Heteroaryl compounds as ligands for IRAK4 degradation

Abstract

The present invention provides compounds and compositions for modulating IRAK4. In some embodiments, the compounds and compositions are provided for the treatment of inflammatory diseases or autoimmune diseases.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority to U.S. Provisional Patent Application No. 63 / 465,602, filed on 11 May 2023, which is incorporated herein by reference in its entirety.

[0002] This disclosure generally relates to compounds and compositions, as well as methods for producing them and the use of said compounds and compositions for treating inflammatory or autoimmune diseases. [Background technology]

[0003] The recruitment of immune cells to the site of injury involves the coordinated interaction of numerous soluble mediators. Several cytokines, including interleukin-1 (IL-1), appear to play a crucial role in these processes. IL-1 triggers pro-inflammatory responses and is involved in tissue degeneration seen in chronic inflammatory states. Therefore, IL-1 is also involved in the process of bone resorption and the regulation of adipose tissue. Thus, IL-1 plays a vital role in many medical conditions, including rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, diabetes, obesity, cancer, and sepsis.

[0004] Treatment of cells with IL-1 induces the formation of a complex consisting of two IL-1 receptor chains, namely IL-1R1 and IL-1RAcP. The resulting heterodimer recruits an adapter molecule called MyD88 and 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 centrally located kinase domain that mediate interactions with adapter proteins of the MyD88 family. 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 Toll / IL-1 receptor (TIR) ​​signaling. Under overexpression conditions, all IRAKs can mediate the activation of nuclear factor κB and the stress-induced mitogen-activated protein kinase (MAPK) signaling cascade. Studies have demonstrated that IRAK4 kinase activity is essential for cytokine production, MAPK activation, and induction of NF-κB regulatory 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 immunoprotection, compounds that modulate IRAK4 function are likely to be useful in treating inflammation, cell proliferation, and immune-related symptoms and diseases associated with IRAK-mediated signaling (e.g., 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 essential process that maintains cellular homeostasis. Through the ubiquitin-proteasome pathway (UPP), damaged, misfolded, or excess proteins are selectively identified and removed. The UPP plays a central role in regulating almost all intracellular processes. Protein ubiquitination is achieved by E3 ubiquitin ligases, which bind to proteins and attach ubiquitin molecules to them, leading to proteasomal degradation of proteins.

[0006] The therapeutic use of UPP is attracting considerable interest (Zhou et al., Mol. Cell 2000, 6, 751-756). One promising treatment method involves using proteolytic chimeric molecules (commonly called PROTACs) to remove unwanted proteins through proteolysis (Scheepstra et al., Comp. Struct. Biotech. J. 2019, 17, 160-176). PROTACs are ligand-targeted degraders that integrate an E3 ligase with the target protein to be degraded. These divalent molecules typically consist of an E3 ligase ligand and a small linker portion that binds to the target protein. PROTACs can position the E3 ligase at the appropriate distance and orientation relative to the target protein, thereby ubiquitinating the target protein. The ubiquitinated target protein is then recognized and degraded by the proteasome.

[0007] Thus, in one aspect, provided herein is a compound targeted to degrade IRAK4.

[0008] (Summary of the Invention) Described herein are, in certain embodiments, compounds and compositions thereof for degrading IRAK4. In various embodiments, the compounds and compositions thereof can be used for the treatment of inflammatory or autoimmune diseases.

[0009] This embodiment can be more fully understood by reference to the detailed description and examples that illustrate non-limiting embodiments.

[0010] Embodiment 1 is of formula (I): [Chemical formula] [Wherein, R 2 is C1-C6 haloalkyl; W is CH or N; X 1 is CH or N; X 2 is CH2, CH, C, N, or NH; Y 1 and Y 2 are independently CH or N; L 1 is -C(O)N(H)-, -C(O)- or C1-C6 alkylene; L 2 [[ID=5​​​​​​​​​​​​​​​​​​​* is L 2 Show the connection points to; ** indicates a bond site to the piperidinyl-2,6-dione group. It is a compound of or a pharmaceutically acceptable salt thereof.

[0011] Embodiment 2 is the compound of Embodiment 1 or a pharmaceutically acceptable salt thereof, wherein the compound is of formula (IIa), (IIb), or (IIc): [ka] That is the case.

[0012] Embodiment 3 is R 1 However, the compound described in Embodiment 1 or 2 or a pharmaceutically acceptable salt thereof is a C1-C3 haloalkyl compound.

[0013] Embodiment 4 is R 1 However, the compound described in Embodiment 3 or a pharmaceutically acceptable salt thereof is -CHF2 or -CF3.

[0014] Embodiment 5 is a compound or a pharmaceutically acceptable salt thereof described in any one of Embodiments 1 to 4, wherein W is CH.

[0015] Implementation 6 is X 1 However, the compound is CH, and is a pharmaceutically acceptable salt thereof, as described in any one of Embodiments 1 to 5.

[0016] Embodiment 7 is X 1 However, N is the compound described in any one of Embodiments 1 to 5 or a pharmaceutically acceptable salt thereof.

[0017] Embodiment 8 is Y 1 However, the compound is CH, and is a pharmaceutically acceptable salt thereof, as described in any one of Embodiments 1 to 7.

[0018] Embodiment 9 is Y 2However, the compound described in any one of Embodiments 1 to 8 or a pharmaceutically acceptable salt thereof is CH.

[0019] Embodiment 10 is L 1 The compound is one of the compounds described in any one of Embodiments 1 to 9 or a pharmaceutically acceptable salt thereof, which is -C(O)N(H)-, -C(O)-, or C1-C3 alkylene.

[0020] Embodiment 11 is L 1 The compound described in Embodiment 10 or a pharmaceutically acceptable salt thereof is -C(O)N(H)-, -C(O)-, -CH2-, or -CH2CH2-.

[0021] Embodiment 12 is L 2 However, the bond is -C(O)-, -NR 2 -, -CH2N(R 2 )-, -CH2- or [ka] and R 2 However, it is H or C1-C3 alkyl. The compound described in any one of Embodiments 1 to 11 or a pharmaceutically acceptable salt thereof.

[0022] Embodiment 13 is R 2 However, the compound described in Embodiment 12 or a pharmaceutically acceptable salt thereof is H or -CH3.

[0023] Embodiment 14 is, [ka] but, [ka] The compound is one of the embodiments described in any one of embodiments 1 to 13 or a pharmaceutically acceptable salt thereof.

[0024] Embodiment 15 is, [ka] but, [ka] The compound is one of the compounds described in any one of Embodiments 1 to 14 or a pharmaceutically acceptable salt thereof.

[0025] Embodiment 16 is, [ka] but, [ka] A compound according to any one of Embodiments 1 to 15 or a pharmaceutically acceptable salt thereof.

[0026] Embodiment 17 is a compound selected from the compounds in Table 1 and their pharmaceutically acceptable salts.

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

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

[0029] Embodiment 20 is a method for treating an inflammatory disease or autoimmune disease, characterized by administering an effective amount of any one of Embodiments 1 to 17 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described in Embodiment 18, to a subject in need of treatment, wherein the inflammatory disease or autoimmune disease may optionally be atopic dermatitis, asthma, lupus, rheumatoid arthritis, familial Mediterranean fever, psoriasis, generalized pustular psoriasis, cryopyrin-associated periodic syndromes, hidradenitis suppurativa, Beckett syndrome, or familial cold autoinflammatory syndrome. Detailed Description of the Invention

[0030] (definition) As used herein, the terms “comprising” and “including” are interchangeable. The terms “comprising” and “including” are construed to identify the presence of any described feature or component mentioned, but do not exclude the presence or addition of one or more features, components, or groups thereof. Furthermore, the terms “comprising” and “including” are also intended to include examples that are encompassed by the term “consisting of.” Consequently, the term “consisting of” may be used instead of the terms “comprising” and “including” to provide more specific embodiments of the present invention.

[0031] The term "consisting of" means that the object has at least 90%, 95%, 97%, 98%, or 99% of the described features or components that make up the object. In another embodiment, the term "consisting of" excludes other features or components from the scope of the subsequent description, except those that are not essential to the technical effect to be achieved.

[0032] As used herein, the term “or” is interpreted as an inclusive “or” meaning any one or any combination thereof. Thus, “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 occur only if the combination of elements, functions, procedures or actions is in any way essential and mutually exclusive.

[0033] In this specification, any concentration range, percentage range, percentage range, or integer range is understood to include any integer within the range described, and, where appropriate, its decimal values ​​(such as one-tenth and one-hundredth of an integer), unless otherwise indicated. Similarly, any numerical ranges mentioned herein relating to any physical characteristics such as polymer subunits, size, or thickness are understood to include all integers within the range described, unless otherwise indicated. Where used herein, the terms “about” and “approximately” mean ±20%, ±10%, ±5%, or ±1% of the indicated range, value, or structure, unless otherwise indicated.

[0034] The "alkyl" group consists of 1 to 10 carbon atoms (C1-C 10Alkyl groups are saturated, partially saturated, or unsaturated linear or branched acyclic hydrocarbons having typically 1 to 8 carbon atoms (C1-C8 alkyl), or, in some embodiments, 1 to 6 carbon atoms (C1-C6 alkyl), 1 to 3 carbon atoms (C1-C3 alkyl), or 2 to 6 carbon atoms (C2-C6 alkyl). In some embodiments, alkyl groups are saturated alkyl groups. Representative saturated alkyl groups include methyl, ethyl, n-propyl, n-butyl, n-pentyl, and n-hexyl; saturated branched alkyl groups include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, neopentyl, tert-pentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, and 2,3-dimethylbutyl. In some embodiments, alkyl groups are unsaturated alkyl groups, also called alkenyl or alkynyl groups. An "alkenyl" group is an alkyl group containing one or more carbon-carbon double bonds. The "alkynyl" group 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). Alkyl groups can be substituted or unsubstituted.Where an alkyl group described herein is said to be “substituted,” it means any substituent as seen in the exemplary compounds and embodiments disclosed herein, as well as halogens; hydroxyl; alkoxy; cycloalkyloxy, aryloxy, heterocyclyloxy, heteroaryloxy, heterocycloalkyloxy, cycloalkylalkyloxy, aralkyloxy, heterocyclylalkyloxy, heteroarylalkyloxy, heterocycloalkylalkyloxy; oxo(=O); amino, alkylamino, cycloalkylamino, arylamino, heterocyclylamino, heteroarylamino, heterocycloalkylamino, cycloalkyl Alkylamino, aralkylamino, heterocyclylalkylamino, heteroaralkylamino, heterocycloalkylalkylamino; imino; imide; amidino; guanidino; enamino; acylamino; sulfonylamino; urea, nitrourea; oxime; hydroxylamino; alkoxyamino; aralkoxyamino; hydrazino; hydrazide; hydrazono; azide; nitro; thio(-SH), alkylthio; =S; sulfinyl; sulfonyl; aminosulfonyl; phosphonate; phosphinyl; acyl; formyl; carboxy; ester; carbamate; amide; cyano; isocyanate; isothiocyanato; cyanate; thiocyanato; or may be substituted with -B(OH)2.In certain embodiments, where an alkyl group described herein is said to be “substituted,” it may be substituted with any substituent as found in the exemplary compounds and embodiments disclosed herein, as well as halogens (chloro, iodo, bromo or fluoro); alkyl; hydroxyl; alkoxy; alkoxyalkyl; amino; alkylamino; carboxy; nitro; cyano; thiol; thioether; imine; imide; amidine; guanidine; enamine; aminocarbonyl; acylamino; phosphonate; phosphine; thiocarbonyl; sulfinyl; sulfone; sulfonamide; ketone; aldehyde; ester; urea; urethane; oxime; hydroxylamine; alkoxyamine; aralkoxyamine; N-oxide; hydrazine; hydrazide; hydrazone; azide; isocyanate; isothiocyanate; cyanate; thiocyanate; B(OH)2 or O(alkyl)aminocarbonyl.

[0035] The "cycloalkyl" group is a saturated or partially saturated cyclic alkyl group (C3-C) having 3 to 10 carbon atoms and having a monocyclic or multiple fused or bridging ring, which can be optionally substituted. 10The cycloalkyl group is a cycloalkyl group. In some embodiments, the cycloalkyl group has 3 to 8 ring carbon atoms (C3-C8 cycloalkyl), but in other embodiments, the number of ring carbon atoms is in the range of 3 to 5 (C3-C5 cycloalkyl), 3 to 6 (C3-C6 cycloalkyl), or 3 to 7 (C3-C7 cycloalkyl). In some embodiments, the cycloalkyl group is a saturated cycloalkyl group. Examples of the saturated cycloalkyl group include monocyclic structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 1-methylcyclopropyl, 2-methylcyclopentyl, 2-methylcyclooctyl, etc., or multiple ring structures or cross-linked 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, and hexadienyl. Cycloalkyl groups can be substituted or unsubstituted. An example of such a substituted cycloalkyl group is cyclohexanol.

[0036] An "aryl" group is an aromatic carbocyclic group (C6-C) having 6 to 14 carbon atoms and consisting of a single ring (e.g., phenyl) or multiple fused rings (e.g., naphthyl or anthryl). 14 This refers to an aryl group. In one embodiment, the aryl group has 6 to 14 carbon atoms (C6-C) in the ring portion of the aryl group. 14 It contains aryl atoms, and in other embodiments, 6 to 12 carbon atoms (C6-C6). 12 Aryl) or 6-10 carbon atoms (C6-C 10This includes aryl groups. Certain aryl groups include phenyl, biphenyl, and naphthyl. The aryl group can be substituted or unsubstituted. The phrase "aryl group" also includes groups containing fused rings, such as fused aromatic-aliphatic ring systems (e.g., indanyl, tetrahydronaphthyl).

[0037] "Halogen" or "halo" refers to fluorine, chlorine, bromine, or iodine.

[0038] "Haloalkyl" refers to an alkyl group substituted with one or more halo groups, as defined above. Examples include trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2-fluoropropyl, and 1,2-dibromoethyl. In some embodiments, the haloalkyl group has 1 to 6 carbon atoms and is substituted with one or more halo groups (C1-C6 haloalkyl), or the haloalkyl group has 1 to 3 carbon atoms and is substituted with one or more halo groups (C1-C3 haloalkyl). The halo groups may all be the same or different. Unless otherwise specified, the haloalkyl group may be substituted as desired.

[0039] A "heteroaryl" group is an aromatic ring system having 1 to 4 heteroatoms as ring atoms in a heteroaromatic ring system, with the remaining atoms being carbon atoms. In some embodiments, the heteroaryl group contains 3 to 6 ring atoms, and in other embodiments, 6 to 9 or even 6 to 10 atoms are contained in the ring portion of the group. Suitable heteroatoms include oxygen, sulfur, and nitrogen. In certain embodiments, the heteroaryl ring system is monocyclic or bicyclic. Non-limiting examples include pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, benzisoxazolyl (e.g., benzo[d]isoxazolyl), thiazolyl, pyrrolyl, pyridadinyl, pyrimidyl, pyrazinyl, thiophenyl, benzothiophenyl, furanil, benzofuranil, indolyl (e.g., indolyl-2-onyl or isoindoline-1-onyl), azaindolyl (pyrrolopyridyl or 1H-pyrrolo[2,3-b]pyridyl), indazolyl, benzimidazolyl (e.g., 1H-benzo[d]imidazolyl), imidazopyridyl (e.g., A Examples of heteroaryl groups include, but are not limited to, zabenzimidazolyl (or 1H-imidazo[4,5-b]pyridyl), pyrazolopyridyl, triazolopyridyl, benzotriazolyl (e.g., 1H-benzo[d][1,2,3]triazolyl), benzoxazolyl (e.g., benzo[d]xazolyl), benzothiazolyl, benzothiadiazolyl, isoxazolopyridyl, thianaphthalenyl, prinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl (e.g., 3,4-dihydroisoquinoline-1(2H)-onyl), tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Heteroaryl groups may be substituted or unsubstituted.

[0040] A "heterocyclyl" group is a non-aromatic cycloalkyl group in which the 1 to 4 ring carbon atoms are independently substituted with heteroatoms selected from O, S, and N. In some embodiments, a heterocyclyl group contains 3 to 10 ring members, while other groups contain 3 to 5, 3 to 6, or 3 to 8 ring members. Heterocyclyls can also be bonded to another group at any ring atom (in other words, any carbon or heteroatom of the heterocycle). Heterocycloalkyl groups can be substituted or unsubstituted. Heterocyclyl groups include saturated and partially saturated ring systems. Furthermore, the term "heterocyclyl" is intended to encompass a non-aromatic ring containing at least one heteroatom, which may be fused with an aryl or heteroaryl ring, regardless of its bonding to the rest of the molecule. The term also includes bridging polycyclic ring systems containing heteroatoms. Representative examples of heterocyclyl groups include azilidinyl, azetidinyl, azepanil, pyrrolidyl, imidazolidinyl (e.g., imidazolidine-4-onyl or imidazolidine-2,4-dionyl), pyrazolidinyl, thiazolidinyl, tetrahydrothiophenyl, tetrahydrofuranil, piperidyl, piperazinyl (e.g., piperazine-2-onyl), morpholinyl, thiomorpholinyl, tetrahydropyranil (e.g., tetrahydro-2H-pyranil), tetrahydrothiopyranil, oxathianil, dithianil, 1,4-dioxaspiro[4.5]decanyl, homopiperazinyl, quinuclidyl, or tetrahydropyrimidine-2(1H)-one. Typical substituted heterocyclyl groups may be monosubstituted or bisubstituted or more. For example, a pyridyl group or morpholinyl group may be 2-, 3-, 4-, 5-, or 6-substituted, or bisubstituted with various substituents as listed below, but is not limited to these.

[0041] Except for alkyl groups, where a group described herein is said to be “substituted,” it may be substituted with any suitable substituent. Exemplary substituents are those found in the exemplary compounds and embodiments disclosed herein, as well as halogens (chloro, iodo, bromo or fluoro); alkyl; hydroxyl; alkoxy; alkoxyalkyl; amino; alkylamino; carboxy; nitro; cyano; thiol; thioether; imine; imide; amidine; guanidine; enamine; aminocarbonyl; acylamino; phosphonate; phosphine; thiocarbonyl; sulfinyl; sulfone; sulfonamide; ketone; aldehyde; ester; urea; urethane; oxime; hydroxylamine; alkoxyamine; aralkoxyamine; N-oxide; hydrazine; hydrazide; hydrazone; azide; isocyanate; isothiocyanate; cyanate; thiocyanate; oxygen (=O); B(OH)2, O(alkyl)aminocarbonyl; monocyclic or condensed These are cycloalkyls (e.g., cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl), which may be non-condensed polycyclic; or heterocyclyls (e.g., pyrrolidyl, piperidyl, piperazinyl, morpholinyl, or thiadinyl), which may be monocyclic or condensed or non-condensed polycyclic; monocyclic or condensed or non-condensed polycyclic aryls or heteroaryls (e.g., phenyl, naphthyl, pyrrolyl, indolyl, furanyl, thiophenyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, triazolyl, tetrazolyl, pyrazolyl, pyridyl, quinolinyl, isoquinolinyl, acridinyl, pyrazinyl, pyridadinyl, pyrimidyl, benzimidazolyl, benzothiophenyl, or benzofuranyl); aryloxys; aralkyloxys; heterocyclyloxys; and heterocyclylalkoxys.

[0042] Alternatively, you can use other commonly used chemical names. For example, divalent groups such as divalent alkyl, divalent phenyl, divalent heteroaryl, and divalent heterocyclyl can also be referred to as alkylene, phenylene, heteroarylene, or heterocyclylene, respectively.

[0043] Embodiments of this disclosure include pharmaceutically acceptable salts, tautomers, isotopologues, and stereoisomers of the compounds provided herein (e.g., compounds of formula (I)).

[0044] As used herein, the term “pharmaceutically acceptable salt” refers to a salt produced 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 the compound of formula (I) include, but are not limited to, metal salts derived from aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc, or organic salts derived from lysine, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methyl-glucamine), and procaine. Suitable non-toxic acids include, but are not limited to, inorganic and organic acids such as acetic acid, alginic acid, anthranilic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethensulfonic acid, formic acid, fumaric acid, furoic acid, galacturonic acid, gluconic acid, glucuronic acid, glutamic acid, glycolic acid, hydrobromic acid, hydrochloride, isethionic acid, lactate, maleate, malate, mandelate, methanesulfonate, mucinic acid, nitric acid, pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, propionic acid, salicylic acid, stearic acid, succinic acid, sulfanilic 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. Specific salts include hydrochloride, formate, and mesylate. Other materials are well known in the relevant technical field; see, for example, Remington's Pharmaceutical Sciences, 18th eds., Mack Publishing, Easton PA (1990) or Remington: The Science and Practice of Pharmacy, 19th eds., Mack Publishing, Easton PA (1995).

[0045] In this specification, unless otherwise stated, the terms “stereoisomer” or “stereoisomerically pure” mean one stereoisomer of a particular compound that substantially does not contain any other stereoisomers of that compound. For example, a stereoisomerically pure compound having one chiral center substantially does not contain the opposite enantiomer of that compound. A stereoisomerically pure compound having two chiral centers substantially does not contain any other diastereomer of that compound. A typical stereoisomerically pure compound is one in which one stereoisomer of the compound is present in greater proportions than about 80% by weight and the other stereoisomer of the compound in less than about 20% by weight; one stereoisomer of the compound is present in greater proportions than about 90% by weight and the other stereoisomer of the compound in less than about 10% by weight; one stereoisomer of the compound is present in greater proportions than about 95% by weight and the other stereoisomer of the compound is present in less than about 5% by weight; or one stereoisomer of the compound is present in greater proportions than about 97% by weight and the other stereoisomer of the compound is present in less than about 3% by weight. The compounds disclosed herein have a chiral center and may exist as racemates, individual enantiomers or diastereomers, and mixtures thereof. All such isomeric forms, including mixtures thereof, are included in the embodiments disclosed herein.

[0046] The use of stereoisomerically pure forms of the compounds disclosed herein and the use of mixtures of those forms are encompassed in the embodiments disclosed herein. For example, mixtures containing equimolar or unequal amounts of enantiomers of a particular compound may be used in the methods and compositions disclosed herein. These isomers can be asymmetrically synthesized or separated using standard techniques such as chiral columns or chiral resolving agents. For example, 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 (McGraw-Hill, NY, 1962); Wilen, SH, Tables of Resolving Agents and Optical Resolutions p. 268 (EL Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN, 1972); Todd, M., Separation Of Enantiomers :Synthetic Methods (Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany, 2014); Toda, F., Enantiomer Separation:Fundamentals and Practical Methods (Springer Science & Business Media, 2007); See Subramanian, G., Chiral Separation Techniques: A Practical Approach (John Wiley & Sons, 2008); and Ahuja, S., Chiral Separation Methods for Pharmaceutical and Biotechnological Products (John Wiley & Sons, 2011).

[0047] It should also be noted that the compounds disclosed herein may include E isomers and Z isomers or mixtures thereof, as well as cis and trans isomers or mixtures thereof. In certain embodiments, the compound is isolated as either an E isomer or a Z isomer. In other embodiments, the compound is a mixture of E and Z isomers.

[0048] A "tautomer" refers to an isomer of a compound that is in equilibrium with it. The concentration of these isomers varies depending on the environment in which the compound exists; for example, it may differ depending on whether the compound is a solid, an organic solution, or an aqueous solution. For example, in aqueous solution, pyrazoles may exhibit the following isomers, which are called tautomers: [ka]

[0049] As will be readily apparent to those skilled in the art, various functional groups and other structures may exhibit tautomerism, and all tautomers of the compound of formula (I) are within the scope of this disclosure.

[0050] Furthermore, it should be noted that the compounds disclosed herein may contain atomic isotopes in proportions not found in nature for one or more atoms. For example, a compound may contain tritium ( 3 H), Iodine-125 ( 125 I), Sulfur 35 ( 35 S) or carbon-14 ( 14 Can it be radiolabeled with radioactive isotopes such as C), or deuterium ( 2 H), carbon-13 ( 13 C) or nitrogen 15 ( 15Isotope enrichment can be achieved by means of N), etc. As used herein, “isotopologue” is an isotope-enriched compound. The term “isotopologue” refers to an atom having an isotope composition other than the natural isotope composition of that atom. The term “isotopologue” may also refer to a compound containing at least one atom having an isotope composition other than the natural isotope composition of that atom. The term “isotopologue” means the amount of each isotope present relative to a given atom. Radiolabeled compounds and isotope-enriched compounds are useful as therapeutic agents, e.g., cancer treatments, 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 radioactive or not, are intended to be included within the scope of the embodiments provided herein. In some embodiments, isototopologues of the compounds disclosed herein are provided, for example, isototopologues are compounds rich in deuterium, carbon-13, and / or nitrogen-15. As used herein, “deuterated” means that at least one hydrogen (H) atom is converted into deuterium (D or 2 This refers to compounds substituted with H, i.e., compounds in which deuterium is concentrated at at least one position.

[0051] Regardless of stereoisomer or isotopic composition, it is understood that each compound disclosed herein may be provided in any form of the pharmaceutically acceptable salts discussed herein. Similarly, it is understood that the isotopic composition may vary independently of the stereoisomer composition of each compound referred to herein. Furthermore, the isotopic composition may vary, but is limited to the elements present in each compound or its salts disclosed herein, or independently of the selection of pharmaceutically acceptable salts of each compound.

[0052] Please note that in the event of any discrepancy between the described structure and its name, the described structure will take precedence.

[0053] As used herein, “treatment” means the overall or partial relief of a disorder, disease or condition, or one or more symptoms associated with a disorder, disease or condition, or the reduction or cessation of further progression or worsening of those symptoms, or the relief or eradication of the cause of the disorder, disease or condition itself. In one embodiment, a disorder is a neurodegenerative disease or its symptoms as described herein.

[0054] As used herein, “prevention” means a method of delaying and / or interfering with the onset, recurrence or progression of the whole or partial disability, disease or condition, a method of preventing a subject from acquiring a disability, disease or condition, or a method of reducing the risk of a subject acquiring a disability, disease or condition. In one embodiment, disability is a neurodegenerative disease or its symptoms as described herein.

[0055] In relation to the compounds disclosed herein, the term “effective amount” means an amount that can treat or prevent the disorder, disease or condition, or symptoms thereof, disclosed herein.

[0056] As used herein, the terms “subject” or “patient” include, but are not limited to, animals such as cattle, monkeys, horses, sheep, pigs, birds, turkeys, quail, cats, dogs, mice, rats, rabbits, or guinea pigs, and are mammals in one embodiment and humans in another embodiment. In one embodiment, the subject is a human being who has or is at risk of having a disease or symptoms mediated by S1P5.

[0057] Various features of the present invention may be described in the context of a single embodiment, but these features may also be provided individually or in any suitable combination. Conversely, the present invention may be described herein in the context of a separate embodiment for clarity, but the present invention may also be implemented in a single embodiment.

[0058] compound In one embodiment, the formula provided herein is (I): [ka] [In the formula, R 1 It is a C1-C6 haloalkyl; W is either CH or N; X 1 is either CH or N; X 2 is CH2, CH, C, N, or NH; Y 1 and Y 2 It is independently either CH or N; L 1 These are -C(O)N(H)-, -C(O)-, or C1-C6 alkylenes; L 2 The bonds are -C(O)- and -NR. 2 -, -CH2N(R 2 )-, -CH2-, or [ka] and; R 2 is H or C1-C6 alkyl; x is either 0 or 1; * is L 2 Show the connection point; ** indicates the bonding site of the piperidinyl-2,6-dione group. It is a compound of or a pharmaceutically acceptable salt thereof.

[0059] In some embodiments, R 1 R is a C1-C6 haloalkyl. In some embodiments, R 1 R is a C1-C6 haloalkyl containing 1 to 13 halogen atoms. In some embodiments, R 1 R is a C1-C3 haloalkyl. In some embodiments, R 1R is a C1-C3 haloalkyl containing 1 to 7 halogen atoms. In some embodiments, R 1 R is -CF3, -CHF2, -CH2F, -CCl3, -CHCl2, -CH2Cl, -CF2Cl, -CFCl2, -CH2CF3, -CH2CHF2, or -CH2CCl3. In some embodiments, R 1 is -CHF2 or -CF3. In some embodiments, R 1 is -CF3. In some embodiments, R 1 It is -CHF2.

[0060] In some embodiments, W is CH or N. In some embodiments, W is CH. In some embodiments, W is N.

[0061] In some embodiments, X 1 is CH or N. In some embodiments, X 1 is CH. In some embodiments, X 1 It is N.

[0062] In some embodiments, X 2 is CH2, CH, C, N, or NH. In some embodiments, X 2 is CH2, CH, or C. In some embodiments, X 2 In some embodiments, X 2 is CH. In some embodiments, X 2 In some embodiments, X 2 is N or NH. In some embodiments, X 2 In some embodiments, X 2 It is NH.

[0063] In some embodiments, Y 1 and Y 2 In some embodiments, Y1 is CH. In some embodiments, Y 1 is N. In some embodiments, Y 2 is CH. In some embodiments, Y 2 is N. In some embodiments, Y 1 is CH and Y 2 is N. In some embodiments, Y 2 is CH and Y 1 is N. In some embodiments, Y 1 and Y 2 are each CH. In some embodiments, Y 1 and Y 2 are each N.

[0064] In some embodiments, L 1 is -C(O)N(H)-, -C(O)- or C1-C6 alkylene. In some embodiments, L 1 is -C(O)N(H)-, -C(O)- or C1-C3 alkylene. In some embodiments, L 1 is -C(O)N(H)-, -C(O)-, -CH2- or -CH2CH2-.

[0065] In some embodiments, L 1 is -C(O)N(H)-. In some embodiments, L 1 is -C(O)-. In some embodiments, L 1 is C1-C3 alkylene. In some embodiments, L 1 is -CH2-, -CH2CH2- or -CH2CH2CH2-. In some embodiments, L 1 is -CH2-. In some embodiments, L 1 is -CH2CH2-. In some embodiments, L 1 is -CH2CH2CH2-.

[0066] In some embodiments, L2 is a bond, -C(O)-, -NR 2 -, -CH2N(R 2 ), -CH2- or

Chem.

Chem.

[0067] In some embodiments, L 2 is a bond. In some embodiments, L 2 is -C(O)-. In some embodiments, L 2 is -NR 2 -. In some embodiments, L 2 is -CH2N(R 2 ). In some embodiments, L 2 is -CH2-. In some embodiments, L 2 is

Chem.

[0068] In some embodiments, R 2 is H or C1-C6 alkyl. In some embodiments, R 2 is H or C1-C3 alkyl. In some embodiments, R 2 is H or -CH3.

[0069] In some embodiments, R 2 is H.

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

[0071] In some embodiments, [ka] teeth, [ka] That is the case.

[0072] In some embodiments, [ka] teeth, [ka] That is the case.

[0073] In some embodiments, x is 0 or 1. In some embodiments, x is 0. In some embodiments, x is 1.

[0074] In some embodiments, [ka] teeth, [ka] That is the case.

[0075] In some embodiments, the compound of formula (I) is of formula (IA): [ka] [In the formula, R 1 , L 1 , L 2 W, X 1 , X 2 , Y 1 , Y 2 *, and ** are as described in formula (I). It is a compound of [the compound].

[0076] In some embodiments, the compound of formula (I) is formula (IBi) or (IB-ii): [ka] (In the formula, R 1 , L 1 W, X 1 and X 2 (This is as stated in equation (I)). It is a compound of [the compound].

[0077] In some embodiments, the compound of formula (I) is formula (IIa), (IIb), (IIc), or (IId): [ka] (In the formula, R 1 , L 1 , L 2 W, X 1 , X 2 , Y 1 and Y 2 (This is as stated in equation (I)). It is a compound of [the compound].

[0078] In some embodiments, the compound of formula (I) is of formula (IIIa), (IIIb) or (IIIc): [Chemical formula] (where R 1 , L 2 , W, X 1 , X 2 , Y 1 , Y 2 , * and ** are as described in formula (I); and n is an integer from 1 to 6) is a compound of

[0079] In some embodiments, the compound of formula (I) is of formula (IVa), (IVb), (IVc), (IVd), (IVe) or (IVf): [Chemical formula] [Chemical formula] (where R 1 , R 2 , L 1 , W, X 1 , X 2 , Y 1 , Y 2 , * and ** are as described in formula (I)) is a compound of

[0080] In some embodiments, the compound of formula (I) is of formula (Va), (Vb), (Vc), (Vd) or (Ve): [Chemical formula] [Chemical formula] (where R 1 , L 1 , L 2 , W, X 1 , X2 , Y 1 , Y 2 (* and ** are as described in formula (I)) It is a compound of [the compound].

[0081] In this specification, any description, variation, embodiment, or aspect of any part may be combined with any description, variation, embodiment, or aspect of any other part, and each combination of descriptions shall be understood to be the same as if they were described specifically and individually. For example, R of formula (I) 1 With respect to all descriptions, variations, embodiments or aspects provided herein are in accordance with W, X 1 , X 2 , Y 1 , Y 2 , L 1 , L 2 , R 2 And may be combined with all descriptions, variations, embodiments or aspects of x, and each combination can be combined in the same way as if it were specifically and individually listed. Furthermore, all descriptions, variations, embodiments or aspects of formula (I) apply equally to other formulas described in detail herein, where applicable, and for all formulas, each and all descriptions, variations, embodiments or aspects are understood to be described as if they were described separately and individually. For example, all descriptions, variations, embodiments, or aspects of formula (I) apply equally to any of the formulas detailed herein, where applicable, such as formulas (IA), (IBi), (IB-ii), (IIa), (IIb), (IIc), (IId), (IIIa), (IIIb), (IIIc), (IVa), (IVb), (IVc), (IVd), (IVe), (IVf), (Va), (Vb), (Vc), (Vd), and (Ve), and each and all descriptions, variations, embodiments, or aspects are described for all formulas as if they were described separately and individually.

[0082] In some embodiments, compounds selected from the compounds in Table 1 or their pharmaceutically acceptable salts are provided. While certain compounds described in this disclosure, including Table 1, are shown as specific stereoisomers and / or non-stereochemical forms, it will be understood that any or all stereochemical forms, including any enantiomer or diastereomer forms, as well as any tautomers or other forms of the compounds in this disclosure, including Table 1, are also described herein.

[0083] [Table 1] [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8]

[0084] In this specification, it is understood that combinations of substituents and / or variables in the described formulas are permissible only if such involvement results in a stable compound.

[0085] Furthermore, all compounds of formula (I) that exist in the form of free base or acid can be converted into their pharmaceutically acceptable salts by treatment with appropriate inorganic or organic bases or acids by methods known to those skilled in the art. The salts of the compounds of formula (I) can be converted into the form of free base or acid by standard techniques.

[0086] Synthesis method The compounds described herein can be prepared using conventional organic synthesis methods and commercially available starting materials or by using the methods provided herein. By way of illustration and not limitation, the compounds of formula (I) can be prepared as outlined in Schemes 1-3, similar to the examples described herein. It should be noted that those skilled in the art understand how to modify the methods described in the exemplary schemes and examples to obtain the desired product.

