WO2021057785A1 - 一种irak抑制剂及其制备方法和用途 - Google Patents

一种irak抑制剂及其制备方法和用途 Download PDF

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WO2021057785A1
WO2021057785A1 PCT/CN2020/117093 CN2020117093W WO2021057785A1 WO 2021057785 A1 WO2021057785 A1 WO 2021057785A1 CN 2020117093 W CN2020117093 W CN 2020117093W WO 2021057785 A1 WO2021057785 A1 WO 2021057785A1
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compound
formula
group
acid
degrees celsius
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PCT/CN2020/117093
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English (en)
French (fr)
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野国中
丁陈利
丁雅雯
贺潜
王朝东
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上海美悦生物科技发展有限公司
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Priority to EP20869953.8A priority Critical patent/EP4015513B1/en
Priority to ES20869953T priority patent/ES2967642T3/es
Application filed by 上海美悦生物科技发展有限公司 filed Critical 上海美悦生物科技发展有限公司
Priority to BR112022001568A priority patent/BR112022001568A2/pt
Priority to LTEPPCT/CN2020/117093T priority patent/LT4015513T/lt
Priority to JP2022519127A priority patent/JP7353474B2/ja
Priority to CA3152167A priority patent/CA3152167C/en
Priority to PE2022000414A priority patent/PE20220944A1/es
Priority to KR1020227005043A priority patent/KR20220035450A/ko
Priority to AU2020352311A priority patent/AU2020352311B2/en
Priority to US17/754,121 priority patent/US20220298139A1/en
Priority to CN202080062424.4A priority patent/CN114391013B/zh
Priority to MX2022003504A priority patent/MX2022003504A/es
Priority to HRP20240122TT priority patent/HRP20240122T1/hr
Publication of WO2021057785A1 publication Critical patent/WO2021057785A1/zh
Priority to IL291158A priority patent/IL291158A/en
Priority to DO2022000054A priority patent/DOP2022000054A/es
Priority to CONC2022/0004978A priority patent/CO2022004978A2/es
Priority to ZA2022/04441A priority patent/ZA202204441B/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings

Definitions

  • the present invention relates to the field of medicinal chemistry, in particular to a compound suitable for the treatment of cancer and inflammatory diseases related to interleukin-1 receptor-associated kinase (IRAK), and more specifically to modulate the function of IRAK-4 Compound.
  • IRAK interleukin-1 receptor-associated kinase
  • Interleukin-1 receptor-associated kinase is a family of serine/threonine protein kinases that exist in the cell. There are four members: IRAK1, IRAK2, IRAK-M and IRAK4. The common features are typical The N-terminal death domain, which mediates the interaction with the MyD88-family adaptor protein and the central kinase domain, in 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 signal transduction pathway.
  • TLR The extracellular part of TLR recognizes pathogen-specific molecules (such as lipopolysaccharides, polypeptides, viruses) DNA, etc.), after binding to the ligand, the cell partly recruits MyD88, etc. to form a complex, activates IRAK1 autophosphorylation, and then activates the downstream serine/threonine kinase TAK1, activates NF- ⁇ B and MAPK signaling pathways, and then produces pro-inflammatory Cytokines, chemokines and destructive enzymes eventually lead to inflammation and mediate innate immunity.
  • IL-1R participates 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).
  • RA Rheumatoid arthritis
  • RA Rheumatoid arthritis
  • T/B lymphocytes T/B lymphocytes
  • macrophages TNF- ⁇ and so on.
  • IRAK4 inhibitors can effectively block the production of the pro-inflammatory cytokine tumor necrosis factor (TNF); in mouse models of collagen-induced arthritis, IRAK4 inhibitors can Significantly inhibit the release of TNF, thereby controlling the progress of the disease; in the MyD88-dependent inflammatory gout mouse model, IRAK4 inhibitors can block leukocyte infiltration in a dose-dependent manner (Priscilla N, et.al.J.Exp.Med. ,2015,13(212):2189-2201).
  • TNF tumor necrosis factor
  • the excessive activation of the IRAK4-dependent TLR/IL-1R signaling pathway is closely related to the occurrence and development of rheumatoid arthritis.
  • Many other studies have also confirmed that the activation of the IRAK4 enzyme is closely related to the occurrence and development of the following diseases, such as tumors. , Gout, systemic lupus erythematosus, multiple sclerosis, metabolic syndrome, atherosclerosis, myocardial infarction, sepsis, inflammatory bowel disease, asthma and allergies (Chaudhary D, et.al., J. Med. Chem. 2015, 58(1): 96-110).
  • the present invention provides a compound represented by the following formula I, its stereoisomers, racemates, tautomers, isotope markers, prodrugs or pharmaceutically acceptable salt:
  • Ring A contains at least one N-containing 5-14 membered heteroaryl group or 5-12 membered heterocyclic group
  • Each R 1 , R 2 , R 3 is independently selected from hydrogen, halogen, CN, OH, or the following groups optionally substituted by one, two or more R: (C 1 -C 12 ) aliphatic hydrocarbon group , Optionally containing one, two or more heteroatoms (C 1 -C 12 ) aliphatic hydrocarbon group, C 3-12 cycloalkyl group, 3-12 membered heterocyclic group, C 6-20 aryl group or 5- 14-membered heteroaryl, -NR a R b ;
  • W is selected from O, S, NH, single bond
  • Each R a and R b is independently selected from H, (C 1 -C 12 ) aliphatic hydrocarbon group;
  • Each R is independently selected from halogen, CN, OH, SH, NR a R b or selected from the following groups optionally substituted by one, two or more R′: (C 1 -C 12 ) aliphatic hydrocarbon group , Optionally containing one, two or more heteroatoms (C 1 -C 12 ) aliphatic hydrocarbon group, C 3-12 cycloalkyl group, 3-12 membered heterocyclic group, C 6-20 aryl group or 5- 14-membered heteroaryl;
  • Each R' is independently selected from halogen, CN, OH, SH, NR a R b ;
  • the "(C 1 -C 12 )aliphatic hydrocarbon group optionally containing one, two or more heteroatoms” may be selected from (C 1 -C 12 )aliphatic hydrocarbon group, (C 1 -C 12 ) Aliphatic sulfhydryl group, (C 1 -C 6 ) aliphatic hydrocarbon group oxy (C 1 -C 6 ) aliphatic hydrocarbon group, (C 1 -C 6 ) aliphatic hydrocarbon group mercapto group (C 1 -C 6 ) aliphatic hydrocarbon group, N-(C 1 -C 3 ) aliphatic hydrocarbyl amine group (C 1 -C 6 ) aliphatic hydrocarbyl group, N,N-bis-(C 1 -C 3 ) aliphatic hydrocarbyl amino group (C 1 -C 6 ) aliphatic hydrocarbon group;
  • the "containing at least one N-containing 5-14 membered heteroaryl group or 5-12 membered heterocyclic group” means that the heteroaryl group or heterocyclic group contains at least one nitrogen atom, and may also contain other groups selected from N One or more heteroatoms of, O, or S, for example, are selected from pyridine, pyrrole, piperidine or tetrahydropyrrole.
  • the (C 1 -C 12 ) aliphatic hydrocarbon group may be selected from (C 1 -C 12 )alkyl, (C 2 -C 12 )alkenyl, (C 2 -C 12 )alkynyl, preferably, the ( C 1 -C 12 )aliphatic hydrocarbon group can be selected from (C 1 -C 6 )alkyl, (C 2 -C 6 )alkenyl, (C 2 -C 6 )alkynyl;
  • halogen is selected from F, Cl, Br, I;
  • the "C 3-12 cycloalkyl group” may be selected from cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.
  • the R 1 , R 2 , R 3 may be independently selected from the following groups optionally substituted by one, two or more R: methyl, ethyl, n-propyl, isopropyl, n-butyl Base, 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
  • formula I in the compound represented by formula I, its stereoisomers, racemates, tautomers, isotope markers, prodrugs or pharmaceutically acceptable salts thereof, formula I
  • the compound shown can be selected from the following structures of Formula Ia, Formula Ib, Formula Ic, Formula Id, and Formula Ie:
  • Formula Ia Formula Ib, Formula Ic, Formula Id, and Formula Ie, R 1 , R 2 , R 3 , m, n, and W are as defined in Formula I.
  • formula I in the compound represented by formula I, its stereoisomers, racemates, tautomers, isotope markers, prodrugs or pharmaceutically acceptable salts thereof, formula I
  • the compound shown can be selected from the following structures:
  • the present invention also provides the compound represented by formula I (including formula Ia-formula Ie), its stereoisomers, racemates, tautomers, isotope markers, prodrugs or pharmaceutically acceptable
  • the preparation method of the salt is not limited to the method described below.
  • the preparation method may include the following steps:
  • (a2) M-3 1 is reacted with R x L, wherein X is selected from the R or R 1 with R 1 is a hydroxyl group is a hydroxyl group A substituted group; and when R x is selected from R 1 with a hydroxyl group, the hydroxyl group is In the case of substituted groups, the reaction needs to further include obtaining the product of formula I under acidic and reducing conditions.
  • the acidic conditions can be selected from HCl
  • the reducing conditions can be selected from sodium borohydride
  • R 1 , R 2 , R 3 , m, and W are as defined in formula I; the L 1 is a leaving group, which can be selected from halogen or OTs.
  • the preparation method may include the following steps:
  • N-1 1 is reacted with R x L, wherein X is selected from the R or R 1 with R 1 is a hydroxyl group is a hydroxyl group A substituted group; and when R x is selected from R 1 with a hydroxyl group, the hydroxyl group is For substituted groups, the reaction needs to further include obtaining N-2 products 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;
  • the reducing agent may be selected from Pd/C;
  • R 1 , R 2 , R 3 , m, and W are as defined in formula I; the L 1 is a leaving group, which can be selected from halogen or OTs.
  • the present invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising the compound represented by formula I of the present invention, its stereoisomers, racemates, tautomers, isotope markers, prodrugs, or pharmaceutically acceptable compounds thereof. Accepted salt.
  • the pharmaceutical composition of the present invention further comprises a therapeutically effective amount of the compound represented by formula I, its stereoisomers, racemates, tautomers, and isotope markers of the present invention , Prodrugs or their pharmaceutically acceptable salts and pharmaceutically acceptable carriers.
  • the present invention further provides the use of the compound represented by formula I, its stereoisomers, racemates, tautomers, isotope markers, prodrugs or pharmaceutically acceptable salts thereof in the preparation of IRAK inhibitors.
  • the present invention further provides compounds represented by formula I, their stereoisomers, racemates, tautomers, isotope markers, prodrugs or pharmaceutically acceptable salts thereof in the preparation of prevention and/or treatment of IRAK mediation Use in medicines for leading diseases or conditions.
  • the IRAK-mediated disease or condition is selected from tumor, gout, systemic lupus erythematosus, multiple sclerosis, metabolic syndrome, atherosclerosis, myocardial infarction, sepsis, inflammatory Diseases such as bowel disease, asthma and allergies.
  • the present invention also provides compounds of formula I, their stereoisomers, racemates, tautomers, isotope markers, prodrugs or their pharmaceutically acceptable salts for preparing prevention and/or treatment and leukocyte
  • compounds of formula I their stereoisomers, racemates, tautomers, isotope markers, prodrugs or their pharmaceutically acceptable salts for preparing prevention and/or treatment and leukocyte
  • interleukin-1 receptor related kinase in medicine for diseases or disorders.
  • the present invention also provides a method for the prevention and/or treatment of IRAK-mediated diseases or disorders, comprising administering a therapeutically effective amount of a compound represented by formula I, its stereoisomers, racemates, Tautomers, isotope markers, prodrugs or pharmaceutically acceptable salts thereof, or the pharmaceutical composition.
  • the IRAK is selected from IRAK4-related kinases.
  • the present invention also provides a method for the prevention and/or treatment of interleukin-1 receptor-related diseases, which comprises administering a therapeutically effective amount of a compound represented by formula I, its stereoisomers, and digestive agents to an individual in need thereof.
  • Rotators, tautomers, isotope labels, prodrugs or pharmaceutically acceptable salts thereof, or the pharmaceutical composition are also provided.
  • the disease or disorder related to the interleukin-1 receptor kinase is selected from tumor, gout, systemic lupus erythematosus, multiple sclerosis, metabolic syndrome, atherosclerosis, myocardial infarction , Sepsis, inflammatory bowel disease, asthma, rheumatoid arthritis, sepsis, autoimmune diseases and allergies and other diseases.
  • the method of the present invention may include administering the compound of the present invention alone, as well as administering the compound of the present invention in combination with one or more other chemotherapeutic agents.
  • the administration of multiple drugs can be performed simultaneously or sequentially.
  • halogen refers to F, Cl, Br, and I. In other words, F, Cl, Br, and I can be described as “halogen" in this specification.
  • aliphatic hydrocarbon group includes saturated or unsaturated, linear or branched chain or cyclic hydrocarbon groups.
  • the type of the aliphatic hydrocarbon group can be selected from alkyl, alkenyl, alkynyl, etc.
  • the carbon atoms of the aliphatic hydrocarbon group The number is preferably from 1 to 12, and can also be from 1 to 10, and a further preferred range is from 1 to 6, specifically including but not limited to the following 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-propeny
  • the "aliphatic hydrocarbon group” may optionally include one, two or more heteroatoms (or construed as optional heteroatoms inserted into the aliphatic hydrocarbon group, optionally CC bonds and CH bonds).
  • Suitable heteroatoms are obvious to those skilled in the art and include, for example, sulfur, nitrogen, oxygen, phosphorus, and silicon.
  • the heteroatom-containing aliphatic hydrocarbon group can be selected from the following groups: (C 1 -C 6 ) aliphatic hydrocarbon group oxy group, (C 1 -C 6 ) aliphatic hydrocarbon group mercapto group, (C 1 -C 6 ) aliphatic hydrocarbon group oxygen Group (C 1 -C 6 ) aliphatic hydrocarbon group, (C 1 -C 6 ) aliphatic hydrocarbon group mercapto group (C 1 -C 6 ) aliphatic hydrocarbon group, N-(C 1 -C 3 ) aliphatic hydrocarbon group amine group (C 1 -C 6) ) Aliphatic hydrocarbon group, N,N-bis-(C 1 -C 3 ) aliphatic hydrocarbon amino (C 1 -C 6 ) aliphatic hydrocarbon group, such as methoxy, ethoxy, propoxy, butoxy, Pentyloxy, methoxymethyl, ethoxymethyl, propoxymethyl, methoxye
  • C 3-12 cycloalkyl should be understood to mean a saturated or unsaturated monovalent monocyclic or bicyclic hydrocarbon ring having 3-12 carbon atoms, preferably “C 3-10 cycloalkyl”.
  • C 3-10 cycloalkyl should be 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 group may be a monocyclic hydrocarbon group, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl or cyclodecyl, or a bicyclic group. Hydrocarbon groups such as decalin ring.
  • 3-12 membered heterocyclic group means a saturated or unsaturated monovalent monocyclic or bicyclic ring, which contains 1-5 heteroatoms independently selected from N, O and S, and heteroatom-containing groups do not have Aromatic, the 3-12 membered heterocyclic group is preferably "3-10 membered heterocyclic group".
  • 3-10 membered heterocyclic group means a saturated monovalent monocyclic or bicyclic ring, which contains 1-5, preferably 1-3 heteroatoms selected from N, O and S.
  • the heterocyclic group may be connected to the rest of the molecule through any of the carbon atoms or the nitrogen atom (if present).
  • the heterocyclic group may include but is not limited to: 4-membered ring, such as azetidinyl, oxetanyl; 5-membered ring, such as tetrahydrofuranyl, tetrahydrothienyl, dioxane Pentenyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl; or 6-membered ring, such as tetrahydropyranyl, piperidinyl, morpholinyl, dithiaalkyl, thiomorpholine Group, piperazinyl, or trithiaalkyl; or 7-membered ring, such as diazacycloheptanyl.
  • 4-membered ring such as azetidinyl, oxetanyl
  • 5-membered ring such as tetrahydrofuranyl, tetrahydrothienyl, dioxane Pen
  • the heterocyclic group may be benzo-fused.
  • the heterocyclic group may be bicyclic, such as but not limited to a 5, 5-membered ring, such as hexahydrocyclopenta[c]pyrrole-2(1H)-yl ring, or a 5, 6-membered bicyclic ring, such as hexahydropyrrole And [1,2-a]pyrazine-2(1H)-yl ring.
  • the ring containing the nitrogen atom may be partially unsaturated, that is, it may contain one or more double bonds, such as but not limited to 2,5-dihydro-1H-pyrrolyl, 4H-[1,3,4]thiadi Azinyl, 4,5-dihydrooxazolyl or 4H-[1,4]thiazinyl, or it may be benzo-fused, such as but not limited to dihydroisoquinolinyl.
  • the 3-12 membered heterocyclic group may be further selected from the following groups:
  • C 6-20 aryl should be understood to preferably mean a monovalent aromatic or partially aromatic monocyclic, bicyclic or tricyclic hydrocarbon ring with 6-20 carbon atoms, preferably “C 6-14 aryl” .
  • the term “C 6-14 aryl” should be understood as preferably meaning a monocyclic, bicyclic or partially aromatic monocyclic or partially aromatic monocyclic or partially aromatic having 6, 7, 8, 9, 10, 11, 12, 13 or 14 carbon atoms
  • a tricyclic hydrocarbon ring (“C 6-14 aryl"), especially a ring having 6 carbon atoms (“C 6 aryl”), such as phenyl; or biphenyl, or one having 9 carbon atoms Ring (“C 9 aryl”), such as indanyl or indenyl, or a ring with 10 carbon atoms (“C 10 aryl”), such as tetrahydronaphthyl, dihydronaphthyl or naphthyl, Either a ring having 13 carbon
  • 5-14 membered heteroaryl should be understood to include monovalent monocyclic, bicyclic or tricyclic aromatic ring systems having 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 ring atoms, especially 5 or 6 or 9 or 10 carbon atoms, and it contains 1-5, preferably 1-3 heteroatoms each independently selected from N, O and S and, additionally in each case The bottom can be benzo-fused.
  • the heteroaryl group is selected from thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiol Diazolyl, thio-4H-pyrazolyl, etc.
  • heterocyclic groups or heteroaryl groups include all possible isomeric forms thereof, such as positional isomers thereof. Therefore, for some illustrative non-limiting examples, pyridinyl or pyridinylene includes pyridin-2-yl, pyridin-2-yl, pyridin-3-yl, pyridin-3-yl, pyridin-4-yl And pyridin-4-yl; thienyl or thienylene includes thiophen-2-yl, thiophen-2-yl, thiophen-3-yl, and thiophen-3-yl.
  • the "3-12 membered heterocyclic group” and “5-14 membered heteroaryl group” may further include N-containing 5-12 membered heterocyclic group or 5-14 membered heteroaryl group, that is, containing N-containing 5-12 membered heterocyclic group or 5-14 membered heteroaryl group.
  • the 5-12 membered heterocyclic group or 5-14 membered heteroaryl group of N can be selected from the corresponding groups within the definition of "3-12 membered heterocyclic group” and "5-14 membered heteroaryl".
  • the compound of the present invention may be chiral, and therefore may exist in various enantiomeric forms. Therefore, these compounds may exist in racemate form or optically active form.
  • the compounds of the present invention or intermediates thereof can be separated into enantiomeric compounds by chemical or physical methods known to those skilled in the art, or used in synthesis in this form. In the case of racemic amines, diastereomers are prepared from the mixture by reaction with optically active resolving reagents.
  • Suitable resolution reagents are optically active acids such as R and S forms of tartaric acid, diacetyl tartaric acid, dibenzoyl tartaric acid, mandelic acid, malic acid, lactic acid, appropriate N-protected amino acids (e.g., N- Benzoyl proline or N-benzenesulfonyl proline) or various optically active camphor sulfonic acids.
  • optically active resolving reagents such as dinitrobenzoylphenylglycine, cellulose triacetate or other carbohydrate derivatives or chiral derivatized methacrylate polymers
  • Suitable eluents for this purpose are aqueous or alcohol-containing solvent mixtures, for example, hexane/isopropanol/acetonitrile.
  • the pharmaceutically acceptable salt may be, for example, an acid addition salt of the compound of the present invention that has a nitrogen atom in the chain or ring and is sufficiently basic, for example, an acid addition salt formed with the following inorganic acids: for example, hydrochloric acid, hydrofluorine Acid, hydrobromic acid, hydroiodic acid, sulfuric acid, pyrosulfuric acid, phosphoric acid or nitric acid, or hydrogen sulfate, or acid addition salts formed with the following organic acids: for example, formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid , Propionic acid, butyric acid, caproic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)benzoic acid, camphor acid, cinnamic acid, cyclopentane Propionic acid, digluconic acid, 3-hydroxy-2-nap
  • an alkali metal salt such as a sodium salt or potassium salt
  • an alkaline earth metal salt such as a calcium salt or a magnesium salt
  • an ammonium salt or salts formed with organic bases that provide physiologically acceptable cations, such as salts formed with sodium ions, potassium ions, N-methylglucamine, dimethylglucamine, ethylglucosamine, Lysine, dicyclohexylamine, 1,6-hexanediamine, ethanolamine, glucosamine, meglumine, sarcosine, serinol, trihydroxymethylaminomethane, aminopropanediol, 1-amino-2 ,3,4-Butanetriol.
