore, compounds of structure (I) are also useful in the treatment of conditions resulting from this activation in host species.

In another embodiment, compounds of structure (I) are useful as inhibitors of the NLRP3 (protein)-NEK7 (protein) interaction. Therefore, the compounds are also useful in the treatment of conditions resulting from the NLRP3-NEK7 association in the host species.

In some embodiments, the compounds of structure (I) are useful for treating human conditions mediated by effectors selected from the group consisting of IL-1β, IL-18, and caspase-1.

Embodiments of the invention also relate to the use of compounds of structure (I) and/or their physiologically acceptable salts for the prophylactic or therapeutic treatment and/or monitoring of diseases that are caused, mediated and/or modulated by the activity of the NLRP3 inflammasome. Furthermore, embodiments of the invention relate to the use of compounds of structure (I) and/or their physiologically acceptable salts for the preparation of a medicament for the prophylactic or therapeutic treatment and/or monitoring of diseases that are caused, mediated and/or modulated by the activity of the NLRP3 inflammasome. In some embodiments, the invention relates to the use of a compound of structure I or its physiologically acceptable salts for the preparation of a medicament for the prophylactic or therapeutic treatment of an NLRP3-mediated disorder.

In another embodiment, the present invention relates to a method of treating inflammatory diseases or conditions mediated by the NLRP3 inflammasome by administering to a patient in need thereof a therapeutically effective amount of a compound of structure (I).

In some embodiments, diseases that can be treated with a compound of structure (I) include type II diabetes, atherosclerosis, Alzheimer's disease, aging, fatty liver, metabolic syndrome, asthma, psoriasis, obesity, acute and chronic tissue damage caused by infection, gout, arthritis, enteritis, hepatitis, peritonitis, silicosis, UV-induced skin sunburn, contact hypersensitivity, sepsis, cancer, neurodegenerative disease, multiple sclerosis and Muckle-Wells syndrome.

In some other embodiments, compounds of structure (I) are used in methods of treating disorders or diseases selected from autoimmune diseases, inflammatory diseases, cardiovascular diseases, neurodegenerative diseases, bacterial and viral infections, allergies, asthma, pancreatitis, multiple organ failure, kidney disease, platelet aggregation, cancer, transplantation, sperm motility, red blood cell deficiency, transplant rejection, lung disease, respiratory diseases, ischemic conditions, and cancer.

In some embodiments, the NEK7-associated disorders treatable with the compound of structure (I) are selected from rheumatoid arthritis, psoriatic arthritis, osteoarthritis, systemic lupus erythematosus, lupus nephritis, ankylosing spondylitis, osteoporosis, systemic sclerosis, multiple sclerosis, psoriasis, type I diabetes, type II diabetes, inflammatory bowel disease (Crohn's disease and ulcerative colitis), hyperimmunoglobulinemia D and periodic fever syndrome, cryopyrin-related periodic syndromes, Schnitzler syndrome, systemic juvenile idiopathic arthritis, adult-onset Still's disease, gout, pseudogout, SAPHO syndrome, Castleman disease, sepsis, stroke, atherosclerosis, celiac disease, DIRA (IL-1 receptor antagonist deficiency), Alzheimer's, Parkinson's disease and cancer.

Also included herein are methods of treatment in which at least one compound of structure (I) is administered in combination with an anti-inflammatory or therapeutic agent. Anti-inflammatory agents include, but are not limited to, NSAIDs, non-specific and specific COX-2 enzyme inhibitors, gold compounds, corticosteroids, methotrexate, tumor necrosis factor (TNF) antagonists, immunosuppressants, and methotrexate. Examples of NSAIDs include, but are not limited to, ibuprofen, flurbiprofen, naproxen and naproxen sodium, diclofenac, combinations of diclofenac sodium and misoprostol, sulindac, oxaprozin, diflunisal, piroxicam, indomethacin, etodolac, fenoprofen calcium, ketoprofen, nabumetone sodium, sulfasalazine, tolmetin sodium, and hydroxychloroquine.

Examples of NSAIDs also include specific COX-2 inhibitors such as celecoxib, valdecoxib, lumiracoxib and/or etoricoxib.

In some embodiments, the anti-inflammatory agent is a salicylate. Salicylates include, but are not limited to, acetylsalicylic acid or aspirin, sodium salicylate, and choline and magnesium salicylates.

An anti-inflammatory drug may also be a corticosteroid. For example, corticosteroids can include cortisone, dexamethasone, methylprednisolone, prednisolone, prednisolone sodium phosphate, or prednisolone.

In other embodiments, the anti-inflammatory agent is a gold compound such as sodium aurothiomalate or auranofin.

The present description also includes embodiments in which the anti-inflammatory agent is a metabolic inhibitor, such as a dihydrofolate reductase inhibitor, such as methotrexate, or a dihydroorotate dehydrogenase inhibitor, such as leflunomide.

Therapeutic agents may also include pain and inflammation agents such as histamine and histamine antagonists, bradykinin and bradykinin antagonists, 5-hydroxytryptamine (serotonin), lipid substances formed during the biotransformation of products of selective hydrolysis of membrane phospholipids, eicosanoids, prostaglandins, thromboxanes, leukotrienes, aspirin, nonsteroidal anti-inflammatory drugs, analgesic-antipyretics, agents that inhibit the synthesis of prostaglandins and thromboxanes, selective inhibitors of inducible cyclooxygenase, selective inhibitors of inducible cyclooxygenase-2, autacoids, paracrine hormones, somatostatin, gastrin, cytokines that mediate interactions involved in humoral and cellular immune responses, autacoids of lipid origin, Eicosanoids, β-adrenergic agonists, ipratropium, glucocorticoids, methylxanthines, sodium channel blockers, opioid receptor agonists, calcium channel blockers, membrane stabilizers, and leukotriene inhibitors.

Other embodiments of the present invention relate to combinations in which at least one anti-inflammatory compound is an anti-monoclonal antibody (such as eculizumab or pexelizumab), a TNF antagonist such as entanercept, or infliximab, which is a monoclonal antibody to TNF-alpha.

Therapeutic agents used in combination with compounds of structure (I) may also include small molecules that inhibit NLRP3 inflammasome activation, such as MCC950, sulforaphane, isoliquiritigenin, β-hydroxybutyrate, flufenamic acid, mefenamic acid, 3,4-methylenedioxy-β-nitrostyrene (MNS), and parthenolide.

Other embodiments of the present invention relate to combinations in which at least one active agent is an immunosuppressive compound, such as an immunosuppressive compound selected from methotrexate, leflunomide, cyclosporine, tacrolimus, azathioprine, and mycophenolate mofetil.

The disclosed compounds of structure (I) can be administered in combination with other known therapeutic agents, including anticancer agents. As used herein, the term "anticancer agent" refers to any agent administered to a patient with cancer for the purpose of treating the cancer.

In some embodiments, the anticancer agents fall into the following categories:

Alkylating agents: such as altretamine, bendamustine, busulfan, carmustine, chlorambucil, chlormethine, cyclophosphamide, dacarbazine, ifosfamide, improsulfan, tosylate, lomustine, melphalan, mitobronitol, mitolactol, nimustine, ranimustine, temozolomide, thiotepa, treosulfan, mechlorethamine, carboquone; apaziquone, fotemustine, glufosfamide, palifosfamide, pipobroman, trofosfamide, uramustine, TH-3024, VAL-0834;

Platinum compounds: such as carboplatin, cisplatin, eptaplatin, miriplatin hydrate, oxaliplatin, lobaplatin, nedaplatin, picoplatin, satraplatin; lobaplatin, nedaplatin, picoplatin, satraplatin;

DNA-modifying agents: such as amrubicin, bizantrene, decitabine, mitoxantrone, procarbazine, tribectedine, clofarabine; amsacrine, brostallicin, pixantrone, laromustine 1,3;

Topoisomerase inhibitors: such as etoposide, irinotecan, razoxan, sobuzoxan, teniposide, topotecan; amonafide, belotecan, elliptinium acetate, voreloxin;

Microtubule modifiers: such as cabazitaxel, docetaxel, eribulin, ixabepilone, paclitaxel, vinblastine, vincristine, vinorelbine, vindesine, vinflunine; fosbretabulin, tesetaxel;

Antimetabolites: such as asparaginase3, azacitidine, calcium levofolinate, capecitabine, cladribine, cytarabine, enocitabine, floxuridine, fludarabine, fluorouracil, gemcitabine, mercaptopurine, methotrexate, nelarabine, pemetrexed, pralatrexate, azathioprine, thioguanine, carmofur; doxifluridine, elacytarabine, raltitrexed, sapacitabine, tegafur2,3, trimetrexate;

Anticancer antibiotics: such as bleomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, levamisole, miltefosine, mitomycin C, romidepsin, streptozocin, valrubicin, zinostatin, zorubicin, daunurobicin, plicamycin; aclarubicin, peptomycin, pirarubicin;

Hormones/Antagonists: such as abarelix, abiraterone, bicalutamide, buserelin, calusterone, chlorotrianisene, degarelix, dexamethasone, estradiol, fluocortolone, fluoxymesterone, flutamide, fulvestrant, goserelin, histrelin, leuprorelin, megestrol, mitotane, nafarelin, nandrolone, nilutamide, octreotide, prednisolone, raloxifene, tamoxifen, thyrotropin alfa, toremifene, trilostane, triptorelin, diethylstilbestrol; acolbifene, danazol, deslorelin, epitiostanol, orteronel, enzalutamide 1,3;

Aromatase inhibitors: such as aminoglutethimide, anastrozole, exemestane, fadrozole, letrozole, testolactone; formestane;

Small molecule kinase inhibitors: such as crizotinib, dasatinib, erlotinib, imatinib, lapatinib, nilotinib, pazopanib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, vemurafenib, bosutinib, gefitinib, axitinib; afatinib, alisertib, dabrafenib, dacomitinib, dinaciclib, dovitinib, enzastaurin, nintedanib, lenvatinib, linifanib, linsitinib, masitinib, midostaurin, motesanib, neratinib, orantinib, perifosine, ponatinib, radotinib, rigosertib, tipifamib, tivantinib, tivozanib, trametinib, pimasertib, brivanib alaninate, cediranib.

In some embodiments, drugs administered in combination with the compounds described herein include any suitable drugs that are conveniently delivered by inhalation, such as analgesics, such as codeine, dihydromorphine, ergotamine, fentanyl, or morphine; anginal drugs, such as diltiazem; antiallergic drugs, such as cromoglycate, ketotifen, or nedocromil; anti-infective drugs, such as cephalosporins, penicillins, streptomycin, sulfonamides, tetracyclines, or pentamidine; antihistamines, such as methapyrilene; anti-inflammatory agents, such as beclomethasone, flunisolide, budesonide, tipredane, triamcinolone acetonide, or fluticasone; antitussives, such as noscapine; Bronchodilators such as ephedrine, adrenaline, fenoterol, formoterol, isoprenaline, metaproterenol, phenylephrine, phenylpropanolamine, pirbuterol, reproterol, rimiterol, salbutamol, salmeterol, terbutaline, isoetharine, tulobuterol, orciprenaline or (-)-4-amino-3,5-dichloro-α-[[[6-[2-(2-pyridinyl)ethoxy]hexyl]amino]methyl]benzenemethanol; diuretics such as amiloride; anticholinergics such as ipratropium, atropine or oxitropium; hormones such as cortisone, hydrocortisone or prednisolone; xanthines such as aminophylline, choline theophylline, lysine theophylline or theophylline; and therapeutic proteins and peptides, such as insulin or glucagon. Those skilled in the art will appreciate that, if necessary, the drugs are used in the form of salts (e.g., alkali metal or amine salts or acid addition salts), or as esters (e.g., lower alkyl esters), or as solvates (e.g., hydrates) to optimize the activity and/or stability of the drug.

The agents disclosed herein, or other suitable agents, are administered depending on the condition being treated. Thus, in some embodiments, one or more compounds of the present invention will be co-administered with other agents as described above. When used in combination therapy, the compounds described herein are administered with a second agent simultaneously or separately. Such combined administration may include the simultaneous administration of two agents in a single dosage form, simultaneous administration in separate dosage forms, and separate administration. That is, a compound described herein and any of the agents described above may be formulated together in a single dosage form and administered simultaneously. Alternatively, a compound of the present invention and any of the agents described above may be administered simultaneously, with both agents in separate dosage forms. In another embodiment, a compound of the present invention may be administered immediately after the administration of any of the agents described above, or vice versa. In some embodiments of the separate administration protocol, a compound of the invention and any of the agents described above are administered within minutes, hours, or days of each other.

In some embodiments, the compounds of structure (I) are administered as monotherapy.

To identify a signal transduction or mechanistic pathway and to detect interactions between different signal transduction pathways, various scientists have developed suitable models or model systems, such as cell culture models and transgenic animal models. Interacting compounds can be used to modulate the signal to determine specific stages of the signal transduction cascade. Compounds of embodiments of the invention can also be used as reagents for testing NEK7-dependent signal transduction pathways in animal and/or cell culture models or in the clinical diseases discussed herein.

The methods of embodiments of the invention can be carried out either in vitro or in vivo . The susceptibility of a particular cell to treatment with compounds of structure (I) can be determined, in particular, using in vitro tests, whether during research or clinical use. Typically, a cell culture is combined with the compound at varying concentrations for a period of time sufficient for the active agents to inhibit NEK7 activity, typically from one hour to one week. In vitro treatment can be carried out using cultured cells from a biopsy sample or cell line.

In some embodiments, the _IC50 of compounds of structure (I) for inhibiting NEK7 was determined by the concentration of the compound required to inhibit 50% of the NEK kinase activity. Compounds of structure (I) exhibit _IC50 activity values of less than about 5 mM, preferably less than about 1 mM, and even more preferably less than about 0.100 mM, as described in more detail in the examples.

The examples and preparations presented below further illustrate and exemplify the compounds of the present invention and methods for preparing and testing such compounds. It should be understood that the scope of the present invention is in no way limited by the scope of the following examples and preparations. In the following examples, as well as throughout the description and claims, molecules with one stereocenter, unless otherwise indicated, exist as a racemic mixture. These molecules with two or more stereocenters, unless otherwise indicated, exist as a racemic mixture of diastereomers. Individual enantiomers/diastereomers can be prepared by methods known to those skilled in the art.

EXAMPLES

The following examples are provided for illustrative purposes.

General methods

All proton NMR experiments were recorded on a Bruker NEO spectrometer equipped with a BBFO probe at 400 MHz. Deuterated solvents contained less than 0.05% v/v tetramethylsilane, which was used as a reference signal (set at 0.00 ppm). When deuterated solvents did not contain tetramethylsilane, peaks of residual non-deuterated solvents were used as a reference signal according to published recommendations ( J. Org. Chem. 1997 , 62(21), 7512–7515). Chemical shifts are expressed in parts per million (ppm, δ units). Spin–spin coupling constants are reported in hertz (Hz). Splitting patterns describe apparent multiplicity and are designated as s (singlet), d (doublet), t (triplet), qv (quartet), m (multiplet), qvt (quintuplet), or bs (broadened singlet).

LC/MS analyses were performed on an Agilent Technologies UHPLC 1290 Infinity II with a G6125 MS detector.

Microwave reactions were carried out using a Monowave 300 from Anton Paar GmbH using standard protocols.

NEK7 enzymatic assay

Casein substrate (from bovine milk, hydrolyzed and partially dephosphorylated mixture of α, β, and κ caseins, obtained from Sigma Aldrich, catalog no. C4765, diluted in distilled water to a final concentration of 1 mg/mL) and full-length recombinant human NEK7 (expressed by baculovirus in Sf9 insect cells using an N-terminal GST tag, obtained from SignalChem, catalog no. N09-10G, 0.1 μg/μL) were mixed in assay buffer (20 mM Hepes pH 7.5, 10 mM MgCl ₂ , 1 mM EGTA, 0.02% Brij35, 0.02 mg/mL BSA, 0.1 mM Na ₃ VO ₄ , 2 mM DTT, 1% DMSO). Test compounds (serial 3-fold dilutions in DMSO from 10 μM to 0.5 nM) or vehicle (1% DMSO) were added to the kinase reaction mixture using acoustic technology (Echo550; nanoliter range). After incubation at room temperature for 20 min, the kinase reaction was initiated by the addition of [ ^33P ]-ATP (specific activity 10 μCi/μL) and the mixture was incubated at room temperature for 2 h. The reaction was then stopped by spotting the reaction mixture onto P81 phosphocellulose paper strips. After washing, the radioactivity of the P81 paper was measured and kinase activity data were expressed as the percentage of remaining kinase activity in the test samples compared to vehicle reactions. _IC50 values and curve fits were generated using Prism (GraphPad Software).

IL-1β release assay

Approximately 1.5 million THP-1 cells were seeded in each well of a 6-well TC plate and incubated with 40 nM PMA in RPMI (10% FBS, 1% Penstrep) for 24 h. The medium was then removed and the cells were maintained in RPMI (10% FBS, 1% Penstrep) for 24 h, after which the medium was removed and the cells were pretreated for 2 h with various concentrations of the compounds of interest (typically serial 3-fold dilutions in RPMI + 5% FBS, concentrations from 1 μM to 0.5 nM) in RPMI (5% FBS). The medium was again removed and the cells were incubated with 250 ng/ml LPS and the compounds of interest (concentrations indicated above) in RPMI (5% FBS) for 2 h. The medium was removed one last time, and the cells were incubated with 20 μM nigericin and the compounds of interest (concentrations indicated above) in Opti-MEM for 30 minutes. The cell medium was then collected, and the amount of cleaved IL-1β was determined using a JESS instrument (Protein Simple) and standard protocols. Cleaved IL-1β antibody was obtained from Cell Signaling (catalog no. 83186S) and used at a 1:20 dilution in Antibody Diluent 2. For chemiluminescent detection, Protein Simple 1x anti-rabbit HRP secondary antibody was used along with Protein Simple luminol and peroxide. The incubation time of the primary antibodies was increased from 30 minutes to 60 minutes.

Abbreviations:

°C (degrees Celsius); ¹ H NMR (proton nuclear magnetic resonance); ACN (acetonitrile); Boc ( tert- butyloxycarbonyl); DCM (dichloromethane); DIPEA ( N,N -diisopropylethylamine); DMAP (4-dimethylaminopyridine); DMF ( N,N -dimethylformamide); DMSO- d ₆ (deuterated dimethyl sulfoxide); eq. (equivalent); EtOAc (ethyl acetate); g (gram); (g) gas; h (hour); HPLC (High performance liquid chromatography); LCMS (liquid chromatography with mass spectrometry); MeOH (methanol); mg (milligram); min (minute); ml (milliliter); mmol (millimole); n. (normal); n -BuOH (1-butanol); Pd(PPh ₃ ) ₄ (palladium tetrakis(triphenylphosphine)); PdCl ₂ (dppf) ([1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloride); sec- (secondary); TBAF (tetra-n-butylammonium fluoride); tert- (tertiary); TFA (trifluoroacetic acid); THF (tetrahydrofuran); TLC (Thin Layer Chromatography); UPLC (Ultra Performance Liquid Chromatography).

