BET protein targeted degradation compounds, methods of making and uses thereof

Abstract

A BET protein targeted degradation compound and a preparation method and use thereof. The application discloses a compound shown in general formula (I), a stereoisomer, a pharmaceutically acceptable salt, a solvate or a prodrug thereof, wherein A, L and B are defined in the application. The application further discloses a preparation method of the compound, a pharmaceutical composition containing the compound, the stereoisomer, the pharmaceutically acceptable salt, the solvate or the prodrug thereof as an active substance, and application of the compound in tumor diseases, wherein the compound with formula (I) is a bromodomain protein (BRD2 / 3 / 4) degradation product for treating cancer, can selectively induce degradation of BET proteins, and preliminary drug activity screening shows that the anti-tumor activity of the compound is better than that of a control, and pharmacodynamic evaluation results show that the compound has a significant anti-tumor effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a class of compounds with BET protein-targeting degradation activity, their stereoisomers, pharmaceutically acceptable salts, solvent compounds or prodrug compounds, pharmaceutical compositions containing such compounds as active substances, and methods for preparing and using the compounds. Background Technology

[0002] Proteolysis-Targeting Chimeras (PROTACs) technology uses a linker to connect the ligand of the target protein with the recruitment ligand of the E3 ubiquitin ligase, driving the ubiquitination and degradation of the target protein based on the proteasome. One of the biggest advantages of PROTACs technology is that it can transform the target from "undruggable" to "drug-friendly," allowing it to capture the target protein through any corner or gap. Compared with inhibitors, PROTACs degradative agents can be administered at lower doses, thus reducing side effects. Furthermore, they can be recycled to some extent in vivo, prolonging the drug's efficacy.

[0003] BET stands for bromodomain and extraterminal domain. The BET family of proteins consists of four members: BRD2, BRD3, BRD4, and BRDT, with BRD4 having three different splice variants. BET family proteins are important epigenetic and transcriptional regulatory proteins. Several important proto-oncogenes, such as MYC, BCL2, and CDK6, are regulated by BET proteins, playing a crucial role in normal cell growth and cell cycle progression. BET family proteins have a close relationship with tumors. For example, BRD4 transposes in midline carcinomas and forms a fusion gene with the NUT gene, thereby promoting tumorigenesis.

[0004] Using PROTACs technology to degrade BET proteins (mainly BRD2 and BRD4) is currently a hot research topic for major pharmaceutical companies and research institutions. PROTACs technology can effectively overcome the weakness of small molecule inhibitors in not being able to completely inhibit the activity of target proteins. By introducing E3 ligases to target and degrade target proteins, it is a "triggered" mechanism of action, which does not require occupying the active site at all times to inhibit the activity of target proteins, as small molecule inhibitors do.

[0005] While PROTACs technology is a powerful tool for anti-tumor drug development, its efficacy and safety still need improvement, and currently no drugs are marketed. More importantly, although more than 600 E3 enzymes have been discovered in the human body, the E3 enzymes used for PROTAC design are limited to those targeting VHL, CRBN, IAPs, and MDM2, thus restricting the targetable degradation substrates. Drugability and how to expand to more E3 enzymes are significant challenges for breakthrough research in this field. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, the present invention aims to provide a novel compound with good anti-tumor activity that targets the degradation of BET protein.

[0007] Another object of the present invention is to provide a pharmaceutical composition having a BET protein-targeting degradation compound.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] This invention provides a compound of formula (I), its stereoisomers, pharmaceutically acceptable salts, solvent compounds, or prodrugs:

[0010]

[0011] A represents the BET protein ligand, B represents the E3 ubiquitin ligase ligand, and L represents the linker chain connecting A and B.

[0012] The structure of the BET protein ligand A is as follows:

[0013] .

[0014] The ligand B of the E3 ubiquitin ligase is the structure shown in formula B1', B2', or B3' or its stereoconfiguration:

[0015]

[0016] Among them, Cy1, Cy2, Cy3, Cy4, Cy5, and Cy6 are the same or different, and are independently replaced by no substitution or optionally by one or more R. a The following groups are substituted: C 6-14 aryl, 3- to 14-membered heterocyclic, 5- to 14-membered heteroaryl;

[0017] R b11 R b12 R b21 R b31 R b32 R b33 R b34 Rb35 R b36 R b37 R b38 R b39 R b310 They may be identical or different, each independently selected from: -H, deuterium, halogen, -OH, -NO2, -NH2, -CN, -COOH, unsubstituted or optionally substituted by one or more R b The following groups are substituted: C 1-10 Alkyl, C 1-10 Alkoxy, C 2-10 alkenyl, C 2-10 alkynyl group, C 1-10 Alkyl acyl, C 1-10 Alkyl acyloxy, C 3-10 Cycloalkyl, 3- to 14-membered heterocyclic groups, C 6-14 Aryl, 5- to 14-membered heteroaryl, C 1-10 Alkylamino, C 1-10 Alkylamide group, C 1-10 alkylsulfonyl, C 6-14 Arylsulfonyl, C 1-10 alkyl thionyl, C 6-14 Arylthionyl;

[0018] R a R b Same or different, each independently selected from: deuterium, halogen, -OH, -NO2, -NH2, -CN, -COOH, -CHO, -C(O)NH2, C 1-10 Alkyl, C 1-10 Alkoxy, C 2-10 alkenyl, C 2-10 alkynyl group, C 3-10 Cycloalkyl.

[0019] According to embodiments of the present invention, Cy1, Cy2, Cy3, Cy4, Cy5, and Cy6 may be the same or different, and are independently replaced without substitution or optionally replaced by one or more R. a The following groups are substituted: C 6-10 aryl, 3- to 10-membered heterocyclic, 5- to 10-membered heteroaryl;

[0020] R b11 R b12 R b21 R b31 R b32 R b33 R b34 R b35 R b36 R b37 R b38 R b39 Rb310 They may be identical or different, each independently selected from: -H, deuterium, halogen, -OH, -NO2, -NH2, -CN, -COOH, unsubstituted or optionally substituted by one or more R b The following groups are substituted: C 1-5 Alkyl, C 1-5 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-5 Alkyl acyl, C 1-5 Alkyl acyloxy, C 3-6 cycloalkyl, 3- to 7-membered heterocyclic groups, C 6-8 Aryl, 5- to 7-membered heteroaryl, C 1-3 Alkylamino, C 1-3 Alkylamide group, C 1-3 alkylsulfonyl, C 6-10 Arylsulfonyl, C 1-3 alkyl thionyl, C 6-10 Arylthionyl;

[0021] R a R b Same or different, each independently selected from: deuterium, halogen, -OH, -NO2, -NH2, -CN, -COOH, -CHO, -C(O)NH2, C 1-5 Alkyl, C 1-5 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 Cycloalkyl.

[0022] Preferably, Cy1, Cy2, and Cy5 may be the same or different, and are each independently selected from:

[0023] ;

[0024] Cy4 is selected from:

[0025] ;

[0026] Cy3 and Cy6 may be the same or different, and are selected independently from:

[0027] .

[0028] More preferably, the ligand B of the E3 ubiquitin ligase has the structure shown below:

[0029] .

[0030] The connecting chain L connecting A and B described in this invention has the structure shown in formulas L1', L2', L3', or L4':

[0031]

[0032] Among them, X is connected to A, and Y is connected to B;

[0033] n1, n2, n3, and n4 may be the same or different, and each is independently selected from integers from 0 to 10;

[0034] m1, m2, m3, and m4 may be the same or different, and each is independently selected from integers from 0 to 10;

[0035] p1 and p2 may be the same or different, and are each independently selected from integers from 0 to 10;

[0036] q1, q2, q3, and q4 may be the same or different, and are each independently selected from integers between 0 and 10;

[0037] X and Y may be the same or different, and are independently selected from: -O-, -S-, -NR-, -CRR'-, -C(O)-, -NHC(O)-, -C(O)NH-, -C(O)O-, -S(O)NH-, -NHS(O)-;

[0038] R1 is selected from: -O-, -S-, -N(R)-, -N(R)N(R')-, -C(O)-, -NHC(O)-, -C(O)NH-, -C(O)O-, -S(O)NH-, -NHS(O)-;

[0039] R2 is selected from: -O-, -S-, -N(R)-, -N(R)N(R')-, -C(O)-, -NHC(O)-, -C(O)NH-, -C(O)O-, -S(O)NH-, -NHS(O)-;

[0040] R3 is selected from: -NRR';

[0041] R4 is selected from: -N(R)-, -N(R)N(R')-, -NHC(O)-, -C(O)NH-, -C(O)O-, -S(O)NH-, -NHS(O)-;

[0042] R and R' may be the same or different, and are independently selected from: H and C. 1-5 Alkyl, C 1-5 Halogenated alkyl, C 1-5 Alkoxy, C 2-5 alkenyl, C 2-5 Alkyne group, P(O)(OR'')(OR''')-(CH2) m -C(O)-, where R'' and R''' are the same or different, and are independently selected from C. 1-5 Alkyl, -H, Halogenated C 1-5 Alkyl, C2-6 alkenyl, C 2-6 Alkyne group, m is selected from integers from 1 to 5;

[0043] Or R, R' and the adjacent N form a 5- to 7-membered heterocyclic group.

[0044] Preferably, n1, n2, n3, and n4 are the same or different, and are each independently selected from integers from 0 to 6;

[0045] m1, m2, m3, and m4 may be the same or different, and each is independently selected from integers from 0 to 5;

[0046] p1 and p2 may be the same or different, and each is independently selected from integers from 0 to 5;

[0047] q1, q2, q3, and q4 may be the same or different, and are each independently selected from integers between 0 and 5;

[0048] X and Y are each independently selected from: -NR-, -CRR'-, -C(O)-, -NHC(O)-, -C(O)NH-;

[0049] R1 is selected from: -N(R)-, -N(R)N(R')-, -C(O)-, -NHC(O)-, -C(O)NH-, -C(O)O-;

[0050] R2 is selected from: -N(R)-, -N(R)N(R')-, -NHC(O)-, -NHS(O)-;

[0051] R3 is selected from: -NRR';

[0052] R4 is selected from: -N(R)N(R')-;

[0053] R and R' may be the same or different, and are independently selected from: H and C. 1-5 Alkyl, C 1-5 Halogenated alkyl, C 1-5 alkoxy, C 2-6 alkenyl, C 2-6 Alkyne group, P(O)(OR'')(OR''')-(CH2) m -C(O)-, where R'' and R''' are the same or different, and are independently selected from C. 1-5 Alkyl group, m is selected from 1 to 3 integers;

[0054] Alternatively, R, R' and the adjacent N form a 6-membered saturated heterocyclic group.

[0055] More preferably, the connecting chain L linking A and B has the structure shown below:

[0056]

[0057] According to embodiments of the present invention, compounds of formula (I) include, but are not limited to, the structures shown below:

[0058]

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[0104] 。

[0105] According to embodiments of the present invention, the compound represented by general formula (I) contains a chiral center and can exist in different stereoisomeric forms. All stereoisomeric forms of the compounds involved in the present invention, including but not limited to their enantiomers, diastereomers, and mixtures thereof, such as racemates and optically active isomers, are included within the scope of the present invention.

[0106] According to embodiments of the present invention, the pharmaceutically acceptable salts of the present invention include, but are not limited to, addition salts formed from the following acids: nitric acid, boric acid, phosphoric acid, hydrochloric acid, hydrobromic acid, sulfuric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, acetic acid, lactic acid, maleic acid, citric acid, trichloroacetic acid, trifluoroacetic acid, salicylic acid, fumaric acid, oxalic acid, tartaric acid, benzoic acid, succinic acid, aspartic acid, glutamic acid, camphoric acid, and ascorbic acid.

[0107] According to embodiments of the present invention, the solvent compound described herein is a combination, physical association, and / or solvation of the compound disclosed herein with solvent molecules, such as a hemisolvate or a monosolvate. The compounds disclosed herein may exist, but are not limited to, as solvated forms in pharmaceutically acceptable solvents such as water, methanol, ethanol, or polyols.

[0108] According to embodiments of the present invention, the prodrug is a compound represented by general formula (I), its stereoisomer, or a pharmaceutically acceptable salt, which may have weak or no activity. After administration, it releases the active drug through enzymatic or non-enzymatic conversion in vivo to exert its therapeutic effect.

[0109] In a second aspect, the present invention provides a pharmaceutical composition comprising an effective amount of a compound of formula (I), a stereoisomer thereof, a pharmaceutically acceptable salt, a solvent compound or a prodrug compound as an active ingredient, one or more pharmaceutically acceptable carriers, a diluent or an excipient.

[0110] The present invention also provides the use of the compound represented by the above formula (I), its stereoisomers, pharmaceutically acceptable salts, solvent compounds or prodrug compounds in a medicament for treating BET protein-induced diseases;

[0111] Furthermore, the application is for targeted degradation of BET protein;

[0112] Furthermore, according to an embodiment of the present invention, the stated use is for treating malignant tumors.

[0113] According to embodiments of the present invention, the pharmaceutical composition can also be used in combination with a second therapeutic active agent to treat malignant tumors. The second therapeutic active agent can be a cytotoxic drug (e.g., paclitaxel, docetaxel, vinblastine, podophyllin, homoharringtonine, cytarabine, gemcitabine), a hormonal drug (e.g., goserelin, phenoxyamine, toremifene, medroxyprogesterone acetate, testosterone propionate, flutamide), a biological response modifier (e.g., interferon, interleukin-2, thymosin), a monoclonal antibody drug (e.g., rituximab injection, trastuzumab injection, bevacizumab), or an adjuvant (e.g., interleukin-11, granisetron hydrochloride, aspirin, fentanyl, pamidronate disodium).

[0114] The beneficial effects of this invention are:

[0115] 1) The compound structure shown in formula (I) of the present invention is a novel structure. The compound is simple and easy to synthesize and can be used as an effective compound for targeted degradation of BET protein.

[0116] 2) The compounds involved in this invention exhibit superior antitumor activity compared to the positive control. Pharmacodynamic evaluation results show that the compounds disclosed in this invention have significant antitumor effects. The compounds disclosed in this invention demonstrate excellent antitumor activity and BET protein-targeted degradation activity, and are expected to be developed into a new generation of BET protein-targeted degradation drugs, filling a gap in domestic drug development. Attached Figure Description

[0117] Figure 1 The diagram illustrates the degradation and apoptosis of BET protein in 22RV1 and Vcap cell lines, using the control compound ARV771 and the compounds AG-1, AG-8, AG-11, and AG-19 disclosed in this invention.

[0118] Figure 2 The tumor growth curves of MV-4-11 cell subcutaneous xenograft mice after treatment with compounds AG-08, AG-19 and AG-01 are shown. The data points represent the mean tumor volume within the group, and the error bars represent the standard error (SEM).

[0119] Figure 3 The data represent the weight changes in MV-4-11 cell subcutaneous xenograft mice after treatment with compounds AG-08, AG-19, and AG-01. Data points represent mean weight within groups, and error bars represent standard errors (SEM).

[0120] Figure 4 The data represent the changes in body weight in MV-4-11 cell subcutaneous xenograft mice after administration of compounds AG-08, AG-19, and AG-01. Data represent mean body weight within groups, and error bars represent standard errors (SEM).

[0121] Figure 5 The image shows the tumor changes in MV-4-11 cell subcutaneous xenograft mice after administration of compounds AG-08, AG-19, and AG-01. The underlined portions indicate complete tumor disappearance.

[0122] Terminology Definitions and Explanations

[0123] Unless otherwise stated, the definitions of groups and terms recorded in this application specification and claims, including definitions as examples, exemplary definitions, preferred definitions, definitions recorded in tables, and definitions of specific compounds in the examples, can be arbitrarily combined and combined with each other. Such combinations and combinations of group definitions and compound structures should be understood as being within the scope of this application specification and / or claims.

[0124] Unless otherwise stated, the numerical ranges described in this specification and claims are equivalent to describing at least each specific integer value therein. For example, the numerical range "1-10" is equivalent to describing each integer value in the numerical range "1-10", namely 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0125] It should be understood that in this article, when describing one, two or more, "more" should refer to integers greater than 2, such as 3 or greater than or equal to 3, such as 3, 4, 5, 6, 7, 8, 9 or 10.

[0126] The term "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0127] Term "C" 1-10 "Alkyl" should be understood as representing a straight-chain or branched saturated monovalent hydrocarbon group having 1 to 10 carbon atoms. For example, "C 1-10 "Alkyl" refers to straight-chain and branched alkyl groups having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. 1-5 "Alkyl" means a straight-chain or branched alkyl group having 1, 2, 3, 4, or 5 carbon atoms. The alkyl group is, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl, or their isomers.

[0128] Term "C" 2-10"Alkenyl" should be understood as referring to a straight-chain or branched monovalent hydrocarbon group containing one or more double bonds and having 2 to 10 carbon atoms. "C" 2-6 "Alkenyl" should be understood to preferably represent a monovalent hydrocarbon group, either linear or branched, containing one, two, or three double bonds and having 2, 3, 4, 5, or 6 carbon atoms. The alkenyl group is, for example, vinyl, allyl, (E)-2-methylvinyl, (Z)-2-methylvinyl, (E)-but-2-enyl, (Z)-but-2-enyl, (E)-but-1-enyl, (Z)-but-1-enyl, pent-4-enyl, and (E)-pent-3-enyl. (Z)-pent-3-enyl, (E)-pent-2-enyl, (Z)-pent-2-enyl, (E)-pent-1-enyl, (Z)-pent-1-enyl, hex-5-enyl, (E)-hex-4-enyl, (Z)-hex-4-enyl, (E)-hex-3-enyl, (Z)-hex-3-enyl, (E)-hex-2-enyl, (Z)-hex-2-enyl, (E)-hex-1-enyl, (Z)-hex-1-enyl Isopropenyl, 2-methylprop-2-enyl, 1-methylprop-2-enyl, 2-methylprop-1-enyl, (E)-1-methylprop-1-enyl, (Z)-1-methylprop-1-enyl, 3-methylbut-3-enyl, 2-methylbut-3-enyl, 1-methylbut-3-enyl, 3-methylbut-2-enyl, (E)-2-methylbut-2-enyl, (Z)-2-methylbut-2-enyl, (E)-1-methylbut- 2-Alkenyl, (Z)-1-methylbut-2-alkenyl, (E)-3-methylbut-1-alkenyl, (Z)-3-methylbut-1-alkenyl, (E)-2-methylbut-1-alkenyl, (Z)-2-methylbut-1-alkenyl, (E)-1-methylbut-1-alkenyl, (Z)-1-methylbut-1-alkenyl, 1,1-dimethylprop-2-alkenyl, 1-ethylprop-1-alkenyl, 1-propylvinyl, 1-isopropylvinyl.

