WO2022052861A1 - 5-取代吲哚3-酰胺衍生物及其制备方法和用途 - Google Patents

5-取代吲哚3-酰胺衍生物及其制备方法和用途 Download PDF

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WO2022052861A1
WO2022052861A1 PCT/CN2021/116256 CN2021116256W WO2022052861A1 WO 2022052861 A1 WO2022052861 A1 WO 2022052861A1 CN 2021116256 W CN2021116256 W CN 2021116256W WO 2022052861 A1 WO2022052861 A1 WO 2022052861A1
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substituted
unsubstituted
alkyl
halogen
cyano
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PCT/CN2021/116256
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French (fr)
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杨胜勇
李琳丽
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成都奥睿药业有限公司
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Priority to AU2021341865A priority Critical patent/AU2021341865A1/en
Priority to US18/025,638 priority patent/US20230365546A1/en
Priority to EP21865915.9A priority patent/EP4212525A1/en
Priority to CA3192271A priority patent/CA3192271A1/en
Priority to JP2023516233A priority patent/JP2023541263A/ja
Publication of WO2022052861A1 publication Critical patent/WO2022052861A1/zh

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Definitions

  • the invention belongs to the technical field of medicine, and particularly relates to a 5-substituted indole 3-amide derivative and a preparation method and application thereof.
  • Programmed necrosis is a new caspase-independent programmed cell death that is different from apoptosis and discovered in recent years. It is regulated by death signals and exhibits a necrosis-like structure. Compared with apoptosis, programmed necrosis does not form apoptotic bodies and does not condense chromatin; compared with necrosis, programmed necrosis is a controlled cell death mode regulated by multiple genes.
  • the caspase inhibitor Z-VAD-FMK After adding the caspase inhibitor Z-VAD-FMK to the in vitro culture system, the use of TNF can induce programmed necrosis of cells. The morphological characteristics of necrosis are cell swelling, rupture and release of cell contents, which in turn cause inflammation and immune response.
  • ligands of TLR3 and TLR4 certain bacteria and virus infections can cause programmed cell necrosis in cells.
  • RIPK1 Receptor interacting protein kinase 1
  • RIPK1 Receptor interacting protein kinase 1
  • a variety of death receptors can trigger upstream signaling of programmed necrosis under conditions of stimulation by inflammatory factors or exogenous infection.
  • RIPK1 and RIPK3 form necrosomes.
  • RIPK3 further recruits MLKL, and the phosphorylated MLKL will self-oligomerize and then migrate to the cell membrane, "punching" the cell membrane, leading to the leakage of cell contents and the disruption of ionization balance, eventually leading to the occurrence of cell necrosis.
  • SIRS Systemic Inflammatory Response Syndrome
  • IBD Inflammatory bowel disease
  • RA rheumatoid arthritis
  • MS multiple sclerosis
  • Activated microglia play a key role in the development of Alzheimer's disease (AD), and RIPK1 is highly expressed in microglia, and RIPK1 inhibitors can effectively protect A ⁇ -induced neuronal cell programs in vitro Sexual necrosis.
  • programmed necrosis is also involved in the development of many neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) and Parkinson's disease (PD).
  • ALS amyotrophic lateral sclerosis
  • FTD frontotemporal dementia
  • PD Parkinson's disease
  • Strilic et al. first revealed in 2016 that tumor cells can induce programmed necrosis of vascular endothelial cells, and then tumor cells pass through the blood vessel wall and achieve distant metastasis through blood circulation, which is an important cause of tumor metastasis.
  • RIPK1 Inhibitors can effectively inhibit tumor metastasis.
  • studies have shown that RIPK1 kinase can promote the differentiation of tolerogenic macrophages in the pancreatic cancer tumor microenvironment, and RIPK1 inhibition can induce the differentiation of immunogenic macrophages in the pancreatic cancer tumor microenvironment, leading to the activation of adaptive immunity. and tumor protection.
  • RIPK1 is an important therapeutic target for cell-programmed necrosis-related diseases such as inflammation, autoimmune diseases, neurodegenerative diseases and tumors, and RIPK1 inhibitors are expected to be potential therapeutic drugs for these diseases.
  • the present invention provides 5-substituted indole 3-amide derivatives, preparation methods and uses thereof.
  • the compounds of the present invention can inhibit RIPK1 kinase in vivo.
  • the kinetic results showed that this series of compounds had good pharmacokinetic properties.
  • the present invention provides a new strategy and means for targeting RIPK1 to treat inflammation, autoimmune disease, neurodegenerative disease, tumor and other related diseases.
  • X 1 and X 3 are independently selected from -CR 6 -, N;
  • R 1 is selected from hydrogen, substituted or unsubstituted C 1 -C 10 alkyl, substituted or unsubstituted C 1 -C 10 ether, substituted or unsubstituted C 3 -C 10 cycloalkyl, substituted or unsubstituted C 1 -C 10 cycloalkyl, substituted or unsubstituted C 1 -C 10 cycloalkyl Unsubstituted 3-10-membered heterocycloalkyl, substituted or unsubstituted C 6 -C 20 aryl, substituted or unsubstituted 3-20-membered heteroaryl; wherein, the substituents are deuterium, C 1- C 10 alkyl, cyano, hydroxyl, carboxyl, halogen or nitro;
  • R 2B is selected from
  • R 2 is selected from C 0 -C 6 alkylene substituted or unsubstituted by one or two R 21 ;
  • R 21 is selected from substituted or unsubstituted C 1 -C 10 alkyl, substituted or unsubstituted C 3 -C 10 cycloalkyl, cyano, hydroxyl, carboxyl, halogen or nitro; wherein, the substituent is cyano, hydroxyl, carboxyl, halogen or nitro;
  • Ring B is selected from C 4 -C 10 aryl substituted or unsubstituted with one, two or three R 22 , 4-10 membered heteroaryl substituted or unsubstituted with one, two or three R 22 , or C 3 -C 10 cycloalkyl substituted or unsubstituted by one, two or three R 22 ; wherein, R 22 is independently selected from substituted or unsubstituted C 1 -C 10 alkyl, substituted or unsubstituted C 1 -C 6 alkoxy, substituted or unsubstituted 4-10 membered heterocycloalkyl, cyano, hydroxyl, carboxyl, halogen or nitro; or, two R 22 are connected to form a substituted or unsubstituted 4- 10-membered heterocycloalkyl; wherein, the substituent is C 1- C 10 alkyl, cyano, hydroxyl, carboxyl, halogen or nitro;
  • R 3 is selected from hydrogen, substituted or unsubstituted C 1 -C 10 alkyl; wherein, the substituent is cyano, hydroxyl, carboxyl, halogen or nitro;
  • R 2B is connected with R 3 to form a substituted or unsubstituted 3-10-membered heterocycloalkyl; wherein, the substituent is C 1 -C 10 alkyl, C 4 -C 10 aryl, one or two R 31 -substituted C 4 -C 10 aryl, cyano, hydroxyl, carboxyl, halogen or nitro; wherein, the R 31 is selected from C 1- C 10 alkyl or halogen;
  • R 4 , R 5 and R 6 are each independently selected from hydrogen, C 1- C 10 alkyl, cyano, hydroxyl, carboxyl, halogen or nitro;
  • Ring A is selected from C 4 -C 10 aryl substituted or unsubstituted with one or two RA , 4-10 membered heteroaryl substituted or unsubstituted with one or two RA; wherein, RA is selected from substituted or unsubstituted amino,
  • the substituent is -R A2 -R A3 , R A4 , C 1 -C 10 alkyl, halogen-substituted C 1 -C 10 alkyl, cyano, hydroxyl, carboxyl, halogen or nitro base; wherein, R A4 is selected from 3-10-membered heterocycloalkyl substituted or unsubstituted by -R A2 -R A3 ;
  • R A1 is selected from substituted or unsubstituted C 1 -C 10 alkyl, substituted or unsubstituted C 1 -C 10 alkenyl, substituted or unsubstituted C 3 -C 10 cycloalkyl, substituted or unsubstituted 3 -10-membered heterocycloalkyl, substituted or unsubstituted C 1 -C 6 alkoxy group, substituted or unsubstituted C 2 -C 6 ether group, substituted or unsubstituted C 2 -C 6 amine group; wherein, The substituents are C 1 -C 10 alkyl, hydroxyl substituted C 1 -C 10 alkyl, C 1 -C 10 ester, 3-10 membered heterocycloalkyl, amino, amino, cyano, hydroxyl , carboxyl, halogen or nitro;
  • R A2 is selected from substituted or unsubstituted C 0 -C 6 alkylene, carbonyl; wherein, the substituent is C 1 -C 10 alkyl, cyano, hydroxyl, carboxyl, halogen or nitro;
  • R A3 is selected from substituted or unsubstituted 3-10-membered heterocycloalkyl; wherein, the substituent is C 1 -C 10 alkyl, cyano, hydroxyl, carboxyl, halogen or nitro.
  • R 1 is selected from hydrogen, substituted or unsubstituted C 1 -C 10 alkyl, substituted or unsubstituted C 3 -C 10 cycloalkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl, substituted or unsubstituted Substituted C6 - C20 aryl, substituted or unsubstituted 3-20-membered heteroaryl; wherein, the substituent is C1- C10 alkyl, cyano, hydroxyl, carboxyl, halogen or nitro ;
  • R 2 is selected from C 0 -C 6 alkylene substituted or unsubstituted by one R 21 ; wherein R 21 is selected from substituted or unsubstituted C 1 -C 10 alkyl, cyano, hydroxyl, carboxyl, halogen or Nitro; wherein, the substituent is cyano, hydroxyl, carboxyl, halogen or nitro;
  • Ring B is selected from C 4 -C 10 aryl substituted or unsubstituted with one, two or three R 22 or 4-10 membered heteroaryl substituted or unsubstituted with one, two or three R 22 ;
  • R 22 is independently selected from substituted or unsubstituted C 1 -C 10 alkyl, substituted or unsubstituted C 1 -C 6 alkoxy, substituted or unsubstituted 4-10-membered heterocycloalkyl, cyano group, hydroxyl, carboxyl, halogen or nitro; or, two R 22 are connected to form a substituted or unsubstituted 4-10-membered heterocycloalkyl; wherein, the substituent is C 1- C 10 alkyl, cyano , hydroxyl, carboxyl, halogen or nitro;
  • R 3 is selected from hydrogen, substituted or unsubstituted C 1 -C 10 alkyl; wherein, the substituent is cyano, hydroxyl, carboxyl, halogen or nitro;
  • R 2 is connected with R 3 to form a substituted or unsubstituted 3-10-membered heterocycloalkyl; wherein, the substituent is a C 1- C 10 alkyl group, a cyano group, a hydroxyl group, a carboxyl group, a halogen or a nitro group;
  • X 1 and X 3 are independently selected from CH, N;
  • Ring A is selected from C 4 -C 10 aryl substituted or unsubstituted with one or two RA , 4-10 membered heteroaryl substituted or unsubstituted with one or two RA; wherein, RA is selected from amino , Substituted or unsubstituted C 1 -C 10 alkyl, substituted or unsubstituted C 4 -C 10 aryl, substituted or unsubstituted 4-10 membered heteroaryl, substituted or unsubstituted 3-10 membered heterocycle Wherein, the substituent is -R A2 -R A3 , R A4 , C 1- C 10 alkyl, cyano, hydroxyl, carboxyl, halogen or nitro; wherein, R A4 is selected from the group consisting of -R A2 -R A3 substituted or unsubstituted 3-10 membered heterocycloalkyl;
  • R A1 is selected from substituted or unsubstituted C 1 -C 10 alkyl, substituted or unsubstituted C 1 -C 10 alkenyl, substituted or unsubstituted C 3 -C 10 cycloalkyl, substituted or unsubstituted 3 -10-membered heterocycloalkyl, substituted or unsubstituted C 1 -C 6 alkoxy, substituted or unsubstituted C 2 -C 6 ether; wherein, the substituent is C 1 -C 10 alkyl, 3-10 membered heterocycloalkyl, amino, amine, cyano, hydroxyl, carboxyl, halogen or nitro;
  • R A2 is selected from substituted or unsubstituted C 0 -C 6 alkylene, carbonyl; wherein, the substituent is C 1 -C 10 alkyl, cyano, hydroxyl, carboxyl, halogen or nitro;
  • R A3 is selected from substituted or unsubstituted 3-10-membered heterocycloalkyl; wherein, the substituent is C 1- C 10 alkyl, cyano, hydroxyl, carboxyl, halogen or nitro.
  • the compound of formula I is represented by formula III or formula IV:
  • R 1 is selected from hydrogen, C 1 -C 4 alkyl
  • R 23 is selected from hydrogen, methyl
  • B ring is selected from phenyl substituted or unsubstituted by one or two R 22 ; wherein, R 22 are independently Selected from F, Cl, cyano, methyl, trifluoromethyl, methoxy or trifluoromethoxy.
  • X 1 and X 3 are independently selected from -CR 6 -, N;
  • the R 1 is selected from hydrogen, substituted or unsubstituted C 1 -C 10 alkyl, substituted or unsubstituted C 3 -C 10 cycloalkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl, substituted or unsubstituted Or unsubstituted C 6 -C 20 aryl, substituted or unsubstituted 3-20-membered heteroaryl; wherein, the substituent is C 1- C 10 alkyl, cyano, hydroxyl, carboxyl, halogen or nitro base;
  • L 3 is selected from substituted or unsubstituted C 1 -C 4 alkylene; wherein, the substituent is C 1 -C 10 alkyl, cyano, hydroxyl, carboxyl, halogen or nitro;
  • R 33 is selected from hydrogen, substituted or unsubstituted C 1 -C 10 alkyl, substituted or unsubstituted C 3 -C 10 cycloalkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl, substituted or unsubstituted Substituted C 6 -C 10 aryl; wherein, the substituent is C 1 -C 10 alkyl, cyano, hydroxyl, carboxyl, halogen or nitro;
  • R 4 , R 5 and R 6 are each independently selected from hydrogen, C 1- C 10 alkyl, cyano, hydroxyl, carboxyl, halogen or nitro;
  • Ring A is selected from C 4 -C 10 aryl substituted or unsubstituted with one or two RA , 4-10 membered heteroaryl substituted or unsubstituted with one or two RA; wherein, RA is selected from amino , Substituted or unsubstituted C 1 -C 10 alkyl, substituted or unsubstituted C 4 -C 10 aryl, substituted or unsubstituted 4-10 membered heteroaryl, substituted or unsubstituted 3-10 membered heterocycle Wherein, the substituent is -R A2 -R A3 , R A4 , C 1- C 10 alkyl, cyano, hydroxyl, carboxyl, halogen or nitro; wherein, R A4 is selected from the group consisting of -R A2 -R A3 substituted or unsubstituted 3-10 membered heterocycloalkyl;
  • R A1 is selected from substituted or unsubstituted C 1 -C 10 alkyl, substituted or unsubstituted C 1 -C 10 alkenyl, substituted or unsubstituted C 3 -C 10 cycloalkyl, substituted or unsubstituted 3 -10-membered heterocycloalkyl, substituted or unsubstituted C 1 -C 6 alkoxy, substituted or unsubstituted C 2 -C 6 ether; wherein, the substituent is C 1 -C 10 alkyl, C 1 -C 10 ester group, 3-10 membered heterocycloalkyl group, amino group, amine group, cyano group, hydroxyl group, carboxyl group, halogen or nitro group;
  • R A2 is selected from substituted or unsubstituted C 0 -C 6 alkylene, carbonyl; wherein, the substituent is C 1 -C 10 alkyl, cyano, hydroxyl, carboxyl, halogen or nitro;
  • R A3 is selected from substituted or unsubstituted 3-10-membered heterocycloalkyl; wherein, the substituent is C 1- C 10 alkyl, cyano, hydroxyl, carboxyl, halogen or nitro.
  • R 33 is selected from phenyl, phenyl substituted with one or two substituents; wherein, the substituents are selected from F, Cl or methyl.
  • X 1 and X 3 are independently selected from -CR 6 -, N;
  • X 3 is selected from CH, N;
  • the R 1 is selected from hydrogen, substituted or unsubstituted C 1 -C 10 alkyl, substituted or unsubstituted C 3 -C 10 cycloalkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl, substituted or unsubstituted Or unsubstituted C 6 -C 20 aryl, substituted or unsubstituted 3-20-membered heteroaryl; wherein, the substituent is C 1- C 10 alkyl, cyano, hydroxyl, carboxyl, halogen or nitro base;
  • L 4 is selected from substituted or unsubstituted C 1 -C 3 alkylene; wherein, the substituent is C 1 -C 10 alkyl, cyano, hydroxyl, carboxyl, halogen or nitro;
  • R 3 is selected from hydrogen, substituted or unsubstituted C 1 -C 10 alkyl; wherein, the substituent is cyano, hydroxyl, carboxyl, halogen or nitro;
  • R 4 , R 5 and R 6 are each independently selected from hydrogen, C 1- C 10 alkyl, cyano, hydroxyl, carboxyl, halogen or nitro;
  • Ring A is selected from C 4 -C 10 aryl substituted or unsubstituted with one or two RA , 4-10 membered heteroaryl substituted or unsubstituted with one or two RA; wherein, RA is selected from amino , Substituted or unsubstituted C 1 -C 10 alkyl, substituted or unsubstituted C 4 -C 10 aryl, substituted or unsubstituted 4-10 membered heteroaryl, substituted or unsubstituted 3-10 membered heterocycle Wherein, the substituent is -R A2 -R A3 , R A4 , C 1- C 10 alkyl, cyano, hydroxyl, carboxyl, halogen or nitro; wherein, R A4 is selected from the group consisting of -R A2 -R A3 substituted or unsubstituted 3-10 membered heterocycloalkyl;
  • R A1 is selected from substituted or unsubstituted C 1 -C 10 alkyl, substituted or unsubstituted C 1 -C 10 alkenyl, substituted or unsubstituted C 3 -C 10 cycloalkyl, substituted or unsubstituted 3 -10-membered heterocycloalkyl, substituted or unsubstituted C 1 -C 6 alkoxy, substituted or unsubstituted C 2 -C 6 ether; wherein, the substituent is C 1 -C 10 alkyl, 3-10 membered heterocycloalkyl, amino, amine, cyano, hydroxyl, carboxyl, halogen or nitro;
  • R A2 is selected from substituted or unsubstituted C 0 -C 6 alkylene, carbonyl; wherein, the substituent is C 1 -C 10 alkyl, cyano, hydroxyl, carboxyl, halogen or nitro;
  • R A3 is selected from substituted or unsubstituted 3-10-membered heterocycloalkyl; wherein, the substituent is C 1- C 10 alkyl, cyano, hydroxyl, carboxyl, halogen or nitro.
  • the L 4 is selected from a C 1 -C 2 alkylene group.
  • the X 3 is selected from CH, N.
  • R 1 is selected from hydrogen, C 1 -C 4 alkyl, and C 3 ether.
  • the R 4 , R 5 and R 6 are independently selected from hydrogen and F, respectively.
  • ring A is selected from 5-9-membered heteroaryl substituted or unsubstituted by one or two RAs, 6 - membered aryl substituted or unsubstituted by one or two RAs; wherein, RAs are selected from Amino, amino substituted by C 1 -C 4 alkyl, amino substituted by -CF 3 , amino substituted by -CHF 2 , Methyl, 5-6-membered heteroaryl substituted or unsubstituted by -R A2 -R A3 , methyl substituted by R A4 , phenyl substituted or unsubstituted by -R A2 -R A3 ; wherein, R A4 Selected from 6-membered heterocycloalkyl substituted or unsubstituted by -R A2 -R A3 ; R A1 is selected from C 2 -C 5 alkyl, Cl substituted C 4 alkyl, 5-membered heterocyclo
  • R A2 is selected from C 0 -C 1 alkylene, carbonyl
  • R A3 is selected from methyl-substituted or unsubstituted 6-membered heterocycloalkyl.
  • the A ring is selected from
  • the A ring is selected from:
  • R 2 is selected from C 0 -C 2 alkylene substituted or unsubstituted by one or two R 21 ; wherein, R 21 is selected from methyl;
  • Ring B is selected from C 6 -C 10 aryl substituted or unsubstituted with one, two or three R 22 , 5-9 membered heteroaryl substituted or unsubstituted with one, two or three R 22 , or C 9 -C 10 cycloalkyl substituted or unsubstituted by one, two or three R 22 ; wherein, R 22 is independently selected from C 1 -C 4 alkyl, methoxy, halogen, halogen substituted methyl group, halogen-substituted methoxy, cyano, nitro, Halogen is selected from F, Cl, Br.
  • R 3 is selected from hydrogen.
  • R 1 is selected from hydrogen, substituted or unsubstituted C 1 -C 10 alkyl, substituted or unsubstituted C 3 -C 10 cycloalkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl, substituted or unsubstituted Substituted C6 - C20 aryl, substituted or unsubstituted 3-20-membered heteroaryl; wherein, the substituent is C1- C10 alkyl, cyano, hydroxyl, carboxyl, halogen or nitro ;
  • R 31 is selected from C 1 -C 6 alkylene
  • R 32 is selected from C 4 -C 10 aryl substituted or unsubstituted by one, two or three R 311 , wherein R 311 is independently selected from C 1 -C 6 alkoxy, or two R 311 are connected together Form 4-10 membered heterocycloalkyl;
  • X 1 and X 3 are independently selected from CH, N;
  • Ring A is selected from C 4 -C 10 aryl substituted or unsubstituted with one or two RA , 4-10 membered heteroaryl substituted or unsubstituted with one or two RA; wherein, RA is selected from amino , Substituted or unsubstituted C 1 -C 10 alkyl, substituted or unsubstituted C 4 -C 10 aryl, substituted or unsubstituted 4-10 membered heteroaryl, substituted or unsubstituted 3-10 membered heterocycle Wherein, the substituent is -R A2 -R A3 , R A4 , C 1- C 10 alkyl, cyano, hydroxyl, carboxyl, halogen or nitro; wherein, R A4 is selected from the group consisting of -R A2 -R A3 substituted or unsubstituted 3-10 membered heterocycloalkyl;
  • R A1 is selected from substituted or unsubstituted C 1 -C 10 alkyl, substituted or unsubstituted C 1 -C 10 alkenyl, substituted or unsubstituted C 3 -C 10 cycloalkyl, substituted or unsubstituted 3 -10-membered heterocycloalkyl, substituted or unsubstituted C 1 -C 6 alkoxy, substituted or unsubstituted C 2 -C 6 ether; wherein, the substituent is C 1 -C 10 alkyl, 3-10 membered heterocycloalkyl, amino, amine, cyano, hydroxyl, carboxyl, halogen or nitro;
  • R A2 is selected from substituted or unsubstituted C 0 -C 6 alkylene, carbonyl; wherein, the substituent is C 1 -C 10 alkyl, cyano, hydroxyl, carboxyl, halogen or nitro;
  • R A3 is selected from substituted or unsubstituted 3-10-membered heterocycloalkyl; wherein, the substituent is C 1- C 10 alkyl, cyano, hydroxyl, carboxyl, halogen or nitro.
  • -R 31 -R 32 are selected from
  • the compound represented by formula I-formula VII is specifically:
  • the present invention also provides a preparation method of the above compound,
  • R 2B , R 4 , R 5 , X 1 , X 2 , X 3 and the A ring are as described above.
  • the synthetic process of described intermediate A is:
  • the raw material B, potassium acetate, pinacol biborate and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex were dissolved in anhydrous dioxane, After the reaction system is replaced with an inert gas, the reaction is completed, and the reaction solution is concentrated, sampled, and subjected to column chromatography to obtain the product;
  • the present invention also provides the use of the above-mentioned compound, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof in the preparation of a RIPK1 inhibitor.
  • the present invention also provides the use of the above compound, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for the treatment of inflammation, immune disease, neurodegenerative disease or tumor.
  • the medicament is used for the treatment of inflammatory reactions, immune diseases, neurodegenerative diseases or tumors associated with programmed cell necrosis.
  • the inflammation is colitis.
  • the present invention also provides a pharmaceutical composition, which is a preparation prepared from the above compound, or a stereoisomer or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable auxiliary materials.
  • Programmed necrosis as defined in the present invention is an active and orderly death mode of cells determined by genes. Specifically, it refers to the suicide protection measures initiated by gene regulation when cells are stimulated by internal and external environmental factors, including the induction and activation of some molecular mechanisms and gene programming, through which non-essential cells or cells that are about to be specialized in the body are removed in this way. TNF, ligands of TLR3 and TLR4, some bacteria, virus infection, etc. can cause programmed cell necrosis.
  • the compounds and derivatives provided in the present invention may be named according to the IUPAC (International Union of Pure and Applied Chemistry) or CAS (Chemical Abstracts Service, Columbus, OH) nomenclature system.