Preparation of synthetic intermediates

Intermediate connection A1

1-cyclopropyl-3-iodo-1 H -pyrazolo[3,4- d ]pyrimidin-4-amine

Copper(II) acetate (0.348 g, 1.916 mmol), 2,2'-bipyridine (0.299 g, 1.916 mmol), and sodium bicarbonate (0.322 g, 3.830 mmol) were added to a stirred solution of 3-iodo- 1H -pyrazolo[3,4- d ]pyrimidin-4-amine (0.500 g, 1.916 mmol) and cyclopropylboronic acid (0.329 g, 3.830 mmol) in dichloroethane (10 mL). The resulting mixture was stirred at 70 °C under oxygen atmosphere for 12 h. After the reaction was complete (as determined by TLC), the reaction mixture was filtered through a pad of diatomaceous earth, which was then washed with DCM (20 mL x 2). The combined filtrates were washed with water (20 mL) and brine (25 mL), dried over _Na2SO4 , _filtered , and concentrated under reduced pressure to give a crude material that was purified by flash chromatography (silica gel 230-400 mesh, eluting with 20 mL in petroleum ether) to give the title compound as an off-white solid (0.24 g, 36% yield). ¹ H NMR (400 MHz, DMSO- d6 ) δ=8.21 (s, 1H), 3.74-3.79 (m, 1H), 1.11-1.15 (m, 2H), 1.04-1.09 (m, 2H). _LCMS : 301.8 [M+H].

Intermediate compound A2

3-iodo-1-isopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-4-amine

_Cs2CO3 ( _12.38 g, 38.31 mmol) and 2-iodopropane (3.60 g, 21.16 mmol) were added to a stirred solution of 3-iodo- 1H -pyrazolo[3,4 -d ]pyrimidin-4-amine (5.00 g, 19.15 mmol) in DMF (25 mL) in a sealed 25 mL tube. The reaction mixture was stirred at 90 °C for 16 h and, after the reaction was complete (as determined by TLC), poured onto crushed ice (50 g) and stirred for 15 min. The resulting solid was filtered, washed with water (2 x 5 mL), and dried to give the title compound as an off-white solid (3.25 g, 56% yield). ¹ H NMR (400 MHz, DMSO- d ₆ ) δ=8.18 (s, 1H), 4.93-4.99 (m, 1H), 1.42 (d, J=6.8 Hz, 6H). LCMS: 303.8 [M+H].

Intermediates A3 - A12

The following intermediates were prepared using a procedure similar to that described for intermediate A2, with the 2-iodopropane replaced by the appropriate reagent (alkyl halide or tosylate), as shown below:

Intermediate connection Structure Reagent ¹ H NMR (400 MHz, DMSO- d ₆ ) LCMS A3 δ=8.21 (s, 1H), 5.89-5.93 (m, 1H), 4.93-5.00 (m, 4H). 318.0 [M+H] A4 δ=8.21 (s, 1H), 5.93-6.03 (m, 1H), 5.16-5.20 (m, 1H), 5.03-5.09 (m, 1H), 4.89-4.91 (m, 2H). 301.8 [M+H] A5 δ=8.19 (s, 1H), 5.20-5.26 (m, 1H), 2.28-2.73 (m, 4H), 1.84-1.86 (m, 2H). 315.8 [M+H] A6 δ=8.21 (s, 1H), 5.38-5.44 (m, 1H), 4.00-4.07 (m, 2H), 3.83-3.88 (m, 2H), 2.32-2.43 (m, 2H). 332.0 [M+H] A7 δ=8.20 (s, 1H), 4.82-4.89 (m, 1H), 3.96-4.00 (m, 2H), 3.48-3.54 (m, 2H), 2.05-2.16 (m, 2H), 1.82-1.86 (m, 2H). 345.9 [M+H] A8 δ=8.21 (s, 1H), 4.65-4.71 (m, 1H), 3.85-3.92 (m, 2H), 3.40-3.44 (m, 2H), 2.08-2.20 (m, 2H), 1.74-1.79 (m, 2H). 346.0 [M+H] A9 δ=8.27 (s, 1H), 5.19-5.21 (m, 1H), 3.35 (br.s, 4H). 352.0 [M+H] A10 Not registered (unpurified material as such was taken to the next stage). 402.0 [M+H] A11 Not registered (unpurified material as such was taken to the next stage). 444.8 [M+H] A12 δ=8.24 (s, 1H), 5.59-5.67 (m, 1H), 3.70-3.75 (m, 2H), 3.44-3.52 (m, 2H), 2.56-2.64 (m, 2H). 379.7 [M+H]

Intermediate connection A13

1-(4-amino-3-iodo-1 H -pyrazolo[3,4- d ]pyrimidin-1-yl)-2-methylpropan-2-ol

NaH ₂ PO ₄ (0.044 g, 0.372 mmol) was added to a mixture of 3-iodo-1 H -pyrazolo[3,4-d]pyrimidin-4-amine (0.100 g, 0.380 mmol), 2,2-dimethyloxirane (0.055 g, 0.760 mmol) and K ₂ CO ₃ (0.050 g, 0.372 mmol) in acetonitrile (3 mL) and water (1 mL) and the resulting solution was microwaved at 150 °C for 1 h. After the reaction was complete (as determined by TLC), the solvents were removed under reduced pressure to give a crude material which was purified by flash chromatography (silica gel 230-400 mesh, eluting with 25% EtOAc in petroleum ether) to give the title compound the title compound as a pale brown solid (0.064 g, 51% yield). ¹ H NMR (400 MHz, DMSO- d ₆ ) δ=8.20 (s, 1H), 4.19 (s, 2H), 1.09 (s, 6H). LCMS: 334.0 [M+H].

Intermediate connection A14

3-iodo-1-(pyridin-4-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-4-amine

The title compound was prepared using a procedure similar to that described for Intermediate A1, starting from 3-iodo- 1H -pyrazolo[3,4- d ]pyrimidin-4-amine (1.00 g, 3.83 mmol) and pyridin-4-ylboronic acid (0.94 g, 7.66 mmol) and obtained as a pale brown solid (0.27 g, 21% yield). LCMS: 338.8 [M+H].

Intermediate connection A15

3-iodo-1-methyl-1 H -pyrazolo[3,4- d ]pyrimidin-4-amine

_Cs2CO3 (0.780 g, 2.394 mmol) and methyl iodide (0.138 _mL , 2.203 mmol) were added to a solution of 3-iodo- 1H -pyrazolo[3,4 -d ]pyrimidin-4-amine (0.500 g, 1.916 mmol) in DMF (3 mL) at 0 °C in a sealed 25 mL tube. The reaction mixture was stirred at 25 °C for 1 h and, after completion of the reaction (as determined by TLC), poured onto crushed ice (50 g) and stirred for 30 min. The resulting solid was filtered, washed with water (2 x 5 mL), and dried to give the title compound as a yellow solid (0.380 g, 67% yield). ¹ H NMR (400 MHz, DMSO- d ₆ ) δ=8.22 (s, 1H), 6.79 (s, 2H), 3.89 (s, 3H). LCMS: 276.0 [M+H].

Intermediate connection A16

1-cyclopropyl-3-iodo-1 H -pyrazolo[4,3- c ]pyridin-4-amine

Step 1: Synthesis of 4-chloro-3-iodo-1H-pyrazolo[4,3-c]pyridine

KOH (1.320 g, 23.0 mmol) and iodine (1.620 g, 12.8 mmol) were added to a solution of 4-chloro-1 H -pyrazolo[4,3- c ]pyridine (1.000 g, 6.4 mmol) in dioxane (10 mL) and the resulting mixture was stirred at 75 °C for 4 h. After the reaction was complete (as determined by TLC), the reaction mixture was filtered through a pad of diatomaceous earth and the filtrate was concentrated under reduced pressure to give a crude material, which was purified by reverse phase column chromatography to give the title compound as a white solid (0.633 g, 63% yield). ¹ H NMR (400 MHz, DMSO- d ₆ ) δ=14.12 (br s, 1H), 8.14 (d, J = 6.0 Hz, 1H), 7.66 (d, J = 5.6 Hz, 1H). LCMS: 279.9 [M+H].

Step 2: Synthesis of 4-chloro-1-cyclopropyl-3-iodo-1H-pyrazolo[4,3-c]pyridine

The title compound was prepared using a procedure similar to that described for Intermediate A1, starting from 4-chloro-3-iodo-1H-pyrazolo[4,3-c]pyridine (0.630 g, 2.20 mmol) and cyclopropylboronic acid (0.329 g, 3.83 mmol) and obtained as a white solid (0.430 g, 60% yield). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ = 8.21 (d, J = 6.0 Hz, 1H), 7.81 (d, J = 5.6 Hz, 1H), 3.84-3.89 (m, 1H), 1.14-1.17 (m, 4H). LCMS: 319.7 [M+H].

Step 3: Synthesis of 1-cyclopropyl-3-iodo-1H-pyrazolo[4,3-c]pyridin-4-amine

A mixture of 4-chloro-1-cyclopropyl-3-iodo-1H-pyrazolo[4,3-c]pyridine (0.20 g) and aqueous ammonium hydroxide (25% in water, 8 mL) was microwaved at 150 °C for 2 h. After the reaction was complete (as determined by TLC), the reaction mixture was concentrated under reduced pressure to give the title compound as an off-white solid (0.19 g, quantitative yield). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ = 8.11 (s, 1H), 7.38 (s, 1H), 6.68 (br s, 2H), 3.48-3.54 (m, 1H), 0.97-0.99 (m, 4H). LCMS: 301.0 [M+H].

Intermediate connection A17

1-(3-(benzyloxy)cyclobutyl)-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine

3-(Benzyloxy)cyclobutyl methanesulfonate (prepared as described in PCT Publication No. WO 2019/092170, 0.491 g, 1.916 mmol) and Cs ₂ CO ₃ (0.624 g, 1.916 mmol) were added to a solution of 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (0.250 g, 0.958 mmol) in DMF (5 mL) and the resulting mixture was stirred at 90 °C for 12 h. After completion of the reaction (as determined by TLC), the reaction mixture was poured into ice-water (50 mL) and extracted with ethyl acetate (2 x 30 mL). The combined organic layers were dried over _Na2SO4 , filtered, and evaporated under reduced pressure to give the title compound as _a pale brown gum (0.400 g, 75% pure LCMS), which was used without further purification. LCMS: 422.0 [M+H]

Intermediate connection A18

3-iodo-1-(1-methylazetidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

The title compound was prepared as described in PCT Publication No. WO 2002/076986.

Intermediate connection A19

1-cyclopropyl-N-(2,4-dimethoxybenzyl)-3-iodo-1H-pyrazolo[4,3-c]pyridin-4-amine

A mixture of 4-chloro-1-cyclopropyl-3-iodo-1H-pyrazolo[4,3-c]pyridine (A16, 0.880 g, 2.75 mmol) and (2,5-dimethoxyphenyl)methanamine (1.245 mL, 8.26 mmol) in n-BuOH (10 mL) was stirred at 110 °C for 12 h. After the reaction was complete (as determined by LCMS), the reaction mixture was concentrated under reduced pressure to give a crude material, which was purified using Isolera (silica gel 230-400 mesh, eluting with 40% EtOAc in petroleum ether) to give the title product as a yellow gum (1.0 g, 80% yield). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ=7.81 (d, J=6.4 Hz, 1H), 7.19 (d, J=8.4 Hz, 1H), 6.87 (d, J=6.0 Hz, 1H), 6.60 (d, J=2.4 Hz, 1H), 6.53-6.56 (m, 1H), 6.45-6.47 (m, 1H), 4.62 (d, J=5.6 Hz, 2H), 3.88 (s, 3H), 3.73 (s, 3H), 3.66-3.70 (m, 1H), 1.01-1.10 (m, 4H). LCMS: 451.0 [M+H].

Intermediate connection A20

2-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethan-1-ol

The title compound was prepared as described in PCT Publication No. WO 2011/119663.

Intermediate connection A21

3-iodo-1-(2-methoxyethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

_K2CO3 (0.397 g, 2.870 mmol) and 1-bromo-2-methoxyethane (0.319 g _, 2.299 mmol) were added to a solution of 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (0.500 g, 1.916 mmol) in DMF (6 mL), and the resulting mixture was stirred at 80°C for 12 h in a sealed tube. After the reaction was complete (as determined by TLC), the reaction mixture was poured into crushed ice (25 g) and extracted with EtOAc (2 x 50 mL). The combined organic extracts _{were dried over Na2SO4 ,} filtered, and concentrated under reduced pressure to give the title product (0.500 g, crude), which was used without further purification. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ=8.20 (s, 1H), 4.43 (t, J=5.6 Hz, 2H), 3.75 (t, J=5.6 Hz, 2H), 3.20 (s, 3H). LCMS: 319.8 [M+H].

Intermediate connection A22

3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

The title compound was prepared as described in USPTO Pub. No. WO 2015/165279.

Intermediate connection A23

3-iodo-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

The title compound was prepared as described in USPTO Pub. No. WO 2018/121228.

Intermediate connection B1

3-(4-amino-3-fluorophenyl)-1-cyclopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine

A mixture of 1-cyclopropyl-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (A1, 0.500 g, 1.66 mmol), 2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (0.433 g, 1.82 mmol) and K₂CO₃(0.688 g, 4.98 mmol) in 1,4-dioxane (25 mL) and water (2.5 mL) was purged with N₂for 10 min. Then Pd(PPh) was added₃)₄ (0.092 g, 0.08 mmol) and the reaction mixture was stirred at 100°C for 16 h. After the reaction was complete (as determined by TLC), the mixture was filtered through a pad of diatomaceous earth, which was then washed with EtOAc (2 x 10 mL). The combined filtrates were concentrated under reduced pressure to give a crude material, which was purified by flash chromatography (silica gel 230-400 mesh, eluting with 2% MeOH in DCM) to give the title compound as a yellow solid (0.46 g, 98% yield).¹H NMR (400 MHz, DMSO-d₆) δ=8.23 (s, 1H), 7.15-7.24 (m, 2H), 6.87-6.91 (m, 1H), 5.47 (br.s, 2H), 3.80-3.84 (m, 1H), 1.18-1.19 (m, 2H), 1.05-1.08 (m, 2H). LCMS: 285.0 [M+H].

Intermediate compound B2

3-(4-aminophenyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine

Step 1: Synthesis of 1-isopropyl-3-(4-nitrophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

The title compound was prepared using a procedure similar to that described for Intermediate B1, starting from 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (A2, 1.887 g, 6.23 mmol) and (4-nitrophenyl)boronic acid (1.56 g, 9.34 mmol) and obtained as a yellow solid (1.242 g, 67% yield). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ = 8.38-8.40 (m, 2H), 8.28 (s, 1H), 7.92-7.95 (m, 2H), 5.07-5.14 (m, 1H), 1.51 (d, J = 6.8 Hz, 6H). LCMS: 299.1 [M+H].

Step 2: Synthesis of 3-(4-aminophenyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine

Iron powder (2.320 g, 41.60 mmol) and ammonium chloride (2.220 g, 41.60 mmol) were added to a stirred solution of 1-isopropyl-3-(4-nitrophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (1.242 g, 4.16 mmol) in ethanol (50 mL) and water (20 mL), and the resulting mixture was heated to 80 °C for 3 h. After the reaction was complete (as determined by TLC), the reaction mixture was filtered through a pad of diatomaceous earth, which was then washed with EtOAc (2 x 25 mL). The combined filtrates were concentrated under reduced pressure, the residue was dissolved in EtOAc (100 mL), washed with brine ( ₂₅ mL), dried over _Na2SO4 , filtered and evaporated under reduced pressure to give the title compound as a pale yellow solid (1.042 g, quantitative yield), which was used further without further purification.

Intermediate connection B3

3-(4-amino-3-fluorophenyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine

Step 1: Synthesis of 3-(3-fluoro-4-nitrophenyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine

The title compound was prepared using a procedure similar to that described for intermediate B1, starting from 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (A2, 0.10 g, 0.32 mmol) and (3-fluoro-4-nitrophenyl)boronic acid (0.71 g, 0.39 mmol) and obtained as a yellow solid (0.07 g, 67% yield). LCMS: 315.1 [M-H].

Step 2: Synthesis of 3-(4-amino-3-fluorophenyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine

The title compound was prepared using a procedure similar to that described for step 2 of intermediate B2, starting from 3-(3-fluoro-4-nitrophenyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (0.07 g, 0.22 mmol) and Fe/NH ₄ Cl, and was obtained as a pale yellow solid (0.09 g, quantitative yield), which was used further without further purification. LCMS: 287.1 [M+H].

Intermediate compound B4

3-(4-aminophenyl)-1-(oxetan-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

Step 1: Synthesis of 3-(4-nitrophenyl)-1-(oxetan-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

The title compound was prepared using a procedure similar to that described for intermediate B1, starting from 3-iodo-1-(oxetan-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (A3, 0.800 g, 2.522 mmol) and (4-nitrophenyl)boronic acid (0.632 g, 3.78 mmol) and obtained as a yellow solid (0.596 g, 76% yield). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ = 8.41-8.43 (m, 2H), 8.30 (s, 1H), 7.99-8.01 (m, 2H), 6.05-6.08 (m, 1H), 4.97-5.12 (m, 4H). LCMS: 311.0 [MH].

Step 2: Synthesis of 3-(4-aminophenyl)-1-(oxetan-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

The title compound was prepared using a procedure similar to that described for step 2 of intermediate B2, starting from 3-(4-nitrophenyl)-1-(oxetan-3-yl)-1H - pyrazolo[3,4 -d ]pyrimidin-4-amine (0.596 g, 1.91 mmol) and Fe/NH ₄ Cl, and was obtained as a pale yellow solid (0.42 g, quantitative yield), which was used further without further purification. LCMS: 283.0 [M+H].

Intermediate connection B5

3-(4-amino-3-fluorophenyl)-1-(oxetan-3-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-4-amine

The title compound was prepared using a procedure similar to that described for Intermediate B1, starting from 3-iodo-1-(oxetan-3-yl) -1H- pyrazolo[3,4- d ]pyrimidin-4-amine (A3, 0.110 g, 0.346 mmol) and 2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (0.099 g, 0.420 mmol) and obtained as a pale yellow gum (0.077 g, 74% yield). LCMS: 317.1 [M+H].

Intermediate connection B6

1-allyl-3-(4-amino-3-fluorophenyl)-1 H -pyrazolo[3,4- d ]pyrimidin-4-amine

The title compound was prepared using a procedure similar to that described for Intermediate B1, starting from 1-allyl-3-iodo- 1H -pyrazolo[3,4- d ]pyrimidin-4-amine (A4, 0.15 g, 0.49 mmol) and 2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (0.13 g, 0.54 mmol) and obtained as a yellow gum (0.13 g, 92% yield). LCMS: 285.0 [M+H].

Intermediate compound B7

3-(4-aminophenyl)-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-4-amine

Step 1: Synthesis of 1-cyclopropyl-3-(4-nitrophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

The title compound was prepared using a procedure similar to that described for intermediate B1, starting from 1-cyclopropyl-3-iodo- 1H -pyrazolo[3,4- d ]pyrimidin-4-amine (A1, 0.110 g, 0.36 mmol) and (4-nitrophenyl)boronic acid (0.067 g, 0.40 mmol), and was obtained as a pale yellow solid (0.060 g, 56% yield). ¹ H NMR (400 MHz, DMSO- d ₆ ) δ=8.37 (d, J =8.8 Hz, 2H), 8.30 (s, 1H), 7.91 (d, J =8.8 Hz, 2H), 3.91-3.94 (m, 1H), 1.23-1.24 (m, 2H), 1.11-1.14 (m, 2H). LCMS: 297.0 [M+H].

Step 2: Synthesis of 3-(4-aminophenyl)-1-cyclopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine

The title compound was prepared using a procedure similar to that described for step 2 of intermediate B2, starting from 1-cyclopropyl-3-(4-nitrophenyl)-1H - pyrazolo[3,4 -d ]pyrimidin-4-amine (0.060 g, 0.2 mmol) and Fe/NH ₄ Cl, and was obtained as a pale yellow solid (0.047 g, 89% yield), which was used further without further purification. LCMS: 266.9 [M+H].

