Compounds targeting degradation of irak4 and uses thereof

Abstract

The present disclosure relates to compounds targeting degradation of IRAK4 and uses thereof, and specifically provides a compound of Formula (I) or a pharmaceutically acceptable salt thereof, which has the substituents and structural features described herein. The present disclosure also describes pharmaceutical compositions comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof, and uses of the compounds of Formula (I) or a pharmaceutically acceptable salt thereof in medicine.

Description

[0001] Cross-references to related applications

[0002] This application claims priority and benefits from the following patent application, the entire contents of which are incorporated herein by reference:

[0003] Chinese invention patent application number 202510197215.5, filed with the State Intellectual Property Office on February 21, 2025. Technical Field

[0004] This disclosure relates to compounds that target and degrade IRAK4, or pharmaceutically acceptable salts thereof, methods of their preparation, pharmaceutical compositions containing such compounds or pharmaceutically acceptable salts thereof, and the use of such compounds or pharmaceutically acceptable salts thereof in the prevention or treatment of IRAK4-mediated diseases or conditions. Background Technology

[0005] Interleukin-1 receptor kinase 4 (IRAK4) is a serine / threonine protein kinase and a core regulator of the innate immune response, playing a crucial role in activating the immune system. Upon binding to its ligands, the IL-1 receptor and Toll-like receptor are activated, subsequently recruiting the intracellular myeloid differentiation factor MyD88. MyD88 further recruits IRAK4 through its N-terminal death domain. IRAK4, after autophosphorylation, recruits and activates IRAK1 and IRAK2, forming the MyD88-IRAK4-IRAK1 / 2 complex. This complex transmits signals downstream to the E3 ubiquitin ligase TNF receptor-associated factor (TRAF6), activating the serine / threonine kinase TAK1, further activating the NF-κB and MAPK signaling pathways, leading to the release of various inflammatory cytokines and proliferation-related factors. Studies have shown that excessive IRAK4 activation is associated with various autoimmune diseases, such as atopic dermatitis, hidradenitis suppurativa, and rheumatoid arthritis. However, IRAK4 deficiency is not fatal, and in adulthood, the risk of bacterial infection is reduced, similar to that of healthy individuals.

[0006] Furthermore, the scaffold function of IRAK4 can also regulate downstream signaling pathways, independent of its kinase function. Studies have shown that mouse macrophages lacking IRAK4 kinase function can still be induced to activate the NF-κB signaling pathway; IRAK4 kinase inhibitors cannot block the IRAK4-mediated NF-κB signaling pathway in human monocytes, suggesting that this may be related to the insufficient efficacy of IRAK4 kinase inhibitors in clinical trials.

[0007] Protein degradation-targeting chimeras (PROTACs) are bifunctional molecules. One end is a small molecule inhibitor that recognizes the target protein, connected by a linker. The other end is an E3 ubiquitin ligase ligand that recognizes E3 ubiquitin ligase, forming a ternary complex. After ubiquitination of the target protein, it is degraded in vivo via the ubiquitin-proteasome pathway. Traditional IRAK4 small molecule inhibitors can only block the kinase activity of IRAK4, while IRAK4 PROTACs can degrade intracellular IRAK4, achieving the biological activity of simultaneously blocking IRAK4 kinase activity and scaffold function, thus more effectively inhibiting the release of downstream inflammatory factors mediated by IRAK4. Therefore, it is necessary to develop novel IRAK4 PROTAC drugs for the treatment of IRAK4-related diseases or conditions. Summary of the Invention

[0008] This disclosure relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0009]

[0010] in:

[0011] R 1 Selected from one or more R a The following groups are substituted: pyridinyl, pyridoneyl, or ;

[0012] R 3 The elements are selected from H, deuterium, halogen, CN, OH, or C1-C4 alkyl, wherein the OH or C1-C4 alkyl group is optionally converted by one or more R. c replace;

[0013] R a Selected from halogens, CN, =O, OH, C1-C4 alkyl, C3-C6 cycloalkyl, 4-7 membered heterocyclic groups, or phenyl groups, wherein the OH, C1-C4 alkyl, C3-C6 cycloalkyl, 4-7 membered heterocyclic groups, or phenyl groups are optionally converted by one or more R groups. 1a replace;

[0014] R 1a R c The groups are independently selected from deuterium, halogens, OH, CN, NH2, =O, C1-C6 alkyl, C3-C6 cycloalkyl, or 4-8 membered heterocyclic groups, wherein the OH, NH2, C1-C6 alkyl, C3-C6 cycloalkyl, or 4-8 membered heterocyclic group is optionally surrounded by one or more R d replace;

[0015] R dSelected from deuterium, halogen, OH, NH2, =O, C1-C3 alkyl, C1-C3 alkoxy, CN, COOH, C(O)(C1-C3 alkyl), CONH2, C(O)O(C1-C3 alkyl), C3-C6 cycloalkyl or 4-6 membered heterocyclic groups;

[0016] The Linker is a connection unit, and its structure is as follows: ,in Representative and Connected keys;

[0017] X 1 X 2 They are selected independently from the bond, -O-, -N(R) 10 )-, -C(O)- or C1-C6 alkylene, R 10 It is H or C1-C6 alkyl;

[0018] Het 1 Het 2 The components are independently selected from bonds, C3-C8 cycloalkyl groups or 4-10 heterocyclic groups, wherein the C3-C8 cycloalkyl groups or 4-10 heterocyclic groups are optionally substituted with halogens or C1-C6 alkyl groups;

[0019] The DIM is ;where R 2 It is selected from hydrogen, halogen, NO2, NH2, OH, COOH, C1-C6 alkyl or C1-C6 alkoxy.

[0020] In some implementation schemes, R 1 Selected from one or more R a The following groups are substituted: pyridone group or .

[0021] In some implementation schemes, R 1 Selected from one or more R a Substituted pyridinone group.

[0022] In some implementation schemes, R a Selected from CN, =O, C1-C4 alkyl, C3-C6 cycloalkyl, or 4-7 membered heterocyclic groups, wherein the C1-C4 alkyl, C3-C6 cycloalkyl, or 4-7 membered heterocyclic group is optionally surrounded by one or more R groups. 1a replace.

[0023] In some implementation schemes, R a Selected from CN, C1-C4 alkyl or cyclopropyl, wherein the C1-C4 alkyl or cyclopropyl group is optionally converted by one or more R 1a replace.

[0024] In some implementation schemes, R1a R c The components are independently selected from deuterium, halogens, OH, CN, NH2, =O, or C1-C6 alkyl groups, wherein the OH, NH2, or C1-C6 alkyl group is optionally converted by one or more R groups. d replace.

[0025] In some implementation schemes, R 1a Selected from halogens, OH, NH2, or C1-C6 alkyl groups, wherein the OH, NH2, or C1-C6 alkyl group is optionally converted by one or more R... d replace.

[0026] In some implementation schemes, R d It is selected from halogens, C1-C3 alkyl, C1-C3 alkoxy, C(O)(C1-C3 alkyl), CONH2 or C(O)O(C1-C3 alkyl).

[0027] In some implementation schemes, R 1a It is a halogen. In some implementations, R 1a It is F.

[0028] In some implementation schemes, R a Selected from methyl, Or CN.

[0029] In some implementation schemes, R 1 Selected from , , or .

[0030] In some implementation schemes, R 1 for .

[0031] In some implementation schemes, R 3 Selected from H, halogen, OH, or C1-C4 alkyl, wherein the OH or C1-C4 alkyl is optionally converted by one or more R c replace.

[0032] In some implementation schemes, R 3 Selected from H or arbitrarily selected by one R c Substituted OH.

[0033] In some implementation schemes, R c Selected from halogens or optionally subjected to one or more R d Substituted C1-C6 alkyl groups.

[0034] In some implementation schemes, R c It is isopropyl.

[0035] In some implementation schemes, R3 It is isopropoxy.

[0036] In some implementation schemes, X 1 X 2 They are independently selected from bonds, -C(O)- or C1-C3 alkylene groups.

[0037] In some implementation schemes, X 1 X 2 They are independently selected from bonds, -C(O)- or methylene.

[0038] In some implementation schemes, X 1 X 2 They are selected independently from the bond or methylene group.

[0039] In some implementation schemes, X 1 Selected from -C(O)- or methylene.

[0040] In some implementation schemes, X 1 It is a methylene group.

[0041] In some implementation schemes, X 2 For key.

[0042] In some implementations, Het 1 Het 2 The components are independently selected from C3-C6 cycloalkyl or 4-8 heterocyclic groups, wherein the C3-C6 cycloalkyl or 4-8 heterocyclic groups are optionally substituted with halogens or C1-C6 alkyl groups.

[0043] In some implementations, Het 1 Het 2 The groups are independently selected from 4-8 membered heterocyclic groups, which are optionally substituted with halogens or C1-C6 alkyl groups.

[0044] In some implementations, Het 1 Het 2 Each of the following groups is independently selected from piperazine, piperidinyl, aziridine, or alkyl groups. The piperazine-based, piperidinyl, azacyclic butyl or Optionally substituted with halogens or C1-C6 alkyl groups.

[0045] In some implementations, Het 1 Het 2 Each is independently selected from piperazine, piperidinyl, or... .

[0046] In some implementations, Het 1 Selected from piperazine or .

[0047] In some implementations, Het 2 It is piperidinyl.

[0048] In some implementations, the Linker structure is ,in Representative and Connected keys.

[0049] In some implementations, the Linker is selected from... or ,in Representative and Connected keys.

[0050] In some implementation schemes, R 2 It is selected from halogens, NO2, NH2, OH, COOH, C1-C6 alkyl or C1-C6 alkoxy groups.

[0051] In some implementation schemes, R 2 It is selected from hydrogen, halogen, C1-C6 alkyl or C1-C6 alkoxy.

[0052] In some implementation schemes, R 2 It is selected from halogens, C1-C6 alkyl groups, or C1-C6 alkoxy groups.

[0053] In some implementation schemes, R 2 Selected from H, F or methoxy.

[0054] In some implementation schemes, R 2 Selected from F or methoxy.

[0055] In some implementations, the DIM is selected from the following structures:

[0056] , , , , or .

[0057] In some implementations, the DIM is .

[0058] In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from the compounds of formula (I-1) or pharmaceutically acceptable salts thereof:

[0059]

[0060] (I-1)

[0061] Among them, Ra Linker and DIM are defined above.

[0062] In some embodiments, the compounds of formula (I) of this disclosure or pharmaceutically acceptable salts thereof are selected from the following compounds or pharmaceutically acceptable salts thereof:

[0063]

[0064]

[0065]

[0066]

[0067]

[0068] or .

[0069] On the other hand, this disclosure provides pharmaceutical compositions comprising a compound represented by formula (I) or formula (I-1) of this disclosure or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable excipients.

[0070] On the other hand, this disclosure provides a method for treating diseases mediated by IRAK4 in mammals, including administering to a mammal, preferably a human, a therapeutically effective amount of a compound represented by formula (I) or formula (I-1) of this disclosure, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0071] On the other hand, this disclosure provides methods for treating tumors, inflammatory diseases, neurodegenerative diseases or autoimmune diseases in mammals, including administering to a mammal, preferably a human, a therapeutically effective amount of a compound of formula (I) or formula (I-1) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0072] On the other hand, this disclosure provides the use of compounds of formula (I) or formula (I-1) or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, in the preparation of medicaments for the prevention or treatment of IRAK4-mediated diseases.