[0129] Term "C" 2-10 "Alkyne" should be understood as referring to a straight-chain or branched monovalent hydrocarbon group containing one or more triple bonds and having 2 to 10 carbon atoms. The term "C"... 2-6 "Alkyne" should be understood to preferably represent a straight or branched monovalent hydrocarbon group containing one, two or more triple bonds and having 2, 3, 4, 5 or 6 carbon atoms (i.e., "C"). 2-6The alkynyl group is, for example, ethynyl, prop-1-alkynyl, prop-2-alkynyl, but-1-alkynyl, but-2-alkynyl, but-3-alkynyl, pent-1-alkynyl, pent-2-alkynyl, pent-3-alkynyl, pent-4-alkynyl, hex-1-alkynyl, hex-2-alkynyl, hex-3-alkynyl, hex-4-alkynyl, hex-5-alkynyl, 1-methylprop-2-alkynyl, 2-methylbut-3-alkynyl, 1-methylbut-3-alkynyl, 1-methylbut-2-alkynyl, 3-methylbut-1-alkynyl, 1-ethylprop-2-alkynyl, 3-methylpent-4-alkynyl, 2-methylpent-4-alkynyl, 1-methylpent-4-alkynyl -Alynyl, 2-methylpentan-3-ynyl, 1-methylpentan-3-ynyl, 4-methylpentan-2-ynyl, 1-methylpentan-2-ynyl, 4-methylpentan-1-ynyl, 3-methylpentan-1-ynyl, 2-ethylbutan-3-ynyl, 1-ethylbutan-3-ynyl, 1-ethylbutan-2-ynyl, 1-propylpropan-2-ynyl, 1-isopropylpropan-2-ynyl, 2,2-dimethylbutan-3-ynyl, 1,1-dimethylbutan-3-ynyl, 1,1-dimethylbutan-2-ynyl, or 3,3-dimethylbutan-1-ynyl. In particular, the ynyl group is ethynyl, propan-1-ynyl, or propan-2-ynyl.

[0130] Term "C" 3-10 "Cycloalkyl" should be understood to refer to saturated monovalent monocyclic, bicyclic (such as fused ring, bridged ring, spiro ring) hydrocarbon rings or tricyclic alkanes, which have 3 to 10 carbon atoms. The term "C" 3-10 "Cycloalkyl" should be understood to refer to a saturated monovalent monocyclic, bicyclic (e.g., bridged, spirocyclic) hydrocarbon ring or tricyclic alkane having 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. The C... 3-10 Cycloalkyl groups can be monocyclic hydrocarbon groups, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, or cyclodecyl; or bicyclic hydrocarbon groups, such as borneolyl, indolyl, hexahydroindolyl, tetrahydronaphthyl, decahydronaphthyl, bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.1]heptenyl, 6,6-dimethylbicyclo[3.1.1]heptyl, 2,6,6-trimethylbicyclo[3.1.1]heptyl, bicyclo[2.2.2]octyl, 2,7-diazaspiro[3,5]nonyl, 2,6-diazaspiro[3,4]octyl; or tricyclic hydrocarbon groups, such as adamantyl.

[0131] Unless otherwise defined, the term "3- to 14-membered heterocyclic group" refers to a saturated or unsaturated non-aromatic ring or ring system, for example, a 3-, 4-, 5-, 6-, or 7-membered monocyclic ring, a 7-, 8-, 9-, 10-, 11-, or 12-membered bicyclic ring (such as a fused ring, bridged ring, or spirocyclic ring), or a 10-, 11-, 12-, 13-, or 14-membered tricyclic ring system, and contains at least one, for example, 1, 2, 3, 4, 5, or more heteroatoms selected from O, S, and N, wherein N and S may optionally be oxidized to various oxidation states to form nitrogen oxides, -S(O)-, or -S(O)2- states. The term "3- to 10-membered heterocyclic group" means a saturated or unsaturated non-aromatic ring or ring system, and contains at least one heteroatom selected from O, S, and N. The heterocyclic group can be attached to the remainder of the molecule by any one of the carbon atoms or a nitrogen atom (if present). The heterocyclic group may include fused or bridged rings and spirocyclic rings. Specifically, the heterocyclic group may include, but is not limited to: 4-membered rings, such as azirrobutyl or oxobutyl; 5-membered rings, such as tetrahydrofuranyl, dioxacyclopentenyl, pyrrolyl, imidazoalkyl, pyrazolealkyl, or pyrrololinyl; or 6-membered rings, such as tetrahydropyranyl, piperidinyl, morpholinyl, dithiaalkyl, thiomorpholinyl, piperazinyl, or trithiaalkyl; or 7-membered rings, such as diazacycloheptyl. Optionally, the heterocyclic group may be benzofused. The heterocyclic group may be bicyclic, for example, but not limited to, 5,5-membered rings, such as hexahydrocyclopenta[c]pyrrolo-2(1H)-yl rings, or 5,6-membered bicyclic rings, such as hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl rings. The heterocyclic group can be partially unsaturated, meaning it can contain one or more double bonds, such as, but not limited to, dihydrofuranyl, dihydropyranyl, 2,5-dihydro-1H-pyrroleyl, 4H-[1,3,4]thiadiazinyl, 1,2,3,5-tetrahydrooxazolyl, or 4H-[1,4]thiazinyl. Alternatively, it can be benzofused, such as, but not limited to, dihydroisoquinolinyl. When the 3- to 14-membered heterocyclic group is linked to other groups to form the compounds of the present invention, the carbon atom on the 3- to 14-membered heterocyclic group can be linked to other groups, or the heterocyclic atom on the ring of the 3- to 14-membered heterocyclic group can be linked to other groups. For example, when the 3- to 14-membered heterocyclic group is selected from piperazineyl, the nitrogen atom on the piperazineyl group can be linked to other groups. Or when the 3- to 14-membered heterocyclic group is selected from piperidinyl, the nitrogen atom on the piperidinyl ring and the carbon atom at its para position can be linked to other groups.

[0132] Term "C" 6-14 "Aryl" should preferably be understood to represent a monocyclic, bicyclic (such as fused ring, bridged ring, spiro ring), or tricyclic hydrocarbon ring having 6 to 14 carbon atoms and possessing monovalent aromaticity or partial aromaticity. It can be a monoaromatic ring or a polyaromatic ring fused together, preferably "C". 6-10 "Aromatic". The term "C"6-10 "Aryl" should be understood to preferably represent a monovalent aromatic or partially aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring ("C") having 6, 7, 8, 9, or 10 carbon atoms. 6-10 Aryl), particularly a ring with 6 carbon atoms (“C6 aryl”), such as phenyl; or biphenyl, or a ring with 9 carbon atoms (“C9 aryl”), such as indenyl or indenyl, or a ring with 10 carbon atoms (“C9 aryl”). 10 Aryl), such as tetrahydronaphthyl, dihydronaphthyl, or naphthyl. C 6-14 Aryl groups include, for example, rings with 13 carbon atoms (“C…”). 13 Aryl groups, such as fluorene groups, or rings with 14 carbon atoms (“C”). 14 Aryl), for example, anthracene. When the C 6-14 When the aryl group is substituted, it can be monosubstituted or polysubstituted. Furthermore, there are no restrictions on the substitution site; for example, it can be ortho, para, or meta substituted.

[0133] The term "5- to 14-membered heteroaryl" should be understood to include monocyclic, bicyclic (e.g., fused, bridged, spirocyclic), or tricyclic aromatic ring systems having 5 to 14 ring atoms and containing 1 to 5 heteroatoms independently selected from N, O, and S. The term "5- to 14-membered heteroaryl" should also be understood to include monocyclic, bicyclic, or tricyclic aromatic ring systems having 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 ring atoms, particularly 5, 6, 9, or 10 carbon atoms, and containing 1 to 5, preferably 1 to 3, heteroatoms independently selected from N, O, and S, and in each case, may be benzo[a]fused. "Heteroaryl" also refers to a group in which the heteroaryl ring is fused with one or more aryl, alicyclic, or heterocyclic rings, wherein the root or point of the connection is on the heteroaryl ring. Non-limiting examples include 1-, 2-, 3-, 5-, 6-, 7- or 8-indazinyl, 1-, 3-, 4-, 5-, 6- or 7-isoindolyl, 2-, 3-, 4-, 5-, 6- or 7-indolyl, 2-, 3-, 4-, 5-, 6- or 7-indazolyl, 2-, 4-, 5-, 6-, 7- or 8-purineyl, 1-, 2-, 3-, 4-, 6-, 7-, 8- or 9-quinazinyl, 2-, 3-, 4-, 5-, 6-, 7- or 8-quinolinyl, 1-, 3-, 4- 5, 6, 7 or 8-isoquinolinyl, 1, 4, 5, 6, 7 or 8-phthalazinyl, 2, 3, 4, 5 or 6-naphthidyl, 2, 3, 5, 6, 7 or 8-quinazolinyl, 3, 4, 5, 6, 7 or 8-cenolinyl, 2, 4, 6 or 7-pteridyl, 1, 2, 3, 4, 5, 6, 7 or 8-4aH carbazole, 1, 2, 3, 4, 5, 6, 7 - or 8-carbazolylcarbazolyl, 1-, 3-, 4-, 5-, 6-, 7-, 8- or 9-carbazolyl, 1-, 2-, 3-, 4-, 6-, 7-, 8-, 9- or 10-phenanthridyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8- or 9-acridyl, 1-, 2-, 4-, 5-, 6-, 7-, 8- or 9-pyridyl, 2-, 3-, 4-, 5-, 6-, 8-, 9- or 10-phenanthrolinel, 1-, 2-, 3-, 4-, 6-, 7-, 8- or 9-phenanthrolinel -Phenazinyl, 1-, 2-, 3-, 4-, 6-, 7-, 8-, 9- or 10-phenthiazinyl, 1-, 2-, 3-, 4-, 6-, 7-, 8-, 9- or 10-phenazinyl, 2-, 3-, 4-, 5-, 6- or 1-, 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-benzisoquinolinyl, 2-, 3-, 4- or thieno[2,3-b]furanyl, 2-, 3-, 5-, 6-, 7-, 8-, 9-, 10- or 11-7H-pyrazin[2,3-c]carbazolyl, 2-, 3-, 5-, 6- or 7-2H-furano[3,2-b]-pyranolyl, 2-, 3-, 4-, 5-, 7- or 8-5H-pyrido[2,3-d]-o-azinyl, 1-, 3- or 5-1H-pyrazolo[4,3-d]-azolel, 2-, 4- or 5-4H-imidazo[4,5-d]thiazolyl, 3-, 5- or 8-pyrazolo[2,3-d]pyridazinyl, 2-, 3-, 5- or 6-imidazo[2,1-b]thiazolyl, 1-, 3-, 6-, 7-, 8- or 9-furano[3,4-c]cenolinyl, 1-, 2-, 3-, 4-, 5-, 6-, 8-, 9-, 10- or 11-4H-pyrido[2,3] -c]carbazolyl, 2-, 3-, 6- or 7-imidazo[1,2-b][1,2,4]triazinyl, 7-benzo[b]thiopheneyl, 2-, 4-, 5-, 6- or 7-benzozolyl, 2-, 4-, 5-, 6- or 7-benzimidazinyl, 2-, 4-, 4-, 5-, 6- or 7-benzothiazolyl, 1-, 2-, 4-, 5-, 6-, 7-, 8- or 9-benzoxapinyl, 2-, 4-, 5-, 6-, 7- or 8-benzoazinyl, 1-, 2-, 3-, 5-, 6-, 7-, 8-, 9-, 10- or 11-4H-pyrrolo[1,2-b][2]benzozapinyl. Typical fused heteroaryl groups include, but are not limited to, 2-, 3-, 4-, 5-, 6-, 7-, or 8-quinolinyl, 1-, 3-, 4-, 5-, 6-, 7-, or 8-isoquinolinyl, 2-, 3-, 4-, 5-, 6-, or 7-indolyl, 2-, 3-, 4-, 5-, 6-, or 7-benzo[b]thiophene, 2-, 4-, 5-, 6-, or 7-benzozozolyl, 2-, 4-, 5-, 6-, or 7-benzimidazolyl, and 2-, 4-, 5-, 6-, or 7-benzothiazolyl. When the 5- to 14-membered heteroaryl groups are linked to other groups to form the compounds of the present invention, the carbon atom on the 5- to 14-membered heteroaryl ring may be linked to other groups, or the heteroatom on the 5- to 14-membered heteroaryl ring may be linked to other groups. When the 5- to 14-membered heteroaryl groups are substituted, they may be monosubstituted or polysubstituted. Furthermore, there are no restrictions on the substitution sites; for example, hydrogen atoms bonded to carbon atoms on the heteroaryl ring can be substituted, or hydrogen atoms bonded to heteroatoms on the heteroaryl ring can be substituted.

[0134] The term "spirocycle" refers to a ring system in which two rings share a single ring atom.

[0135] The term "fused ring" refers to a ring system in which two rings share two cyclic atoms.

[0136] The term "bridged ring" refers to a ring system in which two rings share three or more cyclic atoms.

[0137] Unless otherwise stated, heterocyclic and heteroaryl groups include all possible isomers, such as their positional isomers. Thus, for some illustrative, non-limiting examples, they may include forms in which one, two, or more of the following positions (if present) are substituted or bonded to other groups, including pyridin-2-yl, pyridinoid-2-yl, pyridinoid-3-yl, pyridinoid-3-yl, pyridinoid-4-yl, and pyridinoid-4-yl; thiophene or thiophene groups include thiophene-2-yl, thiophene-2-yl, thiophene-3-yl, and thiophene-3-yl; pyrazol-1-yl, pyrazol-3-yl, pyrazol-4-yl, and pyrazol-5-yl.

[0138] The term "alkylamino" refers to -NH-(alkyl), where alkyl is defined as described above. Non-limiting examples of alkylamino include: methylamino, ethylamino, propylamino, isopropylamino, butylamino, etc.

[0139] The term "alkoxy" refers to -O-(alkyl), where alkyl is defined as described above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, and butoxy. Alkoxy groups can be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkyloxy, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, or heterocycloalkyloxy.

[0140] "Halogenated alkyl" refers to an alkyl group that has been substituted with one or more halogens, wherein the alkyl group is as defined above.

[0141] "alkyl acyl" refers to -C(O)-(alkyl), where alkyl is defined as above.

[0142] "alkyl acyloxy" refers to -OC(O)-(alkyl), where alkyl is defined as above.

[0143] "alkylamide" refers to -NH-C(O)-(alkyl), where alkyl is defined as above.

[0144] “C 1-3 "alkylsulfonyl" refers to -NH-S(O)2-(alkyl), where alkyl is defined as above.

[0145] “C 1-3 "alkylthionyl" refers to -NH-S(O)-(alkyl), where alkyl is defined as above. Detailed Implementation

[0146] The following embodiments further describe the present invention; however, these embodiments are merely illustrative and not intended to limit the scope of the invention.

[0147] Example 1: Preparation of compound D1

[0148]

[0149] Step 1: Preparation of compound D1-2

[0150] 12.4 g (69 mmol) of 3-(4-chlorophenyl)-3-oxypropionitrile, 6.5 mL (69 mmol) of 2-butanone, 6 mL (69 mmol) of morpholine, and 200 mL of ethanol were placed in a 500 L single-necked flask. 2.2 g (69 mmol) of sulfur powder was added to the flask, and the mixture was heated to reflux and stirred for 12 hours. TLC analysis showed that the reaction of the starting material was basically complete. The heating was turned off, and the mixture was cooled to room temperature. The solvent was removed by vacuum distillation. 100 mL of purified water was added to the residue, followed by extraction with ethyl acetate (100 mL * 3). The organic phase was dried over sodium sulfate, filtered, and the solvent was removed by vacuum distillation of the filtrate. The residue was purified by column chromatography (eluent: ethyl acetate: petroleum ether = 4:1) to obtain 12.9 g of product (yield 70%).

[0151] Step 2: Preparation of compound D1-4

[0152] Under a nitrogen atmosphere, 12.9 g (48.6 mmol) of compound D1-2 and 30 g (72.9 mmol) of compound D1-3 were placed in a 1 L single-necked flask, and 60 mL of ethyl acetate was added. Separately, 62 g of 1-propylphosphonic anhydride (50% ethyl acetate solution) and 15.4 g of pyridine were added to the mixture. The mixture was stirred at room temperature for 18 hours under nitrogen protection. 150 mL of isopropyl acetate was added to the reaction system, and 200 mL of hydrochloric acid solution (2.5 N) was added dropwise while stirring. The mixture was stirred vigorously for 30 minutes. The organic phase was separated, and the aqueous phase was extracted with isopropyl acetate (100 mL * 3). The organic phases were combined, washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate by vacuum distillation. The filtrate was repeatedly dissolved and evaporated with isopropyl acetate to obtain 28.5 g of the product (yield 90%, ee 99%).

