Irak inhibitor and preparation method therefor and use thereof

Abstract

Disclosed is a compound represented by formula I, a stereoisomer, racemate, tautomer, isotopic label, prodrug thereof or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition comprising same, a preparation method therefor, and medical use thereof. The structure is as shown in formula I.

Description

IRAK inhibitors and methods of making and using the same

[0001] This application claims priority to the prior application filed on September 24, 2019, with the China National Intellectual Property Office, Patent Application No. 201910906833.7, entitled “IRAK inhibitors and methods of making and using the same”. The entire contents of the application are incorporated herein by reference. TECHNICAL FIELD

[0002] The present application relates to the field of medicinal chemistry, in particular to a compound suitable for treating cancer and inflammatory diseases associated with interleukin-1 receptor-associated kinase (IRAK), and more particularly to a compound that modulates the function of IRAK-4. BACKGROUND

[0003] Interleukin-1 receptor-associated kinases (IRAK) are a family of serine / threonine protein kinases that exist in cells, with four members: IRAK1, IRAK2, IRAK-M and IRAK4, which share the common feature of having a typical N-terminal death domain that mediates interaction between MyD88-family adaptor proteins and the central kinase domain, of which IRAK1 and IRAK4 have kinase activity. IRAK4 is a key factor downstream of the Toll-like receptor (TLR) / interleukin-1 receptor (IL-1R) mediated inflammatory signaling pathway, the extracellular part of TLR recognizes pathogen-specific molecules (such as lipopolysaccharide, polypeptide, viral DNA, etc.), after binding with the ligand, the intracellular part recruits MyD88 to form a complex, activates IRAK1 autophosphorylation, and then activates the downstream serine / threonine kinase TAK1, activates the NF-κB and MAPK signaling pathways, and then produces pro-inflammatory cytokines, chemokines and destructive enzymes, ultimately leading to the production of inflammatory response, mediating innate immunity. IL-1R is involved in host defense and hematopoiesis, and is a bridge connecting innate immunity and acquired immunity. (Flannery, et. al. Biochem. Pharmacol., 2010, 80(12): 1981-1991).

[0004] Rheumatoid arthritis (RA) is a chronic, inflammatory, systemic autoimmune disease, with non-suppurative inflammation of joints and joint tissues as the main feature, mainly manifested as synovial inflammation of joints, eventually leading to various tissues such as cartilage, ligament and tendon of joints and multiple organ damage. Studies have shown that a variety of immune cells are involved in and mediate autoimmune inflammation in RA patients, including T / B lymphocytes, macrophages, neutrophils and the like. At the same time, a large number of studies have proved that cytokines are directly related to RA diseases, such as interleukins (IL-1 / IL-6, etc.), TNF-α and the like.

[0005] Studies have shown that IRAK4 inhibitors can effectively block the production of pro-inflammatory cytokine tumor necrosis factor (TNF) in LPS or CpG induced human leukocytes; in collagen-induced arthritis mouse models, IRAK4 inhibitors can significantly inhibit the release of TNF, thereby controlling the progression of the disease; in MyD88-dependent inflammatory gout mouse models, IRAK4 inhibitors can dose-dependently block leukocyte infiltration (Priscilla N, et.al. J. Exp. Med., 2015, 13(212): 2189-2201).

[0006] Therefore, it can be considered that overactivation of IRAK4-dependent TLR / IL-1R signaling pathway is closely related to the occurrence and development of rheumatoid arthritis, and a large number of studies have also confirmed that IRAK4 enzyme activation is closely related to the occurrence and development of diseases such as tumors, gout, systemic lupus erythematosus, multiple sclerosis, metabolic syndrome, atherosclerosis, myocardial infarction, sepsis, inflammatory bowel disease, asthma and allergy (Chaudhary D, et.al., J. Med. Chem. 2015, 58(1): 96-110).

[0007] SUMMARY

[0008] To solve the problems in the prior art, the present application provides a compound as shown in the following formula I, a stereoisomer, a racemate, a tautomer, an isotopically labeled substance, a prodrug or a pharmaceutically acceptable salt thereof:

[0009]

[0010] wherein,

[0011] Ring A is a 5-14 membered heteroaryl or 5-12 membered heterocyclyl containing at least one N-containing 5-14 membered heteroaryl or 5-12 membered heterocyclyl;

[0012] Each of R1, R2 and R3 is independently selected from hydrogen, halogen, CN, OH or the following group optionally substituted with one, two or more R: (C1-C 12Aliphatic hydrocarbon group, optionally containing one, two or more heteroatoms (C1-C1). 12 aliphatic hydrocarbon group, C 3-12 Cycloalkyl, 3-12 membered heterocyclic groups, C 6-20 Aryl or 5-14 heteroaryl, -NR a R b ;

[0013] W is selected from O, S, NH, and single bonds;

[0014] Each R a R b Independently selected from H, (C1-C 12 Aliphatic hydrocarbon groups;

[0015] Each R is independently selected from halogen, CN, OH, SH, NR. a R b Or selected from the following groups substituted with one, two or more R': (C1-C 12 Aliphatic hydrocarbon group, optionally containing one, two or more heteroatoms (C1-C1). 12 aliphatic hydrocarbon group, C 3-12 Cycloalkyl, 3-12 membered heterocyclic groups, C 6-20 Aryl or 5-14 heteroaryl groups;

[0016] Each R' is independently selected from halogen, CN, OH, SH, NR. a R b ;

[0017] n is selected from 1, 2, or 3; m is selected from 1, 2, 3, 4, 5, or 6. According to an embodiment of the present invention,

[0018] The "optionally containing one, two or more heteroatoms (C1-C)" 12 "Aliphatic hydrocarbon group" can be selected from (C1-C1) 12 (Aliphatic hydrocarbon radicals, (C1-C) 12 (C1-C6) aliphatic hydrocarbon mercapto, (C1-C6) aliphatic hydrocarbon oxygen (C1-C6) aliphatic hydrocarbon, (C1-C6) aliphatic hydrocarbon mercapto (C1-C6) aliphatic hydrocarbon, N-(C1-C3) aliphatic hydrocarbon amino (C1-C6) aliphatic hydrocarbon, N,N-di-(C1-C3) aliphatic hydrocarbon amino (C1-C6) aliphatic hydrocarbon;

[0019] The phrase "containing at least one N-containing 5-14-membered heteroaryl or 5-12-membered heterocyclic group" means that the heteroaryl or heterocyclic group contains at least one nitrogen atom, and may also contain one or more other heteroatoms selected from N, O, or S, such as those selected from pyridine, pyrrole, piperidine, or tetrahydropyrrole.

[0020] The (C1-C) 12 The aliphatic hydrocarbon group can be selected from (C1-C1) 12 )alkyl, (C2-C 12 )alkenyl, (C2-C 12 ) alkynyl group, preferably, the (C1-C 12 The aliphatic hydrocarbon group can be selected from (C1-C6)alkyl, (C2-C6)alkenyl, and (C2-C6)ynyl.

[0021] The "halogen" is selected from F, Cl, Br, and I;

[0022] The "C" 3-12 "Cycloalkyl" can be selected from cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

[0023] According to an embodiment of the present invention,

[0024] R1, R2, and R3 can each be independently selected from the following groups that are optionally substituted with one, two, or more R groups: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, vinyl, 1-propenyl, 2-propenyl, 1-methylvinyl, 1-butenyl, 1-ethylvinyl, 1-methyl-2-propenyl, 2-butenyl, 3-butenyl, 2-methyl-1-propenyl, 2-methyl-2-propenyl, 1-pentenyl, 1-hexenyl, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 1-methyl-2-propynyl, 3-butynyl, 1- Pentynyl, 1-hexynyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, methoxy, ethoxy, propoxy, butoxy, pentoxy, methoxymethyl, ethoxymethyl, propoxymethyl, methoxyethyl, ethoxyethyl, propoxyethyl, methoxypropyl, ethoxypropyl, propoxypropyl, N-methylaminomethyl, N-methylaminoethyl, N-ethylaminoethyl, N,N-dimethylaminomethyl, N,N-dimethylaminoethyl, N,N-diethylaminoethyl, amino, N,N-dimethylamino, N,N-diethylamino, tetrahydropyrrolyl, piperidinyl, pyridyl, pyrazinyl, pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl

[0025]

[0026] The The diagram illustrates the connection sites of the stated groups.

[0027] According to embodiments of the present invention, the compound represented by Formula I, its stereoisomers, racemates, tautomers, isotope labels, prodrugs, or pharmaceutically acceptable salts thereof, wherein the compound represented by Formula I may be selected from the structures of Formulas Ia, Ib, Ic, Id, and Ie:

[0028]

[0029] In the formula Ia, formula Ib, formula Ic, formula Id, formula Ie, R1, R2, R3, m, n, W are as defined in formula I.

[0030] According to an embodiment of the present application, the compound of formula I, stereoisomer, racemate, tautomer, isotopically-labeled, prodrug or pharmaceutically acceptable salt thereof, the compound of formula I can be selected from the following structures:

[0031]

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038]

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052] The present invention also provides methods for preparing compounds of Formula I (including Formulas Ia-Ie), their stereoisomers, racemates, tautomers, isotope labels, prodrugs or pharmaceutically acceptable salts thereof, but is not limited to the methods described below.

[0053] In some embodiments, the preparation method may include the following steps:

[0054]

[0055] (a1) M-1 and M-2 are reacted to generate M-3; the reaction can be carried out in the presence of EDCl, HCl, and pyridine;

[0056] (a2)M-3 and R x L1 reaction, where R x Selected from R1 or R1 containing a hydroxyl group, the hydroxyl group is... Substituted groups; and when R x Selected from R1 containing a hydroxyl group, the hydroxyl group is... When a substituted group is involved, the reaction needs to further include obtaining the product of formula I under acidic and reducing conditions, wherein the acidic conditions can be selected from HCl and the reducing conditions can be selected from sodium borohydride.

[0057] In the steps described, R1, R2, R3, m, and W are as defined in Formula I; L1 is a leaving group, which may be selected from halogens or OTs.

[0058] In some embodiments, the preparation method may include the following steps:

[0059]

[0060] (b1) N-1 and R x L1 reaction, where R x Selected from R1 or R1 containing a hydroxyl group, the hydroxyl group is... Substituted groups; and when R x Selected from R1 containing a hydroxyl group, the hydroxyl group is... When a substituted group is involved, the reaction needs to further include obtaining the N-2 product under acidic and reducing conditions; in this step, the acidic conditions can be selected from HCl, and the reducing conditions can be selected from sodium borohydride.

[0061] (b2) Reduce the N-2 obtained in the previous step to obtain N-3; the reducing agent can be selected from Pd / C;

[0062] (b3) React N-3 and M-2 to obtain formula I.

[0063] In the step, R1, R2, R3, m, W are defined as in Formula I; and L1 is a leaving group, which can be selected from halogen or OTs.

[0064] The present application further provides a pharmaceutical composition comprising a compound of Formula I, stereoisomer, racemate, tautomer, isotopically-labeled, prodrug or pharmaceutically acceptable salt thereof as described herein.

[0065] In some embodiments, the pharmaceutical composition further comprises a therapeutically effective amount of a compound of Formula I, stereoisomer, racemate, tautomer, isotopically-labeled, prodrug or pharmaceutically acceptable salt thereof as described herein and a pharmaceutically acceptable carrier.

[0066] The present application further provides use of a compound of Formula I, stereoisomer, racemate, tautomer, isotopically-labeled, prodrug or pharmaceutically acceptable salt thereof in the preparation of an IRAK inhibitor.

[0067] The present application further provides use of a compound of Formula I, stereoisomer, racemate, tautomer, isotopically-labeled, prodrug or pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and / or treating an IRAK-mediated disease or disorder.

[0068] According to embodiments of the present application, the IRAK-mediated disease or disorder is selected from the group consisting of tumor, gout, systemic lupus erythematosus, multiple sclerosis, metabolic syndrome, atherosclerosis, myocardial infarction, sepsis, inflammatory bowel disease, asthma and allergy, etc.

[0069] The present application further provides use of a compound of Formula I, stereoisomer, racemate, tautomer, isotopically-labeled, prodrug or pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and / or treating a disease or disorder associated with interleukin-1 receptor-associated kinase.

[0070] The present application further provides a method for preventing and / or treating an IRAK-mediated disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of Formula I, stereoisomer, racemate, tautomer, isotopically-labeled, prodrug or pharmaceutically acceptable salt thereof or the pharmaceutical composition.

[0071] In some embodiments, the IRAK is selected from IRAK4-associated kinase.

[0072] The present application also provides a method for preventing and / or treating an interleukin-1 receptor associated disease, comprising administering to an individual in need thereof a therapeutically effective amount of a compound represented by Formula I, a stereoisomer, a racemate, a tautomer, an isotopically-labeled material, a prodrug, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition.

[0073] According to an embodiment of the present application, the disease or disorder associated with interleukin-1 receptor associated kinase is selected from the group consisting of tumor, gout, systemic lupus erythematosus, multiple sclerosis, metabolic syndrome, atherosclerosis, myocardial infarction, sepsis, inflammatory bowel disease, asthma, rheumatoid arthritis, sepsis, autoimmune diseases, and allergy.

[0074] The methods of the present application can include administration of a compound of the present application alone, as well as in combination with one or more other chemotherapeutic agents. Administration of multiple drugs can be simultaneous or sequential.

[0075] Definitions of terms and abbreviations

[0076] Unless otherwise indicated, the definitions of groups and terms in the specification and claims hereof include within their scope numerous isomers, racemates, tautomers, isotopically labeled materials, prodrugs, and pharmaceutically acceptable salts thereof, as well as mixtures thereof, and combinations thereof, as would be apparent to one of ordinary skill in the art. Such combinations and mixtures are within the scope of the present application.

[0077] Numerical ranges in the present specification are defined to include both the end points of the range and every integer within the range. For example, "integers from 0 to 6" is defined to include 0, 1, 2, 3, 4, 5, and 6. "More than three" means three or more.

[0078] The optional substitution described herein encompasses the case of no substitution as well as the case of substitution with one or more substituents, for example, "optionally substituted with one, two, or more R" means that there can be no substitution (no substitution) or one, two, or more R.

[0079] The term "halogen" means F, Cl, Br, and I. In other words, F, Cl, Br, and I can be described as "halogen" in the present specification.

[0080] The term "aliphatic hydrocarbon group" includes saturated or unsaturated, straight-chain or branched-chain, or cyclic hydrocarbon groups, the type of which can be selected from alkyl, alkenyl, alkynyl, etc., the number of carbon atoms of which is preferably 1 to 12, also 1 to 10, and further preferably 1 to 6, and specifically can include, but is not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, ethenyl, 1-propenyl, 2-propenyl, 1-methylethenyl, 1-butenyl, 1-ethylethenyl, 1-methyl-2-propenyl, 2-butenyl, 3-butenyl, 2-methyl-1-propenyl, 2-methyl-2-propenyl, 1-pentenyl, 1-hexenyl, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 1-methyl-2-propynyl, 3-butynyl, 1-pentynyl, 1-hexynyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, or cyclodecyl, or a bicyclic hydrocarbon group such as decalin ring.

[0081] The "aliphatic hydrocarbon group" can optionally contain one, two or more heteroatoms (or interpreted as optional insertion of heteroatoms into optional C-C and C-H bonds of the aliphatic hydrocarbon group). Suitable heteroatoms will be apparent to those skilled in the art and include, for example, sulfur, nitrogen, oxygen, phosphorus and silicon. The aliphatic hydrocarbon group containing a heteroatom can be selected from the following groups: (C1-C6) aliphatic hydrocarbon oxy, (C1-C6) aliphatic hydrocarbon mercapto, (C1-C6) aliphatic hydrocarbon oxy(C1-C6) aliphatic hydrocarbon, (C1-C6) aliphatic hydrocarbon mercapto(C1-C6) aliphatic hydrocarbon, N-(C1-C3) aliphatic hydrocarbon aminyl(C1-C6) aliphatic hydrocarbon, N,N-di-(C1-C3) aliphatic hydrocarbon aminyl(C1-C6) aliphatic hydrocarbon, for example, methoxy, ethoxy, propoxy, butoxy, pentoxy, methoxymethyl, ethoxymethyl, propoxymethyl, methoxyethyl, ethoxyethyl, propoxyethyl, methoxypropyl, ethoxypropyl, propoxypropyl, N-methylaminomethyl, N-methylaminoethyl, N-ethylaminoethyl, N,N-dimethylaminomethyl, N,N-dimethylaminoethyl, N,N-diethylaminoethyl; the "aliphatic hydrocarbon" portion of other groups being as explained above.

