Heteroaryl compounds as ligand-directed degraders of irak4

Abstract

This document provides compounds (I) and compositions thereof for regulating IRAK4. In some embodiments, said compounds and compositions are provided for treating inflammatory or autoimmune diseases.

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 619,913, filed January 11, 2024, the disclosure of which is incorporated herein by reference in its entirety for any purpose. Technical Field

[0002] This disclosure generally relates to compounds, compositions and methods of their preparation, and the use of said compounds and compositions in the treatment of inflammatory or autoimmune diseases. Background Technology

[0003] The recruitment of immune cells to the site of injury involves the coordinated interactions of numerous soluble mediators. Several cytokines have shown key roles in these processes, including interleukin-1 (IL-1). IL-1 generates a pro-inflammatory response and contributes to the tissue degeneration observed in chronic inflammatory conditions. IL-1 is also involved in processes that regulate bone resorption and adipose tissue. Therefore, IL-1 plays a crucial role in a wide range of pathological conditions, including rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, diabetes, obesity, cancer, and sepsis.

[0004] IL-1 treatment of cells induces the formation of a complex consisting of two IL-1 receptor chains, IL-1R1 and IL-1RAcP. The resulting heterodimer recruits an adaptor molecule called MyD88, which binds to IL-1 receptor-associated kinase (IRAK) (Wesche et al., J. Biol. Chem. 1999, 274, 19403-19410; O'Neill et al., J. Leukoc. Biol. 1998, 63, 650-657; Auron, Cytokine Growth Factor Rev. 1998, 9:221-237; and O'Neill, Biochem. Soc. Trans. 2000, 28, 557-563). Four members of the IRAK family have been identified: IRAK1, IRAK2, IRAK3, and IRAK4. These proteins are characterized by a typical N-terminal death domain and a central kinase domain that mediates interactions with MyD88 family adaptor proteins. Of the four members of the mammalian IRAK family, IRAK-4 is considered the “master IRAK.” IRAK-4 is a serine / threonine kinase that plays a crucial role in signal transduction via the Toll / IL-1 receptor (TIR). Under overexpression conditions, all IRAKs can mediate the activation of the nuclear factor-κB and stress-induced mitogen-activated protein kinase (MAPK) signaling cascade. Studies have shown that IRAK4 kinase activity is essential for cytokine production, MAPK activation, and the induction of NF-κB-regulated genes in response to TLR ligands (Koziczak-Holbro M. et al., J. Biol. Chem. 2007, 282, 13552-13560). Given the central role of IRAK4 in Toll-like / IL-1R signaling and immune protection, compounds that modulate IRAK4 function could be used to treat inflammatory, proliferative, and immune-related conditions, as well as diseases associated with IRAK-mediated signal transduction, such as rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, diabetes, obesity, allergic diseases, psoriasis, asthma, graft rejection, cancer, and sepsis.

[0005] Protein degradation is a highly regulated and crucial process for maintaining cellular homeostasis. The selective identification and removal of damaged, misfolded, or excess proteins are achieved through the ubiquitin-proteasome pathway (UPP). UPP is central to regulating almost all cellular processes. Protein ubiquitination is accomplished by E3 ubiquitin ligases, which bind to proteins and add ubiquitin molecules to them, thereby tagging the proteins for proteasome degradation.

[0006] The use of UPPs for therapeutic purposes has attracted considerable interest (Zhou et al., Mol. Cell 2000, 6, 751-756). One promising therapy utilizes proteolytically targeted chimeras (commonly known as PROTACs) to remove unwanted proteins through protein degradation (Scheepstra et al., Comp. Struct. Biotech. J. 2019, 17, 160-176). PROTACs are ligand-directed degradative agents that bind E3 ligases to the target protein to be degraded. These divalent molecules typically consist of E3 ligase ligands, which are linked to a small molecule of the target protein via a linker portion. PROTACs position the E3 ligase at an appropriate distance and orientation from the target protein, allowing the latter to ubiquitinate. The ubiquitinated target protein is then recognized by the proteasome, where it is degraded.

[0007] Therefore, in one respect, this paper provides compounds that target IRAK4 for degradation. Summary of the Invention

[0008] In some embodiments, compounds and compositions thereof for degrading IRAK4 are described herein. In various embodiments, the compounds and compositions thereof can be used to treat inflammatory or autoimmune diseases.

[0009] The embodiments of the present invention can be more fully understood by referring to specific implementation methods and examples intended to illustrate non-limiting embodiments.

[0010] Implementation scheme 1 is a compound of formula (I): (I) Or its pharmaceutically acceptable salt, wherein: A is or ; R 1 It is H or -NH2; L 1 It is -C(O)N(H)- or ; Ring B is a 5- to 6-membered monocyclic heterocyclic group or an 8- to 10-membered fused bicyclic heterocyclic group, wherein the heterocyclic group contains 1-3 nitrogen atoms; Each R 2 It is independently a C1-C6 alkyl or halogroup; n is 0-3; R 3 It is H, C1-C6 alkyl or halogroup; The carbon ring (C) is attached to two adjacent carbon atoms, indicated by an asterisk (*). ;and X is O, CH2, or N(H).

[0011] Implementation scheme 2 is a compound according to implementation scheme 1 or a pharmaceutically acceptable salt thereof, wherein the compound has the formula (I'): (I').

[0012] Implementation scheme 3 is a compound according to implementation scheme 1 or a pharmaceutically acceptable salt thereof, wherein the compound has formula (II): (II).

[0013] Implementation scheme 4 is a compound according to implementation scheme 1 or a pharmaceutically acceptable salt thereof, wherein the compound has formula (III): (III).

[0014] Implementation scheme 5 is a compound or a pharmaceutically acceptable salt thereof according to any one of implementation schemes 1-3, wherein: R 1 It is H.

[0015] Implementation scheme 6 is a compound or a pharmaceutically acceptable salt thereof according to any one of implementation schemes 1-3, wherein: R 1 It is -NH2.

[0016] Implementation scheme 7 is a compound or a pharmaceutically acceptable salt thereof according to any one of implementation schemes 1-6, wherein: L 1 It is -C(O)N(H)-.

[0017] Implementation scheme 8 is a compound or a pharmaceutically acceptable salt thereof according to any one of implementation schemes 1-6, wherein: L 1 yes .

[0018] Implementation scheme 9 is a compound or a pharmaceutically acceptable salt thereof according to any one of implementation schemes 1-8, wherein: Ring B is a 6-membered monocyclic heterocyclic group or an 8-membered fused bicyclic heterocyclic group, wherein the heterocyclic group contains 1-2 nitrogen atoms; and Each R 2 It is independently a C1-C3 alkyl or halogroup.

[0019] Implementation scheme 10 is a compound or a pharmaceutically acceptable salt thereof according to any one of implementation schemes 1-9, wherein: yes .

[0020] Implementation scheme 11 is a compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-10, wherein: R 3 It is an H or C1-C3 alkyl group.

[0021] Implementation scheme 12 is a compound or a pharmaceutically acceptable salt thereof according to any one of implementation schemes 1-11, wherein: X is O.

[0022] Implementation scheme 13 is a compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-11, wherein: X is CH2 or N(H).

[0023] Implementation scheme 14 is a compound or a pharmaceutically acceptable salt thereof according to any one of implementation schemes 1-13, wherein: yes .

[0024] Embodiment 15 is a compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-14, wherein the compound has the formula (IIA), (IIB), (IIC), (IID), (IIE), (IIF), (IIIA), (IIIB), (IIIC), (IIID), (IIIE), or (IIIF): (IIA) (IIB) (IIC) (IID) (IIE) (IIF) (IIIA) (IIIB) (IIIC) (IIID) (IIIE) (IIIF).

[0025] Implementation scheme 16 is the compound or a pharmaceutically acceptable salt thereof as described in implementation scheme 15, wherein: X is O.

[0026] Implementation scheme 17 is a compound selected from the compounds in Table 1 and its pharmaceutically acceptable salt.

[0027] Embodiment 18 is a pharmaceutical composition comprising a compound according to any one of Embodiments 1-17 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0028] Embodiment 19 is a method for modulating the activity of interleukin-1 (IL1) receptor-associated kinase 4 (IRAK4), the method comprising contacting IRAK4 with an effective amount of a compound according to any one of Embodiments 1-17 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to Embodiment 18.

[0029] Implementation Scheme 20 is a method for treating an inflammatory or autoimmune disease in a subject of need, the method comprising administering to the subject an effective amount of a compound or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes 1-17, or a pharmaceutical composition according to Implementation Scheme 18, wherein optionally the inflammatory or autoimmune disease is atopic dermatitis, asthma, lupus, rheumatoid arthritis, familial Mediterranean fever, psoriasis, generalized pustular psoriasis, cryoinflammatory syndrome, hidradenitis suppurativa, Behçet's syndrome, or familial cold autoinflammatory syndrome. Detailed Implementation definition

[0030] As used herein, the terms “comprising” and “including” are used interchangeably. The terms “comprising” and “including” should be interpreted as specifying the presence of the mentioned features or components, but do not exclude the presence or addition of one or more features or components or groups thereof. Additionally, the terms “comprising” and “including” are intended to include examples covered by the term “composed of”. Therefore, the term “composed of” may be used instead of the terms “comprising” and “including” to provide more specific embodiments of the invention.

[0031] The term "composed of" means that the subject matter has at least 90%, 95%, 97%, 98%, or 99% of the features or components that constitute it. In another embodiment, the term "composed of" excludes any other features or components from the scope of any subsequent description, except those that are not necessary for the technical effect to be achieved.

[0032] As used herein, the term "or" should be interpreted as inclusive "or," meaning any one or any combination. Therefore, "A, B, or C" means any of the following: "A; B; C; A and B; A and C; B and C; A, B, and C." Exceptions to this definition only arise when a combination of elements, functions, steps, or actions is inherently mutually exclusive in some way.

[0033] In this specification, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range shall be understood to include any integer within the listed range and (where appropriate) its fractional value (such as one-tenth and one-hundredth of an integer). Furthermore, unless otherwise stated, any numerical ranges relating to any physical characteristic (such as polymer subunits, size, or thickness) described herein shall be understood to include any integer within the stated range. Unless otherwise stated, as used herein, the terms “about” and “approximately” mean ±20%, ±10%, ±5%, or ±1% of the range, value, or structure indicated.

[0034] "alkyl" groups are straight-chain or branched acyclic hydrocarbons (C1-C1) having 1 to 10 carbon atoms, and are either saturated, partially saturated, or unsaturated. 10Alkyl groups typically have 1 to 8 carbon atoms (C1-C8 alkyl), or in some embodiments, 1 to 6 (C1-C6 alkyl), 1 to 3 (C1-C3 alkyl), or 2 to 6 (C2-C6 alkyl) carbon atoms. In some embodiments, the alkyl group is a saturated alkyl group. Representative saturated alkyl groups include -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and -n-hexyl; while saturated branched alkyl groups include -isopropyl, -sec-butyl, -isobutyl, tert-butyl, -isopentyl, -neopentyl, tert-pentyl, -2-methylpentyl, -3-methylpentyl, -4-methylpentyl, -2,3-dimethylbutyl, etc. In some embodiments, the alkyl group is an unsaturated alkyl group, also referred to as alkenyl or ynyl. "Alkenyl" is an alkyl group containing one or more carbon-carbon double bonds. "Yynyl" is an alkyl group containing one or more carbon-carbon triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, allyl, CH=CH(CH3), -CH=C(CH3)2, -C(CH3)=CH2, -C(CH3)=CH(CH3), -C(CH2CH3)=CH2, -C≡CH, -C≡C(CH3), -C≡C(CH2CH3), -CH2C≡CH, CH2C≡C(CH3), and CH2C≡C(CH2CH3), etc. Alkyl groups can be substituted or unsubstituted. When alkyl groups are referred to herein as “substituted,” they can be substituted by any one or more substituents, such as those found in the exemplary compounds and embodiments disclosed herein, as well as halogens; hydroxyl groups; alkoxy groups; cycloalkoxy groups, aryloxy groups, heterocyclic alkoxy groups, heteroaryloxy groups, heterocyclic alkoxy groups, cycloalkylalkoxy groups, arylalkoxy groups, heterocyclic alkoxy groups, heteroarylalkoxy groups, heterocyclic alkylalkoxy groups; oxo groups (=O); amino groups, alkylamino groups, cycloalkylamino groups, arylamino groups, heterocyclic amino groups, heteroarylamino groups, heterocyclic alkylamino groups, cycloalkylalkylamino groups. alkyl, aralkylamino, heterocyclic alkylamino, heterocyclic alkylamino, heterocyclic alkylalkylamino; imino; imino; amido; guanidin; enamino; acylamino; sulfonylamino; urea, nitrourea; oxime; hydroxyamino; alkoxyamino; aralkyloxyamino; hydrazine; acylhydrazine; hydrazone; azide; nitro; thio (-SH), alkylthio; =S; sulfinyl; sulfonyl; aminosulfonyl; phosphonate; phosphonyl; acyl; formyl; carboxyl; ester; carbamate; amide; cyano; isocyanate; isothiocyanate; cyanate; thiocyanate; or -B(OH)2.In some embodiments, when the alkyl groups described herein are referred to as “substituted,” they may be substituted by any one or more substituents such as those found in the exemplary compounds and embodiments disclosed herein, as well as halogens (chlorine, iodine, bromine, or fluorine); alkyl; hydroxyl; alkoxy; alkoxyalkyl; amino; alkylamino; carboxyl; nitro; cyano; thiol; thioether; imine; imide; amidine; guanidine; enamine; aminocarbonyl; acylamino; phosphonate; phosphine; thiocarbonyl; sulfinyl; sulfonamide; ketone; aldehyde; ester; urea; urethane; oxime; hydroxylamine; alkoxyamine; arylalkoxyamine; N-oxide; hydrazine; acylhydrazine; hydrazone; azide; isocyanate; isothiocyanate; cyanate; thiocyanate; B(OH)2 or O(alkyl)aminocarbonyl.

[0035] "alkyl-OH" refers to an unbranched or branched alkyl group as defined above, wherein one or more hydrogen atoms are replaced by -OH. For example, "C1-C6 alkyl-OH" refers to a C1-C6 alkyl group substituted with one or more -OH groups. Alkyl-OH may contain multiple hydroxyl groups attached to the same or multiple carbon atoms.

[0036] "alkyl-CN" refers to an unbranched or branched alkyl group as defined above, wherein one or more hydrogen atoms are replaced by -CN. For example, "C1-C6 alkyl-CN" refers to a C1-C6 alkyl group substituted with one or more -CN groups. Alkyl-CN may contain multiple cyano groups attached to the same or multiple carbon atoms.

[0037] "Alkoxy" is -O-(alkyl), where alkyl is as defined above.

[0038] "Cycloalkyl" refers to saturated or partially saturated cycloalkyl groups having 3 to 10 carbon atoms (C3-C4). 10The cycloalkyl group has a single cyclic ring or multiple fused or bridged rings that may be optionally substituted. In some embodiments, the cycloalkyl group has 3 to 8 cyclic carbon atoms (C3-C8 cycloalkyl), while in other embodiments, the number of cyclic carbon atoms is 3 to 5 (C3-C5 cycloalkyl), 3 to 6 (C3-C6 cycloalkyl), or 3-7 (C3-C7 cycloalkyl). In some embodiments, the cycloalkyl group is a saturated cycloalkyl group. Such saturated cycloalkyl groups include, for example, monocyclic structures (such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 1-methylcyclopropyl, 2-methylcyclopentyl, 2-methylcyclooctyl, etc.), or polycyclic or bridged ring structures (such as 1-bicyclo[1.1.1]pentyl, bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, adamantyl, etc.). In other embodiments, the cycloalkyl group is an unsaturated cycloalkyl group. Examples of unsaturated cycloalkyl groups include cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, hexadienyl, etc. Cycloalkyl groups can be substituted or unsubstituted. Such substituted cycloalkyl groups include, for example, cyclohexanol.

[0039] "Aryl" is an aromatic carbocyclic group (C6-C14) having 6 to 14 carbon atoms, consisting of a monocyclic ring (e.g., phenyl) or multiple fused rings (e.g., naphthyl or anthracene). 14 Aryl group). In some embodiments, the aryl group contains 6-14 carbons (C6-C5) in the ring moiety of the group. 14 aryl), and in other cases 6 to 12 (C6-C) 12 Aryl groups or even 6 to 10 carbon atoms (C6-C) 10 Aryl). Specific aryl groups include phenyl, biphenyl, naphthyl, etc. Aryl groups can be substituted or unsubstituted. The phrase "aryl" also includes groups containing fused rings, such as fused aromatic-aliphatic ring systems (e.g., indanyl, tetrahydronaphthyl, etc.).

[0040] "Halogen" or "halogenated" refers to fluorine, chlorine, bromine, or iodine.

[0041] "Haloalkyl" refers to an alkyl group as defined above that is substituted with one or more halogenated groups as defined above, such as trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl, etc. In some embodiments, the haloalkyl group has one to six carbon atoms and is substituted with one or more halogenated groups (C1-C6 haloalkyl), or the haloalkyl group has one to three carbon atoms and is substituted with one or more halogenated groups (C1-C3 haloalkyl). The halogenated groups may be all the same or different. Unless otherwise specified, the haloalkyl group is optionally substituted.

[0042] A "heteroaryl" is an aromatic ring system having one to four heteroatoms as ring atoms, wherein the remaining atoms are carbon atoms. In some embodiments, the heteroaryl contains 3 to 6 ring atoms in the ring portion of the group, and in others it contains 6 to 9 or even 6 to 10 atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen. In some embodiments, the heteroaryl ring system is monocyclic or bicyclic. Non-limiting examples include, but are not limited to, groups such as: pyrrolo, pyrazol, imidazo, triazol, tetrazol, oxazol, isoxazol, benzo[d]isooxazol (e.g., benzo[d]isooxazol), thiazo, pyrrolo, pyridazin, pyrimidin, pyrazin, thienyl, benzo[thienyl]thio, furanyl, benzo[thio]furanyl, indole (e.g., indole-2-one or isoindoline-1-one), azaindole (pyrrolopyridinyl or 1Hpyrrolo[2,3b]pyridinyl), inzol, benzo[imidazo] (e.g., 1H benzo[d]imidazo), imidazopyridinyl ( For example, azirmoniazole (or 1H imidazo[4,5b]pyridyl), pyrazolopyridyl, triazolopyridyl, benzotriazolyl (e.g., 1H benzo[d][1,2,3]triazolyl), benzooxazolyl (e.g., benzo[d]oxazolyl), benzothiazolyl, benzothiadiazolyl, isoxazolopyridyl, benzothiophenyl, purine, xanthine, adenine, guanine, quinolinyl, isoquinolinyl (e.g., 3,4-dihydroisoquinolin-1(2H)-keto), tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl. The heteroaryl group can be substituted or unsubstituted.

[0043] A "heterocyclic group" is a non-aromatic cycloalkyl group in which one to four ring carbon atoms are independently substituted by heteroatoms selected from O, S, and N. In some embodiments, the heterocyclic group comprises 3 to 10 ring members, while other such groups have 3 to 5, 3 to 6, or 3 to 8 ring members. The heterocyclic group may also be bonded to other groups at any ring atom (i.e., any carbon atom or heteroatom in the heterocycle). The heterocyclic alkyl group may be substituted or unsubstituted. The term heterocyclic group encompasses saturated and partially saturated ring systems. Furthermore, the term heterocyclic group is intended to cover any non-aromatic ring containing at least one heteroatom, which may be fused to an aryl or heteroaryl ring regardless of its attachment to the rest of the molecule. The phrase also includes bridging polycyclic ring systems containing heteroatoms. Representative examples of heterocyclic groups include, but are not limited to, aziridinyl, aziridine, aziridine-heptyl, pyrrolidinyl, imidazolyl (e.g., imidazoline-4-one or imidazoline-2,4-diketoyl), pyrazolyl, thiazolyl, tetrahydrothiopheneyl, tetrahydrofuranyl, piperidinyl, piperazinyl (e.g., piperazin-2-one), morpholinyl, thiomorpholinyl, tetrahydropyranyl (e.g., tetrahydro-2H-pyranyl), tetrahydrothiopyranyl, oxathiayl, dithiayl, 1,4-dioxaspiro[4.5]decyl, homopiperazinyl, quininecycloyl, or tetrahydropyrimidin-2(1H)-one. Representative substituted heterocyclic groups can be monosubstituted or substituted more than once, such as, but not limited to, pyridinyl or morpholinyl, which are 2, 3, 4, 5, or 6 times substituted, or secondary substituted by various substituents (those listed below).

[0044] When groups (other than alkyl) described herein are referred to as “substituted,” they may be substituted by any suitable one or more substituents. Illustrative examples of substituents are those found in exemplary compounds and embodiments, as well as halogens (chlorine, iodine, bromine, or fluorine); alkyl; hydroxyl; alkoxy; alkoxyalkyl; amino; alkylamino; carboxyl; nitro; cyano; thiol; thioether; imine; imide; amidine; guanidine; enamine; aminocarbonyl; amide; phosphonate; phosphine; thiocarbonyl; sulfinyl; sulfonyl; sulfonamide; ketone; aldehyde; ester; urea; urethane; oxime; hydroxylamine; alkoxyamine; arylalkoxyamine; N-oxide; hydrazine; acylhydrazine; hydrazone; azide; isocyanate; isothiocyanate; cyanate; thiocyanate; oxygen (=O); B(OH)2, O(alkyl)aminocarbonyl; cycloalkyl, which may be monocyclic or Fused or unfused polycyclic (e.g., cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl) or heterocyclic groups, which may be monocyclic or fused or unfused polycyclic (e.g., pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, or thiazinyl); monocyclic or fused or unfused polycyclic aryl or heteroaryl (e.g., phenyl, naphthyl, pyrrolidinyl, indolyl, furanyl, thiopheneyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, triazolyl, tetrazolyl, pyrazolyl, pyridinyl, quinolinyl, isoquinolinyl, acridineyl, pyrazinyl, pyridazinyl, pyrimidinyl, benzimidazolyl, benzothiopheneyl, or benzofuranyl) aryloxy; arylalkoxy; heterocyclic oxy; and heterocyclic alkoxy.

[0045] Some commonly used alternative chemical names can be used. For example, divalent groups such as divalent "alkyl", divalent "phenyl", divalent "heteroaryl", and divalent "heterocyclic" can also be called "alkylene", "phenylene", "heteroarylene", or "heterocyclic", respectively.

[0046] The embodiments of this disclosure are intended to cover pharmaceutically acceptable salts, tautomers, isotopes and stereoisomers of the compounds of formula (I) provided herein.

[0047] As used herein, the term "pharmaceutically acceptable salt" refers to a salt prepared from a pharmaceutically acceptable, non-toxic acid or base (including inorganic acids and bases as well as organic acids and bases). Suitable pharmaceutically acceptable base addition salts of compounds of formula (I) include, but are not limited to, metal salts made from aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc, or organic salts made from lysine, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucosamine), and procaine. Suitable non-toxic acids include, but are not limited to, inorganic and organic acids such as acetic acid, alginic acid, aminoanilic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, vinylsulfonic acid, formic acid, fumaric acid, furoic acid, galacturonic acid, gluconic acid, glucuronic acid, glutamic acid, glycolic acid, hydrobromic acid, hydrochloric acid, hydroxyethanesulfonic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, viscous acid, nitric acid, dihydroxynaphthyl acid, pantothenic acid, phenylacetic acid, phosphoric acid, propionic acid, salicylic acid, stearic acid, succinic acid, p-aminobenzenesulfonic acid, sulfuric acid, tartaric acid, and p-toluenesulfonic acid. Specific non-toxic acids include hydrochloric acid, hydrobromic acid, maleic acid, phosphoric acid, sulfuric acid, and methanesulfonic acid. Therefore, specific examples of salts include hydrochloride, formate, and methanesulfonate. Others are well known in the field; see, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing, Easton PA (1990) or Remington: The Science and Practice of Pharmacy, 19th edition, Mack Publishing, Easton PA (1995).

[0048] As used herein and unless otherwise stated, the terms "stereoisomer" or "stereoisomer-pure" mean a stereoisomer of a particular compound that substantially does not contain other stereoisomers of that compound. For example, a stereoisomer-pure compound having one chiral center will substantially not contain the opposite enantiomers of the compound. A stereoisomer-pure compound having two chiral centers will substantially not contain other diastereomers of the compound. Typical stereoisomer-pure compounds comprise, by weight, more than about 80% of one stereoisomer of the compound and less than about 20% of other stereoisomers of the compound, more than about 90% of one stereoisomer of the compound and less than about 10% of other stereoisomers of the compound, more than about 95% of one stereoisomer of the compound and less than about 5% of other stereoisomers of the compound, or more than about 97% of one stereoisomer of the non-compound and less than about 3% of other stereoisomers of the compound. The compounds disclosed herein may have a chiral center and may exist as racemates, individual enantiomers or diastereomers, and mixtures thereof. All such isomeric forms are included in the embodiments disclosed herein, including mixtures thereof.

[0049] The embodiments disclosed herein cover the use of the compounds disclosed herein in their pure stereoisomer forms as well as the use of mixtures of those forms. For example, mixtures comprising equal or unequal amounts of enantiomers of a particular compound can be used in the methods and compositions disclosed herein. These isomers can be asymmetrically synthesized or resolved using standard techniques such as chiral columns or chiral resolving agents. See, e.g., Jacques, J., et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen, SH, et al., Tetrahedron 33:2725 (1977); Eliel, EL, Stereochemistry of Carbon Compounds (McGrawHill, NY, 1962); Wilen, SH, Tables of Resolving Agents and OpticalResolutions page 268 (edited by EL Eliel, Univ. of Notre Dame Press, Notre Dame, IN, 1972); Todd, M., Separation Of Enantiomers : Synthetic Methods (Wiley-VCHVerlag GmbH & Co. KGaA, Weinheim, Germany, 2014); Toda, F., EnantiomerSeparation: Fundamentals and Practical Methods (Springer Science & BusinessMedia, 2007); Subramanian, G. Chiral Separation Techniques: A PracticalApproach (John Wiley & Sons, 2008); Ahuja, S., Chiral Separation Methods for Pharmaceutical and Biotechnological Products (John Wiley & Sons, 2011).

[0050] It should also be noted that the compounds disclosed herein may include E and Z isomers or mixtures thereof, as well as cis and trans isomers or mixtures thereof. In some embodiments, the compound is isolated as an E or Z isomer. In other embodiments, the compound is a mixture of E and Z isomers.

[0051] "Tautomers" refer to compounds that exist in equilibrium as isomers. The concentration of the isomers will depend on the environment in which the compound is found and can vary depending on, for example, whether the compound is a solid or in an organic or aqueous solution. For example, in aqueous solution, pyrazole can exhibit the following isomers, which are referred to as tautomers of each other: .

[0052] As will be readily understood by those skilled in the art, a variety of functional groups and other structures can exhibit tautomerism, and all tautomers of the compounds of formula (I) provided herein are within the scope of this disclosure.

[0053] It should also be noted that the compounds disclosed herein may contain atomic isotopes in non-natural proportions at one or more atoms. For example, the compounds may contain radioactive isotopes (such as tritium). 3 H), Iodine-125 ( 125 I), sulfur 35 ( 35 S) or carbon-14 ( 14 C) radioactive labeling, or it can be done using isotopes (such as deuterium). 2 H), carbon-13 ( 13 C) or nitrogen-15 ( 15 Enriched with N). As used herein, an "isotope" is an isotopically enriched compound. The term "isotopically enriched" means an atom whose isotopic composition differs from that of its natural isotopic composition. "Isotopically enriched" can also refer to a compound containing at least one atom whose isotopic composition differs from that of its natural isotopic composition. The term "isotopic composition" refers to the amount of each isotope present for a given atom. Radiolabeled and isotopically enriched compounds can be used as therapeutic agents (e.g., cancer therapeutic agents), research reagents (e.g., binding assay reagents), and diagnostic agents (e.g., in vivo imaging agents). All isotopic variations of the compounds described herein, whether or not radioactive, are intended to be covered within the scope of the embodiments provided herein. In some embodiments, isotopes of the compounds disclosed herein are provided, for example, isotopes enriched with deuterium, carbon-13, and / or nitrogen-15. As used herein, "deuterated" means that at least one hydrogen (H) has been deuterated (using D or D). 2 H represents a substitute compound, that is, a compound enriched in deuterium at at least one position.