[0087] The compound of formula A can be prepared as outlined in Scheme 1. Under basic conditions such as in the presence of DIPEA, coupling of intermediate a-1 with intermediate a-2 forms intermediate a-3, and deprotection of intermediate a-3 under basic conditions forms intermediate a-4. Subsequently, coupling of intermediate a-4 with intermediate a-5 using TCFH / NMI gives intermediate a-6, which is then deprotected under basic conditions to give intermediate a-7. Intermediate a-7 is then coupled with intermediate a-8 (e.g., using HATU) to give intermediate a-9. Deprotection of a-9 under acidic conditions gives the compound of formula A. [Chemical formula] [wherein, X is halo (e.g., chloro); Pg is a protecting group (e.g., Boc); each R is independently alkyl (e.g., methyl or ethyl); R 1 is as described for formula (I); and [Chemical formula] is [ka] This represents the base of equation (I) corresponding to [the given expression].

[0088] The compound of formula B can be prepared as outlined in Scheme 1. By reducing intermediate a-5 (e.g., using NaBH4), intermediate b-1 was obtained. Then, intermediate b-1 was coupled with intermediate a-4 using TCFH / NMI to obtain intermediate b-2. After oxidation of intermediate b-2, reductive amination was performed with intermediate a-8 (e.g., using IBX followed by NaBH(OAc)3) to produce intermediate b-3. Then, under acidic conditions, b-3 was deprotected to obtain the compound of formula B. [ka] [wherein Pg is a protecting group (e.g., Boc); R is an alkyl group (e.g., methyl or ethyl); R 1 This is as described for equation (I); and [ka] teeth, [ka] This represents the base of equation (I) corresponding to [the given expression].

[0089] The compound of formula C can be prepared as outlined in Scheme 3. Reducing intermediate c-1 (e.g., using NaBH4) yields intermediate c-2, which is then converted to c-3. Next, cyanylation yields intermediate c-4, which is then converted to intermediate c-5 (e.g., using DIBAL-H). Reducing c-5 (e.g., using NaBH4) yields intermediate c-6. Intermediate c-6 is then hydrogenated to obtain intermediate c-7. Intermediate c-7 is coupled with intermediate a-4 using TCFH / NMI to obtain intermediate c-8. After oxidation of intermediate c-8, reductive amination is performed together with intermediate a-8 (e.g., using IBX followed by NaBH(OAc)3) to produce intermediate c-9. Finally, c-9 is deprotected under acidic conditions to obtain the compound of formula C. [Table 9] [In the formula, Pg is a protecting group (e.g., Boc); Lg is a leaving group (e.g., mesylate); R is an alkyl group (e.g., methyl or ethyl); R 1 This is as described for equation (I); and [ka] teeth, [ka] [This represents the base of equation (I) corresponding to this.]

[0090] How to use Embodiments of this disclosure provide a method for modulating IRAK4 in a subject requiring modulation, characterized by administering an effective amount of a compound of formula (I) to the subject. Modulation of IRAK4 (e.g., inhibition or activation) can be evaluated and demonstrated by various methods known in the art. Kits and commercially available assays are available to determine whether and to what extent IRAK4 has been modulated (e.g., inhibited or activated).

[0091] In one embodiment, provided herein is a method for modulating IRAK4, characterized by contacting IRAK4 with an effective amount of the compound of formula (I) or any embodiment or variation thereof. In some embodiments, the compound of formula (I) inhibits IRAK4. In another embodiment, 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.

[0092] In some embodiments, provided herein are methods for targeting and degrading IRAK4, characterized by contacting IRAK4 with an effective amount of a compound of formula (I) or any embodiment or modification thereof.

[0093] In some embodiments, the compound of formula (I) adjusts the activity of IRAK4 to approximately 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) increases the activity of IRAK4 by approximately 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%, and 80-100%. Adjust to 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%.

[0094] Furthermore, provided in certain embodiments of this disclosure is a method for degrading IRAK4 in an object that needs to be degraded, characterized by administering an effective amount of the compound of formula (I) to the object. Degradation of IRAK4 can be evaluated and demonstrated by various methods known in the art. Kits and commercially available assays (e.g., cell line assays) can be used to determine whether and to what extent IRAK4 has been degraded.

[0095] In one embodiment, provided herein is a method for degrading IRAK4, characterized by contacting IRAK4 with an effective amount of the compound of formula (I) or any embodiment or variation thereof. In some embodiments, the compound of formula (I) partially degrades IRAK4. In some embodiments, the compound of formula (I) completely degrades IRAK4.

[0096] 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) contains IRAK4 in amounts of approximately 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%, Decompose into 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%.

[0097] In another embodiment, provided herein is a method for treating an inflammatory disease or autoimmune disease in a subject requiring treatment, comprising administering an effective amount of a compound of formula (I) to the subject. In some embodiments, provided herein is a method for treating an inflammatory disease in a subject requiring treatment, comprising administering an effective amount of a compound of formula (I) to the subject. In some embodiments, provided herein is a method for treating an autoimmune disease in a subject requiring treatment, comprising administering an effective amount of a compound of formula (I) to the subject. In some embodiments, provided herein is a method for preventing an inflammatory disease or autoimmune disease in a subject requiring treatment, comprising administering an effective amount of a compound of formula (I) to the subject. In some embodiments, provided herein is a method for preventing an inflammatory disease in a subject requiring treatment, comprising administering an effective amount of a compound of formula (I) to the subject. In some embodiments, provided herein is a method for preventing an autoimmune disease in a subject requiring treatment, comprising administering an effective amount of a compound of formula (I) to the subject. Non-exclusive examples of inflammatory or autoimmune diseases include atopic dermatitis, asthma, lupus, rheumatoid arthritis, familial Mediterranean fever, psoriasis, generalized pustular psoriasis, cryopyrin-associated periodic syndromes, hidradenitis suppurativa, Beckett syndrome, or familial cold autoinflammatory syndrome.

[0098] In some embodiments, administering a compound of formula (I) to a subject predisposed to inflammatory or autoimmune diseases prevents the subject from developing any symptoms of an inflammatory or autoimmune disease (e.g., tumor growth or metastasis). In some embodiments, administering a compound of formula (I) to a subject that has not yet shown any symptoms of an inflammatory or autoimmune disease prevents the subject from developing any symptoms of an inflammatory or autoimmune disease. In some embodiments, administering a compound of formula (I) to a subject in need of treatment reduces the severity of the inflammatory or autoimmune disease in the subject. In some embodiments, administering a compound of formula (I) to a subject in need of treatment stabilizes the inflammatory or autoimmune disease (prevents or delays its worsening). In some embodiments, administering a compound of formula (I) to a subject in need of treatment delays the onset or recurrence of the inflammatory or autoimmune disease. In some embodiments, administering a compound of formula (I) to a subject in need of treatment slows the progression of the inflammatory or autoimmune disease. In some embodiments, administering a compound of formula (I) to a subject in need of treatment provides partial remission of an inflammatory or autoimmune disease. In some embodiments, administering a compound of formula (I) to a subject in need of treatment provides complete remission of an inflammatory or autoimmune disease. In some embodiments, administering a compound of formula (I) to a subject in need of treatment reduces the dose of one or more other drugs required to treat the inflammatory or autoimmune disease. In some embodiments, administering a compound of formula (I) to a subject in need of treatment enhances the effect of other drugs used to treat the inflammatory or autoimmune disease. In some embodiments, administering a compound of formula (I) to a subject in need of treatment slows the progression of the inflammatory or autoimmune disease. In some embodiments, administering a compound of formula (I) to a subject in need of treatment improves the quality of life of a subject with an inflammatory or autoimmune disease.In some embodiments, administering a compound of formula (I) to a subject in need of treatment extends the survival time of subjects with inflammatory or autoimmune diseases.

[0099] In one embodiment, the foregoing provides a method for preventing a subject predisposed to an inflammatory disease or autoimmune disease from developing any symptoms of an inflammatory disease or autoimmune disease, the method comprising administering a compound of formula (I) to the subject. In some embodiments, the foregoing provides a method for preventing a subject that has not yet shown symptoms of an inflammatory disease or autoimmune disease from developing any symptoms of an inflammatory disease or autoimmune disease, the method comprising administering a compound of formula (I) to the subject.

[0100] In some embodiments, the foregoing provides a method for reducing the severity 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 foregoing provides a method for stabilizing 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 prevents the worsening of an inflammatory or autoimmune disease. In some embodiments, the method slows the worsening of an inflammatory or autoimmune disease.

[0101] In another embodiment, provided herein is a method for delaying the onset or recurrence of an inflammatory or autoimmune disease in a subject, the method comprising administering a compound of formula (I) to the subject.

[0102] In some embodiments, the methods provided herein are for slowing the progression of an inflammatory or autoimmune disease in a subject, the methods comprising administering a compound of formula (I) to the subject. In some embodiments, the methods result in partial remission of the inflammatory or autoimmune disease. In some embodiments, the methods result in complete remission of the inflammatory or autoimmune disease.

[0103] In a further embodiment, provided herein is a method for reducing the dose of one or more other agents 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, provided herein is a method for enhancing the effect of another agent used to treat an inflammatory or autoimmune disease in a subject, the method comprising administering a compound of formula (I) to the subject.

[0104] Furthermore, a method is provided for delaying the progression of inflammatory or autoimmune diseases in subjects, the method comprising administering a compound of formula (I) to the subject. In some embodiments, the method improves the quality of life of subjects with inflammatory or autoimmune diseases. In some embodiments, the method extends the survival time of subjects with inflammatory or autoimmune diseases.

[0105] In another embodiment, provided herein are methods for treating symptoms of an inflammatory or autoimmune disease caused by a disease in a subject requiring treatment, comprising administering an effective amount of the compound of formula (I) to the subject. In some embodiments, provided herein are methods for preventing symptoms of an inflammatory or autoimmune disease caused by a disease in a subject requiring treatment, comprising administering an effective amount of the compound of formula (I) to the subject. In some embodiments, administering the compound of formula (I) to a subject predisposed to a disease that causes symptoms of an inflammatory or autoimmune disease prevents the subject from developing symptoms of the inflammatory or autoimmune disease. In some embodiments, administering the compound of formula (I) to a subject that has not yet developed symptoms of an inflammatory or autoimmune disease related to a disease that causes symptoms of an inflammatory or autoimmune disease can prevent the subject from developing symptoms of the inflammatory or autoimmune disease. In some embodiments, administering the compound of formula (I) to a subject requiring treatment reduces the severity of symptoms of an inflammatory or autoimmune disease caused by a disease in the subject. In some embodiments, administering a compound of formula (I) to a subject in need of treatment stabilizes the symptoms of an inflammatory or autoimmune disease (prevents or delays the worsening of symptoms of an inflammatory or autoimmune disease). In some embodiments, administering a compound of formula (I) to a subject in need of treatment delays the onset or recurrence of symptoms of an inflammatory or autoimmune disease caused by the disease. In some embodiments, administering a compound of formula (I) to a subject in need of treatment slows the progression of symptoms of an inflammatory or autoimmune disease caused by the disease. In some embodiments, administering a compound of formula (I) to a subject in need of treatment results in partial remission of the disease causing symptoms of an inflammatory or autoimmune disease. In some embodiments, administering a compound of formula (I) to a subject in need of treatment results in complete remission of the disease causing symptoms of an inflammatory or autoimmune disease.In some embodiments, administering a compound of formula (I) to a subject in need of treatment reduces the dose of one or more other drugs required to treat a disease causing symptoms of an inflammatory or autoimmune disease. In some embodiments, administering a compound of formula (I) to a subject in need of treatment enhances the effect of other drugs used to treat symptoms of an inflammatory or autoimmune disease. In some embodiments, administering a compound of formula (I) to a subject in need of treatment delays the progression of a disease causing symptoms of an inflammatory or autoimmune disease. In some embodiments, administering a compound of formula (I) to a subject in need of treatment improves the quality of life of a subject with a disease causing symptoms of an inflammatory or autoimmune disease. In some embodiments, administering a compound of formula (I) to a subject in need of treatment extends the survival time of a subject with a disease causing symptoms of an inflammatory or autoimmune disease. In some embodiments, the disease is atopic dermatitis, asthma, lupus, rheumatoid arthritis, familial Mediterranean fever, psoriasis, generalized pustular psoriasis, cryopyrin-associated periodic syndromes, hidradenitis suppurativa, Beckett syndrome, or familial cold autoinflammatory syndrome.

[0106] In some embodiments, compounds of formula (I) are useful for treating diseases selected from atopic dermatitis, asthma, lupus, rheumatoid arthritis, familial Mediterranean fever, psoriasis, generalized pustular psoriasis, cryopyrin-associated periodic syndromes, hidradenitis suppurativa, Beckett syndrome, and familial cold autoinflammatory syndrome.

[0107] Pharmaceutical composition and route of administration The compounds provided herein may be administered orally, topically, or parenterally to a subject by conventional dosage forms, such as capsules, microcapsules, tablets, granules, powders, lozenges, tablets, suppositories, injections, suspensions, syrups, patches, creams, lotions, ointments, gels, sprays, solutions, and emulsions.

[0108] The compounds disclosed herein may be administered orally, topically, or parenterally to a subject in the form of conventional dosage forms, such as capsules, microcapsules, tablets, granules, powders, lozenges, pills, suppositories, injections, suspensions, syrups, patches, creams, lotions, ointments, gels, sprays, solutions, and emulsions. Suitable formulations may include excipients (e.g., sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, or calcium carbonate), binders (e.g., cellulose, methylcellulose, hydroxymethylcellulose, polypropylpyrrolidone, polyvinylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, or starch), disintegrants (e.g., starch, carboxymethylcellulose, hydroxypropyl starch, low-substituted hydroxypropylcellulose, sodium bicarbonate, calcium phosphate, or calcium citrate), and lubricants (e.g., magnesium stearate, light It can be manufactured by conventional methods using conventional organic or inorganic additives such as anhydrous silicic acid, talc, sodium lauryl sulfate, flavoring agents (e.g., citric acid, menthol, glycine, or orange powder), preservatives (e.g., sodium benzoate, sodium bisulfate, 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 present at a level that produces the desired effect; for example, in unit doses for both oral and parenteral administration, it ranges from about 0.005 mg per kg of body weight to about 10 mg per kg of body weight of the subject.

[0109] The dose of the compound of formula (I) administered to a subject can vary over a wide range and is subject to the judgment of the healthcare professional. Generally, the compounds disclosed herein can be administered at doses of approximately 0.001 mg to approximately 10 mg per kg of body weight of the subject, 1 to 4 times a day, but the above dose can be appropriately changed depending on the subject's age, weight, medical condition, and route of administration. In any case, the amount of compound of formula (I) administered will vary depending on factors such as the solubility of the active ingredient, the formulation used, and the route of administration.

[0110] The compound of formula (I) may be administered orally for convenience. 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 administered orally as a solution or suspension, dispersed in water or juice (e.g., apple juice or orange juice) or any other liquid.

[0111] The compounds disclosed herein may also be administered intradermally, intramuscularly, intraperitoneally, percutaneously, intravenously, subcutaneously, intranasally, epidurally, sublingually, intracerebrally, intravaginally, percutaneously, rectally, mucous membrane, or by inhalation, or topically to the ear, nose, eye, or skin. The mode of administration is at the discretion of the healthcare professional and may be partially modified depending on the site of the condition.

[0112] In one embodiment, what is provided herein is a capsule containing a compound of formula (I) that does not contain additional carriers, excipients, or vehicles.

[0113] In another embodiment, provided herein is a composition comprising an effective amount of a compound of formula (I) and a pharmaceutically acceptable carrier or vehicle, wherein the pharmaceutically acceptable carrier or vehicle may include excipients, diluents, or mixtures thereof. In one embodiment, the composition is a pharmaceutical composition.

[0114] The compositions may take the form of tablets, chewable tablets, capsules, solutions, parenteral solutions, lozenges, suppositories, and suspensions. The compositions may be formulated to contain a daily dose, or a suitable portion of a daily dose, in a single tablet or capsule, or in a suitable amount of liquid, as a dosage unit. In one embodiment, the solution is prepared from a water-soluble salt such as hydrochloride. In general, all compositions are manufactured according to known methods in pharmaceutical chemistry. Capsules can be manufactured by mixing the compound of formula (I) with a suitable carrier or diluent and filling a suitable amount of the mixture into a capsule. Common carriers and diluents include, but are not limited to, a wide variety of starches, powdered cellulose, especially crystalline and microcrystalline cellulose, sugars such as fructose, mannitol and sucrose, grain flour and similar edible powders, and other inert powders.

[0115] Tablets can be manufactured by direct compression, wet granulation, or dry granulation. The formulations typically contain not only the compound but also diluents, binders, lubricants, and disintegrants. Typical diluents include, for example, various starches, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts such as sodium chloride, and powdered sugars. Powdered cellulose derivatives are also useful. Typical tablet binders include substances such as starch, gelatin, and sugars (e.g., lactose, fructose, glucose). Natural and synthetic gums such as acacia, alginates, methylcellulose, and polyvinylpyrrolidine are also convenient. Polyethylene glycol, ethylcellulose, and waxes can also function as binders.

[0116] In tablets, a lubricant may be necessary to prevent dye from adhering to the tablet and the tableting machine. The lubricant can be selected from lubricating solids such as talc, magnesium stearate, calcium stearate, stearic acid, and hydrogenated vegetable oils. Tablet disintegrants are substances that swell and disintegrate the tablet upon contact with moisture, releasing compounds. These include starch, clay, cellulose, algins, and gums. More specifically, corn and potato starch, methylcellulose, agar, bentonite, wood cellulose, powdered natural sponge, cation exchange resins, alginic acid, guar gum, citrus pulp, and carboxymethylcellulose, such as sodium lauryl sulfate, can also be used. Tablets may also be coated with sugars as flavoring or odor-modifying agents, or with film-forming protective agents to alter the tablet's solubility. Furthermore, the composition can be formulated as a chewable tablet by using substances such as mannitol in the formulation.

[0117] When administering the compound of formula (I) as a suppository, typical bases can be used. Cocoa butter is a conventional suppository base and can be modified by adding wax to slightly raise its melting point. In particular, water-miscible suppository bases containing polyethylene glycol of various molecular weights are widely used.

[0118] The effects of the compound of formula (I) can be delayed or prolonged by appropriate formulation. For example, pellets of the compound of formula (I) that can dissolve gradually can be prepared and incorporated into tablets or capsules, or as a sustained-release implantable device. This technique also includes preparing pellets with several different dissolution rates and filling a capsule with a mixture of these pellets. The tablets or capsules may be coated with a film that resists dissolution for a predictable period. Even parenteral formulations can be made long-acting by dissolving or suspending the compound of formula (I) in an oily or emulsifying agent that disperses slowly in serum.

[0119] Exemplary Example This disclosure is further illustrated by the following embodiments.

[0120] Embodiment P1. Formula (I): [ka] [In the formula, R 1 It is a C1-C6 haloalkyl; W is either CH or N; X 1 is either CH or N; X 2 is CH2, CH, C, N, or NH; Y 1 and Y 2 It is independently either CH or N; L 1 These are -C(O)N(H)-, -C(O)-, or C1-C6 alkylenes; L 2 The bonds are -C(O)- and -NR. 2 -, -CH2N(R 2 )-, -CH2- or [ka] and; R 2 is H or C1-C6 alkyl; x is either 0 or 1; * is L 2 This indicates the connection points to and ** indicates a bond site to the piperidinyl-2,6-dione group. Compounds of or pharmaceutically acceptable salts thereof.

[0121] Embodiment P2. The compound is of formula (IIa), (IIb), or (IIc): [ka] The compound described in Embodiment P1 or a pharmaceutically acceptable salt thereof.

[0122] Embodiment P3.R 1 The compound or a pharmaceutically acceptable salt thereof described in Embodiment P1 or P2, wherein the compound is a C1-C3 haloalkyl group.

[0123] Embodiment P4.R 1 The compound or a pharmaceutically acceptable salt thereof according to Embodiment P3, wherein the compound is -CHF2 or -CF3.

[0124] Embodiment P5. A compound according to any one of Embodiments P1 to P4 or a pharmaceutically acceptable salt thereof, wherein W is CH.

[0125] Embodiment P6. A compound according to any one of Embodiments P1 to P4 or a pharmaceutically acceptable salt thereof, wherein W is N.

[0126] Embodiment P7.X 1 The compound or a pharmaceutically acceptable salt thereof described in any one of embodiments P1 to P6, wherein CH is present.

[0127] Embodiment P8.X 1 A compound or a pharmaceutically acceptable salt thereof according to any one of embodiments P1 to P6, wherein N is present.

[0128] Embodiment P9. X 2 The compound or a pharmaceutically acceptable salt thereof according to any one of embodiments P1 to P8, wherein the compound is CH2, CH, or C.

[0129] Embodiment P10. X 2 A compound or a pharmaceutically acceptable salt thereof according to any one of embodiments P1 to P8, wherein the compound is N or NH.

[0130] Embodiment P11. Y 1 The compound or a pharmaceutically acceptable salt thereof described in any one of embodiments P1 to P10, wherein CH is present.

[0131] Embodiment P12. Y 1 A compound or a pharmaceutically acceptable salt thereof according to any one of embodiments P1 to P10, wherein N is present.

[0132] Embodiment P13. Y 2 The compound or a pharmaceutically acceptable salt thereof described in any one of embodiments P1 to P12, wherein CH is present.

[0133] Embodiment P14. Y 2 A compound or a pharmaceutically acceptable salt thereof according to any one of embodiments P1 to P12, wherein N is present.

[0134] Embodiment P15. L 1 A compound or a pharmaceutically acceptable salt thereof according to any one of embodiments P1 to P14, wherein the compound is -C(O)N(H)-, -C(O)-, or C1-C3 alkylene.

[0135] Embodiment P16. L 1 The compound described in Embodiment P15 or a pharmaceutically acceptable salt thereof, wherein the compound is -C(O)N(H)-, -C(O)-, -CH2- or -CH2CH2-.

[0136] Embodiment P17. L 2 However, the bond is -C(O)-, -NR 2 -, -CH2N(R 2 )-, -CH2- or [ka] and R 2 The compound or a pharmaceutically acceptable salt thereof according to any one of embodiments P1 to P16, wherein the compound is H or a C1-C3 alkyl group.

[0137] Embodiment P18. R 2 The compound described in Embodiment P17 or a pharmaceutically acceptable salt thereof, wherein the compound is H or -CH3.

[0138] Embodiment P19. [ka] but, [ka] The compounds described in embodiments P1 to P18 or their pharmaceutically acceptable salts.

[0139] Embodiment P20. [ka] but, [ka] A compound or a pharmaceutically acceptable salt thereof as described in any one of embodiments P1 to P19.

[0140] Embodiment P21. A compound according to any one of Embodiments P1 to P20 or a pharmaceutically acceptable salt thereof, wherein x is 0.

[0141] Embodiment P22. A compound according to any one of Embodiments P1 to P20 or a pharmaceutically acceptable salt thereof, wherein x is 1.

[0142] Embodiment P23. [ka] but [ka] A compound or a pharmaceutically acceptable salt thereof as described in any one of embodiments P1 to P20 and P22.

[0143] Embodiment P24. Compounds selected from the compounds in Table 1 and their pharmaceutically acceptable salts.

[0144] Embodiment P25. A pharmaceutical composition comprising a compound described in any one of Embodiments P1 to P24 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0145] Embodiment P26. A method for modulating interleukin-1 (IL1) receptor-related kinase 4 (IRAK4), characterized by contacting IRAK4 with an effective amount of a compound described in any one of Embodiments P1 to P24 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of Embodiment P25.

[0146] Embodiment P27. A method for treating an inflammatory disease or autoimmune disease in a subject requiring treatment, characterized by administering an effective amount of a compound described in any one of Embodiments P1 to P24 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of Embodiment P25, to the subject.

[0147] Embodiment P28. The method according to Embodiment P27, wherein the inflammatory or autoimmune disease is atopic dermatitis, asthma, lupus, rheumatoid arthritis, familial Mediterranean fever, psoriasis, generalized pustular psoriasis, cryopyrin-associated periodic syndromes, sweat gland abscess, Beckett syndrome, or familial cold autoinflammatory syndrome. [Examples]

[0148] The following examples are for illustrative purposes only and are not limiting. The compounds were named using the automated naming tool provided in ChemBiodraw Ultra (Cambridgesoft), which generates systematic names for chemical structures while adhering to the Kahn-Ingold-Prelogue order rule for stereochemistry. Those skilled in the art can modify the procedures described in the illustrated examples to arrive at the target product.

[0149] Salts of the compounds described herein can be prepared by standard methods such as mixing an acid (e.g., TFA, formic acid, or HCl) into the mobile phase during chromatographic purification, or stirring the product after chromatographic purification with an acidic solution (e.g., an aqueous solution of HCl).

[0150] The following abbreviations may be related to this application. abbreviation TIFF2026520336000058.tif207156 TIFF2026520336000059.tif81156

[0151] Synthesis Examples (LCMS method) LCMS method 1 Column: Luna C18 (2) 50 x 3 mm, 3 μm. Temperature: 45°C, Flow rate: 1.5 mL / min, Duration: 2.5 minutes. Mobile phase conditions: Initially 95% H2O + 0.1% FA / 5% MeCN + 0.1% FA, linear gradient to 95% MeCN + 0.1% FA for 1.3 minutes, then held in 95% MeCN + 0.1% FA for 1.2 minutes. MSD: ESI positive.

[0152] LCMS method 2 Column: Luna C18 (2) 50 x 3 mm, 3 μm. Temperature: 45°C, Flow rate: 1.5 mL / min, Duration: 3.5 minutes. Mobile phase conditions: Initially 95% H2O + 0.1% FA / 5% MeCN + 0.1% FA, linear gradient to 95% MeCN + 0.1% FA for 1.3 minutes, then retention in 95% MeCN + 0.1% FA for 2.2 minutes. MSD: ESI positive.

[0153] LCMS method 3 Column: SunFire C18 75 x 4.6 mm, 3.5 um. Temperature: 45°C, Flow rate: 1.5 mL / min, Duration: 6 minutes. Mobile phase conditions: Initially 95% H2O + 0.1% FA / 5% MeCN + 0.1% FA, followed by a linear gradient to 95% MeCN + 0.1% FA for 4 minutes, then a hold on 95% MeCN + 0.1% FA for 2 minutes. MSD: Positive.

[0154] LCMS method 4 Column: XBridge C18 4.6 x 75 mm 5 um. Temperature: 45℃, Flow rate: 1.5 mL / min, Duration: 6 minutes. Mobile phase conditions: Initial gradient 95% NH4HCO3 / 5% MeCN, linear gradient with 95% MeCN for 0-3 minutes, holding with 95% MeCN for 3 minutes.

[0155] Synthesis of intermediates The designation of specific intermediate compounds with numbers such as 1 or 2 is unique to the example described. Therefore, while multiple examples may refer to the same intermediate compound number, such as 1 or 2, the chemical structure of the compound differs between different examples.

[0156] Example I-1. Synthesis of a typical intermediate T-1 [ka] Step 1. Production of methyl 4-methylsulfonyloxycyclohexanecarboxylate (2) Methyl 4-hydroxycyclohexanecarboxylate (1) (5.0 g, 31.61 mmol, 1.0 equivalent) and Et3N (6.61 mL, 47.41 mmol, 1.5 equivalent) were dissolved in CH2Cl2 (158 mL, 0.2 M), to which methanesulfonyl chloride (3.18 mL, 41.09 mmol, 1.3 equivalent) was added dropwise at 0°C. The resulting mixture was stirred at 0°C for 30 minutes. TLC (KMnO4): SM: Rf = 0.25, Product: Rf = 0.30 (60% HCl / heptane). Water was added, and the aqueous layer was extracted with CH2Cl2 (3×). The combined organic layers were washed with 1 M HCl(1×) aqueous solution, dried over Na₂SO₄, filtered, and then concentrated to dryness under reduced pressure to obtain pure 2 (7.47 g, quantitative yield) without any further purification. LC-MS method 1: 99.9% purity at 215 nm; [M-MsOH+H] + = 141.2 m / z, [M+Na] + = 259.2 m / z. 1 H NMR (400 MHz, CDCl3) δ ppm 1.67 - 1.82 (m, 4 H), 1.87 - 1.97 (m, 2 H), 1.97 - 2.07 (m, 2 H), 2.36 - 2.45 (m, 1 H), 3.01 (s, 3 H), 3.68 (s, 3 H), 4.88 - 4.94 (m, 1 H).

[0157] Step 2. Preparation of methyl (1r,4r)-4-(4-nitro-3-(trifluoromethyl)-1H-pyrazole-1-yl)cyclohexane-1-carboxylate (4) 4-nitro-3-(trifluoromethyl)-1H-pyrazole 3 (1.0 g, 5.52 mmol, 1.0 eq.) and methyl 4-methylsulfonyloxycyclohexanecarboxylate 2 (1.3 g, 5.52 mmol, 2.0 eq.) were dissolved in dry DMF (18.41 mL, 0.3 M) and Cs2CO3 (3.6 g, 11.05 mmol, 2.0 eq.) was added. After stirring overnight at 90°C, complete conversion to 4 was shown by LC-MS. The reaction was quenched with water, and the aqueous layer was extracted three times with ELISA. The combined organic layers were washed twice with water and once with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by flash chromatography (eluted with 0-35% MTBE / heptane over 15 CV of 80 g SiO2, followed by elution with 35% MTBE / heptane over 3 CV). The target product was eluted with 30% MTBE / heptane (confirmed by TLC with elution of 60% MTBE / heptane, Rf=0.30, UV + KMnO4). The fractions were combined, concentrated to dryness, and 4 (671 mg, 36% yield) was obtained as a pale yellow solid.

[0158] LC-MS method 1: Retention time: 1.819 min, 99.9% purity at 215 nm, [M+H] + = 322.2. 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.52 (qd, J = 13.0, 3.0 Hz, 2 H), 1.83 (br dd, J = 12.5, 3.1 Hz, 2 H), 2.00 - 2.17 (m, 4 H), 2.42 (tt, J = 12.2, 3.5 Hz, 1 H), 3.61 (s, 3 H), 4.39 (tt, J = 11.9, 3.8 Hz, 1 H), 9.19 (s, 1 H). 19 F NMR (377 MHz, DMSO-d6) δ ppm -61.45 (s, 3 F).

[0159] Step 3. Preparation of methyl 4-[4-amino-3-(trifluoromethyl)pyrazole-1-yl]cyclohexanecarboxylate (T-1) To a solution of methyl 4-[4-nitro-3-(trifluoromethyl)pyrazole-1-yl]cyclohexanecarboxylate 4 (671 mg, 2.09 mmol, 1.0 eq.) in ethyl acetate (13.93 mL, 0.15 M), 10% Pd / C (444 mg, 0.42 mmol, 0.2 eq.) was added. Nitrogen was supplied to the solution for 10 minutes, followed by hydrogen bubbling for 10 minutes. The resulting mixture was stirred under a hydrogen atmosphere (1 atm). After stirring at room temperature for 5 hours, complete conversion to T-1 was demonstrated by LC-MS. The reaction mixture was filtered through a Celite pad and washed with siRNA. The filtrate was concentrated under reduced pressure to obtain T-1 (608 mg, quantitative yield) as a pink solid. The crude product was used in the next step without further purification. LC-MS method 1: Retention time: 1.562 min, 99.9% purity at 215 nm, [M+H] + = 292.2. 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.41 - 1.60 (m, 2 H), 1.64 - 1.81 (m, 2 H), 1.91 - 2.06 (m, 4 H), 2.32 - 2.45 (m, 1 H), 3.60 (s, 3 H), 4.00 - 4.13 (m, 1 H), 4.21 (s, 2 H), 7.22 (s, 1 H). 19 F NMR (377 MHz, DMSO-d6) δ ppm -58.85 (s, 3 F).

[0160] Example I-2. Synthesis of a typical intermediate T-2 [ka]

[0161] Step 1. Production of methyl 4-methylsulfonyloxycyclohexanecarboxylate (2) Under nitrogen, a solution of methyl 4-hydroxycyclohexanecarboxylate 1 (5.0 g, 31.61 mmol, 1.0 eq.) in CH2Cl2 (105 mL, 0.3 M) was cooled to 0°C, and methanesulfonyl chloride (2.69 mL, 34.77 mmol, 1.1 eq.) and triethylamine (5.28 mL, 37.93 mmol, 1.2 eq.) were added, with the latter added dropwise. After stirring at 0°C for 2 hours, complete conversion was shown by TLC (CH2Cl2 / MeOH 5.5:0.5, KMnO4 staining). The reaction was quenched by adding water, the phases were separated, and the aqueous phase was extracted three times with CH2Cl2. The combined organic phases were washed twice with saline, dried on magnesium sulfate, filtered, and concentrated to obtain 2 (7.45 g, 99% yield) as a yellow oil. 1 H NMR (400 MHz, CDCl3) δ ppm 1.67 - 1.84 (m, 4 H), 1.87 - 1.99 (m, 2 H), 2.00 - 2.09 (m, 2 H), 2.36 - 2.46 (m, 1 H), 3.02 (s, 3 H), 3.69 (s, 3 H), 4.87 - 4.96 (m, 1 H).

[0162] Step 2. Preparation of methyl 4-[3-(difluoromethyl)-4-nitropyrazole-1-yl]cyclohexanecarboxylate (4) Under nitrogen, in a heated and dried round-bottom flask, a solution of 2 (1.16 g, 4.91 mmol, 1.0 eq.), 3 (0.80 g, 4.91 mmol, 1.0 eq.), and DMF (12.3 mL, 0.4 M) was stirred at room temperature for 5 minutes, and then Cs2CO3 (3.20 g, 9.81 mmol, 2.0 eq.) was added. The resulting mixture was stirred at 90°C for 16 hours. After the incomplete conversion of 3 was confirmed by LC-MS (Method 1), a second dose of 2 (1.16 g, 4.91 mmol, 1.0 eq.) was added, and the mixture was stirred again at 90°C for 48 hours. LC-MS confirmed that 90% of 3 had been converted. Water was added to quench the reaction. Ethyl acetate was added to separate the phases. The aqueous phase was extracted three times with ethyl acetate, and the combined organic phase was washed once with water and once with saline solution, dried on magnesium sulfate, and concentrated. The residue was incorporated into MTBE and water, and the phases were separated. The organic phase was washed five times with water and once with saline solution, dried on magnesium sulfate, filtered, and concentrated to obtain an orange oily substance. The residue was purified by normal-phase flash chromatography (80 g silica column, elution: 0-30% MTBE / heptane over 10 CV). The fractions were combined and concentrated to obtain impure 4 (883 mg). The residue was then purified by reverse-phase flash chromatography (100 g C18 RediSep Rf Gold column, retention solution (DMSO), elution: 5% MeOH / 0.1% HCOOH over 4 CV, then 5%-100% MeOH / 0.1% HCOOH over 15 CV). The fractions were combined and concentrated to obtain 4 (618 mg, 41% yield) as a white solid.