[0073] On the other hand, this disclosure provides the use of compounds of formula (I) or formula (I-1) or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, in the preparation of medicaments for the prevention or treatment of tumors, inflammatory diseases, neurodegenerative diseases or autoimmune diseases.

[0074] On the other hand, this disclosure provides the use of compounds of formula (I) or formula (I-1) or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, in the prevention or treatment of IRAK4-mediated diseases.

[0075] On the other hand, this disclosure provides the use of compounds of formula (I) or formula (I-1) or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, in the prevention or treatment of tumors, inflammatory diseases, neurodegenerative diseases or autoimmune diseases.

[0076] On the other hand, this disclosure provides compounds of formula (I) or formula (I-1) for the prevention or treatment of IRAK4-mediated diseases, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof.

[0077] On the other hand, this disclosure provides compounds of formula (I) or formula (I-1) or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, for the prevention or treatment of tumors, inflammatory diseases, neurodegenerative diseases or autoimmune diseases.

[0078] In some implementations, IRAK4-mediated diseases are selected from tumors, inflammatory diseases, neurodegenerative diseases, or autoimmune diseases.

[0079] Terminology Definitions and Explanations

[0080] Unless otherwise stated, the terms used in this disclosure have the following meanings: the definitions of groups and terms recorded in this disclosure, including definitions as examples, exemplary definitions, preferred definitions, definitions recorded in tables, definitions of specific compounds in the examples, etc., can be arbitrarily combined and combined with each other. A particular term should not be considered uncertain or unclear unless specifically defined, but should be understood in accordance with its ordinary meaning in the art. When trade names appear herein, they are intended to refer to the corresponding product or its active ingredient.

[0081] In this article, " "" indicates a connection point.

[0082] The term "capable of binding" refers to the ability to bind to a target in a measurable manner (e.g., the ligand of an E3 ubiquitin ligase can form a covalent bond with the cysteine ​​residue of the E3 ubiquitin ligase, etc.).

[0083] The term "ubiquitin ligase" refers to a family of proteins that facilitate the transfer of ubiquitin to specific substrate proteins for targeted degradation. E3 ubiquitin ligases, alone or in combination with E2 ubiquitin ligases, are responsible for transferring ubiquitin to target proteins. Typically, ubiquitin ligases participate in polyubiquitination, where a second ubiquitin is linked to a first ubiquitin; a third ubiquitin to a second ubiquitin, and so on. Polyubiquitination labels the protein for degradation by the proteasome. However, there are also ubiquitination events limited to monoubiquitination, where the ubiquitin ligase adds only a single ubiquitin to the substrate molecule. Monoubiquitinated proteins do not target the proteasome for degradation but can instead alter their cellular location or function, for example, by binding to other proteins with domains capable of binding ubiquitin. Further complicating matters, E3 ubiquitin ligases can target different lysine residues on ubiquitin to create chains.

[0084] The term "target protein" refers to proteins and peptides that have any biological function or activity, including structural, regulatory, hormonal, enzymatic, genetic, immune, contractile, storage, transport, and signal transduction. In some embodiments, target protein refers to a protein or polypeptide that binds to a compound of this disclosure and is degradable.

[0085] The term "tautomer" refers to a functional group isomer resulting from the rapid movement of an atom between two positions within a molecule. The compounds of this disclosure can exhibit tautomerism. Tautomers can exist in two or more interconvertible forms. Tautomers generally exist in equilibrium, and attempts to isolate a single tautomer typically yield a mixture whose physicochemical properties are consistent with those of the mixture of compounds. The equilibrium position depends on the intramolecular chemical characteristics. For example, in many aliphatic aldehydes and ketones such as acetaldehyde, the ketone form is dominant; while in phenols, the enol form is dominant. This disclosure encompasses all tautomeric forms of the compounds.

[0086] The term "stereoisomer" refers to isomers that are produced by different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers and diastereomers.

[0087] The compounds disclosed herein may have asymmetric atoms such as carbon, sulfur, nitrogen, and phosphorus atoms, or asymmetric double bonds. Therefore, the compounds disclosed herein may exist in specific geometric or stereoisomeric forms. Specific geometric or stereoisomeric forms may be cis and trans isomers, E- and Z-type geometric isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)- isomers, (L)- isomers, and racemic mixtures thereof or other mixtures, such as mixtures enriched with enantiomers or diastereomers. All such isomers and mixtures thereof are within the scope of the definition of the compounds disclosed herein. Alkyl groups and other substituents may contain additional asymmetric carbon atoms, asymmetric sulfur atoms, asymmetric nitrogen atoms, or asymmetric phosphorus atoms. All such isomers involved in all substituents and mixtures thereof are also included within the scope of the definition of the compounds disclosed herein. The compounds containing asymmetric atoms disclosed herein can be isolated in optically active pure form or in racemic form. The optically active pure form can be separated from racemic mixtures or synthesized using chiral starting materials or chiral reagents.

[0088] The term "substituted" refers to the substitution of one or more hydrogen atoms on a particular atom by a substituent, which can include deuterium and hydrogen variants, provided that the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is oxo (i.e., =O), it means that two hydrogen atoms are substituted; oxo substitution does not occur on the aromatic group.

[0089] The terms “optional” or “optionally” mean that the event or condition subsequently described may or may not occur, including both the occurrence and non-occurrence of said event or condition. For example, “optionally” substituted with a halogen means that the ethyl group can be unsubstituted (CH2CH3), monosubstituted (CH2CH2F, CH2CH2Cl, etc.), polysubstituted (CHFCH2F, CH2CHF2, CHFCH2Cl, CH2CHCl2, etc.), or fully substituted (CF2CF3, CF2CCl3, CCl2CCl3, etc.). Those skilled in the art will understand that for any group containing one or more substituents, no substitution or substitution pattern that is spatially impossible and / or cannot be synthesized is introduced.

[0090] The term "optionally substituted" means that it may or may not be substituted, unless otherwise specified, and the type and number of substituents may be arbitrary on the basis of chemical feasibility.

[0091] When any variable (e.g., R) a R bWhen a group appears more than once in the composition or structure of a compound, its definition is independent in each case. For example, if a group is surrounded by two R... b Replaced, then each R b Each has its own independent options; for group N(C1-C6 alkyl)2, when C1-C6 alkyl is R b When substituted, the two C1-C6 alkyl groups have independent R groups. b Options.

[0092] When one of the variables is selected as a chemical bond or does not exist, it means that the two groups it is connected to are directly connected. For example, when L in ALZ represents a bond, it means that the structure is actually AZ.

[0093] If the linking group mentioned in this article does not specify its linking direction, then its linking direction is arbitrary. For example, when the structural unit... L in 1 When selected from "C1-C3 alkylene-O", L 1 Both loops Q and R can be connected in a left-to-right direction. 1 Composed of "cyclo-Q-C1-C3 alkylene-OR" 1 Alternatively, rings Q and R can be connected from right to left. 1 Composed of "cyclo-QO-C1-C3 alkylene-R" 1 ".

[0094] C in this article m -C n It refers to having an integer number of carbon atoms, either mn or in the range m to n. For example, "C1-C 10 "" means that the group can have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. Similarly, "m-membered" to "n-membered" indicates that the number of ring atoms is m to n. For example, 5-14-membered rings include 5-membered, 6-membered, 7-membered, 8-membered, 9-membered, 10-membered, 11-membered, 12-membered, 13-membered, and 14-membered rings. It also includes any range from n to m. For example, 5-14-membered rings include 6-14-membered, 6-11-membered, 5-10-membered, 6-10-membered, and 6-8-membered rings.

[0095] The term "alkyl" refers to a compound with the general formula C10. n H 2n+1 The hydrocarbon group is a straight-chain or branched group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12) carbon atoms, more preferably an alkyl group containing 1 to 6 carbon atoms. The term "C1-C" is used. 10"Alkyl" should be understood to mean a straight-chain or branched saturated monovalent hydrocarbon group having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. Specific examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl, etc.; the term "C1-C6 alkyl" is also relevant. The term "C1-C4 alkyl" can be understood as referring to a straight-chain or branched saturated monovalent hydrocarbon group having 1 to 4 carbon atoms. Specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, neopentyl, hexyl, 2-methylpentyl, etc. The term "C1-C3 alkyl" can be understood as referring to a straight-chain or branched saturated monovalent hydrocarbon group having 1 to 3 carbon atoms. 10 "alkyl" can include the range of "C1-C6 alkyl", "C1-C4 alkyl" or "C1-C3 alkyl", and "C1-C6 alkyl" can further include "C1-C4 alkyl" or "C1-C3 alkyl", and "C1-C4 alkyl" can further include "C1-C3 alkyl".

[0096] The term "alkylene" refers to a saturated straight-chain or branched aliphatic hydrocarbon group having two residues derived from the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of a parent alkane. It is a straight-chain or branched group containing 1 to 20 carbon atoms, preferably alkylene containing 1 to 12 carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12), and more preferably alkylene containing 1 to 6 carbon atoms. Non-limiting examples of alkylene include, but are not limited to, methylene, -CH(CH3)-, -CH2CH2-, -CH(CH2CH3)-, -CH2CH(CH3)-, -CH2CH2CH2-, -CH2CH2CH2CH2-, etc. The term "C1-C6 alkylene" can be understood to refer to an alkylene having 1 to 6 carbon atoms. The term "C1-C3 alkylene" can be understood to refer to an alkylene having 1 to 3 carbon atoms. Preferably, “C1-C6 alkylene” may include “C1-C3 alkylene”. The term “heteroalkylene” refers to an alkylene in which one or more -CH2- atoms are replaced by heteroatoms selected from N, O and S; wherein the alkylene is as defined above.

[0097] The term "alkoxy" refers to a monovalent group formed by the loss of a hydrogen atom from a hydroxyl group in straight-chain or branched alcohols; it can be understood as "alkyloxy" or "alkyl-O-", where alkyl is defined as described above. The term "C1-C"... 10 "Alkoxy" can be understood as "C1-C" 10 "alkyloxy" or "C1-C" 10 Alkyl-O-"; the term "C1-C6 alkoxy" can be understood as "C1-C6 alkyloxy" or "C1-C6 alkyl-O-". The "C1-C" 10 "Alkoxy" can include the range of "C1-C6 alkoxy" and "C1-C3 alkoxy", and the "C1-C6 alkoxy" can further include "C1-C3 alkoxy".

[0098] The term "cycloalkyl" refers to a fully saturated carbon ring existing in the form of a monocyclic, fused, bridged, or spirocyclic ring. Unless otherwise indicated, the carbon ring is typically a 3- to 10-membered ring. The term "C3-C" is also used. 10 "Cycloalkyl" should be understood as referring to a saturated monovalent monocyclic, fused, spirocyclic, or bridged ring having 3 to 10 carbon atoms. The term "C3-C8 cycloalkyl" should be understood as referring to a saturated monovalent monocyclic, fused, spirocyclic, or bridged ring having 3 to 8 carbon atoms. The term "C3-C6 cycloalkyl" should be understood as referring to a saturated monovalent monocyclic, fused, spirocyclic, or bridged ring having 3 to 6 carbon atoms, specific examples including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. The term “C5-C9 cycloalkyl” should be understood as referring to a saturated monovalent monocyclic, fused, spirocyclic, or bridged ring having 5 to 9 carbon atoms. The term “C5-C7 cycloalkyl” should be understood as referring to a saturated monovalent monocyclic, fused, spirocyclic, or bridged ring having 5 to 7 carbon atoms. Specific examples of the cycloalkyl group include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, norbornyl (bicyclo[2.2.1]heptyl), bicyclo[2.2.2]octyl, adamantyl, spiro[4.5]decyl, etc. The term “C3-C 10 "Cycloalkyl" can include "C3-C8 cycloalkyl", "C3-C6 cycloalkyl", "C5-C9 cycloalkyl" or "C5-C7 cycloalkyl", the term "C3-C8 cycloalkyl" can include "C3-C6 cycloalkyl" or "C5-C7 cycloalkyl", and the term "C5-C9 cycloalkyl" can include "C5-C7 cycloalkyl".

