WO2022057787A1 - 一类细胞程序性坏死抑制剂及其制备方法和用途 - Google Patents

一类细胞程序性坏死抑制剂及其制备方法和用途 Download PDF

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WO2022057787A1
WO2022057787A1 PCT/CN2021/118212 CN2021118212W WO2022057787A1 WO 2022057787 A1 WO2022057787 A1 WO 2022057787A1 CN 2021118212 W CN2021118212 W CN 2021118212W WO 2022057787 A1 WO2022057787 A1 WO 2022057787A1
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PCT/CN2021/118212
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谭立
李盈
秦嬴
戚春婷
项淮江
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中国科学院上海有机化学研究所
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Priority to EP21868610.3A priority Critical patent/EP4215532A1/en
Priority to CN202180063390.5A priority patent/CN116234810A/zh
Priority to JP2023517810A priority patent/JP2023541973A/ja
Priority to US18/245,676 priority patent/US20230331707A1/en
Publication of WO2022057787A1 publication Critical patent/WO2022057787A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P3/06—Antihyperlipidemics
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
    • C07D231/54—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings condensed with carbocyclic rings or ring systems
    • C07D231/56—Benzopyrazoles; Hydrogenated benzopyrazoles
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D235/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings
    • C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
    • C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
    • C07D235/06—Benzimidazoles; Hydrogenated benzimidazoles with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/10—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/26—Heterocyclic compounds containing purine ring systems with an oxygen, sulphur, or nitrogen atom directly attached in position 2 or 6, but not in both
    • C07D473/32—Nitrogen atom
    • C07D473/34—Nitrogen atom attached in position 6, e.g. adenine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Definitions

  • the invention belongs to the field of small molecule compounds, in particular to a class of inhibitor of programmed cell necrosis and a preparation method and application thereof.
  • Apoptosis is the first elucidated mechanism of programmed cell death. In recent years, programmed necrosis has become a new hot spot in the field of cell death.
  • AD Alzheimer's disease
  • MS multiple sclerosis
  • ALS amyotrophic lateral sclerosis
  • retinal degenerative diseases etc.
  • diseases such as inflammation (enteritis, rheumatoid arthritis, psoriasis, etc.), ischemia-reperfusion injury (cerebral infarction, myocardial infarction, etc.) and pathogen infection, it is accompanied by the important pathological feature of programmed cell necrosis. .
  • lung cancer cells can induce programmed necrosis of specific cells in the blood vessel wall in order to pass through the circulatory system and metastasize; the main component of necrosomes is high in pancreatic cancer. It can induce the expression of chemokine CXCL1 and inhibit the immune response of the body. Therefore, inhibiting the occurrence of programmed cell necrosis is recognized as helpful for the treatment and alleviation of various diseases.
  • tumor necrosis factor alpha is one of the main ways to stimulate programmed necrosis of cells in vivo, and its downstream signaling pathway is also the necrosis signaling pathway with the most clear mechanism.
  • TNF- ⁇ tumor necrosis factor alpha
  • TNF- ⁇ first binds to the receptor TNFR1, induces its trimerization and recruits a series of intracellular factors including TRADD, TRAF2, RIPK1, cIAP1/2 and other proteins, In turn, signaling complex I is formed.
  • Complex I can recruit and activate the IKK ⁇ /IKK ⁇ /IKK ⁇ complex and the NF- ⁇ B pathway, and after dissociation, it partially enters the cytoplasm to form a new protein complex IIa, and then recruits procaspase-8 and other proteins through FADD or TRADD, To activate the downstream caspases caspase-3 and caspase-7 and mediate the occurrence of apoptosis.
  • TNF- ⁇ -induced activation of the kinase protein RIPK1 binds to RIPK3 to form a new complex IIb and induces phosphorylation activation of the latter, thereby phosphorylating the downstream substrate MLKL to promote Its oligomerization eventually disrupts the structure of the cell membrane and leads to necrosis.
  • the K63 ubiquitination of RIPK1 by the E3 ubiquitin ligase cIAP can inhibit the latter’s activation and necrosis;
  • the deubiquitinase CYLD can cleave the K63 ubiquitin chain of RIPK1, thereby activating RIPK1 kinase activity and promoting necrosis
  • the formation of corpuscles ultimately achieves positive regulation of cell necrosis;
  • the adaptor protein SPATA2 promotes the activity of CYLD deubiquitinase and inhibits the NF- ⁇ B and MAPK signaling pathways, thereby positively regulating programmed necrosis;
  • the kinase protein TAK1 regulates programmed necrosis through Phosphorylation of the Ser321 site of RIPK1 inhibits the latter's kinas
  • RIPK1 kinase is recognized as a potential therapeutic target for necroptosis-related diseases.
  • the first-in-class RIPK1 inhibitor Necrostatin-1 (Nec-1) and its analogs have demonstrated clear curative effects on a variety of degenerative diseases, inflammation, cancer and other diseases in preclinical studies.
  • AD Alzheimer's disease
  • PD Parkinson's disease
  • PD Huntington's disease
  • inflammatory bowel disease age-related macular degeneration, etc.
  • PD inflammatory bowel disease
  • It has protective effects against inflammatory bowel disease, autoimmune diseases, bombesin-induced acute pancreatitis and sepsis/systemic inflammatory response syndrome (SIRS); can effectively alleviate ischemic brain injury, ischemic myocardial injury, retinal ischemia/regeneration Perfusion injury, retinal detachment-induced photoreceptor cell necrosis, glaucoma, renal ischemia-reperfusion injury, cisplatin-induced renal injury, and traumatic brain injury; at least partial relief is associated with RIPK1-dependent apoptosis, necrosis, or cytokine production
  • Other diseases including hematological and solid organ malignancies, bacterial and viral infections (including tuberculos)
  • Nec-1 derivatives have entered clinical trials for the treatment of ALS and AD; another class of RIPK1 inhibitor GSK2982772 is also in clinical trials for the treatment of various autoimmune diseases.
  • the existing programmed necrosis inhibitors all have different degrees of defects, such as the in vivo inhibitory activity is still not ideal, the pharmacokinetic properties are not good, the oral bioavailability is low, and some cannot enter the central nervous system through the blood-brain barrier. Nervous system, or it is difficult to carry out preclinical animal experiments due to the inability to effectively inhibit murine RIPK1, these shortcomings limit its further research and clinical application.
  • the purpose of the present invention is to provide a class of RIPK1 inhibitors and/or necroptosis inhibitors with novel structures.
  • Ring A is a substituted or unsubstituted 9-10-membered nitrogen-containing heteroaryl group, wherein the 9-10-membered nitrogen-containing heteroaryl group contains 1, 2, 3 or 4 nitrogen heteroatoms as ring atoms;
  • n 0, 1 or 2;
  • R 4 is each independently selected from the group consisting of H, -CN, halogen, substituted or unsubstituted C 1-6 alkyl, -OR b , -SR b , -N(R b ) 2 , -C(O) -NR 6 -R b , -C(O)-NR 6 -C 1-4 alkylene-N(R b ) 2 , -NR 6 -C(O)-R b ;
  • Each R b is independently selected from the group consisting of H, substituted or unsubstituted C 1-6 alkyl; or two R b and the nitrogen atom to which they are attached together form a substituted or unsubstituted 5-, 6- or 7-membered Heterocycloalkyl, wherein, in addition to the N to which R is attached, the heterocycloalkyl contains 0, 1 or 2 additional heteroatoms as ring atoms;
  • R 6 is selected from the group consisting of H, OH, substituted or unsubstituted C 1-4 alkyl, substituted or unsubstituted C 1-4 alkoxy;
  • Ring B is selected from the group consisting of substituted or unsubstituted C 6-10 aryl, substituted or unsubstituted 5-10 membered heteroaryl;
  • L 1 and L 2 are each independently a divalent group selected from the group consisting of:
  • R 1 and R 2 are each independently selected from the group consisting of H, substituted or unsubstituted C 1-4 alkyl;
  • R 3 is selected from the group consisting of H, OH, substituted or unsubstituted C 1-4 alkyl, substituted or unsubstituted C 1-4 alkoxy;
  • Ring C is None or wherein, W is each independently selected from the group consisting of O, S, C, N, C(R c ), and N(R d );
  • R c is each independently selected from the group consisting of H, CN, halogen, substituted or unsubstituted C 1-6 alkyl
  • R d are each independently selected from the group consisting of H, CN, substituted or unsubstituted C 1-6 alkyl (preferably, R d is substituted or unsubstituted C 1 -6 alkyl, more preferably, R is selected from the group consisting of methyl, ethyl, propyl and butyl);
  • R 3 and the ring atom W located at the vicinal position of the linking position of L 1 and ring C and -C(O)- in L 1 together form a substitution or Unsubstituted 5-, 6- or 7-membered saturated heterocycle (preferably, 6-membered saturated heterocycle); wherein, the saturated heterocycle contains 0, 1 or 2 other as heteroatoms of ring atoms;
  • R 5 is selected from the group consisting of H, OH, substituted or unsubstituted C 1-4 alkyl, substituted or unsubstituted C 1-4 alkoxy;
  • R 3 and R 5 and the atoms to which they are attached together form a substituted or unsubstituted 5-, 6- or 7-membered saturated heterocycle; wherein the saturated Heterocycles contain 0, 1 or 2 additional heteroatoms as ring atoms in addition to the N attached to R3 ;
  • Ring D is selected from the group consisting of a substituted or unsubstituted C 6-10 aromatic ring, and a substituted or unsubstituted 5-10 membered heteroaryl;
  • Each R is independently selected from the group consisting of H, C 1-6 alkyl, haloC 1-6 alkyl, C 1-6 hydroxyalkyl.
  • heteroatoms are each independently selected from the following group: O, N, S; preferably, each is independently O or N.
  • each R 4 is independently selected from the following group: H, -CN, -OR b , -N(R b ) 2 , -C(O)-NR 6 -R b , -C(O )-NR 6 -C 1-4 alkylene-N(R b ) 2 ; preferably, R 4 is each independently selected from the group of: -CN, -OR b , -N(R b ) 2 ; more Preferably, R 4 is each independently selected from the group consisting of -CN, -OC 1-6 alkyl, -NH 2 , -NH(C 1-4 alkyl), or -N(C 1-4 alkyl) 2 .
  • each R 4 is independently selected from the following group: H, substituted or unsubstituted C 1-6 alkyl, -OR b , -SR b , -N(R b ) 2 , -C( O)-NR 6 -R b , -C(O)-NR 6 -C 1-4 alkylene-N(R b ) 2 , -NR 6 -C(O)-R b .
  • ring A is shown in the structure selected from the following group:
  • X 1 , X 2 , X 3 , X 4 , X 5 and X 6 are each independently selected from the group consisting of N and C(R a ); and X 1 , X 2 , X 3 , X 4 , X 5 and at most 3 of X 6 are N;
  • Each R a is independently selected from the group consisting of none, H, substituted or unsubstituted C 1-6 alkyl.
  • X 1 , X 2 , X 3 , X 4 , X 5 and X 6 are each independently selected from the group consisting of C, N and CH.
  • X 3 and X 5 are C(R a ) (preferably, X 3 and X 5 are CH); X 2 , X 4 and X 6 are N; and R 4 is selected from the group consisting of: -N(R b ) 2 , -NR 6 -C(O)-R b (preferably, R 4 is -N(R b ) 2 ; more preferably R 4 is -NH 2 , -NH(C 1-4 alkyl ), or -N(C 1-4 alkyl) 2 ; or,
  • X 6 is N
  • X 1 , X 2 , X 3 and X 5 are C(R a ) (preferably, X 1 , X 2 , X 3 and X 5 are CH)
  • X 4 is N or C (R a ) (preferably X 4 is N or CH); and R 4 is selected from the group consisting of -CN, -OC 1-6 alkyl.
  • ring B is selected from the group consisting of substituted or unsubstituted phenyl, substituted or unsubstituted 5- or 6-membered heteroaryl. In another preferred embodiment, ring B is a phenyl group.
  • L 1 is (the carbonyl moiety in L 1 is attached to ring C) and L 2 is absent (ie -L 1 -L 2 - is ).
  • R 1 and R 2 are each independently selected from the group consisting of H and methyl. In another preferred embodiment, one of R 1 and R 2 is H, and the other is H or substituted or unsubstituted C 1-4 alkyl (preferably, H or methyl).
  • ring C is In another preferred embodiment, ring C is in another preferred embodiment, ring C is L1 is (the carbonyl moiety in L 1 is attached to ring C) and L 2 is none, R 3 is H, or R 3 is substituted with W located at the ortho position where L 1 is attached to ring C and C(O) in L 1 or an unsubstituted 5- or 6-membered (preferably, 6-membered) saturated heterocycle.
  • R 3 forms a substituted or unsubstituted 5- or 6-membered saturated heterocyclic ring together with W located at the ortho position of the linking position of L 1 and ring C and C(O) in L 1 ,
  • the ring C is as shown; preferably, as shown.
  • the ring C is shown in the structure selected from the following group:
  • W 1 is selected from the group consisting of O, S, N(R d );
  • W 2 , W 3 , W 4 , W 5 and W 6 are each independently selected from the group consisting of N and C(R c );
  • R 3 is connected to W 1 , W 2 , W 3 , W 4 or W 5 and L 1 in the ortho position to the position where L 1 is connected to ring C
  • the carbonyl groups of are taken together to form a substituted or unsubstituted 5- or 6-membered saturated heterocycle (preferably, a 6-membered saturated heterocycle).
  • the ring C is shown in the structure selected from the following group: wherein W 1 is N(R d ); W 2 , W 3 , W 4 , and W 5 are each independently selected from the group consisting of N and C(R c );
  • R 3 is connected to W 1 , W 2 , W 3 , W 4 or W 5 and L 1 in the ortho position to the position where L 1 is connected to ring C
  • the carbonyl groups of are taken together to form a substituted or unsubstituted 5- or 6-membered saturated heterocycle (preferably a 5-membered saturated heterocycle).
  • the ring C is selected from the following group:
  • R c is each independently selected from the group consisting of H, CN, halogen (such as Cl), substituted or unsubstituted C 1-6 alkyl
  • R d is each independently selected from the group consisting of H, substituted or unsubstituted C 1-6 alkyl.
  • the ring C is selected from the following group: or, for wherein R 1 and R 2 are as previously defined (preferably, one of R 1 and R 2 is H and the other is H or C 1-4 alkyl (eg, methyl)).
  • R c is each independently selected from the following group: H, CN, F, Cl, Br, C 1-4 alkyl; and/or R d is selected from the following group: H, C 1-4 Alkyl (preferably, R d is C 1-4 alkyl).
  • ring D is phenyl
  • ring A, ring B, ring C, ring D, L 1 , L 2 , R 4 , R 5 and n are independently corresponding groups in the specific compounds in Table A.
  • the compound is shown in formula II;
  • R 3 is selected from the group consisting of H, OH, substituted or unsubstituted C 1-4 alkyl, substituted or unsubstituted C 1-4 alkoxy;
  • R 3 and the ring atom W located at the vicinal position of the attachment position of -C(O)- to ring C and the -C(O)- together form a substituted or unsubstituted 5-, 6- or 7-membered saturated heterocycle (preferably 6-membered saturated heterocycle); wherein the saturated heterocycle contains 0, 1 or 2 additional heteroatoms as ring atoms in addition to the N attached to R3 ;
  • R 5 is selected from the group consisting of H, OH, substituted or unsubstituted C 1-4 alkyl, substituted or unsubstituted C 1-4 alkoxy;
  • Ring A, Ring B, Ring D, W, R 1 , R 2 , R 4 and n are as defined in Formula I.
  • ring A, ring B, ring C, ring D, R 1 , R 2 , R 3 , R 4 , R 5 and n are independently the groups corresponding to the specific compounds in Table A.
  • the compound is shown in formula III;
  • X 1 , X 2 , X 3 , X 4 , X 5 and X 6 are each independently selected from the group consisting of N and C(R a ); and X 1 , X 2 , X 3 , X 4 , X 5 and X At most 3 of 6 are N;
  • Each R a is independently selected from the group consisting of none, H, substituted or unsubstituted C 1-6 alkyl;
  • R 3 is selected from the group consisting of H, OH, substituted or unsubstituted C 1-4 alkyl, substituted or unsubstituted C 1-4 alkoxy;
  • R 3 and the ring atom W located at the vicinal position of the attachment position of -C(O)- to ring C and the -C(O)- together form a substituted or unsubstituted 5-, 6- or 7-membered saturated heterocycle (preferably 6-membered saturated heterocycle); wherein the saturated heterocycle contains 0, 1 or 2 additional heteroatoms as ring atoms in addition to the N attached to R3 ;
  • R 5 is selected from the group consisting of H, OH, substituted or unsubstituted C 1-4 alkyl, substituted or unsubstituted C 1-4 alkoxy;
  • Ring B, Ring D, W, R 1 , R 2 , R 4 and n are as defined in formula I.
  • X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , ring B, ring C, ring D, R 1 , R 2 , R 3 , R 4 , R 5 and n is independently the group corresponding to the specific compound in Table A.
  • the compound is shown in formula IV-1 or IV-2;
  • X 1 , X 2 , X 3 , X 4 , X 5 and X 6 are each independently selected from the group consisting of N and C(R a ); and X 1 , X 2 , X 3 , X 4 , X 5 and X At most 3 of 6 are N;
  • R is selected from the group consisting of H, OH, substituted or unsubstituted C 1-4 alkyl
  • R 3 and the ring atom W and the -C(O)- together form a substituted or unsubstituted 5-, 6- or 7-membered saturated heterocycle (preferably, a 6-membered saturated heterocycle); wherein the saturated heterocycle
  • the ring contains 0, 1 or 2 additional heteroatoms as ring atoms in addition to the N attached to R3 ;
  • R 5 is selected from the group consisting of H, OH, substituted or unsubstituted C 1-4 alkyl
  • Ring B, Ring D, W, Ra , R1, R2, R4 and n are as defined in formula I.
  • X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , ring B, W, ring D, R 1 , R 2 , R 3 , R 4 , R 5 and n are independent Ground is the group corresponding to the specific compound in Table A.
  • ring B and ring D are each independently unsubstituted phenyl, or phenyl substituted by 1 or 2 substituents selected from the group consisting of halogen, C 1-4 alkyl , halogenated C 1-4 alkyl.
  • the compound is selected from Table A.
  • R 3 is selected from the group consisting of H, OH, substituted or unsubstituted C 1-4 alkyl, substituted or unsubstituted C 1-4 alkoxy;
  • Ring A, Ring B, Ring C, Ring D, R c , R 1 , R 2 , R 4 , R 5 and n are as defined in Formula II of the first aspect;
  • the method is method two, and the method two comprises the steps:
  • R 3 forms a substituted or unsubstituted 5-, 6- or 7-membered saturated heterocycle together with the ring atom W at the ortho-position to the linking position of -C(O)- and ring C and -C(O)- in L 1 ;
  • L 3 is a single bond, substituted or unsubstituted C 1-2 alkylene
  • Ring A , Ring B, Ring D, Rc , R1, R2, R4, R5 and n are as defined in Formula II of the first aspect.
  • the inert solvent is selected from the following group: dichloromethane,
  • reaction is carried out in the presence of HATU and N,N-diisopropylethylamine.
  • the method is method three, and the method three comprises the steps:
  • R L is halogen; Preferably I;
  • a pharmaceutical composition comprising (a) a therapeutically effective amount of the compound described in the first aspect, or a pharmaceutically acceptable salt thereof, hydrated compound or solvate; and (b) a pharmaceutically acceptable carrier.
  • a compound as described in the first aspect or a pharmaceutical composition as described in the third aspect in preparation for the treatment or prevention of necroptosis and/or human receptors Use in a medicament for a disease or disorder associated with interacting protein 1 kinase (RIPK1).
  • RIPK1 interacting protein 1 kinase
  • the compound or pharmaceutical composition is used to treat or prevent the disease or disorder by inhibiting human receptor interacting protein 1 kinase (RIPK1).
  • RIPK1 human receptor interacting protein 1 kinase
  • the human receptor interacting protein 1 kinase includes RIPK1 in an inactive (or inactive) state and RIPK1 in an activated state.
  • the compound or pharmaceutical composition can also treat or prevent (especially treat) the disease or disorder by inhibiting the activated human receptor-interacting protein 1 kinase (RIPK1).
  • RIPK1 activated human receptor-interacting protein 1 kinase
  • the inhibition of human receptor-interacting protein 1 kinase includes one or more of the following: inhibiting the activity of RIPK1, or inhibiting the phosphorylation of RIPK1.
  • the compound or pharmaceutical composition treats or prevents the disease or disorder by inhibiting the programmed necrosis pathway.
  • the inhibition of programmed necrosis signaling includes one or more of the following: inhibiting the activity of RIPK1, inhibiting the phosphorylation of RIPK1, or inhibiting the phosphorylation of MLKL.
  • the disease or condition is selected from one or more of the following group: degenerative disease, inflammation, ischemia-reperfusion injury, pathogen infection, Parkinson's disease (PD), Henry's disease macular degeneration, autoimmune disease, retinal detachment-induced photoreceptor cell necrosis, glaucoma, cisplatin-induced renal injury and traumatic brain injury, hyperlipidemia-induced atherosclerosis, RIPK1-dependent apoptosis, necrosis or Other diseases associated with cytokine production, bacterial infections, viral infections, and lysosomal storage disorders.
  • PD Parkinson's disease
  • Henry's disease macular degeneration autoimmune disease
  • retinal detachment-induced photoreceptor cell necrosis glaucoma
  • cisplatin-induced renal injury and traumatic brain injury hyperlipidemia-induced atherosclerosis
  • RIPK1-dependent apoptosis necrosis or Other diseases associated with cytokine production, bacterial infections, viral infections, and lys
  • the degenerative diseases include: eg Alzheimer's disease (AD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), retinal degenerative diseases.
  • AD Alzheimer's disease
  • MS multiple sclerosis
  • ALS amyotrophic lateral sclerosis
  • retinal degenerative diseases eg Alzheimer's disease (AD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), retinal degenerative diseases.
  • the inflammation includes one or more of the following: enteritis, rheumatoid arthritis, psoriasis, retinitis pigmentosa, inflammatory bowel disease, Tinton's disease (PD) inflammation Enteropathy, bombesin-induced acute pancreatitis, sepsis/systemic inflammatory response syndrome (SIRS).
  • the ischemia-reperfusion injury includes one or more of the following: cerebral infarction, myocardial infarction, etc., ischemic brain injury, ischemic myocardial injury, retinal ischemia/reperfusion injury , Renal ischemia-reperfusion injury.
  • the other diseases related to RIPK1-dependent apoptosis, necrosis or cytokine production include one or more of the following: hematological and solid organ malignancies.
  • the viral infection includes one or more of the following diseases or conditions: tuberculosis, influenza, coronavirus infection and pneumonia caused by the same.
  • the lysosomal storage disease includes Gaucher disease.
  • a method of treating or preventing a disease or disorder associated with programmed cell necrosis and/or human receptor interacting protein 1 kinase comprising: The subject is administered a therapeutically effective amount of a compound as described in the first aspect or a pharmaceutical composition as described in the third aspect.
  • RIPK1 programmed cell necrosis and/or human receptor interacting protein 1 kinase
  • a method for inhibiting necroptosis comprising the step of: culturing cells in the presence of the compound according to the first aspect, thereby inhibiting inhibiting necroptosis.
  • the method is non-therapeutic in vitro.
  • a method for inhibiting RIPK1 protein kinase activity comprising the step of contacting RIPK1 protein kinase with the compound of the first aspect, thereby inhibiting RIPK1 protein kinase activity.
  • the method is non-therapeutic in vitro.
  • Figure 1 shows the concentration-activity curves of representative compounds such as QY-10-40 and control compounds in inhibiting necroptosis in FADD-deficient Jurkat Jurkat cells or L929 cells.
  • Figure 2 is a graph representing the concentration-inhibitory effect of compound QY-10-40 on the kinase activity of RIPK1(1-330) protein.
  • Figure 3 shows the effects of QY-10-40, Nec-1s and GSK2982772 at different concentrations on the TNF pathway signal activated by TNF ⁇ combined with SM164 in human or murine cell lines.
  • Figure 4 shows the results of a test of the inhibitory activity of RIPK1 inhibitors on programmed necrosis of RIPK1 persistently activated cells.
  • Figure 5 shows the concentration-time profile of a representative compound QY-10-40 in plasma, as well as pharmacokinetic parameters.
  • Figure 6 shows the results of the effect of representative compound QY-10-40 and control compound Nec-1s on changes in mouse body temperature.
  • Figures 7A and B show the effects of representative compounds QY-10-40, QY-13-19 and control compound Nec-1s on body weight change and organ coefficient (organ weight/body weight) in mice, respectively.
  • Figure 8 shows the co-crystal structure of a representative QY-7-2B with the human RIPK1 protein kinase domain, and demonstrates the difference in the binding mode to RIPK1 protein between the compounds of the present invention and the control compound GSK2982772.
  • the present inventors unexpectedly discovered a class of inhibitors of programmed cell necrosis with novel structure.
  • Said inhibitor of programmed necrosis has excellent RIPK1 inhibitory activity. It can thus be used to prepare pharmaceutical compositions for preventing and/or treating diseases involving cell death, RIPK1 and/or inflammation.
  • the preferred compounds provided by the present invention also have excellent inhibitory activity against activated RIPK1, so compared with existing RIPK1 inhibitors that only inhibit inactivated RIPK1, the compounds provided by the present invention have more rapid improvement or Treatment of diseases or disorders involving cell death and/or RIPK1 inflammation (eg, inflammation). Based on the above findings, the inventors have completed the present invention.
  • alkyl refers to a straight or branched chain alkyl group having the specified number of carbon atoms.
  • C1-6 alkyl refers to a straight or branched chain alkyl group having 1 to 6 carbon atoms.
  • alkyl refers to C 1-4 alkyl.
  • alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, and the like, or similar groups.
  • cycloalkyl refers to a cyclic alkyl group having the specified number of carbon atoms.
  • C 3-8 cycloalkyl refers to a cyclic alkyl group having 1 to 8 carbon atoms.
  • Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl.
  • alkoxy refers to an alkyl group, as defined above, which is attached to the remainder of the molecule through an oxygen atom.
  • C 1-6 alkoxy refers to C 1-6 alkyl-O-.
  • the alkoxy group may include methoxy, ethoxy and isopropoxy.
  • halogen refers to F, Cl, Br and I.
  • haloalkyl refers to an alkyl group (as defined above for alkyl) substituted with a halogen.
  • haloalkyl includes trifluoromethyl, difluoromethyl, trifluoromethoxy, perfluoroethyl, and the like.
  • cycloalkyl refers to a cyclic alkyl group having the specified number of ring atoms (eg, C3-8cycloalkyl refers to a cyclic alkyl group having 3, 4, 5, 6, 7, or 8 ring atoms) And hydrocarbon rings that are fully saturated or have no more than one double bond between the ring tips.
  • Cycloalkyl also refers to bicyclic and polycyclic hydrocarbon rings, such as bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, and the like.
  • heterocycloalkyl refers to a cycloalkyl group having the indicated number of ring atoms and containing one to five heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized , and the nitrogen atom is optionally quaternized.
  • Heterocycloalkyl can be a monocyclic, bicyclic or polycyclic ring system.
  • heterocycloalkyl examples include pyrrolidine, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, piperidine, 1,4-dioxane, morpholine, thiomorpholine, thiomorpholine-S-oxide, thiomorpholine-S,S-oxide, piperazine, pyran, pyridone, 3-pyrroline, thiopyran, pyranone, tetrahydrofuran, tetrahydrothiophene, quinuclidine, etc.
  • a heterocycloalkyl group can be attached to the remainder of the molecule through a ring carbon or a heteroatom.
  • alkylene by itself or as part of another substituent refers to a divalent group derived from an alkane such as -CH2- , -CH2CH2- , -CH2CH2CH2- , -CH 2 CH 2 CH 2 CH 2 -.
  • aryl refers to a polyunsaturated (usually aromatic) hydrocarbon group containing the specified number of ring atoms, which may be monocyclic or polycyclic (eg, bicyclic) fused together or linked covalently. ).
  • heteroaryl refers to an aryl group (or ring) having the specified number of ring atoms and containing 1 to 5 (eg, 1, 2, 3, 4, or 5) heteroatoms selected from N, O, and S.
  • nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom is optionally quaternized; for example, a 5-10 membered heteroaryl (or ring) refers to a group containing 5, 6, 7, 8, 9, or 10 ring atoms the heteroaryl (or ring).
  • a nitrogen-containing heteroaryl group as used herein means that at least one of the contained heteroatoms is a nitrogen heteroatom, preferably, all of the contained heteroatoms are nitrogen heteroatoms.
  • a heteroaryl group can be attached to the remainder of the molecule through a heteroatom.
  • Non-limiting examples of aryl groups include phenyl, naphthyl, and non-limiting examples of heteroaryl groups include pyridyl, pyridazinyl, pyrazinyl, pyrimidinyl, triazinyl, quinolinyl, quinoxalinyl , quinazolinyl, cinnoline, phthalazinyl, benzotriazinyl, purine, benzimidazolyl, benzopyrazolyl, benzotriazolyl, benzisoxazolyl, isobenzofuryl, isoindolyl, indolizine, benzotriazinyl, thienopyridyl, thienopyrimidinyl, pyrazolopyrimidinyl, imidazopyridine, benzothiazolyl, benzofuranyl, benzothienyl, indolyl, quinolinyl, isoquinolinyl, iso
  • aryl when used in combination with other terms (eg, aryloxy, arylthio, aralkyl) includes aryl and heteroaryl rings as defined above.
  • the aforementioned terms will include substituted and unsubstituted forms of the designated groups.
  • a bond from a substituent (usually an R group) to the center of an aromatic ring will be understood to mean a bond that provides a link at any available vertex of the aromatic ring.
  • the description also includes linkages on rings fused to aromatic rings.
  • a bond drawn to the center of the indole benzene moiety would represent a bond to any available vertex of the six- or five-membered ring moiety of the indole.
  • the terms “comprising”, “comprising” or “including” mean that the various ingredients can be used together in the mixture or composition of the present invention.
  • the terms “consisting essentially of” and “consisting of” are encompassed by the term “comprising”.
  • the terms "compound of the present invention”, “necroptosis inhibitor of the present invention”, “RIPK1 inhibitor of the present invention” and “inhibitor of the present invention” are used interchangeably to refer to one aspect of the present invention the compound.
  • the term also includes various crystalline forms, pharmaceutically acceptable salts, hydrates or solvates of the compounds described in the first aspect of the invention.
  • each group is as defined in the first aspect.
  • the compound of the present invention is shown in formula II;
  • each group is as defined in the first aspect.
  • the compound of the present invention is shown in formula III;
  • each group is as defined in the first aspect.
  • the compound of the present invention is shown in formula IV-1 or IV-2,
  • each group is as defined in the first aspect.
  • each group of the compound represented by formula I, formula II, formula III, formula IV-1 or formula IV-2 (such as ring A, ring B, ring C, ring D, L 1 , L 2 , n, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R a , R b , R c , R d , W, W 1 , W 2 , W 3 , W 4 , W 5 , X 1 , X 2 , X 3 , X 4 , X 5 , and X 6 ) are each independently the corresponding groups in the specific compounds in Table A.
  • the compound of the present invention is selected from the compounds in Table A, or a pharmaceutically acceptable salt thereof.
  • pharmaceutically acceptable salts refers to salts of the compounds of the present invention with acids or bases that are suitable for use as medicaments.
  • Pharmaceutically acceptable salts include inorganic and organic salts.
  • a preferred class of salts are the salts of the compounds of the present invention with acids.
  • Acids suitable for forming salts include, but are not limited to, inorganic acids such as hydrochloric, hydrobromic, hydrofluoric, sulfuric, nitric, phosphoric; formic, acetic, trifluoroacetic, propionic, oxalic, malonic, succinic, Organic acids such as fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, and naphthalenesulfonic acid; and Amino acid, phenylalanine, aspartic acid, glutamic acid and other amino acids.
  • inorganic acids such as hydrochloric, hydrobromic, hydrofluoric, sulfuric, nitric, phosphoric
  • formic acetic, trifluoroacetic, propionic,
  • salts are those of the compounds of the invention with bases, such as alkali metal salts (eg, sodium or potassium), alkaline earth metal salts (eg, magnesium or calcium), ammonium salts (eg, lower alkanolammonium salts) salts and other pharmaceutically acceptable amine salts) such as methylamine, ethylamine, propylamine, dimethylamine, trimethylamine, diethylamine, triethylamine, tert-butylamine amine salts, ethylenediamine salts, hydroxyethylamine salts, dihydroxyethylamine salts, trihydroxyethylamine salts, and amine salts formed from morpholine, piperazine, and lysine, respectively.
  • bases such as alkali metal salts (eg, sodium or potassium), alkaline earth metal salts (eg, magnesium or calcium), ammonium salts (eg, lower alkanolammonium salts) salts and other pharmaceutically acceptable amine salt
  • solvate refers to a complex in which a compound of the present invention coordinates with solvent molecules to form a complex in specified proportions.