[0082] The term "C 3-12 Cycloalkyl" is understood to mean a saturated or unsaturated, monovalent, monocyclic or bicyclic hydrocarbon ring having 3 to 12 carbon atoms, preferably "C 3-10 Cycloalkyl". The term "C 3-10 Cycloalkyl" is understood to mean a saturated, monovalent, monocyclic or bicyclic hydrocarbon ring having 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. The C 3-10 Cycloalkyl" can be a monocyclic hydrocarbon group such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl or cyclodecyl, or a bicyclic hydrocarbon group such as decalin ring.

[0083] The term "3-12 membered heterocyclyl" means a saturated or unsaturated, monovalent monocyclic or bicyclic ring which contains 1 to 5 heteroatoms independently selected from N, O and S, the heteroatom containing groups are not aromatic, said 3-12 membered heterocyclyl, preferably "3-10 membered heterocyclyl". The term "3-10 membered heterocyclyl" means a saturated, monovalent monocyclic or bicyclic ring which contains 1 to 5, preferably 1 to 3 heteroatoms selected from N, O and S. Said heterocyclyl group can be attached to the rest of the molecule by any of the carbon atoms or the nitrogen atom, if present. In particular, said heterocyclyl group can include, but is not limited to: 4 membered rings, such as azetidinyl, oxetanyl; 5 membered rings, such as tetrahydrofuranyl, tetrahydrothiophenyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl; or 6 membered rings, such as tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl or trithianyl; or 7 membered rings, such as diazepanyl. Optionally, said heterocyclyl group can be benzo-fused. Said heterocyclyl group can be bicyclic, for example, but not limited to, 5,5 membered rings, such as hexahydrocyclopenta[c]pyrrol-2(lH)-yl ring, or 5,6 membered rings, such as hexahydropyrrolo[l,2-a]pyrazin-2(lH)-yl ring. The nitrogen atom containing rings can be partially unsaturated, i.e. it can contain one or more double bonds, for example, but not limited to, 2,5-dihydro-lH-pyrrolyl, 4H-[l,3,4]thiadiazinyl, 4,5-dihydrooxazolyl or 4H-[l,4]thiazinyl, or it can be benzo-fused, for example, but not limited to, dihydroisoquinolinyl. According to the present application, said 3-12 membered heterocyclyl group can be further selected from the following groups:

[0084]

[0085] The term "C 6-20 aryl" is to be understood as preferably meaning a monovalent aromatic or partially aromatic, monocyclic, bicyclic or tricyclic hydrocarbon ring having 6 to 20 carbon atoms, preferably "C 6-14 aryl". The term "C 6-14 aryl" is to be understood as preferably meaning a monovalent aromatic or partially aromatic, monocyclic, bicyclic or tricyclic hydrocarbon ring having 6, 7, 8, 9, 10, 11, 12, 13 or 14 carbon atoms ("C 6-14 aryl"), in particular a ring having 6 carbon atoms ("C6aryl"), for example phenyl; or a ring having 9 carbon atoms ("C9aryl"), for example indanyl or indenyl; or a ring having 10 carbon atoms ("C 10 aryl"), for example tetrahydronaphthyl, dihydronaphthyl or naphthyl; or a ring having 13 carbon atoms ("C 13 aryl"), for example fluorenyl; or a ring having 14 carbon atoms ("C14 aryl") such as anthryl.

[0086] The term "5-14 membered heteroaryl" is to be understood as including monovalent monocyclic, bicyclic or tricyclic aromatic ring systems having 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 ring atoms, in particular 5 or 6 or 9 or 10 carbon atoms, and which contain 1 to 5, preferably 1 to 3, heteroatoms each independently selected from N, O and S and, in addition, in each case can be benzo-fused. In particular, the heteroaryl group is selected from the group consisting of thienyl, furanyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, thia-4H-pyrazolyl and the like and their benzo derivatives, such as, for example, benzofuranyl, benzothienyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzotriazolyl, indazolyl, indolyl, isoindolyl and the like; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl and the like and their benzo derivatives, such as, for example, quinolinyl, quinazolinyl, isoquinolinyl and the like; or azocinyl, indolizinyl, purinyl and the like and their benzo derivatives; or cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenoxazinyl, phenothiazinyl, phenoxazinyl and the like.

[0087] Unless otherwise indicated, the heterocyclyl or heteroaryl group includes all possible isomeric forms thereof, such as positional isomers. Thus, for some illustrative, non-limiting examples, pyridyl or pyridinylene includes pyrid-2-yl, pyrid-2- ylene, pyrid-3-yl, pyrid-3-ylene, pyrid-4-yl and pyrid-4-ylene; thienyl or thienylene includes thien-2-yl, thien-2-ylene, thien-3-yl and thien-3-ylene.

[0088] In the present context, the "3-12 membered heterocyclyl", "5-14 membered heteroaryl" can further also include N-containing 5-12 membered heterocyclyl or 5-14 membered heteroaryl, i.e. the N-containing 5-12 membered heterocyclyl or 5-14 membered heteroaryl can be selected from the corresponding groups within the definition of the "3-12 membered heterocyclyl", "5-14 membered heteroaryl" term.

[0089] Depending on their molecular structure, the compounds according to the application can be chiral and thus can exist in various enantiomeric forms. The compounds can thus exist in racemic or optically active form. The compounds according to the application or intermediates thereof can be separated into the enantiomeric compounds by chemical or physical methods known to those skilled in the art or used in the synthesis in this form. In the case of racemic amines, the diastereomeric forms are prepared from the mixture by reaction with optically active resolving agents. Examples of suitable resolving agents are optically active acids, such as, for example, the R and S forms of tartaric acid, diacetyl tartaric acid, dibenzoyl tartaric acid, mandelic acid, malic acid, lactic acid, suitable N-protected amino acids, such as, for example, N-benzoyl proline or N-benzylsulfonyl proline, or various optically active camphorsulfonic acids. Chromatographic enantiomeric resolution with the aid of optically active resolving agents, such as, for example, dinitrobenzoyl phenyl glycine, cellulose triacetate or other carbohydrate derivatives or chiral derivatized methacrylate polymers, which are fixed on silica gel, can also be advantageously carried out. Suitable eluents for this purpose are aqueous or alcoholic solvent mixtures, for example, hexane / isopropanol / acetonitrile.

[0090] Pharmaceutically acceptable salts can be, for example, acid addition salts of compounds according to the application which have sufficiently basic nitrogen atoms in a chain or ring, for example, acid addition salts with inorganic acids, such as, for example, hydrochloric acid, hydrofluoric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, pyrosulfuric acid, phosphoric acid or nitric acid, or bisulfuric acid, or with organic acids, such as, for example, formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectinic acid, persulfuric acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalene- disulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptanoic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid, hemisulfuric acid or thiocyanic acid.

[0091] In addition, another suitable pharmaceutically acceptable salt of a compound of the present application having sufficient acidity is an alkali metal salt (e.g., sodium salt or potassium salt), an alkaline earth metal salt (e.g., calcium salt or magnesium salt), an ammonium salt, or a salt with an organic base which provides a physiologically acceptable cation, such as a salt with sodium ion, potassium ion, N-methylglucamine, dimethylglucamine, ethylglucamine, lysine, dicyclohexylamine, 1,6-hexanediamine, ethanolamine, glucosamine, meglumine, sarcosine, serinol, tris-hydroxymethylaminomethane, aminopropanediol, 1-amino-2,3,4-butantriol. As an example, the pharmaceutically acceptable salt includes a salt of the group -COOH with sodium ion, potassium ion, calcium ion, magnesium ion, N-methylglucamine, dimethylglucamine, ethylglucamine, lysine, dicyclohexylamine, 1,6-hexanediamine, ethanolamine, glucosamine, meglumine, sarcosine, serinol, tris-hydroxymethylaminomethane, aminopropanediol, 1-amino-2,3,4-butantriol.

[0092] In addition, a basic nitrogen-containing group can be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl, and diamyl sulfate; long chain halides, such as decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; aralkyl halides, such as benzyl and phenethyl bromides and others. As an example, the pharmaceutically acceptable salt includes a hydrochloride, sulfate, nitrate, bisulfate, hydrobromide, acetate, oxalate, citrate, methanesulfonate, formate, or meglumine salt, and the like.

[0093] Since a compound of the present application can have multiple salt-forming sites, the "pharmaceutically acceptable salt" includes not only salts formed at one salt-forming site of a compound of the present application, but also salts formed at two, three, or all of the salt-forming sites. For this reason, the molar ratio of the radical ion (anion) of the compound of formula (I) to the cation of the base required for salt formation in the "pharmaceutically acceptable salt" can vary within a wide range, for example, it can be 4:1 to 1:4, such as 3:1, 2:1, 1:1, 1:2, 1:3, and the like.

[0094] According to the present application, pharmaceutically acceptable anions include anions derived from inorganic or organic acids. The "inorganic acids" include, but are not limited to, hydrochloric acid, hydrofluoric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, pyrosulfuric acid, phosphoric acid, or nitric acid. The "organic acids" include, but are not limited to, formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectinic acid, persulfuric acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecylsulfic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalene- disulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptanoic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid, hemisulfuric acid, or thiocyanic acid.

[0095] Depending on the position and nature of the various substituents, the compounds of the present application can also comprise one or more asymmetric centers. Asymmetric carbon atoms can exist in the (R) or (S) configuration, and where there is only one asymmetric center, a racemic mixture results, and where there are multiple asymmetric centers, a mixture of diastereomers results. In certain cases, asymmetry can also exist due to hindered rotation about a particular bond, for example, where that central bond connects two substituted aromatic rings of the particular compound. Also, substituents can exist in cis or trans isomeric forms.

[0096] The compounds of the present application also include each of their individual possible stereoisomers, either in the form of single stereoisomers or in the form of any mixture in any proportion of said stereoisomers (for example, R-isomers or S-isomers, or E-isomers or Z-isomers). The separation of single stereoisomers (for example, single enantiomers or single diastereomers) of the compounds of the present application can be achieved by any suitable method of the prior art, for example, chromatography, in particular, for example, chiral chromatography.

[0097] The term "tautomer" refers to isomeric forms of a functional group resulting from the movement of a bonded atom between two positions. The compounds of the present application can exhibit tautomerism. Compounds that tautomerize can exist in two or more interconvertible forms. Proton-shift tautomers result from the migration of a hydrogen atom covalently bonded between two atoms. Tautomers generally exist in equilibrium and attempts to isolate a single tautomer usually result in a mixture whose physical and chemical properties are consistent with a mixture of compounds. The position of equilibrium depends on the chemical properties within the molecule. For example, in many aliphatic aldehydes and ketones, such as acetaldehyde, the keto form predominates; in phenols, the enol form predominates. The present application includes all tautomeric forms of the compounds.

[0098] In the present application, the compounds involved also include isotopically-labeled compounds, which are identical to those recited in Formula I, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the application include isotopes of H, C, N, O, S, F, and CI, such as 2 H, 3 H, 13 C, 11 C, 14 C, 15 N, 18 O, 17 O, 32 P, 35 S, 18 F, and 36 Cl. Compounds of the present application, prodrugs thereof, or pharmaceutically acceptable salts of said compounds or of said prodrugs, which contain the aforementioned isotopes and / or other isotopes of other atoms are within the scope of the present application. Certain isotopically-labeled compounds of the present application, for example, those into which radioactive isotopes such as 3 H, and 14 C, can be useful in drug and / or substrate tissue distribution assays. Tritiated, i.e., 3 H, and carbon-14, i.e., 14 C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium, i.e., 2 H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements, and hence can be preferred in some circumstances. The present application includes compounds of the formula as set forth in the claims wherein deuterium or tritium is substituted for hydrogen. Moreover, the presence of deuterium or tritium in a substituent group is not excluded by the absence of the term deuterium or tritium from the name of the substituent group.

[0099] The term "effective amount" or "therapeutically effective amount" refers to that amount of a compound according to the present application which is sufficient to effect the intended application including, but not limited to, the treatment of a disease as defined below. The therapeutically effective amount can vary depending on the intended application (in vitro or in vivo), or the subject and disease condition being treated, e.g., the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The specific dose will vary depending on the particular compounds chosen, the dosing regimen following the administration, whether it is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried.

[0100] The term "excipient" refers to a pharmaceutically acceptable inert ingredient. Examples of excipient classes include, but are not limited to, binders, disintegrants, lubricants, glidants, stabilizers, fillers, and diluents, etc. Excipients can enhance the handling properties of a pharmaceutical formulation, i.e., make the formulation more amenable to direct compression by increasing flow and / or cohesiveness. Examples of typical pharmaceutically acceptable carriers suitable for use in the above formulations are sugars, such as lactose, sucrose, mannitol and sorbitol; starches, such as corn starch, tapioca starch and potato starch; celluloses and derivatives thereof, such as sodium carboxymethyl cellulose, ethyl cellulose and methyl cellulose; calcium phosphates, such as dicalcium phosphate and tricalcium phosphate; sodium sulfate; calcium sulfate; polyvinylpyrrolidone; polyvinyl alcohol; stearic acid; alkaline earth metal stearates, such as magnesium stearate and calcium stearate; stearic acid; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil and corn oil; nonionic, cationic and anionic surfactants; ethylene glycol polymers; fatty alcohols; and grain hydrolysates and other nontoxic compatible fillers, binders, disintegrants, buffers, preservatives, antioxidants, lubricants, colorants and the like auxiliary agents commonly used in pharmaceutical formulations.

[0101] The term "solvate" refers to those forms of the compounds of the present application which form a complex with solvent molecules in the solid or liquid state by coordination. Hydrates are a particular form of solvates wherein the coordination is with water. In the present application, the preferred solvates are hydrates. Further, the pharmaceutically acceptable solvates (hydrates) of the compounds of the general formula I according to the present application refer to co-crystals and clathrates of the compound I with one or more molecules of water or other solvents in stoichiometric amounts. Solvents which can be used for solvates include, but are not limited to, water, methanol, ethanol, ethylene glycol and acetic acid.

[0102] The term "prodrug" or "pharmaceutical precursor" refers to a compound which in vivo is converted to the aforementioned compound of the general formula or the specific compound. Such conversion is affected by hydrolysis of the prodrug in blood or enzymatic conversion of the prodrug in blood or tissue to the parent structure. The prodrugs of the present application can be esters, and in the present application the esters which can act as prodrugs are benzoic acid esters, aliphatic (C1-24) esters, acyloxymethyl esters, carbonic acid esters, carbamic acid esters and amino acid esters. For example, a compound of the present application which contains a hydroxyl / carboxyl group can be acylated to give a compound in the form of a prodrug. Other prodrug forms include phosphate esters, such as those compounds which are phosphorylated on a hydroxyl group of the parent.

[0103] Reagent English abbreviation Corresponding reagent name

[0104] Beneficial effects

[0105] 1) The present application provides a compound of the general formula I with a novel structure. Experiments have verified that the compound of the present application has a significant inhibitory effect on IRAK4 kinase activity, and has a good selective inhibitory effect on IRAK4 kinase activity relative to other kinases.

[0106] 2) The compound of the present application has good drug safety, wide applicability and low toxic side effects. Experiments have verified that the compound of the present application has a very low inhibition rate on human hERG, has no significant time-dependent inhibition on human CYP3A4, has a moderate plasma protein binding rate on humans, rats and mice, has little interspecies difference, and has no significant inhibitory effect on five CYP subtypes of humans.

[0107] 3) The compound of the present application has a significant inhibitory effect on the release of TNF-α in LPS-induced Balb / c female mice.

[0108] 4) The compound of the present application has good pharmacokinetic characteristics, shows good exposure and retention time in animals, and has a suitable half-life and good drug absorption. DETAILED DESCRIPTION

[0109] The technical solutions of the present application will be further described in detail below in combination with specific examples. It should be understood that the following examples are only illustratively described and explained, and should not be interpreted as limiting the scope of protection of the present application. Any technology realized based on the above description of the present application is covered within the scope of protection intended by the present application.

[0110] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0111] Synthesis of compound 001 of example 1

[0112] Reaction scheme:

[0113]

[0114] 1. Synthesis of compound 3

[0115] To a solution of compound 1 (50 g) in dichloromethane (500 mL) was added DMAP (42.5 g), compound 2 (63.4 g), and triethylamine (63.9 g) sequentially at 15 °C and stirred at 25 °C for 18 hours. To the reaction solution was added dichloromethane (200 mL) and washed with water (300 mL*2), 1M dilute hydrochloric acid (300 mL*3), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 3 (98 g, yield: 99%).