  • substitution means that a hydrogen atom in a molecule is replaced by a different atom or molecule.
  • C a -C b alkyl denotes any alkyl group containing "a" to "b” carbon atoms.
  • C1 - C4 alkyl refers to an alkyl group containing 1-4 carbon atoms.
  • C 0 -C b is used to define an alkylene group, it means that the site may be free of alkylene groups.
  • Alkyl refers to a saturated hydrocarbon chain having the specified number of member atoms.
  • C1 - C6 alkyl refers to an alkyl group having 1 to 6 member atoms, eg, 1 to 4 member atoms.
  • Alkyl groups can be straight or branched. Representative branched alkyl groups have one, two or three branches. Alkyl groups may be optionally substituted with one or more substituents as defined herein.
  • Alkyl groups include methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl and tert-butyl), pentyl (n-pentyl, isopentyl and neopentyl) base) and hexyl. Alkyl groups can also be part of other groups such as C1 - C6alkoxy.
  • Cycloalkyl refers to a saturated or partially saturated cyclic group having 3 to 14 carbon atoms and no ring heteroatoms and having a single ring or multiple rings (including fused, bridged and spiro ring systems).
  • cycloalkyl applies when the point of attachment is at a non-aromatic carbon atom (eg 5,6,7,8,-tetra Hydronaphthalene-5-yl).
  • cycloalkyl includes cycloalkenyl groups such as cyclohexenyl.
  • cycloalkyl groups include, for example, adamantyl, cyclopropyl, cyclobutyl, cyclohexyl, cyclopentyl, cyclooctyl, cyclopentenyl, and cyclohexenyl.
  • (Ca-Cb)alkenyl refers to an alkenyl group having a to b carbon atoms and is intended to include, for example, vinyl, propenyl, isopropenyl, 1,3-butadienyl, and the like.
  • Alkynyl means a straight-chain monovalent hydrocarbon group or a branched-chain monovalent hydrocarbon group containing at least one triple bond.
  • alkynyl is also intended to include those hydrocarbyl groups having one triple bond and one double bond.
  • (C2-C6)alkynyl is intended to include ethynyl, propynyl, and the like.
  • Halogen is fluorine, chlorine, bromine or iodine.
  • Heterocycle and “heterocycloalkyl” refer to a saturated ring or a non-aromatic unsaturated ring containing at least one heteroatom; wherein the heteroatom refers to a nitrogen atom, an oxygen atom, and a sulfur atom;
  • Heteroaryl refers to an aromatic unsaturated ring group containing at least one heteroatom; wherein the heteroatom refers to a nitrogen atom, an oxygen atom, a sulfur atom;
  • Ether group refers to a group formed by removing one H from a carbon atom of an ether compound.
  • C 2 -C 6 ether group means that in the ether compound, the total number of carbon atoms of the hydrocarbon group connected to both ends of the oxygen atom is 2 to 6.
  • Amine group refers to a group formed by removing one H from an amine compound.
  • Amine compounds refer to the compounds formed after the hydrogen atoms in the ammonia molecule are partially or completely replaced by hydrocarbon groups.
  • C 2 -C 6 amine groups refer to the amine compounds, the total number of carbon atoms of the hydrocarbon groups connected to both ends of the oxygen atoms is 2 to 6.
  • Ester group refers to a group formed by removing one H from a carbon atom of an ester compound.
  • Ester compounds refer to the compounds formed by the dehydration of acid and alcohol, which have the functional group of "-COO-".
  • C 1 -C 10 ester group means that in the amine compound, the total number of carbon atoms is 1 to 10.
  • R a and R b are connected to form a heterocyclic ring means that at least one atom in R a and R b is connected by a chemical bond, so that the atom or atom chain to which R a and R b are connected together in the general structure is a part of the skeleton of the ring structure Together with R a and R b to form a heterocycle.
  • Steps include enantiomers and diastereomers.
  • a horizontal line connected to a symbol representing a substituent represents a covalent bond.
  • "-R” means that R is connected to other groups through one covalent single bond
  • “-R-” means that R is connected to other groups through two covalent single bonds
  • “-R A2 -R A3” It means that R A3 is connected to R A2 through one covalent single bond, and R A2 is connected to R A3 and one other group through two covalent single bonds, respectively.
  • pharmaceutically acceptable refers to a carrier, vehicle, diluent, adjuvant, and/or salt formed usually
  • salts and “pharmaceutically acceptable salts” refer to the above-mentioned compounds or their stereoisomers, acid and/or base salts with inorganic and/or organic acids and bases, and also zwitterionic salts (internal). salts), also including quaternary ammonium salts such as alkylammonium salts. These salts can be obtained directly in the final isolation and purification of the compounds. It can also be obtained by mixing the above-mentioned compound, or a stereoisomer thereof, with a certain amount of acid or base as appropriate (for example, an equivalent amount).
  • salts may be precipitated in solution and collected by filtration, recovered after evaporation of the solvent, or obtained by lyophilization after reaction in an aqueous medium.
  • the salts described in the present invention can be hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate of the compound salt, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate.
  • one or more compounds of the present invention may be used in combination with each other.
  • the compounds of the present invention may be used in combination with any other active agent for the preparation of medicaments or pharmaceutical compositions for modulating cellular function or treating diseases. If a group of compounds is used, the compounds may be administered to the subject simultaneously, separately or sequentially.
  • Fig. 1 is the kinase selectivity spectrum of compound 34 of the present invention.
  • Fig. 2 is the kinase selectivity spectrum of compound 94 of the present invention.
  • Example 3 is a graph of cell viability in Example 5.
  • Figure 4 shows the effects of compounds 46 and 94 in Example 6 on the necrotic signaling pathway
  • Figure 5 is the result that compounds 46 and 94 in Example 7 protect the mouse SIRS model induced by TNF ⁇ ;
  • Figure 6 is the concentration-response curve of compound terfenadine (left) and compound 94 (right) in Example 8.
  • Fig. 7 is the comparative experiment result of mouse colon length in embodiment 8.
  • Figure 8 is a concentration-response curve of the inhibitory effect of compound terfenadine (left) and compound 94 (right) on hERG current in Example 10.
  • HSGF254 silica gel plate the specification is The TLC silica gel plate is used in Yucheng Chemical (Shanghai) Co., Ltd.
  • GF254 silica gel plate the specification is Column chromatography uses Qingdao Hailang Silica Gel Desiccant Co., Ltd. Silica gel is used as the carrier.
  • the Flash column uses the Agelfino Claricep Flash amorphous silica purification column.
  • the reagents 1-propylphosphoric anhydride and methylmagnesium bromide in the examples of the present invention were purchased from Shanghai McLean Biochemical Technology Co., Ltd., 4N hydrochloric acid dioxane solution was purchased from Panjin Yanfeng Technology Co., Ltd., and 1M borane Tetrahydrofuran solution, N,N-diisopropylethylamine, (S)-tert-butylsulfinamide were purchased from Adamas Reagent.
  • Other reagents and starting materials were purchased from Shanghai Haohong Biomedical Technology Co., Ltd., or can be Synthesized using methods known in the art. Unless otherwise specified, all the reactions of the present invention are carried out under continuous magnetic stirring, under dry nitrogen or argon, the solvent is a dry solvent, and the reaction temperature is in degrees Celsius.
  • iv 1,4-dioxane/water 10:1, [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex, tricyclohexylphosphorus, carbonic acid Cesium, 100 degrees, 16 hours, yield 75%;
  • the specific operation steps include:
  • iv 1,4-dioxane/water 5:1, [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex, tricyclohexylphosphorus, carbonic acid Cesium, 95 degrees, 12 hours, 75%;
  • the specific operation steps include:
  • the specific operation steps include:
  • Compound 141 can be prepared according to the procedure shown in Method II.
  • Compound 141 can be prepared according to the procedure shown in Method II.
  • Example 5 In vitro kinase assay and protection cell programmed necrosis assay of the compounds of the present invention
  • In vitro kinase assays were performed using the Kinase Profiler service provided by Eurofins.
  • the experimental method is as follows: the small molecule compound to be tested (0.001-10 ⁇ M), the protein kinase to be tested and the buffer containing the substrate, 10 mM magnesium acetate and [ ⁇ - 33 P-ATP] are incubated together, starting by adding Mg ⁇ ATPmix The reaction was terminated by adding 3% phosphate solution to the buffer after a period of incubation at room temperature. Subsequently, 10 ⁇ L of the reaction mixture was quantitatively pipetted onto P30 filter paper, washed three times with 75 mM phosphate solution, and then washed once with methanol.
  • the P30 filter paper was dried and added with scintillation fluid for scintillation counting.
  • the inhibitory activity of the compound was expressed by the half inhibitory concentration IC 50 , and the IC 50 value was obtained by fitting the inhibition rate corresponding to each concentration gradient.
  • DMEM medium was purchased from Gibco company, penicillin and streptomycin were purchased from Hyclone company, TNF ⁇ was purchased from Peprotech company, Smac mimetic and Z-VAD-FMK were purchased from Selleck company, CCK8 was purchased from Medchem Express company.
  • HT-29 cells colon cancer cells
  • DMEM+10% FBS+penic/streptomycin medium fetal calf serum
  • HT-29 cells in the logarithmic growth phase were collected and seeded in a 96-well plate at a specific number per well (the adherent cells were generally 8 ⁇ 10 3 cells/well), and the cells were incubated at 37°C under 5% CO 2 . Incubate overnight in a cell incubator. The next day, the compound to be tested was diluted with medium to the corresponding concentration and added to the corresponding wells of the 96-well plate, with 3 replicate wells for each sample. At the same time, a solvent control group and a blank control group containing only medium were set.
  • Cell necrosis protection rate [(XC 0 )/(CC 0 )] ⁇ 100%
  • C, C 0 and X represent the average absorbance values of solvent control group, blank control group and drug-treated group, respectively.
  • C, C 0 and X represent the average absorbance values of solvent control group, blank control group and drug-treated group, respectively.
  • C, C 0 and X represent the average absorbance values of solvent control group, blank control group and drug-treated group, respectively.
  • Graphpad Prism 5.0 software to fit the cell viability curve and calculate the EC 50 value of the test compound to inhibit programmed cell necrosis.
  • the results are shown in Table 2, where +++++ represents IC 50 or EC 50 ⁇ 0.001 ⁇ M, ++++ represents 0.01 ⁇ M > IC 50 or EC 50 ⁇ 0.001 ⁇ M, +++ represents 0.1 ⁇ M > IC 50 Or EC 50 ⁇ 0.01 ⁇ M, ++ means 1 ⁇ M > IC 50 or EC 50 ⁇ 0.1 ⁇ M, + means IC 50 or EC 50 ⁇ 1 ⁇ M.
  • the preferred compounds 34 and 94 were tested for the single-concentration inhibition rate of Eurofins' existing 422 kinases (including mutants) at a concentration of 10 ⁇ M, and IC 50 tests were performed for the kinases with higher inhibitory activity. The results ( The test is tested by Eurofins and provides activity data) as shown in Table 3 and Table 4 below:
  • the EC 50 value of compound 94 in inhibiting the HT-29 cell necrosis model was 0.012 nM
  • the EC 50 values of its series of compounds 34 and 46 in inhibiting the HT-29 cell necrosis model were 0.117 nM and 0.070, respectively. nM.
  • the above experimental results show that the compound 94 and its series of compounds of the present invention can protect HT-29 cells from TSZ-induced necroptosis.
  • TNF ⁇ was purchased from Peprotech Company
  • Smac mimetic and Z-VAD-FMK were purchased from Selleck Company
  • RIPA lysate was purchased from Biyuntian Institute of Biotechnology
  • cocktail was purchased from MCE Company
  • PMSF protease inhibitor, sodium dodecyl sulfonate SDS and glycine were purchased from Sigma
  • acrylamide, Tris, ammonium persulfate APS and N,N,N',N'-tetramethylethylenediamine TEMED were purchased from Wuhan Sevier Biotechnology Co., Ltd. .
  • Extraction of total cell protein Add the corresponding concentration of compound 94, 46 or blank solvent to the cell supernatant to treat the cells and co-induce with TNF ⁇ /Smac mimetic/Z-VAD-FMK for a certain period of time, discard the supernatant, use pre-cooled Wash three times with PBS or normal saline, then add an appropriate volume of RIPA lysis buffer (containing 1% cocktail and 1% PMSF protease inhibitor), and immediately place it on ice for lysis for 15 minutes. After 15 minutes, the cell lysis buffer is scraped off with a scraper and transferred. Into a 1.5ml EP tube, disrupt the cells with a sonicator.
  • the centrifuge tubes were then centrifuged (12000 rpm, 15 min) in a low temperature high-speed centrifuge to remove cell debris. Then use the BCA method to quantify the protein, and use the protein standard to make a standard curve, calculate the protein concentration of each sample according to the standard curve, and then level the concentration of each group of protein samples by calculation, add 5x protein loading buffer, and place it at 100 °C dry. maintained in a thermostat for 10 min. The samples were then directly loaded for electrophoresis or stored at -20°C after aliquoting. Avoid repeated freezing and thawing of protein samples.
  • the proteins are separated by polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis separation, the protein was fully transferred to the PVDF membrane by the tank wet transfer method, and then the PVDF membrane was placed in 5% nonfat dry milk (prepared with TBS/T) at room temperature for blocking for more than 2 hours. Obtain the PVDF membrane band containing the corresponding protein by molecular weight, dilute the primary antibody according to the dilution ratio recommended in the antibody manual, and incubate the protein band at 4°C overnight.
  • SDS-PAGE polyacrylamide gel electrophoresis
  • each band was taken out, rinsed with TBS/T buffer (5 min, 3 times), then added with 1:5000 diluted HRP-labeled secondary antibody, incubated at 37°C with shaking for 1 h, and then eluted with TBS/T to remove After the excess antibody was evenly dropped on the PVDF membrane, the HRP substrate was placed in a fast gel imaging system to develop and take pictures.
  • TNF ⁇ was purchased from Peprotech Company, castor oil was purchased from MCE Company, and sterile saline was purchased from Chengdu Parkson Kechuang Biotechnology Co., Ltd.
  • the dose of TNF ⁇ for modeling was 500 ⁇ g/kg, which was prepared with sterile physiological saline, and was injected into the model by tail vein.
  • the body temperature of the mice was detected by an infrared electric thermometer.
  • the solvent is 25% ethanol castor oil, 75% normal saline, and the administration method is oral administration.
  • the administration doses of compounds 94, 46 and RIPK1 positive compound GSK2982772 are all 40 mg/kg, and they are administered orally before modeling.
  • the body temperature was recorded every hour and the survival of the mice was recorded. After 11 hours, the survival of the mice at 24 and 48 hours was recorded. Survival curves were fitted with Graphpad Prism 5.0 software.
  • SIRS Systemic Inflammatory Response Syndrome
  • TNF ⁇ -induced SIRS mouse model was established to evaluate the in vivo activity of the compounds of the present invention.
  • the modeling dose of TNF ⁇ was 500 ⁇ g/kg, which was prepared with sterile normal saline and injected into mice by tail vein injection. Because measuring the rectal temperature of mice will cause mechanical damage to the mice and may interfere with the experimental results, an infrared electric thermometer is used to measure the body temperature of the mice and observe the survival situation at the same time.
  • Compounds 94 and 46 were administered at a dose of 40 mg/kg, administered once before modeling.
  • the solvent group (Vehicle) was given the corresponding solvent control.
  • mice in the solvent group developed symptoms such as a continuous drop in body temperature and decreased activity in a short period of time, and began to die within 24 hours.
  • the body temperature of the rest of the mice was relatively normal within 11 hours after TNF ⁇ injection, the vitality of the mice was significantly better than that of the solvent group, and the overall mortality was significantly lower than that of the solvent group , and 94 showed better protective effect than the positive drug GSK2982772. It indicated that compounds 94 and 46 exerted a significant anti-inflammatory effect by inhibiting RIPK1 in vivo, and resisted the death induced by high-dose TNF ⁇ in mice.
  • Example 8 Protective effect of compound 94 of the present invention on DSS-induced mouse IBD model
  • Dextran Sulfate Sodium Salt (Dextran Sulfate Sodium Salt, DSS, 36000-50000KD) was purchased from Shanghai Yisheng Biotechnology Co., Ltd., 0.CT tissue fixative was purchased from Sevier Biological Company, PEG300 and Tween-80 were purchased from MCE Company, DMSO was purchased from Sigma Company.
  • mice weighing 17–20 g Female C57BL/6 mice weighing 17–20 g were used.
  • DSS (2.5% wt/vol) was dissolved in drinking water and administered to mice ad libitum for 7 days (from day 0 to day 7). Fresh DSS solution was replaced on day 2, day 4 and day 6, respectively.
  • All water was changed to normal drinking water, and were randomly divided into groups (7 animals in each group) for oral administration: Vehicle group (10% DMSO, 40% PEG300, 5% Tween-80, 45% saline) , GSK3145095 group (40mg/kg) and compound 94 group (40mg/kg).
  • the control group was given normal drinking water every day, and the corresponding solvent was orally administered from the 7th day.
  • the body weight and survival rate of the mice were recorded daily. Survival curves were fitted with Graphpad Prism 5.0 software.
  • the mice (2 mice each) were sacrificed, and the changes in the length of the colons of the mice were observed.
  • IBD Inflammatory bowel disease
  • UC ulcerative colitis
  • CD Crohn's disease
  • mice in the experimental group developed pathological symptoms such as bloody stool and weight loss after 7 days of modeling, indicating that the modeling was successful.
  • pathological symptoms such as bloody stool and weight loss after 7 days of modeling, indicating that the modeling was successful.
  • the mice in the Vehicle group and GSK3145095 group continued to lose weight, and the mice died, while the weight of the mice in the group administered with Compound 94 gradually recovered, and the weight of the mice in the Group 94 was gradually recovered, and the weight of the mice in the Vehicle group and the GSK3145095 group continued to decrease. Death occurred, and the vitality of the mice was also significantly better than that of the Vehicle group.
  • mice were sacrificed on the 12th day (2 mice in each group), the colon length of the mice in the Vehicle group was significantly shorter than that in the Control group, while the colon length of the mice in the Compound 94 group was longer than that of the Vehicle group, and was similar to that of the mice in the Control group. They were basically equivalent, indicating that compound 94 could alleviate DSS-induced colon shortening in mice, and showed a better therapeutic effect than the positive drug GSK3145095. It indicated that compound 94 exerted a significant anti-inflammatory effect by inhibiting RIPK1 in vivo, and alleviated DSS-induced colitis in mice.
  • Blood was collected through the jugular vein or other suitable methods at the corresponding time, about 0.20 mL of each sample was collected, heparin sodium was anticoagulated, the blood samples were placed on ice after collection, and centrifuged within 1 hour to separate the plasma (centrifugation conditions: centrifugal force 6800g, 6 minutes, 2-8°C).
  • the collected plasma samples were stored in a -80°C refrigerator before analysis, and the remaining plasma samples continued to be temporarily stored in a -80°C refrigerator after the analysis, and the storage period was one month.
  • Example 10 The effect of compound 94 of the present invention on hERG potassium channel
  • Stable HEK-hERG cells were washed with DPBS, digested and separated with Trypsin or Tryple solution, resuspended in culture medium and stored in a centrifuge tube for later use. Before patch-clamp recordings, drop cells into small petri dishes to ensure that the cells are at a certain density and that the cells are individually isolated.
  • hERG currents were recorded using whole-cell patch-clamp technique.
  • the cell suspension was added to a small petri dish and placed on an inverted microscope stage. After the cells have adhered, perfuse with extracellular fluid at a recommended flow rate of 1–2 mL/min.
  • the glass micro-electrode is drawn by a micro-electrode drawing apparatus in two steps, and the resistance value of the glass micro-electrode is 2-5M ⁇ after filling with the liquid in the electrode.
  • the stable drug effect is defined as: the last 5 stimulation strip current values of each concentration administration period change less than 10% of the mean value (when the current is greater than or equal to 200pA) or less than 30% of the mean value (when the current is less than 200pA), it can be considered that is stable, if not, the concentration data will not be taken.
  • Stimulus delivery and signal acquisition were performed by PatchMaster software; the patch clamp amplifier amplifies the signal, and the filter is 10KHz.
  • the concentration-response curve of the inhibitory effect of the positive compound terfenadine on hERG current is shown in Figure 8 .
  • the final concentrations of compound 94 were 0.3, 1, 3, 10 and 30 ⁇ M, and the final concentration of DMSO in the extracellular fluid was 0.3%.
  • the concentration-response curve of the inhibitory effect of compound 94 on hERG current is shown in FIG. 8 .
  • the series of compounds provided by the present invention can protect HT-29 cells from TSZ-induced necroptosis, and have good kinase selectivity. Its principle of action is to inhibit the autophosphorylation of RIPK1, which affects the phosphorylation of its downstream RIPK3 and MLKL, and inhibits programmed necrosis by blocking the programmed necrosis signaling pathway.
  • the series of compounds provided by the invention exert a significant anti-inflammatory effect by inhibiting RIPK1 in vivo, and have no obvious inhibitory effect on the hERG potassium channel current at various concentrations, and will not cause side effects due to the obvious inhibitory effect on the hERG potassium channel current. Therefore, the series of compounds provided by the present invention have the potential to be used as RIPK1 inhibitors and active ingredients of anti-inflammatory drugs.

