Protein degradation targeting chimera molecules and uses thereof

Abstract

The present application provides a protein degradation targeting chimera molecule and application thereof, the protein degradation targeting chimera molecule has the structure shown in formula (I), the protein degradation targeting chimera molecule of the present application can significantly degrade IRAK kinase, which shows efficient, high selective IRAK4 and / or IRAK1 degradation effect, and can be used for treating or preventing diseases related to IRAK4 and / or IRAK1 activity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and relates to a protein degradation targeting chimera molecule and application thereof. BACKGROUND

[0002] Protein kinases are enzymes that are widely present in cells and on cell surfaces. So far, nearly 600 protein kinases have been discovered and identified. They belong to a structurally related protein family, and known members thereof are related to almost all cell signaling activities. The catalytic function of protein kinases is to transfer the γ-phosphoryl group in the ATP molecule to the specific threonine, serine or tyrosine group of a target protein, so as to change the conformation of the target protein, and cause the function of the target protein to change from a static state to an activated state. The signal transmission and regulation involving protein kinases play an extremely important role in the normal functions of cells and organs, including cell growth, differentiation, proliferation, angiogenesis, apoptosis, cytoskeleton arrangement, regulation of metabolic reactions, membrane transport and cell movement. In addition, the non-catalytic function of protein kinases also plays an indispensable role, including allosteric effect, subcellular targeting, protein complex scaffold, protein competitive interaction and DNA binding. On the other hand, however, when a gene mutation or overexpression of protein kinases occurs, the disordered protein kinases can cause various pathological changes, including cancer, inflammation, autoimmune diseases, cardiovascular system and nervous system diseases.

[0003] Interleukin-1 receptor-associated kinase (IRAK) refers to a class of serine / threonine kinases that can bind to interleukin-1 receptor (IL-1R) under the induction of pro-inflammatory cytokine interleukin-1 (IL-1), and plays an important role in the regulation of intracellular signal transduction network for controlling inflammation. Currently, there are IRAK1, IRAK2, IRAK3 (also known as IRAK-M) and IRAK4. The four members of the IRAK family all have a serine and threonine kinase domain (KD) and a conserved death domain (DD). Except for IRAK4, the other three IRAK kinases have a long C-terminal domain. Among them, IRAK1 and IRAK4 play a positive regulatory role in the Toll-like receptor (TLR)-mediated signaling pathway, and IRAK2 and IRAK3 play a negative regulatory role in the TLR-mediated signaling pathway. IRAK4 has both kinase activity and scaffold function, is considered to be the first protein kinase activated downstream of IL-1 receptor and all TLRs except TLR3, and initiates signal transduction in the innate immune system through the rapid activation of IRAK1 and the slower activation of IRAK2, activates the NF-κB and JNK signaling pathways downstream, and plays an important role in human inflammatory response and tumor. Therefore, the IRAK family has a broad clinical application prospect, and can be used as an effective potential target for the treatment of various autoimmune system diseases and tumor diseases.

[0004] Targeted protein degradation (TPD) is an emerging technology in the field of small molecule drug design, which can utilize the cellular protein degradation machinery to modulate the activity and abundance of specific proteins in the body, and attack diseases by targeting the degradation of pathogenic proteins, providing a new strategy for the treatment of various diseases including cancer and neurodegenerative diseases. Compared with traditional small molecule inhibitors, TPD drugs do not need to rely on the active site of the protein to function, have potential advantages in dosage, side effects and targeting "undruggable" proteins, and can overcome the drug resistance of existing small molecule inhibitors, greatly expanding the range of druggable targets. Proteolysis-targeting chimera (PROTAC) is a TPD strategy based on the ubiquitin-proteasome system (UPS). PROTAC is a bifunctional molecule, which contains three parts in structure: a ligand for the protein of interest (POI), a ligand for binding and recruiting E3 ubiquitin ligase, and a linker connecting the two ligands. Mechanistically, the PROTAC molecule can induce the mutual approach of the target protein and the ligase, forming a ternary complex of E3 ligase-PROTAC-target protein, and then through the UPS system, the target protein is ubiquitinated and finally targeted for degradation by the proteasome. PROTAC drug molecules have the advantages of the above TPD, and can work with catalytic amounts, not only inhibiting protein activity, but also degrading the entire protein, thereby disrupting the enzymatic and non-enzymatic functions (such as scaffold functions) or transcriptional functions of the protein. The PROTAC technology greatly broadens the possibilities of drug development in target selection, and shows great prospects in the treatment of diseases such as tumors, autoimmune diseases, neurodegenerative diseases and viral infections.

[0005] Effective and selective PROTAC drug molecules can be used as IRAK degraders to block IRAK-dependent pro-inflammatory signaling, thereby providing therapeutic benefits for diseases such as inflammatory diseases and tumors characterized by dysregulated IRAK kinase activity. Therefore, the development of such molecules is a research focus in the art. SUMMARY

[0006] In view of the deficiencies of the prior art, the purpose of the present application is to provide a protein degradation targeting chimera molecule and its application. The protein degradation targeting chimera molecule of the present application can significantly degrade IRAK kinase, and shows efficient and selective IRAK4 and / or IRAK1 degradation, and can be used for treating or preventing diseases related to IRAK4 and / or IRAK1 activity.

[0007] To achieve the object of the present application, the present application adopts the following technical solutions:

[0008] In a first aspect, the present application provides a protein degradation targeting chimera molecule or a meso, rac, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein the protein degradation targeting chimera molecule has a structure shown in formula (I):

[0009]

[0010] In formula (I), PTM is an IRAK protein targeting moiety, which is a small molecule having inhibitory effect on IRAK kinase activity, and is composed of a small molecule moiety containing an amide and three heteroaromatic rings on the left side of formula (I), ring A and ring B.

[0011] R f is selected from halogen or C1-C3 alkyl, wherein C1-C3 alkyl is unsubstituted or substituted with a deuterium atom and / or a halogen atom; preferably, R f is selected from -CF2CF3, -CF3, -CH3, -CD3, -CF2H, -CF2D, -CFH2 or -CFD2;

[0012] Ring A and ring B are each independently absent or a 4-10 membered cycloalkyl or heterocyclyl group, and contain 0-2 heteroatoms independently selected from N, O and S, and the cycloalkyl or heterocyclyl group is substituted or unsubstituted, and when substituted, the substituents are independently selected from one or at least two groups of deuterium atom, halogen, amino, hydroxyl, thiol, nitro, cyano, carbonyl, carboxyl, ester, alkenyl, alkynyl, alkyl, alkoxy, hydroxyalkyl, alkylthio, alkylamino, cycloalkyl, heterocyclyl, cycloalkoxy, cycloalkylthio, heterocycloalkoxy, heterocycloalkylthio, aryl or heteroaryl;

[0013] LBM is an E3 ubiquitin ligase binding moiety selected from E3 ubiquitin ligase ligands such as CRBN (cereblon), VHL (von Hippel-Lindau), IAP (inhibitor of apoptosis) and MDM2 (murine double minute 2), including but not limited to the following structures:

[0014]

[0015]

[0016] a wavy line represents the point of attachment of a group;

[0017] L is a linker connecting the PTM and the LBM, selected from C1-C 20 alkyl, or C1-C 20 0, 1, 2, 3, 4, 5 or 6 -CH2- groups on alkyl are optionally replaced by one or at least two atoms or groups -O-, -S-, -NR a -, carbonyl, alkenyl, alkynyl, -C(O)R a -, -O(O)CR a -, -C(O)OR a -, -C(O)NR a -, -NHC(O)R a -, -S(O)R a -, -S(O)2R a -, -S(O)NR a -, -S(O)2NR a -, -NHS(O)R a -, -NHS(O)2R a -, -P(O)R a -, -P(O)NR a -, aryl, heteroaryl, cycloalkyl, heterocyclyl, wherein the carbonyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocyclyl are optionally substituted by one or at least two groups selected from a deuterium atom, halogen, amino, hydroxyl, thio, nitro, cyano, carbonyl, carboxyl, ester, alkenyl, alkynyl, alkyl, alkoxy, hydroxyalkyl, alkylthio, alkylamino, cycloalkyl, heterocyclyl, cycloalkoxy, cycloalkylthio, heterocycloalkoxy, heterocycloalkylthio, aryl and heteroaryl; wherein R a are independently selected from H, D, halogen, amino, hydroxyl, thio, nitro, cyano, carbonyl, carboxyl, ester, alkenyl, alkynyl, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, wherein the alkenyl, alkynyl, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl and heteroaryl are optionally substituted by one or at least two groups selected from a deuterium atom, halogen, amino, hydroxyl, thio, nitro, cyano, carbonyl, carboxyl, ester, alkenyl, alkynyl, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl and heteroaryl; or R a form together with the atom to which they are attached a cycloalkyl or heterocyclyl, which is optionally substituted by one or at least two groups selected from a deuterium atom, halogen, amino, hydroxyl, thio, nitro, cyano, carbonyl, carboxyl, ester, alkenyl, alkynyl, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl;

[0018] Preferably, L comprises, but is not limited to, the following structures:

[0019]

[0020]

[0021] x, y and z are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8, and the wavy line represents the point of attachment of the group.

[0022] Preferably, non-limiting embodiments of L include, but are not limited to, L15 and L16 below:

[0023]

[0024] wherein, preferably, x and y are each independently 0, 1, 2, 3, 4 or 5, and the wavy line represents the point of attachment of the group.

[0025] In one preferred embodiment, the protein degradation targeting chimera molecule is a compound of Formula (II):

[0026]

[0027] wherein, R f , L and LBM are as defined in Formula (I).

[0028] In another preferred embodiment, the protein degradation targeting chimera molecule is a compound of Formula (III):

[0029]

[0030] wherein, R f , L and LBM are as defined in Formula (I).

[0031] Exemplary compounds of the present application include, but are not limited to:

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038]

[0039] In a second aspect, the present application provides a pharmaceutical composition comprising the protein degradation targeting chimera molecule as described in the first aspect, or a meso, rac, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.

[0040] In a third aspect, the present application provides use of the protein degradation targeting chimera molecule as described in the first aspect, or a meso, rac, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition as described in the second aspect, in the manufacture of a medicament for treating and / or preventing a disease or disorder associated with IRAK kinase activity.

[0041] Preferably, the disease or disorder associated with IRAK kinase activity is selected from a cancer, a neurodegenerative disease, an autoimmune disease, an inflammatory disease, a viral disease, a cardiovascular disorder, a kidney disease, a liver disease, a metabolic disorder, a genetic disorder, or a proliferative disorder.

[0042] Preferably, the cancer is selected from breast cancer, small cell lung cancer, non-small cell lung cancer, bronchioloalveolar carcinoma, prostate cancer, bile duct cancer, bone cancer, bladder cancer, head and neck cancer, kidney cancer, liver cancer, gastrointestinal tissue cancer, esophageal cancer, ovarian cancer, pancreatic cancer, skin cancer, testicular cancer, thyroid cancer, uterine cancer, cervical and vaginal cancer, leukemia, multiple myeloma, or lymphoma.

[0043] Preferably, the autoimmune disease and inflammatory disease is selected from systemic lupus erythematosus, lupus nephritis, arthritis, psoriasis, Crohn's disease, atopic dermatitis, gout, protein associated periodic syndrome, chronic kidney disease or acute kidney injury, chronic obstructive pulmonary disease, asthma, bronchospasm, or graft versus host disease.

[0044] Preferably, the IRAK kinase is one or a combination of at least two of IRAK1, IRAK2, IRAK3, and IRAK4.

[0045] Terminology

[0046] Unless otherwise indicated, the terms used in the specification and claims have the following meanings.

[0047] In the present application, when referring to a "compound" having a specific structural formula, it is generally also encompassing pharmaceutically acceptable salts, stereoisomers, diastereomers, enantiomers, racemic mixtures, and isotopic derivatives thereof.

[0048] As is known to those skilled in the art, in addition to salts of the compounds, solvates, hydrates are alternative forms in which the compounds can exist, and they can be converted into the stated compounds under certain conditions, and therefore, when a compound is mentioned in the present application, solvates and hydrates thereof are generally included.