[0054] It should be understood that, independent of stereoisomer or isotopic composition, each compound disclosed herein may be provided in the form of any pharmaceutically acceptable salt discussed herein. Similarly, it should be understood that the isotopic composition may vary independently of the stereoisomer composition of each compound mentioned herein. Furthermore, while the isotopic composition is limited to those elements present in the respective compounds or their salts disclosed herein, it may also vary independently of the choice of pharmaceutically acceptable salts of the respective compounds.

[0055] It should be noted that if there is a difference between the described structure and the name of the structure, the described structure will be given more weight.

[0056] As used herein, “treatment” means the complete or partial relief of a disorder, disease, or condition, or one or more symptoms associated with a disorder, disease, or condition, or the slowing or halting of the further progression or worsening of those symptoms, or the relief or eradication of one or more causes of the disorder, disease, or condition itself. In one implementation, the disorder is a neurodegenerative disease or its symptoms as described herein.

[0057] As used herein, “prevention” means the complete or partial delay and / or elimination of the onset, recurrence, or spread of an impairment, disease, or condition; preventing a subject from acquiring an impairment, disease, or condition; or a method for reducing the risk of a subject acquiring an impairment, disease, or condition. In one implementation, an impairment is a neurodegenerative disease or its symptoms as described herein.

[0058] The term “effective amount” in relation to the compounds disclosed herein means an amount that is sufficient to treat or prevent the disorder, disease, or ailment or its symptoms disclosed herein.

[0059] As used herein, the terms “subject” or “patient” include animals, including but not limited to animals such as cattle, monkeys, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs, and in one embodiment includes mammals, and in another embodiment includes humans. In one embodiment, the subject is a person who has or is at risk of having an IRAK4-mediated disease or symptoms thereof.

[0060] While various features of the invention can be described in the context of a single embodiment, these features may also be provided individually or in any suitable combination. Conversely, although the invention may be described herein in the context of separate embodiments for clarity, the invention may also be implemented in a single embodiment. compound

[0061] In one respect, this paper provides a compound of formula (I): (I) Or its pharmaceutically acceptable salt, wherein: A is or ; R 1 It is H or -NH2; L 1 It is -C(O)N(H)- or ; Ring B is a 5- to 6-membered monocyclic heterocyclic group or an 8- to 10-membered fused bicyclic heterocyclic group, wherein the heterocyclic group contains 1-3 nitrogen atoms; Each R 2 It is independently a C1-C6 alkyl or halogroup; n is 0-3; R 3 It is H, C1-C6 alkyl or halogroup; The carbon ring (C) is attached to two adjacent carbon atoms, indicated by an asterisk (*). ;and X is O, CH2, or N(H).

[0062] In some implementation schemes, A is or In some implementations, A is... In some implementations, A is... .

[0063] In some implementation schemes, R 1 It is H or -NH2. In some implementations, R 1 It is H. In some implementations, R 1 It is -NH2.

[0064] In some implementations, L 1 It is -C(O)N(H)- or In some implementations, L 1 It is -C(O)N(H)-. In some implementations, L 1 yes .

[0065] In some embodiments, ring B is a 5- to 6-membered monocyclic heterocyclic group or an 8- to 10-membered fused bicyclic heterocyclic group, wherein the heterocyclic group contains 1-3 nitrogen atoms. In some embodiments, ring B is a 6-membered monocyclic heterocyclic group or an 8-membered fused bicyclic heterocyclic group, wherein the heterocyclic group contains 1-2 nitrogen atoms.

[0066] In some embodiments, ring B is a 5- to 6-membered monocyclic heterocyclic group containing 1-3 nitrogen atoms. In some embodiments, ring B is a 5-membered monocyclic heterocyclic group containing 1-3 nitrogen atoms. In some embodiments, ring B is a 5-membered monocyclic heterocyclic group containing 1-2 nitrogen atoms. In some embodiments, ring B is a 5-membered monocyclic heterocyclic group containing one nitrogen atom. In some embodiments, ring B is a 5-membered monocyclic heterocyclic group containing two nitrogen atoms. In some embodiments, ring B is a 6-membered monocyclic heterocyclic group containing 1-3 nitrogen atoms. In some embodiments, ring B is a 6-membered monocyclic heterocyclic group containing 1-2 nitrogen atoms. In some embodiments, ring B is a 6-membered monocyclic heterocyclic group containing one nitrogen atom. In some embodiments, ring B is a 6-membered monocyclic heterocyclic group containing two nitrogen atoms. In some embodiments, ring B is a pyridinealkyl, piperidinyl, or piperazineyl group.

[0067] In some embodiments, ring B is an 8- to 10-membered fused bicyclic heterocyclic group containing 1-3 nitrogen atoms. In some embodiments, ring B is an 8- to 10-membered fused bicyclic heterocyclic group containing 1-2 nitrogen atoms. In some embodiments, ring B is an 8- to 10-membered fused bicyclic heterocyclic group containing one nitrogen atom. In some embodiments, ring B is an 8- to 10-membered fused bicyclic heterocyclic group containing two nitrogen atoms. In some embodiments, ring B is an 8-membered fused bicyclic heterocyclic group containing 1-2 nitrogen atoms. In some embodiments, ring B is an 8-membered fused bicyclic heterocyclic group containing one nitrogen atom. In some embodiments, ring B is an 8-membered fused bicyclic heterocyclic group containing two nitrogen atoms. In some embodiments, ring B is a 9-membered fused bicyclic heterocyclic group containing 1-2 nitrogen atoms. In some embodiments, ring B is a 9-membered fused bicyclic heterocyclic group containing one nitrogen atom. In some embodiments, ring B is a 9-membered fused bicyclic heterocyclic group containing 2 nitrogen atoms. In some embodiments, ring B is a 10-membered fused bicyclic heterocyclic group containing 1-2 nitrogen atoms. In some embodiments, ring B is a 10-membered fused bicyclic heterocyclic group containing one nitrogen atom. In some embodiments, ring B is a 10-membered fused bicyclic heterocyclic group containing 2 nitrogen atoms.

[0068] In some implementation schemes, ring B is .

[0069] In some implementations, each R 2 Independently, it is a C1-C6 alkyl or halogroup. In some embodiments, each R... 2 Independently, it is a C1-C3 alkyl or halogroup. In some embodiments, each R... 2 It is -CH3 or -CH2CH3 independently.

[0070] In some implementation schemes, R2 It is a C1-C6 alkyl group. In some embodiments, R 2 It is a C1-C3 alkyl group. In some embodiments, R 2 It is methyl, ethyl, n-propyl, or isopropyl. In some embodiments, R 2 It is methyl. In some embodiments, R 2 It is ethyl. In some embodiments, R 2 It is n-propyl. In some implementations, R 2 It is isopropyl.

[0071] In some implementation schemes, R 2 It is a halogenated group. In some implementations, R 2 It is Cl, F, or Br. In some implementations, R 2 It is Cl. In some implementations, R 2 It is F. In some implementations, R 2 It is Br.

[0072] In some implementations, n is 0-3. In some implementations, n is 0 or 1. In some implementations, n is 0. In some implementations, n is 1. In some implementations, n is 2. In some implementations, n is 3.

[0073] In some implementation schemes, yes: .

[0074] In some implementation schemes, R 3 It is H, C1-C6 alkyl, or halogroup. In some embodiments, R 3 It is an H or C1-C3 alkyl group. In some embodiments, R 3 It is H or -CH3.

[0075] In some implementation schemes, R 3 It is H.

[0076] In some implementation schemes, R 3 It is a C1-C6 alkyl group. In some embodiments, R 3 It is a C1-C3 alkyl group. In some embodiments, R 3 It is methyl, ethyl, n-propyl, or isopropyl. In some embodiments, R 3 It is methyl. In some embodiments, R 3 It is ethyl. In some embodiments, R 3 It is n-propyl. In some implementations, R 3 It is isopropyl.

[0077] In some implementation schemes, R3 It is a halogenated group. In some implementations, R 3 It is Cl, F, or Br. In some implementations, R 3 It is Cl. In some implementations, R 3 It is F. In some implementations, R 3 It is Br.

[0078] In some implementations, the ring C is attached to two adjacent carbon atoms indicated by *. And X is O, CH2, or N(H). In some embodiments, X is O. In some embodiments, X is CH2 or N(H). In some embodiments, X is CH2. In some embodiments, X is N(H).

[0079] In some implementation schemes, yes: .

[0080] In some embodiments, the compound of formula (I) is a compound of formula (I'): (I') Among them, A and L 1 Ring B, Ring C, R 2 R 3 And n is as described for equation (I).

[0081] In some embodiments, the compound of formula (I) is the same as the compound of formula (II): (II) Where R 1 L 1 Ring B, Ring C, R 2 R 3 And n is as described for equation (I).

[0082] In some embodiments, the compound of formula (I) is a compound of formula (IIA), (IIB), (IIC), (IID), (IIE), or (IIF): (IIA) (IIB) (IIC) (IID) (IIE) (IIF) Where R 1 R 2 R 3 X and n are as described for equation (I). In some variations, X is O. In some variations, X is CH2. In some variations, X is NH.

[0083] In some embodiments, the compound of formula (I) is a compound of formula (IIA-1), (IIB-1), (IIC-1), (IID-1), (IIE-1), or (IIF-1): (IIA-1) (IIB-1) (IIC-1) (IID-1) (IIE-1) (IIF-1) Where R 1 R 2 R 3 And n is as described for equation (I).

[0084] In some embodiments, the compound of formula (I) is a compound of formula (III): (III) Where L 1 Ring B, Ring C, R 2 R 3 And n is as described for equation (I).

[0085] In some embodiments, the compound of formula (I) is a compound of formula (IIIA), (IIIB), (IIIC), (IIID), (IIIE), or (IIIF): (IIIA) (IIIB) (IIIC) (IIID) (IIIE) (IIIF) Where R 2 R 3 X and n are as described for equation (I). In some variations, X is O. In some variations, X is CH2. In some variations, X is NH.

[0086] In some embodiments, the compound of formula (I) is a compound of formula (IIIA-1), (IIIB-1), (IIIC-1), (IIID-1), (IIIE-1), or (IIIF-1): (IIIA-1) (IIIB-1) (IIIC-1) (IIID-1) (IIIE-1) (IIIF-1) Where R 2 R 3 And n is as described for equation (I).

[0087] In the description herein, it should be understood that each description, variation, embodiment, or aspect of a part may be combined with each description, variation, embodiment, or aspect of other parts, as specifically and separately as each and every combination of descriptions is listed. For example, each description, variation, embodiment, or aspect of A with respect to formula (I) provided herein may be combined with R 1 R 2 R 3 , n, L 1Each description, variation, implementation, or aspect combination of rings B, C, and X is listed as if each and every combination were specifically and separately. It should also be understood that, where applicable, all descriptions, variations, implementations, or aspects of equation (I) are equally applicable to and described as if each and every description, variation, implementation, or aspect were listed separately and separately for all equations. For example, where applicable, all descriptions, variations, embodiments, or aspects of formula (I) are equally applicable to any formula detailed herein, such as formulas (I'), (II), (IIA), (IIB), (IIC), (IID), (IIE), (IIF), (IIA-1), (IIB-1), (IIC-1), (IID-1), (IIE-1), (IIF-1), (III), (IIIA), (IIIB), (IIIC), (IIID), (IIIE), (IIIF), (IIIA-1), (IIIB-1), (IIIC-1), (IIID-1), (IIIE-1), and (IIIF-1), and are described equally as if each and every description, variation, embodiment, or aspect were listed separately and individually for all formulas.

[0088] In some embodiments, compounds selected from those in Table 1 or pharmaceutically acceptable salts thereof are provided. Although some compounds described in this disclosure (including Table 1) are presented in specific stereoisomers and / or non-stereochemical forms, it should be understood that any or all stereochemical forms (including any enantiomers or diastereomers) and any tautomers or other forms of any compound described herein (including Table 1) are described. Table 1. "or1" and "or2" indicate that the absolute stereochemistry is not determined, and the stereochemistry may be as drawn or the opposite of the drawn stereochemistry.

[0089] It should be understood that, in this specification, the combination of substituents and / or variables in the formulas described is permitted only if such contributions produce stable compounds.

[0090] Furthermore, all compounds of formula (I) existing in free base or acid form can be converted into their pharmaceutically acceptable salts by treatment with a suitable inorganic or organic base or acid, using methods known to those skilled in the art. Salts of compounds of formula (I) can be converted into their free base or acid form using standard techniques. Synthesis method

[0091] The compounds described herein can be prepared using conventional organic synthesis and commercially available starting materials or the methods provided herein. By way of example and not limitation, compounds of formula (I) can be prepared as outlined in Schemes 1 and 2 and in the examples set forth herein. It should be noted that those skilled in the art will know how to modify the procedures set forth in the illustrative schemes and examples to obtain the desired products.

[0092] The target-binding moiety (TBM) can be prepared as outlined in Scheme 1. In one synthesis, a copper-catalyzed cycloaddition of a and b to an azide alkyne provides intermediate c, which can be deprotected using, for example, an acid such as p-toluenesulfonic acid to form d and subsequently oxidized (e.g., using IBX) to form TBMe. In a second synthesis, intermediates f and g are coupled using, for example, HATU to provide h, which is then oxidized (e.g., using IBX) to give TBMe. Option 1. Where A is as described for formula (I); and Pg is a protecting group, such as tetrahydropyran (THP).

[0093] TBM A (such as e and i from scheme 1) can be coupled to the cerebellar protein binding moiety (CBM) B under reductive amination conditions to provide a compound of formula (I) as shown in scheme 2. Option 2. Among them, A and L 1 Ring B, R 2 R 3 The rings C and n are as described for equation (I). How to use

[0094] Embodiments of this disclosure provide a method for modulating IRAK4 in a subject in need, the method comprising administering an effective amount of a compound of formula (I) to the subject. Modulation of IRAK4 (e.g., inhibition or activation) can be assessed and demonstrated by a variety of methods known in the art. Kits and commercially available assays can be used to determine whether IRAK4 is modulated (e.g., inhibited or activated) and to what extent.

[0095] In one aspect, this document provides a method for modulating IRAK4, the method comprising contacting IRAK4 with an effective amount of a compound of formula (I) or any embodiment or variant thereof. In some embodiments, the compound of formula (I) inhibits IRAK4. In other embodiments, the compound of formula (I) activates IRAK4. In some embodiments, the compound of formula (I) is an agonist of IRAK4. In some embodiments, the compound of formula (I) is an antagonist of IRAK4.

[0096] In some embodiments, this document provides a method for targeting IRAK4 for degradation, the method comprising contacting IRAK4 with an effective amount of a compound of formula (I) or any embodiment or variant thereof.

[0097] In some embodiments, the compound of formula (I) adjusts the activity of IRAK4 to about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the compound of formula (I) adjusts the activity of IRAK4 to about 1%-100%, 5%-100%, 10%-100%, 15%-100%, 20%-100%, 25%-100%, 30%-100%, 35%-100%, 40%-100%, 45%-100%, 50%-100%, 55%-100%, 60%-100%, 65%-100%, 70%-100%, 75%-100%, 80%-100%, or 85%-100%. 90%-100%, 95%-100%, 5%-95%, 5%-90%, 5%-85%, 5%-80%, 5%-75%, 5%-70%, 5%-65%, 5%-60%, 5%-55%, 5%-50%, 5%-45%, 5%-40%, 5%-35%, 5%-30%, 5%-25%, 5%-20%, 5%-15%, 5%-10%, 10%-90%, 20%-80%, 30%-70%, or 40%-60%.

[0098] In some embodiments of this disclosure, a method for degrading IRAK4 in a subject in need is also provided, the method comprising administering an effective amount of a compound of formula (I) to the subject. The degradation of IRAK4 can be assessed and demonstrated by a variety of methods known in the art. Kits and commercially available assays (including cell-based assays) can be used to determine whether IRAK4 has been degraded and to what extent.

[0099] In one aspect, this document provides a method for degrading IRAK4, the method comprising contacting IRAK4 with an effective amount of a compound of formula (I) or any embodiment or variant thereof. In some embodiments, the compound of formula (I) partially degrades IRAK3. In some embodiments, the compound of formula (I) completely degrades IRAK4.

[0100] In some embodiments, the compound of formula (I) degrades IRAK4 by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the compound of formula (I) degrades IRAK4 by about 1%-100%, 5%-100%, 10%-100%, 15%-100%, 20%-100%, 25%-100%, 30%-100%, 35%-100%, 40%-100%, 45%-100%, 50%-100%, 55%-100%, 60%-100%, 65%-100%, 70%-100%, 75%-100%, 80%-100%, 85%-100%, 90%, 95%, or 100%. 0%-100%, 95%-100%, 5%-95%, 5%-90%, 5%-85%, 5%-80%, 5%-75%, 5%-70%, 5%-65%, 5%-60%, 5%-55%, 5%-50%, 5%-45%, 5%-40%, 5%-35%, 5%-30%, 5%-25%, 5%-20%, 5%-15%, 5%-10%, 10%-90%, 20%-80%, 30%-70%, or 40%-60%.

[0101] In another aspect, this document provides a method for treating an inflammatory or autoimmune disease in a subject of need, the method comprising administering to the subject an effective amount of a compound of formula (I). In some embodiments, this document provides a method for treating an inflammatory disease in a subject of need, the method comprising administering to the subject an effective amount of a compound of formula (I). In some embodiments, this document provides a method for treating an autoimmune disease in a subject of need, the method comprising administering to the subject an effective amount of a compound of formula (I). In some embodiments, this document provides a method for preventing an inflammatory or autoimmune disease in a subject of need, the method comprising administering to the subject an effective amount of a compound of formula (I). In some embodiments, this document provides a method for preventing an inflammatory disease in a subject of need, the method comprising administering to the subject an effective amount of a compound of formula (I). In some embodiments, this document provides a method for preventing an autoimmune disease in a subject of need, the method comprising administering to the subject an effective amount of a compound of formula (I). Non-limiting examples of inflammatory or autoimmune diseases include atopic dermatitis, asthma, lupus, rheumatoid arthritis, familial Mediterranean fever, psoriasis, generalized pustular psoriasis, cryoinflammatory syndrome, hidradenitis suppurativa, Behçet's syndrome, or familial cold autoinflammatory syndrome.

[0102] In some embodiments, the compound of formula (I) is administered to a subject susceptible to inflammatory or autoimmune diseases to prevent the subject from developing any symptoms of the inflammatory or autoimmune disease. In some embodiments, the compound of formula (I) is administered to a subject who has not yet exhibited symptoms of inflammatory or autoimmune diseases to prevent the subject from developing any symptoms of the inflammatory or autoimmune disease. In some embodiments, the compound of formula (I) is administered to a subject in need to reduce the severity of the inflammatory or autoimmune disease in the subject. In some embodiments, the compound of formula (I) is administered to a subject in need to stabilize the inflammatory or autoimmune disease (preventing or delaying the worsening of the inflammatory or autoimmune disease). In some embodiments, the compound of formula (I) is administered to a subject in need to delay the onset or recurrence of the inflammatory or autoimmune disease. In some embodiments, the compound of formula (I) is administered to a subject in need to slow the progression of the inflammatory or autoimmune disease. In some embodiments, the compound of formula (I) is administered to a subject in need to provide partial relief of the inflammatory or autoimmune disease. In some embodiments, administration of the compound of formula (I) to a subject in need provides complete remission of an inflammatory or autoimmune disease. In some embodiments, administration of the compound of formula (I) to a subject in need reduces the dosage of one or more other drugs required to treat an inflammatory or autoimmune disease. In some embodiments, administration of the compound of formula (I) to a subject in need enhances the effect of another drug used to treat an inflammatory or autoimmune disease. In some embodiments, administration of the compound of formula (I) to a subject in need delays the progression of an inflammatory or autoimmune disease. In some embodiments, administration of the compound of formula (I) to a subject in need improves the quality of life of a subject with an inflammatory or autoimmune disease. In some embodiments, administration of the compound of formula (I) to a subject in need prolongs the survival of a subject with an inflammatory or autoimmune disease.

[0103] In one aspect, this document provides a method for preventing a subject susceptible to inflammatory or autoimmune diseases from developing any symptoms of the inflammatory or autoimmune disease, the method comprising administering a compound of formula (I) to the subject. In some embodiments, this document provides a method for preventing a subject who has not yet exhibited symptoms of an inflammatory or autoimmune disease from developing any symptoms of the inflammatory or autoimmune disease, the method comprising administering a compound of formula (I) to the subject.

[0104] In some aspects, this document provides a method for reducing the severity of inflammatory or autoimmune disease in a subject, the method comprising administering a compound of formula (I) to the subject. In some embodiments, this document provides a method for stabilizing inflammatory or autoimmune disease in a subject, the method comprising administering a compound of formula (I) to the subject. In some embodiments, the method prevents the exacerbation of the inflammatory or autoimmune disease. In some embodiments, the method delays the exacerbation of the inflammatory or autoimmune disease.

[0105] In another aspect, this article provides a method for delaying the onset or recurrence of inflammatory or autoimmune diseases in subjects, the method comprising administering a compound of formula (I) to the subject.

[0106] In some embodiments, this document provides a method for slowing the progression of an inflammatory or autoimmune disease in a subject, the method comprising administering a compound of formula (I) to the subject. In some embodiments, the method provides partial remission of the inflammatory or autoimmune disease. In some embodiments, the method provides complete remission of the inflammatory or autoimmune disease.

[0107] In another aspect, this document provides a method for reducing the dosage of one or more other drugs required to treat an inflammatory or autoimmune disease in a subject, the method comprising administering a compound of formula (I) to the subject. In some embodiments, this document provides a method for enhancing the effect of another drug used to treat an inflammatory or autoimmune disease in a subject, the method comprising administering a compound of formula (I) to the subject.

[0108] This article also provides a method for delaying the progression of an inflammatory or autoimmune disease in a subject, the method comprising administering a compound of formula (I) to the subject. In some embodiments, the method improves the quality of life of subjects suffering from inflammatory or autoimmune diseases. In some embodiments, the method prolongs the survival of subjects suffering from inflammatory or autoimmune diseases.

[0109] In another aspect, this document provides a method for treating inflammatory or autoimmune symptoms caused by a disease in a subject in need, the method comprising administering an effective amount of a compound of formula (I) to the subject. In some embodiments, this document provides a method for preventing inflammatory or autoimmune symptoms caused by a disease in a subject in need, the method comprising administering an effective amount of a compound of formula (I) to the subject. In some embodiments, administering a compound of formula (I) to a subject susceptible to a disease causing inflammatory or autoimmune symptoms prevents the subject from developing any inflammatory or autoimmune symptoms. In some embodiments, administering a compound of formula (I) to a subject who has not yet exhibited inflammatory or autoimmune symptoms caused by a disease prevents the subject from developing any inflammatory or autoimmune symptoms. In some embodiments, administering a compound of formula (I) to a subject in need reduces the severity of inflammatory or autoimmune symptoms caused by the disease in the subject. In some embodiments, administering a compound of formula (I) to a subject in need stabilizes the inflammatory or autoimmune symptoms of the disease (preventing or delaying the worsening of the inflammatory or autoimmune symptoms). In some embodiments, administration of the compound of formula (I) to a subject in need delays the onset or recurrence of inflammatory or autoimmune symptoms caused by a disease. In some embodiments, administration of the compound of formula (I) to a subject in need slows the progression of inflammatory or autoimmune symptoms caused by a disease. In some embodiments, administration of the compound of formula (I) to a subject in need provides partial relief of the disease causing inflammatory or autoimmune symptoms. In some embodiments, administration of the compound of formula (I) to a subject in need provides complete relief of the disease causing inflammatory or autoimmune symptoms. In some embodiments, administration of the compound of formula (I) to a subject in need reduces the dosage of one or more other drugs required to treat a disease causing inflammatory or autoimmune symptoms. In some embodiments, administration of the compound of formula (I) to a subject in need enhances the effect of another drug used to treat inflammatory or autoimmune symptoms of a disease. In some embodiments, administration of the compound of formula (I) to a subject in need delays the progression of the disease causing inflammatory or autoimmune symptoms. In some embodiments, administration of the compound of formula (I) to a subject in need improves the quality of life of a subject suffering from a disease that causes inflammatory or autoimmune symptoms. In some embodiments, administration of the compound of formula (I) to a subject in need prolongs the survival of a subject suffering from a disease that causes inflammatory or autoimmune symptoms.In some implementations, the disease is atopic dermatitis, asthma, lupus, rheumatoid arthritis, familial Mediterranean fever, psoriasis, generalized pustular psoriasis, cryoinflammatory syndrome, hidradenitis suppurativa, Behçet's syndrome, or familial cold autoinflammatory syndrome.

[0110] In some implementations, the compound of formula (I) can be used to treat disorders selected from: atopic dermatitis, asthma, lupus, rheumatoid arthritis, familial Mediterranean fever, psoriasis, generalized pustular psoriasis, cryoinflammatory syndrome, hidradenitis suppurativa, Behçet's syndrome, and familial cold autoinflammatory syndrome.

[0111] In some embodiments, the compound of formula (I) can be used to treat cancer. In some embodiments, the cancer is a solid tumor, skin cancer, or lymphoma. In some embodiments, the cancer is squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, hepatocellular carcinoma, renal cell carcinoma, bladder cancer, intestinal cancer, breast cancer, cervical cancer, colon cancer, esophageal cancer, head cancer, kidney cancer, liver cancer, lung cancer, cervical cancer, ovarian cancer, pancreatic cancer, prostate cancer, or stomach cancer, leukemia, benign and malignant lymphoma, Burkitt lymphoma, non-Hodgkin lymphoma, benign and malignant melanoma, myeloproliferative disorders, sarcoma, Ewing sarcoma, angiosarcoma, Kaposi's sarcoma, liposarcoma, sarcoma, peripheral neuroepithelial tumor. Synovial sarcoma, glioma, astrocytoma, oligodendroglioma, ependymoma, glioblastoma, neuroblastoma, ganglioneuroma, ganglioglioma, medulloblastoma, pineal cell carcinoma, meningeal sarcoma, neurofibroma, schwannoma, colorectal cancer, breast cancer, prostate cancer, cervical cancer, uterine cancer, lung cancer, ovarian cancer, testicular cancer, thyroid cancer, astrocytoma, esophageal cancer, pancreatic cancer, gastric cancer, liver cancer, colon cancer, melanoma, carcinosarcoma, Hodgkin's disease, nephroblastoma, or teratoma. Other cancers that can be treated with compounds of formula (I) include, for example, T-lineage acute lymphoblastic leukemia (T-ALL), T-lineage lymphoblastic lymphoma (T-LL), peripheral T-cell lymphoma, adult T-cell leukemia, pre-B ALL, pre-B lymphoma, large B-cell lymphoma, B-cell ALL, Philadelphia chromosome-positive ALL, and Philadelphia chromosome-positive CML.

