Inhibitors of apoptosis and methods of making and using the same

Abstract

The present application provides a kind of cell programmed necrosis inhibitor and its preparation method and use. Specifically, the present application provides a kind of compound, or its pharmaceutically acceptable salt, hydrate or solvate, wherein, the compound is as shown in formula I. The present application also provides the preparation method of the compound and the pharmaceutical composition containing it or its use in treating or preventing diseases or conditions related to cell programmed necrosis and / or human receptor interaction protein 1 kinase (RIPK1).

Description

Technical Field

[0001] This invention belongs to the field of small molecule compounds, specifically relating to a class of cell programmed necrosis inhibitors, their preparation methods, and uses. Background Technology

[0002] During development and aging, the human body is always accompanied by the dynamic regulation of cell proliferation and death. Active cell death is indispensable in physiological activities such as normal development, resistance to pathogenic microorganisms, and maintenance of homeostasis. Its imbalance often leads to various diseases, including developmental deformities, immune system disorders, neurodegenerative diseases, and cancer, and even individual death. Therefore, intervention in programmed cell death is of great significance for disease treatment research. Apoptosis was the first programmed cell death mechanism to be elucidated, and in recent years, programmed cell necrosis has become a new hot topic in the field of cell death. Numerous studies have reported that programmed cell necrosis is an important pathological feature in various degenerative diseases (such as Alzheimer's disease (AD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), retinal degenerative diseases, etc.), inflammatory diseases (enteritis, rheumatoid arthritis, psoriasis, etc.), ischemia-reperfusion injury (cerebral infarction, myocardial infarction, etc.), and pathogen infections. Furthermore, programmed necrosis also participates in the regulation of the tumor microenvironment: lung cancer cells can induce programmed necrosis in specific cells of the blood vessel wall to facilitate passage through the circulatory system and metastasis; the high expression of the main components of necrosomes in pancreatic cancer can induce the expression of the chemokine CXCL1, thereby inhibiting the body's immune response. Therefore, inhibiting the occurrence of programmed necrosis is widely recognized as helpful in the treatment and relief of various diseases.

[0003] Studies have shown that tumor necrosis factor-α (TNF-α) is one of the main pathways stimulating programmed cell death in vivo, and its downstream signaling pathway is currently the most clearly understood necrosis signaling pathway. In the classic TNF-α-induced cell death process, TNF-α first binds to its receptor TNFR1, inducing its trimerization and recruiting a series of intracellular factors—including multiple proteins such as TRADD, TRAF2, RIPK1, and cIAP1 / 2—to form signaling complex I. Complex I can recruit and activate the IKKα / IKKβ / IKKγ complex and the NF-κB pathway, and after dissociation, some enter the cytoplasm to form a new protein complex IIa. This complex then recruits proteins such as procaspase-8 via FADD or TRADD to activate downstream caspase-3 and caspase-7 and mediate apoptosis. In the absence of FADD or with caspase inhibitors, the TNF-α-induced activated 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, ultimately disrupting the cell membrane structure and leading to necrosis.

[0004] Multiple adaptor proteins, ubiquitin ligases, deubiquitinating enzymes, and kinases participate in regulating downstream signaling pathways of TNF-α-induced programmed cell death (CCD). For example, the E3 ubiquitin ligase cIAP inhibits the activation and necrosis process of RIPK1 by K63 ubiquitination; the deubiquitinating enzyme CYLD cleaves the K63 ubiquitin chain of RIPK1, thereby activating RIPK1 kinase activity and promoting the formation of necrosomes, ultimately achieving a positive regulatory effect on cell death; the adaptor protein SPATA2 promotes CYLD deubiquitinating enzyme activity and inhibits NF-κB and MAPK signaling pathways, thereby positively regulating CCD; the kinase protein TAK1 inhibits the kinase activity of RIPK1 by phosphorylating the Ser321 site, thereby negatively regulating CCD; and the deubiquitinating enzyme A20 (TNF-α-induced protein 3) and the adaptor protein TAB2 (TAK1 binding protein) also play a role in CCD regulation. 2) Several regulatory factors also participate in this regulatory process; the kinase protein TBK1 inhibits the activation of RIPK1 by phosphorylating the Thr189 site, and the inactivation mutation of TBK1 during aging is also an important pathogenic risk for neurodegenerative diseases such as ALS and FTD; the adaptor protein Optineurin (OPTN) negatively regulates programmed necrosis by inhibiting RIPK1 kinase activity, and the loss of OPTN in ALS may promote progressive myelination disorder and axonal degeneration. Therefore, it can be seen that in the TNF-α-induced programmed necrosis signaling pathway network, functional abnormalities of multiple regulatory components mediate the occurrence of programmed necrosis through the activation of RIPK1 kinase, a core regulatory factor.

[0005] Therefore, RIPK1 kinase is widely recognized as a potential therapeutic target for diseases related to programmed cell death. First-in-class RIPK1 inhibitors, such as Necrostatin-1 (Nec-1) and its analogues, have demonstrated clear efficacy in preclinical studies against various degenerative diseases, inflammation, and cancer. For example, it has alleviating effects on AD, ALS, MS, Parkinson's disease (PD), Huntington's disease (PD), inflammatory bowel disease, and age-related macular degeneration; it has protective effects against psoriasis, retinitis pigmentosa, inflammatory bowel disease, autoimmune diseases, tadalafil-induced acute pancreatitis, and sepsis / systemic inflammatory response syndrome (SIRS); it can effectively alleviate ischemic brain injury, ischemic myocardial injury, retinal ischemia / reperfusion injury, retinal detachment-induced photoreceptor cell necrosis, glaucoma, renal ischemia-reperfusion injury, cisplatin-induced kidney injury, and traumatic brain injury; it can at least partially alleviate other diseases related to RIPK1-dependent apoptosis, necrosis, or cytokine production, including hematologic and solid organ malignancies, bacterial and viral infections (including tuberculosis, influenza, etc.), and lysosomal storage diseases (especially Gaucher disease). Currently, Nec-1 derivatives have entered clinical trials for the treatment of ALS and AD; another class of RIPK1 inhibitors, GSK2982772, is also in clinical trials for the treatment of various autoimmune diseases. However, existing programmed necrosis inhibitors all have varying degrees of shortcomings, such as insufficient in vivo inhibitory activity, poor pharmacokinetic properties, and low oral bioavailability. Some cannot cross the blood-brain barrier to enter the central nervous system, or they cannot effectively inhibit murine RIPK1, making preclinical animal experiments difficult. These shortcomings limit their further research and clinical application.

[0006] Therefore, developing small-molecule RIPK1 kinase activity inhibitors with high specificity, high activity, and blood-brain barrier penetration has clinical application value is currently a challenge and a hot topic in the research on the treatment of diseases related to programmed cell death. In summary, there is an urgent need in this field for novel RIPK1 inhibitors and / or programmed cell death inhibitors with novel chemical structures and more prominent pharmacokinetic and pharmacodynamic properties as candidate drugs for the prevention and treatment of diseases involving cell death and / or inflammation. Summary of the Invention

[0007] The purpose of this invention is to provide a class of RIPK1 inhibitors and / or cell programmed necrosis inhibitors with novel structures.

[0008] In a first aspect of the invention, a compound, or a pharmaceutically acceptable salt, hydrate or solvate thereof, is provided, characterized in that the compound is as shown in Formula I;

[0009]

[0010] in,

[0011] Ring A is a substituted or unsubstituted 9-10 member nitrogen-containing heteroaryl group, wherein the 9-10 member nitrogen-containing heteroaryl group contains 1, 2, 3 or 4 nitrogen heteroatoms as ring atoms;

[0012] n = 0, 1, or 2;

[0013] R 4 Each is independently selected from the following groups: 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 ;

[0014] Each R b Each is independently selected from the following groups: H, substituted or unsubstituted C. 1-6 Alkyl; or two R b Together with the nitrogen atoms attached to them, they form substituted or unsubstituted 5, 6, or 7-membered heterocyclic alkyl groups, wherein, except for those with R b In addition to the connected N, the heterocyclic alkyl group also contains 0, 1 or 2 other heteroatoms as ring atoms;

[0015] R 6 Selected from the following group: H, OH, substituted or unsubstituted C 1-4 Alkyl, substituted or unsubstituted C 1-4 Alkoxy;

[0016] Cycle B is selected from the following group: substituted or unsubstituted C 6-10 Aryl, substituted or unsubstituted 5-10 heteroaryl groups;

[0017] L 1 and L 2 Each is independently a divalent group selected from the following group:

[0018] none,

[0019] And L 1 and L 2 Not simultaneously equal to none;

[0020] R 1 and R 2 Each is independently selected from the following groups: H, substituted or unsubstituted C.1-4 alkyl;

[0021] R 3 Selected from the following group: H, OH, substituted or unsubstituted C 1-4 Alkyl, substituted or unsubstituted C 1-4 Alkoxy;

[0022] Ring C is none or Among them, W is independently selected from the following groups: O, S, C, N, C(R) c ), and N(R) d ); R c Each is independently selected from the following group: H, CN, halogen, substituted or unsubstituted C. 1-6 Alkyl, R d Each is independently selected from the following groups: H, CN, substituted or unsubstituted C. 1-6 Alkyl (preferably, R) d For substituted or unsubstituted C 1-6 Alkyl, more preferably, R d Selected from the group consisting of methyl, ethyl, propyl, and butyl;

[0023] Or, when ring C is L 1 for (L 1 The carbonyl group is connected to the ring C) and L 2 When there is no time, R 3 With L 1 The ring atoms W and L adjacent to the ring C are 1 The -C(O)- groups in the mixture form substituted or unsubstituted 5, 6, or 7-membered saturated heterocycles (preferably 6-membered saturated heterocycles); wherein, the saturated heterocycle, in addition to being substituted with R, forms a substituted or unsubstituted 5, 6, or 7-membered saturated heterocycle. 3 In addition to the connected N, there are 0, 1 or 2 other heteroatoms that act as ring atoms;

[0024] R 5 Selected from the following group: H, OH, substituted or unsubstituted C 1-4 Alkyl, substituted or unsubstituted C 1-4 Alkoxy;

[0025] Or when ring C is nonexistent and L 2 for (L 1 N(R) 3 When R is connected to ring C, 3 and R 5 And the atoms bonded to them together form substituted or unsubstituted 5, 6, or 7-membered saturated heterocycles; wherein, the saturated heterocycles, in addition to being associated with R, form substituted or unsubstituted 5, 6, or 7-membered saturated heterocycles. 3 In addition to the connected N, there are 0, 1 or 2 other heteroatoms that act as ring atoms;

[0026] Ring D is selected from the following group: substituted or unsubstituted C 6-10 Aromatic rings, and substituted or unsubstituted 5-10 membered heteroaryl groups;

[0027] Unless otherwise specified, the term "substitution" refers to the substitution of a hydrogen atom on a group by one or more (e.g., 1, 2, 3, or 4) substituents selected from the group consisting of: oxo (=O), -CN, halogen (e.g., F, Cl, Br, or I), nitro, C. 1-6 Alkyl, Halogenated C 1-6 Alkyl groups, -OR, -SR, -S(O)2R, -S(=O)2NR2, -NR2, -COOR, and C groups optionally substituted with R. 6-10 aryl, 5-10 heteroaryl groups with 1-3 heteroatoms selected from N, S and O, optionally substituted by R, and C groups optionally substituted by R 3-8 Cycloalkyl, 5-12 membered heterocyclic alkyl groups having 1-3 heteroatoms selected from N, S and O, optionally substituted with R, -C 1-4 Alkylene-C 6-10 aryl, -C optionally substituted by R 1-4 Alkylene – a 5-10 membered heteroaryl group having 1-3 heteroatoms selected from N, S, and O, with -C optionally substituted by R. 1-4 Alkylene-C 3-8 Cycloalkyl, -C optionally substituted with R 1-4 Alkylene – 5-12 membered heterocyclic alkyl groups having 1-3 heteroatoms selected from N, S and O;

[0028] R is independently selected from the following groups: H, C 1-6 Alkyl, Halogenated C 1-6 Alkyl, C 1-6 Hydroxyalkyl.

[0029] In another preferred embodiment, the heteroatoms are each independently selected from the group consisting of O, N, and S; more preferably, each is independently O or N.

[0030] In another preferred embodiment, ring A is configured as shown in the group consisting of:

[0031]

[0032] Among them, X 1 X 2 X 3 X 4 X 5 and X 6 Each is independently selected from the following groups: N and C(R) a ); and X 1 X 2 X3 X 4 X 5 and X 6 At most 3 of them are N;

[0033] R a Each is independently selected from the following groups: none, H, substituted or unsubstituted C. 1-6 alkyl.

[0034] In another preferred example, X 1 X 2 X 3 X 4 X 5 and X 6 Each of them is independently selected from the following groups: C, N, and CH.

[0035] In another preferred embodiment, As shown in the structure selected from the following group:

[0036]

[0037] In another preferred embodiment, ring B is selected from the group consisting of substituted or unsubstituted phenyl groups, substituted or unsubstituted 5- or 6-membered heteroaryl groups.

[0038] In another preferred embodiment, ring B is phenyl.

[0039] In another preferred embodiment, L 1 for (L 1 The carbonyl group is connected to the ring C) and L 2 None (i.e., -L) 1 -L 2 -for ).

[0040] In another preferred embodiment, R 1 and R 2 Each is independently selected from the following group: H and methyl.

[0041] In another preferred embodiment, ring C is L 1 for (L 1 The carbonyl group is connected to the ring C) and L 2 For none, R 3 For H, or R 3 With L 1 W and L, which are adjacent to the position connected to ring C 1 The C(O) in the mixture together form substituted or unsubstituted 6-membered saturated heterocycles.

[0042] In another preferred embodiment, the ring C is as follows: As shown.

[0043] In another preferred embodiment, the ring C is shown as a structure selected from the group consisting of:

[0044]

[0045] Among them, W 1 Selected from the following groups: O, S, N(R) d );W 2 W 3 W 4 W 5 and W 6 Each is independently selected from the following groups: N and C(R) c );

[0046] Or, when L 1 for (L 1 The carbonyl group is connected to the ring C) and L 2 When there is no time, R 3 With L 1 W, which is adjacent to the position connected to ring C 1 W 2 W 3 W 4 or W 5 and L 1 The carbonyl groups in the rings together form substituted or unsubstituted 6-membered saturated heterocycles.

[0047] In another preferred embodiment, the ring C is selected from the group consisting of:

[0048]

[0049] Among them, R c Each is independently selected from the following group: H, CN, halogen, substituted or unsubstituted C. 1-6 Alkyl, R d Each is independently selected from the following groups: H, substituted or unsubstituted C. 1-6 alkyl.

[0050] In another preferred embodiment, R c Each of the following is independently selected: H, CN, F, Cl, Br, C 1-4 Alkyl; and / or R d Selected from the following groups: H, C 1-4 Alkyl (preferably, R) d C 1-4 alkyl).

[0051] In another preferred embodiment, ring D is phenyl.

[0052] In another preferred embodiment, rings A, B, C, D, and L... 1L 2 R 4 R 5 n and n are independent groups corresponding to specific compounds in Table A.

[0053] In another preferred embodiment, the compound is shown as in Formula II;

[0054]

[0055] in,

[0056] Ring C is

[0057] R 3 Selected from the following group: H, OH, substituted or unsubstituted C 1-4 Alkyl, substituted or unsubstituted C 1-4 Alkoxy;

[0058] Or, R 3 The ring atom W adjacent to the -C(O)- ring C- and the -C(O)- ring together form a substituted or unsubstituted 5, 6, or 7-membered saturated heterocycle (preferably a 6-membered saturated heterocycle); wherein, the saturated heterocycle, in addition to being connected to R, forms a substituted or unsubstituted 5, 6, or 7-membered saturated heterocycle. 3 In addition to the connected N, there are 0, 1 or 2 other heteroatoms that act as ring atoms;

[0059] R 5 Selected from the following group: H, OH, substituted or unsubstituted C 1-4 Alkyl, substituted or unsubstituted C 1-4 Alkoxy;

[0060] Ring A, Ring B, Ring D, W, R 1 R 2 R 4 And n is as defined in Equation I.

[0061] In another preferred embodiment, rings A, B, C, D, and R... 1 R 2 R 3 R 4 R 5 n and n are independent groups corresponding to the specific compounds in Table A.

[0062] In another preferred embodiment, the compound is shown as in Formula III;

[0063]

[0064] in,

[0065] X 1 X 2 X 3X 4 X 5 and X 6 Each is independently selected from the following groups: N and C(R) a ); and X 1 X 2 X 3 X 4 X 5 and X 6 At most 3 of them are N;

[0066] R a Each is independently selected from the following groups: none, H, substituted or unsubstituted C. 1-6 alkyl;

[0067] Ring C is

[0068] R 3 Selected from the following group: H, OH, substituted or unsubstituted C 1-4 Alkyl, substituted or unsubstituted C 1-4 Alkoxy;

[0069] Or, R 3 The ring atom W adjacent to the -C(O)- ring C- and the -C(O)- ring together form a substituted or unsubstituted 5, 6, or 7-membered saturated heterocycle (preferably a 6-membered saturated heterocycle); wherein, the saturated heterocycle, in addition to being connected to R, forms a substituted or unsubstituted 5, 6, or 7-membered saturated heterocycle. 3 In addition to the connected N, there are 0, 1 or 2 other heteroatoms that act as ring atoms;

[0070] R 5 Selected from the following group: H, OH, substituted or unsubstituted C 1-4 Alkyl, substituted or unsubstituted C 1-4 Alkoxy;

[0071] Rings B, D, W, and R 1 R 2 R 4 And n is as defined in Equation I.

[0072] In another preferred example, 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 n and n are independent groups corresponding to the specific compounds in Table A.