[0049] The "pharmaceutically acceptable salts" of the present application refer to salts of the compounds of the present application which are within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio, and are effective for their intended use. The salts can be prepared in situ during the final isolation and purification of the compounds of the present application, or by separately reacting a free base or a free acid with a suitable reagent under conditions known in the art. For example, a free base can be reacted with a suitable acid. Examples of pharmaceutically acceptable acid addition salts are salts of an amino group (amine group) with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid, or salts formed by using other methods known in the art such as ion exchange. The pharmaceutically acceptable salts of the present application can be prepared by conventional methods, for example, by dissolving the compound of the present application in a water-miscible organic solvent (e.g., methanol, ethanol, acetone and acetonitrile), adding an excess of an aqueous solution of an organic or inorganic acid to the mixture, so that the salt is precipitated from the resulting mixture, removing the solvent and the remaining free acid, and then isolating the precipitated salt. Other pharmaceutically acceptable salts include sodium alginate, ascorbate, benzenesulfonate, adipate, camsylate, aspartate, benzoate, bisulfate, borate, butyrate, camphorate, citrate, dodecylsulfate, ethanesulfate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, heptanoate, hexanoate, hydroiodide, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, mesylate, 2-napsylate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate and the like.

[0050] The term "solvate" as used herein means the physical association of one or more solvent molecules (whether organic or inorganic) with a compound of the application. The physical association can be due to hydrogen bonding, to ionic bonding, to van der Waals forces or to other physical forces. In certain instances, the solvent molecules can be incorporated in the crystal lattice of the solid state form. Solvates are included within the scope of the present application. Solvates can comprise stoichiometric or non-stoichiometric amounts of the solvent molecules. "Solvate" encompasses both solution-phase and isolatable solvates. Exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates. Methods for preparing solvates are known in the art.

[0051] The term "stereoisomers" as used herein means that the compounds of the application can occur in different spatial or physical arrangements that are not enantiomeric or mirror images of each other. Stereoisomers are distinguishable from each other only by their physical orientations, i.e., "handedness" and cannot be distinguished as mirror images of each other. Stereoisomers include enantiomeric pairs of compounds. "Enantiomers" are pairs of molecules that are non-superimposable mirror images of one another. Stereoisomers also include pairs of compounds that are not enantiomers, where one compound is the mirror image of the other, but the mirror image is not a non-superimposable mirror image. Such non-superimposable mirror images are distinguishable from each other only by their physical orientations and are not enantiomeric pairs. "Diastereomers" are structural isomers that have at least two identical atom groups in common, but are not enantiomeric pairs. Diastereomers have the same molecular formula but differ in the spatial arrangement of their atoms. Diastereomers are not mirror images of each other.

[0052] In particular, the compounds of the application can exist in tautomeric forms that have different hydrogen attachments. For example, a ketone and its enol form are tautomers of the ketone-enol tautomer. Each tautomer and mixtures thereof are included within the scope of the compounds of the application. All enantiomers, diastereomers, racemics, meso, cis, trans, tautomers, geometric isomers, epimers, and mixtures thereof, of all compounds are included within the scope of the application.

[0053] An "isotopic derivative" of the application means a molecule of the compounds of the application that is isotopically labeled. The isotopes that are typically used for isotopic labeling are: 2 H and 3 H; carbon isotopes: 11 C, 13 C and 14 C; chlorine isotopes: 35 Cl and 37 Cl; fluorine isotopes: 18 F; iodine isotopes: 123 I and 125 I; nitrogen isotopes: 13 N and15 N; oxygen isotopes: 15 O, 17 O and 18 O; sulfur isotopes: 35 S. These isotope-labeled compounds can be used to study the distribution of pharmaceutical molecules in tissues. Certain heavy isotopes (such as deuterium) 2 Substitution with H can enhance metabolic stability and prolong the half-life, thereby reducing the dosage and providing therapeutic advantages. Isotope-labeled compounds are generally synthesized from labeled starting materials using known synthetic techniques, just like non-isotope-labeled compounds.

[0054] In this invention, the given chemical formula or name shall encompass all stereo and optical isomers and racemic derivatives containing such isomers. Unless otherwise specified, all chiral (enantiomers and diastereomers) and racemic forms are within the scope of this invention. Numerous geometric isomers, such as C=C double bonds, C=N double bonds, and ring systems, may also be present in the compounds, and all such stable isomers are covered within this invention. This invention describes cis- and trans- (or E- and Z-) geometric isomers of the compounds of this invention, which can be separated into mixtures of isomers or into separate isomeric forms.

[0055] The compounds of this invention can be separated in optically active or racemic form. All methods used to prepare the compounds of this invention and the intermediates therein are considered part of this invention. When preparing enantiomers or diastereomers, they can be separated by conventional methods (e.g., by chromatography or fractional crystallization). It should be understood that all possible tautomer forms are included within this invention. The compounds of this invention are commercially available when they are known in the prior art.

[0056] The term "alkyl" refers to a branched or straight-chain saturated aliphatic hydrocarbon group having a specified number of carbon atoms. The alkyl groups used in this invention are preferably C1-C. 12 Alkyl, C1-C 10alkyl, C1-C8alkyl, more preferably C1-C6alkyl, particularly preferably C1-C4alkyl, especially C1-C3alkyl. For example, "C1-C6alkyl" denotes an alkyl group having from 1 to 6 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl (e.g. n-propyl and i-propyl), butyl (e.g. n-butyl, i-butyl, t-butyl) and pentyl (e.g. n-pentyl, i-pentyl, neopentyl). The alkyl group can be substituted or non-substituted, and when substituted, the substituent(s) can be substituted at any available attachment point, preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, carbonyl, carboxy or carboxylic ester group. For C1-C 12 alkyl, C1-C8alkyl, more preferably C1-C6alkyl, particularly preferably C1-C4alkyl, especially C1-C3alkyl. For example, "C1-C6alkyl" denotes an alkyl group having from 1 to 6 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl (e.g. n-propyl and i-propyl), butyl (e.g. n-butyl, i-butyl, t-butyl) and pentyl (e.g. n-pentyl, i-pentyl, neopentyl). The alkyl group can be substituted or non-substituted, and when substituted, the substituent(s) can be substituted at any available attachment point, preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, carbonyl, carboxy or carboxylic ester group. For C1-C

[0057] The term "alkoxy" means -O-(alkyl) or -O-(non-substituted cycloalkyl). For example, "C1-C6alkoxy" means including C1, C2, C3, C4, C5, C6alkoxy. Preferred alkoxy groups are C1-C 10 alkyl, C1-C8alkyl, more preferably C1-C6alkyl, particularly preferably C1-C4alkyl, especially C1-C3alkyl. For example, "C1-C6alkyl" denotes an alkyl group having from 1 to 6 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl (e.g. n-propyl and i-propyl), butyl (e.g. n-butyl, i-butyl, t-butyl) and pentyl (e.g. n-pentyl, i-pentyl, neopentyl). The alkyl group can be substituted or non-substituted, and when substituted, the substituent(s) can be substituted at any available attachment point, preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, carbonyl, carboxy or carboxylic ester group. For C1-C 10 alkyl, C1-C8alkyl, more preferably C1-C6alkyl, particularly preferably C1-C4alkyl, especially C1-C3alkyl. For example, "C1-C6alkyl" denotes an alkyl group having from 1 to 6 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl (e.g. n-propyl and i-propyl), butyl (e.g. n-butyl, i-butyl, t-butyl) and pentyl (e.g. n-pentyl, i-pentyl, neopentyl). The alkyl group can be substituted or non-substituted, and when substituted, the substituent(s) can be substituted at any available attachment point, preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, carbonyl, carboxy or carboxylic ester group. For C1-C

[0058] The term "alkenyl" refers to an alkyl group as defined above consisting of at least two carbon atoms and at least one carbon-carbon double bond, for example, ethenyl, 1 -propenyl, 2-propenyl, 1 -, 2-, or 3-butenyl, and the like. The alkenyl group can be substituted or unsubstituted, and when substituted, the substituent(s) are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, haloalkyl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

[0059] The term "alkynyl" refers to an alkyl group as defined above consisting of at least two carbon atoms and at least one carbon-carbon triple bond, for example, ethynyl, propynyl, butynyl, and the like. The alkynyl group can be substituted or unsubstituted, and when substituted, the substituent(s) are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, haloalkyl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

[0060] The term "halo" or "halogen" includes fluorine, chlorine, bromine, and iodine. In the present invention, one or more halogens can each be independently selected from fluorine, chlorine, bromine, and iodine.

[0061] The term "haloalkyl" refers to branched and straight-chain saturated aliphatic hydrocarbon groups having the indicated number of carbon atoms and substituted with one or more halogens. Examples of haloalkyl include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, trichloromethyl, pentafluoroethyl, pentachloroethyl, 2,2,2-trifluoroethyl, heptafluoropropyl, and heptafluoropropyl.

[0062] The term "carbonyl" or "oxo" refers to an organic functional group (C=0 or C(O)) consisting of two atoms of carbon and oxygen linked by a double bond.

[0063] The term "benzyl" refers to a -CH2-phenyl group or "Bn".

[0064] The term "hydroxyl" refers to an -OH group.

[0065] The term "amino" refers to -NH2.

[0066] The term "cyano" refers to -CN.

[0067] The term "nitro" refers to -NO2.

[0068] The term "carboxyl" refers to -C(O)OH.

[0069] The term "thiol" refers to -SH.

[0070] The term "ester" or "carboxylic acid ester" means -C(O)O-(alkyl) or -C(O)O-(cycloalkyl), wherein alkyl and cycloalkyl are as defined above.

[0071] The term "acyl" means a compound containing a -C(O)R group, wherein R is alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl.

[0072] The term "cycloalkyl" means a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring comprising from 3 to 20 carbon atoms, preferably C3-C8 cycloalkyl groups for the present application. Monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, and cyclooctyl groups, and polycyclic cycloalkyl groups include, but are not limited to, spirocyclic, fused, and bridged cycloalkyl groups, such as norbornyl groups.

[0073] The term "spirocycloalkyl" means a 5- to 20-membered polycyclic group containing two or more cyclic structures sharing one atom between the rings (referred to as a spiro atom), the rings containing one or more double bonds, but none of the rings having a fully conjugated pi-electron system. Preferably 6- to 14-membered, more preferably 7- to 10-membered. Spirocycloalkyl groups are classified as mono-, bi-, or polycyclic depending on the number of spiro atoms shared between the rings, preferably mono- and bi-cyclic, preferably 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 5-membered, or 5-membered / 6-membered. Non-limiting examples of "spirocycloalkyl" groups include, but are not limited to:

[0074]

[0075] The term "fused cycloalkyl" means a 5- to 20-membered polycyclic group containing two or more cyclic structures sharing a pair of adjacent atoms between the rings, one or more rings can contain one or more double bonds, but none of the rings having a fully conjugated pi-electron system, preferably 6- to 14-membered, more preferably 7- to 10-membered. Fused cycloalkyl groups are classified as bi-, tri-, tetra-, or polycyclic depending on the number of rings comprising the group, preferably bi- or tri-cyclic, more preferably 5-membered / 5-membered or 5-membered / 6-membered bi-cyclic alkyl groups. Non-limiting examples of "fused cycloalkyl" groups include, but are not limited to:

[0076]

[0077] The term "bridged cycloalkyl" refers to a polycyclic group of 5 to 20 members containing two or more cyclic structures which share two non-directly attached atoms with each other, one or more rings can contain one or more double bonds, but no ring has a fully conjugated system of π electrons, preferably 6 to 14 members, more preferably 7 to 10 members. It can be classified as bicyclic, tricyclic, tetracyclic or polycyclic bridged cycloalkyl depending on the number of rings comprising the ring system, preferably bicyclic, tricyclic or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of "bridged cycloalkyl" include, but are not limited to:

[0078]

[0079] The cycloalkyl ring can be fused to an aryl, heteroaryl or heterocyclyl ring, wherein the ring attached to the parent structure is a cycloalkyl, non-limiting examples of which include, but are not limited to:

[0080]

[0081] The cycloalkyl group can be optionally substituted or unsubstituted, when substituted, the substituents are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carbonyl, carboxyl or carboxylate.

[0082] The term "heterocyclyl" or "heterocycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic ring system of cyclic hydrocarbon substituent groups containing 3 to 20 ring atoms, one or more of which are heteroatoms selected from N, O and S (N and S heteroatoms can optionally be oxidized), but excluding ring moieties of -O-O-, -O-S- or -S-S-, the remaining ring atoms being carbon. Preferably, 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; most preferably, 3 to 8 ring atoms, of which 1 to 3 are heteroatoms; most preferably, 5 to 7 ring atoms, of which 1 to 2 or 1 to 3 are heteroatoms. Examples of monocyclic heterocyclyl groups include, but are not limited to, tetrahydrofuranyl, tetrahydrothiophenyl, dihydroimidazolyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl and tetrahydropyranyl, polycyclic heterocyclyl groups include, but are not limited to, spirocyclic, fused and bridged heterocyclyl groups.

