Pyrazinecarboxamide compound and preparation method, pharmaceutical composition and application thereof

Abstract

The invention discloses a pyrazinecarboxamide compound as well as a pharmaceutical composition and application of the pyrazinecarboxamide compound. The pyrazinecarboxamide compound (formula I) provided by the invention can efficiently and highly selectively degrade hematopoietic progenitor cell kinase 1, and has no degradation activity on other proteins of the same family. Compared with a hematopoietic progenitor cell kinase 1 small molecule inhibitor, the hematopoietic progenitor cell kinase 1 small molecule inhibitor can more efficiently stimulate T cell anti-tumor immune response and release effector cell factors, and has higher anti-tumor activity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a pyrazine carboxamide compound, a preparation method, a pharmaceutical composition and application thereof. BACKGROUND

[0002] Immunotherapy has completely changed the way cancer is treated. In particular, immune checkpoint inhibitors targeting the PD-1 and CTLA-4 pathways have shown remarkable clinical effects, but they are only effective for specific patient populations and specific types of tumors, have a low tumor response rate, and most patients do not show sustained symptom relief, and drug resistance occurs after a period of use. Therefore, it is urgent to discover new tumor immunotherapy targets to improve the clinical effects of immune checkpoints.

[0003] Hematopoietic progenitor kinase 1 (HPK1, MAP4K1) is a member of the MAP4K family, which is a Ste20 serine / threonine protein kinase. HPK1 is mainly expressed in hematopoietic cells such as T cells, B cells, neutrophils, dendritic cells (DCs), natural killer (NK) cells and macrophages. HPK1 is a negative regulator of T cell receptor and B cell receptor. After TCR activation, HPK1 in the cytoplasm is recruited to the plasma membrane, and its amino acid residues Y381, S171 and T165 are phosphorylated to obtain fully activated HPK1 kinase. Activated HPK1 phosphorylates the amino acid residue S376 of its linker protein SLP76 to provide a binding site for the negative regulator 14-3-3, promotes the proteasomal degradation of SLP76, and ultimately disrupts the stability of the TCR signaling complex, thereby hindering the downstream kinase signaling pathway that promotes T cell activation and proliferation. In addition to TCR signals, HPK1 also negatively regulates T cell signals through the prostaglandin E2 (PGE2) receptor. Under the stimulation of growth factors, stress, inflammatory factors and differentiation factors, HPK1 can also transmit immunosuppressive signals in B cells, NK cells and dendritic cells. The kinase activity of HPK1 inhibits the immune function of various cells, including CD4 + T cells, CD8 +T cells, NK cells and dendritic cells, and showed that inhibition of HPK1 kinase activity was sufficient to induce anti-tumor immune responses. In addition, inhibition of HPK1 kinase activity can further promote T cell effector function, significantly increasing the therapeutic effect of PD-L1 mAb. HPK1 gene deletion, HPK1 inhibitor or HPK1 protac degrader, can enhance CAR-T cell-based immunotherapy, and show better anti-tumor activity in various preclinical hematological and solid tumor mouse models. Notably, HPK1 knockout and inactive kinase knock-in mice do not show lethal inflammatory responses, while these are significant in the absence of some other immune negative regulators, such as CTLA-4 and Cbl-b, indicating that HPK1 inhibitors have high safety. In summary, HPK1 is a potential new target for effective tumor immunotherapy, and the research and development of HPK1 inhibitors is of great significance to improve the key problems currently faced by tumor immunotherapy.

[0004] There are currently four HPK1 inhibitors in early clinical research, including CFI-402411 by Treadwell Therapeutics, BGB-15025 by BeiGene, PRJ1-3024 by Zhuhai Yufan and NDI-101150 by Nimbus Therapeutics. Although various HPK1 inhibitors have been reported, there is currently no related drug on the market. The main challenge faced by the development of HPK1 inhibitors is that the functions of HPK1 family members are different, but the structural homology is very high, and it is difficult to design highly selective inhibitors.

[0005] Compared with traditional drugs, PROTAC has significant advantages: wider range of action, higher activity, can target "undruggable" targets; improve selectivity, activity and safety; overcome tumor drug resistance, etc. PROTAC can achieve selectivity on some targets that small molecules cannot achieve, so developing HPK1 PROTAC molecules is expected to solve the problems of low selectivity and dose-dependent efficacy of HPK1 inhibitors, and there are few reports of HPK1 PROTAC molecules at present. SUMMARY

[0006] Invention purposes: In view of the low selectivity of HPK1 small molecule inhibitors, the first purpose of the present application is to provide a pyrazine carboxamide HPK1 PROTAC degrader, which can realize the selectivity of small molecules by using PROTAC, and develop a HPK1-targeted PROTAC molecule. The PROTAC of the present application can efficiently and selectively degrade HPK1 protein, and has no degradation activity on other proteins in the same family. Compared with HPK1 small molecule inhibitors, it can more efficiently stimulate T cell antitumor immune response, release effector cytokines, and has stronger antitumor activity. The second purpose of the present application is to provide a preparation method of the compound, the third purpose is to provide a pharmaceutical composition containing the compound, and the fourth purpose is to provide a pharmaceutical application of the compound and the pharmaceutical composition thereof.

[0007] Technical scheme: In order to achieve the above purpose, the present application adopts the following technical scheme:

[0008] The present application provides a pyrazine carboxamide compound as shown in formula I, or a pharmaceutically acceptable salt, enantiomer, diastereoisomer, cis-trans isomer, tautomer, atropisomer, conformational isomer, metabolite, prodrug, solvate or hydrate thereof,

[0009]

[0010] In formula I,

[0011] R 1 is C 1-4 alkyl, C 3-6 cycloalkyl, deuterated (C 1-4 alkyl);

[0012] R 2 is C 1-4 alkyl or deuterated (C 1-4 alkyl);

[0013] B is C 3-10 cycloalkylene, 4-10 membered single ring heterocyclylene, 6-20 membered spiro heterocyclylene, 6-18 membered fused heterocyclylene or 7-10 membered bridged heterocyclylene, wherein the 4-10 membered single ring heterocyclylene, 6-20 membered spiro heterocyclylene, 6-18 membered fused heterocyclylene or 7-10 membered bridged heterocyclylene contains at least 1, 2 or 3 heteroatoms selected from nitrogen, oxygen or sulfur;

[0014] E is

[0015] R 3 is deuterium, halogen, cyano, C 1-4 alkyl, halogenated (C 1-4 alkyl), C 1-4 alkoxy or halogenated (C1-4 alkoxy) ;

[0016] a is 0, 1, 2 or 3;

[0017] W is -CH2-, -C(CH3)2or

[0018] L1is a bond, C 3-10 cycloalkylene, 4-10 membered homocyclylene, 6-20 membered spiroheterocyclylene, 6-18 membered fused heterocyclylene or 7-10 membered bridged heterocyclylene, said 4-10 membered homocyclylene, 6-20 membered spiroheterocyclylene, 6-18 membered fused heterocyclylene or 7-10 membered bridged heterocyclylene having at least one, two or three heteroatoms selected from nitrogen, oxygen or sulfur;

[0019] L2is a bond, -(CH2)r1-, C 3-10 cycloalkylene, 4-10 membered homocyclylene, 6-20 membered spiroheterocyclylene, 6-18 membered fused heterocyclylene or 7-10 membered bridged heterocyclylene, said 4-10 membered homocyclylene, 6-20 membered spiroheterocyclylene, 6-18 membered fused heterocyclylene or 7-10 membered bridged heterocyclylene having at least one, two or three heteroatoms selected from nitrogen, oxygen or sulfur;

[0020] L3is a bond, -(CH2)r2-, C 3-10 cycloalkylene, 4-10 membered homocyclylene, 6-20 membered spiroheterocyclylene, 6-18 membered fused heterocyclylene or 7-10 membered bridged heterocyclylene, said 4-10 membered homocyclylene, 6-20 membered spiroheterocyclylene, 6-18 membered fused heterocyclylene or 7-10 membered bridged heterocyclylene having at least one, two or three heteroatoms selected from nitrogen, oxygen or sulfur;

[0021] L4is a bond, -O-, -(CH2)r3-, -(CH2)r4(C=O)-, C 3-10 cycloalkylene, 4-10 membered homocyclylene, 6-20 membered spiroheterocyclylene, 6-18 membered fused heterocyclylene or 7-10 membered bridged heterocyclylene, said 4-10 membered homocyclylene, 6-20 membered spiroheterocyclylene, 6-18 membered fused heterocyclylene or 7-10 membered bridged heterocyclylene having at least one, two or three heteroatoms selected from nitrogen, oxygen or sulfur;

[0022] r1is 0, 1, 2, 3, 4, 5 or 6; r2is 0, 1, 2, 3, 4, 5 or 6;

[0023] r3is 0, 1, 2, 3, 4, 5 or 6; r4is 0, 1, 2, 3, 4, 5 or 6.

[0024] In some embodiments, R 1 is methyl, ethyl, cyclopropyl, or -CD3.

[0025] In some embodiments, R 2 is methyl, ethyl, or -CD3.

[0026] In some embodiments, B is a piperidylene group, a piperazinylene group,

[0027] In some embodiments, E is

[0028] R 3 is fluorine;

[0029] a is 0 or 1 ;

[0030] W is -CH2- or

[0031] In some embodiments, E is

[0032]

[0033] In some embodiments, L1is a bond, and the left side is connected to L2and the right side is connected to E.

[0034] In some embodiments, L2is a bond, -(CH2) r1 -, C 4-6 cycloalkylene, and the left side is connected to L3and the right side is connected to L1;

[0035] In some embodiments, L3is a bond, -(CH2) r2 -, C 4-6 cycloalkylene, and the left side is connected to L4and the right side is connected to L2;

[0036] In some embodiments, L4is a bond, -(CH2) r3 -, -(CH2) r4 (C=0)-, and the left side is connected to B and the right side is connected to L3.

[0037] In some embodiments, n1is 1, 2, 3, or 4.

[0038] In some embodiments, n2is 1, 2, 3, or 4.

[0039] In some embodiments, n3 is 1, 2, or 3.

[0040] In some embodiments, n4 is 1, 2, or 3.

[0041] In some embodiments, n5 is 1, 2, or 3.

[0042] In some embodiments, n6 is 1, 2, or 3.

[0043] In some embodiments, n7 is 0, 1, 2, or 3.

[0044] In some embodiments, n8 is 0, 1, 2, or 3.

[0045] In some embodiments, n9 is 0, 1, 2, or 3.

[0046] In some embodiments, n10 is 0, 1, 2, or 3.

[0047] In some embodiments, Q is 1 CR Q1 or N, R Q1 is halogen, hydroxy, or C 1-4 alkyl.

[0048] In some embodiments, Q is CH or N.

[0049] In some embodiments, Q is 2 CH or N.

[0050] In some embodiments, Q is 3 CH or N.

[0051] In some embodiments, R a is halogen, -(C=0) or C 1-3 alkyl.

[0052] In some embodiments, c is 0, 1, 2, or 3.

[0053] In some embodiments, r1 is 0, 1, 2, 3, or 4.

[0054] In some embodiments, r2 is 0, 1, 2, 3, or 4.

[0055] In some embodiments, r3 is 0, 1, 2, 3, or 4.

[0056] In some embodiments, r4 is 0, 1, 2, 3, or 4.

[0057] In some embodiments, L1 is a bond, and the left side is connected to L2 and the right side is connected to E.

[0058] In some embodiments, L2is a bond, -(CH2)ri-, and the left side is connected to L3and the right side is connected to Li.

[0059] In some embodiments, L3is a bond, -(CH2)ri-, and the left side is connected to L4and the right side is connected to L2.

[0060] In some embodiments, L4is a bond, -(CH2)ri or -(CH2)ri(C=0)-; and the left side is connected to B and the right side is connected to L3.

[0061] In some embodiments, n1is 1, 2, or 3.

[0062] In some embodiments, n2is 1, 2, or 3.

[0063] In some embodiments, n3is 1 or 2.

[0064] In some embodiments, n4is 1 or 2.

[0065] In some embodiments, n5is 1 or 2.

[0066] In some embodiments, n6is 1 or 2.

[0067] In some embodiments, n7is 0, 1, or 2.

[0068] In some embodiments, n8is 0, 1, or 2.

[0069] In some embodiments, n9is 0, 1, or 2.

[0070] In some embodiments, n10is 0, 1, or 2.

[0071] In some embodiments, Q 1 is CR Q1 or N, R Q1 is fluoro, hydroxy, or methyl.

[0072] In some embodiments, Q is CH or N.

[0073] In some embodiments, Q 2 is CH or N.

[0074] In some embodiments, Q 3 is CH or N.

[0075] In some embodiments, R a is fluoro or -(C=0).

[0076] In some embodiments, c is 0, 1, or 2.

[0077] In some embodiments, r1 is 0, 1, 2, or 3.

[0078] In some embodiments, r2 is 0, 1, 2, or 3.

[0079] In some embodiments, r3 is 0, 1, 2, 3, or 4.

[0080] In some embodiments, r4 is 0, 1, 2, 3, or 4.

[0081] In some embodiments, -L4-L3-L2-L1- is a bond,

[0082]

[0083]

[0084] and the left side is attached to B and the right side is attached to E;

[0085] n1 is 1 or 2; n2 is 1 or 2; n3 is 1 or 2; n4 is 1 or 2; n5 is 1 or 2; n6 is 1 or 2; n7 is 0, 1, or 2; n8 is 0, 1, or 2; n9 is 0, 1, or 2; n10 is 0, 1, or 2;

[0086] Q 1 is N or CR Q1 ; R Q1 is hydrogen or fluorine; Q is CH or N; Q 2 is CH or N; Q 3 is CH or N;

[0087] R a is fluorine; c is 0, 1, or 2;

[0088] r1 is 0, 1, or 2; r2 is 0, 1, or 2; r3 is 0, 1, 2, or 3; r4 is 0, 1, or 2.

[0089] In some embodiments, L1 is a bond,

[0090] and the left side is attached to L2 and the right side is attached to E.

[0091] In some embodiments, L2 is a bond, -(CH2)r1-,

[0092] and the left side is attached to L3 and the right side is attached to L1;

[0093] r1 is 0, 1, or 2.

[0094] In some embodiments, L3is a bond, -(CH2)r 2、

[0095] and the left side is connected to L4and the right side is connected to L2;

[0096] r2is 0, 1, or 2.

[0097] In some embodiments, L4is a bond, -(CH2)r3- or -(CH2)r4(C=0)-; and the left side is connected to L3and the right side is connected to E; r3is 0, 1, 2, or 3; r4is 0, 1, 2, or 3.

[0098] In some embodiments, -L1-L2-L3-L4- is a bond,

[0099]

[0100] and the left side is connected to E and the right side is connected to B;

[0101] r2is 1 or 2;

[0102] r3is 0, 1, 2, or 3;

[0103] r4is 1 or 2.

[0104] In some embodiments, -L1-L2-L3-L4- is a bond,

[0105]

[0106] and the left side is connected to E and the right side is connected to B.

[0107] In some embodiments, -L1-L2-L3-L4- is a bond,

[0108]

[0109] and the left side is connected to E and the right side is connected to B.

[0110] In some embodiments, the pyrazinecarboxamide compound of Formula I or a pharmaceutically acceptable salt, enantiomer, diastereomer, cis-trans isomer, tautomer, atropisomer, conformational isomer, metabolite, prodrug, solvate, deuteride, or hydrate thereof is a compound of Formula I-1, I-2, I-2A', I-3, or I-4, or a pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate, deuteride, or hydrate thereof,

[0111]

[0112] wherein:

[0113] Ring B' is a nitrogen-containing heterocyclyl group;

[0114] Ring L3' is a nitrogen-containing heterocyclyl group;

[0115] R 1 , R 2 , L1, L2, L3, B, E, r2, r3, r4 are as defined above.

[0116] In some embodiments, the compound of Formula I is selected from any one of the following compounds:

[0117]

[0118]

[0119]

[0120] The present application provides a method for preparing a pyrazinecarboxamide compound of Formula I-2, or a pharmaceutically acceptable salt, enantiomer, diastereomer, cis-trans isomer, tautomer, atropisomer, conformational isomer, metabolite, prodrug, solvate or hydrate thereof, comprising the following steps:

[0121]

[0122] under acidic conditions, in the presence of a reducing agent, to obtain the compound of Formula I-2 from the compound of Formula I-2A or a salt thereof and the compound of Formula I-2B;

[0123] The compound of Formula I-2 prepared by the above method is salted with a pharmaceutically acceptable acid to obtain the pharmaceutically acceptable salt.

[0124] wherein, Ring B', Ring L3', R 1 , R 2 , L1, L2, E, r3, r3-1, X are as defined above.

[0125] The present application provides a method for preparing a pyrazinecarboxamide compound of Formula I-2, or a pharmaceutically acceptable salt, enantiomer, diastereomer, cis-trans isomer, tautomer, atropisomer, conformational isomer, metabolite, prodrug, solvate or hydrate thereof, comprising the following steps:

[0126]

[0127] In a solvent, the compound represented by formula I-2A or a salt thereof is subjected to a reduction amination reaction with the compound represented by formula I-2A" under basic conditions to obtain the compound represented by formula I-2A' N The compound represented by formula I-2A is subjected to an Ar substitution reaction to obtain the compound represented by formula I-2A';

[0128] The compound represented by formula I-2A obtained by the above method is subjected to salt formation with a pharmaceutically acceptable acid to obtain the pharmaceutically acceptable salt.

[0129] wherein, the definitions of ring B', ring L3', R 1 , R 2 , E are the same as defined above.

[0130] The present application provides a method for preparing a pyrazinecarboxamide compound represented by formula I-1, or a pharmaceutically acceptable salt, enantiomer, diastereoisomer, cis-trans isomer, tautomer, atropisomer, conformational isomer, metabolite, prodrug, solvate or hydrate thereof, comprising the following steps:

[0131]

[0132] In a solvent, the compound represented by formula I-1A or a salt thereof is subjected to a reduction amination reaction with the compound represented by formula I-1B in the presence of a reducing agent under acidic conditions to obtain the compound represented by formula I-1.

[0133] The compound represented by formula I-1 obtained by the above method is subjected to salt formation with a pharmaceutically acceptable acid to obtain the pharmaceutically acceptable salt.

[0134] wherein, the definitions of ring B', ring L3', R 1 , R 2 , L1, L2, E, r3, r3-1, X are the same as defined above.

[0135] The present application provides a method for preparing a pyrazinecarboxamide compound represented by formula I-3, or a pharmaceutically acceptable salt, enantiomer, diastereoisomer, cis-trans isomer, tautomer, atropisomer, conformational isomer, metabolite, prodrug, solvate or hydrate thereof, comprising the following steps:

[0136]

[0137] In a solvent, the compound represented by formula I-3A or a salt thereof is subjected to a condensation reaction with the compound represented by formula I-3B in the presence of a condensing agent under basic conditions to obtain the compound represented by formula I-3.

[0138] The compound represented by formula I-3 obtained by the above method is subjected to salt formation with a pharmaceutically acceptable acid to obtain the pharmaceutically acceptable salt.

[0139] wherein, the definitions of ring B', ring L3', R 1 , R2 , L1, L2, L3, E, r4 are defined as above.

[0140] The present application provides a method for preparing a pyrazine carboxamide compound shown in formula I-4, or a pharmaceutically acceptable salt, enantiomer, diastereomer, cis-trans isomer, tautomer, atropisomer, conformational isomer, metabolite, prodrug, solvate or hydrate thereof, comprising the following steps:

[0141]

[0142] The compound shown in formula I-4A or a salt thereof is condensed with the compound shown in formula I-4B in a solvent under basic condition in the presence of a condensing agent to obtain the compound shown in formula I-4;

[0143] The compound of formula I-4 prepared by the above method is salted with a pharmaceutically acceptable acid to obtain the pharmaceutically acceptable salt;

[0144] wherein, ring L3', B, R 1 , R 2 , L1, L2, E, r4 are defined as above.

[0145] The above condensation reaction and S N The reagent for providing basic condition in the above substitution reaction of Ar includes organic base and inorganic base, the organic base includes but is not limited to triethylamine, pyridine, N,N-diisopropyl ethylamine (DIPEA), n-butyllithium, diisopropyl lithium, sodium acetate, potassium acetate, sodium tert-butoxide, potassium tert-butoxide or 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU); the inorganic base includes but is not limited to sodium hydride, potassium phosphate, sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide, lithium hydroxide and potassium hydroxide; preferably N,N-diisopropyl ethylamine.