[0116] 2. Synthesis of compound 4

[0117] To a solution of compound 3 (50 g) in tetrahydrofuran (300 mL) was added 1M dilute hydrochloric acid (300 mL) at 15 °C and stirred at 25 °C for 20 hours. Cooled to 0 °C, adjusted to PH = 9 with 1M sodium hydroxide solution, extracted with ethyl acetate (200 mL*3), the extract was washed with saturated sodium chloride solution (300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the residue was slurried with petroleum ether (150 mL) to obtain compound 4 (39 g, yield 91%).

[0118] 3. Synthesis of compound 5 & 6

[0119] To a solution of methyl magnesium bromide (85.8 mL) in tetrahydrofuran (500 mL) was added a solution of compound 4 (34.5 g) in tetrahydrofuran (200 mL) dropwise at -40 °C and stirred at -40 °C for 4 hours. The reaction was quenched with saturated ammonium chloride solution (100 mL), extracted with ethyl acetate (500 mL*3), the extract was washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5:1) to obtain compound 5 (4.3 g, yield 10%), compound 6 (7.0 g, yield 17%) and mixture 12 g.

[0120] Compound 5

[0121] 1H NMR (400 MHz, CDC13): δ 7.79 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 4.52-4.41 (m, 1H), 2.44 (s, 3H), 1.95-1.80 (m, 2H), 1.77-1.61 (m, 4H), 1.46-1.35 (m, 2H), 1.19 (s, 3H).

[0122] Compound 6

[0123] 1 H NMR (400 MHz, CDC13): δ 7.79 (d, J = 8.4 Hz, 2H), 7.33 (d, J = 8.0 Hz, 2H), 4.74-4.64 (m, 1H), 2.44 (s, 3H), 1.92-1.79 (m, 2H), 1.77-1.62 (m, 4H), 1.49-1.38 (m, 2H), 1.23 (s, 3H).

[0124] 4. Synthesis of compound 8

[0125] A mixture solution of nitric acid (1.6 mL, 70%) in concentrated sulfuric acid (1.6 mL, 98%) was added dropwise to a solution of compound 7 (2.0 g) in concentrated sulfuric acid (12 mL, 98%) at -15 °C. After the addition, the mixture was stirred at -15 °C for 2 h. The reaction solution was slowly poured into ice water and stirred for 5 min. The solid was collected by suction filtration, washed with water, and dried under reduced pressure to give compound 8 (2.5 g, yield: 97%).

[0126] 5. Synthesis of compound 9

[0127] Hydrazine hydrate (2.4 mL, 98%) was added to a solution of compound 8 (2.0 g) in DMF (20 mL) at room temperature. After the addition, the mixture was heated to 120 °C and stirred for 16 h. The mixture was slowly poured into ice water and stirred. The solid was collected by suction filtration, washed with water, and concentrated under reduced pressure to give compound 9 (1.3 g, yield: 67%).

[0128] 6. Synthesis of compound 10

[0129] Compound 9 (12.4 g) and palladium on carbon (7 g, 10%) were sequentially added to 400 mL of ethyl acetate at 15 °C. After the addition, the mixture was stirred under hydrogen gas protection at 15 °C for 18 h. The palladium on carbon was filtered off from the reaction solution, and the filtrate was concentrated and evaporated to dryness to give compound 10 (10.4 g, yield 99%).

[0130] 7. Synthesis of compound 12

[0131] 25 °C, EDCI.HCl (2.6 g) was added to a solution of compound 10 (1.5 g) and compound 11 (1.4 g) in Py (15 mL), the reaction was stirred at 25 °C for 16 h. The reaction was concentrated to dryness, the residue was slurried with MeOH / H2O = 20 mL / 20 mL to give compound 12 (1.3 g, yield 48%).

[0132] 8. Synthesis of compound 001, 2-((2-(trans-4-hydroxy-cis-4-methylcyclohexyl)-6- methoxy-2H-indazol-5-yl)carbamoyl)-6-methylpyridine 1-oxide

[0133]

[0134] 25 °C, Cesium carbonate (985 mg) was added to a solution of compound 12 (300 mg) and compound 5 (344 mg) in 5 mL of DMF, the reaction was stirred at 90 °C for 16 h. The reaction was added to 30 mL of water, extracted with ethyl acetate (10 mL*3), the organic phase was concentrated under reduced pressure, the residue was purified by high performance liquid preparative column (CH3CN:H2O (0.1% NH4HCO3) = 15-45%, UV: 214 nm, flow rate: 15 ml / min) to give compound 001 (70 mg, yield 17%).

[0135] 1 H NMR (400 MHz, DMSO-d6): δ 14.16 (s, 1H), 8.78 (s, 1H), 8.34 (s, 1H), 8.32-8.30 (m, 1H), 7.77 (d, J = 7.6 Hz, 1H), 7.58 (t, J = 8.0 Hz, 1H), 7.13 (s, 1H), 4.45 (s, 1H), 4.43-4.40 (m, 1H), 3.95 (s, 3H), 2.53 (s, 3H), 2.09-2.00 (m, 4H), 1.68-1.58 (m, 4H), 1.22 (s, 3H). LCMS: Rt = 3.646 min, [M+H] + = 411.1.

[0136] 9. Synthesis of compound 11

[0137] 25℃, m-CPBA (25 g) was added to a solution of compound 13 (10 g) in 200 mL of DCM, and the reaction was stirred at 25℃ for 16 hours. The reaction was filtered, and the filtrate was quenched with a saturated solution of 15.6 g of sodium sulfite, and the mixture was stirred for 2 hours, extracted, and the aqueous phase was adjusted to pH < 7 with dilute hydrochloric acid, and extracted with DCM (50 mL x 3). The organic phases were combined and concentrated, and the residue was slurried with 300 mL of EA to obtain compound 11 (10.1 g, yield 90%).

[0138] Synthesis of compound 010 of example 2

[0139] Reaction formula:

[0140]

[0141] 1. Synthesis of compound 2

[0142] 15℃, DMAP (42.5 g), TsCl (63.4 g), and triethylamine (63.9 g) were sequentially added to a solution of compound 1 (50 g) in 500 mL of DCM, and the reaction was stirred at 25℃ for 18 hours. DCM (200 mL) was added to the reaction, and it was washed with water (300 mL x 2), 1M dilute hydrochloric acid (300 mL x 3), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 2 (98 g, yield: 99%).

[0143] 2. Synthesis of compound 3

[0144] 15℃, 1M dilute hydrochloric acid (300 mL) was added to a solution of compound 2 (50 g) in 300 mL of THF, and the reaction was stirred at 25℃ for 20 hours. It was cooled to 0℃, adjusted to pH = 9 with 1M sodium hydroxide solution, extracted with ethyl acetate (200 mL x 3), washed with saturated sodium chloride solution (300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was slurried with petroleum ether (150 mL) to obtain compound 3 (39 g, yield 91%).

[0145] 3. Synthesis of compounds 4 & 5

[0146] To a solution of methylmagnesium bromide (85.8 mL) in tetrahydrofuran (500 mL) at -40 °C, a solution of compound 3 (34.5 g) in tetrahydrofuran (200 mL) was added dropwise and the reaction was stirred at -40 °C for 4 h. The reaction was quenched with saturated ammonium chloride solution (100 mL), extracted with ethyl acetate (500 mL x 3), washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel chromatography (petroleum ether: ethyl acetate = 5: 1) to give compound 4 (4.3 g, yield 10%), compound 5 (7.0 g, yield 17%) and mixture 12 g.

[0147] Compound 4

[0148] 1 H NMR (400 MHz, CDCl3): δ 7.79 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 4.52-4.41 (m, 1H), 2.44 (s, 3H), 1.95-1.80 (m, 2H), 1.77-1.61 (m, 4H), 1.46-1.35 (m, 2H), 1.19 (s, 3H).

[0149] Compound 5

[0150] 1 H NMR (400 MHz, CDCl3): δ 7.79 (d, J = 8.4 Hz, 2H), 7.33 (d, J = 8.0 Hz, 2H), 4.74-4.64 (m, 1H), 2.44 (s, 3H), 1.92-1.79 (m, 2H), 1.77-1.62 (m, 4H), 1.49-1.38 (m, 2H), 1.23 (s, 3H).

[0151] 4. Synthesis of compound 7

[0152] m-CPBA (25 g) was added to a solution of compound 6 (10 g) in 200 mL of DCM at 25 °C, and the reaction was stirred at 25 °C for 16 h. The reaction was filtered, the filtrate was quenched with a saturated solution of sodium sulfite 15.6 g, the mixture was stirred for 2 h, extracted, the aqueous phase was adjusted to PH < 7 with dilute hydrochloric acid, and extracted with DCM (50 mL * 3), the organic phases were combined and concentrated, and the residue was slurried with 300 mL of EA to give compound 7 (10.1 g, yield 90%).

[0153] 5. Synthesis of compound 9

[0154] Compound 8 (10 g) was added slowly into 80 mL of concentrated sulfuric acid, which was cooled to -7 Celsius, and stirred for 5 minutes at this temperature, then cooled to -15 Celsius, and potassium nitrate (8.9 g) was added slowly, and stirred for 1 hour at this temperature. The combined reaction solution was poured into 1.2 L of ice water, and the precipitated solid was filtered, and the filter cake was dissolved in 2 L of ethyl acetate, and 4 L of sodium bicarbonate solution was added to make it alkaline, and extracted with ethyl acetate (2 L*3), and the organic phase was concentrated and dried, and the residue was purified by silica gel column (DCM / MeOH=300 / 1) to obtain compound 9 (12.3 g, yield 27%).

[0155] 6. Synthesis of compound 11

[0156] Compound 9 (400 mg), compound 10 (1.81 g) and DIPEA (2.86 g) were added sequentially into 20 mL of DMF solution at room temperature, and the reaction solution was stirred in a muffle at 80 Celsius overnight, and after the reaction was completed, water was added, and extracted with ethyl acetate three times, and concentrated under reduced pressure, and purified by silica gel column (DCM / CH3OH=200 / 1) to obtain compound 11 (570 mg, yield 83%).

[0157] 7. Synthesis of compound 12

[0158] Compound 11 (80 mg) and Pd / C (5 mg) were added sequentially into 10 mL of methanol solution at room temperature, and the reaction solution was stirred under hydrogen protection at 55 Celsius overnight, and after the reaction was completed, filtered, and the filtrate was concentrated under reduced pressure, and purified by silica gel column (DCM / CH3OH=100 / 1) to obtain compound 12 (60 mg, yield 65%).

[0159] 8. Synthesis of compound 13

[0160] EDCI.HCl (950 mg) was added to a solution of compound 12 (580 mg) and compound 7 (505 mg) in Py (11 mL) at 25 Celsius, and the reaction solution was stirred at 40 Celsius for 16 hours. The reaction solution was concentrated and dried, and the residue was purified by silica gel column (PE:EA=1:1) to obtain compound 13 (550 mg, yield 55%).

[0161] 9. Synthesis of compound 010, i.e. 2-((2-(trans-4-hydroxy-cis-4-methylcyclohexyl)-6-dimethylamino-2H-indazol-5-yl)carbamoyl)-6-methylpyridine 1-oxide

[0162]

[0163] 25 °C, cesium carbonate (936 mg) was added to a solution of compound 13 (300 mg) and compound 4 (409 mg) in 6 mL of DMF, the reaction was stirred at 90 °C for 16 h. The reaction was added to 30 mL of water, extracted with ethyl acetate (10 mL*3), the organic phase was concentrated under reduced pressure, the residue was purified by high performance liquid preparative chromatography column (CH3CN:H2O (0.1% NH4HCO3) = 20-70%, UV: 214 nm, flow rate: 15 ml / min) to obtain compound 010 (59 mg, yield 14%).

[0164] 1 H NMR (400 MHz, DMSO-d6): δ 14.01 (s, 1H), 8.80 (s, 1H), 8.34 (s, 1H), 8.33-8.30 (m, 1H), 7.77-7.74 (m, 1H), 7.56 (t, J = 7.6 Hz, 1H), 7.33 (s, 1H), 4.44-4.41 (m, 2H), 2.72 (s, 6H), 2.52 (s, 3H), 2.06-2.01 (m, 4H), 1.68-1.55 (m, 4H), 1.23 (s, 3H). LCMS: Rt = 3.318 min, [M+H] + = 424.2.

[0165] Synthesis of compound 013 of example 3

[0166] Reaction formula:

[0167]

[0168] 1. Synthesis of compound 2

[0169] A mixture solution of nitric acid (1.6 mL, 70%) in concentrated sulfuric acid (1.6 mL, 98%) was added dropwise to a solution of compound 1 (2.0 g) in concentrated sulfuric acid (12 mL, 98%) at -15 °C. After the addition was completed, the mixture was stirred at -15 °C for 2 h. The reaction was slowly poured into ice water and stirred for 5 min, suction filtered, washed with water, and the solid was dried under reduced pressure to give compound 2 (2.5 g, yield: 97%).

[0170] 2. Synthesis of compound 3

[0171] Hydrazine hydrate (2.4 mL, 98%) was added to a solution of compound 2 (2.0 g) in DMF (20 mL) at room temperature. After the addition was completed, the mixture was heated to 120 °C and stirred for 16 h. The mixture was cooled to room temperature, slowly poured into ice water and stirred, suction filtered, the solid was washed with water, and the solid was concentrated under reduced pressure to give compound 3 (1.3 g, yield: 67%).

[0172] 3. Synthesis of compound 4

[0173] Compound 3 (12.4 g) and palladium on carbon (7 g, 10%) were added to 400 mL of ethyl acetate at 15 degrees Celsius. After the addition was complete, the mixture was stirred under hydrogen gas protection at 15 degrees Celsius for 18 hours. The palladium on carbon was filtered off from the reaction solution, and the filtrate was concentrated and evaporated to dryness to obtain compound 4 (10.4 g, yield 99%).

[0174] 4. Synthesis of compound 6

[0175] EDCI.HCl (2.6 g) was added to a solution of compound 4 (1.5 g) and compound 5 (1.4 g) in Py (15 mL) at 25 degrees Celsius. The reaction solution was stirred at 25 degrees Celsius for 16 hours. The reaction solution was concentrated and evaporated to dryness, and the residue was slurried with MeOH / H2O = 20 mL / 20 mL to obtain compound 6 (1.3 g, yield 48%).

[0176] 5. Synthesis of compound 8

[0177] Cesium carbonate (3.3 g) was added to a solution of compound 6 (1 g) and compound 7 (1.3 g) in 20 mL of DMF at 25 degrees Celsius. The reaction solution was stirred at 90 degrees Celsius for 16 hours. The reaction solution was added to 50 mL of water, and ethyl acetate (30 mL*3) was extracted. The organic phase was concentrated under reduced pressure, and the residue was purified by high-performance liquid chromatography (CH3CN:H2O (0.1% NH4HCO3) = 20-60%, UV: 214 nm, flow rate: 15 ml / min) to obtain compound 8 (370 mg, yield 25%).

[0178] 6. Synthesis of compound 9

[0179] Compound 8 (350 mg) was added to 5 mL of 2M dilute hydrochloric acid in 5 mL of dioxane at 25 degrees Celsius. The reaction solution was stirred at 25 degrees Celsius for 16 hours. The reaction solution was adjusted to be alkaline with a sodium carbonate solution, and ethyl acetate (10 mL*3) was extracted. The organic phase was concentrated under reduced pressure to obtain compound 9 (150 mg, yield 48%).

[0180] 7. Synthesis of compound 013, i.e., 2-((2-(trans-4-hydroxycyclohexyl)-6-methoxy-2H-indazol-5-yl)carbamoyl)-6-methylpyridine 1-oxide

[0181]

[0182] Sodium borohydride (25 mg) was added to a solution of compound 9 (130 mg) in 2 mL of methanol at 0 °C, and the reaction was stirred at 25 °C for 2 h. The reaction was quenched with 10 mL of ammonium chloride solution and extracted with ethyl acetate (5 mL*3). The organic phase was concentrated under reduced pressure, and the residue was purified by high performance liquid preparative chromatography (CH3CN:H2O (0.1% NH4HCO3) = 10-60%, UV: 214 nm, flow rate: 15 ml / min) to give compound 013 (54 mg, yield 41%).

[0183] 1 H NMR (400 MHz, DMSO-d6): δ 14.16 (s, 1H), 8.78 (s, 1H), 8.31-8.29 (m, 2H), 7.78-7.76 (m, 1H), 7.58 (t, J = 8.0 Hz, 1H), 7.10 (s, 1H), 4.71 (d, J = 4.4 Hz, 1H), 4.40-4.34 (m, 1H), 3.95 (s, 3H), 3.57-3.50 (m, 1H), 2.53 (s, 3H), 2.09-2.06 (m, 2H), 1.97-1.88 (m, 4H), 1.45-1.34 (m, 2H). LCMS: Rt = 2.541 min, [M+H] + = 397.2.