Intermediate connection B8

3-(4-aminophenyl)-1-cyclobutyl-1H-pyrazolo[3,4- d ]pyrimidin-4-amine

Step 1: Synthesis of 1-cyclobutyl-3-(4-nitrophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

The title compound was prepared using a procedure similar to that described for intermediate B1, starting from 1-cyclobutyl-3-iodo- 1H -pyrazolo[3,4- d ]pyrimidin-4-amine (A5, 0.150 g, 0.47 mmol) and (4-nitrophenyl)boronic acid (0.087 g, 0.52 mmol) and obtained as a pale yellow solid (0.160 g, quantitative yield). ^1H NMR (400 MHz, _DMSO-d6 ) δ=8.34-8.41 (m, 2H), 8.28 (s, 1H), 7.94-7.97 (m, 2H), 5.32-5.43 (m, 1H), 2.65-2.75 (m, 2H), 1.86-1.94 (m, 4H). LCMS: 311.2 [M+H].

Step 2: Synthesis of 3-(4-aminophenyl)-1-cyclobutyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine

The title compound was prepared using a procedure similar to that described for step 2 of intermediate B2, starting from 1-cyclobutyl-3-(4-nitrophenyl)-1H - pyrazolo[3,4 -d ]pyrimidin-4-amine (0.16 g, 0.51 mmol) and Fe/NH ₄ Cl, and was obtained as a pale yellow solid (0.140 g, quantitative yield), which was used further without further purification. LCMS: 281.0 [M+H].

Intermediate connection B9

3-(4-amino-3-fluorophenyl)-1-cyclobutyl-1 H -pyrazolo[3,4-d]pyrimidin-4-amine

The title compound was prepared using a procedure similar to that described for Intermediate B1, starting from 1-cyclobutyl-3-iodo- 1H -pyrazolo[3,4- d ]pyrimidin-4-amine (A5, 0.250 g, 0.793 mmol) and 2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (0.225 g, 0.952 mmol) and obtained as a pale yellow solid (0.100 g, 32% yield). LCMS: 299.1 [M+H].

Intermediate connection B10

3-(4-aminophenyl)-1-(tetrahydrofuran-3-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-4-amine

Step 1: Synthesis of 3-(4-nitrophenyl)-1-(tetrahydrofuran-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

The title compound was prepared using a procedure similar to that described for intermediate B1, starting from 3-iodo-1-(tetrahydrofuran-3-yl)-1H - pyrazolo[3,4 -d] pyrimidin-4-amine (A6, 0.165 g, 0.49 mmol) and (4-nitrophenyl)boronic acid (0.091 g, 0.54 mmol), and was obtained as a pale yellow solid (0.086 g, 53% yield). ¹ H NMR (400 MHz, DMSO- d ₆ ) δ=8.38-8.40 (m, 2H), 8.30 (s, 1H), 7.93-7.95 (m, 2H), 5.52-5.58 (m, 1H), 4.06-4.15 (m, 2H), 3.88-3.99 (m, 2H), 2.50-2.51 (m, 2H). LCMS: 327.2 [M+H].

Step 2: Synthesis of 3-(4-aminophenyl)-1-(tetrahydrofuran-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

The title compound was prepared using a procedure similar to that described for step 2 of intermediate B2, starting from 3-(4-nitrophenyl)-1-(tetrahydrofuran-3-yl) -1H- pyrazolo[3,4 -d ]pyrimidin-4-amine (0.115 g, 0.35 mmol) and Fe/NH ₄ Cl, and was obtained as a pale yellow solid (0.083 g, 80% yield), which was used further without further purification. ^1H NMR (400 MHz, DMSO -d6 ) δ=8.22 (s, _1H ), 7.33 (dd, J =2.0, 6.6 Hz, 2H), 6.71 (dd, J =2.0, 6.4 Hz, 2H), 5.48 (br.s, 2H), 5.44-5.47 (m, 1H), 4.04-4.12 (m, 2H), 3.86-3.94 (m, 2H), 2.34-2.41 (m, 2H). LCMS: 296.9 [M+H].

Intermediate connection B11

3-(4-amino-3-fluorophenyl)-1-(tetrahydrofuran-3-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-4-amine

The title compound was prepared using a procedure similar to that described for intermediate B1, starting from 3-iodo-1-(tetrahydrofuran-3-yl) -1H- pyrazolo[3,4- d ]pyrimidin-4-amine (A6, 0.351 g, 1.00 mmol) and 2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (0.301 g, 1.27 mmol), and obtained as a pale yellow solid (0.200 g, 60% yield). ^1H NMR (400 MHz, _DMSO-d6 ) δ=8.23 (s, 1H), 7.24-7.28 (m, 1H), 7.18-7.21 (m, 1H), 6.88-6.92 (m, 1H), 5.46-5.50 (m, 3H), 4.04-4.12 (m, 2H), 3.87-3.94 (m, 2H), 2.33-2.41 (m, 2H). LCMS: 315.1 [M+H].

Intermediate compound B12

3-(4-aminophenyl)-1-(tetrahydro- 2H -pyran-4-yl) -1H- pyrazolo[3,4 -d ]pyrimidin-4-amine

Step 1: Synthesis of 3-(4-nitrophenyl)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

The title compound was prepared using a procedure similar to that described for intermediate B1, starting from 3-iodo-1-(tetrahydrofuran-3-yl)-1H - pyrazolo[3,4- d ]pyrimidin-4-amine (A7, 0.094 g, 0.273 mmol) and (4-nitrophenyl)boronic acid (0.055 g, 0.328 mmol) and obtained as a pale yellow solid (0.078 g, 84% yield). ¹ H NMR (400 MHz, DMSO- d ₆ ) δ=8.37-8.40 (m, 2H), 8.29 (s, 1H), 7.93-7.96 (m, 2H), 4.96-5.02 (m, 1H), 4.00-4.04 (m, 2H), 3.54-3.59 (m, 2H), 2.16-2.24 (m, 2H), 1.89-1.94 (m, 2H). LCMS: 340.9 [M+H].

Step 2: Synthesis of 3-(4-aminophenyl)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

The title compound was prepared using a procedure similar to that described for step 2 of intermediate B2, starting from 3-(4-nitrophenyl)-1-(tetrahydro- 2H -pyran-4-yl) -1H- pyrazolo[3,4- d ]pyrimidin-4-amine (0.078 g, 0.23 mmol) and Fe/ _NH4Cl , and was obtained as a pale yellow solid (0.052 g, 73% yield), which was used further without further purification. LCMS: 311.1 [M+H].

Intermediate compound B13

3-(4-amino-3-fluorophenyl)-1-(tetrahydro-2 H -pyran-3-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-4-amine

The title compound was prepared using a procedure similar to that described for Intermediate B1, starting from 3-iodo-1-(tetrahydro- 2H -pyran-3-yl) -1H- pyrazolo[3,4- d ]pyrimidin-4-amine (A8, 0.050 g, 0.14 mmol) and 2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (0.040 g, 0.17 mmol) and obtained as a yellow gum (0.015 g, 32% yield). LCMS: 329.2 [M+H].

Intermediate connection B14

3-(4-amino-3-fluorophenyl)-1-(3,3-difluorocyclobutyl)-1 H- pyrazolo[3,4- d ]pyrimidin-4-amine

The title compound was prepared using a procedure similar to that described for Intermediate B1, starting from 1-(3,3-difluorocyclobutyl)-3-iodo- 1H -pyrazolo[3,4- d ]pyrimidin-4-amine (A9, 0.100 g, 0.285 mmol) and 2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (0.810 g, 0.341 mmol) and obtained as a pale yellow solid (0.088 g, 93% yield). LCMS: 334.9 [M+H].

Intermediate connection B15

4-(4-amino-3-(4-amino-3-fluorophenyl)-1 H -pyrazolo[3,4- d ]pyrimidin-1-yl)cyclohexan-1-ol

Step 1: Synthesis of 4-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexan-1-ol

An aqueous HCl solution (1.5 N, 5 mL) was added to a solution of 3-iodo-1-(1,4-dioxaspiro[4.5]decan-8-yl)-1H - pyrazolo[3,4- d ]pyrimidin-4-amine (A10, 0.313 g, 0.780 mmol) and the resulting mixture was stirred at room temperature for 12 h. After completion of the reaction (as determined by UPLC), the solvent was removed under reduced pressure to give a residue (0.232 g, LCMS: 358.0 [M+H]), which was dissolved in THF (5 mL). The resulting solution was cooled to 0°C, _NaBH4 (0.050 g, 1.322 mmol) was added, and the mixture was stirred at room temperature for 1 h. After the reaction was complete (as determined by UPLC), aqueous HCl (1.5 N, 5 mL) was added, and the mixture was extracted with EtOAc (2 x ₁₀ mL). The combined organic layers were dried over _Na2SO4 , filtered, and concentrated under reduced pressure to give the title product as a pale brown gum (0.196 g), which was used without further purification. LCMS: 359.8 [M+H].

Step 2: Synthesis of 4-(4-amino-3-(4-amino-3-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexan-1-ol

The title compound was prepared using a procedure similar to that described for Intermediate B1, starting from 4-(4-amino-3-iodo- 1H -pyrazolo[3,4- d ]pyrimidin-1-yl)cyclohexan-1-ol (0.196 g, 0.550 mmol) and 2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (0.160 g, 0.655 mmol) and obtained as a yellow gum (0.120 g, 64% yield). LCMS: 343.0 [M+H].

Intermediate connection B16

3-(4-amino-3-fluorophenyl)-1-(1-(oxetan-3-yl)piperidin-4-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-4-amine

Step 1: Synthesis of tert-butyl 4-(4-amino-3-(3-fluoro-4-nitrophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carboxylate

The title compound was prepared using a procedure similar to that described for Intermediate B1, starting from tert- butyl 4-(4-amino-3-iodo- 1H -pyrazolo[3,4- d ]pyrimidin-1-yl)piperidine-1-carboxylate (A11, 0.10 g, 0.23 mmol) and (3-fluoro-4-nitrophenyl)boronic acid (0.05 g, 0.27 mmol) and obtained as a yellow solid (0.04 g, 40% yield). LCMS: 458.1 [MH].

Step 2: Synthesis of 3-(3-fluoro-4-nitrophenyl)-1-(piperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

TFA (0.5 mL) was added to a solution of tert- butyl 4-(4-amino-3-(3-fluoro-4-nitrophenyl)-1H - pyrazolo[3,4- d ]pyrimidin-1-yl)piperidine-1-carboxylate (100 mg, 0.218 mmol) in dry DCM (5 mL) and the resulting mixture was stirred at room temperature for 2 h. After completion of the reaction (as determined by UPLC), the solvent was evaporated under reduced pressure to give the title product as a pale brown gum (110 mg), which was used further without further purification. LCMS: 358.1 [MH].

Step 3: Synthesis of 3-(3-fluoro-4-nitrophenyl)-1-(1-(oxetan-3-yl)piperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

Oxetan-3-one (0.020 g, 0.277 mmol) and glacial acetic acid (catalytic amount) were added to a solution of 3-(3-fluoro-4-nitrophenyl)-1-(piperidin-4-yl)-1H - pyrazolo[3,4- d ]pyrimidin-4-amine (0.100 g, 0.280 mmol) in DCM (5 mL), and the resulting mixture was stirred at room temperature for 4 h. Sodium triacetoxyborohydride (0.178 g, 0.840 mmol) was then added, and the resulting mixture was stirred at room temperature for 12 h. After the reaction was complete (as determined by UPLC), the solution was diluted with DCM (5 mL) and washed with 10% aqueous _NaHCO3 (5 mL) and brine (5 mL). The organic layer was dried over _Na2SO4 , _filtered and the solvents were evaporated under reduced pressure to give the title product (110 mg), which was used further without further purification. LCMS: 414.2 [MH].

Step 4: Synthesis of 3-(4-amino-3-fluorophenyl)-1-(1-(oxetan-3-yl)piperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

Iron powder (0.135 g, 2.417 mmol) and ammonium chloride (0.142 g, 2.655 mmol) were added to a solution of 3-(3-fluoro-4-nitrophenyl)-1-(1-(oxetan-3-yl)piperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (0.110 g, 0.266 mmol) in ethanol (5 mL) and water (2 mL), and the resulting mixture was stirred at 80 °C for 2 h. After the reaction was complete (as determined by TLC), the reaction mixture was filtered through a pad of diatomaceous earth, which was then washed with EtOAc (5 mL x 2). The combined filtrates were concentrated under reduced pressure to give a residue, which was dissolved in EtOAC (10 mL), then washed with brine (5 mL). The organic layer was separated, dried _{over Na2SO4} , filtered and concentrated under reduced pressure to give the title compound as an off-white solid (0.05 g, 50% yield), which was used without further purification. LCMS: 383.9 [M+H].

Intermediate connection B17

3-(4-amino-3-(4-amino-3-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)tetrahydrothiophene 1,1-dioxide

The title compound was prepared using a procedure similar to that described for intermediate B1, starting from 3-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)tetrahydrothiophene 1,1-dioxide (A12, 0.08 g, 0.21 mmol) and 2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (0.06 g, 0.25 mmol), and was obtained as a yellow gum (0.03 g, 40% yield). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ=8.26 (s, 1H), 7.27-7.31 (m, 1H), 7.20-7.23 (m, 1H), 6.88-6.93 (m, 1H), 5.69 (br.s, 2H), 5.53 (br.s, 2H), 3.72-3.78 (m, 2H), 3.51-3.56 (m, 2H), 2.61-2.70 (m, 2H). LCMS: 362.8 [M+H].

Intermediate connection B18

1-(4-amino-3-(4-aminophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpropan-2-ol

Step 1: Synthesis of 1-(4-amino-3-(4-nitrophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpropan-2-ol

The title compound was prepared using a procedure similar to that described for intermediate B1, starting from 1-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpropan-2-ol (A13, 0.067 g, 0.201 mmol) and (4-nitrophenyl)boronic acid (0.054 g, 0.302 mmol) and obtained as a yellow solid (0.059 g, 90% yield). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ=8.40 (d, J=7.6 Hz, 2H), 8.30 (s, 1H), 7.94 (d, J=6.8 Hz, 2H), 4.32 (br s, 2H), 1.16 (br s, 6H). LCMS: 328.9 [MH].

Step 2: Synthesis of 1-(4-amino-3-(4-aminophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpropan-2-ol

The title compound was prepared using a procedure similar to that described for step 2 of intermediate B2, starting from 1-(4-amino-3-(4-nitrophenyl)-1H - pyrazolo[3,4- d ]pyrimidin-1-yl)-2-methylpropan-2-ol (0.072 g, 0.219 mmol) and Fe/NH ₄ Cl, and was obtained as a pale yellow solid (0.07 g, quantitative yield), which was used further without further purification. LCMS: 299.0 [M+H].

Intermediate connection B19

1-(4-amino-3-(4-amino-3-fluorophenyl)-1 H -pyrazolo[3,4- d ]pyrimidin-1-yl)-2-methylpropan-2-ol

The title compound was prepared using a procedure similar to that described for Intermediate B1, starting from 1-(4-amino-3-iodo- 1H -pyrazolo[3,4- d ]pyrimidin-1-yl)-2-methylpropan-2-ol (A13, 0.110 g, 0.330 mmol) and 2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (0.094 g, 0.396 mmol) and obtained as a pale yellow solid (0.077 g, 66% yield). LCMS: 317.1 [M+H].

Intermediate connection B20

3-(4-amino-3-fluorophenyl)-1-(pyridin-4-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-4-amine

The title compound was prepared using a procedure similar to that described for Intermediate B1, starting from 3-iodo-1-(pyridin-4-yl)-1H - pyrazolo[3,4- d ]pyrimidin-4-amine (A14, 0.250 g, 0.73 mmol) and 2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (0.193 g, 0.81 mmol) and obtained as a yellow gum (0.140 g, 59% yield). LCMS: 321.9 [M+H].

Intermediate connection B21

3-(4-amino-3-chlorophenyl)-1-isopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-4-amine

Step 1: Synthesis of 3-(3-chloro-4-nitrophenyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine

The title compound was prepared using a procedure similar to that described for intermediate B1, starting from 3-iodo-1-isopropyl- 1H -pyrazolo[3,4- d ]pyrimidin-4-amine (A2, 0.115 g, 0.379 mmol) and 2-(3-chloro-4-nitrophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (0.129 g, 0.455 mmol) and obtained as an off-white solid (0.083 g, 66% yield). ^1H NMR (400 MHz, DMSO- d6 ) δ=8.28 (s, 1H), 8.21-8.23 (m, 1H), 7.95-7.95 (m, 1H), 7.82-7.85 (m, 1H), 5.07-5.13 (m, 1H), 1.51 ( _d , J =6.4 Hz, 6H). LCMS: 332.9 [M+H].

Step 2: Synthesis of 3-(4-amino-3-chlorophenyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine

The title compound was prepared using a procedure similar to that described for step 2 of intermediate B2, starting from 3-(3-chloro-4-nitrophenyl)-1-isopropyl- 1H -pyrazolo[3,4 -d ]pyrimidin-4-amine (0.083 g, 0.25 mmol) and Fe/NH ₄ Cl, and was obtained as a pale yellow solid (0.900 g, quantitative yield), which was used further without further purification. LCMS: 302.9 [M+H].

Intermediate connection B22

3-(4-amino-3-chlorophenyl)-1-methyl-1 H- pyrazolo[3,4- d ]pyrimidin-4-amine

The title compound was prepared using a procedure similar to that described for intermediate B1, starting from 3-iodo-1-methyl- 1H -pyrazolo[3,4- d ]pyrimidin-4-amine (A15, 0.100 g, 0.364 mmol) and 2-chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (0.092 g, 0.364 mmol) and obtained as a yellow solid (0.094 g, 94% yield). ^1H NMR (400 MHz, DMSO _-d6 ) δ =8.23 (s, 1H), 7.45-7.46 (m, 1H), 7.31-7.33 (m, 1H), 6.91-6.93 (m, 1H), 5.68 (br.s, 2H), 3.91 (s, 3H). LCMS: 275.0[M+H].

Intermediate connection B23

3-(4-amino-3-fluorophenyl)-1-methyl-1 H -pyrazolo[3,4- d ]pyrimidin-4-amine

The title compound was prepared using a procedure similar to that described for Intermediate B1, starting from 3-iodo-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (B15, 0.110 g, 0.400 mmol) and 2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (0.104 g, 0.440 mmol) and obtained as a brown gum (0.130 g, 97% yield). LCMS: 259.1 [M+H].

Intermediate connection B24

3-(4-amino-3-fluorophenyl)-1-cyclopropyl-1 H -pyrazolo[4,3- c ]pyridin-4-amine

The title compound was prepared using a procedure similar to that described for intermediate B1, starting from 1-cyclopropyl-3-iodo- 1H- pyrazolo[4,3- c ]pyridin-4-amine (A16, 0.190 g, 0.63 mmol) and 2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (0.180 g, 0.75 mmol), and was obtained as a yellow gum (0.070 g, 39% yield). ^1H NMR (400 MHz, DMSO _-d6 ) δ=7.75-7.76 (m, 1H), 7.21-7.24 (m, 1H), 7.14-7.16 (m, 2H), 6.85-6.87 (m, 1H), 5.78 (br.s, 2H), 5.46 (br.s, 2H), 3.67-3.69 (m, 1H), 1.09-1.10 (m, 4H). LCMS: 317.1 [M+H].