[0163] LC-MS method 1: Retention time: 1.745 min, 99.9% purity at 215 nm, [M+H] + = 304.2. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.45 - 1.60 (m, 2 H), 1.76 - 1.89 (m, 2 H), 2.00 - 2.15 (m, 4 H), 2.36 - 2.47 (m, 1 H), 3.61 (s, 3 H), 4.28 - 4.40 (m, 1 H), 7.14 - 7.47 (m, 1 H), 9.05 (s, 1 H). 19 F NMR (377 MHz, DMSO-d6) δ ppm -117.40 (s, 2 F).

[0164] Step 3. Preparation of methyl 4-[4-amino-3-(difluoromethyl)pyrazole-1-yl]cyclohexanecarboxylate (T-2) Under nitrogen, a solution of 4 (625 mg, 2.06 mmol, 1.0 eq.) and ethyl acetate (20.6 mL, 0.1 M) was degassed at room temperature and sparged with nitrogen for 15 minutes. Pd / C (438 mg, 10% w / w, 0.41 mmol, 0.2 eq.) was added and sparged again for 15 minutes. The mixture was sparged with H2 for 15 minutes, and then the mixture was stirred at room temperature for 16 hours, keeping the needle just above the solvent surface. Complete conversion of 4 was observed by LC-MS (Method 1). The mixture was filtered through Celite, and the filter cake was thoroughly washed with ethyl acetate. The resulting solution was concentrated to obtain T-2 (563 mg, 99% yield) as an orange solid. LC-MS method 1: Retention time: 1.297 min, 99.9% purity at 215 nm, [M+H] + = 274.2. 1 H NMR (400 MHz, CDCl3) δ ppm 1.53 - 1.80 (m, 5 H), 2.12 - 2.42 (m, 9 H), 3.93 - 4.03 (m, 1 H), 6.54 - 6.84 (m, 1 H), 7.07 (s, 1 H). 19F NMR (377 MHz, CDCl3) δ ppm -112.22 (s, 2 F).

[0165] Example I-3. Synthesis of a typical intermediate C-1 [ka] Step 1. Preparation of tert-butyl 4-[[3-(2,6-dioxo-3-piperidyl)anilino]methyl]piperidine-1-carboxylate (2) To a solution of 3-(3-aminophenyl)piperidine-2,6-dione 1 (500 mg, 2.45 mmol) in dried CH2Cl2 (12.24 mL), tert-butyl 4-formylpiperidine-1-carboxylate 2 (548 mg, 2.57 mmol) was added at room temperature. The resulting mixture was stirred at room temperature for 20 minutes, then NaBH(OAc)3 (1.56 g, 7.34 mmol) was added, and the resulting mixture was stirred at room temperature for 30 minutes. MeOH and silica gel were added, and the mixture was concentrated to dryness. Purification by silica gel flash chromatography (gradient elution 0-8% MeOH / CH2Cl2) yielded 3 [882 mg (88%)] as a white solid.

[0166] LCMS method 1: 97.9% (UV 215 nm); Calculated exact mass = 401.23; Actual value [M-Boc+H] + = 302.2 1H NMR (400 MHz, DMSO-d6) δ ppm 0.95 - 1.14 (m, 2 H), 1.39 (s, 9 H), 1.62 - 1.80 (m, 3 H), 1.98 - 2.17 (m, 2 H), 2.39 - 2.48 (m, 1 H), 2.55 - 2.65 (m, 2 H), 2.87 (br s, 2 H), 3.32 (s, 1 H), 3.67 (dd, J = 9.8, 5.4 Hz, 1 H), 3.94 (br d, J = 11.7 Hz, 2 H), 5.65 (br s, 1 H), 6.34 (d, J = 7.3 Hz, 1 H), 6.40 (s, 1 H), 6.45 (br d, J = 8.1 Hz, 1 H), 7.00 (t, J = 7.7 Hz, 1 H), 10.78 (s, 1 H).

[0167] Step 2. Preparation of 3-[3-(4-piperidylmethylamino)phenyl]piperidine-2,6-dione 2,2,2-trifluoroacetate (C-1) To a solution of tert-butyl 4-[[3-(2,6-dioxo-3-piperidyl)anilino]methyl]piperidine-1-carboxylate 3 (882 mg, 2.20 mmol) / CH2Cl2 (10.98 mL), TFA (5.11 mL, 65.9 mmol) was added. The resulting mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated, captured with MeCN / PhMe (3x), then THF / heptane (2x), and vacuum-dried to obtain C-1 [913 mg (99%)] as an off-white semi-solid. LC-MS method 1: 99.9% (UV 215 nm); calculated accurate mass (free base) = 301.18; measured value [M-TFA+H] + = 302.4

[0168] Example I-4. Synthesis of a typical intermediate C-2 [ka]

[0169] Step 1. Preparation of tert-butyl 4-[[4-(2,6-dioxo-3-piperidyl)anilino]methyl]piperidine-1-carboxylate (3) To a solution of 3-(4-aminophenyl)piperidine-2,6-dione 1 (200.0 mg, 0.98 mmol) in dry CH2Cl2 (10 mL), tert-butyl 4-formylpiperidine-1-carboxylate 2 (219.3 mg, 1.03 mmol) was added, and the resulting mixture was stirred at room temperature for 20 minutes. Then, NaBH(OAc)3 (622.65 mg, 2.94 mmol) was added, and the resulting mixture was stirred at room temperature for 20 hours. Complete conversion was shown by LC-MS. MeOH and Silica were added, and the solvent was evaporated under reduced pressure. The crude mixture was purified by normal-phase flash chromatography using 0-10% MeOH / CH2Cl2 (2 CV at 0%, then 15 CV up to 10%). The target fraction eluted impurities (at 3% MeOH) and contained SM. A second reverse-phase flash chromatography purification (80 g C18 gold column, retention solution containing DMSO, elution with MeCN / 0.1% HCOOH, 3 CV at 5% MeCN, then 9 CV to 60% MeCN) yielded 3 (300 mg, 76% yield) as an off-white solid.

[0170] LC-MS method 1: 99.9% purity at 215 nm, ([M-Boc+H] + = 302.2;[M-tBu+H] + =346.2. 1H NMR (400 MHz, DMSO-d6) δ ppm 0.97 - 1.09 (m, 2 H), 1.40 (s, 9 H), 1.73 (br d, J = 12.2 Hz, 3 H), 1.95 - 2.14 (m, 3 H), 2.41 - 2.49 (m, 1 H), 2.56 - 2.66 (m, 2 H), 2.89 (br t, J = 5.7 Hz, 2 H), 3.63 (dd, J = 10.5, 4.9 Hz, 1 H), 3.95 (br d, J = 11.5 Hz, 2 H), 5.63 (t, J = 5.9 Hz, 1 H), 6.52 (d, J = 8.6 Hz, 2 H), 6.89 (d, J = 8.6 Hz, 2 H), 10.73 (s, 1 H).

[0171] Step 2. Preparation of 3-[4-(4-piperidylmethylamino)phenyl]piperidine-2,6-dione dihydrochloride (C-2) In a round-bottom flask, tert-butyl 4-[[4-(2,6-dioxo-3-piperidyl)anilino]methyl]piperidine-1-carboxylate 3 (300.0 mg, 0.75 mmol) was dissolved in CH2Cl2 (5 mL). 4 M HCl / 1,4-dioxane (1.87 mL, 7.47 mmol) was added, and the mixture was stirred at room temperature. After 4 hours, complete conversion was shown by HPLC. The crude product was co-evaporated three times with MeCN and toluene. The compound was purified by reverse-phase flash chromatography (eluted over 10 CV using 5% MeCN / 0.02 M HCl aqueous solution on a 50 g C18 gold column, in a holding solution containing DMSO). The fraction was concentrated to obtain C-2 (275 mg, 89%) as an orange semi-solid.

[0172] LC-MS method 1: 99.9% purity at 215 nm, [M-2HCl+H] + = 302.2. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.22 - 1.38 (m, 2 H), 1.77 - 1.85 (m, 1 H), 1.86 - 1.94 (m, 2 H), 1.95 - 2.03 (m, 1 H), 2.43 (br t, J = 4.5 Hz, 1 H), 2.47 (br t, J = 4.8 Hz, 1 H), 2.57 - 2.69 (m, 1 H), 2.85 (q, J = 11.6 Hz, 2 H), 2.94 (br d, J = 6.4 Hz, 2 H), 3.29 (br d, J = 12.2 Hz, 2 H), 3.65 (dd, J = 10.8, 4.9 Hz, 1 H), 6.57 (br d, J = 8.3 Hz, 2 H), 6.93 (d, J = 8.6 Hz, 2 H), 8.12 - 8.25 (m, 1 H), 8.48 - 8.59 (m, 1 H), 10.74 (s, 1 H).

[0173] Example I-5. Synthesis of a typical intermediate C-3 [ka]

[0174] Step 1. Preparation of tert-butyl 4-[4-(2,6-dioxo-3-piperidyl)benzoyl]piperazine-1-carboxylate (3) A solution of tert-butylpiperazine-1-carboxylate 2 (135.76 mg, 0.7300 mmol), 4-(2,6-dioxo-3-piperidyl)benzoic acid 1 (200 mg, 0.8600 mmol), HATU (489.1 mg, 1.29 mmol), and DIPEA (0.75 mL, 4.29 mmol) in DMF (2.5 mL) was stirred at room temperature for 16 hours. The product precipitated from the solution, and the mixture was filtered, washed several times with nanopure water, and dried to obtain compound 3 (203 mg, 59.0% yield) as a white solid. LCMS method 1: 99% purity at 215 nm, [M+H] += 402.2;[M+H-tBu] + =346.2

[0175] Step 2. Preparation of 3-[4-(piperazine-1-carbonyl)phenyl]piperidine-2,6-dione (C-3) A solution of tert-butyl 4-[4-(2,6-dioxo-3-piperidyl)benzoyl]piperazine-1-carboxylate 3 (203.4 mg, 0.5100 mmol) and trifluoroacetic acid (1.5 mL, 19.59 mmol) in DCM (1.5 mL) was stirred overnight at room temperature. Upon completion, the solvent was evaporated, and the crude product C-3 was used in the next step without purification.

[0176] Example I-6. Synthesis of a typical intermediate C-4 [ka]

[0177] Step 1. Preparation of tert-butyl 4-[4-(2,6-dioxo-3-piperidyl)anilino]piperidine-1-carboxylate (3) To a solution of tert-butyl 4-oxopiperidine-1-carboxylate 2 (195 mg, 0.980 mmol, 1 eq.) and 3-(4-aminophenyl)piperidine-2,6-dione 1 (250 mg, 1.22 mmol, 1.25 eq.) in DCM (4.9 mL), NaBH(OAc)3 (385 mg, 6.12 mmol, 6.2 eq.) was added, followed by AcOH (96 mg, 1.59 mmol, 1.6 eq.). The reaction mixture was stirred at room temperature. After 18 hours, complete conversion was shown by LC-MS. Saline solution was added to the reaction mixture, and the product was extracted three times with siRNA. The combined organic matter was washed with saline solution and dried over Na2SO4 to concentrate to dryness. The residue was then purified by reverse-phase chromatography (30 g C18 gold column, holding solution (DMSO), elution: 5% MeCN / 0.1% HCOOH over 3 CV, followed by 5-60% MeCN / 0.1% HCOOH over 13 CV). The fractions were combined and concentrated to obtain 3 (135 mg, 28% yield) as a white solid.

[0178] LC-MS method 1: 99.9% purity at 215 nm, [M-tBu+H] + = 332.2. 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.17 - 1.27 (m, 2 H), 1.40 (s, 9 H), 1.86 (br d, J = 11.7 Hz, 2 H), 1.95 - 2.12 (m, 3 H), 2.54 - 2.65 (m, 1 H), 2.85 - 2.99 (m, 2 H), 3.35 - 3.43 (m, 1 H), 3.63 (dd, J = 10.8, 5.1 Hz, 1 H), 3.85 (br d, J = 12.5 Hz, 2 H), 5.42 (br d, J = 8.6 Hz, 1 H), 6.54 (d, J = 8.3 Hz, 2 H), 6.89 (d, J = 8.3 Hz, 2 H), 10.72 (s, 1 H).

[0179] Step 2. Preparation of 3-[4-(4-piperidylamino)phenyl]piperidine-2,6-dione; 2,2,2-trifluoroacetic acid (C-4) A solution of tert-butyl 4-[4-(2,6-dioxo-3-piperidyl)anilino]piperidine-1-carboxylate 3 (135 mg, 0.350 mmol, 1 eq.) and trifluoroacetic acid (1.5 mL, 19.6 mmol, 56 eq.) in DCM (1.5 mL) was stirred at room temperature. After 18 hours, complete conversion was shown by LC-MS. Volatile substances were removed under reduced pressure to obtain C-4 (139 mg, quantitative yield) as a white solid.

[0180] LC-MS method 1: 99.9% purity at 215 nm, [M-CF3COOH+H] + = 288.2. 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.45 - 1.58 (m, 2 H), 1.93 - 2.13 (m, 4 H), 2.58 - 2.65 (m, 1 H), 2.94 - 3.07 (m, 2 H), 3.24 - 3.34 (m, 3 H), 3.64 (br dd, J = 11.0, 4.9 Hz, 2 H), 5.55 - 5.73 (m, 1 H), 6.57 (d, J = 8.3 Hz, 2 H), 6.92 (d, J = 8.3 Hz, 2 H), 8.21 - 8.36 (m, 1 H), 8.36 - 8.50 (m, 1 H), 10.73 (s, 1 H). 19 F NMR (377 MHz, DMSO-d6) δ ppm -73.65 (s, 3 F).

[0181] Example I-7. Synthesis of a typical intermediate C-5 [ka]

[0182] Step 1. Preparation of tert-butyl 4-(3-bromophenyl)-4-cyanobutanoate (3) 2-(3-bromophenyl)acetonitrile 1 (4.0 g, 20.4 mmol, 1.0 equiv), tert-butylpropa-2-enoate 2 (2.62 g, 20.4 mmol, 1.0 equiv), benzyltriethylammonium chloride (465 mg, 2.04 mmol, 0.1 equiv), and potassium carbonate (2.82 g, 20.4 mmol, 1.0 equiv) were combined in toluene (20 mL), and the reaction mixture was stirred at 65°C for 3 hours. After the reaction mixture was evaporated to dryness, the crude product was dry-loaded with silica and purified by reverse-phase flash chromatography (5% (3 CV) → 80% MeCN in 0.1% HCOOH(aq), 120 g RediSep Rf Gold (trademark registered) C18Aq, 20 CV, λ = 214~254 nm, 65% MeCN, yielding 3 (2.26 g, 6.97 mmol, 34% yield) as an orange oily substance.

[0183] LC-MS method 1: 99.9% purity at 215 nm, [M+H] + = 324.2. 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.37 - 1.40 (m, 9 H), 1.98 - 2.19 (m, 2 H), 2.20 - 2.37 (m, 2 H), 4.27 (t, J = 7.5 Hz, 1 H), 7.36 - 7.46 (m, 2 H), 7.57 (dt, J = 7.0, 1.9 Hz, 1 H), 7.61 (s, 1 H).

[0184] Step 2. Preparation of tert-butyl 4-[3-(4-tert-butoxy-1-cyano-4-oxo-butyl)anilino]piperidine-1-carboxylate (5) In a sealed tube, tert-butyl 4-(3-bromophenyl)-4-cyanobutanoate 3 (200.0 mg, 0.617 mmol, 1.0 equiv), tert-butyl 4-aminopiperidine-1-carboxylate 4 (154.4 mg, 0.740 mmol, 1.2 equiv), cesium carbonate (402.0 mg, 1.23 mmol, 2.0 equiv), and XPhos (44.1 mg, 0.093 mmol, 0.15 equiv.) were sparged with nitrogen, then anhydrous 1,4-dioxane (2 mL) was added, and the mixture was bubbling with nitrogen under sonication for 15 minutes. Then, Pd2(dba)3 (56.5 mg, 0.062 mmol, 0.1 equiv.) was immediately added, and nitrogen bubbling was continued for a further 5 minutes. The tube was sealed, and the reaction mixture was stirred overnight at 90°C. After the reaction mixture cooled to room temperature, water (5 mL) and DCM (5 mL) were added. The phases were separated, and the aqueous layer was extracted with DCM (3 × 5 mL). The organic layers were combined, dried over MgSO4, filtered, and evaporated under reduced pressure. The crude red oil was dry-loaded onto silica and purified by normal-phase flash chromatography (heptane / siRNA, 100:0~30:70, 24 g RediSep Rf Gold (trademark) Normal-Phase Silica, 15 CV, λ = 254~280 nm, 40~50% siRNA) to obtain 5 (226 mg, 0.456 mmol, 74% yield) as an orange viscous oil. LC-MS method 1: 89.4% purity at 215 nm, [M-2t-Bu+H] + = 332.4.

[0185] Step 3. Preparation of 3-[3-(piperidine-1-ium-4-ylamino)phenyl]piperidine-2,6-dione bisulfate (C-5) 226.0 mg, 0.456 mmol, 1.0 equiv of tert-butyl 4-[3-(4-tert-butoxy-1-cyano-4-oxo-butyl)anilino]piperidine-1-carboxylate 5 (226.0 mg, 0.456 mmol, 1.0 equiv) was dissolved in acetic acid (2.6 mL), and sulfuric acid (90 μL, 1.64 mmol, 3.6 equiv) was added dropwise. The mixture was stirred at 118°C for 4 hours. The acetic acid was co-evaporated with toluene (3 × 10 mL), and the residue was dried under high vacuum. The residue was dissolved in the minimum amount of water and purified by reverse-phase flash chromatography (MeCN was analyzed in 0.1% HCOOH(aq) from 0% (5 CV) to 20%, using a 20 g Claricep (trademark registered) Spherical AQ C18 Column, 20 CV, λ = 214~254 nm, 5~10% MeCN product, broadband), yielding C-5 (100 mg, 0.259 mmol, 57% yield) as a white solid (after lyophilization).

[0186] LC-MS method 1: 99.9% purity at 215 nm, [M+H] + = 288.4. 1 H NMR (400 MHz, DMSO-d6) δ ppm 0.89 - 1.66 (m, 3 H), 1.72 - 2.25 (m, 4 H), 2.71 - 2.97 (m, 2 H), 2.99 - 3.50 (m, 5 H), 3.52 - 4.37 (m, 3 H), 5.32 - 5.86 (m, 1 H), 5.97 - 6.74 (m, 3 H), 6.76 - 7.35 (m, 1 H), 7.62 - 9.08 (m, 2 H), 10.40 - 11.23 (m, 1 H).

[0187] Synthesis of common intermediates C-6, C-9, and C-14 Example I-8. Synthesis of intermediate C-9 [ka]

[0188] Step 1. Preparation of tert-butyl 4-(4-bromophenyl)-4-cyanobutanoate (2) To a solution of 2-(4-bromophenyl)acetonitrile 1 (27.25 g, 139 mmol) in toluene (278 mL), tert-butyl propa-2-enoate (20.36 mL, 139 mmol), N-benzyl-N,N-diethylethaneaminium chloride (3.17 g, 13.9 mmol), and potassium carbonate (19.21 g, 139 mmol) were added. The mixture was stirred under nitrogen at 65°C for 3 hours, cooled to room temperature, and filtered through a frit glass funnel. The mixture was concentrated, and the crude mixture was purified in RP-FC (415 g, C18 RediSep Rf Gold, 5% MeCN / 0.1% formic acid, 1 CV, followed by 5-100% MeCN / 0.1% formic acid, 10 CV) in 5-fold batches to obtain 2 [13.31 g (29.5%)].

[0189] LC-MS method 1: 98.1% purity at 215 nm, [M+H] + = 324.0, [M+Na] + = 346.0. 1 H NMR (400 MHz, CDCl3) δ ppm 1.46 (s, 9 H), 2.15 (q, J = 7.3 Hz, 2 H), 2.32 - 2.49 (m, 2 H), 3.91 (t, J = 7.3 Hz, 1 H), 7.24 (d, J = 8.3 Hz, 2 H), 7.53 (d, J = 8.6 Hz, 2 H).

[0190] Step 2. Preparation of tert-butyl 4-[4-[4-(tert-butoxycarbonylamino)-1-piperidyl]phenyl]-4-cyano-butanoate (3) In a vial containing Cs2CO3 (1.09 g, 3.33 mmol), tert-butyl N-(4-piperidyl)carbamate (400 mg, 2.00 mmol), tert-butyl 4-(4-bromophenyl)-4-cyanobutanoate 2 (540 mg, 1.67 mmol), XPhos (119 mg, 0.250 mmol), and Pd2(dba)3 (152.5 mg, 0.1700 mmol), anhydrous 1,4-dioxane (8.33 mL) was added, and the solution was sparged with nitrogen for 15 minutes. The reaction mixture was heated to 90°C and stirred overnight. The reaction mixture was filtered through Celite, rinsed with ELISA, and concentrated. First, the solution was purified by silica gel flash chromatography (gradient elution, 0-100% ethyl acetate / heptane), followed by C18 reverse-phase chromatography (gradient elution, 5-100% MeCN / 0.1% aq. HCO2H) to obtain 3 [466 mg (63%)] as an orange oily substance.

[0191] LCMS method 2: 99.9% (UV 215 nm); Calculated exact mass = 443.28; Actual value [M+H] + = 444.4 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.38 (s, 18 H), 1.44 (br dd, J = 11.6, 2.6 Hz, 2 H), 1.78 (br d, J = 10.5 Hz, 2 H), 1.90 - 2.09 (m, 2 H), 2.18 - 2.31 (m, 2 H), 2.73 (br t, J = 11.4 Hz, 2 H), 3.33 - 3.48 (m, 1 H), 3.65 (br d, J = 12.7 Hz, 2 H), 4.06 (t, J = 7.5 Hz, 1 H), 6.84 (br d, J = 7.6 Hz, 1 H), 6.94 (d, J = 8.8 Hz, 2 H), 7.16 (d, J = 8.6 Hz, 2 H).

[0192] Step 3. Preparation of 3-[4-(4-amino-1-piperidyl)phenyl]piperidine-2,6-dione sulfate (C-9) To a solution of tert-butyl 4-[4-[4-(tert-butoxycarbonylamino)-1-piperidyl]phenyl]-4-cyanobutanoate 3 (466 mg, 1.05 mmol) in acetic acid (5.25 mL), concentrated sulfuric acid (0.170 mL, 3.15 mmol) was added. The mixture was stirred at 118°C and monitored for completion by HPLC / LCMS (approximately 1 h). The reaction mixture was concentrated, AcOH was removed, and the solution was incorporated using PhMe (3x). It was purified by C18 reversed-phase column chromatography (gradient elution, 5-100% MeCN / 0.1% aq HCO2H), lyophilized, and C-9 [254 mg (84%)] was obtained as a pale orange solid.

[0193] LC-MS method 2: 99.9% (215 nm), calculated accurate mass (free base) = 287.16; measured value [M-H2SO4+H] + = 288.4. 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.48 - 1.63 (m, 2 H), 1.91 (br d, J = 13.0 Hz, 2 H), 1.97 - 2.06 (m, 1 H), 2.08 - 2.20 (m, 1 H), 2.40 - 2.49 (m, 1 H), 2.57 - 2.66 (m, 1 H), 2.74 (br t, J = 12.1 Hz, 2 H), 3.15 (s, 1 H), 3.72 (br dd, J = 10.9, 4.0 Hz, 3 H), 6.90 (br d, J = 8.6 Hz, 2 H), 7.05 (br d, J = 8.3 Hz, 2 H), 7.56 - 9.15 (m, 3 H), 10.77 (br s, 1 H), 1H did not appear.

[0194] Example I-9. Synthesis of intermediate C-14 [ka]

[0195] Step 1. Preparation of tert-butyl 2-[4-(4-tert-butoxy-1-cyano-4-oxo-butyl)phenyl]-2,7-diazaspiro[3.5]nonane-7-carboxylate (3) A suspension of Cs2CO3 (1.04 g, 3.180 mmol, 4 eq.), tert-butyl 2,7-diazaspiro[3.5]nonane-7-carboxylate hydrochloride 1 (251 mg, 0.950 mmol, 1.2 eq.), tert-butyl 4-(4-bromophenyl)-4-cyanobutanoate 2 (258 mg, 0.800 mmol, 1 eq.), XPhos (57 mg, 0.120 mmol, 0.15 eq.), and Pd2(dba)3 (73 mg, 0.080 mmol, 0.1 eq.) in anhydrous 1,4-dioxane (4 mL) was degassed under nitrogen for 15 minutes. The mixture was stirred at 90°C. After 18 hours, complete conversion was shown by LC-MS. The reaction mixture was filtered through a Celite pad and rinsed with toluene. The mother liquor was concentrated to dryness, and the residue was purified by normal-phase flash chromatography (80 g silica column, pre-adsorbed, elution: 0-50% toluene / heptane over 20 CV). The fractions were combined and concentrated to obtain a non-pure product, which was purified by reverse-phase flash chromatography (50 g C18 gold column, holding solution (DMSO), elution: 5% MeCN / 0.1% HCOOH over 3 CV, then 5-100% MeCN / 0.1% HCOOH over 28 CV). The fractions were combined and concentrated to obtain 3 (189 mg, 51% yield) as a colorless oil. LC-MS method 1: 99.9% purity at 215 nm, [M-Boc+H] + = 370.4. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.38 (s, 9 H), 1.40 (s, 9 H), 1.64 - 1.72 (m, 4 H), 1.90 - 2.10 (m, 2 H), 2.21 - 2.27 (m, 2 H), 3.27 - 3.33 (m, 4 H), 3.56 (s, 4 H), 4.03 (t, J = 7.5 Hz, 1 H), 6.43 (d, J = 8.6 Hz, 2 H), 7.14 (d, J = 8.6 Hz, 2 H).

[0196] Step 2. Preparation of 3-[4-(2,7-diazaspiro[3,5]nonan-2-yl)phenyl]piperidine-2,6-dione sulfate (C-14) To a solution of tert-butyl 2-[4-(4-tert-butoxy-1-cyano-4-oxo-butyl)phenyl]-2,7-diazaspiro[3.5]nonane-7-carboxylate 3 (188.9 mg, 0.400 mmol, 1 eq.) in acetic acid (2 mL), concentrated sulfuric acid was added (118 mg, 1.21 mmol, 3 eq.). The mixture was stirred at 118°C. After 80 minutes, complete conversion was shown by LC-MS. The acetic acid was co-evaporated with toluene, and the residue was purified by reverse-phase flash chromatography (20 g C18 Aq column, holding solution (H2O), elution: 0% MeCN / 0.1% HCOOH over 5 CV, then 5 to 10% MeCN / 0.1% HCOOH over 40 CV). The fractions were combined, concentrated, and freeze-dried to obtain C-14 (67 mg, 40% yield) as a white solid sulfate. LC-MS method 1: 99.9% purity at 215 nm, [M-H2SO4+H] + = 314.4. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.87 - 1.93 (m, 4 H), 1.94 - 2.09 (m, 3 H), 2.55 - 2.66 (m, 1 H), 2.99 - 3.07 (m, 4 H), 3.56 (s, 4 H), 3.67 (dd, J = 10.8, 4.9 Hz, 1 H), 6.39 (d, J = 8.3 Hz, 2 H), 6.99 (d, J = 8.6 Hz, 2 H).

[0197] The following compounds were synthesized using the same general route, with a change in amine 1 in step 1 (Table 2). [Table 10]

[0198] Example I-10. Synthesis of a common intermediate C-7 [ka]

[0199] Step 1. Preparation of 3-[4-(hydroxymethyl)phenyl]piperidine-2,6-dione (2) To a solution of 10.7 mL, 0.2 M dry THF (500.0 mg, 2.14 mmol, 1.0 eq.) of BH3DMS complex (0.81 mL, 8.58 mmol, 4.0 eq.) was added at 0°C, and the reaction mixture was stirred with rt. After 16 hours, the reaction was quenched with 1 M HCl under stirring until no more gas was formed. The mixture was concentrated to dryness. The crude product was dissolved in the minimum amount of DMSO and then loaded directly onto a 50 g C18 gold column and purified by reverse-phase flash chromatography using 5-30% MeCN / water (+0.1% formic acid). The pure fractions were combined and concentrated to obtain 2 (114 mg, 23% yield) as a white solid.

[0200] LC-MS method 1: Retention time: 1.137 min, 99.9% purity at 215 nm, [M+H] + = 220.1. 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.99 - 2.08 (m, 1 H), 2.12 - 2.24 (m, 1 H), 2.43 - 2.48 (m, 1 H), 2.66 (ddd, J = 17.1, 11.7, 5.4 Hz, 1 H), 3.83 (br dd, J = 11.4, 5.0 Hz, 1 H), 4.47 (br d, J = 5.1 Hz, 2 H), 5.14 (br t, J = 5.6 Hz, 1 H), 7.17 (d, J = 8.1 Hz, 2 H), 7.27 (br d, J = 8.1 Hz, 2 H), 10.81 (br s, 1 H).

[0201] Step 2. Preparation of tert-butyl 4-[[4-(2,6-dioxo-3-piperidyl)phenyl]methyl]piperazine-1-carboxylate (C-7) To a solution of 2 (114.5 mg, 0.52 mmol, 1.0 eq.) of dried DMSO (2.1 mL, 0.25 M), IBX (190.12 mg, 0.68 mmol, 1.3 eq.) was added. The resulting mixture was stirred overnight at room temperature. To a solution of 3 (82.31 mg, 0.44 mmol, 0.8 eq.) of dried DCE (1.8415 mL, 0.8 M), DIPEA (0.51 mL, 2.95 mmol, 8.0 eq.) was added. The resulting mixture was stirred at room temperature for 15 minutes. After adding NaBH(OAc)3 (273.19 mg, 1.29 mmol, 3.5 eq.), the DMSO solution obtained from the IBX oxidation reaction was added. The resulting mixture was stirred at room temperature. After 16 hours, DCE was removed under reduced pressure. The crude mixture was purified by reverse-phase flash chromatography (30 g C18 gold column, elution: 35-55% MeCN / H2O (0.1% formic acid) over 20 CV). The fraction of interest was concentrated to dryness. Co-evaporation of the oily substance with a heptane / MTBE mixture yielded C-7 (111 mg, 52% yield) as a white solid.

[0202] LC-MS method 1: Retention time: 1.199 min, 96.5% purity at 215 nm, [M+H] + = 388.2. 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.39 (s, 9 H), 2.00 - 2.07 (m, 1 H), 2.12 - 2.24 (m, 2 H), 2.27 - 2.35 (m, 4 H), 2.44 - 2.48 (m, 1 H), 2.60 - 2.72 (m, 2 H), 3.28 - 3.31 (m, 2 H), 3.46 (br s, 2 H), 3.84 (br dd, J = 11.5, 4.9 Hz, 1 H), 7.17 - 7.21 (m, 2 H), 7.26 (br d, J = 8.1 Hz, 2 H), 10.83 (s, 1 H).

[0203] Synthesis of common intermediates C-8, C-10, C-11, and C-13 Example I-11. Synthesis of intermediate C-10 [ka]

[0204] Step 1. Preparation of tert-butyl 4-(3-bromophenyl)-4-cyanobutanoate (3) 2-(3-bromophenyl)acetonitrile 1 (4.0 g, 20.4 mmol, 1.0 equiv), tert-butylpropa-2-enoate 2 (2.62 g, 20.4 mmol, 1.0 equiv), benzyltriethylammonium chloride (465 mg, 2.04 mmol, 0.1 equiv), and potassium carbonate (2.82 g, 20.4 mmol, 1.0 equiv) were combined in toluene (20 mL), and the reaction mixture was then stirred at 65°C for 3 hours. After the reaction mixture was evaporated to dryness, the crude product was dry-loaded with silica and purified by reverse-phase flash chromatography (5% (3 CV) → 80% MeCN in 0.1% HCOOH(aq), 120 g RediSep Rf Gold® C18Aq, 20 CV, λ = 214~254 nm, 65% MeCN). 3 (2.26 g, 6.97 mmol, 34% yield) was obtained as an orange oily substance.

[0205] LC-MS method 1: 99.9% purity at 215 nm, [M+H] + = 324.2. 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.37 - 1.40 (m, 9 H), 1.98 - 2.19 (m, 2 H), 2.20 - 2.37 (m, 2 H), 4.27 (t, J = 7.5 Hz, 1 H), 7.36 - 7.46 (m, 2 H), 7.57 (dt, J = 7.0, 1.9 Hz, 1 H), 7.61 (s, 1 H).

[0206] Step 2. Preparation of tert-butyl 4-[[3-(4-tert-butoxy-1-cyano-4-oxo-butyl)-N-methyl-anilino]methyl]piperidine-1-carboxylate (5) In a sealed tube, tert-butyl 4-(3-bromophenyl)-4-cyanobutanoate 3 (400.0 mg, 1.23 mmol, 1.0 equiv), tert-butyl 4-(methylaminomethyl)piperidine-1-carboxylate 4 (338.0 mg, 1.48 mmol, 1.2 equiv), cesium carbonate (804.0 mg, 2.46 mmol, 2.0 equiv), and XPhos (88.2 mg, 0.185 mmol, 0.15 equiv) were sparged with nitrogen, then anhydrous 1,4-dioxane (4 mL) was added, and the mixture was bubbling with nitrogen for 15 minutes under sonication. Then, Pd2(dba)3 (113.0 mg, 0.123 mmol, 0.1 equiv) was immediately added, and nitrogen bubbling was continued for a further 5 minutes. The tube was sealed, and the reaction mixture was stirred overnight at 90°C. After the reaction mixture cooled to room temperature, water (20 mL) and DCM (20 mL) were added. The phases were separated, and the aqueous layer was extracted with DCM (3 × 20 mL). The organic layers were combined, dried over MgSO4, filtered, and evaporated under reduced pressure. The crude red oily substance was dry-loaded onto silica and purified by normal-phase flash chromatography (product containing heptane / siRNA, 100:0~50:50, 80 g RediSep Rf Gold (trademark registered) Normal-Phase Silica, 15 CV, λ = 254~280 nm, 35~45% siRNA), yielding 5 (316 mg, 0.670 mmol, 54% yield) as a yellow resin.

[0207] LC-MS method 1: 99.9% purity at 215 nm, [M+Na] + = 494.4, [M-2t-Bu+H] + = 360.4, [M-Boc-t-Bu+H] + = 316.4. 1 H NMR (400 MHz, CDCl3) δ ppm 1.11 - 1.22 (m, 2 H), 1.46 (d, J = 3.7 Hz, 18 H), 1.68 (br d, J = 12.3 Hz, 2 H), 1.80 - 1.98 (m, 1 H), 2.10 - 2.25 (m, 2 H), 2.31 - 2.52 (m, 2 H), 2.56 - 2.76 (m, 2 H), 2.99 (s, 3 H), 3.21 (d, J = 7.1 Hz, 2 H), 3.89 (t, J = 7.5 Hz, 1 H), 4.14 (br s, 2 H), 6.58 (s, 1 H), 6.62 (br dd, J = 6.9, 5.1 Hz, 2 H), 7.21 (t, J = 7.9 Hz, 1 H).