[0184] Synthesis of compound 016 and compound 220 of example 4

[0185] Reaction formula:

[0186]

[0187]

[0188] 1. Synthesis of compound 2

[0189] Compound 1 (2 g) and AlCl3(4.13 g) were added to dichloromethane (150 mL) at 18 °C, and the reaction was stirred at 55 °C for 18 h. The reaction was quenched with 50 mL of water, extracted with 150 mL of dichloromethane, and then extracted with ethyl acetate (150 mL*3). The organic phase was concentrated and dried, and the residue was slurried with 30 mL of dichloromethane to give compound 2 (1.6 g, yield 86%).

[0190] 2. Synthesis of compound 3

[0191] 25 °C, potassium carbonate (93 mg) was added to a solution of compound 2 (0.1 g) and iodoethane (105 mg) in 2 mL of DMF, the reaction was stirred at 60 °C for 16 h. The reaction was added to 20 mL of water, extracted with ethyl acetate (5 mL*3), the organic phase was concentrated under reduced pressure, the residue was purified by silica gel column (petroleum ether / ethyl acetate = 2 / 1) to give compound 3 (0.1 g, yield 86%).

[0192] 3. Synthesis of compound 4

[0193] 25 °C, 0.3 g Pd / C was added to a solution of compound 3 (1.1 g) in 100 mL of methanol, the reaction was stirred at 25 °C under the pressure of a hydrogen balloon (760 Torr) for 16 h. The reaction was filtered, the filtrate was rotary evaporated to give compound 4 (0.71 g, yield 76%).

[0194] 4. Synthesis of compound 6

[0195] 25 °C, compound 5 (558 mg) was added to a solution of compound 4 (710 mg) and EDCI (840 mg) in 25 mL of pyridine, the reaction was stirred at 25 °C for 16 h. The reaction was added to 100 mL of water, extracted with ethyl acetate (30 mL*3), the organic phase was concentrated under reduced pressure, the residue was purified by silica gel column (dichloromethane / methanol = 60 / 1) to give compound 6 (0.67 g, yield 54%).

[0196] 5. Synthesis of compound 8

[0197] 25 °C, compound 6 (630 mg) was added to a solution of compound 7 (945 mg) and cesium carbonate (1.97 g) in 25 mL of DMF, the reaction was stirred at 90 °C under nitrogen protection for 16 h. The reaction was added to 100 mL of water, extracted with ethyl acetate (30 mL*3), the organic phase was concentrated under reduced pressure, the residue was purified by high performance liquid preparative chromatography column (CH3CN:H2O (0.1% NH4HCO3) = 5-95%, UV: 214 nm, flow rate: 15 ml / min) to give compound 8 (160 mg, yield 18%).

[0198] 6. Synthesis of compound 9

[0199] 25 °C, compound 8 (180 mg) was dissolved in a mixed solution of 30 mL of dioxane and 10 mL of 2M diluted hydrochloric acid, the reaction was stirred at 45 °C for 16 h. The reaction was adjusted to PH > 7 with saturated sodium bicarbonate, then extracted with ethyl acetate (30 mL*3), the organic phase was rotary evaporated to give compound 9 (180 mg, yield 97%).

[0200] 7. Synthesis of compound 016, 2-((2-(trans-4-hydroxycyclohexyl)-6-ethoxy-2H- indazol-5-yl)carbamoyl)-6-methylpyridine 1-oxide and compound 220, 2-((2-(cis-4- hydroxycyclohexyl)-6-ethoxy-2H-indazol-5-yl)carbamoyl)-6-methylpyridine 1-oxide

[0201]

[0202] Sodium borohydride (50 mg) was added to a solution of compound 9 (180 mg) in 20 mL of methanol at 0 °C and the reaction was stirred at 25 °C for 1 h. The reaction was quenched with 10 mL of ammonium chloride solution and extracted with ethyl acetate (20 mL*3). The organic phase was concentrated under reduced pressure and the residue was purified by high performance liquid chromatography (CH3CN:H2O (0.1% NH4HCO3) = 20-50%, UV: 214 nm, flow rate: 15 ml / min) to give compound 016 (123 mg, yield 68%) with retention time Rt= 11.25 min and compound 220 (30 mg, yield 17%) with retention time Rt= 11.75 min.

[0203] Compound 016

[0204] 1 H NMR (400 MHz, CDC13): δ 14.30 (s, 1H), 8.88 (s, 1H), 8.45 (d, J = 7.6 Hz, 1H), 7.83 (s, 1H), 7.43-7.36 (m, 2H), 7.03 (s, 1H), 4.37-4.30 (m, 1H), 4.24 (q, J = 6.8 Hz, 2H), 3.82-3.80 (m, 1H), 2.63 (s, 3H), 2.28 (d, J = 12.8 Hz, 2H), 2.18 (d, J = 14.8 Hz, 2H), 2.06 (q, J = 13.2 Hz, 2H), 1.65-1.56 (m, 3H), 1.55-1.50 (m, 2H).

[0205] LCMS: Rt = 2.486 min, [M+H] + = 411.2.

[0206] Compound 220

[0207] 1H NMR (400 MHz, CDC13): δ 14.30 (s, 1H), 8.89 (s, 1H), 8.45 (d, J = 7.6 Hz, 1H), 7.89 (s, 1H), 7.43-7.36 (m, 2H), 7.04 (s, 1H), 4.41-4.35 (m, 1H), 4.24 (q, J = 8.8 Hz, 2H), 4.14 (br s, 1H), 2.63 (s, 3H), 2.35 (q, J = 8.8 Hz, 2H), 2.08 (d, J = 8.4 Hz, 2H), 1.98 (d, J = 13.2 Hz, 2H), 1.75 (t, J = 13.2 Hz, 2H), 1.64 (t, J = 6.8 Hz, 3H). LCMS: Rt = 2.642 min, [M+H] + = 411.2.

[0208] Synthesis of compound 025 of example 5

[0209] Reaction scheme:

[0210]

[0211] 1. Synthesis of compound 3

[0212] To a solution of compound 2 (45 mL) in tetrahydrofuran (100 mL) was added dropwise a solution of compound 1 (3 g) in tetrahydrofuran (10 mL) at -40 °C and the reaction was stirred at 0 °C for 8 h, quenched with saturated ammonium chloride solution (200 mL), extracted with ethyl acetate (200 mL x 3), the extract was washed with saturated brine (400 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure, the residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5: 1) to give compound 3 (1.5 g, yield 44%).

[0213] 2. Synthesis of compound 025, 2-((2-(trans-4-hydroxy-cis-4- cyclopropylcyclohexyl)-6-methoxy-2H-indazol-5-yl)carbamoyl)-6-methylpyridine 1-oxide

[0214]

[0215] 25 °C, cesium carbonate (820 mg) was added to a solution of compound 4 (300 mg) and compound 3 (374 mg) in NMP (30 mL), the reaction was stirred at 90 °C for 16 h. Water (100 mL) was added, and the mixture was extracted with ethyl acetate (80 mL*4), and the organic phase was concentrated under reduced pressure. The residue was purified by high performance liquid chromatography (CH3CN:H2O (0.1% NH4HCO3) = 5-95%, UV: 214 nm, flow rate: 15 mL / min) to give compound 025 (79 mg, yield 18%).

[0216] 1 H NMR (400 MHz, CDC13): δ 14.13 (s, 1H), 8.89 (s, 1H), 8.45 (d, J = 12 Hz, 1H), 7.89 (s, 1H), 7.42-7.37 (m, 2H), 7.07 (s, 1H), 4.49-4.41 (m, 1H), 4.05 (s, 3H), 2.64 (s, 3H), 2.38-2.22 (m, 4H), 1.92-1.88 (m, 2H), 1.72-1.64 (m, 2H), 1.31-1.26 (m, 1H), 0.98 (s, 1H), 0.43-0.41 (m, 4H). LCMS: Rt = 3.198, [M+H] + = 437.2.

[0217] Synthesis of compound 163 of example 6

[0218] Reaction scheme:

[0219]

[0220] 1. Synthesis of compound 2

[0221] 11.8 g of p-toluenesulfonyl chloride was added to a solution of compound 1 (6.1 g), triethylamine (14.8 g) and DMAP (7.2 g) in DCM (120 mL) at 15 °C, and the reaction was stirred at 10 °C for 16 h. The reaction was washed with 1 N HCl solution (100 mL*3), and the organic phase was dried and concentrated to give compound 2 (13.1 g, yield 84%).

[0222] 2. Synthesis of compound 9

[0223] To a solution of compound 8 (2.0 g) in concentrated sulfuric acid (12 mL, 98%) was added dropwise a mixture of nitric acid (1.6 mL) in concentrated sulfuric acid (1.6 mL, 98%) at -15 °C. After the addition was complete, the mixture was stirred at -15 °C for 2 h. The reaction was slowly poured into ice water and stirred for 5 min. The solid was collected by suction filtration, washed with water, and dried under reduced pressure to give compound 9 (2.5 g, yield: 97%).

[0224] 3. Synthesis of compound 3

[0225] To a solution of compound 8 (2.0 g) in DMF (20 mL) was added hydrazine hydrate (2.4 mL, 98%) at room temperature. After the addition was complete, the mixture was heated to 120 °C and stirred for 16 h. The mixture was cooled to room temperature and slowly poured into ice water and stirred. The solid was collected by suction filtration, washed with water, and concentrated under reduced pressure to give compound 3 (1.3 g, yield: 67%).

[0226] 4. Synthesis of compound 4

[0227] DIPEA (13.4 g) was added to a solution of compound 3 (4.0 g) and compound 2 (10.7 g) in 80 mL of toluene at 15 °C. The reaction was stirred at 130 °C for 48 h. The reaction was added to 100 mL of water and extracted with ethyl acetate (50 mL*3). The organic phase was concentrated under reduced pressure. The residue was purified by high performance liquid chromatography (CH3CN:H2O (0.1% NH4HCO3) = 10-50%, UV: 214 nm, flow rate: 15 ml / min) to give compound 4 (2.5 g, yield 43%).

[0228] 5. Synthesis of compound 5

[0229] Pd / C (400 mg) was added to a solution of compound 4 (1.3 g) in ethyl acetate (30 mL) at 30 °C. The reaction was stirred under a hydrogen balloon for 16 h. The reaction was combined and filtered. The filtrate was concentrated under reduced pressure to give compound 5 (1.9 g, yield 95%).

[0230] 6. Synthesis of compound 163, 2-((2-(3-hydroxy-3-methylbutyl)-6-methoxy-2H- indazol-5-yl)carbamoyl)-6-methylpyridine 1-oxide

[0231]

[0232] 30 °C, HATU (273 mg), Et3N (145 mg) were added to a solution of compound 5 (120 mg) and compound 6 (81 mg) in DMF (2 mL), the reaction was stirred at 30 °C for 18 h, the reaction was concentrated under reduced pressure, the crude was purified by HPLC prep column (CH3CN: H2O (0.1% NH4HCO3) = 5-95%, UV: 214 nm, flow rate: 15 ml / min) to give compound 163 (110 mg, 60%).

[0233] 1 H NMR (400 MHz, DMSO-d6): δ 14.13 (s, 1H), 8.78 (s, 1H), 8.31-8.28 (m, 2H), 7.77-7.75 (m, 1H), 7.60-7.55 (m, 1H), 7.09 (s, 1H), 4.50 (s, 1H), 4.44-4.40 (m, 2H), 3.93 (s, 3H), 2.53 (s, 3H), 2.04-2.00 (m, 2H), 1.15 (s, 6H). LCMS: Rt = 2.784 min, [M+H] + = 385.2.

[0234] Synthesis of compound 284 of example 7

[0235] Reaction scheme:

[0236]

[0237] 1. Synthesis of compound 2

[0238] 25 °C, p-toluenesulfonyl chloride (2.3 g) was added to a solution of compound 1 (1 g), triethylamine (2.9 g) and DMAP (1.4 g) in DCM (20 mL), the reaction was stirred at 25 °C for 16 h. The reaction was washed with 1 N HC1 solution (200 mL*3), the organic phase was dried and concentrated to give compound 2 (2.1 g, yield 75%).

[0239] 2. Synthesis of compound 284, 2-((2-cyclopentyl-6-methoxy-2H-indazol-5-yl)carbamoyl)-6- methylpyridine 1-oxide

[0240]

[0241] 25 °C, cesium carbonate (1.6 g) was added to a solution of compound 3 (500 mg) and compound 2 (485 mg) in 10 mL of DMF, the reaction was stirred at 90 °C for 16 h. The reaction was added to 50 mL of water, extracted with ethyl acetate (30 mL*3), the organic phase was concentrated under reduced pressure, the residue was purified by high performance liquid preparative column (CH3CN:H2O (0.1% NH4HCO3) = 25-60%, UV: 214 nm, flow rate: 15 ml / min) to give compound 284 (123 mg, yield 20%).

[0242] 1 H NMR (400 MHz, DMSO-d6): δ 14.15 (s, 1H), 8.78 (s, 1H), 8.32-8.29 (m, 2H), 7.77-7.75 (m, 1H), 7.57 (t, J = 7.6 Hz, 1H), 7.13 (s, 1H), 4.97-4.90 (m, 1H), 3.96 (s, 3H), 2.53 (s, 3H), 2.22-2.13 (m, 2H), 2.10-2.01 (m, 2H), 1.92-1.82 (m, 2H), 1.74-1.65 (m, 2H). LCMS: Rt = 3.562 min, [M+H] + = 367.2.

[0243] Synthesis of compound 285

[0244] Reaction scheme:

[0245]

[0246] 1. Synthesis of compound 285, 2-((2-cyclohexyl-6-methoxy-2H-indazol-5-yl)carbamoyl)-6- methylpyridine 1-oxide

[0247]

[0248] 25 °C, cesium carbonate (1.6 g) was added to a solution of compound 3 (500 mg) and compound 2 (485 mg) in 10 mL of DMF, the reaction was stirred at 90 °C for 16 h. The reaction was added to 50 mL of water, extracted with ethyl acetate (30 mL*3), the organic phase was concentrated under reduced pressure, the residue was purified by high performance liquid preparative column (CH3CN:H2O (0.1% NH4HCO3) = 25-60%, UV: 214 nm, flow rate: 15 ml / min) to give compound 284 (123 mg, yield 20%).

[0249] 1H NMR (400 MHz, DMSO-d6): δ 14.15 (s, 1H), 8.79 (s, 1H), 8.32-8.29 (m, 2H), 7.78-7.76 (m, 1H), 7.58 (t, J = 8.0 Hz, 1H), 7.11 (s, 1H), 4.40-4.34 (m, 1H), 3.95 (s, 3H), 2.53 (s, 3H), 2.11-2.07 (m, 2H), 1.90-1.80 (m, 4H), 1.70 (d, J = 12.8 Hz, 1H), 1.50-1.40 (m, 2H), 1.31-1.23 (m, 1H). LCMS: Rt = 3.971 min, [M+H] + = 381.2.

[0250] Synthesis of compound 286

[0251] Reaction scheme:

[0252]

[0253] 1. Synthesis of compound 2

[0254] DAST (6 g) was added to a solution of compound 1 (2 g) in DCM (70 mL) at 0 °C, the reaction was stirred at 25 °C for 3 h. The reaction was poured into 50 mL ice water, extracted with dichloromethane (30 mL*2), the organic phase was concentrated under reduced pressure, the residue was purified by silica gel column (PE:EA = 10:1) to give compound 2 (1.8 g, yield 82%).

[0255] 2. Synthesis of compound 286, 2-((2-(4,4-difluorocyclohexyl)-6-methoxy-2H-indazol-5- yl)carbamoyl)-6-methylpyridine 1-oxide

[0256]

[0257] Cesium carbonate (1.6 g) was added to a solution of compound 3 (500 mg) and compound 2 (731 mg) in 10 mL of NMP at 25 °C, the reaction was stirred at 90 °C for 16 h. The reaction was added to 30 mL water, extracted with ethyl acetate (10 mL*3), the organic phase was concentrated under reduced pressure, the residue was purified by high performance liquid preparation column (CH3CN:H2O (0.1% NH4HCO3) = 40-70%, UV: 214 nm, flow rate: 15 ml / min) to give compound 286 (129 mg, yield 18%).

[0258] 1H NMR (400 MHz, DMSO-d6): δ 14.17 (s, 1H), 8.80 (s, 1H), 8.35 (s, 1H), 8.32-8.29 (m, 1H), 7.78-7.76 (m, 1H), 7.58 (t, J = 8.0 Hz, 1H), 7.13 (s, 1H), 4.68-4.57 (m, 1H), 3.97 (s, 3H), 2.53 (s, 3H), 2.32-2.07 (m, 8H). LCMS: Rt = 3.692 min, [M+H] + = 417.2.