[0083] The term "spiroheterocyclyl" refers to a polycyclic group of 5 to 20 members containing two or more cyclic structures sharing a single atom between the rings, containing one or more double bonds in the ring but no ring having a fully conjugated pi-electron system, and wherein one or more ring atoms are heteroatoms selected from N, O, and S (N and S heteroatoms can optionally be oxidized), with the remaining ring atoms being carbon. Preferably, 6 to 14 members, more preferably 7 to 10 members. Spiroheterocyclyl groups are classified as mono-, bi-, or polycyclic depending on the number of rings sharing a spiro atom between the rings, preferably mono- and bi-cyclic. More preferably, 4-member / 4-member, 4-member / 5-member, 4-member / 6-member, 5-member / 5-member, or 5-member / 6-member mono- spiroheterocyclyl groups. Non-limiting examples of "spiroheterocyclyl" groups include, but are not limited to:

[0084]

[0085] The term "fused heterocyclyl" refers to a polycyclic group of 5 to 20 members containing two or more cyclic structures sharing a pair of adjacent atoms between the rings, one or more rings can contain one or more double bonds but no ring having a fully conjugated pi-electron system, wherein one or more ring atoms are heteroatoms selected from N, O, and S (N and S heteroatoms can optionally be oxidized), with the remaining ring atoms being carbon. Preferably, 6 to 14 members, more preferably 7 to 10 members. Fused heterocyclyl groups are classified as bi-, tri-, tetra-, or polycyclic depending on the number of rings, preferably bi- or tri-cyclic, more preferably 5-member / 5-member or 5-member / 6-member bi-cyclic fused heterocyclyl groups. Non-limiting examples of "fused heterocyclyl" groups include, but are not limited to:

[0086]

[0087] The term "bridged heterocyclyl" refers to a polycyclic group of 5 to 20 members containing two or more cyclic structures sharing two non-adjacent atoms between the rings, one or more rings can contain one or more double bonds but no ring having a fully conjugated pi-electron system, wherein one or more ring atoms are heteroatoms selected from N, O, and S (N and S heteroatoms can optionally be oxidized), with the remaining ring atoms being carbon. Preferably, 6 to 14 members, more preferably 7 to 10 members. Bridged heterocyclyl groups are classified as bi-, tri-, tetra-, or polycyclic depending on the number of rings, preferably bi-, tri-, or tetra-cyclic, more preferably bi- or tri-cyclic. Non-limiting examples of "bridged heterocyclyl" groups include, but are not limited to:

[0088]

[0089] The heterocyclyl ring can be fused to an aryl, heteroaryl, or cycloalkyl ring, wherein the ring that is attached to the parent structure is a heterocyclyl, non-limiting examples of which include, but are not limited to:

[0090]

[0091] Heterocyclyl can be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, haloalkyl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carbonyl, carboxyl, or carboxylate.

[0092] The term "aryl" refers to a monocyclic, bicyclic, or tricyclic ring system having a total of 6 to 14 ring atoms, wherein at least one ring in the system is aromatic and wherein each ring in the system contains 3 to 7 ring atoms. In certain embodiments of the application, "aryl" refers to an aromatic ring system, including, but not limited to, phenyl, naphthyl, biphenyl, indanyl, 1-naphthyl, 2-naphthyl, and tetrahydronaphthyl. Aryl groups of the present application are preferably C6-Ci2aryl. In certain embodiments of the application, "aryl" refers to a phenyl group. 10 Heterocyclyl can be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, haloalkyl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carbonyl, carboxyl, or carboxylate.

[0093] The term "heteroaryl" means a stable 3-, 4-, 5-, 6-, or 7-membered aromatic monocyclic or 7-, 8-, 9-, 10-membered aromatic bicyclic or polycyclic heterocyclic ring which is fully or partially unsaturated and which contains carbon atoms and 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S (N and S heteroatoms can be optionally oxidized). Nitrogen atoms are substituted or unsubstituted (i.e., N or NR, where R is H or, if defined, another substituent). The heterocyclic ring can be attached through any heteroatom or carbon atom that results in a stable structure. Heteroaryl groups as described herein can be substituted on carbon or nitrogen atoms, if the resulting compound is stable. Nitrogen atoms in the heterocycle can optionally be quaternized. Preferably, when the total number of S and O atoms in the heterocycle exceeds one, then these heteroatoms are not adjacent one another. Preferably, the total number of S and O atoms in the heterocycle is not more than one. When the term "heterocycle" is used, it is intended to encompass heteroaryl groups. Examples of heteroaryl groups include, but are not limited to, acridinyl, imidazolyl, furanyl, thienyl, oxazolyl, thiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, indolyl, indolizinyl, indazolyl, pyrimidinyl, phenazinyl, piperazinyl, piperidinyl, purinyl, pyranyl, pyrazinyl, pyrrolyl, and quinolinyl. The term "heteroaryl" can also include biaryl structures formed from the above defined "aryl," "heterocycle," or "cycloalkyl" groups with monocyclic "heteroaryl" groups, such as, but not limited to, "-phenylbipyridyl-," "-phenylbipyrimidinyl-," "-pyridylbinaphthyl-," "-pyrimidylbinaphthyl-," and "-pyridylbipyrimidinyl-," where the present application also includes spiro, fused, and bridged compounds containing, for example, the above heterocycles.

[0094] "Optional" or "optionally" as used herein means that the subsequently described event or circumstance can or can not occur, such that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "heterocycle optionally substituted with alkyl" means that alkyl can or can not be present, such that the description includes instances where the heterocycle is substituted with alkyl and instances where the heterocycle is not substituted with alkyl.

[0095] "Substituted" or "substitution" as used herein means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3, of a group are each, independently of one another, replaced with a corresponding number of substituents, provided that the normal valence is maintained and that the substitution results in a stable compound. It goes without saying that the substituents are only in their possible chemical positions, which a person skilled in the art is able to determine (experimentally or theoretically) as possible or impossible without undue effort. For example, an amino or hydroxyl group with a free hydrogen can not be stable when bound to a carbon atom with an unsaturated (e.g., olefinic) bond.

[0096] A "pharmaceutical composition" means a mixture of one or more of the compounds described herein or physiologically / pharmaceutically acceptable salts or prodrugs thereof with other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of the active ingredient to a subject, and to facilitate absorption of the active ingredient, thereby facilitating biological activity.

[0097] Compared with the prior art, the present application has the following beneficial effects:

[0098] The protein degradation targeting chimera molecule of the present application can significantly degrade IRAK kinase, which shows high-efficiency and high-selectivity IRAK4 and / or IRAK1 degradation, and can be used for treating or preventing diseases related to IRAK4 and / or IRAK1 activity. BRIEF DESCRIPTION OF DRAWINGS

[0099] Figure 1 A graph of the degradation test results of the compound on IRAK4 in THP-1 cells;

[0100] Figure 2 A graph of the test results of the effect of the compound on the downstream signal molecules of the TLR signaling pathway in THP-1 cells. DETAILED DESCRIPTION

[0101] The technical solutions of the present application are further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments are only used to help understand the present application and should not be regarded as specific limitations on the present application.

[0102] The embodiments of the present application, including the descriptions provided in the embodiments, are intended to illustrate the implementation of the present application, and are not intended to limit the scope of any claims. According to the present application, those skilled in the art will understand that many changes can be made to the specific embodiments disclosed without departing from the spirit and scope of the present application and still obtain the same or similar results.

[0103] Unless otherwise specified, all materials / reagents are obtained from commercial suppliers without further purification. The structures of the compounds in the following examples are characterized and determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS).

[0104] 1 H NMR spectra were recorded at room temperature on a Bruker Avance 400MHz spectrometer, with deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), deuterated methanol (CD3OD) or deuterium water (D2O) as the determination solvent. The chemical shift value (δ) is in ppm, with tetramethylsilane (TMS) or residual solvent peak as the internal standard, and the coupling constant (J) is in hertz (Hz), 1Multiplicity of the peak types in the H NMR spectra is abbreviated as follows: s (singlet), d (doublet), t (triplet), q (quartet), qn (quintet), m (multiplet), br (broad).

[0105] The instrument used for liquid chromatography-mass spectrometry (LC-MS) was Shimadzu LCMS-2020, and the instrument used for preparative high-performance liquid chromatography (Prep-HPLC) was Bonna-Agela FLEXA FL-H100G. The thin layer chromatography (TLC) used thin layer chromatography silica gel plates, model Yantai Huanghai HSGF254 thin layer chromatography silica gel plates, with a size of 2.5 x 8 cm and a coating thickness of 0.2 ± 0.03 mm for reaction monitoring, and a size of 20 x 20 cm and a coating thickness of 0.4-0.5 mm for separation and purification. The silica gel column chromatography used silica gel, model 100-200 mesh or 200-300 mesh silica gel from Qingdao Gulf Fine Chemicals.

[0106] Synthesis of intermediate 1: 6-(1H-pyrazol-3-yl)picolinic acid (Int-1)

[0107]

[0108] Synthesis route:

[0109]

[0110] To a round bottom flask, 1-(tetrahydropyranyl)-1H-pyrazole-5-boronic acid pinacol ester Int-1a (15.3 g, 55.0 mmol), 6-bromo-2-pyridinecarboxylic acid Int-1b (10.1 g, 50.0 mmol), tetrakis(triphenylphosphine)palladium (1.0 g, 0.86 mmol), sodium carbonate (10.6 g, 100.0 mmol), 1,4-dioxane (300 mL) and water (60 mL) were added in sequence, the bottle was replaced with nitrogen three times quickly, then the reaction liquid was warmed to 80 °C and stirred for 16 hours. TLC and LC-MS showed that the reaction was complete, the reaction liquid was cooled to room temperature, then quenched with water (300 mL), extracted with ethyl acetate (300 mL), the aqueous phase was acidified with dilute hydrochloric acid (4N) to pH = 3, a large amount of solid was precipitated, then stirred at room temperature for half an hour, filtered, and the filter cake was washed with water, then dried to obtain compound Int-1 (9.0 g, yield 95%) as a yellow solid.

[0111] MS (ES + ): m / z 190.0 [M+H] + .

[0112] Synthesis of intermediate 2: 3-(difluoromethyl)-4-nitro-1H-pyrazole (Int-2)

[0113]

[0114] Synthesis route:

[0115]

[0116] Step 1: Synthesis of 1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-3-carbaldehyde (Int-2c)

[0117] To a round bottom flask was added 1H-pyrazole-3-carbaldehyde Int-2a (19.2 g, 200.0 mmol), 3,4-dihydro-2H-pyran Int-2b (17.7 g, 210.0 mmol), p-toluenesulfonic acid (2.4 g, 14.0 mmol) and tetrahydrofuran (400 mL) successively at room temperature, the reaction solution was kept at 70 °C reflux and stirred for 2 hours. TLC and LC-MS showed that the reaction was complete, after the reaction solution was cooled to room temperature, the solvent was removed by concentration under reduced pressure, then purified by silica gel column chromatography (6% ethyl acetate in petroleum ether as eluent) again to give compound Int-2c (34.0 g, yield 94%).

[0118] MS (ES + ): m / z 181.1 [M+H] + .

[0119] Step 2: Synthesis of 3-(difluoromethyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole (Int-2d)

[0120] To a solution of compound Int-2c (18.0 g, 100.0 mmol) in dichloromethane (300 mL) was added diethylamine sulfide trifluoride (DAST, 40.2 g, 250.0 mmol) dropwise slowly under 0 °C ice bath, the reaction solution was kept at 0 °C ice bath and stirred for 5 hours. TLC and LC-MS showed that the reaction was complete, the reaction mixture was poured into water (200 mL), the pH was adjusted to neutral with 5% aqueous sodium bicarbonate solution, then extracted with dichloromethane (200 mL x 3). The combined organic phase was washed with saturated brine (600 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure to give compound Int-2d (20.0 g) as a crude product, which was used directly in the next step reaction.

[0121] MS (ES + ): m / z 203.1 [M+H] + .

[0122] Step 3: Synthesis of 3-(difluoromethyl)-1H-pyrazole (Int-2e)

[0123] To a solution of compound Int-2d (20.0 g) crude product in 1,4-dioxane (100 mL) was added concentrated hydrochloric acid (30 mL) slowly dropwise at room temperature, the reaction was kept stirring at 90 °C for 2 h. TLC and LC-MS showed the reaction was complete, the reaction mixture was poured into ice water (400 mL), extracted with ethyl acetate (300 mL x 3). The combined organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give compound Int-2e (12.0 g) crude product, which was used directly in the next reaction.