[0208] Step 3. Preparation of 3-[3-[methyl(piperidine-1-ium-4-ylmethyl)amino]phenyl]piperidine-2,6-dione bisulfate (C-10)] 316.0 mg, 0.670 mmol of tert-butyl 4-[[3-(4-tert-butoxy-1-cyano-4-oxo-butyl)-N-methyl-anilino]methyl]piperidine-1-carboxylate 5 (316.0 mg, 0.670 mmol) was dissolved in acetic acid (4 mL), and then sulfuric acid (133 μL, 2.43 mmol, 3.6 equiv) was added dropwise. The mixture was stirred at 118°C for 2 hours. The acetic acid was co-evaporated with toluene (3 × 5 mL), and the residue was dried under high vacuum. The residue was dissolved in the minimum amount of water and purified by reverse-phase flash chromatography (0% (5 CV) → 40% MeCN in 0.1% HCOOH(aq), 40 g Claricep (trademark registered) Spherical AQ C18, 20 CV, λ = 214~254 nm, product containing 20~25% MeCN, multiband), to obtain C-10 (187.5 mg, 0.453 mmol, 68% yield) as a yellow solid.

[0209] LC-MS method 1: 99.9% purity at 215 nm, [M-H2SO4+H] + = 316.4. 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.33 (q, J = 11.4 Hz, 2 H), 1.74 (br d, J = 12.7 Hz, 2 H), 1.88 - 1.98 (m, 1 H), 1.99 - 2.10 (m, 1 H), 2.10 - 2.26 (m, 1 H), 2.39 - 2.48 (m, 1 H), 2.63 (ddd, J = 17.1, 11.2, 5.5 Hz, 1 H), 2.76 (br t, J = 11.9 Hz, 2 H), 2.90 (s, 3 H), 3.12 - 3.25 (m, 4 H), 3.74 (br dd, J = 10.6, 4.9 Hz, 1 H), 6.44 (br d, J = 7.5 Hz, 1 H), 6.51 (br s, 1 H), 6.58 (br d, J = 8.4 Hz, 1 H), 7.11 (t, J = 7.8 Hz, 1 H), 8.33 (s, 1 H), 10.80 (br s, 1 H).

[0210] The following compounds were synthesized using the same general route, with a change in amine 4 in step 2 (Table 3). [ka]

[0211] Example I-12. Synthesis of a common intermediate C-12 [ka]

[0212] Step 1. Preparation of tert-butyl 4-(4-bromophenyl)-4-cyanobutanoate (2) To a solution of 2-(4-bromophenyl)acetonitrile 1 (27.25 g, 139 mmol) in toluene (278 mL), tert-butyl propa-2-enoate 2 (20.36 mL, 139 mmol), N-benzyl-N,N-diethylethaneaminium chloride (3.17 g, 13.9 mmol), and potassium carbonate (19.21 g, 139 mmol) were added. The mixture was stirred under nitrogen at 65°C for 3 hours, then cooled to room temperature and filtered through a frit glass funnel. The mixture was concentrated, and the crude mixture was purified in 5 batches using RP-FC (415 g, C18 RediSep Rf Gold, 5% MeCN / 0.1% formic acid, 1 CV, followed by 5-100% MeCN / 0.1% formic acid, 10 CV) to obtain 3 [13.31 g (29.5%)]. LC-MS method 1: 98.1% purity at 215 nm, [M+H] + = 324.0, [M+Na] + = 346.0. 1 H NMR (400 MHz, CDCl3) δ ppm 1.46 (s, 9 H), 2.15 (q, J = 7.3 Hz, 2 H), 2.32 - 2.49 (m, 2 H), 3.91 (t, J = 7.3 Hz, 1 H), 7.24 (d, J = 8.3 Hz, 2 H), 7.53 (d, J = 8.6 Hz, 2 H).

[0213] Step 2. Preparation of tert-butyl 4-[4-(4-tert-butoxy-1-cyano-4-oxo-butyl)phenyl]-3,6-dihydro-2H-pyridine-1-carboxylate (5) In a 250 mL round-bottom flask, a solution of tert-butyl 4-(4-bromophenyl)-4-cyanobutanoate 3 (1.0 g, 3.08 mmol), tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydro-2H-pyridine-1-carboxylate 4 (1.15 g, 3.70 mmol), Cs2CO3 (3.02 g, 9.25 mmol), and tricyclohexylphosphine (86.5 mg, 0.31 mmol) in 1,4-dioxane (24.7 mL) and water (6.2 mL) was sparged with nitrogen for 5 minutes, and then Pd2(dba)3 (34.62 mg, 0.15 mmol) was added to the solution. The solution was sparged again for 5 minutes, and the mixture was stirred at 90°C for 4 hours. The mixture was diluted with ethyl acetate, filtered over Celite, and the filtrate was concentrated to dryness. It was then purified by SiO2 normal-phase chromatography (gradient elution, 0-20% ethyl acetate / heptane) to obtain 5 [1.49 g (quantitative)] as a light brown oily substance.

[0214] LC-MS method 1: 90.0% purity at 215 nm, [M-Boc+H] + = 327.2. 1 H NMR (400 MHz, CDCl3) δ ppm 1.25 - 1.29 (m, 2 H), 1.44 - 1.47 (m, 9 H), 1.50 (s, 9 H), 2.17 (q, J = 7.3 Hz, 2 H), 2.52 (br s, 2 H), 3.65 (t, J = 5.6 Hz, 2 H), 3.93 - 3.99 (m, 1 H), 4.09 (br d, J = 2.4 Hz, 2 H), 5.99 - 6.14 (m, 1 H), 7.29 - 7.34 (m, 2 H), 7.37 - 7.42 (m, 2 H).

[0215] Step 3. Preparation of 3-[4-(1,2,3,6-tetrahydropyridine-4-yl)phenyl]piperidine-2,6-dione sulfate (6) To a solution of tert-butyl 4-[4-(4-tert-butoxy-1-cyano-4-oxo-butyl)phenyl]-3,6-dihydro-2H-pyridine-1-carboxylate 5 (1.31 g, 3.07 mmol) in acetic acid (15.4 mL), concentrated sulfuric acid (0.49 mL, 9.21 mmol) was added. The mixture was stirred at 118 °C for 0.75 hours. The reaction mixture was concentrated, and the crude mixture was purified by C18 RediSep Rf Gold reverse-phase chromatography (gradient elution, 0% MeCN / 0.1% HCO2H aqueous solution, 10 CV, followed by 0-100% MeCN / 0.1% HCO2H aqueous solution, 10 CV) to obtain 6 [1.03 g (91% yield)] as a white solid.

[0216] LC-MS method 1: 99.9% purity at 215 nm, [M-H2SO4+H] + = 271.2. 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.99 - 2.06 (m, 1 H), 2.12 - 2.26 (m, 1 H), 2.57 - 2.73 (m, 3 H), 3.15 (s, 1 H), 3.27 (br t, J = 5.9 Hz, 2 H), 3.71 (br s, 2 H), 3.87 (dd, J = 11.5, 4.9 Hz, 1 H), 4.74 (br d, J = 15.2 Hz, 1 H), 6.18 (br s, 1 H), 7.23 (d, J = 8.1 Hz, 2 H), 7.43 (d, J = 8.3 Hz, 2 H), 8.24 (br s, 1 H), 9.84 (br s, 2 H), 10.85 (br s, 1 H).

[0217] Step 4. Preparation of 3-[4-(4-piperidyl)phenyl]piperidine-2,6-dione formate (C12) To a solution of sulfuric acid, 3-[4-(1,2,3,6-tetrahydropyridine-4-yl)phenyl]piperidine-2,6-dione 6 (220 mg, 0.60 mmol) in 1,4-dioxane (8.10 mL) and water (0.41 mL), Pd / C (189.46 mg, 0.18 mmol) was added at room temperature, and the air in the flask was replaced with hydrogen by bubbling with H2. The reaction mixture was stirred at room temperature under hydrogen until the reaction was complete (approximately overnight). The mixture was filtered through Celite, rinsed with MeCN, and concentrated under reduced pressure to obtain C-12 [135 mg (70%)] as an off-white solid.

[0218] LC-MS method 1: 80.9% purity (at 215 nm), [M-HCOOH+H] + = 273.4. 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.69 - 1.79 (m, 2 H), 1.89 - 2.07 (m, 4 H), 2.11 - 2.24 (m, 1 H), 2.64 - 2.70 (m, 1 H), 2.75 - 2.84 (m, 1 H), 2.90 - 3.00 (m, 2 H), 3.26 - 3.29 (m, 1 H), 3.33 - 3.36 (m, 1 H), 3.83 (dd, J = 11.5, 5.1 Hz, 1 H), 7.19 (s, 4 H), 10.83 (s, 1 H).

[0219] Example I-13. Synthesis of a common intermediate C-15 [ka]

[0220] Step 1. Preparation of tert-butyl 4-(5-bromo-2-pyridyl)-4-cyanobutanoate (3) Solutions of 1 (350 mg, 1.78 mmol, 1 eq.) and 2 (0.27 mL, 1.87 mmol, 1.05 eq.) in THF (8.88 mL, 0.2 M) were cooled to 0°C (ice bath). t-BuOK (19.93 mg, 0.18 mmol, 0.1 eq.) was added, and the reaction mixture was stirred overnight at 0°C (the ice bath was maintained, and the temperature was slowly raised to rt). The reaction mixture was concentrated to dryness. The crude product was dissolved in the minimum amount of DMSO and injected into a C18 RediSep Rf Gold column. Purification was performed using a 5-100% MeCN / water (0.1% formic acid) gradient-50 g gold column. The product was eluted with 62% MeCN. All fractions containing the target compound were collected, concentrated, and allowed to dry to obtain fraction 3 (128 mg, 22% yield) as a pale yellow solid.

[0221] LC-MS method 1: Retention time: 1.860 min, 99.9% purity at 215 nm, [M-tBu+H] + = 269.0. 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.39 (s, 9 H), 2.15 (q, J = 7.3 Hz, 2 H), 2.29 - 2.36 (m, 2 H), 4.43 (t, J = 7.2 Hz, 1 H), 7.46 (d, J = 7.8 Hz, 1 H), 8.14 (dd, J = 8.3, 2.4 Hz, 1 H), 8.75 (d, J = 2.4 Hz, 1 H).

[0222] Step 2. Preparation of tert-butyl 4-[5-[4-(tert-butoxycarbonylamino)-1-piperidyl]-2-pyridyl]-4-cyanobutanoate (5) In a sealed tube, solutions of 3 (128.3 mg, 0.39 mmol, 1 eq.), 4 (94.82 mg, 0.47 mmol, 1.2 eq.), and Cs2CO3 (257.09 mg, 0.79 mmol, 2 eq.) in 1,4-dioxane (1.97 mL, 0.2 M) were added. The reaction mixture was bubbling with nitrogen for 10 minutes. Then, XPhos (37.62 mg, 0.08 mmol, 0.2 eq.) and Pd2(dba)3·CHCl3 (40.83 mg, 0.04 mmol, 0.1 eq.) were added, and the mixture was bubbling with nitrogen for another 10 minutes, followed by stirring overnight at 110°C. The reaction mixture was filtered on a Celite pad (rinsed with siRNA). The filtrate was concentrated to dryness. The crude product was dissolved in the minimum amount of DMSO. Purification was performed using a 5-100% MeCN / water (+0.1% formic acid) - 50 g RediSep Rf Gold column. The pure fractions were combined, and the solvent was evaporated to dryness to obtain 5 (71 mg, 40% yield) as a brown oily substance.

[0223] LCMS method 1: Retention time: 1.868 min, [M+H] + = 445.2. 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.39 (s, 18 H), 1.42 - 1.51 (m, 2 H), 1.80 (br d, J = 10.8 Hz, 2 H), 2.09 - 2.13 (m, 2 H), 2.26 - 2.31 (m, 2 H), 2.77 - 2.86 (m, 2 H), 3.38 - 3.48 (m, 1 H), 3.69 - 3.76 (m, 2 H), 4.21 (t, J = 7.3 Hz, 1 H), 6.86 (br d, J = 7.8 Hz, 1 H), 7.23 (d, J = 8.6 Hz, 1 H), 7.35 (dd, J = 8.8, 2.9 Hz, 1 H), 8.29 (d, J = 2.9 Hz, 1 H).

[0224] Step 3. Preparation of 3-[5-(4-amino-1-piperidyl)-2-pyridyl]piperidine-2,6-dione sulfate (C-15) A solution of 5 (71.2 mg, 0.16 mmol, 1 eq.) and concentrated sulfuric acid (34.15 μL, 0.64 mmol, 4 eq.) in acetic acid (0.800 mL, 0.2 M) was stirred at 118°C for 60 minutes. The reaction mixture was concentrated to dryness to obtain C-15 (160 mg, quantitative yield) as a brown oily substance. The product was used in the next step without purification.

[0225] LC-MS method 1: Retention time: 0.207 min, 38.7% purity at 215 nm, [M-H2SO4+H] + = 289.2. 1 H NMR (400 MHz, D2O:heavy water) δ ppm 1.63 (qd, J = 12.3, 4.0 Hz, 2 H), 1.97 (s, 6 H), 2.03 - 2.10 (m, 2 H), 2.22 - 2.42 (m, 2 H), 2.74 - 2.79 (m, 1 H), 2.97 - 3.08 (m, 2 H), 3.41 (tt, J = 11.6, 4.1 Hz, 1 H), 3.80 - 3.93 (m, 2 H), 7.65 (d, J = 9.3 Hz, 1 H), 7.94 (dd, J = 9.3, 2.9 Hz, 1 H), 8.19 (d, J = 2.9 Hz, 1 H).

[0226] Example I-14. Synthesis of a common intermediate C-16 [ka]

[0227] Step 1. Preparation of tert-butyl (S)-3-(4-(2,6-bis(benzyloxy)pyridine-3-yl)phenyl)piperidine-1-carboxylate (3) In a sealed tube, NaHCO3 (0.1 mL, 0.7700 mmol), tert-butyl (S)-3-(4-bromophenyl)piperidine-1-carboxylate 1 (87.24 mg, 0.2600 mmol), 2,6-dibenzyloxy-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine 2 (128.4 mg, 0.3100 mmol), and Pd(PPh3)4 (29.63 mg, 0.0300 mmol) were added, and nitrogen was flowed through the tube for 5 minutes. A mixture of 1,4-dioxane (2.0512 mL) and water (0.5128 mL) (degassed by sparging with nitrogen for 10 minutes) was added, and the mixture was sparged with nitrogen for a further 5 minutes. The vial was sealed, and the reaction mixture was heated overnight at 90°C. Ethyl acetate and water were added to the reaction mixture to separate the phases. The aqueous phase was extracted three times with ethyl acetate, and the combined organic phase was washed once with brine, dried on magnesium sulfate, filtered, and evaporated under reduced pressure. The substance was purified by normal-phase chromatography using a gradient of heptane and ethyl acetate (0% to 30% over 20 CV). The pure fractions were combined, evaporated under reduced pressure, and dried under high vacuum to obtain 3 (180.2 mg) as a yellow oily substance. LC-MS method 2: RT = 2.506 min; Purity: 93.0% at 215 nm

[0228] Step 2. Preparation of tert-butyl (3S)-3-(4-(2,6-dioxopiperidine-3-yl)phenyl)piperidine-1-carboxylate (4) A solution of tert-butyl (S)-3-(4-(2,6-bis(benzyloxy)pyridine-3-yl)phenyl)piperidine-1-carboxylate 3 (124.7 mg, 0.2300 mmol) in THF (0.8087 mL) and ethanol (0.8087 mL) was degassed for 15 minutes, then Pd(OH)2 (25.55 mg, 0.0400 mmol) was added and the mixture was sparged for a further 5 minutes. The reactor was then mounted on an apparatus capable of generating a positive hydrogen pressure (approximately 80 psi) and heated overnight at 60°C. When complete conversion to the product was observed by LC-MS, the solvent was evaporated, and the crude product was purified by reverse-phase chromatography using 0.1% FA water and acetonitrile (5% to 100% over 20 CV). The pure fractions were combined, evaporated, and dried under high vacuum to obtain 4 (55 mg). LC-MS method 1: RT = 1.765 min; Purity: 99.9% at 215 nm; [M+Na] + :396.2;[Mt-Bu] + :317.2;[M-Boc] + :274.2.

[0229] Step 3. Preparation of 3-(4-((S)-piperidine-3-yl)phenyl)piperidine-2,6-dione (C-16) To a reaction flask, tert-butyl (3S)-3-[4-(2,6-dioxo-3-piperidyl)phenyl]piperidine-1-carboxylate 4 (84.2 mg, 0.2300 mmol) and HCl / dioxane (2.5 mL, 10 mmol) were added, and the solution was stirred at room temperature for 2 hours. Upon completion, confirmation by LC-MS was performed, and the volatile substances were evaporated under reduced pressure. This was followed by three co-evaporations using acetonitrile to obtain C-16 (80.7 mg) as a purple solid. LCMS method 1: RT = 0.961 min; Purity: 99.9% at 215 nm; [M+H] + :273.2.

[0230] Compound C-16i was obtained using the other enantiomer of tert-butyl 3-(4-bromophenyl)piperidine-1-carboxylate.

[0231] Example I-15. Synthesis of a common intermediate C-17 [ka]

[0232] Step 1. Preparation of tert-butyl N-[1-[5-(cyanomethyl)-2-pyridyl]-4-piperidyl]carbamate (3) 2-(6-chloro-3-pyridyl)acetonitrile 1 (1.5 g, 9.83 mmol, 1 eq.) and tert-butyl N-(4-piperidyl)carbamate 2 (2.95 g, 14.75 mmol, 1.5 eq.) were dissolved in a 1:8 toluene / DMSO (0.9 / 7.9 mL) solution, to which DIPEA (8.56 mL, 49.15 mmol, 5 eq.) was added at room temperature. The resulting mixture was stirred in an oil bath at 130°C. After 72 hours, LC-MS showed that all components had been converted. Toluene was removed under reduced pressure, and the residue was purified by reverse-phase flash chromatography (150 g C18 RediSep Rf Gold column, holding solution (DMSO), elution: 5% MeOH / 0.1% HCOOH over 5 CV, then 5% to 100% MeOH / 0.1% HCOOH over 20 CV, then 100% MeOH / 0.1% HCOOH over 3 CV). The fractions were combined and concentrated to obtain 3 (1.81 g, 50% yield) as an off-white solid.

[0233] LC-MS method 1: 85.8% purity at 215 nm, [M+H] + = 317.2. 1H NMR (400 MHz, CDCl3-d) δ ppm 1.46 (s, 9 H), 1.53 - 1.57 (m, 2 H), 2.04 - 2.14 (m, 2 H), 3.04 - 3.18 (m, 2 H), 3.58 - 3.66 (m, 2 H), 4.27 (d, J = 12.0 Hz, 2 H), 4.47 (br s, 1 H), 6.76 (d, J = 8.3 Hz, 1 H), 7.51 - 7.61 (m, 1 H), 8.11 (s, 1 H).

[0234] Step 2. Preparation of tert-butyl 4-[6-[4-(tert-butoxycarbonylamino)-1-piperidyl]-3-pyridyl]-4-cyanobutanoate (5) To a solution of tert-butyl N-[1-[5-(cyanomethyl)-2-pyridyl]-4-piperidyl]carbamate 3 (0.91 g, 2.86 mmol, 1 eq.) cooled to 0°C in THF (14.30 mL), NaOtBu (286 μL, 0.2900 mmol, 0.1 eq.) was added, followed by the direct addition of tert-butyl propa-2-enoate 4 (440 μL, 3.0 mmol, 1.05 eq.). The reaction mixture was then slowly warmed to room temperature. After 16 hours at room temperature, completion was indicated by LC-MS. The reaction mixture was evaporated to dryness, and the residue was purified by reverse-phase flash chromatography (100 g C18 RediSep Rf Gold column, holding solution (DMSO), elution: 5% MeCN / 0.1% HCOOH over 5 CV, then 100% MeCN / 0.1% HCOOH over 20 CV, then 100% MeCN / 0.1% HCOOH over 3 CV, with the target product eluted at approximately 70% MeCN). The fractions were combined and concentrated to obtain 5 (224 mg, 17% yield) as a yellow oily substance.

[0235] LC-MS method 1: 99.9% purity at 215 nm, [M+H] + = 445.2. 1H NMR (400 MHz, CDCl3-d) δ ppm 1.33 - 1.55 (m, 18 H), 1.58 - 1.63 (m, 1 H), 1.98 - 2.20 (m, 4 H), 2.32 - 2.49 (m, 2 H), 2.94 - 3.06 (m, 2 H), 3.63 - 3.78 (m, 1 H), 3.81 - 3.88 (m, 1 H), 4.13 - 4.31 (m, 2 H), 4.40 - 4.53 (m, 1 H), 6.68 (d, J = 8.8 Hz, 1 H), 7.45 (dd, J = 8.8, 2.7 Hz, 1 H), 8.09 (d, J = 2.7 Hz, 1 H)

[0236] Step 3. Preparation of 3-[6-(4-amino-1-piperidyl)-3-pyridyl]piperidine-2,6-dione bisulfate (C-17) A solution of tert-butyl 4-[6-[4-(tert-butoxycarbonylamino)-1-piperidyl]-3-pyridyl]-4-cyanobutanoate 5 (256.3 mg, 0.58 mmol, 1 eq.) and concentrated sulfuric acid (92 μL, 1.73 mmol, 3 eq.) in glacial acetic acid (99 μL, 1.73 mmol, 3 eq.) was stirred at 120°C (pre-heated in an oil bath). After 1 hour at this temperature, completion was indicated by LC-MS. The reaction mixture was then evaporated to dryness and co-evaporated three times using toluene. The residue was purified by reverse-phase flash chromatography (100 g C18 RediSep Rf Gold column, holding solution (water), elution: 0% MeCN / 0.1% HCOOH over 15 CV, then 0 to 100% MeCN / 0.1% HCOOH over 5 CV, then 100% MeCN / 0.1% HCOOH over 3 CV, the target product eluted at approximately 0% MeCN). The fractions were combined and concentrated to obtain C-17 (90 mg, 40% yield) as a white solid.

[0237] LC-MS method 1: 99.9% purity at 254 nm, [M-H2SO4+H] += 289.2. 1 H NMR (400 MHz, DMSO-d6 ) δ ppm 1.36 - 1.47 (m, 2 H), 1.84 - 2.00 (m, 3 H), 2.10 - 2.23 (m, 1 H), 2.61 - 2.74 (m, 1 H), 2.80 - 2.94 (m, 2 H), 3.17 - 3.24 (m, 2 H), 3.73 (dd, J = 12.3, 4.8 Hz, 1 H), 4.29 (d, J = 12.7 Hz, 2 H), 6.85 (d, J = 8.8 Hz, 1 H), 7.40 (dd, J = 8.7, 2.3 Hz, 1 H), 7.96 (d, J = 2.2 Hz, 1 H), 8.26 (s, 2 H), 10.82 (br s, 1 H).

[0238] Synthesis of common intermediates C-18, C-19, C-20, and C-21 Example I-16. Synthesis of common intermediates C-19 and C-20 [ka]

[0239] Step 1. Preparation of tert-butyl N-[4-[5-(cyanomethyl)-2-pyridyl]cyclohexa-3-en-1-yl]carbamate (3) In a sealed tube, 2-(6-chloro-3-pyridyl)acetonitrile 1 (375.0 mg, 2.46 mmol, 1.0 equiv), tert-butyl N-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)cyclohexa-3-en-1-yl]carbamate 2 (1.00 g, 2.95 mmol, 1.2 equiv), and sodium bicarbonate (619.4 mg, 7.37 mmol, 3.0 equiv) were mixed with 1,4-dioxane (4 mL) and water (1 mL). The mixture was then bubbling with nitrogen for 30 minutes under sonication. Subsequently, Pd(PPh3)4 (284.0 mg, 0.246 mmol, 0.1 equiv) was immediately added, and nitrogen bubbling was continued for a further 10 minutes. The tube was sealed, and the reaction mixture was stirred overnight at 90°C. After the reaction mixture cooled to room temperature, water (10 mL) and DCM (10 mL) were added. The phases were separated, and the aqueous layer was extracted with DCM (3 × 10 mL). The organic layers were combined, dried over MgSO4, filtered, and evaporated under reduced pressure. The crude product was dry-loaded onto silica and purified by normal-phase flash chromatography (heptane / siRNA, 100:0~40:60, 80 g RediSep Rf Gold® Normal-Phase Silica, 20 CV, λ = 254~280 nm, 48~56% siRNA) to obtain 3 (513 mg, 1.63 mmol, 67% yield) as a white solid.

[0240] LC-MS method 1: 99.9% purity at 215 nm, [M+H] + = 314.2, [Mt-Bu+H] + = 258.2. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.39 (s, 9 H), 1.46 - 1.62 (m, 1 H), 1.85 - 1.99 (m, 1 H), 2.11 (ddd, J = 12.6, 9.2, 3.2 Hz, 1 H), 2.36 - 2.48 (m, 2 H), 2.66 (br d, J = 17.6 Hz, 1 H), 3.53 (br s, 1 H), 4.06 (s, 2 H), 6.63 (br s, 1 H), 6.85 (br d, J = 7.3 Hz, 1 H), 7.55 (d, J = 8.2 Hz, 1 H), 7.72 (dd, J = 8.3, 2.4 Hz, 1 H), 8.47 (d, J = 1.8 Hz, 1 H).

[0241] Step 2. Preparation of trans-tert-butyl N-[4-[5-(cyanomethyl)-2-pyridyl]cyclohexyl]carbamate (trans-4 / cis-4) tert-butyl N-[4-[5-(cyanomethyl)-2-pyridyl]cyclohexa-3-en-1-yl]carbamate 3 (510.0 mg, 1.63 mmol, 1.0 equiv) was dissolved in ethyl acetate (32 mL), and the solution was degassed by bubbling with nitrogen under sonication for 20 minutes. Pd / C 5% w / w (346 mg, 0.163 mmol, 0.1 equiv) was added, and the mixture was further degassed by bubbling with nitrogen under sonication for 20 minutes. The nitrogen balloon was replaced with a hydrogen-filled balloon, and the reaction mixture was bubbling with this for 10 minutes. The reaction mixture was then stirred overnight under a static hydrogen atmosphere. The reaction mixture was filtered through Celite to remove the catalyst, and the Celite was thoroughly washed with ELISA. The solvent was evaporated under reduced pressure to obtain a cis / trans mixture of the product as a gray crystalline solid (500 mg, 1(55:45 cis / trans ratio by 1H NMR). The cis / trans mixture was subjected to SFC chiral separation to obtain trans-4 (213 mg, 0.663 mmol, 41% yield) and cis-4 (250 mg, 0.771 mmol, 47% yield) as white solids (88% total yield). trans-4. LC-MS method 3: 98.2% purity at 215 nm, [Mt-Bu+H] + = 260.2, [M+H] + = 316.2, [M+Na] + = 339.2. HPLC (chiral) method 1: 100% purity at 215 nm 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.21 - 1.35 (m, 2 H), 1.38 (s, 9 H), 1.48 - 1.63 (m, 2 H), 1.79 - 1.96 (m, 4 H), 2.53 - 2.65 (m, 1 H), 3.19 - 3.31 (m, 1 H), 4.03 (s, 2 H), 6.76 (br d, J = 7.7 Hz, 1 H), 7.31 (d, J = 8.1 Hz, 1 H), 7.68 (dd, J = 8.1, 2.1 Hz, 1 H), 8.44 (d, J = 1.6 Hz, 1 H). cis-4. LC-MS method 3: 97.3% purity at 215 nm, [M+H] + = 316.2, [M+Na] + = 339.1, [Mt-Bu+H] + = 260.2. HPLC (chiral) method 1: 100% purity at 215 nm. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.39 (s, 9 H), 1.50 - 1.63 (m, 4 H), 1.63 - 1.75 (m, 2 H), 1.82 - 2.00 (m, 2 H), 2.70 (br t, J = 10.5 Hz, 1 H), 3.63 (br s, 1 H), 4.04 (s, 2 H), 6.85 (br d, J = 4.8 Hz, 1 H), 7.38 (d, J = 8.1 Hz, 1 H), 7.71 (dd, J = 8.0, 1.9 Hz, 1 H), 8.44 (s, 1 H).

[0242] Step 3. Preparation of trans-tert-butyl 4-[6-[4-(tert-butoxycarbonylamino)cyclohexyl]-3-pyridyl]-4-cyanobutanoate (trans-6) trans-tert-butyl N-[4-[5-(cyanomethyl)-2-pyridyl]cyclohexyl]carbamate trans-4 (181.0 mg, 0.563 mmol, 1.0 equiv) and tert-butyl propa-2-enoate 5 (93 μL, 0.620 mmol, 1.1 equiv) were dissolved in ethanol (5.0 mL) and 1,4-dioxane (0.5 mL). Then, a solution of benzyltrimethylammonium hydroxide 40% w / w / MeOH (51 μL, 0.113 mmol, 0.2 equiv) was added, and the reaction mixture was stirred at 80°C for 4 hours. After evaporating the reaction mixture to dryness, the crude product was dry-loaded with silica and purified by normal-phase flash chromatography (heptane / siRNA, 100:0~40:60, 24 g RediSep Rf Gold® Normal-Phase Silica, 25 CV, λ = 254~280 nm, 40~48% siRNA for product, 52~60% siRNA for starting material) to obtain trans-6 (64 mg, 0.116 mmol, 21% yield) as a white solid, and trans-4 (85 mg, 0.269 mmol, 48% yield) was recovered as a white solid. LC-MS method 1: 80.4% purity at 215 nm, [M+H] + = 444.2.

[0243] Step 4. Preparation of trans-[4-[5-(2,6-dioxo-3-piperidyl)-2-pyridyl]cyclohexyl]ammonium bisulfate (C-20) trans-tert-butyl 4-[6-[4-(tert-butoxycarbonylamino)cyclohexyl]-3-pyridyl]-4-cyanobutanoate trans-6 (150.0 mg, 0.288 mmol, 1.0 equiv) was dissolved in acetic acid (5.7 mL), and sulfuric acid (63.0 μL, 1.15 mmol, 4.0 equiv) was added dropwise. The mixture was stirred at 118°C for 4 hours. The acetic acid was co-evaporated with toluene (3 × 10 mL), and the residue was dried under high vacuum. The residue was dissolved in the minimum amount of water and purified by reverse-phase flash chromatography (MeCN in 0.1% HCOOH(aq) from 0% (5 CV) to 20%, 40 g Claricep (trademark registered) Spherical AQ C18 Column, 20 CV, λ = 214~254 nm, 0% MeCN, broadband), and trans-7 (109 mg, 0.227 mmol, 79% yield) was obtained as a white solid sulfate after lyophilization. LC-MS method 4: 80.4% purity at 215 nm, [M+H] + = 288.1.

[0244] The following compounds were synthesized using the same general route and post-separation (Cis / Trans) ring geometry, with a modification to starting material 1 (SM-1) in step 1 (Table 4). [Table 11]

[0245] Synthesis of the final compound General method for final product 1 Example S1. Synthesis of P-1 [ka]

[0246] Step 1. Preparation of ethyl 5-[(3R,5R)-3-(tert-butoxycarbonylamino)-5-fluoro-1-piperidyl]pyrazolo[1,5-a]pyrimidine-3-carboxylate (3) To a solution of ethyl 5-chloropyrazolo[1,5-a]pyrimidine-3-carboxylate 1 (800 mg, 3.55 mmol, 1.0 eq.) in MeCN (17.7 mL), DIPEA (1.54 mL, 8.86 mmol, 2.5 eq.) and tert-butyl N-[(3R,5R)-5-fluoro-3-piperidyl]carbamate 2 (1.01 g, 4.61 mmol, 1.3 eq.) were added. After stirring at 60°C over the weekend, complete conversion to 3 was shown by LC-MS. The solvent was removed under reduced pressure, and the residue was vacuum-dried to obtain 3 (1.44 g, quantitative yield) as a white solid. The crude product was used in the next step without further purification.

[0247] LC-MS method 1: Retention time: 1.656 min, 99.9% purity at 215 nm, [M+H] + = 408.2. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.37 - 1.45 (m, 9 H), 1.56 - 1.92 (m, 1 H), 2.09 - 2.20 (m, 1 H), 2.56 - 2.71 (m, 1 H), 2.76 - 3.03 (m, 1 H), 3.08 - 3.19 (m, 1 H), 3.29 (s, 1 H), 3.37 (br s, 1 H), 3.56 - 3.73 (m, 2 H), 4.11 - 4.25 (m, 2 H), 4.41 - 4.73 (m, 1 H), 4.99 (br s, 1 H), 5.11 (br s, 1 H), 6.85 (br d, J = 8.1 Hz, 1 H), 7.10 (br d, J = 7.8 Hz, 1 H), 8.22 (s, 1 H), 8.73 (d, J = 7.8 Hz, 1 H). 19 F NMR (377 MHz, DMSO-d6) δ ppm -184.32 (s, 1 F).

[0248] Step 2. Preparation of 5-[(3R,5R)-3-(tert-butoxycarbonylamino)-5-fluoro-1-piperidyl]pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (4) To a solution of ethyl 5-[(3R,5R)-3-(tert-butoxycarbonylamino)-5-fluoro-1-piperidyl]pyrazolo[1,5-a]pyrimidine-3-carboxylate 3 (1.44 mg, 3.53 mmol, 1.0 eq) / THF (5.89 mL) and methanol (5.89 mL), a solution of LiOH·H2O (1.48 g, 35.34 mmol, 10.0 eq.) / water (5.89 mL) was added. After stirring at 60°C for 18 hours, complete conversion to the target product 4 was shown by LC-MS. The reaction mixture was concentrated under reduced pressure to remove THF / MeOH, and the crude mixture was diluted with water. Under vigorous stirring, the mixture was acidified with 6 N HCl aqueous solution to pH=3 (precipitate formation). The suspension was filtered through a Buchner funnel, and the solid was rinsed with water. The solid was dried overnight in a stove under vacuum to obtain 4 (1.40 g, quantitative yield) as a white solid. The crude product was used in the next step without further purification.

[0249] LC-MS method 1: Retention time: 1.487 min, 99.9% purity at 215 nm, [M+H] + = 380.1. 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.41 (s, 9 H), 1.66 - 1.95 (m, 1 H), 2.05 - 2.26 (m, 1 H), 2.92 - 3.11 (m, 1 H), 3.28 - 3.30 (m, 1 H), 3.34 - 3.49 (m, 1 H), 3.59 - 3.74 (m, 1 H), 4.52 - 4.75 (m, 1 H), 4.99 (br s, 1 H), 5.11 (br s, 1 H), 6.82 (br d, J = 7.6 Hz, 1 H), 7.12 (br d, J = 7.8Hz, 1H), 8.19(s, 1H), 8.73 (d, J = 7.8 Hz, 1 H). 19 F NMR (377 MHz, DMSO-d6) δ ppm-184.32 (s, 1 F).