[0099] The term "heterocyclic group" refers to a fully saturated or partially saturated (not aromatic as a whole) monovalent monocyclic, fused, spirocyclic, or bridged ring group containing 1 to 5 heteroatoms or heterogroups (i.e., groups containing heteroatoms) in its ring atoms. The "heteroatoms or heterogroups" include, but are not limited to, nitrogen (N), oxygen (O), sulfur (S), phosphorus (P), boron (B), -S(=O)2-, -S(=O)-, and optionally substituted -NH-, -S(=O)(=NH)-, -C(=O)NH-, -C(=NH)-, -S(=O)2NH-, S(=O)NH-, or -NHC(=O)NH-, etc., which typically contain 3 to 20 ring atoms. The term "4-14 membered heterocyclic group" refers to a heterocyclic group with 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 ring atoms, and whose ring atoms contain 1 to 5 independent heteroatoms or heterogroups selected from those described above. "4-14 membered heterocyclic group" can include "6-14 membered heterocyclic group", "6-11 membered heterocyclic group", "6-10 membered heterocyclic group", "6-8 membered heterocyclic group", "4-10 membered heterocyclic group", "4-8 membered heterocyclic group", "4-7 membered heterocyclic group", "4-6 membered heterocyclic group", "5-10 membered heterocyclic group", "5-9 membered heterocyclic group", "5-8 membered heterocyclic group", or "5-7 membered heterocyclic group". The term "5-10 membered heterocyclic group" can include "5-9 membered heterocyclic group," "5-8 membered heterocyclic group," "5-7 membered heterocyclic group," "6-10 membered heterocyclic group," or "6-8 membered heterocyclic group." The term "4-10 membered heterocyclic group"... This refers to heterocyclic groups with 4, 5, 6, 7, 8, 9, or 10 ring atoms, and whose ring atoms contain 1 to 5 independently selected heteroatoms or heteroatomic groups as described above. "4-10 membered heterocyclic groups" include "4-8 membered heterocyclic groups," "4-7 membered heterocyclic groups," or "4-6 membered heterocyclic groups." Specific examples of 4-membered heterocyclic groups include, but are not limited to, nitrogen-containing heterocyclic butyl or oxocyclic butyl; specific examples of 5-membered heterocyclic groups include, but are not limited to, tetrahydrofuranyl, dioxacyclopentenyl, pyrrolyl, imidazoyl, pyrazolyl, pyrrolinyl, 4,5-dihydrooxazolyl, or 2,5-dihydro-1H-pyrrolyl; specific examples of 6-membered heterocyclic groups include, but are not limited to, tetrahydropyrrolyl... The heterocyclic group may be uranyl, piperidinyl, morpholinyl, dithiaalkyl, thiomorpholinyl, piperazinyl, trithiaalkyl, tetrahydropyridinyl, or 4H-[1,3,4]thiadiazinyl; specific examples of 7-membered heterocyclic groups include, but are not limited to, diazacycloheptyl. The heterocyclic group may also be a bicyclic group, wherein specific examples of 5,5-membered bicyclic groups include, but are not limited to, hexahydrocyclopentano[c]pyrrolo-2(1H)-yl; specific examples of 5,6-membered bicyclic groups include, but are not limited to, hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl, 5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazinyl, or 5,6,7,8-tetrahydroimidazo[1,5-a]pyrazinyl.Optionally, the heterocyclic group can be a benzofused cyclic group of the above-mentioned 4-7 membered heterocyclic groups, and specific examples include, but are not limited to, dihydroisoquinolinyl. "4-10 membered heterocyclic group" can include "5-10 membered heterocyclic group", "5-9 membered heterocyclic group", "5-8 membered heterocyclic group", "5-7 membered heterocyclic group", "5-6 membered heterocyclic group", "6-10 membered heterocyclic group", "6-8 membered heterocyclic group", "4-8 membered heterocyclic group", "4-7 membered heterocyclic group", "4-6 membered heterocyclic group", "4-10 membered heterocyclic alkyl group", "5-10 membered heterocyclic alkyl group", "4-7 membered heterocyclic alkyl group", "5-6 membered heterocyclic alkyl group", "6-8 membered heterocyclic alkyl group", etc. "4-7 membered heterocyclic group" can further include "4-6 membered heterocyclic group", "5-7 membered heterocyclic group", "5-6 membered heterocyclic group", "4-7 membered heterocyclic alkyl group", "4-6 membered heterocyclic alkyl group", "5-7 membered heterocyclic alkyl group", "5-6 membered heterocyclic alkyl group", etc. Although some bicyclic heterocyclic groups in this disclosure contain a benzene ring or a heteroaromatic ring, the heterocyclic groups as a whole are still non-aromatic.

[0100] The term "heterocyclic alkyl" refers to a fully saturated monovalent cyclic group that exists in the form of a monocyclic, fused, bridged, or spirocyclic ring, wherein the ring atoms contain 1 to 5 heteroatoms or heteroatomic groups (i.e., atomic groups containing heteroatoms). The "heteroatoms or heteroatomic groups" include, but are not limited to, nitrogen (N), oxygen (O), sulfur (S), phosphorus (P), boron (B), -S(=O)2-, -S(=O)-, and optionally substituted -NH-, -S(=O)(=NH)-, -C(=O)NH-, -C(=NH)-, -S(=O)2NH-, S(=O)NH-, or -NHC(=O)NH-, etc., which typically contain 3 to 20 ring atoms. The term "3-10 membered heterocyclic alkyl" refers to a heterocyclic alkyl group having 3, 4, 5, 6, 7, 8, 9 or 10 ring atoms, and containing 1 to 5 independent heteroatoms or heterogroups selected from those described above. "3-10 membered heterocyclic alkyl" includes "3-8 membered heterocyclic alkyl", wherein specific examples of 4 membered heterocyclic alkyl include, but are not limited to, acridine, oxadiazolyl, or thiobutylcycloyl; specific examples of 5 membered heterocyclic alkyl include, but are not limited to, tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolidinyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, imidazolyl, or tetrahydropyrazolyl; specific examples of 6 membered heterocyclic alkyl include, but are not limited to, piperidinyl, tetrahydropyranyl, tetrahydrothiaranyl, morpholinyl, piperazine, 1,4-thiaoxalyl, 1,4-dioxane, thiomorpholinyl, 1,3-dithiaalkyl, or 1,4-dithiaalkyl; and specific examples of 7 membered heterocyclic alkyl include, but are not limited to, azirheptanyl, oxaheptanyl, or thioheptanyl.

[0101] The term "halogen" or "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0102] The term "hydroxyl group" refers to the -OH group.

[0103] The term "cyano" refers to the -CN group.

[0104] The term "amino" refers to the -NH2 group.

[0105] The term "nitro" refers to the -NO2 group.

[0106] The term “therapeutic effective amount” means the amount of a compound of this disclosure used to treat or prevent a particular disease, condition, or disorder; (ii) to reduce, improve, or eliminate one or more symptoms of a particular disease, condition, or disorder; or (iii) to prevent or delay the onset of one or more symptoms of a particular disease, condition, or disorder described herein. The amount of a compound of this disclosure constituting a “therapeutic effective amount” varies depending on the compound, the disease state and its severity, the route of administration, and the age of the mammal to be treated, but may routinely be determined by a person skilled in the art based on their own knowledge and this disclosure.

[0107] The term "pharmaceutical acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0108] The term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt of an acid or base, including salts formed by a compound with an inorganic or organic acid, and salts formed by a compound with an inorganic or organic base.

[0109] The term "pharmaceutical composition" refers to a mixture of one or more compounds of this disclosure or their salts with pharmaceutically acceptable excipients. The purpose of a pharmaceutical composition is to facilitate the administration of the compounds of this disclosure to an organism.

[0110] The term "pharmaceuticalally acceptable excipient" refers to excipients that do not cause significant irritation to the organism and do not impair the biological activity and properties of the active compound. Suitable excipients are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, etc.

[0111] The word “comprise” or “comprise” and its English variants such as comprises or comprising should be understood in an open, non-exclusive sense, meaning “including but not limited to”.

[0112] This disclosure also includes compounds of this disclosure that are identical to those described herein, but in which one or more atoms are labeled with isotopes whose atomic weights or mass numbers differ from those commonly found in nature. Examples of isotopes that can be incorporated into compounds of this disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as... 2 H, 3 H, 11 C 13 C 14 C 13 N、 15 N、 15 O、 17 O、 18 O、 31 P, 32 P, 35 S, 18 F, 123 I, 125 I and 36 Cl, etc.

[0113] Compounds of this disclosure labeled with certain isotopes (e.g., using...) 3 H and 14 C-labeling can be used in the analysis of compound and / or substrate tissue distribution. Tritiumization (i.e., 3 H) and carbon-14 (i.e. 14 C) Isotopes are particularly preferred due to their ease of preparation and detectability. Positron-emitting isotopes, such as... 15 O、 13 N、 11 C and 18 F can be used in positron emission tomography (PET) studies to determine substrate occupancy. The isotopically labeled compounds of this disclosure can typically be prepared by replacing the unlabeled reagent with an isotopically labeled reagent using a procedure similar to those disclosed in the schemes and / or examples below.

[0114] The pharmaceutical compositions disclosed herein can be prepared by combining the compounds of this disclosure with suitable pharmaceutically acceptable excipients, for example, in solid, semi-solid, liquid or gaseous formulations, such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, suppositories, injections, inhalers, gels, microspheres and aerosols.

[0115] Typical routes of administration of the compounds disclosed herein, or their pharmaceutically acceptable salts, or pharmaceutical compositions thereof, include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, vaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, and intravenous administration.

[0116] The pharmaceutical compositions disclosed herein can be manufactured using methods well known in the art, such as conventional mixing, dissolving, granulation, emulsification, freeze drying, etc.

[0117] In some embodiments, the pharmaceutical composition is in an oral form. For oral administration, the pharmaceutical composition can be formulated by mixing the active compound with pharmaceutically acceptable excipients well known in the art. These excipients enable the compounds of this disclosure to be formulated into tablets, pills, lozenges, sugar-coated tablets, capsules, liquids, gels, pastes, suspensions, etc., for oral administration to patients.

[0118] Solid oral compositions can be prepared using conventional mixing, filling, or tableting methods. For example, they can be obtained by mixing the active compound with solid excipients, optionally milling the resulting mixture, adding other suitable excipients if necessary, and then processing the mixture into granules to obtain the core of a tablet or sugar-coated formulation. Suitable excipients include, but are not limited to, binders, diluents, disintegrants, lubricants, flow aids, or flavoring agents.

[0119] The pharmaceutical composition may also be suitable for parenteral administration, such as in suitable unit dosage forms of sterile solutions, suspensions or lyophilized products.