  • Hydrophilate refers to a complex formed by the coordination of a compound of the present invention with water.
  • the compounds of the present invention also include prodrugs of the compounds described in the first aspect.
  • prodrug includes a class of compounds of formula (I) which may be biologically active or inactive by themselves and undergo a metabolic or chemical reaction in the human body when administered in an appropriate manner, or A salt or solution of a compound of formula (I).
  • the prodrugs include (but are not limited to) carboxylate, carbonate, phosphate, nitrate, sulfate, sulfone, sulfoxide, amino compounds, carbamates, azo compounds of the compounds , phosphoramide, glucoside, ether, acetal and other forms.
  • the preparation methods of the compounds of the present invention are described in more detail below, but these specific methods do not constitute any limitation of the present invention.
  • the compounds of the present invention can also be conveniently prepared by optionally combining various synthetic methods described in this specification or known in the art, and such combinations can be easily performed by those skilled in the art to which the present invention pertains.
  • the preparation method of the compound of formula II provided by the present invention is method one, and the method one comprises the steps:
  • R 3 is selected from the group consisting of H, OH, substituted or unsubstituted C 1-4 alkyl, substituted or unsubstituted C 1-4 alkoxy;
  • Ring A, Ring B, Ring C, Ring D, R c , R 1 , R 2 , R 4 , R 5 and n are as defined in Formula II.
  • the preparation method of the compound of formula II provided by the present invention is method two, and the method two comprises the steps:
  • R 3 forms a substituted or unsubstituted 5-, 6- or 7-membered saturated heterocycle together with the ring atom W at the ortho-position to the linking position of -C(O)- and ring C and -C(O)- in L 1 ;
  • L 3 is a single bond, substituted or unsubstituted C 1-2 alkylene
  • Ring A Ring B, Ring D, Rc , R1, R2, R4, R5 and n are as defined in Formula II .
  • the active group such as N-H, etc.
  • the compound of formula II of the present invention is obtained by removing the protecting group.
  • compositions and methods of administration are provided.
  • the compound of the present invention Since the compound of the present invention has excellent inhibitory effect on the activity of RIPK1 and/or the inhibitory activity on programmed necrosis of cells, the compound of the present invention and its various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or Solvates, etc., and pharmaceutical compositions containing the compound of the present invention as the main active ingredient can be used for the treatment, prevention and alleviation of programmed necrosis of cells and/or human receptor interacting protein 1 kinase (RIPK1) (such as its activity or expression level) ) related diseases or conditions.
  • RIPK1 human receptor interacting protein 1 kinase
  • the preferred compounds of the present invention not only have an inhibitory effect on RIPK1 in an inactive state so as to prevent the activation or activation of RIPK1 in related diseases or disorders (such as inflammation), but also effectively inhibit activated RIPK1 to enable more Rapidly achieve therapeutic or interventional effects on related diseases/disorders (eg, inflammation).
  • the compounds of the present invention are useful in the treatment of the following diseases or conditions: degenerative diseases (eg Alzheimer's disease (AD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), retinal degenerative diseases, etc.), inflammation (enteritis, rheumatoid arthritis, psoriasis, etc.), ischemia-reperfusion injury (cerebral infarction, myocardial infarction, etc.) and pathogen infection, etc.; or, Parkinson's disease (PD), Huntington's disease (PD) inflammatory bowel disease, age-related macular degeneration, etc., psoriasis, retinitis pigmentosa, inflammatory bowel disease, autoimmune disease, bombesin-induced acute pancreatitis and sepsis/systemic inflammatory response syndrome (SIRS), ischemic brain injury, ischemic myocardial injury, retinal ischemia/reperfusion injury, retinal detachment-induced photo
  • degenerative diseases
  • the pharmaceutical composition of the present invention comprises the compound of the present invention or a pharmacologically acceptable salt thereof and a pharmacologically acceptable excipient or carrier within a safe and effective amount.
  • the "safe and effective amount” refers to: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects.
  • the pharmaceutical composition contains 0.1-1000 mg of the present compound/dose, more preferably 0.5-500 mg of the present compound/dose.
  • the "one dose” is a capsule or tablet.
  • “Pharmaceutically acceptable carrier” refers to one or more compatible solid or liquid filler or gelling substances which are suitable for human use and which must be of sufficient purity and sufficiently low toxicity. "Compatibility” as used herein means that the components of the composition can be admixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds.
  • Examples of pharmaceutically acceptable carrier moieties include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid) , magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as Tween), wetting Agents (such as sodium lauryl sulfate), colorants, flavors, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.
  • cellulose and its derivatives such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.
  • gelatin such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate,
  • the mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include, but are not limited to: oral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical.
  • a particularly preferred mode of administration is oral.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
  • the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with (a) fillers or compatibilizers, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders such as, for example, hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, For example, glycerol; (d) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) Absorption accelerators such as quaternary amine compounds; (g) wetting agents such as cetyl alcohol and glyceryl monostea
  • Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials well known in the art. They may contain opacifying agents, and the release of the active compound or compounds in such compositions may be in a certain part of the digestive tract in a delayed manner. Examples of embedding components that can be employed are polymeric substances and waxes. If desired, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures.
  • liquid dosage forms may contain inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances, and the like.
  • inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylform
  • compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
  • Suspensions in addition to the active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances and the like.
  • suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances and the like.
  • compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.
  • Dosage forms for topical administration of the compounds of this invention include ointments, powders, patches, sprays and inhalants.
  • the active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required if necessary.
  • the compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.
  • a safe and effective amount of the compound of the present invention is suitable for mammals (such as human beings) in need of treatment, and the dose is the effective dose considered pharmaceutically, for a 60kg body weight, the daily dose is
  • the administration dose is usually 0.2 to 1000 mg, preferably 0.5 to 500 mg.
  • the specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.
  • the compounds of the present invention have excellent inhibitory activity against RIPK1 kinase.
  • the compounds of the present invention can effectively inhibit the programmed cell necrosis pathway (such as the phosphorylation of RIPK1 itself and the phosphorylation of the downstream protein MLKL).
  • the compound of the present invention has high bioavailability, and within 24 hours after administration, the blood drug concentration is above the effective concentration most of the time.
  • the compounds of the present invention can effectively reduce the inflammatory response (especially caused by TNF ⁇ ).
  • the compound of the present invention can alleviate the inflammatory response induced by the abnormal increase in the level of TNF ⁇ caused by the immune response during the coronavirus infection.
  • the compound of the present invention can effectively inhibit RIPK1 which is already in an activated state, so the intervention effect on related inflammation can be realized more quickly.
  • the compounds of the present invention have very strong inhibitory activity on both human and murine RIPK1, so there is no need to use expensive and limited primate models in preclinical tests, which is very beneficial to its expansion in different disease models application.
  • Methyl 2-(4-iodophenyl)acetate (QY-5-23): Dissolve 4-iodophenylacetic acid (500mg, 1.91mmol) in 5ml methanol, add 5 drops of concentrated sulfuric acid dropwise at room temperature, Mix and stir evenly in a 15ml pressure bottle, slowly heat up to 65°C, and stir at reflux for 5h.
  • N-methyl-1H-benzo[d]imidazole-5-carboxamide (QY-5-14): methyl benzimidazole-5-carboxylate (1.40 g, 7.94 mmol) was added to a 150 ml sealed tube, added The ethanol solution of methylamine (33%wt, 40ml) was heated under reflux and stirred for 24h. After the reaction was completed, the ethanol was removed by rotary evaporation to obtain a yellow-brown blocky solid, which was directly carried out to the next step without separation and purification. ESI-MS m/z 175.9(M+H) + .
  • N-methyl-1-(4-(2-(methyl(2,3,5-trifluorobenzyl)amino)-2-oxoethyl)phenyl)-1H-benzo[d]imidazole -5-formamide (QY-5-40): add QY-5-36 (192mg, 0.46mmol) into a 15ml pressure bottle, dissolve 3ml dimethyl sulfoxide, stir well and add QY-5-14 ( 88mg, 0.51mmol), cuprous iodide (44mg, 0.23mmol), cesium carbonate (298mg, 0.92mmol), 4,7-dimethoxy-1,10 phenoline (33mg, 0.14mmol), nitrogen protection After heating to 100 °C reaction overnight.
  • N-methoxy-N-methyl-2-phenylacetamide (QY-5-81): dissolve phenylacetic acid (1.0 g, 7.34 mmol) in 15 ml dichloromethane in a 30 ml pressure bottle, add HATU (3.6 g, 9.54 mmol), dimethylhydroxylamine hydrochloride (787 mg, 8.07 mmol) was added to the stirred reaction solution; N,N-diisopropylethylamine (2.84 g, 22.0 mmol) was added dropwise at room temperature mmol), and continued to stir the reaction for 5.5 h. The reaction was monitored in real time by LC-MS.
  • N-tert-butyl(4-(5-(methylcarbamoyl)-1H-benzo[d]imidazol 1-yl)benzyl)carbamate (QY-6-2): QY-5 -101 (200mg, 0.60mmol) was added to a 15ml pressure bottle, 3ml of dimethyl sulfoxide was dissolved, and after stirring evenly, QY-5-14 (105mg, 0.60mmol), cuprous iodide (57mg, 0.30mmol) were added, Cesium carbonate (390 mg, 1.20 mmol), 4,7-dimethoxy-1,10 phenoline (43 mg, 0.18 mmol), heated to 100° C. overnight under nitrogen protection.
  • Ethyl 5-benzyl-1,2,4-oxadiazole-3-carboxylate (QY-7-4): Dissolve QY-6-97 (84 mg, 0.34 mmol) in 3 ml N,N-dimethylformaldehyde In the formamide, transfer to an 8ml pressure bottle, mix and stir evenly, and then gradually heat up to 140 °C.
  • ESI-MS m/z 233.1(M+H) + .
  • Ethyl 5-benzyl-1,3,4-oxadiazole-2-carboxylate (QY-7-23): stir and mix QY-7-21 in 10ml dichloromethane in a 30ml pressure bottle , p-toluenesulfonyl chloride (760mg, 3.99mmol) was dissolved in 4ml of dichloromethane and added dropwise to the reaction solution, stirring was continued at room temperature, the reaction was monitored by thin layer chromatography, and the reaction was stopped after stirring for 6h.
  • reaction system was transferred to a separatory funnel, washed with 80ml of distilled water, extracted with ethyl acetate (40ml*3), combined with the organic phases, washed with saturated sodium chloride (15ml*2), and dried over anhydrous sodium sulfate.
  • ESI-MS m/z 273.1(M+H) + .
  • Ethyl 5-benzyloxazole-2-carboxylate (QY-8-25): in a 15ml pressure bottle, dissolve QY-8-15 (206mg, 0.83mmol) in 4ml acetonitrile, phosphorus pentoxide ( 587 mg, 4.1 mmol) was mixed with 4 ml of acetonitrile and added dropwise to the reaction solution, stirred vigorously and slowly heated to 70°C. LC-MS real-time monitoring, no raw material peak after 3h reaction, stop the reaction.
  • ESI-MS m/z 232.0(M+H) + .
  • N-(4-(4-Amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)benzyl)-5-benzyloxazole-2-carboxamide (QY-8-30): QY-8-28 (0.39mmol) was dissolved in an 8ml pressure-resistant bottle containing 4ml of dichloromethane, HATU (177mg, 0.47mmol) was added, and QY-8-22 (220mg, 0.62mmol) was added to the stirred reaction liquid; N,N-diisopropylethylamine (200 mg, 1.6 mmol) was added dropwise at room temperature, and the reaction was continued to stir for 6 h. The reaction was monitored in real time by LC-MS.
  • N -Boc-2-methylalanine (320mg, 1.6mmol) was dissolved in a 15ml pressure bottle containing 8ml of dichloromethane, HATU (652mg, 1.7mmol) was added, QY-7-64 (230mg, 1.4 mmol) was added to the stirred reaction solution; N,N-diisopropylethylamine (810 mg, 6.3 mmol) was added dropwise at room temperature, and stirring was continued at room temperature.
  • the reaction was monitored in real time by LC-MS. After 5h, the raw materials were consumed.
  • ESI-MS m/z 247.1(M+H) + .
  • Ethyl 2-ethoxy-2-iminoacetate (ZSQ-13-19): ethyl cyanoformate (9.9 ml, 100 mmol) was added to a 250 ml round-bottomed flask, mixed with ethanol (23.0 ml, 400 mmol) and stirred After dissolving evenly, the reaction system was reduced to 0 ° C, and acetyl chloride (14.2 ml, 200 mmol was added dropwise to the reaction solution, kept at 0 ° C and continued to stir overnight after the addition was complete. After the reaction was complete, a blocky white solid was obtained, and suction filtration was obtained to obtain a thick The product can be directly carried on to the next step without separation and purification. ESI-MS m/z 146.0(M+H) + .
  • Ethyl 2H-tetrazole-5-carboxylate (QY-8-50): The compound ethyl cyanoformate (1.0 g, 10.0 mmol) was dissolved in 12 ml of pyridine, mixed and stirred well in a 75 ml sealed tube, at room temperature 1.76 ml of trifluoroacetic acid was slowly added dropwise under the conditions, and after stirring for 10 min, sodium azide (700 mg, 10.8 mmol) was added, the temperature was slowly raised to 60° C., and the reaction was stopped after 24 h.
  • Ethyl 2-benzyl-2H-tetrazole-5-carboxylate (QY-8-55): stir and mix QY-8-50 (10.0 mmol) in 10 ml of N,N-dimethylformamide , potassium carbonate (4.2 g, 30.0 mmol) was added to the reaction solution, benzyl bromide (1.7 g, 10.0 mmol) was added dropwise to the reaction system under vigorous stirring at room temperature, and the reaction was stopped after stirring overnight. The potassium carbonate was removed by diatomaceous earth suction filtration, and the organic solvent was removed by spin drying.
  • N-(4-(4-Amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)benzyl)-2-benzyl-2H-tetrazole-5-carboxamide (QY-8- 58): Dissolve QY-8-56 (0.40mmol) in 3ml dichloromethane in an 8ml pressure bottle, add HATU (182mg, 0.48mmol), QY-8-22 (141mg, 0.40mmol); slowly at room temperature N,N-Diisopropylethylamine (259 mg, 2.0 mmol) was added dropwise and the reaction continued to stir. The reaction was monitored in real time by LC-MS. After 4 hours, the raw materials were consumed.
  • N-Boc-4-iodobenzylamine (330 mg, 1.0 mmol) was added In a 15ml pressure bottle, after 3ml of dimethyl sulfoxide was dissolved evenly, add adenine (135mg, 1.0mmol), cuprous iodide (95mg, 0.5mmol), cesium carbonate (652mg, 2.0mmol), 4,7- Dimethoxy-1,10 Pheroline (48 mg, 0., 2 mmol) was heated to 100°C under nitrogen protection and reacted overnight.
  • N-(4-(6-Amino-9H-purin-9-yl)benzyl)-5-benzyl-1-methyl-1H-pyrazole-3-carboxamide (QY-8-43): in In an 8ml pressure bottle, dissolve QY-6-103B (150mg, 0.69mmol) in 8ml dichloromethane, add HATU (316mg, 0.83mmol), and add QY-8-36 (166mg, 0.69mmol) to the stirred mixture. In the reaction solution; N,N-diisopropylethylamine (313 mg, 2.43 mmol) was added dropwise at room temperature, and the reaction was continued to stir for 4 h. LC-MS monitors the reaction in real time.
  • the reaction was monitored in real time by LC-MS.
  • the reactant was completely consumed, it was transferred to a separatory funnel, washed with 5ml of distilled water, extracted with dichloromethane (5ml*3), combined with the organic phases, washed with saturated sodium chloride (5ml*2), anhydrous
  • ESI-MS m/z 442.0(M+H) + .
  • N-(4-(4-Amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)benzyl)-5-benzyl-1-methyl-1H-pyrazole-3-carboxamide (QY-10-21): Dissolve QY-6-103B (271 mg, 1.25 mmol) in 15 ml dichloromethane in a 100 ml round-bottomed flask, add HATU (570 mg, 1.50 mmol), and mix QY-8-22 ( 300 mg, 1.25 mmol) was added to the stirred reaction solution; N,N-diisopropylethylamine (645 mg, 5.0 mmol) was added dropwise at room temperature, and the reaction was continued to stir for 3.5 h.
  • ESI-MS m/z 245.1(M+H) + .
  • N-(4-(4-Amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)benzyl)-5-benzyl-1-methyl-1H-pyrazole-4-carboxamide (QY-10-39): QY-10-56 (0.31mmol) was dissolved in 3ml dichloromethane in an 8ml pressure bottle, HATU (141mg, 0.37mmol), QY-8-22 (74mg, 0.31mmol) were added ) into the reaction solution; N,N-diisopropylethylamine (160 mg, 1.24 mmol) was added dropwise at room temperature.
  • ESI-MS m/z 142.1(M+H) + .
  • Methyl 5-benzyl-1-methyl-1H-1,2,3-triazole-4-carboxylate (QY-12-95): add 5ml toluene into a 15ml pressure bottle, QY-12-88 (305mg, 2.16mmol), stir well and add palladium acetate (24mg, 0.11mmol), triphenylphosphine (113mg, 0.43mmol), potassium carbonate (746mg, 5.40mmol), pivalic acid (66mg, 0.65mmol), Benzyl chloride (274 mg, 2.16 mmol) was added dropwise, and the mixture was heated to 100° C. and reacted overnight under nitrogen protection.
  • Benzyl-N-(4-iodobenzyl)-1-methyl-1H-1,2,3-triazole-4-carboxamide (QY-13-17):QY-13-15 (0.75mmol) Dissolve in 5ml dichloromethane in a 15ml pressure bottle, add HATU (341mg, 0.90mmol) and 4-iodobenzylamine (174mg, 0.75mmol), and dropwise add N,N-diisopropylethylamine ( 289 mg, 2.24 mmol), stirring was continued at room temperature.
  • N-(4-(6-Amino-9H-purin-9-yl)benzyl)-5-benzyl-1-methyl-1H-1,2,3-triazole-4-carboxamide (QY- 13-19): add QY-13-17 (153mg, 0.35mmol) into an 8ml pressure bottle, after 3ml of dimethyl sulfoxide is dissolved evenly, add adenine (57mg, 0.42mmol), cuprous iodide (34mg , 0.18 mmol), cesium carbonate (346 mg, 1.1 mmol), 4,7-dimethoxy-1,10 phenoline (17 mg, 0.07 mmol), heated to 100° C. overnight under nitrogen protection.
  • Methylhydrazine hydrochloride (QY-12-67): Weigh 1-BOC-2-methylhydrazine (7.31g, 50.0mmol) and transfer it into a 100ml round-bottomed flask, slowly add 30ml of dioxane under ice bath conditions The hydrogen chloride solution of the ring was kept stirring in an ice bath for 30 min, and the stirring was continued at room temperature overnight. When the raw materials were consumed, the reaction was stopped. 30 ml of dichloromethane was added to dilute the reaction solution, and all organic solvents were removed by rotary evaporation to obtain a milky white solid powder, which could be directly carried out to the next step without separation and purification.
  • ESI-MS m/z 250.2(M+H) + .
  • ESI-MS m/z 315.2(M+H) + .
  • control compound used in the test example is as follows
  • the biological assay protocol used was: the effect of compounds on TNF-induced necroptosis in FADD (Fas-associated death domain)-depleted Jurkat cells and L929 cells.
  • cell types closely related to the RIP1 pathway were selected, namely FADD-deficient Jurkat cells (human peripheral blood leukemia T cell line) and L929 cells , using two different stimulation methods: tumor necrosis factor (TNF ⁇ ) alone, or TNF ⁇ combined with mitochondria-derived cysteine aspartate activator (SMAC) SM164, calculated by detecting chemiluminescence values cell viability, thereby deriving the biological activity of the compound to inhibit programmed cell necrosis.
  • TNF ⁇ tumor necrosis factor
  • SMAC mitochondria-derived cysteine aspartate activator
  • FADD-deficient Jurkat cells FADD-deficient Jurkat cells (human peripheral blood leukemia T cell line) were cultured in vitro, and after growing to the logarithmic growth phase, the cells were collected, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cell concentration was adjusted to 2.5 ⁇ 10 5 /mL, cells were seeded into 384-well plates, 40 ⁇ l per well. SM164 (50 nM) diluted in cell culture medium and 5 ⁇ L of each compound were added to the corresponding wells.
  • TNF ⁇ 50 ng/mL
  • cell culture medium 5 ⁇ L of TNF ⁇ (50 ng/mL) diluted in cell culture medium was added to each well of the stimulation group, and the culture group was added to the culture group. base 5 ⁇ L.
  • 15 ⁇ l of Cell Titer-Glo solution was added to each well, incubated at room temperature for 30 min, and luminescence was detected to measure the level of intracellular ATP.
  • Control wells with unstimulated DMSO were taken as 100% cell viability.
  • L929 cells out-cultured L929 cells (mouse fibroblasts) were digested and diluted to 6.25 ⁇ 10 4 /ml, and the cells were seeded into 384-well plates, 40 ⁇ l per well. Place in a cell incubator (37°C, 5% CO2) for 12h. 5 ⁇ L of SM164 (500 nM) diluted in cell culture medium and 5 ⁇ L of each compound were added to the corresponding wells. After pretreatment at 37°C for 1 h, 5 ⁇ L of TNF ⁇ (500 ng/mL) diluted in cell culture medium was added to each well in the stimulation group, and 5 ⁇ L of TNF ⁇ (500 ng/mL) diluted in cell culture medium was added to the control group.
  • SM164 500 nM
  • the activity of inhibiting programmed necrosis was stronger than that of the clinical inhibitor GSK2982772; on murine L929 cells in which GSK2982772 was almost inactivated, the preferred compounds of the present invention QY10-40, QY-11-76 and QY-11-102A/B Several other compounds were still able to inhibit programmed necrosis well.
  • QY10-40 basically completely inhibited TNF ⁇ -induced necroptosis in FADD-deficient Jurkat cells or L929 cells at a concentration of more than 1 nM, and did not exhibit cytotoxicity at a 1000-fold effective inhibitory concentration (1 ⁇ M).
  • the adopted biological assay protocol is: the half-life of the test compound in human or murine liver microsomes.
  • Liver microsomes contain a variety of enzymes involved in drug metabolism, especially cytochrome P-450, and are therefore one of the major tissues for drug metabolism in vivo.
  • the stability of a compound in liver microsomes has a certain correlation with its in vivo pharmacokinetic stability. Therefore, by testing the half-life of different compounds in human or murine liver microsomes, the relative stability of the compounds in vivo can be roughly predicted.
  • the test results are shown in Table 2, and the specific methods are as follows:
  • the binding mode of the compounds to RIPK1 is obtained by the IC 50 of the compounds of the present invention inhibiting the RIPK1 kinase activity in the environment of different concentrations of ATP.
  • Nec-1s a known non-competitive inhibitor of RIPK1 ATP, was used as a control.
  • the process of in vitro kinase reaction is related to the substrate concentration.
  • the effect of substrate-competitive inhibitors will change drastically with the change of substrate concentration, while the effect of substrate-non-competitive inhibitors does not change with the change of substrate concentration.
  • ATP-competitive inhibitors of kinases inhibit kinase activity by competing with ATP for the binding site, and ATP-competitive kinase inhibitors are generally less specific due to the conservation of ATP binding sites. In contrast, ATP noncompetitive kinase inhibitors generally have higher specificity.
  • RIPK1(1-330) protein at a final concentration of 2 ⁇ M and ATP (1X kinase buffer) at the corresponding concentration were added to a 384-well plate with a final concentration of 5 ⁇ L. At least 3 sub-wells were set in each group, and the reaction was performed at 37°C for 2 h. Add 5 ⁇ L of ADP-Glo reagent, which is used to stop the kinase reaction and remove residual ATP from the reaction system. Let stand at room temperature for 40 min. 10 ⁇ L of Kinase Detection Reagent for converting ADP to ATP and luciferase and luciferin for detecting ATP were introduced into the system.
  • the bioassay protocol employed was to test the effect of compounds on key signals in the TNF ⁇ -induced FADD-deficient Jurkat or L929 cell necroptosis pathway using the known RIPK1 ATP non-competitive inhibitor Nec-1s and the clinical inhibitor GSK2982772 as comparison.
  • Necroptosis induced by TNF ⁇ is mediated by a complex called Necrosome (ComplexIIb), which includes TRADD, FADD, cysteine protease 8 (Caspase-8), RIPK1, RIPK3 and MLKL.
  • the kinase activity of RIPK1 has been shown to play a critical role in the regulation of programmed cell necrosis.
  • RIPK1 is activated by autophosphorylation, of which serine 166 is one of the main phosphorylation sites.
  • oligomeric MLKL translocates to the cell membrane and mediates cell death by calcium influx.
  • the compounds of the present invention were used to treat FADD-deficient Jurkat or L929 cells, and then TNF ⁇ was added to induce the program After sexual necrosis, cells were harvested and detected by western blot for phosphorylation of RIPK1 and oligomerization of MLKL.
  • FADD-deficient Jurkat cells or L929 cells were cultured in vitro, and after growing to the logarithmic growth phase, the cells were collected, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cell concentration was adjusted to 1X10 6 /mL.
  • a 12-well cell culture plate 1 ml of cells was added to each well, and 0.2 ⁇ L of DMSO solution with a concentration of 50 mM drug or pure DMSO control was added to each well for 1 hour of pretreatment.
  • TNF ⁇ 100 ⁇ g/ml TNF ⁇ (PBS solution) was added to each group, placed in a cell incubator (37 °C, 5% CO2) for 4 hours, and then centrifuged at 3000 rpm for 3 min to collect cells and washed with pre-cooled PBS solution for two days. all over. Remove the supernatant as much as possible, add 200 ⁇ L of RIPA cell lysate to the cell pellet, place it on a shaker at 4°C for 30 minutes, centrifuge at 15,000 rpm and 4°C for 15 minutes, and take the supernatant cell lysate.
  • PBS solution 100 ⁇ g/ml TNF ⁇
  • the transferred PC membrane was placed in 5% nonfat dry milk (TBST solution) for blocking for 2h, and incubated with the corresponding primary antibody for 12h at four degrees. Washed with TBST 3 times, 10 min each time. Incubate with the corresponding secondary antibody for 2 h at room temperature. Washed with TBST three times, 10 min each time. Incubate with ECL luminescent solution and detect the luminescent signal.
  • TBST solution 5% nonfat dry milk
  • the biological test protocol used was: the effect of compounds on the programmed necrosis of RIPK1 S161E mutant MEFs cells (mouse embryonic fibroblasts).
  • the intracellular RIPK1 has been highly activated, and the reported RIPK1 inhibitors are more inclined to inhibit the newly synthesized RIPK1 kinase protein that has not been activated, and the inhibition of the highly activated RIPK1 is weak, so it has a negative effect on the RIPK1.
  • the effect of intervention on associated inflammation was also weaker and slower.
  • MEFs cells stably expressing RIPK1 S161E (simulating the activation state after RIPK1 is phosphorylated) were selected, and TNF ⁇ -SM164-zVAD (Caspase Inhibitor, used to inhibit apoptosis) (TSZ) combined to induce programmed cell necrosis, the cell viability was calculated by detecting the chemiluminescence value, so as to obtain the biological activity of the compound to inhibit programmed cell necrosis.
  • TNF ⁇ -SM164-zVAD Caspase Inhibitor, used to inhibit apoptosis
  • RIPK1-replenished MEFs cells WT-RIPK1, S161A-RIPK1 and S161E-RIPK1 were replenished in RIPK1 stably knocked-out cells, stably expressed and cultured to the logarithmic growth phase.
  • the cells were collected and centrifuged at 1000 rpm for 5 min. Discard the supernatant, adjust the cell concentration to 5 ⁇ 10 4 /mL, and inoculate the cells into a 384-well plate, 40 ⁇ L per well.
  • SM164 100nM
  • 5 ⁇ L of zVAD 50 ⁇ M
  • compound diluted in cell culture medium were added to the corresponding wells.
  • each well of the stimulation group was added with TNF diluted in cell culture medium. (50ng/mL) 5 ⁇ L, and the control group was added with 5 ⁇ L of culture medium. After culturing in a cell incubator (37° C., 5% CO 2 ) for 12 h, 15 ⁇ L of Cell Titer-Glo solution was added to each well, incubated at room temperature for 30 min, and luminescence was detected to measure the level of intracellular ATP. Unstimulated DMSO control wells (Ctrl) were taken as 100% cell viability. Compound EC50 values were calculated using Prism Graphpad statistical software. The results are shown in Figure 4.
  • the adopted biological test protocol is: drug metabolism test of the compound in vivo in mice.
  • the representative compound QY-10-40 was selected, and a single dose of gavage (PO, 10 mg/kg) or intravenous injection (IV, 1 mg/kg) was used.
  • mice Tail vein injection of tumor necrosis factor TNF ⁇ resulted in the development of a systemic inflammatory response syndrome in mice, resulting in hypothermia and death in mice.
  • the known RIPK1 inhibitor Nec-1s was used as a control, and the clinical inhibitor GSK2982772 had poor inhibitory activity on murine RIPK1, so it was not used.
  • Systemic inflammatory response syndrome also known as inflammatory storm, refers to a systemic non-specific inflammatory response caused by infectious or non-infectious injuries such as severe infection, multiple trauma, burns, ischemia-reperfusion, and acute pancreatitis. Under this condition, a large amount of inflammatory factors will be released, and in severe cases, the body will lose control of the inflammatory response, resulting in multiple organ failure and even death.
  • the adopted biological test protocol RIPK1 K612R/K612R gene mutant mice were used as model mice to test the effects of representative compounds QY-10-40 or QY-13-19 on spontaneous enteritis.
  • Nec-1s a known RIPK1 inhibitor, was used as a control.
  • the K612R mutation results in decreased intracellular levels of RIPK1 but aberrant activation, making it sensitive to programmed necrosis and caspase-1 activation and in response to TLRs signaling.
  • RIPK1 K612R/K612R mice exhibited age-dependent decreased expression and abnormal activation of RIPK1, spontaneous intestinal inflammation, and splenomegaly.
  • mice were dissolved in 0.5% carboxymethyl cellulose according to the required concentration one day in advance, and sonicated overnight.
  • the mice were given a gavage volume of 0.1 ml/10 g body weight, their body weight was measured three times a week, and samples were collected at the end of the experiment.
  • the effects of compounds were calculated using Prism Graphpad statistical software.
  • Figures 7A and B show the effects of representative compounds QY-10-40, QY-13-19 and control compound Nec-1s on body weight change and organ coefficient (organ weight/body weight) in mice, respectively.
  • the experimental results show that the compound QY-10-40 or QY-13-19 can inhibit the effect of K612R mutation-induced digestive system inflammation on the body weight and spleen organ coefficient of mice, and the efficacy is at a lower dose (25mg/kg). Better than the control compound Nec-1s (50 mg/kg).
  • the recombinant human RIPK1 catalytic domain truncated protein (containing amino acid residues 1-294 and C23A, C127A, C233A, C240A quadruple mutations) was expressed and purified by insect cell Sf9, and concentrated to about 9 mg/mL, QY-7-2B (final concentration 2.5 mM) was added to induce co-crystal formation by hanging drop method and steam diffusion method at 18°C.
  • the crystal diffraction data of RIPK1 and QY-7-2B co-crystal were collected by X-ray diffraction method, and the resolution was determined by data analysis as The electron cloud distribution structure inside the co-crystal was simulated, and the three-dimensional binding model of the RIPK1 recombinant protein molecule and the small molecule QY-7-2B at the atomic level was simulated by software such as COOT and PHENIX.
  • Figure 8 shows the co-crystal structure of a representative QY-7-2B with the human RIPK1 protein kinase domain, and demonstrates the difference in the binding mode to RIPK1 protein between the compounds of the present invention and the control compound GSK2982772.
  • both QY-7-2B and GSK2982772 occupy the hydrophobic allosteric pocket near the ATP-binding pocket of the kinase domain of RIPK1, and promote the Leu157 configuration near the pocket to remain outward, so that RIPK1 is stabilized in an inactive state.
  • a part of QY-7-2B extends to the hinge region of the ATP-binding pocket and forms a hydrogen bond with the Met95 residue, which further stabilizes the interaction with RIPK1, thereby enhancing its inhibitory effect on the latter.