[0146] The condensing agent in the above reaction includes but is not limited to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-dicyclohexyl carbodiimide (DCC), N,N'-diisopropyl carbodiimide (DIC), O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azobenzotriazole (HOAt), O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), benzotriazole-1-yl-oxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) or benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP); preferably HATU, BOP or PyBOP.

[0147] In the above synthesis scheme, the reagent providing acidic condition includes, but is not limited to, hydrogen chloride, 1,4-dioxane solution of hydrogen chloride, 1,4-dioxane solution of hydrochloric acid, trifluoroacetic acid, formic acid, acetic acid, glacial acetic acid, hydrochloric acid, concentrated sulfuric acid, methanesulfonic acid, nitric acid, phosphoric acid, p-toluenesulfonic acid; preferably acetic acid or glacial acetic acid.

[0148] Specifically, in the above reductive amination reaction, the reagent providing acidic condition can be directly added acid reagent; and / or added basic salt reagent, the acid generated in the reaction process from the acid brought in by the basic salt and the acidic salt form of the starting material. The basic salt includes, but is not limited to, sodium acetate, anhydrous sodium acetate, preferably anhydrous sodium acetate; the acidic salt of the starting material is preferably hydrochloride or trifluoroacetate; specifically, when the starting material is in the form of an acidic salt, the way to provide acidic condition can be: adding reagent sodium acetate, and forming acetic acid in the reaction process from sodium acetate and the acid in the starting material.

[0149] In the above synthesis scheme, the reducing agent includes, but is not limited to, sodium triacetoxyborohydride, sodium borohydride, lithium borohydride, sodium borohydride acetate, sodium cyanoborohydride and sodium acetylborohydride, etc., preferably sodium triacetoxyborohydride or sodium cyanoborohydride.

[0150] The solvent used in the reaction of the above step includes, but is not limited to, pyridine, ethylene glycol, dimethyl ether, acetic acid, methanol, ethanol, acetonitrile, n-butanol, toluene, tetrahydrofuran, dichloromethane, petroleum ether, ethyl acetate, n-hexane, dimethyl sulfoxide, 1,4-dioxane, water, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, 1,2-dibromoethane and mixtures thereof.

[0151] The present application provides a pyrazine carboxamide compound as shown in formula I, or a pharmaceutically acceptable salt, enantiomer, diastereomer, cis-trans isomer, tautomer, atropisomer, conformational isomer, metabolite, prodrug, solvate or hydrate thereof for use in the preparation of a medicament for modulating the ubiquitination and degradation of hematopoietic progenitor kinase 1.

[0152] The present application provides a pyrazine carboxamide compound as shown in formula I, or a pharmaceutically acceptable salt, enantiomer, diastereomer, cis-trans isomer, tautomer, atropisomer, conformational isomer, metabolite, prodrug, solvate or hydrate thereof for use in the preparation of a medicament for treating and / or preventing cancer.

[0153] In some embodiments, the cancer is one or more of bone cancer, lung cancer, gastric cancer, colorectal cancer, membrane adenocarcinoma, breast cancer, prostate cancer, liver cancer, pancreatic cancer, brain cancer, bladder cancer, cervical cancer, stomach cancer, kidney cancer, head and neck cancer, thyroid cancer, esophageal cancer, lymphoma, leukemia, or skin cancer.

[0154] The present application provides a pharmaceutical composition comprising a pyrazine carboxamide compound as shown in Formula I, or a pharmaceutically acceptable salt, enantiomer, diastereomer, cis-trans isomer, tautomer, atropisomer, conformational isomer, metabolite, prodrug, solvate or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

[0155] In some embodiments, in the pharmaceutical composition, the pyrazine carboxamide compound, or a pharmaceutically acceptable salt, enantiomer, diastereomer, cis-trans isomer, tautomer, atropisomer, conformational isomer, metabolite, prodrug, solvate or hydrate thereof, is in a therapeutically effective amount.

[0156] The present application provides a use of a pharmaceutical composition in the preparation of a medicament for modulating the ubiquitination and degradation of hematopoietic progenitor kinase 1.

[0157] The present application provides a use of a pharmaceutical composition in the preparation of a medicament for treating and / or preventing cancer.

[0158] In some embodiments, the cancer is one or more of bone cancer, lung cancer, gastric cancer, colorectal cancer, membrane adenocarcinoma, breast cancer, prostate cancer, liver cancer, pancreatic cancer, brain cancer, bladder cancer, cervical cancer, stomach cancer, kidney cancer, head and neck cancer, thyroid cancer, esophageal cancer, lymphoma, leukemia, or skin cancer.

[0159] The pharmaceutically acceptable carrier can be an excipient widely used in the field of pharmaceutical production. The excipient is mainly used to provide a safe, stable and functional pharmaceutical composition, and can also provide a method for the active ingredient to be dissolved at the desired rate after the subject is administered, or to promote the active ingredient to be effectively absorbed after the subject is administered with the composition. The pharmaceutical excipient can be an inert filler, or provide a certain function, such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient of the composition. The pharmaceutical excipient can include one or more of the following excipients: binding agent, suspending agent, emulsifying agent, diluent, filler, granulating agent, adhesive, disintegrating agent, lubricant, anti-adhesion agent, flow aid, wetting agent, gelling agent, absorption delaying agent, dissolution inhibitor, enhancer, adsorbent, buffer, chelating agent, preservative, colorant, flavoring agent and sweetening agent.

[0160] The pharmaceutical compositions of the present application can be prepared according to the disclosure using any method known to those of skill in the art. For example, conventional mixing, dissolving, granulating, emulsifying, levigating, encapsulating, entrapping or lyophilizing processes.

[0161] The pharmaceutical compositions described herein can be administered in any form including injection (intravenous), mucosally, orally (both solid and liquid formulations), inhalation, ocularly, rectally, topically, or parenterally (infusion, injection, implantation, subcutaneous, intravenous, intraarterial, intramuscular). The pharmaceutical compositions of the present application can also be in a controlled or sustained release formulation (e.g., liposome or microsphere). Examples of solid oral formulations include, but are not limited to, powders, capsules, caplets, soft gel capsules, and tablets. Examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical formulations include, but are not limited to, creams, gels, ointments, pastes, patches, poultices, foams, lotions, drops, or serums. Examples of formulations for parenteral administration include, but are not limited to, solutions for injection, dry powder formulations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical compositions described herein include, but are not limited to, eye drops and other ophthalmic formulations; aerosols, such as nasal sprays or inhalers; liquid dosage forms suitable for parenteral administration; suppositories; and lozenges.

[0162] The compounds of the present disclosure can exist in particular stereoisomeric forms. The term "isomers" refers to compounds having the same molecular formula but different structures. These include cis- and trans- (or E- and Z-) isomers, (-)- and (+)- enantiomeric forms, (R)- and (S)- enantiomeric forms, diastereomers, (D)- and (L)- isomers, tautomers, atropisomers, conformational isomers, and mixtures thereof (e.g., racemates, mixtures of diastereomers). Substituents in the compounds of the present disclosure can be present in additional asymmetric atoms. All such stereoisomers, as well as mixtures thereof, are included in the scope of the present disclosure. The optically active (-)- and (+)- isomers, (R)- and (S)- enantiomeric forms, and (D)- and (L)- isomers can be prepared by chiral synthesis, chiral reagents, or other conventional techniques. One isomer of a compound of the present disclosure can be prepared by asymmetric synthesis or by chiral auxiliary, or, when a basic functional group (e.g., amino) or an acidic functional group (e.g., carboxyl) is present in the molecule, by forming a diastereomeric salt with an appropriate optically active acid or base, followed by separation of the diastereomeric salt by conventional means and liberation of the pure enantiomer by conventional methods known in the art. Additionally, separation of enantiomers and diastereomers is typically accomplished by chromatography.

[0163] In the chemical structures of the compounds of the present disclosure, the bond " / " indicates that the configuration is not specified, i.e., if chiral isomers are present in the chemical structure, the bond " / " can be either or or both. or both configurations.

[0164] The term "pharmaceutically acceptable salt" means a salt of a compound of the present application which is found to possess the specific substituents of the compounds of the present application which is prepared from a relatively non-toxic acid or base. Alkali addition salts are obtained by contacting the free form of such compounds in solution or in a suitable inert solvent, with a sufficient amount of a base to produce the desired salt. Pharmaceutically acceptable alkali addition salts include sodium, potassium, calcium, ammonium, organic amino or magnesium salts or similar salts. Acid addition salts are obtained by contacting the free form of such compounds in solution or in a suitable inert solvent, with a sufficient amount of an acid to produce the desired salt. Examples of pharmaceutically acceptable acid addition salts include mineral acid salts such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid (to form carboxylate or bicarbonate salts), phosphoric acid (to form phosphate, monohydrogenphosphate, dihydrogenphosphate salts), sulfuric acid (to form sulfate or bisulfate salts), hydriodic acid, phosphorous acid, and the like; and organic acid salts such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and the like; salts with amino acids such as arginine, lysine and the like, salts with organic acids such as gluconic acid, glucose

[0165] The "pharmaceutically acceptable salts" of the present application can be synthesized from the parent compound that contains an acidic or basic moiety by conventional chemical methods. Generally, such salts are prepared either by contacting the free acid or base forms of these compounds with a sufficient amount of the appropriate base or acid in water or in an organic solvent or in a mixture of both to produce the desired salt. Generally, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred.

[0166] The term "metabolite" means a pharmacologically active product produced through metabolism in the body of a compound of formula I or salt thereof. Such products can result, for example, from the oxidation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, glucuronation, enzymatic cleavage, and the like of the administered compound. Accordingly, the present application includes metabolites of compounds of the present application, including those produced by contact of a compound of the present application with a mammal for a period of time sufficient to yield a metabolic product thereof.

[0167] Identification of metabolites is typically performed by preparing a radiolabelled isotope of a compound of the application, administering it parenterally to an animal such as rat, mouse, guinea pig, monkey, or to man, at a dose that will result in a detectable amount of the compound in the blood or other biological fluid, waiting sufficient time for metabolism to occur (typically about 30 seconds to 30 hours) and isolating the transformation products from the urine, blood or other biological sample. These products are readily isolated because of the label (or otherwise by utilizing antibodies capable of binding to the epitope present on the metabolite). The structure of the metabolite is determined in a conventional manner, e.g., by MS, LC / MS or NMR analysis. In general, the analysis of metabolites is performed in the same way as a conventional drug metabolism study as is known to those skilled in the art. As long as the metabolite products are not otherwise found in the body, they are useful in the determination of the therapeutic dosage range of a compound of the application. The compounds of the application can contain unnatural proportions of atomic isotopes at one or more of the atoms in a compound, for example, at any position of the compounds. For example, the compounds can be radiolabeled with radioactive isotopes, such as for example tritium ( 3 H), iodine-125 ( 125 I) or carbon-14 ( 14 C). All isotopic variations of the compounds of the present application, whether radioactive or not, are encompassed within the scope of the present application.

[0168] In addition to salt forms, the compounds of this application can exist in a variety of

[0169] Those skilled in the art will appreciate that the use of in the structural formulae of the groups described herein refers to the corresponding group attached to the rest of the compound of Formula I by the position indicated in the group.

[0170] The term "halogen" means fluorine, chlorine, bromine or iodine.

[0171] The term "alkyl" means a straight or branched chain alkyl group having the number of carbon atoms indicated. Examples of alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, i-butyl or sec-butyl.

[0172] The term "deuteroalkyl" refers to an alkyl group, as defined above, substituted with one or more deuterium. Examples of deuteroalkyl groups include, but are not limited to, -CD3.

[0173] The term "alkoxy" refers to -0-(alkyl), wherein alkyl is as defined above. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy.

[0174] The term "haloalkyl" refers to an alkyl group, as defined above, substituted with one or more halogen. Examples of haloalkyl groups include, but are not limited to, trifluoromethyl, trifluoroethyl, difluoromethyl, difluoroethyl.

[0175] The term "haloalkoxy" refers to an alkoxy group, as defined above, substituted with one or more halogen. Examples of haloalkoxy groups include, but are not limited to, trifluoromethoxy, trifluoroethoxy.

[0176] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic all-carbon ring having 3 to 20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) ring atoms (i.e., 3 to 20 membered cycloalkyl). The cycloalkyl group preferably has 3 to 12 ring atoms (i.e., 3 to 12 membered cycloalkyl) or 3 to 10 ring atoms (i.e., 3 to 10 membered cycloalkyl), more preferably 3 to 8 ring atoms (i.e., 3 to 8 membered cycloalkyl), most preferably 3 to 6 ring atoms (i.e., 3 to 6 membered cycloalkyl), and even more preferably 5 or 6 ring atoms (i.e., 5 or 6 membered cycloalkyl). The term "cycloalkylene" refers to a divalent radical formed by removing an additional hydrogen atom from the above "cycloalkyl" groups. Examples of cycloalkylene groups include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene.

[0177] The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic heterocycle (i.e., monocyclic heterocyclyl) or a polycyclic heterocyclic ring system (i.e., polycyclic heterocyclyl) having at least one (e.g., 1, 2, 3, or 4) heteroatom(s) selected from the group consisting of nitrogen, oxygen, and sulfur (said nitrogen can optionally be oxidized, i.e., form a nitro oxide; said sulfur can optionally be oxidized, i.e., form a sulfoxide or sulfone, but not -O-O-, -O-S-, or -S-S-) in the ring and having from 4 to 20 (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) ring atoms (i.e., 4- to 20-membered heterocyclyl), including 4- to 10-membered monoheterocyclyl, 6- to 20-membered spiroheterocyclyl, 6- to 18-membered fusedheterocyclyl, 7- to 10-membered bridgedheterocyclyl. The heterocyclyl group preferably has from 4 to 16 ring atoms (i.e., 4- to 16-membered heterocyclyl), of which 1 or 2 are nitrogen atoms; further preferably from 4 to 11 ring atoms (i.e., 4- to 11-membered heterocyclyl), of which 1 or 2 are nitrogen atoms. "Spiroheterocyclyl" refers to a polycyclic heterocyclic ring system sharing one atom (referred to as the spiro atom) between the rings, which can contain one or more double bonds in the ring and which has at least one (e.g., 1, 2, 3, or 4) heteroatom(s) selected from the group consisting of nitrogen, oxygen, and sulfur (said nitrogen can optionally be oxidized, i.e., form a nitro oxide; said sulfur can optionally be oxidized, i.e., form a sulfoxide or sulfone, but not -O-O-, -O-S-, or -S-S-) in the ring, provided that at least one monocyclic heterocyclyl is present and the point of attachment is on the monocyclic heterocyclyl, which has from 6 to 20 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) ring atoms (i.e., 6- to 20-membered spiroheterocyclyl). The spiroheterocyclyl group preferably has from 6 to 14 ring atoms (i.e., 6- to 14-membered spiroheterocyclyl), more preferably from 7 to 14 ring atoms (i.e., 7- to 14-membered spiroheterocyclyl) or from 7 to 11 ring atoms (i.e., 7- to 11-membered spiroheterocyclyl). The spiroheterocyclyl group includes mono- and polyspirocyclyl (e.g., dispirocyclyl and the like), preferably monospirocyclyl or dispirocyclyl, more preferably 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 4-membered, 5-membered / 5-membered, 5-membered / 6-membered, 6-membered / 4-membered, 6-membered / 5-membered, 6-membered / 6-membered, 6-membered / 7-membered, 7-membered / 5-membered, or 7-membered / 6-membered monospirocyclyl."Fused heterocyclic group" refers to a polycyclic heterocyclic system in which two adjacent atoms are shared between the rings. The ring may contain one or more double bonds and at least one (e.g., 1, 2, 3 or 4) heteroatom selected from nitrogen, oxygen and sulfur (the nitrogen may optionally be oxidized to form nitrogen oxides; the sulfur may optionally be oxidized to form sulfoxides or sulfones, but excluding -OO-, -OS- or -SS-). It is a monocyclic heterocyclic group fused with one or more monocyclic heterocyclic groups, or a monocyclic heterocyclic group fused with one or more cycloalkyl, aryl or heteroaryl groups, wherein the bonding point is on the monocyclic heterocyclic group and has 6 to 18 (e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) ring atoms (i.e., 6 to 20 membered fused heterocyclic group). The fused heterocyclic group is preferably a fused heterocyclic group having 6 to 14 ring atoms (i.e., a 6 to 14-membered fused heterocyclic group), more preferably a fused heterocyclic group having 7 to 10 ring atoms (i.e., a 7 to 10-membered fused heterocyclic group). The fused heterocyclic group includes bicyclic and polycyclic fused heterocyclic groups (such as tricyclic fused heterocyclic groups, tetracyclic fused heterocyclic groups, etc., preferably bicyclic or tricyclic fused heterocyclic groups, more preferably 3 / 4, 3 / 5, 3 / 6, 4 / 4, 4 / 5, 4 / 6, 5 / 3, 5 / 4, 5 / 5, 5 / 6, 5 / 7, 6 / 3, 6 / 4, 6 / 5, 6 / 6, 6 / 6). / 7-membered, 7 / 5-membered, or 7 / 6-membered bicyclic fused heterocyclic groups. The term "bridged heterocyclic group" refers to a polycyclic heterocyclic system in which two non-directly connected atoms are shared between the rings. The rings may contain one or more double bonds, and the rings contain at least one (e.g., 1, 2, 3, or 4) heteroatoms selected from nitrogen, oxygen, and sulfur (the nitrogen may optionally be oxidized, i.e., forming nitrogen oxides; the sulfur may optionally be oxidized, i.e., forming sulfoxides or sulfones, but excluding -OO-, -OS-, or -SS-), having 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) ring atoms (i.e., 5 to 20-membered bridged heterocyclic groups). The bridged heterocyclic group is preferably a bridged heterocyclic group having 6 to 14 ring atoms (i.e., 6 to 14-membered bridged heterocyclic groups), more preferably a bridged heterocyclic group having 7 to 10 ring atoms (i.e., 7 to 20-membered bridged heterocyclic groups). (10-membered bridged heterocyclic group). Based on the number of constituent rings, they can be classified into bicyclic bridged heterocyclic groups and polycyclic bridged heterocyclic groups (such as tricyclic bridged heterocyclic groups, tetracyclic bridged heterocyclic groups, etc.), with bicyclic or tricyclic bridged heterocyclic groups being preferred. The term "hypocyclic group" refers to a divalent group formed by removing another hydrogen atom from the aforementioned "heterocyclic alkyl" group, including monocyclic heterocyclic alkyl hypocarbonyl hypocarbonyl, spirocyclic heterocyclic alkyl hypocarbonyl, fused heterocyclic alkyl hypocarbonyl, and bridged heterocyclic alkyl hypocarbonyl. Examples include, but are not limited to, those mentioned above.

[0178]

[0179] The above-mentioned preferred conditions can be combined arbitrarily to obtain preferred examples of the present application without departing from the common knowledge in the art.

[0180] The reagents and raw materials used in the present application are commercially available.

[0181] Advantages: Compared with the prior art, the present application has the following remarkable advantages:

[0182] The pyrazine carboxamide compound designed in the present application can efficiently and selectively degrade HPK1 protein (DC 50 to reach the level of nanomolar concentration), and has no degradation activity on proteins such as GLK and HGK in the same family; at the same time, it can more efficiently stimulate T cell anti-tumor immune response (EC 50 to reach the level of nanomolar concentration), and has a good therapeutic effect on cancer. DETAILED DESCRIPTION

[0183] The present application will be further described by way of examples below, but the present application is not limited in the scope of the examples. The experimental methods in the following examples without specific conditions are selected according to conventional methods and conditions, or according to the instructions of the goods.

[0184] Example 1: Synthesis of intermediate 3

[0185]

[0186] a) Dissolve raw material 1 (5.0 g, 25.38 mmol) in tetrahydrofuran solution (50 mL), add 60% sodium hydride (1.5 g, 38.07 mmol) portionwise at 0°C, stir at 0°C for 0.5 h, then add CD3I (4.41 g, 30.45 mmol) to the reaction system, and react at room temperature for 12 h. After detecting the end of the reaction by TLC, quench the reaction with saturated aqueous ammonium chloride solution (50 mL), extract with ethyl acetate three times, wash the combined organic layer with saturated brine three times, dry over anhydrous sodium sulfate, filter, and concentrate under reduced pressure, then purify by column chromatography to obtain intermediate 2 (3.24 g, 60%). MS (ESI, m / z): 214 (M + +1).