Intermediate connection B25

3-(4-aminophenyl)-1-cyclopropyl-1 H -pyrazolo[4,3- c ]pyridin-4-amine

Step 1: Synthesis of 1-cyclopropyl-3-(4-nitrophenyl)-1H-pyrazolo[4,3-c]pyridin-4-amine

The title compound was prepared using a procedure similar to that described for intermediate B1, starting from 1-cyclopropyl-3-iodo- 1H -pyrazolo[4,3 -c ]pyridin-4-amine (A16, 0.190 g, 0.63 mmol) and (4-nitrophenyl)boronic acid (0.126 g, 0.75 mmol) and obtained as a pale yellow solid (0.090 g, 50% yield). ¹ H NMR (400 MHz, DMSO- d ₆ ) δ=8.36-8.39 (m, 2H), 7.93-7.96 (m, 2H), 7.84 (d, J =6.0 Hz, 1H), 6.95 (d, J =6.0 Hz, 1H), 5.98 (br.s, 2H), 3.78-3.81 (m, 1H), 1.13-1.16 (m, 4H). LCMS: 296.1 [M+H].

Step 2: Synthesis of 3-(4-aminophenyl)-1-cyclopropyl-1H-pyrazolo[4,3-c]pyridin-4-amine

The title compound was prepared using a procedure similar to that described for step 2 of intermediate B2, starting from 1-cyclopropyl-3-(4-nitrophenyl)-1H-pyrazolo[4,3-c]pyridin-4-amine (0.093 g, 0.31 mmol) and Fe/NH ₄ Cl, and was obtained as a pale yellow solid (0.052 g, 63% yield), which was used further without further purification. LCMS: 266.0 [M+H].

Intermediate connection B26

3-(4-amino-3-fluorophenyl)-1-(3-(benzyloxy)cyclobutyl)-1 H -pyrazolo[3,4- d ]pyrimidin-4-amine

A mixture of 1-(3-(benzyloxy)cyclobutyl)-3-iodo- 1H -pyrazolo[3,4 -d ]pyrimidin-4-amine (A17, 0.400 g, 0.712 mmol), 2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (0.186 g, 0.783 mmol) and _K2CO3 (0.197 g, 1.424 mmol) in dioxane (7 mL), ethanol (3 mL) and water (3 mL) was purged with _N2 for 10 _min . _PdCl2 (dppf) (0.026 g, 0.036 mmol) was then added and the resulting mixture was purged with _N2 again for 10 min, then stirred at 80°C for 16 h in a sealed tube. After the reaction was complete (as determined by TLC and LCMS), the reaction mixture was filtered through a pad of diatomaceous earth, which was then washed with EtOAc (3 x 10 mL x 3). The combined filtrates were concentrated under reduced pressure to give a crude material, which was purified by flash chromatography (silica gel 230-400 mesh, eluting with 4% MeOH in DCM) to give the title compound as a pale brown solid (0.179 g, 62% yield). ^1H NMR (400 MHz, _DMSO-d6 ) δ=8.21 (s, 1H), 7.20-7.40 (m, 7H), 6.89-6.93 (m, 1H), 5.42-5.48 (m, 3H), 4.47-4.48 (m, 3H), 2.74-2.83 (m, 2H), 2.60 (s, 2H). LCMS: 405.1 [M+H].

Intermediate connection B27

3-(4-amino-3-methylphenyl)-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-4-amine

The title compound was prepared using a procedure similar to that described for intermediate B26, starting from 1-cyclopropyl-3-iodo- 1H -pyrazolo[3,4- d ]pyrimidin-4-amine (A1, 0.250 g, 0.830 mmol) and 2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (0.213 g, 0.913 mmol) and obtained as a brown gum (0.22 g), which was used further without further purification. LCMS: 281.3 [M+H]

Intermediate connection B28

3-(4-amino-3-fluorophenyl)-1-(1-methylazetidin-3-yl)-1 H- pyrazolo[3,4- d ]pyrimidin-4-amine

The title compound was prepared following a procedure similar to that described for intermediate B26, starting from 3-iodo-1-(1-methylazetidin-3-yl) -1H- pyrazolo[3,4- d ]pyrimidin-4-amine (A18, 0.380 g, 1.151 mmol) and 2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (0.300 g, 1.266 mmol) and obtained as a brown solid (0.26 g, 48% yield). LCMS: 314.4 [M+H].

Intermediate connection B29

3-(4-amino-2,5-difluorophenyl)-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-4-amine

The title compound was prepared following a procedure similar to that described for Intermediate B26, starting from 1-cyclopropyl-3-iodo- 1H- pyrazolo[3,4- d ]pyrimidin-4-amine (A1, 0.165 g, 0.548 mmol) and 2,5-difluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (prepared as described in PCT Publication No. WO 2020/172093, 0.300 g, 1.266 mmol) and obtained as a pale yellow solid (0.051 g, 27% yield). LCMS: 303.2 [M+H].

Intermediate connection B30

(2-amino-5-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)phenyl)methanol

The title compound was prepared following a procedure similar to that described for Intermediate B26, starting from 1-cyclopropyl-3-iodo- 1H- pyrazolo[3,4- d ]pyrimidin-4-amine (A1, 0.250 g, 0.830 mmol) and (2-amino-5-(4,4,5,5-tetramethyl-1,3-dioxolan-2-yl)phenyl)methanol (prepared as described in PCT Publication No. WO 2011/8130628, 0.207 g, 0.830 mmol) and obtained as a pale pink solid (0.122 g, 47% yield). LCMS: 297.1 [M+H].

Intermediate connection B31

3-(6-aminopyridin-3-yl)-1-cyclopropyl- N- (2,4-dimethoxybenzyl)-1H - pyrazolo[4,3- c ]pyridin-4-amine

The title compound was prepared following a procedure similar to that described for intermediate B26, starting from 1-cyclopropyl- N- (2,4-dimethoxybenzyl)-3-iodo- 1H -pyrazolo[4,3- c ]pyridin-4-amine (A19, 0.300 g, 0.666 mmol) and 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine (0.147 g, 0.666 mmol) and obtained as a brown gum (0.11 g, 30% yield). LCMS: 416.9 [M+H].

Intermediate connection B32

2-(4-amino-3-(4-amino-3-fluorophenyl)-1 H -pyrazolo[3,4- d ]pyrimidin-1-yl)ethan-1-ol

The title compound was prepared following a procedure similar to that described for intermediate B26, starting from 2-(4-amino-3-iodo- 1H -pyrazolo[3,4- d ]pyrimidin-1-yl)ethan-1-ol (A20, 0.420 g, 1.377 mmol) and 2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (0.327 g, 1.379 mmol) and obtained as a brown solid (0.390 g, 92% yield). LCMS: 289.1 [M+H].

Intermediate connection B33

3-(4-amino-3-fluorophenyl)-1-(2-methoxyethyl)-1 H -pyrazolo[3,4- d ]pyrimidin-4-amine

The title compound was prepared following a procedure similar to that described for intermediate B26, starting from 3-iodo-1-(2-methoxyethyl) -1H- pyrazolo[3,4- d ]pyrimidin-4-amine (A21, 0.620 g, 1.943 mmol) and 2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (0.461 g, 1.943 mmol) and obtained as a brown solid (0.42 g), which was used without further purification. LCMS: 302.9 [M+H].

Intermediate connection B34

3-(4-amino-3-fluorophenyl)-1-cyclopropyl- N -(2,4-dimethoxybenzyl)-1 H -pyrazolo[4,3- c ]pyridin-4-amine

The title compound was prepared following a procedure similar to that described for intermediate B26, starting from 1-cyclopropyl- N- (2,4-dimethoxybenzyl)-3-iodo- 1H -pyrazolo[4,3- c ]pyridin-4-amine (A19, 0.600 g, 1.333 mmol) and 2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (0.316 g, 1.333 mmol), and was obtained as a brown solid (0.43 g, 72% yield). ^1H NMR (400 MHz, DMSO _- d6 ) δ=7.82 (d, J =6.0 Hz, 1H), 7.07-7.19 (m, 3H), 6.83-6.89 (m, 2H), 6.51 (d, J =2.0 Hz, 1H), 6.40-6.43 (m, 1H), 5.62 (t, J =5.6 Hz, 1H), 5.49 (br.s, 2H), 4.49 (d, J =5.6 Hz, 2H), 3.65-3.72 (m, 7H), 1.08-1.09 (m, 4H). LCMS: 434.2 [M+H].

Intermediate connection B35

3-(6-aminopyridin-3-yl)-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-4-amine

The title compound was prepared following a procedure similar to that described for intermediate B26, starting from 1-cyclopropyl-3-iodo- 1H- pyrazolo[3,4- d ]pyrimidin-4-amine (A1, 0.250 g, 0.830 mmol) and 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine (0.183 g, 0.830 mmol) and obtained as a brown solid (0.085 g, 38% yield). LCMS: 268.6 [M+H].

Intermediate connection B36

3-(4-amino-3-fluorophenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1 H- pyrazolo[3,4- d ]pyrimidin-4-amine

The title compound was prepared following a procedure similar to that described for intermediate B26, starting from 3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (A22, 0.500 g, 1.278 mmol) and 2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (0.454 g, 1.917 mmol), and was obtained as a brown solid (0.16 g, 30% yield). ^1H NMR (400 MHz, DMSO _-d6 ) δ=8.26 (s, 1H), 7.20-7.27 (m, 2H), 6.88-6.93 (m, 1H), 5.62 (s, 2H), 5.52 (br.s, 2H), 3.62 (t, J =8.0 Hz, 2H), 0.84 (t, J =8.0 Hz, 2H), -0.12 (s, 9H). LCMS: 375.4 [M+H].

Intermediate connection B37

3-(4-amino-3-fluorophenyl)-1-(1-methylpiperidin-4-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-4-amine

The title compound was prepared following a procedure similar to that described for intermediate B26, starting from 3-iodo-1-(1-methylpiperidin-4-yl) -1H- pyrazolo[3,4- d ]pyrimidin-4-amine (A23, 0.180 g, 0.503 mmol) and 2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (0.131 g, 0.553 mmol) and obtained as a brown solid (0.22 g, 72% yield). LCMS: 342.2 [M+H].

General method for the synthesis of carbamates Intermediates C

Pyridine (1.2 eq) and phenyl chloroformate (1.5 eq) were added to a solution of the amine (1.0 eq) in THF (10 vol) at 0 °C. The reaction mixture was allowed to warm to 25 °C and stirred for 12 h. After the reaction was complete (as determined by TLC), the mixture was diluted with EtOAc (10 mL) and washed with brine (5 mL). The organic layer was dried over Na ₂ SO ₄ , filtered, and concentrated under reduced pressure to give a crude material, which was purified by flash chromatography (silica gel 230-400 mesh, eluting with 10-20% EtOAc in petroleum ether) to give the desired carbamate.

The following carbamates were prepared using the above general method.

Carbamate Structure Source material ¹ H NMR/LCMS C1 403.1 [M+H] C2 389.1 [M+H] C3 405.2 [M+H] C4 261.0 [M+H] C5 261.0 [M+H] C6 312.9 [M+H] C7 312.9 [M+H] C8 315.1 [M+H] C9 265.1 [M+H] C10 259.1 [M+H] C11 278.0 [M+H] C12 277.1 [M+H] C13 277.1 [M+H] C14 270.9 [M-H] C15 273.0 [M-H] C16 247.1 [M+H] C17 233.0 [M+H] C18 261.0 [M+H] C19 272.5 [M+H] C20 272.5 [M+H] C21 349.6 [M+H] C22 274.9 [M+H]

Note: The amines used to synthesize the carbamates are commercially available or were synthesized using literature methods as follows:

3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-amine (C6 precursor) and 5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-amine (C7 precursor) were synthesized from 1-(trifluoromethyl)cyclopropane-1-carboxylic acid methyl ester as described in Synthesis 2013, 45, 171-173.

3-(1,1,1-trifluoro-2-methylpropan-2-yl)isoxazol-5-amine (precursor C8) and 3-(2-fluoropropan-2-yl)isoxazol-5-amine (precursor C9) were synthesized from methyl 3,3,3-trifluoro-2,2-dimethylpropanoate and methyl 2-fluoro-2-methylpropionate, respectively, according to the method described in Synthesis 2013, 45, 171-173.

2-(5-Aminoisoxazol-3-yl)-2-methylpropanenitrile (precursor C20) was synthesized from 2,2-dimethyl-3-oxopentanedinitrile as described in J. Med. Chem. 2012, 55, 1082–1105.

3-((( tert- butyldimethylsilyl)oxy)methyl)isoxazol-5-amine (precursor C21) was synthesized from 4-(( tert- butyldiphenylsilyl)oxy)-3-oxobutanenitrile as described in PCT Publication No. WO 2013/104561.

3-( Tertiary- butyl)-4-methylisoxazol-5-amine (precursor C22) was synthesized from 2,4,4-trimethyl-3-oxopentanenitrile as described in PCT Publication No. WO 2012/019015.

Synthesis of 3-(1-methylcyclobutyl)isoxazol-5-amine (C19 precursor):

NH ₂ OH H ₂ SO ₄ (0.699 g, 4.25 mmol) was added to a stirred solution of 3-(1-methylcyclobutyl)-3-oxopropanenitrile (prepared as described in PCT Publication No. WO 2017/060874, 0.500 g, 3.86 mmol) and sodium hydroxide (0.170 g, 4.25 mmol) in EtOH (10 mL) and water (10 mL). The pH of the resulting mixture was adjusted to 7.5 with aqueous NaOH (1 M) and the reaction mixture was stirred at 80°C for 15 h. After the reaction was complete (as determined by TLC), the reaction mixture was concentrated under reduced pressure to give a residue that was taken up in DCM (25 mL), washed with water (10 mL), dried _{over Na2SO4} , filtered, and concentrated under reduced pressure. The resulting crude material was purified by flash chromatography (silica gel 230-400 mesh, eluting with 40% EtOAc in petroleum ether) to give the title product as an off-white solid (0.110 g, 19% yield). ¹ H NMR (400 MHz, CDCl ₃ ) δ=5.04 (s, 1H), 2.43-2.49 (m, 2H), 1.96-2.02 (m, 4H), 1.50 (s, 3H). LCMS: 153.2 [M+H].

GETTING EXAMPLES

General procedure for the preparation of urea for the synthesis of examples 1-72

Method A - Triethylamine (2.0 equiv) was added to a mixture of amine intermediate D (1.0 equiv) and carbamate intermediate E (1.0 equiv) in THF (10 vol) and the resulting mixture was stirred at 60°C for 12 h in a sealed tube. After completion of the reaction (as determined by LCMS), the reaction mixture was concentrated under reduced pressure to afford a crude material, which was purified by preparative reverse-phase HPLC to afford the desired product.

Method B - DMAP (0.05 equiv) and DIPEA (1.5 equiv) were added to a solution of amine intermediate D (1.0 equiv) and carbamate intermediate E (1.0 equiv) in THF (10 vol), and the resulting mixture was stirred at 60°C for 12 h in a sealed tube. After completion of the reaction (as determined by LCMS), the reaction mixture was concentrated under reduced pressure to yield a crude material, which was purified by preparative reverse-phase HPLC to yield the desired product.

The following compounds were prepared using the general procedures described above.

Example 1

1-(4-(4-amino-1-isopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)phenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea

The title compound was prepared according to the general procedure for the preparation of urea (method A) starting from 3-(4-aminophenyl)-1-isopropyl- 1H- pyrazolo[3,4 -d ]pyrimidin-4-amine (B2, 0.250 g, 0.93 mmol) and phenyl (3-(tert-butyl)isoxazol-5-yl)carbamate (C4, 0.242 g, 0.93 mmol) and obtained as an off-white solid (0.150 g, 37% yield). ¹ H NMR (400 MHz, DMSO -d6 ) δ=10.15 (br.s, 1H), 9.09 (br.s, 1H), 8.24 (br.s, 1H), 7.55-7.66 (m, 4H), 6.10 (s, 1H), 5.03-5.10 ₍ m, 1H), 1.49 (d, J =6.4 Hz, 6H), 1.27 (s, 9H). LCMS: 435.3 [M+H].

Example 2

1-(4-(4-amino-1-(oxetan-3-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)phenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 3-(4-aminophenyl)-1-(oxetan-3-yl)-1H - pyrazolo[3,4 -d ]pyrimidin-4-amine (B4, 0.210 g, 0.744 mmol) and phenyl (3-( tert-butyl )isoxazol-5-yl)carbamate (C4, 0.193 g, 0.744 mmol) and obtained as an off-white solid (0.053 g, 16% yield). ^1H NMR (400 MHz, DMSO _-d6 ) δ=10.23 (br.s, 1H), 9.15 (br.s, 1H), 8.31 (s, 1H), 7.74 (s, 4H), 6.16 (s, 1H), 6.06-6.10 (m, 1H), 5.16 (t, J =6.4 Hz, 2H), 5.05 (t, J =6.8 Hz, 2H), 1.33 (s, 9H). LCMS: 449.2[M+H].

Example 3

1-(4-(4-amino-1-(oxetan-3-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)phenyl)-3-(5-( tert- butyl)isoxazol-3-yl)urea

The title compound was prepared according to the general procedure for the preparation of urea (method A) starting from 3-(4-aminophenyl)-1-(oxetan-3-yl)-1H - pyrazolo[3,4 -d ]pyrimidin-4-amine (B4, 0.070 g, 0.248 mmol) and phenyl (5-( tert- butyl)isoxazol-3-yl)carbamate (C5, 0.065 g, 0.248 mmol) and obtained as an off-white solid (0.035 g, 32% yield). ¹ H NMR (400 MHz, DMSO -d6 ) δ=9.59 (br.s, 1H), 9.07 (br.s, 1H), 8.26 (s, 1H), 7.66 (br.s, 4H), 6.53 (s, 1H), 5.98-6.04 ₍ m, 1H), 5.09 (t, J =6.4 Hz, 2H), 5.00 (t, J =6.8 Hz, 2H), 1.31 (s, 9H). LCMS: 449.2[M+H].

Example 4

1-(4-(4-amino-1-(tetrahydro-2 H -pyran-4-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)phenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 3-(4-aminophenyl)-1-(tetrahydro- 2H -pyran-4-yl) -1H- pyrazolo[3,4 -d ]pyrimidin-4-amine (B12, 0.05 g, 0.16 mmol) and phenyl (3-( tert-butyl )isoxazol-5-yl)carbamate (C4, 0.042 g, 0.16 mmol) and obtained as an off-white solid (0.013 g, 20% yield). ^1H NMR (400 MHz, DMSO _-d6 ) δ=10.13 (br.s, 1H), 9.08 (br.s, 1H), 8.25 (s, 1H), 7.61-7.67 (m, 4H), 6.10 (s, 1H), 4.90-4.98 (m, 1H), 4.00-4.03 (m, 2H), 3.53-3.58 (m, 2H), 2.19-2.26 (m, 2H), 1.87-1.91 (m, 2H), 1.27 (s, 9H). LCMS: 477.1[M+H].

Example 5

1-(4-(4-amino-1-(tetrahydrofuran-3-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)phenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 3-(4-aminophenyl)-1-(tetrahydrofuran-3-yl)-1H - pyrazolo[3,4 -d ]pyrimidin-4-amine (B10, 0.083 g, 0.28 mmol) and phenyl (3-( tert-butyl )isoxazol-5-yl)carbamate (C4, 0.072 g, 0.28 mmol) and obtained as a white solid (0.048 g, 36% yield). ^1H NMR (400 MHz, DMSO _-d6 ) δ=10.17 (br.s, 1H), 9.08 (br.s, 1H), 8.26 (s, 1H), 7.60-7.67 (m, 4H), 6.10 (s, 1H), 5.49-5.52 (m, 1H), 4.07-4.14 (m, 2H), 3.89-3.97 (m, 2H), 2.38-2.43 (m, 2H), 1.27 (s, 9H). LCMS: 463.0[M+H].