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Abstract

5-取代吲哚3-酰胺衍生物制备方法及其用途，属于医药领域。提供了式Ⅰ所示的化合物或其药学上可接受的盐。该类化合物能够显著抑制RIPK1激酶的活力，而且选择性较高，安全性良好，是一种RIPK1激酶抑制剂，可以作为炎症、免疫性疾病、肿瘤和神经退行性疾病等的潜在治疗药物。TNFα诱导的SIRS模型实验证明，化合物能够在体内发挥抑制RIPK1激酶的作用，药代动力学结果显示该系列化合物具有良好药代动力学性质，为靶向RIPK1的疾病治疗提供了一种新的策略和手段。

Description

5-取代吲哚3-酰胺衍生物及其制备方法和用途 技术领域
本发明属于医药技术领域，具体涉及5-取代吲哚3-酰胺衍生物及其制备方法和用途。
背景技术
细胞程序性坏死(necroptosis/programmed necrosis)是近年来发现的一种不同于凋亡的、新的caspase非依赖性的细胞程序性死亡方式。它受死亡信号调控，呈现坏死样结构特点。与凋亡相比，细胞程序性坏死不形成凋亡小体，染色质不凝聚；与坏死相比，细胞程序性坏死是受多种基因调控的受控细胞死亡方式。体外培养体系中加入caspase抑制剂Z-VAD-FMK后，采用TNF可诱导细胞发生程序性坏死，细胞呈现坏死的形态特征为细胞肿胀、破裂、释放细胞内容物，继而引起炎症和免疫应答。除TNF外，TLR3和TLR4的配体，某些细菌、病毒感染等均可引起细胞发生细胞程序性坏死。
受体相互作用蛋白激酶1(RIPK1)是一种具有特异性丝氨酸/苏氨酸激酶活性的蛋白，具有类似于其他蛋白激酶的N-末端激酶结构域，但具有不同的结合结构域。研究表明，RIPK1是细胞程序性坏死的关键调控因子，它通过RIPK1/RIPK3/MLKL信号转导轴调控细胞程序性坏死。多种死亡受体，例如TNFR、FAS、TRAILR和Toll样受体等，可在受到炎症因子刺激或外源性感染的条件下触发细胞程序性坏死上游信号。在cIAPs和caspase活性受限的情况下，RIPK1与RIPK3形成坏死小体(necrosome)。RIPK3会进一步招募MLKL，被磷酸化的MLKL会自我寡聚化然后迁移到细胞膜，对细胞膜进行“打孔”，导致细胞内容物外泄和电离平衡破坏，最终导致细胞坏死的发生。
细胞程序性坏死与炎症、自身免疫性疾病、神经退行性疾病和肿瘤等相关疾病的发生发展密切相关。例如，研究表明，细胞程序性坏死是导致全身性炎症反应综合征(Systemic Inflammatory Response Syndrome，SIRS)的重要原因。SIRS是由于感染或非感染因素作用于机体，引起机体失控而自我破坏的全身炎症反应。危重病人常因机体代偿性抗炎反应能力降低及代谢功能紊乱，易引发SIRS。而TNFα诱导的SIRS疾病模型在长期的研究中被证明与RIPK1依赖的necroptosis高度相关。炎症性肠病(Inflammatory bowel  diseases,IBD)是指由于环境、遗传、感染、免疫等原因引起的异常免疫介导的肠道炎症，是一种慢性、非特异性肠炎性疾病。其发病机理尚不清楚，但肠道上皮细胞过度凋亡、肠黏膜屏障受损、肠上皮细胞通透性提高被认为是IBD发生的原因之一，有研究表明细胞程序性坏死在IBD的发病机制中起重要作用。此外，细胞程序性坏死在类风湿性关节炎(RA)、银屑病和多发性硬化症(MS)等多种自身免疫性疾病的发病机制中也起着重要作用。活化的小神经胶质细胞在阿尔兹海默症(AD)发生过程中起关键作用，而RIPK1在小神经胶质细胞中高表达，RIPK1抑制剂则能够在体外有效保护Aβ诱导的神经元细胞程序性坏死。除了阿尔兹海默症外，程序性坏死也参与肌萎缩性脊髓侧索硬化症(ALS)、额颞痴呆(FTD)和帕金森(PD)等众多神经退行性疾病的发生发展。Strilic等人于2016年首次揭示了肿瘤细胞可以诱导血管内皮细胞发生程序性坏死，继而肿瘤细胞穿过血管壁、并通过血液循环实现远端转移，这是导致肿瘤转移的重要原因，实验表明RIPK1抑制剂能有效的抑制肿瘤转移。此外，有研究表明RIPK1激酶可以促进胰腺癌肿瘤微环境中的耐受性巨噬细胞分化，RIPK1抑制则可以使胰腺癌肿瘤微环境中的免疫原性巨噬细胞分化，从而导致适应性免疫激活和肿瘤保护。
综上所述，RIPK1是炎症、自身免疫性疾病、神经退行性疾病和肿瘤等细胞程序性坏死相关疾病的重要治疗靶标，RIPK1抑制剂有望成为这些疾病的潜在治疗药物。
发明内容
针对需要开发新的抗细胞程序性坏死相关疾病药物的问题，本发明提供5-取代吲哚3-酰胺衍生物及其制备方法和用途，本发明化合物能够在体内发挥抑制RIPK1激酶的作用，药代动力学结果显示该系列化合物具有良好药代动力学性质。本发明为靶向RIPK1治疗炎症、自身免疫性疾病、神经退行性疾病和肿瘤等相关疾病提供了一种新的策略和手段。
式I所示的化合物、或其立体异构体、或其药学上可接受的盐：
Figure PCTCN2021116256-appb-000001
其中，
X 1、X 3独立选自-CR 6-、N；
X 2选自-NR 1-、-CH＝CH-；
其中，R 1选自氢、取代或未取代的C 1-C 10烷基、取代或未取代的C 1-C 10醚基、取代或未取代的C 3-C 10环烷基、取代或未取代的3-10元杂环烷基、取代或未取代的C 6-C 20芳基、取代或未取代的3-20元杂芳基；其中，所述取代基是氘、C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
R 2B选自
Figure PCTCN2021116256-appb-000002
其中，R 2选自被一个或两个R 21取代或未取代的C 0-C 6亚烷基；其中，R 21选自取代或未取代的C 1-C 10烷基、取代或未取代的C 3-C 10环烷基、氰基、羟基、羧基、卤素或硝基；其中，所述取代基为氰基、羟基、羧基、卤素或硝基；
B环选自被一个、两个或三个R 22取代或未取代的C 4-C 10芳基，被一个、两个或三个R 22取代或未取代的4-10元杂芳基或者被一个、两个或三个R 22取代或未取代的C 3-C 10环烷基；其中，R 22分别独立选自取代或未取代的C 1-C 10烷基、取代或未取代的C 1-C 6烷氧基、取代或未取代的4-10元杂环烷基、氰基、羟基、羧基、卤素或硝基；或者，两个R 22相连形成取代或未取代的4-10元杂环烷基；其中，所述取代基为C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
R 3选自氢、取代或未取代的C 1-C 10烷基；其中，所述取代基为氰基、羟基、羧基、卤素或硝基；
或者R 2B与R 3相连形成取代或未取代的3-10元杂环烷基；其中，所述取代基为C 1-C 10烷基、C 4-C 10芳基、一个或两个R 31取代的C 4-C 10芳基、氰基、羟基、羧基、卤素或硝基；其中，所述R 31选自C 1-C 10烷基或卤素；
R 4、R 5和R 6分别独立选自氢、C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
A环选自被一个或两个R A取代或未取代的C 4-C 10芳基、一个或两个R A取代或未取代的4-10元杂芳基；其中，R A选自取代或未取代的氨基、
Figure PCTCN2021116256-appb-000003
取代或未取代的C 1-C 10烷基、取代或未取代的 C 4-C 10芳基、取代或未取代的4-10元杂芳基、取代或未取代的3-10元杂环烷基、；其中，所述取代基为-R A2-R A3、R A4、C 1-C 10烷基、卤素取代的C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；其中，R A4选自被-R A2-R A3取代或未取代的3-10元杂环烷基；
R A1选自取代或未取代的C 1-C 10烷基、取代或未取代的C 1-C 10烯基、取代或未取代的C 3-C 10环烷基、取代或未取代的3-10元杂环烷基、取代或未取代的C 1-C 6烷氧基、取代或未取代的C 2-C 6醚基、取代或未取代的C 2-C 6胺基；其中，所述取代基为C 1-C 10烷基、羟基取代的C 1-C 10烷基、C 1-C 10酯基、3-10元杂环烷基、氨基、胺基、氰基、羟基、羧基、卤素或硝基；
R A2选自取代或未取代的C 0-C 6亚烷基、羰基；其中，所述取代基为C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
R A3选自取代或未取代的3-10元杂环烷基；其中，所述取代基为C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基。
优选的，所述式I的化合物如式Ⅱ所示：
Figure PCTCN2021116256-appb-000004
其中，
R 1选自氢、取代或未取代的C 1-C 10烷基、取代或未取代的C 3-C 10环烷基、取代或未取代的3-10元杂环烷基、取代或未取代的C 6-C 20芳基、取代或未取代的3-20元杂芳基；其中，所述取代基是C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
R 2选自被一个R 21取代或未取代的C 0-C 6亚烷基；其中，R 21选自取代或未取代的C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；其中，所述取代基为氰基、羟基、羧基、卤素或硝基；
B环选自被一个、两个或三个R 22取代或未取代的C 4-C 10芳基或者被一个、两个或三个R 22取代或未取代的4-10元杂芳基；其中，R 22分别独立选自取代或未取代的C 1-C 10烷基、取代或未取代的C 1-C 6烷氧基、取代或未取代的4-10元杂环烷基、氰基、羟基、羧基、卤素或硝基；或者，两个R 22相连 形成取代或未取代的4-10元杂环烷基；其中，所述取代基为C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
R 3选自氢、取代或未取代的C 1-C 10烷基；其中，所述取代基为氰基、羟基、羧基、卤素或硝基；
或者R 2与R 3相连形成取代或未取代的3-10元杂环烷基；其中，所述取代基为C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
X 1、X 3独立选自CH、N；
A环选自被一个或两个R A取代或未取代的C 4-C 10芳基、一个或两个R A取代或未取代的4-10元杂芳基；其中，R A选自氨基、
Figure PCTCN2021116256-appb-000005
取代或未取代的C 1-C 10烷基、取代或未取代的C 4-C 10芳基、取代或未取代的4-10元杂芳基、取代或未取代的3-10元杂环烷基、；其中，所述取代基为-R A2-R A3、R A4、C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；其中，R A4选自被-R A2-R A3取代或未取代的3-10元杂环烷基；
R A1选自取代或未取代的C 1-C 10烷基、取代或未取代的C 1-C 10烯基、取代或未取代的C 3-C 10环烷基、取代或未取代的3-10元杂环烷基、取代或未取代的C 1-C 6烷氧基、取代或未取代的C 2-C 6醚基；其中，所述取代基为C 1-C 10烷基、3-10元杂环烷基、氨基、胺基、氰基、羟基、羧基、卤素或硝基；
R A2选自取代或未取代的C 0-C 6亚烷基、羰基；其中，所述取代基为C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
R A3选自取代或未取代的3-10元杂环烷基；其中，所述取代基为C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基。
优选的，所述式I的化合物如式Ⅲ或式Ⅳ所示：
Figure PCTCN2021116256-appb-000006
其中，R 1选自氢、C 1-C 4烷基；R 23选自氢、甲基；B环选自被一个、两个R 22取代或未取代的苯基；其中，R 22分别独立选自F、Cl、氰基、甲基、三氟甲基、甲氧基或三氟甲氧基。
优选的，所述式I的化合物如式Ⅴ所示：
Figure PCTCN2021116256-appb-000007
其中，
X 1、X 3独立选自-CR 6-、N；
X 2选自-NR 1-、-CH＝CH-；
所述R 1选自氢、取代或未取代的C 1-C 10烷基、取代或未取代的C 3-C 10环烷基、取代或未取代的3-10元杂环烷基、取代或未取代的C 6-C 20芳基、取代或未取代的3-20元杂芳基；其中，所述取代基是C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
L 3选自取代或未取代的C 1-C 4亚烷基；其中，所述取代基为C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
R 33选自氢、取代或未取代的C 1-C 10烷基、取代或未取代的C 3-C 10环烷基、取代或未取代的3-10元杂环烷基、取代或未取代的C 6-C 10芳基；其中，所述取代基为C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
R 4、R 5和R 6分别独立选自氢、C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
A环选自被一个或两个R A取代或未取代的C 4-C 10芳基、一个或两个R A取代或未取代的4-10元杂芳基；其中，R A选自氨基、
Figure PCTCN2021116256-appb-000008
取代或未取代的C 1-C 10烷基、取代或未取代的C 4-C 10芳基、取代或未取代的4-10元杂芳基、取代或未取代的3-10元杂环烷基、；其中，所述取代基为-R A2-R A3、R A4、C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；其中，R A4选自被-R A2-R A3取代或未取代的3-10元杂环烷基；
R A1选自取代或未取代的C 1-C 10烷基、取代或未取代的C 1-C 10烯基、取代或未取代的C 3-C 10环烷基、取代或未取代的3-10元杂环烷基、取代或未取代的C 1-C 6烷氧基、取代或未取代的C 2-C 6醚基；其中，所述取代基为C 1- C 10烷基、C 1-C 10酯基、3-10元杂环烷基、氨基、胺基、氰基、羟基、羧基、卤素或硝基；
R A2选自取代或未取代的C 0-C 6亚烷基、羰基；其中，所述取代基为C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
R A3选自取代或未取代的3-10元杂环烷基；其中，所述取代基为C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基。
优选的，R 33选自苯基、被一个或两个取代基取代的苯基；其中，所述取代基选自F、Cl或甲基。
优选的，所述式I的化合物如式Ⅵ所示：
Figure PCTCN2021116256-appb-000009
其中，
X 1、X 3独立选自-CR 6-、N；
X 2选自-NR 1-、-CH＝CH-；
X 3选自CH、N；
所述R 1选自氢、取代或未取代的C 1-C 10烷基、取代或未取代的C 3-C 10环烷基、取代或未取代的3-10元杂环烷基、取代或未取代的C 6-C 20芳基、取代或未取代的3-20元杂芳基；其中，所述取代基是C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
L 4选自取代或未取代的C 1-C 3亚烷基；其中，所述取代基为C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
R 3选自氢、取代或未取代的C 1-C 10烷基；其中，所述取代基为氰基、羟基、羧基、卤素或硝基；
R 4、R 5和R 6分别独立选自氢、C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
A环选自被一个或两个R A取代或未取代的C 4-C 10芳基、一个或两个R A 取代或未取代的4-10元杂芳基；其中，R A选自氨基、
Figure PCTCN2021116256-appb-000010
取代或未取代的C 1-C 10烷基、取代或未取代的C 4-C 10芳基、取代或未取代的4-10元杂芳基、取代或未取代的3-10元杂环烷基、；其中，所述取代基为-R A2-R A3、R A4、C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；其中，R A4选自被-R A2-R A3取代或未取代的3-10元杂环烷基；
R A1选自取代或未取代的C 1-C 10烷基、取代或未取代的C 1-C 10烯基、取代或未取代的C 3-C 10环烷基、取代或未取代的3-10元杂环烷基、取代或未取代的C 1-C 6烷氧基、取代或未取代的C 2-C 6醚基；其中，所述取代基为C 1-C 10烷基、3-10元杂环烷基、氨基、胺基、氰基、羟基、羧基、卤素或硝基；
R A2选自取代或未取代的C 0-C 6亚烷基、羰基；其中，所述取代基为C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
R A3选自取代或未取代的3-10元杂环烷基；其中，所述取代基为C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基。
优选的，所述L 4选自的C 1-C 2的亚烷基。
优选的，所述X 3选自CH、N。
优选的，R 1选自氢、C 1-C 4烷基、C 3醚基。
优选的，所述R 4、R 5和R 6分别独立选自氢、F。
优选的，A环选自被一个或两个R A取代或未取代的5-9元杂芳基、被一个或两个R A取代或未取代的6元芳基；其中，R A选自氨基、被C 1-C 4烷基取代的氨基、被-CF 3取代的氨基、被-CHF 2取代的氨基、
Figure PCTCN2021116256-appb-000011
Figure PCTCN2021116256-appb-000012
甲基、被-R A2-R A3取代或未取代的5-6元杂芳基、被R A4取代的甲基、被-R A2-R A3取代或未取代的苯基；其中，R A4选自被-R A2-R A3取代或未取代的6元杂环烷基；R A1选自C 2-C 5烷基、Cl取代的C 4烷基、5元杂环烷基取代的甲基、乙烯基、N,N-二甲基胺基取代的乙烯基、C 3-C 6环烷基、一个或两个F取代的C 3-C 6环烷基、4-6元杂环烷基、羟基取代的4-6元杂环烷基、-CH 2OH取代的4-5元杂环烷基、甲基取代的6元杂环烷基、甲氧基、F 取代的甲氧基、C 2-C 3醚基、羟基取代的丙胺基；
R A2选自C 0-C 1亚烷基、羰基；
R A3选自甲基取代或未取代的6元杂环烷基。
优选的，A环选自
Figure PCTCN2021116256-appb-000013
Figure PCTCN2021116256-appb-000014
其中L 1选自S或NH；L 2分别独立选自CH或N。
优选的，A环选自：
Figure PCTCN2021116256-appb-000015
Figure PCTCN2021116256-appb-000016
优选的，R 2选自被一个或两个R 21取代或未取代的C 0-C 2亚烷基；其中，R 21选自甲基；
B环选自被一个、两个或三个R 22取代或未取代的C 6-C 10芳基，被一个、两个或三个R 22取代或未取代的5-9元杂芳基或者被一个、两个或三个R 22取 代或未取代的C 9-C 10环烷基；其中，R 22分别独立选自C 1-C 4烷基、甲氧基、卤素、卤素取代的甲基、卤素取代的甲氧基、氰基、硝基、
Figure PCTCN2021116256-appb-000017
卤素选自F、Cl、Br。
优选的，R 3选自氢。
优选的，所述式I的化合物如式Ⅶ所示：
Figure PCTCN2021116256-appb-000018
其中，
R 1选自氢、取代或未取代的C 1-C 10烷基、取代或未取代的C 3-C 10环烷基、取代或未取代的3-10元杂环烷基、取代或未取代的C 6-C 20芳基、取代或未取代的3-20元杂芳基；其中，所述取代基是C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
R 31选自C 1-C 6亚烷基；
R 32选自被一个、两个或三个R 311取代或未取代的C 4-C 10芳基，其中，R 311分别独立选自C 1-C 6烷氧基，或者两个R 311相连形成4-10元杂环烷基；
X 1、X 3独立选自CH、N；
A环选自被一个或两个R A取代或未取代的C 4-C 10芳基、一个或两个R A取代或未取代的4-10元杂芳基；其中，R A选自氨基、