  • an alkali metal salt such as a sodium salt or potassium salt
  • an alkaline earth metal salt such as a calcium salt or a magnesium salt
  • an ammonium salt or salts formed with organic bases that provide physiologically acceptable cations, such as salts formed with sodium ions, potassium ions, N-methyl
  • the pharmaceutically acceptable salt includes the salt formed by the group -COOH with the following substances: sodium ion, potassium ion, calcium ion, magnesium ion, N-methylglucamine, dimethylglucamine, Ethyl glucosamine, lysine, dicyclohexylamine, 1,6-hexanediamine, ethanolamine, glucosamine, meglumine, sarcosine, serinol, trishydroxymethylaminomethane, aminopropanediol , 1-Amino-2,3,4-butanetriol.
  • basic nitrogen-containing groups can be quaternized with the following reagents: lower alkyl halides, such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dialkyl sulfates, such as sulfuric acid Dimethyl, diethyl sulfate, dibutyl sulfate and dipentyl sulfate; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; aralkyl Halides such as benzyl and phenethyl bromide.
  • lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides
  • dialkyl sulfates such as sulfuric acid Dimethyl, diethyl sulfate, dibutyl sulfate and dipentyl
  • pharmaceutically acceptable salts include hydrochloride, sulfate, nitrate, bisulfate, hydrobromide, acetate, oxalate, citrate, methanesulfonate, formate, or Meglumine salt and so on.
  • the "pharmaceutically acceptable salt” includes not only the salt formed at one salt-forming site of the compound of the present invention, but also 2, 3 or all of them.
  • the salt formed at the salt-forming site can be varied within a relatively large range, for example, it can be 4 :1 ⁇ 1:4, such as 3:1, 2:1, 1:1, 1:2, 1:3, etc.
  • pharmaceutically acceptable anions include anions selected from the ionization of inorganic acids or organic acids.
  • the "inorganic acid” includes, but is not limited to, hydrochloric acid, hydrofluoric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, pyrosulfuric acid, phosphoric acid, or nitric acid.
  • the "organic acid” includes but is not limited to formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, caproic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid , 2-(4-Hydroxybenzoyl)benzoic acid, camphor acid, cinnamic acid, cyclopentane propionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, hexanoic 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,
  • the compounds of the present invention may also contain one or more asymmetric centers.
  • Asymmetric carbon atoms can exist in the (R) or (S) configuration. When there is only one asymmetric center, a racemic mixture is produced, and when multiple asymmetric centers are contained, a diastereomeric mixture is obtained. In some cases, there may be asymmetry due to hindered rotation around a specific bond, for example, the central bond connects two substituted aromatic rings of a specific compound.
  • the substituent may also exist in a cis- or trans-isomeric form.
  • the compounds of the present invention also include all possible stereoisomers of each, which is a single stereoisomer or the stereoisomer (for example, R-isomer or S-isomer, or E-isomer or Z-isomer) in the form of any mixture in any ratio.
  • a single stereoisomer (e.g., single enantiomer or single diastereomer) of the compound of the present invention can be achieved by any suitable prior art method (e.g., chromatography, especially, e.g., chiral chromatography) Separation.
  • tautomer refers to an isomer of a functional group resulting from the rapid movement of an atom in a molecule at two positions.
  • the compounds of the present invention may exhibit tautomerism.
  • Tautomeric compounds can exist in two or more mutually convertible species.
  • Proton shift tautomers result from the migration of covalently bonded hydrogen atoms between two atoms.
  • Tautomers generally exist in an equilibrium form. An attempt to separate a single tautomer usually produces a mixture whose physical and chemical properties are consistent with a mixture of compounds. The position of equilibrium depends on the chemical properties of the molecule.
  • the ketone type is dominant; in phenol, the enol type is dominant.
  • the present invention encompasses all tautomeric forms of the compound.
  • the involved compounds also include isotopically-labeled compounds.
  • the isotopically-labeled compounds are the same as those shown in Formula I, but one or more of the atoms are different from the usual atomic mass or mass number. Naturally occurring atomic mass or mass number atomic substitution.
  • isotopes that can be incorporated into the compounds of the present invention include isotopes of H, C, N, O, S, F, and Cl, such as 2 H, 3 H, 13 C, 11 C, 14 C, 15 N, 18 O, respectively. , 17 O, 32 P, 35 S, 18 F and 36 Cl.
  • the compounds of the present invention containing the above-mentioned isotopes and/or other isotopes of other atoms, prodrugs thereof, or pharmaceutically acceptable salts of the compounds or prodrugs are within the scope of the present invention.
  • the replacement of heavier isotopes can provide certain therapeutic advantages derived from higher metabolic stability (for example, increased in vivo half-life or reduced dosage requirements), and therefore can be used in certain Some cases are preferred.
  • the compounds of the present invention as claimed in the claims can be specifically limited to be substituted with deuterium or tritium.
  • the absence of the term deuterium or tritium in the hydrogen appearing in the substituent does not mean that deuterium or tritium is excluded, but deuterium or tritium may also be included in the same way.
  • the term "effective amount” or “therapeutically effective amount” refers to the amount of the compound of the present invention sufficient to achieve the intended application (including but not limited to the treatment of diseases as defined below).
  • the therapeutically effective amount may vary depending on the following factors: the intended application (in vitro or in vivo), or the subject to be treated and the disease condition such as the weight and age of the subject, the severity of the disease condition and the mode of administration, etc. It can be easily determined by a person of ordinary skill in the art.
  • the specific dosage will vary depending on the following factors: the particular compound selected, the dosing regimen on which it is based, whether it is administered in combination with other compounds, the timing of administration, the tissue to be administered, and the physical delivery system carried.
  • excipients refers to pharmaceutically acceptable inert ingredients.
  • examples of the types of excipients include, without limitation, binders, disintegrants, lubricants, glidants, stabilizers, fillers, diluents, and the like. Excipients can enhance the handling characteristics of the pharmaceutical preparation, that is, make the preparation more suitable for direct compression by increasing fluidity and/or adhesion.
  • Examples of typical pharmaceutically acceptable carriers suitable for the above formulations are: sugars, such as lactose, sucrose, mannitol, and sorbitol; starches, such as corn starch, tapioca starch, and potato starch; cellulose and its derivatives, For example, 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; hard Fatty acid alkaline earth metal salts, 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; B Glycol polymers; fatty alcohols; and grain hydrolyzed solids and other non-toxic compatible fillers, binders, dis
  • solvate refers to those forms of the compound of the present invention, which form a complex by coordination with solvent molecules in a solid or liquid state. Hydrates are a specific form of solvates in which the coordination is carried out with water. In the present invention, the preferred solvate is a hydrate. Further, the pharmaceutically acceptable solvate (hydrate) of the compound of the general formula I of the present invention refers to the co-crystal and clathrate formed by the compound I and one or more stoichiometric molecules of water or other solvents. Solvents that can be used for solvates include, but are not limited to: water, methanol, ethanol, ethylene glycol, and acetic acid.
  • prodrug represents the conversion of a compound into a compound represented by the aforementioned general formula or specific compound in vivo. Such conversion is affected by the hydrolysis of the prodrug in the blood or the enzymatic conversion of the prodrug into the maternal structure in the blood or tissues.
  • the prodrugs of the present invention can be esters.
  • esters can be used as prodrugs including phenyl esters, aliphatic (C1-24) esters, acyloxymethyl esters, carbonates, carbamates and amino acids. Esters.
  • a compound in the present invention contains a hydroxyl/carboxyl group, that is, it can be acylated to obtain a compound in the form of a prodrug.
  • Other prodrug forms include phosphate esters, for example, these phosphate ester compounds are obtained by phosphorylation of the parent hydroxyl group.
  • the present invention provides a compound of general formula I with a novel structure. It has been verified by experiments that the compound of the present invention has a significant inhibitory effect on the activity of IRAK4 kinase, and compared with other kinases, it has a good selective inhibitory effect on the activity of IRAK4 kinase. effect;
  • the compound of the present invention has good medication safety, wide applicability and low toxic and side effects.
  • Experimental verification shows that the compound of the present invention has a very low inhibitory rate on human hERG, and has no obvious time-dependent inhibition of human CYP3A4.
  • the plasma protein binding rate of human, rat, and mouse is moderate, and the difference between species is small. At the same time, it has no obvious inhibitory effect on the five human CYP subtypes;
  • the compound of the present invention has a significant inhibitory effect on the release of TNF- ⁇ from Balb/c female mice induced by LPS;
  • the compound of the present invention has good pharmacokinetic characteristics, shows good exposure and retention time in animals, and has a suitable half-life and good drug absorption.
  • the raw materials and reagents used in the following examples are all commercially available products, or can be prepared by known methods.
  • a mixed solution of nitric acid (1.6 mL, 70%) and 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 minus 15 degrees Celsius.
  • the mixed system was stirred at minus 15 degrees Celsius for 2 hours, the reaction solution was slowly poured into ice water and stirred for 5 minutes, filtered with suction, washed with water, and the solid was collected and dried under reduced pressure to obtain compound 8 (2.5 g, yield: 97%).
  • Compound 010 is 2-((2-(trans-4-hydroxy-cis-4-methylcyclohexyl)-6-dimethylamino-2H-indazol-5-yl)carbamoyl) Synthesis of -6-methylpyridine 1-oxide
  • a mixed solution of nitric acid (1.6 mL, 70%) and 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 minus 15 degrees Celsius.
  • the mixed system was stirred at minus 15 degrees Celsius for 2 hours, the reaction solution was slowly poured into ice water and stirred for 5 minutes, filtered with suction, washed with water, and the solid was collected and dried under reduced pressure to obtain compound 2 (2.5 g, yield: 97%).
  • Compound 016 is 2-((2-(trans-4-hydroxycyclohexyl)-6-ethoxy-2H-indazol-5-yl)carbamoyl)-6-picoline 1-oxidation
  • Compound 220 is 2-((2-(cis-4-hydroxycyclohexyl)-6-ethoxy-2H-indazol-5-yl)carbamoyl)-6-picoline 1-oxidation
  • Compound 025 is 2-((2-(trans-4-hydroxy-cis-4-cyclopropylcyclohexyl)-6-methoxy-2H-indazol-5-yl)carbamoyl) Synthesis of -6-methylpyridine 1-oxide
  • Compound 163 is 2-((2-(3-hydroxy-3-methylbutyl)-6-methoxy-2H-indazol-5-yl)carbamoyl)-6-methylpyridine 1 -Synthesis of oxides
  • HATU (273mg) and Et 3 N (145mg) were added to the DMF (2mL) solution of compound 5 (120mg) and compound 6 (81mg) at 30 degrees Celsius, and the reaction was stirred at 30 degrees Celsius for 18 hours.
  • Compound 284 is the synthesis of 2-((2-cyclopentyl-6-methoxy-2H-indazol-5-yl)carbamoyl)-6-picoline 1-oxide
  • Compound 286 is 2-((2-(4,4-difluorocyclohexyl)-6-methoxy-2H-indazol-5-yl)carbamoyl)-6-picoline 1-oxidation Synthesis
  • Compound 287 is 2-((2-(cis-4-hydroxy-trans-4-methylcyclohexyl)-6-dimethylamino-2H-indazol-5-yl)carbamoyl) Synthesis of -6-methylpyridine 1-oxide
  • Compound 015 is 2-((2-(trans-4-hydroxycyclohexyl)-6-methoxy-2H-indazol-5-yl)carbamoyl)-6-isopropylpyridine 1- Oxide and compound 288 namely 2-((2-(cis-4-hydroxycyclohexyl)-6-methoxy-2H-indazol-5-yl)carbamoyl)-6-isopropylpyridine 1 -Synthesis of oxides
  • Compound 014 is 2-((2-(trans-4-hydroxycyclohexyl)-6-methoxy-2H-indazol-5-yl)carbamoyl)-6-cyclopropylpyridine 1- Oxide and compound 218 namely 2-((2-(cis-4-hydroxycyclohexyl)-6-methoxy-2H-indazol-5-yl)carbamoyl)-6-cyclopropylpyridine 1 -Synthesis of oxides
  • Compound 019 is 2-((2-(trans-4-hydroxycyclohexyl)-6-cyclopropylmethoxy-2H-indazol-5-yl)carbamoyl)-6-picoline 1 -Oxide and compound 292 namely 2-((2-(cis-4-hydroxycyclohexyl)-6-cyclopropylmethoxy-2H-indazol-5-yl)carbamoyl)-6-methyl Synthesis of pyridine 1-oxide
  • Compound 291 is 2-((2-(trans-4-cyano-cis-4-methylcyclohexyl)-6-methoxy-2H-indazol-5-yl)carbamoyl) Synthesis of -6-methylpyridine 1-oxide
  • Compound 002 is 2-((2-(trans-4-hydroxy-cis-4-methylcyclohexyl)-6-methoxy-2H-indazol-5-yl)carbamoyl)- Synthesis of 6-cyclopropylpyridine 1-oxide
  • Potassium hydroxide (4.58g) was added to the ethanol/water (40mL/10mL) solution of compound 3 (1.3g), and the reaction was stirred at 90 degrees Celsius for 16 hours.
  • the pH was adjusted to 6 with 1M dilute hydrochloric acid and ethyl acetate (100 mL ⁇ 3), the extract was washed with water (50 mL), saturated brine (100 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain compound 4 (1.12 g, yield 77%).
  • Acetic acid (30.6g) was added to the ethanol solution (350 mL) of compound 9 (0.17mol) at 25°C, compound 8 (22g) was stirred at 25°C for 10 minutes, and refluxed at 80°C for 16 hours. After the reaction was completed, the reaction solution was partially concentrated. Add water (70mL) for beating, filter to obtain a solid, add ethanol (200mL) and heat to reflux to dissolve the solid, cool to room temperature, add n-heptane (200mL) for beating for 2 hours, filter two steps to obtain compound 7 (35g, yield 67% ).
  • Compound 175 is 2-((2-(trans-4-hydroxy-cis-4-ethylcyanocyclohexyl)-6-methoxy-2H-indazol-5-yl)carbamoyl) Synthesis of -6-methylpyridine 1-oxide
  • Compound 176 is 2-((2-(trans-4-hydroxy-cis-4-ethylcyanocyclohexyl)-6-methoxy-2H-indazol-5-yl)carbamoyl) Synthesis of 6-Cyclopropylpyridine 1-oxide
  • Compound 042 is 2-((2-(trans-4-hydroxy-cis-4-methylcyclohexyl)-6-methoxy-2H-indazol-5-yl)carbamoyl)- Synthesis of 6-Methoxypyridine 1-oxide
  • Example 21 Compound B is 2-((2-(trans-4-hydroxy-cis-4-methylcyclohexyl)-6-methoxy-2H-indazol-5-yl)carbamoyl) Synthesis of -6-methylpyridine
  • test examples are used to further explain the present invention, but these test examples are not meant to limit the scope of the present invention.
  • Compound B in the biological test example is the structure synthesized in Example 21
  • Test Example 1 Determination of the inhibitory effect of the compound of the present invention on the activity of human IRAK4 kinase
  • the inhibitory activity of the compound against IRAK4 at the Km concentration of ATP was measured in the IRAK4MSA (Mobility-Shift Assay, mobility detection technology of microfluidic chip technology) described below.
  • the final concentration is 1nM; ATP ATP (Sigma, Product Number: A7699-1G) The final concentration is 37 ⁇ M; the substrate used for the kinase reaction is 5-FAM (5-carboxyfluorescein) labeled polypeptide (5-FAM-IPTSPITTTYFFFKKK-COOH), the substrate peptide FAM- P8 (GL Biochem, article number: 112396), the final concentration is 5 ⁇ M.
  • a 500 ⁇ M compound solution was prepared with 100% DMSO, and 10 concentration gradients were diluted 4-fold with 100% DMSO, and then further diluted 10-fold with compound buffer (50mM HEPES, pH 7.5, 0.00015% Brij-35) , Prepare the compound intermediate dilution solution containing 10% DMSO, the final concentration of the compound is in the range of 10 ⁇ M-0.04nM, and transfer 5 ⁇ l to a black 384-well plate.
  • compound buffer 50mM HEPES, pH 7.5, 0.00015% Brij-35
  • the kinase IRAK4 was diluted with kinase buffer (50mM HEPES, pH 7.5, 0.00015% Brij-35, 2mM DTT) into a 2.5nM IRAK4 solution, and transferred 10 ⁇ l to a 384-well plate, and incubated with the compound for 10-15 minutes.
  • kinase buffer 50mM HEPES, pH 7.5, 0.00015% Brij-35, 2mM DTT
  • reaction buffer 50 mM HEPES, pH 7.5, 0.00015% Brij-35, 10 mM MgCl 2
  • Reading Caliper EZ Reader (PerkinElmer) conversion of phosphorylated substrate, so as to calculate the inhibition rate of the compound of IRAK4, IC 50 was calculated with XL-fit software.
  • test results show that the compounds of the examples of the present invention have a good inhibitory effect on IRAK4 kinase activity, and the IC 50 value is less than 100 nM, preferably less than 30 nM.
  • some exemplary compound activity values are as follows:
  • the inhibitory IC 50 value of the compound of the present invention on human IRAK4 kinase activity is shown in Table 1.
  • Compound ID IC 50 (nM) Compound B 30.0 001 6.0 002 3.7 010 11.0 013 7.6 014 2.5 015 5.4 016 8.1 019 8.1 025 13.0 163 23.0 175 7.1 176 4.3 187 9.2 218 8.3 220 14.0 284 28.0 285 5.9 286 14.0 287 13.0 288 13.0 289 1.7 291 14.0
  • Test Example 2 Determination of the inhibitory effect of the compound of the present invention on human IRAK1 kinase activity
  • Test Example 1 This test is used to evaluate the inhibitory effect of compounds on human IRAK1 kinase activity.
  • the main test materials are the same as those in Test Example 1.
  • the inhibitory activity of the compound against IRAK1 at the Km concentration of ATP was measured in the IRAK1 MSA (Mobility-Shift Assay, mobility detection technology of microfluidic chip technology) described below.
  • IRAK1 MSA Mobility-Shift Assay, mobility detection technology of microfluidic chip technology
  • N-terminal GST glutathione-S-transferase
  • human IRAK1 as the enzyme (GST-IRAK1, kinase IRAK1, Carna)
  • the final concentration is 3nM
  • the final concentration of ATP (Sigma) is 97 ⁇ M
  • the substrate used for the kinase reaction is 5-FAM (5-carboxyfluorescein) labeled polypeptide (5-FAM-IPTSPITTTYFFFKKK-COOH), and the substrate peptide FAM-P8 (GL Biochem), with a final concentration of 5 ⁇ M.
  • a 500 ⁇ M compound solution was prepared with 100% DMSO, and 10 concentration gradients were diluted 4-fold with 100% DMSO, and then further diluted 10-fold with compound buffer (50mM HEPES, pH 7.5, 0.00015% Brij-35) , Prepare the compound intermediate dilution solution containing 10% DMSO, the final concentration of the compound is in the range of 10 ⁇ M-0.04nM, and transfer 5 ⁇ l to a black 384-well plate.
  • compound buffer 50mM HEPES, pH 7.5, 0.00015% Brij-35
  • the kinase IRAK1 was diluted with kinase buffer (50mM HEPES, pH 7.5, 0.00015% Brij-35, 2mM DTT) into a 7.5nM IRAK1 solution, and transferred 10 ⁇ l to a 384-well plate, and incubated with the compound for 10-15 minutes.
  • kinase buffer 50mM HEPES, pH 7.5, 0.00015% Brij-35, 2mM DTT
  • reaction buffer 50mM HEPES, pH 7.5, 0.00015% Brij-35, 10mM MgCl2
  • Use Caliper EZ Reader PerkinElmer to read the conversion rate of substrate phosphorylation to calculate the compound's inhibition rate of IRAK1, and use XL-fit software to calculate IC50.
  • test results show that the compounds of the examples of the present invention have significant selective inhibitory activity against IRAK4, and the IC50 (nM) ratio of IRAK1 to IRAK4 is greater than 500, preferably greater than 200.
  • IC50 (nM) ratio of IRAK1 to IRAK4 is greater than 500, preferably greater than 200.
  • some exemplary compound activity values are as follows: The inhibitory IC 50 values of the compounds of the present invention on human IRAK1 kinase activity are shown in Table 2.
  • the HEK-293 cell line stably expressing the hERG potassium channel was used for experimental detection.