[0187] b) Weigh intermediate 2 (1.0 g, 4.7 mmol), methanesulfonic acid [n-butyldi(1-adamantyl)phosphine](2-amino-1,1'-biphenyl-2-yl)palladium(II) (0.4 g, 0.5 mmol), n-butyldi(1-adamantyl)phosphine (0.2 g, 0.5 mmol), 4,4,4',4',5,5'-hexamethyl-2,2'-bis(1,3,2-dioxaborane) (3.0 g, 11.8 mmol), and potassium acetate (1.4 g, 14.22 mmol) into a double-necked flask. Under argon atmosphere, add anhydrous cyclopentyl methyl ether (24 mL) to the reaction system and react at 100 °C for 12 h. After the reaction is complete, cool to room temperature and filter with diatomaceous earth. Collect the filtrate, concentrate it to obtain crude intermediate 3, and proceed directly to the next step. MS (ESI, m / z): 262 (M + +1).

[0188] Example 2: Synthesis of Intermediate 5

[0189]

[0190] The synthesis method of intermediate 5 is the same as that in step b of Example 1, except that intermediate 2 is replaced with raw material 4. MS(ESI,m / z):259(M + +1).

[0191] Example 3: Synthesis of Intermediate 14

[0192]

[0193] a) Starting material 6 (11.0 g, 50.0 mmol) and methylamine hydrochloride (12.6 g, 0.2 mol) were dissolved in methanol (25 mL). Triethylamine (25.5 mL, 0.2 mol) was then added dropwise to the reaction system, and the mixture was reacted at room temperature for 24 h. After the reaction was complete, methanol was removed by rotary evaporation under reduced pressure. 50 mL of water was added, and the mixture was stirred for 15 minutes. The mixture was then filtered, and the filter cake was dried to obtain intermediate 7 (9.4 g, 87%). MS (ESI, m / z): 217 (M + +1).

[0194] b) Weigh intermediate 7 (3.7 g, 16.9 mmol) and sodium nitrite (1.2 g, 17.8 mmol), and slowly add pyridinium hydrofluoride (7.47 mL) dropwise to the reaction system at -10 °C. Transfer to room temperature and stir for 2 h. After the reaction is complete, add water (30 mL) to quench the reaction, stir for 15 minutes, filter, take the filter cake, dry it to obtain intermediate 8 (3.4 g, 92%). 1H NMR (400 MHz, Chloroform-d) δ 5.92 (s, 1H), 3.94 (s, 3H), 3.12 (d, J = 5.0 Hz, 3H). MS (ESI, m / z): 220 (M + +1).

[0195] c) Intermediate 8 (481.8 mg, 2.2 mmol), Intermediate 5 (2.2 mmol) and PdCl2(PPh3)2 (140.2 mg, 0.2 mmol) and cesium fluoride (1 g, 6.6 mmol) were dissolved in 1,4-dioxane (7.5 mL) and water (2.5 mL) under argon, and stirred at 95 °C for 8 h. After the reaction was completed, the reaction solution was cooled to room temperature, concentrated, extracted with ethyl acetate three times, the combined organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated, and the organic phase was purified by column chromatography to obtain Intermediate 9 (499.0 mg, 72%) 1 H NMR (400 MHz, Chloroform-d) δ 7.87 (s, 1H), 7.65-7.60 (m, 2H), 7.45-7.36 (m, 2H), 3.84 (s, 3H), 3.83 (s, 3H), 2.94 (d, J = 4.8 Hz, 3H). MS (ESI, m / z): 316 (M + +1).

[0196] d) Intermediate 9 (1.4 g, 4.6 mmol) and raw material 10 (1.5 g, 5.5 mmol) were dissolved in DMF (4.0 mL), and DIPEA (1.2 g, 9.1 mmol) was slowly added to the reaction system under stirring, and the reaction was carried out at 100 °C for 10 h. After the reaction was completed, water (15 mL) was added to the reaction system to quench the reaction, extracted with ethyl acetate three times, the combined organic layer was washed with saturated brine three times, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure, and purified by column chromatography to obtain light yellow solid 11 (1.1 g, 43%) 1H NMR (400 MHz, Chloroform-d) δ 10.46 (s, 1H), 7.91 (s, 1H), 7.81-7.75 (m, 3H), 7.73 (dd, J = 6.9, 1.7 Hz, 1H), 7.48-7.40 (m, 2H), 7.20-7.14 (m, 2H), 4.24 (s, 2H), 3.93 (s, 3H), 3.89 (s, 3H), 3.08 (d, J = 4.7 Hz, 3H), 2.80 (t, J = 12.5 Hz, 2H), 2.62 (tt, J = 12.1, 3.6 Hz, 1H), 1.84 (d, J = 12.7 Hz, 2H), 1.67-1.58 (m, 2H), 1.48 (s, 9H). MS (ESI, m / z): 572 (M + +1).

[0197] e) Intermediate 11 (856.5 mg, 1.5 mmol) was dissolved in methanol (2.5 mL) and tetrahydrofuran (2.5 mL), and after the compound was completely dissolved, 2 M aqueous potassium hydroxide solution (5 mL) was slowly added dropwise, and the reaction was performed at 50°C for 3 h. After the reaction was completed, the solvent was removed by distillation under reduced pressure, and the pH was adjusted to 4 with 2 M aqueous HCl solution, and a brownish yellow solid was precipitated, which was suction filtered and the filter cake was collected and dried to obtain crude intermediate 12, which was directly used in the next step. MS (ESI, m / z): 558 (M + +1).

[0198] f) Intermediate 12 (779.8 mg, 1.4 mmol), ammonium chloride (144.5 mg, 2.7 mmol), and BOP (884.0 mg, 2.0 mmol) were added. Anhydrous DMF (5.0 mL) was added to the system under argon, and DIPEA (1.1 g, 8.2 mmol) was added to the reaction system under stirring, and the reaction was performed at room temperature for 4 h. After the reaction was completed, the reaction was quenched with pure water (10 mL), extracted with ethyl acetate three times, and the organic layer was combined and washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain light yellow solid intermediate 13 (762.8 mg, 98%) 1H NMR (300 MHz, DMSO-d6) δ 11.43 (s, 1H), 8.35 (s, 1H), 8.17 (d, J = 4.8 Hz, 1H), 7.80 - 7.58 (m, 5H), 7.53 - 7.35 (m, 2H), 7.20 (d, J = 8.4 Hz, 2H), 4.07 (d, J = 11.9 Hz, 2H), 3.91 (s, 3H), 2.95 (d, J = 4.5 Hz, 3H), 2.80 (s, 2H), 2.64 (t, J = 12.0 Hz, 1H), 1.76 (d, J = 13.0 Hz, 2H), 1.55 - 1.43 (m, 2H), 1.42 (s, 9H). MS (ESI, m / z): 557 (M + +1).

[0199] g) Dissolve 13 (278 mg, 0.5 mmol) in dichloromethane (1 mL), slowly add trifluoroacetic acid (1 mL) into the reaction system, stir the reaction at room temperature for 3 h. After the reaction is completed, concentrate the reaction solution and add appropriate amount of ether to quench, stir for 15 min, then filter and collect the filter cake, dry to obtain the trifluoroacetate salt of yellow solid intermediate 14. (377.3 mg, 95%). 1 H NMR (300 MHz, DMSO-d6) δ 11.43 (s, 1H), 8.35 (s, 1H), 8.17 (d, J = 4.8 Hz, 1H), 7.80 - 7.58 (m, 5H), 7.53 - 7.35 (m, 2H), 7.20 (d, J = 8.4 Hz, 2H), 4.07 (d, J = 11.9 Hz, 2H), 3.91 (s, 3H), 2.95 (d, J = 4.5 Hz, 3H), 2.80 (s, 2H), 2.64 (t, J = 12.0 Hz, 1H), 1.76 (d, J = 13.0 Hz, 2H), 1.55 - 1.43 (m, 2H), 1.42 (s, 9H). MS (ESI, m / z): 557 (M + +1).

[0200] Example 4: Synthesis of intermediates 16a and 16b

[0201]

[0202] a) Intermediate 14 (400.0 mg, 0.87 mmol) was dissolved in dry acetonitrile (5.0 mL) under argon protection, DIPEA (0.5 mL) was added at 0 °C, and tert-butyl bromoacetate (171 mg, 0.87 mmol) was added dropwise slowly at 0 °C for 1 h. After the reaction was completed, it was concentrated under reduced pressure and diluted with water, extracted with ethyl acetate three times, the combined organic layers were washed with saturated NaCl solution, dried over anhydrous Na2SO4, filtered and concentrated, and purified by column chromatography to give intermediate 15a (447.0 mg, 45%). 1 H NMR (400 MHz, Chloroform-d) δ 11.01 (s, 1H), 7.95 (s, 1H), 7.79 (d, J = 8.5 Hz, 2H), 7.69 (d, J = 5.2 Hz, 1H), 7.66 (dd, J = 5.6, 3.0 Hz, 1H), 7.58 (s, 1H), 7.49 - 7.42 (m, 2H), 7.19 (d, J = 8.4 Hz, 2H), 3.92 (s, 3H), 3.24 - 3.03 (m, 7H), 2.40 (d, J = 70.9 Hz, 4H), 1.86 (s, 3H), 1.49 (s, 9H). MS (ESI, m / z): 571 (M + +1). In the same way, only the starting material was replaced with tert-butyl bromoacetate to give intermediate 15b. MS (ESI, m / z): 585 (M + +1).

[0203] b) Intermediate 15a (387.4 mg, 1 mmol) was dissolved in dimethyl ether (2 mL), and trifluoroacetic acid (148 μί) was added, and the mixture was stirred at 25 °C for 8 h. The mixture was directly rotary evaporated to give crude intermediate 16a, which was directly used in the next step. MS (ESI, m / z): 515 (M + +1). In the same way, only the starting material was replaced with tert-butyl bromoacetate to give intermediate 16b. MS (ESI, m / z): 529 (M + +1).

[0204] Example 5: Synthesis of intermediate 25

[0205]

[0206] a) Intermediate 17 (3.0 g, 21.3 mmol), piperidin-4-ylmethanol (2.94 g, 25.5 mmol) and potassium carbonate (5.9 g, 42.5 mmol) were dissolved in DMF (35 mL) under argon protection, and the mixture was stirred at 80 °C for 12 h. After the reaction was completed, the reaction mixture was cooled to room temperature, water (100 mL) was added to precipitate the product, and the mixture was filtered and the filter cake was washed with water. The filter cake was dried to give intermediate 18 (3.7 g, 73%). MS (ESI, m / z): 237 (M + +1).

[0207] b) Intermediate 18 (500.0 mg, 2.12 mmol) and sodium bicarbonate (355.6 mg, 4.2 mmol) were dissolved in DCM (10 mL), and Dess-Martin periodinane (1.1 g, 2.5 mmol) was added portionwise to the reaction mixture at 0 °C. The mixture was stirred at room temperature for 1 h. After the reaction was completed, the reaction mixture was quenched with 20% Na2S2O3 aqueous solution (15 mL), extracted with DCM three times, and the combined organic phase was washed with saturated NaCl solution three times to remove water, dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by column chromatography to give intermediate 19 (184.9, 79%). MS (ESI, m / z): 235 (M + +1).

[0208] c) Intermediate 19 (200.0 mg, 853.8 μmol), p-toluenesulfonic acid (14.7 mg, 85.4 μmol) and propanediol (324.8 mg, 4.3 mmol) were dissolved in toluene (6 mL), and the mixture was stirred at 130 °C for 6 h. After the reaction was completed, the reaction mixture was cooled to room temperature, the solvent was concentrated under reduced pressure, and the residue was diluted with water and extracted with ethyl acetate three times. The combined organic phase was washed with saturated NaCl solution, dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by column chromatography to give intermediate 20 (177 mg, 71%). 1 H NMR (400 MHz, Chloroform-d) δ 8.16 - 8.02 (m, 2H), 6.86 - 6.76 (m, 2H), 4.31 (d, J = 5.4 Hz, 1H), 4.15 - 4.07 (m, 2H), 3.96 (dq, J = 13.3, 2.2, 1.6 Hz, 2H), 3.81 - 3.70 (m, 2H), 2.93 (td, J = 12.8, 2.7 Hz, 2H), 2.14 - 2.00 (m, 1H), 1.94 - 1.74 (m, 3H), 1.51 - 1.32 (m, 3H). MS (ESI, m / z): 293 (M + +1).

[0209] d) Intermediate 20 (210.0 mg, 718.4 μmol) was dissolved in methanol (2 mL), Pd / C (20% in Water, 42 mg, 0.2 mmol,) was added to the reaction system, and the reaction was carried out under hydrogen at room temperature for 12 h. After the reaction was completed, the reaction system was filtered and concentrated to give crude intermediate 21, which was directly used in the next step. MS (ESI, m / z): 263 (M + +1).

[0210] e) The synthesis method of intermediate 22 was the same as that in step d of Example 3, except that the raw material 10 was replaced by intermediate 21. MS (ESI, m / z): 558 (M + +1).

[0211] f) The synthesis method of intermediate 23 was the same as that in step e of Example 3, except that the intermediate 11 was replaced by intermediate 22. MS (ESI, m / z): 544 (M + +1).

[0212] g) The synthesis method of intermediate 24 was the same as that in step f of Example 3, except that the intermediate 12 was replaced by intermediate 23. MS (ESI, m / z): 543 (M + +1).

[0213] h) Intermediate 24 (380 mg, 0.7 mmol), sodium formate (95.3 mg, 1.4 mmol) were weighed into a two-necked flask, formic acid (5 mL) was added to the reaction system under nitrogen protection, and the reaction was carried out at 70°C for 4 h. After the reaction was completed, water was added to dilute the reaction system, which was then extracted with dichloromethane three times. The combined organic phase was washed with saturated sodium chloride three times to remove water, dried over anhydrous Na2SO4, filtered and concentrated, and purified by column chromatography to give intermediate 25 (257.5 mg, 76%). MS (ESI, m / z): 485 (M + +1).

[0214] Example 6: Synthesis of intermediate 26

[0215]

[0216] The synthesis method of intermediate 14 in Reference Example 3 was used, except that the raw material 10 in step d was replaced by 4-(4-aminophenyl)piperazine-1-carboxylic acid tert-butyl ester. MS (ESI, m / z): 458 (M + +1).

[0217] Example 7: Synthesis of intermediate 27

[0218]

[0219] The synthetic method of intermediate 14 in Reference Example 3 was followed except that the starting material 10 in step d was replaced with 3-(4-aminophenyl)azetidine-1- carboxylate tert-butyl ester. MS (ESI, m / z): 429 (M+1). + +1).

[0220] Example 8: Synthesis of intermediate 28

[0221]

[0222] The synthetic method of intermediate 14 in Reference Example 3 was followed except that the starting material 10 in step d was replaced with 6-(4-aminophenyl)-2,6-diazaspiro[3.3]heptane- 2-carboxylate tert-butyl ester. MS (ESI, m / z): 470 (M+1). + +1).

[0223] Example 9: Synthesis of intermediate 29

[0224]

[0225] The synthetic method of intermediate 14 in Reference Example 3 was followed except that the starting material 10 in step d was replaced with 2-(4-aminophenyl)-2,7-diazaspiro[3.5]nonane-7- carboxylate tert-butyl ester. MS (ESI, m / z): 498 (M+1). + +1).

[0226] Example 10: Synthesis of intermediate 30

[0227]

[0228] The synthetic method of intermediate 14 in Reference Example 3 was followed except that the starting material 10 in step d was replaced with 2-(4-aminophenyl)-2,7-diazaspiro[3.5]nonane-7- carboxylate tert-butyl ester. MS (ESI, m / z): 526 (M+1). + +1).

[0229] Example 11: Synthesis of intermediate 31

[0230]

[0231] The synthetic method of intermediate 14 in Reference Example 3 was followed except that the starting material 10 in step d was replaced with 5-(4-aminophenyl)hexahydropyrrolo[3,4-c]pyrrole-2(1H)- carboxylate tert-butyl ester. MS (ESI, m / z): 484 (M+1). + +1).

[0232] Example 12: Synthesis of intermediate 32

[0233]

[0234] The synthesis of intermediate 14 in Reference Example 3 was followed, except that methylamine hydrochloride in step a was replaced by deuterated methylamine hydrochloride. MS (ESI, m / z): 460 (M + + 1).

[0235] Example 13: Synthesis of intermediate 33

[0236]

[0237] The synthesis of intermediate 14 in Reference Example 3 was followed, except that methylamine hydrochloride in step a was replaced by deuterated methylamine hydrochloride, and intermediate 5 in step c was replaced by intermediate 3. MS (ESI, m / z): 463 (M + + 1).

[0238] Example 14: Synthesis of intermediate 34

[0239]

[0240] The synthesis of intermediate 14 in Reference Example 3 was followed, except that intermediate 5 in step c was replaced by intermediate 3. MS (ESI, m / z): 460 (M + + 1).

[0241] Example 15: Synthesis of intermediates 38, 41 and 44

[0242]

[0243] a) The starting material 35 (1.2 g, 12.0 mmol) and the starting material 36 (1.9 g, 10.9 mmol) were dissolved in anhydrous dichloromethane (50 mL), and after stirring at room temperature for 4 h, sodium triacetoxyborohydride (4.6 g, 21.8 mmol) was added, and stirring was continued at room temperature for 16 h. After the reaction was completed, the reaction solution was filtered with diatomite, and the filtrate was taken and concentrated. After purification by column chromatography, intermediate 37 (697.0 mg, 25%) was obtained. MS (ESI, m / z): 257 (M + + 1). In the same way, intermediate 40 was obtained by replacing the starting material 36 with the starting material 39. MS (ESI, m / z): 271 (M + + 1). Intermediate 43 was obtained by replacing the starting material 35 with the starting material 42. MS (ESI, m / z): 271 (M + + 1).

[0244] b) Intermediate 37 (650 mg, 2.5 mmol) was dissolved in dichloromethane (6 mL), 4M HCl in dioxane (1.5 mL) was added at 0 °C, and the reaction was stirred at room temperature for 4 h. After the reaction was completed, the reaction solution was rotary evaporated to obtain the hydrochloride salt of intermediate 38. MS (ESI, m / z): 157 (M + +1). The same method was used to obtain intermediate 41 by replacing intermediate 37 with intermediate 40. MS (ESI, m / z): 171 (M + +1). Intermediate 44 was obtained by replacing intermediate 37 with starting material 43. MS (ESI, m / z): 171 (M + +1).

[0245] Example 16: Synthesis of intermediate 51

[0246]

[0247] a) Starting material 45 was prepared by using the method disclosed in the patent application “WO2022232536 specification page 98, example 1”. Starting material 45 (2.0 g, 5.9 mmol), starting material 46 (5.5 g, 17.7 mmol) and Pd(t-Bu3P)2 (603.0 mg, 1.18 mmol) were dissolved in 1,4 dioxane (18 mL) and water (6 mL), and DIPEA (1.9 g, 14.8 mmol) was added to the reaction system under nitrogen protection. The reaction was stirred at 100 °C for 8 h. After the reaction was completed, the reaction solution was filtered with celite, and the filtrate was concentrated. The purified intermediate 47 (1.5 g, 58%) was obtained by column chromatography. MS (ESI, m / z): 440 (M + +1).

[0248] b) Intermediate 47 (1.2 g, 2.7 mmol) was dissolved in DMF (15 mL), and 10% Pd / C (227.9 mg, 2.7 mmol) was added to the reaction system in batches, and the reaction was stirred at room temperature for 8 h under hydrogen condition. After the reaction was completed, the reaction solution was filtered with celite, and the filtrate was concentrated. The purified intermediate 48 (952.6 mg, 80%) was obtained by column chromatography. MS (ESI, m / z): 442 (M + +1).

[0249] c) The synthesis method was referred to step b of example 14, and the hydrochloride salt of intermediate 49 was obtained by replacing intermediate 37 with intermediate 48. MS (ESI, m / z): 342 (M + +1).

[0250] d) Refer to the step a of the synthetic example 4, replace the intermediate 14 with intermediate 49 to obtain the intermediate 50. MS (ESI, m / z): 456 (M + +1).