[0259] Synthesis of compound 287

[0260] Reaction Scheme:

[0261]

[0262] 1. Synthesis of compound 287, 2-((2-(cis-4-hydroxy-trans-4-methylcyclohexyl)-6- dimethylamino-2H-indazol-5-yl)carbamoyl)-6-methylpyridine 1-oxide

[0263]

[0264] Cesium carbonate (800 mg) was added to a solution of compound 1 (255 mg) and compound 2 (350 mg) in 5 mL of NMP at 25 °C, and the reaction was stirred at 90 °C for 16 h. The reaction was added to 30 mL of water, extracted with ethyl acetate (10 mL*3), and the organic phase was concentrated under reduced pressure. The residue was purified by high performance liquid preparative chromatography column (CH3CN:H2O (0.1% NH4HCO3) = 35-60%, UV: 214 nm, flow rate: 15 ml / min) to give compound 287 (51 mg, yield 15%).

[0265] 1 H NMR (400 MHz, CDCl3): δ 14.03 (s, 1H), 8.92 (s, 1H), 8.46 (d, J = 8.0 Hz, 1H), 7.89 (s, 1H), 7.41-7.35 (m, 3H), 4.37-4.31 (m, 1H), 2.84 (s, 6H), 2.63 (s, 3H), 2.37-2.27 (m, 2H), 2.11-2.05 (m, 2H), 1.93-1.85 (m, 2H), 1.61-1.58 (m, 2H), 1.33 (s, 3H). LCMS: Rt = 3.298 min, [M+H] + = 424.3.

[0266] Synthesis of compound 015 and compound 288 of example 11

[0267] Reaction formula:

[0268]

[0269] 1. Synthesis of compound 3

[0270] Compound 1 (5 g), compound 2 (26 g) and cesium carbonate (29 g) were added to DMF (400 mL) at 20 degrees Celsius, replaced with nitrogen, stirred at 90 degrees Celsius for 24 hours, cooled to 20 degrees Celsius, added water (800 mL), extracted with ethyl acetate (800 mL x 3), the organic phase was washed with saturated sodium chloride solution (500 mL x 3), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1:1), and then slurried with MTBE (50 mL) to obtain compound 3 (1.2 g, yield 14%).

[0271] 2. Synthesis of compound 4

[0272] Pd / C (0.1 g) was added to a solution of compound 3 (1.1 g) in ethyl acetate (200 mL) at 25 degrees Celsius, stirred under a hydrogen balloon for 16 hours, filtered, and concentrated under reduced pressure to obtain compound 4 (901 mg, yield 90%).

[0273] 3. Synthesis of compound 6

[0274] Compound 5 (900 mg) was dissolved in dichloromethane (20 mL), and mCPBA (2.5 g) was slowly added, and the reaction solution was stirred at 25 degrees Celsius for 16 hours. After the residue was quenched with an aqueous sodium sulfite solution and then adjusted to a pH of less than 7 with dilute hydrochloric acid, it was extracted with dichloromethane (50 mL x 3), and the organic phase was concentrated and purified by column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain compound 6 (0.68 g, yield 69%).

[0275] 4. Synthesis of compound 7

[0276] To a solution of compound 6 (164 mg) and compound 4 (250 mg) in DMF (10 mL) was added HATU (376 mg) and DIPEA (128 mg) at 25 degrees Celsius, and stirred for 16 hours. The reaction phase was added with water (100 mL), extracted with EA (20 mL x 3), and the extract was washed with saturated sodium chloride solution (200 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography (dichloromethane:methanol = 40:1) to obtain compound 7 (480 mg, yield 99%).

[0277] 5. Synthesis of compound 8

[0278] 0°C, to a solution of compound 7 (480 mg) in dioxane (10 mL) was added 4 M HC1 (10 mL), the reaction was stirred at 30°C for 16 h, cooled to 0°C, adjusted to pH = 8 with saturated NaHC03solution, extracted with EA (40 mL x 5), washed with saturated NaCl (200 mL), dried over anhydrous Na2S04, filtered, and concentrated under reduced pressure to give compound 8 (435 mg, yield 99%).

[0279] 7. Synthesis of compound 015, 2-((2-(trans-4-hydroxycyclohexyl)-6-methoxy-2H- indazol-5-yl)carbamoyl)-6-isopropylpyridine 1-oxide and compound 288, 2-((2-(cis-4- hydroxycyclohexyl)-6-methoxy-2H-indazol-5-yl)carbamoyl)-6-isopropylpyridine 1- oxide

[0280]

[0281] 0°C, to a solution of compound 7 (480 mg) in dioxane (10 mL) was added 4 M HC1 (10 mL), the reaction was stirred at 30°C for 16 h, cooled to 0°C, adjusted to pH = 8 with saturated NaHC03solution, extracted with EA (40 mL x 5), washed with saturated NaCl (200 mL), dried over anhydrous Na2S04, filtered, and concentrated under reduced pressure to give compound 8 (435 mg, yield 99%).

[0282] Compound 015

[0283] 1 H NMR (400 MHz, CDC13): δ 14.18 (s, 1H), 8.88 (s, 1H), 8.44 (d, J = 5.2 Hz, 1H), 7.83 (s, 1H), 7.47-7.40 (m, 2H), 7.05 (s, 1H), 4.34-4.33 (m, 1H), 4.04 (s, 3H), 3.99-3.95 (m, 1H), 3.82-3.80 (m, 1H), 2.30-2.26 (m, 2H), 2.19-2.16 (m, 2H), 2.05 (q, J = 9.2 Hz, 2H), 1.60-1.50 (m, 2H), 1.36 (s, 3H). 1.35 (s, 3H). LCMS: Rt = 3.531, [M+H] + = 425.2.

[0284] Compound 288

[0285] 1 H NMR (400 MHz, CDC13): δ 14.17 (s, 1H), 8.88 (s, 1H), 8.44 (d, J = 5.2 Hz, 1H), 7.89 (s, 1H), 7.47-7.40 (m, 2H), 7.06 (s, 1H), 4.39-4.37 (m, 1H), 4.14 (s, 1H), 4.05 (s, 3H), 3.99-3.95 (m, 1H), 2.38-2.34 (m, 2H), 2.09-2.04 (m, 2H), 2.01-1.96 (m, 2H), 1.80-1.76 (m, 2H), 1.36 (s, 3H). 1.35 (s, 3H). LCMS: Rt = 3.472, [M+H] + = 425.2.

[0286] Synthesis of compound 014 and compound 218 of example 12

[0287] Reaction scheme:

[0288]

[0289] 1. Synthesis of compound 3

[0290] Pd(dppf)Cl2(113 mg) and K3PO4(13.4 g) were added to a solution of compound 1 (7.3 g) and compound 2 (6.5 g) in toluene (70 mL), replaced with nitrogen, and the reaction solution was stirred at 100 degrees Celsius for 16 h. After cooling, the residue was purified by thin layer column chromatography (petroleum ether: ethyl acetate = 20: 1) to obtain compound 3 (1.2 g, yield 20%).

[0291] 2. Synthesis of compound 4

[0292] m-CPBA (5.7 g) was added to a solution of compound 3 (1.1 g) in 100 mL of DCM at 25 degrees Celsius, and the reaction solution was stirred at 25 degrees Celsius for 48 h. The reaction solution was quenched with a saturated solution of 2.4 g of sodium sulfite, the organic phase was washed with saturated sodium bicarbonate (100 mL x 3), the organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 4 (1.1 g, yield 92%).

[0293] 3. Synthesis of compound 5

[0294] Compound 4 (1.2 g) and LiOH.H2O (730 mg) were added to THF / H2O (30 mL / 10 mL) successively, and the system was replaced with nitrogen. After addition was completed, the mixture was stirred at 30°C for 4 hours. Water (50 mL) was added, and the aqueous phase was slowly adjusted to pH 7 with 1N HCl, extracted with ethyl acetate (100 mL x 3), washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, filtered, and dried under reduced pressure to obtain compound 5 (844 mg, yield 84%).

[0295] 4. Synthesis of compound 7

[0296] To a solution of compound 6 (450 mg) and compound 5 (319 mg) in pyridine (35 mL) was added DECI (427 mg) at 25°C, and stirred for 16 hours. It was concentrated to dryness under reduced pressure, water (100 mL) was added, extracted with DCM (100 mL x 3), and the extract was washed with saturated sodium chloride solution (200 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was slurried with ethyl acetate (10 mL) to obtain compound 7 (314 mg, yield 45%).

[0297] 5. Synthesis of compound 8

[0298] To a solution of compound 7 (289 mg) in tetrahydrofuran (25 mL) was added 4M hydrochloric acid (250 mL) at 0°C, and stirred at 30°C for 16 hours. It was cooled to 0°C, adjusted to pH 8 with saturated NaHCO3 solution, extracted with DCM (40 mL x 5), and the extract was washed with saturated NaCl (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 8 (249 mg, yield 95%).

[0299] 6. Synthesis of compound 014, 2-((2-(trans-4-hydroxycyclohexyl)-6-methoxy-2H-indazol-5-yl)carbamoyl)-6-cyclopropylpyridine 1-oxide, and compound 218, 2-((2-(cis-4-hydroxycyclohexyl)-6-methoxy-2H-indazol-5-yl)carbamoyl)-6-cyclopropylpyridine 1-oxide

[0300]

[0301] Sodium borohydride (67 mg) was added to a solution of compound 8 (249 mg) in 20 mL of methanol at 0 °C, stirred at 30 °C for 16 h. The reaction solution was adjusted to pH = 7 with saturated ammonium chloride, extracted with DCM (40 mL x 5), the extract was washed with saturated NaCl (200 mL), concentrated under reduced pressure, and the residue was purified by high performance liquid preparative chromatography column (CH3CN:H2O (0.1% NH4HCO3) = 30-70%, UV: 214 nm, flow rate: 15 ml / min) to give compound 014 (108 mg, yield 43%) with retention time Rt= 7.99 min and compound 218 (14 mg, yield 5%) with retention time Rt= 8.50 min.

[0302] Compound 014

[0303] 1 H NMR (400 MHz, CDC13): δ 14.26 (s, 1H), 8.87 (s, 1H), 8.39 (d, J = 8.0 Hz, 1H), 7.82 (s, 1H), 7.39-7.35 (m, 1H), 7.04 (s, 2H), 4.37-4.29 (m, 1H), 4.04 (s, 3H), 3.83-3.75 (m, 1H), 2.88-2.80 (m, 1H), 2.28-2.25 (m, 2H), 2.18-2.15 (m, 2H), 2.09-2.00 (m, 2H), 1.58-1.48 (m, 2H), 1.29-1.26 (m, 2H), 0.84-0.82 (m, 2H).

[0304] LCMS: Rt = 3.312, [M+H] + = 423.2.

[0305] Compound 218

[0306] 1 H NMR (400 MHz, CDC13): δ 14.26 (s, 1H), 8.87 (s, 1H), 8.39 (d, J = 8.0 Hz, 1H), 7.82 (s, 1H), 7.39-7.35 (m, 1H), 7.04 (s, 2H), 4.37-4.29 (m, 1H), 4.04 (s, 3H), 3.83-3.75 (m, 1H), 2.88-2.80 (m, 1H), 2.28-2.25 (m, 2H), 2.18-2.15 (m, 2H), 2.09-2.00 (m, 2H), 1.58-1.48 (m, 2H), 1.29-1.26 (m, 2H), 0.84-0.82 (m, 2H).

[0307] LCMS: Rt = 2.807, [M+H] + = 423.2.

[0308] Synthesis of compound 187 of example 13

[0309] Reaction scheme:

[0310]

[0311] 1. Synthesis of compound 2

[0312] To a solution of compound 1 (50 g) in dichloromethane (500 mL) was added DMAP (42.5 g), TsCl (63.4 g), and triethylamine (63.9 g) sequentially at 15 °C and stirred at 25 °C for 18 h. To the reaction mixture was added dichloromethane (200 mL) and washed with water (300 mL x 2), 1 M dilute hydrochloric acid (300 mL x 3), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 2 (98 g. Yield: 99%).

[0313] 2. Synthesis of compound 3

[0314] To a solution of compound 2 (50 g) in tetrahydrofuran (300 mL) was added 1 M dilute hydrochloric acid (300 mL) at 15 °C and stirred at 25 °C for 20 h. Cooled to 0 °C, adjusted to pH = 9 with 1 M sodium hydroxide solution, extracted with ethyl acetate (200 mL x 3), washed the extract with saturated sodium chloride solution (300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was slurried with petroleum ether (150 mL) to give compound 3 (39 g, yield 91%).

[0315] 3. Synthesis of compound 5

[0316] To a solution of compound 4 (5.0 g) in tetrahydrofuran (100 mL) was added compound 3 (74.6 mL) dropwise at -40 °C and stirred at -40 °C for 4 h. TLC showed that the reaction was complete, the reaction was quenched with saturated ammonium chloride solution (50 mL), extracted with ethyl acetate (100 mL x 3), washed the extract with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5: 1) to give compound 5 (900 mg, yield: 16%).

[0317] 4. Synthesis of compound 7

[0318] Add 80 mL of concentrated sulfuric acid into a 1 L flask, stir for 5 minutes at -12 Celsius (part of the concentrated sulfuric acid is in ice state), then slowly add compound 6 (10 g) at this temperature, keep the temperature no fluctuation, stir for 5 minutes at this temperature, then slowly add the mixture of nitric acid 8 mL and concentrated sulfuric acid 8 mL at -12 Celsius, keep the temperature and stir for 1.5 hours. Monitor the reaction by TLC. Slowly pour the mixture into ice water (keep the temperature low), stir for 20 minutes, then filter, wash with water, and dry under reduced pressure to obtain compound 7 (13 g, yield: 100%).

[0319] 5. Synthesis of compound 8

[0320] Dissolve compound 7 (30 g) in 450 mL of DMF, slowly add hydrazine hydrate 36.3 mL (98%) at 0 Celsius, then stir the mixture at 120 Celsius for 18 hours. After the reaction is complete, slowly pour the cooled reaction solution into ice water, stir for 10 minutes, then filter, wash with water, and dry under reduced pressure to obtain compound 8 (20 g, yield: 69%).

[0321] 6. Synthesis of compound 9

[0322] Add compound 8 (10 g) and 5 g of palladium on carbon (10%) to 200 mL of ethyl acetate, respectively, and stir the reaction solution under hydrogen protection at 20 Celsius for 16 hours. After the reaction is complete, add diatomite to filter out the palladium on carbon, concentrate and dry the filtrate to obtain compound 9 (8 g, yield: 94%).

[0323] 7. Synthesis of compound 11

[0324] Add m-CPBA (25 g) to a 200 mL DCM solution of compound 12 (10 g) at 25 Celsius, and stir the reaction solution at 25 Celsius for 16 hours. Filter the reaction solution, quench the filtrate with a saturated solution of 15.6 g of sodium sulfite, stir the mixture for 2 hours, extract, adjust the aqueous phase to pH < 7 with dilute hydrochloric acid, extract with DCM (50 mL*3), combine the organic phases, concentrate, and slurry the residue with 300 mL of EA to obtain compound 11 (10.1 g, yield 90%).

[0325] 8. Synthesis of compound 10

[0326] Add EDCI.HCl (2.6 g) to a Py (15 mL) solution of compound 9 (1.5 g) and compound 11 (1.4 g) at 25 Celsius, and stir the reaction solution at 25 Celsius for 16 hours. Concentrate and dry the reaction solution, and slurry the residue with MeOH / H2O = 20 mL / 20 mL to obtain compound 10 (1.3 g, yield 48%).

[0327] 9. Synthesis of compound 187, 2-((2-(trans-4-hydroxy-cis-4-ethynylcyclohexyl)-6- methoxy-2H-indazol-5-yl)carbamoyl)-6-methylpyridine 1-oxide

[0328]

[0329] 30 °C, compound 10 (300 mg), compound 5 (444 mg) and cesium carbonate (820 mg) were added successively to NMP (10 mL), and the reaction was stirred at 90 °C for 18 h. LCMS showed the reaction was completed. The reaction was quenched by water (15 mL) at 30 °C, extracted with ethyl acetate (10 mL x 3), washed with saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by high performance liquid preparative chromatography (CH3CN:H2O (0.1% NH4HCO3) = 5-90%, UV: 214 nm, flow rate: 15 ml / min) to give compound 187 (85 mg, yield 20%).