[0073] In another preferred embodiment, the compound is shown as in formula IV-1 or IV-2;

[0074]

[0075] in,

[0076] X 1 X 2 X 3 X 4 X 5 and X 6 Each is independently selected from the following groups: N and C(R) a ); and X 1 X 2 X 3 X 4 X 5 and X 6 At most 3 of them are N;

[0077] R 3 Selected from the following group: H, OH, substituted or unsubstituted C 1-4 alkyl;

[0078] Or, R 3 Together with the ring atom W and the -C(O)-, it forms a substituted or unsubstituted 5, 6, or 7-membered saturated heterocycle (preferably a 6-membered saturated heterocycle); wherein, the saturated heterocycle, in addition to being substituted with R, forms a substituted or unsubstituted 5, 6, or 7-membered saturated heterocycle. 3 In addition to the connected N, there are 0, 1 or 2 other heteroatoms that act as ring atoms;

[0079] R 5 Selected from the following group: H, OH, substituted or unsubstituted C 1-4 alkyl;

[0080] Rings B, D, W, and R a R 1 R 2 R 4 And n is as defined in Equation I.

[0081] In another preferred example, 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 n and n are independent groups corresponding to the specific compounds in Table A.

[0082] In another preferred embodiment, ring B and ring D are each independently an unsubstituted phenyl group, or a phenyl group substituted with one or two substituents selected from the group consisting of: halogen, C 1-4 Alkyl, Halogenated C 1-4 alkyl.

[0083] In another preferred embodiment, the compound is selected from Table A.

[0084] In a second aspect of the invention, a method for preparing a compound as shown in Formula II is provided, wherein,

[0085] i. The preparation method is Method 1, and Method 1 includes the following steps:

[0086] In an inert solvent, the compound shown in formula II-2A and the compound shown in formula II-2B are reacted to give the compound shown in formula II-2C.

[0087]

[0088] in,

[0089] R 3 Selected from the following group: H, OH, substituted or unsubstituted C 1-4 Alkyl, substituted or unsubstituted C 1-4 Alkoxy;

[0090] 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;

[0091] or,

[0092] ii. The method is method two, and method two includes the following steps:

[0093] a) In an inert solvent, the compound shown in formula II-2A and the compound shown in formula II-2B are reacted to give the compound shown in formula II-2C;

[0094]

[0095] b) In an inert solvent, react the compound shown in formula II-2C to form the compound shown in formula II.

[0096]

[0097] in,

[0098] R 3The ring atom W and L located adjacent to the -C(O)- ring C bond position. 1 The -C(O)- groups in the rings together form substituted or unsubstituted 5, 6, or 7-membered saturated heterocycles;

[0099] L 3 C is a single bond, substituted or unsubstituted 1-2 Alkylene;

[0100] Saturated heterocycles, ring A, ring B, ring D, R c R 1 R 2 R 4 R 5 And n is as defined in Formula II of the first aspect.

[0101] In another preferred embodiment, the inert solvent is selected from the group consisting of: dichloromethane,

[0102] In another preferred embodiment, the reaction is carried out in the presence of HATU and N,N-diisopropylethylamine.

[0103] In a third aspect of the invention, a pharmaceutical composition is provided comprising (a) a therapeutically effective amount of the compound as described in the first aspect, or a pharmaceutically acceptable salt, hydrate, or solvate thereof; and (b) a pharmaceutically acceptable carrier.

[0104] In a fourth aspect of the invention, the use of a compound as described in the first aspect or a pharmaceutical composition as described in the third aspect in the preparation of a medicament for treating or preventing diseases or conditions associated with programmed cell death and / or human receptor-interacting protein 1 kinase (RIPK1).

[0105] In another preferred embodiment, the compound or pharmaceutical composition treats or prevents the disease or condition by inhibiting human receptor-interacting protein 1 kinase (RIPK1).

[0106] In another preferred embodiment, the human receptor-interacting protein 1 kinase (RIPK1) includes RIPK1 in an inactivated (or unactivated) state and RIPK1 in an activated state.

[0107] In another preferred embodiment, the compound or pharmaceutical composition may also treat or prevent (especially treat) the disease or condition by inhibiting the activated human receptor-interacting protein 1 kinase (RIPK1).

[0108] In another preferred embodiment, the inhibitor of human receptor-interacting protein 1 kinase (RIPK1) includes one or more of the following: inhibiting the activity of RIPK1 or inhibiting the phosphorylation of RIPK1.

[0109] In another preferred embodiment, the compound or pharmaceutical composition treats or prevents the disease or condition by inhibiting the programmed necrosis signaling pathway.

[0110] In another preferred embodiment, the inhibition of the programmed cell death signaling pathway includes one or more of the following: inhibition of RIPK1 activity, inhibition of RIPK1 phosphorylation, or inhibition of MLKL phosphorylation.

[0111] In another preferred embodiment, the disease or condition is selected from one or more of the following groups: degenerative diseases, inflammation, ischemia-reperfusion injury, pathogen infection, Parkinson's disease (PD), age-related macular degeneration, autoimmune diseases, retinal detachment-induced photoreceptor cell necrosis, glaucoma, cisplatin-induced kidney injury and traumatic brain injury, hyperlipidemia-related atherosclerosis, other diseases related to RIPK1-dependent apoptosis, necrosis or cytokine production, bacterial infection, viral infection and lysosomal storage diseases.

[0112] In another preferred embodiment, the degenerative diseases include, for example, Alzheimer's disease (AD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), and retinal degenerative diseases.

[0113] In another preferred embodiment, the inflammation includes one or more of the following: enteritis, rheumatoid arthritis, psoriasis, retinitis pigmentosa, inflammatory bowel disease, Tinton's disease (PD) inflammatory bowel disease, dermal induced acute pancreatitis, and sepsis / systemic inflammatory response syndrome (SIRS).

[0114] In another preferred embodiment, the ischemia-reperfusion injury includes one or more of the following: cerebral infarction, myocardial infarction, ischemic brain injury, ischemic myocardial injury, retinal ischemia / reperfusion injury, and renal ischemia-reperfusion injury.

[0115] In another preferred embodiment, the other diseases associated with RIPK1-dependent apoptosis, necrosis, or cytokine production include one or more of the following: hematologic and solid organ malignancies.

[0116] In another preferred embodiment, the viral infection includes one or more of the following diseases or conditions: tuberculosis, influenza, coronavirus infection and pneumonia caused by it.

[0117] In another preferred embodiment, the lysosomal storage disease includes Gaucher disease.

[0118] In a fifth aspect of the invention, a method for treating or preventing diseases or conditions associated with programmed cell death and / or human receptor-interacting protein 1 kinase (RIPK1) is provided, the method comprising: administering to a subject a therapeutically effective amount of a compound as described in the first aspect or a pharmaceutical composition as described in the third aspect.

[0119] In a sixth aspect of the invention, a method for inhibiting programmed cell death is provided, comprising the step of: culturing cells in the presence of the compound as described in the first aspect, thereby inhibiting programmed cell death.

[0120] In another preferred embodiment, the method is non-therapeutic in vitro.

[0121] In a seventh aspect of the invention, a method for inhibiting RIPK1 protein kinase activity is provided, comprising the steps of contacting RIPK1 protein kinase with a compound as described in the first aspect, thereby inhibiting RIPK1 protein kinase activity.

[0122] In another preferred embodiment, the method is non-therapeutic in vitro.

[0123] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0124] Figure 1 The concentration-activity curves of representative compounds such as QY-10-40 and control compounds inhibiting programmed necrosis in FADD-deficient Jurkat or L929 cells are shown.

[0125] Figure 2 This is a concentration-inhibition curve representing the kinase activity of compound QY-10-40 on RIPK1(1-330) protein.

[0126] Figure 3 The effects of QY-10-40, Nec-1s, and GSK2982772 at different concentrations on TNF pathway signaling activated by TNFα in combination with SM164 were shown. Figure 4 The results of the assay show the inhibitory activity of RIPK1 inhibitors against programmed cell death continuously activated by RIPK1.

[0127] Figure 5 The concentration-time curves of the representative compound QY-10-40 in plasma, as well as its pharmacokinetic parameters, are shown.

[0128] Figure 6 The results show the effects of the representative compound QY-10-40 and the control compound Nec-1s on changes in body temperature in mice.

[0129] Figure 7 The co-crystallization structure of the representative QY-7-2B and the human RIPK1 protein kinase domain is shown, and the differences in the binding mode of the series of compounds of the present invention and the control compound GSK2982772 with the RIPK1 protein are demonstrated. Detailed Implementation

[0130] Through long-term and in-depth research, the inventors unexpectedly discovered a novel class of programmed cell death inhibitors. These inhibitors exhibit excellent RIPK1 inhibitory activity. Therefore, they can be used to prepare pharmaceutical compositions for the prevention and / or treatment of diseases involving cell death, RIPK1, and / or inflammation. In particular, the preferred compounds provided by this invention also exhibit excellent inhibitory activity against activated RIPK1, thus, compared to existing RIPK1 inhibitors that only inhibit inactivated RIPK1, the compounds provided by this invention provide a more rapid improvement or treatment of diseases or conditions involving cell death and / or RIPK1-related inflammation (such as inflammation). Based on the above findings, the inventors completed this invention.

[0131] the term

[0132] Unless otherwise expressly stated, the terms used in this invention and herein have the following meanings:

[0133] As used herein, the term "alkyl" refers to a straight-chain or branched alkyl group having a specified number of carbon atoms. For example, C 1-6 Alkyl groups are straight-chain or branched alkyl groups having 1 to 6 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, etc., or similar groups.

[0134] As used herein, the term "cycloalkyl" refers to a cyclic alkyl group having a specified number of carbon atoms. For example, "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, and cyclooctyl.

[0135] As used herein, the term "alkoxy" refers to an alkyl group as defined above, which is attached to the remainder of the molecule via an oxygen atom. For example, C 1-6 Alkoxy refers to C 1-6 Alkyl-O-. Preferably, C 1-6 Alkoxy groups can include methoxy, ethoxy, and isopropoxy groups.

[0136] As used in this article, the term "halogen" refers to F, Cl, Br, and I.

[0137] As used herein, the term "haloalkyl" refers to an alkyl group substituted with a halogen (the definition of an alkyl group is as described above). Preferably, haloalkyl groups include trifluoromethyl, difluoromethyl, trifluoromethoxy, perfluoroethyl, etc.

[0138] As used herein, the term "cycloalkyl" refers to a ring having a specified number of ring atoms (e.g., C10, C20, C30, C40, C50, C60, C7 ... 3-8 Cycloalkyl refers to a cyclic alkyl group having 3, 4, 5, 6, 7, or 8 ring atoms and being a fully saturated hydrocarbon ring or having no more than one double bond between the ring apexes. "Cycloalkyl" also refers to bicyclic and polycyclic hydrocarbon rings, such as bicyclic [2.2.1]heptane, bicyclic [2.2.2]octane, etc. The term "heterocyclic alkyl" or "heterocyclic alkyl" refers to a cycloalkyl group having a specified 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. Heterocyclic alkyl groups can be monocyclic, bicyclic, or polycyclic systems. Non-limiting examples of heterocyclic alkyl groups include pyrrolidine, imidazoline, pyrazolidine, butyrolactam, valproic acid, imidazolidinone, hydantoin, dioxolane, benzodiimide, piperidine, 1,4-dioxane, morpholine, thiomorpholine, thiomorpholine-S-oxide, thiomorpholine-S,S-oxide, piperazine, pyran, pyridone, 3-pyrrolidine, thiaran, pyranone, tetrahydrofuran, tetrahydrothiophene, quinine ring, etc. Heterocyclic alkyl groups can be attached to the rest of the molecule via a cyclic carbon or heteroatom.

[0139] The term "alkylene" itself, or as part of another substituent, refers to a divalent group derived from an alkane, such as -CH2CH2CH2CH2-.

[0140] Unless otherwise stated, the term "aryl" refers to a polyunsaturated (usually aromatic) hydrocarbon group containing a specified number of ring atoms, which can be monocyclic or fused together or covalently linked polycyclic (e.g., bicyclic). The term "heteroaryl" refers to an aryl group (or ring) having a specified number of ring atoms and containing 1 to 5 (e.g., 1, 2, 3, 4, or 5) heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom is optionally quaternized; for example, 5-10 membered heteroaryl (or ring) refers to a heteroaryl group (or ring) containing 5, 6, 7, 8, 9, or 10 ring atoms. As used herein, a nitrogen-containing heteroaryl group means that at least one of the heteroatoms is a nitrogen heteroatom, preferably, all of the heteroatoms are nitrogen heteroatoms. Heteroaryl groups can be attached to the rest of the molecule via heteroatoms. Non-limiting examples of aryl groups include phenyl and naphthyl, while non-limiting examples of heteroaryl groups include pyridyl, pyrazinyl, pyrazinyl, pyrimidinyl, triazinyl, quinolinyl, quinoxalinyl, quinazolinyl, terpineyl, phthalazinyl, benzotriazinyl, purine, benzimidazolyl, benzopyrazolyl, benzotriazolyl, benzoisoxazolyl, and isobenzofuranyl. ryl), isoindolyl, indene, benzotriazinyl, thienopyridyl, thienopyrimidinyl, pyrazolopyrimidinyl, imidazopyridine, benzothiazolyl, benzofuranyl, benzothiaphenyl, indolyl, quinolinyl, isoquinolinyl, isothiazolyl, pyrazolyl, indazoleyl, pteridinyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiadiazolyl, pyrroleyl, thiazolyl, furanyl, thiopheneyl, etc. The substituents in each of the above aryl and heteroaryl ring systems are selected from the group of acceptable substituents listed below.

[0141] For the sake of brevity, when the term "aryl" is used in combination with other terms (such as aryloxy, arylthio, arylalkyl), it includes aryl and heteroaryl rings as defined above.

[0142] In some embodiments, the terms above (such as "alkyl", "aryl", and "heteroaryl") will include substituted and unsubstituted forms of the specified groups. Optional substituents among these groups include, for example, oxo (=O), -CN, halogens (such as F, Cl, Br, or I), C... 1-6 Alkyl, Halogenated C 1-6 Alkyl, -OR, -SR, -S(O)2R, -S(=O)2NR2, -NR2, -COOR, or C substituted with one or more Rs. 6-10 Aryl, 5-10 heteroaryl, C 3-8 Cycloalkyl, 5-12 membered heterocyclic alkyl, arylalkyl (e.g., -C) 1-4 Alkylene-C 6-10 aryl), heteroarylalkyl such as -C 1-4 alkylene-5-10-membered heteroaryl, cycloalkylalkyl such as -C1-4 Alkylene-C 3-8 Cycloalkyl, heterocycloalkyl, alkyl such as -C 1-4 Alkylene-5-12-membered heterocyclic alkyl.

[0143] For the compounds presented herein, a bond from a substituent (typically an R group) to the center of an aromatic ring (e.g., benzene, pyridine, etc.) will be understood as a bond that provides a connection at any available vertex of the aromatic ring. In some embodiments, this description also includes connections on the ring fused to the aromatic ring. For example, a bond drawn to the center of the indole benzene moiety would represent a bond connected to any available vertex of the six- or five-membered ring moiety of indole.

[0144] In this invention, the terms "containing," "comprising," or "including" indicate that various ingredients may be used together in the mixtures or compositions of this invention. Therefore, the terms "consistent with..." and "composed of..." are included in the term "containing."

[0145] Active ingredients

[0146] As used herein, the terms "compound of the invention," "inhibitor of programmed cell necrosis of the invention," "RIPK1 inhibitor of the invention," and "inhibitor of the invention" are used interchangeably to refer to compounds as described in one aspect of the invention. The term also includes various crystalline forms, pharmaceutically acceptable salts, hydrates, or solvates of the compounds described in the first aspect of the invention.

[0147] In another preferred embodiment, the compound of the present invention is shown in Formula I;

[0148]

[0149] The groups are as defined in the first aspect.

[0150] In another preferred embodiment, the compound of the present invention is shown in Formula II;

[0151]

[0152] The groups are as defined in the first aspect.

[0153] In another preferred embodiment, the compound of the present invention is shown in Formula III;

[0154]

[0155] The groups are as defined in the first aspect.

[0156] In another preferred embodiment, the compounds of the present invention are shown as those of formula IV-1 or IV-2.

[0157]

[0158] The groups are as defined in the first aspect.

[0159] In another preferred embodiment, each group of the compound represented by Formula I, Formula II, Formula III, Formula IV-1 or Formula IV-2 is independently the corresponding group in the specific compound in Table A.

[0160] In another preferred embodiment, the compounds of the present invention are selected from the compounds in Table A, or pharmaceutically acceptable salts thereof.

[0161] The term "pharmaceutically acceptable salt" refers to a salt formed by the compounds of the present invention with an acid or base that is suitable for use as a medicine. Pharmaceutically acceptable salts include both inorganic and organic salts. A preferred class of salts are those formed by the compounds of the present invention with an acid. Acids suitable for forming salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, 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 acids such as proline, phenylalanine, aspartic acid, and glutamic acid. Another preferred class of salts are salts formed by the compounds of the present invention with a base, such as alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., magnesium or calcium salts), ammonium salts (such as lower alkanol ammonium salts and other pharmaceutically acceptable amine salts), such as methylamine salts, ethylamine salts, propylamine salts, dimethylamine salts, trimethylamine salts, diethylamine salts, triethylamine salts, tert-butylamine salts, ethylenediamine salts, hydroxyethylamine salts, dihydroxyethylamine salts, trihydroxyethylamine salts, and amine salts formed from morpholine, piperazine, and lysine, respectively.

[0162] The term "solvate" refers to a complex formed by the coordination of the compound of the present invention with solvent molecules in a specific ratio. "Hydrate" refers to a complex formed by the coordination of the compound of the present invention with water.