  • Amplifier purchased from HEKA (Germany), model EPC10
  • Micromanipulator purchased from Sutter Instruments (USA), model MP225
  • Electrode drawing instrument purchased from Sutter Instruments (USA), model P97
  • Microscope purchased from Nikon, model TE300
  • Capillary glass tube purchased from Sutter Instruments (USA), model BF150-86-10
  • Patch clamp detection Under an inverted microscope, manipulate the glass electrode micromanipulator (micromanipulation) to contact the recording electrode with the cell, and then give a negative pressure to promote the formation of a G ⁇ seal on the cell. After forming the G ⁇ seal, perform fast capacitance compensation, and then continue to give negative pressure to suck through the cell membrane to form a whole-cell recording mode. In the whole cell recording mode, perform slow capacitance compensation and record the values of membrane capacitance and series resistance.
  • the voltage stimulation scheme of the cell hERG potassium current is as follows: the cell membrane clamp voltage is -80mV, then it is depolarized from -80mV to +30mV for 2.5 seconds, and then quickly maintained at -50mV for 4 seconds to stimulate the tail current of the hERG channel. Collect data repeatedly every 10 seconds. Use -50mV as leakage current detection.
  • C represents the concentration of the test compound, IC 50 inhibition concentration representatives, h for Hill coefficient.
  • the curve fitting and the calculation of IC 50 are done using GraphPad Prism 5.0 software.
  • the test results show that the compound of the embodiment of the present invention has a very low inhibitory rate on human hERG, and may even be significantly better than that of the comparative compound A.
  • the hERG inhibition (30 ⁇ M) is less than 50%, preferably less than 30%.
  • some exemplary compound inhibition rate values are as follows:
  • Test Example 4 Determination of TDI (Time Dependent Inhibition) data of the compound of the present invention
  • the purpose of this experimental study is to study the time-dependent inhibitory effect of the compound on the human P450 metabolizing enzyme CYP3A4.
  • the human mixed liver microsomes used in this experiment are from Corning Corporation.
  • the test compound will be incubated with human liver microsomes and the probe substrate midazolam (CYP3A4), and the test compound will be set to 30 ⁇ M.
  • the reaction will be initiated by the addition of the coenzyme NADPH.
  • the internal standard has been dissolved in the acetonitrile in advance.
  • the supernatant is collected by centrifugation.
  • the characteristic metabolite 1-hydroxy-midazolam (CYP3A4) in the supernatant was analyzed by the LC-MS/MS method.
  • Selective inhibitors (Verapamil vs. CYP3A4) will serve as a positive control.
  • test results show that the compounds of the examples of the present invention have no obvious time-dependent inhibition of human CYP3A4.
  • the TDI values of some exemplary compounds are as follows:
  • the purpose of this test is to determine the plasma protein binding rate (PPB) data of the test compound.
  • the concentration of the test compound or reference compound in the final dosing matrix is 1 ⁇ M and the DMSO content is 0.2%.
  • Collect the 0-hour sample add 25 ⁇ L of the compound-containing matrix to a blank 96-well collection plate and store at -20°C.
  • sample preparation At the end of the culture (5 hours), sample preparation:
  • test compound and reference compound dosing side samples take 25 ⁇ L of dosing side sample, add 25 ⁇ L of blank buffer solution and mix. Add 200 ⁇ L of ACN containing internal standard, shake at 600 rpm for 10 minutes, and then centrifuge at 5594 g for 15 minutes on a centrifuge.
  • 0-hour samples test compound and reference compound. 0-hour samples: remelted at 37°C and mixed with the same volume (25 ⁇ L) of the corresponding matrix (blank buffer solution). Add 200 ⁇ L of ACN containing internal standard, shake at 600 rpm for 10 minutes, and then centrifuge at 5594 g for 15 minutes on a centrifuge.
  • the test results show that the compound of the present invention has a moderate binding rate to plasma proteins of humans, rats, and mice, and the difference between species is very small, and may even be significantly smaller than that of comparative compound A.
  • the PPB of some exemplary compounds The data is as follows:
  • Test Example 6 The inhibitory effect of the compound of the present invention on the release of TNF- ⁇ from Balb/c female mice induced by LPS
  • the female Balb/c mice were randomly divided into several groups, each with 4 mice.
  • the groups included normal control + vehicle group, model + vehicle group, model + positive drug group, and other model + test drug groups.
  • Normal control animals received intraperitoneal injection of normal saline (10ml/kg), and model animals received LPS stimulation (Sigma product number L2630, intraperitoneal injection, 10mL/kg, 0.2mg/kg).
  • LPS stimulation Sigma product number L2630, intraperitoneal injection, 10mL/kg, 0.2mg/kg.
  • the test drug was sequentially added with DMSO, Solutol, and 10mM PBS to make a solution or turbid solution with the required concentration of administration.
  • the final volume ratio of each component of the solvent DMSO, Solutol, and 10mM PBS was 5:15:80.
  • Each experimental group was given corresponding intragastric administration (10ml/kg) at the set dose 16h before LPS (or saline) stimulation.
  • Animals in each group were euthanized with CO2 1.5h after stimulation, and blood was collected from the heart. The obtained whole blood is not anticoagulant.
  • the quantification of TNF ⁇ is determined by the TNF ⁇ ELISA kit, according to the manufacturer's instructions.
  • A450 absorbance readings using a microplate reader SpectraMax i3x (Molecular Device) detector to calculate the inhibition rate of the compound, IC 50 was calculated using GraphPad Prism 7.0 software.
  • the test results show that the compounds of the examples of the present invention have a significant inhibitory effect on the release of TNF- ⁇ from Balb/c female mice induced by LPS, and the inhibition rate is greater than 50%, preferably greater than 70%, specifically, some exemplary compounds
  • the inhibition rate is as follows:
  • Test Example 7 Determining the inhibition of the compound of the present invention on the five main CYP450 enzyme subtypes of human liver microsomes
  • the purpose of this experimental study is to study the inhibitory effects of the test compounds on the five main human P450 metabolic enzymes, namely CYP1A2, 2C9, 2C19, 2D6 and 3A4-M.
  • the human mixed liver microsomes used in this experiment are from Corning Corporation.
  • the test compound (Compound 14) will interact with human liver microsomes and five probe substrates (phenacetin for CYP1A2, diclofenac for CYP2C9, mephenytoin for CYP2C19, dextromethorphan for CYP2D6, and midazolam for CYP2C9.
  • CYP3A4-M is a mixed substrate) for co-incubation (see the table below), and 7 concentration points will be set for the test compound.
  • the reaction will be initiated by the addition of the coenzyme NADPH. Add ice acetonitrile containing internal standard to the incubation system to stop the reaction. After the protein is precipitated, the supernatant is collected by centrifugation.
  • Characteristic metabolites in the supernatant acetaminophen versus CYP1A2, 4-hydroxydiclofenac versus CYP2C9, 4-hydroxymephenytoin versus CYP2C19, dextrorphan versus CYP2D6, 1-hydroxy-midazolam versus CYP3A4 -M
  • acetaminophen versus CYP1A2, 4-hydroxydiclofenac versus CYP2C9, 4-hydroxymephenytoin versus CYP2C19, dextrorphan versus CYP2D6, 1-hydroxy-midazolam versus CYP3A4 -M Analyzed by LC-MS/MS method.
  • the selective inhibitor Kertoconazole vs. CYP3A4-M
  • All incubations were performed in parallel.
  • the test results show that the compound of the example of the present invention has no obvious inhibitory effect on the five human CYP subtypes, and the inhibitory effect on the three subtypes 1A2, 2C9, and 2C19 is significantly less than that of the comparative compound A.
  • some The inhibition rates of exemplary compounds are as follows:
  • Test Example 8 PK analysis test of the compound of the present invention on rats
  • mice pharmacokinetics test of the preferred embodiment of the present invention was carried out using male SPF-grade SD rats (Shanghai Xipuer-Bikai Experimental Animal Co., Ltd.).
  • Administration method single oral administration or single intravenous injection
  • Sample processing 0.2 mL of venous blood was collected, the blood sample was placed on ice after collection, and plasma was separated by centrifugation (centrifugation conditions: 8000 rpm, 6 minutes, 4°C). The collected plasma was stored at -80°C before analysis.
  • Internal standard working solution Pipette a certain amount of tolbutamide internal standard stock solution with a concentration of 645,000ng/mL into a volumetric flask, dilute to the mark with methanol and mix well to obtain a concentration of 50ng/mL Internal standard working solution.
  • Sample pretreatment Take 50 ⁇ L of plasma sample into a 1.5mL centrifuge tube, add 250 ⁇ L of internal standard solution (blank without internal standard supplemented with the same volume of methanol), vortex to mix, centrifuge at 14,000 rpm for 5 minutes, and take 200 ⁇ L on top The clear solution was added to the 96-well sample injection plate, and the LC-MS/MS sample was injected and analyzed.
  • Mobile phase A solution is 0.1% formic acid aqueous solution, B solution is 0.1% formic acid acetonitrile solution
  • the data processing system is Analyst software (American Applied Biosystems, software version number 1.5.5).
  • test results show that the compounds of the examples of the present invention all show good pharmacokinetic characteristics in mice, show good exposure and retention time in animals, and have suitable half-life and good drug absorption. Yes, the pharmacokinetic data of some exemplary compounds are shown below:

Abstract

公开了一种式I所示的化合物、其立体异构体、消旋体、互变异构体、同位素标记物、前药或其药学上可接受的盐，以及包含其的药物组合物，其制备方法，及其医药用途，所述结构如式I。

Description

一种IRAK抑制剂及其制备方法和用途
本申请要求2019年9月24日向中国国家知识产权局提交的，专利申请号为201910906833.7，发明名称为“一种IRAK抑制剂及其制备方法和用途”的在先申请的优先权。所述申请的全文通过引用的方式结合于本申请中。
技术领域
本发明涉及药物化学领域，具体的涉及一种适用于治疗癌症和与白细胞介素-1受体相关激酶(IRAK)相关的炎性疾病的化合物，并且更具体地是调节IRAK-4的功能的化合物。
背景技术
白细胞介素-1受体相关激酶(IRAK)是存在于细胞内的一类丝/苏氨酸蛋白激酶家族，有四个成员：IRAK1，IRAK2，IRAK-M和IRAK4，共同特征是具有典型的N-末端死亡结构域，该结构域介导与MyD88-家族衔接蛋白和位于中心的激酶结构域之间的相互作用，其中IRAK1和IRAK4具有激酶活性。IRAK4是Toll样受体(TLR)/白介素-1受体(IL-1R)介导的炎症信号转导通路下游的关键因子，TLR细胞外部分识别病原特异性分子(如脂多糖，多肽，病毒DNA等)，与配体结合后，细胞内部分招募MyD88等形成复合体，激活IRAK1自磷酸化，进而活化下游丝氨酸/苏氨酸激酶TAK1，激活NF-κB及MAPK信号通路，随后产生促炎细胞因子、趋化因子和破坏性酶，最终导致产生炎症反应，介导先天性免疫。IL-1R参与宿主防御和造血，是连接先天免疫和获得性免疫的桥梁。(Flannery,et.al.Biochem.Pharmacol.,2010,80(12):1981-1991)。
类风湿性关节炎(rheumatoid arthritis,RA)是一种慢性、炎性、系统性的自身免疫性疾病,以关节和关节组织非化脓性炎症为主要特征,主要表现为关节滑膜炎,终致关节的软骨、韧带、肌腱等各种组织以及多脏器损害。研究显示，在RA患者中有多种免疫细胞参与并介导了自身免疫性炎症，其中包括T/B淋巴细胞、巨噬细胞、中性粒细胞等。同时也有大量研究证明细胞因子与RA疾病直接关联，如白介素类(IL-1/IL-6等)，TNF-α等。
研究表明，在LPS或CpG诱导的人白细胞中，IRAK4抑制剂能够有效地阻断促炎细胞因子肿瘤坏死因子(TNF)的产生；在胶原蛋白诱导关节炎的小鼠模型中，IRAK4抑制剂能够显著抑制TNF的释放，从而控制疾病的进程；在MyD88依赖性炎症性痛风小鼠模型中，IRAK4抑制剂能够剂量依赖性地阻断白细胞浸润(Priscilla N,et.al.J.Exp.Med.,2015,13(212):2189-2201)。
因此可以认为，IRAK4依赖性的TLR/IL-1R信号通路的过度激活与类风湿性关节炎发生发展密切相关，另多项研究也证实，IRAK4酶活化与以下疾病的发生发展密切相关，如肿瘤、痛风、系统性红斑狼疮、多发性硬化症、代谢综合症、动脉粥样硬化、心肌梗死、脓血症、炎症性肠病、哮喘和过敏等疾病(Chaudhary D,et.al.,J.Med.Chem.2015,58(1):96-110)。
发明内容
为解决现有技术中存在的问题，本发明提供如下式I所示的化合物、其立体异构体、消旋体、互变异构体、同位素标记物、前药或其药学上可接受的盐：
Figure PCTCN2020117093-appb-000001
其中，
环A为含有至少一个含N的5-14元杂芳基或5-12元杂环基；
每个R 1、R 2、R 3各自独立的选自氢、卤素、CN、OH或任选被一个、两个或更多个R取代的如下基团：(C 1-C 12)脂肪烃基，任选地包含一个、两个或更多个杂原子(C 1-C 12)脂肪烃基，C 3-12环烷基、3-12元杂环基、C 6-20芳基或5-14元杂芳基、-NR aR b；
W选自O，S，NH，单键；
每个R a、R b独立的选自H、(C 1-C 12)脂肪烃基；
每个R独立的选自卤素、CN、OH、SH、NR aR b或选自任选被一个、两个或更多个R’取代的如下基团：(C 1-C 12)脂肪烃基，任选地包含一个、两个或更多个杂原子(C 1-C 12)脂肪烃基，C 3-12环烷基、3-12元杂环基、C 6-20芳基或5-14元杂芳基；
每个R’独立的选自卤素、CN、OH、SH、NR aR b；
n选自1、2、3；m选自1、2、3、4、5、6。根据本发明的实施方案，
所述“任选地包含一个、两个或更多个杂原子的(C 1-C 12)脂肪烃基”可以选自(C 1-C 12)脂肪烃基氧基、(C 1-C 12)脂肪烃基巯基，(C 1-C 6)脂肪烃基氧基(C 1-C 6)脂肪烃基、(C 1-C 6)脂肪烃基巯基(C 1-C 6)脂肪烃基、N-(C 1-C 3)脂肪烃基胺基(C 1-C 6)脂肪烃基、N,N-二-(C 1-C 3)脂肪烃基胺基(C 1-C 6)脂肪烃基；
所述“含有至少一个含N的5-14元杂芳基或5-12元杂环基”是指所述杂芳基或杂环基中至少含有一个氮原子，还可以含有其他选自N、O、或S的一个或多个杂原子，例如选自吡啶，吡咯，哌啶或四氢吡咯等。
所述(C 1-C 12)脂肪烃基可以选自(C 1-C 12)烷基、(C 2-C 12)烯基、(C 2-C 12)炔基，优选的，所述(C 1-C 12)脂肪烃基可以选自(C 1-C 6)烷基、(C 2-C 6)烯基、(C 2-C 6)炔基；
所述“卤素”选自F、Cl、Br、I；
所述“C 3-12环烷基”可以选自环丙基、环丁基、环戊基或环己基。
根据本发明的实施方案，