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Abstract

本发明提供了一类细胞程序性坏死抑制剂及其制备方法和用途。具体地，本发明提供了一种化合物，或其药学上可接受的盐、水合物或溶剂化物，其中，所述的化合物如式I所示。本发明还提供可该化合物的制备方法以及含其的药物组合物或其在治疗或预防与细胞程序性坏死和/或人受体相互作用蛋白1激酶(RIPK1)相关的疾病或病症中的用途。

Description

一类细胞程序性坏死抑制剂及其制备方法和用途 技术领域
本发明属于小分子化合物领域，具体涉及一类细胞程序性坏死抑制剂及其制备方法和用途。
背景技术
在发育与衰老的过程中，人体内总是伴随着细胞增殖与死亡的动态调节。细胞的主动性死亡在正常发育、抵抗病原微生物入侵、维持内环境稳态等生理活动中不可或缺，其失调往往导致发育畸形、免疫系统疾病、神经退行性疾病及癌症等多种疾病，乃至个体的死亡。因此，对细胞程序性死亡进行进行干预，对于疾病治疗研究有着重要的意义。凋亡是首个被阐明的细胞程序性死亡机制，近年来，细胞程序性坏死成为细胞死亡领域的新热点。已有多项研究报道，在多种退行性疾病(如阿尔茨海默氏症(AD)、多发性硬化症(MS)、肌萎缩脊髓侧索硬化症(ALS)、视网膜退行性疾病等)、炎症(肠炎、风湿性关节炎、银屑病等)、缺血再灌注损伤(脑梗、心肌梗死等)以及病原体感染等多种疾病中，都伴随有细胞程序性坏死这一重要病理特征。此外，细胞程序性坏死还参与肿瘤微环境的调控：肺癌细胞能诱导血管壁特定细胞发生程序性坏死，以便通过循环系统并发生转移；坏死小体(Necrosome)的主要成分在胰腺癌中的高表达，能诱导趋化因子CXCL1的表达进而抑制机体的免疫响应。因此，抑制细胞程序性坏死的发生，被公认有助于多种疾病的治疗与缓解。
研究表明，肿瘤坏死因子α(TNF-α)是刺激体内细胞发生程序性坏死的主要途径之一，其下游信号通路也是当前机制最为明确的坏死信号通路。在经典的TNF-α诱导细胞坏死过程中，TNF-α首先结合受体TNFR1，诱导其三聚化并招募胞内一系列因子——包括TRADD、TRAF2、RIPK1、cIAP1/2等多个蛋白，进而形成信号复合物I。复合物I能招募并激活IKKα/IKKβ/IKKγ复合物以及NF-κB通路，并在解离后部分进入胞质，形成新的蛋白复合物IIa，再通过FADD或TRADD招募procaspase-8等蛋白，以激活下游的半胱天冬酶caspase-3与caspase-7并介导凋亡的发生。在FADD缺失或caspase抑制剂用药的情况下，TNF-α诱导激活的激酶蛋白RIPK1会与RIPK3结合形成新的复合物IIb，并诱导后者的磷酸化激活，从而磷酸化下游底物MLKL以促使其寡聚，最终扰乱细胞膜的结构并导致坏死的发生。
多个衔接蛋白、泛素连接酶、去泛素化酶和激酶蛋白参与调控着TNF-α诱导细胞程序性坏死的下游信号通路。例如：E3泛素连接酶cIAP对RIPK1的K63泛素化能抑制后者的激活及坏死的进程；去泛素化酶CYLD可以剪切RIPK1的K63泛素链，进而激活RIPK1激酶活性并促进坏死小体的形成，最终实现对细胞坏死的正向调控作用；衔接蛋白SPATA2促进CYLD去泛素化酶活性，并抑制NF-κB和MAPK信号通路，进而正向调控程 序性坏死；激酶蛋白TAK1通过磷酸化RIPK1的Ser321位点抑制后者激酶活性，进而负向调控程序性坏死，而去泛素化酶A20(TNF-αiniduced protein 3)、衔接蛋白TAB2(TAK1 binding protein 2)等多个调控因子也参与到此调控过程；激酶蛋白TBK1则通过磷酸化RIPK1的Thr189位点抑制后者的激活，在衰老过程中TBK1的失活性突变也是ALS、FTD等神经退行性疾病的重要致病风险；衔接蛋白Optineurin(OPTN)通过抑制RIPK1激酶活性负向调控程序性坏死，而在ALS中丧失OPTN则可能促发进行性髓鞘形成障碍和轴突退行性病变。由此可见，TNF-α诱导细胞程序性坏死的信号通路网络中，多个调控组件的功能性异常都通过RIPK1激酶这一核心调控因子的激活来介导程序性坏死的发生。
因此，RIPK1激酶被公认是细胞程序性坏死相关疾病的潜在治疗靶标。首创型RIPK1抑制剂Necrostatin-1(Nec-1)及其类似物在临床前研究中，已经对多种退行性疾病、炎症、癌症等疾病展示出明确的疗效。例如，对AD、ALS、MS、帕金森氏症(PD)、亨廷顿氏症(PD)炎性肠病、老年性黄斑变性等具有缓解作用；对银屑病、色素性视网膜炎、炎症性肠病、自身免疫性疾病、蛙皮素诱导的急性胰腺炎和败血症/全身炎症反应综合症(SIRS)具有保护作用；能有效缓解缺血性脑损伤、缺血性心肌损伤、视网膜缺血/再灌注损伤、视网膜脱离诱导的感光细胞坏死、青光眼、肾缺血再灌注损伤、顺铂诱导的肾损伤和创伤性脑损伤；至少部分缓解由RIPK1依赖性细胞凋亡、坏死或细胞因子生成所相关的其他疾病，包括血液和实体器官恶性肿瘤、细菌感染和病毒感染(包括结核病、流感等)和溶酶体贮积症(尤其是戈谢病)。当前，Nec-1衍生物已进入治疗ALS和AD的临床试验当中；另一类RIPK1抑制剂GSK2982772也正处于治疗多种自身免疫性疾病的临床试验当中。然而，现有的程序性坏死抑制剂均存在着不同程度的缺陷，如活体抑制活性仍不够理想、药代性质不佳、口服生物利用度低等，还有一些无法透过血脑屏障进入中枢神经系统，或是因不能有效抑制鼠源RIPK1而难以实施临床前动物试验，这些缺点均限制了其进一步的研究与临床应用。
因此，研发出具有临床应用价值的高特异性、高活性并且具有血脑屏障穿透性的小分子RIPK1激酶活性抑制剂，这是目前治疗细胞程序性坏死相关疾病治疗研究中的难点与热点。综上所述，本领域迫切需要具有新颖化学结构、药代药效性质更突出的新型RIPK1抑制剂和/或细胞程序性坏死抑制剂，以作为预防和治疗涉及细胞死亡和/或炎症疾病的候选药物。
发明内容
本发明的目的就是提供一类具有新颖结构的RIPK1抑制剂和/或细胞程序性坏死抑制剂。
在本发明的第一方面中，提供了一种化合物，或其药学上可接受的盐、水合物或溶剂化物，其特征在于，所述的化合物如式I所示；
Figure PCTCN2021118212-appb-000001
其中，
环A为取代或未取代的9-10元含氮杂芳基，其中所述9-10元含氮杂芳基含有1、2、3或4个氮杂原子作为环原子；
n＝0、1或2；
R 4各自独立地选自下组：H、-CN、卤素、取代或未取代的C 1-6烷基、-O-R b、-S-R b、-N(R b) 2、-C(O)-NR 6-R b、-C(O)-NR 6-C 1-4亚烷基-N(R b) 2、-NR 6-C(O)-R b；
各个R b各自独立地选自下组：H、取代或未取代的C 1-6烷基；或者两个R b以及与它们相连的氮原子共同形成取代或未取代的5、6或7元杂环烷基，其中，除了与R b相连的N外，所述杂环烷基还含有0、1或2个另外的作为环原子的杂原子；
R 6选自下组：H、OH、取代或未取代的C 1-4烷基、取代或未取代的C 1-4烷氧基；
环B选自下组：取代或未取代的C 6-10芳基，取代或未取代的5-10元杂芳基；
L 1和L 2各自独立地为选自下组的二价基团：
无、
Figure PCTCN2021118212-appb-000002
并且L 1和L 2不同时为无；
R 1和R 2各自独立地选自下组：H、取代或未取代的C 1-4烷基；
R 3选自下组：H、OH、取代或未取代的C 1-4烷基、取代或未取代的C 1-4烷氧基；
环C为无或
Figure PCTCN2021118212-appb-000003
其中，W各自独立地选自下组：O、S、C、N、C(R c)、和N(R d)；R c各自独立地选自下组：H、CN、卤素、取代或未取代的C 1-6烷基，R d各自独立地选自下组：H、CN、取代或未取代的C 1-6烷基(较佳地，R d为取代或未取代的C 1-6烷基，更佳地，R d选自下组：甲基、乙基、丙基和丁基)；
或者，当环C为
Figure PCTCN2021118212-appb-000004
L 1为
Figure PCTCN2021118212-appb-000005
(L 1中羰基部分与环C连接)且L 2为无时，R 3与位于L 1与环C连接位置邻位的环原子W以及L 1中的-C(O)-共同形成取代或未取代的5、6或7元饱和杂环(较佳地，6元饱和杂环)；其中，所述饱和杂环除了与R 3相连的N外还含有0、1或2个另外的作为环原子的杂原子；
R 5选自下组：H、OH、取代或未取代的C 1-4烷基、取代或未取代的C 1-4烷氧基；
或者当环C为无且L 2为
Figure PCTCN2021118212-appb-000006
(L 1中N(R 3)部分与环C连接)时，R 3和R 5 以及与它们相连的原子共同形成取代或未取代的5、6或7元饱和杂环；其中，所述饱和杂环除了与R 3相连的N外还含有0、1或2个另外的作为环原子的杂原子；
环D选自下组：取代或未取代的C 6-10芳环、和取代或未取代的5-10元杂芳基；
除非特别说明，所述的取代指基团上的氢原子被一个或者多个(例如1、2、3、或4个等)选自下组的取代基所取代：氧代(＝O)、-CN、卤素(如F、Cl、Br或I)、硝基、C 1-6烷基、卤代C 1-6烷基、-OR、-SR、-S(O) 2R、-S(＝O) 2NR 2、-NR 2、-COOR、被R任选取代的C 6-10芳基、被R任选取代的具有1-3个选自N、S和O的杂原子的5-10元杂芳基、被R任选取代的C 3-8环烷基、被R任选取代的具有1-3个选自N、S和O的杂原子的5-12元杂环烷基、被R任选取代的-C 1-4亚烷基-C 6-10芳基、被R任选取代的-C 1-4亚烷基-具有1-3个选自N、S和O的杂原子的5-10元杂芳基、被R任选取代的-C 1-4亚烷基-C 3-8环烷基、被R任选取代的-C 1-4亚烷基-具有1-3个选自N、S和O的杂原子的5-12元杂环烷基；
R各自独立地选自下组：H、C 1-6烷基、卤代C 1-6烷基、C 1-6羟烷基。
在另一优选例中，所述的杂原子各自独立地选自下组：O、N、S；较佳地，各自独立地为O或N。
在另一优选例中，R 4各自独立地选自下组：H、-CN、-O-R b、-N(R b) 2、-C(O)-NR 6-R b、-C(O)-NR 6-C 1-4亚烷基-N(R b) 2；较佳地，R 4各自独立地选自下组：-CN、-O-R b、-N(R b) 2；更佳地，R 4各自独立地选自下组：-CN、-O-C 1-6烷基、-NH 2、-NH(C 1-4烷基)、或-N(C 1-4烷基) 2。
在另一优选例中，R 4各自独立地选自下组：H、取代或未取代的C 1-6烷基、-O-R b、-S-R b、-N(R b) 2、-C(O)-NR 6-R b、-C(O)-NR 6-C 1-4亚烷基-N(R b) 2、-NR 6-C(O)-R b。
在另一优选例中，环A如选自下组的结构所示：
Figure PCTCN2021118212-appb-000007
其中，X 1、X 2、X 3、X 4、X 5和X 6各自独立地选自下组：N和C(R a)；并且X 1、X 2、X 3、X 4、X 5和X 6中至多3个为N；
R a各自独立地选自下组：无、H、取代或未取代的C 1-6烷基。
在另一优选例中，X 1、X 2、X 3、X 4、X 5和X 6各自独立地选自下组：C、N和CH。
在另一优选例中，
Figure PCTCN2021118212-appb-000008
为
Figure PCTCN2021118212-appb-000009
较佳地，如选自下组的结构所示：
Figure PCTCN2021118212-appb-000010
在另一优选例中，
Figure PCTCN2021118212-appb-000011
为
Figure PCTCN2021118212-appb-000012
其中，X 3和X 5为C(R a)(较佳地，X 3和X 5为CH)；X 2、X 4和X 6为N；和R 4选自下组：-N(R b) 2、-NR 6-C(O)-R b(较佳地，R 4为-N(R b) 2；更佳地R 4为-NH 2、-NH(C 1-4烷基)、或-N(C 1-4烷基) 2；或者，
在另一优选例中，
Figure PCTCN2021118212-appb-000013
为
Figure PCTCN2021118212-appb-000014
其中，X 6为N，X 1、X 2、X 3和X 5为C(R a)(较佳地，X 1、X 2、X 3和X 5为CH)；X 4为N或C(R a)(较佳地X 4为N或CH)；和R 4选自下组：-CN、-O-C 1-6烷基。
在另一优选例中，环B选自下组：取代或未取代的苯基、取代或未取代的5或6元杂芳基。在另一优选例中，环B为苯基。
在另一优选例中，L 1为
Figure PCTCN2021118212-appb-000015
(L 1中羰基部分与环C连接)且L 2为无(即-L 1-L 2-为
Figure PCTCN2021118212-appb-000016
)。
在另一优选例中，R 1和R 2各自独立地选自下组：H和甲基。在另一优选例中，R 1和R 2之一为H，另一个为H或取代或未取代的C 1-4烷基(较佳地，为H或甲基)。
在另一优选例中，环C为
Figure PCTCN2021118212-appb-000017
在另一优选例中，环C为
Figure PCTCN2021118212-appb-000018
L 1为
Figure PCTCN2021118212-appb-000019
(L 1中羰基部分与环C连接)且L 2为无，R 3为H，或者R 3与位于L 1与环C连接位置邻位的W以及L 1中的C(O)共同形成取代或未取代的5或6元(较佳地，6元)饱和杂环。
在另一优选例中，R 3与位于L 1与环C连接位置邻位的W以及L 1中的C(O)共同形成取代或未取代的5或6元饱和杂环时，
Figure PCTCN2021118212-appb-000020
为
Figure PCTCN2021118212-appb-000021
Figure PCTCN2021118212-appb-000022
较佳地，为
Figure PCTCN2021118212-appb-000023
在另一优选例中，所述的环C如
Figure PCTCN2021118212-appb-000024
所示；较佳地，如
Figure PCTCN2021118212-appb-000025
所示。
在另一优选例中，所述的环C如选自下组的结构所示：
Figure PCTCN2021118212-appb-000026
其中，W 1选自下组：O、S、N(R d)；W 2、W 3、W 4、W 5和W 6各自独立地选自下组：N和C(R c)；
或者，当L 1为
Figure PCTCN2021118212-appb-000027
(L 1中羰基部分与环C连接)且L 2为无时，R 3与位于L 1与环C连接位置邻位的W 1、W 2、W 3、W 4或W 5以及L 1中的羰基共同形成取代或未取代的5或6元饱和杂环(较佳地，6元饱和杂环)。
在另一优选例中，所述的环C如选自下组的结构所示：
Figure PCTCN2021118212-appb-000028
其中，W 1为N(R d)；W 2、W 3、W 4、和W 5各自独立地选自下组：N和C(R c)；
或者，当L 1为
Figure PCTCN2021118212-appb-000029
(L 1中羰基部分与环C连接)且L 2为无时，R 3与位于L 1与环C连接位置邻位的W 1、W 2、W 3、W 4或W 5以及L 1中的羰基共同形成取代或未取代的5或6元饱和杂环(较佳地5元饱和杂环)。
在另一优选例中，所述的环C选自下组：
Figure PCTCN2021118212-appb-000030
其中，R c各自独立地选自下组：H、CN、卤素(如Cl)、取代或未取代的C 1-6烷基，R d各自独立地选自下组：H、取代或未取代的C 1-6烷基。
在另一优选例中，所述的环C选自下组：
Figure PCTCN2021118212-appb-000031
或者，
Figure PCTCN2021118212-appb-000032
为
Figure PCTCN2021118212-appb-000033
其中R 1和R 2如前定义(较佳地，R 1和R 2之一为H且另一个为H或C 1-4烷基(如甲基))。
在另一优选例中，R c各自独立地选自下组：H、CN、F、Cl、Br、C 1-4烷基；和/或R d选自下组：H、C 1-4烷基(较佳地，R d为C 1-4烷基)。
在另一优选例中，环D为苯基。
在另一优选例中，环A、环B、环C、环D、L 1、L 2、R 4、R 5和n独立地为表A中具体化合物中对应的基团。
在另一优选例中，所述的化合物如式II所示；
Figure PCTCN2021118212-appb-000034
其中，
环C为
Figure PCTCN2021118212-appb-000035
R 3选自下组：H、OH、取代或未取代的C 1-4烷基、取代或未取代的C 1-4烷氧基；
或者，R 3与位于-C(O)-与环C连接位置邻位的环原子W以及该-C(O)-共同形成取代或未取代的5、6或7元饱和杂环(较佳地，6元饱和杂环)；其中，所述饱和杂环除了与R 3相连的N外还含有0、1或2个另外的作为环原子的杂原子；
R 5选自下组：H、OH、取代或未取代的C 1-4烷基、取代或未取代的C 1-4烷氧基；
环A、环B、环D、W、R 1、R 2、R 4和n如式I中所定义。
在另一优选例中，环A、环B、环C、环D、R 1、R 2、R 3、R 4、R 5和n独立地为表A中具体化合物所对应的基团。
在另一优选例中，所述的化合物如式III所示；
Figure PCTCN2021118212-appb-000036
其中，
X 1、X 2、X 3、X 4、X 5和X 6各自独立地选自下组：N和C(R a)；并且X 1、X 2、X 3、X 4、X 5和X 6中至多3个为N；
R a各自独立地选自下组：无、H、取代或未取代的C 1-6烷基；
环C为
Figure PCTCN2021118212-appb-000037
R 3选自下组：H、OH、取代或未取代的C 1-4烷基、取代或未取代的C 1-4烷氧基；
或者，R 3与位于-C(O)-与环C连接位置邻位的环原子W以及该-C(O)-共同形成取代或未取代的5、6或7元饱和杂环(较佳地，6元饱和杂环)；其中，所述饱和杂环除了与R 3相连的N外还含有0、1或2个另外的作为环原子的杂原子；
R 5选自下组：H、OH、取代或未取代的C 1-4烷基、取代或未取代的C 1-4烷氧基；
环B、环D、W、R 1、R 2、R 4和n如式I中所定义。
在另一优选例中，X 1、X 2、X 3、X 4、X 5、X 6、环B、环C、环D、R 1、R 2、R 3、R 4、R 5和n独立地为表A中具体化合物所对应的基团。
在另一优选例中，所述的化合物如式IV-1或IV-2所示；
Figure PCTCN2021118212-appb-000038
其中，
X 1、X 2、X 3、X 4、X 5和X 6各自独立地选自下组：N和C(R a)；并且X 1、X 2、X 3、X 4、X 5和X 6中至多3个为N；
R 3选自下组：H、OH、取代或未取代的C 1-4烷基；
或者，R 3与环原子W以及该-C(O)-共同形成取代或未取代的5、6或7元饱和杂环(较佳地，6元饱和杂环)；其中，所述饱和杂环除了与R 3相连的N外还含有0、1或2个另外的作为环原子的杂原子；
R 5选自下组：H、OH、取代或未取代的C 1-4烷基；
环B、环D、W、R a、R 1、R 2、R 4和n如式I中所定义。
在另一优选例中，X 1、X 2、X 3、X 4、X 5、X 6、环B、W、环D、R 1、R 2、R 3、R 4、R 5和n独立地为表A中具体化合物所对应的基团。
在另一优选例中，环B和环D各自独立地为未取代的苯基，或者为被1或2个选自下组的取代基所取代的苯基：卤素、C 1-4烷基、卤代C 1-4烷基。
在另一优选例中，所述的化合物选自表A。
在本发明的第二方面中，提供了一种如第一方面所述化合物的制备方法，其中，所述化合物如式II所示，其中，
i.所述的制备方法为方法一，并且所述的方法一包括步骤：
在惰性溶剂中，使如式II-2A所示化合物和如式II-2B所示化合物反应，得到如式II-2C所示化合物；
Figure PCTCN2021118212-appb-000039
其中，
R 3选自下组：H、OH、取代或未取代的C 1-4烷基、取代或未取代的C 1-4烷氧基；
环A、环B、环C、环D、R c、R 1、R 2、R 4、R 5和n如第一方面中式II中所定义；
或者，
ii.所述方法为方法二，并且所述方法二包括步骤：
a)在惰性溶剂中，使如式II-2A所示化合物和如式II-2B所示化合物反应，得到如式II-2C所示化合物；
Figure PCTCN2021118212-appb-000040
和
b)在惰性溶剂中，使如式II-2C所示化合物反应形成如式II所示化合物
Figure PCTCN2021118212-appb-000041
其中，
R 3与位于-C(O)-与环C连接位置邻位的环原子W以及L 1中的-C(O)-共同形成取代或未取代的5、6或7元饱和杂环；
L 3为单键、取代或未取代的C 1-2亚烷基；
饱和杂环、环A、环B、环D、R c、R 1、R 2、R 4、R 5和n如第一方面中式II中所定义。
在另一优选例中，所述的惰性溶剂选自下组：二氯甲烷、
在另一优选例中，所述的反应在HATU和N,N-二异丙基乙胺存在下进行。
在另一优选例中，所述的化合物如式III所示，其中，
iii.所述方法为方法三，并且所述方法三包括步骤：
a)在惰性溶剂中，使如式III-A所示化合物和如式III-B所示化合物反应，得到如式III所示化合物；
Figure PCTCN2021118212-appb-000042
其中，R L为卤素；较佳地为I；
X 1、X 2、X 3、X 4、X 5、X 6、环B、环C、环D、R 1、R 2、R 3、R 4、R 5和如第一方面中定义。
在本发明的第三方面中，提供了一种药物组合物，所述的药物组合物包括(a)治疗有效量的如第一方面所述的化合物、或其药学上可接受的盐、水合物或溶剂化物；和(b)药学上可接受的载体。
在本发明的第四方面中，提供了一种如第一方面所述的化合物或如第三方面所述的药物组合物在制备用于治疗或预防与细胞程序性坏死和/或人受体相互作用蛋白1激酶(RIPK1)相关的疾病或病症的药物中的用途。
在另一优选例中，所述的化合物或药物组合物通过抑制人受体相互作用蛋白1激酶(RIPK1)来治疗或预防所述的疾病或病症。
在另一优选例中，所述的人受体相互作用蛋白1激酶(RIPK1)包括失活(或未激活)状态的RIPK1和激活状态的RIPK1。
在另一优选例中，所述的化合物或药物组合物还可通过抑制激活状态的人受体相互作用蛋白1激酶(RIPK1)来治疗或预防(尤其是治疗)所述的疾病或病症。
在另一优选例中，所述的抑制人受体相互作用蛋白1激酶(RIPK1)包括下述的一种或多种：抑制RIPK1的活性、或抑制RIPK1的的磷酸化。
在另一优选例中，所述的化合物或药物组合物通过抑制细胞程序性坏死号通路来治疗或预防所述的疾病或病症。
在另一优选例中，所述的抑制细胞程序性坏死号通路包括下述的一种或多种：抑制RIPK1的活性、抑制RIPK1的的磷酸化、或抑制MLKL的磷酸化。
在另一优选例中，所述的疾病或病症选自下组中的一种或多种：退行性疾病、炎症、缺血再灌注损伤、病原体感染、帕金森氏症(PD)、亨老年性黄斑变性、自身免疫性疾病、视网膜脱离诱导的感光细胞坏死、青光眼、顺铂诱导的肾损伤和创伤性脑损伤、高血脂所致动脉粥样硬化、由RIPK1依赖性细胞凋亡、坏死或细胞因子生成相关的其他疾病、细菌感染、病毒感染和溶酶体贮积症。
在另一优选例中，所述的退行性疾病包括：如阿尔茨海默氏症(AD)、多发性硬化症(MS)、肌萎缩脊髓侧索硬化症(ALS)、视网膜退行性疾病。
在另一优选例中，所述的炎症包括以下的一种或多种：肠炎、风湿性关节炎、银屑 病、色素性视网膜炎、炎症性肠病、廷顿氏症(PD)炎性肠病、蛙皮素诱导的急性胰腺炎、败血症/全身炎症反应综合症(SIRS)。
在另一优选例中，所述的缺血再灌注损伤包括以下的一种或多种：脑梗、心肌梗死等、缺血性脑损伤、缺血性心肌损伤、视网膜缺血/再灌注损伤、肾缺血再灌注损伤。
在另一优选例中，所述由RIPK1依赖性细胞凋亡、坏死或细胞因子生成相关的其他疾病包括以下的一种或多种：血液和实体器官恶性肿瘤。
在另一优选例中，所述病毒感染包括包括以下的一种或多种疾病或病症：结核病、流感、冠状病毒感染及其所致肺炎。
在另一优选例中，所述溶酶体贮积症包括戈谢病。
在本发明的第五方面，提供了一种治疗或预防与细胞程序性坏死和/或人受体相互作用蛋白1激酶(RIPK1)相关的疾病或病症的方法，所述的方法包括：向需要的对象施用治疗有效量的如第一方面所述的化合物或如第三方面所述的药物组合物。
在本发明的第六方面中，提供了一种抑制细胞程序性坏死的方法，包括步骤：在如第一方面的所述的化合物的存在下，培养细胞，从而抑制抑制细胞程序性坏死。
在另一优选例中，所述的方法是体外非治疗性的。
在本发明的第七方面中，提供了一种抑制RIPK1蛋白激酶活性的方法，包括步骤：使RIPK1蛋白激酶与如第一方面的所述的化合物接触，从而抑制RIPK1蛋白激酶活性。
在另一优选例中，所述的方法是体外非治疗性的。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了代表性化合物QY-10-40等以及对照化合物抑制FADD缺陷Jurkat Jurkat细胞或L929细胞发生程序性坏死的浓度-活性曲线。
图2是代表化合物QY-10-40对RIPK1(1-330)蛋白的激酶活性的浓度-抑制效果曲线图。
图3显示了QY-10-40、Nec-1s和GSK2982772在不同浓度下，在人源或鼠源细胞株中，对TNFα联用SM164所激活的TNF通路信号的影响。图4显示了RIPK1抑制剂对RIPK1持续激活细胞的程序性坏死的抑制活性测试的结果。
图5显示了代表性化合物QY-10-40在血浆中的浓度时间曲线，以及药代动力学参数。
图6显示了代表性化合物QY-10-40及对照化合物Nec-1s对小鼠体温变化的影响结果。
图7A和B分别显示了代表性化合物QY-10-40、QY-13-19及对照化合物Nec-1s对小鼠体重变化和脏器系数(脏器重量/体重)的影响结果。
图8显示了代表性QY-7-2B与人源RIPK1蛋白激酶域的共结晶结构，并展示了本发明 系列化合物与对照化合物GSK2982772在和RIPK1蛋白结合模式上的不同。
具体实施方式
本发明人经过长期而深入的研究，意外地发现一类结构新颖的细胞程序性坏死抑制剂。所述的细胞程序性坏死抑制剂具有优异的RIPK1抑制活性。因此可以用于制备预防和/或治疗涉及细胞死亡、RIPK1和/或炎症的疾病的药物组合物。特别地，本发明提供的优选化合物对已激活的RIPK1也具有优异的抑制活性，因此相比仅抑制失活的RIPK1的现有的RIPK1抑制剂而言，本发明提供的化合物具有更快速改善或治疗涉及细胞死亡和/或RIPK1炎症的疾病或病症(如炎症)。基于上述发现，发明人完成了本发明。
术语
除非明确另外指出，根据本发明和本文所用的术语具有以下含义：
如本文所用，术语“烷基”指具有指定碳原子数的直链或支链烷基。例如C 1-6烷基是指具有1到6个碳原子的直链或支链烷基。优选地，烷基是指C 1-4烷基。烷基的实例包括但不限于：甲基、乙基、丙基、异丙基、丁基、异丁基、仲丁基、叔丁基等，或类似基团。
如本文所用，术语“环烷基”指具有指定碳原子数的环状烷基。例如，“C 3-8环烷基”指具有1～8个碳原子的环状烷基。环烷基的实例包括但不限于：环丙基、环丁基、环戊基、环己基、环庚基、环辛基。
如本文所用，术语“烷氧基”指上文定义的烷基，其通过氧原子连接至分子的剩余部分。例如，C 1-6烷氧基是指C 1-6烷基-O-。优选地，烷氧基可包括甲氧基、乙氧基和异丙氧基。
如本文所用，术语“卤素”指F、Cl、Br和I。
如本文所用，术语“卤代烷基”指被卤素取代的烷基(烷基的定义如前所述)。优选地，卤代烷基包括三氟甲基、二氟甲基、三氟甲氧基、全氟乙基等。
如本文所用，术语“环烷基”是指具有指定环原子数(例如，C 3-8环烷基是指具有3、4、5、6、7或8个环原子的环状烷基)并且完全饱和的或在环顶之间具有不超过一个双键的烃环。“环烷基”也指双环和多环烃环，例如双环[2.2.1]庚烷、双环[2.2.2]辛烷等。术语“杂环烷基”或“杂环烷基”是指具有指定环原子数且含有一至五个选自N、O和S的杂原子的环烷基，其中氮和硫原子任选被氧化，且氮原子任选被季铵化。杂环烷基可以是单环、双环或多环体系。杂环烷基的非限制性例子包括吡咯烷、咪唑烷、吡唑烷、丁内酰胺、戊内酰胺、咪唑烷酮、乙内酰脲、二氧戊环、苯邻二甲酰亚胺、哌啶、1,4-二噁烷、吗啉、硫代吗啉、硫代吗啉-S-氧化物、硫代吗啉-S,S-氧化物、哌嗪、吡喃、吡啶酮、3-吡咯啉、噻喃、吡喃酮、四氢呋喃、四氢噻吩、奎宁环等。杂环烷基可以经环碳或杂原子连接于分子的其余部分。
术语“亚烷基”本身或作为另一取代基的一部分是指衍生自烷烃的二价基团，例如 -CH 2-、-CH 2CH 2-、-CH 2CH 2CH 2-、-CH 2CH 2CH 2CH 2-。
除非另有表述，术语“芳基”表示含指定环原子数的多不饱和的(通常芳香性)的烃基，其可以是单环或稠合在一起或共价连接的多环(如二环)。术语"杂芳基"是指具有指定环原子数且含有1至5个(如1、2、3、4或5个)选自N、O、和S的杂原子的芳基(或环)，其中氮和硫原子任选被氧化，氮原子任选被季铵化；例如，5-10元杂芳基(或环)是指含5、6、7、8、9或10个环原子的杂芳基(或环)。如本文所用含氮杂芳基是指所含的杂原子中至少一个为氮杂原子，优选地，所含的所有杂原子均为氮杂原子。杂芳基可通过杂原子连接于分子的其余部分。芳基的非限制性例子包括苯基、萘基，而杂芳基的非限制性例子包括吡啶基、哒嗪基、吡嗪基、嘧啶基、三嗪基、喹啉基、喹喔啉基、喹唑啉基、噌啉基、酞嗪基、苯并三嗪基(benzotriazinyl)、嘌呤基、苯并咪唑基、苯并吡唑基、苯并三唑基、苯并异噁唑基、异苯并呋喃基(isobenzofuryl)、异吲哚基、中氮茚基、苯并三嗪基、噻吩并吡啶基、噻吩并嘧啶基、吡唑并嘧啶基、咪唑并吡啶、苯并噻唑基、苯并呋喃基、苯并噻吩基、吲哚基、喹啉基、异喹啉基、异噻唑基、吡唑基、吲唑基、蝶啶基、咪唑基、三唑基、四唑基、噁唑基、异噁唑基、噻二唑基、吡咯基、噻唑基、呋喃基、噻吩基等等。以上芳基和杂芳基环系统各自的取代基选自下述可接受的取代基的组。
为简洁起见，当术语“芳基”与其它术语(例如芳氧基，芳硫基，芳烷基)组合使用时，包括如上所定义的芳基和杂芳基环。
在一些实施例中，上述术语(如“烷基”，“芳基”和“杂芳基”)将包括指定基团的取代和未取代形式。这些基团中可选的取代基包括，例如，氧代(＝O)、-CN、卤素(如F、Cl、Br或I)、C 1-6烷基、卤代C 1-6烷基、-OR、-SR、-S(O) 2R、-S(＝O) 2NR 2、-NR 2、-COOR、或者被一个或多个R任选取代的C 6-10芳基、5-10元杂芳基、C 3-8环烷基、5-12元杂环烷基、芳基烷基(如-C 1-4亚烷基-C 6-10芳基)、杂芳基烷基如-C 1-4亚烷基-5-10元杂芳基、环烷基烷基如-C 1-4亚烷基-C 3-8环烷基、杂环烷基烷基如-C 1-4亚烷基-5-12元杂环烷基。