[0250] Step 3. Preparation of methyl 4-[4-[[5-[(3R,5R)-3-(tert-butoxycarbonylamino)-5-fluoro-1-piperidyl]pyrazolo[1,5-a]pyrimidine-3-carbonyl]amino]-3-(trifluoromethyl)pyrazole-1-yl]cyclohexanecarboxylate (5) To a solution of 5-[(3R,5R)-3-(tert-butoxycarbonylamino)-5-fluoro-1-piperidyl]pyrazolo[1,5-a]pyrimidine-3-carboxylic acid 4 (330 mg, 0.87 mmol, 1.0 eq.) and methyl 4-[4-amino-3-(trifluoromethyl)pyrazole-1-yl]cyclohexanecarboxylate T-1 (255 mg, 0.87 mmol, 1.0 eq.) in MeCN (4.37 mL, 0.2 M), NMI (208 μL, 2.62 mmol, 3.0 eq.) was added, followed by TCFH (368 mg, 1.31 mmol, 1.5 eq.). The resulting mixture was stirred at room temperature. After stirring for 1 hour, complete conversion to 7 was shown by LC-MS. Nanopure water was added to the reaction mixture. The suspension was sonicated and filtered through a Buchner funnel. The solid was rinsed with nanopure water and dried under high vacuum for 2 hours to obtain 5 (279 mg, 46% yield) as a white solid. The crude product was used without further purification.

[0251] LC-MS method 1: Retention time: 1.812 min, 99.9% purity at 215 nm, [M+H] + = 653.2. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.30 (br s, 9 H), 1.49 - 1.62 (m, 3 H), 1.75 - 1.92 (m, 4 H), 2.01 - 2.11 (m, 5 H), 3.40 - 3.57 (m, 1 H), 3.63 (s, 3 H), 4.32 (br t, J = 10.9 Hz, 1 H), 4.50 - 4.64 (m, 1 H), 4.96 - 5.18 (m, 1 H), 6.92 (br d, J = 5.5 Hz, 1 H), 7.09 (br s, 1 H), 8.30 (s, 1 H), 8.41 (br s, 1 H), 8.82 (br d, J = 7.8 Hz, 1 H), 9.22 (br s, 1 H). 2H was not observed. 19 F NMR (377 MHz, DMSO-d6) δ ppm -185.73 - -184.64 (m, 1 F), -59.01 (br s, 3 F).

[0252] Step 4. Preparation of 4-[4-[[5-[(3R,5R)-3-(tert-butoxycarbonylamino)-5-fluoro-1-piperidyl]pyrazolo[1,5-a]pyrimidine-3-carbonyl]amino]-3-(trifluoromethyl)pyrazole-1-yl]cyclohexanecarboxylic acid (6) To a solution of methyl 4-[4-[[5-[(3R,5R)-3-(tert-butoxycarbonylamino)-5-fluoro-1-piperidyl]pyrazolo[1,5-a]pyrimidine-3-carbonyl]amino]-3-(trifluoromethyl)pyrazole-1-yl]cyclohexanecarboxylate 5 (279 mg, 0.43 mmol, 1.0 eq.) in THF (2.14 mL, 0.1 M), a solution of LiOH·H2O (180 mg, 4.27 mmol, 10.0 eq.) in water (2.14 mL, 0.1 M) was added, and the resulting mixture was stirred at room temperature. After stirring overnight, complete conversion to 6 was shown by LC-MS. The reaction mixture was concentrated under reduced pressure, and the residue was suspended in nanopure water. Under vigorous stirring at 0°C, aqueous 6N HCl was added until the pH became 3. The solid was filtered through a Buchner funnel, rinsed with nanopure water, and dried overnight under high vacuum to obtain 6 (232 mg, 83% yield) as a white solid. The product was used in the next step without further purification.

[0253] LC-MS method 1: Retention time: 1.667 min, 99.9% purity at 215 nm, [M+H] + = 639.2. 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.29 (br s, 9 H), 1.43 - 1.61 (m, 3 H), 1.68 - 1.93 (m, 4 H), 1.97 - 2.14 (m, 5 H), 2.25 - 2.39 (m, 1 H), 3.39 - 3.75 (m, 2 H), 4.30 (br t, J = 11.7 Hz, 1 H), 4.50 - 4.68 (m, 1 H), 4.89 - 5.19 (m, 1 H), 6.91 (br s, 1 H), 7.10 (br s, 1 H), 8.29 (br s, 1 H), 8.41 (br s, 1 H), 8.81 (br d, J = 6.1 Hz, 1 H), 9.21 (br s, 1 H), 12.15 (br s, 1 H). 19F NMR (377 MHz, DMSO-d6) δ ppm -186.50 - -181.97 (m, 1 F), -59.01 (br s, 3 F).

[0254] Step 5. Preparation of tert-butyl N-[(3R,5R)-1-[3-[[1-[4-[4-[[3-(2,6-dioxo-3-piperidyl)anilino]methyl]piperidine-1-carbonyl]cyclohexyl]-3-(trifluoromethyl)pyrazole-4-yl]carbamoyl]pyrazolo[1,5-a]pyrimidine-5-yl]-5-fluoro-3-piperidyl]carbamate (7) To a solution of 3-[3-(4-piperidylmethylamino)phenyl]piperidine-2,6-dione 2,2,2-trifluoroacetate C-1 (44.8 mg, 0.090 mmol) and 4-[4-[[5-[(3R,5R)-3-(tert-butoxycarbonylamino)-5-fluoro-1-piperidyl]pyrazolo[1,5-a]pyrimidine-3-carbonyl]amino]-3-(trifluoromethyl)pyrazole-1-yl]cyclohexanecarboxylic acid 6 (38 mg, 0.060 mmol) in DMF (0.500 mL), DIPEA (103.6 μL, 0.600 mmol) was added. The reaction mixture was stirred at room temperature for 5 minutes, and then HATU (27.15 mg, 0.070 mmol) was added. The resulting mixture was stirred overnight at room temperature. The crude reaction mixture was loaded onto a C18 reverse-phase column for purification (30 g C18 gold column, gradient elution, 50-65% MeCN / 0.1% HCO2H aqueous solution), yielding 7 [40 mg (73%)] as a pale yellow solid.

[0255] LCMS method 1: 99.9% (UV 215 nm); Calculated exact mass = 921.43; Actual value [M+H] + = 922.2. 1H NMR (400 MHz, DMSO-d6) δ ppm 0.93 - 1.05 (m, 1 H), 1.08 - 1.16 (m, 1 H), 1.29 (br s, 9 H), 1.50 - 1.67 (m, 2 H), 1.78 (br s, 5 H), 1.82 - 1.97 (m, 4 H), 2.02 - 2.15 (m, 6 H), 2.40 - 2.46 (m, 1 H), 2.57 - 2.68 (m, 1 H), 2.76 (br t, J = 11.2 Hz, 1 H), 2.90 (br s, 2 H), 2.93 - 3.09 (m, 2 H), 3.37 - 3.57 (m, 1 H), 3.68 (br dd, J = 9.7, 5.3 Hz, 2 H), 4.04 (br d, J = 13.7 Hz, 1 H), 4.24 - 4.37 (m, 1 H), 4.37 - 4.45 (m, 1 H), 4.47 - 4.68 (m, 1 H), 4.97 - 5.17 (m, 1 H), 5.68 (br s, 1 H), 6.35 (br d, J = 7.3 Hz, 1 H), 6.41 (s, 1 H), 6.46 (br d, J = 8.3 Hz, 1 H), 6.91 (br d, J = 6.8 Hz, 1 H), 7.01 (t, J = 7.7 Hz, 1 H), 7.09 (br d, J = 3.7 Hz, 1 H), 8.30 (s, 1 H), 8.40 (br s, 1 H), 8.82 (br d, J = 7.8 Hz, 1 H), 9.22 (s, 1 H), 10.78 (s, 1 H). 19 F NMR (377 MHz, DMSO-d6) δ ppm-59.00 (s, 1 F).

[0256] Step 6. Preparation of N-[1-[4-[4-[[3-(2,6-dioxo-3-piperidyl)anilino]methyl]piperidine-1-carbonyl]cyclohexyl]-3-(trifluoromethyl)pyrazole-4-yl]-5-[(3R,5R)-3-amino-5-fluoro-1-piperidyl]pyrazolo[1,5-a]pyrimidine-3-carboxamide hydrochloride (P-1) A solution of tert-butyl N-[(3R,5R)-1-[3-[[1-[4-[4-[[3-(2,6-dioxo-3-piperidyl)anilino]methyl]piperidine-1-carbonyl]cyclohexyl]-3-(trifluoromethyl)pyrazole-4-yl]carbamoyl]pyrazolo[1,5-a]pyrimidine-5-yl]-5-fluoro-3-piperidyl]carbamate 7 (40 mg, 0.040 mmol) in 4.0 M HCl / 1,4-dioxane (1.63 mL, 6.51 mmol) was stirred at room temperature (approximately 1 hour) until the reaction was complete, as determined by HPLC analysis. The solution was concentrated and purified by C18 reverse-phase flash chromatography (gradient elution, 5-40% MeCN / 0.02 M HCl aqueous solution). After lyophilization, P-1 [26.41 mg (73%)] was obtained as a white solid in the form of the HCl salt. LCMS method 3: 98.8% (UV 215 nm); Calculated exact mass (free base) = 821.37; Observed value [M-HCl+H] + = 822.3. 1H NMR (400 MHz, DMSO-d6) δ ppm 0.94 - 1.22 (m, 2 H), 1.48 - 1.67 (m, 2 H), 1.72 - 2.23 (m, 13 H), 2.34 - 2.47 (m, 1 H), 2.56 - 2.68 (m, 2 H), 2.71 - 2.83 (m, 1 H), 2.89 - 3.07 (m, 3 H), 3.23 - 3.57 (m, 4 H), 3.99 - 4.10 (m, 1 H), 4.29 - 4.47 (m, 2 H), 4.54 - 4.84 (m, 2 H), 5.12 (d, J = 46.7 Hz, 1 H), 6.34 - 6.79 (m, 3 H), 6.91 (d, J = 8.1 Hz, 1 H), 7.03 - 7.15 (m, 1 H), 8.07 - 8.38 (m, 4 H), 8.45 (s, 1 H), 8.93 (d, J = 7.8 Hz, 1 H), 9.24 (s, 1 H), 10.81 (s, 1 H), 1H was not clearly defined. 19 F NMR (377 MHz, DMSO d6) δ ppm -184.88 (s, 1 F), -58.78 (s, 3 F).

[0257] Example S2. Synthesis of P-2 [ka]

[0258] Step 5. Preparation of tert-butyl N-[(3R,5R)-1-[3-[[1-[4-[4-[[4-(2,6-dioxo-3 piperidyl)anilino]methyl]piperidine-1-carbonyl]cyclohexyl]-3-(trifluoromethyl)pyrazole-4-yl]carbamoyl]pyrazolo[1,5-a]pyrimidine-5-yl]-5-fluoro-3-piperidyl]carbamate (2) DIPEA (0.11 mL, 0.63 mmol) was added to a solution of 3-[4-(4-piperidylmethylamino)phenyl]piperidine-2,6-dione dihydrochloride C-2 (47.13 mg, 0.09 mmol) and 4-[4-[[5-[(3R,5R)-3-(tert-butoxycarbonylamino)-5-fluoro-1piperidyl]pyrazolo[1,5-a]pyrimidine-3-carbonyl]amino]-3-(trifluoromethyl)pyrazole-1-yl]cyclohexanecarboxylic acid 1 (intermediate 6 obtained from the synthesis of P-1) (40.0 mg, 0.06 mmol) in DMF (1 mL). The mixture was stirred at room temperature for 5 minutes, and then HATU (28.58 mg, 0.08 mmol) was added. The mixture was stirred at room temperature for 16 hours, and complete conversion to the target product was shown by LC-MS. The mixture was directly injected into a 50 g C18 gold column and purified by reverse-phase flash chromatography (retention solution containing DMSO, elution with MeCN / 0.1% HCOOH, 4 CV at 5% MeCN; 1 CV up to 55% MeCN; 15 CV up to 70% MeCN). The product eluted at 61% MeCN. The fraction was concentrated to obtain 2 (49.3 mg, 85% yield) as an orange solid.

[0259] LC-MS method 1: 99.9% purity at 215 nm, [M+H] + = 922.2, [M+2H] 2+ = 461.8. 1H NMR (400 MHz, DMSO-d6) δ ppm 0.95 - 1.04 (m, 1 H), 1.07 - 1.17 (m, 1 H), 1.28 (br s, 9 H), 1.48 - 1.65 (m, 3 H), 1.72 - 1.84 (m, 5 H), 1.85 - 1.97 (m, 4 H), 1.98 - 2.07 (m, 4 H), 2.09 - 2.18 (m, 2 H), 2.40 - 2.47 (m, 2 H), 2.56 - 2.66 (m, 1 H), 2.71 - 2.81 (m, 1 H), 2.91 (br d, J = 5.6 Hz, 2 H), 2.97 - 3.06 (m, 1 H), 3.60 - 3.71 (m, 2 H), 4.01 - 4.09 (m, 1 H), 4.28 - 4.36 (m, 1 H), 4.38 - 4.46 (m, 1 H), 4.99 - 5.15 (m, 1 H), 5.62 - 5.68 (m, 1 H), 6.53 (d, J = 8.3 Hz, 2 H), 6.87 - 6.95 (m, 3 H), 7.11 (br s, 1 H), 8.30 (br s, 1 H), 8.42 (br s, 1 H), 8.80 (br d, J = 7.8 Hz, 1 H), 9.22 (br s, 1 H), 10.73 (br s, 1 H).

[0260] Step 6. Preparation of N-[1-[4-[4-[[4-(2,6-dioxo-3-piperidyl)anilino]methyl]piperidine-1-carbonyl]cyclohexyl]-3-(trifluoromethyl)pyrazole-4-yl]-5-[(3R,5R)-3-amino-5-fluoro-1-piperidyl]pyrazolo[1,5-a]pyrimidine-3-carboxamide hydrochloride (P-2) tert-butyl N-[(3R,5R)-1-[3-[[1-[4-[4-[[4-(2,6-dioxo-3-piperidyl)anilino]methyl]piperidine-1-carbonyl]cyclohexyl]-3-(trifluoromethyl)pyrazole-4-yl]carbamoyl]pyrazolo[1,5-a]pyrimidine-5-yl]-5-fluoro-3-piperidyl]carbamate 2 (49.3 mg, 0.05 mmol) was dissolved in 4 M HCl / 1,4-dioxane (2.01 mL, 8.02 mmol). The solution was stirred at rt for 30 minutes. Complete conversion was shown by LC-MS. The solvent was evaporated under reduced pressure, and the crude product was purified by reverse-phase flash chromatography (50 g C18 gold column, holding solution containing DMSO, eluted with MeCN / 0.02 M HCl, 3.5 CV with 5% MeCN; 10 CV to 35% MeCN). The fractions were combined, concentrated, and lyophilized to obtain P-2 (36.4 mg, 82% yield) as a white solid, yielding the hydrochloride salt.

[0261] LC-MS method 3: 98.7% purity at 215 nm, [M-HCl+H] + = 822.4;[M-HCl+2H] 2+ = 411.7. 1H NMR (400 MHz, DMSO-d6) δ ppm 0.96 - 1.22 (m, 2 H), 1.48 - 1.68 (m, 2 H), 1.74 - 2.20 (m, 12 H), 2.35 - 2.47 (m, 1 H), 2.55 - 2.68 (m, 1 H), 2.71 - 2.81 (m, 1 H), 2.96 - 3.08 (m, 3 H), 3.24 - 3.61 (m, 3 H), 3.69 - 3.78 (m, 2 H), 4.00 - 4.09 (m, 2 H), 4.28 - 4.48 (m, 2 H), 4.54 - 4.84 (m, 2 H), 5.06 (d, J = 46.5 Hz, 1 H), 6.76 - 7.16 (m, 5 H), 8.21 - 8.38 (m, 4 H), 8.45 (s, 1 H), 8.93 (d, J = 7.8 Hz, 1 H), 9.24 (s, 1 H), 10.78 (s, 1 H). 19 F NMR (377 MHz, DMSO-d6) δ ppm -185.00 (s, 1 F), -58.75 (s, 3 F).

[0262] Example S3. Synthesis of P-15 [ka]

[0263] Step 3. Preparation of 5-methyl 4-[4-[[5-[(3R,5R)-3-(tert-butoxycarbonylamino)-5-fluoro-1-piperidyl]pyrazolo[1,5-a]pyrimidine-3-carbonyl]amino]-3-(difluoromethyl)pyrazole-1-yl]cyclohexanecarboxylate (2) Methyl 4-[4-amino-3-(difluoromethyl)pyrazole-1-yl]cyclohexanecarboxylate T-2 (239 mg, 0.870 mmol, 1.0 equivalent) and 5-[(3R,5R)-3-(tert-butoxycarbonylamino)-5-fluoro-1-piperidyl]pyrazolo[1,5-a]pyrimidine-3-carboxylic acid 1 (intermediate 4 obtained from the synthesis of P-1) (332 mg, 0.870 mmol, 1.0 equivalent) were dissolved in MeCN (4.37 mL, 0.2 M), to which NMI (208 μL, 2.62 mmol, 3.0 equivalent), followed by TCFH (368 mg, 1.31 mmol, 1.5 equivalent). The resulting mixture was stirred at room temperature for 1 hour. Nanopure water was added to the reaction mixture. The suspension was sonicated and then filtered through a Buchner funnel. The solid was washed with nanopure water and dried under high vacuum to obtain pure 2 (466 mg, 84% yield) without further purification.

[0264] LC-MS method 1: 99.9% purity at 215 nm, [M+H] + = 635.2 m / z. 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.34 (s, 9 H), 1.40 - 1.44 (m, 1 H), 1.48 - 1.59 (m, 2 H), 1.76 - 1.94 (m, 3 H), 1.98 - 2.17 (m, 5 H), 2.36 - 2.45 (m, 1 H), 2.98 - 3.12 (m, 1 H), 3.37 - 3.52 (m, 1 H), 3.62 (s, 3 H), 3.64 - 3.71 (m, 1 H), 4.20 - 4.29 (m, 1 H), 4.58 - 4.80 (m, 1 H), 4.94 - 5.12 (m, 1 H), 6.85 - 6.95 (m, 1 H), 7.02 - 7.23 (m, 2 H), 8.23 ​​- 8.39 (m, 2 H), 8.81 (d, J = 7.8 Hz, 1 H), 9.31 (s, 1 H). 19F NMR (377 MHz, DMSO-d6) δ ppm -184.13 (s, 1 F), -111.44 (s, 2 F).

[0265] Step 4. Preparation of 4-[3-(difluoromethyl)-4-[[5-[(3R,5R)-3-(tert-butoxycarbonylamino)-5-fluoro-1-piperidyl]pyrazolo[1,5-a]pyrimidine-3-carbonyl]amino]pyrazole-1-yl]cyclohexanecarboxylic acid (3) To a solution of methyl 4-[3-(difluoromethyl)-4-[[5-[(3R,5R)-3-(tert-butoxycarbonylamino)-5-fluoro-1-piperidyl]pyrazolo[1,5-a]pyrimidine-3-carbonyl]amino]pyrazole-1-yl]cyclohexanecarboxylate 2 (466 mg, 0.730 mmol, 1.0 equivalent) in THF (2.45 mL, 0.15 M), a solution of LiOH·H2O (308 mg, 7.34 mmol, 10 equivalents) / water (2.45 mL, 0.15 M) was added. The resulting mixture was stirred overnight at room temperature. The solvent was removed under reduced pressure, and the residue was suspended in nanopure water and sonicated. Under vigorous stirring at 0°C, a 6 M aqueous HCl solution was added until the pH became 3. The solid was filtered through a Buchner funnel and rinsed with nanopure water to obtain pure 3 (362 mg, 79% yield).

[0266] LC-MS method 1: 99.9% purity at 215 nm, [M+H] + = 621.3 m / z. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.34 (s, 9 H), 1.39 - 1.43 (m, 1 H), 1.47 - 1.57 (m, 2 H), 1.72 - 1.86 (m, 3 H), 2.00 - 2.15 (m, 5 H), 2.26 - 2.35 (m, 1 H), 2.99 - 3.13 (m, 1 H), 3.42 - 3.52 (m, 2 H), 3.63 - 3.72 (m, 1 H), 4.17 - 4.30 (m, 1 H), 4.95 - 5.12 (m, 1 H), 6.85 - 6.95 (m, 1 H), 7.03 - 7.23 (m, 2 H), 8.28 (s, 1 H), 8.34 (s, 1 H), 8.81 (d, J = 8.1 Hz, 1 H), 9.31 (s, 1 H), 12.09 - 12.23 (m, 1 H). 19 F NMR (377 MHz, DMSO-d6) δ ppm -184.09 (s, 1 F), -111.31 - (s, 2 F).

[0267] Step 5. Preparation of tert-butyl N-[(3R,5R)-1-[3-[[3-(difluoromethyl)-1-[4-[[1-[4-(2,6-dioxo-3-piperidyl)phenyl]-4-piperidyl]carbamoyl]cyclohexyl]pyrazole-4-yl]carbamoyl]pyrazolo[1,5-a]pyrimidine-5-yl]-5-fluoro-3-piperidyl]carbamate (4) 4-[4-[[5-[(3R,5R)-3-(tert-butoxycarbonylamino)-5-fluoro-1-piperidyl]pyrazolo[1,5-a]pyrimidine-3-carbonyl]amino]-3-(difluoromethyl)pyrazole-1-yl]cyclohexanecarboxylic acid 3 (60 mg, 0.10 mmol), 3-[4-(4-amino-1-piperidyl)phenyl]piperidine-2,6-dione sulfate C-9 (55.9 mg, 0.150 mmol), and DIPEA (0.17 mL, 0.97 mmol) were mixed in DMF (1.0 mL) with HATU (55.14 mg, 0.1500 mmol). The solution was stirred overnight at room temperature, and HPLC analysis was performed to reach 68% completion. Additional 3-[4-(4-amino-1-piperidyl)phenyl]piperidine-2,6-dione sulfate 3 (18.6 mg, 0.75 mmol) and HATU (18.4 mg, 0.75 mmol) were added, and the reaction mixture was stirred at room temperature for a further 3 hours to complete the process. The reaction mixture was diluted with H2O, and the mixture was stirred in an ice bath for 1 hour to form a fine precipitate. The aqueous suspension was extracted with DCM (3x), and the suspended precipitate in the aqueous solution was filtered, and the cake was rinsed with H2O. The white solid was suspended in H2O, frozen, and freeze-dried overnight, and 4 [46 mg (48%)] was used in the next step without further purification. HPLC: 90.4% (215 nm). LCMS method 2: Calculated exact mass = 889.42, actual value [M+2H] 2+ = 446.0.

[0268] Step 6. Preparation of 5-[(3R,5R)-3-amino-5-fluoro-1-piperidyl]-N-[3-(difluoromethyl)-1-[4-[[1-[4-(2,6-dioxo-3-piperidyl)phenyl]-4-piperidyl]carbamoyl]cyclohexyl]pyrazole-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide hydrochloride (P-15) A solution of tert-butyl N-[(3R,5R)-1-[3-[[3-(difluoromethyl)-1-[4-[[1-[4-(2,6-dioxo-3-piperidyl)phenyl]-4-piperidyl]carbamoyl]cyclohexyl]pyrazole-4-yl]carbamoyl]pyrazolo[1,5-a]pyrimidine-5-yl]-5-fluoro-3-piperidyl]carbamate 4 (46 mg, 0.050 mmol) in 4.0 M HCl / 1,4-dioxane (3.5 mL, 0.050 mmol) was stirred at room temperature (approximately 1.5 h) until the reaction was complete, as determined by HPLC analysis. The reaction mixture was concentrated to dryness, purified by C18 reverse-phase chromatography (gradient elution, 5-100% MeCN / 20 mM HCl aqueous solution), and lyophilized to obtain P-15 [13.58 mg (37%)] as a white solid.

[0269] LCMS method 3: 99.9% (215 nm); Calculated exact mass (free base) = 789.37; Actual value [M-HCl+H] + = 790.3. 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.55 - 1.67 (m, 3 H), 1.70 - 1.82 (m, 3 H), 1.83 - 2.26 (m, 12 H), 2.35 - 2.41 (m, 1 H), 2.63 - 2.72 (m, 1 H), 3.25 - 3.52 (m, 4 H), 3.57 - 3.66 (m, 2 H), 3.76 - 3.83 (m, 2 H), 4.21 - 4.31 (m, 1 H), 4.47 - 4.71 (m, 1 H), 4.73 - 4.97 (m, 1 H), 5.11 (d, J = 48.0 Hz, 1 H), 6.89 (d, J = 8.0 Hz, 1 H), 6.97 - 7.35 (m, 5 H), 7.79 - 8.00 (m, 1 H), 8.15 - 8.28 (m, 3 H), 8.33 (s, 1 H), 8.39 (s, 1 H), 8.93 (d, J = 7.6 Hz, 1 H), 9.33 (s, 1 H), 10.81 (s, 1 H). 19 F NMR (377 MHz, DMSO- d6) δ ppm -185.26 (s, 1 F), -111.21 (s, 2 F).

[0270] Example S4. Synthesis of P-16 [ka] Step 5. Preparation of tert-butyl N-[(3R,5R)-1-[3-[[3-(difluoromethyl)-1-[4-[4-[[3-(2,6-dioxo-3-piperidyl)-N-methyl-anilino]methyl]piperidine-1-carbonyl]cyclohexyl]pyrazole-4-yl]carbamoyl]pyrazolo[1,5-a]pyrimidine-5-yl]-5-fluoro-3-piperidyl]carbamate (2) 4-[3-(difluoromethyl)-4-[[5-[(3R,5R)-3-(tert-butoxycarbonylamino)-5-fluoro-1-piperidyl]pyrazolo[1,5-a]pyrimidine-3-carbonyl]amino]pyrazole-1-yl]cyclohexanecarboxylic acid 1 (intermediate 3 obtained from the synthesis of P-15) (70.0 mg, 0.113 mmol, 1.0 equiv) and 3-[3-[methyl(piperidine-1-ium-4-ylmethyl)amino]phenyl]piperidine-2,6-dione bisulfate C-10 (60.6 mg, 0.147 mmol, 1.3 equiv) were dissolved in DMF (0.4 mL), and then DIPEA (196 μL, 1.13 mmol, 10 equiv) and HATU (51.5 mg, 0.135 mmol, 1.2 equiv) were added sequentially. The reaction mixture was stirred overnight at room temperature. Further amine (0.3 equivol) and HATU (0.6 equivol) were added to the reaction mixture, and it was stirred for a further 1 hour at room temperature. The reaction mixture was injected directly into a column for reverse-phase flash column chromatography (5% (3 CV) → 70% MeCN in 0.1% HCOOH (aq), 50 g RediSep Rf Gold® C18Aq, 20 CV, λ = 214~254 nm, 60% MeCN to obtain the product). The resulting fraction was evaporated to obtain 8 (88.4 mg, 0.096 mmol, 85% yield) as a white solid.

[0271] LC-MS method 1: 99.9% purity at 215 nm, [M+H] + = 919.4, [M+2H] 2+ = 459.8. 1H NMR (400 MHz, DMSO-d6) δ ppm 0.85 - 1.19 (m, 4 H), 1.34 (br s, 9 H), 1.51 - 1.66 (m, 4 H), 1.66 - 1.99 (m, 8 H), 1.99 - 2.09 (m, 4 H), 2.10 - 2.25 (m, 2 H), 2.26 - 2.44 (m, 2 H), 2.54 - 2.79 (m, 2 H), 2.99 - 3.13 (m, 1 H), 3.25 (br s, 2 H), 3.34 - 3.55 (m, 1 H), 3.56 - 3.72 (m, 1 H), 3.73 - 3.87 (m, 1 H), 3.89 - 4.09 (m, 1 H), 4.25 (br d, J = 9.0 Hz, 1 H), 4.32 - 4.51 (m, 1 H), 4.52 - 4.92 (m, 1 H), 4.93 - 5.20 (m, 1 H), 6.27 - 6.80 (m, 2) H), 6.89 (br d, J = 4.9 Hz, 1 H), 6.97 - 7.43 (m, 3 H), 8.14 (s, 1 H), 8.22 - 8.56 (m, 2 H), 8.82 (br d, J = 7.0 Hz, 1 H), 9.32 (br s, 1 H), 10.82 (br s, 1 H). 19 F NMR (377 MHz, DMSO-d6) δ ppm -186.81 - -182.66 (m, 1 F), -114.66 - -107.40 (m, 2 F).

[0272] Step 6. Preparation of 5-((3R,5R)-3-amino-5-fluoropiperidine-1-yl)-N-(3-(difluoromethyl)-1-((1r,4r)-4-(4-(((3-(2,6-dioxopiperidine-3-yl)phenyl)(methyl)amino)methyl)piperidine-1-carbonyl)cyclohexyl)-1H-pyrazole-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide hydrochloride (P-16) 4.0 M HCl / 1,4-dioxane (3.40 mL, 13.6 mmol, 150 equiv) was added to tert-butyl N-[(3R,5R)-1-[3-[[3-(difluoromethyl)-1-[4-[4-[[3-(2,6-dioxo-3-piperidyl)-N-methyl-anilino]methyl]piperidine-1-carbonyl]cyclohexyl]pyrazole-4-yl]carbamoyl]pyrazolo[1,5-a]pyrimidine-5-yl]-5-fluoro-3-piperidyl]carbamate 8 (83.4 mg, 0.091 mmol, 1.0 equiv), and the mixture was sonicated for 30 minutes and then stirred for 1 hour. The solvent was evaporated to dryness, and the residue was purified by reverse-phase flash chromatography (5% (3 CV) → 50% MeCN in 0.02 M HCl(aq), 50 g RediSep Rf Gold® C18Aq, 15 CV, λ = 214~254 nm, 32~35% MeCN to obtain the product). The fraction containing the product was evaporated to dryness, then co-evaporated with water (3 × 10 mL) to completely remove all traces of residual HCl, and the residue was freeze-dried overnight to obtain P-16 (54.29 mg, 0.0645 mmol, 71% yield) as a white solid.

[0273] LC-MS method 3: 97.1% purity at 215 nm, [M-HCl+H] + = 818.4, [M-HCl+2H] 2+ = 409.8. 1H NMR (400 MHz, DMSO-d6) δ ppm 0.86 - 1.30 (m, 2 H), 1.38 - 1.95 (m, 10 H), 1.95 - 2.13 (m, 4 H), 2.14 - 2.29 (m, 1 H), 2.41 (br d, J = 10.3 Hz, 2 H), 2.60 - 2.78 (m, 2 H), 2.84 - 3.12 (m, 4 H), 3.18 - 3.56 (m, 5 H), 3.74 - 3.91 (m, 1 H), 3.98 (br d, J = 12.7 Hz, 1 H), 4.25 (br t, J = 11.7 Hz, 1 H), 4.36 (br d, J = 9.9 Hz, 1 H), 4.52 - 4.75 (m, 1 H), 4.76 - 4.98 (m, 1 H), 5.10 (d, J = 46.6 Hz, 1 H), 6.56 - 7.73 (m, 6 H), 8.32 (s, 1 H), 8.38 (s, 1 H), 8.50 (br s, 3 H), 8.92 (d, J = 7.8 Hz, 1 H), 9.33 (s, 1 H), 10.85 (br s, 1 H). 19 F NMR (377 MHz, DMSO-d6) δ ppm -184.65 (s, 1 F), -111.30 (s, 2 F).

[0274] Example S5. Synthesis of P-32 [ka]

[0275] Step 5. Preparation of tert-butyl N-[(3R,5R)-1-[3-[[3-(difluoromethyl)-1-[4-[[1-[5-(2,6-dioxo-3-piperidyl)-2-pyridyl]-4-piperidyl]carbamoyl]cyclohexyl]pyrazole-4-yl]carbamoyl]pyrazolo[1,5-a]pyrimidine-5-yl]-5-fluoro-3-piperidyl]carbamate (2) 4-[3-(difluoromethyl)-4-[[5-[(3R,5R)-3-(tert-butoxycarbonylamino)-5-fluoro-1-piperidyl]pyrazolo[1,5-a]pyrimidine-3-carbonyl]amino]pyrazole-1-yl]cyclohexanecarboxylic acid 1 (intermediate 3 obtained from the synthesis of P-15) (144.7 mg, 0.23 mmol, 1 eq.) was dissolved in DMF (4.65 mL), to which DIPEA (0.41 mL, 2.33 mmol, 1 eq.) and HATU (115.13 mg, 0.30 mmol, 1.3 eq.) were added. After 10 minutes at room temperature, 3-[6-(4-amino-1-piperidyl)-3-pyridyl]piperidine-2,6-dione bisulfate C-17 (90 mg, 0.23 mmol, 1 eq.) was added, and the reaction mixture was stirred at room temperature. After 16 hours, LC-MS indicated completion. The reaction mixture was purified by reverse-phase flash chromatography (50 g C18 RediSep Rf Gold column, holding solution (DMF / DMSO), elution: 0% MeCN / 0.1% HCOOH over 3 CV, then 0 to 30% MeCN / 0.1% HCOOH over 15 CV, then 0 to 100% MeCN / 0.1% HCOOH over 5 CV, then 100% MeCN / 0.1% HCOOH over 3 CV; the target product eluted at approximately 30% MeCN). The fractions were combined and concentrated to obtain 2 (57 mg, 16% yield) as a pale yellow solid.

[0276] LC-MS method 1: 58.4% purity at 215 nm, [M+H] + = 891.2. 1H NMR (400 MHz, DMSO-d6 ) δ ppm 1.16 - 1.27 (m, 2 H), 1.34 (br s, 9 H), 1.52 - 1.63 (m, 2 H), 1.64 - 1.91 (m, 8 H), 1.93 - 2.06 (m, 3 H), 2.10 - 2.21 (m, 3 H), 2.60 - 2.74 (m, 1 H), 2.88 - 2.99 (m, 2 H), 3.00 - 3.17 (m, 1 H), 3.42 (br s, 1 H), 3.44 - 3.61 (m, 2 H), 3.62 - 3.86 (m, 2 H), 4.11 - 4.28 (m, 3 H), 4.50 - 4.86 (m, 1 H), 4.93 - 5.13 (m, 1 H), 6.70 - 6.90 (m, 2 H), 6.91 - 7.26 (m, 2 H), 7.34 - 7.49 (m, 1 H), 7.74 (d, J = 7.6 Hz, 1 H), 7.95 (d, J = 2.4 Hz, 1 H), 8.25 - 8.31 (m, 1 H), 8.34 (br s, 1 H), 8.81 (d, J = 7.8 Hz, 1 H), 9.31 (br s, 1 H), 10.80 (s, 1 H). 19 F NMR (377 MHz, DMSO-d6) δ ppm -183.86 (br s, 1 F), -111.28 (br s, 2 F).