Example 6

1-(4-(4-amino-1-isopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-chlorophenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 3-(4-amino-3-chlorophenyl)-1-isopropyl- 1H -pyrazolo[3,4 -d ]pyrimidin-4-amine (B21, 0.080 g, 0.264 mmol) and phenyl (3-( tert-butyl )isoxazol-5-yl)carbamate (C4, 0.067 g, 0.264 mmol) and obtained as an off-white solid (0.003 g, 3% yield). ¹ H NMR (400 MHz, DMSO- ₆ ) δ=10.85 (br.s, 1H), 8.66 (br.s, 1H), 8.35 (d, J =8.4 Hz, 1H), 8.25 (s, 1H), 7.72 (d, J =2.0 Hz, 1H), 7.61-7.64 (m, 1H), 6.12 (s, 1H), 5.04-5.10 (m, 1H), 1.50 (d, J =6.8 Hz, 6H), 1.28 (s, 9H). LCMS: 469.1[M+H].

Example 7

1-(4-(4-amino-1-cyclobutyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)phenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 3-(4-aminophenyl)-1-cyclobutyl- 1H- pyrazolo[3,4 -d ]pyrimidin-4-amine (B8, 0.140 g, 0.49 mmol) and phenyl (3-( tert-butyl )isoxazol-5-yl)carbamate (C4, 0.130 g, 0.49 mmol) and obtained as an off-white solid (0.067 g, 29% yield). ^1H NMR (400 MHz, DMSO _-d6 ) δ=10.15 (br.s, 1H), 9.09 (br.s, 1H), 8.24 (s, 1H), 7.62-7.68 (m, 4H), 6.10 (s, 1H), 5.32-5.36 (m, 1H), 2.69-2.74 (m, 2H), 2.39-2.42 (m, 2H), 1.87-1.89 (m, 2H), 1.27 (s, 9H). LCMS: 447.2[M+H].

Example 8

1-(4-(4-amino-1-cyclobutyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 3-(4-aminophenyl)-1-cyclobutyl- 1H- pyrazolo[3,4 -d ]pyrimidin-4-amine (B9, 0.100 g, 0.33 mmol] and phenyl (3-( tert- butyl)isoxazol-5-yl)carbamate (C4, 0.087 g, 0.33 mmol) and obtained as a pale yellow solid (0.005 g, 4% yield). ¹ H NMR (400 MHz, DMSO- d ₆ ) δ = 9.10 (br s, 1H), 8.23-8.28 (m, 2H), 7.47-7.53 (m, 2H), 6.90 (br s, 1H), 6.09 (s, 1H), 5.29-5.37 (m, 1H), 2.67-2.73 (m, 2H), 2.38-2.41 (m, 2H), 1.85-1.89 (m, 2H), 1.26 (s, 9H). LCMS: 465.1[M+H].

Example 9

1-(4-(4-amino-1-(2-hydroxy-2-methylpropyl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)phenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 1-(4-amino-3-(4-aminophenyl)-1H - pyrazolo[3,4- d ]pyrimidin-1-yl)-2-methylpropan-2-ol (B18, 0.119 g, 0.4 mmol) and phenyl (3-( tert-butyl )isoxazol-5-yl)carbamate (C4, 0.103 g, 0.4 mmol) and obtained as an off-white solid (0.017 g, 9% yield). ¹ H NMR (400 MHz, DMSO -d6 ) δ=9.44 (br.s, 1H), 8.24 (s, 1H), 7.59-7.68 (m, 4H), 6.07 (s, 1H), 4.81 (br.s, 1H), 4.27 ₍ br.s, 2H), 1.26 (s, 9H), 1.15 (s, 6H). LCMS: 465.1[M+H].

Example 10

1-(4-(4-amino-1-(2-hydroxy-2-methylpropyl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 1-(4-amino-3-(4-amino-3-fluorophenyl) -1H- pyrazolo[3,4 -d ]pyrimidin-1-yl)-2-methylpropan-2-ol (B19, 0.077 g, 0.243 mmol) and phenyl (3-( tert-butyl )isoxazol-5-yl)carbamate (C4, 0.063 g, 0.243 mmol) and obtained as an off-white solid (0.013 g, 11% yield). ¹ H NMR (400 MHz, CD ₃ OD) δ=8.33-8.37 (m, 1H), 8.29 (s, 1H), 7.53-7.59 (m, 2H), 6.19 (s, 1H), 4.42 (s, 2H), 1.35 (s, 9H), 1.29 (s, 6H). LCMS: 483.3[M+H].

Example 11

1-(4-(4-amino-1-isopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 3-(4-amino-3-fluorophenyl)-1-isopropyl- 1H -pyrazolo[3,4 -d ]pyrimidin-4-amine (B3, 0.200 g, 0.69 mmol) and phenyl (3-( tert- butyl)isoxazol-5-yl)carbamate (C4, 0.182 g, 0.69 mmol) and obtained as an off-white solid (0.011 g, 3% yield). ¹ H NMR (400 MHz, DMSO -d6 ) δ=10.40 (br.s, 1H), 8.93 (br.s, 1H), 8.24-8.30 (m, 2H), 7.47-7.52 (m, 2H), 6.95 ₍ br.s, 2H), 6.11 (s, 1H), 5.03-5.10 (m, 1H), 1.49 (d, J =6.4 Hz, 6H), 1.27 (s, 9H). LCMS: 453.2[M+H]

Example 12

1-(4-(4-amino-1-(oxetan-3-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-( tert-butyl )isoxazol-5-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 3-(4-amino-3-fluorophenyl)-1-(oxetan-3-yl) -1H- pyrazolo[3,4- d ]pyrimidin-4-amine (B5, 0.200 g, 0.66 mmol) and phenyl (3-( tert-butyl )isoxazol-5-yl)carbamate (C4, 0.173 g, 0.66 mmol) and obtained as an off-white solid (0.027 g, 10% yield). ^1H NMR (400 MHz, DMSO _-d6 ) δ=8.97 (br.s, 1H), 8.29-8.33 (m, 1H), 8.25 (s, 1H), 7.52-7.59 (m, 2H), 6.11 (s, 1H), 5.98-6.05 (m, 1H), 5.01-5.11 (m, 2H), 4.98-4.99 (m, 2H), 1.27 (s, 9H). LCMS: 467.1[M+H].

Example 13

1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea

The title compound was prepared according to the general procedure for the preparation of urea (method A) starting from 3-(4-amino-3-fluorophenyl)-1-cyclopropyl- 1H- pyrazolo[3,4 -d ]pyrimidin-4-amine (B1, 0.150 g, 0.52 mmol) and phenyl (3-( tert-butyl )isoxazol-5-yl)carbamate (C4, 0.137 g, 0.52 mmol) and obtained as a white solid (0.035 g, 15% yield). ^1H NMR (400 MHz, DMSO -d6 ) δ=10.42 (br.s, 1H), 8.98 (br.s, 1H), 8.25-8.29 (m, 2H), 7.44-7.50 (m, 2H), 6.95 ₍ br.s, 2H), 6.10 (s, 1H), 3.85-3.87 (m, 1H), 1.27 (s, 9H), 1.20-1.23 (m, 2H), 1.08-1.10 (m, 2H). LCMS: 451.2[M+H].

Example 14

1-(4-(4-amino-1-(1-(oxetan-3-yl)piperidin-4-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 3-(4-amino-3-fluorophenyl)-1-(1-(oxetan-3-yl)piperidin-4-yl) -1H- pyrazolo[3,4- d ]pyrimidin-4-amine (B16, 0.050 g, 0.13 mmol) and phenyl (3-( tert- butyl)isoxazol-5-yl)carbamate (C4, 0.034 g, 0.13 mmol) and obtained as an off-white solid (0.006 g, 8% yield). ¹ H NMR (400 MHz, CD ₃ OD) δ=8.32-8.36 (m, 1H), 8.27 (s, 1H), 7.51-7.57 (m, 2H), 6.19 (s, 1H), 4.65-4.82 (m, 5H), 3.61-3.64 (m, 1H), 2.98-3.01 (m, 2H), 2.37-2.44 (m, 2H), 2.13-2.19 (m, 2H), 2.04-2.07 (m, 2H), 1.35 (s, 9H). LCMS: 550.0[M+H].

Example 15

1-(4-(4-amino-1-(4-hydroxycyclohexyl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-( tert-butyl )isoxazol-5-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 4-(4-amino-3-(4-amino-3-fluorophenyl) -1H -pyrazolo[3,4- d ]pyrimidin-1-yl)cyclohexan-1-ol (B15, 0.075 g, 0.22 mmol) and phenyl (3-( tert-butyl )isoxazol-5-yl)carbamate (C4, 0.057 g, 0.22 mmol) and obtained as an off-white solid (0.007 g, 6% yield). ¹ H NMR (400 MHz, CD ₃ OD) δ=8.32-8.36 (m, 1H), 8.27 (s, 1H), 7.50-7.55 (m, 2H), 6.19 (s, 1H), 4.74-4.80 (m, 1H), 3.72-3.73 (m, 1H), 2.14-2.21 (m, 4H), 2.03-2.06 (m, 2H), 1.55-1.59 (m, 2H), 1.35 (s, 9H). LCMS: 509.2[M+H].

Example 16

1-(4-(4-amino-1-(3,3-difluorocyclobutyl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 3-(4-amino-3-fluorophenyl)-1-(3,3-difluorocyclobutyl) -1H- pyrazolo[3,4 -d ]pyrimidin-4-amine (B14, 0.110 g, 0.33 mmol) and phenyl (3-( tert-butyl )isoxazol-5-yl)carbamate (C4, 0.086 g, 0.33 mmol) and obtained as an off-white solid (0.020 g, 13% yield). ^1H NMR (400 MHz, DMSO _-d6 ) δ=9.28 (br.s, 1H), 8.26-8.30 (m, 2H), 7.48-7.55 (m, 2H), 7.08 (br.s, 1H), 6.07 (s, 1H), 5.28-5.33 (m, 1H), 3.25-3.34 (m, 4H), 1.27 (s, 9H). LCMS: 501.1[M+H].

Example 17

1-(4-(4-amino-1-(pyridin-4-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-( tert-butyl )isoxazol-5-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 3-(4-amino-3-fluorophenyl)-1-(pyridin-4-yl) -1H- pyrazolo[3,4 -d ]pyrimidin-4-amine (B20, 0.140 g, 0.43 mmol) and phenyl (3-( tert-butyl )isoxazol-5-yl)carbamate (C4, 0.113 g, 0.43 mmol) and obtained as an off-white solid (0.006 g, 3% yield). ¹ H NMR (400 MHz, DMSO _-d6 ) δ=10.44 (br.s, 1H), 9.00 (br.s, 1H), 8.73-8.74 (m, 2H), 8.34-8.47 (m, 4H), 7.59-7.68 (m, 2H), 6.13 (s, 1H), 1.28 (s, 9H). LCMS: 488.2[M+H].

Example 18

1-(4-(4-amino-1-(1,1-dioxidotetrahydrothiophen-3-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 3-(4-amino-3-(4-amino-3-fluorophenyl)-1H - pyrazolo[3,4- d ]pyrimidin-1-yl)tetrahydrothiophene 1,1-dioxide (B17, 0.030 g, 0.082 mmol) and phenyl (3-( tert- butyl)isoxazol-5-yl)carbamate (C4, 0.021 g, 0.082 mmol) and obtained as a white solid (0.003 g, 7% yield). ¹ H NMR (400 MHz, CD ₃ OD) δ=8.31-8.36 (m, 2H), 7.54-7.60 (m, 2H), 6.19 (s, 1H), 5.78-5.80 (m, 1H), 3.58-3.63 (m, 3H), 2.78-2.89 (m, 2H), 1.32 (s, 9H). LCMS: 529.2[M+H].

Example 19

1-(4-(4-amino-1-(tetrahydro-2 H -pyran-3-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 3-(4-amino-3-fluorophenyl)-1-(tetrahydro- 2H -pyran-3-yl)-1H - pyrazolo[3,4- d ]pyrimidin-4-amine (B13, 0.015 g, 0.045 mmol) and phenyl (3-( tert- butyl)isoxazol-5-yl)carbamate (C4, 0.011 g, 0.045 mmol) and obtained as an off-white solid (0.002 g, 9% yield). ¹ H NMR (400 MHz, CD ₃ OD) δ=8.54 (s, 1H), 8.35 (d, J =7.2 Hz, 1H), 8.28 (s, 1H), 7.50-7.56 (m, 2H), 6.19 (s, 1H), 4.60 (s, 1H), 3.87-4.05 (m, 3H), 3.50-3.58 (m, 1H), 2.38-2.42 (m, 1H), 2.21-2.23 (m, 1H), 1.92-2.03 (m, 2H), 1.35 (s, 9H). LCMS: 495.1[M+H].

Example 20

1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)phenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea

The title compound was prepared according to the general procedure for the preparation of urea (method A) starting from 3-(4-aminophenyl)-1-cyclopropyl- 1H- pyrazolo[3,4 -d ]pyrimidin-4-amine (B7, 0.100 g, 0.38 mmol) and phenyl (3-( tert-butyl )isoxazol-5-yl)carbamate (C4, 0.100 g, 0.38 mmol) and obtained as a white solid (0.045 g, 27% yield). ^1H NMR (400 MHz, DMSO _-d6 ) δ=10.11 (br.s, 1H), 9.10 (br.s, 1H), 8.26 (s, 1H), 7.58-7.65 (m, 4H), 6.09 (s, 1H), 3.83-3.89 (m, 1H), 1.24 (s, 9H), 1.19-1.23 (m, 2H), 1.06-1.11 (m, 2H). LCMS: 433.2[M+H].

Example 21

1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 3-(4-amino-3-fluorophenyl)-1-cyclopropyl- 1H- pyrazolo[3,4 -d ]pyrimidin-4-amine (B1, 0.100 g, 0.31 mmol) and phenyl (3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)carbamate (C6, 0.091 g, 0.31 mmol) and obtained as an off-white solid (0.020 g, 12% yield). ¹ H NMR (400 MHz, DMSO -d6 ) δ=10.63 (br.s, 1H), 8.92 (br.s, 1H), 8.25-8.29 (m, 2H), 7.44-7.51 (m, 2H), 6.87 (br.s, _2H ), 6.20 (s, 1H), 3.84-3.89 (m, 1H), 1.45-1.76 (m, 2H), 1.36-1.41 (m, 2H), 1.19-1.23 (m, 2H), 1.06-1.11 (m, 2H). LCMS: 503.1[M+H].

Example 22

1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(1,1,1-trifluoro-2-methylpropan-2-yl)isoxazol-5-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 3-(4-amino-3-fluorophenyl)-1-cyclopropyl- 1H- pyrazolo[3,4 -d ]pyrimidin-4-amine (B1, 0.050 g, 0.17 mmol) and phenyl (3-(1,1,1-trifluoro-2-methylpropan-2-yl)isoxazol-5-yl)carbamate (C8, 0.045 g, 0.17 mmol) and obtained as an off-white solid (0.004 g, 4% yield). ¹ H NMR (400 MHz, DMSO -d6 ) δ=10.62 (br.s, 1H), 8.96 (br.s, 1H), 8.25-8.28 (m, 2H), 7.45-7.52 (m, 2H), 6.99 ₍ br.s, 2H), 6.25 (s, 1H), 3.84-3.90 (m, 1H), 1.53 (s, 6H), 1.19-1.23 (m, 2H), 1.10-1.12 (m, 2H). LCMS: 505.1[M+H].

Example 23

1-(4-(4-amino-1-(oxetan-3-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(5-( tert- butyl)isoxazol-3-yl)urea

The title compound was prepared according to the general procedure for the preparation of urea (method A) starting from 3-(4-amino-3-fluorophenyl)-1-(oxetan-3-yl) -1H- pyrazolo[3,4 -d ]pyrimidin-4-amine (B5, 0.150 g, 0.5 mmol) and phenyl (5-( tert-butyl )isoxazol-3-yl)carbamate (C5, 0.130 g, 0.5 mmol) and obtained as an off-white solid (0.015 g, 6% yield). ¹ H NMR (400 MHz, DMSO _-d6 ) δ=10.00 (br.s, 1H), 9.09 (br.s, 1H), 8.32-8.37 (m, 1H), 8.26 (s, 1H), 7.51-7.59 (m, 2H), 6.80 (br.s, 2H), 6.53 (s, 1H), 5.99-6.04 (m, 1H), 5.10 (t, J =6.4 Hz, 2H), 5.00 (t, J =6.4 Hz, 2H), 1.31 (s, 9H). LCMS: 467.2[M+H].

Example 24

1-(4-(4-amino-1-(tetrahydrofuran-3-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 3-(4-amino-3-fluorophenyl)-1-(tetrahydrofuran-3-yl) -1H- pyrazolo[3,4- d ]pyrimidin-4-amine (B11, 0.200 g, 0.59 mmol) and phenyl (3-( tert- butyl)isoxazol-5-yl)carbamate (C4, 0.154 g, 0.59 mmol) and obtained as a white solid (0.030 g, 10% yield), 1H NMR (400 MHz, DMSO- d ₆ ) δ = 10.40 (br s, 1H), 8.92 (br s, 1H), 8.26-8.31 (m, 2H), 7.47-7.54 (m, 2H), 6.80 (br.s, 2H), 6.11 (s, 1H), 5.47-5.53 (m, 1H), 4.05-4.13 (m, 2H), 3.87-3.96 (m, 2H), 2.40-2.41 (m, 2H), 1.27 (s, 9H). LCMS: 481.2[M+H].

Example 25

1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 3-(4-amino-3-fluorophenyl)-1-cyclopropyl- 1H- pyrazolo[3,4 -d ]pyrimidin-4-amine (B1, 0.070 g, 0.24 mmol) and phenyl (5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)carbamate (C7, 0.077 g, 0.24 mmol) and obtained as an off-white solid; (0.013 mg, 11% yield). ^1H NMR (400 MHz, DMSO _-d6 ) δ=11.78 (br.s, 1H), 8.76 (s, 1H), 7.43-7.45 (m, 2H), 6.84-6.88 (m, 2H), 5.46 (s, 2H), 3.91-3.97 (m, 1H), 1.49-1.54 (m, 4H), 1.19-1.22 (m, 2H), 1.10-1.15 (m, 2H). LCMS: 503.1[M+H].

Example 26

1-(4-(1-allyl-4-amino-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(5-( tert- butyl)isoxazol-3-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 1-allyl 3-(4-amino-3-fluorophenyl) -1H- pyrazolo[3,4 -d ]pyrimidin-4-amine (B6, 0.130 g, 0.45 mmol) and phenyl (5-( tert-butyl )isoxazol-3-yl)carbamate (C5, 0.119 g, 0.45 mmol) and obtained as an off-white solid; (0.012 g, 6% yield). ¹ H NMR (400 MHz, DMSO -d6 ) δ=10.46 (br.s, 1H), 9.52 (br.s, 1H), 8.24-8.28 (m, 2H), 7.45-7.50 (m, 2H), 6.95 ₍ br.s, 2H), 6.52 (s, 1H), 6.00-6.10 (m, 1H), 5.18-5.21 (m, 2H), 5.13 (d, J =1.6 Hz, 2H), 1.31 (s, 9H). LCMS: 451.2[M+H].