[0153] Step 3: Preparation of compound D1-5

[0154] 28.5 g (43.2 mmol) of compound D1-4 was placed in a 500 mL single-necked flask, and 100 mL of isopropyl acetate was added. 70 g of piperazine was also added. The mixture was stirred at room temperature for 12 hours. The mixture was filtered through diatomaceous earth. 100 mL of 5% hydrochloric acid solution was added to the filtrate, and the mixture was stirred vigorously for 30 minutes. The organic phase was separated, and the aqueous phase was extracted with isopropyl acetate (100 mL x 3). The combined organic phases were washed successively with 10% potassium carbonate aqueous solution and saturated brine. The organic phase was dried over sodium sulfate and filtered. 10 g of p-toluenesulfonic acid monohydrate was added to the filtrate, followed by 150 mL of methyl tert-butyl ether. A solid precipitated. The mixture was stirred at room temperature for another 8 hours. The mixture was then filtered, and the filter cake was washed with methyl tert-butyl ether (10 mL x 3). The cake was dried under vacuum to obtain 21.6 g of the product (yield 82%, ee 98%).

[0155] Step 4: Preparation of compound D1-6

[0156] 21.6 g (35.5 mmol) of compound D1-5 was placed in a 500 mL single-necked flask, and 100 mL of isopropyl acetate was added. 100 mL of a 10% potassium carbonate aqueous solution was also added. The mixture was stirred vigorously for 2 hours, filtered, and the organic phase was separated. The aqueous phase was extracted with isopropyl acetate (50 mL x 3). The organic phases were combined and dried over sodium sulfate. After filtration, 5 mL of acetic acid was added to the filtrate. A water separator was installed, and the mixture was heated to reflux at 90 °C for 6 hours. The solvent was removed under reduced pressure until a solid precipitated. Crystallization was completed by standing at room temperature. The filtrate was filtered, and the filter cake was dried under vacuum with isopropyl acetate (10 mL x 3) to obtain 10.5 g of product (yield 71%, ee 99%).

[0157] Step 5: Preparation of compound D1-7

[0158] 10.5 g (25 mmol) of compound D1-6 was placed in a 500 mL single-necked flask, and 150 mL of dry tetrahydrofuran was added to dissolve it. The mixture was cooled to -78 °C, and 27.5 mL of potassium tert-butoxide (1 M tetrahydrofuran solution) was added dropwise to the system. The mixture was slowly restored to room temperature and stirred for 30 minutes. The temperature was further lowered to -78 °C, and 4.5 mL (30 mmol) of diethyl chlorophosphate was added. The mixture was slowly restored to -10 °C. 2.8 g (37.5 mmol) of acetylhydrazine was added, and the mixture was stirred for 1 hour at room temperature. 20 mL of n-butanol was added, and the mixture was stirred at 90 °C for 1 hour. The solvent was removed by vacuum distillation, and the residue was purified by column chromatography (eluting ethyl acetate: petroleum ether = 2:1) to give 10 g of product (yield 88%).

[0159] Step 6: Preparation of compound D1

[0160] 10 g (22 mmol) of compound D1-7 was placed in a 500 mL single-necked flask, and 250 mL of trifluoroacetic acid / dichloromethane (40%) was added. The mixture was stirred at room temperature for 8 hours. The solvent was removed from the filtrate under reduced pressure, and 100 mL of purified water was added. Ethyl acetate (100 mL * 3) was added for extraction. The organic phase was dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: dichloromethane:methanol = 15:1) to give 8.4 g of product (yield 95%).

[0161] Example 2: Preparation of compound P1

[0162]

[0163] Step 1: Preparation of compound P1-2

[0164] Take 10 g (55 mmol) of p-bromobenzonitrile, 10.9 g (110 mmol) of 4-methylthiazole, 11 g (110 mmol) of potassium acetate, and 123 mg (0.55 mmol) of palladium acetate in a 500 mL single-necked flask, add 100 mL of DMA, purge with nitrogen three times, heat and stir at 120 °C for 3 hours under nitrogen protection; TLC showed that the reaction of the starting material was basically complete, turn off the heating, cool to room temperature, add 600 mL of water, extract with ethyl acetate (200 mL * 3), wash the organic phase with saturated brine, dry with sodium sulfate, filter, evaporate the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluting agent: ethyl acetate: petroleum ether = 1:5) to obtain 8.25 g (yield 75%) of product;

[0165] Step 2: Preparation of compound P1-3

[0166] 8.25 g (41.3 mmol) of compound P1-2 was placed in a 500 mL single-necked flask, and 300 mL of tetrahydrofuran was added. Under nitrogen protection, the mixture was cooled to 0 °C. 4.7 g (124 mmol) of lithium aluminum tetrahydrofuran was carefully added to the flask. The mixture was heated to 60 °C and stirred for 3 hours. TLC analysis showed that the reaction was essentially complete. The heating was turned off, and the mixture was cooled to 0 °C. 10 mL of water was slowly added dropwise to quench the reaction. Sodium sulfate dodecahydrate was added, and the mixture was stirred. The mixture was extracted with ethyl acetate (100 mL x 5). The organic phase was dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: dichloromethane:methanol = 10:1) to obtain 4.2 g (50% yield) of the product.

[0167] Step 3: Preparation of compound P1-4

[0168] 5.7 g (24.8 mmol) of Boc-L-hydroxyproline, 9.4 g (24.8 mmol) of HATU, and 5.4 g (41.4 mmol) of DIEA were placed in a 500 mL single-necked flask, 100 mL of LDM was added, and the mixture was stirred at room temperature for 1 hour. 4.2 g (20.7 mmol) of compound P1-3 was then added to the mixture, and the mixture was stirred at room temperature overnight. TLC analysis showed that the reaction was essentially complete. 400 mL of water was added, and the mixture was extracted with ethyl acetate (200 mL x 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: dichloromethane:methanol = 20:1) to obtain 4.7 g (55% yield) of the product.

[0169] Step 4: Preparation of compound P1-5

[0170] 4.7 g (11.3 mmol) of compound P1-4 was placed in a 250 mL single-necked flask, 80 mL of hydrochloric acid methanol solution was added, and the mixture was stirred at room temperature for 2 hours. TLC showed that the reaction of the starting material was basically complete. The solvent was removed by vacuum distillation, and the mixture was distilled multiple times with dichloromethane (100 mL * 3) to obtain 3.9 g of hydrochloride of compound P1-5, which was directly used in the next step of the reaction.

[0171] Step 5: Preparation of compound P1-6

[0172] 3.1 g (13.6 mmol) of N-Boc-L-tert-leucine, 5.2 g (13.6 mmol) of HATU, and 2.9 g (22.4 mmol) of DIEA were placed in a 500 mL single-necked flask, 100 mL of LDMF was added, and the mixture was stirred at room temperature for 1 hour. Separately, 3.9 g (11.2 mmol) of compound P1-5 hydrochloride was added to the mixture, and the mixture was stirred at room temperature overnight. TLC analysis showed that the reaction was essentially complete. 400 mL of water was added, and the mixture was extracted with ethyl acetate (200 mL * 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: ethyl acetate: petroleum ether = 2:1) to obtain 3.6 g (61% yield) of the product.

[0173] Step 6: Preparation of compound P1

[0174] Take 3.6 g (6.8 mmol) of compound P1-6 into a 250 mL single-necked flask, add 80 mL of hydrochloric acid methanol solution, stir at room temperature for 2 hours, and TLC shows that the starting material is basically completely reacted. Remove the solvent under reduced pressure and distill multiple times with dichloromethane (100 mL * 3) to obtain compound P1 3.1 g, which can be directly used in the next step of the reaction.

[0175] Example 3: Preparation of compound L1

[0176]

[0177] Step 1: Preparation of compound L1-2

[0178] 8.6 g of sodium hydride (216 mmol, 60%) was placed in a 500 mL single-necked flask, and 200 mL of tetrahydrofuran was added. Under nitrogen protection, the mixture was cooled to 0 °C. Separately, 15 g (144 mmol) of 1,5-pentanediol was dissolved in 100 mL of tetrahydrofuran and slowly added to the above system. The mixture was stirred for 1 hour. Separately, 24.6 g (144 mmol) of benzyl bromide was added to the above system, and the mixture was brought back to room temperature and stirred for 8 hours. TLC analysis showed that the reaction of the starting material was basically complete. The reaction was quenched with 50 mL of ice water, extracted with ethyl acetate (200 mL * 3), and the organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: ethyl acetate: petroleum ether = 1:1) to obtain 25.1 g (90% yield) of product.

[0179] Step 2: Preparation of compound L1-3

[0180] 25.1 g (129.4 mmol) of compound L1-2, 50.4 g (258.8 mmol) of tert-butyl bromoacetate, and 41.7 g (129.4 mmol) of tetrabutylammonium bromide were placed in a 1 L single-necked flask. 300 mL of dichloromethane was added, along with 300 mL of 35% sodium hydroxide solution. The mixture was stirred overnight at room temperature. TLC analysis showed that the reaction of the starting material was basically complete. 300 mL of water was added, and the mixture was extracted with dichloromethane (200 mL * 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluting agent: ethyl acetate: petroleum ether = 1:5) to obtain 15.5 g (yield 39%) of the product.

[0181] Step 3: Preparation of compound L1-4

[0182] Take 15.5 g (50 mmol) of compound L1-3 into a 500 mL single-necked flask, add 200 mL of methanol, dissolve to obtain a clear solution, add 1.5 g of palladium on carbon catalyst into the solution, stir at room temperature for 12 hours under hydrogen atmosphere, and TLC shows that the starting material has basically reacted completely. Filter, concentrate and dry the filtrate, and use it directly for the next step of the reaction.

[0183] Step 4: Preparation of compound L1-5

[0184] 11 g of compound L1-4 (crude product) was placed in a 500 mL single-necked flask, and 100 mL of dichloromethane was added to dissolve it to obtain a clear solution. 8 g of pyridine (101 mmol) was added, and 10.5 g of p-toluenesulfonyl chloride (55 mmol) was dissolved in 20 mL of dichloromethane and added dropwise to the above system. After the addition was complete, the mixture was stirred at room temperature for 3 hours. TLC showed that the reaction of the starting material was basically complete. 300 mL of water was added, and the mixture was extracted with dichloromethane (200 mL * 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluting agent: ethyl acetate: petroleum ether = 1:4) to obtain 14.9 g (yield 80%) of product.

[0185] Step 5: Preparation of compound L1-6

[0186] 14.9 g (40 mmol) of compound L1-5 was placed in a 500 mL single-necked flask, and 80 mL of DMF was added to dissolve it to obtain a clear solution. 8.9 g (48 mmol) of potassium phthalimide was added to the above system, and the mixture was heated to 80 °C and stirred for 16 hours. TLC analysis showed that the reaction of the starting material was basically complete. 700 mL of methyl tert-butyl ether was added, and a solid precipitated. The mixture was filtered, and the methyl tert-butyl ether was removed from the filtrate under reduced pressure. 500 mL of water was added, and the mixture was extracted with ethyl acetate (100 mL * 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: ethyl acetate: petroleum ether = 1:3) to obtain 10 g (72% yield) of the product.

[0187] Step 6: Preparation of compound L1

[0188] 10 g (28.8 mmol) of compound L1-6 was placed in a 500 mL single-necked flask, and 200 mL of methanol was added to dissolve it, yielding a clear solution. 2.5 g (80%, 57.6 mmol) of hydrazine hydrate was then added to the solution, and the mixture was heated to 80 °C and stirred for 6 hours. TLC analysis showed that the reaction proceeds were essentially complete, and the solvent was removed under reduced pressure. 200 mL of methyl tert-butyl ether was added, and the mixture was stirred for 1 hour. The mixture was filtered, and the filter cake was washed with methyl tert-butyl ether (50 mL x 3). The filtrate was concentrated and dried, and used directly in the next reaction step.

[0189] Example 4: Preparation of compound L2

[0190]

[0191] Step 1: Preparation of compound L2-2

[0192] 20 g (102 mmol) of compound L2-1, 39.8 g (204 mmol) of tert-butyl bromoacetate, and 32.8 g (102 mmol) of tetrabutylammonium bromide were placed in a 1 L single-necked flask. 300 mL of dichloromethane was added, along with 300 mL of 35% sodium hydroxide solution. The mixture was stirred overnight at room temperature. TLC analysis showed that the reaction of the starting material was basically complete. 300 mL of water was added, and the mixture was extracted with dichloromethane (200 mL * 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: ethyl acetate: petroleum ether = 1:5) to obtain 13.6 g (yield 43%) of the product.

[0193] Step 2: Preparation of compound L2-3

[0194] Take 13.6 g (43.9 mmol) of compound L2-2 into a 500 mL single-necked flask, add 200 mL of methanol, dissolve to obtain a clear solution, add 1.4 g of palladium on carbon catalyst into the solution, stir at room temperature for 12 hours under hydrogen atmosphere, TLC shows that the starting material has basically reacted completely, filter, concentrate and dry the filtrate, and use it directly for the next step of the reaction;

[0195] Step 3: Preparation of compound L2-4

[0196] 9.7 g (crude product) of compound L2-3 was placed in a 500 mL single-necked flask, and 100 mL of dichloromethane was added to dissolve it to obtain a clear solution. 7 g (88 mmol) of pyridine was added, and 8.3 g (43.9 mmol) of p-toluenesulfonyl chloride was dissolved in 20 mL of dichloromethane and added dropwise to the above system. After the addition was complete, the mixture was stirred at room temperature for 3 hours. TLC showed that the reaction of the starting material was basically complete. 300 mL of water was added, and the mixture was extracted with dichloromethane (200 mL * 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluting agent: ethyl acetate: petroleum ether = 1:4) to obtain 13.5 g (yield 82%) of product.

[0197] Step 4: Preparation of compound L2-5

[0198] 13.5 g (36 mmol) of compound L2-4 was placed in a 500 mL single-necked flask, and 100 mL of LDM was added to dissolve it to obtain a clear solution. 7.2 g (43.2 mmol) of benzyl hydrazide carboxylate was added to the above system, and the mixture was heated to 80 °C and stirred for 16 hours. TLC analysis showed that the reaction of the starting material was basically complete. 700 mL of methyl tert-butyl ether was added, and a solid precipitated. The mixture was filtered, and the methyl tert-butyl ether was removed from the filtrate under reduced pressure. 500 mL of water was added, and the mixture was extracted with ethyl acetate (100 mL * 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: ethyl acetate: petroleum ether = 1:3) to obtain 8.6 g (65% yield) of the product.

[0199] Step 5: Preparation of compound L2-6

[0200] 8.6 g (23.4 mmol) of compound L2-5 and 6.56 g (46.8 mmol) of potassium carbonate were placed in a 500 mL single-necked flask, 100 mL of LDM was added, and 3.9 g (28 mmol) of iodomethane was added to the flask. The mixture was heated to 90 °C and stirred for 16 hours. TLC showed that the reaction of the starting material was basically complete. 600 mL of water was added, and the mixture was extracted with ethyl acetate (100 mL * 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: ethyl acetate: petroleum ether = 1:4) to obtain 4.6 g (yield 51%) of product.

[0201] Step 6: Preparation of compound L2

[0202] Take 4.6 g (17.6 mmol) of compound L2-6 in a 250 mL single-necked flask, add 100 mL of methanol, dissolve to obtain a clear solution, add 0.5 g of palladium on carbon catalyst to it, stir at room temperature for 12 hours under hydrogen atmosphere, and TLC shows that the starting material has basically reacted completely. Filter, concentrate and dry the filtrate, and use it directly for the next step of the reaction.

[0203] Example 5: Preparation of compound L3

[0204]

[0205] Step 1: Preparation of compound L3-2

[0206] 24.3 g (126 mmol) of compound L3-1, 19.9 g (151 mmol) of tert-butyl hydrazide carboxylate, and 0.1 g (0.63 mmol) of p-toluenesulfonic acid were placed in a 500 mL single-necked flask. 200 mL of dichloromethane was added to dissolve the mixture and a clear solution was obtained. 23.9 g (630 mmol) of sodium borohydride was added in portions to the solution, and the mixture was stirred overnight at room temperature. TLC analysis showed that the reaction of the starting material was basically complete. The reaction was quenched with 20 mL of ice water, and 300 mL of water was added. The mixture was extracted with dichloromethane (200 mL * 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluting agent: ethyl acetate: petroleum ether = 1:1) to obtain 28.4 g (78% yield) of the product.

[0207] Step 2: Preparation of compound L3-3

[0208] 28.4 g (91.9 mmol) of compound L3-2 and 26.7 g (194 mmol) of potassium carbonate were placed in a 500 mL single-necked flask, and 300 mL of LDM was added. 15.5 g (110 mmol) of iodomethane was added to the flask. The mixture was heated to 90 °C and stirred for 16 hours. TLC showed that the reaction of the starting material was basically complete. 1500 mL of water was added, and the mixture was extracted with ethyl acetate (100 mL * 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluting agent: ethyl acetate: petroleum ether = 1:4) to obtain 13.4 g (yield 45%) of product.

[0209] Step 3: Preparation of compound L3-4

[0210] 13.4 g (41.5 mmol) of compound L3-3 was placed in a 500 mL single-necked flask, and 100 mL of hydrochloric acid-methanol solution was added. The mixture was stirred at room temperature for 2 hours. TLC showed that the reaction of the starting material was basically complete. The solvent was removed by vacuum distillation, and the mixture was distilled multiple times with dichloromethane (100 mL * 3) to obtain 9.3 g of benzyl (2-(1-methylhydrazyl)ethyl)carbamate hydrochloride, which was directly used in the next step of the reaction.