[0163] Furthermore, the compounds of the present invention also include prodrugs of the compounds described in the first aspect. The term "prodrug" includes compounds that are themselves biologically active or inactive, and which, when taken by an appropriate method, are metabolized or chemically reacted in the human body to form compounds of formula (I), or salts or solutions of compounds of formula (I). The prodrugs include (but are not limited to) carboxylic acid esters, carbonates, phosphate esters, nitrate esters, sulfate esters, sulfone esters, sulfoxide esters, amino compounds, carbamates, azo compounds, phosphoramides, glucosides, ethers, acetals, etc.

[0164] Preparation method

[0165] 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 on 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, such combinations being readily performed by those skilled in the art.

[0166] In one specific embodiment, the method for preparing the compound of formula II provided by the present invention is method one, and method one includes the following steps:

[0167] In an inert solvent, the compound shown in formula II-2A and the compound shown in formula II-2B are reacted to give the compound shown in formula II-2C.

[0168]

[0169] in,

[0170] R 3 Selected from the following group: H, OH, substituted or unsubstituted C 1-4 Alkyl, substituted or unsubstituted C 1-4 Alkoxy;

[0171] Ring A, Ring B, Ring C, Ring D, R c R 1 R 2 R 4 R 5 And n is as defined in Equation II.

[0172] In one specific embodiment, the method for preparing the compound of formula II provided by the present invention is method two, and method two includes the following steps:

[0173] a) In an inert solvent, the compound shown in formula II-2A and the compound shown in formula II-2B are reacted to give the compound shown in formula II-2C;

[0174]

[0175] b) In an inert solvent, react the compound shown in formula II-2C to form the compound shown in formula II.

[0176]

[0177] in,

[0178] R 3 The ring atom W and L located adjacent to the -C(O)- ring C bond position. 1 The -C(O)- groups in the rings together form substituted or unsubstituted 5, 6, or 7-membered saturated heterocycles;

[0179] L3 C is a single bond, substituted or unsubstituted 1-2 Alkylene;

[0180] Saturated heterocycles, ring A, ring B, ring D, R c R 1 R 2 R 4 R 5 And n is as defined in Equation II.

[0181] It should be understood that the active groups (such as NH) in Method 1 or Method 2 described above may be protected during the reaction process, and the compound of Formula II of the present invention can be obtained by removing the protecting group thereafter.

[0182] Pharmaceutical Compositions and Administration

[0183] Because the compounds of the present invention possess excellent inhibitory activity against RIPK1 and / or against programmed cell death, the compounds of the present invention and their various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and pharmaceutical compositions containing the compounds of the present invention as the main active ingredient can be used to treat, prevent, and alleviate diseases or conditions associated with programmed cell death and / or human receptor-interacting protein 1 kinase (RIPK1) (such as its activity or expression level). In particular, preferred compounds of the present invention not only inhibit inactivated RIPK1, thereby preventing the activation or reactivation of RIPK1 in related diseases or conditions (such as inflammation), but also effectively inhibit activated RIPK1, thereby enabling more rapid treatment or intervention for related diseases / conditions (such as inflammation). According to existing technology, the compounds of this invention can be used to treat the following diseases or conditions: degenerative diseases (such as 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 infections, etc.; or, Parkinson's disease (PD), Huntington's disease (PD), inflammatory bowel disease, age-related macular degeneration, psoriasis, retinitis pigmentosa, inflammatory bowel disease, autoimmune diseases, and acute pancreatitis induced by frog skin extract. Inflammation and sepsis / systemic inflammatory response syndrome (SIRS), ischemic brain injury, ischemic myocardial injury, retinal ischemia / reperfusion injury, retinal detachment-induced photoreceptor cell necrosis, glaucoma, renal ischemia-reperfusion injury, cisplatin-induced kidney injury and traumatic brain injury, atherosclerosis due to hyperlipidemia, other diseases related to RIPK1-dependent apoptosis, necrosis or cytokine production, including hematologic and solid organ malignancies, bacterial and viral infections (including tuberculosis, influenza, coronavirus infection and pneumonia caused by it), and lysosomal storage diseases (especially Gaucher disease).

[0184] The pharmaceutical compositions of the present invention comprise, within a safe and effective range, the compound of the present invention or a pharmacologically acceptable salt thereof, and a pharmacologically acceptable excipient or carrier. "Safe and effective range" refers to an amount of the compound sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 0.1-1000 mg of the compound of the present invention per dose, more preferably, 0.5-500 mg of the compound of the present invention per dose. Preferably, "one dose" is one capsule or tablet.

[0185] "Pharmaceutically acceptable carriers" refers to one or more compatible solid or liquid fillers or gelling substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with and with the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carriers 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, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween), wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0186] There are no particular limitations on the administration methods of the compounds or pharmaceutical compositions of the present invention. Representative administration methods include (but are not limited to): oral, rectal, parenteral (intravenous, intramuscular, or subcutaneous), and topical administration. Oral administration is particularly preferred.

[0187] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following components: (a) fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, and sodium carbonate; (e) slowing agents, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. Buffers may also be included in capsules, tablets, and pills.

[0188] Solid dosage forms such as tablets, sugar pills, capsules, pellets, and granules can be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the active compound or compound from such compositions can be delayed in a portion of the digestive tract. Examples of encapsulating components that can be used are polymeric substances and waxes. If necessary, the active compound may also be formed into microcapsules with one or more of the excipients described above.

[0189] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances.

[0190] In addition to these inert diluents, the composition may also contain auxiliaries such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and fragrances.

[0191] In addition to the active compound, the suspension may contain suspending agents such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0192] 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.

[0193] Dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays, and inhalers. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be necessary.

[0194] The compounds of this invention can be administered alone or in combination with other pharmaceutically acceptable compounds.

[0195] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to the mammal (such as a human) requiring treatment. The dosage at the time of administration is the pharmaceutically considered effective dose. For a person weighing 60 kg, the daily dose is typically 0.2–1000 mg, preferably 0.5–500 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skill of a skilled physician.

[0196] The main advantages of this invention include:

[0197] (a) The compounds of the present invention have excellent inhibitory activity against programmed cell death.

[0198] (b) The compounds of the present invention have excellent metabolic stability.

[0199] (c) The compounds of the present invention have excellent inhibitory activity against RIPK1 kinase.

[0200] (d) The compounds of the present invention can effectively inhibit programmed cell death pathways (such as phosphorylation of RIPK1 itself and phosphorylation of downstream protein MLKL).

[0201] (e) The compounds of the present invention have high bioavailability, and the blood drug concentration remains above the effective concentration for most of the time within 24 hours after administration.

[0202] (f) The compounds of the present invention can effectively reduce inflammatory responses (especially those caused by TNFα).

[0203] (g) The compounds of the present invention can alleviate the inflammatory response induced by abnormally high TNFα levels due to immune response during coronavirus infection.

[0204] (h) The compounds of the present invention can effectively inhibit RIPK1 in an activated state, thus achieving the intervention effect on related inflammation more quickly.

[0205] (i) The compounds of the present invention have very strong inhibitory activity against both human and mouse RIPK1, so they do not require expensive and limited primate models in preclinical trials, which is very beneficial for their application in different disease models.

[0206] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0207] Synthesis Examples

[0208] Method 1:

[0209] Synthesis of compound QY-5-23:

[0210]

[0211] 2-(4-Iodophenyl)methyl acetate (QY-5-233): 4-Iodophenylacetic acid (500 mg, 1.91 mmol) was dissolved in 5 mL of methanol. At room temperature, 5 drops of concentrated sulfuric acid were added dropwise, and the mixture was stirred thoroughly in a 15 mL pressure-resistant flask. The mixture was slowly heated to 65 °C and refluxed with stirring for 5 h. After the reaction was complete, saturated sodium bicarbonate solution was added dropwise in an ice bath to quench the reaction. The mixture was then transferred to a separatory funnel and extracted with ethyl acetate (10 mL * 3). The organic phases were combined and washed with saturated sodium chloride (5 mL * 2). The organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to remove the solvent, yielding 600 mg of a pale yellow oily crude product. This crude product was directly used for the next reaction without further purification. ESI-MS m / z 276.9 (M+H) + .

[0212] Synthesis of compound QY-5-14:

[0213]

[0214] N-Methyl-1H-benzo[d]imidazolium-5-carboxamide (QY-5-14): Methyl benzimidazole-5-carboxylate (1.40 g, 7.94 mmol) was added to a 150 mL sealed tube, followed by an ethanol solution of methylamine (33% wt, 40 mL). The mixture was heated under reflux and stirred for 24 h until the reaction was complete. The ethanol was removed by rotary evaporation, yielding a yellowish-brown lumpy solid. This solid was directly used in the next reaction without further purification. ESI-MS m / z 175.9 (M+H) + .

[0215] Synthesis of compound QY-5-25:

[0216]

[0217] 2-(4-(5-(methylcarbamoyl)-1H-benzo[d]imidazol-1-yl)phenyl)methyl acetate (QY-5-25): In a 15 ml pressure-resistant bottle, add 5 ml of dimethyl sulfoxide, QY-5-23 (600 mg, 2.17 mmol), and stir until dissolved. Then add QY-5-14 (314 mg, 1.81 mmol), cuprous iodide (172 mg, 0.91 mmol), cesium carbonate (1.18 g, 3.62 mmol), and 4,7-dimethoxy-1,10-phenoxyline (87 mg, 0.36 mmol). Under nitrogen protection, heat to 100 °C and react overnight. After the reactants are consumed, filter to remove insoluble solids, and then purify by C18 reversed-phase chromatography (water:acetonitrile = 0-80%) to obtain 486 mg of dark brown oily liquid, yield 83%. ESI-MS m / z 324.1 (M+H) +

[0218] Synthesis of compound QY-5-34:

[0219]

[0220] 2-(4-(5-(methylcarbamoyl)-1H-benzo[d]imidazol-1-yl)phenyl)acetic acid (QY-5-34):

[0221] QY-5-25 (486 mg, 1.50 mmol) was dissolved in 5 mL of tetrahydrofuran and transferred to a 15 mL pressure-resistant bottle. Lithium hydroxide monohydrate (126 mg, 3.0 mmol) was dissolved in 5 mL of distilled water and added dropwise to the reaction solution under ice bath conditions. After mixing and stirring until homogeneous, the mixture was gradually brought to room temperature. The reaction was monitored in real time by LC-MS. After 2 h, the reaction was completed and transferred to a 100 mL round-bottom flask. The organic solvent was removed by rotary evaporation, and the reaction system was dissolved in methanol. The mixture was purified by C18 reversed-phase chromatography (water:acetonitrile = 0-80%) to give 292 mg of dark green foamy solid, with a yield of 63%. ESI-MS m / z 308.0 (M+H) - .

[0222] Synthesis of compound QY-5-35:

[0223]

[0224] 1-(4-(2-(((1H-indol-3-yl)methyl)amino)-2-oxoethyl)phenyl)-N-methyl-1H-benzo[d]imidazolium-5-carboxamide (QY-5-35): QY-5-34 (38 mg, 0.12 mmol) was dissolved in 1.5 ml of dichloromethane in an 8 ml pressure-resistant bottle. HATU (56 mg, 0.147 mmol) was added. Indol-3-methylamine (21 mg, 0.15 mmol) was dissolved in 1 ml of dichloromethane and added dropwise to the stirred reaction solution. N,N-diisopropylethylamine (40 mg, 0.31 mmol) was added dropwise under ice bath conditions. The mixture was stirred for 10 min, gradually restored to room temperature, and then stirred for 4 h. LC-MS was used for real-time monitoring. After the reaction was complete, 5 ml of dichloromethane was added to dilute the reaction solution. The solution was transferred to a separatory funnel, washed with 5 ml of distilled water, and extracted with dichloromethane (5 ml * 3). The organic phases were combined, washed with saturated sodium chloride (5 ml * 2), and the resulting organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to remove dichloromethane. The solution was then purified by C18 reversed-phase chromatography (water:acetonitrile = 0-80%) to give 29 mg of a pale yellow oily liquid, with a yield of 55%. ESI-MS m / z 438.1 (M+H) + .

[0225] Method 2:

[0226] Synthesis of compound QY-5-30:

[0227]

[0228] (4-Iodophenyl)-N-(2,3,5-trifluorobenzyl)acetamide (QY-5-30): 2,3,5-trifluorobenzylamine (200 mg, 1.24 mmol) was dissolved in 7 ml of dichloromethane in a 15 ml pressure-resistant bottle. HATU (566 mg, 1.49 mmol) was added. p-Iodophenylacetic acid (390 mg, 1.49 mmol) was dissolved in 1 ml of dichloromethane and added dropwise to the stirred reaction solution. N,N-diisopropylethylamine (400 mg, 3.1 mmol) was added dropwise under ice bath conditions. The mixture was stirred for 10 min, and after gradually returning to room temperature, the reaction was stirred for 6 h. LC-MS was used for real-time monitoring. After the raw materials were consumed, 15 ml of dichloromethane was added to dilute the reaction solution. The solution was transferred to a separatory funnel, washed with 15 ml of distilled water, and extracted with dichloromethane (15 ml * 3). The organic phases were combined, washed with saturated sodium chloride (10 ml * 2), and the resulting organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to remove dichloromethane. The solution was then purified by C18 reversed-phase chromatography (water:acetonitrile = 0-80%) to give 400 mg of white solid, with a yield of 80%. ESI-MS m / z 406.0 (M+H) + .

[0229] Synthesis of compound QY-5-36:

[0230]

[0231] 2-(4-Iodophenyl)-N-methyl-N-(2,3,5-trifluorobenzyl)acetamide (QY-5-36): QY-5-30 (300 mg, 0.74 mmol) and 6 mL of tetrahydrofuran were added to a 15 mL pressure-resistant flask and mixed thoroughly. Sodium hydride (30 mg, 0.74 mmol) was slowly added to the reaction solution under ice bath conditions, and the mixture was stirred for 10 min. Iodomethane (126 mg, 0.89 mmol) was slowly added dropwise to the reaction solution under ice-salt bath conditions. The mixture was gradually brought to room temperature and stirred continuously. LC-MS was used to monitor the reaction until all reactants were consumed. The reaction was quenched with distilled water, and tetrahydrofuran was removed by rotary evaporation. The reaction system was dissolved in methanol, filtered through a membrane, and purified by C18 reversed-phase chromatography (water:acetonitrile = 0-80%) to obtain 192 mg of a pale yellow oily liquid, with a yield of 62%. ESI-MS m / z 419.9 (M+H) + .

[0232] Synthesis of compound QY-5-40:

[0233]

[0234] N-Methyl-1-(4-(2-(methyl(2,3,5-trifluorobenzyl)amino)-2-oxoethyl)phenyl)-1H-benzo[d]imidazolium-5-carboxamide (QY-5-40): Add QY-5-36 (192 mg, 0.46 mmol) to a 15 ml pressure-resistant bottle, dissolve in 3 ml of dimethyl sulfoxide, stir well, then add QY-5-14 (88 mg, 0.51 mmol), cuprous iodide (44 mg, 0.23 mmol), cesium carbonate (298 mg, 0.92 mmol), and 4,7-dimethoxy-1,10-phenoxyline (33 mg, 0.14 mmol). Under nitrogen protection, heat to 100 °C and react overnight. After the reactants were consumed, the solid insoluble matter was removed by filtration through a membrane filter, followed by purification using a C18 reversed-phase chromatography column (water:acetonitrile = 0-80%) to yield 143 mg of a yellowish-brown solid, with a yield of 67%. ESI-MS m / z 467.1 (M+H) + .

[0235] Method 3:

[0236] Synthesis of compound QY-5-62:

[0237]

[0238] 1-(4-(2-(benzyl(hydroxy)amino)-2-oxoethyl)phenyl)-N-methyl-1H-benzo[d]imidazolium-5-carboxamide (QY-5-62): QY-5-34 (150 mg, 0.49 mmol) was dissolved in 4 ml of dichloromethane and added to an 8 ml pressure-resistant bottle. HATU (221 mg, 0.58 mmol) was added, and N-benzylhydroxylamine hydrochloride (93 mg, 0.58 mmol) was added to the stirred reaction solution. N,N-diisopropylethylamine (250 mg, 1.94 mmol) was added dropwise under ice bath conditions. The mixture was gradually brought to room temperature and stirred for 3 h. LC-MS was used for real-time monitoring. After the reactants were consumed, 5 ml of dichloromethane was added to dilute the reaction solution. The solution was transferred to a separatory funnel, washed with 4 ml of distilled water, and extracted with dichloromethane (5 ml * 3). The organic phases were combined, washed with saturated sodium chloride (5 ml * 2), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to remove dichloromethane. The solution was then purified by separation on a C18 reversed-phase column (water:acetonitrile = 20%-90%) to give 85 mg of white solid, with a yield of 42%. ESI-MS m / z 415.1 (M+H) + .

[0239] Method 4:

[0240] Synthesis of compound QY-5-81:

[0241]

[0242] N-Methoxy-N-methyl-2-phenylacetamide (QY-5-81): In a 30 mL pressure-resistant flask, phenylacetic acid (1.0 g, 7.34 mmol) was dissolved in 15 mL of dichloromethane. HATU (3.6 g, 9.54 mmol) was added, followed by dimethylhydroxylamine hydrochloride (787 mg, 8.07 mmol) to the stirred reaction solution. N,N-diisopropylethylamine (2.84 g, 22.0 mmol) was added dropwise at room temperature, and the reaction was continued with stirring for 5.5 h. The reaction was monitored in real time by LC-MS. After the reactants were completely consumed, the mixture was transferred to a separatory funnel, washed with 25 mL of distilled water, extracted with dichloromethane (20 mL * 3), and the organic phases were combined. The mixture was washed with saturated sodium chloride (10 mL * 2), dried over anhydrous sodium sulfate, filtered, and the dichloromethane was removed by rotary evaporation. The solution was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 0-100%) to give 1.23 g of a pale yellow transparent liquid, with a yield of 94%. ESI-MS m / z 180.1(M+H) + .