Figure PCTCN2021116256-appb-000019
取代或未取代的C 1-C 10烷基、取代或未取代的C 4-C 10芳基、取代或未取代的4-10元杂芳基、取代或未取代的3-10元杂环烷基、；其中，所述取代基为-R A2-R A3、R A4、C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；其中，R A4选自被-R A2-R A3取代或未取代的3-10元杂环烷基；
R A1选自取代或未取代的C 1-C 10烷基、取代或未取代的C 1-C 10烯基、取代或未取代的C 3-C 10环烷基、取代或未取代的3-10元杂环烷基、取代或未 取代的C 1-C 6烷氧基、取代或未取代的C 2-C 6醚基；其中，所述取代基为C 1-C 10烷基、3-10元杂环烷基、氨基、胺基、氰基、羟基、羧基、卤素或硝基；
R A2选自取代或未取代的C 0-C 6亚烷基、羰基；其中，所述取代基为C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
R A3选自取代或未取代的3-10元杂环烷基；其中，所述取代基为C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基。
优选的，-R 31-R 32选自
Figure PCTCN2021116256-appb-000020
优选的，式I-式Ⅶ所示化合物具体为：
Figure PCTCN2021116256-appb-000021
Figure PCTCN2021116256-appb-000022
Figure PCTCN2021116256-appb-000023
Figure PCTCN2021116256-appb-000024
Figure PCTCN2021116256-appb-000025
Figure PCTCN2021116256-appb-000026
Figure PCTCN2021116256-appb-000027
Figure PCTCN2021116256-appb-000028
Figure PCTCN2021116256-appb-000029
Figure PCTCN2021116256-appb-000030
Figure PCTCN2021116256-appb-000031
Figure PCTCN2021116256-appb-000032
Figure PCTCN2021116256-appb-000033
Figure PCTCN2021116256-appb-000034
Figure PCTCN2021116256-appb-000035
Figure PCTCN2021116256-appb-000036
Figure PCTCN2021116256-appb-000037
Figure PCTCN2021116256-appb-000038
Figure PCTCN2021116256-appb-000039
本发明还提供上述化合物的制备方法，
通过如下反应步骤进行：
Figure PCTCN2021116256-appb-000040
其中，R 2B、R 4、R 5、X 1、X 2、X 3和A环如上所述。
优选的，所述中间体A的合成过程为：
将原料A、醋酸钾、联硼酸频那醇酯和[1,1’-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物溶于无水二氧六环，用惰性气体置换反应体系后反应，反应完毕，将反应液浓缩、拌样、柱层析，得到产品；
所述中间体A与原料B合成式I化合物的过程为：
将中间体A、原料B、[1,1’-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物、、三环己基磷膦和、碳酸铯溶于二氧六环/水混合液中，用惰性气体置换反应体系后反应，反应完毕，将反应液浓缩、拌样、柱层析，得到产品；
所述中间体B的合成过程为：
将原料B、醋酸钾、联硼酸频那醇酯和[1,1’-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物溶于无水二氧六环，用惰性气体置换反应体系后反应，反应完毕，将反应液浓缩、拌样、柱层析，得到产品；
所述所述中间体B与原料A合成式I化合物的过程为：
将中间体B、原料A、[1,1’-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物、三环己基磷膦和碳酸铯溶于二氧六环/水混合液中，用惰性气体置换反应体系后反应，反应完毕，将反应液浓缩、拌样、柱层析，得到产品。
本发明还提供上述化合物、或其立体异构体、或其药学上可接受的盐在制备RIPK1抑制剂中的用途。
本发明还提供上述化合物、或其立体异构体、或其药学上可接受的盐在制备用于治疗炎症、免疫性疾病、神经退行性疾病或肿瘤的药物中的用途。
优选的，所述药物用于治疗细胞程序性坏死相关的炎症反应、免疫性疾病、神经退行性疾病或肿瘤。
优选的，所述炎症为结肠炎。
本发明还提供一种药物组合物，它是以上述化合物、或其立体异构体、或其药学上可接受的盐，加上药学上可接受的辅料制备而成的制剂。本发明所定义的程序性细胞坏死是一个由基因决定的细胞主动的有序的死亡方式。具体指细胞遇到内、外环境因子刺激时，受基因调控启动的自杀保护措施，包括一些分子机制的诱导激活和基因编程，通过这种方式去除体内非必需细胞或即将发生特化的细胞。TNF、TLR3和TLR4的配体、某些细菌、病毒感染等均可引起程序性细胞坏死。
本发明中提供的化合物和衍生物可以根据IUPAC(国际纯粹与应用化学联合会)或CAS(化学文摘服务社，Columbus，OH)命名系统命名。
关于本发明的使用术语的定义：除非另有说明，本文中基团或者术语提供的初始定义适用于整篇说明书的该基团或者术语；对于本文没有具体定义的术语，应该根据公开内容和上下文，给出本领域技术人员能够给予它们的含义。
“取代”是指分子中的氢原子被其它不同的原子或分子所替换。
碳氢基团中碳原子含量的最小值和最大值通过前缀表示，例如，前缀C a-C b烷基表明任何含“a”至“b”个碳原子的烷基。因此，例如，“C 1-C 4烷基”是指包含1-4个碳原子的烷基。特别地，用“C 0-C b”限定亚烷基时，表示该部位可以是无亚烷基。
“烷基”是指具有指定数目的成员原子的饱和烃链。例如，C 1-C 6烷基是指具有1至6个成员原子，例如1至4个成员原子的烷基基团。烷基基团可以是直链或支链的。代表性的支链烷基基团具有一个、两个或三个支链。烷基基团可任选地被一个或多个如本文所定义的取代基取代。烷基包括甲基、 乙基、丙基(正丙基和异丙基)、丁基(正丁基、异丁基和叔丁基)、戊基(正戊基、异戊基和新戊基)和己基。烷基基团也可以是其他基团的一部分，所述其他基团为例如C 1-C 6烷氧基。
“环烷基”是指具有3至14个碳原子且没有环杂原子且具有单个环或多个环(包括稠合、桥连和螺环体系)的饱和或部分饱和的环状基团。对于具有不含环杂原子的芳族和非芳族环的多环体系，当连接点位于非芳族碳原子时，适用术语“环烷基”(例如5,6,7,8,-四氢化萘-5-基)。术语“环烷基”包括环烯基基团，诸如环己烯基。环烷基基团的实例包括例如，金刚烷基、环丙基、环丁基、环己基、环戊基、环辛基、环戊烯基和环己烯基。
“烯基”是指具有2至10个碳原子和在一些实施方案中2至6个碳原子或2至4个碳原子且具有至少1个乙烯基不饱和位点(>C＝C<)的直链或支链烃基基团。例如，(Ca-Cb)烯基是指具有a至b个碳原子的烯基基团并且意在包括例如乙烯基、丙烯基、异丙烯基、1,3-丁二烯基等。
“炔基”是指含有至少一个三键的直链一价烃基或支链一价烃基。术语“炔基”还意在包括具有一个三键和一个双键的那些烃基基团。例如，(C2-C6)炔基意在包括乙炔基、丙炔基等。
“卤素”为氟、氯、溴或碘。
“杂环”、“杂环烷基”指包含至少一个杂原子的饱和环或非芳香性的不饱和环；其中杂原子指氮原子、氧原子、硫原子；
“杂芳基”指包含至少一个杂原子的芳香性不饱和环基；其中杂原子指氮原子、氧原子、硫原子；
“醚基”是指醚类化合物的碳原子去掉一个H后形成的基团。C 2-C 6醚基是指所述醚类化合物中，氧原子两端连接的烃基的碳原子总数为2至6个。
“胺基”是指胺类化合物去掉一个H后形成的基团。胺类化合物是指氨分子中的氢原子部分或全部被烃基取代后形成的化合物，C 2-C 6胺基是指所述胺类化合物中，氧原子两端连接的烃基的碳原子总数为2至6个。
“酯基”是指酯类化合物的碳原子去掉一个H后形成的基团。酯类化合物是指酸与醇失水后形成的化合物，其具有“-COO-”的官能团。C 1-C 10酯基是指所述胺类化合物中，碳原子总数为1至10个。
“R a和R b相连形成杂环”指R a和R b中分别至少有一个原子通过化学键连接，使得通式结构中R a和R b共同连接的原子或原子链作为环结构的一部分骨架与R a和R b共同构成杂环。
“立体异构体”包括对映异构体和非对映异构体。
在本发明中，与代表取代基的符号连接的横线表示共价键。例如：“-R”是指R通过一个共价单键与其他基团连接；“-R-”是指R通过两个共价单键与其他基团连接；“-R A2-R A3”是指R A3通过一个共价单键与R A2连接，R A2通过两个共价单键分别与R A3和一个其他基团连接。
术语“药学上可接受的”是指某载体、运载物、稀释剂、辅料，和/或所形成的盐通常
在化学上或物理上与构成某药物剂型的其它成分相兼容，并在生理上与受体相兼容。
术语“盐”和“可药用的盐”是指上述化合物或其立体异构体，与无机和/或有机酸和碱形成的酸式和/或碱式盐，也包括两性离子盐(内盐)，还包括季铵盐，例如烷基铵盐。这些盐可以是在化合物的最后分离和纯化中直接得到。也可以是通过将上述化合物，或其立体异构体，与一定数量的酸或碱适当(例如等当量)进行混合而得到。这些盐可能在溶液中形成沉淀而以过滤方法收集，或在溶剂蒸发后回收而得到，或在水介质中反应后冷冻干燥制得。本发明中所述盐可以是化合物的盐酸盐、硫酸盐、枸橼酸盐、苯磺酸盐、氢溴酸盐、氢氟酸盐、磷酸盐、乙酸盐、丙酸盐、丁二酸盐、草酸盐、苹果酸盐、琥珀酸盐、富马酸盐、马来酸盐、酒石酸盐或三氟乙酸盐。
在某些实施方式中，本发明的一种或多种化合物可以彼此联合使用。也可选择将本发明的化合物与任何其它的活性试剂结合使用，用于制备调控细胞功能或治疗疾病的药物或药物组合物。如果使用的是一组化合物，则可将这些化合物同时、分别或有序地对受试对象进行给药。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1为本发明化合物34的激酶选择性谱图；
图2为本发明化合物94的激酶选择性谱图；
图3为实施例5中细胞生存率曲线图；
图4为实施例6中化合物46和94对坏死信号通路的影响；
图5为实施例7中化合物46和94保护了TNFα诱导的小鼠SIRS模型的结果；
图6为实施例8中化合物特非那定(左)和化合物94(右)浓度-反应曲线。
图7为实施例8中小鼠结肠长度的对比实验结果；
图8为实施例10中化合物特非那定(左)和化合物94(右)对hERG电流的抑制作用浓度-反应曲线。
具体实施方式
下面参照实施例进一步阐释本公开。对本公开的具体示例性实施方案的描述是为了说明和例证的目的。这些描述并非想将本公开限定为所公开的精确形式，并且很显然，根据本申请说明书的教导，可以进行很多改变和变化。对示例性实施例进行选择和描述的目的在于解释本公开的特定原理及其实际应用，从而使得本领域的技术人员能够实现并利用本公开的各种不同的示例性实施方案以及各种不同的选择和改变。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
NMR的测定是用BRUKER 400MR DD2核磁仪，测定溶剂为氘代二甲基亚砜(DMSO-d6)，氘代甲醇(CD3OD)和氘代氯仿(CDCl3)，内标为四甲基硅烷(TMS)。液质联用色谱LC-MS的测定用Agilent 1200 Infinity II–InfinityLab LC/MSD质谱仪。HPLC的测定使用Agilent 1200 Infinity II高压液相色谱仪(Sunfire C18 5um 150x 4.6mm色谱柱；)。薄层层析硅胶板使用烟台江友硅胶开发有限公司HSGF254硅胶板，规格是
Figure PCTCN2021116256-appb-000041
TLC硅胶板使用于成化工(上海)有限公司GF254硅胶板，规格是
Figure PCTCN2021116256-appb-000042
Figure PCTCN2021116256-appb-000043
柱层析使用青岛海浪硅胶干燥剂有限公司
Figure PCTCN2021116256-appb-000044
目硅胶为载体。Flash柱使用艾杰尔飞诺美Claricep Flash无定形硅胶纯化柱。本发明实施例中的试剂1-丙基磷酸酐,甲基溴化镁是从上海麦克林生化科技有限公司购买，4N盐酸二氧六环溶液从盘锦研峰科技有限公司购买，1M硼烷的四氢呋喃溶液、N,N-二异丙基乙胺、(S)-叔丁基亚磺酰胺是从阿达玛斯试剂购买其他试剂和起始原料从上海皓鸿生物医药科技有限公司购买，或者可以采 用本领域已知的方法来合成。在无特殊说明的情况下，本发明的所有反应均在连续的磁力搅拌下，在干燥氮气或氩气下进行，溶剂为干燥溶剂，反应温度单位为摄氏度。
实施例1本发明化合物34制备(方法Ⅰ)。
Figure PCTCN2021116256-appb-000045
i N,N-二甲基甲酰胺，(S)-1-(3-氟苯基)乙胺，1-羟基苯并三唑，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐，N,N-二异丙基乙胺，65度，8小时，产率85％。
ii吡啶，环丙甲酰氯，三乙胺，0℃到常温，6小时，产率90％；
iii无水二氧六环,[1,1’-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物，醋酸钾，联硼酸频那醇酯，95度，24小时,产率60％；
iv 1,4-二氧六环/水＝10：1，[1,1’-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物，三环己基磷，碳酸铯,100度,16小时,产率75％；
具体操作步骤包括：
(S)-5-溴-N-(1-(3-氟苯基)乙基)-1H-吲哚-3-酰胺(中间体2)的制备：
将5-溴吲哚-3-甲酸(4.5g,18.8mmol)、1-羟基苯并三唑(HOBT)(3g,21.6mmol)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI)(4.3g,23.4mmol)溶于60毫升N,N-二甲基甲酰胺中，然后加入N,N-二异丙基乙胺(3.6mL,23.4mmol)，常温活化羧酸0.5小时后，加入(S)-1-(3-氟苯基)乙胺(2.8 g,19.8mmol)并将反应加热至65度反应8小时，TLC监控反应完成后，将反应液浓缩，用二氯甲烷和硫代硫酸钠溶液进行萃取，有机相水洗、饱和氯化钠溶液洗涤后加入硫酸钠干燥，抽滤干燥后经柱层析得到中间体2(5.7g，白色固体)，产率为85％。
1H NMR(400MHz,DMSO-d 6)δ11.46(s,1H),8.35(s,1H),8.19(d,J＝5.9Hz,1H),7.96(d,J＝2.5Hz,1H),7.25(d,J＝7.7Hz,1H),7.11–7.02(m,2H),7.00(s,1H),6.97(d,J＝7.0Hz,1H),5.21–5.11(m,1H),1.47(d,J＝7.1Hz,3H)
N-(6-溴苯并[d]噻唑-2-基)环丙甲酰胺(中间体4)的制备：
将1克中间体3(1.0g,4.4mmol)溶于15毫升干燥吡啶中，加入1.4mL三乙胺(575mg,5.7mmol)，在0度条件下缓慢滴入370μL环丙甲酰氯(499mg,4.8mmol)，温度逐渐升至室温反应6小时，直接将反应液浓缩、用二氯甲烷和饱和碳酸钠溶液进行萃取，有机相水洗、饱和氯化钠溶液洗涤后加入硫酸钠干燥，柱层析纯化得中间体4(1.2g，淡黄色固体)，产率为90％。 1H NMR(400MHz,CDCl 3)δ9.01(s,1H),8.11(d,J＝6.9Hz,1H),7.55(s,1H),7.02(d,J＝5.4Hz,1H),1.23(d,J＝11.0Hz,1H),1.16(dt,J＝7.4 3.9Hz,2H),0.93(td,J＝7.0,3.9Hz,2H).
N-(6-(频那醇硼酸酯)苯并[d]噻唑-2-基)环丙甲酰胺(中间体5)的制备：
将中间体4(10g,33.8mmol)、醋酸钾(6.6g,67.6mmol)、12克联硼酸频那醇酯(12g,47.2mmol)和2.8克[1,1’-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(2.8g,3.4mmol)溶于200毫升无水二氧六环中，用氮气将反应体系置换三次后，置于95度反应24小时，石油醚：乙酸乙酯＝20：1展开剂监控反应，反应完毕，将反应液浓缩、拌样、柱层析，选用石油醚：乙酸乙酯＝80：1洗脱液纯化得到中间体5(6.9g，白色固体)，产率为60％。 1H NMR(400MHz,DMSO-d 6)δ12.01(s,1H),8.45(d,J＝6.7Hz,1H),8.03(s,1H),7.87(d,J＝5.7Hz,1H),1.21(d,J＝11.2Hz,1H),1.33(s,12H),1.11(dt,J＝7.4 3.9Hz,2H),0.99(td,J＝7.0,3.9Hz,2H).
(S)-5-(2-(环丙甲酰胺)苯并[d]噻唑-6-基)-N-(1-(3-氟苯基)乙基)-1H-吲哚-3-甲酰胺(化合物34)的制备：
将中间体2(400mg,1.1mmol)、中间体5(580mg,1.7mmol)、133 毫克[1,1’-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(133mg,0.16mmol)、41毫克三环己基膦(45mg,0.16mmol)和780毫克碳酸铯(780mg,2.4mmol)溶于100毫升1，4-二氧六环/水(10：1)的混合液中，将反应体系用氮气置换三次后置于100度反应16小时，乙酸乙酯：石油醚＝1：1展开剂监控反应，TLC监控反应完毕，将反应液浓缩、拌样、柱层析，选用乙酸乙酯：石油醚＝5:1洗脱液纯化出化合物34(318mg，类白色固体)，产率为58％。
1H NMR(400MHz,DMSO)δ12.63(s,1H),11.66(s,1H),8.43(s,1H),8.31(d,J＝7.9Hz,1H),8.21(d,J＝2.4Hz,1H),7.81–7.75(m,2H),7.70(dd,J＝8.5,1.6Hz,2H),7.53(d,J＝8.1Hz,2H),7.37(dd,J＝14.1,8.1Hz,2H),7.25(t,J＝9.4Hz,2H),7.09–7.00(m,1H),5.75(s,1H),5.27–5.15(m,1H),2.06(d,J＝11.0Hz,1H),0.96(d,J＝5.1Hz,4H).
实施例2本发明化合物94的制备(方法Ⅱ)。
Figure PCTCN2021116256-appb-000046
i N,N-二甲基甲酰胺，碘甲烷，碳酸铯，80度，8小时，90％。
ii甲醇，水，氢氧化钠，60度，3小时，95％；
iii N,N-二甲基甲酰胺，(S)-1-(3-氟苯基)乙胺，1-羟基苯并三唑，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐，N,N-二异丙基乙胺，65度，8小时，85％。
iv 1,4-二氧六环/水＝5：1，[1,1’-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物，三环己基磷，碳酸铯,95度,12小时,75％；
v吡啶，环丙甲酰氯，三乙胺，0℃到常温，6小时，90％；
vi 1,4-二氧六环/水＝10：1，[1,1’-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物，三环己基磷，碳酸铯,95度,12小时,75％；
具体操作步骤包括：
甲基5-溴-1-甲基-1H-吲哚-3-甲酸(中间体2)的制备：
将中间体1(1.0g,3.9mmol)，碘甲烷(700mg,4.9mmol)和碳酸铯(1.8g,5.5mmol)溶于30毫升N，N-二甲基甲酰胺中，氮气保护下80度反应8小时，乙酸乙酯：石油醚＝1:3展开剂监控反应，反应完毕，直接将反应液浓缩、拌样、柱层析。乙酸乙酯：石油醚＝1:1洗脱液纯化得到中间体2(937mg，白色固体)，产率为90％。
1H NMR(400MHz,DMSO-d 6)δ8.18(s,1H),8.11(d,J＝1.9Hz,1H),7.55(d,J＝8.7Hz,1H),7.41(dd,J＝8.7,1.9Hz,1H),3.86(s,3H),3.81(s,3H).
5-溴-1-甲基-1H-吲哚-3-甲酸(中间体3)的制备：