对于本文提供的化合物，从取代基(通常为R基团)到芳香环(例如苯，吡啶等)的中心的键将被理解为是指在芳香环的任何可用顶点提供连接的键。在一些实施例中，该描述也包括稠合在芳环上的环上的连接。例如，绘制到吲哚苯部分的中心的键将表示与吲哚的六元或五元环部分的任何可用顶点连接的键。
本发明中，术语“含有”、“包含”或“包括”表示各种成分可一起应用于本发明的混合物或组合物中。因此，术语“主要由...组成”和“由...组成”包含在术语“含有”中。
活性成分
如本文所用，术语“本发明化合物”、“本发明的细胞程序性坏死抑制剂”、“本发明的RIPK1抑制剂”和“本发明的抑制剂”可以互换使用是指如本发明一方面所述的化合物。该术语还包括本发明第一方面所述的化合物的各种晶型形式、药学上可接受的 盐、水合物或溶剂合物。
在另一优选例中，本发明化合物如式I所示；
Figure PCTCN2021118212-appb-000043
其中，各基团如第一方面中定义。
在另一优选例中，本发明化合物如式II所示；
Figure PCTCN2021118212-appb-000044
其中，各基团如第一方面中定义。
在另一优选例中，本发明化合物如式III所示；
Figure PCTCN2021118212-appb-000045
其中，各基团如第一方面中定义。
在另一优选例中，本发明化合物如式IV-1或IV-2所示，
Figure PCTCN2021118212-appb-000046
其中，各基团如第一方面中定义。
在另一优选例中，如式I、式II、式III、式IV-1或式IV-2所示的化合物的各基团(如环A、环B、环C、环D、L 1、L 2、n、R 1、R 2、R 3、R 4、R 5、R 6、R a、R b、R c、R d、W、W 1、W 2、W 3、W 4、W 5、X 1、X 2、X 3、X 4、X 5、和X 6)各自独立地为表A中具体化合物中对应的基团。
在另一优选例中，本发明化合物选自表A的化合物，或其药学上可接受的盐。
其中，术语“药学上可接受的盐”指本发明化合物与酸或碱所形成的适合用作药物的盐。药学上可接受的盐包括无机盐和有机盐。一类优选的盐是本发明化合物与酸形成的盐。适合形成盐的酸包括但并不限于：盐酸、氢溴酸、氢氟酸、硫酸、硝酸、磷酸等无机酸；甲酸、乙酸、三氟乙酸、丙酸、草酸、丙二酸、琥珀酸、富马酸、马来酸、乳 酸、苹果酸、酒石酸、柠檬酸、苦味酸、苯甲酸、甲磺酸、乙磺酸、对甲苯磺酸、苯磺酸、萘磺酸等有机酸；以及脯氨酸、苯丙氨酸、天冬氨酸、谷氨酸等氨基酸。另一类优选的盐是本发明化合物与碱形成的盐，例如碱金属盐(例如钠盐或钾盐)、碱土金属盐(例如镁盐或钙盐)、铵盐(如低级的烷醇铵盐以及其它药学上可接受的胺盐)，例如甲胺盐、乙胺盐、丙胺盐、二甲基胺盐、三甲基胺盐、二乙基胺盐、三乙基胺盐、叔丁基胺盐、乙二胺盐、羟乙胺盐、二羟乙胺盐、三羟乙胺盐，以及分别由吗啉、哌嗪、赖氨酸形成的胺盐。
术语“溶剂合物”指本发明化合物与溶剂分子配位形成特定比例的配合物。“水合物”是指本发明化合物与水进行配位形成的配合物。
此外，本发明化合物还包括第一方面所述的化合物的前药。术语“前药”包括其本身可以是具有生物学活性的或非活性的，当用适当的方法服用后，其在人体内进行代谢或化学反应而转变成式(I)的一类化合物，或式(I)的一个化合物所组成的盐或溶液。所述的前药包括(但不局限于)所述化合物的羧酸酯、碳酸酯、磷酸酯、硝酸酯、硫酸酯、砜酯、亚砜酯、氨基化合物、氨基甲酸盐、偶氮化合物、磷酰胺、葡萄糖苷、醚、乙缩醛等形式。
制备方法
下面更具体地描述本发明的化合物的制备方法，但这些具体方法不对本发明构成任何限制。本发明化合物还可以任选将在本说明书中描述的或本领域已知的各种合成方法组合起来而方便地制得，这样的组合可由本发明所属领域的技术人员容易地进行。
在一个具体实施例中，本发明提供的式II化合物的制备方法为方法一，并且所述的方法一包括步骤：
在惰性溶剂中，使如式II-2A所示化合物和如式II-2B所示化合物反应，得到如式II-2C所示化合物；
Figure PCTCN2021118212-appb-000047
其中，
R 3选自下组：H、OH、取代或未取代的C 1-4烷基、取代或未取代的C 1-4烷氧基；
环A、环B、环C、环D、R c、R 1、R 2、R 4、R 5和n如式II中所定义。
在一个具体实施例中，本发明提供的式II化合物的制备方法为方法二，并且所述方法二包括步骤：
a)在惰性溶剂中，使如式II-2A所示化合物和如式II-2B所示化合物反应，得到如式II-2C所示化合物；
Figure PCTCN2021118212-appb-000048
和
b)在惰性溶剂中，使如式II-2C所示化合物反应形成如式II所示化合物
Figure PCTCN2021118212-appb-000049
其中，
R 3与位于-C(O)-与环C连接位置邻位的环原子W以及L 1中的-C(O)-共同形成取代或未取代的5、6或7元饱和杂环；
L 3为单键、取代或未取代的C 1-2亚烷基；
饱和杂环、环A、环B、环D、R c、R 1、R 2、R 4、R 5和n如式II中所定义。
应当理解，上述方法一或方法二的中的活性基团(如N-H等)在反应过程中可以是被保护的，之后通过脱去保护基获得本发明的式II化合物。
药物组合物和施用方法
由于本发明化合物具有优异的对RIPK1的活性的抑制作用和/或对细胞程序性坏死的抑制活性，因此本发明化合物及其各种晶型，药学上可接受的无机或有机盐，水合物或溶剂合物等，以及含有本发明化合物为主要活性成分的药物组合物可用于治疗、预防以及缓解与细胞程序性坏死和/或人受体相互作用蛋白1激酶(RIPK1)(如其活性或表达量)相关的的疾病或病症。特别地，本发明的优选化合物不仅对失活状态的RIPK1具有抑制作用从而可以在相关疾病或病症(如炎症)中预防RIPK1的活化或被激活，而且还能有效抑制已激活的RIPK1从而能够更快速地实现对相关疾病/病症(如炎症)的治疗或干预作用。根据现有技术，本发明化合物可用于治疗以下疾病或病症：退行性疾病(如阿尔茨海默氏症(AD)、多发性硬化症(MS)、肌萎缩脊髓侧索硬化症(ALS)、视网膜退行性疾病等)、炎症(肠炎、风湿性关节炎、银屑病等)、缺血再灌注损伤(脑梗、心肌梗死等)以及病原体感染等；或者，帕金森氏症(PD)、亨廷顿氏症(PD)炎性肠病、老年性黄斑变性等、银屑病、色素性视网膜炎、炎症性肠病、自身免疫性疾病、蛙皮素诱导的急性胰腺炎和败血症/全身炎症反应综合症(SIRS)、缺血性脑损伤、缺血性心肌损伤、视网膜缺血/再灌注损伤、视网膜脱离诱导的感光细胞坏死、青光眼、肾缺血再灌注损伤、顺铂诱导的肾损伤和创伤性脑损伤、高血脂导致的动脉粥样硬化、由RIPK1依赖性细胞凋亡、坏死或细胞因子生成所相关的其他疾病，包括血液和实体器官恶性肿瘤、细菌感染和病毒感染(包括结核病、流感、冠状病毒感染及其所致肺炎等)和溶酶体贮积症(尤其是戈谢病)。
本发明的药物组合物包含安全有效量范围内的本发明化合物或其药理上可接受的盐及药理上可以接受的赋形剂或载体。其中“安全有效量”指的是：化合物的量足以明显改善病情，而不至于产生严重的副作用。通常，药物组合物含有0.1-1000mg本发明化合物/剂，更佳地，含有0.5-500mg本发明化合物/剂。较佳地，所述的“一剂”为一个胶囊或药片。
“药学上可以接受的载体”指的是：一种或多种相容性固体或液体填料或凝胶物质，它们适合于人使用，而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组份能和本发明的化合物以及它们之间相互掺和，而不明显降低化合物的药效。药学上可以接受的载体部分例子有纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如吐温)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。
本发明化合物或药物组合物的施用方式没有特别限制，代表性的施用方式包括(但并不限于)：口服、直肠、肠胃外(静脉内、肌肉内或皮下)、和局部给药。特别优选的施用方式是口服。
用于口服给药的固体剂型包括胶囊剂、片剂、丸剂、散剂和颗粒剂。在这些固体剂型中，活性化合物与至少一种常规惰性赋形剂(或载体)混合，如柠檬酸钠或磷酸二钙，或与下述成分混合：(a)填料或增容剂，例如，淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸；(b)粘合剂，例如，羟甲基纤维素、藻酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖和阿拉伯胶；(c)保湿剂，例如，甘油；(d)崩解剂，例如，琼脂、碳酸钙、马铃薯淀粉或木薯淀粉、藻酸、某些复合硅酸盐、和碳酸钠；(e)缓溶剂，例如石蜡；(f)吸收加速剂，例如，季胺化合物；(g)润湿剂，例如鲸蜡醇和单硬脂酸甘油酯；(h)吸附剂，例如，高岭土；和(i)润滑剂，例如，滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠，或其混合物。胶囊剂、片剂和丸剂中，剂型也可包含缓冲剂。
固体剂型如片剂、糖丸、胶囊剂、丸剂和颗粒剂可采用包衣和壳材制备，如肠衣和其它本领域公知的材料。它们可包含不透明剂，并且，这种组合物中活性化合物或化合物的释放可以延迟的方式在消化道内的某一部分中释放。可采用的包埋组分的实例是聚合物质和蜡类物质。必要时，活性化合物也可与上述赋形剂中的一种或多种形成微胶囊形式。
用于口服给药的液体剂型包括药学上可接受的乳液、溶液、悬浮液、糖浆或酊剂。除了活性化合物外，液体剂型可包含本领域中常规采用的惰性稀释剂，如水或其它溶剂，增溶剂和乳化剂，例知，乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1,3-丁二醇、二甲基甲酰胺以及油，特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油或这些物质的混合物等。
除了这些惰性稀释剂外，组合物也可包含助剂，如润湿剂、乳化剂和悬浮剂、甜味剂、矫味剂和香料。
除了活性化合物外，悬浮液可包含悬浮剂，例如，乙氧基化异十八烷醇、聚氧乙烯山梨醇和脱水山梨醇酯、微晶纤维素、甲醇铝和琼脂或这些物质的混合物等。
用于肠胃外注射的组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。
用于局部给药的本发明化合物的剂型包括软膏剂、散剂、贴剂、喷射剂和吸入剂。活性成分在无菌条件下与生理上可接受的载体及任何防腐剂、缓冲剂，或必要时可能需要的推进剂一起混合。
本发明化合物可以单独给药，或者与其他药学上可接受的化合物联合给药。
使用药物组合物时，是将安全有效量的本发明化合物适用于需要治疗的哺乳动物(如人)，其中施用时剂量为药学上认为的有效给药剂量，对于60kg体重的人而言，日给药剂量通常为0.2～1000mg，优选0.5～500mg。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
本发明的主要优点包括：
(a)本发明化合物具有优异的对细胞程序性坏死的抑制活性。
(b)本发明化合物具有优异的代谢稳定性。
(c)本发明化合物具有优异的RIPK1激酶的抑制活性。
(d)本发明化合物能够有效地抑制细胞程序性坏死通路(如RIPK1自身的磷酸化以及下游蛋白MLKL的磷酸化)。
(e)本发明化合物的生物利用度高，给药后24h内，大部分时间血药浓度均在有效浓度以上。
(f)本发明化合物能够有效减少炎症反应(尤其是有TNFα引起的)。
(g)本发明化合物能够缓解冠状病毒感染过程中，因免疫反应导致TNFα水平异常增高进而诱导的炎症反应。
(h)本发明化合物能够有效抑制已处于激活状态的RIPK1，因此能更快地实现对相关炎症的干预效果。
(i)本发明化合物对人源和鼠源RIPK1均有非常强的抑制活性，因此在临床前试验中不需要利用昂贵且有限的灵长类动物模型，非常利于其在不同疾病模型上的拓展应用。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
合成实施例
方法1：
化合物QY-5-23的合成：
Figure PCTCN2021118212-appb-000050
2-(4-碘苯基)醋酸甲酯(QY-5-23)：将4-碘苯乙酸(500mg，1.91mmol)溶解于5ml甲醇中，室温条件下，滴入5滴浓硫酸，在15ml耐压瓶中混合搅拌均匀，缓慢升温至65℃，回流搅拌5h。待反应完成后，冰浴滴加饱和碳酸氢钠溶液淬灭反应，转移至分液漏斗中，乙酸乙酯萃取(10ml*3)，合并有机相，饱和氯化钠洗(5ml*2)，得有机相经无水硫酸钠干燥，过滤并旋干除去溶剂，得600mg淡黄色油状液体粗产物，不经分离纯化直接进行下步反应。ESI-MS m/z 276.9(M+H) +.
化合物QY-5-14的合成：
Figure PCTCN2021118212-appb-000051
N-甲基-1H-苯并[d]咪唑-5-甲酰胺(QY-5-14):将苯并咪唑-5-甲酸甲酯(1.40g，7.94mmol)加入150ml封管中，加入甲胺的乙醇溶液(33％wt,40ml)，加热回流搅拌24h，待反应完全，旋转蒸发仪旋干除去乙醇，得黄褐色块状固体，不经分离纯化直接进行下步反应。ESI-MS m/z 175.9(M+H) +.
化合物QY-5-25的合成：
Figure PCTCN2021118212-appb-000052
2-(4-(5-(甲基氨基甲酰基)-1H-苯并[d]咪唑-1-基)苯基)醋酸甲酯(QY-5-25)：15ml耐压瓶中，依次加入5ml二甲基亚砜，QY-5-23(600mg，2.17mmol)，搅拌溶解均匀后加入QY-5-14(314mg，1.81mmol)，碘化亚铜(172mg，0.91mmol)，碳酸铯(1.18g，3.62mmol)，4,7-二甲氧基-1,10菲洛琳(87mg,0.36mmol)，氮气保护后加热至100℃反应过夜。待反应物消耗完毕，滤膜过滤除去固体不溶物后C18反相色谱柱分离纯化(水：乙腈＝0-80％)得486mg黑褐色油状液体，产率83％。ESI-MS m/z 324.1(M+H) +
化合物QY-5-34的合成：
Figure PCTCN2021118212-appb-000053
2-(4-(5-(甲基氨基甲酰基)-1H-苯并[d]咪唑-1-基)苯基)醋酸(QY-5-34)：
将QY-5-25(486mg，1.50mmol)溶解于5ml四氢呋喃中，转移至15ml耐压瓶，氢氧 化锂一水合物(126mg，3.0mmol)溶解于5ml蒸馏水中，冰浴条件下滴加至反应液中，混合搅拌均匀后逐渐回至室温，LC-MS实时监测，2h反应完全，转移至100ml圆底烧瓶中，旋转蒸发除去有机溶剂后，甲醇溶解反应体系，C18反相色谱柱分离纯化(水：乙腈＝0-80％)得292mg墨绿色泡沫状固体，产率63％。ESI-MS m/z 308.0(M+H) -.
化合物QY-5-35的合成：
Figure PCTCN2021118212-appb-000054
1-(4-(2-(((1H-吲哚-3-基)甲基)氨基)-2-氧代乙基)苯基)-N-甲基-1H-苯并[d]咪唑-5-甲酰胺(QY-5-35)：QY-5-34(38mg，0.12mmol)在8ml耐压瓶内溶解于1.5ml二氯甲烷中，加入HATU(56mg，0.147mmol)，将吲哚-3-甲胺(21mg，0.15mmol)溶解于1ml二氯甲烷中，滴加入搅拌均的反应液中；冰浴条件下滴加N,N-二异丙基乙胺(40mg，0.31mmol)，搅拌10min，逐渐恢复至室温后搅拌反应4h。LC-MS实时监测，待反应完成后，加入5ml二氯甲烷稀释反应液，转移至分液漏斗中，5ml蒸馏水洗，二氯甲烷萃取(5ml*3)，合并有机相，饱和氯化钠洗(5ml*2)，后处理得有机相经无水硫酸钠干燥，过滤并旋干除去二氯甲烷，C18反相色谱柱分离纯化(水：乙腈＝0-80％)得29mg淡黄色油状液体，产率55％。ESI-MS m/z 438.1(M+H) +.
方法2：
化合物QY-5-30的合成：
Figure PCTCN2021118212-appb-000055
(4-碘苯基)-N-(2,3,5-三氟苄基)乙酰胺(QY-5-30)：2,3,5-三氟苄胺(200mg，1.24mmol)在15ml耐压瓶内溶解于7ml二氯甲烷中，加入HATU(566mg，1.49mmol)，将对碘苯乙酸(390mg，1.49mmol)溶解于1ml二氯甲烷中，滴加入搅拌均的反应液中；冰浴条件下滴加N,N-二异丙基乙胺(400mg，3.1mmol)，搅拌10min，逐渐恢复至室温后搅拌反应6h。LC-MS实时监测，待原料消耗完毕后，加入15ml二氯甲烷稀释反应液，转移至分液漏斗中，15ml蒸馏水洗，二氯甲烷萃取(15ml*3)，合并有机相，饱和氯化钠洗(10ml*2)，后处理得有机相经无水硫酸钠干燥，过滤并旋干除去二氯甲烷，C18反相色谱柱分离纯化(水：乙腈＝0-80％)得400mg白色固体，产率80％。ESI-MS m/z 406.0(M+H) +.
化合物QY-5-36的合成：
Figure PCTCN2021118212-appb-000056
2-(4-碘苯基)-N-甲基-N-(2,3,5-三氟苄基)乙酰胺(QY-5-36)：将QY-5-30(300mg，0.74mmol)及6ml四氢呋喃加入15ml耐压瓶中混合搅拌均匀，冰浴条件下缓慢加入氢化钠(30mg，0.74mmol)至反应液中，搅拌10min；将碘甲烷(126mg，0.89mmol)冰盐浴条件下缓慢滴至反应液中，逐渐回至室温后继续搅拌。LC-MS实时监测至反应物消耗完全，加入蒸馏水淬灭反应后旋转蒸发仪除去四氢呋喃，甲醇溶解反应体系，过滤膜后C18反相色谱柱分离纯化(水：乙腈＝0-80％)得192mg淡黄色油状液体，产率62％。ESI-MS m/z 419.9(M+H) +.
化合物QY-5-40的合成：
Figure PCTCN2021118212-appb-000057
N-甲基-1-(4-(2-(甲基(2,3,5-三氟苄基)氨基)-2-氧代乙基)苯基)-1H-苯并[d]咪唑-5-甲酰胺(QY-5-40)：将QY-5-36(192mg，0.46mmol)加入15ml耐压瓶中，3ml二甲基亚砜溶解，搅拌均匀后加入QY-5-14(88mg，0.51mmol)，碘化亚铜(44mg，0.23mmol)，碳酸铯(298mg，0.92mmol)，4,7-二甲氧基-1,10菲洛琳(33mg,0.14mmol)，氮气保护后加热至100℃反应过夜。待反应物消耗完毕，滤膜过滤除去固体不溶物后C18反相色谱柱分离纯化(水：乙腈＝0-80％)得143mg黄褐色固体，产率67％。ESI-MS m/z 467.1(M+H) +.
方法3：
化合物QY-5-62的合成：
Figure PCTCN2021118212-appb-000058
1-(4-(2-(苄基(羟基)氨基)-2-氧代乙基)苯基)-N-甲基-1H-苯并[d]咪唑-5-甲酰胺(QY-5-62)：QY-5-34(150mg，0.49mmol)溶解于4ml二氯甲烷后加入8ml耐压瓶中，加入HATU(221mg，0.58mmol)，将N-苄基羟胺盐酸盐(93mg，0.58mmol)加入搅拌均的反应液中；冰浴条件下滴加N,N-二异丙基乙胺(250mg，1.94mmol)，逐渐恢复至室温后搅拌反应3h。LC-MS实时监测，待反应物消耗完毕，加入5ml二氯甲烷稀释反应液，转移至分液漏斗，4ml蒸馏水洗，二氯甲烷萃取(5ml*3)，合并有机相，饱和氯化钠洗涤(5ml*2)，无水硫酸钠干燥有机相，过滤并旋干除去二氯甲烷，C18反相色谱柱分离纯化(水：乙腈＝20％-90％)得85mg白色固体，产率42％。ESI-MS m/z 415.1(M+H) +.
方法4：
化合物QY-5-81的合成：
Figure PCTCN2021118212-appb-000059
N-甲氧基-N-甲基-2-苯基乙酰胺(QY-5-81)：在30ml耐压瓶中将苯乙酸(1.0g，7.34mmol)溶解于15ml二氯甲烷中，加入HATU(3.6g，9.54mmol)，将二甲羟胺盐酸盐(787mg，8.07mmol)加入搅拌均的反应液中；室温条件下滴加N,N-二异丙基乙胺(2.84g，22.0mmol)，继续搅拌反应5.5h。LC-MS实时监测反应，待反应物消耗完全，转移至分液漏斗，25ml蒸馏水洗，二氯甲烷萃取(20ml*3)，合并有机相，饱和氯化钠洗涤(10ml*2)，无水硫酸钠干燥有机相，过滤并旋蒸除去二氯甲烷，硅胶柱层析分离纯化(石油醚：乙酸乙酯＝0-100％)得1.23g淡黄色透明液体，产率94％。ESI-MS m/z 180.1(M+H) +.
化合物QY-5-84的合成：
Figure PCTCN2021118212-appb-000060
1-苯基丙烷-2-酮(QY-5-84)：在30ml耐压瓶中将QY-5-81(1.03g，5.75mmol)溶解于15ml四氢呋喃中，700rp持续搅拌，0摄氏度下缓慢滴加甲基溴化镁(6.90ml，6.90mmol)，滴加完毕后逐渐回至室温继续搅拌。LC-MS实时监测反应，待反应完全后停止反应。向反应体系缓慢滴入1N盐酸淬灭反应，乙酸乙酯萃取(20ml*3),合并有机相，饱和食盐水洗涤(10ml*1)，无水硫酸钠干燥，旋转蒸发仪除去有机溶剂，硅胶柱层析分离纯化(石油醚：乙酸乙酯＝0-60％)得609mg无色透明液体，产率79％。
化合物QY-5-87的合成：
Figure PCTCN2021118212-appb-000061
2,4-二氧代-5-苯基戊酸乙酯(QY-5-87)：将QY-5-84(509mg，3.79mmol)混匀至10ml超干四氢呋喃中，加至30ml耐压瓶搅拌；氢化钠(152mg，3.80mmo)溶解于5ml超干四氢呋喃中，0℃条件下逐滴加入悬浊液中,零摄氏度下混匀后将草酸二乙酯(665mg，4.55mmol)加至反应体系中，逐渐回至室温后持续搅拌3h后停止反应。零摄氏度滴加蒸馏水淬灭反应，旋干移除超干四氢呋喃，加入10ml乙酸乙酯稀释反应体系，转移至分液漏斗，10ml蒸馏水洗后加乙酸乙酯萃取(15ml*3)，合并有机相后经饱和食盐水洗涤(10ml*2)，无水硫酸钠干燥，过滤并旋干移除乙酸乙酯，C18反相色谱柱分离纯化(水：乙腈＝20％-90％)得495mg黄褐色液体，产率56％。ESI-MS m/z 235.1(M+H) +.
化合物QY-5-90的合成：
Figure PCTCN2021118212-appb-000062
5-苄基异噁唑-3-羧酸乙酯(QY-5-90)：将QY-5-87(250mg，1.07mmol)加入4ml无水乙醇后转移至15ml耐压瓶中混合搅拌均匀，室温下缓缓加入盐酸羟胺(111mg，1.60mmol)至反应体系中，升温至回流后持续搅拌反应过夜。待反应物消耗完全，旋干移除有机溶剂，硅胶柱层析分离纯化(石油醚：乙酸乙酯＝0-35％)得180mg橙黄色透明液体，产率73％。ESI-MS m/z 232.1.1(M+H) +.
化合物QY-5-91的合成：
Figure PCTCN2021118212-appb-000063
5-苄基异噁唑-3-羧酸(QY-5-91)：将QY-5-90(360mg，1.56mmol)溶解于4ml乙醇中，转移至15ml耐压瓶，氢氧化钾(437mg，7.78mmol)溶解于3ml蒸馏水中，冰浴条件下滴加至反应液中，混合搅拌均匀后逐渐回至室温，加热至80℃，LC-MS实时监测，4h反应完全，加入饱和亚硫酸氢钾体调节PH至酸性，一并旋干后甲醇溶解反应体系，过滤膜后经C18反相色谱柱分离纯化(水：乙腈＝0-80％)得258mg白色固体粉末，产率82％。ESI-MS m/z 202.1(M+H) -.
化合物QY-5-101的合成：
Figure PCTCN2021118212-appb-000064
(4-碘苄基)氨基甲酸叔丁基酯氨基甲酸酯(QY-5-101)：将对碘苄胺(800mg，3.43mmol)溶解于10ml二氯甲烷中，转移至15ml耐压瓶，二碳酸二叔丁酯(749mg，3.43mmol)缓慢滴加至反应液中，加入4-二甲氨基吡啶(126mg，1.03mmol)，室温搅拌，LC-MS实时监测，3.5h反应完全，一并旋干反应体系，硅胶柱层析分离纯化(石油醚：乙酸乙酯＝0-40％)得925mg白色固体，产率81％。ESI-MS m/z 277.0(M+H) +.
化合物QY-6-2的合成：
Figure PCTCN2021118212-appb-000065
N-叔丁基(4-(5-(甲基氨基甲酰基)-1H-苯并[d]咪唑1-基)苄基)氨基甲酸酯(QY-6-2):将QY-5-101(200mg，0.60mmol)加入15ml耐压瓶中，3ml二甲基亚砜溶解，搅拌均匀后加入QY-5-14(105mg，0.60mmol)，碘化亚铜(57mg，0.30mmol)，碳酸铯(390mg，1.20mmol)，4,7-二甲氧基-1,10菲洛琳(43mg,0.18mmol)，氮气保护后加热至100℃反应过夜。待反应物消耗完毕，滤膜过滤除去固体不溶物后C18反相色谱柱分离纯化(水：乙腈＝0-80％)得203mg黄褐色油状液体，产率89％。ESI-MS m/z 381.1(M+H) +.
化合物QY-6-6的合成：
Figure PCTCN2021118212-appb-000066
1-(4-(氨基甲基)苯基)-N-甲基-1H-苯并[d]咪唑-5-甲酰胺(QY-6-6)：将QY-6-2(203mg，0.53mmol)混匀于5ml二氯甲烷中，转移至8ml耐压瓶中，室温条件下，滴加三氟乙酸1.0ml至反应液中，混合搅拌均匀。LC-MS实时监测，3.5h后原料消耗完毕，转移至圆底烧瓶，每次加入20ml二氯甲烷,旋干移去有机溶剂混合物，重复4-6次，此步反应产物无需分离纯化可直接用于后续合成。ESI-MS m/z 281.0(M+H) +.
化合物QY-6-16的合成：
Figure PCTCN2021118212-appb-000067
5-苄基-N-(4-(5-(甲基氨基甲酰基)-1H-苯并[d]咪唑-1-基)苄基)异噁唑-3-甲酰胺(QY-6-16)：在8ml耐压瓶中将QY-6-6(70mg，0.25mmol)溶解于2ml二氯甲烷中，加入HATU(114g，0.30mmol)，将QY-5-91(50mg，0.25mmol)加入搅拌均的反应液中；室温条件下滴加N,N-二异丙基乙胺(97mg，0.75mmol)，继续搅拌反应4h。LC-MS实时监测反应，待反应物消耗完全，转移至分液漏斗，5ml蒸馏水洗，二氯甲烷萃取(5ml*3)，合并有机相，饱和氯化钠洗涤(5ml*2)，无水硫酸钠干燥有机相，过滤并旋蒸除去二氯甲烷，滤膜过滤后经C18反相色谱柱分离纯化(水：乙腈＝0-80％)得116mg黄褐色油状液体，产率52％。ESI-MS m/z 466.1(M+H) +.
方法5：
化合物QY-6-98的合成：
Figure PCTCN2021118212-appb-000068
5-苄基-1-乙基甲基-1H-吡唑-3-羧酸乙酯(QY-6-98)：在15ml耐压瓶中将QY-5-87(250mg，1.07mmol)搅拌混匀于7ml无水乙醇中，室温加入甲基肼盐酸盐(101mg，1.21mmol)，逐渐升温至90℃，薄层色谱监测反应，继续搅拌4h后停止反应。转至旋转蒸发仪除去有机溶剂，硅胶柱层析分离纯化(石油醚：乙酸乙酯＝0-60％)得两种构型产物，产物A：104mg橙黄色液体，产率40％；产物B：62mg橙黄色液体，产率24％。ESI-MS  m/z 245.2(M+H) +.
化合物QY-6-103B的合成：
Figure PCTCN2021118212-appb-000069
6-苄基-1-甲基-1H-吡唑-3-羧酸(QY-6-103B):将QY-6-98(62mg，0.25mmol)溶解于2ml四氢呋喃中，转移至8ml耐压瓶，氢氧化锂一水合物(27mg，0.63mmol)溶解于1ml蒸馏水中，冰浴条件下滴加至反应液中，混合搅拌均匀后逐渐回至室温，LC-MS实时监测，搅拌过夜后停止反应，旋转蒸发除去有机溶剂后，甲醇溶解反应体系，C18反相色谱柱分离纯化(水：乙腈＝0-80％)得41mg白色固体，产率76％。ESI-MS m/z 215.1(M+H) -.
化合物QY-7-2B的合成：
Figure PCTCN2021118212-appb-000070
1-(4-((5-苄基-1-甲基-1H-吡唑-3-甲酰胺基)甲基)苯基)-N-甲基-1H-苯并[d]咪唑-5-甲酰胺(QY-7-2B)：在8ml耐压瓶中将QY-6-103B(15mg，0.07mmol)溶解于1ml二氯甲烷中，加入HATU(32mg，0.09mmol)，将QY-6-6(20mg，0.07mmol)加入搅拌均的反应液中；室温条件下滴加N,N-二异丙基乙胺(23g，0.18mmol)，继续搅拌反应4h。LC-MS实时监测反应，待反应物消耗完全，转移至分液漏斗，2ml蒸馏水洗，二氯甲烷萃取(2ml*3)，合并有机相，饱和氯化钠洗涤(2ml*2)，无水硫酸钠干燥有机相，过滤并旋蒸除去二氯甲烷，硅胶柱层析分离纯化(石油醚：乙酸乙酯＝0-100％)得19mg淡黄褐色液体，产率59％。ESI-MS m/z 479.1(M+H) +.
方法6：
化合物QY-6-97的合成：
Figure PCTCN2021118212-appb-000071
(E)-2-(羟亚氨基)-2-(2-苯基乙酰胺基)醋酸乙酯(QY-6-97)：苯乙酸(200mg，1.47mmol)在15ml耐压瓶内溶解于7ml二氯甲烷中，加入HATU(669mg，1.76mmol)，将2-羟胺基-2-亚氨基乙酸乙酯(233mg，1.76mmol)溶解于2ml二氯甲烷中，滴加入搅拌均的反应液中；室温下滴加N,N-二异丙基乙胺(569mg，4.41mmol)，搅拌10min，逐渐恢复至室温后搅拌反应6h。LC-MS实时监测，待反应完成后，加入10ml二氯甲烷稀释反应液，转移至分液漏斗中，5ml蒸馏水洗，二氯甲烷萃取(5ml*3)，合并有机相，饱和氯化钠洗(5ml*2)，后处理得有机相经无水硫酸钠干燥，过滤并旋干除去二氯甲烷， 硅胶柱层析分离纯化(石油醚：乙酸乙酯＝0-80％)得84mg白色固体粉末，产率23％。ESI-MS m/z 251.1(M+H) +.
化合物QY-7-4的合成：
Figure PCTCN2021118212-appb-000072
乙基5-苄基-1,2,4-噁二唑-3-羧酸酯(QY-7-4)：将QY-6-97(84mg，0.34mmol)溶解于3mlN,N-二甲基甲酰胺中，转移至8ml耐压瓶中，混合搅拌均匀后逐渐升温至140℃。LC-MS实时监测，2h反应完全；C18反相色谱柱分离纯化(水：乙腈＝0-100％)得64mg淡黄色油状液体，产率81％。ESI-MS m/z 233.1(M+H) +.