[0120] The dosage of compounds or compositions used in the treatments described in this disclosure will generally vary depending on the severity of the disease, the patient’s weight, and the relative efficacy of the compound. However, as a general guideline, a suitable daily dose of the compounds of general formula (I) described herein is from 0.01 mg / kg to 1000 mg / kg.

[0121] The compounds disclosed herein can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments of this disclosure. Detailed Implementation

[0122] The following detailed description of specific implementation schemes illustrates the contents of this disclosure, but does not imply any adverse limitation thereof. Various specific implementation schemes of this disclosure have been described in detail herein, and it will be apparent to those skilled in the art that various changes and modifications can be made to these specific implementation schemes without departing from the spirit and scope of this disclosure.

[0123] The chemical reactions in the specific embodiments of this disclosure are carried out in a suitable solvent, which must be suitable for the chemical changes and the reagents and materials required by this disclosure. To obtain the compounds of this disclosure, it is sometimes necessary for those skilled in the art to modify or select the synthesis steps or reaction flow based on existing embodiments.

[0124] All reagents used in this disclosure are commercially available and can be used without further purification.

[0125] Unless otherwise stated, proportions expressed for mixed solvents are volume-based. Unless otherwise stated, % refers to wt%.

[0126] The structure of the compound was determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shifts are measured in units of 10⁻⁶. -6 (ppm). The solvents used for NMR determination included deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, D₂O, CF₃COOD, etc., with tetramethylsilane (TMS) as the internal standard; "IC 50 "Half-inhibition concentration" refers to the concentration at which half of the maximum inhibitory effect is achieved.

[0127] Explanation of terms or abbreviations:

[0128] t-Bu: tert-butyl; t-BuNO2: tert-butyl nitrite; TEA: triethylamine; THF: tetrahydrofuran; MeOH: methanol; DCM: dichloromethane; DMF: N,N-dimethylformamide; DIEA: N,N-diisopropylethylamine; HATU: O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethylurea hexafluorophosphate; Boc: tert-butyloxycarbonyl; STAB / NaBH(OAc)3: triethylamine Sodium acetoxyborohydride; AcOH / HOAc: acetic acid; MeCN / ACN: acetonitrile; TFA: trifluoroacetic acid; Pd(dppf)Cl2: 1,1-bis(diphenylphosphine)ferrocene palladium dichloride; dioxane: 1,4-dioxane; toluene / PhMe: toluene; Raney-Ni: Raney nickel; DMSO: dimethyl sulfoxide; M: mol / L; Py: pyridine; PE: petroleum ether; EA: ethyl acetate.

[0129] The eluent described below may be a mixture of two or more solvents, with the ratio being the volume ratio of each solvent.

[0130] Synthesis of intermediate P: 5-isopropoxy-2-(piperazin-1-yl)-N-(pyrazolo[1,5-a]pyrimidin-3-yl)benzo[d]thiazol-6-carboxamide

[0131]

[0132] Step 1: Synthesis of 1-(4-bromo-3-isopropoxyphenyl)thiourea (A-4)

[0133] 4-Bromo-3-isopropoxyaniline (A-3, 19.8 g, 86.05 mmol) and phosgene (11.9 g, 103.26 mmol) were dissolved in toluene (200 mL), and the mixture was heated to 100 °C and stirred for 1 hour, with the reaction monitored by TLC. After the reaction was complete, the reaction solution was cooled to room temperature and diluted with tetrahydrofuran (200 mL). Ammonia gas was then introduced into the reaction solution with stirring at room temperature for approximately 30 min, and the reaction was monitored by TLC to indicate completeness. The solvent was removed by concentration under reduced pressure, and the residue was separated by silica gel column chromatography (petroleum ether / ethyl acetate = 2 / 1) to give the title compound (20.5 g).

[0134] MS m / z (ESI): 289.0 [M+H] +

[0135] Step 2: Synthesis of 6-bromo-5-isopropoxybenzo[d]thiazol-2-amine (A-5)

[0136] At room temperature, 1-(4-bromo-3-isopropoxyphenyl)thiourea (A-4, 5.0 g, 17.29 mmol) was dissolved in acetic acid (50 mL), and a solution of liquid bromine (3.3 g, 20.63 mmol) dissolved in acetic acid (10 mL) was slowly added dropwise with stirring. The mixture was heated to 100 °C and stirred for 1 hour. After the reaction was complete, the reaction solution was cooled to room temperature, concentrated, and diluted with ethyl acetate (200 mL) and water (50 mL). The pH was then adjusted to 8-9 with 1 N sodium hydroxide solution. The organic phase was collected, washed with saturated brine, and dried over anhydrous sodium sulfate. The solvent was removed by concentration under reduced pressure, and the residue was separated by silica gel column chromatography (petroleum ether / ethyl acetate = 2 / 1) to give the title compound (2.8 g).

[0137] MS m / z (ESI): 287.0 [M+H] +

[0138] Step 3: Synthesis of 6-bromo-2-chloro-5-isopropoxybenzo[d]thiazole (A-6)

[0139] At room temperature, 6-bromo-5-isopropoxybenzo[d]thiazol-2-amine (A-5, 1.4 g, 4.87 mmol) and copper chloride (1.0 g, 7.46 mmol) were dissolved in acetonitrile (20 mL). Tert-butyl nitrite (754 mg, 7.31 mmol) was slowly added dropwise with stirring. The mixture was stirred at room temperature for 30 min, then heated to 65 °C and stirred for 1 h. After the reaction was complete, the reaction solution was cooled to room temperature, diluted with water (50 mL), and extracted with ethyl acetate (30 mL × 3). The organic phase was collected, washed with saturated brine, and dried over anhydrous sodium sulfate. The solvent was removed by concentration under reduced pressure, and the residue was separated by silica gel column chromatography (petroleum ether / ethyl acetate = 20 / 1) to give the title compound (1.23 g).

[0140] MS m / z (ESI): 305.9 [M+H] +

[0141] Step 4: Synthesis of 4-(6-bromo-5-isopropoxybenzo[d]thiazolyl)piperazine-1-carboxylic acid tert-butyl ester (B-1)

[0142] 6-Bromo-2-chloro-5-isopropoxybenzo[d]thiazole (A-6, 530 mg, 1.73 mmol), piperazine-1-carboxylic acid tert-butyl ester (322 mg, 1.73 mmol), and potassium carbonate (478 mg, 3.46 mmol) were dissolved in N,N-dimethylformamide (5 mL) at room temperature, and the mixture was heated to 80°C and stirred for 4 hours. After the reaction was complete, the reaction solution was cooled to room temperature, diluted with water (50 mL), and then extracted with ethyl acetate (30 mL × 3). The organic phase was collected, washed with saturated brine, and dried over anhydrous sodium sulfate. The solvent was removed by concentration under reduced pressure, and the residue was separated by silica gel column chromatography (petroleum ether / ethyl acetate = 20 / 1) to give the title compound (700 mg).

[0143] MS m / z (ESI): 456.1 [M+H] +

[0144] Step 5: Synthesis of methyl 2-(4-(tert-butoxycarbonyl)piperazin-1-yl)-5-isopropoxybenzo[d]thiazol-6-carboxylate (B-2)

[0145] At room temperature, 4-(6-bromo-5-isopropoxybenzo[d]thiazolyl)piperazine-1-carboxylic acid tert-butyl ester (B-1, 713 mg, 1.56 mmol), triethylamine (316 mg, 3.12 mmol), and [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride (117 mg, 0.16 mmol) were dissolved in methanol (40 mL). The mixture was purged three times with a carbon monoxide bag, and the mixture was refluxed at 80 °C with stirring for 16 h. After the reaction was complete, the reaction solution was cooled to room temperature, concentrated under reduced pressure to remove the solvent, and the residue was separated by silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 1) to give the title compound (247 mg).

[0146] MS m / z (ESI): 436.2 [M+H] +

[0147] Step 6: Synthesis of 2-(4-(tert-butoxycarbonyl)piperazin-1-yl)-5-isopropoxybenzo[d]thiazol-6-carboxylic acid (B-3)

[0148] Methyl 2-(4-(tert-butoxycarbonyl)piperazin-1-yl)-5-isopropoxybenzo[d]thiazol-6-carboxylate (B-2, 272 mg, 0.625 mmol) and lithium hydroxide monohydrate (50 mg, 1.19 mmol) were dissolved in tetrahydrofuran / methanol / water (8 / 2 / 2 mL) at room temperature and stirred for 16 h. After the reaction was complete, the mixture was diluted with water (30 mL), the pH was adjusted to 4-5 with 1 N hydrochloric acid, and the mixture was extracted with ethyl acetate (20 mL × 3). The organic phase was collected, washed with saturated brine, and dried over anhydrous sodium sulfate. The solvent was removed by concentration under reduced pressure, and the residue was separated by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 1) to give the title compound (215 mg).

[0149] MS m / z (ESI): 422.2 [M+H] +

[0150] Step 7: Synthesis of 4-(5-isopropoxy-6-(pyrazolo[1,5-a]pyrimidin-3-ylcarbamoyl)benzo[d]thiazolyl)piperazine-1-carboxylic acid tert-butyl ester (P-1)

[0151] At room temperature, 2-(4-(tert-butoxycarbonyl)piperazin-1-yl)-5-isopropoxybenzo[d]thiazol-6-carboxylic acid (B-3, 150 mg, 0.356 mmol), pyrazolo[1,5-a]pyrimidine-3-amine hydrochloride (73 mg, 0.427 mmol), O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethylurea hexafluorophosphate (162 mg, 0.427 mmol), and N,N-diisopropylethylamine (184 mg, 1.424 mmol) were dissolved in N,N-dimethylformamide (5 mL), and the mixture was stirred at room temperature for 40 h. After the reaction was complete, the mixture was diluted with water (50 mL), extracted with ethyl acetate (20 mL * 3), and the organic phase was collected, washed with saturated brine, and dried over anhydrous sodium sulfate. The filtrate was filtered, concentrated, and separated by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 1) to give the title compound (165 mg).

[0152] MS m / z (ESI): 538.2 [M+H] +

[0153] Step 8: Synthesis of 5-isopropoxy-2-(piperazin-1-yl)-N-(pyrazolo[1,5-a]pyrimidin-3-yl)benzo[d]thiazol-6-carboxamide (intermediate P)

[0154] At room temperature, 4-(5-isopropoxy-6-(pyrazolo[1,5-a]pyrimidin-3-ylcarbamoyl)benzo[d]thiazo-2-yl)piperazine-1-carboxylic acid tert-butyl ester (P-1, 165 mg, 0.307 mmol) was dissolved in dichloromethane (2 mL), and then slowly added dropwise to a solution of dioxane hydrochloride (2 mL, 4N). The mixture was stirred at room temperature for 2 h. After the reaction was complete, the solution was filtered and freeze-dried to give the crude title compound (130 mg).

[0155] MS m / z (ESI): 438.2 [M+H] +

[0156] Synthesis of intermediate Q: N-(1-((1S,2R)-2-fluorocyclopropyl)-2-oxo-1,2-dihydropyridin-3-yl)-5-isopropoxy-2-(piperazin-1-yl)benzo[d]thiazol-6-carboxamide

[0157]

[0158] Following the synthetic method of intermediate P, the pyrazolo[1,5-a]pyrimidine-3-amine hydrochloride was replaced with 3-amino-1-((1S,2R)-2-fluorocyclopropyl)pyridin-2(1H)-one, and intermediate Q was prepared by the same method.