[0251] e) Refer to the step b of the synthetic example 4, replace the intermediate 15a with intermediate 49 to obtain the intermediate 51. MS (ESI, m / z): 400 (M + +1).

[0252] Example 17: Synthesis of intermediate 52

[0253]

[0254] Refer to the synthesis of intermediate 51 in the reference example 16, replace the raw material 46 in step a with tert-butyl 3,3-difluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6- dihydropyridine-1(2H)-carboxylate to obtain the intermediate 52. MS (ESI, m / z): 436 (M + +1).

[0255] Example 18: Synthesis of compound S1

[0256]

[0257] a) Refer to the method disclosed in the patent application “WO2022243365 specification page 23, example 2” to prepare the raw material 53. Dissolve 53 (830 mg, 3.0 mmol) and raw material 54 (262.8 mg, 3.6 mmol) in 1,4-dioxane (15 mL), add DIPEA (580.5 mg, 4.5 mmol) to the reaction system, stir at 90°C overnight. After the reaction is completed, the reaction solution is concentrated under reduced pressure, diluted with water, extracted with ethyl acetate three times, the combined organic layer is washed with saturated NaCl solution, dried over anhydrous Na2SO4, filtered and concentrated, purified by column chromatography to obtain the corresponding intermediate 55 (246.8 mg, 25%). MS (ESI, m / z): 330 (M + +1).

[0258] b) Refer to the step b of the synthetic example 5, replace the corresponding raw material to obtain the intermediate 56. MS (ESI, m / z): 328 (M + +1).

[0259] c) Intermediate 14 (100 mg, 0.2 mmol), intermediate 56 (245.2 mg, 0.75 mmol) were dissolved in anhydrous methanol (0.5 mL) and anhydrous dichloromethane (0.5 mL), 1 drop of acetic acid was added to the reaction system under the condition of nitrogen protection, after stirring at room temperature for 15 minutes, sodium triacetoxyborohydride (88.4 mg, 0.4 mmol) and sodium cyanoborohydride (1.3 mg, 0.02 mmol) were added to the reaction system, and the reaction was carried out at 50°C for 1h. After the reaction was completed, it was quenched with saturated sodium bicarbonate solution, extracted with dichloromethane for three times, the combined organic layer was washed with saturated NaCl solution, dried over anhydrous Na2SO4, filtered and concentrated, and purified by column chromatography to give compound S1 (29 mg, 19%). 1 H NMR (600 MHz, Chloroform-d) δ 11.03 (s, 1H), 8.19 (s, 1H), 7.95 (s, 1H), 7.85-7.76 (m, 2H), 7.75-7.54 (m, 4H), 7.45 (s, 2H), 7.24-7.09 (m, 2H), 6.79 (s, 1H), 6.50 (d, J = 7.7 Hz, 1H), 5.21 (s, 1H), 4.99-4.87 (dd, J = 12.2, 5.3 Hz, 1H), 4.18 (t, J = 7.3 Hz, 2H), 3.92 (s, 3H), 3.79-3.70 (m, 2H), 3.51-3.44 (m, 1H), 3.16-2.96 (m, 5H), 2.94-2.67 (m, 5H), 2.51 (s, 1H), 2.10-2.09 (m, 1H), 1.87 (s, 4H). MS (ESI, m / z): 782 (M + +1).

[0260] Example 19: Synthesis of compound S2

[0261]

[0262] The synthesis method is referred to the synthesis method of compound S1 in Example 18, except that the raw material 54 in step a is replaced by 2-azaspiro[3.3]heptan-6-ol (54a) to obtain compound S2. 1H NMR (400 MHz, Chloroform-d) δ 11.03 (s, 1H), 8.06 (s, 1H), 7.95 (s, 1H), 7.80 (d, J = 8.5 Hz, 2H), 7.72-7.63 (m, 3H), 7.58 (s, 1H), 7.46 (d, J = 5.2 Hz, 2H), 7.28 (s, 1H), 7.20 (d, J = 8.5 Hz, 2H), 7.05 (d, J = 10.9 Hz, 1H), 5.21 (s, 1H), 4.94 (dd, J = 12.3, 5.3 Hz, 1H), 4.00-3.92 (m, 5H), 3.10 (d, J = 4.6 Hz, 3H), 3.08-2.66 (m, 9H), 2.69 (p, J = 6.8 Hz, 1H), 2.52 (s, 1H), 2.14-2.09 (m, 1H), 2.01-1.98 (m, 4H), 1.92-1.80 (m, 4H), 1.52-1.44 (m, 4H). MS (ESI, m / z): 836 (M + +1).

[0263] Example 20: Synthesis of compound S3

[0264]

[0265] The synthesis method refers to that of compound S1 in Example 18, except that the raw material 54 in step a is replaced by 7-azaspiro[3.5]nonan-2-ol (54b) to obtain compound S3. 1 H NMR (400 MHz, Chloroform-d) δ 11.03 (s, 1H), 8.06 (s, 1H), 7.95 (s, 1H), 7.80 (d, J = 8.5 Hz, 2H), 7.72-7.63 (m, 3H), 7.58 (s, 1H), 7.46 (d, J = 5.2 Hz, 2H), 7.28 (s, 1H), 7.20 (d, J = 8.5 Hz, 2H), 7.05 (d, J = 10.9 Hz, 1H), 5.21 (s, 1H), 4.94 (dd, J = 12.3, 5.3 Hz, 1H), 4.00-3.92 (m, 5H), 3.10 (d, J = 4.6 Hz, 3H), 3.08-2.66 (m, 9H), 2.69 (p, J = 6.8 Hz, 1H), 2.52 (s, 1H), 2.14-2.09 (m, 1H), 2.01-1.98 (m, 4H), 1.92-1.80 (m, 4H), 1.52-1.44 (m, 4H). MS (ESI, m / z): 836 (M + +1).

[0266] Example 21: Synthesis of compound S4

[0267]

[0268] The synthesis method is referred to the synthesis method of compound S1 in Example 18, except that the raw material 54 in step a is replaced by 2-azaspiro[3.5]nonan-7-ol (54c) to obtain compound S4. 1 H NMR (600 MHz, Chloroform-d) δ 11.03 (s, 1H), 8.19 (s, 1H), 7.95 (s, 1H), 7.85-7.76 (m, 2H), 7.75-7.54 (m, 4H), 7.45 (s, 2H), 7.24-7.09 (m, 2H), 6.78 (d, J = 2.1 Hz, 1H), 6.51 (dd, J = 8.3, 2.2 Hz, 1H), 5.21 (s, 1H), 4.93 (dd, J = 12.1, 5.4 Hz, 1H), 4.18 (t, J = 7.3 Hz, 2H), 3.92 (s, 3H), 3.79-3.70 (m, 2H), 3.16-2.68 (m, 10H), 2.52 (s, 1H), 2.10-2.09 (m, 2H), 1.71-1.68 (m, 4H), 1.52-1.43 (m, 4H). MS (ESI, m / z): 836 (M + +1).

[0269] Example 22: Synthesis of compound S5

[0270]

[0271] The synthetic method refers to that of compound S1 in Example 18, except that the raw material 54 in step a is replaced by piperidin-4-ol (54d) to obtain compound S5. δ 11.03 (s, 1H), 8.06 (s, 1H), 7.95 (s, 1H), 7.80 (d, J = 8.5 Hz, 2H), 7.73-7.63 (m, 3H), 7.58 (s, 1H), 7.46 (d, J = 5.2 Hz, 2H), 7.28 (s, 1H), 7.20 (d, J = 8.5 Hz, 2H), 7.05 (d, J = 10.9 Hz, 1H), 5.21 (s, 1H), 4.94 (dd, J = 12.3, 5.3 Hz, 1H), 4.02-3.91 (s, 5H), 3.10 (d, J = 4.6 Hz, 3H), 3.08-2.64 (m, 9H), 2.73 (p, J = 5.8 Hz, 1H), 2.30-2.19 (m, 2H), 2.17-2.08 (m, 1H), 2.01-1.91 (m, 2H), 1.90-1.58 (m, 6H). MS (ESI, m / z): 796 (M + +1).

[0272] Example 23: Synthesis of compound S6

[0273]

[0274] The synthetic method refers to that of compound S1 in Example 18, except that the raw material 54 in step a is replaced by piperidin-4-ol (54d) to obtain compound S5. δ 11.03 (s, 1H), 8.06 (s, 1H), 7.95 (s, 1H), 7.80 (d, J = 8.5 Hz, 2H), 7.73-7.63 (m, 3H), 7.58 (s, 1H), 7.46 (d, J = 5.2 Hz, 2H), 7.28 (s, 1H), 7.20 (d, J = 8.5 Hz, 2H), 7.05 (d, J = 10.9 Hz, 1H), 5.21 (s, 1H), 4.94 (dd, J = 12.3, 5.3 Hz, 1H), 4.02-3.91 (s, 5H), 3.10 (d, J = 4.6 Hz, 3H), 3.08-2.64 (m, 9H), 2.73 (p, J = 5.8 Hz, 1H), 2.30-2.19 (m, 2H), 2.17-2.08 (m, 1H), 2.01-1.91 (m, 2H), 1.90-1.58 (m, 6H). MS (ESI, m / z): 796 (M 1 H NMR (400 MHz, Chloroform-d) δ 11.03 (s, 1H), 8.06 (s, 1H), 7.95 (s, 1H), 7.80 (d, J = 8.5 Hz, 2H), 7.73-7.63 (m, 3H), 7.58 (s, 1H), 7.46 (d, J = 5.2 Hz, 2H), 7.28 (s, 1H), 7.20 (d, J = 8.5 Hz, 2H), 7.05 (d, J = 10.9 Hz, 1H), 5.21 (s, 1H), 4.94 (dd, J = 12.3, 5.3 Hz, 1H), 4.02-3.91 (s, 5H), 3.10 (d, J = 4.6 Hz, 3H), 3.08-2.64 (m, 9H), 2.55-2.30 (m, 1H), 2.38-2.19 (m, 2H), 2.17-2.08 (m, 1H), 2.01-1.91 (m, 2H), 1.89-1.77 (m, 3H), 1.39-1.26 (m, 4H). MS (ESI, m / z): 810 (M + +1).

[0275] Example 24: Synthesis of compound S7

[0276]

[0277] The synthesis method refers to that of compound S1 in Example 18, except that the raw material 54 in step a is replaced by azetidine-3-methanol (54f) to obtain compound S7. 1 H NMR (600 MHz, Chloroform-d) δ 11.03 (s, 1H), 8.19 (s, 1H), 7.95 (s, 1H), 7.87-7.53 (m, 6H), 7.45 (s, 2H), 7.18 (s, 2H), 6.79 (s, 1H), 6.51 (s, 1H), 5.21 (s, 1H), 4.93 (dd, J = 12.1, 5.4 Hz, 1H), 4.18 (t, J = 7.3 Hz, 2H), 3.92 (s, 3H), 3.79-3.70 (m, 2H), 3.18-2.94 (m, 6H), 3.93-2.65 (m, 5H), 2.51 (s, 1H), 2.26-2.09 (m, 3H), 1.93-1.80 (m, 4H). MS (ESI, m / z): 782 (M + +1).

[0278] Example 25: Synthesis of compound S8

[0279]

[0280] The synthesis method refers to that of compound S1 in Example 18, except that the raw material 54 in step a is replaced by (S)-pyrrolidine-3-methanol (54g) to obtain compound S8. 1H NMR (600 MHz, Chloroform-d) δ 11.03 (s, 1H), 8.52 (s, 1H), 7.93 (s, 1H), 7.79 (d, J = 8.1 Hz, 2H), 7.76 (q, J = 4.7 Hz, 1H), 7.64 (dd, J = 11.7, 7.4 Hz, 2H), 7.58 (s, 1H), 7.44 (d, J = 6.5 Hz, 2H), 7.19 (d, J = 8.2 Hz, 2H), 6.94 (d, J = 2.3 Hz, 1H), 6.66 (d, J = 8.3 Hz, 1H), 5.37 (s, 1H), 4.93 (dd, J = 12.4, 5.4 Hz, 1H), 3.89 (s, 3H), 3.57 (t, J = 8.7 Hz, 1H), 3.49 - 3.43 (m, 1H), 3.38 (q, J = 8.4 Hz, 1H), 3.20 - 3.15 (m, 1H), 3.14 - 3.05 (m, 4H), 2.99 (d, J = 11.1 Hz, 1H), 2.92 - 2.58 (m, 5H), 2.50 - 2.38 (m, 3H), 2.24 - 2.03 (m, 4H), 1.89 - 1.75 (m, 4H). MS (ESI, m / z): 796 (M + +1).

[0281] Example 26: Synthesis of compound S9

[0282]

[0283] The synthesis method refers to that of compound S1 in Example 18, except that the raw material 54 in step a is replaced by (R)-pyrrolidine-3-methanol (54h) to obtain compound S9. 1H NMR (400 MHz, Chloroform-d) δ 11.03 (s, 1H), 8.11 (s, 1H), 7.95 (s, 1H), 7.81 (d, J = 8.4 Hz, 2H), 7.71 (d, J = 4.4 Hz, 1H), 7.68 - 7.62 (m, 2H), 7.58 (s, 1H), 7.49 - 7.43 (m, 2H), 7.21 (d, J = 8.4 Hz, 2H), 6.97 (s, 1H), 6.69 (d, J = 9.1 Hz, 1H), 5.23 (s, 1H), 4.94 (dd, J = 12.0, 5.3 Hz, 1H), 3.92 (s, 3H), 3.64 - 3.56 (m, 1H), 3.54 - 3.47 (m, 1H), 3.41 (q, J = 8.6, 8.0 Hz, 1H), 3.25 - 3.17 (m, 1H), 3.14 - 3.05 (m, 4H), 3.00 (d, J = 4.9 Hz, 1H), 2.94 - 2.58 (m, 5H), 2.56 - 2.37 (m, 3H), 2.27 - 2.03 (m, 4H), 1.84 (m, 4H). MS (ESI, m / z): 796 (M + +1).

[0284] Example 27: Synthesis of compound S10

[0285]

[0286] The synthesis method refers to that of compound S1 in Example 18, except that the raw material 54 in step a is replaced by 2-(piperidin-4-yl)ethan-1-ol (54i) to obtain compound S10. 1H NMR (400 MHz, Chloroform-d) δ 11.03 (s, 1H), 8.06 (s, 1H), 7.95 (s, 1H), 7.80 (d, J = 8.5 Hz, 2H), 7.73-7.63 (m, 3H), 7.58 (s, 1H), 7.46 (d, J = 5.2 Hz, 2H), 7.28 (s, 1H), 7.20 (d, J = 8.5 Hz, 2H), 7.05 (d, J = 10.9 Hz, 1H), 5.21 (s, 1H), 4.94 (dd, J = 12.3, 5.3 Hz, 1H), 4.02-3.91 (s, 5H), 3.10 (d, J = 4.6 Hz, 3H), 3.08-2.64 (m, 9H), 2.52 (s, 1H), 2.38-2.19 (m, 2H), 2.17-2.08 (m, 1H), 2.01-1.91 (m, 2H), 1.89-1.77 (m, 2H), 1.55-1.43 (m, 3H), 1.39-1.26 (m, 4H). MS (ESI, m / z): 824 (M + +1).

[0287] Example 28: Synthesis of compound S11

[0288]

[0289] The synthesis method refers to that of compound S1 in Example 18, except that the raw material 54 in step a is replaced by 3-(piperazin-4-yl)propan-1-ol (54j) to obtain compound S11. 1 H NMR (400 MHz, Chloroform-d) δ 11.03 (s, 1H), 8.06 (s, 1H), 7.95 (s, 1H), 7.80 (d, J = 8.5 Hz, 2H), 7.73-7.63 (m, 3H), 7.58 (s, 1H), 7.46 (d, J = 5.2 Hz, 2H), 7.28 (s, 1H), 7.20 (d, J = 8.5 Hz, 2H), 7.05 (d, J = 10.9 Hz, 1H), 5.21 (s, 1H), 4.94 (dd, J = 12.3, 5.3 Hz, 1H), 3.93 (s, 3H), 3.39 (t, J = 5.7 Hz, 3H), 3.10 (d, J = 4.6 Hz, 3H), 3.08-2.64 (m, 9H), 2.72-2.66 (m, 2H), 2.52 (s, 1H), 2.48-2.32 (m, 2H), 2.17-2.08 (m, 1H), 2.01-1.91 (m, 2H), 1.89-1.77 (m, 2H), 1.62-1.53 (m, 2H). MS (ESI, m / z): 839 (M+ +1).

[0290] Example 29: Synthesis of compound S12

[0291]

[0292] The synthesis method refers to that of compound S1 in Example 18, except that the raw material 54 in step a is replaced by 3-(piperidin-4-yl)propan-1-ol (54k) to obtain compound S12. 1 H NMR (400 MHz, Chloroform-d) δ 11.03 (s, 1H), 8.06 (s, 1H), 7.95 (s, 1H), 7.80 (d, J = 8.5 Hz, 2H), 7.73-7.63 (m, 3H), 7.58 (s, 1H), 7.46 (d, J = 5.2 Hz, 2H), 7.28 (s, 1H), 7.20 (d, J = 8.5 Hz, 2H), 7.05 (d, J = 10.9 Hz, 1H), 5.21 (s, 1H), 4.94 (dd, J = 12.3, 5.3 Hz, 1H), 4.02-3.91 (s, 5H), 3.10 (d, J = 4.6 Hz, 3H), 3.08-2.64 (m, 9H), 2.52 (s, 1H), 2.38-2.19 (m, 2H), 2.17-2.08 (m, 1H), 2.01-1.91 (m, 2H), 1.89-1.77 (m, 3H), 1.49-1.26 (m, 9H). MS (ESI, m / z): 838 (M + +1).

[0293] Example 30: Synthesis of compound S13

[0294]

[0295] The synthesis method refers to that of compound S1 in Example 18, except that the raw material 54 in step a is replaced by (3-fluoroazetidin-3-yl)methanol (54l) to obtain compound S13. 1H NMR (400 MHz, Chloroform-d) δ 11.03 (s, 1H), 8.19 (s, 1H), 7.95 (s, 1H), 7.87-7.53 (m, 6H), 7.45 (s, 2H), 7.18 (s, 2H), 6.79 (s, 1H), 6.51 (s, 1H), 5.21 (s, 1H), 4.93 (dd, J = 12.1, 5.4 Hz, 1H), 4.38-4.22 (m, 2H), 4.13-4.01 (m, 2H), 3.92 (s, 3H), 3.18-2.94 (m, 7H), 3.93-2.65 (m, 5H), 2.53 (s, 1H), 2.17-2.09 (m, 1H), 1.93-1.80 (m, 4H). MS (ESI, m / z): 800 (M + +1).

[0296] Example 31: Synthesis of compound S14

[0297]

[0298] The synthesis method refers to that of compound S1 in Reference Example 18, except that the raw material 54 in step a is replaced by (R)-(3-fluoropyrrolidin-3-yl)methanol (54m) to obtain compound S14. 1 H NMR (400 MHz, Chloroform-d) δ 11.03 (s, 1H), 8.19 (s, 1H), 7.95 (s, 1H), 7.87-7.53 (m, 6H), 7.45 (s, 2H), 7.18 (s, 2H), 6.79 (s, 1H), 6.51 (s, 1H), 5.21 (s, 1H), 4.93 (dd, J = 12.1, 5.4 Hz, 1H), 4.38-4.22 (m, 2H), 4.13-4.01 (m, 2H), 3.92 (s, 3H), 3.18-2.94 (m, 7H), 3.93-2.65 (m, 5H), 2.53 (s, 1H), 2.17-2.09 (m, 1H), 1.93-1.80 (m, 4H). MS (ESI, m / z): 800 (M + +1).