[0330] 1 H NMR (400 MHz, CDC13): δ 14.14 (s, 1H), 8.89 (s, 1H), 8.46 (dd, J1= 7.6 Hz, J2= 2.8 Hz, 1H), 7.87 (s, 1H), 7.45-7.37 (m, 2H), 7.07 (s, 1H), 4.44-4.33 (m, 1H), 4.06 (s, 3H), 2.69-2.60 (m, 4H), 2.33-2.27 (m, 4H), 2.25-2.17 (m, 2H), 1.90-1.79 (m, 2H). LCMS: Rt = 3.162 min, [M+H] + = 421.2

[0331] Synthesis of compound 019 and compound 292

[0332] Reaction scheme:

[0333]

[0334] 1. Synthesis of compound 3

[0335] PPh3(15 g) was added to a solution of compound 1 (7 g) and compound 2 (3.37 g) in THF (200 mL) under ice bath. After the solution was stirred for 10 min, DIAD (3.1 g) was added slowly to the solution. The reaction was stirred at 30 °C for 18 h. Water (50 mL) was added to the reaction solution. The solution was extracted with ethyl acetate (40 mL*4). The organic phase was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE / EA = 10 / 1 to PE / EA = 2 / 1) to give compound 3 (6.0 g, yield 66%).

[0336] 2. Synthesis of compound 4

[0337] Pd / C (1.0 g, 10%) was added to a solution of compound 3 (6.5 g) in ethyl acetate (300 mL) at 15 °C. The reaction was stirred at 30 °C under hydrogen balloon (760 Torr) for 18 h. The reaction solution was filtered and concentrated under reduced pressure to give compound 4 (4.5 g, yield 80%).

[0338] 3. Synthesis of compound 6

[0339] EDCI.HC1 (2.1 g) was added to a solution of compound 4 (1.5 g) and compound 5 (1.1 g) in pyridine (30 mL) at 25 °C. The reaction was stirred at 25 °C for 16 h. The reaction solution was concentrated and dried. The residue was purified by silica gel column (PE:EA = 1:1) to give compound 6 (810 mg, yield 32%).

[0340] 4. Synthesis of compound 8

[0341] Cesium carbonate (2.3 g) was added to a solution of compound 6 (810 mg) and compound 7 (1.1 g) in 15 mL of DMF at 25 °C. The reaction was stirred at 90 °C for 16 h. The reaction solution was added to 50 mL of water. The solution was extracted with ethyl acetate (30 mL*3). The organic phase was concentrated under reduced pressure. The residue was purified by high performance liquid preparation column (CH3CN:H2O (0.1% NH4HCO3) = 30-55%, UV: 214 nm, flow rate: 15 ml / min) to give compound 8 (320 mg, yield 28%).

[0342] 5. Synthesis of compound 9

[0343] 4 mL of 2M diluted hydrochloric acid was added to a solution of compound 8 (320 mg) in 4 mL of dioxane at 25 °C. The reaction was stirred at 25 °C for 16 h. The reaction solution was adjusted to alkaline with sodium bicarbonate solution. The solution was extracted with ethyl acetate (10 mL*2). The organic phase was concentrated under reduced pressure to give compound 9 (250 mg, yield 86%).

[0344] 6. Synthesis of compound 019, 2-((2-(trans-4-hydroxycyclohexyl)-6- cyclopropylmethoxy-2H-indazol-5-yl)carbamoyl)-6-methylpyridine 1-oxide and compound 292, 2-((2-(cis-4-hydroxycyclohexyl)-6-cyclopropylmethoxy-2H-indazol-5- yl)carbamoyl)-6-methylpyridine 1-oxide

[0345]

[0346] Sodium borohydride (44 mg) was added to a solution of compound 7 (250 mg) in 5 mL of methanol at 0 °C and the reaction was stirred at 25 °C for 2 hours. The reaction was quenched with 10 mL of ammonium chloride solution and extracted with ethyl acetate (5 mL*3). The organic phase was concentrated under reduced pressure and the residue was purified by high performance liquid chromatography (CH3CN:H2O (0.1% NH4HCO3) = 35-60%, UV: 214 nm, flow rate: 15 ml / min) to give compound 019 (77 mg, yield 31%) with retention time Rt= 10.7 min and compound 292 (12 mg, yield 5%) with retention time Rt= 11.1 min.

[0347] Compound 019:

[0348] 1 H NMR (400 MHz, DMSO-d6): δ 14.31 (s, 1H), 8.78 (s, 1H), 8.31-8.27 (m, 2H), 7.77-7.75 (m, 1H), 7.56 (t, J = 7.6 Hz, 1H), 7.07 (s, 1H), 4.69 (s, 1H), 4.40-4.32 (m, 1H), 4.02 (d, J = 6.8 Hz, 1H), 3.56-3.51 (m, 1H), 2.52 (s, 3H), 2.09-2.05 (m, 2H), 1.97-1.87 (m, 4H), 1.44-1.34 (m, 3H), 0.66-0.61 (m, 2H), 0.48-0.45 (m, 2H). LCMS: Rt= 3.391 min, [M+H] + = 437.2.

[0349] Compound 292:

[0350] 1H NMR (400 MHz, DMSO-d6): δ 14.31 (s, 1H), 8.79 (s, 1H), 8.31-8.29 (m, 2H), 7.77-7.75 (m, 1H), 7.57 (t, J = 7.6 Hz, 1H), 7.08 (s, 1H), 4.49 (d, J = 6.8 Hz, 1H), 4.40-4.34 (m, 1H), 4.02 (d, J = 6.8 Hz, 2H), 3.87 (s, 1H), 2.52 (s, 3H), 2.33-2.22 (m, 2H), 1.56-1.75 (m, 4H), 1.66-1.60 (m, 2H), 1.41-1.34 (m, 1H), 0.65-0.61 (m, 2H), 0.49-0.45 (m, 2H). LCMS: Rt = 3.101 min, [M+H] + = 437.2.

[0351] Synthesis of compound 291

[0352] Reaction scheme:

[0353]

[0354] 1. Synthesis of compound 2

[0355] Compound 1 (800 mg) was dissolved in 10 mL of tetrahydrofuran at 0 degree Celsius, LiHMDS (1 M THF solution, 5.50 mL) was slowly added dropwise at 0 degree Celsius, after stirring at 0 degree Celsius for 60 minutes, iodomethane (680 mg) was slowly added to the reaction solution, and the reaction was carried out at this temperature for 1.5 hours. After the reaction was completed, the reaction was quenched by adding saturated ammonium chloride solution (10 mL) to the reaction solution, then extracted with ethyl acetate (25 mL x 2), concentrated under reduced pressure, and the residue was purified by silica gel column (petroleum ether: ethyl acetate = 7: 1) to obtain compound 2 (420 mg. Yield: 55%).

[0356] 2. Synthesis of compound 3

[0357] Compound 2 (700 mg) and 3M HCl (18 mL) were sequentially added to a tetrahydrofuran (18 mL) solution at 0 degree Celsius, and stirred at 50 degrees Celsius for 5 hours. After the reaction was completed, 3M aqueous sodium hydroxide solution was added to the reaction solution to adjust pH = 8, then extracted with dichloromethane (20 mL x 2), concentrated under reduced pressure, and the residue was purified by silica gel column (petroleum ether: ethyl acetate = 4: 1) to obtain compound 3 (420 mg, yield: 78%).

[0358] 3. Synthesis of compound 4

[0359] 25 °C, compound 3 (390 mg) was dissolved in 8 mL of ethanol, and a solution of sodium borohydride (112 mg) in ethanol (1 mL) was added dropwise to the reaction at -70 °C, and stirred for 1 h at -70 °C. After the reaction was completed, 8 mL of water was added to quench the reaction, and then extracted with ethyl acetate (15 mL x 2), and the organic phase was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1 : 1) to obtain compound 4 (280 mg, yield: 66%).

[0360] 4. Synthesis of compound 5

[0361] 28 °C, compound 4 (250 mg), TosCl (406 mg), DMAP (261 mg) and triethylamine (0.5 mL) were sequentially added to dichloromethane (8 mL), and stirred for 18 h at 28 °C. After the reaction was completed, the reaction was concentrated and dried, and the residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 6: 1) to obtain compound 5 (370 mg, yield: 64%).

[0362] 5. Synthesis of compound 291, 2-((2-(trans-4-cyano-cis-4-methylcyclohexyl)-6-methoxy-2H-indazol-5-yl)carbamoyl)-6-methylpyridine 1-oxide

[0363]

[0364] 25 °C, compound 5 (296 mg), compound 6 (350 mg) and cesium carbonate (808 mg) were added to DMF (6 mL), and after the addition, the mixture was heated to 90 °C and stirred for 18 h. After the reaction was completed, 10 mL of water was added to quench the reaction, and extracted twice with 40 mL of ethyl acetate, and the organic phase was concentrated under reduced pressure, and the residue was purified by high performance liquid chromatography (CH3CN: H2O (0.1% NH4HCO3) = 5-95%, UV: 214 nm, flow rate: 15 ml / min) to obtain compound 291 (80 mg, yield 19%).

[0365] 1 H NMR (400 MHz, CDC13): δ 14.14 (s, 1H), 8.88 (s, 1H), 8.45 (d, J = 7.6 Hz, 1H), 7.87 (s, 1H), 7.44-7.38 (m, 2H), 7.06 (s, 1H), 4.53-4.50 (m, 1H), 4.06 (s, 3H), 2.63 (s, 3H), 2.46-2.39 (m, 2H), 2.28-2.20 (m, 2H), 2.02-1.88 (m, 4H), 1.45 (s, 3H). LCMS: Rt = 3.310 min, [M+H]+ = 420.2

[0366] Synthesis of compound 002

[0367] Reaction formula:

[0368]

[0369] 1. Synthesis of compound 3

[0370] Compound 2 (1.0 g), compound 1 (0.91 g), EDCI (1.6 g) were added to pyridine (15 mL) at 28 degrees Celsius and stirred for 18 hours at 28 degrees Celsius, after the reaction was completed, concentrated under reduced pressure, the residue was slurried with methanol and water to obtain compound 3 (1.0 g, yield: 56%).

[0371] 2. Synthesis of compound 002, 2-((2-(trans-4-hydroxy-cis-4-methylcyclohexyl)-6- methoxy-2H-indazol-5-yl)carbamoyl)-6-cyclopropylpyridine 1-oxide

[0372]

[0373] Compound 3 (500 mg), compound 4 (675 mg), cesium carbonate (1.26 g) were sequentially added to DMF (10 mL) at 25 degrees Celsius and stirred for 18 hours at 90 degrees Celsius, after the reaction was completed, cooled to 25 degrees Celsius, quenched with water (5 mL), extracted with ethyl acetate (15 mL x 3), the extract was washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was subjected to high performance liquid chromatography (CH3CN:H2O = 20-45%, UV: 214 nm, flow rate 15 mL / min) to obtain compound 002 (130 mg, yield 19%).

[0374] 1 H NMR (400 MHz, CDCl3): δ 14.22 (s, 1H), 8.89 (s, 1H), 8.40 (dd, J1= 2.0 Hz, J2= 8.0 Hz, 1H), 7.87 (s, 1H), 7.37 (t, J = 8.0 Hz, 1H), 7.14-7.01 (m, 2H), 4.46-4.34 (m, 1H), 4.05 (s, 3H), 2.91-2.81 (m, 1H), 2.30-2.08 (m, 4H), 1.93-1.82 (m, 2H), 1.76-1.69 (m, 2H), 1.39 (s, 3H), 1.32-1.23 (m, 2H), 0.89-0.76 (m, 2H). LCMS: Rt = 2.859 min, [M+H]+ = 437.2

[0375] Synthesis of compound 289

[0376] Reaction formula:

[0377]

[0378] 1. Synthesis of compound 3

[0379] Compound 1 (5.0 g), compound 2 (2.3 g), cesium carbonate (13.4 g) and Pd2(dba)3 (0.25 g), BINAP (0.51 g) were sequentially added to toluene (100 mL) at 26 °C, and the reaction was stirred at 80 °C for 18 hours under nitrogen protection. After the reaction was completed, the temperature was lowered to 26 °C, water (100 mL) was added, and extraction was performed with ethyl acetate (200 mL x 3). The extract was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 10:1) to obtain compound 3 (1.3 g, yield 30%).

[0380] 2. Synthesis of compound 4

[0381] To a solution of compound 3 (1.3 g) in ethanol / water (40 mL / 10 mL), potassium hydroxide (4.58 g) was added, and the reaction was stirred at 90 °C for 16 hours. The pH was adjusted to 6 with 1M dilute hydrochloric acid, and extraction was performed with ethyl acetate (100 mL x 3). The extract was washed with water (50 mL) and saturated brine (100 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain compound 4 (1.12 g, yield 77%).

[0382] 3. Synthesis of compound 5

[0383] To a solution of compound 4 (360 mg) in dichloromethane (50 mL) at 26 °C, compound m-CPBA (1.86 g) was added and the reaction was stirred at 26 °C for 3 days. After the reaction was completed, filtration was performed, a saturated solution of sodium sulfite was added to the filtrate, the pH was adjusted to <7 with dilute hydrochloric acid, and stirring was performed at 26 °C for 2 hours. Extraction was performed with dichloromethane (200 mL x 3), the extract was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by preparative plate (dichloromethane:methanol = 20:1) to obtain compound 5 (60 mg, yield 15%).

[0384] 4. Synthesis of compound 289, i.e., 2-((2-(trans-4-hydroxy-cis-4-methylcyclohexyl)-6-methoxy-2H-indazol-5-yl)carbamoyl)-6-cyclopropylaminopyridine 1-oxide

[0385]

[0386] Compound 5 (49 mg), compound 6 (69 mg), HATU (118 mg), DIPEA (66 mg) were added into DMF (5 mL) in turn, and the reaction was stirred at 25 °C for 18 h. The reaction was concentrated under reduced pressure, and the residue was purified by high performance liquid preparative chromatography (CH3CN:H2O = 30-95%, UV: 214 nm, flow rate 15 mL / min) to give compound 289 (95 mg, yield 83%).

[0387] 1 H NMR (400 MHz, CDC13): δ 14.29 (s, 1H), 8.89 (s, 1H), 7.86 (t, J = 6.4 Hz, 2H), 7.41 (t, J = 8.0 Hz, 1H), 7.15 (s, 2H), 7.07 (s, 1H), 4.43-4.38 (m, 1H), 4.04 (s, 3H), 2.61 (br s, 1H), 2.25-2.13 (m, 4H), 1.89-1.85 (m, 1H), 1.75-1.64 (m, 4H), 1.39 (s, 3H), 0.93-0.90 (m, 2H), 0.74-0.72 (m, 2H). LCMS: Rt = 3.308 min, [M+H] + = 452.2.

[0388] 5. Synthesis of compound 9

[0389] To a solution of compound 10 (34 g) in ethanol (350 mL) was added NaN3(13.2 g) at 0 °C, and the reaction was stirred at 25 °C for 16 h. The reaction was directly used in the next step.

[0390] 6. Synthesis of compound 7

[0391] To a solution of compound 9 (0.17 mol) in ethanol (350 mL) was added acetic acid (30.6 g) at 25 °C, and compound 8 (22 g) was added and stirred at 25 °C for 10 min. The reaction was refluxed at 80 °C for 16 h. After the reaction was completed, the reaction solution was partially concentrated, water (70 mL) was added to slurry, and the solid was filtered. The solid was dissolved by heating reflux in ethanol (200 mL), cooled to room temperature, and n-heptane (200 mL) was added to slurry for 2 h. Compound 7 (35 g, yield 67%) was obtained by filtration.

[0392] 7. Preparation of compound 6

[0393] Pd / C (150 mg) was added to a solution of compound 7 (300 mg) in ethyl acetate (50 mL) at 25 °C and the reaction was stirred at 25 °C for 16 hours. After the reaction was completed, it was filtered and concentrated under reduced pressure to obtain compound 6 (260 mg, yield 96%).

[0394] Synthesis of compound 175

[0395] Reaction scheme:

[0396]

[0397] 1. Synthesis of compound 175, 2-((2-(trans-4-hydroxy-cis-4-ethylnitrile cyclohexyl)-6-methoxy-2H-indazol-5-yl)carbamoyl)-6-methylpyridine 1-oxide

[0398]

[0399] Cesium carbonate (1.4 g) was added to a solution of compound 1 (520 mg) and compound 2 (806 mg) in 10 mL of DMF at 25 °C, and the reaction was stirred at 90 °C for 16 hours. The reaction was poured into 50 mL of water, extracted with ethyl acetate (30 mL*3), and the organic phase was concentrated under reduced pressure. The residue was purified by high performance liquid preparative chromatography column (CH3CN:H2O (0.1% NH4HCO3) = 20-40%, UV: 214 nm, flow rate: 15 ml / min) to obtain compound 175 (64 mg, yield 8%).