[0159] MS m / z (ESI): 472.2 [M+H] +

[0160] Example 1: Synthesis of 2-(4-(1-(2-((S)-2,6-dioxopiperidin-3-yl)-3-oxoisoindoline-5-yl)piperidin-4-yl)methyl)piperazin-1-yl)-N-(1-(1S, 2R)-2-fluorocyclopropyl)-2-oxo-1,2-dihydropyridin-3-yl)-5-isopropoxybenzothiazol-6-carboxamide (Compound 1)

[0161]

[0162] Step 1: Synthesis of methyl 2-cyano-5-(4-(dimethoxymethyl)piperidin-1-yl)benzoate (1c)

[0163] Methyl 2-cyano-5-fluorobenzoate (240 g, 1.34 mol) was dissolved in N,N-dimethylformamide (2.4 L), followed by the addition of N,N-diisopropylethylamine (350 mL) and 4-(dimethoxymethyl)piperidine (234.6 g, 1.47 mol). The solution was then dissolved in 100 mL of water. o Stirred at C for 12 h, the reaction was completed according to LCMS. Water (7.2 L) was added to the reaction solution, and a solid precipitated. The solid was filtered, and the filter cake was slurried with methyl tert-butyl ether (1.2 L), filtered, and dried to obtain the target compound (338 g, yield: 79.25%).

[0164] MS m / z (ESI): 318.9 [M+H] +

[0165] Step 2: Synthesis of methyl 5-[4-(dimethoxymethyl)piperidin-1-yl]-2-carboxybenzoate (1d)

[0166] Methyl 2-cyano-5-(4-(dimethoxymethyl)piperidin-1-yl)benzoate (1 c, 325 g, 1.02 mol) was dissolved in water (1.7 L), followed by the addition of sodium hypophosphite monohydrate (324 g, 3.06 mol), pyridine (658 mL), and acetic acid (642 mL). After the addition was complete, Raney nickel (299.6 g, 5.1 mol) was slowly added at room temperature. The temperature was then raised to 70°C. oStirred at C for 12 h. LCMS showed the reaction was complete. The reaction mixture was filtered through a diatomaceous earth pad, the filter cake was washed with methanol (1 L), methanol was removed by rotary evaporation, and the mixture was extracted twice with dichloromethane. The organic phase was washed once with saturated sodium bicarbonate, then washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain the crude product. The crude product was slurried with PE / EA = 5 / 1-3 / 1 and the solvent was removed by rotary evaporation. The product was then slurried with petroleum ether:ethyl acetate = 20:1 to obtain the target compound (183 g, yield: 55.78%).

[0167] MS m / z (ESI): 322.2 [M+H] +

[0168] Step 3: Synthesis of tert-butyl (S)-5-amino-4-(6-(4-(dimethoxymethyl)piperidin-1-yl)-1-oxoisoindoline-2-yl)-5-oxovalerate (1f)

[0169] (S)-4,5-diamino-5-oxopentanoic acid tert-butyl ester (8.5 g, 42.01 mmol) was dissolved in N,N-dimethylformamide (100 mL) and dichloromethane (100 mL), followed by the addition of methyl 5-[4-(dimethoxymethyl)piperidin-1-yl]-2-carboxybenzoate (1 d, 9.0 g, 28.01 mmol) and acetic acid (3.36 g, 56.1 mmol). The solution was then heated to 25 °C. o Stir at C for 30 minutes, then add sodium triacetoxyborohydride (17.81 g, 84.02 mol) and heat at 25°C. o The reaction was carried out overnight at C. Water was added to the reaction solution, and the mixture was extracted with dichloromethane and dried over anhydrous sodium sulfate to obtain the crude product. Finally, the crude product was slurried (petroleum ether: ethyl acetate = 5:1) to obtain the target compound (12.0 g, yield: 90.10%).

[0170] MS m / z (ESI): 476.3 [M+H] +

[0171] Step 4: Synthesis of (S)-5-amino-4-(6-(4-formylpiperidin-1-yl)-1-oxoisoindoline-2-yl)-5-oxovaleric acid (1 g)

[0172] (S)-5-amino-4-(6-(4-(dimethoxymethyl)piperidin-1-yl)-1-oxoisoindoline-2-yl)-5-oxovalerate tert-butyl ester (1f, 5.0 g, 10.51 mmol) was dissolved in dichloromethane (100 mL), and trifluoroacetic acid (11.99 g, 105.14 mmol) was added. The solution was heated to 25 °C. oThe reaction was carried out overnight. After removing the solvent from the reaction solution by rotary evaporation, acetonitrile (100 mL) was added, and the solution was adjusted to a weakly alkaline state (pH: 7-8) with N,N-diisopropylethylamine. The solution was filtered, and the solvent was removed from the filtrate by rotary evaporation to obtain the target compound (4.2 g, yield: 99.51%).

[0173] MS m / z (ESI): 374.2 [M+H] +

[0174] Step 5: Synthesis of (S)-1-(2-(2,6-dioxopiperidin-3-yl)-3-oxoisoindoline-5-yl)piperidine-4-carboxaldehyde (1f)

[0175] (S)-5-amino-4-(6-(4-formylpiperidin-1-yl)-1-oxoisoindoline-2-yl)-5-oxopentanoic acid (1 g, 4.2 g, 11.25 mmol) was dissolved in acetonitrile (150 mL), and N,N-diisopropylethylamine (2.91 g, 22.5 mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethylurea hexafluorophosphate (5.09 g, 13.5 mmol) were added. The solution was heated to 25 °C. o C was reacted overnight. The reaction solution was poured into water, extracted with ethyl acetate, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, the solvent was removed by rotary evaporation, and the crude product was purified by direct mixing with column chromatography (dichloromethane:acetonitrile = 1:1). The solvent was removed by rotary evaporation to obtain the target product (1.8 g, yield: 45.03%).

[0176] MS m / z (ESI): 356.2 [M+H] +

[0177] Step 6: Synthesis of 2-(4-(1-(2-((S)-2,6-dioxopiperidin-3-yl)-3-oxoisoindoline-5-yl)piperidin-4-yl)methyl)piperazin-1-yl)-N-(1-(1S, 2R)-2-fluorocyclopropyl)-2-oxo-1,2-dihydropyridin-3-yl)-5-isopropoxybenzothiazol-6-carboxamide (Compound 1)

[0178] N-(1-(1S, 2R)-2-fluorocyclopropyl)-2-oxo-1,2-dihydropyridin-3-yl)-5-isopropoxy-2-(piperazin-1-yl)benzo[d]thiazolyl-6-carboxamide (intermediate Q, 200 mg, 0.42 mmol) was dissolved in dichloromethane (4 mL) and methanol (0.4 mL), followed by the addition of N,N-diisopropylethylamine (109.63 mg, 0.84 mmol). After stirring for 0.5 hours, acetic acid (38.21 mg, 0.63 mmol) was added, and after stirring for another 0.5 hours, (S)-1-(2-(2,6-dioxopiperidin-3-yl)-3-oxoisoindoline-5-yl)piperidin-4-carboxaldehyde (1f, 150.73 mg, 0.42 mmol) was added. Add mmol), stir for 0.5 hours, then add sodium triacetoxyborohydride (314.62 mg, 1.48 mmol), stir at room temperature for 12 hours, add water to the reaction solution, and extract with dichloromethane. Adjust the pH of the organic phase to weakly alkaline with saturated sodium bicarbonate aqueous solution, wash with saturated brine, dry with anhydrous sodium sulfate, remove solvent by rotary evaporation to obtain crude product, and finally slurry (acetonitrile / methanol = 20:1) to obtain the target compound (140 mg, yield: 40.7%, enantiomeric ratio: 97.2:2.8).

[0179] MS m / z (ESI): 811.3 [M+H] +

[0180] 1H NMR (400 MHz, DMSO-d6) δ 10.97 (s, 1H), 10.88 (s, 1H), 8.531– 8.49(m, 1H), 8.42 (s, 1H), 7.42 – 7.38 (m, 2H), 7.32 (s, 1H), 7.27 – 7.26 (m,1H), 7.16 (s, 1H), 6.33 – 6.30 (m, 1H), 5.12 – 5.02 (m, 3H), 4.35 – 4.31 (m,1H), 4.22 – 4.18 (m, 1H), 3.78 – 3.75 (m, 2H), 3.62 – 3.34 (m, 7H), 2.96 –2.86 (m, 1H), 2.79 – 2.68 (m, 2H), 2.65 – 2.54 (m, 1H), 2.41 – 2.31 (m, 1H), 2.31 – 2.17 (m, 2H), 2.03 – 1.95 (m, 1H), 1.84 – 1.81 (m, 3H), 1.67 – 1.55 (m, 1H), 1.50 – 1.48 (m, 6H), 1.26 – 1.23 (m, 1H).

[0181] Examples 2-8 Synthesis of Compounds 2-8

[0182] Referring to the corresponding embodiments described in PCT / CN2024 / 124493 (the contents of which are incorporated herein by reference in their entirety), compounds 2-8 were synthesized by replacing the corresponding raw material 1A with raw material A in the table below.

[0183]

[0184] Biological activity and related property test examples

[0185] Test Example 1: HiBiT Method for Detecting the Degradation Effect of Compounds on IRAK4 in THP-1-HiBiT-IRAK4 Overexpressing Cells

[0186] THP-1-HiBiT-IRAK4 cells (constructed by Shanghai Medicilon) were seeded in 96-well flat-bottomed white plates (Corning, catalog number: 3917) at a density of 60,000 cells / 25 µL / well. Then, 25 µL / well of a concentration gradient compound was added (final compound concentration: initial concentration 5 µM, 5-fold serial dilution, for a total of 8 concentration points). After mixing, the mixture was incubated at 37 ℃ in a 5% CO2 incubator for 24 h. The experimental groups were as follows: sample treatment group (cells + concentration gradient compound), solvent control group (cells + solvent control), and blank control group (culture medium only). 50 µL of Nano-Glo HiBiT Lytic Detection buffer (Promega, catalog number: N3040) was added, and the mixture was incubated at room temperature with shaking (350 rpm) for 10 min. The fluorescence value (RLU) was then read using a PE Envision microplate reader. The degradation rate % of the compound on IRAK4 is calculated as follows: (1 - (RLU value of sample treatment group - RLU value of blank control group) / (RLU value of solvent control group - RLU value of blank control group)) × 100%. The degradation rate and maximum degradation rate D of the compound at each concentration are then calculated. max The degradation activity curve of the compound was obtained by nonlinear fitting of compound concentration-degradation rate using Graphpad Prism 9 software, and the half-maximum degradation concentration (DC) of the compound was calculated. 50 D max The larger the value, the higher the DC 50 The smaller the value, the stronger the degradation activity of the compound. The D value of the test compound... max and DC 50 The values ​​are shown in Table 1.