[0243] Synthesis of compound QY-5-84:

[0244]

[0245] 1-Phenylaceton-2-one (QY-5-84): QY-5-81 (1.03 g, 5.75 mmol) was dissolved in 15 mL of tetrahydrofuran in a 30 mL pressure-resistant flask. The mixture was stirred continuously at 700 rpm, and methyl magnesium bromide (6.90 mL, 6.90 mmol) was slowly added dropwise at 0 °C. After the addition was complete, the mixture was gradually brought to room temperature with continued stirring. The reaction was monitored in real-time by LC-MS, and stopped after completion. The reaction was quenched by slowly adding 1 N hydrochloric acid. The mixture was extracted with ethyl acetate (20 mL * 3), and the organic phases were combined. The mixture was washed with saturated brine (10 mL * 1), dried over anhydrous sodium sulfate, and the organic solvent was removed by rotary evaporation. The mixture was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 0-60%) to give 609 mg of a colorless, transparent liquid, with a yield of 79%.

[0246] Synthesis of compound QY-5-87:

[0247]

[0248] Ethyl 2,4-dioxo-5-phenylpentanoate (QY-5-87): Mix QY-5-84 (509 mg, 3.79 mmol) into 10 ml of ultra-dry tetrahydrofuran, add to a 30 ml pressure-resistant bottle and stir; dissolve sodium hydride (152 mg, 3.80 mmol) in 5 ml of ultra-dry tetrahydrofuran, add it dropwise to the suspension at 0 °C, mix well at 0 °C, then add diethyl oxalate (665 mg, 4.55 mmol) to the reaction system, gradually return to room temperature, and continue stirring for 3 h before stopping the reaction. The reaction was quenched dropwise with distilled water at 0°C. The ultra-dry tetrahydrofuran was removed by rotary evaporation. The reaction mixture was diluted with 10 ml of ethyl acetate, transferred to a separatory funnel, washed with 10 ml of distilled water, and extracted with ethyl acetate (15 ml x 3). The combined organic phases were washed with saturated brine (10 ml x 2), dried over anhydrous sodium sulfate, filtered, and the ethyl acetate was removed by rotary evaporation. The mixture was then purified by C18 reversed-phase chromatography (water:acetonitrile = 20%-90%) to give 495 mg of a yellowish-brown liquid, with a yield of 56%. ESI-MS m / z 235.1 (M+H) + .

[0249] Synthesis of compound QY-5-90:

[0250]

[0251] 5-Benzylisoxazole-3-carboxylic acid ethyl ester (QY-5-90): QY-5-87 (250 mg, 1.07 mmol) was added to 4 mL of anhydrous ethanol and then transferred to a 15 mL pressure-resistant flask. The mixture was stirred thoroughly. Hydroxylamine hydrochloride (111 mg, 1.60 mmol) was slowly added to the reaction system at room temperature. The mixture was heated to reflux and stirred overnight. After the reactants were completely consumed, the organic solvent was removed by rotary evaporation. The mixture was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 0-35%) to give 180 mg of an orange-yellow transparent liquid, with a yield of 73%. ESI-MS m / z 232.1.1 (M+H) + .

[0252] Synthesis of compound QY-5-91:

[0253]

[0254] 5-Benzylisoxazole-3-carboxylic acid (QY-5-91): QY-5-90 (360 mg, 1.56 mmol) was dissolved in 4 mL of ethanol and transferred to a 15 mL pressure-resistant bottle. Potassium hydroxide (437 mg, 7.78 mmol) was dissolved in 3 mL of distilled water and added dropwise to the reaction solution under ice bath conditions. After mixing and stirring thoroughly, the mixture was gradually brought to room temperature and heated to 80 °C. The reaction was monitored in real time by LC-MS for 4 h until complete. Saturated potassium bisulfite was added to adjust the pH to acidic, and the mixture was evaporated to dryness. The reaction system was then dissolved in methanol, filtered through a membrane, and purified by C18 reversed-phase chromatography (water:acetonitrile = 0-80%) to obtain 258 mg of white solid powder, with a yield of 82%. ESI-MS m / z 202.1 (M+H) - .

[0255] Synthesis of compound QY-5-101:

[0256]

[0257] (4-Iodobenzyl)carbamate tert-butyl ester (QY-5-101): p-Iodobenzylamine (800 mg, 3.43 mmol) was dissolved in 10 mL of dichloromethane and transferred to a 15 mL pressure-resistant bottle. Di-tert-butyl dicarbonate (749 mg, 3.43 mmol) was slowly added dropwise to the reaction solution. 4-Dimethylaminopyridine (126 mg, 1.03 mmol) was added, and the mixture was stirred at room temperature. The reaction was monitored in real-time by LC-MS. The reaction was completed in 3.5 h. The entire reaction mixture was then evaporated to dryness and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 0-40%) to give 925 mg of a white solid, yield 81%. ESI-MS m / z 277.0 (M+H) + .

[0258] Synthesis of compound QY-6-2:

[0259]

[0260] N-tert-butyl(4-(5-(methylcarbamoyl)-1H-benzo[d]imidazol-1-yl)benzyl)carbamate (QY-6-2): QY-5-101 (200 mg, 0.60 mmol) was added to a 15 ml pressure-resistant bottle, dissolved in 3 ml of dimethyl sulfoxide, and stirred until homogeneous. Then, QY-5-14 (105 mg, 0.60 mmol), cuprous iodide (57 mg, 0.30 mmol), cesium carbonate (390 mg, 1.20 mmol), and 4,7-dimethoxy-1,10-phenoxyline (43 mg, 0.18 mmol) were added. Under nitrogen protection, the mixture was heated to 100 °C and reacted overnight. After the reactants were completely consumed, the mixture was filtered through a membrane to remove insoluble solids, and then purified by C18 reversed-phase chromatography (water:acetonitrile = 0-80%) to obtain 203 mg of a yellowish-brown oily liquid, with a yield of 89%. ESI-MS m / z 381.1 (M+H) + .

[0261] Synthesis of compound QY-6-6:

[0262]

[0263] 1-(4-(aminomethyl)phenyl)-N-methyl-1H-benzo[d]imidazolium-5-carboxamide (QY-6-6): QY-6-2 (203 mg, 0.53 mmol) was mixed in 5 mL of dichloromethane and transferred to an 8 mL pressure-resistant bottle. At room temperature, 1.0 mL of trifluoroacetic acid was added dropwise to the reaction solution, and the mixture was stirred thoroughly. LC-MS was used for real-time monitoring. After 3.5 h, the reactants were completely consumed and transferred to a round-bottom flask. 20 mL of dichloromethane was added each time, and the mixture was evaporated to dryness to remove the organic solvent mixture. This process was repeated 4-6 times. The product from this step does not require separation and purification and can be directly used for subsequent synthesis. ESI-MS m / z 281.0 (M+H) + .

[0264] Synthesis of compound QY-6-16:

[0265]

[0266] 5-Benzyl-N-(4-(5-(methylcarbamoyl)-1H-benzo[d]imidazol-1-yl)benzyl)isoxazole-3-carboxamide (QY-6-16): In an 8 ml pressure-resistant bottle, QY-6-6 (70 mg, 0.25 mmol) was dissolved in 2 ml of dichloromethane, and HATU (114 g, 0.30 mmol) was added. QY-5-91 (50 mg, 0.25 mmol) was added to the stirred reaction solution. N,N-diisopropylethylamine (97 mg, 0.75 mmol) was added dropwise at room temperature, and the reaction was stirred for 4 h. The reaction was monitored in real time by LC-MS. Once the reactants were completely consumed, the mixture was transferred to a separatory funnel, washed with 5 ml of distilled water, extracted with dichloromethane (5 ml x 3), and the organic phases were combined. The mixture was washed with saturated sodium chloride (5 ml x 2), dried over anhydrous sodium sulfate, filtered, and the dichloromethane was removed by rotary evaporation. After filtration through a membrane, the solution was purified by C18 reversed-phase chromatography (water:acetonitrile = 0-80%) to obtain 116 mg of a yellowish-brown oily liquid, with a yield of 52%. ESI-MS m / z 466.1 (M+H) + .

[0267] Method 5:

[0268] Synthesis of compound QY-6-98:

[0269]

[0270] 5-Benzyl-1-ethylmethyl-1H-pyrazole-3-carboxylic acid ethyl ester (QY-6-98): QY-5-87 (250 mg, 1.07 mmol) was mixed with 7 mL of anhydrous ethanol in a 15 mL pressure-resistant bottle. Methylhydrazine hydrochloride (101 mg, 1.21 mmol) was added at room temperature. The temperature was gradually increased to 90 °C, and the reaction was monitored by thin-layer chromatography. The reaction was stopped after stirring for 4 h. The organic solvent was removed by rotary evaporation, and the product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 0-60%) to give two configurations: Product A: 104 mg orange-yellow liquid, yield 40%; Product B: 62 mg orange-yellow liquid, yield 24%. ESI-MS m / z 245.2 (M+H) + .

[0271] Synthesis of compound QY-6-103B:

[0272]

[0273] 6-Benzyl-1-methyl-1H-pyrazole-3-carboxylic acid (QY-6-103B): QY-6-98 (62 mg, 0.25 mmol) was dissolved in 2 mL of tetrahydrofuran and transferred to an 8 mL pressure-resistant bottle. Lithium hydroxide monohydrate (27 mg, 0.63 mmol) was dissolved in 1 mL of distilled water and added dropwise to the reaction solution under ice bath conditions. After mixing and stirring until homogeneous, the mixture was gradually brought to room temperature. The reaction was monitored in real time by LC-MS. After stirring overnight, the reaction was stopped. The organic solvent was removed by rotary evaporation, and the reaction system was dissolved in methanol. The mixture was purified by C18 reversed-phase chromatography (water:acetonitrile = 0-80%) to give 41 mg of white solid, yield 76%. ESI-MS m / z 215.1 (M+H) - .

[0274] Synthesis of compound QY-7-2B:

[0275]

[0276] 1-(4-((5-benzyl-1-methyl-1H-pyrazole-3-carboxamido)methyl)phenyl)-N-methyl-1H-benzo[d]imidazolium-5-carboxamide (QY-7-2B): In an 8 ml pressure-resistant bottle, QY-6-103B (15 mg, 0.07 mmol) was dissolved in 1 ml dichloromethane, and HATU (32 mg, 0.09 mmol) was added. QY-6-6 (20 mg, 0.07 mmol) was added to the stirred reaction solution. N,N-diisopropylethylamine (23 g, 0.18 mmol) was added dropwise at room temperature, and the reaction was stirred for 4 h. The reaction was monitored in real time by LC-MS. Once the reactants were completely consumed, the mixture was transferred to a separatory funnel, washed with 2 ml of distilled water, extracted with dichloromethane (2 ml x 3), and the organic phases were combined. The mixture was washed with saturated sodium chloride (2 ml x 2), dried over anhydrous sodium sulfate, filtered, and the dichloromethane was removed by rotary evaporation. The solution was then purified by silica gel column chromatography (petroleum ether:ethyl acetate = 0-100%) to give 19 mg of a pale yellowish-brown liquid, with a yield of 59%. ESI-MS m / z 479.1 (M+H) + .

[0277] Method 6:

[0278] Synthesis of compound QY-6-97:

[0279]

[0280] (E)-2-(hydroxyimino)-2-(2-phenylacetamido)ethyl acetate (QY-6-97): Phenylacetic acid (200 mg, 1.47 mmol) was dissolved in 7 ml of dichloromethane in a 15 ml pressure-resistant bottle. HATU (669 mg, 1.76 mmol) was added. Ethyl 2-hydroxyamino-2-iminoacetate (233 mg, 1.76 mmol) was dissolved in 2 ml of dichloromethane and added dropwise to the well-stirred reaction solution. N,N-diisopropylethylamine (569 mg, 4.41 mmol) was added dropwise at room temperature. The mixture was stirred for 10 min, and then gradually brought to room temperature before stirring for 6 h. LC-MS was used for real-time monitoring. After the reaction was complete, 10 ml of dichloromethane was added to dilute the reaction solution. The solution was transferred to a separatory funnel, washed with 5 ml of distilled water, and extracted with dichloromethane (5 ml * 3). The organic phases were combined, washed with saturated sodium chloride (5 ml * 2), and the resulting organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to remove dichloromethane. The solution was then purified by silica gel column chromatography (petroleum ether: ethyl acetate = 0-80%) to give 84 mg of white solid powder, with a yield of 23%. ESI-MS m / z 251.1 (M+H) + .

[0281] Synthesis of compound QY-7-4:

[0282]

[0283] Ethyl 5-benzyl-1,2,4-oxadiazole-3-carboxylic acid ester (QY-7-4): QY-6-97 (84 mg, 0.34 mmol) was dissolved in 3 mL of N,N-dimethylformamide, transferred to an 8 mL pressure-resistant bottle, mixed and stirred thoroughly, and then gradually heated to 140 °C. The reaction was monitored in real-time by LC-MS, and was completed in 2 h. Purification was achieved by C18 reversed-phase chromatography (water:acetonitrile = 0-100%) to obtain 64 mg of a pale yellow oily liquid, with a yield of 81%. ESI-MS m / z 233.1 (M+H) + .

[0284] Synthesis of compound QY-7-13:

[0285]

[0286] 5-Benzyl-1,2,4-oxadiazole-3-carboxylic acid (QY-7-13): QY-7-4 (51 mg, 0.22 mmol) was dissolved in 2 mL of tetrahydrofuran and transferred to an 8 mL pressure-resistant bottle. Lithium hydroxide monohydrate (14 mg, 0.33 mmol) was dissolved in 1 mL of distilled water and added dropwise to the reaction solution under ice bath conditions. After mixing and stirring thoroughly, the mixture was gradually brought to room temperature. The reaction was monitored in real time by LC-MS. After 4 h, the reaction was stopped, and all solvents in the reaction system were removed by rotary evaporation. The crude product could be directly used for subsequent synthesis. ESI-MS m / z 203.1 (M+H) - .

[0287] Synthesis of compound QY-7-14:

[0288]

[0289] 5-Benzyl-N-(4-(5-(methylcarbamoyl)-1H-benzo[dimidazol-1-yl)benzyl)-1,2,4-oxadiazole-3-carboxamide (QY-7-14): QY-7-13 (0.22 mmol) was dissolved in 3 ml of dichloromethane in a 15 ml pressure-resistant bottle, HATU (83 mg, 0.22 mmol) was added, and QY-6-6 (51 mg, 0.18 mmol) was added to the stirred reaction solution; N,N-diisopropylethylamine (59 mg, 0.46 mmol) was added dropwise at room temperature, and the reaction was stirred at room temperature for 4 h. LC-MS was used for real-time monitoring. After the reaction was complete, 10 ml of dichloromethane was added to dilute the reaction solution. The solution was transferred to a separatory funnel, washed with 5 ml of distilled water, and extracted with dichloromethane (5 ml * 3). The organic phases were combined, washed with saturated sodium chloride (5 ml * 2), and the resulting organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to remove dichloromethane. After filtration through a membrane, the solution was purified by C18 reversed-phase chromatography (water:acetonitrile = 0-90%) to obtain 35 mg of a yellowish-brown oily liquid, with a yield of 42%. ESI-MS m / z 467.1 (M+H) + .

[0290] Method 7:

[0291] Synthesis of compound QY-7-21:

[0292]

[0293] 2-O-2-(2-(2-phenylacetylacetyl)hydrazino)ethyl acetate (QY-7-21): Phenylacetylhydrazine (600 mg, 3.99 mmol) was dissolved uniformly in 30 mL of dichloromethane in a 100 mL round-bottom flask. Oxaloyl chloride monoethyl ester (600 mg, 4.39 mmol) was slowly added dropwise under ice bath conditions. The reaction system was gradually brought to room temperature with continuous stirring. LC-MS was used for real-time monitoring. The reaction was stopped after 3 hours when no starting material peak was observed. The reaction was quenched with distilled water, washed with 10 mL of distilled water, extracted with dichloromethane (15 mL * 3), and the organic phases were combined. The mixture was washed with saturated brine (10 mL * 1), dried over anhydrous sodium sulfate, and the organic solvent was removed by rotary evaporation to obtain a white solid crude product that could be directly used for subsequent reactions. ESI-MS m / z 251.0 (M+H) + .

[0294] Synthesis of compound QY-7-23:

[0295]

[0296] 5-Benzyl-1,3,4-oxadiazole-2-carboxylic acid ethyl ester (QY-7-23): QY-7-21 was stirred and mixed in 10 ml of dichloromethane in a 30 ml pressure-resistant flask. Toluenesulfonyl chloride (760 mg, 3.99 mmol) was dissolved in 4 ml of dichloromethane and added dropwise to the reaction mixture. The mixture was stirred continuously at room temperature, and the reaction was monitored by thin-layer chromatography. The reaction was stopped after stirring for 6 h. The mixture was transferred to a separatory funnel, washed with 10 ml of distilled water, and extracted with dichloromethane (10 ml * 3). The organic phases were combined, washed with saturated ammonium chloride (5 ml * 2), and saturated sodium chloride (5 ml * 2). The resulting organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to remove dichloromethane. The solution was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 0-40%) to give 384 mg of a yellowish-brown liquid. The two-step reaction yield was 41%. ESI-MS m / z 233.1 (M+H) + .