[0277] Step 6. Manufacturing of N-[3-(difluoromethyl)-1-[4-[[1-[5-(2,6-dioxo-3-piperidyl)-2-pyridyl]-4-piperidyl]carbamoyl]cyclohexyl]pyrazole-4-yl]-5-[(3R,5R)-3-amino-5-fluoro-1-piperidyl]pyrazolo[1,5-a]pyrimidine-3-carboxamide hydrochloride (P-32) To tert-butyl N-[(3R,5R)-1-[3-[[3-(difluoromethyl)-1-[4-[[1-[5-(2,6-dioxo-3-piperidyl)-2-pyridyl]-4-piperidyl]carbamoyl]cyclohexyl]pyrazole-4-yl]carbamoyl]pyrazolo[1,5-a]pyrimidine-5-yl]-5-fluoro-3-piperidyl]carbamate 2 (57 mg, 0.05 mmol, 1 eq.), a solution of 4 M HCl / 1,4-dioxane (0.32 mL, 1.3 mmol, 27 eq.) was added. The mixture was stirred at room temperature. After 1 hour, complete conversion was shown by LC-MS. The volatile substances were evaporated under vacuum. The resulting residue was purified by reverse-phase flash chromatography (30 g C18 RediSep Rf Gold column, holding solution (water), elution: 5% MeCN / 0.2 M HCl over 5 CV, then 5 to 20% MeCN / 0.2 M HCl over 15 CV, then 20% MeCN / 0.2 M HCl over 5 CV, then 20 to 100% MeCN / 0.2 M HCl over 2 CV, then 100% MeCN / 0.2% HCOOH over 7 CV). The fractions were combined and concentrated to obtain P-32 (37.3 mg, 95% yield) as a pale yellow solid hydrochloride.

[0278] LC-MS method 3: 97.4% purity at 215 nm, [M-HCl+2H] 2+ = 396.2;[M-HCl+H] + = 791.4. 1H NMR (400 MHz, DMSO-d6 ) δ ppm 1.40 - 1.66 (m, 3 H), 1.67 - 1.82 (m, 2 H), 1.83 - 1.93 (m, 4 H), 1.94 - 2.02 (m, 1 H), 2.02 - 2.14 (m, 3 H), 2.15 - 2.45 (m, 3 H), 2.55 - 2.75 (m, 3 H), 3.27 - 3.56 (m, 5 H), 3.87 - 3.96 (m, 1 H), 3.99 (dd, J = 13.1, 4.8 Hz, 1 H), 4.14 - 4.32 (m, 3 H), 4.56 - 4.70 (m, 1 H), 4.80 - 4.92 (m, 1 H), 5.05 - 5.14 (m, 1 H), 6.91 (d, J = 8.1 Hz, 1 H), 7.14 (t, J = 53.8 Hz, 1 H), 7.46 (d, J = 9.5 Hz, 1 H), 7.82 - 8.03 (m, 3 H), 8.32 (s, 1 H), 8.35 - 8.52 (m, 4 H), 8.93 (d, J = 8.1 Hz, 1 H), 9.33 (s, 1 H), 10.95 (s, 1 H). 19 F NMR (377 MHz, DMSO-d6) δ ppm -184.64 (br s, 1 F), -111.27 (s, 2 F).

[0279] The following compounds were synthesized using the same general route, with modifications to the intermediate (TX) in step 3 and the CBM (CX) in step 5 (Table 5). [Table 12] [Table 13] [Table 14] [Table 15] [Table 16] [Table 17] [Table 18]

[0280] General method for final product 2 Example S6. Synthesis of P-5 [ka]

[0281] Step 1. Preparation of [4-[4-amino-3-(trifluoromethyl)pyrazole-1-yl]cyclohexyl]methanol (2) To a solution of methyl 4-[4-amino-3-(trifluoromethyl)pyrazole-1-yl]cyclohexanecarboxylate T-1 (325 mg, 1.12 mmol, 1 eq.) in THF (3.7 mL) and ethanol (7.4 mL), CaCl2 (248 mg, 2.23 mmol, 2 eq.) and then NaBH4 (169 mg, 4.46 mmol, 4 eq.) were added at 0°C. After the addition, the reaction mixture was stirred at room temperature. After 18 hours, complete conversion was shown by LC-MS. Water was added, and the reaction mixture was stirred at room temperature. After 1 hour, the aqueous layer was extracted three times with siRNA. The organic matter was washed with saline solution, dried over Na2SO4, and concentrated to obtain 2 (283 mg, 96% yield) as a pink oil.

[0282] LC-MS method 1: 99.9% purity at 215 nm, [M+H] + = 264.2. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.04 (qd, J = 12.8, 2.9 Hz, 2 H), 1.34 - 1.47 (m, 1 H), 1.63 (qd, J = 12.6, 3.4 Hz, 2 H), 1.83 (br d, J = 11.5 Hz, 2 H), 1.94 - 1.99 (m, 2 H), 3.24 (t, J = 5.7 Hz, 2 H), 3.95 - 4.07 (m, 1 H), 4.18 (s, 2 H), 4.45 (t, J = 5.3 Hz, 1 H), 7.23 (s, 1 H).

[0283] Step 2. Preparation of tert-butyl N-[(3R,5R)-5-fluoro-1-[3-[[1-[4-(hydroxymethyl)cyclohexyl]-3-(trifluoromethyl)pyrazole-4-yl]carbamoyl]pyrazolo[1,5-a]pyrimidine-5-yl]-3-piperidyl]carbamate (4) [4-[4-amino-3-(trifluoromethyl)pyrazole-1-yl]cyclohexyl]methanol 2 (283 mg, 1.07 mmol, 1 eq.), 5-[(3R,5R)-3-(tert-butoxycarbonylamino)-5-fluoro-1-piperidyl]pyrazolo[1,5-a]pyrimidine-3-carboxylic acid 3 (intermediate 4 obtained from the synthesis of P-1) (408 mg, 1.07 mmol, 1 eq.), and NMI (298 μL, 3.76 mmol, 3.5 eq.) were mixed in MeCN (5.4 mL) with TCFH (362 mg, 1.29 mmol, 1.2 eq.). The reaction mixture was stirred at room temperature. After 18 hours, complete conversion was shown by LC-MS. Water was added, and the reaction mixture was stirred at 0°C. After 30 minutes, the solid was filtered through a Buchner funnel, rinsed with a water / MeCN mixture, and then dried under high vacuum to obtain 4 (554 mg, 61% yield) as a brown solid.

[0284] LC-MS method 1: 73.7% purity at 215 nm, [M+H] + = 625.2. 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.02 - 1.15 (m, 2 H), 1.29 (br s, 9 H), 1.69 - 1.93 (m, 6 H), 1.99 - 2.16 (m, 5 H), 3.26 (br t, J = 5.0 Hz, 2 H), 3.39 - 3.56 (m, 1 H), 3.58 - 3.72 (m, 1 H), 4.23 (br t, J = 11.4 Hz, 1 H), 4.47 (br t, J = 4.9 Hz, 1 H), 4.51 - 4.64 (m, 1 H), 4.97 - 5.15 (m, 1 H), 6.91 (br d, J = 5.6 Hz, 1 H), 7.10 (br s, 1 H), 8.29 (s, 1 H), 8.40 (br s, 1 H), 8.81 (br d, J = 7.6 Hz, 1 H), 9.21 (br s, 1 H). 19 F NMR (377 MHz, DMSO-d6) δ ppm -185.41 - -184.01 (m, 1 F), -58.98 (br s, 3 F).

[0285] Step 3. Preparation of tert-butyl N-[(3R,5R)-1-[3-[[1-[4-[[4-[4-(2,6-dioxo-3-piperidyl)anilino]-1-piperidyl]methyl]cyclohexyl]-3-(trifluoromethyl)pyrazole-4-yl]carbamoyl]pyrazolo[1,5-a]pyrimidine-5-yl]-5-fluoro-3-piperidyl]carbamate (5) Preparation of Solution A: To a solution of tert-butyl N-[(3R,5R)-5-fluoro-1-[3-[[1-[4-(hydroxymethyl)cyclohexyl]-3-(trifluoromethyl)pyrazole-4-yl]carbamoyl]pyrazolo[1,5-a]pyrimidine-5-yl]-3-piperidyl]carbamate 4 (100 mg, 0.160 mmol, 1 eq.) in anhydrous DMSO (1.5 mL), IBX (58 mg, 0.210 mmol, 1.3 eq.) was added. The resulting mixture was stirred at room temperature. After 18 hours, LC-MS demonstrated complete conversion to tert-butyl N-[(3R,5R)-5-fluoro-1-[3-[[1-(4-formylcyclohexyl)-3-(trifluoromethyl)pyrazole-4-yl]carbamoyl]pyrazolo[1,5-a]pyrimidine-5-yl]-3-piperidyl]carbamate.

[0286] 3-[4-(4-piperidylamino)phenyl]piperidine-2,6-dione 2,2,2-trifluoroacetic acid C-4 (77 mg, 0.190 mmol, 1.2 eq.) was dissolved in anhydrous DCE (1.6 mL) and DIPEA (0.22 mL, 1.28 mmol, 8 eq.) was added. The resulting mixture was stirred at room temperature. After 15 minutes, NaBH(OAc)3 (51 mg, 0.240 mmol, 1.5 eq.) was added, followed by solution A, and the reaction mixture was stirred at room temperature. After 1 hour, complete conversion was shown by LC-MS. Volatile substances were removed under reduced pressure, and the residue was purified by reverse-phase flash chromatography (30 g C18 gold column, holding solution (DMSO), elution: 5% MeCN / 0.1% HCOOH over 3 CV, then 5 to 35% MeCN / 0.1% HCOOH over 5 CV, then 35 to 40% MeCN / 0.1% HCOOH over 5 CV, then 40 to 100% MeCN / 0.1% HCOOH over 5 CV). The fractions were combined and concentrated to obtain 5 (59.2 mg, 36% yield) as a white solid. This was used directly for the next step. LC-MS method 1: 87.2% purity at 215 nm, [M+H]+ = 894.4.

[0287] Step 4. Preparation of N-[1-[4-[[4-[4-(2,6-dioxo-3-piperidyl)anilino]-1-piperidyl]methyl]cyclohexyl]-3-(trifluoromethyl)pyrazole-4-yl]-5-[(3R,5R)-3-amino-5-fluoro-1-piperidyl]pyrazolo[1,5-a]pyrimidine-3-carboxamide hydrochloride (P-5) A solution of tert-butyl N-[(3R,5R)-1-[3-[[1-[4-[[4-(2,6-dioxo-3-piperidyl)anilino]-1-piperidyl]methyl]cyclohexyl]-3-(trifluoromethyl)pyrazole-4-yl]carbamoyl]pyrazolo[1,5-a]pyrimidine-5-yl]-5-fluoro-3-piperidyl]carbamate 5 (59.2 mg, 0.066 mmol, 1 eq.) in 4M HCl and a solution of 1,4-dioxane (3.5 mL, 14.0 mmol, 210 eq.) was stirred at room temperature. After 1 hour, complete conversion was shown by LC-MS. The solvent was removed under reduced pressure, and the residue was purified by reverse-phase flash chromatography (30 g C18 gold column, holding solution (DMSO), elution: 5% MeCN / 0.02 M HCl over 3 CV, then 5 to 30% MeCN / 0.02 M HCl over 17 CV). The fractions were combined and concentrated. The residue was purified by preparative HPLC (MeCN / 0.2 M HCl). The fractions were combined, concentrated, and lyophilized to obtain P-5 (21.29 mg, 40% yield) as a white solid hydrochloride.

[0288] LC-MS method 3: 99.9% purity at 215 nm, [M-HCl+H] + = 794.4. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.16 - 1.28 (m, 3 H), 1.79 - 2.03 (m, 8 H), 2.06 - 2.15 (m, 5 H), 2.35 - 2.46 (m, 2 H), 2.57 - 2.69 (m, 2 H), 2.94 - 3.08 (m, 3 H), 3.21 - 3.62 (m, 7 H), 3.65 - 3.71 (m, 1 H), 4.28 - 4.38 (m, 1 H), 4.51 - 4.87 (m, 2 H), 5.12 (d, J = 48.0 Hz, 1 H), 6.60 - 6.73 (m, 2H), 6.92 (d, J = 7.8 Hz, 1 H), 6.97 (d, J = 7.5 Hz, 2 H), 8.28 - 8.37 (m, 3 H), 8.46 (s, 1 H), 8.94 (d, J = 8.0 Hz, 1 H), 9.24 (s, 1 H), 9.53 - 9.73 (m, 1 H), 10.76 (s, 1 H). 19 F NMR (377 MHz, DMSO-d6) δ ppm -185.01 (s, 1 F), -58.97 (s, 3 F).

[0289] Example S7. Synthesis of P-6 [ka]

[0290] Step 3. Preparation of tert-butyl N-[(3R,5R)-1-[3-[[1-[4-[[4-[[3-(2,6-dioxo-3-piperidyl)anilino]methyl]-1-piperidyl]methyl]cyclohexyl]-3-(trifluoromethyl)pyrazole-4-yl]carbamoyl]pyrazolo[1,5-a]pyrimidine-5-yl]-5-fluoro-3-piperidyl]carbamate (2) To a solution of tert-butyl N-[(3R,5R)-5-fluoro-1-[3-[[1-[4-(hydroxymethyl)cyclohexyl]-3-(trifluoromethyl)pyrazole-4-yl]carbamoyl]pyrazolo[1,5-a]pyrimidine-5-yl]-3-piperidyl]carbamate 1 (intermediate 4 for the synthesis of P-5) (100 mg, 0.16 mmol) in dry DMSO (1.6 mL), IBX (58.3 mg, 0.21 mmol) was added. The resulting mixture was stirred overnight at room temperature. In the second flask, crude 3-[3-(4-piperidylmethylamino)phenyl]piperidine-2,6-dionetrifluoroacetate C-1 (80.1 mg, 0.19 mmol) was suspended in dry DCE (1.6 mL, 6 mL), and DIPEA (223 μL, 1.28 mmol) was added. The resulting mixture was stirred at room temperature for 0.25 hours. NaBH(OAc)3 (44.3 mg, 0.21 mmol) was then added. The solution containing the aldehyde intermediate was then added dropwise via a cannula. The resulting mixture was stirred at room temperature for 1 hour. The DCE was then evaporated under reduced pressure. The resulting DMSO solution was loaded directly onto a 30 g gold C18 Isco chromatography column. Elution was performed using a MeCN / 0.1% formic acid aqueous solution (40-60%). The product was eluted with a 45% MeCN / 0.1% formic acid aqueous solution. The target fractions were combined and concentrated to obtain fraction 2 (75 mg, 52% yield) as a pale yellow solid.

[0291] LC-MS method 3: 98.9% purity at 215 nm, [M+H] + = 908.5, [M+2H] 2+ = 454.8. 1H NMR (400 MHz, DMSO-d6) δ ppm 0.97 - 1.25 (m, 6 H), 1.47 - 1.69 (m, 3 H), 1.70 - 1.83 (m, 5 H), 1.83 - 2.00 (m, 6 H), 2.00 - 2.16 (m, 8 H), 2.20 (br d, J = 6.8 Hz, 2 H), 2.55 - 2.69 (m, 2 H), 2.82 - 3.00 (m, 5 H), 3.38 - 3.57 (m, 2 H), 3.58 - 3.72 (m, 3 H), 4.24 (br t, J = 11.4 Hz, 1 H), 4.46 - 4.67 (m, 1 H), 4.97 - 5.15 (m, 1 H), 5.58 - 5.70 (m, 1 H), 6.34 (d, J = 7.3 Hz, 1 H), 6.40 (s, 1 H), 6.45 (br d, J = 8.3 Hz, 1 H), 6.91 (br d, J = 7.3 Hz, 1 H), 7.00 (t, J = 7.7 Hz, 1 H), 7.05 - 7.17 (m, 1 H), 8.29 (s, 1 H), 8.39 (br s, 1 H), 8.82 (br d, J = 8.1 Hz, 1 H), 9.21 (s, 1 H), 10.78 (s, 1 H).

[0292] Step 4. Preparation of 5-((3R,5R)-3-amino-5-fluoropiperidine-1-yl)-N-(1-((1r,4r)-4-((4-(((3-(2,6-dioxopiperidine-3-yl)phenyl)amino)methyl)piperidine-1-yl)methyl)cyclohexyl)-3-(trifluoromethyl)-1H-pyrazole-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide hydrochloride (P-6) In a round-bottom flask, tert-butyl N-[(3R,5R)-1-[3-[[1-[4-[[4-[[3-(2,6-dioxo-3-piperidyl)anilino]methyl]-1-piperidyl]methyl]cyclohexyl]-3-(trifluoromethyl)pyrazole-4-yl]carbamoyl]pyrazolo[1,5-a]pyrimidine-5-yl]-5-fluoro-3-piperidyl]carbamate 2 (75 mg, 0.08 mmol) was dissolved in 4 M HCl solution / dioxane (3.0 mL, 12.00 mmol). The solution was stirred at room temperature for 0.5 hours. The solvent was then evaporated under reduced pressure. The residue was dissolved in water, and the solution was loaded onto a 30 g gold C18 Isco chromatography column. Elution was performed using MeCN / 0.02 M HCl aqueous solution (5-40%). The product was eluted with a 25% MeCN / 0.02 M HCl aqueous solution. The desired fraction was combined and concentrated to obtain the product, but its purity was only 92.4% (LCMS method 3). The impure product was further purified by preparative HPLC. The desired fraction was combined and concentrated. The residue was dissolved in water and freeze-dried to obtain P-6 (29 mg, 42% yield) as a white solid hydrochloride.

[0293] LC-MS method 3: 99.9% purity at 215 nm, [M-HCl+H] + = 808.4, [M-HCl+2H] 2+ = 404.8, [M-HCl+3H]3 + =270.2. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.09 - 1.31 (m, 2 H), 1.51 - 2.18 (m, 15 H), 2.35 - 2.47 (m, 2 H), 2.54 - 2.65 (m, 2 H), 2.82 - 3.14 (m, 5 H), 3.16 - 3.60 (m, 5 H), 3.72 (dd, J = 10.0, 5.1 Hz, 1 H), 4.26 - 4.39 (m, 2 H), 4.53 - 4.90 (m, 2 H), 5.12 (d, J = 45.7 Hz, 1 H), 6.28 - 6.78 (m, 3 H), 6.93 (d, J = 7.6 Hz, 1 H), 7.01 - 7.16 (m, 1 H), 8.29 - 8.56 (m, 4 H), 8.94 (d, J = 7.8 Hz, 1 H), 9.24 (s, 1 H), 9.47 - 9.86 (m, 1 H), 10.81 (s, 1 H). 19 F NMR (377 MHz, DMSO-d6) δ ppm -184.84 (s, 1 F), -58.65 (s, 3 F).

[0294] Example S8. Synthesis of P-19 [ka]

[0295] Step 1. Preparation of [4-[4-amino-3-(difluoromethyl)pyrazole-1-yl]cyclohexyl]methanol (2) To a solution of methyl 4-[4-amino-3-(difluoromethyl)pyrazole-1-yl]cyclohexanecarboxylate T-2 (1.23 g, 4.5 mmol, 1.0 eq.) in THF (15.0 mL, 0.1 M) and ethanol (30.0 mL), CaCl2 (1.0 g, 9.0 mmol, 2.0 eq.) and then NaBH4 (0.68 g, 18.0 mmol, 4.0 eq.) were added at 0°C. The resulting mixture was stirred overnight and then heated to room temperature. After stirring overnight, water was added, and the reaction mixture was stirred at room temperature for 1 hour. The aqueous phase was extracted with SiO(3x). The organic layer was washed with saline solution, dried over Na2SO4, and concentrated to dryness to obtain [4-[4-amino-3-(difluoromethyl)pyrazole-1-yl]cyclohexyl]methanol 2 (1.1 g, 99% yield) as an orange oil. LC-MS method 1: Retention time: 0.989 min, 99.0% purity at 215 nm, [M+H] + = 246.2. 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.04 (qd, J = 12.9, 3.2 Hz, 2 H), 1.32 - 1.43 (m, 1 H), 1.62 (qd, J = 12.6, 3.3 Hz, 2 H), 1.82 (br d, J = 11.5 Hz, 2 H), 1.93 - 1.98 (m, 2 H), 3.24 (t, J = 5.7 Hz, 2 H), 3.94 (tt, J = 11.9, 3.8 Hz, 1 H), 4.00 - 4.06 (m, 2 H), 4.44 (t, J = 5.3 Hz, 1 H), 6.72 - 7.02 (m, 1 H), 7.14 (s, 1 H). 19 F NMR (377 MHz, DMSO-d6) δ ppm -111.21 (s, 2 F).

[0296] Step 2. Preparation of tert-butyl N-[(3r,5r)-1-[3-[[3-(difluoromethyl)-1-[4-(hydroxymethyl)cyclohexyl]pyrazole-4-yl]carbamoyl]pyrazolo[1,5-a]pyrimidine-5-yl]-5-fluoro-3-piperidyl]carbamate (4) [4-[4-amino-3-(difluoromethyl)pyrazole-1-yl]cyclohexyl]methanol 2 (511.8 mg, 1.88 mmol, 1.5 eq.), 5-[(3r,5r)-3-(tert-butoxycarbonylamino)-5-fluoro-1-piperidyl]pyrazolo[1,5-a]pyrimidine-3-carboxylic acid 3 (intermediate 4 obtained from the synthesis of P-1) (475.0 mg, 1.25 mmol, 1.0 eq.), and NMI (362.72 μL, 4.58 mmol, 3.6 eq.) were mixed in MeCN (12.5 mL, 0.1 M) with TCFH (440.43 mg, 1.57 mmol, 1.2 eq.). The resulting mixture was stirred overnight at room temperature. Water was added, and the reaction mixture was stirred at room temperature for 1 hour. The solid was filtered through a Buchner funnel and rinsed with a water / MeCN mixture. Purification by reverse-phase chromatography (50 g C18 RediSep Rf Gold column, holding solution (DMSO), elution: 5% MeOH / 0.1% HCOOH over 4 CV, then 5% to 100% MeOH / 0.1% HCOOH over 15 CV). The pure fractions were combined and concentrated under reduced pressure to obtain tert-butyl N-[(3r,5r)-1-[3-[[3-(difluoromethyl)-1-[4-(hydroxymethyl)cyclohexyl]pyrazole-4-yl]carbamoyl]pyrazolo[1,5-a]pyrimidine-5-yl]-5-fluoro-3-piperidyl]carbamate 4 (421 mg, 55% yield) as a brown solid.

[0297] LC-MS method 1: Retention time: 1.629 min, 99.9% purity at 215 nm, [M+H] + = 607.2. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.03 - 1.15 (m, 2 H), 1.34 (br s, 9 H), 1.64 - 1.80 (m, 3 H), 1.82 - 1.93 (m, 3 H), 1.99 - 2.07 (m, 2 H), 2.08 - 2.18 (m, 1 H), 2.95 - 3.14 (m, 1 H), 3.26 (t, J = 5.7 Hz, 2 H), 3.37 - 3.52 (m, 1 H), 3.62 - 3.71 (m, 1 H), 4.12 - 4.21 (m, 1 H), 4.47 (t, J = 5.3 Hz, 1 H), 4.69 (br s, 1 H), 4.95 - 5.12 (m, 1 H), 6.86 - 6.94 (m, 1 H), 7.01 - 7.22 (m, 2 H), 8.28 (s, 1 H), 8.34 (br s, 1 H), 8.82 (d, J = 7.8 Hz, 1 H), 9.31 (br s, 1 H). 19 F NMR (377 MHz, DMSO-d6) δ ppm -186.02 - -179.79 (m, 1F), -113.10 -108.69 (m, 2F).

[0298] Step 3. Preparation of tert-butyl N-[(3R,5R)-1-[3-[[3-(difluoromethyl)-1-[4-[[4-[[3-(2,6-dioxo-3-piperidyl)-N-methyl-anilino]methyl]-1-piperidyl]methyl]cyclohexyl]pyrazole-4-yl]carbamoyl]pyrazolo[1,5-a]pyrimidine-5-yl]-5-fluoro-3-piperidyl]carbamate (5) To a solution of 4 (150.0 mg, 0.127 mmol, 1.0 eq.) in dry DMSO (1.4 mL, 0.1 M), IBX (64.0 mg, 0.152 mmol, 1.2 eq.) was added. The resulting mixture was stirred overnight at room temperature. Separately, a solution of C-10 (1.0 mL, 0.1400 mmol, 1.1 eq.) in DCE (1 mL) and DIPEA (215.66 μL, 1.24 mmol, 10 eq.) was stirred at room temperature for 10 minutes, and the DMSO solution containing oxidized 4 was added. After stirring for 10 minutes, NaBH(OAc)3 (34.18 mg, 0.1600 mmol, 1.3 eq.) was added. The resulting mixture was stirred at room temperature for 1 hour. The DCE was removed under reduced pressure. The crude mixture was purified by column chromatography C18 (5 CV at 5%; 15 CV up to 35%; 15 CV up to 35% MeCN / H2O (0.1% formic acid)). The fraction of interest was concentrated to dryness to obtain 5 (122 mg, 99% yield) as a yellow solid.

[0299] LC-MS method 1: Retention time: 1.508 min, purity 35.3% at 215 nm, [M+2H] 2+ = 452.6. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.01 - 1.09 (m, 2 H), 1.11 - 1.12 (m, 2 H), 1.22 - 1.42 (m, 9 H), 1.58 - 1.83 (m, 6 H), 1.85 - 1.96 (m, 3 H), 2.00 - 2.08 (m, 2 H), 2.09 - 2.25 (m, 4 H), 2.42 - 2.49 (m, 2 H), 2.57 - 2.66 (m, 1 H), 2.88 - 2.95 (m, 3 H), 3.16 - 3.22 (m, 2 H), 3.38 - 3.49 (m, 2 H), 3.63 - 3.71 (m, 1 H), 3.75 (br dd, J = 10.5, 4.9 Hz, 1 H), 4.12 - 4.23 (m, 2 H), 4.98 - 5.12 (m, 2 H), 6.42 (br d, J = 7.3 Hz, 1 H), 6.51 (s, 1 H), 6.54 - 6.59 (m, 1 H), 6.86 - 6.94 (m, 1 H), 7.04 - 7.15 (m, 2 H), 7.18 - 7.26 (m, 1 H), 7.43 - 7.50 (m, 1 H), 7.65 - 7.70 (m, 1 H), 7.96 (d, J = 7.8 Hz, 1 H), 8.15 (s, 1 H), 8.27 - 8.31 (m, 1 H), 8.32 - 8.37 (m, 1 H), 8.82 (br d, J = 8.1 Hz, 1 H), 9.32 (br d, J = 3.2 Hz, 1 H), 10.80 (s, 1 H). 19 F NMR (377 MHz, DMSO-d6) δ ppm -184.86 (s, 1 F), -111.10 (s, 2 F).

[0300] Step 4. Manufacturing of N-[3-(difluoromethyl)-1-[4-[[4-[[3-(2,6-dioxo-3-piperidyl)-N-methyl-anilino]methyl]-1-piperidyl]methyl]cyclohexyl]pyrazole-4-yl]-5-[(3R,5R)-3-amino-5-fluoro-1-piperidyl]pyrazolo[1,5-a]pyrimidine-3-carboxamide dihydrochloride (P-19) In a round-bottom flask, 5 (60 mg, 0.07 mmol) was dissolved in 4 M HCl / 1,4-dioxane (2.49 mL, 9.96 mmol, 150 eq.) solution. The mixture was stirred at room temperature for 1.5 hours. The solvent was removed under reduced pressure. Water was added to the crude product, and the resulting solution was loaded directly onto a 30 g C18 gold column and purified with 0.02 M MeCN / HCl (3 CV to 5%, 15 CV to 35%). The product was eluted with 28% MeCN. The fractions were combined, concentrated, and lyophilized to obtain P-19 (19.09 mg, 35% yield) as a white solid dihydrochloride.

[0301] LC-MS method 3: Retention time: 2.478 min, 98.8% purity at 215 nm, [M-2HCl+H] + = 804.3, [M-2HCl+H] 2+ = 402.8. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.12 - 1.28 (m, 2 H), 1.52 - 2.11 (m, 14 H), 2.12 - 2.25 (m, 1 H), 2.35 - 2.48 (m, 3 H), 2.57 - 2.65 (m, 1 H), 2.78 - 2.98 (m, 6 H), 3.04 - 3.58 (m, 7 H), 3.72 - 3.78 (m, 1 H), 4.18 - 4.29 (m, 1 H), 4.50 - 4.97 (m, 2 H), 5.10 (d, J = 47.0 Hz, 1 H), 6.39 - 6.72 (m, 3H), 6.84 - 6.95 (m, 1 H), 6.96 - 7.30 (m, 2 H), 8.19 - 8.35 (m, 4 H), 8.39 (s, 1 H), 8.94 (d, J = 7.8 Hz, 1 H), 9.21 - 9.60 (m, 2 H), 10.80 (s, 1 H). 19 F NMR (377 MHz, DMSO-d6) δ ppm -184.67 (s, 1 F), -111.29 (s, 2 F).

[0302] The following compounds were synthesized using the same general route, with modifications to the intermediate (TX) in step 1 and the CBM (CX) in step 3 (Table 6). [Table 19] [Table 20] [Table 21] [Table 22]

[0303] General method for the final product 3 Example S9. Synthesis of P-11 [ka]

[0304] Step 1. Preparation of 2-oxabicyclo[3.2.2]nonane-3-one(2) To a solution of 1 (800 mg, 6.44 mmol, 1.0 eq.) of dried DCM (42.9 mL, 0.15 M), NaHCO3 (541.15 mg, 6.44 mmol, 1.0 eq.) and mCPBA (1.5 g, 6.44 mmol, 1.0 eq.) were added at 0°C. The resulting mixture was stirred for 4 hours and then heated to room temperature. The reaction mixture was diluted with DCM. The organic material was washed once with 1N NaOH aqueous solution and once with saline solution, dried over Na2SO4, and concentrated to dryness. The crude mixture was purified by flash chromatography eluting with a 40-75% HCl / heptane gradient. The target fraction (eluted with 50% siRNA / heptane, Rf = 0.2 on TLC eluted with 50% siRNA / heptane, vanillin stained (grayish-purple spots)) was concentrated and allowed to dry to obtain 2 (478 mg, yield 52%) as a white solid. LC-MS method 1: Retention time: 1.292 min, 99.9% purity at 200 nm, [M+H] + = 141.1. 1 H NMR (400 MHz, CDCl3) δ ppm 1.72 - 1.96 (m, 6 H), 2.04 - 2.10 (m, 1 H), 2.17 - 2.27 (m, 2 H), 2.87 (d, J = 4.2 Hz, 2 H), 4.50 - 4.55 (m, 1 H).

[0305] Step 2. Preparation of methyl 2-(4-hydroxycyclohexyl)acetate (3) MeONa (1.09 g, 20.15 mmol, 5.0 eq.) was added to a methanol (20.15 mL, 0.2 M) solution of 2 (565 mg, 4.03 mmol, 1.0 eq.) of 2.2 mg. The resulting mixture was stirred at room temperature for 1 hour. TLC indicated completion of the reaction (eluted with 70% MTBE / heptane and confirmed with vanillin: Rf SM = 0.3 (gray / purple spot), Rf EP = 0.4 (purple spot)). MeOH was evaporated under reduced pressure. The residue was dissolved in MTBE and saturated NH4Cl aqueous solution was added. The aqueous phase was extracted 3 times with MTBE. The organic layer was dried over Na2SO4 and concentrated to dryness to obtain 3 (258 mg, 37% yield). 1 H NMR (400 MHz, CDCl3) δ ppm 1.38 - 1.65 (m, 8 H), 1.68 - 1.78 (m, 2 H), 1.82 - 1.94 (m, 1 H), 2.26 (d, J = 7.3 Hz, 2 H), 3.68 (s, 3 H), 3.96 - 4.03 (m, 1 H).

[0306] Step 3. Preparation of methyl 2-(4-methylsulfonyloxycyclohexyl)acetate (4) To a solution of 3 (258 mg, 1.5 mmol, 1.0 eq.) and TEA (313.2 μL, 2.25 mmol, 1.5 eq.) in DCM (7.50 mL, 0.2 M), methanesulfonyl chloride (150.73 μL, 1.95 mmol, 1.3 eq.) was added dropwise at 0°C. The resulting mixture was stirred at 0°C for 30 minutes. Completion of the reaction was indicated by TLC (elution with 60% siRNA / heptane and confirmation with KMnO4, Rf SM = 0.35, Rf = 0.50). Water was added, and the aqueous phase was extracted three times with DCM. The organic layer was washed once with 1 N aqueous HCl solution, dried over Na2SO4, and concentrated to dryness to obtain 4 (375 mg, 99% yield) as a pale yellow liquid. 1H NMR (400 MHz, CDCl3) δ ppm 1.36 - 1.50 (m, 2 H), 1.61 - 1.72 (m, 4 H), 1.83 - 1.95 (m, 1 H), 2.03 - 2.11 (m, 2 H), 2.26 (d, J = 7.1 Hz, 2 H), 3.01 (s, 3 H), 3.67 - 3.68 (m, 3 H), 4.97 (br s, 1 H).

[0307] Step 4. Preparation of methyl 2-[4-[4-nitro-3-(trifluoromethyl)pyrazole-1-yl]cyclohexyl]acetate (6) To a solution of 4 (207.36 mg, 0.83 mmol, 1.0 eq.) and 5 (150 mg, 0.83 mmol, 1.0 eq.) in dried DMF (2.76 mL), Cs2CO3 (539.82 mg, 1.66 mmol, 2.0 eq.) was added. The resulting mixture was stirred at 90°C for 24 hours. An additional 4 (207.36 mg, 0.83 mmol, 1.0 eq.) was added, and the resulting mixture was stirred at 90°C for 72 hours. Water was added to the reaction mixture. The aqueous phase was extracted three times with siRNA. The organic layer was washed three times with water and once with brine, dried over Na2SO4, and concentrated to dryness. The crude mixture was purified by flash column chromatography eluting with 0 to 35% MTBE / heptane (20 CV), followed by 35% MTBE / heptane (6 CV). The target fraction (confirmed by TLC eluting with 35% MTBE / heptane and then 60% MTBE / heptane, Rf = 0.30, UV + KMnO4) was concentrated to dryness to obtain fraction 6 (103 mg, yield 37%) as a pale yellow solid.

[0308] LC-MS method 1: Retention time: 1.853 min, 99.9% purity at 215 nm, [M+H] + = 336.2. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.14 - 1.27 (m, 3 H), 1.73 - 1.86 (m, 5 H), 2.07 - 2.11 (m, 1 H), 2.26 (d, J = 6.8 Hz, 2 H), 3.60 (s, 3 H), 4.32 (tt, J = 11.7, 3.5 Hz, 1 H), 9.20 (s, 1 H). 19 F NMR (377 MHz, DMSO-d6) δ ppm -61.43 (s, 3 F).