[0211] Step 4: Preparation of compound L3-6

[0212] 11 g (62.5 mmol) of compound L3-5 was placed in a 500 mL single-necked flask, and 100 mL of dichloromethane was added to dissolve it to obtain a clear solution. 9.9 g (125 mmol) of pyridine was added, and 11.9 g (62.5 mmol) of p-toluenesulfonyl chloride was dissolved in 20 mL of dichloromethane and added dropwise to the above system. After the addition was complete, the mixture was stirred at room temperature for 3 hours. TLC showed that the reaction of the starting material was basically complete. 300 mL of water was added, and the mixture was extracted with dichloromethane (200 mL * 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluting agent: ethyl acetate: petroleum ether = 1:3) to obtain 15.5 g (75% yield) of product.

[0213] Step 5: Preparation of compound L3-7

[0214] 15.1 g (45.7 mmol) of compound L3-6 was placed in a 500 mL single-necked flask, and 100 mL of LDM was added to dissolve it to obtain a clear solution. 9.3 g (crude product) of compound L3-4 was added to the above system, and the mixture was heated to 80 °C and stirred for 16 hours. TLC analysis showed that the reaction of the starting material was basically complete. 700 mL of methyl tert-butyl ether was added, and a solid precipitated. The mixture was filtered, and the methyl tert-butyl ether was removed from the filtrate under reduced pressure. 500 mL of water was added, and the mixture was extracted with ethyl acetate (100 mL * 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: ethyl acetate: petroleum ether = 1:3) to obtain 9.8 g (62% yield) of the product.

[0215] Step 6: Preparation of compound L3-8

[0216] 9.8 g (25.7 mmol) of compound L3-7 and 7.1 g (51.4 mmol) of potassium carbonate were placed in a 500 mL single-necked flask, 150 mL of LDM was added, and 4.3 g (30.8 mmol) of iodomethane was added to the flask. The mixture was heated to 90 °C and stirred for 16 hours. TLC showed that the reaction of the starting material was basically complete. 800 mL of water was added, and the mixture was extracted with ethyl acetate (100 mL * 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluting agent: ethyl acetate: petroleum ether = 1:3) to obtain 4.9 g (yield 49%) of product.

[0217] Step 7: Preparation of compound L3

[0218] Take 4.9 g (12.4 mmol) of compound L3-8 into a 250 mL single-necked flask, add 100 mL of methanol, dissolve to obtain a clear solution, add 0.5 g of palladium on carbon catalyst into the solution, stir at room temperature for 12 hours under hydrogen atmosphere, and TLC shows that the starting material has basically reacted completely. Filter, concentrate and dry the filtrate, and use it directly for the next step of the reaction.

[0219] Example 6: Preparation of compound L4

[0220]

[0221] Step 1: Preparation of compound L4-2

[0222] 20 g (144 mmol) of compound L4-1 and 18.1 g (216 mmol) of 3,4-dihydro-2H-pyran were placed in a 500 mL single-necked flask, and 200 mL of dichloromethane was added. 2.5 g (14.4 mmol) of p-toluenesulfonic acid was also added. The mixture was stirred overnight at room temperature. TLC analysis showed that the reaction proceeds were essentially complete. 300 mL of water was added, and the mixture was extracted with dichloromethane (100 mL x 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: ethyl acetate: petroleum ether = 1:30) to obtain 29.5 g (92% yield) of the product.

[0223] Step 2: Preparation of compound L4-3

[0224] 29.5 g (132 mmol) of compound L4-2 and 16.7 g (110 mmol) of 2-benzyloxyethanol were placed in a 500 mL single-necked flask, and 200 mL (50%) sodium hydroxide solution was added. 5.3 g (16.5 mmol) of tetrabutylammonium bromide was also added. The mixture was stirred at 70 °C for 6 hours. TLC analysis showed that the reaction was essentially complete. 800 mL of water was added, and the mixture was extracted with ethyl acetate (100 mL * 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: ethyl acetate: petroleum ether = 1:10) to obtain 24.6 g (76% yield) of the product.

[0225] Step 3: Preparation of compound L4-4

[0226] 24.6 g (84 mmol) of compound L4-3 was placed in a 500 mL single-necked flask, 200 mL of methanol was added, and 100 mL of 1 M hydrochloric acid solution was added to the flask. The mixture was stirred at room temperature for 6 hours. TLC showed that the reaction of the starting material was basically complete. The solvent was removed by vacuum distillation, and 400 mL of water was added. The mixture was extracted with ethyl acetate (100 mL * 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed by vacuum distillation of the filtrate to obtain 17.6 g (96% yield) of product, which was directly used in the next reaction.

[0227] Step 4: Preparation of compound L4-5

[0228] 17.6 g (84 mmol) of compound L4-4, 32.8 g (168 mmol) of tert-butyl bromoacetate, and 27 g (84 mmol) of tetrabutylammonium bromide were placed in a 1 L single-necked flask. 300 mL of dichloromethane was added, along with 300 mL of 35% sodium hydroxide solution. The mixture was stirred overnight at room temperature. TLC analysis showed that the reaction of the starting material was basically complete. 300 mL of water was added, and the mixture was extracted with dichloromethane (200 mL * 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: ethyl acetate: petroleum ether = 1:4) to obtain 12.5 g (yield 46%) of the product.

[0229] Step 5: Preparation of compound L4-6

[0230] Take 12.5 g (38.6 mmol) of compound L4-5 into a 250 mL single-necked flask, add 100 mL of methanol, dissolve to obtain a clear solution, add 1.2 g of palladium on carbon catalyst into the solution, stir at room temperature for 12 hours under hydrogen atmosphere, TLC shows that the starting material has basically reacted completely, filter, concentrate and dry the filtrate, and use it directly for the next step of the reaction;

[0231] Step 6: Preparation of compound L4-7

[0232] 9.1 g of compound L4-6 (crude product) was placed in a 500 mL single-necked flask, and 100 mL of dichloromethane was added to dissolve it to obtain a clear solution. 6.2 g (80 mmol) of pyridine was added, and 8.3 g (42 mmol) of p-toluenesulfonyl chloride was dissolved in 20 mL of dichloromethane and added dropwise to the above system. After the addition was complete, the mixture was stirred at room temperature for 3 hours. TLC showed that the reaction of the starting material was basically complete. 300 mL of water was added, and the mixture was extracted with dichloromethane (200 mL * 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluting agent: ethyl acetate: petroleum ether = 1:5) to obtain 11.4 g (yield 76%) of product.

[0233] 11.4 g (29.4 mmol) of the intermediate was placed in a 500 mL single-necked flask, and 80 mL of LDM was added to dissolve it to obtain a clear solution. 6.5 g (35.2 mmol) of potassium phthalimide was added to the above system, and the mixture was heated to 80 °C and stirred for 16 hours. TLC analysis showed that the reaction of the starting material was basically complete. 700 mL of methyl tert-butyl ether was added, and a solid precipitated. The mixture was filtered, and the methyl tert-butyl ether was removed from the filtrate under reduced pressure. 500 mL of water was added, and the mixture was extracted with ethyl acetate (100 mL * 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: ethyl acetate: petroleum ether = 1:4) to obtain 8 g (75% yield) of the product.

[0234] Step 7: Preparation of compound L4

[0235] 8 g (22 mmol) of compound L4-7 was placed in a 500 mL single-necked flask, and 150 mL of methanol was added to dissolve it, yielding a clear solution. 1.8 g (80%, 44 mmol) of hydrazine hydrate was then added to the solution, and the mixture was heated to 80 °C and stirred for 6 hours. TLC analysis showed that the reaction proceeds were essentially complete, and the solvent was removed under reduced pressure. 200 mL of methyl tert-butyl ether was added, and the mixture was stirred for 1 hour. The mixture was filtered, and the filter cake was washed with methyl tert-butyl ether (50 mL x 3). The filtrate was concentrated and dried, and used directly in the next reaction step.

[0236] Example 7: Preparation of compound L5

[0237]

[0238] Step 1: Preparation of compound L5-2

[0239] 10 g (45.5 mmol) of compound L5-1, 7.4 g (45.5 mmol) of N-hydroxyphthalimide, and 17.9 g (68.3 mmol) of triphenylphosphine were placed in a 500 mL single-necked flask. 200 mL of tetrahydrofuran was added, and the mixture was purged with nitrogen three times. The temperature was lowered to below 0 °C, and 11.9 g (68.3 mmol) of DEAD was added dropwise under a nitrogen atmosphere. After the addition was complete, the mixture was stirred overnight at room temperature. TLC showed that the reaction of the starting material was basically complete. 500 mL of water was added, and the mixture was extracted with ethyl acetate (100 mL * 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: ethyl acetate: petroleum ether = 1:8) to obtain 14.1 g (yield 85%) of the product.

[0240] Step 2: Preparation of compound L5

[0241] 14.1 g (38.6 mmol) of compound L5-2 was placed in a 500 mL single-necked flask, and 150 mL of methanol was added to dissolve it, yielding a clear solution. 3.1 g (80%, 77.2 mmol) of hydrazine hydrate was added to the solution, and the mixture was heated to 80 °C and stirred for 6 hours. TLC analysis showed that the reaction proceeds were essentially complete, and the solvent was removed under reduced pressure. 200 mL of methyl tert-butyl ether was added, and the mixture was stirred for 1 hour. The mixture was filtered, and the filter cake was washed with methyl tert-butyl ether (50 mL x 3). The filtrate was concentrated and dried, and used directly in the next reaction step.

[0242] Example 8: Preparation of compound L6

[0243]

[0244] Step 1: Preparation of compound L6-2

[0245] 20 g (90 mmol) of compound L6-1 was placed in a 500 mL single-necked flask, and 100 mL of dichloromethane was added to dissolve it to obtain a clear solution. 28.4 g (360 mmol) of pyridine and 1.1 g (9 mmol) of DMAP were added. Separately, 38.6 g (198 mmol) of p-toluenesulfonyl chloride was dissolved in 50 mL of dichloromethane and added dropwise to the above system. After the addition was complete, the mixture was stirred at room temperature for 3 hours. TLC showed that the reaction of the starting material was basically complete. 300 mL of water was added, and the mixture was extracted with dichloromethane (200 mL * 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluting agent: ethyl acetate: petroleum ether = 1:8) to obtain 29.1 g (61% yield) of product.

[0246] Step 2: Preparation of compound L6-3

[0247] 29.1 g (55 mmol) of compound L6-2 was placed in a 500 mL single-necked flask, and 80 mL of LDM was added to dissolve it to obtain a clear solution. 22.4 g (121 mmol) of potassium phthalimide was added to the above system, and the mixture was heated to 80 °C and stirred for 16 hours. TLC analysis showed that the reaction of the starting material was basically complete. 700 mL of methyl tert-butyl ether was added, and a solid precipitated. The mixture was filtered, and the methyl tert-butyl ether was removed from the filtrate under reduced pressure. 500 mL of water was added, and the mixture was extracted with ethyl acetate (100 mL * 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: ethyl acetate: petroleum ether = 1:8) to obtain 17.7 g (67% yield) of the product.

[0248] Step 3: Preparation of compound L6

[0249] 17.7 g (36.8 mmol) of compound L6-3 was placed in a 500 mL single-necked flask, and 150 mL of methanol was added to dissolve it, yielding a clear solution. 3.0 g (80%, 73.8 mmol) of hydrazine hydrate was added to the solution, and the mixture was heated to 80 °C and stirred for 6 hours. TLC analysis showed that the reaction proceeds were essentially complete. The solvent was removed by vacuum evaporation. 200 mL of methyl tert-butyl ether was added, and the mixture was stirred for 1 hour. The mixture was filtered, and the filter cake was washed with methyl tert-butyl ether (50 mL x 3). The filtrate was concentrated and dried, and used directly in the next reaction step.

[0250] Example 9: Preparation of compound L7

[0251]

[0252] Step 1: Preparation of compound L7-2

[0253] 10 g (62.5 mmol) of compound L7-1, 10.2 g (62.5 mmol) of N-hydroxyphthalimide, and 24.6 g (93.8 mmol) of triphenylphosphine were placed in a 500 mL single-necked flask. 200 mL of tetrahydrofuran was added, and the mixture was purged with nitrogen three times. The temperature was lowered to below 0 °C, and 16.3 g (93.8 mmol) of DEAD was added dropwise under a nitrogen atmosphere. After the addition was complete, the mixture was stirred overnight at room temperature. TLC showed that the reaction of the starting material was basically complete. 500 mL of water was added, and the mixture was extracted with ethyl acetate (100 mL * 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: ethyl acetate: petroleum ether = 1:6) to obtain 15.3 g (80% yield) of the product.

[0254] Step 2: Preparation of compound L7

[0255] 15.3 g (50.2 mmol) of compound L7-2 was placed in a 500 mL single-necked flask, and 150 mL of methanol was added to dissolve it, yielding a clear solution. 4.1 g (80%, 100.6 mmol) of hydrazine hydrate was added to the solution, and the mixture was heated to 80 °C and stirred for 6 hours. TLC analysis showed that the reaction proceeds were essentially complete, and the solvent was removed under reduced pressure. 200 mL of methyl tert-butyl ether was added, and the mixture was stirred for 1 hour. The mixture was filtered, and the filter cake was washed with methyl tert-butyl ether (50 mL x 3). The filtrate was concentrated and dried, and used directly in the next reaction step.

[0256] Example 10: Preparation of compound L8

[0257]

[0258] Step 1: Preparation of compound L8-2

[0259] 10 g (62.5 mmol) of compound L8-1, 10.2 g (62.5 mmol) of N-hydroxyphthalimide, and 24.6 g (93.8 mmol) of triphenylphosphine were placed in a 500 mL single-necked flask. 200 mL of tetrahydrofuran was added, and the mixture was purged with nitrogen three times. The temperature was lowered to below 0 °C, and 16.3 g (93.8 mmol) of DEAD was added dropwise under a nitrogen atmosphere. After the addition was complete, the mixture was stirred overnight at room temperature. TLC showed that the reaction of the starting material was basically complete. 500 mL of water was added, and the mixture was extracted with ethyl acetate (100 mL * 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: ethyl acetate: petroleum ether = 1:6) to obtain 15.3 g (yield 80%) of the product.

[0260] Step 2: Preparation of compound L8-3

[0261] 15.3 g (50.2 mmol) of compound L8-2 was placed in a 250 mL single-necked flask, 150 mL of dichloromethane was added, and the mixture was cooled to below 0 °C. 30 mL of trifluoroacetic acid was then added dropwise, and the mixture was stirred at room temperature for 6 hours. TLC analysis showed that the reaction was essentially complete. 200 mL of water was added, and the mixture was extracted with dichloromethane (100 mL x 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: dichloromethane:methanol = 10:1) to obtain 8.2 g (66% yield) of the product.

[0262] Step 3: Preparation of compound L8-4

[0263] Take 8.2 g (33 mmol) of compound L8-3, 17.1 g (45 mmol) of HATU, and 7.7 g (60 mmol) of DIEA into a 500 mL single-necked flask, add 100 mL of LDMF, stir at room temperature for 1 hour, then add 4.4 g (30 mmol) of tert-butyl 2-(aminooxy)acetate and stir at room temperature overnight. TLC showed that the reaction of the starting material was basically complete. Add 400 mL of water and extract with ethyl acetate (200 mL * 3). Wash the organic phase with saturated brine, dry with sodium sulfate, filter, evaporate the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluting agent: ethyl acetate: petroleum ether = 1:6) to obtain 7.7 g (yield 68%) of product.

[0264] Step 4: Preparation of compound L8

[0265] 7.7 g (20.4 mmol) of compound L8-4 was placed in a 250 mL single-necked flask, and 100 mL of methanol was added to dissolve it, yielding a clear solution. Separately, 1.6 g (80%, 40.8 mmol) of hydrazine hydrate was added to the solution, and the mixture was heated to 80 °C and stirred for 6 hours. TLC analysis showed that the reaction proceeds were essentially complete, and the solvent was removed under reduced pressure. 200 mL of methyl tert-butyl ether was added, and the mixture was stirred for 1 hour. The mixture was filtered, and the filter cake was washed with methyl tert-butyl ether (50 mL x 3). The filtrate was concentrated and dried, and used directly in the next reaction step.

[0266] Example 11: Preparation of compound L9

[0267]

[0268] Step 1: Preparation of compound L9-2

[0269] 10 g (21.4 mmol) of compound L9-1, 10.1 g (26.7 mmol) of HATU, and 4.6 g (35.6 mmol) of DIEA were placed in a 500 mL single-necked flask. 100 mL of LDMF was added, and the mixture was stirred at room temperature for 1 hour. 2.3 g (17.8 mmol) of tert-butyl 2-(aminooxy)acetate was added to the mixture, and the mixture was stirred at room temperature overnight. TLC analysis showed that the reaction was basically complete. 400 mL of water was added, and the mixture was extracted with ethyl acetate (200 mL * 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: ethyl acetate: petroleum ether = 1:5) to obtain 7.2 g (70% yield) of the product.

[0270] Step 2: Preparation of compound L9-3

[0271] 7.2 g (12.4 mmol) of compound L9-2 was placed in a 250 mL single-necked flask, 100 mL of dichloromethane was added, and 10.5 g (124 mmol) of piperidine was added to the flask. The mixture was stirred overnight at room temperature. TLC showed that the reaction of the starting material was basically complete. The solvent was removed from the filtrate under reduced pressure, 200 mL of water was added, and the mixture was extracted with dichloromethane (200 mL * 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: dichloromethane: methanol = 15:1) to obtain 4.6 g (yield 92%) of product.

[0272] Step 3: Preparation of compound L9-4

[0273] 4.6 g (12.8 mmol) of compound L9-3 and 2.5 g (12.8 mmol) of diethylphosphoacetic acid were placed in a 250 mL single-necked flask. 100 mL of dichloromethane, 0.16 g (1.3 mmol) of DMAP, and 2.4 g (12.8 mmol) of EDCI were added. The mixture was stirred overnight at room temperature. TLC showed that the reaction of the starting material was basically complete. 200 mL of water was added, and the mixture was extracted with dichloromethane (200 mL * 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluting agent: ethyl acetate: petroleum ether = 1:8) to obtain 3.1 g (yield 45%) of product.