[0124] MS (ES + ): m / z 119.0 [M+H] + .

[0125] Step 4: Synthesis of 3-(difluoromethyl)-4-nitro-1H-pyrazole (Int-2)

[0126] To a solution of compound Int-2e (12.0 g) crude product in concentrated sulfuric acid (120 mL) was added concentrated nitric acid (36 mL) slowly dropwise at 0 °C ice bath, then the reaction was warmed to 120 °C and stirred for 3 h. TLC and LC-MS showed the reaction was complete, the reaction mixture was slowly poured into ice water (600 mL), directly extracted with ethyl acetate (400 mL x 3). The combined organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure, then purified by silica gel column chromatography (petroleum ether containing 9% ethyl acetate as eluent) to give compound Int-2 (11.0 g, 67% yield over three steps) as a yellow solid.

[0127] MS (ES + ): m / z 164.0 [M+H] + .

[0128] Intermediate 3: Synthesis of tert-butyl 4-(3-(4-amino-3-(difluoromethyl)-1H-pyrazol-1- yl)azetidin-1-yl)piperidine-1-carboxylate (Int-3)

[0129]

[0130] Synthesis route:

[0131]

[0132] Step 1: Synthesis of tert-butyl 3-(p-tolylsulfonyloxy)azetidine-1-carboxylate (Int-3b)

[0133] To a solution of N-Boc-3-hydroxyazetidine Int-3a (50.0 g, 288.7 mmol) in dichloromethane (500 mL) was added p-toluenesulfonyl chloride (54.9 g, 288.0 mmol) and pyridine (27.4 g, 347.0 mmol) at 0 °C under ice-bath. After the reaction solution was naturally warmed to room temperature, it was stirred for 36 h. TLC and LC-MS showed the reaction was complete. The reaction mixture was poured into water (500 mL), and the aqueous phase was extracted with dichloromethane (500 mL x 2). The combined organic phase was washed with 5% aqueous sodium bicarbonate solution and saturated brine successively, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was then purified by silica gel column chromatography (petroleum ether containing 25% ethyl acetate as eluent) to give compound Int-3b (86.5 g, yield 92%).

[0134] MS (ESI): m / z 328.1 [M+H] + ): m / z 328.1 [M+H] + .

[0135] Step 2: Synthesis of tert-butyl 3-(3-(difluoromethyl)-4-nitro-1H-pyrazol-1-yl)azetidine-1-carboxylate (Int-3c)

[0136] To a round-bottom flask was added compound Int-2 (16.3 g, 100.0 mmol), Int-3b (36.0 g, 110.0 mmol), potassium carbonate (20.7 g, 150.0 mmol) and dimethyl sulfoxide (150 mL) successively at room temperature. The reaction solution was warmed to 100 °C and stirred for 16 h. TLC and LC-MS showed the reaction was complete. After the reaction solution was cooled to room temperature, it was quenched with water (400 mL) and extracted with ethyl acetate (300 mL x 3). The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was then purified by silica gel column chromatography (petroleum ether containing 25% ethyl acetate as eluent) to give compound Int-3c (19.5 g, yield 92%).

[0137] MS (ESI): m / z 328.1 [M+H] + ): m / z 328.1 [M+H] + .

[0138] Step 3: Synthesis of tert-butyl 4-(3-(3-(difluoromethyl)-4-nitro-1H-pyrazol-1-yl)azetidin-1-yl)piperidine-1-carboxylate (Int-3d)

[0139] To a solution of compound Int-3c (15.9 g, 50.0 mmol) in dichloromethane (100 mL) was added trifluoroacetic acid (30 mL) dropwise at room temperature. The reaction was stirred at room temperature for 1 h. TLC and LC-MS showed the reaction was complete. The reaction was concentrated under reduced pressure to remove dichloromethane solvent and trifluoroacetic acid to give a light yellow solid. The crude product after concentration was dissolved in methanol (200 mL), the solution was adjusted to weak basicity (pH = 8) with triethylamine, then N-Boc-4-piperidone (10.0 g, 50.0 mmol) and palladium on carbon (10% wet powder, 50 wt% moisture, 4.2 g) were added to the solution, and a hydrogen balloon was added. After the bottle was replaced with hydrogen three times, the reaction was stirred at room temperature for 16 h under the protection of hydrogen. TLC and LC-MS showed the reaction was complete. The palladium on carbon solid was removed by suction filtration, the filter cake was washed with methanol, and the filtrate was concentrated under reduced pressure, then purified by silica gel column chromatography (petroleum ether containing 25% ethyl acetate as eluent) to give compound Int-3d (18.4 g, yield 92%).

[0140] MS (ES + ): m / z 402.2 [M+H] + .

[0141] Step 4: Synthesis of tert-butyl 4-(3-(4-amino-3-(difluoromethyl)-1H-pyrazol-1-yl)azetidin-1- yl)piperidine-1-carboxylate (Int-3)

[0142] To a solution of compound Int-3d (10.0 g, 24.9 mmol) in methanol (50 mL) was added palladium on carbon (10% wet powder, 50 wt% moisture, 2.1 g) and a hydrogen balloon was added. After the bottle was replaced with hydrogen three times, the reaction was stirred at room temperature for 4 h under the protection of hydrogen. TLC and LC-MS showed the reaction was complete. The palladium on carbon solid was removed by suction filtration, the filter cake was washed with methanol, and the filtrate was concentrated under reduced pressure, then purified by silica gel column chromatography (dichloromethane containing 9% methanol as eluent) to give compound Int-3 (7.9 g, yield 85%) as a white solid.

[0143] MS (ES + ): m / z 372.2 [M+H] + .

[0144] Intermediate 4: Synthesis of N-(3-(difluoromethyl)-1-(1-(piperidin-4-yl)azetidin-3-yl)-1H-pyrazol-4-yl)-6-(1H-pyrazol-3-yl)picolinamide (Int-4)

[0145]

[0146] Synthesis route:

[0147]

[0148] Step 1: Synthesis of tert-butyl 4-(3-(4-(6-(1H-pyrazol-3-yl)picolinamido)-3- (difluoromethyl)-1H-pyrazol-1-yl)azetidin-1-yl)piperidine-1-carboxylate (Int-4a)

[0149] To a solution of compound Int-1 (3.8 g, 20.0 mmol) and Int-3 (7.4 g, 20.0 mmol) in dichloromethane (100 mL) was added 4-dimethylaminopyridine (DMAP, 4.9 g, 40.0 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 4.2 g, 22.0 mmol) at room temperature. The reaction was stirred at room temperature for 12 h. TLC and LC-MS showed the reaction was complete. The reaction mixture was poured into water (100 mL) and extracted with dichloromethane (100 mL x 3). The organic phases were combined, washed with saturated brine (200 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure, then purified by silica gel column chromatography (9% methanol in dichloromethane as eluent) to give compound Int-4a (9.4 g, 87% yield) as a light yellow solid.

[0150] MS (ES + ): m / z 543.3 [M+H] + .

[0151] Step 2: Synthesis of N-(3-(difluoromethyl)-1-(1-(piperidin-4-yl)azetidin-3-yl)-1H- pyrazol-4-yl)-6-(1H-pyrazol-3-yl)picolinamide (Int-4)

[0152] To a solution of compound Int-4a (2.7 g, 5.0 mmol) in dichloromethane (20 mL) was added hydrogen chloride in 1,4-dioxane (4 N, 25 mL) dropwise at room temperature. The reaction was stirred at room temperature for 2 h. TLC and LC-MS showed the reaction was complete. The reaction was concentrated under reduced pressure to remove solvent and volatile matter to give a yellow solid crude. The concentrated crude was dissolved in methanol (50 mL), and potassium carbonate (13.8 g, 100.0 mmol) was added to the solution. The reaction was stirred at room temperature for 1 h, and the solid was removed by filtration. The filtrate was concentrated under reduced pressure, then purified by silica gel column chromatography (9% methanol in dichloromethane as eluent) to give compound Int-4 (2.1 g, 95% yield) as a white solid.

[0153] MS (ES +): m / z 443.2 [M+H] + .

[0154] Synthesis of Intermediate 5: 3-(4-bromo-3-methyl-2-oxo-2,3-dihydro-lH- benzo[d]imidazol-l-yl)piperidine-2,6-dione (Int-5)

[0155]

[0156] Synthesis route:

[0157]

[0158] To a suspension of 7-bromo-l-methyl-l,3-dihydro-2H-benzo[d]imidazol-2-one Int-5a (3.0 g, 13.2 mmol) in tetrahydrofuran (15 mL) was added dropwise a solution of lithium bis(trimethylsilyl)amide (LiHMDS) in tetrahydrofuran (1.0 M, 33 mL) slowly at room temperature, the dropwise addition time was 15 minutes, the reaction was stirred at room temperature for 1 hour. Then the reaction was added dropwise to a solution of 3-bromopiperidine-2,6-dione Int-5b (5.0 g, 26.4 mmol) in tetrahydrofuran (30 mL) slowly at room temperature, the dropwise addition time was 10 minutes, the resulting reaction mixture was warmed to 60 °C and stirred for 4 hours. TLC and LC-MS showed the reaction was complete, the reaction mixture was poured into ice water (100 mL), acidified with dilute hydrochloric acid (2 N) to pH = 2, a large amount of solid precipitated, filtered, and the filter cake was washed with water to give compound Int-5 (2.85 g, yield 64%) as a gray solid.

[0159] MS (ES + ): m / z 338.0 [M+H] + .

[0160] Synthesis of Intermediate 6: N-(l-(azetidin-3-yl)-3-(difluoromethyl)-lH-pyrazol-4-yl)- 6-(lH-pyrazol-3-yl)picolinamide (Int-6)

[0161]

[0162] Synthesis route:

[0163]

[0164] Steps 1, 2 and 3 were the same as the synthesis route and method of Intermediate 3 Step 4, Intermediate 4 Step 1 and Intermediate 4 Step 2 respectively, except that compound Int-3d was replaced with equivalent amount of compound Int-3c, to produce Intermediate 6, compound Int-6, as a white solid.

[0165] MS (ES + ): m / z 360.1 [M+H] + .

[0166] Example 1: Synthesis of N-(3-(difluoromethyl)-1-(1-(1-((S)-14-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-15,15-dimethyl-12-oxo-3,6,9-trioxa-13-azahexadecyl)piperidin-4-yl)azetidin-3-yl)-1H-pyrazol-4-yl)-6-(1H-pyrazol-3-yl)picolinamide (1)

[0167]

[0168] Synthesis route:

[0169]

[0170] Step 1: Synthesis of tert-butyl 3-(2-(2-(2-(4-(3-(4-(6-(1H-pyrazol-3-yl)picolinamido)-3-(difluoromethyl)-1H-pyrazol-1-yl)azetidin-1-yl)piperidin-1-yl)ethoxy)ethoxy)ethoxy)propanoate (1b)

[0171] A mixture of compound Int-4 (619 mg, 1.40 mmol), potassium carbonate (387 mg, 2.80 mmol) and tert-butyl 3-(2-(2-(2-(p-tolylsulfonyloxy)ethoxy)ethoxy)ethoxy)propanoate 1a (636 mg, 1.47 mmol) was dissolved in N,N-dimethylformamide (5.0 mL) at room temperature, and the reaction was heated to 80 °C and stirred for 5 hours. TLC and LC-MS showed that the reaction was complete. After the reaction was cooled to room temperature, the reaction mixture was poured into water (10 mL) and extracted with ethyl acetate (10 mL x 3). The organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure, and then purified by silica gel column chromatography (10% methanol in dichloromethane as eluent) to give compound 1b (826 mg, yield 84%) as a light yellow solid.

[0172] MS (ES + ): m / z 703.3 [M+H] + .

[0173] Step 2: Synthesis of 3-(2-(2-(2-(4-(3-(4-(6-(lH-pyrazol-3-yl)picolinamido)-3- (difluoromethyl)-lH-pyrazol-l-yl)azetidin-l-yl)piperidin-l-yl)ethoxy)ethoxy)ethoxy)propanoic acid (lc)

[0174] To a solution of compound lb (200 mg, 0.28 mmol) in dichloromethane (3 mL) was added trifluoroacetic acid (0.4 mL) at room temperature, the reaction was stirred at room temperature for 2 h. TLC and LC-MS showed the reaction was complete, the solution was adjusted to weak alkaline (pH = 8) with triethylamine, the reaction was concentrated under reduced pressure, then purified by silica gel column chromatography (10% methanol in dichloromethane as eluent) to give compound lc (163 mg, yield 90%) as a white solid which was hygroscopic.