[0309] Step 5. Preparation of methyl 2-[4-[4-amino-3-(trifluoromethyl)pyrazole-1-yl]cyclohexyl]acetate (7) Nitrogen was supplied to a solution of 6 (103 mg, 0.31 mmol, 1.0 eq.) ethyl acetate (3 mL, 0.08 M) and methanol (1 mL, 0.08 M) and bubbled for 5 minutes. Then, 10% Pd / C (65.39 mg, 0.06 mmol, 20 mol%) was added, and nitrogen was supplied to the solution and bubbled for another 5 minutes. Then, hydrogen was supplied to the solution and bubbled for 5 minutes, after which the resulting mixture was stirred under a hydrogen atmosphere (1 atm) for 3 hours. The reaction mixture was filtered through Celite and rinsed with ELISA. The filtrate was concentrated under reduced pressure. The crude mixture was purified by reverse-phase flash chromatography (30 g C18 gold column, elution: 45-70% MeOH / H2O (0.1% formic acid) over 20 CV). The target fraction was eluted with 60% MeOH / H2O (0.1% formic acid), concentrated to dryness, and 7 (64 mg, 68% yield) was obtained as a brown oily substance. LC-MS method 1: Retention time: 1.618 min, 99.9% purity at 215 nm, [M+H] + = 306.1. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.06 - 1.22 (m, 2 H), 1.60 - 1.84 (m, 5 H), 1.91 - 2.03 (m, 2 H), 2.24 (d, J = 6.6 Hz, 2 H), 3.59 (s, 3 H), 4.00 (ddd, J = 11.9, 8.2, 3.9 Hz, 1 H), 4.20 (br s, 2 H), 7.23 (s, 1 H). 19 F NMR (377 MHz, DMSO-d6) δ ppm -58.82 (s, 3 F).

[0310] Step 6. Production of 2-[4-[4-amino-3-(trifluoromethyl)pyrazole-1-yl]cyclohexyl]ethanol (8) To a solution of methyl 7 (64.0 mg, 0.210 mmol, 1 eq.) in THF (0.70 mL, 0.1 M) and ethanol (1.40 mL, 0.1 M), CaCl2 (46.53 mg, 0.420 mmol, 2.0 eq.), followed by NaBH4 (31.72 mg, 0.840 mmol, 4.0 eq.) was added at 0°C. The resulting mixture was stirred overnight and warmed to room temperature. After stirring overnight, NaBH4 (10 mg, 0.210 mmol, 1.0 eq.) was added at 0°C. The resulting mixture was stirred at 0°C for 1 hour, then stirred at room temperature for 20 hours. Next, water was added, and the reaction mixture was stirred at room temperature for 1 hour. The aqueous phase was extracted three times with SiO2. The organic substance was washed with saline solution, dried on Na2SO4, and concentrated to dryness to obtain 8 (49 mg, 77% yield) as a pale orange oily substance.

[0311] LC-MS method 1: Retention time: 1.412 min, 91.9% purity at 215 nm, [M+H] + = 278.1. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.01 - 1.10 (m, 2 H), 1.31 - 1.47 (m, 3 H), 1.56 - 1.70 (m, 2 H), 1.81 (br d, J = 12.2 Hz, 2 H), 1.95 (br s, 2 H), 3.40 - 3.51 (m, 2 H), 3.93 - 4.01 (m, 1 H), 4.18 (s, 2 H), 4.33 (t, J = 5.1 Hz, 1 H), 7.22 (s, 1 H). 19 F NMR (377 MHz, DMSO-d6) δ ppm -58.80 (s, 3 F).

[0312] Step 7. Preparation of tert-butyl N-[(3R,5R)-5-fluoro-1-[3-[[1-[4-(2-hydroxyethyl)cyclohexyl]-3-(trifluoromethyl)pyrazole-4-yl]carbamoyl]pyrazolo[1,5-a]pyrimidine-5-yl]-3-piperidyl]carbamate (10) To a solution of 8 (49.65 mg, 0.160 mmol, 1.25 eq.) and 9 (intermediate 4 obtained from the synthesis of P-1) (50 mg, 0.130 mmol, 1.0 eq.) in MeCN (1 mL, 1.3 M), NMI (31.32 μL, 0.400 mmol, 3.5 eq.) was added, followed by TCFH (55.47 mg, 0.200 mmol, 1.5 eq.). The resulting mixture was stirred overnight at room temperature. Nanopure water was added, and the resulting suspension was stirred at 0°C for 30 minutes. The solid was filtered through a Buchner funnel, rinsed with water, co-evaporated with MeCN, and dried under high vacuum to obtain 10 (81 mg, 69% yield) as a brown solid.

[0313] LC-MS method 1: Retention time: 1.720 min, 72.2% purity at 215 nm, [M+H] + = 639.2. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.06 (br s, 16 H), 1.60 - 1.91 (m, 6 H), 1.99 - 2.20 (m, 4 H), 3.41 - 3.49 (m, 2 H), 3.60 - 3.71 (m, 1 H), 4.19 - 4.29 (m, 1 H), 4.35 (br t, J = 5.0 Hz, 1 H), 4.50 - 4.61 (m, 1 H), 4.98 - 5.14 (m, 1 H), 6.82 - 6.97 (m, 1 H), 7.06 - 7.19 (m, 1 H), 8.39 (br s, 1 H), 8.82 (br d, J = 8.1 Hz, 1 H), 9.21 (br s, 1 H). 19 F NMR (377 MHz, DMSO-d6) δ ppm -185.35 - -183.28 (m, 1 F), -58.99 (s, 3 F).

[0314] Step 8. Preparation of tert-butyl N-[(3R,5R)-1-[3-[[1-[4-[2-[4-[4-(2,6-dioxo-3-piperidyl)phenyl]piperazine-1-yl]ethyl]cyclohexyl]-3-(trifluoromethyl)pyrazole-4-yl]carbamoyl]pyrazolo[1,5-a]pyrimidine-5-yl]-5-fluoro-3-piperidyl]carbamate (11) To a solution of 10 (81 mg, 0.13 mmol, 1.0 eq.) in DMSO (1 mL, 1.3 M), IBX (46.17 mg, 0.16 mmol, 1.3 eq.) was added. The resulting mixture was stirred overnight at room temperature. To a solution of a second C-6 (56.01 mg, 0.15 mmol, 1.2 eq.) in DCM (1 mL), DIPEA (87.39 μL, 0.50 mmol, 4.0 eq.) was added. The resulting mixture was stirred at room temperature for 5 minutes. Next, NaBH(OAc)3 (34.62 mg, 0.16 mmol, 1.3 eq.) was added, followed by the DMSO solution obtained from the oxidation of IBX. The reaction mixture was stirred at room temperature for 1 hour. DCE was evaporated under reduced pressure. The crude mixture was purified by column chromatography C18 (30 g C18 gold column, elution): purified by 5 to 50% MeCN / H2O (0.1% formic acid) over 15 CV. The fraction of interest (eluted with 40% MeCN / H2O (0.1% formic acid)) was concentrated to dryness to obtain 11 (39 mg, 34% yield) as a yellow semi-solid.

[0315] LC-MS method 1: Retention time: 1.457 min, 98.5% purity at 215 nm, [M+H] + = 894.2. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.07 - 1.45 (m, 15 H), 1.68 - 1.92 (m, 6 H), 1.96 - 2.16 (m, 6 H), 2.36 - 2.42 (m, 2 H), 2.56 - 2.64 (m, 2 H), 3.12 (br s, 5 H), 3.45 - 3.55 (m, 1 H), 3.58 - 3.68 (m, 1 H), 3.72 (br dd, J = 10.8, 4.4 Hz, 1 H), 4.19 - 4.28 (m, 1 H), 4.52 - 4.63 (m, 1 H), 4.97 - 5.15 (m, 2 H), 6.83 - 6.95 (m, 3 H), 7.00 - 7.14 (m, 3 H), 8.14 (s, 1 H), 8.29 (s, 1 H), 8.39 (br s, 1 H), 8.82 (br d, J = 7.3 Hz, 1 H), 9.21 (br d, J = 1.0 Hz, 1 H), 10.77 (s, 1 H). 19 F NMR (377 MHz, DMSO-d6) δ ppm -184.67 (s, 1 F), -58.99 (s, 3 F).

[0316] Step 9. Preparation of 5-((3R,5R)-3-amino-5-fluoropiperidine-1-yl)-N-(1-((1r,4r)-4-(2-(4-(4-(2,6-dioxopiperidine-3-yl)phenyl)piperazine-1-yl)ethyl)cyclohexyl)-3-(trifluoromethyl)-1H-pyrazole-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide hydrochloride (P-11) A 4 M HCl solution of tert-butyl 11 (39 mg, 0.04 mmol, 1.0 eq.) and a dioxane (1.64 mL, 6.54 mmol, 150 eq.) solution were stirred at room temperature for 30 minutes. The crude mixture was purified by C18 column chromatography (30 g C18 gold column, elution: 5-40% MeCN / H2O (0.02 M in HCl) over 15 CV). The fraction of interest (eluted with 32% MeCN / H2O (0.02 M in HCl)) was concentrated to dryness and lyophilized to obtain P-11 (21.3 mg, 61% yield) as a white solid.

[0317] LC-MS method 3: Retention time: 1.917 min, 99.9% purity at 215 nm, [M-HCl+H] + = 794.4, [M-HCl+2H] 2+ = 397.8. 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.09 - 1.28 (m, 2 H), 1.37 - 1.50 (m, 1 H), 1.64 - 2.23 (m, 11 H), 2.34 - 2.48 (m, 2 H), 2.59 - 2.72 (m, 1 H), 3.03 - 3.25 (m, 6 H), 3.26 - 3.34 (m, 1 H), 3.41 - 3.67 (m, 4 H), 3.71 - 3.90 (m, 3 H), 4.25 - 4.38 (m, 1 H), 4.47 - 4.97 (m, 2 H), 5.12 (d, J = 46.0 Hz, 1 H), 6.92 (d, J = 7.8 Hz, 1 H), 6.97 (d, J = 8.8 Hz, 2 H), 7.12 (d, J = 8.6 Hz, 2 H), 8.28 - 8.53 (m, 4 H), 8.94 (d, J = 7.8 Hz, 1 H), 9.23 (s, 1 H), 10.40 (br s, 1 H), 10.79 (s, 1 H). 19F NMR (377 MHz, DMSO-d6) δ ppm -184.93 (s, 1 F), 58.81 (s, 3 F).

[0318] Example S10. Synthesis of P-21 [ka]

[0319] Step 1. Preparation of [4-[3-(difluoromethyl)-4-nitropyrazole-1-yl]cyclohexyl]methanol (2) To a 1:2 solution of methyl 4-[3-(difluoromethyl)-4-nitropyrazole-1-yl]cyclohexanecarboxylate 1 (intermediate 4 obtained from the synthesis of T-2) (730.0 mg, 2.26 mmol, 1 eq.) in THF (3.8 mL):ethanol (7.5 mL), CaCl2 (501.2 mg, 4.52 mmol, 2 eq.) and then NaBH4 (341.6 mg, 9.03 mmol, 4 eq.) were added at 0°C. The resulting mixture was stirred at rt. After 16 hours at room temperature, all conversions were shown by LC-MS. The reaction was quenched by adding water dropwise to the reaction mixture and stirring at room temperature for 1 hour. The product was then extracted three times with ELISA, the organic layers were washed once with water and once with brine, and finally dried over MgSO4. HCl was removed under reduced pressure, and the residue was purified by normal-phase flash chromatography (80 g silica column, retention solution (DCM), elution: 0% HCl / heptane over 3 CV, then 0 to 100% HCl / heptane over 10 CV, then 100% HCl / heptane over 3 CV (the target product eluted around 50% HCl)). The fractions were combined and concentrated to obtain 2 (371.9 mg, 58% yield) as a colorless oil.

[0320] LC-MS method 1: 97.5% purity at 215 nm, [M+H] + = 276.2. 1H NMR (400 MHz, CDCl3-d) δ ppm 1.14 - 1.30 (m, 3 H), 1.70 - 1.87 (m, 2 H), 1.97 - 2.09 (m, 2 H), 2.25 - 2.34 (m, 2 H), 3.54 (d, J = 6.1 Hz, 2 H), 4.18 (tt, J = 12.1, 3.9 Hz, 1 H), 7.10 (t, J = 53.3 Hz, 1 H), 8.22 (s, 1 H). 19 F NMR (377 MHz, CDCl3-d) δ ppm -117.64 (d, J = 53.13, 2 F).

[0321] Step 2. Preparation of [4-[3-(difluoromethyl)-4-nitropyrazole-1-yl]cyclohexyl]methylmethanesulfonate (3) [4-[3-(difluoromethyl)-4-nitropyrazole-1-yl]cyclohexyl]methanol 2 (428.5 mg, 1.52 mmol) and Et3N (0.28 mL, 1.98 mmol) in DCM (7.62 mL) were mixed dropwise with MsCl (0.13 mL, 1.68 mmol) at 0°C. The reaction mixture was then slowly warmed to room temperature and stirred at this temperature. After 3 hours, TLC (3:7 heptane / siRNA) indicated completion of the reaction. The reaction mixture was then partitioned with water and siRNA. The phases were separated, and the organic phase was washed three times with water, once with 1 N hydrochloric acid, and once with brine, and then dried over MgSO4. siRNA was removed under reduced pressure to obtain 3 (529 mg, 93% yield) as a pale yellow oil. The product was used in the next step without purification. LC-MS method 1: 94.8% purity at 215 nm, [M+H] + = 354.0. 1H NMR (400 MHz, CDCl3-d) δ ppm 1.22 - 1.37 (m, 3 H), 1.80 - 1.90 (m, 2 H), 2.05 - 2.13 (m, 2 H), 2.27 - 2.36 (m, 2 H), 3.04 (s, 3 H), 4.08 - 4.14 (m, 2 H), 4.15 - 4.24 (m, 1 H), 7.11 (t, J = 53.4 Hz, 1 H), 8.22 (s, 1 H). 19 F NMR (377 MHz, CDCl3-d) δ ppm -117.64 (d, J = 53.13, 2 F).

[0322] Step 3. Preparation of 2-[4-[3-(difluoromethyl)-4-nitropyrazole-1-yl]cyclohexyl]acetaldehyde (4) A solution of [4-[3-(difluoromethyl)-4-nitropyrazole-1-yl]cyclohexyl]methylmethanesulfonate 3 (529.0 mg, 1.5 mmol, 1 eq.) and NaCN (183.4 mg, 3.74 mmol, 2.5 eq.) in DMSO (7.49 mL) was heated to 50°C and stirred at this temperature. After 16 hours at 50°C, LC-MS indicated that the reaction was complete. The reaction mixture was cooled to rt and quenched with water with vigorous stirring. Then, siRNA was added and the product was extracted twice with siRNA. The combined organic layers were washed once with concentrated NaHCO3 aqueous solution and once with saline. Finally, the solution of the product was dried over MgSO4 to remove siRNA under reduced pressure, and 4 (342 mg, 79% yield) was obtained as a yellow oil.

[0323] LC-MS method 1: 98.8% purity at 215 nm, [M+H] + = 285.2. 1H NMR (400 MHz, CDCl3-d) δ ppm 1.30 - 1.46 (m, 2 H), 1.76 - 1.96 (m, 3 H), 2.06 - 2.16 (m, 2 H), 2.29 - 2.37 (m, 2 H), 2.37 - 2.40 (m, 2 H), 4.20 (tt, J = 12.0, 3.9 Hz, 1 H), 7.12 (t, J = 54.2 Hz, 1 H), 8.22 (s, 1 H). 19 F NMR (377 MHz, CDCl3-d) δ ppm -117.68 (s, 2 F).

[0324] Step 4. Preparation of 2-[4-[3-(difluoromethyl)-4-nitropyrazole-1-yl]cyclohexyl]acetaldehyde (5) To a solution of 2-[4-[3-(difluoromethyl)-4-nitropyrazole-1-yl]cyclohexyl]acetonitrile 4 (342 mg, 1.18 mmol, 1 eq.) in DCM (5.88 mL), a solution of 1 M DIBAL-H in DCM (3.53 mL, 3.53 mmol, 3 eq.) was added at -78°C. The reaction mixture was then stirred at the same temperature. After 2 hours, LC-MS indicated that the reaction was complete. The reaction was slowly quenched with Rochelle salt solution at -78°C and stirred at rt for 1 hour. The product was then extracted three times with siRNA, and the combined organic layers were washed twice with 1 M hydrochloric acid and once with saline. Finally, the solution of the product was dried over MgSO4, and siRNA was removed under reduced pressure to obtain 5 (264 mg, 78% yield) as a yellow oil.

[0325] LC-MS method 1: 99.9% purity at 215 nm, [M+H] + = 288.2. 1H NMR (400 MHz, DMSO-d6 ) δ ppm 1.36 (q, J = 6.5 Hz, 2 H), 1.75 (qd, J = 12.5, 3.2 Hz, 2 H), 1.85 (d, J = 12.5 Hz, 2 H), 2.02 - 2.15 (m, 2 H), 3.40 - 3.50 (m, 2 H), 4.19 - 4.32 (m, 1 H), 4.32 - 4.40 (m, 1 H), 7.30 (t, J = 52.3 Hz, 1 H), 9.06 (s, 1 H). 19 F NMR (377 MHz, DMSO-d6) δ ppm -117.36 (s, 2 F).

[0326] Step 5. Production of 2-[4-[3-(difluoromethyl)-4-nitropyrazole-1-yl]cyclohexyl]ethanol (6) To a solution of 2-[4-[3-(difluoromethyl)-4-nitropyrazole-1-yl]cyclohexyl]acetaldehyde 5 (264 mg, 0.92 mmol, 1 eq.) in methanol (4.59 mL), NaBH4 (69.52 mg, 1.84 mmol) was added at 0°C. The resulting mixture was stirred at room temperature. After 16 hours, LC-MS indicated that the reaction was complete. Water was added, and the mixture was stirred at room temperature for 1 hour. The product was extracted three times with siRNA, and the combined organic layers were washed twice with brine. Finally, the solution of the product was dried over MgSO4, and siRNA was removed under reduced pressure to obtain 6 (216 mg, 68% yield) as a yellow oil.

[0327] LC-MS method 1: 83.3% purity at 215 nm, [M+H] + = 290.2 1H NMR (400 MHz, DMSO-d6 ) δ ppm 1.03 - 1.15 (m, 2 H), 1.36 (q, J = 6.5 Hz, 2 H), 1.41 - 1.52 (m, 1 H), 1.69 - 1.81 (m, 2 H), 1.85 (br d, J = 12.5 Hz, 2 H), 2.02 - 2.12 (m, 2 H), 3.37 - 3.52 (m, 2 H), 4.22 - 4.32 (m, 1 H), 4.33 - 4.39 (m, 1 H), 7.30 (t, J = 53.2 Hz, 1 H), 9.06 (s, 1 H). 19 F NMR (377 MHz, DMSO-d6) δ ppm -117.36 (s, 2 F).

[0328] Step 6. Production of 2-[4-[4-amino-3-(difluoromethyl)pyrazole-1-yl]cyclohexyl]ethanol (7) 2-[4-[3-(difluoromethyl)-4-nitropyrazole-1-yl]cyclohexyl]ethanol 6 (216.1 mg, 0.75 mmol, 1 eq.) was dissolved in siRNA (3.74 mL) and bubbling with N2 for 5 minutes. Then, 10% Pd / C (238.5 mg, 0.22 mmol, 0.3 eq.) was added, and the solution was bubbling with N2 for another 5 minutes. Then, the solution was bubbling with H2 for 5 minutes, and the resulting mixture was stirred at room temperature under 1 atm of H2. After 3 hours, LC-MS indicated that the reaction was complete. The solution was filtered through a Celite pad and washed thoroughly with siRNA. Finally, the filtrate was concentrated under reduced pressure to obtain 7 (169 mg, 81% yield) as a yellow oil. This product was used in the next step without purification. LC-MS method 1: 92.9% purity at 215 nm, [M+H] + = 260.2.

[0329] Step 7. Preparation of tert-butyl N-[(3R,5R)-1-[3-[[3-(difluoromethyl)-1-[4-(2-hydroxyethyl)cyclohexyl]pyrazole-4-yl]carbamoyl]pyrazolo[1,5-a]pyrimidine-5-yl]-5-fluoro-3-piperidyl]carbamate (9) TCFH (168.6 mg, 0.60 mmol, 1.2 eq.) was added at 0°C to a solution of 2-[4-[4-amino-3-(difluoromethyl)pyrazole-1-yl]cyclohexyl]ethanol 7 (168.8 mg, 0.65 mmol, 1.3 eq.), 5-[(3R,5R)-3-(tert-butoxycarbonylamino)-5-fluoro-1-piperidyl]pyrazolo[1,5-a]pyrimidine-3-carboxylic acid 8 (intermediate 4 obtained from the synthesis of P-1) (190 mg, 0.50 mmol, 1 eq.), and NMI (0.14 mL, 1.75 mmol, 3.5 eq.) in MeCN (2.50 mL). The reaction mixture was then stirred at room temperature. After 16 hours, LC-MS indicated that the reaction was complete. MeCN was removed under reduced pressure, and the residue was purified by reverse-phase flash chromatography (30 g C18 column, holding solution (DMSO), elution: 5% MeOH / 0.1% HCOOH over 3 CV, then 5 to 100% MeOH / 0.1% HCOOH over 15 CV, then 100% MeOH / 0.1% HCOOH over 3 CV (the target product eluted at approximately 80% MeCN)). The fractions were combined and concentrated to obtain 9 (155.6 mg, 50% yield) as an off-white solid.

[0330] LC-MS method 1: 99.9% purity at 215 nm, [M+H] + = 621.2 1H NMR (400 MHz, DMSO-d6 ) δ ppm 1.04 - 1.17 (m, 3 H), 1.29 - 1.44 (m, 12 H), 1.65 - 1.77 (m, 3 H), 1.80 - 1.93 (m, 3 H), 1.97 - 2.07 (m, 2 H), 2.10 - 2.18 (m, 1 H), 2.94 - 3.14 (m, 1 H), 3.36 - 3.53 (m, 3 H), 3.61 - 3.72 (m, 1 H), 4.11 - 4.23 (m, 1 H), 4.31 - 4.40 (m, 1 H), 4.94 - 5.13 (m, 1 H), 6.83 - 6.90 (m, 1 H), 6.91 - 7.23 (m, 2 H), 8.28 (s, 1 H), 8.33 (br s, 1 H), 8.82 (d, J = 8.1 Hz, 1 H), 9.31 (br s, 1 H). 19 F NMR (377 MHz, DMSO-d6) δ ppm -184.27 - -183.87 (m, 1 F), -112.01 - -110.10 (m, 2 F).

[0331] Step 8. Preparation of tert-butyl N-[(3R,5R)-1-[3-[[3-(difluoromethyl)-1-[4-[2-[4-[4-(2,6-dioxo-3-piperidyl)phenyl]piperazine-1-yl]ethyl]cyclohexyl]pyrazole-4-yl]carbamoyl]pyrazolo[1,5-a]pyrimidine-5-yl]-5-fluoro-3-piperidyl]carbamate (10) To a solution of tert-butyl N-[(3R,5R)-1-[3-[[3-(difluoromethyl)-1-[4-(2-hydroxyethyl)cyclohexyl]pyrazole-4-yl]carbamoyl]pyrazolo[1,5-a]pyrimidine-5-yl]-5-fluoro-3-piperidyl]carbamate 9 (140 mg, 0.23 mmol, 1 eq.) in dried DMSO (2.2 mL), IBX (75.8 mg, 0.27 mmol, 1.2 eq.) was added. The resulting mixture was stirred at room temperature. After 16 hours, LC-MS showed that all of the product had been converted to aldehydes. To a solution of 3-(4-piperazine-1-ylphenyl)piperidine-2,6-dione dihydrochloride C-6 (43.0 mg, 0.12 mmol, 1.1 eq.) in DCE (1.0 mL), DIPEA (196 μL, 1.13 mmol, 10 eq.) was added. The mixture was stirred at room temperature for 10 minutes, and then the aldehyde solution was added. After stirring for another 10 minutes, NaBH(OAc)3 (31.1 mg, 0.15 mmol, 1.3 eq.) was added. The resulting mixture was then stirred at room temperature. After 1 hour, LC-MS indicated that the reaction was complete. DCE was removed under reduced pressure, and the residue was purified by reverse-phase flash chromatography (50 g C18 column, holding solution (DMSO), elution: 5% MeCN / 0.1% HCOOH over 3 CV, then 5 to 35% MeCN / 0.1% HCOOH over 15 CV, then 35 to 100% MeCN / 0.1% HCOOH over 1 CV, then 100% MeCN / 0.1% HCOOH over 3 CV (the target product eluted at approximately 30% MeCN)). The fractions were combined and concentrated to obtain 10 (86 mg, 87% yield) as a white solid. The product was used in the next step without purification. LC-MS method 1: 99.9% purity at 215 nm, [M+H] + = 876.4

[0332] Step 9. Preparation of N-[3-(difluoromethyl)-1-[4-[2-[4-[4-(2,6-dioxo-3-piperidyl)phenyl]piperazine-1-yl]ethyl]cyclohexyl]pyrazole-4-yl]-5-[(3R,5R)-3-amino-5-fluoro-1-piperidyl]pyrazolo[1,5-a]pyrimidine-3-carboxamide formate (P-21) tert-butyl N-[(3R,5R)-1-[3-[[3-(difluoromethyl)-1-[4-[2-[4-[4-(2,6-dioxo-3-piperidyl)phenyl]piperazine-1-yl]ethyl]cyclohexyl]pyrazole-4-yl]carbamoyl]pyrazolo[1,5-a]pyrimidine-5-yl]-5-fluoro-3-piperidyl]carbamate 10 (86 mg, 0.10 mmol, 1 eq.) was dissolved in 4 M HCl / 1,4-dioxane (3.68 mL, 14.73 mmol, 150 eq.). The mixture was stirred at room temperature. After 5 hours, LC-MS indicated that the reaction was complete. Volatile substances were evaporated under vacuum. The resulting residue was purified by reverse-phase flash chromatography (30 g C18 RediSep Rf Gold column, holding solution (water), elution: 5% MeCN / 0.02 M HCl over 5 CV, then 5 to 20% MeCN / 0.02 M HCl over 15 CV, then 20% MeCN / 0.02 M HCl over 5 CV, then 20 to 100% MeCN / 0.02 M HCl over 2 CV, then 100% MeCN / 0.02 M HCl over 7 CV). The fractions were combined, concentrated, and lyophilized to obtain P-21 (24.1 mg, 31% yield) as a white solid formate.

[0333] LC-MS method 3: 98.7% purity at 215 nm, [M-HCOOH+2H] 2+ = 388.7;[M-HCOOH+H] + = 776.5. 1H NMR (400 MHz, DMSO-d6 ) δ ppm 1.02 - 1.24 (m, 2 H), 1.32 - 1.49 (m, 3 H), 1.54 - 1.81 (m, 3 H), 1.83 - 1.94 (m, 2 H), 1.97 - 2.25 (m, 5 H), 2.30 - 2.40 (m, 2 H), 2.41 - 2.49 (m, 2 H), 2.58 - 2.70 (m, 1 H), 2.86 - 2.97 (m, 1 H), 2.99 - 3.07 (m, 1 H), 3.08 - 3.16 (m, 4 H), 3.21 - 3.54 (m, 4 H), 3.72 (dd, J = 10.9, 5.0 Hz, 1 H), 4.13 - 4.24 (m, 1 H), 4.26 - 4.88 (m, 2 H), 5.01 (d, J = 47.0 Hz, 1 H), 6.80 - 6.96 (m, 3 H), 6.98 - 7.31 (m, 3 H), 8.20 (s, 1 H), 8.27 (s, 1 H), 8.38 (s, 1 H), 8.81 (d, J = 7.8 Hz, 1 H), 9.33 (s, 1 H), 10.77 (s, 1 H). 19 F NMR (377 MHz, DMSO-d6) δ ppm -182.92 (s, 1 F), -111.03 (s, 2 F).

[0334] Example S11. Synthesis of P-23 [ka]

[0335] Step 8. Preparation of tert-butyl N-[(3R,5R)-1-[3-[[3-(difluoromethyl)-1-[4-[2-[2-[4-(2,6-dioxo-3-piperidyl)phenyl]-2,7-diazaspiro[3.5]nonane-7-yl]ethyl]cyclohexyl]pyrazole-4-yl]carbamoyl]pyrazolo[1,5-a]pyrimidine-5-yl]-5-fluoro-3-piperidyl]carbamate (2) Preparation of Solution A: To a solution of tert-butyl N-[(3R,5R)-1-[3-[[3-(difluoromethyl)-1-[4-(2-hydroxyethyl)cyclohexyl]pyrazole-4-yl]carbamoyl]pyrazolo[1,5-a]pyrimidine-5-yl]-5-fluoro-3-piperidyl]carbamate 1 (intermediate 9 obtained from the synthesis of P-21) (60 mg, 0.097 mmol, 1 eq.) in anhydrous DMSO (0.6 mL), IBX (33 mg, 0.116 mmol, 1.2 eq.) was added. The resulting mixture was stirred at room temperature. After 18 hours, LC-MS demonstrated complete conversion to tert-butyl N-[(3R,5R)-1-[3-[[3-(difluoromethyl)-1-[4-(2-oxoethyl)cyclohexyl]pyrazole-4-yl]carbamoyl]pyrazolo[1,5-a]pyrimidine-5-yl]-5-fluoro-3-piperidyl]carbamate.

[0336] To a solution of C-14 (65 mg, 0.160 mmol, 1.6 eq.) of 3-[4-(2,7-diazaspiro[3,5]nonan-2-yl)phenyl]piperidine-2,6-dione sulfate, DIPEA (0.17 mL, 0.970 mmol, 10 eq.) was added in DCE (2 mL). The reaction mixture was stirred at room temperature for 10 minutes, after which solution A was added. After 10 minutes, NaBH(OAc)3 (26.7 mg, 0.130 mmol, 1.3 eq.) was added, and the reaction mixture was stirred at room temperature. After 72 hours, complete conversion was shown by LC-MS. Volatile substances were removed under reduced pressure, and the residue was purified by reverse-phase flash chromatography (30 g C18 gold column, holding solution (DMSO), elution: 5% MeCN / 0.1% HCOOH over 3 CV, then 5 to 35% MeCN / 0.1% HCOOH over 8 CV, then 35% MeCN / 0.1% HCOOH over 3 CV, then 35 to 100% MeCN / 0.1% HCOOH over 6 CV). The fractions were combined and concentrated to obtain 2 (46.3 mg, 52% yield) as a white solid.

[0337] LC-MS method 1: 99.0% purity at 215 nm, [M+H] + = 917.4. 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.07 - 1.17 (m, 2 H), 1.28 - 1.43 (m, 13 H), 1.69 - 1.79 (m, 7 H), 1.80 - 1.91 (m, 3 H), 1.95 - 2.17 (m, 7 H), 2.28 - 2.40 (m, 6 H), 2.57 - 2.65 (m, 1 H), 3.51 (s, 4 H), 3.69 (br dd, J = 11.0, 4.9 Hz, 2 H), 4.13 - 4.22 (m, 1 H), 4.95 - 5.12 (m, 1 H), 6.38 (d, J = 8.6 Hz, 2 H), 6.89 (br d, J = 7.8 Hz, 1 H), 6.99 (d, J = 8.6 Hz, 2 H), 7.03 - 7.14 (m, 1 H), 8.16 (s, 1 H), 8.28 (s, 1 H), 8.33 (br s, 1 H), 8.82 (d, J = 8.1 Hz, 1 H), 9.31 (br s, 1 H), 10.74 (s, 1 H).

[0338] Step 9. Preparation of N-[3-(difluoromethyl)-1-[4-[2-[2-[4-(2,6-dioxo-3-piperidyl)phenyl]-2,7-diazaspiro[3,5]nonane-7-yl]ethyl]cyclohexyl]pyrazole-4-yl]-5-[(3R,5R)-3-amino-5-fluoro-1-piperidyl]pyrazolo[1,5-a]pyrimidine-3-carboxamide formate (P-23) A 4 M HCl solution of tert-butyl N-[(3R,5R)-1-[3-[[3-(difluoromethyl)-1-[4-[2-[2-[4-(2,6-dioxo-3-piperidyl)phenyl]-2,7-diazaspiro[3.5]nonane-7-yl]ethyl]cyclohexyl]pyrazole-4-yl]carbamoyl]pyrazolo[1,5-a]pyrimidine-5-yl]-5-fluoro-3-piperidyl]carbamate 6 (46.3 mg, 0.051 mmol, 1 eq.) in a 1,4-dioxane (4 mL, 16.000 mmol, 310 eq.) solution was stirred at room temperature. After 18 hours, complete conversion was shown by HPLC. The solvent was removed under reduced pressure, and the residue was purified by reverse-phase flash chromatography (30 g C18 gold column, holding solution (H2O), elution: 5% MeCN / 0.02 M HCl over 3 CV, then 5 to 20% MeCN / 0.02 M HCl over 15 CV). The fractions were combined and concentrated. The residue was purified by preparative HPLC (0.1% formic acid / MeCN / H2O). The fractions were combined, concentrated, and lyophilized to obtain P-23 (18.5 mg, 44% yield) as a white solid formate.

[0339] LC-MS method 3: 98.7% purity at 215 nm, [M-HCOOH+H] + = 816.5. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.04 - 1.20 (m, 2 H), 1.29 - 1.43 (m, 3 H), 1.56 - 1.78 (m, 6 H), 1.82 - 1.93 (m, 2 H), 1.94 - 2.24 (m, 5 H), 2.28 - 2.41 (m, 5 H), 2.41 - 2.48 (m, 1 H), 2.55 - 2.70 (m, 1 H), 2.85 - 2.98 (m, 1 H), 3.03 - 3.10 (m, 1 H), 3.30 - 3.42 (m, 5 H), 3.48 - 3.52 (m, 4 H), 3.69 (dd, J = 10.9, 5.0 Hz, 1 H), 4.13 - 4.24 (m, 1 H), 4.29 - 4.55 (m, 1 H), 4.60 - 4.85 (m, 1 H), 5.01 (d, J = 48.0 Hz, 1 H), 6.38 (d, J = 8.6 Hz, 2 H), 6.92 (d, J = 8.1 Hz, 1 H), 6.95 - 7.31 (m, 3 H), 8.19 (s, 1 H), 8.28 (s, 1 H), 8.37 (s, 1 H), 8.81 (d, J = 8.1 Hz, 1 H), 9.33 (s, 1 H), 10.74 (s, 1 H). 19 F NMR (377 MHz, DMSO-d6) δ ppm -183.46 (s, 1 F), -110.98 (s, 2 F).