化合物QY-7-13的合成：
Figure PCTCN2021118212-appb-000073
5-苄基-1,2,4-噁二唑-3-羧酸(QY-7-13)：将QY-7-4(51mg，0.22mmol)溶解于2ml四氢呋喃中，转移至8ml耐压瓶，氢氧化锂一水合物(14mg，0.33mmol)溶解于1ml蒸馏水中，冰浴条件下滴加至反应液中，混合搅拌均匀后逐渐回至室温，LC-MS实时监测，4h后停止反应，旋转蒸发除去反应体系所有溶剂，粗产品可直接进行后续合成。ESI-MS m/z 203.1(M+H) -.
化合物QY-7-14的合成：
Figure PCTCN2021118212-appb-000074
5-苄基-N-(4-(5-(甲基氨基甲酰基)-1H-苯并[d咪唑-1-基)苄基)-1,2,4-噁二唑-3-甲酰胺(QY-7-14)：QY-7-13(0.22mmol)在15ml耐压瓶内溶解于3ml二氯甲烷中，加入HATU(83mg，0.22mmol)，将QY-6-6(51mg，0.18mmol)加入搅拌均的反应液中；室温下滴加N,N-二异丙基乙胺(59mg，0.46mmol)，室温搅拌反应4h。LC-MS实时监测，待反应完成后，加入10ml二氯甲烷稀释反应液，转移至分液漏斗中，5ml蒸馏水洗，二氯甲烷萃取(5ml*3)，合并有机相，饱和氯化钠洗(5ml*2)，后处理得有机相经无水硫酸钠干燥，过滤并旋干除去二氯甲烷，滤膜过滤后经C18反相色谱柱分离纯化(水：乙腈＝0-90％)得35mg黄褐色油状液体，产率42％。ESI-MS m/z 467.1(M+H) +.
方法7：
化合物QY-7-21的合成：
Figure PCTCN2021118212-appb-000075
2-氧代-2-(2-(2-苯基乙酰基乙酰基)肼基)醋酸乙酯(QY-7-21)：在100ml圆底烧瓶中 将苯乙酰肼(600mg，3.99mmol)溶解均匀于30ml二氯甲烷中，冰浴条件下缓慢滴入草酰氯单乙酯(600mg，4.39mmol)，将反应体系逐渐回至室温持续搅拌。LC-MS实时监测，3h后无原料峰，停止反应。加入蒸馏水淬灭反应，加10ml蒸馏水洗，二氯甲烷萃取(15ml*3),合并有机相，饱和食盐水洗涤(10ml*1)，无水硫酸钠干燥，旋转蒸发仪除去有机溶剂得白色固体粗产品可直接进行后续反应。ESI-MS m/z 251.0(M+H) +.
化合物QY-7-23的合成：
Figure PCTCN2021118212-appb-000076
5-苄基-1,3,4-噁二唑-2-羧酸乙酯(QY-7-23)：在30ml耐压瓶中将QY-7-21搅拌混匀于10ml二氯甲烷中，对甲基苯磺酰氯(760mg，3.99mmol)溶解于4ml二氯甲烷后滴加至反应液中，室温下持续搅拌，薄层色谱监测反应，继续搅拌6h后停止反应。转移至分液漏斗中，10ml蒸馏水洗，二氯甲烷萃取(10ml*3)，合并有机相，饱和氯化铵洗涤(5ml*2)，饱和氯化钠洗涤(5ml*2)，后处理得有机相经无水硫酸钠干燥，过滤并旋干除去二氯甲烷，硅胶柱层析分离纯化(石油醚：乙酸乙酯＝0-40％)得384mg黄褐色液体，两步反应产率41％。ESI-MS m/z 233.1(M+H) +.
化合物QY-7-28的合成：
Figure PCTCN2021118212-appb-000077
5-苄基-1,3,4-噁二唑-2-羧酸(QY-7-28)：将QY-7-23(56mg，0.24mmol)溶解于1ml甲醇中，转移至8ml耐压瓶，氢氧化锂一水合物(10mg，0.24mmol)溶解于0.5ml蒸馏水中，冰浴条件下滴加至反应液中，混合搅拌均匀后逐渐回至室温，LC-MS实时监测，1.5h后停止反应，旋转蒸发除去反应体系所有溶剂，滤膜过滤后经C18反相色谱柱分离纯化(水：乙腈＝0-30％)得33mg白色固体，产率67％。ESI-MS m/z 203.0(M+H) -.
化合物QY-7-32的合成：
Figure PCTCN2021118212-appb-000078
5-苄基-N-(4-(5-(甲基氨基甲酰基)-1H-苯并[d咪唑-1-基)苄基)-1,3,4-噁二唑-2-甲酰胺(QY-7-32)：QY-7-28(33mg，0.16mmol)溶解于盛有2ml二氯甲烷的8ml耐压瓶中，加入HATU(74mg，0.19mmol)，将QY-6-6(45mg，0.16mmol)加入搅拌均的反应液中；室温条件下滴加N,N-二异丙基乙胺(52mg，0.40mmol)，继续搅拌反应3.5h。LC-MS实时监测反应，待原料消耗完毕，将反应体系转移至分液漏斗，5ml蒸馏水洗，二氯甲烷萃取(5ml*3)，合并有机相，饱和氯化钠洗涤(5ml*2)，无水硫酸钠干燥有机相，过滤并旋蒸除去二氯甲烷，滤膜过滤后经C18反相色谱柱分离纯化(水：乙腈＝0-80％)得 15mg白色固体，产率21％。ESI-MS m/z 467.0(M+H) +.
方法8：
化合物QY-7-65的合成：
Figure PCTCN2021118212-appb-000079
N-甲基-1-(4-(2-oxo-2-(2-苯基吡咯烷基-1-基)乙基)苯基)-1H-苯并[d]咪唑-5-甲酰胺(QY-7-65)：QY-5-34(49mg，0.16mmol)溶解于盛有2ml二氯甲烷的8ml耐压瓶中，加入HATU(69mg，0.18mmol)，将QY-7-64(20mg，0.14mmol)加入搅拌均的反应液中；室温条件下滴加N,N-二异丙基乙胺(72mg，0.56mmol)，继续搅拌反应3h。LC-MS实时监测反应，待反应结束后将反应体系转移至分液漏斗，5ml蒸馏水洗，二氯甲烷萃取(5ml*3)，合并有机相，饱和氯化钠洗涤(5ml*2)，无水硫酸钠干燥有机相，旋蒸除去二氯甲烷，滤膜过滤后经C18反相色谱柱分离纯化(水：乙腈＝0-80％)得19mg黄褐色固体，产率31％。ESI-MS m/z 439.1(M+H) +.
方法9：
化合物QY-9-15的合成：
Figure PCTCN2021118212-appb-000080
1-苄基-1H-吡唑-3-羧酸乙酯(QY-9-15)：在250ml圆底烧瓶中将3-乙氧羰基吡唑(6.0g，42.8mmol)搅拌混匀于90ml乙腈中，加入碳酸钾(17.8g，128.4mmol)至反应液中，室温剧烈搅拌下将溴化苄(8.8g，51.4mmol)滴加至反应体系，LC-MS监测反应，继续搅拌8h后停止反应。硅藻土抽滤除去无机盐，旋干除去有机溶剂，硅胶柱层析分离纯化(石油醚：乙酸乙酯＝0-50％)得6.54g白色固体，产率66％。ESI-MS m/z 231.1(M+H) +.
化合物QY-9-19的合成：
Figure PCTCN2021118212-appb-000081
1-苄基-4-溴-1H-吡唑-3-羧酸乙酯(QY-9-19)：在250ml圆底烧瓶中将QY-9-15(6.4g，27.8mmol)溶解于100ml乙腈中，搅拌均匀后室温缓慢滴加液溴(6.7g，41.7mmol)至反应体系，搅拌过夜后停止反应。缓慢滴入3M硫代硫酸钠溶液淬灭反应，搅拌15min，旋蒸除去乙腈，乙酸乙酯萃取(20ml*3),合并有机相，硫代硫酸钠溶液洗涤(10ml*1)，饱和食盐水洗涤(10ml*1)，无水硫酸钠干燥，旋转蒸发仪除去乙酸乙酯，硅胶柱层析分离纯化(石油醚：乙酸乙酯＝0-40％)得5.17g白色固体，产率60％。ESI-MS m/z 309.0(M+H) +.
化合物QY-9-26的合成：
Figure PCTCN2021118212-appb-000082
乙基(E)-1-苄基-4-(2-乙氧基乙烯基)-1H-吡唑-3-羧酸酯(QY-9-26)：QY-9-19(2.50g，8.1mmol)溶解于30ml乙二醇二甲醚及5ml蒸馏水后加入150ml封管中，加入Pd(dppf)Cl2(0.59g，0.81mmol)，碳酸铯(5.80g，17.8mmol)，(E)-1-乙氧乙烯基-2-硼酸频那醇酯(3.20g，16.2mmol)溶解于10ml 1,2-二氯乙烷后滴加入剧烈搅拌的反应液中，氮气保护后逐渐加热至90℃反应过夜。待反应物消耗完毕，将反应体系转移至分液漏斗，80ml蒸馏水洗，乙酸乙酯萃取(40ml*3)，合并有机相，饱和氯化钠洗涤(15ml*2)，无水硫酸钠干燥有机相，旋蒸除去有机溶剂，硅胶柱层析分离纯化(石油醚：乙酸乙酯＝0-60％)得2.10g白色固体，产率87％。ESI-MS m/z 301.1(M+H) +.
化合物QY-9-28的合成：
Figure PCTCN2021118212-appb-000083
1-苄基-4-(2-乙基氧代乙基)-1H-吡唑-3-羧酸乙酯(QY-9-28)：QY-9-26(2.10g，7.0mmol)溶解于30ml四氢呋喃后加入100ml圆底烧瓶中，冰浴条件下滴加6M盐酸水溶液10ml至反应液中，搅拌2h后补加15ml 6M盐酸水溶液。LC-MS实时监测反应，待反应物消耗完毕后加入饱和碳酸氢钠溶液调节PH至弱碱性，将反应体系转移至分液漏斗，乙酸乙酯萃取(20ml*3)，合并有机相，饱和氯化钠洗涤(10ml*2)，无水硫酸钠干燥有机相，旋蒸除去乙酸乙酯，硅胶柱层析分离纯化(石油醚：乙酸乙酯＝0-50％)得800mg淡黄色透明液体，产率42％。ESI-MS m/z 273.1(M+H) +.
化合物QY-7-98的合成：
Figure PCTCN2021118212-appb-000084
1-苄基-4-(2-((4-(5-(甲基氨基甲酰基)-1H-苯并[d咪唑-1-基)苄基)氨基)乙基)-1H-吡唑-3-羧酸乙酯(QY-7-98):将QY-9-28(316mg，1.2mmol)混匀至6ml 1,2-二氯乙烷中，加至30ml耐压瓶搅拌；加入QY-6-6(270mg，0.96mmo)，冰醋酸(279mg，4.6mmol)搅拌20min后加入三乙酰氧基硼氢化钠(492mg，2.3mmol)，室温下搅拌过夜后原料消耗完毕。冰浴条件下滴加蒸馏水淬灭反应，滤膜过滤后经C18反相色谱柱分离纯化(水：乙腈＝0-80％)得128mg淡黄色固体，产率25％。ESI-MS m/z 537.1(M+H) +.
化合物QY-8-7的合成：
Figure PCTCN2021118212-appb-000085
(4-((2-苄基-7-氧代-2,4,5,7-四氢四氢-6H-吡唑并[3,4-c]吡啶-6-基)甲基)苯基)-N-甲基-1H-苯并[d]咪唑-5-甲酰胺(QY-8-7)：将QY-7-98(64mg，0.12mmol)混匀至7ml二甲苯中，转移至15ml耐压瓶搅拌；室温下缓慢滴加三甲基铝(0.36mmo)至反应液中，氮气保护后逐渐升温至120℃，搅拌过夜停止反应。待反应液缓缓回至室温后滴加蒸馏水淬灭反应，加入罗氏盐溶液搅拌30min，加入10ml乙酸乙酯稀释反应体系，转移至分液漏斗，乙酸乙酯萃取(10ml*3)，合并有机相，饱和氯化钠洗涤(10ml*2)，无水硫酸钠干燥，过滤并旋蒸除去有机溶剂，滤膜过滤后经高效液相色谱HPLC分离纯化(水：乙腈＝10-60％)得8mg白色固体，产率14％。ESI-MS m/z 491.1(M+H) +.
方法10：
化合物QY-8-15的合成：
Figure PCTCN2021118212-appb-000086
2-氧代-2-((2-氧代-3-苯丙基)氨基)醋酸乙酯(QY-8-15)：在30ml耐压瓶中将1-氨基-3-苯丙基-2-酮盐酸盐(500mg，2.7mmol)搅拌混匀于15ml二氯甲烷中，冰浴条件下将三乙胺(817mg，8.1mmol)滴加入反应体系，搅拌10min后缓慢滴入草酰氯单乙酯(734mg，5.4mmol)，逐渐回至室温后搅拌过夜。LC-MS监测原料消耗完毕后停止反应。转移至分液漏斗中，蒸馏水洗(10ml*2)，二氯甲烷萃取(15ml*3)，合并有机相，饱和氯化钠洗涤(5ml*2)，后处理得有机相经无水硫酸钠干燥，过滤并旋干除去二氯甲烷，硅胶柱层析分离纯化(石油醚：乙酸乙酯＝0-50％)得206mg黄褐色固体，产率31％。ESI-MS m/z 250.2(M+H) +.
化合物QY-8-25的合成：
Figure PCTCN2021118212-appb-000087
5-苄基噁唑-2-羧酸乙酯(QY-8-25)：在15ml耐压瓶中将QY-8-15(206mg，0.83mmol)溶解于4ml乙腈中，五氧化二磷(587mg，4.1mmol)混匀于4ml乙腈后滴加至反应液，剧烈搅拌并缓慢升温至70℃。LC-MS实时监测，反应3h后无原料峰，停止反应。待降至室温后将反应体系缓慢滴入零摄氏度饱和食盐水中，搅拌5min，乙酸乙酯萃取(10ml*3),合并有机相，饱和NaHCO3洗涤(5ml*1)，饱和食盐水洗涤(5ml*1)，无机盐干燥剂干燥，旋转蒸发仪除去有机溶剂，硅胶柱层析分离纯化(石油醚：乙酸乙酯＝0-30％)得90mg淡黄色透明液体，产率47％。ESI-MS m/z 232.0(M+H) +.
化合物QY-8-28的合成：
Figure PCTCN2021118212-appb-000088
5-苄基噁唑-2-羧酸(QY-8-28)：将QY-8-25(90mg，0.34mmol)溶解于2ml四氢呋喃中，转移至8ml耐压瓶，氢氧化锂一水合物(33mg，0.78mmol)溶解于0.5ml蒸馏水中，冰浴条件下滴加至反应液中，混合搅拌均匀后逐渐回至室温，LC-MS实时监测，6h后停止反应，旋转蒸发除去反应体系所有溶剂，粗产品可直接进行后续合成。ESI-MS m/z 202.1(M+H) -.
化合物QY-8-20的合成：
Figure PCTCN2021118212-appb-000089
(4-(4-氨基-7H-吡咯并[2,3-d]嘧啶-7-基)苄基)氨基甲酸叔丁酯(QY-8-20)：将N-Boc-4-碘苄胺(2.48g，7.5mmol)加入30ml耐压瓶中，15ml二甲基亚砜溶解均匀后，加入4-氨基-7H-吡咯(2,3-d)嘧啶(1.0g，7.5mmol)，碘化亚铜(711mg，3.7mmol)，碳酸铯(4.86g，14.9mmol)，4,7-二甲氧基-1,10菲洛琳(358mg，1.5mmol)，氮气保护后加热至100℃反应过夜。待反应物消耗完毕，滤膜过滤除去固体不溶物后C18反相色谱柱分离纯化(水：乙腈＝0-80％)得1.94g浅绿色油状液体，产率77％。ESI-MS m/z 340.1(M+H) +.
化合物QY-8-22的合成：
Figure PCTCN2021118212-appb-000090
7-(4-(氨基甲基)苯基)-7H-吡咯并[2,3-d]嘧啶-4-氨(QY-8-22)：将QY-8-20(1.94g，5.7mmol)混匀于20ml二氯甲烷中，转移至100ml圆底烧瓶中，室温条件下，滴加三氟乙酸2.0ml至反应液中，混合搅拌均匀。LC-MS实时监测，搅拌过夜后原料消耗完毕。每次加入20ml二氯甲烷,旋干移去有机溶剂混合物，重复4-6次，此步粗产物可不经分离纯化直接用于后续合成。ESI-MS m/z 240.1(M+H) +.
化合物QY-8-30的合成：
Figure PCTCN2021118212-appb-000091
N-(4-(4-氨基-7H-吡咯并[2,3-d]嘧啶-7-基)苄基)-5-苄基噁唑-2-甲酰胺(QY-8-30)： QY-8-28(0.39mmol)溶解于盛有4ml二氯甲烷的8ml耐压瓶中，加入HATU(177mg，0.47mmol)，将QY-8-22(220mg，0.62mmol)加入搅拌均的反应液中；室温条件下滴加N,N-二异丙基乙胺(200mg，1.6mmol)，继续搅拌反应6h。LC-MS实时监测反应，待反应结束后将反应体系转移至分液漏斗，10ml蒸馏水洗，二氯甲烷萃取(15ml*2)，合并有机相，饱和氯化钠洗涤(5ml*2)，无水硫酸钠干燥有机相，旋蒸除去二氯甲烷，滤膜过滤后经C18反相色谱柱分离纯化(水：乙腈＝0-80％)得38mg黄褐色油状液体，产率23％。ESI-MS m/z 425.2(M+H) +.
方法11：
化合物QY-8-33的合成：
Figure PCTCN2021118212-appb-000092
(2-甲基-1-氧代-1-(2-(对甲基苯基)吡咯烷-1-基)丙-2-基)氨基甲酸叔丁酯(QY-8-33)：N-Boc-2-甲基丙氨酸(320mg，1.6mmol)溶解于盛有8ml二氯甲烷的15ml耐压瓶中，加入HATU(652mg，1.7mmol)，将QY-7-64(230mg，1.4mmol)加入搅拌均的反应液中；室温条件下滴加N,N-二异丙基乙胺(810mg，6.3mmol)，室温下继续搅拌。LC-MS实时监测反应，5h后原料消耗完毕，将反应体系转移至分液漏斗，蒸馏水洗(10ml*3)，二氯甲烷萃取(15ml*3)，合并有机相，饱和氯化钠洗涤(10ml*2)，无水硫酸钠干燥有机相，旋蒸除去二氯甲烷，硅胶柱层析分离纯化(石油醚：乙酸乙酯＝0-70％)得202mg淡黄色固体，产率41％。ESI-MS m/z 247.1(M+H) +.
化合物QY-8-39的合成：
Figure PCTCN2021118212-appb-000093
3-氨基-2-甲基-1-(2-(对甲基苯基)吡咯烷-1-基)丙-1-酮(QY-8-39)：将QY-8-33(202mg，0.58mmol)混匀于4ml二氯甲烷中，转入15ml耐压瓶中，室温条件下，滴加三氟乙酸0.4ml至反应液中，混合搅拌均匀。LC-MS实时监测，3h后原料消耗完毕。将反应体系转入100ml圆底烧瓶中，每次加入10ml二氯甲烷,旋转蒸发除去有机溶剂混合物，重复3-4次，此步粗产物可不经分离纯化直接用于后续合成。ESI-MS m/z 247.1(M+H) +.
化合物QY-8-42的合成：
Figure PCTCN2021118212-appb-000094
N-甲基-1-(4-(2-((2-甲基-1-氧代-1-(2-(对甲基苯基)吡咯烷-1-基)丙-2-基)氨基)-2-氧代乙基)苯基)-1H-苯并[d]咪唑-5-甲酰胺(QY-8-42)：在8ml耐压瓶中将QY-8-39(0.29mmol)溶解于3ml二氯甲烷，加入HATU(221mg，0.58mmol)，QY-5-34(90mg， 0.29mmol)；室温下缓慢滴加N,N-二异丙基乙胺(150mg，1.2mmol)，继续搅拌反应5h。LC-MS实时监测反应，待反应物消耗完全，加入5ml二氯甲烷稀释反应液，转移至分液漏斗，10ml蒸馏水洗，二氯甲烷萃取(10ml*3)，合并有机相，饱和氯化钠洗涤(10ml*2)，无水硫酸钠干燥有机相，过滤并旋蒸除去二氯甲烷，滤膜过滤后经C18反相色谱柱分离纯化(水：乙腈＝0-80％)得48mg黄褐色油状液体，产率31％。ESI-MS m/z 538.1(M+H) +.
方法12：
化合物ZSQ-13-19的合成：
Figure PCTCN2021118212-appb-000095
2-乙氧基-2-亚氨醋酸乙酯(ZSQ-13-19):将氰基甲酸乙酯(9.9ml，100mmol)加入250ml圆底烧瓶中，与乙醇(23.0ml，400mmol)混合搅拌溶解均匀后将反应体系降至0℃，逐滴滴加乙酰氯(14.2ml，200mmol至反应液，滴加完毕后保持0℃继续搅拌过夜。反应完全后得到块状白色固体，抽滤得到粗产物可不经分离纯化直接进行下步反应。ESI-MS m/z 146.0(M+H) +.
化合物ZSQ-13-20的合成：
Figure PCTCN2021118212-appb-000096
(Z)-2-氨基-2-(2-(2-苯基乙酰基)亚肼基(hydrazono))醋酸乙酯(ZSQ-13-20)将ZSQ-13-19(7.26g，50mmol)加入250ml圆底烧瓶中，加入50ml乙醇混合搅拌溶解均匀后加入苯乙酰肼(7.51g，50mmol)，持续搅拌下加入40ml乙醚，室温搅拌过夜后停止反应，抽滤得到粗产物可不经分离纯化直接进行下步反应。ESI-MS m/z 250.1(M+H) +.
化合物QY-8-49的合成：
Figure PCTCN2021118212-appb-000097
1-乙酰基-5-苄基-1H-1,2,4-三唑-3-羧酸乙酯(QY-8-49):将化合物ZSQ(1.0g，4.0mmol)溶解于5ml醋酸酐中，在15ml耐压瓶中混合搅拌均匀后缓慢升温至140℃，剧烈搅拌3h。LC-MS监测至原料反应完，待反应体系回至室温后缓慢滴入饱和碳酸氢钠水溶液淬灭反应，搅拌15min后转移至分液漏斗，乙酸乙酯萃取(20ml*3)，合并有机相，饱和氯化钠洗涤(10ml*2)，无水硫酸钠干燥有机相，过滤并旋蒸除去乙酸乙酯，硅胶柱层析分离纯化(石油醚：乙酸乙酯＝0-40％)得280mg淡黄色透明油状液体，产率26％。ESI-MS m/z 274.1(M+H) +.
化合物QY-8-52的合成：
Figure PCTCN2021118212-appb-000098
5-苄基-1H-1,2,4-三唑-3-羧酸(QY-8-52)：将QY-8-52(280mg，1.0mmol)溶解于4ml四氢呋喃中，转移至8ml耐压瓶，氢氧化锂一水合物(147mg，3.5mmol)溶解于1ml蒸馏水中，冰浴条件下滴加至反应液中，混合搅拌均匀后逐渐回至室温，过夜搅拌后LC-MS监测原料反应完全，旋转蒸发除去反应体系所有溶剂，粗产品可不经纯化直接进行后续合成。ESI-MS m/z 203.9(M+H) +.
化合物QY-8-60的合成：
Figure PCTCN2021118212-appb-000099
N-(4-(4-氨基-7H-吡咯并[2,3-d]嘧啶-7-基)苄基)-5-苄基-1H-1,2,4-三唑-3-甲酰胺(QY-8-60)：在8ml耐压瓶中将QY-8-52(0.40mmol)溶解于3ml二氯甲烷中，向反应液中加入HATU(182mg，0.48mmol)，QY-8-22(155mg，0.44mmol)；室温下缓慢滴加N,N-二异丙基乙胺(258mg，2.0mmol)，继续搅拌反应4h。LC-MS监测至反应物消耗完全，停止反应，加入5ml二氯甲烷稀释反应液，转移至分液漏斗，10ml蒸馏水洗，二氯甲烷萃取(10ml*3)，合并有机相，饱和氯化钠洗涤(10ml*2)，无水硫酸钠干燥有机相，过滤并旋蒸除去二氯甲烷，滤膜过滤后经C18反相色谱柱分离纯化(水：乙腈＝0-80％)得66mg黄褐色液体，产率39％。ESI-MS m/z 425.2(M+H) +.
方法13：
化合物QY-8-50的合成：
Figure PCTCN2021118212-appb-000100
乙基2H-四唑-5-羧酸酯(QY-8-50)：将化合物氰基甲酸乙酯(1.0g，10.0mmol)溶解于12ml吡啶中，在75ml封管中混合搅拌均匀，室温条件下缓慢滴入三氟乙酸1.76ml，搅拌10min后加入叠氮化钠(700mg，10.8mmol)，缓慢升温至60℃，24h后停止反应。硅藻土抽滤除去叠氮化钠，乙酸乙酯(10ml*3)冲洗硅藻土，得有机相经旋转蒸发仪除去，得到粗产物不经分离纯化可直接进行下一步。ESI-MS m/z 143.0(M+H) +.
化合物QY-8-55的合成：
Figure PCTCN2021118212-appb-000101
乙基2-苄基-2H-四唑-5-羧酸酯(QY-8-55)：将QY-8-50(10.0mmol)搅拌混匀于 10ml N,N-二甲基甲酰胺中，加入碳酸钾(4.2g，30.0mmol)至反应液中，室温剧烈搅拌下将溴化苄(1.7g，10.0mmol)滴加至反应体系，搅拌过夜后停止反应。硅藻土抽滤除去碳酸钾，旋干除去有机溶剂，滤膜过滤后经C18反相色谱柱分离纯化(水：乙腈＝0-100％)得550mg黄褐色油状液体，两步反应产率24％。ESI-MS m/z 233.1(M+H) +.
化合物QY-8-56的合成：
Figure PCTCN2021118212-appb-000102
2-苄基-2H-四唑-5-羧酸(QY-8-56)：将QY-8-55(100mg，0.43mmol)溶解于3ml四氢呋喃中，转移至8ml耐压瓶，氢氧化锂一水合物(27mg，0.65mmol)溶解于0.8ml蒸馏水中，室温下滴加至反应液中，LC-MS实时监测反应进程，2h后原料消耗完毕，旋转蒸发除去反应体系所有溶剂，粗产品可不经纯化直接进行后续合成。ESI-MS m/z 205.1(M+H) +.
化合物QY-8-58的合成：
Figure PCTCN2021118212-appb-000103
N-(4-(4-氨基-7H-吡咯并[2,3-d]嘧啶-7-基)苄基)-2-苄基-2H-四唑-5-甲酰胺(QY-8-58)：在8ml耐压瓶中将QY-8-56(0.40mmol)溶解于3ml二氯甲烷，加入HATU(182mg，0.48mmol)，QY-8-22(141mg，0.40mmol)；室温下缓慢滴加N,N-二异丙基乙胺(259mg，2.0mmol)，继续搅拌反应。LC-MS实时监测反应，4h后原料消耗完毕，加入5ml二氯甲烷稀释反应液，转移至分液漏斗，10ml蒸馏水洗，二氯甲烷萃取(10ml*3)，合并有机相，饱和氯化钠洗涤(10ml*2)，无水硫酸钠干燥有机相，过滤并旋蒸除去二氯甲烷，滤膜过滤后经C18反相色谱柱分离纯化(水：乙腈＝0-60％)得140mg黄褐色固体，产率82％。ESI-MS m/z 426.1(M+H) +.
方法14：
化合物QY-8-29的合成：
Figure PCTCN2021118212-appb-000104
(4-(6-氨基-9H-嘌呤-9-基)苄基)氨基甲酸叔丁基酯(QY-8-29):将N-Boc-4-碘苄胺(330mg，1.0mmol)加入15ml耐压瓶中，3ml二甲基亚砜溶解均匀后，加入腺嘌呤(135mg，1.0mmol)，碘化亚铜(95mg，0.5mmol)，碳酸铯(652mg，2.0mmol)，4,7-二甲氧基-1,10菲洛琳(48mg，0.,2mmol)，氮气保护后加热至100℃反应过夜。待反应物消耗完毕，滤膜过滤除去固体不溶物后C18反相色谱柱分离纯化(水：乙腈＝0-80％)得287mg 绿白色固体，产率84％。ESI-MS m/z 341.2(M+H) +.
化合物QY-8-36的合成：
Figure PCTCN2021118212-appb-000105
9-(4-(氨基甲基)苯基)-9H-嘌呤-6氨(QY-8-36):将QY-8-29(286g，0.84mmol)混匀于3ml二氯甲烷中，在15ml耐压瓶中瓶中室温滴加三氟乙酸0.5ml至反应液中，混合搅拌均匀。LC-MS实时监测，搅拌过夜后原料消耗完毕。每次加入20ml二氯甲烷,旋干移去有机溶剂混合物，重复3-4次，此步粗产物可不经分离纯化直接用于后续合成。ESI-MS m/z241.2(M+H) +.
化合物QY-8-43的合成：
Figure PCTCN2021118212-appb-000106
N-(4-(6-氨基-9H-嘌呤-9-基)苄基)-5-苄基-1-甲基-1H-吡唑-3-甲酰胺(QY-8-43):在8ml耐压瓶中将QY-6-103B(150mg，0.69mmol)溶解于8ml二氯甲烷中，加入HATU(316mg，0.83mmol)，将QY-8-36(166mg，0.69mmol)加入搅拌均的反应液中；室温条件下滴加N,N-二异丙基乙胺(313mg，2.43mmol)，继续搅拌反应4h。LC-MS实时监测反应，待反应物消耗完全，转移至分液漏斗，10ml蒸馏水洗，二氯甲烷萃取(15ml*3)，合并有机相，饱和氯化钠洗涤(10ml*2)，无水硫酸钠干燥有机相，过滤并旋蒸除去二氯甲烷，硅胶柱层析分离纯化(二氯甲烷：甲醇＝0-25％)得142mg白色固体，产率47％。ESI-MS m/z 439.1(M+H) +.
方法15：
化合物QY-9-69的合成:
Figure PCTCN2021118212-appb-000107
O-N-(4-(4-氨基-7H-吡咯并[2,3-d]嘧啶-7-基)苄基)-2-(3-(三氟甲氧基)苯基)乙酰胺(QY-9-69):在8ml耐压瓶中将QY-8-22(59mg，0.25mmol)溶解于3ml二氯甲烷中，加入HATU(125mg，0.33mmol)，将3-三氟甲氧基苯乙酸(60mg，0.25mmol)加入搅拌均的反应液中；室温条件下滴加N,N-二异丙基乙胺(129mg，1.0mmol)，继续搅拌反应3.5h。LC-MS实时监测反应，待反应物消耗完全，转移至分液漏斗，5ml蒸馏水洗，二氯甲烷萃取(5ml*3)，合并有机相，饱和氯化钠洗涤(5ml*2)，无水硫酸钠干燥有机相，过滤并旋蒸除去二氯甲烷，硅胶柱层析分离纯化(二氯甲烷：甲醇＝0-30％)得88mg淡黄色油状液体，产率80.0％。ESI-MS m/z 442.0(M+H) +.
方法16：
化合物QY-9-10的合成:
Figure PCTCN2021118212-appb-000108
2-(2-氟吡啶-3-基)-2-氧代醋酸乙酯(QY-9-10):将2-氟吡啶(5.0g，51.5mmol)加入250ml圆底烧瓶中,加入120ml四氢呋喃后溶解搅拌均匀，氮气置换保护后将反应体系转移至-78℃下，缓慢滴加二异丙基氨基锂(30.4ml，61.8mmol)至反应液中，滴加完毕后加入草酸二乙酯(9.0g，61.8mmol)，逐渐回至室温后继续搅拌2h，LC-MS实时监测反应至反应物消耗完全，加入饱和氯化铵淬灭反应，将反应体系转至分液漏斗，乙酸乙酯萃取(20ml*3)，合并有机相，饱和氯化钠洗涤(15ml*2)，无水硫酸钠干燥有机相，过滤并旋蒸除去乙酸乙酯，硅胶柱层析分离纯化(石油醚：乙酸乙酯＝0-40％)得3.54g亮黄色油状液体，产率34.9％。ESI-MS m/z 198.0(M+H) +.
化合物QY-9-18的合成:
Figure PCTCN2021118212-appb-000109
1H-吡唑并[3,4-b]吡啶-3-羧酸乙酯(QY-9-18)：将QY-9-10(2.87g，14.6mmol)加入100ml圆底烧瓶中,加入25ml N-甲基吡咯烷酮后溶解搅拌均匀，冰浴条件下缓慢滴加水合肼(0.87g，17.5mmol)至反应液中，保持0℃搅拌20min，逐渐回至室温后加热至80℃搅拌过夜，LC-MS监测反应物消耗完全，加入蒸馏水，滤膜过滤后经C18反相色谱柱分离纯化(水：乙腈＝0-80％)得1.37g黄红色固体，产率49％。ESI-MS m/z 192.0(M+H) +.