Example 27

1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(2-fluoropropan-2-yl)isoxazol-5-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 3-(4-amino-3-fluorophenyl)-1-cyclopropyl- 1H -pyrazolo[3,4 -d ]pyrimidin-4-amine (B1, 0.050 g, 0.176 mmol) and phenyl (3-(2-fluoropropan-2-yl)isoxazol-5-yl)carbamate (C9, 0.047 g, 0.176 mmol) and obtained as an off-white solid (0.007 mg, 9% yield). ^1H NMR (400 MHz, DMSO _-d6 ) δ=8.32-8.35 (m, 1H), 8.25 (s, 1H), 7.32-7.37 (m, 2H), 5.92 (s, 1H), 3.83-3.89 (m, 1H), 1.66 (s, 3H), 1.61 (s, 3H), 1.20-1.24 (m, 2H), 1.07-1.09 (m, 2H). LCMS: 455.1[M+H].

Example 28

1-(4-(4-amino-1-methyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-chlorophenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 3-(4-amino-3-chlorophenyl)-1-methyl- 1H- pyrazolo[3,4 -d ]pyrimidin-4-amine (B22, 0.068 g, 0.248 mmol) and phenyl (3-( tert- butyl)isoxazol-5-yl)carbamate (C4, 0.064 g, 0.248 mmol) as an off-white solid; (0.005 g, 4% yield). ^1H NMR (400 MHz, CD3OD) δ=8.70-8.68 (m, 1H), 7.47-7.67 (m, 2H), 7.00-7.02 (m, 1H), 6.20 (s, 1H), 4.10 (s, 3H), 1.34 (s, 9H). LCMS: 441.1[M+H].

Example 29

1-(4-(4-amino-1-methyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 3-(4-amino-3-fluorophenyl)-1-methyl- 1H- pyrazolo[3,4 -d ]pyrimidin-4-amine (B23, 0.052 g, 0.201 mmol) and phenyl (3-( tert- butyl)isoxazol-5-yl)carbamate (C4, 0.052 g, 0.201 mmol) and obtained as an off-white solid; (0.005 g, 6% yield). ¹ H NMR (400 MHz, CD ₃ OD) δ=10.60 (br.s, 1H), 9.05 (br.s, 1H), 8.25-8.30 (m, 2H), 7.45-7.51 (m, 2H), 6.90 (s, 2H), 6.09 (s, 1H), 3.95 (s, 3H), 1.27 (s, 9H). LCMS: 423.1[MH].

Example 30

1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[4,3- c ]pyridin-3-yl)-2-fluorophenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 3-(4-amino-3-fluorophenyl)-1-cyclopropyl- 1H -pyrazolo[4,3 -c ]pyridin-4-amine (B24, 0.070 g, 0.24 mmol) and phenyl (3-( tert-butyl )isoxazol-5-yl)carbamate (C4, 0.064 g, 0.24 mmol) and obtained as an off-white solid (0.004 g, 4% yield). ¹ H NMR (400 MHz, DMSO _-d6 ) δ=10.52 (br.s, 1H), 9.09 (br.s, 1H), 8.23-8.27 (m, 1H), 7.80 (s, 1H), 7.44-7.53 (m, 2H), 6.90 (d, J =6.0 Hz, 1H), 6.10 (s, 1H), 5.86 (br.s, 2H), 3.72-3.74 (m, 1H), 1.27 (s, 9H), 1.12-1.13 (m, 4H). LCMS: 450.2[M+H].

Example 31

1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[4,3-c]pyridin-3-yl)phenyl)-3-(3-( tert- butyl)isoxazol-5-yl)urea

The title compound was prepared according to the general procedure for the preparation of urea (method A) starting from 3-(4-aminophenyl)-1-cyclopropyl- 1H- pyrazolo[4,3- c ]pyridin-4-amine (B25, 0.052 g, 0.2 mmol) and phenyl (3-( tert-butyl )isoxazol-5-yl)carbamate (C4, 0.05 g, 0.2 mmol) and obtained as a white solid (0.007 g, 8% yield). ¹ H NMR (400 MHz, DMSO _-d6 ) δ=10.85 (br.s, 1H), 9.68 (br.s, 1H), 7.78 (br.s, 1H), 7.66 (d, J =8.4 Hz, 2H), 7.56 (d, J =8.4 Hz, 2H), 6.90 (d, J =6.0 Hz, 1H), 6.08 (s, 1H), 5.75 (br.s, 2H), 3.68-3.73 (m, 1H), 1.26 (s, 9H), 1.11-1.12 (m, 4H). LCMS: 432.3[M+H].

Example 32

1-(4-(4-amino-1-cyclopropyl- 1H- pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(1-methylcyclopropyl)isoxazol-5-yl)

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 3-(4-amino-3-fluorophenyl)-1-cyclopropyl- 1H- pyrazolo[3,4 -d ]pyrimidin-4-amine (B1, 0.050 g, 0.176 mmol) and phenyl (3-(1-methylcyclopropyl)isoxazol-5-yl)carbamate (C10, 0.045 g, 0.176 mmol) and obtained as an off-white solid (0.009 g, 11% yield). ^1H NMR (400 MHz, DMSO -d6 ) δ=10.29 (br.s, 1H), 8.90 (br.s, 1H), 8.24-8.28 (m, 2H), 7.44-7.51 (m, 2H), 6.94 ₍ br.s, 2H), 5.86 (s, 1H), 3.84-3.89 (m, 1H), 1.38 (s, 3H), 1.19-1.22 (m, 2H), 1.08-1.11 (m, 2H), 0.94-0.97 (m, 2H), 0.82-0.85 (m, 2H). LCMS: 449.1 [M+H].

Example 33

1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(5-( tert- butyl)-1,3,4-thiadiazol-2-yl)

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 3-(4-amino-3-fluorophenyl)-1-cyclopropyl- 1H- pyrazolo[3,4 -d ]pyrimidin-4-amine (B1, 0.050 g, 0.176 mmol) and phenyl (5-( tert- butyl)-1,3,4-thiadiazol-2-yl)carbamate (C11, 0.049 g, 0.176 mmol) and obtained as an off-white solid (0.012 mg, 14% yield). ^1H NMR (400 MHz, DMSO _-d6 ) δ=8.67 (br.s, 1H), 8.33-8.41 (m, 1H), 8.25 (s, 1H), 7.34-7.39 (m, 2H), 6.87 (br.s, 2H), 3.83-3.87 (m, 1H), 1.34 (s, 9H), 1.20-1.23 (m, 2H), 1.06-1.11 (m, 2H). LCMS: 468.2 [M+H].

Example 34

1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(4-( tert- butyl)thiazol-2-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 3-(4-amino-3-fluorophenyl)-1-cyclopropyl- 1H- pyrazolo[3,4 -d ]pyrimidin-4-amine (B1, 0.050 g, 0.176 mmol) and phenyl (4-( tert- butyl)thiazol-2-yl)carbamate (C12, 0.049 g, 0.176 mmol) and obtained as an off-white solid (0.004 g, 5% yield). ¹ H NMR (400 MHz, CD ₃ OD) δ=8.45-8.49 (m, 1H), 8.40 (s, 1H), 7.50-7.58 (m, 2H), 6.65 (s, 1H), 3.96-4.02 (m, 1H), 1.31-1.40 (m, 11H), 1.21-1.25 (m, 2H). LCMS: 467.2 [M+H].

Example 35

1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-( tert- butyl)isothiazol-5-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 3-(4-amino-3-fluorophenyl)-1-cyclopropyl- 1H- pyrazolo[3,4 -d ]pyrimidin-4-amine (B1, 0.050 g, 0.176 mmol) and phenyl (3-( tert- butyl)isothiazol-5-yl)carbamate (C13, 0.049 g, 0.176 mmol) and obtained as an off-white solid (0.016 g, 19% yield). ^1H NMR (400 MHz, DMSO -d6 ) δ=11.42 (br.s, 1H), 8.51 (br.s, 1H), 8.26 (s, 1H), 8.07-8.11 (m, 1H), 7.39-7.43 (m, 2H), 6.70 ( _s , 1H), 3.84-3.87 (m, 1H), 1.21-1.26 (m, 11H), 1.08-1.10 (m, 2H). LCMS: 465.1 [MH].

Example 36

1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(5-( tert- butyl)-1,3,4-oxadiazol-2-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from phenyl (4-(4-amino-1-cyclopropyl- 1H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)carbamate (C3, 0.050 g, 0.124 mmol) and 5-( tert- butyl)-1,3,4-oxadiazol-2-amine (0.017 g, 0.124 mmol) and obtained as an off-white solid (0.002 g, 4% yield). ¹ H NMR (400 MHz, CD ₃ OD) δ=8.37-8.43 (m, 2H), 7.46-7.62 (m, 2H), 3.93-3.95 (m, 1H), 1.45 (s, 9H), 1.31-1.38 (m, 2H), 1.20-1.23 (m, 2H). LCMS: 451.9 [M+H].

Example 37

1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-( tert- butyl)-1,2,4-thiadiazol-5-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from phenyl (4-(4-amino-1-cyclopropyl- 1H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)carbamate (C3, 0.050 g, 0.124 mmol) and 3-( tert- butyl)-1,2,4-thiadiazol-5-amine (0.019 g, 0.124 mmol) and obtained as an off-white solid (0.001 g, 1% yield). ¹ H NMR (400 MHz, CD ₃ OD) δ=8.27-8.39 (m, 2H), 7.51-7.56 (m, 2H), 3.80-3.86 (m, 1H), 1.40 (s, 9H), 1.31-1.33 (m, 2H), 1.17-1.21 (m, 2H). LCMS: 468.0 [M+H].

Example 38

1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(1-( tert- butyl)-1 H -1,2,4-triazol-3-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from phenyl (4-(4-amino-1-cyclopropyl- 1H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)carbamate (C3, 0.050 g, 0.124 mmol) and 1-( tert- butyl) -1H- 1,2,4-triazol-3-amine (0.017 g, 0.124 mmol) and obtained as an off-white solid (0.009 g, 15% yield). ^1H NMR (400 MHz, DMSO _-d6 ) δ=10.80 (br.s, 1H), 10.21 (br.s, 1H), 8.55 (s, 1H), 8.42-8.46 (m, 1H), 8.26 (s, 1H), 7.44-7.52 (m, 2H), 6.94 (br.s, 2H), 3.83-3.88 (m, 1H), 1.57 (s, 9H), 1.19-1.23 (m, 2H), 1.06-1.11 (m, 2H). LCMS: 451.0 [M+H].

Example 39

1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(trifluoromethyl)isoxazol-5-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 3-(4-amino-3-fluorophenyl)-1-cyclopropyl- 1H- pyrazolo[3,4-d]pyrimidin-4-amine (B1, 0.050 g, 0.176 mmol) and phenyl (3-(trifluoromethyl)isoxazol-5-yl)carbamate (C14, 0.048 g, 0.176 mmol) and obtained as a white solid (0.004 g, 5% yield). ¹ H NMR (400 MHz, DMSO -d6 ) δ=11.11 (br.s, 1H), 8.97 (br.s, 1H), 8.23-8.27 (m, 2H), 7.46-7.52 (m, 2H), 6.95 ₍ br.s, 2H), 6.53 (s, 1H), 3.84-3.88 (m, 1H), 1.19-1.24 (m, 2H), 1.07-1.12 (m, 2H). LCMS: 462.9 [M+H].

Example 40

1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(pentan-3-yl)isoxazol-5-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 3-(4-amino-3-fluorophenyl)-1-cyclopropyl- 1H- pyrazolo[3,4 -d ]pyrimidin-4-amine (B1, 0.05 g, 0.176 mmol) and phenyl (3-(pentan-3-yl)isoxazol-5-yl)carbamate (C15, 0.048 g, 0.176 mmol) and obtained as a pale brown solid (0.006 g, 7% yield). ¹ H NMR (400 MHz, DMSO -d6 ) δ=10.44 (br.s, 1H), 8.97 (br.s, 1H), 8.26-8.29 (m, 2H), 7.44-7.50 (m, 2H), 6.81 (br.s, _2H ), 6.00 (s, 1H), 3.84-3.86 (m, 1H), 1.51-1.66 (m, 4H), 1.08-1.21 (m, 4H), 0.79-0.82 (m, 6H). LCMS: 465.0 [M+H].

Example 41

1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-isopropylisoxazol-5-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 3-(4-amino-3-fluorophenyl)-1-cyclopropyl- 1H- pyrazolo[3,4 -d ]pyrimidin-4-amine (B1, 0.050 g, 0.176 mmol) and phenyl (3-isopropylisoxazol-5-yl)carbamate (C16, 0.043 g, 0.176 mmol) and obtained as an off-white solid (0.007 g, 9% yield). ¹ H NMR (400 MHz, DMSO- d6 ) δ=10.49 (br.s, 1H), 9.01 (br.s, 1H), 8.27-8.32 (m, 2H), 7.44-7.52 (m, 2H), 6.07 (s, 1H), 3.87-3.92 ₍ m, 1H), 2.90-2.97 (m, 1H), 1.18-1.24 (m, 8H), 1.10-1.12 (m, 2H). LCMS: 437.0 [M+H].

Example 42

1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-ethylisoxazol-5-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 3-(4-amino-3-fluorophenyl)-1-cyclopropyl- 1H- pyrazolo[3,4 -d ]pyrimidin-4-amine (B1, 0.050 g, 0.176 mmol) and phenyl (3-ethylisoxazol-5-yl)carbamate (C17, 0.041 g, 0.176 mmol) and obtained as an off-white solid (0.006 g, 8% yield). ¹ H NMR (400 MHz, DMSO -d6 ) δ=10.44 (br.s, 1H), 8.94 (br.s, 1H), 8.26-8.30 (m, 2H), 7.44-7.51 (m, 2H), 6.99 ₍ br.s, 2H), 6.05 (s, 1H), 3.84-3.89 (m, 1H), 2.53-2.59 (m, 2H), 1.18-1.21 (m, 5H), 1.08-1.10 (m, 2H). LCMS: 423.0 [M+H].

Example 43

1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-( sec- butyl)isoxazol-5-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 3-(4-amino-3-fluorophenyl)-1-cyclopropyl- 1H- pyrazolo[3,4 -d ]pyrimidin-4-amine (B1, 0.080 g, 0.281 mmol) and phenyl (3-( sec- butyl)isoxazol-5-yl)carbamate (C18, 0.073 g, 0.281 mmol) and obtained as a white solid (0.004 g, 4% yield). ^1H NMR (400 MHz, DMSO _-d6 ) δ=10.42 (br.s, 1H), 8.96 (br.s, 1H), 8.26-8.33 (m, 2H), 7.44-7.52 (m, 2H), 6.04 (s, 1H), 3.87-3.92 (m, 1H), 2.68-2.73 (m, 1H), 1.54-1.60 (m, 2H), 1.08-1.24 (m, 7H), 0.82-0.84 (m, 3H). LCMS: 451.1 [M+H].

Example 44

1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(1-methylcyclobutyl)isoxazol-5-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 3-(4-amino-3-fluorophenyl)-1-cyclopropyl- 1H- pyrazolo[3,4 -d ]pyrimidin-4-amine (B1, 0.100 g, 0.352 mmol) and phenyl (3-(1-methylcyclobutyl)isoxazol-5-yl)carbamate (C19, 0.096 g, 0.352 mmol) and obtained as a white solid (0.002 g, 1% yield). ¹ H NMR (400 MHz, CD ₃ OD) δ=8.35-8.41 (m, 2H), 7.49-7.56 (m, 2H), 6.16 (s, 1H), 3.98-4.02 (m, 1H), 2.46-2.51 (m, 2H), 1.96-2.13 (m, 4H), 1.54 (s, 3H), 1.23-1.38 (m, 4H). LCMS: 463.1 [M+H].

Example 45

1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[4,3- c ]pyridin-3-yl)-2-fluorophenyl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 3-(4-amino-3-fluorophenyl)-1-cyclopropyl- 1H -pyrazolo[4,3 -c ]pyridin-4-amine (B24, 0.160 g, 0.565 mmol) and phenyl (3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)carbamate (C6, 0.176 g, 0.565 mmol) and obtained as an off-white solid (0.012 g, 4% yield). ^1H NMR (400 MHz, DMSO _- d6 ) δ=10.69 (br.s, 1H), 8.97 (br.s, 1H), 8.23-8.27 (m, 1H), 7.80 (d, J =6.0 Hz, 1H), 7.46-7.54 (m, 2H), 6.91 (d, J =6.0 Hz, 1H), 6.22 (s, 1H), 5.85 (br.s, 2H), 3.72-3.74 (m, 1H), 1.39-1.49 (m, 4H), 1.12-1.13 (m, 4H). LCMS: 502.2 [M+H].

Example 46

1-(4-(4-amino-1-(3-hydroxycyclobutyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea

Step 1: Synthesis of 1-(4-(4-amino-1-(3-(benzyloxy)cyclobutyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea

The title compound was prepared according to the general procedure for the preparation of urea (method B) starting from 3-(4-amino-3-fluorophenyl)-1-(3-(benzyloxy)cyclobutyl) -1H- pyrazolo[3,4 -d ]pyrimidin-4-amine (B26, 0.090 g, 0.223 mmol) and phenyl (3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)carbamate (C6, 0.076 g, 0.245 mmol) and obtained as a brown gum (70 mg), which was used without further purification. LCMS: 623.4 [M+H].

Step 2: Synthesis of 1-(4-(4-amino-1-(3-hydroxycyclobutyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea

Boron trichloride (1 M in DCM, 0.899 mL, 0.899 mmol) was added dropwise to a solution of 1-(4-(4-amino-1-(3-(benzyloxy)cyclobutyl)-1H - pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea (0.070 g, 0.112 mmol) in dry DCM (5 mL) at -60 °C and the resulting mixture was stirred at 0 °C for 3 h. After completion of the reaction (as determined by TLC and LCMS), the reaction mixture was cooled to -70 °C, neutralized with aqueous ammonia (25% in water), and extracted with DCM (10 mL x 2). The combined organic layers _{were dried over Na2SO4 ,} filtered and concentrated under reduced pressure to give a crude material which was purified by preparative HPLC (eluting with 1% TFA in water:ACN) to give the title product as a white solid (0.009 g, 15% yield). ¹ H NMR (400 MHz, _CD3OD ) δ=8.34-8.38 (m, 2H), 7.53-7.61 (m, 2H), 6.34 (s, 1H), 5.61-5.64 (m, 1H), 4.73-4.79 (m, 1H), 2.94-3.01 (m, 2H), 2.57-2.63 (m, 2H), 1.39-1.48 (m, 4H). LCMS: 533.7 [M+H].

Example 47

1-(5-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)pyridin-2-yl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)

Step 1: Synthesis of 1-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine (0.100 g, 0.454 mmol) and phenyl (3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)carbamate (C6, 0.142 g, 0.454 mmol) and obtained as a brown gum (150 mg) which was used without further purification. LCMS: 439.5 [M+H].

Step 2: Synthesis of 1-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea

The title compound was prepared following a procedure similar to that described for intermediate B26, starting from 1-cyclopropyl-3-iodo- 1H- pyrazolo[3,4- d ]pyrimidin-4-amine (A1, 0.100 g, 0.332 mmol) and 1-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea (0.146 g, 0.332 mmol), and was obtained as an off-white solid (0.013 g, 8% yield). ^1H NMR (400 MHz, DMSO- d6 ) δ=11.68 (br.s, 1H), 9.93 (br.s, 1H), 8.53 (d, J =2.0 Hz, 1H), 8.28 (s, 1H), 8.02-8.05 (m, _1H ), 7.72 (d, J =8.4 Hz, 1H), 6.27 (s, 1H), 3.86-3.90 (m, 1H), 1.10-1.49 (m, 8H). LCMS: 486.2 [M+H].