[0297] Synthesis of compound QY-7-28:

[0298]

[0299] 5-Benzyl-1,3,4-oxadiazole-2-carboxylic acid (QY-7-28): QY-7-23 (56 mg, 0.24 mmol) was dissolved in 1 mL of methanol and transferred to an 8 mL pressure-resistant bottle. Lithium hydroxide monohydrate (10 mg, 0.24 mmol) was dissolved in 0.5 mL of distilled water and added dropwise to the reaction solution under ice bath conditions. After mixing and stirring until homogeneous, the mixture was gradually brought to room temperature. The reaction was monitored in real time by LC-MS. After 1.5 h, the reaction was stopped. All solvents in the reaction system were removed by rotary evaporation. The mixture was filtered through a C18 reversed-phase chromatography column (water:acetonitrile = 0-30%) to obtain 33 mg of white solid, with a yield of 67%. ESI-MS m / z 203.0 (M+H) - .

[0300] Synthesis of compound QY-7-32:

[0301]

[0302] 5-Benzyl-N-(4-(5-(methylcarbamoyl)-1H-benzo[dimidazol-1-yl)benzyl)-1,3,4-oxadiazole-2-carboxamide (QY-7-32): QY-7-28 (33 mg, 0.16 mmol) was dissolved in an 8 ml pressure-resistant bottle containing 2 ml of dichloromethane, and HATU (74 mg, 0.19 mmol) was added. QY-6-6 (45 mg, 0.16 mmol) was added to the stirred reaction solution. N,N-diisopropylethylamine (52 mg, 0.40 mmol) was added dropwise at room temperature, and the reaction was stirred for 3.5 h. The reaction was monitored in real time by LC-MS. After the starting materials were consumed, the reaction system was transferred to a separatory funnel, washed with 5 ml of distilled water, extracted with dichloromethane (5 ml * 3), and the organic phases were combined. The mixture was washed with saturated sodium chloride (5 ml * 2), dried over anhydrous sodium sulfate, filtered, and the dichloromethane was removed by rotary evaporation. After filtration through a membrane, the solution was purified by C18 reversed-phase chromatography (water:acetonitrile = 0-80%) to give 15 mg of white solid, with a yield of 21%. ESI-MS m / z 467.0 (M+H) + .

[0303] Method 8:

[0304] Synthesis of compound QY-7-65:

[0305]

[0306] N-Methyl-1-(4-(2-oxo-2-(2-phenylpyrrolyl-1-yl)ethyl)phenyl)-1H-benzo[d]imidazolium-5-carboxamide (QY-7-65): QY-5-34 (49 mg, 0.16 mmol) was dissolved in an 8 ml pressure-resistant bottle containing 2 ml of dichloromethane. HATU (69 mg, 0.18 mmol) was added, and QY-7-64 (20 mg, 0.14 mmol) was added to the stirred reaction solution. N,N-diisopropylethylamine (72 mg, 0.56 mmol) was added dropwise at room temperature, and the reaction was stirred for 3 h. The reaction was monitored in real time by LC-MS. After the reaction was complete, the reaction system was transferred to a separatory funnel, washed with 5 ml of distilled water, extracted with dichloromethane (5 ml * 3), the organic phases were combined, washed with saturated sodium chloride (5 ml * 2), dried over anhydrous sodium sulfate, and dichloromethane was removed by rotary evaporation. After filtration through a C18 reversed-phase column, the solution was purified (water:acetonitrile = 0-80%) to give 19 mg of a yellowish-brown solid, with a yield of 31%. ESI-MS m / z 439.1 (M+H) + .

[0307] Method 9:

[0308] Synthesis of compound QY-9-15:

[0309]

[0310] 1-Benzyl-1H-pyrazole-3-carboxylic acid ethyl ester (QY-9-15): In a 250 mL round-bottom flask, 6.0 g (42.8 mmol) of 3-ethoxycarbonylpyrazole was stirred and mixed into 90 mL of acetonitrile. Potassium carbonate (17.8 g, 128.4 mmol) was added to the reaction solution. Benzyl bromide (8.8 g, 51.4 mmol) was added dropwise to the reaction system under vigorous stirring at room temperature. The reaction was monitored by LC-MS. After stirring for 8 h, the reaction was stopped. The inorganic salts were removed by diatomaceous earth filtration, and the organic solvent was removed by rotary evaporation. The product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 0-50%) to give 6.54 g of white solid, yield 66%. ESI-MS m / z 231.1 (M+H) + .

[0311] Synthesis of compound QY-9-19:

[0312]

[0313] 1-Benzyl-4-bromo-1H-pyrazole-3-carboxylic acid ethyl ester (QY-9-19): In a 250 mL round-bottom flask, QY-9-15 (6.4 g, 27.8 mmol) was dissolved in 100 mL of acetonitrile. After stirring, liquid bromine (6.7 g, 41.7 mmol) was slowly added dropwise to the reaction system at room temperature. The reaction was stopped after stirring overnight. The reaction was quenched by slowly adding 3 M sodium thiosulfate solution. After stirring for 15 min, acetonitrile was removed by rotary evaporation, and the mixture was extracted with ethyl acetate (20 mL * 3). The organic phases were combined, washed with sodium thiosulfate solution (10 mL * 1), washed with saturated brine (10 mL * 1), dried over anhydrous sodium sulfate, and ethyl acetate was removed by rotary evaporation. The mixture was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 0-40%) to give 5.17 g of white solid, yield 60%. ESI-MS m / z 309.0 (M+H) + .

[0314] Synthesis of compound QY-9-26:

[0315]

[0316] Ethyl (E)-1-benzyl-4-(2-ethoxyvinyl)-1H-pyrazole-3-carboxylic acid ester (QY-9-26): QY-9-19 (2.50 g, 8.1 mmol) was dissolved in 30 ml of 1,2-dichloroethane and 5 ml of distilled water, and then added to a 150 ml sealed tube. Pd(dppf)Cl2 (0.59 g, 0.81 mmol), cesium carbonate (5.80 g, 17.8 mmol), and (E)-1-ethoxyvinyl-2-boronic acid pinacol ester (3.20 g, 16.2 mmol) were dissolved in 10 ml of 1,2-dichloroethane and then added dropwise to the vigorously stirred reaction solution. Under nitrogen protection, the mixture was gradually heated to 90 °C and reacted overnight. After the reactants were consumed, the reaction system was transferred to a separatory funnel, washed with 80 ml of distilled water, extracted with ethyl acetate (40 ml * 3), the organic phases were combined, washed with saturated sodium chloride (15 ml * 2), dried over anhydrous sodium sulfate, the organic solvent was removed by rotary evaporation, and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 0-60%) to give 2.10 g of white solid, yield 87%. ESI-MS m / z 301.1 (M+H) + .

[0317] Synthesis of compound QY-9-28:

[0318]

[0319] 1-Benzyl-4-(2-ethyloxoethyl)-1H-pyrazole-3-carboxylic acid ethyl ester (QY-9-28): QY-9-26 (2.10 g, 7.0 mmol) was dissolved in 30 mL of tetrahydrofuran and added to a 100 mL round-bottom flask. Under ice bath conditions, 10 mL of 6M hydrochloric acid aqueous solution was added dropwise to the reaction mixture. After stirring for 2 h, 15 mL of 6M hydrochloric acid aqueous solution was added. The reaction was monitored in real time by LC-MS. After the reactants were consumed, saturated sodium bicarbonate solution was added to adjust the pH to weakly alkaline. The reaction system was transferred to a separatory funnel and extracted with ethyl acetate (20 mL * 3). The organic phases were combined, washed with saturated sodium chloride (10 mL * 2), dried over anhydrous sodium sulfate, and the ethyl acetate was removed by rotary evaporation. The mixture was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 0-50%) to give 800 mg of a pale yellow transparent liquid, with a yield of 42%. ESI-MS m / z 273.1 (M+H) + .

[0320] Synthesis of compound QY-7-98:

[0321]

[0322] 1-Benzyl-4-(2-((4-(5-(methylcarbamoyl)-1H-benzo[dimidazol-1-yl)benzyl)amino)ethyl)-1H-pyrazole-3-carboxylic acid ethyl ester (QY-7-98): QY-9-28 (316 mg, 1.2 mmol) was mixed into 6 mL of 1,2-dichloroethane and added to a 30 mL pressure-resistant flask with stirring. QY-6-6 (270 mg, 0.96 mmol) and glacial acetic acid (279 mg, 4.6 mmol) were added and stirred for 20 min. Sodium triacetoxyborohydride (492 mg, 2.3 mmol) was added and stirred overnight at room temperature until the reactants were consumed. The reaction was quenched dropwise with distilled water under ice bath conditions. After filtration through a filter membrane, the product was purified by C18 reversed-phase chromatography (water:acetonitrile = 0-80%) to give 128 mg of a pale yellow solid, with a yield of 25%. ESI-MS m / z 537.1 (M+H) + .

[0323] Synthesis of compound QY-8-7:

[0324]

[0325] (4-((2-benzyl-7-oxo-2,4,5,7-tetrahydrotetrahydro-6H-pyrazolo[3,4-c]pyridin-6-yl)methyl)phenyl)-N-methyl-1H-benzo[d]imidazolium-5-carboxamide (QY-8-7): Mix QY-7-98 (64 mg, 0.12 mmol) into 7 ml xylene, transfer to a 15 ml pressure-resistant bottle and stir; slowly add trimethylaluminum (0.36 mmol) dropwise to the reaction solution at room temperature, and gradually raise the temperature to 120 °C under nitrogen protection, stirring overnight to stop the reaction. After the reaction solution was allowed to slowly return to room temperature, distilled water was added dropwise to quench the reaction. Roche salt solution was added and stirred for 30 min. The reaction mixture was diluted with 10 ml of ethyl acetate, transferred to a separatory funnel, and extracted with ethyl acetate (10 ml * 3). The organic phases were combined, washed with saturated sodium chloride (10 ml * 2), dried over anhydrous sodium sulfate, filtered, and the organic solvent was removed by rotary evaporation. After filtration through a filter membrane, the mixture was purified by high-performance liquid chromatography (HPLC) (water:acetonitrile = 10-60%) to give 8 mg of white solid, yield 14%. ESI-MS m / z 491.1 (M+H) + .

[0326] Method 10:

[0327] Synthesis of compound QY-8-15:

[0328]

[0329] 2-Oxo-2-((2-oxo-3-phenylpropyl)amino)ethyl acetate (QY-8-15): 1-Amino-3-phenylpropyl-2-one hydrochloride (500 mg, 2.7 mmol) was mixed with water in 15 ml of dichloromethane in a 30 ml pressure-resistant flask. Triethylamine (817 mg, 8.1 mmol) was added dropwise to the reaction system under ice bath conditions. After stirring for 10 min, oxaloyl chloride monoethyl ester (734 mg, 5.4 mmol) was slowly added dropwise. The mixture was gradually brought to room temperature and stirred overnight. The reaction was stopped after the starting materials were consumed, as monitored by LC-MS. The extract was transferred to a separatory funnel, washed with distilled water (10 ml * 2), extracted with dichloromethane (15 ml * 3), and the organic phases were combined. The organic phase was washed with saturated sodium chloride (5 ml * 2), and then dried over anhydrous sodium sulfate. The mixture was filtered and evaporated to dryness to remove dichloromethane. The final product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 0-50%) to give 206 mg of a yellowish-brown solid, in 31% yield. ESI-MS m / z 250.2 (M+H) + .

[0330] Synthesis of compound QY-8-25:

[0331]

[0332] 5-Benzyloxazol-2-carboxylic acid ethyl ester (QY-8-25): QY-8-15 (206 mg, 0.83 mmol) was dissolved in 4 ml acetonitrile in a 15 ml pressure-resistant flask. Phosphorus pentoxide (587 mg, 4.1 mmol) was mixed into 4 ml acetonitrile and added dropwise to the reaction solution. The mixture was stirred vigorously and slowly heated to 70 °C. LC-MS was used for real-time monitoring. After 3 hours, no starting material peak was observed, and the reaction was stopped. The mixture was allowed to cool to room temperature, and the reaction system was slowly added dropwise to 0 °C saturated brine. The mixture was stirred for 5 min, extracted with ethyl acetate (10 ml * 3), and the organic phases were combined. The mixture was washed with saturated NaHCO3 (5 ml * 1), then with saturated brine (5 ml * 1), dried with an inorganic salt drying agent, and the organic solvent was removed by rotary evaporation. The mixture was then purified by silica gel column chromatography (petroleum ether: ethyl acetate = 0-30%) to give 90 mg of a pale yellow transparent liquid, with a yield of 47%. ESI-MS m / z 232.0 (M+H) + .

[0333] Synthesis of compound QY-8-28:

[0334]

[0335] 5-Benzyloxazole-2-carboxylic acid (QY-8-28): QY-8-25 (90 mg, 0.34 mmol) was dissolved in 2 mL of tetrahydrofuran and transferred to an 8 mL pressure-resistant bottle. Lithium hydroxide monohydrate (33 mg, 0.78 mmol) was dissolved in 0.5 mL of distilled water and added dropwise to the reaction solution under ice bath conditions. After mixing and stirring thoroughly, the mixture was gradually brought to room temperature. The reaction was monitored in real time by LC-MS. After 6 h, the reaction was stopped, and all solvents in the reaction system were removed by rotary evaporation. The crude product could be directly used for subsequent synthesis. ESI-MS m / z 202.1 (M+H) - .

[0336] Synthesis of compound QY-8-20:

[0337]

[0338] (4-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)benzyl)tert-butyl carbamate (QY-8-20): N-Boc-4-iodobenzylamine (2.48 g, 7.5 mmol) was added to a 30 ml pressure-resistant bottle, and after being dissolved evenly in 15 ml of dimethyl sulfoxide, 4-amino-7H-pyrrolo(2,3-d)pyrimidin (1.0 g, 7.5 mmol), cuprous iodide (711 mg, 3.7 mmol), cesium carbonate (4.86 g, 14.9 mmol), and 4,7-dimethoxy-1,10-phenololine (358 mg, 1.5 mmol) were added. After nitrogen protection, the mixture was heated to 100 °C and reacted overnight. After the reactants were consumed, the mixture was filtered through a membrane to remove insoluble solids, and then purified by C18 reversed-phase chromatography (water:acetonitrile = 0-80%) to obtain 1.94 g of a light green oily liquid, with a yield of 77%. ESI-MS m / z 340.1 (M+H) + .

[0339] Synthesis of compound QY-8-22:

[0340]

[0341] 7-(4-(aminomethyl)phenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amino (QY-8-22): QY-8-20 (1.94 g, 5.7 mmol) was mixed in 20 mL of dichloromethane and transferred to a 100 mL round-bottom flask. At room temperature, 2.0 mL of trifluoroacetic acid was added dropwise to the reaction mixture, and the mixture was stirred until homogeneous. The reaction mixture was monitored in real-time by LC-MS. After stirring overnight, the reactants were completely consumed. Each time, 20 mL of dichloromethane was added, and the mixture was evaporated to dryness to remove the organic solvent mixture. This process was repeated 4-6 times. The crude product from this step can be used directly for subsequent synthesis without further purification. ESI-MS m / z 240.1 (M+H) + .

[0342] Synthesis of compound QY-8-30:

[0343]

[0344] N-(4-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)benzyl)-5-benzyloxazol-2-carboxamide (QY-8-30): QY-8-28 (0.39 mmol) was dissolved in an 8 ml pressure-resistant bottle containing 4 ml of dichloromethane. HATU (177 mg, 0.47 mmol) was added, and QY-8-22 (220 mg, 0.62 mmol) was added to the stirred reaction solution. N,N-diisopropylethylamine (200 mg, 1.6 mmol) was added dropwise at room temperature, and the reaction was stirred for 6 h. The reaction was monitored in real time by LC-MS. After the reaction was complete, the reaction system was transferred to a separatory funnel, washed with 10 ml of distilled water, extracted with dichloromethane (15 ml * 2), the organic phases were combined, washed with saturated sodium chloride (5 ml * 2), dried over anhydrous sodium sulfate, and dichloromethane was removed by rotary evaporation. After filtration through a C18 reversed-phase column, the solution was purified (water:acetonitrile = 0-80%) to obtain 38 mg of a yellowish-brown oily liquid, with a yield of 23%. ESI-MS m / z 425.2 (M+H) + .

[0345] Method 11:

[0346] Synthesis of compound QY-8-33:

[0347]

[0348] (2-Methyl-1-oxo-1-(2-(p-methylphenyl)pyrrolidin-1-yl)propyl-2-yl)tert-butyl carbamate (QY-8-33): N-Boc-2-methylalanine (320 mg, 1.6 mmol) was dissolved in a 15 ml pressure-resistant bottle containing 8 ml of dichloromethane, HATU (652 mg, 1.7 mmol) was added, and QY-7-64 (230 mg, 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 5 hours, the starting material was consumed, and the reaction system was transferred to a separatory funnel. The mixture was washed with distilled water (10 ml x 3), extracted with dichloromethane (15 ml x 3), and the organic phases were combined. The organic phase was washed with saturated sodium chloride (10 ml x 2), dried over anhydrous sodium sulfate, and dichloromethane was removed by rotary evaporation. The mixture was then purified by silica gel column chromatography (petroleum ether: ethyl acetate = 0-70%) to give 202 mg of a pale yellow solid, with a yield of 41%. ESI-MS m / z 247.1 (M+H) + .

[0349] Synthesis of compound QY-8-39:

[0350]

[0351] 3-Amino-2-methyl-1-(2-(p-methylphenyl)pyrrolidin-1-yl)prop-1-one (QY-8-39): QY-8-33 (202 mg, 0.58 mmol) was mixed in 4 mL of dichloromethane and transferred to a 15 mL pressure-resistant flask. At room temperature, 0.4 mL of trifluoroacetic acid was added dropwise to the reaction mixture, and the mixture was stirred thoroughly. The reaction mixture was monitored in real-time by LC-MS. After 3 hours, the reactants were completely consumed. The reaction mixture was then transferred to a 100 mL round-bottom flask, with 10 mL of dichloromethane added each time. The organic solvent mixture was removed by rotary evaporation. This process was repeated 3-4 times. The crude product from this step can be used directly for subsequent synthesis without further purification. ESI-MS m / z 247.1 (M+H) + .