所述R 1、R 2、R 3可以各自独立的选自任选被一个、两个或更多个R取代的如下基团：甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、正戊基、异戊基、新戊基、正己基、乙烯基、1-丙烯基、2-丙烯基、1-甲基乙烯基、1-丁烯基、1-乙基乙烯基、1-甲基-2-丙烯基、2-丁烯基、3-丁烯基、2-甲基-1-丙烯基、2-甲基-2-丙烯基、1-戊烯基、1-己烯基、乙炔基，1-丙炔基、2-丙炔基、1-丁炔基、1-甲基-2-丙炔基、3-丁炔基、1-戊炔基、1-己炔基、环丙基、环丁基、环戊基、环己基、甲氧基，乙氧基，丙氧基，丁氧基，戊氧基，甲氧基甲基，乙氧 基甲基、丙氧基甲基、甲氧基乙基、乙氧基乙基、丙氧基乙基、甲氧基丙基、乙氧基丙基、丙氧基丙基、N-甲基胺甲基、N-甲基胺乙基、N-乙基胺乙基、N,N-二甲基胺甲基、N,N-二甲基胺乙基、N,N-二乙基胺乙基、氨基、N,N-二甲基氨基、N,N-二乙基氨基、四氢吡咯基、哌啶基、吡啶基，吡嗪基、吡咯基，咪唑基、吡唑基、恶唑基、异恶唑基、
Figure PCTCN2020117093-appb-000002
所述
Figure PCTCN2020117093-appb-000003
示意所述基团的连接位点。
根据本发明的实施方案，所述式I所示的化合物、其立体异构体、消旋体、互变异构体、同位素标记物、前药或其药学上可接受的盐中，式I所示的化合物可以选自如下式Ia、式Ib、式Ic、式Id、式Ie结构：
Figure PCTCN2020117093-appb-000004
所述式Ia、式Ib、式Ic、式Id、式Ie中，R 1，R 2，R 3，m，n，W如式I所定义。
根据本发明的实施方案，所述式I所示的化合物、其立体异构体、消旋体、互变异构体、 同位素标记物、前药或其药学上可接受的盐中，式I所示的化合物可以选自如下结构：
Figure PCTCN2020117093-appb-000005
Figure PCTCN2020117093-appb-000006
Figure PCTCN2020117093-appb-000007
Figure PCTCN2020117093-appb-000008
Figure PCTCN2020117093-appb-000009
Figure PCTCN2020117093-appb-000010
Figure PCTCN2020117093-appb-000011
Figure PCTCN2020117093-appb-000012
Figure PCTCN2020117093-appb-000013
Figure PCTCN2020117093-appb-000014
Figure PCTCN2020117093-appb-000015
Figure PCTCN2020117093-appb-000016
Figure PCTCN2020117093-appb-000017
Figure PCTCN2020117093-appb-000018
Figure PCTCN2020117093-appb-000019
Figure PCTCN2020117093-appb-000020
Figure PCTCN2020117093-appb-000021
Figure PCTCN2020117093-appb-000022
Figure PCTCN2020117093-appb-000023
Figure PCTCN2020117093-appb-000024
Figure PCTCN2020117093-appb-000025
本发明还提供所述式I所示的化合物(包括式Ia-式Ie)、其立体异构体、消旋体、互变异构体、同位素标记物、前药或其药学上可接受的盐的制备方法，但不仅限于以下描述的方法。
在一些实施方式中，所述制备方法可以包括如下步骤：
Figure PCTCN2020117093-appb-000026
(a1)采用M-1与M-2生成M-3；所述反应可以在EDCl.HCl、吡啶存在条件下进行；
(a2)M-3与R xL 1反应，其中R x选自R 1或带有羟基的R 1中羟基被
Figure PCTCN2020117093-appb-000027
取代的基团；且当R x选自带有羟基的R 1中羟基被
Figure PCTCN2020117093-appb-000028
取代的基团时，反应需进一步包括经酸性、还原条件得到式I产品，所述酸性条件可以选自HCl，还原条件可以选自硼氢化钠；
所述步骤中，R 1，R 2，R 3，m，W如式I所定义；所述L 1为离去基团，可以选自卤素或OTs。
在一些实施方式中，所述制备方法可以包括如下步骤：
Figure PCTCN2020117093-appb-000029
(b1)N-1与R xL 1反应，其中R x选自R 1或带有羟基的R 1中羟基被
Figure PCTCN2020117093-appb-000030
取代的基团；且当R x选自带有羟基的R 1中羟基被
Figure PCTCN2020117093-appb-000031
取代的基团时，反应需进一步包括经酸性、还原条件得到N-2产品；该步骤中，所述酸性条件可以选自HCl，还原条件可以选自硼氢化钠；
(b2)将上步反应得到的N-2还原得到N-3；所述还原剂可以选自Pd/C；
(b3)将N-3和M-2反应得到式I。
所述步骤中，R 1，R 2，R 3，m，W如式I所定义；所述L 1为离去基团，可以选自卤素或OTs。
本发明进一步提供一种药物组合物，其包含本发明所述式I所示的化合物、其立体异构体、消旋体、互变异构体、同位素标记物、前药或其药学上可接受的盐。
在一些实施方案中，本发明所述的药物组合物进一步包含治疗有效量的本发明所述式I所示的化合物、其立体异构体、消旋体、互变异构体、同位素标记物、前药或其药学上可接受的盐和药学上可接受的载体。
本发明进一步提供式I所示的化合物、其立体异构体、消旋体、互变异构体、同位素标记物、前药或其药学上可接受的盐在制备IRAK抑制剂中的用途。
本发明进一步提供式I所示的化合物、其立体异构体、消旋体、互变异构体、同位素标记物、前药或其药学上可接受的盐在制备预防和/或治疗IRAK介导的疾病或病症的药物中的用途。
根据本发明的实施方案，所述IRAK介导的疾病或病症选自肿瘤、痛风、系统性红斑狼疮、多发性硬化症、代谢综合症、动脉粥样硬化、心肌梗死、脓血症、炎症性肠病、哮喘和过敏等疾病。
本发明还提供式I所示的化合物、其立体异构体、消旋体、互变异构体、同位素标记物、前药或其药学上可接受的盐在制备预防和/或治疗与白细胞介素-1受体相关激酶的疾病或病症的药物中的用途。
本发明还提供一种IRAK介导的疾病或病症的预防和/或治疗方法，包括向有此需要的个体施用治疗有效量的式I所示的化合物、其立体异构体、消旋体、互变异构体、同位素标记物、前药或其药学上可接受的盐或所述药物组合物。
在一些实施方案中，所述IRAK选自IRAK4相关激酶。
本发明还提供一种与白细胞介素-1受体相关疾病的预防和/或治疗方法，包括向有此需要的个体施用治疗有效量的式I所示的化合物、其立体异构体、消旋体、互变异构体、同位素标记物、前药或其药学上可接受的盐或所述药物组合物。
根据本发明的实施方案，所述与白细胞介素-1受体相关激酶的疾病或病症选自肿瘤、痛风、系统性红斑狼疮、多发性硬化症、代谢综合症、动脉粥样硬化、心肌梗死、脓血症、炎症性肠病、哮喘、类风湿性关节炎、败血症、自身免疫性疾病和过敏等疾病。
本发明的方法可包括单独给予本发明化合物、以及将本发明化合物与一种或多种其它化学治疗剂组合给药。多种药物的给药可以同时或相继进行。
术语定义和缩写
除非另有说明，本申请说明书和权利要求书中记载的基团和术语定义，包括其作为实例的定义、示例性的定义、优选的定义、表格中记载的定义、实施例中具体化合物的定义等，可以彼此之间任意组合和结合。这样的组合和结合后的基团定义及化合物结构，应当属于本申请说明书记载的范围内。
本申请说明书和权利要求书记载的数值范围，当该数值范围被定义为“整数”时，应当理解为记载了该范围的两个端点以及该范围内的每一个整数。例如，“0～6的整数”应当理解为记载了0、1、2、3、4、5和6的每一个整数。“更多个”表示三个或三个以上。
本文所述任选的被取代基所取代的情形涵盖了无取代以及被一个或多个取代基所取代的情形，例如“任选被一个、两个或更多个R取代”意味着可以不被R取代(无取代)或被一个、两个或更多个R取代。
术语“卤素”指F、Cl、Br和I。换言之，F、Cl、Br和I在本说明书中可描述为“卤素”。
术语“脂肪烃基”包括饱和或不饱和，直链或支链的链状或环状烃基，所述脂肪烃基的类型可选自烷基、烯基、炔基等，所述脂肪烃基的碳原子数优选为1～12，还可以为1～10，进一步的优选范围为1～6，具体可包括但不限于如下基团：甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、正戊基、异戊基、新戊基、正己基、乙烯基、1-丙烯基、2-丙烯基、 1-甲基乙烯基、1-丁烯基、1-乙基乙烯基、1-甲基-2-丙烯基、2-丁烯基、3-丁烯基、2-甲基-1-丙烯基、2-甲基-2-丙烯基、1-戊烯基、1-己烯基、乙炔基，1-丙炔基，2-丙炔基，1-丁炔基，1-甲基-2-丙炔基，3-丁炔基，1-戊炔基、1-己炔基、环丙基、环丁基、环戊基和环己基；
所述“脂肪烃基”可以任选地包含一个、两个或更多个杂原子(或解释为任选地杂原子插入至脂肪烃基中任选地C-C键和C-H键)。适宜的杂原子对于本领域技术人员而言是显而易见的，并且包括例如硫、氮、氧、磷和硅。所述包含杂原子的脂肪烃基基团可选自以下基团：(C 1-C 6)脂肪烃基氧基、(C 1-C 6)脂肪烃基巯基，(C 1-C 6)脂肪烃基氧基(C 1-C 6)脂肪烃基、(C 1-C 6)脂肪烃基巯基(C 1-C 6)脂肪烃基、N-(C 1-C 3)脂肪烃基胺基(C 1-C 6)脂肪烃基、N,N-二-(C 1-C 3)脂肪烃基胺基(C 1-C 6)脂肪烃基，例如可以为甲氧基，乙氧基，丙氧基，丁氧基，戊氧基，甲氧基甲基，乙氧基甲基、丙氧基甲基、甲氧基乙基、乙氧基乙基、丙氧基乙基、甲氧基丙基、乙氧基丙基、丙氧基丙基、N-甲基胺甲基、N-甲基胺乙基、N-乙基胺乙基、N,N-二甲基胺甲基、N,N-二甲基胺乙基、N,N-二乙基胺乙基；其他基团中所含“脂肪烃基”部分同上述解释。
术语“C 3-12环烷基”应理解为表示饱和或不饱和的一价单环或双环烃环，其具有3-12个碳原子，优选“C 3-10环烷基”。术语“C 3-10环烷基”应理解为表示饱和的一价单环或双环烃环，其具有3、4、5、6、7、8、9或10个碳原子。所述C 3-10环烷基可以是单环烃基，如环丙基、环丁基、环戊基、环己基、环庚基、环辛基、环壬基或环癸基，或者是双环烃基如十氢化萘环。
术语“3-12元杂环基”意指饱和或不饱和的一价单环或双环，其包含1-5个独立选自N、O和S的杂原子，含杂原子的基团不具有芳香性，所述3-12元杂环基，优选“3-10元杂环基”。术语“3-10元杂环基”意指饱和的一价单环或双环，其包含1-5个，优选1-3个选自N、O和S的杂原子。所述杂环基可以通过所述碳原子中的任一个或氮原子(如果存在的话)与分子的其余部分连接。特别地，所述杂环基可以包括但不限于：4元环，如氮杂环丁烷基、氧杂环丁烷基；5元环，如四氢呋喃基、四氢噻吩基、二氧杂环戊烯基、吡咯烷基、咪唑烷基、吡唑烷基、吡咯啉基；或6元环，如四氢吡喃基、哌啶基、吗啉基、二噻烷基、硫代吗啉基、哌嗪基或三噻烷基；或7元环，如二氮杂环庚烷基。任选地，所述杂环基可以是苯并稠合的。所述杂环基可以是双环的，例如但不限于5,5元环，如六氢环戊并[c]吡咯-2(1H)-基环，或者5,6元双环，如六氢吡咯并[1,2-a]吡嗪-2(1H)-基环。含氮原子的环可以是部分不饱和的，即它可以包含一个或多个双键，例如但不限于2,5-二氢-1H-吡咯基、4H-[1,3,4]噻二嗪基、4,5-二氢噁唑基或4H-[1,4]噻嗪基，或者，它可以是苯并稠合的，例如但不限于二氢异喹啉基。根据本发明，所述3-12元杂环基可以进一步选自如下基团：
Figure PCTCN2020117093-appb-000032
术语“C 6-20芳基”应理解为优选表示具有6-20个碳原子的一价芳香性或部分芳香性的单环、双环或三环烃环，优选“C 6-14芳基”。术语“C 6-14芳基”应理解为优选表示具有6、7、8、9、10、11、12、13或14个碳原子的一价芳香性或部分芳香性的单环、双环或三环烃环(“C 6-14芳基”)，特别是具有6个碳原子的环(“C 6芳基”)，例如苯基；或联苯基，或者是具有9个碳 原子的环(“C 9芳基”)，例如茚满基或茚基，或者是具有10个碳原子的环(“C 10芳基”)，例如四氢化萘基、二氢萘基或萘基，或者是具有13个碳原子的环(“C 13芳基”)，例如芴基，或者是具有14个碳原子的环(“C 14芳基”)，例如蒽基。
术语“5-14元杂芳基”应理解为包括这样的一价单环、双环或三环芳族环系：其具有5、6、7、8、9、10、11、12、13或14个环原子，特别是5或6或9或10个碳原子，且其包含1-5个，优选1-3各独立选自N、O和S的杂原子并且，另外在每一种情况下可为苯并稠合的。特别地，杂芳基选自噻吩基、呋喃基、吡咯基、噁唑基、噻唑基、咪唑基、吡唑基、异噁唑基、异噻唑基、噁二唑基、三唑基、噻二唑基、噻-4H-吡唑基等以及它们的苯并衍生物，例如苯并呋喃基、苯并噻吩基、苯并噁唑基、苯并异噁唑基、苯并咪唑基、苯并三唑基、吲唑基、吲哚基、异吲哚基等；或吡啶基、哒嗪基、嘧啶基、吡嗪基、三嗪基等，以及它们的苯并衍生物，例如喹啉基、喹唑啉基、异喹啉基等；或吖辛因基、吲嗪基、嘌呤基等以及它们的苯并衍生物；或噌啉基、酞嗪基、喹唑啉基、喹喔啉基、萘啶基、蝶啶基、咔唑基、吖啶基、吩嗪基、吩噻嗪基、吩噁嗪基等。
除非另有说明，杂环基或杂芳基包括其所有可能的异构形式，例如其位置异构体。因此，对于一些说明性的非限制性实例，吡啶基或亚吡啶基包括吡啶-2-基、亚吡啶-2-基、吡啶-3-基、亚吡啶-3-基、吡啶-4-基和亚吡啶-4-基；噻吩基或亚噻吩基包括噻吩-2-基、亚噻吩-2-基、噻吩-3-基和亚噻吩-3-基。
在本文中，所述“3-12元杂环基”、“5-14元杂芳基”还可以进一步包括含N的5-12元杂环基或5-14元杂芳基，即含N的5-12元杂环基或5-14元杂芳基可选自“3-12元杂环基”、“5-14元杂芳基”术语定义范围内的相应基团。
根据其分子结构，本发明的化合物可以是手性的，因此可能存在各种对映异构体形式。因而这些化合物可以以消旋体形式或光学活性形式存在。本发明的化合物或其中间体可以通过本领域技术人员公知的化学或物理方法分离为对映异构体化合物，或者以此形式用于合成。在外消旋的胺的情况中，通过与光学活性的拆分试剂反应，从混合物制得非对映异构体。适当的拆分试剂的示例是光学活性的酸，例如R和S形式的酒石酸、二乙酰酒石酸、二苯甲酰酒石酸、扁桃酸、苹果酸、乳酸、适当的N-保护的氨基酸(例如N-苯甲酰脯氨酸或N-苯磺酰基脯氨酸)或各种光学活性的樟脑磺酸。借助光学活性的拆分试剂(例如固定在硅胶上的二硝基苯甲酰基苯基甘氨酸、三乙酸纤维素或其它碳水化合物的衍生物或手性衍生化的异丁烯酸酯聚合物)，也可有利地进行色谱对映体拆分。用于此目的的适当的洗脱剂是含水或含醇的溶剂混合物，例如，己烷/异丙醇/乙腈。
药学上可接受的盐可以是例如在链或环中具有氮原子的具有足够碱性的本发明的化合物的酸加成盐，例如与如下无机酸形成的酸加成盐：例如盐酸、氢氟酸、氢溴酸、氢碘酸、硫酸、焦硫酸、磷酸或硝酸，或硫酸氢盐、或者与如下有机酸形成的酸加成盐：例如甲酸、乙酸、乙酰乙酸、丙酮酸、三氟乙酸、丙酸、丁酸、己酸、庚酸、十一烷酸、月桂酸、苯甲酸、水杨酸、2-(4-羟基苯甲酰基)苯甲酸、樟脑酸、肉桂酸、环戊烷丙酸、二葡糖酸、3-羟基-2-萘甲酸、烟酸、扑酸、果胶酯酸、过硫酸、3-苯基丙酸、苦味酸、特戊酸、2-羟基乙磺酸、衣康酸、氨基磺酸、三氟甲磺酸、十二烷基硫酸、乙磺酸、苯磺酸、对甲苯磺酸、甲磺酸、2-萘磺酸、萘二磺酸、樟脑磺酸、柠檬酸、酒石酸、硬脂酸、乳酸、草酸、丙二酸、琥珀酸、苹果酸、己二酸、藻酸、马来酸、富马酸、D-葡糖酸、扁桃酸、抗坏血酸、葡庚酸、甘油磷 酸、天冬氨酸、磺基水杨酸、半硫酸或硫氰酸。
另外，具有足够酸性的本发明的化合物的另一种适合的药学上可接受的盐是碱金属盐(例如钠盐或钾盐)、碱土金属盐(例如钙盐或镁盐)、铵盐，或与提供生理学可接受的阳离子的有机碱形成的盐，例如与如下物质形成的盐：钠离子、钾离子、N-甲基葡糖胺、二甲基葡糖胺、乙基葡糖胺、赖氨酸、二环己基胺、1,6-己二胺、乙醇胺、葡糖胺、葡甲胺、肌氨酸、丝氨醇、三羟基甲基氨基甲烷、氨基丙二醇、1-氨基-2,3,4-丁三醇。作为实例，所述药学上可接受的盐包括基团-COOH与如下物质形成的盐：钠离子、钾离子、钙离子、镁离子、N-甲基葡糖胺、二甲基葡糖胺、乙基葡糖胺、赖氨酸、二环己基胺、1,6-己二胺、乙醇胺、葡糖胺、葡甲胺、肌氨酸、丝氨醇、三羟基甲基氨基甲烷、氨基丙二醇、1-氨基-2,3,4-丁三醇。
另外，碱性含氮基团可用如下试剂季铵化：低级烷基卤化物，例如甲基、乙基、丙基和丁基氯化物、溴化物和碘化物；硫酸二烷基酯，例如硫酸二甲酯、硫酸二乙酯、硫酸二丁酯和硫酸二戊酯；长链卤化物，例如癸基、月桂基、肉豆蔻基和硬脂基氯化物、溴化物和碘化物；芳烷基卤化物如苄基和苯乙基溴化物等。作为实例，药学上可接受的盐包括盐酸盐、硫酸盐、硝酸盐、硫酸氢盐、氢溴酸盐、醋酸盐、草酸盐、柠檬酸盐、甲磺酸盐、甲酸盐或葡甲胺盐等。
由于本发明的化合物可存在多个成盐位点，所述“药学上可接受的盐”不仅包括本发明化合物其中1个成盐位点上形成的盐，而且还包括其中2、3或全部成盐位点上形成的盐。为此，所述“药学上可接受的盐”中式(I)化合物与成盐所需的酸的根离子(阴离子)或碱的阳离子摩尔比可以在较大的范围内变化，例如可以是4:1～1:4，如3:1、2:1、1:1、1:2、1:3等。
根据本发明，药学上可接受的阴离子包括选自由无机酸或有机酸电离生成的阴离子。所述“无机酸”包括但不限于盐酸、氢氟酸、氢溴酸、氢碘酸、硫酸、焦硫酸、磷酸或硝酸。所述“有机酸”包括但不限于甲酸、乙酸、乙酰乙酸、丙酮酸、三氟乙酸、丙酸、丁酸、己酸、庚酸、十一烷酸、月桂酸、苯甲酸、水杨酸、2-(4-羟基苯甲酰基)苯甲酸、樟脑酸、肉桂酸、环戊烷丙酸、二葡糖酸、3-羟基-2-萘甲酸、烟酸、扑酸、果胶酯酸、过硫酸、3-苯基丙酸、苦味酸、特戊酸、2-羟基乙磺酸、衣康酸、氨基磺酸、三氟甲磺酸、十二烷基硫酸、乙磺酸、苯磺酸、对甲苯磺酸、甲磺酸、2-萘磺酸、萘二磺酸、樟脑磺酸、柠檬酸、酒石酸、硬脂酸、乳酸、草酸、丙二酸、琥珀酸、苹果酸、己二酸、藻酸、马来酸、富马酸、D-葡糖酸、扁桃酸、抗坏血酸、葡庚酸、甘油磷酸、天冬氨酸、磺基水杨酸、半硫酸或硫氰酸。
根据不同取代基的位置和性质，本发明的化合物还可以包含一个或多个不对称中心。不对称碳原子可以(R)或(S)构型存在，仅有一个不对称中心时，产生外消旋混合物，含有多个不对称中心时，得到非对映异构体混合物。在某些情况下，由于围绕特定键的旋转受阻还可能存在不对称性，例如该中心键连接特定化合物的两个被取代的芳族环。并且，取代基还可以顺式或反式异构的形式存在。
本发明化合物还包括其各自所有可能的立体异构体，其是单一立体异构体或所述立体异构体(例如R-异构体或S-异构体，或者E-异构体或Z-异构体)的任意比例的任意混合物的形式。可通过任意适合的现有技术方法(例如色谱法，特别是例如手性色谱法)实现本发明的化合物的单一立体异构体(例如单一对映异构体或单一非对映异构体)的分离。
术语“互变异构体”是指因分子中某一原子在两个位置迅速移动而产生的官能团异构体。本发明化合物可表现出互变异构现象。互变异构的化合物可以存在两种或多种可相互转化的 种类。质子移变互变异构体来自两个原子之间共价键合的氢原子的迁移。互变异构体一般以平衡形式存在，尝试分离单一互变异构体时通常产生一种混合物，其理化性质与化合物的混合物是一致的。平衡的位置取决于分子内的化学特性。例如，在很多脂族醛和酮如乙醛中，酮型占优势；而在酚中，烯醇型占优势。本发明包含化合物的所有互变异构形式。
在本发明中，所涉及的化合物亦包括经同位素标记的化合物，所述经同位素标记的化合物与式I中所示的那些相同，但是其中一或多个原子被原子质量或质量数不同于通常天然存在的原子质量或质量数的原子替代。可掺入本发明的化合物的同位素的实例包括H、C、N、O、S、F及Cl的同位素，分别诸如 2H、 3H、 13C、 11C、 14C、 15N、 18O、 17O、 32P、 35S、 18F及 36Cl。含有上述同位素和/或其他原子的其他同位素的本发明的化合物、其前药、或者所述化合物或所述前药的药学上可接受的盐在本发明的范围内。本发明的某些经同位素标记的化合物，例如掺入放射性同位素(诸如 3H和 14C)的化合物可用于药物和/或底物组织分布测定。氚(即 3H)和碳14(即 14C)同位素因易于制备和可检测性而成为特别优选的。再者，以较重的同位素(诸如氘，即 2H)替代可提供源自更高的代谢稳定性的某些治疗优势(例如增加的体内半衰期或减少的剂量需求)，并因此可在某些情况下是优选的。如权利要求所请求保护的本发明化合物可特别地限定以氘或氚替代。此外，取代基中出现的氢未单独列明术语氘或氚并不表示排除氘或氚，而是同样也可以包含氘或氚。
术语“有效量”或者“治疗有效量”是指足以实现预期应用(包括但不限于如下定义的疾病治疗)的本发明所述化合物的量。治疗有效量可以取决于以下因素而改变：预期应用(体外或者体内)，或者所治疗的受试者和疾病病症如受试者的重量和年龄、疾病病症的严重性和给药方式等，其可以由本领域普通技术人员容易地确定。具体剂量将取决于以下因素而改变：所选择的特定化合物、所依据的给药方案、是否与其它化合物组合给药、给药的时间安排、所给药的组织和所承载的物理递送系统。
术语“辅料”是指可药用惰性成分。赋形剂种类的实例非限制性地包括粘合剂、崩解剂、润滑剂、助流剂、稳定剂、填充剂和稀释剂等。赋形剂能增强药物制剂的操作特性，即通过增加流动性和/或粘着性使制剂更适于直接压缩。适用于上述制剂的典型的药学上可接受的载体的实例为：糖类，例如乳糖、蔗糖、甘露醇和山梨醇；淀粉类，例如玉米淀粉、木薯淀粉和土豆淀粉；纤维素及其衍生物，例如羧甲基纤维素钠，乙基纤维素和甲基纤维素；磷酸钙类，例如磷酸二钙和磷酸三钙；硫酸钠；硫酸钙；聚乙烯吡咯烷酮；聚乙烯醇；硬脂酸；硬脂酸碱土金属盐，例如硬脂酸镁和硬脂酸钙；硬脂酸；植物油类，例如花生油、棉籽油、芝麻油、橄榄油和玉米油；非离子、阳离子和负离子表面活性剂；乙二醇聚合物；脂肪醇类；和谷物水解固形物以及其它无毒的可相容的填充剂、粘合剂、崩解剂、缓冲剂、防腐剂、抗氧剂、润滑剂、着色剂等在药物制剂中常用到的辅料。
术语“溶剂化物”是本发明的化合物的那些形式，其以固体或液体的状态通过与溶剂分子的配位作用形成配合物。水合物是溶剂化物的特定形式，其中配位作用是与水进行。在本发明中，优选的溶剂化物是水合物。进一步的，本发明通式I化合物的药学上可接受的溶剂化物(水合物)是指化合物I与化学计量学的一个或多个分子的水或其他溶剂形成的共晶和包合物。可用于溶剂化物的溶剂包括但不限于：水、甲醇、乙醇、乙二醇和醋酸。
术语“前药”或称为“药物前体”，代表化合物在体内转化为前述通式或具体化合物所示的化合物。这样的转化受前体药物在血液中水解或在血液或组织中经酶转化为母体结构的影响。 本发明前药可以是酯，在本发明中酯可以作为前药的有苯酯类，脂肪族(C1-24)酯类，酰氧基甲基酯类，碳酸酯，氨基甲酸酯类和氨基酸酯类。例如本发明里的一个化合物包含羟基/羧基，即可以将其酰化得到前体药物形式的化合物。其他的前药形式包括磷酸酯，如这些磷酸酯类化合物是经母体上的羟基磷酸化得到的。
试剂英文缩写对应的试剂名称
Figure PCTCN2020117093-appb-000033
有益效果
1)本发明提供了一种具有新颖结构的通式I化合物，经实验验证，本发明化合物对于IRAK4激酶活性有明显的抑制作用，且相对于其他激酶，针对IRAK4激酶活性具有良好的选择性抑制作用；
2)本发明化合物具有良好的用药安全性、广泛的适用性和较低的毒副作用，经实验验证，本发明化合物对人hERG的抑制率很低，对于人CYP3A4无明显的时间依赖性抑制，对人、大鼠、小鼠的血浆蛋白结合率适中，种属间的差异很小，同时，对人的5种CYP亚型均无明显的抑制作用；
3)本发明化合物对LPS诱导的Balb/c雌性小鼠释放TNF-α具有明显的抑制作用；
4)本发明化合物具有良好的药代动力学特征，在动物体内显示出良好的暴露量和滞留时间，且具有适宜的半衰期和良好的药物吸收性。
具体实施方式
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解，下列实 施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过已知方法制备。
实施例1化合物001的合成
反应式:
Figure PCTCN2020117093-appb-000034
1.化合物3的合成
15摄氏度下依次向化合物1(50g)的二氯甲烷溶液(500mL)中加入DMAP(42.5g)，化合物2(63.4g)，和三乙胺(63.9g)，并在25摄氏度下搅拌反应18小时。向反应液中加入二氯甲烷(200mL)用水洗涤(300mL*2)，1M的稀盐酸(300mL*3)洗涤，有机相用无水硫酸钠干燥，过滤，减压浓缩得到化合物3(98g，收率：99％)。
2.化合物4的合成
15摄氏度下向化合物3(50g)的四氢呋喃溶液(300mL)中加入1M稀盐酸(300mL)并在25摄氏度下搅拌反应20小时。冷却到0摄氏度，用1M的氢氧化钠溶液调到PH＝9,用乙酸乙酯(200mL×3)萃取，萃取液用饱和氯化钠溶液(300mL)洗涤，无水硫酸钠干燥,过滤,减压浓缩，残留物用石油醚(150mL)打浆得化合物4(39g,收率91％)。
3.化合物5&6的合成
-40摄氏度下向甲基溴化镁(85.8mL)的四氢呋喃溶液(500mL)中滴加化合物4(34.5g)的四氢呋喃溶液(200mL)，并在-40摄氏度下搅拌反应4小时，用饱和氯化铵溶液(100mL)淬灭反应，用乙酸乙酯(500mL×3)萃取，萃取液用饱和食盐水(300mL)洗涤,无水硫酸钠干燥，过滤，减压浓缩，残留物用硅胶色谱柱纯化(石油醚:乙酸乙酯＝5:1)得化合物5(4.3g,收率10％)，化 合物6(7.0g,收率17％)和混合物12g。
化合物5
1H NMR(400MHz,CDCl 3):δ7.79(d,J＝8.0Hz,2H),7.32(d,J＝8.4Hz,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)。
化合物6
1H NMR(400MHz,CDCl 3):δ7.79(d,J＝8.4Hz,2H),7.33(d,J＝8.0Hz,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)。
4.化合物8的合成
零下15摄氏度下向化合物7(2.0g)的浓硫酸(12mL，98％)溶液中滴加硝酸(1.6mL,70％)的浓硫酸(1.6mL,98％)混合溶液。加完后混合体系零下15摄氏度搅拌2小时，将反应液缓慢倾倒入冰水中并搅拌5分钟，抽滤，用水洗涤，收集固体减压干燥得到化合物8(2.5g，收率：97％)。
5.化合物9的合成
室温下向化合物8(2.0g)的DMF(20mL)溶液中加入水合肼(2.4mL,98％)，加完后混合体系加热到120摄氏度搅拌16小时，冷却到室温，混合体系慢慢倒入冰水中并搅拌，抽滤，用水洗涤固体，收集固体减压浓缩得到化合物9(1.3g,收率：67％)。
6.化合物10的合成
15摄氏度下将化合物9(12.4g)和钯碳(7g,10％)依次加入到400mL乙酸乙酯中。加完后混合体系在15摄氏度氢气保护下搅拌18小时，将反应液中钯碳滤掉后将滤液浓缩蒸干得到化合物10(10.4g,收率99％)。
7.化合物12的合成
25摄氏度下将EDCI.HCl(2.6g)加到化合物10(1.5g)和化合物11(1.4g)的Py(15mL)溶液中，反应液25摄氏度搅拌16小时。反应液浓缩蒸干，残留物通过MeOH/H 2O＝20mL/20mL打浆得到化合物12(1.3g，收率48％)。
8.化合物001即2-((2-(反式-4-羟基-顺式-4-甲基环己基)-6-甲氧基-2H-吲唑-5-基)氨甲酰基)-6-甲基吡啶1-氧化物的合成
Figure PCTCN2020117093-appb-000035
25摄氏度下将碳酸铯(985mg)加入到化合物12(300mg)和化合物5(344mg)的5mL的DMF溶液中，反应液90摄氏度搅拌16小时。反应液加到30mL水中，乙酸乙酯萃取(10mL*3)，有机相减压浓缩，残余物通过高效液相制备色谱柱(CH 3CN:H 2O(0.1％NH 4HCO 3)＝15-45％,UV:214nm,流速:15ml/min)纯化得到化合物001(70mg,收率17％)。
1H NMR(400MHz,DMSO-d6):δ14.16(s,1H),8.78(s,1H),8.34(s,1H),8.32-8.30(m,1H),7.77(d,J＝7.6Hz,1H),7.58(t,J＝8.0Hz,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＝3.646min,[M+H] +＝411.1.