[0274] Step 4: Preparation of compound L9

[0275] 3.1 g (5.8 mmol) of compound L9-4 was placed in a 250 mL single-necked flask, and 80 mL of hydrochloric acid-methanol solution was added. The mixture was stirred at room temperature for 2 hours. TLC showed that the reaction of the starting material was basically complete. The solvent was removed by vacuum distillation, and the mixture was distilled multiple times with dichloromethane (100 mL * 3) to obtain 2.7 g of tert-butyl 2-(6-amino-2-(2-(diethoxyphosphoryl)acetamide)hexamethylene)acetate hydrochloride, which was directly used in the next step of the reaction.

[0276] Example 12: Preparation of compound L10

[0277]

[0278] Step 1: Preparation of compound L10-2

[0279] 10 g (48.1 mmol) of compound L10-1, 8.0 g (57.7 mmol) of 3-bromo-1-propanol, and 13.3 g (96.2 mmol) of potassium carbonate were placed in a 500 mL single-necked flask. 200 mL of LDM was added, and the mixture was heated to 90 °C and stirred overnight. TLC analysis showed that the reaction of the starting material was basically complete. 800 mL of water was added, and the mixture was extracted with ethyl acetate (200 mL * 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: ethyl acetate: petroleum ether = 1:5) to give 6.7 g (yield 52%) of product.

[0280] Step 2: Preparation of compound L10-3

[0281] 6.7 g (25.2 mmol) of compound L10-2, 9.8 g (50.4 mmol) of tert-butyl bromoacetate, and 8.1 g (25.2 mmol) of tetrabutylammonium bromide were placed in a 500 mL single-necked flask. 100 mL of dichloromethane was added, along with 100 mL of 35% sodium hydroxide solution. The mixture was stirred overnight at room temperature. TLC analysis showed that the reaction of the starting material was basically complete. 300 mL of water was added, and the mixture was extracted with dichloromethane (200 mL * 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: ethyl acetate: petroleum ether = 1:4) to obtain 3.9 g (yield 41%) of the product.

[0282] Step 3: Preparation of compound L10

[0283] Take 3.9 g (10.3 mmol) of compound L10-3 in a 250 mL single-necked flask, add 100 mL of methanol, dissolve to obtain a clear solution, add 1.1 g of palladium on carbon catalyst to it, stir at room temperature for 12 hours under hydrogen atmosphere, and TLC shows that the starting material has basically reacted completely. Filter, concentrate and dry the filtrate, and use it directly for the next step of the reaction.

[0284] Example 13: Preparation of compound L11

[0285]

[0286] Step 1: Preparation of compound L11-2

[0287] 4.3 g (69.8 mmol) of 1,2-ethylene glycol was added to a 500 mL single-necked flask, along with 200 mL of tetrahydrofuran. The mixture was kept under nitrogen protection and cooled to 0 °C. Sodium hydride 31 (60%, 76.8 mmol) was added to the flask, and the mixture was brought back to room temperature and stirred for 3 hours. 115 g (58 mmol) of L11-11 was added to the flask, and the mixture was heated to 60 °C and stirred overnight. TLC analysis showed that the reaction was essentially complete. 400 mL of water was added, and the mixture was extracted with ethyl acetate (200 mL x 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: ethyl acetate: petroleum ether = 1:2) to obtain 9.4 g (68% yield) of the product.

[0288] Step 2: Preparation of compound L11-3

[0289] 9.4 g (39.3 mmol) of compound L11-2 was placed in a 250 mL single-necked flask, and 100 mL of dichloromethane was added to dissolve it to obtain a clear solution. 6.2 g (78.6 mmol) of pyridine was added, and 8.3 g (42 mmol) of p-toluenesulfonyl chloride was dissolved in 20 mL of dichloromethane and added dropwise to the above system. After the addition was complete, the mixture was stirred at room temperature for 3 hours. TLC showed that the reaction of the starting material was basically complete. 300 mL of water was added, and the mixture was extracted with dichloromethane (200 mL * 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluting agent: ethyl acetate: petroleum ether = 1:5) to obtain 13.0 g (yield 84%) of product.

[0290] Step 3: Preparation of compound L11-4

[0291] 13.0 g (33.1 mmol) of compound L11-3 was placed in a 500 mL single-necked flask, and 80 mL of DMF was added to dissolve it to obtain a clear solution. 5.84 g (39.7 mmol) of 2-aminooxyacetic acid tert-butyl ester was added to the above system, and the mixture was heated to 90 °C and stirred for 16 hours. TLC analysis showed that the reaction of the starting material was basically complete. 400 mL of water was added, and the mixture was extracted with ethyl acetate (200 mL * 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: ethyl acetate: petroleum ether = 1:5) to obtain 8.1 g (66% yield) of the product.

[0292] Step 4: Preparation of compound L11-5

[0293] 8.1 g (22 mmol) of compound L11-4 and 6.1 g (44 mmol) of potassium carbonate were placed in a 250 mL single-necked flask, and 100 mL of LDM was added. 3.7 g (26.4 mmol) of iodomethane was also added. The mixture was heated to 90 °C and stirred for 16 hours. TLC analysis showed that the reaction was essentially complete. 600 mL of water was added, and the mixture was extracted with ethyl acetate (100 mL x 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: ethyl acetate: petroleum ether = 1:4) to obtain 4.8 g (57% yield) of the product.

[0294] Step 5: Preparation of compound L11

[0295] Take 4.8 g (12.6 mmol) of compound L11-5 into a 250 mL single-necked flask, add 100 mL of methanol, dissolve to obtain a clear solution, add 0.5 g of palladium on carbon catalyst into the solution, stir at room temperature for 12 hours under hydrogen atmosphere, and TLC shows that the starting material has basically reacted completely. Filter, concentrate and dry the filtrate, and use it directly for the next step of the reaction.

[0296] Example 14: Preparation of compound L12

[0297]

[0298] Step 1: Preparation of compound L12-2

[0299] 10 g (45.5 mmol) of compound L12-1, 10.6 g (54.5 mmol) of tert-butyl bromoacetate, and 12.6 g (91 mmol) of potassium carbonate were placed in a 500 mL single-necked flask. 200 mL of DMF was added, and the mixture was heated to 90 °C and stirred overnight. TLC analysis showed that the reaction of the starting material was basically complete. 800 mL of water was added, and the mixture was extracted with ethyl acetate (200 mL * 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: ethyl acetate: petroleum ether = 1:3) to obtain 11.2 g (74% yield) of the product.

[0300] Step 2: Preparation of compound L12-3

[0301] Take 11.2 g (33.5 mmol) of compound L12-2 into a 250 mL single-necked flask, add 100 mL of methanol, dissolve to obtain a clear solution, add 1.1 g of palladium on carbon catalyst into the solution, stir at room temperature for 12 hours under hydrogen atmosphere, TLC shows that the starting material has basically reacted completely, filter, concentrate and dry the filtrate, and use it directly for the next step of the reaction;

[0302] Step 3: Preparation of compound L12-4

[0303] Take 6.7 g of compound L12-3 (crude product), 10.3 g (40.2 mmol) of N-benzyloxycarbonyl-3-bromoethylamine, and 9.3 g (67 mmol) of potassium carbonate in a 500 mL single-necked flask, add 150 mL of LDM, heat to 90 °C and stir overnight; TLC showed that the reaction of the starting material was basically complete, add 800 mL of water, extract with ethyl acetate (200 mL * 3), wash the organic phase with saturated brine, dry with sodium sulfate, filter, evaporate the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluting agent: ethyl acetate: petroleum ether = 1:5) to obtain 8.0 g (yield 63%) of product;

[0304] Step 4: Preparation of compound L12-5

[0305] Take 8.0 g (21.2 mmol) of compound L12-4 in a 250 mL single-necked flask, add 100 mL of methanol, dissolve to obtain a clear solution, add 0.8 g of palladium on carbon catalyst to it, stir at room temperature for 12 hours under hydrogen atmosphere, TLC shows that the starting material has basically reacted completely, filter, concentrate and dry the filtrate, and use it directly for the next step of the reaction;

[0306] Step 5: Preparation of compound L12-6

[0307] Take 5.7 g (25.2 mmol) of 2-((((benzyloxy)carbonyl)amino)oxy)acetic acid, 12.0 g (31.5 mmol) of HATU, and 5.4 g (42 mmol) of DIEA into a 500 mL single-necked flask, add 100 mL of LDMF, stir at room temperature for 1 hour, and then add 5.2 g (21 mmol) of L12-5 to the flask and stir at room temperature overnight. TLC showed that the reaction of the starting material was basically complete. Add 600 mL of water and extract with ethyl acetate (200 mL * 3). Wash the organic phase with saturated brine, dry with sodium sulfate, filter, evaporate the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluting agent: ethyl acetate: petroleum ether = 1:4) to obtain 6.2 g (yield 66%) of product.

[0308] Step 6: Preparation of compound L12

[0309] Take 6.2 g (13.8 mmol) of compound L12-6 in a 250 mL single-necked flask, add 100 mL of methanol, dissolve to obtain a clear solution, add 0.6 g of palladium on carbon catalyst to it, stir at room temperature for 12 hours under hydrogen atmosphere, TLC shows that the starting material has basically reacted completely, filter, concentrate and dry the filtrate, and use it directly for the next step of the reaction.

[0310] Example 15: Preparation of compound AG-01

[0311]

[0312] Step 1: Preparation of intermediate AG-01-1

[0313] Take 100 mg (0.25 mmol) of D1, 131 mg (0.35 mmol) of HATU, and 60 mg (0.46 mmol) of DIEA into a 50 mL single-necked flask, add 20 mL of LDM, and stir at room temperature for 1 hour; separately add 49 mg (0.23 mmol) of L1 to the flask, and stir at room temperature overnight; TLC showed that the reaction of the starting material was basically complete, add 100 mL of water, extract with ethyl acetate (50 mL * 3), wash the organic phase with saturated brine, dry with sodium sulfate, filter, evaporate the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluent: ethyl acetate: petroleum ether = 1:5) to obtain 88 mg (yield 64%) of product;

[0314] Step 2: Preparation of intermediate AG-01-2

[0315] Take 88 mg (0.15 mmol) of intermediate AG-01-1 in a 50 mL single-necked flask, add 10 mL of dichloromethane, cool to below 0 °C, and add 3 mL of trifluoroacetic acid dropwise. Stir at room temperature for 6 hours. TLC showed that the reaction of the starting material was basically complete. Add 20 mL of water and extract with dichloromethane (20 mL * 3). Wash the organic phase with saturated brine, dry with sodium sulfate, filter, remove the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluent: dichloromethane: methanol = 10:1) to obtain 69 mg (yield 86%) of intermediate AG-01-2.

[0316] Step 3: Preparation of compound AG-01

[0317] Take 69 mg (0.13 mmol) of intermediate AG-01-2, 66 mg (0.17 mmol) of HATU, and 31 mg (0.24 mmol) of DIEA into a 50 mL single-necked flask, add 10 mL of DMF, and stir at room temperature for 1 hour; separately add 51 mg (0.12 mmol) of P1 into the flask, and stir at room temperature overnight; TLC showed that the reaction of the starting material was basically complete, add 100 mL of water, extract with ethyl acetate (50 mL * 3), wash the organic phase with saturated brine, dry with sodium sulfate, filter, evaporate the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluting agent: ethyl acetate: petroleum ether = 1:4) to obtain 62 mg (yield 54%) of AG-01. 1HNMR(400MHz, d6-DMSO): δ = 9.06(s, 1H), 8.08(s, 3H), 7.76-7.65(d, 2H), 7.56-7.43(d, 4H), 7.33-7.25(d, 2H), 4.71-4.63(m,4H), 4.55(s, 1H), 4.43-4.36(m, 3H), 3.72-3.49(m, 8H), 3.12-2.79(m, 4H), 2.53(s, 3H), 2.48-2.43(m, 2H), 2.40(s, 3H), 2.34(s, 3H), 2.25(s, 3H), 1.52-1.41(m, 4H), 1.32-1.29(m, 2H), 0.96(s, 9H), [M+H] + m / z = 957.8.

[0318] Example 16: Preparation of compound AG-02

[0319]

[0320] Step 1: Preparation of intermediate AG-02-1

[0321] Take 112 mg (0.28 mmol) of D1, 144 mg (0.38 mmol) of HATU, and 65 mg (0.5 mmol) of DIEA into a 50 mL single-necked flask, add 20 mL of DMF, and stir at room temperature for 1 hour; separately add 44 mg (0.25 mmol) of L7 into the flask, and stir at room temperature overnight; TLC showed that the reaction of the starting material was basically complete, add 100 mL of water, extract with ethyl acetate (50 mL * 3), wash the organic phase with saturated brine, dry with sodium sulfate, filter, evaporate the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluent: ethyl acetate: petroleum ether = 1:5) to obtain 95 mg (yield 68%) of product;

[0322] Step 2: Preparation of intermediate AG-02-2

[0323] Take 95 mg (0.17 mmol) of intermediate AG-02-1 in a 50 mL single-necked flask, add 10 mL of dichloromethane, cool to below 0 °C, and add 3 mL of trifluoroacetic acid dropwise. Stir at room temperature for 6 hours. TLC showed that the reaction of the starting material was basically complete. Add 20 mL of water and extract with dichloromethane (20 mL * 3). Wash the organic phase with saturated brine, dry with sodium sulfate, filter, remove the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluent: dichloromethane: methanol = 10:1) to obtain 75 mg (yield 88%) of intermediate AG-02-2.

[0324] Step 3: Preparation of compound AG-02

[0325] Take 75 mg (0.15 mmol) of intermediate AG-02-2, 78 mg (0.20 mmol) of HATU, and 36 mg (0.28 mmol) of DIEA into a 50 mL single-necked flask, add 10 mL of LDMF, and stir at room temperature for 1 hour; separately add 59 mg (0.14 mmol) of P1 into the flask, and stir at room temperature overnight; TLC showed that the reaction of the starting material was basically complete, add 100 mL of water, extract with ethyl acetate (50 mL * 3), wash the organic phase with saturated brine, dry with sodium sulfate, filter, evaporate the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluting agent: ethyl acetate: petroleum ether = 1:4) to obtain 73 mg (yield 59%) of AG-02. 1 HNMR(400MHz, d6-DMSO): δ = 9.08(s,1H), 8.05(s,3H), 7.73-7.64(d, 2H), 7.52-7.44(d, 4H), 7.38-7.29(d, 2H), 4.77-4.65(m, 1H),4.58(s, 1H), 4.45-4.38(m, 3H), 3.77-3.46(m, 6H), 3.15-2.77(m, 2H), 2.55(s,3H), 2.42(s, 3H), 2.36(s, 3H), 2.24(s, 3H), 1.55-1.47(m, 4H), 1.35-1.28(m,2H), 0.91(s, 9H), [M+H] + m / z = 915.6.

[0326] Example 17: Preparation of compound AG-03

[0327]

[0328] Step 1: Preparation of intermediate AG-03-1

[0329] Take 112 mg (0.28 mmol) of D1, 144 mg (0.38 mmol) of HATU, and 65 mg (0.5 mmol) of DIEA into a 50 mL single-necked flask, add 20 mL of DMF, and stir at room temperature for 1 hour; separately add 62 mg (0.25 mmol) of L2 to the flask, and stir at room temperature overnight; TLC showed that the reaction of the starting materials was basically complete, add 100 mL of water, extract with ethyl acetate (50 mL * 3), wash the organic phase with saturated brine, dry with sodium sulfate, filter, evaporate the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluting agent: ethyl acetate: petroleum ether = 1:5) to obtain 99 mg (yield 63%) of product;

[0330] Step 2: Preparation of intermediate AG-03-2

[0331] 99 mg (0.16 mmol) of intermediate AG-03-1 was placed in a 50 mL single-necked flask, 10 mL of dichloromethane was added, and the mixture was cooled to below 0 °C. 3 mL of trifluoroacetic acid was then added dropwise, and the mixture was stirred at room temperature for 6 hours. TLC analysis showed that the reaction was essentially complete. 20 mL of water was added, and the mixture was extracted with dichloromethane (20 mL x 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: dichloromethane:methanol = 10:1) to obtain 74 mg (82% yield) of intermediate AG-03-2.

[0332] Step 3: Preparation of compound AG-03

[0333] Take 74 mg (0.13 mmol) of intermediate AG-03-2, 68 mg (0.18 mmol) of HATU, and 31 mg (0.24 mmol) of DIEA into a 50 mL single-necked flask, add 10 mL of DMF, and stir at room temperature for 1 hour; separately add 51 mg (0.12 mmol) of P1 to the flask, and stir at room temperature overnight; TLC showed that the reaction of the starting material was basically complete, add 100 mL of water, extract with ethyl acetate (50 mL * 3), wash the organic phase with saturated brine, dry with sodium sulfate, filter, evaporate the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluting agent: ethyl acetate: petroleum ether = 1:4) to obtain 72 mg (yield 61%) of AG-03. 1 HNMR(400MHz, d6-DMSO): δ = 9.09(s, 1H), 8.10(s, 3H), 7.78-7.62(d, 2H), 7.55-7.45(d, 4H), 7.38-7.27(d, 2H), 4.73-4.64(m,1H),4.58(s, 1H), 4.44-4.33(m, 3H), 3.75-3.45(m, 6H), 3.14-2.77(m, 4H), 2.55(s, 3H), 2.44-2.43(m, 2H), 2.41(s, 3H), 2.33(s, 3H), 2.29(s, 3H), 2.24(s,3H), 1.55-1.47(m, 4H), 1.34-1.27(m, 2H), 0.93(s, 9H), [M+H] + m / z = 988.5.