[0400] 1 H NMR (400 MHz, DMSO-d6): δ 14.16 (s, 1H), 8.79 (s, 1H), 8.37 (s, 1H), 8.32-8.29 (m, 1H), 7.78-7.76 (m, 1H), 7.58 (t, J = 8.0 Hz, 1H), 7.11 (s, 1H), 5.20 (s, 1H), 4.49-4.45 (m, 1H), 3.95 (s, 3H), 2.82 (s, 2H), 2.53 (s, 3H), 2.14-2.01 (m, 4H), 1.84-1.80 (m, 2H), 1.71-1.64 (m, 2H). LCMS: Rt = 9.367 min, [M+H] + = 436.2

[0401] Synthesis of compound 176

[0402] Reaction scheme:

[0403]

[0404] 1. Synthesis of compound 176, 2-((2-(trans-4-hydroxy-cis-4-ethylnitrile cyclohexyl)-6-methoxy-2H-indazol-5-yl)carbamoyl)-6-cyclopropyl pyridine 1-oxide

[0405]

[0406] 25℃, to compound 8 (420 mg), compound 9 (601 mg), cesium carbonate (1.06 g) in DMF (10 mL) was added successively, and the reaction was stirred at 90℃ for 16 h. After the reaction was completed, the reaction was quenched by adding water (5 mL), extracted with ethyl acetate (10 mL x 3), washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and then purified by high performance liquid chromatography (CH3CN:H2O = 25-55%, UV: 214 nm, flow rate 15 mL / min) and then by preparative plate (dichloromethane:methanol = 20:1) to give compound 176 (25 mg, yield 4%).

[0407] 1 H NMR (400 MHz, CDC13): δ 14.25 (s, 1H), 8.89 (s, 1H), 8.39 (dd, J1= 2.0 Hz, J2= 8.0 Hz, 1H), 7.86 (s, 1H), 7.38 (t, J = 8.0 Hz, 1H), 7.09-7.03 (m, 2H), 4.53-4.43 (m, 1H), 4.05 (s, 3H), 2.90-2.81 (m, 1H), 2.76 (s, 2H), 2.33-2.17 (m, 4H), 2.13-2.03 (m, 2H), 1.99-1.93 (m, 1H), 1.89-1.73 (m, 2H), 1.30-1.26 (m, 2H), 0.88-0.80 (m, 2H). LCMS: Rt = 3.553 min, [M+H] + = 462.2

[0408] Synthesis of compound 042

[0409] Reaction scheme:

[0410]

[0411] 1. Synthesis of compound 2

[0412] To a solution of compound 1 (5.0 g) in dichloromethane (50 mL) was added compound m-CPBA (13.3 g) at 25 °C and stirred for 18 h at 25 °C. Filtered, to the filtrate was added saturated aqueous sodium sulfite (8.2 g) and stirred for 2 h at 25 °C, extracted with dichloromethane (50 mL x 3), washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, the residue was purified by trituration with ethyl acetate to give compound 2 (600 mg, yield 11%)

[0413] 2. Synthesis of compound 042, 2-((2-(trans-4-hydroxy-cis-4-methylcyclohexyl)-6- methoxy-2H-indazol-5-yl)carbamoyl)-6-methoxypyridine 1-oxide

[0414]

[0415] To a solution of compound 2 (92 mg), compound 4 (150 mg), HATU (311 mg), triethylamine (165 mg) in DMF (5 mL) was added at 25 °C and stirred for 16 h at 25 °C. After completion of the reaction, the reaction was quenched with water (5 mL), extracted with ethyl acetate (5 mL x 3), the extract was washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, the residue was purified by high performance liquid chromatography (CH3CN: H2O = 10-40%, UV: 214 nm, flow rate 15 mL / min) to give compound 042 (96 mg, yield 41%).

[0416] 1 H NMR (400 MHz, CDCl3): δ 14.15 (s, 1H), 8.89 (s, 1H), 8.22 (dd, J1= 2.0 Hz, J2= 8.0 Hz, 1H), 7.86 (s, 1H), 7.49 (t, J = 8.0 Hz, 1H), 7.11-7.02 (m, 2H), 4.44-4.34 (m, 1H), 4.16 (s, 3H), 4.03 (s, 3H), 2.29-2.08 (m, 4H), 1.91-1.82 (m, 2H), 1.73-1.69 (m, 2H), 1.39 (s, 3H). LCMS: Rt = 2.713 min, [M+H] + = 427.2

[0417] Example 21 Synthesis of compound B, 2-((2-(trans-4-hydroxy-cis-4-methylcyclohexyl)-6- methoxy-2H-indazol-5-yl)carbamoyl)-6-methylpyridine

[0418]

[0419] Compound 1 (150 mg), compound 2 (75 mg), HATU (249 mg), and DIPEA (141 mg) were added sequentially to DMF (5 mL) at 25°C, and the mixture was stirred at 25°C for 16 hours. Water (50 mL) was added, and the mixture was extracted with ethyl acetate (10 mL × 3). The extract was washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by preparative high-performance liquid chromatography (CH3CN:H2O = 30-95%, UV: 214 nm, flow rate 15 mL / min) to give 170 mg of white solid, with a yield of 79%.

[0420] 1H NMR (400MHz, CDCl3): δ10.82(s,1H),8.85(s,1H),8.10(d,J=7.6Hz,1H),7.87(s,1H),7.78(t,J=7.6Hz,1H), 7.32(d,J=7.2Hz,1H),7.08(s,1H),4.43-4.37(m,1H),4.03(s,3H),2.66(s,3H),2.27-2.13(m,4H),1.89(br s,1H),1.76-1.68(m,4H),1.40(s,3H).LCMS: Rt=3.604min, [M+H]+=395.2.

[0421] Biological evaluation

[0422] The following test cases are used to further explain the present invention, but these test cases are not intended to limit the scope of the present invention.

[0423] The structure of compound A in the biological test example:

[0424]

[0425] Compound B in the biological test examples has the structure synthesized in Example 21.

[0426]

[0427] Test Example 1: Determining the inhibitory effect of the compound of the present invention on human IRAK4 kinase activity.

[0428] Main test materials

[0429] ATP (Sigma, product number: A7699-1G)

[0430] DMSO (Sigma, part number D2650)

[0431] EDTA (Sigma, part number: E5134)

[0432] HEPES (Sigma, Cat. No. V900477-500G)

[0433] DTT (Sigma, Cat. No. D0632-25g)

[0434] Brij-35 (Sigma, Cat. No. B4184)

[0435] 96-well plate (Corning, Cat. No. 3365)

[0436] 384-well plate (Corning, Cat. No. 3573)

[0437] Experimental procedure

[0438] The IRAK4 inhibitory activity of the compounds at the Km concentration of ATP was measured in the IRAK4 MSA (Mobility-Shift Assay, a microfluidic chip technology) described below.

[0439] The recombinant fusion protein of N-terminal GST (Glutathione-S-transferase) and human IRAK4 was used as the enzyme (GST-IRAK4, kinase IRAK4 (Carna, Cat. No. 09-145)) with a final concentration of 1 nM; ATP ATP (Sigma, Cat. No. A7699-1G) with a final concentration of 37 μM; the substrate for the kinase reaction was a 5-FAM (5-carboxyfluorescein) labeled polypeptide (5-FAM-IPTSPITTTYFFFKKK-COOH), and the substrate peptide FAM-P8 (GL Biochem, Cat. No. 112396) with a final concentration of 5 μM.

[0440] In this test, the compound solution was prepared with 100% DMSO at 500 μM, and 10 concentration gradients were diluted 4 times with 100% DMSO, and further diluted 10 times with the compound buffer (50 mM HEPES, pH 7.5, 0.00015% Brij-35) to prepare the compound intermediate dilution solution containing 10% DMSO, and the final concentration of the compound was in the range of 10 μM-0.04 nM, and 5 μl was transferred to a black 384-well plate.

[0441] The kinase IRAK4 was diluted with the kinase buffer (50 mM HEPES, pH 7.5, 0.00015% Brij-35, 2 mM DTT) to a solution of 2.5 nM IRAK4, and 10 μl was transferred to the 384-well plate for incubation with the compound for 10-15 minutes.

[0442] Substrate and ATP were diluted to 12.5 μM and 92.5 μM, respectively, in reaction buffer (50 mM HEPES, pH 7.5, 0.00015% Brij-35, 10 mM MgCl2). 10 μl was transferred to a 384-well plate, the reaction was initiated, and incubated at 28 °C for 1 hour. The reaction was terminated by transferring 25 μl of 50 mM EDTA to the 384-well plate.

[0443] The conversion of substrate phosphorylation was read by Caliper EZ Reader (PerkinElmer), and the inhibition rate of IRAK4 was calculated. The IC50 was calculated by XL-fit software. 50 .

[0444] The test results show that the compounds of the present application have good inhibitory effect on IRAK4 kinase activity, and the IC 50 value is less than 100 nM, preferably less than 30 nM. Specifically, the activity values of some exemplary compounds are shown as follows:

[0445] The IC 50 values of the compounds of the present application on human IRAK4 kinase activity are shown in Table 1,

[0446] Table 1 IC 50

[0447] Compound ID IC 50 (nM) Compound B 30.00 16.00 23.70 101.00 137.60 142.50 155.40 168.10 198.10 2513.01 6323.01 757.11 764.31 879.22 188.32 2014.02 8438.02 855.92 8614.02 8713.02 8813.02 891.72 914.0

[0448] 29214

[0449] Test Example 2, Determination of the inhibitory effect of the compounds of the present application on human IRAK1 kinase activity

[0450] This test is used to evaluate the inhibitory effect of the compounds on human IRAK1 kinase activity, and the main test materials are the same as those in Test Example 1.

[0451] The IRAKI inhibitory activity of the compounds at the Km concentration of ATP was measured in IRAKI MSA (Mobility-Shift Assay, a microfluidic chip technology) described below. The recombinant fusion protein of N-terminal GST (glutathione-S-transferase) and human IRAKI was used as the enzyme (GST-IRAKI, kinase IRAKI, Carna) with a final concentration of 3 nM; ATP (Sigma) with a final concentration of 97 μM; the substrate for the kinase reaction was a 5-FAM (5-carboxyfluorescein) labeled polypeptide (5-FAM-IPTSPITTTYFFFKKK-COOH), substrate peptide FAM-P8 (GL Biochem) with a final concentration of 5 μM.

[0452] In this assay, the compound solution was prepared at 500 μM with 100% DMSO, and 10 concentration gradients were diluted 4-fold with 100% DMSO, and further diluted 10-fold with the compound buffer (50 mM HEPES, pH 7.5, 0.00015% Brij-35) to prepare the compound intermediate dilution solution containing 10% DMSO, and the final concentration of the compound was in the range of 10 μM-0.04 nM, and 5 μl was transferred to a black 384-well plate.

[0453] The kinase IRAKI was diluted to an IRAKI solution of 7.5 nM with the kinase buffer (50 mM HEPES, pH 7.5, 0.00015% Brij-35, 2 mM DTT), and 10 μl was transferred to a 384-well plate to be incubated with the compound for 10-15 minutes.

[0454] The substrate and ATP were diluted to 12.5 μM and 242.5 μM, respectively, with the reaction buffer (50 mM HEPES, pH 7.5, 0.00015% Brij-35, 10 mM MgCl2). 10 μl was transferred to a 384-well plate to start the reaction, and reacted at 28 degrees Celsius for 1 hour. 25 μl of 50 mM EDTA was transferred to the 384-well plate to terminate the reaction. The conversion rate of the substrate phosphorylation was read by Caliper EZ Reader (PerkinElmer), and the inhibition rate of the compound on IRAKI was calculated, and the IC50 was calculated by XL-fit software.

[0455] The test results show that the compounds of the embodiments of the present application have significant selective inhibitory activity on IRAK4, and the IC50 (nM) ratio of IRAK1 to IRAK4 is greater than 500, preferably greater than 200. Specifically, the activity values of some exemplary compounds are shown as follows: the inhibition IC50 of the compounds of the present application on human IRAKI kinase activity 50 The values are shown in Table 2.

[0456] Table 2 IC50 values for human IRAK1 kinase activity 50

[0457] Compound ID IRAK1 IC 50 (nM) IRAK1 IC 50 (nM) IRAK4 IC 50 (nM) 163 299 313 0.100 140 396 73.2

[0458] From the data in Table 2, it can be seen that the compounds of the present application have significant selectivity for human IRAK4 over human IRAK1 activity.

[0459] Test Example 3: hERG assay for the compounds of the present application

[0460] This assay was used to evaluate the cardiac safety of the compounds. The experiment was performed using a HEK-293 cell line stably expressing the hERG potassium channel.

[0461] Instrumentation:

[0462] Amplifier: EPC10 from HEKA (Germany)

[0463] Micro-manipulator: MP225 from Sutter Instruments (USA)

[0464] Electrode puller: P97 from Sutter Instruments (USA)

[0465] Microscope: TE300 from Nikon

[0466] Capillary glass tubing: BF150-86-10 from Sutter Instruments (USA)

[0467] Data acquisition and analysis software: PatchMaster, Igor Pro 6.0 and GraphPad Prism 5.0

[0468] Experimental procedure

[0469] The test compound stock solution was diluted with DMSO to 0.3 mM, 1 mM and 3 mM, respectively. The test compound stock solution was diluted with extracellular solution (140 mM NaCl, 3.5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM Glucose, 10 mM HEPES, 1.25 mM NaH2PO4, pH 7.4 adjusted with NaOH) to get 0.3 μM, 1 μM, 3 μM, 10 μM, 30 μM test compound working solution. All test compound working solutions were sonicated for 20 min.

[0470] Patch-clamp recording: Under the inverted microscope, the glass electrode micro-manipulator (micromanip) was used to contact the recording electrode to the cell, then negative pressure was applied to promote the cell to form GΩ seal. After the GΩ seal was formed, fast capacitance compensation was performed, then negative pressure was continuously applied to suck the cell membrane to form the whole-cell recording mode. In the whole-cell recording mode, slow capacitance compensation was performed and the values of membrane capacitance and series resistance were recorded.

[0471] The voltage stimulation protocol for hERG potassium current was as follows: the cell membrane was clamped at -80 mV, then depolarized from -80 mV to +30 mV for 2.5 seconds, then quickly held at -50 mV for 4 seconds to evoke the tail current of hERG channel. The data was collected every 10 seconds. The leakage current was detected at -50 mV.

[0472] The cell-seeded coverslips were placed in the recording chamber of the inverted microscope, and the negative control and test compound were perfused from low concentration to high concentration through the recording chamber by gravity perfusion to quickly act on the cells. In the recording, the external solution was continuously circulated by a vacuum pump. The current detected in each cell of the negative control was used as the control group of the cell itself. Each drug concentration was applied for 5 minutes or until the current was stable. All experiments were performed at room temperature.

[0473] Data analysis:

[0474] First, the current after each drug concentration was standardized Then the corresponding inhibition rate was calculated The basic statistics, including mean (Mean), standard deviation (SD), standard error (SE) and repeat number (n), were calculated for each concentration. The dose-dependent curve was fitted by the following equation, and the half-inhibitory concentration (IC 50 ) of the test compound was calculated:

[0475] where C represents the concentration of the test compound, IC 50 represents the half-inhibitory concentration, and h represents the Hill coefficient. Curve fitting and IC 50The calculations were performed using GraphPad Prism 5.0 software.

[0476] The test results show that the compounds of the embodiments of the present application have very low inhibition rate on human hERG, and even can be significantly better than the comparative compound A, the inhibition on hERG (30 μM) is less than 50%, preferably less than 30%. Specifically, the inhibition rate values of some exemplary compounds are shown as follows:

[0477] Table 3 Inhibition on hERG at 30 μM

[0478] Compound ID Inhibition on hERG (30 μM) A 27.10% ± 1.74% 163 8.73% ± 1.37% 001 5.09% ± 2.43%

[0479] As can be seen from the data in Table 3, the compounds of the present application have low inhibition rate on human hERG, and have certain advantages over compound A.

[0480] Test Example 4: Determination of TDI (Time-Dependent Inhibition) data of the compounds of the present application

[0481] The purpose of the present experiment is to study the time-dependent inhibition of the compounds on human P450 metabolic enzyme CYP3A4. The human mixed liver microsomes used in the present experiment are from the United States Corning Company.

[0482] The test compound will be co-incubated with human liver microsomes and the probe substrate midazolam (CYP3A4), and the test compound will be set to 30 μM. The reaction will be started by the addition of coenzyme NADPH. Acetonitrile is added to the incubation system to terminate the reaction, and the internal standard has been previously dissolved in acetonitrile. After protein precipitation, centrifugation is performed to obtain the supernatant. The characteristic metabolite 1-hydroxy-midazolam (CYP3A4) in the supernatant is analyzed by LC-MS / MS method. Finally, the influence of the test compound on the generation of these characteristic metabolites is studied according to the obtained data. Selective inhibitors (verapamil on CYP3A4) will be used as positive controls.