9.化合物11的合成
25摄氏度下将m-CPBA(25g)加到化合物13(10g)的200mL DCM溶液中，反应液25摄氏度搅拌16小时。反应液过滤，滤液用15.6g亚硫酸钠配成的饱和溶液淬灭，混合溶液搅拌2小时，萃取，水相用稀盐酸调至PH<7，DCM萃取(50mL*3)，有机相合并浓缩，残余物用300mL EA打浆得到化合物11(10.1g,收率90％)。
实施例2化合物010的合成
反应式:
Figure PCTCN2020117093-appb-000036
1.化合物2的合成
15摄氏度下依次向化合物1(50g)的二氯甲烷溶液(500mL)中加入DMAP(42.5g)，TsCl(63.4g)，和三乙胺(63.9g)，并在25摄氏度下搅拌反应18小时。向反应液中加入二氯甲烷(200mL)用水洗涤(300mL*2)，1M的稀盐酸(300mL*3)洗涤，有机相用无水硫酸钠干燥，过滤，减压浓缩得到化合物2(98g。收率：99％)。
2.化合物3的合成
15摄氏度下向化合物2(50g)的四氢呋喃溶液(300mL)中加入1M稀盐酸(300mL)并在25摄氏度下搅拌反应20小时。冷却到0摄氏度,用1M的氢氧化钠溶液调到PH＝9,用乙酸乙酯(200mL×3)萃取,萃取液用饱和氯化钠溶液(300mL)洗涤,无水硫酸钠干燥,过滤,减压浓缩，残留物用石油醚(150mL)打浆得化合物3(39g,收率91％)。
3.化合物4&5的合成
-40摄氏度下向甲基溴化镁(85.8mL)的四氢呋喃溶液(500mL)中滴加化合物3(34.5g)的四氢呋喃溶液(200mL)，并在-40摄氏度下搅拌反应4小时,用饱和氯化铵溶液(100mL)淬灭反应, 用乙酸乙酯(500mL×3)萃取，萃取液用饱和食盐水(300mL)洗涤,无水硫酸钠干燥，过滤，减压浓缩,残留物用硅胶色谱柱纯化(石油醚:乙酸乙酯＝5:1)得化合物4(4.3g,收率10％)，化合物5(7.0g,收率17％)和混合物12g。
化合物4
1H NMR(400MHz,CDCl 3):δ7.79(d,J＝8.0Hz,2H),7.32(d,J＝8.4Hz,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)。
化合物5
1H NMR(400MHz,CDCl 3):δ7.79(d,J＝8.4Hz,2H),7.33(d,J＝8.0Hz,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)。
4.化合物7的合成
25摄氏度下将m-CPBA(25g)加到化合物6(10g)的200mL DCM溶液中，反应液25摄氏度搅拌16小时。反应液过滤，滤液用15.6g亚硫酸钠配成的饱和溶液淬灭，混合溶液搅拌2小时，萃取，水相用稀盐酸调至PH<7，DCM萃取(50mL*3)，有机相合并浓缩，残余物用300mL EA打浆得到化合物7(10.1g,收率90％)。
5.化合物9的合成
将80mL浓硫酸加入到500mL三口瓶，冷却至零下7摄氏度，缓慢加入化合物8(10g)，在此温度下搅拌5分钟后，冷却至零下15摄氏度，缓慢加入硝酸钾(8.9g)，在此温度下搅拌1小时。合并的反应液倒入1.2L冰水中，将析出的固体过滤，滤饼溶于2L乙酸乙酯，加入4L碳酸氢钠溶液调至碱性，乙酸乙酯萃取(2L*3)，有机相浓缩蒸干，残留物通过硅胶柱(DCM/MeOH＝300/1)纯化得到化合物9(12.3g，收率27％)。
6.化合物11的合成
室温下将化合物9(400mg)，化合物10(1.81g)和DIPEA(2.86g),依次加入到20mL的DMF溶液中，反应液在闷罐中80摄氏度搅拌过夜，反应结束后加入水，乙酸乙酯萃取三次，减压浓缩通过硅胶柱纯化(DCM/CH 3OH＝200/1)得到化合物11(570mg，收率83％)。
7.化合物12的合成
室温下将化合物11(80mg)和Pd/C(5mg)依次加入到10mL的甲醇溶液中，反应液在氢气保护下55℃搅拌过夜，反应结束后过滤，滤液减压浓缩后通过硅胶柱纯化(DCM/CH 3OH＝100/1)得到化合物12(60mg，收率65％)。
8.化合物13的合成
25摄氏度下将EDCI.HCl(950mg)加到化合物12(580mg)和化合物7(505mg)的Py(11mL)溶液中，反应液40摄氏度搅拌16小时。反应液浓缩蒸干，残余物通过硅胶柱(PE:EA＝1:1)纯化得到化合物13(550mg,收率55％)。
9.化合物010即2-((2-(反式-4-羟基-顺式-4-甲基环己基)-6-二甲基氨基-2H-吲唑-5-基)氨甲酰基)-6-甲基吡啶1-氧化物的合成
Figure PCTCN2020117093-appb-000037
25摄氏度下将碳酸铯(936mg)加入到化合物13(300mg)和化合物4(409mg)的6mL 的DMF溶液中，反应液90摄氏度搅拌16小时。反应液加到30mL水中，乙酸乙酯萃取(10mL*3)，有机相减压浓缩，残余物通过高效液相制备色谱柱(CH 3CN:H 2O(0.1％NH 4HCO 3)＝20-70％,UV:214nm,流速:15ml/min)纯化得到化合物010(59mg,收率14％)。
1H NMR(400MHz,DMSO-d 6):δ14.01(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＝7.6Hz,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＝3.318min,[M+H] +＝424.2.
实施例3化合物013的合成
反应式：
Figure PCTCN2020117093-appb-000038
1.化合物2的合成
零下15摄氏度下向化合物1(2.0g)的浓硫酸(12mL，98％)溶液中滴加硝酸(1.6mL,70％)的浓硫酸(1.6mL,98％)混合溶液。加完后混合体系零下15摄氏度搅拌2小时，将反应液缓慢倾倒入冰水中并搅拌5分钟，抽滤，用水洗涤，收集固体减压干燥得到化合物2(2.5g,收率：97％)。
2.化合物3的合成
室温下向化合物2(2.0g)的DMF(20mL)溶液中加入水合肼(2.4mL,98％)，加完后混合体系加热到120摄氏度搅拌16小时，冷却到室温，混合体系慢慢倒入冰水中并搅拌，抽滤，用水洗涤固体，收集固体减压浓缩得到化合物3(1.3g,收率：67％)。
3.化合物4的合成
15摄氏度下将化合物3(12.4g)和钯碳(7g,10％)依次加入到400mL乙酸乙酯中。加完后混合体系在15摄氏度氢气保护下搅拌18小时，将反应液中钯碳滤掉后将滤液浓缩蒸干得到化合物4(10.4g,收率99％)。
4.化合物6的合成
25摄氏度下将EDCI.HCl(2.6g)加到化合物4(1.5g)和化合物5(1.4g)的Py(15mL)溶液中，反应液25摄氏度搅拌16小时。反应液浓缩蒸干，残留物通过MeOH/H 2O＝20mL/20mL打浆得到化合物6(1.3g，收率48％)。
5.化合物8的合成
25摄氏度下将碳酸铯(3.3g)加入到化合物6(1g)和化合物7(1.3g)的20mL的DMF溶液中，反应液90摄氏度搅拌16小时。反应液加到50mL水中，乙酸乙酯萃取(30mL*3)，有机相减压浓缩，残余物通过高效液相制备色谱柱(CH 3CN:H 2O(0.1％NH 4HCO 3)＝20-60％,UV:214nm,流速:15ml/min)纯化得到化合物8(370mg,收率25％)。
6.化合物9的合成
25摄氏度下向化合物8(350mg)的5mL二氧六环溶液中加入5mL 2M稀盐酸，反应液25摄氏度搅拌16小时。反应液用碳酸钠溶液调至碱性，乙酸乙酯萃取(10mL*3)，有机相减压浓缩得到化合物9(150mg,收率48％)。
7.化合物013即2-((2-(反式-4-羟基环己基)-6-甲氧基-2H-吲唑-5-基)氨甲酰基)-6-甲基吡啶1-氧化物的合成
Figure PCTCN2020117093-appb-000039
0摄氏度下将硼氢化钠(25mg)加到化合物9(130mg)的2mL甲醇溶液中，反应液25摄氏度搅拌2小时。反应液用10mL氯化铵溶液淬灭，乙酸乙酯萃取(5mL*3)，有机相减压浓缩，残余物通过高效液相制备色谱柱(CH 3CN:H 2O(0.1％NH 4HCO 3)＝10-60％,UV:214nm,流速:15ml/min)纯化得到化合物013(54mg,收率41％)。
1H NMR(400MHz,DMSO-d 6):δ14.16(s,1H),8.78(s,1H),8.31-8.29(m,2H),7.78-7.76(m,1H),7.58(t,J＝8.0Hz,1H),7.10(s,1H),4.71(d,J＝4.4Hz,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＝2.541min,[M+H] +＝397.2.
实施例4化合物016和化合物220的合成
反应式:
Figure PCTCN2020117093-appb-000040
Figure PCTCN2020117093-appb-000041
1.化合物2的合成
18摄氏度下将化合物1(2g)和AlCl 3(4.13g)依次加入到二氯甲烷(150mL)中，反应液55摄氏度搅拌18小时。反应液用50mL水淬灭后用150mL二氯甲烷萃取后再用乙酸乙酯萃取(150mL*3)，有机相浓缩蒸干，残留物用30mL二氯甲烷打浆得到化合物2(1.6g，收率86％)。
2.化合物3的合成
25摄氏度下将碳酸钾(93mg)加入到化合物2(0.1g)和碘乙烷(105mg)的2mL的DMF溶液中，反应液60摄氏度搅拌16小时。反应液加到20mL水中，乙酸乙酯萃取(5mL*3)，有机相减压浓缩，残余物通过硅胶柱纯化(石油醚/乙酸乙酯＝2/1)得到化合物3(0.1g,收率86％)。
3.化合物4的合成
25摄氏度下向化合物3(1.1g)的100mL甲醇溶液中加入0.3g Pd/C，反应液25摄氏度,一个氢气球压力下(760Torr)搅拌16小时。反应液过滤，滤液旋干得到化合物4(0.71g,收率76％)。
4.化合物6的合成
25摄氏度下将化合物5(558mg)加入到化合物4(710mg)和EDCI(840mg)的25mL的吡啶溶液中，反应液25摄氏度搅拌16小时。反应液加到100mL水中，乙酸乙酯萃取(30mL*3)，有机相减压浓缩，残余物通过硅胶柱纯化(二氯甲烷/甲醇＝60/1)得到化合物6(0.67g,收率54％)。
5.化合物8的合成
25摄氏度下将化合物6(630mg)加入到化合物7(945mg)和碳酸铯(1.97g)的25mL的DMF溶液中，反应液氮气保护下，90摄氏度搅拌16小时。将反应液加到100mL水中，乙酸乙酯萃取(30mL*3)，有机相减压浓缩，残余物通过高效液相制备色谱柱(CH 3CN:H 2O(0.1％NH 4HCO 3)＝5-95％,UV:214nm,流速:15ml/min)纯化得到化合物8(160mg,收率18％)。
6.化合物9的合成
25摄氏度下将化合物8(180mg)溶于30mL二氧六环和10mL的2M的稀盐酸混合溶液中，反应液45摄氏度下搅拌16小时。反应液用饱和碳酸氢钠调PH>7,再用乙酸乙酯萃取(30mL*3)，有机相旋干得到化合物9(180mg,收率97％)。
7.化合物016即2-((2-(反式-4-羟基环己基)-6-乙氧基-2H-吲唑-5-基)氨甲酰基)-6-甲基吡啶1- 氧化物和化合物220即2-((2-(顺式-4-羟基环己基)-6-乙氧基-2H-吲唑-5-基)氨甲酰基)-6-甲基吡啶1-氧化物的合成
Figure PCTCN2020117093-appb-000042
0摄氏度下将硼氢化钠(50mg)加到化合物9(180mg)的20mL甲醇溶液中，反应液25摄氏度搅拌1小时。反应液用10mL氯化铵溶液淬灭，乙酸乙酯萃取(20mL*3)，有机相减压浓缩，残余物通过高效液相制备色谱柱(CH 3CN:H 2O(0.1％NH 4HCO 3)＝20-50％,UV:214nm,流速:15ml/min)纯化得到保留时间Rt＝11.25min的化合物016(123mg,收率68％),保留时间Rt＝11.75min的化合物220(30mg,收率17％)。
化合物016
1H NMR(400MHz,CDCl 3):δ14.30(s,1H),8.88(s,1H),8.45(d,J＝7.6Hz,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＝6.8Hz,2H),3.82-3.80(m,1H),2.63(s,3H),2.28(d,J＝12.8Hz,2H),2.18(d,J＝14.8Hz,2H),2.06(q,J＝13.2Hz,2H),1.65-1.56(m,3H),1.55-1.50(m,2H).
LCMS:Rt＝2.486min,[M+H] +＝411.2.
化合物220
1H NMR(400MHz,CDCl 3):δ14.30(s,1H),8.89(s,1H),8.45(d,J＝7.6Hz,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＝8.8Hz,2H),4.14(br s,1H),2.63(s,3H),2.35(q,J＝8.8Hz,2H),2.08(d,J＝8.4Hz,2H),1.98(d,J＝13.2Hz,2H),1.75(t,J＝13.2Hz,2H),1.64(t,J＝6.8Hz,3H).LCMS:Rt＝2.642min,[M+H] +＝411.2.
实施例5化合物025的合成
反应式:
Figure PCTCN2020117093-appb-000043
1.化合物3的合成
-40摄氏度下向化合物2(45mL)的四氢呋喃溶液(100mL)中滴加化合物1(3g)的四氢呋喃溶液(10mL)，并在0摄氏度下搅拌反应8小时，用饱和氯化铵溶液(200mL)淬灭反应，用乙酸乙酯(200mL×3)萃取，萃取液用饱和食盐水(400mL)洗涤，无水硫酸钠干燥，过滤，减压浓缩，残留物用硅胶色谱柱纯化(石油醚:乙酸乙酯＝5:1)得到化合物3(1.5g,收率44％)。
2.化合物025即2-((2-(反式-4-羟基-顺式-4-环丙基环己基)-6-甲氧基-2H-吲唑-5-基)氨甲酰基)-6-甲基吡啶1-氧化物的合成
Figure PCTCN2020117093-appb-000044
25摄氏度下将碳酸铯(820mg)加到化合物4(300mg)和化合物3(374mg)的NMP(30mL)溶液中，反应液90摄氏度搅拌16h。冷却加入水(100mL)，乙酸乙酯萃取(80mL*4)，有机相减压浓缩，残留物高效液相制备色谱柱(CH 3CN:H 2O(0.1％NH 4HCO 3)＝5-95％，UV:214nm，流速：15mL/min)得到化合物025(79mg,收率18％)。
1H NMR(400MHz,CDCl 3):δ14.13(s,1H),8.89(s,1H),8.45(d,J＝12Hz,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.