[0175] MS (ES + ): m / z 647.3 [M+H] + .

[0176] Step 3: Synthesis of N-(3-(difluoromethyl)-l-(l-(l-((S)-14-((2S,4R)-4-hydroxy-2-((4-(4- methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-l-carbonyl)-15,15-dimethyl-12-oxo-3,6,9- trioxa-13-azahexadecyl)piperidin-4-yl)azetidin-3-yl)-lH-pyrazol-4-yl)-6-(lH-pyrazol-3- yl)picolinamide (1)

[0177] To a solution of compound lc (71 mg, 0.11 mmol), (2S,4R)-l-((S)-2-amino-3,3-dimethylbutanoyl)- 4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride Id (56 mg, 0.12 mmol), l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 42 mg, 0.22 mmol), 1-hydroxybenzotriazole (HOBt, 30 mg, 0.22 mmol) and N,N- diisopropylethylamine (DIPEA, 84 mg, 0.65 mmol) in dichloromethane (5 mL) was stirred at room temperature for 15 h. TLC and LC-MS showed the reaction was complete, the reaction was concentrated under reduced pressure, then purified by Prep-HPLC (20-80% acetonitrile in water as mobile phase) to give compound 1 (40 mg, yield 34%) as a white solid.

[0178] MS (ES + ): m / z 1059.5 [M+H] + .

[0179] 1 H NMR (400 MHz, CDC13): δ 10.48 (s, 1H), 8.66 (s, 1H), 8.43 (s, 1H), 8.11 (d, J = 7.6 Hz, 1H), 8.08 (d, J = 7.9 Hz, 1H), 7.91 (t, J = 7.8 Hz, 1H), 7.67 (d, J = 1.8 Hz, 1H), 7.60 (t, J = 5.7 Hz, 1H), 7.32 (s, 4H), 6.98 (d, J = 1.8 Hz, 1H), 6.89 (t, J = 54.7 Hz, 1H), 4.94 (qn, J = 6.9 Hz, 1H), 4.73 (t, J = 8.0 Hz, 1H), 4.57 - 4.50 (m, 3H), 4.33 (dd, J = 15.0, 5.3 Hz, 1H), 4.09 (d, J = 11.2 Hz, 1H), 3.78 (t, J = 7.3 Hz, 2H), 3.71 (t, J = 5.5 Hz, 2H), 3.64 - 3.55 (m, 11H), 3.45 (t, J = 7.1 Hz, 2H), 2.91 - 2.84 (m, 2H), 2.58 - 2.55 (m, 2H), 2.53 - 2.42 (m, 6H), 2.26 - 2.00 (m, 5H), 1.72 - 1.67 (m, 2H), 1.44 - 1.36 (m, 2H), 0.95 (s, 9H).

[0180] Example 2: Synthesis of N-(3-(difluoromethyl)-l-(l-(l-(3-(l-(2,6-dioxopiperidin-3-yl)-3-methyl-2-oxo-2,3-dihydro-lH- benzo[d]imidazol-4-yl)prop-2-yn-l-yl)piperidin-4-yl)azetidin-3-yl)-lH-pyrazol-4-yl)-6-(lH-pyrazol-3- yl)picolinamide (2)

[0181]

[0182] Synthesis route:

[0183]

[0184] Step 1: Synthesis of 3-(3-methyl-2-methyl-4-(3-(tetrahydro-2H-pyran-2-yl)oxy)prop-l-yn-l-yl)-2,3-dihydro-lH- benzo[d]imidazol-l-yl)piperidine-2,6-dione (2b)

[0185] To a round bottom flask, compound Int-5 (676 mg, 2.0 mmol), bis(triphenylphosphine)palladium(II) dichloride (70 mg, 0.1 mmol), cuprous iodide (19 mg, 0.1 mmol), triethylamine (607 mg, 6.0 mmol), 2-(prop-2-yn-1-yloxy)tetrahydro-2H-pyran 2a (420 mg, 3.0 mmol) and N,N-dimethylformamide (10 mL) were added successively at room temperature, the reaction solution was kept at 80 °C for 15 h with stirring after the tube was purged with nitrogen for 3 times quickly. TLC and LC-MS showed the reaction was complete, the reaction solution was cooled to room temperature, quenched with water (20 mL) and extracted with ethyl acetate (15 mL x 3). The combined organic phase was washed with saturated brine (30 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure, then purified by silica gel column chromatography (5% methanol in dichloromethane as eluent) to give compound 2b (690 mg, yield 87%) as a light yellow oil.

[0186] MS (ES + ): m / z 398.1 [M+H] + .

[0187] Step 2: Synthesis of 3-(4-(3-hydroxyprop-1-yn-1-yl)-3-methyl-2-oxo-2,3-dihydro-1H- benzo[d]imidazol-1-yl)piperidine-2,6-dione (2c)

[0188] To a solution of compound 2b (300 mg, 0.75 mmol) in tetrahydrofuran (5 mL) was added acetic acid (5 mL) and water (3 mL) at room temperature, the reaction solution was kept at 50 °C for 3 h with stirring. TLC and LC-MS showed the reaction was complete, the pH was adjusted to neutral with saturated aqueous sodium bicarbonate solution, then extracted with dichloromethane (15 mL x 3). The combined organic phase was washed with saturated brine (30 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure, then purified by silica gel column chromatography (5% methanol in dichloromethane as eluent) to give compound 2c (184 mg, yield 78%) as a light yellow solid.

[0189] MS (ES + ): m / z 314.1 [M+H] + .

[0190] Step 3: Synthesis of 3-(1-(2,6-dioxopiperidin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1H- benzo[d]imidazol-4-yl)prop-2-ynal (2d)

[0191] To a solution of compound 2c (170 mg, 0.54 mmol) in dichloromethane (5 mL) and N,N-diisopropyl ethylamine (1 mL) was added Dess-Martin Periodinane (254 mg, 0.60 mmol) at room temperature. The reaction was stirred at 40 °C for 2 h. TLC and LC-MS showed the reaction was complete. The reaction was quenched with water (10 mL), adjusted to neutral with saturated aqueous sodium bicarbonate solution, and extracted with dichloromethane (10 mL x 3). The combined organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (eluted with 5% methanol in dichloromethane) to give compound 2d (151 mg, 90% yield) as a light yellow solid.

[0192] MS (ESI): m / z 312.1 [M+H] + ): m / z 312.1 [M+H] + .

[0193] Step 4: Synthesis of N-(3-(difluoromethyl)-1-(1-(1-(3-(1-(2,6-dioxopiperidin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-4-yl)prop-2-yn-1-yl)piperidin-4-yl)azetidin-3-yl)-1H-pyrazol-4-yl)-6-(1H-pyrazol-3-yl)picolinamide (2)

[0194] To a round-bottom flask was added compound 2d (124 mg, 0.40 mmol), compound Int-4 (177 mg, 0.40 mmol), tetrahydrofuran (4 mL), and methanol (6 mL) sequentially at room temperature. The reaction was stirred at 50 °C for 1 h. Then sodium cyanoborohydride (76 mg, 1.21 mmol) and acetic acid (0.5 mL) were added to the reaction. The reaction was stirred at 50 °C for another 2 h. TLC and LC-MS showed the reaction was complete. The reaction was quenched with water (10 mL), adjusted to neutral with saturated aqueous sodium bicarbonate solution, and extracted with dichloromethane (10 mL x 3). After the combined organic phase was concentrated under reduced pressure, Prep-HPLC separation and purification (30-80% acetonitrile in water as the mobile phase) gave compound 2 (21 mg, 7% yield) as a white solid.

[0195] MS (ESI): m / z 738.3 [M+H] + ): m / z 738.3 [M+H] + .

[0196] 1H NMR (400 MHz, DMSO-d6): δ 13.86 (minor), 13.24 (major) (s, 1H), 11.13 (major), 11.06 (minor) (s, 1H), 10.59 (minor), 10.44 (major) (s, 1H), 8.51 (major), 8.31 (minor) (s, 1H), 8.24 - 8.14 (m, 1H), 8.10 (t, J = 7.6 Hz, 1H), 8.02 (d, J = 7.9 Hz, 1H), 7.92 (major), 7.66 (minor) (s, 1H), 7.45 - 6.94 (m, 5H), 5.39 (major), 5.31 (minor) (dd, J = 12.4, 5.0 Hz, 1H), 5.10 (qn, J = 7.0 Hz, 1H), 3.78 (t, J = 6.9 Hz, 2H), 3.66 (major), 3.59 (minor) (s, 3H), 3.61 (s, 2H), 2.90 - 2.84 (m, 2H), 2.75 - 2.61 (m, 2H), 2.35 - 2.27 (m, 3H), 2.07 - 1.94 (m, 2H), 1.78 - 1.71 (m, 2H), 1.47 - 1.22 (m, 4H).

[0197] Example 3: Synthesis of N-(3-(difluoromethyl)-1-(1-(1-(4-(1-(2,6-dioxopiperidin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-4-yl)but-3-yn-1-yl)piperidin-4-yl)azetidin-3-yl)-1H-pyrazol-4-yl)-6-(1H-pyrazol-3-yl)picolinamide (3)

[0198]

[0199] Synthetic route:

[0200]

[0201] Step 1: Synthesis of 3-(4-(4-hydroxybut-1-yn-1-yl)-3-methyl-2-oxo-2,3-dihydro-1H- benzo[d]imidazol-1-yl)piperidine-2,6-dione (3b)

[0202] The same synthetic route and method as in Example 2, Step 1, except replacing 2-(prop-2-yn-1-yloxy)tetrahydro-2H-pyran 2a with an equivalent amount of 3-butyn-1-ol 3a to produce compound 3b.

[0203] MS (ES+ ): m / z 328.1 [M+H] + .

[0204] Step 2: Synthesis of 3-(4-(4-bromobut-1-yne-1-yl)-3-methyl-2-oxo-2,3-dihydro-1H- benzo[d]imidazol-1-yl)piperidine-2,6-dione (3c)

[0205] To a solution of compound 3b (60 mg, 0.18 mmol) in dichloromethane (5 mL) was added carbon tetrabromide (120 mg, 0.36 mmol) at room temperature, after the flask was purged with nitrogen for three times, the reaction solution was stirred at room temperature for 1 hour. Then the reaction solution was placed in an ice bath at 0 °C, a solution of triphenylphosphine (96 mg, 0.36 mmol) in dichloromethane (2 mL) was added dropwise slowly, the reaction solution was stirred in an ice bath for half an hour, and then warmed to 50 °C for 3 hours. TLC and LC-MS showed that the reaction was complete, water (10 mL) was added to quench, and then extracted with dichloromethane (10 mL x 3). The combined organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure, and then purified by silica gel column chromatography (5% methanol in dichloromethane as eluent) to give compound 3c (23 mg, 33% yield) as a light yellow solid.

[0206] MS (ES + ): m / z 390.0 [M+H] + .

[0207] Step 3: Synthesis of N-(3-(difluoromethyl)-1-(1-(1-(4-(1-(2,6-dioxopiperidin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-4-yl)but-3-yn-1-yl)piperidin-4-yl)azetidin-3-yl)-1H-pyrazol-4-yl)-6-(1H-pyrazol-3-yl)picolinamide (3) To a solution of compound 3c (20 mg, 0.05 mmol) and compound Int-4 (23 mg, 0.05 mmol) in N,N-dimethylformamide (1 mL) was added N,N-diisopropylethylamine (DIPEA, 40 mg, 0.31 mmol) at room temperature, the reaction solution was stirred at 70 °C for 15 hours. TLC and LC-MS showed that the reaction was complete, after the reaction solution was cooled to room temperature, water (5 mL) was added to quench, and then extracted with dichloromethane (5 mL x 3). After the combined organic phase was concentrated under reduced pressure, it was separated and purified by Prep-HPLC (30-80% acetonitrile in water as mobile phase) to give compound 3 (5 mg, 13% yield) as a white solid.

[0208] MS (ES + ): m / z 752.4 [M+H]+ .