[0483] The test results show that the compounds of the embodiments of the present application have no significant time-dependent inhibition on human CYP3A4, and the TDI values of some exemplary compounds are shown as follows:

[0484] Table 4 Time-dependent inhibition (TDI) on human CYP3A4 at 30 μM

[0485] Compound ID TDI (3A4, 30 μM) 001 -3.68% 014 +3.85%

[0486] Test Example 5: Determination of plasma protein binding rate (PPB) data of the compounds of the present application

[0487] The purpose of this experiment is to determine the plasma protein binding rate (PPB) data of the test compound.

[0488] In the PPB experiment, the final concentration of the test compound or reference compound in the dosing matrix is 1 μM with 0.2% DMSO content.

[0489] Collect 0 hour sample: Take 25 μL of the compound-containing matrix and add to a blank 96-well collection plate and store at -20°C.

[0490] Prepare the equilibrium dialysis set-up. Add 100 μL of buffer to the receiving side of the equilibrium dialysis plate. Add 100 μL of the compound- or reference compound-containing dosing matrix to the dosing side of the equilibrium dialysis plate. Place the prepared equilibrium dialysis plate in a 37°C shaker at 60 rpm for 5 hours.

[0491] At the end of the incubation (5 hours), prepare the samples:

[0492] Prepare the receiving side sample: Take 25 μL from the receiving side and mix in a 96-well sample collection plate with 25 μL of the appropriate matrix (blank plasma). Add 200 μL of ACN containing the internal standard and shake at 600 rpm for 10 minutes, then centrifuge at 5594 g for 15 minutes in a centrifuge.

[0493] Prepare the dosing side sample: Take 25 μL of the dosing side sample and mix with 25 μL of blank buffer solution. Add 200 μL of ACN containing the internal standard and shake at 600 rpm for 10 minutes, then centrifuge at 5594 g for 15 minutes in a centrifuge.

[0494] Prepare the 0 hour sample: Take 25 μL of the 0 hour sample and mix with 25 μL of the appropriate matrix (blank buffer solution) at 37°C. Add 200 μL of ACN containing the internal standard and shake at 600 rpm for 10 minutes, then centrifuge at 5594 g for 15 minutes in a centrifuge.

[0495] After centrifugation of all samples, take 50 μL of the supernatant and mix with 50 μL of ultrapure water. The samples are sent for liquid chromatography-mass spectrometry analysis.

[0496] The test results show that the plasma protein binding rate of the compound of the present application to humans, rats and mice is moderate, and the interspecies difference is very small, and can even be significantly less than the comparative compound A. Specifically, the PPB data of some exemplary compounds are as follows:

[0497] Table 5 Plasma protein binding rate (PPB) data

[0498]

[0499] Test Example 6, Inhibition of LPS-induced TNF-α release in Balb / c female mice by the compounds of the present application

[0500] Experimental procedure

[0501] Female Balb / c mice were randomly divided into several groups, 4 mice in each group, including normal control + vehicle group, model + vehicle group, model + positive drug group and other model + test drug group. The normal control group of animals received intraperitoneal injection of normal saline (10 ml / kg), and the model animals received LPS stimulation (Sigma, L2630, intraperitoneal injection, 10 mL / kg, 0.2 mg / kg). In the experiment, the test drugs were added into DMSO, Solutol and 10 mM PBS to prepare solutions or suspensions of the required concentration for administration. The final volume ratio of each component of the vehicle, DMSO, Solutol and 10 mM PBS, was 5:15:80. Each experimental group was given corresponding gavage (10 ml / kg) at the set dose 16 h before LPS (or saline) stimulation, and each group of animals was euthanized with CO2 1.5 h after stimulation, and heart blood was collected. The obtained whole blood was not anticoagulated, and after standing in wet ice for 1.5 h, it was centrifuged at 2000g, 4°C for 10 min to separate serum. The serum was stored at -80°C for TNFα determination. The quantification of TNFα was completed by TNFα ELISA kit according to the manufacturer's instructions. The absorbance A450 was detected by enzyme marker SpectraMax i3x (Molecular Device), and the inhibition rate of the compound was calculated, and the IC50 was calculated by GraphPad Prism 7.0 software 50 .

[0502] The test results show that the compounds of the present application have obvious inhibitory effect on the release of TNF-α in LPS-induced Balb / c female mice, and the inhibition rate is greater than 50%, preferably greater than 70%, and specifically, the inhibition rates of some exemplary compounds are as follows:

[0503] Table 6 Inhibition rate of TNF-α release in LPS-induced Balb / c female mice

[0504] Compound ID Inhibition rate of TNF-α % 0137 6.29 0017 4.00 1637 1.56 1678.71

[0505] Test Example 7, Determination of the inhibition of the compounds of the present application on five major CYP450 enzyme subtypes of human liver microsomes

[0506] The purpose of this experiment is to study the inhibitory effect of the test compound on five major human P450 metabolic enzymes, CYP1A2, 2C9, 2C19, 2D6 and 3A4-M. The human mixed liver microsomes used in this experiment are from the United States Corning Company. The test compound (compound 14) will be co-incubated with human liver microsomes and five probe substrates (phenacetin for CYP1A2, diclofenac for CYP2C9, phenytoin for CYP2C19, dextromethorphan for CYP2D6, midazolam for CYP3A4-M, which is a mixed substrate) (see the table below), and the test compound will be set at 7 concentration points. The reaction will be started by the addition of coenzyme NADPH. The reaction is terminated by adding ice acetonitrile containing internal standards to the incubation system. After protein precipitation, centrifugation is performed to obtain the supernatant. The characteristic metabolites in the supernatant (acetaminophen for CYP1A2, 4-hydroxydiclofenac for CYP2C9, 4-hydroxyphenytoin for CYP2C19, dextrorphan for CYP2D6, 1-hydroxy-midazolam for CYP3A4-M) are analyzed by LC-MS / MS method. Finally, the effect of the test compound on the generation of these characteristic metabolites is studied according to the data obtained. Selective inhibitors (ketoconazole for CYP3A4-M) will be used as positive controls. All incubations are performed in parallel.

[0507] The test results show that the compounds of the embodiments of the present application have no significant inhibitory effect on the five CYP subtypes of humans, and the inhibitory effect on 1A2, 2C9, 2C19, 3 subtypes is significantly smaller than that of the comparative compound A. Specifically, the inhibition rates of some exemplary compounds are as follows:

[0508] Table 7 Inhibition (IC50, nM) of the compounds of the present application on five major CYP450 enzyme subtypes CYP1A2, 2C9, 2C19, 2D6 and 3A4 of human liver microsomes 50 ,nM)

[0509]

[0510] Test Example 8, PK analysis test of the compounds of the present application on rats

[0511] The mouse pharmacokinetic test of the preferred embodiments of the present application is carried out using male SPF SD rats (Shanghai Xipu-Bike Experimental Animal Co., Ltd.).

[0512] Dosing method: single gavage oral administration or single intravenous injection

[0513] Sampling points: 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 24 hours after administration

[0514] Sample processing: 0.2 mL of venous blood was collected and placed on ice after blood sample collection, and plasma was separated by centrifugation (centrifugation conditions: 8000 rpm, 6 min, 4℃). The collected plasma was stored at -80℃ before analysis.

[0515] Internal standard working solution: a certain amount of tolubutamide internal standard stock solution with a concentration of 645,000 ng / mL was taken into a certain volume of volumetric flask, and then methanol was added to the mark after mixing, to prepare an internal standard working solution with a concentration of 50 ng / mL.

[0516] Sample pretreatment: 50 μL of plasma sample was taken into a 1.5 mL centrifuge tube, 250 μL of internal standard solution (blank without internal standard, supplemented with the same volume of methanol) was added, vortexed and mixed, centrifuged at 14000 rpm for 5 min, and 200 μL of supernatant was taken and added to a 96-well sample injection plate for LC-MS / MS injection analysis.

[0517] Liquid phase conditions:

[0518] Chromatographic column: ACQUITY UPLC BEH C18 1.7 μm (50 mm x 2.10 mm)

[0519] Mobile phase: A liquid is 0.1% formic acid aqueous solution, B liquid is 0.1% formic acid acetonitrile solution

[0520] Flow rate: 0.5 mL / min

[0521] The data processing system is Analyst software (American Applied Biosystems Corporation, software version number 1.5.5).

[0522] The test results show that the compounds of the embodiments of the present application all exhibit good pharmacokinetic characteristics on mice, show good exposure and retention time in animals, and have suitable half-life and good drug absorption. Specifically, the pharmacokinetic data of some exemplary compounds are as follows:

[0523] Table 8 Pharmacokinetic data of different compounds in ICR mice after single gavage oral administration

[0524]

[0525] The embodiments of the present application are described above. However, the present application is not limited to the above-mentioned embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A compound of formula I, its stereoisomers, racemates, tautomers, isotope-labeled compounds, prodrugs or pharmaceutically acceptable salts thereof: Wherein, Ring A is a 5-14 membered heteroaryl or 5-12 membered heterocyclic group containing at least one N; Each of R1, R2, and R3 is independently selected from hydrogen, halogen, CN, OH, or the following groups optionally substituted with one, two, or more Rs: (C1-C 12 ) an aliphatic hydrocarbon group, optionally containing one, two, or more heteroatoms (C1-C 12 ) an aliphatic hydrocarbon group, C 3-12 cycloalkyl, 3- to 12-membered heterocyclic group, C 6-20 aryl or 5- to 14-membered heteroaryl, -NR a R b ; W is selected from O, S, NH, single bond; Each R a 、R b is independently selected from H, (C1-C 12 ) aliphatic hydrocarbon group; Each R is independently selected from halogen, CN, OH, SH, NR a R b or is selected from the following groups optionally substituted by one, two or more R': (C1-C 12 ) aliphatic hydrocarbon group, optionally containing one, two or more heteroatoms (C1-C 12 ) aliphatic hydrocarbon group, C 3-12 cycloalkyl, 3- to 12-membered heterocyclic group, C 6-20 aryl or 5- to 14-membered heteroaryl; Each R’ is independently selected from halogen, CN, OH, SH, NR a R b ; n is selected from 1, 2, 3; m is selected from 1, 2, 3, 4, 5, 6.

2. A compound of formula I, its stereoisomers, racemates, tautomers, isotope-labeled compounds, prodrugs or pharmaceutically acceptable salts thereof according to claim 1, characterized in that: The "optionally containing one, two or more heteroatoms (C1-C 12 ) aliphatic hydrocarbon group" may be selected from (C1-C 12 ) aliphatic hydrocarbon group oxy, (C1-C 12 ) aliphatic hydrocarbon group mercapto, (C1-C6) aliphatic hydrocarbon group oxy (C1-C6) aliphatic hydrocarbon group, (C1-C6) aliphatic hydrocarbon group mercapto (C1-C6) aliphatic hydrocarbon group, N-(C1-C3) aliphatic hydrocarbon group amino (C1-C6) aliphatic hydrocarbon group, N,N-di-(C1-C3) aliphatic hydrocarbon group amino (C1-C6) aliphatic hydrocarbon group; The "5-14 membered heteroaryl or 5-12 membered heterocyclic group containing N" is selected from pyridine, pyrrole, piperidine, pyrrolidine. The (C1-C 12 ) aliphatic hydrocarbon group may be selected from (C1-C 12 ) alkyl, (C2-C 12 ) alkenyl, (C2-C 12 ) alkynyl. Preferably, the (C1-C 12 ) aliphatic hydrocarbon group may be selected from (C1-C6) alkyl, (C2-C6) alkenyl, (C2-C6) alkynyl; The "halogen" is selected from F, Cl, Br, I; The "C 3-12 cycloalkyl" may be selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.

3. A compound of formula I, its stereoisomers, racemates, tautomers, isotope-labeled compounds, prodrugs or pharmaceutically acceptable salts thereof according to claim 1 or 2, characterized in that: R1, R2, R3 may each independently be selected from the following groups optionally substituted with one, two or more Rs: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, vinyl, 1-propenyl, 2-propenyl, 1-methylethenyl, 1-butenyl, 1-ethylethenyl, 1-methyl-2-propenyl, 2-butenyl, 3-butenyl, 2-methyl-1-propenyl, 2-methyl-2-propenyl, 1-pentenyl, 1-hexenyl, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 1-methyl-2-propynyl, 3-butynyl, 1-pentynyl, 1-hexynyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, methoxy, ethoxy, propoxy, butoxy, pentyloxy, methoxymethyl, ethoxymethyl, propoxymethyl, methoxyethyl, ethoxyethyl, propoxyethyl, methoxypropyl, ethoxypropyl, propoxypropyl, N-methylaminomethyl, N-methylaminoethyl, N-ethylaminoethyl, N,N-dimethylaminomethyl, N,N-dimethylaminoethyl, N,N-diethylaminoethyl, amino, N,N-dimethylamino, N,N-diethylamino, pyrrolidinyl, piperidinyl, pyridyl, pyrazinyl, pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, The above-mentioned Indicate the connection sites of the groups.

4. A compound of formula I, its stereoisomers, racemates, tautomers, isotope-labeled compounds, prodrugs or pharmaceutically acceptable salts thereof according to any one of claims 1-3, characterized in that: Among the compounds of formula I, their stereoisomers, racemates, tautomers, isotope-labeled compounds, prodrugs or their pharmaceutically acceptable salts, the compounds of formula I may be selected from the following structures of formula Ia, formula Ib, formula Ic, formula Id, formula Ie: In formula Ia, formula Ib, formula Ic, formula Id, formula Ie, R1, R2, R3, m, n, W are as defined in formula I.

5. A compound of formula I, its stereoisomers, racemates, tautomers, isotope-labeled compounds, prodrugs or pharmaceutically acceptable salts thereof according to any one of claims 1-4, characterized in that, The compounds of formula I may be selected from the following structures:

6. A process for preparing a compound of formula I as claimed in any one of claims 1 - 5, its stereoisomers, racemates, tautomers, isotope - labeled compounds, prodrugs or pharmaceutically acceptable salts thereof, characterized in that, The preparation method includes the following steps: (a1) React M-1 with M-2 to form M-3; the reaction can be carried out in the presence of EDCl.HCl and pyridine; (a2) M-3 reacts with R x in the L1 reaction, where R x is selected from R1 or the hydroxyl group in R1 with a hydroxyl group is Substituted group; and when R x is selected from those in which the hydroxyl group in R1 with a hydroxyl group is When substituting groups, the reaction further needs to include obtaining the product of formula I through acidic and reduction conditions. The acidic conditions can be selected from HCl, and the reduction conditions can be selected from sodium borohydride; In the above steps, R1, R2, R3, m, W are as defined in formula I; L1 is a leaving group and can be selected from halogen or OTs.

7. A process for preparing a compound of formula I as claimed in any one of claims 1 - 5, its stereoisomers, racemates, tautomers, isotope - labeled compounds, prodrugs or pharmaceutically acceptable salts thereof, characterized in that, The preparation method includes the following steps: (b1)N-1 reacts with R x in the reaction of L1, where R x is selected from R1 or the hydroxyl group in R1 with a hydroxyl group is Substituted group; and when R x is selected from those in which the hydroxyl group in R1 with a hydroxyl group is When substituting groups, the reaction further needs to include obtaining N-2 through acidic and reduction conditions; in this step, the acidic conditions can be selected from HCl, and the reduction conditions can be selected from sodium borohydride; (b2) Reduce N-2 obtained from the above reaction to obtain N-3; the reducing agent can be selected from Pd / C; (b3) React N-3 with M-2 to obtain Formula I. In the said step, R1, R2, R3, m, and W are as defined in Formula I; the said L1 is a leaving group and can be selected from halogen or OTs.

8. A pharmaceutical composition comprising a compound of formula I as described in any one of claims 1 - 5, its stereoisomers, racemates, tautomers, isotopically labeled compounds, prodrugs or pharmaceutically acceptable salts thereof.

9. Use of a compound of formula I as described in any one of claims 1 - 5, its stereoisomers, racemates, tautomers, isotopically labeled compounds, prodrugs or pharmaceutically acceptable salts thereof, or the pharmaceutical composition according to claim 8, in the preparation of a medicament for preventing and / or treating IRAK - mediated diseases or disorders.

10. The use according to claim 9, wherein The said disease or disorder is selected from diseases such as tumors, gout, systemic lupus erythematosus, multiple sclerosis, metabolic syndrome, atherosclerosis, myocardial infarction, sepsis, inflammatory bowel disease, asthma, rheumatoid arthritis, and allergies.