将中间体2(507mg,1.9mmol)溶于20毫升甲醇中，加入10毫升2mol/L的氢氧化钠溶液，60度条件下搅拌反应3小时，乙酸乙酯：石油醚＝1:1展开剂监控反应，反应结束后直接将反应液浓缩、加水稀释后调节pH＝4,析出固体，固体抽滤干燥得到中间体3(432mg，白色固体),产率为90％，未经进一步纯化直接进行下一步反应。
(S)-5-溴-N-(1-(3-苯基)乙基)-1-甲基-1H-吲哚-3-甲酰胺(中间体4)的制备：
将中间体3(987mg,3.9mmol)、1-羟基苯并三唑(HOBT)(806mg,5.8mmol)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI)(1.0g,5.8mmol)溶于20毫升N,N-二甲基甲酰胺中，然后加入N,N-二异丙基乙胺(1.0g,7.8mmol)，常温活化羧酸0.5小时后，加入(S)-1-(3-氟苯基)乙胺(650mg,4.6 mmol),并将反应加热至65度反应8小时，TLC监控反应完成后，将反应液浓缩，用二氯甲烷和硫代硫酸钠溶液进行萃取，有机相水洗、饱和氯化钠溶液洗涤后加入硫酸钠干燥，抽滤后柱层析得到中间体4(1.2g，白色固体)，产率为84％。
1H NMR(400MHz,DMSO)δ8.34(d,J＝8.3Hz,1H),8.26(d,J＝1.9Hz,1H),8.19(s,1H),7.49(d,J＝8.8Hz,1H),7.37–7.30(m,2H),7.22(t,J＝9.0Hz,2H),7.05(d,J＝9.3Hz,1H),5.17(d,J＝7.5Hz,1H),3.84(s,3H),1.46(d,J＝7.1Hz,3H).
(S)-N-(1-(3-苯基)乙基)-1-甲基-5-(频那醇硼酸酯)-1H-吲哚-3-甲酰胺(中间体5)的制备：
将中间体4(2.0g,5.3mmol)、醋酸钾(1.1g,11.2mmol)、联硼酸频那醇酯(1.7g,6.9mmol)和[1,1’-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(410mg,0.5mmol)溶于50毫升无水二氧六环，用氮气将反应体系置换三次后，置于95度反应12小时，反应完毕，将反应液垫硅藻土过滤，浓缩、拌样、柱层析，选用石油醚：乙酸乙酯＝1：1洗脱液纯化出中间体5(1.3g，淡黄色固体)，产率为60％。
1H NMR(400MHz,DMSO-d 6)δ8.55(s,1H),8.33(d,J＝7.9Hz,1H),8.14(s,1H),7.48(t,J＝6.8Hz,2H),7.36(dd,J＝14.1,8.0Hz,1H),7.22(t,J＝9.5Hz,2H),7.06–6.99(m,1H),5.19–5.11(m,1H),3.84(s,3H),1.47(d,J＝7.1Hz,3H),1.30(s,12H).
N-(7-溴-[1,2,4]三唑并[1,5-a]吡啶-2-基)环丙甲酰胺(中间体7)的制备：
将中间体6(1.0g,4.7mmol)溶于15毫升干燥吡啶中，加入1.4mL三乙胺(949mg,9.4mmol)，在0度条件下缓慢滴入环丙甲酰氯(541mg,5.2mmol)，温度逐渐升至室温反应6小时，直接将反应液浓缩、用二氯甲烷和饱和碳酸钠溶液进行萃取，有机相水洗、饱和氯化钠溶液洗涤后加入硫酸钠干燥，柱层析纯化得到中间体7(1.2g，黄色固体)，产率为90％。
1H NMR(400MHz,CDCl3)δ9.25(s,1H),8.40(d,J＝7.1Hz,1H),7.82(s,1H),7.09(d,J＝5.9Hz,1H),1.27(d,J＝11.5Hz,1H),1.21(dt,J＝7.6,3.9Hz,2H),0.96(td,J＝7.0,3.9Hz,2H).
(S)-5-(2-(环丙甲酰胺)-[1,2,4]三唑并[1,5-a]吡啶-7-基)-N-(1-(3-苯基)乙 基)-1-甲基-1H-吲哚-3-甲酰胺(化合物94)的制备：
将中间体7(280mg,1.0mmol)、中间体5(422mg,1.0mmol)、[1,1’-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(85mg,0.1mmol)、三环己基膦(28mg,0.1mmol)和碳酸铯(390mg,1.2mmol)溶于100毫升1，4-二氧六环/水(10：1)的混合液中，将反应体系用氮气置换三次后置于100度反应12小时，乙酸乙酯：石油醚＝3：1展开剂监控反应，与原料极性相比，产物点极性增大，TLC监控反应完毕，将反应液浓缩、拌样、柱层析，选用乙酸乙酯：石油醚＝10:1洗脱液纯化出化合物94(372mg，白色固体)，产率为75％。
1H NMR(400MHz,DMSO)δ11.04(s,1H),8.83(d,J＝7.1Hz,1H),8.53(s,1H),8.37(d,J＝8.0Hz,2H),8.22(s,1H),7.86(s,1H),7.72(d,J＝8.6Hz,1H),7.66(d,J＝8.7Hz,1H),7.46–7.40(m,1H),7.37(dd,J＝14.2,7.8Hz,2H),7.25(t,J＝9.7Hz,2H),7.05(t,J＝8.6Hz,1H),5.22(dd,J＝14.3,6.9Hz,1H),3.90(s,3H),2.07(s,1H),0.96(d,J＝5.3Hz,4H).
实施例3本发明化合物142制备(方法Ⅲ)
Figure PCTCN2021116256-appb-000047
i 2,4,6-三丙基-1,3,5,2,4,6-三氧三磷酸-2,4,6-三氧化物，吡啶，80℃，12小时，产率80％。
具体操作步骤包括：
(S)-5-(2-氨基-[1,2,4]三唑并[1,5-a]吡啶-7-基)-N-(1-(3-氟苯基)乙基)-1-甲基-1H-吲哚-3-甲酰胺(141)的制备：
按方法II所示操作步骤可制备获得化合物141。
1H NMR(400MHz,DMSO-d 6)δ8.55(d,J＝7.0Hz,1H),8.48(d,J＝1.8Hz,1H),8.37(d,J＝8.0Hz,1H),8.21(s,1H),7.73–7.60(m,2H),7.55(d,J＝2.0Hz,1H),7.37(q,J＝7.4Hz,1H),7.32–7.12(m,3H),7.04(td,J＝8.7,2.6Hz,1H),5.99(s,2H),5.20(q,J＝7.3Hz,1H),3.89(s,3H),1.49(d,J＝7.1Hz,3H).
5-(2-(2,2-二氟环丙烷-1-甲酰胺)-[1,2,4]三唑并[1,5-a]吡啶-7-基)-N-((S)-1-(3-氟苯基)乙基)-1-甲基-1H-吲哚-3-甲酰胺(142)的制备：
将化合物141(150mg,0.35mmol)与的2，2-二氟环丙烷羧酸(85mg,0.70mmol)溶于15毫升干燥吡啶中，室温下加入2,4,6-三丙基-1,3,5,2,4,6-三氧三磷酸-2,4,6-三氧化物(229mg,50％的乙酸乙酯溶液)，温度逐渐升至80℃反应6小时，直接将反应液浓缩，用二氯甲烷拌样，柱层析纯化得到142(149mg，淡黄色固体)，产率为80％。
1H NMR(400MHz,DMSO-d 6)δ11.41(s,1H),8.93(d,J＝7.0Hz,1H),8.60(d,J＝1.8Hz,1H),8.45(d,J＝8.1Hz,1H),8.29(s,1H),7.96(d,J＝1.9Hz,1H),7.80(dd,J＝8.6,1.9Hz,1H),7.72(d,J＝8.7Hz,1H),7.53(dd,J＝7.2,2.0Hz,1H),7.46–7.39(m,1H),7.35–7.28(m,2H),7.15–7.05(m,1H),5.27(t,J＝7.4Hz,1H),3.95(d,J＝13.0Hz,3H),2.10(d,J＝7.1Hz,1H),1.55(d,J＝7.0Hz,3H),1.31–1.18(m,2H).
实施例4本发明化合物162的制备(方法IV)
Figure PCTCN2021116256-appb-000048
i氯甲酸甲酯，N,N-二异丙基乙胺，二氯甲烷，室温，6小时，90％。
ii(R)-3-吡咯烷醇，吡啶，100度，16小时，80％；
(S)-5-(2-氨基-[1,2,4]三唑并[1,5-a]吡啶-7-基)-N-(1-(3-氟苯基)乙基)-1-甲基-1H-吲哚-3-甲酰胺(141)的制备：
按方法II所示操作步骤可制备获得化合物141。
1H NMR(400MHz,DMSO-d 6)δ8.55(d,J＝7.0Hz,1H),8.48(d,J＝1.8Hz,1H),8.37(d,J＝8.0Hz,1H),8.21(s,1H),7.73–7.60(m,2H),7.55(d,J＝2.0Hz,1H),7.37(q,J＝7.4Hz,1H),7.32–7.12(m,3H),7.04(td,J＝8.7,2.6Hz,1H),5.99(s,2H),5.20(q,J＝7.3Hz,1H),3.89(s,3H),1.49(d,J＝7.1Hz,3H).
甲基(S)-(7-(3-((1-(3-氟苯基)乙基)氨甲酰基)-1-甲基-1H- 吲哚-5-基)-(1,2,4]三唑并[1,5-a]吡啶-2-基)氨基甲酸酯(166)的制备：
将中间体141(513mg,1.2mmol)加入烧瓶中，依次加入10毫升二氯甲烷，1毫升N,N-二异丙基乙胺(310mg,2.4mmol)和氯甲酸甲酯(131mg,1.4mmol)，室温条件下搅拌反应6小时，二氯甲烷：甲醇＝20:1展开剂监控反应，反应完毕，直接将反应液浓缩、拌样，硅胶柱纯化得到166(525mg，淡黄色固体)，产率为90％。
1H NMR(400MHz,DMSO-d 6)δ10.55(s,1H),8.89–8.80(m,1H),8.53(s,1H),8.39(d,J＝8.0Hz,1H),8.23(s,1H),7.86(s,1H),7.78–7.63(m,2H),7.50–7.35(m,2H),7.25(t,J＝9.7Hz,2H),7.04(d,J＝10.0Hz,1H),5.22(s,1H),3.91(s,3H),3.70(s,3H),1.50(d,J＝7.1Hz,3H).
N-((S)-1-(3-氟苯基)乙基)-5-(2-((R)-3-羟基吡咯烷-1-甲酰胺)-[1,2,4]三唑并[1,5-a]吡啶-7-基)-1-甲基-1H-吲哚-3-甲酰胺(162)的制备：
将100毫克中间体166(100mg,0.2mmol)溶于吡啶中，加入50毫克(R)-3-吡咯烷醇(52mg,0.6mmol)，反应加热至100度反应16小时，二氯甲烷：甲醇＝20:1展开剂监控反应，反应完毕，将反应液浓缩，经过反相色谱柱纯化得到162(86mg，白色固体)，产率为80％。
1H NMR(400MHz,DMSO-d 6)δ9.23(s,1H),8.80(d,J＝7.1Hz,1H),8.53(s,1H),8.41(d,J＝8.0Hz,1H),8.24(s,1H),7.82(s,1H),7.72(dd,J＝8.6,1.9Hz,1H),7.66(d,J＝8.6Hz,1H),7.45–7.33(m,2H),7.29–7.20(m,2H),7.05(ddd,J＝10.3,8.2,2.7Hz,1H),5.22(q,J＝7.3Hz,1H),4.98(d,J＝3.4Hz,1H),4.30(s,1H),3.90(s,3H),3.49(s,3H),3.31(s,1H),1.96–1.88(m,1H),1.81(s,1H),1.49(d,J＝7.0Hz,3H).
本发明其他化合物均可采用与实施例1-4相似的方法进行合成，采用的合成方法及结构表征如下表所示：
表1本发明化合物的合成方法及其结构表征
Figure PCTCN2021116256-appb-000049
Figure PCTCN2021116256-appb-000050
Figure PCTCN2021116256-appb-000051
Figure PCTCN2021116256-appb-000052
Figure PCTCN2021116256-appb-000053
Figure PCTCN2021116256-appb-000054
Figure PCTCN2021116256-appb-000055
Figure PCTCN2021116256-appb-000056
Figure PCTCN2021116256-appb-000057
Figure PCTCN2021116256-appb-000058
Figure PCTCN2021116256-appb-000059
Figure PCTCN2021116256-appb-000060
Figure PCTCN2021116256-appb-000061
Figure PCTCN2021116256-appb-000062
Figure PCTCN2021116256-appb-000063
Figure PCTCN2021116256-appb-000064
Figure PCTCN2021116256-appb-000065
Figure PCTCN2021116256-appb-000066
Figure PCTCN2021116256-appb-000067
Figure PCTCN2021116256-appb-000068
Figure PCTCN2021116256-appb-000069
Figure PCTCN2021116256-appb-000070
Figure PCTCN2021116256-appb-000071
Figure PCTCN2021116256-appb-000072
Figure PCTCN2021116256-appb-000073
Figure PCTCN2021116256-appb-000074
Figure PCTCN2021116256-appb-000075
Figure PCTCN2021116256-appb-000076
Figure PCTCN2021116256-appb-000077
Figure PCTCN2021116256-appb-000078
Figure PCTCN2021116256-appb-000079
Figure PCTCN2021116256-appb-000080
Figure PCTCN2021116256-appb-000081
Figure PCTCN2021116256-appb-000082
Figure PCTCN2021116256-appb-000083
Figure PCTCN2021116256-appb-000084
Figure PCTCN2021116256-appb-000085
Figure PCTCN2021116256-appb-000086
Figure PCTCN2021116256-appb-000087
Figure PCTCN2021116256-appb-000088
Figure PCTCN2021116256-appb-000089
Figure PCTCN2021116256-appb-000090
Figure PCTCN2021116256-appb-000091
Figure PCTCN2021116256-appb-000092
Figure PCTCN2021116256-appb-000093
Figure PCTCN2021116256-appb-000094
Figure PCTCN2021116256-appb-000095
Figure PCTCN2021116256-appb-000096
Figure PCTCN2021116256-appb-000097
Figure PCTCN2021116256-appb-000098
Figure PCTCN2021116256-appb-000099
Figure PCTCN2021116256-appb-000100
Figure PCTCN2021116256-appb-000101
Figure PCTCN2021116256-appb-000102
Figure PCTCN2021116256-appb-000103
Figure PCTCN2021116256-appb-000104
Figure PCTCN2021116256-appb-000105
Figure PCTCN2021116256-appb-000106
Figure PCTCN2021116256-appb-000107
Figure PCTCN2021116256-appb-000108
Figure PCTCN2021116256-appb-000109
Figure PCTCN2021116256-appb-000110
Figure PCTCN2021116256-appb-000111
Figure PCTCN2021116256-appb-000112
Figure PCTCN2021116256-appb-000113
Figure PCTCN2021116256-appb-000114
Figure PCTCN2021116256-appb-000115
Figure PCTCN2021116256-appb-000116
Figure PCTCN2021116256-appb-000117
Figure PCTCN2021116256-appb-000118
Figure PCTCN2021116256-appb-000119
Figure PCTCN2021116256-appb-000120
Figure PCTCN2021116256-appb-000121
Figure PCTCN2021116256-appb-000122
Figure PCTCN2021116256-appb-000123
Figure PCTCN2021116256-appb-000124
Figure PCTCN2021116256-appb-000125
Figure PCTCN2021116256-appb-000126
Figure PCTCN2021116256-appb-000127
Figure PCTCN2021116256-appb-000128
Figure PCTCN2021116256-appb-000129
Figure PCTCN2021116256-appb-000130
Figure PCTCN2021116256-appb-000131
Figure PCTCN2021116256-appb-000132
Figure PCTCN2021116256-appb-000133
Figure PCTCN2021116256-appb-000134
Figure PCTCN2021116256-appb-000135
Figure PCTCN2021116256-appb-000136
Figure PCTCN2021116256-appb-000137
Figure PCTCN2021116256-appb-000138
Figure PCTCN2021116256-appb-000139
Figure PCTCN2021116256-appb-000140
Figure PCTCN2021116256-appb-000141
Figure PCTCN2021116256-appb-000142
实施例5本发明化合物体外激酶实验和保护细胞程序性坏死实验
体外激酶试验采用Eurofins公司提供的Kinase Profiler服务完成。实验方法如下：将待测小分子化合物(0.001-10μM)、待测蛋白激酶与包含底物、10mM醋酸镁和[γ- 33P-ATP]的缓冲液共同孵育，通过加入Mg\ATPmix以开始反应，在室温下孵育一段时间后，向缓冲液中加入3％的磷酸盐溶液以终止反应。随后定量吸取10μL反应混合液滴在P30滤纸上，并用75mM的磷酸盐溶液清洗3次，再用甲醇清洗一次，将P30滤纸晾干后加入闪烁液进行闪烁计数。化合物的抑制活性用半数抑制浓度IC 50来表示，IC 50值由各浓度梯度对应的抑制率拟合得到。
体外保护HT-29细胞程序性坏死实验由自己完成。
1)实验材料
DMEM培养基购自Gibco公司，青、链霉素购自Hyclone公司，TNFα购自Peprotech公司，Smac mimetic和Z-VAD-FMK购自Selleck公司，CCK8购自Medchem Express公司。
2)实验方法
HT-29细胞(结肠癌细胞)采用DMEM+10％FBS+青/链霉素培养基培 养。实验时，收集处于对数生长期的HT-29细胞，以每孔特定数目(贴壁细胞一般为8×10 3cells/well)接种于96孔板中，在37℃、5％CO 2的细胞培养箱中培养过夜。次日，用培养基稀释待测化合物到相应浓度并加入到96孔板相应孔中，每个样品3个复孔，同时设置溶剂对照组和只含培养基的空白对照组，将加药后的细胞用TNFα/Smac mimetic/Z-VAD-FMK联合诱导24h，再每孔加入10μl CCK8溶液，并在细胞培养箱中孵育1-3h，随后用酶标仪在495nm波长下检测吸光度，并根据以下公式计算药物对细胞的坏死保护率：细胞坏死保护率＝[(X-C 0)/(C-C 0)]×100％