[0112] In some implementations, cancers include breast cancer, colorectal cancer, non-small cell lung cancer, ovarian cancer, kidney cancer, sarcoma, melanoma, head and neck cancer, hepatocellular carcinoma, thyroid cancer, multidrug-resistant leukemia, lymphoma, multiple myeloma, esophageal cancer, colorectal cancer, pancreatic cancer, mesothelioma, carcinoma (e.g., adenocarcinoma, including esophageal adenocarcinoma), sarcoma (e.g., spindle cell sarcoma, liposarcoma, leiomyosarcoma, abdominal leiomyosarcoma, sclerosing epithelioid sarcoma), and melanoma (e.g., metastatic malignant melanoma).

[0113] In some implementations, compounds of formula (I) can be used to treat fibrosis, such as interstitial pulmonary fibrosis, cystic fibrosis, progressive pulmonary fibrosis, and idiopathic pulmonary fibrosis. Pharmaceutical Compositions and Routes of Administration

[0114] The compounds provided herein can be administered to subjects orally, topically, or parenterally in conventional formulations such as capsules, microcapsules, tablets, granules, powders, lozenges, pills, suppositories, injections, suspensions, syrups, patches, creams, lotions, ointments, gels, sprays, solutions, and emulsions.

[0115] The compounds disclosed herein can be administered to subjects orally, topically, or parenterally in conventional formulations such as capsules, microcapsules, tablets, granules, powders, lozenges, pills, suppositories, injections, suspensions, syrups, patches, creams, lotions, ointments, gels, sprays, solutions, and emulsions. Suitable formulations can be prepared using conventional organic or inorganic additives, such as excipients (e.g., sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, or calcium carbonate), binders (e.g., cellulose, methylcellulose, hydroxymethylcellulose, polypropylene pyrrolidone, polyvinylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, or starch), and disintegrants (e.g., starch, carboxymethylcellulose, hydroxypropyl starch, low-substituted hydroxypropylcellulose, sodium bicarbonate, calcium phosphate, or...). The pharmaceutical composition may contain: calcium citrate, lubricants (e.g., magnesium stearate, light anhydrous silicate, talc, or sodium lauryl sulfate), flavoring agents (e.g., citric acid, menthol, glycine, or orange powder), preservatives (e.g., sodium benzoate, sodium bisulfite, methylparaben, or propylparaben), stabilizers (e.g., citric acid, sodium citrate, or acetic acid), suspending agents (e.g., methylcellulose, polyvinylpyrrolidone, or aluminum stearate), dispersants (e.g., hydroxypropyl methylcellulose), diluents (e.g., water), and base waxes (e.g., cocoa butter, white petrolatum, or polyethylene glycol). The effective amount of the compound of formula (I) in the pharmaceutical composition may be at a level at which the desired effect is achieved.

[0116] For convenience, the compound of formula (I) can be administered orally. In one embodiment, when administered orally, the compound of formula (I) is administered with food and water. In another embodiment, the compound of formula (I) is dispersed in water or fruit juice (e.g., apple juice or orange juice) or any other liquid and administered orally as a solution or suspension.

[0117] The compounds disclosed herein can also be administered intradermally, intramuscularly, intraperitoneally, transdermally, intravenously, subcutaneously, intranasally, epidurally, sublingually, intracerebrally, intravaginally, transdermally, rectally, or via inhalation or local application to the ear, nose, eye, or skin. The method of administration is determined by the healthcare practitioner and may depend in part on the site of the medical condition.

[0118] In one embodiment, this document provides capsules containing a compound of formula (I) without additional carriers, excipients or mediators.

[0119] In another embodiment, this document provides a composition comprising an effective amount of a compound of formula (I) and a pharmaceutically acceptable carrier or mediator, wherein the pharmaceutically acceptable carrier or mediator may include excipients, diluents, or mixtures thereof. In one embodiment, the composition is a pharmaceutical composition.

[0120] The composition may be in the form of tablets, chewable tablets, capsules, solutions, parenteral solutions, lozenges, suppositories, and suspensions. The composition may be formulated into a convenient portion containing a daily dose or a daily dose in the form of a dosage unit, which may be a single tablet or capsule or a convenient volume of liquid. In one embodiment, the solution is prepared from a water-soluble salt such as hydrochloride. Typically, all compositions are prepared according to methods known in medicinal chemistry. Capsules may be prepared by mixing a compound of formula (I) with a suitable carrier or diluent and filling an appropriate amount of the mixture into a capsule. Commonly used carriers and diluents include, but are not limited to, inert powdered substances such as many different kinds of starch, powdered cellulose (especially crystalline and microcrystalline cellulose), sugars (such as fructose, mannitol, and sucrose), cereal flours, and similar edible powders.

[0121] Tablets can be prepared by direct compression, wet granulation, or dry granulation. Their formulations typically incorporate diluents, binders, lubricants, disintegrants, and compounds. Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or calcium sulfate, inorganic salts (such as sodium chloride), and powdered sugar. Powdered cellulose derivatives are also useful. Typical tablet binders are substances such as starch, gelatin, and sugars (such as lactose, fructose, glucose, etc.). Natural and synthetic gums are also readily available, including gum arabic, alginate, methylcellulose, polyvinylpyrrolidone, etc. Polyethylene glycol, ethylcellulose, and waxes can also be used as binders.

[0122] Lubricants may be needed in tablet formulations to prevent tablets and punches from sticking to the dye. Lubricants can be selected from smooth solids such as talc, magnesium stearate and calcium stearate, stearic acid, and hydrogenated vegetable oils. Tablet disintegrants are substances that swell upon wetting to break up the tablet and release the compound. They include starch, clay, cellulose, alginate, and gums. More specifically, substances such as corn and potato starch, methylcellulose, agar, bentonite, lignocellulose, powdered natural sponges, cation exchange resins, alginate, guar gum, citrus pomace, and carboxymethyl cellulose, as well as sodium lauryl sulfate, can be used. Tablets can be coated with sugar as a flavoring agent and sealant, or with film-forming protectants that alter the dissolution properties of the tablet. Compositions can also be formulated into chewable tablets, for example, by using substances such as mannitol in the formulation.

[0123] When compounds of formula (I) need to be administered in suppository form, typical matrices can be used. Cocoa butter is a conventional suppository matrix, which can be modified by adding waxes to slightly increase its melting point. In particular, water-miscible suppository matrices containing polyethylene glycol of various molecular weights are widely used.

[0124] The action of compounds of formula (I) can be delayed or prolonged by appropriate formulations. For example, slow-dissolving pellets of compounds of formula (I) can be prepared and incorporated into tablets or capsules, or used as sustained-release implantable devices. The technique also includes preparing pellets with several different dissolution rates and filling capsules with mixtures of pellets. Tablets or capsules can be coated with a membrane that resists dissolution for a predictable period of time. Even parenteral formulations can be made into long-acting formulations by dissolving or suspending compounds of formula (I) in an oily or emulsified medium, allowing them to be slowly dispersed in serum. Exemplary Implementation

[0125] This disclosure is further described through the following implementation scheme.

[0126] Implementation scheme P1. A compound of formula (I) (I) Or its pharmaceutically acceptable salt, wherein: A is or ; R 1 It is H or -NH2; L 1 It is -C(O)N(H)- or ; Ring B is a 5- to 6-membered monocyclic heterocyclic group or an 8- to 10-membered fused bicyclic heterocyclic group, wherein the heterocyclic group contains 1-3 nitrogen atoms; Each R2 It is independently a C1-C6 alkyl or halogroup; n is 0-3; R 3 It is H, C1-C6 alkyl or halogroup; The carbon ring (C) is attached to two adjacent carbon atoms, indicated by an asterisk (*). ;and X is O, CH2, or N(H).

[0127] Implementation Scheme P2. The compound or a pharmaceutically acceptable salt thereof according to Implementation Scheme P1, wherein the compound has the formula (I'): (I').

[0128] Implementation Scheme P3. The compound or a pharmaceutically acceptable salt thereof according to Implementation Scheme P1, wherein the compound has formula (II): (II).

[0129] Implementation Scheme P4. The compound or a pharmaceutically acceptable salt thereof according to Implementation Scheme P1, wherein the compound has formula (III): (III).

[0130] Implementation Scheme P5. The compound or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes P1-P3, wherein: R 1 It is H.

[0131] Implementation Scheme P6. The compound or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes P1-P3, wherein: R 1 It is -NH2.

[0132] Implementation Scheme P7. The compound or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes P1-P6, wherein: L 1 It is -C(O)N(H)-.

[0133] Implementation Scheme P8. The compound or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes P1-P6, wherein: L 1 yes .

[0134] Implementation Scheme P9. The compound or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes P1-P8, wherein: Ring B is a 6-membered monocyclic heterocyclic group or an 8-membered fused bicyclic heterocyclic group, wherein the heterocyclic group contains 1-2 nitrogen atoms.

[0135] Implementation Scheme P10. The compound or a pharmaceutically acceptable salt thereof as described in Implementation Scheme P9, wherein: Ring B is .

[0136] Implementation Scheme P11. The compound or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes P1-P10, wherein: n is 0 or 1.

[0137] Implementation Scheme P12. The compound or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes P1-P11, wherein: Each R 2 It is independently a C1-C3 alkyl or halogroup.

[0138] Implementation Scheme P13. The compound or a pharmaceutically acceptable salt thereof as described in Implementation Scheme P12, wherein: Each R 2 It is -CH3 or -CH2CH3 independently.

[0139] Implementation Scheme P14. The compound or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes P1-P13, wherein: yes .

[0140] Implementation Scheme P15. The compound or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes P1-P14, wherein: R 3 It is an H or C1-C3 alkyl group.

[0141] Implementation Scheme P16. The compound or a pharmaceutically acceptable salt thereof as described in Implementation Scheme P15, wherein: R 3 It is H or -CH3.

[0142] Implementation Scheme P17. The compound or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes P1-P16, wherein: X is O.

[0143] Implementation Scheme P18. The compound or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes P1-P16, wherein: X is CH2 or N(H).

[0144] Implementation Scheme P19. The compound or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes P1-P18, wherein: yes .

[0145] Implementation Scheme P20. A compound or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes P1-P3, P5, P6, P8-P17 and P19, wherein the compound has the formula (IIA), (IIB), (IIC), (IID), (IIE) or (IIF): (IIA) (IIB) (IIC) (IID) (IIE) (IIF).

[0146] Implementation Scheme P21. A compound or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes P1, P2, P4-P6, P8-P17 and P19, wherein the compound has the formula (IIIA), (IIIB), (IIIC), (IIID), (IIIE) or (IIIF): (IIIA) (IIIB) (IIIC) (IIID) (IIIE) (IIIF).

[0147] Implementation Scheme P22. The compound or a pharmaceutically acceptable salt thereof as described in Implementation Scheme P20 or P21, wherein: X is O.

[0148] Implementation plan P23. A compound selected from the compounds in Table 1 and its pharmaceutically acceptable salt.

[0149] Implementation Scheme P24. A pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes P1-P23 and a pharmaceutically acceptable excipient.

[0150] Implementation Scheme P25. A method for modulating the activity of interleukin-1 (IL1) receptor-associated kinase 4 (IRAK4), the method comprising contacting IRAK4 with an effective amount of a compound according to any one of Implementation Schemes P1-P23 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to Implementation Scheme P24.

[0151] Implementation Scheme P26. A method for treating an inflammatory or autoimmune disease in a subject of need, the method comprising administering to the subject an effective amount of a compound or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes P1-P23, or a pharmaceutical composition according to Implementation Scheme P24.

[0152] Implementation Plan P27. The method described in Implementation Plan P26, wherein the inflammatory or autoimmune disease is atopic dermatitis, asthma, lupus, rheumatoid arthritis, familial Mediterranean fever, psoriasis, generalized pustular psoriasis, cold inflammatory cyclic syndrome, hidradenitis suppurativa, Behçet's syndrome, or familial cold autoinflammatory syndrome. Example

[0153] The following examples are presented by way of illustration and not limitation. Compounds were named using an automatic name generation tool provided in ChemBiodraw Ultra (Cambridgesoft), which generates systematic names of chemical structures and supports the Cahn-Ingold-Prelog rule for stereochemistry. Those skilled in the art can modify the procedures illustrated in the illustrative examples to obtain desired products.

[0154] Salts of the compounds described herein can be prepared by standard methods, such as including an acid (e.g., TFA, formic acid, or HCl) in the mobile phase during chromatographic purification, or by stirring the product with an acid solution (e.g., an aqueous HCl solution) after chromatographic purification.

[0155] As used in some chemical structures provided in the following examples, the use of "or1" or "or2" to specify a particular atom indicates the absolute stereochemistry of which the indicated atom is not determined.

[0156] The following abbreviations may be relevant to this application. abbreviation ACN or MeCN: Acetonitrile AcOH: Acetic acid ADMP: 2-Azide-1,3-Dimethylimidazoline hexafluorophosphate aq: water-based Boc: tert-butoxycarbonyl CAM: Cerium ammonium molybdate cat.: catalyst CBM: Cerebellum protein binding region conc.: concentrated CV: Column volume DCM: Dichloromethane DHP: dihydropyran DIBAL-H: Diisobutylaluminum hydride DIPEA: N,N-Diisopropylethylamine DMAP: 4-Dimethylaminopyridine DMF: Dimethylformamide DMP: Des Martin periodoylene DMSO: Dimethyl sulfoxide eq.: equivalent EtOAc: Ethyl acetate h: hours HATU = 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate hept.: heptane IBX: 2-Iodooxybenzoic acid MeOH: Methanol min: minutes MTBE: Methyl tert-butyl ether OAc: Acetate PdCl2(dppf): bis(diphenylphosphino)ferrocene)palladium(II) dichloride Pd2(dba)3:tris(dibenzylacetone)dipalladium(0) petroleum ether: petroleum ether PhMe: Toluene PPTS: Pyridonium p-toluenesulfonate Preparative HPLC: Preparative high-performance liquid chromatography. PTSA or pTSA: p-Toluenesulfonic acid RT: Room temperature RuPhos Pd G3: (2-Dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II)methanesulfonate RuPhos Pd G4: [dicyclohexyl(2',6'-diisopropoxy-2-biphenyl)phosphine-κP](methanesulfonate-κO)[2'-(methylamino-κN)-2-biphenyl-κC] 2 ]palladium sat.: saturated SFC: Supercritical Fluid Chromatography TBAF: Tetrabutylammonium fluoride TBM: Target Binding Part TFA: Trifluoroacetic acid TFAA: Trifluoroacetic anhydride THF: Tetrahydrofuran TLC: Thin-layer chromatography UPLC: Ultra-high performance liquid chromatography Xantphos: (SP-4-3)-[[5-(diphenylphosphino)-9,9-dimethyl-9H-xanthan-4-yl]diphenylphosphino-κP](methanesulfonate-κO)[2'-(methylamino-κN)[1,1'-biphenyl]-2-yl-κC]-palladium Synthesis Examples Procedure for joint synthesis

[0157] The intermediate compounds mentioned in each embodiment are numbered only for that embodiment. For example, intermediate 1' in embodiment S1 and intermediate 1' in embodiment S2 are described in different embodiments and therefore refer to different compounds. Example S1: Preparation of intermediate alkynes 2-(2-((1r,4r)-4-ethynylcyclohexyl)ethoxy)tetrahydro-2H-pyran (L-1) and 2-((1r,4r)-4-ethynylcyclohexyl)ethanol-1-ol (L-2)

[0158] Step 1'. Preparation of [4-[[tert-butyl(diphenyl)silyl]oxymethyl]cyclohexyl]methanol 2': Imidazole (0.85 g, 12.48 mmol, 0.6 equivalent) was added to a solution of [4-(hydroxymethyl)cyclohexyl]methanol 1' (3 g, 20.8 mmol, 1 equivalent) in dry DMF (83.2 mL, 0.25 M), followed by the addition of tert-butylchlorodiphenylsilane (2.7 mL, 10.4 mmol, 0.5 equivalent). After stirring at room temperature for 18 h, TLC (25% EtOAc in heptane, showing UV + KMnO4) showed complete conversion. A semi-salt aqueous solution (40 mL) was added to the reaction mixture. The aqueous phase was extracted with EtOAc (2 x 50 mL). The organic matter was washed with semi-salt water (4 x 25 mL), dried over Na2SO4, and concentrated to dryness. The residue was dry-packed and purified by normal-phase rapid chromatography (80 g silica column, elution: 0 to 30% EtOAc / heptane, 10 CV, approximately 20% EtOAc in the product). The fractions were combined and concentrated to give 2' (6.48 g, 43% yield) as a colorless oil.

[0159] 1 H NMR (400 MHz, chloroform-d): δ ppm 0.82 - 1.02 (m, 4 H), 1.03 - 1.07 (m,9 H), 1.38 - 1.55 (m, 2 H), 1.66 - 1.88 (m, 4 H), 2.04 (br d, J = 3.2 Hz, 2H), 3.42 - 3.49 (m, 3 H), 7.32 - 7.44 (m, 6 H), 7.65 (d, J = 6.6 Hz, 3 H), 7.70 - 7.75 (m, 1 H).

[0160] Step 2'. Preparation of [4-[[tert-butyl(diphenyl)silyl]oxymethyl]cyclohexyl]methylmethanesulfonate 3': Add [4-[[tert-butyl(diphenyl)silyl]oxymethyl]cyclohexyl]methanol 2' (6.48 g, 8.93 mmol, 1 equivalent) to a round-bottom flask (44.65 mL, 0.2 M). The flask was then cooled to 0ºC, followed by the addition of methanesulfonyl chloride (0.76 mL, 9.82 mmol, 1.1 equivalent) and triethylamine (1.62 mL, 11.61 mmol, 1.3 equivalent). After 2 h, TLC (heptane / EtOAc; 10 / 90) showed complete conversion of the starting material to the desired product (KMnO4 chromogenic agent). The mixture was partitioned between EtOAc and water. The organic phase was washed once with water, once with 1 N HCl, dried over magnesium sulfate, filtered, and evaporated under reduced pressure to give 3' as a colorless solid (3.65 g, 89% yield). The crude product was used directly in the next reaction as is.

[0161] 1 H NMR (400 MHz, chloroform-d): δ ppm 1.03 (br s, 3 H), 1.07 (br d, J =12.7 Hz, 9 H), 1.45 - 1.56 (m, 1 H), 1.64 - 1.77 (m, 1 H), 1.83 - 1.90 (m, 3H), 3.01 (s, 3 H), 3.48 (d, J = 6.1 Hz, 2 H), 4.05 (d, J = 6.6 Hz, 2 H), 7.35- 7.47 (m, 6 H), 7.64 - 7.69 (m, 3 H), 7.71 - 7.79 (m, 1 H). 1 The two protons are not obvious in H NMR.

[0162] Step 3'. Preparation of 2-[4-[[tert-butyl(diphenyl)silyl]oxymethyl]cyclohexyl]acetonitrile 4': Add [4-[[tert-butyl(diphenyl)silyl]oxymethyl]cyclohexyl]methylmethanesulfonate 3' (3.65 g, 7.92 mmol, 1 equivalent) and NaCN (1.19 g, 18.21 mmol, 2.3 equivalent) to a round-bottom flask in DMSO (12.2 mL, 0.65 M). The reaction mixture was then stirred at 50ºC. After an overnight period, TLC (heptane / EtOAc; 50 / 50) showed complete conversion of the starting material to compound 4' (KMnO4 chromogenic agent). The reaction mixture was poured into an Erlenmeyer flask containing crushed ice and stirred until all the ice melted. The resulting solid was separated by filtration and dissolved in a minimal amount of ethyl acetate, washed with NaHCO3 and brine, and evaporated to dryness. The residue was dry-packed and purified by normal-phase rapid chromatography (80 g silica column, elution: 5 to 100% EtOAc / heptane, 15 CV). The fractions were combined and concentrated to give 4' (2.35 g, 76% yield) as a colorless oil.

[0163] 1 H NMR (400 MHz, chloroform-d): δ ppm 0.83 - 0.91 (m, 1 H), 0.98 - 1.05 (m, 2 H), 1.05 - 1.09 (m, 9 H), 1.09 - 1.18 (m, 2 H), 1.46 - 1.56 (m, 1 H), 1.58- 1.65 (m, 1 H), 1.89 (br t, J = 11.9 Hz, 3 H), 2.27 (d, J = 6.6 Hz, 2 H), 3.48 (d, J = 5.9 Hz, 2 H), 7.37 - 7.46 (m, 6 H), 7.64 - 7.69 (m, 3 H), 7.71 -7.75 (m, 1 H).

[0164] Step 4'. Preparation of 2-[4-[[tert-butyl(diphenyl)silyl]oxymethyl]cyclohexyl]acetaldehyde 5': 2.35 g (6 mmol, 1 equivalent) of 2-[4-[[tert-butyl(diphenyl)silyl]oxymethyl]cyclohexyl]acetonitrile 4' in DCM (10.7 mL, 0.56 M) was added to a round-bottom flask, and the reaction mixture was cooled to -78ºC. Then, DIBAL-H (1 M solution in DCM) (17.99 mL, 17.99 mmol, 3 equivalents) was added to the cooled reaction mixture, and the reaction was stirred at -78ºC. After 4 h, 40 mL of 4 M HCl was carefully added, and the solution was slowly warmed to RT. Stirring was continued at RT for 10 min, and the mixture was partitioned between ethyl acetate and 1 M HCl. The organic phase was washed with water, dried over magnesium sulfate, and concentrated under reduced pressure. The residue was dry-packed and purified by normal-phase rapid chromatography (40 g silica column, elution: 1:3 heptane:EtOAc). The fractions were combined and concentrated to give 5' (803 mg, 34% yield) as a colorless oil, which was used as is in the next reaction.

[0165] Step 5'. Preparation of 2-[4-[[tert-butyl(diphenyl)silyl]oxymethyl]cyclohexyl]ethanol 6': 2-[4-[[tert-butyl(diphenyl)silyl]oxymethyl]cyclohexyl]acetaldehyde 5' (800 mg, 2.03 mmol, 1 equivalent) in EtOH (10.2 mL, 0.2 M) was added to a round-bottom flask at 0ºC. NaBH4 (192.26 mg, 5.08 mmol, 2.5 equivalent) was added to the reaction mixture and the reaction was stirred at 0ºC. After 4 h, Rochelle salt was added at 0ºC, and the solution was then heated to room temperature. The organic layer was extracted twice with EtOAc, washed twice with brine, and dried over MgSO4. The residue was dry-packed and purified by normal-phase rapid chromatography (40 g silica column, elution: 0 to 30% EtOAc / heptane, 15 CV, approximately 20% EtOAc in the product). The fractions were combined and concentrated to give 6' (353.7 mg, 44% yield) as a colorless oil.

[0166] 1H NMR (400 MHz, chloroform-d): δ ppm 0.93 - 1.03 (m, 4 H), 1.06 (s, 9 H), 1.13 - 1.20 (m, 1 H), 1.31 - 1.41 (m, 1 H), 1.50 (q, J = 6.8 Hz, 3 H), 1.74 -1.86 (m, 4 H), 3.47 (d, J = 6.1 Hz, 2 H), 3.67 - 3.73 (m, 2 H), 7.36 - 7.45 (m, 6 H), 7.67 (dd, J = 7.8, 1.5 Hz, 4 H).

[0167] Step 6'. Preparation of tert-butyl-diphenyl-[[4-(2-tetrahydropyran-2-yloxyethyl)cyclohexyl]methoxy]silane 7': PPTS (205.28 mg, 0.82 mmol, 0.2 equivalents) and DHP (0.93 mL, 10.21 mmol, 2.5 equivalents) were added to a solution of 2-[4-[[tert-butyl(diphenyl)silyl]oxymethyl]cyclohexyl]ethanol 6' (1.62 g, 4.08 mmol, 1 equivalent) in DCM (25.06 mL, 0.16 M). The reaction was stirred at room temperature. After 24 h, TLC showed complete conversion of the starting material 6'. The reaction mixture was concentrated to dryness, and the residue was packed dry and purified by normal-phase rapid chromatography (80 g silica column, elution: 0 to 30% EtOAc / heptane, 15 CV, approximately 20% EtOAc in the product). The fractions were combined and concentrated to give 7' (1.79 g, 91% yield), which was a colorless oil.

[0168] 1H NMR (400 MHz, chloroform-d): δ ppm 0.84 - 0.91 (m, 3 H), 0.93 - 1.00 (m, 3 H), 1.03 - 1.11 (m, 9 H), 1.29 - 1.39 (m, 2 H), 1.50 - 1.55 (m, 3 H), 1.56- 1.63 (m, 2 H), 1.70 (br s, 5 H), 3.40 - 3.45 (m, 1 H), 3.47 (d, J = 6.1 Hz, 2 H), 3.48 - 3.55 (m, 1 H), 3.76 - 3.84 (m, J = 9.7, 7.2, 7.2 Hz, 1 H), 3.84- 3.92 (m, 1 H), 4.55 - 4.61 (m, 1 H), 7.35 - 7.45 (m, 6 H), 7.64 - 7.76 (m, 4 H).

[0169] Step 7'. Preparation of [4-(2-tetrahydropyran-2-yloxyethyl)cyclohexyl]methanol 8': At room temperature, 1 M TBAF solution (14.89 mL, 14.89 mmol, 4 equivalents) in tert-butyl-diphenyl-[[4-(2-tetrahydropyran-2-yloxyethyl)cyclohexyl]methoxy]silane 7' (1.79 g, 3.72 mmol, 1 equivalent) in THF (4.65 mL, 0.8 M) with stirring was added. After 5 h, TLC showed complete conversion of the starting material 7'. The solvent was removed under reduced pressure, and the residue was purified by normal-phase rapid chromatography (80 g gold column, solid sediment, elution 0 to 40% EtOAc / heptane, 10 CV) (CAM was used as the TLC staining agent). The fractions were combined and concentrated to give 8' as a colorless oil (577.5 mg, 58% yield).

[0170] 1 H NMR (400 MHz, chloroform-d): δ ppm 0.90 - 1.01 (m, 4 H), 1.33 - 1.46 (m, 2 H), 1.48 - 1.63 (m, 7 H), 1.68 - 1.76 (m, 1 H), 1.77 - 1.89 (m, 5 H), 3.39-3.54 (m, 4H), 3.77-3.91 (m, 2H), 4.56-4.60 (m, 1H).

[0171] Step 8'. Preparation of 4-(2-Tetrahydropyran-2-yloxyethyl)cyclohexaneformaldehyde 9': At 0ºC, DMP (2.47 g, 5.81 mmol, 2 equivalents) and water (3 drops) were added to a stirred solution of [4-(2-tetrahydropyran-2-yloxyethyl)cyclohexyl]methanol 8' (704.5 mg, 2.91 mmol, 1 equivalent) in DCM (36.34 mL, 0.04 M) and THF (36.34 mL, 0.04 M). After 2.5 h, TLC showed complete conversion of the starting material 8'. The solvent was removed under reduced pressure without a heating bath, absorbed with EtOAc, washed with saturated NaHCO3, extracted with EtOAc (3x), and then washed with brine. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was then purified by normal-phase rapid chromatography (24 g column, solid sediment, elution of 0 to 30% EtOAc / heptane, 10 CV). The fractions were combined and concentrated to give 9' (459.5 mg, 66% yield) as a colorless solid.

[0172] 1 H NMR (400 MHz, DMSO-d6): δ ppm 0.92 - 1.01 (m, 2 H), 1.08 - 1.22 (m, 2 H), 1.26 - 1.36 (m, 1 H), 1.38 - 1.53 (m, 6 H), 1.55 - 1.64 (m, 1 H), 1.65- 1.83 (m, 3 H), 1.84 - 1.94 (m, 2 H), 2.15 - 2.25 (m, 1 H), 3.32 - 3.45 (m, 2 H), 3.62 - 3.77 (m, 2 H), 4.49 - 4.56 (m, 1 H), 9.55 (d, J = 1.0 Hz, 1 H).