化合物QY-9-21的合成:
Figure PCTCN2021118212-appb-000110
5-溴-1H-吡唑并[3,4-b]吡啶e-3-羧酸乙酯(QY-9-21)：将QY-9-18(1.37g，7.17mmol)与乙酸钠(3.53g，43.0mmol)混匀至25ml冰醋酸中，加至100ml圆底烧瓶中搅拌均匀，室温下逐滴加入液溴(3.43g，21.5mmol),加热至100℃后搅拌8h，LC-MS监测反应物消耗完全，逐渐回至室温后停止反应。加入3M硫代硫酸钠溶液淬灭反应，转移至分液漏斗，乙酸乙酯萃取(15ml*3)，合并有机相，蒸馏水洗涤(10ml*2)，饱和食盐水洗涤(10ml*2)，无水硫酸钠干燥，过滤并旋干除去乙酸乙酯，硅胶柱层析分离纯化(石油醚：乙酸乙酯＝0-25％)得0.26g白色固体，产率13.4％。ESI-MS m/z 269.9(M+H) +.
化合物QY-9-29的合成:
Figure PCTCN2021118212-appb-000111
5-溴-1-(四氢-2H-吡喃-2-基)-1H-吡唑并[3,4-b]吡啶e-3-羧酸乙酯(QY-9-29)：QY-9-21(255mg，0.94mmol)溶解于7ml二氯甲烷后转入15ml耐压瓶中，加入3,4-二氢-2H-吡喃(159mg，1.89mmol)，将对甲苯磺酸吡啶(48mg，0.19mmol)加入搅拌均的反应液中；加热至60℃持续搅拌5h，LC-MS实时监测，待反应物消耗完毕，直接旋干除去二氯甲烷，硅胶柱层析分离纯化(石油醚：乙酸乙酯＝0-15％)得269mg白色固体，产率80.8％。ESI-MS m/z 354.0(M+H) +.
化合物QY-9-51的合成:
Figure PCTCN2021118212-appb-000112
5-碘-1-(四氢-2H-吡喃-2-基)-1H-吡唑并[3,4-b]吡啶e-3-羧酸乙酯(QY-9-51)：将QY-9-29(390mg，1.10mmol)加入15ml耐压瓶中，5ml 1,4-二氧六环溶解均匀后，加入碘化钠(436mg，2.91mmol)，碘化亚铜(34mg，0.18mmol)，(1S,2S)-(+)-N,N'-二甲基-1,2-环己二胺(38mg，0.27mmol)，加热至130℃，微波反应3h，LC-MS监测反应原料消耗完全。滤膜过滤除去固体不溶物后C18反相色谱柱分离纯化(水：乙腈＝5-100％)得359mg黄褐色固体，产率81.3％。ESI-MS m/z 402.0(M+H) +.
化合物QY-9-57的合成:
Figure PCTCN2021118212-appb-000113
5-(per氟乙基)-1-(四氢-2H-吡喃-2-基)-1H-吡唑并[3,4-b]吡啶-3-羧酸乙酯(QY-9-57)：将QY-9-51(316mg，0.79mmol)加入15ml耐压瓶中，4ml二甲基亚砜溶解均匀，加入(五氟乙基)三甲基硅烷(604mg，3.14mmol)，碘化亚铜(600mg，3.15mmol)，搅拌均匀后缓慢加热至80℃反应过夜，LC-MS监测反应原料消耗完全。滤膜过滤除去固体不溶物后C18反相色谱柱分离纯化(水：乙腈＝5-100％)得106mg无色透明油状液体，产率34.1％。ESI-MS m/z 394.1(M+H) +.
化合物QY-9-74的合成:
Figure PCTCN2021118212-appb-000114
5-(全氟乙基)-1-(四氢-2H-吡喃-2-基)-1H-吡唑并[3,4-b]吡啶e-3-羧酸(QY-9-74)：将QY-9-57(46mg，0.12mmol)溶解于2ml四氢呋喃中，转移至8ml耐压瓶，氢氧化锂一水合物(24mg，0.47mmol)溶解于0.5ml蒸馏水中，冰浴条件下滴加至反应液中，混合搅拌均匀后逐渐回至室温，LC-MS监测，搅拌过夜后反应完全；旋转蒸发除去四氢呋喃与水后，油泵抽干不经分离纯化直接进行下一步反应。ESI-MS m/z 364.1(M+H) -.
化合物QY-9-76的合成:
Figure PCTCN2021118212-appb-000115
N-(4-(6-氨基-9H-嘌呤-9-基)苄基)-5-(全氟乙基)-1-(四氢-2H-吡喃-2-基)-1H-吡唑并[3,4-b]吡啶e-3-甲酰胺(QY-9-76)：QY-9-74(0.12mmol)在8ml耐压瓶内溶解于3ml二氯甲烷中，加入HATU(54mg，0.14mmol)与QY-8-36(31mg，0.13mmol)，室温下滴加N,N-二异丙基乙胺(60mg，0.47mmol)，室温条件下持续搅拌。LC-MS实时监测，待反应完成后，加入5ml二氯甲烷稀释反应液，转移至分液漏斗中，5ml蒸馏水洗，二氯甲烷萃取(5ml*3)，合并有机相，饱和氯化钠洗(5ml*2)，后处理得有机相经无水硫酸钠干燥，过滤并旋干除去二氯甲烷，硅胶柱层析分离纯化(二氯甲烷：甲醇(氨)＝0-20％)得52mg黄褐色固体，产率76％。ESI-MS m/z 588.2(M+H) +.
化合物QY-9-77的合成:
Figure PCTCN2021118212-appb-000116
N-(4-(6-氨基-9H-嘌呤-9-基)苄基)-5-(全氟乙基)-1H-吡唑并[3,4-b]吡啶e-3-甲酰胺(QY-9-77)：将QY-9-76(52mg，0.09mmol)混匀于2ml二氯甲烷中，转移至8ml耐压瓶中，室温条件下，滴加三氟乙酸0.4ml至反应液中，混合搅拌均匀。LC-MS实时监测，1.5h后原料消耗完毕，转移至圆底烧瓶，加入10ml二氯甲烷,旋干移去有机溶剂混合物，重复3-4次，经HPLC分离纯化，得38mg白色固体，产率87％。ESI-MS m/z 504.1(M+H) +.
方法17：
化合物QY-10-21的合成:
Figure PCTCN2021118212-appb-000117
N-(4-(4-氨基-7H-吡咯并[2,3-d]嘧啶-7-基)苄基)-5-苄基-1-甲基-1H-吡唑-3-甲酰胺(QY-10-21)：在100ml圆底烧瓶中将QY-6-103B(271mg，1.25mmol)溶解于15ml二氯甲烷中，加入HATU(570mg，1.50mmol)，将QY-8-22(300mg，1.25mmol)加入搅拌均的反应液中；室温条件下滴加N,N-二异丙基乙胺(645mg，5.0mmol)，继续搅拌反应3.5h。LC-MS实时监测反应至底物消耗完全，转移至分液漏斗，10ml蒸馏水洗，二氯甲烷萃取(15ml*3)，合并有机相，饱和氯化钠洗涤(10ml*2)，无水硫酸钠干燥有机相，过滤并旋蒸除去二氯甲烷，硅胶柱层析分离纯化(二氯甲烷：甲醇(氨)＝0-25％)得344mg淡黄色油状液体，产率63％。ESI-MS m/z 438.2(M+H) +.
方法18：
化合物QY-10-47的合成:
Figure PCTCN2021118212-appb-000118
5-苄基-1-甲基-1H-吡唑-4-羧酸乙酯(QY-10-47)：75ml封管中，依次加入15ml甲苯，1-甲基吡唑-4-羧酸乙酯(960mg，6.23mmol)，搅拌均匀后加入醋酸钯(139mg，0.62mmol)，三苯基膦(327mg，1.25mmol)，碳酸钾(2.15g，15.6mmol)，特戊酸(191mg，1.87mmol)，滴入氯化苄(946mg，7.47mmol)，氮气保护后加热至100℃反应过夜。LC-MS监测反应，反应原料消耗完毕，加入乙酸乙酯稀释反应液，转移至分液漏斗后加入蒸馏水洗涤，乙酸乙酯萃取(10ml*3)，合并有机相，饱和氯化钠洗涤(10ml*2)，无水硫酸钠干燥有机相，过滤并旋蒸除去有机溶剂，硅胶柱层析分离纯化(石油醚：乙酸乙酯＝0-40％)得190mg淡黄色油状液体，产率12.5％。ESI-MS m/z 245.1(M+H) +.
化合物QY-10-56的合成:
Figure PCTCN2021118212-appb-000119
5-苄基-1-甲基-1H-吡唑-4-羧酸(QY-10-56)：将QY-10-47(190mg，0.78mmol)溶解于5ml四氢呋喃中，转移至15ml耐压瓶，氢氧化锂一水合物(130mg，3.11mmol)溶解于2ml蒸馏水中，冰浴条件下滴加至反应液中，混合搅拌均匀后逐渐回至室温，LC-MS实时监测，搅拌过夜反应完全，转移至100ml圆底烧瓶中，旋转蒸发除去有机溶剂和水，油泵抽干不经分离纯化直接进行下步反应。ESI-MS m/z 215.1(M+H) -.
化合物QY-10-39的合成:
Figure PCTCN2021118212-appb-000120
N-(4-(4-氨基-7H-吡咯并[2,3-d]嘧啶-7-基)苄基)-5-苄基-1-甲基-1H-吡唑-4-甲酰胺(QY-10-39)：QY-10-56(0.31mmol)在8ml耐压瓶内溶解于3ml二氯甲烷中，加入HATU(141mg，0.37mmol)，QY-8-22(74mg，0.31mmol)至反应液中；室温下滴加N,N-二异丙基乙胺(160mg，1.24mmol)。LC-MS实时监测，4h后原料消耗完毕，加入10ml二氯甲烷稀释反应液，转移至分液漏斗中，5ml蒸馏水洗，二氯甲烷萃取(10ml*3)，合并有机相，饱和氯化钠洗(5ml*2)，后处理得有机相经无水硫酸钠干燥，过滤并旋干除去二氯甲烷，硅胶柱层析分离纯化(二氯甲烷：甲醇(氨)＝0-20％)得89mg白色固体，产率66％。ESI-MS m/z 438.2(M+H) +.
方法19：
化合物QY-12-88的合成:
Figure PCTCN2021118212-appb-000121
1-甲基-1H-1,2,3-三唑-4-羧酸甲酯(QY-12-88)：在100ml圆底烧瓶中将1,2,3-三氮唑-4-甲酸甲酯(1.27g，10.0mmol)溶解于20mlN,N-二甲基甲酰胺中，室温加入碳酸钾(0.83g，6.0mmol)，冰浴条件下滴加碘甲烷(1.50g，10.5mmol)至反应液中，搅拌1h后逐渐回至室温继续搅拌16h。LC-MS实时监测底物消耗完全，停止反应。旋蒸除去有机溶剂后加二氯甲烷稀释反应液，转移至分液漏斗，蒸馏水洗(10ml*3)，二氯甲烷萃取(15ml*3)，合并有机相，饱和氯化钠洗涤(10ml*2)，无水硫酸钠干燥有机相，过滤并旋蒸除去二氯甲烷，硅胶柱层析分离纯化(石油醚：乙酸乙酯＝10-90％)得305mg白色粉末状固体，产率22％。ESI-MS m/z 142.1(M+H) +.
化合物QY-12-95的合成:
Figure PCTCN2021118212-appb-000122
5-苄基-1-甲基-1H-1,2,3-三唑-4-羧酸甲酯(QY-12-95)：15ml耐压瓶中，加入5ml甲苯，QY-12-88(305mg，2.16mmol)，搅拌均匀后加入醋酸钯(24mg，0.11mmol)，三苯基膦(113mg，0.43mmol)，碳酸钾(746mg，5.40mmol)，特戊酸(66mg，0.65mmol)，滴入氯化苄(274mg，2.16mmol)，氮气保护后加热至100℃反应过夜。待反应原料消耗完毕，旋蒸除去甲苯，乙酸乙酯稀释反应液，硅藻土抽滤后旋蒸除去有机溶剂，硅胶柱层析分离纯化(石油醚：乙酸乙酯＝0-80％)得173mg淡黄色透明油状液体，产率35％。ESI-MS m/z 232.1(M+H) +.
化合物QY-13-15的合成:
Figure PCTCN2021118212-appb-000123
5-苄基-1-甲基-1H-1,2,3-三唑-4-羧酸(QY-13-15):将QY-12-95(173mg，0.75mmol)溶解于3ml四氢呋喃中，转移至8ml耐压瓶，氢氧化锂一水合物(63mg，1.50mmol)溶解于0.8ml蒸馏水中，室温下滴加至反应液中，LC-MS实时监测反应进程，5h后原料消耗完毕，旋转蒸发除去反应体系所有溶剂，粗产品可不经纯化直接进行后续合成。ESI-MS m/z 218.1(M+H) +.
化合物QY-13-17的合成:
Figure PCTCN2021118212-appb-000124
苄基-N-(4-碘苄基)-1-甲基-1H-1,2,3-三唑-4-甲酰胺(QY-13-17):QY-13-15 (0.75mmol)在15ml耐压瓶内溶解于5ml二氯甲烷中，加入HATU(341mg，0.90mmol)与4-碘苄胺(174mg，0.75mmol)，室温下滴加N,N-二异丙基乙胺(289mg，2.24mmol)，室温条件下持续搅拌。LC-MS实时监测，待反应完成后，加入15ml二氯甲烷稀释反应液，转移至分液漏斗中，10ml蒸馏水洗，二氯甲烷萃取(10ml*3)，合并有机相，饱和氯化钠洗(10ml*2)，后处理得有机相经无水硫酸钠干燥，过滤并旋干除去二氯甲烷，硅胶柱层析分离纯化(石油醚：乙酸乙酯＝10-100％)得153mg白色泡沫固体，产率47％。ESI-MS m/z 433.1(M+H) +.
化合物QY-13-19的合成:
Figure PCTCN2021118212-appb-000125
N-(4-(6-氨基-9H-嘌呤-9-基)苄基)-5-苄基-1-甲基-1H-1,2,3-三唑-4-甲酰胺(QY-13-19):将QY-13-17(153mg，0.35mmol)加入8ml耐压瓶中，3ml二甲基亚砜溶解均匀后，加入腺嘌呤(57mg，0.42mmol)，碘化亚铜(34mg，0.18mmol)，碳酸铯(346mg，1.1mmol)，4,7-二甲氧基-1,10菲洛琳(17mg，0.07mmol)，氮气保护后加热至100℃反应过夜。待反应物消耗完毕，滤膜过滤除去固体不溶物后C18反相色谱柱分离纯化(水：乙腈＝10-100％)得55mg白色固体，产率35％。ESI-MS m/z 440.1(M+H) +.
方法20：
化合物QY-13-10的合成:
Figure PCTCN2021118212-appb-000126
(S)-(1-(4-溴苯基)乙基)氨基甲酸叔丁酯(QY-13-10):将(S)-1-(4-溴苯)乙胺(2.20g，11.0mmol)溶解于50ml四氢呋喃中，转移至200ml圆底烧瓶中，将二碳酸二叔丁酯(2.18g，10.0mmol)缓慢滴加至反应液中，室温搅拌过夜，监测反应完全，一并旋干反应体系，硅胶柱层析分离纯化(石油醚：乙酸乙酯＝0-40％)得2.50g白色固体，产率83％。ESI-MS m/z 300.1(M+H) +.
化合物QY-13-22的合成:
Figure PCTCN2021118212-appb-000127
(S)-(1-(4-碘苯基)乙基)氨基甲酸叔丁酯(QY-13-22):30ml耐压瓶中，依次加入10ml 1,4-二氧六环，QY-13-10(1.50g，5.0mmol)，搅拌溶解均匀后加入碘化钠(1.50g，10.0mmol)，碘化亚铜(48mg，0.25mmol)，(1S,2S)-(+)-N,N'-二甲基-1,2-环己二胺(71mg，0.50mmol)，氮气保护后加热至110℃搅拌24h。待反应物消耗完毕，滤膜过滤除去固体不溶物后旋走有机溶剂，硅胶柱层析分离纯化(二氯甲烷：乙酸乙酯＝0-30％)得1.62g黄白色固体，产率93％。ESI-MS m/z 348.2(M+H) +
化合物QY-13-23的合成:
Figure PCTCN2021118212-appb-000128
(S)-(1-(4-(6-氨基-9H-嘌呤-9-基)苯基)乙基)氨基甲酸叔丁酯(QY-13-23):
将QY-13-22(1.62g，4.66mmol)加入40ml耐压瓶中，25ml二甲基亚砜溶解均匀后，加入腺嘌呤(755mg，5.59mmol)，碘化亚铜(444mg，2.33mmol)，碳酸铯(2.39g，7.34mmol)，4,7-二甲氧基-1,10菲洛琳(112mg，0.47mmol)，氮气保护后加热至100℃反应过夜。待反应物消耗完毕，滤膜过滤除去固体不溶物后C18反相色谱柱分离纯化(水：乙腈＝10-100％)得646mg黄白色固体，产率39％。ESI-MS m/z 355.1(M+H) +.
化合物QY-13-28的合成:
Figure PCTCN2021118212-appb-000129
(S)-9-(4-(1-氨乙基)苯基)-9H-嘌呤-6-胺(QY-13-28)：将QY-13-23(1.45g，4.08mmol)混匀于20ml二氯甲烷中，转移至100ml圆底烧瓶中，室温条件下，滴加三氟乙酸2.0ml至反应液中，混合搅拌均匀。LC-MS实时监测，4h后原料消耗完毕，加入20ml二氯甲烷,旋干移去有机溶剂混合物，重复3-4次，所得产物可不经纯化直接进行下一步反应。ESI-MS m/z 255.2(M+H) +.
化合物QY-12-67的合成:
Figure PCTCN2021118212-appb-000130
甲基肼盐酸盐(QY-12-67):称取1-BOC-2-甲基肼(7.31g，50.0mmol)转入100ml圆底烧瓶中，冰浴条件下缓慢加入30ml二氧六环的氯化氢溶液，保持冰浴搅拌30min，继续室温搅拌过夜，待原料消耗完毕，停止反应。加入二氯甲烷30ml稀释反应液，旋蒸除去所有有机溶剂，得乳白色固体粉末，可不经分离纯化直接进行下步反应。
化合物QY-12-69的合成:
Figure PCTCN2021118212-appb-000131
1-亚苄基-2-甲基肼(QY-12-69):将苯甲醛(1.38g，13.0mmol)加入100ml圆底烧瓶中,加入24ml四氢呋喃后溶解搅拌均匀，室温下加入QY-12-67(1.60g，19.5mmol)至反应液中，加热至70℃搅拌4h，薄层色谱监测反应原料消耗完全，停止反应，滤膜过滤除去滤渣，所得粗产物直接进行下步反应。ESI-MS m/z 135.1(M+H) +.
化合物QY-12-66的合成:
Figure PCTCN2021118212-appb-000132
(E)-2-氰基-3-羟基-4-苯基丁-2-烯酸乙酯(QY-12-66):在250ml圆底烧瓶中将氰基乙酸乙酯(5.65g，50.0mmol)溶解均匀于100ml四氢呋喃中，冰浴条件下缓慢分批加入氢化钠(60％wt)(2.0g，50.0mmol)，将反应体系逐渐回至室温搅拌10min后将苯乙酰氯(6.44g，42.0mmol)混匀于20ml四氢呋喃中滴入上述反应液。薄层色谱监测，4.5h后无原料剩余，停止反应。加入蒸馏水淬灭反应，旋蒸除去四氢呋喃后加入50ml二氯甲烷稀释反应液，加20ml蒸馏水洗，二氯甲烷萃取(20ml*3),合并有机相，饱和食盐水洗涤(10ml*1)，无水硫酸钠干燥，旋转蒸发仪除去有机溶剂后，经硅胶柱层析分离纯化(石油醚：乙酸乙酯＝10-30％)得4.16g橙黄色油状液体，产率43％。
化合物QY-12-70的合成:
Figure PCTCN2021118212-appb-000133
(E)-3-氯-2-氰基-4-苯基丁-2-烯酸乙酯(QY-12-70):将QY-12-66(2.08g，9.0mmol)溶解均匀于10ml三氯氧磷中，冰浴条件下缓慢滴加三乙胺(1.37g，13.5mmol)至反应液，将反应体系逐渐回至室温搅拌5min后加热至88℃。薄层色谱监测，1h后无原料剩余，停止反应。将反应体系缓慢滴入冰水中淬灭反应，转移至分液漏斗后加入乙酸乙酯萃取(40ml*3),合并有机相，1N稀盐酸洗涤(10ml*2)，饱和食盐水洗涤(10ml*1)，无水硫酸钠干燥，旋转蒸发仪除去有机溶剂后，经硅胶柱层析分离纯化(石油醚：乙酸乙酯＝0-35％)得950mg淡黄色油状液体，产率42％。ESI-MS m/z 250.2(M+H) +.
化合物QY-12-73的合成:
Figure PCTCN2021118212-appb-000134
3-氨基-5-苄基-1-甲基-1H-吡唑-4-羧酸乙酯(QY-12-73):将QY-12-69(1.41g，10.5mmol)与QY-12-70(1.86g，7.5mmol)溶解均匀于21ml四氢呋喃中，室温搅拌4h 后旋蒸除去有机溶剂；加入21ml乙醇搅拌混匀反应体系，随后加入2.4ml 12N浓盐酸，加热回流搅拌过夜，LC-MS监测反应完毕，停止反应。旋蒸除去所有溶剂，经硅胶柱层析分离纯化(二氯甲烷：甲醇(氨)＝0-20％)得1.05g黄褐色泡沫固体，产率54％。ESI-MS m/z 260.2(M+H) +.
化合物QY-12-78的合成:
Figure PCTCN2021118212-appb-000135
5-苄基-3-碘-1-甲基-1H-吡唑-4-羧酸乙酯(QY-12-78):将QY-12-73(350mg，1.35mmol)溶解均匀于3ml乙腈与6ml蒸馏水中，冰浴条件下滴入浓硫酸(265mg，2.7mmol)，搅拌10min后将亚硝酸钠(102mg，1.48mmol)溶解于3ml蒸馏水中缓慢滴入上述反应体系，冰浴搅拌5min后缓慢回至室温搅拌1h；将碘化钾(672mg，4.05mmol)溶解于3ml蒸馏水中，冰浴条件下向反应体系滴加碘化钾水溶液，保持0℃搅拌1h，LC-MS监测反应完毕，停止反应。加入硫代硫酸钠溶液淬灭反应，后转移至分液漏斗加入乙酸乙酯萃取(20ml*3),合并有机相，蒸馏水洗涤(10ml*1)，饱和食盐水洗涤(10ml*2)，无水硫酸钠干燥，旋转蒸发除去有机溶剂后，硅胶柱层析分离纯化(石油醚：乙酸乙酯＝0-50％)得150mg黄色固体，产率30％。ESI-MS m/z 371.1(M+H) +.
化合物QY-12-79的合成:
Figure PCTCN2021118212-appb-000136
ethyl(E)-5-苄基-3-(2-乙氧基乙烯基)-1-甲基-1H-吡唑-4-羧酸(QY-12-79):
QY-12-78(150mg，0.41mmol)溶解于2ml乙二醇二甲醚及0.4ml蒸馏水后加入8ml耐压瓶中，加入Pd(dppf)Cl2(30mg，0.04mmol)，碳酸铯(294mg，0.90mmol)，(E)-1-乙氧乙烯基-2-硼酸频那醇酯(161mg，0.81mmol)，混合搅拌均匀后，氮气保护反应体系并逐渐加热至90℃反应过夜。LC-MS监测反应完成，停止反应，滤膜过滤反应液后经C18反相色谱柱分离纯化(水：乙腈＝10-100％)得112mg黄褐色固体，产率87％。ESI-MS m/z 315.2(M+H) +.
化合物QY-12-84的合成:
Figure PCTCN2021118212-appb-000137
5-苄基-1-甲基-3-(2-氧乙基)-1H-吡唑-4-羧酸乙酯(QY-12-84):
QY-12-79(112g，0.36mmol)溶解于2.0ml四氢呋喃后加入8ml耐压瓶中，冰浴条件下滴加6M盐酸水溶液1.5ml至反应液中，室温搅拌过夜。LC-MS监测至反应完全后 加入饱和碳酸氢钠溶液调节PH至弱碱性，将反应体系转移至分液漏斗，乙酸乙酯萃取(5ml*3)，合并有机相，饱和氯化钠洗涤(5ml*2)，无水硫酸钠干燥有机相，旋蒸除去有机溶剂，得粗产品直接进行下步反应。ESI-MS m/z 287.1(M+H) +.
化合物QY-13-32的合成:
Figure PCTCN2021118212-appb-000138
(S)-3-(2-((1-(4-(6-氨基-9H-嘌呤-9-基)苯基)ethyl)氨基)乙基)-5-苄基-1-甲基-1H-吡唑-4-羧酸乙酯(QY-13-32):将QY-12-84(1.7mmol)混匀至10ml 1,2-二氯乙烷中，加至30ml耐压瓶搅拌；加入QY-13-28(1.08g，4.24mmo)，冰醋酸(306mg，5.1mmol)搅拌20min后加入三乙酰氧基硼氢化钠(899mg，4.24mmol)，室温搅拌过夜,LC-MS监测反应完全，停止反应。旋蒸除去有机溶剂，二甲基亚砜溶混匀反应体系后滤膜过滤，C18反相色谱柱分离纯化(水：乙腈＝10-100％)得586mg黄绿色泡沫状固体，产率66％。ESI-MS m/z 525.3(M+H) +.
化合物QY-13-33的合成:
Figure PCTCN2021118212-appb-000139
(5-(S)-5-(1-(4-(6-氨基-9H-嘌呤-9-基)苯基)ethyl)-3-苄基-2-甲基-2,5,6,7-四氢-4H-吡唑并[4,3-c]吡啶-4-酮(QY-13-33):将QY-13-32(262mg，0.50mmol)混匀至20ml二甲苯中，转移至40ml耐压瓶搅拌；室温下缓慢滴加三甲基铝(1.50mmo)至反应液中，氮气保护后逐渐升温至120℃，搅拌过夜停止反应。待反应液缓缓回至室温后滴加蒸馏水淬灭反应，加入罗氏盐溶液搅拌30min，加入20ml乙酸乙酯稀释反应体系，转移至分液漏斗，乙酸乙酯萃取(60ml*3)，合并有机相，饱和氯化钠洗涤(20ml*2)，无水硫酸钠干燥，过滤并旋蒸除去有机溶剂，经硅胶柱层析分离纯化(二氯甲烷：甲醇(氨)＝0-20％)得105mg橙黄色油状液体，产率44％。ESI-MS m/z 479.2(M+H) +.
按照上述方法通过替换不同的合成底物，得到如表A中所示的化合物：
表A
Figure PCTCN2021118212-appb-000140
Figure PCTCN2021118212-appb-000141
Figure PCTCN2021118212-appb-000142
Figure PCTCN2021118212-appb-000143
Figure PCTCN2021118212-appb-000144
Figure PCTCN2021118212-appb-000145
表A中所示化合物的NMR数据如下：
QY-5-35： 1H NMR(400MHz,DMSO-d6)δ10.93(s,1H),8.83(s,1H),8.55(dd,J＝7.2,4.5Hz,1H),8.42(m,1H),8.31(d,J＝1.1Hz,0.5H),8.10(s,0.5H),7.88–7.84(m,1H),7.69–7.63(m,2H),7.54(m,3H),7.36(d,J＝8.1Hz,1H),7.27(t,J＝2.2Hz,1H),7.12–7.05(m,1H),7.01–6.95(m,1H),4.45–4.43(m,2H),3.57(d,J＝5.9Hz,2H),2.81(dd,J＝12.4,4.5Hz,3H)；ESI-MS m/z 438.0(M+H) +.
QY-5-40： 1H NMR(400MHz,DMSO-d6)δ8.79(s,1H),8.53(d,J＝4.6Hz,1H),8.31(s,1H),7.90(dd,J＝8.6,1.6Hz,1H),7.70–7.62(m,4H),7.51(d,J＝8.5Hz,3H),4.63(s,2H),3.96(s,2H),3.13(s,3H),2.83(d,J＝4.5Hz,3H)；ESI-MS m/z 466.2(M+H) +.
QY-5-62： 1H NMR(400MHz,DMSO-d6)δ10.13(s,1H),8.79(d,J＝4.6Hz,1H),8.58–8.51(m,1H),8.31(d,J＝1.1Hz,1H),7.90(m,1H),7.66(dd,J＝11.9,5.2Hz,3H),7.54(m,2H),7.32(m,5H),4.74(s,2H),3.92(s,2H),2.81(dd,J＝11.6,4.5Hz,3H)；ESI-MS m/z 415.2(M+H) +.
QY-6-15： 1H NMR(400MHz,DMSO-d6)δ10.97(d,J＝9.0Hz,1H),8.76(m,1H),8.59–8.50(m,1H),8.30(d,J＝1.1Hz,1H),8.06(m,3H),7.85–7.83(m,1H),7.72(dd,J＝8.8,6.7Hz, 2H),7.41–7.27(m,5H),6.73(d,J＝2.1Hz,1H),4.28(s,2H),2.81(m,3H)；ESI-MS m/z 452.1(M+H) +.
QY-6-16： 1H NMR(400MHz,DMSO-d6)δ9.43(q,J＝6.1Hz,1H),8.82(m,1H),8.54(d,J＝4.6Hz,1H),8.30(d,J＝1.1Hz,1H),8.08(s,1H),7.90–7.84(m,1H),7.67(dd,J＝8.1,2.0Hz,2H),7.57(t,J＝9.1Hz,2H),7.40–7.24(m,5H),6.59(d,J＝2.4Hz,1H),4.53(t,J＝5.6Hz,2H),4.23(s,2H),2.80(m,3H)；ESI-MS m/z 466.2(M+H) +.
QY-7-2A：1H NMR(400MHz,DMSO-d6)δ9.06(t,J＝4.9Hz,1H),8.90(m,1H),8.56(d,J＝4.5Hz,1H),8.31(d,J＝1.1Hz,1H),7.91–7.86(m,1H),7.69(t,J＝8.1Hz,3H),7.56(t,J＝9.1Hz,2H),7.26(m,5H),6.68(d,J＝3.1Hz,1H),4.50(t,J＝5.5Hz,2H),4.03(d,J＝1.5Hz,3H),3.89(s,2H),2.80(dd,J＝13.9,4.5Hz,3H)；ESI-MS m/z 479.1(M+H) +.
QY-7-2B： 1H NMR(400MHz,DMSO-d6)δ8.85(s,1H),8.77(t,J＝6.4Hz,1H),8.55(d,J＝4.5Hz,1H),8.31(d,J＝1.1Hz,1H),7.91–7.86(m,1H),7.72–7.63(m,3H),7.56(t,J＝9.3Hz,2H),7.34(t,J＝7.3Hz,2H),7.26(m,3H),6.37(d,J＝1.7Hz,1H),4.49(t,J＝5.7Hz,2H),4.07(s,2H),3.78(s,3H),2.80(m,3H)；ESI-MS m/z 479.1(M+H) +.
QY-7-14： 1H NMR(400MHz,DMSO-d6)δ9.67(t,J＝6.1Hz,1H),8.81(m,1H),8.54(d,J＝4.5Hz,1H),8.30(d,J＝1.1Hz,1H),7.90–7.84(m,1H),7.73–7.65(m,3H),7.59(t,J＝9.1Hz,2H),7.41–7.32(m,5H),4.55(t,J＝5.6Hz,2H),4.46(s,2H),2.80(dd,J＝13.9,4.5Hz,3H)；ESI-MS m/z 467.0(M+H) +.
QY-7-32： 1H NMR(400MHz,DMSO-d6)δ9.96(d,J＝6.0Hz,1H),8.75(d,J＝12.2Hz,1H),8.51(s,1H),8.08(d,J＝12.0Hz,1H),7.84(d,J＝12.0Hz,2H),7.74–7.54(m,4H),7.42–7.28(m,4H),4.57(s,2H),4.39(d,J＝12.1Hz,2H),2.80(d,J＝7.7Hz,3H)；ESI-MS m/z 467.1(M+H) +.
QY-7-65： 1H NMR(400MHz,DMSO-d6)δ8.78(s,1H),8.57–8.51(m,1H),8.07(d,J＝14.7Hz,1H),7.86–7.81(m,2H),7.67(d,J＝8.4Hz,1H),7.54(dd,J＝12.0,8.5Hz,2H),7.41(t,J＝7.4Hz,1H),7.23(m,5H),5.27(d,J＝6.4Hz,0.5H),5.09(dd,J＝7.9,2.3Hz,0.5H),3.90(m,2H),3.72–3.58(m,2H),2.79(d,J＝4.1Hz,3H),2.44–2.20(m,2H),1.83(dd,J＝7.5,2.9Hz,2H)；ESI-MS m/z 439.1(M+H) +.