其中，C、C 0和X分别代表溶剂对照组、空白对照组和药物处理组的平均吸光度值。最后，使用Graphpad Prism 5.0软件拟合细胞生存率曲线并计算出待测化合物抑制细胞程序性坏死的EC 50值。结果如表2所示，其中，+++++代表IC 50或EC 50<0.001μM，++++代表0.01μM>IC 50或EC 50≧0.001μM，+++代表0.1μM>IC 50或EC 50≧0.01μM，++代表1μM>IC 50或EC 50≧0.1μM，+代表IC 50或EC 50≧1μM。
表2本发明化合物对RIPK1激酶抑制活性和保护细胞程序性坏死活性
化合物编号 RIPK1(IC 50) HT-29(EC 50)
1 +++ +++
2 +++ ++
3 +++ +++
4 +++ +++
5 ++ +
6 ++ +++
7 +++ +++
8 +++ ++++
9 +++ ++++
10 ++ ++
11 ++ +
12 ++ +
13 +++ ++++
14 + +
15 +++ +++
16 +++ +++
17 +++ +++
18 +++ ++++
19 +++ ++++
20 + +
21 +++ ++++
22 +++ ++++
23 ++ ++++
24 ++ +++
25 +++ +++++
26 ++ +
27 +++ +++
28 ++ +
29 ++ ++
30 +++ +++++
31 ++ ++
32 +++ ++++
33 +++ +++++
34 +++ +++++
35 ++ +++
36 ++ ++
37 +++ ++++
38 +++ ++++
39 ++ +++
40 ++ +++
41 ++ ++
42 ++ +
43 + +
44 ++ +++
45 +++ +++
46 +++ +++++
47 +++ ++++
48 +++ ++++
49 +++ +++
50 +++ +++
51 +++ +++
52 ++ +++
53 ++ ++
54 +++ +++++
55 +++ ++++
56 +++ ++++
57 ++ +++
58 ++ ++
59 +++ ++++
60 + +
61 + +
62 ++ ++++
63 +++ +++
64 ++ +++
65 ++ +++
66 + +
67 + +
68 + +
69 + +
70 + ++
71 + +
72 + +
73 + +
74 ++ ++
75 + ++
76 ++ ++
77 ++ ++
78 + ++
79 ++ +++
80 ++ +++
81 +++ ++
82 +++ ++
83 ++ ++
84 ++ ++
85 + +
86 + +
87 + +
88 +++ ++++
89 +++ ++++
90 ++ ++++
91 ++ ++
92 ++ ++
93 + +
94 ++++ +++++
95 ++ ++
96 +++ +++
97 + +
98 + +
99 + +
100 + +
101 +++ ++
102 ++ ++
103 +++ ++
104 ++++ +++++
105 +++ ++++
106 ++++ +++++
107 +++ ++++
108 ++++ ++++
109 ++++ +++++
110 +++++ +++++
111 ++++ +++++
112 +++ ++++
113 ++++ ++++
114 +++ ++++
115 ++++ +++
116 ++++ +++
117 +++ +++
118 +++ ++++
119 ++++ ++++
120 ++++ +++++
121 +++ ++++
122 ++++ +++++
123 +++ ++++
124 +++ ++++
125 + +
126 +++ +++
127 +++ +++++
128 ++ ++
129 +++ ++++
130 +++ ++++
131 +++ +++++
132 ++ ++
133 +++ +++++
134 + ++
135 +++ ++++
136 ++ +++
137 +++ +++++
138 + +
139 +++ +++
140 ++ +++
141 +++ ++++
142 +++ +++++
143 +++ +++++
144 +++ +++++
145 ++++ +++++
146 ++++ +++++
147 +++ ++++
148 + +
149 +++ +++++
150 +++ ++++
151 ++++ +++++
152 +++ +++
153 ++ ++
154 +++ ++++
155 +++ ++++
156 +++ +++++
157 +++ ++++
158 ++++ +++++
159 ++ ++
160 +++ +++
161 ++++ +++++
162 +++ +++
163 ++ +++
164 ++ ++++
165 +++ +++++
166 +++ +++++
167 +++ +++
168 +++ +++++
169 ++++ +++++
170 ++++ +++++
171 ++++ +++++
172 + ++
173 ++++ +++++
174 ++++ +++++
175 +++ +++
176 ++ ++
177 +++ +++
178 ++++ ++++
179 +++++ +++++
180 ++++ +++++
181 ++ ++
182 ++++ +++++
183 ++ ++
184 ++ ++
185 ++++ +++++
186 ++ ++
187 +++++ +++++
188 ++ ++
189 ++ ++
190 ++++ ++++
191 +++++ +++++
192 +++++ +++++
193 +++ +++
194 +++++ +++++
195 +++++ +++++
196 +++ +++++
197 ++++ +++++
198 +++ ++++
199 ++++ +++++
200 +++ +++++
201 +++ ++++
202 + +++
203 +++ +++++
204 +++ +++++
205 +++ +++++
206 ++++ +++++
207 ++ ++++
208 +++ +++++
209 ++++ +++++
210 +++ +++++
211 ++++ +++++
212 ++++ +++++
213 ++++ +++++
214 + +
将优选化合物34和94在浓度为10μM的条件下对Eurofins公司现有的422种激酶(包含突变体)进行单浓度抑制率的测试，针对具有较高抑制活性的激酶进行IC 50测试，结果(试验由Eurofins公司测试并提供活性数据)如下表3、表4所示：
表3化合物34对422个激酶的单浓度抑制活性(10μM)
Figure PCTCN2021116256-appb-000143
Figure PCTCN2021116256-appb-000144
Figure PCTCN2021116256-appb-000145
Figure PCTCN2021116256-appb-000146
Figure PCTCN2021116256-appb-000147
Figure PCTCN2021116256-appb-000148
表4化合物94对422个激酶的单浓度抑制活性(10μM)
Figure PCTCN2021116256-appb-000149
Figure PCTCN2021116256-appb-000150
Figure PCTCN2021116256-appb-000151
Figure PCTCN2021116256-appb-000152
Figure PCTCN2021116256-appb-000153
Figure PCTCN2021116256-appb-000154
根据表3和表4的数据绘制了化合物34和94的选择性图谱，(绘制流程及操作参考http://bcb.med.usherbrooke.ca/kinomerenderLig.php)，如图1、图2所示。其中，红色圆形斑点表示有明显抑制作用的激酶，红色斑点的半径越大，说明对激酶的抑制作用越高，反之越小。由表3、表4和图1、图2可以看出化合物34和94具有良好的激酶选择性。
根据图3所示，化合物94抑制HT-29细胞程序性坏死模型的EC 50值为0.012nM，其系列化合物34和46抑制HT-29细胞程序性坏死模型的EC 50值分别为0.117nM、0.070nM。以上实验结果表明，本发明化合物94及其系列化合物能够保护HT-29细胞免受TSZ诱导的坏死样凋亡。
实施例6本发明化合物94及46对RIPK1信号通路的调控
1)实验材料
TNFα购自Peprotech公司，Smac mimetic和Z-VAD-FMK购自Selleck公司，RIPA裂解液购自碧云天生物技术研究所，cocktail购自MCE公司，PMSF蛋白酶抑制剂、十二烷基磺酸钠SDS和甘氨酸购自Sigma公司，丙烯酰胺、三羟甲基氨基甲烷Tris、过硫酸铵APS和N，N，N'，N'-四甲基乙二胺TEMED均购自武汉赛维尔生物科技有限公司。
2)实验方法
细胞总蛋白的提取：在细胞上清中加入相应浓度的化合物94、46或空白溶剂处理细胞并用TNFα/Smac mimetic/Z-VAD-FMK联合诱导一定时间后，弃尽上清，用预冷的PBS或生理盐水洗三次，再加入适当体积的RIPA裂解液(含1％cocktail和1％PMSF蛋白酶抑制剂)，立即置于冰上平放裂解15min，15min后将细胞裂解液用刮刀刮下转移至1.5ml的EP管中，用超声破碎仪破碎细胞。随后将离心管置于低温高速离心机中离心(12000rpm，15min)以除去细胞碎片。再用BCA法进行蛋白定量，并用蛋白标准品制作标准曲线，根据标准曲线计算各样品蛋白浓度，再通过计算将各组蛋白样品浓 度调平，加入5x蛋白上样缓冲液后置于100℃干式恒温器中保持10min。随后直接上样电泳或分装后保存于-20℃备用。蛋白样品避免反复冻融。
蛋白样品制备好后，使用聚丙烯酰胺凝胶电泳(SDS-PAGE)分离蛋白，聚丙烯酰胺凝胶制胶配方如表5所示，一般采用10％的分离胶进行分离。电泳分离后，用槽式湿转法将蛋白充分转移至PVDF膜上，然后将PVDF膜置于5％的脱脂奶粉(脱脂奶粉用TBS/T配制)中室温封闭2h以上，按照所需蛋白的分子量取得含相应蛋白的PVDF膜带，按抗体说明书推荐的稀释比例稀释一抗并在4℃孵育蛋白条带过夜。次日，取出各条带，用TBS/T缓冲液漂洗(5min，3次)后加入1:5000稀释的HRP标记的二抗，在37℃下摇动孵育1h，随后用TBS/T洗脱除去过量抗体，在PVDF膜上均匀滴加HRP底物后，置于快速凝胶成像系统中显影并拍照。
表5 SDS-PAGE中分离胶和浓缩胶配方
Figure PCTCN2021116256-appb-000155
3)实验结果
结果如图4所示，化合物94和46通过抑制RIPK1的自身磷酸化，影响了它下游RIPK3和MLKL的磷酸化，且这种抑制活性具有浓度依赖效应，与细胞水平的保护活性一致，说明化合物94和46通过阻断程序性坏死信号通路发挥抑制程序性坏死的作用。
实施例7本发明化合物94及46对TNFα诱导的小鼠SIRS模型的保护作用
1)实验材料
TNFα购自Peprotech公司，蓖麻油购自MCE公司，无菌生理盐水购自成都百盛科创生物科技有限公司。
2)实验方法
TNFα的造模剂量为500μg/kg，用无菌生理盐水配制，通过尾静脉注射 方式造模。小鼠的体温通过红外电动体温枪来检测。溶剂为25％乙醇蓖麻油、75％生理盐水，给药方式为口服，化合物94、46和RIPK1阳性化合物GSK2982772的给药剂量均为40mg/kg，造模前口服给药。每小时记录一次体温并记录小鼠的生存情况，11h后再记录24h和48h小鼠的生存情况。用Graphpad Prism 5.0软件拟合生存期曲线。
3)实验结果
为了验证化合物94和46是否在体内也具有抗RIPK1和抑制程序性坏死的活性，本实验选择了经典炎症模型SIRS。全身性炎症反应综合征(Systemic Inflammatory Response Syndrome，SIRS)是由于感染或非感染因素作用于机体，引起机体失控而自我破坏的全身炎症反应。危重病人常因机体代偿性抗炎反应能力降低及代谢功能紊乱，易引发SIRS。而TNFα诱导的SIRS疾病模型在长期的研究中被证明与RIPK1依赖的细胞凋亡与程序性坏死高度相关。
本实验建立了TNFα诱导的SIRS小鼠模型，以评价本发明化合物的体内活性。TNFα的造模剂量为500μg/kg，用无菌生理盐水配制，通过尾静脉注射方式进入小鼠体内。因为测小鼠的直肠温度会对小鼠造成机械性损伤，可能对实验结果产生干扰，故采用红外电动体温枪来测量小鼠的体温，同时观察生存情况。化合物94和46的给药剂量为40mg/kg，造模前给药一次。溶剂组(Vehicle)给予相应溶剂对照。
实验结果如图5所示，尾静脉注射TNFα后，溶剂组小鼠在短时间内产生体温持续下降，活动减少等症状，并在24h开始死亡。而给予化合物94和46的组别除个别小鼠外，其余的小鼠在TNFα注射后的11h内体温相对正常，小鼠的活力明显优于溶剂组，整体死亡率与溶剂组相比显著降低，且94呈现出比阳性药GSK2982772更好的保护效果。说明化合物94和46通过在体内抑制RIPK1发挥了显著的抗炎作用，抵抗了高剂量TNFα诱导小鼠所产生的死亡。
实施例8本发明化合物94对DSS诱导的小鼠IBD模型的保护作用
1)实验材料
葡聚糖硫酸钠盐(Dextran Sulfate Sodium Salt,DSS,36000-50000KD)购自上海翊圣生物科技有限公司，0.C.T组织固定液购自赛维尔生物公司，PEG300和吐温-80购自MCE公司，DMSO购自Sigma公司。
2)实验方法
使用重17–20g的雌性C57BL/6小鼠。实验组将DSS(2.5％wt/vol)溶于饮用水中给小鼠随意服用7天(从第0天到第7天)。分别在第2天、第4天和第6天更换新鲜的DSS溶液。第7天将所有的水换为正常的饮用水，并随机分组(每组7只)口服给药：Vehicle组(10％DMSO、40％PEG300、5％吐温-80、45％生理盐水)，GSK3145095组(40mg/kg)和化合物94组(40mg/kg)。Control组每天给予正常的饮用水，且从第7天开始口服给予相应的溶剂。每天记录小鼠的体重和存活率。用Graphpad Prism 5.0软件拟合生存期曲线。第12天处死小鼠(各2只)，观察小鼠结肠的长度变化。
3)实验结果
为了验证化合物94是否在体内自身免疫性疾病模型中也具有抗RIPK1和抑制程序性坏死的活性，本实验选择了经典结肠炎模型IBD。炎症性肠病(Inflammatory bowel diseases,IBD)是指由于环境、遗传、感染、免疫等原因引起的异常免疫介导的肠道炎症，是一种慢性、非特异性肠炎性疾病，其发病机理尚不清楚。IBD的主要病理类型是溃疡性结肠炎(UC)和克罗恩病(CD)。虽然IBD的病因尚不清楚，但肠道上皮细胞过度凋亡、肠黏膜屏障受损、肠上皮细胞通透性提高被认为是IBD发生的原因之一。有研究表明坏死在IBD的发病机制中起重要作用。
实验组小鼠在造模7天后，出现血便、体重下降等病理症状，说明造模成功。如图6所示，造模7天后随机分组给药，Vehicle组和GSK3145095组体重持续下降，并出现小鼠死亡，而给予化合物94的组别小鼠体重逐步回升，且到第14天也未出现死亡，小鼠的活力也明显优于Vehicle组。如图7所示，第12天处死小鼠(各2只)，Vehicle组小鼠结肠长度较Control组明显缩短，而化合物94组小鼠结肠长度比Vehicle组长，与Control组小鼠结肠长度基本相当，表明化合物94可以缓解DSS诱导的小鼠结肠缩短现象，且呈现出比阳性药GSK3145095更好的治疗效果。说明化合物94通过在体内抑制RIPK1发挥了显著的抗炎作用，缓解了DSS诱导的小鼠结肠炎。
实施例9本发明代表性化合物的体内药代动力学研究
1)实验方法
准确称取适量的代表性化合物，用终体积5％DMSO、40％PEG400和55％saline溶解，涡旋或超声使充分混匀，得到1mg/mL的澄清给药溶液，根据体重，计算大鼠给药量(10mg/kg)，并通过静脉或灌胃口服给药。给药后在不同时间进行大鼠采血，采血时间点为：静脉给药后0.083h，0.25h， 0.5h，1h，2h，4h，8h，24h；灌胃口服给药后0.25h，0.5h，1h，2h，4h，6h，8h，24h。在相应时间经颈静脉或其它合适方式采血，每个样品采集约0.20mL，肝素钠抗凝，血液样本采集后放置于冰上，并于1小时内离心分离血浆(离心条件：离心力6800g，6分钟，2-8℃)。采集的血浆样本在分析前存放于-80℃冰箱内，分析后剩余血浆样本继续于-80℃冰箱暂存，保存期限为一个月。
通过不同时间点的血药浓度数据，运用WinNonlin计算药代动力学参数，如AUC(0-t)，T1/2，C max，T max和MRT等。进行血浆药物浓度-时间曲线绘制时，BLQ均记为0。进行药代参数计算时，给药前的浓度按照0计算；C max之前的BLQ(包括“No peak”)按照0计算；C max之后出现的BLQ(包括“No peak”)一律不参与计算。本实验由上海美迪西完成。
2)实验结果
结果见表6，可以看出，单次口服给药代表性化合物10mg/kg后，大鼠对六个代表性化合物的口服吸收均较好，口服生物利用度最高为60.85％。从初步药代动力学实验可以看出，化合物单次给药后在体内蓄积的药物浓度可以达到抑制RIPK1和坏死信号通路的半数抑制浓度值，说明该系列化合物是疫类一类具有开发前景的RIPK1抑制剂。
表6代表性化合物经口服给药后大鼠体内药代动力学参数
Figure PCTCN2021116256-appb-000156
实施例10本发明化合物94对hERG钾离子通道的影响
1)实验方法
将稳定HEK-hERG细胞用DPBS进行冲洗，用Trypsin或Tryple溶液进行消化分离，用培养基重悬细胞并存于离心管中备用。膜片钳记录之前，将细胞滴加于小培养皿中，确保细胞具有一定密度且细胞呈单个分离状态。
采用全细胞膜片钳技术记录hERG电流。取细胞悬液加于小培养皿中，置于倒置显微镜载物台上。待细胞贴壁后，用细胞外液灌流，推荐流速为1– 2mL/min。玻璃微电极由微电极拉制仪两步拉制，充灌电极内液后其入水电阻值为2-5MΩ。
建立全细胞记录模式后，保持钳制电位为-80mV。给予去极化电压至+60mV持续850ms，然后复极化至-50mV维持1275ms引出hERG尾电流。这样一组脉冲程序每15秒钟重复一次，贯穿整个实验。
电流稳定后采用从低浓度到高浓度胞外连续灌流给药的方式。从低浓度开始，持续灌流至药效稳定，然后进行下一浓度的灌流。本实验将分别测试化合物94(0.3，1，3，10，30μM)和阳性化合物特非那定对hERG尾电流的阻断效应(N≥2)。
药效稳定的定义为：每个浓度给药阶段的最后5个刺激条电流值变化小于均值的10％(当电流大于等于200pA时)或小于均值的30％(当电流小于200pA时)可认为是稳定的，若不稳定，将不采取该浓度数据。
通过PatchMaster软件进行刺激发放及信号采集；膜片钳放大器放大信号，滤波为10KHz。
使用FitMaster，EXCEL，Graphpad Prism和SPSS 21.0等进行进一步数据分析和曲线拟合。数据均以均值、标准差表示。
在数据处理中，判断对hERG的阻断效应时，将尾电流的峰值和其基线进行校正。用尾流的抑制率表示不同浓度下各化合物的作用。抑制率％＝100×(给药前尾电流峰值-给药后尾电流峰值)/给药前尾电流峰值。所有细胞各个浓度的抑制率的SD≤15，作为可接受标准(除异常数据外)。
IC 50数值由Hill方程进行拟合所得(如果适用)：
Figure PCTCN2021116256-appb-000157
本实验由上海美迪西完成。
2)实验结果
阳性化合物特非那定对hERG电流的抑制作用浓度-反应曲线见图8。结果显示，特非那定对hERG钾通道的作用具有浓度依赖性，由Hill方程拟合得出特非那定对hERG电流抑制作用的IC 50值为0.045μM(N＝3)，与文献报道相符。
化合物94最终浓度为0.3、1、3、10和30μM，细胞外液中DMSO最 终浓度为0.3％。化合物94对hERG电流的抑制作用浓度-反应曲线见图8。在本实验条件下，化合物94在各检测浓度(0.3、1、3、10和30μM)下对hERG钾通道电流均无明显抑制作用，在30μM时对hERG钾通道电流的平均抑制率为3.40％(N＝3)，故化合物94对hERG电流抑制作用的IC 50值大于30μM。这表明化合物94不会对hERG钾通道电流产生明显抑制作用而引起副作用。
综上所述，本发明提供的系列化合物能够保护HT-29细胞免受TSZ诱导的坏死样凋亡，且具有良好的激酶选择性。其作用原理是抑制RIPK1的自身磷酸化，影响了它下游RIPK3和MLKL的磷酸化，通过阻断程序性坏死信号通路发挥了抑制程序性坏死的作用。发明提供的系列化合物通过在体内抑制RIPK1发挥了显著的抗炎作用，且在各浓度下对hERG钾通道电流均无明显抑制作用，不会由于对hERG钾通道电流产生明显抑制作用而引起副作用。因而，本发明提供的系列化合物具有作为RIPK1抑制剂和抗炎药物的活性成分而进行应用的潜力。