[0299] Example 32: Synthesis of compound S15

[0300]

[0301] The synthetic method refers to that of Compound S1 in Reference Example 18, except that the raw material 54 in step a is replaced by (S)-(3-fluoropyrrolidin-3-yl)methanol (54n) to obtain Compound S15. 1 H NMR (400 MHz, Chloroform-d) δ 11.03 (s, 1H), 8.11 (s, 1H), 7.95 (s, 1H), 7.81 (d, J = 8.4 Hz, 2H), 7.71 (d, J = 4.4 Hz, 1H), 7.68 - 7.62 (m, 2H), 7.58 (s, 1H), 7.49 - 7.43 (m, 2H), 7.21 (d, J = 8.4 Hz, 2H), 6.97 (s, 1H), 6.69 (d, J = 9.1 Hz, 1H), 5.23 (s, 1H), 4.94 (dd, J = 12.0, 5.3 Hz, 1H), 3.92 (s, 3H), 3.64 - 3.56 (m, 1H), 3.54 - 3.47 (m, 1H), 3.41 (q, J = 8.6, 8.0 Hz, 1H), 3.25 - 3.17 (m, 1H), 3.14 - 2.90 (m, 5H), 2.96 - 2.62 (m, 7H), 2.56 - 2.48 (s, 3H), 1.84 (m, 4H). MS (ESI, m / z): 814 (M + +1).

[0302] Example 33: Synthesis of Compound S16

[0303]

[0304] The synthetic method refers to that of Compound S1 in Reference Example 18, except that the raw material 54 in step a is replaced by (4-fluoropiperidin-4-yl)methanol (54o) to obtain Compound S16. 1H NMR (400 MHz, Chloroform-d) δ 11.03 (s, 1H), 8.06 (s, 1H), 7.95 (s, 1H), 7.80 (d, J = 8.5 Hz, 2H), 7.73-7.63 (m, 3H), 7.58 (s, 1H), 7.46 (d, J = 5.2 Hz, 2H), 7.28 (s, 1H), 7.20 (d, J = 8.5 Hz, 2H), 7.05 (d, J = 10.9 Hz, 1H), 5.21 (s, 1H), 4.94 (dd, J = 12.3, 5.3 Hz, 1H), 4.32-4.11 (m, 2H), 3.91 (s, 3H), 3.44-3.30 (m, 2H), 3.10 (d, J = 4.6 Hz, 3H), 3.08-2.64 (m, 7H), 2.66-2.58 (m, 2H), 2.55-2.40 (m, 1H), 2.17-2.09 (m, 3H), 1.98-1.88 (m, 2H), 1.78-1.63 (m, 4H). MS (ESI, m / z): 828 (M + +1).

[0305] Example 34: Synthesis of compound S17

[0306]

[0307] a) The synthesis method of intermediate 58 is the same as that of intermediate 55 in Example 18, except that the raw material 54 in step a is replaced by raw material 57 to obtain intermediate 58. MS (ESI, m / z): 443 (M + +1).

[0308] b) The synthesis method of intermediate 59 is the same as that of intermediate 38 in Example 15, except that the intermediate 37 in step b is replaced by intermediate 58 to obtain the hydrochloride salt of intermediate 59. MS (ESI, m / z): 343 (M + +1).

[0309] c) Intermediate 16a (154.2 mg, 0.3 mmol), raw material intermediate 59 (94.1 mg, 0.28 mmol) and BOP (165.8 mg, 0.38 mmol) were dissolved in anhydrous DMF (1.5 mL), and DIPEA (116.1 mg, 0.9 mmol) was added to the reaction system under argon protection. The reaction was carried out at room temperature for 4 h. After the reaction was completed, water was added for dilution, and ethyl acetate was extracted three times. The organic phases were combined and washed with saturated NaCl solution, dried over anhydrous Na2SO4, and filtered. The salt was removed by suction filtration, and the concentrated under reduced pressure was purified by column chromatography to obtain compound S17 (75.4 mg, 30%). 1HNMR (400 MHz, Chloroform-d) δ 11.03 (s, 1H), 8.06 (s, 1H), 7.95 (s, 1H), 7.80 (d, J = 8.5 Hz, 2H), 7.73-7.63 (m, 3H), 7.58 (s, 1H), 7.46 (d, J = 5.2 Hz, 2H), 7.28 (s, 1H), 7.20 (d, J = 8.5 Hz, 2H), 7.05 (d, J = 10.9 Hz, 1H), 5.21 (s, 1H), 4.94 (dd, J = 12.3, 5.3 Hz, 1H), 3.92 (s, 3H), 3.51-3.45 (m, 2H), 3.38-3.20 (m, 2H), 3.15 (t, J = 5.3 Hz, 2H), 3.10 (d, J = 4.6 Hz, 3H), 3.02-2.67 (m, 11H), 2.55-2.43 (m, 1H), 2.17-2.00 (m, 3H), 1.89-1.78 (m, 4H). MS (ESI, m / z): 853 (M + +1).

[0310] Example 35: Synthesis of compound S18

[0311]

[0312] The synthesis method refers to that of compound S17 in Example 34, except that 16a in step c is replaced by intermediate 16b to obtain compound S18. 1 HNMR (400 MHz, Chloroform-d) δ 11.03 (s, 1H), 8.06 (s, 1H), 7.95 (s, 1H), 7.80 (d, J = 8.5 Hz, 2H), 7.73-7.63 (m, 3H), 7.58 (s, 1H), 7.46 (d, J = 5.2 Hz, 2H), 7.28 (s, 1H), 7.20 (d, J = 8.5 Hz, 2H), 7.05 (d, J = 10.9 Hz, 1H), 5.21 (s, 1H), 4.94 (dd, J = 12.3, 5.3 Hz, 1H), 3.92 (s, 3H), 3.51-3.45 (m, 2H), 3.38-3.20 (m, 2H), 3.15 (t, J = 5.3 Hz, 2H), 3.10 (d, J = 4.6 Hz, 3H), 3.02-2.67 (m, 11H), 2.55-2.43 (m, 1H), 2.17-2.00 (m, 3H), 1.89-1.78 (m, 4H). MS (ESI, m / z): 853 (M + +1).

[0313] Example 36: Synthesis of compound S19

[0314]

[0315] The synthetic method refers to that of compound S17 in Example 34, except that the starting material in Example 34, step c is replaced by intermediate 14 and intermediate 51 to obtain compound S19. 1 H NMR (400 MHz, Chloroform-d) δ 11.03 (s, 1H), 8.06 (s, 1H), 7.95 (s, 1H), 7.80 (d, J = 8.5 Hz, 2H), 7.73-7.63 (m, 3H), 7.58 (s, 1H), 7.46 (d, J = 5.2 Hz, 2H), 7.28 (s, 1H), 7.20 (d, J = 8.5 Hz, 2H), 7.05 (d, J = 10.9 Hz, 1H), 5.21 (s, 1H), 4.94 (dd, J = 12.3, 5.3 Hz, 1H), 3.92 (s, 3H), 3.10 (d, J = 4.6 Hz, 3H), 3.08-2.63 (m, 13H), 2.60-2.40 (m, 2H), 2.17-2.00 (m, 1H), 1.92-1.76 (m, 8H). MS (ESI, m / z): 838 (M + +1).

[0316] Example 37: Synthesis of compound S20

[0317]

[0318] The synthetic method refers to that of compound S17 in Example 34, except that the starting material in Example 34, step c is replaced by intermediate 14 and intermediate 52 to obtain compound S20. 1 H NMR (400 MHz, Chloroform-d) δ 11.03 (s, 1H), 8.06 (s, 1H), 7.95 (s, 1H), 7.80 (d, J = 8.5 Hz, 2H), 7.73-7.63 (m, 3H), 7.58 (s, 1H), 7.46 (d, J = 5.2 Hz, 2H), 7.28 (s, 1H), 7.20 (d, J = 8.5 Hz, 2H), 7.05 (d, J = 10.9 Hz, 1H), 5.21 (s, 1H), 4.94 (dd, J = 12.3, 5.3 Hz, 1H), 3.92 (s, 3H), 3.18-2.63 (m, 17H), 2.50-2.40 (m, 1H), 2.17-2.00 (m, 1H), 1.92-1.76 (m, 6H). MS (ESI, m / z): 874 (M + +1).

[0319] Example 38: Synthesis of compound S21

[0320]

[0321] The synthesis of compound S21 is the same as that of compound S1, except that the raw material 53 in Example 18 is replaced by raw material 60 (prepared by the method disclosed in the patent application “Synthesis of intermediate compound R 1A-1 in the specification of WO2023088245, page 48), and the raw material 54 is replaced by azetidin-3-methanol to obtain compound S21. 1 H NMR (600 MHz, Chloroform-d) δ 11.02 (s, 1H), 8.07 (s, 1H), 7.95 (s, 1H), 7.79 (d, J = 7.9 Hz, 2H), 7.74-7.62 (m, 2H), 7.58 (s, 1H), 7.45 (d, J = 6.4 Hz, 3H), 7.17 (dd, J = 15.9, 7.3 Hz, 3H), 6.59 (d, J = 8.3 Hz, 1H), 5.19 (s, 1H), 4.93 (dd, J = 12.7, 5.3 Hz, 1H), 4.42 (s, 2H), 3.94 (d, J = 17.0 Hz, 4H), 3.10 (d, J = 3.7 Hz, 3H), 3.05-2.92 (m, 3H), 2.91-2.60 (m, 6H), 2.54-2.44 (m, 1H), 2.13 (d, J = 8.8 Hz, 3H), 1.89-1.74 (m, 4H). MS (ESI, m / z): 782 (M + +1).

[0322] Example 39: Synthesis of compound S22

[0323]

[0324] The synthesis of compound S22 is the same as that of compound S1, except that the raw material 53 in Example 18 is replaced by raw material 60, and the raw material 54 is replaced by pyrrolidin-3-methanol to obtain compound S22. 1H NMR (600 MHz, Chloroform-d) δ 11.01 (s, 1H), 8.18 (s, 1H), 7.95 (s, 1H), 7.79 (d, J = 7.7 Hz, 2H), 7.68 (d, J = 21.1 Hz, 2H), 7.58 (s, 1H), 7.45 (s, 3H), 7.19 (d, J = 7.1 Hz, 3H), 6.96 (d, J = 8.7 Hz, 1H), 5.26 (s, 1H), 4.95 (dd, J = 12.4, 5.5 Hz, 1H), 3.91 (s, 3H), 3.77 - 3.59 (m, 3H), 3.44 (s, 1H), 3.10 (m, 5H), 2.97 - 2.37 (m, 8H), 2.15 (m, 4H), 1.86 (m, 4H). MS (ESI, m / z): 796 (M + +1).

[0325] Example 40: Synthesis of compound S23

[0326]

[0327] The synthesis of compound S23 is the same as that of compound S1, except that the starting material 54 in Example 18 is replaced by piperidin-4-methanol, and the intermediate 14 is replaced by intermediate 26 to obtain compound S23. MS (ESI, m / z): 811 (M + +1).

[0328] Example 41: Synthesis of compound S24

[0329]

[0330] The synthesis of compound S24 is the same as that of compound S1, except that the starting material 54 in Example 18 is replaced by azetidin-3-methanol, and the intermediate 14 in step c is replaced by intermediate 26 to obtain compound S24. 1H NMR (400 MHz, Chloroform-d) δ 10.87 (s, 1H), 8.08 (s, 1H), 7.94 (s, 1H), 7.75 (d, J = 9.0 Hz, 2H), 7.70 - 7.63 (m, 3H), 7.57 (s, 1H), 7.47 - 7.43 (m, 2H), 6.98 - 6.90 (m, 3H), 6.69 (dd, J = 8.5, 2.2 Hz, 1H), 5.20 (s, 1H), 4.93 (dd, J = 12.1, 5.4 Hz, 1H), 3.91 (s, 3H), 3.59 (dd, J = 10.1, 7.2 Hz, 1H), 3.51 (m, 1H), 3.47 - 3.38 (m, 1H), 3.26 - 3.14 (m, 5H), 3.08 (d, J = 4.5 Hz, 3H), 2.90 - 2.58 (m, 8H), 2.52 - 2.41 (m, 2H), 2.26 - 2.18 (m, 1H), 2.15 - 2.08 (m, 1H), 1.88 - 1.80 (m, 1H). MS (ESI, m / z): 797 (M+1). + +1).

[0331] Example 42: Synthesis of compound S25

[0332]

[0333] The synthesis of compound S25 is the same as that of compound S1, except that the starting material 54 in step a of Example 18 is replaced by (S)-pyrrolidine-3-methanol and the intermediate 14 in step c is replaced by intermediate 26 to give compound S25. 1 H NMR (400 MHz, Chloroform-d) δ 10.87 (s, 1H), 8.08 (s, 1H), 7.94 (s, 1H), 7.75 (d, J = 9.0 Hz, 2H), 7.70 - 7.63 (m, 3H), 7.57 (s, 1H), 7.47 - 7.43 (m, 2H), 6.98 - 6.90 (m, 3H), 6.69 (dd, J = 8.5, 2.2 Hz, 1H), 5.20 (s, 1H), 4.93 (dd, J = 12.1, 5.4 Hz, 1H), 3.91 (s, 3H), 3.59 (dd, J = 10.1, 7.2 Hz, 1H), 3.51 (m, 1H), 3.47 - 3.38 (m, 1H), 3.26 - 3.14 (m, 5H), 3.08 (d, J = 4.5 Hz, 3H), 2.90 - 2.58 (m, 8H), 2.52 - 2.41 (m, 2H), 2.26 - 2.18 (m, 1H), 2.15 - 2.08 (m, 1H), 1.88 - 1.80 (m, 1H). MS (ESI, m / z): 797 (M+1). + +1).

[0334] Example 43: Synthesis of compound S26

[0335]

[0336] The synthesis of compound S26 was the same as that of compound S1 in Example 18, except that the starting material 54 in step a of Example 18 was replaced by (R)-pyrrolidin-3- methanol and the intermediate 14 in step c was replaced by intermediate 26 to give compound S26. 1 HNMR (400 MHz, Chloroform-d) δ 10.87 (s, 1H), 8.08 (s, 1H), 7.94 (s, 1H), 7.75 (d, J = 9.0 Hz, 2H), 7.70 - 7.63 (m, 3H), 7.57 (s, 1H), 7.47 - 7.43 (m, 2H), 6.98 - 6.90 (m, 3H), 6.69 (dd, J = 8.5, 2.2 Hz, 1H), 5.20 (s, 1H), 4.93 (dd, J = 12.1, 5.4 Hz, 1H), 3.91 (s, 3H), 3.59 (dd, J = 10.1, 7.2 Hz, 1H), 3.51 (m, 1H), 3.47 - 3.38 (m, 1H), 3.26 - 3.14 (m, 5H), 3.08 (d, J = 4.5 Hz, 3H), 2.90 - 2.58 (m, 8H), 2.52 - 2.41 (m, 2H), 2.26 - 2.18 (m, 1H), 2.15 - 2.08 (m, 1H), 1.88 - 1.80 (m, 1H). MS (ESI, m / z): 797 (M + +1).

[0337] Example 44: Synthesis of compound S27

[0338]

[0339] The synthesis of compound S27 was the same as that of compound S19 in Example 36, except that the intermediate 14 in Example 36 was replaced by intermediate 26 to give compound S27. 1H NMR (400 MHz, Chloroform-d) δ 10.87 (s, 1H), 8.08 (s, 1H), 7.94 (s, 1H), 7.75 (d, J = 9.0 Hz, 2H), 7.70-7.63 (m, 3H), 7.57 (s, 1H), 7.47-7.43 (m, 2H), 6.98-6.90 (m, 3H), 6.69 (dd, J = 8.5, 2.2 Hz, 1H), 5.20 (s, 1H), 4.93 (dd, J = 12.1, 5.4 Hz, 1H), 3.91 (s, 3H), 4.07-3.96 (m, 4H), 3.92 (t, 2H), 3.54-3.26 (m, 2H), 3.08 (d, J = 4.5 Hz, 3H), 2.90-2.58 (m, 9H), 2.50-2.40 (m, 1H), 2.15-2.08 (m, 1H), 1.98-1.76 (m, 4H). MS (ESI, m / z): 839 (M + +1).

[0340] Example 45: Synthesis of compound S28

[0341]

[0342] The synthesis of compound S28 is the same as that of compound S19 in Example 36, except that intermediate 51 in Example 36 is replaced by intermediate 52, and intermediate 14 is replaced by intermediate 26 to obtain compound S28. 1 H NMR (400 MHz, Chloroform-d) δ 10.87 (s, 1H), 8.08 (s, 1H), 7.94 (s, 1H), 7.75 (d, J = 9.0 Hz, 2H), 7.70-7.63 (m, 3H), 7.57 (s, 1H), 7.47-7.43 (m, 2H), 6.98-6.90 (m, 3H), 6.69 (dd, J = 8.5, 2.2 Hz, 1H), 5.20 (s, 1H), 4.93 (dd, J = 12.1, 5.4 Hz, 1H), 3.91 (s, 3H), 4.07-3.96 (m, 4H), 3.92 (t, 2H), 3.54-3.26 (m, 2H), 3.08 (d, J = 4.5 Hz, 3H), 3.03-2.50 (m, 10H), 2.15-2.08 (m, 1H), 1.98-1.86 (m, 2H). MS (ESI, m / z): 875 (M + +1).

[0343] Example 46: Synthesis of compound S29

[0344]

[0345] a) The synthesis of intermediate 62 was the same as intermediate 55, except that the starting material 54 in step a of example 18 was replaced by starting material 61 to give intermediate 62. MS (ESI, m / z): 483 (M + +1).

[0346] b) The synthesis of intermediate 63 was the same as intermediate 38, except that the intermediate 37 in step b of example 15 was replaced by intermediate 62 to give the hydrochloride salt of intermediate 63. MS (ESI, m / z): 383 (M + +1).

[0347] c) The synthesis of compound S29 was the same as compound S1, except that the intermediate 14 in step c of example 18 was replaced by intermediate 63, and intermediate 56 was replaced by intermediate 25 to give compound S29. 1 H NMR (400 MHz, Chloroform-d) δ 10.86 (s, 1H), 8.30 (s, 1H), 7.94 (s, 1H), 7.76-7.71 (m, 2H), 7.69 (d, J = 8.5 Hz, 1H), 7.64 (dt, J = 7.7, 3.9 Hz, 2H), 7.56 (s, 1H), 7.47-7.43 (m, 2H), 7.29 (d, J = 2.3 Hz, 1H), 7.05 (dd, J = 8.6, 2.4 Hz, 1H), 6.98-6.93 (m, 2H), 5.25 (s, 1H), 4.94 (dd, J = 12.2, 5.4 Hz, 1H), 4.11-4.02 (m, 2H), 3.91 (s, 3H), 3.82-3.77 (m, 2H), 3.64 (dt, J = 12.1, 3.6 Hz, 2H), 3.07 (d, J = 4.6 Hz, 3H), 2.95-2.63 (m, 8H), 2.50-2.40 (m, 1H), 2.29 (d, J = 7.1 Hz, 2H), 2.16-2.08 (m, 1H), 1.89-1.78 (m, 4H), 1.68-1.57 (m, 1H), 1.43-1.32 (m, 4H). MS (ESI, m / z): 851 (M + +1).

[0348] Example 47: Synthesis of compound S30

[0349]

[0350] The synthesis of compound S30 was the same as compound S29, except that the intermediate 61 in step a of example 46 was replaced by intermediate 57 to give compound S30. 1H NMR (400 MHz, Chloroform-d) δ 10.86 (s, 1H), 8.30 (s, 1H), 7.94 (s, 1H), 7.76-7.71 (m, 2H), 7.69 (d, J = 8.5 Hz, 1H), 7.64 (dt, J = 7.7, 3.9 Hz, 2H), 7.56 (s, 1H), 7.47-7.43 (m, 2H), 7.29 (d, J = 2.3 Hz, 1H), 7.05 (dd, J = 8.6, 2.4 Hz, 1H), 6.98-6.93 (m, 2H), 5.25 (s, 1H), 4.94 (dd, J = 12.2, 5.4 Hz, 1H), 3.91 (s, 3H), 3.64 (dt, J = 12.1, 3.6 Hz, 2H), 3.43 (t, J = 5.0 Hz, 3H), 3.07 (d, J = 4.6 Hz, 3H), 2.95-2.63 (m, 5H), 2.58 (t, J = 5.1 Hz, 4H), 2.29 (d, J = 7.1 Hz, 2H), 2.16-2.08 (m, 1H), 1.68-1.57 (m, 1H), 1.43-1.32 (m, 4H). MS (ESI, m / z): 811 (M + +1).