[0173] Step 9'. Preparation of 2-[2-(4-ethynylcyclohexyl)ethoxy]tetrahydropyran L-1: 4-(2-tetrahydropyran-2-yloxyethyl)cyclohexaneformaldehyde 9' (459.5 mg, 1.91 mmol, 1 equivalent) and K2CO3 (494.18 mg, 3.82 mmol, 2 equivalents) were added to a flame-dried round-bottom flask. Vacuum was applied, and the flask was then filled with nitrogen (repeated three times). MeOH (9.56 mL, 0.2 M) was then added, and the reaction mixture was stirred at room temperature under nitrogen for 20 min. Then, Bestmann-Ohira reagent (4.4 mL, 2.29 mmol, 1.2 equivalents) (10% in acetonitrile) was added to the reaction mixture. The resulting mixture was stirred at room temperature under a nitrogen atmosphere. After 4 h, TLC (eluting with 10% EtOAc in heptane) showed complete conversion of the starting material 9'. The residue was dried and then purified by normal-phase rapid chromatography (24 g column, solid sediment, elution of 0 to 10% EtOAc / heptane, 10 CV). The fractions were combined and concentrated to give L-1 (275.7 mg, 57% yield) as a colorless oil.

[0174] 1 H NMR (400 MHz, chloroform-d): δ ppm 0.88 - 0.98 (m, 2 H), 1.33 - 1.45 (m, 3 H), 1.48 - 1.62 (m, 7 H), 1.68 - 1.76 (m, 1 H), 1.77 - 1.88 (m, 3 H), 1.95- 2.03 (m, 2 H), 2.14 - 2.24 (m, 1 H), 3.36 - 3.56 (m, 2 H), 3.75 - 3.90 (m, 2 H), 4.55 - 4.59 (m, 1 H).

[0175] Step 10'. Preparation of 4-(2-Tetrahydropyran-2-yloxyethyl)cyclohexaneformaldehyde L-2: PTSA (3.39 mg, 0.02 mmol, 0.1 equivalent) was added to a stirred solution of 2-[2-(4-ethynylcyclohexyl)ethoxy]tetrahydropyran L-1 (50 mg, 0.2 mmol, 1 equivalent) in MeOH (1.97 mL, 0.1 M). After 1 h, TLC showed complete conversion of the starting material L-1. The reaction mixture was quenched with triethylamine, and the mixture was concentrated under reduced pressure. The residue was then purified by normal-phase rapid chromatography (24 g gold column, solid sediment, elution 0 to 30% EtOAc / heptane, 10 CV) (using CAM as TLC developer). The fractions were combined and concentrated to give L-2 (18 mg, 60% yield) as a colorless oil.

[0176] 1 H NMR (400 MHz, chloroform-d): δ ppm 0.88 - 1.01 (m, 2 H), 1.30 - 1.50 (m, 6 H), 1.73 - 1.82 (m, 2 H), 1.95 - 2.03 (m, 2 H), 2.03 - 2.07 (m, 1 H), 2.13- 2.24 (m, 1 H), 3.68 (t, J = 6.6 Hz, 2 H). Example S2: Preparation of intermediate 2-((1r,4r)-4-aminocyclohexyl)ethanol-1-ol (L-3)

[0177] LCMS Method 1. Kinetex XB-C18 75 x 3.0 mm 2.6 μm, Temperature: RT, Flow Rate: 1.5 mL / min, Run Time: 5.5 min, Mobile Phase Conditions: Mobile Phase A: 0.1% TFA in H2O, Mobile Phase B: 0.1% TFA in ACN, Gradient: Initially 95% mobile phase A and 5% mobile phase B, linear gradient over 4.0 min, changing to 5% mobile phase A and 95% mobile phase B. MSD positive ions.

[0178] Step 1'. Preparation of tert-butyl carbamate 2': A solution of 10 g (38.1 mmol) of 2-(1r,4r)-4-((tert-butoxycarbonyl)amino)cyclohexyl)acetic acid 1' in THF (250 mL) was stirred at 0ºC under a nitrogen atmosphere for 15 min. BH3·DMS (7.23 mL, 76 mmol) was added fractionally to the stirred solution at 0ºC under a nitrogen atmosphere. The reaction mixture was then stirred at 0ºC for 15 min and further stirred at room temperature under a nitrogen atmosphere for 2 h. The reaction mixture was concentrated under reduced pressure to give a crude product, which was diluted with cold water and extracted with EtOAc. The combined organic layers were washed with aqueous brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a crude product. The crude product was purified by rapid column chromatography (using silica gel (230-400 mesh) with 40%-50% ethyl acetate in petroleum ether) to give tert-butyl ((1r,4r)-4-(2-hydroxyethyl)cyclohexyl)carbamate 2' (7.21 g, 78% yield) as a white solid.

[0179] LCMS Method 1 Retention time: 2.315 min, [M+H-56] + = 188.2.

[0180] Step 2'. Preparation of 2-((1r,4r)-4-aminocyclohexyl)ethanol-1-ol 3': 4.0 M HCl (36.0 mL, 144 mmol) in dioxane was added to a stirred solution of ((1r,4r)-4-(2-hydroxyethyl)cyclohexyl)carbamate tert-butyl ester 2' (7.0 g, 28.8 mmol) in DCM (100 mL), and the reaction mixture was stirred at 25ºC under a nitrogen atmosphere for 30 min. The solvent was evaporated under reduced pressure to give 2-((1r,4r)-4-aminocyclohexyl)ethanol-1-ol HCl 3' (5.465 g) as a white solid, which was used for the next step without further purification.

[0181] LCMS Method 1: Retention time: 0.493 min, [M+H] + = 144.2.

[0182] 1H NMR (400 MHz, DMSO-d6): δ ppm 0.88 – 1.00 (m, 2H), 1.25 – 1.38 (m,5H), 1.69 – 1.80 (m, 2H), 1.90 – 1.99 (m, 2H), 2.83 – 2.93 (m, 1H), 3.41 (t,J = 6.4 Hz, 2H), 8.08 (s, 2H). Program for TBM synthesis Example S3: Preparation of 1-(4-(((R)-1-cyanoethyl)amino)-5-(4-((1r,4R)-4-(2-oxoethyl)cyclohexyl)-1H-1,2,3-triazol-1-yl)pyridin-2-yl)-1H-pyrazolo[3,4-b]pyridine-5-carboxynitrile (A-1)

[0183] LCMS Method 1. Column: Kinetex XB-C18 75 x 3.0 mm, 2.6 μm. Temperature: 45ºC, Flow rate: 1.0 mL / min, Run time: 5.0 min. Mobile phase conditions: Mobile phase A: 5.0 mm ammonium formate, pH 3.3: CH3CN (98:02), Mobile phase B: CH3CN: buffer (98:02), Gradient: Initially 80% mobile phase A and 20% mobile phase B were linearly gradiented to 100% mobile phase B over 4.0 min, then held for 0.6 min. MSD positive ions.

[0184] UPLC Method 2. Column: Zorbax SB-C18 50 x 2.1 mm, 1.8 μm. Temperature: 45ºC, Flow rate: 0.7 mL / min, Run time: 3.0 min. Mobile phase conditions: Mobile phase A: 5.0 mM ammonium acetate: CH3CN (95:05), Mobile phase B: CH3CN: 5.0 mM ammonium acetate (95:05), Gradient: Initially 80% mobile phase A and 20% mobile phase B were linearly gradiented to 100% mobile phase B over 2.0 min, then held for 0.3 min. MSD positive ions.

[0185] Step 1'. Preparation of (R)-2-((2-chloro-5-nitropyridin-4-yl)amino)propionamide 3': Under N2 atmosphere and at RT, (R)-2-aminopropionamide hydrochloride 2' (15.49 g, 124 mmol, 1.2 equivalents) was added to a stirred solution of 2,4-dichloro-5-nitropyridinium 1' (20.0 g, 104 mmol, 1.0 equivalent) in acetonitrile (200 mL), followed by the addition of DIPEA (54.3 mL, 311 mmol, 3.0 equivalents). The reaction was then heated to 40ºC for 6 h. The reactants were cooled to RT, poured into ice-cold water (500 mL), and extracted with ethyl acetate (3 x 700 mL). The combined organic layers were washed with brine (2 x 250 mL), dried over sodium sulfate, and concentrated under reduced pressure to give (R)-2-((2-chloro-5-nitropyridin-4-yl)amino)propionamide 3' (27 g, 97% yield) as a pale yellow solid.

[0186] LCMS Method 1: Retention time: 0.876 min, 91.45% purity, at 220 nm, [M+H] + = 245.0.

[0187] 1 H NMR (300 MHz, DMSO-d6): δ ppm 1.42 (d, J = 8.8 Hz, 3H), 4.34 - 4.39 (m, 1H), 6.90 (s, 1H), 7.46 (s, 1H), 7.69 (s, 1H), 8.66 - 8.68 (m, 1H), 8.93(s, 1H).

[0188] Step 2'. Preparation of (R)-2-((2-chloro-5-nitropyridin-4-yl)amino)propionitrile 4': At 0ºC, pyridine (81 mL, 1004 mmol, 10.0 equivalent) was added to a stirred solution of (R)-2-((2-chloro-5-nitropyridin-4-yl)amino)propionamide 3' (27.0 g, 100 mmol, 1.0 equivalent) in DCM (270 mL) over a 10 min period, followed by the addition of trifluoroacetic anhydride (70.9 mL, 502 mmol, 5.0 equivalent). The reaction mixture was then slowly warmed to room temperature and stirred for 2 h. The reaction mixture was slowly quenched with ice-cold water (300 mL) and extracted with DCM (2 x 300 mL). The combined organic layers were washed with 1.5 N HCl solution (2 x 200 mL) and brine (2 x 250 mL), dried over sodium sulfate and concentrated under reduced pressure to give (R)-2-((2-chloro-5-nitropyridin-4-yl)amino)propionitrile 4' (21 g, 89% yield) as a pale yellow solid.

[0189] LCMS Method 1: Retention time: 1.583 min, 96.3% purity, at 220 nm, [M+H] + = 227.0.

[0190] 1 H NMR (400 MHz, DMSO-d6): δ ppm 1.68 (d, J = 7.2 Hz, 3H), 5.18 - 5.22(m, 1H), 7.35 (s, 1H), 8.42 (d, J = 8.4 Hz, 1H), 8.95 (s, 1H).

[0191] Step 3'. Preparation of (R)-1-(4-((1-cyanoethyl)amino)-5-nitropyridin-2-yl)-1H-pyrazolo[3,4-b]pyridin-5-carboxylonitrile 6': Under N2 atmosphere, zinc acetate (0.917 g, 5.00 mmol, 0.6 equivalent) and K2CO3 (2.301 g, 16.65 mmol, 2.0 equivalent) were added to a stirred solution of (R)-2-((2-chloro-5-nitropyridin-4-yl)amino)propionitrile 4' (1.965 g, 8.33 mmol, 1.0 equivalent) and 1H-pyrazolo[3,4-b]pyridin-5-carboxylonitrile 5' (1.2 g, 8.33 mmol, 1.0 equivalent) in 1,4-dioxane (70 mL) and purged for 10 min. Then, under a N2 atmosphere, Xantphos (0.482 g, 0.833 mmol, 0.1 equivalent) and Pd2(dba)3 (0.381 g, 0.416 mmol, 0.05 equivalent) were added and purged for another 5 min. The reaction mixture was then stirred at 100ºC for 16 h. The reaction was cooled to RT and filtered through a diatomaceous earth bed, and the residue (filter cake) was washed with DCM (500 mL). The filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by column chromatography (using silica gel (230-400 mesh) with 80% ethyl acetate in petroleum ether) to give (R)-1-(4-((1-cyanoethyl)amino)-5-nitropyridin-2-yl)-1H-pyrazolo[3,4-b]pyridin-5-carboxynitrile 6' (1.5 g, 53.9% yield) as a yellow solid.

[0192] 1 H NMR (400 MHz, DMSO-d6): δ ppm 1.77 (d, J = 7.2 Hz, 3H), 5.21 - 5.25(m, 1H), 7.86 (s, 1H), 8.53 (d, J = 7.6 Hz, 1H), 8.78 (s, 1H), 9.03 - 9.11(m, 2H), 9.22(s, 1H).

[0193] Step 4'. Preparation of (R)-1-(5-amino-4-((1-cyanoethyl)amino)pyridin-2-yl)-1H-pyrazolo[3,4-b]pyridin-5-carboxylonitrile 7': Iron (1.253 g, 22.44 mmol) and ammonium chloride (0.600 g, 11.22 mmol) were added to a stirred solution of (R)-1-(4-((1-cyanoethyl)amino)-5-nitropyridin-2-yl)-1H-pyrazolo[3,4-b]pyridin-5-carboxylonitrile 6' (1.5 g, 4.49 mmol) in 100 mL ethanol:water (7:3) and stirred at 85ºC for 6 h. The reaction mixture was cooled to RT and filtered through a diatomaceous earth bed, the bed was washed with DCM (500 mL) and concentrated under reduced pressure to give the crude product. The crude product was dissolved in water and stirred at RT for 10 min. The crude product was then filtered through a Buchner funnel, washed with water, and dried to give (R)-1-(5-amino-4-((1-cyanoethyl)amino)pyridin-2-yl)-1H-pyrazolo[3,4-b]pyridin-5-carboxynitrile 7' (1.2 g, 61.5% yield) as a brown solid, which was used for the next step without further purification.

[0194] UPLC Method 2: Retention time: 0.561 min, [M+H] + = 305.0.

[0195] Step 5'. Preparation of (R)-1-(5-azido-4-((1-cyanoethyl)amino)pyridin-2-yl)-1H-pyrazolo[3,4-b]pyridin-5-carboxylonitrile 8': ADMP (2.81 g, 9.86 mmol, 2.5 equivalents) and DMAP (1.204 g, 9.86 mmol, 2.5 equivalents) were added to a stirred solution of (R)-1-(5-amino-4-((1-cyanoethyl)amino)pyridin-2-yl)-1H-pyrazolo[3,4-b]pyridin-5-carboxylonitrile 7' (1.2 g, 3.94 mmol, 1.0 equivalent) in acetonitrile (20 mL) and stirred for 16 h at RT under a nitrogen atmosphere. The reaction mixture was treated with ice-cold water (100 mL) and extracted with ethyl acetate (3 x 150 mL). The combined organic layers were dried over sodium sulfate and then concentrated under reduced pressure to give (R)-1-(5-azido-4-((1-cyanoethyl)amino)pyridin-2-yl)-1H-pyrazolo[3,4-b]pyridine-5-carboxynitrile 8' (1.3 g, 56.8% yield) as a viscous liquid, which was used for the next step without further purification.

[0196] LCMS Method 1: Retention time: 1.484 min, [M+H] += 331.0.

[0197] Step 6'. Preparation of 1-(4-(((R)-1-cyanoethyl)amino)-5-(4-((1r,4R)-4-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)cyclohexyl)-1H-1,2,3-triazol-1-yl)pyridin-2-yl)-1H-pyrazolo[3,4-b]pyridin-5-carboxylonitrile 9': At room temperature, 2-(2-(((1r,4r)-4-ethynylcyclohexyl)ethoxy)tetrahydro-2H-pyran L-1 (338) was added to a stirred solution of (R)-1-(5-azido-4-((1-cyanoethyl)amino)pyridin-2-yl)-1H-pyrazolo[3,4-b]pyridin-5-carboxylonitrile 8' (430 mg, 1.302 mmol) in acetone (6.0 mL). The reaction mixture was first added (mg, 1.432 mmol), followed by sodium ascorbate (129 mg, 0.651 mmol), and then a solution of copper(II) sulfate pentahydrate (65 mg, 0.26 mmol) in H2O (1.0 mL). The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was treated with water (100 mL) and extracted with ethyl acetate (3 x 75 mL). The combined organic layers were washed with brine (10 mL), dried over sodium sulfate, and concentrated under reduced pressure to give the crude product. The crude compound was purified by column chromatography (using silica gel (230-400 mesh) with 80%-90% ethyl acetate / petroleum ether) to give 1-(4-(((R)-1-cyanoethyl)amino)-5-(4-((1r,4R)-4-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)cyclohexyl)-1H-1,2,3-triazol-1-yl)pyridin-2-yl)-1H-pyrazolo[3,4-b]pyridin-5-carboxylonitrile 9' (220 mg, 20.2% yield).

[0198] LCMS Method 1: Retention time 3.078 min, 67.93% purity, at 220 nm, [M+H] + = 567.0.

[0199] Step 7'. Preparation of 1-(4-(((R)-1-cyanoethyl)amino)-5-(4-((1r,4R)-4-(2-hydroxyethyl)cyclohexyl)-1H-1,2,3-triazol-1-yl)pyridin-2-yl)-1H-pyrazolo[3,4-b]pyridin-5-carboxylonitrile 10': Under RT, 1-(4-(((R)-1-cyanoethyl)amino)-5-(4-((1r,4R)-4-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)cyclohexyl)-1H-1,2,3-triazol-1-yl)pyridin-2-yl)-1H-pyrazolo[3,4-b]pyridin-5-carboxylonitrile 9' (220 mg, 0.388 mmol) was added to MeOH (4.0 mmol) pTSA (73.8 mg, 0.388 mmol) was added to the stirred solution in mL and stirred for 2 h. The reaction mixture was treated with saturated sodium bicarbonate solution (30 mL) and extracted with DCM (2 x 40 mL). The organic layer was washed with brine (25.0 mL), dried over sodium sulfate, and concentrated under reduced pressure to give crude product 1-(4-(((R)-1-cyanoethyl)amino)-5-(4-((1r,4R)-4-(2-hydroxyethyl)cyclohexyl)-1H-1,2,3-triazol-1-yl)pyridin-2-yl)-1H-pyrazolo[3,4-b]pyridin-5-carboxynitrile 10' (130 mg) as a brown solid, which was used for the next step without further purification.

[0200] LCMS Method 1: Retention time: 1.994 min, [M+H] + = 483.2.

[0201] Step 8'. Preparation of 1-(4-(((R)-1-cyanoethyl)amino)-5-(4-((1r,4R)-4-(2-oxoethyl)cyclohexyl)-1H-1,2,3-triazol-1-yl)pyridin-2-yl)-1H-pyrazolo[3,4-b]pyridin-5-carboxylonitrile A-1: ​​Under RT, IBX (151 mg, 0.539 mmol, 2.0 equivalent) was added to 1-(4-(((R)-1-cyanoethyl)amino)-5-(4-((1r,4R)-4-(2-hydroxyethyl)cyclohexyl)-1H-1,2,3-triazol-1-yl)pyridin-2-yl)-1H-pyrazolo[3,4-b]pyridin-5-carboxylonitrile 10' (130 mg, 0.269 mmol ... mmol (1.0 equivalent) was added to a solution of DMSO (3 mL). The resulting solution was stirred at RT for 2 h. The reaction mixture was diluted with ethyl acetate (15 mL) and washed with ice-cold water, aqueous sodium bicarbonate solution, and then with aqueous salt solution. The mixture was dried over sodium sulfate and concentrated under reduced pressure to give a crude product 1-(4-(((R)-1-cyanoethyl)amino)-5-(4-((1r,4R)-4-(2-oxoethyl)cyclohexyl)-1H-1,2,3-triazol-1-yl)pyridin-2-yl)-1H-pyrazolo[3,4-b]pyridine-5-carboxynitrile A-1 (90 mg) as a brown solid, which was used for the next step without further purification.

[0202] LCMS Method 1: Retention time: 2.297 min, [M+H] + = 481.0. Example S4: Preparation of 6-amino-1-(4-(((R)-1-cyanoethyl)amino)-5-(4-((1r,4R)-4-(2-oxoethyl)cyclohexyl)-1H-1,2,3-triazol-1-yl)pyridin-2-yl)-1H-pyrazolo[3,4-b]pyridine-5-carboxylonitrile (A-2)

[0203] LCMS Method 1. Kinetex XB - C18, 75 x 3.0 mm, 2.6 μm, temperature: RT, flow rate: 1.0 mL / min, run time: 5 min. Mobile phase conditions: Mobile phase A: 5.0 mm ammonium formate, pH 3.3: CH3CN (98:02), Mobile phase B: CH3CN: 5.0 mm ammonium formate, pH 3.3 (98:02), gradient: a linear gradient from the initial 80% mobile phase A and 20% mobile phase B to 100% mobile phase B over 4.0 min. MSD positive ions.

[0204] LCMS Method 2.Aquity Uplc BEH-C18, 50 x 3.0 mm, 1.7 μm, temperature: RT, flow rate: 1.0 mL / min, run time: 5 min, mobile phase conditions: mobile phase A: 0.1% TFA in H2O, mobile phase B: 0.1% TFA in ACN, gradient: linear gradient change from initial 80% mobile phase A and 20% mobile phase B to 100% mobile phase B over 3.0 min. MSD positive ions.

[0205] Step 1'. Preparation of (R)-2-((2-chloro-5-nitropyridin-4-yl)amino)acrylamide 3': Under nitrogen atmosphere and at RT, (R)-2-aminopropionamide hydrochloride 2' (38.7 g, 311 mmol) and DIPEA (113.0 mL, 648 mmol) were added to a stirred solution of 2,4-dichloro-5-nitropyridinium 1' (50.0 g, 259 mmol) in acetonitrile (500 mL), and the resulting solution was stirred at 45ºC for 2 h. The solution was then cooled to room temperature and concentrated under reduced pressure (70% by volume). Finally, cold water (500 mL) was added to the reaction mixture and stirred for 10 min to give a pale yellow solid. The solid was filtered, washed with water and dried to give a pure compound (R)-2-((2-chloro-5-nitropyridin-4-yl)amino)propionamide 3' (55.0 g, 86% yield) as a pale yellow solid.

[0206] LCMS Method 1: Retention time: 0.88 min, 99.29% purity, at 220 nm, [M+H] + = 245.0.

[0207] Step 2'. Preparation of (R)-2-((2-chloro-5-nitropyridin-4-yl)amino)propionitrile 4': Pyridine (179.0 mL, 2207 mmol) was slowly added to a stirred solution of (R)-2-((2-chloro-5-nitropyridin-4-yl)amino)propionamide 3' (54.0 g, 221 mmol) in DCM (500 mL) at 0ºC for 10 min. Then, TFAA (156.0 mL, 1104 mmol) was added dropwise (over 30 min) at 0ºC and stirred at RT for 3 h. The reaction mixture was cooled to 0ºC and treated with ice-cold water (500 mL). The reaction mixture was extracted with DCM (3 x 300 mL), and the combined organic layers were washed with 1.0 N HCl (200 mL), followed by washing with a brine solution (500 mL), dried over sodium sulfate, and concentrated under reduced pressure to give the crude product. The crude product was washed with MTBE (3 x 200 mL) to give a pale yellow solid and dried to give a pure compound (R)-2-((2-chloro-5-nitropyridin-4-yl)amino)propionitrile 4' (46.0 g, 92% yield) as a pale yellow solid.

[0208] LCMS Method 1: Retention time: 1.75 min, 99.94% purity, at 220 nm, [MH] + = 225.0.

[0209] Step 3'. Preparation of (R)-2-((5-nitro-2-(1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-4-yl)amino)propionitrile 6': Under nitrogen atmosphere and at RT, 1H-pyrazolo[3,4-d]pyrimidin 5' (5.83 g, 48.5 mmol), zinc acetate (8.10 g, 44.1 mmol), and DIPEA (46.2 mL, 265 mmol) were added to a stirred solution of (R)-2-((2-chloro-5-nitropyridin-4-yl)amino)propionitrile 4' (10.0 g, 44.1 mmol) in dioxane (80 mL). Pd2(dba)3 (2.02 g, 2.206 mmol) was added, and the reaction mixture was degassed for 15 min. The reaction mixture was stirred at 85ºC for 16 h. The reaction mixture was cooled to RT, filtered through a diatomaceous earth bed, and concentrated under reduced pressure to give a crude product. The crude product was purified by rapid silica gel column chromatography (using 10% methanol / DCM) to give the compound (R)-2-((5-nitro-2-(1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-4-yl)amino)propionitrile 6' (7.0 g, 48.3% yield) as a yellow solid.

[0210] LCMS Method 2: Retention time: 2.33 min, 94.01% purity, at 220 nm, [M+H] + = 311.

[0211] Step 4'. Preparation of (R)-2-((5-amino-2-(1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-4-yl)amino)propionitrile 7': Iron (3.15 g, 56.4 mmol) and ammonium chloride (3.02 g, 56.4 mmol) were added to a stirred solution of (R)-2-((5-nitro-2-(1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-4-yl)amino)propionitrile 6' (7.0 g, 22.56 mmol) in 150 mL ethanol:water (4:1) and stirred at 85ºC for 4 h. The reaction mixture was cooled to RT, treated with ice-cold saturated sodium bicarbonate solution (50 mL) and extracted with 20% methanol (3 x 200 mL) in DCM. The combined organic layers were dried over sodium sulfate and concentrated under reduced pressure to give crude compound (R)-2-((5-amino-2-(1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-4-yl)amino)propionitrile 7' (3.0 g), which was used for the next step without further purification.

[0212] LCMS Method 1: Retention Time: 2.20 min, [M+H] + = 281.2

[0213] Step 5'. Preparation of (R)-2-((5-azido-2-(1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-4-yl)amino)propionitrile 8': At room temperature, ADMP (2.195 g, 26.8 mmol) and DMAP (1.961 g, 16.05 mmol) were added to a stirred solution of (R)-2-((5-amino-2-(1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-4-yl)amino)propionitrile 7' (3.0 g, 10.70 mmol) in acetonitrile (30 mL) and stirred for 16 h under a nitrogen atmosphere. The reaction mixture was treated with ice-cold water and extracted with ethyl acetate (3 x 100 mL). The combined organic layers were washed with brine, dried over sodium sulfate, and then concentrated under reduced pressure to give compound (R)-2-((5-azido-2-(1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-4-yl)amino)propionitrile 8' (4.0 g), which was used directly in the next step without further purification.

[0214] LCMS Method 1: Retention time: 1.15 min, [M+H] + = 305.

[0215] Step 6'. Preparation of (2R)-2-((2-(1H-pyrazolo[3,4-d]pyrimidin-1-yl)-5-(4-((1r,4R)-4-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)cyclohexyl)-1H-1,2,3-triazol-1-yl)pyridin-4-yl)amino)propionitrile 9': At room temperature, 2-(2-(((1r,4r)-4-ethynylcyclohexyl)ethoxy)tetrahydro-2H-pyran L-1 (3.40 g) was added to a stirred solution of (R)-2-((5-azido-2-(1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyridin-4-yl)amino)propionitrile 8' (4.0 g, 13.06 mmol) in 55.0 mL acetone:water (11:1) (3.40 g). The reaction mixture was prepared with sodium ascorbate (0.536 g, 6.53 mmol) and copper(II) sulfate pentahydrate (1.630 g, 6.53 mmol). The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was treated with water and extracted with ethyl acetate (3 x 40 mL). The combined organic layers were dried over sodium sulfate and concentrated under reduced pressure to give the crude product. The crude product was purified by column chromatography (using silica gel (230-400 mesh) and eluted with 70%-90% ethyl acetate in petroleum ether) to give the compound (2R)-2-((2-(1H-pyrazolo[3,4-d]pyrimidin-1-yl)-5-(4-((1r,4R)-4-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)cyclohexyl)-1H-1,2,3-triazol-1-yl)pyridin-4-yl)amino)propionitrile 9' (1.6 g, 18.69% yield) as a yellow solid.

[0216] LCMS Method 1: Retention time: 2.72 min, 82.87% purity, at 220 nm, [M+H] + = 543.2.