Claims (25)

  1. 式I所示的化合物、或其立体异构体、或其药学上可接受的盐：
    Figure PCTCN2021116256-appb-100001
    其中，
    X 1、X 3独立选自-CR 6-、N；
    X 2选自-NR 1-、-CH＝CH-；
    其中，R 1选自氢、取代或未取代的C 1-C 10烷基、取代或未取代的C 1-C 10醚基、取代或未取代的C 3-C 10环烷基、取代或未取代的3-10元杂环烷基、取代或未取代的C 6-C 20芳基、取代或未取代的3-20元杂芳基；其中，所述取代基是氘、C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
    R 2B选自
    Figure PCTCN2021116256-appb-100002
    其中，R 2选自被一个或两个R 21取代或未取代的C 0-C 6亚烷基；其中，R 21选自取代或未取代的C 1-C 10烷基、取代或未取代的C 3-C 10环烷基、氰基、羟基、羧基、卤素或硝基；其中，所述取代基为氰基、羟基、羧基、卤素或硝基；
    B环选自被一个、两个或三个R 22取代或未取代的C 4-C 10芳基，被一个、两个或三个R 22取代或未取代的4-10元杂芳基或者被一个、两个或三个R 22取代或未取代的C 3-C 10环烷基；其中，R 22分别独立选自取代或未取代的C 1-C 10烷基、取代或未取代的C 1-C 6烷氧基、取代或未取代的4-10元杂环烷基、氰基、羟基、羧基、卤素或硝基；或者，两个R 22相连形成取代或未取代的4-10元杂环烷基；其中，所述取代基为C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
    R 3选自氢、取代或未取代的C 1-C 10烷基；其中，所述取代基为氰基、羟基、羧基、卤素或硝基；
    或者R 2B与R 3相连形成取代或未取代的3-10元杂环烷基；其中，所述取代基为C 1-C 10烷基、C 4-C 10芳基、一个或两个R 31取代的C 4-C 10芳基、氰基、 羟基、羧基、卤素或硝基；其中，所述R 31选自C 1-C 10烷基或卤素；
    R 4、R 5和R 6分别独立选自氢、C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
    A环选自被一个或两个R A取代或未取代的C 4-C 10芳基、一个或两个R A取代或未取代的4-10元杂芳基；其中，R A选自取代或未取代的氨基、
    Figure PCTCN2021116256-appb-100003
    取代或未取代的C 1-C 10烷基、取代或未取代的C 4-C 10芳基、取代或未取代的4-10元杂芳基、取代或未取代的3-10元杂环烷基、；其中，所述取代基为-R A2-R A3、R A4、C 1-C 10烷基、卤素取代的C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；其中，R A4选自被-R A2-R A3取代或未取代的3-10元杂环烷基；
    R A1选自取代或未取代的C 1-C 10烷基、取代或未取代的C 1-C 10烯基、取代或未取代的C 3-C 10环烷基、取代或未取代的3-10元杂环烷基、取代或未取代的C 1-C 6烷氧基、取代或未取代的C 2-C 6醚基、取代或未取代的C 2-C 6胺基；其中，所述取代基为C 1-C 10烷基、羟基取代的C 1-C 10烷基、C 1-C 10酯基、3-10元杂环烷基、氨基、胺基、氰基、羟基、羧基、卤素或硝基；
    R A2选自取代或未取代的C 0-C 6亚烷基、羰基；其中，所述取代基为C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
    R A3选自取代或未取代的3-10元杂环烷基；其中，所述取代基为C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基。
  2. 按照权利要求1所述的化合物、或其立体异构体、或其药学上可接受的盐，其特征在于：所述式I的化合物如式Ⅱ所示：
    Figure PCTCN2021116256-appb-100004
    其中，
    R 1选自氢、取代或未取代的C 1-C 10烷基、取代或未取代的C 3-C 10环烷基、取代或未取代的3-10元杂环烷基、取代或未取代的C 6-C 20芳基、取代或未取 代的3-20元杂芳基；其中，所述取代基是C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
    R 2选自被一个R 21取代或未取代的C 0-C 6亚烷基；其中，R 21选自取代或未取代的C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；其中，所述取代基为氰基、羟基、羧基、卤素或硝基；
    B环选自被一个、两个或三个R 22取代或未取代的C 4-C 10芳基或者被一个、两个或三个R 22取代或未取代的4-10元杂芳基；其中，R 22分别独立选自取代或未取代的C 1-C 10烷基、取代或未取代的C 1-C 6烷氧基、取代或未取代的4-10元杂环烷基、氰基、羟基、羧基、卤素或硝基；或者，两个R 22相连形成取代或未取代的4-10元杂环烷基；其中，所述取代基为C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
    R 3选自氢、取代或未取代的C 1-C 10烷基；其中，所述取代基为氰基、羟基、羧基、卤素或硝基；
    或者R 2与R 3相连形成取代或未取代的3-10元杂环烷基；其中，所述取代基为C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
    X 1、X 3独立选自CH、N；
    A环选自被一个或两个R A取代或未取代的C 4-C 10芳基、一个或两个R A取代或未取代的4-10元杂芳基；其中，R A选自氨基、
    Figure PCTCN2021116256-appb-100005
    取代或未取代的C 1-C 10烷基、取代或未取代的C 4-C 10芳基、取代或未取代的4-10元杂芳基、取代或未取代的3-10元杂环烷基、；其中，所述取代基为-R A2-R A3、R A4、C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；其中，R A4选自被-R A2-R A3取代或未取代的3-10元杂环烷基；
    R A1选自取代或未取代的C 1-C 10烷基、取代或未取代的C 1-C 10烯基、取代或未取代的C 3-C 10环烷基、取代或未取代的3-10元杂环烷基、取代或未取代的C 1-C 6烷氧基、取代或未取代的C 2-C 6醚基；其中，所述取代基为C 1-C 10烷基、3-10元杂环烷基、氨基、胺基、氰基、羟基、羧基、卤素或硝基；
    R A2选自取代或未取代的C 0-C 6亚烷基、羰基；其中，所述取代基为C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
    R A3选自取代或未取代的3-10元杂环烷基；其中，所述取代基为C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基。
  3. 按照权利要求1或2所述的化合物、或其立体异构体、或其药学上可接受的盐，其特征在于：所述式I的化合物如式Ⅲ或式Ⅳ所示：
    Figure PCTCN2021116256-appb-100006
    其中，R 1选自氢、C 1-C 4烷基；R 23选自氢、甲基；B环选自被一个、两个R 22取代或未取代的苯基；其中，R 22分别独立选自F、Cl、氰基、甲基、三氟甲基、甲氧基或三氟甲氧基。
  4. 按照权利要求1所述的化合物、或其立体异构体、或其药学上可接受的盐，其特征在于：所述式I的化合物如式Ⅴ所示：
    Figure PCTCN2021116256-appb-100007
    其中，
    X 1、X 3独立选自-CR 6-、N；
    X 2选自-NR 1-、-CH＝CH-；
    所述R 1选自氢、取代或未取代的C 1-C 10烷基、取代或未取代的C 3-C 10环烷基、取代或未取代的3-10元杂环烷基、取代或未取代的C 6-C 20芳基、取代或未取代的3-20元杂芳基；其中，所述取代基是C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
    L 3选自取代或未取代的C 1-C 4亚烷基；其中，所述取代基为C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
    R 33选自氢、取代或未取代的C 1-C 10烷基、取代或未取代的C 3-C 10环烷基、取代或未取代的3-10元杂环烷基、取代或未取代的C 6-C 10芳基；其中，所述取代基为C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
    R 4、R 5和R 6分别独立选自氢、C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
    A环选自被一个或两个R A取代或未取代的C 4-C 10芳基、一个或两个R A 取代或未取代的4-10元杂芳基；其中，R A选自氨基、
    Figure PCTCN2021116256-appb-100008
    取代或未取代的C 1-C 10烷基、取代或未取代的C 4-C 10芳基、取代或未取代的4-10元杂芳基、取代或未取代的3-10元杂环烷基、；其中，所述取代基为-R A2-R A3、R A4、C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；其中，R A4选自被-R A2-R A3取代或未取代的3-10元杂环烷基；
    R A1选自取代或未取代的C 1-C 10烷基、取代或未取代的C 1-C 10烯基、取代或未取代的C 3-C 10环烷基、取代或未取代的3-10元杂环烷基、取代或未取代的C 1-C 6烷氧基、取代或未取代的C 2-C 6醚基；其中，所述取代基为C 1-C 10烷基、C 1-C 10酯基、3-10元杂环烷基、氨基、胺基、氰基、羟基、羧基、卤素或硝基；
    R A2选自取代或未取代的C 0-C 6亚烷基、羰基；其中，所述取代基为C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
    R A3选自取代或未取代的3-10元杂环烷基；其中，所述取代基为C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基。
  5. 按照权利要求4所述的化合物，其特征在于：
    R 33选自苯基、被一个或两个取代基取代的苯基；其中，所述取代基选自F、Cl或甲基。
  6. 按照权利要求1所述的化合物、或其立体异构体、或其药学上可接受的盐，其特征在于：所述式I的化合物如式Ⅵ所示：
    Figure PCTCN2021116256-appb-100009
    其中，
    X 1、X 3独立选自-CR 6-、N；
    X 2选自-NR 1-、-CH＝CH-；
    X 3选自CH、N；
    所述R 1选自氢、取代或未取代的C 1-C 10烷基、取代或未取代的C 3-C 10环烷基、取代或未取代的3-10元杂环烷基、取代或未取代的C 6-C 20芳基、取代或未取代的3-20元杂芳基；其中，所述取代基是C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
    L 4选自取代或未取代的C 1-C 3亚烷基；其中，所述取代基为C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
    R 3选自氢、取代或未取代的C 1-C 10烷基；其中，所述取代基为氰基、羟基、羧基、卤素或硝基；
    R 4、R 5和R 6分别独立选自氢、C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
    A环选自被一个或两个R A取代或未取代的C 4-C 10芳基、一个或两个R A取代或未取代的4-10元杂芳基；其中，R A选自氨基、
    Figure PCTCN2021116256-appb-100010
    取代或未取代的C 1-C 10烷基、取代或未取代的C 4-C 10芳基、取代或未取代的4-10元杂芳基、取代或未取代的3-10元杂环烷基、；其中，所述取代基为-R A2-R A3、R A4、C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；其中，R A4选自被-R A2-R A3取代或未取代的3-10元杂环烷基；
    R A1选自取代或未取代的C 1-C 10烷基、取代或未取代的C 1-C 10烯基、取代或未取代的C 3-C 10环烷基、取代或未取代的3-10元杂环烷基、取代或未取代的C 1-C 6烷氧基、取代或未取代的C 2-C 6醚基；其中，所述取代基为C 1-C 10烷基、3-10元杂环烷基、氨基、胺基、氰基、羟基、羧基、卤素或硝基；
    R A2选自取代或未取代的C 0-C 6亚烷基、羰基；其中，所述取代基为C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
    R A3选自取代或未取代的3-10元杂环烷基；其中，所述取代基为C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基。
  7. 按照权利要求6所述的化合物、或其立体异构体、或其药学上可接受的盐，其特征在于：所述L 4选自的C 1-C 2的亚烷基。
  8. 按照权利要求6所述的化合物、或其立体异构体、或其药学上可接受的盐，其特征在于：所述X 3选自CH、N。
  9. 按照权利要求1-4或6任一项所述的化合物、或其立体异构体、或其药学上可接受的盐，其特征在于：R 1选自氢、C 1-C 4烷基、C 3醚基。
  10. 按照权利要求1、4或6任一项所述的化合物、或其立体异构体、或其药学上可接受的盐，其特征在于：所述R 4、R 5和R 6分别独立选自氢、F。
  11. 按照权利要求1-4或6任一项所述的化合物、或其立体异构体、或其药学上可接受的盐，其特征在于：
    A环选自被一个或两个R A取代或未取代的5-9元杂芳基、被一个或两个R A取代或未取代的6元芳基；其中，R A选自氨基、被C 1-C 4烷基取代的氨基、被-CF 3取代的氨基、被-CHF 2取代的氨基、
    Figure PCTCN2021116256-appb-100011
    甲基、被-R A2-R A3取代或未取代的5-6元杂芳基、被R A4取代的甲基、被-R A2-R A3取代或未取代的苯基；其中，R A4选自被-R A2-R A3取代或未取代的6元杂环烷基；R A1选自C 2-C 5烷基、Cl取代的C 4烷基、5元杂环烷基取代的甲基、乙烯基、N,N-二甲基胺基取代的乙烯基、C 3-C 6环烷基、一个或两个F取代的C 3-C 6环烷基、4-6元杂环烷基、羟基取代的4-6元杂环烷基、-CH 2OH取代的4-5元杂环烷基、甲基取代的6元杂环烷基、甲氧基、F取代的甲氧基、C 2-C 3醚基、羟基取代的丙胺基；
    R A2选自C 0-C 1亚烷基、羰基；
    R A3选自甲基取代或未取代的6元杂环烷基。
  12. 按照权利要求11所述的化合物、或其立体异构体、或其药学上可接受的盐，其特征在于：
    A环选自
    Figure PCTCN2021116256-appb-100012
    Figure PCTCN2021116256-appb-100013
    Figure PCTCN2021116256-appb-100014
    其中L 1选自S或NH；L 2分别独立选自CH或N。
  13. 按照权利要求12所述的化合物、或其立体异构体、或其药学上可接受的盐，其特征在于：
    A环选自：
    Figure PCTCN2021116256-appb-100015
  14. 按照权利要求1或2所述的化合物、或其立体异构体、或其药学上可接受的盐，其特征在于：
    R 2选自被一个或两个R 21取代或未取代的C 0-C 2亚烷基；其中，R 21选自甲基；
    B环选自被一个、两个或三个R 22取代或未取代的C 6-C 10芳基，被一个、两个或三个R 22取代或未取代的5-9元杂芳基或者被一个、两个或三个R 22取代或未取代的C 9-C 10环烷基；其中，R 22分别独立选自C 1-C 4烷基、甲氧基、卤素、卤素取代的甲基、卤素取代的甲氧基、氰基、硝基、
    Figure PCTCN2021116256-appb-100016
    卤素选自F、Cl、Br。
  15. 按照权利要求1或2所述的化合物、或其立体异构体、或其药学上可接受的盐，其特征在于：R 3选自氢。
  16. 按照权利要求1或2所述的化合物、或其立体异构体、或其药学上可接受的盐，其特征在于：所述式I的化合物如式Ⅶ所示：
    Figure PCTCN2021116256-appb-100017
    其中，
    R 1选自氢、取代或未取代的C 1-C 10烷基、取代或未取代的C 3-C 10环烷基、取代或未取代的3-10元杂环烷基、取代或未取代的C 6-C 20芳基、取代或未取代的3-20元杂芳基；其中，所述取代基是C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
    R 31选自C 1-C 6亚烷基；
    R 32选自被一个、两个或三个R 311取代或未取代的C 4-C 10芳基，其中，R 311分别独立选自C 1-C 6烷氧基，或者两个R 311相连形成4-10元杂环烷基；
    X 1、X 3独立选自CH、N；
    A环选自被一个或两个R A取代或未取代的C 4-C 10芳基、一个或两个R A取代或未取代的4-10元杂芳基；其中，R A选自氨基、
    Figure PCTCN2021116256-appb-100018
    取代或未取代的C 1-C 10烷基、取代或未取代的C 4-C 10芳基、取代或未取代的4-10元杂芳基、取代或未取代的3-10元杂环烷基、；其中，所述取代基为-R A2-R A3、R A4、C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；其中，R A4选自被-R A2-R A3取代或未取代的3-10元杂环烷基；
    R A1选自取代或未取代的C 1-C 10烷基、取代或未取代的C 1-C 10烯基、取代或未取代的C 3-C 10环烷基、取代或未取代的3-10元杂环烷基、取代或未取代的C 1-C 6烷氧基、取代或未取代的C 2-C 6醚基；其中，所述取代基为C 1-C 10烷基、3-10元杂环烷基、氨基、胺基、氰基、羟基、羧基、卤素或硝基；
    R A2选自取代或未取代的C 0-C 6亚烷基、羰基；其中，所述取代基为C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基；
    R A3选自取代或未取代的3-10元杂环烷基；其中，所述取代基为C 1-C 10烷基、氰基、羟基、羧基、卤素或硝基。
  17. 按照权利要求16所述的化合物、或其立体异构体、或其药学上可接 受的盐，其特征在于：
    -R 31-R 32选自
    Figure PCTCN2021116256-appb-100019
  18. 按照权利要求1-17任一项所述的化合物、或其立体异构体、或其药学上可接受的盐，其特征在于：式I-式Ⅶ所示化合物具体为：
    Figure PCTCN2021116256-appb-100020
    Figure PCTCN2021116256-appb-100021
    Figure PCTCN2021116256-appb-100022
    Figure PCTCN2021116256-appb-100023
    Figure PCTCN2021116256-appb-100024
    Figure PCTCN2021116256-appb-100025
    Figure PCTCN2021116256-appb-100026
    Figure PCTCN2021116256-appb-100027
    Figure PCTCN2021116256-appb-100028
    Figure PCTCN2021116256-appb-100029
    Figure PCTCN2021116256-appb-100030
    Figure PCTCN2021116256-appb-100031
    Figure PCTCN2021116256-appb-100032
    Figure PCTCN2021116256-appb-100033
    Figure PCTCN2021116256-appb-100034
    Figure PCTCN2021116256-appb-100035
    Figure PCTCN2021116256-appb-100036
    Figure PCTCN2021116256-appb-100037
    Figure PCTCN2021116256-appb-100038
  19. 按照权利要求1-18任一项所述的化合物的制备方法，其特征在于，通过如下反应步骤进行：
    Figure PCTCN2021116256-appb-100039
    其中，R 2B、R 4、R 5、X 1、X 2、X 3和A环如权利要求1-17任一项所述。
  20. 按照权利要求19所述的制备方法，其特征在于：
    所述中间体A的合成过程为：
    将原料A、醋酸钾、联硼酸频那醇酯和[1,1’-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物溶于无水二氧六环，用惰性气体置换反应体系后反应，反应完毕，将反应液浓缩、拌样、柱层析，得到产品；
    所述中间体A与原料B合成式I化合物的过程为：
    将中间体A、原料B、[1,1’-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物、、三环己基磷膦和、碳酸铯溶于二氧六环/水混合液中，用惰性气体置换反应体系后反应，反应完毕，将反应液浓缩、拌样、柱层析，得到产品；
    所述中间体B的合成过程为：
    将原料B、醋酸钾、联硼酸频那醇酯和[1,1’-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物溶于无水二氧六环，用惰性气体置换反应体系后反应， 反应完毕，将反应液浓缩、拌样、柱层析，得到产品；
    所述所述中间体B与原料A合成式I化合物的过程为：
    将中间体B、原料A、[1,1’-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物、三环己基磷膦和碳酸铯溶于二氧六环/水混合液中，用惰性气体置换反应体系后反应，反应完毕，将反应液浓缩、拌样、柱层析，得到产品。
  21. 按照权利要求1-18任一项所述的化合物、或其立体异构体、或其药学上可接受的盐在制备RIPK1抑制剂中的用途。
  22. 按照权利要求1-18任一项所述的化合物、或其立体异构体、或其药学上可接受的盐在制备用于治疗炎症、免疫性疾病、神经退行性疾病或肿瘤的药物中的用途。
  23. 按照权利要求22所述的用途，其特征在于：所述药物用于治疗细胞程序性坏死相关的炎症反应、免疫性疾病、神经退行性疾病或肿瘤。
  24. 按照权利要求22或23所述的用途，其特征在于：所述炎症为结肠炎。
  25. 一种药物组合物，其特征在于：它是以权利要求1-18任一项所述的化合物、或其立体异构体、或其药学上可接受的盐，加上药学上可接受的辅料制备而成的制剂。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11767322B2 (en) 2020-10-19 2023-09-26 Bristol-Myers Squibb Company Triazolopyridinyl compounds as kinase inhibitors
WO2023180976A1 (en) * 2022-03-25 2023-09-28 1ST Biotherapeutics, Inc. Indazoles as hematopoietic progenitor kinase 1 (hpk1) inhibitors and methods for using same