[0351] Example 48: Synthesis of compound S31

[0352]

[0353] The synthesis of compound S30 is the same as that of compound S29, except that intermediate 63 in step c of Example 46 is replaced by intermediate 49 to give compound S31. 1H NMR (400 MHz, Chloroform-d) δ 10.86 (s, 1H), 8.30 (s, 1H), 7.94 (s, 1H), 7.76-7.71 (m, 2H), 7.69 (d, J = 8.5 Hz, 1H), 7.64 (dt, J = 7.7, 3.9 Hz, 2H), 7.56 (s, 1H), 7.47-7.43 (m, 2H), 7.29 (d, J = 2.3 Hz, 1H), 7.05 (dd, J = 8.6, 2.4 Hz, 1H), 6.98-6.93 (m, 2H), 5.25 (s, 1H), 4.94 (dd, J = 12.2, 5.4 Hz, 1H), 3.91 (s, 3H), 3.64 (dt, J = 12.1, 3.6 Hz, 2H), 3.07 (d, J = 4.6 Hz, 3H), 2.95-2.63 (m, 9H), 2.50-2.40 (m, 1H), 2.29 (d, J = 7.1 Hz, 2H), 2.16-2.08 (m, 1H), 1.89-1.78 (m, 4H), 1.68-1.57 (m, 1H), 1.43-1.32 (m, 4H). MS (ESI, m / z): 810 (M + +1).

[0354] Example 49: Synthesis of compound S32

[0355]

[0356] The synthesis of compound S32 is the same as that of compound S29, except that the intermediate 61 in step a of Example 46 is replaced by tert-butyl 2,7-diazaspiro[3.5]nonane-2-carboxylate to give compound S32. 1H NMR (400 MHz, Chloroform-d) δ 10.86 (s, 1H), 8.30 (s, 1H), 7.94 (s, 1H), 7.76-7.71 (m, 2H), 7.69 (d, J = 8.5 Hz, 1H), 7.64 (dt, J = 7.7, 3.9 Hz, 2H), 7.56 (s, 1H), 7.47-7.43 (m, 2H), 7.29 (d, J = 2.3 Hz, 1H), 7.05 (dd, J = 8.6, 2.4 Hz, 1H), 6.98-6.93 (m, 2H), 5.25 (s, 1H), 4.94 (dd, J = 12.2, 5.4 Hz, 1H), 3.91 (s, 3H), 3.78-3.67 (m, 2H), 3.64 (dt, J = 12.1, 3.6 Hz, 2H), 3.07 (d, J = 4.6 Hz, 3H), 2.95-2.63 (m, 11H), 2.50-2.40 (m, 3H), 2.16-2.08 (m, 1H), 1.89-1.68 (m, 8H). MS (ESI, m / z): 851 (M + +1).

[0357] Example 50: Synthesis of compound S33

[0358]

[0359] The synthesis of compound S33 was the same as that of compound S1 except that the starting material 54 in Example 18, step a was replaced by intermediate 38 to give compound S33. δ 11.03 (s, 1H), 8.06 (s, 1H), 7.95 (s, 1H), 7.80 (d, J = 8.5 Hz, 2H), 7.73-7.63 (m, 3H), 7.58 (s, 1H), 7.46 (d, J = 5.2 Hz, 2H), 7.28 (s, 1H), 6.78 (d, J = 2.1 Hz, 1H), 6.51 (dd, J = 8.3, 2.2 Hz, 1H), 5.21 (s, 1H), 4.94 (dd, J = 12.3, 5.3 Hz, 1H), 4.22-4.13 (m, 2H), 3.93 (s, 3H), 3.78-3.70 (m, 2H), 3.10 (d, J = 4.6 Hz, 3H), 3.08-2.64 (m, 13H), 2.50-2.40 (m, 1H), 2.17-2.08 (m, 1H), 1.98-1.88 (m, 4H), 1.78-1.65 (m, 4H). MS (ESI, m / z): 851 (M + +1).

[0360] Example 51: Synthesis of compound S34

[0361]

[0362] The synthesis of compound S34 is the same as that of compound S1, except that the starting material 54 in Example 18, step a is replaced by intermediate 38, and the intermediate 14 in step c is replaced by intermediate 27 to give compound S34. The synthesis of compound S33 is the same as that of compound S1, except that the starting material 54 in Example 18, step a is replaced by intermediate 38 to give compound S33. δ 11.03 (s, 1H), 8.06 (s, 1H), 7.95 (s, 1H), 7.80 (d, J = 8.5 Hz, 2H), 7.73-7.63 (m, 3H), 7.58 (s, 1H), 7.46 (d, J = 5.2 Hz, 2H), 7.28 (s, 1H), 6.78 (d, J = 2.1 Hz, 1H), 6.51 (dd, J = 8.3, 2.2 Hz, 1H), 5.21 (s, 1H), 4.94 (dd, J = 12.3, 5.3 Hz, 1H), 4.22-4.13 (m, 2H), 3.93 (s, 3H), 3.78-3.70 (m, 2H), 3.56-3.43 (m, 2H), 3.10-2.64 (m, 15H), 2.17-2.08 (m, 1H), 1.78-1.65 (m, 4H). MS (ESI, m / z): 823 (M + +1).

[0363] Example 52: Synthesis of compound S35

[0364]

[0365] The synthetic method of compound S35 is the same as that of compound S1, except that the raw material 54 in step a of Example 18 is replaced by intermediate 44 to obtain compound S35. δ 11.03 (s, 1H), 8.06 (s, 1H), 7.95 (s, 1H), 7.80 (d, J = 8.5 Hz, 2H), 7.73-7.63 (m, 3H), 7.58 (s, 1H), 7.46 (d, J = 5.2 Hz, 2H), 7.28 (s, 1H), 6.78 (d, J = 2.1 Hz, 1H), 6.51 (dd, J = 8.3, 2.2 Hz, 1H), 5.21 (s, 1H), 4.94 (dd, J = 12.3, 5.3 Hz, 1H), 4.22-4.13 (m, 2H), 3.93 (s, 3H), 3.78-3.70 (m, 2H), 3.10 (d, J = 4.6 Hz, 3H), 3.08-2.64 (m, 12H), 2.50-2.40 (m, 1H), 2.20-2.08 (m, 4H), 1.98-1.88 (m, 4H), 1.54-1.44 (m, 4H). MS (ESI, m / z): 865 (M + +1).

[0366] Example 53: Synthesis of compound S36

[0367]

[0368] The synthetic method of compound S36 is the same as that of compound S1, except that the raw material 54 in step a of Example 18 is replaced by intermediate 41 to obtain compound S36. δ 11.03 (s, 1H), 8.06 (s, 1H), 7.95 (s, 1H), 7.80 (d, J = 8.5 Hz, 2H), 7.73-7.63 (m, 3H), 7.58 (s, 1H), 7.46 (d, J = 5.2 Hz, 2H), 7.28 (s, 1H), 6.78 (d, J = 2.1 Hz, 1H), 6.51 (dd, J = 8.3, 2.2 Hz, 1H), 5.21 (s, 1H), 4.94 (dd, J = 12.3, 5.3 Hz, 1H), 4.22-4.13 (m, 2H), 3.93 (s, 3H), 3.78-3.70 (m, 2H), 3.10 (d, J = 4.6 Hz, 3H), 2.98-2.64 (m, 14H), 2.50-2.40 (m, 2H), 2.17-2.08 (m, 1H), 1.98-1.88 (m, 4H), 1.78-1.65 (m, 4H). MS (ESI, m / z): 865 (M + +1).

[0369] Example 54: Synthesis of compound S37

[0370]

[0371] Intermediate 28 (140.7 mg, 0.3 mmol) and intermediate 29 (140.7 mg, 0.3 mmol) were dissolved in 1,4-dioxane (1.5 mL), and DIPEA (193.5 mg, 1.5 mmol) was added to the reaction system. The reaction was stirred at 90 °C overnight. After the reaction was completed, the reaction solution was concentrated under reduced pressure, diluted with water, extracted with ethyl acetate three times, and the combined organic layers were washed with saturated NaCl solution, dried over anhydrous Na2SO4, filtered and concentrated, and purified by column chromatography to obtain compound S38 (43.5 mg, 24%). δ 11.03 (s, 1H), 8.06 (s, 1H), 7.95 (s, 1H), 7.80 (d, J = 8.5 Hz, 2H), 7.73-7.63 (m, 3H), 7.58 (s, 1H), 7.46 (d, J = 5.2 Hz, 2H), 7.28 (s, 1H), 7.28 (d, J = 2.1 Hz, 1H), 7.05 (dd, J = 8.3, 2.2 Hz, 1H), 5.21 (s, 1H), 4.94 (dd, J = 12.3, 5.3 Hz, 1H), 4.32-4.03 (m, 4H), 3.93 (s, 3H), 3.82-3.65 (m, 4H), 3.10 (d, J = 4.6 Hz, 3H), 2.17-2.08 (m, 1H). MS (ESI, m / z): 726 (M+1). + +1).

[0372] Example 55: Synthesis of compound S38

[0373]

[0374] The synthesis method of compound S38 is the same as that of compound S37, except that intermediate 28 in Example 54 is replaced by intermediate 29 to obtain compound S38. 1H NMR (600 MHz, Chloroform-d) δ 11.03 (s, 1H), 8.19 (s, 1H), 7.95 (s, 1H), 7.85-7.76 (m, 2H), 7.75-7.54 (m, 4H), 7.45 (s, 2H), 7.24-7.09 (m, 2H), 6.79 (s, 1H), 6.50 (d, J = 7.7 Hz, 1H), 5.21 (s, 1H), 4.99-4.87 (dd, J = 12.2, 5.3 Hz, 1H), 3.92 (s, 3H), 3.61 (s, 3H), 3.58-3.43 (m, 4H), 3.20-2.96 (m, 4H), 2.94-2.67 (m, 3H), 2.27-2.10 (m, 1H), 1.60-1.56 (t, J = 5.3 Hz, 4H). MS (ESI, m / z): 754 (M + +1).

[0375] Example 56: Synthesis of compound S39

[0376]

[0377] The synthesis of compound S39 is the same as that of compound S37, except that intermediate 28 in Example 54 is replaced by intermediate 30 to obtain compound S39. 1 H NMR (600 MHz, Chloroform-d) δ 11.03 (s, 1H), 8.19 (s, 1H), 7.95 (s, 1H), 7.85-7.76 (m, 2H), 7.75-7.54 (m, 4H), 7.45 (s, 2H), 7.24-7.09 (m, 2H), 6.79 (s, 1H), 6.50 (d, J = 7.7 Hz, 1H), 5.21 (s, 1H), 4.99-4.87 (dd, J = 12.2, 5.3 Hz, 1H), 3.92 (s, 3H), 3.71-3.53 (m, 4H), 3.20-2.96 (m, 3H), 2.94-2.67 (m, 3H), 2.46 (t, J = 5.3 Hz, 4H), 2.27-2.10 (m, 1H), 1.61-1.41 (m, 8H). MS (ESI, m / z): 782 (M + +1).

[0378] Example 57: Synthesis of compound S40

[0379]

[0380] Compound S40 is synthesized in the same way as compound S37; simply replace intermediate 28 in Example 54 with intermediate 31 to obtain compound S40. 1 H NMR(600MHz,Chloroform-d)δ11.03(s,1H),8.19(s,1H),7.95(s,1H),7.85-7.76(m,2H),7.75-7. 54(m,4H),7.45(s,2H),7.24-7.09(m,2H),6.79(s,1H),6.50(d,J=7.7Hz,1H),5.21(s,1H),4.99- 4.87(dd,J=12.2,5.3Hz,1H),3.92(s,3H),3.69-3.61(m,2H),3.45-3.37(m,2H),3.20-3.08(m,3H ),3.04-2.67(m,5H),2.53-2.51(m,2H),2.46-2.41(m,2H),2.27-2.10(m,1H).MS(ESI,m / z):740(M + +1).

[0381] Example 58: Synthesis of compound S41

[0382]

[0383] Compound S41 is synthesized in the same way as compound S37. Compound S41 can be obtained simply by replacing intermediate 28 in Example 54 with intermediate 14. 1 H NMR(400MHz,Chloroform-d)δ10.87(s,1H),8.08(s,1H),7.94(s,1H),7.75(d,J=9.0Hz ,2H),7.70-7.63(m,3H),7.57(s,1H),7.47-7.43(m,2H),6.98-6.90(m,3H),6.69(dd,J =8.5,2.2Hz,1H),5.20(s,1H),4.93(dd,J=12.1,5.4Hz,1H),3.91(s,3H),3.26-3.14(m ,4H),3.08(d,J=4.5Hz,3H),2.93-2.58(m,7H),2.13-2.04(m,1H).MS(ESI,m / z):714(M + +1).

[0384] Example 59: Synthesis of compound S42

[0385]

[0386] Compound S42 is synthesized in the same way as compound S1. Simply replace the starting material 54 in step a of Example 18 with (R)-pyrrolidine-3-methanol and the intermediate 14 in step c with intermediate 28 to obtain compound S42. 1 HNMR(400MHz,Chloroform-d)δ10.87(s,1H),8.08(s,1H),7.94(s,1H),7.75(d,J=9.0Hz,2H),7.70-7.63(m,3H),7 .57(s,1H),7.47-7.43(m,2H),6.98-6.90(m,3H),6.69(dd,J=8.5,2.2Hz,1H),5.20(s,1H),4.93(dd,J=12.1,5.4Hz ,1H),3.91(s,3H),3.64-3.56(m,5H),3.54-3.47(m,1H),3.44-3.38(m,1H),3.26-3.14(m,1H),3.08-3.03(m,7H), 2.93-2.58(m,4H),2.54-2.42(m,2H),2.28-2.18(m,1H),2.17-2.08(m,1H),1.92-1.80(m,1H).MS(ESI,m / z):809(M + +1).

[0387] Example 60: Synthesis of compound S43

[0388]

[0389] Compound S43 is synthesized in the same way as compound S1. Simply replace the starting material 54 in step a of Example 18 with (S)-pyrrolidine-3-methanol and replace intermediate 14 in step c with intermediate 28 to obtain compound S43. 1HNMR(600MHz,Chloroform-d)δ10.87(s,1H),8.32(s,1H),7.93(d,J=2.5Hz,1H),7.74(d,J=8.2Hz,2H),7.70(s,1H),7.6 4(t,J=6.6Hz,2H),7.56(s,1H),7.49-7.39(m,2H),6.97-6.88(m,3H),6.67(d,J=8.4Hz,1H),5.20(s,1H),4.93(dd,J=12. 0,5.9Hz,1H),3.90(d,J=2.4Hz,3H),3.61-3.58(m,5H),3.49(s,1H),3.46-3.33(m,1H),3.22-3.12(m,1H),3.07-3.03(m ,7H),2.92-2.55(m,4H),2.52-2.41(m,2H),2.26-2.17(m,1H),2.14-2.08(m,1H),1.88-1.79(m,1H).MS(ESI,m / z):809(M + +1).

[0390] Example 61: Synthesis of compound S44

[0391]

[0392] Compound S44 is synthesized in the same way as compound S1. Simply replace the raw material 54 in step a of Example 18 with 4-hydroxymethylpiperidine and replace intermediate 14 in step c with intermediate 28 to obtain compound S44. 1 HNMR(400MHz,Chloroform-d)δ11.03(s,1H),8.06(s,1H),7.95(s,1H),7.80(d,J=8.5Hz,2H),7.72-7. 63(m,3H),7.58(s,1H),7.50-7.44(m,2H),7.28(d,J=2.4Hz,1H),7.20(d,J=8.5Hz,2H),7.05(dd,J=8.6 ,2.4Hz,1H),5.21(s,1H),4.94(dd,J=12.3,5.3Hz,1H),3.96(d,J=13.1Hz,2H),3.92(s,3H),3.61(s,4H ),3.18-2.66(m,12H),2.29(s,2H),2.12(dd,J=9.4,3.9Hz,1H),2.01-1.83(m,5H).MS(ESI,m / z):823(M + +1).

[0393] Example 62: Synthesis of compound S45

[0394]

[0395] Compound S45 is synthesized in the same way as compound S1. Simply replace the raw material 54 in step a of Example 18 with 3-azacyclobutane-methanol and replace intermediate 14 in step c with intermediate 31 to obtain compound S45. 1 H NMR(600MHz,Chloroform-d)δ11.03(s,1H),8.19(s,1H),7.95(s,1H),7.85-7.76(m,2H),7.75-7.54(m,4H ),7.45(s,2H),7.24-7.09(m,2H),6.79(s,1H),6.50(d,J=7.7Hz,1H),5.21(s,1H),4.99-4.87(dd,J=12.2, 5.3Hz,1H),4.18(t,J=7.3Hz,2H),3.92(s,3H),3.74(m,2H),3.69-3.61(m,2H),3.45-3.37(m,2H),3.20-3. 08(m,4H),3.04-2.67(m,5H),2.53-2.51(m,2H),2.46-2.41(m,2H),2.27-2.10(m,3H).MS(ESI,m / z):809(M + +1).

[0396] Example 63: Synthesis of compound S46

[0397]

[0398] Compound S46 is synthesized in the same way as compound S1. Simply replace the starting material 54 in step a of Example 18 with 3-azacyclobutane-methanol, and replace intermediate 14 in step c with intermediate 30 to obtain compound S46. 1H NMR(600MHz,Chloroform-d)δ11.03(s,1H),8.19(s,1H),7.95(s,1H),7.85-7.76(m,2H),7.75-7.5 4(m,4H),7.45(s,2H),7.24-7.09(m,2H),6.79(s,1H),6.50(d,J=7.7Hz,1H),5.21(s,1H),4.99-4.8 7(dd,J=12.2,5.3Hz,1H),4.18(t,J=7.3Hz,2H),3.92(s,3H),3.74-3.53(m,6H),3.20-2.96(m,4H), 2.94-2.67(m,3H),2.46(t,J=5.3Hz,4H),2.27-2.10(m,3H),1.61-1.41(m,8H).MS(ESI,m / z):851(M + +1).

[0399] Example 64: Synthesis of compound S47

[0400]

[0401] Compound S47 is synthesized in the same way as compound S1. Simply replace raw material 53 in step a of Example 18 with raw material 64 (prepared by the method disclosed in Example 23 on page 319 of the specification of patent application "WO2020012337"), and replace raw material 54 with 4-hydroxymethylpiperidine to obtain compound S47. 1 H NMR(400MHz,Chloroform-d)δ11.03(s,1H),8.06(s,1H),7.95(s,1H),7.80(d,J=8.5Hz,2H),7.72-7 .63(m,3H),7.58(s,1H),7.50-7.44(m,2H),7.28(d,J=2.4Hz,1H),7.20(d,J=8.5Hz,2H),5.21(s,1H) ,4.94(dd,J=12.3,5.3Hz,1H),3.96(d,J=13.1Hz,2H),3.92(s,3H),3.18-2.66(m,12H),2.52(s,1H) ,2.29(s,2H),2.12(dd,J=9.4,3.9Hz,1H),2.01-1.83(m,5H),1.39-1.25(m,4H).MS(ESI,m / z):828(M + +1).

[0402] Example 65: Synthesis of compound S48

[0403]

[0404] a) The method disclosed in patent application "Synthesis of Example 2 on page 303 of the specification in WO2020012337" was used to prepare raw material 65. 65 (2.0 g, 6.0 mmol), 4-piperidinylmethanol (712.7 mg, 6.0 mmol), Cs₂CO₃ (4.0 g, 12.7 mmol), and PEPPSI-IPr (602.0 mg, 0.6 mmol) were dissolved in 1,4-dioxane (3 mL) solution. The reaction was carried out at 100 °C for 12 h under argon protection. After the reaction, the reaction solution was filtered through diatomaceous earth, the filtrate was concentrated, and purified by column chromatography to obtain intermediate 66 (1.2 g, 57%). MS (ESI, m / z): 358 (M + +1).

[0405] b) Same as step b in Example 5, but by changing the corresponding raw materials, intermediate 67 can be obtained. MS(ESI,m / z):356 (M + +1).

[0406] c) Compound S48 is synthesized using the same steps as compound S1 in Example 18, except that the corresponding raw materials are replaced to obtain compound S48. 1 H NMR(400MHz,Chloroform-d)δ11.03(s,1H),8.06(s,1H),7.95(s,1H),7.80(d,J=8.5Hz,2H),7.72-7.63 (m,3H),7.58(s,1H),7.56-7.51(m,2H),7.20(d,J=8.5Hz,2H),7.05-7.01(m,2H),5.21(s,1H),5.11(dd, J=13.4,5.1Hz,1H),4.55-4.33(m,2H),3.96(d,J=13.1Hz,2H),3.92(s,3H),3.18-2.66(m,12H),2.52(s, 1H),2.29(s,2H),2.12(dd,J=9.4,3.9Hz,1H),2.01-1.83(m,5H),1.39-1.25(m,4H).MS(ESI,m / z):796(M + +1).