[0217] Step 7'. Preparation of 6-amino-1-(4-(((R)-1-cyanoethyl)amino)-5-(4-((1r,4R)-4-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)cyclohexyl)-1H-1,2,3-triazol-1-yl)pyridin-2-yl)-1H-pyrazolo[3,4-b]pyridine-5-carboxynitrile 10': Under RT, to (2R)-2-((2-(1H-pyrazolo[3,4-d]pyrimidin-1-yl)-5-(4-((1r,4R)-4-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)cyclohexyl)-1H-1,2,3-triazol-1-yl)pyridin-4-yl)amino)propionitrile 9' (1.1 g, 2.027 g) DIPEA (2.83 mL, 16.22 mmol) was added to a stirred solution of 2-propanol (10 mL). The reaction mixture was warmed to 80ºC and stirred for 10 min, followed by the addition of malononitrile (0.453 mL, 10.14 mmol) at the same temperature and stirring at 80ºC for another 6 h. The reaction mixture was cooled to RT, filtered, and the solid was washed with 2-propanol (2 x 10 mL) and dried to give the crude compound 6-amino-1-(4-(((R)-1-cyanoethyl)amino)-5-(4-((1r,4R)-4-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)cyclohexyl)-1H-1,2,3-triazol-1-yl)pyridin-2-yl)-1H-pyrazolo[3,4-b]pyridin-5-carboxynitrile 10' (1.0 g), which was used directly for the next step without further purification.

[0218] LCMS Method 1: Retention time: 2.23 min, [M+H] + = 582.2.

[0219] Step 8'. Preparation of 6-amino-1-(4-(((R)-1-cyanoethyl)amino)-5-(4-((1r,4R)-4-(2-hydroxyethyl)cyclohexyl)-1H-1,2,3-triazol-1-yl)pyridin-2-yl)-1H-pyrazolo[3,4-b]pyridin-5-carboxylonitrile 11': Under RT, 6-amino-1-(4-(((R)-1-cyanoethyl)amino)-5-(4-((1r,4R)-4-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)cyclohexyl)-1H-1,2,3-triazol-1-yl)pyridin-2-yl)-1H-pyrazolo[3,4-b]pyridin-5-carboxylonitrile 10' (800 mg, 1.375 mg) pTSA (131 mg, 0.688 mmol) was added to a stirred solution in MeOH and stirred for 1 h. The reaction mixture was diluted with 20% methanol in DCM (50 mL) and treated with saturated sodium bicarbonate solution (20 mL). The organic layer was collected, washed with brine, dried over sodium sulfate, and concentrated under reduced pressure to give the crude product. The crude product was washed with MTBE (3 x 20 mL) to give compound 6-amino-1-(4-(((R)-1-cyanoethyl)amino)-5-(4-((1r,4R)-4-(2-hydroxyethyl)cyclohexyl)-1H-1,2,3-triazol-1-yl)pyridin-2-yl)-1H-pyrazolo[3,4-b]pyridin-5-carboxynitrile 11' (450 mg, 61.1% yield).

[0220] Step 9'. Preparation of 6-amino-1-(4-(((R)-1-cyanoethyl)amino)-5-(4-((1r,4R)-4-(2-oxoethyl)cyclohexyl)-1H-1,2,3-triazol-1-yl)pyridin-2-yl)-1H-pyrazolo[3,4-b]pyridin-5-carboxylonitrile A-2: Under RT, IBX (760 mg, 2.71 mmol) was added to a stirred solution of 6-amino-1-(4-(((R)-1-cyanoethyl)amino)-5-(4-((1r,4R)-4-(2-hydroxyethyl)cyclohexyl)-1H-1,2,3-triazol-1-yl)pyridin-2-yl)-1H-pyrazolo[3,4-b]pyridin-5-carboxylonitrile 11' (450 mg, 0.904 mmol) in DMSO (5 mL). The mixture was stirred for 2 h. The reaction mixture was quenched with sodium bicarbonate solution and extracted with ethyl acetate (3 x 20 mL). The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated under reduced pressure to give compound 6-amino-1-(4-(((R)-1-cyanoethyl)amino)-5-(4-((1r,4R)-4-(2-oxoethyl)cyclohexyl)-1H-1,2,3-triazol-1-yl)pyridin-2-yl)-1H-pyrazolo[3,4-b]pyridin-5-carboxynitrile A-2 (400 mg), which was used for the next step without further purification. Example S5: Preparation of 6-(5-cyano-1H-pyrazolo[3,4-b]pyridin-1-yl)-4-(((R)-1-cyanoethyl)amino)-N-((1r,4R)-4-(2-oxoethyl)cyclohexyl)nicotinamide (A-3)

[0221] LCMS Method 1. Column: Luna C18 (2) 50 x 3 mm, 3 μm. Temperature: 45ºC, Flow rate: 1.5 mL / min, Run time: 2.5 min. Mobile phase conditions: Initial 95% H2O 0.1% FA / 5% MeCN 0.1% FA, linear gradient change to 95% MeCN 0.1% FA over 1.3 min, then held at 95% MeCN 0.1% FA for 1.2 min. MSD: ESI positive ion.

[0222] LCMS Method 2.Kinetex XB-C18, 75 x 3.0 mm, 2.6 μm. Temperature: RT, Flow rate: 1.0 mL / min, Run time: 5.0 min. Mobile phase conditions: Mobile phase A: 5.0 mm ammonium formate, pH 3.3: CH3CN (98:02), Mobile phase B: CH3CN in 5.0 mm ammonium formate (98:02), Gradient: A linear gradient from initial 80% mobile phase A and 20% mobile phase B to 100% mobile phase B over 4.6 min. MSD positive ions.

[0223] Step 1'. Preparation of tert-butyl 6-chloro-4-[[(1R)-2-amino-1-methyl-2-oxo-ethyl]amino]pyridine-3-carboxylate 3': Under nitrogen atmosphere, DIPEA (2.81 mL, 16.122 mmol, 4.0 equivalents) was added to a solution of (2R)-2-aminopropionamide hydrochloride 2' (1.5 g, 12.091 mmol, 3.0 equivalents) and tert-butyl 4,6-dichloropyridine-3-carboxylate 1' (1.0 g, 4.030 mmol, 1.0 equivalents) in MeCN (20 mL, 0.2 M). The reaction mixture was stirred at 65ºC for 96 h. The reaction mixture was cooled to room temperature. Acetonitrile was removed under high vacuum. DCM and water were added, and the phases were separated. The aqueous phase was extracted three times with DCM. The combined organic phases were washed once with brine, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by normal-phase rapid chromatography (40 g silica column, elution: 0 to 10% CH2Cl2 / CH3OH, 15 CV, product exit at 5% CH3OH). The fractions were combined and concentrated to give tert-butyl 6-chloro-4-[[(1R)-2-amino-1-methyl-2-oxo-ethyl]amino]pyridine-3-carboxylate 3' (1 g, 83% yield) as a white solid.

[0224] LCMS Method 1: Retention time: 1.601 min, 99.9% purity, at 215 nm, [M+H] + = 300.2.

[0225] 1 H NMR (400 MHz, DMSO-d6): δ ppm 1.36 (d, J = 6.8 Hz, 3 H), 1.55 (s, 9H), 4.15 (t, J = 6.8 Hz, 1 H), 6.57 (s, 1 H), 7.31 (br s, 1 H), 7.62 (br s, 1H), 8.44 (d, J = 6.8 Hz, 1 H), 8.50 (s, 1 H).

[0226] Step 2'. Preparation of tert-butyl 6-chloro-4-[[(1R)-1-cyanoethyl]amino]pyridine-3-carboxylate 4': At -15ºC, tert-butyl 6-chloro-4-[[(1R)-2-amino-1-methyl-2-oxo-ethyl]amino]pyridine-3-carboxylate 3' (1.0 g, 3.336 mmol, 1.0 equivalent) and pyridine (2.7 mL, 33.361 mmol, 10.0 equivalent) in DCM (19.0 mL) were added dropwise to a solution of 6-chloro-4-[[(1R)-2-amino-1-methyl-2-oxo-ethyl]amino]pyridine-3-carboxylate 3' (1.0 g, 3.336 mmol, 1.0 equivalent) in DCM (11.4 mL, 0.1 M) (0.9 mL, 6.672 mmol, 2.0 equivalent). The resulting mixture was warmed to room temperature and stirred for 2.5 h. The mixture was washed with 1 M HCl (3 x 30 mL), saturated sodium bicarbonate (1 x 40 mL), and brine (1 x 40 mL). The organic layer was dried over Na2SO4, filtered, and concentrated. The residue was purified by normal-phase rapid chromatography (40 g silica gel column, elution: 0 to 5% CH2Cl2 / CH3OH, 15 CV, product exit at 1.0% CH3OH). The fractions were combined and concentrated to give tert-butyl 6-chloro-4-[[(1R)-1-cyanoethyl]amino]pyridine-3-carboxylate 4' (0.76 g, 84% yield) as a white solid.

[0227] LCMS Method 1: Retention time: 1.833 min, 98.7% purity, at 215 nm, [M+H] + = 282.2.

[0228] 1 H NMR (400 MHz, DMSO-d6): δ ppm 1.56 (s, 9 H), 1.66 (d, J = 7.1 Hz, 3H), 5.01 (quin, J = 7.1 Hz, 1 H), 7.06 (s, 1 H), 8.19 (d, J = 7.6 Hz, 1 H), 8.59 (s, 1 H).

[0229] Step 3'. Preparation of 6-(5-cyanopyrazolo[3,4-b]pyridin-1-yl)-4-[[(1R)-1-cyanoethyl]amino]pyridine-3-carboxylic acid tert-butyl ester 6': 6-chloro-4-[[(1R)-1-cyanoethyl]amino]pyridine-3-carboxylic acid tert-butyl ester 4' (760.0 mg, 2.697 mmol, 1.0 equivalent), 1H-pyrazolo[3,4-b]pyridine-5-carboxylonitrile 5' (388.8 mg, 2.697 mmol, 1.0 equivalent), XantPhos (0.234 g, 0.404 mmol, 0.15 equivalent), and Pd2(dba)3·CHCl3 (418.8 mg, 0.404 mmol, 0.3 M) in 1,4-dioxane (9.0 mL, 0.3 M) were added to a dry, sealed tube. 494.9 mg (2.697 mmol, 1.0 equivalent) and Zn(OAc)2 (494.9 mg, 2.697 mmol, 1.0 equivalent). Nitrogen gas was then bubbled through the mixture for 15 minutes. The tube was sealed and the mixture was stirred at 105ºC for 16 hours. The reactants were filtered through diatomaceous earth, washed with DCM, and the filtrate was evaporated. The residue was subjected to normal-phase rapid chromatography (40 g silica column, elution: 0 to 10% DCM / MeOH, 15 CV, product exit at 5% MeOH). The fractions of the future autonomous peaks were combined and concentrated under reduced pressure to give 6-(5-cyanopyrazolo[3,4-b]pyridin-1-yl)-4-[[(1R)-1-cyanoethyl]amino]pyridine-3-carboxylic acid tert-butyl ester 6' (906 mg, 77% yield) as an orange solid. Contaminated with XantPhosO2.

[0230] LCMS Method 1: Retention time: 1.781 min, 88% purity, at 215 nm, [M+H] + = 390.2.

[0231] 1 H NMR (400 MHz, DMSO-d6): δ ppm 1.60 (s, 9 H), 1.72 (br d, J = 6.8Hz, 3 H), 4.99 - 5.10 (m, 1 H), 7.60 (s, 1 H), 8.30 (br d, J = 7.1 Hz, 1 H), 8.71 (s, 1 H), 8.87 (s, 1 H), 9.05 (dd, J = 12.1, 1.8 Hz, 1 H), 11.96 (br s,1 H).

[0232] Step 4'. Preparation of 6-(5-cyanopyrazolo[3,4-b]pyridin-1-yl)-4-[[(1R)-1-cyanoethyl]amino]pyridine-3-carboxylic acid 7': To a solution of 6-(5-cyanopyrazolo[3,4-b]pyridin-1-yl)-4-[[(1R)-1-cyanoethyl]amino]pyridine-3-carboxylic acid tert-butyl ester 6' (1.17 g, 3.025 mmol, 1.0 equivalent) in DCM (8.9 mL, 0.3 M), TFA (11.6 mL, 45.376 mmol, 15 equivalent) was added. The reaction was stirred at room temperature for 16 hours. The solvent was removed under vacuum, and the residue was co-evaporated with PhMe (3 times), followed by MeCN (2 times), to give a yellow semi-solid. H₂O (10 mL) / MeCN (2 mL) / EtOAc (2 mL) was added, and the mixture was sonicated for 3–5 min. The solvent was evaporated to near dryness, forming a precipitate, which was filtered and washed with MTBE to give 182 mg of 7'. Analysis of the remaining solution showed a mixture of 7' and byproducts. The product was purified by reversed-phase chromatography (using a 50G C18 column, elution: 5% MeOH / 0.1% HCOOH, 4 CV, then 5% to 75% MeOH / 0.1% HCOOH, 15 CV, product exiting at 48% MeOH). The pure fractions were combined and concentrated under reduced pressure to give 6-(5-cyanopyrazolo[3,4-b]pyridin-1-yl)-4-[[(1R)-1-cyanoethyl]amino]pyridine-3-carboxylic acid 7' (315 mg, 29% yield) as a white solid.

[0233] LCMS Method 1: Retention time: 1.372 min, 99.9% purity, at 215 nm, [M+H] + = 334.2.

[0234] 1 H NMR (400 MHz, DMSO-d6): δ ppm 1.71 (d, J = 6.8 Hz, 3 H), 4.99 -5.10 (m, 1 H), 7.59 (s, 1 H), 8.55 (br d, J = 7.6 Hz, 1 H), 8.72 (s, 1 H),8.88 (s, 1 H), 9.04 (d, J = 2.0 Hz, 1 H), 9.07 (d, J = 2.0 Hz, 1 H), 13.63(br s, 1 H).

[0235] Step 5'. Preparation of 6-(5-cyano-1H-pyrazolo[3,4-b]pyridin-1-yl)-4-(((R)-1-cyanoethyl)amino)-N-((1r,4R)-4-(2-hydroxyethyl)cyclohexyl)nicotinamide 8': At room temperature, under a nitrogen atmosphere, HATU (5.99 g, 15.75 mmol) and DIPEA (9.17 mL, 52.5 mmol) were added to a stirred solution of (R)-6-(5-cyano-1H-pyrazolo[3,4-b]pyridin-1-yl)-4-((1-cyanoethyl)amino)hydrochloric acid 7' (3.50 g, 10.50 mmol) in DCM (30 mL). The reaction mixture was stirred at room temperature under a nitrogen atmosphere for 10 minutes, and 2-((1r,4r)-4-aminocyclohexyl)ethanol-1-ol L-3 (2.256 g, 15.75 mmol) was added. The reaction mixture was then stirred at RT for 8 h. The reaction mixture was concentrated under reduced pressure, and the crude product was treated with cold water (250 mL) and extracted with ethyl acetate (2 x 300 mL). The organic layers were washed with brine, and the combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (60-120 mesh), and the desired product was eluted with 40% ethyl acetate / petroleum ether to give 6-(5-cyano-1H-pyrazolo[3,4-b]pyridin-1-yl)-4-(((R)-1-cyanoethyl)amino)-N-((1r,4R)-4-(2-hydroxyethyl)cyclohexyl)nicotinamide 8' (4.62 g).

[0236] LCMS Method 1: Retention time: 2.309 min, [M+H] + = 459.2

[0237] Step 6'. Preparation of 6-(5-cyano-1H-pyrazolo[3,4-b]pyridin-1-yl)-4-(((R)-1-cyanoethyl)amino)-N-((1r,4R)-4-(2-oxoethyl)cyclohexyl)nicotinamide A-3: At room temperature and under a nitrogen atmosphere, IBX (1710 mg, 2.75 mmol) was added participle-by-parts to a stirred solution of 6-(5-cyano-1H-pyrazolo[3,4-b]70pyridin-1-yl)-4-(((R)-1-cyanoethyl)amino)-N-((1r,4R)-4-(2-hydroxyethyl)cyclohexyl)nicotinamide 8' (500 mg, 0.916 mmol) in DMSO (10 mL), and the reaction mixture was stirred at room temperature for 4 h. The reaction mixture was quenched with saturated sodium bicarbonate solution and extracted with ethyl acetate (3 x 100 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to give 6-(5-cyano-1H-pyrazolo[3,4-b]pyridin-1-yl)-4-((R)-1-cyanoethyl)amino)-N-((1r,4R)-4-(2-oxoethyl)cyclohexyl)nicotinamide A-3 (330.0 mg) as a pale yellow solid. The crude product was used for the next step without further purification.

[0238] LCMS Method 2 Retention time: 1.940 min, [M+H] + = 457.2

[0239] 1 H NMR (400 MHz, CDCl3): δ ppm 1.06 – 1.19 (m, 2H), 1.33 – 1.47 (m,2H), 1.67 (d, J = 7.2 Hz, 3H), 1.72 – 1.97 (m, 4H), 2.32 – 2.39 (m, 2H), 3.70– 3.81 (m, 1H), 4.90 – 5.00 (m, 1H), 7.48 (s, 1H), 8.52 – 8.68 (m, 1H), 8.69– 8.85 (m, 3H), 9.01 – 9.11 (m, 2H), 9.67 – 9.72 (m, 1H). Example S6: Preparation of 5-chloro-6-((4-((((R)-1-cyanoethyl)amino)-5-(4-((1r,4R)-4-(2-oxoethyl)cyclohexyl)-1H-1,2,3-triazol-1-yl)pyridin-2-yl)amino)nicotinonitrile (A-4)

[0240] LCMS Method 1. Kinetex XB-C18, 50 x 4.6 mm, 5.0 μm. Temperature: RT, flow rate: 1.0 mL / min, run time: 5.5 min. Mobile phase conditions: Mobile phase A: 0.1% TFA in H2O, mobile phase B: 0.1% TFA in ACN, gradient: initial 95% mobile phase A and 5% mobile phase B linearly changed to 95% mobile phase B over 2.5 min. MSD positive ions.

[0241] LCMS Method 2. Kinetex XB-C18, 75 x 3.0 mm, 2.6 μm. Temperature: RT, Flow rate: 1.0 mL / min, Run time: 5.0 min. Mobile phase conditions: Mobile phase A: 5.0 mm ammonium formate, pH 3.3: CH3CN (98:02), Mobile phase B: CH3CN: 5.0 mm ammonium formate, pH 3.3 (98:02), Gradient: A linear gradient from initial 98% mobile phase A and 2% mobile phase B to 100% mobile phase B over 4 min. MSD positive ions.

[0242] Step 1'. Preparation of (R)-5-chloro-6-((4-(((1-cyanoethyl)amino)-5-nitropyridin-2-yl)amino)nicotinonitrile 3': Pd2(dba)3 (0.315 g, 0.344 mmol) was added to a stirred solution of (R)-2-((2-chloro-5-nitropyridin-4-yl)amino)propionitrile 1' (2.5 g, 8.60 mmol) and 6-amino-5-chloronicotinonitrile 2' (1.32 g, 8.60 mmol) in 1,4-dioxane (25 mL) and purged with nitrogen for 5 min. Xantphos (0.398 g, 0.688 mmol), followed by zinc acetate (0.474 g, 2.58 mmol) and K2CO3 (2.97 g, 21.5 mmol) was added to the reaction mixture and purged with nitrogen for 2 min. The reaction mixture was stirred at 100ºC under a nitrogen atmosphere for 16 h. The reaction mixture was then filtered through a diatomaceous earth bed, which was washed with 5% MeOH / DCM (500 mL). The filtrate was then transferred to a separatory funnel containing water (400 mL) and extracted with 5% MeOH / DCM (2 x 250 mL). The combined organic extracts were washed with brine (100 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The resulting solid was washed with ethyl acetate (50 mL), filtered, and dried under vacuum to give (R)-5-chloro-6-((4-(((1-cyanoethyl)amino)-5-nitropyridin-2-yl)amino)nicotinonitrile 3' (2.13 g, 60.6% yield) as a brown solid. It was used for the next step without further purification.

[0243] LCMS Method 1: Retention time: 2.59 min, [M+H] + = 344.1.

[0244] 1 H NMR (400 MHz, DMSO-d6): δ ppm 1.74 (d, J = 6.8 Hz, 3H), 4.96 – 5.04(m, 1H), 7.78 (s, 1H), 8.34 (d, J = 7.2 Hz, 1H), 8.54 (d, J = 1.6 Hz, 1H), 8.71 (d, J = 1.6 Hz, 1H), 9.00 (s, 1H), 9.58 (brs, 1H).

[0245] Step 2'. Preparation of (R)-6-((5-amino-4-((1-cyanoethyl)amino)pyridin-2-yl)amino)-5-chloronicotinonitrile 4': 10% palladium on carbon (0.547 g) was added to a stirred solution of (R)-5-chloro-6-((4-((1-cyanoethyl)amino)-5-nitropyridin-2-yl)amino)nicotinonitrile 3' (2.10 g, 5.14 mmol) in 1,4-dioxane (20 mL). The resulting reaction mixture was stirred at room temperature for 72 h under a hydrogen atmosphere. The reaction mixture was filtered through a diatomaceous earth bed, and the diatomaceous earth bed was washed with 5% MeOH / DCM (150 mL). The filtrate was evaporated under reduced pressure to give (R)-6-((5-amino-4-((1-cyanoethyl)amino)pyridin-2-yl)amino)-5-chloronicotinonitrile 4' as a brown solid (1.8 g, 76% yield), which was used for the next step without further purification.

[0246] LCMS Method 1: Retention time: 1.92 min, [M+H] + = 314.1.

[0247] Step 3. Preparation of (R)-6-((5-azido-4-((1-cyanoethyl)amino)pyridin-2-yl)amino)-5-chloronicotinonitrile 5': DMF (2.5 mL) was added to a stirred solution of (R)-6-((5-amino-4-((1-cyanoethyl)amino)pyridin-2-yl)amino)-5-chloronicotinonitrile 4' (500 mg, 1.08 mmol) and ADMP (649 mg, 2.16 mmol) in acetonitrile (10 mL). Then, DMF (264 mg, 2.161 mmol) was added to the reaction mixture, and the reaction mixture was stirred at room temperature for 16 h. The reaction mixture was then transferred to an ice-filled conical flask, and the solution was stirred at room temperature for 30 min, producing a brown solid. The solid was filtered, washed with ice-cold water, and evaporated to dryness to give (R)-6-((5-azido-4-((1-cyanoethyl)amino)pyridin-2-yl)amino)-5-chloronicotinonitrile 5' as a brown solid (513 mg, 0.907 mmol, 84% yield). It was used for the next step without further purification.

[0248] LCMS Method 2: Retention time: 1.742 min, [M+H] + = 340.0.

[0249] Step 4'. Preparation of 1-(4-(((R)-1-cyanoethyl)amino)-5-(4-((1r,4R)-4-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)cyclohexyl)-1H-1,2,3-triazol-1-yl)pyridin-2-yl)-1H-pyrazolo[3,4-b]pyridine-5-carboxynitrile 6': 2-((1r,4r)-4-ethynylcyclohexyl)ethanol-1-ol 2' (110 mg, 0.721 mmol) was added to a stirred solution of ((R)-6-((5-azido-4-((1-cyanoethyl)amino)pyridin-2-yl)amino)-5-chloronicotinonitrile 5' (360 mg, 0.721 mmol) in acetone (5.0 mL), followed by the addition of sodium ascorbate (71.4 mL). A solution of copper(II) sulfate pentahydrate (65.4 mg, 0.360 mmol) in H2O (0.556 mL) was added to the reaction mixture at room temperature. After stirring at room temperature for 2 h, the reaction mixture was concentrated using a rotary evaporator, and the residue was diluted with water (10 mL) and stirred at room temperature for 15 min. The brown solid was filtered, washed with water, and evaporated to dryness to give 5-chloro-6-((4-((((R)-1-cyanoethyl)amino)-5-(4-((1r,4R)-4-(2-hydroxyethyl)cyclohexyl)-1H-1,2,3-triazol-1-yl)pyridin-2-yl)amino)nicotinonitrile 6' (350 mg) as a brown solid. The crude product was used for the next step without further purification.

[0250] LCMS Method 1: Retention time: 2.037 min, [M+H] + = 492.3.

[0251] Step 5'. 5-Chloro-6-((4-((((R)-1-cyanoethyl)amino)-5-(4-((1r,4R)-4-(2-oxoethyl)cyclohexyl)-1H-1,2,3-triazol-1-yl)pyridin-2-yl)amino)nicotinonitrile A-4: Under a nitrogen atmosphere at room temperature, add IBX (384 mg, 1.372 mmol) to a stirred solution of 5-chloro-6-((4-((((R)-1-cyanoethyl)amino)-5-(4-(((1r,4R)-4-(2-hydroxyethyl)cyclohexyl)-1H-1,2,3-triazol-1-yl)pyridin-2-yl)amino)nicotinonitrile 6' (600 mg, 0.549 mmol) in DMSO (4 mL) and stir the reaction mixture at room temperature for 4 h. The reactants were treated with 10% NaHCO3 solution (30 mL) and stirred at room temperature for 15 min. The brown solid was filtered, washed with water, and evaporated to dryness to give 5-chloro-6-((4-((((R)-1-cyanoethyl)amino)-5-(4-((1r,4R)-4-(2-oxoethyl)cyclohexyl)-1H-1,2,3-triazol-1-yl)pyridin-2-yl)amino)nicotinonitrile A-4 (600 mg) as a brown solid. The crude product was used for the next step without further purification.

[0252] LCMS Method 2: Retention time: 2.748 min, [M+H] + = 490.0. Program for CBM synthesis Example S7: Preparation of 6-(piperazin-1-yl)-2H-spiro[benzofuran-3,3'-piperidine]-2',6'-dione (C-1 and C-2)

[0253] LCMS Method 1. Kinetex XB-C18, 75 x 3.0 mm, 2.6 μm. Temperature: RT, Flow rate: 1.0 mL / min, Run time: 4.7 min. Mobile phase conditions: Mobile phase A: 5.0 mm ammonium formate, pH 3.3: CH3CN (98:02), Mobile phase B: CH3CN in 5.0 mm ammonium formate (98:02), Gradient: A linear gradient from initial 80% mobile phase A and 20% mobile phase B to 100% mobile phase B over 4.6 min. MSD positive ions.

[0254] LCMS Method 2.Kinetex XB-C18, 50 x 4.6 mm, 5.0 μm. Temperature: RT, flow rate: 1.0 mL / min, run time: 5.5 min. Mobile phase conditions: Mobile phase A: 0.1% TFA in H2O, mobile phase B: 0.1% TFA in ACN, gradient: initial 95% mobile phase A and 5% mobile phase B linearly changed to 95% mobile phase B over 2.5 min. MSD positive ions.

[0255] Step 1'. Preparation of ethyl 6-bromobenzofuran-3-carboxylate 2': Add tetrafluoroborate diethyl ether complex (0.572 mL, 4.23 mmol) to a stirred solution of 4-bromo-2-hydroxybenzaldehyde 1' (8.5 g, 42.3 mmol) in CH2Cl2 (1.0 mL), followed by dropwise addition of ethyl diazonium ether solution in toluene (27.0 mL, 42.3 mmol) to the reaction mixture, and the reaction should not be carried out above 38ºC. Once the gas release stops, concentrate the reaction mixture using a rotary evaporator and add H2SO4 (0.3 to 0.5 mL) to the mixture while stirring. After 5 to 10 min, dilute the mixture with CH2Cl2 (5 to 10 mL) and quench the H2SO4 with saturated NaHCO3 solution. The reaction mixture was extracted with DCM, and the combined organic layers were dried over sodium sulfate, filtered, concentrated, and the crude compound was purified by column chromatography (using silica gel (230-400 mesh) with 0-5% ethyl acetate / petroleum ether) to give ethyl 6-bromobenzofuran-3-carboxylate 2' (5.5 g, 48.3% yield) as a pale yellow solid.