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115197118B (zh) * 2022-06-21 2024-01-23 贵州医科大学 一种3,3双取代硫化氧化吲哚衍生物的合成方法

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998006715A1 (en) * 1996-08-09 1998-02-19 Smithkline Beecham Corporation Novel piperazine containing compounds
WO2003082868A1 (en) * 2002-03-28 2003-10-09 Eisai Co., Ltd. 7-azaindoles as inhibitors of c-jun n-terminal kinases for the treatment of neurodegenerative disorders
WO2011050245A1 (en) * 2009-10-23 2011-04-28 Yangbo Feng Bicyclic heteroaryls as kinase inhibitors
CN106535890A (zh) * 2014-03-20 2017-03-22 萨穆梅德有限公司 5‑取代的吲唑‑3‑羧酰胺及其制备和应用
WO2018075858A1 (en) * 2016-10-21 2018-04-26 Samumed, Llc Methods of using indazole-3-carboxamides and their use as wnt/b-catenin signaling pathway inhibitors
CN108368110A (zh) * 2015-12-07 2018-08-03 普莱希科公司 用于激酶调节的化合物和方法以及其适应症
WO2019215075A1 (en) * 2018-05-07 2019-11-14 Aziende Chimiche Riunite Angelini Francesco - A.C.R.A.F. S.P.A. 1h-indazole-3-carboxamide compounds as glycogen synthase kinase 3 beta inhibitors
CN111434661A (zh) * 2019-01-11 2020-07-21 爱科诺生物医药股份有限公司 具有细胞坏死抑制活性的芳香杂环化合物及其应用
WO2020150552A2 (en) * 2019-01-17 2020-07-23 Samumed, Llc Methods of treating cartilage disorders through inhibition of clk and dyrk

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998006715A1 (en) * 1996-08-09 1998-02-19 Smithkline Beecham Corporation Novel piperazine containing compounds
WO2003082868A1 (en) * 2002-03-28 2003-10-09 Eisai Co., Ltd. 7-azaindoles as inhibitors of c-jun n-terminal kinases for the treatment of neurodegenerative disorders
WO2011050245A1 (en) * 2009-10-23 2011-04-28 Yangbo Feng Bicyclic heteroaryls as kinase inhibitors
CN106535890A (zh) * 2014-03-20 2017-03-22 萨穆梅德有限公司 5‑取代的吲唑‑3‑羧酰胺及其制备和应用
CN108368110A (zh) * 2015-12-07 2018-08-03 普莱希科公司 用于激酶调节的化合物和方法以及其适应症
WO2018075858A1 (en) * 2016-10-21 2018-04-26 Samumed, Llc Methods of using indazole-3-carboxamides and their use as wnt/b-catenin signaling pathway inhibitors
WO2019215075A1 (en) * 2018-05-07 2019-11-14 Aziende Chimiche Riunite Angelini Francesco - A.C.R.A.F. S.P.A. 1h-indazole-3-carboxamide compounds as glycogen synthase kinase 3 beta inhibitors
CN111434661A (zh) * 2019-01-11 2020-07-21 爱科诺生物医药股份有限公司 具有细胞坏死抑制活性的芳香杂环化合物及其应用
WO2020150552A2 (en) * 2019-01-17 2020-07-23 Samumed, Llc Methods of treating cartilage disorders through inhibition of clk and dyrk

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11767322B2 (en) 2020-10-19 2023-09-26 Bristol-Myers Squibb Company Triazolopyridinyl compounds as kinase inhibitors
WO2023180976A1 (en) * 2022-03-25 2023-09-28 1ST Biotherapeutics, Inc. Indazoles as hematopoietic progenitor kinase 1 (hpk1) inhibitors and methods for using same

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