[0187] Table 1. D of the compounds disclosed herein max and DC 50

[0188]

[0189] Test Example 2: FACS method for detecting the degradation effect of the compound on IRAK4 in human peripheral blood mononuclear cells (hPBMCs)

[0190] Human PBMC cells (Hycell, W-HPB100C) were seeded in 96-well U-plates (Corning, catalog number: 3799) at a density of 800,000 cells / 100 µL / well. Then, 100 µL / well of a concentration gradient compound was added (final concentration: initial concentration 500 nM, 3-fold serial dilution, for a total of 10 concentration points), and the mixture was incubated at 37 °C in a 5% CO2 incubator for 24 h. After incubation, the cells in each well were mixed, centrifuged at 350 g at 4 °C for 5 min, and the supernatant was discarded. The cell pellet was resuspended in 200 µL / well PBS (Cytiva, catalog number: SH30256.01), centrifuged at 350 g at 4 °C for 5 min, and the supernatant was discarded. After incubating with LIVE / DEAD Fixable Near-IR Dead Cell dye (Thermofisher, catalog number: L10119) at 4°C for 10 min, add 100 µL / well of staining buffer (0.5% BSA + PBS) (BSA sourced from Sigma, catalog number: V900933-100G), centrifuge at 350 g for 5 min (4°C), and discard the supernatant. Add 100 µL / well of Fix Buffer I (BD, catalog number: 557870) and fix at 37°C for 10 min. After fixation, add 100 µL / well of staining buffer, centrifuge at 350 g for 5 min (4°C), and discard the supernatant. Add 100 µL / well of Perm buffer III (BD, catalog number: 558050) and incubate on ice for 30 min. After cell permeabilization, add 100 µL / well of staining buffer, centrifuge at 400 g for 5 min (4°C), and discard the supernatant. Add 50 µL / well Human TruStain FcX (Biolegend, catalog number: 422302) and incubate at room temperature for 15 min. Add 50 µL / well antibody staining solution (containing 4 µL / well PE-anti-human IRAK4 antibody (BD, catalog number: 560303)) and mix well, then incubate at room temperature for 1 h. After staining, add 100 µL / well staining buffer to the cells, centrifuge at 400 g at room temperature for 5 min, discard the supernatant, resuspend the cells in 120 µL / well staining buffer, and analyze the live cells using a flow cytometer (BD, Canto II). The mean fluorescence intensity (MFI) of IRAK4 was analyzed using Flowjo V10 and Graphpad Prism 9 software. The degradation rate % of the compound for IRAK4 was calculated as (1 - (MFI value of the sample treatment group / MFI value of the solvent control group)) × 100%. The degradation rate and maximum degradation rate D of the compound at each concentration were also calculated. maxThe degradation activity curve of the compound was obtained by nonlinear fitting of compound concentration-degradation rate using Graphpad Prism 9 software, and the maximum degradation rate (D) of the compound was calculated. max ), half-maximal degradation concentration (DC) 50 D max The larger the value, the higher the DC 50 The smaller the value, the stronger the degradation activity of the compound. The D value of the test compound... max and DC 50 The values ​​are shown in Table 2.

[0191] Table 2. Dermatological activity of the disclosed compounds in human PBMC cells. max and DC 50

[0192]

[0193] Test Example 3: Determination of the metabolic stability of the disclosed compound in liver microsomes

[0194] I. Experimental Materials and Instruments

[0195] 1. Sources of liver microsomes: Human liver microsomes (Corning 452117), CD-1 mouse liver microsomes (XENOTECHM1000)

[0196] 2. Na2HPO4 (Shanghai Test No. 20040618)

[0197] 3. KH2PO4 (Shanghai Test No. 10017608)

[0198] 4. NADPH (Roche diagnostics gmbh 10107824001)

[0199] 5. Tolbutamide (Sigma-aldrich T0891-100G)

[0200] 6. Positive control compound verapamil (Aladdin V303890-100mg)

[0201] 7. AB5500+ Liquid Chromatography-Mass Spectrometry (LC-MS) System

[0202] II. Experimental Procedure

[0203] 1. Preparation of 100 mM phosphate-buffered saline (PBS): Weigh 7.098 g Na₂HPO₄ and dissolve it in 500 mL of pure water by sonication to obtain solution A. Weigh 3.400 g KH₂PO₄ and dissolve it in 250 mL of pure water by sonication to obtain solution B. Place solution A on a stirrer and slowly add solution B until the pH reaches 7.4 to prepare a 100 mM PBS buffer.

[0204] 2. Preparation of the reaction system

[0205] Prepare the reaction system according to the table below:

[0206] 3. Incubate the reaction system in a 37 °C water bath for 10 minutes. Add 40 μL of 10 mM NADPH solution (NADPH dissolved in 100 mM phosphate buffer) to the reaction system, bringing the final NADPH concentration to 1 mM. Use 40 μL of phosphate buffer instead of NADPH solution as a negative control. The negative control is used to eliminate the influence of the compound's own chemical stability.

[0207] 4. The reaction was initiated by adding 4 μL of the disclosed compound (100 μM) and the positive control compound verapamil to the reaction system. The final concentration of the compound was 1 μM.

[0208] 5. After thorough mixing in a vortex mixer at 0.5, 15, 30, 45, and 60 minutes, 50 µL of the incubated sample was taken out each time, and the reaction was terminated with 4 times the volume of ice-cold acetonitrile containing the internal standard (50 ng / mL tolbutamide). The sample was centrifuged at 3,220 g for 45 minutes. After centrifugation, 90 μL of the supernatant was transferred to a sample plate, and 150 μL of ultrapure water was added and mixed well for LC-MS / MS analysis.

[0209] All data were calculated using Microsoft Excel software. Peak areas were detected by extracting ion spectra. The in vitro half-life (t0.05) of the parent drug was determined by linearly fitting the natural logarithm of the elimination percentage of the parent drug to time. 1 / 2 ).

[0210] In vitro half-life (t 1 / 2 ) Calculated using slope (k):

[0211] in vitro t 1 / 2 = 0.693 / k

[0212] The test data are shown in Table 3, indicating that the compound disclosed herein has good liver microsomal stability.

[0213] Table 3. Half-life values ​​of the compounds disclosed herein in liver microsomes

[0214]

[0215] Test Example 4: Determination of the plasma protein binding rate of the disclosed compounds

[0216] I. Experimental Materials and Instruments

[0217] 1. CD1 mouse plasma (BioIVT)

[0218] 2. Human plasma (from Healthy Asia volunteers at Weifang High-tech Industrial Development Zone People's Hospital (collected at the hospital and with ethical approval))

[0219] 3. Na2HPO4 (Sigma S5136-500G)

[0220] 4. NaH2PO4 (Sigma S3139-500G)

[0221] 5. NaCl (Sigma S5886-IKG)

[0222] 6. Positive control compound ketoconazole (Sigma Lot# SLBB5223V)

[0223] 7. Alprazolam (Ceriliant FE12172003)

[0224] 8. Rabelor (NIFDC 100484-201001)

[0225] 9. Ketoprofen (NIFDC 100337-201104)

[0226] 10. Polycarbonate centrifuge tubes (BECKMAN, 343778)

[0227] 11. High-speed centrifugal rotor (BECKMAN, MLA-150)

[0228] 12. Optima™ MAX-XP Ultracentrifuge (BECKMAN)

[0229] II. Experimental Procedure

[0230] 1. Prepare DMSO working solutions of the test compound and positive control compound at a concentration of 1 mM.

[0231] 2. The water bath, incubator, and ultracentrifuge were all set to 37 ℃, and the rotor was pre-cultured in the 37 ℃ incubator for about 30 minutes.

[0232] 3. Thaw the frozen plasma in a water bath at room temperature (store at -80 °C), centrifuge at 3,220 g for 10 minutes to remove clots, and collect the supernatant into a new tube. Check and record the pH value of the plasma.

[0233] 4. Pre-incubate the plasma in a 37°C water bath for 5 minutes.

[0234] 5. Add 3 μL of 1 mM working solution to 2997 μL of preheated plasma to obtain a spiked plasma sample with a final concentration of 1 μM.

[0235] 6. Transfer 1 mL of spiked plasma sample to two ultracentrifuge tubes (n=2). Equilibrate the ultracentrifuge tubes into a preheated rotor and incubate at 37°C and 5% CO2 for 30 minutes. After incubation, centrifuge the ultracentrifuge tubes at 37°C and 800,000 g for 3 hours.

[0236] 7. Transfer 50 μL of spiked plasma sample into six 0.6 mL tubes. Incubate the samples at 37°C and 5% CO2 for 0, 0.5, and 3.5 hours. At the specified time points, add 50 μL of PBS, mix thoroughly, and then add 400 μL of acetonitrile containing internal standards (15S, 100 nM alprazolam, 500 nM labetalol, and 2 μM tyroprofen) to precipitate the protein. Vortex the sample for 5 minutes, and then centrifuge at 20,000 g for 15 minutes. The sample at 0.5 hours is a non-centrifuged control.

[0237] 8. After ultracentrifugation, remove 50 μL from the center of the ultracentrifuge tube as the ultracentrifuged sample, and then add 50 μL of blank plasma to the sample. Then, process the sample in the same way as the non-centrifuged control sample. Transfer 100 μL of supernatant to the sample plate, add 100 μL of ultrapure water, mix well, and use for LC-MS / MS analysis.

[0238] 9. Data Analysis: All calculations were performed using Microsoft Excel. The following calculations show the percentage of binding between the test compound and the control compound:

[0239] Free concentration % = (peak area ratio) 超速离心后样品 / peak area ratio 非离心对照样品 ) × 100%

[0240] Binding rate % = 100% - Free rate %

[0241] The experimental results are shown in Table 4.

[0242] Table 4. Protein binding rates of the disclosed compounds in plasma.

[0243]

[0244] Test Example 5: Determination of membrane permeability and transport properties of the disclosed compounds

[0245] The membrane permeability and transport properties of the compounds disclosed herein were determined using the following experimental methods.

[0246] I. Experimental Materials and Instruments

[0247] 1. Caco-2 cells (ATCC)

[0248] 2. HEPES (Solarbio 804D049), penicillin / streptomycin (Solarbio 20200109), Trypsin / EDTA (Solarbio), PBS (Solarbio 20200620)

[0249] 3. Fetal bovine serum (FBS) (Sigma WXBD0055V), Fluorescein (Sigma MKCJ3738), NaHCO3 (Sigma SLBZ4647)

[0250] 4. Hank's balanced salt solution (HBSS) (Gibco 2085528), non-essential amino acids (NEAA) (Gibco 2211548), and Trypsin / EDTA (Gibco 2120732)

[0251] 5. High-glucose DMEM medium (Corning 20319014)

[0252] 6. 96-well HTS transwell plate (Corning, 3391)

[0253] 7. Resistance meter (Millipore, Millicell) ® ERS-2)

[0254] 8. Cellometer ® Vision (Nexcelom Bioscience)

[0255] 9. Infinite 200 PRO microplate reader (Tecan, Infinite M200PRO)

[0256] 10. Positive control compounds: metoprolol (Sinopharm 100084-201403), erythromycin (MCE 84550), and cimetidine (Sinopharm 100158-201406).

[0257] 11. Kanamycin (Merck QR11795)

[0258] 12. Triple Quad™ 5500+ LC-MS / MS System (AB Inc)

[0259] 13. CO2 Incubator (Thermo 371)

[0260] 14. T-75 culture flask (Thermo 156499)

[0261] II. Experimental Procedure

[0262] 1. Caco-2 cell culture

[0263] 1) Preparation of transport buffer (HBSS containing 25 mM HEPES, pH 7.4): Accurately weigh 5.958 g HEPES and 0.35 g NaHCO3, add 900 mL of pure water to dissolve them, then add 100 mL of 10×HBSS and stir well. Adjust the pH to 7.4 with sodium hydroxide and filter.

[0264] 2) Preparation of Caco-2 cell culture medium: FBS, penicillin, streptomycin, kanamycin and NEAA were added to high glucose DMEM medium to prepare a cell culture medium containing 10% FBS, 0.1 mg / mL streptomycin, 100 units of penicillin, 0.6 μg / mL kanamycin and 1×NEAA.