[0407] Example 66: Synthesis of compound S49

[0408]

[0409] Compound S49 is synthesized in the same way as compound S48. Simply replace raw material 65 in step a of Example 65 with raw material 68 (prepared by the method disclosed in Example 10 of the specification on page 98 of patent application "WO2024077244") to obtain compound S49. 1 H NMR(400MHz,Chloroform-d)δ11.03(s,1H),8.06(s,1H),7.95(s,1H),7.80(d,J=8.5Hz,2H),7.72-7.63 (m,3H),7.58(s,1H),7.56-7.51(m,2H),7.20(d,J=8.5Hz,2H),7.05-7.01(m,2H),5.25(dd,J=13.4,5.1H z,1H),5.21(s,1H),4.55-4.33(m,2H),3.96(d,J=13.1Hz,2H),3.92(s,3H),3.18-2.66(m,12H),2.52(s, 1H),2.29(s,2H),2.12(dd,J=9.4,3.9Hz,1H),2.01-1.83(m,5H),1.39-1.25(m,4H).MS(ESI,m / z):796(M + +1).

[0410] Example 67: Synthesis of compound S50

[0411]

[0412] Compound S50 is synthesized in the same way as compound S48. Simply replace raw material 65 in step a of Example 65 with raw material 69 (which is prepared by the method disclosed in Example C48018 on page 88 of the specification in patent application WO2021126973) to obtain compound S50. 1H NMR(400MHz,Chloroform-d)δ11.03(s,1H),8.06(s,1H),7.95(s,1H),7.80(d,J=8.5Hz,2H),7.72-7.63(m,3H),7.58 (s,1H),7.50(s,1H),7.43(d,J=1.8Hz,1H),7.18(d,J=8.5Hz,1H),7.20(d,J=8.5Hz,2H),7.15(dd,J=8.6,2.4Hz,1H) ,5.25(dd,J=13.4,5.1Hz,1H),5.21(s,1H),4.55-4.33(m,2H),3.96(d,J=13.1Hz,2H),3.92(s,3H),3.18-2.66(m,12 H),2.52(s,1H),2.29(s,2H),2.12(dd,J=9.4,3.9Hz,1H),2.01-1.83(m,5H),1.39-1.25(m,4H).MS(ESI,m / z):796(M + +1).

[0413] Example 68: Synthesis of compound S51

[0414]

[0415] Compound S51 is synthesized in the same way as compound S47, except that intermediate 14 in step c of Example 64 is replaced with intermediate 26 to obtain compound S51. 1 H NMR(400MHz,Chloroform-d)δ11.03(s,1H),8.06(s,1H),7.95(s,1H),7.80(d,J=8.5Hz,2H),7.72-7.6 3(m,3H),7.58(s,1H),7.48-7.44(m,2H),7.28(d,J=2.4Hz,1H),6.98(d,J=8.5Hz,2H),5.21(s,1H),4.9 4(dd,J=12.3,5.3Hz,1H),3.96(d,J=13.1Hz,2H),3.92(s,3H),3.19(t,J=5.4Hz,4H),3.18-2.66(m,12 H),2.29(s,2H),2.12(dd,J=9.4,3.9Hz,1H),2.01-1.93(m,1H),1.39-1.25(m,4H).MS(ESI,m / z):829(M + +1).

[0416] Example 69: Synthesis of compound S52

[0417]

[0418] Compound S52 is synthesized in the same way as compound S49. Simply replace intermediate 14 in step c of Example 66 with intermediate 26 to obtain compound S52. 1 H NMR(400MHz,Chloroform-d)δ11.03(s,1H),8.06(s,1H),7.95(s,1H),7.80(d,J=8.5Hz,2H),7.72-7.63(m ,3H),7.58(s,1H),7.56-7.51(m,2H),7.05-7.01(m,2H),6.98(d,J=8.5Hz,2H),5.25(dd,J=13.4,5.1Hz,1H ),5.21(s,1H),4.55-4.33(m,2H),3.96(d,J=13.1Hz,2H),3.92(s,3H),3.19(t,J=5.4Hz,4H),3.18-2.66(m ,12H),2.29(s,2H),2.12(dd,J=9.4,3.9Hz,1H),2.01-1.93(m,1H),1.39-1.25(m,4H).MS(ESI,m / z):797(M + +1).

[0419] Example 70: Synthesis of compound S53

[0420]

[0421] a) Same as step a in Example 65, except that starting material 65 is replaced with starting material 69 and 4-piperidinylmethanol is replaced with starting material 57 to obtain intermediate 70. MS(ESI,m / z):429(M + +1).

[0422] b) Same as step b in Example 15, except that intermediate 37 is replaced with intermediate 70 to obtain the hydrochloride salt of intermediate 71. MS(ESI,m / z):329(M + +1).

[0423] c) Same as step c in Example 18, except that intermediate 14 is replaced with intermediate 71 and intermediate 56 is replaced with intermediate 25 to obtain compound S53. 1H NMR(400MHz,Chloroform-d)δ10.86(s,1H),8.30(s,1H),7.94(s,1H),7.76-7.71(m,2H),7.69(d,J=8.5Hz,1H),7.64(dt,J=7.7,3.9Hz,2H),7. 56(s,1H),7.50(s,1H),7.43(d,J=1.8Hz,1H),7.18(d,J=8.5Hz,1H),7.15(dd,J=8.6,2.4Hz,1H),6.98-6.93(m,2H),5.25(dd,J=13.4,5.1Hz,1H ),5.25(s,1H),4.55-4.33(m,2H),3.91(s,3H),3.64(dt,J=12.1,3.6Hz ,2H),3.43(t,J=5.0Hz,3H),3.07(d,J=4.6Hz,3H),2.95-2.63(m,5H),2 .58(t,J=5.1Hz,3H),2.29(d,J=7.1Hz,2H),2.13(m,1H),2.04-1.97(m,1H),1.89(dd,J=9.4,5.7Hz,2H),1.43-1.32(m,4H).MS(ESI,m / z):797(M + +1).

[0424] Example 71: Synthesis of compound S54

[0425]

[0426] Compound S54 is synthesized in the same way as compound S53; simply replace raw material 69 in Example 70 with raw material 68 to obtain compound S54. 1HNMR(400MHz,Chloroform-d)δ10.86(s,1H),8.30(s,1H),7.94(s,1H),7.76-7.71(m,2H),7.69(d,J=8.5Hz,1H),7.64(dt,J=7.7,3 .9Hz,2H),7.56(s,1H),7.54-7.51(m,2H),7.05-7.01(m,2H),6.98-6.93(m,2H),5.25(dd,J=13.4,5.1Hz,1H),5.25(s,1H),4.55-4. 33(m,2H),3.91(s,3H),3.64(dt,J=12.1,3.6Hz,2H),3.43(t,J=5.0Hz,3H),3.07(d,J=4.6Hz,3H),2.95-2.63(m,5H),2.58(t,J=5. 1Hz,3H),2.29(d,J=7.1Hz,2H),2.13(m,1H),2.04-1.97(m,1H),1.89(dd,J=9.4,5.7Hz,2H),1.43-1.32(m,4H).MS(ESI,m / z):797(M + +1).

[0427] Example 72: Synthesis of compound S55

[0428]

[0429] a) Same as step a in Example 18, except that raw material 53 is replaced with raw material 64, and raw material 54 is replaced with raw material 57 to obtain intermediate 72. MS(ESI,m / z):461(M + +1).

[0430] b) Same as step b in Example 15, except that intermediate 37 is replaced with intermediate 72 to obtain the hydrochloride salt of intermediate 73. MS(ESI,m / z):361(M + +1).

[0431] c) Same as step c in Example 18, except that intermediate 14 is replaced with intermediate 73 and intermediate 56 is replaced with intermediate 25 to obtain compound S55. 1H NMR(400MHz,Chloroform-d)δ10.86(s,1H),8.30(s,1H),7.94(s,1H),7.76-7.71(m,2H),7.69(d,J=8.5Hz,1H),7.64(dt,J=7.7 ,3.9Hz,2H),7.56(s,1H),7.47-7.43(m,2H),7.29(d,J=2.3Hz,1H),6.98-6.93(m,2H),5.25(s,1H),4.94(dd,J=12.2,5.4Hz,1H ),3.91(s,3H),3.64(dt,J=12.1,3.6Hz,2H),3.43(t,J=5.0Hz,3H),3.07(d,J=4.6Hz,3H),2.95-2.63(m,5H),2.58(t,J=5.1Hz, 3H),2.29(d,J=7.1Hz,2H),2.13(m,1H),2.04-1.97(m,1H),1.89(dd,J=9.4,5.7Hz,2H),1.43-1.32(m,4H).MS(ESI,m / z):829(M + +1).

[0432] Example 73: Synthesis of compound S56

[0433]

[0434] Compound S56 is synthesized in the same way as compound S6. Simply replace intermediate 14 in step c of Example 23 with intermediate 32 to obtain compound S56. 1 H NMR(400MHz,Chloroform-d)δ11.03(s,1H),8.06(s,1H),7.95(s,1H),7.80(d,J=8.5Hz,2H),7.72-7.63(m, 3H),7.58(s,1H),7.50-7.44(m,2H),7.28(d,J=2.4Hz,1H),7.20(d,J=8.5Hz,2H),7.05(dd,J=8.6,2.4Hz,1 H),5.21(s,1H),4.94(dd,J=12.3,5.3Hz,1H),3.96(d,J=13.1Hz,2H),3.92(s,3H),3.18-2.66(m,9H),2.52 (s,1H),2.29(s,2H),2.12(dd,J=9.4,3.9Hz,1H),2.01-1.83(m,5H),1.39-1.25(m,4H).MS(ESI,m / z):813(M ++1).

[0435] Example 74: Synthesis of compound S57

[0436]

[0437] Compound S57 is synthesized in the same way as compound S6. Simply replace intermediate 14 in step c of Example 23 with intermediate 33 to obtain compound S57. 1 H NMR(400MHz,Chloroform-d)δ11.03(s,1H),8.06(s,1H),7.95(s,1H),7.80(d,J=8.5Hz,2H),7.72-7.63 (m,3H),7.58(s,1H),7.50-7.44(m,2H),7.28(d,J=2.4Hz,1H),7.20(d,J=8.5Hz,2H),7.05(dd,J=8.6,2. 4Hz,1H),5.21(s,1H),4.94(dd,J=12.3,5.3Hz,1H),3.96(d,J=13.1Hz,2H),3.18-2.66(m,9H),2.52(s, 1H),2.29(s,2H),2.12(dd,J=9.4,3.9Hz,1H),2.01-1.83(m,5H),1.39-1.25(m,4H).MS(ESI,m / z):816(M + +1).

[0438] Example 75: Synthesis of compound S58

[0439]

[0440] Compound S58 is synthesized in the same way as compound S6. Simply replace intermediate 14 in step c of Example 23 with intermediate 34 to obtain compound S58. 1H NMR(400MHz,Chloroform-d)δ11.03(s,1H),8.06(s,1H),7.95(s,1H),7.80(d,J=8.5Hz,2H),7.72-7.63 (m,3H),7.58(s,1H),7.50-7.44(m,2H),7.28(d,J=2.4Hz,1H),7.20(d,J=8.5Hz,2H),7.05(dd,J=8.6,2. 4Hz,1H),5.21(s,1H),4.94(dd,J=12.3,5.3Hz,1H),3.96(d,J=13.1Hz,2H),3.18-2.66(m,12H),2.52(s, 1H),2.29(s,2H),2.12(dd,J=9.4,3.9Hz,1H),2.01-1.83(m,5H),1.39-1.25(m,4H).MS(ESI,m / z):813(M + +1).

[0441] Example 76: Synthesis of compound S59

[0442]

[0443] Compound S59 is synthesized in the same way as compound S8; simply replace intermediate 14 in Example 25 with intermediate 33 to obtain compound S59. 1 H NMR (400MHz, Chloroform-d) δ11.03(s,1H),8.21(s,1H),7.95(s,1H),7.80(d,J=8.5Hz,2H),7.72(d,J=4.5Hz,1H),7.65(dd,J=8.5,4 .1Hz,2H),7.58(s,1H),7.50-7.41(m,2H),7.20(d,J=8.5Hz,2H),6.96(d,J=1.8Hz,1H),6.69(dd,J=8.5,1.9Hz,1H),5.26(s,1H),4.9 3(dd,J=12.1,5.4Hz,1H),3.63-3.54(m,1H),3.49(td,J=9.0,8.1,3.9Hz,1H),3.41(q,J=7.9Hz,1H),3.20(dd,J=9.9,7.3Hz,1H),3.1 0-3.05(m,1H),3.00(d,J=6.0Hz,1H),2.95-2.59(m,5H),2.53-2.37(m,3H),2.27-2.07(m,3H),1.90-1.77(m,5H).MS(ESI,m / z):802(M+ +1).

[0444] Example 77: Synthesis of compound S60

[0445]

[0446] The synthesis method of compound S60 is the same as that of compound S8. Compound S60 can be obtained by simply replacing intermediate 14 in step c of Example 25 with intermediate 32. 1 H NMR (400MHz, Chloroform-d) δ11.03(s,1H),8.21(s,1H),7.95(s,1H),7.80(d,J=8.5Hz,2H),7.72(d,J=4.5Hz,1H),7.65(dd,J=8.5,4.1H z,2H),7.58(s,1H),7.50-7.41(m,2H),7.20(d,J=8.5Hz,2H),6.96(d,J=1.8Hz,1H),6.69(dd,J=8.5,1.9Hz,1H),5.26(s,1H),4.93(dd,J =12.1,5.4Hz,1H),3.91(s,3H),3.63-3.54(m,1H),3.49(td,J=9.0,8.1,3.9Hz,1H),3.41(q,J=7.9Hz,1H),3.20(dd,J=9.9,7.3Hz,1H),3 .10-3.05(m,1H),3.00(d,J=6.0Hz,1H),2.95-2.59(m,5H),2.53-2.37(m,3H),2.27-2.07(m,3H),1.90-1.77(m,5H).MS(ESI,m / z):799(M + +1).

[0447] Example 78: Synthesis of compound S61

[0448]

[0449] The synthesis method of compound S61 is the same as that of compound S8. Compound S61 can be obtained by simply replacing intermediate 14 in step c of Example 25 with intermediate 34. 1H NMR (400MHz, Chloroform-d) δ11.03(s,1H),8.21(s,1H),7.95(s,1H),7.80(d,J=8.5Hz,2H),7.72(d,J=4.5Hz,1H),7.65(dd,J=8.5,4 .1Hz,2H),7.58(s,1H),7.50-7.41(m,2H),7.20(d,J=8.5Hz,2H),6.96(d,J=1.8Hz,1H),6.69(dd,J=8.5,1.9Hz,1H),5.26(s,1H),4.9 3(dd,J=12.1,5.4Hz,1H),3.63-3.54(m,1H),3.49(td,J=9.0,8.1,3.9Hz,1H),3.41(q,J=7.9Hz,1H),3.20(dd,J=9.9,7.3Hz,1H),3.1 3-3.05(m,4H),3.00(d,J=6.0Hz,1H),2.95-2.59(m,5H),2.53-2.37(m,3H),2.27-2.07(m,3H),1.90-1.77(m,5H).MS(ESI,m / z):799(M + +1).

[0450] Example 79: Synthesis of compound S62

[0451]

[0452] The synthesis method of compound S62 is the same as that of compound S9. Compound S62 can be obtained by simply replacing intermediate 14 in step c of Example 26 with intermediate 33. 1H NMR(400MHz,Chloroform-d)δ11.03(s,1H),8.11(s,1H),7.95(s,1H),7.81(d,J=8.4Hz,2H),7.74-7.62(m,3H),7.58(s ,1H),7.49-7.43(m,2H),7.21(d,J=8.4Hz,2H),6.97(d,J=2.2Hz,1H),6.69(dd,J=8.5,2.2Hz,1H),5.23(s,1H),4.94(d d,J=12.0,5.3Hz,1H),3.64-3.56(m,1H),3.54-3.47(m,1H),3.46-3.38(m,1H),3.25-3.17(m,1H),3.09-3.03(m,1H),3 .00(d,J=10.2Hz,1H),2.95-2.59(m,5H),2.57-2.36(m,3H),2.23-2.08(m,3H),1.93-1.76(m,5H).MS(ESI,m / z):802(M + +1).

[0453] Example 80: Synthesis of compound S63

[0454]

[0455] The synthesis method of compound S63 is the same as that of compound S9. Compound S63 can be obtained by simply replacing intermediate 14 in step c of Example 26 with intermediate 32. 1H NMR(400MHz,Chloroform-d)δ11.03(s,1H),8.11(s,1H),7.95(s,1H),7.81(d,J=8.4Hz,2H),7.74-7.62(m,3H),7.58(s,1 H),7.49-7.43(m,2H),7.21(d,J=8.4Hz,2H),6.97(d,J=2.2Hz,1H),6.69(dd,J=8.5,2.2Hz,1H),5.23(s,1H),4.94(dd,J=1 2.0,5.3Hz,1H),3.92(s,3H),3.64-3.56(m,1H),3.54-3.47(m,1H),3.46-3.38(m,1H),3.25-3.17(m,1H),3.09-3.03(m,1H ),3.00(d,J=10.2Hz,1H),2.95-2.59(m,5H),2.57-2.36(m,3H),2.23-2.08(m,3H),1.93-1.76(m,5H).MS(ESI,m / z):799(M + +1).

[0456] Example 81: Synthesis of compound S64

[0457]

[0458] Compound S63 is synthesized in the same way as compound S9. Compound S64 can be obtained simply by replacing intermediate 14 in step c of Example 26 with intermediate 34. 1H NMR(400MHz,Chloroform-d)δ11.03(s,1H),8.11(s,1H),7.95(s,1H),7.81(d,J=8.4Hz,2H),7.74-7.62(m,3H),7.58(s,1 H),7.49-7.43(m,2H),7.21(d,J=8.4Hz,2H),6.97(d,J=2.2Hz,1H),6.69(dd,J=8.5,2.2Hz,1H),5.23(s,1H),4.94(dd,J=1 2.0,5.3Hz,1H),3.92(s,3H),3.64-3.56(m,1H),3.54-3.47(m,1H),3.46-3.38(m,1H),3.25-3.17(m,1H),3.09-3.03(m,1H ),3.00(d,J=10.2Hz,1H),2.95-2.59(m,5H),2.57-2.36(m,3H),2.23-2.08(m,3H),1.93-1.76(m,5H).MS(ESI,m / z):799(M + +1).

[0459] Example 82: Synthesis of compound S65

[0460]

[0461] Compound S65 was synthesized using the same method as compound S25 in Example 42, except that intermediate 26 in step c of Example 42 was replaced with intermediate 74 to obtain compound S65. MS (ESI, m / z): 803 (M + +1).

[0462] The synthesis method of intermediate 74 is the same as that of intermediate 14 in Example 3. The intermediate 74 can be obtained by simply replacing methylamine hydrochloride in step a of Example 3 with deuterated methylamine hydrochloride and replacing intermediate 5 in step c with intermediate 3. 1H NMR(600MHz,Chloroform-d)δ10.87(s,1H),8.32(s,1H),7.93(d,J=2.5Hz,1H),7.74(d,J=8.2Hz,2H),7.70 (s,1H),7.64(t,J=6.6Hz,2H),7.56(s,1H),1.49-7.39(m,2H),6.97-6.88(m,3H),6.67(d,J=8.4Hz,1H),4.9 3(dd,J=12.0,5.9Hz,1H),3.58(t,J=8.9Hz,1H),3.49(s,1H),3.46-3.33(m,1H),3.22-3.12(m,5H),2.92-2 .55(m,8H),2.52-2.41(m,2H),2.26-2.17(m,1H),2.14-2.08(m,1H),1.88-1.79(m,1H).MS(ESI,m / z):464(M + +1).