[0352] Synthesis of compound QY-8-42:

[0353]

[0354] N-Methyl-1-(4-(2-((2-methyl-1-oxo-1-(2-(p-methylphenyl)pyrrolidin-1-yl)propyl-2-yl)amino)-2-oxoethyl)phenyl)-1H-benzo[d]imidazolium-5-carboxamide (QY-8-42): In an 8 ml pressure-resistant bottle, QY-8-39 (0.29 mmol) was dissolved in 3 ml of dichloromethane, and HATU (221 mg, 0.58 mmol) and QY-5-34 (90 mg, 0.29 mmol) were added. N,N-diisopropylethylamine (150 mg, 1.2 mmol) was slowly added dropwise at room temperature, and the reaction was stirred for 5 h. The reaction was monitored in real time by LC-MS. Once the reactants were completely consumed, 5 ml of dichloromethane was added to dilute the reaction solution. The solution was transferred to a separatory funnel, washed with 10 ml of distilled water, and extracted with dichloromethane (10 ml * 3). The organic phases were combined, washed with saturated sodium chloride (10 ml * 2), dried over anhydrous sodium sulfate, filtered, and the dichloromethane was removed by rotary evaporation. After filtration through a membrane, the solution was purified by C18 reversed-phase chromatography (water:acetonitrile = 0-80%) to obtain 48 mg of a yellowish-brown oily liquid, with a yield of 31%. ESI-MS m / z 538.1 (M+H) + .

[0355] Method 12:

[0356] Synthesis of compound ZSQ-13-19:

[0357]

[0358] Ethyl 2-ethoxy-2-iminoacetate (ZSQ-13-19): Ethyl cyanoformate (9.9 ml, 100 mmol) was added to a 250 ml round-bottom flask and mixed with ethanol (23.0 ml, 400 mmol) until dissolved. The reaction system was then cooled to 0 °C. Acetyl chloride (14.2 ml, 200 mmol) was added dropwise to the reaction solution. After the addition was complete, the mixture was stirred overnight at 0 °C. After the reaction was complete, a white solid in lumps was obtained. The crude product was obtained by filtration and can be directly used for the next reaction without further purification. ESI-MS m / z 146.0 (M+H) + .

[0359] Synthesis of compound ZSQ-13-20:

[0360]

[0361] (Z)-2-amino-2-(2-(2-phenylacetyl)hydrazono)ethyl acetate (ZSQ-13-20): ZSQ-13-19 (7.26 g, 50 mmol) was added to a 250 mL round-bottom flask. 50 mL of ethanol was added, and the mixture was stirred until dissolved. Then, phenylacetylhydrazine (7.51 g, 50 mmol) was added, and 40 mL of diethyl ether was added while stirring continuously. The reaction was stopped after stirring overnight at room temperature. The crude product was obtained by filtration and can be directly used for the next reaction without further purification. ESI-MS m / z 250.1 (M+H) + .

[0362] Synthesis of compound QY-8-49:

[0363]

[0364] Ethyl 1-acetyl-5-benzyl-1H-1,2,4-triazole-3-carboxylic acid (QY-8-49): Compound ZSQ (1.0 g, 4.0 mmol) was dissolved in 5 mL of acetic anhydride. The mixture was stirred thoroughly in a 15 mL pressure-resistant flask, and the temperature was slowly raised to 140 °C and stirred vigorously for 3 h. LC-MS was monitored until the reaction was complete. After the reaction system returned to room temperature, saturated sodium bicarbonate aqueous solution was slowly added dropwise to quench the reaction. After stirring for 15 min, the mixture was transferred to a separatory funnel and extracted with ethyl acetate (20 mL * 3). The organic phases were combined, washed with saturated sodium chloride (10 mL * 2), dried over anhydrous sodium sulfate, filtered, and the ethyl acetate was removed by rotary evaporation. The solution was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 0-40%) to give 280 mg of a pale yellow transparent oily liquid, with a yield of 26%. ESI-MS m / z 274.1 (M+H) + .

[0365] Synthesis of compound QY-8-52:

[0366]

[0367] 5-Benzyl-1H-1,2,4-triazole-3-carboxylic acid (QY-8-52): QY-8-52 (280 mg, 1.0 mmol) was dissolved in 4 mL of tetrahydrofuran and transferred to an 8 mL pressure-resistant bottle. Lithium hydroxide monohydrate (147 mg, 3.5 mmol) was dissolved in 1 mL of distilled water and added dropwise to the reaction solution under ice bath conditions. After mixing and stirring thoroughly, the mixture was gradually brought to room temperature and stirred overnight. LC-MS was used to monitor the reaction until complete. All solvents in the reaction system were removed by rotary evaporation. The crude product can be directly used for subsequent synthesis without purification. ESI-MS m / z 203.9 (M+H) + .

[0368] Synthesis of compound QY-8-60:

[0369]

[0370] N-(4-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)benzyl)-5-benzyl-1H-1,2,4-triazol-3-carboxamide (QY-8-60): In an 8 ml pressure-resistant bottle, QY-8-52 (0.40 mmol) was dissolved in 3 ml of dichloromethane. HATU (182 mg, 0.48 mmol) and QY-8-22 (155 mg, 0.44 mmol) were added to the reaction solution. N,N-diisopropylethylamine (258 mg, 2.0 mmol) was slowly added dropwise at room temperature, and the reaction was stirred for 4 h. LC-MS was monitored until the reactants were completely consumed, at which point the reaction was stopped. 5 mL of dichloromethane was added to dilute the reaction solution, which was then transferred to a separatory funnel. The solution was washed with 10 mL of distilled water, extracted with dichloromethane (10 mL x 3), and the organic phases were combined. The mixture was washed with saturated sodium chloride (10 mL x 2), dried over anhydrous sodium sulfate, filtered, and the dichloromethane was removed by rotary evaporation. The purified solution was then filtered through a C18 reversed-phase column (water:acetonitrile = 0-80%) to yield 66 mg of a yellowish-brown liquid, with a yield of 39%. ESI-MS m / z 425.2 (M+H) + .

[0371] Method 13:

[0372] Synthesis of compound QY-8-50:

[0373]

[0374] Ethyl 2H-tetrazole-5-carboxylic acid ester (QY-8-50): Ethyl cyanoformate (1.0 g, 10.0 mmol) was dissolved in 12 mL of pyridine. The mixture was stirred thoroughly in a 75 mL sealed tube. 1.76 mL of trifluoroacetic acid was slowly added dropwise at room temperature. After stirring for 10 min, sodium azide (700 mg, 10.8 mmol) was added. The mixture was slowly heated to 60 °C, and the reaction was stopped after 24 h. Sodium azide was removed by diatomaceous earth filtration. The diatomaceous earth was washed with ethyl acetate (10 mL * 3). The organic phase was removed by rotary evaporation. The crude product was directly used for the next step without further purification. ESI-MS m / z 143.0 (M+H) + .

[0375] Synthesis of compound QY-8-55:

[0376]

[0377] Ethyl 2-benzyl-2H-tetrazole-5-carboxylic acid ester (QY-8-55): QY-8-50 (10.0 mmol) was stirred and mixed 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. The reaction was stopped after stirring overnight. The potassium carbonate was removed by diatomaceous earth filtration, and the organic solvent was removed by rotary evaporation. The solution was filtered through a C18 reversed-phase chromatography column (water:acetonitrile = 0-100%) to obtain 550 mg of a yellowish-brown oily liquid. The yield of the two-step reaction was 24%. ESI-MS m / z 233.1 (M+H) + .

[0378] Synthesis of compound QY-8-56:

[0379]

[0380] 2-Benzyl-2H-tetrazole-5-carboxylic acid (QY-8-56): QY-8-55 (100 mg, 0.43 mmol) was dissolved in 3 mL of tetrahydrofuran and transferred to an 8 mL pressure-resistant bottle. Lithium hydroxide monohydrate (27 mg, 0.65 mmol) was dissolved in 0.8 mL of distilled water and added dropwise to the reaction solution at room temperature. The reaction progress was monitored in real time by LC-MS. After 2 h, the starting materials were consumed. All solvents in the reaction system were removed by rotary evaporation. The crude product can be directly used for subsequent synthesis without purification. ESI-MS m / z 205.1 (M+H) + .

[0381] Synthesis of compound QY-8-58:

[0382]

[0383] 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 (0.40 mmol) in 3 ml dichloromethane in an 8 ml pressure-resistant bottle, add HATU (182 mg, 0.48 mmol) and QY-8-22 (141 mg, 0.40 mmol); slowly add N,N-diisopropylethylamine (259 mg, 2.0 mmol) at room temperature, and continue stirring the reaction. The reaction was monitored in real time by LC-MS. After 4 hours, the starting material was consumed. 5 ml of dichloromethane was added to dilute the reaction solution, which was then transferred to a separatory funnel. The solution was washed with 10 ml of distilled water, extracted with dichloromethane (10 ml * 3), and the organic phases were combined. The solution was washed with saturated sodium chloride (10 ml * 2), dried over anhydrous sodium sulfate, filtered, and the dichloromethane was removed by rotary evaporation. After filtration through a membrane, the solution was purified by C18 reversed-phase chromatography (water:acetonitrile = 0-60%) to obtain 140 mg of a yellowish-brown solid, with a yield of 82%. ESI-MS m / z 426.1 (M+H) + .

[0384] Method 14:

[0385] Synthesis of compound QY-8-29:

[0386]

[0387] (4-(6-amino-9H-purin-9-yl)benzyl)tert-butyl carbamate (QY-8-29): N-Boc-4-iodobenzylamine (330 mg, 1.0 mmol) was added to a 15 mL pressure-resistant bottle, dissolved in 3 mL of dimethyl sulfoxide, followed by the addition of adenine (135 mg, 1.0 mmol), cuprous iodide (95 mg, 0.5 mmol), cesium carbonate (652 mg, 2.0 mmol), and 4,7-dimethoxy-1,10-phenoxyl (48 mg, 0.2 mmol). The mixture was heated to 100 °C overnight under nitrogen protection. After the reactants were consumed, the mixture was filtered to remove insoluble solids, and then purified by C18 reversed-phase chromatography (water:acetonitrile = 0-80%) to give 287 mg of a greenish-white solid, yield 84%. ESI-MS m / z 341.2 (M+H) + .

[0388] Synthesis of compound QY-8-36:

[0389]

[0390] 9-(4-(aminomethyl)phenyl)-9H-purine-6-amino (QY-8-36): Mix QY-8-29 (286 g, 0.84 mmol) in 3 mL of dichloromethane. Add 0.5 mL of trifluoroacetic acid dropwise to the reaction mixture in a 15 mL pressure-resistant flask at room temperature, and stir until homogeneous. Monitor the reaction mixture in real-time by LC-MS. After stirring overnight, the reactants are consumed. Add 20 mL of dichloromethane each time, evaporate to dryness, and remove the organic solvent mixture. Repeat 3-4 times. The crude product from this step can be used directly for subsequent synthesis without further purification. ESI-MS m / z 241.2 (M+H) + .

[0391] Synthesis of compound QY-8-43:

[0392]

[0393] N-(4-(6-amino-9H-purin-9-yl)benzyl)-5-benzyl-1-methyl-1H-pyrazole-3-carboxamide (QY-8-43): In an 8 ml pressure-resistant bottle, QY-6-103B (150 mg, 0.69 mmol) was dissolved in 8 ml of dichloromethane, and HATU (316 mg, 0.83 mmol) was added. QY-8-36 (166 mg, 0.69 mmol) was added to the stirred reaction solution. N,N-diisopropylethylamine (313 mg, 2.43 mmol) was added dropwise at room temperature, and the reaction was stirred for 4 h. The reaction was monitored in real time by LC-MS. Once the reactants were completely consumed, the mixture was transferred to a separatory funnel, washed with 10 ml of distilled water, extracted with dichloromethane (15 ml * 3), and the organic phases were combined. The mixture was washed with saturated sodium chloride (10 ml * 2), dried over anhydrous sodium sulfate, filtered, and the dichloromethane was removed by rotary evaporation. The resulting product was purified by silica gel column chromatography (dichloromethane:methanol = 0-25%) to give 142 mg of a white solid, with a yield of 47%. ESI-MS m / z 439.1 (M+H) + .

[0394] Method 15:

[0395] Synthesis of compound QY-9-69:

[0396]

[0397] ON-(4-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)benzyl)-2-(3-(trifluoromethoxy)phenyl)acetamide (QY-9-69): In an 8 ml pressure-resistant bottle, QY-8-22 (59 mg, 0.25 mmol) was dissolved in 3 ml of dichloromethane, HATU (125 mg, 0.33 mmol) was added, and 3-trifluoromethoxyphenylacetic acid (60 mg, 0.25 mmol) was added to the stirred reaction solution; N,N-diisopropylethylamine (129 mg, 1.0 mmol) was added dropwise at room temperature, and the reaction was stirred for 3.5 h. The reaction was monitored in real time by LC-MS. Once the reactants were completely consumed, the mixture was transferred to a separatory funnel, washed with 5 ml of distilled water, extracted with dichloromethane (5 ml x 3), and the organic phases were combined. The mixture was washed with saturated sodium chloride (5 ml x 2), dried over anhydrous sodium sulfate, filtered, and the dichloromethane was removed by rotary evaporation. The solution was then purified by silica gel column chromatography (dichloromethane:methanol = 0-30%) to give 88 mg of a pale yellow oily liquid, with a yield of 80.0%. ESI-MS m / z 442.0 (M+H) + .

[0398] Method 16:

[0399] Synthesis of compound QY-9-10:

[0400]

[0401] 2-(2-Fluoropyridin-3-yl)-2-oxoethyl acetate (QY-9-10): Add 5.0 g (51.5 mmol) of 2-fluoropyridine to a 250 ml round-bottom flask, add 120 ml of tetrahydrofuran, dissolve and stir until homogeneous. After nitrogen purging, transfer the reaction system to -78 °C. Slowly add 30.4 ml (61.8 mmol) of lithium diisopropylaminoacetate to the reaction solution. After the addition is complete, add 9.0 g (61.8 mmol) of diethyl oxalate. The mixture was gradually brought to room temperature and stirred for 2 hours. The reaction was monitored in real-time by LC-MS until all reactants were consumed. Saturated ammonium chloride was added to quench the reaction. The reaction mixture was transferred to a separatory funnel and extracted with ethyl acetate (20 ml * 3). The organic phases were combined and washed with saturated sodium chloride (15 ml * 2). The organic phase was dried over anhydrous sodium sulfate, filtered, and the ethyl acetate was removed by rotary evaporation. The mixture was then purified by silica gel column chromatography (petroleum ether:ethyl acetate = 0-40%) to give 3.54 g of a bright yellow oily liquid, with a yield of 34.9%. ESI-MS m / z 198.0 (M+H) + .

[0402] Synthesis of compound QY-9-18:

[0403]

[0404] 1H-pyrazolo[3,4-b]pyridine-3-carboxylic acid ethyl ester (QY-9-18): QY-9-10 (2.87 g, 14.6 mmol) was added to a 100 mL round-bottom flask, followed by the addition of 25 mL of N-methylpyrrolidone. The mixture was stirred until dissolved and homogeneous. Hydrazine hydrate (0.87 g, 17.5 mmol) was slowly added dropwise to the reaction solution under ice bath conditions. The mixture was stirred at 0 °C for 20 min, gradually cooled to room temperature, and then heated to 80 °C with stirring overnight. LC-MS was used to monitor complete consumption of the reactants. Distilled water was added, and the mixture was filtered through a membrane and purified by C18 reversed-phase chromatography (water:acetonitrile = 0-80%) to obtain 1.37 g of a yellow-red solid, with a yield of 49%. ESI-MS m / z 192.0 (M+H) + .

[0405] Synthesis of compound QY-9-21:

[0406]

[0407] 5-Bromo-1H-pyrazolo[3,4-b]pyridine ε-3-carboxylic acid ethyl ester (QY-9-21): QY-9-18 (1.37 g, 7.17 mmol) and sodium acetate (3.53 g, 43.0 mmol) were mixed into 25 ml of glacial acetic acid and added to a 100 ml round-bottom flask. The mixture was stirred until homogeneous. Liquid bromine (3.43 g, 21.5 mmol) was added dropwise at room temperature. The mixture was heated to 100 °C and stirred for 8 h. The reaction was stopped after the reactants were completely consumed by LC-MS and the temperature was gradually lowered to room temperature. The reaction was quenched with 3M sodium thiosulfate solution, transferred to a separatory funnel, and extracted with ethyl acetate (15 ml x 3). The organic phases were combined, washed with distilled water (10 ml x 2), washed with saturated brine (10 ml x 2), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to remove ethyl acetate. The mixture was then purified by silica gel column chromatography (petroleum ether:ethyl acetate = 0-25%) to give 0.26 g of white solid, yield 13.4%. ESI-MS m / z 269.9 (M+H) + .

[0408] Synthesis of compound QY-9-29:

[0409]

[0410] 5-Bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyridine ethyl 3-carboxylate (QY-9-29): QY-9-21 (255 mg, 0.94 mmol) was dissolved in 7 mL of dichloromethane and transferred to a 15 mL pressure-resistant flask. 3,4-Dihydro-2H-pyran (159 mg, 1.89 mmol) was added, followed by pyridine p-toluenesulfonic acid (48 mg, 0.19 mmol) to the stirred reaction mixture. The mixture was heated to 60 °C and stirred continuously for 5 h, monitored in real-time by LC-MS. After the reactants were completely consumed, the dichloromethane was removed by rotary evaporation. The mixture was then purified by silica gel column chromatography (petroleum ether: ethyl acetate = 0-15%) to give 269 mg of white solid, yield 80.8%. ESI-MS m / z 354.0 (M+H) + .