[0209] 1 H NMR (400 MHz, DMSO-d6): δ 13.85 (minor), 13.22 (major) (s, 1H), 11.12 (major), 11.03 (minor) (s, 1H), 10.59 (minor), 10.44 (major) (s, 1H), 8.51 (major), 8.30 (minor) (s, 1H), 8.24 - 8.17 (m, 1H), 8.11 (t, J = 7.4 Hz, 1H), 8.02 (d, J = 8.1 Hz, 1H), 7.92 (major), 7.65 (minor) (s, 1H), 7.45 - 6.95 (m, 5H), 5.38 (major), 5.32 (minor) (dd, J = 12.6, 5.4 Hz, 1H), 5.07 (qn, J = 6.5 Hz, 1H), 3.70 (t, J = 7.3 Hz, 2H), 3.67 (s, 3H), 2.90 - 2.80 (m, 3H), 2.71 - 2.56 (m, 5H), 2.36 - 2.27 (m, 1H), 2.16 - 1.97 (m, 4H), 1.71 - 1.65 (m, 2H), 1.24 - 1.16 (m, 4H).

[0210] Example 4: Synthesis of N-(3-(difluoromethyl)-1-(1-(1-(5-(1-(2,6-dioxopiperidin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-4-yl)pent-4-yn-1-yl)piperidin-4-yl)azetidin-3-yl)-1H-pyrazol-4-yl)-6-(1H-pyrazol-3-yl)picolinamide (4)

[0211]

[0212] Synthetic route:

[0213]

[0214] Steps 1, 2 and 3 are the same as the synthetic route and method of steps 1, 3 and 4 of Example 2, except that 2-(prop-2-yn-1-yloxy)tetrahydro-2H-pyran 2a is replaced with equivalent amount of 4-pentyn-1-ol 4a, to produce compound 4 as a white solid.

[0215] MS (ES + ): m / z 766.4 [M+H] + .

[0216] 1 H NMR (400 MHz, DMSO-d6): δ 13.88 (minor), 13.23 (major) (s, 1H), 11.11 (major), 11.02 (minor) (s, 1H), 10.61 (minor), 10.44 (major) (s, 1H), 8.52 (major), 8.32 (minor) (s, 1H), 8.23 - 8.15 (m, 1H), 8.10 (t, J = 7.3 Hz, 1H), 8.02 (d, J = 8.3 Hz, 1H), 7.92 (major), 7.65 (minor) (s, 1H), 7.43 - 6.90 (m, 5H), 5.39 (major), 5.32 (minor) (dd, J = 12.8, 5.5 Hz, 1H), 5.09 (qn, J = 6.8 Hz, 1H), 3.73 (t, J = 7.0 Hz, 2H), 3.65 (major), 3.58 (minor) (s, 3H), 2.94 - 2.84 (m, 2H), 2.77 - 2.63 (m, 4H), 2.61 - 2.53 (m, 4H), 2.34 - 2.32 (m, 1H), 2.05 - 1.95 (m, 2H), 1.91 - 1.74 (m, 4H), 1.46 - 1.23 (m, 4H).

[0217] Example 5: Synthesis of N-(3-(difluoromethyl)-1-(1-(1-(6-(1-(2,6-dioxopiperidin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-4-yl)hex-5-yn-1-yl)piperidin-4-yl)azetidin-3-yl)-1H-pyrazol-4-yl)-6-(1H-pyrazol-3-yl)picolinamide (5)

[0218]

[0219] Synthetic route:

[0220]

[0221] Steps 1, 2 and 3 are the same as the synthetic route and method of steps 1, 3 and 4 of Example 2, except that 2-(prop-2-yn-1-yloxy)tetrahydro-2H-pyran 2a is replaced with equivalent amount of 5-hexyn-1-ol 5a, to produce compound 5 as a white solid.

[0222] MS (ES + ): m / z 780.4 [M+H] + .

[0223] 1 H NMR (400 MHz, DMSO-d6): δ 10.49 (s, 1H), 8.43 (s, 1H), 8.17 (d, J = 7.6 Hz, 1H), 8.11 (t, J = 7.7 Hz, 1H), 8.03 (d, J = 7.6 Hz, 1H), 7.83 (s, 1H), 7.46 - 6.91 (m, 5H), 5.37 (dd, J = 12.8, 5.8 Hz, 1H), 5.06 (qn, J = 6.8 Hz, 1H), 3.68 (t, J = 6.8 Hz, 2H), 3.64 (s, 3H), 2.93 - 2.84 (m, 2H), 2.78 - 2.59 (m, 4H), 2.33 - 2.27 (m, 3H), 2.18 - 2.10 (m, 2H), 2.04 - 1.90 (m, 4H), 1.68 - 1.55 (m, 4H), 1.23 - 1.14 (m, 4H).

[0224] Example 6: Synthesis of N-(3-(difluoromethyl)-1-(1-(1-(7-(1-(2,6-dioxopiperidin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-4-yl)hept-6-yn-1-yl)piperidin-4-yl)azetidin-3-yl)-1H-pyrazol-4-yl)-6-(1H-pyrazol-3-yl)picolinamide (6)

[0225]

[0226] Synthetic route:

[0227]

[0228] Steps 1, 2 and 3 are the same as the synthetic route and method of steps 1, 3 and 4 of Example 2, except that 2-(prop-2-yn-1-yloxy)tetrahydro-2H-pyran 2a is replaced with equivalent amount of 6-heptyne-1-ol 6a, to produce compound 6 as a white solid.

[0229] MS (ES + ): m / z 794.4 [M+H] + .

[0230] 1H NMR (400 MHz, DMSO-d6): δ 13.87 (minor), 13.24 (major) (s, 1H), 11.12 (major), 11.02 (minor) (s, 1H), 10.60 (minor), 10.43 (major) (s, 1H), 8.51 (major), 8.30 (minor) (s, 1H), 8.24 - 8.16 (m, 1H), 8.11 (t, J = 7.4 Hz, 1H), 8.03 (d, J = 8.0 Hz, 1H), 7.92 (major), 7.67 (minor) (s, 1H), 7.41 - 6.91 (m, 5H), 5.38 (major), 5.32 (minor) (dd, J = 12.3, 5.5 Hz, 1H), 5.06 (qn, J = 6.9 Hz, 1H), 3.68 (t, J = 7.1 Hz, 2H), 3.64 (major), 3.57 (minor) (s, 3H), 2.95 - 2.82 (m, 2H), 2.81 - 2.58 (m, 4H), 2.34 - 2.25 (m, 3H), 2.19 - 2.11 (m, 2H), 2.05 - 1.95 (m, 2H), 1.69 - 1.55 (m, 4H), 1.51 - 1.37 (m, 4H), 1.29 - 1.17 (m, 4H).

[0231] Example 7: Synthesis of N-(3-(difluoromethyl)-1-(1-(1-(2-(3-((2-(2,6-dioxopiperidin-3- yl)-1-oxoisoindolin-4-yl)amino)-3-oxopropoxy)ethyl)piperidin-4-yl)azetidin-3-yl)-1H- pyrazol-4-yl)-6-(1H-pyrazol-3-yl)picolinamide (7)

[0232]

[0233] Synthesis route:

[0234]

[0235] Step 1: Synthesis of 3-(2-bromoethoxy)-N-(2-(2,6-dioxopiperidin-3-yl)-1- oxoisoindolin-4-yl)propanamide (7b)

[0236] A mixture of lenalidomide 7a (259 mg, 1.0 mmol), 3-(2-bromoethoxy)propanoic acid 7b (295 mg, 1.5 mmol) and l-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (EDCI, 383 mg, 2.0 mmol) was dissolved in N,N- dimethylformamide (5.0 mL) at room temperature, the reaction was heated to 40 °C and stirred for 15 h. TLC and LC-MS showed the reaction was complete, the reaction mixture was poured into water (10 mL) and extracted with ethyl acetate (15 mL x 3). The organic phase was combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure, then purified by silica gel column chromatography (5% methanol in dichloromethane as eluent) to give compound 7c (153 mg, 35% yield) as a white solid.

[0237] MS (ES + ): m / z 438.0 [M+H] + .

[0238] Step 2: Synthesis of N-(3-(difluoromethyl)-l-(l-(l-(2-(3-((2-(2,6-dioxopiperidin-3-yl)-l- oxoisoindolin-4-yl)amino)-3-oxopropoxy)ethyl)piperidin-4-yl)azetidin-3-yl)-lH- pyrazol-4-yl)-6-(lH-pyrazol-3-yl)picolinamide (7)

[0239] To a solution of compound 7c (22 mg, 0.05 mmol) and compound Int-4 (23 mg, 0.05 mmol) in N,N-dimethylformamide (1 mL) was added N,N- diisopropylethylamine (DIPEA, 26 mg, 0.20 mmol) at room temperature, the reaction was kept at 80 °C and stirred for 15 h. TLC and LC-MS showed the reaction was complete, after the reaction was cooled to room temperature, quenched with water (5 mL) and extracted with dichloromethane (5 mL x 3). The organic phase was combined and concentrated under reduced pressure, then purified by Prep-HPLC (30-80% acetonitrile in water as mobile phase) to give compound 7 (5 mg, 14% yield) as a white solid.

[0240] MS (ES + ): m / z 800.4 [M+H] + .

[0241] 1H NMR (400 MHz, DMSO-d6): δ 11.04 (s, 1H), 10.52 (s, 1H), 9.86 (s, 1H), 8.38 (s, 1H), 8.19 - 8.14 (m, 2H), 8.11 (t, J = 7.7 Hz, 1H), 8.03 (d, J = 7.8 Hz, 1H), 7.97 - 7.81 (m, 2H), 7.51 - 7.42 (m, 2H), 7.24 (t, J = 54.1 Hz, 1H), 7.02 (d, J = 1.8 Hz, 1H), 5.04 (qn, J = 6.7 Hz, 1H), 4.78 - 4.66 (m, 1H), 4.52 - 4.31 (m, 2H), 3.71 - 3.62 (m, 4H), 3.54 - 3.49 (m, 4H), 2.79 - 2.73 (m, 2H), 2.68 - 2.60 (m, 3H), 2.48 - 2.44 (m, 2H), 2.12 - 1.96 (m, 6H), 1.61 - 1.52 (m, 2H), 1.19 - 1.08 (m, 2H).

[0242] Example 8: Synthesis of N-(3-(difluoromethyl)-1-(1-(1-(3-(2-((3-(1-(2,6-dioxopiperidin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-4-yl)prop-2-yn-1-yl)oxy)ethoxy)propanoyl)piperidin-4-yl)azetidin-3-yl)-1H-pyrazol-4-yl)-6-(1H-pyrazol-3-yl)picolinamide (8)

[0243]

[0244] Synthesis route:

[0245]

[0246] Step 1: Synthesis of tert-butyl 3-(2-((3-(1-(2,6-dioxopiperidin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-4-yl)prop-2-yn-1-yl)oxy)ethoxy)propanoate (8b)

[0247] Into a round bottom flask, compound Int-5 (338 mg, 1.0 mmol), bis(triphenylphosphine)palladium(II) dichloride (35 mg, 0.05 mmol), cuprous iodide (10 mg, 0.05 mmol), triethylamine (304 mg, 3.0 mmol), tert-butyl 3-(2-(prop-2-yn-1-yloxy)ethoxy)propanoate 8a (342 mg, 1.5 mmol) and N,N-dimethylformamide (10 mL) were added sequentially at room temperature, the flask was purged with nitrogen for 3 times quickly, then the reaction was stirred at 80 °C for 15 h. TLC and LC-MS showed the reaction was complete, the reaction was cooled to room temperature, quenched with water (20 mL), extracted with ethyl acetate (15 mL x 3). The organic phase was washed with saturated brine (30 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure, then purified by silica gel column chromatography (5% methanol in dichloromethane as eluent) to give compound 8b (388 mg, 80% yield) as a light yellow solid.

[0248] MS (ESI): m / z 508.2 [M+Na] + ): m / z 508.2 [M+Na] + .

[0249] Step 2: Synthesis of 3-(2-((3-(1-(2,6-dioxopiperidin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1H- benzo[d]imidazol-4-yl)prop-2-yn-1-yl)oxy)ethoxy)propanoic acid (8c)

[0250] Into a round bottom flask, compound 8b (49 mg, 0.1 mmol) was dissolved in formic acid (2 mL) at room temperature, the reaction was stirred at room temperature for 15 h. TLC and LC-MS showed the reaction was complete, the reaction was directly concentrated under reduced pressure, then purified by Prep-TLC (5% methanol in dichloromethane as eluent) to give compound 8c (30 mg, 70% yield) as a light yellow solid.

[0251] MS (ESI): m / z 508.2 [M+Na] + ): m / z 508.2 [M+Na] + .