[0463] Example 83: Synthesis of compound S66

[0464]

[0465] Compound S66 is synthesized in the same way as compound S26 in Example 43. Simply replace intermediate 26 in step c of Example 43 with intermediate 74 to obtain compound S66. 1 H NMR(400MHz,Chloroform-d)δ10.87(s,1H),8.08(s,1H),7.94(s,1H),7.75(d,J=9.0Hz,2H),7.70-7.63(m ,3H),7.57(s,1H),7.47-7.43(m,2H),6.98-6.90(m,3H),6.69(dd,J=8.5,2.2Hz,1H),5.20(s,1H),4.93(dd ,J=12.1,5.4Hz,1H),3.64-3.56(m,1H),3.54-3.47(m,1H),3.47-3.38(m,1H),3.26-3.14(m,5H),2.93-2. 58(m,8H),2.54-2.42(m,2H),2.28-2.18(m,1H),2.17-2.08(m,1H),1.92-1.80(m,1H).MS(ESI,m / z):803(M + +1).

[0466] Example 84: Evaluation of the activity of the compounds of the present invention against HPK1 kinase

[0467] 1. Experimental Methods

[0468] The HPK1 kinase domain (1-307aa) was dissolved in a kinase buffer (20mM HEPES pH 7.5, 10mM MgCl2, 1mM EGTA, 0.5mM TCEP, 0.01% BriJ-35, 0.05% BSA) and added to a 96-well plate to a final concentration of 20 nM. Then, serially diluted test compounds of the present invention (10 concentration points for each compound) were added. The HPK1 kinase was pre-incubated with the compounds of the present invention for 30 min. Then, 20 μL of substrate solution (final concentration of 0.5 mg / mL SLP76-SH2 domain and 10 μM ATP) was added to each well to initiate the enzymatic reaction. The reaction was incubated at room temperature for 1 h. The inhibition rate of the compounds of the present invention against HPK1 kinase activity at each concentration was determined according to the Luminescent Kinase Assay Kit (Beyotime Biotechnology Company) instructions. The IC50 of the inhibitory activity of the compounds of the present invention against HPK1 kinase was calculated using GraphPad fitting. 50 Values. The experimental results are shown in Table 1.

[0469] 2. Experimental Results

[0470] Table 1. Kinase inhibitory activity of the compounds of the present invention against HPK1 protein (IC50). 50 nM)

[0471]

[0472]

[0473] As shown in Table 1, using compound 7 of the example disclosed on page 114 of patent WO2023001794 as a positive control, the compound of the present invention has essentially the same activity as the positive control. This indicates that the compound of the present invention basically maintains HPK1 inhibitory activity and has good binding to HPK1 protein.

[0474] Example 85: Evaluation of the degradation activity and selectivity of the compounds of the present invention against HPK1 protein.

[0475] 1. Experimental Methods

[0476] (1) Cell collection: Jurkat cells were cultured adherently for 12 hours, and then different concentrations of some compounds of the present invention were co-incubated with the cells for 12 hours.

[0477] (2) Protein extraction: Discard the original culture medium, wash the cells three times with pre-cooled PBS, add an appropriate amount of lysis buffer, mix well and place on ice for lysis for 30 min, centrifuge at 12000 rpm for 5 min at 4℃, transfer the supernatant after centrifugation to a new centrifuge tube for quantification.

[0478] (3) Protein quantification: Protein was quantified using the BCA method. Solution A and solution B were mixed at a ratio of 50:1 to obtain the BCA working solution. 100 μL of BCA working solution, 9 μL of triple-distilled water, and 1 μL of protein sample were added to a 96-well plate, mixed, and incubated at 37°C for 30 min. The absorbance was measured using a full-wavelength microplate reader, and the protein concentration was calculated based on the standard curve. The quantified protein was mixed with 5× loading buffer at a ratio of 4:1, denatured in boiling water for 8 min, cooled on ice, and stored at -20°C for later use.

[0479] (4) Prepare SDS-PAGE gel. The voltage of the stacking gel is 90V and the voltage of the separating gel is 120V. At 4℃, the membrane is transferred at 100V for 1 hour. After the transfer is completed, the protein membrane is immediately placed in the prepared 5% BSA solution and shaken slowly on a shaker. The membrane is then blocked at room temperature for 1 hour.

[0480] (5) Refer to the instructions of HPK1 Antibody (4472SCST), GLK (D1L4G) RabbitmAb (92427SCST), HGK Antibody (3485SCST), RabbitmAb (92711TCST), and β-actin Mouse Monoclonal antibody (BE0021 easybio) to dilute the primary antibody at the appropriate ratio, incubate overnight at 4°C with gentle shaking, and then wash 3 times with PBST for 10 min each time.

[0481] (6) Select Goat Anti-Rabbit IgG(H&L)-HRP Conjugated (BE0101easybio) or Goat Anti-Mouse IgG(H&L)-HRP Conjugated (BE0102 easybio) according to the primary antibody. Refer to the instructions for the secondary antibody, dilute the secondary antibody at an appropriate ratio, and incubate at room temperature with gentle shaking for 1 hour. Wash with PBST 3 times, 10 min each time.

[0482] (7) Add ECL luminescent solution, observe the results using a developer, quantify the Western blotting results using ImageJ software, calculate the protein degradation rates of HPK1, GLK, and HGK by comparing with the control group without added compounds, and calculate the degradation rates of DC using GraphPad fitting. 50The maximum degradation rate (Dmax) of HPK1 was the degradation rate at a concentration of 1 μM. The experimental results are shown in Table 2.

[0483] 2. Experimental Results

[0484] Table 2. Degradation activity and selectivity of the compounds of this invention against HPK1.

[0485]

[0486]

[0487] As shown in Table 2, the compounds of the present invention have good degradation activity against HPK1 protein, while the compounds of the present invention have no degradation activity against GLK and HGK proteins of the same family at 10 μM. This indicates that the compounds of the present invention can efficiently and selectively degrade HPK1 protein.

[0488] Example 86: Evaluation of T cell cytokine release

[0489] 1. Experimental Methods

[0490] HPK1 kinase inhibits the release of T cell effector cytokines; therefore, the ability of the compound of this invention to enhance T cell cytokine release was detected by ELISA. Human PBMCs were thawed and resuspended in RPMI 1640 (Gibco) medium containing 10% bovine placental serum, and the cells were seeded in 96-well plates (1×10⁻⁶). 5 ( / well). Serially diluted compounds of the present invention were added to 96-well plates and incubated at 37°C, 5% CO2 for 1 h. PBMCs were activated with 100 μL of anti-CD3 / CD28 antibody (final concentration 1 μg / mL), and then incubated at 37°C, 5% CO2 for 48 h. The supernatant was diluted 8-fold with PBS, and 100 μL of the diluted supernatant was used to detect the release of IL-2 at each concentration of the compound of the present invention using a human IL-2 ELISA kit (4abio). The EC50 of the compound of the present invention was calculated using GraphPad fitting. 50 Values. The experimental results are shown in Table 3.

[0491] 2. Experimental Results

[0492] Table 3. Results of the assay for the activity of the compounds of the present invention in stimulating the release of IL-2 cytokine from PBMCs.

[0493]

[0494] As shown in Table 3, using compound 7 of the example disclosed on page 114 of patent WO2023001794 as a positive control, the compound of the present invention significantly enhanced the release of IL-2 from PBMC cells, and was superior to the positive control compound. This indicates that the compound of the present invention can enhance the release of T cell effector cytokines by degrading HPK1 protein, thereby restoring or enhancing the anti-tumor immune response.

Claims

1. A pyrazinamide compound of formula I, or a pharmaceutically acceptable salt, enantiomer, diastereomer, cis-trans isomer, tautomer, trans-blocker isomer, conformational isomer, metabolite, prodrug, solvate, deuterated product, or hydrate thereof, characterized in that, In Formula I: R 1 C 1-4 Alkyl, C 3-6 cycloalkyl or deuterated (C 1-4 alkyl); R 2 C 1-4 Alkyl or deuterated (C 1-4 alkyl); B is C 3-10 Cycloalkyl, 4-10 membered monocyclic heterocyclic, 6-20 membered spirocyclic, 6-18 membered fused heterocyclic or 7-10 membered bridged heterocyclic, wherein the 4-10 membered monocyclic, 6-20 membered spirocyclic, 6-18 membered fused heterocyclic or 7-10 membered bridged heterocyclic contains at least one, two or three heteroatoms selected from nitrogen, oxygen or sulfur; E is R 3 For deuterium, halogen, cyano, C 1-4 Alkyl, halogenated (C 1-4 Alkyl), C 1-4 Alkoxy or halogenated (C 1-4 Alkyl groups); a can be 0, 1, 2, or 3; W is -CH2-, -C(CH3)2 or L1 represents a chemical bond, C 3-10 Cycloalkylene, 4-10-membered monocyclic heterocyclic, 6-20-membered spirocyclic, 6-18-membered fused heterocyclic or 7-10-membered bridged heterocyclic, wherein the 4-10-membered monocyclic, 6-20-membered spirocyclic, 6-18-membered fused heterocyclic or 7-10-membered bridged heterocyclic contains at least one, two or three heteroatoms selected from nitrogen, oxygen or sulfur; L2 is a chemical bond. -(CH2)r1-、C 3-10 Cycloalkylene, 4-10-membered monocyclic heterocyclic, 6-20-membered spirocyclic, 6-18-membered fused heterocyclic or 7-10-membered bridged heterocyclic, wherein the 4-10-membered monocyclic, 6-20-membered spirocyclic, 6-18-membered fused heterocyclic or 7-10-membered bridged heterocyclic contains at least one, two or three heteroatoms selected from nitrogen, oxygen or sulfur; L3 is a chemical bond. -(CH2)r2-、C 3-10 Cycloalkylene, 4-10-membered monocyclic heterocyclic, 6-20-membered spirocyclic, 6-18-membered fused heterocyclic or 7-10-membered bridged heterocyclic, wherein the 4-10-membered monocyclic, 6-20-membered spirocyclic, 6-18-membered fused heterocyclic or 7-10-membered bridged heterocyclic contains at least one, two or three heteroatoms selected from nitrogen, oxygen or sulfur; L4 is a chemical bond. -O-、-(CH2)r3-、-(CH2)r4(C=O)-、C 3-10 Cycloalkylene, 4-10-membered monocyclic heterocyclic, 6-20-membered spirocyclic, 6-18-membered fused heterocyclic or 7-10-membered bridged heterocyclic, wherein the 4-10-membered monocyclic, 6-20-membered spirocyclic, 6-18-membered fused heterocyclic or 7-10-membered bridged heterocyclic contains at least one, two or three heteroatoms selected from nitrogen, oxygen or sulfur; r1 is 0, 1, 2, 3, 4, 5 or 6; r2 is 0, 1, 2, 3, 4, 5 or 6; r3 is 0, 1, 2, 3, 4, 5 or 6; r4 can be 0, 1, 2, 3, 4, 5, or 6.

2. The pyrazinamide compound according to claim 1, or its pharmaceutically acceptable salt, enantiomer, diastereomer, cis-trans isomer, tautomer, trans-blocking isomer, conformational isomer, metabolite, prodrug, solvate, deuterated product, or hydrate, characterized in that, R 1 It can be methyl, ethyl, cyclopropyl, or -CD3; And / or, R 2 It can be methyl, ethyl, or -CD3; And / or, B is piperidinyl, piperazineyl, 3. The pyrazinamide compound according to claim 1, or its pharmaceutically acceptable salt, enantiomer and diastereomer, cis-trans isomer, tautomer, trans-blocking isomer, conformational isomer, metabolite, prodrug, solvate, deuterated product, or hydrate, characterized in that, E is R 3 It is fluorine; a is 0 or 1; W is -CH2- or 4. The pyrazinamide compound according to claim 1, or its pharmaceutically acceptable salt, enantiomer, diastereomer, cis-trans isomer, tautomer, trans-blocking isomer, conformational isomer, metabolite, prodrug, solvate, deuterated product, or hydrate, characterized in that, L1 is a chemical bond. And its left side is connected to L2, and its right side is connected to E; And / or, L2 is a chemical bond, -(CH2) r1 -、C 4-6 Cycloalkylene Furthermore, the left side is connected to L3, and the right side is connected to L1; And / or, L3 is a chemical bond, -(CH2) r2 -、C 4-6 Cycloalkylene Furthermore, it is connected to L4 on the left and to L2 on the right. And / or, L4 is a chemical bond, -(CH2) r3 -、-(CH2) r4 (C=O)-、 Furthermore, it is connected to B on the left and to L3 on the right. And / or, n1 is 1, 2, 3 or 4; And / or, n2 is 1, 2, 3 or 4; And / or, n3 is 1, 2 or 3; And / or, n4 is 1, 2 or 3; And / or, n5 is 1, 2 or 3; And / or, n6 is 1, 2 or 3; And / or, n7 is 0, 1, 2 or 3; And / or, n8 is 0, 1, 2, or 3; And / or, n9 is 0, 1, 2 or 3; And / or, n10 is 0, 1, 2 or 3; And / or, Q 1 For CR Q1 Or N, R Q1 Halogen, hydroxyl or C 1-4 alkyl; And / or, Q is CH or N; And / or, Q 2 For CH or N; And / or, Q 3 For CH or N; And / or, R a Halogen, -(C=O) or C 1-3 alkyl; And / or, c is 0, 1, 2 or 3; And / or, r1 is 0, 1, 2, 3 or 4; And / or, r2 is 0, 1, 2, 3 or 4; And / or, r3 is 0, 1, 2, 3 or 4; And / or, r4 is 0, 1, 2, 3 or 4.

5. The pyrazinamide compound according to claim 1, or its pharmaceutically acceptable salt, enantiomer, diastereomer, cis-trans isomer, tautomer, trans-blocking isomer, conformational isomer, metabolite, prodrug, solvate, deuterated product, or hydrate, characterized in that, L1 is a chemical bond. And its left side is connected to L2, and its right side is connected to E; And / or, L2 is a chemical bond, -(CH2)r1-, Furthermore, the left side is connected to L3, and the right side is connected to L1; And / or, L3 is a chemical bond, -(CH2)r2, Furthermore, it is connected to L4 on the left and to L2 on the right. And / or, L4 is a chemical bond, -(CH2)r3 or -(CH2)r4(C=O)-; and the left side is connected to B, and the right side is connected to L3; And / or, n1 is 1, 2 or 3; And / or, n2 is 1, 2 or 3; And / or, n3 is 1 or 2; And / or, n4 is 1 or 2; And / or, n5 is 1 or 2; And / or, n6 is 1 or 2; And / or, n7 is 0, 1, or 2; And / or, n8 is 0, 1, or 2; And / or, n9 is 0, 1, or 2; And / or, n10 is 0, 1, or 2; And / or, Q 1 For CR Q1 Or N, R Q1 It can be fluorine, hydroxyl, or methyl; And / or, Q is CH or N; And / or, Q 2 For CH or N; And / or, Q 3 For CH or N; And / or, R a It is fluorine or -(C=O); And / or, c is 0, 1, or 2; And / or, r1 is 0, 1, 2 or 3; And / or, r2 is 0, 1, 2 or 3; And / or, r3 is 0, 1, 2, 3 or 4; And / or, r4 is 0, 1, 2, 3 or 4.

6. The pyrazinamide compound according to claim 1, or its pharmaceutically acceptable salt, enantiomer, diastereomer, cis-trans isomer, tautomer, trans-blocking isomer, conformational isomer, metabolite, prodrug, solvate, deuterated product, or hydrate, characterized in that, -L4-L3-L2-L1- represents a chemical bond. And its left side is connected to B, and its right side is connected to E; n1 is 1 or 2; n2 is 1 or 2; n3 is 1 or 2; n4 is 1 or 2; n5 is 1 or 2; n6 is 1 or 2; n7 is 0, 1, or 2; n8 is 0, 1, or 2; n9 can be 0, 1, or 2; n10 is 0, 1, or 2; Q 1 For N or CR Q1 R Q1 It is hydrogen or fluorine; Q is CH or N; Q 2 For CH or N; Q 3 For CH or N; R a It is fluorine; c is 0, 1, or 2; r1 is 0, 1, or 2; r2 is 0, 1, or 2; r3 can be 0, 1, 2, or 3; r4 can be 0, 1, or 2.

7. The pyrazinamide compound according to claim 1, or its pharmaceutically acceptable salt, enantiomer, diastereomer, cis-trans isomer, tautomer, trans-blocking isomer, conformational isomer, metabolite, prodrug, solvate, deuterated product, or hydrate, characterized in that, L1 is a chemical bond. And its left side is connected to L2, and its right side is connected to E; And / or, L2 is a chemical bond, -(CH2)r1-, Furthermore, the left side is connected to L3, and the right side is connected to L1; r1 is 0, 1, or 2; And / or, L3 is a chemical bond, -(CH2)r 2、 Furthermore, it is connected to L4 on the left and to L2 on the right. r2 is 0, 1, or 2; And / or, L4 is a chemical bond, -(CH2)r3- or -(CH2)r4(C=O)-; and the left side is connected to L3, and the right side is connected to E; r3 can be 0, 1, 2, or 3; r4 can be 0, 1, 2, or 3.

8. The pyrazinamide compound according to claim 1, or its pharmaceutically acceptable salt, enantiomer, diastereomer, cis-trans isomer, tautomer, trans-blocking isomer, conformational isomer, metabolite, prodrug, solvate, deuterated product, or hydrate, characterized in that, -L1-L2-L3-L4- represents a chemical bond. And its left side is connected to E, and its right side is connected to B; r2 is 1 or 2; r3 can be 0, 1, 2, or 3; r4 is 1 or 2.

9. The pyrazinamide compound according to claim 1, or its pharmaceutically acceptable salt, enantiomer, diastereomer, cis-trans isomer, tautomer, trans-blocking isomer, conformational isomer, metabolite, prodrug, solvate, deuterated product, or hydrate, characterized in that, It is a compound represented by formula I-1, I-2, I-2A', I-3 or I-4, or a pharmaceutically acceptable salt, enantiomer, diastereomer, cis-trans isomer, tautomer, trans-blocker isomer, conformational isomer, metabolite, prodrug, solvate, deuterated product or hydrate thereof. in: Ring B' is a nitrogen-containing heterocyclic group; Ring L3' is a nitrogen-containing heterocyclic group; R 1 R 2 The definitions of L1, L2, L3, B, E, r2, r3, and r4 are as described in any one of claims 1-8.

10. The pyrazinamide compound according to claim 1, or its pharmaceutically acceptable salt, enantiomer, diastereomer, cis-trans isomer, tautomer, trans-blocking isomer, conformational isomer, metabolite, prodrug, solvate, deuterated product, or hydrate, characterized in that, Compounds selected from any of the following:

11. A method for preparing a pyrazinamide compound of formula I-2 as described in claim 9, or a pharmaceutically acceptable salt, enantiomer, diastereomer, cis-trans isomer, tautomer, transisomer, conformational isomer, metabolite, prodrug, solvate, or hydrate thereof, characterized in that, Includes the following steps: In a solvent, the compound of formula I-2A or its salt reacts with the compound of formula I-2B via a reductive amination reaction to obtain the compound of formula I-2; The compound of formula I-2 obtained by the above method is salted with a pharmaceutically acceptable acid to obtain the pharmaceutically acceptable salt. in: r3-1 is either 0 or 1; X is a single bond or CH; Ring B', Ring L3', R 1 R 2 The definitions of L1, L2, E, and r3 are as described in any one of claims 1-8.

12. A pharmaceutical composition comprising a therapeutically effective amount of a pyrazinamide compound as described in any one of claims 1-10, or a pharmaceutically acceptable salt, enantiomer, diastereomer, cis-trans isomer, tautomer, transisomer, conformational isomer, metabolite, prodrug, solvate, or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

13. The use of a pyrazine carboxamide compound according to any one of claims 1-10, or a pharmaceutically acceptable salt, enantiomer, diastereomer, cis-trans isomer, tautomer, transisomer, conformational isomer, metabolite, prodrug, solvate, or hydrate thereof, or the use of the pharmaceutical composition according to claim 12 in the preparation of a medicament for regulating the ubiquitination and degradation of hematopoietic progenitor cell kinase 1.

14. The use of a pyrazine carboxamide compound according to any one of claims 1-10, or a pharmaceutically acceptable salt, enantiomer, diastereomer, cis-trans isomer, tautomer, transisomer, conformational isomer, metabolite, prodrug, solvate, or hydrate of the thereof, or the use of the pharmaceutical composition of claim 12 in the preparation of a medicament for the treatment and / or prevention of cancer.

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