实施例6化合物163的合成
反应式：
Figure PCTCN2020117093-appb-000045
1.化合物2的合成
15摄氏度下将对甲苯磺酰氯(11.8g)加入到化合物1(6.1g),三乙胺(14.8g)和DMAP(7.2g)的DCM(120mL)溶液中,反应液10摄氏度搅拌16小时。反应液用1N HCl溶液洗涤(100mL*3)，有机相干燥浓缩，得到化合物2(13.1g,收率84％)。
2.化合物9的合成
零下15摄氏度下向化合物8(2.0g)的浓硫酸(12mL，98％)溶液中滴加硝酸(1.6mL)的浓硫酸(1.6mL,98％)混合溶液。加完后混合体系零下15摄氏度搅拌2小时，将反应液缓慢倾倒入冰水中并搅拌5分钟，抽滤，用水洗涤，收集固体减压干燥得到化合物9(2.5g,收 率：97％)。
3.化合物3的合成
室温下向化合物8(2.0g)的DMF(20mL)溶液中加入水合肼(2.4mL,98％)，加完后混合体系加热到120摄氏度搅拌16小时，冷却到室温，混合体系慢慢倒入冰水中并搅拌，抽滤，用水洗涤固体，收集固体减压浓缩得到化合物3(1.3g,收率：67％)。
4.化合物4的合成
15摄氏度下将DIPEA(13.4g)加入到化合物3(4.0g)和化合物2(10.7g)的80mL的甲苯溶液中，反应液130摄氏度搅拌48小时，反应液加到100mL水中，乙酸乙酯萃取(50mL*3)，有机相减压浓缩，残余物通过高效液相制备色谱柱(CH3CN:H2O(0.1％NH4HCO3)＝10-50％,UV:214nm,流速:15ml/min)纯化得到化合物4(2.5g,收率43％)。
5.化合物5的合成
30摄氏度下将400mg的Pd/C加入到化合物4(1.3g)的乙酸乙酯(30mL)溶液中，反应在一个氢气球压力下搅拌16h,反应液合并过滤，减压浓缩，得到化合物5(1.9g，收率95％)。
6.化合物163即2-((2-(3-羟基-3-甲基丁基)-6-甲氧基-2H-吲唑-5-基)氨基甲酰基)-6-甲基吡啶1-氧化物的合成
Figure PCTCN2020117093-appb-000046
30摄氏度下将HATU(273mg),Et 3N(145mg)加入到化合物5(120mg)和化合物6(81mg)的DMF(2mL)溶液中，反应30摄氏度搅拌18小时，反应液减压浓缩，粗品用高效液相制备色谱柱(CH3CN:H2O(0.1％NH4HCO3)＝5-95％,UV:214nm,流速:15ml/min)纯化得到化合物163(110mg,60％).
1H NMR(400MHz,DMSO-d 6):δ14.13(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＝2.784min,[M+H] +＝385.2.
实施例7化合物284的合成
反应式:
Figure PCTCN2020117093-appb-000047
1.化合物2的合成
25摄氏度下将对甲苯磺酰氯(2.3g)加入到化合物1(1g),三乙胺(2.9g)和DMAP(1.4g)的DCM(20mL)溶液中，反应液25摄氏度搅拌16小时。反应液用1N HCl溶液洗涤(200mL*3)，有机相干燥浓缩，得到化合物2(2.1g，收率75％)。
2.化合物284即2-((2-环戊基-6-甲氧基-2H-吲唑-5-基)氨基甲酰基)-6-甲基吡啶1-氧化物的合成
Figure PCTCN2020117093-appb-000048
25摄氏度下将碳酸铯(1.6g)加入到化合物3(500mg)和化合物2(485mg)的10mL的DMF溶液中，反应液90摄氏度搅拌16小时。反应液加到50mL水中，乙酸乙酯萃取(30mL*3)，有机相减压浓缩，残余物通过高效液相制备色谱柱(CH 3CN:H 2O(0.1％NH 4HCO 3)＝25-60％,UV:214nm,流动速率:15ml/min)纯化得到化合物284(123mg,收率20％)。
1H NMR(400MHz,DMSO-d 6):δ14.15(s,1H),8.78(s,1H),8.32-8.29(m,2H),7.77-7.75(m,1H),7.57(t,J＝7.6Hz,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＝3.562min,[M+H] +＝367.2.
实施例8化合物285的合成
反应式:
Figure PCTCN2020117093-appb-000049
1.化合物285即2-((2-环己基-6-甲氧基-2H-吲唑-5-基)氨基甲酰基)-6-甲基吡啶1-氧化物的合成
Figure PCTCN2020117093-appb-000050
25摄氏度下将碳酸铯(1.3g)加入到化合物2(400mg)和化合物1(511mg)的8mL的NMP溶液中，反应液90摄氏度搅拌16小时。反应液加到30mL水中，乙酸乙酯萃取(10mL*3)，有机相减压浓缩，残余物通过高效液相制备色谱柱(CH 3CN:H 2O(0.1％NH 4HCO 3)＝25-75％,UV:214nm,流速:15ml/min)纯化得到化合物285(68mg,收率13％)。
1H NMR(400MHz,DMSO-d 6):δ14.15(s,1H),8.79(s,1H),8.32-8.29(m,2H),7.78-7.76(m,1H),7.58(t,J＝8.0Hz,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＝12.8Hz,1H),1.50-1.40(m,2H),1.31-1.23(m,1H).LCMS:Rt＝3.971min,[M+H] +＝381.2.
实施例9化合物286的合成
反应式:
Figure PCTCN2020117093-appb-000051
1.化合物2的合成
0摄氏度下将DAST(6g)加入到化合物1(2g)的DCM(70mL)溶液中,反应液25摄氏度搅拌3小时。反应液倒入50mL冰水中，二氯甲烷萃取(30mL*2)，有机相减压浓缩，残留物通过硅胶柱(PE:EA＝10:1)纯化得到化合物2(1.8g,收率82％)。
2.化合物286即2-((2-(4,4-二氟环己基)-6-甲氧基-2H-吲唑-5-基)氨基甲酰基)-6-甲基吡啶1-氧化物的合成
Figure PCTCN2020117093-appb-000052
25摄氏度下将碳酸铯(1.6g)加入到化合物3(500mg)和化合物2(731mg)的10mL的NMP溶液中，反应液90摄氏度搅拌16小时。反应液加到30mL水中，乙酸乙酯萃取(10mL*3)，有机相减压浓缩，残余物通过高效液相制备色谱柱(CH 3CN:H 2O(0.1％NH 4HCO 3)＝40-70％,UV:214nm,流速:15ml/min)纯化得到化合物286(129mg,收率18％)。
1H NMR(400MHz,DMSO-d 6):δ14.17(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＝8.0Hz,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＝3.692min,[M+H] +＝417.2.
实施例10化合物287的合成
反应式:
Figure PCTCN2020117093-appb-000053
1.化合物287即2-((2-(顺式-4-羟基-反式-4-甲基环己基)-6-二甲基氨基-2H-吲唑-5-基)氨甲酰基)-6-甲基吡啶1-氧化物的合成
Figure PCTCN2020117093-appb-000054
25摄氏度下将碳酸铯(800mg)加入到化合物1(255mg)和化合物2(350mg)的5mL的NMP溶液中，反应液90摄氏度搅拌16小时。反应液加到30mL水中，乙酸乙酯萃取(10mL*3)，有机相减压浓缩，残余物通过高效液相制备色谱柱(CH 3CN:H 2O(0.1％NH 4HCO 3)＝35-60％,UV:214nm,流速:15ml/min)纯化得到化合物287(51mg,收率15％)。
1H NMR(400MHz,CDCl 3):δ14.03(s,1H),8.92(s,1H),8.46(d,J＝8.0Hz,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＝3.298min,[M+H] +＝424.3.
实施例11化合物015和化合物288的合成
反应式：
Figure PCTCN2020117093-appb-000055
1.化合物3的合成
20摄氏度下依次将化合物1(5g)，化合物2(26g)和碳酸铯(29g)加入到DMF(400 mL)，置换氮气，90摄氏度下搅拌24小时，冷却至20摄氏度，加水(800mL)，乙酸乙酯(800mL×3)萃取，有机相用饱和氯化钠溶液(500mL×3)洗，无水硫酸钠干燥，过滤，减压浓缩，残留物用硅胶色谱柱纯化(石油醚：乙酸乙酯＝1：1)，MTBE(50mL)打浆，得到化合物3(1.2g，收率14％)。
2.化合物4的合成
25摄氏度下将0.1g Pd/C加入到化合物3(1.1g)的乙酸乙酯(200mL)溶液中，氢气球下搅拌16h，过滤，减压浓缩，得到化合物4(901mg，收率90％)。
3.化合物6的合成
将化合物5(900mg)溶于二氯甲烷(20mL)溶液中，慢慢加入mCPBA(2.5g)，反应液25摄氏度搅拌16h。过滤，残留物用亚硫酸钠水溶液淬灭后再用稀盐酸调节PH<7，用二氯甲烷萃取(50mL*3)，有机相浓缩并用(石油醚：乙酸乙酯＝1：1)过柱纯化得到化合物6(0.68g，收率69％)。
4.化合物7的合成
在25摄氏度下向化合物6(164mg)和化合物4(250mg)的DMF(10mL)溶液中加入HATU(376mg)和DIPEA(128mg)搅拌16h。反应相加水(100mL)，EA(20mL×3)萃取，萃取液用饱和氯化钠溶液(200mL)洗，无水硫酸钠干燥，过滤，减压浓缩，残留物用(二氯甲烷：甲醇＝40：1)过柱纯化打浆得到化合物7(480mg，收率99％)。
5.化合物8的合成
0摄氏度下，向化合物7(480mg)的二氧六环溶液(10mL)中加入4M盐酸(10mL)，30摄氏度下搅拌反应16小时，冷却到0摄氏度,用饱和NaHCO 3溶液调到pH＝8，EA(40mL×5)萃取，饱和NaCl(200mL)洗,无水硫酸钠干燥,过滤,减压浓缩得化合物8(435mg,收率99％)。
7.化合物015即2-((2-(反式-4-羟基环己基)-6-甲氧基-2H-吲唑-5-基)氨甲酰基)-6-异丙基吡啶1-氧化物和化合物288即2-((2-(顺式-4-羟基环己基)-6-甲氧基-2H-吲唑-5-基)氨甲酰基)-6-异丙基吡啶1-氧化物的合成
Figure PCTCN2020117093-appb-000056
0摄氏度下将硼氢化钠(118mg)加到化合物8(435mg)的20mL甲醇溶液中，30摄氏度搅拌16小时。反应液饱和氯化铵调到pH＝7，DCM(40mL×5)萃取，饱和NaCl(200mL)洗，减压浓缩，残余物通过高效液相制备色谱柱(CH 3CN:H 2O(0.1％NH 4HCO 3)＝30-70％,UV:214nm,流速:15ml/min)纯化得到保留时间Rt＝28.13min的化合物015(171mg,收率39％)；保留时间Rt＝29.25min的化合物288(44mg,收率10％)。
化合物015
1H NMR(400MHz,CDCl 3):δ14.18(s,1H),8.88(s,1H),8.44(d,J＝5.2Hz,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＝9.2Hz,2H),1.60-1.50(m,2H),1.36(s,3H).1.35(s,3H).LCMS:Rt＝3.531,[M+H] +＝425.2.
化合物288
1H NMR(400MHz,CDCl 3):δ14.17(s,1H),8.88(s,1H),8.44(d,J＝5.2Hz,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.
实施例12化合物014和化合物218的合成
反应式：
Figure PCTCN2020117093-appb-000057
1.化合物3的合成
将Pd(dppf)Cl 2(113mg)与K 3PO 4(13.4g)加到化合物1(7.3g)和化合物2(6.5g)的甲苯(70mL)溶液中，置换氮气，反应液100摄氏度搅拌16h。冷却，减压浓缩，残留物用薄层柱色谱(石油醚：乙酸乙酯＝20：1)纯化得到化合物3(1.2g，收率20％)。
2.化合物4的合成
25摄氏度下将m-CPBA(5.7g)加到化合物3(1.1g)的100mL DCM溶液中，反应液25摄氏度搅拌48小时，反应液用2.4g亚硫酸钠配成的饱和溶液淬灭，有机相用饱和碳酸氢钠(100mL×3)洗涤，有机相用无水硫酸钠干燥，过滤，减压浓缩得到化合物4(1.1g,收率92％)。
3.化合物5的合成
依次将化合物4(1.2g)和LiOH.H 2O(730mg)加入到THF/H 2O(30mL/10mL)中,置换氮气。加完后混合体系30℃搅拌4小时。加水(50mL)，水相用1N HCl缓慢调节pH至7，乙酸乙酯萃取(100mL×3)，用饱和食盐水洗涤(200mL)，无水硫酸钠干燥，抽滤，减压干燥得到化合物5(844mg,收率84％)。
4.化合物7的合成
在25℃下向化合物6(450mg)和化合物5(319mg)的吡啶(35mL)溶液中加入DECI(427mg)，搅拌16h。减压浓缩至干，加水(100mL)，DCM(100mL×3)萃取，萃取液用饱和氯化钠溶液(200mL)洗，无水硫酸钠干燥，过滤，减压浓缩，残留物用乙酸乙酯(10mL) 打浆得到化合物7(314mg，收率45％)。
5.化合物8的合成
0摄氏度下,向化合物7(289mg)的四氢呋喃溶液(25mL)中加入4M盐酸(250mL)，30摄氏度下搅拌反应16小时，冷却到0摄氏度,用饱和NaHCO 3溶液调到pH＝8，DCM(40mL×5)萃取，萃取液用饱和NaCl(200mL)洗,无水硫酸钠干燥,过滤,减压浓缩得化合物8(249mg,收率95％)。
6.化合物014即2-((2-(反式-4-羟基环己基)-6-甲氧基-2H-吲唑-5-基)氨甲酰基)-6-环丙基吡啶1-氧化物和化合物218即2-((2-(顺式-4-羟基环己基)-6-甲氧基-2H-吲唑-5-基)氨甲酰基)-6-环丙基吡啶1-氧化物的合成
Figure PCTCN2020117093-appb-000058
0摄氏度下将硼氢化钠(67mg)加到化合物8(249mg)的20mL甲醇溶液中，30摄氏度搅拌16小时。反应液饱和氯化铵调到pH＝7，DCM(40mL×5)萃取，萃取液用饱和NaCl(200mL)洗，减压浓缩，残余物通过高效液相制备色谱柱(CH 3CN:H 2O(0.1％NH 4HCO 3)＝30-70％,UV:214nm，流动速率:15ml/min)纯化得到保留时间Rt＝7.99min的化合物014(108mg,收率43％)；保留时间Rt＝8.50min的化合物218(14mg,收率5％)。
化合物014
1H NMR(400MHz,CDCl 3):δ14.26(s,1H),8.87(s,1H),8.39(d,J＝8.0Hz,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).
LCMS:Rt＝3.312,[M+H] +＝423.2.
化合物218
1H NMR(400MHz,CDCl 3):δ14.20(s,1H),8.88(s,1H),8.40(d,J＝8.0Hz,1H),7.80(s,1H),7.38-7.26(m,1H),7.06(s,2H),4.69(s,1H),4.41-4.35(m,1H),4.14(s,1H),4.05(s,3H),2.89-2.80(m,1H),2.41-2.31(m,2H),2.08-2.04(m,2H),2.00-1.96(m,2H),1.80-1.72(m,2H),1.28-1.26(m,2H),0.84-0.83(m,2H).
LCMS:Rt＝2.807,[M+H] +＝423.2.
实施例13化合物187的合成
反应式：
Figure PCTCN2020117093-appb-000059
1.化合物2的合成
15摄氏度下依次向化合物1(50g)的二氯甲烷溶液(500mL)中加入DMAP(42.5g)，TsCl(63.4g)，和三乙胺(63.9g)，并在25摄氏度下搅拌反应18小时。向反应液中加入二氯甲烷(200mL)用水洗涤(300mL×2)，1M的稀盐酸(300mL×3)洗涤，有机相用无水硫酸钠干燥，过滤，减压浓缩得到化合物2(98g。收率：99％)。
2.化合物3的合成
15摄氏度下向化合物2(50g)的四氢呋喃溶液(300mL)中加入1M稀盐酸(300mL)并在25摄氏度下搅拌反应20小时。冷却到0摄氏度,用1M的氢氧化钠溶液调到pH＝9,用乙酸乙酯(200mL×3)萃取,萃取液用饱和氯化钠溶液(300mL)洗涤,无水硫酸钠干燥,过滤,减压浓缩，残留物用石油醚(150mL)打浆得化合物3(39g,收率91％)。
3.化合物5的合成
-40摄氏度下向化合物4(5.0g)的四氢呋喃(100mL)中滴加化合物3(74.6mL)并在-40摄氏度下搅拌反应4小时，TLC显示反应完毕，用饱和氯化铵溶液(50mL)淬灭反应，乙酸乙酯(100mL×3)萃取，萃取液用饱和氯化钠溶液(50mL)洗涤，无水硫酸钠干燥,过滤,减压浓缩，残留物用硅胶色谱柱(石油醚：乙酸乙酯＝5:1)纯化得化合物5(900mg，收率：16％)。
4.化合物7的合成
将80mL浓硫酸加到1L的三口瓶中，零下12摄氏度搅拌5分钟(部分浓硫酸结冰状态)，然后在此温度下尽可能的慢慢加入化合物6(10g)保持温度无大幅度波动，此温度下搅拌五分钟，然后在零下12摄氏度左右缓慢滴加硝酸8mL和浓硫酸8mL的混合液，保持温度搅拌1.5小时。点板监测原料反应完全。将混合液慢慢倒入冰水中(保持低温)搅拌20分钟然后过滤，经过水洗涤后减压蒸干得到化合物7(13g,收率：100％)。
5.化合物8的合成
将化合物7(30g)溶解到450mL的DMF中，0摄氏度下慢慢滴入水合肼36.3mL(98％)，然后混合液在120摄氏度搅拌18小时。反应完全后将冷却后的反应液慢慢倒入冰水中，搅拌10分钟然后过滤，经过水洗涤后减压蒸干得到化合物8(20g,收率：69％)。
6.化合物9的合成
将化合物8(10g)和5g钯碳(10％)依次加入到200mL乙酸乙酯中，20摄氏度下反应液在氢气保护下搅拌16小时。反应完全后加入硅藻土将钯碳滤掉，滤液浓缩干燥得到化合物9(8g,收率：94％)。
7.化合物11的合成
25摄氏度下将m-CPBA(25g)加到化合物12(10g)的200mL DCM溶液中，反应液25摄氏度搅拌16小时。反应液过滤，滤液用15.6g亚硫酸钠配成的饱和溶液淬灭，混合溶液搅拌2小时，萃取，水相用稀盐酸调至pH<7，DCM萃取(50mL*3)，有机相合并浓缩，残余物用300mL EA打浆得到化合物11(10.1g,收率90％)。
8.化合物10的合成
25摄氏度下将EDCI.HCl(2.6g)加到化合物9(1.5g)和化合物11(1.4g)的Py(15mL)溶液中，反应液25摄氏度搅拌16小时。反应液浓缩蒸干，残留物通过MeOH/H 2O＝20mL/20mL打浆得到化合物10(1.3g，收率48％)。
9.化合物187即2-((2-(反式-4-羟基-顺式-4-乙炔基环己基)-6-甲氧基-2H-吲唑-5-基)氨甲酰基)-6-甲基吡啶1-氧化物的合成
Figure PCTCN2020117093-appb-000060
30摄氏度下依次向NMP(10mL)中加入化合物10(300mg)，化合物5(444mg)和碳酸铯(820mg)，并在90摄氏度下搅拌反应18小时，LCMS检测反应完毕，降至30摄氏度加水(15mL)淬灭反应，用乙酸乙酯(10mL×3)萃取,萃取液用饱和氯化钠溶液(10mL)洗涤,无水硫酸钠干燥,过滤,减压浓缩，残留物用高效液相制备色谱柱(CH 3CN:H 2O(0.1％NH 4HCO 3)＝5-90％,UV:214nm,流速:15ml/min)纯化得到化合物187(85mg,收率20％)。
1H NMR(400MHz,CDCl 3):δ14.14(s,1H),8.89(s,1H),8.46(dd,J  1＝7.6Hz，J  2＝2.8Hz,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＝3.162min,[M+H] +＝421.2
实施例14化合物019和化合物292的合成
反应式：
Figure PCTCN2020117093-appb-000061
1.化合物3的合成
冰浴下将PPh 3(15g)加入到化合物1(7g)和化合物2(3.37g)的THF(200mL)溶液中，反应液搅拌10min后，将DIAD(3.1g)慢慢滴加到反应液中，反应在30摄氏度下搅拌18h,向反应液加水50mL，乙酸乙酯萃取(40mL*4)，有机相减压浓缩，残留物用硅胶色谱柱纯化(PE/EA＝10/1 to PE/EA＝2/1)得到化合物3(6.0g，收率66％)。
2.化合物4的合成
15摄氏度下将Pd/C(1.0g，10％)加入到化合物3(6.5g)的乙酸乙酯(300mL)溶液中，反应在30摄氏度、氢气球下(760Torr)搅拌18小时，反应液过滤，减压浓缩，得到化合物4(4.5g，收率80％)。
3.化合物6的合成
25摄氏度下将EDCI.HCl(2.1g)加到化合物4(1.5g)和化合物5(1.1g)的吡啶(30mL)溶液中，反应液在25摄氏度搅拌16小时。反应液浓缩蒸干，残留物通过硅胶柱(PE:EA＝1:1)纯化得到化合物6(810mg，收率32％)。
4.化合物8的合成
25摄氏度下将碳酸铯(2.3g)加入到化合物6(810mg)和化合物7(1.1g)的15mL的DMF溶液中，反应液90摄氏度搅拌16小时。反应液加到50mL水中，乙酸乙酯萃取(30mL*3)，有机相减压浓缩，残余物通过高效液相制备色谱柱(CH 3CN:H 2O(0.1％NH 4HCO 3)＝30-55％,UV:214nm,流速:15ml/min)纯化得到化合物8(320mg,收率28％)。
5.化合物9的合成
25摄氏度下向化合物8(320mg)的4mL二氧六环溶液中加入4mL 2M稀盐酸，反应液25摄氏度搅拌16小时。反应液用碳酸氢钠溶液调至碱性，乙酸乙酯萃取(10mL*2)，有机相减压浓缩得到化合物9(250mg,收率86％)。
6.化合物019即2-((2-(反式-4-羟基环己基)-6-环丙甲氧基-2H-吲唑-5-基)氨甲酰基)-6-甲基吡啶1-氧化物和化合物292即2-((2-(顺式-4-羟基环己基)-6-环丙甲氧基-2H-吲唑-5-基)氨甲酰基)-6-甲基吡啶1-氧化物的合成
Figure PCTCN2020117093-appb-000062
0摄氏度下将硼氢化钠(44mg)加到化合物7(250mg)的5mL甲醇溶液中，反应液25摄氏度搅拌2小时。反应液用10mL氯化铵溶液淬灭，乙酸乙酯萃取(5mL*3)，有机相减压浓缩，残余物通过高效液相制备色谱柱(CH 3CN:H 2O(0.1％NH 4HCO 3)＝35-60％,UV:214nm,流速:15ml/min)纯化得到保留时间Rt＝10.7min的化合物019(77mg,收率31％)和保留时间Rt＝11.1min的化合物292(12mg,收率5％)。
化合物019：
1H NMR(400MHz,DMSO-d6):δ14.31(s,1H),8.78(s,1H),8.31-8.27(m,2H),7.77-7.75(m,1H),7.56(t,J＝7.6Hz,1H),7.07(s,1H),4.69(s,1H),4.40-4.32(m,1H),4.02(d,J＝6.8Hz,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＝3.391min,[M+H] +＝437.2.