[0334] Example 18: Preparation of compound AG-04

[0335]

[0336] Step 1: Preparation of intermediate AG-04-1

[0337] Take 112 mg (0.28 mmol) of D1, 144 mg (0.38 mmol) of HATU, and 65 mg (0.5 mmol) of DIEA into a 50 mL single-necked flask, add 20 mL of LDM, and stir at room temperature for 1 hour; separately add 65 mg (0.25 mmol) of L3 to the flask, and stir at room temperature overnight; TLC showed that the reaction of the starting material was basically complete, add 100 mL of water, extract with ethyl acetate (50 mL * 3), wash the organic phase with saturated brine, dry with sodium sulfate, filter, evaporate the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluent: ethyl acetate: petroleum ether = 1:5) to obtain 106 mg (yield 66%) of product;

[0338] Step 2: Preparation of intermediate AG-04-2

[0339] 106 mg (0.17 mmol) of intermediate AG-04-1 was placed in a 50 mL single-necked flask, 10 mL of dichloromethane was added, and the mixture was cooled to below 0 °C. 3 mL of trifluoroacetic acid was then added dropwise, and the mixture was stirred at room temperature for 6 hours. TLC analysis showed that the reaction was essentially complete. 20 mL of water was added, and the mixture was extracted with dichloromethane (20 mL x 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: dichloromethane:methanol = 10:1) to obtain 86 mg (83% yield) of intermediate AG-04-2.

[0340] Step 3: Preparation of compound AG-04

[0341] Take 86 mg (0.15 mmol) of intermediate AG-04-2, 76 mg (0.20 mmol) of HATU, and 34 mg (0.26 mmol) of DIEA into a 50 mL single-necked flask, add 10 mL of LDMF, and stir at room temperature for 1 hour; separately add 56 mg (0.13 mmol) of P1 into the flask, and stir at room temperature overnight; TLC showed that the reaction of the starting material was basically complete, add 100 mL of water, extract with ethyl acetate (50 mL * 3), wash the organic phase with saturated brine, dry with sodium sulfate, filter, evaporate the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluting agent: ethyl acetate: petroleum ether = 1:4) to obtain 78 mg (yield 60%) of AG-04. 1HNMR(400MHz, d6-DMSO): δ = 9.11(s, 1H), 8.12(s,3H), 7.81-7.72(d, 2H), 7.59-7.47(d, 4H), 7.36-7.24(d, 2H), 4.77-4.65(m, 1H),4.55(s, 1H), 4.49-4.38(m, 3H), 3.73-3.46(m, 6H), 3.18-2.79(m, 4H), 2.57(s,3H), 2.42-2.46(m, 2H), 2.40(s, 3H), 2.36(s, 6H), 2.31(s, 3H), 2.26(s, 3H),1.52-1.48(m, 4H), 1.33-1.26(m, 2H), 0.95(s, 9H), [M+H] + m / z = 1001.3.

[0342] Example 19: Preparation of compound AG-05

[0343]

[0344] Step 1: Preparation of intermediate AG-05-1

[0345] Take 112 mg (0.28 mmol) of D1, 144 mg (0.38 mmol) of HATU, and 65 mg (0.5 mmol) of DIEA into a 50 mL single-necked flask, add 20 mL of LDM, and stir at room temperature for 1 hour; separately add 93 mg (0.42 mmol) of L6 to the flask, and stir at room temperature overnight; TLC showed that the reaction of the starting material was basically complete, add 100 mL of water, extract with ethyl acetate (50 mL * 3), wash the organic phase with saturated brine, dry with sodium sulfate, filter, evaporate the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluent: ethyl acetate: petroleum ether = 1:5) to obtain 91 mg (yield 46%) of product;

[0346] Step 2: Preparation of compound AG-05

[0347] Take 91 mg (0.11 mmol) of intermediate AG-05-1, 52 mg (0.12 mmol) of P1, and 44 mg (0.15 mmol) of triphosgene in a 50 mL single-necked flask, add 10 mL of dichloromethane, cool to 0 °C and stir for 0.5 hours; separately add 26 mg (0.20 mmol) of DIEA dropwise, stir overnight at room temperature; TLC showed that the reaction of the starting material was basically complete, the solvent was removed from the filtrate under reduced pressure, 100 mL of water was added, and the mixture was extracted with ethyl acetate (50 mL * 3), the organic phase was washed with saturated brine, dried over sodium sulfate, filtered, the solvent was removed from the filtrate under reduced pressure, and the residue was purified by column chromatography (eluting agent: ethyl acetate: petroleum ether = 1:4) to obtain 68 mg (yield 62%) of AG-05. 1 HNMR(400MHz,d6-DMSO): δ = 9.03(s, 1H), 8.05(s, 3H), 7.72-7.63(d, 2H), 7.58-7.46(d, 4H),7.35-7.28(d, 2H), 4.75-4.64(m, 1H), 4.58(s, 1H), 4.42-4.34(m, 3H), 3.78-3.51(m, 12H), 3.17-2.72(m, 6H), 2.60(s, 3H), 2.52-2.48(m, 4H), 2.42(s, 3H), 2.37(s, 3H), 2.22(s, 3H), 1.56-1.44(m, 4H), 1.35-1.22(m, 2H), 0.99(s, 9H), [M+H] + m / z = 1060.3.

[0348] Example 20: Preparation of compound AG-06

[0349]

[0350] Step 1: Preparation of intermediate AG-06-1

[0351] Take 112 mg (0.28 mmol) of D1, 144 mg (0.38 mmol) of HATU, and 65 mg (0.5 mmol) of DIEA into a 50 mL single-necked flask, add 20 mL of LDM, and stir at room temperature for 1 hour; separately add 48 mg (0.25 mmol) of L8 into the flask, and stir at room temperature overnight; TLC showed that the reaction of the starting material was basically complete, add 100 mL of water, extract with ethyl acetate (50 mL * 3), wash the organic phase with saturated brine, dry with sodium sulfate, filter, evaporate the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluent: ethyl acetate: petroleum ether = 1:5) to obtain 109 mg (yield 69%) of product;

[0352] Step 2: Preparation of intermediate AG-06-2

[0353] 109 mg (0.17 mmol) of intermediate AG-06-1 was placed in a 50 mL single-necked flask, 10 mL of dichloromethane was added, and the mixture was cooled to below 0 °C. 3 mL of trifluoroacetic acid was then added dropwise, and the mixture was stirred at room temperature for 6 hours. TLC analysis showed that the reaction was essentially complete. 20 mL of water was added, and the mixture was extracted with dichloromethane (20 mL x 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: dichloromethane:methanol = 10:1) to obtain 75 mg (76% yield) of intermediate AG-06-2.

[0354] Step 3: Preparation of compound AG-06

[0355] Take 86 mg (0.15 mmol) of intermediate AG-06-2, 76 mg (0.20 mmol) of HATU, and 34 mg (0.26 mmol) of DIEA into a 50 mL single-necked flask, add 10 mL of DMF, and stir at room temperature for 1 hour; separately add 56 mg (0.13 mmol) of P1 into the flask, and stir at room temperature overnight; TLC showed that the reaction of the starting material was basically complete, add 100 mL of water, extract with ethyl acetate (50 mL * 3), wash the organic phase with saturated brine, dry with sodium sulfate, filter, evaporate the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluting agent: ethyl acetate: petroleum ether = 1:4) to obtain 83 mg (yield 64%) of AG-06. 1 HNMR(400MHz, d6-DMSO): δ = 9.09(s, 1H), 8.06(s, 3H), 7.71-7.63(d, 2H), 7.52-7.45(d, 4H), 7.39-7.28(d, 2H), 4.82-4.73(m,1H), 4.62(s, 1H), 4.54-4.46(m, 3H), 3.88-3.56(m, 6H), 3.25-2.99(m, 4H), 2.68(s, 3H), 2.53-2.49(m, 2H), 2.43(s, 3H), 2.33(s, 3H), 2.28(s, 3H), 1.56-1.47(m, 4H), 1.38-1.25(m, 2H), 0.93(s, 9H), [M+H] + m / z = 988.6.

[0356] Example 21: Preparation of compound AG-07

[0357]

[0358] Step 1: Preparation of intermediate AG-07-1

[0359] Take 112 mg (0.28 mmol) of D1, 144 mg (0.38 mmol) of HATU, and 65 mg (0.5 mmol) of DIEA into a 50 mL single-necked flask, add 20 mL of DMF, and stir at room temperature for 1 hour; separately add 37 mg (0.25 mmol) of 2-aminooxyacetic acid tert-butyl ester, and stir at room temperature overnight; TLC showed that the reaction of the starting material was basically complete, add 100 mL of water, extract with ethyl acetate (50 mL * 3), wash the organic phase with saturated brine, dry with sodium sulfate, filter, evaporate the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluent: ethyl acetate: petroleum ether = 1:5) to obtain 94 mg (yield 71%) of product;

[0360] Step 2: Preparation of intermediate AG-07-2

[0361] Take 94 mg (0.18 mmol) of intermediate AG-07-1 in a 50 mL single-necked flask, add 10 mL of dichloromethane, cool to below 0 °C, and add 3 mL of trifluoroacetic acid dropwise. Stir at room temperature for 6 hours. TLC showed that the reaction of the starting material was basically complete. Add 20 mL of water and extract with dichloromethane (20 mL * 3). Wash the organic phase with saturated brine, dry with sodium sulfate, filter, evaporate the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluent: dichloromethane: methanol = 10:1) to obtain 65 mg (yield 77%) of intermediate AG-07-2.

[0362] Step 3: Preparation of compound AG-07

[0363] Take 86 mg (0.14 mmol) of intermediate AG-07-2, 71 mg (0.19 mmol) of HATU, and 31 mg (0.24 mmol) of DIEA into a 50 mL single-necked flask, add 10 mL of LDMF, and stir at room temperature for 1 hour; separately add 52 mg (0.12 mmol) of P1 to the flask, and stir at room temperature overnight; TLC showed that the reaction of the starting material was basically complete, add 100 mL of water, extract with ethyl acetate (50 mL * 3), wash the organic phase with saturated brine, dry with sodium sulfate, filter, evaporate the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluting agent: ethyl acetate: petroleum ether = 1:4) to obtain 67 mg (yield 61%) of AG-07; 1HNMR(400MHz, d6-DMSO) δ = 9.11(s, 1H), 8.03(s,3H), 7.82-7.68(d, 2H), 7.61-7.56(d, 4H), 7.43-7.33(d, 2H), 4.77-4.68(m, 1H),4.59(s, 1H), 4.47-4.35(m, 3H), 3.22-2.83(m, 2H), 2.63(s, 3H), 2.51-2.47(m,2H), 2.38(s, 3H), 2.26(s, 3H), 2.12(s, 3H), 1.58-1.44(m, 4H), 1.28-1.15(m,2H), 0.92(s, 9H), [M+H] + m / z = 887.3.

[0364] Example 22: Preparation of compound AG-08

[0365]

[0366] Step 1: Preparation of intermediate AG-08-1

[0367] Take 228 mg (0.57 mmol) of D1, 296 mg (0.78 mmol) of HATU, and 134 mg (1.04 mmol) of DIEA into a 50 mL single-necked flask, add 20 mL of LDM, and stir at room temperature for 1 hour; separately add 76 mg (0.52 mmol) of 2-aminooxyacetic acid tert-butyl ester, and stir at room temperature overnight; TLC showed that the reaction of the starting material was basically complete, add 100 mL of water, extract with ethyl acetate (50 mL * 3), wash the organic phase with saturated brine, dry with sodium sulfate, filter, evaporate the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluent: ethyl acetate: petroleum ether = 1:5) to obtain 201 mg (yield 73%) of product;

[0368] Step 2: Preparation of intermediate AG-08-2

[0369] 201 mg (0.38 mmol) of intermediate AG-08-1 was placed in a 50 mL single-necked flask, 10 mL of dichloromethane was added, and the mixture was cooled to below 0 °C. 3 mL of trifluoroacetic acid was then added dropwise, and the mixture was stirred at room temperature for 6 hours. TLC analysis showed that the reaction was essentially complete. 20 mL of water was added, and the mixture was extracted with dichloromethane (20 mL x 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: dichloromethane:methanol = 10:1) to obtain 142 mg (79% yield) of intermediate AG-08-2.

[0370] Step 3: Preparation of intermediate AG-08-3

[0371] 142 mg (0.38 mmol) of intermediate AG-08-2, 193 mg (0.51 mmol) of HATU, and 88 mg (0.68 mmol) of DIEA were placed in a 50 mL single-necked flask, and 20 mL of LDMF was added. The mixture was stirred at room temperature for 1 hour. Separately, 51 mg (0.34 mmol) of 2-aminooxyacetic acid tert-butyl ester was added to the mixture, and the mixture was stirred at room temperature overnight. TLC analysis showed that the reaction of the starting material was basically complete. 100 mL of water was added, and the mixture was extracted with ethyl acetate (50 mL * 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluting agent: ethyl acetate: petroleum ether = 1:5) to obtain 139 mg (yield 68%) of the product.

[0372] Step 4: Preparation of compound AG-08-4

[0373] 139 mg (0.25 mmol) of intermediate AG-08-3 was placed in a 50 mL single-necked flask, 10 mL of dichloromethane was added, and the mixture was cooled to below 0 °C. 3 mL of trifluoroacetic acid was then added dropwise, and the mixture was stirred at room temperature for 6 hours. TLC analysis showed that the reaction of the starting material was basically complete. 20 mL of water was added, and the mixture was extracted with dichloromethane (20 mL * 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: dichloromethane: methanol = 10:1) to obtain 106 mg (76% yield) of intermediate AG-08-4.

[0374] Step 5: Preparation of compound AG-08

[0375] Take 106 mg (0.19 mmol) of intermediate AG-08-4, 103 mg (0.27 mmol) of HATU, and 46 mg (0.36 mmol) of DIEA into a 50 mL single-necked flask, add 10 mL of DMF, and stir at room temperature for 1 hour; separately add 75 mg (0.18 mmol) of P1 to the flask, and stir at room temperature overnight.

[0376] TLC analysis showed that the reaction of the raw materials was basically complete. 100 mL of water was added, and the mixture was extracted with ethyl acetate (50 mL * 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: ethyl acetate: petroleum ether = 1:4) to obtain 100 mg (yield 58%) of AG-08. 1HNMR(400MHz, d6-DMSO): δ = 9.05(s, 1H),8.06(s, 3H), 7.81-7.64(d, 2H), 7.55-7.48(d, 4H), 7.40-7.36(d, 2H), 4.71-4.62(m, 1H), 4.62(s, 1H), 4.54-4.43(m, 3H), 3.35-3.16(m, 4H), 2.84(s, 3H), 2.65-2.53(m, 2H), 2.47(s, 3H), 2.32(s, 3H), 2.24(s, 3H), 1.66-1.51(m, 4H), 1.35-1.27(m, 2H), 1.01(s, 9H), [M+H] + m / z = 960.2.

[0377] Example 23: Preparation of compound AG-09

[0378]

[0379] Step 1: Preparation of intermediate AG-09-1

[0380] Take 112 mg (0.28 mmol) of D1, 144 mg (0.38 mmol) of HATU, and 65 mg (0.5 mmol) of DIEA into a 50 mL single-necked flask, add 20 mL of LDM, and stir at room temperature for 1 hour; separately add 59 mg (0.25 mmol) of L5 to the flask, and stir at room temperature overnight; TLC showed that the reaction of the starting material was basically complete, add 100 mL of water, extract with ethyl acetate (50 mL * 3), wash the organic phase with saturated brine, dry with sodium sulfate, filter, evaporate the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluent: ethyl acetate: petroleum ether = 1:5) to obtain 94 mg (yield 61%) of product;

[0381] Step 2: Preparation of intermediate AG-09-2

[0382] Take 94 mg (0.15 mmol) of intermediate AG-09-1 in a 50 mL single-necked flask, add 10 mL of dichloromethane, cool to below 0 °C, and add 3 mL of trifluoroacetic acid dropwise. Stir at room temperature for 6 hours. TLC showed that the reaction of the starting material was basically complete. Add 20 mL of water and extract with dichloromethane (20 mL * 3). Wash the organic phase with saturated brine, dry with sodium sulfate, filter, remove the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluent: dichloromethane: methanol = 10:1) to obtain 69 mg (yield 81%) of intermediate AG-09-2.

[0383] Step 3: Preparation of compound AG-09

[0384] Take 69 mg (0.12 mmol) of intermediate AG-09-2, 63 mg (0.17 mmol) of HATU, and 28 mg (0.22 mmol) of DIEA into a 50 mL single-necked flask, add 10 mL of DMF, and stir at room temperature for 1 hour; separately add 48 mg (0.11 mmol) of P1 into the flask, and stir at room temperature overnight; TLC showed that the reaction of the starting material was basically complete, add 100 mL of water, extract with ethyl acetate (50 mL * 3), wash the organic phase with saturated brine, dry with sodium sulfate, filter, evaporate the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluent: ethyl acetate: petroleum ether = 1:4) to obtain 60 mg (yield 56%) of AG-09. 1 HNMR(400MHz,d6-DMSO): δ = 9.06(s, 1H), 8.12(s, 3H), 7.77-7.65(d, 2H), 7.58-7.47(d, 4H), 7.35-7.24(d, 2H), 4.75-4.67(m,1H), 4.52(s, 1H), 4.41-4.34(m, 3H), 3.74-3.43(m, 6H), 3.17-2.75(m, 4H), 2.57(s, 3H), 2.47-2.41(m, 2H), 2.35(s, 3H), 2.24(s, 3H), 2.17(s, 3H), 2.06(s,3H), 1.59-1.45(m, 4H), 1.33-1.25(m, 2H), 0.98(s, 9H), [M+H] + m / z = 975.7.