Example 48

1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-methylphenyl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea

The title compound was prepared according to the general procedure for the preparation of urea (method B) starting from 3-(4-amino-3-methylphenyl)-1-cyclopropyl- 1H -pyrazolo[3,4-d]pyrimidin-4-amine (B27, 0.110 g, 0.392 mmol) and phenyl (3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)carbamate (C6, 0.135 g, 0.432 mmol) and obtained as a pale brown solid (0.051 g, 26% yield). ¹ H NMR (400 MHz, CD ₃ OD) δ=8.41 (s, 1H), 7.95-7.97 (m, 1H), 7.52-7.57 (m, 2H), 6.31 (s, 1H), 3.98-4.01 (m, 1H), 2.41 (s, 3H), 1.21-1.48 (m, 8H). LCMS: 499.1 [M+H].

Example 49

1-(4-(4-amino-1-(1-methylazetidin-3-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea

The title compound was prepared according to the general procedure for the preparation of urea (method B) starting from 3-(4-amino-3-fluorophenyl)-1-(1-methylazetidin-3-yl)-1H - pyrazolo[3,4- d ]pyrimidin-4-amine (B28, 0.130 g, 0.278 mmol) and phenyl (3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)carbamate (C6, 0.095 g, 0.306 mmol) and obtained as a white solid (0.010 g, 7% yield). ¹ H NMR (400 MHz, CD ₃ OD) δ=8.35-8.39 (m, 1H), 8.30 (s, 1H), 7.58-7.65 (m, 2H), 6.35 (s, 1H), 5.74-5.78 (m, 1H), 4.38-4.48 (m, 4H), 2.92 (s, 3H), 1.40-1.49 (m, 4H). LCMS: 532.2 [M+H].

Example 50

1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2,5-difluorophenyl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea

The title compound was prepared according to the general procedure for the preparation of urea (method B) starting from 3-(4-amino-2,5-difluorophenyl)-1-cyclopropyl- 1H- pyrazolo[3,4 -d ]pyrimidin-4-amine (B29, 0.050 g, 0.165 mmol) and phenyl (3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)carbamate (C6, 0.0516 g, 0.165 mmol) and obtained as an off-white solid (0.002 g, 3% yield). ^1H NMR (400 MHz, DMSO -d6 ) δ=10.60 (br.s, 1H), 9.08 (br.s, 1H), 8.17 (s, 1H), 8.03-8.08 (m, 1H), 7.37-7.42 (m, 1H), 6.16 ( _s , 1H), 3.78-3.82 (m, 1H), 1.32-1.42 (m, 4H), 1.02-1.20 (m, 4H). LCMS: 521.3 [M+H].

Example 51

1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-(hydroxymethyl)phenyl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method B) starting from (2-amino-5-(4-amino-1-cyclopropyl- 1H -pyrazolo[3,4 -d ]pyrimidin-3-yl)phenyl)methanol (B30, 0.120 g, 0.405 mmol)) and phenyl (3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)carbamate (C6, 0.126 g, 0.405 mmol) and obtained as an off-white solid (0.025 g, 12% yield). ^1H NMR (400 MHz, DMSO -d6 ) δ=11.14 (br.s, 1H), 8.65 (br.s, 1H), 8.38 (s, 1H), 8.06-8.08 (m, 1H), 7.54-7.62 (m, 2H), 6.19 ( _s , 1H), 4.60 (s, 2H), 3.90-3.94 (m, 1H), 1.24-1.48 (m, 8H). LCMS: 515.2 [M+H].

Example 52

1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ] pyrimidin- 3-yl)-2-fluorophenyl)-3-(3-(2-cyanopropan-2-yl)isoxazol-5-yl)urea

The title compound was prepared according to the general procedure for the preparation of urea (method B) starting from 3-(4-amino-3-fluorophenyl)-1-cyclopropyl- 1H -pyrazolo[3,4 -d ]pyrimidin-4-amine (B1, 0.120 g, 0.422 mmol) and phenyl (3-(2-cyanopropan-2-yl)isoxazol-5-yl)carbamate (C20, 0.126 g, 0.464 mmol) and obtained as an off-white solid (0.029 mg, 14% yield). ¹ H NMR (400 MHz, CD ₃ OD) δ=8.30-8.37 (m, 2H), 7.50-7.55 (m, 2H), 6.35 (s, 1H), 3.80-3.86 (m, 1H), 1.78 (s, 6H), 1.17-1.37 (m, 4H). LCMS: 462.2 [M+H].

Example 53

1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(hydroxymethyl)isoxazol-5-yl)urea

Step 1: Synthesis of 1-(4-(4-amino-1-cyclopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(((tert-butyldimethylsilyl)oxy)methyl)isoxazol-5-yl)urea

The title compound was prepared according to the general procedure for the preparation of urea (method B) starting from 3-(4-amino-3-fluorophenyl)-1-cyclopropyl- 1H -pyrazolo[3,4 -d ]pyrimidin-4-amine (B1, 0.100 g, 0.352 mmol) and phenyl (3-((( tert- butyldimethylsilyl)oxy)methyl)isoxazol-5-yl)carbamate (C21, 0.135 g, 0.387 mmol) and obtained as a white solid (0.020 g, 8% yield). ¹ H NMR (400 MHz, CD ₃ OD) δ=8.30-8.36 (m, 2H), 7.49-7.54 (m, 2H), 6.24 (s, 1H), 4.73 (s, 2H), 3.80-3.85 (m, 1H), 1.29-1.33 (m, 2H), 1.16-1.21 (m, 2H), 0.99 (s, 9H), 0.12 (s, 6H). LCMS: 539.3 [M+H].

Step 2: Synthesis of 1-(4-(4-amino-1-cyclopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(hydroxymethyl)isoxazol-5-yl)urea

TBAF (1 M in THF, 0.037 mL, 0.037 mmol) was added to a solution of 1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-((( tert- butyldimethylsilyl)oxy)methyl)isoxazol-5-yl)urea (0.020 g, 0.037 mmol) in THF (3 mL) at 0 °C and the resulting mixture was stirred at 25 °C for 1 h. After completion of the reaction (as determined by TLC), the reaction mixture was concentrated under reduced pressure to give a crude material, which was purified by preparative HPLC (ELSD method, eluting with 1% TFA in a mixture of water and CAN) to give the title compound as a white solid (0.005 g, 31% yield, TFA salt). ^1H NMR (400 MHz, DMSO -d6 ) δ=δ 10.46 (br.s, 1H), 8.98 (br.s, 1H), 8.27-8.35 (m, 2H), 7.45-7.53 (m, 2H), 6.14 (s, 1H), 4.44 ₍ s, 2H), 3.87-3.92 (m, 1H), 1.10-1.23 (m, 4H). LCMS: 425.2 [M+H].

Example 54

1-(5-(4-amino-1-cyclopropyl-1 H -pyrazolo[4,3- c ]pyridin-3-yl)pyridin-2-yl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea

Step 1: Synthesis of 1-(5-(1-cyclopropyl-4-((2,4-dimethoxybenzyl)amino)-1H-pyrazolo[4,3-c]pyridin-3-yl)pyridin-2-yl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method B) starting from 3-(6-aminopyridin-3-yl)-1-cyclopropyl- N -(2,4-dimethoxybenzyl)-1 H -pyrazolo[4,3- c ]pyridin-4-amine (B31, 0.050 g, 0.120 mmol) and phenyl (3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)carbamate (C6, 0.037 g, 0.120 mmol) and obtained as an off-white gum (0.012 g, 15% yield). LCMS: 635.2 [M+H].

Step 2: Synthesis of 1-(5-(4-amino-1-cyclopropyl-1H-pyrazolo[4,3-c]pyridin-3-yl)pyridin-2-yl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea

Triethylsilane (0.1 mL, 0.626 mmol) and TFA (0.1 mL, 1.298 mmol) were added to a solution of 1-(5-(1-cyclopropyl-4-((2,4-dimethoxybenzyl)amino)-1H - pyrazolo[4,3- c ]pyridin-3-yl)pyridin-2-yl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea (0.012 g, 0.019 mmol) in DCM (2 mL) at 0 °C and the resulting mixture was stirred at 25 °C for 12 h. After completion of the reaction (as determined by LCMS), the reaction mixture was concentrated under reduced pressure to give a crude material, which was purified by preparative HPLC (eluting with 10 mM NH ₄ OAc in ACN), to give the title product as an off-white solid (0.001 g, 10% yield). ¹ H NMR (400 MHz, CD ₃ OD) δ=8.64 (d, J =1.6 Hz, 1H), 8.09-8.11 (m, 1H), 7.80 (d, J =6.0 Hz, 1H), 7.47-7.49 (m, 1H), 7.04 (d, J =6.4 Hz, 1H), 6.39 (s, 1H), 3.80-3.86 (m, 1H), 0.90-1.50 (m, 8H). LCMS: 485.4 [M+H].

Example 55

1-(4-(4-amino-1-(2-hydroxyethyl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea

The title compound was prepared according to the general procedure for the preparation of urea (method B) starting from 2-(4-amino-3-(4-amino-3-fluorophenyl) -1H- pyrazolo[3,4 -d ]pyrimidin-1-yl)ethan-1-ol (B32, 0.11 g, 0.382 mmol) and phenyl (3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)carbamate (C6, 0.10 g, 0.320 mmol) and obtained as an off-white solid (0.002 g, 1% yield). ¹ H NMR (400 MHz, DMSO -d6 ) δ=10.66 (br.s, 1H), 8.99 (br.s, 1H), 8.26-8.30 (m, 2H), 7.48-7.54 (m, 2H), 6.94 ₍ br.s, 2H), 6.22 (s, 1H), 4.90 (br.s, 1H), 4.39 (t, J =6.0 Hz, 2H), 3.85 (t, J =6.0 Hz, 2H), 1.39-1.49 (m, 4H). LCMS: 507.2 [M+H].

Example 56

1-(4-(4-amino-1-(2-hydroxyethyl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)urea

The title compound was prepared according to the general procedure for the preparation of urea (method B) starting from 2-(4-amino-3-(4-amino-3-fluorophenyl) -1H- pyrazolo[3,4 -d ]pyrimidin-1-yl)ethan-1-ol (B32, 0.100 g, 0.347 mmol) and phenyl (5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)carbamate (C7, 0.108 g, 0.347 mmol) and obtained as an off-white solid (0.004 g, 3% yield). ¹ H NMR (400 MHz, CD ₃ OD) δ=8.36-8.41 (m, 2H), 7.52-7.59 (m, 2H), 6.83 (s, 1H), 4.58 (t, J =5.6 Hz, 2H), 4.07 (t, J =5.2 Hz, 2H), 1.49-1.59 (m, 4H). LCMS: 507.2 [M+H].

Example 57

1-(4-(4-amino-1-(2-methoxyethyl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea

The title compound was prepared according to the general procedure for the preparation of urea (method B) starting from 3-(4-amino-3-fluorophenyl)-1-(2-methoxyethyl) -1H- pyrazolo[3,4 -d ]pyrimidin-4-amine (B33, 0.125 g, 0.413 mmol) and phenyl (3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)carbamate (C6, 0.129 g, 0.413 mmol) and obtained as an off-white solid (0.020 g, 9% yield). ¹ H NMR (400 MHz, DMSO -d6 ) δ=10.61 (br.s, 1H), 8.98 (br.s, 1H), 8.25-8.29 (m, 2H), 7.48-7.54 (m, 2H), 6.96 ₍ br.s, 2H), 6.22 (s, 1H), 4.50 (t, J =5.2 Hz, 2H), 3.82 (t, J =5.2 Hz, 2H), 3.23 (s, 3H), 1.38-1.47 (m, 4H). LCMS: 521.2 [M+H].

Example 58

1-(4-(4-amino-1-(2-methoxyethyl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)urea

The title compound was prepared according to the general procedure for the preparation of urea (method B) starting from 3-(4-amino-3-fluorophenyl)-1-(2-methoxyethyl) -1H- pyrazolo[3,4 -d ]pyrimidin-4-amine (B33, 0.125 g, 0.413 mmol) and phenyl (5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)carbamate (C7, 0.129 g, 0.413 mmol) and obtained as an off-white solid (0.015 g, 7% yield). ¹ H NMR (400 MHz, DMSO _-d6 ) δ=10.07 (br.s, 1H), 9.00 (br.s, 1H), 8.31-8.35 (m, 2H), 7.47-7.55 (m, 2H), 6.92 (s, 1H), 4.53 (br.s, 2H), 3.83 (t, J =5.2 Hz, 2H), 3.23 (s, 3H), 1.51-1.52 (m, 4H). LCMS: 521.2 [M+H].

Example 59

1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-methylphenyl)-3-(5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)urea

The title compound was prepared according to the general procedure for the preparation of urea (method B) starting from 3-(4-amino-3-methylphenyl)-1-cyclopropyl- 1H- pyrazolo[3,4 -d ]pyrimidin-4-amine (B27, 0.100 g, 0.357 mmol) and phenyl (5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)carbamate (C7, 0.123 g, 0.392 mmol) and obtained as a white solid (0.026 g, 15% yield). ¹ H NMR (400 MHz, CD ₃ OD) δ=8.40 (s, 1H), 8.01-8.03 (m, 1H), 7.51-7.57 (m, 2H), 6.74 (s, 1H), 3.97-4.01 (m, 1H), 2.42 (s, 3H), 1.49-1.59 (m, 4H), 1.21-1.38 (m, 4H). LCMS: 499.5 [M+H].

Example 60

1-(4-(4-amino-1-(3-hydroxycyclobutyl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)urea

Step 1: Synthesis of 1-(4-(4-amino-1-(3-(benzyloxy)cyclobutyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorophenyl)-3-(5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)urea

The title compound was prepared according to the general procedure for the preparation of urea (method B) starting from 3-(4-amino-3-fluorophenyl)-1-(3-(benzyloxy)cyclobutyl)-1H - pyrazolo[3,4 -d ]pyrimidin-4-amine (B26, 0.090 g, 0.223 mmol) and phenyl (5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)carbamate (C7, 0.076 g, 0.245 mmol) and obtained as a brown gum (70 mg), which was used without further purification. LCMS: 623.4 [M+H].

Step 2: Synthesis of 1-(4-(4-amino-1-(3-hydroxycyclobutyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorophenyl)-3-(5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)urea

Boron trichloride (1 M in DCM, 0.899 mL, 0.899 mmol) was added dropwise to a solution of 1-(4-(4-amino-1-(3-(benzyloxy)cyclobutyl)-1H - pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)urea (0.070 g, 0.112 mmol) in dry DCM (5 mL) at -60 °C and the resulting mixture was left at 0 °C for 3 h. After completion of the reaction (as determined by TLC and LCMS), the reaction mixture was cooled to -70 °C, neutralized with aqueous ammonia (25% in water), and extracted with DCM (2 x 10 mL). The combined organic layers _{were dried over Na2SO4 ,} filtered and concentrated under reduced pressure to give a crude material which was purified by preparative HPLC (eluting with 1% TFA in water:ACN) to give the title product as a white solid (0.009 g, 15% yield). ¹ H NMR (400 MHz, _CD3OD ) δ=8.36-8.40 (m, 2H), 7.53-7.61 (m, 2H), 6.82 (s, 1H), 5.62-5.69 (m, 1H), 4.74-4.80 (m, 1H), 2.94-2.99 (m, 2H), 2.58-2.63 (m, 2H), 1.49-1.58 (m, 4H). LCMS: 533.7 [M+H].

Example 61

1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[4,3- c ]pyridin-3-yl)-2-fluorophenyl)-3-(5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)urea

Step 1: Synthesis of 1-(4-(1-cyclopropyl-4-((2,4-dimethoxybenzyl)amino)-1H-pyrazolo[4,3-c]pyridin-3-yl)-2-fluorophenyl)-3-(5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method B) starting from 3-(4-amino-3-fluorophenyl)-1-cyclopropyl- N- (2,4-dimethoxybenzyl)-1H - pyrazolo[4,3- c ]pyridin-4-amine (B34, 0.220 g, 0.508 mmol) and phenyl (5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)carbamate (C7, 0.158 g, 0.508 mmol) and obtained as a brown gum (0.093 g, 28% yield) which was used without further purification. LCMS: 652.3 [M+H].

Step 2: Synthesis of 1-(4-(4-amino-1-cyclopropyl-1H-pyrazolo[4,3-c]pyridin-3-yl)-2-fluorophenyl)-3-(5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)urea

Triethylsilane (0.1 mL) and TFA (0.1 mL) were added to a solution of 1-(4-(1-cyclopropyl-4-((2,4-dimethoxybenzyl)amino)-1H - pyrazolo[4,3- c ]pyridin-3-yl)-2-fluorophenyl)-3-(5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)urea (0.093 g, 0.143 mmol) in DCM (2 mL) at 0 °C and the resulting mixture was stirred at 25 °C for 12 h. After the reaction was complete (as determined by LCMS), the reaction mixture was concentrated under reduced pressure to give a crude material, which was purified by preparative HPLC (eluting with 0.1% TFA in water and ACN) to afford the title product as a white solid (0.050 g, 68% yield). ¹ H NMR (400 MHz, DMSO _- d6 ) δ=10.10 (br.s, 1H), 9.06 (br.s, 1H), 8.33-8.37 (m, 1H), 7.89 (br.s, 2H), 7.83 (d, J =7.2 Hz, 1H), 7.54-7.57 (m, 1H), 7.44-7.47 (m, 1H), 7.32 (d, J =7.2 Hz, 1H), 6.91 (s, 1H), 3.94-3.96 (m, 1H), 1.51-1.56 (m, 4H), 1.19-1.20 (m, 4H). LCMS: 502.5 [M+H].

Example 62

1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2,5-difluorophenyl)-3-(5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)urea

The title compound was prepared according to the general procedure for the preparation of urea (method B) starting from 3-(4-amino-2,5-difluorophenyl)-1-cyclopropyl- 1H- pyrazolo[3,4 -d ]pyrimidin-4-amine (B29, 0.100 g, 0.331 mmol) and phenyl (5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)carbamate (C7, 0.103 g, 0.331 mmol) and obtained as an off-white solid (0.002 g, 1% yield). ¹ H NMR (400 MHz, CD ₃ OD) δ=8.24-8.29 (m, 2H), 7.38-7.43 (m, 1H), 6.85 (s, 1H), 3.80-3.85 (m, 1H), 1.47-1.59 (m, 4H), 1.20-1.33 (m, 4H). LCMS: 521.2 [M+H].

Example 63

1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-(hydroxymethyl)phenyl)-3-(5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method B) starting from (2-amino-5-(4-amino-1-cyclopropyl- 1H -pyrazolo[3,4 -d ]pyrimidin-3-yl)phenyl)methanol (B30, 0.086 g, 0.290 mmol) and phenyl (5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)carbamate (C7, 0.091 g, 0.290 mmol) and obtained as an off-white solid (0.004 g, 3% yield). ^1H NMR (400 MHz, DMSO -d6 ) δ=10.39 (br.s, 1H), 8.58 (br.s, 1H), 8.27 (s, 1H), 8.06-8.08 (m, 1H), 7.52-7.62 (m, 2H), 6.89 ( _s , 1H), 5.45 (t, J =5.6 Hz, 1H), 4.59 (d, J =5.6 Hz, 2H), 3.84-3.89 (m, 1H), 1.50-1.57 (m, 4H), 1.20-1.23 (m, 4H). LCMS: 515.2 [M+H].