[0340] Example S12. Synthesis of P-24

change

[0341] Step 8. Preparation of tert-butyl N-[(3R,5R)-1-[3-[[3-(difluoromethyl)-1-[4-[2-[4-[3-(2,6-dioxo-3-piperidyl)anilino]-1-piperidyl]ethyl]cyclohexyl]pyrazole-4-yl]carbamoyl]pyrazolo[1,5-a]pyrimidine-5-yl]-5-fluoro-3-piperidyl]carbamate (2) IBX (37.9 mg, 0.135 mmol, 1.2 equiv) was added to tert-butyl N-[(3R,5R)-1-[3-[[3-(difluoromethyl)-1-[4-(2-hydroxyethyl)cyclohexyl]pyrazole-4-yl]carbamoyl]pyrazolo[1,5-a]pyrimidine-5-yl]-5-fluoro-3-piperidyl]carbamate 1 (intermediate 9 obtained from the synthesis of P-21) (70.0 mg, 0.113 mmol, 1.0 equiv) in DMSO (1 mL). The reaction mixture was then stirred overnight at room temperature under a nitrogen atmosphere. DIPEA (196 μL, 1.13 mmol, 10.0 equiv) was added to a suspension of 3-[3-(piperidine-1-ium-4-ylamino)phenyl]piperidine-2,6-dione bisulfate C-5 (47.8 mg, 0.124 mmol, 1.1 equiv) in DCE (1 mL), and the mixture was stirred for 10 minutes under a nitrogen atmosphere. Then, a DMSO solution containing the pre-prepared aldehyde (oxidized 7) (69.7 mg, 0.113 mmol, 1.0 equiv) was added to the reaction mixture, and stirring was continued for a further 30 minutes. Finally, sodium triacetoxyborohydride (31.0 mg, 0.146 mmol, 1.3 equiv) was added all at once. The reaction mixture was stirred overnight at room temperature under a nitrogen atmosphere. The volatile substance was evaporated under reduced pressure, and the remaining DMSO solution was directly injected into a reverse-phase flash chromatography column (5% (3 CV) → 50% MeCN in 0.1% HCOOH (aqueous solution), 30g RediSep Rf Gold® C18Aq, 20 CV, λ = 214~254 nm, 36~41% MeCN for product). The fraction was co-evaporated with MeCN to obtain 2 (86 mg, 0.072 mmol, 64% yield) as a white solid.

[0342] LC-MS method 1: 75.0% purity at 215 nm, [M+H] + = 890.6, [M+2H] 2+ = 446.0. 1H NMR (400 MHz, DMSO-d6) δ ppm 0.96 - 1.20 (m, 4 H), 1.34 (br s, 9 H), 1.39 - 1.44 (m, 2 H), 1.72 - 1.87 (m, 8 H), 1.95 - 2.07 (m, 4 H), 2.09 - 2.22 (m, 2 H), 2.42 - 2.47 (m, 3 H), 2.58 - 2.72 (m, 1 H), 2.98 - 3.09 (m, 1 H), 3.40 (br d, J = 13.7 Hz, 2 H), 3.52 (s, 4 H), 3.66 (br d, J = 3.7 Hz, 1 H), 3.73 (br dd. H), 6.31 (br d, J = 7.8 Hz, 1 H), 6.49 (d, J = 7.5 Hz, 1 H), 6.83 - 6.96 (m, 1 H), 7.02 - 7.25 (m, 3 H), 8.28 (s, 1 H), 8.33 (br s, 1 H), 8.82 (d, J = 7.8 Hz, 1 H), 9.31 (br s, 1 H), 10.79 (s, 1 H). 19 F NMR (377 MHz, DMSO-d6) δ ppm -186.03 - -182.66 (m, 1 F), -114.14 - -108.43 (m, 2 F).

[0343] Step 9. Preparation of 5-((3R,5R)-3-amino-5-fluoropiperidine-1-yl)-N-(3-(difluoromethyl)-1-((1r,4r)-4-(2-(4-((3-(2,6-dioxopiperidine-3-yl)phenyl)amino)piperidine-1-yl)ethyl)cyclohexyl)-1H-pyrazole-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide dihydrochloride (P-24) 4.0 M HCl / 1,4-dioxane (2.62 mL, 10.5 mmol, 150 equiv) was added to tert-butyl N-[(3R,5R)-1-[3-[[3-(difluoromethyl)-1-[4-[2-[4-[3-(2,6-dioxo-3-piperidyl)anilino]-1-piperidyl]ethyl]cyclohexyl]pyrazole-4-yl]carbamoyl]pyrazolo[1,5-a]pyrimidine-5-yl]-5-fluoro-3-piperidyl]carbamate 2 (83.0 mg, 0.070 mmol, 1.0 equiv), and the mixture was sonicated for 30 minutes and then stirred for 1 hour. The solvent was evaporated to dryness, and the residue was purified by reverse-phase flash chromatography (5% (3 CV) → 50% MeCN in 0.02 M HCl(aq), 50 g RediSep Rf Gold® C18Aq, 15 CV, λ = 214~254 nm, 24~30% MeCN, product, broadband) to obtain a non-pure product (69 mg). After preparative HPLC purification, a pure product was obtained (MeCN / HCl(aq)): the fraction containing the product was evaporated to dryness, then co-evaporated with water (3 × 10 mL) to completely remove any trace of residual HCl, and the residue was freeze-dried overnight to obtain P-24 (36.38 mg, 0.0458 mmol, 65% yield) as a white solid dihydrochloride.

[0344] LC-MS method 3: 99.4% purity at 215 nm, [M-2HCl+H] + = 790.4, [M-2HCl+2H] 2+ = 395.7. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.05 - 1.27 (m, 2 H), 1.31 - 1.52 (m, 1 H), 1.56 - 1.70 (m, 2 H), 1.70 - 1.82 (m, 2 H), 1.82 - 1.99 (m, 5 H), 2.00 - 2.25 (m, 7 H), 2.34 - 2.47 (m, 1 H), 2.59 - 2.76 (m, 1 H), 2.85 - 3.19 (m, 4 H), 3.20 - 3.43 (m, 4 H), 3.44 - 3.66 (m, 3 H), 3.66 - 3.93 (m, 1 H), 4.15 - 4.32 (m, 1 H), 4.55 - 4.74 (m, 1 H), 4.77 - 4.97 (m, 1 H), 5.10 (d, J = 45.8 Hz, 1 H), 6.52 - 6.97 (m, 4 H), 6.99 - 7.31 (m, 2 H), 8.27 - 8.40 (m, 2 H), 8.41 - 8.64 (m, 3 H), 8.93 (d, J = 7.8 Hz, 1 H), 9.33 (s, 1 H), 10.36 - 10.70 (m, 1 H), 10.76 - 10.93 (m, 1 H). 19 F NMR (377 MHz, DMSO-d6) δ ppm -184.60 (s, 1 F), -111.27 (s, 2 F).

[0345] The following compounds were synthesized using the same general route, with a modification to CBM(CX) in step 8 (Table 7). [Table 23] [Table 24] [Table 25] [Table 26]

[0346] Method 4 for final product Example S13. Synthesis of P-27 [ka]

[0347] Step 1. Preparation of tert-butyl N-[2-[4-[4-(2,6-dioxo-3-piperidyl)phenyl]piperazine-1-yl]ethyl]carbamate (3) C-6 (1.0 g, 2.89 mmol, 1.0 equiv) of 3-(4-piperazine-1,4-diium-1-ylphenyl)piperidine-2,6-dione dichloride was added to DCM (25 mL), then to triethylamine (2.42 mL, 17.3 mmol, 6.0 equiv), and the mixture was stirred until a clear solution was obtained. tert-butyl N-(2-oxoethyl)carbamate 2 (532.3 mg, 3.18 mmol, 1.1 equiv) was added, and the reaction mixture was stirred at room temperature for 30 minutes. Then, sodium triacetoxyborohydride (757.2 mg, 3.47 mmol, 1.2 equiv) was added all at once, and stirring was continued under a nitrogen atmosphere for 24 hours. tert-butyl N-(2-oxoethyl)carbamate 2 (532.3 mg, 3.18 mmol, 1.1 equiv) and a second sodium triacetoxyborohydride (757.2 mg, 3.47 mmol, 1.2 equiv) were added to the reaction mixture, and stirring was continued for a further 24 hours under a nitrogen atmosphere. The reaction mixture was quenched with water (25 mL) to separate the phases, and the aqueous phase was extracted with DCM (3 × 25 mL). The organic layer was washed with brine (25 mL), dried over MgSO4, and evaporated to dryness. The crude product was dry-loaded onto silica and purified by normal-phase flash chromatography (DCM / MeOH, 100:0~90:10, 120 g cartridge, 15 CV, λ= 254~280 nm, product at 5~8% MeOH) to obtain product 3 (the product also contains a hemiaminal adduct of the product and the aldehyde starting material, which is suitable as a starting material for the next step; 1.37 g, 2.63 mmol, 91% yield) as a white solid. LC-MS method 1: 80.1% purity at 215 nm, [M+H] + = 417.4 (hemiaminal by-product: [M+H] + = 576.4). 1H NMR (400 MHz, DMSO-d6) δ ppm 1.04 (d, J = 6.0 Hz, 1 H), 1.37 (s, 9 H), 1.93 - 2.06 (m, 1 H), 2.06 - 2.20 (m, 1 H), 2.36 (br t, J = 6.6 Hz, 2 H), 2.42 - 2.48 (m, 1 H), 2.52 - 2.55 (m, 2 H), 2.55 - 2.72 (m, 2 H), 2.95 - 3.21 (m, 6 H), 3.72 (br dd, J = 10.9, 4.8 Hz, 1 H), 6.55 - 6.75 (m, 1 H), 6.88 (br d, J = 8.7 Hz, 2 H), 7.04 (br d, J = 8.6 Hz, 2 H), 10.77 (s, 1 H).

[0348] Step 2. Preparation of 2-[4-[4-(2,6-dioxo-3-piperidyl)phenyl]piperazine-1-ium-1-yl]ethylammonium dichloride (4) 4.0 M HCl / 1,4-dioxane (24.1 mL, 96.3 mmol, 50.0 equiv) was added to tert-butyl N-[2-[4-[4-(2,6-dioxo-3-piperidyl)phenyl]piperazine-1-yl]ethyl]carbamate 3 (1.37 g, 2.63 mmol, 1.0 equiv), and the mixture was stirred for 1 hour. The volatile substances were evaporated under reduced pressure to obtain the crude product as an off-white solid. The crude product was dissolved in the minimum amount of water and purified by reverse-phase flash chromatography (MeCN / 0.02 M HCl(aq) from 0% (5 CV) to 30%, 120 g Claricep (trademark registered) Spherical AQ C18 Column, 15 CV, λ = 214~254 nm, first broadband at 5-9% MeCN and second sharp band at 9-11% MeCN, both containing only the target product). After evaporating the fraction to dryness, the residue was co-evaporated with water (3 × 25 mL) to remove all residual HCl, and the mixture was freeze-dried to isolate 4 (1.13 g, 2.45 mmol, 93% yield) as a brown solid.

[0349] LC-MS method 4: 84.3% purity at 215 nm, [M-2HCl+H] + = 317.2. 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.94 - 2.04 (m, 1 H), 2.07 - 2.22 (m, 1 H), 2.40 - 2.49 (m, 1 H), 2.57 - 2.72 (m, 1 H), 3.20 (br s, 4 H), 3.31 - 3.39 (m, 3 H), 3.41 - 3.51 (m, 3 H), 3.67 (br s, 3 H), 6.98 (d, J = 8.8 Hz, 2 H), 7.11 (d, J = 8.7 Hz, 2 H), 8.49 (br s, 3 H), 10.78 (s, 1 H), 11.37 (br s, 1 H).

[0350] Step 3. Preparation of 1-cyclopenta-3-en-1-yl-3-(difluoromethyl)-4-nitropyrazole (7) 3-(difluoromethyl)-4-nitro-1H-pyrazole 5 (750.0 mg, 4.60 mmol, 1.0 equiv), cyclopenta-3-en-1-ol 6 (464.2 mg, 5.52 mmol, 1.2 equiv), and triphenylphosphine (1.81 g, 6.90 mmol, 1.5 equiv) were dissolved in THF (9 mL). The mixture was cooled to 0°C, and then diisopropyl azodicarboxylate (1.35 mL, 6.90 mmol, 1.5 equiv) was added dropwise. The reaction mixture was stirred at 0°C for 1 hour, and then stirred overnight at room temperature under a nitrogen atmosphere. The volatile substance was evaporated under reduced pressure, and the residue was purified by normal-phase flash chromatography (heptane / siRNA, 100:0~75:25, 80 g RediSep Rf Gold® Normal-Phase Silica, 15 CV, λ = 254~280 nm, 8 to 9% siRNA for undesirable minor positional isomers, 17 to 21% siRNA for the desired major positional isomer), yielding 7 (755 mg, 3.23 mmol, 70% yield) as an orange oily substance (minor positional isomers were not isolated). LC-MS method 1: 98.0% purity at 215 nm, [M+H] + = 230.1. 1 H NMR (400 MHz, DMSO-d6) δ ppm 2.71 (dd, J = 15.6, 4.5 Hz, 2H), 2.91 (dd, J = 15.7, 8.4 Hz, 2H), 5.19 (tt, J = 8.4, 4.5 Hz, 1H), 5.78 (m, 2H), 7.30 (t, J = 52.9 Hz, 1H), 9.03 (s, 1H). 19 F NMR (377 MHz, DMSO-d6) δ ppm -117.35 (d, J = 52.9 Hz, 2 F).

[0351] Step 4. Preparation of (1s,2r)-4-[3-(difluoromethyl)-4-nitropyrazole-1-yl]cyclopentan-1,2-diol (8) Osmium tetroxide 4% w / w / H2O (205 μL, 0.032 mmol, 0.01 equiv) was added to a solution of 1-cyclopenta-3-en-1-yl-3-(difluoromethyl)-4-nitropyrazole 7 (755.0 mg, 3.23 mmol, 1.0 equiv) and NMO (467.9 mg, 3.87 mmol, 1.2 equiv) in THF (5 mL) and tert-butanol (10 mL). The reaction mixture was then stirred at room temperature for 3 hours. The mixture was evaporated to dryness under reduced pressure, and the residue was dissolved in DCM, dry-loaded with silica, and purified by normal-phase flash chromatography (DCM / MeOH, 100:0 to 90:10, 80 g RediSep Rf Gold® Normal-Phase Silica, 15 CV, λ = 254~280 nm, 6~7% MeOH to obtain the product), yielding 8 (620 mg, 2.35 mmol, 73% yield) as an orange oily substance.

[0352] LC-MS method 1: 99.9% purity at 215 nm, [M+H] + = 264.2. 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.97 - 2.09 (m, 2H), 2.10 - 2.20 (m, 2H), 4.09 - 4.18 (m, 2H), 4.70 (d, J = 4.2 Hz, 2H), 5.06 (tt, J = 8.8, 6.2 Hz, 1H), 7.30 (t, J = 53.0 Hz, 1H), 9.08 (s, 1H). 19 F NMR (377 MHz, DMSO-d6) δ ppm -117.34 (d, J = 53.0 Hz, 2 F).

[0353] Step 5. Preparation of 3-[3-(difluoromethyl)-4-nitropyrazole-1-yl]pentanedial (9) Sodium periodate (604.0 mg, 2.82 mmol, 1.2 equiv) was added to a solution of (1s,2r)-4-[3-(difluoromethyl)-4-nitropyrazole-1-yl]cyclopentan-1,2-diol 8 (620.0 mg, 2.35 mmol, 1.0 equiv) / THF (8 mL) and water (4 mL), and the reaction mixture was stirred overnight. After evaporating the solvent to dryness, the residue was taken into DCM (15 mL) and dried over MgSO4 overnight to dehydrate the product. The reaction mixture was filtered, and the volatile substances were evaporated to dryness under reduced pressure to obtain 9 (20:80 dialdehyde / cyclic monohydrate; 598 mg, 2.17 mmol, 92% yield) as a yellow viscous solid. LC-MS method 1: 99.9% purity at 215 nm, [M+H] + = 262.1. 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.81 - 2.46 (m, 2 H), 3.03 - 3.26 (m, 2 H), 4.59 - 5.15 (m, 1 H), 5.19 - 5.69 (m, 1 H), 7.08 - 7.56 (m, 1 H), 8.97 - 9.30 (m, 1 H), 9.59 (s, 1 H). 19 F NMR (377 MHz, DMSO-d6) δ ppm -117.84 - -117.23 (m, 2 F).

[0354] Step 6. Preparation of 3-[4-[4-[2-[4-[3-(difluoromethyl)-4-nitropyrazole-1-yl]piperidine-1-ium-1-yl]ethyl]piperazine-4-ium-1-yl]phenyl]piperidine-2,6-dione diformate (10) 2-[4-[4-(2,6-dioxo-3-piperidyl)phenyl]piperazine-1-ium-1-yl]ethylammonium dichloride 4 (729.9 mg, 1.63 mmol, 1.0 equiv) was suspended in DCE (30 mL) under a nitrogen atmosphere, and then DIPEA (2.27 mL, 13.0 mmol, 8.0 equiv) was added. The mixture was sonicated for 20 minutes to form a suspension, promoting the partial dissolution of the starting material. 3-[3-(difluoromethyl)-4-nitropyrazole-1-yl]pentanedial 9 (80% monohydrate, 540 mg, 1.96 mmol, 1.2 equiv) was added, and the reaction mixture was stirred for 10 minutes. Sodium triacetoxyborohydride (1.04 g, 4.89 mmol, 3.0 equiv) was added all at once, and the reaction mixture was stirred overnight under a nitrogen atmosphere. Another sodium triacetoxyborohydride (1.04 g, 4.89 mmol, 3.0 equiv) was added to the reaction mixture, and this was stirred overnight under a nitrogen atmosphere. The reaction mixture was quenched with water (30 mL) and extracted with DCM (3 × 30 mL). The combined organic layers were dried over MgSO4 and concentrated under reduced pressure. The crude product was purified by reverse-phase flash chromatography (5% (3 CV) → 30% MeCN in 0.1% HCOOH(aq), 150 g RediSep Rf Gold® C18Aq, 20 CV, λ = 214~254 nm, 18~21% MeCN). After evaporation and lyophilization of the fraction, 10 (540 mg, 0.787 mmol, 48% yield) was obtained as an orange solid diformate.

[0355] LC-MS method 1: 92.6% purity at 215 nm, [M-2HCOOH+H] + = 546.2, [M-2HCOOH+2H] 2+ = 273.6. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.74 - 2.28 (m, 10 H), 2.52 - 2.82 (m, 8 H), 3.04 (br s, 3 H), 3.46 - 4.55 (m, 2 H), 4.58 - 6.11 (m, 1 H), 6.60 - 7.12 (m, 4 H), 7.14 - 7.62 (m, 1 H), 8.16 (br s, 2 H), 9.08 (br s, 1 H), 10.77 (br s, 1 H). 19 F NMR (377 MHz, DMSO-d6) δ ppm -117.44 (br d, J = 53.1 Hz, 2 F).

[0356] Step 7. Preparation of 3-[4-[4-[2-[4-[4-amino-3-(difluoromethyl)pyrazole-1-yl]-1-piperidyl]ethyl]piperazine-1-yl]phenyl]piperidine-2,6-dione (11) 3-[4-[4-[2-[4-[3-(difluoromethyl)-4-nitropyrazole-1-yl]piperidine-1-ium-1-yl]ethyl]piperazine-4-ium-1-yl]phenyl]piperidine-2,6-dione diformate 10 (493.0 mg, 0.772 mmol, 1.0 equiv) was dissolved in ethyl acetate (40 mL), and the solution was then placed in a hydrogenation reactor and degassed by nitrogen bubbling for 20 minutes. After adding Pd / C 5% w / w (164.4 mg, 0.077 mmol, 0.1 equiv), the mixture was further degassed by nitrogen bubbling for 20 minutes. The reactor was pressurized with hydrogen at 50 psi, and the reaction mixture was stirred at room temperature for 72 hours. The reaction mixture was filtered through Celite to remove the catalyst, and the Celite was thoroughly washed with 1:1 MeOH / DCM (5 × 20 mL). The volatile substances were evaporated to obtain 11 (391 mg, 0.758 mmol, 98% yield) as an orange solid.

[0357] LC-MS method 1: 99.9% purity at 215 nm, [M+H] += 516.2, [M+2H] 2+ = 258.8. 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.76 - 1.96 (m, 4 H), 1.97 - 2.18 (m, 4 H), 2.40 - 2.47 (m, 2 H), 2.47 (s, 2 H), 2.53 - 2.58 (m, 4 H), 2.58 - 2.71 (m, 1 H), 2.96 (br d, J = 11.6 Hz, 2 H), 3.04 - 3.15 (m, 4 H), 3.17 (d, J = 4.8 Hz, 1 H), 3.72 (dd, J = 11.0, 4.9 Hz, 1 H), 3.89 - 4.18 (m, 3 H), 6.72 - 7.02 (m, 3 H), 7.04 (br d, J = 8.7 Hz, 2 H), 7.17 (s, 1 H), 10.77 (s, 1 H). 19 F NMR (377 MHz, DMSO-d6) δ ppm -111.35 (d, J = 54.5 Hz, 2 F).

[0358] Step 8. Preparation of tert-butyl N-[(3R,5R)-1-[3-[[3-(difluoromethyl)-1-[1-[2-[4-[4-(2,6-dioxo-3-piperidyl)phenyl]piperazine-1-yl]ethyl]-4-piperidyl]pyrazole-4-yl]carbamoyl]pyrazolo[1,5-a]pyrimidine-5-yl]-5-fluoro-3-piperidyl]carbamate (13) TCFH (32.6 mg, 0.116 mmol, 1.2 equiv) was added to a portion of a MeCN (1.9 mL) solution of 5-[(3R,5R)-3-(tert-butoxycarbonylamino)-5-fluoro-1-piperidyl]pyrazolo[1,5-a]pyrimidine-3-carboxylic acid 12 (intermediate 4 obtained from the synthesis of P-1) (36.8 mg, 0.097 mmol, 1.0 equiv), 3-[4-[4-[2-[4-[4-amino-3-(difluoromethyl)pyrazole-1-yl]-1-piperidyl]ethyl]piperazin-1-yl]phenyl]piperidine-2,6-dione 11 (50.0 mg, 0.097 mmol, 1.0 equiv), and NMI (27 μL, 0.339 mmol, 3.5 equiv). Subsequently, the reaction mixture was stirred overnight at room temperature. The volatile substances were evaporated under reduced pressure, and the crude product was then dissolved in DMSO and injected directly into the column for reverse-phase flash column chromatography purification (5% (3 CV) → 50% MeCN in 0.1% HCOOH(aq), 50 g RediSep Rf Gold® C18Aq, 20 CV, λ = 214~254 nm, 30~35% MeCN for product). The fraction was co-evaporated with MeCN to obtain 13 (45 mg, 0.051 mmol, 53% yield) as a white solid.

[0359] LC-MS method 1: 99.9% purity at 215 nm, [Mt-Bu+H] + = 821.0, [M+2H] 2+ = 439.2, [Mt-Bu+2H] 2+ = 411.2. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.34 (br s, 9 H), 1.70 - 2.05 (m, 7 H), 2.05 - 2.26 (m, 5 H), 2.55 - 2.70 (m, 6 H), 3.01 (br d, J = 10.4 Hz, 4 H), 3.11 (br s, 4 H), 3.58 - 3.78 (m, 4 H), 4.06 - 4.44 (m, 2 H), 4.47 - 4.91 (m, 1 H), 5.03 (d, J = 46.2 Hz, 1 H), 6.89 (br d, J = 8.1 Hz, 3 H), 6.98 - 7.33 (m, 4 H), 8.29 (s, 1 H), 8.35 (br s, 1 H), 8.81 (br d, J = 7.7 Hz, 1 H), 9.31 (br s, 1 H), 10.76 (s, 1 H). 19 F NMR (377 MHz, DMSO-d6) δ ppm -186.27 - -181.60 (m, 1 F), -114.92 - -107.65 (m, 2 F).

[0360] Step 9. Preparation of 5-((3R,5R)-3-amino-5-fluoropiperidine-1-yl)-N-(3-(difluoromethyl)-1-(1-(2-(4-(4-(2,6-dioxopiperidine-3-yl)phenyl)piperazine-1-yl)ethyl)piperidine-4-yl)-1H-pyrazole-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide trihydrochloride (P-27) 4.0 M HCl / 1,4-dioxane (1.84 mL, 7.35 mmol, 150 equiv) was added to tert-butyl N-[(3R,5R)-1-[3-[[3-(difluoromethyl)-1-[1-[2-[4-[4-(2,6-dioxo-3-piperidyl)phenyl]piperazine-1-yl]ethyl]-4-piperidyl]pyrazole-4-yl]carbamoyl]pyrazolo[1,5-a]pyrimidine-5-yl]-5-fluoro-3-piperidyl]carbamate 13 (43.0 mg, 0.049 mmol, 1.0 equiv), and the mixture was sonicated for 30 minutes and then stirred for 1 hour. The solvent was evaporated to dryness, and the residue was purified by reverse-phase flash chromatography (5% (3 CV) → 30% MeCN in 0.02 M HCl(aq), 50 g RediSep Rf Gold® C18Aq, 20 CV, λ = 214~254 nm, 17~20% MeCN, product, broadband). The fraction containing the product was evaporated to dryness, and then co-evaporated with water (3 × 10 mL) to completely remove any trace of residual HCl. The residue was freeze-dried overnight to obtain P-27 (17.29 mg, 0.021 mmol, 44% yield) as a white solid trihydrochloride.

[0361] LC-MS method 3: 95.9% purity at 215 nm, [M-3HCl+H] + = 777.5, [M-3HCl+2H] 2+ = 389.3. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.88 - 2.04 (m, 2 H), 2.05 - 2.23 (m, 2 H), 2.27 - 2.39 (m, 4 H), 2.39 - 2.43 (m, 1 H), 2.44 - 2.47 (m, 1 H), 2.59 - 2.72 (m, 1 H), 3.06 - 3.27 (m, 6 H), 3.27 - 3.44 (m, 4 H), 3.44 - 3.51 (m, 2 H), 3.67 - 3.72 (m, 2 H), 3.74 - 3.80 (m, 2 H), 3.84 - 3.96 (m, 3 H), 4.47 - 4.74 (m, 2 H), 4.75 - 5.00 (m, 1 H), 5.10 (d, J = 46.5 Hz, 1 H), 6.90 (d, J = 7.9 Hz, 1 H), 7.00 (d, J = 8.6 Hz, 2 H), 7.05 - 7.41 (m, 3 H), 8.16 - 8.42 (m, 4 H), 8.47 (s, 1 H), 8.94 (d, J = 7.8 Hz, 1 H), 9.37 (s, 1 H), 10.54 - 11.05 (m, 3 H). 19 F NMR (377 MHz, DMSO-d6) δ ppm -184.62 (s, 1 F), -113.10 - -109.99 (m, 2 F).

[0362] Table 27

[0363] examples of biology Example B1. BIOCHEM method of biochemistry of IRAK4 PhosphoSens The PhosphoSens® biochemical assay was performed according to the distributor's instructions (AssayQuant Technologies Inc., Marlborough, MA). A 1.25 X stock solution of IRAK4 (PV4002, ThermoFisher Scientific, Waltham, MA), a 5 X stock solution of ATP, and the Sox-conjugated peptide substrate AQT0326 (CSKS-AQT0326B, AssayQuant Technologies) were prepared in 1X kinase reaction buffer [containing 50 mM HEPES (pH 7.5), 0.01% Brij-35, 0.5 mM EGTA, 20 mM MgCl2, and 1 mM DTT]. To obtain final concentrations of 200 μM ATP and 10 μM peptide substrate, a mixture of ATP (10 μL) and substrate solution was added to a Corning 3574 384-well white unbound surface microtiter plate (containing 0.5 μL of serially diluted test compounds prepared in DMSO). The enzyme solution (40 μL) with a final IRAK4 concentration of 1 nM was added to initiate the reaction, and the λ was measured every 71 seconds for 240 minutes at room temperature using a BioTek Synergy H4 plate reader. EX 360 / λ EM Monitoring was performed using 485 (Agilent Technologies, Santa Clara, CA). The initial linear portion of the progression curve was fitted according to a linear equation to determine the slope, and converted to % inhibition relative to the 100% activity value of the control that was not treated with the inhibitor. IC of each compound 50 The values ​​were obtained by fitting a % inhibitor-compound concentration curve using Dotmatics software (Dotmatics, Bishops Stortford, Hertfordshire, England).

[0364] Example B2. Reagent preparation Cell culture media were prepared in a sterile tissue culture hood by adding 10% FBS and 1% penicillin-streptomycin to 500 mL of RPMI1640 medium (without phenol red). The medium was filtered through a Nalgene Bottle Top Filter and stored at 4°C.

[0365] Cell titer Glo (CTG) buffer and substrate (CellTiter-Glo Luminescent Cell Viability Assay, Promega Ref. # G7573) were stored at -20°C. 100 mL of CTG buffer was warmed in a bead bath and added to a bottle containing the CTG substrate in a tissue culture hood. The solution was mixed homogenously using a pipette. The CTG reagent was dispensed into 15 mL Falcon tubes and stored at -20°C.

[0366] For a homogeneous time-resolved fluorescence (HTRF) assay, the Cisbio HTRF kit was used, containing the following: Resolved buffer #1 4X, Blocking reagent #3 100X, 20X antibody 1 (anti-IRAK4 d2), 20X antibody 2 (Anti-IRAK4 k), and detection buffer.

[0367] The 4X decomposition buffer was stored at 4°C. To use as a 1X decomposition buffer, the 4X solution was diluted with deionized water (distilled water, Gibco Cat. # 15230279) and 100X blocking reagent in a volume ratio of 1:3:0.04.

[0368] Aliquots of 20X antibody solution were stored at -80°C, and the detection buffer was stored at 4°C. To use as a 1X antibody solution, aliquots of 20X antibody solution were diluted with detection buffer at a volume ratio of 1:19.

[0369] Example B3. Procedure for homogeneous time-resolved fluorescence (ALE THP1 HTRF) of advanced fatty acid oxidation end product THP1. Cells were lysed with shaking at room temperature for 45 minutes. A BCA protein assay was performed and normalized to the target total protein concentration using 1X lysis buffer. Next, a 1X antibody solution was prepared by adding 380 μL of detection buffer to an aliquot of 20 μL of 20X antibody solution and mixing thoroughly. The 1X antibody solutions were combined in a 1:1 ratio and vortexed briefly. 20 μL of 1X anti-IRAK4-k antibody solution was maintained as the control well. 384-well plates (ProxiPlate-384 Plus, Perkin Elmer Cat.# 6008289) were loaded by adding 4 μL of the mixed antibody solution to empty wells using a single-channel repeater. Using a multi-channel repeater, 16 μL of decomposition solution was added per well, and all formed foam was defoamed using a 20 μL pipette tip and the end of a Kimwipe. Each control was prepared in 3-row configuration in 10 columns. Buffer controls were prepared in wells A10, B10, and C10 by adding 16 μL of degradation buffer and 4 μL of detection buffer. Cryptate controls were prepared in wells D10, E10, and F10 by adding 16 μL of degradation buffer, 2 μL of detection buffer, and 2 μL of 1 X anti-IRAK4-k antibody solution. Negative controls were prepared in wells G10, H10, and I10 by adding 16 μL of degradation buffer and 4 μL of mixed antibody solution. The plate was sealed with a clear seal and covered with an aluminum lid. The plate was centrifuged at 800 g for 5 minutes and incubated overnight in the dark at room temperature. The next day, the plate was centrifuged at 800 g for 5 minutes. Samples were analyzed using a plate reader (Envision, PerkinElmer) with the Desnor 384 HTRF program.

[0370] A summary of the ALE THP1 HTRF data for the tested compounds is shown in Table 9 below. [Table 28]

[0371] Although the present invention is described in some detail by examples and embodiments for the purpose of clarity of understanding, these descriptions and embodiments should not be construed as limiting the scope of the invention. All disclosures of patent and scientific documents cited herein are expressly incorporated herein in their entirety by reference.

Claims

1. Equation (I): 【Chemistry 1】 [In the formula, R 1 C 1 -C 6 It is a haloalkyl; W is either CH or N; X 1 is either CH or N; X 2 CH 2 , CH, C, N, or NH; Y 1 and Y 2 These are independently CH or N; L 1 is -C(O)N(H)-, -C(O)- or C 1 -C 6 is alkylene; L 2 The bonds are -C(O)- and -NR. 2 -, -CH 2 N(R 2 )-,-CH 2 -or 【Chemistry 2】 And; R 2 is H or C 1 -C 6 It is alkyl; x is either 0 or 1; * is L 2 The connection points are shown for and ** indicates a bond site to the piperidinyl-2,6-dione group. Compounds of or pharmaceutically acceptable salts thereof.

2. The compound is of formula (IIa), (IIb), or (IIc): 【Transformation 3】 The compound according to claim 1 or a pharmaceutically acceptable salt thereof.

3. R 1 However, C 1 -C 3 A compound according to claim 1 or 2, which is a haloalkyl compound, or a pharmaceutically acceptable salt thereof.

4. R 1 However, -CHF 2 or -CF 3 The compound according to claim 3 or a pharmaceutically acceptable salt thereof.

5. A compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, wherein W is CH.

6. X 1 A compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, wherein the compound is CH.

7. X 1 A compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, wherein N is present.

8. Y 1 A compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof, wherein the compound is CH.

9. Y 2 A compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, wherein the compound is CH.

10. L 1 However, -C(O)N(H)-, -C(O)- or C 1 -C 3 A compound according to any one of claims 1 to 9, which is an alkylene, or a pharmaceutically acceptable salt thereof.

11. L 1 However, -C(O)N(H)-, -C(O)-, -CH 2 - or -CH 2 CH 2 - The compound according to claim 10 or a pharmaceutically acceptable salt thereof.

12. L 2 However, the bond is -C(O)-, -NR 2 -, -CH 2 N(R 2 )-,-CH 2 -or 【Chemistry 4】 and R 2 However, H or C 1 -C 3 A compound according to any one of claims 1 to 11, which is alkyl, or a pharmaceutically acceptable salt thereof.

13. R 2 However, H or -CH 3 The compound according to claim 12 or a pharmaceutically acceptable salt thereof. 【Request Item 14】 【Chemistry 5】 but, 【Transformation 6】 A compound according to any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof. 【Request Item 15】 【Chemistry 7】 but, 【Transformation 8】 A compound according to any one of claims 1 to 14 or a pharmaceutically acceptable salt thereof. 【Request Item 16】 【Chemistry 9】 but, 【Chemistry 10】 A compound according to any one of claims 1 to 15 or a pharmaceutically acceptable salt thereof.

17. A compound and its pharmaceutically acceptable salt, selected from the compounds and their pharmaceutically acceptable salts listed in Table 1.

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

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

20. A method for treating an inflammatory disease or autoimmune disease in a subject requiring treatment, characterized by administering an effective amount of a compound according to any one of claims 1 to 17 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 18, to the subject, wherein the inflammatory disease or autoimmune disease may optionally be atopic dermatitis, asthma, lupus, rheumatoid arthritis, familial Mediterranean fever, psoriasis, generalized pustular psoriasis, cryopyrin-associated periodic syndromes, hidradenitis suppurativa, Beckett syndrome, or familial cold autoinflammatory syndrome.

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