QY-8-7： 1H NMR(400MHz,DMSO-d6)δ8.73(d,J＝13.6Hz,1H),8.51(d,J＝4.5Hz,1H),8.30(d,J＝1.1Hz,1H),7.85(m,1H),7.77(s,1H),7.68(dt,J＝8.4,6.6Hz,3H),7.57(t,J＝8.6Hz,2H),7.39–7.29(m,5H),5.37(s,2H),4.76(d,J＝5.0Hz,2H),3.56(m,2H),2.84–2.76(m,5H)；ESI-MS m/z 491.1(M+H) +.
QY-8-30： 1H NMR(400MHz,DMSO-d6)δ9.49(t,J＝6.3Hz,1H),8.60(s,2H),8.34(s,1H),7.75(d,J＝3.6Hz,1H),7.66(d,J＝8.5Hz,2H),7.48(d,J＝8.5Hz,2H),7.31(m,5H),7.16(s,1H),7.05(d,J＝3.6Hz,1H),4.47(d,J＝6.2Hz,2H),4.14(s,2H)；ESI-MS m/z 425.2(M+H) +.
QY-8-31： 1H NMR(400MHz,DMSO-d6)δ9.62(t,J＝6.2Hz,1H),8.60(s,2H),8.34(s,1H),7.77(d,J＝3.6Hz,1H),7.71–7.65(m,2H),7.50(d,J＝8.5Hz,2H),7.42–7.27(m,5H),7.05(d,J＝3.6Hz,1H),4.51(d,J＝6.2Hz,2H),4.45(s,2H)；ESI-MS m/z 426.1(M+H) +.
QY-8-42： 1H NMR(400MHz,DMSO-d6)δ8.81(s,1H),8.53(d,J＝8.6Hz,2H),8.30(d,J＝1.2Hz,1H),7.85(s,1H),7.64(m,5H),7.07–6.90(m,4H),4.95(d,J＝5.7Hz,1H),3.64(d,J＝7.2Hz,2H),3.51(m,2H),2.80(dd,J＝12.2,4.5Hz,3H),2.23(d,J＝3.2Hz,3H),2.00(m,1H),1.66(m,2H),1.47(d,J＝5.5Hz,1H),1.40(s,3H),1.33(s,3H)；ESI-MS m/z 538.1(M+H) +.
QY-8-43： 1H NMR(400MHz,DMSO-d6)δ8.72(t,J＝6.3Hz,1H),8.67(s,1H),8.33(s,1H),7.76(d,J＝8.5Hz,2H),7.49(d,J＝8.5Hz,2H),7.34(t,J＝7.3Hz,2H),7.28–7.19(m,3H),6.36(s,1H),4.46(d,J＝6.3Hz,2H),4.06(s,2H),3.77(s,3H)；ESI-MS m/z 439.1(M+H) +.
QY-8-48： 1H NMR(400MHz,DMSO-d6)δ9.38(t,J＝6.2Hz,1H),8.60(s,2H),8.34(s,1H),7.76(d,J＝3.6Hz,1H),7.70–7.64(m,2H),7.49(d,J＝8.5Hz,2H),7.38–7.26(m,5H),7.05(d,J＝3.6Hz,1H),6.57(s,1H),4.49(d,J＝6.2Hz,2H),4.22(s,2H)；ESI-MS m/z 425.2(M+H) +.
QY-8-58： 1H NMR(400MHz,DMSO-d6)δ9.69(t,J＝6.2Hz,1H),8.77(s,2H),8.34(s,1H),7.76(d,J＝3.6Hz,1H),7.67(d,J＝8.5Hz,2H),7.51(d,J＝8.5Hz,2H),7.44–7.38(m,5H),7.05(d,J＝3.6Hz,1H),6.03(s,2H),4.54(d,J＝6.2Hz,2H)；ESI-MS m/z 426.1(M+H) +.
QY-8-60： 1H NMR(400MHz,DMSO-d6)δ8.33(s,1H),7.74(d,J＝3.6Hz,1H),7.66(d,J＝8.4Hz,2H),7.49(d,J＝8.4Hz,2H),7.34–7.20(m,6H),7.03(d,J＝3.6Hz,1H),4.48(d,J＝6.2Hz,2H),4.11(s,2H)；ESI-MS m/z 425.2(M+H) +.
QY-9-33： 1H NMR(400MHz,DMSO-d6)δ8.70(t,J＝6.3Hz,1H),8.36(s,1H),7.78(d,J＝3.6Hz,1H),7.64(d,J＝8.3Hz,2H),7.48(d,J＝8.3Hz,2H),7.33(d,J＝6.9Hz,2H),7.25(t,J＝6.8Hz,3H),7.07(d,J＝3.6Hz,1H),6.37(s,1H),4.46(d,J＝6.2Hz,2H),4.13–4.06(m,4H),1.23(d,J＝7.2Hz,3H)；ESI-MS m/z 452.1(M+H) +.
QY-9-34： 1H NMR(400MHz,DMSO-d6)δ8.74–8.67(m,2H),8.35(s,1H),7.78–7.72(m,2H),7.51(d,J＝8.5Hz,2H),7.36–7.31(m,2H),7.25(dd,J＝7.2,5.4Hz,3H),6.37(s,1H),4.46(d,J＝6.2Hz,2H),4.10(m,4H),1.23(d,J＝7.1Hz,3H)；ESI-MS m/z 453.1(M+H) +.
QY-9-41： 1H NMR(400MHz,DMSO-d6)δ8.36(s,1H),7.81–7.76(m,2H),7.69(d,J＝8.5Hz,2H),7.50(d,J＝8.5Hz,2H),7.38–7.28(m,5H),7.07(d,J＝3.6Hz,1H),5.37(s,2H),4.72(s,2H),3.52(t,J＝6.6Hz,2H),2.77(t,J＝6.6Hz,2H)；ESI-MS m/z 450.1(M+H) +.
QY-9-50： 1H NMR(400MHz,DMSO-d6)δ8.71(s,1H),8.35(s,1H),7.84–7.76(m,3H),7.53(d,J＝8.5Hz,2H),7.39–7.28(m,5H),5.37(s,2H),4.73(s,2H),3.52(t,J＝6.6Hz,2H),2.77(t,J＝6.6Hz,2H)；ESI-MS m/z 451.2(M+H) +.
QY-9-69： 1H NMR(400MHz,DMSO-d6)δ8.74(t,J＝5.9Hz,1H),8.36(s,1H),7.78(d,J＝3.6Hz,1H),7.69–7.60(m,2H),7.44(m,3H),7.35–7.27(m,2H),7.25(dd,J＝8.2, 1.0Hz,1H),7.07(d,J＝3.6Hz,1H),4.35(d,J＝5.9Hz,2H),3.59(s,2H)；ESI-MS m/z 442.1(M+H) +.
QY-9-70： 1H NMR(400MHz,DMSO-d6)δ8.75(t,J＝5.9Hz,1H),8.71(s,1H),8.35(s,1H),7.78(d,J＝8.5Hz,2H),7.49–7.42(m,3H),7.35–7.29(m,2H),7.25(dd,J＝8.2,1.0Hz,1H),4.36(d,J＝5.9Hz,2H),3.59(s,2H)；ESI-MS m/z 443.1(M+H +.
QY-9-77： 1H NMR(400MHz,DMSO-d6)δ14.82(s,1H),9.51(t,J＝6.3Hz,1H),8.93(d,J＝2.1Hz,1H),8.80(d,J＝2.0Hz,1H),8.68(s,1H),8.32(s,1H),7.80(d,J＝8.5Hz,2H),7.59(d,J＝8.5Hz,2H),4.59(d,J＝6.2Hz,2H)；ESI-MS m/z 504.1(M+H) +.
QY-10-21： 1H NMR(400MHz,DMSO-d6)δ8.75(s,1H),8.35(s,1H),7.77(d,J＝3.6Hz,1H),7.64(d,J＝8.5Hz,2H),7.47(d,J＝8.5Hz,2H),7.34(t,J＝7.3Hz,2H),7.27–7.21(m,3H),7.06(d,J＝3.6Hz,1H),6.36(s,1H),4.44(d,J＝6.0Hz,2H),4.06(s,2H),3.77(s,3H)；ESI-MS m/z 438.1(M+H) +.
QY-10-39： 1H NMR(400MHz,DMSO-d6)δ8.71(t,J＝6.0Hz,1H),8.35(s,1H),7.99(s,1H),7.78(d,J＝3.6Hz,1H),7.70–7.65(m,2H),7.49(d,J＝8.5Hz,2H),7.31–7.26(m,2H),7.19(m,3H),7.07(d,J＝3.6Hz,1H),4.50(d,J＝5.8Hz,2H),4.46(s,2H),3.66(s,3H)；ESI-MS m/z 438.1(M+H) +.
QY-10-40： 1H NMR(400MHz,DMSO-d6)δ8.76–8.66(m,2H),8.35(s,1H),7.99(s,1H),7.82–7.77(m,2H),7.52(d,J＝8.6Hz,2H),7.31–7.25(m,2H),7.21–7.17(m,3H),4.51(d,J＝5.8Hz,2H),4.46(s,2H),3.66(s,3H)；ESI-MS m/z 439.1(M+H) +.
QY-10-65： 1H NMR(400MHz,DMSO-d6)δ8.76–8.68(m,2H),8.36(s,1H),8.04(s,1H),7.82–7.77(m,2H),7.53(d,J＝8.5Hz,2H),7.31–7.25(m,2H),7.19(dd,J＝7.3,3.5Hz,3H),4.51(d,J＝5.7Hz,2H),4.48(s,2H),3.98(q,J＝7.2Hz,2H),1.07(t,J＝7.2Hz,3H)；ESI-MS m/z 453.2(M+H) +.
QY-10-73： 1H NMR(400MHz,DMSO-d6)δ8.85–8.72(m,2H),8.52(d,J＝4.5Hz,1H),8.31(d,J＝1.2Hz,1H),8.01(d,J＝3.3Hz,1H),7.90–7.84(m,1H),7.68(q,J＝7.5Hz,3H),7.58(m,2H),7.32–7.26(m,2H),7.22–7.17(m,3H),4.54(t,J＝5.2Hz,2H),4.47(s,2H),3.66(s,3H),2.81(m,3H)；ESI-MS m/z 479.1(M+H) +.
QY-11-68A： 1H NMR(400MHz,DMSO-d6)δ8.79(m,2H),8.73(s,1H),8.08(s,1H),8.01(s,1H),7.87(s,2H),7.68(d,J＝8.4Hz,2H),7.59(d,J＝8.4Hz,2H),7.32–7.26(m,2H),7.20(dd,J＝5.1,2.8Hz,3H),4.55(d,J＝5.9Hz,2H),4.47(s,2H),3.66(s,3H),3.66–3.46(m,8H),3.32(d,J＝5.7Hz,2H),3.13(s,2H)；ESI-MS m/z 578.2(M+H) +.
QY-11-68B： 1H NMR(400MHz,DMSO-d6)δ8.78(m,2H),8.70(s,1H),8.36(d,J＝1.2Hz,1H),8.01(s,1H),7.89(dd,J＝8.6,1.5Hz,1H),7.72–7.65(m,3H),7.57(d,J＝8.4Hz,2H),7.32–7.26(m,2H),7.21–7.16(m,3H),4.54(d,J＝6.0Hz,2H),4.47(s,2H),4.02(m,2H),3.68(s,2H),3.66(s,3H),3.58(m,4H),3.37(d,J＝5.6Hz,2H),3.17(s,2H)；ESI-MS m/z 578.2 (M+H) +.
QY-11-69A： 1H NMR(400MHz,DMSO-d6)δ8.77(t,J＝6.1Hz,1H),8.73(s,1H),8.66(s,1H),8.07(s,1H),8.01(s,1H),7.85(d,J＝0.8Hz,2H),7.68(d,J＝8.4Hz,2H),7.59(d,J＝8.4Hz,2H),7.32–7.26(m,2H),7.22–7.17(m,3H),4.55(d,J＝5.9Hz,2H),4.47(s,2H),3.84(s,8H),3.66(s,3H),3.50(d,J＝5.4Hz,2H),2.90(s,2H),2.78(s,3H)；ESI-MS m/z 591.2(M+H) +.
QY-11-69B： 1H NMR(400MHz,DMSO-d6)δ8.76(t,J＝6.1Hz,1H),8.71(s,1H),8.67(s,1H),8.34(d,J＝1.2Hz,1H),8.01(s,1H),7.89(m,1H),7.70–7.65(m,3H),7.57(m,2H),7.29(m,2H),7.19(dd,J＝5.0,2.8Hz,3H),4.54(d,J＝5.9Hz,2H),4.47(s,2H),4.06(s,8H),3.66(s,3H),3.56(d,J＝5.7Hz,2H),3.01(s,2H),2.81(s,3H)；ESI-MS m/z 591.2(M+H) +.
QY-11-74A： 1H NMR(400MHz,DMSO-d6)δ8.77(t,J＝5.9Hz,2H),8.72(s,1H),8.08(s,1H),8.01(s,1H),7.87(s,2H),7.67(d,J＝8.5Hz,2H),7.59(d,J＝8.5Hz,2H),7.31–7.25(m,2H),7.20(m,3H),4.55(d,J＝6.0Hz,2H),4.47(s,2H),3.66(s,3H),3.61(d,J＝5.8Hz,2H),3.26(m,2H),2.84(d,J＝4.8Hz,6H)；ESI-MS m/z 536.2(M+H) +.
QY-11-74B： 1H NMR(400MHz,DMSO-d6)δ8.76(t,J＝5.8Hz,2H),8.70(s,1H),8.36(d,J＝1.2Hz,1H),8.01(s,1H),7.89(dd,J＝8.6,1.5Hz,1H),7.71–7.65(m,3H),7.57(d,J＝8.5Hz,2H),7.29(dd,J＝10.4,4.4Hz,2H),7.19(dd,J＝5.0,2.8Hz,3H),4.54(d,J＝6.0Hz,2H),4.47(s,2H),3.66(s,3H),3.64(s,2H),3.31(dd,J＝11.5,5.8Hz,2H),2.88(d,J＝4.8Hz,6H)；ESI-MS m/z 536.2(M+H) +.
QY-11-75： 1H NMR(400MHz,DMSO-d6)δ9.13(s,1H),8.75(t,J＝6.1Hz,1H),8.02(s,1H),7.83(d,J＝8.2Hz,2H),7.53(d,J＝8.2Hz,2H),7.44(s,1H),7.34(s,1H),7.28(m,2H),7.20(d,J＝7.4Hz,3H),4.58(d,J＝6.0Hz,2H),4.48(s,2H),4.01(s,3H),3.83(s,3H),3.67(s,3H)；ESI-MS m/z 494.1(M+H) +.
QY-11-76
1H NMR(400MHz,DMSO-d6)δ8.92(s,1H),8.76(t,J＝6.1Hz,1H),8.01(s,1H),7.71(d,J＝8.5Hz,2H),7.58(d,J＝8.5Hz,2H),7.35(s,1H),7.32–7.25(m,2H),7.22–7.16(m,3H),7.10(s,1H),4.54(d,J＝6.0Hz,2H),4.47(s,2H),3.86(s,3H),3.81(s,3H),3.66(s,3H)；ESI-MS m/z 482.1(M+H) +.
QY-11-77： 1H NMR(400MHz,DMSO-d6)(400MHz,DMSO)δ8.80–8.74(m,2H),8.61(s,2H),8.00(s,1H),7.75(d,J＝7.1Hz,1H),7.65(d,J＝8.5Hz,2H),7.58(d,J＝8.5Hz,2H),7.31–7.25(m,2H),7.19(t,J＝6.3Hz,3H),7.08(d,J＝7.1Hz,1H),4.54(d,J＝6.0Hz,2H),4.46(s,2H),3.66(s,3H)；ESI-MS m/z 438.1(M+H) +.
QY-11-78： 1H NMR(400MHz,DMSO-d6)δ8.69(t,J＝6.1Hz,1H),8.45(s,1H),8.39(s,1H),8.07(d,J＝8.6Hz,2H),7.99(s,1H),7.48(d,J＝8.6Hz,2H),7.28(m,2H),7.19(m,3H),4.49(d,J＝5.9Hz,2H),4.46(s,2H),3.66(s,3H)；ESI-MS m/z 439.1(M+H) +.
QY-11-79： 1H NMR(400MHz,DMSO-d6)δ11.99(s,1H),8.71(t,J＝6.0Hz,1H),8.33(d,J＝5.1Hz,1H),8.00(s,1H),7.77(d,J＝8.2Hz,2H),7.62–7.57(m,1H),7.49(d,J＝8.2Hz,2H),7.33–7.24(m,3H),7.23–7.16(m,3H),6.67(dd,J＝3.5,1.8Hz,1H),4.53(d,J＝6.0Hz,2H),4.47(s,2H),3.66(s,3H)；ESI-MS m/z 422.1(M+H) +.
QY-11-80： 1H NMR(400MHz,DMSO-d6)δ12.55(s,1H),8.91(s,1H),8.74(t,J＝6.0Hz,1H),8.13(d,J＝8.3Hz,2H),8.01(s,1H),7.79–7.73(m,1H),7.54(d,J＝8.3Hz,2H),7.28(m,2H),7.23–7.17(m,3H),6.96(m,1H),4.55(d,J＝5.9Hz,2H),4.47(s,2H),3.66(s,3H)；ESI-MS m/z 423.2(M+H) +.
QY-11-102A： 1H NMR(400MHz,DMSO-d6)δ9.02(s,1H),8.75(t,J＝6.0Hz,1H),8.00(d,J＝2.3Hz,1H),7.71–7.67(m,2H),7.57(dd,J＝8.6,2.1Hz,3H),7.34(d,J＝2.3Hz,1H),7.30–7.26(m,2H),7.21–7.17(m,3H),7.08–7.05(m,1H),4.54(d,J＝5.9Hz,2H),4.47(s,2H),3.85(s,3H),3.80(s,3H)；ESI-MS m/z 452.1(M+H) +.
QY-11-102B： 1H NMR(400MHz,DMSO-d6)δ8.82(s,1H),8.75(t,J＝6.0Hz,1H),8.01(s,1H),7.75–7.66(m,3H),7.57(d,J＝8.5Hz,2H),7.28(m,2H),7.22–7.16(m,3H),7.09–7.00(m,2H),4.54(d,J＝6.0Hz,2H),4.47(s,2H),3.80(s,3H),3.66(s,3H)；ESI-MS m/z 452.1(M+H) +.
QY-11-104： 1H NMR(400MHz,DMSO-d6)δ9.26(s,1H),8.72(t,J＝6.0Hz,1H),8.47(q,J＝4.3Hz,1H),8.33(s,1H),8.07(d,J＝8.6Hz,2H),8.00(s,1H),7.79–7.72(m,2H),7.52(d,J＝8.6Hz,2H),7.29(m,2H),7.20(m,3H),4.52(d,J＝6.0Hz,2H),4.47(s,2H),3.66(s,3H),2.82(d,J＝4.5Hz,3H)；ESI-MS m/z 479.1(M+H) +.
QY-12-4： 1H NMR(400MHz,DMSO-d6)δ8.87(s,1H),8.70(t,J＝6.0Hz,1H),8.00(t,J＝4.3Hz,3H),7.63(m,1H),7.48(m,2H),7.27(d,J＝7.2Hz,2H),7.20(m,3H),7.04–6.97(m,2H),4.50(d,J＝6.0Hz,2H),4.47(s,2H),3.80(s,3H),3.66(s,3H)；ESI-MS m/z 452.1(M+H) +.
QY-12-31： 1H NMR(400MHz,DMSO-d6)δ8.54(s,1H),8.46(d,J＝7.9Hz,1H),8.19(s,1H),8.08(s,1H),7.87–7.76(m,2H),7.60–7.50(m,2H),7.39(s,2H),7.32–7.22(m,2H),7.22–7.10(m,3H),5.20(p,J＝7.2Hz,1H),4.50–4.24(m,2H),3.64(s,3H),1.50(d,J＝7.1Hz,3H)；ESI-MS m/z 453.1(M+H) +.
QY-12-47： 1H NMR(400MHz,DMSO-d6)δ8.81–8.67(m,2H),8.53(q,J＝4.5Hz,1H),8.31(d,J＝1.5Hz,1H),7.89(dd,J＝8.6,1.7Hz,1H),7.70–7.58(m,3H),7.54–7.44(m,2H),7.32(dd,J＝8.1,6.5Hz,2H),7.29–7.20(m,1H),7.14–7.04(m,2H),6.90(dd,J＝6.0,4.3Hz,1H),5.77(t,J＝4.1Hz,1H),5.59(s,2H),4.48(d,J＝6.0Hz,2H),2.82(d,J＝4.5Hz,3H)；ESI-MS m/z 482.2(M+H) +.
QY-12-51： 1H NMR(400MHz,DMSO-d6)δ9.19(t,J＝6.1Hz,1H),8.56(s,1H),8.20(s,1H),7.87–7.67(m,2H),7.47–7.38(m,4H),7.38–7.23(m,3H),7.14–7.05(m,3H),7.01(d,J＝4.3Hz,1H),5.76(s,2H),4.48(d,J＝6.0Hz,2H)；ESI-MS m/z 449.1(M+H) +.
QY-12-90： 1H NMR(400MHz,DMSO-d6)δ8.72(s,1H),8.35(s,1H),7.93–7.73(m,2H),7.64–7.50(m,2H),7.37–7.26(m,4H),7.26–7.16(m,1H),6.01(q,J＝7.0Hz,1H),4.40(d,J＝3.3Hz,2H),3.64(s,3H),3.14(m,2H),2.74(m,2H),1.59(d,J＝7.1Hz,3H)；ESI-MS m/z 479.2(M+H) +.
QY-12-97： 1H NMR(400MHz,DMSO-d6)δ8.70(s,1H),8.48(d,J＝7.9Hz,1H),8.35(s,1H),8.08(s,1H),7.84–7.69(m,2H),7.64–7.53(m,2H),7.31–7.22(m,2H),7.21–7.08(m,3H),5.20(p,J＝7.1Hz,1H),4.57–4.20(m,2H),3.65(s,3H),1.51(d,J＝7.1Hz,3H)；ESI-MS m/z 453.1(M+H) +.
QY-12-100： 1H NMR(400MHz,DMSO-d6)δ8.50(s,1H),8.45(t,J＝6.0Hz,1H),8.14(s,1H),7.83–7.69(m,2H),7.49–7.38(m,2H),7.35(s,2H),7.23(dd,J＝8.0,6.6Hz,2H),7.18–7.07(m,3H),4.44(d,J＝6.0Hz,2H),4.22(s,2H),3.59(s,3H)；ESI-MS m/z 473.2(M+H) +.
QY-12-102： 1H NMR(400MHz,DMSO-d6)δ8.70(s,1H),8.35(s,1H),8.10(d,J＝6.6Hz,2H),7.78–7.64(m,2H),7.62–7.48(m,2H),7.26(dd,J＝8.0,6.8Hz,2H),7.20–7.14(m,1H),7.14–7.08(m,2H),4.33(s,2H),3.65(s,3H),1.70(s,6H)；ESI-MS m/z 467.2(M+H) +.
QY-13-19： 1H NMR(400MHz,DMSO-d6)δ9.30(t,J＝6.3Hz,1H),8.71(s,1H),8.35(s,1H),7.83–7.70(m,2H),7.55(d,J＝8.3Hz,2H),7.37–7.26(m,2H),7.27–7.14(m,3H),4.54(d,J＝6.3Hz,2H),4.47(s,2H),3.90(s,3H)；ESI-MS m/z 440.2(M+H) +.
QY-13-29： 1H NMR(400MHz,DMSO-d6)δ8.56(s,1H),8.46(d,J＝7.8Hz,1H),8.20(s,1H),7.89–7.77(m,2H),7.58(d,J＝8.5Hz,2H),7.42(s,2H),7.28(m,2H),7.24–7.19(m,1H),7.19–7.12(m,2H),5.14(p,J＝7.1Hz,1H),4.37–4.06(m,2H),3.67(s,3H),1.47(d,J＝7.0Hz,3H)；ESI-MS m/z 487.1(M+H) +.
QY-13-31： 1H NMR(400MHz,DMSO-d6)δ8.55(s,1H),8.48(d,J＝7.9Hz,1H),8.19(s,1H),8.08(s,1H),7.86–7.74(m,2H),7.60–7.51(m,2H),7.41(s,2H),7.27(dd,J＝8.0,6.8Hz,2H),7.20–7.12(m,3H),5.20(p,J＝7.2Hz,1H),4.56–4.22(m,2H),3.65(s,3H),1.50(d,J＝7.1Hz,3H)；ESI-MS m/z 453.2(M+H) +.
QY-13-33： 1H NMR(400MHz,DMSO-d6)δ8.59(s,1H),8.21(s,1H),7.98–7.76(m,2H),7.55(d,J＝8.4Hz,2H),7.43(s,2H),7.37–7.27(m,4H),7.26–7.16(m,1H),6.01(q,J＝7.0Hz,1H),4.48–4.32(m,2H),3.64(s,3H),3.51(m,1H),3.14(m,1H),2.86–2.62(m,2H),1.58(d,J＝7.1Hz,3H)；ESI-MS m/z 479.1(M+H) +.
QY-13-50： 1H NMR(400MHz,DMSO-d6)δ9.09(d,J＝8.3Hz,1H),8.71(s,1H),8.35(s,1H),7.82–7.71(m,2H),7.70–7.55(m,2H),7.32–7.25(m,2H),7.23–7.13(m,3H),5.26(p,J＝7.2Hz,1H),4.57–4.29(m,2H),3.89(s,3H),1.56(d,J＝7.1Hz,3H)；ESI-MS m/z 454.2(M+H) +.
QY-15-59： 1H NMR(400MHz,DMSO-d6)δ9.27(t,J＝6.3Hz,1H),8.85(s,0.35H),8.80(s,0.65H),8.36(s,0.65H),8.16(s,0.35H),7.96(d,J＝8.4Hz,0.35H),7.77(d,J＝8.5Hz,0.65H),7.74–7.64(m,3H),7.60(d,J＝8.2Hz,2H),7.30(t,J＝7.5Hz,2H),7.22(m,3H),4.57(d,J＝6.3Hz,2H),4.47(s,2H),3.90(s,3H)；ESI-MS m/z 448.1(M+H) +.
ET-0934: 1H NMR(400MHz,DMSO-d6)δ9.27(t,J＝6.4Hz,1H),8.65(s,1H),8.35–8.15(m,1H),7.68(d,J＝8.2Hz,2H),7.63–7.51(m,3H),7.30(t,J＝7.4Hz,2H),7.22(d,J＝8.2Hz,3H),4.56(d,J＝6.3Hz,2H),4.47(s,2H),3.90(s,3H),3.87(s,3H)；ESI-MS m/z 454.1(M+H) +.
ET-0935: 1H NMR(400MHz,DMSO-d6)δ9.23(t,J＝6.4Hz,1H),8.81(s,1H),8.18(d,J＝2.6Hz,1H),7.98–7.78(m,3H),7.54(d,J＝8.1Hz,2H),7.30(t,J＝7.5Hz,2H),7.23(d,J＝6.3Hz,3H),4.54(d,J＝6.3Hz,2H),4.47(s,2H),3.89(d,J＝2.8Hz,6H)；ESI-MS m/z 454.1(M+H) +.
ZSQ-13-35： 1H NMR(400MHz,DMSO-d6)δ9.24(t,J＝6.2Hz,1H),8.68(d,J＝3.6Hz,2H),8.50(d,J＝4.5Hz,1H),8.29(d,J＝1.1Hz,1H),7.86(dd,J＝8.6,1.6Hz,1H),7.67–7.62(m,3H),7.56(d,J＝8.5Hz,2H),7.41–7.32(m,5H),5.66(s,2H),4.54(d,J＝6.2Hz,2H),2.82(d,J＝4.5Hz,3H)；ESI-MS m/z 466.1(M+H) +.
ZSQ-13-36： 1H NMR(400MHz,DMSO-d6)δ9.72(t,J＝6.2Hz,1H),8.76(s,1H),8.54–8.49(m,1H),8.30(d,J＝1.1Hz,1H),7.88(dd,J＝8.6,1.6Hz,1H),7.70–7.65(m,3H),7.59(d,J＝8.5Hz,2H),7.42–7.39(m,5H),6.03(d,J＝4.5Hz,2H),4.57(d,J＝6.2Hz,2H),2.82(d,J＝4.5Hz,3H)；ESI-MS m/z 467.2(M+H) +.
ZSQ-13-46： 1H NMR(400MHz,DMSO-d6)δ9.20(s,1H),8.73–8.59(m,2H),8.34(d,J＝1.0Hz,1H),7.97(dd,J＝8.6,1.5Hz,1H),7.76–7.65(m,3H),7.53(t,J＝9.5Hz,2H),7.35–7.20(m,6H),4.41(t,J＝5.8Hz,2H),3.53(d,J＝2.8Hz,2H),2.82(m,3H)；ESI-MS m/z 466.1(M+H) +.
ZSQ-15-48： 1H NMR(400MHz,DMSO-d6)δ8.69(t,J＝6.3Hz,1H),8.45(s,1H),8.39(s,1H),8.06–8.00(m,2H),7.46(d,J＝8.6Hz,2H),7.34(dd,J＝8.2,6.4Hz,2H),7.27–7.21(m,3H),6.36(s,1H),4.44(d,J＝6.3Hz,2H),4.06(s,2H),3.77(s,3H)；ESI-MS m/z 439.2(M+H) +.
ZSQ-15-67： 1H NMR(400MHz,DMSO-d6)δ8.73(t,J＝6.3Hz,1H),8.55(d,J＝4.6Hz,1H),8.49(s,1H),8.41(s,1H),7.96(dd,J＝8.9,1.5Hz,1H),7.85(d,J＝8.9Hz,1H),7.72(d,J＝8.4Hz,2H),7.50(d,J＝8.5Hz,2H),7.34(t,J＝7.3Hz,2H),7.25(dd,J＝8.8,7.3Hz,3H),6.37(s,1H),4.47(d,J＝6.3Hz,2H),4.07(s,2H),3.78(s,3H),2.82(d,J＝4.5Hz,3H)；ESI-MS m/z 479.1(M+H) +.
ZSQ-15-68： 1H NMR(400MHz,DMSO-d6)δ8.73(t,J＝6.0Hz,1H),8.38(d,J＝4.5Hz, 1H),8.20(d,J＝1.3Hz,1H),8.00(s,1H),7.75–7.68(m,2H),7.60–7.49(m,5H),7.28(dd,J＝9.3,5.6Hz,2H),7.20(d,J＝6.6Hz,3H),6.80(d,J＝3.3Hz,1H),4.52(d,J＝6.0Hz,2H),4.48(s,2H),3.66(s,3H),2.80(d,J＝4.4Hz,3H)；ESI-MS m/z 478.2(M+H) +.
ZSQ-16-3： 1H NMR(400MHz,DMSO-d6)δ8.77(t,J＝6.3Hz,1H),8.37(s,2H),7.74(d,J＝3.4Hz,1H),7.59(d,J＝6.8Hz,1H),7.52(s,4H),7.37–7.31(m,2H),7.25(m,4H),6.97(d,J＝7.2Hz,1H),6.36(s,1H),4.47(d,J＝6.3Hz,2H),4.07(s,2H),3.77(s,3H)；ESI-MS m/z 437.1(M+H) +.
XHJ-2-88： 1H NMR(400MHz,DMSO-d6)δ9.56(q,J＝6.3Hz,1H),9.09(m,1H),8.66–8.56(m,1H),8.34(s,1H),7.93(t,J＝8.4Hz,1H),7.75–7.55(m,5H),7.32(m,5H),7.18(s,1H),4.53(t,J＝5.8Hz,2H),4.15(s,2H),2.81(m,3H)；ESI-MS m/z 466.1(M+H) +.
XHJ-4-36： 1H NMR(400MHz,DMSO-d6)δ8.72(q,J＝6.1Hz,1H),8.63(d,J＝6.9Hz,1H),8.50(d,J＝4.5Hz,1H),8.29(d,J＝1.2Hz,1H),7.85(dd,J＝8.6,1.5Hz,1H),7.81(s,1H),7.65–7.63(m,2H),7.55(t,J＝8.6Hz,2H),7.34(dd,J＝9.4,5.5Hz,2H),7.25(dd,J＝7.1,5.2Hz,3H),6.38(d,J＝1.8Hz,1H),4.49(t,J＝5.4Hz,2H),4.10(m,4H),2.80(m,3H),1.25–1.22(m,3H)；ESI-MS m/z 493.1(M+H) +.