[0265] 3) Culture the cells in a T-75 culture flask at 37 °C and 5% CO2. When the cells reach 80-90% confluence, discard the culture medium. Wash the cells with 5 mL PBS, add 1.5 mL Trypsin / EDTA, and then incubate at 37 °C for 5-10 minutes until the cells detach in a quicksand-like manner. Finally, neutralize the Trypsin / EDTA with Caco-2 cell culture medium.

[0266] 4) Centrifuge the cell suspension at 120 xg for 10 minutes and discard the supernatant.

[0267] 5) Resuspend the cells in cell culture medium and adjust the density to 6.86 × 10⁻⁶. 5 Cell suspension with cells / mL.

[0268] 2. Caco-2 cell seeding

[0269] 1) Add 50 μL of culture medium to each well of the Transwell chamber, add 25 mL of Caco-2 cell culture medium to the bottom layer, and preheat in a 37 °C, 5% CO2 incubator for 1 hour.

[0270] 2) Add 50 μL of cell suspension to each well of the preheated Transwell chamber, resulting in a final seeding density of 2.4 × 10⁻⁶ cells / well. 5 cells / cm².

[0271] 3) Incubate for 14-18 days, changing the culture medium every other day, and changing the culture medium within 48 hours after the initial inoculation. The culture medium must be changed the day before the experiment.

[0272] 3. Assess the integrity of the monolayer cell membrane

[0273] 1) After 14 days of cell culture, the cells fused and differentiated, ready for transport experiments.

[0274] 2) Measure the resistance of the single-layer film using a resistance meter and record the resistance of each hole.

[0275] 3) After the measurement is completed, re-incubate the Transwell culture plate.

[0276] 4) Calculate the TEER value:

[0277] TEER value = TEER (Ω) measurement × membrane area (cm²) 2 )

[0278] The electrical resistance of a single cell membrane is <230 Ω·cm 2 This indicates that the single-layer cell membrane has poor density and cannot be used in experiments.

[0279] 4. Transport experiment

[0280] 1) Dilute the 10 mM DMSO stock solution of the disclosed compound or positive control compound with DMSO to obtain a 2 mM stock solution, and then dilute the 2 mM stock solution with transfer buffer to obtain a 10 μM working solution of the disclosed compound or positive control compound.

[0281] 2) Remove the Caco-2 cell plate from the incubator, then wash the Transwell culture plate twice with preheated transport buffer, and incubate it in a 37 °C incubator for 30 minutes.

[0282] 3) To determine the transport rate of the compound from the top to the base (A→B), 108 μL of the compound's working solution was added to the Transwell chamber (top). Simultaneously, 8 μL of sample was immediately removed from the top and transferred to 72 μL of transport buffer. 240 μL of stop solution containing an internal standard was added to terminate the transport and serve as the initial top sample. At the same time, 300 μL of transport buffer was added to the receiving end (base). The experiment used a two-sample setup.

[0283] 4) To determine the transport rate of the compound from the base end to the top end (B→A), 308 μL of the compound's working solution was added to the receiving end (base end). Simultaneously, 8 μL of sample was immediately removed from the base end and transferred to 72 μL of transport buffer. 240 μL of stop solution containing an internal standard was added to terminate the transport and serve as the initial base end sample. At the same time, 300 μL of transport buffer was added to the Transwell chamber (top end). The experiment used a two-sample setup.

[0284] 5) Incubate the cell culture plate in a 37 °C CO2 incubator for 2 hours.

[0285] 6) After the transport experiment, take 8 μL of sample from the dosing end (i.e., the top end in the A→B direction and the base end in the B→A direction) and add it to 72 μL of transport buffer. Then, add 240 μL of stop solution containing internal standard to terminate the transport. Take 80 μL of sample from the receiving end (i.e., the base end in the A→B direction and the top end in the B→A direction) and add it to 240 μL of stop solution containing internal standard. Vortex at 1000 rpm for 10 minutes and centrifuge at 3,220 g for 30 minutes. Take 100 μL of supernatant into the sample plate, add 100 μL of ultrapure water and mix well for LC-MS / MS analysis.

[0286] 7) After the transport experiment, measure the fluorescence value. Prepare a 10 mM fluorescein stock solution with water, then dilute it to 100 μM with transport buffer. Add 100 μL of fluorescein solution to the top of the Transwell chamber, and add 300 μL of transport buffer to the bottom end. Incubate at 37 °C in a CO2 incubator for 30 minutes. Transfer 80 μL of the solution from the bottom end to a 96-well plate, and measure the cell fluorescence value using a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 530 nm (to check membrane integrity).

[0287] The fluorescence value of the Caco-2 cell monolayer membrane is calculated using the following formula:

[0288] LY Leakage = {I acceptor ×0.3 / (I acceptor ×0.3+I donor ×0.1)} × 100%

[0289] I acceptor The fluorescence density on the receiving side (0.3 mL), I donor This refers to the fluorescence density on the administration side (0.1 mL). LY > 1.0% indicates poor monolayer cell membrane compactness, and the corresponding result will be excluded from the evaluation.

[0290] Determine the peak areas of the compound on the administration and reception sides. Calculate the apparent permeability coefficient (P0) of the compound. app(Unit: cm / s) and Efflux ratio (ER):

[0291] P app ={V A ×[drug] acceptor / (Area×incubation time×[drug] initial dono}

[0292] V A The volume of the receiving solution is 0.3 mL for A→B and 0.1 mL for B→A. Area is the area of ​​the Transwell-96-well plate membrane (0.143 cm²). 2 ); incubation time is the incubation period (unit: s).

[0293]

[0294] P app (B-A) P represents the apparent permeability from the basal end to the apex. app (A-B) The apparent permeability coefficient is measured from the top to the base.

[0295] The calculated apparent permeability coefficient and efflux ratio of the compound disclosed herein are shown in Table 5, indicating that the compound disclosed herein has good Caco-2 permeability.

[0296] Table 5. Apparent permeability and efflux ratio of the compounds disclosed herein

[0297]

[0298] Test Example 6: Inhibitory effect of the disclosed compound on the activities of CYP2C9, CYP2D6, and CYP3A4 enzymes

[0299] The inhibition of CYP2C9, CYP2D6, and CYP3A4 enzyme activities by the compounds of this invention was determined using the following experimental method.

[0300] Experimental materials and instruments

[0301] 1. Human liver microsomes (Corning 452117)

[0302] 2. Na2HPO4 (Shanghai Test No. 20040618)

[0303] 3. KH2PO4 (Shanghai Test No. 10017608)

[0304] 4. NADPH (Roche diagnostics gmbh 10107824001)

[0305] 5. Positive substrates: diclofenac sodium (Dalian Meilun MB1277), dextromethorphan hydrobromide (Dalian Meilun MB1568), and midazolam (Cerilliant M-908).

[0306] 6. Positive inhibitors: sulfamethoxazole (Dalian Meilun MB3276), quinidine (Dalian Meilun MB1702), and ketoconazole (Dalian Meilun MB1132).

[0307] 7. Tolbutamide (Sigma-aldrich T0891-100G)

[0308] 8. AB Sciex Triple Quad 5500+ Liquid Chromatography-Mass Spectrometry (LC-MS / MS) System

[0309] II. Experimental Procedure

[0310] 1. Preparation of 100 mM phosphate-buffered saline (PBS): Weigh 7.098 g Na₂HPO₄ and dissolve it in 500 mL of pure water by sonication to obtain solution A. Weigh 3.400 g KH₂PO₄ and dissolve it in 250 mL of pure water by sonication to obtain solution B. Slowly add solution B to solution A on a stirrer until the pH reaches 7.4 to prepare a 100 mM PBS buffer.

[0311] 2. Prepare a 10 mM NADPH solution using 100 mM PBS buffer. Dilute 10 mM of the stock solution of the disclosed compounds with DMSO to obtain 200× concentration working solutions of the compounds (10000, 3333.3, 1111.1, 370.37, 123.46, 41.15, 13.7, 0 μM). Dilute the positive inhibitor stock solution with DMSO to obtain 200× concentration positive inhibitor working solutions (sulfamethoxazole, 1000, 300, 100, 30, 10, 3, 0 μM; quinidine / ketoconazole, 100, 30, 10, 3, 1, 0.3, 0 μM). Prepare 200× concentration substrate working solutions (1200 μM diclofenac, 400 μM dextromethorphan, and 200 μM midazolam) using water, acetonitrile, or acetonitrile / methanol.

[0312] 3. Take 2 μL of 20 mg / mL liver microsomal solution, 1 μL of substrate working solution, 1 μL of compound working solution, and 176 μL of PBS buffer, mix well, and pre-incubate in a 37 °C water bath for 10 minutes. For the positive control group, add 1 μL of positive inhibitor working solution instead of the compound working solution. Simultaneously, pre-incubate together with 10 mM NADPH solution in a 37 °C water bath for 10 minutes. After 10 minutes, add 20 μL of NADPH to each well to start the reaction, incubating at 37 °C for 5 minutes (CYP2C9), 20 minutes (CYP2D6), or 5 minutes (CYP3A4). All incubated samples should be in duplicate. After the appropriate incubation time, add 400 μL of ice-cold methanol containing the internal standard (50 ng / mL tolbutamide) to all samples to terminate the reaction. Vortex to mix, and centrifuge at 3220 g, 4 °C for 30 minutes. After centrifugation, transfer 50 μL of supernatant to the sample plate, add 100 μL of ultrapure water and mix well for LC-MS / MS analysis.

[0313] The IC50 values ​​of the compound of this invention against CYP2C9, CYP2D6, and CYP3A4 were calculated using Excel XLfit 5.3.1.3. 50 The test data showed that the compound disclosed in this paper has no significant inhibitory effect on CYP2C9, CYP2D6, and CYP3A4.

[0314] Test Example 7: Determination of the potential inhibitory effect of the disclosed compounds on the voltage-gated potassium ion channel hERG.

[0315] This experiment used the Chinese hamster ovary (CHO) cell line (B'SYS GmbH) stably expressing the hERG potassium channel. hERG-CHO cells were clamped in a whole-cell voltage-clamp pattern using a SyncroPatch 384i / 384 (Nanion) automated patch-clamp system, and hERG currents were induced by appropriate voltages. The test compounds were then tested: cells were first perfused six times with extracellular fluid containing 0.1% DMSO. The measured stable hERG current was used as the baseline, and the baseline current value was the mean of five stable sampling points (tail current magnitude). 空白 After the hERG current stabilized, five concentration gradients of the test compound (0.37, 1.11, 3.33, 10, and 30 µM) were perfused around the cells. The cells were allowed to fully react with the compound for 10 minutes while hERG currents were recorded simultaneously. Once the current stabilized, five stable hERG current values ​​were read, and their average was taken as the final current value at the specific concentration (tail current magnitude). 化合物Cisapride (Sigma (C4740)) was used as a positive control for simultaneous assays at six concentrations (0.001, 0.004, 0.012, 0.037, 0.111, and 0.333 µM) to verify the stability of the test cells and the accuracy of the results. After testing the compounds, 450 nM of Dopiride (Beijing Yipulis Technology Development Co., Ltd., D525700) was added to all test cells to completely suppress their currents, serving as a complete positive control for these cells (tail current size). 阳性对照 Finally, the tail current suppression rate is calculated using the following formula: {Tail current suppression rate (%) = [1 - (tail current magnitude)]} 化合物 - Tail current magnitude 阳性对照 ) / (tail current magnitude) 空白 - Tail current magnitude 阳性对照 The dose-response curve was then fitted using Graphpad Prism 8.0 software, and the IC was calculated. 50 value.