[0385] Example 24: Preparation of compound AG-10

[0386]

[0387] Step 1: Preparation of intermediate AG-10-1

[0388] Take 100 mg (0.25 mmol) of D1, 131 mg (0.35 mmol) of HATU, and 60 mg (0.46 mmol) of DIEA into a 50 mL single-necked flask, add 20 mL of DMF, and stir at room temperature for 1 hour; separately add 57 mg (0.23 mmol) of L10 to the flask, and stir at room temperature overnight; TLC showed that the reaction of the starting material was basically complete, add 100 mL of water, extract with ethyl acetate (50 mL * 3), wash the organic phase with saturated brine, dry with sodium sulfate, filter, evaporate the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluting agent: ethyl acetate: petroleum ether = 1:5) to obtain 82 mg (yield 57%) of product;

[0389] Step 2: Preparation of intermediate AG-10-2

[0390] Take 82 mg (0.13 mmol) of intermediate AG-10-1 in a 50 mL single-necked flask, add 10 mL of dichloromethane, cool to below 0 °C, and add 3 mL of trifluoroacetic acid dropwise. Stir at room temperature for 6 hours. TLC showed that the reaction of the starting material was basically complete. Add 20 mL of water and extract with dichloromethane (20 mL * 3). Wash the organic phase with saturated brine, dry with sodium sulfate, filter, remove the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluent: dichloromethane: methanol = 10:1) to obtain 63 mg (yield 84%) of intermediate AG-10-2.

[0391] Step 3: Preparation of compound AG-10

[0392] Take 63 mg (0.11 mmol) of intermediate AG-10-2, 57 mg (0.15 mmol) of HATU, and 26 mg (0.20 mmol) of DIEA into a 50 mL single-necked flask, add 10 mL of LDMF, and stir at room temperature for 1 hour; separately add 43 mg (0.10 mmol) of P1 into the flask, and stir at room temperature overnight; TLC showed that the reaction of the starting material was basically complete, add 100 mL of water, extract with ethyl acetate (50 mL * 3), wash the organic phase with saturated brine, dry with sodium sulfate, filter, evaporate the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluting agent: ethyl acetate: petroleum ether = 1:4) to obtain 55 mg (yield 56%) of AG-10.

[0393] 1HNMR(400MHz,d6-DMSO): δ = 9.12(s, 1H), 8.16(s, 3H), 7.88-7.75(d, 2H), 7.62-7.51(d, 4H), 7.44-7.35(d, 2H), 4.79-4.65(m, 1H), 4.46(s, 1H), 4.35-4.24(m, 3H), 3.85-3.61(m, 8H), 3.33(s, 3H), 3.23-2.88(m, 4H), 2.68(s, 3H), 2.53-2.49(m, 2H), 2.41(s, 3H), 2.35(s, 3H), 2.23(s, 3H), 1.58-1.43(m, 4H), 1.26-1.18(m, 2H), 0.91(s, 9H), [M+H] + m / z = 986.4.

[0394] Example 25: Preparation of compound AG-11

[0395]

[0396] Step 1: Preparation of intermediate AG-11-1

[0397] Take 112 mg (0.28 mmol) of D1, 144 mg (0.38 mmol) of HATU, and 65 mg (0.5 mmol) of DIEA into a 50 mL single-necked flask, add 20 mL of LDM, and stir at room temperature for 1 hour; separately add 62 mg (0.25 mmol) of L11 to the flask, and stir at room temperature overnight; TLC showed that the reaction of the starting material was basically complete, add 100 mL of water, extract with ethyl acetate (50 mL * 3), wash the organic phase with saturated brine, dry with sodium sulfate, filter, evaporate the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluent: ethyl acetate: petroleum ether = 1:5) to obtain 99 mg (yield 63%) of product;

[0398] Step 2: Preparation of intermediate AG-11-2

[0399] Take 99 mg (0.16 mmol) of intermediate AG-11-1 in a 50 mL single-necked flask, add 10 mL of dichloromethane, cool to below 0 °C, and add 3 mL of trifluoroacetic acid dropwise. Stir at room temperature for 6 hours. TLC showed that the reaction of the starting material was basically complete. Add 20 mL of water and extract with dichloromethane (20 mL * 3). Wash the organic phase with saturated brine, dry with sodium sulfate, filter, remove the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluent: dichloromethane: methanol = 10:1) to obtain 79 mg (yield 86%) of intermediate AG-11-2.

[0400] Step 3: Preparation of compound AG-11

[0401] Take 79 mg (0.14 mmol) of intermediate AG-11-2, 74 mg (0.20 mmol) of HATU, and 34 mg (0.26 mmol) of DIEA into a 50 mL single-necked flask, add 10 mL of DMF, and stir at room temperature for 1 hour; separately add 54 mg (0.13 mmol) of P1 to the flask, and stir at room temperature overnight; TLC showed that the reaction of the starting material was basically complete, add 100 mL of water, extract with ethyl acetate (50 mL * 3), wash the organic phase with saturated brine, dry with sodium sulfate, filter, evaporate the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluent: ethyl acetate: petroleum ether = 1:4) to obtain 71 mg (yield 55%) of AG-11.

[0402] 1 HNMR(400MHz, d6-DMSO): δ = 9.09(s, 1H), 8.10(s, 3H), 7.78-7.62(d,2H), 7.55-7.45(d, 4H), 7.38-7.27(d, 2H), 4.73-4.64(m, 1H), 4.58(s, 1H), 4.44-4.33(m, 3H), 3.75-3.45(m, 6H), 3.14-2.77(m, 4H), 2.55(s, 3H), 2.44-2.43(m,2H), 2.41(s, 3H), 2.33(s, 3H), 2.29(s, 3H), 2.24(s, 3H), 1.55-1.47(m, 4H),1.34-1.27(m, 2H), 0.93(s,9H), [M+H] + m / z = 988.5.

[0403] Example 26: Preparation of compound AG-12

[0404]

[0405] Step 1: Preparation of intermediate AG-12-1

[0406] Take 112 mg (0.28 mmol) of D1, 144 mg (0.38 mmol) of HATU, and 65 mg (0.5 mmol) of DIEA into a 50 mL single-necked flask, add 20 mL of LDM, and stir at room temperature for 1 hour; separately add 79 mg (0.25 mmol) of L12 into the flask, and stir at room temperature overnight; TLC showed that the reaction of the starting materials was basically complete, add 100 mL of water, extract with ethyl acetate (50 mL * 3), wash the organic phase with saturated brine, dry with sodium sulfate, filter, evaporate the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluent: ethyl acetate: petroleum ether = 1:5) to obtain 99 mg (yield 57%) of product;

[0407] Step 2: Preparation of intermediate AG-12-2

[0408] Take 99 mg (0.16 mmol) of intermediate AG-12-1 in a 50 mL single-necked flask, add 10 mL of dichloromethane, cool to below 0 °C, and add 3 mL of trifluoroacetic acid dropwise. Stir at room temperature for 6 hours. TLC showed that the reaction of the starting material was basically complete. Add 20 mL of water and extract with dichloromethane (20 mL * 3). Wash the organic phase with saturated brine, dry with sodium sulfate, filter, remove the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluent: dichloromethane: methanol = 10:1) to obtain 83 mg (yield 81%) of intermediate AG-12-2.

[0409] Step 3: Preparation of compound AG-12

[0410] Take 83 mg (0.13 mmol) of intermediate AG-12-2, 68 mg (0.18 mmol) of HATU, and 31 mg (0.24 mmol) of DIEA into a 50 mL single-necked flask, add 10 mL of LDMF, and stir at room temperature for 1 hour; separately add 51 mg (0.12 mmol) of P1 into the flask, and stir at room temperature overnight; TLC showed that the reaction of the starting material was basically complete, add 100 mL of water, extract with ethyl acetate (50 mL * 3), wash the organic phase with saturated brine, dry with sodium sulfate, filter, evaporate the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluting agent: ethyl acetate: petroleum ether = 1:4) to obtain 73 mg (yield 58%) of AG-12.

[0411] 1HNMR(400MHz, d6-DMSO): δ = 9.08(s, 1H), 8.04(s, 3H), 7.82-7.77(d,2H), 7.62-7.48(d, 4H), 7.32-7.23(d, 2H), 4.88-4.72(m, 1H), 4.66(s, 1H), 4.53-4.46(m, 3H), 3.85-3.46(m, 8H), 3.27-2.84(m, 6H), 2.76(s, 3H), 2.68-2.56(m,4H), 2.48(s, 3H), 2.39(s, 3H), 2.28(s, 3H), 1.63-1.47(m, 4H), 1.31-1.25(m,2H), 0.96(s,9H), [M+H] + m / z = 1056.8.

[0412] Example 27: Preparation of compound AG-13

[0413]

[0414] Step 1: Preparation of intermediate AG-13-1

[0415] Take 112 mg (0.28 mmol) of D1, 144 mg (0.38 mmol) of HATU, and 65 mg (0.5 mmol) of DIEA into a 50 mL single-necked flask, add 20 mL of DMF, and stir at room temperature for 1 hour; separately take 109 mg (0.25 mmol) of L9 and add it to the flask, and stir at room temperature overnight; TLC showed that the reaction of the starting material was basically complete, add 100 mL of water, extract with ethyl acetate (50 mL * 3), wash the organic phase with saturated brine, dry with sodium sulfate, filter, evaporate the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluent: ethyl acetate: petroleum ether = 1:5) to obtain 123 mg (yield 60%) of product;

[0416] Step 2: Preparation of intermediate AG-13-2

[0417] 123 mg (0.15 mmol) of intermediate AG-13-1 was placed in a 50 mL single-necked flask, 10 mL of dichloromethane was added, and the mixture was cooled to below 0 °C. 3 mL of trifluoroacetic acid was then added dropwise, and the mixture was stirred at room temperature for 6 hours. TLC analysis showed that the reaction was essentially complete. 20 mL of water was added, and the mixture was extracted with dichloromethane (20 mL x 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: dichloromethane:methanol = 10:1) to obtain 89 mg (78% yield) of intermediate AG-13-2.

[0418] Step 3: Preparation of compound AG-13

[0419] Take 89 mg (0.12 mmol) of intermediate AG-13-2, 63 mg (0.17 mmol) of HATU, and 28 mg (0.22 mmol) of DIEA into a 50 mL single-necked flask, add 10 mL of DMF, and stir at room temperature for 1 hour; separately add 46 mg (0.11 mmol) of P1 into the flask, and stir at room temperature overnight; TLC showed that the reaction of the starting material was basically complete, add 100 mL of water, extract with ethyl acetate (50 mL * 3), wash the organic phase with saturated brine, dry with sodium sulfate, filter, evaporate the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluting agent: ethyl acetate: petroleum ether = 1:4) to obtain 85 mg (yield 66%) of AG-13.

[0420] 1 HNMR(400MHz, d6-DMSO): δ = 9.02(s, 1H), 8.03(s, 3H), 7.71-7.62(d,2H), 7.53-7.43(d, 4H), 7.34-7.24(d, 2H), 4.74-4.68(m, 1H), 4.57(s, 1H), 4.47-4.35(m, 6H), 3.75-3.47(m, 8H), 3.17-2.77(m, 4H), 2.58(s, 3H), 2.47-2.41(m,4H), 2.38(s, 3H), 2.31(s, 3H), 2.27(s, 3H), 1.57-1.45(m, 4H), 1.36-1.18(m,8H), 0.97(s,9H), [M+H] + m / z = 1177.7.

[0421] Example 28: Preparation of compound P2

[0422]

[0423] Step 1: Preparation of compound P2-2

[0424] 39.64 g (198 mmol) of compound P2-1 was placed in a 500 mL single-necked flask, and 300 mL of dichloromethane was added. 30.07 g (297 mmol) of triethylamine was also added to the flask. The mixture was cooled to 0 °C and stirred. 51.58 g (238 mmol) of Boc anhydride was dissolved in 100 mL of dichloromethane and added dropwise to the above system. The mixture was stirred at room temperature for 3 hours. TLC analysis showed that the reaction of the starting material was essentially complete. 300 mL of water was added, and the mixture was extracted with dichloromethane (200 mL * 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was precipitated by methanol / water (10:1) to obtain 49.8 g (84% yield) of the product.

[0425] Step 2: Preparation of compound P2-3

[0426] 10.8 g (36 mmol) of compound P2-2, 7.2 g (72 mmol) of 4-methylthiazole, 7.1 g (72 mmol) of potassium acetate, and 80 mg (0.36 mmol) of palladium acetate were placed in a 500 mL single-necked flask. 50 mL of DMA was added, and the mixture was purged with nitrogen three times. The mixture was heated and stirred at 120 °C for 3 hours under nitrogen protection. TLC showed that the reaction of the starting material was basically complete. The heating was turned off, and the mixture was cooled to room temperature. 600 mL of water was added, and the mixture was extracted with ethyl acetate (200 mL * 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: ethyl acetate: petroleum ether = 1:4) to obtain 6.6 g (yield 58%) of product.

[0427] Step 3: Preparation of compound P2-4

[0428] Take 6.6 g (20.8 mmol) of compound P2-3 into a 500 mL single-necked flask, add 100 mL of hydrochloric acid methanol solution, and stir at room temperature for 2 hours; TLC showed that the reaction of the starting material was basically complete, the solvent was removed by vacuum distillation, and the mixture was distilled multiple times with dichloromethane (100 mL * 3) to obtain 4.8 g of hydrochloride of compound P2-4, which was directly used in the next step of the reaction.

[0429] Step 4: Preparation of compound P2-5

[0430] 5.5 g (23.8 mmol) of Boc-L-hydroxyproline, 10.9 g (28.6 mmol) of HATU, and 9.2 g (71.4 mmol) of DIEA were placed in a 500 mL single-necked flask, 100 mL of LDM was added, and the mixture was stirred at room temperature for 1 hour. 5.2 g (23.8 mmol) of compound P2-4 was then added to the flask, and the mixture was stirred at room temperature overnight. TLC analysis showed that the reaction proceeds were essentially complete. 400 mL of water was added, and the mixture was extracted with ethyl acetate (200 mL x 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: dichloromethane:methanol = 20:1) to obtain 6.4 g (63% yield) of the product.

[0431] Step 5: Preparation of compound P2-6

[0432] Take 6.4 g (14.8 mmol) of compound P2-5 into a 250 mL single-necked flask, add 80 mL of hydrochloric acid methanol solution, and stir at room temperature for 2 hours; TLC showed that the reaction of the starting material was basically complete, the solvent was removed by vacuum distillation, and the solution was distilled multiple times with dichloromethane (100 mL * 3) to obtain 3.9 g of P2-6 hydrochloride, which was directly used in the next step of the reaction;

[0433] Step 6: Preparation of compound P2-7

[0434] 4.1 g (17.5 mmol) of N-Boc-L-tert-leucine, 10 g (26.3 mmol) of HATU, and 6.8 g (52.5 mmol) of DIEA were placed in a 500 mL single-necked flask, and 100 mL of DMF was added. The mixture was stirred at room temperature for 1 hour. 5.8 g (17.5 mmol) of the hydrochloride of compound P2-6 was then added to the mixture, and the mixture was stirred at room temperature overnight. TLC analysis showed that the reaction proceeds were essentially complete. 600 mL of water was added, and the mixture was extracted with ethyl acetate (200 mL x 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: ethyl acetate: petroleum ether = 1:1) to obtain 6.2 g (65% yield) of the product.

[0435] Step 7: Preparation of compound P2

[0436] Take 6.2 g (11.4 mmol) of compound P2-7 into a 250 mL single-necked flask, add 100 mL of hydrochloric acid methanol solution, and stir at room temperature for 2 hours; TLC showed that the reaction of the starting material was basically complete, remove the solvent under reduced pressure, and distill multiple times with dichloromethane (100 mL * 3) to obtain 5.2 g of hydrochloride salt of compound P2, which was directly used in the next step of the reaction.

[0437] Preparation of compounds AG-14 to AG-26: Refer to the reaction procedures in Examples 15 to 27 for specific reaction conditions.

[0438]

[0439]

[0440]

[0441] .

[0442] Example 29: Preparation of compound AG-27

[0443]

[0444] Step 1: Preparation of intermediate AG-27-1

[0445] Take 100 mg (0.25 mmol) of D1, 131 mg (0.35 mmol) of HATU, and 60 mg (0.46 mmol) of DIEA into a 50 mL single-necked flask, add 20 mL of LDM, and stir at room temperature for 1 hour; separately add 49 mg (0.23 mmol) of L1 to the flask, and stir at room temperature overnight; TLC showed that the reaction of the starting material was basically complete, add 100 mL of water, extract with ethyl acetate (50 mL * 3), wash the organic phase with saturated brine, dry with sodium sulfate, filter, evaporate the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluent: ethyl acetate: petroleum ether = 1:5) to obtain 88 mg (yield 64%) of product;

[0446] Step 2: Preparation of intermediate AG-27-2

[0447] Take 88 mg (0.15 mmol) of intermediate AG-27-1 in a 50 mL single-necked flask, add 10 mL of dichloromethane, cool to below 0 °C, and add 3 mL of trifluoroacetic acid dropwise. Stir at room temperature for 6 hours. TLC showed that the reaction of the starting material was basically complete. Add 20 mL of water and extract with dichloromethane (20 mL * 3). Wash the organic phase with saturated brine, dry with sodium sulfate, filter, remove the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluent: dichloromethane: methanol = 10:1) to obtain 69 mg (yield 86%) of intermediate AG-27-2.