[0252] Step 3: Synthesis of N-(3-(difluoromethyl)-1-(1-(1-(3-(2-((3-(1-(2,6-dioxopiperidin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-4-yl)prop-2-yn-1-yl)oxy)ethoxy)propanoyl)piperidin-4-yl)azetidin-3-yl)-1H-pyrazol-4-yl)-6-(1H-pyrazol-3-yl)picolinamide (8)

[0253] To a solution of compound 8c (21 mg, 0.05 mmol) and compound Int-4 (23 mg, 0.05 mmol) in N,N-dimethylformamide (1 mL) was added 2-(7-azabenzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 29 mg, 0.075 mmol) and N,N-diisopropylethylamine (DIPEA, 13 mg, 0.10 mmol) at room temperature. The reaction was stirred at room temperature for 15 h. TLC and LC-MS showed the reaction was complete. The reaction was quenched with water (5 mL) and extracted with dichloromethane (5 mL x 3). The organic phases were combined and concentrated under reduced pressure. Purification was performed by Prep-HPLC (30-80% acetonitrile in water as mobile phase) to give compound 8 (11 mg, 26% yield) as a white solid.

[0254] MS (ES + ): m / z 854.3 [M+H] + .

[0255] 1 H NMR (400 MHz, DMSO-d6): δ 13.93 (minor), 13.27 (major) (s, 1H), 11.11 (s, 1H), 10.62 (minor), 10.45 (major) (s, 1H), 8.50 (major), 8.38 (minor) (s, 1H), 8.22-8.15 (m, 1H), 8.10 (t, J = 7.6 Hz, 1H), 8.03 (d, J = 8.2 Hz, 1H), 7.91 (major), 7.68 (minor) (s, 1H), 7.39-6.93 (m, 5H), 5.40 (minor), 5.32 (major) (t, J = 4.7 Hz, 1H), 5.06 (qn, J = 6.7 Hz, 1H), 4.46 (minor), 4.44 (major) (s, 2H), 4.02-3.94 (m, 1H), 3.76-3.61 (m, 9H), 3.59-3.50 (m, 7H), 2.96-2.79 (m, 2H), 2.56 (t, J = 6.7 Hz, 2H), 2.40-2.32 (m, 1H), 2.07-1.95 (m, 2H), 1.70-1.58 (m, 2H), 1.19-1.03 (m, 2H).

[0256] Example 9: Synthesis of N-(3-(difluoromethyl)-1-(1-(1-(5-(1-(2,6-dioxopiperidin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-4-yl)pent-4-ynoyl)piperidin-4-yl)azetidin-3-yl)-1H-pyrazol-4-yl)-6-(1H-pyrazol-3-yl)picolinamide (9)

[0257]

[0258] Synthetic route:

[0259]

[0260] Step 1: Synthesis of tert-butyl 5-(1-(2,6-dioxopiperidin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1H- benzo[d]imidazol-4-yl)pent-4-ynoate (8b)

[0261] Compound Int-5 (338 mg, 1.0 mmol), bis(triphenylphosphine)palladium(II) dichloride (35 mg, 0.05 mmol), cuprous iodide (10 mg, 0.05 mmol), triethylamine (304 mg, 3.0 mmol), tert-butyl 4-pentynoate 9a (231 mg, 1.5 mmol) and N,N-dimethylformamide (10 mL) were added into a round bottom flask sequentially at room temperature, after the tube was replaced with nitrogen three times quickly, the reaction solution was kept at 80 °C and stirred for 15 hours. TLC and LC-MS showed that the reaction was complete, after the reaction solution was cooled to room temperature, water (20 mL) was added for quenching, extracted with ethyl acetate (15 mL x 3). The organic phase was washed with saturated brine (30 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure, then purified by silica gel column chromatography (5% methanol in dichloromethane as eluent) to give compound 9b (341 mg, yield 83%) as a light yellow solid.

[0262] MS (ES + ): m / z 412.2 [M+H] + .

[0263] Step 2: Synthesis of 5-(1-(2,6-dioxopiperidin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-4- yl)pent-4-ynoic acid (9c)

[0264] Compound 9b (82 mg, 0.2 mmol) was dissolved in formic acid (3 mL) at room temperature, and the reaction was stirred at room temperature for 15 h. TLC and LC-MS showed the reaction was complete, and the reaction was directly concentrated under reduced pressure, and purified by Prep-TLC (10% methanol in dichloromethane as eluent) to give compound 9c (43 mg, yield 61%) as a light yellow solid.

[0265] MS (ES + ): m / z 356.1 [M+H] + .

[0266] Step 3: Synthesis of N-(3-(difluoromethyl)-1-(1-(1-(5-(1-(2,6-dioxopiperidin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-4-yl)pent-4-ynoyl)piperidin-4-yl)azetidin-3-yl)-1H-pyrazol-4-yl)-6-(1H-pyrazol-3-yl)picolinamide (9)

[0267] To a solution of compound 9c (18 mg, 0.05 mmol) and compound Int-4 (23 mg, 0.05 mmol) in N,N-dimethylformamide (1 mL) was added 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 29 mg, 0.075 mmol) and N,N-diisopropylethylamine (DIPEA, 13 mg, 0.10 mmol) at room temperature, and the reaction was stirred at room temperature for 15 h. TLC and LC-MS showed the reaction was complete, and the reaction was quenched by water (5 mL) and extracted with dichloromethane (5 mL x 3). The organic phases were combined and concentrated under reduced pressure, and purified by Prep-HPLC (30-80% acetonitrile in water as mobile phase) to give compound 9 (7 mg, yield 18%) as a white solid.

[0268] MS (ES + ): m / z 780.3 [M+H] + .

[0269] 1H NMR (400 MHz, DMSO-d6): d 13.84 (minor), 13.23 (major) (s, 1H), 11.11 (minor), 10.99 (major) (s, 1H), 10.47 (s, 1H), 8.54 (major), 8.34 (minor) (s, 1H), 8.25 - 8.14 (m, 1H), 8.11 (t, J = 8.1 Hz, 1H), 8.03 (d, J = 8.4 Hz, 1H), 7.91 (major), 7.69 (minor) (s, 1H), 7.40 - 6.89 (m, 5H), 5.40 - 5.10 (m, 2H), 4.17 - 4.05 (m, 2H), 3.88 - 3.76 (m, 2H), 3.62 - 3.59 (m, 2H), 3.54 (s, 3H), 3.15 - 3.08 (m, 2H), 2.94 - 2.83 (m, 2H), 2.72 - 2.63 (m, 6H), 2.06 - 1.97 (m, 1H), 1.84 - 1.68 (m, 2H), 1.21 - 1.05 (m, 2H).

[0270] Example 10: Synthesis of N-(3-(difluoromethyl)-1-(1-(6-(1-(2,6-dioxopiperidin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-4-yl)hex-5-yn-1-yl)azetidin-3-yl)-1H-pyrazol-4-yl)-6-(1H-pyrazol-3-yl)picolinamide (10)

[0271]

[0272] Synthesis route:

[0273]

[0274] The same as the synthesis route and method of Example 5, except that the compound Int-4 of step 3 is replaced with equivalent amount of compound Int-6, compound 10 is prepared as a white solid.

[0275] MS (ES + ): m / z 697.3 [M+H] + .

[0276] 1H NMR (400 MHz, DMSO-d6): δ 13.85 (minor), 13.24 (major) (s, 1H), 11.11 (minor), 11.00 (major) (s, 1H), 10.58 (minor), 10.46 (major) (s, 1H), 8.51 (major), 8.28 (minor) (s, 1H), 8.23 - 8.16 (m, 1H), 8.10 (t, J = 7.6 Hz, 1H), 8.03 (d, J = 8.2 Hz, 1H), 7.91 (major), 7.69 (minor) (s, 1H), 7.41 - 6.89 (m, 5H), 5.37 (minor), 5.32 (major) (t, J = 4.6 Hz, 1H), 5.08 (qn, J = 6.6 Hz, 1H), 3.70 (t, J = 7.0 Hz, 2H), 3.65 (s, 1H), 3.58 (s, 3H), 3.51 (s, 1H), 2.54 (t, J = 6.9 Hz, 2H), 2.02 - 1.97 (m, 3H), 1.64 - 1.58 (m, 2H), 1.52 - 1.43 (m, 3H), 1.35 - 1.29 (m, 2H).

[0277] Example 11: Synthesis of N-(3-(difluoromethyl)-l-(l-(2-(3-((2-(2,6-dioxopiperidin-3- yl)-l-oxoisoindolin-4-yl)amino)-3-oxopropoxy)ethyl)azetidin-3-yl)-lH-pyrazol-4-yl)-6-(lH- pyrazol-3-yl)picolinamide (11)

[0278]

[0279] Synthesis route:

[0280]

[0281] The same as the synthesis route and method of Example 7, except that the compound Int-4 of step 2 is replaced with equivalent amount of compound Int-6, compound 11 is prepared as a white solid.

[0282] MS (ES + ): m / z 717.3 [M+H] + .

[0283] 1H NMR (400 MHz, DMSO-d6): δ 11.02 (s, 1H), 10.49 (s, 1H), 9.87 (s, 1H), 8.40 (s, 1H), 8.21 (s, 1H), 8.17 (d, J = 7.8 Hz, 1H), 8.11 (t, J = 7.7 Hz, 1H), 8.03 (d, J = 7.5 Hz, 1H), 7.83 (s, 1H), 7.78 (d, J = 7.0 Hz, 1H), 7.51 - 7.44 (m, 2H), 7.22 (t, J = 54.1 Hz, 1H), 7.02 (d, J = 1.9 Hz, 1H), 5.13 (dd, J = 13.3, 5.0 Hz, 1H), 5.03 (qn, J = 6.5 Hz, 1H), 4.41 - 4.30 (m, 2H), 3.73 - 3.68 (m, 4H), 3.58 - 3.56 (m, 4H), 2.95 - 2.86 (m, 1H), 2.68 - 2.58 (m, 4H), 2.38 - 2.27 (m, 1H), 2.06 - 1.94 (m, 2H).

[0284] Example 12: Synthesis of N-(3-(difluoromethyl)-1-(1-(3-(2-((3-(1-(2,6-dioxopiperidin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-4-yl)prop-2-yn-1-yl)oxy)ethoxy)propanoyl)azetidin-3-yl)-1H-pyrazol-4-yl)-6-(1H-pyrazol-3-yl)picolinamide (12)

[0285]

[0286] Synthesis route:

[0287]

[0288] The same as the synthesis route and method of Example 8, except that the compound Int-4 of step 3 is replaced with equivalent amount of compound Int-6, compound 12 is prepared as a white solid.

[0289] MS (ES + ): m / z 771.4 [M+H] + .

[0290] 1H NMR (400 MHz, DMSO-d6): δ 13.85 (minor), 13.24 (major) (s, 1H), 11.11 (s, 1H), 10.48 (major), 10.42 (minor) (s, 1H), 8.51 (major), 8.35 (minor) (s, 1H), 8.22 - 8.14 (m, 1H), 8.10 (t, J = 7.5 Hz, 1H), 8.03 (d, J = 8.0 Hz, 1H), 7.90 (major), 7.69 (minor) (s, 1H), 7.44 - 6.92 (m, 5H), 5.38 (major), 5.32 (minor) (dd, J = 12.5, 5.5 Hz, 1H), 4.63 (t, J = 8.4 Hz, 1H), 4.48 - 4.41 (m, 3H), 4.34 (t, J = 9.1 Hz, 1H), 4.16 - 4.12 (m, 1H), 3.67 - 3.61 (m, 6H), 3.60 - 3.54 (m, 4H), 2.95 - 2.55 (m, 2H), 2.38 (t, J = 6.3 Hz, 2H), 2.07 - 1.94 (m, 2H).

[0291] Example 13: Synthesis of N-(3-(difluoromethyl)-l-(l-(5-(l-(2,6-dioxopiperidin-3-yl)-3- methyl-2-oxo-2,3-dihydro-lH-benzo[d]imidazol-4-yl)pent-4-ynoyl)azetidin-3-yl)-lH- pyrazol-4-yl)-6-(lH-pyrazol-3-yl)picolinamide (13)

[0292]

[0293] Synthesis route:

[0294]

[0295] The same as the synthesis route and method of Example 9, except that the compound Int-4 of step 3 is replaced with equivalent amount of compound Int-6, compound 13 is prepared as a white solid.

[0296] MS (ES + ): m / z 697.2 [M+H] + .