化合物292：
1H NMR(400MHz,DMSO-d6):δ14.31(s,1H),8.79(s,1H),8.31-8.29(m,2H),7.77-7.75(m,1H),7.57(t,J＝7.6Hz,1H),7.08(s,1H),4.49(d,J＝6.8Hz,1H),4.40-4.34(m,1H),4.02(d,J＝6.8Hz,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＝3.101min,[M+H] +＝437.2.
实施例15化合物291的合成
反应式：
Figure PCTCN2020117093-appb-000063
1.化合物2的合成
0摄氏度下将化合物1(800mg)溶解到10mL四氢呋喃中，在0摄氏度下慢慢将LiHMDS(1M的THF溶液，5.50mL)慢慢滴加进去，0摄氏度下搅拌60分钟后向反应液中慢慢加碘甲烷(680mg)，此温度下反应1.5小时。反应完毕后向反应液中加入氯化铵饱和溶液(10mL)淬灭反应，然后用乙酸乙酯(25mL×2)萃取，减压浓缩，残留物用硅胶柱纯化(石油醚：乙酸乙酯＝7：1)得到化合物2(420mg。收率：55％)。
2.化合物3的合成
0摄氏度下依次将化合物2(700mg)和3M HCl(18mL)加入到四氢呋喃(18mL)溶液中，50摄氏度搅拌5小时。反应完毕后，向反应液中加入3M氢氧化钠水溶液调节pH＝8，然后用二氯甲烷(20mL×2)萃取，减压浓缩，残留物用硅胶柱纯化(石油醚：乙酸乙酯＝4：1)得到化合物3(420mg，收率：78％)。
3.化合物4的合成
25摄氏度下将化合物3(390mg)溶解到8mL乙醇中，零下70摄氏度下将硼氢化钠(112mg)的乙醇(1mL)溶液滴入到反应液中，零下70摄氏度搅拌1小时。反应完毕后向反应液中加入8mL水淬灭反应，然后用乙酸乙酯(15mL×2)萃取，有机相减压浓缩，残留物用硅胶色谱柱纯化(石油醚：乙酸乙酯＝1：1)得到化合物4(280mg，收率：66％)。
4.化合物5的合成
28摄氏度下依次将化合物4(250mg)，TosCl(406mg)，DMAP(261mg)和三乙胺(0.5mL)加入到二氯甲烷(8mL)中，在28摄氏度下搅拌反应18小时。反应完毕后将反应液浓缩蒸干，残留物用硅胶色谱柱纯化(石油醚：乙酸乙酯＝6：1)得到化合物5(370mg，收率：64％)。
5.化合物291即2-((2-(反式-4-氰基-顺式-4-甲基环己基)-6-甲氧基-2H-吲唑-5-基)氨甲酰基)-6-甲基吡啶1-氧化物的合成
Figure PCTCN2020117093-appb-000064
25摄氏度下将化合物5(296mg)，化合物6(350mg)和碳酸铯(808mg)加入到DMF(6mL)中，加完后混合体系加热到90摄氏度并搅拌18小时。反应完毕后加10mL水淬灭反应，用40mL乙酸乙酯萃取两次，有机相减压浓缩，残留物用高效液相制备色谱(CH 3CN:H 2O(0.1％NH 4HCO 3)＝5-95％,UV:214nm,流速:15ml/min)得到化合物291(80mg，收率19％)。
1H NMR(400MHz,CDCl 3):δ14.14(s,1H),8.88(s,1H),8.45(d,J＝7.6Hz,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＝3.310min,[M+H] +＝420.2
实施例16化合物002的合成
反应式：
Figure PCTCN2020117093-appb-000065
1.化合物3的合成
28摄氏度下将化合物2(1.0g)，化合物1(0.91g)，EDCI(1.6g)加入到吡啶(15mL)中，并在28摄氏度下搅拌18小时，反应完成后，减压浓缩，残留物用甲醇和水打浆得化合物3(1.0g，收率：56％)。
2.化合物002即2-((2-(反式-4-羟基-顺式-4-甲基环己基)-6-甲氧基-2H-吲唑-5-基)氨甲酰基)-6-环丙基吡啶1-氧化物的合成
Figure PCTCN2020117093-appb-000066
25摄氏度下依次向DMF(10mL)中加入化合物3(500mg)，化合物4(675mg)，碳酸铯(1.26g)，并在90摄氏度下搅拌反应18小时，反应完成后，冷却到25摄氏度，加水(5mL)淬灭反应，用乙酸乙酯(15mL×3)萃取，萃取液用饱和食盐水(10mL)洗涤，无水硫酸钠干燥，过滤，减压浓缩，残留物用高效液相制备色谱(CH 3CN:H 2O＝20-45％，UV:214nm，流速15mL/min)得到化合物002(130mg，收率19％)。
1H NMR(400MHz,CDCl 3):δ14.22(s,1H),8.89(s,1H),8.40(dd,J 1＝2.0Hz，J 2＝8.0Hz，1H),7.87(s,1H),7.37(t,J＝8.0Hz,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＝2.859min,[M+H] +＝437.2
实施例17化合物289的合成
反应式：
Figure PCTCN2020117093-appb-000067
1.化合物3的合成
26摄氏度下依次将化合物1(5.0g)，化合物2(2.3g)，碳酸铯(13.4g)和Pd 2(dba) 3(0.25g)，BINAP(0.51g)加入到甲苯(100mL)中，并在氮气保护下80摄氏度搅拌反应18小时，反应完毕，降温到26摄氏度，加水(100mL)，用乙酸乙酯(200mL×3)萃取，萃取液用饱和食盐水(100mL)洗涤，无水硫酸钠干燥，减压浓缩，残留物用硅胶色谱柱(石油醚：乙酸乙酯＝10:1)纯化得到化合物3(1.3g，收率30％)。
2.化合物4的合成
向化合物3(1.3g)的乙醇/水(40mL/10mL)溶液中加入氢氧化钾(4.58g)，并在90摄氏度下搅拌反应16小时，用1M稀盐酸调PH＝6，用乙酸乙酯(100mL×3)萃取，萃取液用水(50mL)，饱和食盐水(100mL)洗涤，无水硫酸钠干燥，减压浓缩得到化合物4(1.12g，收率77％)。
3.化合物5的合成
26摄氏度下向化合物4(360mg)的二氯甲烷溶液(50mL)中加入化合物m-CPBA(1.86g)并在26℃下搅拌反应3天。反应完毕后，过滤，滤液中加入亚硫酸钠的饱和溶液，用稀盐酸调节pH<7，在26℃下搅拌2小时，用二氯甲烷(200mL×3)萃取，萃取液经饱和食盐水(100mL)洗涤，无水硫酸钠干燥，过滤，减压浓缩，残留物制备板纯化(二氯甲烷：甲醇＝20:1)纯化得到化合物5(60mg，收率15％)。
4.化合物289即2-((2-(反式-4-羟基-顺式-4-甲基环己基)-6-甲氧基-2H-吲唑-5-基)氨甲酰基)-6-环丙基氨基吡啶1-氧化物的合成
Figure PCTCN2020117093-appb-000068
将化合物5(49mg)，化合物6(69mg)，HATU(118mg)，DIPEA(66mg)，依次加入到DMF(5mL)中，并在25摄氏度下搅拌反应18小时，减压浓缩，残留物用高效液相制备色谱(CH 3CN:H 2O＝30-95％，UV:214nm，流速15mL/min)纯化后得到化合物289(95mg，收率83％)。
1H NMR(400MHz,CDCl 3):δ14.29(s,1H),8.89(s,1H),7.86(t,J＝6.4Hz,2H),7.41(t,J＝8.0Hz,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＝3.308min,[M+H] +＝452.2.
5.化合物9的合成
0℃下向化合物10(34g)的乙醇溶液(350mL)中加入NaN 3(13.2g)，在25℃下搅拌反应16小时，反应完后直接投入下一步。
6.化合物7的合成
25℃下向化合物9(0.17mol)的乙醇溶液(350mL)中加入醋酸(30.6g)，化合物8(22g)25℃搅拌10分钟，80℃回流反应16h，反应完全后，反应液部分浓缩，加入水(70mL)进行打浆，过滤得固体，固体加入乙醇(200mL)加热回流溶解，冷却至室温，加入正庚烷(200mL)打浆2小时，过滤两步得化合物7(35g，收率67％)。
7.化合物6的制备
25℃下向化合物7(300mg)的乙酸乙酯溶液(50mL)中加入Pd/C(150mg)并在25℃下搅拌反应16小时。反应完成后，过滤，减压浓缩得到化合物6(260mg，收率96％)。
实施例18化合物175的合成
反应式：
Figure PCTCN2020117093-appb-000069
1.化合物175即2-((2-(反式-4-羟基-顺式-4-乙氰基环己基)-6-甲氧基-2H-吲唑-5-基)氨甲酰基)-6-甲基吡啶1-氧化物的合成
Figure PCTCN2020117093-appb-000070
25摄氏度下将碳酸铯(1.4g)加入到化合物1(520mg)和化合物2(806mg)的10mL的DMF溶液中，反应液90摄氏度搅拌16小时。反应液倒入50mL水中，乙酸乙酯萃取(30mL*3)，有机相减压浓缩，残余物通过高效液相制备色谱柱 (CH 3CN:H 2O(0.1％NH 4HCO 3)＝20-40％,UV:214nm,流速:15ml/min)纯化得到化合物175(64mg,收率8％)。
1H NMR(400MHz,DMSO-d 6):δ14.16(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＝8.0Hz,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＝9.367min,[M+H] +＝436.2
实施例19化合物176的合成
反应式：
Figure PCTCN2020117093-appb-000071
1.化合物176即2-((2-(反式-4-羟基-顺式-4-乙氰基环己基)-6-甲氧基-2H-吲唑-5-基)氨甲酰基)-6-环丙基吡啶1-氧化物的合成
Figure PCTCN2020117093-appb-000072
25摄氏度下依次向DMF(10mL)中加入化合物8(420mg)，化合物9(601mg)，碳酸铯(1.06g，并在90摄氏度下搅拌反应16小时，反应完毕，冷却到25摄氏度，加水(5mL)淬灭反应，用乙酸乙酯(10mL×3)萃取，萃取液用饱和食盐水(10mL)洗涤，无水硫酸钠干燥，过滤，减压浓缩，残留物用高效液相制备色谱(CH 3CN:H 2O＝25-55％，UV:214nm，流速15mL/min)纯化后再用制备板(二氯甲烷：甲醇＝20:1)纯化得到化合物176(25mg，收率4％)。
1H NMR(400MHz,CDCl 3):δ14.25(s,1H),8.89(s,1H),8.39(dd,J 1＝2.0Hz，J 2＝8.0Hz，1H),7.86(s,1H),7.38(t,J＝8.0Hz,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＝3.553min,[M+H] +＝462.2
实施例20化合物042的合成
反应式：
Figure PCTCN2020117093-appb-000073
1.化合物2的合成
25℃下向化合物1(5.0g)的二氯甲烷溶液(50mL)中加入化合物m-CPBA(13.3g) 并在25℃下搅拌反应18小时。过滤，滤液中加入亚硫酸钠(8.2g)的饱和水溶液，在25℃下搅拌2小时，用二氯甲烷(50mL×3)萃取，萃取液用饱和食盐水(50mL)洗涤，无水硫酸钠干燥，过滤，减压浓缩，残留物用乙酸乙酯打浆纯化得到化合物2(600mg，收率11％)
2.化合物042即2-((2-(反式-4-羟基-顺式-4-甲基环己基)-6-甲氧基-2H-吲唑-5-基)氨甲酰基)-6-甲氧基吡啶1-氧化物的合成
Figure PCTCN2020117093-appb-000074
25℃下向DMF(5mL)中加入化合物2(92mg)，化合物4(150mg)，HATU(311mg)，三乙胺(165mg)并在25℃下搅拌反应16小时，反应完成后，加水(5mL)淬灭反应，用乙酸乙酯(5mL×3)萃取，萃取液用饱和食盐水(10mL)洗涤，无水硫酸钠干燥，减压浓缩，残留物用高效液相制备色谱(CH 3CN:H 2O＝10-40％，UV:214nm，流速15mL/min)得到化合物042(96mg，收率41％)。
1H NMR(400MHz,CDCl 3):δ14.15(s,1H),8.89(s,1H),8.22(dd,J 1＝2.0Hz，J 2＝8.0Hz，1H),7.86(s,1H),7.49(t,J＝8.0Hz,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＝2.713min,[M+H] +＝427.2
实施例21化合物B即2-((2-(反式-4-羟基-顺式-4-甲基环己基)-6-甲氧基-2H-吲唑-5-基)氨甲酰基)-6-甲基吡啶的合成
Figure PCTCN2020117093-appb-000075
25摄氏度下依次向DMF(5mL)中加入化合物1(150mg)，化合物2(75mg)，HATU(249mg)，DIPEA(141mg)，并在25摄氏度下搅拌反应16小时，加水(50mL)，用乙酸乙酯(10mL×3)萃取，萃取液用饱和食盐水(10mL)洗涤，无水硫酸钠干燥，过滤，减压浓缩，残留物用高效液相制备色谱(CH 3CN:H 2O＝30-95％，UV:214nm，流速15mL/min)纯化后得到白色固体170mg，收率79％。
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.