[0256] 1 H NMR (400 MHz, CDCl3): δ ppm 1.44 (t, J = 7.2 Hz, 3H), 4.43 (q, J =7.2 Hz, 2H), 7.50 (dd, J = 8.2 Hz and 1.6 Hz, 1H), 7.74 (d, J = 1.6 Hz, 1H), 7.95 (d, J = 8.4 Hz, 1H), 8.24 (s, 1H).

[0257] Step 2'. Preparation of tert-butyl piperazine-1-carboxylate 4': Tert-butyl piperazine-1-carboxylate 3' (2.492 g, 13.38 mmol) was added to a stirred solution of ethyl 6-bromobenzofuran-3-carboxylate 2' (3.0 g, 11.15 mmol) in DMF (90 mL), followed by the addition of Cs₂CO₃ (7.26 g, 22.30 mmol). The resulting reaction mixture was degassed with nitrogen for 5 min, and then RuPhos Pd G₃ (0.466 g, 0.557 mmol) was added. The reaction mixture was heated to 90°C. o C. Stir in a sealed glass vial for 6 h. Cool the reaction mixture to room temperature and concentrate under reduced pressure to give the crude product. The crude product was then purified by silica gel column chromatography (using 15%-20% ethyl acetate in petroleum ether) to give 4'-(3-(ethoxycarbonyl)benzofuran-6-yl)piperazine-1-carboxylic acid tert-butyl ester (1.8 g, 4.52 mmol, 40.5% yield) as a pale yellow solid.

[0258] LCMS Method 2: Retention time: 2.837 min, [M+H] + = 374.4.

[0259] Step 3'. Preparation of tert-butyl 4-(3-(methoxycarbonyl)-2,3-dihydrobenzofuran-6-yl)piperazine-1-carboxylate 5': Magnesium (366 mg, 15.06 mmol) was added fractionally to a stirred solution of tert-butyl 4-(3-(ethoxycarbonyl)benzofuran-6-yl)piperazine-1-carboxylate 4' (600 mg, 1.506 mmol) in MeOH (40 mL) at 0–20ºC under a nitrogen atmosphere. The reaction mixture was then heated at RT and stirred for 48 h. The reaction mixture was quenched with an aqueous solution of ammonium chloride (100 mL) and concentrated to remove methanol. The crude product was then extracted with ethyl acetate (3 x 100 mL). The separated organic layer was washed with brine (40 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by column chromatography (using 13%-15% ethyl acetate in petroleum ether) to give tert-butyl 4-(3-(methoxycarbonyl)-2,3-dihydrobenzofuran-6-yl)piperazine-1-carboxylate 5' (1.45 g, 3.93 mmol, 87% yield) as a white solid.

[0260] LCMS Method 2: Retention time: 2.55 min, [M+H] + = 363.2.

[0261] Step 4'. Preparation of tert-butyl 4-(3-(2-cyanoethyl)-3-(methoxycarbonyl)-2,3-dihydrobenzofuran-6-yl)piperazine-1-carboxylate 6': Under RT, benzyltriethylammonium chloride (0.089 g, 0.392 mmol) was added to a stirred solution of tert-butyl 4-(3-(methoxycarbonyl)-2,3-dihydrobenzofuran-6-yl)piperazine-1-carboxylate 5' (1.450 g, 3.92 mmol) and K2CO3 (1.084 g, 7.84 mmol) in toluene (40 mL), followed by acrylonitrile (0.416 g, 7.84 mmol). The reaction mixture was then heated to 90ºC and stirred for 3 h. The reaction mixture was quenched with water (50 mL) and extracted with ethyl acetate (3 x 50.0 mL). The combined organic layers were then dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give tert-butyl 4-(3-(2-cyanoethyl)-3-(methoxycarbonyl)-2,3-dihydrobenzofuran-6-yl)piperazine-1-carboxylate 6' (1.7 g, 3.56 mmol, 91% yield), a viscous brown liquid. The product was used for the next step without further purification.

[0262] LCMS Method 2: Retention time: 2.615 min, [M+H] + = 416.3.

[0263] Step 5'. Preparation of 6-(piperazin-1-yl)-2H-spiro[benzofuran-3,3'-piperidine]-2',6'-dione, 2 AcOH 7': H2SO4 (0.353 mL, 6.63 mmol) was added to a stirred solution of 4-(3-(2-cyanoethyl)-3-(methoxycarbonyl)-2,3-dihydrobenzofuran-6-yl)piperazin-1-carboxylic acid tert-butyl ester 6' (1.7 g, 3.31 mmol) in AcOH (40.0 mL). The resulting reaction mixture was heated to 120 °C. o C and stirred for 12 h. The reaction mixture was concentrated under reduced pressure to give crude 6-(piperazin-1-yl)-2H-spiro[benzofuran-3,3'-piperidine]-2',6'-dione, 2 AcOH, 7' (2.0 g) as a viscous brown liquid, which was used for the next step without further purification.

[0264] LCMS Method 2: Retention time: 1.262 min, [M+H] + = 302.2.

[0265] Step 6'. Preparation of tert-butyl 4-(2',6'-dioxo-2H-spiro[benzofuran-3,3'-piperidine]-6-yl)piperazine-1-carboxylate 8': DIPEA (8.0 mL, 45.8 mmol) was added to a stirred solution of 6-(piperazine-1-yl)-2H-spiro[benzofuran-3,3'-piperidine]-2',6'-dione, 2AcOH, 7' (2.0 g) in acetonitrile (30.0 mL) and stirred for 5 min. Then, ditert-butyl dicarbonate (3.36 mL, 4.47 mmol) was added to the reaction mixture, and the resulting reaction mixture was stirred at RT for 4 h. The reaction mixture was diluted with water (50 mL) and extracted with DCM (3 x 100 mL). The combined organic layers were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (using silica gel (230-400 mesh) with 60% ethyl acetate / petroleum ether) to give tert-butyl 4-(2',6'-dioxo-2H-spiro[benzofuran-3,3'-piperidin]-6-yl)piperazine-1-carboxylate 8' (650 mg, 1.587 mmol, 54.8% yield) as a white solid.

[0266] LCMS Method 2: Retention time: 2.319 min, [M+H] + = 402.2.

[0267] Step 7'. Chiral SFC resolution of 4-(2',6'-dioxo-2H-spiro[benzofuran-3,3'-piperidine]-6-yl)piperazine-1-carboxylic acid tert-butyl ester 9a' (peak 1) and 4-(2',6'-dioxo-2H-spiro[benzofuran-3,3'-piperidine]-6-yl)piperazine-1-carboxylic acid tert-butyl ester 9b' (peak 2): 750 mg of the racemic compound was purified by chiral SFC to give 4-(2',6'-dioxo-2H-spiro[benzofuran-3,3'-piperidine]-6-yl)piperazine-1-carboxylic acid tert-butyl ester 9a' (240 mg). mg, peak 1) and tert-butyl 4-(2',6'-dioxo-2H-spiro[benzofuran-3,3'-piperidin]-6-yl)piperazine-1-carboxylate 9b' (260 mg, peak 2).

[0268] SFC method: YMC Cellulose SC, 250 x 4.6 mm, 5 μm, flow rate: 3.0 mL / min. Co-solvent: 50.0% IPA.

[0269] Peak 1 (RT 4.843 min, ee 99.95%): 4-(2',6'-dioxo-2H-spiro[benzofuran-3,3'-piperidine]-6-yl)piperazine-1-carboxylic acid tert-butyl ester 9a'.

[0270] 1 H NMR (400 MHz, DMSO-d6): δ ppm 1.41 (s, 9H), 2.08 – 2.14 (m, 2H), 2.50 – 2.74 (m, 2H), 3.05 – 3.08 (m, 4H), 3.35 – 3.43 (m, 4H), 4.39 (d, J =9.2 Hz, 1H), 4.81 (d, J = 9.2 Hz, 1H), 6.43 – 6.46 (m, 2H), 7.04 (d, J = 8.8Hz, 1H), 10.95 (s, 1H).

[0271] LCMS Method 2: Retention time: 2.321 min, 96.06% purity, at 220 nm, [M+H] + = 402.2.

[0272] Peak 2 (RT 5.995 min, ee 99.82%): 4-(2',6'-dioxo-2H-spiro[benzofuran-3,3'-piperidine]-6-yl)piperazine-1-carboxylic acid tert-butyl ester 9b'.

[0273] 1 H NMR (400 MHz, DMSO-d6): δ ppm 1.42 (s, 9H), 2.06 – 2.16 (m, 2H), 2.60 – 2.68 (m, 2H), 3.06 – 3.09 (m, 4H), 3.41 – 3.44 (m, 4H), 4.40 (d, J =9.2 Hz, 1H), 4.81 (d, J = 9.2 Hz, 1H), 6.44 – 6.46 (m, 2H), 7.04 – 7.06 (m,1H), 10.96 (s, 1H).

[0274] LCMS Method 2: Retention time: 2.322 min, 96.59% purity, at 220 nm, [M+H] + = 402.2.

[0275] Step 8a'. Preparation of 6-(piperazin-1-yl)-2H-spiro[benzofuran-3,3'-piperidine]-2',6'-dione.2HCl C-1: Under RT, 4.0N HCl (4.0 mL, 16 mmol) in 1,4-dioxane was added to a stirred solution of tert-butyl 4-(2',6'-dioxo-2H-spiro[benzofuran-3,3'-piperidine]-6-yl)piperazin-1-carboxylate 9a' (peak 1) (230 mg, 0.55 mmol) in DCM (5 mL). The resulting reaction mixture was stirred under RT for 6 h. The reaction mixture was concentrated under reduced pressure to give crude 6-(piperazin-1-yl)-2H-spiro[benzofuran-3,3'-piperidine]-2',6'-dione.2HCl C-1 (peak 1) (200 mg) as a grayish-white solid, which was used for the next step without further purification.

[0276] LCMS Method 2: Retention time: 1.284 min, [M+H] + = 302.2.

[0277] Step 8b'. Preparation of 6-(piperazin-1-yl)-2H-spiro[benzofuran-3,3'-piperidine]-2',6'-dione.2HCl C-2: Under RT, 4.0 NHCl (4.0 mL, 16 mmol) in 1,4-dioxane was added to a stirred solution of tert-butyl piperazine-1-carboxylate 9b' (peak 2) (250 mg, 0.601 mmol) in DCM (5 mL). The resulting reaction mixture was stirred under RT for 6 h. The reaction mixture was concentrated under reduced pressure to give crude 6-(piperazin-1-yl)-2H-spiro[benzofuran-3,3'-piperidine]-2',6'-dione, 2 HCl C-2 (peak 2) (210 mg) as a grayish-white solid, which was used for the next step without further purification.

[0278] LCMS Method 2: Retention time: 1.305 min, [M+H] + = 302.1.

[0279] The CBM intermediates prepared by the procedure described in Example S7 are summarized in Table 2. Table 2. Example S8: Preparation of 6-(piperidin-4-yl)-2H-spiro[benzofuran-3,3'-piperidin]-2',6'-dione (C-11 and C-12)

[0280] LCMS Method 1. Kinetex XB-C18, 50 x 4.6 mm, 5.0 μm. Temperature: RT, flow rate: 1.0 mL / min, run time: 5.5 min. Mobile phase conditions: Mobile phase A: 0.1% TFA in H2O, mobile phase B: 0.1% TFA in ACN, gradient: initial 95% mobile phase A and 5% mobile phase B linearly changed to 95% mobile phase B over 2.5 min. MSD positive ions.

[0281] LCMS Method 2. Aquity Uplc BEH C18, 50 x 2.1 mm, 1.7 μm. Temperature: RT, Flow rate: 0.7 mL / min, Run time: 2.5 min. Mobile phase conditions: Mobile phase A: 0.1% TFA in H2O, Mobile phase B: 0.1% TFA in ACN, Gradient: Initially 95% mobile phase A and 5% mobile phase B, linearly gradient to 98% mobile phase B over 1.5 min, then held for 0.5 min. MSD positive ions.

[0282] LCMS Method 3. Column: Kinetex XB-C18 75 x 3.0 mm, 2.6 μm. Temperature: RT, Flow rate: 1.0 mL / min, Run time: 5.0 min. Mobile phase conditions: Mobile phase A: 5.0 mm ammonium formate, pH 3.3: CH3CN (98:02), Mobile phase B: CH3CN: buffer (98:02), Gradient: Initially 40% mobile phase A and 60% mobile phase B were linearly gradiented to 100% mobile phase B over 3.5 min, then held for 1.5 min. MSD positive ions.

[0283] LCMS Method 4. Column: Kinetex XB-C18 75 x 3.0 mm, 2.6 μm. Temperature: RT, Flow rate: 1.0 mL / min, Run time: 5.0 min. Mobile phase conditions: Mobile phase A: 5.0 mm ammonium formate, pH 3.3: CH3CN (98:02), Mobile phase B: CH3CN: buffer (98:02), Gradient: Initially 20% mobile phase A and 80% mobile phase B were linearly gradiented to 100% mobile phase B over 4.0 min, then held for 0.6 min. MSD positive ions.

[0284] Step 1'. Preparation of tert-butyl 4-(3-(ethoxycarbonyl)benzofuran-6-yl)-3,6-dihydropyridine-1(2H)-carboxylate 3': Under RT, K2CO3 (835 mg, 6.04 mmol) and tert-butyl 4-(3-(ethoxycarbonyl)benzofuran-6-yl)-3,6-dihydropyridine-1(2H)-carboxylate 2' (822 mg, 2.66 mmol) were added to a stirred solution of ethyl 6-bromobenzofuran-3-carboxylate 1' (650 mg, 2.416 mmol) in 1,4-dioxane (10 mL) and water (1.11 mL), and the reaction mixture was degassed for 10 min. Then PdCl2 (dppf) (177 mg, 0.242 mmol) was added and the mixture was stirred at 100ºC for 16 h. The reaction mixture was filtered through a diatomaceous earth bed and washed with ethyl acetate (3 x 20 mL). The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous Na₂SO₄, filtered, and subsequently concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (using silica gel (230-400 mesh) and eluted with 25% ethyl acetate in petroleum ether) to give tert-butyl 4-(3-(ethoxycarbonyl)benzofuran-6-yl)-3,6-dihydropyridine-1(2H)-carboxylate 3' (600 mg, 63.3% yield) as a pale yellow liquid.

[0285] LCMS Method 3: Retention time: 2.96 min, [M+H-100] + = 272.1.

[0286] 1 H NMR (400 MHz, DMSO-d6): δ ppm 1.35 (t, J = 7.2 Hz, 3H), 1.44 (s,9H), 2.50 -2.54 (m, 2H), 3.57 (t, J = 5.6 Hz, 2H), 4.03 - 4.04 (m, 2H), 4.32- 4.38 (m, 2H), 6.27 (s, 1H), 7.53 -7.55 (m, 1H), 7.75 (s, 1H), 7.91 (d, J =8.4 Hz, 1H), 8.74 (s, 1H).

[0287] Step 2'. Preparation of tert-butyl 4-(3-(ethoxycarbonyl)-2,3-dihydrobenzofuran-6-yl)piperidine-1-carboxylate 4': Under a hydrogen atmosphere (balloon pressure), 10% Pd / C (344 mg) was added to a stirred solution of tert-butyl 4-(3-(ethoxycarbonyl)-2,3-dihydrobenzofuran-6-yl)-3,6-dihydropyridine-1(2H)-carboxylate 3' (600 mg, 1.615 mmol) in ethyl acetate (10 mL) and stirred at 25ºC for 16 h. The reaction mixture was filtered through a diatomaceous earth bed and washed with ethyl acetate (3 x 20 mL). The combined organic layers were concentrated under reduced pressure to give crude tert-butyl 4-(3-(ethoxycarbonyl)-2,3-dihydrobenzofuran-6-yl)piperidine-1-carboxylate 4' (650 mg, 100% yield), which was used for the next step without further purification.

[0288] LCMS Method 4: Retention time: 3.50 min, [M+H-100] + = 276.0.

[0289] Step 3'. Preparation of tert-butyl 4-(3-(2-cyanoethyl)-3-(ethoxycarbonyl)-2,3-dihydrobenzofuran-6-yl)piperidine-1-carboxylate 5': Acrylonitrile (170 mg, 3.20 mmol) and benzyltriethylammonium chloride (72.8 mg, 0.320 mmol) were added to a stirred solution of tert-butyl 4-(3-(ethoxycarbonyl)-2,3-dihydrobenzofuran-6-yl)piperidine-1-carboxylate 4' (600 mg, 1.598 mmol) in toluene (15 mL), followed by the addition of K2CO3 (442 mg, 3.20 mmol). The reaction mixture was stirred at 80ºC for 16 h. The reaction mixture was quenched with ice-cold water (10 mL) and extracted with ethyl acetate (3 x 10 mL). The combined organic layers were washed with an aqueous salt solution, dried over sodium sulfate, and concentrated under reduced pressure to give crude tert-butyl 4-(3-(2-cyanoethyl)-3-(ethoxycarbonyl)-2,3-dihydrobenzofuran-6-yl)piperidine-1-carboxylate 5' (700 mg, 84% yield), which was used for the next step without further purification.

[0290] LCMS Method 4: Retention time: 2.48 min, [M+H-56] + = 373.2.

[0291] Step 4'. Preparation of 6-(piperidin-4-yl)-2H-spiro[benzofuran-3,3'-piperidin]-2',6'-dione 6': Add H2SO4 (0.373 mL, 7.00 mmol) to a stirred solution of 4-(3-(2-cyanoethyl)-3-(ethoxycarbonyl)-2,3-dihydrobenzofuran-6-yl)piperidin-1-carboxylic acid tert-butyl ester 5' (600 mg, 1.400 mmol) in acetic acid (5 mL) and heat at 120 °C. o The mixture was stirred at C for 16 h. The reaction mixture was concentrated under reduced pressure and the residual acid was removed by co-distillation with ACN (10 mL) and toluene (10 mL) to give crude 6-(piperidin-4-yl)-2H-spiro[benzofuran-3,3'-piperidin]-2',6'-dione 6' (500 mg, 81% yield) as a pale yellow liquid, which was used for the next step without further purification.

[0292] LCMS Method 2: Retention time: 0.85 min, [M+H] + = 301.0.

[0293] Step 5'. Preparation of tert-butyl 4-(2',6'-dioxo-2H-spiro[benzofuran-3,3'-piperidine]-6-yl)piperidine-1-carboxylate 7': at 0 ºC DIPEA (3.49 mL, 19.98 mmol) and Boc anhydride (0.928 mL, 4.00 mmol) were added to a stirred solution of 6-(piperidin-4-yl)-2H-spiro[benzofuran-3,3'-piperidin]-2',6'-dione 6' (600 mg, 1.998 mmol) in acetonitrile (10 mL), and the mixture was stirred for 2 h. The reaction mixture was diluted with ethyl acetate (20 mL), cold water (20 mL) was added, and the mixture was extracted with ethyl acetate (3 x 20 mL). The combined organic layers were washed with aqueous brine, dried over sodium sulfate, and then concentrated under reduced pressure to give the crude product. The crude product was purified by column chromatography (using silica gel (230-400 mesh) and eluted with 65% ethyl acetate in petroleum ether) to give tert-butyl 4-(2',6'-dioxo-2H-spiro[benzofuran-3,3'-piperidin]-6-yl)piperidin-1-carboxylate 7' (300 mg) (a mixture of enantiomers) as a pale yellow solid.

[0294] LCMS Method 4: Retention time: 2.63 min, [M+H-56] + = 345.0.

[0295] Step 6'. Chiral SFC resolution of tert-butyl piperidine-1-carboxylate 8a' (peak 01) and 4-(2',6'-dioxo-2H-spiro[benzofuran-3,3'-piperidine]-6-yl)piperidine-1-carboxylate 8b' (peak 02): The enantiomers (500 mg) of 4-(2',6'-dioxo-2H-spiro[benzofuran-3,3'-piperidine]-6-yl)piperidine-1-carboxylate 7' were purified by SFC to give pure 8a' (peak 01) (100 mg, 12.27% yield) and 8b' (peak 02) (200 mg, 24.16% yield) as a grayish-white solid.

[0296] SFC separation: YMC Cellulose-SC, 250 mm x 4.6 x 5 μm, flow rate: 3.0 mL / min, co-solvent: 40.0%.

[0297] Peak 01: 8a' (Rt 4.21 min, ee 100%).

[0298] LCMS Method 4: Retention time: 2.58 min, [M+H-56] + = 345.2.

[0299] 1 H NMR (400 MHz, DMSO-d6): δ ppm 1.44 – 1.47 (m, 11H), 1.70 – 1.73 (m,2H), 2.13 – 2.17 (m, 2H), 2.60 – 2.77 (m, 5H), 4.03 – 4.06 (m, 2H), 4.43 (d,J = 9.2 Hz, 1H), 4.80 (d, J = 9.2 Hz, 1H), 6.75 – 6.77 (m, 2H), 7.14 (d, J =8.4 Hz, 1H), 11.00 (s, 1H).

[0300] Peak 02: 8b' (Rt 4.81 min, ee 98.74%).

[0301] LCMS Method 4: Retention time: 2.64 min, [M+H-100] + = 301.2.

[0302] 1H NMR (400 MHz, DMSO-d6): δ ppm 1.38 - 1.47 (m, 11H), 1.70 - 1.73 (m,2H), 2.13 - 2.17 (m, 2H), 2.60 - 2.82 (m, 5H), 4.03 - 4.06 (m, 2H), 4.43 (d,J = 9.2 Hz, 1H), 4.80 (d, J = 9.2 Hz, 1H), 6.75 - 6.77 (m, 2H), 7.14 (d, J =8.4 Hz, 1H), 11.00 (s, 1H).

[0303] Step 7'. Preparation of 6-(piperidin-4-yl)-2H-spiro[benzofuran-3,3'-piperidin]-2',6'-dione, HCl C-11: Under RT, 4.0 M HCl (0.624 mL, 2.497 mmol) in 1,4-dioxane was added to a stirred solution of 4-(2',6'-dioxo-2H-spiro[benzofuran-3,3'-piperidin]-6-yl)piperidin-1-carboxylic acid tert-butyl ester 8a' (100 mg, 0.250 mmol) in DCM (2 mL) and stirred for 1 h. The reaction mixture was concentrated under reduced pressure and the residual acid was removed by co-distillation with ACN (10 mL) and toluene (10 mL) to give crude 6-(piperidin-4-yl)-2H-spiro[benzofuran-3,3'-piperidin]-2',6'-dione, HCl C-11 (100 mg), which was used for the next step without further purification.

[0304] LCMS Method 1: Retention time: 1.61 min, [M+H] + = 301.3.

[0305] Step 8'. Preparation of 6-(piperidin-4-yl)-2H-spiro[benzofuran-3,3'-piperidin]-2',6'-dione, HCl C-12: Under RT, 4.0 M HCl (0.437 mL, 1.748 mmol) in 1,4-dioxane was added to a stirred solution of 4-(2',6'-dioxo-2H-spiro[benzofuran-3,3'-piperidin]-6-yl)piperidin-1-carboxylic acid tert-butyl ester 8b' (140 mg, 0.350 mmol) in DCM (5 mL) and stirred for 1 h. The reaction mixture was concentrated under reduced pressure and the residual acid was removed by co-distillation with ACN (10 mL) and toluene (10 mL) to give crude 6-(piperidin-4-yl)-2H-spiro[benzofuran-3,3'-piperidin]-2',6'-dione, HCl C-12 (peak-02) (131 mg), which was used for the next step without further purification.

[0306] LCMS Method 1: Retention time: 1.56 min, [M+H] + = 301.1. Example S9: Preparation of 5-methyl-6-(piperazin-1-yl)-2H-spiro[benzofuran-3,3'-piperidine]-2',6'-dione (C-13 and C-14)

[0307] LCMS Method 1. Kinetex XB-C18, 75 x 3.0 mm, 2.6 μm. Temperature: RT, Flow rate: 1.0 mL / min, Run time: 5.5 min. Mobile phase conditions: Mobile phase A: 0.1% TFA in H2O, Mobile phase B: 0.1% TFA in ACN, Gradient: Initially 95% mobile phase A and 5% mobile phase B, linear gradient change to 95% mobile phase B over 4.5 min. MSD positive ions.

[0308] LCMS Method 2. Kinetex XB-C18, 75 x 3.0 mm, 2.6 μm. Temperature: RT, Flow rate: 1.0 mL / min, Run time: 5.0 min. Mobile phase conditions: Mobile phase A: 5.0 mm ammonium formate, pH 3.3: CH3CN (98:02), Mobile phase B: CH3CN in 5.0 mm ammonium formate (98:02), Gradient: A linear gradient from the initial 80% mobile phase A and 20% mobile phase B to 100% mobile phase B over 4.06 min. MSD positive ions.

[0309] LCMS Method 3.Aquity Uplc BEH C18 50 x 2.1 mm, 1.7 μm. Temperature: RT, Flow rate: 0.7 mL / min, Run time: 2.0 min. Mobile phase conditions: Mobile phase A: 0.1% TFA in H2O, Mobile phase B: 0.1% TFA in ACN, Gradient: Initially 95% mobile phase A and 40% mobile phase B, linear gradient change to 98% mobile phase B over 2.0 min. MSD positive ions.

[0310] Step 1'. Preparation of ethyl 6-bromo-5-methylbenzofuran-3-carboxylate 2': Tetrafluoroborate diethyl ether complex (0.063 mL, 0.465 mmol) was added dropwise to a stirred solution of 4-bromo-2-hydroxy-5-methylbenzaldehyde 1' (1.0 g, 4.65 mmol) in DCM (1.0 mL), followed by a solution of ethyl diazonyl acetate in toluene (7.90 mL, 9.30 mmol). The reaction was not permitted to proceed above 38ºC. After stirring at room temperature for 1 h, the tetrafluoroborate diethyl ether complex (0.063 mL, 0.465 mmol) and the solution of ethyl diazonyl acetate in toluene (7.90 mL, 9.30 mmol) were added to the reaction mixture and stirred for another 1 h. Once gas escaping ceased, the reaction mixture was concentrated using a rotary evaporator, and H2SO4 (0.3 to 0.5 mL) was added to the mixture while stirring. After 5 to 10 min, the mixture was diluted with CH2Cl2 (5 to 10 mL), and the H2SO4 was quenched with saturated NaHCO3 solution. The reaction mixture was extracted with DCM, the combined organic layers were dried over sodium sulfate, filtered, concentrated, and the crude compound was purified by column chromatography (using silica gel (230-400 mesh) with 0-5% ethyl acetate / petroleum ether) to give ethyl 6-bromo-5-methylbenzofuran-3-carboxylate 2' (900 mg, 68.4% yield) as a pale yellow solid.

[0311] 1 H NMR (400 MHz, CDCl3): δ ppm 1.44 (t, J = 7.2 Hz, 3H), 2.54 (s, 3H), 4.43 (q, J = 6.8 Hz, 2H), 7.77 (s, 1H), 7.95 (s, 1H), 8.21 (s, 1H).

[0312] Step 2'. Preparation of 4'-(3-(ethoxycarbonyl)-5-methylbenzofuran-6-yl)piperazine-1-carboxylate tert-butyl ester 4': Under a nitrogen atmosphere at room temperature, cesium carbonate (1.726 g, 5.30 mmol) was added to a stirred solution of ethyl 6-bromo-5-methylbenzofuran-3-carboxylate 2' (0.900 g, 3.18 mmol) and tert-butyl piperazine-1-carboxylate 3' (0.987 g, 5.30 mmol) in 1,4-dioxane (10.0 mL). The reaction mixture was degassed with nitrogen for 10 min, and RuPhos Pd G3 (0.222 g, 0.265 mmol) was added under positive pressure nitrogen flow, followed by further stirring at 100ºC for 6 h. The reaction mixture was cooled to room temperature, quenched with saturated ammonium chloride solution, and extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (using silica gel (230-400 mesh) with 0-20% ethyl acetate / petroleum ether) to give 4'-(3-(ethoxycarbonyl)-5-methylbenzofuran-6-yl)piperazine-1-carboxylic acid tert-butyl ester 4' (600 mg, 53.6% yield) as a yellow liquid.