[0297] 1H NMR (400 MHz, DMSO-d6): δ 11.11 (s, 1H), 10.49 (s, 1H), 8.49 (s, 1H), 8.18 (d, J = 7.6 Hz, 1H), 8.11 (t, J = 7.7 Hz, 1H), 8.04 (d, J = 7.4 Hz, 1H), 7.84 (s, 1H), 7.39 - 6.93 (m, 5H), 5.47 - 5.31 (m, 2H), 4.66 (t, J = 8.4 Hz, 1H), 4.50 - 4.47 (m, 1H), 4.37 (t, J = 9.1 Hz, 1H), 4.22 - 4.16 (m, 1H), 3.66 (s, 3H), 2.96 - 2.84 (m, 2H), 2.76 - 2.59 (m, 4H), 2.06 - 1.95 (m, 2H).

[0298] Compounds 14-25 were synthesized according to the above examples.

[0299] Bioassay 1. ADP-Glo assay for IRAK1 and IRAK4 kinase activity

[0300] Prepare 2x ATP / substrate solution and 2x kinase solution in kinase reaction buffer (50 mM Hepes, 10 mM MgCl2, 0.01% Brij-35, 1 mM EGTA, 2 mM DTT, H2O) respectively, Transfer compound dilutions 40Nal to 384 assay plates using a T-sonicator, centrifuge and add 2 μL of 2x kinase solution, centrifuge at 1000 rpm for 1 min, incubate at 25 °C for 10 min. Add 2 μL of 2x ATP / substrate solution to the 384 assay plates, centrifuge at 1000 rpm for 1 min, incubate at 25 °C for 60 min. Add 4 μL of ADP-Glo reagent per well, centrifuge at 1000 rpm for 1 min, incubate at 25 °C for 40 min. Add 8 μL of kinase detection reagent per well, centrifuge at 1000 rpm for 1 min, incubate at 25 °C for 40 min. Read chemiluminescence signal on a BMG plate reader, calculate the inhibition of kinase activity using the following formula:

[0301] % Inhibition = (Positive control well mean - compound well) / (Positive control well mean - negative control well mean) x 100

[0302] The half maximal inhibitory concentration (IC50) of compounds against enzyme activity was fitted by non-linear regression equation (dose response-variable slope) in GraphPad 8.0.

[0303] Non-linear regression equation: Y = Bottom + (Top-Bottom) / (1 + 10^((LogIC 50X) x HillSlope

[0304] X: Log of compound concentration

[0305] Y: % Inhibition

[0306] HillSlope: Slope of the curve.

[0307] Bottom: Y value of the bottom plateau.

[0308] Top: Y value of the top plateau.

[0309] Table 1 below provides the IC50 range of some compounds for IRAK1 and IRAK4 kinase activity inhibition: A: < 1 nM; B: 1-10 nM; C: > 10 nM.

[0310] Table 1 Compounds for IRAK1 and IRAK4 kinase activity inhibition

[0311]

[0312] Bioassay 2. Degradation of IRAK4 in cells

[0313] The cell line THP-1 used in the experiment was purchased from Wuhan Ponsay Life Science Co., Ltd. and cultured in RPMI-1640 containing 10% fetal bovine serum (0.05 mM β-mercaptoethanol and 1% P / S). The logarithmic phase cells were collected by centrifugation, resuspended with culture medium, counted, and the cell density was adjusted to 1 x 10 6 / mL, 2 mL per well, and incubated in a 37°C, 5% CO2 incubator for 30 minutes. IRAK4 degradation in THP-1 cells, 20 mM compound stock solution was diluted with DMSO to 10000 µM, 5000 µM, 1000 µM, 200 µM, 40 µM, 8 µM, 1.6 µM, 0.32 µM, 0.064 µM. 2 µL per well was added to the corresponding concentration well, and the final concentration was 20 µM, 10 µM, 5 µM, 1 µM, 200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM, 0.064 nM, respectively, and incubated in a 37°C, 5% CO2 incubator for 24 hours. After 24 hours of treatment with the corresponding concentration of compound, the cells were collected by centrifugation, and the supernatant was discarded. The cell pellet was lysed with RIPA lysis buffer (protease inhibitor) on ice for 15 minutes, centrifuged at 13000 rpm at 4°C for 30 minutes, and the supernatant was collected to measure the protein concentration. The protein concentration was adjusted to 3 mg / mL, 5x SDS-PAGE loading buffer was added, and the sample was heated at 95°C for 7 minutes. SDS-PAGE electrophoresis was performed by Bio-Rad Mini-PROTEAN Tetra, and the protein imaging of IRAK4 in the sample was performed by LI-COR Odyssey dual-color infrared fluorescence gel imaging system. The degradation rate of the compound on IRAK4 was calculated by semi-quantification with Image J.

[0314] Band intensity’ = Band intensity IRAK4 / Band intensity GAPDH

[0315] Degradation rate% = (1 - Band intensity’ 化合物 / Band intensity’ DMSO ) x 100

[0316] Wherein GAPDH represents glyceraldehyde-3-phosphate dehydrogenase.

[0317] Figure 1 Some degradation of IRAK4 in THP-1 cells by some compounds is provided, as shown in the following table 1: Figure 1 ( Figure 1 The last column marked “—” in the table represents the negative control, and in the figure, the two concentrations of 1.6 nM and 8 nM in compound 3 were reversed during operation, which has been additionally marked with a red box and red text) It can be seen from the figure that compounds 2-6 and 8-10 have obvious degradation effect on IRAK4 in THP-1 cells, and show Hook effect at high concentration.

[0318] The following table 2 provides the DC50 interval of some compounds for IRAK4 degradation in THP-1 cells: A: <1 nM; B: 1-10 nM; C: 11-100 nM; D: 101-1000 nM; E: >1000 nM.

[0319] Table 2 shows the effect of compounds on the concentration-degradation of IRAK4 in THP-1.

[0320]

[0321] 3. Effects of biological assay on the secretion of TNF-α and IL-6 by human PBMCs.

[0322] Human PBMCs used in the experiment were purchased from Miaoshun (Shanghai) Biotechnology Co., Ltd., donor number DS232998. After thawing, the cryopreserved human PBMCs were immediately resuspended in RPMI-1640 (2% FBS and 1% P / S), counted, and the cell density was adjusted to 5 × 10⁶ cells / mL. 5 90 μL of the compound was seeded per well in a 96-well U-shaped plate and incubated at 37°C with 5% CO2 for 30 minutes. 0.5 μL of 10 mM DMSO stock solution was added to 49.5 μL of RPMI-1640 (2% FBS and 1% P / S) to dilute to 100 μM working solution. This solution was then diluted five-fold with RPMI-1640 (2% FBS and 1% P / S). 0.5 μL of DMSO was added to 49.5 μL of RPMI-1640 (2% FBS and 1% P / S) to prepare the working solution for the DMSO control wells. 10 μL of both the compound working solution and the DMSO working solution were added to the corresponding wells, centrifuged at 600 rpm for 30 seconds, and incubated overnight at 37°C with 5% CO2. LPS was diluted to a working concentration of 2.5 μg / mL with RPMI-1640 (2% FBS and 1% P / S). 25 μL of LPS working solution was added to each well, centrifuged at 600 rpm for 30 seconds, and incubated at 37°C with 5% CO2 for 4–6 hours (hTNF-α) or 24 hours (hIL-6). After the incubation period, the mixture was centrifuged at 1000 rpm for 5 minutes, and the supernatant was transferred to another 96-well plate for storage. The supernatant was diluted three-fold for ELISA analysis of hTNF-α and hIL-6. The concentrations of hTNF-α and hIL-6 in the supernatant of the corresponding wells were calculated according to the ELISA kit instructions. The inhibitory effect of the compounds on the secretion of hTNF-α and hIL-6 by LPS-stimulated human PBMCs was calculated using the following formula.

[0323] Inhibition rate % = (1 - (compound wells - background wells) / (negative control wells - background wells)) × 100

[0324] Compound pores: Cells + Compounds + LPS Stimulation

[0325] Background wells: Cells + DMSO control

[0326] Negative control wells: Cells + DMSO control + LPS stimulation

[0327] The following Table 3 provides the IC50 range of some compounds for the inhibition of LPS-stimulated human PBMCs to secrete hTNF-a and hIL-6: A: <1 nM; B: 1-10 nM; C: 11-100 nM; D: 101-1000 nM; E: >1000 nM.

[0328] Table 3 Inhibition of LPS-stimulated human PBMCs to secrete hTNF-a and hIL-6 by compounds

[0329]

[0330] Bioassay 4. Effect on TLR signaling pathway

[0331] The cell line THP-1 used in the experiment was purchased from Wuhan Ponsay Life Science Co., Ltd. and cultured in RPMI-1640 containing 10% fetal bovine serum (0.05 mM β-mercaptoethanol and 1% P / S). The logarithmic phase cells were collected by centrifugation, resuspended with culture medium, counted, and the cell density was adjusted to 1 x 10^6 / mL, 4 mL was inoculated per well, and incubated in a 37°C, 5% CO2 incubator for 30 minutes. The 10 mM compound stock solution was diluted with DMSO to 1000 μM, 100 μM, 10 μM, 1 μM, 0.1 μM, and 0.01 μM. 4 μL was added to each well to the corresponding concentration well, and the final concentration was 1000 nM, 100 nM, 10 nM, 1 nM, 0.1 nM, and 0.01 nM, respectively, and incubated in a 37°C, 5% CO2 incubator for 24 hours. After 24 hours, 20 μL / well of 1 mg / mL LPS was added to the corresponding wells, and incubated in a 37°C, 5% CO2 incubator for 30 minutes. After 30 minutes, the cells were collected by centrifugation, and the supernatant was discarded. The cell pellet was lysed with RIPA lysis buffer (protease inhibitors and phosphatase inhibitors) on ice for 15 minutes, centrifuged at 4°C, 13000 rpm for 30 minutes, the supernatant was collected, the protein concentration was measured, the protein concentration was adjusted to 4 mg / mL, 5x SDS-PAGE loading buffer was added, and heated at 95°C for 7 minutes. The protein content of Phospho-p38 MAPK (Thr180 / Tyr182), Phospho-ERK1 / 2 (Thr202 / Tyr204), and NF-κB p65 Phospho (Ser529) in the sample was detected by SDS-PAGE and WB.

[0332] The detection results are shown in Table 4, and it can be seen from Table 4 that the compound 2 capable of degrading IRAK4 can inhibit the MAPK and NFκB signaling pathways downstream of TLR / IL1R. Figure 2 Figure 2

[0333] ​​Applicants state that the protein degradation targeting chimera molecules and uses thereof of the present application are illustrated by the above examples, but the present application is not limited to the above examples, i.e., it is not meant that the present application must rely on the above examples to be implemented. It should be understood by those skilled in the art that any improvement on the present application, equivalent replacement of the raw materials selected by the present application, addition of auxiliary ingredients, selection of specific modes, etc. all fall within the protection scope and disclosure scope of the present application.

Claims

1. A protein degradation-targeting chimera molecule, or a pharmaceutically acceptable salt thereof, characterized in that, The protein degradation targeting chimera molecule is a compound represented by general formula (III): ； Among them, R f Selected from -CF2H; LBM is selected from: ； L is the following structure: or ; x is 1, 2, 3, 4 or 5, y is selected from 0, and the wavy line represents the linking site of the group.

2. The protein degradation targeting chimera molecule or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The protein degradation targeting chimera molecule is any one of the following compounds: 。 3. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the protein degradation targeting chimera molecule or a pharmaceutically acceptable salt thereof according to claim 1 or 2, and optionally a pharmaceutically acceptable carrier.

4. Use of the protein degradation targeting chimera molecule or a pharmaceutically acceptable salt thereof according to claim 1 or 2, or the pharmaceutical composition according to claim 3 in the preparation of a medicament for treating leukemia, multiple myeloma, lymphoma or pancreatic cancer.

5. Use of the protein degradation targeting chimera molecule or a pharmaceutically acceptable salt thereof according to claim 1 or 2, or the pharmaceutical composition according to claim 3 in the preparation of a medicament for treating an autoimmune disease or an inflammatory disease.

6. Use according to claim 5, characterized in that, The autoimmune disease or the inflammatory disease is selected from systemic lupus erythematosus, arthritis, psoriasis, Crohn's disease, atopic dermatitis, protein-related periodic syndrome, chronic obstructive pulmonary disease, asthma or graft-versus-host disease.

7. Use according to claim 5, characterized in that, The autoimmune disease or the inflammatory disease is selected from lupus nephritis or gout.

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