[0448] Step 3: Preparation of compound AG-27

[0449] Take 69 mg (0.13 mmol) of intermediate AG-27-2, 66 mg (0.17 mmol) of HATU, and 31 mg (0.24 mmol) of DIEA into a 50 mL single-necked flask, add 10 mL of LDMF, and stir at room temperature for 1 hour; separately add 86 mg (0.12 mmol) of compound P3 into the flask, and stir at room temperature overnight; TLC showed that the reaction of the starting material was basically complete, add 100 mL of water, extract with ethyl acetate (50 mL * 3), wash the organic phase with saturated brine, dry with sodium sulfate, filter, evaporate the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluent: ethyl acetate: petroleum ether = 1:4) to obtain 79 mg (yield 53%) of AG-27. 1 HNMR(400MHz, d6-DMSO): δ = 8.08(s, 3H),7.76-7.69(d, 2H), 7.57-7.48(d, 2H), 7.40-7.35(m, 5H), 5.11-5.06(m, 1H), 4.71-4.67(m, 1H), 4.55-4.52(m, 2H), 4.28-4.17(m, 1H), 3.95-3.90(m, 1H), 3.77-3.72(m, 1H), 3.66-3.60(m, 3H), 3.52-3.48(m, 6H), 3.42-3.40(s, 6H), 3.33-3.30(s,6H), 3.12-2.95(m, 6H), 2.73-2.66(m, 3H), 2.48-2.40(m, 5H), 2.31-2.26(m, 6H),2.02-1.96(m, 4H), 1.53-1.46(m, 6H), 1.33-1.26(m, 5H), 1.12-1.06(m, 3H), 0.96-0.90(m, 18H), [M+H] + m / z = 1245.2.

[0450] Preparation of compounds AG-28 to AG-40: Refer to the reaction process in Example 29 for specific reaction conditions.

[0451]

[0452]

[0453]

[0454]

[0455]

[0456] Example 30: Preparation of compound P4

[0457]

[0458] Step 1: Preparation of compound P4-2

[0459] 15.3 g (93.3 mmol) of compound P2-1 and 37.7 g (373 mmol) of triethylamine were placed in a 500 mL single-necked flask, and 200 mL of acetic acid was added. The mixture was heated to 90 °C and stirred for 2 hours. 18 g (93.3 mmol) of 3-nitrophthalic acid was added to the mixture, and the mixture was heated to 90 °C and stirred for 24 hours. TLC showed that the starting materials had basically reacted completely. The mixture was cooled to room temperature, and the reaction solution was poured into 1 L of water, resulting in a large amount of precipitate. The mixture was filtered, the filter cake was washed with water (50 mL * 3), and dried by forced air to obtain 20.6 g (73% yield) of the product.

[0460] Step 2: Preparation of compound P4

[0461] Take 20.6 g (68 mmol) of compound P2-2 into a 500 mL single-necked flask, add 150 mL of methanol, dissolve to obtain a clear solution, add 2.1 g of palladium on carbon catalyst into it, stir at room temperature for 12 hours under hydrogen atmosphere, TLC shows that the starting material has basically reacted completely, filter, concentrate and dry the filtrate, and use it directly for the next step of the reaction;

[0462] Example 31: Preparation of compound AG-41

[0463]

[0464] Step 1: Preparation of intermediate AG-01-1

[0465] 100 mg (0.25 mmol) of compound D1, 131 mg (0.35 mmol) of HATU, and 60 mg (0.46 mmol) of DIEA were placed in a 50 mL single-necked flask, and 20 mL of DMF was added. The mixture was stirred at room temperature for 1 hour. 49 mg (0.23 mmol) of compound L1 was added to the flask, and the mixture was stirred at room temperature overnight. TLC analysis showed that the reaction proceeds were essentially complete. 100 mL of water was added, and the mixture was extracted with ethyl acetate (50 mL x 3). The organic phase was washed with saturated brine, dried over sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by column chromatography (eluent: ethyl acetate: petroleum ether = 1:5) to obtain 88 mg (64% yield) of the product.

[0466] Step 2: Preparation of intermediate AG-01-2

[0467] Take 88 mg (0.15 mmol) of intermediate AG-01-1 in a 50 mL single-necked flask, add 10 mL of dichloromethane, cool to below 0 °C, and add 3 mL of trifluoroacetic acid dropwise. Stir at room temperature for 6 hours. TLC showed that the reaction of the starting material was basically complete. Add 20 mL of water and extract with dichloromethane (20 mL * 3). Wash the organic phase with saturated brine, dry with sodium sulfate, filter, remove the solvent from the filtrate under reduced pressure, and purify the residue by column chromatography (eluent: dichloromethane: methanol = 10:1) to obtain 69 mg (yield 86%) of intermediate AG-01-2.

[0468] Step 3: Preparation of compound AG-41

[0469] Take 69 mg (0.13 mmol) of intermediate AG-01-2 in a 50 mL single-necked flask, add 10 mL of dichloromethane, and cool the system to 0 °C. Add 2 mL (26 mmol) of thionyl chloride dropwise to the above system. After the addition is complete, return to room temperature and stir for 3 hours. Evaporate the solvent under reduced pressure, and then distill multiple times with dichloromethane (50 mL * 3). Add 15 mL of dichloromethane to obtain an acyl chloride intermediate solution. Add 71 mg (0.26 mmol) of compound P4 and 40 mg (0.39 mmol) of triethylamine to the above system, and stir at room temperature for 4 hours. TLC analysis shows that the reaction of the starting material is basically complete. Add 100 mL of water and 100 mL of ethyl acetate. Wash with 1 M hydrochloric acid solution and saturated brine, respectively. Dry the organic layer with anhydrous sodium sulfate, filter, and evaporate the solvent under reduced pressure. Purify the residue by column chromatography (eluent: ethyl acetate: petroleum ether = 1:4) to obtain 71 mg (yield 57%) of AG-41. 1 HNMR(400MHz, d6-DMSO): δ = 10.02(s,1H), 8.65-8.61(d, 1H), 8.05(s, 1H), 7.86-7.74(m, 3H), 7.62-7.57(m, 3H), 7.25-7.18(s, 1H), 4.65-4.62(m, 1H), 4.56-4.52(m, 1H), 4.33-4.26(s, 2H), 3.36-3.22(m, 2H), 3.05-2.96(m, 3H), 2.73-2.66(m, 1H), 2.48-2.43(s, 3H), 2.31-2.26(s, 3H), 2.22-2.20(s, 3H), 2.16-2.03(m, 4H), 1.53-1.46(m, 4H), 1.12-1.06(m, 2H),[M+H] + m / z = 800.3.

[0470] Preparation of compounds AG-42 to AG-54: Refer to the reaction process in Example 31 for specific reaction conditions.

[0471]

[0472]

[0473]

[0474] .

[0475] Biological evaluation:

[0476] Example 32: In vitro cell assay of the compounds of the present invention

[0477] 1. Materials

[0478] Cell lines:

[0479] LNCap human prostate cancer cells were derived from the Chinese Academy of Sciences Cell Bank.

[0480] 22RV1 human prostate cancer cells were derived from the Chinese Academy of Sciences Cell Bank.

[0481] Vcap human prostate cancer cells were derived from the Chinese Academy of Sciences Cell Bank.

[0482] Reagents and consumables:

[0483] CellTiter-Lumi™ Luminescent Cell Viability Assay Kit

[0484] 384-well cell culture plate (Cat#3765 Corning)

[0485] Fetal bovine serum (Cat#10099141 gibco)

[0486] Culture medium (gibco)

[0487] Test samples: ARV771, the compound to be tested in this invention.

[0488] Reagent preparation

[0489] Table 1 Preparation of culture medium

[0490]

[0491] Preparation of test samples: The compound was dissolved in 5 mM DMSO and stored at -80℃ for later use.

[0492] IC 50 -Cell viability assay procedure

[0493] 1) Collect cells in the logarithmic growth phase, count them, resuspend them in complete culture medium, adjust the cell concentration to an appropriate level, and seed them in 384-well plates. Add 40 μL of phosphate-buffered saline solution to the outer wells of the 384-well plate, and add 36 μL of the cell suspension to the remaining wells of each plate. Then, incubate the 384-well plates overnight in a CO2 incubator. Observe cell growth under a microscope and take photos for archiving.

[0494] 2) Perform serial dilutions of the test compound. Dilute each compound or 10 concentration gradients and add 4 μL / well to a 384-well plate. The final concentration of the compound should start from 10 μM and be serially diluted 3 times for a total of 10 concentration points. Each concentration should be replicated three times.

[0495] 3) Place the cell plate in a carbon dioxide incubator and incubate for 72 hours at 37°C and 5% CO2 concentration;

[0496] 4) Add 40 μL / well of CellTiter-Lumi™ luminescence assay reagent to the cell plate and shake at room temperature in the dark for 10 minutes to stabilize the luminescence signal;

[0497] 5) Use SpectraMax Id5 Multi-Mode Reader to perform chemiluminescence detection and calculate the inhibition rate based on the chemiluminescence intensity.

[0498] Data analysis

[0499] The inhibition rate of drug on tumor cell growth = (A C -A S ) / A C *100%

[0500] A C Chemiluminescence intensity of the negative control

[0501] A S Chemiluminescence intensity of the sample

[0502] Using software to develop ICs 50 Curve fitting and calculation of IC 50 value.

[0503] Experimental Results

[0504] This experiment tested the inhibitory effects of 55 compounds on three cell lines. The final concentrations of the compounds ranged from 10 μM to 0.5 nM, with three-fold serial dilutions, for a total of 10 assay points. The table below shows the IC50 values ​​of each compound in different cell lines. 50 (nM) values ​​(1000nm <++++; 500nm <+++≤1000nm; 100nm <++≤500nm; 0.1nm <+≤100nm).

[0505] Table 2. Inhibitory effects of the compounds involved in this invention on two cell lines.

[0506]

[0507]

[0508] .

[0509] Example 33: Test of BRD protein degradation levels

[0510] 1. Materials

[0511] Cell lines: 22Rv1, Vcap.

[0512] Reagents and consumables:

[0513] 6-well culture plate

[0514] Culture media (DMEM, RPMI)

[0515] Fetal bovine serum (FBS)

[0516] Glutamine (Glutamax, Glu)

[0517] Sodium pyruvate (SP)

[0518] Penicillin-Streptomycin Bivalent Antibody

[0519] RIPA pyrolysis fluid

[0520] Cocktail protease inhibitors

[0521] Protease inhibitors (PMSF)

[0522] 30% Acr-Bis (29:1)

[0523] 1M Tris-HCl, pH 8.8

[0524] 1M Tris-HCl, pH 6.8

[0525] 10% SDS

[0526] Ammonium persulfate substitute (APS substitute)

[0527] TEMED

[0528] Western transfer solution

[0529] QuickBlock Western Sealing Solution

[0530] QuickBlock Western primary antibody dilution solution

[0531] SDS-PAGE protein loading buffer (5X, odorless)

[0532] QuickBlock Western secondary antibody dilution solution

[0533] TBS (10X)

[0534] PageRuler™ Prestained Protein Ladder, 10 to 180 kDa

[0535] SDS-PAGE electrophoresis buffer (Tris-Gly, 10X)

[0536] BCA Protein Concentration Assay Kit (Enhanced Version)

[0537] BRD4

[0538] BRD3

[0539] BRD2

[0540] β acting

[0541] metal bath

[0542] Fully automated chemiluminescence / gel imaging system

[0543] Tested compounds: ARV771, AG-01, AG-08, AG-11, AG-19.

[0544] Experimental steps

[0545] 3.1 Reagent Preparation

[0546] Table 3 Preparation of culture medium

[0547]

[0548] Preparation of cell cryopreservation solution

[0549] 90% FBS + 10% DMSO

[0550] Preparation of compound storage solutions

[0551] Dilute the compound with DMSO to a final concentration of 5 mM.

[0552] Antibody preparation

[0553] Table 4 Antibody Configuration

[0554]

[0555] 3.2 Experimental Procedure

[0556] After all reagents are prepared, confirm that everything is ready before starting to add samples;

[0557] 1) Collect cells in the logarithmic growth phase, count them, resuspend them in complete culture medium, adjust the cell concentration to a suitable level, seed them in 6-well plates, and incubate the plates overnight in a CO2 incubator. Observe cell growth under a microscope; when the cell confluence reaches approximately 80%, take a photograph for record-keeping.

[0558] 2) Remove the culture medium from the 6-well plate and add 900 μL of fresh culture medium to each well;

[0559] 3) Perform serial dilutions of the test compounds. Dilute each compound into 5 concentration gradients and add 100 μL / well to a 6-well plate. The final concentration of the compound should start from 100 nM and be serially diluted 3-fold, for a total of 5 concentration points and one negative control.

[0560] 4) Place the cell plate in a carbon dioxide incubator and incubate for 16 hours at 37°C and 5% CO2 concentration;

[0561] 5) Observe under a microscope and take photos for archiving;

[0562] 6) Remove the culture medium, add 1 ml of PBS to each well and wash once, remove the culture medium, add 300-500 μl / well of RIPA (containing 1% PMSF and 1% cocktail), lyse on ice for 15 min, scrape off the cell lysate from each well with a cell scraper and collect it into an EP tube;

[0563] 7) Centrifuge at 12000 r / min, 4℃ for 15 min;

[0564] 8) Transfer the supernatant to a new EP tube;

[0565] 9) Use the BCA kit to determine the protein concentration of whole cell lysate, and add an appropriate amount of PBS to adjust the protein concentration of a group of samples to be basically consistent;

[0566] 10) After adding an appropriate amount of 5* protein loading, boil the sample at 100℃ for 10 minutes;

[0567] 11) Select an appropriate separating gel concentration based on the molecular weight of the protein being tested;

[0568] 12) Electrophoresis: 85V constant voltage electrophoresis for 30 min, 135V constant voltage electrophoresis for 70 min;

[0569] 13) Transfer: 400mA constant current for 100min

[0570] 14) Rinse with TBST for 5 min, then seal with rapid sealing solution for 15 min;

[0571] 15) Rinse with TBST for 5 min, add the prepared primary antibody according to the ratio, and incubate overnight at 4°C;

[0572] 16) Rinse 3 times with TBST, 5 minutes each time;

[0573] 17) Add the corresponding secondary antibody and incubate at room temperature for 60 minutes;

[0574] 18) Rinse 3 times with TBST, 5 minutes each time;

[0575] 19) ECL color development, photograph and save using chemiluminescence analyzer.

[0576] Experimental Results

[0577] See the appendix for experimental results. Figure 1 .

[0578] Example 34: In vivo pharmacodynamic testing

[0579] This invention uses BALB / c Nude mice as a tumor-bearing experimental model. In accordance with the requirements of the "Guidelines for Pharmacodynamics of Antitumor Drugs" and the "Technical Guidelines for Non-Clinical Research of Cytotoxic Antitumor Drugs", the invention observes whether the preferred compound of this patent has an inhibitory effect on the growth of human myeloid monocytic leukemia MV-4-11 and MOLM16 cell subcutaneous xenografts in nude mice and the intensity of such effect.

[0580] Groups and Dosing Regimens

[0581] Model control group: Intraperitoneal injection of an equal volume of solvent once a day, observed for 10 to 21 days depending on the tumor condition;

[0582] ARV-771 group: intraperitoneal injection, 30 mg / kg, once daily;

[0583] AG-08 group: Intraperitoneal injection, 15 mg / kg, once daily;

[0584] AG-08 group: Intraperitoneal injection, 30 mg / kg, once every other day;

[0585] AG-01 group: Intraperitoneal injection, 30 mg / kg, once daily;

[0586] AG-19 group: intraperitoneal injection, 30 mg / kg, once daily.

[0587] test animals

[0588] Source, strain, line: BALB / c Nude mice, provided by Zhejiang Vital River Laboratory Animal Technology Co., Ltd. Experimental animal production license: SCXK(Zhe) 2019-0001; Certificate number: 20210326Abzz0619000692; Age: 4-5W; Gender: male; Number of animals: 8 per group, 24 in total.

[0589] Animal model establishment

[0590] Collect the cultured human myelomonocytic leukemia MV-4-11 cell suspension with a concentration of 1X10 7 cells / mL and inoculate 0.2 mL per mouse subcutaneously into the right axilla;

[0591] The transplanted tumors of mice were measured for the diameter of the transplanted tumors with a vernier caliper. After 10 days of inoculation, when the tumors grew to an average volume of 140 mm 3 , the animals were randomly divided into groups of 8. At the same time, the mice in each group started to be administered drugs. The drug administration plan is shown in the group and drug administration plan. The method of measuring the tumor diameter was used (the calculation formula for tumor volume is: V = 0.5a × b 2 , where a and b represent the long diameter and short diameter of the tumor respectively), and the anti-tumor effect of the test sample was dynamically observed. After the experiment, the mice were sacrificed immediately, and the tumor masses were surgically removed and weighed.

[0592] Statistical analysis

[0593] The mean values are expressed as mean±SEM, and the inter-group analysis was performed by t-test for statistical analysis. GraphPad Prism 8.0.2 was used to statistically analyze the results.

[0594] Table 5 Tumor volumes at different time points in each group

[0595]

[0596] Table 6 Evaluation of the anti-tumor efficacy of AG-08, AG-19 and AG-01 on the MV-4-11 transplanted tumor model of human myelomonocytic leukemia cells (calculated based on the tumor volume on the 21st day after drug administration)

[0597]

Claims

1. A compound represented by formula (I): in, A represents the BET protein ligand, B represents the E3 ubiquitin ligase ligand, and L represents the linker chain connecting A and B. These three parts are linked by chemical bonds; the compound of formula (I) is selected from: 。 2. A pharmaceutical composition, characterized in that... The active ingredient comprises an effective amount of the compound of formula (I) as claimed in claim 1, and one or more pharmaceutically acceptable carriers, diluents or excipients.

3. Use of the compound of formula (I) according to claim 1 in the preparation of a drug for treating prostate cancer and leukemia.

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