XHJ-4-48： 1H NMR(400MHz,DMSO-d6)δ8.85–8.70(m,2H),8.54(d,J＝4.4Hz,1H),8.07(s,1H),7.91–7.80(m,2H),7.70–7.64(m,2H),7.61–7.51(m,2H),7.34(m,2H),7.30–7.19(m,3H),6.37(d,J＝1.9Hz,1H),4.49(t,J＝5.6Hz,2H),4.03(m,4H),2.80(dd,J＝14.7,4.5Hz,3H),1.67(dd,J＝14.6,7.4Hz,2H),0.80(t,J＝7.4Hz,3H)；ESI-MS m/z 507.1(M+H) +.
ZSQ-20-94： 1H NMR(400MHz,DMSO-d6)δ9.15(t,J＝6.1Hz,1H),8.69(s,1H),8.34(s,1H),8.20(s,2H),7.82–7.76(m,2H),7.57(d,J＝2.0Hz,1H),7.50–7.43(m,2H),7.34–7.22(m,3H),7.17–7.11(m,2H),6.97(d,J＝2.1Hz,1H),5.75(s,2H),4.50(d,J＝6.1Hz,2H)；ESI-MS m/z 425.1(M+H) +.
ZSQ-20-106： 1H NMR(400MHz,DMSO-d6)δ9.14(t,J＝6.0Hz,1H),8.74(s,1H),8.38(s,3H),7.84–7.76(m,2H),7.72(s,1H),7.51–7.43(m,2H),7.35–7.25(m,3H),7.14(dd,J＝7.6,1.9Hz,2H),5.50(s,2H),4.53(d,J＝6.0Hz,2H)；ESI-MS m/z 459.0(M+H) +.
ZSQ-21-1： 1H NMR(400MHz,DMSO-d6)δ8.96–8.87(m,1H),8.70(s,1H),8.35(s,1H),8.23(s,2H),7.82–7.75(m,2H),7.69(d,J＝4.4Hz,1H),7.48–7.40(m,2H),7.35–7.23(m,3H),7.17–7.09(m,2H),5.57(s,2H),4.51(d,J＝6.1Hz,2H)；ESI-MS m/z 443.1(M+H) +.
ZSQ-21-2： 1H NMR(400MHz,DMSO-d6)δ8.70(s,1H),8.34(s,1H),8.21(s,2H),7.82(d,J＝8.4Hz,2H),7.52(d,J＝8.3Hz,2H),7.47(s,1H),7.33(dd,J＝7.9,6.4Hz,2H),7.29–7.21(m,3H),5.72(s,2H),4.75(s,2H),3.58(t,J＝6.8Hz,2H),2.80(t,J＝6.8Hz,2H)；ESI-MSm/z 451.1(M+H) +.
ZSQ-21-8： 1H NMR(400MHz,DMSO-d6)δ9.42(t,J＝6.0Hz,1H),8.61(s,1H),8.31(s,1H),8.25(s,1H),7.86–7.79(m,2H),7.66(s,2H),7.49–7.44(m,2H),7.38–7.28(m,3H),7.25 –7.18(m,2H),5.93(s,2H),4.51(s,2H)；ESI-MS m/z 426.1(M+H) +.
ZSQ-21-13： 1H NMR(400MHz,DMSO-d6)δ8.77(s,1H),8.66(s,1H),8.32(s,1H),8.17(s,2H),7.85(s,1H),7.77(d,J＝8.0Hz,2H),7.50(d,J＝8.1Hz,2H),7.43–7.31(m,4H),5.28(s,2H),4.48(d,J＝5.4Hz,2H)；ESI-MS m/z 425.2(M+H) +.
ZSQ-21-14： 1H NMR(400MHz,DMSO-d6)δ9.21(t,J＝6.0Hz,1H),8.78(s,1H),8.68(s,1H),8.34(s,1H),8.18(s,2H),7.99(d,J＝1.2Hz,1H),7.82–7.72(m,2H),7.47–7.40(m,2H),7.39–7.29(m,3H),7.28–7.20(m,2H),5.68(s,2H),4.48(d,J＝6.0Hz,2H)；ESI-MS m/z 425.2(M+H) +.
ZSQ-21-18： 1H NMR(400MHz,DMSO-d6)δ8.83(t,J＝6.1Hz,1H),8.68(s,1H),8.34(s,1H),8.20(s,2H),7.81–7.73(m,2H),7.47–7.40(m,2H),7.35–7.27(m,2H),7.27–7.20(m,1H),7.10–6.92(m,3H),6.29(d,J＝4.1Hz,1H),5.72(s,2H),4.45(d,J＝6.0Hz,2H)；ESI-MS m/z 458.1(M+H) +.
ZSQ-21-30： 1H NMR(400MHz,DMSO-d6)δ8.72(t,J＝6.1Hz,1H),8.60(s,1H),8.25(s,1H),7.82–7.77(m,2H),7.69(s,2H),7.44(m,2H),7.35–7.30(m,2H),7.28–7.23(m,1H),7.11(d,J＝7.2Hz,2H),6.88(dd,J＝5.9,4.3Hz,1H),5.75(t,J＝4.1Hz,1H),5.58(s,2H),4.45(d,J＝6.1Hz,2H)；ESI-MS m/z 442.1(M+H) +.
ZSQ-22-96： 1H NMR(400MHz,DMSO-d6)δ8.71(s,1H),8.35(s,1H),7.87–7.73(m,2H),7.60–7.46(m,2H),7.37–7.26(m,4H),7.25–7.17(m,1H),4.74(s,2H),4.39(s,2H),3.65(s,3H),3.57(d,J＝6.7Hz,2H),2.83(t,J＝6.7Hz,2H)；ESI-MS m/z 465.2(M+H) +.
生物测试例
测试例中所用的对照化合物结构如下
Figure PCTCN2021118212-appb-000146
Figure PCTCN2021118212-appb-000147
生物测试例1 RIPK1抑制剂对细胞程序性坏死的抑制活性测试
所采用的的生物测试方案为：化合物对TNF诱导的FADD(Fas相关死亡结构域)缺失的Jurkat细胞和L929细胞程序性坏死的影响。
为了在细胞水平上验证本发明中的化合物对细胞程序性坏死的抑制作用，选取与RIP1通路有紧密相关性的细胞类型，即FADD缺陷Jurkat细胞(人源外周血白血病T细胞株)和L929细胞，分别采用两种不同的刺激方式：单独使用肿瘤坏死因子(TNFα)，或者将TNFα与线粒体衍生的半胱氨酸天冬氨酸激活剂(SMAC)SM164联用，通过检测化学发光值来计算细胞活力，从而得出化合物抑制细胞程序性坏死的生物活性。
方法：FADD缺陷Jurkat细胞：体外培养FADD缺陷Jurkat细胞(人源外周血白血病T细胞株)，生长至对数生长期后，收集细胞，1000rpm离心5min，弃上清，调整细胞浓度至2.5×10 5/mL，将细胞接种至384孔板中，每孔40μl。在相应的孔中加入使用细胞培养基稀释的SM164(50nM)和化合物各5μL，37℃预处理1h后，刺激组每孔加入细胞培养基稀释的TNFα(50ng/mL)5μL，对照组加入培养基5μL。放置细胞培养箱(37℃，5％CO2)中培养14h后，每孔加入15μl Cell Titer-Glo溶液，室温孵育30min，检测化学发光值(luminescence)以衡量细胞内ATP水平。以未刺激DMSO对照孔为100％细胞活力。L929细胞：外培养L929细胞(小鼠成纤维细胞)消化后稀释至6.25×10 4/ml，将细胞接种到384孔板中，每孔40μl。放置细胞培养箱(37℃，5％CO2)中培养12h。在相应的孔中加入使用细胞培养基稀释的SM164(500nM)和化合物各5μL，37℃预处理1h后，刺激组每孔加入细胞培养基稀释的TNFα(500ng/mL)5μL，对照组加入培养基5μL，放置细胞培养箱(37℃，5％CO2)中培养14h后，每孔加入15μl Cell Titer-Glo溶液，室温孵育30min，检测化学发光值(luminescence)以衡量细胞内ATP水平。以未刺激DMSO对照孔为100％细胞活力。运用Prism Graphpad统计软件计算化合物EC 50值。结果如图1和表1所示。
表1 RIPK1抑制剂对细胞程序性坏死的抑制活性测试结果
Figure PCTCN2021118212-appb-000148
Figure PCTCN2021118212-appb-000149
图1和表1的实验结果表明：无论是在TNFα单独刺激还是联用SM164刺激的情况下，本发明的优选化合物如QY-10-40、QY-11-76、QY-11-102A/B、QY-12-100、QY-13-19、QY-13-31、QY-13-33、QY-15-59和ET-0935等多个化合物均在人源的FADD缺陷Jurkat细胞上，展现出比临床抑制剂GSK2982772更强的抑制程序坏死的活性；而在GSK2982772几乎失活的鼠源L929细胞上，本发明的优选化合物QY10-40，QY-11-76和QY-11-102A/B等多个化合物依然能够很好地抑制程序性坏死发生。QY10-40在1nM浓度以上基本能完全抑制TNFα诱导的FADD缺陷Jurkat细胞或L929细胞的程序性坏死，且在1000倍有效抑 制浓度下(1μM)，并未展现出细胞毒性。
生物测试例2 RIPK1抑制剂的肝微粒体稳定性测试
所采用的的生物测试方案为：测试化合物在人源或鼠源肝微粒体中的半衰期。
肝微粒体含有多种参与药物代谢的酶，尤其是细胞色素P-450，因此是活体中药物代谢的主要组织之一。化合物在肝微粒体中的稳定性，与其活体药代稳定性有一定的相关性。因此，通过测试不同化合物在人源或鼠源肝微粒体中的半衰期，可以大致地预测化合物在体内的相对稳定性。测试结果如表2所示，具体方法如下：
方法：取0.75μL、0.5mM的样品(待测化合物，和对照品维拉帕米)，9.38μL、20mg/mL肝微粒体(Human and Mouse)加入到239.88μL PBS中(冰上)，分别分装成30μL/管的样本，放到37℃的恒温水浴锅中孵育5min，然后全部取出。在零时刻对照组样品中先加入90μL冰乙腈，然后依次加入15μL 3mM的NADPH溶液，一起放入37℃恒温水浴锅中孵育，待5min、10min、20min、30min、60min后分别取出相应样品，并加入90μL冰乙腈淬灭。所有样品10000rpm离心10min，取上清液放入液相瓶中待LC-MS分析。
表2 RIPK1抑制剂的肝微粒体稳定性测试结果
Figure PCTCN2021118212-appb-000150
实验结果表明：QY-10-40等多个代表性RIPK1抑制剂在人源或鼠源肝微粒体中均展现出了较好的稳定性。
生物测试例3 测试代表性化合物QY-10-40对RIPK1蛋白激酶活性的影响。
所采用的的生物测试方案：测试化合物QY-10-40对RIPK1(1-330)蛋白的激酶活性的影响。体外纯化的RIPK1(1-330)蛋白保留完的激酶活性域，并且保持着良好的激酶活性。通过本发明中的化合物在不同浓度的ATP环境中抑制RIPK1激酶活性的IC 50，得出化合物与RIPK1的结合方式。使用已知的RIPK1 ATP非竞争型抑制剂Nec-1s作为对照。
体外激酶反应的过程与底物浓度有关，底物竞争性的抑制剂的作用效果会随底物浓度的变化而剧烈变化，而底物非竞争抑制剂的效果并不随底物浓度的变化而变化。激酶的ATP竞争型抑制剂通过与ATP竞争结合位点来抑制激酶活性，由于ATP结合位点的保守性，ATP竞争型激酶抑制剂通常特异性较差。相反，ATP非竞争型激酶抑制剂一般具有较高的特异性。
方法：在384孔板中分别加入终浓度为2μM的RIPK1(1-330)蛋白和相应浓度的ATP(1X kinase buffer)，终浓度5μL，每组至少设置3个副孔，37℃反应2h。加入5μL ADP-Glo试剂，该试剂用于停止激酶反应并且去除反应体系中残留的ATP。室温静置40min。加入10μL激酶检测试剂(Kinase Detection Reagent)，该试剂用于将ADP转变为ATP并且在体系中引入检测ATP的荧光素酶(luciferase)和萤光素(luciferin)。室温反应1h。使用7500快速实时PCR系统(Fast Real-Time PCR System)检测发光。运用Prism Graphpad统计软件计算化合物抑制激酶反应的IC 50。实验结果请见图2。
实验结果表明：代表化合物QY-10-40对RIPK1激酶展示出浓度依赖性的有效抑制，其有效半抑制浓度为219nM，优于对照的Nec-1s(有效半抑制浓度为499nM)。
生物测试例4：代表性抑制剂对细胞程序性坏死通路的影响。
所采用的的生物测试方案为：测试化合物对TNFα诱导的FADD缺陷Jurkat或L929细胞程序性坏死通路中关键信号的影响，使用已知的RIPK1 ATP非竞争型抑制剂Nec-1s与临床抑制剂GSK2982772作为对照。
由TNFα诱导的Necroptosis是通过被称作Necrosome(ComplexIIb)的复合物介导信号传递的，这个复合物包括TRADD，FADD，半胱氨酸蛋白酶8(Caspase-8)，RIPK1，RIPK3和MLKL。RIPK1的激酶活性被证明在调控细胞程序性坏死过程中发挥关键性作用。在细胞程序性坏死过程中，RIPK1会发生自磷酸化激活，其中第166位的丝氨酸是主要的磷酸化位点之一。细胞程序性坏死发生后，寡聚化的MLKL转移到细胞膜，通过钙流入的方式介导细胞死亡。
为了验证本发明中的化合物是否通过抑制RIPK1的活性来抑制细胞程序性死亡，并检测细胞程序性坏死通路信号的变化，使用本发明中的化合物处理FADD缺陷Jurkat或L929细胞，再加入TNFα诱导程序性坏死，后收集细胞并使用western blot检测RIPK1的磷酸化和MLKL的寡聚化。
方法：体外培养FADD缺陷Jurkat细胞或L929细胞，生长至对数生长期后，收集细胞，1000rpm离心5min，弃上清，调整细胞浓度至1X10 6/mL。在12孔细胞培养板中，每孔加入1ml细胞，每孔加入0.2μL浓度为50mM药物的DMSO溶液或纯DMSO对照，预处理1个小时。刺激组每组加入0.5μL浓度为100μg/ml的TNFα(PBS溶液)，放置细胞培养箱(37℃，5％CO2)中培养4小时后，3000rpm离心3min收集细胞并使用预冷PBS溶液清洗两遍。尽量去除上清，向细胞沉淀中加入200μL RIPA细胞裂解液，置于4℃摇床裂解30min后，15000rpm 4℃离心15min，取上清的细胞裂解液。使用BCA蛋白定量试剂盒检测每组蛋白含量，并使用PIPA裂解液调齐蛋白量，使终体积为100μL。样品进行western-blot鉴定。Western-blot：向100μL细胞裂解液中加入25μL 5X蛋白loading buffer，95℃加热10min。待样品冷却后，使用SDS-PAGE(9％)凝胶60V进行电泳，30min后切换至120V直至前沿条带电泳至凝胶底端。使用turbo半干转系统，恒定电流0.2A转移80分钟，将凝胶中的蛋白转移至孔径大小为0.2μL的PC膜上。转移后的PC膜置于5％脱脂奶粉(TBST溶液)封闭2h，使用相应的一抗在四度孵育12h。TBST清洗3次，每次10min。使用相应二抗室温孵育2h。TBST清洗三次，每次10min。使用ECL发光液孵育并检测发光信号。实验结果如图3所示
实验结果表明：代表性化合物QY-10-40在1.6nM下即能有效地抑制Jurkat细胞中RIPK1自身的磷酸化以及下游蛋白MLKL的磷酸化，其抑制效果略优于GSK2982772，远优于Nec-1s；在L929细胞中，QY-10-40远优于另外两者。
生物测试例5 RIPK1抑制剂对细胞程序性坏死的抑制活性测试
所采用的的生物测试方案为：化合物对RIPK1 S161E突变的MEFs细胞(小鼠胚胎成纤维细胞)程序性坏死的影响。
在相关炎症发展的严重时期，细胞内的RIPK1已被高度激活，而已报道的RIPK1抑制剂更倾向于抑制新合成的尚未被激活的RIPK1激酶蛋白，对已高度激活的RIPK1抑制较弱，因而对相关炎症的干预效果也更弱、更慢。为了在细胞水平上验证本发明中的化合物对已高度激活的RIPK1激酶的抑制作用，选取稳定表达RIPK1 S161E(模拟RIPK1被磷酸化后的激活状态)的MEFs细胞，采用TNFα-SM164-zVAD(Caspase抑制剂，用于抑制凋亡发生)(TSZ)联用诱导细胞程序性坏死，通过检测化学发光值来计算细胞活力，从而得出化合物抑制细胞程序性坏死的生物活性。
方法：RIPK1回补的MEFs细胞：在RIPK1稳定敲除的细胞中分别回补WT-RIPK1,S161A-RIPK1以及S161E-RIPK1,稳定表达并培养至对数生长期后，收集细胞，1000rpm离心5min，弃上清，调整细胞浓度至5X10 4/mL，将细胞接种至384孔板中，每孔40μL，。在相应的孔中加入使用细胞培养基稀释的SM164(100nM),zVAD(50μM)和化合物各5μL，37℃化预处理1h后，刺激组每孔加入细胞培养基稀释的TNF细胞培养基稀释的(50ng/mL)5μL，对照组加入培养基5μL。放置细胞培养箱(37℃，5％CO 2)中培养12h后，每孔加入15μLCell Titer-Glo溶液，室温孵育30min，检测化学发光值 (luminescence)以衡量细胞内ATP水平。以未刺激DMSO对照孔(Ctrl)为100％细胞活力。运用Prism Graphpad统计软件计算化合物EC 50值。结果如图4所示。
实验结果表明：在表达RIPK1 WT的MEFs细胞中，代表性化合物QY-10-40、QY-11-76和QY11-77(均为600nM)与Nec-1s、GSK2982772(均为10μM)均能有效抑制程序性坏死的发生。在表达RIPK1 S161E突变体的MEFs细胞中，代表性化合物QY-10-40、QY-11-76和QY11-77在600nM下，即能显著地抑制表达在刺激下发生的程序性坏死；与此相对，Nec-1s在10μM浓度下没有表现出抑制，GSK2982772仅表现出了微弱抑制。因此，代表化合物对已高度激活的RIPK1的抑制效果远优于另两者。
生物测试例6 代表性化合物QY-10-40的药代动力学性质
所采用的的生物测试方案为：化合物在小鼠活体上的药物代谢测试。
为了在活体上验证本发明中的化合物对激酶组的选择性，选取代表化合物QY-10-40，利用灌胃(PO，10mg/kg)或静脉注射(IV，1mg/kg)的单次给药方式，测试化合物在小鼠(n＝3)上的药代动力学性质。实验结果如图5所示。
结果表明，QY-10-40单次用药后，在小鼠体内迅速达到峰值，并展示出较好的生物利用度(F)和半衰期(T 1/2)，在灌胃用药10mg/kg后24h中，大部分时间血药浓度能稳定保持在有效抑制浓度(10nM)之上。
生物测试例7 代表性化合物QY-10-40对TNFα引起的系统性炎症反应综合征的影响
所采用的的生物测试方案：尾静脉注射肿瘤坏死因子TNFα会导致小鼠系统性炎症反应综合征的发生，导致小鼠体温下降并死亡。测试代表性化合物QY-10-40对TNFα引发的系统性炎症反应综合征的影响，监测小鼠体温变化和死亡情况。使用已知的RIPK1抑制剂Nec-1s作为对照，临床抑制剂GSK2982772对鼠源RIPK1抑制活性很差，故不使用。
系统性炎症反应综合征也称为炎症风暴，是指严重感染、多发性创伤、烧伤、缺血再灌注、急性胰腺炎等感染性或非感染性伤害导致的全身非特异性炎症反应。该条件下会有大量的炎症因子释放，严重情况导致机体对炎症反应失控，造成多器官功能衰竭、甚至死亡。
方法：待测化合物提前一天根据所需浓度溶解于0.5％羧甲基纤维素，超声过夜。每只小鼠灌胃200μl，20min后，通过尾静脉注射10mg TNFα(溶解于125μl PBS中)。注射后用红外体温仪监测小鼠体温变化。运用Prism Graphpad统计软件计算化合物的影响。实验结果如图6所示。
实验结果表明，在没有抑制剂参与的情况下，小鼠注射TNFα6小时以内即全部死亡；化合物QY-10-40能够在低剂量下(2mg/kg)即可有效抵抗TNFα引起的体温降低、炎症反应及死亡，且在相同剂量下效果优于Nec-1s。
生物测试例8 代表性化合物对小鼠RIPK1 K612R/K612R突变所诱导消化系统慢性炎症的影响
所采用的的生物测试方案：采用RIPK1 K612R/K612R基因突变鼠作为模型鼠，测试代表性化合物QY-10-40或QY-13-19对自发性肠道炎的影响。使用已知的RIPK1抑制剂Nec-1s作为对照。
K612R突变可导致细胞内RIPK1水平下降但异常激活，使其对程序性坏死和caspase-1激活敏感，并响应TLRs信号传导。RIPK1 K612R/K612R小鼠会出现年龄依赖性的RIPK1表达降低和异常激活、自发性肠道炎症和脾肿大等反应。
方法：待测化合物提前一天根据所需浓度溶解于0.5％羧甲基纤维素，超声过夜。小鼠灌胃体积为0.1ml/10g体重，每周测三次体重，在实验终点收集样本。运用Prism Graphpad统计软件计算化合物的影响。
图7A和B分别显示了代表性化合物QY-10-40、QY-13-19及对照化合物Nec-1s对小鼠体重变化和脏器系数(脏器重量/体重)的影响结果。
实验结果表明，化合物QY-10-40或QY-13-19能够抑制K612R突变所致的消化系统炎症对小鼠体重以及脾脏脏器系数的影响，且在更低剂量下(25mg/kg)疗效优于对照化合物Nec-1s(50mg/kg)。
生物测试例9 代表性化合物QY-7-2B与人员重组RIPK1蛋白的共结晶及其结构解析
方法：通过昆虫细胞Sf9表达并纯化经重组的人源RIPK1的催化域截断型蛋白(包含氨基酸残基1-294及C23A、C127A、C233A、C240A四重突变)，并浓缩至约9mg/mL，加入QY-7-2B(终浓度2.5mM)，在18℃下通过悬滴法与蒸汽扩撒法诱导共结晶形成。利用X射线衍射法采集RIPK1与QY-7-2B共晶的晶体衍射数据，经由数据分析确定出分辨率为
Figure PCTCN2021118212-appb-000151
的共晶内部的电子云分布结构，并通过COOT和PHENIX等软件模拟出RIPK1重组蛋白分子与小分子QY-7-2B在原子水平上的三维结合模型。
图8显示了代表性QY-7-2B与人源RIPK1蛋白激酶域的共结晶结构，并展示了本发明系列化合物与对照化合物GSK2982772在和RIPK1蛋白结合模式上的不同。具体地，QY-7-2B与GSK2982772都占据了RIPK1激酶域ATP结合口袋附近的疏水变构口袋，并促使该口袋附近的Leu157构型保持朝外，使得RIPK1被稳定在采取了非激活状态。与GSK2982772不同的是，QY-7-2B的一部分延伸至ATP结合口袋的铰链区，与Met95残基形成了氢键作用，进一步稳定了与RIPK1的相互结合，进而增强了对后者的抑制效果。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (11)

  1. 一种化合物，或其药学上可接受的盐、水合物或溶剂化物，其特征在于，所述的化合物如式I所示；
    Figure PCTCN2021118212-appb-100001
    其中，
    环A为取代或未取代的9-10元含氮杂芳基，其中所述9-10元含氮杂芳基含有1、2、3或4个氮杂原子作为环原子；
    n＝0、1或2；
    R 4各自独立地选自下组：H、CN、卤素、取代或未取代的C 1-6烷基、-O-R b、-S-R b、-N(R b) 2、-C(O)-NR 6-R b、-C(O)-NR 6-C 1-4亚烷基-N(R b) 2、-NR 6-C(O)-R b；
    各个R b各自独立地选自下组：H、取代或未取代的C 1-6烷基；或者两个R b以及与它们相连的氮原子共同形成取代或未取代的5、6或7元杂环烷基，其中，除了与R b相连的N外，所述杂环烷基还含有0、1或2个另外的作为环原子的杂原子；
    R 6选自下组：H、OH、取代或未取代的C 1-4烷基、取代或未取代的C 1-4烷氧基；
    环B选自下组：取代或未取代的C 6-10芳基，取代或未取代的5-10元杂芳基；
    L 1和L 2各自独立地为选自下组的二价基团：
    无、
    Figure PCTCN2021118212-appb-100002
    并且L 1和L 2不同时为无；
    R 1和R 2各自独立地选自下组：H、取代或未取代的C 1-4烷基；
    R 3选自下组：H、OH、取代或未取代的C 1-4烷基、取代或未取代的C 1-4烷氧基；
    环C为无或
    Figure PCTCN2021118212-appb-100003
    其中，W各自独立地选自下组：O、S、C、N、C(R c)、和N(R d)；R c各自独立地选自下组：H、CN、卤素、取代或未取代的C 1-6烷基，R d各自独立地选自下组：H、CN、取代或未取代的C 1-6烷基；
    或者，当环C为
    Figure PCTCN2021118212-appb-100004
    L 1为
    Figure PCTCN2021118212-appb-100005
    且L 2为无时，R 3与位于L 1与环C连接位置邻位的环原子W以及L 1中的-C(O)-共同形成取代或未取代的5、6或7元饱和杂环；其中，所述饱和杂环除了与R 3相连的N外还含有0、1或2个另外的作为环原子的杂原子；
    R 5选自下组：H、OH、取代或未取代的C 1-4烷基、取代或未取代的C 1-4烷氧基；
    或者当环C为无且L 2为
    Figure PCTCN2021118212-appb-100006
    时，R 3和R 5以及与它们相连的原子共同形成取代或未取代的5、6或7元饱和杂环；其中，所述饱和杂环除了与R 3相连的N外还含有0、1或2个另外的作为环原子的杂原子；
    环D选自下组：取代或未取代的C 6-10芳环、和取代或未取代的5-10元杂芳基；
    除非特别说明，所述的取代指基团上的氢原子被一个或者多个(例如1、2、3、或4个等)选自下组的取代基所取代：氧代(＝O)、-CN、卤素、硝基、C 1-6烷基、卤代C 1-6烷基、-OR、-SR、-S(O) 2R、-S(＝O) 2NR 2、-NR 2、-COOR、被R任选取代的C 6-10芳基、被R任选取代的具有1-3个选自N、S和O的杂原子的5-10元杂芳基、被R任选取代的C 3-8环烷基、被R任选取代的具有1-3个选自N、S和O的杂原子的5-12元杂环烷基、被R任选取代的-C 1-4亚烷基-C 6-10芳基、被R任选取代的-C 1-4亚烷基-具有1-3个选自N、S和O的杂原子的5-10元杂芳基、被R任选取代的-C 1-4亚烷基-C 3-8环烷基、被R任选取代的-C 1-4亚烷基-具有1-3个选自N、S和O的杂原子的5-12元杂环烷基；
    R各自独立地选自下组：H、C 1-6烷基、卤代C 1-6烷基、C 1-6羟烷基。
  2. 如权利要求1所述的化合物，其特征在于，R 4各自独立地选自下组：H、取代或未取代的C 1-6烷基、-O-R b、-S-R b、-N(R b) 2、-C(O)-NR 6-R b、-C(O)-NR 6-C 1-4亚烷基-N(R b) 2、-NR 6-C(O)-R b。
  3. 如权利要求1或2所述的化合物，其特征在于，所述的化合物如式II所示；
    Figure PCTCN2021118212-appb-100007
    其中，
    环C为
    Figure PCTCN2021118212-appb-100008
    R 3选自下组：H、OH、取代或未取代的C 1-4烷基、取代或未取代的C 1-4烷氧基；
    或者，R 3与位于-C(O)-与环C连接位置邻位的环原子W以及该-C(O)-共同形成取代或未取代的5、6或7元饱和杂环；其中，所述饱和杂环除了与R 3相连的N外还含有0、1或2个另外的作为环原子的杂原子；
    R 5选自下组：H、OH、取代或未取代的C 1-4烷基、取代或未取代的C 1-4烷氧基；
    环A、环B、环D、W、R 1、R 2、R 4和n如式I中所定义。
  4. 如权利要求1或2所述的化合物，其特征在于，所述的化合物如式III所示；
    Figure PCTCN2021118212-appb-100009
    其中，
    X 1、X 2、X 3、X 4、X 5和X 6各自独立地选自下组：N和C(R a)；并且X 1、X 2、X 3、X 4、X 5和X 6中至多3个为N；
    R a各自独立地选自下组：无、H、取代或未取代的C 1-6烷基；
    环C为
    Figure PCTCN2021118212-appb-100010
    R 3选自下组：H、OH、取代或未取代的C 1-4烷基、取代或未取代的C 1-4烷氧基；
    或者，R 3与位于-C(O)-与环C连接位置邻位的环原子W以及该-C(O)-共同形成取代或未取代的5、6或7元饱和杂环；其中，所述饱和杂环除了与R 3相连的N外还含有0、1或2个另外的作为环原子的杂原子；
    R 5选自下组：H、OH、取代或未取代的C 1-4烷基、取代或未取代的C 1-4烷氧基；
    环B、环D、W、R 1、R 2、R 4和n如式I中所定义。
  5. 如权利要求1或2所述的化合物，其特征在于，所述的化合物如式IV-1或IV-2所示；
    Figure PCTCN2021118212-appb-100011
    其中，
    X 1、X 2、X 3、X 4、X 5和X 6各自独立地选自下组：N和C(R a)；并且X 1、X 2、X 3、X 4、X 5和X 6中至多3个为N；
    R 3选自下组：H、OH、取代或未取代的C 1-4烷基；
    或者，R 3与环原子W以及该-C(O)-共同形成取代或未取代的5、6或7元饱和杂环；其中，所述饱和杂环除了与R 3相连的N外还含有0、1或2个另外的作为环原子的杂原子；
    R 5选自下组：H、OH、取代或未取代的C 1-4烷基；
    环B、环D、W、R a、R 1、R 2、R 4和n如式I中所定义。
  6. 如权利要求5所述的化合物，其特征在于，环B和环D各自独立地为未取代的苯基，或者为被1或2个选自下组的取代基所取代的苯基：卤素、C 1-4烷基、卤代C 1-4烷基。
  7. 如权利要求1或2所述的化合物，其特征在于，所述的化合物选自表A：
    表A
    Figure PCTCN2021118212-appb-100012
    Figure PCTCN2021118212-appb-100013
    Figure PCTCN2021118212-appb-100014
    Figure PCTCN2021118212-appb-100015
    Figure PCTCN2021118212-appb-100016
    Figure PCTCN2021118212-appb-100017
  8. 一种如权利要求3所述的化合物的制备方法，其特征在于，
    i.所述的制备方法为方法一，并且所述的方法一包括步骤：
    在惰性溶剂中，使如式II-2A所示化合物和如式II-2B所示化合物反应，得到如式II-2C所示化合物；
    Figure PCTCN2021118212-appb-100018
    其中，
    R 3选自下组：H、OH、取代或未取代的C 1-4烷基；
    环A、环B、环C、环D、R c、R 1、R 2、R 4、R 5和n如式II中所定义；
    或者，
    ii.所述方法为方法二，并且所述方法二包括步骤：
    a)在惰性溶剂中，使如式II-2A所示化合物和如式II-2B所示化合物反应，得到如式II-2C所示化合物；
    Figure PCTCN2021118212-appb-100019
    b)在惰性溶剂中，使如式II-2C所示化合物反应形成如式II所示化合物
    Figure PCTCN2021118212-appb-100020
    其中，
    R 3与位于-C(O)-与环C连接位置邻位的环原子W以及L 1中的-C(O)-共同形成取代或未取代的5、6或7元饱和杂环；
    L 3为单键、取代或未取代的C 1-2亚烷基；
    饱和杂环、环A、环B、环D、R c、R 1、R 2、R 4、R 5和n如式II中所定义。
  9. 一种药物组合物，其特征在于，所述的药物组合物包括(a)治疗有效量的如权利要求1或2所述的化合物、或其药学上可接受的盐、水合物或溶剂化物；和(b)药学上可接受的载体。
  10. 如权利要求1或2所述的化合物或如权利要求9所述的药物组合物在制备用于治疗或预防与细胞程序性坏死和/或人受体相互作用蛋白1激酶(RIPK1)相关的疾病或病症的药物中的用途。
  11. 如权利要求10所述的用途，其特征在于，所述的疾病或病症选自下组中的一种或多种：退行性疾病、炎症、缺血再灌注损伤、病原体感染、帕金森氏症(PD)、亨老年性黄斑变性、自身免疫性疾病、视网膜脱离诱导的感光细胞坏死、青光眼、顺铂诱导的肾损伤和创伤性脑损伤、高血脂所致动脉粥样硬化、由RIPK1依赖性细胞凋亡、坏死或细胞因子生成相关的其他疾病、细菌感染、病毒感染和溶酶体贮积症。
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