[0316] Test data indicate that the disclosed compound hERG has poor inhibitory activity and low potential safety risks in the future.

[0317] Test Example 8: Detection of the pharmacokinetic properties of the disclosed compounds in mice.

[0318] Experimental materials

[0319] 1. Healthy adult male CD-1 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0320] 2. PEG400 (polyethylene glycol 400), HP-β-CD (hydroxypropyl-β-cyclodextrin), hydrochloric acid, tolbutamide, methanol, acetonitrile, propylene glycol and formic acid were purchased from Merck (USA).

[0321] 3. K2EDTA anticoagulant tubing was purchased from Jiangsu Xinkang Medical Equipment Co., Ltd.

[0322] II. Experimental Methods

[0323] 1. Drug preparation: Weigh a certain amount of the disclosed compound and dissolve it in 20% PEG400 + 4% 1M HCl + 76% (20% HP-β-CD) to prepare a 1 mg / mL solution for gavage administration. The gavage solution is then diluted with 20% HP-β-CD to 0.2 mg / mL for intravenous administration.

[0324] 2. Administration methods: Gavage group: CD-1 mice were administered the drug via gavage without fasting, at a dose of 10 mg / kg. Intravenous group: CD-1 mice were administered the drug via intravenous injection without fasting, at a dose of 1 mg / kg.

[0325] 3. Animal experiments: After oral or intravenous administration to mice, blood samples of more than 20 µL were collected from the orbital cavity at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8 and 24 h after administration and added to K2EDTA anticoagulant tubes. The plasma was separated by centrifugation at 12000 rpm, 4 ℃ for 5 minutes and stored at -20℃.

[0326] 4. Determination of the content of the analyte compound in mouse plasma after gavage administration of different concentrations of the drug: The sample was thawed at room temperature and vortexed for 1 min; 10 µL was quantitatively transferred to a 2 mL 96-well plate, 50 µL of internal standard solution (10 ng / mL tolbutamide acetonitrile solution) and 50 µL of precipitant (acetonitrile:methanol 7:3) were added, and the mixture was shaken (1000 rpm × 3 min); centrifuged (4000 rpm × 15 min), and 80 µL of the supernatant was transferred to a 2 mL 96-well plate, 80 µL of diluent (water) was added, and the mixture was shaken well (1000 rpm × 3 min). Quantitative detection was performed using an LC-MS / MS system (AB Sciex Triple Quad 6500+).

[0327] 5. Data Processing: Pharmacokinetic parameters were calculated using the non-compartmental model statistical moment method with Phoenix WinNonlin 8.0 software (Certara, USA). The test data are shown in Table 6, indicating that the compound disclosed herein has good oral absorption in mice.

[0328] Table 6. Pharmacokinetic data of the compounds disclosed herein in CD-1 mice.

[0329]

[0330] Test Example 9: Detection of the pharmacokinetic properties of the disclosed compound in rats.

[0331] Experimental materials

[0332] 1. Healthy adult male SD rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0333] 2. PEG400 (polyethylene glycol 400), HP-β-CD (hydroxypropyl-β-cyclodextrin), hydrochloric acid, tolbutamide, methanol, acetonitrile, propylene glycol and formic acid were purchased from Merck (USA).

[0334] 3. K2EDTA anticoagulant tubing was purchased from Jiangsu Xinkang Medical Equipment Co., Ltd.

[0335] II. Experimental Methods

[0336] 1. Drug preparation: Weigh a certain amount of the disclosed compound, dissolve it in 20% PEG400 + 4% 1M HCl + 76% (20% HP-β-CD) to prepare a 1 mg / mL solution for oral administration.

[0337] 2. Administration method: SD rats were administered the drug via gavage after fasting, with a dosage of 10 mg / kg.

[0338] 3. Animal experiments: After oral administration to rats, 100 µL of blood was collected from the jugular vein at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8 and 24 h after administration and added to an anticoagulant tube. The plasma was separated by centrifugation at 12000 rpm, 4 ℃ for 5 minutes and stored at -20 ℃.

[0339] 4. Determination of the content of the target compound in rat plasma after gavage administration of different concentrations of the drug: The sample was thawed at room temperature and vortexed for 1 min; 10 µL was quantitatively transferred to a 2 mL 96-well plate, 50 µL of internal standard solution (10 ng / mL tolbutamide acetonitrile solution) and 50 µL of precipitant (acetonitrile:methanol 7:3) were added, and the mixture was shaken (1000 rpm × 3 min); centrifuged (4000 rpm × 15 min), and 80 µL of the supernatant was transferred to a 2 mL 96-well plate, 80 µL of diluent (water) was added, and the mixture was shaken well (1000 rpm × 3 min). Quantitative detection was performed using an LC-MS / MS system (AB Sciex Triple Quad 6500+).

[0340] 5. Data Processing: Pharmacokinetic parameters were calculated using the non-compartmental model statistical moment method with Phoenix WinNonlin 8.0 software (Certara, USA). The test data are shown in Table 7, indicating that the compound disclosed herein has good oral absorption in rats.

[0341] Table 7. Pharmacokinetic data of the disclosed compounds in SD rats

[0342]

Claims

1. The compound represented by formula (I) or a pharmaceutically acceptable salt thereof, in: R 1 Selected from one or more R a The following groups are substituted: pyridinyl, pyridoneyl, or ; R 3 Selected from H, deuterium, halogen, CN, OH, or C1-C4 alkyl, wherein the OH or C1-C4 alkyl is optionally converted by one or more R c replace; R a Selected from halogens, CN, =O, OH, C1-C4 alkyl, C3-C6 cycloalkyl, 4-7 membered heterocyclic groups, or phenyl groups, wherein the OH, C1-C4 alkyl, C3-C6 cycloalkyl, 4-7 membered heterocyclic groups, or phenyl groups are optionally converted by one or more R groups. 1a replace; R 1a R c The groups are independently selected from deuterium, halogens, OH, CN, NH2, =O, C1-C6 alkyl, C3-C6 cycloalkyl, or 4-8 membered heterocyclic groups, wherein the OH, NH2, C1-C6 alkyl, C3-C6 cycloalkyl, or 4-8 membered heterocyclic group is optionally surrounded by one or more R d replace; R d Selected from deuterium, halogen, OH, NH2, =O, C1-C3 alkyl, C1-C3 alkoxy, CN, COOH, C(O)(C1-C3 alkyl), CONH2, C(O)O(C1-C3 alkyl), C3-C6 cycloalkyl or 4-6 membered heterocyclic groups; The Linker is a connection unit, and its structure is as follows: ,in Representative and Connected keys; X 1 X 2 They are selected independently from the bond, -O-, -N(R) 10 )-, -C(O)- or C1-C6 alkylene, R 10 It is H or C1-C6 alkyl; Het 1 Het 2 The components are independently selected from bonds, C3-C8 cycloalkyl groups or 4-10 heterocyclic groups, wherein the C3-C8 cycloalkyl groups or 4-10 heterocyclic groups are optionally substituted with halogens or C1-C6 alkyl groups; The DIM is ;where R 2 It is selected from hydrogen, halogen, NO2, NH2, OH, COOH, C1-C6 alkyl or C1-C6 alkoxy.

2. The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, wherein, R 1 Selected from one or more R a The following groups are substituted: pyridone group or Or, R 1 Selected from one or more R a Substituted pyridinone group; or, R 1 Selected from , , or Or, R 1 for .

3. The compound of formula (I) according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein, R a Selected from CN, =O, C1-C4 alkyl, C3-C6 cycloalkyl, or 4-7 membered heterocyclic groups, wherein the C1-C4 alkyl, C3-C6 cycloalkyl, or 4-7 membered heterocyclic group is optionally surrounded by one or more R groups. 1a Replace; or, R a Selected from CN, C1-C4 alkyl or cyclopropyl, wherein the C1-C4 alkyl or cyclopropyl group is optionally converted by one or more R 1a Replace; or, R a Selected from methyl, Or CN.

4. The compound of formula (I) according to any one of claims 1-3, or a pharmaceutically acceptable salt thereof, wherein, R 3 Selected from H, halogen, OH, or C1-C4 alkyl, wherein the OH or C1-C4 alkyl is optionally converted by one or more R c Replace; or, R 3 Selected from H or arbitrarily selected by one R c Substituted OH; or, R 3 It is isopropoxy.

5. The compound of formula (I) according to any one of claims 1-4, or a pharmaceutically acceptable salt thereof, wherein, X 1 X 2 Each is independently selected from bonds, -C(O)- or C1-C3 alkylene groups; or, X 1 X 2 Each is independently selected from the bond, -C(O)-, or methylene; or, X 1 X 2 Each is independently selected from either a bond or a methylene group; or, X 1 Selected from -C(O)- or methylene; or, X 1 Methylene; or, X 2 For key.

6. The compound of formula (I) according to any one of claims 1-5, or a pharmaceutically acceptable salt thereof, wherein, Het 1 Het 2 The components are independently selected from C3-C6 cycloalkyl or 4-8 heterocyclic groups, wherein the C3-C6 cycloalkyl or 4-8 heterocyclic groups are optionally substituted with halogens or C1-C6 alkyl groups; or, Het 1 Het 2 The groups are independently selected from 4-8 membered heterocyclic groups, which are optionally substituted with halogens or C1-C6 alkyl groups; or, Het 1 Het 2 Each of the following groups is independently selected from piperazine, piperidinyl, aziridine, or alkyl groups. The piperazine-based, piperidinyl, azacyclic butyl or Optionally substituted with halogens or C1-C6 alkyl groups; or, Het 1 Het 2 Each is independently selected from piperazine, piperidinyl, or... Or, Het 1 Selected from piperazine or Or, Het 2 It is piperidinyl.

7. The compound of formula (I) according to any one of claims 1-6, or a pharmaceutically acceptable salt thereof, wherein, The Linker structure is as follows: ,in Representative and A connected key; or, the Linker is selected from... or ,in Representative and Connected keys.

8. The compound of formula (I) according to any one of claims 1-7, or a pharmaceutically acceptable salt thereof, wherein, R 2 Selected from halogens, NO2, NH2, OH, COOH, C1-C6 alkyl or C1-C6 alkoxy groups; or, R 2 Selected from hydrogen, halogen, C1-C6 alkyl, or C1-C6 alkoxy; or, R 2 Selected from halogens, C1-C6 alkyl groups, or C1-C6 alkoxy groups; or, R 2 Selected from H, F, or methoxy; or, R 2 Selected from F or methoxy.

9. The compound of formula (I) according to any one of claims 1-8, or a pharmaceutically acceptable salt thereof, wherein, The DIM is selected from the following structures: , , , , or .

10. The compound of formula (I) according to any one of claims 1-9, or a pharmaceutically acceptable salt thereof, wherein, The compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from the following compounds of formula (I-1) or pharmaceutically acceptable salts thereof: （I-1） Among them, R a Linker and DIM are as defined in any one of claims 1-9.

11. Selected from the following compounds or their pharmaceutically acceptable salts: or .

12. A pharmaceutical composition comprising a compound of formula (I) according to any one of claims 1-11 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

13. Use of any compound of claims 1-11 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 12 in the preparation of a medicament for the prevention or treatment of IRAK4-mediated diseases; preferably, the IRAK4-mediated diseases are selected from tumors, inflammatory diseases, neurodegenerative diseases or autoimmune diseases.

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