[0411] Synthesis of compound QY-9-51:

[0412]

[0413] 5-Iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyridine ε-3-carboxylic acid ethyl ester (QY-9-51): QY-9-29 (390 mg, 1.10 mmol) was added to a 15 ml pressure-resistant bottle. After dissolving 1,4-dioxane in 5 ml, sodium iodide (436 mg, 2.91 mmol), cuprous iodide (34 mg, 0.18 mmol), and (1S,2S)-(+)-N,N'-dimethyl-1,2-cyclohexanediamine (38 mg, 0.27 mmol) were added. The mixture was heated to 130 °C and microwaved for 3 h. The reaction mixture was monitored by LC-MS to ensure complete consumption of the reactants. After removing the insoluble solids by filtration, the mixture was purified by C18 reversed-phase chromatography (water:acetonitrile = 5-100%) to obtain 359 mg of a yellowish-brown solid, with a yield of 81.3%. ESI-MS m / z 402.0(M+H) + .

[0414] Synthesis of compound QY-9-57:

[0415]

[0416] 5-(perfluoroethyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyridine-3-carboxylic acid ethyl ester (QY-9-57): QY-9-51 (316 mg, 0.79 mmol) was added to a 15 mL pressure-resistant bottle, dissolved in 4 mL of dimethyl sulfoxide, and then (pentafluoroethyl)trimethylsilane (604 mg, 3.14 mmol) and cuprous iodide (600 mg, 3.15 mmol) were added. After stirring, the mixture was slowly heated to 80 °C and reacted overnight. LC-MS was used to monitor the complete consumption of the reactants. After filtering to remove insoluble solids, the mixture was purified by C18 reversed-phase chromatography (water:acetonitrile = 5-100%) to obtain 106 mg of a colorless, transparent, oily liquid, with a yield of 34.1%. ESI-MS m / z 394.1 (M+H) + .

[0417] Synthesis of compound QY-9-74:

[0418]

[0419] 5-(perfluoroethyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyridine-3-carboxylic acid (QY-9-74): QY-9-57 (46 mg, 0.12 mmol) was dissolved in 2 mL of tetrahydrofuran and transferred to an 8 mL pressure-resistant bottle. Lithium hydroxide monohydrate (24 mg, 0.47 mmol) was dissolved in 0.5 mL of distilled water and added dropwise to the reaction solution under ice bath conditions. After mixing and stirring until homogeneous, the mixture was gradually brought to room temperature. The reaction was monitored by LC-MS and stirred overnight until complete. After removing tetrahydrofuran and water by rotary evaporation, the mixture was dried by pumping dryness and proceeded directly to the next step of the reaction without separation and purification. ESI-MS m / z 364.1 (M+H) - .

[0420] Synthesis of compound QY-9-76:

[0421]

[0422] N-(4-(6-amino-9H-purin-9-yl)benzyl)-5-(perfluoroethyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyridine-3-carboxamide (QY-9-76): Dissolve QY-9-74 (0.12 mmol) in 3 ml of dichloromethane in an 8 ml pressure-resistant bottle, add HATU (54 mg, 0.14 mmol) and QY-8-36 (31 mg, 0.13 mmol), and add N,N-diisopropylethylamine (60 mg, 0.47 mmol) dropwise at room temperature while stirring continuously at room temperature. LC-MS was used for real-time monitoring. After the reaction was complete, 5 ml of dichloromethane was added to dilute the reaction solution. The solution was transferred to a separatory funnel, washed with 5 ml of distilled water, and extracted with dichloromethane (5 ml * 3). The organic phases were combined, washed with saturated sodium chloride (5 ml * 2), and the resulting organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to remove dichloromethane. The solution was then purified by silica gel column chromatography (dichloromethane:methanol (ammonia) = 0-20%) to give 52 mg of a yellowish-brown solid, with a yield of 76%. ESI-MS m / z 588.2 (M+H) + .

[0423] Synthesis of compound QY-9-77:

[0424]

[0425] N-(4-(6-amino-9H-purin-9-yl)benzyl)-5-(perfluoroethyl)-1H-pyrazolo[3,4-b]pyridine-3-carboxamide (QY-9-77): QY-9-76 (52 mg, 0.09 mmol) was mixed in 2 mL of dichloromethane and transferred to an 8 mL pressure-resistant bottle. At room temperature, 0.4 mL of trifluoroacetic acid was added dropwise to the reaction mixture, and the mixture was stirred until homogeneous. LC-MS was used for real-time monitoring. After 1.5 h, the reactants were consumed and transferred to a round-bottom flask. 10 mL of dichloromethane was added, and the mixture was evaporated to dryness to remove the organic solvent mixture. This process was repeated 3-4 times. The mixture was purified by HPLC to obtain 38 mg of white solid, with a yield of 87%. ESI-MS m / z 504.1 (M+H) + .

[0426] Method 17:

[0427] Synthesis of compound QY-10-21:

[0428]

[0429] N-(4-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)benzyl)-5-benzyl-1-methyl-1H-pyrazole-3-carboxamide (QY-10-21): In a 100 ml round-bottom flask, QY-6-103B (271 mg, 1.25 mmol) was dissolved in 15 ml dichloromethane, and HATU (570 mg, 1.50 mmol) was added. 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 stirred for 3.5 h. The reaction was monitored in real time by LC-MS until the substrate was completely consumed. The mixture was transferred to a separatory funnel, washed with 10 ml of distilled water, extracted with dichloromethane (15 ml * 3), and the organic phases were combined. The mixture was washed with saturated sodium chloride (10 ml * 2), dried over anhydrous sodium sulfate, filtered, and the dichloromethane was removed by rotary evaporation. The solution was then purified by silica gel column chromatography (dichloromethane:methanol (ammonia) = 0-25%) to give 344 mg of a pale yellow oily liquid, with a yield of 63%. ESI-MS m / z 438.2 (M+H) + .

[0430] Method 18:

[0431] Synthesis of compound QY-10-47:

[0432]

[0433] 5-Benzyl-1-methyl-1H-pyrazole-4-carboxylic acid ethyl ester (QY-10-47): In a 75 ml sealed tube, add 15 ml of toluene, 1-methylpyrazole-4-carboxylic acid ethyl ester (960 mg, 6.23 mmol), stir well, then add palladium acetate (139 mg, 0.62 mmol), triphenylphosphine (327 mg, 1.25 mmol), potassium carbonate (2.15 g, 15.6 mmol), tervastatin (191 mg, 1.87 mmol), and dropwise add benzyl chloride (946 mg, 7.47 mmol). Under nitrogen protection, heat to 100 °C and react overnight. The reaction was monitored by LC-MS. After the reactants were consumed, the reaction solution was diluted with ethyl acetate, transferred to a separatory funnel, washed with distilled water, and extracted with ethyl acetate (10 ml * 3). The organic phases were combined, washed with saturated sodium chloride (10 ml * 2), dried over anhydrous sodium sulfate, filtered, and the organic solvent was removed by rotary evaporation. The solution was then purified by silica gel column chromatography (petroleum ether:ethyl acetate = 0-40%) to give 190 mg of a pale yellow oily liquid, with a yield of 12.5%. ESI-MS m / z 245.1 (M+H) + .

[0434] Synthesis of compound QY-10-56:

[0435]

[0436] 5-Benzyl-1-methyl-1H-pyrazole-4-carboxylic acid (QY-10-56): QY-10-47 (190 mg, 0.78 mmol) was dissolved in 5 mL of tetrahydrofuran and transferred to a 15 mL pressure-resistant bottle. Lithium hydroxide monohydrate (130 mg, 3.11 mmol) was dissolved in 2 mL of distilled water and added dropwise to the reaction solution under ice bath conditions. After mixing and stirring until homogeneous, the mixture was gradually brought to room temperature. LC-MS was used for real-time monitoring. The reaction was stirred overnight until complete. The solution was transferred to a 100 mL round-bottom flask, and the organic solvent and water were removed by rotary evaporation. The mixture was then dried using an oil pump and proceeded directly to the next reaction without further separation and purification. ESI-MS m / z 215.1 (M+H) - .

[0437] Synthesis of compound QY-10-39:

[0438]

[0439] N-(4-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)benzyl)-5-benzyl-1-methyl-1H-pyrazole-4-carboxamide (QY-10-39): Dissolve QY-10-56 (0.31 mmol) in 3 ml dichloromethane in an 8 ml pressure-resistant bottle, add HATU (141 mg, 0.37 mmol) and QY-8-22 (74 mg, 0.31 mmol) to the reaction solution; add N,N-diisopropylethylamine (160 mg, 1.24 mmol) dropwise at room temperature. LC-MS real-time monitoring was performed. After 4 hours, the raw material was consumed. 10 ml of dichloromethane was added to dilute the reaction solution, which was then transferred to a separatory funnel. The mixture was washed with 5 ml of distilled water and extracted with dichloromethane (10 ml * 3). The organic phases were combined and washed with saturated sodium chloride (5 ml * 2). The resulting organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to remove dichloromethane. The solution was then purified by silica gel column chromatography (dichloromethane:methanol (ammonia) = 0-20%) to give 89 mg of white solid, with a yield of 66%. ESI-MS m / z 438.2 (M+H) + .

[0440] By substituting different synthetic substrates using the method described above, compounds as shown in Table A were obtained:

[0441] Table A

[0442]

[0443]

[0444]

[0445]

[0446]

[0447] The NMR data for the compounds shown in Table A are as follows:

[0448] QY-5-35: 1 H 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) + .

[0449] QY-5-40: 1 H 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) + .

[0450] QY-5-62: 1 H 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 / z415.2(M+H) + .

[0451] QY-6-15: 1 H 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) + .

[0452] QY-6-16: 1 H 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) + .

[0453] 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) + .

[0454] 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) + .

[0455] QY-7-14: 1 H 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) + .

[0456] QY-7-32: 1 H 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) + .

[0457] 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) + .

[0458] QY-8-7: 1 H 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) + .

[0459] QY-8-30: 1 H 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 / z425.2(M+H) + .

[0460] 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) + .

[0461] QY-8-42: 1 H 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) + .

[0462] QY-8-43: 1 H 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) + .

[0463] 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 / z425.2(M+H) + .

[0464] QY-8-58: 1 H 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) + .

[0465] QY-8-60: 1 H 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) + .

[0466] 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) + .

[0467] QY-9-34: 1 H 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) + .

[0468] QY-9-41: 1 H 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) + .

[0469] 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) + .

[0470] QY-9-69: 1 H 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) + .

[0471] QY-9-70: 1 H 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 + .

[0472] QY-9-77: 1 H 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) + .

[0473] QY-10-21:1 H 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) + .

[0474] QY-10-39: 1 H 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) + .

[0475] QY-10-40: 1 H 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) + .

[0476] 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) + .

[0477] QY-10-73: 1 H 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) + .

[0478] QY-11-68A: 1 H 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) + .

[0479] 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-MSm / z 578.2(M+H) + .

[0480] QY-11-69A: 1 H 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 / z591.2(M+H) + .

[0481] QY-11-69B: 1 H 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) + .

[0482] QY-11-74A: 1 H 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) + .

[0483] QY-11-74B: 1 H 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) + .

[0484] QY-11-75: 1 H 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) + .

[0485] QY-11-76

[0486] 1 H 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) + .

[0487] QY-11-77: 1 H 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) + .

[0488] QY-11-78: 1 H 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) + .

[0489] 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) + .

[0490] QY-11-80: 1 H 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) + .

[0491] QY-11-102A: 1 H 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) + .

[0492] 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 / z452.1(M+H) + .

[0493] QY-11-104: 1 H 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) + .

[0494] QY-12-4: 1 H 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) + .

[0495] 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) + .

[0496] ZSQ-13-36: 1 H 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) + .

[0497] ZSQ-13-46: 1 H 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 / z466.1(M+H) + .

[0498] 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) + .

[0499] ZSQ-15-67: 1 H 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) + .

[0500] ZSQ-15-68: 1 H 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) + .

[0501] 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) + .

[0502] XHJ-2-88: 1 H 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) + .

[0503] XHJ-4-36: 1 H 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) + .

[0504] 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) + .

[0505] ZSQ-20-94: 1 H 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-MSm / z 425.1(M+H) + .

[0506] ZSQ-20-106: 1 H 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) + .

[0507] 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) + .

[0508] ZSQ-21-2: 1 H 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-MS m / z 451.1(M+H) + .

[0509] ZSQ-21-8: 1 H 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) + .

[0510] ZSQ-21-13: 1 H 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)+ .

[0511] ZSQ-21-14: 1 H 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 / z425.2(M+H) + .

[0512] ZSQ-21-18: 1 H 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) + .

[0513] ZSQ-21-30: 1 H 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) + .

[0514] Biological test cases

[0515] The structure of the control compound used in the test example is as follows:

[0516]

[0517] Biological Test Example 1: Inhibitory Activity Test of RIPK1 Inhibitors on Programmed Cell Necrosis

[0518] The biological assay used was to investigate the effect of the compound on TNF-induced programmed necrosis in Jurkat and L929 cells lacking the FADD (Fas-related death domain).

[0519] To verify the inhibitory effect of the compounds in this invention on programmed cell death at the cellular level, cell types closely related to the RIP1 pathway, namely FADD-deficient Jurkat cells (human peripheral blood leukemia T cell line) and L929 cells, were selected. Two different stimulation methods were used: tumor necrosis factor (TNFα) alone, or TNFα in combination with mitochondrial-derived cysteine ​​aspartate activator (SMAC) SM164. Cell viability was calculated by detecting chemiluminescence values, thereby determining the biological activity of the compounds in inhibiting programmed cell death.

[0520] Methods: FADD-deficient Jurkat cells: FADD-deficient Jurkat cells (human peripheral blood leukemia T cell line) were cultured in vitro. After reaching the logarithmic growth phase, cells were collected, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cell concentration was adjusted to 2.5 × 10⁻⁶ cells / mL. 5 Cells were seeded into 384-well plates at a concentration of 40 μL / mL. 5 μL each of SM164 (50 nM) diluted with cell culture medium and the compound were added to the corresponding wells. After pretreatment at 37°C for 1 h, 5 μL of TNFα (50 ng / mL) diluted with cell culture medium was added to each well in the stimulation group, and 5 μL of culture medium was added to each well in the control group. After culturing in a cell culture incubator (37°C, 5% CO2) for 14 h, 15 μL of Cell Titer-Glo solution was added to each well, and the cells were incubated at room temperature for 30 min. Chemiluminescence was measured to assess intracellular ATP levels. The unstimulated DMSO control wells were considered 100% cell viability. L929 cells: Exogenous L929 cells (mouse fibroblasts) were digested and diluted to 6.25 × 10⁻⁶. 4Cells were seeded at a concentration of 40 μl / ml into 384-well plates. Cells were incubated for 12 h at 37°C (5% CO2). 5 μl each of SM164 (500 nM) diluted with cell culture medium and the compound were added to the corresponding wells. After pretreatment at 37°C for 1 h, 5 μl of TNFα (500 ng / mL) diluted with cell culture medium was added to each well in the stimulation group, and 5 μl of culture medium was added to each well in the control group. Cells were incubated for 14 h at 37°C (5% CO2). Then, 15 μl of CellTiter-Glo solution was added to each well, and the cells were incubated at room temperature for 30 min. Chemiluminescence was measured to assess intracellular ATP levels. The unstimulated DMSO control wells were considered 100% viable. The EC50 of the compound was calculated using Prism Graphpad statistical software. 50 Value. Result as follows Figure 1 As shown in Table 1.

[0521] Table 1. Results of the inhibitory activity test of RIPK1 inhibitors against programmed cell death.

[0522]

[0523]

[0524]

[0525] Figure 1 The experimental results in Table 1 show that, regardless of whether TNFα stimulation was performed alone or in combination with SM164 stimulation, the preferred compounds QY-10-40, QY-11-76, and QY-11-102A / B of this invention exhibited stronger inhibitory activity against programmed necrosis in human FADD-deficient Jurkat cells than the clinical inhibitor GSK2982772. Furthermore, in mouse L929 cells where GSK2982772 was almost inactivated, several compounds, including the preferred compounds QY10-40, QY-11-76, and QY-11-102A / B of this invention, still effectively inhibited programmed necrosis. QY10-40 at concentrations above 1 nM essentially completely inhibited TNFα-induced programmed necrosis in FADD-deficient Jurkat or L929 cells, and at a 1000-fold effective inhibitory concentration (1 μM), it did not exhibit cytotoxicity.

[0526] Biological Test Example 2: Liver Microsomal Stability Test of RIPK1 Inhibitor

[0527] The biological assay protocol used was to test the half-life of the compound in human or mouse liver microsomes.

[0528] Hepatic microsomes contain various enzymes involved in drug metabolism, especially cytochrome P-450, making them one of the main tissues for drug metabolism in vivo. The stability of compounds in hepatic microsomes is correlated with their in vivo pharmacokinetic stability. Therefore, by testing the half-life of different compounds in human or mouse hepatic microsomes, the relative stability of compounds in vivo can be roughly predicted. The test results are shown in Table 2, and the specific methods are as follows:

[0529] Methods: 0.75 μL of the sample (0.5 mM of the test compound and verapamil, the reference standard) and 9.38 μL of liver microsomes (Human and Mouse) at 20 mg / mL were added to 239.88 μL of PBS (on ice). The solutions were aliquoted into 30 μL tubes and incubated in a 37°C water bath for 5 min. At time zero, 90 μL of ice-cold acetonitrile was added to the control sample, followed by 15 μL of 3 mM NADPH solution. All samples were incubated in a 37°C water bath for 5, 10, 20, 30, and 60 min, and quenched with 90 μL of ice-cold acetonitrile. All samples were centrifuged at 10,000 rpm for 10 min, and the supernatant was transferred to a liquid chromatography-mass spectrometry (LC-MS) bottle for analysis.