[0313] LCMS Method 1: Retention time: 3.589 min, [M+H-56] + = 333.9.

[0314] Step 3'. Preparation of tert-butyl piperazine-1-carboxylate 5': Magnesium (563 mg, 23.17 mmol) was added to a stirred solution of tert-butyl piperazine-1-carboxylate 4' (600 mg, 1.545 mmol) in MeOH (15.0 mL) at room temperature under a nitrogen atmosphere, and the reaction mixture was stirred for 16 h at room temperature. The reaction mixture was quenched with an aqueous solution of ammonium chloride (100 mL) and concentrated to remove methanol. The crude product was extracted with ethyl acetate (3 x 100 mL), and the organic layer was washed with brine (40 mL). The combined organic layers were dried over anhydrous sodium sulfate and filtered, then concentrated under reduced pressure to give a crude product, tert-butyl 4-(3-(methoxycarbonyl)-5-methyl-2,3-dihydrobenzofuran-6-yl)piperazine-1-carboxylate 5' (585 mg, 91% yield), as a yellow liquid. The crude product was used for the next step without further purification.

[0315] LCMS Method 2: Retention time: 3.281 min, [M+H] + = 377.4.

[0316] Step 4'. Preparation of tert-butyl piperazine-1-carboxylate 6': At room temperature, benzyltriethylammonium chloride (35.2 mg, 0.154 mmol) was added to a stirred solution of 4-(3-(2-cyanoethyl)-3-(methoxycarbonyl)-5-methyl-2,3-dihydrobenzofuran-6-yl)piperazine-1-carboxylate 5' (581 mg, 1.543 mmol), acrylonitrile (0.203 mL, 3.09 mmol), and K2CO3 (427 mg, 3.09 mmol) in toluene (10.0 mL), and the reaction mixture was then stirred at 90ºC for 16 h. The reaction mixture was treated with water (50 mL) and extracted with ethyl acetate (3 x 50.0 mL). The combined organic layers were then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (using silica gel (230-400 mesh) with 0-20% ethyl acetate / petroleum ether) to give 4-(3-(2-cyanoethyl)-3-(methoxycarbonyl)-5-methyl-2,3-dihydrobenzofuran-6-yl)piperazine-1-carboxylic acid tert-butyl ester 6' (450 mg, 59.1% yield) as a yellow liquid.

[0317] LCMS Method 2: Retention time: 3.193 min, [M+H] + = 430.4.

[0318] Step 5'. Preparation of 5-methyl-6-(piperazin-1-yl)-2H-spiro[benzofuran-3,3'-piperidine]-2',6'-dione 7': At room temperature, H2SO4 (0.112 mL, 2.095 mmol) was added dropwise to a stirred solution of 4-(3-(2-cyanoethyl)-3-(methoxycarbonyl)-5-methyl-2,3-dihydrobenzofuran-6-yl)piperazin-1-carboxylic acid tert-butyl ester 6' (450 mg, 1.048 mmol) in acetic acid (5.0 mL), and the reaction mixture was stirred at 120ºC for 16 h. The reaction mixture was concentrated under reduced pressure to give crude 5-methyl-6-(piperazin-1-yl)-2H-spiro[benzofuran-3,3'-piperidine]-2',6'-dione, 2-sulfate 7' (600 mg) as a viscous brown liquid. The crude product was used in the next step without further purification.

[0319] LCMS Method 3: Retention time: 0.388 min, [M+H] + = 316.0.

[0320] Step 6'. Preparation of tert-butyl 4-(5-methyl-2',6'-dioxo-2H-spiro[benzofuran-3,3'-piperidine]-6-yl)piperazine-1-carboxylate 8': At room temperature, under positive pressure of nitrogen, DIPEA (3.07 mL, 17.59 mmol) was added dropwise to a stirred solution of 5-methyl-6-(piperazine-1-yl)-2H-spiro[benzofuran-3,3'-piperidine]-2',6'-dione, 2-sulfate 7' (600 mg, 1.173 mmol) in acetonitrile (5.0 mL) until the solution became alkaline. Then, Boc anhydride (1.362 mL, 5.86 mmol) was added dropwise, and the reaction mixture was stirred at room temperature for 4 h. The reaction mixture was treated with water (50 mL) and extracted with ethyl acetate (3 x 100 mL). The combined organic layers were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (using silica gel (230-400 mesh) with 0-60% ethyl acetate / petroleum ether) to give tert-butyl 4-(5-methyl-2',6'-dioxo-2H-spiro[benzofuran-3,3'-piperidin]-6-yl)piperazine-1-carboxylate 8' (340 mg, 64.2% yield) as a grayish-white solid.

[0321] LCMS Method 3: Retention Time: 1.345 min, [M+H] + = 416.0.

[0322] Step 7'. Chiral SFC resolution of 4-(5-methyl-2',6'-dioxo-2H-spiro[benzofuran-3,3'-piperidine]-6-yl)piperazine-1-carboxylic acid tert-butyl ester 9a' (peak 1) and 4-(5-methyl-2',6'-dioxo-2H-spiro[benzofuran-3,3'-piperidine]-6-yl)piperazine-1-carboxylic acid tert-butyl ester 9b' (peak 2): 340 mg of the racemic compound was purified by chiral SFC to give 4-(5-methyl-2',6'-dioxo-2H-spiro[benzofuran-3,3'-piperidine]-6-yl)piperazine-1-carboxylic acid tert-butyl ester 9a' (120 mg). mg, peak 1) and tert-butyl 4-(5-methyl-2',6'-dioxo-2Hspiro[benzofuran-3,3'-piperidin]-6-yl)piperazine-1-carboxylate 9b' (130 mg, peak 2).

[0323] SFC method: YMC Cellulose-SC, 250 mm x 4.6 x 5 μm, flow rate: 3.0 mL / min (1:1 ACN and IPA), co-solvent: 40.0%, BPR: 100.0 bar, BPR temperature: 50°C o C.

[0324] Peak 01: RT 3.083 min, ee 100%.

[0325] LCMS Method 2: Retention time: 2.767 min, [M+H] + = 416.2.

[0326] 1 H NMR (400 MHz, DMSO-d6): δ ppm 1.22 – 1.31 (m, 1H), 1.42 (s, 9H), 2.10 – 2.19 (m, 5H), 2.70 – 2.79 (m, 4H), 3.40 – 3.50 (m, 4H), 4.49 (d, J =9.2 Hz, 1H), 4.79 (d, J = 9.2 Hz, 1H), 6.55 (s, 1H), 7.02 (s, 1H), 10.98 (s,1H).

[0327] Peak 02: RT 3.797 min, ee 99.73%.

[0328] LCMS Method 1: Retention time: 2.767 min, [M+H] + = 416.2.

[0329] Step 8'. Preparation of 5-methyl-6-(piperazin-1-yl)-2H-spiro[benzofuran-3,3'-piperidine]-2',6'-dione, 2HCl C-13: At room temperature and under a nitrogen atmosphere, tert-butyl piperazine-1-carboxylate 9' (0.120 g, 0.289 mmol) was added dropwise to a stirred solution of 1,4-dioxane in 10 mL of DCM with 4.0 N hydrogen chloride solution (2.166 mL, 8.66 mmol) and the reaction mixture was stirred at room temperature for 3 h. The reaction mixture was concentrated under reduced pressure to give crude 5-methyl-6-(piperazin-1-yl)-2H-spiro[benzofuran-3,3'-piperidine]-2',6'-dione as a grayish-white solid, 2 HCl C13 (120 mg). The crude product was used for the next step without further purification.

[0330] LCMS Method 2: Retention time: 1.993 min, [M+H] + = 316.2. Example S10: Preparation of 7-(piperazin-1-yl)-2H-spiro[benzofuran-3,3'-piperidine]-2',6'-dione (C-15 and C-16)

[0331] LCMS Method 1. Kinetex XB-C18, 75 x 3.0 mm, 2.6 μm. Temperature: RT, Flow rate: 1.0 mL / min, Run time: 4.7 min. Mobile phase conditions: Mobile phase A: 5.0 mm ammonium formate, pH 3.3: CH3CN (98:02), Mobile phase B: CH3CN in 5.0 mm ammonium formate (98:02), Gradient: Initially 60% mobile phase A and 40% mobile phase B, linear gradient change to 100% mobile phase B over 3.5 min, then held for 1.0 min. MSD positive ions.

[0332] LCMS Method 2. Kinetex XB-C18, 75 x 3.0 mm, 2.6 μm. Temperature: RT, Flow rate: 1.0 mL / min, Run time: 5.5 min. Mobile phase conditions: Mobile phase A: 0.1% TFA in H2O, Mobile phase B: 0.1% TFA in ACN, Gradient: Initially 95% mobile phase A and 5% mobile phase B were linearly gradiented to 95% mobile phase B over 2.5 min, then held for 2.0 min. MSD positive ions.

[0333] LCMS Method 3. Aquity Uplc BEH C18, 50 x 2.1 mm, 1.7 μm. Temperature: RT, Flow rate: 0.7 mL / min, Run time: 2.0 min. Mobile phase conditions: Mobile phase A: 0.1% TFA in H2O, Mobile phase B: 0.1% TFA in ACN, Gradient: Initially 80% mobile phase A and 20% mobile phase B, linearly changed to 98% mobile phase B over 1.5 min, then held for 0.5 min. MSD positive ions.

[0334] LCMS Method 4.Kinetex XB-C18, 75 x 3.0 mm, 2.6 μm. Temperature: RT, Flow rate: 1.0 mL / min, Run time: 4.7 min. Mobile phase conditions: Mobile phase A: 5.0 mm ammonium formate, pH 3.3: CH3CN (98:02), Mobile phase B: CH3CN in 5.0 mm ammonium formate (98:02), Gradient: Initially 80% mobile phase A and 20% mobile phase B, linear gradient change to 100% mobile phase B over 4.0 min, then held for 0.6 min. MSD positive ions.

[0335] Step 1'. Preparation of ethyl 7-bromobenzofuran-3-carboxylate 2': Tetrafluoroborate diethyl ether complex (0.235 mL, 1.741 mmol) was added dropwise to a stirred solution of 3-bromo-2-hydroxybenzaldehyde 1' (3.5 g, 17.41 mmol) in DCM (15 mL), followed by dropwise addition of diazoethyl acetate (29.6 mL, 34.8 mmol, 15% in toluene) and stirring at 35ºC. Once bubbling stopped, the reaction mixture was concentrated under reduced pressure and 0.5 mL of concentrated H2SO4 catalyst was added and stirred for 16 h. The reaction mixture was diluted with ethyl acetate (30 mL) and the H2SO4 was quenched with saturated NaHCO3 solution. The reaction mixture was extracted with ethyl acetate (3 x 30 mL), and the combined organic layers were collected, dried over sodium sulfate, filtered, concentrated, and the crude compound was purified by column chromatography (using silica gel (230-400 mesh), with 0-5% DCM / petroleum ether) to give ethyl 7-bromobenzofuran-3-carboxylate 2' (700 mg, 9.83% yield) as a pale yellow liquid.

[0336] 1 H NMR (400 MHz, DMSO-d6): δ ppm 1.36 (t, J = 7.2 Hz, 3H), 4.36 (q, J= 7.2 Hz, 2H), 7.37 (t, J = 7.2 Hz, 1H), 7.68 (dd, J = 1.2, 8.0 Hz, 1H), 7.98 (dd, J = 1.2 Hz and 8.0 Hz, 1H), 8.88 (s, 1H).

[0337] Step 2'. Preparation of 4'-(3-(ethoxycarbonyl)benzofuran-7-yl)piperazine-1-carboxylate tert-butyl ester 4': Under a nitrogen atmosphere at room temperature, Cs₂CO₃ (1816 mg, 5.57 mmol) was added to a stirred solution of 7-bromobenzofuran-3-carboxylate ethyl ester 2' (750 mg, 2.79 mmol) and piperazine-1-carboxylate tert-butyl ester 3' (519 mg, 2.79 mmol) in 1,4-dioxane (2 mL). The reaction mixture was degassed with nitrogen for 10 min, and RuPhos-Pd-G₃ (233 mg, 0.279 mmol) was added, followed by stirring at 100ºC for 16 h. The reaction mixture was cooled to room temperature, quenched with saturated ammonium chloride solution (10 mL), and extracted with ethyl acetate (3 x 30 mL). The combined organic layers were collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. This crude product was purified by column chromatography (using silica gel (230-400 mesh) with 30% ethyl acetate / petroleum ether) to give 4'-(3-(ethoxycarbonyl)benzofuran-7-yl)piperazine-1-carboxylic acid tert-butyl ester 4' (600 mg, 55.2% yield) as a pale yellow liquid.

[0338] LCMS Method 1: Retention time: 2.83 min, [M+H] + = 375.2.

[0339] Step 3'. Preparation of tert-butyl 4-(3-(methoxycarbonyl)-2,3-dihydrobenzofuran-7-yl)piperazine-1-carboxylate 5': In 0 ºC Magnesium (1.93 g, 16.02 mmol) was added to a stirred solution of 4-(3-(ethoxycarbonyl)benzofuran-7-yl)piperazin-1-carboxylate 4' (600 mg, 1.602 mmol) in methanol (10 mL) under a nitrogen atmosphere, and the reaction mixture was stirred at room temperature for 16 h. The reaction mixture was quenched with anhydrous ammonium chloride (50 mL) and concentrated to remove methanol. The crude product was extracted with ethyl acetate (3 x 50 mL), and the organic layer was washed with brine (40 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product 4-(3-(methoxycarbonyl)-2,3-dihydrobenzofuran-7-yl)piperazin-1-carboxylate 5' (500 mg, 76% yield) as a pale yellow liquid. The crude product was used for the next step without further purification.

[0340] LCMS Method 2: Retention Time: 3.00 min, [M+H] + = 363.2.

[0341] Step 4'. Preparation of tert-butyl 4-(3-(2-cyanoethyl)-3-(methoxycarbonyl)-2,3-dihydrobenzofuran-7-yl)piperazine-1-carboxylate 6': At room temperature, under positive pressure of nitrogen, benzyltriethylammonium chloride (75 mg, 0.331 mmol) was added to a stirred solution of 4-(3-(methoxycarbonyl)-2,3-dihydrobenzofuran-7-yl)piperazine-1-carboxylate 5' (600 mg, 1.656 mmol), acrylonitrile (132 mg, 2.483 mmol), and K2CO3 (458 mg, 3.31 mmol) in toluene (10 mL), and the reaction mixture was then stirred at 100ºC for 16 h. The reaction mixture was cooled to RT, treated with cold water (20 mL), and extracted with ethyl acetate (3 x 30.0 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give crude tert-butyl 4-(3-(2-cyanoethyl)-3-(methoxycarbonyl)-2,3-dihydrobenzofuran-7-yl)piperazine-1-carboxylate 6' (600 mg, 70.9% yield). The crude product was used for the next step without further purification.

[0342] LCMS Method 4: Retention Time: 3.00 min, [M+H] + = 416.2.

[0343] Step 5'. Preparation of 7-(piperazin-1-yl)-2H-spiro[benzofuran-3,3'-piperidine]-2',6'-dione 7': At room temperature, H2SO4 (0.160 mL, 3.01 mmol) was added dropwise to a stirred solution of 4-(3-(2-cyanoethyl)-3-(methoxycarbonyl)-2,3-dihydrobenzofuran-7-yl)piperazin-1-carboxylic acid tert-butyl ester 6' (500 mg, 1.203 mmol) in acetic acid (10.0 mL). The reaction mixture was stirred at 120ºC for 16 h. The reaction mixture was concentrated under reduced pressure to give crude sulfate (500 mg) of 7-(piperazin-1-yl)-2H-spiro[benzofuran-3,3'-piperidine]-2',6'-dione 7' as a dark brown oil, which was used for the next step without further purification.

[0344] LCMS Method 3: Retention time: 0.389 min, [M+H] + = 302.0.

[0345] Step 6'. Preparation of tert-butyl 4-(2',6'-dioxo-2H-spiro[benzofuran-3,3'-piperidin]-7-yl)piperazine-1-carboxylate 8': at 0 ºCUnder positive pressure of nitrogen, DIPEA (2.65 mL, 15.18 mmol) was added to a stirred solution of 7-(piperazin-1-yl)-2H-spiro[benzofuran-3,3'-piperidine]-2',6'-dione 7' (500 mg, 1.518 mmol) in acetonitrile (5 mL) until the solution became alkaline. Boc anhydride (0.705 mL, 3.04 mmol) was then added, and the reaction mixture was stirred at room temperature for 16 h. The reaction mixture was treated with water (20 mL) and extracted with ethyl acetate (3 x 20 mL). The combined organic layers were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product. The crude product was purified by column chromatography (using silica gel (230-400 mesh) with 65% ethyl acetate / petroleum ether) to give a mixture of enantiomers of tert-butyl 4-(2',6'-dioxo-2H-spiro[benzofuran-3,3'-piperidin]-7-yl)piperazine-1-carboxylate 8' as a pale yellow solid (250 mg, 37.5% yield).

[0346] 1 H NMR (400 MHz, DMSO- d6): δ ppm 1.41 (s, 9H), 2.14 - 2.18 (m, 2H), 2.63 - 2.68 (m, 2H), 2.95 - 3.07 (m, 4H), 3.42 - 3.46 (m, 4H), 4.50 (d, J =9.2 Hz, 1H), 4.82 (d, J = 8.8 Hz, 1H), 6.76 - 6.83 (m, 3H), 11.02 (s, 1H).

[0347] Step 7'. Chiral SFC resolution of 4-(2',6'-dioxo-2H-spiro[benzofuran-3,3'-piperidine]-7-yl)piperazine-1-carboxylic acid tert-butyl ester 9a' (peak 1) and 4-(2',6'-dioxo-2H-spiro[benzofuran-3,3'-piperidine]-7-yl)piperazine-1-carboxylic acid tert-butyl ester 9b' (peak 2): 250 mg of the racemic compound was purified by chiral SFC to give 4-(2',6'-dioxo-2H-spiro[benzofuran-3,3'-piperidine]-7-yl)piperazine-1-carboxylic acid tert-butyl ester 9a' (peak 2). mg, peak 01) and tert-butyl 4-(2',6'-dioxo-2H-spiro[benzofuran-3,3'-piperidin]-7-yl)piperazine-1-carboxylate 9b' (77 mg, peak 02).

[0348] SFC method: Cellulose-1, 250 mm x 4.6 x 5 μm, flow rate: 3.0 mL / min, co-solvent: 30.0%.

[0349] Peak 01: Rt: 3.189 min, ee 100%.

[0350] LCMS Method 2: Retention time: 3.01 min, [M+H] + = 402.9.

[0351] 1 H NMR (400 MHz, DMSO- d6): δ ppm 1.41 (s, 9H), 2.14 - 2.18 (m, 2H), 2.63 - 2.68 (m, 2H), 2.95 - 3.07 (m, 4H), 3.44 - 3.45 (m, 4H), 4.50 (d, J =9.2 Hz, 1H), 4.82 (d, J = 9.2 Hz, 1H), 6.77 - 6.83 (m, 3H), 11.02 (s, 1H).

[0352] Peak 02: Rt: 4.107 min, ee 99.83%.

[0353] LCMS Method 4: Retention Time: 2.38 min, [M+H] + = 402.2.

[0354] 1 H NMR (400 MHz, DMSO- d6): δ ppm 1.42 (s, 9H), 2.14 - 2.18 (m, 2H), 2.63 - 2.68 (m, 2H), 2.97 - 3.07 (m, 4H), 3.04 - 3.45 (m, 4H), 4.50 (d, J =9.2 Hz, 1H), 4.82 (d, J = 9.2 Hz, 1H), 6.77 - 6.83 (m, 3H), 11.02 (s, 1H).

[0355] Step 8a'. Preparation of 7-(piperazin-1-yl)-2H-spiro[benzofuran-3,3'-piperidine]-2',6'-dione, HCl C-15: At room temperature, under positive pressure of nitrogen, 4.0 M HCl (0.187 mL, 0.747 mmol) in dioxane was added dropwise to a stirred solution of 9a' (60 mg, 0.149 mmol) of 4-(2',6'-dioxo-2H-spiro[benzofuran-3,3'-piperidine]-7-yl)piperazin-1-carboxylate in DCM (3 mL) and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure to give crude 7-(piperazin-1-yl)-2H-spiro[benzofuran-3,3'-piperidine]-2',6'-dione, HCl C-15 (50 mg, 97% yield) as a pale yellow solid. The crude product was used in the next step without further purification.

[0356] LCMS Method 4: Retention Time: 0.68 min, [M+H] + = 302.0.

[0357] Step 8b'. Preparation of 7-(piperazin-1-yl)-2H-spiro[benzofuran-3,3'-piperidine]-2',6'-dione, HCl C-16: At room temperature, under positive pressure of nitrogen, 4.0 M HCl (0.46 mL, 1.86 mmol) in dioxane was added dropwise to a stirred solution of 9b' (75 mg, 0.187 mmol) of 4-(2',6'-dioxo-2H-spiro[benzofuran-3,3'-piperidine]-7-yl)piperazin-1-carboxylate in DCM (5 mL) and the reaction mixture was stirred at room temperature for 3 h. The reaction mixture was concentrated under reduced pressure to give crude 7-(piperazin-1-yl)-2H-spiro[benzofuran-3,3'-piperidine]-2',6'-dione, HCl C-16 (88 mg). The crude product was used for the next step without further purification.

[0358] LCMS Method 1: Retention time: 0.76 min, [M+H] + = 302.0. Example S11: Preparation of 5-((R)-2-methylpiperazin-1-yl)-2,3-dihydrospiro[indene-1,3'-piperidine]-2',6'-dione (C-17 and C-18)

[0359] LCMS Method 1.Kinetex XB-C18, 75 x 3.0 mm, 2.6 μm. Temperature: RT, Flow rate: 1.0 mL / min, Run time: 5.0 min. Mobile phase conditions: Mobile phase A: 5.0 mm ammonium formate, pH 3.3: CH3CN (98:02), Mobile phase B: CH3CN in 5.0 mm ammonium formate (98:02), Gradient: A linear gradient from the initial 80% mobile phase A and 20% mobile phase B to 100% mobile phase B over 4.06 min. MSD positive ions.

[0360] LCMS Method 2. Aquity Uplc BEH C18 50 x 3.0 mm, 1.7 μm. Temperature: RT, Flow rate: 0.7 mL / min, Run time: 2.0 min. Mobile phase conditions: Mobile phase A: 0.1% TFA in H2O, Mobile phase B: 0.1% TFA in ACN, Gradient: Initially 80% mobile phase A and 20% mobile phase B, linear gradient changes to 98% mobile phase B within 2.0 min. MSD positive ions.

[0361] LCMS Method 3. Kinetex XB-C18, 75 x 3.0 mm, 2.6 μm. Temperature: RT, Flow rate: 1.0 mL / min, Run time: 5.0 min. Mobile phase conditions: Mobile phase A: 0.1% TFA in H2O, Mobile phase B: 0.1% TFA in ACN, Gradient: Initially 95% mobile phase A and 5% mobile phase B, linear gradient change to 95% mobile phase B over 4.0 min. MSD positive ions.

[0362] Step 1'. Preparation of tert-butyl 3-(5-bromo-1-cyano-2,3-dihydro-1H-indene-1-yl)propionate 2': At room temperature, K2CO3 (933 mg, 6.75 mmol) and benzyltriethylammonium chloride (77 mg, 0.338 mmol) were added to a stirred solution of 5-bromo-2,3-dihydro-1H-indene-1-carboxynitrile 1' (750 mg, 3.38 mmol) and tert-butyl acrylate (0.989 mL, 6.75 mmol) in toluene (2.0 mL), and the reaction mixture was stirred at 90ºC for 20 h. The reaction mixture was treated with water (100 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic layers were then dried over sodium sulfate, filtered, and concentrated under reduced pressure to give crude tert-butyl 3-(5-bromo-1-cyano-2,3-dihydro-1H-inden-1-yl)propionate 2' (1.05 g, 72.8% yield). The crude product was used for the next step without further purification.

[0363] LCMS Method 1: Retention time: 3.640 min, [M+H] + = 350.0 and [M+H+2] + = 352.0.

[0364] Step 2'. Preparation of tert-butyl (3R)-4-(1-(3-(tert-butoxy)-3-oxopropyl)-1-cyano-2,3-dihydro-1H-indene-5-yl)-3-methylpiperazine-1-carboxylate 4': At room temperature, under positive pressure of nitrogen, cesium carbonate (1.860 g, 5.71 mmol) was added to a stirred solution of tert-butyl 3-(5-bromo-1-cyano-2,3-dihydro-1H-indene-1-yl)propionate 2' (1.0 g, 2.86 mmol) and (R)-1-Boc-3-methylpiperazine 3' (0.858 g, 4.28 mmol) in 1,4-dioxane (1.0 mL). The reaction mixture was then degassed with nitrogen for 5 min, followed by the addition of RuPhos Pd G4 (0.243 g / mL). The reaction mixture was stirred at 100ºC for 16 h. The reaction mixture was cooled to room temperature, quenched with saturated ammonium chloride solution, and extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (using silica gel (230-400 mesh) with 0-20% ethyl acetate / petroleum ether) to give (3R)-4-(1-(3-(tert-butoxy)-3-oxopropyl)-1-cyano-2,3-dihydro-1H-inden-5-yl)-3-methylpiperazine-1-carboxylic acid tert-butyl 4' (1.00 g, 61.5% yield) as a yellow liquid.

[0365] LCMS Method 1: Retention time: 3.946 min, [M+H] + = 470.2.

[0366] Step 3'. Preparation of 5-((R)-2-methylpiperazin-1-yl)-2,3-dihydrospiro[indene-1,3'-piperidine]-2',6'-dione 5': At room temperature, sulfuric acid (0.340 mL, 6.39 mmol) was added to a stirred solution of (3R)-4-(1-(3-(tert-butoxy)-3-oxopropyl)-1-cyano-2,3-dihydro-1H-indene-5-yl)-3-methylpiperazin-1-carboxylic acid tert-butyl ester 4' (1.0 g, 2.129 mmol) in AcOH (10.0 mL), and the reaction mixture was stirred at 120ºC for 24 h. The reaction mixture was concentrated under reduced pressure to give crude 5-((R)-2-methylpiperazin-1-yl)-2,3-dihydrospiro[indene-1,3'-piperidine]-2',6'-dione, 5' sulfate (750 mg, 112% yield), a brown liquid. The crude product was used for the next step without further purification.

[0367] LCMS Method 2: Retention time: 0.378 min, [M+H] + = 314.0.

[0368] Step 4'. Preparation of (3R)-4-(2',6'-dioxo-2,3-dihydrospiro[indene-1,3'-piperidine]-5-yl)-3-methylpiperazine-1-carboxylic acid tert-butyl ester 6': DIPEA (5.60 mL, 32.1 mmol) was added to a stirred solution of 5-((R)-2-methylpiperazine-1-yl)-2,3-dihydrospiro[indene-1,3'-piperidine]-2',6'-dione disulfate 5' (0.670 g, 2.138 mmol) in acetonitrile (2.0 mL) until the solution became alkaline. Boc anhydride (2.98 mL, 12.83 mmol) was then added at room temperature, and the reaction mixture was stirred at room temperature for 4 h. The reaction mixture was treated with water (50 mL) and extracted with ethyl acetate (3 x 100 mL). The combined organic layers were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (using silica gel (230-400 mesh) with 0-60% ethyl acetate / petroleum ether) to give (3R)-4-(2',6'-dioxo-2,3-dihydrospiro[indene-1,3'-piperidine]-5-yl)-3-methylpiperazine-1-carboxylic acid tert-butyl ester 6' (370.4 mg, 39.0% yield) as a grayish-white solid.