生物学评价
以下测试例用于进一步解释本发明，但这些测试例并非意味着限制本发明的范围。
生物测试例中的化合物A结构：
Figure PCTCN2020117093-appb-000076
生物测试例中的化合物B为实施例21合成的结构
Figure PCTCN2020117093-appb-000077
测试例1、测定本发明化合物对人IRAK4激酶活性的抑制作用
主要试验材料
ATP(Sigma，货号：A7699-1G)
DMSO(Sigma，货号D2650)
EDTA(Sigma，货号：E5134)
HEPES(Sigma，货号：V900477-500G)
DTT(Sigma，货号：D0632-25g)
Brij-35(Sigma，货号：B4184)
96孔板(Corning，货号：3365)
384孔板(Corning，货号：3573)
实验步骤
化合物在ATP的Km浓度时对IRAK4抑制活性，在下文描述的IRAK4MSA(Mobility-Shift Assay，微流体芯片技术的迁移率检测技术)中进行测量。
使用N-末端GST(谷胱甘肽-S-转移酶)和人IRAK4的重组融合蛋白作为酶(GST-IRAK4，激酶IRAK4(Carna，货号：09-145))，终浓度为1nM；ATP ATP(Sigma，货号：A7699-1G)终浓度为37μM；用于激酶反应的底物为5-FAM(5-羧基荧光素)标记的多肽(5-FAM-IPTSPITTTYFFFKKK-COOH)，底物肽FAM-P8(GL Biochem，货号：112396)，终浓度为5μM。
在该试验中，用100％DMSO配制成500μM的化合物溶液，并用100％DMSO 4倍稀释10个浓度梯度，再用化合物缓冲液(50mM HEPES，pH 7.5，0.00015％Brij-35)进一步稀释10倍，配成含10％DMSO的化合物中间稀释溶液，化合物终浓度在10μM-0.04nM范围内，转移5μl至黑色384孔板中。
将激酶IRAK4用激酶缓冲液(50mM HEPES，pH 7.5，0.00015％Brij-35，2mM DTT) 稀释成2.5nM的IRAK4溶液，并转移10μl至384孔板中，与化合物共孵育10-15分钟。
将底物和ATP分别用反应缓冲液(50mM HEPES，pH 7.5，0.00015％Brij-35，10mM MgCl 2)稀释成12.5μM和92.5μM。转移10μl至384孔板，起始反应，并于28摄氏度反应1小时。转移25μl 50mM EDTA至384孔板，终止反应。
用Caliper EZ Reader(PerkinElmer)读取底物磷酸化的转化率，从而计算化合物对IRAK4的抑制率，用XL-fit软件计算IC 50。
测试结果表明，本发明实施例化合物对IRAK4激酶活性均有较好的抑制作用，IC 50值小于100nM，优选为小于30nM。具体的，一些示例性的化合物活性数值如下所示：
本发明化合物对人IRAK4激酶活性的抑制IC 50值如表1所示，
表1对人IRAK4激酶活性的IC 50
化合物ID IC 50(nM)
化合物B 30.0
001 6.0
002 3.7
010 11.0
013 7.6
014 2.5
015 5.4
016 8.1
019 8.1
025 13.0
163 23.0
175 7.1
176 4.3
187 9.2
218 8.3
220 14.0
284 28.0
285 5.9
286 14.0
287 13.0
288 13.0
289 1.7
291 14.0
292 14
测试例2、测定本发明化合物对人IRAK1激酶活性的抑制作用
本试验用于评价化合物对人IRAK1激酶活性的抑制作用，主要试验材料同测试例1。
化合物在ATP的Km浓度时对IRAK1抑制活性，在下文描述的IRAK1 MSA(Mobility-Shift Assay，微流体芯片技术的迁移率检测技术)中进行测量。使用N-末端GST(谷胱甘肽-S-转移酶)和人IRAK1的重组融合蛋白作为酶(GST-IRAK1，激酶IRAK1，Carna)，终浓度为3nM；ATP(Sigma)终浓度为97μM；用于激酶反应的底物为5-FAM(5-羧基荧光素)标记的多肽(5-FAM-IPTSPITTTYFFFKKK-COOH)，底物肽FAM-P8(GL Biochem)，终浓度为5μM。
在该试验中，用100％DMSO配制成500μM的化合物溶液，并用100％DMSO 4倍稀释10个浓度梯度，再用化合物缓冲液(50mM HEPES，pH 7.5，0.00015％Brij-35)进一步稀释10倍，配成含10％DMSO的化合物中间稀释溶液，化合物终浓度在10μM-0.04nM范围内，转移5μl至黑色384孔板中。
将激酶IRAK1用激酶缓冲液(50mM HEPES，pH 7.5，0.00015％Brij-35，2mM DTT)稀释成7.5nM的IRAK1溶液，并转移10μl至384孔板中，与化合物共孵育10-15分钟。
将底物和ATP分别用反应缓冲液(50mM HEPES，pH 7.5，0.00015％Brij-35，10mM MgCl2)稀释成12.5μM和242.5μM。转移10μl至384孔板，起始反应，并于28摄氏度反应1小时。转移25μl 50mM EDTA至384孔板，终止反应。用Caliper EZ Reader(PerkinElmer)读取底物磷酸化的转化率，从而计算化合物对IRAK1的抑制率，用XL-fit软件计算IC50。
测试结果表明，本发明实施例化合物对IRAK4具有显著的选择性抑制活性，IRAK1与IRAK4的IC50(nM)比值大于500，优选为大于200。具体的，一些示例性的化合物活性数值如下所示：本发明化合物对人IRAK1激酶活性的抑制IC 50值如表2所示。
表2对人IRAK1激酶活性的IC 50
化合物ID IRAK1IC 50(nM) IRAK1IC 50(nM)/IRAK4IC 50(nM)
163 2993 130.1
001 4039 673.2
从表2中数据可以看出，对比人IRAK1活性，本发明化合物对人IRAK4具有显著的选择性。
测试例3、测定本发明化合物的hERG试验
本试验用于评价化合物的心脏安全性,采用了hERG钾通道稳定表达的HEK-293细胞系进行实验检测。
仪器信息：
放大器：购自HEKA(Germany)，型号EPC10
微操纵器：购自Sutter Instruments(USA)，型号MP225
电极拉制仪：购自Sutter Instruments(USA)，型号P97
显微镜：购自Nikon，型号TE300
毛细玻璃管：购自Sutter Instruments(USA)，型号BF150-86-10
数据采集和分析软件：PatchMaster，Igor Pro 6.0以及GraphPad Prism 5.0
实验方法
用DMSO将测试化合物储液依次稀释为0.3mM,1mM以及3mM的稀释液。用细胞外液(140mM NaCl,3.5mM KCl,1mM MgCl2,2mM CaCl2,10mM Glucose,10mM HEPES,1.25mM NaH2PO4,NaOH调节pH＝7.4)稀释待测试化合物储液，得到浓度为0.3μΜ,1μΜ,3μM,10μΜ,30μΜ的待测试化合物工作液，所有待测试化合物工作液进行超声20min。
膜片钳检测:在倒置显微镜下，操纵玻璃电极微操纵器(微操)将记录电极接触到细胞，然后给予负压作用，促进细胞形成GΩ封接。形成GΩ封接后，进行快速电容补偿，然后持续给予负压作用，吸破细胞膜，形成全细胞记录模式。在全细胞记录模式下，进行慢速电容补偿并记录膜电容及串联电阻的数值。
细胞hERG钾电流的电压刺激方案如下：细胞膜钳制电压为-80mV，然后由-80mV除极至+30mV维持2.5秒，然后迅速保持在-50mV维持4秒，激发出hERG通道的尾电流。每隔10秒重复采集数据。以-50mV为漏电流检测。
将种植细胞的盖玻片，放置于倒置显微镜的记录小室中，阴性对照以及测试化合物利用重力灌流的方法从低浓度到高浓度，依次流经记录小室从而快速作用于细胞。在记录中，利用真空泵进行外液的持续循环。以每个细胞阴性对照中，检测到的电流，作为细胞自己的对照组。每个药物浓度作用5分钟或者至电流稳定。所有实验在室温下进行。
数据分析:
首先标准化每一个药物浓度作用后的电流
Figure PCTCN2020117093-appb-000078
然后计算对应的抑制率
Figure PCTCN2020117093-appb-000079
对每一个浓度计算基本统计量，包括平均数(Mean)，保准差(SD)，标准误差(SE)以及重复例数(n)。用以下的方程拟合剂量依赖曲线，并计算测试化合物的半抑制浓度(IC 50)：
其中C代表测试化合物浓度，IC 50代表半抑制浓度，h代表希尔系数。曲线拟合以及IC 50的计算利用GraphPad Prism 5.0软件完成。
测试结果表明，本发明实施例化合物对人hERG的抑制率很低，甚至可以显著优于对比化合物A，对于hERG的抑制(30μM)小于50％，优选地，小于30％。具体的，一些示例性的化合物抑制率数值如下所示：
表3 30M浓度下对hERG的抑制
化合物ID hERG抑制(30M)
A 27.10％±1.74％
163 8.73％±1.37％
001 5.09％±2.43％
从表3中数据可以看出，本发明化合物对人hERG的抑制率低，且对化合物A有一定优 势。
测试例4、测定本发明化合物的TDI(时间依赖性抑制)数据
本实验研究的目的是研究化合物对人P450代谢酶CYP3A4的时间依赖性抑制作用。本实验利用的人的混合肝微粒体来源于美国Corning公司。
受试化合物将与人肝微粒体以及探针底物咪达唑仑(CYP3A4)进行共孵育，受试化合物将设置为30μM。反应将由辅酶NADPH的加入来启动。在孵育体系中加入乙腈来终止反应，乙腈中已事先溶有内标。蛋白沉淀后，离心取上清。上清液中的特征性代谢物1-羟基-咪达唑仑(CYP3A4)由LC-MS/MS方法分析。最后根据所得数据来研究受试化合物对这些特征性代谢物生成的影响。选择性抑制剂(维拉帕米对CYP3A4)将会作为阳性对照。
测试结果表明，本发明实施例化合物对人CYP3A4无明显的时间依赖性抑制，具体的，一些示例性的化合物的TDI数值如下所示：
表4 30μM浓度下对人CYP3A4的时间依赖性抑制(TDI)
化合物ID TDI(3A4，30μM)
001 -3.68％
014 +3.85％
测试例5、测定本发明化合物的血浆蛋白结合率(PPB)数据
本试验的目的是测定受试化合物的血浆蛋白结合率(PPB)数据。
在PPB试验中，最终给药基质中含受试化合物或参照化合物的浓度为1μM DMSO含量0.2％。
收集0小时样品：取25μL含化合物的基质加入到空白的96孔收集板中，储存于-20℃。
准备平衡透析装置。将100μL缓冲液加入到平衡透析板的接受侧。再将100μL含化合物或参照化合物的给药基质加入到平衡透析板的给药侧。将准备好的平衡透析板置于37℃摇床中以60转/分钟速度振摇5小时。
在培养结束时(5小时)，进行样品制备：
制备接受侧样品：由接受侧的样品取出25μL，置于96孔样品收集板中，加入25μL相应的基质(空白血浆)混合。加入200μL含内标的ACN，于600转/分钟振荡10分钟，然后在离心机上采用5594g离心15分钟。
制备给药侧样品：受试化合物及参照化合物给药侧样品：取25μL给药侧样品，加入25μL空白缓冲溶液混合。加入200μL含内标的ACN，于600转/分钟振荡10分钟，然后在离心机上采用5594g离心15分钟。
制备0小时样品：受试化合物及参照化合物0小时样品：37℃复融，与相同体积(25μL)相应的基质(空白缓冲溶液)混合。加入200μL含内标的ACN，于600转/分钟振荡10分钟，然后在离心机上采用5594g离心15分钟。
所有样品离心后，取50μL上清加入50μL超纯水混合，样品送至液质联用分析。
测试结果表明，本发明实施例化合物对人、大鼠、小鼠的血浆蛋白结合率适中，种属间的差异很小，甚至可以显著小于对比化合物A，具体的，一些示例性的化合物的PPB数据如下所示：
表5血浆蛋白结合率(PPB)数据
Figure PCTCN2020117093-appb-000080
测试例6、本发明化合物对LPS诱导的Balb/c雌性小鼠释放TNF-α的抑制作用
实验步骤
将雌性Balb/c小鼠随机分成若干组，每组4只，组别包括正常对照+溶媒组、模型+溶媒组、模型+阳性药组及其它模型+测试药组。正常对照组动物接受腹腔注射生理盐水(10ml/kg)，模型动物接收LPS刺激(Sigma货号L2630,腹腔注射,10mL/kg，0.2mg/kg)。实验中测试药依次加入DMSO,Solutol,10mM PBS制成所需给药浓度的溶液或浊液，溶媒各成分DMSO、Solutol、10mM PBS的终体积比为5:15:80。各实验组按设定剂量在LPS(或saline)刺激前16h进行相应的灌胃给药(10ml/kg)，各组动物在刺激后1.5h用CO2安乐死，进行心脏采血。所得全血不抗凝，于湿冰中静置1.5h后2000g,4℃离心10min分离血清。血清-80℃冻存备TNFα测定。TNFα的定量通过TNFαELISA试剂盒，按制造商的使用说明书完成测定。吸光度A450的读值用酶标仪SpectraMax i3x(Molecular Device)检测，从而计算化合物对的抑制率，用GraphPad Prism 7.0软件计算IC 50。
测试结果表明，本发明实施例化合物对LPS诱导的Balb/c雌性小鼠释放TNF-α具有明显的抑制作用，抑制率大于50％，优选的，大于70％，具体的，一些示例性的化合物的抑制率如下所示：
表6 LPS诱导的Balb/c雌性小鼠释放TNF-α的抑制率
化合物ID TNF-α的抑制率％
013 76.29
001 74.00
163 71.56
016 78.71
测试例7、测定本发明化合物对人肝微粒体五种主要CYP450酶亚型的抑制
本实验研究的目的是研究受试化合物对5种主要人P450代谢酶，即CYP1A2、2C9、2C19、2D6和3A4-M的抑制作用进行研究。本实验利用的人的混合肝微粒体来源于美国Corning公司。受试化合物(化合物14)将与人肝微粒体以及五种探针底物(非那西丁对CYP1A2，双氯芬酸对CYP2C9，美芬妥英对CYP2C19，右美沙芬对CYP2D6，咪达唑仑对CYP3A4-M，为混合底物)进行共孵育(见下表)，受试化合物将设置7个浓度点。反应将由辅酶NADPH的加入来启动。在孵育体系中加入含有内标的冰乙腈来终止反应。蛋白沉淀后，离心取上清。上清液中的特征性代谢物(对乙酰氨基酚对CYP1A2，4-羟基双氯芬酸对CYP2C9，4-羟基美芬妥英对CYP2C19，右啡烷对CYP2D6，1-羟基-咪达唑仑对CYP3A4-M)由LC-MS/MS方法分析。最后根据所得数据来研究受试化合物对这些特征性代谢物生成的影响。选择性抑制剂将(酮康唑对CYP3A4-M)会作为阳性对照。所有的孵育均平行1份进行。
测试结果表明，本发明实施例化合物对人的5种CYP亚型均无明显的抑制作用，且在1A2、2C9、2C19这3个亚型上的抑制作用显著小于对比化合物A，具体的，一些示例性的化合物的抑制率如下所示：
表7对人肝微粒体五种主要CYP450酶亚型CYP1A2、2C9、2C19、2D6和3A4的抑制(IC 50,nM)
Figure PCTCN2020117093-appb-000081
测试例8、本发明化合物对大鼠的PK分析测试
本发明优选实施例的小鼠药物代谢动力学试验，采用雄性SPF级的SD大鼠(上海西普尔-必凯实验动物有限公司)进行。
给药方式：单次灌胃口服给药或单次静脉注射
取样点：给药后0.083,0.25,0.5,1,2,4,6,8,24小时
样品处理：静脉采血0.2mL，血液样本采集后置于冰上，离心分离血浆(离心条件：8000转/分钟，6分钟，4℃)。收集的血浆分析前存放于-80℃。
内标工作液：吸取一定量的浓度为645,000ng/mL的甲苯磺丁脲内标储备液至一定体积的容量瓶中，用甲醇定容至刻度后混匀，制得浓度为50ng/mL的内标工作溶液。
样品前处理：取50μL血浆样品至1.5mL离心管中，加入250μL内标溶液(空白不加内标补加相同体积的甲醇)，涡旋混匀，14000转/分钟离心5分钟，取200μL上清液加入到96孔进样板中，LC-MS/MS进样分析。
液相条件：
色谱柱：ACQUITY UPLC BEH C18 1.7μm(50mm×2.10mm)
移动相：A液为0.1％甲酸水溶液，B液为0.1％甲酸乙腈溶液
流速：0.5mL/min
数据处理系统为Analyst软件(美国应用生物系统公司，软件版本号1.5.5)。
测试结果表明，本发明实施例化合物在小鼠上均表现出良好的药代动力学特征，在动物体内显示出良好的暴露量和滞留时间，且具有适宜的半衰期和良好的药物吸收性，具体的，一些示例性的化合物的药代动力学数据如下所示：
表8单次灌胃口服给予ICR小鼠不同化合物的药代动力学研究数据
Figure PCTCN2020117093-appb-000082
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种式I所示的化合物、其立体异构体、消旋体、互变异构体、同位素标记物、前药或其药学上可接受的盐：
    Figure PCTCN2020117093-appb-100001
    其中，
    环A为含有至少一个含N的5-14元杂芳基或5-12元杂环基；
    每个R 1、R 2、R 3各自独立的选自氢、卤素、CN、OH或任选被一个、两个或更多个R取代的如下基团：(C 1-C 12)脂肪烃基，任选地包含一个、两个或更多个杂原子(C 1-C 12)脂肪烃基，C 3-12环烷基、3-12元杂环基、C 6-20芳基或5-14元杂芳基、-NR aR b；
    W选自O，S，NH，单键；
    每个R a、R b独立的选自H、(C 1-C 12)脂肪烃基；
    每个R独立的选自卤素、CN、OH、SH、NR aR b或选自任选被一个、两个或更多个R’取代的如下基团：(C 1-C 12)脂肪烃基，任选地包含一个、两个或更多个杂原子(C 1-C 12)脂肪烃基，C 3-12环烷基、3-12元杂环基、C 6-20芳基或5-14元杂芳基；
    每个R’独立的选自卤素、CN、OH、SH、NR aR b；
    n选自1、2、3；m选自1、2、3、4、5、6。
  2. 一种根据权利要求1所述的式I所示的化合物、其立体异构体、消旋体、互变异构体、同位素标记物、前药或其药学上可接受的盐，其特征在于：所述“任选地包含一个、两个或更多个杂原子(C 1-C 12)脂肪烃基”可以选自(C 1-C 12)脂肪烃基氧基、(C 1-C 12)脂肪烃基巯基，(C 1-C 6)脂肪烃基氧基(C 1-C 6)脂肪烃基、(C 1-C 6)脂肪烃基巯基(C 1-C 6)脂肪烃基、N-(C 1-C 3)脂肪烃基胺基(C 1-C 6)脂肪烃基、N,N-二-(C 1-C 3)脂肪烃基胺基(C 1-C 6)脂肪烃基；
    所述“含N的5-14元杂芳基或5-12元杂环基”选自吡啶，吡咯，哌啶，四氢吡咯。
    所述(C 1-C 12)脂肪烃基可以选自(C 1-C 12)烷基、(C 2-C 12)烯基、(C 2-C 12)炔基，优选的，所述(C 1-C 12)脂肪烃基可以选自(C 1-C 6)烷基、(C 2-C 6)烯基、(C 2-C 6)炔基；
    所述“卤素”选自F、Cl、Br、I；
    所述“C 3-12环烷基”可以选自环丙基、环丁基、环戊基、环己基。
  3. 一种根据权利要求1或2所述的式I所示的化合物、其立体异构体、消旋体、互变异构体、同位素标记物、前药或其药学上可接受的盐，其特征在于：
    所述R 1、R 2、R 3可以各自独立的选自任选被一个、两个或更多个R取代的如下基团：甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、正戊基、异戊基、新戊基、正己基、 乙烯基、1-丙烯基、2-丙烯基、1-甲基乙烯基、1-丁烯基、1-乙基乙烯基、1-甲基-2-丙烯基、2-丁烯基、3-丁烯基、2-甲基-1-丙烯基、2-甲基-2-丙烯基、1-戊烯基、1-己烯基、乙炔基，1-丙炔基、2-丙炔基、1-丁炔基、1-甲基-2-丙炔基、3-丁炔基、1-戊炔基、1-己炔基、环丙基、环丁基、环戊基、环己基、甲氧基，乙氧基，丙氧基，丁氧基，戊氧基，甲氧基甲基，乙氧基甲基、丙氧基甲基、甲氧基乙基、乙氧基乙基、丙氧基乙基、甲氧基丙基、乙氧基丙基、丙氧基丙基、N-甲基胺甲基、N-甲基胺乙基、N-乙基胺乙基、N,N-二甲基胺甲基、N,N-二甲基胺乙基、N,N-二乙基胺乙基、氨基、N,N-二甲基氨基、N,N-二乙基氨基、四氢吡咯基、哌啶基、吡啶基，吡嗪基、吡咯基，咪唑基、吡唑基、恶唑基、异恶唑基、
    Figure PCTCN2020117093-appb-100002
    所述
    Figure PCTCN2020117093-appb-100003
    示意所述基团的连接位点。
  4. 一种根据权利要求1-3任一项所述的式I所示的化合物、其立体异构体、消旋体、互变异构体、同位素标记物、前药或其药学上可接受的盐，其特征在于：
    所述式I所示的化合物、其立体异构体、消旋体、互变异构体、同位素标记物、前药或其药学上可接受的盐中，式I所示的化合物可以选自如下式Ia、式Ib、式Ic、式Id、式Ie结构：
    Figure PCTCN2020117093-appb-100004
    Figure PCTCN2020117093-appb-100005
    所述式Ia、式Ib、式Ic、式Id、式Ie中，R 1，R 2，R 3，m，n，W如式I所定义。
  5. 一种根据权利要求1-4任一项所述的式I所示的化合物、其立体异构体、消旋体、互变异构体、同位素标记物、前药或其药学上可接受的盐，其特征在于，所述式I所示的化合物可以选自如下结构：
    Figure PCTCN2020117093-appb-100006
    Figure PCTCN2020117093-appb-100007
    Figure PCTCN2020117093-appb-100008
    Figure PCTCN2020117093-appb-100009
    Figure PCTCN2020117093-appb-100010
    Figure PCTCN2020117093-appb-100011
    Figure PCTCN2020117093-appb-100012
    Figure PCTCN2020117093-appb-100013
    Figure PCTCN2020117093-appb-100014
    Figure PCTCN2020117093-appb-100015
    Figure PCTCN2020117093-appb-100016
    Figure PCTCN2020117093-appb-100017
    Figure PCTCN2020117093-appb-100018
    Figure PCTCN2020117093-appb-100019
    Figure PCTCN2020117093-appb-100020
    Figure PCTCN2020117093-appb-100021
    Figure PCTCN2020117093-appb-100022
    Figure PCTCN2020117093-appb-100023
    Figure PCTCN2020117093-appb-100024
    Figure PCTCN2020117093-appb-100025
    Figure PCTCN2020117093-appb-100026
    Figure PCTCN2020117093-appb-100027
  6. 一种根据权利要求1-5任一项所述的式I所示的化合物、其立体异构体、消旋体、互变异构体、同位素标记物、前药或其药学上可接受的盐的制备方法，其特征在于，所述制备方法包括如下步骤：
    Figure PCTCN2020117093-appb-100028
    (a1)采用M-1与M-2生成M-3；所述反应可以在EDCl.HCl、吡啶存在条件下进行；
    (a2)M-3与R xL 1反应，其中R x选自R 1或带有羟基的R 1中羟基被
    Figure PCTCN2020117093-appb-100029
    取代的基团；且当R x选自带有羟基的R 1中羟基被
    Figure PCTCN2020117093-appb-100030
    取代的基团时，反应需进一步包括经酸性、还原条件得到式I产品，所述酸性条件可以选自HCl，还原条件可以选自硼氢化钠；
    所述步骤中，R 1，R 2，R 3，m，W如式I所定义；所述L 1为离去基团，可以选自卤素或OTs。
  7. 一种根据权利要求1-5任一项所述的式I所示的化合物、其立体异构体、消旋体、互变异构体、同位素标记物、前药或其药学上可接受的盐的制备方法，其特征在于，所述制备方法包括如下步骤：
    Figure PCTCN2020117093-appb-100031
    (b1)N-1与R xL 1反应，其中R x选自R 1或带有羟基的R 1中羟基被
    Figure PCTCN2020117093-appb-100032
    取代的基团；且当R x选自带有羟基的R 1中羟基被
    Figure PCTCN2020117093-appb-100033
    取代的基团时，反应需进一步包括经酸性、还原条件得到N-2产品；该步骤中，所述酸性条件可以选自HCl，还原条件可以选自硼氢化钠；
    (b2)将上步反应得到的N-2还原得到N-3；所述还原剂可以选自Pd/C；
    (b3)将N-3和M-2反应得到式I。
    所述步骤中，R 1，R 2，R 3，m，W如式I所定义；所述L 1为离去基团，可以选自卤素或OTs。
  8. 一种药物组合物，包含权利要求1-5任一项所述的一种式I所示的化合物、其立体异构体、消旋体、互变异构体、同位素标记物、前药或其药学上可接受的盐。
  9. 根据权利要求1-5任一项所述的一种式I所示的化合物、其立体异构体、消旋体、互变异构体、同位素标记物、前药或其药学上可接受的盐或权利要求8所述的药物组合物在制备预防和/或治疗IRAK介导的疾病或病症的药物中的用途。
  10. 根据权利要求9所述的用途，其特征在于，所述疾病或病症选自肿瘤、痛风、系统性红斑狼疮、多发性硬化症、代谢综合症、动脉粥样硬化、心肌梗死、脓血症、炎症性肠病、哮喘、类风湿性关节炎和过敏等疾病。
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