Example 64

1-(5-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)pyridin-2-yl)-3-(5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)urea

The title compound was prepared according to the general procedure for the preparation of urea (method B) starting from 3-(6-aminopyridin-3-yl)-1-cyclopropyl- 1H- pyrazolo[3,4 -d ]pyrimidin-4-amine (B35, 0.085 g, 0.318 mmol) and 1 phenyl (5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)carbamate (C7, 0.099 g, 0.318 mmol) and obtained as an off-white solid (0.003 g, 2% yield). ¹ H NMR (400 MHz, DMSO -d6 ) δ=11.03 (br.s, 1H), 9.83 (br.s, 1H), 8.51 (br.s, 1H), 8.27 (s, 1H), 8.01-8.04 (m, 1H), 7.72-7.74 ₍ m, 1H), 6.97 (s, 1H), 3.86-3.88 (m, 1H), 1.49-1.58 (m, 4H), 1.12-1.24 (m, 4H). LCMS: 486.1 [M+H].

Example 65

1-(4-(4-amino-1-cyclopropyl-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-( tert- butyl)-4-methylisoxazol-5-yl)urea

The title compound was prepared according to the general procedure for the preparation of urea (method B) starting from 3-(4-amino-3-fluorophenyl)-1-cyclopropyl- 1H -pyrazolo[3,4 -d ]pyrimidin-4-amine (B1, 0.100 g, 0.352 mmol) and phenyl (3-( tert-butyl )-4-methylisoxazol-5-yl)carbamate (C22, 0.096 g, 0.350 mmol) and obtained as a white solid (0.013 g, 8% yield). ¹ H NMR (400 MHz, CD ₃ OD) δ=8.39 (s, 1H), 8.22-8.26 (m, 1H), 7.46-7.55 (m, 2H), 3.94-3.97 (m, 1H), 2.09 (s, 3H), 1.35-1.39 (m, 11H), 1.21-1.23 (m, 2H). LCMS: 465.2 [M+H].

Example 66

1-(4-(4-amino-1-(2-hydroxy-2-methylpropyl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea

The title compound was prepared according to the general procedure for the preparation of urea (method B) starting from 1-(4-amino-3-(4-amino-3-fluorophenyl)-1H - pyrazolo[3,4 -d ]pyrimidin-1-yl)-2-methylpropan-2-ol (B19, 0.100 g, 0.316 mmol) and phenyl (3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)carbamate (C6, 0.099 g, 0.316 mmol) and obtained as a white solid (0.033 g, 19% yield). ¹ H NMR (400 MHz, DMSO -d6 ) δ=10.63 (br.s, 1H), 8.97 (br.s, 1H), 8.26-8.30 (m, 2H), 7.48-7.54 (m, 2H), 6.96 ₍ br.s, 2H), 6.22 (s, 1H), 4.77 (br.s, 1H), 4.28 (s, 2H), 1.39-1.49 (m, 4H), 1.15 (s, 6H). LCMS: 535.3 [M+H].

Example 67

1-(4-(4-amino-1-(2-hydroxy-2-methylpropyl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)urea

The title compound was prepared according to the general procedure for the preparation of urea (method B) starting from 1-(4-amino-3-(4-amino-3-fluorophenyl)-1H - pyrazolo[3,4 -d ]pyrimidin-1-yl)-2-methylpropan-2-ol (B19, 0.100 g, 0.316 mmol) and phenyl (5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)carbamate (C7, 0.099 g, 0.316 mmol) and obtained as a white solid (0.022 g, 13% yield). ^1H NMR (400 MHz, DMSO -d6 ) δ=10.08 (br.s, 1H), 9.04 (br.s, 1H), 8.42 (s, 1H), 8.31-8.35 (m, 1H), 7.47-7.55 (m, 2H), 6.91 ( _s , 1H), 4.31 (s, 2H), 1.48-1.58 (m, 4H), 1.17 (s, 6H). LCMS: 535.3 [M+H].

Example 68

1-(4-(4-amino-1-(1-methylazetidin-3-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)urea

The title compound was prepared according to the general procedure for the preparation of urea (method B) starting from 3-(4-amino-3-fluorophenyl)-1-(1-methylazetidin-3-yl)-1H - pyrazolo[3,4- d ]pyrimidin-4-amine (B28, 0.130 g, 0.278 mmol) and phenyl (5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)carbamate (C7, 0.095 g, 0.306 mmol) and obtained as a white solid (0.006 mg, 4% yield). ¹ H NMR (400 MHz, CD ₃ OD δ=8.26-8.41 (m, 2H), 7.56-7.64 (m, 2H), 6.84 (s, 1H), 5.71-5.74 (m, 1H), 4.29-4.90 (m, 4H), 2.85 (s, 3H), 1.52-1.59 (m, 4H). LCMS: 532.3 [M+H].

Example 69

1-(4-(4-amino-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea

Step 1: Synthesis of 1-(4-(4-amino-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 3-(4-amino-3-fluorophenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H - pyrazolo[3,4- d ]pyrimidin-4-amine (B36, 0.06 g, 0.160 mmol) and phenyl (3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)carbamate (C6, 0.05 g, 0.160 mmol) and obtained as a pale brown solid (80 mg), which was used without further purification. LCMS: 593.3 [M+H].

Step 2: Synthesis of 1-(4-(4-amino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea

HCl (g) in dioxane (4 M, 1 mL, 0.135 mmol) was added to a solution of 1-(4-(4-amino-1-((2-(trimethylsilyl)ethoxy)methyl) -1H- pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea (0.08 g, 0.135 mmol) in DCM (5 mL) at 0 °C and the resulting mixture was stirred at 25 °C for 12 h. After the reaction was complete (as determined by UPLC), the reaction mixture was concentrated under reduced pressure to give a crude material, which was purified by preparative HPLC (eluting with 10 mM NH ₄ OAc in ACN) to afford the title product as an off-white solid. white solid (0.003 g, 5% yield), 1H NMR (400 MHz, CD ₃ OD) δ=8.30-8.34 (m, 1H), 8.24 (s, 1H), 7.49-7.54 (m, 2H), 6.31 (s, 1H), 1.37-1.46 (m, 4H). LCMS: 463.3 [M+H].

Example 70

1-(4-(4-amino-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)urea

Step 1: Synthesis of 1-(4-(4-amino-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorophenyl)-3-(5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)urea

The title compound was prepared according to the General Procedure for the Preparation of Urea (Method A) starting from 3-(4-amino-3-fluorophenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H - pyrazolo[3,4- d ]pyrimidin-4-amine (B36, 0.06 g, 0.160 mmol) and phenyl (5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)carbamate (C7, 0.05 g, 0.160 mmol) and obtained as a pale brown solid (80 mg), which was used without further purification. LCMS: 593.3 [M+H].

Step 2: Synthesis of 1-(4-(4-amino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorophenyl)-3-(5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)urea

HCl (g) in dioxane (4 M, 1 mL, 0.135 mmol) was added to a solution of 1-(4-(4-amino-1-((2-(trimethylsilyl)ethoxy)methyl) -1H- pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)urea (0.08 g, 0.135 mmol) in DCM (5 mL) at 0 °C and the resulting mixture was stirred at 25 °C for 12 h. After the reaction was complete (as determined by UPLC), the reaction mixture was concentrated under reduced pressure to give a crude material which was purified by preparative HPLC (eluting with 10 mM NH ₄ OAc in CAN) to afford the title product as an off-white solid. white solid (0.007 g, 11% yield), 1H NMR (400 MHz, _DMSO-d6 ) δ=13.61 (br.s, 1H), 10.04 (br.s, 1H), 8.96 (br.s, 1H), 8.27-8.30 (m, 1H), 8.22 (s, 1H), 7.46-7.53 (m, 2H), 6.92 (s, 1H), 1.51-1.58 (m, 4H). LCMS: 463.3 [M+H].

Example 71

1-(4-(4-amino-1-(1-methylpiperidin-4-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)urea

The title compound was prepared according to the general procedure for the preparation of urea (method B) starting from 3-(4-amino-3-fluorophenyl)-1-(1-methylpiperidin-4-yl)-1H - pyrazolo[3,4- d ]pyrimidin-4-amine (B37, 0.100 g, 0.179 mmol) and phenyl (3-(1-(trifluoromethyl)cyclopropyl)isoxazol-5-yl)carbamate (C6, 0.061 g, 0.197 mmol) and obtained as a white solid (0.008 g, 8% yield). ¹ H NMR (400 MHz, CD ₃ OD) δ=8.34-8.38 (m, 2H), 7.52-7.57 (m, 2H), 6.35 (s, 1H), 5.12-5.17 (m, 1H), 3.73-3.76 (m, 2H), 3.33-3.39 (m, 2H), 3.00 (s, 3H), 2.59-2.62 (m, 2H), 2.35-2.39 (m, 2H), 1.45-1.49 (m, 4H). LCMS: 560.2 [M+H].

Example 72

1-(4-(4-amino-1-(1-methylpiperidin-4-yl)-1 H -pyrazolo[3,4- d ]pyrimidin-3-yl)-2-fluorophenyl)-3-(5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)urea

The title compound was prepared according to the general procedure for the preparation of urea (method B) starting from 3-(4-amino-3-fluorophenyl)-1-(1-methylpiperidin-4-yl)-1H - pyrazolo[3,4- d ]pyrimidin-4-amine (B37, 0.100 g, 0.179 mmol) and phenyl (5-(1-(trifluoromethyl)cyclopropyl)isoxazol-3-yl)carbamate (C7, 0.061 g, 0.197 mmol) and obtained as a brown solid (0.004 g, 4% yield). ¹ H NMR (400 MHz, CD ₃ OD) δ=8.36-8.40 (m, 2H), 7.51-7.56 (m, 2H), 6.82 (s, 1H), 5.10-5.18 (m, 1H), 3.73-3.76 (m, 2H), 3.37-3.39 (m, 2H), 3.00 (s, 3H), 2.59-2.62 (m, 2H), 2.35-2.38 (m, 2H), 1.39-1.59 (m, 4H). LCMS: 560.3 [M+H].

Biological example 1

Biochemical analysis of compounds

Representative compounds were tested for inhibitory activity against NEK7 and IL-1β release according to the procedures described above. The results are presented in the table below.

Table 2. Activity of representative compounds No. NEK7 IC ₅₀ (nM) Il-1β release IC ₅₀ (nM) 1 ** - 2 *** - 3 *** - 4 ** - 5 *** - 6 * - 7 *** - 8 **** - 9 ** - 10 **** - 11 **** - 12 **** +++ 13 **** ++++ 14 **** ++++ 15 **** - 16 * - 17 ** - 18 *** - 19 ** - 20 **** - 21 **** ++++ 22 *** - 23 **** - 24 **** - 25 **** +++ 26 *** - 27 * - 28 * - 29 **** - 30 **** ++++ 31 * - 32 **** +++ 33 * ++ 34 * - 35 * - 36 * - 37 * - 38 * - 39 *** - 40 ** - 41 **** - 42 *** - 43 *** - 44 *** - 45 *** +++ 46 **** - 48 ** - 49 * - 50 ** - 52 **** - 53 * - 54 *** - 55 **** - 57 *** - 58 *** - 59 * - 60 **** - 61 * - 62 ** - 63 * - 64 *** - 66 *** - 67 ** - 68 * - For IC ₅₀ activity of NEK7 in Table 2:
* IC ₅₀ greater than 501 nM
**IC ₅₀ range from 301-500 nM
*** IC ₅₀ range from 151-300 nM
**** IC ₅₀ less than 150 nM
For IC ₅₀ of IL-1β activity in Table 2:
+ IC ₅₀ greater than 501 nM
++ IC ₅₀ range of 201-500 nM
+++ IC ₅₀ of 51-200 nM
++++ IC ₅₀ less than 50 nM
- means that the value was not defined

The various embodiments described above may be combined to provide additional embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referenced in this specification and/or listed in the application brief, including but not limited to U.S. Provisional Patent Application No. 63/036,244, filed June 8, 2020, U.S. Provisional Patent Application No. 63/170,776, filed April 5, 2021, and U.S. Provisional Patent Application No. 63/185,257, filed May 6, 2021, are incorporated herein by reference in their entireties. Aspects of the embodiments may be modified, if necessary, to utilize the concepts of the various patents, applications, and publications to provide additional embodiments.

These and other modifications may be made to the embodiments in view of the detailed description provided above. In general, the terms used in the following claims should not be construed as limiting the claims to the specific embodiments disclosed in the description and claims, but should be construed as including all possible embodiments, as well as the full scope of equivalents to which the claims entitle. Accordingly, the claims are not limited by the description.

Claims (61)

1. A compound having the following structure (I): or a pharmaceutically acceptable salt thereof, where: A is _C6 aryl, _C6 cycloalkyl, or 6-membered monocyclic heteroaryl having one nitrogen heteroatom, each of which is optionally substituted with one or two ^R5 ; X represents N or CH; Y represents NH; R ¹ is H; R ² is H, C ₃ -C ₈ cycloalkyl, 4-6-membered heterocyclyl having one ring heteroatom selected from nitrogen, oxygen and sulfur, or 6-membered heteroaryl having one ring heteroatom selected from nitrogen, each of which is optionally substituted with one further substituent selected from halogen, hydroxyl and C ₁ -C ₆ alkyl; or R ² is methyl, ethyl or 2-methylpropyl, each of which is optionally substituted with one further substituent selected from hydroxyl, C ₂ -C ₆ alkenyl or C ₁ -C ₆ alkoxy; R ³ is a heteroaryl selected from isoxazolyl, 1,3,4-oxadiazolyl, 1,2,4-triazolyl, thiazolyl, isothiazolyl and 1,2,4-thiadiazolyl, each of which is optionally substituted with one substituent selected from the group consisting of C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, C _1-2 haloalkylC ₃ cycloalkyl and C _1-2 alkylC _3-4 cycloalkyl; R ⁴ is H; and R ⁵ in each occurrence independently represents halogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ hydroxylalkyl. 2. A compound according to claim 1, wherein R ² is C ₃ -C ₆ cycloalkyl, 4-6-membered heterocyclyl having one ring heteroatom selected from nitrogen, oxygen and sulfur, or 6-membered heteroaryl having one ring heteroatom selected from nitrogen, each of which is optionally substituted with one more substituent selected from halogen, hydroxyl and C ₁ -C ₆ alkyl. 3. A compound according to claim 1 or 2, wherein R ² is C ₃ -C ₄ cycloalkyl or 4-6 membered heterocyclyl having one ring heteroatom selected from nitrogen, oxygen and sulfur, each of which is optionally substituted with another substituent selected from halogen, hydroxyl and C ₁ -C ₆ alkyl. 4. A compound according to any one of claims 1 to 3, wherein R ² is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, each of which is optionally substituted with another substituent selected from halogen, hydroxyl and C ₁ -C ₆ alkyl. 5. A compound according to any one of claims 1 to 3, wherein R ² is oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, azetidinyl, pyrrolidinyl or dioxidotetrahydrothiophenyl, each of which is optionally substituted with a further substituent selected from halogen, hydroxyl and C ₁ -C ₆ alkyl. 6. A compound according to any one of claims 1 to 3, wherein R ² is pyridinyl optionally substituted with another substituent selected from halogen, hydroxyl and C ₁ -C ₆ alkyl. 7. A compound according to any one of claims 1 to 6, wherein R ² is unsubstituted. 8. A compound according to any one of claims 1 to 6, wherein R ² is substituted by one or more of hydroxyl, methyl, methoxy and fluorine. 9. The compound according to claim 1, wherein R ² has one of the following structures: 10. A compound according to claim 1, wherein R ³ is isoxazolyl optionally substituted with one substituent selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, C _{1 -2} haloalkylC ₃ cycloalkyl and C _1-2 alkylC _3-4 cycloalkyl. 11. A compound according to claim 1, wherein R ³ is thiazolyl optionally substituted with one substituent selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, C _1-2 haloalkylC ₃ cycloalkyl and C _1-2 alkylC _3-4 cycloalkyl. 12. A compound according to claim 1, wherein R ³ is isothiazolyl optionally substituted with one substituent selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, C _1-2 haloalkylC ₃ cycloalkyl and C _1-2 alkylC _3-4 cycloalkyl. 13. A compound according to claim 1, wherein R ³ is 1,2,4-thiadiazolyl optionally substituted with one substituent selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, C _1-2 haloalkylC ₃ cycloalkyl and C _1-2 alkylC _3-4 cycloalkyl. 14. A compound according to claim 1, wherein R ³ is 1,3,4-oxadiazolyl optionally substituted with one substituent selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, C _1-2 haloalkylC ₃ cycloalkyl and C _1-2 alkylC _3-4 cycloalkyl. 15. A compound according to claim 1, wherein R ³ is 1,2,4-triazolyl optionally substituted with one substituent selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, C _1-2 haloalkylC ₃ cycloalkyl and C _1-2 alkylC _3-4 cycloalkyl. 16. A compound according to any one of claims 1-15, wherein R ³ is substituted with C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, C _1-2 haloalkylC ₃ cycloalkyl or C _{1-2 alkylC 3-4} cycloalkyl. 17. A compound according to any one of claims 1-16, wherein R ³ has one of the following structures: 18. A compound according to any one of claims 1 to 17, wherein X is N. 19. A compound according to any one of claims 1 to 18, wherein X is CH. 20. The compound according to claim 1, wherein A is phenyl. 21. The compound of claim 1, wherein A is pyridinyl. 22. A compound according to any one of claims 1 to 21, wherein A is unsubstituted. 23. A compound according to any one of claims 1 to 21, wherein A is substituted by one or more R ⁵ . 24. The compound according to claim 23, wherein R ⁵ is a halogen. 25. The compound of claim 24, wherein the halogen is fluorine or chlorine. 26. The compound of claim 23, wherein R ⁵ is C ₁ -C ₆ alkyl. 27. The compound according to claim 26, wherein C ₁ -C ₆ alkyl is methyl. 28. The compound of claim 23, wherein R ⁵ is C ₁ -C ₆ hydroxylalkyl. 29. The compound of claim 28, wherein the C ₁ -C ₆ hydroxylalkyl is -CH ₂ OH. 30. A compound according to any one of paragraphs 1-29, wherein A has one of the following structures: 31. The compound of claim 1, wherein the compound has one of the following structures: or a pharmaceutically acceptable salt thereof. 32. The compound of any one of claims 1-31, wherein the compound is an NLRP3 inflammasome modulator. 33. The compound of any one of claims 1-32, wherein the compound is an NEK7 inhibitor. 34. A pharmaceutical composition for inhibiting NEK7 and/or modulating the activity of the NLRP3 inflammasome, comprising an effective amount of a compound according to any one of claims 1-33 and a pharmaceutically acceptable carrier, diluent or excipient. 35. A method of treating an NLRP3-mediated disorder, comprising administering a therapeutically effective amount of a compound according to any one of claims 1-33 or a pharmaceutical composition according to claim 34 to a subject in need thereof. 36. The method of claim 35, wherein the disorder is selected from autoimmune diseases, inflammatory diseases, cardiovascular diseases, neurodegenerative diseases, allergies, asthma, pancreatitis, multiple organ failure, kidney disease, platelet aggregation, cancer, transplantation, sperm motility, transplant rejection, lung disease, respiratory diseases, and ischemic conditions. 37. The method of claim 35 or 36, wherein the disorder is selected from type II diabetes, atherosclerosis, Alzheimer's disease, fatty liver, asthma, psoriasis, obesity, acute and chronic tissue injury caused by infection, gout, arthritis, macular degeneration, enteritis, hepatitis, peritonitis, silicosis, UV sunburn, contact hypersensitivity, sepsis, cancer, neurodegenerative disease, multiple sclerosis, and Muckle-Wells syndrome.