[0369] LCMS Method 3: Retention time: 2.243 min, [M+H] + = 414.1.

[0370] Step 5'. Chiral SFC resolution of (3R)-4-(2',6'-dioxo-2,3-dihydrospiro[indene-1,3'-piperidine]-5-yl)-3-methylpiperazine-1-carboxylic acid tert-butyl ester 7a' (peak 1) and (3R)-4-(2',6'-dioxo-2,3-dihydrospiro[indene-1,3'-piperidine]-5-yl)-3-methylpiperazine-1-carboxylic acid tert-butyl ester 7b' (peak 2): 370 mg of the racemic compound was purified by chiral SFC to give (3R)-4-(2',6'-dioxo-2,3-dihydrospiro[indene-1,3'-piperidine]-5-yl)-3-methylpiperazine-1-carboxylic acid tert-butyl ester 7a' (170 mg). mg, peak 1) and (3R)-4-(2',6'-dioxo-2,3-dihydrospiro[indene-1,3'-piperidin]-5-yl)-3-methylpiperazine-1-carboxylic acid tert-butyl ester 7b' (170 mg, peak 2).

[0371] SFC method: YMC Cellulose-SC, 250 mm x 4.6 x 5 μm, flow rate: 3.0 mL / min (1:1 ACN and IPA), co-solvent: 40.0%, BPR: 100.0 bar, BPR temperature: 50°C o C.

[0372] Peak 01: RT 6.240 min, ee 100%.

[0373] LCMS Method 3: Retention time: 2.245 min, [M+H] + = 414.1.

[0374] 1 H NMR (400 MHz, CDCl3): δ ppm 1.03 (d, J = 6.4 Hz, 3H), 1.51 (s, 9H), 2.00 – 2.15 (m, 2H), 2.18 – 2.26 (m, 1H), 2.68 – 2.77 (m, 3H), 2.98 – 3.23(m, 5H), 3.50 – 3.40 (m, 1H), 3.74 – 3.88 (m, 3H), 6.73 – 6.80 (m, 1H), 6.83(s, 1H), 6.99 – 7.04 (m, 1H), 7.89 (s, 1H).

[0375] Peak 02: RT 7.488 min, ee 99.667%.

[0376] LCMS Method 3: Retention time: 2.237 min, [M+H] += 414.1.

[0377] 1 H NMR (400 MHz, CDCl3): δ ppm 0.89 (d, J = 6.4 Hz, 3H), 1.42 (s, 9H), 1.85 – 1.94 (m, 1H), 2.02 – 2.19 (m, 2H), 2.45 – 2.50 (m, 1H), 2.52 – 2.58(m, 1H), 2.59 – 2.70 (m, 1H), 2.87 – 2.98 (m, 3H), 2.99 – 3.29 (m, 3H), 3.65– 3.72 (m, 1H), 3.80 – 3.95 (m, 2H), 6.73 (dd, J = 8.4 and 2.0 Hz, 1H), 6.80 –6.85 (m, 1H), 6.97 (d, J = 8.4 Hz, 1H), 10.77 (s, 1H).

[0378] Step 6'. Preparation of 5-((R)-2-methylpiperazin-1-yl)-2,3-dihydrospiro[indene-1,3'-piperidine]-2',6'-dione C17: At room temperature and under a nitrogen atmosphere, 4.0 N hydrochloric acid solution (2.72 mL, 10.88 mmol) of 1,4-dioxane was added dropwise to a stirred solution of (3R)-4-(2',6'-dioxo-2,3-dihydrospiro[indene-1,3'-piperidine]-5-yl)-3-methylpiperazin-1-carboxylic acid tert-butyl ester 7' (0.150 g, 0.363 mmol) in DCM (5 mL) and the reaction mixture was stirred at room temperature for 3 h. The reaction mixture was concentrated under reduced pressure to give crude 5-((R)-2-methylpiperazin-1-yl)-2,3-dihydrospiro[indene-1,3'-piperidine]-2',6'-dione as a grayish-white solid, 2 HCl C-17 (150.0 mg, 90% yield). The crude product was used for the next step without further purification.

[0379] LCMS Method 3: Retention time: 1.754 min, [M+H] + = 314.2. Example S12: Preparation of 6-(piperazin-1-yl)spiro[indoline-3,3'-piperidine]-2',6'-dione (C-19 and C-20)

[0380] LCMS Method 1.Kinetex XB-C18, 75 x 3.0 mm, 2.6 μm. Temperature: RT, Flow rate: 1.0 mL / min, Run time: 5.0 min. Mobile phase conditions: Mobile phase A: 5.0 mm ammonium formate, pH 3.3: CH3CN (98:02), Mobile phase B: CH3CN in 5.0 mm ammonium formate (98:02), Gradient: A linear gradient from initial 80% mobile phase A and 20% mobile phase B to 100% mobile phase B over 4.5 min. MSD positive ions.

[0381] LCMS Method 2. Aquity Uplc BEH C18 50 x 3.0 mm, 1.7 μm. Temperature: RT, Flow rate: 0.7 mL / min, Run time: 2.0 min. Mobile phase conditions: Mobile phase A: 0.1% TFA in H2O, Mobile phase B: 0.1% TFA in ACN, Gradient: Initially 80% mobile phase A and 20% mobile phase B, linear gradient changes to 98% mobile phase B within 2.0 min. MSD positive ions.

[0382] LCMS Method 3. Aquity Uplc BEH C18 50 x 3.0 mm, 1.7 μm. Temperature: RT, Flow rate: 0.7 mL / min, Run time: 2.6 min. Mobile phase conditions: Mobile phase A: 5.0 mm ammonium formate, pH 3.3: CH3CN (98:02), Mobile phase B: CH3CN in 5.0 mm ammonium formate (98:02), Gradient: Over 2.0 min, an initial linear gradient from 80% mobile phase A and 20% mobile phase B to 98% mobile phase B. MSD positive ions.

[0383] Step 1'. Preparation of 1-(tert-butyl)3-methyl 6-bromo-1H-indole-1,3-dicarboxylate 2': DMAP (0.063 g, 0.512 mmol) was added to a stirred solution of methyl 6-bromo-1H-indole-3-carboxylate 1' (1.3 g, 5.12 mmol) in acetonitrile (10.23 ml) at room temperature under a nitrogen atmosphere, followed by the addition of Boc anhydride (1.782 ml, 7.67 mmol), and the reaction was stirred at room temperature for 3 h. The reaction mixture was treated with water and extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (using silica gel (230-400 mesh), with 0-30% ethyl acetate / petroleum ether) to give 1-(tert-butyl)3-methyl 6-bromo-1H-indole-1,3-dicarboxylate 2' (1.7 g, 91% yield) as an off-white solid.

[0384] LCMS Method 1: Retention time: 1.177 min, [M+H] + = 254.

[0385] Step 2'. Preparation of 1-(tert-butyl)-3-methyl-6-bromoindoline-1,3-dicarboxylate 3': Magnesium (0.933 g, 38.4 mmol) was added to a stirred solution of 1-(tert-butyl)-3-methyl-6-bromo-1H-indoline-1,3-dicarboxylate 2' (1.7 g, 4.80 mmol) in MeOH (20 mL) and DCM (5.00 mL) at 0ºC under a nitrogen atmosphere. The reaction mixture was allowed to slowly reach room temperature and stirred for 6 h. The reaction mixture was quenched with an aqueous solution of ammonium chloride (100 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude product 1-(tert-butyl)-3-methyl-6-bromoindoline-1,3-dicarboxylate 3' (1.6 g, 94% yield) was used for the next step without further purification.

[0386] LCMS Method 2: Retention time: 1.379 min, [M+H-100] + = 256.0.

[0387] Step 3'. Preparation of 1-(tert-butyl)-3-methyl-6-bromo-3-(2-cyanoethyl)indoline-1,3-dicarboxylate 4': At room temperature, benzyltriethylammonium chloride (102 mg, 0.449 mmol) was added to a stirred solution of 1-(tert-butyl)-3-methyl-6-bromoindoline-1,3-dicarboxylate 3' (1600 mg, 4.49 mmol), acrylonitrile (0.591 mL, 8.98 mmol), and K2CO3 (1242 mg, 8.98 mmol) in toluene (10 mL). The reaction mixture was then stirred at 90ºC for 3 h. The crude reaction mixture was treated with water and extracted with ethyl acetate (3 x 50 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated. The crude product 1-(tert-butyl)3-methyl-6-bromo-3-(2-cyanoethyl)indoline-1,3-dicarboxylate 4' (1.488 g, 81% yield) was used for the next step without further purification.

[0388] LCMS Method 2: Retention time: 1.327 min, [M+H+2-100] + = 311.2.

[0389] Step 4'. Preparation of (3R)-4-(2',6'-dioxo-2,3-dihydrospiro[indene-1,3'-piperidine]-5-yl)-3-methylpiperazine-1-carboxylate tert-butyl ester 6': At room temperature, under positive pressure of nitrogen, cesium carbonate (2369 mg, 7.27 mmol) was added to a stirred solution of 1-(tert-butyl)-3-methyl-6-bromo-3-(2-cyanoethyl)indoline-1,3-dicarboxylate 4' (1488 mg, 3.64 mmol) and piperazine-1-carboxylate tert-butyl ester 5' (1016 mg, 5.45 mmol) in 1,4-dioxane (10.0 mL). The reaction mixture was then degassed with nitrogen for 5 min, followed by the addition of RuPhos Pd G4 (309 mg, 0.364 mmol). The reaction mixture was then stirred at 100ºC for 16 h. The reaction mixture was treated with water (50 mL) and extracted with ethyl acetate (3 x 100 mL). The combined organic layers were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (using silica gel (230-400 mesh) with 0-60% ethyl acetate / petroleum ether) to give 1-(tert-butyl)3-methyl 6-(4-(tert-butoxycarbonyl)piperazin-1-yl)-3-(2-cyanoethyl)indoline-1,3-dicarboxylate 6' (1.176 g, 34.6% yield) as a brown semi-solid.

[0390] LCMS Method 3: Retention time: 1.669 min, [M+H] + = 515.2.

[0391] Step 5'. Preparation of 6-(piperazin-1-yl)spiro[indoline-3,3'-piperidine]-2',6'-diketone sulfate 7': At room temperature, under a nitrogen atmosphere, sulfuric acid (0.242 mL, 4.55 mmol) was added dropwise to a stirred solution of 1-(tert-butyl)3-methyl 6-(4-(tert-butoxycarbonyl)piperazin-1-yl)-3-(2-cyanoethyl)indoline-1,3-dicarboxylate 6' (1.17 g, 2.274 mmol) in acetic acid (10.0 mL), and the reaction mixture was stirred at 120ºC for 3 h. The crude reaction mixture was concentrated under reduced pressure and co-distilled with acetonitrile (3 x 20 mL) to give crude 6-(piperazin-1-yl)spiro[indoline-3,3'-piperidine]-2',6'-diketone sulfate 7' (1.0 g, 74.0% yield) as a brown viscous liquid. The crude product was used in the next step without further purification.

[0392] LCMS Method 2: Retention time: 0.44 min, [M+H] + = 301.2.

[0393] Step 6'. Preparation of tert-butyl 6-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2',6'-dioxospiro[indoline-3,3'-piperidine]-1-carboxylate 8': At room temperature and under a nitrogen atmosphere, boc anhydride (0.390 mL, 1.682 mmol) and DIPEA (1.469 mL, 8.41 mmol) were added to a stirred solution of 6-(piperazin-1-yl)spiro[indoline-3,3'-piperidine]-2',6'-dione sulfate 7' (1.0 g, 1.682 mmol) in acetonitrile (10 mL), and the reaction mixture was stirred at room temperature for 16 h. The reaction mixture was treated with water and extracted with ethyl acetate (3 x 50 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (using silica gel (230-400 mesh) with 0-80% ethyl acetate / petroleum ether) to give tert-butyl 6-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2',6'-dioxospiro[indoline-3,3'-piperidine]-1-carboxylate 8' (355 mg, 38.4% yield) as a grayish-white solid.

[0394] LCMS Method 3: Retention Time: 1.494 min, [M+H] + = 501.2.

[0395] Step 7'. Chiral SFC resolution of tert-butyl 6-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2',6'-dioxospi[indoline-3,3'-piperidine]-1-carboxylate 9a' (peak 1) and tert-butyl 6-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2',6'-dioxospi[indoline-3,3'-piperidine]-1-carboxylate 9b' (peak 2): 355 mg of the racemic compound was purified by chiral SFC to give tert-butyl 6-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2',6'-dioxospi[indoline-3,3'-piperidine]-1-carboxylate 9a' (150 mg). mg, peak 1) and tert-butyl 6-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2',6'-dioxospiro[indoline-3,3'-piperidine]-1-carboxylate 9b' (160 mg, peak 2).

[0396] SFC method: (R,R)-WHELK-01 250 mm x 4.6 x 5 μm, flow rate: 3.0 mL / min (1:1 ACN and IPA), co-solvent: 40.0%, BPR: 100.0 bar, BPR temperature: 50°C o C.

[0397] Peak 01: RT 2.304 min, ee 100%.

[0398] LCMS Method 1: Retention time: 2.895 min, [M+H] + = 501.3.

[0399] 1 H NMR (400 MHz, CDCl3): δ ppm 1.42 (s, 9H), 1.52 (br s, 9H), 2.04 -2.19 (m, 2H), 2.52 - 2.56 (m, 2H), 3.00 - 3.15 (m, 4H), 3.73 - 3.86 (m, 2H), 4.26 (br d, J = 11.01 Hz, 1H), 6.57 (dd, J = 8.25, 2.25 Hz, 1H), 7.04 (d, J =8.50 Hz, 1H), 7.39 – 7.49 (m, 1H), 10.96 (s, 1H).

[0400] Peak 02: RT 3.723 min, ee 99.8%.

[0401] LCMS Method 1: Retention time: 2.899 min, [M+H] + = 501.3.

[0402] 1 H NMR (400 MHz, CDCl3): δ ppm 1.42 (s, 9H), 1.52 (br s, 9H), 2.10 -2.15 (m, 2H), 2.62 - 2.68 (m, 2H), 3.01 - 3.10 (m, 4H), 3.43 - 3.50 (m, 4H), 3.84 (br d, J = 11.6 Hz, 1H), 4.27 (br d, J = 11.6 Hz, 1H), 6.57 (dd, J =8.25, 2.25 Hz, 1H), 7.04 (d, J = 8.50 Hz, 1H), 7.35 – 7.50 (m, 1H), 10.96 (s, 1H).

[0403] Step 8'. Preparation of 5-((R)-2-methylpiperazin-1-yl)-2,3-dihydrospiro[indole-1,3'-piperidine]-2',6'-dione C-19: To a stirred solution of 6-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2',6'-dioxospiro[indoline-3,3'-piperidine]-1-carboxylic acid tert-butyl ester 9a' (150 mg, 0.300 mmol) in DCM (10 mL), hydrochloric acid in dioxane (0.375 mL, 1.498 mmol) was added and stirred at room temperature for 3 h. The reaction mixture was concentrated under reduced pressure to give crude 6-(piperazin-1-yl)spiro[indoline-3,3'-piperidine]-2',6'-dione HCl 10' (145 mg, 116% yield) as a grayish-white solid. The crude product was used for the next step without further purification.

[0404] LCMS Method 1: Retention time: 0.993 min, [M+H] + = 301.2. Synthesis of the final compound using TBM and CBM molecules Example S13: Preparation of 1-(4-(((R)-1-cyanoethyl)amino)-5-(4-((1r,4R)-4-(2-(4-(2',6'-dioxo-2H-spiro[benzofuran-3,3'-piperidin]-6-yl)piperazin-1-yl)ethyl)cyclohexyl)-1H-1,2,3-triazol-1-yl)pyridin-2-yl)-1H-pyrazolo[3,4-b]pyridine-5-carboxynitrile P-1.

[0405] LCMS Method 1. Kinetex XB-C18, 75 x 3.0 mm, 2.6 μm. Temperature: RT, Flow rate: 1.0 mL / min, Run time: 5.5 min. Mobile phase conditions: Mobile phase A: 0.1% TFA in H2O, Mobile phase B: 0.1% TFA in ACN, Gradient: Initially 95% mobile phase A and 5% mobile phase B, linear gradient change to 95% mobile phase B over 4.0 min. MSD positive ions.

[0406] Step 1'. Preparation of 1-(4-(((R)-1-cyanoethyl)amino)-5-(4-((1r,4R)-4-(2-(4-(2',6'-dioxo-2H-spiro[benzofuran-3,3'-piperidin]-6-yl)piperazin-1-yl)ethyl)cyclohexyl)-1H-1,2,3-triazol-1-yl)pyridin-2-yl)-1H-pyrazolo[3,4-b]pyridine-5-carboxylonitrile P-1: To 1-(4-(((R)-1-cyanoethyl)amino)-5-(4-((1r,4R)-4-(2-oxoethyl)cyclohexyl)-1H-1,2,3-triazol-1-yl)pyridin-2-yl)-1H-pyrazolo[3,4-b]pyridine-5-carboxylonitrile A-1 (100 6-(piperazin-1-yl)-2H-spiro[benzofuran-3,3'-piperidine]-2',6'-dione, 2 HCl C-1 (67.6 mg, 0.177 mmol) was added to a stirred solution in DMSO (3.0 mL). The reaction mixture was stirred at room temperature for 30 min, and sodium triacetoxyborohydride (107 mg, 0.506 mmol) was added under a nitrogen atmosphere, followed by stirring at room temperature for 1 h. The reaction mixture was treated with water and extracted with ethyl acetate, and the combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude compound was purified by preparative HPLC to give 1-(4-(((R)-1-cyanoethyl)amino)-5-(4-((1r,4R)-4-(2-(4-(2',6'-dioxo-2Hspiro[benzofuran-3,3'-piperidin]-6-yl)piperazin-1-yl)ethyl)cyclohexyl)-1H-1,2,3-triazol-1-yl)pyridin-2-yl)-1H-pyrazolo[3,4-b]pyridine-5-carboxylonitrile P-1 (30 mg, 17.28% yield) as a grayish-white solid.

[0407] Preparative HPLC method: X-Bridge C18, 150 x 19 mm, 5 µm, mobile phase A: 0.1% TFA in H2O, mobile phase B: ACN, flow rate: 15.0 mL / min.

[0408] LCMS Method 1: Retention time: 2.123 min, [M+H] + = 766.2.

[0409] 1H NMR (400 MHz, DMSO-d6): δ ppm 1.12-1.28 (m, 2H), 1.48 (s, 1H), 1.45-1.59 (m, 2H), 1.61 (d, J = 6.80 Hz, 3H), 1.65 (m, 2H), 1.84-1.90 (m,2H), 2.17-2.13 (m, 4H), 2.66-2.68 (m, 1H), 2.73-2.81 (m, 2H), 2.97 (t, J =8.8 Hz, 2H), 3.12 (q, J = 8.0 Hz, 2H), 3.25-3.23 (m, 2H), 3.58 (d, J = 11.60Hz, 2H), 3.84 (d, J = 12.80 Hz, 2H), 4.43 (d, J = 9.20 Hz, 1H), 4.82 (d, J =9.20 Hz, 1H), 5.02 (t, J = 7.2 Hz, 1H), 6.57-6.50 (m, 2H), 7.20-7.08 (m, 2H), 7.67 (s, 1H), 8.36 (s, 1H), 8.41 (s, 1H), 8.72 (s, 1H), 9.06 (d, J = 2.00 Hz, 2H), 10.98 (s, 1H).

[0410] Table 3 summarizes the compounds prepared using the procedure described in Example S13. Biological Examples Example B1. Reagent Preparation

[0411] Cell culture medium was prepared under sterile conditions in a tissue culture hood by adding 10% FBS and 1% penicillin-streptomycin to 500 mL of phenol red-free RPMI 1640 medium. The medium was filtered through a Nalgene top filter and stored at 4ºC.

[0412] Store Cell Titer Glo (CTG) buffer and substrate (CellTiter-Glo luminescent cell viability assay, Promega Ref. #G7573) at -20ºC. Warm 100 mL of CTG buffer in a bead bath in a tissue culture hood and add it to the CTG substrate vial. Mix the solution with a pipette until homogeneous. Aliquot the CTG reagent into 15 mL Falcon tubes and store at -20ºC.

[0413] For homogeneous time-resolved fluorescence (HTRF) assays, the Cisbio HTRF kit was used, which contains: lysis buffer #1 4X, blocking reagent #3 100X, 20X antibody 1 (anti-IRAK4 d2), 20X antibody 2 (anti-IRAK4 k), and detection buffer.

[0414] Store the 4X lysis buffer at 4ºC. For use as a 1X lysis buffer, dilute the 4X solution with deionized water (distilled water, Gibco catalog number 15230279) and 100X blocking reagent at a volume ratio of 1:3:0.04.

[0415] Store aliquots of the 20X antibody solution at -80ºC and the detection buffer at 4ºC. For use as a 1X antibody solution, dilute aliquots of the 20X antibody solution with the detection buffer at a volume ratio of 1:19. Example B2. THP1 homogeneous time-resolved fluorescence (THP1 HTRF) procedure

[0416] THP1 plates were prepared as follows. Two copies of each plate were prepared for CTG assay. Two THP-1 T-175 flasks were used to plate one 384-well plate. Cells were collected from the two T-175 flasks into 200 mL centrifuge tubes and centrifuged at 1200 rpm for 10 min. The supernatant was completely removed, and the cells were resuspended in 7.5 mL of cell culture medium. 25 μL of culture medium and 30 μL of trypan blue staining were added to 5 μL of the cell suspension. Cells were counted twice using a counters (automated cell counter, Thermo Fisher Scientific), and viability and live cell counts were recorded. The actual cell count is 6 times the live cell count. Based on the count, a cell suspension was prepared in colorless RPMI medium to a final concentration of 7.5e6 cells / mL. Using the standard multidrop combi kit, 20 μL of the 7.5e6 cells / mL cell stock solution was added to the entire 384-well plate, except for the upper half of column 1 (negative control). The multidrop combi is always pre-rinsed to achieve a stable flow rate and washed with 20 mL of deionized water followed by 20 mL of ethanol after use. Cells are incubated in a 5% CO2 incubator at 37ºC.

[0417] Dosing was performed using the following method. 20 nL of the compound in DMSO was added to both the dosing plate and the CTG plate using an Echo (Beckman Coulter) and the plates were incubated at 37ºC for 18 h. Before administration, the compound plate was rotated at 1200 rpm for 1 min. The compound plate was sealed and stored appropriately during incubation.

[0418] Following drug administration, HTRF assays were performed using the following method. The treatment plate (IRAK4_HTRF) was centrifuged at 600 g for 10 min. 13 µL of cell culture medium was removed using a CyBio Felix automated liquid processor (Analytikjena). 7.5 μL of lysis buffer was added to all columns, and an additional 7.5 μL of lysis buffer was added to column 1 (upper half, negative control). The plate was incubated on a shaker at room temperature for 2 h. The plate was centrifuged at 600 g for 5 min. Custom-made 20X IRAK4 antibodies obtained from CISBIO anti-IRAK4-d2 (recipient Ab) and anti-IRAK4-K (donor Ab) were diluted to a 1X antibody mixture. Using a multichannel repeater, 4 μL of the 1X antibody mixture was added to each well. The plate was centrifuged at 600 g for 5 min and incubated in the dark at room temperature for 18 h. The next day (18 hours later), the HTRF assay plate was centrifuged at 600 g for 5 min and read at 665 / 615 nm using an Envision plate reader.

[0419] Next, a CTG assay was performed to assess cell death using the following method: Add 20 μL of CTG reagent to all wells and cover the plates. Place the plates on a shaker for 1–2 min and incubate in the dark at room temperature for 20 min (not exceeding 30 min). Centrifuge the plates at 1200 rpm for 1 min and analyze the luminescence using a plate reader (Envision, PerkinElmer).

[0420] A summary of the THP1 HTRF data for the tested compounds is provided in Table 4 below. Table 4. THP1 HTRF results of the compounds ND = Undetermined

[0421] Although the invention has been described in detail by way of illustration and example for purposes of clarity, the description and examples should not be construed as limiting the scope of the invention. All disclosures of patents and scientific literature cited herein are expressly incorporated herein by reference in their entirety.

Claims

1. A compound of formula (I) Or its pharmaceutically acceptable salt, wherein: A is or ; R 1 It is H or -NH2; L 1 It is -C(O)N(H)- or ; Ring B is a 5- to 6-membered monocyclic heterocyclic group or an 8- to 10-membered fused bicyclic heterocyclic group, wherein the heterocyclic group contains 1-3 nitrogen atoms; Each R 2 It is independently a C1-C6 alkyl or halogroup; n is 0-3; R 3 It is H, C1-C6 alkyl or halogroup; A ring C is attached to two adjacent carbon atoms, indicated by an asterisk (*). ;and X is O, CH2, or N(H).

2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound has the formula (I'): 。 3. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound has formula (II): 。 4. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound has formula (III): 。 5. The compound according to any one of claims 1-3, or a pharmaceutically acceptable salt thereof, wherein: R 1 It is H.

6. The compound according to any one of claims 1-3, or a pharmaceutically acceptable salt thereof, wherein: R 1 It is -NH2.

7. The compound according to any one of claims 1-6, or a pharmaceutically acceptable salt thereof, wherein: L 1 It is -C(O)N(H)-.

8. The compound according to any one of claims 1-6, or a pharmaceutically acceptable salt thereof, wherein: L 1 yes .

9. The compound according to any one of claims 1-8, or a pharmaceutically acceptable salt thereof, wherein: Ring B is a 6-membered monocyclic heterocyclic group or an 8-membered fused bicyclic heterocyclic group, wherein the heterocyclic group contains 1-2 nitrogen atoms; and Each R 2 It is independently a C1-C3 alkyl or halogroup.

10. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-9, wherein: yes .

11. The compound according to any one of claims 1-10, or a pharmaceutically acceptable salt thereof, wherein: R 3 It is an H or C1-C3 alkyl group.

12. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-11, wherein: X is O.

13. The compound according to any one of claims 1-11, or a pharmaceutically acceptable salt thereof, wherein: X is CH2 or N(H).

14. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-13, wherein: yes .

15. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-14, wherein the compound has the formula (IIA), (IIB), (IIC), (IID), (IIE), (IIF), (IIIA), (IIIB), (IIIC), (IIID), (IIIE), or (IIIF): 。 16. The compound of claim 15 or a pharmaceutically acceptable salt thereof, wherein: X is O.

17. A compound selected from the compounds in Table 1 and a pharmaceutically acceptable salt thereof.

18. A pharmaceutical composition comprising a compound according to any one of claims 1-17 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

19. A method for modulating the activity of interleukin-1 (IL1) receptor-associated kinase 4 (IRAK4), the method comprising contacting IRAK4 with an effective amount of a compound according to any one of claims 1-17 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to claim 18.

20. A method for treating an inflammatory or autoimmune disease in a subject in need, the method comprising administering to the subject an effective amount of a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-17, or a pharmaceutical composition according to claim 18, optionally wherein the inflammatory or autoimmune disease is atopic dermatitis, asthma, lupus, rheumatoid arthritis, familial Mediterranean fever, psoriasis, generalized pustular psoriasis, cryoinflammatory syndrome, hidradenitis suppurativa, Behçet's syndrome, or familial cold autoinflammatory syndrome.

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