[0530] Table 2. Results of liver microsomal stability assay for RIPK1 inhibitors.

[0531]

[0532]

[0533] Experimental results show that several representative RIPK1 inhibitors, such as QY-10-40, exhibit good stability in human or mouse liver microsomes.

[0534] Biological test example 3 tested the effect of the representative compound QY-10-40 on the activity of RIPK1 protein kinase.

[0535] The biological assay used was to test the effect of compound QY-10-40 on the kinase activity of RIPK1(1-330) protein. The in vitro purified RIPK1(1-330) protein retained its complete kinase active domain and maintained good kinase activity. The IC50 values ​​of the compounds in this invention inhibiting RIPK1 kinase activity in different ATP environments were determined. 50 The binding mechanism of the compound to RIPK1 was determined. The known RIPK1 ATP non-competitive inhibitor Nec-1s was used as a control.

[0536] The in vitro kinase reaction process is related to substrate concentration. The efficacy of substrate-competitive inhibitors varies drastically with substrate concentration, while the efficacy of substrate-non-competitive inhibitors remains unchanged. ATP-competitive inhibitors of kinases inhibit kinase activity by competing with ATP for binding sites. Due to the conservation of ATP binding sites, ATP-competitive kinase inhibitors generally have poor specificity. Conversely, ATP-non-competitive kinase inhibitors generally have high specificity.

[0537] Methods: RIPK1(1-330) protein and ATP (1X kinase buffer) at a final concentration of 2 μM and 5 μL, respectively, were added to 384-well plates. At least three auxiliary wells were included in each group. The reaction was carried out at 37°C for 2 h. 5 μL of ADP-Glo ​​reagent was added to stop the kinase reaction and remove residual ATP from the reaction system. The plate was incubated at room temperature for 40 min. 10 μL of kinase detection reagent was added to convert ADP to ATP and introduce luciferase and luciferin, which are used to detect ATP. The reaction was carried out at room temperature for 1 h. Luminescence was detected using a 7500 Fast Real-Time PCR System. The IC50 of the compounds inhibiting the kinase reaction was calculated using Prism Graphpad statistical software. 50 Please see the experimental results. Figure 2 .

[0538] Experimental results showed that the representative compound QY-10-40 exhibited concentration-dependent effective inhibition of RIPK1 kinase, with an effective half-maximum inhibitory concentration (WMC) of 219 nM, which was superior to the control compound Nec-1s (WMC of 499 nM).

[0539] Biological Test Example 4: The effect of representative inhibitors on the programmed necrosis pathway in cells.

[0540] The biological assay protocol used was as follows: the effects of the compound on key signals in the programmed necrosis pathway of TNFα-induced FADD-deficient Jurkat or L929 cells were tested, with known RIPK1 ATP non-competitive inhibitors Nec-1s and clinical inhibitor GSK2982772 used as controls.

[0541] TNFα-induced necroptosis is mediated by a complex called the Necrosome (Complex IIb), which includes TRADD, FADD, caspase-8, RIPK1, RIPK3, and MLKL. The kinase activity of RIPK1 has been shown to play a crucial role in regulating programmed cell death. During programmed cell death, RIPK1 undergoes autophosphorylation activation, with serine at position 166 being one of the major phosphorylation sites. Following programmed cell death, oligomerized MLKL translocates to the cell membrane, mediating cell death via calcium influx.

[0542] To verify whether the compounds in this invention inhibit programmed cell death by inhibiting RIPK1 activity and to detect changes in programmed cell death pathway signals, FADD-deficient Jurkat or L929 cells were treated with the compounds in this invention, followed by the addition of TNFα to induce programmed cell death. Cells were then collected and Western blot was used to detect RIPK1 phosphorylation and MLKL oligomerization.

[0543] Methods: FADD-deficient Jurkat cells or L929 cells were cultured in vitro and grown to the logarithmic growth phase. Cells were then collected, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cell concentration was adjusted to 1 x 10⁻⁶ cells / mL. 6 / mL. In a 12-well cell culture plate, 1 ml of cells were added to each well, along with 0.2 μL of 50 mM DMSO solution or pure DMSO control, and pretreated for 1 hour. For the stimulation group, 0.5 μL of 100 μg / ml TNFα (PBS solution) was added to each well. The plates were incubated in a cell culture incubator (37°C, 5% CO2) for 4 hours, then centrifuged at 3000 rpm for 3 min to collect cells and washed twice with pre-cooled PBS solution. The supernatant was removed as much as possible, and 200 μL of RIPA cell lysis buffer was added to the cell pellet. The cells were incubated at 4°C on a shaker for 30 min, then centrifuged at 15000 rpm at 4°C for 15 min, and the supernatant cell lysis buffer was collected. The protein content of each group was detected using a BCA protein quantification kit, and the protein level was adjusted to a final volume of 100 μL using PIPA lysis buffer. Western blot analysis was performed on the samples. Western blot: Add 25 μL of 5X protein loading buffer to 100 μL of cell lysis buffer and heat at 95 °C for 10 min. After the sample cools, perform SDS-PAGE (9%) gel electrophoresis at 60 V for 30 min, then switch to 120 V until the leading band reaches the bottom of the gel. Using a turbo semi-dry transfer system, transfer the protein from the gel to a 0.2 μL PC membrane at a constant current of 0.2 A for 80 min. Block the transferred PC membrane in 5% skim milk powder (TBST solution) for 2 h, and incubate with the appropriate primary antibody at 4 °C for 12 h. Wash three times with TBST for 10 min each time. Incubate with the appropriate secondary antibody at room temperature for 2 h. Wash three times with TBST for 10 min each time. Incubate with ECL chemiluminescence buffer and detect the luminescence signal. Experimental results are as follows. Figure 3 As shown

[0544] Experimental results show that the representative compound QY-10-40 can effectively inhibit the phosphorylation of RIPK1 itself and the phosphorylation of downstream protein MLKL in Jurkat cells at 1.6 nM. Its inhibitory effect is slightly better than GSK2982772 and much better than Nec-1s. In L929 cells, QY-10-40 is far superior to the other two.

[0545] Biological Test Example 5: Inhibitory Activity Test of RIPK1 Inhibitor on Programmed Cell Necrosis

[0546] The biological assay used was to investigate the effect of the compound on programmed necrosis of RIPK1 S161E mutant MEFs (mouse embryonic fibroblasts).

[0547] During the severe stages of related inflammation, intracellular RIPK1 is highly activated. Reported RIPK1 inhibitors tend to inhibit newly synthesized, unactivated RIPK1 kinase proteins, showing weaker inhibition of highly activated RIPK1, thus resulting in a weaker and slower intervention effect on related inflammation. To verify the inhibitory effect of the compounds in this invention on highly activated RIPK1 kinase at the cellular level, MEF cells stably expressing RIPK1 S161E (mimicking the activated state of phosphorylated RIPK1) were selected. Programmed cell death was induced using TNFα-SM164-zVAD (a caspase inhibitor used to inhibit apoptosis) (TSZ). Cell viability was calculated by detecting chemiluminescence values, thereby determining the biological activity of the compounds in inhibiting programmed cell death.

[0548] Methods: RIPK1-replenished MEFs: WT-RIPK1, S161A-RIPK1, and S161E-RIPK1 were reinfused into RIPK1-stably knocked-out cells, respectively. After stable expression and culture to the logarithmic growth phase, cells were collected, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cell concentration was adjusted to 5 x 10⁻⁶ cells / mL. 4 Cells were seeded into 384-well plates at a concentration of 40 μL / mL. 5 μL each of SM164 (100 nM), zVAD (50 μM), and the compound diluted with cell culture medium were added to the corresponding wells. After pretreatment at 37°C for 1 h, 5 μL of TNF diluted with cell culture medium (50 ng / mL) was added to each well in the stimulation group, and 5 μL of culture medium was added to each well in the control group. After culturing in a cell culture incubator (37°C, 5% CO2) for 12 h, 15 μL of Cell Titer-Glo solution was added to each well, and the cells were incubated at room temperature for 30 min. Chemiluminescence was measured to assess intracellular ATP levels. The unstimulated DMSO control well (Ctrl) was considered 100% cell viability. The EC50 of the compound was calculated using Prism Graphpad statistical software. 50 Value. Result as follows Figure 4 As shown.

[0549] Experimental results showed that in MEF cells expressing RIPK1 WT, representative compounds QY-10-40, QY-11-76, and QY11-77 (all at 600 nM), along with Nec-1s and GSK2982772 (all at 10 μM), effectively inhibited programmed necrosis. In MEF cells expressing the RIPK1 S161E mutant, representative compounds QY-10-40, QY-11-76, and QY11-77 at 600 nM significantly inhibited stimulated programmed necrosis; in contrast, Nec-1s showed no inhibition at 10 μM, and GSK2982772 showed only weak inhibition. Therefore, the representative compounds showed significantly better inhibitory effects on highly activated RIPK1 than the other two.

[0550] Pharmacokinetic properties of the representative compound QY-10-40 (Example 6)

[0551] The biological testing protocol used was: drug metabolism assay of the compound in live mice.

[0552] To verify the selectivity of the compounds of this invention for the kinase community in vivo, the representative compound QY-10-40 was selected, and its pharmacokinetic properties in mice (n=3) were tested using a single administration route, either by gavage (PO, 10 mg / kg) or intravenous injection (IV, 1 mg / kg). The experimental results are as follows: Figure 5 As shown.

[0553] The results showed that QY-10-40 rapidly reached peak efficacy in mice after a single dose, exhibiting good bioavailability (F) and half-life (T). 1 / 2 After oral administration of 10 mg / kg, the blood drug concentration remained stable above the effective inhibitory concentration (10 nM) for most of the 24 hours.

[0554] Biological test example 7: Effect of the representative compound QY-10-40 on TNFα-induced systemic inflammatory response syndrome.

[0555] The biological assay protocol employed involved tail vein injection of tumor necrosis factor-alpha (TNFα) to induce systemic inflammatory response syndrome (SIRS) in mice, leading to hypothermia and death. The effect of the representative compound QY-10-40 on TNFα-induced SIRS was tested, and changes in mouse body temperature and mortality were monitored. Known RIPK1 inhibitors, such as Nec-1s, were used as controls. The clinical inhibitor GSK2982772 was not used due to its poor inhibitory activity against murine RIPK1.

[0556] Systemic inflammatory response syndrome, also known as cytokine storm, refers to a systemic nonspecific inflammatory response caused by infectious or non-infectious injuries such as severe infection, multiple traumas, burns, ischemia-reperfusion injury, and acute pancreatitis. Under these conditions, a large number of inflammatory factors are released, and in severe cases, the body's inflammatory response becomes uncontrolled, leading to multiple organ failure and even death.

[0557] Methods: The test compounds were dissolved in 0.5% carboxymethyl cellulose at the required concentration one day in advance and sonicated overnight. Each mouse was administered 200 μl by gavage, and 20 min later, 10 mg TNFα (dissolved in 125 μl PBS) was injected via tail vein. Body temperature changes in mice were monitored using an infrared thermometer after injection. The effects of the compounds were calculated using Prism Graphpad statistical software. Experimental results are shown below. Figure 6 As shown.

[0558] Experimental results showed that, without the participation of inhibitors, all mice injected with TNFα died within 6 hours; compound QY-10-40 could effectively resist the hypothermia, inflammatory response and death caused by TNFα at a low dose (2mg / kg), and its effect was better than Nec-1s at the same dose.

[0559] Biological Test Example 8: Co-crystallization and structural analysis of representative compound QY-7-2B with recombinant RIPK1 protein from human subjects.

[0560] Methods: Recombinant human RIPK1 catalytic domain-truncate protein (containing amino acid residues 1-294 and quadruple mutations C23A, C127A, C233A, and C240A) was expressed and purified in insect Sf9 cells and concentrated to approximately 9 mg / mL. QY-7-2B (final concentration 2.5 mM) was added, and co-crystallization was induced at 18 °C using a hanging drop method and steam diffusion method. X-ray diffraction data of the RIPK1 / QY-7-2B co-crystallization were collected, and the resolution was determined by data analysis. The electron cloud distribution structure inside the eutectic was analyzed, and a three-dimensional binding model of the RIPK1 recombinant protein molecule and the small molecule QY-7-2B at the atomic level was simulated using software such as COOT and PHENIX.

[0561] Figure 7The co-crystallization structure of the representative QY-7-2B and the human RIPK1 protein kinase domain is shown, and the differences in the binding modes of the compounds of this invention and the control compound GSK2982772 with the RIPK1 protein are demonstrated. Specifically, both QY-7-2B and GSK2982772 occupy the hydrophobic allosteric pocket near the ATP-binding pocket of the RIPK1 kinase domain, causing the Leu157 conformation near this pocket to remain outward, thus stabilizing RIPK1 in an inactive state. Unlike GSK2982772, a portion of QY-7-2B extends into the hinge region of the ATP-binding pocket, forming hydrogen bonds with the Met95 residues, further stabilizing the binding with RIPK1 and thus enhancing the inhibitory effect on the latter.

[0562] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A compound, or a pharmaceutically acceptable salt thereof, characterized in that, The compound is shown as Formula II; wherein, selected from the group consisting of: Ring B is R 1 and R 2 are each independently selected from the group consisting of H, C 1-4 alkyl; The ring C is selected from the group consisting of: wherein R c each independently is selected from the group consisting of H, CN, halogen, C 1-6 alkyl, R d each independently is C 1-6 alkyl; R 3 selected from the group consisting of H, OH, C 1-4 alkyl; R 5 selected from the group consisting of H, OH, C 1-4 alkyl, C 1-4 alkoxy; Ring D is selected from the group consisting of substituted or unsubstituted C 6-10 an aryl ring, and a substituted or unsubstituted 5-10 membered heteroaryl; wherein C 6-10 the aryl ring is phenyl, and the 5-10 membered heteroaryl is pyridyl; and said substitution means that a hydrogen atom on the group is replaced by one or more substituents selected from the group consisting of -CN, halogen, C 1-6 alkyl, halogenated C 1-6 alkyl; and the compound is not:

2. The compound of claim 1, wherein R 1 and R 2 are each independently selected from the group consisting of H and methyl.

3. The compound of claim 1, wherein Ring D is selected from the group consisting of: substituted or unsubstituted C 6-10 aromatic ring; wherein C 6-10 the aromatic ring is phenyl.

4. The compound of claim 1, wherein Ring D is unsubstituted phenyl, or is phenyl substituted with 1 or 2 substituents selected from the group consisting of halo, C 1-4 alkyl, haloC 1-4 alkyl.

5. A process for preparing a compound of claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, The method comprises the step of: reacting a compound shown as Formula II-2A and a compound shown as Formula II-2B in an inert solvent to obtain a compound shown as Formula II-2C; wherein, Ring B, Ring C, Ring D, R c , R 1 , R 2 , R 3 and R 5 are as defined in Formula II.

6. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises (a) a therapeutically effective amount of a compound shown as Formula II, or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier; wherein Ring B, Ring C, Ring D, R 1 , R 2 , R 3 and R 5 as defined in any one of claims 1 to 4.

7. Use of a compound shown as Formula II, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 6, in the manufacture of a medicament for treating or preventing a disease or disorder associated with necroptosis and / or receptor-interacting protein 1 kinase; wherein, Ring B, Ring C, Ring D, R 1 , R 2 , R 3 and R 5 as defined in any one of claims 1 to 4.

8. Use according to claim 7, characterized in that, The disease or disorder is selected from one or more of the group consisting of: a degenerative disease, inflammation, ischemia-reperfusion injury, pathogen infection, Parkinson's disease, age-related macular degeneration, autoimmune disease, photoreceptor cell necrosis induced by retinal detachment, glaucoma, cisplatin-induced kidney injury, and traumatic brain injury, atherosclerosis caused by hyperlipidemia, blood and solid organ malignancies, bacterial infection, viral infection, and lysosomal storage disease.

9. The use of claim 8, wherein, The degenerative disease comprises: Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis, retinal degenerative disease; The inflammation comprises one or more of: intestinal inflammation, rheumatoid arthritis, psoriasis, retinitis pigmentosa, inflammatory bowel disease, Huntington's disease inflammatory bowel disease, frog skin induced acute pancreatitis, sepsis / systemic inflammatory response syndrome; The ischemia-reperfusion injury comprises one or more of: cerebral infarction, myocardial infarction, ischemic brain injury, ischemic myocardial injury, retinal ischemia / reperfusion injury, renal ischemia-reperfusion injury; The viral infection comprises one or more of the following diseases or disorders: tuberculosis, influenza, coronavirus infection, and pneumonia caused by the same; The lysosomal storage disease comprises Gaucher disease.

10. A method for inhibiting necroptosis, comprising the step of: culturing a cell in the presence of a compound shown as Formula II, or a pharmaceutically acceptable salt thereof, thereby inhibiting necroptosis; and the method is not a method for diagnosis or treatment of a disease; wherein, ring A, ring B, ring C, ring D, R 1 , R 2 , R 3 , R 4 , R 5 and n are as defined in any one of claims 1 to 4.

11. A method for inhibiting RIPK1 protein kinase activity, comprising the step of: contacting a RIPK1 protein kinase with a compound shown as Formula II, or a pharmaceutically acceptable salt thereof, thereby inhibiting RIPK1 protein kinase activity; and the method is not a method for diagnosis or treatment of a disease. wherein ring A, ring B, ring C, ring D, R 1 , R 2 , R 3 , R 4 , R 5 and n are as defined in any one of claims 1 to 4.

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